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Biocatalytic, enantioenriched primary amination of tertiary C–H bonds

Nature Catalysis (2024)Cite this article

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A preprint version of the article is available at ChemRxiv.

Abstract

Intermolecular functionalization of tertiary C–H bonds to construct fully substituted stereogenic carbon centres represents a formidable challenge: without the assistance of directing groups, state-of-the-art catalysts struggle to introduce chirality to racemic tertiary sp3-carbon centres. Direct asymmetric functionalization of such centres is a worthy reactivity and selectivity goal for modern biocatalysis. Here we present an engineered nitrene transferase (P411-TEA-5274), derived from a bacterial cytochrome P450, that is capable of aminating tertiary C–H bonds to provide chiral α-tertiary primary amines with high efficiency (up to 2,300 total turnovers) and selectivity (up to >99% enantiomeric excess). The construction of fully substituted stereocentres with methyl and ethyl groups underscores the enzyme’s remarkable selectivity. A comprehensive substrate scope study demonstrates the biocatalyst’s compatibility with diverse functional groups and tertiary C–H bonds. Mechanistic studies explain how active-site residues distinguish between the enantiomers and enable the enzyme to perform this transformation with excellent enantioselectivity.

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Fig. 1: Direct enantioselective functionalization of C(sp3)–H bonds.
Fig. 2: Directed evolution for enzymatic primary amination of tertiary C–H bonds.
Fig. 3: Substrate scope study.
Fig. 4: Mechanistic studies of enantioenriched enzymatic primary amination of tertiary C–H bonds.
Fig. 5: Molecular dynamics simulations of iron-haem-catalysed tertiary C–H amination.

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Data availability

Crystallographic data are available free of charge from the Cambridge Crystallographic Data Centre under no. CCDC 2287786 (3f derivative (S)-N-(2-(4-methoxyphenyl)butan-2-yl)benzamide). The original materials and data that support the findings of this study are available within the paper and its Supplementary Information or can be obtained from the corresponding author upon reasonable request.

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Acknowledgements

This work is supported by the National Institute of General Medical Science of the National Institutes of Health (grant no. R01GM138740). Support by National Science Foundation Division of Chemistry (grant no. CHE-2153972 to K.N.H.) and the Alexander von Humboldt-Foundation (Feodor Lynen Fellowship, T.R.) is gratefully acknowledged. We thank S. C. Virgil for the maintenance of the Caltech Center for Catalysis and Chemical Synthesis (3CS). We thank M. Shahgoli for mass spectrometry assistance. We thank D. Vander Velde for the maintenance of the Caltech nuclear magnetic resonance facility. We thank M. K. Takase and L. M. Henling for assistance with X-ray crystallographic data collection. We also thank S. Brinkmann-Chen for the helpful discussions and comments on the manuscript.

Author information

Author notes

  1. Torben Rogge

    Present address: Institut für Chemie, Technische Universität Berlin, Berlin, Germany

  2. These authors contributed equally: Runze Mao, Shilong Gao, Zi-Yang Qin.

Authors and Affiliations

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

    Runze Mao, Shilong Gao, Zi-Yang Qin, Sophia J. Wu, Zi-Qi Li, Anuvab Das & Frances H. Arnold

  2. Department of Chemistry and Biochemistry, University of California, Los Angeles, CA, USA

    Torben Rogge & K. N. Houk

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Contributions

R.M. conceptualized and designed the project under the guidance of F.H.A. R.M. and S.G. carried out the initial screening of haem proteins. R.M., S.G. and S.J.W. performed the directed evolution experiments, with support from Z.-Y.Q. for the validation. R.M., S.G. and Z.-Y.Q. investigated the substrate scope and reaction mechanism, with support from Z.-Q.L. T.R. carried out the computational studies with K.N.H. providing guidance. A.D. conducted crystallization of a 3f derivative. R.M. and F.H.A. wrote the manuscript with input from all authors.

Corresponding author

Correspondence to Frances H. Arnold.

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Nature Catalysis thanks Bernhard Hauer, Nicholas Turner and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Supplementary information

Supplementary Information

Supplementary Methods 1–23, Tables 1–8, Figs. 1–26 and References.

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Supplementary Data 1

Compound 3f derivative.cif Crystallographic data for compound 3f derivative. CCDC reference 2287786.

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Mao, R., Gao, S., Qin, ZY. et al. Biocatalytic, enantioenriched primary amination of tertiary C–H bonds. Nat Catal (2024). https://doi.org/10.1038/s41929-024-01149-w

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