The extracellular matrix (ECM) provides a supportive framework for neurons and glia in the healthy central nervous system (CNS). However, extensive ECM remodeling occurs after CNS insult or injury, and in neuroinflammation. Upregulation and deposition of growth-inhibitory ECM components results in failures in neuronal generation and resolution of harmful inflammation. Chondroitin sulfate proteoglycans (CSPGs), which consist of chondroitin sulfate (CS) polysaccharide chains attached to a core proteoglycan, have been identified as the predominant inhibitory component of this dysregulated ECM, impeding critical repair processes and contributing to prolonged inflammation. Furthermore, CSPGs exert their inhibitory and immunomodulatory effects through their polysaccharide side chains. These CS chains undergo further modification by the addition of sulfate groups, giving rise to various sulfation patterns, or motifs. Recognition of these motifs by protein ligands and cell-surface receptors enable CSPGs to modulate the activity of downstream signaling pathways and regulate neuronal growth and immune processes. Thus, understanding the structure-function relationships of CS glycosaminoglycans (GAGs) will enable development of strategies to promote neuroregeneration and repair after injury and in neurodegenerative diseases.
\r\n\r\nCurrent strategies to target CSPGs and CS GAGs broadly act on all sulfation patterns. This has limited efforts to decode the biological functions of individual sulfation motifs. Herein, we describe our efforts to comprehensively examine the shifts in the CS sulfation composition profile following spinal cord contusion injury. We develop a novel single-chain variable fragment (scFv) selective for the disulfated CS-E motif, and demonstrate its ability to rescue CSPG-mediated inhibition of neurite outgrowth on primary cortical neurons.
\r\n\r\nFurthermore, we provide the first evidence that the CS-E sulfation motif directly modulates\r\nthe activity of microglia, and that 4-O-sulfated CS exacerbates pro-inflammatory immune cell activation and conversion of T cells to a pathogenic phenotype in the experimental autoimmune encephalomyelitis (EAE) model of neuroinflammation. Together, our results contribute toward a greater understanding of the role of ECM components in disease processes and provide insights into the role of specific CS sulfation motifs in neuroinflammation and regeneration.
", "doi": "10.7907/gkge-0e55", "publication_date": "2026", "thesis_type": "phd", "thesis_year": "2026" }, { "id": "thesis:17291", "collection": "thesis", "collection_id": "17291", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05292025-004444146", "type": "thesis", "title": "Biomolecular Engineering of Gas Vesicles with Thiol Functionality", "author": [ { "family_name": "Schrunk", "given_name": "Erik Tao", "orcid": "0009-0003-2002-7411", "clpid": "Schrunk-Erik-Tao" } ], "thesis_advisor": [ { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" } ], "thesis_committee": [ { "family_name": "Tirrell", "given_name": "David A.", "orcid": "0000-0003-3175-4596", "clpid": "Tirrell-D-A" }, { "family_name": "Wang", "given_name": "Kaihang", "orcid": "0000-0001-7657-8755", "clpid": "Wang-Kaihang" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "orcid": "0000-0001-5661-1714", "clpid": "Hsieh-Wilson-L-C" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Therapies involving the administration of engineered cells, such as CAR-T cell therapy and the delivery of genetically modified gut microbes, have enjoyed clinical success and increasing interest in recent years. While these therapies continue to show great promise, the opacity of tissue precludes the use of light in the observation and potential manipulation of these engineered cells as they carry out their functions within the body. To access these cells non-invasively in deep tissue requires the use of imaging modalities that do not involve light, of which ultrasound (US) is especially appealing due to its relatively low cost, safety, and widespread availability. Engineered cells can exhibit US contrast by expressing gas vesicles (GVs), air-filled polymeric proteinaceous nanostructures; GVs have already been used as acoustic reporters for gut colonization, tumor cell activity, and more.
\r\n \r\nWhereas GVs are most notable for their acoustic properties, we set out to further expand the function of GVs by chemically modifying them at the genetic level. Our goals were twofold: we wished to equip the external, solution-facing side of GVs with a unique chemical handle; and we wished to hide a reactive group within the internal, air-facing side of GVs that could only be revealed when the GV structures are irreversibly collapsed. To accomplish both these goals, we chose to incorporate cysteine into the shell of GVs because cysteine\u2019s thiol side chain is chemically unique among all natural amino acids and because wild-type GVs do not contain cysteine in their shells. We set up a cysteine scanning mutant library of the GV shell protein, GvpA/GvpA1, and screened for cysteine-tolerant mutations in the gene. Through this process, we discovered cysteine substitutions that furnished thiol groups facing both the GV exterior and interior.
\r\n\r\nThe GV-exterior-facing cysteines were leveraged to develop a modified GvpA that contains the bioorthogonal six amino acid tetracysteine tag, or TC tag. The TC tag reacts with the membrane-permeable molecule FlAsH, which becomes fluorescent upon reaction. We used TC-tagged GvpA, or tcGvpA, to express GVs in HEK 293T cells, and used confocal microscopy of FlAsH to study those GVs. Notably, we only substituted a small percentage of GvpA to tcGvpA, leaving the rest of the GvpA as wild type; to our knowledge, this is the only report of a polymeric proteinaceous structure that employs this chimeric assembly approach being successfully expressed and labeled with FlAsH. The microscopy results from this study were used to generate three-dimensional renderings that provided insights into the size and positioning of GV clusters expressed within HEK 293T cells.
\r\n\r\nSecond, we identified several interior-facing cysteine mutants to the GV shell protein GvpA1, which we used to develop \u201cSonoCages\u201d: chemical entities whose reactivity is gated by US. We purified GVs with one mutation from our screen, V47C, and reacted them with monobromobimane (mBBr), a fluorogenic, thiol-reactive molecule. The mutant GVs only reacted with mBBr after treatment with US, which collapsed the GVs and exposed their hydrophobic interiors to the bulk solution. Thus, we had developed thiol-bearing SonoCages whose cysteines could only engage in reactions after US-mediated collapse of the GVs\u2014a process we call \u201csono-uncaging\u201d in analogy to photo-uncaging. We further demonstrated the utility of SonoCages by preparing a hydrogel containing SonoCages and mBBr and using US to create fluorescent patterns corresponding to regions of GV collapse.
\r\n \r\nThe work presented in this thesis not only demonstrates the functionalization of the GV interior and exterior, but also establishes a framework through which further modifications can be performed. Whereas we used cysteine as our reactive chemical of choice, other amino acids (including non-canonical amino acids) could be used to explore a much wider library of reactivities. The vast potential of GV chemical modification, along with the amazing results from the rest of the Shapiro Lab and in labs across the world, serves as a reminder that GVs and GV-based technologies are not just a bubble (pun intended)\u2014they are going to be around for a long time.
", "doi": "10.7907/49mr-3744", "publication_date": "2025", "thesis_type": "phd", "thesis_year": "2025" }, { "id": "thesis:17221", "collection": "thesis", "collection_id": "17221", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05122025-153409748", "primary_object_url": { "basename": "Flesch Thesis.pdf", "content": "final", "filesize": 48300552, "license": "other", "mime_type": "application/pdf", "url": "/17221/1/Flesch Thesis.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Asymmetric Transformations from Palladium Enolates and Progress Toward the Total Synthesis of Hypermoin A", "author": [ { "family_name": "Flesch", "given_name": "Kaylin Nicole", "orcid": "0000-0002-8582-2614", "clpid": "Flesch-Kaylin-Nicole" } ], "thesis_advisor": [ { "family_name": "Stoltz", "given_name": "Brian M.", "orcid": "0000-0001-9837-1528", "clpid": "Stoltz-B-M" } ], "thesis_committee": [ { "family_name": "Reisman", "given_name": "Sarah E.", "orcid": "0000-0001-8244-9300", "clpid": "Reisman-S-E" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "orcid": "0000-0001-5661-1714", "clpid": "Hsieh-Wilson-L-C" }, { "family_name": "Nelson", "given_name": "Hosea M.", "orcid": "0000-0002-4666-2793", "clpid": "Nelson-H-M" }, { "family_name": "Stoltz", "given_name": "Brian M.", "orcid": "0000-0001-9837-1528", "clpid": "Stoltz-B-M" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Research in the Stoltz group is centered around developing new methodologies for the asymmetric formation of stereocenters and the application of these technologies in complex natural product total synthesis. Herein we describe the development of new enantioselective transformations from Pd enolate intermediates and efforts toward the total synthesis of hypermoin A. Chapter 1 reports the development of an asymmetric intramolecular decarboxylative [4+2] cycloaddition from a catalytically generated chiral Pd enolate, forging four contiguous stereocenters in a single transformation. Mechanistic studies including quantum mechanics calculations, Eyring analysis, and KIE studies offer insight into the reaction mechanism. Appendix 2 discloses efforts toward the development of an asymmetric intermolecular decarboxylative double Michael addition. Chapter 2 describes an enantioselective cyclization of Pd enolates and isocyanates to form spirocyclic \u03b3-lactams. This reaction proceeds under mild reaction conditions and utilizes a novel Meldrum\u2019s acid derivative to achieve catalyst turnover, delivering enantioenriched products in up to 97% yield and 96% ee. Chapter 3 outlines the ongoing progress toward the total synthesis of hypermoin A. A [4+2] cycloaddition and ring expansion strategy has been developed in a model system to form the key [3.2.2] bicycle and current efforts are dedicated to the application of this sequence in a more complex setting.", "doi": "10.7907/jfcn-4r24", "publication_date": "2025", "thesis_type": "phd", "thesis_year": "2025" }, { "id": "thesis:17340", "collection": "thesis", "collection_id": "17340", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06012025-173559572", "type": "thesis", "title": "Design and Application of Complexity Generating Strategies and Transformations in Natural Product Synthesis", "author": [ { "family_name": "Stegner", "given_name": "Andrea Anna Therese", "orcid": "0000-0001-9408-3967", "clpid": "Stegner-Andrea-Anna-Therese" } ], "thesis_advisor": [ { "family_name": "Reisman", "given_name": "Sarah E.", "orcid": "https://orcid.org/0000-0001-8244-9300", "clpid": "Reisman-S-E" } ], "thesis_committee": [ { "family_name": "Robb", "given_name": "Maxwell J.", "orcid": "0000-0002-0528-9857", "clpid": "Robb-M-J" }, { "family_name": "Stoltz", "given_name": "Brian M.", "orcid": "0000-0001-9837-1528", "clpid": "Stoltz-B-M" }, { "family_name": "Wilson", "given_name": "Linda C.", "orcid": "0000-0001-5661-1714", "clpid": "Hsieh-Wilson-L-C" }, { "family_name": "Reisman", "given_name": "Sarah E.", "orcid": "0000-0001-8244-9300", "clpid": "Reisman-S-E" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Rapid and efficient chemical synthesis of complex molecules is critical for enabling studies of their biological function and therapeutic potential. Complexity generation can be achieved by: (1) the development of selective transformations that rapidly construct complex scaffolds from simple precursors, and (2) the application of strategic retrosynthetic disconnections that maximize the efficiency of a synthesis i.e., via convergent fragment coupling strategies. We disclose efforts to address these aims by combining reaction design with the identification of strategic disconnections.
\r\n\r\nToward the first aim, a diastereoselective dearomative pyridine cyclization that forges the tetracyclic core of the matrine-type lupin alkaloids in a single step from commodity feedstocks was developed. This reaction, paired with a C15-selective oxidation cascade and late-stage isomerization, enabled the first total synthesis of (\u2013)-sophoridine and the shortest syntheses to date of (+)-matrine, (+)-isomatrine, (+)-allomatrine, and (+)- isosophoridine.
\r\n\r\nTo address the second aim, a convergent strategy for the synthesis of 6,7-seco-ent- kauranoids via strategic, transition metal-catalyzed C-C bond formation was developed. Both a Ni-catalyzed sp\u00b2\u2013sp\u00b3 and a dual Ni/Pd-catalyzed sp\u00b2\u2013sp\u00b2 coupling were developed and enabled efficient union of complex fragments, the latter yielding 1,3 dienes that underwent divergent annulation reactions to form various complex ring systems. These transformations provided the complete skeleton of isorosthin A and advanced intermediates en route to isodocarpin and secoexertifolin A.
", "doi": "10.7907/w4xa-hy67", "publication_date": "2025", "thesis_type": "phd", "thesis_year": "2025" }, { "id": "thesis:17257", "collection": "thesis", "collection_id": "17257", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05212025-221836479", "type": "thesis", "title": "Advancing Stimulated Raman Scattering Microscopy through Deep Learning and Gel-Based Tissue Engineering", "author": [ { "family_name": "Lin", "given_name": "Li-En", "orcid": "0000-0003-3086-6991", "clpid": "Lin-Li-En" } ], "thesis_advisor": [ { "family_name": "Wei", "given_name": "Lu", "orcid": "0000-0001-9170-2283", "clpid": "Wei-Lu" } ], "thesis_committee": [ { "family_name": "Okumura", "given_name": "Mitchio", "orcid": "0000-0001-6874-1137", "clpid": "Okumura-M" }, { "family_name": "Van Valen", "given_name": "David A.", "orcid": "0000-0001-7534-7621", "clpid": "Van-Valen-D" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "orcid": "0000-0001-5661-1714", "clpid": "Hsieh-Wilson-L-C" }, { "family_name": "Wei", "given_name": "Lu", "orcid": "0000-0001-9170-2283", "clpid": "Wei-Lu" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Stimulated Raman scattering (SRS) microscopy is a highly effective label-free imaging method for investigating the molecular composition of biological systems. Its broader use has been held back by spatial resolution, imaging speed, and large-scale tissue imaging compatibility. Breaking these limitations requires an integrated approach beyond the development of optical hardware. This thesis introduces a compilation of techniques that leverage gel-based tissue engineering and deep learning to enhance the capabilities of SRS microscopy.
\r\n \r\nThe first chapter, Gel-Enabled Super-Resolution Label-Free Volumetric Vibrational Imaging, introduces VISTA, a sample-expansion vibrational imaging technique that achieves label-free super-resolution imaging of protein-dense biological structures with resolution as fine as 78 nm. By enabling isotropic expansion and protein retention, VISTA allows for high-throughput, unbiased volumetric imaging without labeling, with further enhancement using deep learning-based component prediction.
\r\n\r\nThe second chapter, High-Resolution Imaging of In Vivo Protein Aggregates, applies VISTA to image amyloid-beta and polyQ aggregates in biological samples with high specificity. Combined with segmentation using convolutional neural networks, this technique is capable of mapping aggregate structure and microenvironments, enabling new insights into neurodegenerative disease pathology.
\r\n\r\nThe third chapter, High-Throughput Volumetric Mapping Facilitated by Active Tissue SHRINK, introduces SHRINK, a hydrogel-based sample shrinkage method that isotropically shrinks tissue while maintaining structural integrity. Active shrinkage enhances imaging throughput and signal sensitivity and enables rapid, large-scale, three-dimensional whole-organ mapping with SRS microscopy.
\r\n\r\nThe fourth chapter, Deep Learning-Augmented Metabolic Profiling in Live Neuronal Cultures, presents a tandem deep learning platform for live-cell metabolic imaging. By integrating a recurrent convolutional neural network and U-Net segmentation model with deuterium-labeled metabolic tracing, this platform enables non-invasive, high-speed profiling of lipid, protein, glucose, and water metabolism in neuronal subtypes under physiological and pathological conditions.
\r\n\r\nThese developments represent multidimensional strategies that expand the application of SRS microscopy to high-resolution, high-throughput, and dynamic imaging in a variety of biological systems. The integration of deep learning and gel-based tissue engineering techniques opens new avenues for SRS microscopy to explore complex biological questions.
", "doi": "10.7907/9azz-r140", "publication_date": "2025", "thesis_type": "phd", "thesis_year": "2025" }, { "id": "thesis:17441", "collection": "thesis", "collection_id": "17441", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06102025-162952847", "type": "thesis", "title": "Stereoselective Synthesis of \u03b1- and \u03b2-Functionalized Carbonyl Derivatives: Allylic Alkylation of Dialkyl Malonates, Total Synthesis of Hunterine A, and Progress Toward the Synthesis of Kuroshine A", "author": [ { "family_name": "Hicks", "given_name": "Elliot Frederick", "orcid": "0009-0004-8965-0460", "clpid": "Hicks-Elliot-Frederick" } ], "thesis_advisor": [ { "family_name": "Stoltz", "given_name": "Brian M.", "orcid": "0000-0001-9837-1528", "clpid": "Stoltz-B-M" } ], "thesis_committee": [ { "family_name": "Reisman", "given_name": "Sarah E.", "orcid": "0000-0001-8244-9300", "clpid": "Reisman-S-E" }, { "family_name": "Robb", "given_name": "Maxwell J.", "orcid": "0000-0002-0528-9857", "clpid": "Robb-M-J" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "orcid": "0000-0001-5661-1714", "clpid": "Hsieh-Wilson-L-C" }, { "family_name": "Stoltz", "given_name": "Brian M.", "orcid": "0000-0001-9837-1528", "clpid": "Stoltz-B-M" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Research in the Stoltz group generally focuses on the synthesis of complex molecules enabled by the discovery and application of new synthetic methodologies. The majority of the research described in this thesis encompasses variations on this unifying central theme.
\r\n \r\nChapter 1 focuses on the development of new asymmetric methodology for the synthesis of stereogenic all-carbon quaternary centers through the application of iridium catalysis. A key advance was the use of malonate nucleophiles and bis-alkyl substituted electrophiles, two inputs commonly employed in allylic alkylation chemistry that had yet to be utilized for the synthesis of quaternary carbon centers.
\r\n \r\nChapter 2 describes the development and execution of a synthetic strategy for the asymmetric construction of a rearranged monoterpene indole alkaloid, hunterine A. Key to the assembly of this molecule were an enantioselective desymmetrization, aza-Cope/Mannich rearrangement, azide-alkene dipolar cycloaddition, and regioselective aziridine opening.
\r\n \r\nChapter 3 details an investigation born out of an observation of a regioselective aziridine opening during the course of the synthesis of hunterine A. This research focuses on the synthesis of sterically unbiased fused bicyclic N-aryl aziridines and their reactivity in a variety of ring opening reactions.
\r\n\r\nChapter 4 and Appendix 7 describe ongoing efforts toward the synthesis of Kuroshine A and other highly oxidized zoanthamine alkaloids. The synthetic strategies described focus on evaluating formation of the ABC ring system of the molecule via a masked ortho-benzoquinone cycloaddition.
", "doi": "10.7907/fs03-xy64", "publication_date": "2025", "thesis_type": "phd", "thesis_year": "2025" }, { "id": "thesis:16280", "collection": "thesis", "collection_id": "16280", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:01162024-161326655", "primary_object_url": { "basename": "TDilanyan_Thesis.pdf", "content": "final", "filesize": 7266593, "license": "other", "mime_type": "application/pdf", "url": "/16280/4/TDilanyan_Thesis.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Open-Source Custom Beads for Single-Cell Transcriptomics", "author": [ { "family_name": "Dilanyan", "given_name": "Taleen Gaied", "orcid": "0000-0002-3131-3259", "clpid": "Dilanyan-Taleen-Gaied" } ], "thesis_advisor": [ { "family_name": "Pachter", "given_name": "Lior S.", "orcid": "0000-0002-9164-6231", "clpid": "Pachter-L" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "orcid": "0000-0001-5661-1714", "clpid": "Hsieh-Wilson-L-C" }, { "family_name": "Pachter", "given_name": "Lior S.", "orcid": "0000-0002-9164-6231", "clpid": "Pachter-L" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Open-source single-cell genomics technologies have helped democratize single-cell genomics and expedite method development. Methods such as inDrops and Drop-seq for single-cell RNA-seq preceded popular technologies such as the 10x Genomics\u2019 Chromium platform, however despite initial enthusiasm for open-source methods, their popularity has waned. A major reason has been the lack of availability of low-cost, customizable beads, which are essential for microfluidics based single-cell RNA-seq. We address this challenge by introducing a new method for producing barcoded hydrogel beads for single-cell RNA-seq called HiPER (High-throughput PER-barcoded hydrogel beads) that allows for increasing the diversity of barcode sequences, reducing manufacturing cost, and that can be readily adapted to custom applications. HiPER barcodes are decoupled from the capture sequences and can therefore be configured to capture RNA, DNA, or tailored for specific-gene enrichment.", "doi": "10.7907/v52p-gf80", "publication_date": "2024", "thesis_type": "phd", "thesis_year": "2024" }, { "id": "thesis:16342", "collection": "thesis", "collection_id": "16342", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:03292024-213352165", "primary_object_url": { "basename": "Wang_thesis_final.pdf", "content": "final", "filesize": 12860605, "license": "other", "mime_type": "application/pdf", "url": "/16342/3/Wang_thesis_final.pdf", "version": "v7.0.0" }, "type": "thesis", "title": "Chemo-Selective Proteomics for Discovery of Polymicrobial Interactions", "author": [ { "family_name": "Wang", "given_name": "Grace Zimu", "orcid": "0000-0002-0938-304X", "clpid": "Wang-Grace-Zimu" } ], "thesis_advisor": [ { "family_name": "Tirrell", "given_name": "David A.", "orcid": "0000-0003-3175-4596", "clpid": "Tirrell-D-A" } ], "thesis_committee": [ { "family_name": "Shapiro", "given_name": "Mikhail G.", "orcid": "0000-0002-0291-4215", "clpid": "Shapiro-M-G" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "orcid": "0000-0001-5661-1714", "clpid": "Hsieh-Wilson-L-C" }, { "family_name": "Tirrell", "given_name": "David A.", "orcid": "0000-0003-3175-4596", "clpid": "Tirrell-D-A" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The future of microbiome research lies in our ability to engineer polymicrobial interactions toward improved host health outcomes, which requires a fundamental molecular understanding of how microbial species sense and respond to ecological competition. Chronic respiratory infection by polymicrobial communities is the leading cause of mortality and morbidity in people living with cystic fibrosis (CF). My thesis work adapts chemo-selective proteomics to dissect molecular mechanisms that drive interspecies dynamics between two notorious opportunistic pathogens dominating chronic CF infection, Pseudomonas aeruginosa and Staphylococcus aureus.
\r\n\r\nIn Chapter 1, I introduce bioorthogonal noncanonical amino acid tagging (BONCAT)-based comparative proteomics, focusing on time-resolved, cell-specific, and cellular state-selective proteomic applications in the dissection of complex microbial systems. In Chapter 2, I discuss a new usage of time-resolved BONCAT\r\nto monitor immediate competition-sensing responses in interbacterial warfare. While coinfection by the Gram-negative Pseudomonas aeruginosa and the Gram-positive Staphylococcus aureus is associated with poor patient outcomes, the interspecies interactions responsible for such decline remain unknown. We\r\ndiscovered that P. aeruginosa senses S. aureus secreted cytotoxic peptides from a distance and preempts potential competition through activation of type six secretion system (T6SS). P. aeruginosa enhances such competition-sensing-induced antagonism through concomitant attraction toward S. aureus peptides, effectively reducing cellular distances between neighboring species and providing a competitive advantage. In Chapter 3, I discuss a new usage of cell-selective BONCAT to target protein synthesis analysis of the lowabundance organism, S. aureus, in a coculture environment predominated by P. aeruginosa. P. aeruginosa robustly outcompetes S. aureus, and conventional shotgun proteomics, which is biased toward highly abundant proteins on principle, could only identify and quantify less than 5% of total protein synthesis by S. aureus in coculture. We demonstrate that chemical enrichment affords a more than 12-fold increase in total protein abundances synthesized by S. aureus. About 50% of protein \u201chits\u201d with statistically significant changes in expression were not detected in pre-enrichment lysates, highlighting BONCAT as a powerful strategy that facilitates high-resolution proteomic analysis of low-abundance organisms in polymicrobial communities.
", "doi": "10.7907/1vw0-gt98", "publication_date": "2024", "thesis_type": "phd", "thesis_year": "2024" }, { "id": "thesis:16236", "collection": "thesis", "collection_id": "16236", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:11032023-041429778", "primary_object_url": { "basename": "Jiajun Du Thesis 110323.pdf", "content": "final", "filesize": 33583941, "license": "other", "mime_type": "application/pdf", "url": "/16236/6/Jiajun Du Thesis 110323.pdf", "version": "v8.0.0" }, "type": "thesis", "title": "Vibrational Imaging for Chemical Biology: from Label-Free to Molecular probes", "author": [ { "family_name": "Du", "given_name": "Jiajun", "orcid": "0000-0003-2693-834X", "clpid": "Du-Jiajun" } ], "thesis_advisor": [ { "family_name": "Wei", "given_name": "Lu", "orcid": "0000-0001-9170-2283", "clpid": "Wei-Lu" } ], "thesis_committee": [ { "family_name": "Tirrell", "given_name": "David A.", "orcid": "0000-0003-3175-4596", "clpid": "Tirrell-D-A" }, { "family_name": "Stoltz", "given_name": "Brian M.", "orcid": "0000-0001-9837-1528", "clpid": "Stoltz-B-M" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "orcid": "0000-0001-5661-1714", "clpid": "Hsieh-Wilson-L-C" }, { "family_name": "Wei", "given_name": "Lu", "orcid": "0000-0001-9170-2283", "clpid": "Wei-Lu" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Since the invention of stimulated Raman scattering (SRS) microscopy in 2008, vibrational imaging is increasingly recognized as a powerful tool for biological investigation. As the most suitable far field vibrational imaging modality for live biological studies, SRS microscopy is taking the lead role within its vibrational counterparts with desired sensitivity and image quality. The totally different mechanism of generating vibration signals from fluorescence signals determines the special features of vibrational imaging. Bond vibration originating signals provide inherent optical contrast for every molecule and the quantitative manner allows straightforward quantification. Since the inception, SRS microscopy has achieved large success in label-free imaging. Label-free imaging avoids tedious labeling step and has the least perturbation to the biological samples but with limited sensitivity and specificity. The introducing of labeling starting about 10 years ago opens up a new avenue for SRS microscopy to tackle the fundamental limitations of label-free approaches. Whether to use label-free or molecular probes for SRS microscopy depends on the specific studies. This thesis aims to utilize SRS microscopy (both label-free and minimally labeling) for metabolic study and develop new molecular probes for SRS microscopy.
\r\n\r\nWe start from comparing different vibrational imaging modality and fluorescence imaging and conclude that SRS is the best vibrational imaging technique for biological samples. Then we discuss the features of label-free, bioorthogonal labeling and super-multiplexed SRS imaging. The minimally perturbative triple bond tagging and isotope labeling makes SRS especially suitable for tracking metabolites and accessing metabolic pathways. Furthermore, we also summarize the design principles for functional Raman imaging probe development based on their spectroscopic signatures. (Chapter 1).
\r\n\r\nNon-invasively probing metabolites within single live cells is highly desired but challenging. We explored Raman spectro-microscopy towards spatially-resolved single cell metabolomics, with the specific goal of identifying druggable metabolic susceptibilities from a series of patient-derived melanoma cell lines. The chemical composition analysis of single cell and single organelle lipid droplets identified the fatty acid synthesis pathway and lipid mono-unsaturation as druggable susceptibility. More importantly we revealed that inhibiting lipid mono-unsaturation leads to cellular apoptosis accompanied by the formation of phase-separated intracellular membrane domains. (Chapter 2).
\r\n\r\nNext, we established a first-in-class design of multi-color photoactivatable Raman probes for subcellular imaging and tracking. The fast photochemically generated alkynes from cyclopropenones enable background-free Raman imaging with desired photocontrollable features. After necessary molecule engineering to improve the biocompatibility and sensitivity, we generated organelle-specific probes for targeting mitochondria, lipid droplets, endoplasmic reticulum, and lysosomes. Multiplexed photoactivated imaging and tracking at both subcellular and single-cell levels was also demonstrated to monitor the dynamic migration and interactions of the cellular contents. (Chapter 3).
\r\n\r\nFurther improvement of the Raman signal with molecular probes is a central topic for Raman imaging. Recently developed electronic preresonance (epr) probes boost Raman signals and pushed SRS sensitivity close to that offered by confocal fluorescence microscopy. To guide the development of even stronger Raman probes and fill the final gap between epr-SRS probes and single molecule imaging, the structure-function relationship of epr-SRS probes is indispensable. We therefore used ab initio approach employing the displaced harmonic oscillator (DHO) model for calculating the epr-SRS signals, which proves to provide a consistent agreement between simulated and experimental SRS intensities of various triple-bond bearing epr-SRS probes. The theory also allows us to illustrate how the observed intensity differences between molecular scaffolds stem from the coupling strength between the electronic excitation and the targeted vibrational mode. Utilizing the discovered structure-function relationship of epr-SRS probes, we engineered MARS palette for higher sensitivity. With chemical modification to improve Raman mode displacement or enhance transition dipole moment or adjust detuning, we enhance the signal of alkynyl pyronins and nitrile pyronins, setting the current sensitivity records for small molecule far-field Raman probes. (Chapter 4 and 5).
", "doi": "10.7907/dm1y-r078", "publication_date": "2024", "thesis_type": "phd", "thesis_year": "2024" }, { "id": "thesis:14987", "collection": "thesis", "collection_id": "14987", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:07252022-075028264", "primary_object_url": { "basename": "2023_Thesis_Hao-Hsuan_Hsieh_Final.pdf", "content": "final", "filesize": 29274358, "license": "other", "mime_type": "application/pdf", "url": "/14987/1/2023_Thesis_Hao-Hsuan_Hsieh_Final.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Coordination Between Mammalian Nascent Protein Targeting and Cotranslational Chaperones", "author": [ { "family_name": "Hsieh", "given_name": "Hao-Hsuan", "orcid": "0000-0001-9629-5832", "clpid": "Hsieh-Hao-Hsuan" } ], "thesis_advisor": [ { "family_name": "Shan", "given_name": "Shu-ou", "orcid": "0000-0002-6526-1733", "clpid": "Shan-Shu-ou" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Shan", "given_name": "Shu-ou", "orcid": "0000-0002-6526-1733", "clpid": "Shan-Shu-ou" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "orcid": "0000-0001-5661-1714", "clpid": "Hsieh-Wilson-L-C" }, { "family_name": "Voorhees", "given_name": "Rebecca M.", "orcid": "0000-0003-1640-2293", "clpid": "Voorhees-R-M" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Protein biogenesis starts with ribosome synthesizing nascent polypeptide chain. Ribosome is a major hub for multiple pathways including membrane targeting, chaperones, chemical modification, and quality control. All these pathways need to coordinate with each other spatially on the ribosomal surface and temporally within the translation elongation window. Accumulating data start to point to more intricate interaction and coordination between different pathways beyond the simple competition traditionally presumed.
\r\n\r\nIn Chapter 1, I demonstrate the coordination between a cotranslational chaperone, NAC, and the ER targeting machinery, SRP. NAC and SRP can bind to the same ribosome simultaneously despite overlapping binding sites, allowing NAC to change conformation of SRP specifically to the NC sequences. This allostery enhances the specificity of SRP-SR association, explaining the long-observed effect of NAC modulating ER targeting specificity.
\r\n\r\nIn Chapter 2, I dig deeper into the mechanism of NAC regulating SRP. Based on cryo-EM structures, NAC domain sits on top of the ribosomal tunnel exit, potentially sensing the identity of NC, and is anchored by positively charged NAC\u03b2 N-terminal tail. NAC-UBA domain is the key to recruiting SRP and coordinating the substrate handover to SRP.
\r\n\r\nIn Chapter 3, I focus on the cotranslational HSP40/HSP70 system of RAC. Ribosome binding of RAC stimulates its cochaperone activity to activate HSP70 ATP hydrolysis. Ribosome sensing by RAC is related to the NBD of HSPA14. RAC-stimulated HSP70 engagement to NC keeps it in a folding-competent unfolded state before HSP70 dissociation.
\r\n\r\nTaken together, this study advances the experimental and theoretical tools to studying cotranslational pathways associated with the mammalian ribosome and demonstrates the interesting question of coordination between cotranslational pathways.
", "doi": "10.7907/x61p-vf31", "publication_date": "2023", "thesis_type": "phd", "thesis_year": "2023" }, { "id": "thesis:16058", "collection": "thesis", "collection_id": "16058", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06022023-190747966", "type": "thesis", "title": "Enantioselective Syntheses of Tetrahydroisoquinolines (THIQs) via Iridium-Catalyzed Asymmetric Hydrogenation and Progress Toward the Total Synthesis of (+)-Cyanocycline A", "author": [ { "family_name": "Kim", "given_name": "Alexia Nahyun", "orcid": "0000-0002-4060-8892", "clpid": "Kim-Alexia-Nahyun" } ], "thesis_advisor": [ { "family_name": "Stoltz", "given_name": "Brian M.", "orcid": "0000-0001-9837-1528", "clpid": "Stoltz-B-M" } ], "thesis_committee": [ { "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" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "orcid": "0000-0001-5661-1714", "clpid": "Hsieh-Wilson-L-C" }, { "family_name": "Stoltz", "given_name": "Brian M.", "orcid": "0000-0001-9837-1528", "clpid": "Stoltz-B-M" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Described herein are two reviews and three projects related to the asymmetric syntheses of tetrahydroisoquinoline (THIQs) alkaloids, and the progress toward the total synthesis of (+)-cyanocycline A. In Chapter 1, a review of the development of asymmetric methodologies for the preparation of enantioenriched N-heteroarenes is detailed. In Chapter 2, the development of an iridium-catalyzed enantio- and diastereoselective hydrogenation of 1,3-disubstituted isoquinolines to achieve cis-THIQs is reported. Chapter 3 describes the iridium-catalyzed asymmetric hydrogenation of 1,3-disubstituted isoquinolines that can afford trans-THIQs in a single transformation. Preliminary mechanistic insights to the iridium-catalyzed asymmetric hydrogenation method using deuterium experiments are detailed.
\r\n\r\nChapter 4 details a comprehensive review of the advances in the total syntheses of complex THIQ alkaloids from 2000 \u2013 2020, ranging from simple benzyl THIQ natural products to complex THIQ alkaloids such as Ecteinascidin-743. In Chapter 5, efforts toward the total synthesis of (+)-cyanocycline A are described, harnessing a non- biomimetic synthetic route through a convergent cross-coupling of two heterocyclic fragments followed by a global hydrogenation event.
", "doi": "10.7907/4xty-8711", "publication_date": "2023", "thesis_type": "phd", "thesis_year": "2023" }, { "id": "thesis:14966", "collection": "thesis", "collection_id": "14966", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:07032022-182356199", "primary_object_url": { "basename": "ChienIYang_Thesis_final_2023.pdf", "content": "final", "filesize": 13334113, "license": "other", "mime_type": "application/pdf", "url": "/14966/6/ChienIYang_Thesis_final_2023.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "Mechanism and Function of Nascent Protein Modification in Bacteria", "author": [ { "family_name": "Yang", "given_name": "Chien-I", "orcid": "0000-0001-8606-5013", "clpid": "Yang-Chien-I" } ], "thesis_advisor": [ { "family_name": "Shan", "given_name": "Shu-ou", "orcid": "0000-0002-6526-1733", "clpid": "Shan-Shu-ou" } ], "thesis_committee": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "orcid": "0000-0003-1464-2461", "clpid": "Dougherty-D-A" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "orcid": "0000-0001-5661-1714", "clpid": "Hsieh-Wilson-L-C" }, { "family_name": "Varshavsky", "given_name": "Alexander J.", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A-J" }, { "family_name": "Shan", "given_name": "Shu-ou", "orcid": "0000-0002-6526-1733", "clpid": "Shan-Shu-ou" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Newly synthesized proteins undergo multiple modifications to ensure proper biogenesis and acquire their functions. N-terminal methionine excision (NME), mediated by the sequential actions of peptide deformylase (PDF) and methionine aminopeptidase (MAP), is an essential and the most prevalent N-terminal protein modification in the bacterial proteome. Despite the extensive studies on enzymatic catalysis, how NME impacts various cellular functions and how the enzymes achieve timing and selectivity under complex cellular conditions have been long-standing puzzles.
\r\n\r\nIn this work, we use a combination of biochemical analyses, computational modeling, and in vivo measurements to investigate the molecular mechanisms and physiological functions of cotranslational NME reactions. We show that the interactions between the ribosome, the nascent chain, the NME enzymes, and other ribosome-associated protein biogenesis factors dramatically remodel the kinetics and specificity of NME reactions under physiological conditions. In addition, we apply time-resolved, system-wide analyses on the translatome and steady-state proteome to study how the inhibition of PDF influences diverse cellular pathways in bacteria. The results unveil the impact of NME on the biogenesis of nascent proteins and highlight the role of the membrane in coupling the biochemical activities of NME enzymes to cellular physiology.
", "doi": "10.7907/qhz7-a383", "publication_date": "2023", "thesis_type": "phd", "thesis_year": "2023" }, { "id": "thesis:14566", "collection": "thesis", "collection_id": "14566", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:04292022-203100439", "type": "thesis", "title": "Palladium-Catalyzed Cascade Cyclizations in Natural Product Synthesis: Synthetic Studies of Noraugustamine and Falcatin A", "author": [ { "family_name": "Holman", "given_name": "Karli Rose", "orcid": "0000-0001-6424-9479", "clpid": "Holman-Karli-Rose" } ], "thesis_advisor": [ { "family_name": "Reisman", "given_name": "Sarah E.", "orcid": "0000-0001-8244-9300", "clpid": "Reisman-S-E" } ], "thesis_committee": [ { "family_name": "Fu", "given_name": "Gregory C.", "orcid": "0000-0002-0927-680X", "clpid": "Fu-G-C" }, { "family_name": "Reisman", "given_name": "Sarah E.", "orcid": "0000-0001-8244-9300", "clpid": "Reisman-S-E" }, { "family_name": "Stoltz", "given_name": "Brian M.", "orcid": "0000-0001-9837-1528", "clpid": "Stoltz-B-M" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "orcid": "0000-0001-5661-1714", "clpid": "Hsieh-Wilson-L-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Palladium-catalyzed cascade cyclizations present a powerful strategy for the rapid assembly of polycyclic skeletal frameworks, enabling the efficient synthesis of bioactive and structurally complex natural products. Herein, we review the field of palladium-catalyzed cascade cyclizations in natural product synthesis and describe our application of these transformations toward the total syntheses of noraugustamine and falcatin A.
\r\n\r\nOur approach to the Amaryllidaceae alkaloid noraugustamine was driven by the simultaneous disconnection of a C\u2013C and a C\u2013N bond, with the aim of forming both bonds and two of the target\u2019s six rings in a single step. A radical cascade cyclization delivered noraugustamine but displayed poor regioselectivity for 6-exo-trig versus 7-endo-trig cyclization. Improved regioselectivity was achieved using a palladium-catalyzed Heck cyclization, leading to the development of a novel oxidative Heck/aza-Wacker cascade forming both of the desired bonds with good yield and selectivity. This transformation and the general lessons taken from this work should find broad utility in the design of cascade cyclizations toward alkaloids of similar complexity.
\r\n\r\nWe also investigated a palladium-catalyzed carboetherification cascade toward the synthesis of the central five- and seven-membered rings of the myrsinane diterpene falcatin A. In this case, competitive C\u2013O coupling, olefin insertion, and cyclopropanation hindered our efforts to develop the proposed transformation in a simplified model system. A stereoselective bromoetherification and a nickel-catalyzed Nozaki\u2013Hiyama\u2013Kishi reaction were ultimately successful, forming the targeted rings. Efforts to synthesize a fully elaborated cyclization substrate, translate the key steps from the model system, and complete the synthesis of falcatin A are ongoing.
", "doi": "10.7907/9r6z-tw08", "publication_date": "2022", "thesis_type": "phd", "thesis_year": "2022" }, { "id": "thesis:14355", "collection": "thesis", "collection_id": "14355", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:09072021-162545226", "primary_object_url": { "basename": "CRL_Thesis_Short.pdf", "content": "final", "filesize": 13889808, "license": "other", "mime_type": "application/pdf", "url": "/14355/1/CRL_Thesis_Short.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "The Modular Synthesis and Functionalization of Cyclic Compounds Using Modern Methods", "author": [ { "family_name": "Lacker", "given_name": "Caitlin Rebecca", "orcid": "0000-0003-2531-2636", "clpid": "Lacker-Caitlin-Rebecca" } ], "thesis_advisor": [ { "family_name": "Reisman", "given_name": "Sarah E.", "orcid": "0000-0001-8244-9300", "clpid": "Reisman-S-E" } ], "thesis_committee": [ { "family_name": "Fu", "given_name": "Gregory C.", "orcid": "0000-0002-0927-680X", "clpid": "Fu-G-C" }, { "family_name": "Reisman", "given_name": "Sarah E.", "orcid": "0000-0001-8244-9300", "clpid": "Reisman-S-E" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "orcid": "0000-0001-5661-1714", "clpid": "Hsieh-Wilson-L-C" }, { "family_name": "Stoltz", "given_name": "Brian M.", "orcid": "0000-0001-9837-1528", "clpid": "Stoltz-B-M" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Accessing libraries of similar compounds quickly is important in the pharmaceutical industry, as it allows for the expedient investigation of a wide variety of parameters. An efficient strategy to access compounds of interest is to start from a single intermediate containing an interesting or pharmaceutically active structure and decorating it with varying functionality to generate a library of related compounds. Cross-coupling is a powerful tool for this type of divergent, modular approach.
\r\n\r\nHerein, we discuss several strategies geared towards the synthesis of small libraries of compounds of interest. First, a modular approach towards a library of enantioenriched trans cyclobutanes is discussed. This strategy allows for the synthesis of diverse substrates from a single enantioenriched intermediate, and this approach was applied to the synthesis of the small molecule (+)-rumphellaone A. Finally, the development of an enantioselective nickel-catalyzed photoredox cross-coupling to form N-(hetero)benzylic azoles in collaboration with researchers at Merck is discussed.
", "doi": "10.7907/61ky-5w83", "publication_date": "2022", "thesis_type": "phd", "thesis_year": "2022" }, { "id": "thesis:13672", "collection": "thesis", "collection_id": "13672", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:04102020-135244808", "primary_object_url": { "basename": "Gu_Nina_thesis.pdf", "content": "final", "filesize": 25088141, "license": "other", "mime_type": "application/pdf", "url": "/13672/20/Gu_Nina_thesis.pdf", "version": "v12.0.0" }, "type": "thesis", "title": "Synthesis, Characterization, and Reactivity of Thiolate-Supported Metalloradicals", "author": [ { "family_name": "Gu", "given_name": "Nina Xiao", "orcid": "0000-0002-4637-8418", "clpid": "Gu-Nina-Xiao" } ], "thesis_advisor": [ { "family_name": "Peters", "given_name": "Jonas C.", "clpid": "Peters-J-C" } ], "thesis_committee": [ { "family_name": "Agapie", "given_name": "Theodor", "clpid": "Agapie-T" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" }, { "family_name": "Fu", "given_name": "Gregory C.", "clpid": "Fu-G-C" }, { "family_name": "Peters", "given_name": "Jonas C.", "clpid": "Peters-J-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Reactive metalloradical intermediates have been implicated in both biological and synthetic catalyst systems for small molecule activation processes, including proton reduction and ammonia oxidation. Towards a greater mechanistic understanding of such transformations on well-defined model complexes, this thesis explores relevant H\u2013H and N\u2013N bond-forming reactions mediated by trivalent Fe and Ni species, as well as catalytic N\u2013N bond cleavage mediated by an open-shell VFe bimetallic complex. First, a pair of thiolate-supported, S = \u00bd iron and nickel hydrides are synthesized and spectroscopically characterized at low temperatures (Chapters 2, 3). Paramagnetic iron and nickel hydrides have been proposed as catalytic intermediates of [NiFe] hydrogenase and nitrogenase, but characterization of such molecular species are limited. For both the FeIII and NiIII hydride complexes described herein, spin delocalization onto the thiolate ligand is proposed to stabilize the formal 3+ metal oxidation state. Furthermore, both the FeIII\u2013H and NiIII\u2013H species are demonstrated to undergo the bimolecular reductive elimination of dihydrogen upon warming, albeit with distinct activation parameters consistent with different proposed pathways for H\u2013H bond formation. Chapter 4 expands upon the H\u2013H bond forming chemistry demonstrated on the Ni system to demonstrate related N\u2013N bond formation from an analogous NiIII\u2013NH2 species, resulting in the formation of a NiII2(N2H4) complex. Given the diverse mechanistic possibilities for the overall 6e-/6H+ transformation to oxidize ammonia to dinitrogen, identification of the active M(NHx) intermediate and pathway for N\u2013N bond formation is a central mechanistic question. While the homocoupling of M\u2013NH2 species to form hydrazine has been hypothesized as the key N\u2013N bond forming step in ammonia oxidation systems, stoichiometric examples of this transformation from M\u2013NH2 complexes are rare. Lastly, Chapter 5 details the synthesis of a heterobimetallic VFe complex featuring a bridging thiolate, inspired by the structure of the VFe nitrogenase cofactor. This VFe species is demonstrated to be an active catalyst for the disproportionation of hydrazine to dinitrogen and ammonia. Notably, the heterobimetallic complex is appreciably more active than monometallic analogues of the individual V and Fe sites, suggesting that bimetallic cooperativity may facilitate the observed catalysis.
", "doi": "10.7907/8zwz-gh20", "publication_date": "2020", "thesis_type": "phd", "thesis_year": "2020" }, { "id": "thesis:13697", "collection": "thesis", "collection_id": "13697", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05052020-172131873", "primary_object_url": { "basename": "[FINAL]SSFThesis_Compiled.pdf", "content": "final", "filesize": 11556184, "license": "other", "mime_type": "application/pdf", "url": "/13697/1/[FINAL]SSFThesis_Compiled.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "Development of a Modular Strategy Towards the Total Synthesis of (+)-Pleuromutilin and Progress Towards the Synthesis of (\u2013)-Merrilactone A", "author": [ { "family_name": "Feng", "given_name": "Sean S. L.", "orcid": "0000-0002-8095-6402", "clpid": "Feng-Sean-S-L" } ], "thesis_advisor": [ { "family_name": "Reisman", "given_name": "Sarah E.", "clpid": "Reisman-S-E" } ], "thesis_committee": [ { "family_name": "Stoltz", "given_name": "Brian M.", "clpid": "Stoltz-B-M" }, { "family_name": "Fu", "given_name": "Gregory C.", "clpid": "Fu-G-C" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" }, { "family_name": "Reisman", "given_name": "Sarah E.", "clpid": "Reisman-S-E" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Natural products have long stood as a rich source of biologically relevant molecules bearing highly functionalized and complex architectures. On one hand, they are a focal point for the development of new therapeutic agents owing to their inherent biological activities. On the other, they serve as an exciting testing ground for existing synthetic methodologies and provide opportunities for the development of new reactions.
\r\n\r\nHerein, we describe a modular strategy that was employed for the total synthesis of the antibiotic (+)-pleuromutilin. Key features of our synthesis include (1) the development of a highly stereoselective SmI\u2082-mediated ketyl radical cyclization to establish the central eight-membered ring and (2) a modular crotylation reaction to install the eight-membered ring\u2019s backbone that permits full control over the stereochemistry at C12 as desired. During our synthetic studies, a transannular [1,5]-hydrogen atom transfer reaction that affects a stereospecific redox relay to set the C10 stereocenter was serendipitously uncovered. This strategy enabled the completion of a concise total synthesis of (+)-pleuromutilin, proceeding in 18 steps. To demonstrate the modularity of our synthetic approach, the same strategy was readily applied to the synthesis of (+)-12-epi-pleuromutilin with no reoptimization, providing a new platform for the preparation of fully synthetic derivatives that may hold promise as broad-spectrum antibiotics.
\r\n\r\nThis report also highlights the work we have conducted in the development of a synthetic strategy towards (\u2013)-merrilactone A. We detail our investigation of a Pd-catalyzed asymmetric allylic alkylation reaction that rapidly constructs the D-ring bearing the C5 and C6 vicinal quaternary centers. Potential paths forward to complete the synthesis of this neurotropic natural product leveraging this advanced intermediate will also be discussed.
", "doi": "10.7907/chfe-0q83", "publication_date": "2020", "thesis_type": "phd", "thesis_year": "2020" }, { "id": "thesis:13815", "collection": "thesis", "collection_id": "13815", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06112020-001257003", "primary_object_url": { "basename": "JohnThompson_Thesis_Final.pdf", "content": "final", "filesize": 14848581, "license": "other", "mime_type": "application/pdf", "url": "/13815/1/JohnThompson_Thesis_Final.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "Chemical Tools for Studying O-GlcNAc Glycosylation at the Systems Level", "author": [ { "family_name": "Thompson", "given_name": "John Warren Lenzi", "orcid": "0000-0003-0061-4996", "clpid": "Thompson-John-Warren-Lenzi" } ], "thesis_advisor": [ { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "orcid": "0000-0001-5661-1714", "clpid": "Hsieh-Wilson-L-C" } ], "thesis_committee": [ { "family_name": "Hoelz", "given_name": "Andre", "orcid": "0000-0003-0923-3284", "clpid": "Hoelz-A" }, { "family_name": "Chan", "given_name": "David C.", "orcid": "0000-0002-0191-2154", "clpid": "Chan-D-C" }, { "family_name": "Pachter", "given_name": "Lior S.", "orcid": "0000-0002-9164-6231", "clpid": "Pachter-L" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "orcid": "0000-0001-5661-1714", "clpid": "Hsieh-Wilson-L-C" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "The addition of O-linked \u03b2-N-acetylglucosamine (O-GlcNAc) to intracellular serine and threonine residues is a ubiquitous post-translational modification (PTM) found in all higher eukaryotes. Like other PTMs, it is finely regulated in response to stimuli and dysregulated in multiple diseases. However, unlike other PTMs, methods to detect and profile the dynamics of O-GlcNAc glycosylation are still in their infancy. Herein, we discuss the background, development, and application of new chemical tools that have allowed for some of the first systems-level investigations of O-GlcNAcylation in different cells, organ systems, and disease states. We also significantly advance established techniques for the detection and monitoring of O-GlcNAc on proteins of interest. Using these new techniques, we first uncover a novel O-GlcNAcylation site on Cdk5 and show that this site can dynamically regulate Cdk5 activity in the context of neurodegenerative disease. Next, we apply novel chemical, mass spectrometric, and computational tools to, for the first time, uncover cellular networks engaged by O-GlcNAcylation in vivo. Finally, we undertake the systematic optimization of mass spectrometry based O-GlcNAcomics and use these new insights to significantly advance our understanding of O-GlcNAcylation dynamics in metabolic diseases of the liver. Overall, the techniques developed and data generated herein are closing the methodological and intellectual gaps between the study of O-GlcNAc glycosylation and that of other PTMs.
", "doi": "10.7907/gx3z-k069", "publication_date": "2020", "thesis_type": "phd", "thesis_year": "2020" }, { "id": "thesis:11562", "collection": "thesis", "collection_id": "11562", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05292019-144516804", "type": "thesis", "title": "Structural and Biochemical Studies of Enzymes in Bacterial Glycobiology", "author": [ { "family_name": "Yun", "given_name": "Hyun Gi", "orcid": "0000-0002-3508-5791", "clpid": "Yun-Hyun-Gi" } ], "thesis_advisor": [ { "family_name": "Clemons", "given_name": "William M.", "orcid": "0000-0002-0021-889X", "clpid": "Clemons-W-M" } ], "thesis_committee": [ { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "orcid": "0000-0001-5661-1714", "clpid": "Hsieh-Wilson-L-C" }, { "family_name": "Shan", "given_name": "Shu-ou", "orcid": "0000-0002-6526-1733", "clpid": "Shan-Shu-ou" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Clemons", "given_name": "William M.", "orcid": "0000-0002-0021-889X", "clpid": "Clemons-W-M" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The speed that bacterial pathogens gain resistance to antibiotics is alarming. Designing new antibacterial agents is urgent, but it requires understanding their bacterial targets at the molecular level to achieve high specificity and potency. In this thesis, I discuss the structural and biochemical investigations of three potential protein targets for antibiotics. The first is a UDP-Glc/GlcNAc 4-epimerase, called Gne, from the human pathogen Campylobacter jejuni. This enzyme is the sole source of N-acetylgalactosamine (GalNAc) in C. jejuni, which is a common component in three major glycoconjugates decorating the cell surface and is critical for pathogenesis. The second target protein is an integral membrane protein, called MraY, which catalyzes the transfer of phospho-N-acetylmuramyl (MurNAc) pentapeptide to a lipid carrier, undecaprenyl phosphate (C55-P), producing Lipid I in the peptidoglycan biosynthesis pathway. In the following step, a peripheral protein called MurG catalyzes transferring N-acetylglucosamine (GlcNAc) to Lipid I and produces Lipid II, which provides the first building block of the peptidoglycan layer. Peptidoglycan is uniquely bacterial, with MraY and MurG both being essential for cell viability; therefore, they are attractive targets for the development of antibacterial agents and work toward their structures is presented. Finally, MraY from Escherichia coli is the target for the lysis protein E from phage \u03a6X174.Efforts toward elucidating the EcMraY-E complexstructure are demonstrated here. In total, this thesis provides important data toward a full mechanistic understanding of these important antibacterial targets.", "doi": "10.7907/VKH3-XX98", "publication_date": "2019", "thesis_type": "phd", "thesis_year": "2019" }, { "id": "thesis:10382", "collection": "thesis", "collection_id": "10382", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:08182017-161833257", "primary_object_url": { "basename": "MDavis_THESIS_ASSEMBLED_FINAL.pdf", "content": "final", "filesize": 7374405, "license": "other", "mime_type": "application/pdf", "url": "/10382/1/MDavis_THESIS_ASSEMBLED_FINAL.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Computational Studies of Noncovalent Interactions in Ligand-Gated Ion Channels \u2013 and - Synthesis and Characterization of Red and Near Infrared Cyanine Dyes", "author": [ { "family_name": "Davis", "given_name": "Matthew Robert", "orcid": "0000-0002-6374-4555", "clpid": "Davis-Matthew-Robert" } ], "thesis_advisor": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" } ], "thesis_committee": [ { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" }, { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Cai", "given_name": "Long", "clpid": "Cai-Long" }, { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "This thesis is presented in two parts. The first part, Chapter 2, 3, and 4, offers a series of studies on noncovalent interactions in Ligand Gation Ion Channels (LGICs). The second part describes a series of studies involving the synthesis and characterization of cyanine dyes. The common thread in this work is the use of Density Functional Theory (DFT) to study chemical-scale phenomenon. Chapter 1 offers a brief introduction to DFT and a comparison with other traditional computational chemistry methodology; Hartree-Fock (HF). Summaries of the use of DFT to study both noncovalent interations and electronically excited states are also presented. In addition, the author comments on the correct application of DFT.
\r\n\r\nChapter 2 details a computational study of the cation-\u03c0 interaction of complex cations to substituted benzenes and indoles. The cation-\u03c0 interaction is the electrostatic interaction between a cation and the negative electrostatic potential on an aromatic ring originating from its large permanent quadrupole moment. This chapter, in addition to establishing the correct computational parameters, establishes a large set of substituent effects with which to study cation-\u03c0 interactions in vivo. These binding energy values are compared to previous applications of cation-\u03c0 binding energies from our lab, and it was found that the derived binding energies are sufficiently accurate.
\r\n\r\nChapter 3 applies the foundational knowledge from the previous chapter to study cation-\u03c0 interactions of cationic ligands to multiple aromatics. This is a common motif in vivo known as the aromatic box. Using this methodology, it is established that cation binding in this form is cooperative. Further, many aromatic boxes from crystal structures were evaluated energetically.
\r\n\r\nChapter 4 describes work to develop a new amino acid to study hydrogen bonds in Xenopus laevis oocytes. These fluorinated aliphatic amino acids inductively attenuate the hydrogen bond accepting ability of the carbonyl. This new strategy was used to probe for a hydrogen bond between the indole NH \u03b14 TrpB and a backbone carbonyl associated with L119 on the \u03b22 subunit of the \u03b14\u03b22 nicotinic acetylcholine receptor (nAChR). The fluorinated amino acids were validated computationally and with NMR studies. This new strategy showed that the \u03b14-\u03b22 interfacial hydrogen prediction was false.
\r\n\r\nChapter 5 describes the synthesis and characterization of a series of meso-aromatic-acetylene cyanine dyes which feature a very large Stokes shift. Synthesis of the dyes features a key Sonagashira reaction. These dyes are investigated photophysically and computationally using time dependent DFT (TDDFT). The mechanism for this Stokes shift is an excitation to the S2 state, relaxation to the S1 state, and normal cyanine fluorescence.
\r\n\r\nChapter 6 describes three separate strategies to construct a cyanine-based photocage to release drugs in vivo using an ortho-quinone methide strategy. One strategy utilized an acetylene-aromatic cyanine dye much like those described in Chapter 5, the second utilized an ethynyl-trimethylphenyl cation dye, and the third a photoinduced electron transfer cyanine dye. None of these strategies produced a usable photocage. The failure of these strategies are ascribed to both the short excited state lifetime of cyanine dyes and the direction of the transition dipole moment.
\r\n\r\nFinally, three appendices are presented. Appendix A describes early work to synthesize and characterize a meso-hydroxy substituted Cy5 dye. Appendix B offers many of the same computations as Chapters 2 and 3 using HF instead of DFT. Appendix C describes orbital mixing of cyanine dyes from Chapter 5 using HF instead of DFT.
", "doi": "10.7907/Z9XK8CQF", "publication_date": "2018", "thesis_type": "phd", "thesis_year": "2018" }, { "id": "thesis:10615", "collection": "thesis", "collection_id": "10615", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:12192017-155744407", "primary_object_url": { "basename": "FinalThesis.pdf", "content": "final", "filesize": 83182527, "license": "other", "mime_type": "application/pdf", "url": "/10615/83/FinalThesis.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "Elucidating the Role of O-GlcNAc Glycosylation in Neurobiology and Neurodegeneration", "author": [ { "family_name": "Jensen", "given_name": "Elizabeth Hwang", "orcid": "0000-0002-6177-4304", "clpid": "Jensen-Elizabeth-Hwang" } ], "thesis_advisor": [ { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" } ], "thesis_committee": [ { "family_name": "Dervan", "given_name": "Peter B.", "clpid": "Dervan-P-B" }, { "family_name": "Rothenberg", "given_name": "Ellen V.", "clpid": "Rothenberg-E-V" }, { "family_name": "Prober", "given_name": "David A.", "clpid": "Prober-D-A" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" } ], "local_group": [ { "literal": "div_bbe" } ], "abstract": "O-GlcNAc glycosylation is a dynamic, inducible post-translational modification (PTM) essential for neuronal homeostasis and found on proteins associated with neurodegenerative diseases such as \u03b1-synuclein, amyloid precursor protein, and tau. Intracellularly, O-GlcNAc modification is cycled by two enzymes in mammalian cells: O-GlcNAc transferase (OGT) appends O-GlcNAc to serine or threonine residues and O-GlcNAcase (OGA) removes O-GlcNAc. OGT modifies over 1000 different proteins, but the lack of a well-defined consensus sequence or substrate structural constraints has hampered efforts to predict sites a priori. Furthermore, the identification of O-GlcNAc modification sites has been obstructed by the difficulty of enriching and detecting O-GlcNAc using traditional biochemical methods. Here, we established and employed biological and chemical tools to illuminate the role of O-GlcNAc in neuronal function.
\r\n\r\nIn Chapter 2, we sought to determine the role of O-GlcNAc in learning, memory, and neurodegeneration. Deletion of the OGT gene causes early postnatal lethality in mice, complicating efforts to study O-GlcNAc glycosylation in mature neuronal function and dysfunction. We demonstrated that the loss of OGT in the forebrain of adult mice (OGT cKO) leads to progressive neurodegeneration, including neuronal death, neuroinflammation, hyperphosphorylated tau, amyloidogenic A\u03b2-peptides, and memory deficits. In the hippocampus, we showed that OGT ablation lead to the upregulation of neuroinflammatory genes and the downregulation of cholesterol biosynthetic genes. Additionally, a gene network analysis (WGCNA), qPCR, and immunohistochemistry (IHC) revealed that loss of O-GlcNAc perturbed cell cycle progression in the hippocampal neurons. In the hippocampus, we identified increased neuroinflammatory gene transcription in OGT cKO mice and both tau neurofibrillary tangle (NFT)-forming and amyloid-forming Alzheimer\u2019s disease (AD) mouse models. However, only OGT cKO and NFT-forming mice displayed decreased synaptic gene expression, suggesting that NFT formation and OGT cKO compromise hippocampal synaptic transcription. These studies indicate that O-GlcNAcylation regulates pathways vital for the maintenance of neuronal health and suggest that dysfunctional O-GlcNAc signaling may be an important contributor to neurodegenerative diseases.
\r\n\r\nIn order to understand the critical O-GlcNAc-mediated neuronal functions that underlie OGT cKO dysfunction, we next developed and utilized novel biological and chemical tools in order to identify key OGT interactors and substrates in the brain in Chapter 3. Due to the lack of a well-defined OGT substrate sequence and structural constraints, OGT is believed to obtain its substrate specificity through its interactome where specific interactors target OGT to specific substrates. In order to identify these interactors, we used CRISPR/Cas9 to generate a novel mouse with a minimally tagged OGT in order to identify the endogenous OGT brain interactome using tandem affinity purification and MS methods. The preliminary OGT brain interactome consisted of previously identified OGT interactors and substrates as well as novel interactors. The identified OGT interactors were enriched for ribosomal and cytoskeletal proteins in addition to axonal, dendritic, and neuronal cell body proteins, implicating OGT as a pivotal mediator of neuronal structure and function.
\r\n\r\nIn addition to the OGT interactome, we sought to uncover OGT\u2019s substrates or the O-GlcNAcome. We developed an improved approach to quantitatively label and enrich O-GlcNAcylated proteins for site identification. Chemoenzymatic labeling followed by Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) installed a new MS-compatible linker designed for facile purification and release of O-GlcNAcylated proteins for downstream MS analysis. We validated the approach by identifying several established O-GlcNAc sites on the proteins \u03b1-crystallin and OGT as well as discovering new, previously unreported sites on both proteins. Notably, these novel sites on OGT lie in key functional domains of OGT, underscoring how this site identification method can reveal important biological insights into protein activity and regulation.
\r\n\r\nFinally, in Chapters 4 and 5, we focus on the post-translational modification (PTM) code on a specific transcription factor (TF), CREB (cAMP response element binding protein). CREB regulates memory formation through its transcriptional control of neuronal metabolism, activity, differentiation, development, and survival. CREB phosphorylation at serine 133 has been previously shown to enhance CREB-mediated transcription while CREB glycosylation at serine 40 has been shown to decrease CREB-mediated transcription. However, the exact gene networks modulated by and potential interplay between CREB glycosylation and phosphorylation have not been explored. Through differential expression analysis with glycosylation-deficient (S40A) and phosphorylation-deficient (S133A) CREB mutants, we showed that CREB O-GlcNAcylation is important for neuronal activity and excitability while phosphorylation at serine 133 regulated the expression of genes involved in neuronal differentiation. Using WGCNA, we demonstrated that CREB O-GlcNAcylation at serine 40 and phosphorylation at serine 133 mediate mutually exclusive gene networks. The glycosylation-deficient mutant enhanced neuronal activity- and excitotoxicity-related gene networks while the phosphorylation-deficient mutant perturbed neuronal differentiation and amino and fatty acid metabolism-related gene networks. Our work sheds light on the regulation of CREB through PTMs to modulate neuronal function and delineate the roles of O-GlcNAcylation and phosphorylation in modulating neuronal excitability and neuronal development and metabolism respectively. Altogether, these studies demonstrate that O-GlcNAc modification is a critical mediator of neuronal homeostasis and neurodegeneration.
", "doi": "10.7907/Z9JD4TZ9", "publication_date": "2018", "thesis_type": "phd", "thesis_year": "2018" }, { "id": "thesis:10717", "collection": "thesis", "collection_id": "10717", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:02132018-133402404", "primary_object_url": { "basename": "GMM_Thesis.pdf", "content": "final", "filesize": 8654115, "license": "other", "mime_type": "application/pdf", "url": "/10717/1/GMM_Thesis.pdf", "version": "v16.0.0" }, "type": "thesis", "title": "Eph Receptor Clustering by Chondroitin Sulfate Inhibits Axon Regeneration", "author": [ { "family_name": "Miller", "given_name": "Gregory Martin", "clpid": "Miller-Gregory-Martin" } ], "thesis_advisor": [ { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" } ], "thesis_committee": [ { "family_name": "Hoelz", "given_name": "Andre", "clpid": "Hoelz-A" }, { "family_name": "Goddard", "given_name": "William A., III", "clpid": "Goddard-W-A-III" }, { "family_name": "Dervan", "given_name": "Peter B.", "clpid": "Dervan-P-B" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Chondroitin sulfate proteoglycans (CSPGs) play important roles in the developing and mature nervous system, where they guide axons, maintain stable connections, restrict synaptic plasticity, and prevent axon regeneration following CNS injury. The chondroitin sulfate glycosaminoglycan (CS GAG) chains that decorate CSPGs are essential for their functions. Through these sugar chains, CSPGs are able to bind and regulate the activity of a diverse range of proteins and through these interactions can regulate neuronal growth. These CS-protein interactions depend on specific sulfation patterns within the CS GAG chains, and accordingly, particular CS sulfation motifs are upregulated during development, in the mature nervous system, and in response to CNS injury. Thus, spatiotemporal regulation of CS GAG biosynthesis may provide an important mechanism to control the functions of CSPGs and modulate intracellular signaling pathways. Here, we will discuss these sulfation-dependent processes and highlight how the CS sugars on CSPGs contribute to neuronal growth, axon guidance, and plasticity in the nervous system.
\r\n\r\nChondroitin sulfate proteoglycans (CSPGs) are a major barrier to regenerating axons in the central nervous system (CNS), exerting their inhibitory effect through their polysaccharide side chains. Chondroitin sulfate (CS) potently inhibits axon regeneration through modulation of inhibitory signaling pathways induced by carbohydrate binding to protein ligands and receptors. Here, we identify a novel carbohydrate-protein interaction between CS and EphA4 that inhibits axon regrowth. We characterize the mechanism of activation and demonstrate how carbohydrate binding induces phosphorylation of the intracellular kinase domain through clustering of cell surface EphA4. Collectively, our studies present a novel mechanism of EphA4 activation by CS independent of the canonical ephrin ligands and uncover the role of this interaction in inhibition of neurite regrowth after injury. Our results underscore a mechanism of action by which carbohydrates can function as direct, activating ligands for protein receptors and provide mechanistic insights into the inhibition of axon growth by CS following injury to the CNS.
\r\n\r\nChondroitin sulfate proteoglycans (CSPGs) regulate neuronal plasticity, as well as axon regeneration and guidance through their ability to bind protein ligands and cell surface receptors. In this way, extracellular CSPGs can modulate the activity of intracellular signaling pathways. Here, a computational analysis of EphA4-CS interactions is performed to characterize the importance of key arginine and lysine residues towards CS binding, and to identify structural differences in CS-A, CS-C, CS-D, and CS-E docking to EphA4. Carbohydrate-induced Eph receptor clustering could be a general mechanism of Eph receptor activation. To identify additional Eph receptors that interact with CS, CS-E was docked to all EphA and EphB family members to predict relative binding affinities. The relative strengths of the predicted binding energies are: EphB4 > EphA8 > EphA1 > EphA3 > EphB1 > EphB3 > EphA7 > EphA5 > EphA4 > EphA6 > EphB2 > EphB6 > EphA2. In addition, the arginine and lysine residues that mediate CS binding are identified for each Eph receptor. These computational predictions provide mechanistic insights into Eph receptor activation by chondroitin sulfate and have implications for inhibition of axon regeneration following injury to the nervous system and axon guidance during development.
", "doi": "10.7907/Z9RJ4GPT", "publication_date": "2018", "thesis_type": "phd", "thesis_year": "2018" }, { "id": "thesis:10731", "collection": "thesis", "collection_id": "10731", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:02232018-152758526", "type": "thesis", "title": "Interplay of Proton Transfer, Electron Transfer and Proton-Coupled Electron Transfer in Transition Metal Mediated Nitrogen Fixation", "author": [ { "family_name": "Matson", "given_name": "Benjamin David", "orcid": "0000-0001-5733-0893", "clpid": "Matson-Benjamin-David" } ], "thesis_advisor": [ { "family_name": "Peters", "given_name": "Jonas C.", "clpid": "Peters-J-C" } ], "thesis_committee": [ { "family_name": "Agapie", "given_name": "Theodor", "clpid": "Agapie-T" }, { "family_name": "Miller", "given_name": "Thomas F.", "clpid": "Miller-T-F" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" }, { "family_name": "Peters", "given_name": "Jonas C.", "clpid": "Peters-J-C" } ], "local_group": [ { "literal": "Resnick Sustainability Institute" }, { "literal": "div_chem" } ], "abstract": "Mitigation of the hydrogen evolution reaction (HER) is a key challenge in selective small molecule reduction catalysis, including the nitrogen (N2) reduction reactions (N2RR) using H+/e- currency. Here we explore, via DFT calculations, three iron model systems, P3EFe (E = B, Si, C), known to mediate both N2RR and HER, but with different selectivity depending on the identity of the auxiliary ligand. It is shown that the respective efficiencies of these systems for N2RR trend with the predicted N\u2013H bonds strengths of two putative hydrazido intermediates of the proposed catalytic cycle, P3EFe(NNH2)+ and P3EFe(NNH2). Bimolecular proton-coupled electron transfer (PCET) from intermediates with weak N\u2013H bonds is posited as a major source of H2 instead of more traditional scenarios that proceed via metal hydride intermediates and proton transfer/electron transfer (PT/ET) pathways.
\r\n\r\nStudies on our most efficient molecular iron catalyst, [P3BFe]+, reveal that the interaction of acid and reductant, Cp*2Co, is critical to achieve high efficiency for NH3, leading to the demonstration of electrocatalytic N2RR. Stoichiometric reactivity shows that Cp*2Co is required to observe productive N\u2013H bond formation with anilinium triflate acids under catalytic conditions. A study of substituted anilinium triflate acids demonstrates a strong correlation between pKa and the efficiency for NH3, which DFT studies attribute to the kinetics and thermodynamics of Cp*2Co protonation. These results contribute to the growing body of evidence suggesting that metallocenes should be considered as more than single electron transfer reagents in the proton-coupled reduction of small molecule substrates and that ring-functionalized metallocenes, believed to be intermediates on the background HER pathway, can play a critical role in productive bond-forming steps.
", "doi": "10.7907/Z9MS3R0Z", "publication_date": "2018", "thesis_type": "phd", "thesis_year": "2018" }, { "id": "thesis:10364", "collection": "thesis", "collection_id": "10364", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:08042017-162439257", "primary_object_url": { "basename": "MakVictor_Thesis2017_FINAL.pdf", "content": "final", "filesize": 70506194, "license": "other", "mime_type": "application/pdf", "url": "/10364/85/MakVictor_Thesis2017_FINAL.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "Development of Synthetic Strategies for the Total Synthesis of Ent-Kauranoid and Diterpenoid Alkaloid Natural Products", "author": [ { "family_name": "Mak", "given_name": "Victor Wei-Dek", "clpid": "Mak-Victor-Wei-Dek" } ], "thesis_advisor": [ { "family_name": "Reisman", "given_name": "Sarah E.", "clpid": "Reisman-S-E" } ], "thesis_committee": [ { "family_name": "Stoltz", "given_name": "Brian M.", "clpid": "Stoltz-B-M" }, { "family_name": "Reisman", "given_name": "Sarah E.", "clpid": "Reisman-S-E" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" }, { "family_name": "Fu", "given_name": "Gregory C.", "clpid": "Fu-G-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "As part of an ongoing synthetic effort directed towards biologically active ent-kauranoid natural products, the preparation of two structurally unique natural products, (\u2013)-trichorabdal A and (\u2013)-longikaurin E, is presented. The syntheses intercept an early intermediate from the synthetic route towards the rearranged natural product (\u2013)-maoecrystal Z, and thus, represents a unified synthetic strategy to access structurally unique ent-kauranoids. Specifically, the syntheses are enabled by a palladium-mediated oxidative cyclization of a silyl ketene acetal to install a key quaternary center within the bicyclo[3.2.1]octane unit, as well as a reductive cyclization of an aldehyde-lactone to construct the oxabicyclo[2.2.2]octane motif of (\u2013)-longikaurin E.
\r\n\r\nA synthetic strategy to access C19-diterpenoid alkaloids, specifically of the aconitine type, is presented. These highly bridged polycyclic natural products are generally characterized by a substituted piperidyl ring bridging a hydrindane framework that is further attached to a bicyclo[3.2.1]octane. The synthetic strategy relies on the enantioselective synthesis of two bicyclic fragments, which are coupled in a convergent fashion through a 1,2-addition/semipinacol rearrangement sequence to forge a sterically hindered quaternary center. Efficient access to late stage intermediates has enabled the synthesis of the aconitine carbocyclic core, with appropriate functionality for advancement to a selective voltage-gated K+ channel blocker, talatisamine. Additionally, the synthetic strategy described herein is well applicable to the synthesis of related denudatine and napelline type C20-diterpenoid alkaloids.
", "doi": "10.7907/Z9ST7N04", "publication_date": "2018", "thesis_type": "phd", "thesis_year": "2018" }, { "id": "thesis:10967", "collection": "thesis", "collection_id": "10967", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05292018-232454800", "type": "thesis", "title": "Synthesis, NMR Solution Structure, and Neuritogenic Activity of Chondroitin Sulfate D and E", "author": [ { "family_name": "Yang", "given_name": "Kuang-Wei", "clpid": "Yang-Kuang-Wei" } ], "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": "Goddard", "given_name": "William A., III", "clpid": "Goddard-W-A-III" }, { "family_name": "Reisman", "given_name": "Sarah E.", "clpid": "Reisman-S-E" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Chondroitin sulfate are ubiquitously expressed linear, sulfated polysaccharides that play critical roles in neuronal development and regeneration growth factor signaling, morphogenesis, and virus invasion. The diverse sulfation patterns presented by chondroitin sulfate has been suggested to regulate its activity, but the structural complexity and heterogeneity have hampered the understanding of structure-activity relationship. Therefore, we envisioned that chemically synthesized chondroitin sulfate oligosaccharide may provide a unique opportunity to specifically study the functions of sulfation patterns.
\r\n\r\nHere, we report the synthesis of a CS-D and CS-E tetrasaccharide in a step-efficient manner. By generating a disaccharide precursor from hydrolysis of polysaccharides, we were able to streamline the synthesis and reduce the number of steps by one-third comparing to the traditional synthesis without losing versatility of the synthetic route and functionality of the final product. With the structurally defined molecules, we were able to determine the NMR solution structure of CS-D and CS-E. In this work, we accomplished the first structural study of CS-D tetrasaccharide and the most thorough study of CS-E to date. Furthermore, we also discovered the existence of a second conformer in CS-D, which is the first time for such behavior to be observed experimentally in chondroitin sulfate. The electrostatic potential surface constructed based on the NMR structure presented unique structural features that may allow proteins to interact specifically.
\r\n\r\nThe CS-D and CS-E tetrasaccharide, along with a CS-D disaccharide, was\r\ninvestigated for their neuritogenic activity. We discovered that the CS-D tetrasaccharide specifically stimulates dendritic growth whereas the CS-E tetrasaccharide preferentially promoted axonal growth, revealing the potential critical role chondroitin sulfate with specific sulfation patterns may play in the nervous system. The lack of activity of the CS-D disaccharide suggested that the minimum motif required for activity of CS-D is a tetrasaccharide.
\r\n", "doi": "10.7907/TPD7-FB87", "publication_date": "2018", "thesis_type": "phd", "thesis_year": "2018" }, { "id": "thesis:9930", "collection": "thesis", "collection_id": "9930", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:10032016-103410952", "type": "thesis", "title": "Discovery and Development of Small-Molecule Modulators for the Sulfation of Glycosaminoglycans and Studying the Role of O-GlcNAc on CREB through Semisynthesis", "author": [ { "family_name": "Cheung", "given_name": "Sheldon Ting Fong", "clpid": "Cheung-Sheldon-Ting-Fong" } ], "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": "Dervan", "given_name": "Peter B.", "clpid": "Dervan-P-B" }, { "family_name": "Clemons", "given_name": "William M.", "clpid": "Clemons-W-M" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Glycosaminoglycans (GAGs) are sulfated polysaccharides that play key roles in many cellular processes, ranging from viral invasion and cancer metastasis to neuronal development. Their diverse biological activities stem from their complex sulfation patterns, which are tightly regulated in vivo. For instance, the GAG chondroitin sulfate (CS) has been shown to undergo regiochemical sulfation during development and after spinal cord injury. However, few tools exist to modulate specific GAG sulfation patterns and study their importance in different biological contexts. Here, we identified the first cell-permeable small molecule that can selectively inhibit GAG sulfotransferases and modify the fine structure of GAGs. We demonstrate that the inhibitor reduces GAG sulfation in vitro and in cells and reverses CS-E-mediated inhibition of neuronal outgrowth. This small molecule may serve as a useful lead compound or chemical tool for studying the importance of CS and other GAGs in normal biology and disease.
\r\n\r\nThe \u03b2-N-acetyl-D-glucosamine (O-GlcNAc) post-translational modification plays a major role in many diseases such as cancer, diabetes, and neurodegenerative disorders, but much is still unknown about its molecular-level influence on protein structure and function. Although post-translational modifications have been known to induce important structural changes in proteins, notably, no structures of O-GlcNAcylated proteins exist. The challenge of obtaining homogeneous glycoproteins bearing the GlcNAc sugar at defined sites has hindered the structural and biochemical studies of this modification. Here we have utilized a semisynthetic approach to generate a homogeneously O-GlcNAcylated form of cyclic-AMP response element binding protein (CREB) for structural and functional studies.
", "doi": "10.7907/Z9PR7T0K", "publication_date": "2017", "thesis_type": "phd", "thesis_year": "2017" }, { "id": "thesis:10335", "collection": "thesis", "collection_id": "10335", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06122017-230026717", "type": "thesis", "title": "DarwinDock and GAG-Dock: Methods and Applications for Small Molecule Docking", "author": [ { "family_name": "Griffith", "given_name": "Adam Reid", "clpid": "Griffith-Adam-Reid" } ], "thesis_advisor": [ { "family_name": "Goddard", "given_name": "William A., III", "clpid": "Goddard-W-A-III" } ], "thesis_committee": [ { "family_name": "Heath", "given_name": "James R.", "clpid": "Heath-J-R" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" }, { "family_name": "Goddard", "given_name": "William A., III", "clpid": "Goddard-W-A-III" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Computational modeling is an effective tool in studying complex biological systems. Docking of small molecule ligands in particular is useful both in understanding the functioning of proteins as well as in the development of pharmaceuticals. Together with experiment, modeling can often provide a thorough picture of a given system. Computation can often provide details that are difficult or impossible to determine experimentally, while experiments provide guidance on what calculations are useful or interesting. Our goal is to extend computational modeling, specifically ligand docking, to systems not previously possible, such as the challenging glycosaminoglycan (GAG) systems. In order to do this it was first necessary to develop an automatic way of performing docking without extensive user input and experimental knowledge to narrow the list of candidate poses. DarwinDock represents our efforts in this respect. It is a method for small-molecule docking that separates pose generation and scoring into separate stages, which allows for complete binding site sampling followed by efficient, hierarchical sampling. Our convergence criteria for complete sampling allows for diverse systems to be studied without prior knowledge of how large a set of poses needs to be to span a given binding site, making the procedure more automatic. We also replace bulky, nonpolar residues with alanine, which we refer to as \"alanization\". This allows the ligand to interact more closely with polar sidechains, which help to orient the ligand. Additionally, alanization reduces the impact of incorrect sidechain placement on ligand placement, a concern that sometimes requires user intervention. With DarwinDock working for standard small molecules, it was then necessary to modify the procedure to work on challenging GAG ligands, which are large and have strong negative charges. A modification to DarwinDock \u2013 GAG-Dock \u2013 allows the method to be applied to GAGs and protein surface interactions. GAGs are large, linear polysaccharides with strong negative charge. They typically interact with the surfaces of proteins, rather than the cavities favored by most small-molecule drugs. GAG-Dock systematically samples the protein surface for unknown binding sites and modifies the pose generation to allow for large, surface-interacting ligands. GAG-Dock allowed us to study several systems important for neuronal development and answer interesting questions posed by experiment. Finally, we needed a way to validate our predictions for GAG binding sites. We used a systematic approach to identify sets of beneficial mutations to the GAG binding sites by building up from individual in silico mutations. Standard mutation experiments typically employ large mutations, such as arginine to alanine, which decrease or destroy binding. However, such information is not always definitive, as large mutations can have wide-ranging effects beyond direct protein-ligand interactions. Mutations that increase binding, however, are less ambiguous because they must form new interactions with the ligand in order to affect binding energies or affinity. Therefore, we have identified and proposed sets of mutations for our GAG predictions for PTPs, NgR1, NgR3, and EphB3. We encourage our experimentalist colleagues to try these mutations and validate our predictions.
", "doi": "10.7907/Z91Z42GS", "publication_date": "2017", "thesis_type": "phd", "thesis_year": "2017" }, { "id": "thesis:10267", "collection": "thesis", "collection_id": "10267", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06042017-183300784", "type": "thesis", "title": "Discovering Biological Roles of Glycosaminoglycans and Protein O-GlcNAcylation Using Chemical Tools", "author": [ { "family_name": "Griffin", "given_name": "Matthew Everett", "orcid": "0000-0001-9549-4418", "clpid": "Griffin-Matthew-Everett" } ], "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": "Ismagilov", "given_name": "Rustem F.", "clpid": "Ismagilov-R-F" }, { "family_name": "Sternberg", "given_name": "Paul W.", "clpid": "Sternberg-P-W" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Carbohydrates surround nearly every cell in the human body. Glycosaminoglycans like chondroitin sulfate and heparan sulfate on the cell surface regulate protein ligand engagement and receptor activation to control a variety of biological processes including development, angiogenesis, and neuronal growth. These polysaccharides exert activity through protein binding to their diverse chemical structures. Therefore, the development of methods to tailor glycosaminoglycan populations at the cell surface with defined structures could provide novel approaches to control biological activity. Herein, two new methods to engineer the cell surface glycocalyx with known glycosaminoglycans are reported. Together, these methods provide complementary short- and long-term approaches to change carbohydrate structures at the cell surface and guide neuronal growth and stem cell differentiation. It is also critical to identify unknown protein-carbohydrate interactions that underlie biological phenomena. Studies delineating novel GAG interactions with an orphan receptor and related soluble ligands are reported herein as well as work towards understanding the biological functions of these newly discovered interactions. These results showcase the utility of chemical biology and biochemical tools to discover and modulate various GAG-protein interactions in diverse biological systems.
\r\n\r\nWithin the cell, thousands of proteins are modified by O-GlcNAc glycosylation, a process that is uniquely catalyzed by a single transferase and hydrolase pair unlike many other post-translational modifications. O-GlcNAcylation functions in many biological contexts including transcription, translation, proteostasis, and metabolism. Key to understanding its effects on these physiological phenomena is the discovery of O-GlcNAc modification sites. However, due to a number of technical challenges, O-GlcNAc proteomics has not progressed nearly as quickly as phosphoproteomics. Thus, developing new methods to enrich O-GlcNAcylated substrates and map modification sites is critical to unravel the myriad functions of O-GlcNAc. Herein, a labeling approach using a chemically cleavable tag is reported as an improved method to capture and release O-GlcNAcylated substrates. Unlike other methods, the cleavable Dde tag is quantitatively removed under mild, neutral conditions and leaves a minimal residual tag on the O-GlcNAcylated peptide to be analyzed. Moreover, the Dde linker outcompetes a previously used UV-cleavable tag both at the protein and peptide enrichment levels. Together, these results highlight the potential usefulness of this method to illuminate novel roles of O-GlcNAcylation in diverse systems.
\r\n", "doi": "10.7907/Z9610XCH", "publication_date": "2017", "thesis_type": "phd", "thesis_year": "2017" }, { "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:9846", "collection": "thesis", "collection_id": "9846", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06052016-231830980", "primary_object_url": { "basename": "Quigley_Thesis_Final_BQ.pdf", "content": "final", "filesize": 4483574, "license": "other", "mime_type": "application/pdf", "url": "/9846/20/Quigley_Thesis_Final_BQ.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "Understanding Selectivity and Activity in Z-selective Metathesis with Cyclometallated Ru-based Catalysts ", "author": [ { "family_name": "Quigley", "given_name": "Brendan Liam", "clpid": "Quigley-Brendan-Liam" } ], "thesis_advisor": [ { "family_name": "Grubbs", "given_name": "Robert H.", "clpid": "Grubbs-R-H" } ], "thesis_committee": [ { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" }, { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" }, { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Grubbs", "given_name": "Robert H.", "clpid": "Grubbs-R-H" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "With the advent of well-defined highly active catalysts, olefin metathesis has become a powerful tool for the formation of carbon\u2013carbon double bonds in a variety of settings. However, these traditional catalysts preferentially form the E-alkene product. Recent efforts have yielded several families of Z-selective metathesis catalysts, including a family of Ru complexes with cyclometallated NHC ligands developed in our group. The work in this thesis describes efforts to develop an improved understanding of the catalyst features that govern activity and selectivity in the cyclometallated Ru-based catalysts, as well as to expand the scope of reactivity in these systems.
\r\n\r\nChapter 1 provides an outline of the key features that govern selectivity in cross metathesis applications.
\r\n\r\nChapter 2 describes the application of cyclometallated Ru-based catalysts in Z-selective cross metathesis (CM) of allylic-substituted olefins. Efficient CM is demonstrated in the case of acrolein acetals providing a new route to access Z-\u03b1,\u03b2-unsaturated acetals and aldehydes. For a variety of other allylic-substituted olefins, reactivity was lower but could be correlated with the structure of the catalyst and substrate. The implications of the observed reactivity are discussed and contextualized with regard to reactivity of previous metathesis catalysts.
\r\n\r\nChapter 3 describes the development of a series of cyclometallated Z-selective metathesis with varying N-aryl groups that allow elucidation of the key catalyst features that govern activity and selectivity in these systems. The synthesis of the catalyst series is described, including several strategies employed to circumvent unexpected side-reactions. The second part of the chapter focuses on the dynamic behavior of the catalysts in solution and studies of an unusual C\u2013H\u00b7\u00b7\u00b7\u00b7F\u2013C intramolecular interaction observed in some of these catalysts. Finally, the reactivity of these catalysts in a variety of CM applications are discussed, which allows for development of a refined model of how the N-aryl group affects Z-selectivity and activity in these catalysts systems and how this varies across different classes of substrate.
", "doi": "10.7907/Z96W984J", "publication_date": "2016", "thesis_type": "phd", "thesis_year": "2016" }, { "id": "thesis:9039", "collection": "thesis", "collection_id": "9039", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06292015-212932175", "primary_object_url": { "basename": "Martinez_Thomas_2016_Thesis.pdf", "content": "final", "filesize": 29641943, "license": "other", "mime_type": "application/pdf", "url": "/9039/1/Martinez_Thomas_2016_Thesis.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Investigations of Pyrrole-Imidazole Polyamide Effects on DNA Replication", "author": [ { "family_name": "Martinez", "given_name": "Thomas Farid", "orcid": "0000-0002-4011-8164", "clpid": "Martinez-Thomas-Farid" } ], "thesis_advisor": [ { "family_name": "Dervan", "given_name": "Peter B.", "clpid": "Dervan-P-B" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Shan", "given_name": "Shu-ou", "clpid": "Shan-Shu-ou" }, { "family_name": "Campbell", "given_name": "Judith L.", "clpid": "Campbell-J-L" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" }, { "family_name": "Dervan", "given_name": "Peter B.", "clpid": "Dervan-P-B" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Pyrrole\u2013Imidazole polyamides are programmable, cell-permeable small molecules that bind in the minor groove of double-stranded DNA sequence-specifically. Polyamide binding has been shown to alter the local helical structure of DNA, disrupt protein-DNA interactions, and modulate endogenous gene expression. Py\u2013Im polyamides targeted to the androgen receptor-DNA interface have been observed to decrease expression of androgen-regulated genes, upregulate p53, and induce apoptosis in a hormone-sensitive prostate cancer cell line. Here we report that androgen response element (ARE)-targeted polyamides induced DNA replication stress in a hormone-insensitive prostate cancer cell line. The ATR checkpoint kinase was activated in response to this stress, causing phosphorylation of MCM2, and FANCD2 was monoubiquitinated. Surprisingly, little single-stranded DNA was exhibited, and the ATR targets RPA2 and Chk1 were not phosphorylated. We conclude that polyamide induces relatively low level replication stress, and suggest inhibition of the replicative helicase as a putative mechanism based on in vitro assays. We also demonstrate polyamide-induced inhibition of DNA replication in cell free extracts from X. laevis oocytes. In this system, inhibition of chromatin decondensation is observed, preventing DNA replication initiation. Finally, we show that Py-Im polyamides targeted to the ARE and ETS binding sequence downregulate AR- and ERG-driven signaling in a prostate cancer cell line harboring the TMPRSS2-ERG fusion. In a mouse xenograft model, ARE-targeted polyamide treatment reduced growth of the tumor.
", "doi": "10.7907/Z9RF5RZ2", "publication_date": "2016", "thesis_type": "phd", "thesis_year": "2016" }, { "id": "thesis:8977", "collection": "thesis", "collection_id": "8977", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06032015-135024245", "primary_object_url": { "basename": "ACWang_Thesis_060315.pdf", "content": "final", "filesize": 5348339, "license": "other", "mime_type": "application/pdf", "url": "/8977/1/ACWang_Thesis_060315.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Investigating the Role of O-GlcNAc Glycosylation in Neurodegeneration", "author": [ { "family_name": "Wang", "given_name": "Andrew Chih-Kae", "clpid": "Wang-Andrew-Chih-Kae" } ], "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": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" }, { "family_name": "Shan", "given_name": "Shu-ou", "clpid": "Shan-Shu-ou" }, { "family_name": "Chan", "given_name": "David C.", "clpid": "Chan-D-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "O-GlcNAc glycosylation of nuclear and cytosolic proteins is an essential post-translational modification implicated in many diseases, from cancer to diabetes. Importantly, many important neuronal proteins are also O-GlcNAc modified, and aberrant O-GlcNAcylation of these proteins may contribute to the pathology of neurodegenerative diseases although these mechanisms have not been well defined. Here we investigated the role of O-GlcNAc glycosylation in the brain, utilizing both chemistry and molecular biology to study O-GlcNAc transferase (OGT), the enzyme that adds the sugar modification. To evaluate the role of OGT in adult neurons, we generated a forebrain-specific conditional knockout of OGT (OGT cKO) in mice. Although indistinguishable from wild-type littermates at birth, after three weeks we observe progressive neurodegeneration in OGT cKO mice. Hallmarks of Alzheimer\u2019s disease, including neuronal loss, neuroinflammation, behavioral deficits, hyperphosphorylated tau, and amyloid beta peptide accumulation, are observed. Furthermore, decreases in OGT protein levels were found in human AD brain tissue, suggesting that altered O-GlcNAcylation likely contributes to neurodegenerative diseases in humans. This model is one of a few mouse models that recapitulate AD phenotypes without mutating and overexpressing human tau, amyloid precursor protein, or presenilin, highlighting the essential role of OGT in neurodegenerative pathways.
\r\n\r\nGiven the importance of OGT in the brain, we further investigated the regulation of the OGT enzyme by phosphorylation. We found that phosphorylation of OGT near its C-terminus reduces its activity in cancer cells, and have developed phosphorylation-specific antibodies to aid mechanistic studies. Furthermore, mutation of this phosphorylation site on OGT, followed by overexpression in neurons was shown to enhance neurite outgrowth, demonstrating a functional consequence for this site. Thus phosphorylation of OGT inhibits its activity and enhances neurite outgrowth, and current studies aim to characterize the signaling pathway that regulates OGT phosphorylation in neurons.
", "doi": "10.7907/Z97W695K", "publication_date": "2015", "thesis_type": "phd", "thesis_year": "2015" }, { "id": "thesis:8869", "collection": "thesis", "collection_id": "8869", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05192015-131629900", "primary_object_url": { "basename": "Corey-Reeves-2015-FullThesis.pdf", "content": "final", "filesize": 33285414, "license": "other", "mime_type": "application/pdf", "url": "/8869/134/Corey-Reeves-2015-FullThesis.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Strategies for the Stereoselective Synthesis of Carbon Quaternary Centers via Transition Metal-Catalyzed Alkylation of Enolate Compounds", "author": [ { "family_name": "Reeves", "given_name": "Corey Michael", "clpid": "Reeves-Corey-Michael" } ], "thesis_advisor": [ { "family_name": "Stoltz", "given_name": "Brian M.", "clpid": "Stoltz-B-M" } ], "thesis_committee": [ { "family_name": "Dervan", "given_name": "Peter B.", "clpid": "Dervan-P-B" }, { "family_name": "Reisman", "given_name": "Sarah E.", "clpid": "Reisman-S-E" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" }, { "family_name": "Stoltz", "given_name": "Brian M.", "clpid": "Stoltz-B-M" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Notwithstanding advances in modern chemical methods, the selective installation of sterically encumbered carbon stereocenters, in particular all-carbon quaternary centers, remains an unsolved problem in organic chemistry. The prevalence of all-carbon quaternary centers in biologically active natural products and pharmaceutical compounds provides a strong impetus to address current limitations in the state of the art of their generation. This thesis presents four related projects, all of which share in the goal of constructing highly-congested carbon centers in a stereoselective manner, and in the use of transition-metal catalyzed alkylation as a means to address that goal.
\r\n\r\nThe first research described is an extension of allylic alkylation methodology previously developed in the Stoltz group to small, strained rings. This research constitutes the first transition metal-catalyzed enantioselective \u03b1-alkylation of cyclobutanones. Under Pd-catalysis, this chemistry affords all\u2013carbon \u03b1-quaternary cyclobutanones in good to excellent yields and enantioselectivities.
\r\n\r\nNext is described our development of a (trimethylsilyl)ethyl \u03b2-ketoester class of enolate precursors, and their application in palladium\u2013catalyzed asymmetric allylic alkylation to yield a variety of \u03b1-quaternary ketones and lactams. Independent coupling partner synthesis engenders enhanced allyl substrate scope relative to allyl \u03b2-ketoester substrates; highly functionalized \u03b1-quaternary ketones generated by the union of our fluoride-triggered \u03b2-ketoesters and sensitive allylic alkylation coupling partners serve to demonstrate the utility of this method for complex fragment coupling.
\r\n\r\nLastly, our development of an Ir-catalyzed asymmetric allylic alkylation of cyclic \u03b2-ketoesters to afford highly congested, vicinal stereocenters comprised of tertiary and all-carbon quaternary centers with outstanding regio-, diastereo-, and enantiocontrol is detailed. Implementation of a subsequent Pd-catalyzed alkylation affords dialkylated products with pinpoint stereochemical control of both chiral centers. The chemistry is then extended to include acyclic \u03b2-ketoesters and similar levels of selective and functional group tolerance are observed. Critical to the successful development of this method was the employment of iridium catalysis in concert with N-aryl-phosphoramidite ligands.
\r\n", "doi": "10.7907/Z9VX0DGH", "publication_date": "2015", "thesis_type": "phd", "thesis_year": "2015" }, { "id": "thesis:8812", "collection": "thesis", "collection_id": "8812", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:04052015-200117829", "primary_object_url": { "basename": "JSK_Thesis_Full.pdf", "content": "final", "filesize": 37266574, "license": "other", "mime_type": "application/pdf", "url": "/8812/31/JSK_Thesis_Full.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Targeting DNA Repeat Sequences with Py-Im Polyamides", "author": [ { "family_name": "Kang", "given_name": "JeenJoo Sophia", "clpid": "Kang-JeenJoo-Sophia" } ], "thesis_advisor": [ { "family_name": "Dervan", "given_name": "Peter B.", "clpid": "Dervan-P-B" } ], "thesis_committee": [ { "family_name": "Grubbs", "given_name": "Robert H.", "clpid": "Grubbs-R-H" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" }, { "family_name": "Heath", "given_name": "James R.", "clpid": "Heath-J-R" }, { "family_name": "Dervan", "given_name": "Peter B.", "clpid": "Dervan-P-B" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Hairpin pyrrole-imdazole polyamides are cell-permeable, sequence-programmable oligomers that bind in the minor groove of DNA. This thesis describes studies of Py-Im polyamides targeted to biologically important DNA repeat sequences for the purpose of modulating disease states. Design of a hairpin polyamide that binds the CG dyad, a site of DNA methylation that can become dysregulated in cancer, is described. We report the synthesis of a DNA methylation antagonist, its sequence specificity and affinity informed by Bind-n-Seq and iteratively designed, which improves inhibitory activity in a cell-free assay by 1000-fold to low nanomolar IC50. Additionally, a hairpin polyamide targeted to the telomeric sequence is found to trigger a slow necrotic-type cell death with the release of inflammatory molecules in a model of B cell lymphoma. The effects of the polyamide are unique in this class of oligomers; its effects are characterized and a functional assay of phagocytosis by macrophages is described. Additionally, hairpin polyamides targeted to pathologically expanded CTG\u2022CAG triplet repeat DNA sequences, the molecular cause of myotonic dystrophy type 1, are synthesized and assessed for toxicity. Lastly, ChIP-seq of Hypoxia-Inducible Factor is performed under hypoxia-induced conditions. The study results show that ChIP-seq can be employed to understand the genome-wide perturbation of Hypoxia-Inducible Factor occupancy by a Py-Im polyamide.", "doi": "10.7907/Z93R0QSQ", "publication_date": "2015", "thesis_type": "phd", "thesis_year": "2015" }, { "id": "thesis:8403", "collection": "thesis", "collection_id": "8403", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:05272014-224131184", "primary_object_url": { "basename": "Sheng_Thesis_Final.pdf", "content": "final", "filesize": 36633278, "license": "other", "mime_type": "application/pdf", "url": "/8403/1/Sheng_Thesis_Final.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Tunable Heparan Sulfate Glycomimetics for Modulating Chemokine Activity", "author": [ { "family_name": "Sheng", "given_name": "Gloria J.", "clpid": "Sheng-Gloria-J" } ], "thesis_advisor": [ { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "orcid": "0000-0001-5661-1714", "clpid": "Hsieh-Wilson-L-C" } ], "thesis_committee": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "orcid": "0000-0003-1464-2461", "clpid": "Dougherty-D-A" }, { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" }, { "family_name": "Mazmanian", "given_name": "Sarkis K.", "orcid": "0000-0003-2713-1513", "clpid": "Mazmanian-S-K" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "orcid": "0000-0001-5661-1714", "clpid": "Hsieh-Wilson-L-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Heparan sulfate (HS) glycosaminoglycans participate in critical biological processes by modulating the activity of a diverse set of protein binding partners. Such proteins include all known members of the chemokine superfamily, which are thought to guide the migration of distinct subsets of immune cells through their interactions with HS proteoglycans on endothelial cell surfaces. Animal-derived heparin polysaccharides have been shown to reduce inflammation levels through the inhibition of HS-chemokine interactions; however, the clinical usage of heparin as an anti-inflammatory drug is hampered by its anticoagulant activity and potential risk for side effects, such as heparin-induced thrombocytopenia (HIT).
\r\n\r\nHere, we describe an expedient, divergent synthesis to prepare defined glycomimetics of HS that recapitulate the macromolecular structure and biological activity of natural HS glycosaminoglycans. Our synthetic approach uses a core disaccharide precursor to generate a library of four differentially sulfated polymers. We show that a trisulfated glycopolymer antagonizes the chemotactic activities of pro-inflammatory chemokine RANTES with similar potency as heparin polysaccharide, without potentiating the anticoagulant activities of antithrombin III. The same glycopolymer also inhibited the homeostatic chemokine SDF-1 with significantly more efficacy than heparin. Our work offers a general strategy for modulating chemokines and dissecting the pleiotropic functions of HS/heparin through the presentation of defined sulfation motifs within multivalent polymeric scaffolds.
", "doi": "10.7907/JQ2Z-EN67", "publication_date": "2014-06-12", "thesis_type": "phd", "thesis_year": "2014" }, { "id": "thesis:7920", "collection": "thesis", "collection_id": "7920", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:07302013-124522822", "primary_object_url": { "basename": "Yang-Fei-2014-Thesis.pdf", "content": "final", "filesize": 4984667, "license": "other", "mime_type": "application/pdf", "url": "/7920/37/Yang-Fei-2014-Thesis.pdf", "version": "v8.0.0" }, "type": "thesis", "title": "Antitumor Activity of Py-lm Polyamides", "author": [ { "family_name": "Yang", "given_name": "Fei", "clpid": "Yang-Fei" } ], "thesis_advisor": [ { "family_name": "Dervan", "given_name": "Peter B.", "clpid": "Dervan-P-B" } ], "thesis_committee": [ { "family_name": "Dervan", "given_name": "Peter B.", "clpid": "Dervan-P-B" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" }, { "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": "Molecules that inhibit DNA dependent processes are the most commonly used agents for the treatment of cancer. The genotoxicity associated with their mechanisms of action, unfortunately, make them extremely toxic to the patient and cancer cells alike. The work presented in this thesis outlines the development of Py-Im polyamides as non-genotoxic DNA-targeted antitumor molecules that interfere with RNA polymerase II elongation. We initially characterized the pharmacokinetic profiles of two hairpin polyamides to establish their bioavailability in the serum and tissues after a single administration. We next determined the molecular mechanism that contributes to toxicity of a hairpin polyamide in human prostate cancer cells in cell culture and we demonstrated antitumor effects of the compound against LNCaP xenografts in mice. Finally, we conducted animal toxicity experiments on 4 polyamides that vary on the gamma-turn with respect to the substitution of amino and acetamide groups at the alpha and beta positions. From this study we identified a second generation compound that retains antitumor activity with significantly reduce animal toxicity. This work sets the foundation for the development of Py-Im polyamides as DNA targeted therapeutics for the treatment of advanced prostate cancer.", "doi": "10.7907/VCN2-H580", "publication_date": "2014", "thesis_type": "phd", "thesis_year": "2014" }, { "id": "thesis:7956", "collection": "thesis", "collection_id": "7956", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:09162013-140043647", "primary_object_url": { "basename": "Joshua Brown Thesis.pdf", "content": "final", "filesize": 7545983, "license": "other", "mime_type": "application/pdf", "url": "/7956/1/Joshua Brown Thesis.pdf", "version": "v9.0.0" }, "type": "thesis", "title": "Investigation of Receptors for the Modulation of Neuronal Growth by Chondroitin Sulfate", "author": [ { "family_name": "Brown", "given_name": "Joshua Micah", "clpid": "Brown-Joshua-Micah" } ], "thesis_advisor": [ { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" } ], "thesis_committee": [ { "family_name": "Chan", "given_name": "David C.", "clpid": "Chan-D-C" }, { "family_name": "Patterson", "given_name": "Paul H.", "clpid": "Patterson-P-H" }, { "family_name": "Clemons", "given_name": "William M.", "clpid": "Clemons-W-M" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "A major obstacle to neural regeneration after injury in the central nervous system (CNS) is the environment encountered by injured axons. This environment is inhibitory due to proteins expressed by the CNS myelin as well as molecules present in the glial scar. Experimental results have implicated chondroitin sulfate proteoglycans (CSPGs) as major inhibitors of axonal regeneration after CNS injury, but until recently, the mechanisms of this inhibition were not well understood. Furthermore, the complex nature of the chondroitin sulfate (CS) chains made it difficult to study their contribution to CSPG function. This thesis describes a specific carbohydrate epitope, CS-E, that is primarily responsible for the inhibition of CNS axonal regrowth in the presence of CSPGs. We show that removal or blocking of the CS-E motif via genetic elimination of the enzyme responsible for generating CS-E or a monoclonal antibody that binds specifically to the CS-E motif significantly reduces the inhibitory activity of CSPGs on axon growth. Furthermore, we show that CS-E functions as a protein recognition element to engage receptors, including the transmembrane protein tyrosine phosphatase PTP\u03c3, which had been previously established to be a receptor for CSPGs. Finally, we show that the protein tyrosine kinase receptor EphA4 is a novel receptor for the CS-E motif, and as with PTP\u03c3, neurons deficient in EphA4 exhibit reduced inhibition by CS-E. Our results demonstrate that a specific sugar epitope within chondroitin sulfate polysaccharides directs important physiological processes, and establish the importance of the chemical structure of CS chains in modulating the activity of CSPGs in vivo. The identification of receptors that mediate the inhibitory effect of CS-E advances our understanding of the mechanisms of axon regeneration following injury to the CNS when CS-E expression is upregulated. These findings provide us with the opportunity to develop therapies for the recovery of axonal outgrowth after damage to the nervous system, which in conjunction with blocking approaches targeting the CS motif, can provide a powerful strategy for allowing recovery after injury to the CNS.", "doi": "10.7907/Z90G3H45", "publication_date": "2014", "thesis_type": "phd", "thesis_year": "2014" }, { "id": "thesis:8034", "collection": "thesis", "collection_id": "8034", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:12022013-141841545", "primary_object_url": { "basename": "NavarroRaul_FullThesis_2014.pdf", "content": "final", "filesize": 16823427, "license": "other", "mime_type": "application/pdf", "url": "/8034/205/NavarroRaul_FullThesis_2014.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "New Strategies for the Total Synthesis of Aza-Propellane Natural Products", "author": [ { "family_name": "Navarro", "given_name": "Raul", "clpid": "Navarro-Raul" } ], "thesis_advisor": [ { "family_name": "Reisman", "given_name": "Sarah E.", "clpid": "Reisman-S-E" } ], "thesis_committee": [ { "family_name": "Stoltz", "given_name": "Brian M.", "clpid": "Stoltz-B-M" }, { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" }, { "family_name": "Reisman", "given_name": "Sarah E.", "clpid": "Reisman-S-E" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The propellane alkaloids comprise a large class of natural products that possess varying degrees of structural complexity and biological activity. The earliest of these to be isolated was acutumine, a chlorinated alkaloid that has been shown to exhibit selective T-cell cytotoxicity and antiamnesic properties. Alternatively, the hasubanan family of natural products has garnered considerable attention from the synthetic community in part due to its structural similarities to morphine. While these alkaloids have been the subject of numerous synthetic studies over the last forty years, very few enantioselective total syntheses have been reported to date.
\r\n\r\nAs part of a research program directed towards the synthesis of various alkaloid natural products, we have developed a unified strategy for the preparation of the hasubanan and acutumine alkaloids. Specifically, a highly diastereoselective 1,2-addition of organometallic reagents to benzoquinone-derived tert-butanesulfinimines was established, which provides access to enantioenriched 4-aminocyclohexadienone products. This methodology enabled the enantioselective construction of functionalized dihydroindolones, which were found to undergo intramolecular Friedel-Crafts conjugate additions to furnish the propellane cores of several hasubanan alkaloids. As a result of these studies, the first enantioselective total syntheses of 8-demethoxyrunanine and cepharatines A, C, and D were accomplished in 9-11 steps from commercially available starting materials.
\r\n\r\nMore recent efforts have focused on applying the sulfinimine methodology to the synthesis of a more structurally complex propellane alkaloid, acutumine. Extensive studies have determined that a properly functionalized dihydroindolone undergoes a photochemical [2+2] cycloaddition followed by a lactone fragmentation/Dieckmann cyclization to establish the carbocyclic framework of the natural product. The preparation of more appropriately oxidized propellane intermediates is currently under investigation, and is anticipated to facilitate our synthetic endeavors toward acutumine.
\r\n\r\n", "doi": "10.7907/XQ0M-NT53", "publication_date": "2014", "thesis_type": "phd", "thesis_year": "2014" }, { "id": "thesis:8170", "collection": "thesis", "collection_id": "8170", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:03252014-222127174", "primary_object_url": { "basename": "Yeoman-John-2014-FullThesis-Final.pdf", "content": "final", "filesize": 23014759, "license": "other", "mime_type": "application/pdf", "url": "/8170/44/Yeoman-John-2014-FullThesis-Final.pdf", "version": "v10.0.0" }, "type": "thesis", "title": "A Unified Strategy to Ent-Kauranoid Natural Products: Total Syntheses of (-)-Maoecrystal Z, (-)-Trichorabdal A, and (-)-Longikaurin E", "author": [ { "family_name": "Yeoman", "given_name": "John Thomas Schafer", "clpid": "Yeoman-John-Thomas-Schafer" } ], "thesis_advisor": [ { "family_name": "Reisman", "given_name": "Sarah E.", "clpid": "Reisman-S-E" } ], "thesis_committee": [ { "family_name": "Stoltz", "given_name": "Brian M.", "clpid": "Stoltz-B-M" }, { "family_name": "Reisman", "given_name": "Sarah E.", "clpid": "Reisman-S-E" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" }, { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The diterpenoid constituents of the Isodon plants have attracted reasearchers interested in both their chemical structures and biological properties for more than a half-century. In recent years, the isolations of new members displaying previously unprecedented ring systems and highly selective biological properties have piqued interest from the synthetic community in this class of natural products.
\r\n\r\nReported herein is the first total synthesis of such a recently isolated diterpenoid, (\u2013)-maoecrystal Z. The principal transformations implemented in this synthesis include two highly diastereoselective radical cyclization reactions: a Sm(II)-mediated reductive cascade cyclization, which forms two rings and establishes four new stereocenters in a single step, and a Ti(III)-mediated reductive epoxide-acrylate coupling that yields a functionalized spirolactone product, which forms a core bicycle of maoecrystal Z.
\r\n\r\nThe preparation of two additional ent-kauranoid natural products, (\u2013)-trichorabdal A and (\u2013)-longikaurin E, is also described from a derivative of this key spirolactone. These syntheses are additionally enabled by the palladium-mediated oxidative cyclization reaction of a silyl ketene acetal precursor that is used to install the bridgehead all-carbon quaternary stereocenter and bicyclo[3.2.1]octane present in each natural product. These studies have established a synthetic relationship among three architecturally distinct ent-kaurane diterpenoids and have forged a path for the preparation of interesting unnatural ent-kauranoid structural analogs for more thorough biological study.
", "doi": "10.7907/5TDN-7M11", "publication_date": "2014", "thesis_type": "phd", "thesis_year": "2014" }, { "id": "thesis:7843", "collection": "thesis", "collection_id": "7843", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06052013-123658997", "primary_object_url": { "basename": "Krishnamurthy_Chithra_2013_Thesis_full.pdf", "content": "final", "filesize": 4832972, "license": "other", "mime_type": "application/pdf", "url": "/7843/37/Krishnamurthy_Chithra_2013_Thesis_full.pdf", "version": "v10.0.0" }, "type": "thesis", "title": "Chemical Probes to Study Fucosylated Glycans", "author": [ { "family_name": "Krishnamurthy", "given_name": "Chithra", "clpid": "Krishnamurthy-Chithra" } ], "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": "Shan", "given_name": "Shu-ou", "clpid": "Shan-Shu-ou" }, { "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": "Fucosylated glycans have many critical biological roles, from leukocyte adhesion to host-microbe interactions. However, a molecular level understanding of these sugars has been lacking, in part due to the chemical and structural diversity of glycans that make them challenging to study. In order to gain a deeper understanding of fucosylated glycans, we have explored the use of chemical probes to study these structures. In Chapters 1 and 2, we apply a metabolic labeling technique for the investigation of fucosylated glycans in neurons, where they have been implicated in learning and memory processes. However, the molecular mechanisms by which these sugars influence neuronal processes are not well understood, and only a handful of fucosylated glycoproteins have been identified. In order to facilitate our understanding of these processes, we exploit non-natural fucose analogs to identify the fucose proteome in rat cortical neurons, identifying proteins involved in cell adhesion, neuronal signaling, and synaptic transmission. Moreover, we track fucosylated glycoproteins in hippocampal neurons, and show that fucosylated glycoproteins localize to the Golgi, axons, and dendrites, and are enriched in synapses. In Chapter 4, we report a new chemoenzymatic strategy for the sensitive detection of the Fuc\u03b1(1-2)Gal epitope, which has been implicated in tumorigenesis as a potential biomarker of cancer progression. We demonstrate that the approach is highly selective for the Fuc\u03b1(1-2)Gal motif, detects a variety of complex glycans and glycoproteins, and can be used to profile the relative abundance of the motif on live cells, discriminating malignant from normal cells. These approaches represent new potential applications and strategies for the investigation of fucosylated glycans, and expand the technologies available for understanding the roles of this important class of carbohydrates in physiology and disease.", "doi": "10.7907/Z99021SG", "publication_date": "2013", "thesis_type": "phd", "thesis_year": "2013" }, { "id": "thesis:7444", "collection": "thesis", "collection_id": "7444", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:01272013-150337805", "primary_object_url": { "basename": "medium.jpg", "content": "", "filesize": 6725, "license": "other", "mime_type": "image/png", "url": "/7444/1/medium.jpg", "version": "v2.0.0" }, "type": "thesis", "title": "Discovery of New Roles for Chondroitin Sulfate in Neurotrophin Signaling and Retinotopic Development", "author": [ { "family_name": "Rogers", "given_name": "Claude Joseph", "clpid": "Rogers-Claude-Joseph" } ], "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": "Shan", "given_name": "Shu-ou", "clpid": "Shan-Shu-ou" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Zinn", "given_name": "Kai George", "clpid": "Zinn-K-G" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Chondroitin sulfate (CS), a member of the glycosaminoglycan family of linear polysaccharides, is involved in the formation and maintenance of neuronal networks. CS has dual roles in regulating neuronal morphology: promoting or inhibiting neuronal outgrowth, depending on the context. A single sulfated epitope, CS-E, is capable of inducing both types of activity.
\r\n\r\nMembers of the neurotrophin (NT) family of growth factors are required for CS- E-induced neurite outgrowth in hippocampal neurons. Here, we demonstrate that CS is capable of forming ternary complexes with NTs and their receptors. These complexes were discovered using a novel, carbohydrate microarray-based approach that allows for the rapid screening of such interactions. To support these findings, we computationally determined the CS-E-binding site of the complexes, suggesting a structural basis for the interaction. In addition, we showed that CS-E is capable of attenuating NT signaling in cells, consistent with our computational and microarray data. This is the first demonstration that CS-E is involved in NT signaling and that CS is capable of supporting multimeric signaling complexes.
\r\n\r\nIn addition to stimulating growth factor signaling, CS has been known to repulsively guide retinal ganglion cell (RGC) axons for over twenty years. However, its function in vivo is unknown. RGCs are the only neuron type that transmits visual information to the brain, and their guidance, which maps a topographic projection of the retina to the superior colliculus (SC), is tightly regulated. Here, we show that CS-E is required for the proper formation of this topographic order. CS-E, but not the other major sulfation patterns, is a repellent guidance cue for RGC axons, with a graded activity profile from low to high along the dorsal-ventral axis of the retina, congruent with EphB3 expression. EphB3 binds specifically to CS-E with physiologically relevant affinity, and is required for CS-E-mediated guidance. CS-E-null mice have defects in topographic mapping in which ventral axons form ectopic termina- tions medial to their correct location in the SC. These results indicate that CS is a repulsive guidance cue required to map the dorsal-ventral axis of the retina along the lateral-medial axis of the SC. This is the first report of a non-protein topographical\r\nguidance cue.
", "doi": "10.7907/Z9N58JCD", "publication_date": "2013", "thesis_type": "phd", "thesis_year": "2013" }, { "id": "thesis:7204", "collection": "thesis", "collection_id": "7204", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:09132012-234333515", "primary_object_url": { "basename": "dropbox.cgi_get=1&key=6e0eff88ef58320721332530c754ba2c", "content": "final", "filesize": 8549662, "license": "other", "mime_type": "application/pdf", "url": "/7204/1/dropbox.cgi_get=1&key=6e0eff88ef58320721332530c754ba2c", "version": "v4.0.0" }, "type": "thesis", "title": "Applications of Computational Protein Design to Red Fluorescent Proteins", "author": [ { "family_name": "Moore", "given_name": "Matthew Michaels", "clpid": "Moore-Matthew-Michaels" } ], "thesis_advisor": [ { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" } ], "thesis_committee": [ { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "orcid": "0000-0001-5661-1714", "clpid": "Hsieh-Wilson-L-C" }, { "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": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The research comprising this thesis is presented in three chapters divided into two parts. Part One, Chapters 2 and 3, of the thesis focuses on the application of rational design and CPD to the core residues of FPs. Chapter 2 applies CPD to the well known red fluorescent protein mCherry. Design hypotheses in this work were driven by a desire to red-shift the fluorescence emission of the parent protein. Chapter 3 takes the most successful results from the mCherry system and attempts to applies these results to the far-red FP mPlum. These two proteins, mCherry and mPlum, share a directed evolution parent, mRFP1. Part Two, consisting of Chapter 4, presents the beginnings of a comprehensive study into the applications CPD for designing FP surfaces. The system used is based on DsRed, the oligomeric parent of monomer mCherry. Professional and personal acknowledgments conclude the thesis.", "doi": "10.7907/6876-A570", "publication_date": "2013", "thesis_type": "phd", "thesis_year": "2013" }, { "id": "thesis:7818", "collection": "thesis", "collection_id": "7818", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06022013-204012683", "primary_object_url": { "basename": "Oh_YoungIn_2013_Thesis.pdf", "content": "final", "filesize": 5502870, "license": "other", "mime_type": "application/pdf", "url": "/7818/1/Oh_YoungIn_2013_Thesis.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Synthesis and Biological Activity of Anticoagulant Heparan Sulfate Glycopolymers ", "author": [ { "family_name": "Oh", "given_name": "Young In", "clpid": "Oh-Young-In" } ], "thesis_advisor": [ { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" } ], "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": "Clemons", "given_name": "William M.", "clpid": "Clemons-W-M" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Heparin has been used as an anticoagulant drug for more than 70 years. The global distribution of contaminated heparin in 2007, which resulted in adverse clinical effects and over 100 deaths, emphasizes the necessity for safer alternatives to animal-sourced heparin. The structural complexity and heterogeneity of animal-sourced heparin not only impedes safe access to these biologically active molecules, but also hinders investigations on the significance of structural constituents at a molecular level. Efficient methods for preparing new synthetic heparins with targeted biological activity are necessary not only to ensure clinical safety, but to optimize derivative design to minimize potential side effects. Low molecular weight heparins have become a reliable alternative to heparin, due to their predictable dosages, long half-lives, and reduced side effects. However, heparin oligosaccharide synthesis is a challenging endeavor due to the necessity for complex protecting group manipulation and stereoselective glycosidic linkage chemistry, which often result in lengthy synthetic routes and low yields. Recently, chemoenzymatic syntheses have produced targeted ultralow molecular weight heparins with high-efficiency, but continue to be restricted by the substrate specificities of enzymes.
\r\n\r\nTo address the need for access to homogeneous, complex glycosaminoglycan structures, we have synthesized novel heparan sulfate glycopolymers with well-defined carbohydrate structures and tunable chain length through ring-opening metathesis polymerization chemistry. These polymers recapitulate the key features of anticoagulant heparan sulfate by displaying the sulfation pattern responsible for heparin\u2019s anticoagulant activity. The use of polymerization chemistry greatly simplifies the synthesis of complex glycosaminoglycan structures, providing a facile method to generate homogeneous macromolecules with tunable biological and chemical properties. Through the use of in vitro chromogenic substrate assays and ex vivo clotting assays, we found that the HS glycopolymers exhibited anticoagulant activity in a sulfation pattern and length-dependent manner. Compared to heparin standards, our short polymers did not display any activity. However, our longer polymers were able to incorporate in vitro and ex vivo characteristics of both low-molecular-weight heparin derivatives and heparin, displaying hybrid anticoagulant properties. These studies emphasize the significance of sulfation pattern specificity in specific carbohydrate-protein interactions, and demonstrate the effectiveness of multivalent molecules in recapitulating the activity of natural polysaccharides.
", "doi": "10.7907/E16S-6T52", "publication_date": "2013", "thesis_type": "phd", "thesis_year": "2013" }, { "id": "thesis:7816", "collection": "thesis", "collection_id": "7816", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06012013-114632816", "primary_object_url": { "basename": "Wibowo_Thesis_2015_FinalEdit.pdf", "content": "final", "filesize": 2961019, "license": "other", "mime_type": "application/pdf", "url": "/7816/38/Wibowo_Thesis_2015_FinalEdit.pdf", "version": "v7.0.0" }, "type": "thesis", "title": "Identification of Fucose-\u03b1(1-2)-Galactose Binding Proteins in the Mammalian Brain", "author": [ { "family_name": "Wibowo", "given_name": "Arif", "clpid": "Wibowo-Arif" } ], "thesis_advisor": [ { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" } ], "thesis_committee": [ { "family_name": "Dervan", "given_name": "Peter B.", "clpid": "Dervan-P-B" }, { "family_name": "Beauchamp", "given_name": "Jesse L.", "clpid": "Beauchamp-J-L" }, { "family_name": "Grubbs", "given_name": "Robert H.", "clpid": "Grubbs-R-H" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Fucose-\u03b1(1-2)-galactose (Fuc\u03b1(1-2)Gal) carbohydrates have been implicated in cognitive functions. However, the underlying molecular mechanisms that govern these processes are not well understood. While significant progress has been made toward identifying glycoconjugates bearing this carbohydrate epitope, a major challenge remains the discovery of interactions mediated by these sugars. Here, we employ the use of multivalent glycopolymers to enable the proteomic identification of weak affinity, low abundant Fuc\u03b1(1-2)Gal-binding proteins (i.e. lectins) from the brain. End-biotinylated glycopolymers containing photoactivatable crosslinkers were used to capture and enrich potential Fuc\u03b1(1-2)Gal-specific lectins from rat brain lysates. Candidate lectins were tested for their ability to bind Fuc\u03b1(1-2)Gal, and the functional significance of the interaction was investigated for one such candidate, SV2a, using a knock-out mouse system. Our results suggest an important role for this glycan-lectin interaction in facilitating synaptic changes necessary for neuronal communication. This study highlights the use of glycopolymer mimetics to discover novel lectins and identify functional interactions between fucosyl carbohydrates and lectins in the brain.", "doi": "10.7907/ETC4-N802", "publication_date": "2013", "thesis_type": "phd", "thesis_year": "2013" }, { "id": "thesis:7883", "collection": "thesis", "collection_id": "7883", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06102013-121027424", "primary_object_url": { "basename": "Bennett-N-BThesis.pdf", "content": "final", "filesize": 118366833, "license": "other", "mime_type": "application/pdf", "url": "/7883/79/Bennett-N-BThesis.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "Palladium-Catalyzed Asymmetric Allylic Alkylation: Insights, Application Toward Cyclopentanoid and Cycloheptanoid Molecules, and the Total Synthesis of Several Daucane Sesquiterpenes ", "author": [ { "family_name": "Bennett", "given_name": "Nathan Bruce", "clpid": "Bennett-Nathan-Bruce" } ], "thesis_advisor": [ { "family_name": "Stoltz", "given_name": "Brian M.", "clpid": "Stoltz-B-M" } ], "thesis_committee": [ { "family_name": "Reisman", "given_name": "Sarah E.", "clpid": "Reisman-S-E" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" }, { "family_name": "Fu", "given_name": "Gregory C.", "clpid": "Fu-G-C" }, { "family_name": "Virgil", "given_name": "Scott C.", "clpid": "Virgil-S-C" }, { "family_name": "Stoltz", "given_name": "Brian M.", "clpid": "Stoltz-B-M" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The asymmetric construction of quaternary stereocenters is a topic of great interest in the organic chemistry community given their prevalence in natural products and biologically active molecules. Over the last decade, the Stoltz group has pursued the synthesis of this challenging motif via a palladium-catalyzed allylic alkylation using chiral phosphinooxazoline (PHOX) ligands. Recent results indicate that the alkylation of lactams and imides consistently proceeds with enantioselectivities substantially higher than any other substrate class previously examined in this system. This observation prompted exploration of the characteristics that distinguish these molecules as superior alkylation substrates, resulting in newfound insights and marked improvements in the allylic alkylation of carbocyclic compounds.
\r\n\r\nGeneral routes to cyclopentanoid and cycloheptanoid core structures have been developed that incorporate the palladium-catalyzed allylic alkylation as a key transformation. The unique reactivity of \u03b1-quaternary vinylogous esters upon addition of hydride or organometallic reagents enables divergent access to \u03b3-quaternary acylcyclopentenes or cycloheptenones through respective ring contraction or carbonyl transposition pathways. Derivatization of the resulting molecules provides a series of mono-, bi-, and tricyclic systems that can serve as valuable intermediates for the total synthesis of complex natural products.
\r\n\r\nThe allylic alkylation and ring contraction methodology has been employed to prepare variably functionalized bicyclo[5.3.0]decane molecules and enables the enantioselective total syntheses of daucene, daucenal, epoxydaucenal B, and 14-p-anisoyloxydauc-4,8-diene. This route overcomes the challenge of accessing \u03b2-substituted acylcyclopentenes by employing a siloxyenone to effect the Grignard addition and ring opening in a single step. Subsequent ring-closing metathesis and aldol reactions form the hydroazulene core of these targets. Derivatization of a key enone intermediate allows access to either the daucane sesquiterpene or sphenobolane diterpene carbon skeletons, as well as other oxygenated scaffolds.
\r\n", "doi": "10.7907/ZP3K-A474", "publication_date": "2013", "thesis_type": "phd", "thesis_year": "2013" }, { "id": "thesis:7845", "collection": "thesis", "collection_id": "7845", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:06052013-151748060", "primary_object_url": { "basename": "Nguyen_Thang_2013_Thesis.pdf", "content": "final", "filesize": 11272797, "license": "other", "mime_type": "application/pdf", "url": "/7845/1/Nguyen_Thang_2013_Thesis.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "A Study of Protein Targeting Reveals Insights into Mitigating Protein Aggregation", "author": [ { "family_name": "Nguyen", "given_name": "Thang Xuan", "clpid": "Nguyen-Thang-Xuan" } ], "thesis_advisor": [ { "family_name": "Shan", "given_name": "Shu-ou", "clpid": "Shan-Shu-ou" } ], "thesis_committee": [ { "family_name": "Clemons", "given_name": "William M.", "clpid": "Clemons-W-M" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" }, { "family_name": "Chan", "given_name": "David C.", "clpid": "Chan-D-C" }, { "family_name": "Shan", "given_name": "Shu-ou", "clpid": "Shan-Shu-ou" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "A unique chloroplast Signal Recognition Particle (SRP) in green plants is primarily dedicated to the post-translational targeting of light harvesting chlorophyll-a/b binding (LHC) proteins. Our study of the thermodynamics and kinetics of the GTPases of the system demonstrates that GTPase complex assembly and activation are highly coupled in the chloroplast GTPases, suggesting they may forego the GTPase activation step as a key regulatory point. This reflects adaptations of the chloroplast SRP to the delivery of their unique substrate protein. Devotion to one highly hydrophobic family of proteins also may have allowed the chloroplast SRP system to evolve an efficient chaperone in the cpSRP43 subunit. To understand the mechanism of disaggregation, we showed that LHC proteins form micellar, disc-shaped aggregates that present a recognition motif (L18) on the aggregate surface. Further molecular genetic and structure-activity analyses reveal that the action of cpSRP43 can be dissected into two steps: (i) initial recognition of L18 on the aggregate surface; and (ii) aggregate remodeling, during which highly adaptable binding interactions of cpSRP43 with hydrophobic transmembrane domains of the substrate protein compete with the packing interactions within the aggregate. We also tested the adaptability of cpSRP43 for alternative substrates, specifically in attempts to improve membrane protein expression and inhibition of amyloid beta fibrillization. These preliminary results attest to cpSRP43\u2019s potential as a molecular chaperone and provides the impetus for further engineering endeavors to address problems that stem from protein aggregation.", "doi": "10.7907/JA4R-TE24", "publication_date": "2013", "thesis_type": "phd", "thesis_year": "2013" }, { "id": "thesis:6859", "collection": "thesis", "collection_id": "6859", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:03212012-034245395", "primary_object_url": { "basename": "01_-_Intro_-_FINAL.pdf", "content": "final", "filesize": 334443, "license": "other", "mime_type": "application/pdf", "url": "/6859/1/01_-_Intro_-_FINAL.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Aryne Annulation Reactions Toward the Synthesis of Heterocyclic Molecules", "author": [ { "family_name": "Gilmore", "given_name": "Christopher Dennis", "clpid": "Gilmore-Christopher-Dennis" } ], "thesis_advisor": [ { "family_name": "Stoltz", "given_name": "Brian M.", "clpid": "Stoltz-B-M" } ], "thesis_committee": [ { "family_name": "Bercaw", "given_name": "John E.", "clpid": "Bercaw-J-E" }, { "family_name": "Dervan", "given_name": "Peter B.", "clpid": "Dervan-P-B" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" }, { "family_name": "Stoltz", "given_name": "Brian M.", "clpid": "Stoltz-B-M" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The last decade has seen an outgrowth in the development of synthetic methodologies exploiting benzyne. The unique ability of this reactive intermediate to directly furnish ortho-difuntionalized aromatic systems first stoked interest in this research group as a possible partner in asymmetric arylation reactions. Since our initial forays, we have expanded our synthetic strategies to include bond insertions, cycloadditions, condensations, and multicomponent reactions. The first project discussed in this volume is the development of an aryne annulation strategy for constructing common, synthetically useful heterocyclic structures in a convergent manner. We have developed a convergent approach to indoles and indolines. Likewise, through an orthogonal functional group intallation upon an enamine substrate, isoquinolines, quinolines, and isoquinolones can all be accessed as well. In this manner, we have been able to generate an array of functionalized heterocycles, including some that are prohibited by traditional means of synthesis. We have also begun to understand some of the reactivity trends in this context for the elusive aryne reaction partner. The development of the aryne annulation strategy for the synthesis of isoquinolines directly led to the shortest reported total synthesis of the opiate alkaloid papaverine, and the tetrahydroisoquinoline anticancer antibiotic quinocarcin. Our more recent, ongoing efforts toward the synthesis of the bis-tetrahydroisoquinoline antitumor molecule jorumycin and its many structural relatives are detailed herein. Jorumycin has been targeted through a combination of aryne annulation and acyl-alkylation/condensation methodologies aimed at the synthesis of a functionalized bis-isoquinoline intermediate. Reduction of this key bis-isoquinoline to a bis-tetrahydroisoquinoline and subsequent lactamization will provide the pentacyclic core of jorumycin and related natural products in only three steps from simple isoquinoline building blocks. The final project described is the development of several different aryne multicomponent reactions to form novel carbo- and heterocyclic scaffolds, including iminoisobenzfurans, iminoindenones, dibenzoketocaprolactams, and 2-quinolones.\r\n", "doi": "10.7907/C8F7-DQ34", "publication_date": "2012", "thesis_type": "phd", "thesis_year": "2012" }, { "id": "thesis:6204", "collection": "thesis", "collection_id": "6204", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:12142010-165109760", "primary_object_url": { "basename": "Muzikar_Thesis.pdf", "content": "final", "filesize": 10132799, "license": "other", "mime_type": "application/pdf", "url": "/6204/8/Muzikar_Thesis.pdf", "version": "v7.0.0" }, "type": "thesis", "title": "Repression of DNA-Binding-Dependent Glucocorticoid Receptor-Mediated Gene Expression", "author": [ { "family_name": "Muzikar", "given_name": "Katy Ann", "clpid": "Muzikar-Katy-Ann" } ], "thesis_advisor": [ { "family_name": "Dervan", "given_name": "Peter B.", "clpid": "Dervan-P-B" } ], "thesis_committee": [ { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" }, { "family_name": "Dervan", "given_name": "Peter B.", "clpid": "Dervan-P-B" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Gene expression is controlled by transcription factors that regulate the rates at which genes are expressed either by recruiting or inhibiting protein complexes that bind to the promoters or enhancers of target genes. Molecules that can specifically modulate these protein-DNA interfaces show promise as tools for understanding gene regulation pathways and may have application in human medicine. Hairpin pyrrole-imidazole polyamides are programmable oligomers that bind the DNA minor groove in a sequence-specific manner with affinities comparable to those of natural DNA-binding proteins. These cell-permeable small molecules have been shown to enter the nuclei of live cells, disrupt protein-DNA interactions, and downregulate endogenous gene expression. This thesis describes the use of polyamides to modulate gene expression in order to probe gene regulation mechanisms of several different biologically relevant systems. A polyamide is designed to target the glucocorticoid receptor transcription factor DNA binding site located in the promoter of the glucocorticoid-induced leucine zipper gene. This polyamide is shown to bind with high affinity to the promoter sequence, modulate the expression of this gene, and disrupt the binding of the protein to the gene\u2019s promoter. Examination of the global effects of this polyamide on mRNA transcription is used to elucidate a list of genes that are regulated by a glucocorticoid receptor protein-DNA dependent mechanism. Also in this thesis, the specificities of a Cy3-labeled polyamide known to downregulate expression of the Vascular Endothelial Growth Factor is examined using DNA microarrays composed of hairpins harboring all 524,800 unique 10 base pair DNA sequences. We experimentally verify the correlation of Cy3 fluorescence intensity with quantitative DNase I footprint-derived binding affinities. Additionally, progress is made towards the polyamide-mediated inhibition of Myc/Max transcription factor gene regulation. ", "doi": "10.7907/FKCV-KP50", "publication_date": "2011", "thesis_type": "phd", "thesis_year": "2011" }, { "id": "thesis:6223", "collection": "thesis", "collection_id": "6223", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:01132011-095304695", "primary_object_url": { "basename": "011611_Complete_Thesis.pdf", "content": "final", "filesize": 29546795, "license": "other", "mime_type": "application/pdf", "url": "/6223/55/011611_Complete_Thesis.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "New Tools for Studying O-G1cNAc Glycosylation and Chondroitin Sulfate Proteoglycans and Studies on the Roles of O-G1cNAc Glycosylation on the Transcription Factor CREB", "author": [ { "family_name": "Clark", "given_name": "Peter Michael", "clpid": "Clark-Peter-Michael" } ], "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": "Dervan", "given_name": "Peter B.", "clpid": "Dervan-P-B" }, { "family_name": "Kennedy", "given_name": "Mary B.", "clpid": "Kennedy-M-B" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The addition and removal of the monosaccharide N-acetyl-D-glucosamine (GlcNAc) to serine and threonine residues of proteins has emerged as a critical regulator of cellular processes. However, studies of O-GlcNAc in such complex systems as the brain have been limited, in part due to the lack of tools. Here we report the development of new tools for studying O-GlcNAc, and the application of these and other tools for studying the roles of O-GlcNAc in the brain.
\r\n\r\nWorking from a previously established chemoenzymatic method, we designed an isotopic labeling strategy for probing the dynamics of O-GlcNAc glycosylation using quantitative proteomics. With this tool, we show that O-GlcNAc is dynamically modulated on specific proteins by excitatory stimulation of the brain in vivo. Separately, we improved this chemoenzymatic strategy by integrating [3+2] azide-alkyne cycloaddition chemistry to attach biotin and fluorescent tags to O-GlcNAc residues. These tags allow for the direct fluorescence detection, proteomic analysis, and cellular imaging of O-GlcNAc modified proteins. With this strategy, we identified over 146 novel glycoproteins from the mammalian brain.
\r\n\r\nThe transcription factor cAMP-response element binding protein (CREB) is critical for numerous functions in the brain, including neuronal survival, neuronal development, synaptic plasticity, and long-term memory. We show that CREB is highly glycosylated in the brain and discover new glycosylation sites on CREB in neurons. One of these sites is dynamically modulated and is important for regulating CREB. Removal of this glycosylation site alters CREB-mediated functions in vitro and in vivo. These studies are the first demonstration that O-glycosylation at a specific site on a specific protein is critical for neuronal function and behavior.
\r\n\r\nChondroitin sulfates (CS) are sulfated linear polysaccharides important in neuronal development and viral invasion. Depending on their sulfation patterns, CS molecules differ dramatically in their functions. We developed a computational method to model the structure and function of CS. Using this approach, we show that different CS tetrasaccharides have distinct solution structures. We also modeled the CS binding site on a variety of proteins and discovered that CS may be important in modulating protein-protein interactions.
", "doi": "10.7907/FTJS-8M82", "publication_date": "2011", "thesis_type": "phd", "thesis_year": "2011" }, { "id": "thesis:5059", "collection": "thesis", "collection_id": "5059", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-12182008-145714", "primary_object_url": { "basename": "Final_thesis_whole.pdf", "content": "final", "filesize": 9685569, "license": "other", "mime_type": "application/pdf", "url": "/5059/9/Final_thesis_whole.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Identification and Characterization of the Plasticity-Relevant Fucose-\u03b1(1-2) Galactose Glycoproteome from Mouse Brain", "author": [ { "family_name": "Murrey", "given_name": "Heather Elizabeth", "clpid": "Murrey-Heather-Elizabeth" } ], "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": "Chan", "given_name": "David C.", "clpid": "Chan-D-C" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" }, { "family_name": "Patterson", "given_name": "Paul H.", "clpid": "Patterson-P-H" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Fuc\u03b1(1-2)Gal carbohydrates have been implicated in cognitive processes such as learning and memory. However, a molecular level understanding of their functions has been lacking. This thesis describes multiple chemical and biological approaches that we have undertaken to elucidate the molecular mechanisms by which fucosyl sugars mediate neuronal communication. We demonstrate that Fuc\u03b1(1-2)Gal carbohydrates play an important role in the regulation of synaptic proteins and neuronal morphology. We identify synapsins Ia and Ib as prominent Fuc\u03b1(1-2)Gal glycoproteins in rat hippocampus, and fucosylation protects synapsin I from proteolytic degradation by the calcium-activated protease calpain. Synapsin fucosylation has important consequences on neuronal growth and morphology, with defucosylation leading to stunted neurites and delayed synapse formation. In addition, we identify the Fuc\u03b1(1-2)Gal proteome from mouse olfactory bulb using lectin affinity chromatography. We discover four major classes of Fuc\u03b1(1-2)Gal glycoproteins, including the immunoglobulin superfamily of cell adhesion molecules, ion channels and solute carriers/transporters, ATP-binding proteins, and synaptic vesicle-associated proteins. Protein fucosylation is regulated by FUT1 in mouse olfactory bulb, and olfactory bulb development is impaired in FUT1-deficient mice. In particular, FUT1 KO animals exhibit defects in the olfactory nerve and glomerular layers of olfactory sensory neurons expressing the fucosylated cell adhesion molecules NCAM and OCAM. Lastly, we explore the molecular mechanisms of protein fucosylation by metabolic labeling with alkynyl- and azido-fucose derivatives. We demonstrate that fucosylated glycoconjugates are present along both axons and dendrites of developing neuronal cultures, as well as in the Golgi body. We identify the fucosylated proteome from cultured cortical neurons, and demonstrate that proteins such as NCAM, the MARCKS family of proteins, and the inositol 1,4,5 triphosphate receptor are fucosylated. In addition, we can label fucosylated glycans in vivo, which will have important consequences for studies on the dynamics of protein fucosylation in living animals. Cumulatively, our studies suggest important functional roles for fucosyl-carbohydrates in the nervous system, and implicate an extended role for fucose in the molecular mechanisms that may underlie synaptic plasticity and neuronal development.\r\n", "doi": "10.7907/QG38-9P18", "publication_date": "2009", "thesis_type": "phd", "thesis_year": "2009" }, { "id": "thesis:5196", "collection": "thesis", "collection_id": "5196", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05232009-204358", "primary_object_url": { "basename": "00CompleteThesis.pdf", "content": "final", "filesize": 23112891, "license": "other", "mime_type": "", "url": "/5196/1/00CompleteThesis.pdf", "version": "v12.0.0" }, "type": "thesis", "title": "Discovery of Aminoacyl-tRNA Synthetase Mutants for the Incorporation of Non Canonical Amino Acids into Proteins", "author": [ { "family_name": "Tanrikulu", "given_name": "Ismet \u00c7ag\u02d8ler", "orcid": "0000-0002-7165-0399", "clpid": "Tanrikulu-Ismet-\u00c7ag\u02d8ler" } ], "thesis_advisor": [ { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" }, { "family_name": "Goddard", "given_name": "William A., III", "clpid": "Goddard-W-A-III" } ], "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": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" }, { "family_name": "Goddard", "given_name": "William A., III", "clpid": "Goddard-W-A-III" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Efficient in vivo incorporation of a noncanonical amino acid into proteins often requires engineering new aminoacyl-tRNA synthetase (AARS) activity into the cell, usually by modifying a natural aaRS. Although experimental methods, relying on mutagenesis and library screening, have identified many successful mutant aaRS-substrate pairs in the recent years, computational approaches have reported only a few successes. Here we compare the results of computational and experimental screens of an E. coli methionyl-tRNA synthetase (MetRS) saturation-mutagenesis library for binding (in silico), and activation and cell-surface display (in vivo) of azidonorleucine (Anl).
\r\n\r\nThree positions (L13, Y260, and H301) in the methionine binding pocket of E. coli MetRS were randomized, and the resulting library was screened for MetRS activity toward Anl, based on a screening strategy previously established in our group. This strategy relies on the introduction of reactive side chains into surface-exposed sites on outer-membrane protein C (OmpC), and their subsequent labeling with reactive, fluorescent probes. We have discovered a large diversity of MetRS mutants that allow the incorporation of Anl into proteins in vivo. The extent of OmpC expression and the amount of available Anl during the screen have substantial effects on the outcome of the screens. In addition to displaying improved activities toward Anl, identified mutants also show an improved discrimination against Met. We have shown that the degree of cell-surface labeling in vivo correlates well with the measured rates of Anl activation in vitro, which reflects the success of the screen design.
\r\n\r\nComputational analysis of the experimentally identified mutants revealed a good agreement between computed binding energies and in vitro activation data. To better test the computation model, we performed an in silico screen for Anl binding on a saturation-mutagenesis library comparable to the experimental library. Computational screen predominantly selects mutants that interact with Anl through hydrogen bonds, whereas the hydrophobic residues are selected more often by the experimental screen. We identify that experimental mutants try to optimize packing at the Y260 and H301 sites, but not at the L13 site. We discuss possible explanations for these results. Combined results from computation and experiments suggest the importance of various factors in ligand recognition and in vivo selection of MetRS mutants. We explore the implications of these factors to the future efforts in the engineering of new MetRS activities.
\r\n", "doi": "10.7907/E7D5-HN96", "publication_date": "2009", "thesis_type": "phd", "thesis_year": "2009" }, { "id": "thesis:2222", "collection": "thesis", "collection_id": "2222", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05282009-131419", "primary_object_url": { "basename": "main.pdf", "content": "final", "filesize": 4341788, "license": "other", "mime_type": "application/pdf", "url": "/2222/1/main.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "A Monte Carlo-Based Torsion Construction Algorithm for Ligand Design", "author": [ { "family_name": "Kekenes-Huskey", "given_name": "Peter Michael", "orcid": "0000-0001-7286-3022", "clpid": "Kekenes-Huskey-Peter-Michael" } ], "thesis_advisor": [ { "family_name": "Goddard", "given_name": "William A., III", "clpid": "Goddard-W-A-III" } ], "thesis_committee": [ { "family_name": "Heath", "given_name": "James R.", "clpid": "Heath-J-R" }, { "family_name": "Goddard", "given_name": "William A., III", "clpid": "Goddard-W-A-III" }, { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "A wealth of computational strategies [1,2,3,4,] is available for predicting the binding site and affinities of a putative ligand inside a target receptor. Although numerous techniques focus on the orientation of ligands or fragments thereof, few methods have delved into improving the accuracy of generating reliable ligand conformations within predicted binding modes. In an effort to comprehensively sample the torsion space available to a flexible ligand and focus on low-energy conformations, a recursive, Metropolis Monte Carlo (MC)-based rotamer design protocol has been developed. This approach recursively samples adjacent rotatable bonds from a defined anchor and directs the search along low-energy pathways, such that high-affinity conformations of the ligand can be identified. Furthermore, this program applies spatial constraints within the search that restrict the solutions to structurally dissimilar conformations, thus encouraging a diverse solution set. The performance of moleculeGL has been evaluated for a set of 55 co-crystals, for which the number of rotatable bonds ranged from 2 to 32. Approximately 80 percent of the structures are predicted within 2.0 A2 root mean square deviations (RMSD) with respect to the crystal structure, starting from an arbitrary ligand conformation. This level of accuracy suggests the program's applicability to the design of pharmacaphore substituents, for which the position of a chemically active pharmacaphore is well-known.", "doi": "10.7907/A1MQ-3116", "publication_date": "2009", "thesis_type": "phd", "thesis_year": "2009" }, { "id": "thesis:1255", "collection": "thesis", "collection_id": "1255", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-04022009-144521", "primary_object_url": { "basename": "bolin_thesis_final.pdf", "content": "final", "filesize": 6046031, "license": "other", "mime_type": "application/pdf", "url": "/1255/1/bolin_thesis_final.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "A Combined Experimental and Computational Study of Ligand Effects on C-H Bond Activation by Palladium and Platinum Complexes", "author": [ { "family_name": "Lin", "given_name": "Bo-Lin", "clpid": "Lin-Bo-Lin" } ], "thesis_advisor": [ { "family_name": "Bercaw", "given_name": "John E.", "clpid": "Bercaw-J-E" } ], "thesis_committee": [ { "family_name": "Grubbs", "given_name": "Robert H.", "clpid": "Grubbs-R-H" }, { "family_name": "Bercaw", "given_name": "John E.", "clpid": "Bercaw-J-E" }, { "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": "Abnormally large kinetic hydrogen/deuterium isotope effects (KIEs, ~ 20) are measured for the protonolysis of several dimethylpalladium(II) complexes with various bidentate ligands by trifluoroethanol (TFE) at room temperature. Analyses of semiclassical KIE theory suggest that the occurrence of hydrogen tunneling needs to be invoked in order to explain these KIE values, which is further supported by the KIE-temperature-dependence study for the protonolysis of (dppe)Pd(CH\u2083)\u2082 by CF\u2083CD\u2082OD/CF\u2083CH\u2082OH.
\r\n\r\nDensity functional theory (DFT) computation suggests that protonation at the MII-C bond is kinetically preferred over protonation at the metal center for the protonolysis of (COD)Pt(CH\u2083)\u2082 by TFA and the dimethylpalladium(II) complexes by TFE in dichloroethane. The computation further indicates the significant contribution of hydrogen tunneling in the abnormally large KIEs observed experimentally.
\r\n\r\nThe monomethylpalladium(II) complex, (COD)Pd(CH\u2083)Cl (COD = 1,5-cyclooctadiene), undergoes both benzene C-H activation and migratory insertion of olefin, with the former faster than the latter, at room temperature under the assistance of an anionic \u03b2-diketiminate ligand, to yield \u03b7\u00b3-(2-R-cyclooctenyl)palladium(II) \u03b2-diketiminate (R = methyl or phenyl).
\r\n\r\nDFT computation result suggests that bisindolide-type ligands and carbenearyl-type ligands are likely to lead to faster benzene C-H bond activation as well as lower relative VIII barrier heights of the C-H bond activation versus the insertion of olefins than those in monomethyl palladium(II) with \u03b2-diketiminate.
\r\n\r\nSeveral pyridine-like ligands were found to improve Pd(OAc)\u2082-catalyzed allylic oxidation of allylbenzene to cinnamyl acetate by p-benzoquinone in acetic acid. The best ligand examined, bipyrimidine, was used to identify the catalyst precursor for this system, (bipyrimidine)Pd(OAc)\u2082, which was fully characterized. Mechanistic studies suggest the reaction takes place through disproportionation of (bipyrimidine)Pd(OAc)\u2082 to form a bipyrimidine-bridged dimer, which reacts with olefin to form a PdII-olefin adduct, followed by allylic C-H activation to produce (\u03b7\u00b3-allyl)PdII species. The (\u03b7\u00b3-allyl)PdII intermediate undergoes a reversible acetate attack to generate a Pd\u2070-(allyl acetate) adduct, which subsequently reacts with p-benzoquinone to release allyl acetate and regenerate (bipyrimidine)Pd(OAc)\u2082. No KIE is observed for the competition experiment between allylbenzene-d\u2080 and allylbenzene-d\u2085(CD\u2082=CD-CD\u2082-C\u2086H\u2085), suggesting that allylic C-H activation is not rate determining. Catalytic allylic acetoxylations of other terminal olefins as well as cyclohexene were also effected by (bipyrimidine)Pd(OAc)\u2082.
\r\n", "doi": "10.7907/PD2Z-QZ56", "publication_date": "2009", "thesis_type": "phd", "thesis_year": "2009" }, { "id": "thesis:1536", "collection": "thesis", "collection_id": "1536", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-04282009-211225", "primary_object_url": { "basename": "00CompleteThesis.pdf", "content": "final", "filesize": 21277358, "license": "other", "mime_type": "application/pdf", "url": "/1536/1/00CompleteThesis.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Synthesis and Structural Studies of Cyclic Py-Im Polyamides", "author": [ { "family_name": "Chenoweth", "given_name": "David Michael", "orcid": "0000-0002-0819-4669", "clpid": "Chenoweth-David-Michael" } ], "thesis_advisor": [ { "family_name": "Dervan", "given_name": "Peter B.", "clpid": "Dervan-P-B" } ], "thesis_committee": [ { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" }, { "family_name": "Stoltz", "given_name": "Brian M.", "clpid": "Stoltz-B-M" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Dervan", "given_name": "Peter B.", "clpid": "Dervan-P-B" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The work presented in this thesis is focused on the molecular recognition of DNA by minor groove binding polyamides. Methods and strategies for the solution-phase synthesis of hairpin and cyclic pyrrole-imidazole polyamides are presented with optimized protocols requiring little to no chromatography. These synthetic strategies have led to the design of cyclic polyamides targeted to the androgen response element and are shown to be biologically active and cell permeable in cell culture experiments in addition their binding affinities rival that of most polyamide architectures. The structural elucidation of an \u03b1-amino-turn-linked cyclic polyamide is presented at 1.17 \u00c5 resolution providing insight into the detailed molecular recognition process and allosteric modulation responsible for the inhibition of transcription factor-DNA binding. Additionally, structural elucidation of a \u03b2-amino-turn-linked cyclic polyamide, highlighting the conformational differences compared to the \u03b1-amino-turn linked structure is presented. A structural basis for the inability of polyamides to bind dsRNA is also proposed based on biophysical, structural, and modeling data. In addition to these studies a new class of programmable oligomers targeting the DNA sequence 5\u2019-WGGGGW-3\u2019 were shown to inhibit DNA binding of the Nf-kB transcription factor by EMSA gel shift. Compounds synthesized in this study were found to possess unique fluorescent properties with the ability to modulate their fluorescence by binding their targeted dsDNA, leading to sequence specific fluorescent detection reagents. Efforts toward the templated-assembly of polyamides using higher-order DNA structure (NCP) are also reported and the development of a new pro-fluorescent class of heterocycle, which has the potential to be used as a chemical reporter of ligation events is described.\r\n", "doi": "10.7907/PZEC-VA33", "publication_date": "2009", "thesis_type": "phd", "thesis_year": "2009" }, { "id": "thesis:42", "collection": "thesis", "collection_id": "42", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-01062009-133603", "primary_object_url": { "basename": "ALE_Thesis.pdf", "content": "final", "filesize": 25714013, "license": "other", "mime_type": "application/pdf", "url": "/42/1/ALE_Thesis.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Investigating Structure-Function Relationships in Ion Channels Using Unnatural Amino Acids", "author": [ { "family_name": "Eastwood", "given_name": "Amy Lynn", "clpid": "Eastwood-Amy-Lynn" } ], "thesis_advisor": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" }, { "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": "Dervan", "given_name": "Peter B.", "clpid": "Dervan-P-B" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Ion channels are proteins that traverse the cell membrane and form gated pores that open and close in response to various stimuli. In order to experimentally probe aspects of ion channel functionality, we performed subtle structure function studies using the in vivo nonsense suppression method, which allows for the incorporation of synthetically accessible unnatural amino acids and hydroxy acids into an ion channel at a site of interest.
\r\n\r\nFluorinated aromatic amino acids are good probes for a cation-\u03c0 interaction because fluorine substituents reduce the binding affinity of the aromatic for a cation in a linear, step-wise fashion. In collaboration with Professor Richard Horn at the Thomas Jefferson University, we substituted a series of fluorinated phenylalanines for important tyrosines in the Shaker B K\u207a channel and experimentally determined that TEA was binding to the residues through a cation-\u03c0 interaction. We also determined that Ca\u00b2\u207a binds to and blocks the NaV1.4 channel through a cation-\u03c0 interaction with a tyrosine at the top of the pore of this channel. We found that tetrodotoxin, another channel blocker, also binds to this same residue through a cation-\u03c0 interaction. Finally, we proved that lidocaine and other local anesthetics bind to a phenylalanine at the bottom of the pore of this channel through a cation-\u03c0 interaction.
\r\n\r\nAn important aspect of our work is the development of unnatural amino acids that can be used in the study of ion channels through the in vivo nonsense-suppression methodology. We determined that D-amino acids could not be incorporated into ion channels using this method. We synthesized several novel fluorescent-MTS reagents to be used in FRET studies. We probed the sterics around phenylalanines using the unnatural amino acid 3,5-dimethylphenylalanine. We also attempted to incorporate 4-amino-phenylalanine, but, unfortunately, we never saw the enhanced binding of a cationic ligand that was our expected phenotype.
\r\n\r\nFinally, we also designed and synthesized two \u03b1-hydroxy acids capable of site-specific proteolysis upon UV irradiation. We used a tripeptide model system to isolate and characterize the cleavage fragments, proving that these two residues are indeed capable of site-specific proteolysis through the predicted mechanism.
\r\n", "doi": "10.7907/86B5-SV57", "publication_date": "2009", "thesis_type": "phd", "thesis_year": "2009" }, { "id": "thesis:4333", "collection": "thesis", "collection_id": "4333", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-10302008-123855", "primary_object_url": { "basename": "00fullthesis.pdf", "content": "final", "filesize": 23301278, "license": "other", "mime_type": "application/pdf", "url": "/4333/1/00fullthesis.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Understanding the Chemical Basis of Neuronal Development and Communication: I. The Role of Fucose \u03b1(1-2) Galactose Carbohydrates in Neuronal Growth. II. Structure-Function Analysis of Chondroitin Sulfate in the Brain", "author": [ { "family_name": "Gama", "given_name": "Cristal Ivette", "clpid": "Gama-Cristal-Ivette" } ], "thesis_advisor": [ { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" } ], "thesis_committee": [ { "family_name": "Campbell", "given_name": "Judith L.", "clpid": "Campbell-J-L" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" }, { "family_name": "Kennedy", "given_name": "Mary B.", "clpid": "Kennedy-M-B" }, { "family_name": "Patterson", "given_name": "Paul H.", "clpid": "Patterson-P-H" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Although carbohydrates are known to participate in many processes including inflammation and cancer metastasis, their functional roles are only beginning to be understood on a molecular level. Unlike DNA and proteins, carbohydrate structures are not template-encoded and are challenging to detect in vivo and manipulate for structure-function analyses. New tools are needed to complement biochemical and genetic approaches to advance our understanding of carbohydrates and their physiological roles. We seek to understand the roles of carbohydrates in regulating the structure and function of proteins in the brain. Our focus is on two modifications that are important in neuronal communication and development: fucosylation (Part I) and chondroitin sulfate modifications (Part II).
\r\n\r\nIn Part I, we describe our progress in elucidating the molecular mechanisms by which fucosyl saccharides regulate neuronal communication. Previous studies have shown that preventing formation of fucose\u03b1(1-2)galactose saccharides causes reversible amnesia in animals, suggesting that these sugars play essential roles in learning and memory. However, proteins expressing the fucose\u03b1(1-2)galactose epitope or proteins binding this epitope have not been identified. Using chemical probes, we established that fucose\u03b1(1-2)galactose associated proteins participate in a novel carbohydrate-mediated pathway for regulating neuronal growth. Additionally, we found that fucose\u03b1(1-2)galactose glycoproteins are prevalent in developing brain and that synapsin Ia/Ib are the major fucose\u03b1(1-2)galactose glycoproteins in adult brain. In our attempts to identify Fuc\u03b1(1-2)Gal lectins, we have established that multivalent polymers enhance our ability to capture and characterize such proteins.
\r\n\r\nIn Part II, we describe our efforts toward understanding the role of chondroitin sulfate glycosaminoglycans in neuronal development. Chondroitin sulfate glycosaminoglycans are structurally complex and heterogeneous in nature, thus hampering efforts to understand their precise biological roles. It is thought that chondroitin sulfate activity is dictated by a sulfation code, where distinct sulfation sequences are spatially and temporally regulated. We have developed a chemical approach to evaluate the structure-activity relationship of chondroitin sulfate as it effects neuronal growth. We generated the first synthetic library of well-defined chondroitin sulfate oligosaccharides containing various sulfation sequences and have demonstrated that the chondroitin sulfate-E sequence is a stimulatory motif that promotes the growth of several neuron types. Moreover, we determined that chondroitin sulfate-E stimulation was facilitated through activation of the midkine/PTP\u03b6 and BDNF/TrkB pathways.
", "doi": "10.7907/CDGH-MJ49", "publication_date": "2009", "thesis_type": "phd", "thesis_year": "2009" }, { "id": "thesis:4398", "collection": "thesis", "collection_id": "4398", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-11032008-191338", "primary_object_url": { "basename": "09_Thesis.pdf", "content": "final", "filesize": 2188603, "license": "other", "mime_type": "application/pdf", "url": "/4398/9/09_Thesis.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Completion of a Programmable DNA-Binding Small Molecule Library", "author": [ { "family_name": "Hsu", "given_name": "Carey Frank", "clpid": "Hsu-Carey- Frank" } ], "thesis_advisor": [ { "family_name": "Dervan", "given_name": "Peter B.", "orcid": "0000-0001-8852-7306", "clpid": "Dervan-P-B" } ], "thesis_committee": [ { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "orcid": "0000-0001-5661-1714", "clpid": "Hsieh-Wilson-L-C" }, { "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" }, { "family_name": "Stoltz", "given_name": "Brian M.", "orcid": "0000-0001-9837-1528", "clpid": "Stoltz-B-M" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Hairpin pyrrole-imidazole (Py-Im) polyamides are programmable oligomers that bind the DNA minor groove in a sequence-specific manner with affinities comparable to those of natural DNA-binding proteins. These cell-permeable small molecules have been shown to enter the nuclei of live cells and downregulate endogenous gene expression. We complete here a library of 27 hairpin Py-Im polyamides that bind 7-base-pair sequences of the general form 5\u2019-WWGNNNW-3\u2019 (where W = A or T, N = W, G, or C). A table of binding affinities and sequence contexts for this completed 27-member library has been assembled for the benefit of the chemical biology community interested in molecular control of transcription. Quantitative fluorescence-based methods have been developed to determine the nuclear concentration of polyamide-fluorescein conjugates in cell culture. Confocal laser scanning microscopy and flow cytometry techniques are utilized to plot calibration curves, from which the nuclear concentration can be interpolated. Although confocal microscopy and flow cytometry generate disparate values, taken together these experiments suggest that the polyamide concentration inside the cell nucleus is lower than it is outside the cell. To further our understanding of C-terminal tail linkage effects on sequence specificity, the equilibrium association constants of hairpin polyamide conjugates were measured by quantitative DNase I footprint titration experiments. These results indicate that linkers and functional R groups on the tails of hairpin polyamide conjugates have recognition properties that should be considered in the design of these molecules to target DNA binding sites. Furthermore, these \u03b2-alanine-C\u2083-linked polyamide conjugates are shown to decrease hypoxia-inducible transcription of vascular endothelial growth factor (VEGF) in cultured HeLa cells. In addition, polyamide conjugates designed to target the Oct4 octamer DNA element modulate the expression levels of Oct4-driven genes in P19 mouse embryonal carcinoma cells and R1 mouse embryonic stem (ES) cells.\r\n", "doi": "10.7907/PG0R-3X44", "publication_date": "2009", "thesis_type": "phd", "thesis_year": "2009" }, { "id": "thesis:2223", "collection": "thesis", "collection_id": "2223", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05282009-145527", "primary_object_url": { "basename": "15_Thesis.pdf", "content": "final", "filesize": 3614145, "license": "other", "mime_type": "", "url": "/2223/15/15_Thesis.pdf", "version": "v8.0.0" }, "type": "thesis", "title": "Microarray and Genome-wide Sequencing Approaches to Characterizing DNA Binding Molecules", "author": [ { "family_name": "Puckett", "given_name": "James William", "clpid": "Puckett-James-William" } ], "thesis_advisor": [ { "family_name": "Dervan", "given_name": "Peter B.", "orcid": "0000-0001-8852-7306", "clpid": "Dervan-P-B" } ], "thesis_committee": [ { "family_name": "Grubbs", "given_name": "Robert H.", "orcid": "0000-0002-0057-7817", "clpid": "Grubbs-R-H" }, { "family_name": "Dervan", "given_name": "Peter B.", "orcid": "0000-0001-8852-7306", "clpid": "Dervan-P-B" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "orcid": "0000-0001-5661-1714", "clpid": "Hsieh-Wilson-L-C" }, { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Hairpin and linear beta-alanine linked pyrrole-imidazole (Py-Im) polyamides are programmable oligomers that bind the DNA minor groove sequence-specifically with affinities comparable to those of DNA-binding proteins found in nature. These small molecules have been observed to localize within the nucleus of living cells and modulate endogenous gene expression. Herein, we demonstrate the utility of a linear beta-alanine linked pyrrole-imidazole polyamide to upregulate frataxin mRNA and protein expression in a cell line derived from a Friedreich\u2019s Ataxia patient. We examine the binding affinities and specificities of additional linear beta-alanine linked polyamides. We examine binding specificities of a Cy3-fluorescently labeled version of the frataxin\u00ad expression modulating-polyamide and a Cy3-labeled polyamide known to downregulate expression of the Vascular Endothelial Growth Factor using DNA microarrays composed of hairpins harboring all 524,800 unique 10 bp DNA sequences. We experimentally verify the correlation of Cy3 fluorescence intensity with quantitative DNase I footprint derived binding affinities. We find that Cy3 dye placement on the polyamide tail versus labeling of an internal pyrrole does not significantly alter DNA specificity. Finally, we examine the genome-wide binding preferences of Androgen Receptor (AR) in LNCaP cells using ChIP-Seq (chromatin immunoprecipitation followed by high-throughput DNA sequencing). We observe the canonical ARE motif to be present in a majority of the immunoprecipitated binding regions. We observe a secondary sequence motif that may be the dimerization of AR with a forkhead protein, an interaction known in the literature but without a defined sequence motif. We also define AR occupancy with respect to location in and about known genes. We correlate gene expression profiles from mRNA microarray data with the ChIP-Seq data.", "doi": "10.7907/E3NT-SC46", "publication_date": "2009", "thesis_type": "phd", "thesis_year": "2009" }, { "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:2483", "collection": "thesis", "collection_id": "2483", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-06062008-103127", "primary_object_url": { "basename": "CACThesis080606c.pdf", "content": "final", "filesize": 7455624, "license": "other", "mime_type": "application/pdf", "url": "/2483/1/CACThesis080606c.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Self-Assembled Monolayers for the Study of Biological Targets ", "author": [ { "family_name": "Canaria", "given_name": "Christie Anne", "clpid": "Canaria-Christie-Anne" } ], "thesis_advisor": [ { "family_name": "Fraser", "given_name": "Scott E.", "clpid": "Fraser-S-E" } ], "thesis_committee": [ { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" }, { "family_name": "Asthagiri", "given_name": "Anand R.", "clpid": "Asthagiri-A-R" }, { "family_name": "Collier", "given_name": "C. Patrick", "clpid": "Collier-C-P" }, { "family_name": "Lansford", "given_name": "Rusty", "clpid": "Lansford-R" }, { "family_name": "Fraser", "given_name": "Scott E.", "clpid": "Fraser-S-E" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Understanding the interactions of biological molecules with solid supports is vital for the development of detection systems and assay platforms. These relationships are frequently quite complex, involving hydrophobic interactions, electrostatic interactions, van der Waals forces, and covalent chemical bonds. We can exploit these interactions in a solid support device by modifying the surface substrate with thin films and monolayers. Self-assembled monolayers (SAMs) are powerful tools for functionalizing and imparting chemical character to surfaces. In this thesis, alkylthiol reagents are utilized to build SAMs on gold (Au) substrates. This work characterizes and studies monolayer formation. In addition, I use SAMs to generate surfaces specific for binding proteins, DNA, and cells. The popular biotin-streptavidin motif is used to demonstrate protein binding, as well as characterize monolayer composition as a result of solvent effects. Novel reagent syntheses are presented for both biotinylated alkylthiols and triethylene-glycol alkylthiols. Together, these two reagents generate substrates which bind specific proteins, while repelling non-specific ones. An additional reagent, \u201cDMT-coated controlled porous glass (CPG),\u201d was designed and synthesized for the generation of custom sequence oligonucleotides. Phosphoramidite syntheses using this modified CPG yield oligos with a 3\u2019 alkylthiol modification. SAMs generated with this reagent demonstrate specific binding of complement strands. Both electrochemical techniques and restriction enzymes (where appropriate) provide methods for releasing monolayer-bound species. Lastly, I employ SAMs to generate substrates amenable to cell capture and cell adhesion. Binding B- and T-cell lymphocytes is achieved, demonstrating SAM-coated Au as a substrate for cell panning. Chemokine vascular endothelial growth factor (VEGF) is also bound to SAMs, generating surfaces amenable to cell adhesion and motility. Cells plated on higher surface concentrations of VEGF migrate faster, and I show the effect is specific to cells with VEGF receptors. Overall, this thesis explores the formation and utilization of SAMs for capturing and studying biological targets. The findings here may be transferred in the future into bio-sensing devices and arrays.", "doi": "10.7907/39DF-7V92", "publication_date": "2008", "thesis_type": "phd", "thesis_year": "2008" }, { "id": "thesis:4105", "collection": "thesis", "collection_id": "4105", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-10152007-175458", "primary_object_url": { "basename": "Beginning.pdf", "content": "final", "filesize": 161532, "license": "other", "mime_type": "application/pdf", "url": "/4105/2/Beginning.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Alpha-Diaminobutyric Acid-Linked Hairpin Polyamide-Alklylator Conjugates ", "author": [ { "family_name": "Tsai", "given_name": "Sherry Mon-Yue", "clpid": "Tsai-Sherry-Mon-Yue" } ], "thesis_advisor": [ { "family_name": "Dervan", "given_name": "Peter B.", "clpid": "Dervan-P-B" } ], "thesis_committee": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" }, { "family_name": "Dervan", "given_name": "Peter B.", "clpid": "Dervan-P-B" }, { "family_name": "Goddard", "given_name": "William A., III", "clpid": "Goddard-W-A-III" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The ability to control gene expression through the use of DNA sequence-specific, cell-permeable molecules holds therapeutic promise. Pyrrole-imidazole polyamides are a class of synthetic ligands that can be programmed to bind a broad repertoire of DNA sequences with affinities and specificities comparable to natural DNA-binding proteins. These ligands are generally linked via a turn moiety, resulting in a \u2018hairpin\u2019 structure. Conjugation of polyamides to the non-specific DNA alkylator chlorambucil produces molecules capable of the sequence-specific alkylation of DNA that can arrest gene transcription. We have identified \u03b1-diaminobutyric acid (\u03b1-DABA) as a new turn moiety that can give polyamide-chlorambucil conjugates distinctive biological properties in cellular and small animal models; this may be due to their increased DNA alkylation specificities relative to the standard \u03b3-DABA-linked conjugates. A general characterization of \u03b1-DABA-linked polyamides and their conjugates is reported.
\r\n\r\nAlso described is the development of a modular synthesis of chondroitin sulfate (CS) glycosaminoglycans \u2014 a class of linear, sulfated oligosaccharides that play critical roles in neuronal development, cell division, and spinal cord injury. CS structure in vivo is complex and heterogeneous, hampering efforts to understand its precise biological roles. Access to CS molecules of precisely defined structures is critical to understanding their structure-function relationships. The reported synthetic route is capable of accessing CS structures of defined lengths and sulfation motifs, providing a new approach to understanding these important molecules.
\r\n", "doi": "10.7907/3S8Y-YD79", "publication_date": "2008", "thesis_type": "phd", "thesis_year": "2008" }, { "id": "thesis:2682", "collection": "thesis", "collection_id": "2682", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-06212007-155330", "primary_object_url": { "basename": "01Title_and_Contents.pdf", "content": "final", "filesize": 31136, "license": "other", "mime_type": "application/pdf", "url": "/2682/1/01Title_and_Contents.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Endogenous Gene Regulation by DNA Binding Polyamides", "author": [ { "family_name": "Nickols", "given_name": "Nicholas George", "orcid": "0000-0002-3131-9212", "clpid": "Nickols-Nicholas-George" } ], "thesis_advisor": [ { "family_name": "Dervan", "given_name": "Peter B.", "orcid": "0000-0001-8852-7306", "clpid": "Dervan-P-B" } ], "thesis_committee": [ { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" }, { "family_name": "Parker", "given_name": "Carl Stevens", "orcid": "0000-0001-9795-4211", "clpid": "Parker-C-S" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "orcid": "0000-0001-5661-1714", "clpid": "Hsieh-Wilson-L-C" }, { "family_name": "Dervan", "given_name": "Peter B.", "orcid": "0000-0001-8852-7306", "clpid": "Dervan-P-B" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Cells integrate stimuli through networks of proteins that affect programs of gene expression. The expression of genes is controlled by transcription activators and repressors that recruit or inhibit the recruitment of transcription complexes to the promoters of target genes. Molecules that can specifically modulate these protein-DNA interfaces could have significant applications in human medicine. Polyamides containing N-methylimidazole (Im) and N-methylpyrrole (Py) comprise a class of programmable DNA-binding ligands capable of binding to a broad repertoire of DNA sequences with affinities and specificities comparable to those of natural DNA-binding proteins. Numerous polyamide-dye conjugates localize in the nuclei of cultured cells. Multiple studies have shown that polyamides are capable of displacing DNA binding proteins from their target sites in a sequence specific manner. Early experiments in viral systems showed that polyamides can be active in cell culture. This thesis describes the use of polyamides to modulate the activity of two transcription activators: hypoxia inducible factor 1 (HIF-1) and androgen receptor (AR). Direct inhibition of the HIF-1-DNA and AR-DNA interfaces has relevance to human cancer. A polyamide that targets the hypoxia response element (HRE) binds its target site with high affinity and inhibits the expression of Vascular Endothelial Growth Factor (VEGF) and other HIF-1 induced genes in cultured cells. Similarly, a polyamide that targets the androgen response element binds its target site with high affinity and inhibits the expression of prostate specific antigen (PSA) and other androgen-induced genes in cultured cells. In both systems, the inhibition of target genes is consistent with a decrease in the occupancy of the transcription factor at relevant loci. The genome-wide effect of the HRE-targeted polyamide was compared to that of siRNA against HIF-1\u03b1. Remarkably, a comparable number of genes were affected by the HRE targeted polyamide as by the siRNA. The effect of the ARE targeted polyamide was compared to the synthetic antiandrogen bicalutamide. The polyamide and bicalutamide had a comparable effect on PSA expression, and genome-wide expression analysis reveals that both affected a similar number of transcripts. In addition, HIF-1\u03b1 siRNA and bicalutamide affected nearly all genes induced by hypoxia or androgen, respectively, while the HRE- and ARE- targeted polyamides affected a subset of each pathway that could be consistent with the DNA binding preferences of the polyamides. The data suggest that polyamides can be used to modulate the activity of selected transcription factor pathways.", "doi": "10.7907/YH4W-4H67", "publication_date": "2008", "thesis_type": "phd", "thesis_year": "2008" }, { "id": "thesis:5035", "collection": "thesis", "collection_id": "5035", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-12172007-134752", "primary_object_url": { "basename": "(Full)RMMThesis.pdf", "content": "final", "filesize": 45005243, "license": "other", "mime_type": "application/pdf", "url": "/5035/11/(Full)RMMThesis.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "Applications of Palladium-Catalyzed Enantioselective Decarboxylative Alkylation in Natural Products Total Synthesis", "author": [ { "family_name": "McFadden", "given_name": "Ryan Michael", "clpid": "McFadden-Ryan-Michael" } ], "thesis_advisor": [ { "family_name": "Stoltz", "given_name": "Brian M.", "clpid": "Stoltz-B-M" } ], "thesis_committee": [ { "family_name": "Grubbs", "given_name": "Robert H.", "clpid": "Grubbs-R-H" }, { "family_name": "Stoltz", "given_name": "Brian M.", "clpid": "Stoltz-B-M" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" }, { "family_name": "Goddard", "given_name": "William A., III", "clpid": "Goddard-W-A-III" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The catalytic enantioselective preparation of all-carbon quaternary stereocenters within rings via alkylation is a major challenge in synthetic organic chemistry. Many important natural products and biologically active pharmaceuticals contain this motif. We have developed palladium-catalyzed decarboxylative alkylations capable of generating all-carbon quaternary stereocenters in good yield with high enantioselectivity.
\r\n\r\nAlkylated products are readily elaborated to synthetically useful cyclic scaffolds. The enantioselective decarboxylative alkylation is thus utilized to prepare intermediates previously reported in the total syntheses of classic natural products. Herein, we disclose modern formal syntheses of (\u2013)-Thujopsene, (-\u2013)-Dysidiolide, and (\u2013)-Aspidospermine.
\r\n\r\nThe longer-term goal was to apply this new enantioselective catalysis to the total syntheses of natural products with novel carbocyclic architectures. Our methodology is demonstrated during the first protecting group-free enantioselective total synthesis of (+)-dichroanone, a 4a-methyltetrahydrofluorene. The [6-5-6] tricyclic natural products family has members with important biological activity, and our route to (+)-dichroanone may provide general access to related compounds. During our synthetic endeavors, a novel Kumada-benzannulation approach to the aromatic portion of (+)-dichroanone was developed, along with a unique synthesis of a hydroxy-p-benzoquinone from a phenol. The absolute stereochemistry of the natural product was verified for the first time during our total synthesis.
\r\n\r\nSignificant progress has been made toward the total synthesis of the marine meroterpenoid liphagal, a potent and selective phosphatidylinositol 3-kinase alpha inhibitor. The enantioselective decarboxylative alkylation has been employed, and an acetylene [2 + 2] photoaddition / ring-opening sequence is used to construct the 7-membered ring. New understanding about the reactivity of [6-7] bicyclic scaffolds has been gathered, and the information applied during preparation of liphagal\u2019s benzofuran motif. Our efforts have led to a functionally diverse array of liphagal analogues, which may be used for structure-activity-relationship studies with phosphatidylinositol 3-kinases.
\r\n", "doi": "10.7907/GQ1E-RW28", "publication_date": "2008", "thesis_type": "phd", "thesis_year": "2008" }, { "id": "thesis:2554", "collection": "thesis", "collection_id": "2554", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-06112008-094638", "primary_object_url": { "basename": "Otey_FinalThesis.pdf", "content": "final", "filesize": 7056815, "license": "other", "mime_type": "application/pdf", "url": "/2554/1/Otey_FinalThesis.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Structural and Functional Exploration of an Artificial Family of Cytochromes P450", "author": [ { "family_name": "Otey", "given_name": "Christopher Richard", "clpid": "Otey-Christopher-Richard" } ], "thesis_advisor": [ { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" } ], "thesis_committee": [ { "family_name": "Rees", "given_name": "Douglas C.", "orcid": "0000-0003-4073-1185", "clpid": "Rees-D-C" }, { "family_name": "Dougherty", "given_name": "Dennis A.", "orcid": "0000-0003-1464-2461", "clpid": "Dougherty-D-A" }, { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "orcid": "0000-0001-5661-1714", "clpid": "Hsieh-Wilson-L-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Protein families are comprised of numerous sequences that adopt a similar three-dimensional structure and functional properties. The superfamily of cytochromes P450 are an excellent example of a common structural scaffold being utilized for a variety of biological functions. This functional diversity is achieved in Nature through millions of years of evolution to create new and diverse sequences. We have used site-directed recombination guided by the computation algorithm SCHEMA to create an artificial family of cytochromes P450 in the laboratory. Members of this family possess unique properties such as altered activity profiles, increased thermostability and the ability to accept new substrates.
\r\n\r\nWe developed screening tools for the rapid analysis of hundreds of individual P450s. These high-throughput assays include the 4-aminoantipyrine (4-AAP) assay which is capable of detecting the hydroxylation of an aromatic ring. High-throughput carbon monoxide binding facilitates the rapid detection of P450s that correctly incorporate a heme cofactor and are thus properly folded and potentially functional. Finally, a substrate binding assay which measures a spectral shift that occurs when a substrate binds in a P450 active site is described.
\r\n\r\nFourteen double-crossover chimeras created from the bacterial P450s CYP102A1 and CYP102A2 were constructed to calibrate the P450 scaffold for SCHEMA, a computational algorithm used to minimize structural disruption in chimeric proteins. We found that only chimeras with high levels of structural disruption as measured by SCHEMA were unfolded. Among the fourteen chimeras we also observed three different activity profiles based on peroxygenase kinetic assays with the substrates p-nitrophenoxydodecanoic acid (12-pNCA), 2-phenoxyethanol and allyloxybenzene.
\r\n\r\nWe applied this calibration to create an artificial family comprising ~3,000 chimeric heme P450 proteins that correctly fold and incorporate a heme cofactor by recombining three cytochromes P450 at seven crossover locations chosen to minimize structural disruption. Members of this protein family differ from any known sequence at an average of 72 and by as many as 109 amino acids. Most (>73%) of the properly folded chimeric P450 heme proteins are catalytically active peroxygenases; some are more thermostable than the parent proteins. A multiple sequence alignment of 955 chimeras, including both folded and not, was analyzed using logistic regression analysis (LRA) to identify key structural contributions to cytochrome P450 heme incorporation and peroxygenase activity and suggests possible structural differences between parents CYP102A1 and CYP102A2.
\r\n\r\nThirty-four members of this artificial family were assayed for functional diversity on a set of eight substrates. P450 chimeras were able to exceed the parents in total activity on all eight substrates and were grouped into five different groups based on activity profiles using K-means clustering. Activity profiles on eight substrates were then performed in high throughput to produce a data set of 330 chimeras. The mean percent standard deviation of the activity assays showed the reproducibility of these high-throughput data and further analysis may reveal information about sequence-structure-function relationships.
\r\n\r\nThe products of the catalytic reactions of four chimeric P450s with substrates of human P450s, some of which are drug compounds, were analyzed by HPLC in order to determine their identity. Chimeras were able to produce authentic human metabolites of chlorzoxazone, zoxazolamine and propranolol, showed peroxidase acitivty on 4-aminobiphenyl and produced an unknown product with tolbutamide. Finally, the peroxygenase activity of a mutant P450 heme domain is able to be further altered and enhanced using directed evolution. After two rounds of directed evolution and screening with the 4-AAP assay, we found mutants with altered substrate specificities and an overall enhancement of activity.
\r\n\r\nThe design and high-throughput methodologies described here can be used to create artificial protein families and to discover new and useful protein sequences. Like natural protein families, artificial protein families can be used to identify regions of protein sequence and structure that are important for folding and function. This is especially useful for analyzing protein families with few members or for validating tools for structure prediction and for protein sequence-structure-function analysis. Artificial protein families are also rich in sequence diversity and can provide sources of novel protein function. Using the high-throughput methodologies described here, chimeric P450s with enhanced activity, altered activity profiles, and the ability to hydroxylate drug-like compounds to produce authentic human metabolites were discovered in our artificial family of P450s. These methodologies will hopefully be extended to the study of other protein families and to the creation and discovery of increasingly valuable protein catalysts.
", "doi": "10.7907/MZQM-N011", "publication_date": "2008", "thesis_type": "phd", "thesis_year": "2008" }, { "id": "thesis:840", "collection": "thesis", "collection_id": "840", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-03022007-083916", "primary_object_url": { "basename": "Khidekel_Upload.pdf", "content": "final", "filesize": 7306956, "license": "other", "mime_type": "application/pdf", "url": "/840/1/Khidekel_Upload.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "A Chemoenzymatic Strategy toward Understanding O-GlcNAc Glycosylation in the Brain", "author": [ { "family_name": "Khidekel", "given_name": "Nelly", "clpid": "Khidekel-Nelly" } ], "thesis_advisor": [ { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "orcid": "0000-0001-5661-1714", "clpid": "Hsieh-Wilson-L-C" } ], "thesis_committee": [ { "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": "Schuman", "given_name": "Erin Margaret", "orcid": "0000-0002-7053-1005", "clpid": "Schuman-E-M" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "orcid": "0000-0001-5661-1714", "clpid": "Hsieh-Wilson-L-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Posttranslational modification to proteins represents a fundamental mechanism by which protein function is extended and elaborated. In the brain, modifications such as phosphorylation play critical roles in mediating neuronal communication and development. Unique among carbohydrate modifications is the addition of a single monosaccharide, N-acetyl-D-glucosamine, to serine and threonine residues of proteins (O-GlcNAc glycosylation). The modification shares intriguing features with phosphorylation, including its intracellular and dynamic nature. The enzyme responsible for adding the modification to proteins is necessary for life at the single cell level and O-GlcNAc glycosylation has been linked to nutrient sensing, gene expression, and in the brain, to neurodegeneration. Despite tantalizing evidence for the modification\u2019s importance, understanding O-GlcNAc glycosylation has been hampered by insufficient strategies to study it at single-protein level as well as across the proteome. Here we describe the development of a new, chemoenzymatic strategy to facilitate the discovery of O-GlcNAc proteins, as well as the first studies aimed at understanding O-GlcNAc proteome-wide, in the brain.
\r\n\r\nOur approach capitalizes on an engineered enzyme and synthetic unnatural substrate to specifically 'tag' O-GlcNAc-modified proteins for rapid and sensitive detection. We applied the methodology to the discovery of low-abundance, endogenous O-GlcNAc proteins from cells. We also combined the approach with mass spectrometry for the isolation of O-GlcNAc peptides and the mapping of glycosylation sites, the first step toward functional analysis of the modification. Overall, our efforts led to the identification of nearly fifty new O-GlcNAc proteins, several of which serve as targets for mechanistic study. Many of the proteins function in the control of transcription and translation, highlighting the proposed role for O-GlcNAc in regulating gene expression. Additionally, we provide evidence that O-GlcNAc glycosylation is particularly prevalent on proteins at the nerve terminal, or synaptosome, where it may function to control vesicle cycling and neurotransmitter release. Finally, our work has also led to the first bioanalytical, quantitative assays for O-GlcNAc dynamics in both cells and tissue. Here, we have shown that O-GlcNAc is reversible in neuronal tissue and can respond rapidly and robustly to neuronal stimulation in vivo.
", "doi": "10.7907/MT8P-JB95", "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:298", "collection": "thesis", "collection_id": "298", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-01232007-144117", "primary_object_url": { "basename": "Zhang_Kechun_2007.pdf", "content": "final", "filesize": 2960289, "license": "other", "mime_type": "application/pdf", "url": "/298/11/Zhang_Kechun_2007.pdf", "version": "v6.0.0" }, "type": "thesis", "title": "Engineering Protein-Based Materials Through Coiled-Coil Motifs", "author": [ { "family_name": "Zhang", "given_name": "Kechun", "orcid": "0000-0002-7139-2799", "clpid": "Zhang-Kechun" } ], "thesis_advisor": [ { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" } ], "thesis_committee": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" }, { "family_name": "Asthagiri", "given_name": "Anand R.", "clpid": "Asthagiri-A-R" }, { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Natural biomaterials are highly organized from the molecular to the macroscopic level in a hierarchical manner, requiring synthetic technologies to achieve this level of complexity. A biosynthetic approach to material design has emerged as an attractive option. In particular, proteins represent a promising class of molecules for creating new materials due to their determined sequence and structure. The research described in this thesis focuses on engineering protein-based materials using coiled-coil motifs. The coiled coil is a common protein architecture consisting of two or more \u03b1-helices wrapped around one another to form a supercoil. Despite its simple conformation, the coiled-coil motif plays diverse roles in biological systems functioning as sensors, recognition elements, scaffolds, levers, rotating arms and springs.
\r\n\r\nFirst, a designed parallel heterodimeric leucine zipper pair was used as the protein capture domain to construct an artificial polypeptide scaffold for surface functionalization. By using a mutant E. coli phenylalanyl-tRNA synthetase, the photoreactive amino acid para-azidophenylalanine was incorporated. This protein polymer was spin-coated and photocrosslinked to octyltrichlorosilane-treated surfaces. The resulting protein films were shown to immobilize recombinant proteins through association of coiled coil heterodimer. Furthermore, in conjunction with microfluidic chips that were specifically designed for on-chip mixing using laminar flow, gradients of leucine zipper tagged proteins were formed in the microchannels and immobilized on the engineered protein films. This provides a general technique for producing surface-bound multicomponent gradients. The adhesion of human umbilical vein endothelial cells cultured on a surface-bound gradient of cell binding ligands generated by this technique was examined. In addition, to generate protein walkers that have different lateral mobility rates on a surface, several variants of the leucine zipper pair with tunable heterodimerization affinities were designed and synthesized to allow diversity in the association strength of proteins linked to a surface.
\r\n\r\nThe coiled-coil motif was also used to construct protein hydrogels. Hydrogels formed from a triblock artificial protein bearing dissimilar helical coiled-coil end domains (P and A) erode more than one hundred fold slower than hydrogels formed from those bearing the same end domains (either P or A). The reduced erosion rate is a consequence of the fact that looped chains are suppressed because P and A tend not to associate with each other. Thus, by harnessing selective molecular recognition, discrete aggregation number and orientational discrimination of coiled-coil protein domains, the erosion rate of hydrogels can be tuned over several orders of magnitude.
\r\n\r\nFinally, a biosynthetic approach was developed to control and probe cooperativity in multiunit biomotor assemblies by linking molecular motors to artificial protein scaffolds using the heterodimeric leucine zipper pair. This approach provides precise control over spatial and elastic coupling between motors. Cooperative interactions between monomeric kinesin-1 motors attached to protein scaffolds enhance hydrolysis activity and microtubule gliding velocity. However, these interactions are not influenced by changes in the elastic properties of the scaffold, distinguishing multimotor transport from that powered by unorganized monomeric motors. These results highlight the role of supramolecular architecture in determining mechanisms of collective transport.
", "doi": "10.7907/324A-2K76", "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:4983", "collection": "thesis", "collection_id": "4983", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-12132006-122200", "primary_object_url": { "basename": "Tully_Thesis_Part_I.pdf", "content": "final", "filesize": 23918309, "license": "other", "mime_type": "application/pdf", "url": "/4983/1/Tully_Thesis_Part_I.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Synthesis and Biological Activity of Chondroitin Sulfate Biopolymers", "author": [ { "family_name": "Tully", "given_name": "Sarah Erin", "clpid": "Tully-Sarah-Erin" } ], "thesis_advisor": [ { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "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" }, { "family_name": "Dervan", "given_name": "Peter B.", "clpid": "Dervan-P-B" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Chondroitin sulfate glycosaminoglycans are ubiquitously expressed linear, sulfated polysaccharides involved in cell growth, neuronal development and spinal cord injury. The different sulfation motifs presented by chondroitin sulfate may regulate its activity, but efforts to understand the precise biological roles of this glycosaminoglycan have been hampered by its complexity and heterogeneity. Here, we report the synthesis of well-defined chondroitin sulfate oligosaccharides through a convergent approach that permits installation of sulfate groups at precise positions along the carbohydrate backbone, biological evaluation of the synthetic molecules, and generation of antibodies that recognize the distinct sulfation motifs.
\r\n\r\nUsing the chondroitin sulfate oligosaccharide library, we demonstrate that specific sulfation patterns act as molecular recognition elements for growth factors, and modulate neuronal growth. We identified a chondroitin sulfate tetrasaccharide, CS-E, which stimulates the growth and differentiation of multiple neuron types. Through use of carbohydrate microarrays, we found that the CS-E tetrasaccharide binds to a variety of proteins involved in promoting neurite outgrowth. A CS-E disaccharide, an unsulfated tetrasaccharide, and three other sulfated tetrasaccharides, CS-A, CS-C, and CS-R, were also investigated, and showed little effect on neurite outgrowth and reduced growth factor binding compared to the CS-E tetrasaccharide. These studies represent the first, direct investigations into the structure-activity relationships of chondroitin sulfate using homogeneous synthetic molecules, define a tetrasaccharide as a minimal motif required for function, and reveal the importance of sulfation in chondroitin sulfate bioactivity.
\r\n", "doi": "10.7907/c6pj-np35", "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: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:5248", "collection": "thesis", "collection_id": "5248", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-08182005-120859", "primary_object_url": { "basename": "Thesis.pdf", "content": "final", "filesize": 17633298, "license": "other", "mime_type": "application/pdf", "url": "/5248/15/Thesis.pdf", "version": "v7.0.0" }, "type": "thesis", "title": "Development of the Enantioselective Oxidation of Secondary Alcohols and Natural Products Total Synthesis", "author": [ { "family_name": "Bagdanoff", "given_name": "Jeffrey Thomas", "clpid": "Bagdanoff-Jeffrey-Thomas" } ], "thesis_advisor": [ { "family_name": "Stoltz", "given_name": "Brian M.", "clpid": "Stoltz-B-M" } ], "thesis_committee": [ { "family_name": "Grubbs", "given_name": "Robert H.", "clpid": "Grubbs-R-H" }, { "family_name": "Stoltz", "given_name": "Brian M.", "clpid": "Stoltz-B-M" }, { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Oxidation is a fundamental process in chemistry and biology. In synthetic chemistry, there are several methods for the asymmetric oxidation of organic substrates. Classically, these methods have focused on the delivery of a heteroatom from a reagent or catalyst to a prochiral substrate. What have historically been underdeveloped are enantioselective oxidation methods that do not involve the transfer of a heteroatom, but rather are defined by the enantioselective dehydrogenation of an organic substrate. This type of oxidative transformation was investigated using a palladium(II) catalyst system.
\r\n\r\nA palladium-catalyzed oxidative kinetic resolution of secondary alcohols was developed. Key features of the catalytic system include the use of (\u2013)-sparteine as the source of chiral relay, and molecular oxygen as the sole stoicheometric oxidant. Under the described catalytic system, a number of benzylic and allylic alcohols have been oxidized in an enantioselective manner, to provide a ketone and residual alcohol in high enantiomeric excess and excellent yield.
\r\n\r\nSubsequent to the original system, the systematic investigation of a number of mechanistic hypotheses involving the role of exogenous bases and H-bonding additives prompted the discovery of new reaction conditions displaying greatly enhanced reactivity, selectivity, atom economy, and generality. The net result of these improvements was a catalytic system effective in oxidative desymmetrization of a number of complex meso-diols. Ultimately, these advances have permitted our method to be applied towards a number of synthetic endeavors, including the key step in the total synthesis of the natural product alkaloid (\u2013)-lobeline.
\r\n", "doi": "10.7907/Z94F1NQK", "publication_date": "2006", "thesis_type": "phd", "thesis_year": "2006" }, { "id": "thesis:839", "collection": "thesis", "collection_id": "839", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-03022006-172355", "primary_object_url": { "basename": "Cashin-Amanda-thesis-2006.pdf", "content": "final", "filesize": 13486878, "license": "other", "mime_type": "application/pdf", "url": "/839/49/Cashin-Amanda-thesis-2006.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Chemical Scale Investigations of Drug-Receptor Interactions at the Nicotinic Acetylcholine Receptor", "author": [ { "family_name": "Cashin", "given_name": "Amanda Leigh", "clpid": "Cashin-Amanda-Leigh" } ], "thesis_advisor": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" } ], "thesis_committee": [ { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" }, { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" }, { "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": "Biological signaling pathways employ a vast array of integral membrane proteins that process and interpret the chemical, electrical, and mechanical signals that are delivered to cells. Among these proteins, ligand gated ion channels (LGIC) are therapeutic targets for Alzheimer\u2019s disease, Schizophrenia, drug addiction, and learning and memory. High-resolution structural data on neuroreceptors are only just becoming available, yet the functional importance of particular structural features can be challenging.
\r\n\r\nThe primary focus of the present work is to gain a chemical scale understanding of the ligand-receptor binding determinants of LGICs. In particular, these studies explore drug-receptor interactions at the nicotinic acetylcholine receptor (nAChR), the most extensively studied members of the Cys-loop family of LGICs. The present study utilizes in vivo nonsense suppression methodology to perform chemical scale investigations of nAChR agonist activity.
\r\n\r\nThe binding of three distinct agonists\u2013-acetylcholine (ACh), nicotine, and epibatidine--to the nicotinic acetylcholine receptor (nAChR) has been probed using unnatural amino acid mutagenesis. ACh makes a cation-pi interaction with Trp a149, while nicotine employs a hydrogen bond to a backbone carbonyl in the same region of the agonist binding site. The nicotine analogue epibatidine achieves its high potency by taking advantage of both the cation-pi interaction and the backbone hydrogen bond.
\r\n\r\nNonsense suppression was also utilized to probe the importance of residues outside of the binding box in nAChR function. These studies demonstrate a structural role of the highly conserved aD89 residue in stabilizing the agonist binding site near aW149. In addition to outer shell residue, aK145 is shown to be important for proper nAChR function. In combination with additional evidence from other recent advances, this site is proposed to be important in initiating the nAChR channel gating pathway.
\r\n\r\nResidues outside the aromatic binding site were also examined through computational protein design studies. Results from these studies identify outer shell mutations 116Q and 57R (AChBP numbering) that enhance nAChR specificity for nicotine, over ACh and epibatidine compared to wild-type receptors.
\r\n\r\nFinally, a series of cationic polyamides were shown to enhance polyamide affinity while maintaining specificity by varying the number, relative spacing, and linker length of aminoalkyl side chains.
", "doi": "10.7907/RX9T-1069", "publication_date": "2006", "thesis_type": "phd", "thesis_year": "2006" }, { "id": "thesis:3265", "collection": "thesis", "collection_id": "3265", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-08292005-210930", "primary_object_url": { "basename": "TW_Thesis.pdf", "content": "final", "filesize": 2367366, "license": "other", "mime_type": "application/pdf", "url": "/3265/1/TW_Thesis.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "A Chemical-Scale Study on the Ligand-Binding Site of a Serotonin-Gated Ion Channel", "author": [ { "family_name": "Mu", "given_name": "Tingwei", "orcid": "0000-0002-6419-9296", "clpid": "Mu-Tingwei" } ], "thesis_advisor": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" } ], "thesis_committee": [ { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" }, { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Goddard", "given_name": "William A., III", "clpid": "Goddard-W-A-III" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Signal transmission is a combination of electrical and chemical processes. Upon binding neurotransmitters, ligand-gated ion channels open to allow ion flux, which converts chemical signals to electrical signals. In this thesis, experiments in conjunction with computations are utilized to study the mechanism of the ligand-binding process.
\r\n \r\nThe target receptor is a serotonin-gated chloride channel, the MOD-1 receptor. From the viewpoint of a chemist, we explore the specific orientation of the agonist inside the binding pocket and the specific non-covalent interactions responsible for binding. In Chapter 3, computational chemistry is used to build a homology model of MOD-1 using the acetylcholine binding protein template. We proceed to dock the agonist into the binding pocket. The binding pattern from the model provides guidance for the ensuing experimental studies.
\r\n\r\nUnnatural amino acid mutagenesis is a powerful tool to modify the structure of the protein at the chemical level. Systematic perturbations can be introduced at a specific amino acid. Therefore, specific non-covalent interactions, such as hydrogen bonding and cation-pi interactions can be probed. In Chapter 2, we prove that cation-pi interactions between the agonist serotonin and Trp 226 in loop C of MOD-1 play a key role in binding the ligand. Surprisingly, this cation-pi site in MOD-1 is different from that in the serotonin type 3 receptor although these two receptors both bind serotonin, and they are highly homologous. In Chapter 4, we further show that hydrogen bonds between serotonin and Gln 228 and Asn 223 in MOD-1 are important in the binding process. Both conventional and unnatural amino acid mutagenesis are used in conjunction with serotonin analogues. The results from these thorough structure-function studies confirm aspects of the hydrogen bond pattern described in the model.
\r\n\r\nIn Chapter 5, we apply another strategy called the tethered agonist approach to further probe the agonist binding site. This is another elegant example of the effectiveness of the nonsense suppression method.
", "doi": "10.7907/VSEF-4856", "publication_date": "2006", "thesis_type": "phd", "thesis_year": "2006" }, { "id": "thesis:3201", "collection": "thesis", "collection_id": "3201", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-08232005-174620", "primary_object_url": { "basename": "00-Tobias_Whole_Thesis.pdf", "content": "final", "filesize": 5273145, "license": "other", "mime_type": "application/pdf", "url": "/3201/1/00-Tobias_Whole_Thesis.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Directed Evolution of Biosynthetic Pathways to Carotenoids with Unnatural Carbon Backbones", "author": [ { "family_name": "Tobias", "given_name": "Alexander Vincent", "orcid": "0000-0002-5866-5254", "clpid": "Tobias-Alexander-Vincent" } ], "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": "Asthagiri", "given_name": "Anand R.", "orcid": "0000-0002-4925-7523", "clpid": "Asthagiri-A-R" }, { "family_name": "Newman", "given_name": "Dianne K.", "orcid": "0000-0003-1647-1918", "clpid": "Newman-D-K" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "orcid": "0000-0001-5661-1714", "clpid": "Hsieh-Wilson-L-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Over the course of evolution, nature continually discovers new small molecules through the alteration of biosynthetic enzymes and pathways by mutation and gene transfer. Hundreds of these natural products have proven indispensable to medicine, culture, and technology, greatly contributing to increases in the length and quality of human lives. Chemists have found that the \"chemical space\" surrounding natural products is especially rich in functional molecules, and synthesis of natural product analogs has uncovered many with new or improved properties.
\r\n\r\nInspired by nature's search algorithm, we and others have conducted our own evolution of carotenoid biosynthetic pathways in the laboratory. Chapter 1 comprehensively reviews the motivations, accomplishments, and challenges of this research area as of early 2005, and describes in detail how biosynthetic routes to dozens of new carotenoids have been established.
\r\n\r\nTo expand the number of carotenoid backbones beyond the C30 and C40 carbon scaffolds that give rise to the ~700 known natural carotenoids, we subjected a carotenoid synthase, the enzyme responsible for carotenoid backbone synthesis, to directed evolution. Chapter 2 describes the evolution of the C30 carotenoid synthase CrtM from Staphylococcus aureus for the ability to synthesize C40 carotenoids. This work also resulted in novel carotenoids with C35 backbones. We later found that some of the CrtM mutants generated in this laboratory evolution experiment, as well as several second-generation variants, are also capable of synthesizing unnatural C45 and C50 carotenoid backbones when supplied with appropriate prenyl diphosphate precursors.
\r\n\r\nChapter 3 describes the creation of full-fledged pathways to carotenoid pigments based on the C45 and C50 scaffolds. Coexpression of the carotenoid desaturase CrtI from Erwinia uredovora resulted in the biosynthesis of at least 10 new C45 and C50 carotenoids with different systems of conjugated double bonds. We also present evidence of an unnatural asymmetric C40 carotenoid pathway beginning with the condensation of farnesyl diphosphate (FPP, C15PP) and farnesylgeranyl diphosphate (FGPP, C25PP). In addition to clarifying how CrtM and CrtI achieve their product specificities, this work also sheds light on the molecular mechanisms used by evolution to access new chemical diversity and the selective pressures that have shaped natural product biosynthesis.
", "doi": "10.7907/WF0Q-2J98", "publication_date": "2006", "thesis_type": "phd", "thesis_year": "2006" }, { "id": "thesis:974", "collection": "thesis", "collection_id": "974", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-03172005-145045", "primary_object_url": { "basename": "CSN_entire_thesis.pdf", "content": "final", "filesize": 8197462, "license": "other", "mime_type": "application/pdf", "url": "/974/7/CSN_entire_thesis.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Engineering the Mechanical Properties of Ocular Tissues", "author": [ { "family_name": "Nickerson", "given_name": "Charles Sellers", "clpid": "Nickerson-Charles-Sellers" } ], "thesis_advisor": [ { "family_name": "Kornfield", "given_name": "Julia A.", "clpid": "Kornfield-J-A" } ], "thesis_committee": [ { "family_name": "Tirrell", "given_name": "David A.", "clpid": "Tirrell-D-A" }, { "family_name": "Kornfield", "given_name": "Julia A.", "clpid": "Kornfield-J-A" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" }, { "family_name": "Grubbs", "given_name": "Robert H.", "clpid": "Grubbs-R-H" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Age and disease can cause changes in the mechanical properties of ocular tissues, which compromise visual acuity and can lead to blindness. Thus, there is great interest in understanding the mechanical properties of ocular tissues and in developing appropriate therapeutic strategies. The goal of this thesis is to discover and manipulate the molecular mechanisms that determine the bulk physical properties of the vitreous and the cornea. Both tissues are ordered biocomposites of fibrous collagen embedded in soft matrices of proteoglycans (PGs) and glycosaminoglycans (GAGs). The hydration state, mole fraction, and organization of these components determine the mechanical properties of their respective tissues. We describe the use of a novel \"cleat geometry\" for rheometry in shear to quantitatively measure the rheological properties of vitreous and other soft biomaterials. Where as the mechanical strength of these tissues has traditionally been attributed to their collagenous components, results from coordinated mechanical and biochemical analyses suggest that PGs and GAGs also make significant contributions. We hypothesize that hyaluronan contributes to the mechanical properties of the vitreous through a Donnan swelling mechanism that induces a state of tension in the collagen network. We hypothesize that the PG/GAG matrix plays a significant role in the mechanics of the cornea by restricting the translation and deformation of the collagen fibrils.
\r\n\r\nOur therapeutic goal in the vitreous is liquefaction: we seek pharmacological agents capable of separating the vitreous from the retina and destabilizing the network without damaging the adjacent tissues (retina and lens). We present evidence that using urea to disrupt hydrogen bonds may provide clinical benefits by inducing posterior vitreous detachment and reducing the shear modulus of the vitreous.
\r\n\r\nOur therapeutic goal in the cornea is to stabilize its mechanical properties against the softening associated with keratoconus. We report a clinically-relevant rise in the modulus of corneas treated with glyceraldehyde to induce crosslinking through glycation. We hypothesize that the mechanically-relevant crosslinks are those that change the properties of the soft PG/GAG matrix and its coupling to the collagen fibrils, rather than the much more numerous crosslinks that form within fibrils.
", "doi": "10.7907/0R13-2109", "publication_date": "2006", "thesis_type": "phd", "thesis_year": "2006" }, { "id": "thesis:3742", "collection": "thesis", "collection_id": "3742", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-09242004-095741", "primary_object_url": { "basename": "ElectronicThesis.pdf", "content": "final", "filesize": 7845852, "license": "other", "mime_type": "application/pdf", "url": "/3742/1/ElectronicThesis.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Design and Development of New Enantioselective Organocatalytic Transformations, a Two-Step Synthesis of Carbohydrates, and Progress toward the Total Synthesis of Callipeltoside C", "author": [ { "family_name": "Northrup", "given_name": "Alan Bowers", "clpid": "Northrup-Alan-Bowers" } ], "thesis_advisor": [ { "family_name": "MacMillan", "given_name": "David W. C.", "clpid": "MacMillan-D-W-C" } ], "thesis_committee": [ { "family_name": "Dervan", "given_name": "Peter B.", "clpid": "Dervan-P-B" }, { "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 LUMO-lowering activation of a,b-unsaturated ketones has been accomplished through the development of a new imidazolidinone organocatalyst. That new imidazolidinone catalyst provided the first enantioselective catalytic Diels-Alder reaction with simple ketone dienophiles. Significantly, that catalyst is able to activate both cyclic and acyclic a, b-unsaturated ketones in this cycloaddition process.
\r\n\r\nA new strategy for the synthesis of two privileged structural motifs, the polyketide and polyglycolate architectures, has been developed based on the direct aldehyde aldol reaction. Two different catalysts are presented that are capable of performing the enantioselective direct aldol cross coupling of two distinct aldehyde components. Imidazolidinones have been shown for the first time to initiate the HOMO-raising activation of both saturated and a, b-unsaturated aldehyde substrates. Using an imidazolidinone catalyst, the first direct enantioselective catalytic aldol coupling of two aldehydes is described and provides synthetically valuable b-hydroxy dimethylacetals. Later, proline was found to be an exceptionally effective catalyst for the direct aldehyde aldol reaction. In contrast to imidazolidinone catalysts, proline affords b-hydroxyaldehyde products that are primed for use directly in subsequent aldol reactions.
\r\n\r\nUtilizing those direct aldehyde aldol methodologies, a two-step synthesis of 2,4,6-O-protected carbohydrates has been developed. Importantly, this modular strategy is capable of producing highly enantioenriched differentially protected forms of glucose, mannose, allose, mannosamine, as well as unnatural hexose derivatives. Furthermore, this method for sugar synthesis has been applied to the construction of differentially protected 13C6-labeled glucose, mannose, and allose in just four steps from labeled ethylene glycol.
\r\n\r\nThe enantioselective catalytic direct aldehyde aldol reaction was further applied toward the total synthesis of the marine natural product callipeltoside C. Several key fragments have been successfully synthesized and coupled to form macrolactone precursors. Nozaki-Hiyama-Kishi ring closure across the C-9/C-10 bond, however, affords exclusively the undesired C-9 epimer. Therefore, completion of the total synthesis will require a revised order for fragment assembly.
\r\n", "doi": "10.7907/g84s-an43", "publication_date": "2005", "thesis_type": "phd", "thesis_year": "2005" }, { "id": "thesis:2053", "collection": "thesis", "collection_id": "2053", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05252005-143409", "primary_object_url": { "basename": "MMTTitle.pdf", "content": "final", "filesize": 32869, "license": "other", "mime_type": "application/pdf", "url": "/2053/13/MMTTitle.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "The Molecular Recognition of DNA by Novel Heterocycles", "author": [ { "family_name": "Marques", "given_name": "Michael Anthony", "clpid": "Marques-Michael-Anthony" } ], "thesis_advisor": [ { "family_name": "Dervan", "given_name": "Peter B.", "orcid": "0000-0001-8852-7306", "clpid": "Dervan-P-B" } ], "thesis_committee": [ { "family_name": "Stoltz", "given_name": "Brian M.", "orcid": "0000-0001-9837-1528", "clpid": "Stoltz-B-M" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "orcid": "0000-0001-5661-1714", "clpid": "Hsieh-Wilson-L-C" }, { "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": "With a rapid movement toward personalized genetic medicine, tailoring treatment to individual patient needs based on their genetic code is becoming an important goal. The ability to develop small molecules capable of reprogramming the cellular machinery at the genetic level is one approach to the difficult challenge of treating diseases that result from aberrant gene expression. Inspired by the architecture of the natural products netropsin and distamycin, polyamides are capable of binding the DNA minor groove with high affinity and fidelity. Originally composed of five-membered heterocyclic carboxamides, polyamides have evolved in both form and function. A search has been initiated to develop new DNA specific oligomers that have different electronic and geometric properties. Alteration of these properties may lead to a new class of compounds, capable of targeting DNA sequences that have previously been shown to be difficult to recognize. Second-generation compounds containing novel heterocyclic recognition elements, within the context of both 5-membered heterocyclic carboxamides and fused 6-5 benzimidazole analogues, have recently been developed. These molecules have successful DNA recognition profiles as well as favorable cell uptake properties, important considerations when searching for effective pharmacophores. These new classes of rationally designed oligomers offer one approach to the challenging problem of regulating gene expression.", "doi": "10.7907/GHEJ-A648", "publication_date": "2005", "thesis_type": "phd", "thesis_year": "2005" }, { "id": "thesis:2051", "collection": "thesis", "collection_id": "2051", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05252005-100011", "primary_object_url": { "basename": "00ATPK2005Thesis.pdf", "content": "final", "filesize": 21858313, "license": "other", "mime_type": "application/pdf", "url": "/2051/1/00ATPK2005Thesis.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "DNA-Templated Dimerizations of Minor Groove-Binding Polyamides", "author": [ { "family_name": "Poulin-Kerstien", "given_name": "Adam Thomas", "clpid": "Poulin-Kerstien-Adam-Thomas" } ], "thesis_advisor": [ { "family_name": "Dervan", "given_name": "Peter B.", "orcid": "0000-0001-8852-7306", "clpid": "Dervan-P-B" } ], "thesis_committee": [ { "family_name": "Mayo", "given_name": "Stephen L.", "orcid": "0000-0002-9785-5018", "clpid": "Mayo-S-L" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "orcid": "0000-0001-5661-1714", "clpid": "Hsieh-Wilson-L-C" }, { "family_name": "Dervan", "given_name": "Peter B.", "orcid": "0000-0001-8852-7306", "clpid": "Dervan-P-B" }, { "family_name": "Grubbs", "given_name": "Robert H.", "orcid": "0000-0002-0057-7817", "clpid": "Grubbs-R-H" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Polyamides have emerged as a class of small molecules capable of binding the minor groove of DNA with high affinity and sequence specificity that have potential applications in molecular biology and human medicine. In efforts towards the use of polyamides in living cells, we report research directed towards DNA-templated formations of polyamide dimers. We find that formation of polyamide dimers, linked both turn-to-turn and turn-to-tail, can be templated via a 1,3-dipolar cycloaddition using a targeted sequence of DNA. The dimer products formed in situ may prove to have interesting biological effects.
\r\n\r\nAlso reported in this thesis are several uses of polyamides as molecular tools. We find that polyamide-biotin conjugates are able to selectively bind and capture targeted pieces of DNA via streptavidin-coated magnetic beads, effectively enriching mixtures of DNA fragments in the fragment of interest. Such molecules may find utility in the identification of DNA-protein complexes. In a second utility we report the use of polyamide-maleimide and chlorambucil conjugates to impart sequence specificity on nonspecific DNA enzymes for crystallographic studies.
", "doi": "10.7907/em7t-g660", "publication_date": "2005", "thesis_type": "phd", "thesis_year": "2005" }, { "id": "thesis:2005", "collection": "thesis", "collection_id": "2005", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05242005-172213", "primary_object_url": { "basename": "00_Introduction.pdf", "content": "final", "filesize": 126053, "license": "other", "mime_type": "application/pdf", "url": "/2005/1/00_Introduction.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Investigations of Ion Channel Structure and Function. I. Studies of Nicotine Binding to the Acetylcholine Receptor. II. Development of Tools for Studying Learning and Memory with Unnatural Amino Acids", "author": [ { "family_name": "Petersson", "given_name": "Ernest James", "orcid": "0000-0003-3854-9210", "clpid": "Petersson-Ernest-James" } ], "thesis_advisor": [ { "family_name": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" } ], "thesis_committee": [ { "family_name": "Goddard", "given_name": "William A., III", "clpid": "Goddard-W-A-III" }, { "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": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "This dissertation can be divided into two main sections:
\r\n\r\nI. In previous studies, we have used fluorinated tryptophan derivatives to conclusively identify a cation-pi interaction with Trp 149 in the binding of acetylcholine (ACh) to the muscle-type nicotinic acetylcholine receptor (nAChR). We have incorporated mimics of ACh, termed tethered agonists, in the binding site to produce self-activating channels. Using tertiary tethered agonists that would only become cations and activate the channel when protonated, we identified a perturbed pKa for the binding pocket, which has implications for the binding of tertiary agonists like nicotine (Nic). It has been shown that Nic does not participate in a straight-forward cation-pi interaction as ACh does. We have examined a hydrogen bond between the Nic pyrrolidine N-H and the backbone carbonyl of Trp149 by introducing an ester linkage at this point, weakening the carbonyl H-bond accepting ability. Calculations performed on hydrogen bound complexes of ACh, Nic, and the Nic analog epibatidine (Epi) explain the trends observed for ligand activation of the nAChR. Expanding upon this study, we have performed molecular dynamics (MD) simulations of models of the ligand binding domain of the nAChR. Ligand-bound structures from these simulations have been taken on to quantum mechanical/molecular mechanical (QMMM) calculations to model the effects of unnatural amino acid mutations in an environment that simulates the full nAChR binding pocket.
\r\n\r\nII. The nAChR is essential to neurotransmission at the junction between nerve and muscle cells, and it plays an important role in many central nervous system processes. However, its role in learning and memory is limited, at least in our current molecular models of these events. In a sense, the formation of a memory consists of the strengthening of some synaptic connections and the weakening of others. These processes, termed long term potentiation (LTP) and depression (LTD) respectively, are primarily governed by modifications to glutamate receptors (GluRs). We have developed tools for studying the mechanism and timecourse of these modifications (of phosphorylation in particular), and we have demonstrated the first incorporation of unnatural amino acids into a GluR.
", "doi": "10.7907/yhty-re87", "publication_date": "2005", "thesis_type": "phd", "thesis_year": "2005" }, { "id": "thesis:2819", "collection": "thesis", "collection_id": "2819", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-07072004-222212", "primary_object_url": { "basename": "cjb-thesis.pdf", "content": "final", "filesize": 2586913, "license": "other", "mime_type": "application/pdf", "url": "/2819/7/cjb-thesis.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Investigations in Enantioselective Catalysis. Development of Novel Asymmetric Organocatalytic Reactions", "author": [ { "family_name": "Borths", "given_name": "Christopher J.", "clpid": "Borths-Christopher-J" } ], "thesis_advisor": [ { "family_name": "MacMillan", "given_name": "David W. C.", "clpid": "MacMillan-D-W-C" } ], "thesis_committee": [ { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" }, { "family_name": "Stoltz", "given_name": "Brian M.", "clpid": "Stoltz-B-M" }, { "family_name": "MacMillan", "given_name": "David W. C.", "clpid": "MacMillan-D-W-C" }, { "family_name": "Peters", "given_name": "Jonas C.", "clpid": "Peters-J-C" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "A new strategy for the catalysis of organic transformations using iminium ion activation has been developed. Using this strategy, the first asymmetric organocatalytic Diels-Alder reaction has been developed. This methodology has demonstrated the possibility of an imidazolidinone salt to function as an effective asymmetric catalyst for a wide variety of chemical transformations.
\r\n\r\nThe iminium ion activation strategy has also proved successful for conjugate additions, and an asymmetric organocatalytic Mukaiyama-Michael reaction has been developed using the principles of LUMO-lowering catalysis. A more reactive and selective chiral imidazolidinone catalyst was developed, and this secondary amine has extended the range of transformations possible with iminium ion catalysis.
\r\n\r\nProgress has been made towards the development of an enantioselective organocatalytic alpha-oxidation of ketones. Proline catalysis has been demonstrated to effectively catalyze the asymmetric alpha-oxidation of cyclohexanone, but extension of this methodology to other ketones has not been successful. These studies have further demonstrated the utility of proline as a catalyst, and provide a platform for the extension of HOMO-raising catalysis to other organic transformations.
\r\n", "doi": "10.7907/bhq4-ep12", "publication_date": "2005", "thesis_type": "phd", "thesis_year": "2005" }, { "id": "thesis:735", "collection": "thesis", "collection_id": "735", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-02242005-174252", "primary_object_url": { "basename": "FinalThesis.pdf", "content": "final", "filesize": 178174, "license": "other", "mime_type": "application/pdf", "url": "/735/1/FinalThesis.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Iminium and Enamine Activation: Methods for Enantioselective Organocatalysis", "author": [ { "family_name": "Brown", "given_name": "Sean Pomeroy", "clpid": "Brown-Sean-Pomeroy" } ], "thesis_advisor": [ { "family_name": "MacMillan", "given_name": "David W. C.", "clpid": "MacMillan-D-W-C" } ], "thesis_committee": [ { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" }, { "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": "Lewis", "given_name": "Nathan Saul", "clpid": "Lewis-N-S" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Further development of an organocatalytic LUMO-lowering activation strategy utilizing chiral imidazolidinone salts has been described. Enantioselective catalytic Friedel-Crafts alkylations of furans and thiophenes have been achieved with good yields and high levels of enantioselectivity. Furthermore, this methodology has been utilized to access enantioenriched a-chiral esters.
\r\n\r\nThe organocatalytic iminium activation strategy has been applied to the development of an enantioselective Mukaiyama-Michael reaction for the construction of the g-butenolide architecture. This reaction is viable due to imidazolidinone catalysts' ability to partition silyloxyfurans to react through an unprecedented 1,4-addition manifold to a,b-unstaturated aldehydes. This Mukaiyama-Michael methodology has also been extended to provide access to a-amino acids by use of silyloxyoxazoles.
\r\n\r\nEnamine activation of aldehydes has provided the first direct asymmetric a-oxidation of carbonyls. This proline catalyzed HOMO-raising activation strategy affords high levels of reaction efficiency and enantioselectivity. Moreover, the function of proline solubility has been investigated to explain an unusual kinetic and enantioselective profile.
\r\n\r\nThe imidazolidinone framework, developed for iminium activation, was also demonstrated to participate in enamine activation of aldehydes to perform the enantioselective a-chlorination of aldehydes. A first generation catalyst provided good yields and high enantioselectivities at -30 \u00b0C. Design of a second generation catalyst afforded high levels of reaction efficiency and enantioselectivity at ambient temperature.
", "doi": "10.7907/SPVW-N190", "publication_date": "2005", "thesis_type": "phd", "thesis_year": "2005" }, { "id": "thesis:4123", "collection": "thesis", "collection_id": "4123", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-10172004-184202", "primary_object_url": { "basename": "Best_Thesis_Complete.pdf", "content": "final", "filesize": 6576686, "license": "other", "mime_type": "application/pdf", "url": "/4123/9/Best_Thesis_Complete.pdf", "version": "v4.0.0" }, "type": "thesis", "title": "Localization of DNA-Binding Polyamides in Living Cells", "author": [ { "family_name": "Best", "given_name": "Timothy Patrick", "clpid": "Best-Timothy-Patrick" } ], "thesis_advisor": [ { "family_name": "Dervan", "given_name": "Peter B.", "clpid": "Dervan-P-B" } ], "thesis_committee": [ { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" }, { "family_name": "Parker", "given_name": "Carl Stevens", "clpid": "Parker-C-S" }, { "family_name": "Dervan", "given_name": "Peter B.", "clpid": "Dervan-P-B" }, { "family_name": "Fraser", "given_name": "Scott E.", "clpid": "Fraser-S-E" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "Regulation of the processing of genes into nucleic acids and proteins is a substantial goal in medicine. Small molecules that could enter cells, localize to the nucleus, and bind chromosomal DNA sequence-specifically and with high affinity would be important tools for gene regulation. Pyrrole-imidazole polyamides are small molecules that bind the minor groove of DNA in a sequence-specific fashion according to a set of pairing rules, and with affinities rivaling natural transcription factors. Several in vitro experiments have shown that by directly competing with transcription factors for binding sites in gene promoter regions, polyamides can act to inhibit transcription of those genes. Polyamides bearing transcription activation domains can bind to promoter regions, recruit the transcriptional machinery to the gene, and activate transcription in vitro. Attempts to reproduce these results in vivo were largely unsuccessful, perhaps due to poor cellular trafficking properties of polyamides and polyamide-peptide conjugates.
\r\n\r\nIt was found that polyamides bearing the Bodipy fluorophore localize primarily to the cytoplasm of cells, or were excluded from cells altogether. In attempts to overcome this quality, peptides shown to improve cellular trafficking were appended to the polyamides. These peptides were generally not successful at inducing uptake, and were in many cases toxic to the cells. Small molecules were also appended to polyamides, likewise to improve uptake properties, but met with limited success. Surprisingly, the addition of a fluorescein or fluorescein-like fluorophore to polyamides permit them to localize to the nuclei of all cell lines tested, in a molecular content- and shape-dependent manner. This technology has been applied to several in vivo experiments, including the inhibition of androgen receptor binding to its cognate element in gene promoter regions.
", "doi": "10.7907/MQ0D-8P65", "publication_date": "2005", "thesis_type": "phd", "thesis_year": "2005" }, { "id": "thesis:387", "collection": "thesis", "collection_id": "387", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-01282004-181526", "primary_object_url": { "basename": "JJMW_thesis.pdf", "content": "final", "filesize": 26165225, "license": "other", "mime_type": "application/pdf", "url": "/387/7/JJMW_thesis.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Design and Development of New Enantioselective Catalytic Reactions and Progress towards the Total Synthesis of Callipeltoside", "author": [ { "family_name": "Wiener", "given_name": "John Jacob Moely", "clpid": "Wiener-John-Jacob-Moely" } ], "thesis_advisor": [ { "family_name": "MacMillan", "given_name": "David W. C.", "clpid": "MacMillan-D-W-C" } ], "thesis_committee": [ { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" }, { "family_name": "MacMillan", "given_name": "David W. C.", "clpid": "MacMillan-D-W-C" }, { "family_name": "Grubbs", "given_name": "Robert H.", "clpid": "Grubbs-R-H" }, { "family_name": "Marcus", "given_name": "Rudolph A.", "clpid": "Marcus-R-A" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The development of a new enantioselective catalytic anti aldol reaction is described. In this Lewis acid-catalyzed process, a chiral metal-ligand enolate complex is accessed through soft-enolization and reacts with an aldehyde to form aldol adducts in good enantioselectivity and anti diastereoselection. Mechanistic studies confirm the non-Mukaiyama pathway involving a reactive metal enolate species. Investigations have shown that the choice of amine base has a remarkable effect on the mechanism and outcome of the reaction.
\r\n\r\nThe development of the first enantioselective organocatalytic [1,3]-dipolar cycloaddition reaction is also reported. In this imidazolidinone-catalyzed process, nitrones react with a,b-unsaturated aldehydes to form chiral isoxazolidines in excellent yield, enantioselectivity, and diastereoselection. The scope of this process appears quite general with respect to both the nitrone and aldehyde components of the reaction. A second-generation imidazolidinone catalyst offers improved reaction rates and selectivities and has also facilitated the development of the first exo selective organocatalytic [1,3]-dipolar and Diels-Alder cycloaddition reactions.
\r\n\r\nA synthetic approach towards the marine natural product callipeltoside A is described. The synthesis relies upon rapid construction of the stereochemical backbone through a novel tandem amino-sulfide acyl-Claisen rearrangement. Subsequent elaboration towards the macrolide has involved a highly diastereoselective reductive opening of a spirocyclic intermediate, highly diastereoselective Ireland Claisen rearrangement, and synthesis of the tetrahydropyran moiety through a palladium catalyzed carbonylative cyclization. Completion of the synthesis has yet to be achieved due to difficulties in removal of a benzyl ether protecting group.
", "doi": "10.7907/KA4F-XJ24", "publication_date": "2004", "thesis_type": "phd", "thesis_year": "2004" }, { "id": "thesis:1893", "collection": "thesis", "collection_id": "1893", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05202004-174324", "primary_object_url": { "basename": "ALL_THESIS_2.pdf", "content": "final", "filesize": 8738128, "license": "other", "mime_type": "application/pdf", "url": "/1893/1/ALL_THESIS_2.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Prediction of Structure, Function, and Spectroscopic Properties of G-Protein-Coupled Receptors: Methods and Applications", "author": [ { "family_name": "Trabanino", "given_name": "Rene Jouvanni", "clpid": "Trabanino-Rene-Jouvanni" } ], "thesis_advisor": [ { "family_name": "Goddard", "given_name": "William A., III", "clpid": "Goddard-W-A-III" } ], "thesis_committee": [ { "family_name": "Campbell", "given_name": "Judith L.", "clpid": "Campbell-J-L" }, { "family_name": "Rees", "given_name": "Douglas C.", "clpid": "Rees-D-C" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" }, { "family_name": "Goddard", "given_name": "William A., III", "clpid": "Goddard-W-A-III" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "G-protein-coupled receptors are of great pharmaceutical interest, comprising the majority of targets for currently marketed drugs. The theme of my thesis is the development of the structure prediction method, MembStruk, for the superfamily of G-protein-coupled receptors. The first part of this thesis focuses on the methods and their validation. There are several steps involved in MembStruk that are detailed and tested for membrane proteins with known structures in the first few chapters (Chapters 2-6). Specifically, the first principles methods for predicting the transmembrane helical ranges and the helix hydrophobic centers are tested. The program for predicting the transmembrane helical ranges, TM2ndS, ranks in the top two when comparing performance with other top prediction methods. And because it is based on general principles, it can be applied robustly for membrane protein families for which little structural information is available. The simulation of the EC-II closing is also tested on bovine rhodopsin. The use of the MembStruk method on bovine rhodopsin as a validation case is presented in detail (Chapter 2). The large majority (71%) of the residues involved in binding in rhodopsin are predicted and the protein structure itself is 2.84 \u00c5 coordinate root mean square error in the transmembrane main chain atoms from the crystal structure.
\r\n\r\nThe second part of the thesis discusses applications on various G-protein-coupled receptor systems. The application of the MembStruk method to other peptide chemokine G-protein-coupled receptors like CCR1 and CCR5 is discussed in Chapter 9. The fundamental scientific problems of G-protein-coupled receptor modulation of absorption and relaxation properties of a bound chromophore (retinal) are addressed and results are presented for the predictions of these properties.
\r\n\r\nThe prediction of structure and function of G-protein-coupled receptors would allow for structure-based drug design and a rational approach to reducing drug cross-reactivity across receptor families.
", "doi": "10.7907/VHED-4063", "publication_date": "2004", "thesis_type": "phd", "thesis_year": "2004" }, { "id": "thesis:3678", "collection": "thesis", "collection_id": "3678", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-09212004-154502", "primary_object_url": { "basename": "Robinson_ne_2004.pdf", "content": "final", "filesize": 18675174, "license": "other", "mime_type": "application/pdf", "url": "/3678/1/Robinson_ne_2004.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Investigations of Peptide and Protein Deamidation", "author": [ { "family_name": "Robinson", "given_name": "Noah Edward", "clpid": "Robinson-Noah-Edward" } ], "thesis_advisor": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Beauchamp", "given_name": "Jesse L.", "clpid": "Beauchamp-J-L" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" }, { "family_name": "Richards", "given_name": "John H.", "clpid": "Richards-J-H" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "This work concerns the peptide and protein deamidation of asparaginyl, Asn, and glutaminyl, Gln, residues to aspartyl and glutamyl residues, respectively. This is accompanied by a change in charge and increase of one unit in mass. Isomerization also often takes place. This reaction--which occurs spontaneously and non-enzymatically in many proteins under physiological conditions--has been hypothesized to be a general molecular clock, which could be used as a timer for protein turnover, development, or aging, and for the controlled conversion of one protein into another. Since this hypothesis was first proposed, amide molecular clocks have been demonstrated in several biological systems. These include the timing of protein turnover in rat cytochrome c and aldolase; the counting of individual enzyme molecule catalytic cycles in triosephosphate isomerase; and the resetable time-dependent monitoring of DNA repair in Bcl-XL.
\r\n\r\nThe objective of this work is to increase the understanding of peptide and protein deamidation, especially the prediction of deamidation rates for specific amides in proteins. This capability has been developed for Asn residues in proteins of known three-dimensional structure, and the sequence information required to do this for Gln residues has also been obtained. This work provides stability information to protein chemists and needed information to those who continue to investigate the molecular clock hypothesis.
\r\n\r\nThe problem was attacked in two phases. The first consisted of experimental work to determine the rate of deamidation as a function of primary sequence near the amide. All 400 possible peptides of the type GlyXxxAsnYyyGly were synthesized and the deamidation rates of 323 of these were measured. An additional 67 asparaginyl containing peptides that had a variety of lengths and forms were also measured in order to assess the efficacy of pentapeptides in this work. Also, a similar set of 400 peptides were synthesized with Gln in place of Asn. The deamidation rates of 52 of the Gln peptides have been measured and, from these rates, the rates of the other Gln peptides have been inferred. With the addition of rates for 34 peptides with blocked Cys, a total of 476 individual rate experiments on peptides have been carried out in 0.15 M Tris buffer at pH 7.4, 37.00 [degrees]C. Additional rate experiments were conducted on solution effects and on properties of the experimental system.
\r\n\r\nThe second phase involved using experimental data from the literature--on relative instabilities of specific amides in proteins under specified solvent conditions and on protein three-dimensional structures--to quantify the effect that secondary, tertiary, and quaternary protein structure has on primary structure deamidation rates.
\r\n\r\nFactors that determine deamidation rates include structural components as well as electronic effects. It happens that the primary sequence component is so large that it is possible to separate it from the rest of the problem and measure it alone. The secondary, tertiary, and quaternary effects were then considered. It was found that, on average, 60% of the rate is accounted for by primary sequence, while the remaining 40% is ascribable to secondary, tertiary, and quaternary structure in those proteins for which suitable deamidation observations are available. For all proteins, the estimated values are 50% and 50%. For individual amides, these percentages vary over a wide range.
\r\n\r\nA procedure was developed through which the quantitative deamidation rate of any amide in a protein for which the three-dimensional structure is known can be calculated. These estimated rates are accurate to within about a factor of two or better for most amides with half-times less than 100 days. They can be used to predict the fastest amides in a protein to greater than 95% reliability. This reliability is remarkable, considering the fact that deamidation half-times vary from less than 1 day to over 100 years in 37.00 [degrees]C, pH 7.4, 0.15 M Tris buffer. The procedure was calibrated with only relative amide instabilities. It was then evaluated with absolute deamidation rates.
\r\n\r\nDetails of this work are given in the pages which follow. The result was a computerized procedure through which deamidation rates for every Asn of every protein in the PDB Protein Database were calculated and analyzed.
\r\n\r\nThis work provides two discoveries of biological importance.
\r\n\r\nFirst, the assumption of genetic determination of deamidation rates, which has until now been dependent upon scattered and largely qualitative observations, has been placed upon firm quantitative foundation with a thorough understanding of the effects of primary, secondary, and tertiary structure.
\r\n\r\nSecond, this quantitative understanding has permitted reliable estimation of the distribution functions of deamidation rates in thousands of proteins. These rates show that a substantial fraction of proteins are genetically programmed to deamidate in biologically relevant timed intervals even though most of the genetically available structures deamidate more slowly. This quantitative preference for fast deamidation times relative to slow deamidation times is an entirely new discovery. The fact that this property is present within the thousands of proteins of currently known three-dimensional structure markedly strengthens the molecular clock hypothesis.
\r\n", "doi": "10.7907/JHAP-JG47", "publication_date": "2004", "thesis_type": "phd", "thesis_year": "2004" }, { "id": "thesis:1599", "collection": "thesis", "collection_id": "1599", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05042004-203854", "primary_object_url": { "basename": "Preface.pdf", "content": "final", "filesize": 277016, "license": "other", "mime_type": "application/pdf", "url": "/1599/8/Preface.pdf", "version": "v5.0.0" }, "type": "thesis", "title": "Structure and Function Studies of the Human Dopamine Receptors", "author": [ { "family_name": "Kalani", "given_name": "M. Yashar S.", "orcid": "0000-0002-5923-1255", "clpid": "Kalani-M-Yashar-S" } ], "thesis_advisor": [ { "family_name": "Goddard", "given_name": "William A., III", "clpid": "Goddard-W-A-III" } ], "thesis_committee": [ { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Goddard", "given_name": "William A., III", "clpid": "Goddard-W-A-III" }, { "family_name": "Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" }, { "family_name": "Marcus", "given_name": "Rudolph A.", "clpid": "Marcus-R-A" }, { "family_name": "Vaidehi", "given_name": "Nagarajan", "clpid": "Vaidehi-N" }, { "family_name": "Patterson", "given_name": "Paul H.", "clpid": "Patterson-P-H" }, { "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": "Dopamine neurotransmitter and its receptors play a critical role in cell signaling process responsible for information transfer in neurons functioning in the nervous system. Development of improved therapeutics for such disorders as Parkinson's and schizophrenia would be significantly enhanced with the availability of the three-dimensional (3-D) structure for the dopamine receptors and of the binding site for dopamine and other agonists and antagonists. In this thesis, I report the 3-D structures of the 5 subtypes of the human dopamine receptors, predicted from primary sequence using first principles theoretical and computational techniques. I use the term \"first principles\" to mean that we do not use the high resolution crystal structure of rhodopsin as a template, nor do we use homology modeling or threading of any kind to determine the structure. Predicting the binding sites, and the relative binding affinities of endogenous ligands and various pharmaceuticals to the 5 receptors validates the predicted structures. These structures correctly predict the critical residues for binding dopamine and several antagonists, identified by mutation studies and give relative binding affinities that correlate well with experiment. The predicted binding site for dopamine and agonists is located between transmembrane helices (TM) 3, 4, 5, and 6, while the best antagonists bind to a site involving TM helices 2, 3, 4, 6, and 7 with minimal contacts to TM 5. We identify characteristic differences between the binding sites of agonists and antagonists, as well as factors that cause differential binding to the 5 subtypes of the human dopamine receptors.
\r\n\r\nThis thesis consists of five chapters that have, or will shortly result in publications. The first chapter is a brief introduction to the field, the motivation for the project, my scientific contributions, and contribution of others on the team. Chapter two introduces the methods and their successes at reproducing experimentally known results for the human D2 dopamine receptor; it discusses, in great detail, the active site of pharmaceutical agonists and antagonists to the human D2 dopamine receptor, and highlights the strengths and shortcomings of homology modeling for membrane bound proteins; this chapter will be submitted for publication to the Journal of Molecular Biology. Chapter three reports the results of a blind study performed in collaboration with Aventis Pharmaceuticals. For this study, we were provided with the two-dimensional structure of 9 antagonists and were asked to predict their binding sites, binding affinities, and to explain the differential binding of the ligands to the human D2 and D3 dopamine receptors and the human a1A adrenergic receptor. The results of this study are in preparation for submission to the Journal of Medicinal Chemistry. Chapters four and five of the thesis give preliminary results of comparative studies of the agonist and antagonist binding sites of the five subtypes of the human dopamine receptors. Chapter 6 contains results of another blind study on the G2A receptor with Professor Owen Witte.
\r\n\r\nIn addition to the six main chapters, this thesis contains 6 independent appendices that report results of similar studies in other systems. The first 2 appendices are work that has already been published. The remaining 4 appendices will shortly result in publications, but at this time, they are not publication worthy; these appendices represent data that has been analyzed but has not been written in paper format.
\r\n\r\nIn addition, I would like to make note of the studies that I have conducted on the 9 subtypes of the human adrenergic receptors with Mr. Peter Freddolino, the 4 human histamine receptors that were conducted with Mr. Freddolino and Mr. Maziyar Kalani, and the 4 G2A-like lipid receptors conducted with Mr. Rene Trabanino, Dr. Radu, Dr. Yang, and Professor Owen Witte of the Howard Hughes Medical Institute at the David Geffen School of Medicine at the University of California, Los Angeles.
", "doi": "10.7907/T6NV-7W30", "publication_date": "2004", "thesis_type": "phd", "thesis_year": "2004" }, { "id": "thesis:1693", "collection": "thesis", "collection_id": "1693", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05092003-135830", "primary_object_url": { "basename": "title_TOC.pdf", "content": "final", "filesize": 85190, "license": "other", "mime_type": "application/pdf", "url": "/1693/6/title_TOC.pdf", "version": "v3.0.0" }, "type": "thesis", "title": "Detection of DNA by Sequence Specific Fluorescent Polyamides", "author": [ { "family_name": "Rucker", "given_name": "Victor Clay", "clpid": "Rucker-Victor-Clay" } ], "thesis_advisor": [ { "family_name": "Rutledge", "given_name": "David B.", "clpid": "Rutledge-D-B" } ], "thesis_committee": [ { "family_name": "Grubbs", "given_name": "Robert H.", "clpid": "Grubbs-R-H" }, { "family_name": "Gray", "given_name": "Harry B.", "clpid": "Gray-H-B" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "clpid": "Hsieh-Wilson-L-C" }, { "family_name": "Dervan", "given_name": "Peter B.", "clpid": "Dervan-P-B" }, { "family_name": "Rutledge", "given_name": "David B.", "clpid": "Rutledge-D-B" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "This thesis covers the use of hairpin polyamides to achieve, most notably, HIV-1 LTR gene regulation and fluorescent detection of double stranded DNA. In Chapter 2 we discuss our collaboration with Professor David Margolis to study integrated HIV-1 latency in quiescent T-lymphocytes. Understanding latency in HIV-1 infection is of paramount importance for developing anti-HIV-1 therapeutics. Chapter 3 deals with the characterization of a special case of 2-beta-2 polyamide binding in the minor groove, and we discuss the use of (S)-2,4-diaminobutyric acid to influence polyamide specificity and orientation. In Chapter 4 we present data concerning the use of hairpin polyamides that, when unbound to DNA, quench the fluorescence of the xanthene fluorphore to which they are covalently attached. We cover experiments aimed at exploring the uses of this fluorescence phenomenon to optically detect double stranded DNA in a sequence specific manner, an issue of great importance as shown in the literature by the numerous denaturing assays for oligo detection by hybridization. In Chapter 5, the fruits of a collaboration with Alexander Dunn of Professor Gray's group, we attempt to define the mechanism whereby polyamides quench tetramethyl rhodamine fluorescence.", "doi": "10.7907/93KR-9543", "publication_date": "2003", "thesis_type": "phd", "thesis_year": "2003" }, { "id": "thesis:2990", "collection": "thesis", "collection_id": "2990", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-07312002-120928", "primary_object_url": { "basename": "lintongli-thesis.pdf", "content": "final", "filesize": 1959356, "license": "other", "mime_type": "application/pdf", "url": "/2990/1/lintongli-thesis.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "The Tethered Agonist Approach to Mapping Ion-Channel Proteins: Toward a Structural Model for the Agonist-Binding Site of the Nicotinic Acetylcholine Receptor", "author": [ { "family_name": "Li", "given_name": "Lintong", "clpid": "Li-Lintong" } ], "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": "Dougherty", "given_name": "Dennis A.", "clpid": "Dougherty-D-A" }, { "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": "Chan", "given_name": "Sunney I.", "clpid": "Chan-S-I" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "The integral membrane proteins of neurons and other excitable cells are generally resistant to high-resolution structural tools. In this thesis we present our efforts to probe the structure of the agonist-binding site of the nicotinic acetylcholine receptor (nAChR) using the tethered agonist approach, which combines chemical synthesis, the nonsense suppression methodology for unnatural amino acid incorporation and electrophysiology.
\r\n\r\nIn Chapter 2, we present the results of incorporating a series of tethered quaternary ammonium derivatives of tyrosine into the nAChR using the in vivo nonsense suppression methodology for incorporating unnatural amino acids site-specifically. At three sites, a constitutively active receptor results, but the pattern of activation as a function of chain length is different. At position alpha149, there is a clear preference for a three-carbon tether, while at position alpha93 tethers of 2-5 carbons are comparably effective. At position gamma55/delta57, all tethers except the shortest one can activate the receptor. Based on these and other data, a model for the binding site of the receptor can be developed by analogy to the acetylcholine esterase crystal structure.
\r\n\r\nIn Chapter 3, we report evidence that the N-terminal extracellular domain of nAChR is closely related to acetylcholine binding protein (AChBP), whose crystal structure was solved in May 2001. Based on the model obtained from docking acetylcholine into the structure of AChBP, we designed and incorporated a new tethered agonist, lysyl-carbamylcholine. Incorporation of this tethered agonist at several positions produced constitutively active receptors, with significant activity seen at alpha192, alpha193, and gamma119/delta121. These results demonstrated that the loop E residue gamma119/delta121 on the complementary subunit is very near the agonist-binding site. We also investigated the role of an intersubunit hydrogen bond, which was seen in the crystal structure of AChBP. Incorporation of tryptophan analogs that abolish the hydrogen bonding abilities slowed the desensitization of the receptor, which implied that this hydrogen bond might play a key role in the allosteric transitions of desensitization.
\r\n\r\nIn Chapter 4, we describe our efforts to prepare a short tethered agonist and the results of incorporating it into nAChR at alpha198 by chemical modification of cysteine mutants introduced by nonsense suppression methodology. Methanethiosulphonate ethyltrimethylammonium (MTSET) modification resulted in constitutive activity, which suggested the closeness of alpha198 to the agonist-binding site.
\r\n\r\nIn Chapter 5, methods in molecular biology, electrophysiology and molecular docking, and the synthesis of amino acids and dinucleotide dCA-amino acids are summarized.
", "doi": "10.7907/AMDZ-XE24", "publication_date": "2003", "thesis_type": "phd", "thesis_year": "2003" }, { "id": "thesis:1760", "collection": "thesis", "collection_id": "1760", "cite_using_url": "https://resolver.caltech.edu/CaltechETD:etd-05132002-124824", "primary_object_url": { "basename": "aycfuThesis.pdf", "content": "final", "filesize": 6107091, "license": "other", "mime_type": "application/pdf", "url": "/1760/1/aycfuThesis.pdf", "version": "v2.0.0" }, "type": "thesis", "title": "Microfabricated Fluorescence-Activated Cell Sorters (\u00b5FACS) for Screening Bacterial Cells", "author": [ { "family_name": "Fu", "given_name": "Anne Yen-Chen", "clpid": "Fu-Anne-Yen-Chen" } ], "thesis_advisor": [ { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" } ], "thesis_committee": [ { "family_name": "Tirrell", "given_name": "David A.", "orcid": "0000-0003-3175-4596", "clpid": "Tirrell-D-A" }, { "family_name": "Anderson", "given_name": "W. French", "orcid": "0000-0003-4043-1684", "clpid": "Anderson-W-F" }, { "family_name": "Arnold", "given_name": "Frances Hamilton", "orcid": "0000-0002-4027-364X", "clpid": "Arnold-F-H" }, { "family_name": "Dervan", "given_name": "Peter B.", "orcid": "0000-0001-8852-7306", "clpid": "Dervan-P-B" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "orcid": "0000-0001-5661-1714", "clpid": "Hsieh-Wilson-L-C" }, { "family_name": "Quake", "given_name": "Stephen R.", "clpid": "Quake-S-R" } ], "local_group": [ { "literal": "div_chem" } ], "abstract": "In this thesis, I have developed elastomeric microfabricated cell sorting devices using a micromachining technology, \"soft lithography\". Inexpensive elastomeric microfabricated devices were designed to replace flow chambers in conventional fluorescence-activatived cell sorters (FACS). Sorting of cells and other particles was accomplished via different means of flow control. My early work of cell sorting on these devices was accomplished using electrokinetic flow. However, in order to alleviate problems associated with electrokinetic flow, the microfabricated cell sorter was integrated with microvalves and micropumps for pneumatic actuation control. The integrated cell sorter has better capabilities of fine-tuning the flow control, manipulating single cells and is less harmful to the cells than electrokinetic flow. Substantial enrichments of beads and cells were accomplished on these devices. Novel sorting algorithms, which can only be implemented in microfabricated devices, were also demonstrated. Compared with conventional FACS, these microfabricated devices allow for more sensitive optical detection for bacterial cells and DNA, innovative sorting schemes and are disposable to eliminate any cross-contamination from previous runs. Ultimately, these elastomeric microfluidic flow cells provide an inexpensive, robust and effective way to perform cell sorting and can be used as stand-alone devices or as a part of an integrated system for diagnostics and/or cytometric measurements.
\r\n\r\nPresently, the microfabricated cell sorter is enjoying new applications in various fields for high throughput screening, including directed evolution, digital genetic circuits, microbiology and cell biology of gene expression and regulation. In addition, this sorter is not limited only to the detection of optical signals. I have attached the sorter to a high resolution magnetometer, a superconducting quantum interference device (SQUID) microscope, to obtain cytometric data of the magnetic field strengths of magnetotactic bacteria as they flowed through the device.
\r\n\r\nThis thesis lays down the foundation for future work in cell sorting and single cell analysis. Time-course measurements of a single cell for kinetic studies can be implemented using novel sorting schemes. Sample dispensing and any downstream analysis, such as cell lysis and/or polymerase chain reaction, can be carried out immediately after the cells have been sorted. The sorter could also be incorporated with other technologies to measure cellular magnetic or electrical properties. We anticipate that an integrated lab-on-chip, where cell sorting is one of the steps of a complete analysis system, is not far off.
", "doi": "10.7907/6HKD-RQ61", "publication_date": "2002", "thesis_type": "phd", "thesis_year": "2002" }, { "id": "thesis:8130", "collection": "thesis", "collection_id": "8130", "cite_using_url": "https://resolver.caltech.edu/CaltechTHESIS:03122014-104807866", "type": "thesis", "title": "Stereochemically Modified Polyamides for Recognition in the Minor Groove of DNA", "author": [ { "family_name": "Herman", "given_name": "David Matthew", "clpid": "Herman-David-Matthew" } ], "thesis_advisor": [ { "family_name": "Hoffmann", "given_name": "Michael R.", "orcid": "0000-0001-6495-1946", "clpid": "Hoffmann-M-R" } ], "thesis_committee": [ { "family_name": "Parker", "given_name": "Carl Stevens", "orcid": "0000-0001-9795-4211", "clpid": "Parker-C-S" }, { "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" }, { "family_name": "Hsieh-Wilson", "given_name": "Linda C.", "orcid": "0000-0001-5661-1714", "clpid": "Hsieh-Wilson-L-C" }, { "family_name": "Hoffmann", "given_name": "Michael R.", "orcid": "0000-0001-6495-1946", "clpid": "Hoffmann-M-R" } ], "local_group": [ { "literal": "div_biol" } ], "abstract": "The design of synthetic molecules that recognize specific sequences of DNA\r\nis an ongoing challenge in molecular medicine. Cell-permeable small molecules\r\ntargeting predetermined DNA sequences offer a potential approach for offsetting\r\nthe abnormal effects of misregulated gene-expression. Over the past twenty years,\r\nProfessor Peter B. Dervan has developed a set of pairing rules for the rational design\r\nof minor groove binding polyamides containing pyrrole (Py), imidazole (Im), and\r\nhydroxypyrrole (Hp). Polyamides have illustrated the capability to permeate cells\r\nand inhibit transcription of specific genes in vivo. This provides impetus to identify\r\nstructural elements that expand the repetoire of polyamide motifs with recognition\r\nproperties comparable to naturally occurring DNA binding proteins. Through the\r\nintroduction of chiral amino acids, we have developed chiral polyamides with\r\nstereochemically regulated binding characteristics. In addition, chiral substituents\r\nhave facilitated the development of new polyamide motifs that broaden binding site\r\nsizes targetable by this class of ligands.", "doi": "10.7907/vsf2-fe75", "publication_date": "2001", "thesis_type": "phd", "thesis_year": "2001" } ]