Abstract
The formation of C–N bonds—of great importance to the pharmaceutical industry—can be facilitated enzymatically using nucleophilic and nitrene transfer mechanisms. However, neither natural nor engineered enzymes are known to generate and control nitrogen-centred radicals, which serve as valuable species for C–N bond formation. Here we use flavin-dependent ‘ene’-reductases with an exogenous photoredox catalyst to selectively generate amidyl radicals within the protein active site. These enzymes are engineered through directed evolution to catalyse 5-exo, 6-endo, 7-endo, 8-endo, and intermolecular hydroamination reactions with high levels of enantioselectivity. Mechanistic studies suggest that radical initiation occurs via an enzyme-gated mechanism, where the protein thermodynamically activates the substrate for reduction by the photocatalyst. Molecular dynamics studies indicate that the enzymes bind substrates using non-canonical binding interactions, which may serve as a handle to further manipulate reactivity. This approach demonstrates the versatility of these enzymes for controlling the reactivity of high-energy radical intermediates and highlights the opportunity for synergistic catalyst strategies to unlock previously inaccessible enzymatic functions.
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The data that support the findings in this study are available in this article and the Supplementary Information. Source data are provided with this paper.
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Acknowledgements
The research reported here was supported by the National Institutes of Health National Institute of General Medical Sciences (R01 GM127703). Transient absorption spectroscopy studies were supported by the Division of Chemical Sciences, Geosciences, and Biosciences, Office of Basic Energy Sciences of the US Department of Energy (DOE) through grant DE-SC0019370. We thank the Princeton Catalysis Initiative and Merck Sharp & Dohme LLC, a subsidiary of Merck & Co., Inc., Rahway, NJ, USA, for financial support. This work made use of the Cornell University NMR Facility, which is supported, in part, by the NSF though MRI Award CHE-1531632. D.G.O. acknowledges support from the Postgraduate Scholarships Doctoral Program of the Natural Sciences and Engineering Research Council of Canada. We thank K. Meihaus, P. Clayman, K. Biegasiewicz and Y. Liu for assistance in preparing this manuscript, and T. Qiao for assistance with density functional theory calculations.
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T.K.H. conceived and directed the project. Y.Y. and J.C. performed and analysed the experiments. D.G.O. performed the transient absorption spectroscopy experiments. D.G.O. and G.D.S. analysed and interpreted the spectroscopy results. D.V. performed the MD simulations. D.V. and C.K.P. analysed and interpreted the MD simulations. All authors discussed the results and commented on the manuscript.
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Ye, Y., Cao, J., Oblinsky, D.G. et al. Using enzymes to tame nitrogen-centred radicals for enantioselective hydroamination. Nat. Chem. 15, 206–212 (2023). https://doi.org/10.1038/s41557-022-01083-z
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DOI: https://doi.org/10.1038/s41557-022-01083-z
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