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An enzymatic platform for the asymmetric amination of primary, secondary and tertiary C(sp3)–H bonds

Nature Chemistry volume 11, pages 987–993 (2019)Cite this article

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Abstract

The ability to selectively functionalize ubiquitous C–H bonds streamlines the construction of complex molecular architectures from easily available precursors. Here we report enzyme catalysts derived from a cytochrome P450 that use a nitrene transfer mechanism for the enantioselective amination of primary, secondary and tertiary C(sp3)–H bonds. These fully genetically encoded enzymes are produced and function in bacteria, where they can be optimized by directed evolution for a broad spectrum of enantioselective C(sp3)–H amination reactions. These catalysts can aminate a variety of benzylic, allylic and aliphatic C–H bonds in excellent enantioselectivity with access to either antipode of product. Enantioselective amination of primary C(sp3)–H bonds in substrates that bear geminal dimethyl substituents furnished chiral amines that feature a quaternary stereocentre. Moreover, these enzymes enabled the enantioconvergent transformation of racemic substrates that possess a tertiary C(sp3)–H bond to afford products that bear a tetrasubstituted stereocentre, a process that has eluded small-molecule catalysts. Further engineering allowed for the enantioselective construction of methyl–ethyl stereocentres, which is notoriously challenging in asymmetric catalysis.

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Fig. 1: The three major types of asymmetric C(sp3)–H functionalization and our envisioned biocatalytic C(sp3)–H amination.
Fig. 2: Enantioselective amination of secondary C(sp3)–H bonds.
Fig. 3: Amination of unactivated secondary C(sp3)–H bonds.
Fig. 4: Engineered P411Diane variants for the enantioselective amination of primary and tertiary C(sp3)–H bonds.
Fig. 5: Mechanistic insight.
Fig. 6: Free energy profile of the iron porphyrin-catalysed C(sp3)–H amination.

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Data availability

All data necessary to support the paper’s conclusions are available in the main text and the Supplementary Information. Solid-state structures of 2a, 4a, 5a and 5f are available free of charge from the Cambridge Crystallographic Data Centre under reference nos CCDC 1905551, 1905553, 1905552 and 1905554. 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 was supported by the NSF (grant nos MCB-1513007 for F.H.A. and CHE-1654122 for P.L.). Y.Y. thanks the National Institutes of Health for a postdoctoral fellowship (grant no. 1F32GM133126-01). Calculations were performed at the Center for Research Computing at the University of Pittsburgh. We thank R. K. Zhang, K. Chen, D. C. Miller, D. K. Romney (Caltech) and Y. Wang (University of Pittsburgh) for helpful discussions and comments on the manuscript, L. Henling for X-ray diffraction analysis and S. Virgil for assistance with chiral supercritical fluid chromatography analysis.

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Authors and Affiliations

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

    Yang Yang, Inha Cho & Frances H. Arnold

  2. Department of Chemistry, University of Pittsburgh, Pittsburgh, PA, USA

    Xiaotian Qi & Peng Liu

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Contributions

Y.Y. designed the overall research with F.H.A. providing guidance. Y.Y. and I.C. designed and performed the initial screening of haem proteins and directed evolution experiments. Y.Y. designed and performed the substrate scope study and mechanistic study. X.Q. carried out the computational studies with P.L. providing guidance. Y.Y. and F.H.A. wrote the manuscript with the input of all other authors.

Corresponding authors

Correspondence to Peng Liu or Frances H. Arnold.

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Competing interests

A provisional patent application (inventors Y.Y. and I.C.) has been filed through the California Institute of Technology. The provisional patent covers the development and application of engineered cytochromes P450 for the synthesis of chiral diamine derivatives by C–H amination.

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Supplementary information

Supplementary information.

NMR spectra

1H, 13C and 19F NMR spectra.

X-ray crystal structure of 2a

Crystallographic data for compound 2a.

X-ray crystal structure of 4a

Crystallographic data for compound 4a.

X-ray crystal structure of 5a

Crystallographic data for compound 5a.

X-ray crystal structure of 5f

Crystallographic data for compound 5f.

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Yang, Y., Cho, I., Qi, X. et al. An enzymatic platform for the asymmetric amination of primary, secondary and tertiary C(sp3)–H bonds. Nat. Chem. 11, 987–993 (2019). https://doi.org/10.1038/s41557-019-0343-5

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