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Enzymatic assembly of carbon–carbon bonds via iron-catalysed sp3 C–H functionalization

Naturevolume 565pages6772 (2019) | Download Citation


Although abundant in organic molecules, carbon–hydrogen (C–H) bonds are typically considered unreactive and unavailable for chemical manipulation. Recent advances in C–H functionalization technology have begun to transform this logic, while emphasizing the importance of and challenges associated with selective alkylation at a sp3 carbon1,2. Here we describe iron-based catalysts for the enantio-, regio- and chemoselective intermolecular alkylation of sp3 C–H bonds through carbene C–H insertion. The catalysts, derived from a cytochrome P450 enzyme in which the native cysteine axial ligand has been substituted for serine (cytochrome P411), are fully genetically encoded and produced in bacteria, where they can be tuned by directed evolution for activity and selectivity. That these proteins activate iron, the most abundant transition metal, to perform this chemistry provides a desirable alternative to noble-metal catalysts, which have dominated the field of C–H functionalization1,2. The laboratory-evolved enzymes functionalize diverse substrates containing benzylic, allylic or α-amino C–H bonds with high turnover and excellent selectivity. Furthermore, they have enabled the development of concise routes to several natural products. The use of the native iron-haem cofactor of these enzymes to mediate sp3 C–H alkylation suggests that diverse haem proteins could serve as potential catalysts for this abiological transformation, and will facilitate the development of new enzymatic C–H functionalization reactions for applications in chemistry and synthetic biology.

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This work was supported by the National Science Foundation (NSF), Division of Molecular and Cellular Biosciences (grant MCB-1513007). R.K.Z. acknowledges support from the NSF Graduate Research Fellowship (grant DGE-1144469) and the Donna and Benjamin M. Rosen Bioengineering Center. X.H. is supported by a Ruth L. Kirschstein National Institutes of Health Postdoctoral Fellowship (grant F32GM125231). L.W. received support from the Austrian Marshall Plan Foundation. We thank A. Z. Zhou for experimental assistance; N. W. Goldberg, S. C. Hammer, K. E. Hernandez, Z. Jia, A. M. Knight, G. Kubik, R. D. Lewis, C. K. Prier, D. K. Romney and J. Zhang for discussions; S. Virgil, M. Shahgholi and D. VanderVelde for analytical support; and B. Stoltz for use of polarimeter and gas chromatography equipment.

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Nature thanks B. de Bruin, N. Turner and T. Ward for their contribution to the peer review of this work.

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Author notes

    • Lena Wohlschlager

    Present address: Institute of Food Technology, University of Natural Resources and Life Sciences, Vienna, Austria

    • Hans Renata

    Present address: Department of Chemistry, The Scripps Research Institute, Jupiter, FL, USA


  1. Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA, USA

    • Ruijie K. Zhang
    • , Kai Chen
    • , Xiongyi Huang
    • , Lena Wohlschlager
    • , Hans Renata
    •  & Frances H. Arnold


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R.K.Z. designed the overall research with F.H.A. providing guidance. R.K.Z. and H.R. designed and conducted the initial screening of haem proteins; R.K.Z. and L.W. performed the directed evolution experiments. R.K.Z., K.C. and X.H. designed and performed the substrate scope studies. R.K.Z. and F.H.A. wrote the manuscript with input from all authors.

Competing interests

A provisional patent application has been filed through the California Institute of Technology based on the results presented here.

Corresponding author

Correspondence to Frances H. Arnold.

Supplementary information

  1. Supplementary Information

    This file contains experimental procedures, information about enzyme variants, Supplementary Figures 1–15, Supplementary Tables 1–13, and additional data. Please see the table of contents for details.

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