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Dual-function enzyme catalysis for enantioselective carbon–nitrogen bond formation

Nature Chemistry volume 13, pages 1166–1172 (2021)Cite this article

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Abstract

Chiral amines can be made by insertion of a carbene into an N–H bond using two-catalyst systems that combine a transition metal-based carbene-transfer catalyst and a chiral proton-transfer catalyst to enforce stereocontrol. Haem proteins can effect carbene N–H insertion, but asymmetric protonation in an active site replete with proton sources is challenging. Here we describe engineered cytochrome P450 enzymes that catalyse carbene N–H insertion to prepare biologically relevant α-amino lactones with high activity and enantioselectivity (up to 32,100 total turnovers, >99% yield and 98% e.e.). These enzymes serve as dual-function catalysts, inducing carbene transfer and promoting the subsequent proton transfer with excellent stereoselectivity in a single active site. Computational studies uncover the detailed mechanism of this new-to-nature enzymatic reaction and explain how active-site residues accelerate this transformation and provide stereocontrol.

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Fig. 1: Asymmetric carbene N–H insertion using small-molecule catalysts and enzymes.
Fig. 2: Screen for enzymatic N–H insertion with a haem protein collection and identification of A264S as the key mutation for achieving high activity and selectivity.
Fig. 3: Computational modelling elucidates the origins of enantioselectivity of carbene transfer into N–H bonds catalysed by P411-L6.
Fig. 4: Enantioselective carbene N–H insertion of secondary and primary anilines and aliphatic amines.
Fig. 5: Investigation of the catalytic efficiency of L7_FL and scale-up of asymmetric N–H insertion reactions.

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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. X-ray crystal structures of 3e (CCDC 2065484) and 3l (CCDC 2065489) are available free of charge from the Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif. 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. Source data are provided with this paper.

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Acknowledgements

This work was supported by the National Science Foundation Division of Molecular and Cellular Biosciences (grant 2016137 to F.H.A.), the US Army Research Office Institute for Collaborative Biotechnologies (cooperative agreement W911NF-19-2-0026 to F.H.A.), the Spanish Ministry of Science and Innovation MICINN (grant PID2019-111300GA-I00 to M.G.-B.) and the Generalitat de Catalunya AGAUR Beatriu de Pinós H2020 MSCA-Cofund (2018-BP-00204 project to M.G.-B.). K.C. thanks the Resnick Sustainability Institute at Caltech for fellowship support. The computer resources at MinoTauro and the Barcelona Supercomputing Center BSC-RES are acknowledged (RES-QSB-2020-2-0016). We thank D. C. Miller, S. Brinkmann-Chen, R. Lal and T. Zeng for helpful discussions and comments on the manuscript. We further thank M. Shahgholi for high-resolution mass spectrometry analysis and M. K. Takase for X-ray crystallographic analysis.

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Author notes

  1. Kai Chen

    Present address: Innovative Genomics Institute, University of California, Berkeley, CA, USA

Authors and Affiliations

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

    Zhen Liu, Andrew Z. Zhou, Kai Chen & Frances H. Arnold

  2. Institut de Química Computacional i Catàlisi and Departament de Química, Universitat de Girona, Girona, Spain

    Carla Calvó-Tusell & Marc Garcia-Borràs

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Contributions

Z.L. and K.C. conceived and designed the overall project with F.H.A. providing guidance. Z.L. and A.Z.Z. designed and performed the initial screening of haem proteins and the substrate scope study. C.C.-T. and M.G.-B. carried out the computational studies. Z.L., K.C., M.G.-B. and F.H.A. wrote the manuscript with the input of all authors.

Corresponding authors

Correspondence to Kai Chen, Marc Garcia-Borràs or Frances H. Arnold.

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Competing interests

K.C., Z.L. and A.Z.Z. are inventors on a US Patent Application (invention title, Diverse Carbene Transferase Enzyme Catalysts Derived from a P450 Enzyme; application no., 17/200,394) filed by the California Institute of Technology, which covers lactone-carbene N–H insertion with engineered P450 enzymes. The patent was filed on 12 March 2021. The remaining authors declare no competing interests.

Additional information

Peer review information Nature Chemistry thanks Sabine Flitsch, Sason Shaik 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 Figs. 1–21, Tables 1–17, experimental data, procedural details, synthesis and characterization data, NMR spectra, X-ray crystallographic data and computational modelling data.

Supplementary Data 1

crystallographic data of compound 3e; CCDC 2065484.

Supplementary Data 2

crystallographic data of compound 3l; CCDC 2065489.

Source data

Source Data Fig. 2

Statistical source data of 40 previously reported variants.

Source Data Fig. 4

Statistical source data of substrate scope.

Source Data Fig. 5

Statistical source data of OD test.

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Liu, Z., Calvó-Tusell, C., Zhou, A.Z. et al. Dual-function enzyme catalysis for enantioselective carbon–nitrogen bond formation. Nat. Chem. 13, 1166–1172 (2021). https://doi.org/10.1038/s41557-021-00794-z

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