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Regio- and stereoselectivity of P450-catalysed hydroxylation of steroids controlled by laboratory evolution

Abstract

A current challenge in synthetic organic chemistry is the development of methods that allow the regio- and stereoselective oxidative C–H activation of natural or synthetic compounds with formation of the corresponding alcohols. Cytochrome P450 enzymes enable C–H activation at non-activated positions, but the simultaneous control of both regio- and stereoselectivity is problematic. Here, we demonstrate that directed evolution using iterative saturation mutagenesis provides a means to solve synthetic problems of this kind. Using P450 BM3(F87A) as the starting enzyme and testosterone as the substrate, which results in a 1:1 mixture of the 2β- and 15β-alcohols, mutants were obtained that are 96–97% selective for either of the two regioisomers, each with complete diastereoselectivity. The mutants can be used for selective oxidative hydroxylation of other steroids without performing additional mutagenesis experiments. Molecular dynamics simulations and docking experiments shed light on the origin of regio- and stereoselectivity.

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Figure 1: Mechanism- and structure-based choice of sites for saturation mutagenesis.
Figure 2: Computational results of MD simulations and dockings.

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Acknowledgements

This work was supported by the Fonds der Chemischen Industrie. The authors thank F. Schulz, S. Bastian, J. Drone and D. Bougioukou for discussions, H. Hinrichs and A. Deege for HPLC analyses and C. Farès for NMR analyses.

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S.K. and F.E.Z. performed the experimental work. S.K. and F.E.Z. evaluated the data. J.P.A. carried out the theoretical calculations and analyses. J.P.A. and S.K. performed the kinetics and coupling calculations. M.T.R., S.K. and J.P.A. wrote the manuscript.

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Correspondence to Manfred T. Reetz.

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The authors declare no competing financial interests.

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Kille, S., Zilly, F., Acevedo, J. et al. Regio- and stereoselectivity of P450-catalysed hydroxylation of steroids controlled by laboratory evolution. Nature Chem 3, 738–743 (2011). https://doi.org/10.1038/nchem.1113

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