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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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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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.
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.
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 Figs. 1–21, Tables 1–17, experimental data, procedural details, synthesis and characterization data, NMR spectra, X-ray crystallographic data and computational modelling data.
crystallographic data of compound 3e; CCDC 2065484.
crystallographic data of compound 3l; CCDC 2065489.
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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