Nicotine addiction, opioid use disorder, and COVID-19 have made lasting impacts on every aspect of society. These are complicated conditions, and studies in these fields will likely continue for decades, if not centuries. Here, we make contributions to each of these issues using electrophysiology and microscopy. The first chapter goes into the motivation behind this thesis and the major experiments I used in my graduate career. In the second chapter, we introduce a new amino acid into the mouse muscle nicotinic acetylcholine receptor in an attempt to understand the dynamics of receptor activation. In the third chapter, we continue the Lester lab\u2019s work on the neuroscientific effects of menthol and how it plays a role in nicotine addiction. We found the binding site for menthol on the \u03b14\u03b22 nicotinic acetylcholine receptor, which continues our hypothesis that the neuroscientific effects of menthol are detrimental to cigarette smokers. Fortunately, partly because of our studies, mentholated nicotine products are being phased out of the United States. The fourth and fifth chapters investigate \u03bc-opioid receptor trafficking, both the trafficking from the endoplasmic reticulum and endocytosis from the plasma membrane. Both of these events play a role in inducing opioid use disorder and increasing the danger of using opioids. We hope that these studies will help other researchers understand opioid use disorder and fight the opioid epidemic. Finally, we studied the effects of SARS-COV-2 proteins on epithelial sodium channels. These channels are important for regulating lung fluid levels where their improper function may cause pulmonary edema. Pulmonary edema has been observed in COVID-19 patients. Altogether, we believe that we have made meaningful impacts on these important health concerns in this thesis. We look forward to how the scientific communities continue to build on our results.
", "doi": "10.7907/pdtj-8238", "publication_date": "2022", "thesis_type": "phd", "thesis_year": "2022" }, { "id": "thesis:13989", "collection": "thesis", "collection_id": "13989", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:11032020-013753331", "primary_object_url": { "basename": "PhD Thesis_Maiko Obana.pdf", "content": "final", "filesize": 21540937, "license": "other", "mime_type": "application/pdf", "url": "/13989/1/PhD Thesis_Maiko Obana.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Genetically Encoded 3,4-Ethylenedioxythiophene (EDOT) Functionality for Fabrication of Protein-Based Conductive Polymers", "author": [ { "family_name": "Obana", "given_name": "Maiko", "orcid": "0000-0003-4150-0055", "clpid": "Obana-Maiko" } ], "thesis_advisor": [ { "family_name": "Tirrell", "given_name": "David A.", "orcid": "0000-0003-3175-4596", "clpid": "Tirrell-D-A" } ], "thesis_committee": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" }, { "family_name": "Kornfield", "given_name": "Julia A.", "orcid": "0000-0001-6746-8634", "clpid": "Kornfield-J-A" }, { "family_name": "Robb", "given_name": "Maxwell J.", "orcid": "0000-0002-0528-9857", "clpid": "Robb-M-J" }, { "family_name": "Tirrell", "given_name": "David A.", "orcid": "0000-0003-3175-4596", "clpid": "Tirrell-D-A" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Genetic code expansion provides powerful strategies to improve the properties of protein-based materials. One novel application of this technique is to genetically incorporate an electroactive functional group into proteins, which can be subsequently polymerized into conductive polymers, enabling fabrication of various protein\u2013conductive polymer hybrids that are widely applicable in bioelectronics. To this end, we developed a technique to incorporate an amino acid bearing 3,4-ethylenedioxythiophene (EDOT), the monomer precursor of a well-known conductive polymer PEDOT.
\r\n\r\nIn Chapter 1, we review the basics of protein-based materials and genetic code expansion technology. We also highlight some examples where genetic code expansion was used for the development of protein-based materials. Finally, we discuss applications of protein and peptide-based materials in bioelectronics.
\r\n\r\nIn Chapter 2, we describe our effort to incorporate an amino acid bearing EDOT group (EDOT-Ala) designed as an analogue of aromatic canonical amino acids. We synthesized EDOT-Ala in three steps of organic reaction, and evaluated the activity of known aminoacyl-tRNA synthetase (aaRS) variants for EDOT-Ala. In addition, we performed evolution of aaRS for EDOT-Ala using two different evolution techniques: cell viability- based approach and phage-assisted approach. Although the evolution experiment did not yield an aaRS variant that can incorporate EDOT-Ala, the results presented in this chapter provide valuable information for engineering of aaRS and incorporation of non-canonical amino acids (ncAA) with bulky functional groups.
\r\n\r\nIn Chapter 3, we describe the incorporation of another EDOT-functionalized amino acid (EDOT-Lys) designed as an analogue of a canonical amino acid pyrrolysine (Pyl). When we co-expressed a GFP reporter and a mutant pyrrolysyl-tRNA synthetase (PylRS) in E. coli in the presence of EDOT-Lys, the cells exhibited strong fluorescence as an indication of successful incorporation of EDOT-Lys into GFP. We further confirmed the incorporation using MALDI-TOF mass spectrometry.
\r\n\r\nIn Chapter 4, we describe the electropolymerization of a model protein XTEN that carries genetically incorporated EDOT-Lys (XTEN-E49am). We performed electropolymerization of XTEN-E49am in the presence of a self-doping EDOT monomer (EDOT-S) by cyclic voltammetry. The solution formed dark blue solids immediately after the potential cycles. The composition of the product was determined by FT-IR spectroscopy, suggesting that one protein is found per 12.5 monomer units of PEDOT. In addition, we investigated the effect of amino acids located adjacent to EDOT-Lys. Although the presence of cysteine (Cys), lysine (Lys), methionine (Met), arginine (Arg), and tryptophan (Trp) located adjacent to EDOT-Lys had an impact on the electropolymerization of model peptides, XTEN proteins carrying these adjacent residues (XTEN-E49am-G50Z; Z = Cys, Lys, Met, Arg, Trp) were electropolymerized with EDOT-S without noticeable effect from these adjacent residues, indicating that the EDOT-Lys residues in proteins undergo electropolymerization with EDOT-S in different chemical environments.
\r\n\r\nIn Chapter 5, we describe the oxidative chemical polymerization of XTEN proteins and model peptides. When XTEN-E49am was polymerized with EDOT-S by addition of ammonium persulfate (APS) and iron(III) chloride (FeCl3), the solution yielded dark blue solids. To evaluate the reactivity of the EDOT-Lys residue in the protein, we reacted the protein with an end-capped EDOT derivative (EDOT-cap). MALDI-TOF mass spectrometry revealed the appearance of new peaks corresponding to the addition of one or two EDOT-caps to the protein, suggesting that EDOT-Lys residue in the protein can react with EDOT derivatives. We also investigated the effect of adjacent amino acids using a series of model peptides. In polymerization using APS without FeCl3 catalyst, peptides carrying basic amino acids (His, Lys, Arg) adjacent to EDOT-Lys showed enhanced polymerization compared to the ones carrying neutral and acidic adjacent residues. All the tested peptides polymerized well when FeCl3 was added as a catalyst. The results presented in this chapter provide valuable insights into synthesis of protein\u2013PEDOT conjugates via oxidative chemical polymerization.
", "doi": "10.7907/a9qc-cp16", "publication_date": "2021", "thesis_type": "phd", "thesis_year": "2021" }, { "id": "thesis:13870", "collection": "thesis", "collection_id": "13870", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:09082020-225341960", "primary_object_url": { "basename": "Proofread_v_Threatt-thesis-compiled.pdf", "content": "final", "filesize": 11152640, "license": "other", "mime_type": "application/pdf", "url": "/13870/1/Proofread_v_Threatt-thesis-compiled.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "In Vivo Activity of Rhodium Metalloinsertors and Exploration of Drug Delivery Systems", "author": [ { "family_name": "Threatt", "given_name": "Stephanie Denise", "orcid": "0000-0002-2303-2166", "clpid": "Threatt-Stephanie-Denise" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" }, { "family_name": "Dougherty", "given_name": "Dennis A.", "orcid": "0000-0003-1464-2461", "clpid": "Dougherty-D-A" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Synold", "given_name": "Timothy", "orcid": "0000-0002-4075-2544", "clpid": "Synold-Timothy" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Rhodium metalloinsertors are octahedral complexes developed to selectively target the mismatches and insertions/deletions (indels) that result from mismatch repair (MMR) deficient cancers. By incorporating particularly wide, aromatic, inserting ligands, these complexes are able to detect thermodynamically destabilized mismatch sites via a binding mode known as metalloinsertion, in which the inserting ligand binds DNA via the minor groove and results in ejection of the destabilized mismatched base pair. In vitro analyses of metalloinsertors have found that these complexes are selectively cytotoxic towards MMR-deficient cancer cells compared to MMR-proficient cells. Furthermore, the newest family of Rh-O metalloinsertors, which includes [Rh(phen)(chrysi)(PPO)]\u00b2\u207a (Rh-PPO), displays preferential cytotoxicities in the nanomolar range, which is significantly more potent than first generation metalloinsertors and many standard of care chemotherapeutics. Given the high level of potency and selectivity of Rh-O metalloinsertors, further clinical development of these complexes has been pursued.
\r\n\r\nHere, we present the first preclinical mouse evaluation of a rhodium metalloinsertor as an anticancer agent. The Rh-O metalloinsertor Rh-PPO was evaluated in the HCT116 colorectal cancer xenograft tumor model alongside saline and oxaliplatin controls. Intraperitoneal studies with Rh-PPO showed significant decreases in tumor volumes over time and final tumor weights, indicating Rh-PPO has notable anticancer activity. Additionally, Rh-PPO treatment resulted in a noteworthy increase in the length of mouse survival that was on par with the FDA approved chemotherapeutic oxaliplatin. Pharmacokinetic analyses revealed rapid absorption of Rh-PPO in plasma with notable accumulation in the liver compared to tumors. Importantly, intratumoral metalloinsertor administration resulted in enhanced anticancer effects, which points to a need for more selective delivery methods in order to further metalloinsertor development.
\r\n\r\nIn order to target cancerous cells with still higher selectivity, routes to metalloinsertor antibody drug conjugate (ADC) designs were explored. By attaching Rh-O metalloinsertors to an antibody specific to cancer-associated antigens, our complexes may become even more specifically directed to induce selective cytotoxicity in diseased cells. Three ADC drug linkers that incorporate maleimide groups into the N^O coordinating ligand of a Rh-O metalloinsertor were designed, synthesized, and characterized. These complexes were evaluated for their cellular potency and selectivity toward MMR-deficient cancer cells. Studies revealed that functionalization of the hydroxyl-containing ancillary ligand resulted in decreased potency and abolished preferential cytotoxicity, contrary to previous studies that assessed modifications of this ligand.
\r\n\r\nLiposomal formulations of Rh-PPO were also explored to further target metalloinsertors to malignant cells. Liposomal drug encapsulations have a demonstrated ability to decrease systemic toxicity and increase tumor drug uptake; therefore, the biological activity of Rh-PPO liposomal formulations was explored. Four distinct Rh-PPO liposome formation methods were developed and the resulting liposomes were assessed for their encapsulation efficiency, cellular toxicity, and stability. Remote loaded Rh-PPO liposomes were found to display the most promising chemical and biological characteristics, although additional optimization of encapsulation procedures is necessary for further preclinical evaluation of this metalloinsertor drug delivery approach.
\r\n\r\nAs metalloinsertors continue preclinical assessment and development, a greater understanding of their mechanism of action is imperative. Biological studies with Rh-PPO and the fluorescent analogue RhPPO-Cy3 have shown that DNA damage from metalloinsertor treatment involves the formation of DNA double strand breaks near metalloinsertor-mismatch binding sites. Furthermore, the DNA damage response, including recruitment of pH2AX and Rad51 proteins, becomes activated in response to Rh-PPO treatment. In order to further elucidate the unique mechanism of action of Rh-O metalloinsertors, which involves both metalloinsertor enantiomers binding to DNA mismatches and displaying biological activity, structural studies are ongoing. X-ray crystallography and microelectron diffraction (microED) techniques have been used in attempts to obtain a high resolution structure of Rh-O metalloinsertors bound to DNA mismatch sites. Gaining these structural insights will be critical to understanding the increased cytotoxic selectivity and uniquely high potency of these second generation metalloinsertor complexes.
\r\n\r\nThe experiments detailed in this thesis have advanced the preclinical development of rhodium metalloinsertors. The ability of Rh-O metalloinsertors to decrease tumor growth in vivo has been established. Additionally, liposomal and ADC metalloinsertor drug formulations have been pursued as drug delivery systems, and the biological mechanisms relevant to metalloinsertor activity have been analyzed. Additional efforts to study rhodium metalloinsertors will continue to advance these promising chemotherapeutics as novel, targeted treatments for MMR-deficient cancers.
", "doi": "10.7907/dmqv-ed54", "publication_date": "2021", "thesis_type": "phd", "thesis_year": "2021" }, { "id": "thesis:13800", "collection": "thesis", "collection_id": "13800", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06082020-163907557", "primary_object_url": { "basename": "Silva_RebekahMB_2020.pdf", "content": "final", "filesize": 21105463, "license": "other", "mime_type": "application/pdf", "url": "/13800/2/Silva_RebekahMB_2020.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Attributes of the [4Fe4S] Cofactor Coordinated by UvrC, a DNA Repair Enzyme", "author": [ { "family_name": "Silva", "given_name": "Rebekah Miriam Brawer", "orcid": "0000-0002-9144-4939", "clpid": "Silva-Rebekah-Miriam-Brawer" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Protein-bound iron sulfur clusters are critical in cells and allow proteins to carry out many essential functions as electron carriers, catalysts for challenging organic reactions, and sensors of cellular environments. A wide range of protein families are known to coordinate iron sulfur clusters, and a growing category includes proteins involved in maintenance of the genome. Within the last three decades, iron sulfur clusters have been demonstrated to be important for enzymes that function in DNA repair, DNA replication, and transcription pathways. To date, iron sulfur clusters in the cubane [4Fe4S] geometry with all cysteine ligands have been exclusively reported for DNA repair and replication enzymes. In contrast to enzymes where the cofactor is necessary for active site chemistry or directly-linked to protein function, the [4Fe4S] cluster in the overwhelming majority of repair and replication enzymes is not involved in the catalytic modification of DNA substrates. Rather, the role of the cofactor appears to vary in function from protein to protein, and has been demonstrated to be important for protein stability, in the assembly of multisubunit proteins, and for substrate recognition, among other roles. Through investigations of the redox chemistry of the cofactor, our group has found that these enzymes participate in DNA-mediated charge transport chemistry, the process through which electrons rapidly migrate through well-stacked, duplex DNA. Long-range, DNA-mediated redox signaling provides a means of rapid communication among DNA-processing proteins for organizing repair and replication activities across the nucleus.
\r\n\r\nNotably, the first observations of the [4Fe4S] cofactor associated with repair and replications enzymes has consistently occurred well after the first biochemical studies of these enzymes. In some cases, the demonstration of a [4Fe4S] center has taken place decades later after initial work. Some proteins have required use of anaerobic methods in order to detect the cofactor, perhaps explaining why in some cases the metal center had eluded observation. Analysis of protein sequences might be expected to help accelerate identification of new iron sulfur centers in repair and replication enzymes. However, even with the abundance of sequencing data available in the post-genomic era, prediction of a metal center based on sequences alone has been challenging. This is in large part because the spacing of the coordinating cysteine residues can be quite irregular, leading to a weak bioinformatic signature.
\r\n\r\nIdentifying proteins with overlooked [4Fe4S] cofactors poses an exciting challenge, and there are some elegant examples in the literature where data from genetics assays has been used in combination with careful sequence analysis to predict and discover iron sulfur centers in repair and replication enzymes. Described here is the evolution of our studies on one well-known repair enzyme from Escherichia coli, UvrC. UvrC is part of the nucleotide excision repair pathway in the Bacteria domain which is responsible for addressing the wide class of bulky, helix-distorting lesions that can form after exposure to sources such as ultraviolet light, cigarette smoke, chemotherapeutics, and protein-DNA crosslinks. UvrC, an excision nuclease with two distinct active sites that incise the phosphodiester backbone on either side of the site of damage, has been historically challenging to study. Given how essential UvrC is in repairing damaged substrates, new insight has been greatly needed.
\r\n\r\nThrough integration of several key reports from the literature regarding the sequence of UvrC and evidence that pointed to a cofactor from genetics assays, our group predicted that UvrC is a [4Fe4S] protein. Development of a new overexpression system and an anaerobic purification method allowed for isolation of UvrC in holo form. We used spectroscopic techniques to confirm that the cluster type was [4Fe4S], and a combination of spectroscopy and chromatography to demonstrate that the UvrC-bound cofactor is susceptible to oxidative degradation. We also found that loss of the cofactor, either through aerobic degradation or mutation of coordinating cysteines, is associated with aggregation of apoprotein. Importantly, in its holo form with the cofactor bound, UvrC forms high affinity complexes with duplexed DNA substrates; the apparent dissociation constants to well-matched and damaged duplex substrates are 100 \u00b1 20 nM and 80 \u00b1 30 nM, respectively. This high affinity DNA binding contrasts reports made for isolated protein lacking the cofactor. Moreover, using DNA electrochemistry, we find that the cluster coordinated by UvrC is redox-active and participates in DNA-mediated charge transport chemistry with DNA-bound midpoint potential of 90 mV vs. NHE.
\r\n\r\nThe work detailed in this dissertation has highlighted how critical the [4Fe4S] center is for UvrC, where the cofactor has been implicated in protein stabilization, substrate binding, and redox signaling on DNA. Handling an apo form of UvrC may have led to the previous challenges catalogued by researchers. Through the development of entirely new methods to study UvrC under anaerobic conditions, many opportunities are now available to study UvrC and the NER pathway anew in vitro and in vivo. Such work will contribute additional insight on how iron sulfur clusters are essential for enzymes that maintain genomic integrity.
\r\n\r\n", "doi": "10.7907/r0j6-jk09", "publication_date": "2020", "thesis_type": "phd", "thesis_year": "2020" }, { "id": "thesis:13667", "collection": "thesis", "collection_id": "13667", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:04022020-212557295", "type": "thesis", "title": "Mechanisms of Phenazine-Mediated Extracellular Electron Transfer by Pseudomonas aeruginosa", "author": [ { "family_name": "Saunders", "given_name": "Scott Harrison", "orcid": "0000-0003-4224-9106", "clpid": "Saunders-Scott-Harrison" } ], "thesis_advisor": [ { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" } ], "thesis_committee": [ { "family_name": "Meyerowitz", "given_name": "Elliot M.", "orcid": "0000-0003-4798-5153", "clpid": "Meyerowitz-E-M" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" }, { "family_name": "Murray", "given_name": "Richard M.", "orcid": "0000-0002-5785-7481", "clpid": "Murray-R-M" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "Extracellular electron transfer (EET), the process whereby cells access electron acceptors or donors that reside many cell lengths away, enables metabolic activity by microorganisms, particularly under oxidant-limited conditions that occur in multicellular bacterial biofilms. Although different mechanisms underpin this process in individual organisms, a potentially widespread strategy involves extracellular electron shuttles, redox-active metabolites that are secreted and recycled by diverse bacteria. Here, I first review general aspects of the electron shuttling strategy, such as the chemical diversity and potential distribution of electron shuttle producers and users, and the costs associated with electron shuttle biosynthesis. Then I address the long-standing question: how do these electron shuttles catalyze electron transfer within biofilms without being lost to the environment? I show that phenazine electron shuttles mediate efficient EET through interactions with extracellular DNA (eDNA) in Pseudomonas aeruginosa biofilms, which are important in nature and disease. Retention of pyocyanin (PYO) and phenazine carboxamide in the biofilm matrix is facilitated by binding to eDNA. In vitro, different phenazines can exchange electrons in the presence or absence of DNA and phenazines can participate directly in redox reactions through DNA; the biofilm eDNA can also support rapid electron transfer between redox-active intercalators. Electrochemical measurements of biofilms indicate that retained PYO supports an efficient redox cycle with rapid EET and slow loss from the biofilm. Together, these results establish that eDNA facilitates phenazine metabolic processes in P. aeruginosa biofilms, suggesting a model for how extracellular electron shuttles achieve retention and efficient EET in biofilms.
", "doi": "10.7907/P4Z5-5445", "publication_date": "2020", "thesis_type": "phd", "thesis_year": "2020" }, { "id": "thesis:11325", "collection": "thesis", "collection_id": "11325", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:12212018-140003184", "type": "thesis", "title": "Exploring the Biological Activity of Rhodium Metalloinsertors", "author": [ { "family_name": "Boyle", "given_name": "Kelsey Melinda", "orcid": "0000-0002-6728-8403", "clpid": "Boyle-Kelsey-Melinda" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Rhodium metalloinsertors are a unique family of potential anticancer agents that have been show to bind selectively to thermodynamically destabilized DNA base pair mismatches, abasic sites, and insertions/deletions (indels) in vitro. These metalloinsertors are also able to target mismatches in cells: metalloinsertors preferentially kill mismatch repair (MMR)-deficient cancer cells, which have a relative abundance of uncorrected DNA mismatches and indels, over MMR-proficient cells, which can repair these lesions. As such, these complexes have shown great promise as a potential treatment strategy for MMR-deficient cancers, which are often resistant to classic chemotherapies.
\r\n\r\nRecently, a new class of metalloinsertors that bear a rhodium-oxygen bond was synthesized and shown to have remarkable potency and selectivity towards MMR-deficient cells. We have discovered many key differences between first generation metalloinsertors and these new Rh-O metalloinsertors: (1) the MMR-selectivity of first generation metalloinsertors is heavily influenced by ancillary ligand bulk and lipophilicity, whereas the MMR-selectivity of Rh-O metalloinsertors is strong regardless of ancillary ligand properties, (2) first generation metalloinsertors have toxicities in the micromolar range while Rh-O metalloinsertors have toxicities in the nanomolar range, and (3) first generation metalloinsertors can only bind DNA via the \u0394-enantiomer while Rh-O metalloinsertors can bind DNA via both the \u0394- and \u039b-enantiomers. Excitingly, the improved potency and selectivity of these \"Rh-O\" metalloinsertors brings them into a realm of clinical relevance.
\r\n\r\nHere we examine the basis for the improved potency and selectivity of these new Rh-O metalloinsertors. A family of six Rh-O metalloinsertors that vary in the steric bulk and lipophilicity of an ancillary ligand was synthesized and characterized. Regardless of ancillary ligand identity, these Rh-O metalloinsertors exhibit nanomolar or low-micromolar toxicities and all preferentially target MMR-deficient cancer cells over MMR-proficient cells. Notably, the off-target accumulation of these metalloinsertors in mitochondria is very low. This cellular distribution is in stark contrast with first generation metalloinsertors in which increased ligand lipophilicity led to increased mitochondrial uptake and ultimately non-selective mitochondrial-mediated cell death. We believe robust selectivity of these complexes is retained in part due to their low off-target accumulation in the mitochondria, which is further complemented by the low dosing requirements of these potent therapeutic agents.
\r\n\r\nOur studies also suggest the high potency of these complexes may be due to a difference in DNA-binding abilities, which is supported by observed differences in which enantiomers can bind to DNA mismatches, differences in ligand buckling at physiological pH, and lipophilicity of the therapeutics, with Rh-O metalloinsertors being dramatically more lipophilic than their first generation counterparts. To better understand the structural basis for this increased potency, crystallographic experiments are underway. A first generation metalloinsertor was previously crystallized with mismatched DNA, and the structure was pivotal in identifying the DNA binding mode of metalloinsertion. Using similar methods, we are working to produce a high-resolution crystal structure of an Rh-O metalloinsertor with mismatched DNA in order to gain structural insights into the increased potency of these new complexes. A significant difference in DNA binding could result in different biological activation of proteins and overall higher potency of these Rh-O metalloinsertors.
\r\n\r\nFinally, as metalloinsertors are moved towards pre-clinical study, understanding their biological activity in diverse cell culture experiments is essential. We examined a metalloinsertor and the FDA approved chemotherapeutic agent cisplatin in 27 diverse colorectal cancer cell lines. The comparison of these drugs revealed the metalloinsertor to be on average five times more potent than cisplatin in this panel. The potency of the metalloinsertor in different cell lines spanned nearly three orders of magnitude and correlated with whole-cell uptake of rhodium. Additionally, a fluorescent metalloinsertor conjugate was used to quantify the number of lesions in DNA that could be targeted by metalloinsertion, a result that correlated well with the potency of a metalloinsertor across several cell lines, consistent with DNA mismatches as the effective biological target of the metalloinsertor.
\r\n\r\nThe experiments described within this thesis have allowed us to gain a better understanding of the biological activity of rhodium metalloinsertors. We have established that Rh-O metalloinsertors are distinct from first generation metalloinsertors, and that these new metalloinsertors can serve as highly tunable, potent, and mismatch-selective anticancer agents. Furthermore, this potency is observed across diverse cell lines and has been shown to correlate with the number of genomic DNA lesions that can be bound by metalloinsertion. The unique biological activity of these complexes makes them ideal candidates for the treatment of MMR-deficient cancers, and the potency and tunability of Rh-O metalloinsertors will allow for the development of previously unattainable diagnostic and therapeutic tools for MMR-deficiencies.
", "doi": "10.7907/1KNM-Y111", "publication_date": "2019", "thesis_type": "phd", "thesis_year": "2019" }, { "id": "thesis:11519", "collection": "thesis", "collection_id": "11519", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05142019-181954795", "primary_object_url": { "basename": "JCB_Thesis_Compiled.pdf", "content": "final", "filesize": 24161328, "license": "other", "mime_type": "application/pdf", "url": "/11519/67/JCB_Thesis_Compiled.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "Reaction Development for the Total Syntheses of the Terpenoid Natural Products (+)-Psiguadial B, (+)-Rumphellaone A, and (\u2013)-Isodocarpin", "author": [ { "family_name": "Beck", "given_name": "Jordan Casey", "orcid": "0000-0003-0898-5644", "clpid": "Beck-Jordan-Casey" } ], "thesis_advisor": [ { "family_name": "Reisman", "given_name": "Sarah E.", "orcid": "0000-0001-8244-9300", "clpid": "Reisman-S-E" } ], "thesis_committee": [ { "family_name": "Stoltz", "given_name": "Brian M.", "orcid": "0000-0001-9837-1528", "clpid": "Stoltz-B-M" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" }, { "family_name": "Robb", "given_name": "Maxwell J.", "orcid": "0000-0002-0528-9857", "clpid": "Robb-M-J" }, { "family_name": "Reisman", "given_name": "Sarah E.", "orcid": "0000-0001-8244-9300", "clpid": "Reisman-S-E" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The de novo synthesis of bioactive natural products provides an opportunity to learn more about the mechanism of bioactivity and to develop novel chemistry that is of interest to the synthetic community. Herein, we describe our strategy for the total synthesis of the trans-fused cyclobutane containing meroterpenoid (+)-psiguadial B. Key to this strategy was the development of a photochemical Wolff Rearrangement with asymmetric ketene aminolysis. A palladium-catalyzed C\u2013H alkenylation is used to build structural complexity, and we use two different epimerization strategies to perform an enantiodivergent synthesis of (+)-psiguadial B.
\r\n \r\nThis strategy was explored further and applied to the synthesis of chiral cyclobutanes through a 1,2-difunctionalization strategy, wherein a C\u2013H arylation forges one carbon-carbon bond and a subsequent decarboxylative cross-coupling enables functionalization at the adjacent carbon. This strategy enabled the asymmetric total synthesis of (+)-rumphellaone A in 9 steps.
\r\n \r\nThis report also highlights the work we have conducted in the development of a unified strategy for the enmein-type ent-kauranoid natural product, (\u2013)-isodocarpin. We detail our investigation of a convergent cross-electrophile coupling as a means to build the core of (\u2013)-isodocarpin. We also discuss our development of a 1,2-addition/semi-Pinacol rearrangement strategy for the preparation of all-carbon quaternary centers, which can be elaborated to enmein-type ent-kauranoid natural product scaffolds.
\r\n", "doi": "10.7907/78A5-K715", "publication_date": "2019", "thesis_type": "phd", "thesis_year": "2019" }, { "id": "thesis:11282", "collection": "thesis", "collection_id": "11282", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:11262018-103442842", "primary_object_url": { "basename": "AZ_thesis_FINAL.pdf", "content": "final", "filesize": 12244069, "license": "other", "mime_type": "application/pdf", "url": "/11282/1/AZ_thesis_FINAL.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Investigations of DNA-Mediated Redox Signaling Between E.coli DNA Repair Pathways", "author": [ { "family_name": "Zhou", "given_name": "Andy", "orcid": "0000-0003-3383-0855", "clpid": "Zhou-Andy" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The 4Fe4S cluster has been identified in various DNA-processing proteins spanning a variety of biological functions and all domains of life. Recently, a novel functional role for the cluster has been identified for proteins in DNA repair and replication as a redox switch for DNA binding. Human DNA primase utilizes this redox switch to coordinate primer handoff in replication. The enzymatic activity of DNA polymerase \u03b4 is tuned by the redox-switch, allowing for a fast and reversible regulation of replication in response to oxidative stress. In all cases, the redox of the 4Fe4S cluster is achieved through DNA-mediated charge transport (CT), the ability for DNA to carry charge through its \u03c0-stack. Due to the reliance of this phenomena on the \u03c0-stacking of the nitrogenous bases, DNA CT is sensitive to DNA lesions and mismatches and can proceed over long molecular distances if the DNA is well-stacked. Given this powerful biological phenomena, new inter-protein signaling interactions have been identified with important downstream consequences for genome fidelity. Here, we investigate the ways DNA-mediated charge transport between DNA processing enzymes results in efficient DNA repair or prevention of DNA-damage.
\r\n\r\nFirst, we investigated Dps, a bacterial ferritin that protects DNA from oxidative stress and implicated in bacterial survival and virulence. Dps iron sites can scavenge diffusing oxidants directly but additionally electrons and electron holes can be rapidly transported through the base-pair \u03c0-stack though DNA CT, thus providing an additional mechanism of genome protection by Dps. Using X-band EPR, we monitored formation of mononuclear high-spin Fe(III) sites of low symmetry as a gauge of effective Dps protection via oxidation of its iron sites. Using poly(dGdC)2 or poly(dAdT)2 DNA, we uncovered the dependence of DNA protection by Dps to the formation of guanine radical intermediates. Oxidation of Dps iron sites depended on the presence of the W52 residue. Point mutations of W52 revealed its involvement in an electron transfer (ET) pathway for the oxidation of the Dps iron sites. Finally, we investigated the in vivo consequences of the Dps W52 residue by complementing knockout Dps E.coli with plasmids expressing WT, W52A, or W52Y Dps and applying oxidative stress to the cells through hydrogen peroxide treatment. These assays further demonstrated the ability of Dps to protect the E.coli genome from harmful oxidants DNA-mediated electron transfer processes.
\r\n\r\nSecond, we assessed the redox properties of EndoIII and MutY, two base excision repair glycosylases containing 4Fe4S clusters, in the presence and absence of DNA. Previous work has shown these proteins to have a midpoint redox potential around 80mV vs. NHE when bound to DNA with a positive shift in potential in the absence of DNA. However, electrochemical details that define this midpoint potential have not been uncovered. Using a pyrolytic graphite edge electrode, we measured the midopoint potential of point mutations of EndoIII where point charges are flipped near the cluster (K208E, Y205H, and E200K) in the absence of DNA. Our measurements suggest that a change in a single point charge is not enough to shift the 4Fe4S cluster midpoint potential dramatically. Addition of a poly-L-glutamate polyanion introduced a slight negative shift (~20mV), but with the introduction of DNA a large negative shift was observed (70mV). Overall, binding to the DNA polyanion is the dominant effect in tuning the redox potential of the 4Fe4S cluster, helping to explain why all DNA binding proteins with 4Fe4S clusters studied to date have similar DNA-bound potentials.
\r\n\r\nWith these similar DNA-bound potentials, inter-protein redox signaling should occur. Previous works have demonstrated DNA-mediated redox signaling such as EndoIII signaling to DinG helicase, involved in R-loop maturation, increasing cellular survival by resolving deleterious R-loops. Additionally, different cluster- containing repair proteins of different functions and domains of life have been shown using atomic force microscopy (AFM) to localize to DNA mismatches through a redox switch for DNA-binding affinity. Given a DNA-mediated redox signaling system to scan the genome for lesions, the expression levels of these proteins may play a role in defining the scanning efficiency. We identified that the EndoIII E.coli knockout strain was sensitive to UV irradiation. This implies that EndoIII assists the nucleotide excision repair (NER) pathway via DNA-mediated redox signaling. However, knockout of MutY, another 4Fe4S glycosylase, does not impart the same UV sensitivity, and thus suggests key differences between MutY and EndoIII that define effective DNA-mediated redox signaling. Thus, the effect of protein expression level on the efficiency of DNA-mediated redox signaling was investigated using inducible protein expression of EndoIII to rescue UV-sensitivity. Using both plasmid-based and genome integrated constructs, we uncovered that low amounts of EndoIII expression were enough to rescue the growth defect, and overexpression of WT EndoIII leads to a greater defect caused by excess non-specific enzyme activity. These findings further informed investigation of this unique protein signaling interaction between EndoIII and NER protein UvrC.
\r\n\r\nWith proper EndoIII rescue plasmids, we further characterized the DNA- mediated redox signaling interaction between EndoIII and UvrC. Using UV-irradiation of genetic knockout strains and growth curve analysis, we demonstrate that EndoIII expression is essential for efficient repair of UV-induced DNA lesions, as measured through quantitative changes in growth lag-time when wild-type or mutant EndoIII is present in the cell. Electrochemical analysis of EndoIII point mutants quantify the DNA-CT inefficiencies that lead to the observed phenotypes. EndoIII, a BER repair protein, assists the NER pathway in the repair of UV-induced DNA lesions via DNA-mediated redox signaling. These results give evidence of a new signaling crosstalk between two distinct DNA repair pathways.
", "doi": "10.7907/G7NF-S349", "publication_date": "2019", "thesis_type": "phd", "thesis_year": "2019" }, { "id": "thesis:10893", "collection": "thesis", "collection_id": "10893", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05152018-150143251", "primary_object_url": { "basename": "Del_Castillo_Trevor_2018_thesis.pdf", "content": "final", "filesize": 5086155, "license": "other", "mime_type": "application/pdf", "url": "/10893/109/Del_Castillo_Trevor_2018_thesis.pdf", "version": "v8.0.0" }, "type": "thesis", "title": "The Quest for Electrocatalytic Nitrogen Fixation with a Molecular Catalyst and What We Learned Along the Way", "author": [ { "family_name": "Del Castillo", "given_name": "Trevor James", "orcid": "0000-0001-5120-1935", "clpid": "Del-Castillo-Trevor-James" } ], "thesis_advisor": [ { "family_name": "Peters", "given_name": "Jonas C.", "clpid": "Peters-J-C" } ], "thesis_committee": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" }, { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Peters", "given_name": "Jonas C.", "clpid": "Peters-J-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "This report details research into the mechanism and operating principles underlying the nitrogen fixation efficacy of a tris(phosphine)borane iron complex (P3BFe). The data presented provide what is to our knowledge the first unambiguous demonstration of electrocatalytic nitrogen fixation by a molecular catalyst and contribute to a growing body of evidence that metallocenes may play multiple roles during reductive catalysis.
", "doi": "10.7907/CM5B-RG20", "publication_date": "2018", "thesis_type": "phd", "thesis_year": "2018" }, { "id": "thesis:10739", "collection": "thesis", "collection_id": "10739", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:03012018-094939210", "primary_object_url": { "basename": "PLB Thesis_complete.pdf", "content": "final", "filesize": 9981102, "license": "other", "mime_type": "application/pdf", "url": "/10739/73/PLB Thesis_complete.pdf", "version": "v10.0.0" }, "type": "thesis", "title": "Elucidating the Role of [4Fe4S] Clusters in DNA Replication and Repair Proteins", "author": [ { "family_name": "Bartels", "given_name": "Phillip Leon", "orcid": "0000-0002-9688-6592", "clpid": "Bartels-Phillip-Leon" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Campbell", "given_name": "Judith L.", "clpid": "Campbell-J-L" }, { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" } ], "local_group": [ { "literal": "Kavli Nanoscience Institute" }, { "literal": "div_chem" } ], "abstract": "[4Fe4S] clusters, redox cofactors, have been discovered in DNA processing enzymes ranging from bacterial base excision repair glycosylases to eukaryotic DNA polymerases. Bacterial repair proteins are activated toward redox activity when bound to DNA and can take advantage of DNA-mediated charge transport (DNA CT) to search the genome for lesions. DNA CT involves the rapid transport of charges through the \u03c0-stacked base pairs and is sharply attenuated in the presence of lesions, mismatches, or other stacking perturbations. Thus, [4Fe4S] repair proteins use this chemistry to rapidly redistribute to target lesions and communicate with one another over long distances.
\r\n\r\nThe general function of [4Fe4S] clusters in bacterial DNA repair has received much attention, but previous efforts have left several critical questions unanswered. First, while the redox potential of these proteins is affected by DNA binding, the relative importance of the negatively-charged DNA, the protein environment surrounding the cluster, and solvent has remained unclear. Second, the importance of [4Fe4S] clusters and DNA CT to human disease has never been directly addressed. The biological consequences of this chemistry are certainly a pressing issue, as numerous disease-relevant mutations in the human homologues of well-studied repair proteins have been recorded. Finally, the existence of [4Fe4S] clusters in eukaryotic DNA replication proteins in general, and in the B-family DNA polymerases in particular, was entirely unexpected. The function of the [4Fe4S] cluster in replication proteins was far from obvious, and the functional differences from repair proteins made them difficult to explain even in the context of CT signaling. Herein, these questions have been addressed using a combination of electrochemical, spectroscopic, and biochemical approaches.
\r\n\r\nFirst, we describe the use of pyrolytic graphite edge electrodes (PGE) and S K-edge X-ray absorption spectroscopy (XAS) to address the influence of protein environment, DNA, and solvation on the [4Fe4S] cluster redox potential in the bacterial base excision repair glycosylases endonuclease III (EndoIII) and MutY. The PGE surface is rough and favorable for protein binding; electron transfer can be further enhanced in the presence of carbon nanotubes. Electrochemical signals for EndoIII and MutY in the absence of DNA are large and reproducible, and a potential shift upon DNA binding is observed. With respect to studying proteins in the absence of DNA, the PGE electrode represents a significant advance over previously used highly-oriented pyrolytic graphite (HOPG), which is hydrophobic and difficult to prepare. To test the effect of protein environment on redox potential, a series of EndoIII point mutants were prepared in which the charge within 5 \u00c5 of the cluster was reversed or added in. None of these mutations induced a significant shift in redox potential relative to wild type, arguing that DNA electrostatics are the dominant factor in potential modulation. In parallel, XAS studies were performed on EndoIII and MutY in the presence and absence of DNA, and in the presence and absence of solvent. Ligating cysteinyl thiols and inorganic S atoms in the [4Fe4S] cluster absorb at different intensities in XAS depending on solvent environment and local electrostatics; these changes, in turn, directly correlate to redox potential. By XAS, DNA was found to induce a significant shift in absorbance, and thus potential; the removal of solvent had a smaller effect. Together, these studies provide new approaches for the study of DNA-binding [4Fe4S] proteins and reveal the critical role of DNA in tuning the redox potential.
\r\n\r\nSecond, we report on a novel mutation in human MUTYH identified from a colorectal cancer patient and confirmed to be pathological. MUTYH is responsible for repairing certain lesions induced by oxidative stress and is thus frequently implicated in cancer. This new variant, C306W, contains a mutation in one of the cysteines that ligates the [4Fe4S] cluster. Electrochemistry, activity and DNA binding assays, and spectroscopic analyses were performed for C306W alongside wild type MUTYH and two other disease-relevant mutants, Y179C and G396D, with an unaltered cluster environment. From this work, it is now clear that C306W can still bind a cluster, but it is susceptible to oxidative degradation to the [3Fe4S]+ state upon redox signaling in an aerobic environment. Consequently, enzymatic activity is very low, and DNA binding is poor. Overall, this represents the first complete characterization of the [4Fe4S] cluster in a human homologue of MutY, and the first demonstration of pathology resulting from a mutation that primarily affects the [4Fe4S] cluster.
\r\n\r\nMoving into DNA replication proteins, we report on the characterization of the [4Fe4S] cluster in yeast DNA polymerase (Pol) \u03b4, the eukaryotic lagging strand polymerase. Pol \u03b4 shows reversible electrochemical signals at a midpoint potential indistinguishable from EndoIII under the same conditions, and EPR spectroscopy confirms use of the [4Fe4S]3+/2+ couple. The electrochemical signal is attenuated on DNA containing an abasic site or a CA mismatch, confirming that Pol \u03b4 is capable of DNA-mediated signaling. Bulk electrolysis and photooxidation were used to oxidize Pol \u03b4 under anaerobic conditions, and activity assays were carried out using oxidized or untreated protein. Oxidation stalls replication activity, while electrochemical reduction of oxidized samples restores activity to untreated levels. These results thus reveal that cluster oxidation serves as a reversible switch regulating Pol \u03b4 activity, suggesting an in vivo role in responding to replication stress, especially oxidative stress. In an effort to address these possibilities, we have carried out preliminary efforts in the characterization of two potentially CT-deficient mutants, W1053A and Y1078A. Both mutants were found to be too structurally unstable to proceed with in vivo experiments, but they can serve to guide future efforts in this direction.
\r\n\r\nFinally, a strategy to examine charge transport through RPA-bound single-stranded DNA is reported. RPA is the eukaryotic single-stranded binding protein and forms a protective coat around vulnerable unwound DNA at replication forks. Given the importance of redox signaling in replication proteins, we aimed to use photooxidation experiments to determine if CT through RPA is a viable pathway; if so, this would open up a large set of long-range transfer pathways to [4Fe4S] proteins in replication. These efforts are ongoing, but the experimental strategy and initial efforts are discussed.
", "doi": "10.7907/Z9H1307G", "publication_date": "2018", "thesis_type": "phd", "thesis_year": "2018" }, { "id": "thesis:10732", "collection": "thesis", "collection_id": "10732", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:02232018-173939270", "type": "thesis", "title": "Iron, Cobalt, and Nickel Metalloboranes: Reactivity, Catalysis, N2 Activation and Stabilization of Reactive N2Hx Ligands", "author": [ { "family_name": "Nesbit", "given_name": "Mark Allen", "orcid": "0000-0002-5642-9303", "clpid": "Nesbit-Mark-Allen" } ], "thesis_advisor": [ { "family_name": "Peters", "given_name": "Jonas C.", "clpid": "Peters-J-C" } ], "thesis_committee": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Fu", "given_name": "Gregory C.", "clpid": "Fu-G-C" }, { "family_name": "Agapie", "given_name": "Theodor", "clpid": "Agapie-T" }, { "family_name": "Peters", "given_name": "Jonas C.", "clpid": "Peters-J-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The reactivity of Fe and Co compounds supported by a bisphosphinoborane (DPB) ligand ([(DPB)Fe]2(N2) and (DPB)Co(N2)) towards E-H bonds (E = C, N, S, O, Si) is reported along with the catalytic hydrosilylation of ketones and aldehydes. The Fe and Co compounds displayed a mix of 1-electron and 2-electron chemistry. In some cases [(DPB)Fe]2(N2) and (DPB)Co(N2) facilitated oxidative addition of the E-H bond across the M-B interaction, and in others evolution of H2 giving a 1-electron oxidized complex of the general form (DPB)M(E) was observed. The reaction of Ph2SiH2 with (DPB)Co(N2) was found to be reversible, similar to the previously reported related nickel complex (PhDPBMes)Ni. The reactivity of these Fe and Co compounds is compared to previously reported Ni compounds supported by a similar ligand which catalyze olefin hydrogenation and hydrosilylation of substituted benzaldehydes.
\r\n\r\nThe synthesis and metalation with nickel of two new variants of the DPB ligand (DP*BPh and DP*BMes) is described. The primary modification introduced in DP*BPh and DP*BMes is the incorporation of a tertiary amine moiety into the secondary coordination sphere. This was done with the hypothesis that the amine moiety might act as a proton shuttle and facilitate proton reduction or hydrogen oxidation electrocatalysis. The process of screening these compounds for activity as proton reduction and hydrogen oxidation catalysts is also discussed. Additionally, the stoichiometric reactivity of [(DP*BPh)Ni]2(N2) and (DP*BMes)Ni(N2) with H2 was studied. We observed that [(DP*BPh)Ni]2(N2) slowly decomposed to an unidentified mixture of products while (DP*BMes)Ni(N2) dimerized to form a phosphine bridged Ni-borohydride dimer [(DP*BMesH)Ni]2. [(DP*BPh)Ni]2(N2) and (DP*BMes)Ni(N2) were also tested as precatalysts for olefin hydrogenation and found to be less active that their previously reported counterpart (PhDPBMes)Ni. [(DP*BPh)Ni]2(N2) and (DP*BMes)Ni(N2) correspondingly showed no activity for hydrogenation of polar substrates such as ketones, aldehydes, or CO2.
\r\n\r\nLastly, the synthesis of a new trisphosphinoborane ligand (ArP3B) with bulky aryl substituents on the phosphines and its metalation with Fe is described. The anionic-N2 adduct [(ArP3B)Fe(N2)][Na(12-C-4)2] was observed to react with H+ sources to generate the first observed parent iron-diazenido (ArP3B)Fe(NNH) and an iron-hydrazido(2-) [(ArP3B)Fe(NNH2)]+. [(ArP3B)Fe(NNH2)]+ was found to have similar spectroscopic properties to the previously reported [(TPB)Fe(NNH2)]+. A thorough characterization of [(ArP3B)Fe(N2)][Na(12-C-4)2], (ArP3B)Fe(NNH), and [(ArP3B)Fe(NNH2)]+ by a variety of continuous wave and pulsed ERP techniques is presented along with 57Fe M\u00f6ssbauer data. The new (ArP3B)Fe system was also canvassed for activity as a catalyst for conversion of N2 to NH3 and found to yield substoichiometric amounts of NH3 in the presence of KC8 and HBArF24\u20222Et2O while no NH3 was observed using CoCp*2 and [H2NPh2][OTf].
", "doi": "10.7907/Z9G15Z28", "publication_date": "2018", "thesis_type": "phd", "thesis_year": "2018" }, { "id": "thesis:11071", "collection": "thesis", "collection_id": "11071", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06112018-200314076", "primary_object_url": { "basename": "EOB Thesis 062018c.pdf", "content": "final", "filesize": 11150005, "license": "other", "mime_type": "application/pdf", "url": "/11071/1/EOB Thesis 062018c.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "Redox Signaling in Eukaryotic DNA Replication and Repair", "author": [ { "family_name": "O'Brien", "given_name": "Elizabeth", "orcid": "0000-0003-2889-1688", "clpid": "O'Brien-Elizabeth" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "local_group": [ { "literal": "Kavli Nanoscience Institute" }, { "literal": "div_chem" } ], "abstract": "DNA-mediated charge transport chemistry (DNA CT) offers an intriguing regulatory mechanism in biology, as it is long-range, rapid, and sensitive to mismatches and perturbations to base stacking. DNA-processing enzymes in all three domains of life moreover have been shown to contain [4Fe4S] clusters, commonly redox cofactors. Bacterial [4Fe4S] repair proteins have been shown to signal one another using long-range DNA-mediated charge transport (DNA CT), facilitating the redistribution to damaged genomic DNA in cells. The role of metabolically expensive, [4Fe4S] cluster cofactors in eukaryotic systems, however, was less clear than in prokaryotes.
\r\n\r\nHere we examine the chemical role of the [4Fe4S] cluster in eukaryotic DNA primase and the human base excision repair glycosylase, MUTYH. The primase cluster functions as a redox switch regulating DNA binding and redox signaling activity in humans and yeast. Yeast moreover require the primase redox switch for viability. Human MUTYH, a bifunctional glycosylase which repairs oxidative DNA lesions, performs DNA-mediated redox signaling, similarly to the bacterial homologue MutY. The MUTYH mutation which destabilizes the [4Fe4S] cluster during redox signaling, C306W, promotes degradation and loss of activity, associated with hereditary colorectal cancer.
\r\n\r\nTo assess the redox role of the human primase [4Fe4S] cluster, we perform anaerobic DNA electrochemistry on the [4Fe4S] domain of human primase (p58C), which independently binds DNA. On DNA-modified Au electrodes, we compare the redox activity of electrochemically oxidized and electrochemically reduced p58C. Oxidized [4Fe4S]3+ p58C is electrochemically active, and reduced [4Fe4S]2+ p58C state is redox-inert. This redox-driven switch is electrochemically reversible, and is mediated by a triad of conserved tyrosines between the DNA binding interface and [4Fe4S] cluster. Mutation of residues Y309, Y345, and Y347 to phenylalanine causes attenuation of redox switching on DNA. Single-atom mutations in the redox pathway moreover compromise initiation and truncation of primer synthesis but do not affect RNA polymerase activity. We find that primase truncation is gated by DNA CT in vitro; a single mismatch in the nascent primer abrogates truncation of primase products. As\r\nprimase is tethered to DNA polymerase \u03b1, a putative [4Fe4S] enzyme to which primase hands off the RNA-primed template, we propose that DNA-mediated signaling between primase and polymerase \u03b1 chemically regulates this handoff during the first steps of replication.
\r\n\r\nEukaryotic primase must bind both DNA and nucleotide triphosphates (NTPs) in order to convert to active form. Using DNA electrochemistry we show that p58C, and full-length DNA primase, display a robust, semi-reversible NTP-dependent signal on DNA, centered near 150mV vs. NHE. This signal is dependent on the tyrosine redox pathway. The presence of reversible redox activity at a physiological potential when primase is bound to DNA and NTPs suggests that reversible redox switching from the [4Fe4S]2+ to the [4Fe4S]3+ state is important for the activity of primase during replication.
\r\n\r\nThe cluster serves as a redox switch governing DNA binding in yeast primase, just as in human primase. Mutation of tyrosines 395 and 397 in yeast primase moreover, alters the same electron transfer chemistry as the mutation of their orthologues, Y345 and Y347, respectively, alters in human primase. Although these tyrosines are arranged differently in the yeast and human proteins, they perform the same reaction to affect the switch. The single-atom Y395F mutation causes some sensitivity to chemically induced oxidative stress in yeast, and single-residue mutation Y397L confers lethality in yeast cells. A constellation of tyrosines for protein-DNA electron transfer mediates the redox switch in eukaryotic primases, regulates the affinity for RNA-primed DNA template, and is required for primase function in vivo.
\r\n\r\nWe finally characterize a novel mutation in the [4Fe4S] human base excision repair protein, MUTYH, which destabilizes the cluster environment and has pathogenic consequences. The MUTYH C306W mutation alters one of the cysteines coordinating the cluster to tryptophan. This mutation moreover is associated with hereditary colorectal cancer and causes defective DNA binding and enzymatic activity. We perform DNA electrochemistry on WT MUTYH, as well as C306W and two cancer-associated mutants, Y197C and G396D, which have an unaltered cluster environment. MUTYH variants participate in redox signaling, but C306W is destabilized upon oxidation from the [4Fe4S]2+ to the [4Fe4S]3+ state during signaling on DNA, leading to degradation to a [3Fe4S]+ cluster and loss of DNA binding and activity. A [4Fe4S] human DNA repair enzyme performs redox signaling on DNA; dysregulation of this signaling activity is linked to tumorigenesis.
\r\n", "doi": "10.7907/KGCP-SD98", "publication_date": "2018", "thesis_type": "phd", "thesis_year": "2018" }, { "id": "thesis:10354", "collection": "thesis", "collection_id": "10354", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:07282017-141924517", "primary_object_url": { "basename": "TJZ Thesis Final Proofread and Corrected.pdf", "content": "final", "filesize": 7024906, "license": "other", "mime_type": "application/pdf", "url": "/10354/23/TJZ Thesis Final Proofread and Corrected.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Magnetic Field Effects and Biophysical Studies on DNA Charge Transport and Repair", "author": [ { "family_name": "Zwang", "given_name": "Theodore Joseph", "clpid": "Zwang-Theodore-Joseph" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Miller", "given_name": "Thomas F.", "clpid": "Miller-T-F" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "DNA-mediated charge transport (DNA CT) is well established in both ground and excited state systems. Although theoretical models are still being developed, it is clear that the integrity of the extended \u03c0-stack of the aromatic heterocycles, the nucleic acid bases, plays a critical role. Electron donors and acceptors must be electronically well coupled into the \u03c0-stack, typically via intercalation. Perturbations that distort the \u03c0-stack, such as single-base mismatches, abasic sites, base lesions, and protein binding that kinks the double helix, attenuate DNA CT dramatically.
\r\n\r\nThis thesis encompasses work that first aims to understand how DNA duplex structure informs characteristics of DNA CT and then continues to develop an understanding of the role these structural features play in biological systems. To contextualize these advancements, this first chapter outlines foundational work that has shown ways that DNA structure influences its ability to conduct charge.
\r\n\r\nNext, experiments were conducted on magnetized DNA-modified electrodes to explore spin-selective electron transport through hydrated duplex DNA. These results show that the two spins migrate through duplex DNA with a different yield and that spin selectivity requires charge transport through the DNA duplex. Significantly, shifting the same duplex DNA between right-handed B- and left-handed Z-forms leads to a diode-like switch in spin selectivity; which spin moves more efficiently through the duplex depends upon the DNA helicity. With DNA, the supramolecular organization of chiral moieties, rather than the chirality of the individual monomers, determines the selectivity in spin, and thus a conformational change can switch the spin selectivity.
\r\n\r\nThis exquisite spin selectivity begged the question: how might biology take advantage of such a spin filter? Photolyase and cryptochromes both have been shown to exhibit magnetosensitive chemistry nearby a DNA binding pocket, and photolyase had previously been shown capable of DNA CT. Thus, electrochemical studies were conducted to monitor the repair of cyclobutane pyrimidine dimer lesions by E coli photolyase and truncated A Thaliana Cryptochrome 1 with an applied magnetic field. We find that the yield of dimer repair is dependent on the strength and angle of the applied magnetic field even when using magnetic fields weaker than 1 Gauss, though spin selective DNA CT is not involved. These data illustrate how cyclobutane dimer repair could be used in a biological compass that is informed by the angles of Earth\u2019s magnetic field.
\r\n\r\nNext DNA-mediated electrochemistry and atomic force microscopy studies were used to describe a role for redox active [4Fe4S] clusters in DNA-mediated charge transport signaling. DNA-modified electrochemistry shows that the [4Fe4S] cluster of DNA-bound DinG, an ATP-dependent helicase that repairs R-loops, is redox-active at cellular potentials and ATP hydrolysis increases DNA-mediated redox signaling. Atomic force microscopy experiments demonstrate that DinG and Endonuclease III, a base excision repair enzyme, cooperate at long range using DNA charge transport to redistribute to regions of DNA damage. These data are then described using an equilibrium model which elucidates fundamental characteristics of this redox chemistry that allow DNA CT to coordinate the activities of DNA repair enzymes across the genome.
\r\n\r\nThe importance of the oxidation state of the redox-active [4Fe4S] cluster in the DNA damage detection process is then further explored. Together, these results show that the reduction and oxidation of [4Fe4S] clusters through DNA-mediated charge transport facilitates long-range signaling between [4Fe4S] repair proteins. The redox-modulated change in DNA-binding affinity regulates the ability of [4Fe4S] repair proteins to collaborate in the lesion detection process.
", "doi": "10.7907/Z9TT4P4H", "publication_date": "2018", "thesis_type": "phd", "thesis_year": "2018" }, { "id": "thesis:10253", "collection": "thesis", "collection_id": "10253", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06022017-112043547", "primary_object_url": { "basename": "HunterBryan2017thesis.pdf", "content": "final", "filesize": 22940679, "license": "other", "mime_type": "application/pdf", "url": "/10253/73/HunterBryan2017thesis.pdf", "version": "v16.0.0" }, "type": "thesis", "title": "Fuels and Materials from Sunlight and Water", "author": [ { "family_name": "Hunter", "given_name": "Bryan Michael", "orcid": "0000-0001-8559-9304", "clpid": "Hunter-Bryan-Michael" } ], "thesis_advisor": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" } ], "thesis_committee": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Rossman", "given_name": "George Robert", "clpid": "Rossman-G-R" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" } ], "local_group": [ { "literal": "Resnick Sustainability Institute" }, { "literal": "div_chem" } ], "abstract": "The urgency to develop new technologies that harness energy and natural feedstocks in a sustainable fashion has never been more apparent. With global power consumption growing at an exponential rate, only one resource is truly capable of powering the planet: the sun. Sunlight is reliable, clean, and free.
\r\n\r\nSignificant resources have been pledged to develop and refine solar energy devices that convert photons into electricity (i.e. photovoltaics), but the sun\u2019s intermittency and the poor overlap of solar irradiance with global power demand a different strategy. In light of these limitations, we have proposed a device which converts solar energy into reduced chemical fuels (e.g. dihydrogen or methane) that can be indefinitely stored and easily transported. In principle, the only required inputs are sunlight, an earth-abundant feedstock such as carbon dioxide, protons (H+), and reducing equivalents (e-). The source of these protons and electrons must be abundant and ubiquitous\u2014we chose water.
\r\n\r\nDespite the 2-billion-year history of plants performing water oxidation to produce molecular oxygen, protons, and electrons (Photosystem II), our understanding of this complex 4H+/4e- process has been severely limited. Only recently have high-performing, earth-abundant heterogeneous electrocatalysts been reported that can be scaled up to make functioning devices.
\r\n\r\nThis dissertation describes progress on both the synthetic and mechanistic fronts in developing earth-abundant heterogeneous water oxidation catalysts for solar-driven water splitting. We have synthesized nanoparticulate Ni-Fe catalysts with the highest measured activity on flat electrodes to date. We carefully characterized these materials spectroscopically to determine that edge-site iron was active in catalysis. We then undertook novel in-situ spectroelectrochemical techniques in non-aqueous media to identify the active iron species, which is surprisingly a cis-dioxo-iron(VI) corner site. The data also indicate that geminal iron-oxo coupling may be the operative mechanism of O-O bond formation, a new scheme with potential biological relevance.
\r\n\r\nFinally, we have expanded our goal to include sustainably reducing other feedstocks, such as carbon dioxide and hydrocarbons. In doing so, we aim to make pharmaceuticals, polymers, and other high-value products from sunlight and water.
", "doi": "10.7907/Z9FQ9TNB", "publication_date": "2017", "thesis_type": "phd", "thesis_year": "2017" }, { "id": "thesis:10329", "collection": "thesis", "collection_id": "10329", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06092017-062335915", "type": "thesis", "title": "Targeting DNA Mismatches with Luminescent Ruthenium Complexes", "author": [ { "family_name": "Boynton", "given_name": "Adam Nathaniel", "clpid": "Boynton-Adam-Nathaniel" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "DNA base pair mismatches occur naturally in cells, typically as a result of errors during replication. Cells have evolved a DNA damage response pathway called mismatch repair (MMR) that identifies and corrects base pair mismatches in newly synthesized DNA. However, proteins involved in MMR can undergo mutations, rendering them incapable of correcting mismatches. Such deficiencies in MMR leads to an increase in genetic mutations and are associated with several forms of cancer. Because a higher mismatch frequency serves as an early indicator of cancer progression, DNA mismatches are a promising target in the design of small molecule therapeutics and diagnostics. In this context, transition metal complexes are prime candidates, owing to their\u00a0valuable spectroscopic and photophysical properties and versatile coordination sphere geometries. Our laboratory focuses on generating octahedral rhodium and ruthenium complexes that selectively target DNA mismatches. A class of rhodium complexes bearing sterically expansive planar ligands bind DNA mismatches with high selectivity and exhibit preferential cytotoxicity towards MMR-deficient cancer cells. These compounds bind to DNA through metalloinsertion, in which the bulky ligand inserts into the duplex at the thermodynamically destabilized mismatch site, displacing the mismatched bases into the DNA groove.
\r\n\r\nHerein we describe recent advances in the development of luminescent ruthenium complexes that selectively probe DNA mismatches. We demonstrate that [Ru(Me4phen)2(dppz)]2+ (Me4phen = 3,4,7,8-tetramethyl-1,10-phenanthroline; dppz = dipyrido[3,2-a:2\u2019,3\u2019-c]phenazine) is a DNA \u201clight switch\u201d that exhibits a significantly brighter steady-state emission in the presence of a DNA duplex containing a mismatch relative to completely well-matched DNA. Importantly, the bulky Me4phen ancillary ligands discourage deep intercalation of dppz between well-matched base pairs, and instead, [Ru(Me4phen)2(dppz)]2+ favors metalloinsertion at thermodynamically destabilized mismatches. [Ru(Me4phen)2(dppz)]2+ possesses a higher binding affinity towards a DNA mismatch relative to well-matched base pairs, and furthermore exhibits a longer excited-state emission lifetime when bound to a mismatch compared to that when intercalated at well-matched sites; both of these observations contribute to the dramatic steady-state emission enhancement detected with the mismatched DNA duplex. Additionally, we reveal that the right-handed delta (\u2206) isomer of [Ru(Me4phen)2(dppz)]2+ is the enantiomer which imparts all mismatch selectivity, consistent with the handedness of B-form DNA.
\r\n\r\nAnother mismatch-specific luminescent probe presented in this work is [Ru(bpy)2(BNIQ)]2+ (bpy = 2,2\u2019-bipyridine; BNIQ = benzo[c][1,7]naphthyridine-1-isoquinoline). In contrast to [Ru(Me4phen)2(dppz)]2+, the BNIQ complex exploits a bulky inserting ligand that selectively undergoes metalloinsertion at a DNA mismatch. This compound too exhibits a brighter steady-state emission in the presence of a mismatched duplex compared to entirely well-matched DNA, which we attribute to the fact that [Ru(bpy)2(BNIQ)]2+ possesses nearly a 500-fold higher binding affinity for the mismatch site compared to well-matched base pairs. Taken together, [Ru(Me4phen)2(dppz)]2+ and [Ru(bpy)2(BNIQ)]2+ represent two different yet valid approaches in the rational design of mismatch-specific small molecules, one based on ancillary ligand functionalization and the other on incorporating a sterically expansive inserting ligand.
\r\n\r\nA third approach towards the design of mismatch-specific luminescent ruthenium probes that is briefly explored here is the modification of the intercalating dppz ligand of [Ru(bpy)2(dppz)]2+. Bearing a dppz ligand substituted with four methyl groups, [Ru(bpy)2(tmdppz)]2+ (tmdppz = 3,4,7,8-tetramethyl dipyridophenazine) shows no luminescence discrimination between mismatched and well-matched duplexes. This observation ostensibly arises from the fact that the appended methyl groups shield the dppz phenazine nitrogen atoms from interactions with water when intercalated within the DNA.
\r\n\r\nWith mismatch-specific luminescent metalloinsertors such as [Ru(Me4phen)2(dppz)]2+ in hand, we have commenced biological investigations to see whether these compounds can serve as luminescent proxies for rhodium metalloinsertors in MMR-deficient cancer cells. Confocal microscopy of HCT116N and HCT116O cells reveals that [Ru(Me4phen)2(dppz)]2+ does preferentially localize to mitochondria, unlike potent cell-selective rhodium complexes such as [Rh(chrysi)(phen)(PPO)]2+ (PPO = 2-(pyridine-2-yl)propan-2-ol; chrysi = 5,6-chrysenequinone diimine); however, [Ru(Me4phen)2(dppz)]2+ shows some degree of nuclear entry. Here our goal is the application of the mismatch-specific luminescent probe in co-localization experiments to investigate what proteins are involved in the DNA damage response that is activated upon metalloinsertor binding in cellulo.
\r\n\r\nThe work presented here expands beyond the study of luminescent ruthenium complexes. Amino acid conjugates of the earlier-generation rhodium metalloinsertor [Rh(HDPA)2(chrysi)]3+ (HDPA = 2,2\u2019-dipyridylamine) were synthesized. While these conjugates exhibit mismatch binding affinities comparable to other rhodium metalloinsertors, they lose cell-selective biological activity, which may arise from altered uptake and/or sub-cellular localization. Finally, preliminary investigations were conducted on [Re(CO)3(pyOEt)(dppn)]+ (pyOEt = ethyl 3-(pyridin-4-yl)propanoate; dppn = benzodipyridophenazine) and [Ru(CN)(tpy)(dppz)]+ (tpy = terpyridine; CN = cyano), which were designed as IR-active probes to study the kinetics of DNA-mediated charge transport (CT) by time-resolved infrared (TRIR) spectroscopy. While these complexes do not possess the desired spectral TRIR properties as originally intended, steady-state luminescence experiments do suggest that this donor-acceptor pair is capable of undergoing DNA-mediated electron transfer.
\r\n\r\nAltogether, this work demonstrates the versatility of transition metal complexes as non-covalent probes for DNA. Importantly, through the rational modification of their three-dimensional ligand scaffold, one can achieve site-specific recognition of clinically relevant biomarkers such as DNA mismatches.
", "doi": "10.7907/Z9CF9N5M", "publication_date": "2017", "thesis_type": "phd", "thesis_year": "2017" }, { "id": "thesis:9853", "collection": "thesis", "collection_id": "9853", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06072016-160630609", "type": "thesis", "title": "Agonist Binding Studies at Two Subtypes of the Nicotinic Acetylcholine Receptor Involved in Parkinson\u2019s Disease and Addiction", "author": [ { "family_name": "Post", "given_name": "Michael Robert", "orcid": "0000-0002-3214-7619", "clpid": "Post-Michael-Robert" } ], "thesis_advisor": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" } ], "thesis_committee": [ { "family_name": "Reisman", "given_name": "Sarah E.", "clpid": "Reisman-S-E" }, { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Neuronal nicotinic acetylcholine receptors (nAChR) consist of pentameric ligand gated ion channels that typically regulate the release of neurotransmitter. This group of receptors is made of many subunits that combine into pentamers to form different subtypes, with each subtype having a unique pharmacology, function, and localization in the nervous system. The \u03b16\u03b22 subtype is found predominantly in the dopaminergic pathways in the brain, and is therefore a promising target for addiction and Parkinson\u2019s disease. A major goal in treating these disorders is to develop subtype-selective agonists, and advanced knowledge of the binding site which sits at the \u03b16-\u03b22 subunit interface is critical. This thesis dissertation describes high precision, chemical scale structure-function studies designed to probe specific interactions between a variety of agonists and the amino acids which make up the \u03b16\u03b22 binding site.
\r\n\r\nBefore these studies, which utilize nonsense suppression-based non-canonical amino acid mutagenesis, could be conducted, a heterologous expression system for \u03b16\u03b22 had to be developed.
\r\n\r\nChapter 2 details four reporter mutations that allow high expression levels of \u03b16\u03b22 in Xenopus oocytes. Further work presented in this chapter characterizes a variety of compounds at this subtype including acetylcholine, the endogenous agonist, nicotine, and TC299423, a promising drug candidate designed to be \u03b16-selective.
\r\n\r\nChapters 3 and 4 discuss the structure-function studies used to probe for binding interactions of acetylcholine, nicotine, and TC299423 with the \u03b16-\u03b22 interface. Fluorination series were executed to probe for cation-\u03c0 interactions with TrpB, TyrA, and TyrC2, all sites of the \u03b16 face. Of the nine possible agonist-side chain interactions, the only functionally important cation-\u03c0 interaction was found between acetylcholine and TrpB, suggesting the subtype has a unique pharmacology. Studies utilizing \u03b1-hydroxy acids were then performed to determine whether these agonists make a functional hydrogen bond between their amine NH and the backbone carbonyl associated with TrpB. Here, nicotine was found to make a strong hydrogen bond, whose energy was quantified via double-mutant cycle analysis, but TC299423 was not.
\r\n\r\nChapter 5 further explores TC299423 at the \u03b14\u03b22 subtype. Experiments here showed that TC299423 makes a dual cation-\u03c0 interaction with both TrpB and TyrC2. Further studies revealed this dual cation-\u03c0 effect to be true for several secondary amines, and a structure-function study with nornicotine established this as a general feature for secondary amines.
\r\n\r\nChapter 6 describes work done to probe for a hydrogen bond between the indole NH of \u03b14 TrpB and a backbone carbonyl associated with L119 on the \u03b22 subunit. This study required development of a new strategy to probe for hydrogen bonds as the amino acid sequence does not allow for \u03b1-hydroxy substitution. Instead, a fluorinated side chain strategy was used to inductively attenuate the hydrogen bond accepting ability of the carbonyl, and it proved the \u03b14-\u03b22 interfacial hydrogen prediction false.
\r\n\r\nFinally two appendices suggest possible avenues to explore with the new \u03b16\u03b22 expression system. Appendix A describes work done to determine whether there is cross-talk between \u03b16\u03b22 and P2X receptors. Appendix B details initial investigations on the effects of ethanol and other alcohols on the function of \u03b16\u03b22.
", "doi": "10.7907/Z9GT5K50", "publication_date": "2016", "thesis_type": "phd", "thesis_year": "2016" }, { "id": "thesis:9703", "collection": "thesis", "collection_id": "9703", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05052016-123724002", "primary_object_url": { "basename": "Chaubard_Jean-Luc_Thesis_2016.pdf", "content": "final", "filesize": 18091047, "license": "other", "mime_type": "application/pdf", "url": "/9703/1/Chaubard_Jean-Luc_Thesis_2016.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "Development of Chemoenzymatic Labeling Approaches for the Detection of Fucosylated Biomarkers", "author": [ { "family_name": "Chaubard", "given_name": "Jean-Luc", "clpid": "Chaubard-Jean-Luc" } ], "thesis_advisor": [ { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" } ], "thesis_committee": [ { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Lester", "given_name": "Henry A.", "clpid": "Lester-H-A" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" }, { "family_name": "Heath", "given_name": "James R.", "clpid": "Heath-J-R" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Protein fucosylation regulates a diverse set of physiological functions such as memory and learning, development, and disease pathogenesis. However, our current understanding of these processes is far behind that of other post-translational modifications, such as phosphorylation. This is, in part, due to the lack of tools available for the study of this important protein modification. To address this need, I have developed novel chemoenzymatic methods that enable the labeling and detection of unique forms of fucosylation, specifically fucose-\u03b1(1-2)-galactose (Fuc\u03b1(1-2)Gal) and core fucose. Additionally, novel glycosyltransferase assays were developed in-house to aid in the future development of both new and existing chemoenzymatic approaches.
\r\n\r\nI have demonstrated that the approach to detect Fuc\u03b1(1-2)Gal is highly selective for this disaccharide motif, detects a variety of complex glycans and glycoproteins, and can be used to profile the relative abundance of this motif on live cells, discriminating malignant from normal cells. I have also shown that the chemoenzymatic detection of core fucose exhibits superior specificity towards this glycan on a variety of complex N-glycans and when compared to current fucose-specific lectins. Further, the approach is amenable to detection of core fucosylated glycans from multiple biological settings, can be exploited as an antibody-conjugation method, and can be integrated into a diagnostic platform for the profiling of protein specific core fucosylation levels. These approaches represent new potential strategies for biomarker identification and expand the technologies available for understanding the role of these important fucosylated glycans in physiology and disease.
\r\n", "doi": "10.7907/Z9K35RN1", "publication_date": "2016", "thesis_type": "phd", "thesis_year": "2016" }, { "id": "thesis:9063", "collection": "thesis", "collection_id": "9063", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:07192015-214603085", "primary_object_url": { "basename": "DNA CT signaling within the cell_MAG_2015.pdf", "content": "final", "filesize": 4584274, "license": "other", "mime_type": "application/pdf", "url": "/9063/1/DNA CT signaling within the cell_MAG_2015.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "DNA-Mediated Charge Transport Signaling Within the Cell", "author": [ { "family_name": "Grodick", "given_name": "Michael Andrew", "clpid": "Grodick-Michael-Andrew" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "DNA possesses the curious ability to conduct charge longitudinally through the \u03c0-stacked base pairs that reside within the interior of the double helix. The rate of charge transport (CT) through DNA has a shallow distance dependence. DNA CT can occur over at least 34 nm, a very long molecular distance. Lastly, DNA CT is exquisitely sensitive to disruptions, such as DNA damage, that affect the dynamics of base-pair stacking. Many DNA repair and DNA-processing enzymes are being found to contain 4Fe-4S clusters. These co-factors have been found in glycosylases, helicases, helicase-nucleases, and even enzymes such as DNA polymerase, RNA polymerase, and primase across the phylogeny. The role of these clusters in these enzymes has remained elusive. Generally, iron-sulfur clusters serve redox roles in nature since, formally, the cluster can exist in multiple oxidation states that can be accessed within a biological context. Taken together, these facts were used as a foundation for the hypothesis that DNA-binding proteins with 4Fe-4S clusters utilize DNA-mediated CT as a means to signal one another to scan the genome as a first step in locating the subtle damage that occurs within a sea of undamaged bases within cells.
\r\n\r\nHerein we describe a role for 4Fe-4S clusters in DNA-mediated charge transport signaling among EndoIII, MutY, and DinG, which are from distinct repair pathways in E. coli. The DinG helicase is an ATP-dependent helicase that contains a 4Fe-4S cluster. To study the DNA-bound redox properties of DinG, DNA-modified electrochemistry was used to show that the 4Fe-4S cluster of DNA-bound DinG is redox-active at cellular potentials, and shares the 80 mV vs. NHE redox potential of EndoIII and MutY. ATP hydrolysis by DinG increases the DNA-mediated redox signal observed electrochemically, likely reflecting better coupling of the 4Fe-4S cluster to DNA while DinG unwinds DNA, which could have interesting biological implications. Atomic force microscopy experiments demonstrate that DinG and EndoIII cooperate at long range using DNA charge transport to redistribute to regions of DNA damage. Genetics experiments, moreover, reveal that this DNA-mediated signaling among proteins also occurs within the cell and, remarkably, is required for cellular viability under conditions of stress. Knocking out DinG in CC104 cells leads to a decrease in MutY activity that is rescued by EndoIII D138A, but not EndoIII Y82A. DinG, thus, appears to help MutY find its substrate using DNA-mediated CT, but do MutY or EndoIII aid DinG in a similar way? The InvA strain of bacteria was used to observe DinG activity, since DinG activity is required within InvA to maintain normal growth. Silencing the gene encoding EndoIII in InvA results in a significant growth defect that is rescued by the overexpression of RNAseH, a protein that dismantles the substrate of DinG, R-loops. This establishes signaling between DinG and EndoIII. Furthermore, rescue of this growth defect by the expression of EndoIII D138A, the catalytically inactive but CT-proficient mutant of EndoIII, is also observed, but expression of EndoIII Y82A, which is CT-deficient but enzymatically active, does not rescue growth. These results provide strong evidence that DinG and EndoIII utilize DNA-mediated signaling to process DNA damage. This work thus expands the scope of DNA-mediated signaling within the cell, as it indicates that DNA-mediated signaling facilitates the activities of DNA repair enzymes across the genome, even for proteins from distinct repair pathways.
\r\n\r\nIn separate work presented here, it is shown that the UvrC protein from E. coli contains a hitherto undiscovered 4Fe-4S cluster. A broad shoulder at 410 nm, characteristic of 4Fe-4S clusters, is observed in the UV-visible absorbance spectrum of UvrC. Electron paramagnetic resonance spectroscopy of UvrC incubated with sodium dithionite, reveals a spectrum with the signature features of a reduced, [4Fe-4S]+1, cluster. DNA-modified electrodes were used to show that UvrC has the same DNA-bound redox potential, of ~80 mV vs. NHE, as EndoIII, DinG, and MutY. Again, this means that these proteins are capable of performing inter-protein electron transfer reactions. Does UvrC use DNA-mediated signaling to facilitate the repair of its substrates?
\r\n\r\nUvrC is part of the nucleotide excision repair (NER) pathway in E. coli and is the protein within the pathway that performs the chemistry required to repair bulky DNA lesions, such as cyclopyrimidine dimers, that form as a product of UV irradiation. We tested if UvrC utilizes DNA-mediated signaling to facilitate the efficient repair of UV-induced DNA damage products by helping UvrC locate DNA damage. The UV sensitivity of E. coli cells lacking DinG, a putative signaling partner of UvrC, was examined. Knocking out DinG in E. coli leads to a sensitivity of the cells to UV irradiation. A 5-10 fold reduction in the amount of cells that survive after irradiation with 90 J/m2 of UV light is observed. This is consistent with the hypothesis that UvrC and DinG are signaling partners, but is this signaling due to DNA-mediated CT? Complementing the knockout cells with EndoIII D138A, which can also serve as a DNA CT signaling partner, rescues cells lacking DinG from UV irradiation, while complementing the cells with EndoIII Y82A shows no rescue of viability. These results indicate that there is cross-talk between the NER pathway and DinG via DNA-mediated signaling. Perhaps more importantly, this work also establishes that DinG, EndoIII, MutY, and UvrC comprise a signaling network that seems to be unified by the ability of these proteins to perform long range DNA-mediated CT signaling via their 4Fe-4S clusters.
\r\n\r\n\r\n", "doi": "10.7907/Z9F769GX", "publication_date": "2016", "thesis_type": "phd", "thesis_year": "2016" }, { "id": "thesis:9007", "collection": "thesis", "collection_id": "9007", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06062015-192618908", "primary_object_url": { "basename": "Anna Arnold_2015_Thesis full version.pdf", "content": "final", "filesize": 35527821, "license": "other", "mime_type": "application/pdf", "url": "/9007/143/Anna Arnold_2015_Thesis full version.pdf", "version": "v7.0.0" }, "type": "thesis", "title": "Investigations of DNA-Mediated Protein Oxidation", "author": [ { "family_name": "Arnold", "given_name": "Anna Ruth", "clpid": "Arnold-Anna-Ruth" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "DNA charge transport (CT) involves the efficient transfer of electrons or electron holes through the DNA π-stack over long molecular distances of at least 100 base-pairs. Despite this shallow distance dependence, DNA CT is sensitive to mismatches or lesions that disrupt π-stacking and is critically dependent on proper electronic coupling of the donor and acceptor moieties into the base stack. Favorable DNA CT is very rapid, occurring on the picosecond timescale. Because of this speed, electron holes equilibrate along the DNA π-stack, forming a characteristic pattern of DNA damage at low oxidation potential guanine multiplets. Furthermore, DNA CT may be used in a biological context. DNA processing enzymes with 4Fe4S clusters can perform DNA-mediated electron transfer (ET) self-exchange reactions with other 4Fe4S cluster proteins, even if the proteins are quite dissimilar, as long as the DNA-bound [4Fe4S]3+/2+ redox potentials are conserved. This mechanism would allow low copy number DNA repair proteins to find their lesions efficiently within the cell. DNA CT may also be used biologically for the long-range, selective activation of redox-active transcription factors. Within this work, we pursue other proteins that may utilize DNA CT within the cell and further elucidate aspects of the DNA-mediated ET self-exchange reaction of 4Fe4S cluster proteins.
\r\n\r\nDps proteins, bacterial mini-ferritins that protect DNA from oxidative stress, are implicated in the survival and virulence of pathogenic bacteria. One aspect of their protection involves ferroxidase activity, whereby ferrous iron is bound and oxidized selectively by hydrogen peroxide, thereby preventing formation of damaging hydroxyl radicals via Fenton chemistry. Understanding the specific mechanism by which Dps proteins protect the bacterial genome could inform the development of new antibiotics. We investigate whether DNA-binding E. coli Dps can utilize DNA CT to protect the genome from a distance. An intercalating ruthenium photooxidant was employed to generate oxidative DNA damage via the flash-quench technique, which localizes to a low potential guanine triplet. We find that Dps loaded with ferrous iron, in contrast to Apo-Dps and ferric iron-loaded Dps which lack available reducing equivalents, significantly attenuates the yield of oxidative DNA damage at the guanine triplet. These data demonstrate that ferrous iron-loaded Dps is selectively oxidized to fill guanine radical holes, thereby restoring the integrity of the DNA. Luminescence studies indicate no direct interaction between the ruthenium photooxidant and Dps, supporting the DNA-mediated oxidation of ferrous iron-loaded Dps. Thus DNA CT may be a mechanism by which Dps efficiently protects the genome of pathogenic bacteria from a distance.
\r\n\r\nFurther work focused on spectroscopic characterization of the DNA-mediated oxidation of ferrous iron-loaded Dps. X-band EPR was used to monitor the oxidation of DNA-bound Dps after DNA photooxidation via the flash-quench technique. Upon irradiation with poly(dGdC)2, a signal arises with g = 4.3, consistent with the formation of mononuclear high-spin Fe(III) sites of low symmetry, the expected oxidation product of Dps with one iron bound at each ferroxidase site. When poly(dGdC)2 is substituted with poly(dAdT)2, the yield of Dps oxidation is decreased significantly, indicating that guanine radicals facilitate Dps oxidation. The more favorable oxidation of Dps by guanine radicals supports the feasibility of a long-distance protection mechanism via DNA CT where Dps is oxidized to fill guanine radical holes in the bacterial genome produced by reactive oxygen species.
\r\n\r\nWe have also explored possible electron transfer intermediates in the DNA-mediated oxidation of ferrous iron-loaded Dps. Dps proteins contain a conserved tryptophan residue in close proximity to the ferroxidase site (W52 in E. coli Dps). In comparison to WT Dps, in EPR studies of the oxidation of ferrous iron-loaded Dps following DNA photooxidation, W52Y and W52A mutants were deficient in forming the characteristic EPR signal at g = 4.3, with a larger deficiency for W52A compared to W52Y. In addition to EPR, we also probed the role of W52 Dps in cells using a hydrogen peroxide survival assay. Bacteria containing W52Y Dps survived the hydrogen peroxide challenge more similarly to those containing WT Dps, whereas cells with W52A Dps died off as quickly as cells without Dps. Overall, these results suggest the possibility of W52 as a CT hopping intermediate.
\r\n\r\nDNA-modified electrodes have become an essential tool for the study of the redox chemistry of DNA processing enzymes with 4Fe4S clusters. In many cases, it is necessary to investigate different complex samples and substrates in parallel in order to elucidate this chemistry. Therefore, we optimized and characterized a multiplexed electrochemical platform with the 4Fe4S cluster base excision repair glycosylase Endonuclease III (EndoIII). Closely packed DNA films, where the protein has limited surface accessibility, produce EndoIII electrochemical signals sensitive to an intervening mismatch, indicating a DNA-mediated process. Multiplexed analysis allowed more robust characterization of the CT-deficient Y82A EndoIII mutant, as well as comparison of a new family of mutations altering the electrostatics surrounding the 4Fe4S cluster in an effort to shift the reduction potential of the cluster. While little change in the DNA-bound midpoint potential was found for this family of mutants, likely indicating the dominant effect of DNA-binding on establishing the protein redox potential, significant variations in the efficiency of DNA-mediated electron transfer were apparent. On the basis of the stability of these proteins, examined by circular dichroism, we proposed that the electron transfer pathway in EndoIII can be perturbed not only by the removal of aromatic residues but also through changes in solvation near the cluster.
\r\n\r\nWhile the 4Fe4S cluster of EndoIII is relatively insensitive to oxidation and reduction in solution, we have found that upon DNA binding, the reduction potential of the [4Fe4S]3+/2+ couple shifts negatively by approximately 200 mV, bringing this couple into a physiologically relevant range. Demonstrated using electrochemistry experiments in the presence and absence of DNA, these studies do not provide direct molecular evidence for the species being observed. Sulfur K-edge X-ray absorbance spectroscopy (XAS) can be used to probe directly the covalency of iron-sulfur clusters, which is correlated to their reduction potential. We have shown that the Fe-S covalency of the 4Fe4S cluster of EndoIII increases upon DNA binding, stabilizing the oxidized [4Fe4S]3+ cluster, consistent with a negative shift in reduction potential. The 7% increase in Fe-S covalency corresponds to an approximately 150 mV shift, remarkably similar to DNA electrochemistry results. Therefore we have obtained direct molecular evidence for the shift in 4Fe4S reduction potential of EndoIII upon DNA binding, supporting the feasibility of our model whereby these proteins can utilize DNA CT to cooperate in order to efficiently find DNA lesions inside cells.
\r\n\r\nIn conclusion, in this work we have explored the biological applications of DNA CT. We discovered that the DNA-binding bacterial ferritin Dps can protect the bacterial genome from a distance via DNA CT, perhaps contributing to pathogen survival and virulence. Furthermore, we optimized a multiplexed electrochemical platform for the study of the redox chemistry of DNA-bound 4Fe4S cluster proteins. Finally, we have used sulfur K-edge XAS to obtain direct molecular evidence for the negative shift in 4Fe4S cluster reduction potential of EndoIII upon DNA binding. These studies contribute to the understanding of DNA-mediated protein oxidation within cells.
", "doi": "10.7907/Z9ZW1HVN", "publication_date": "2015", "thesis_type": "phd", "thesis_year": "2015" }, { "id": "thesis:8968", "collection": "thesis", "collection_id": "8968", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06022015-104348919", "primary_object_url": { "basename": "AGWeidmannThesisCompiled.pdf", "content": "final", "filesize": 47094104, "license": "other", "mime_type": "application/pdf", "url": "/8968/1/AGWeidmannThesisCompiled.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Biological Activity of Rhodium Metalloinsertors and the Design of Bifunctional Conjugates", "author": [ { "family_name": "Weidmann", "given_name": "Alyson Gloria", "orcid": "0000-0003-3876-2847", "clpid": "Weidmann-Alyson-Gloria" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Stoltz", "given_name": "Brian M.", "clpid": "Stoltz-B-M" }, { "family_name": "Peters", "given_name": "Jonas C.", "clpid": "Peters-J-C" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The Barton laboratory has established that octahedral rhodium complexes bearing the sterically expansive 5,6-chrysene diimine ligand can target thermodynamically destabilized sites, such as base pair mismatches, in DNA with high affinity and selectivity. These complexes approach DNA from the minor groove, ejecting the mismatched base pairs from the duplex in a binding mode termed metalloinsertion. In recent years, we have shown that these metalloinsertor complexes also exhibit cytotoxicity preferentially in cancer cells that are deficient in the mismatch repair (MMR) machinery.
\r\n\r\nHere, we establish that a sensitive structure-activity relationship exists for rhodium metalloinsertors. We studied the relationship between the chemical structures of metalloinsertors and their effect on biological activity for ten complexes with similar DNA binding affinities, but wide variation in their lipophilicity. Drastic differences were observed in the selectivities of the complexes for MMR-deficient cells. Compounds with hydrophilic ligands were highly selective, exhibiting preferential cytotoxicity in MMR-deficient cells at low concentrations and short incubation periods, whereas complexes with lipophilic ligands displayed poor cell-selectivity. It was discovered that all of the complexes localized to the nucleus in concentrations sufficient for mismatch binding; however, highly lipophilic complexes also exhibited high mitochondrial uptake. Significantly, these results support the notion that mitochondrial DNA is not the desired target for our metalloinsertor complexes; instead, selectivity stems from targeting mismatches in genomic DNA.
\r\n\r\nWe have also explored the potential for metalloinsertors to be developed into more complex structures with multiple functionalities that could either enhance their overall potency or impart mismatch selectivity onto other therapeutic cargo. We have constructed a family of bifunctional metalloinsertor conjugates incorporating cis-platinum, each unique in its chemical structure, DNA binding interactions, and biological activity. The study of these complexes in MMR-deficient cells has established that the cell-selective biological activity of rhodium metalloinsertors proceeds through a critical cellular pathway leading to necrosis.
\r\n\r\nWe further explored the underlying mechanisms surrounding the biological response to mismatch recognition by metalloinsertors in the genome. Immunofluorescence assays of MMR-deficient and MMR-proficient cells revealed that a critical biomarker for DNA damage, phosphorylation of histone H2AX (\u03b3H2AX) rapidly accumulates in response to metalloinsertor treatment, signifying the induction of double strand breaks in the genome. Significantly, we have discovered that our metalloinsertor complexes selectively inhibit transcription in MMR-deficient cells, which may be a crucial checkpoint in the eventual breakdown of the cell via necrosis. Additionally, preliminary in vivo studies have revealed the capability of these compounds to traverse the complex environments of multicellular organisms and accumulate in MMR-deficient tumors. Our ever-increasing understanding of metalloinsertors, as well as the development of new generations of complexes both monofunctional and bifunctional, enables their continued progress into the clinic as promising new chemotherapeutic agents.
", "doi": "10.7907/Z9RX991X", "publication_date": "2015", "thesis_type": "phd", "thesis_year": "2015" }, { "id": "thesis:8967", "collection": "thesis", "collection_id": "8967", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06022015-103853129", "primary_object_url": { "basename": "ALFurstThesisCompiled.pdf", "content": "final", "filesize": 25945747, "license": "other", "mime_type": "application/pdf", "url": "/8967/1/ALFurstThesisCompiled.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "DNA-Mediated Charge Transport Devices for Protein Detection", "author": [ { "family_name": "Furst", "given_name": "Ariel Lesa", "orcid": "0000-0001-9583-9703", "clpid": "Furst-Ariel-Lesa" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Agapie", "given_name": "Theodor", "clpid": "Agapie-T" }, { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Detection of biologically relevant targets, including small molecules, proteins, DNA, and RNA, is vital for fundamental research as well as clinical diagnostics. Sensors with biological elements provide a natural foundation for such devices because of the inherent recognition capabilities of biomolecules. Electrochemical DNA platforms are simple, sensitive, and do not require complex target labeling or expensive instrumentation. Sensitivity and specificity are added to DNA electrochemical platforms when the physical properties of DNA are harnessed. The inherent structure of DNA, with its stacked core of aromatic bases, enables DNA to act as a wire via DNA-mediated charge transport (DNA CT). DNA CT is not only robust over long molecular distances of at least 34 nm, but is also especially sensitive to anything that perturbs proper base stacking, including DNA mismatches, lesions, or DNA-binding proteins that distort the \u03c0-stack. Electrochemical sensors based on DNA CT have previously been used for single-nucleotide polymorphism detection, hybridization assays, and DNA-binding protein detection. Here, improvements to (i) the structure of DNA monolayers and (ii) the signal amplification with DNA CT platforms for improved sensitivity and detection are described.
\r\n\r\nFirst, improvements to the control over DNA monolayer formation are reported through the incorporation of copper-free click chemistry into DNA monolayer assembly. As opposed to conventional film formation involving the self-assembly of thiolated DNA, copper-free click chemistry enables DNA to be tethered to a pre-formed mixed alkylthiol monolayer. The total amount of DNA in the final film is directly related to the amount of azide in the underlying alkylthiol monolayer. DNA monolayers formed with this technique are significantly more homogeneous and lower density, with a larger amount of individual helices exposed to the analyte solution. With these improved monolayers, significantly more sensitive detection of the transcription factor TATA binding protein (TBP) is achieved.
\r\n\r\nUsing low-density DNA monolayers, two-electrode DNA arrays were designed and fabricated to enable the placement of multiple DNA sequences onto a single underlying electrode. To pattern DNA onto the primary electrode surface of these arrays, a copper precatalyst for click chemistry was electrochemically activated at the secondary electrode. The location of the secondary electrode relative to the primary electrode enabled the patterning of up to four sequences of DNA onto a single electrode surface. As opposed to conventional electrochemical readout from the primary, DNA-modified electrode, a secondary microelectrode, coupled with electrocatalytic signal amplification, enables more sensitive detection with spatial resolution on the DNA array electrode surface. Using this two-electrode platform, arrays have been formed that facilitate differentiation between well-matched and mismatched sequences, detection of transcription factors, and sequence-selective DNA hybridization, all with the incorporation of internal controls.
\r\n\r\nFor effective clinical detection, the two working electrode platform was multiplexed to contain two complementary arrays, each with fifteen electrodes. This platform, coupled with low density DNA monolayers and electrocatalysis with readout from a secondary electrode, enabled even more sensitive detection from especially small volumes (4 \u03bcL per well). This multiplexed platform has enabled the simultaneous detection of two transcription factors, TBP and CopG, with surface dissociation constants comparable to their solution dissociation constants.
\r\n\r\nWith the sensitivity and selectivity obtained from the multiplexed, two working electrode array, an electrochemical signal-on assay for activity of the human methyltransferase DNMT1 was incorporated. DNMT1 is the most abundant human methyltransferase, and its aberrant methylation has been linked to the development of cancer. However, current methods to monitor methyltransferase activity are either ineffective with crude samples or are impractical to develop for clinical applications due to a reliance on radioactivity. Electrochemical detection of methyltransferase activity, in contrast, circumvents these issues. The signal-on detection assay translates methylation events into electrochemical signals via a methylation-specific restriction enzyme. Using the two working electrode platform combined with this assay, DNMT1 activity from tumor and healthy adjacent tissue lysate were evaluated. Our electrochemical measurements revealed significant differences in methyltransferase activity between tumor tissue and healthy adjacent tissue.
\r\n\r\nAs differential activity was observed between colorectal tumor tissue and healthy adjacent tissue, ten tumor sets were subsequently analyzed for DNMT1 activity both electrochemically and by tritium incorporation. These results were compared to expression levels of DNMT1, measured by qPCR, and total DNMT1 protein content, measured by Western blot. The only trend detected was that hyperactivity was observed in the tumor samples as compared to the healthy adjacent tissue when measured electrochemically. These advances in DNA CT-based platforms have propelled this class of sensors from the purely academic realm into the realm of clinically relevant detection.
\r\n", "doi": "10.7907/Z9KH0K88", "publication_date": "2015", "thesis_type": "phd", "thesis_year": "2015" }, { "id": "thesis:8927", "collection": "thesis", "collection_id": "8927", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05292015-144036736", "primary_object_url": { "basename": "Christopher_Bruno_Marotta_2015-0522-Thesis.pdf", "content": "final", "filesize": 14106595, "license": "other", "mime_type": "application/pdf", "url": "/8927/1/Christopher_Bruno_Marotta_2015-0522-Thesis.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Structure-Function Studies of Nicotinic Acetylcholine Receptors Using Selective Agonists and Positive Allosteric Modulators", "author": [ { "family_name": "Marotta", "given_name": "Christopher Bruno", "orcid": "0000-0002-3110-0819", "clpid": "Marotta-Christopher-Bruno" } ], "thesis_advisor": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Miller", "given_name": "Thomas F.", "clpid": "Miller-T-F" }, { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "This dissertation primarily describes chemical-scale studies of nicotinic acetylcholine receptors (nAChRs) in order to better understand ligand-receptor selectivity and allosteric modulation influences during receptor activation. Electrophysiology coupled with canonical and non-canonical amino acids mutagenesis is used to probe subtle changes in receptor function.
\r\n\t\r\nThe first half of this dissertation focuses on differential agonist selectivity of \u03b14\u03b22-containing nAChRs. The \u03b14\u03b22 nAChR can assemble in alternative stoichiometries as well as assemble with other accessory subunits. Chapter 2 identifies key structural residues that dictate binding and activation of three stoichiometry-dependent \u03b14\u03b22 receptor ligands: sazetidine-A, cytisine, and NS9283. These do not follow previously suggested hydrogen-bonding patterns of selectivity. Instead, three residues on the complementary subunit strongly influence binding ability of a ligand and receptor activation. Chapter 3 involves isolation of a \u03b15\u03b14\u03b22 receptor-enriched population to test for a potential alternative agonist binding location at the \u03b15 \u03b14 interface. Results strongly suggest that agonist occupation of this site is not necessary for receptor activation and that the \u03b15 subunit only incorporates at the accessory subunit location.
\r\n\t\r\nThe second half of this dissertation seeks to identify residue interactions with positive allosteric modulators (PAMs) of the \u03b17 nAChR. Chapter 4 focuses on methods development to study loss of potentiation of Type I PAMs, which indicate residues vital to propagation of PAM effects and/or binding. Chapter 5 investigates \u03b17 receptor modulation by a Type II PAM (PNU 120596). These results show that PNU 120596 does not alter the agonist binding site, thus is relegated to influencing only the gating component of activation. From this, we were able to map a potential network of residues from the agonist binding site to the proposed PNU 120596 binding site that are essential for receptor potentiation.
", "doi": "10.7907/Z9V122Q9", "publication_date": "2015", "thesis_type": "phd", "thesis_year": "2015" }, { "id": "thesis:8769", "collection": "thesis", "collection_id": "8769", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:02122015-130640999", "type": "thesis", "title": "DNA-Mediated Oxidation of Transcription Factor p53", "author": [ { "family_name": "Schaefer", "given_name": "Kathryn Nicole", "orcid": "0000-0003-0908-3191", "clpid": "Schaefer-Kathryn-Nicole" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Deshaies", "given_name": "Raymond Joseph", "clpid": "Deshaies-R-J" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Transcription factor p53 is the most commonly altered gene in human cancer. As a redox-active protein in direct contact with DNA, p53 can directly sense oxidative stress through DNA-mediated charge transport. Electron hole transport occurs with a shallow distance dependence over long distances through the \u03c0-stacked DNA bases, leading to the oxidation and dissociation of DNA-bound p53. The extent of p53 dissociation depends upon the redox potential of the response element DNA in direct contact with each p53 monomer. The DNA sequence dependence of p53 oxidative dissociation was examined by electrophoretic mobility shift assays using radiolabeled oligonucleotides containing both synthetic and human p53 response elements with an appended anthraquinone photooxidant. Greater p53 dissociation is observed from DNA sequences containing low redox potential purine regions, particularly guanine triplets, within the p53 response element. Using denaturing polyacrylamide gel electrophoresis of irradiated anthraquinone-modified DNA, the DNA damage sites, which correspond to locations of preferred electron hole localization, were determined. The resulting DNA damage preferentially localizes to guanine doublets and triplets within the response element. Oxidative DNA damage is inhibited in the presence of p53, however, only at DNA sites within the response element, and therefore in direct contact with p53. From these data, predictions about the sensitivity of human p53-binding sites to oxidative stress, as well as possible biological implications, have been made. On the basis of our data, the guanine pattern within the purine region of each p53-binding site determines the response of p53 to DNA-mediated oxidation, yielding for some sequences the oxidative dissociation of p53 from a distance and thereby providing another potential role for DNA charge transport chemistry within the cell.
\r\n\r\nTo determine whether the change in p53 response element occupancy observed in vitro also correlates in cellulo, chromatin immunoprecipition (ChIP) and quantitative PCR (qPCR) were used to directly quantify p53 binding to certain response elements in HCT116N cells. The HCT116N cells containing a wild type p53 were treated with the photooxidant [Rh(phi)2bpy]3+, Nutlin-3 to upregulate p53, and subsequently irradiated to induce oxidative genomic stress. To covalently tether p53 interacting with DNA, the cells were fixed with disuccinimidyl glutarate and formaldehyde. The nuclei of the harvested cells were isolated, sonicated, and immunoprecipitated using magnetic beads conjugated with a monoclonal p53 antibody. The purified immounoprecipiated DNA was then quantified via qPCR and genomic sequencing. Overall, the ChIP results were significantly varied over ten experimental trials, but one trend is observed overall: greater variation of p53 occupancy is observed in response elements from which oxidative dissociation would be expected, while significantly less change in p53 occupancy occurs for response elements from which oxidative dissociation would not be anticipated.
\r\n\r\nThe chemical oxidation of transcription factor p53 via DNA CT was also investigated with respect to the protein at the amino acid level. Transcription factor p53 plays a critical role in the cellular response to stress stimuli, which may be modulated through the redox modulation of conserved cysteine residues within the DNA-binding domain. Residues within p53 that enable oxidative dissociation are herein investigated. Of the 8 mutants studied by electrophoretic mobility shift assay (EMSA), only the C275S mutation significantly decreased the protein affinity (KD) for the Gadd45 response element. EMSA assays of p53 oxidative dissociation promoted by photoexcitation of anthraquinone-tethered Gadd45 oligonucleotides were used to determine the influence of p53 mutations on oxidative dissociation; mutation to C275S severely attenuates oxidative dissociation while C277S substantially attenuates dissociation. Differential thiol labeling was used to determine the oxidation states of cysteine residues within p53 after DNA-mediated oxidation. Reduced cysteines were iodoacetamide labeled, while oxidized cysteines participating in disulfide bonds were 13C2D2-iodoacetamide labeled. Intensities of respective iodoacetamide-modified peptide fragments were analyzed using a QTRAP 6500 LC-MS/MS system, quantified with Skyline, and directly compared. A distinct shift in peptide labeling toward 13C2D2-iodoacetamide labeled cysteines is observed in oxidized samples as compared to the respective controls. All of the observable cysteine residues trend toward the heavy label under conditions of DNA CT, indicating the formation of multiple disulfide bonds potentially among the C124, C135, C141, C182, C275, and C277. Based on these data it is proposed that disulfide formation involving C275 is critical for inducing oxidative dissociation of p53 from DNA.
\r\n", "doi": "10.7907/Z9BV7DJQ", "publication_date": "2015", "thesis_type": "phd", "thesis_year": "2015" }, { "id": "thesis:8392", "collection": "thesis", "collection_id": "8392", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05232014-201621983", "type": "thesis", "title": "Computational Design of Self-Assembling Proteins and Protein-DNA Nanowires", "author": [ { "family_name": "Mou", "given_name": "Yun", "clpid": "Mou-Yun" } ], "thesis_advisor": [ { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" } ], "thesis_committee": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" }, { "family_name": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" }, { "family_name": "Clemons", "given_name": "William M.", "orcid": "0000-0002-0021-889X", "clpid": "Clemons-W-M" }, { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Computational protein design (CPD) is a burgeoning field that uses a physical-chemical or knowledge-based scoring function to create protein variants with new or improved properties. This exciting approach has recently been used to generate proteins with entirely new functions, ones that are not observed in naturally occurring proteins. For example, several enzymes were designed to catalyze reactions that are not in the repertoire of any known natural enzyme. In these designs, novel catalytic activity was built de novo (from scratch) into a previously inert protein scaffold. In addition to de novo enzyme design, the computational design of protein-protein interactions can also be used to create novel functionality, such as neutralization of influenza. Our goal here was to design a protein that can self-assemble with DNA into nanowires. We used computational tools to homodimerize a transcription factor that binds a specific sequence of double-stranded DNA. We arranged the protein-protein and protein-DNA binding sites so that the self-assembly could occur in a linear fashion to generate nanowires. Upon mixing our designed protein homodimer with the double-stranded DNA, the molecules immediately self-assembled into nanowires. This nanowire topology was confirmed using atomic force microscopy. Co-crystal structure showed that the nanowire is assembled via the desired interactions. To the best of our knowledge, this is the first example of a protein-DNA self-assembly that does not rely on covalent interactions. We anticipate that this new material will stimulate further interest in the development of advanced biomaterials.", "doi": "10.7907/Z9PG1PPV", "publication_date": "2014", "thesis_type": "phd", "thesis_year": "2014" }, { "id": "thesis:7951", "collection": "thesis", "collection_id": "7951", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:09102013-094001578", "primary_object_url": { "basename": "mui_timothy_2014_thesis.pdf", "content": "final", "filesize": 24873791, "license": "other", "mime_type": "application/pdf", "url": "/7951/1/mui_timothy_2014_thesis.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Expanding the Repertoire of DNA-Mediated Signaling in DNA Repair", "author": [ { "family_name": "Mui", "given_name": "Timothy Paul", "clpid": "Mui-Timothy-Paul" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Campbell", "given_name": "Judith L.", "clpid": "Campbell-J-L" }, { "family_name": "Reisman", "given_name": "Sarah E.", "clpid": "Reisman-S-E" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "DNA damage is extremely detrimental to the cell and must be repaired to protect the genome. DNA is capable of conducting charge through the overlapping \u03c0-orbitals of stacked bases; this phenomenon is extremely sensitive to the integrity of the \u03c0-stack, as perturbations attenuate DNA charge transport (CT). Based on the E. coli base excision repair (BER) proteins EndoIII and MutY, it has recently been proposed that redox-active proteins containing metal clusters can utilize DNA CT to signal one another to locate sites of DNA damage.
\r\n\t\r\nTo expand our repertoire of proteins that utilize DNA-mediated signaling, we measured the DNA-bound redox potential of the nucleotide excision repair (NER) helicase XPD from Sulfolobus acidocaldarius. A midpoint potential of 82 mV versus NHE was observed, resembling that of the previously reported BER proteins. The redox signal increases in intensity with ATP hydrolysis in only the WT protein and mutants that maintain ATPase activity and not for ATPase-deficient mutants. The signal increase correlates directly with ATP activity, suggesting that DNA-mediated signaling may play a general role in protein signaling. Several mutations in human XPD that lead to XP-related diseases have been identified; using SaXPD, we explored how these mutations, which are conserved in the thermophile, affect protein electrochemistry.
\r\n\r\nTo further understand the electrochemical signaling of XPD, we studied the yeast S. cerevisiae Rad3 protein. ScRad3 mutants were incubated on a DNA-modified electrode and exhibited a similar redox potential to SaXPD. We developed a haploid strain of S. cerevisiae that allowed for easy manipulation of Rad3. In a survival assay, the ATPase- and helicase-deficient mutants show little survival, while the two disease-related mutants exhibit survival similar to WT. When both a WT and G47R (ATPase/helicase deficient) strain were challenged with different DNA damaging agents, both exhibited comparable survival in the presence of hydroxyurea, while with methyl methanesulfonate and camptothecin, the G47R strain exhibits a significant change in growth, suggesting that Rad3 is involved in repairing damage beyond traditional NER substrates. Together, these data expand our understanding of redox-active proteins at the interface of DNA repair.
\r\n", "doi": "10.7907/WXQM-CJ32", "publication_date": "2014", "thesis_type": "phd", "thesis_year": "2014" }, { "id": "thesis:8220", "collection": "thesis", "collection_id": "8220", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05062014-151417635", "primary_object_url": { "basename": "NHD THESIS Full.pdf", "content": "final", "filesize": 13805012, "license": "other", "mime_type": "application/pdf", "url": "/8220/1/NHD THESIS Full.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Studies of the Serotonin Type 3A Receptor and the Chemical Preparation of tRNA", "author": [ { "family_name": "Duffy", "given_name": "Noah Hanville", "clpid": "Duffy-Noah-Hanville" } ], "thesis_advisor": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" } ], "thesis_committee": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Stoltz", "given_name": "Brian M.", "clpid": "Stoltz-B-M" }, { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" }, { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "This thesis describes studies surrounding a ligand-gated ion channel (LGIC): the serotonin type 3A receptor (5-HT3AR). Structure-function experiments using unnatural amino acid mutagenesis are described, as well as experiments on the methodology of unnatural amino acid mutagenesis. Chapter 1 introduces LGICs, experimental methods, and an overview of the unnatural amino acid mutagenesis.
\r\n\r\nIn Chapter 2, the binding orientation of the clinically available drugs ondansetron and granisetron within 5-HT3A is determined through a combination of unnatural amino acid mutagenesis and an inhibition based assay. A cation-\u03c0 interaction is found for both ondansetron and granisetron with a specific tryptophan residue (Trp183, TrpB) of the mouse 5-HT3AR, which establishes a binding orientation for these drugs.
\r\n\r\nIn Chapter 3, further studies were performed with ondansetron and granisetron with 5-HT3A. The primary determinant of binding for these drugs was determined to not include interactions with a specific tyrosine residue (Tyr234, TyrC2). In completing these studies, evidence supporting a cation-\u03c0 interaction of a synthetic agonist, meta-chlorophenylbiguanide, was found with TyrC2.
\r\n\r\nIn Chapter 4, a direct chemical acylation strategy was implemented to prepare full-length suppressor tRNA mediated by lanthanum(III) and amino acid phosphate esters. The derived aminoacyl-tRNA is shown to be translationally competent in Xenopus oocytes.
\r\n\r\nAppendix A.1 gives details of a pharmacological method for determining the equilibrium dissociation constant, KB, of a competitive antagonist with a receptor, known as Schild analysis. Appendix A.2 describes an examination of the inhibitory activity of new chemical analogs of the 5-HT3A antagonist ondansetron. Appendix A.3 reports an organic synthesis of an intermediate for a new unnatural amino acid. Appendix A.4 covers an additional methodological examination for the preparation of amino-acyl tRNA.
", "doi": "10.7907/X1YA-DM13", "publication_date": "2014", "thesis_type": "phd", "thesis_year": "2014" }, { "id": "thesis:8050", "collection": "thesis", "collection_id": "8050", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:01102014-204136086", "primary_object_url": { "basename": "wiggenhorn_david_craig_2014_thesis.pdf", "content": "final", "filesize": 11340130, "license": "other", "mime_type": "application/pdf", "url": "/8050/1/wiggenhorn_david_craig_2014_thesis.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Nanostructured Tungsten Trioxide Photoanodes for Solar Energy Conversion", "author": [ { "family_name": "Wiggenhorn", "given_name": "David Craig", "clpid": "Wiggenhorn-David-Craig" } ], "thesis_advisor": [ { "family_name": "Lewis", "given_name": "Nathan Saul", "clpid": "Lewis-N-S" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Okumura", "given_name": "Mitchio", "clpid": "Okumura-M" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Lewis", "given_name": "Nathan Saul", "clpid": "Lewis-N-S" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Nanostructured tungsten trioxide (WO3) photoelectrodes are potential candidates for the anodic portion of an integrated solar water-splitting device that generates hydrogen fuel and oxygen from water. These nanostructured materials can potentially offer improved performance in photooxidation reactions compared to unstructured materials because of enhancements in light scattering, increases in surface area, and their decoupling of the directions of light absorption and carrier collection. To evaluate the presence of these effects and their contributions toward energy conversion efficiency, a variety of nanostructured WO3 photoanodes were synthesized by electrodeposition within nanoporous templates and by anodization of tungsten foils. A robust fabrication process was developed for the creation of oriented WO3 nanorod arrays, which allows for control nanorod diameter and length. Films of nanostructured WO3 platelets were grown via anodization, the morphology of the films was controlled by the anodization conditions, and the current-voltage performance and spectral response properties of these films were studied. The observed photocurrents were consistent with the apparent morphologies of the nanostructured arrays. Measurements of electrochemically active surface area and other physical characteristics were correlated with observed differences in absorbance, external quantum yield, and photocurrent density for the anodized arrays. The capability to quantify these characteristics and relate them to photoanode performance metrics can allow for selection of appropriate structural parameters when designing photoanodes for solar energy conversion.", "doi": "10.7907/0CKV-SM88", "publication_date": "2014", "thesis_type": "phd", "thesis_year": "2014" }, { "id": "thesis:8032", "collection": "thesis", "collection_id": "8032", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:11202013-144350295", "primary_object_url": { "basename": "GleasonRohrer2013thesis.pdf", "content": "final", "filesize": 2166776, "license": "other", "mime_type": "application/pdf", "url": "/8032/1/GleasonRohrer2013thesis.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Measurement of the Band Bending and Surface Dipole at Chemically Functionalized Si(111)/Vacuum Interfaces", "author": [ { "family_name": "Gleason-Rohrer", "given_name": "David Charles", "clpid": "Gleason-Rohrer-David-Charles" } ], "thesis_advisor": [ { "family_name": "Lewis", "given_name": "Nathan Saul", "clpid": "Lewis-N-S" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Brunschwig", "given_name": "Bruce S.", "clpid": "Brunschwig-B-S" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Goddard", "given_name": "William A., III", "clpid": "Goddard-W-A-III" }, { "family_name": "Lewis", "given_name": "Nathan Saul", "clpid": "Lewis-N-S" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The core-level energy shifts observed using X-ray photoelectron spectroscopy (XPS) have been used to determine the band bending at Si(111) surfaces terminated with Si-Br, Si-H, and Si-CH3 groups, respectively. The surface termination influenced the band bending, with the Si 2p3/2 binding energy affected more by the surface chemistry than by the dopant type. The highest binding energies were measured on Si(111)-Br (whose Fermi level was positioned near the conduction band at the surface), followed by Si(111)-H, followed by Si(111)-CH3 (whose Fermi level was positioned near mid-gap at the surface). Si(111)-CH3 surfaces exposed to Br2(g) yielded the lowest binding energies, with the Fermi level positioned between mid-gap and the valence band. The Fermi level position of Br2(g)-exposed Si(111)-CH3 was consistent with the presence of negatively charged bromine-containing ions on such surfaces. The binding energies of all of the species detected on the surface (C, O, Br) shifted with the band bending, illustrating the importance of isolating the effects of band bending when measuring chemical shifts on semiconductor surfaces. The influence of band bending was confirmed by surface photovoltage (SPV) measurements, which showed that the core levels shifted toward their flat-band values upon illumination. Where applicable, the contribution from the X-ray source to the SPV was isolated and quantified. Work functions were measured by ultraviolet photoelectron spectroscopy (UPS), allowing for calculation of the sign and magnitude of the surface dipole in such systems. The values of the surface dipoles were in good agreement with previous measurements as well as with electronegativity considerations. The binding energies of the adventitious carbon signals were affected by band bending as well as by the surface dipole. A model of band bending in which charged surface states are located exterior to the surface dipole is consistent with the XPS and UPS behavior of the chemically functionalized Si(111) surfaces investigated herein.
", "doi": "10.7907/PD7F-P488", "publication_date": "2014", "thesis_type": "phd", "thesis_year": "2014" }, { "id": "thesis:8212", "collection": "thesis", "collection_id": "8212", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05042014-135648744", "primary_object_url": { "basename": "Daeffler May 2 2014.pdf", "content": "final", "filesize": 21926468, "license": "other", "mime_type": "application/pdf", "url": "/8212/1/Daeffler May 2 2014.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Functional Evaluation of Noncovalent Interactions in Neuroreceptors and Progress Toward the Expansion of Unnatural Amino Acid Methodology", "author": [ { "family_name": "Daeffler", "given_name": "Kristina Nicole-McCleary", "clpid": "Daeffler-Kristina-Nicole-McCleary" } ], "thesis_advisor": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" } ], "thesis_committee": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Heath", "given_name": "James R.", "clpid": "Heath-J-R" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "This dissertation primarily describes chemical-scale studies of G protein-coupled receptors and Cys-loop ligand-gated ion channels to better understand ligand binding interactions and the mechanism of channel activation using recently published crystal structures as a guide. These studies employ the use of unnatural amino acid mutagenesis and electrophysiology to measure subtle changes in receptor function.
\r\n\r\nIn chapter 2, the role of a conserved aromatic microdomain predicted in the D3 dopamine receptor is probed in the closely related D2 and D4 dopamine receptors. This domain was found to act as a structural unit near the ligand binding site that is important for receptor function. The domain consists of several functionally important noncovalent interactions including hydrogen bond, aromatic-aromatic, and sulfur-\u03c0 interactions that show strong couplings by mutant cycle analysis. We also assign an alternate interpretation for the linear fluorination plot observed at W6.48, a residue previously thought to participate in a cation-\u03c0 interaction with dopamine.
\r\n\r\nChapter 3 outlines attempts to incorporate chemically synthesized and in vitro acylated unnatural amino acids into mammalian cells. While our attempts were not successful, method optimizations and data for nonsense suppression with an in vivo acylated tRNA are included. This chapter is aimed to aid future researchers attempting unnatural amino acid mutagenesis in mammalian cells.
\r\n\r\nChapter 4 identifies a cation-\u03c0 interaction between glutamate and a tyrosine residue on loop C in the GluCl\u03b2 receptor. Using the recently published crystal structure of the homologous GluCl\u03b1 receptor, other ligand-binding and protein-protein interactions are probed to determine the similarity between this invertebrate receptor and other more distantly related vertebrate Cys-loop receptors. We find that many of the interactions previously observed are conserved in the GluCl receptors, however care must be taken when extrapolating structural data.
\r\n\r\nChapter 5 examines inherent properties of the GluCl\u03b1 receptor that are responsible for the observed glutamate insensitivity of the receptor. Chimera synthesis and mutagenesis reveal the C-terminal portion of the M4 helix and the C-terminus as contributing to formation of the decoupled state, where ligand binding is incapable of triggering channel gating. Receptor mutagenesis was unable to identify single residue mismatches or impaired protein-protein interactions within this domain. We conclude that M4 helix structure and/or membrane dynamics are likely the cause of ligand insensitivity in this receptor and that the M4 helix has an role important in the activation process.
", "doi": "10.7907/ST7S-DB65", "publication_date": "2014", "thesis_type": "phd", "thesis_year": "2014" }, { "id": "thesis:7960", "collection": "thesis", "collection_id": "7960", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:09192013-114842407", "primary_object_url": { "basename": "Pheeney_Thesis Complied 09-19-2013.pdf", "content": "final", "filesize": 4478398, "license": "other", "mime_type": "application/pdf", "url": "/7960/1/Pheeney_Thesis Complied 09-19-2013.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Multiplexed DNA-Mediated Electrochemistry", "author": [ { "family_name": "Pheeney", "given_name": "Catrina Gale", "clpid": "Pheeney-Catrina-Gale" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Heath", "given_name": "James R.", "clpid": "Heath-J-R" }, { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The aromatic core of double helical DNA possesses the unique and remarkable ability to form a conduit for electrons to travel over exceptionally long molecular distances. This core of \u03c0-stacked nucleobases creates an efficient pathway for charge transfer to proceed that is exquisitely sensitive to even subtle perturbations. Ground state electrochemistry of DNA-modified electrodes has been one of the major techniques used both to investigate and to harness the property of DNA-mediated charge transfer. DNA-modified electrodes have been an essential tool for both gaining insights into the fundamental properties of DNA and, due to the exquisite specificity of DNA-mediated charge transfer for the integrity of the \u03c0-stack, for use in next generation diagnostic sensing. Here, multiplexed DNA-modified electrodes are used to (i) gain new insights on the electrochemical coupling of metalloproteins to the DNA \u03c0-stack with relevance to the fundaments of in vivo DNA-mediated charge transfer and (ii) enhance the overall sensitivity of DNA-mediated reduction for use in the detection of low abundance diagnostic targets.
\r\n\r\nFirst, Methylene Blue (MB\u2032) was covalently attached to DNA through a flexible C12 alkyl linker to yield a new redox reporter for DNA electrochemistry measurements with enhanced sensitivity. Tethered, intercalated MB\u2032 was reduced through DNA-mediated charge transport. The redox signal intensity for MB\u2032-dT-C12-DNA was found to be at least 3 fold larger than that of previously used Nile Blue (NB)-dT-DNA, which is coupled to the base stack via direct conjugation. The signal attenuation, due to an intervening mismatch, and therefore the degree of DNA-mediated reduction, does, however, depend on the DNA film morphology and the backfilling agent used to passivate the surface. These results highlight two possible mechanisms for the reduction of MB\u2032 on the DNA-modified electrode that are distinguishable by their kinetics: reduction mediated by the DNA base pair stack and direct surface reduction of MB\u2032 at the electrode. The extent of direct reduction at the surface can be minimized by overall DNA assembly conditions.
\r\n\r\nNext, a series of intercalation-based DNA-mediated electrochemical reporters were developed, using a flexible alkane linkage to validate and explore their DNA-mediated reduction. The general mechanism for the reduction of distally bound redox active species, covalently tethered to DNA through flexible alkyl linkages, was established to be an intraduplex DNA-mediated pathway. MB, NB, and anthraquinone were covalently tethered to DNA with three different covalent linkages. The extent of electronic coupling of the reporter was shown to correlate with the DNA binding affinity of the redox active species, supporting an intercalative mechanism. These electrochemical signals were shown to be exceptionally sensitive to a single intervening \u03c0-stack perturbation, an AC mismatch, in a densely packed DNA monolayer, which further supports that the reduction is DNA-mediated. Finally, this DNA-mediated reduction of MB occurs primarily via intra- rather than inter duplex intercalation, as probed through varying the proximity and integrity of the neighboring duplex DNA.\r\nFurther gains to electrochemical sensitivity of our DNA-modified devices were then achieved through the application of electrocatalytic signal amplification using these solvent accessible intercalative reporters, MB-dT-C8, and hemoglobin as a novel electron sink. Electrocatalysis offers an excellent means of electrochemical signal amplification, yet in DNA based sensors, its application has been limited due to strict assembly conditions. We describe the use of hemoglobin as a robust and effective electron sink for electrocatalysis in DNA sensing on low density DNA films. Protein shielding of the heme redox center minimizes direct reduction at the electrode surface and permits assays on low density DNA films. Electrocatalysis of MB that is covalently tethered to the DNA by a flexible alkyl linkage allows for efficient interactions with both the base stack and hemoglobin. Consistent suppression of the redox signal upon incorporation of single CA mismatch in the DNA oligomer demonstrates that both the unamplified and the electrocatalytically amplified redox signals are generated through DNA-mediated charge transport. Electrocatalysis with hemoglobin is robust: it is stable to pH and temperature variations. The utility and applicability of electrocatalysis with hemoglobin is demonstrated through restriction enzyme detection, and an enhancement in sensitivity permits femtomole DNA sampling.
\r\n\r\nFinally, we expanded the application of our multiplexed DNA-modified electrodes to the electrochemical characterization of DNA-bound proteins containing [4Fe-4S] clusters. DNA-modified electrodes have become an essential tool for the characterization of the redox chemistry of DNA repair proteins that contain redox cofactors. Multiplexed analysis of EndonucleaseIII (EndoIII), a DNA repair protein containing a [4Fe-4S] cluster known to be accessible via DNA-mediated charge transport, elucidated subtle differences in the electrochemical behavior as a function of DNA morphology. DNA-bound EndoIII is seen to have two different electron transfer pathways for reduction, either through the DNA base stack or through direct surface reduction. Closely packed DNA films, where the protein has limited surface accessibility, produce electrochemical signals reflecting electron transfer that is DNA-mediated. The electrochemical comparison of EndoIII mutants, including a new family of mutations altering the electrostatics surrounding the [4Fe-4S] cluster, was able to be quantitatively performed. While little change in the midpoint potential was found for this family of mutants, significant variations in the efficiency of DNA-mediated electron transfer were apparent. Based on the stability of these proteins, examined by circular dichroism, we propose that the electron transfer pathway can be perturbed not only by the removal of aromatic residues, but also through changes in solvation near the cluster.
\r\n", "doi": "10.7907/PCF7-9669", "publication_date": "2014", "thesis_type": "phd", "thesis_year": "2014" }, { "id": "thesis:8412", "collection": "thesis", "collection_id": "8412", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05282014-143107089", "primary_object_url": { "basename": "thesis_final.pdf", "content": "final", "filesize": 8258010, "license": "other", "mime_type": "application/pdf", "url": "/8412/1/thesis_final.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Design, Synthesis, and Biological Activity of Rhodium Metalloinsertors", "author": [ { "family_name": "Komor", "given_name": "Alexis Christine", "clpid": "Komor-Alexis-Christine" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Peters", "given_name": "Jonas C.", "clpid": "Peters-J-C" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Deficiencies in the mismatch repair (MMR) pathway are associated with several types of cancers, as well as resistance to commonly used chemotherapeutics. Rhodium metalloinsertors have been found to bind DNA mismatches with high affinity and specificity in vitro, and also exhibit cell-selective cytotoxicity, targeting MMR-deficient cells over MMR-proficient cells.
\r\n \r\nHere we examine the biological fate of rhodium metalloinsertors bearing dipyridylamine ancillary ligands. These complexes are shown to exhibit accelerated cellular uptake which permits the observation of various cellular responses, including disruption of the cell cycle and induction of necrosis, which occur preferentially in the MMR-deficient cell line. These cellular responses provide insight into the mechanisms underlying the selective activity of this novel class of targeted anti-cancer agents.
\r\n\r\nIn addition, ten distinct metalloinsertors with varying lipophilicities are synthesized and their mismatch binding affinities and biological activities studied. While they are found to have similar binding affinities, their cell-selective antiproliferative and cytotoxic activities vary significantly. Inductively coupled plasma mass spectrometry (ICP-MS) experiments show that all of these metalloinsertors localize in the nucleus at sufficient concentrations for binding to DNA mismatches. Furthermore, metalloinsertors with high rhodium localization in the mitochondria show toxicity that is not selective for MMR-deficient cells. This work supports the notion that specific targeting of the metalloinsertors to nuclear DNA gives rise to their cytotoxic and antiproliferative activities that are selective for cells deficient in MMR.
\r\n\r\nTo explore further the basis of the unique selectivity of the metlloinsertors in targeting MMR-deficient cells, experiments were conducted using engineered NCI-H23 lung adenocarcinoma cells that contain a doxycycline-inducible shRNA which suppresses the expression of the MMR gene MLH1. Here we use this new cell line to further validate rhodium metalloinsertors as compounds capable of differentially inhibiting the proliferation of MMR-deficient cancer cells over isogenic MMR-proficient cells. General DNA damaging agents, such as cisplatin and etoposide, in contrast, are less effective in the induced cell line defective in MMR.
\r\n\r\nFinally, we describe a new subclass of metalloinsertors with enhanced potency and selectivity, in which the complexes show Rh-O coordination. In particular, it has been found that both \u0394 and \u039b enantiomers of [Rh(chrysi)(phen)(DPE)]2+ bind to DNA with similar affinities, suggesting a possible different binding conformation than previous metalloinsertors. Remarkably, all members of this new family of compounds have significantly increased potency in a range of cellular assays; indeed, all are more potent than the FDA-approved anticancer drugs cisplatin and MNNG. Moreover, these activities are coupled with high levels of selectivity for MMR-deficient cells.
\r\n", "doi": "10.7907/KPCY-JS09", "publication_date": "2014", "thesis_type": "phd", "thesis_year": "2014" }, { "id": "thesis:7892", "collection": "thesis", "collection_id": "7892", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06112013-202017350", "primary_object_url": { "basename": "Heather R Williamson Thesis library.pdf", "content": "final", "filesize": 4523391, "license": "other", "mime_type": "application/pdf", "url": "/7892/1/Heather R Williamson Thesis library.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Engineering Multi Step Electron Tunneling Systems in Proteins", "author": [ { "family_name": "Williamson", "given_name": "Heather R.", "clpid": "Williamson-Heather-R" } ], "thesis_advisor": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" } ], "thesis_committee": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Stoltz", "given_name": "Brian M.", "clpid": "Stoltz-B-M" }, { "family_name": "Agapie", "given_name": "Theodor", "clpid": "Agapie-T" }, { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Multi-step electron tunneling, or \u201chopping,\u201d has become a fast-developing research field with studies ranging from theoretical modeling systems, inorganic complexes, to biological systems. In particular, the field is exploring hopping mechanisms in new proteins and protein complexes, as well as further understanding the classical biological hopping systems such as ribonuclease reductase, DNA photolyases, and photosystem II. Despite the plethora of natural systems, only a few biologically engineered systems exist. Engineered hopping systems can provide valuable information on key structural and electronic features, just like other kinds of biological model systems. Also, engineered systems can harness common biologic processes and utilize them for alternative reactions. In this thesis, two new hopping systems are engineered and characterized.
\r\n\r\nThe protein Pseudomonas aeruginosa azurin is used as a building block to create the two new hopping systems. Besides being well studied and amenable to mutation, azurin already has been used to successfully engineer a hopping system. The two hopping systems presented in this thesis have a histidine-attached high potential rhenium 4,7-dimethyl-1,10-phenanthroline tricarbonyl [Re(dmp)(CO)3] + label which, when excited, acts as the initial electron acceptor. The metal donor is the type I copper of the azurin protein. The hopping intermediates are all tryptophan, an amino acid mutated into the azurin at select sites between the photoactive metal label and the protein metal site. One system exhibits an inter-molecular hopping through a protein dimer interface; the other system undergoes intra-molecular multi-hopping utilizing a tryptophan \u201cwire.\u201d The electron transfer reactions are triggered by excitation of the rhenium label and monitored by UV-Visible transient absorption, luminescence decays measurements, and time-resolved Infrared spectroscopy (TRIR). Both systems were structurally characterized by protein X-ray crystallography.
", "doi": "10.7907/BRZJ-YZ76", "publication_date": "2013", "thesis_type": "phd", "thesis_year": "2013" }, { "id": "thesis:7695", "collection": "thesis", "collection_id": "7695", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05102013-152319367", "primary_object_url": { "basename": "mccaig_thesis_2013.pdf", "content": "final", "filesize": 17744381, "license": "other", "mime_type": "application/pdf", "url": "/7695/32/mccaig_thesis_2013.pdf", "version": "v10.0.0" }, "type": "thesis", "title": "Resonant Nanocantilever Chemical Vapor Sensors", "author": [ { "family_name": "McCaig", "given_name": "Heather Catherine", "clpid": "McCaig-Heather-Catherine" } ], "thesis_advisor": [ { "family_name": "Lewis", "given_name": "Nathan Saul", "clpid": "Lewis-N-S" } ], "thesis_committee": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Lewis", "given_name": "Nathan Saul", "clpid": "Lewis-N-S" }, { "family_name": "Roukes", "given_name": "Michael Lee", "clpid": "Roukes-M-L" }, { "family_name": "Kornfield", "given_name": "Julia A.", "clpid": "Kornfield-J-A" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Chemical vapor sensors are used in a wide variety of fields such as security, environmental monitoring, the food and beverage industry, and healthcare to detect disease biomarkers on exhaled breath. An electronic nose is composed of an array of cross-responsive chemical vapor sensors, in which every sensor responds to a varying degree to each chemical vapor, creating a \"fingerprint\" for that vapor. Incorporating an electronic nose into a highly-miniaturized vapor detection system, capable of bringing near laboratory-quality analysis into the field, requires the use of extremely small, fast, and sensitive sensors. One option is resonant nanocantilevers, which respond to changes in mass and stiffness by shifts in resonant frequency, and are capable of detecting mass-loading at the attogram (10-18 g) level in ambient conditions.
\r\n\r\nTo determine whether nanocantilevers can be used in an electronic nose, an array of five nanocantilevers, wherein each sensor was coated with a different dropcast polymer film (2-10 nm thick), were exposed to seven chemical vapors with a range of functional groups. The array successfully discriminated between all vapors, indicating that sensor responses were dominated by vapor absorption into polymer films, and not by non-specific physisorption. The thinness of the polymer film, combined with the small vapor capture area of the nanocantilevers, resulted in lower sensitivity than desired, limiting their effectiveness. To overcome this challenge, surface initiated atom transfer radical polymerization (SI-ATRP) was used to grow a 100 nm thick, uniform films of poly(methylmethacrylate) (PMMA), poly(methyl acrylate) (PMA), and poly(n-butyl methacrylate) (PBMA) on nanocantilevers. The thick polymer films absorbed more vapor, significantly increasing nanocantilever sensitivity. To determine the relative roles of mass loading and stiffness change on nanocantilever sensor response, SI-ATRP was combined with chromium masking, enabling polymer film growth to be localized to either the clamped end (sensitive to stiffness) or the free end (sensitive to mass-loading) of the nanocantilevers. These experiments revealed that changes in stiffness, induced by vapor absorption into the polymer films, dominated the sensor responses, and not mass-loading as was initially assumed. This work demonstrated that an array resonant nanocantilevers can be successfully used a sensitive, nanoscale electronic nose.
", "doi": "10.7907/Z957191Q", "publication_date": "2013", "thesis_type": "phd", "thesis_year": "2013" }, { "id": "thesis:7446", "collection": "thesis", "collection_id": "7446", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:01282013-083506263", "primary_object_url": { "basename": "Muren_Natalie_2013_combined_chapters.pdf", "content": "final", "filesize": 5695199, "license": "other", "mime_type": "application/pdf", "url": "/7446/1/Muren_Natalie_2013_combined_chapters.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "DNA-Mediated Charge Transport for Long-Range Sensing and Protein Detection", "author": [ { "family_name": "Muren", "given_name": "Natalie Bloom", "clpid": "Muren-Natalie-Bloom" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" }, { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The structural core of DNA, a continuous stack of aromatic heterocycles\u2014the base pairs\u2014that extends down the helical axis, gives rise to the fascinating electronic properties of this molecule that is so critical for life. This \u03c0-stacked structure facilitates a unique form of charge conduction, termed DNA-mediated charge transport (DNA CT). Experiments with diverse platforms, in solution, on surfaces, and with single molecules, collectively provide a broad and consistent perspective on the essential characteristics of this chemistry. Notably, DNA CT can proceed over long molecular distances, but is remarkably sensitive to perturbations in base pair stacking. These characteristics suggest that DNA CT may be used for long-range sensing both in nature and in nanoelectronic applications. Here, measurements of DNA CT with surface and single molecule platforms are used to (i) determine how ground state DNA CT varies over regimes of increasing distance and (ii) apply this chemistry to the electrical detection of DNA-binding proteins.
\r\n\r\nFirst, the design and fabrication of multiplexed, DNA-modified electrodes on silicon chips is reported. These lithographically patterned chips with 16 individually addressable gold electrodes allow for the measurement of DNA CT with four different types of DNA, side by side on the same surface, with four-fold redundancy. Discrimination of DNA with a single base mismatch and detection of sequence-specific restriction enzyme activity are both achieved with these chips. Scaling of these devices to microelectrode dimensions is also demonstrated. Importantly, these chips show greater reproducibility and consistency than commercially available rod electrodes. This greater signal quality, combined with the capacity to examine different samples side by side, opens the door for more complex applications of this platform.
\r\n\r\nThe fully developed, multiplexed chips are first used to compare DNA CT over short and long distance regimes. DNA is evaluated in this context because the efficacy of a long-range sensor, in either nature or nanoelectronics, is determined largely by its capacity to facilitate CT in a manner that is minimally affected by the CT distance. DNA CT over 34 nm in 100-mer monolayers is found to yield electrochemical signals that are comparable in size to shorter 17-mer DNA. Signal attenuation from a single base-pair mismatch in the 100-mer is also comparable to that for 17-mers, and confirms that CT in these 100-mer films is DNA-mediated. Efficient cleavage by a restriction enzyme indicates that the 100-mer DNA adopts a native, upright conformation. The alkanethiol linker used to anchor the DNA to the electrode is found to limit the electron-transfer rate for both DNA lengths. Thus the impact of increasing the CT distance on DNA CT is too small to be resolved by this platform, even over 34 nm. These measurements put DNA among the longest and most conductive molecular wires reported to date.
\r\n\r\nNext, DNA CT with multiplexed chips is extended to the electrochemical detection of methyltransferases, proteins that are attractive targets because of their prominent role in the initial stages of many types of cancer. Electrochemical detection of binding and activity by these proteins is achieved by two different methods. First, DNA-binding and base-flipping by these proteins disrupts the DNA \u03c0-stack and may be used for direct \u201csignal OFF\u201d detection. Using this method, the concentration- and cofactor- dependence of SssI methyltransferase, the bacterial analog of human methyltransferases, are examined. Second, methylation-conferred protection of DNA against cutting by a restriction enzyme may be used for \u201csignal ON\u201d detection of methyltransferase activity. With this approach, the use of both unmethylated and hemimethylated DNA substrates is demonstrated for the sensitive detection of both bacterial (SssI) and human (Dnmt1) methyltransferase activity. Importantly, the electrochemical format of these assays requires minimal equipment, is low cost, and may be easily applied to high throughput studies, making it an accessible option for a variety of research and clinical settings.
\r\n\r\nAlongside work with this surface, electrochemical platform, a single molecule, carbon nanotube-DNA (CNT-DNA) platform is also used to evaluate DNA CT over increasing distances and to detect protein binding. CNT-DNA devices consist of a single molecule of DNA that is made to bridge a gap cut in a CNT covalently, such that current flow through the device is DNA-mediated. Upon introduction of DNA bridges of varying length (15-mer, 60-mer, and 100-mer), the device resistance is minimally affected, echoing the result of long distance electrochemistry experiments. These devices are also used to detect SssI methyltransferase binding by the direct \u201csignal OFF\u201d method used with multiplexed chips; DNA-binding and base-flipping disrupts DNA CT and shuts off current flow through the device. CNT-DNA devices are used to electronically measure the sequence-specific, cofactor-dependent, and reversible binding of SssI. DNA methylation catalyzed by SssI is also detected based on its alteration of the protein-binding affinity of the device. This detection approach, which relies on DNA as both a recognition element and electrical transducer, represents a unique strategy for the specific, single molecule detection of protein binding and activity.
\r\n", "doi": "10.7907/6KG5-KQ87", "publication_date": "2013", "thesis_type": "phd", "thesis_year": "2013" }, { "id": "thesis:7775", "collection": "thesis", "collection_id": "7775", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05302013-155904165", "primary_object_url": { "basename": "FordNicole2013thesis.pdf", "content": "final", "filesize": 5837787, "license": "other", "mime_type": "application/pdf", "url": "/7775/1/FordNicole2013thesis.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "Capturing Protein Dynamics with Time-Resolved Luminescence Spectroscopy", "author": [ { "family_name": "Ford", "given_name": "Nicole Danielle Bouley", "clpid": "Ford-Nicole-Danielle-Bouley" } ], "thesis_advisor": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" } ], "thesis_committee": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Miller", "given_name": "Thomas F.", "clpid": "Miller-T-F" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Winkler", "given_name": "Jay Richmond", "clpid": "Winkler-J-R" }, { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The presented doctoral research utilizes time-resolved spectroscopy to characterize protein dynamics and folding mechanisms. We resolve millisecond-timescale folding by coupling time-resolved fluorescence energy transfer (trFRET) to a continuous flow microfluidic mixer to obtain intramolecular distance distributions throughout the folding process. We have elucidated the folding mechanisms of two cytochromes---one that exhibits two-state folding (cytochrome
We have also investigated intrachain contact dynamics in unfolded cytochrome
In addition, we have explored the pathway dependence of electron tunneling rates between metal sites in proteins. Our research group has converted cytochrome
Deficiencies in DNA mismatch repair (MMR) have been implicated in the development of several forms of cancers, and MMR-deficient cells tend to be resistant to commonly employed cancer therapeutics such as cisplatin. Mismatch-targeting metalloinsertors developed in our laboratory have shown great promise as therapeutic and diagnostic agents for MMR-deficient cancers. In this work, we examine fundamental aspects of binding interactions of octahedral rhodium and ruthenium complexes to DNA mismatches, and strive to develop a luminescent sensor for mismatches inside cells.
\r\n\r\nWe first demonstrate that the mismatch binding affinity of rhodium metalloinsertors directly correlates with their antiproliferative effect against MMR-deficient colorectal carcinoma cells. Smaller ancillary ligands on the rhodium center facilitate binding to mismatches via metalloinsertion from the narrow minor groove of DNA. Complexes with higher mismatch binding affinity in turn selectively inhibit the growth of MMR-deficient cells compared to MMR-proficient ones. This correlation suggests that DNA mismatches are indeed the biological target of rhodium metalloinsertors inside cells.
\r\n\r\nBesides rhodium metalloinsertors, luminescent ruthenium complexes are found to bind DNA mismatches as well. Mismatch binding is accompanied by enhanced luminescence intensity. We determined two crystal structures of \u0394-Ru(bpy)2dppz2+ bound to oligonucleotide duplexes. For an oligonucleotide containing AA mismatches, the atomic-resolution structure revealed that the ruthenium complex binds to DNA mismatches also through metalloinsertion: the complex inserts a planar ligand into the mismatched site from the minor groove, ejecting the mismatched bases out of the helix. Several binding geometries of the complex intercalated between well-matched DNA were also observed.
\r\n\r\nTo improve the mismatch selectivity of luminescent ruthenium complexes, we tethered the complexes to organic dye molecules in an effort to amplify mismatch-associated luminescence signal through resonance energy transfer. We also modified the structure of the inserting ligand in an attempt to improve the binding affinity to mismatches over well-matched DNA. Coupling mismatch binding to luminescence response has proved most challenging in these endeavors.
\r\n\r\nFinally, we venture into the realm of RNA. Unlike their nonspecific binding to DNA, ruthenium complexes bind poorly to well-matched RNA but quite avidly to RNA mismatches. As a result, mismatched RNA produces a higher luminescence signal from bound ruthenium. We subsequently applied the ruthenium complex to image RNA mismatches inside live HeLa cells using fluorescence microscopy.
", "doi": "10.7907/4TM6-BR89", "publication_date": "2012", "thesis_type": "phd", "thesis_year": "2012" }, { "id": "thesis:7072", "collection": "thesis", "collection_id": "7072", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05242012-110320685", "type": "thesis", "title": "DNA-mediated Charge Transport in a Biological Context: Cooperation among Metalloproteins to Find Lesions in the Genome", "author": [ { "family_name": "Sontz", "given_name": "Pamela Alisa", "clpid": "Sontz-Pamela-Alisa" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Clemons", "given_name": "William M.", "clpid": "Clemons-W-M" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Damaged bases in DNA are known to lead to errors in replication and transcription, compromising the integrity of the genome. A molecular wire, DNA conducts charge with shallow distance dependence, yet mismatches and lesions attenuate this process. We have proposed a model where repair proteins, containing redox-active [4Fe4S] clusters, utilize DNA charge transport (CT) to scan the genome for lesions. Based on this model, proteins are predicted to redistribute onto strands where DNA CT is inhibited. Using single-molecule atomic force microscopy (AFM) we have probed the redistribution of EndoIII, a base excision repair protein that contains a [4Fe4S] cluster. Consistent with the model, we find a redistribution of EndoIII onto DNA strands (3.8 kbp) containing C:A mismatch, which is not a specific substrate of EndoIII but inhibits CT. Proteins with mutations making them deficient in DNA-mediated CT do not similarly redistribute onto mismatched strands.
\r\n\r\nVarious DNA-binding proteins, such as those involved in repair and pathways that maintain the integrity of DNA, have been found to contain FeS domains and other redox cofactors. We are discovering proteins from alternate repair pathways that may also utilize DNA CT to find damage. XPD, a 5\u2032-3\u2032 helicase involved in nucleotide excision repair, contains a conserved [4Fe4S] cluster and exhibits a DNA-bound redox potential that indicates it is able to carry out DNA CT. In AFM studies, we observe also the redistribution of XPD onto strands containing a mismatch. We further demonstrate that an XPD mutant, L325V, defective in carrying out DNA CT, does not redistribute onto mismatched strands.
\r\n\r\nDNA CT between distinct repair proteins bound to DNA was also probed by AFM. When XPD and EndoIII are mixed together, they coordinate in relocalizing onto mismatched strands. However, when a CT-deficient mutant of either repair protein is combined with the CT-proficient repair partner, no relocalization occurs. These data not only indicate a general link between the ability of a repair protein to carry out DNA CT and its ability to redistribute onto DNA strands near lesions but also provide evidence for coordinated DNA CT between repair proteins in their search for damage in the genome.
\r\n", "doi": "10.7907/9YT4-7181", "publication_date": "2012-05-07", "thesis_type": "phd", "thesis_year": "2012" }, { "id": "thesis:6708", "collection": "thesis", "collection_id": "6708", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:10062011-140224145", "primary_object_url": { "basename": "Thesis_final_wmg.pdf", "content": "final", "filesize": 4162574, "license": "other", "mime_type": "application/pdf", "url": "/6708/1/Thesis_final_wmg.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Regulation of Wild-Type and Mutant p53 through DNA-mediated Charge Transport", "author": [ { "family_name": "Geil", "given_name": "Wendy Mercer", "clpid": "Geil-Wendy-Mercer" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Beauchamp", "given_name": "Jesse L.", "clpid": "Beauchamp-J-L" }, { "family_name": "Shan", "given_name": "Shu-ou", "clpid": "Shan-Shu-ou" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The global transcription factor p53 controls many cellular processes, including the cellular response to oxidative stress. It had been determined that that dissociation of wild type p53 from its promoter site can occur upon DNA-mediated oxidation. In this work, we use site-directed mutagenesis to construct charge-deficient mutants of p53; the chemistry of DNA-mediated oxidation of p53 was examined using these mutants.
\r\n\r\nThe control point for p53 oxidation through DNA-mediated charge transport (DNA CT) is cysteine 275. Using differential thiol labeling and detection of modified peptides with mass spectrometry, we demonstrated that cysteines 124 and 141 in superstable p53 form a terminal disulfide bond upon DNA-mediated oxidation. This leads to a conformational change that inhibits DNA from binding by p53. The disulfide formed between cysteines 124 and 141 is a result of a series of disulfide bond exchange across the protein from the DNA base stack.
\r\n\r\nWe also investigated the dependence of p53 oxidation on DNA sequences. ESMA analysis of biologically derived p53 recognition sequences with varying quantities of guanine doublets and triplets showed efficient p53 oxidation to depend on the presence of low energy GG or GGG sites. Moreover, consistent results were found with biologically derived promoter sequences. Sequence S100A2, with guanine triplets on the same strand, showed the most oxidation of p53, followed by ODC1 and caspase-1. We confirmed these sequence-specific effects by measuring the change in expression level of the genes after induction of DNA CT in vivo. S100A2 mRNA levels decreased after photooxidant and light treatment, reflecting the oxidation and dissociation of p53 from the S100A2 site. However, caspase-1 and ODC1 mRNA levels remained the same, indicating less DNA-mediated p53 oxidation.
\r\n\r\nThe results from this study illustrate how protein oxidation at a distance through DNA CT contributes to cellular signaling. This oxidative signaling can control how p53 regulates gene expression under oxidative stress, and this signaling may be disrupted in cancerous cells.
\r\n", "doi": "10.7907/0NVZ-QC23", "publication_date": "2012", "thesis_type": "phd", "thesis_year": "2012" }, { "id": "thesis:6721", "collection": "thesis", "collection_id": "6721", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:10282011-143721555", "primary_object_url": { "basename": "Olmon2012thesis.pdf", "content": "final", "filesize": 6459459, "license": "other", "mime_type": "application/pdf", "url": "/6721/1/Olmon2012thesis.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "Investigating DNA-Mediated Charge Transport by Time-Resolved Spectroscopy", "author": [ { "family_name": "Olmon", "given_name": "Eric Daniel", "clpid": "Olmon-Eric-Daniel" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Miller", "given_name": "Thomas F.", "clpid": "Miller-T-F" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "In all organisms, oxidation threatens the integrity of the genome. Numerous studies have suggested that DNA-mediated charge transport (CT) may play an important role in the sequestration, detection, and repair of oxidative damage. To fully understand the mechanism of DNA-mediated CT, it is necessary to characterize transient intermediates that arise during the reaction and to determine the lifetimes of these intermediates. Time-resolved spectroscopy is the most appropriate experimental method for such observations. Each intermediate has a characteristic spectrum. By observing time-dependent changes in the absorption spectrum of the sample, it is therefore possible to determine what species are present at a particular time and how long it exists in solution. Experiments presented here involve the use of time-resolved spectroscopy to better understand the process of DNA-mediated CT.
\r\n\r\nThe study of DNA-mediated CT requires a robust and consistent method for triggering the CT reaction. The metal complexes that have traditionally been used for this purpose provide several advantages over organic phototriggers: they are synthetically versatile, they are stable in solution, they exhibit rich photophysics, and many are strong photooxidants. However, the spectroscopic features used to follow the photochemical processes triggered by these probes are generally broad optical bands. These can be difficult to resolve in samples that contain several absorbing species. For this reason, we have developed a Re photooxidant bearing a set of vibrationally active carbonyl ligands that can be covalently tethered to DNA. Unlike many absorption bands in the visible range, the vibrational absorption bands of these ligands are narrow, well-resolved, and specific. Such probes can be used to follow the complex photophysical pathways observed in biochemical systems with good precision, making them useful for the study of DNA-mediated CT.
\r\n\r\nSpecifically, the complex [Re(CO)3(dppz)(py′-OR)]+ (dppz = dipyrido[3,2-a:2′,3′-c]-phenazine; py′-OR = 4-functionalized pyridine) offers IR sensitivity and can oxidize DNA directly from the excited state. The behavior of several covalent and noncovalent Re-DNA constructs was monitored by time-resolved IR (TRIR) and UV/visible spectroscopies, as well as biochemical methods, confirming the ability of the complex to trigger long-range oxidation of DNA. Optical excitation of the complex leads to population of metal-to-ligand charge transfer excited states and at least two distinct intraligand charge transfer excited states. Several experimental observations are consistent with charge injection by excited Re*. These include similarity between TRIR spectra and the spectrum of reduced Re observed by spectroelectrochemistry, the appearance of a guanine radical signal in TRIR spectra, and the eventual formation of permanent guanine oxidation products. The majority of reactivity occurs on the ultrafast time scale, although processes dependent on slower conformational motions of DNA, such as the accumulation of oxidative damage at guanine, are also observed.
\r\n\r\nThe photooxidation activity of this Re complex was compared directly to that of other metallointercalators that have been used previously in our laboratory to oxidize DNA. The complexes [Rh(phi)2(bpy′)]3+ (phi = 9,10-phenanthrenequinone diimine; bpy′ = 4-methyl-4′-(butyric acid)-2,2′-bipyridine), [Ir(ppy)2(dppz′)]+ (ppy = 2-phenylpyridine; dppz′ = 6-(dipyrido[3,2-a:2′,3′-c]phenazin-11-yl)hex-5-ynoic acid), and [Re(CO)3(dppz)(py′-OH)]+ (py′-OH = 3-(pyridin-4-yl)-propanoic acid) were each covalently tethered to DNA. Biochemical studies show that upon irradiation, the three complexes oxidize guanine by long-range DNA-mediated CT with the efficiency: Rh > Re > Ir. Comparison of spectra obtained by spectroelectrochemistry after bulk reduction of the free metal complexes with those obtained by transient absorption (TA) spectroscopy of the conjugates suggests that excitation of the conjugates at 355 nm results in the formation of the reduced metal states. Electrochemical experiments and kinetic analysis of the TA decays verify that the primary factors responsible for the trend observed in the guanine oxidation yield of the three complexes are the thermodynamic driving force for CT, variations in the efficiency of back electron transfer, and coupling to DNA.
\r\n\r\nThe ability of redox-active DNA-binding proteins to act as hole sinks in DNA-mediated CT systems was also studied by time-resolved spectroscopy. Such experiments are designed to provide support for the utilization of DNA-mediated CT in biological systems. In studies involving the cell cycle regulator p53, photoexcitation results in the formation of a weak transient band at 405 nm. This band, which is not observed in samples lacking the protein, resembles the primary spectral feature of the tyrosine cation radical. Although the signal is weak and reproducibility is inconsistent, these results suggest that photolysis of the sample leads to DNA-mediated oxidation of tyrosine in p53. Similar experiments were conducted on the transcriptional activator SoxR. Here, the presence of dithionite, required in solution to keep the protein reduced, complicates the photochemistry of the system considerably. Regardless, a weak absorbance at 418 nm that develops following photolysis at 355 nm provides evidence for the DNA-mediated oxidation of the protein. The behavior of the base excision repair protein endonuclease III was also observed in the presence of DNA and metal complex oxidants. In flash-quench studies, addition of the protein results in the formation of a strong negative signal at 410 nm in TA traces. In studies involving direct photooxidation by Rh, Ir, and Re complexes, no new transients are detected upon the addition of protein, but changes in the intensities of the resultant TA spectra and in the steady-state absorbance spectra following photolysis indicate that DNA-mediated oxidation of the protein may be taking place.
\r\n\r\nThe experiments described here comprise several new developments in the story of DNA-mediated CT. First, proof of concept has been given for a valuable new vibrationally-active Re probe. Further modifications on the characteristics of this complex and further study by time-resolved vibrational spectroscopy will allow us to observe DNA-mediated CT with high spectral resolution. Second, comparison between this Re probe and established photooxidants shows that the Re complex is a strong photooxidant in its own right and that this complex can be added to our growing toolbox of CT phototriggers. Third, time-resolved studies involving redox-active proteins have provided preliminary direct evidence for the ability of these proteins to serve as CT probes themselves. Further refinement of the experimental methods used in these experiments will allow us to observe such processes with greater sensitivity, increasing our knowledge of the mechanism and applications of DNA-mediated CT.
\r\n", "doi": "10.7907/6GDE-2707", "publication_date": "2012", "thesis_type": "phd", "thesis_year": "2012" }, { "id": "thesis:6728", "collection": "thesis", "collection_id": "6728", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:11042011-173102026", "primary_object_url": { "basename": "APBlum-Thesis.pdf", "content": "final", "filesize": 9303119, "license": "other", "mime_type": "application/pdf", "url": "/6728/13/APBlum-Thesis.pdf", "version": "v7.0.0" }, "type": "thesis", "title": "Structure-Function Studies of Nicotinic Acetylcholine Receptors Using Unnatural Amino Acids and Synthetic Agonist Analogs ", "author": [ { "family_name": "Blum", "given_name": "Angela Patricia", "clpid": "Blum-Angela-Patricia" } ], "thesis_advisor": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" } ], "thesis_committee": [ { "family_name": "Grubbs", "given_name": "Robert H.", "clpid": "Grubbs-R-H" }, { "family_name": "Dervan", "given_name": "Peter B.", "clpid": "Dervan-P-B" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "This dissertation primarily describes structure-function studies of the prototypical Cys-loop ligand-gated ion channel, the nicotinic acetylcholine receptors (nAChRs).
\r\n\r\nAgonists that bind nAChRs, including acetylcholine, nicotine, and the smoking cessation drug varenicline, share one of the longest-known, best-studied pharmacophores, consisting of a cationic N and a hydrogen bond acceptor. A major theme of this thesis is concerned with defining the nAChR residues that bind the nicotinic pharmacophore. Chapters 2 and 3 establish that a hydrogen bond links the pharmacophore\u2019s hydrogen bond acceptor to a backbone NH in the protein. The establishment of this interaction, and the disproval of other predicted interactions, represents the completion of the nicotinic pharmacophore binding model. Chapter 4 uses this model to characterize how the nAChR differentiates between stereoisomers of an agonist.
\r\n\r\nChapter 5 describes functional studies of a vicinal disulfide that has played a pivotal role in a number of pioneering studies of nAChRs. Despite its historical importance, the functional role of this disulfide has not been defined. We identify a speculative role for the vicinal disulfide that involves the formation of a functionally important network of hydrogen bonds.
\r\n\r\nChapter 6 outlines three strategies for the photochemical cleavage of protein and peptide backbones using unnatural amino acids. One of these strategies is based on a selenide-mediated cleavage of a backbone ester moiety. Model studies establish the viability of this chemistry and suggest that it could be a useful tool for protein structure-function studies.
\r\n\r\nChapter 7 concerns preliminary work from a collaboration with laboratories from USC and Caltech that is aimed at developing small-molecule treatments for vision loss associated with photoreceptor degeneration. The initial goal of this project is to develop a photosensitive small molecule that can activate a voltage-gated potassium channel.
\r\n\r\nThe final chapter discusses work that was done in the Grubbs lab at Caltech in which a strategy for preparing N-heterocyclic carbene-containing metal complexes was developed.
\r\n \t\r\n\r\n", "doi": "10.7907/KFPV-JN09", "publication_date": "2012", "thesis_type": "phd", "thesis_year": "2012" }, { "id": "thesis:6826", "collection": "thesis", "collection_id": "6826", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:02172012-141718925", "primary_object_url": { "basename": "NPuskar_Title.pdf", "content": "", "filesize": 131293, "license": "other", "mime_type": "application/pdf", "url": "/6826/1/NPuskar_Title.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Structure-Function Studies of Nicotinic Acetylcholine Receptors Using Unnatural Amino Acids", "author": [ { "family_name": "Puskar", "given_name": "Nyssa Leigh", "clpid": "Puskar-Nyssa-Leigh" } ], "thesis_advisor": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" } ], "thesis_committee": [ { "family_name": "Dervan", "given_name": "Peter B.", "clpid": "Dervan-P-B" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Lester", "given_name": "Henry A.", "clpid": "Lester-H-A" }, { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "This dissertation primarily describes structure-function studies of the nicotinic acetylcholine receptors (nAChRs). These studies use a combination of unnatural amino acid mutagenesis and electrophysiology to determine the specific molecular interactions required for neurotransmitter binding to nAChRs. Chapter 2 examines the mode of agonist activation for the \u03b14\u03b22 nAChR, the receptor responsible for nicotine addiction. This study investigates the molecular interactions that differentiate the \u03b14\u03b22 receptor from other receptor subtypes and endow it with the ability to mediate nicotine addiction. We report that the high affinity for nicotine at the \u03b14\u03b22 receptor is a result of a strong cation-\u03c0 interaction and a strengthened backbone hydrogen bond to a conserved tryptophan (TrpB) of this receptor. We also establish that a point mutation just four residues away from TrpB appears to influence the shape of the agonist binding site, such that it can differentiate the agonist binding mode of the \u03b14\u03b22 and muscle-type receptors. Chapter 3 extends studies of the point mutation near TrpB, termed the \u201cloop B glycine.\u201d We examine the muscle-type, \u03b14\u03b22, and \u03b17 subtypes and show that the identity of this residue strongly correlates with agonist potency. Low-potency receptor subtypes have a glycine at the loop B site, while high-potency receptors have a lysine at this site. We establish that mutation of this residue can to convert a low-potency receptor to a high-potency receptor and vice versa. Chapter 4 investigates the agonist binding mechanism of the \u03b14\u03b24 receptor. We show both ACh and nicotine make a strong cation-\u03c0 interaction to TrpB, and nicotine makes a strong hydrogen bond to the backbone carbonyl of TrpB. Additionally, chimeric \u03b2 subunits are used to examine the influence of the complementary binding component on receptor pharmacology for the \u03b14\u03b22 and \u03b14\u03b24 receptors. Last, chapter 5 is a methodology-based project focused on optimizing the incorporation of unnatural amino acids into mammalian cells. Using HEK293T cells, we successfully suppressed an amber stop codon using HSAS, an in vivo aminoacylated tRNA. Additional studies will pursue the viability of in vitro aminoacylated tRNAs for nonsense suppression in mammalian cells.", "doi": "10.7907/YK2P-K088", "publication_date": "2012", "thesis_type": "phd", "thesis_year": "2012" }, { "id": "thesis:6257", "collection": "thesis", "collection_id": "6257", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:02272011-102614437", "primary_object_url": { "basename": "VJS-Thesis.pdf", "content": "final", "filesize": 4696579, "license": "other", "mime_type": "application/pdf", "url": "/6257/1/VJS-Thesis.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Investigations of C-H Activation and the Conversion of Methanol to Triptane", "author": [ { "family_name": "Scott", "given_name": "Valerie J.", "clpid": "Scott-Valerie-J" } ], "thesis_advisor": [ { "family_name": "Bercaw", "given_name": "John E.", "clpid": "Bercaw-J-E" }, { "family_name": "Labinger", "given_name": "Jay A.", "clpid": "Labinger-J-A" } ], "thesis_committee": [ { "family_name": "Stoltz", "given_name": "Brian M.", "clpid": "Stoltz-B-M" }, { "family_name": "Labinger", "given_name": "Jay A.", "clpid": "Labinger-J-A" }, { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Bercaw", "given_name": "John E.", "clpid": "Bercaw-J-E" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Broadly speaking, this thesis represents research towards understanding the mechanisms and important species related to small molecule conversion, namely methane to methanol and methanol to higher hydrocarbons. The first section is on understanding the catalytic formation of methanol from methane, with specific interest in using gold (Au). While this transformation is known to occur catalytically, very little is understood about how it happens. To study this reaction, well-defined Au-complexes were synthesized and reactions relevant to the possible catalytic cycles were examined. In doing so, the first simple Au(III)-monoalkyl complex was generated and characterized: (Idipp)AuI2Me, where Idipp = 1,3-bis(2,6-diisopropylphenyl)imidazol-2-ylidene). Kinetics experiments demonstrated that the complex reductively eliminates methyl iodide, which is relevant to the functionalization step in CH activation. At low concentrations of iodide, the reductive elimination happens faster, from an unobserved 3-coordinate intermediate. However, at high iodide concentrations, the pathway is still consistent with reductive elimination, but from a 5-coordinate intermediate. This is in contrast to the related platinum-system, as well as to density functional theory calculations done on the Au-system.
\r\n \r\nThe second section studies the C-H activation step alone by close examination of the microscopic reverse: protonation of a metal-alkyl. It had previously been noted that the observed kinetic isotope effects (KIEs) were unusually high for the protonolysis of a few Pd complexes and one Pt complex. It was hypothesized that these high KIEs and involvement of quantum mechanical tunneling may indicate a change in the mechanism of the protonolysis reaction, from protonation at the metal center and reductive coupling to direct protonation of the M-Me bond. The experiments described here were designed to explicitly test this theory and demonstrated that no correlation can be made between mechanism and tunneling.
\r\n\r\nThe third section is focused on the study of the conversion of methanol to highly branched alkanes that make good fuel additives, namely 2,2,3-trimethylbutane (triptane), amidst other alkanes, olefins, and aromatics. Catalyzed by ZnI2 or InI3 at high temperatures, the reaction is hydrogen deficient: aromatics are formed as unsaturated by-products necessary for alkane generation. While the product distributions are somewhat different for the two different catalysts, the general mechanism is the same. While typical InI3 reactions generate more alkanes, more aromatics, and fewer olefins than ZnI2 reactions, longer reaction times and higher temperatures make the ZnI2 reaction look like the InI3 profile. Furthermore, InI3 can activate alkanes; it was found that InI3 can \u201cupgrade\u201d other alkanes with methanol. Notably, a 1:1 mixture of 2,3-dimethylbutane and methanol can be converted into triptane with good selectivity and little aromatic formation; ZnI2 can carry out similar chemistry at higher temperatures. Quantification of the iodine-containing products in each reaction mixture was attempted because of its relevance to the system\u2019s industrial viability and found that these concentrations were significantly higher than would be acceptable in an industrial setting.
\r\n", "doi": "10.7907/P0A0-JV28", "publication_date": "2011-02-14", "thesis_type": "phd", "thesis_year": "2011" }, { "id": "thesis:6506", "collection": "thesis", "collection_id": "6506", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06072011-185553467", "primary_object_url": { "basename": "Ahmad_FinalThesis.pdf", "content": "final", "filesize": 9905679, "license": "other", "mime_type": "application/pdf", "url": "/6506/1/Ahmad_FinalThesis.pdf", "version": "v8.0.0" }, "type": "thesis", "title": "Microfluidics Platforms for Quantitative, Multiplexed Protein Detection", "author": [ { "family_name": "Ahmad", "given_name": "Habibullah", "clpid": "Ahmad-Habibullah" } ], "thesis_advisor": [ { "family_name": "Heath", "given_name": "James R.", "clpid": "Heath-J-R" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Scherer", "given_name": "Axel", "clpid": "Scherer-A" }, { "family_name": "Heath", "given_name": "James R.", "clpid": "Heath-J-R" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "This thesis describes the development of microfluidic platforms that enable cheap, facile, rapid, and multi-parameter protein sensing. The first section of this work describes two strategies for high density DNA microarray patterning: microcontact printing and flow patterning. A protocol is provided for micron-scale alignment of multiple PDMS stamps to a single substrate, and a simple strategy to allow very low aspect-ratio stamping is enumerated.
\r\n\r\nThe second section describes the formation of high density antibody microarrays using flow patterned DNA microarrays in conjunction with DEAL chemistry, and applies these microarrays to biological measurements. The platform\u2019s performance is first characterized using a human chorionic gonadotropin assay, and is subsequently used to stratify 22 cancer patients from frozen serum samples by quantifying the levels of twelve serum proteins. A microfluidic plasma separation device is then detailed to allow for similar measurements from fresh finger pricks of blood.
\r\n\r\nThe third section of this work outlines improvements to the flow patterning platform through two alternate schemes: covalent attachment and DMSO patterning. Both protocols are shown to dramatically increase the consistency of microarray elements across a single chip when compared to the initial method. Theoretical simulations are used to describe the mechanism by which DMSO enhances patterning consistency.
\r\n\r\nThe fourth section describes the design and fabrication of a robotics system that is capable of autonomously interfacing and manipulating PDMS substrates, and its application to producing barcode microarrays. The resulting substrates show unprecedented consistency from chip to chip, and we demonstrate through massively parallel single-cell measurements that data derived from different substrates is statistically indistinguishable.
\r\n\r\nFinally, we introduce an integrated software and hardware package designed to facilitate and automate microfluidic control at the laboratory level. We further provide the technical details of a related system which optimizes and comprehensively automates microfluidic blood assays such that even non-technical users who have never worked with microfluidics can regularly obtain the same standard of data that is produced in the lab.
\r\n", "doi": "10.7907/04A6-YT31", "publication_date": "2011", "thesis_type": "phd", "thesis_year": "2011" }, { "id": "thesis:6464", "collection": "thesis", "collection_id": "6464", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05272011-113632785", "primary_object_url": { "basename": "Thesis.pdf", "content": "final", "filesize": 10925182, "license": "other", "mime_type": "application/pdf", "url": "/6464/1/Thesis.pdf", "version": "v7.0.0" }, "type": "thesis", "title": "The Biological Activity of Rhodium Metalloinsertors", "author": [ { "family_name": "Ernst", "given_name": "Russell J.", "clpid": "Ernst-Russell-J" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" }, { "family_name": "Phillips", "given_name": "Robert B.", "orcid": "0000-0003-3082-2809", "clpid": "Phillips-R" }, { "family_name": "Deshaies", "given_name": "Raymond Joseph", "orcid": "0000-0002-3671-9354", "clpid": "Deshaies-R-J" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Mismatches in DNA occur naturally during replication and as a result of endogenous DNA damaging agents, but the mismatch repair (MMR) pathway acts to correct mismatches before subsequent rounds of replication. The loss of MMR carries dire consequences, including increased mutation rates, carcinogenesis, and resistance to a variety of clinical anti-cancer agents, such as cisplatin and DNA alkylators. Rhodium metalloinsertors previously developed in our laboratory bind to DNA mismatches with high affinity and specificity, and represent a promising strategy to target mismatches in cells. Thus, uncorrected mismatches can be exploited to provide a basis of discrimination between MMR-deficient, cancerous cells and MMR-proficient, healthy cells.
\r\n\r\nHere we describe the application of rhodium metalloinsertors to inhibit cellular proliferation selectively in MMR-deficient cells compared to those that are MMR-proficient. The colorectal carcinoma cell lines HCT116N and HCT116O serve as an isogenic model system for MMR deficiency. We show that the \u0394-isomer of an octahedral rhodium complex containing a bulky chelate ligand for insertion into a DNA mismatch is active both in targeting base mismatches in vitro and in inhibiting DNA synthesis selectively in the HCT116O cell line.
\r\n\r\nA family of derivative complexes with varying ancillary ligands has also been synthesized, and both DNA mismatch binding affinities and anti-proliferative activities against the HCT116 cell lines have been determined. DNA photocleavage experiments reveal that all complexes bind to the mismatched sites with high specificities; DNA binding affinities to oligonucleotides containing single base CA and CC mismatches, obtained through photocleavage titration or competition, vary from 104 to 108 M-1 for the series of complexes. Significantly, binding affinities are found to be inversely related to ancillary ligand size and directly related to differential inhibition of the HCT116 cell lines. The observed trend in binding affinity is consistent with the metalloinsertion mode where the complex binds from the minor groove with ejection of mismatched base pairs. The correlation between binding affinity and targeting of the MMR-deficient cell line suggests that rhodium metalloinsertors exert their selective biological effects on MMR-deficient cells through mismatch binding in vivo.
\r\n\r\nIn particular, rhodium metalloinsertors bearing dipyridylamine ancillary ligands are shown to exhibit accelerated cellular uptake. This increased uptake allows us to observe additional cellular responses to these agents, including disruption of the cell cycle, monitored by flow cytometry assays, and induction of necrosis, monitored by dye exclusion and caspase inhibition assays, that also occur preferentially in the HCT116O cell line. Finally, these cellular responses provide insight into the mechanisms underlying the selective activity of this novel class of targeted anti-cancer agents, and are consistent with the idea that repair proteins are activated in response to DNA mismatch binding.
\r\n", "doi": "10.7907/3K6K-JX55", "publication_date": "2011", "thesis_type": "phd", "thesis_year": "2011" }, { "id": "thesis:6375", "collection": "thesis", "collection_id": "6375", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05042011-174816761", "primary_object_url": { "basename": "CAWthesis.pdf", "content": "final", "filesize": 24168443, "license": "other", "mime_type": "application/pdf", "url": "/6375/9/CAWthesis.pdf", "version": "v10.0.0" }, "type": "thesis", "title": "Tuning Nitric Oxide Synthase: Investigating the Thiolate \"Push\" and No Release", "author": [ { "family_name": "Whited", "given_name": "Charlotte A.", "clpid": "Whited-Charlotte-A" } ], "thesis_advisor": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" } ], "thesis_committee": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Agapie", "given_name": "Theodor", "clpid": "Agapie-T" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "All heme thiolate enzymes have conserved hydrogen bonding networks surrounding the axial thiolate ligand. In order to understand the role of this proximal hydrogen bonding network in nitric oxide synthases (NOS), three mutants of the NOS enzyme from Geobacillus stearothermophilus were expressed and characterized. The wild type enzyme has a tryptophan residue at position 70 that \u03c0-stacks with the porphyrin ring and donates a long hydrogen-bonding interaction to the thiolate ligand of the heme iron. The native Trp was replaced with His, Phe, and Tyr. These three residues were selected to investigate the two effects of the Trp, H-bonding and Pi-stacking. Several different spectroscopic techniques were used to investigate the stability and properties of these mutant enzymes. The identity of each mutant was confirmed by mass spectrometry. Both UV-visible absorption and circular dichroism spectroscopies were used to assess the stability of the new proteins. It was shown using binding assays, generation of the ferrous-CO species, and redox titrations that the \u03c3-donating abilities of the thiolate are increased after removal of the hydrogen bonding group in the Trp. Finally, electron paramagnetic resonance spectroscopy and Evans method nuclear magnetic resonance spectroscopy were used to characterize the spin state of the iron center in each mutant, reflecting the increased \u03c3-donating capabilities of the thiolate upon removal of the hydrogen bonding group. The reduction potential of wild type and W70H were determined by chemical titration to be -362 and -339 mV vs. NHE, respectively. This is the first report of the reduction potential of any bacterial nitric oxide synthase.
\r\n\r\nThe reactivity of each the wild type enzyme and the three new mutants was tested using stopped-flow mixing coupled with UV-visible absorption spectroscopy and the Griess Assay. Autoxidation rates measured by stopped-flow suggest that the Tyr and Phe mutants do indeed have significantly more negative reduction potentials, but that the His mutant is particularly slow to oxidize. The Griess Assays showed that all four enzymes produce nitrite in solution, when provided with substrate, cofactor and hydrogen peroxide (as a source of reducing equivalents). In single turnover experiments, however, only three of the four enzymes showed evidence of ferric-NO production. The His mutant showed no intermediate absorbance near 440 nm (which would be indicative of ferric-NO formation), suggesting that it releases NO- rather than the radical species NO\u2219. The role of this hydrogen bond is concluded to be an electronic one, rather than playing any part in positioning the heme. It prevents formation of the inactive P420 species, and tunes the reduction potential to one high enough to be reduced by a reductase but low enough to still deliver an electron to the redox active cofactor, tetrahydrobiopterin, at the end of catalysis.
\r\n\r\nThe rate at which NO is released by each NOS enzyme varies greatly among isoforms and species, over nearly two orders of magnitude. One residue (an isoleucine located above the heme in bacterial enzymes) involved in the gating of NO release has been previously identified by Stuehr. However, this single residue does not account for the entirety of the differences among the forms of NOS. Another residue, a histidine at position 134 in NOS from Geobacillus stearothermophilus (gsNOS), was hypothesized to also participate in gating NO release based on an observed correlation between rates of NO release and the bulk of side chains at this position. Each single point mutation, H134S and I223V, and the double mutant were expressed in gsNOS and their reactivity toward the diatomic molecules CO and NO were studied. CO rebinding was investigated using laser flash photolysis and NO release using stopped flow UV-visible spectroscopy. The presence of both monomer and dimer was observed in solution, and position 134 was shown to be another key residue in gating NO release. Wild type gsNOS contains both the bulkier Ile223 and His134 and has the slowest measured NO release (0.039 s-1) of all NOS enzymes. A new, more accurate kinetics model for turnover is proposed. Each single mutation increased NO release substantially, while the double mutant has a rate constant of 1.0 s-1, nearly as fast as mammalian iNOS at 2.3 s-1, identifying position 134 as another important factor determining rate constants for NO release.
\r\n", "doi": "10.7907/PZAY-WQ64", "publication_date": "2011", "thesis_type": "phd", "thesis_year": "2011" }, { "id": "thesis:6359", "collection": "thesis", "collection_id": "6359", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:04272011-115528661", "primary_object_url": { "basename": "Ophir_Vermesh_-_PhD_Thesis_(Final).pdf", "content": "final", "filesize": 24522869, "license": "other", "mime_type": "application/pdf", "url": "/6359/1/Ophir_Vermesh_-_PhD_Thesis_(Final).pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Highly Informative Analytical Platforms for Rapid, Non-Invasive Diagnosis and Stratification of Patients with Cancer", "author": [ { "family_name": "Vermesh", "given_name": "Ophir", "clpid": "Vermesh-Ophir" } ], "thesis_advisor": [ { "family_name": "Heath", "given_name": "James R.", "clpid": "Heath-J-R" } ], "thesis_committee": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Heath", "given_name": "James R.", "clpid": "Heath-J-R" }, { "family_name": "Beauchamp", "given_name": "Jesse L.", "clpid": "Beauchamp-J-L" }, { "family_name": "Lewis", "given_name": "Nathan Saul", "clpid": "Lewis-N-S" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "As the tissue that contains the largest representation of the human proteome, blood is the most important fluid for clinical diagnostics. However, although changes of plasma protein profiles reflect physiological or pathological conditions associated with many human diseases, only a handful of plasma proteins are routinely used in clinical tests. Reasons for this include the intrinsic complexity of the plasma proteome, the heterogeneity of human diseases and the rapid degradation of proteins in sampled blood. The first part of this thesis reports an integrated microfluidic system, the integrated blood barcode chip (IBBC) that can sensitively sample a large panel of protein biomarkers over broad concentration ranges and within 10 minutes of sample collection. It enables on-chip blood separation and rapid measurement of a panel of plasma proteins from quantities of whole blood as small as those obtained by a finger prick. The device holds potential for inexpensive, noninvasive and informative clinical diagnoses, particularly in point-of-care settings.
\r\n\r\nProteomic approaches, on which the IBBC platform is based, have shown great promise in recent years for correctly classifying and diagnosing cancer patients. However, no large antibody-based microarray studies have yet been conducted to evaluate and validate plasma molecular signatures for detection of glioblastoma and monitoring of its response to therapy. In the second part of this thesis, plasma samples from 46 glioblastoma patients (72 total samples) are compared with those of 47 healthy controls with respect to the plasma levels of 35 different proteins known to be generally associated with tumor growth, survival, invasion, migration, and immune regulation. Average-linkage hierarchical clustering of the patient data stratified the two groups effectively, permitting accurate assignment of test samples into either GBM or healthy control groups with a sensitivity and specificity as high as 90% and 94%, respectively (when test samples within unbiased clusters were removed). The accuracy of these assignments improved (sensitivity and specificity as high as 94% and 96%, respectively) when the cluster analysis was repeated on increasingly trimmed sets of proteins that exhibited the most statistically significant (p < 0.05) differential expression. The diagnostic accuracy was also higher for test samples that fell into more homogeneous clusters. Intriguingly, test samples that fell within perfectly homogeneous clusters (all members belonging to the same group) could be diagnosed with 100% accuracy. Using the same 35-protein panel, we then analyzed plasma samples from GBM patients who were treated with the chemotherapeutic drug Avastin (Bevacizumab) in an effort to stratify patients based on treatment-responsiveness. Specifically, we compared 52 samples from (25) patients who exhibited tumor recurrence with 51 samples from (21) patients who did not exhibit recurrence. Again, several proteins were highly differentially expressed and cluster analysis provided effective stratification of patients between these two groups (sensitivity and specificity of 90% and 96%, respectively).
\r\n", "doi": "10.7907/40BR-TW68", "publication_date": "2011-06-10", "thesis_type": "phd", "thesis_year": "2011" }, { "id": "thesis:6414", "collection": "thesis", "collection_id": "6414", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05202011-180941490", "primary_object_url": { "basename": "main.pdf", "content": "final", "filesize": 32486201, "license": "other", "mime_type": "application/pdf", "url": "/6414/1/main.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "Light Trapping in Plasmonic Solar Cells", "author": [ { "family_name": "Ferry", "given_name": "Vivian Eleanor", "clpid": "Ferry-Vivian-Eleanor" } ], "thesis_advisor": [ { "family_name": "Atwater", "given_name": "Harry Albert", "clpid": "Atwater-H-A" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Lewis", "given_name": "Nathan Saul", "clpid": "Lewis-N-S" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Atwater", "given_name": "Harry Albert", "clpid": "Atwater-H-A" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Subwavelength nanostructures enable the manipulation and molding of light in nanoscale dimensions. By controlling and designing the complex dielectric function and nanoscale geometry we can affect the coupling of light into specific active materials and tune macroscale properties such as reflection, transmission, and absorption. Most solar cell systems face a trade-off with decreasing semiconductor thickness: reducing the semiconductor volume increases open circuit voltages, but also decreases the absorp- tion and thus the photocurrent. Light trapping is particularly critical for thin-film amorphous Si (a-Si:H) solar cells, which must be made less than optically thick to enable complete carrier collection. By enhancing absorption in a given semiconductor volume, we can achieve high efficiency devices with less than 100 nm of active region.
\r\n\r\nIn this thesis we explore the use of designed plasmonic nanostructures to couple incident sunlight into localized resonant modes and propagating waveguide modes of an ultrathin semiconductor for enhanced solar-to-electricity conversion. We begin by developing computational tools to analyze incoupling from sunlight to guided modes across the solar spectrum and a range of incident angles. We then show the potential of this method to result in absorption enhancements beyond the limits for thick film solar cells. The second part of this thesis describes the integration of plasmonic nanos- tructures with a-Si:H solar cells, showing that designed nanostructures can lead to enhanced photocurrent over randomly textured light trapping surfaces, and develops a computational model to accurately simulate the absorption in these structures. The final chapter discusses the fabrication of a high-efficiency (9.5%) solar cell with a less than 100 nm absorber layer and broadband, angle isotropic photocurrent enhance- ment. Moreover, we discuss general design rules where light trapping nanopatterns are defined by their spatial coherence spectral density.
", "doi": "10.7907/AMD4-Q845", "publication_date": "2011", "thesis_type": "phd", "thesis_year": "2011" }, { "id": "thesis:6277", "collection": "thesis", "collection_id": "6277", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:04032011-125158842", "primary_object_url": { "basename": "Complete_Thesis.pdf", "content": "final", "filesize": 6687006, "license": "other", "mime_type": "", "url": "/6277/12/Complete_Thesis.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "DNA-Mediated Charge Transfer Between [4Fe-4S] Cluster Glycosylases", "author": [ { "family_name": "Romano", "given_name": "Christine Anne", "clpid": "Romano-Christine-Anne" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The work performed herein describes three proteins: Uracil DNA glycosylase (UDG) from Archaeoglobus fulgidus, MutY, and Endonuclease III (EndoIII) from Escherichia coli. They are DNA repair glycosylases that contain [4Fe-4S] clusters. While the catalytic mechanisms of many BER enzymes have been studied in detail, questions remain about how these enzymes search the vast amount of cellular DNA to find their substrates, and why some require a [4Fe-4S] cluster. The iron-sulfur cluster is not necessary for catalysis, and it only displays a physiologically relevant midpoint potential when bound to DNA. We have proposed that UDG, MutY, and EndoIII use their [4Fe-4S] clusters to participate in DNA-mediated charge transport (CT), and that these proteins mediate long-range electrochemical signaling in order to detect DNA damage.
\r\n\r\nThis scheme for DNA damage detection assumes that CT occurs efficiently between the DNA helix and the [4Fe-4S] cluster of the bound protein. In order for efficient CT to occur, a pathway of amino acids must be present that facilitates CT between the DNA and the iron-sulfur cluster. For each of the enzymes mentioned, this pathway was explored through mutagenesis. In UDG, MutY, and EndoIII, several amino acids thought to be important for CT were mutated and the resulting proteins were characterized biochemically. Their CT capabilities were analyzed by cyclic voltammetry on DNA-modified electrodes. In these experiments, the mutants\u2019 signal intensities were quantified and compared to those of wild-type enzyme. An attenuated signal relative to wild-type protein may indicate that the mutant is deficient in CT and that the targeted amino acid is part of the protein-DNA CT pathway in the native enzyme. Many mutants were also screened by enzymatic assays and circular dichroism spectroscopy to further characterize their DNA-binding properties and structural stability.
\r\n\r\nThe A. fulgidus UDG mutants examined, C17H, C85S, and C101S, all contained mutations in the cysteine residues that ligate the [4Fe-4S] cluster. These mutants were designed to determine how the iron-sulfur cluster coordination environment affects protein-DNA CT. The mutants exhibited varying signal strengths relative to WT UDG on DNA-modified electrodes. C85S produced a weaker signal, indicating a CT deficiency. The signal intensity from C101S was within error of that of WT, and the signal from C17H was larger than that of WT, possibly indicating that this mutant is less structurally stable than WT UDG.
\r\n \r\nIn E. coli MutY, position Y82 aligns with Y165 in MUTYH, a residue in which mutations have been found in many colorectal cancer patients. To better understand the correlation between protein-DNA CT and colorectal cancer, the MutY mutants Y82C and Y82L were prepared and characterized. Y82C exhibited a CT deficiency relative to WT MutY, whereas Y82L did not. These data indicate that Y82 forms part of the CT pathway in native E. coli MutY, but that other long-chain amino acids, such as leucine, can also mediate CT efficiently at this position.
\r\n \r\nSeveral different mutants of E. coli EndoIII were examined. First, the Y82 position was targeted, since the aligning MUTYH residue has been found mutated in colorectal cancer patients and because this residue is located near the protein-DNA interface. Five mutations were made at or near the Y82 position, and their cyclic voltammetry signals demonstrated that aromatic amino acids best mediate CT at this position. Other residues towards the interior of the protein, Y75, Y55, and F30 were also mutated to alanines. These mutants exhibited CT deficiencies, implicating the residues as part of a potential CT pathway. Residues W178 and Y185, located near the [4Fe-4S] cluster of EndoIII, were also mutated to alanines. The resulting mutants produced larger signals than that of WT EndoIII. These mutants were later shown by circular dichroism spectroscopy to be less stable structurally than WT EndoIII. All of the mutants mentioned exhibited enzymatic properties similar to those of WT, suggesting that they are able to bind DNA and excise damage nucleobases as well as the native enzyme. Several of these mutants were also used in a mutagenesis-based experiment to assay how EndoIII variants help MutY search for DNA lesions, although data from these experiments showed no significant differences in mutation rate between strains expressing different EndoIII variants.
\r\n \r\nIn total, the mutagenesis studies performed here helped determine the characteristics of BER enzymes that enable them to mediate DNA-protein CT. All these enzymes must contain a stable, well-protected metallocluster that charge can access through a series of CT-facilitating amino acids. In discovering several residues important for protein-DNA CT in UDG, MutY, and EndoIII, we have strengthened support for the hypothesis that these enzymes facilitate DNA-mediated CT in vivo. These enzymes may in fact be part of a much larger array of redox-active DNA-binding proteins that communicate electrochemically to help each other detect and repair DNA lesions inside the cell.
\r\n", "doi": "10.7907/63TC-FN74", "publication_date": "2011-06-10", "thesis_type": "phd", "thesis_year": "2011" }, { "id": "thesis:6231", "collection": "thesis", "collection_id": "6231", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:01242011-170306838", "primary_object_url": { "basename": "Saouma_final.pdf", "content": "final", "filesize": 12129295, "license": "other", "mime_type": "application/pdf", "url": "/6231/1/Saouma_final.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Iron Mediated Reduction Schemes for Dinitrogen and Carbon Dioxide", "author": [ { "family_name": "Saouma", "given_name": "Caroline Thalia Abdunnur", "clpid": "Saouma-Caroline-Thalia-Abdunnur" } ], "thesis_advisor": [ { "family_name": "Peters", "given_name": "Jonas C.", "clpid": "Peters-J-C" } ], "thesis_committee": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Okumura", "given_name": "Mitchio", "clpid": "Okumura-M" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Bercaw", "given_name": "John E.", "clpid": "Bercaw-J-E" }, { "family_name": "Peters", "given_name": "Jonas C.", "clpid": "Peters-J-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Several mono- and diiron species that coordinate NxHy ligands have been prepared and studied, to serve as structural, spectroscopic, and/or reactivity mimics to intermediates to an alternating reduction scheme for N\u2082 (i.e., Mn-N\u2261N \u2192 Mn-HN=NH \u2192 Mn-H\u2082N-NH\u2082 \u2192 Mn + 2 NH\u2083). The reaction between [PhBPR\u2083]FeMe ([PhBPR\u2083] = (PhB(CH\u2082PR\u2082)\u2083-; R = Ph, CH\u2082Cy) and hydrazine affords {[PhBPR\u2083]Fe}\u2082(\u03bc-\u03b7\u00b9: \u03b7\u00b9-N\u2082H\u2084)(\u03bc\u00b2- \u03b7\u00b2:N\u2082H\u2082). In one instance (R = Ph), the stepwise oxidation of coordinated hydrazine to diazene, and diazene to dinitrogen is achieved, giving {[PhBPPh\u2083]Fe}\u2082(\u03bc-\u03b7\u00b9:\u03b7\u00b9-N\u2082H\u2082)(\u03bc-\u03b7 2: \u03b7 2-N2H2) and {[PhBPPh\u2083]Fe}\u2082(\u03bc-NH)\u2082, respectively.
\r\n\t\r\nAs an extension to this work, a family of complexes which feature the same auxiliary ligands (i.e., [PhBPCH2Cy\u2083]Fe(OAc)), that are all iron(II), and that only differ in the oxidation state of the nitrogenous ligand has also been prepared: {[PhBPCH2Cy\u2083]Fe(OAc)}\u2082(\u03bc-N\u2082), {[PhBPCH2Cy\u2083]Fe(OAc)}\u2082(\u03bc-N\u2082H\u2082), {[PhBPCH2Cy\u2083]Fe(OAc)}\u2082(\u03bc-N\u2082H\u2084), and {[PhBPCH2Cy\u2083]Fe(OAc)(NH\u2083).
\r\n\t\r\nTo determine whether similar species could be isolated at a single iron site, the coordination chemistry of the more crowded \u201c[PhBPmter3]Fe\u201d fragment was investigated and compared to that of the \u201c[PhBPPh3]Fe\u201d scaffold. Treatment of [PhBPmter3]FeMe with hydrazine generates the unusual 5-coordinate hydrazido complex, [PhBPmter3]Fe(\u03bc2-N2H3), which features an Fe=N \u03c0 bond. Both 5- and 6-coordinate iron complexes that coordinate hydrazine were also synthesized, and the oxidation of these hydrazine and hydrazido(-) species was explored. In most instances, oxidation results in disproportionation of the N2Hy ligand, and [PhBPR3]Fe(NH3)(OAc) (R = Ph, mter) is isolated.
\r\n\t\r\nA 5-coordinate diiron diazene redox pair of complexes, {[PhBPPh3]Fe(CO)}2(\u03bc-\u03b71:\u03b71-N2H2)0/- was also prepared and studied. The electronic structure of the Fe-NH-NH-Fe core in these complexes is unusual in that it features a highly activated diazene ligand, which is unprecedented for mid-to-late transition metals. Combined structural, spectroscopic, and computation studies indicate that there is much \u03c0-covalency within the Fe-NH-NH-Fe core, which has a similar electronic structure as butadiene.
\r\n\t\r\nWith regards to CO2 reduction, the ability of iron(I) to mediate the one- and two- electron reductions of CO2 was explored. The reaction between [PhBPCH2Cy3]Fe(PCy)3 and CO2 is solvent dependent, with oxalate formation to generate {[PhBPCH2Cy3]Fe}2(\u03bc-\u03b72:\u03b72-oxalato) being favored in THF, and decarbonylation to give {[PhBPCH2Cy3]Fe}2(\u03bc-O)(\u03bc-CO) occurring exclusively in MeCy. Studies aimed at understanding this unusual solvent-induced selectivity are presented.
\r\n", "doi": "10.7907/46C3-BY97", "publication_date": "2011", "thesis_type": "phd", "thesis_year": "2011" }, { "id": "thesis:6205", "collection": "thesis", "collection_id": "6205", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:12152010-142322737", "primary_object_url": { "basename": "Thesis-Complete.pdf", "content": "final", "filesize": 2602190, "license": "other", "mime_type": "application/pdf", "url": "/6205/1/Thesis-Complete.pdf", "version": "v10.0.0" }, "type": "thesis", "title": "Chemistry and Electronics of the Ge(111) Surface", "author": [ { "family_name": "Knapp", "given_name": "David", "clpid": "Knapp-David" } ], "thesis_advisor": [ { "family_name": "Lewis", "given_name": "Nathan Saul", "clpid": "Lewis-N-S" } ], "thesis_committee": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Okumura", "given_name": "Mitchio", "clpid": "Okumura-M" }, { "family_name": "Atwater", "given_name": "Harry Albert", "clpid": "Atwater-H-A" }, { "family_name": "Lewis", "given_name": "Nathan Saul", "clpid": "Lewis-N-S" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The halogenation/alkylation procedure that has been proven to chemically and electrically passivate the Si(111) surface has been adapted for application to Ge(111). Removal of the Ge(111) surface oxide with 6\u20139 M HF(aq), followed by exposure to Br2 vapor, then alkylmagnesium or alkyllithium reagents yields air stable surfaces with surface recombination velocities (SRVs) as low as 40 cm/sec\u22121 at \ufb02at-band conditions. Surface charges with a density on the order of 1012 cm\u22122 cause a negative surface potential of almost 300 mV in n-type CH3 -Ge(111) samples prepared with this method. The oxidized surface shows a strongly positive surface potential in atmospheric conditions. A negative surface potential is also present in CH3 -Si(111), but the wider bandgap prevents this from causing inversion conditions in extrinsic samples. Ge(111) surfaces alkylated with a larger organic group, such as ethyl or decyl, displayed a weaker surface potential and higher surface recombination velocity as the surface was brought near \ufb02at-band. Mercury contacts to alkylated n-type substrates form rectifying junctions with barrier heights of 0.6 \u00b1 0.1 eV. Contacts to p-type substrates or to oxidized n-type substrates show no measurable recti\ufb01cation. X-ray photoelectron spectroscopy (XPS) con\ufb01rms that the area concentration of surface-bound carbon on CH3 -Ge(111) surfaces is equal to that of CH3 -Si(111) surfaces. Other passivation methods were less successful.
\r\n\r\nEvery atop Ge atom of an ideal CH3-Ge(111) should be capped and the Ge-C bonds should be directed normal to the surface plane. Infrared absorption spectroscopy (IRAS) of methyl-terminated surfaces prepared from HF-etched precursors did not display distinguishable absorption peaks, but if the Ge substrate is \ufb01rst treated with an anisotropic etch before the HF etch, IRAS con\ufb01rms the methyl group orientation with the polarization-dependent \u201cumbrella\u201d mode absorption at 1232 cm\u22121 and a polarization-independent rocking mode at 755 cm\u22121. Well-ordered CH3-Ge(111) surfaces displayed less surface charging while maintaining the low SRVs, indicating that such surfaces are successfully passivated.
\r\n\r\n", "doi": "10.7907/CGA1-QJ35", "publication_date": "2011", "thesis_type": "phd", "thesis_year": "2011" }, { "id": "thesis:5284", "collection": "thesis", "collection_id": "5284", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:09252009-142627047", "primary_object_url": { "basename": "thesis.pdf", "content": "final", "filesize": 2524728, "license": "other", "mime_type": "application/pdf", "url": "/5284/1/thesis.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Gas-Phase Terahertz Spectroscopy and the Study of Complex Interstellar Chemistry", "author": [ { "family_name": "Braakman", "given_name": "Rogier", "orcid": "0000-0002-4485-8450", "clpid": "Braakman-Rogier" } ], "thesis_advisor": [ { "family_name": "Blake", "given_name": "Geoffrey A.", "orcid": "0000-0003-0787-1610", "clpid": "Blake-G-A" } ], "thesis_committee": [ { "family_name": "Okumura", "given_name": "Mitchio", "orcid": "0000-0001-6874-1137", "clpid": "Okumura-M" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" }, { "family_name": "Marcus", "given_name": "Rudolph A.", "orcid": "0000-0001-6547-1469", "clpid": "Marcus-R-A" }, { "family_name": "Blake", "given_name": "Geoffrey A.", "orcid": "0000-0003-0787-1610", "clpid": "Blake-G-A" } ], "local_group": [ { "literal": "Astronomy Department" }, { "literal": "div_chem" } ], "abstract": "Terahertz spectroscopy holds great promise in the advancement of the field of astrochemistry. The sensitive observation of interstellar THz radiation is expected to lower detection limits and allow the study of larger and more complex species than is currently possible at millimeter wavelengths, which will place further constraints on chemical models and permit a direct comparison to the organic compounds seen in carbonaceous chondrites. With the successful recent launch of the Herschel Space Telescope, which will give high-fidelity access to interstellar THz radiation for the first time, and the completion of the Atacama Large Millimeter Array (ALMA) by 2013, the THz astronomy era is upon us. Unfortunately, laboratory THz spectroscopy presents significant challenges and will be soon be lagging behind the newly available observational platforms. Technologies to extend the capabilities of high-resolution spectroscopic systems into the THz domain are actively being pursued on many fronts, but affordable systems that are broadly tunable, sensitive and achieve the necessary resolution are not yet available. The work in this thesis should therefore be seen as part of the effort in the transition from centimeter-/millimeter-wave to THz spectroscopy that is currently taking place in the astrochemistry community.
\r\n\r\nAs part of this thesis, observational searches for the complex organics hydroxyacetone (CH\u2083COCH\u2082OH), 2-cyanoethanol (OHCH\u2082CH\u2082CN) and methoxyacetonitrile (CH\u2083OCH\u2082CN) were attempted at millimeter wavelengths. The unsuccessful nature of these searches highlight the current limits of studying interstellar chemistry using pure rotational spectroscopy. The characterization of the laboratory spectra of these molecules is nonetheless important as it will aid in the assignment and description of the rotational substructure and band shapes of their THz torsional spectra, features that may allow their interstellar detection; and this thesis presents methods by which such complex spectra may be rapidly and efficiently collected and fit using automated spectrometers and modern software tools.
\r\n\r\nThe description of the spectrum of hydroxyacetone is furthermore of interest due to the presence of the very low barrier to internal rotation in this molecule. Many interstellar compounds, both known and potential future targets, have functional groups capable of internal rotation in their structure; and so the effort in understanding the complex effects of the low barrier rotor in this case will benefit the general effort to further understand internal rotation.
\r\n\r\nIn searching for new interstellar molecules, both at millimeter wavelengths and at higher THz frequencies, characterization of the complete spectra of known interstellar molecules is of great importance to allow substraction of their contribution to observational spectra. In this thesis, the ground-state rotational spectrum of methanol, the most important \"interstellar weed\", is catalogued and described in detail through most of the THz region that will be accessible with Herschel and ALMA.
\r\n\r\nLastly, as part of the effort to increase the sensitivity of THz spectrometers, the use of Fabry-Perot cavities at these frequencies is explored. Such resonant cavities hold the potential to significantly increase the possible path lengths in spectroscopic system and to allow novel and sensitive detection techniques. Optimal configurations and the limits on achievable path lengths and Q-factors of such cavities are discussed, as are the possible extensions of Fourier Transform MicroWave (FT-MW) techniques to THz frequencies.
", "doi": "10.7907/R2KK-E302", "publication_date": "2010", "thesis_type": "phd", "thesis_year": "2010" }, { "id": "thesis:5430", "collection": "thesis", "collection_id": "5430", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:12092009-103613738", "primary_object_url": { "basename": "HCTai_thesis.pdf", "content": "final", "filesize": 6888578, "license": "other", "mime_type": "application/pdf", "url": "/5430/1/HCTai_thesis.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Characterization of the Brain Proteasome and its Interacting Proteins and their Regulation by Neuronal Activity", "author": [ { "family_name": "Tai", "given_name": "Hwan-Ching", "orcid": "0000-0003-0668-9163", "clpid": "Tai-Hwan-Ching" } ], "thesis_advisor": [ { "family_name": "Schuman", "given_name": "Erin Margaret", "orcid": "0000-0002-7053-1005", "clpid": "Schuman-E-M" } ], "thesis_committee": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "orcid": "0000-0003-1464-2461", "clpid": "Dougherty-D-A" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" }, { "family_name": "Zinn", "given_name": "Kai George", "orcid": "0000-0002-6706-5605", "clpid": "Zinn-K-G" }, { "family_name": "Parker", "given_name": "Carl Stevens", "orcid": "0000-0001-9795-4211", "clpid": "Parker-C-S" }, { "family_name": "Schuman", "given_name": "Erin Margaret", "orcid": "0000-0002-7053-1005", "clpid": "Schuman-E-M" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Despite the importance of proteasome-mediated proteolysis in synaptic plasticity, protein quality control, and cell regulation, little is known about proteasome composition and regulation in the brain. This thesis represents the first detailed study of mammalian brain proteasomes. Using a new affinity purification method, 26S proteasomes were isolated from the cytosolic and the synaptic compartments of the rat cortex. The proteins associated with the 26S proteasome were purified and analyzed by tandem mass spectrometry. A total of 30 proteasome-interacting proteins were identified in the brain. Several differences were seen in the spectrum of proteasome-associated proteins in the cytosol and the synaptosome. For example, the proteasome-associated protein ECM29 was found only in the cytosolic 26S proteasome, and the ubiquitin-binding factor TAX1BP1 only in the synaptic 26S proteasome. These findings allowed for further investigations into the interplay between proteasome regulation and synaptic plasticity.
\r\n\r\nNeuronal exposure to the neurotransmitter NMDA caused the degradation of 19S particles, resulting in lower levels of 26S proteasomes. The levels of ubiquitin conjugates also decreased, as did two proteasome-bound ubiquitin ligases, UBE3A/E6-AP and HUWE1/ARF-BP1, both of which have been linked to neurogenetic disorders associated with mental retardation. Thus, in the brain, proteasomes have a characteristic set of associated proteins that may serve as regulators or cofactors. Moreover, the content and pattern of associated proteins can vary with synaptic activity, in a manner likely to influence synaptic plasticity.
", "doi": "10.7907/DGMA-1K68", "publication_date": "2010", "thesis_type": "phd", "thesis_year": "2010" }, { "id": "thesis:5278", "collection": "thesis", "collection_id": "5278", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-09032009-105125", "primary_object_url": { "basename": "thesis_jenelle_bray.pdf", "content": "final", "filesize": 3782190, "license": "other", "mime_type": "application/pdf", "url": "/5278/1/thesis_jenelle_bray.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "The Development and Application of Computational Methods for the Prediction of G Protein-Coupled Receptor Structures", "author": [ { "family_name": "Bray", "given_name": "Jenelle Kiara", "clpid": "Bray-Jenelle-Kiara" } ], "thesis_advisor": [ { "family_name": "Goddard", "given_name": "William A., III", "clpid": "Goddard-W-A-III" } ], "thesis_committee": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Marcus", "given_name": "Rudolph A.", "clpid": "Marcus-R-A" }, { "family_name": "Goddard", "given_name": "William A., III", "clpid": "Goddard-W-A-III" }, { "family_name": "Clemons", "given_name": "William M.", "clpid": "Clemons-W-M" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Computational methods for the prediction of G protein-coupled receptor (GPCR) structures were applied to serotonin receptors, and new methods were developed to predict an orphan GPCR structure. First, the MembStruk procedure was used to predict the structures of the serotonin 2b and 2c receptors. Ligand binding sites for agonists and antagonists were predicted for both receptors. In addition, the SAR data for a series of psilocybin analogs bound to serotonin 2c were predicted. There was good agreement with binding and mutagenesis experiments.
\r\n\r\nA new structure prediction procedure called SuperBiHelix was developed to predict an ensemble of low-lying structures. SuperBiHelix samples the tilt and sweep angles of the transmembrane helices along with the rotation of the helices along the helical axes. The procedure was validated on the \u03b22-adrenergic receptor and A2A adenosine receptor crystal structures. This procedure was then used to predict the structure of GPR88, an orphan receptor. GPR88 has been identified as a novel target for psychiatric disorders. Three lipids were predicted to bind to GPR88. The head group of a lipid would bind to R113(3) and R116(3) at the extracellular side of the receptor. The lipid tail would bind in an aliphatic pocket in the TM2-TM3-TM6-TM7 region. The predicted bound complexes offer good suggestions for binding and mutagenesis experiments that could help validate the proposed structures.
\r\n", "doi": "10.7907/1655-ES74", "publication_date": "2010", "thesis_type": "phd", "thesis_year": "2010" }, { "id": "thesis:5499", "collection": "thesis", "collection_id": "5499", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:01062010-122905478", "primary_object_url": { "basename": "CAP_thesis.pdf", "content": "final", "filesize": 11296575, "license": "other", "mime_type": "application/pdf", "url": "/5499/8/CAP_thesis.pdf", "version": "v8.0.0" }, "type": "thesis", "title": "The Cellular Uptake of Luminescent Ruthenium Complexes", "author": [ { "family_name": "Puckett", "given_name": "Cindy Ann", "clpid": "Puckett-Cindy-Ann" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" }, { "family_name": "Lewis", "given_name": "Nathan Saul", "clpid": "Lewis-N-S" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Transition metal complexes have enormous potential as diagnostic and therapeutic agents, but their internalization and distribution in living cells are only poorly understood. Here, we perform one of the few systematic explorations of the uptake efficiency and mechanism of a class of metal complexes: luminescent dipyridophenazine (dppz) complexes of ruthenium(II). Substitution of the ancillary ligands permits variation in the overall complex charge, size, and hydrophobicity. We find that internalization of these complexes occurs mostly through passive diffusion, driven by the membrane potential, and that hydrophobicity, rather than size, is the most important determinant of compound accumulation. Across different cell types with all compounds, mostly uneven cytoplasmic staining is observed with near exclusion from the nucleus. Conjugation to cell-penetrating peptides, such as D-octaarginine, increases uptake efficiency, but leads to trapping in endosomes below a threshold concentration. Above this threshold concentration, substantial staining of the nucleus as well as the cytosol is observed. An appended fluorescein tag lowers the threshold concentration, indicating the importance of payload to the internalization and distribution of cell-penetrating peptides. Shorter peptides, including the nuclear targeting signal RrRK (where r = D-arginine), are also studied, though none have as high a degree of uptake nor as low a threshold concentration as the octaarginine conjugate. These studies provide a basis for the future design and optimization of metal complexes for biological application. ", "doi": "10.7907/2484-1405", "publication_date": "2010", "thesis_type": "phd", "thesis_year": "2010" }, { "id": "thesis:5269", "collection": "thesis", "collection_id": "5269", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-06162009-143222", "primary_object_url": { "basename": "FullThesis1.pdf", "content": "final", "filesize": 73920847, "license": "other", "mime_type": "application/pdf", "url": "/5269/11/FullThesis1.pdf", "version": "v7.0.0" }, "type": "thesis", "title": "Investigations into the Generality of Metalloinsertion at DNA Defects", "author": [ { "family_name": "Zeglis", "given_name": "Brian Matthew", "clpid": "Zeglis-Brian-Matthew" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" }, { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Metalloinsertors are substitutionally inert, octahedral transition metal complexes that bind to thermodynamically destabilized mismatched sites in duplex DNA with high affinity and selectivity. The complexes approach DNA from the minor groove, eject the mismatched bases into the major groove, and replace the displaced bases in the helical \u03c0-stack with their own sterically expansive ligands. Herein, we describe a series of five investigations aimed at elucidating the generality of metalloinsertion at DNA defects.
\r\n\r\nIn an effort to develop a diagnostic for mismatched DNA, a bifunctional, mismatch-specific conjugate with rhodium metalloinsertor and fluorophore subunits has been constructed. A proof-of-concept conjugate was successfully produced that displays an almost fourfold fluorescence enhancement in the presence of mismatched versus matched DNA.
\r\n\r\nTo investigate the range of metal complexes capable of mismatch-specific metalloinsertion, a ruthenium bisdipyridyl complex bearing the heptacyclic eilatin ligand has been synthesized and characterized. Electrophoresis competition experiments illustrate that the complex does display mismatch-preferential, though not necessarily mismatch-selective, binding.
\r\n\r\nTo probe the generality of metalloinsertion at other common thermodynamically destabilized DNA defects, the binding of rhodium metalloinsertors at abasic sites and single base bulges has been studied. It was determined that metalloinsertors bind abasic sites with high affinity and specificity, without regard to the identity of the unpaired base and with little dependence on the sequence context of the defect. Single base bulge recognition proved more elusive, with both the identity of the unpaired base and the sequence context influencing recognition.
\r\n\r\nTo determine the structural generality of metalloinsertion, single crystal X-ray diffraction was employed to determine the structure of \u0394-Rh(bpy)2(chrysi)3+ bound to an oligonucleotide duplex containing two A\u2022A mismatches. Two structures were obtained at <2 \u00c5 resolution, and each provides an archetypical picture of metalloinsertion: the bulky rhodium complex inserts into the mismatched site from the minor groove, ejecting the mismatched bases and replacing the displaced base pair with its own sterically expansive ligand.
\r\n\r\nFinally, two mismatch-specific conjugates have been designed for chemotherapeutic applications: a metalloinsertor-oxaliplatin conjugate for the selective delivery of platinum chemotherapeutics to mismatch repair deficient cells and a metalloinsertor-Auger electron emitter conjugate for the selective irradiation of mismatch-containing DNA.
", "doi": "10.7907/0YRQ-3W54", "publication_date": "2010", "thesis_type": "phd", "thesis_year": "2010" }, { "id": "thesis:5583", "collection": "thesis", "collection_id": "5583", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:03092010-121643566", "primary_object_url": { "basename": "Thesis.pdf", "content": "final", "filesize": 5771024, "license": "other", "mime_type": "application/pdf", "url": "/5583/11/Thesis.pdf", "version": "v8.0.0" }, "type": "thesis", "title": "Rapid Construction of Protein Capture Agents with Chemically Designed Stability and Antibody-Like Recognition Properties", "author": [ { "family_name": "Agnew", "given_name": "Heather Dawn", "clpid": "Agnew-Heather-Dawn" } ], "thesis_advisor": [ { "family_name": "Heath", "given_name": "James R.", "clpid": "Heath-J-R" } ], "thesis_committee": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Grubbs", "given_name": "Robert H.", "clpid": "Grubbs-R-H" }, { "family_name": "Davis", "given_name": "Mark E.", "clpid": "Davis-M-E" }, { "family_name": "Heath", "given_name": "James R.", "clpid": "Heath-J-R" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "This thesis describes technologies for the rapid and scalable production of high-affinity, high-specificity protein capture agents which possess the affinities and specificities of antibodies, but also exhibit improved chemical, biochemical, and physical stability. I will discuss how the chemical flexibility of comprehensive, one-bead-one-compound (OBOC) libraries of oligopeptides may be combined with iterative in situ click chemistry to select multi-ligand capture agents. Large OBOC libraries form the basis of individual peptide ligands, and also permit chemically designed stability through the incorporation of artificial (azide or acetylene) and non-natural amino acid building blocks. The in situ click chemistry method then utilizes the target protein as the catalyst, or template, for assembling its own biligand via formation of a 1,2,3-triazole linkage between two individual ligands (azide and acetylene). This process can be repeated to produce triligands, tetraligands, and other higher-order multi-ligands with an accompanying increase in affinity and specificity through cooperative interactions. Once found, multi-ligand capture agents can be produced in gram amounts via conventional synthetic methods such as the Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC). This is a general and robust strategy for the inexpensive, high-throughput construction of protein capture agents that can be exploited to detect protein biomarkers in multi-parameter clinical diagnostic assays.
\r\n\r\nWhile high-affinity protein capture agents represent a significant technology advance, they are just one component of what is necessary for highly multiplexed measurements of protein biomarkers. It is also important to develop or optimize the actual assay platforms that can enable sensitive multi-parameter protein measurements using these capture agents. Silicon nanowire (SiNW) nanoelectronic sensors can provide quantitative, label-free multi-parameter measurements of protein biomarkers in real time. However, SiNW sensors can be challenging to deploy because unprotected Si forms a native oxide layer that can significantly reduce the detection sensitivity of the nanowire sensors via dielectric shielding. Another technical challenge is the development of chemistries which allow for the selective encoding of nanowire surfaces with the capture agents. To overcome these challenges, the final part of this thesis presents a general method to functionalize organic and biological molecules on highly passivated Si(111) surfaces with minimal surface oxidation.
", "doi": "10.7907/1HJG-AQ59", "publication_date": "2010", "thesis_type": "phd", "thesis_year": "2010" }, { "id": "thesis:5908", "collection": "thesis", "collection_id": "5908", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06022010-164832965", "primary_object_url": { "basename": "Thesis_master.pdf", "content": "final", "filesize": 6629180, "license": "other", "mime_type": "application/pdf", "url": "/5908/1/Thesis_master.pdf", "version": "v7.0.0" }, "type": "thesis", "title": "The Development of Br\u00f8nsted Acid Catalysis Technologies and Mechanistic Investigations Therein", "author": [ { "family_name": "Carrera", "given_name": "Diane Elizabeth", "clpid": "Carrera-Diane-Elizabeth" } ], "thesis_advisor": [ { "family_name": "MacMillan", "given_name": "David W. C.", "clpid": "MacMillan-D-W-C" } ], "thesis_committee": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Dervan", "given_name": "Peter B.", "clpid": "Dervan-P-B" }, { "family_name": "Stoltz", "given_name": "Brian M.", "clpid": "Stoltz-B-M" }, { "family_name": "MacMillan", "given_name": "David W. C.", "clpid": "MacMillan-D-W-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The enantioselective reductive amination of ketones with Hantzsch ester has been achieved through Br\u00f8nsted acid catalysis. A novel triphenylsilyl substituted BINOL-derived phosphoric acid catalyst has been developed for this transformation, imparting high levels of selectivity when used with methyl ketones and aromatic amines. A stereochemical model for the observed selectivity based on torsional effects has been developed through molecular modeling and is further supported by a single crystal x-ray structure of an imine-catalyst complex.
\r\n\r\nMechanistic studies have revealed the importance of catalyst buffering and drying agent on reaction efficiency while a Hammett analysis of acetophenone derivatives offers insight into the key factors involved in the enantiodetermining step. Kinetic studies have shown that imine reduction is rate-determining and follows Michaelis-Menten kinetics. Determination of the Eyring parameters for the imine reduction has also been accomplished and suggests that the phosphoric acid catalyst behaves in a bifunctional manner by activating both the imine electrophile and the Hantzsch ester nucleophile.
\r\n\r\nThe intermolecular addition of vinyl, aromatic, and heteroaromatic potassium trifluoroborate salts to non-activating imines and enamines can also be accomplished through Br\u00f8nsted acid activation. This analog of the Petasis reaction shows a wide substrate scope and is amenable to use with a variety of carbamate protected nitrogen electrophiles in the first example of metal-free 1,2-additions of trifluoroborate nucleophiles. The mechanistic underpinnings of benzyl trifluoroborate addition has also been explored and, in contrast to what is seen with \u03c0-nucleophilic species, appears to proceed through an intramolecular alkyl-transfer mechanism.
", "doi": "10.7907/ZGS8-QT92", "publication_date": "2010", "thesis_type": "phd", "thesis_year": "2010" }, { "id": "thesis:5500", "collection": "thesis", "collection_id": "5500", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:01062010-125002622", "primary_object_url": { "basename": "JCGThesis.pdf", "content": "final", "filesize": 14836624, "license": "other", "mime_type": "", "url": "/5500/7/JCGThesis.pdf", "version": "v10.0.0" }, "type": "thesis", "title": "Exploring DNA-Mediated Charge Transport with Fast Radical Traps", "author": [ { "family_name": "Genereux", "given_name": "Joseph Charles", "orcid": "0000-0002-5093-7710", "clpid": "Genereux-Joseph-Charles" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Marcus", "given_name": "Rudolph A.", "clpid": "Marcus-R-A" }, { "family_name": "Zewail", "given_name": "Ahmed H.", "clpid": "Zewail-A-H" }, { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The \u03c0-stack of DNA is competent for mediating charge transport (CT), both by single-step and multi-step mechanisms. The yield of long-range single-step CT from photoexcited 2-aminopurine to guanine across adenine tracts has a shallow, periodic distance dependence, with increasing amplitude and decreasing slope with temperature. To measure total CT yield, herein we employ the fast radical traps N2-cyclopropylguanine (CPG), and N6-cyclopropyladenine (CPA), which are similar to the unmodified bases, but undergo rapid decomposition upon oxidation. We find that decomposition of CPG by a photoexcited rhodium intercalator across an adenine tract has similar periodic distance dependence to quenching of 2-aminopurine by guanine, and the same temperature dependence as well. In contrast, decomposition of CPG by photoexcited 2-aminopurine is monotonic with respect to adenine tract length, and also competes with back electron transfer (BET). Eliminating BET by separating 2-aminopurine from the adenine tract with three high-potential inosines restores the non-monotonic distance dependence. We also determined decomposition of CPA along adenine tracts by photoexcited rhodium, and found the CT yield to be distance-independent, demonstrating that the periodicity associated with guanine oxidation is with respect to adenine tract length, not donor-acceptor separation. This length-dependent periodicity, and the associated temperature dependence, support a model of conformational gating in the formation of CT-active domains along the DNA.
\r\n\r\nDNA-mediated electrochemistry is facile in self-assembled monolayers on electrodes, and redox-active dyes are reduced through the DNA \u03c0-stack at potentials far lower than those of the individual bases. Since cytosine is the most readily reduced base, we incorporated CPC into DNA monolayers to assay for bridge occupation, and CPC decomposition was not observed.
\r\n\r\nTo explore the relative contributions of single-step and multi-step mechanisms to CT yield across adenine tracts, we compared quantum yields previously collected from 2-aminopurine fluorescence quenching experiments to those of CPG decomposition. For seven or eight intervening adenines, single-step CT accounts for the entire CT yield, while for four to six adenines, multi-step CT is the dominant mechanism. We interrupted multi-step CT by substituting CPA for an adenine on the bridge, and found the total CT yield across five or six intervening adenines is lowered to the single-step CT yield. Blocking single-step CT by replacing the terminal guanine with redox-inactive inosine does not affect CPA decomposition on the bridge. These results imply that single-step and multi-step CT processes are not in direct competition for these assemblies, consistent with the model of conformationally gated CT-active states.
\r\n", "doi": "10.7907/R0CF-GF80", "publication_date": "2010", "thesis_type": "phd", "thesis_year": "2010" }, { "id": "thesis:5618", "collection": "thesis", "collection_id": "5618", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:03192010-175848794", "primary_object_url": { "basename": "PEL_Thesis_Caltech_rev2.pdf", "content": "final", "filesize": 2328398, "license": "other", "mime_type": "application/pdf", "url": "/5618/1/PEL_Thesis_Caltech_rev2.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Activation of Transcription from a Distance: Investigations on the Oxidation of SoxR by DNA-Mediated Charge Transport\r ", "author": [ { "family_name": "Lee", "given_name": "Paul Eulehwann", "clpid": "Lee-Paul-Eulehwann" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Campbell", "given_name": "Judith L.", "clpid": "Campbell-J-L" }, { "family_name": "Clemons", "given_name": "William M.", "clpid": "Clemons-W-M" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "In enteric bacteria, the cellular response to oxidative stress caused by superoxide is activated by soxR, which encodes a redox-active transcription factor that contains a [2Fe2S] cluster and binds DNA with high affinity. Here we describe how SoxR may detect global changes in oxidative stress while bound to DNA at a single location through DNA-mediated charge transport. A unique property of DNA is its ability to delocalize charge along its base stack, allowing oxidative damage to be funneled to specific sites of low oxidation potential. Charge transport also has the potential to access proteins with redox-active moieties.
\r\n\r\nElectrochemical studies presented here demonstrate that the redox couple of the [2Fe2S] clusters of SoxR can be accessed through the DNA, and that when the protein is bound to DNA, is shifted almost 0.5 V positive to its potential measured in solution in the absence of DNA. SoxR in its reduced form is found to inhibit guanine damage by repairing guanine radicals formed in DNA by the use of various photoactive metallointercalators, by donating an electron from one of its [2Fe2S]\u207a clusters and filling the guanine radical hole. RT-PCR is used to monitor the amount of soxS mRNA produced in cells that have taken up the DNA binding photooxidant [Rh(phi)2bpy]\u00b3\u207a and are treated with light. Cells thus treated to generate guanine radicals express soxS, evidence that SoxR is being oxidized. An in vitro assay is furthermore used to examine directly the DNA-mediated oxidation of SoxR by measuring its transcriptional activity. [Rh(phi)2bpy']\u00b3\u207a, tethered to DNA 80 bp from the soxS promoter, induces transcription by activating SoxR upon irradiation. These results demonstrate not only that guanine radicals can act to oxidize SoxR, but that the resulting oxidized, DNA-bound protein is biologically active. Thus, transcription can be activated from a distance through DNA-mediated charge transport.
\r\n\r\nThe ability of DNA to conduct charge along its base stack allows offers a general strategy for DNA-mediated signaling of oxidative stress, as it allows information about oxidative events to be transmitted quickly and directly to the proteins responsible for turning on the genes necessary for cell survival.
\r\n", "doi": "10.7907/44P4-C408", "publication_date": "2010", "thesis_type": "phd", "thesis_year": "2010" }, { "id": "thesis:5790", "collection": "thesis", "collection_id": "5790", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05102010-102555148", "type": "thesis", "title": "Geochemical Mechanisms of Biomineralization from Analysis of Deep-Sea and Laboratory Cultured Corals", "author": [ { "family_name": "Gagnon", "given_name": "Alexander C.", "clpid": "Gagnon-Alexander-C" } ], "thesis_advisor": [ { "family_name": "Adkins", "given_name": "Jess F.", "clpid": "Adkins-J-F" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" } ], "thesis_committee": [ { "family_name": "Blake", "given_name": "Geoffrey A.", "clpid": "Blake-G-A" }, { "family_name": "Eiler", "given_name": "John M.", "clpid": "Eiler-J-M" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Adkins", "given_name": "Jess F.", "clpid": "Adkins-J-F" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The ocean is a major component of global heat transport and represents a large exchangeable reservoir of CO\u2082. The importance of these effects on climate can be quantified with records of ocean temperature, chemistry and dynamics spanning past climate change. One approach to reconstruct past ocean conditions relies on the chemical composition of CaCO\u2083 skeletons from coral. Despite the utility of these geochemical proxies, several lines of evidence suggest that biomineralization, the process corals use to build their skeletons, also influences composition, complicating the interpretation of past records. Coral grown under constant environmental conditions, either collected from the deep-sea or cultured in the laboratory, are used to quantify and spatially map the effects of biomineralization on skeletal composition.
\r\n\r\nIn modern deep-sea coral, Mg/Ca increases with decreasing Sr/Ca in most the skeleton, consistent with closed-system (Rayleigh) precipitation. Results also show composition strongly follows skeletal architecture. Centers of calcification (COCs) are small regions of disorganized crystals thought to be the initial stage of skeletal extension. Unlike the rest of the skeleton, Mg/Ca ratios vary more than two fold within the COCs while Sr/Ca is near constant. Our data provide new constraints on a number of possible mechanisms for this effect.
\r\n\r\nIn a complementary set of experiments the nanoSIMS, a new instrument capable of accurate sub-micron compositional analysis, is applied to adult cultured surface coral (1) mapping the pattern of metal ion incorporation in new growth and showing that the calcifying fluid is likely in direct exchange with seawater; and (2) testing the sensitivity of Me/Ca ratios to aragonite saturation \u03a9. Despite a large range of \u03a9 and calcification rates, the average Sr/Ca of nanoSIMS spot measurements in cultured coral are within 1.2% (2 sigma std. dev. of the 5 means). These data suggest that temperature is a more significant control on Sr/Ca than aragonite saturation between \u03a9 = 2.5--5. Within the framework of a closed-system (Rayleigh) model for biomineralization the results constrain explanations for the sensitivity of coral calcification rates to ocean acidification, improving our understanding of how anthropogenic CO\u2082 will impact coral reefs.
", "doi": "10.7907/N1MW-8Q84", "publication_date": "2010", "thesis_type": "phd", "thesis_year": "2010" }, { "id": "thesis:5261", "collection": "thesis", "collection_id": "5261", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-12102008-101354", "primary_object_url": { "basename": "Acknowledgements.pdf", "content": "final", "filesize": 95334, "license": "other", "mime_type": "application/pdf", "url": "/5261/1/Acknowledgements.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Electrical Detection of DNA Binding Proteins", "author": [ { "family_name": "Gorodetsky", "given_name": "Alon A.", "clpid": "Gorodetsky-Alon-A" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Heath", "given_name": "James R.", "clpid": "Heath-J-R" }, { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Lewis", "given_name": "Nathan Saul", "clpid": "Lewis-N-S" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The base pair stack of double helical DNA has proven to be an effective medium for charge transport. The \u03c0-stacked DNA base pairs can mediate charge transport (CT) chemistry over distances as long as 20 nm, and the reaction is exquisitely sensitive to DNA sequence-dependent conformation and dynamics. This sensitivity to perturbations in DNA structure and base pair stacking makes DNA-mediated charge transport chemistry an ideal methodology for the electrical detection of base mismatches, lesions, and protein binding. Efforts toward expanding the scope of electrochemistry at DNA-modified surfaces for biosensing applications are presented here.", "doi": "10.7907/VPKN-CV38", "publication_date": "2009", "thesis_type": "phd", "thesis_year": "2009" }, { "id": "thesis:2966", "collection": "thesis", "collection_id": "2966", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-07222008-144633", "primary_object_url": { "basename": "Exploration_of_Physisorbed_Monolayers_for_Molecular_Scale_Surface_Patterning.pdf", "content": "final", "filesize": 7546114, "license": "other", "mime_type": "application/pdf", "url": "/2966/5/Exploration_of_Physisorbed_Monolayers_for_Molecular_Scale_Surface_Patterning.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Exploration of Physisorbed Monolayers for Molecular-Scale Surface Patterning", "author": [ { "family_name": "Papadantonakis", "given_name": "Kimberly Marshall", "orcid": "0000-0002-9900-5500", "clpid": "Papadantonakis-Kimberly-Marshall" } ], "thesis_advisor": [ { "family_name": "Lewis", "given_name": "Nathan Saul", "clpid": "Lewis-N-S" } ], "thesis_committee": [ { "family_name": "Okumura", "given_name": "Mitchio", "clpid": "Okumura-M" }, { "family_name": "Brunschwig", "given_name": "Bruce S.", "clpid": "Brunschwig-B-S" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Bercaw", "given_name": "John E.", "clpid": "Bercaw-J-E" }, { "family_name": "Lewis", "given_name": "Nathan Saul", "clpid": "Lewis-N-S" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Many simple organic molecules, such as straight-chain alkanes and simple aromatics, spontaneously assemble into highly ordered monolayers at solid\u2013liquid interfaces. These monolayers are composed of molecules that lie flat at the interface without forming chemical bonds to the surface of the solid. These monolayer structures are highly ordered and produce patterns with features on the scale of just a single nanometer in length. The exploitation of this physisorption phenomenon may provide a promising route toward an inexpensive nanometer-scale surface patterning technique. However, two fundamental challenges must be overcome before physisorbed monolayers can be useful in surface-patterning applications: (1) absence of control over the particular pattern formed by the molecules; and (2) pattern impermanence.
\r\n\r\nThis document opens with an introductory chapter that contains background on physisorbed monolayers and a brief description of scanning tunneling microscopy, the experimental technique which is commonly used to study monolayers. The second and third chapters present details on the results of experiments with a monolayer templating technique. This templating technique involves replacement of the molecules comprising a monolayer of either normal alkanes or symmetrical thioethers by symmetrical ethers. The ethers are forced to conform to the structure of the existing template monolayer, which differs from the structure of an ether monolayer formed in the absence of the template. The monolayer templating technique offers researchers a limited method for exercising control over the surface patterns formed by particular molecules.
\r\n\r\nThe challenge of pattern impermanence is addressed in the fourth chapter of this document. The molecules comprising physisorbed monolayers are free to exchange with molecules in the solution contacting the surface, thus the orientation of the monolayer structure within a particular surface region can change with time. A technique analogous to traditional lithographic methods that may allow physisorbed monolayers to be used for permanent surface patterning is described. The technique would employ physisorbed monolayers as surface masks while other molecular species chemically bond to regions of the surface left uncovered by the masking monolayer. Descriptions of the progress made toward the development of the patterning technique, and of the substantial challenges encountered during efforts to develop such a patterning method close the chapter.
\r\n", "doi": "10.7907/JZCK-B721", "publication_date": "2009", "thesis_type": "phd", "thesis_year": "2009" }, { "id": "thesis:5240", "collection": "thesis", "collection_id": "5240", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-07252008-141614", "primary_object_url": { "basename": "Full_Thesis_PRE.pdf", "content": "final", "filesize": 5692021, "license": "other", "mime_type": "application/pdf", "url": "/5240/7/Full_Thesis_PRE.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "The Selective Oligomerization of Ethylene Using Chromium Diphosphine Catalysts and the Synthesis and Reactivity of Group 7 Carbonyl Derivatives Relevant to Synthesis Gas Conversion", "author": [ { "family_name": "Elowe", "given_name": "Paul Richard", "clpid": "Elowe-Paul-Richard" } ], "thesis_advisor": [ { "family_name": "Bercaw", "given_name": "John E.", "clpid": "Bercaw-J-E" } ], "thesis_committee": [ { "family_name": "Bercaw", "given_name": "John E.", "clpid": "Bercaw-J-E" }, { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The work presented in this thesis explores two distinct fields of organometallic chemistry with a common goal of selectively transforming cheap and abundant feedstocks to value-added chemicals using homogeneous catalysts.
\r\n\r\nChapter 1 presents the synthesis and characterization of a series of bis(diphenylphosphino)amine ligands and their corresponding chromium(III) trichloride complexes. The isolated chromium complexes are precursors to highly active catalysts for the selective oligomerization of ethylene to 1-hexene and 1-octene. The unique feature of the ligands presented herein is the presence of coordinating functionalities tethered to the nitrogen backbone. These act as hemilabile donors, which stabilize the active species and/or transition states during catalysis. This increased stability leads to more productive catalysts. Furthermore, important solvent and additive effects have been investigated. While reactions in non-polar solvents exhibit poor activity at lower ethylene pressures, those in more polar solvents are highly active and generate very little undesired polymer. Varying the solvent has a significant impact on 1-hexene/1-octene selectivity as well. Experiments with potentially coordinating additives result in a higher tendency for 1-octene formation. An investigation of catalyst decomposition is also discussed.
\r\n\r\nChapter 2 presents synthetic, structural and reactivity studies on a series of Group 7 carbonyl derivatives relevant to synthesis gas conversion. Reduction of the carbonyl precursors with a hydride source generates the corresponding formyl species. This reaction is facilitated when more electrophilic carbonyl complexes are employed. Neutral and cationic Fischer carbene complexes were prepared by the reaction of the formyl species with boranes and alkyltriflates, respectively. Further reduction of Group 7 methoxycarbenes with a hydride leads to the formation of a reactive methoxymethyl species. Dimethyl ether release is obtained from treatment of a manganese methoxymethyl species with a hydride. Moreover, subjecting manganese methoxymethyl complexes to an atmosphere of CO generates acyl complexes via migratory insertion. Preliminary mechanistic details are presented.
\r\n", "doi": "10.7907/S7NA-Y054", "publication_date": "2009", "thesis_type": "phd", "thesis_year": "2009" }, { "id": "thesis:674", "collection": "thesis", "collection_id": "674", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-02182009-100346", "primary_object_url": { "basename": "pricewhelan_21809.pdf", "content": "final", "filesize": 7043779, "license": "other", "mime_type": "application/pdf", "url": "/674/1/pricewhelan_21809.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Physiology and Mechanisms of Pyocyanin Reduction in Pseudomonas aeruginosa", "author": [ { "family_name": "Price-Whelan", "given_name": "Alexa Mari", "orcid": "0000-0001-7587-7534", "clpid": "Price-Whelan-Alexa-Mari" } ], "thesis_advisor": [ { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" } ], "thesis_committee": [ { "family_name": "Leadbetter", "given_name": "Jared R.", "orcid": "0000-0002-7033-0844", "clpid": "Leadbetter-J-R" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Meyerowitz", "given_name": "Elliot M.", "orcid": "0000-0003-4798-5153", "clpid": "Meyerowitz-E-M" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "The opportunistic pathogen Pseudomonas aeruginosa excretes redox-active small molecules called phenazines. This thesis addresses the possibility that the phenazine pyocyanin acts as an electron acceptor for energy metabolism and exerts beneficial effects on P. aeruginosa physiology. The effects of phenazine production and exposure on P. aeruginosa strain PA14 were examined by comparing the physiological status of the wild type to a mutant defective in phenazine production. Quantification of intracellular NADH and NAD+ pools revealed a more reduced intracellular redox state in the phenazine-null mutant compared to the wild type, consistent with the capacity of P. aeruginosa to reduce pyocyanin. High-performance liquid chromatography of culture metabolites showed that the wild type excreted pyruvate in late stationary phase, indicating that pyocyanin alters flux through central metabolic pathways.
\r\n\r\nWe set out to identify mechanisms allowing P. aeruginosa to catalyze pyocyanin redox cycling. Through a genetic screen, we found two loci required for full pyocyanin-dependent ferric citrate reduction activity in P. aeruginosa PA14: (1) the gene gpsA, encoding the soluble glycerol-3-phosphate dehydrogenase (GpsA), and (2) the operon fbcFBC, encoding the respiratory cytochrome bc1 complex. Mutants lacking functional GpsA had oxidized cytoplasms and may be defective in pyocyanin reduction due to a lack of sufficient NADH. In contrast, mutants lacking a functional cytochrome bc1 complex produced ample reducing power for pyocyanin reduction, raising the possibility that the cytochrome bc1 complex directly catalyzes pyocyanin reduction.
\r\n\r\nPyocyanin has previously been shown to affect the development of P. aeruginosa colonies on agar surfaces: phenazine-null mutants form wrinkled (rugose) colonies, while the wild type forms smooth colonies. Using this colony biofilm assay, we showed that the \u0394gpsA mutant forms rugose colonies, consistent with a role for pyocyanin reduction in stimulating smooth colony formation. Modulation of electron acceptor availability through nitrate addition to the medium promoted smooth colony formation in rugose mutants. These results imply that rugosity is an adaptation to electron acceptor limitation.
\r\n\r\nThe work in this thesis has provided insight into the physiological relevance of pyocyanin reduction in P. aeruginosa, mechanisms controlling intracellular redox state in bacteria, and mechanisms that may contribute to P. aeruginosa virulence.
\r\n", "doi": "10.7907/N42E-M534", "publication_date": "2009", "thesis_type": "phd", "thesis_year": "2009" }, { "id": "thesis:1655", "collection": "thesis", "collection_id": "1655", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05062009-173812", "primary_object_url": { "basename": "MTW-Thesis.pdf", "content": "final", "filesize": 10507124, "license": "other", "mime_type": "application/pdf", "url": "/1655/1/MTW-Thesis.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Synthetic and Mechanistic Studies of Small-Molecule Activation at Low-Valent Iron, Cobalt, and Iridium Centers", "author": [ { "family_name": "Whited", "given_name": "Matthew Thomas", "orcid": "0000-0002-1193-9078", "clpid": "Whited-Matthew-Thomas" } ], "thesis_advisor": [ { "family_name": "Grubbs", "given_name": "Robert H.", "clpid": "Grubbs-R-H" }, { "family_name": "Peters", "given_name": "Jonas C.", "clpid": "Peters-J-C" } ], "thesis_committee": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" }, { "family_name": "Grubbs", "given_name": "Robert H.", "clpid": "Grubbs-R-H" }, { "family_name": "Agapie", "given_name": "Theodor", "clpid": "Agapie-T" }, { "family_name": "Peters", "given_name": "Jonas C.", "clpid": "Peters-J-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The preparation of transition-metal systems for catalytic multielectron transformations of small molecules remains a significant challenge for synthetic chemists. The realization of new transformations often depends critically on the design of frameworks capable of stabilizing unusual oxidation states and molecular geometries, providing a frontier molecular-orbital landscape that is well suited to interact with the molecules of interest. This thesis has sought to address two particularly noteworthy challenges in the field of small-molecule activation, dinitrogen reduction and C\u2013H bond functionalization, through judicious ligand choice and design.
\r\n\r\nChapters 2 and 3 describe the syntheses of new tri- and tetradentate hybrid ligands incorporating a single X-type donor (amido or silyl) and multiple phosphine donors designed to stabilize low oxidation states at iron and cobalt and support dinitrogen reduction and other multielectron redox transformations. While the amidophosphine ligands do allow access to unusual monovalent iron and cobalt complexes, the isolation of dinitrogen adducts supported by these ligands remains elusive and the weakness of the silicon\u2013nitrogen bond makes the complexes prone to decomposition. In contrast, the tris(phosphino)silyl ligands presented in Chapter 3 afford straightforward access to the first terminally bonded dinitrogen complexes of monovalent iron, and the structure of these and related complexes are described along with preliminary experiments showing that protonolysis of the iron(I)\u2013dinitrogen complexes produces hydrazine in reasonable stoichiometric yields.
\r\n\r\nChapters 4 through 7 address the functionalization of ether and amine C\u2013H bonds by a double C\u2013H activation route. Chapter 4 describes the investigation of reactivity of low-valent, pincer-supported iridium species with a variety of ethers, leading to a number of selective C\u2013H, C\u2013C, and C\u2013O bond cleavage events, affording in several cases iridium carbene complexes by double C\u2013H activation and loss of dihydrogen.
\r\n\r\nChapter 5 presents an exploration of the electronic structure of the unusual square-planar iridium(I) alkoxycarbenes and their nucleophilic activation of several heterocumulene substrates, leading to multiple-bond metathesis events promoted by metal- rather than ligand-initiated reactivity. Chapter 6 describes the discovery of new atom and group transfer reactions from diazo reagents to the alkoxycarbenes and the implementation of these reactions in an unprecedented catalytic cycle for C=E bond formation by multiple C\u2013H activations.
\r\n\r\nChapter 7 explores the related reactivity of a low-valent pincer iridium complex with methyl amines and the reactivity of the resulting iridium(III) dihydrido aminocarbenes, which is shown to diverge substantially from that observed for the iridium(I) carbene species.
\r\n", "doi": "10.7907/4N3R-5194", "publication_date": "2009", "thesis_type": "phd", "thesis_year": "2009" }, { "id": "thesis:1517", "collection": "thesis", "collection_id": "1517", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-04262009-232200", "primary_object_url": { "basename": "thesis_all.pdf", "content": "final", "filesize": 3994474, "license": "other", "mime_type": "application/pdf", "url": "/1517/8/thesis_all.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Nonlinear Polymeric Architectures via Olefin Metathesis", "author": [ { "family_name": "Gorodetskaya", "given_name": "Irina A.", "clpid": "Gorodetskaya-Irina-A" } ], "thesis_advisor": [ { "family_name": "Grubbs", "given_name": "Robert H.", "clpid": "Grubbs-R-H" } ], "thesis_committee": [ { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Heath", "given_name": "James R.", "clpid": "Heath-J-R" }, { "family_name": "Grubbs", "given_name": "Robert H.", "clpid": "Grubbs-R-H" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The research presented in this thesis focuses on application of olefin metathesis to the construction of hyperbranched and cyclic macromolecules. The olefin metathesis reaction is briefly reviewed in Chapter 1, along with its applications in polymer synthesis. A very mild, simple, and modular, olefin metathesis-based hyperbranched polymerization route is presented in Chapter 2. This method utilizes the cross metathesis selectivity of the functional group tolerant N-heterocyclic carbene ruthenium catalyst towards different types of alkenes, and it can be applied to the polymerization of many easily prepared ABn monomers. Moreover, the same method can be used to post-synthetically functionalize such polymers for realization of their substrate carrying potential. Chapter 3 describes one such functionalization example\u2014a pyrene analyte is attached to a metathesis derived hyperbranched polymer. This modification of the polymer provides insight into its solution structure relative to a linear analog. In addition, molecular weight control of the metathesis hyperbranched polymerization is discussed in detail in Chapter 4. The careful choice of the catalysts loading and the use of a multifunctional core are found to be important parameters in preparation of polymers which span a range of molecular weights.
\r\n\r\nEven well-established materials, such as polyethylene, can benefit from olefin metathesis and the unusual polymeric architectures it can efficiently create. For example, a cyclic polymer which lacks end groups, as opposed to having many end groups like a hyperbranched polymer, is expected to possess unique physical properties. The preparation of cyclic and linear polyethylenes and the study of their relative rheological properties are described in Chapter 5. The polymerization methodology outlined in this chapter takes advantage of ring-expansion metathesis polymerization\u2014a facile method for the synthesis of cyclic macromolecules. Some efforts directed at molecular weight control of this cyclic polymerization are also discussed.
\r\n", "doi": "10.7907/WRYR-4674", "publication_date": "2009", "thesis_type": "phd", "thesis_year": "2009" }, { "id": "thesis:878", "collection": "thesis", "collection_id": "878", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-03052008-065324", "primary_object_url": { "basename": "ConnorTOC.pdf", "content": "final", "filesize": 154276, "license": "other", "mime_type": "application/pdf", "url": "/878/8/ConnorTOC.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "N-Terminal Modification and Codon Reassignment with Non-Canonical Amino Acids in Proteins", "author": [ { "family_name": "Connor", "given_name": "Rebecca Elizabeth", "clpid": "Connor-Rebecca-Elizabeth" } ], "thesis_advisor": [ { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" } ], "thesis_committee": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Varshavsky", "given_name": "Alexander J.", "clpid": "Varshavsky-A-J" }, { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" }, { "family_name": "Shan", "given_name": "Shu-ou", "clpid": "Shan-Shu-ou" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Proteins are ubiquitous macromolecules that effect and control all the processes of life from reproduction to respiration to physical motion. These diverse molecules also provide physical structure and defensive mechanisms. The twenty canonical amino acids can be found in virtually every protein; however, in some organisms, the set of endogenous amino acids also contains residues outside the \u201ccanon,\u201d such as pyrrolysine, selenocysteine, and formylmethionine. Although a range of chemistries is available through natural side-chain diversity, some functionalities such as halogens, ketones, azides, alkenes, and alkynes are not found in nature. The introduction of a broader range of chemical functionality into proteins and protein-based materials through the use of non-canonical amino acids represents a challenging goal for protein engineering. The persistence of all the amino acids throughout protein sequences also presents a challenge for biochemical modification at a particular location. The insertion of a non-natural amino acid at a single location on a protein can allow specific modification without further affecting the natural protein sequence. The focus of this thesis is on a new method for the post-translational site-specific introduction of non-canonical amino acids to the N-terminus of proteins in vitro and progress made towards developing a complementary in vivo method using the E. coli L,F-transferase. Addition of non-proteinogenic functionality to the coat proteins of M13 bacteriophage using non-canonical amino acids is also explored.\r\n", "doi": "10.7907/ERVA-Q969", "publication_date": "2008", "thesis_type": "phd", "thesis_year": "2008" }, { "id": "thesis:1757", "collection": "thesis", "collection_id": "1757", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05122008-105452", "primary_object_url": { "basename": "08_Full_Thesis.pdf", "content": "final", "filesize": 2552947, "license": "other", "mime_type": "application/pdf", "url": "/1757/9/08_Full_Thesis.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Electron Tunneling and Hopping Through Proteins", "author": [ { "family_name": "Shih", "given_name": "Crystal", "clpid": "Shih-Crystal" } ], "thesis_advisor": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Winkler", "given_name": "Jay Richmond", "clpid": "Winkler-J-R" } ], "thesis_committee": [ { "family_name": "Lewis", "given_name": "Nathan Saul", "clpid": "Lewis-N-S" }, { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Richards", "given_name": "John H.", "clpid": "Richards-J-H" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Long-range electron tunneling is a central component of processes that are essential for biological function. While many studies have been made to understand electron transfer in proteins, biologically efficient electron transfer at distances exceeding 25 \u00c5 remains unobserved in these experiments and hence unresolved. It is proposed that long-range electron transfer is in actuality multistep electron tunneling. What is reported in this thesis is the design, synthesis, and study of many protein systems for the purpose of studying multistep electron tunneling in azurin.
\r\n\r\nIn each system, a histidine has been introduced on the protein for attachment of a high-potential ruthenium or rhenium sensitizer ([Ru(trpy)(tfmbpy)]2+ or [Re(dmp)(CO)3]+); a nitrotyrosine, tryptophan, or tyrosine is placed between the two metal centers on the tunneling pathway. The electron transfer is triggered with the excitation of the metal label with laser light, and the kinetics are monitored, for the most part, by time-resolved UV-VIS spectroscopy.
\r\n\r\nThe first system to empirically demonstrate multistep electron tunneling in proteins was discovered; ultrafast electron transfer is observed between the copper and rhenium centers in the Re124/W122 system; the system was structurally characterized and studied by time-resolved UV-VIS and IR spectroscopies. A two-step tunneling model is proposed; the data sets for the different methods utilized are all in excellent agreement with the model.
\r\n\r\nSystematic perturbations were made to the working hopping system. It was discovered that nitrotyrosine can participate as an intermediate, but studies to demonstrate its participation in multistep tunneling are not yet fully realized. A second hopping system was discovered in the development of the Re126/W122 system.
\r\n", "doi": "10.7907/0E5A-1W62", "publication_date": "2008", "thesis_type": "phd", "thesis_year": "2008" }, { "id": "thesis:1654", "collection": "thesis", "collection_id": "1654", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05062008-171457", "primary_object_url": { "basename": "00thesis.pdf", "content": "final", "filesize": 4852909, "license": "other", "mime_type": "application/pdf", "url": "/1654/1/00thesis.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Characterizing \u03b1-Synuclein Membrane Bound Structure", "author": [ { "family_name": "Lai", "given_name": "Bert Tsunyin", "clpid": "Lai-Bert-Tsunyin" } ], "thesis_advisor": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Winkler", "given_name": "Jay Richmond", "clpid": "Winkler-J-R" } ], "thesis_committee": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Richards", "given_name": "John H.", "clpid": "Richards-J-H" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "A feature of Parkinson's disease is the presence of fibrillar protein deposits composed mostly of \u03b1-synuclein and calcium ions in the brain\u2019s substantia nigra region. Although \u03b1-synuclein is natively unfolded, the N-terminal region of the protein is highly helical in the presence of membrane mimics, such as acidic phospholipid vesicles and SDS micelles. The C-terminal region of \u03b1-synuclein is known to bind to calcium ions and modulates aggregation. In this thesis, the structure of \u03b1-synuclein variants, incorporated with tryptophan and 3-nitrotyrosine as donor and energy acceptor pairs, have been characterized in the presence of SDS micelles, small unilammelar vesicles, and calcium ions by various techniques. Distance distributions extracted from time-resolved fluorescence energy-transfer measurements provide site-specific information on the protein conformations. In addition, similar studies using mutants linked to early onset Parkinson\u2019s disease were also performed to investigate the structural effect caused by these mutations. Furthermore, single tryptophan mutants have been designed as fluorescent reporters. The locations of these different tryptophan residues in the bilayer were probed by lipids labeled with bromine and dinitrophenol quenchers. Finally, preliminary studies of the intramolecular structure of \u03b1-synuclein aggregates have been carried out, while elucidation of intermolecular \u03b1-synuclein aggregate structures was made possible by the synthesis of new dyes that allow for long-range fluorescent energy transfer.\r\n", "doi": "10.7907/ZWH6-3B84", "publication_date": "2008", "thesis_type": "phd", "thesis_year": "2008" }, { "id": "thesis:5247", "collection": "thesis", "collection_id": "5247", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-08142007-151304", "primary_object_url": { "basename": "thesis2.pdf", "content": "final", "filesize": 7088139, "license": "other", "mime_type": "application/pdf", "url": "/5247/1/thesis2.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Ruthenium Olefin Metathesis Complexes: Catalyst Development and Mechanistic Studies", "author": [ { "family_name": "Anderson", "given_name": "Donde R.", "clpid": "Anderson-Donde-R" } ], "thesis_advisor": [ { "family_name": "Grubbs", "given_name": "Robert H.", "clpid": "Grubbs-R-H" } ], "thesis_committee": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Stoltz", "given_name": "Brian M.", "clpid": "Stoltz-B-M" }, { "family_name": "Bercaw", "given_name": "John E.", "clpid": "Bercaw-J-E" }, { "family_name": "Grubbs", "given_name": "Robert H.", "clpid": "Grubbs-R-H" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The field of olefin metathesis has grown to include organometallic chemists who strive to develop more efficient catalysts and to understand their mechanism of activity and decomposition, synthetic organic chemists who construct complex molecules utilizing existing catalysts and continually find challenging reactions in need of more efficient catalysts, and polymer chemists who utilize current catalysts to synthesize polymers with an ever-widening array of functional groups and structures in a controlled manner. This thesis describes the exploration of new ligands for olefin metathesis catalysts and the investigation of the model compounds of olefin metathesis reaction intermediates.
\r\n\r\nChapter 2 describes the synthesis, characterization, activity and kinetic selectivity of ruthenium olefin metathesis complexes bearing cyclic (alkyl)(amino)carbenes (CAACs). The activity of phosphine-free CAAC-ruthenium complexes is significantly affected by steric interactions. By decreasing the steric bulk of the ligand, a new catalyst with activity comparable to that of existing NHC-ruthenium (N-heterocyclic carbene) complexes has been synthesized. Additionally, these complexes exhibit unusual E/Z-diastereoselectivity and ethenolysis selectivity relative to previously studied NHC-ruthenium complexes.
\r\n\r\nChapter 3 describes the exploration of 3- and 6-membered carbenes as ligands for ruthenium olefin metathesis complexes. Stable silver-cyclopropenylidene adducts were synthesized and utilized as carbene transfer reagents in the presence of ruthenium precursors. Although good conversions were observed, isolation of cyclopropenylidene-ruthenium complexes was unsuccessful. Ruthenium complexes of 6-membered \u2018borazine\u2019-like carbenes were isolated, characterized and evaluated for ring-closing metathesis activity.
\r\n\r\nChapter 4 describes the development of a model system to study ruthenium-olefin complexes relevant to the mechanism of olefin metathesis. Upon addition of the ligand precursor 1,2-divinylbenzene to (H\u2082IMes)(py\u2082)(Cl)\u2082Ru=CHPh (H\u2082IMes = 1,3-dimesityl-4,5-dihydroimidazol-2-ylidene), two ruthenium-olefin adducts are formed. Based on \u00b9H NMR spectroscopy experiments and X-ray crystallographic analysis, the solution phase and solid-state structure of these complexes is assigned. Exploration of the generality of these observations through variation of the N-heterocyclic carbene ligand and the ligand precursor are also presented.
\r\n\r\nAppendix 1 describes the screening of transitional-metal salts and ligands for the non-oxidative hydration of styrene. Appendix 2 describes the investigation of a prior report of intramolecular olefin hydroalkoxylation with ruthenium, copper and silver salts. Appendix 3 describes the evaluation of chiral NHCs as ligands for ruthenium and rhodium hydrosilylation catalysts. Appendix 4 describes the investigation of tin(II) halides as ligands for ruthenium olefin metathesis catalysts. Appendix 5 contains X-ray crystallographic analysis parameters of the structures presented in this thesis.
\r\n", "doi": "10.7907/6B6N-2V77", "publication_date": "2008", "thesis_type": "phd", "thesis_year": "2008" }, { "id": "thesis:2266", "collection": "thesis", "collection_id": "2266", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05292008-230310", "primary_object_url": { "basename": "Finalthesis.pdf", "content": "final", "filesize": 1038907, "license": "other", "mime_type": "application/pdf", "url": "/2266/3/Finalthesis.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Methodologies for the Rapid Synthesis of Hexoses and Their Application Towards a Differentially-Protected Chondroitin Sulfate Tetrasaccharide", "author": [ { "family_name": "Saliba", "given_name": "Katie Rose", "clpid": "Saliba-Katie-Rose" } ], "thesis_advisor": [ { "family_name": "MacMillan", "given_name": "David W. C.", "clpid": "MacMillan-D-W-C" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" } ], "thesis_committee": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Parker", "given_name": "Carl Stevens", "clpid": "Parker-C-S" }, { "family_name": "MacMillan", "given_name": "David W. C.", "clpid": "MacMillan-D-W-C" }, { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Carbohydrates play many roles in biology, but their study has been hindered by the paucity of methods available to rapidly access hexoses. In 2004, the MacMillan laboratory published a two-step aldol methodology that allows access to the erythrohexoses allose, glucose, and mannose. Described herein is the development of two methodologies to access hexoses. First, the two-step aldol methodology for accessing the erythrohexoses was expanded to allow access to a differentially-protected mannosamine and gulose. Also described is the discovery of a one-step aldol methodology for accessing hexoses, which has allowed access to a protected allose and gulose.
\r\n\r\nThis methodology was applied to the synthesis of a differentially-protected chondroitin sulfate di- and tetrasaccharide. Chondroitin sulfate is a complex linear polysaccharide composed of alternating glucuronic acid and galactosamine residues that are heterogeneously sulfated. Combining the aldol methodology with a Cerny epoxide methodology developed in the Hsieh-Wilson laboratory, a core disaccharide was accessed. Model studies confirmed each position could be accessed selectively. Elaboration of this disaccharide to the protected tetrasaccharide was hindered by an unfavorable rearrangement during the tetrasaccharide coupling, so a second core disaccharide was synthesized. This core disaccharide was elaborated to a common intermediate to confirm that it should still allow selective access to each position, and then the disaccharide was elaborated towards the desired protected tetrasaccharide.
", "doi": "10.7907/HWFJ-P813", "publication_date": "2008", "thesis_type": "phd", "thesis_year": "2008" }, { "id": "thesis:2408", "collection": "thesis", "collection_id": "2408", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-06022008-092549", "primary_object_url": { "basename": "thesis_final_052708.pdf", "content": "final", "filesize": 18265663, "license": "other", "mime_type": "", "url": "/2408/13/thesis_final_052708.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "DNA-Mediated Charge Transport in DNA Repair", "author": [ { "family_name": "Boal", "given_name": "Amie Kathleen", "orcid": "0000-0002-1234-8472", "clpid": "Boal-Amie-Kathleen" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Parker", "given_name": "Carl Stevens", "clpid": "Parker-C-S" }, { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Lewis", "given_name": "Nathan Saul", "clpid": "Lewis-N-S" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The double-helical structure of deoxyribonucleic acid (DNA) imparts upon this important biological molecule both the ability to store genetic information within a cell and also the capacity to serve as medium for charge transport. DNA-mediated charge transport is now a very well-studied phenomenon but biological roles for these reactions have not been explored. It has been demonstrated that DNA-mediated charge transport can funnel oxidative DNA damage to sites of low oxidation potential in a number of biologically relevant environments ranging from reconstituted nucleosome core particles, to isolated nuclei and mitochondria from HeLa cells. DNA-mediated charge transport may also play a role in transcriptional activation or repression as modulated by redox-active transcription factors. Here we examine how DNA-mediated charge migration could also provide a pathway for protein-protein communication among DNA repair enzymes, a pathway that might serve as a scheme for rapid lesion detection inside the cell.", "doi": "10.7907/CMWB-6C92", "publication_date": "2008", "thesis_type": "phd", "thesis_year": "2008" }, { "id": "thesis:4186", "collection": "thesis", "collection_id": "4186", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-10192007-190231", "primary_object_url": { "basename": "Jordan_Katz_Thesis2007.pdf", "content": "final", "filesize": 10752286, "license": "other", "mime_type": "application/pdf", "url": "/4186/1/Jordan_Katz_Thesis2007.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Metal Oxide-Based Photoelectrochemical Cells for Solar Energy Conversion", "author": [ { "family_name": "Katz", "given_name": "Jordan E.", "orcid": "0000-0002-6242-2124", "clpid": "Katz-Jordan-E" } ], "thesis_advisor": [ { "family_name": "Lewis", "given_name": "Nathan Saul", "clpid": "Lewis-N-S" } ], "thesis_committee": [ { "family_name": "Okumura", "given_name": "Mitchio", "clpid": "Okumura-M" }, { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Lewis", "given_name": "Nathan Saul", "clpid": "Lewis-N-S" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "In order to address the need for CO2-free energy, recent trends in global CO2 emissions and energy production are analyzed, and the photoelectrochemical properties of two types of metal oxide-based solar cells are presented.
\r\n\r\nThe effects of potential-determining cations (Li+, H+) in the electrolyte of TiO2-based dye-sensitized solar cells, using Ru(H2L\u2019)2(NCS)2, where H2L\u2019 is 4,4\u2019-dicarboxylic acid-2,2\u2019bipyridine, as a sensitizer was investigated using current density vs potential (J-E), spectrochronocoulometric, and spectroscopic methods. Photoelectrochemical cells with lower concentrations of the cations Li+ and H+ had increased open-circuit voltages (Voc), and decreased short-circuit current densities (Jsc). Spectrochronocoulometric methods indicated that the energy of states in TiO2 shifted by approximately -1 V when in contact with electrolytes lacking small cations. Spectral response measurements indicated that the loss of photocurrent was accompanied by a nearly monotonic drop in the external quantum yield across all wavelengths.
\r\n\r\nTransient absorption spectroscopy was used to measure the kinetics of interfacial electron transfer of the same system. No dependence was observed on the ultrafast dynamics of electron injection on cations used in ClO4--based solutions. However, in solutions of TBA+ with I3-/I-, femtosecond, but not picosecond, dynamics were observed. In contrast, for solutions with Li+ and ClO4-, I- or I-/I3-, both femtosecond and picosecond dynamics were observed. Nanosecond-resolved spectroscopy results show that the absence of small cations did not affect the rate of recombination, while the regeneration rate of [RuIII(H2L\u2019)2(NCS)2]+ was decreased. Results indicate that both the ground and excited state reduction potentials of the sensitizer shift as a function of small cations in solution, along with the energy of states in TiO2. The efficiency of electron injection is thus largely unchanged; rather a decrease in the regeneration rate accounts for the loss of Jsc.
\r\n\r\nFinally, a novel, high-throughput, combinatorial approach for the synthesis and screening of mixed-metal oxides for use as water-splitting photocatalysts was developed. The methodology relies on inkjet printing to form quantitative mixtures of aqueous metal oxide precursors. After pyrolysis, the photoelectrochemical properties of metal oxides can be fully characterized in an automated high-speed system, including measurement of the Voc and J-E curves.
\r\n", "doi": "10.7907/29VB-HV45", "publication_date": "2008", "thesis_type": "phd", "thesis_year": "2008" }, { "id": "thesis:2763", "collection": "thesis", "collection_id": "2763", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-06282007-105808", "type": "thesis", "title": "DNA-Mediated Hole and Electron Transport", "author": [ { "family_name": "Shao", "given_name": "Fangwei", "orcid": "0000-0003-2007-3920", "clpid": "Shao-Fangwei" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" }, { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Marcus", "given_name": "Rudolph A.", "clpid": "Marcus-R-A" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Since the elucidation of the double helical structure of DNA, it has been proposed that the dynamic [pi]-stacking base pair array may mediate charge migration, hole transport (HT), and electron transport (ET). In this thesis work, both DNA-mediated HT and ET are investigated to explore their mechanisms by using kinetically fast electron/hole traps: cyclopropylamine-substituted bases, especially N4-cyclopropylcytosine (CPC), and N2-cyclopropylguanine (CPG). Both biochemical reaction with a variety of photooxidants and electrochemistry show that the modified bases, CPC and CPG, have similar redox properties as the natural DNA bases and are irreversible kinetic traps by ring opening on the picosecond time scale.
\r\n\r\nIn DNA assemblies containing either [Rh(phi)2(bpy')]3+ (Rh) or an anthraquinone derivative (AQ), two high energy photooxidants, appreciable oxidative damage at a distant CPC is observed, which shows that hole migration must involve also the higher energy pyrimidine bases. The damage yield is modulated by lower energy guanine sites on the same or complementary strand. Significantly, the efficiency in trapping at CPC is similar to that at flanking CPG. Thus, HT is not simply a function of the relative energies of the isolated bases, but instead may require orbital mixing among the bases. Hole migration through DNA involves occupation of all the DNA bases with radical delocalization.
\r\n\r\nThe oxidation of CPC via distant photooxidants has been found also to be sensitive to intervening structure and sequences. AQ-modified DNA assemblies of identical base composition but different base sequence have been probed. Single and double base substitutions within A-tracts modulate CPC decomposition. In fact, the entire sequence within the DNA assembly is seen to govern CPC oxidation, not simply the bases intervening between CPC and the tethered photooxidant.
\r\n\r\nThese data are reconciled in a mechanistic model of conformationally gated hole transport through delocalized DNA domains. Oxidation of CPG separated from a tethered photooxidant by A-tracts with a series of lengths over 50 A exhibits a nonmonotonically periodic distance dependence and shows that the domain sizes in the A-tract is 4-5 base pairs. Sequence-dependent DNA structure and dynamics are essential to the transient formation of the domains and hole propagation among the domains. This dynamic, delocalized model provides a basis to reconcile and exploit DNA HT chemistry.
\r\n\r\nJust as long-range hole transport through DNA has now been established, DNA-mediated electron transport has not been as well characterized. Three iridium complexes have therefore been designed in order to initiate both photooxidative and photoreductive reaction of DNA and allow direct comparison between the two. Redox potentials of excited Ir complexes are determined by both triplet energy (E0-0) and ground state redox potentials. Two of the iridium complexes prepared have excited state potentials that are suffcient to oxidize purines, but not pyrimidines. The excited state oxidation potentials of three Ir complexes are around -1.0 V and would be able to reduce DNA pyrimidines. Both CPC and CPG in DNA can be decomposed by photoirradiation with the noncovalently bound iridium complexes. In particular, two of the complexes have the potential to probe oxidation of purines and reduction of pyrimidines in DNA.
\r\n\r\nStudies were also conducted using one of the iridium complexes covalently tethered to DNA oligonucleotides. Hence the metal complex serves as both a photooxidant and photoreductant in the study of DNA-mediated hole and electron transport. In the Ir-tethered DNA assemblies, a metal complex stabilizes the DNA duplex through its intercalative, functionalized dppz ligand. Cyclopropylamine-substituted bases, CPC and CPG, are used as kinetic fast electron and hole traps to probe the resulting charge migration processes after direct photoirradiation of the assemblies. Reductive decomposition of CPC via ET as well as the oxidation of CPG via HT is observed. Thus, the iridium tethered DNA containing cyclopropylamine-substituted bases provides a unique model system to explore the two DNA-mediated charge transport processes through the same DNA bridges. For the first time, ET and HT can be initialized by the same photoredox probe employing the identical electronic interaction mode with DNA.
\r\n\r\nA flash quench technique was also applied to Iridium-tethered DNA in order to generate the ground state photoreductant and initiate photoreduction using 5'-bromo-uridine (BrU) as the electron trap. Efficiencies of BrU reduction in Ir-DNA upon flash quench technique was found to be comparable to that of CPG oxidation upon direct photoirradiation of Ir-DNA. Furthermore, in Ir-tethered DNA assemblies containing CPG or BrU as either the hole or electron trap, the sequence dependence of HT versus ET through an A-tract was examed. When CPG and BrU are placed in either purine or pyrimidine strands in A-tract, decomposition of both modified bases are observed. Thus, transient electron occupancy during ET, as well as hole occupancy during HT, are distributed onto both purine and pymidine strands in A-tract. Additionally, BrU decomposes in a more efficient fashion when it is located on a thyime-containing strands, which indicates that DNA-mediated ET prefers to pyrimidine strands rather than purine strands.
\r\n", "doi": "10.7907/VZ0J-W403", "publication_date": "2008", "thesis_type": "phd", "thesis_year": "2008" }, { "id": "thesis:3337", "collection": "thesis", "collection_id": "3337", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-09052006-214421", "type": "thesis", "title": "Targeting Human Telomerase RNA via Biochemical and in vitro Selection Methods", "author": [ { "family_name": "Ueda", "given_name": "Christine Terumi", "clpid": "Ueda-Christine-Terumi" } ], "thesis_advisor": [ { "family_name": "Roberts", "given_name": "Richard W.", "clpid": "Roberts-R-W" } ], "thesis_committee": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Dervan", "given_name": "Peter B.", "clpid": "Dervan-P-B" }, { "family_name": "Deshaies", "given_name": "Raymond Joseph", "clpid": "Deshaies-R-J" }, { "family_name": "Roberts", "given_name": "Richard W.", "clpid": "Roberts-R-W" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Telomerase is an enzyme responsible for the maintenance of eukaryotic chromosome ends. The two main components required for activity are a protein subunit, the human telomerase reverse transcriptase (hTERT), and an RNA subunit, the human telomerase RNA (hTR). While telomerase is not active in most normal human cells, roughly 85% to 90% of oncogenic cells display increased telomerase activity. An understanding of the biochemistry of telomerase will aid in the development of molecules that will lead to antitumor-specific therapies. The first part of the work presented here describes a biochemical analysis of an RNA-RNA interaction between two catalytically important domains of hTR. The interactions were characterized via mobility-shift assay, mutation analysis, and UV cross-linking experiments. The data argue for a revised model for the structure of hTR, and point to possible three-dimensional contacts present in the telomerase complex. The next part of this thesis describes an in vitro selection against the catalytically important RNA stem-loop P6.1 in hTR. The in vitro selection was performed using mRNA display, which allows us to isolate RNA-binding peptides from libraries containing trillions of unique sequences. Unexpectedly, the selected peptide binds with high affinity and specificity to the relatively rare dimeric form of the P6.1 stem-loop rather than the more abundant monomeric conformation. Characterization of this novel RNA-peptide interaction was performed via circular dichroism, steady-state fluorescence, mobility-shift assay, and surface plasmon resonance. The data highlight the power of mRNA display to isolate high affinity ligands from large libraries of molecules.", "doi": "10.7907/2NK8-KK06", "publication_date": "2007", "thesis_type": "phd", "thesis_year": "2007" }, { "id": "thesis:1580", "collection": "thesis", "collection_id": "1580", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05022007-055147", "primary_object_url": { "basename": "thesisfinal.pdf", "content": "final", "filesize": 1380455, "license": "other", "mime_type": "application/pdf", "url": "/1580/1/thesisfinal.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Interfacial Electron-Transfer Reactions at Semiconductor Electrodes", "author": [ { "family_name": "Hamann", "given_name": "Thomas William", "orcid": "0000-0001-6917-7494", "clpid": "Hamann-Thomas-William" } ], "thesis_advisor": [ { "family_name": "Lewis", "given_name": "Nathan Saul", "clpid": "Lewis-N-S" } ], "thesis_committee": [ { "family_name": "Lewis", "given_name": "Nathan Saul", "clpid": "Lewis-N-S" }, { "family_name": "Blake", "given_name": "Geoffrey A.", "clpid": "Blake-G-A" }, { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Differential capacitance versus potential and current density vs. potential measurements were used to determine the energetics and kinetics, respectively, of the interfacial electron-transfer processes of n-type ZnO electrodes in contact with aqueous solutions. The electron-transfer rate constant, ket, vs. driving force was investigated employing a series of non-adsorbing, one-electron, outer-sphere redox couples with formal reduction potentials spanning approximately 900 mV in the band-gap region. The data were well-fit by a parabola generated using classical Marcus theory with a reorganization energy, \u03bb, of 0.67 eV. The dependence of ket on \u03bb was determined using a series of compounds with similar formal reduction potentials, but reorganization energies that span approximately 1 eV. The interfacial electron-transfer rate constant decreases as the reorganization energy of the acceptor species increases and a plot of the logarithm of the electron-transfer rate constant vs. (\u03bb+\u0394G0'2/4\u03bbKBT is linear with a slope of \u2248-1. Changes in solution pH were used to shift the band-edge positions of ZnO electrodes relative to solution-based electron acceptors having pH-independent redox potentials. This strategy allowed investigation of the pH-induced driving-force dependence of ket in the normal and inverted regions. It was further found that introduction of the tert-butyl functionality on osmium tris-bipyridyl decreased the self-exchange rate constant, determined from NMR line-broadening measurements, by a factor of 50 and the interfacial electron-transfer rate constant by 100, compared to that of the analogous methyl-substituted complex. The results indicate that the tert-butyl group can act as a spacer on an outer-sphere redox couple to significantly decrease the electronic coupling of the electron-transfer reaction both in self-exchange and interfacial electron-transfer processes. Methyl-terminated, n-type, (111)-oriented Si surfaces in contact with an electron acceptor having a pH-independent redox potential were used to verify that the band edges of the modified Si electrode were fixed with respect to changes in solution pH. These results, taken together, provide strong evidence that interfacial electron-transfer rate constants at semiconductor electrodes are in excellent agreement with the predictions of a Marcus-type model of interfacial electron-transfer reactions.", "doi": "10.7907/mc8e-x717", "publication_date": "2007", "thesis_type": "phd", "thesis_year": "2007" }, { "id": "thesis:2024", "collection": "thesis", "collection_id": "2024", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05242007-151027", "primary_object_url": { "basename": "FullThesis.pdf", "content": "final", "filesize": 9456838, "license": "other", "mime_type": "application/pdf", "url": "/2024/7/FullThesis.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Silicon Nanowires as Biological Sensors and Highly Efficient Thermoelectric Materials", "author": [ { "family_name": "Bunimovich", "given_name": "Yuri Leonid", "orcid": "0000-0002-7920-8781", "clpid": "Bunimovich-Yuri-Leonid" } ], "thesis_advisor": [ { "family_name": "Heath", "given_name": "James R.", "clpid": "Heath-J-R" } ], "thesis_committee": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Heath", "given_name": "James R.", "clpid": "Heath-J-R" }, { "family_name": "Beauchamp", "given_name": "Jesse L.", "clpid": "Beauchamp-J-L" }, { "family_name": "Roukes", "given_name": "Michael Lee", "clpid": "Roukes-M-L" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Silicon nanowires are of significant interest because of their novel properties which afford new functions. Here, we study silicon nanowires fabricated via a well established top down approach called superlattice nanowire pattern transfer (SNAP). In the first part of the thesis, nanowires are utilized for biological sensing of DNA and proteins in an electrolyte solution. Important electronic and surface properties are considered as means to optimize the device sensitivity. The removal of silicon-oxide interface is shown to improve the limit of detection by two orders of magnitude. The sensitivity can be further improved by the reduction of the doping level to 1017 cm-3. In this way, sub-femtomolar concentration of oligonucleotides in physiological conditions can be detected. While the Debye screening is circumvented by the electrostatic adsorption of primary DNA on the amine-terminated monolayer, the detection of proteins is limited by the size of the antibodies. In low ionic strength solution, ~10\u00b5M, human IL2 cytokine is detectable at 1 to 10pM concentrations. Furthermore, a model is developed which allows the determination of kinetic parameters and absolute analyte concentrations from the real-time resistance of the nanowires. This model is consistent with Langmuir model, and could, in principle, be used to determine the amount of low abundance biological molecules at concentrations below those detectable with other label-free methods, such as surface plasmon resonance technique. In addition, a novel electrochemical technique is developed which allows the spatially-selective functionalization of silicon nanowires and the construction of a small library of proteins. In the second part, the discovery of highly efficient thermoelectric materials based on silicon nanowires is discussed. A relatively simple, scalable, and single component system of silicon nanowires with figure of merit of ~1 at room temperature is developed. ZT can be tuned at various temperatures to exceed unity by varying nanowire size and/or impurity doping level. Such enhancement in ZT compared to the bulk value is achieved by significantly perturbing the phonon-mediated heat transport in a nanowire. Decreased thermal conductivities and longer lifetimes of long-wavelength phonons in a nanowire are major reasons for an increased thermoelectric efficiency of these structures.", "doi": "10.7907/EY6H-XK94", "publication_date": "2007", "thesis_type": "phd", "thesis_year": "2007" }, { "id": "thesis:2205", "collection": "thesis", "collection_id": "2205", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05282007-225441", "primary_object_url": { "basename": "keathesis75.pdf", "content": "final", "filesize": 2877981, "license": "other", "mime_type": "application/pdf", "url": "/2205/1/keathesis75.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Fundamental Mechanisms and Biological Applications of DNA-Mediated Charge Transport", "author": [ { "family_name": "Augustyn", "given_name": "Katherine Emily", "clpid": "Augustyn-Katherine-Emily" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" }, { "family_name": "Collier", "given_name": "C. Patrick", "orcid": "0000-0002-8198-793X", "clpid": "Collier-C-P" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The Pi-stacked array of heterocylic aromatic DNA base pairs provides an intriguing medium for facilitating the transport of migrating charges. The mechanism of hole transport through this dynamic molecule has been extensively investigated using a wide range of techniques. In particular, our group has taken advantage of the octahedral metal complexes of rhodium (III) and ruthenium (II) to probe charge transport reactions through DNA at long range. These intercalating photooxidants, which are extremely well coupled to the DNA ?-stack, can provide us with mechanistic information through a variety of biochemical and spectroscopic techniques. Here we continue to investigate the mechanism of DNA-mediated charge transport on fast time scales using a variety of hole traps and photooxidants and examine this interesting chemistry in a biological context. DNA-mediated charge transport across three different adenine tracts lengths is monitored using a probe interior to the bridge, N6-cyclopropyladenine, CPA. Upon oxidation, the cyclopropylamine-subsituted deoxynucleoside decomposes rapidly, and the efficiency of decomposition can be used as a kinetically fast measurement of hole occupancy. This trap, incorporated serially across the bridge, can be oxidized by a distally bound photooxidant, [Rh(phi)2(bpy\u2019)]3+ (phi = 9,10-phenanthrenequinone diimmine) without significant attenuation in yield over a distance of 5 nm. These results are consistent with complete delocalization across the DNA bridge. Photooxidation of N2-cyclopropylguanine, CPG, within duplex DNA is used to probe DNA charge transport reactions initiated by the covalently bound photooxidants, [Rh(phi)2(bpy\u2019)]3+ and anthraquinone. Duplexes containing the photooxidant separated from the CPG trap by an increasing number of intervening bases are examined in order to probe DNA charge transport reactions with this kinetically fast hole trap as a function of distance and sequence. Charge transport events through sequences containing various length adenine tracts as well as most mixed sequence bridges do not simply decay exponentially nor geometrically as a function of distance. In particular, for variable-length A-tracts, decomposition decreases in a periodic fashion with increasing distance between the photooxidant and the trap; the period is ~4-5 base pairs. Results obtained from charge injection studies using 2-aminopurine as a fluorescent probe have shown a similar periodic distance dependence. These periodicities are not observed in measurements of oxidative DNA damage using double guanine sites as a slow, irreversible hole trap. Thus, CT through DNA must be probed on multiple time scales to provide mechanistic information. These results are consistent with our model for DNA CT through transient delocalized DNA domains defined by sequence-dependent base pair dynamics. While mechanistic investigations are critical for a fundamental understanding of how charges migrate through DNA, it is important to consider the biological consequences of this process. A biological role for DNA-mediated CT has been investigated in the context of the transcription factor, p53, a tumor suppressor protein involved in myriad cellular pathways such as apoptosis and growth arrest. DNA assemblies containing an anthraquinone photooxidant tethered to the 5\u2019 end of sequences containing p53 binding sites were constructed to examine the binding affinity as a function of photooxidation. We demonstrate that through photoinduced DNA-mediated CT, the p53 protein becomes oxidized and exhibits differential binding for various promoter sequence including Gadd45, p21, and Mdm2. Additionally, insertion of a mismatch intervening between the photooxidant and the p53 binding site serves to attenuate this change in binding affinity associated with photooxidation. MALDI-TOF mass spectrometric analysis of p53 tryptic digests following irradiation of the DNA bound protein provides further evidence that a chemical change occurs, consistent with oxidation of a cysteine residue in the DNA binding domain. Dipyridophenazine complexes of ruthenium (II) have been used extensively to spectroscopically investigate DNA-mediated charge transport. A novel tris heteroleptic dipyridophenazine complex of ruthenium (II), [{Ru(phen)(dppz)(bpy\u2019-his)}{Ru(NH3)5}]5+, containing a covalently tethered ruthenium pentaammine quencher coordinated through a bridging histadine has been synthesized and characterized spectroscopically and biochemically in a DNA environment and in organic solvent. Capable of undergoing intramolecular photoinduced electron transfer, the steady-state and time-resolved luminescence measurements indicate that the tethered-quencher complex is quenched relative to the parent complexes [Ru(phen)(dppz)(bpy\u2019]2+ and [Ru(phen)(dppz)(bpy\u2019-his)]2+ in DNA and acetonitrile. Intercalated into guanine containing DNA, [{Ru(phen)(dppz)(bpy\u2019-his)}{Ru(NH3)5}]5+, upon excitation and intramolecular quenching, is capable of injecting charge into the duplex as evidenced by EPR detection of guanine radicals. DNA-mediated charge transport is also evidenced using a kinetically fast cyclopropylamine-substituted base as a hole trap that undergoes irreversible oxidative ring opening on the picosecond time scale. Guanine oxidation is not observed in measurements using guanine radical as a slow, irreversible hole trap indicating that back electron transfer reactions are competitive with hole injection into the duplex. Moreover, transient absorption measurements reveal a novel photophysical reaction pathway for [{Ru(phen)(dppz)(bpy\u2019-his)}{Ru(NH3)5}]5+ in the presence of DNA, competitive with the intermolecular flash-quench process. These results illustrate the remarkable redox chemistry occurring within a bimolecular ruthenium complex intercalated in duplex DNA.", "doi": "10.7907/AYHS-7Q13", "publication_date": "2007", "thesis_type": "phd", "thesis_year": "2007" }, { "id": "thesis:2245", "collection": "thesis", "collection_id": "2245", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05292007-061922", "primary_object_url": { "basename": "14_PRE_CHAPTERS.pdf", "content": "final", "filesize": 214398, "license": "other", "mime_type": "application/pdf", "url": "/2245/14/14_PRE_CHAPTERS.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Refolding a \u03b2-Barrel Membrane Protein\r \r ", "author": [ { "family_name": "Arjara", "given_name": "Gitrada", "clpid": "Arjara-Gitrada" } ], "thesis_advisor": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Richards", "given_name": "John H.", "clpid": "Richards-J-H" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Richards", "given_name": "John H.", "clpid": "Richards-J-H" }, { "family_name": "Clemons", "given_name": "William M.", "clpid": "Clemons-W-M" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The field of membrane protein folding is relatively new compared to soluble protein folding. This thesis describes spectroscopy investigations of the refolding and dynamics of a \u03b2-barrel membrane protein. The amphiphilic, \u03b2-barrel outer membrane protein A (OmpA) refolds and inserts directly into a lipid vesicle or micelle from a denatured state in aqueous urea solution. Spectroscopic probes used to study this system are native tryptophans located at positions 7, 15, 57, 102, and 143. Steady-state and time-resolved fluorescence measurements were performed using single tryptophan mutants of full-length OmpA (325 residues) and the truncated, transmembrane domain (176 residues). Both full-length and truncated mutants exhibit similar tryptophan emission lifetimes, suggesting that the transmembrane microenvironment is not greatly perturbed by the presence of the C-terminus.
\r\n\r\nWhile the microenvironments of folded full-length and truncated OmpA appear similar, the dynamics of refolding at each tryptophan position exhibit subtle differences when the C-terminus is present. Specifically, we observe that tryptophan-102, which faces the pore interior, inserts and folds the fastest while tryptophan-7, which does not cross the bilayer, is the slowest. Fluorescence anisotropy decays also indicate that tryptophan-7 is the most flexible residue compared to the other tryptophans. Temperature studies below the lipid gel-liquid transition temperature have also been performed. In the lipid gel phase, OmpA adsorbs to the surface of the vesicles but contains immediate \u03b2-sheet structure upon folding as well as very hydrophobic tryptophan environments. It is still uncertain from ensemble measurements whether this species is a true intermediate.
\r\n\r\nFluorescence energy transfer kinetics have successfully determined the intramolecular distance between tryptophan-7 and cysteine-175 labeled with a dansyl fluorophore. These results reveal that the barrel ends of OmpA come into contact early in the refolding process and remain close together up to the final assembly of the barrel. We also have evidence that the adsorbed species at low temperatures is not an intermediate in the folding pathway since no energy transfer is observed for this species. These spectroscopic investigations have provided the foundation for further fundamental studies to dissect the molecular mechanism of the folding pathway of OmpA as well as other integral membrane proteins.
", "doi": "10.7907/4PSM-AS02", "publication_date": "2007", "thesis_type": "phd", "thesis_year": "2007" }, { "id": "thesis:2250", "collection": "thesis", "collection_id": "2250", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05292007-183523", "primary_object_url": { "basename": "Title.pdf", "content": "final", "filesize": 97822, "license": "other", "mime_type": "application/pdf", "url": "/2250/15/Title.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "Identification and Functional Analysis of O-G1cNAc Glycosylation on the Transcription Factor cAMP-Response Element Binding Protein", "author": [ { "family_name": "Lamarre-Vincent", "given_name": "Nathan", "clpid": "Lamarre-Vincent-Nathan" } ], "thesis_advisor": [ { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" } ], "thesis_committee": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" }, { "family_name": "Parker", "given_name": "Carl Stevens", "clpid": "Parker-C-S" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The survival and development of organisms requires the ability of cells to communicate with the environment and with surrounding cells. This demand has led to the evolution of a number of methods used for communication. Chief among these is the ability to modify protein function with post-translational modifications (PTMs). PTMs allow cells to use a single protein for a variety of tasks and link protein activity with a specific environmental or cellular cue. Modification of transcription factors has arisen as a key model for the study of PTMs and their effects on cell processes. PTMs modulate transcriptional activity required for key processes such as development, differentiation and cell survival.
\r\n\r\nThe eukaryotic transcription factor cAMP-response element binding protein (CREB) is a transcription factor that confers dynamic control of a number of cellular processes including neuronal and pancreatic cell survival, gluconeogenesis and neuronal long-term potentiation. CREB is activated by phosphorylation of single serine residue. The observation that a number of kinase signaling cascades converge on CREB has led to the question of how cells deal with the apparent loss of signal identity that occurs as a result of this convergence. In this thesis I describe the identification, characterization and functional analysis of a novel PTM of CREB, O-GlcNAc glycosylation, that provides an additional level of control of CREB activity. CREB glycosylation moderates phosphorylation-dependent CREB activity and reduces CREB-dependent gene expression in pancreatic [beta]-cells, and as a result promotes [beta]-cell death, as observed in type II diabetes. CREB glycosylation offers us an example of how cells use multiple PTMs to control protein function and how dysfunction in the regulation of these modifications may contribute to disease states.
", "doi": "10.7907/ZEXB-6889", "publication_date": "2007", "thesis_type": "phd", "thesis_year": "2007" }, { "id": "thesis:2974", "collection": "thesis", "collection_id": "2974", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-07242006-134454", "primary_object_url": { "basename": "FullThesis.pdf", "content": "final", "filesize": 4851131, "license": "other", "mime_type": "application/pdf", "url": "/2974/7/FullThesis.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Application of Iminium Activation Technologies To Natural Product Synthesis: Total Synthesis of the Spiculisporic Acids, Progress Towards the Total Synthesis of Cylindrocyclophane F, and Formal Synthesis of Cylindrocyclophane A", "author": [ { "family_name": "Goodwin", "given_name": "Nicole Cathleen", "orcid": "0000-0002-6093-7175", "clpid": "Goodwin-Nicole-Cathleen" } ], "thesis_advisor": [ { "family_name": "MacMillan", "given_name": "David W. C.", "clpid": "MacMillan-D-W-C" } ], "thesis_committee": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "MacMillan", "given_name": "David W. C.", "clpid": "MacMillan-D-W-C" }, { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The first enantioselective, catalytic vinylogous Mukaiyama-Michael reaction of siloxyfurans with simple alpha,beta-unsaturated aldehydes has been reported using chiral imidazolidinones. This methodology provides access to enantioenriched gamma-butenolides, a privileged motif in organic synthesis. The utility of this organocatalytic Mukaiyama-Michael reaction was highlighted by the total syntheses of (--)-spiculisporic acid and (--)-5-epi-spiculisporic acid.
\r\n\r\nInvestigations into the total syntheses of cylindrocyclophanes A and F necessitated the development of a novel B-alkyl Suzuki cross-coupling of trimethylanilinium salts using a nickel(0) catalyst and bulky phosphine ligand. This methodology study revealed a very competitive nickel-catalyzed demethylation pathway, which produced dimethylaniline byproducts. A possible explanation for this side reaction is discussed. This technology was applied to a dimerization strategy for the C\u2082-symmetric cylindrocyclophane F. Synthesis of a dimerization precursor included an enantioselective organocatalytic 1,4-addition of 3,5-dimethoxy-N,N-dimethylaniline into an \u03b1,\u03b2-unsaturated aldehyde. However, the B-alkyl Suzuki cross-coupling was unsuccessful in promoting a dimerization.
\r\n\r\nNext, the synthesis of cylindrocyclophane A was explored using an alternative ring-closing metathesis dimerization strategy. A dimerization precursor was to be assembled via the cross-coupling of trimethylanilinium salts with potassium (vinyl)trifluoroborate salts, whose syntheses featured an organocatalytic 1,4-conjugate reduction of a \u03b2,\u03b2-disubstituted enal. This cross-coupling strategy revealed olefin isomerization as a major side-reaction in the nickel-catalyzed Suzuki dimerization, making this route a non-productive approach to the natural product.
\r\n\r\nLastly, formal synthesis of cylindrocyclophane A was accomplished using (i) a nickel-catalyzed Stille cross-coupling of an activated vinyl stannane with a judiciously chosen trimethylanilinium salt and (ii) an asymmetric palladium-catalyzed allylic alkylation of an acyclic ketone. The latter represents the first example of application of the Pd\u2082(dba)\u2083/t-Bu-PHOX catalyst system to effect an asymmetric allylic alkylation on an acyclic system with good stereoselectivity. This route constituted a formal synthesis of cyclindrocyclophane A in eight linear steps, making it more efficient than the published route to the same advanced intermediate reported by Smith, which was synthesized in eleven steps.
", "doi": "10.7907/C7D7-XN63", "publication_date": "2007", "thesis_type": "phd", "thesis_year": "2007" }, { "id": "thesis:3300", "collection": "thesis", "collection_id": "3300", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-08312006-131856", "primary_object_url": { "basename": "Thesis.pdf", "content": "final", "filesize": 6311994, "license": "other", "mime_type": "application/pdf", "url": "/3300/1/Thesis.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Electrochemical Studies of Electron Transfer in DNA Films with Covalently Tethered Intercalators", "author": [ { "family_name": "Liu", "given_name": "Tao", "clpid": "Liu-Tao" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Lewis", "given_name": "Nathan Saul", "clpid": "Lewis-N-S" }, { "family_name": "Collier", "given_name": "C. Patrick", "clpid": "Collier-C-P" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The base stack within double-helical DNA provides an efficient pathway for charge transport. This DNA-mediated charge transport chemistry has been shown to be exquisitely sensitive to minor perturbations in DNA structure and base stacking both in solution and on surfaces. As a result, electrochemical studies on DNA-modified electrodes may provide a novel approach to the development of sensitive, but inexpensive DNA sensing devices. Using intercalated, covalently bound daunomycin as a redox probe, we have examined ground state charge transport in DNA films on gold electrodes. DNA-mediated electron transfer is found to occur over a distance as long as 100 \u00c5 in the film. Moreover, while the introduction of one or even two breaks in the sugar-phosphate backbone yields no detectable effect on electron transfer, a CA base-pair mismatch significantly attenuates the electron transfer yield. These results confirm that the base pair stack is the pathway for DNA mediated charge transfer, not the sugar-phosphate backbone. Based on these studies, we have developed a method to electrochemically monitor the trapping of double- stranded DNA with a 6-base overhang on a gold electrode modified with double- stranded DNA probes containing a complementary overhang. The trapping of the double-stranded target can be monitored by the reduction of daunomycin crosslinked to the target. A CA mismatch in the target duplex can also be detected by the diminished reduction signal.
\r\n\r\nIt has been shown that the electronic coupling between an intercalator and the pi-stack of DNA is critically important for the reduction of the intercalator through DNA-mediated charge transport. Using covalently tethered anthraquinone derivatives as the redox probe, we have investigated also the influence of the binding mode of the intercalator on its reduction in DNA films. The results of these studies underscore the importance of direct interaction between the redox probe and the pi-stack in order to observe efficient DNA-mediated electrochemistry through DNA films. These studies have also shown that the covalent linkage has a significant effect on the intercalation of the probe to the base stack of DNA.
\r\n\r\nIn an effort to develop a redox probe that has effective electronic coupling with the pi-stack while being covalently tethered to a DNA strand with a stable linkage, we have crosslinked Nile blue, a redox active DNA intercalator with high DNA binding affinities, to the 5\u2019-end of oligonucleotides. The covalently tethered Nile blue is shown to be sensitive probe for DNA-mediated electron transfer on the gold surface. An intervening CA mismatch has been detected in both tightly packed and loosely packed films of DNA\u2013Nile blue conjugate. We have also coupled the reduction of Nile blue to an electrocatalytic cycle involving freely diffusing ferricyanide, which significantly enhances the sensitivity to intervening mismatches. Furthermore, using Nile blue as the covalently tethered probe, we have developed a method for DNA mismatch detection that eliminates sample modification and has a potential for high throughput assays. These studies may provide a practical approach to diagnostic devices for mutation detection with high sensitivity and low expense.
", "doi": "10.7907/MMSW-4A41", "publication_date": "2007", "thesis_type": "phd", "thesis_year": "2007" }, { "id": "thesis:3788", "collection": "thesis", "collection_id": "3788", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-09262006-134259", "primary_object_url": { "basename": "YHN-Thesis.pdf", "content": "final", "filesize": 8579300, "license": "other", "mime_type": "application/pdf", "url": "/3788/1/YHN-Thesis.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Wiring Inducible Nitric Oxide Synthase", "author": [ { "family_name": "Nguyen", "given_name": "Yen Hoang Le", "clpid": "Nguyen-Yen-Hoang-Le" } ], "thesis_advisor": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" } ], "thesis_committee": [ { "family_name": "Dervan", "given_name": "Peter B.", "clpid": "Dervan-P-B" }, { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Winkler", "given_name": "Jay Richmond", "clpid": "Winkler-J-R" }, { "family_name": "Peters", "given_name": "Jonas C.", "clpid": "Peters-J-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The \"wires project\" in the Gray group has been focused on characterizing short-lived intermediates of Fe heme enzyme catalytic cycles by designing and synthesizing photosensitizers (wires) that bind to the protein active site with high affinity. The heart of this thesis is on rhenium channel binding and ruthenium surface binding wires for inducible nitric oxide synthase (iNOS). Binding and inhibition studies were conducted, electron transfer (ET) kinetics were studied, and iNOS catalytic activity was assayed for nitric oxide (NO) production.
\r\n\r\nBoth channel and surface binding wires bind to iNOS with low micro molar affinity. Channel binding wires bind at the active site, closely interacting with the protoporphyrin IX iron heme (Fe heme). Characteristic spectral shifts of Fe heme perturbation were observed. The surface binding wires bind presumably at the hydrophobic patch of the oxygenase domain where the reductase domain was proposed to dock during electron transfer processes. The surface binding wire interacts closely with the Fe heme from the surface of the protein, but still close to where spectral shifts of the Fe heme were observed; however, the surface binding wire does not displace other channel binding wires, indicative of a second binding site.
\r\n\r\nUpon photo-excitation of all rhenium wires, the resting state Fe(III) heme is reduced to Fe(II) heme in less than 10 ns, characterized by transient absorption spectroscopy. This ET rate is orders of magnitude faster than Fe(III) reduction by the reductase domain (kET = 1 s-1) under biological conditions. In some cases, the wires were observed to ligate the Fe(II), creating a six-coordinate Fe(II) complex. The fully coordinated Fe(II) species is prevented from binding oxygen, and the catalytic mechanism is terminated. Another electron cannot be injected, and there is no production of NO. In the cases where the wire was shorter, ligation of the Fe(II) species was not observed. The Fe(II) remains five-coordinate, leaving room for oxygen to bind and for the mechanism to continue. In this case, NOS catalytic activity was assayed for the production of NO by photo-excitation of the wires. Complications of photodecomposition of NO indicators presented a challenge in data analysis. It is possible that a very small amount of NO was produced by photo-excitation of the wire; unfortunately, nothing definitely can be concluded. A new method to assay for NOS catalytic activity was proposed.
\r\n\r\nBoth channel and surface binding wires led to many insights on substrate binding modes at the protein active site and on the surface; ET mechanisms were redefined, including amino acid radicals participating in electron transfer processes; and future directions for new wire were designed with hopes of accomplishing the long standing goal of characterizing high-valent Fe species.
", "doi": "10.7907/fgsb-4m91", "publication_date": "2007", "thesis_type": "phd", "thesis_year": "2007" }, { "id": "thesis:1836", "collection": "thesis", "collection_id": "1836", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05162006-201134", "primary_object_url": { "basename": "Thesis.pdf", "content": "final", "filesize": 2008856, "license": "other", "mime_type": "application/pdf", "url": "/1836/1/Thesis.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Novel Reactivity at Iron Centers Supported by Poly(phosphino)borate Ligands", "author": [ { "family_name": "Thomas", "given_name": "Christine Marie", "orcid": "0000-0001-5009-0479", "clpid": "Thomas-Christine-Marie" } ], "thesis_advisor": [ { "family_name": "Peters", "given_name": "Jonas C.", "clpid": "Peters-J-C" } ], "thesis_committee": [ { "family_name": "Stoltz", "given_name": "Brian M.", "clpid": "Stoltz-B-M" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Peters", "given_name": "Jonas C.", "clpid": "Peters-J-C" }, { "family_name": "Grubbs", "given_name": "Robert H.", "clpid": "Grubbs-R-H" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The reactivity of the iron(II) alkyl species [PhBPiPr3]FeMe ([PhBPiPr3] = PhB(CH2PiPr2)3-) towards Si-H bonds is presented. Reaction of [PhBPiPr3]FeMe with primary aryl silanes results in the unusual \u03b73 silane adducts [PhBPiPr3]Fe(H)(\u03b73-H2SiMeR). X-ray crystallography, Mossbauer spectroscopy, and theoretical calculations confirm this structural assignment; however, solution NMR experiments suggest a degree of fluxionality in solution.
\r\n\r\nLow valent, tris(phosphino)borate iron platforms have been shown to facilitate the activation of white phosphorus, P4. The iron(I) precursors {[PhBPiPr3]Fe}2(\u03bc-N2) and [PhBPPh3]Fe(PPh3) react with P4 to quantitatively generate {[PhBPiPr3]Fe}2(\u03bc-P4) and {[PhBPPh3]Fe}2(\u03bc-P4), respectively. These unique iron(II) dimers bridged by square P42- units have been characterized structurally and spectroscopically, and their reactivity has been examined. A simplified electronic structure calculation is presented to aid in discussion of bonding within these complexes.
\r\n\r\nMotivated by the versatility of the tris(phosphino)borate ligands, a new family of tripodal hybrid bis(phosphino)pyrazolylborate ligands, [PhBPtBu2(pz')]- ([PhBPtBu2(pz')]- = PhB(CH2PtBu2)2(pz')-), has been prepared and characterized. The synthesis, spectroscopy, and solid-state structures of four-coordinate, pseudo-tetrahedral iron(II) and cobalt(II) halide complexes supported by these ligands is presented. To compare the electron-releasing ability of these ligands with their [PhBPR3] analogues, the cyclic voltammetry of these complexes is introduced. Potential routes to a terminal cobalt or iron nitride complex via extrusion of N2 from coordinated azide and metathesis with the N-atom transfer reagent Li(dbabh) are investigated.
\r\n\r\nReduction of the [PhBPtBu2(pz')]MX halide complexes in the presence of excess phosphine generates low valent [PhBPtBu2(pz')]MI(PMe3) precursors. These precursors react with organic azides to generate cobalt(III) and iron(III) imides. Initial reactivity studies indicate that these imides are more moderately more reactive than the corresponding tris(phosphino)borate complexes. The electrochemistry of the [PhBPtBu2(pz')]FeIII(NR) imides features a quasi-reversible to fully reversible oxidation event, dependent on choice of pyrazolyl substituents and scan rate. This oxidation can be achieved chemically to generate the isolable cationic iron(IV) imides, [PhBPtBu2(pz')]FeIV(NR)+. The structural and spectroscopic characterization of these highly unusual complexes is discussed.
\r\n", "doi": "10.7907/AGQC-8E07", "publication_date": "2006", "thesis_type": "phd", "thesis_year": "2006" }, { "id": "thesis:3820", "collection": "thesis", "collection_id": "3820", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-09292005-152710", "primary_object_url": { "basename": "Thesis1.pdf", "content": "final", "filesize": 4307223, "license": "other", "mime_type": "application/pdf", "url": "/3820/1/Thesis1.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Chemical and Electrical Passivation of Single Crystal Silicon Surfaces Through Covalently Bound Organic Monolayers", "author": [ { "family_name": "Nemanick", "given_name": "Eric Joseph", "orcid": "0000-0002-4650-6491", "clpid": "Nemanick-Eric-Joseph" } ], "thesis_advisor": [ { "family_name": "Lewis", "given_name": "Nathan Saul", "clpid": "Lewis-N-S" } ], "thesis_committee": [ { "family_name": "Blake", "given_name": "Geoffrey A.", "clpid": "Blake-G-A" }, { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Lewis", "given_name": "Nathan Saul", "clpid": "Lewis-N-S" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The formation of and passivation by alkyl monolayers on Si(111) and Si(100) surfaces was studied. Crystalline Si(111) and Si(100) surfaces were alkylated in a two-step chlorination/alkylation process using both straight chain alkyl groups CH\u2083, C\u2082H\u2085, C\u2084H\u2089, and C\u2088H\u2081\u2087, as well sterically bulky alkyl groups such as (CH\u2083)\u2082CH-(iso-propyl), (CH\u2083)\u2083C-(tert-butyl), and C\u2086H\u2085-(phenyl) moieties to form well defined alkyl monolayers on the surface. X-ray photoelectron spectroscopic (XPS) data in the C 1s region of such surfaces exhibited a low energy emission at 283.6 binding eV, consistent with carbon bonded to Si, demonstrating a Si-C covalent bond. The C 1s XPS data indicated that larger alkyl groups were present at lower coverages than methyl groups on both the CH\u2083-terminated Si(111) surfaces as well as CH\u2083-terminated Si(100) surfaces. Despite the lower monolayer percent coverage, no Cl was detected after alkylation on either surface. Functionalization with even the bulky alkyl groups effectively inhibited the oxidation of the Si surfaces in air and produced low (< 100 cm s\u207b\u00b9) surface recombination velocities, indicating a low density of electronically active surface trap states. Transmission infrared spectroscopy indicated that the Si(111) surfaces were partially H-terminated after the functionalization reaction for groups larger than CH\u2083. High resolution soft X-ray photoelectron spectroscopic (SXPS) measurements of the Si 2p region of the alkylated Si(111) and Si(100) surfaces show monolayer coverage of SiCl and SiCH\u2083 on the Si(111) surface, with mixtures of species on H-term Si(111) as well as H-term Si(100) surfaces. Bulkier alkyl groups such as C\u2082H\u2085 and phenyl on both Si(111) and Si(100) surfaces as well as on CH\u2083-terminated Si(100) show broad Si 2p peaks with binding shifts indicative of hybrid surfaces composed of both Si-R groups and SiH/SiH\u2082 species. To model surface packing of these alkyl groups on the Si(111) surface, molecular dynamics modeling was employed using Cerius. The energies for various packing densities and packing patterns were calculated and referenced versus the reactant energy for each surface. From this it was concluded that 100% of the Si(111) surface should be CH\u2083-terminated, with lower packing densities for C\u2082H\u2085 between 50-80%, 50-66.7% for C\u2088H\u2081\u2087, 33.3-40% for tert-butyl, 33.3-50% for iso-propyl, and 50-66.7% for phenyl. A single electron transfer (SET) mechanism for the reaction of alkyl Grignards with the Cl-terminated surface is proposed. Application of a reducing potential, -2.5 V vs. Ag\u207a/Ag, to Cl-terminated Si(111) electrodes in tetrahydrofuran resulted in the complete elimination of Cl, as measured by XPS. The data are consistent with a mechanism in which the reaction of alkyl Grignard reagents with the Cl-terminated Si(111) surfaces involves electron transfer from the Grignard reagent to the Si, loss of chloride to solution, and subsequent reaction between the resultant silicon radical and alkyl radical to form a silicon-carbon bond. Sites sterically hindered by neighboring alkyl groups abstract a H atom to produce Si-H bonds on the surface.
", "doi": "10.7907/GNSW-GJ84", "publication_date": "2006", "thesis_type": "phd", "thesis_year": "2006" }, { "id": "thesis:2755", "collection": "thesis", "collection_id": "2755", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-06282006-103230", "primary_object_url": { "basename": "jmfinalthesis.pdf", "content": "final", "filesize": 9371133, "license": "other", "mime_type": "application/pdf", "url": "/2755/1/jmfinalthesis.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Single Mammalian Cell Gene Expression Analysis Using Microfluidics", "author": [ { "family_name": "Marcus", "given_name": "Joshua Scott", "clpid": "Marcus-Joshua-Scott" } ], "thesis_advisor": [ { "family_name": "Quake", "given_name": "Stephen R.", "orcid": "0000-0002-1613-0809", "clpid": "Quake-S-R" } ], "thesis_committee": [ { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" }, { "family_name": "Heath", "given_name": "James R.", "orcid": "0000-0001-5356-4385", "clpid": "Heath-J-R" }, { "family_name": "Campbell", "given_name": "Judith L.", "orcid": "0000-0001-8291-5551", "clpid": "Campbell-J-L" }, { "family_name": "Quake", "given_name": "Stephen R.", "orcid": "0000-0002-1613-0809", "clpid": "Quake-S-R" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "Single cell gene expression studies hold great promise for deciphering the ubiquitous heterogeneity present in biological organisms. Although much progress has been made in the field, tools to study gene expression (specific and global) in single cells are generally lacking. This thesis describes the development of novel microfluidic technologies and processes capable of processing single cells to first strand cDNA in a parallel fashion, thereby filling a void in the single cell biology field. The author then utilizes the technology to probe for transcriptional noise in ubiquitous genes present in single mammalian cells. The noise measured far exceeds any measurement reported to this date, and was shown to be attenuated during the G2 stage of the cell cycle. The work presented here is first hand proof that technological innovation is a key component in undertaking novel science.", "doi": "10.7907/M3GA-PT31", "publication_date": "2006", "thesis_type": "phd", "thesis_year": "2006" }, { "id": "thesis:2655", "collection": "thesis", "collection_id": "2655", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-06202006-113417", "primary_object_url": { "basename": "Ceres_PhD_Thesis.pdf", "content": "final", "filesize": 12510610, "license": "other", "mime_type": "application/pdf", "url": "/2655/1/Ceres_PhD_Thesis.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Electron Transfer at DNA-Modified Electrodes", "author": [ { "family_name": "Ceres", "given_name": "Donato Marino", "clpid": "Ceres-Donato-Marino" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Lewis", "given_name": "Nathan Saul", "orcid": "0000-0001-5245-0538", "clpid": "Lewis-N-S" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" }, { "family_name": "Heath", "given_name": "James R.", "orcid": "0000-0001-5356-4385", "clpid": "Heath-J-R" }, { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The DNA pi stack provides an efficient pathway for transport of electron and electron holes. Ground-state electron transport is furthermore extremely sensitive to subtle DNA structural perturbations, such as a single base mismatch, that alter pi-stacking. As a result, DNA-modified electrodes have allowed the development of highly sensitive diagnostic devices for the detection of base mismatches, lesions, and mutations. We have been able to apply DNA-mediated charge transduction, using a methylene blue/ferricyanide electrocatalytic cycle, in a DNA chip format for the detection of a single base mismatches at a microelectrode. Electrocatalysis is detected at DNA-modified electrodes down to 40 um electrode in diameter, where 108 DNA molecules are responsible for the electron transduction. This exquisite sensitivity both for mismatch detection irrespective of sequence context and to a small number of molecules is an important requisite for the development of a device able to detect multiple genetic variations in the absence of DNA amplification.
\r\n\r\nWe have also investigated in detail the electrochemical properties of DNA films. DNA is a highly charged molecule and, when self-assembled on a gold surface in a dense array, its properties are similar to those of polyelectrolyte films. We have found that the structure of the DNA film is sensitive to ion concentration and identity. Variations of the electrostatic potential across the film can sensitively affect both thermodynamics and kinetics of redox reporters incorporated in the film. Methylene blue reduction in the DNA film occurs via a two electron, one proton process. The Pourbaix diagram is linear in the case of a monovalent anionic buffer, while it is curved in phosphate buffer. Electron transfer kinetics are also affected by the relative concentration of divalent anions: at low pH the film is compressed in the linker portion and the rate of electron transfer is faster. Based on this understanding of the electrostatic balance inside the DNA film, a new analytical tool for monitoring hybridization events on gold surfaces has been developed using electrochemical impedance spectroscopy of ferricyanide.
\r\n\r\nIn order to explore the electron transport properties of DNA films mechanistically scanning tunneling microscopy (STM) has also been employed. These experiments provide a first opportunity to examine DNA conductivity under physiological conditions. These STM experiments on DNA films show that DNA, when perpendicularly oriented with respect to the surface, is coupled to the STM tip and the local density of states contribute to the measured tunneling current. At positive biases, when the surface is positive, the DNA is tilted towards the surface and as a result decoupled from the tip; the DNA appears \"transparent\" and the underlying surface instead is imaged. Also important is the integrity of the base stack. When the percentage of DNA duplexes containing a single base mismatch in the film is increased, the conductivity of the film decreases. The STM tip, being held at a constant current, approaches the DNA film until, at a critical mismatch content, the tip must penetrate the film and image resolution is lost. The current versus voltage characteristic of the DNA film has furthermore been determined through a new scanning tunneling spectroscopic technique that provides highly stable and reproducible measurements. We find that DNA duplex films under physiological conditions exhibit negative differential resistance, which is a feature that is typical of resonant electron tunneling via energetically localized molecular orbitals. This observation provides an experimental evidence for the existence of localized states within the DNA HOMO-LUMO gap that can be responsible for the ground state electron transport observed in electrochemical experiments.
", "doi": "10.7907/vdyv-zf71", "publication_date": "2006", "thesis_type": "phd", "thesis_year": "2006" }, { "id": "thesis:5249", "collection": "thesis", "collection_id": "5249", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-08222005-162934", "primary_object_url": { "basename": "Intro.pdf", "content": "final", "filesize": 69725, "license": "other", "mime_type": "application/pdf", "url": "/5249/1/Intro.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Application of Transition Metal Catalysis to Small Molecule Synthesis", "author": [ { "family_name": "Morrill", "given_name": "Christie", "clpid": "Morrill-Christie" } ], "thesis_advisor": [ { "family_name": "Grubbs", "given_name": "Robert H.", "clpid": "Grubbs-R-H" } ], "thesis_committee": [ { "family_name": "Stoltz", "given_name": "Brian M.", "clpid": "Stoltz-B-M" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Peters", "given_name": "Jonas C.", "clpid": "Peters-J-C" }, { "family_name": "Grubbs", "given_name": "Robert H.", "clpid": "Grubbs-R-H" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Over the past decade, transition metal catalysis has developed into a new field in organic synthesis, enabling numerous synthetic transformations that were previously not feasible. This thesis describes the application of both ruthenium and rhenium catalysis to the synthesis of several classes of small molecules. Ruthenium-catalyzed ring-opening cross-metathesis of five- through eight-membered ring cycloolefins was investigated for the synthesis of functionalized dienes (Chapter 1). Unsubstituted, trisubstituted, and allyl-substituted cycloolefins were studied. Regioselective reactions could be achieved with the use of unsymmetrical cycloolefins. Ruthenium-catalyzed cross-metathesis was explored for the synthesis of both di- and trisubstituted vinyl boronates (Chapter 2). These reactions proceeded efficiently for a wide variety of functionalized alkenes and generally exhibited high E-stereoselectivity. The resultant vinyl boronate products were stereoselectively converted into both Z-vinyl bromides and E-vinyl iodides. The rhenium-catalyzed 1,3-isomerization of allylic alcohols was employed in the synthesis of various allylic alcohols (Chapter 3). Two different strategies were developed to promote high product selectivity in these reactions: conjugated product synthesis and N,O-bis(trimethylsilyl)acetamide-promoted product trapping. These reactions enabled the synthesis of allylic alcohols with conjugated or non-conjugated, di- or trisubstituted, and electron-rich or electron-deficient alkene components. Partial chirality transfer was observed during the 1,3-isomerization of certain enantioenriched allylic alcohols. The fundamental reaction properties observed during these studies were all consistent with the operation of a mechanism involving a chair-like transition state, which contains a partially cationic allyl moiety, as the primary reaction pathway.", "doi": "10.7907/QRWG-BM33", "publication_date": "2006", "thesis_type": "phd", "thesis_year": "2006" }, { "id": "thesis:5254", "collection": "thesis", "collection_id": "5254", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-10142005-105810", "primary_object_url": { "basename": "THallpages.pdf", "content": "final", "filesize": 3185125, "license": "other", "mime_type": "application/pdf", "url": "/5254/1/THallpages.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Investigating Imidazolidinone Catalysts: Enantioselective Organocatalytic Diels-Alder Reactions, Conjugate Additions to Access Non-Natural \u221d-Amino Acids, and Bimodal Catalyst Activation for the Development of Organo-Cascade Reactions", "author": [ { "family_name": "Larsen", "given_name": "Catharine Ho\u00e0ng-Mai", "clpid": "Larsen-Catharine-Ho\u00e0ng-Mai" } ], "thesis_advisor": [ { "family_name": "MacMillan", "given_name": "David W. C.", "clpid": "MacMillan-D-W-C" } ], "thesis_committee": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "MacMillan", "given_name": "David W. C.", "clpid": "MacMillan-D-W-C" }, { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The MacMillan group focuses on the development of new strategies that harness the power of simple organic compounds to catalyze asymmetric reactions. To this end, we have designed amine catalysts which activate alpha,beta-unsaturated aldehydes via the reversible formation of chiral iminium ions (in analogy to LUMO-lowering activation by reversible metal-substrate complexation). Kinetic studies highlight the importance of the acid co-catalyst and identified a more reactive imidazolidinone catalyst complex, which improved enantioselectivities and vastly expanded the substrate scope of the first highly enantioselective organocatalytic Diels\u2013Alder reaction. Exploration of the crucial components of catalyst architecture led to the development of the second-generation imidazolidinone that not only catalyzes cycloadditions, but a variety of other reactions of aldehydes with excellent selectivity.
\r\n\r\nComplementary to the 1,2-addition observed with Lewis acids, the alternative mode of activation offered by iminium catalysis allows for 1,4-addition of heterocycles to alpha,beta-unsaturated aldehydes. Using a chiral amine catalyst, the first asymmetric conjugate addition of oxazoles generates protected quaternary alpha-amino acids with an adjacent tertiary stereocenter, a widely applicable motif in biology, materials science, and medicine. Finally, having demonstrated that imidazolidinones can activate both electrophiles (LUMO-lowering) and nucleophiles (HOMO-raising), these iminium and enamine catalysis cycles can be linked for tandem nucleophilic addition/electrophilic trapping of enals. In a single synthetic operation, this enantioselective conjugate addition/alpha-halogenation sequence takes achiral starting materials and selectively connects them, creating multiple stereocenters across the newly formed bonds.
\r\n", "doi": "10.7907/2WS6-B577", "publication_date": "2006", "thesis_type": "phd", "thesis_year": "2006" }, { "id": "thesis:2528", "collection": "thesis", "collection_id": "2528", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-06092006-062410", "primary_object_url": { "basename": "thesis3.pdf", "content": "final", "filesize": 7605036, "license": "other", "mime_type": "application/pdf", "url": "/2528/1/thesis3.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Synthesis and Applications of Bulky Rhodium(III) Intercalators for the Recognition of DNA Mismatches", "author": [ { "family_name": "Hart", "given_name": "Jonathan Ross", "orcid": "0000-0002-3905-225X", "clpid": "Hart-Jonathan-Ross" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" }, { "family_name": "Peters", "given_name": "Jonas C.", "orcid": "0000-0002-6610-4414", "clpid": "Peters-J-C" }, { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The recognition of DNA base mismatches is of considerable interest for both the diagnosis and treatment of mismatch repair-deficient cancers. Two new mismatch recognition complexes have been synthesized. The first, [Rh(bpy)2(phzi)]3+ (phzi=benzo[a]phenazine-5,6-quinone diimine), recognizes DNA mismatches with high specificity and affinity, 1 x 107 Mm-1, two orders of magnitude stronger than [Rh(bpy)2(chrysi)]3+ (chrysi=chrysene-5,6-quinone diimine), the parent complex that binds single thermodynamically-destabilized base-mismatch sites in duplex DNA. The second, [Rh(bqdi)2(chrysi)]3+, is able to recognize more stable mismatches such as the G-G mismatch.
\r\n\r\nThese complexes have been applied in a variety of ways. A method has been developed for the discovery of new single nucleotide polymorphisms, SNPs, within a sequence of interest amplified from pooled genomic DNA. SNPs are readily detected using these mismatch selective molecules without false positives; allele frequencies as low as 0.05 can be detected.
\r\n\r\nUpon photoexcitation, the rhodium(III) diimine complexes cleave DNA by hydrogen atom abstraction from the sugar to yield 3'-phosphate terminated DNA that is inactive for enzymatic modification. This 3'-phosphate can be removed using T4-polynucleotide kinase opening up the possibility of enzymatic modification at the site of rhodium cleavage. The cleavage site can be fluorescently labeled. Terminal transferase can also be used to attach a homopolymer tail tagging the damage site, allowing the amplification of the DNA up to the damaged site.
\r\n\r\nThis assay can also be employed towards the development of early cancer diagnostics. Some cancers are deficient in the repair of DNA base mismatches. As a consequence, these cells have an increased number of mismatches within their genome. These mismatches in extracted genomic DNA were cleaved using mismatch-specific rhodium complexes. The cleavage sites were labeled with radioactivity, allowing the number of mismatch sites to be quantitated. A significant number of sites were cleaved in the mismatch repair deficient DU145 cell line, 1 base/3000 bp, while no sites were cleaved in the mismatch repair proficient cell line SW620. This method may present a new method for the detection of mismatch repair deficiency.
\r\n\r\nThese mismatch-specific complexes also are shown to have an antiproliferative effect on mismatch repair deficient cell lines. Mismatch repair deficiency is a contributing factor in both hereditary and sporadic human cancers. Both [Rh(bpy)2(chrysi)]Cl3 and [Rh(bpy)2(phzi)]Cl3 show a stronger antiproliferative effect against MMR deficient cells than proficient cells. Effects of stereoisomers, incubation time, and UV irradiation are also demonstrated.
", "doi": "10.7907/sh3b-2f25", "publication_date": "2006", "thesis_type": "phd", "thesis_year": "2006" }, { "id": "thesis:5205", "collection": "thesis", "collection_id": "5205", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05272005-103515", "primary_object_url": { "basename": "0.pdf", "content": "final", "filesize": 158766, "license": "other", "mime_type": "application/pdf", "url": "/5205/1/0.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Coordination Chemistry from Trigonally Coordinated Iron Platforms: Chemistry Relevant to Dinitrogen Reduction", "author": [ { "family_name": "Betley", "given_name": "Theodore Alexander", "orcid": "0000-0001-5946-9629", "clpid": "Betley-Theodore-Alexander" } ], "thesis_advisor": [ { "family_name": "Peters", "given_name": "Jonas C.", "clpid": "Peters-J-C" } ], "thesis_committee": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Stoltz", "given_name": "Brian M.", "clpid": "Stoltz-B-M" }, { "family_name": "Bercaw", "given_name": "John E.", "clpid": "Bercaw-J-E" }, { "family_name": "Peters", "given_name": "Jonas C.", "clpid": "Peters-J-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The synthesis for a sterically encumbered, strong-field tris(diisopropylphosphino)borate ligand, [PhBPiPr3] ([PhBPiPr3] = [PhB(CH2PiPr2)3]-), is reported to probe aspects of its conformational and electronic characteristics within a host of complexes. To this end, the Tl(I) complex, [PhBPiPr3]Tl, was synthesized, characterized, and used to install the [PhBPiPr3] ligand onto complexes of Fe, Co, and Ru. The spectroscopic, electrochemical, magnetic, and structural features of these complexes are compared with similar examples.
\r\n\r\nTrigonally coordinated \"[PhBPiPr3]M\" platforms (M = Fe, Co) support both pi-acidic (N2) and pi-basic (NR2-) ligands at a fourth binding site. Methylation of monomeric [M0(N2)-] species successfully derivatizes the beta-N atom of the N2 ligand and affords the diazenido product [MII(N2Me)]. Addition of RN3 to MI(N2)MI results in oxidative nitrene transfer to generate [PhBPiPr3]M\u2261NR with concomitant N2 release.
\r\n\r\nA tetrahedrally coordinated L3Fe-Nx platform that accommodates both terminal nitride (L3FeIV\u2261N) and dinitrogen (L3FeI-N2-FeIL3) functionalities is described. The diamagnetic L3FeIV\u2261N species featured has been characterized in solution under ambient conditions by multinuclear NMR (1H, 31P, and 15N) and infrared spectroscopy. The terminal nitride complex oxidatively couples to generate the previously reported L3FeI-N2-FeIL3 species.
\r\n\r\nThe [PhBPiPr3] ligand can support a single iron or cobalt center in a pseudo-tetrahedral environment in which dinitrogen is bound in the fourth coordination site. Zero-valent metal-dinitrogen complexes have the general formula, [[PhBPiPr3]M(mu-N2)]2[Mg2+], while bridging structures can also be obtained as neutral [MI]\u2014N2\u2014[MI] or as anionic [(M)2(N2)]- species. The nature of the structural distortions observed in both [M(mu-N2)]2[Mg2+] and [Mn]\u2014N2\u2014[Mn] complexes are described. Magnetic characterization of the neutral and mixed-valence dimeric complexes reveal the complexes remain ferromagnetically coupled over all temperatures investigated.
\r\n\r\nThe coordination chemistry of group VIII metals featuring the bis(8-quinolinyl)amine (HBQA) ligand is presented. The electrochemical behavior of Fe, Ru, and Os complexes bearing the BQA ligand is reported and compared to related ligand platforms. Halide and phosphine ligand exchange reactions are examined from complexes of the type (BQA)MX(PR3)2 (M = Ru, Os). Carbonyl and dinitrogen complexes of Ru and Os are prepared from halide abstraction from divalent Ru and Os precursors. The spectroscopic and structural features of these complexes are compared with similar examples.
\r\n", "doi": "10.7907/PY23-ME66", "publication_date": "2005", "thesis_type": "phd", "thesis_year": "2005" }, { "id": "thesis:1721", "collection": "thesis", "collection_id": "1721", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05112005-100825", "primary_object_url": { "basename": "050511_final_thesis.pdf", "content": "final", "filesize": 2183003, "license": "other", "mime_type": "application/pdf", "url": "/1721/1/050511_final_thesis.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "P450 BM3 Electrochemistry and Electrocatalysis", "author": [ { "family_name": "Udit", "given_name": "Andrew K.", "orcid": "0000-0002-4454-7687", "clpid": "Udit-Andrew-K" } ], "thesis_advisor": [ { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" }, { "family_name": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" } ], "thesis_committee": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" }, { "family_name": "Collier", "given_name": "C. Patrick", "orcid": "0000-0002-8198-793X", "clpid": "Collier-C-P" }, { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" }, { "family_name": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Cytochromes P450 catalyze monooxygenations of relatively inert substrates. The significance of this activity in physiology and industry has inspired many to capture this activity in vitro. However, practical applications of P450s will continue to be hindered by the need for reducing equivalents from NAD(P)H. While electrochemical methods provide a potential solution, the difficulty in achieving good electronic coupling to the heme remains an enormous obstacle.
\r\n\r\nFlavocytochrome P450 BM3 is soluble, well-characterized, and easily manipulated, making it a good target for in vitro applications. Bioelectrocatalysis was first attempted with holo BM3 using a novel electrochemical mediator, 1,1\u2019-dicarboxycobaltocene (Mred). Absorption spectroscopy confirmed electron transfer (ET) from Mred to the cofactors, while electrolyses resulted in Mred-mediated hydroxylation of lauric acid by both the holo (16.5 nmol product / nmol enzyme / min) and heme proteins (hBM3) (1.8 nmol product / nmol enzyme / min).
\r\n\r\nSubsequent bioelectrocatalysis was attempted with the more stable hBM3. We achieved direct electrochemistry of hBM3 by wiring it through engineered surface Cys387 to a basal plane graphite electrode (BPG) with 1-pyreneiodoacetamide (Py). AFM images revealed that only pyrene-wired enzyme molecules adsorb to BPG. ko for the BPG-Py-hBM3 system was 650 \u00b1 50/s. Rotated-disk electrode (RDE) experiments show that the BPG-Py-hBM3 system catalyzes the four-electron reduction of dioxygen to water. Analogous experiments were performed with enzyme labeled at Cys62, which is spatially adjacent to Cys387 but does not provide a similar well-coupled through-bond pathway to the heme. Surprisingly, the Cys62 mutant showed similar electrode kinetics, demonstrating that the pyrene tether does not provide a unique pathway, but probably anchors the protein onto the electrode surface in a favorable docking mode for ET.
\r\n\r\nExtensive electrochemical characterization of hBM3 was conducted in various surfactant films on BPG. Cyclic voltammetry of hBM3 in SDS films revealed the Fe(III/II) redox couple at -330 mV (vs. Ag/AgCl, pH 7.4), and ko of 40/s. Although voltammetry confirmed catalytic dioxygen reduction by Fe(II), substrate oxidation was not observed.
\r\n\r\nVoltammetry of hBM3 mutant 1-12G in DDAPSS films revealed Fe(III/II) (-202 mV) and Fe(II/I) (-1082 mV) redox couples of the heme (pH 7). Catalytic activity included dioxygen reduction by Fe(II), and reductive dehalogenation by Fe(I). Voltammetry on hBM3 in DDAPSS revealed that hBM3 and 1-12G display distinct redox properties. Absorption spectra in solution showed the Fe(III) Soret at 418 nm for hBM3, and a split Soret for 1-12G at 390 and 418 nm. Voltammetry of the proteins within DDAPSS films revealed nearly identical Fe(III/II) potentials (~ -200 mV), but significant differences in ko, 250 vs. 30 per second, and Fe(III/II) \u2013 CO potentials, -140 mV vs. -115 mV, for hBM3 vs. 1-12G. Catalytic dioxygen reduction by the proteins on RDEs was analyzed using Levich and Koutecky-Levich treatments. Calculated values of n, 2.7 vs. 4.7, and kobs, 1400000 vs. 100000/M/s, for hBM3 vs. 1-12G suggest that the two proteins differ strikingly in their reaction with dioxygen.
\r\n\r\nUsing the prototypical cytochrome P450 CAM (CAM), we attempted to generate high-valent species of P450 in DDAB films. Performing rapid-scan (50 V/s) voltammetry revealed a couple (E) at 831 mV. E was not observed at scan rates less than 30 V/s at room temperature; however, at four degrees Celsius E could be reversibly generated at 1 V/s. E was found to be sensitive to imidazole in solution and to variations in pH, suggesting that the redox reaction is occurring at the metal center (i.e., Fe(IV/III)) rather than at the porphyrin macrocycle. Electrolyses revealed that the electrochemically generated high-valent species is only capable of performing S-oxidation, converting thioanisole to methyl phenyl sulfoxide.
", "doi": "10.7907/wr4v-d345", "publication_date": "2005", "thesis_type": "phd", "thesis_year": "2005" }, { "id": "thesis:1359", "collection": "thesis", "collection_id": "1359", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-04122005-152030", "primary_object_url": { "basename": "JJWeaver_Final_Thesis.pdf", "content": "final", "filesize": 1882093, "license": "other", "mime_type": "application/pdf", "url": "/1359/11/JJWeaver_Final_Thesis.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Corroles", "author": [ { "family_name": "Weaver", "given_name": "Jeremy John", "clpid": "Weaver-Jeremy-John" } ], "thesis_advisor": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" } ], "thesis_committee": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Collier", "given_name": "C. Patrick", "clpid": "Collier-C-P" }, { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Richards", "given_name": "John H.", "clpid": "Richards-J-H" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Corroles, porphyrin analogues, are the center of a rapidly growing field of research. By virtue of a missing meso-carbon, corroles retain the aromaticity of porphyrins, but become tribasic ligands in place of the dibasic porphyrin. This thesis is a study of synthetic methods for corroles, as well as their photophysical properties and potential applications.
\r\n\r\nNew synthetic methodologies for the free-base molecules have been used to obtain corroles with pentafluorophenyl meso substituents in both water-soluble and non-water-soluble forms. Closed-shell metallocorrole complexes have been synthesized by introducing Ga(III) and Sn(IV) ions into the macrocycle. Likewise, an open shell transition metal corrole utilizing Mn(III) has been made. Problems arising from making a third type of closed shell metallocorrole by introduction of In(III) are also discussed. Among other characterizations of these complexes, Gouterman\u2019s four-orbital model for porphyrins is reinterpreted under the reduced symmetry of the corrole macrocycle to explain the absorption and singlet emission spectra of the molecules. Evidence of a triplet excited state is also presented.
\r\n\r\nThe application of corrole complexes to other aspects of chemistry is then examined in two different areas. The interactions of the water-soluble corroles with human serum albumin were investigated to assess their usefulness as diagnostic agents and drugs for cancer research. These highly colored compounds have also been introduced as the dye component of dye-sensitized solar cells, and various aspects of the cells, including overall efficiency, have been tested.
\r\n\r\nThis thesis concludes with a summary of results obtained from collaborations on the interactions of corroles in cellular systems and synthetic attempts toward new types of water-soluble corroles, including an imidazole substituted chromium corrole and a sulfonated manganese nitrido corrole.
", "doi": "10.7907/8DS8-QS11", "publication_date": "2005", "thesis_type": "phd", "thesis_year": "2005" }, { "id": "thesis:1875", "collection": "thesis", "collection_id": "1875", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05192005-234308", "primary_object_url": { "basename": "WBBThesis.pdf", "content": "final", "filesize": 4368721, "license": "other", "mime_type": "application/pdf", "url": "/1875/1/WBBThesis.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Ultrafast Photoreduction of Nitric Oxide Synthase by Electron Tunneling Wires", "author": [ { "family_name": "Bittner", "given_name": "Wendy Belliston", "clpid": "Bittner-Wendy-Belliston" } ], "thesis_advisor": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" } ], "thesis_committee": [ { "family_name": "Bercaw", "given_name": "John E.", "clpid": "Bercaw-J-E" }, { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Richards", "given_name": "John H.", "clpid": "Richards-J-H" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The Gray group has a long-standing interest in the study of methods for rapid delivery of electrons to enzyme active sites. This thesis describes picosecond to nanosecond reduction of the heme active site of the inducible nitric oxide synthase oxygenase domain (iNOSoxy) bound to Re- and Ru-diimine electron-tunneling wires. The Re wires have the form [(4,7-dimethylphenanthroline)ReI(CO)3L]+ where L is a perfluorinated biphenyl bridge connecting a rhenium-ligated imidazole or aminopropylimidazole to a distal imidazole (F8bp-im (1) and C3-F8bp-im (2)) or F (F9bp (3) and C3-F9bp (4)). All four bind tightly (micromolar to nanomolar Kd) in the active site channel of iNOSoxy. Upon excitation with 355 nm light, the bound rhenium of 1, 2, or 4 is quenched in fewer than 200 ps, possibly by electron donation from a nearby tryptophan residue. When a through-bond pathway from the rhenium to the heme iron exists, the active site Fe(III) is then reduced to Fe(II) within 300 ps, approximately ten orders of magnitude faster than the naturally occurring reduction. The Ru-diimine wire, [(4, 4\u2019, 5, 5\u2019-tetramethylbipyridine)2Ru(bpyF9bp)]2+ (5), also binds tightly to iNOSoxy. The binding of 5 is independent of tetrahydrobiopterin, arginine, imidazole, and 1, indicating that tmRu-F9bp resides on the surface of the enzyme. Reductive flash-quench studies have shown that the bound wire is capable of reducing the imidazole-bound active-site heme in approximately 50 ns, fully seven orders of magnitude faster than the comparable in vivo process. This work represents the first demonstration of electron-tunneling wires that specifically target and rapidly reduce an enzyme without blocking the active site channel.", "doi": "10.7907/e82v-jn92", "publication_date": "2005", "thesis_type": "phd", "thesis_year": "2005" }, { "id": "thesis:2092", "collection": "thesis", "collection_id": "2092", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05262005-123044", "primary_object_url": { "basename": "thesis_all.pdf", "content": "final", "filesize": 3677757, "license": "other", "mime_type": "application/pdf", "url": "/2092/1/thesis_all.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Chemical Characterization and Charge Carrier Dynamics of Crystalline Silicon(III) Surfaces Modified with Surface-Bound Organic Functional Groups", "author": [ { "family_name": "Webb", "given_name": "Lauren J.", "orcid": "0000-0001-9999-5500", "clpid": "Webb-Lauren-J" } ], "thesis_advisor": [ { "family_name": "Lewis", "given_name": "Nathan Saul", "clpid": "Lewis-N-S" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Lewis", "given_name": "Nathan Saul", "clpid": "Lewis-N-S" }, { "family_name": "Marcus", "given_name": "Rudolph A.", "clpid": "Marcus-R-A" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Investigations of the chemical structure and charge carrier properties of alkylated crystalline silicon(111) surfaces are presented. Hydrogen-terminated Si(111) surfaces were alkylated with a series of saturated hydrocarbons through a chlorination/alkylation procedure and characterized using surface-sensitive techniques. High-resolution soft X-ray photoelectron spectroscopy (SXPS) identified 1 monolayer of H, Cl, and C on the H-, Cl- and CH3-terminated surfaces, respectively. Surfaces functionalized with bulkier alkyls showed Si 2p binding energy shifts that suggest that unalkylated Si atoms are bonded to hydrogen. Alkylated Si(111) surfaces were further characterized using transmission infrared spectroscopy (TIRS). The Si-Cl stretching and bending motions were identified at 583 and 528 cm-1, respectively. On the methyl-terminated Si(111) surface, a CH3 symmetrical bending vibrational mode at 1257 cm-1 polarized perpendicular to the surface was observed. On the C2H5-terminated surface, a Si-H stretching and bending motion at 2080 and 627 cm-1, respectively, were observed, confirming that unalkylated Si atoms are terminated with H atoms. Structural morphology of the CH3-terminated Si(111) surface was investigated by low energy electron diffraction (LEED), which found that this surface retained a flat, unreconstructed (1 x 1) structure. Scanning tunneling microscopy (STM) images of CH3-Si(111) were obtained at 4.7 and 77 K. The functionalized surface preserved the atomically flat morphology of freshly etched H-Si(111), and at 4.7 K individual methyl H atoms were clearly resolved for the first time.
\r\n\r\nElectronic passivation of the alkylated Si(111) surface was investigated through measurement of surface charge carrier recombination velocities (S), through time-resolved radio frequency (rf) photoconductivity decay methods. While unpassivated H- and Cl-terminated surfaces reacted rapidly in an air ambient to yield S>1400 cm s-1, alkylated surfaces preserved S<200 cm s-1 even when exposed to air for a period of weeks.
\r\n\r\nFinally, the two-step chlorination/alkylation route was compared to three other Si(111) surface functionalization techniques: (1) chlorination with Cl2(g) followed by alkylation with an alkylmagnesium halide reagent, (2) Lewis acid-mediated reduction of a terminal alkene, and (3) anodization of the H-terminated Si(111) surface in diethyl ether containing 3.0 M CH3MgI. The chemical properties and charge carrier recombination rates of each surface were measured as a function of time exposed to air.
", "doi": "10.7907/DM8E-JA05", "publication_date": "2005", "thesis_type": "phd", "thesis_year": "2005" }, { "id": "thesis:976", "collection": "thesis", "collection_id": "976", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-03172005-154741", "primary_object_url": { "basename": "Andrei_Deev_thesis.pdf", "content": "final", "filesize": 2323497, "license": "other", "mime_type": "application/pdf", "url": "/976/1/Andrei_Deev_thesis.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Cavity Ringdown Spectroscopy of Atmospherically Important Radicals", "author": [ { "family_name": "Deev", "given_name": "Andrei", "clpid": "Deev-Andrei" } ], "thesis_advisor": [ { "family_name": "Okumura", "given_name": "Mitchio", "clpid": "Okumura-M" } ], "thesis_committee": [ { "family_name": "Beauchamp", "given_name": "Jesse L.", "clpid": "Beauchamp-J-L" }, { "family_name": "Blake", "given_name": "Geoffrey A.", "clpid": "Blake-G-A" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Okumura", "given_name": "Mitchio", "clpid": "Okumura-M" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Many radicals, due to their electronic structure, have low-lying electronic states transitions to which lie in the near-IR. They often carry more information about the molecules than the transitions in the UV. However, these transitions even in the most important atmospheric radicals have not been thoroughly investigated due to their weakness and low attainable concentrations of radicals. This thesis describes the application of cavity ringdown spectroscopy to detection of near-IR states of some atmospherically important radicals.
\r\n\r\nThe near-IR cavity ringdown spectrometer constructed for these experiments is described in detail and characterized. The pulsed near-IR laser radiation was generated by sequential Raman shifting of the output of a tunable dye laser in hydrogen. The constructed multi-pass Raman cell extended the tunable range of the available dye laser continuously from the visible to 6000 cm\u207b\u00b9 with 0.15 cm\u207b\u00b9 resolution. The sensitivity of the instrument is \u22480.5 % of the mirror loss.
\r\n\r\nThe near-IR \u00c3 \u2190 X~ transition in peroxy radicals offers detection specificity for at least small radicals. The sensitivity of this transition to hydrogen atom substitution has been explored. The spectra of chloro-ethyl, -propyl, -butyl and -butenyl peroxy radicals in the 7000-8600 cm\u207b\u00b9 region are reported. The origin bands of the electronic transition were found to be shifted by 200 cm\u207b\u00b9 to the red. The spectra have more complex structure than those of unsubstituted homologues. DFT calculations predicted multiple conformers for C\u2082H\u2084ClO\u2082 and C\u2083H\u2086ClO\u2082 with the energies within 2 kcal/mol. Tentative assignment of the C\u2082H\u2084ClO\u2082 spectrum is presented. The integrated cross-section for the transition in chloro-ethyl peroxy radical is estimated from the known rate of self-reaction.
\r\n\r\nThe first full absorption spectrum of the \"dark\" \u00c3 \u00b2E' \u2190 X~ \u00b2A'\u2082 transition of the nitrate radical NO\u2083 in the 6000-10700 cm\u207b\u00b9 region is reported. \u03bd\u2082, \u03bd\u2083, and \u03bd\u2084 progressions and several combination bands are assigned. A more accurate estimate for the position of the \"dark\" origin is given. Analysis of the partially resolved rotational contours suggests that NO\u2083 undergoes static Jahn-Teller distortion in some of the vibronic states.
", "doi": "10.7907/YQBN-RX97", "publication_date": "2005", "thesis_type": "phd", "thesis_year": "2005" }, { "id": "thesis:2349", "collection": "thesis", "collection_id": "2349", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-06012004-115510", "primary_object_url": { "basename": "Title.pdf", "content": "final", "filesize": 384405, "license": "other", "mime_type": "application/pdf", "url": "/2349/8/Title.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Ligand Design, Coordination Chemistry, and Mechanistic Studies of (Phosphino)Borates and their Platinum, Nickel, and Copper Complexes", "author": [ { "family_name": "Thomas", "given_name": "John Christopher", "clpid": "Thomas-John-Christopher" } ], "thesis_advisor": [ { "family_name": "Peters", "given_name": "Jonas C.", "clpid": "Peters-J-C" } ], "thesis_committee": [ { "family_name": "Bercaw", "given_name": "John E.", "clpid": "Bercaw-J-E" }, { "family_name": "Stoltz", "given_name": "Brian M.", "clpid": "Stoltz-B-M" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Peters", "given_name": "Jonas C.", "clpid": "Peters-J-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Synthetic methods are presented for the preparation of various substituted bis(phosphino)borates. A relatively general protocol based on the delivery of a nucleophilic phosphine-containing carbanion to a borane electrophile has been developed. Preparative methods for the synthesis of substituted diarylchloroboranes from dimethyldiaryltin reagents provide the borane electrophiles. Methyldialkyl- or methyldiarylphosphines are selectively deprotonated at the phosphine-methyl using alkyl lithium bases to form the carbanion nucleophiles. The reaction of diverse phosphine-containing carbanions with diarylchloroboranes results in bis(phosphino)borates selectively substituted at the borate, at the phosphine, or at both positions. In addition to the generated lithium salts of the bis(phosphino)borates, cation-exchange protocols provide methods for preparing ammonium and thallium bis(phosphino)borate salts. Structural data for some of these derivatives are presented.
\r\n\r\nThe electronic properties of transition metals coordinated by bis(phosphino)borates are explored through NMR and IR spectroscopies. The spectroscopic features of platinum(II) dimethyl and methyl carbonyl complexes are examined for trends based on the substitution pattern of the (phosphino)borate ligand. These trends indicate that phosphine substituents have a more significant impact than borate substituents on electronics of the metal center. Structural and spectroscopic comparisons of structurally similar platinum(II) dimethyl and methyl carbonyl complexes indicate that the anionic bis(phosphino)borate ligand renders platinum(II) more electron-rich than structurally similar neutral phosphine donors. Related spectroscopic studies of anionic and neutral molybdenum(0) tetracarbonyl complexes provide results analogous to those found when comparing neutral and cationic platinum(II) systems.
\r\n\r\nComparative studies on the ligand exchange and benzene C-H activation chemistry of structurally similar platinum(II) complexes convey the similarities and differences between zwitterionic and cationic systems. Examination of THF ligand self-exchange by magnetization transfer shows a change in mechanism between the neutral and cationic species. Both bis(phosphino)borate-ligated and neutral bis(phosphine) platinum methyl solvento complexes undergo a benzene C-H activation to form the corresponding phenyl solvento complex; however, the rates of reaction and ultimate products differ. Extensive isotopic studies indicate that the zwitterionic system forms observable intermediates prior to benzene C-H activation, some of which are attributable to ligand metalation processes.
\r\n\r\nStructural and spectroscopic studies of a phenyl-substituted tris(phosphino)borate on platinum are presented. Alkyl- and hydride-containing platinum(II) and platinum(IV) species have been synthesized. The structural and spectroscopic features of these complexes are compared to related tris(pyrazolyl)borate systems on platinum.
\r\n\r\nCoordination and reaction chemistry of an isopropyl-substituted tris(phosphino)borate on nickel are discussed. Complexes in the Ni(II), Ni(I), and Ni(0) oxidation states have been prepared. This system is compared through structural, spectroscopic, and electrochemical methods to related phenyl-substituted tris(phosphino)borate chemistry on nickel. Reactivity studies aimed at preparing Ni(III) and Ni(IV) complexes containing metal-ligand multiple bonds through group-transfer reactions are presented. Theoretical studies using density functional methods are used to probe several target species containing multiply-bonded ligands.
\r\n\r\nThe coordination chemistry of copper(I) is explored using bis(phosphino)borates. Both aryl- and alkyl-substituted bis(phosphino)borates provide access to copper(I) complexes. A tert-butyl-substituted bis(phosphino)borate is particularly useful for preparing a family of three-coordinate compounds. The spectroscopic and structural features of these complexes are compared with similar, previously described examples.
", "doi": "10.7907/GCAQ-8D59", "publication_date": "2004", "thesis_type": "phd", "thesis_year": "2004" }, { "id": "thesis:2140", "collection": "thesis", "collection_id": "2140", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05272004-213231", "primary_object_url": { "basename": "THESIS.pdf", "content": "final", "filesize": 14968159, "license": "other", "mime_type": "application/pdf", "url": "/2140/1/THESIS.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Femtosecond Time-Resolved Spectroscopy of Anionic Systems: Dynamics of Mesoscopic Solvation and Gas-Phase Organic Reactions", "author": [ { "family_name": "Paik", "given_name": "Daniel Hern", "clpid": "Paik-Daniel-Hern" } ], "thesis_advisor": [ { "family_name": "Zewail", "given_name": "Ahmed H.", "clpid": "Zewail-A-H" } ], "thesis_committee": [ { "family_name": "McKoy", "given_name": "Basil Vincent", "clpid": "McKoy-B-V" }, { "family_name": "Zewail", "given_name": "Ahmed H.", "clpid": "Zewail-A-H" }, { "family_name": "Collier", "given_name": "C. Patrick", "clpid": "Collier-C-P" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "This thesis work presents the femtosecond time-resolved spectroscopy of anionic systems ranging from gas-phase organic molecules to the finite-sized molecular clusters. The subject matter in this thesis is twofold: Mesoscopic solvation of anionic clusters and transition state dynamics of neutral organic molecules.
\r\n\r\nSolvation dynamics in the finite sized clusters were investigated at the molecular level of details. The main objective of the cluster study was to follow the evolution of cluster properties as function of cluster size. Ultrafast processes exhibited in the cluster systems were investigated by utilizing the femtosecond time-resolved anion photoelectron spectroscopy. The bond rupture and solvent evaporation of homogeneous and heterogeneous clusters were studied, and the correlation between the dissociation rates and the cluster size is obtained. Gas-phase analogues of solvated systems were studied, and the key steps involved in the solvation dynamics are highlighted.
\r\n\r\nDirect probing of the transition state dynamics in the ground state is studied with femtosecond time resolution. The transition state of the ring inversion reaction of cyclooctatetraene was directly accessed by the vertical detachment from the planar anion. The subsequent nuclear motion was then probed by ionization mass spectrometry. The oscillatory feature observed in the transients reflects trajectories of motion (resonance) along the reaction coordinate, and comparison with theory is reported. This work demonstrated the applicability of the charge reversal scheme to a complex organic system and suggests the possibility of studying ground-state thermal organic reactions with their transition states resolved in real time.
", "doi": "10.7907/W687-V550", "publication_date": "2004", "thesis_type": "phd", "thesis_year": "2004" }, { "id": "thesis:1856", "collection": "thesis", "collection_id": "1856", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05182004-181406", "primary_object_url": { "basename": "TitlePage.pdf", "content": "final", "filesize": 48514, "license": "other", "mime_type": "application/pdf", "url": "/1856/2/TitlePage.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Oxidative DNA Damage by Long-Range Charge Transport", "author": [ { "family_name": "Delaney", "given_name": "Sarah", "orcid": "0000-0002-8366-3808", "clpid": "Delaney-Sarah" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Bercaw", "given_name": "John E.", "clpid": "Bercaw-J-E" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Ever since the elucidation of the double helical structure of DNA, it has been proposed that the stack of base pairs within the double helix may mediate charge transport (CT) reactions. In fact, CT through DNA can result in chemistry at a distance, yielding oxidative DNA damage at a site remote from the bound oxidant. DNA CT chemistry depends upon coupling within the stacked base pair array, and this chemistry is remarkably sensitive to sequence-dependent DNA structure and dynamics. Using a variety of octahedral transition metal complexes, DNA CT has been probed to explore mechanistic considerations and biological possibilities.
\r\n\r\nInteractions with DNA by a family of ruthenium(II) complexes bearing the dipyridophenazine (dppz) ligand or its derivatives have been examined. An intercalative binding mode has been established based on luminescence enhancements in the presence of DNA, excited state quenching, fluorescence polarization values and enantioselectivity. Oxidative damage to DNA by these complexes using the flash/quench method has also been examined. A direct correlation between the amount of guanine oxidation obtained via DNA CT and the strength of intercalative binding was observed. These results support the importance of close association and intercalation for DNA-mediated CT. Electronic access to the DNA base pairs, provided by intercalation of the oxidant, is a prerequisite for efficient CT through the DNA pi-stack.
\r\n\r\nUsing polypyridyl ruthenium complexes, a reductive flash/quench scheme in DNA has also been explored. The flash/quench scheme previously utilized in DNA studies involves an oxidative quencher and allows for examination of electronic hole transport through DNA. In contrast, a reductive flash/quench technique would allow for direct observation of electron transport through the base stack. In our studies, p-methoxydimethylaniline and potassium iodide have proven to be effective reductive quenchers of dipyridophenazine complexes of ruthenium. However, by transient absorption spectroscopy, high performance liquid chromatography, gel electrophoresis, and electron paramagnetic resonance we are unable to observe any DNA reduction products with the ruthenium complexes examined. Rates of back electron transfer may in fact be faster than trapping of the anion radical, thus hindering observation of long-range damage.
\r\n\r\nThe oxidative flash/quench technique was applied in probing DNA CT in a range of DNA assemblies containing a tethered ruthenium intercalator and methylindole (M), a low potential nucleobase analog, where radical formation at a distance as a function of DNA sequence could be examined both by laser spectroscopy and biochemical methods. Hole injection and subsequent formation of the methylindole radical cation were observed at a distance of over 30 \u0160at rates > 10e7 s-1 in assemblies containing no guanine bases intervening the ruthenium intercalator and GMG oxidation site. Radical yield was, however, strikingly sensitive to an intervening base mismatch; no significant methylindole radical formation was evident with an intervening AA mismatch. Also critical is the sequence at the injection site; this sequence determines initial hole localization and hence the probability of hole propagation. With guanine rather than inosine near the site of hole injection, decreased yields of radicals and long-range oxidative damage are observed. The presence of the low energy guanine site in this case serves to localize the hole and increase the probability of back reaction at the injection site therefore diminishing CT through the base pair stack.
\r\n\r\nDNA assemblies containing a pendant dppz complex of Ru(II) along with two oxidative traps, a site containing the nucleoside analog methylindole (5?-GMG-3?) and a 5?-GGG-3? site, were constructed to explore charge equilibration across the base pair stack. In these assemblies the base radicals form with a rate of 10e7 s-1. Interestingly, the rate of base radical formation does not change upon the addition of a second radical trap, the 5?-GGG-3? site; however the yield of methylindole oxidation is significantly lower. This observation indicates that the 5?-GGG-3? site is effective in competing for the migrating charge and provides a second trapping site. Importantly, switching the orientation of the two trapping sites does not affect the yield of oxidized products at either site. Therefore, in DNA both forward and reverse charge transport occur so as to provide equilibration across the duplex on a time scale that is fast compared to trapping at a particular site. Further evidence of charge equilibration results from incorporating an intervening base-stacking perturbation and monitoring the fate of the injected charge. These experiments underscore the dynamic nature of DNA charge transport and reveal the importance of considering radical propagation in both directions along the DNA duplex.
\r\n\r\nDNA conjugates containing adjacent duplex and guanine quadruplex assemblies have been designed to explore CT into quadruplex architectures. The quadruplex assemblies have been characterized structurally using circular dichroism and by assaying for chemical protection. Using an intercalating rhodium photooxidant, noncovalently bound or tethered to the duplex end, oxidizing radicals are found to be trapped in the folded quadruplex. Damage is observed almost exclusively at the external tetrads of the quadruplex. Little damage of the center tetrad is observed, due most likely to lowered efficiency of radical trapping within the quadruplex core. This pattern of damage is distinct from that observed for repetitive G sequences within duplex DNA. The data indicate, furthermore, that in the conjugates examined, the guanine quadruplex provides a more effective trap than a 5?-GG-3? guanine doublet within duplex DNA. Additionally, within these assemblies, sufficient base-base overlap must exist at the duplex/quadruplex junction to allow for charge migration. This funneling of damage to the quadruplex, as well as the unique pattern of damage within the quadruplex, requires consideration with respect to the analysis of oxidative DNA damage within the cell.
", "doi": "10.7907/9Q0X-TZ17", "publication_date": "2004", "thesis_type": "phd", "thesis_year": "2004" }, { "id": "thesis:1744", "collection": "thesis", "collection_id": "1744", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05122004-052751", "primary_object_url": { "basename": "title.pdf", "content": "final", "filesize": 44549, "license": "other", "mime_type": "application/pdf", "url": "/1744/7/title.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Structure and Reactivity of Metal Complexes Bound to DNA", "author": [ { "family_name": "Bhattacharya", "given_name": "Pratip K.", "orcid": "0000-0002-0625-252X", "clpid": "Bhattacharya-Pratip-K" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Lewis", "given_name": "Nathan Saul", "orcid": "0000-0001-5245-0538", "clpid": "Lewis-N-S" }, { "family_name": "Roberts", "given_name": "Richard W.", "orcid": "0000-0002-8587-5097", "clpid": "Roberts-R-W" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" }, { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Establishing correlations among structure, dynamics and reactivity is a fundamental problem in biological chemistry. Here, this problem is explored in the context of the design and reactivity of different metallointercalators bound to DNA.
\r\n\r\nFirst, the effects of intervening mismatches on DNA structure, dynamics and DNA charge transport reactivity is examined. The \u03c0-stacked DNA base pairs mediate charge transport chemistry over long molecular distances in a reaction that is exquisitely sensitive to DNA sequence dependent conformation and dynamics. To examine the long-range charge transport as a function of intervening base mismatches, a series of DNA oligonucleotides were synthesized that incorporate a ruthenium intercalator, [Ru(phen)(bpy')(dppz)]\u00b2\u207a (phen = 1,10 phenanthroline; bpy' = 4-butyric acid-4'-methylbipyridine; dppz = dipyrido[3,2-a:2',3'-c]phenazine) linked covalently to the 5' terminus of one strand and containing two 5'-GG-3' sites in the complementary strand. Single base mismatches were introduced between the two guanine doublet steps, and the efficiency of transport through the mismatches was determined through measurements of the ratio of oxidative damage at the guanine doublets distal versus proximal to the intercalated ruthenium oxidant. Differing relative extents of guanine oxidation were observed for the different mismatches. The damage ratio of oxidation at the distal versus proximal site for the duplexes containing different mismatches varies in the order GC ~ GG ~ GT ~ GA > AA > CC ~ TT ~ CA ~ CT. The extent of distal/proximal guanine oxidation in different mismatch-containing duplexes was then compared with the helical stability of the duplexes, electrochemical data for intercalator reduction on different mismatch-containing DNA films, and base-pair lifetimes for oligomers containing the different mismatches derived from \u00b9H NMR measurements of the imino proton exchange rates. The exchange kinetics of the imino protons were measured from selective longitudinal relaxation times, and the effect of the mismatch was observed on the base pair lifetime up to a distance of two neighboring base pairs. The overall order of base-pair lifetimes in the selected sequence context of the base pair was as follows: GC > GG > AA > CC > TT. While a clear correlation is evident both with helix stability and electrochemical data monitoring reduction of an intercalator through DNA films, guanine damage ratios was found to correlate most closely with base-pair lifetimes. These results underscore the importance of base dynamics in modulating long-range charge transport through the DNA base-pair stack.
\r\n\r\nIn a related \u00b9H NMR structural study of the ruthenium intercalator, [Ru(phen)(bpy')(dppz)]\u00b2\u207a covalently tethered to a short eightmer DNA duplex, d(ACGAGCAC)\u2022d(GTICTCGT) with a nine carbon linker, the type of construct used in charge transport experiments, a very fast exchange was observed. Comparison of the NOESY data obtained from the NMR study of this system and control samples comprising of the duplex with only linker and the duplex alone, led to the conclusion that the nine carbon linker is positioned between the second and fourth bases from the point of its origin. The absence of any site specificity of the metal complex in the oligonucleotide complicates the structural characterization by NMR study. This led us to conceive of a more general strategy of obtaining structural information of metal complexes that bind non-specifically to DNA based on paramagnetic NMR.
\r\n\r\nThe selective paramagnetic relaxation of oligonucleotide proton resonances of two short self-complementary oligonucleotides; d(GTCGAC)\u2082 and d(GTGCAC)\u2082 by Ni(phen)\u2082(L)\u00b2\u207a where L= dipyridophenazine (dppz), dipyrido[3,2-d:2',3'-f]quinoxaline (dpq) and phenanthrenequinone (phi) was examined to obtain structural insight into the non-covalent binding of these metal complexes to DNA. In the oligonucleotide d(GTCGAC)\u2082, preferential broadening of the G1H8, G4H8, T2H6, and C3H6 proton resonances was observed with Ni(phen)\u2082(dppz)\u00b2\u207a, Ni(phen)\u2082(dpq)\u00b2\u207a and Ni(phen)\u2082(phi)\u00b2\u207a. In the case of the sequence d(GTGCAC)\u2082, where the central two bases are juxtaposed from the previous one, preferential broadening was observed instead for the A5H2 proton resonance. Thus, a subtle change in the sequence of the oligonucleotide can cause significant change in the binding location of the metal complex in the oligonucleotide. Owing to comparable changes for all metal complexes and sequences in broadening of the thymine methyl proton resonances, the switch in preferential broadening was attributed to a change in site location within the oligomer rather than to an alteration of groove location. Therefore, even for DNA-binding complexes of low sequence-specificity, distinct variations in binding as a function of sequence are apparent and can be monitored using paramagnetic probes.
\r\n\r\nFinally, \u00b9H NMR spectroscopy was employed to study the binding of [Rh(bpy)\u2082chrysi]\u00b3\u207a (chrysi = 5,6-chrysenquinone diimine), a metal complex which specifically targets mismatches, to a ninemer oligonucleotide d(GCCTCAGGC)\u2082 containing centrally placed CC mismatch. Evidence supports intercalation by the metal complex within the mismatch site (i) upfield chemical shifts and significant broadening of the chrysi resonances and (ii) an increase in duplex melting temperature in the presence of the metal complex. To simplify the NMR spectra, the \u0394 isomer of [Rh(d\u2088\u208bbpy)\u2082chrysi]\u00b3\u207a was employed in NMR experiments with DNA. A break in the connectivity in the NOE walk is observed between T\u2084 and C\u2085, thereby marking the binding site of the metal complex at the CC mismatch. Intermolecular NOE's place the metal complex in the major groove of the oligonucleotide.
\r\n\r\nThus through a series of experiments in this thesis, attempts have been made to correlate the structure and dynamics of metal complexes bound to DNA. Truly, metal complexes bound to DNA provide an interesting system to study structure, function and dynamics in a single package.
", "doi": "10.7907/ZANY-J082", "publication_date": "2004", "thesis_type": "phd", "thesis_year": "2004" }, { "id": "thesis:1305", "collection": "thesis", "collection_id": "1305", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-04082004-161537", "primary_object_url": { "basename": "thesis.pdf", "content": "final", "filesize": 5218662, "license": "other", "mime_type": "application/pdf", "url": "/1305/1/thesis.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Fundamental Aspects of DNA-Mediated Charge Transport", "author": [ { "family_name": "Williams", "given_name": "Tashica Tr\u00e9shun", "clpid": "Williams-Tashica-Tr\u00e9shun" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Marcus", "given_name": "Rudolph A.", "clpid": "Marcus-R-A" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The \u03c0-stacked array of DNA base pairs has fascinated scientists since its structural delineation. Here are described fundamental studies to probe how this \u03c0-stacked array mediates DNA charge transport (CT). Intercalators, such as dipyridophenazine (dppz) complexes of ruthenium and phenanthrenequinone diimine (phi) complexes of rhodium, serve as powerful and systematic probes of DNA CT in these studies.
\r\n\r\nIn a series of rhodium-tethered DNA assemblies, with varying A/T sequences intervening between guanine doublet sites (5'-GG-3'), long-range oxidative DNA damage is examined. The guanine doublet sites are known as sites of low oxidation potential in DNA, and the rhodium complex serves as a spatially separated, potent photooxidant. Although these studies are inconsistent with a mechanism involving guanine hopping and tunneling through A/T sequences, these data illustrate that the sequence of bases is an important determinant in attenuating oxidative damage yields of CT. Based on these data, we propose hopping among domains defined by sequence-dependent structure. Additional studies are also described using rhodium-tethered DNA assemblies to examine how different ionic distributions around the DNA duplex modulate DNA CT. In the rhodium-DNA conjugates, differences in long-range oxidative damage yield were observed depending on the position of pendent charges on the oligomer.
\r\n\r\nA direct comparison of DNA CT utilizing a variety of oxidants has also been performed. CT is assayed both through determination of the yield of oxidative guanine damage and, in derivative DNA assemblies, by analysis of the yield of a faster oxidative trapping reaction, ring-opening of N2-cyclopropylguanine (CPG) within the DNA duplex. We find clear differences in oxidative damage ratios at the distal versus proximal 5'-GG-3' depending upon the photooxidant employed. There is also a correlation seen between absolute yield of oxidative damage and distal/proximal damage ratio; photooxidants that produce higher distal/proximal damage ratios have lower yields. These differences observed among photooxidants as well as the complex distance dependence are attributed to differences in rates of back electron transfer (BET).
\r\n\r\nA study of the overall effect of bridge energetics on DNA CT has also been performed by constructing rhodium-DNA assemblies containing varying numbers of inosine, a guanine base analog with a higher oxidation potential, between two 5'-GG-3' sites. For the rhodium conjugates, only a slight diminution in distal oxidative yield with increasing distance is observed, suggesting direct charge injection by rhodium into higher energy sites of the intervening bridge.
\r\n\r\nThese results, taken together, provide insight into salient parameters that govern DNA CT, in particular how energetics, charge distribution, and sequence-dependent DNA structure and dynamics modulate charge migration through DNA.
", "doi": "10.7907/83ec-2t89", "publication_date": "2004", "thesis_type": "phd", "thesis_year": "2004" }, { "id": "thesis:1511", "collection": "thesis", "collection_id": "1511", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-04262004-184449", "primary_object_url": { "basename": "chapter0titlepages.pdf", "content": "final", "filesize": 40219, "license": "other", "mime_type": "application/pdf", "url": "/1511/1/chapter0titlepages.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Chemical Effects of Acoustic Cavitation", "author": [ { "family_name": "Lesko", "given_name": "Timothy Michael", "clpid": "Lesko-Timothy-Michael" } ], "thesis_advisor": [ { "family_name": "Hoffmann", "given_name": "Michael R.", "clpid": "Hoffmann-M-R" } ], "thesis_committee": [ { "family_name": "McKoy", "given_name": "Basil Vincent", "clpid": "McKoy-B-V" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Hoffmann", "given_name": "Michael R.", "clpid": "Hoffmann-M-R" }, { "family_name": "Lewis", "given_name": "Nathan Saul", "clpid": "Lewis-N-S" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "A novel high-frequency, high-power, pilot-plant scale sonochemical reactor was developed and used to study the degradation of organic pollutants in aqueous solutions. The degradation rates of trichloroethylene, dichloromethane, and phenol were found to exceed those of similar frequency, small-scale bench reactors by factors ranging from 2.5 to 7. Experiments with 10 \u00b5M methyl orange in the large reactor operating at different total volumes exhibited a linear dependence between the observed sonolytic rate constants and the applied power density. Likewise, steady-state \u2022OH-radical (aq) in each reactor were calculated and shown to correlate with the applied power density in the vessel.
\r\n\r\nThe sonochemical decomposition of phenol was further studied in a bench-scale ultrasound reactor combination with ozonolysis. The addition of ozone during sonication did not affect the first-order degradation rate constants of phenol compared to the linear combination of separate sonication and ozonation experiments. However, enhancement of the degradation rates of the total organic carbon (TOC) by 43% was observed for sonolytic ozonation compared to the separate sonication and ozonolysis experiments. The synergistic action of O\u2083 (aq) and ultrasound enhanced oxalate degradation rates 16-fold compared to the simple linear addition of the two independent systems. Several degradation pathways are considered which may account for the rate enhancements observed when ultrasonic irradiation is applied concurrently with ozonolysis.
\r\n\r\nIn addition, the decomposition of aqueous ozone in the presence of hydrogen peroxide was investigated. H\u2082O\u2082 enhances the reactivity of O\u2083 (aq) by reactions that remain obscure. Several free-radical degradation mechanisms for O\u2083 decomposition correctly predict the ozone-decay kinetics in pure water but vastly overestimate reaction rates in the presence of H\u2082O\u2082. Results from solvent deuteration experiments in neat water are compatible with a chain-process driven by electron transfer and/or O-transfer processes. However, the large kinetic isotope effect (KIE) found in the O\u2083/H\u2082O\u2082 system provides compelling evidence for an elementary reaction (O\u2083 + HO\u2082\u207b) involving H-O\u2082\u207b bond cleavage and does not support appreciable radical production from the O\u2083 + HO\u2082\u207b reaction. The magnitude of the observed KIE is consistent with a hydride transfer process yielding a closed-shell trioxide HO\u2083\u207b, the conjugate anion of H\u2082O\u2083.
", "doi": "10.7907/TY3J-F564", "publication_date": "2004", "thesis_type": "phd", "thesis_year": "2004" }, { "id": "thesis:20", "collection": "thesis", "collection_id": "20", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-01042004-210542", "primary_object_url": { "basename": "title&chpt1.pdf", "content": "final", "filesize": 4552547, "license": "other", "mime_type": "application/pdf", "url": "/20/5/title&chpt1.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Chemical-Scale Manipulation of Ion Channels: in vivo Nonsense Suppression and Targeted Disulfide Crosslinking", "author": [ { "family_name": "Zacharias", "given_name": "Niki Marie", "orcid": "0000-0002-9364-6016", "clpid": "Zacharias-Niki-Marie" } ], "thesis_advisor": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" }, { "family_name": "Lester", "given_name": "Henry A.", "clpid": "Lester-H-A" } ], "thesis_committee": [ { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" }, { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" }, { "family_name": "Lester", "given_name": "Henry A.", "clpid": "Lester-H-A" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Roberts", "given_name": "Richard W.", "clpid": "Roberts-R-W" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The study of the three-dimensional shape and structure-function relationships of ion channels is a very challenging field of research. Ion channels are integral-membrane proteins that when open allow ions to flux across the cell membrane. The structure and function of ion channels are dependent on the cell membrane that surrounds them. Because an ion channel must be embedded in a cell membrane, many techniques used to probe the structure of soluble proteins cannot be used in the study of ion channels.
\r\n\r\nOne versatile technique that has been shown to be quite valuable in the structure-function studies of ion channels is the in vivo nonsense suppression method for unnatural amino acid incorporation. This technique allows one to site-specifically incorporate an unnatural amino acid or hydroxy acid into a protein in a living cell. To date more than 60 amino acids and hydroxy acids have been incorporated into proteins using in vivo nonsense suppression. The method has been shown to accommodate a wide variety of unnatural amino acids and hydroxy acids. Chapter One will discusses the in vivo nonsense suppression method in greater detail.
\r\n\r\nA key component of this work is the design and synthesis of new unnatural amino acids that have novel properties. Chapter 2 discusses the synthesis and uses of 5-(o-nitrobenzyl)selenyl-2-hydroxypentanoic acid (NBSeOH). NBSeOH is used to site-specifically cleave a peptide backbone. The o-nitrobenzyl protecting group is photochemically removed to reveal a selenium anion. The selenium anion then initiates an intramolecular SN2 displacement that cleaves the backbone of the protein. Preliminary data reveals that NBSeOH can be incorporated into a protein in vivo and in vitro, and photolysis of proteins and peptides containing NBSeOH does lead to protein backbone cleavage.
\r\n\r\nChapter 4 discusses how the in vivo nonsense suppression method was used to incorporate unnatural amino acids containing a quaternary ammonium moiety to mimic the quaternary ammonium on acetylcholine. These unnatural amino acids were used to probe the nicotinic acetylcholine receptor?s binding site. These unnatural amino acids are called tethered agonists because when they were incorporated into four different positions on the nicotinic acetylcholine receptor partial opening of the channel occurred even when agonist was not present. These tethered agonists were used to obtain distance information about where acetylcholine binds within the receptor.
\r\n\r\nAnother technique used to probe the structure of ion channels is targeted disulfide crosslinking. In the targeted disulfide crosslinking method, cysteine residues are introduced at various locations throughout a protein and oxidized to see whether disulfide bond formation can occur. Since only cysteine residues close in space will form a disulfide bond, this method can reveal fine structural aspects of a protein. The method was used to study the pore lining structure of the nicotinic acetylcholine receptor. Several cysteine mutants were made using mutagenesis and then studied in functional channels expressed in Xenopus oocytes. The channels were then exposed to oxidizing agents, and the ability of these mutant channels to form disulfide bonds was evaluated. Chapter 3 describes the work dealing with the targeted disulfide crosslinking experiments in the nicotinic acetylcholine receptor.
", "doi": "10.7907/y4ay-ph28", "publication_date": "2004", "thesis_type": "phd", "thesis_year": "2004" }, { "id": "thesis:2442", "collection": "thesis", "collection_id": "2442", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-06042004-153530", "primary_object_url": { "basename": "Elizabeth_I_Mayo.pdf", "content": "final", "filesize": 7155748, "license": "other", "mime_type": "application/pdf", "url": "/2442/1/Elizabeth_I_Mayo.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Kinetics and Thermodynamics of Dye (Group VIII Metal)\u2013Sensitized Nanocrystalline Titanium Dioxide Photoelectrodes", "author": [ { "family_name": "Mayo", "given_name": "Elizabeth Idonia", "clpid": "Mayo-Elizabeth-Idonia" } ], "thesis_advisor": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Lewis", "given_name": "Nathan Saul", "clpid": "Lewis-N-S" } ], "thesis_committee": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Stoltz", "given_name": "Brian M.", "clpid": "Stoltz-B-M" }, { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Lewis", "given_name": "Nathan Saul", "clpid": "Lewis-N-S" }, { "family_name": "Goddard", "given_name": "William A., III", "clpid": "Goddard-W-A-III" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "This thesis reports a comprehensive series of experiments involving complementary kinetics and thermodynamic measurements directed at isolating the important individual reactions in dye-sensitized nanocrystalline titanium dioxide solar cells (DSSCs). These experiments were done in conjunction with steady-state photoelectrochemical measurements; a combination which allowed a greater understanding of the overall mechanisms and driving forces of these systems.
\r\n\r\nAlternative two-electron redox couples were studied and efficiency increases of >40% were achieved when compared to similar systems using iodide/triiodide. Surface treatment with carboxylic acids minimized direct reduction of the redox couple by electrons in the titanium dioxide, and interestingly, the photocurrent also increased resulting in overall efficiency increases as high as 20%. Bridging ligands were used in an attempt to minimize recombination of the injected electrons with the resulting oxidized dyes, but DSSCs with these sensitizers showed poor conversion efficiencies and no distance dependence for injection or recombination was observed. The lack of distance dependence was attributed to the flexible single carboxyl anchoring group. To further investigate the effect of binding mode, a series of carboxyl-modified ruthenium bipyridyl sensitizers were studied. A single carboxyl anchoring group resulted in unstable DSSCs due to enhanced desorption as well as poor photon-to-current conversion efficiencies. These dyes injected efficiently into TiO\u2082 on the nanosecond timescale, and regeneration of the oxidized sensitizers competed effectively with recombination. Consequently, individual kinetics measurements could not explain the decreased steady-state performance. The regeneration rates of these dyes in solution were found to rapid, approaching the diffusion controlled limit. The regeneration rate was dependent on the number and electron-withdrawing nature of the pendant groups, with the rate decreasing with increasing number of electron withdrawing substituents. Iridium dyes with cyclometalating ligands were shown to be efficient sensitizers in DSSCs, with quantum yields on the order of a ruthenium analogue having similar spectral overlap. Overall, the repeated inconsistencies between the steady-state behavior and the measured individual kinetics processes indicate that the current kinetic model is insufficient to accurately predict photoelectrochemical behavior.
", "doi": "10.7907/E9WY-1N05", "publication_date": "2004", "thesis_type": "phd", "thesis_year": "2004" }, { "id": "thesis:5202", "collection": "thesis", "collection_id": "5202", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05262004-173345", "primary_object_url": { "basename": "Choi-T-L-2004-thesis.pdf", "content": "final", "filesize": 2032578, "license": "other", "mime_type": "application/pdf", "url": "/5202/23/Choi-T-L-2004-thesis.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Olefin Metathesis: a Versatile Tool for the Synthesis of Small to Large Molecules", "author": [ { "family_name": "Choi", "given_name": "Tae-Lim", "orcid": "0000-0001-9521-6450", "clpid": "Choi-Tae-Lim" } ], "thesis_advisor": [ { "family_name": "Grubbs", "given_name": "Robert H.", "clpid": "Grubbs-R-H" } ], "thesis_committee": [ { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" }, { "family_name": "Stoltz", "given_name": "Brian M.", "clpid": "Stoltz-B-M" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Grubbs", "given_name": "Robert H.", "clpid": "Grubbs-R-H" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "In olefin metathesis, the designing of better catalysts has been the key to the success of its utility. Throughout the history of olefin metathesis research, the development of new and improved catalysts has brought new applications and new strucures that are accessible by olefin metathesis routes. With the developmet of highly active catalyst containing an N-heterocyclic carbene, the field of olefin metathesis is currently in a period of renaissance opening up the versatile synthesis of both small orgainc molecules to macromolecules. Following four chapters describle recent applications towards the synthesis of molecules with various sizes.\r\n", "doi": "10.7907/FD0X-D313", "publication_date": "2004", "thesis_type": "phd", "thesis_year": "2004" }, { "id": "thesis:4188", "collection": "thesis", "collection_id": "4188", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-10202002-002307", "primary_object_url": { "basename": "Thesis.pdf", "content": "final", "filesize": 13085158, "license": "other", "mime_type": "application/pdf", "url": "/4188/11/Thesis.pdf", "version": "v7.0.0" }, "type": "thesis", "title": "I. Structure-Function Analysis of the Mechanosensitive Channel of Large Conductance. II. Design of Novel Magnetic Materials using Crystal Engineering", "author": [ { "family_name": "Maurer", "given_name": "Joshua Ahab", "orcid": "0000-0002-6663-0721", "clpid": "Maurer-Joshua-Ahab" } ], "thesis_advisor": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" } ], "thesis_committee": [ { "family_name": "Grubbs", "given_name": "Robert H.", "clpid": "Grubbs-R-H" }, { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" }, { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" }, { "family_name": "Lester", "given_name": "Henry A.", "clpid": "Lester-H-A" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Davis", "given_name": "Mark E.", "clpid": "Davis-M-E" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The work presented here encompasses two distinct areas, with the first section addressing structure-function relationships in the mechanosensitive channel of large conductance (MscL) from bacteria. A high-throughput fluorescent screening technique has been developed for the E. coli homologue of MscL. This technique has been applied to a large library of random E. coli MscL mutations to provide insights into channel function. Additionally, attempts have been made to characterize the functionally important regions of MscL and comparisons have been made between the E. coli and M. tuberculosis homologues of MscL.
\r\n\r\nThe second section addresses the design of novel magnetic materials. The guanidinium sulfonate \"Ward lattice\" from crystal engineering has been used to develop a new family of frustrated magnetic materials.
", "doi": "10.7907/DNFY-8G73", "publication_date": "2003", "thesis_type": "phd", "thesis_year": "2003" }, { "id": "thesis:404", "collection": "thesis", "collection_id": "404", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-01302003-225031", "primary_object_url": { "basename": "title.pdf", "content": "final", "filesize": 27363, "license": "other", "mime_type": "application/pdf", "url": "/404/9/title.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Catalysts for Olefin Metathesis: Ruthenium Alkylidene Complexes with Phosphine and N-Heterocyclic Carbene Ligands", "author": [ { "family_name": "Trnka", "given_name": "Tina Maria", "clpid": "Trnka-Tina-Maria" } ], "thesis_advisor": [ { "family_name": "Grubbs", "given_name": "Robert H.", "clpid": "Grubbs-R-H" } ], "thesis_committee": [ { "family_name": "Bercaw", "given_name": "John E.", "clpid": "Bercaw-J-E" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Dervan", "given_name": "Peter B.", "clpid": "Dervan-P-B" }, { "family_name": "Grubbs", "given_name": "Robert H.", "clpid": "Grubbs-R-H" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The objectives of the work described in this dissertation were twofold: (1) to further improve the activity and selectivity of ruthenium-based olefin metathesis catalysts, and (2) to obtain a better understanding of how these catalysts operate.
\r\n\r\nThe first problem was addressed by varying the ligand sphere within the L2X2Ru=CHR framework. Chapter 2 explores the metathesis of directly functionalized olefins, such as 1,1-difluoroethylene and acrylonitrile. Detailed studies revealed that ruthenium alkylidene complexes react readily with these olefins but stop after a single turnover of the catalytic cycle. This effect is caused by electronically deactivating carbene substituents, which dramatically decrease the rate of phosphine dissociation from the metal center and thus prevent catalyst re-initiation.
\r\n\r\nChapter 3 describes complexes where the L ligands are phosphines, N-heterocyclic carbenes (NHCs), imidazoles, or pyridines, where the X ligands are chlorides, and where the carbene moiety is either benzylidene or a cyclic moiety. Improved catalytic activity and selectivity were achieved with complexes containing a combination of phosphine and NHC ligands. The reactivity and stability profiles of these species can be tuned through the stereoelectronic properties of the NHC. To facilitate the use of NHCs in organometallic applications, a synthetic route was developed that employs NHC adducts to protect the reactive carbene centers.
\r\n\r\nChapter 4 describes the reactions of ruthenium alkylidene complexes with alkynes. In the majority of cases, the metathesis polymerization of alkynes is unsuccessful because of competing reactions to form eta3-vinylcarbene and eta5-cyclopentadienyl derivatives. The eta3-vinylcarbene complexes are particularly interesting as models for the olefin-bound intermediate in the olefin metathesis catalytic cycle, and their structures demonstrate that it is possible for the chloride ligands to adopt a cis arrangement that places one of the chlorides trans to the L donor ligand.
\r\n\r\nThe studies in Chapter 5 explore the stereoelectronic properties of phosphine and NHC ligands and provide valuable insights about electronic structure and bonding. This information was obtained by a variety of techniques, including structure-activity studies, kinetics, x-ray crystallography, heteronuclear NMR, infrared spectroscopy, and gas-phase UV photoelectron spectroscopy.
", "doi": "10.7907/PP5B-E858", "publication_date": "2003", "thesis_type": "phd", "thesis_year": "2003" }, { "id": "thesis:2038", "collection": "thesis", "collection_id": "2038", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05252003-231704", "primary_object_url": { "basename": "00Title_Page.pdf", "content": "final", "filesize": 127141, "license": "other", "mime_type": "application/pdf", "url": "/2038/1/00Title_Page.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Spectroscopic Characterization of DNA-Mediated Charge Transfer", "author": [ { "family_name": "Treadway", "given_name": "Christopher Ryan", "clpid": "Treadway-Christopher-Ryan" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Peters", "given_name": "Jonas C.", "clpid": "Peters-J-C" }, { "family_name": "Marcus", "given_name": "Rudolph A.", "clpid": "Marcus-R-A" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The DNA double helix provides a well-characterized molecular pi stack in which charge transfer rates and efficiencies may be examined. We investigated the phenomenon in a series of DNA duplexes modified with various photo- and redox-active species.
\r\n\r\nCharge transfer dynamics through DNA were distance independent for a series of duplexes modified with 7-deazaguanine and a covalently attached ethidium chromophore. The decay times were grouped into two components: (1) injection of charge into the DNA base stack and (2) a correlated charge transfer corresponding to the reorientation of ethidium within the duplex.
\r\n\r\nUsing the modified bases 2-aminopurine and 7-deazaguanine, intrastrand charge transfer through DNA was observed on the picosecond timescale. Charge transfer rates and quenching yields were also dependent on the reaction?s driving force and the composition of the intervening base stack.
\r\n\r\nThe efficiency of photooxidation of 7-deazaguanine by a ruthenium(II) intercalator in DNA over 7?14 angstroms was shallow and dependent on the chirality of the covalently attached metallointercalator. Nanosecond to subnanosecond decay rates were measured, and the spectroscopic signature of a charge transfer intermediate was observed.
\r\n\r\nA series of ruthenium(II) intercalators with high oxidation potentials was created. Redox reactivity of the compounds with DNA did not correlate directly with oxidation potential and was dependent on DNA binding and luminescence quenching abilities. Thus, redox potential may not be used as the sole predictor of reactivity with the base stack of DNA.
\r\n\r\nFinally, the binding of ruthenium(II) and rhodium(III) intercalators to DNA was investigated with CD and NMR spectroscopies. Data confirmed that metallointercalators do not preferentially bind next to each other along the double helix. Hence, direct contact of reactants is not responsible for fast and efficient charge transfer between metallointercalators bound noncovalently to DNA.
\r\n\r\nThese studies have provided direct measurements of the dynamics of DNA-mediated charge transfer and proven, once again, that the DNA pi stack facilitates fast and efficient charge transfer. Most importantly, the stacking and dynamics of the reactants and DNA bases were found to affect the charge transfer behavior.
", "doi": "10.7907/KMWD-4M75", "publication_date": "2003", "thesis_type": "phd", "thesis_year": "2003" }, { "id": "thesis:3194", "collection": "thesis", "collection_id": "3194", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-08222002-162219", "primary_object_url": { "basename": "ack,abs,TOC.pdf", "content": "final", "filesize": 162916, "license": "other", "mime_type": "application/pdf", "url": "/3194/1/ack,abs,TOC.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Electrochemical Sensors Based on DNA-Mediated Charge Transport Chemistry", "author": [ { "family_name": "Boon", "given_name": "Elizabeth Marshall", "orcid": "0000-0003-1891-839X", "clpid": "Boon-Elizabeth-Marshall" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" }, { "family_name": "Anson", "given_name": "Fred C.", "clpid": "Anson-F-C" }, { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The base pair stack within double helical DNA provides an effective medium for charge transport. The \u03c0-stacked DNA base pairs mediate charge transport chemistry over long molecular distances in a reaction that is exquisitely sensitive to DNA sequence-dependent conformation and dynamics. This sensitivity to minor perturbations in DNA structure and base stacking makes DNA-mediated charge transport chemistry an ideal platform for DNA sensing. Electrochemical methods through DNA-modified electrode surfaces that exploit this sensitivity for efficient biosensing are described. Gold electrodes are modified with DNA double helices and used to monitor the electrochemistry of bound redox-active intercalators. The efficiency of electrochemical reduction of the intercalated redox-probe, in a DNA-mediated reaction, provides an indicator of base stacking within the surface-bound duplexes. Perfectly stacked DNA is capable of mediating the electrochemical reduction, while duplexes containing \u03c0-stacking perturbations, such as single base mismatches, do not support current flow to the intercalator.
\r\n\r\nAll single base mismatches, including thermodynamically stable GT and GA mismatches, as well as many common base damage products can be detected within DNA and DNA/RNA hybrid duplexes using this assay. Moreover, mismatches can be detected as a small percentage of a perfectly matched film, making it possible to detect mutations associated with genetic disorders in only a small fraction of cells. This assay is also compatible with DNA based chip technology. Furthermore, electrochemistry at DNA films is found to provide a novel and sensitive method for probing protein dependent changes in DNA structure and enzymatic reactions.
\r\n\r\nThe efficient transport of charge through self-assembled monolayers of thiol-terminated duplexes on gold therefore offers an extremely sensitive probe for the integrity of DNA sequences. Completely new approaches to single base mismatch detection as well as assaying protein-DNA interactions and reactions on surfaces are now available. This technology is generally applicable as a tool for directly measuring base pair stacking in nucleic acid duplexes.
", "doi": "10.7907/PFXM-7M76", "publication_date": "2003", "thesis_type": "phd", "thesis_year": "2003" }, { "id": "thesis:768", "collection": "thesis", "collection_id": "768", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-02262003-004639", "primary_object_url": { "basename": "CH-0-Prf.pdf", "content": "final", "filesize": 174648, "license": "other", "mime_type": "application/pdf", "url": "/768/1/CH-0-Prf.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Spatial, Temporal, and Chemical Aspects of Vapor Detection Using Conductive Composite Chemically Sensitive Resistors", "author": [ { "family_name": "Briglin", "given_name": "Shawn Michael", "clpid": "Briglin-Shawn-Michael" } ], "thesis_advisor": [ { "family_name": "Lewis", "given_name": "Nathan Saul", "clpid": "Lewis-N-S" } ], "thesis_committee": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Blake", "given_name": "Geoffrey A.", "clpid": "Blake-G-A" }, { "family_name": "Okumura", "given_name": "Mitchio", "clpid": "Okumura-M" }, { "family_name": "Lewis", "given_name": "Nathan Saul", "clpid": "Lewis-N-S" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "We have investigated the vapor response properties of chemically sensitive thin film resistors prepared from conductor-insulator composites. A new sensor type was developed from alkylamine-capped gold nanocrystals, and films of this composite, which are composed of nanometer-scale gold cores separated by regions of insulating alkylamine chains, exhibit small reversible resistance increases upon exposure to vapors such as water, acetone, or toluene. However, these films exhibit large irreversible resistance decreases in exposure to vapors possessing the thiol (-SH) functionality. The resistance change is shown useful for determination of the mercaptan concentration, and readily permits the detection of methylmercaptan at concentrations as low as 4 ppb (parts per billion), and hydrogen sulfide at concentrations as low as 9 ppm (parts per million). We have also investigated the geometric, spatial, and temporal response properties of chemically sensitive resistors prepared from polymer-carbon black composites in exposure to common organic vapors. The reversible resistance responses of these detectors were evaluated with short rise-time pulses of vapor, and detectors formed from very thin (< 200 nm) films of polyethylene-co-vinyl acetate (PEVA)-carbon black composites produced steady-state responses within 17 ms for methanol exposures and within 90 ms for toluene, acetone, or n-hexane. In accord with Fickian diffusion, the response times of PEVA-carbon black detectors were proportional to the square of the film thicknesses in the range 510 nm to 5700 nm, and the response vs. time profiles were well fit by a simple finite difference model based on Fickian diffusion. The temporal response also provides useful information for the identification or discrimination of solvent vapors beyond that available solely in the steady-state response employed in previous studies of this sensor type. We also demonstrate that there is an optimum detector volume to produce the highest signal/noise ratio for a given composite when exposed to a fixed volume of analyte vapor, and we show that useful information and optimizations can be obtained from the spatiotemporal response profile of an analyte moving in a controlled path across an array of chemically identical, but spatially nonequivalent, detectors.\r\n", "doi": "10.7907/XHF4-YX71", "publication_date": "2003", "thesis_type": "phd", "thesis_year": "2003" }, { "id": "thesis:6782", "collection": "thesis", "collection_id": "6782", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:01262012-090632523", "primary_object_url": { "basename": "Copeland_kd_2002.pdf", "content": "final", "filesize": 75650200, "license": "other", "mime_type": "application/pdf", "url": "/6782/1/Copeland_kd_2002.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "The Reactions of Metallointercalator-Peptide Conjugates and DNA", "author": [ { "family_name": "Copeland", "given_name": "Kimberly Davis", "clpid": "Copeland-Kimberly-Davis" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" }, { "family_name": "Parker", "given_name": "Carl Stevens", "clpid": "Parker-C-S" }, { "family_name": "Roberts", "given_name": "Richard W.", "clpid": "Roberts-R-W" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "A family of metallointercalator-peptide conjugates for reaction with DNA has been constructed. In these chimeras, the metallointercalator provides binding affinity for\r\nDNA and the peptide contributes reactivity. With the goal of creating an artificial nuclease, we have tethered metal-binding peptides to a sequence neutral intercalator,\r\n[Rh(phi)_2bpy']^(3+) (phi = phenanthrenequinone diimine, bpy' = 4-butyric acid-4' -methyl-2,2'-bipyridine). This is a general strategy, and we have observed Zn^(2+)-promoted\r\ncleavage of plasmid DNA with widely different peptides: a designed helical peptide with histidine residues, and a hairpin peptide modeled after the active site of the BamHI\r\nendonuclease. To optimize the peptide composition of our artificial nuclease we created a library of 16,000 conjugates, but no new active conjugates were identified with this combinatorial strategy. To achieve oxidative cleavage of the DNA backbone we have also used our intercalator-peptide conjugates to deliver copper to DNA. Tethered peptides containing histidine residues promote oxidative strand scission in DNA restriction fragments and oligonucleotides in the presence of Cu^(2+) and a reducing agent. Importantly, by comparing the photocleavage pattern of the rhodium intercalator with the copper cleavage pattern of the metal-binding peptide, the interactions of the conjugate with DNA could be dissected. Finally, short peptides were tethered to [Ru(phen)(bpy')(dppz)]^(2+) \r\n(phen = 1,10-phenanthroline, dppz = dipyridophenazine) to\r\ncreate fluorescent DNA crosslinking agents. Through a flash-quench reaction, the ruthenium intercalator generates guanine radicals in a DNA duplex. These guanine\r\nradicals can react with water or oxygen, but also with tethered peptides to produce pennanent DNA-peptide crosslinks. The DNA-peptide crosslinks were detected by gel\r\nelectrophoresis and absorbance measurements, and characterized by mass spectrometry. Although they have low affinity for DNA, untethered peptides could also be crosslinked to DNA using the ruthenium chemistry. The peptide composition influences conjugate binding and the extent and pattern of crosslinking; indeed, positively charged residues were essential for effective crosslinking. Although the flexibility of our tethered peptides\r\nis an obstacle to the rational design of reactive conjugates, we have demonstrated that peptides can mediate a variety of reactions if delivered to DNA by metallointercalators.", "doi": "10.7907/MR9T-7W57", "publication_date": "2002", "thesis_type": "phd", "thesis_year": "2002" }, { "id": "thesis:1968", "collection": "thesis", "collection_id": "1968", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05232005-084837", "primary_object_url": { "basename": "Villahermosa_r_2002.pdf", "content": "final", "filesize": 7455022, "license": "other", "mime_type": "application/pdf", "url": "/1968/1/Villahermosa_r_2002.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Electron Tunneling Through Phenylene Bridges", "author": [ { "family_name": "Villahermosa", "given_name": "Randy Michael", "clpid": "Villahermosa-Randy-Michael" } ], "thesis_advisor": [ { "family_name": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" } ], "thesis_committee": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" }, { "family_name": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" }, { "family_name": "Lewis", "given_name": "Nathan Saul", "orcid": "0000-0001-5245-0538", "clpid": "Lewis-N-S" }, { "family_name": "Marcus", "given_name": "Rudolph A.", "orcid": "0000-0001-6547-1469", "clpid": "Marcus-R-A" }, { "family_name": "Winkler", "given_name": "Jay Richmond", "orcid": "0000-0002-4453-9716", "clpid": "Winkler-J-R" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "A series of donor-bridge-acceptor (D-B-A) complexes, [(bpy)2Ru(bpy-(XY)n-G)](PF6)2 (where n = 2 to 5, bpy = 2,2'-bipyridine, XY = 2,5-xylene, and G is 3-ethynyl-4-methoxyN, N-dimethylaniline; abbreviated [Ru-(XY)n-G]2+), were designed, synthesized, and characterized to study electron tunneling through phenylene bridges. [Ru-(XY)n -G]2+ is a versatile D-8-A scaffold that exhibits a strong correlation between xylene conformation and electron transfer properties.
\r\n\r\n[Ru-(XY)n -G](PF6)2 was assembled from three components in a convergent process. Stepwise oligomer growth produced the well-defined bridging ligand, bpy-(XY)n; bipyridine was regioselectively functionalized with a xylene oligomer through multiple cycles of palladium-catalyzed cross-couplings. The donor, synthesized separately, was joined to bpy-(XY)n using an alkyne linkage. Metalation with a (bpy)2Ru fragment finished assembly of the D-8-A complex. The D-B-A series was analyzed with mass spectrometry- and NMR.
\r\n\r\nSpectroscopic, electrochemical, and spectroelectrochemical characterizations of [Ru-(XY)n -G]2+ indicate no significant electronic or chemical difference among the members of the series. UV-visible absorption spectra, with a metal to ligand charge transfer (MLCT) band maximum of 460 nm, resemble the model complexes [Ru(bpy)3]2+ and [Ru-XY-TMS]2+ (where TMS = trimethylsilyl). Representative cyclic voltammograms of [Ru-(XY)3-G]2+ contain reversible redox couples for Ru111m and Gw+, with potentials of 1.24 and 0.59 V (vs. Ag/AgCl). Spectroelectrochemical traces, displaying loss of MLCT intensity and increased absorption centered at 520 nm, indicate the formation of [Ru111-(XY)n -G+]4+.
\r\n\r\nThe flash-quench technique was used to measure the electron transfer rates for [Rum\u00b7(XY)11-G] 3+-+ [Ruu-(XY)n-G+]3+. The rates, 9.0 \u00b1 0.3 x 106, 2 \u00b1 1x105, and \u00b7 6 ::: 1 103 for n == 3 through 5, have a strong dependence on donor-acceptor distance. Estimates of the donor-acceptor distance were used to determine a distance decay constant, J), of 0.84 A-1 \u2022 The typical 13-value for electron tunneling through phenylenes is\r\n0.4 A-1 \u2022 The unusually high ~-value for [Ru-(XY)n -G]2+ is attributed to near-orthogonal dihedral angles between adjacent xylene rings. UV-visible spectra, cyclic voltammograms, and structural information, from molecular modeling calculations and a crystal structure, all support a near-orthogonal twist angle.
\r\n\r\nThe versatility of [Ru-(XY)n-G]2+ as a molecular scaffold was demonstrated in studies on electron transfer reactions in nanocrystalline Ti02 solar cells. Modified to incorporate a terminal carboxyl group, [Ru-(XY)n -BA]2+ (where BA : 4wethynyl-benzoic acid) was synthesized and used as a Ti02 solar cell dye. Utilizing the flash-quench method, the second-order rate constant for dye regeneration with r- ([Rum\u00b7(XY)n-BA] 3+-+ [Ru-(XY)11-BA]2+) in homogenous fluid solution was 1.5 x 1010 M'1s-1 for all three dyes in the series n=O to 2.
", "doi": "10.7907/qxxp-m840", "publication_date": "2002", "thesis_type": "phd", "thesis_year": "2002" }, { "id": "thesis:6797", "collection": "thesis", "collection_id": "6797", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:01312012-114925650", "primary_object_url": { "basename": "Nunez_me_2002.pdf", "content": "final", "filesize": 51836776, "license": "other", "mime_type": "application/pdf", "url": "/6797/1/Nunez_me_2002.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Oxidation of DNA by Long-Range Charge Transport", "author": [ { "family_name": "Nu\u00f1ez", "given_name": "Megan Elizabeth", "clpid": "Nu\u00f1ez-Megan-Elizabeth" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Roberts", "given_name": "Richard W.", "clpid": "Roberts-R-W" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Ever since the double helical structure of DNA was elucidated, it has been proposed that charge might move through the stacked base pairs of the double helix because of the electronic coupling of the \u03c0 orbitals of the nucleotide bases with neighboring bases. Here it is demonstrated that electronic \"holes\" generated\r\nby a one-electron oxidation of DNA can result in permanent lesions on guanine bases up to 200 \u00c5 away from the intercalating oxidant as a result of such charge\r\nmigration. Both rhodium and ruthenium complexes, covalently tethered to the 5' end of a double-stranded oligonucleotide and intercalated into the base stack, can with photoactivation promote oxidation of guanines in 5'-GG-3' sites over this distance. Since charges can move efficiently through the DNA oligonucleotides, it was important to characterize this reaction in more detail, and to extend observations of charge transport through DNA to larger and more complicated DNA assemblies that more closely mimic its structure in vivo.
\r\n\r\nLong-range oxidative damage to guanine doublets in DNA is shown to compete for oxidation with other reactions, such as the repair of thymine dimers. When both thymine dimer lesions and guanine doublets are present, both can be\r\noxidized by a photoexcited rhodium complex, although each in lower yield than in the absence of the other. While the 5-GG-3' may represent the thermodynamically favored site for oxidative reaction, repair of the thymine dimer appears to be kinetically more favorable. Therefore electronic \"holes\" generated on genomic DNA might not of necessity cause DNA damage, but could also be funneled onto proteins or other oxidizible sites.
\r\n\r\nUsing a variety of intercalating photooxidants targeted to a specific site on a restriction fragment by an appended triplex-forming oligonucleotide, the upper distance limits and sequence effects on long-range charge transfer through DNA were examined. Charge migration occurs in both directions from the intercalator and on both DNA strands of the target, but the oxidation is significantly more\r\nefficient to the 3' side of the triplex, over 25-38 base pairs. When intercalators were tethered directly to the 5' terminus of the triplex-forming strand as opposed\r\nto the center, significant amounts of oxidative damage was generated only in the immediate vicinity of the intercalation site, suggesting that the base stack is\r\ndistorted at the 5' end of the triplex region in the duplex/triplex junction. Targeting of photooxidative damage by triplex formation extends previous studies of long-range charge transport to significantly longer DNA sequences through a strategy that does not require covalent attachment of the photooxidant to the DNA being probed.
\r\n\r\nWithin eukaryotic cells most DNA is packaged as nucleosome core particles, made up of ~146 base pairs of DNA wrapped around a core of histone proteins. Photoexcited rhodium complexes were also used to explore charge\r\ntransport through DNA within these structures. Although histone proteins inhibit intercalation of a noncovalent rhodium complex, they do not prevent oxidation of\r\n5'-GG-3' sites, the signature of oxidative charge transport through DNA. Furthermore, some of these sites are not directly accessible to a solution-bound oxidant due to his tones in the major groove, and thus they must be oxidized from a distance. Therefore, although the structure of the nucleosome core particle generally protects DNA from damage from solution-borne molecules, it does not protect the DNA from charge transfer damage through the base pair stack. In\r\nsupport of this assertion, guanine bases within nucleosomal DNA were oxidized at a distance of over 23 base pairs from a covalently-tethered rhodium intercalator.
\r\n\r\nThe environment within the cell nucleus contains a variety of other proteins and small molecules that could potentially influence the migration of charge through DNA. Using the rhodium photochemistry, the oxidation of\r\nguanine by photoexcited rhodium complexes inside of nuclei from cultured human cells was examined and compared with the oxidative damage on bare genomic DNA. Oxidation occurs preferentially at the 5'-guanine of 5'-GG-3' sites, indicative of base damage by DNA-mediated charge transport chemistry. Moreover, oxidative damage occurs at protein-bound sites which are inaccessible to rhodium. Thus, on transcriptionally active DNA within the cell nucleus, DNA-mediated charge transport acts to induce base damage from a distance. Direct interaction of an oxidant is not necessary to generate a base lesion at a specific\r\nsite within the nucleus.
\r\n\r\nAll of these observations indicate that charges can migrate along DNA within the cell. These observations require a reconsideration of cellular mechanisms for DNA damage and repair, and present new avenues for exploration in the design of DNA-based drugs and therapies.
", "doi": "10.7907/ZQ5T-9Z15", "publication_date": "2002", "thesis_type": "phd", "thesis_year": "2002" }, { "id": "thesis:1296", "collection": "thesis", "collection_id": "1296", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-04072008-151825", "primary_object_url": { "basename": "Chong_sh_2001.pdf", "content": "final", "filesize": 4476745, "license": "other", "mime_type": "application/pdf", "url": "/1296/1/Chong_sh_2001.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Ultrafast Dynamics of Barrier Crossing: Step-Wise Solvation Effect on Isomerization of Trans-Stilbene in Alkane Clusters", "author": [ { "family_name": "Chong", "given_name": "Sing Hwa", "clpid": "Chong-Sing-Hwa" } ], "thesis_advisor": [ { "family_name": "Zewail", "given_name": "Ahmed H.", "clpid": "Zewail-A-H" }, { "family_name": "Bercaw", "given_name": "John E.", "clpid": "Bercaw-J-E" } ], "thesis_committee": [ { "family_name": "Okumura", "given_name": "Mitchio", "orcid": "0000-0001-6874-1137", "clpid": "Okumura-M" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" }, { "family_name": "McKoy", "given_name": "Basil Vincent", "clpid": "McKoy-B-V" }, { "family_name": "Zewail", "given_name": "Ahmed H.", "clpid": "Zewail-A-H" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The ultrafast spectroscopic study of molecular clusters in supersonic beams can provide valuable information on the structure, energetics and dynamics of molecular aggregates in gas phase and in solution. This information will shed light on important issues such as how molecules interact, how energy flows in solvated systems, and how chemical reactions progress.
\r\n\r\nAlthough microscopic friction and solvation in barrier crossing reactions is of fundamental importance in molecular dynamics, their roles are not well understood. This is mainly due to the fact that few comprehensive investigations of this subject have been performed. In this thesis, the detail studies of a prototypical barrier crossing reaction\u2014the photoisomerization of jet-cooled trans-stilbene\u2014in size-selected n-alkane clusters, using the picosecond pump-probe ionization TOF mass spectrometry and transient technique, are reported. The microcanonical nonradiative decay rate constants at the S1 manifold for trans-stilbene-hexanen and trans-stilbene-octanen (n = 1, 2) complexes, including certain deuterated variants, were measured as a function of excitation energy, with the energy range defined by tuning the pump wavelength from the 0-0 transition of trans-stilbene to ~3200 cm-1 higher in energy. The experimental results were modeled with standard RRKM theory, nonadiabatic RRKM theory and Kramers-type theory for microcanonical systems. It was found that the excess energy dependence results could be accounted for very well by the nonadiabatic RRKM theory, from which analysis the barriers to isomerization for all of the trans-stilbene n-alkane clusters were found to be lower than that of the parent molecule by ~50%. The analysis revealed that not only can the differences in the rates among the four trans-stilbene-hexane1 isotopic species studied (combinations of trans-stilbene-h12 and -d12 with n-hexane-h14 and -d14) be attributed to energy friction, a term describing how energy is \"drained away\" from the reaction coordinate as a result of the change in the vibrational density of states, but the reduction in the nonradiative rates of the 1:2 complexes, relative to that of the 1:1 complexes, can also be attributed to the same energy friction.
\r\n\r\nFinally, in the same studies, the cluster binding energies of trans-stilbene-hexanen and trans-stilbene-octanen were also determined.
", "doi": "10.7907/5m8y-hz17", "publication_date": "2001", "thesis_type": "phd", "thesis_year": "2001" }, { "id": "thesis:4706", "collection": "thesis", "collection_id": "4706", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-12012008-142143", "primary_object_url": { "basename": "Sanford_ms_2001.pdf", "content": "final", "filesize": 10451013, "license": "other", "mime_type": "application/pdf", "url": "/4706/1/Sanford_ms_2001.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Synthetic and Mechanistic Investigations of Ruthenium Olefin Metathesis Catalysts", "author": [ { "family_name": "Sanford", "given_name": "Melanie Sarah", "orcid": "0000-0001-9342-9436", "clpid": "Sanford-Melanie-Sarah" } ], "thesis_advisor": [ { "family_name": "Grubbs", "given_name": "Robert H.", "orcid": "0000-0002-0057-7817", "clpid": "Grubbs-R-H" } ], "thesis_committee": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" }, { "family_name": "Grubbs", "given_name": "Robert H.", "orcid": "0000-0002-0057-7817", "clpid": "Grubbs-R-H" }, { "family_name": "MacMillan", "given_name": "David W. C.", "orcid": "0000-0003-3352-4532", "clpid": "MacMillan-D-W-C" }, { "family_name": "Bercaw", "given_name": "John E.", "clpid": "Bercaw-J-E" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The ruthenium-based catalysts (PCy[subscript 3])[subscript 2](Cl)[subscript 2]Ru=CHPh (1) and (IMesH[subscript 2])(PCy[subscript 3])(Cl)[subscript 2]Ru=CHPh (2) [IMesH[subscript 2] = 1,3-dimesityl-4,5-dihydroimidazol-2-ylidene] show high olefin metathesis activity in the presence of most common functional groups and have been widely used in synthetic chemistry. This thesis describes mechanistic, structural, and synthetic studies aimed at understanding the reactivity of these complexes, and at developing new olefin metathesis catalysts with superior properties.
\r\n\r\nChapter 2 details the effects of ligand variation on the mechanism and activity of ruthenium-based olefin metathesis catalysts. A series of ruthenium complexes of the general formula (L)(PR[subscript 3])(X)[subscript 2]Ru=CHR[superscript 1] were prepared, and the influence of the ancillary ligands L,X, R, and R[superscript 1] on the rates of phosphine dissociation and initiation as well as on the overall catalytic activity was examined.
\r\n\r\nChapter 3 describes the synthesis of a series of ruthenium benzylidenes containing N-heterocyclic carbene ligands. The new complexes, of the general formula (IMesH[subscript 2])(X)[subscript m](L)[subscript n]Ru=CHPh, were prepared using a variety of synthetic methods, and the bis-pyridine adduct (IMesH[subscript 2])(Cl)[subscript 2](C[subscript 5]H[subscript 5]N)[subscript 2]Ru=CHPh served as a particularly valuable synthon in these systems. Several of these compounds were characterized by X-ray crystallography, and the barriers to benzylidene and N-heterocyclic carbene rotation were determined using [superscript 1]H NMR spectroscopy.
\r\n\r\nChapter 4 describes the preparation of a series of four-coordinate ruthenium benzylidenes that serve as analogues of the 14-electron olefin metathesis intermediate (L)(Cl)[subscript 2]Ru=CHPh. These coordinatively unsaturated species have the general formula (L)(OR)[subscript 2]Ru=CHPh, and are stabilized by sterically bulky and \u03c0-donating alkoxide ligands, such as tert-butoxide, hexafluoro-tert-butoxide, and perfluoro-tert-butoxide. The new compounds were characterized by X-ray crystallography, and their reactivity with incoming ligands, including substituted alcohols, phenols, carboxylates, and pyridine, was investigated. In addition, their olefin metathesis activities were examined in the presence and absence of HCl co-catalyst.
\r\n\r\nChapters 5 and 6 describe the synthesis and characterization of neutral and cationic tris(pyrazolyl)borate ruthenium complexes. The new complexes were characterized by NMR spectroscopy and X-ray crystallography, and their olefin metathesis activities were explored.
", "doi": "10.7907/Q96P-VK05", "publication_date": "2001", "thesis_type": "phd", "thesis_year": "2001" }, { "id": "thesis:8148", "collection": "thesis", "collection_id": "8148", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:03192014-141855635", "primary_object_url": { "basename": "Brandow_cg_2001.pdf", "content": "final", "filesize": 38024719, "license": "other", "mime_type": "application/pdf", "url": "/8148/1/Brandow_cg_2001.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Zirconocenes as Models for Homogeneous Ziegler-Natta Olefin Polymerization Catalysts", "author": [ { "family_name": "Brandow", "given_name": "Christopher Graham", "clpid": "Brandow-Christopher-Graham" } ], "thesis_advisor": [ { "family_name": "Goddard", "given_name": "William A., III", "orcid": "0000-0003-0097-5716", "clpid": "Goddard-W-A-III" } ], "thesis_committee": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" }, { "family_name": "Goddard", "given_name": "William A., III", "orcid": "0000-0003-0097-5716", "clpid": "Goddard-W-A-III" }, { "family_name": "Bercaw", "given_name": "John E.", "clpid": "Bercaw-J-E" }, { "family_name": "Grubbs", "given_name": "Robert H.", "orcid": "0000-0002-0057-7817", "clpid": "Grubbs-R-H" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Using density functional theory, we studied the fundamental steps of olefin \r\npolymerization for zwitterionic and cationic Group IV ansa-zirconocenes and a neutral ansa-\r\nyttrocene. Complexes [H2E(C5H4)2ZrMe]n (n = 0: E = BH2 (1), BF2 (2), AlH2(3); n = +: E = CH2(4), SiH2(5)) and \r\nH2Si(C5H4)2YMe were used as computational models. The largest\r\ndifferences among these three classes of compounds were the strength of olefin binding and the \r\nstability of the \u03b2-agostic alkyl intermediate towards \u03b2-hydrogen elimination. We investigated \r\nthe effect of solvent on the reaction energetics for land 5. We found that in benzene the \r\nenergetics became very similar except that a higher olefin insertion barrier was\r\ncalculated for 1. The calculated anion affinity of [CH3BF3]- was weaker towards 1 than 5. The\r\ncalculated olefin binding depended primarily on the charge of the ansa linker, and the olefin \r\ninsertion barrier was found to decrease steadily in the following order: [H2C(C5H4)2ZrMe]+ > [F2B(C5H4)2ZrMe] \u2248 [H2B(C5H4)2ZrMe] > [H2Si(C5H4)2ZrMe]+ > \r\n[H2Al(C5H4)2ZrMe].
\r\n\r\n\r\nWe prepared ansa-zirconocene dicarbonyl complexes Me2ECp2Zr(CO)2 (E = Si, C), and\r\nt-butyl substituted complexes (t-BuCp)2Zr(CO)2, Me2E(t-BuCp)2Zr(CO)2 (E = Si, C),\r\n(Me2Si)2(t-BuCp)2Zr(CO)2 as well as analogous zirconocene complexes. Both the reduction\r\npotentials and carbonyl stretching frequencies follow the same order: Me2SiCp2ZrCl2>\r\nMe2CCp2ZrCl2> Cp2ZrCl2> (Me2Si)2Cp2ZrCl2. This ordering is a result of both the donating\r\nabilities of the cyclopentadienyl substituents and the orientation of the cyclopentadiene rings.\r\nAdditionally, we prepared a series of analogous cationic zirconocene complexes\r\n[LZrOCMe3][MeB(C6F5)3] (L = CP2, Me2SiCp2, Me2CCP2, (Me2Si)2Cp2) and studied the kinetics of anion dissociation. We found that the enthalpy of anion dissociation increased from 10.3 kcal\u2022mol-1 to 17.6 kcal\u2022mol-1 as exposure of the zirconium center increased.
\r\n\r\nWe also prepared series of zirconocene complexes bearing 2,2-dimethyl-2-sila-4-pentenyl substituents (and methyl-substituted olefin variants). Methide abstraction with B(C6F5) results in reversible coordination of the tethered olefin to the cationic zirconium center. The kinetics of olefin dissociation have been examined using NMR methods, and the effects of ligand variation for unlinked, singly [SiMe2]-linked and doubly [SiMe2]-linked bis(cyclopentadienyl) arrangements has been compared (\u0394G\u2021 for olefin dissociation varies from 12.8 to 15.6 kcal\u2022mol-1). Methide abstraction from 1,2-(SiMe2)2(\u03b75-C5H3)2Zr(CH3)-(CH2CMe2CH2CH = CH2) results in rapid \u03b2-allyl elimination with loss of isobutene yielding the allyl cation [{1,2-(SiMe2)2(\u03b75-C5H3)2Zr(\u03b73-CH2CH=CH2)]+.
\r\n", "doi": "10.7907/kxb2-wp19", "publication_date": "2001", "thesis_type": "phd", "thesis_year": "2001" }, { "id": "thesis:8157", "collection": "thesis", "collection_id": "8157", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:03212014-095851500", "primary_object_url": { "basename": "odom 2001.pdf", "content": "final", "filesize": 39129217, "license": "other", "mime_type": "application/pdf", "url": "/8157/1/odom 2001.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "The Application of Metallointercalators in Recognition of and Charge Transport in Nucleic Acids", "author": [ { "family_name": "Odom", "given_name": "Duncan T.", "orcid": "0000-0001-6201-5599", "clpid": "Odom-Duncan-T" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" }, { "family_name": "Parker", "given_name": "Carl Stevens", "orcid": "0000-0001-9795-4211", "clpid": "Parker-C-S" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Metal complexes that utilize the 9,10-phenanthrene quinone diimine (phi) moiety\r\nbind to DNA through the major groove. These metallointercalators can recognize DNA\r\nsites and perform reactions on DNA as a substrate. The site-specific metallointercalator\r\n\u039b-1-Rh(MGP)_2phi^(5+) competitively disrupts the major groove binding of a transcription\r\nfactor, yAP-1, from an oligonucleotide that contains a common binding site. The\r\ndemonstration that metal complexes can prevent transcription factor binding to DNA site-specifically\r\nis an important step in using metallointercalators as therapeutics.
\r\n\r\nThe distinctive photochemistry of metallointercalators can also be applied to\r\npromote long range charge transport in DNA. Experiments using duplexes with regions\r\n4 to 10 nucleotides long containing strictly adenine and thymine sequences of varying\r\norder showed that radical migration is more dependent on the sequence of bases, and\r\nless dependent on the distance between the guanine doublets. This result suggests that\r\nmechanistic proposals of long range charge transport must involve all the bases.
\r\n\r\nRNA/DNA hybrids show charge migration to guanines from a remote site, thus\r\ndemonstrating that nucleic acid stacking other than B-form can serve as a radical bridge.\r\nDouble crossover DNA assemblies also provide a medium for charge transport at\r\ndistances up to 100 \u00c5 from the site of radical introduction by a tethered metal complex.\r\nThis radical migration was found to be robust to mismatches, and limited to individual,\r\nelectronically distinct base stacks. In single DNA crossover assemblies, which have\r\nconsiderably greater flexibility, charge migration proceeds to both base stacks due to\r\nconformational isomers not present in the rigid and tightly annealed double crossovers.
\r\n\r\nFinally, a rapid, efficient, gel-based technique was developed to investigate\r\nthymine dimer repair. Two oligonucleotides, one radioactively labeled, are photoligated\r\nvia the bases of a thymine-thymine interface; reversal of this ligation is easily visualized\r\nby gel electrophoresis. This assay was used to show that the repair of thymine dimers\r\nfrom a distance through DNA charge transport can be accomplished with different\r\nphotooxidants.
\r\n\r\nThus, nucleic acids that support long range charge transport have been shown to\r\ninclude A-track DNA, RNA/DNA hybrids, and single and double crossovers, and a\r\nmethod for thymine dimer repair detection using charge transport was developed.\r\nThese observations underscore and extend the remarkable finding that DNA can serve a\r\nmedium for charge transport via the heteroaromatic base stack.
\r\n\r\n", "doi": "10.7907/yz1f-w961", "publication_date": "2001", "thesis_type": "phd", "thesis_year": "2001" }, { "id": "thesis:8158", "collection": "thesis", "collection_id": "8158", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:03212014-101732487", "primary_object_url": { "basename": "Royea_wj_2001.pdf", "content": "final", "filesize": 28591395, "license": "other", "mime_type": "application/pdf", "url": "/8158/1/Royea_wj_2001.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Investigations of Charge-Carrier Dynamics at Semiconductor/Liquid lnterfaces", "author": [ { "family_name": "Royea", "given_name": "William Joseph", "clpid": "Royea-William-Joseph" } ], "thesis_advisor": [ { "family_name": "Lewis", "given_name": "Nathan Saul", "orcid": "0000-0001-5245-0538", "clpid": "Lewis-N-S" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" }, { "family_name": "Marcus", "given_name": "Rudolph A.", "orcid": "0000-0001-6547-1469", "clpid": "Marcus-R-A" }, { "family_name": "Lewis", "given_name": "Nathan Saul", "orcid": "0000-0001-5245-0538", "clpid": "Lewis-N-S" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Theoretical and experimental investigations of charge-carrier dynamics at\r\nsemiconductor/liquid interfaces, specifically with respect to interfacial electron transfer\r\nand surface recombination, are presented.
\r\n\r\nFermi's golden rule has been used to formulate rate expressions for charge transfer\r\nof delocalized carriers in a nondegenerately doped semiconducting electrode to localized,\r\nouter-sphere redox acceptors in an electrolyte phase. The treatment allows comparison\r\nbetween charge-transfer kinetic data at metallic, semimetallic, and semiconducting\r\nelectrodes in terms of parameters such as the electronic coupling to the electrode, the\r\nattenuation of coupling with distance into the electrolyte, and the reorganization energy\r\nof the charge-transfer event. Within this framework, rate constant values expected at\r\nrepresentative semiconducting electrodes have been determined from experimental data\r\nfor charge transfer at metallic electrodes. The maximum rate constant (i.e., at optimal\r\nexoergicity) for outer-sphere processes at semiconducting electrodes is computed to be in\r\nthe range 10-17-10-16 cm4 s-1, which is in excellent agreement with prior theoretical\r\nmodels and experimental results for charge-transfer kinetics at semiconductor/liquid\r\ninterfaces.
\r\n\r\nDouble-layer corrections have been evaluated for semiconductor electrodes in both\r\ndepletion and accumulation conditions. In conjuction with the Gouy-Chapman-Stern\r\nmodel, a finite difference approach has been used to calculate potential drops at a\r\nrepresentative solid/liquid interface. Under all conditions that were simulated, the\r\ncorrection to the driving force used to evaluate the interfacial rate constant was\r\ndetermined to be less than 2% of the uncorrected interfacial rate constant.
\r\n\r\nPhotoconductivity decay lifetimes have been obtained for Si(111) in contact with\r\nsolutions of CH3OH or tetrahydrofuran containing one-electron oxidants. Silicon\r\nsurfaces in contact with electrolyte solutions having Nernstian redox potentials > 0 V vs.\r\nSCE exhibited low effective surface recombination velocities regardless of the different\r\nsurface chemistries. The formation of an inversion layer, and not a reduced density of\r\nelectrical trap sites on the surface, is shown to be responsible for the long charge-carrier\r\nlifetimes observed for these systems. In addition, a method for preparing an air-stable,\r\nlow surface recombination velocity Si surface through a two-step, chlorination/alkylation\r\nreaction is described.
", "doi": "10.7907/gc45-ed38", "publication_date": "2001", "thesis_type": "phd", "thesis_year": "2001" }, { "id": "thesis:11371", "collection": "thesis", "collection_id": "11371", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:01312019-135136732", "type": "thesis", "title": "Reactions of Heme Proteins to Solutions and Crystals", "author": [ { "family_name": "Tezcan", "given_name": "Faik Akif", "orcid": "0000-0002-4733-6500", "clpid": "Tezcan-Faik-Akif" } ], "thesis_advisor": [ { "family_name": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" }, { "family_name": "Bercaw", "given_name": "John E.", "clpid": "Bercaw-J-E" }, { "family_name": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" }, { "family_name": "Winkler", "given_name": "Jay Richmond", "orcid": "0000-0002-4453-9716", "clpid": "Winkler-J-R" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "To assess the effects of heme solvation and iron ligation on reduction potentials in c-type cytochromes, we have examined the redox and ligand-binding properties of microperoxidase-8 (MP8). Methionine-, histidine- and amine-coordination to MP8 were found to account for 130, -40 and -10-mV shifts in the Fe(III/II)-potential, respectively. Our finding that reduction potentials increase with decreasing heme-surface exposure suggests that the protein matrix can further tune the reduction potential by 500 mV through water exclusion from the heme pocket.
\r\n\r\nThe 410-mV upshift in the cytochrome c (cyt c) potential as the heme cofactor is moved from a highly-solvated environment to the protein interior signals a 10-kcal/mol greater stability of the reduced form. Consequently, there exists a range of denaturant concentrations where Fe(II)-cyt c is folded and Fe(III)-cyt c is unfolded. Electron injection into the oxidized protein in this range triggers the folding reaction. Using NADH as a redox photosensitizer, cyt c folding can be initiated within 100 \u00b5s. Our results suggest that the folding of cyt c is rate-limited by ligand-substitution events on the iron center.
\r\n\r\nDue to an increased barrier to ligand substitution, folding of Co(III)-substituted cyt cis 5 orders of magnitude slower than Fe-cyt c. The slow folding kinetics of Co(III)-cyt c have allowed the convenient study of protein dynamics with a variety of spectroscopic techniques, revealing previously unresolved folding pathways involving Lys- and His-misligated populations of the unfolded molecule and extremely long-lived folding intermediates.
\r\n\r\nFactors that control electron flow between proteins are not well understood, owing to uncertainties in the relative orientations and structures of the reactants during the short time that tunneling occurs. To circumvent this ambiguity, we have measured the kinetics of electron transfer (ET) between native and Zn-substituted tuna cyt c molecules in crystals of known structure. ET rates (320 s-1 for *Zn-cyt c \u2192 Fe(III)-cyt c; 2000 s-1 for Fe(II)-cyt c \u2192 Zn-cyt c+) over a Zn-Fe distance of 24.1 \u00c5 closely match those for intraprotein ET over similar donor-acceptor separations. Our results indicate that van der Waals interactions and water mediated H-bonds provide effective electronic coupling across a protein-protein interface.
", "doi": "10.7907/rrqd-9v38", "publication_date": "2001", "thesis_type": "phd", "thesis_year": "2001" }, { "id": "thesis:17666", "collection": "thesis", "collection_id": "17666", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:09042025-153842319", "primary_object_url": { "basename": "Shogren-Knaak_MA_2000.pdf", "content": "final", "filesize": 51871805, "license": "other", "mime_type": "application/pdf", "url": "/17666/1/Shogren-Knaak_MA_2000.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Incorporating Function into \u03b2\u03b2\u03b1-Motif Peptide Scaffolds", "author": [ { "family_name": "Shogren-Knaak", "given_name": "Michael Aaron", "orcid": "0000-0001-6526-3977", "clpid": "Shogren-Knaak-Michael-Aaron" } ], "thesis_advisor": [ { "family_name": "Imperiali", "given_name": "Barbara", "orcid": "0000-0002-5749-7869", "clpid": "Imperiali-B" } ], "thesis_committee": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" }, { "family_name": "Imperiali", "given_name": "Barbara", "orcid": "0000-0002-5749-7869", "clpid": "Imperiali-B" }, { "family_name": "Dougherty", "given_name": "Dennis A.", "orcid": "0000-0003-1464-2461", "clpid": "Dougherty-D-A" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The generation of functional biomolecules constitutes an important and\r\nchallenging goal in bioorganic chemistry. Efforts to incorporate chemical\r\nfunctionality into a \u03b2\u03b2\u03b1-motif (BBA) peptide scaffold are presented. In the course\r\nof this work we have utilized this system to help determine to what extent folded\r\npeptide scaffolds can support chemical function, which strategies are best suited\r\nto the discovery of functional peptides, and what can be learned about biological\r\ncatalysis. To address these issues several different strategies involving the BBA\r\npeptide scaffold have been explored.
\r\n\r\nInitial efforts focused on using the BBA peptide scaffold to modulate the\r\nreactivity of a non-natural pyridoxamine-associated amino acid (Pam). The Pam\r\nresidue was incorporated at three different positions in the BBA peptide, and\r\ndifferent potential general acid and general base residues were included. These\r\npeptides, BP1-BP6, showed improved transamination rates and stereoselectivity\r\nrelative to a model pyridoxamine compound. Additionally, one peptide, BPS,\r\nmaintained many of the secondary and super-secondary structural features of\r\nthe BBA peptide scaffold. A second generation of Pam-containing peptides,\r\npeptides CBP01-CBP18, was constructed based on BPS to investigate the effects\r\nof systematic amino acid changes in the vicinity of the pyridoxamine\r\nfunctionality. These peptides also exhibited enhanced rates of transamination\r\nand stereoselectivity and showed clear trends in stereoselectivity as a function of\r\nthe amino acid substitutions. Finally, peptide systems utilizing other\r\nmechanisms of influencing pyridoxamine-mediated transamination were\r\ninvestigated.
\r\n\r\nSubsequent work, undertaken in collaboration with Kevin McDonnell,\r\nsought to address some of the limitations of the Pam amino acid approach. This\r\neffort focused on developing high-throughput techniques for finding BBA\r\npeptides capable of mediating aldol condensation. A library of over 100,000\r\ndifferent BBA peptides, the CPLB peptides, was designed to incorporate one of\r\nseveral possible basic residues within a core of many potential residues. These\r\npeptides were synthesized and assayed for their ability to sequester a fluorescent\r\nprobe, DMED, which resembles the reaction intermediates in aldol condensation.\r\nThe \"winners\" of this screen exhibited a high degree of sequence similarity,\r\nexhibiting \u03b2-diaminoproprionic acid (Dap) exclusively as the basic residue, and\r\nfavoring aromatic, hydrophobic amino acids as the neighboring residues.\r\nStructural and functional characterization of one of these \"winners,\" peptide\r\nCPLB-A2, was performed. Despite the inclusion of the Dap group within the\r\nhydrophobic core, this peptide appeared to be a monomeric species with a high\r\ndegree of the secondary and super-secondary structure expected of a BBA\r\npeptide. This peptide demonstrated an enhanced ability to sequester DMED\r\nrelative to Dap-containing model peptides and had the capacity to generate an\r\nenamineone reaction intermediate.
\r\n\r\nIn the course of these efforts, techniques were created to aid in the\r\npurification and characterization of N-terminally capped peptides. An affinity\r\npurification method involving a reversible biotin-based capping group was\r\ndeveloped to aid in the isolation of N-terminal glycolyl-capped peptides. This\r\nmethod proved practical for the efficient parallel purification of peptides. A\r\ncapping method involving an \u03b1-chloroacetyl group was developed to generate\r\nN-terminally capped peptides that were not only stable to standard assay\r\nconditions but also capable of being transformed to a form compatible with\r\nEdman sequencing. This method proved to be effective for the identification of\r\nunknown N-terminally capped peptides.
", "doi": "10.7907/wg2w-q908", "publication_date": "2000", "thesis_type": "phd", "thesis_year": "2000" }, { "id": "thesis:10783", "collection": "thesis", "collection_id": "10783", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:03302018-093456214", "primary_object_url": { "basename": "Kielkopf_CL_2000.pdf", "content": "final", "filesize": 46362175, "license": "other", "mime_type": "application/pdf", "url": "/10783/1/Kielkopf_CL_2000.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Structural Basis of DNA Recognition by Synthetic Ligands", "author": [ { "family_name": "Kielkopf", "given_name": "Clara Louise", "clpid": "Kielkopf-Clara-Louise" } ], "thesis_advisor": [ { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" } ], "thesis_committee": [ { "family_name": "Dervan", "given_name": "Peter B.", "clpid": "Dervan-P-B" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Roberts", "given_name": "Richard W.", "clpid": "Roberts-R-W" }, { "family_name": "Bjorkman", "given_name": "Pamela J.", "clpid": "Bjorkman-P-J" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "The DNA double helix presents functional groups in the major and minor grooves that can be used by ligands for readout of the base pair sequence. The overall flexibility and shape also distinguishes various sequences. Although the information displayed in the major groove is more diverse, small DNA-binding polyamides predictably distinguish all four base pairs in the minor groove. In this work, the structural basis of this recognition has been studied using x-ray crystallographic techniques, and is described for G\u2022C (Chapter 2) and T\u2022A base pairs (Chapter 3-4). The polyamides directly read the DNA sequence using a combination of specific hydrogen bonds and shape selection. A second class of ligands called intercalators bind to DNA via the major groove by slipping a planar aromatic ligand between the bases. Although this shape-selective DNA binding is relatively nonspecific, the substitution of the ancillary ligands on octahedral metallointercalators allows specific sequences to be recognized in the major groove. The high resolution crystal structure of a designed metal complex bound to its target site reveals the nature of these specific contacts, as well as the precise stacking of the ligand between the bases. A detailed understanding of specific DNA recognition by small molecules is important for their further development as tools for molecular biology and medicine. These structures reveal how synthetic ligands can distinguish all four base pairs in both the major and the minor grooves of DNA.", "doi": "10.7907/73K1-K290", "publication_date": "2000", "thesis_type": "phd", "thesis_year": "2000" }, { "id": "thesis:13596", "collection": "thesis", "collection_id": "13596", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:11222019-095315401", "primary_object_url": { "basename": "scholl-m-2000.pdf", "content": "final", "filesize": 4431025, "license": "other", "mime_type": "application/pdf", "url": "/13596/1/scholl-m-2000.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Expanding the Scope of Ruthenium-Based Olefin Metathesis Catalysts", "author": [ { "family_name": "Scholl", "given_name": "Matthias", "clpid": "Scholl-Matthias" } ], "thesis_advisor": [ { "family_name": "Grubbs", "given_name": "Robert H.", "clpid": "Grubbs-R-H" } ], "thesis_committee": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Goddard", "given_name": "William A., III", "clpid": "Goddard-W-A-III" }, { "family_name": "Grubbs", "given_name": "Robert H.", "clpid": "Grubbs-R-H" }, { "family_name": "Peters", "given_name": "Jonas C.", "clpid": "Peters-J-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The development of well-defined ruthenium alkylidene (PCy\u2083)\u2082Cl\u2082Ru=CHPh brought about a revolution in the area of olefin metathesis. The objective of the work presented here is to expand the scope of ruthenium-based olefin metathesis catalysts such as (PCy\u2083)\u2082Cl\u2082Ru=CHPh through the development of novel synthetic organic methods for ring-closing metathesis as well as through modification of the ligand sphere of the ruthenium complexes.
\r\n\r\nChapter 2 describes the application of ruthenium alkylidenes to the catalysis of polycyclization reactions. Several acyclic precursors have been synthesized and reacted with (PCy\u2083)\u2082Cl\u2082Ru=CHPh. These precursors vary in topology and contain acetylenic and/or cycloolefinic metathesis relays. The cyclization reactions proceed in good yields to produce polycyclic polyenes.
\r\n\r\nChapter 3 focuses on the synthesis of racemic and enantiopure targets containing the 6,8-dioxabicycIo [3.2.1]octane skeleton using an intramolecular ruthenium-catalyzed ring-closing metathesis reaction as the key step. The natural product frontalin is synthesized in racemic and enantiopure forms and in excellent yields using this methodology.
\r\n\r\nChapter 4 outlines the preparation of a novel imidazolylidene-substituted ruthenium-based complex starting from (PCy\u2083)\u2082RuCl\u2082(=CHPh). The N-heterocyclic carbene-substituted olefin metathesis initiator exhibits increased ring-closing metathesis activity at elevated temperature compared to that of the parent complex (PCy\u2083)\u2082Cl\u2082Ru(=CHPh). Di-, tri-, and tetra-substituted cycloolefins are successfully prepared from corresponding diene precursors in moderate to excellent yields.
\r\n\r\nChapter 5 describes the preparation of a new family of 1,3-dimesityl-4,5-dihydroimidazol-2-ylidene-substituted ruthenium-based complexes. These air and water tolerant systems exhibit an increased ring-closing metathesis activity at elevated temperature when compared to that of the parent complex (PCy\u2083)\u2082Cl\u2082Ru(=CHPh) as well as to the complexes disclosed previously in Chapter 4. In many instances the activity of these new complexes also rivals or exceeds that of the alkoxy-imido molybdenum-based olefin metathesis catalysts. Applications of chiral N-heterocyclic carbene ruthenium complexes to asymmetric ring-closing metathesis are also briefly discussed.
\r\n\r\nFinally, the synthesis of the Schiff base-substituted ruthenium carbene complexes on a solid support is described in Chapter 6. The activities of the supported complexes are compared to those of their unsupported counterparts. The newly prepared systems are found to be highly stable to air, moisture, and temperature, and exhibit increased catalytic activity in acidic media.
", "doi": "10.7907/55x3-h359", "publication_date": "2000", "thesis_type": "phd", "thesis_year": "2000" }, { "id": "thesis:17853", "collection": "thesis", "collection_id": "17853", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:02022026-212838189", "primary_object_url": { "basename": "Dmochowski_IJ_2000.pdf", "content": "final", "filesize": 64940562, "license": "other", "mime_type": "application/pdf", "url": "/17853/1/Dmochowski_IJ_2000.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Probing Cytochrome P450 with Sensitizer-Linked Substrates", "author": [ { "family_name": "Dmochowski", "given_name": "Ivan Julian", "orcid": "0000-0001-7162-1347", "clpid": "Dmochowski-Ivan-Julian" } ], "thesis_advisor": [ { "family_name": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" } ], "thesis_committee": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" }, { "family_name": "Dervan", "given_name": "Peter B.", "orcid": "0000-0001-8852-7306", "clpid": "Dervan-P-B" }, { "family_name": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The covalent attachment of the photosensitizer [Ru(bpy)3]2+ to a substrate\r\nconstitutes a powerful new method for probing the steric and electronic properties of\r\nburied enzyme active sites. Particularly important targets are oxygenases (cytochromes\r\nP450) involved in drug metabolism and many disease states. The crystal structure of a\r\nP450:Ru-adamantyl complex reveals that the substrate moiety gains access to the active\r\ncenter via a deep channel and rests above the heme much like the natural substrate\r\ncamphor. This structure also identifies significant P450 conformational changes\r\nassociated with substrate binding for the first time: the channel opens via a 6-\u00c5 loop\r\nmovement. Turnover studies show that activity is not significantly diminished in the\r\nP450:Ru-adamantyl complex. Preliminary light-activated substrate turnover experiments\r\nalso show promise.
\r\n\r\nThe binding of Ru-substrates to P450 was measured by time-resolved\r\nluminescence and UV-vis assays. These molecules specifically recognize submicromolar\r\ncytochrome P450cam in the presence of other heme proteins. In the P450:Ru-substrate\r\nconjugates, energy transfer to the heme dramatically accelerates the Ru-luminescence\r\ndecay. Quantifying the fraction of quenched Ru2+* provides an accurate method for\r\ndetermining dissociation constants. In addition, for the P450:Ru-adamantyl complex,\r\nForster analysis of the energy-transfer kinetics yields a Ru-Fe distance (21 \u00c5) virtually\r\nthe same as that measured in the crystal structure. Similar analysis for other Ru-substrates\r\nof varying length shows a narrow range of Ru-Fe distances, indicating\r\nfavorable association of the {Ru(bpy)3}2+ moiety with the protein. The binding of the \u039b\r\nand \u0394 enantiomers of Ru-C9-Ad to P450 was measured, and the results, KD(\u0394/\u039b) ~2,\r\nsuggest that the bipyridyl ligands interact with aromatic residues at the mouth of the\r\nsubstrate channel. Thus, enantiospecific interactions may be exploited in the design of\r\nenzyme-metallosubstrate conjugates.
\r\n\r\nOxidative and reductive quenching of P450:[Ru-(CH2)n-substrate]2+* conjugates in\r\nsolution triggers the injection of holes and electrons from ruthenium to the heme on submillisecond\r\ntime scales. In order to accelerate electron transfer, conjugated\r\n(perfluorobiphenyl-bridged) Ru-probes were synthesized which bind P450 strongly (KD<\r\n1 \u03bcM). Photoexcitation of conjugated Ru-imidazole-P450 complexes reduces the heme\r\non submicrosecond time scales, opening new avenues for the study of short-lived enzyme\r\nintermediates.
", "doi": "10.7907/89h6-qd11", "publication_date": "2000", "thesis_type": "phd", "thesis_year": "2000" }, { "id": "thesis:17619", "collection": "thesis", "collection_id": "17619", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:08132025-181322821", "primary_object_url": { "basename": "Bremer_RE_2000.pdf", "content": "final", "filesize": 48189409, "license": "other", "mime_type": "application/pdf", "url": "/17619/1/Bremer_RE_2000.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Inhibition of DNA Major Groove Binding Proteins by Hairpin Polyamides", "author": [ { "family_name": "Bremer", "given_name": "Ryan Elwood", "clpid": "Bremer-Ryan-Elwood" } ], "thesis_advisor": [ { "family_name": "Hoffmann", "given_name": "Michael R.", "orcid": "0000-0001-6495-1946", "clpid": "Hoffmann-M-R" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Hoffmann", "given_name": "Michael R.", "orcid": "0000-0001-6495-1946", "clpid": "Hoffmann-M-R" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" }, { "family_name": "Dervan", "given_name": "Peter B.", "orcid": "0000-0001-8852-7306", "clpid": "Dervan-P-B" }, { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Small molecules that bind to any predetermined DNA sequence in the human\r\ngenome are potentially useful tools for molecular biology and human medicine.\r\nPolyamides containing N-methylimidazole (Im) and N-methylpyrrole (Py) are cell\r\npermeable small molecules that bind DNA according to a set of \"pairing rules\" with\r\naffinities and specificites similar to many naturally occurring DNA binding proteins.\r\nIm/Py polyamides offer a general approach to the chemical regulation of gene expression,\r\nprovided inhibition of DNA binding for a variety of transcription factor families can be\r\nachieved. Polyamides bound in the minor groove have been shown to co-occupy the DNA\r\nhelix with proteins in the major groove. We demonstrate here that polyamides containing\r\na positively charged moiety directed to the DNA backbone can effectively inhibit DNA\r\nbinding by an exclusively major groove protein, potentially by competing with the\r\npositively charged protein side chains for contacts to the negatively charged phosphate\r\nbackbone. The requisite positive patch can be achieved with a naturally derived C-terminal\r\nArg-Pro-Arg tripeptide (Chapter 2) or a simple synthetic diaminoalkyl chain\r\ndelivered from the N-1 of a single pyrrole residue (Chapter 3). The functional repertoire\r\nof poly amides as synthetic ligands for the control of transcription factor binding has been\r\nexpanded to include proteins which bind exclusively in the major groove of DNA. The\r\nbroad targetable sequence repertoire of polyamides, coupled with the ubiquity of\r\nbackbone contacts in protein recognition of DNA, make phosphate neutralization by a\r\npositive patch a promising approach for inhibition of major groove transcription factors.\r\nOther investigations into polyamide:DNA recognition have afforded N=aminoalkylpyrrole-\r\ncontaining polyamides that offer enhanced affinity without\r\ncompromising specificity (Chapter 3), desmethylpyrrole-containing polyamides that\r\nincrease water solubility while retaining subnanomolar affinity (Chapter 4 ), and structural\r\ninsight into the lower affinities observed with Hp-containing polyamides (Chapter 5).", "doi": "10.7907/0rz0-ah38", "publication_date": "2000", "thesis_type": "phd", "thesis_year": "2000" }, { "id": "thesis:3784", "collection": "thesis", "collection_id": "3784", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-09262005-133922", "primary_object_url": { "basename": "Blackwell_he_1999.pdf", "content": "final", "filesize": 8482978, "license": "other", "mime_type": "application/pdf", "url": "/3784/1/Blackwell_he_1999.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "New synthetic applications for ring-closing metathesis and cross-metathesis employing well-defined ruthenium alkylidenes", "author": [ { "family_name": "Blackwell", "given_name": "Helen Elizabeth", "clpid": "Blackwell-H-E" } ], "thesis_advisor": [ { "family_name": "Grubbs", "given_name": "Robert H.", "clpid": "Grubbs-R-H" } ], "thesis_committee": [ { "family_name": "Grubbs", "given_name": "Robert H.", "clpid": "Grubbs-R-H" }, { "family_name": "Imperiali", "given_name": "Barbara", "clpid": "Imperiali-B" }, { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "NOTE: Text or symbols not renderable in plain ASCII are indicated by [...]. Abstract is included in .pdf document.\n\nThe development of well-defined ruthenium alkylidene [...] (1) has revolutionized the application of the olefin metathesis reaction in organic synthesis. This thesis describes: (1) the application of ring-closing metathesis (RCM) to the synthesis of constrained cyclic peptides, (2) the conformational analyses of these peptide architectures, and (3) the development of new selective cross-metathesis (CM) methodology employing alkylidene 1.\n\nChapter 2 describes the initial application of RCM to the synthesis of cyclic amino acids and peptide [...]. Introduction of olefin functionality into peptidic structures proved straightforward, and treatment with ruthenium alkylidene 1 generated cyclic amino acids and dipeptides in excellent yields. Tetrapeptide diene analogs of naturally-occurring disulfide [...] proved to be robust substrates for RCM, and the macrocyclic olefin products could be prepared either in solution or on solid support.\n\nChapter 3 describes the application of RCM to the synthesis of macrocyclic hexapeptide [Beta]-sheets. The acyclic hexapeptide diene frameworks were modeled after hexapeptide disulfides reported to adopt [Beta]-sheet conformations. Treatment with ruthenium alkylidene 1, however, afforded only low yields of the desired 20-membered macrocycles: conformational analyses suggested that the hexapeptides adopted a helical rather than [Beta]-sheet conformation in the apolar solvents in which RCM was performed. These peptide systems were redesigned in Chapter 4 so that macrocyclization was favored when the peptides adopted a helical conformation. A series of macrocyclic heptapeptides were prepared in high yield via RCM which were shown to adopt [...]-helical conformations both in solution and in the solid state.\n\nChapter 5 describes new CM methodology involving the coupling of terminal olefins with symmetrically disubstituted olefins to generate heterodimeric cross-products in excellent yields and with high trans selectivity. CM was employed in an initial self-metathesis step to synthesize a variety of disubstituted olefins with diverse functionality, which were then further processed in the CM with terminal olefins. Finally, a new CM application was introduced involving the metathesis of acrolein acetal derivatives with terminal olefins to generate [...] aldehydes.\n", "doi": "10.7907/XBZ4-CV61", "publication_date": "1999", "thesis_type": "phd", "thesis_year": "1999" }, { "id": "thesis:16275", "collection": "thesis", "collection_id": "16275", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:01092024-222740445", "primary_object_url": { "basename": "Kim_CH_1999.pdf", "content": "final", "filesize": 67561800, "license": "other", "mime_type": "application/pdf", "url": "/16275/1/Kim_CH_1999.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Site-Specific Chemical Modification and Crosslinking Studies of U6 snRNA in the Yeast spliceosome", "author": [ { "family_name": "Kim", "given_name": "Chang Hee", "clpid": "Kim-Chang-Hee" } ], "thesis_advisor": [ { "family_name": "Abelson", "given_name": "John N.", "clpid": "Abelson-J-N" } ], "thesis_committee": [ { "family_name": "Campbell", "given_name": "Judith L.", "clpid": "Campbell-J-L" }, { "family_name": "Abelson", "given_name": "John N.", "clpid": "Abelson-J-N" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" }, { "family_name": "Parker", "given_name": "Carl Stevens", "orcid": "0000-0001-9795-4211", "clpid": "Parker-C-S" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Pre-mRNA splicing is an essential step in eukaryotic gene expression. Most eukaryotic genes are interrupted by sequences, called introns, that do not code for sequences in the final protein gene product. Pre-mRNA splicing is the process by which introns are removed and the coding sequences, called exons, are joined together following transcription of a gene into RNA. The spliced mRNA subsequently acts as the coding template for protein synthesis. Pre-mRNA splicing occurs in a large ribonucleoprotein particle called the spliceosome which includes five essential small nuclear RNAs (snRNAs) and more than 50 different proteins. It is thought that the active site of the spliceosome consists of U2, U6 and U5 snRNAs. In order to probe the structure of the catalytic site, we have done an extensive site-specific chemical modification and crosslinking studies of U6 RNA in the yeast spliceosome. From an extensive screen of site-specific 2'-deoxy modifications, we have found four that block the first step of splicing, yet are able to assemble spliceosome complexes which precede the reactive spliceosome, suggesting that the 2'-hydroxyls at these nucleotides may be required for catalysis. In the crosslinking experiments, we found crosslinks between 4-thioU placed in the central conserved sequences in U6 with the 5' splice site of the pre-mRNA, which participates in the first chemical step of splicing. We were able to determine the order of this crosslink by doing the experiment in yeast mutant spliceosomes which were blocked in well-defined stages of splicing. We also found that a conserved nucleotide in the 3' stem-loop of U6 crosslinks to a nucleotide in a region of the actin pre-mRNA intron which is required for its splicing in vitro. We also found crosslinks to snRNAs -- U2 and U4. Certain positions in U6 can crosslink first to U4 snRNA, then subsequently to the pre-mRNA, consistent with the notion that the spliceosome is a dynamic machine.", "doi": "10.7907/cfw0-z473", "publication_date": "1999", "thesis_type": "phd", "thesis_year": "1999" }, { "id": "thesis:16275", "collection": "thesis", "collection_id": "16275", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:01092024-222740445", "primary_object_url": { "basename": "Kim_CH_1999.pdf", "content": "final", "filesize": 67561800, "license": "other", "mime_type": "application/pdf", "url": "/16275/1/Kim_CH_1999.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Site-Specific Chemical Modification and Crosslinking Studies of U6 snRNA in the Yeast spliceosome", "author": [ { "family_name": "Kim", "given_name": "Chang Hee", "clpid": "Kim-Chang-Hee" } ], "thesis_advisor": [ { "family_name": "Abelson", "given_name": "John N.", "clpid": "Abelson-J-N" } ], "thesis_committee": [ { "family_name": "Campbell", "given_name": "Judith L.", "clpid": "Campbell-J-L" }, { "family_name": "Abelson", "given_name": "John N.", "clpid": "Abelson-J-N" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" }, { "family_name": "Parker", "given_name": "Carl Stevens", "orcid": "0000-0001-9795-4211", "clpid": "Parker-C-S" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Pre-mRNA splicing is an essential step in eukaryotic gene expression. Most eukaryotic genes are interrupted by sequences, called introns, that do not code for sequences in the final protein gene product. Pre-mRNA splicing is the process by which introns are removed and the coding sequences, called exons, are joined together following transcription of a gene into RNA. The spliced mRNA subsequently acts as the coding template for protein synthesis. Pre-mRNA splicing occurs in a large ribonucleoprotein particle called the spliceosome which includes five essential small nuclear RNAs (snRNAs) and more than 50 different proteins. It is thought that the active site of the spliceosome consists of U2, U6 and U5 snRNAs. In order to probe the structure of the catalytic site, we have done an extensive site-specific chemical modification and crosslinking studies of U6 RNA in the yeast spliceosome. From an extensive screen of site-specific 2'-deoxy modifications, we have found four that block the first step of splicing, yet are able to assemble spliceosome complexes which precede the reactive spliceosome, suggesting that the 2'-hydroxyls at these nucleotides may be required for catalysis. In the crosslinking experiments, we found crosslinks between 4-thioU placed in the central conserved sequences in U6 with the 5' splice site of the pre-mRNA, which participates in the first chemical step of splicing. We were able to determine the order of this crosslink by doing the experiment in yeast mutant spliceosomes which were blocked in well-defined stages of splicing. We also found that a conserved nucleotide in the 3' stem-loop of U6 crosslinks to a nucleotide in a region of the actin pre-mRNA intron which is required for its splicing in vitro. We also found crosslinks to snRNAs -- U2 and U4. Certain positions in U6 can crosslink first to U4 snRNA, then subsequently to the pre-mRNA, consistent with the notion that the spliceosome is a dynamic machine.", "doi": "10.7907/cfw0-z473", "publication_date": "1999", "thesis_type": "phd", "thesis_year": "1999" }, { "id": "thesis:9922", "collection": "thesis", "collection_id": "9922", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:09122016-085802807", "type": "thesis", "title": "Synthesis of an \u03b1,3-Dehydrotoluene Biradical Precursor with DNA Cleaving Activity and Studies Directed Toward the Total Synthesis of Tetracycline", "author": [ { "family_name": "Parrish", "given_name": "Cynthia Ann", "clpid": "Parrish-Cynthia-Ann" } ], "thesis_advisor": [ { "family_name": "Myers", "given_name": "Andrew G.", "clpid": "Myers-A-G" } ], "thesis_committee": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" }, { "family_name": "Grubbs", "given_name": "Robert H.", "orcid": "0000-0002-0057-7817", "clpid": "Grubbs-R-H" }, { "family_name": "Imperiali", "given_name": "Barbara", "orcid": "0000-0002-5749-7869", "clpid": "Imperiali-B" }, { "family_name": "Myers", "given_name": "Andrew G.", "clpid": "Myers-A-G" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The synthesis and characterization of a model of the enediyne antibiotics is\r\ndescribed. The prepared conjugate consists of an \u03b1,3-dehydrotoluene biradical precursor\r\ntethered to an N-methylpyrrolecarboxamide minor groove binding element. The conjugate\r\nis shown to bind and cleave DNA with sequence selectivity. The binding domain is shown\r\nto localize the allene-ene-yne effector domain for sequence-selective DNA cleavage at\r\nmicromolar concentrations of substrate. The time course of DNA cleavage parallels the rate\r\nof cyclization of the bioconjugate in organic solvent to form an \u03b1,3-dehydrotoluene\r\nbiradical. These results indicate that the (Z)-allene-ene-yne functional group is a viable\r\neffector domain for the cleavage of DNA upon mild thermal activation.
\r\n\r\nSynthetic studies directed toward a concise and versatile synthesis of the antibiotic\r\ntetracycline are described. A strategy based on an isobenzofuran Diels-Alder cycloaddition\r\nto assemble the two halves of tetracycline is presented. The synthesis of the phthalide lefthand\r\nhalf is shown in five steps with 56% overall yield from commercially available\r\nstarting materials. Several isobenzofuran Diels-Alder reactions are described that model\r\nthe proposed condensation of the two halves of tetracycline. Specifically, a thermal DielsAlder\r\nreaction is successfully demonstrated with an enone dienophile containing an a-ester\r\nfunctional group. The synthesis of 6-dimethylaminomethyl-2,2-dimethyl-1,3-dioxin-4-one\r\nas a protected and fully functionalized right-hand half of the A ring of tetracycline is\r\ndescribed. Strategies are discussed that aim to utilize this substrate in the synthesis of the\r\nright-hand half of tetracycline. A novel and potentially rapid route involving intermediate\r\n2,4- or 2,5-cyclohexadienones from the oxidation of phenol precursors is briefly\r\nexamined.
", "doi": "10.7907/3m0y-w259", "publication_date": "1999", "thesis_type": "phd", "thesis_year": "1999" }, { "id": "thesis:16203", "collection": "thesis", "collection_id": "16203", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:10062023-220054560", "primary_object_url": { "basename": "Wilhelm_TE_1999.pdf", "content": "final", "filesize": 70575762, "license": "other", "mime_type": "application/pdf", "url": "/16203/1/Wilhelm_TE_1999.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Easier and More Efficient Methods for the Generation of Metathesis Catalysts: Investigations into Group VI and VII", "author": [ { "family_name": "Wilhelm", "given_name": "Thomas Edward", "clpid": "Wilhelm-Thomas-Edward" } ], "thesis_advisor": [ { "family_name": "Grubbs", "given_name": "Robert H.", "orcid": "0000-0002-0057-7817", "clpid": "Grubbs-R-H" } ], "thesis_committee": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" }, { "family_name": "Bercaw", "given_name": "John E.", "clpid": "Bercaw-J-E" }, { "family_name": "Goddard", "given_name": "William A., III", "orcid": "0000-0003-0097-5716", "clpid": "Goddard-W-A-III" }, { "family_name": "Grubbs", "given_name": "Robert H.", "orcid": "0000-0002-0057-7817", "clpid": "Grubbs-R-H" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Chapter 1:
\r\n\r\nA high yield procedure for generating the ruthenium hydride complexes\r\nRu(H)(H2)Cl(PR3)2 (R=Cyclohexyl, cyclopentyl, isopropyl) in very high yield is\r\npresented. Following a novel insertion-elimination pathway, these hydrides can\r\nreact with propargyl or vinyl halides to make metathesis active vinyl and alkyl\r\ncarbene species with the general formulas (PR3)2Cl2Ru=CH-CH=CR'2 and\r\n(PR3)2Cl2Ru=CHR', respectively. Tertiary propargyl chlorides like 3-chloro-3-\r\nmethyl-1-butyne work best, yielding Ru-vinyl carbenes in extremely high yield.\r\nAn alternate route is to first add an alkyne, and then add HCl to give similar\r\nspecies.
\r\n\r\nIn attempting to learn about the insertion-elimination mechanism, the\r\ncompounds M(H)Cl(CO)(PR3)2 (M=Ru, Os; R=cyclohexyl, isopropyl) were found\r\nto react with 3-chloro-3-methyl-1-butyne to produce the metathesis inactive\r\ncarbenes with general formula cis-Cl2-trans(PCy3)2(CO)M=CHCH=CMe2. Kinetics\r\nofRu(H)Cl(CO)(PiPr3)2 can only be analyzed qualitatively, but from all of\r\nthe available data a mechanism is proposed for the insertion of hydrides into\r\nalkynes and rearrangement to give carbenes. The compounds\r\nM(H)Cl(CO)2(PR3)2 (M=Ru, Os; R=cyclohexyl, isopropyl) show no alkyne insertion.
\r\n\r\nThe osmium analogs Os(H)3Cl(PCy3)2 and (PCy3)2Cl2Os=CH=CH=CMe2\r\nwere investigated for the ability to generate carbenes. The osmium carbene,\r\nhowever, rapidly transforms to the hydrido-carbyne species (PCy3)2Cl2Os(H)(=CCH=\r\nCMe2). It appears that additional stabilization of the osmium system will\r\nbe necessary to prevent such rearrangement.
\r\n\r\nIt is also presented that (PCy3)2Cl2Ru=CHR' reacts with dihydrogen to\r\ngive H3CR', Ru(H)2(C1)2(PCy3)2, and Ru(H)(H2)Cl(PCy3)2. Theoretically, all\r\nRu(H)2(Cl)2(PCy3)2 can be converted to Ru(H)(H2)Cl(PCy3)2. It is thus possible\r\nto go from hydrides to carbenes, and back to hydrides.
\r\n\r\nChapter 2:
\r\n\r\nComplexes of the type M(O)Cl2(PR3)3 (M=W, Mo; R3=PMePh2, PMe2Ph) were\r\nsynthesized using literature procedures, and shown to react with 3,3-\r\ndiphenylcyclopropene to give the \u03b72-olefin complexes M(O)Cl2(PR3)2(\u03b72-\r\ndiphenylcyclopropene). Spectroscopic data suggest a distorted octahedral structure\r\nfor both, with the oxo ligand in the axial position with the olefin cis to it\r\nand the two mutually trans phosphines in the equatorial plane, which was confirmed\r\nfor M=W with an x-ray diffraction study. The olefin complexes react\r\nwith suitable alkoxides to give the oxo-carbene species M(O)(OR)2(PR3)(=CHCH=\r\nCPh2), the first known single component tungsten and molybdenum oxo-alkylidene\r\nmetathesis catalysts, in which the phosphine is readily displaced\r\nwith THF. For these complexes, spectroscopic data suggest a distorted trigonal\r\nbipyramid with the oxo, alkylidene, and one alkoxide ligand in the equatorial\r\nplane, which was confirmed for M=W by a diffraction experiment. These\r\nalkylidene species are active in olefin metathesis reactions, showing comparable\r\nactivity to similar arylimido complexes previously described; polymerization\r\ndata is presented for norbornene and cyclooctene. In addition, the olefin\r\ncomplexes were shown to be active in olefin metathesis at elevated temperatures.
", "doi": "10.7907/3d4p-py55", "publication_date": "1999", "thesis_type": "phd", "thesis_year": "1999" }, { "id": "thesis:7206", "collection": "thesis", "collection_id": "7206", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:09172012-133347649", "primary_object_url": { "basename": "Doleman_bj_1999.pdf", "content": "final", "filesize": 25978440, "license": "other", "mime_type": "application/pdf", "url": "/7206/1/Doleman_bj_1999.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Study and development of an \"electronic nose\" and comparison with mammalian olfaction", "author": [ { "family_name": "Doleman", "given_name": "Brett James", "clpid": "Doleman-B-J" } ], "thesis_advisor": [ { "family_name": "Lewis", "given_name": "Nathan Saul", "clpid": "Lewis-N-S" } ], "thesis_committee": [ { "family_name": "Lewis", "given_name": "Nathan Saul", "clpid": "Lewis-N-S" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Marcus", "given_name": "Rudolph A.", "clpid": "Marcus-R-A" }, { "family_name": "Anson", "given_name": "Fred C.", "clpid": "Anson-F-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Arrays of broadly responsive vapor detectors (i.e., electronic noses) are receiving\r\nan increasing amount of scientific attention for their potential as analytical devices, as\r\nmodels for studying mammalian olfaction, and perhaps for someday ultimately duplicating\r\nor surpassing the mammalian olfactory sense. Herein, research was primarily focused on\r\nan electronic nose composed of an array of carbon black-polymer composite detectors\r\nwhile arrays of tin oxide detectors and organic conducting polymer detectors were used\r\nonly in a comparison study. The research determined the odorant resolving power of\r\nelectronic nose sensor arrays, explored the dependence of the electronic nose array\r\nresponse intensity on odorant vapor pressure, compared the odorant detection thresholds\r\nand odorant classification properties of the electronic nose to the mammalian olfactory\r\nsense, and attempted to predict human odor quality judgements using electronic nose\r\ndetector responses.
\r\n\r\nThe Fisher linear discriminant statistical metric was utilized to quantify the\r\nperformance of arrays composed of carbon black-insulating polymer composite detectors,\r\ntin oxide detectors and bulk conducting organic polymer detectors in resolving nineteen\r\nodorant vapors. The odorant resolving power of the sensor arrays as a function of the\r\nchemical composition of the detectors and the number of detectors they contained was\r\nstudied. The results provided insights into optimizing the chemical diversity and size of a\r\nchemical vapor sensor array for various tasks.
\r\n\r\nResponse data were collected for a carbon black-polymer composite electronic nose\r\narray during exposure to homologous series of 1-alcohol and n-alkane odorants. The mean\r\nresponse intensity of the electronic nose detectors, and the response intensity of the most\r\nstrongly-driven set of electronic nose detectors, was essentially constant for members of a\r\nchemically homologous odorant series when the concentration of each odorant in the gas\r\nphase was maintained at a constant fraction of the odorant's vapor pressure. A similar\r\ntrend is observed in human odor detection threshold values for these same odorants. The\r\ndata imply that the trends in detector responses and human detection thresholds can be\r\nunderstood based on the thermodynamic tendency to establish a relatively constant\r\nconcentration of sorbed odorant into each of the polymeric films of the electronic nose and\r\ninto the olfactory epithelium of humans at a constant fraction of the odorant's vapor\r\npressure.
\r\n\r\nExperiments were performed to compare the detection thresholds and trends in\r\ndiscrimination abilities of the electronic nose to those of the mammalian olfactory sense,\r\nand to develop models predicting human odor quality judgements from electronic nose\r\ndetector responses. The detection thresholds for the electronic nose and the human nose\r\nwere compared for series of n-alkanes and 1-alcohols. Trends in the odorant-discriminating\r\nabilities of an electronic nose and mammalian noses were compared for\r\nseries of esters, alcohols and carboxylic acids. Electronic nose response data were\r\ncollected for a diverse set of odorants which had previously been quantitatively\r\ncharacterized by human panelists according to many categories of odor quality. The\r\nresponses of the electronic nose detectors were then used in attempts at predicting the\r\nhuman odor quality judgements.
", "doi": "10.7907/R6QQ-NX09", "publication_date": "1999", "thesis_type": "phd", "thesis_year": "1999" }, { "id": "thesis:17685", "collection": "thesis", "collection_id": "17685", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:09172025-215507596", "primary_object_url": { "basename": "Wu_S_1999.pdf", "content": "final", "filesize": 60275951, "license": "other", "mime_type": "application/pdf", "url": "/17685/1/Wu_S_1999.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Development of Broadly Tunable Parametric Light Sources and Their Application to Alkali Metal - Small Molecule Cluster Spectroscopy", "author": [ { "family_name": "Wu", "given_name": "Sheng", "clpid": "Wu-Sheng" } ], "thesis_advisor": [ { "family_name": "Blake", "given_name": "Geoffrey A.", "orcid": "0000-0003-0787-1610", "clpid": "Blake-G-A" } ], "thesis_committee": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" }, { "family_name": "Blake", "given_name": "Geoffrey A.", "orcid": "0000-0003-0787-1610", "clpid": "Blake-G-A" }, { "family_name": "Hoffmann", "given_name": "Michael R.", "orcid": "0000-0001-6495-1946", "clpid": "Hoffmann-M-R" }, { "family_name": "McKoy", "given_name": "Basil Vincent", "clpid": "McKoy-B-V" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Widely tunable laser sources based on second order nonlinear optical (NLO) conversions\r\nand covering the 200 nm to 3 \u03bcm region have been constructed. Considerable\r\neffort is devoted to achieving reasonable linewidth and efficiency, two critical parameters\r\nfor the practical use of NLO materials in spectroscopic and other experiments.\r\nUsing these devices, the ionization behavior of alkali metal - small molecule clusters\r\nis explored. In particular, the spectroscopic results are used to investigate the physics\r\nof salvation between potassium atoms and ions with small molecules.
\r\n\r\nRecent advances in the fabrication of nonlinear optical materials have led to the\r\ndiscovery of several promising crystal candidates for high power applications. These\r\nadvances, coupled with the rapid progress in high power pulsed pump laser technology,\r\nhave made practically possible widely tunable laser sources based on nonlinear\r\noptical conversions. Three major devices have been developed that provide continuous\r\ncoverage from 200 nm to 3 \u03bcm, and which combine reasonable power efficiency\r\nwith a range of linewidths suitable for various spectroscopic measurements.
\r\n\r\nThe design philosophy throughout is to create practical devices that are as simple\r\nand durable as possible. For example, a compact, low cost OPO based on type II\r\nphase matching in BBO and off-the-shelf optics has been fabricated. The type II BBQ\r\nOPO is fully computer controlled, and provides wide tunability (\u2248 410 nm to 3 \u03bcm)\r\nand relatively narrow bandwidth (\u2248 1 cm-1) in the same package. To generate narrow\r\nbandwidth radiation, e.g., close to the transform limit, an extremely simple, unidirectional\r\nOptical Parametric Generator/Optical Parametric Amplifier (OPG/OPA)\r\nis designed based on an intense nanosecond (ns) pump source. Compared with other\r\nCW laser-seeded optical parametric devices, this OPG/OPA design combines simple\r\noperation with a remarkable insensitivity to the matching of the seed laser frequency\r\nto the OPO cavity mode. Numerical models are used to fully characterize the NLO\r\nbehavior of these cavities, and to optimize their performance.
\r\n\r\nFinally, in order to produce UV harmonics of the fundamental oscillators, a\r\nbroadly tunable harmonic generator which effectively covers the 190 to 420 nm region\r\nhas been constructed. The thermal dephasing problem in high average UV power generation\r\nwith NLO crystals is fully explored for the first time, and a new method of\r\novercoming this thermal dephasing problem is proposed and tested. For each of the\r\ndevices, several experiments had been carried out to characterize their performance,\r\nparticularly their wide tunability, ease of operation, and high power scalability.
\r\n\r\nMost importantly for this thesis, fundamental research on the interaction between\r\npotassium and small molecules such as water, ammonia, and benzene has been carried\r\nout with the above mentioned laser sources by collecting the Photo-Ionization\r\nEfficiency (PIE) ion yield and Zero Kinetic Energy Electron - Pulsed Field Ionization\r\n(ZEKE-PFI) spectra of neutral alkali metal-solvent clusters. The tunable sources\r\nmade it possible to rapidly map out the ionization potentials of the different clusters,\r\nand to thereby estimate the binding energies of the neutral and ionic clusters; while\r\nthe ZEKE-PFI spectra of K(NH3) reveal, as expected, a considerable increase in the\r\nangular anisotropy of the intermolecular forces in the charged clusters. Interestingly,\r\nthe measured pair-pair interaction energies of the dimers stand in contradiction to the\r\nion selectivity achieved by biological systems. Many body forces therefore must contribute\r\nsubstantially to the subtle balancing of forces that operate in the condensed\r\nphase, and the results indicate the importance of further characterization of large clusters,\r\nespecially those with mixed constituents such as K(C6H6)n(H2O)m. Additional\r\napplications in fields as diverse as atmospheric science and molecular astrophysics can\r\nbe expected as NLO materials and processes are further refined.
", "doi": "10.7907/ztvp-j735", "publication_date": "1999", "thesis_type": "phd", "thesis_year": "1999" }, { "id": "thesis:16233", "collection": "thesis", "collection_id": "16233", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:11012023-164245860", "primary_object_url": { "basename": "Dias_ELK_1998.pdf", "content": "final", "filesize": 53677508, "license": "other", "mime_type": "application/pdf", "url": "/16233/1/Dias_ELK_1998.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Ruthenium-Based Olefin Metathesis Catalysts: Synthesis, Mechanism, and Activity", "author": [ { "family_name": "Dias", "given_name": "Eric Lee Kuiokalani", "clpid": "DIas-Eric-Lee-Kuiokalani" } ], "thesis_advisor": [ { "family_name": "Grubbs", "given_name": "Robert H.", "orcid": "0000-0002-0057-7817", "clpid": "Grubbs-R-H" } ], "thesis_committee": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "orcid": "0000-0003-1464-2461", "clpid": "Dougherty-D-A" }, { "family_name": "Grubbs", "given_name": "Robert H.", "orcid": "0000-0002-0057-7817", "clpid": "Grubbs-R-H" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" }, { "family_name": "Bercaw", "given_name": "John E.", "clpid": "Bercaw-J-E" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Several ruthenium-based olefin metathesis catalysts of the formula (PR\u2083)\u2082X\u2082Ru=CHCHCPh\u2082 have been synthesized, and relative catalyst activities were determined by monitoring the ring-closing metathesis of the acyclic diene diethyl diallylmalonate. The following order of increasing activity was determined: X = I < Br < Cl, and PR\u2083 = PPh\u2083 << P\u2071Pr\u2082Ph < PCy\u2082Ph < P\u2071Pr\u2083 < PCy\u2083. Additional studies were conducted with the catalyst (PCy\u2083)\u2082Cl\u2082Ru=CH\u2082 to probe the mechanism of olefin metathesis by this class of catalysts. The data support a scheme in which there are two competing pathways: the dominant one in which a phosphine dissociates from the ruthenium center, and a minor one in which both phosphines remain bound. Higher catalyst activities could be achieved by the addition of CuCl to the reaction.
\r\n\r\nThe carbenes (PCy\u2083)\u2082Cl\u2082Ru=CHR (R = CHCPh\u2082, Ph) react with the bridged-chloride dimers [(p-cymene)RuCl\u2082]\u2082, [(p-cymene)OsCl\u2082]\u2082, and [(\u1d57Bu\u2082Cp)RhCl\u2082]\u2082 to quantitatively form bimetallic, bridged-chloride ruthenium carbenes and and one equivalent of each corresponding piano-stool complex. In the ring-opening metathesis polymerization (ROMP) of 1,5-cyclooctadiene, catalyst activity was found to increase in the order M = Ru < Os < Rh for the ancillary metal centers, with all of the bimetallic catalysts having higher activities. The kinetics of ROMP were studied, and the data support an associative mechanism of olefin metathesis.
\r\n\r\nReaction of excess pyridine with (PCy\u2083)\u2082(Cl)\u2082Ru=CHPh produces the stable, bis(pyridine) adduct (PCy\u2083)(pyr)\u2082(Cl)\u2082Ru(CHPh). In solution, an equilibrium is established with the mono(pyridine) adduct (PCy\u2083)(pyr)(Cl)\u2082Ru(CHPh). Reaction of thallium salts of ,B-diketonates with (PCy\u2083)\u2082ChRu=CHR (R = CHCPh\u2082, Ph) produces the complexes (PCy\u2083)(L)\u2082Ru(CHR) (L = acac, \u1d57Bu\u2082acac; R = CHCPh\u2082, Ph). While the pyridine complexes are stable and completely initiate RCM, the propagating methylidene is very unstable, decomposing as fast as, or faster than, it is formed. The \u03b2-diketonate complexes initiate ROMP in the presence of HCl and RCM in the presence of CuCl.
", "doi": "10.7907/30mh-mq42", "publication_date": "1998", "thesis_type": "phd", "thesis_year": "1998" }, { "id": "thesis:17605", "collection": "thesis", "collection_id": "17605", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:08082025-165811908", "primary_object_url": { "basename": "Hastings_CA_1998.pdf", "content": "final", "filesize": 79636336, "license": "other", "mime_type": "application/pdf", "url": "/17605/1/Hastings_CA_1998.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Part A. Studies Directed Toward the Total Synthesis of Chebulagic Acid. Part B. DNA Recognition by Metallointercalator-Peptide Conjugates", "author": [ { "family_name": "Hastings", "given_name": "Curtis Asa", "clpid": "Hastings-Curtis-Asa" } ], "thesis_advisor": [ { "family_name": "Carreira", "given_name": "Erick Moran", "orcid": "0000-0003-1472-490X", "clpid": "Carreira-E-M" } ], "thesis_committee": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "orcid": "0000-0003-1464-2461", "clpid": "Dougherty-D-A" }, { "family_name": "Goddard", "given_name": "William A., III", "orcid": "0000-0003-0097-5716", "clpid": "Goddard-W-A-III" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" }, { "family_name": "Carreira", "given_name": "Erick Moran", "orcid": "0000-0003-1472-490X", "clpid": "Carreira-E-M" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Part A. The development of asymmetric allene/enone and allene/enoate\r\nintramolecular [2+2]-photocycloadditions is described. Irradiation of optically active\r\nallenes (89-92% ee) appended to enones and enoates afforded alkylidenecyclobutane\r\nphotoadducts with high levels of asymmetric induction (83 -100%) derived exclusively\r\nfrom the allene fragment. Substrates examined include allenyl alcohols appended to 1,3-\r\ncyclopentanedione, 1,3-cyclohexanedione, and 4-hydroxycoumarin. The absolute sense of\r\ninduction in these reactions was determined by photocycloaddition of an allene containing\r\nan internal stereochemical label. The exa-methylenecyclobutanes obtained upon irradiation\r\nof allene-coumarins were isolated as single olefin diastereomers. A model for the high\r\nlevels of enantioinduction observed in these transformations is presented.
\r\n\r\nThe asymmetric intramolecular allene/enoate [2+2]-photocycloaddition was applied\r\nto the synthesis of the topoisomerase I inhibitor chebulagic acid. The synthetic utility of\r\nthis reaction was demonstrated by the preparation of an advanced intermediate containing\r\nall of the stereochemical information present in the chebulic acid fragment of chebulagic\r\nacid. In the course of these synthetic studies an unusual 8-alkynyl lactone photoproduct\r\nwas obtained upon photocyloaddition of a substrate that was expected to afford a [7.6.6.4]tetracyclic\r\nsystem. A 1,5-hydrogen shift in a biradical intermediate was implicated in the\r\nformation of this product by isotopic labeling studies; the mechanistic implications of these\r\nresults for enantioselectivity in photochemical reactions of optically active allenes tethered\r\nto enones and enoates are discussed.
\r\n\r\nPart B. The DNA recognition properties of peptide conjugates of\r\nphenenthrenequinone diimine complexes of rhodium(III) have been studied. The structural\r\nand thermodynamic basis for the 5'-CCA-3'-selectivity of the metallointercalator-peptide\r\nconjugate [Rh(phi)2(phen')]3+ -AANVAIAAWERAA-CONH2 was investigated. A protocol\r\nfor measuring dissociation constants of DNA cleaving ligands by cleavage titration is\r\ndescribed. Using this protocol, the energetic contribution of the peptide to sequenceselective\r\nbinding was assessed, and evidence for the origin of the enhanced sequenceselectivity\r\nobserved at elevated temperature was obtained. Micromolar quantities of [\u0394Rh-(phi)2(phen')]3+-AANVAIAAWERAA-CONH2 were synthesized to examine the\r\nstructure of the metallointercalator-peptide conjugate and the metallointercalator-peptide\r\nconjugate\u2022DNA complex by NMR. NMR results for the metallointercalator-peptide\r\nconjugate in the absence of DNA are reported.
\r\n\r\nA family of peptide conjugates of [Rh(phi)2(phen')]3+(phi = 9, 10-\r\nphenanthrenequinone diimine, phen' = 5-(amidoglutaryl)-1,10-phenanthroline) was also\r\nsynthesized. The peptide sequences were obtained by single amino acid modification of the\r\n5'-CCA-3'-selective metallointercalator-peptide conjugate [Rh(phi)2(phen')]3+-\r\nAANVAIAAWERAA-CONH2 to explore the correlation between the amino acid sequence\r\nof the peptide and the nucleotide sequence of the DNA target. Changing the position of the\r\nglutamate at position 10 in the sequence of the appended peptide resulted in the\r\nidentification of a 5'-ACA-3'-selective metallointercalator-peptide conjugate,\r\n[Rh(phi)2(phen')3+-AANVAEAAWARAA-CONH2. Locating the glutamate on one face of\r\na putative \u03b1-helix was found to be essential for sequence specificity; peptide conjugates\r\nwith the glutamate at positions 7, 8, 12, and 13 did not afford sequence-selective DNA\r\nrecognition. Further amino acid substitutions were made at positions 6 and 10. Mutating\r\nthe glutamate at position 6 to arginine caused complex changes in the recognition\r\ncharacteristics of the resulting conjugate. To probe the interactions that give rise to\r\nsequence specificity, we have measured thermodynamic dissociation constants for these\r\nsequence-selective metallointercalator-peptide conjugates and [Rh(phi)2(phen')]3+.
\r\n\r\nThe observed sequence preferences are consistent with the model of Sardesai et al.\r\nfor the sequence selectivity of Rh(phi)2(phen')]3+-AANVAIAAWERAA-CONH2. This\r\nmodel states that sequence-specific DNA recognition requires the peptide to adopt an \u03b1-helical\r\nconformation, and that Glu10 makes a critical base-specific contact with the 5'-\r\nterminal cytosine of the recognition sequence. Using the additional sequence-selectivity\r\ndata, this model is refined. This refined model suggests that recognition of the central C\u2022G\r\nbase pair of the 5'-CCA-3' recognition sequence of [Rh]-E10 is accomplished by Ile6\r\nthrough shape-selection, and that recognition of the 5' -terminal A\u2022T base pair of the 5' -\r\nACA-3' recognition sequence of [Rh]-E6 is accomplished by van der Waals contacts\r\nbetween alanine and thymine methyl groups. The implications of these results for the de\r\nnova design of sequence-selective DNA binding peptides are discussed.
", "doi": "10.7907/e4be-bd57", "publication_date": "1998", "thesis_type": "phd", "thesis_year": "1998" }, { "id": "thesis:17792", "collection": "thesis", "collection_id": "17792", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:12102025-233557591", "primary_object_url": { "basename": "Lim_AC_1998.pdf", "content": "final", "filesize": 60742266, "license": "other", "mime_type": "application/pdf", "url": "/17792/1/Lim_AC_1998.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Investigation of RNA Tertiary Structure and Function by Transition Metal Complexes", "author": [ { "family_name": "Lim", "given_name": "Ai Ching", "clpid": "Lim-Ai-Ching" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" }, { "family_name": "Goddard", "given_name": "William A., III", "orcid": "0000-0003-0097-5716", "clpid": "Goddard-W-A-III" }, { "family_name": "Parker", "given_name": "Carl Stevens", "orcid": "0000-0001-9795-4211", "clpid": "Parker-C-S" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Phenanthrenequinone diimine (phi) complexes of rhodium(III) were employed to\r\nprobe RNA secondary and tertiary structure. These complexes bind via intercalation in\r\nopen major grooves of RNA and upon irradiation promote strand scission. By probing\r\nboth synthetic and natural molecules containing a variety of tertiary motifs, a systematic and\r\npredictive understanding of the factors involved in RNA recognition by these complexes is\r\nsought. The metal complex Rh(phen)2phi3+ (phen = 1,10-phenanthroline) recognizes and\r\ncleaves synthetic triple helices selectively over double helices. The cleavage sites are\r\ndependent upon maximizing overlap between the phi ligand and the basepairs, and\r\nminimizing charge repulsion between the metal complex and protonated bases. These\r\ncleavage sites have proven useful in explaining rhodium complex cleavage in natural\r\nsystems such as tRNAPhe. With these complexes, we also seek to investigate the\r\ndifferences and similarities in RNA and DNA secondary and tertiary folding, by probing\r\nthe tertiary structure of tDNAPhe compared to tRNAPhe. These complexes have elucidated\r\nthe B-form nature of the DNA duplex as well as the tertiary folding of the DNA molecule,\r\nthus shedding light on the feasibility of using DNA analogs of RNA for structural studies.\r\nThese shape selective probes have also been applied to probe the tertiary structure of HIV\r\nand BIV (TAR (trans-activation response) RNAs. \u0394-Rh(phen)2phi3+ binds with high\r\naffinity (Kb= 6.1 \u00b1 1.3 x 105 M-1) and specificity to sites at and across from a bulge\r\nregion which is the recognition element for the binding of the Tat (trans-activating) peptide.\r\nImportantly, the metal complex recognizes an RNA base-triple the formation of which is\r\nnecessary for transactivation. Derivatives of Rh(phen)2phi3+, Rh(MGP)2phi5+(MGP = 4-\r\nguanidylmethy 1-1, 10-phenanthroline) and Rh(GEB)2phi5+ (GEB = 4-(2-guanidylethyl)-4'methy\r\n1-2,2'-bipyridine) where guanidinium moieties have been added to the ancillary\r\nligands of the rhodium complex, show enhanced affinity and selectivity for HIV and BIV\r\nRNA sequences. This is due to the guanidinium moieties mimicking the arginine side\r\nchains on the native Tat peptide, and making non-specific contacts with the phosphate\r\nbackbone of the RNA. However, even without these functionalities, shape-selection,\r\nmatching the shape of the small metal complex to its nucleic acid target, provides sufficient\r\nselective stabilization for RNA site discrimination. Indeed, these complexes compete\r\neffectively with the specific Tat peptides for their binding sites on their respective TAR\r\nRNAs. These complexes therefore employ shape selection to recognize structural\r\nvariations along the RNA polymer which are important for protein recognition. Shape-selective\r\nrecognition could also be applied to the design of novel small molecules to target\r\nnucleic acid sites with high site-selectivity, in the development of molecules to inhibit\r\nprotein recognition, and, potentially, in the design of new chemotherapeutics.", "publication_date": "1998", "thesis_type": "phd", "thesis_year": "1998" }, { "id": "thesis:11084", "collection": "thesis", "collection_id": "11084", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06142018-101514702", "primary_object_url": { "basename": "Maughon_BR_1998.pdf", "content": "final", "filesize": 55184505, "license": "other", "mime_type": "application/pdf", "url": "/11084/1/Maughon_BR_1998.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Synthesis of Functionalized Polymers by Ring-Opening Metathesis Polymerization (ROMP)", "author": [ { "family_name": "Maughon", "given_name": "Bob Robinson, Jr.", "clpid": "Maughon-Bob-Robinson-Jr" } ], "thesis_advisor": [ { "family_name": "Grubbs", "given_name": "Robert H.", "clpid": "Grubbs-R-H" } ], "thesis_committee": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Carreira", "given_name": "Erick Moran", "clpid": "Carreira-E-M" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "In Chapter 1, the ROMP of 5-methacrylate-1-cyclooctene and the copolymerization\r\nof this monomer with cyclooctadiene using the initiator (PCy3)2Cl2Ru=CHCH=CPh2 were\r\ninvestigated to produce polymers with cross-linkable side-chains. The impact of\r\nconcentration, monomer to initiator ratio, and the amount of inhibitor in the polymerization\r\nwas examined. These polymers were cross-linked through the methacrylate side-chains\r\nwith either thermal or photochemical initiation, and the incorporation of these polymers into\r\npoly(methyl methacrylate) (PMMA) to produce AB cross-linked materials was\r\naccomplished. A comparison of the physical properties of PMMA and these new materials\r\ndemonstrated that these materials had higher thermal stability and solvent resistance than\r\npure PMMA.
\r\n\r\n\r\nAs an extension of the work presented in Chapter 1, Chapter 2 illustrated an\r\nalternative approach for the preparation of cross-linkable polymers by ROMP. The\r\nsynthesis of ring-opening metathesis polymerization chain transfer agents bearing\r\nmethacrylate and epoxide end-functionality was accomplished. In the presence of these\r\nchain transfer agents, cyclooctadiene was polymerized via a ruthenium benzylidene\r\ninitiator, (PCy3)Cl2Ru=CHPh, to produce telechelic poly(butadiene)s with either\r\nmethacrylate or epoxide end groups. The impact of initiator concentration, reaction time,\r\nand temperature on the polymer yield and chain transfer agent incorporation was examined.\r\nControl over the polymer molecular weight through the cyclooctadiene/chain transfer agent\r\nratio was demonstrated providing for a range of telechelic poly(butadiene) molecular\r\nweights. Successful cross-linking of these polymers by thermal or photochemical initiation\r\nin the case of the bis(methacrylate)-functionalized telechelic poly(butadiene)s or through\r\nacid catalysis in the case of the bis(epoxide)-functionalized telechelic poly(butadiene)s was\r\naccomplished.
\r\n\r\n\r\nIn an effort to further explore the functional group tolerance of the ruthenium-based\r\nmetathesis initiators developed in our group, the investigation presented in Chapter 3\r\nencompassed the synthesis and living ring-opening metathesis polymerization (ROMP) of\r\nsubstituted cyclobutenes with the functional group tolerant polymerization initiators\r\n(PCy3)2Cl2Ru=CHCH=CPh2 and (PCy3)2Cl2Ru=CHPh. Synthetic methodology was\r\ndeveloped for the synthesis of a wide variety of 3-functionalized cyclobutenes containing\r\nether, ester, alcohol, amine, amide, and carboxylic acid substituents. Coordination of these\r\nfunctional groups to the propagating carbene was observed resulting in the formation of a\r\nchelated propagating species with concomitant loss of one phosphine ligand from the metal\r\ncenter. Studies aimed at understanding this chelation and its effect on the polymerization\r\nwere undertaken. Based on these results, the synthesis of a series of functionalized\r\ncyclobutenes was accomplished which minimized this chelation and allowed for living\r\npolymerizations. A new class of functionalized poly(butadiene) homopolymers and\r\ndiblock copolymers was synthesized and the thermal properties analyzed by\r\nthermogravimetric analysis and differential scanning calorimetry.
\r\n\r\n\r\nIn Chapter 4, the effect of backbone flexibility on the mesomorphic behavior of\r\nside-chain liquid crystalline polymers synthesized by ring-opening metathesis\r\npolymerization was investigated. The synthesis of norbornene and cyclobutene monomers\r\ncontaining a p-nitrostilbene moiety as the mesogenic group and polymerization of these\r\nmonomers with the metathesis initiator (PCy3)2Cl2Ru=CHPh to produce side-chain liquid\r\ncrystalline polymers with low polydispersities and defined molecular weights was\r\naccomplished. The relatively rigid poly(norbornene)s displayed enantiotropic nematic\r\nmesomorphism with glass transitions from 44-64\u00b0C and isotropization temperatures\r\nbetween 108-121\u00b0C, whereas the more flexible poly(butadiene)s showed enantiotropic\r\nsmectic A mesomorphism with glass transition temperatures from 14-31\u00b0C and\r\nisotropization temperatures between 74-111\u00b0C. A diblock copolymer containing a 1:1\r\nmixture of the poly(norbornene) and poly(butadiene) backbones also exhibited a smectic A\r\nmesophase. The dependence of the degree of polymerization and flexible spacer length on\r\nthe phase transitions of these systems was determined demonstrating stabilization of the\r\nmesophase by both increasing molecular weight and flexible spacer length.
\r\n\r\n\r\nA short chapter on the development of methodology for an improved synthesis of\r\n3-methyl-3-phenylcyclopropene was included in Appendix 1. This research was\r\ninvestigated in hopes of developing a more facile and inexpensive procedure for the\r\npreparation of this compound than has been previously reported. Phase transfer catalyzed\r\ndichlorocarbene addition to \u03b1-methylstyrene followed by a selective catalytic Bu3SnH\r\nreduction resulted in the 1-chloro-2-methyl-2-phenylcyclopropane intermediate in excellent\r\nyield. Base-induced elimination of this compound resulted in the desired 3-methyl-3-phenylcyclopropene. \r\nThis approach allowed for the preparation of this cyclopropene on\r\nlarge scale utilizing inexpensive reagents.
\r\n\r\n\r\n\r\n\r\n", "doi": "10.7907/txks-pt47", "publication_date": "1998", "thesis_type": "phd", "thesis_year": "1998" }, { "id": "thesis:17615", "collection": "thesis", "collection_id": "17615", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:08112025-225322656", "primary_object_url": { "basename": "Arkin_MR_1997.pdf", "content": "final", "filesize": 88219433, "license": "other", "mime_type": "application/pdf", "url": "/17615/1/Arkin_MR_1997.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Investigations of DNA-Mediated Electron Transfer Reactions with Metallointercalators", "author": [ { "family_name": "Arkin", "given_name": "Michelle R.", "orcid": "0000-0002-9366-6770", "clpid": "Arkin-Michelle-R" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Marcus", "given_name": "Rudolph A.", "orcid": "0000-0001-6547-1469", "clpid": "Marcus-R-A" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" }, { "family_name": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" }, { "family_name": "Lewis", "given_name": "Nathan Saul", "orcid": "0000-0001-5245-0538", "clpid": "Lewis-N-S" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The array of \u03c0-stacked base pairs in DNA represents a novel medium for electron transfer\r\nreactions, and metallointercalators have served as useful tools to study this chemistry. Ultrafast\r\nkinetic measurements indicate that photoinduced electron transfer reactions between\r\nM(phen)2(X2dppz)2+ (M = Ru, Os; dppz = dipyrido[3,2-a:2,3-c]phenazine; X = H, CH3) [M(II)]\r\nand Rh(phi)2bpy3+ (phi = phenanthrenequinone diimine) [Rh(III)] can occur with rates> 3 x 1010\r\ns-1. Recombination reactions between M(III) and Rh(II) are also very fast (~ 1010 s-1), and rates\r\nare found to be independent of the loading of \u0394-Rh(phi)2bpy3+ on DNA. However, reaction rates\r\nand efficiencies are highly sensitive to i) the structure and chirality of intercalators and ii) the\r\nsequence and conformation of the DNA double helix. Photoinduced reactions between Ru(II) and\r\nRh(III) bound to the DNA helix and to SOS micelles, which lack the ordered \u03c0-stacked array, are\r\nalso compared. In contrast to DNA, quenching in micelles occurs by diffusion. The details of\r\nintercalation and DNA sequence are thus found to be important characteristics of DNA-mediated\r\nET reactions.
\r\n\r\nTo study long-range reactions through DNA, metallointercalator-DNA conjugates have\r\nbeen prepared. Rh(III) and novel trisheteroleptic complexes of Ru(II) are tethered to the 5'-termini\r\nof oligonucleotides by solid- and solution-phase methods, and these complexes have provided\r\nspectroscopic and photochemical tools to characterize chimeric structures. In addition to\r\nexperiments in which DNA serves as a molecular bridge connecting donor and acceptor, the double\r\nhelix may also serve as a reactant in electron transfer chemistry. Ru(III) oxidants have been\r\ngenerated in situ by a flash-quench methodology and have been found, by transient absorption\r\nspectroscopy, to oxidize G residues in DNA. Furthermore, using a tethered Ru(III)-DNA\r\nconjugate, oxidation products are observed 37 \u00c5 from the metallointercalator. These investigations\r\nof DNA-mediated electron transfer reactions contribute to our understanding of oxidative damage\r\nin DNA and may lead to a novel class of DNA-based biosensors.
", "doi": "10.7907/4ggg-6x62", "publication_date": "1997", "thesis_type": "phd", "thesis_year": "1997" }, { "id": "thesis:17590", "collection": "thesis", "collection_id": "17590", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:08042025-205701232", "primary_object_url": { "basename": "Johann_TW_1997.pdf", "content": "final", "filesize": 76248947, "license": "other", "mime_type": "application/pdf", "url": "/17590/1/Johann_TW_1997.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Sequence-Specific Inhibition of DNA Polymerase by Phenanthrene Quinone Diimine Complexes of Rhodium(III)", "author": [ { "family_name": "Johann", "given_name": "Timothy Wilmot", "orcid": "0000-0003-2212-9684", "clpid": "Johann-Timothy-Wilmot" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Baldeschwieler", "given_name": "John D.", "clpid": "Baldeschwieler-J-D" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" }, { "family_name": "Okumura", "given_name": "Mitchio", "orcid": "0000-0001-6874-1137", "clpid": "Okumura-M" }, { "family_name": "Parker", "given_name": "Carl Stevens", "orcid": "0000-0001-9795-4211", "clpid": "Parker-C-S" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The DNA binding characteristics of several phenanthrenequinone\r\ndiimine (phi) complexes of rhodium (III) as well as their ability to inhibit\r\nfunctionally DNA polymerase have been investigated. Affinity constants\r\nhave been determined to be 5x107 M-1 and 1x108 M-1 for \u0394 and \u039b 1Rh(\r\nMGP)2phi5+ binding to the DNA sequences 5'-CATCTG-3' and 5'-\r\nCATATG-3' respectively. The exchange rate, at 21\u00b0C, has been determined to\r\nbe 16 s-1 for the binding of 1-\u039b-Rh(MGP)2phi5+ to 5'-CATATG-3' through the\r\nuse of variable temperature 1H-NMR. Similar 1H-NMR experiments were\r\ncarried out to determine the kinetics of the interaction of 1-\u0394-Rh(MGP)2phi5+\r\nwith a duplex DNA of the sequence 5'-CGCATCTGAC-3'. 1-\u039b-Rh(\r\nMGP)2phi5+, 1-\u0394-Rh(MGP)2phi5+, and Rh(MT)phi3+, which binds to 5'-\r\nTGCA-3', were found to be potent sequence-specific inhibitors of DNA\r\npolymerase. All of these complexes bind to DNA through intercalation. In\r\nexperiments where two templates competed for extension by DNA\r\npolymerase, these complexes were shown to inhibit the extension of\r\ntemplates containing their binding sequences as compared to control\r\ntemplates. Furthermore, in direct competition experiments containing two\r\ntemplates, where each contained a binding sequence for a different metal\r\ncomplex, the relative activity of DNA polymerase on each template was\r\n\"tuned\" by the addition of metal complex specific for that template. \u0394-Rh(\r\nDPB)2phi3+ was also found to be a potent inhibitor of DNA polymerase,\r\nbut not in a template-specific manner. The relative potency of sequence- specific\r\ninhibition shown by 1-\u039b-Rh(MGP)2phi5+, 1-\u039b-Rh(MGP)2phi5+, and\r\nRh(MT)2phi3+ was compared to the binding kinetics, complex size, complex\r\ncharge, binding affinity and binding induced DNA distortion for these\r\ncomplexes. Greater DNA distortion was found to correlate with greater\r\ninhibition. These studies have shown that these molecules not only bind to\r\nDNA in a sequence-specific manner, but can functionally inhibit enzymatic\r\nreactions in a sequence-specific manner as well.", "doi": "10.7907/fb9e-rw88", "publication_date": "1997", "thesis_type": "phd", "thesis_year": "1997" }, { "id": "thesis:17590", "collection": "thesis", "collection_id": "17590", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:08042025-205701232", "primary_object_url": { "basename": "Johann_TW_1997.pdf", "content": "final", "filesize": 76248947, "license": "other", "mime_type": "application/pdf", "url": "/17590/1/Johann_TW_1997.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Sequence-Specific Inhibition of DNA Polymerase by Phenanthrene Quinone Diimine Complexes of Rhodium(III)", "author": [ { "family_name": "Johann", "given_name": "Timothy Wilmot", "orcid": "0000-0003-2212-9684", "clpid": "Johann-Timothy-Wilmot" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Baldeschwieler", "given_name": "John D.", "clpid": "Baldeschwieler-J-D" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" }, { "family_name": "Okumura", "given_name": "Mitchio", "orcid": "0000-0001-6874-1137", "clpid": "Okumura-M" }, { "family_name": "Parker", "given_name": "Carl Stevens", "orcid": "0000-0001-9795-4211", "clpid": "Parker-C-S" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The DNA binding characteristics of several phenanthrenequinone\r\ndiimine (phi) complexes of rhodium (III) as well as their ability to inhibit\r\nfunctionally DNA polymerase have been investigated. Affinity constants\r\nhave been determined to be 5x107 M-1 and 1x108 M-1 for \u0394 and \u039b 1Rh(\r\nMGP)2phi5+ binding to the DNA sequences 5'-CATCTG-3' and 5'-\r\nCATATG-3' respectively. The exchange rate, at 21\u00b0C, has been determined to\r\nbe 16 s-1 for the binding of 1-\u039b-Rh(MGP)2phi5+ to 5'-CATATG-3' through the\r\nuse of variable temperature 1H-NMR. Similar 1H-NMR experiments were\r\ncarried out to determine the kinetics of the interaction of 1-\u0394-Rh(MGP)2phi5+\r\nwith a duplex DNA of the sequence 5'-CGCATCTGAC-3'. 1-\u039b-Rh(\r\nMGP)2phi5+, 1-\u0394-Rh(MGP)2phi5+, and Rh(MT)phi3+, which binds to 5'-\r\nTGCA-3', were found to be potent sequence-specific inhibitors of DNA\r\npolymerase. All of these complexes bind to DNA through intercalation. In\r\nexperiments where two templates competed for extension by DNA\r\npolymerase, these complexes were shown to inhibit the extension of\r\ntemplates containing their binding sequences as compared to control\r\ntemplates. Furthermore, in direct competition experiments containing two\r\ntemplates, where each contained a binding sequence for a different metal\r\ncomplex, the relative activity of DNA polymerase on each template was\r\n\"tuned\" by the addition of metal complex specific for that template. \u0394-Rh(\r\nDPB)2phi3+ was also found to be a potent inhibitor of DNA polymerase,\r\nbut not in a template-specific manner. The relative potency of sequence- specific\r\ninhibition shown by 1-\u039b-Rh(MGP)2phi5+, 1-\u039b-Rh(MGP)2phi5+, and\r\nRh(MT)2phi3+ was compared to the binding kinetics, complex size, complex\r\ncharge, binding affinity and binding induced DNA distortion for these\r\ncomplexes. Greater DNA distortion was found to correlate with greater\r\ninhibition. These studies have shown that these molecules not only bind to\r\nDNA in a sequence-specific manner, but can functionally inhibit enzymatic\r\nreactions in a sequence-specific manner as well.", "doi": "10.7907/fb9e-rw88", "publication_date": "1997", "thesis_type": "phd", "thesis_year": "1997" }, { "id": "thesis:17543", "collection": "thesis", "collection_id": "17543", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:07212025-032307531", "primary_object_url": { "basename": "Kenyon_CN_1997.pdf", "content": "final", "filesize": 58673989, "license": "other", "mime_type": "application/pdf", "url": "/17543/1/Kenyon_CN_1997.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Time-Resolved and Steady-State Investigations of Carrier Dynamics at the Semiconductor/Liquid Interface", "author": [ { "family_name": "Kenyon", "given_name": "Christopher Neil", "clpid": "Kenyon-Christopher-Neil" } ], "thesis_advisor": [ { "family_name": "Lewis", "given_name": "Nathan Saul", "orcid": "0000-0001-5245-0538", "clpid": "Lewis-N-S" } ], "thesis_committee": [ { "family_name": "Anson", "given_name": "Fred C.", "clpid": "Anson-F-C" }, { "family_name": "Lewis", "given_name": "Nathan Saul", "orcid": "0000-0001-5245-0538", "clpid": "Lewis-N-S" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" }, { "family_name": "Blake", "given_name": "Geoffrey A.", "orcid": "0000-0003-0787-1610", "clpid": "Blake-G-A" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Fundamental investigations of carrier transport and charge transfer at the\r\nsemiconductor/liquid interface are presented. The application of both steady-state and time-resolved\r\nmethods to these studies is discussed.
\r\n\r\nSmall signal photocurrent transients have been measured for n-Si/CH3OH-dimethylferrocene\r\n(Me2Fc)+/0/Pt and n-Si/Au/CH3OH-Me2Fc+/0/Pt interfaces. The\r\nphotocurrent transients for these interfaces decayed in less than 10 \u03bcs, and were limited by the\r\nseries resistance of the cell in combination with the space charge capacitance of the\r\nsemiconductor. An equivalent circuit model is presented and physically justified in order to\r\nexplain this behavior, and to elucidate the conditions under which photocurrent transients at\r\nsemiconductor electrodes can be expected to yield information regarding the faradaic charge\r\ntransfer rate across the semiconductor/liquid interface. To provide additional support for\r\nthe equivalent circuit representation, transient photocurrent responses are also presented for\r\nn-Si/Pt/NaOH(aq)/Ni(OH)2//Ni, n-TiO2//NaOH(aq)/Ni(OH)2//Ni and n-TiO2//NaOH(aq)Fe(CN)63-/4-fNi(OH)2/Ni contacts.
\r\n\r\nStudies are reported on the behaviors of thin, nearly intrinsically doped Si\r\nelectrodes having interdigitated n+ and p+ back contact points. An analysis of the factors\r\ngoverning the photocurrent directionality and photovoltage in these samples is presented.\r\nAdditionally the back contact geometry has been exploited to perform measurements of the\r\nopen circuit potential of either electrons or holes while the other carrier type was under\r\nelectrical control. In combination with current density-voltage measurements of carriers\r\npassing through the back contact points, these data allowed a comparison of the behavior of\r\na given carrier type when generated by an applied bias (i.e., as majority carriers) relative to\r\ntheir behavior when generated with band gap illumination of the solid (as minority\r\ncarriers). The results have been used to validate certain key predictions of the quasi-Fermi\r\nlevel concept in photo-electrochemistry. In addition, digital simulations that include two-dimensional\r\nrepresentations of the charge density distribution and of the current fluxes in\r\nthe solid have been utilized to provide a quantitative understanding of the observed\r\nexperimental behavior.
\r\n\r\nThe application of time-resolved photoluminescence to the study of InP interfaces is\r\ndescribed. Photoluminescence decay profiles for etched n-type and p-type InP have been\r\nrecorded. These data provide support for a bulk-recombination limited PL lifetime in p-InP,\r\nwhile that of n-InP is evidenced to be dominated by radiative recombination.\r\nAdditional PL decay data are reported for a series of InP/liquid contacts. InP/CH3CN\r\njunctions having Me2Fc+/0, decamethylferrocene+/0 (Me10Fc+/0), methyl viologen\r\n(MV)2+/+, and cobaltocene (COCp2)+/0 as acceptor species have been studied. Quantitative\r\ninformation on the rate constant for charge transfer could not be obtained from these\r\nstudies, but upper limits are suggested, and promising systems for further study are\r\nidentified.
", "doi": "10.7907/ef6e-0w18", "publication_date": "1997", "thesis_type": "phd", "thesis_year": "1997" }, { "id": "thesis:17543", "collection": "thesis", "collection_id": "17543", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:07212025-032307531", "primary_object_url": { "basename": "Kenyon_CN_1997.pdf", "content": "final", "filesize": 58673989, "license": "other", "mime_type": "application/pdf", "url": "/17543/1/Kenyon_CN_1997.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Time-Resolved and Steady-State Investigations of Carrier Dynamics at the Semiconductor/Liquid Interface", "author": [ { "family_name": "Kenyon", "given_name": "Christopher Neil", "clpid": "Kenyon-Christopher-Neil" } ], "thesis_advisor": [ { "family_name": "Lewis", "given_name": "Nathan Saul", "orcid": "0000-0001-5245-0538", "clpid": "Lewis-N-S" } ], "thesis_committee": [ { "family_name": "Anson", "given_name": "Fred C.", "clpid": "Anson-F-C" }, { "family_name": "Lewis", "given_name": "Nathan Saul", "orcid": "0000-0001-5245-0538", "clpid": "Lewis-N-S" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" }, { "family_name": "Blake", "given_name": "Geoffrey A.", "orcid": "0000-0003-0787-1610", "clpid": "Blake-G-A" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Fundamental investigations of carrier transport and charge transfer at the\r\nsemiconductor/liquid interface are presented. The application of both steady-state and time-resolved\r\nmethods to these studies is discussed.
\r\n\r\nSmall signal photocurrent transients have been measured for n-Si/CH3OH-dimethylferrocene\r\n(Me2Fc)+/0/Pt and n-Si/Au/CH3OH-Me2Fc+/0/Pt interfaces. The\r\nphotocurrent transients for these interfaces decayed in less than 10 \u03bcs, and were limited by the\r\nseries resistance of the cell in combination with the space charge capacitance of the\r\nsemiconductor. An equivalent circuit model is presented and physically justified in order to\r\nexplain this behavior, and to elucidate the conditions under which photocurrent transients at\r\nsemiconductor electrodes can be expected to yield information regarding the faradaic charge\r\ntransfer rate across the semiconductor/liquid interface. To provide additional support for\r\nthe equivalent circuit representation, transient photocurrent responses are also presented for\r\nn-Si/Pt/NaOH(aq)/Ni(OH)2//Ni, n-TiO2//NaOH(aq)/Ni(OH)2//Ni and n-TiO2//NaOH(aq)Fe(CN)63-/4-fNi(OH)2/Ni contacts.
\r\n\r\nStudies are reported on the behaviors of thin, nearly intrinsically doped Si\r\nelectrodes having interdigitated n+ and p+ back contact points. An analysis of the factors\r\ngoverning the photocurrent directionality and photovoltage in these samples is presented.\r\nAdditionally the back contact geometry has been exploited to perform measurements of the\r\nopen circuit potential of either electrons or holes while the other carrier type was under\r\nelectrical control. In combination with current density-voltage measurements of carriers\r\npassing through the back contact points, these data allowed a comparison of the behavior of\r\na given carrier type when generated by an applied bias (i.e., as majority carriers) relative to\r\ntheir behavior when generated with band gap illumination of the solid (as minority\r\ncarriers). The results have been used to validate certain key predictions of the quasi-Fermi\r\nlevel concept in photo-electrochemistry. In addition, digital simulations that include two-dimensional\r\nrepresentations of the charge density distribution and of the current fluxes in\r\nthe solid have been utilized to provide a quantitative understanding of the observed\r\nexperimental behavior.
\r\n\r\nThe application of time-resolved photoluminescence to the study of InP interfaces is\r\ndescribed. Photoluminescence decay profiles for etched n-type and p-type InP have been\r\nrecorded. These data provide support for a bulk-recombination limited PL lifetime in p-InP,\r\nwhile that of n-InP is evidenced to be dominated by radiative recombination.\r\nAdditional PL decay data are reported for a series of InP/liquid contacts. InP/CH3CN\r\njunctions having Me2Fc+/0, decamethylferrocene+/0 (Me10Fc+/0), methyl viologen\r\n(MV)2+/+, and cobaltocene (COCp2)+/0 as acceptor species have been studied. Quantitative\r\ninformation on the rate constant for charge transfer could not be obtained from these\r\nstudies, but upper limits are suggested, and promising systems for further study are\r\nidentified.
", "doi": "10.7907/ef6e-0w18", "publication_date": "1997", "thesis_type": "phd", "thesis_year": "1997" }, { "id": "thesis:17541", "collection": "thesis", "collection_id": "17541", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:07182025-180545892", "primary_object_url": { "basename": "Lin_SC_1997.pdf", "content": "final", "filesize": 38508661, "license": "other", "mime_type": "application/pdf", "url": "/17541/1/Lin_SC_1997.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "The Molecular Recognition of DNA by Rhodium(III)-Zinc Finger Peptide Chimeras", "author": [ { "family_name": "Lin", "given_name": "Susanne Chosein", "clpid": "Lin-Susanne-Chosein" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" }, { "family_name": "Dougherty", "given_name": "Dennis A.", "orcid": "0000-0003-1464-2461", "clpid": "Dougherty-D-A" }, { "family_name": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Covalent chimeras of zinc finger peptide domains with\r\nphenanthrenequinone diimine (phi) complexes of rhodium (III) have been\r\ndesigned, synthesized and their DNA recognition characteristics examined. The\r\nrhodium complex binds in the major groove of DNA by intercalation and allows\r\nthe attached peptide to interact with DNA in a sequence-specific manner.\r\nChimeras of [Rh(phi)2(bpy')]3+ (bpy' = 4-(4-carboxybutyl), 4'-methyl-2,2'bipyridine)\r\nand [Rh(phi)2(phen')]3+ (phen' = (5-amidoglutaryl)-1,10-phen-anthroline)\r\nand four different zinc finger peptides (Sp1 finger 2 and 3, ADR1b and\r\nADR1b-Ala) have been successfully synthesized using solid phase coupling\r\nmethodology. Electronic spectroscopy showed the rhodium complex and\r\npeptide to be essentially independent units. A method to successfully fold the\r\npeptide portion of the chimera with zinc has been developed, and 1H HMR\r\nspectroscopy has been used to confirm folding. The resultant chimeras bind\r\ntightly to DNA, and the rhodium intercalator promotes DNA cleavage with\r\nphotoactivation. Analysis of the DNA sites targeted by the chimeras on DNA\r\nrestriction fragments have demonstrated that the peptide can direct new\r\nrecognition. Variations in the rhodium complexes and peptides resulted in\r\ndifferences in specificity as seen by photocleavage. Studies on smaller\r\noligonucleotides containing the recognition sequences have shown the rhodium -\r\nSp1-2 chimera to bind with affinities of 107-108 M-1 for its target sites. Hence,\r\nformation of rhodium(III) - zinc finger chimeras provide a route to establish\r\nhigh affinity DNA binding by a single zinc finger domain. At some sites, the\r\nrhodium complex and zinc finger appeared to bind independently to adjacent\r\nsegments. For the [Rh(phi)2(phen')]3+ - Sp1 - 2 chimeras, a strong high affinity\r\nsite (Ka greater than or equal to 108 M-1) was observed, where it was postulated that the rhodium\r\ncomplex and zinc finger bind to the opposite strands of the GCG binding site in a\r\ncooperative fashion. These rhodium (III) - zinc finger chimeras represent a new\r\nroute to examine the specific interactions of a single zinc finger with DNA in\r\nchemical detail and provide the basis to build a family of sequence-specific DNA\r\nbinding molecules.", "doi": "10.7907/mss5-wf97", "publication_date": "1997", "thesis_type": "phd", "thesis_year": "1997" }, { "id": "thesis:17534", "collection": "thesis", "collection_id": "17534", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:07162025-181821401", "primary_object_url": { "basename": "Singer_RA_1997.pdf", "content": "final", "filesize": 55951942, "license": "other", "mime_type": "application/pdf", "url": "/17534/1/Singer_RA_1997.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Design of Novel Titanium(IV) Schiff Base Complexes for Catalytic, Enantioselective Aldol Additions to Aldehydes", "author": [ { "family_name": "Singer", "given_name": "Robert Alan", "orcid": "0000-0001-9730-1261", "clpid": "Singer-Robert-Alan" } ], "thesis_advisor": [ { "family_name": "Carreira", "given_name": "Erick Moran", "orcid": "0000-0003-1472-490X", "clpid": "Carreira-E-M" } ], "thesis_committee": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "orcid": "0000-0003-1464-2461", "clpid": "Dougherty-D-A" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" }, { "family_name": "Carreira", "given_name": "Erick Moran", "orcid": "0000-0003-1472-490X", "clpid": "Carreira-E-M" }, { "family_name": "Myers", "given_name": "Andrew G.", "clpid": "Myers-A-G" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "A novel chiral Schiff base ligand derived from 2-amino-2'-hydroxy-1, 1'-binaphthyl\r\nand 3-bromo-5-tbutylsalicylaldehyde has been prepared. When binding the chiral ligand to\r\ntitanium(IV) with 3,5-di-fbutylsalicylic acid, the complex formed functions as an efficient\r\ncatalyst for the Mukaiyama aldol addition reaction. Using only 1-2 mol% of the catalyst,\r\nsilyl ketene acetal additions to aldehydes were carried out in good chemical yield and in\r\nexcellent levels of enantioselectivity. Unsaturated aldehydes tended to produce adducts in\r\n95-99% ee, while aliphatic aldehyde products were typically obtained in 94-95% ee.
\r\n\r\nThe methodology was extended to include dienolate additions to aldehydes by\r\nutilizing silyl enol ethers of dioxinones. Optimal selectivities in the dienolate additions\r\nwere obtained with unsaturated, unbranched aldehydes (90-94% ee). Aromatic and\r\naliphatic aldehydes were usually isolated in 80-84% ee. Since many of the adducts were\r\ncrystalline solids, the optical purity was enhanced by recrystallization. By heating the\r\ndioxinone adduct in the presence of an alcohol or amine, the products were transformed to\r\nmore useful \u03b2-ketoesters or \u03b2-ketoamides.
\r\n\r\nTo demonstrate the utility of the methodology developed, the asymmetric addition\r\nreactions have been applied to the total synthesis of (R)-epinephrine and macrolactin A.\r\nAfter carrying out an enantioselective acetate addition to 3,4-dimethoxybenzaldehyde in\r\n95% ee, the \u03b2-hydroxyester was converted to the amino-alcohol by a Hoffman\r\nrearrangement and a reduction. After deprotecting the catechol, (R)-epinephrine was\r\nobtained in 5 steps overall. Dienolate additions to \u03b2-stannylpropenal were employed to\r\nprepare two key fragments of macrolactin A. The convergent route involved stitching\r\ntogether three fragments with a Stille coupling and a Horner-Emmons olefination.\r\nMacrocyclization was accomplished by an intramolecular Stille coupling.
", "doi": "10.7907/w287-z238", "publication_date": "1997", "thesis_type": "phd", "thesis_year": "1997" }, { "id": "thesis:8089", "collection": "thesis", "collection_id": "8089", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:02202014-095838875", "primary_object_url": { "basename": "Yang-bh-1997.pdf", "content": "final", "filesize": 35168728, "license": "other", "mime_type": "application/pdf", "url": "/8089/1/Yang-bh-1997.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Use of pseudoephedrine as a practical chiral auxiliary for asymmetric synthesis", "author": [ { "family_name": "Yang", "given_name": "Bryant H.", "clpid": "Yang-Bryant-H" } ], "thesis_advisor": [ { "family_name": "Myers", "given_name": "Andrew G.", "clpid": "Myers-A-G" } ], "thesis_committee": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" }, { "family_name": "Carreira", "given_name": "Erick Moran", "clpid": "Carreira-E-M" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Myers", "given_name": "Andrew G.", "clpid": "Myers-A-G" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The use of pseudoephedrine as a practical chiral auxiliary for asymmetric synthesis is describe. Both enantiomers of pseudoephedrine are inexpensive commodity chemicals and can be N-acylated in high yields to form tertiary amides. In the presence of lithium chloride, the enolates of the corresponding pseudoephedrine amides undergo highly diastereoselective a1kylations with a wide range of alkyl halides to afford \u03b1-substituted products in high yields. These products can then be transformed in a single operation into highly enantiomerically enriched carboxylic acids, alcohols, and aldehydes. Lithium amidotrihydroborate (LAB) is shown to be a powerful reductant for the selective reduction of tertiary amides in general and pseudoephedrine amides in particular to form primary alcohols.
\t\r\n", "doi": "10.7907/1K2S-P110", "publication_date": "1997", "thesis_type": "phd", "thesis_year": "1997" }, { "id": "thesis:17528", "collection": "thesis", "collection_id": "17528", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:07152025-200013414", "primary_object_url": { "basename": "Mines_GA_1997.pdf", "content": "final", "filesize": 47880549, "license": "other", "mime_type": "application/pdf", "url": "/17528/1/Mines_GA_1997.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Cytochrome c : Folding Triggered by Electron Transfer. Rates of Heme Oxidation and Reduction at High Driving Forces", "author": [ { "family_name": "Mines", "given_name": "Gary Alan", "clpid": "Mines-Gary-Alan" } ], "thesis_advisor": [ { "family_name": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" }, { "family_name": "Chan", "given_name": "Sunney I.", "orcid": "0000-0002-5348-2723", "clpid": "Chan-S-I" } ], "thesis_committee": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" }, { "family_name": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" }, { "family_name": "Chan", "given_name": "Sunney I.", "orcid": "0000-0002-5348-2723", "clpid": "Chan-S-I" }, { "family_name": "Goddard", "given_name": "William A., III", "orcid": "0000-0003-0097-5716", "clpid": "Goddard-W-A-III" }, { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Rates of various intramolecular heme oxidations and reductions in a series of\r\nclosely related RuL2(X)(His33)cytochromes c [L = bipyridine or phenanthroline\r\nderivatives; X = imidazole (im) or cyanide (CN-)] have been measured over a freeenergy\r\nrange of 0.54 to 1.89 eV. The driving-force dependence of Fe2+\u2192Ru3+ electron\r\ntransfer (ET) is well described by semiclassical ET theory with a coupling-limited rate\r\n(kmax) of 2.8 x 106 s-1 and a reorganization energy of 0.74 eV. As predicted by theory,\r\nthe rate of an exergonic (-\u0394G\u00b0 = 1.3 eV) heme reduction reaction,\r\n*Ru2+(bpy)2(im)(His)\u2192Fe3+, falls in the inverted region (k = 2.0 x 105 s-1). In contrast,\r\nthe rates of three highly exergonic heme reductions, *Ru2+(phen)2(CN)(His)\u2192Fe3+ (3.1\r\nx 105 s-1; 1.4 eV), Ru+(4,4'-(CONH(C2H5))2-bpy)2(im)(His)\u2192Fe3+ (2.3 x 105 s-1; 1.44\r\neV), and Ru+(phen)2(CN)(His)\u2192Fe3+ (4.5 x 105 s-1; 1.89 eV), are much higher than\r\nexpected for reactions directly to ground-state products. Agreement with theory is\r\ngreatly improved by assuming that an electronically excited ferroheme\r\n(Fe2+\u2192*Fe2+ ~ 1.05 eV) is the initial product in each of these reactions.
\r\n\r\nIn a separate investigation, rates of folding of ferrocytochromes c from horse\r\n(h-cyt c) and yeast (y-cyt c) were measured over a range of denaturant concentrations\r\n(guanidine hydrochloride, GuHCl) and folding free energies (\u0394Gf) using a new ET\r\ntriggering technique. The backbone structures of the two homologs are similar, but y-cyt\r\nc is ~ 15 kJ mol-1 less stable than h-cyt c and is unfolded at concentrations of GuHCl ~ 1.5\r\nM lower than for h-cyt c. Activation free energies exhibit a linear dependence on GuHCl\r\nand \u0394Gf for both proteins, with folding rates decreasing with increasing concentration of\r\nGuHCl (less negative \u0394Gf). At a given denaturant concentration, the folding rates for y-cyt\r\nc are about an order of magnitude slower than those for h-cyt c, but when the folding\r\nfree energies are matched, folding rates of the two homologs are comparable.
", "doi": "10.7907/rtx8-jn96", "publication_date": "1997", "thesis_type": "phd", "thesis_year": "1997" }, { "id": "thesis:17681", "collection": "thesis", "collection_id": "17681", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:09152025-212928780", "primary_object_url": { "basename": "Terbrueggen_R_1996.pdf", "content": "final", "filesize": 65415187, "license": "other", "mime_type": "application/pdf", "url": "/17681/1/Terbrueggen_R_1996.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Exploring the Direct and Indirect Readout of DNA with Phenanthrenequinone Diimine Complexes of Rhodium(III)", "author": [ { "family_name": "Terbrueggen", "given_name": "Robert Henry", "clpid": "Terbrueggen-Robert-Henry" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" }, { "family_name": "Baldeschwieler", "given_name": "John D.", "clpid": "Baldeschwieler-J-D" }, { "family_name": "Lewis", "given_name": "Nathan Saul", "orcid": "0000-0001-5245-0538", "clpid": "Lewis-N-S" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Phenanthrenequinone diimine (phi) complexes of rhodium(III) have been\r\ndesigned and characterized in order to investigate the principles of direct and\r\nindirect readout of double helical DNA The metallointercalator l-Rh(MGP)2phi5+\r\n(MGP = 4-guanidylmethyl-1,10-phenanthroline) binds via intercalation in the\r\nmajor groove of DNA and upon irradiation promotes DNA strand scission. The\r\n\u039b-enantiomer, \u039b-1-Rh(MGP)2phi5+, binds at subnanomolar concentrations to the\r\n6 base pair sequence, 5'-CATATG-3', with enantiospecificity. An essential feature\r\nof this recognition is the sequence-specific unwinding of the DNA helix which\r\npermits direct contacts between guanidinium functionalities on the metal complex\r\nand guanine residues. Deazaguanine substitutions were used to establish direct\r\ncontacts between the N7 nitrogen atoms of guanine and the guanidinium moiety on\r\nthe metal complex. Through an assay developed to test for sequence-specific DNA\r\nunwinding, a 70 \u00b1 10 degrees unwinding of the sequence 5'-CATATG-3' upon\r\nbinding by \u039b-1-Rh(MGP)2phi5+ was established. Thus, the sequence-dependent\r\ntwistability of DNA plays an important role in determining the sequence specificity\r\nof the complex. The \u0394-enantiomer, \u0394-1-Rh(MGP)2phi5+, binds preferentially to\r\nthe 6 base pair sequence, 5'-CATCTG-3'. The hierarchy of recognition sites\r\ndetermined in photocleavage studies on oligonucleotides suggests that DNA\r\nrecognition by this complex also involves sequence specific contacts by the\r\nguanidinium functionalities. Photocleavage studies indicate additional similarities\r\nin the recognition of \u0394 and \u039b-1-Rh(MGP)2phi5+. Both enantiomers of 1-\r\nRh(MGP)2phi5+ display increased binding specificity relative to the parent\r\ncomplex, Rh(phen)2phi3+. The exchange rates of both enantiomers are also\r\ndecreased at least a 1000-fold relative to Rh(phen)2phi3+. Studies in which the\r\nlength of the linker arm between the core of the metal complex and the guanidinium\r\nmoiety was varied demonstrate that proper orientation of the guanidinium moiety is\r\nan essential feature of complex specificity. As the length of the linker arm\r\nincreases, the binding specificity of the complex decreases. DNA recognition\r\nstudies with Rh(APB)2phi5+ (APB= 4-(3-aminopropyl)-4'-2,2'-bipyridine) have\r\ndemonstrated that the amino moiety can also be used to alter the sequence\r\nspecificity of phi complexes of rhodium(III), although the sequence specificity of\r\nthis complex is reduced greatly as compared with \u0394- and \u039b-1-Rh(MGP)2phi5+.\r\nThis work therefore demonstrates that the guanidinium moiety may be used to\r\nenhance both the binding affinity and specificity of phi complexes of rhodium(III).\r\nIn mimicking DNA binding proteins, molecules which recognize their binding sites\r\nthrough direct and indirect readout of the DNA can be designed. Importantly, this\r\nstudy highlights a new structural element of DNA recognition, the sequence-dependent\r\ntwistability of the DNA helix. This sequence-dependent twistability may\r\nbe an essential feature of the recognition of sequences by DNA-binding proteins\r\nand may be powerfully exploited in future design.", "doi": "10.7907/yv86-3710", "publication_date": "1996", "thesis_type": "phd", "thesis_year": "1996" }, { "id": "thesis:14040", "collection": "thesis", "collection_id": "14040", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:12222020-175451626", "primary_object_url": { "basename": "forman-je_1996.pdf", "content": "final", "filesize": 143036862, "license": "other", "mime_type": "application/pdf", "url": "/14040/1/forman-je_1996.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Non-Covalent Interactions in Aqueous Media: Molecular Recognition Studies Through Circular Dichroism and Self-Assembly of Discrete Aggregates", "author": [ { "family_name": "Forman", "given_name": "Jonathan Eric", "clpid": "Forman-Jonathan-Eric" } ], "thesis_advisor": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" } ], "thesis_committee": [ { "family_name": "Myers", "given_name": "Andrew G.", "clpid": "Myers-A-G" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The application of circular dichroism (CD) spectroscopy to the study of molecular recognition phenomena in chiral water-soluble cyclophane hosts is described. The CD method produces results that complement and expand upon previous NMR studies. This includes allowing the measurement of larger binding constants by allowing studies to be carried out at lower concentrations.
\r\n\r\nUsing the excitonic chirality method, these studies have provided a means of assigning the absolute stereochemistry of the ethenoanthracene building blocks used in preparation of the hosts. This information, along with an x-ray structure of one of the cyclophane molecules, has provided important information concerning host structure. The x-ray structure and CD spectral changes observed on guest binding have also served to provide direct experimental evidence for binding conformations of the hosts.
\r\n\r\nThe chiral hosts have been shown to induce CD in achiral chromophoric guests. Analysis of this induced CD using INDO/S and coupled-oscillator calculations has provided valuable information concerning the conformations of the bound guest. These data complement information obtained in NMR studies (D values) and provide additional insights into the important factors that govern the binding event.
\r\n\r\nFinally, preliminary studies of self-assembling systems in aqueous media are reported. These studies employ etheno- and ethanoanthracene based structures designed to form aggregates with well defined order and discrete stoichiometries. These molecules are designed to aggregate through hydrophobic forces. The aggregate is kept from becoming a micelle using polar groups strategically placed to complement one another within the assembling structure.
", "doi": "10.7907/ce8j-qk19", "publication_date": "1996", "thesis_type": "phd", "thesis_year": "1996" }, { "id": "thesis:3383", "collection": "thesis", "collection_id": "3383", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-09082006-114255", "primary_object_url": { "basename": "Campisi_d_1996.pdf", "content": "final", "filesize": 7329538, "license": "other", "mime_type": "application/pdf", "url": "/3383/1/Campisi_d_1996.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Transition metal complexes as probes of DNA sequence-dependent structure", "author": [ { "family_name": "Campisi", "given_name": "Donna", "clpid": "Campisi-D" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Lewis", "given_name": "Nathan Saul", "clpid": "Lewis-N-S" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Different transition metal complexes have been applied in probing variations in the structure of double helical DNA. The following probes, which all bind DNA noncovalently, have been utilized: Ru(phen)3(2)+, Ru(TMP)3(2)+, Rh(phen)2phi3+, Rh(TMP)2phi3+, Rh(dmbpy)2phi3+, Ru(phen)2dppz2+, Ru(bpy)2dppz2+, and Rh(bpy)2dppz3+ (phen = 1,10 phenanthroline; TMP = 3,4,7,8,-tetramethyl- 1,10-phenanthroline; phi = 9,10-phenanthrenequinone diimine; dmbpy = 5,5'-dimethylbipyridyl; bpy = bipyridyl; dppz = dipyrido[3,2-a;2',3'-c]phenazine). The local structure recognized by [Delta]-Rh(phen)2phi3+ has been defined by comparisons of photocleavage data on crystallographically characterized oligonucleotides with their structural parameters. A quantitative correlation has been determined between [Delta]-Rh(phen)2phi3+ photocleavage and extent of openness in the major groove due to differential propeller twisting, or interpurine angle. Therefore, [Delta]-Rh(phen)2phi3+ has been developed as a probe of DNA propeller twisting in solution. Differences in reaction pathway partitioning between enantiomers of Rh(phen)2phi3+ are attributed to differing extent of shape complementarity with DNA binding sites. Rh(TMP)2phi3+ has been explored in probing DNA mismatches in solution. Both [Delta]-Rh(phen)2phi3+ and Rh(TMP)2phi3+ sensitively mark local structural perturbations in an oligonucleotide, arising from substitution of a CG base pair with TG and AG mismatches. Rh(phen)2phi3+ and Ru(TMP)3(2) have also been applied in probing structural variations in the context of a long DNA strand. A C7 stretch is targeted by Ru(TMP)3(2), an A DNA probe and Rh(phen)2phi3+, a B DNA probe. These results indicate this sequence is heteronomous, containing wide major and minor grooves. [Delta]- and [Lambda]-Rh(phen)2phi3+ also discriminate structural differences between bent and nonbent DNA fragments. Variations in metal complex-DNA interactions have also been examined by a gel electrophoretic mobility assay. Intercalator size, hydrophobicity of ancillary ligands, metal complex charge, and chirality all influence the extent of DNA retardation. Taken together, these studies demonstrate that transition metal complexes can be profitably and uniquely applied towards exploring DNA structural heterogeneity.", "doi": "10.7907/kay4-sz63", "publication_date": "1996", "thesis_type": "phd", "thesis_year": "1996" }, { "id": "thesis:8062", "collection": "thesis", "collection_id": "8062", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:02042014-091755725", "primary_object_url": { "basename": "Bren_kl_1996.pdf", "content": "final", "filesize": 60833701, "license": "other", "mime_type": "application/pdf", "url": "/8062/1/Bren_kl_1996.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Structurally Engineered Cytochromes c with Novel Ligand-Binding Properties", "author": [ { "family_name": "Bren", "given_name": "Kara Lynne", "orcid": "0000-0002-8082-3634", "clpid": "Bren-Kara-Lynne" } ], "thesis_advisor": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" } ], "thesis_committee": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Richards", "given_name": "John H.", "clpid": "Richards-J-H" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Semisynthesis of horse heart cytochrome c and site-directed mutagenesis of Saccharomyces cerevisiae (S. c.) iso-1-cytochrome c have been utilized to substitute Ala for the cytochrome c heme axial ligand Met80 to yield ligand-binding proteins (horse heart Ala80cyt c and S. c. Ala80cyt c) with spectroscopic properties remarkably similar to those of myoglobin. Both species of Fe(II)Ala80cyt c form exceptionally stable dioxygen complexes with autoxidation rates 10-30x smaller and O2 binding constants ~ 3x greater than those of myoglobin. The resistance of O2-Fe(II)Ala80cyt c to autoxidation is attributed in part to protection of the heme site from solvent as exhibited by the exceptionally slow rate of CO binding to the heme as well as the low quantum yield of CO photodissociation.
\r\n\r\nUV/vis, EPR, and paramagnetic NMR spectroscopy indicate that at pH 7 the Fe(III)Ala80cyt c heme is low-spin with axial His-OH- coordination and that below pH ~6.5, Fe(III)Ala80cyt c is high-spin with His-H2O heme ligation. Significant differences in the pH dependence of the 1H NMR spectra of S. c. Fe(III)Ala80cyt c compared to wild-type demonstrate that the axial ligands influence the conformational energetics of cytochrome c.
\r\n\r\n1H NMR spectroscopy has been utilized to determine the solution structure of the cyanide derivative of S. c. Fe(III)Ala80cyt c. 82% of the resonances in the 1H NMR spectrum of S. c. CN-Fe(III)Ala80cyt c have been assigned through 1D and 2D experiments. The RMSD values after restrained energy minimization of the family of 17 structures obtained from distance geometry calculations are 0.68 \u00b1 0.11 \u00c5 for the backbone and 1.32 \u00b1 0.14 \u00c5 for all heavy atoms. The solution structure indicates that a tyrosine in the \"distal\" pocket of CN-Fe(III)Ala80cyt c forms a hydrogen bond with the Fe(III)-CN unit, suggesting that it may play a role analogous to that of the distal histidine in myoglobin in stabilizing the dioxygen adduct.
", "doi": "10.7907/D8HZ-E792", "publication_date": "1996", "thesis_type": "phd", "thesis_year": "1996" }, { "id": "thesis:4022", "collection": "thesis", "collection_id": "4022", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-10102006-144012", "primary_object_url": { "basename": "Anderson_kk_1996.pdf", "content": "final", "filesize": 15826297, "license": "other", "mime_type": "application/pdf", "url": "/4022/1/Anderson_kk_1996.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "An Improved Model for One-Dimensional Polaronic Ferromagnetism: Poly-Meta-Phenylenefuchsone", "author": [ { "family_name": "Anderson", "given_name": "Kraig Knute", "clpid": "Anderson-Kraig-Knute" } ], "thesis_advisor": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "orcid": "0000-0003-1464-2461", "clpid": "Dougherty-D-A" } ], "thesis_committee": [ { "family_name": "Grubbs", "given_name": "Robert H.", "orcid": "0000-0002-0057-7817", "clpid": "Grubbs-R-H" }, { "family_name": "Dougherty", "given_name": "Dennis A.", "orcid": "0000-0003-1464-2461", "clpid": "Dougherty-D-A" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" }, { "family_name": "Okumura", "given_name": "Mitchio", "orcid": "0000-0001-6874-1137", "clpid": "Okumura-M" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The design, reductive doping, and magnetic characterization of poly-meta-phenylenefuchsone, an improved model for one-dimensional polaronic ferromagnetism, are described. Previous work demonstrated that delocalized radical cations, when linked through appropriate topologies, can exhibit ferromagnetic, or high spin interactions between unpaired electron spins. Consideration of these examples led to the choice of the radical anion of 2,6-di-tert-butylfuchsone as a spin containing unit, due to its relative stability, solubility, spin density, and ease of generation. Electrochemical doping of its polymers is more convenient and effective than chemical doping, resulting in a substantial increase in magnetic properties relative to previous models. The magnetic results are aided by dilution of the doped polymer in a diamagnetic solid, which is interpreted as reducing intermolecular antiferromagnetic interactions. The significant results of this model system provide clear directions for future designs, including improving solubility, spin density, doping efficiency, and defect suppression.
\r\n\r\nA tetraphenoxyl analog to existing quintet tetraradical A was envisioned. The synthesis and oxidation of its precursor tetraphenol I are described, as well as a number of simpler analogs. No evidence of high spin interactions was observed. Instead, the X-ray crystal structure of the product indicates that the oxidized tetraphenol undergoes ring closure of its central cyclobutane ring to form a bicyclobutane, which rapidly rearranges to a ring opened butadiene. The novel feature of this known rearrangement in the current system is that it occurs readily, even under very mild conditions.
", "doi": "10.7907/25a7-sf85", "publication_date": "1996", "thesis_type": "phd", "thesis_year": "1996" }, { "id": "thesis:11902", "collection": "thesis", "collection_id": "11902", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:11082019-094749072", "primary_object_url": { "basename": "jenkins-y-1996.pdf", "content": "final", "filesize": 6480454, "license": "other", "mime_type": "application/pdf", "url": "/11902/1/jenkins-y-1996.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Dipyridophenazine Complexes of Ruthenium(II) as Luminescent Reporters of DNA", "author": [ { "family_name": "Jenkins", "given_name": "Yonchu", "clpid": "Jenkins-Yonchu" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Imperiali", "given_name": "Barbara", "clpid": "Imperiali-B" }, { "family_name": "Richards", "given_name": "John H.", "clpid": "Richards-J-H" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The search for novel diagnostics for DNA detection has generated interest in the potential applications of polypyridyl complexes of ruthenium(II). Recently, it was reported that dipyridophenazine complexes of ruthenium(II) may serve as molecular light switches for DNA. These complexes, which bind DNA avidly through intercalation, show no luminescence in aqueous solutions. Upon intercalation into double-helical DNA and the concomitant protection of the phenazine ring from quenching by interaction with water, intense photoluminescence is apparent. The light switch effect as a function of nucleic acid sequence and conformation was examined for [Ru(phen)2dppz]2+ and [Ru(bpy)2dppz]2+. The emission properties of these complexes were found to be extremely sensitive to the nature of the intercalation environment with strong correlations between the luminescence parameters and the level of water protection afforded by the double helix. In order to impart sequence specificity to the light switch effect, various methods have been developed for appending a functionalized [Ru(phen)2dppz]2+ to the 5' terminus of oligonucleotides, both on the solid support and in solution. Assays for analyzing the structural integrity of the resulting conjugates are described. These ruthenated oligonucleotides can serve as enzyme substrates, enabling the construction of long metalated oligonucleotides not easily prepared using chemical synthesis. In order to evaluate their utility as useful DNA diagnostics, a series of ruthenated oligonucleotides were synthesized and their photophysical properties characterized. Biochemical analysis of oligonucleotide duplexes containing ruthenated strands showed no significant structural perturbation of the duplex as a result of the ruthenium modification. The overall results of this investigation suggest that an oligonucleotide functionalized with a dppz complex of ruthenium may be used to target single-stranded DNA in a sequence-specific fashion and that this derivative could be extremely valuable in the development of novel hybridization probes for both heterogeneous and homogeneous assays.
", "doi": "10.7907/n4jx-hp63", "publication_date": "1996", "thesis_type": "phd", "thesis_year": "1996" }, { "id": "thesis:4213", "collection": "thesis", "collection_id": "4213", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-10222007-132632", "primary_object_url": { "basename": "Sardesai_ny_1995.pdf", "content": "final", "filesize": 17895094, "license": "other", "mime_type": "application/pdf", "url": "/4213/1/Sardesai_ny_1995.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Rhodium intercalators as novel peptide delivery systems to the major groove of DNA : towards the design of artificial repressors", "author": [ { "family_name": "Sardesai", "given_name": "Niranjan Y.", "clpid": "Sardesai-N-Y" } ], "thesis_advisor": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" } ], "thesis_committee": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Bercaw", "given_name": "John E.", "clpid": "Bercaw-J-E" }, { "family_name": "Richards", "given_name": "John H.", "clpid": "Richards-J-H" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "NOTE: Text or symbols not renderable in plain ASCII are indicated by [...]. Abstract is included in .pdf document.\n\nPhenanthrenequinone diimine (phi) complexes of rhodium(III) bearing tethered peptides have been designed to serve as metallointercalating anchors to deliver peptide side chain functionalities for DNA recognition in the major groove. Metal-peptide \ncomplexes containing 11-15 amino acid residues were prepared using two complementary synthetic strategies: by direct coupling of a pendant carboxylate on the coordinatively saturated rhodium complex, [...] (phen'=5-amidoglutaryl-1, 10-phenanthroline), to the N-terminus of a resin-bound peptide in a manner analogous to the chain-elongation step in solid phase peptide synthesis; or by coupling phen' containing the pendant carboxylate to the resin-bound peptide, followed by coordination of [...] to the bidentate chelator attached to the peptide. With coordination complexes which are stable to peptide deprotection and cleavage conditions from the resin, the solid phase synthetic strategies prove convenient to apply. The metal-peptide complexes have been characterized by amino acid analysis, electronic spectroscopy, circular dichroism and mass spectrometry, where a novel pattern of peptide fragmentation facilitates the detailed sequence analysis of the appended peptide. All the metal-peptide complexes bind and, with photoactivation, cleave DNA with evidence of major groove chemistry. Significantly, the DNA site-specificity is seen to depend on the peptide side-chain functional groups. In one series, a single glutamate at position 10 is found to be essential in directing DNA site-recognition to the sequence 5'-CCA-3'. Methylation of the glutamate side chain or single ElOQ, E1OD, E1OA mutations abolish this selectivity. The glutamate is essential to maintain [...]-helicity in the peptide and make base specific contacts, thereby providing a glutamate switch for site-specific DNA recognition. A second series, based on the recognition helix of the phage 434 repressor, reproduces operator binding. Photocleavage and MPE-Fe footprint analysis indicates that these metal-peptide complexes bind to the 5'-ACAA-3' operator sequences as monomers at\n10 nM concentration and differentiate between operator site variants. These studies represent a new strategy to create an array of metal-peptide complexes with differing sequence specificity for DNA and suggest a route to the construction of small molecules that function as artificial repressors.\n", "doi": "10.7907/0v7r-fp28", "publication_date": "1995", "thesis_type": "phd", "thesis_year": "1995" }, { "id": "thesis:4052", "collection": "thesis", "collection_id": "4052", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-10122007-091509", "primary_object_url": { "basename": "Johnson_rd_1995.pdf", "content": "final", "filesize": 10056797, "license": "other", "mime_type": "application/pdf", "url": "/4052/1/Johnson_rd_1995.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Multivalent Protein Binding to Metal-Complexing Materials: Applications to Synthetic Receptors and Affinity Chromatography", "author": [ { "family_name": "Johnson", "given_name": "Robert David", "clpid": "Johnson-Robert-David" } ], "thesis_advisor": [ { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" } ], "thesis_committee": [ { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" }, { "family_name": "Gray", "given_name": "Harry B.", "orcid": "0000-0002-7937-7876", "clpid": "Gray-H-B" }, { "family_name": "Barton", "given_name": "Jacqueline K.", "orcid": "0000-0001-9883-1600", "clpid": "Barton-J-K" }, { "family_name": "Brady", "given_name": "John F.", "orcid": "0000-0001-5817-9128", "clpid": "Brady-J-F" }, { "family_name": "Davis", "given_name": "Mark E.", "orcid": "0000-0001-8294-1477", "clpid": "Davis-M-E" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "NOTE: Text or symbols not renderable in plain ASCII are indicated by [...]. Abstract is included in .pdf document.\r\n\r\n\r\nThis investigation demonstrates that proteins have the capability to bind \r\nsimultaneously to multiple transition metals of metal-complexing materials. This finding \r\nhas important implications for the design of novel materials for protein recognition. Our \r\napproach to protein recognition, based on intermolecular metal-to-ligand interactions, \r\nmatches a protein's unique pattern of histidines with a complementary arrangement of \r\ntransition metal complexes.\r\n\r\n A model system is used to demonstrate the validity of this approach in the \r\nsimplest case by matching the distance between metal ions of rationally designed bis-metal [...] \"receptors\" to that between imidazoles of bis-imidazole \"targets.\" This \r\nsystem additionally demonstrates how other features of receptor design can influence \r\nbinding selectivity. A 2D NMR procedure is developed to measure directly protein \r\nsurface histidine binding to copper complexes, and subsequently demonstrates that the \r\nlocal environment of the histidine and the structure of the copper complex can modulate \r\nindividual copper-histidine interactions. Thus it may indeed be possible to design metal-containing receptors which are able to form simultaneous metal-ligand bonds with a \r\nspecific arrangement of protein metal-coordinating groups.\r\n\r\n There are two important obstacles preventing a similarly detailed description of \r\nprotein binding to metal-complexing surfaces: protein adsorption may involve binding to \r\none or more metal sites, and a detailed description of the geometry of surface metal sites \r\nwould be hopelessly complex. We can, however, apply the microscopic concept of \r\nsimultaneous metal-ligand interactions to interpret the macroscopic phenomena of \r\nprotein partitioning in immobilized metal affinity chromatography (IMAC). In this \r\ncontext, the ability of commercially available IMAC materials to support multiple \r\nprotein-surface interactions is shown to be dependent on three factors: the number of\r\nhistidines on the protein (as manipulated by site directed mutagenesis), the number of \r\ndeprotonated amino groups on the protein (pH control), and the density of binding sites \r\non the surface (copper loading). These results demonstrate that a realistic description of \r\nprotein binding in IMAC must consider a heterogeneous population of surface binding \r\nsites. In IMAC this is shown to be conveniently expressed by the Temkin isotherm, \r\nmaking it an instructive model to explore heterogeneity displayed by other \r\nchromatographic materials and by biological systems.", "doi": "10.7907/6kdk-t643", "publication_date": "1995", "thesis_type": "phd", "thesis_year": "1995" }, { "id": "thesis:2596", "collection": "thesis", "collection_id": "2596", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-06142007-132218", "primary_object_url": { "basename": "Chang_tk_1991.pdf", "content": "final", "filesize": 8356149, "license": "other", "mime_type": "application/pdf", "url": "/2596/1/Chang_tk_1991.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Gene synthesis, expression, and mutagenesis of azurin", "author": [ { "family_name": "Chang", "given_name": "Thomas Kyu-Young", "clpid": "Chang-T-K" } ], "thesis_advisor": [ { "family_name": "Richards", "given_name": "John H.", "clpid": "Richards-J-H" }, { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" } ], "thesis_committee": [ { "family_name": "Barton", "given_name": "Jacqueline K.", "clpid": "Barton-J-K" }, { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Richards", "given_name": "John H.", "clpid": "Richards-J-H" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "A synthetic gene for the blue copper protein Pseudomonas aeruginosa azurin has been constructed using a novel, stepwise method. This method is designed to synthesize a gene of any size reliably by building only controlled amounts of the gene in each step. Another advantage of this method is that the intermediate plasmids, which are typically present in picomolar amounts, can be amplified through plasmid preparation for editing. Using this approach, the azurin gene was synthesized in five steps.\n\nBoth the synthetic genes for azurin and Populus nigra, var italica plastocyanin have been expressed in E. coli. These expressions have been achieved by using a synthetic Shine-Dalgano sequence and the signal sequence for azurin, which directs the transport of the expressed proteins to the periplasmic space of E. coli. The membrane translocation not only facilitates the purification of azurin and plastocyanin but also seems to be required for the proper folding of these proteins. In contrast to these successful expressions, earlier efforts to express plastocyanin in the cytoplasm of E. coli, either directly or as a fusion protein, have been unsuccessful at yielding folded plastocyanin.\n\nSite-saturation cassette mutagenesis was performed in azurin at Methionine 121, one of the four ligands to the copper. Variants that contain each of the other nineteen amino acids as well as the amber stop codon have been identified. Surprisingly, all the variants are stable, as judged by Western blot. Furthermore, all mutants that have been isolated at this position (Asn, Asp, Gly, His, Ile, Leu, Val) have the characteristic blue absorption near 600 nm. Despite such similarity with the wild-type azurin, these mutants seem to have a more flexible copper center. They can lose or incorporate copper at a faster rate than the wild-type protein.\n\nThese results, along with those from EPR experiments, suggest that while Methionine 121 is important in giving stability to the copper center and in tuning the redox potential, its contribution to azurin's spectroscopic properties is small when copper is coordinated to the site. Its spectroscopic contribution apparently becomes more significant when nickel or cobalt resides in the copper site, for the electronic spectra of these derivatives differ markedly from that of azurin with copper. It is likely that the copper centers of the mutants are flexible enough to accommodate the preferred geometries of nickel and cobalt, whereas it is more rigid in the wild-type. Finally, several mutants of azurin are proposed that are designed to probe systematically specific aspects of the biological electron transfer mechanism.", "doi": "10.7907/795e-hp65", "publication_date": "1991", "thesis_type": "phd", "thesis_year": "1991" } ]