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Directed Evolution of a Cytochrome P450 Carbene Transferase for Selective Functionalization of Cyclic Compounds

Brandenberg, Oliver F. and Chen, Kai and Arnold, Frances H. (2019) Directed Evolution of a Cytochrome P450 Carbene Transferase for Selective Functionalization of Cyclic Compounds. Journal of the American Chemical Society, 141 (22). pp. 8989-8995. ISSN 0002-7863. https://resolver.caltech.edu/CaltechAUTHORS:20190509-152409265

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Abstract

Transfers of carbene moieties to heterocycles or cyclic alkenes to obtain C(sp^2)–H alkylation or cyclopropane products are valuable transformations for synthesis of pharmacophores and chemical building blocks. Through their readily tunable active-site geometries, hemoprotein “carbene transferases” could provide an alternative to traditional transition metal catalysts by enabling heterocycle functionalizations with high chemo-, regio-, and stereocontrol. However, carbene transferases accepting heterocyclic substrates are scarce; the few enzymes capable of heterocycle or cyclic internal alkene functionalization described to date are characterized by low turnovers or depend on artificially introduced, costly iridium–porphyrin cofactors. We addressed this challenge by evolving a cytochrome P450 for highly efficient carbene transfer to indoles, pyrroles, and cyclic alkenes. We first developed a spectrophotometric high-throughput screening assay based on 1-methylindole C3-alkylation that enabled rapid analysis of thousands of P450 variants and comprehensive directed evolution via random and targeted mutagenesis. This effort yielded a P450 variant with 11 amino acid substitutions and a large deletion of the non-catalytic P450 reductase domain, which chemoselectively C_3-alkylates indoles with up to 470 turnovers per minute and 18 000 total turnovers. We subsequently used this optimized alkylation variant for parallel evolution toward more challenging heterocycle carbene functionalizations, including C_2/C_3 regioselective pyrrole alkylation, enantioselective indole alkylation with ethyl 2-diazopropanoate, and cyclic internal alkene cyclopropanation. The resulting set of efficient biocatalysts showcases the tunability of hemoproteins for highly selective functionalization of cyclic targets and the power of directed evolution to enhance the scope of new-to-nature enzyme catalysts.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1021/jacs.9b02931DOIArticle
ORCID:
AuthorORCID
Brandenberg, Oliver F.0000-0001-5662-1234
Chen, Kai0000-0002-3325-3536
Arnold, Frances H.0000-0002-4027-364X
Additional Information:© 2019 American Chemical Society. Received: March 17, 2019; Published: May 9, 2019. This work was supported by a grant from the National Science Foundation, Division of Molecular and Cellular Biosciences, to F.H.A. (NSF MCB-1513007). O.F.B. acknowledges support from the Deutsche Forschungsgemeinschaft (DFG grant BR 5238/1-1) and the Swiss National Science Foundation (SNF grant P300PA-171225). K.C. thanks the Resnick Sustainability Institute at Caltech for fellowship support. The authors are grateful to Dr. Christopher Prier for help with initial experimental design and comments on the manuscript. We also thank Dr. David Romney and Anders Knight for helpful comments and discussions. The authors declare no competing financial interest.
Group:Resnick Sustainability Institute
Funders:
Funding AgencyGrant Number
NSFMCB-1513007
Deutsche Forschungsgemeinschaft (DFG)BR 5238/1-1
Swiss National Science Foundation (SNSF)P300PA-171225
Resnick Sustainability InstituteUNSPECIFIED
Subject Keywords:Biocatalysis, Cytochrome P450, Carbene Transfer, Indole Alkylation, Cyclopropanation
Issue or Number:22
Record Number:CaltechAUTHORS:20190509-152409265
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20190509-152409265
Official Citation:Directed Evolution of a Cytochrome P450 Carbene Transferase for Selective Functionalization of Cyclic Compounds. Oliver F. Brandenberg, Kai Chen, and Frances H. Arnold. Journal of the American Chemical Society 2019 141 (22), 8989-8995. DOI: 10.1021/jacs.9b02931
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:95383
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:09 May 2019 22:52
Last Modified:03 Oct 2019 21:12

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