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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 M. and Arnold, Frances H. (2023) Chemodivergent C(sp³)-H and C(sp²)-H cyanomethylation using engineered carbene transferases. Nature Catalysis, 6 (2). pp. 152-160. ISSN 2520-1158. PMCID PMC9983643. doi:10.1038/s41929-022-00908-x.

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The ubiquity of C–H bonds presents an opportunity to efficiently elaborate and build complexity in organic molecules. Methods for selective functionalization, however, must differentiate among multiple, chemically similar C–H bonds: enzymes are attractive because they can be finely tuned using directed evolution to achieve divergent reaction outcomes. Here we present engineered enzymes that effect a new-to-nature C–H alkylation (C–H carbene insertion) with distinct selectivities: cytochrome P450-based carbene transferases deliver an α-cyanocarbene either 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 were needed to adjust the enzyme’s chemoselectivity. Structural studies of the C(sp³)–H alkylase reveal an active-site helical disruption, which alters the structure and electrostatics of the substrate-binding pocket compared to the native enzyme. Overall, this work demonstrates advantages of using highly tuneable enzymes as C–H functionalization catalysts for divergent molecular derivatization.

Item Type:Article
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URLURL TypeDescription ReadCube access
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, Nayla M.0000-0002-2979-5818
Arnold, Frances H.0000-0002-4027-364X
Additional Information:This work is supported by the National Institute of General Medical Science of the NIH (grant no. R01GM138740). E.A. is supported by a Ruth Kirschstein NIH Postdoctoral Fellowship (grant no. F32GM143799). R.M. is supported by Swiss National Science Foundation (grant no. P2ELP2_195118). N.J.P. acknowledges support from Merck and the Helen Hay Whitney Foundation through the Merck-HHWF Fellowship. We thank D. C. Rees for providing space and resources to carry out the crystallography studies and for valuable discussion, and S. C. Virgil, J. T. Kaiser and M. Shahgholi for analytical assistance. We also thank S. Brinkman-Chen, J. L. Kennemur, Z. Liu and D. C. Miller for helpful discussions and comments on the manuscript. We thank D. and J. 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 US Department of Energy and the National Institutes of Health. 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.M.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 analysed 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. Data availability. All data necessary to support the paper’s conclusions are available in the main text and Supplementary Information or from the authors upon reasonable request. The haem-domain structure of P411-PFA is available through the PDB 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. The authors declare no competing interests.
Funding AgencyGrant Number
NIH Postdoctoral FellowshipF32GM143799
Swiss National Science Foundation (SNSF)P2ELP2_195118
Helen Hay Whitney FoundationUNSPECIFIED
Gordon and Betty Moore FoundationUNSPECIFIED
Caltech Beckman InstituteUNSPECIFIED
Issue or Number:2
PubMed Central ID:PMC9983643
Record Number:CaltechAUTHORS:20230209-988069100.21
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Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:119183
Deposited By: Research Services Depository
Deposited On:17 Mar 2023 15:17
Last Modified:17 Mar 2023 15:17

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