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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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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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.
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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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DOI: https://doi.org/10.1038/nchem.2285
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