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Enzymatic hydroxylation of an unactivated methylene C–H bond guided by molecular dynamics simulations

Nature Chemistry volume 7, pages 653–660 (2015)Cite this article

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Abstract

The hallmark of enzymes from secondary metabolic pathways is the pairing of powerful reactivity with exquisite site selectivity. The application of these biocatalytic tools in organic synthesis, however, remains under-utilized due to limitations in substrate scope and scalability. Here, we report how the reactivity of a monooxygenase (PikC) from the pikromycin pathway is modified through computationally guided protein and substrate engineering, and applied to the oxidation of unactivated methylene C–H bonds. Molecular dynamics and quantum mechanical calculations were used to develop a predictive model for substrate scope, site selectivity and stereoselectivity of PikC-mediated C–H oxidation. A suite of menthol derivatives was screened computationally and evaluated through in vitro reactions, where each substrate adhered to the predicted models for selectivity and conversion to product. This platform was also expanded beyond menthol-based substrates to the selective hydroxylation of a variety of substrate cores ranging from cyclic to fused bicyclic and bridged bicyclic compounds.

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Figure 1: Selected oxidation products of (–)-menthol and calculated bond dissociation energies for the corresponding C–H bonds (in kcal mol−1).
Figure 2: Substrate anchoring mechanism employed by P450 PikC.
Figure 3: Substrate binding and tertiary structure of PikC variants observed in MD simulations.
Figure 4: Stereoselectivity in the C4–H abstraction catalysed by PikC.
Figure 5: Site-selective oxidation with PikCD50ND176QE246A-RhFRED.

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Acknowledgements

This work was supported by the National Science Foundation under the CCI Center for Selective C−H Functionalization (CHE-1205646) and the National Institutes of Health (NIH R01 grant GM078553 to D.H.S., J.M. and L.M.P. and grant GM075962 to K.N.H.). Calculations were performed using the Extreme Science and Engineering Discovery Environment (XSEDE), which is funded by the NSF (OCI-1053575), and the UCLA Institute of Digital Research and Education (IDRE). The authors also acknowledge the Life Sciences Research Foundation for a postdoctoral fellowship (to A.R.H.N.) and the University of Michigan Chemistry–Biology Interface (CBI) training programme (GM008597, to S.N.). J. Stachowski is thanked for discussions.

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Authors and Affiliations

  1. Life Sciences Institute, University of Michigan, Ann Arbor, 48109, Michigan, USA

    Alison R. H. Narayan & David H. Sherman

  2. Department of Chemistry, University of California, Los Angeles, 90095, California, USA

    Gonzalo Jiménez-Osés, Peng Liu, Raghunath O. Ramabhadran, Yun-Fang Yang, Lawrence R. Furan, Zhe Li & K. N. Houk

  3. Department of Medicinal Chemistry, University of Michigan, Ann Arbor, 48109, Michigan, USA

    Solymar Negretti, John Montgomery & David H. Sherman

  4. Department of Chemistry, University of Michigan, Ann Arbor, 48109, Michigan, USA

    Wanxiang Zhao, Michael M. Gilbert, John Montgomery & David H. Sherman

  5. Skaggs School of Pharmacy & Pharmaceutical Sciences, University of California, San Diego, 92093, California, USA

    Larissa M. Podust

  6. Department of Microbiology & Immunology, University of Michigan, Ann Arbor, 48109, Michigan, USA

    David H. Sherman

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  1. Alison R. H. Narayan
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Contributions

A.R.H.N. and G.J-O. contributed equally to this work. A.R.H.N., G.J-O., P.L., S.N., J.M., K.N.H. and D.H.S. conceived, designed and supervised the project. G.J-O., P.L., R.O.R., Y-F.Y., L.R.F. and Z.L. performed the computational experiments. A.R.H.N., S.N., W.Z. and M.M.G. synthesized substrates and characterized products. A.R.H.N. conducted the biochemical experiments. A.R.H.N., G.J-O., P.L., L.M.P., J.M., K.N.H. and D.H.S. wrote and edited the manuscript.

Corresponding authors

Correspondence to John Montgomery, K. N. Houk or David H. Sherman.

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Narayan, A., Jiménez-Osés, G., Liu, P. et al. Enzymatic hydroxylation of an unactivated methylene C–H bond guided by molecular dynamics simulations. Nature Chem 7, 653–660 (2015). https://doi.org/10.1038/nchem.2285

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