[ { "id": "https://authors.library.caltech.edu/records/0y901-fg157", "eprint_id": 122031, "eprint_status": "archive", "datestamp": "2023-08-20 16:48:27", "lastmod": "2023-12-13 16:39:02", "type": "monograph", "metadata_visibility": "show", "creators": { "items": [ { "id": "Mao-Runze", "name": { "family": "Mao", "given": "Runze" }, "orcid": "0000-0003-4678-7251" }, { "id": "Wackelin-Daniel-J", "name": { "family": "Wackelin", "given": "Daniel J." } }, { "id": "Jamieson-Cooper-S", "name": { "family": "Jamieson", "given": "Cooper S." }, "orcid": "0000-0002-6076-6230" }, { "id": "Rogge-Torben", "name": { "family": "Rogge", "given": "Torben" }, "orcid": "0000-0002-4519-4596" }, { "id": "Gao-Shilong", "name": { "family": "Gao", "given": "Shilong" }, "orcid": "0000-0003-2808-6283" }, { "id": "Das-Anuvab", "name": { "family": "Das", "given": "Anuvab" }, "orcid": "0000-0002-9344-4414" }, { "id": "Taylor-Doris-Mia", "name": { "family": "Taylor", "given": "Doris Mia" }, "orcid": "0000-0001-6854-7844" }, { "id": "Houk-Kendall-N", "name": { "family": "Houk", "given": "K. N." }, "orcid": "0000-0002-8387-5261" }, { "id": "Arnold-F-H", "name": { "family": "Arnold", "given": "Frances H." }, "orcid": "0000-0002-4027-364X" } ] }, "title": "Enantio- and Diastereoenriched Enzymatic Synthesis of 1,2,3-Polysubstituted Cyclopropanes from (Z/E)-Trisubstituted Enol Acetates", "ispublished": "unpub", "full_text_status": "public", "note": "This work is licensed under a Creative Commons Attribution 4.0 International License, which allows reusers to distribute, remix, adapt, and build upon the material in any medium or format, so long as attribution is given to the creator. The license allows for commercial use. \n\nSupport by the National Science Foundation Division of Molecular and Cellular Biosciences (MCB-2016137 to F.H.A) and the National Science Foundation Division of Chemistry (CHE-1764328 to K.N.H.) is gratefully acknowledged. R.M. acknowledges support from the Swiss National Science Foundation (SNSF) Early Mobility Postdoctoral Fellowship (P2ELP2_195118). D.J.W. acknowledges support fromthe National Science Foundation Graduate Research Fellowship (DGE-1745301). C.S.J. acknowledges support from the Ruth L. Kirchstein NIH Postdoctoral Fellowship (1F32GM145009). T.R. acknowledges support from the Alexander von Humboldt-Foundation (Feodor Lynen fellowship). We thank Dr. Scott C. Virgil for the maintenance of the Caltech Center for Catalysis and Chemical Synthesis (3CS). We thank Dr. Michael K. Takase and Lawrence M. Henling for assistance with X-ray crystallographic data collection. We thank Dr. Mona Shahgoli for mass spectrometry assistance. We thank Dr. David VanderVelde for the maintenance of the Caltech NMR facility. We also thank Dr. Sabine Brinkmann-Chen and Dr. Zhen Liu for their helpful discussions and comments on the manuscript. Calculations were performed on the Hoffman2 cluster at the University of California, Los Angeles. Anton 2 computer time was provided by the Pittsburgh Supercomputing Center (PSC) through Grant R01GM116961 from the National Institutes of Health. The Anton 2 machine at PSC was generously made available by D.E. Shaw Research. \n\nAuthor contributions. R.M. conceived and designed the overall project with F.H.A. providing guidance. R.M. performed the initial screening of heme proteins and the directed evolution experiments. R.M. and D.J.W. studied the substrate scope. R.M., D.J.W. and S.G. synthesized the olefin substrates 1; R.M. and D.J.W. synthesized diazoacetonitrile 2a. C.S.J. and T.R. carried out the computational studies with K.N.H providing guidance. A.D. conducted crystallization of 3j and 3k. D.M.T. performed sequencing of SSM libraries based on IC-G3. R.M., D.J.W., C.S.J. and F.H.A wrote the manuscript with input from all authors. \n\nThe authors declare no competing interests.\n\n
Submitted - nihpp-rs2802333v1.pdf
Supplemental Material - 520dc4f8c4bf764a8e35237d.pdf
", "abstract": "In nature and synthetic chemistry, stereoselective [2+1] cyclopropanation is the most prevalent strategy for the synthesis of chiral cyclopropanes, a class of key pharmacophores in pharmaceuticals and bioactive natural products. One of the most extensively studied reactions in the organic chemist's arsenal, stereoselective [2+1] cyclopropanation, largely relies on the use of stereodefined olefins, which require elaborate laboratory synthesis or tedious separation to ensure high stereoselectivity. Here we report engineered hemoproteins derived from a bacterial cytochrome P450 that catalyze the synthesis of chiral 1,2,3-polysubstituted cyclopropanes, regardless of the stereopurity of the olefin substrates used. Cytochrome P450_(BM3) variant IC-G3 exclusively converts (Z)-enol acetates to enantio- and diastereoenriched cyclopropanes and in our model reaction delivers a leftover (E)-enol acetate with 98% stereopurity, using whole Escherichia coli cells. IC-G3 was further engineered with a single mutation to enable the biotransformation of (E)-enol acetates to \u03b1-branched ketones with high levels of enantioselectivity while simultaneously catalyzing the cyclopropanation of (Z)-enol acetates with excellent activities and selectivities. We conducted docking studies and molecular dynamics simulations to understand how active-site residues distinguish between the substrate isomers and enable the enzyme to perform these distinct transformations with such high selectivities. Computational studies suggest the observed enantio- and diastereoselectivities are achieved through a stepwise pathway. These biotransformations streamline the synthesis of chiral 1,2,3-polysubstituted cyclopropanes from readily available mixtures of (Z/E)-olefins, adding a new dimension to classical cyclopropanation methods.", "date": "2023-06-29", "date_type": "published", "id_number": "CaltechAUTHORS:20230628-257200000.36", "official_url": "https://resolver.caltech.edu/CaltechAUTHORS:20230628-257200000.36", "rights": "No commercial reproduction, distribution, display or performance rights in this work are provided.", "funders": { "items": [ { "agency": "NSF", "grant_number": "MCB-2016137" }, { "agency": "NSF", "grant_number": "CHE-1764328" }, { "agency": "Swiss National Science Foundation (SNSF)", "grant_number": "P2ELP2_195118" }, { "agency": "NSF Graduate Research Fellowship", "grant_number": "DGE-1745301" }, { "agency": "NIH Postdoctoral Fellowship", "grant_number": "1F32GM145009" }, { "agency": "Alexander von Humboldt Foundation" }, { "agency": "NIH", "grant_number": "R01GM116961" } ] }, "local_group": { "items": [ { "id": "Division-of-Biology-and-Biological-Engineering" } ] }, "doi": "10.21203/rs.3.rs-2802333/v1", "pmcid": "PMC10120758", "primary_object": { "basename": "nihpp-rs2802333v1.pdf", "url": "https://authors.library.caltech.edu/records/0y901-fg157/files/nihpp-rs2802333v1.pdf" }, "related_objects": [ { "basename": "520dc4f8c4bf764a8e35237d.pdf", "url": "https://authors.library.caltech.edu/records/0y901-fg157/files/520dc4f8c4bf764a8e35237d.pdf" } ], "resource_type": "monograph", "pub_year": "2023", "author_list": "Mao, Runze; Wackelin, Daniel J.; et el." }, { "id": "https://authors.library.caltech.edu/records/ftmh1-h8573", "eprint_id": 120397, "eprint_status": "archive", "datestamp": "2023-08-20 08:32:21", "lastmod": "2023-12-13 16:38:36", "type": "monograph", "metadata_visibility": "show", "creators": { "items": [ { "id": "Athavale-Soumitra-V", "name": { "family": "Athavale", "given": "Soumitra V." }, "orcid": "0000-0001-5620-6997" }, { "id": "Gao-Shilong", "name": { "family": "Gao", "given": "Shilong" }, "orcid": "0000-0003-2808-6283" }, { "id": "Das-Anuvab", "name": { "family": "Das", "given": "Anuvab" }, "orcid": "0000-0002-9344-4414" }, { "id": "Chandra-Mallojjala-Sharath", "name": { "family": "Chandra Mallojjala", "given": "Sharath" }, "orcid": "0000-0003-0446-792X" }, { "id": "Alfonzo-Edwin", "name": { "family": "Alfonzo", "given": "Edwin" }, "orcid": "0000-0002-4112-8751" }, { "id": "Long-Yueming", "name": { "family": "Long", "given": "Yueming" } }, { "id": "Hirschi-Jennifer-S", "name": { "family": "Hirschi", "given": "Jennifer S." }, "orcid": "0000-0002-3470-0561" }, { "id": "Arnold-F-H", "name": { "family": "Arnold", "given": "Frances H." }, "orcid": "0000-0002-4027-364X" } ] }, "title": "Enzymatic Nitrogen Insertion into Unactivated C\u2013H Bonds", "ispublished": "unpub", "full_text_status": "public", "note": "The content is available under CC BY NC ND 4.0 License. \n\nWe thank Dr. Zhen Liu for assistance with CalB reactions, Dr. Michael K. Takashe for assistance with collecting X-ray single crystal data, Ziyang Qin for assistance with synthesis and Dr. Mona Shahgholi for HRMS analysis. \n\nThis research is supported by the Department of Energy (DOE250078) to F.H.A.and by a Ruth Kirschstein NIH Postdoctoral Fellowship (F32GM143799) to E.A.. Financial support from the XSEDE Science Gateway Program (under the NSF grant number ACI-1548562, CHE180061 and CHE210031) (J.S.H. and M.S.C.) and the National Institutes of Health under R15 GM142103 (J.S.H.) is acknowledged.\n\nSubmitted - enzymatic-nitrogen-insertion-into-unactivated-c-h-bonds.pdf
Supplemental Material - si-enzymatic-nitrogen-insertion-into-unactivated-c-h-bonds.pdf
", "abstract": "Selective functionalization of aliphatic C\u2013H bonds, ubiquitous in molecular structures, could allow ready access to diverse chemical products. While enzymatic oxygenation of C\u2013H bonds is well established, the analogous enzymatic nitrogen functionalization is still unknown; nature is reliant on pre-oxidized compounds for nitrogen incorporation. Likewise, synthetic methods for selective nitrogen derivatization of unbiased C\u2013H bonds remain elusive. In this work, new-to-nature heme-containing nitrene transferases were used as starting points for the directed evolution of enzymes to selectively aminate and amidate unactivated C(sp3)\u2013H sites. The desymmetrization of methyl- and ethylcyclohexane with divergent site selectivity is offered as demonstration. The evolved enzymes in these lineages are highly promiscuous and show activity towards a wide array of substrates, providing a foundation for further evolution of nitrene transferase function. Computational studies and kinetic isotope effects (KIEs) are consistent with a stepwise radical pathway involving an irreversible, enantiodetermining hydrogen atom transfer (HAT), followed by a lower-barrier diastereoselectivity determining radical rebound step. In-enzyme molecular dynamics (MD) simulations reveal a predominantly hydrophobic pocket with favorable dispersion interactions with the substrate. By offering a direct path from saturated precursors, these enzymes present a new biochemical logic for accessing nitrogen-containing compounds.", "date": "2023-03-29", "date_type": "published", "id_number": "CaltechAUTHORS:20230324-534348000.8", "official_url": "https://resolver.caltech.edu/CaltechAUTHORS:20230324-534348000.8", "rights": "No commercial reproduction, distribution, display or performance rights in this work are provided.", "funders": { "items": [ { "agency": "Department of Energy (DOE)", "grant_number": "DE-SC0021141" }, { "agency": "NIH Postdoctoral Fellowship", "grant_number": "F32GM143799" }, { "agency": "NSF", "grant_number": "OAC-1548562" }, { "agency": "NSF", "grant_number": "CHE-180061" }, { "agency": "NSF", "grant_number": "CHE-210031" }, { "agency": "NIH", "grant_number": "R15 GM142103" } ] }, "local_group": { "items": [ { "id": "Division-of-Biology-and-Biological-Engineering" } ] }, "doi": "10.26434/chemrxiv-2022-w8sg3", "primary_object": { "basename": "enzymatic-nitrogen-insertion-into-unactivated-c-h-bonds.pdf", "url": "https://authors.library.caltech.edu/records/ftmh1-h8573/files/enzymatic-nitrogen-insertion-into-unactivated-c-h-bonds.pdf" }, "related_objects": [ { "basename": "si-enzymatic-nitrogen-insertion-into-unactivated-c-h-bonds.pdf", "url": "https://authors.library.caltech.edu/records/ftmh1-h8573/files/si-enzymatic-nitrogen-insertion-into-unactivated-c-h-bonds.pdf" } ], "resource_type": "monograph", "pub_year": "2023", "author_list": "Athavale, Soumitra V.; Gao, Shilong; et el." }, { "id": "https://authors.library.caltech.edu/records/nmjvx-xnk18", "eprint_id": 120391, "eprint_status": "archive", "datestamp": "2023-08-20 08:27:02", "lastmod": "2023-12-13 16:38:34", "type": "monograph", "metadata_visibility": "show", "creators": { "items": [ { "id": "Calv\u00f3-Tusell-Carla", "name": { "family": "Calv\u00f3-Tusell", "given": "Carla" }, "orcid": "0000-0003-2681-8460" }, { "id": "Liu-Zhen-CHEM", "name": { "family": "Liu", "given": "Zhen" } }, { "id": "Chen-Kai", "name": { "family": "Chen", "given": "Kai" } }, { "id": "Arnold-F-H", "name": { "family": "Arnold", "given": "Frances H." }, "orcid": "0000-0002-4027-364X" }, { "id": "Garcia-Borr\u00e0s-Marc", "name": { "family": "Garcia-Borr\u00e0s", "given": "Marc" }, "orcid": "0000-0001-9458-1114" } ] }, "title": "Reversing the enantioselectivity of enzymatic carbene N\u2013H insertion through mechanism-guided protein engineering", "ispublished": "unpub", "full_text_status": "public", "note": "The content is available under CC BY NC ND 4.0 License. \n\nThis work was supported by the Spanish MICINN (Ministerio de Ciencia e Innovaci\u00f3n) PID2019-111300GA-I00 project (M.G.B), the Ram\u00f3n y Cajal program via the RYC2020-028628-I fellowship (M.G.B), and the NSF Division of Molecular and Cellular Biosciences (grant 2016137 to F.H.A.). K.C. thanks the Life Sciences Research Foundation for funding support. We thank Dr. Sabine Brinkmann-Chen, Dr. Ferran Feixas, and Dr. Cooper S. Jamieson for helpful discussions and comments on the manuscript.\n\nSupplemental Material - supporting-information.pdf
", "abstract": "In this work, we report a computationally driven approach to access enantiodivergent enzymatic carbene N\u2013H bond insertions catalyzed by P411 enzyme variants. Computational modeling was employed to guide engineering efforts to control the accessible conformations of a key lactone-carbene (LAC) intermediate in the enzyme active site by installing a new H-bond anchoring point. By combining MD simulations and protein engineering, a reversed (R-selective) P411 enzyme variant, L5_FL-B3, was obtained in a single round of semi-rational directed evolution. L5_FL-B3 accepts a broad scope of amine substrates with excellent yields (up to >99%), high efficiency (up to 12,300 TTN) and good enantiocontrol (up to 7:93 er), which complements the previously engineered S-selective P411-L7_LF variant.", "date": "2023-03-29", "date_type": "published", "id_number": "CaltechAUTHORS:20230324-284945000.5", "official_url": "https://resolver.caltech.edu/CaltechAUTHORS:20230324-284945000.5", "rights": "No commercial reproduction, distribution, display or performance rights in this work are provided.", "funders": { "items": [ { "agency": "Ministerio de Ciencia e Innovaci\u00f3n (MICINN)", "grant_number": "PID2019-111300GA-I00" }, { "agency": "Ram\u00f3n y Cajal Programme", "grant_number": "RYC2020-028628-I" }, { "agency": "NSF", "grant_number": "MCB-2016137" }, { "agency": "Life Sciences Research Foundation" } ] }, "local_group": { "items": [ { "id": "Division-of-Biology-and-Biological-Engineering" } ] }, "doi": "10.26434/chemrxiv-2022-f02xh", "primary_object": { "basename": "supporting-information.pdf", "url": "https://authors.library.caltech.edu/records/nmjvx-xnk18/files/supporting-information.pdf" }, "related_objects": [ { "basename": "reversing-the-enantioselectivity-of-enzymatic-carbene-n-h-insertion-through-mechanism-guided-protein-engineering.pdf", "url": "https://authors.library.caltech.edu/records/nmjvx-xnk18/files/reversing-the-enantioselectivity-of-enzymatic-carbene-n-h-insertion-through-mechanism-guided-protein-engineering.pdf" } ], "resource_type": "monograph", "pub_year": "2023", "author_list": "Calv\u00f3-Tusell, Carla; Liu, Zhen; et el." }, { "id": "https://authors.library.caltech.edu/records/zgx46-49973", "eprint_id": 120311, "eprint_status": "archive", "datestamp": "2023-08-20 08:39:58", "lastmod": "2023-12-13 16:38:38", "type": "monograph", "metadata_visibility": "show", "creators": { "items": [ { "id": "Danelius-Emma", "name": { "family": "Danelius", "given": "Emma" }, "orcid": "0000-0002-7322-9661" }, { "id": "Porter-Nicholas-J", "name": { "family": "Porter", "given": "Nicholas J." }, "orcid": "0000-0002-9803-5310" }, { "id": "Unge-Johan", "name": { "family": "Unge", "given": "Johan" } }, { "id": "Arnold-F-H", "name": { "family": "Arnold", "given": "Frances H." }, "orcid": "0000-0002-4027-364X" }, { "id": "Gonen-Tamir", "name": { "family": "Gonen", "given": "Tamir" }, "orcid": "0000-0002-9254-4069" } ] }, "title": "MicroED structure of a protoglobin reactive carbene intermediate", "ispublished": "unpub", "full_text_status": "public", "note": "The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license. \n\nE.D. thanks The Wenner-Gren Foundations for their support through the Wenner-Gren Postdoctoral Fellowship. This study was supported by the National Institutes of Health P41GM136508. The Gonen laboratory is supported by funds from the Howard Hughes Medical Institute. N.J.P. thanks Merck and the Helen Hay Whitney Foundation for their support through the Merck-Helen Hay Whitney Foundation Postdoctoral Scholarship. This publication is based on work supported by the United States Army Research Office under Contract W911NF-19-0026 for the Institute for Collaborative Biotechnologies and the G. Harold and Leila Y. Mathers Charitable Foundation. \n\nAuthor contributions. N.P. conducted protein expression and crystallization experiments. E.D. prepared the samples and conducted MicroED data collection. E.D. and J.U. analyzed the data and solved the structures. E.D., J.U., N.P, F.A, and T.G. took part in preparation of the manuscript. \n\nThe authors have declared no competing interest.\n\nSubmitted - 2022.10.18.512604v1.full.pdf
", "abstract": "Microcrystal electron diffraction (MicroED) is an emerging technique which has shown great potential for describing new chemical and biological molecular structures. [1] Several important structures of small molecules, natural products and peptides have been determined usingab initiomethods. [2] However, only a couple of novel protein structures have thus far been derived by MicroED. [3, 4] Taking advantage of recent technological advances including higher acceleration voltage and using a low-noise detector in counting mode, we have determined the first structure of anAeropyrum pernixprotoglobin (ApePgb) variant by MicroED using an AlphaFold2 model for phasing. The structure revealed that mutations introduced during directed evolution enhance carbene transfer activity by reorienting an alphahelix ofApePgb into a dynamic loop making the catalytic active site more readily accessible. After exposing the tiny crystals to substrate, we also trapped the reactive iron-carbenoid intermediate involved in this engineeredApePgb's new-to-nature activity, a challenging carbene transfer from a diazirine via a putative metallo-carbene. The bound structure discloses how an enlarged active site pocket stabilizes the carbene bound to the heme iron and, presumably, the transition state for formation of this key intermediate. This work demonstrates that improved MicroED technology and the advancement in protein structure prediction now enables investigation of structures that were previously beyond reach.", "date": "2023-03-22", "date_type": "published", "id_number": "CaltechAUTHORS:20230322-101583000.17", "official_url": "https://resolver.caltech.edu/CaltechAUTHORS:20230322-101583000.17", "rights": "No commercial reproduction, distribution, display or performance rights in this work are provided.", "funders": { "items": [ { "agency": "Wenner-Gren Foundation" }, { "agency": "NIH", "grant_number": "P41GM136508" }, { "agency": "Howard Hughes Medical Institute (HHMI)" }, { "agency": "Merck" }, { "agency": "Helen Hay Whitney Foundation" }, { "agency": "Army Research Office", "grant_number": "W911NF-19-0026" }, { "agency": "G. Harold and Leila Y. Mathers Charitable Foundation" } ] }, "local_group": { "items": [ { "id": "Division-of-Biology-and-Biological-Engineering" } ] }, "doi": "10.1101/2022.10.18.512604", "primary_object": { "basename": "2022.10.18.512604v1.full.pdf", "url": "https://authors.library.caltech.edu/records/zgx46-49973/files/2022.10.18.512604v1.full.pdf" }, "resource_type": "monograph", "pub_year": "2023", "author_list": "Danelius, Emma; Porter, Nicholas J.; et el." }, { "id": "https://authors.library.caltech.edu/records/43w5j-8se58", "eprint_id": 117922, "eprint_status": "archive", "datestamp": "2023-08-20 08:38:47", "lastmod": "2023-12-13 16:39:53", "type": "monograph", "metadata_visibility": "show", "creators": { "items": [ { "id": "Zhang-Juner", "name": { "family": "Zhang", "given": "Juner" }, "orcid": "0000-0001-8181-1187" }, { "id": "Maggiolo-Ailiena-O", "name": { "family": "Maggiolo", "given": "Ailiena O." }, "orcid": "0000-0003-1707-5060" }, { "id": "Alfonzo-Edwin", "name": { "family": "Alfonzo", "given": "Edwin" }, "orcid": "0000-0002-4112-8751" }, { "id": "Mao-Runze", "name": { "family": "Mao", "given": "Runze" }, "orcid": "0000-0003-4678-7251" }, { "id": "Porter-Nicholas-J", "name": { "family": "Porter", "given": "Nicholas J." }, "orcid": "0000-0002-9803-5310" }, { "id": "Abney-Nayla-M", "name": { "family": "Abney", "given": "Nayla" }, "orcid": "0000-0002-2979-5818" }, { "id": "Arnold-F-H", "name": { "family": "Arnold", "given": "Frances H." }, "orcid": "0000-0002-4027-364X" } ] }, "title": "Chemodivergent C(sp\u00b3)\u2013H and C(sp\u00b2)\u2013H Cyanomethylation Using Engineered Carbene Transferases", "ispublished": "unpub", "full_text_status": "public", "note": "The content is available under CC BY NC ND 4.0 License. \n\nThis work is supported by the National Institute of General Medical Science of the NIH (R01-GM138740). E.A. is supported by a Ruth Kirschstein NIH Postdoctoral Fellowship (F32GM143799), R.M. is supported by Swiss National Science Foundation (P2ELP2_195118). N.J.P. acknowledges support from Merck and the Helen Hay Whitney Foundation through the Merck-HHWF Fellowship. We thank Prof. Douglas C. Rees for providing space and resources to carry out the crystallography studies and for valuable discussion, and Dr. Scott C. Virgil, Dr. Jens T. Kaiser, and Dr. Mona Shahgholi for analytical assistance. We also thank Dr. Sabine Brinkman-Chen, Dr. Jennifer L. Kennemur, Dr. Zhen Liu, and Dr. David C. Miller for helpful discussions and comments on the manuscript. We thank Donald and Judith Voet, the Gordon and Betty Moore Foundation, and the Beckman Institute for their generous support of the Molecular Observatory at Caltech. We thank the staff at Beamline 12-2, Stanford Synchrotron Radiation Lightsource (SSRL). SSRL operations are supported by the U.S. Department of Energy and the National Institutes of Health. \n\nAuthors Contributions. J.Z. designed the overall research with F.H.A. providing guidance. J.Z. designed and conducted the initial screening of haem proteins; J.Z. and N.A. performed the directed evolution experiments. J.Z., E.A., and R.M. designed and performed the substrate scope studies and analysis. A.O.M. obtained and analyzed the X-ray crystal structure of the engineered proteins with N.J.P. providing help. J.Z. and F.H.A. wrote the manuscript with input from all authors. \n\nThe authors declare no competing interests. \n\nData availability. All data necessary to support the paper's conclusions are available in the main text and the Supplementary Information. The haem domain structure of P411-PFA is available through the Protein Data Bank ID 8DSG. Plasmids encoding the enzymes reported in this study are available for research purposes from F.H.A. under a material transfer agreement with the California Institute of Technology.\n\nSubmitted - chemodivergent-c-sp3-h-and-c-sp2-h-cyanomethylation-using-engineered-carbene-transferases.pdf
", "abstract": "The ubiquity of C\u2013H bonds presents an attractive opportunity to elaborate and build complexity in organic molecules. Methods for selective functionalization, however, often must differentiate among multiple chemically similar and, in some cases indistinguishable, C\u2013H bonds within the same molecule. An advantage of enzymes is that they can be finely tuned using directed evolution to achieve control over divergent C\u2013H functionalization pathways. Here, we present engineered enzymes that effect a new-to-nature C\u2013H alkylation (C\u2013H carbene insertion) with unparalleled selectivity: two complementary carbene C\u2013H transferases derived from a cytochrome P450 from Bacillus megaterium deliver an \u03b1-cyanocarbene into the \u03b1-amino C(sp\u00b3)\u2013H bonds or the ortho-arene C(sp\u00b2)\u2013H bonds of N-substituted arenes. These two transformations proceed via different mechanisms, yet only minimal changes to the protein scaffold (nine mutations, less than 2% of the sequence) were needed to adjust the enzyme's control over the site-selectivity of cyanomethylation. The X-ray crystal structure of the selective C(sp\u00b3)\u2013H alkylase, P411-PFA, reveals an unprecedented helical disruption which alters the shape and electrostatics in the enzyme active site. Overall, this work demonstrates the advantages of using enzymes as C\u2013H functionalization catalysts for divergent molecular derivatization.", "date": "2022-11-21", "date_type": "published", "id_number": "CaltechAUTHORS:20221121-680850000.2", "official_url": "https://resolver.caltech.edu/CaltechAUTHORS:20221121-680850000.2", "rights": "No commercial reproduction, distribution, display or performance rights in this work are provided.", "funders": { "items": [ { "agency": "NIH", "grant_number": "R01GM138740" }, { "agency": "NIH Postdoctoral Fellowship", "grant_number": "F32GM143799" }, { "agency": "Swiss National Science Foundation (SNSF)", "grant_number": "P2ELP2_195118" }, { "agency": "Helen Hay Whitney Foundation" }, { "agency": "Gordon and Betty Moore Foundation" }, { "agency": "Caltech Beckman Institute" } ] }, "local_group": { "items": [ { "id": "Division-of-Biology-and-Biological-Engineering" } ] }, "doi": "10.26434/chemrxiv-2022-z6pkw", "primary_object": { "basename": "chemodivergent-c-sp3-h-and-c-sp2-h-cyanomethylation-using-engineered-carbene-transferases.pdf", "url": "https://authors.library.caltech.edu/records/43w5j-8se58/files/chemodivergent-c-sp3-h-and-c-sp2-h-cyanomethylation-using-engineered-carbene-transferases.pdf" }, "resource_type": "monograph", "pub_year": "2022", "author_list": "Zhang, Juner; Maggiolo, Ailiena O.; et el." }, { "id": "https://authors.library.caltech.edu/records/x4pkf-x3638", "eprint_id": 101613, "eprint_status": "archive", "datestamp": "2023-08-19 18:40:22", "lastmod": "2023-12-13 16:39:35", "type": "monograph", "metadata_visibility": "show", "creators": { "items": [ { "id": "Goldberg-Nathaniel-W", "name": { "family": "Goldberg", "given": "Nathaniel" }, "orcid": "0000-0003-0201-8856" }, { "id": "Knight-Anders-M", "name": { "family": "Knight", "given": "Anders M." }, "orcid": "0000-0001-9665-8197" }, { "id": "Zhang-Ruijie-K", "name": { "family": "Zhang", "given": "Ruijie" }, "orcid": "0000-0002-7251-5527" }, { "id": "Arnold-F-H", "name": { "family": "Arnold", "given": "Frances H." }, "orcid": "0000-0002-4027-364X" } ] }, "title": "Nitrene Transfer Catalyzed by a Non-Heme Iron Enzyme and Enhanced by Non-Native Small-Molecule Cofactors", "ispublished": "unpub", "full_text_status": "public", "note": "LICENCE: CC BY-NC-ND 4.0. \n\nSubmission date: 30.10.2019; First online date Posted date: 05.11.2019. \n\nThis work was supported by the National Science Foundation (NSF) Division of Molecular and Cellular Biosciences (grant MCB-1513007). N. W. G., A. M. K., and R. K. Z. acknowledge support from the NIH training grants NIH T32 GM07616 (N. W. G.) and NIH T32 GM112592 (A. M. K., R. K. Z.) and NSF Graduate Research Fellowship DGE-1144469 (A. M. K, R. K. Z.). We thank Sabine Brinkmann-Chen for critical reading of the manuscript and Noah P. Dunham, S. B. Jennifer Kan, and Benjamin J. Levin for helpful discussions. We thank Professor Hans Renata and Professor Harry Gray for generously sharing plasmids. \n\nA provisional patent has been filed through the California Institute of Technology based on the results presented here.\n\nSupplemental Material - EFE_SI_20191028.pdf
", "abstract": "Transition-metal catalysis is a powerful tool for the construction of chemical bonds. Here we show that a non-heme iron enzyme can catalyze olefin aziridination and nitrene C\u2013H insertion, and that these activities can be improved by directed evolution. The non-heme iron center allows for facile modification of the primary coordination sphere by addition of metal-coordinating molecules, enabling control over enzyme activity and selectivity using small molecules.", "date": "2020-02-27", "date_type": "published", "id_number": "CaltechAUTHORS:20200227-102506840", "official_url": "https://resolver.caltech.edu/CaltechAUTHORS:20200227-102506840", "rights": "No commercial reproduction, distribution, display or performance rights in this work are provided.", "funders": { "items": [ { "agency": "NSF", "grant_number": "MCB-1513007" }, { "agency": "NIH Predoctoral Fellowship", "grant_number": "T32 GM07616" }, { "agency": "NIH Predoctoral Fellowship", "grant_number": "T32 GM112592" }, { "agency": "NSF Graduate Research Fellowship", "grant_number": "DGE-1144469" } ] }, "local_group": { "items": [ { "id": "Division-of-Biology-and-Biological-Engineering" } ] }, "doi": "10.26434/chemrxiv.10062044.v1", "primary_object": { "basename": "EFE_SI_20191028.pdf", "url": "https://authors.library.caltech.edu/records/x4pkf-x3638/files/EFE_SI_20191028.pdf" }, "related_objects": [ { "basename": "Nitrene_Transfer_Catalyzed_by_a_Non-Heme_Iron_Enzyme_and_Enhanced_by_Non-Native_Small-Molecule_Cofactors_v1.pdf", "url": "https://authors.library.caltech.edu/records/x4pkf-x3638/files/Nitrene_Transfer_Catalyzed_by_a_Non-Heme_Iron_Enzyme_and_Enhanced_by_Non-Native_Small-Molecule_Cofactors_v1.pdf" } ], "resource_type": "monograph", "pub_year": "2020", "author_list": "Goldberg, Nathaniel; Knight, Anders M.; et el." }, { "id": "https://authors.library.caltech.edu/records/6ydv9-z7f07", "eprint_id": 93736, "eprint_status": "archive", "datestamp": "2023-08-19 14:19:11", "lastmod": "2023-10-20 17:22:38", "type": "monograph", "metadata_visibility": "show", "creators": { "items": [ { "id": "Cho-Inha", "name": { "family": "Cho", "given": "Inha" }, "orcid": "0000-0002-7564-5378" }, { "id": "Jia-Zhi-Jun", "name": { "family": "Jia", "given": "Zhi-Jun" }, "orcid": "0000-0002-5143-4875" }, { "id": "Arnold-F-H", "name": { "family": "Arnold", "given": "Frances H." }, "orcid": "0000-0002-4027-364X" } ] }, "title": "Genetically Tunable Enzymatic C\u2012H Amidation for Lactam Synthesis", "ispublished": "unpub", "full_text_status": "public", "note": "Preprint submitted on 12.02.2019, 21:02 and posted on 13.02.2019, 09:02. \n\nDeclaration of Conflict of Interest: Caltech has filed a provisional patent application (CIT-8187-P). \n\nWe thank K. Chen, S. Brinkmann-Chen, D. C. Miller and D. K. Romney for comments on the manuscript; X. Huang for helpful discussions; K. Chen for the parent variant E10FA; The Caltech Center for Catalysis and Chemical Synthesis and the Caltech Mass Spectrometry Laboratory for analytical support. \n\nFunding: Supported by NSF Division of Molecular and Cellular Biosciences grant MCB-1513007; Joseph J. Jacobs Institute for Molecular Engineering for Medicine (I.C.); and Deutsche Forschungsgemeinschaft (JI 289/1-1 to Z.-J.J.). \n\nAuthor contributions: I.C. and Z.-J.J. designed the research and performed the experiments with guidance from F.H.A.. I.C., Z.-J.J. and F.H.A. wrote the manuscript. \n\nCompeting interests: Caltech has filed a provisional patent application (CIT-8187-P); and Data and materials availability: All data are available in the main text or the supplementary materials.\n\nSubmitted - Cho_Genetically_Tunable_Enzymatic.pdf
", "abstract": "A major challenge in carbon\u2012hydrogen (C\u2012H) bond functionalization is to have the catalyst control precisely where a reaction takes place. Here we report engineered cytochrome P450 enzymes that perform unprecedented enantioselective C\u2012H amidation reactions and control the site selectivity to divergently construct \u03b2-, \u03b3- and \u03b4-lactams, completely overruling the inherent reactivities of the C\u2012H bonds. The enzymes, expressed in Escherichia coli cells, accomplish this abiological carbon\u2012nitrogen (C\u2012N) bond formation via reactive iron-bound carbonyl nitrenes generated from nature-inspired acyl-protected hydroxamate precursors. This transformation is exceptionally efficient (up to 1,020,000 total turnovers) and selective (up to 25:1 regioselectivity and 96% enantiomeric excess), and can be performed easily on preparative scale.", "date": "2019-03-12", "date_type": "published", "publisher": "Caltech Library", "id_number": "CaltechAUTHORS:20190312-131401661", "official_url": "https://resolver.caltech.edu/CaltechAUTHORS:20190312-131401661", "rights": "No commercial reproduction, distribution, display or performance rights in this work are provided.", "funders": { "items": [ { "agency": "NSF", "grant_number": "MCB-1513007" }, { "agency": "Jacobs Institute for Molecular Engineering for Medicine" }, { "agency": "Deutsche Forschungsgemeinschaft (DFG)", "grant_number": "JI 289/1-1" } ] }, "local_group": { "items": [ { "id": "Jacobs-Institute-for-Molecular-Engineering-for-Medicine" } ] }, "primary_object": { "basename": "Cho_Genetically_Tunable_Enzymatic.pdf", "url": "https://authors.library.caltech.edu/records/6ydv9-z7f07/files/Cho_Genetically_Tunable_Enzymatic.pdf" }, "resource_type": "monograph", "pub_year": "2019", "author_list": "Cho, Inha; Jia, Zhi-Jun; et el." } ]