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Enantioselective oxidation of unactivated C–H bonds in cyclic amines by iterative docking-guided mutagenesis of P450BM3 (CYP102A1)

Nature Synthesis volume 1, pages 936–945 (2022)Cite this article

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Abstract

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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Fig. 1: Synthetic applications of enantioselective oxidation of cyclic amines by engineered P450BM3.
Fig. 2: Molecular structure of two cyclic amine oxidation products determined by single-crystal X-ray diffraction.
Fig. 3: IDGM of P450BM3 for the selective (S)-β oxidation of N-Ips-azepane (2).
Fig. 4: IDGM for (S)-endo oxidation of N-Boc-7-azabicyclo[2.2.1]heptane (5).

Data availability

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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Acknowledgements

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.

Author information

Authors and Affiliations

  1. Department of Chemistry, University of Oxford, Inorganic Chemistry Laboratory, Oxford, UK

    Yuan Zhang, Yushu Li, Ellie Jaques & Luet L. Wong

  2. Oxford Suzhou Centre for Advanced Research, Suzhou, P.R. China

    Ziyue Xiong, Jeremy Robertson & Luet L. Wong

  3. Department of Chemistry, University of Oxford, Chemistry Research Laboratory, Oxford, UK

    Mary Wilson, Kirsten E. Christensen, Pol Hernández-Lladó & Jeremy Robertson

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Contributions

Y.Z. performed enzyme and substrate engineering, activity screening, preparative-scale substrate oxidations, product characterization, MD simulations, substrate docking and docking-guided mutagenesis, and wrote the paper. Z.X. performed the stereoselective synthesis of anisodamine and wrote the paper. Y.L., E.J. and P.H.-L. carried out activity screening and product characterization experiments. M.W. performed activity screening, product characterization and steps in the synthesis of anisodamine. K.E.C. determined the crystal structures to assign the absolute configurations of two products and wrote the paper. J.R. conceived and guided the project, designed the synthesis of anisodamine and wrote the paper. L.L.W. conceived and guided the project, designed the steps in IDGM and wrote the paper.

Corresponding authors

Correspondence to Kirsten E. Christensen, Jeremy Robertson or Luet L. Wong.

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Peer review information

Nature Synthesis thanks Rita Bernhardt, Rudi Fasan, Nicholas Turner and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary handling editor Alison Stoddart, in collaboration with the Nature Synthesis team.

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Supplementary information

Supplementary Information

Supplementary Methods, Figs. 1–27, Tables 1–17, product characterization data and NMR spectra.

Supplementary Data 1

Crystallographic data for compound 4b, CCDC 2159261.

Supplementary Data 2

Structure factors for 4b, CCDC 2159261.

Supplementary Data 3

Crystallographic data for compound 6a, CCDC 2159262.

Supplementary Data 4

Structure factors for compound 6a, CCDC 2159262.

Source data

Source Data Fig. 2

Original Ortep figures for crystal structures.

Source Data Fig. 3

Source data for Fig. 3a,b.

Source Data Fig. 3

Original ray-traced figures from Pymol for Fig. 3c–f.

Source Data Fig. 4

Source data for Fig. 4a and Fig. 4b.

Source Data Fig. 4

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

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