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Chemodivergent C(sp3)–H and C(sp2)–H cyanomethylation using engineered carbene transferases

Nature Catalysis volume 6, pages 152–160 (2023)Cite this article

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Abstract

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.

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Fig. 1: Reaction design.
Fig. 2: Crystallographic studies of P411-PFA (PDB 8DSG).
Fig. 3: Directed evolution of a selective arene C–H cyanomethylase.
Fig. 4: Substrate scope study.

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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.

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Acknowledgements

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.

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  1. Juner Zhang

    Present address: Department of Chemistry, Princeton University, Princeton, NJ, USA

  2. Nayla M. Abney

    Present address: Department of Bioengineering, Stanford University, Stanford, CA, USA

Authors and Affiliations

  1. Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA, USA

    Juner Zhang, Ailiena O. Maggiolo, Edwin Alfonzo, Runze Mao, Nicholas J. Porter, Nayla M. Abney & Frances H. Arnold

  2. Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA

    Frances H. Arnold

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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.

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Correspondence to Frances H. Arnold.

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Supplementary Figs. 1–9, Tables 1–8, Methods, Notes 1–6, NMR spectra and references.

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Zhang, J., Maggiolo, A.O., Alfonzo, E. et al. Chemodivergent C(sp3)–H and C(sp2)–H cyanomethylation using engineered carbene transferases. Nat Catal 6, 152–160 (2023). https://doi.org/10.1038/s41929-022-00908-x

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