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Chemodivergent C(sp³)–H and C(sp²)–H Cyanomethylation Using Engineered Carbene Transferases

Zhang, Juner and Maggiolo, Ailiena O. and Alfonzo, Edwin and Mao, Runze and Porter, Nicholas J. and Abney, Nayla and Arnold, Frances H. (2022) Chemodivergent C(sp³)–H and C(sp²)–H Cyanomethylation Using Engineered Carbene Transferases. . (Unpublished) https://resolver.caltech.edu/CaltechAUTHORS:20221121-680850000.2

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Abstract

The ubiquity of C–H 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–H 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–H functionalization pathways. Here, we present engineered enzymes that effect a new-to-nature C–H alkylation (C–H carbene insertion) with unparalleled selectivity: two complementary carbene C–H transferases derived from a cytochrome P450 from Bacillus megaterium deliver an α-cyanocarbene into the α-amino C(sp³)–H bonds or the ortho-arene C(sp²)–H 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(sp³)–H 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–H functionalization catalysts for divergent molecular derivatization.


Item Type:Report or Paper (Discussion Paper)
Related URLs:
URLURL TypeDescription
https://doi.org/10.26434/chemrxiv-2022-z6pkwDOIDiscussion Paper
ORCID:
AuthorORCID
Zhang, Juner0000-0001-8181-1187
Maggiolo, Ailiena O.0000-0003-1707-5060
Alfonzo, Edwin0000-0002-4112-8751
Mao, Runze0000-0003-4678-7251
Porter, Nicholas J.0000-0002-9803-5310
Abney, Nayla0000-0002-2979-5818
Arnold, Frances H.0000-0002-4027-364X
Alternate Title:Chemodivergent C(sp3)–H and C(sp2)–H Cyanomethylation Using Engineered Carbene Transferases
Additional Information:The content is available under CC BY NC ND 4.0 License. This 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. Authors 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. The authors declare no competing interests. Data 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.
Funders:
Funding AgencyGrant Number
NIHR01GM138740
NIH Postdoctoral FellowshipF32GM143799
Swiss National Science Foundation (SNSF)P2ELP2_195118
Helen Hay Whitney FoundationUNSPECIFIED
Gordon and Betty Moore FoundationUNSPECIFIED
Caltech Beckman InstituteUNSPECIFIED
DOI:10.26434/chemrxiv-2022-z6pkw
Record Number:CaltechAUTHORS:20221121-680850000.2
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20221121-680850000.2
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:117922
Collection:CaltechAUTHORS
Deposited By: George Porter
Deposited On:21 Nov 2022 20:22
Last Modified:21 Nov 2022 20:22

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