Selective oxidation of ring C–H bonds is an attractive route to functionalized cyclic amines, which are versatile intermediates in drug synthesis and important fragment molecules in drug discovery. Here we report a combined substrate and enzyme engineering approach to achieve enantioselective functionalization of all unactivated C–H bonds of azepane, azocane, 7-azabicyclo[2.2.1]heptane and 8-azaspiro[4.5]decane by cytochrome P450BM3 (CYP102A1). Different N-modifying groups provide product diversity at high enantioselectivity (up to 99% e.e.) from a panel of just 48 variants of P450BM3. Substrate docking into molecular-dynamics-simulated structures of enzyme variants is shown to be useful for designing mutations to increase enantioselectivity by disfavouring binding poses leading to the unwanted enantiomer, and to increase enzymatic activity by disfavouring non-productive poses from ten or so variants per generation. The synthetic application of remote C–H activation within cyclic amines is exemplified by the synthesis of anisodamine via enantioselective hydroxylation of N-Boc-nortropinone.
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All data supporting the findings of this study are available within the paper and the Supplementary Information. Source data are provided with this paper. Crystallographic data for the structures reported in this Article have been deposited at the Cambridge Crystallographic Data Centre, under deposition numbers CCDC 2159261 ((R)-4b) and 2159262 ((S)-6a). Copies of the data can be obtained free of charge via https://www.ccdc.cam.ac.uk/structures/.
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This work was supported by the Biotechnology and Biological Sciences Research Council, UK (BB/V003445/1). Y.Z. acknowledges a University of Oxford–China Scholarship Council Graduate Scholarship. M.W. and P.H.-L. acknowledge the EPSRC Centre for Doctoral Training in Synthesis for Biology and Medicine (EP/L015838/1) for graduate studentships. For the purpose of open access, the authors have applied a creative commons attribution (CC BY) licence to any author-accepted manuscript version arising.
The authors declare no competing interests.
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Supplementary Methods, Figs. 1–27, Tables 1–17, product characterization data and NMR spectra.
Crystallographic data for compound 4b, CCDC 2159261.
Structure factors for 4b, CCDC 2159261.
Crystallographic data for compound 6a, CCDC 2159262.
Structure factors for compound 6a, CCDC 2159262.
Original Ortep figures for crystal structures.
Source data for Fig. 3a,b.
Original ray-traced figures from Pymol for Fig. 3c–f.
Source data for Fig. 4a and Fig. 4b.
Original ray-traced figures from Pymol for Fig. 4c,d.
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Zhang, Y., Xiong, Z., Li, Y. et al. Enantioselective oxidation of unactivated C–H bonds in cyclic amines by iterative docking-guided mutagenesis of P450BM3 (CYP102A1). Nat. Synth 1, 936–945 (2022). https://doi.org/10.1038/s44160-022-00166-6