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Genetically programmed chiral organoborane synthesis

Nature volume 552, pages 132–136 (2017)Cite this article

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Abstract

Recent advances in enzyme engineering and design have expanded nature’s catalytic repertoire to functions that are new to biology1,2,3. However, only a subset of these engineered enzymes can function in living systems4,5,6,7. Finding enzymatic pathways that form chemical bonds that are not found in biology is particularly difficult in the cellular environment, as this depends on the discovery not only of new enzyme activities, but also of reagents that are both sufficiently reactive for the desired transformation and stable in vivo. Here we report the discovery, evolution and generalization of a fully genetically encoded platform for producing chiral organoboranes in bacteria. Escherichia coli cells harbouring wild-type cytochrome c from Rhodothermus marinus8 (Rma cyt c) were found to form carbon–boron bonds in the presence of borane–Lewis base complexes, through carbene insertion into boron–hydrogen bonds. Directed evolution of Rma cyt c in the bacterial catalyst provided access to 16 novel chiral organoboranes. The catalyst is suitable for gram-scale biosynthesis, providing up to 15,300 turnovers, a turnover frequency of 6,100 h–1, a 99:1 enantiomeric ratio and 100% chemoselectivity. The enantiopreference of the biocatalyst could also be tuned to provide either enantiomer of the organoborane products. Evolved in the context of whole-cell catalysts, the proteins were more active in the whole-cell system than in purified forms. This study establishes a DNA-encoded and readily engineered bacterial platform for borylation; engineering can be accomplished at a pace that rivals the development of chemical synthetic methods, with the ability to achieve turnovers that are two orders of magnitude (over 400-fold) greater than those of known chiral catalysts for the same class of transformation9,10,11. This tunable method for manipulating boron in cells could expand the scope of boron chemistry in living systems.

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Figure 1: Discovery, evolution and characterization of a bacterial catalyst for borylation.
Figure 2: Scope of chiral organoborane production in E. coli.
Figure 3: Expanding the generality and utility of biological borylation.

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Acknowledgements

This work was supported in part by the National Science Foundation, Office of Chemical, Bioengineering, Environmental and Transport Systems SusChEM Initiative (grant CBET-1403077), the Gordon and Betty Moore Foundation through grant GBMF2809 to the Caltech Programmable Molecular Technology Initiative, and the Jacobs Institute for Molecular Engineering for Medicine at Caltech. X.H. is supported by a Ruth L. Kirschstein National Institutes of Health Postdoctoral Fellowship (F32GM125231). We thank O. F. Brandenberg, S. Brinkmann-Chen, T. Hashimoto, R. D. Lewis, and D. K. Romney for discussions and/or comments on the manuscript, and N. W. Goldberg and A. Zutshi for experimental assistance. We are grateful to S. Virgil, N. Torian, M. K. Takase and L. Henling for analytical support, and H. Gray for providing the pEC86 plasmid.

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Author notes

  1. S. B. Jennifer Kan and Xiongyi Huang: These authors contributed equally to this work.

Authors and Affiliations

  1. Division of Chemistry and Chemical Engineering, California Institute of Technology, 1200 East California Boulevard, MC 210-41, Pasadena, 91125, California, USA

    S. B. Jennifer Kan, Xiongyi Huang, Yosephine Gumulya, Kai Chen & Frances H. Arnold

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Contributions

S.B.J.K. and X.H. designed the research with guidance from F.H.A. S.B.J.K., X.H., Y.G. and K.C. performed the experiments and analysed the data. S.B.J.K., X.H. and F.H.A. wrote the manuscript with input from all authors.

Corresponding author

Correspondence to Frances H. Arnold.

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Competing interests

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

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Reviewer Information Nature thanks M. Fischbach and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Figure 1 Examples of boron-containing natural products.

Extended Data Figure 2 Summary of known catalytic systems for metal–carbenoid insertion reactions of boranes.

a, Rh2(esp)2-catalysed borylation of diazo esters with NHC-boranes27. b, Cu(MeCN)4PF6-catalysed borylation of diazo esters with phosphine-borane9. c, [Rh(C2H4)2Cl]2-catalysed borylation of diazo esters with amine-borane10. d, Cu(MeCN)4PF6-catalysed borylation of CF3-substituted (diazomethyl)benzene with phosphine-borane11. e, Rh2(R-BTPCP)4-catalysed borylation using alkynes as carbene precursors42. f, I2-catalysed borylation of diazo esters with NHC-boranes43.

Extended Data Figure 3 Effect of biological borylation on E. coli cell viability.

Cell viability assay was performed in biological triplicate, see Methods section for experimental protocol.

Extended Data Table 1 Preliminary borylation experiments with haem and haem proteins using NHC-borane (1) and Me-EDA (2) as substrates
Full size table
Extended Data Table 2 Biosynthesis of organoboranes 3 and 9 via serial substrate addition
Full size table
Extended Data Table 3 Directed evolution of whole-cell Rma cyt c for improved enantioselectivity in the biosynthesis of organoboranes 17, (R)-18 and (S)-18
Full size table

Supplementary information

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Supplementary Data

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Supplementary Data

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Kan, S., Huang, X., Gumulya, Y. et al. Genetically programmed chiral organoborane synthesis. Nature 552, 132–136 (2017). https://doi.org/10.1038/nature24996

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