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Genetically programmed chiral organoborane synthesis

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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References

  1. Renata, H., Wang, Z. J. & Arnold, F. H. Expanding the enzyme universe: accessing non-natural reactions by mechanism-guided directed evolution. Angew. Chem. Int. Ed. 54, 3351–3367 (2015)

    Article  CAS  Google Scholar 

  2. Hyster, T. K. & Ward, T. R. Genetic optimization of metalloenzymes: enhancing enzymes for non-natural reactions. Angew. Chem. Int. Ed. 55, 7344–7357 (2016)

    Article  CAS  Google Scholar 

  3. Hammer, S. C., Knight, A. M. & Arnold, F. H. Design and evolution of enzymes for non-natural chemistry. Curr. Opin. Green Sustainable Chem. 7, 23–30 (2017)

    Article  Google Scholar 

  4. Coelho, P. S. et al. A serine-substituted P450 catalyzes highly efficient carbene transfer to olefins in vivo. Nat. Chem. Biol. 9, 485–487 (2013)

    Article  CAS  Google Scholar 

  5. Jeschek, M. et al. Directed evolution of artificial metalloenzymes for in vivo metathesis. Nature 537, 661–665 (2016)

    Article  CAS  ADS  Google Scholar 

  6. Kan, S. B. J., Lewis, R. D., Chen, K. & Arnold, F. H. Directed evolution of cytochrome c for carbon–silicon bond formation: Bringing silicon to life. Science 354, 1048–1051 (2016)

    Article  CAS  ADS  Google Scholar 

  7. Tinoco, A., Steck, V., Tyagi, V. & Fasan, R. Highly diastereo- and enantioselective synthesis of trifluoromethyl-substituted cyclopropanes via myoglobin-catalyzed transfer of trifluoromethylcarbene. J. Am. Chem. Soc. 139, 5293–5296 (2017)

    Article  CAS  Google Scholar 

  8. Stelter, M. et al. A novel type of monoheme cytochrome c: biochemical and structural characterization at 1.23 A resolution of Rhodothermus marinus cytochrome c. Biochemistry 47, 11953–11963 (2008)

    Article  CAS  Google Scholar 

  9. Cheng, Q.-Q., Zhu, S.-F., Zhang, Y.-Z., Xie, X.-L. & Zhou, Q.-L. Copper-catalyzed B–H bond insertion reaction: a highly efficient and enantioselective C–B bond-forming reaction with amine–borane and phosphine–borane adducts. J. Am. Chem. Soc. 135, 14094–14097 (2013)

    Article  CAS  Google Scholar 

  10. Chen, D., Zhang, X., Qi, W.-Y., Xu, B. & Xu, M.-H. Rhodium(i)-catalyzed asymmetric carbene insertion into B–H bonds: highly enantioselective access to functionalized organoboranes. J. Am. Chem. Soc. 137, 5268–5271 (2015)

    Article  CAS  Google Scholar 

  11. Hyde, S. et al. Copper-catalyzed insertion into heteroatom–hydrogen bonds with trifluorodiazoalkanes. Angew. Chem. Int. Ed. 55, 3785–3789 (2016)

    Article  CAS  Google Scholar 

  12. Irschik, H., Schummer, D., Gerth, K., Höfle, G. & Reichenbach, H. The tartrolons, new boron-containing antibiotics from a myxobacterium, Sorangium cellulosum J. Antibiot. 48, 26–30 (1995)

    Article  CAS  Google Scholar 

  13. Wolkenstein, K., Sun, H., Falk, H. & Griesinger, C. Structure and absolute configuration of Jurassic polyketide-derived spiroborate pigments obtained from microgram quantities. J. Am. Chem. Soc. 137, 13460–13463 (2015)

    Article  CAS  Google Scholar 

  14. Chen, X. et al. Structural identification of a bacterial quorum-sensing signal containing boron. Nature 415, 545–549 (2002)

    Article  CAS  ADS  Google Scholar 

  15. Elshahawi, S. I. et al. Boronated tartrolon antibiotic produced by symbiotic cellulose-degrading bacteria in shipworm gills. Proc. Natl Acad. Sci. USA 110, E295–E304 (2013)

    Article  CAS  Google Scholar 

  16. Dembitsky, V. M., Al Quntar, A. A. & Srebnik, M. Natural and synthetic small boron-containing molecules as potential inhibitors of bacterial and fungal quorum sensing. Chem. Rev. 111, 209–237 (2011)

    Article  CAS  Google Scholar 

  17. Prier, C. K., Zhang, R. K., Buller, A. R., Brinkmann-Chen, S. & Arnold, F. H. Enantioselective, intermolecular benzylic C–H amination catalysed by an engineered iron-haem enzyme. Nat. Chem. 9, 629–634 (2017)

    Article  CAS  Google Scholar 

  18. Das, B. C. et al. Boron chemicals in diagnosis and therapeutics. Future Med. Chem. 5, 653–676 (2013)

    Article  CAS  Google Scholar 

  19. Miyaura, N. & Suzuki, A. Palladium-catalyzed cross-coupling reactions of organoboron compounds. Chem. Rev. 95, 2457–2483 (1995)

    Article  CAS  Google Scholar 

  20. Leonori, D. & Aggarwal, V. K. Lithiation–borylation methodology and its application in synthesis. Acc. Chem. Res. 47, 3174–3183 (2014)

    Article  CAS  Google Scholar 

  21. Leonori, D. & Aggarwal, V. K. Stereospecific couplings of secondary and tertiary boronic esters. Angew. Chem. Int. Ed. 54, 1082–1096 (2015)

    Article  CAS  Google Scholar 

  22. Tehfe, M. A. et al. A water-compatible NHC-borane: Photopolymerizations in water and rate constants for elementary radical reactions. ACS Macro Lett. 1, 92–95 (2012)

    Article  CAS  Google Scholar 

  23. Handa, S., Wang, Y., Gallou, F. & Lipshutz, B. H. Sustainable Fe–ppm Pd nanoparticle catalysis of Suzuki-Miyaura cross-couplings in water. Science 349, 1087–1091 (2015)

    Article  CAS  ADS  Google Scholar 

  24. Chang, M. C. Y., Pralle, A., Isacoff, E. Y. & Chang, C. J. A selective, cell-permeable optical probe for hydrogen peroxide in living cells. J. Am. Chem. Soc. 126, 15392–15393 (2004)

    Article  CAS  Google Scholar 

  25. Halo, T. L., Appelbaum, J., Hobert, E. M., Balkin, D. M. & Schepartz, A. Selective recognition of protein tetraserine motifs with a cell-permeable, pro-fluorescent bis-boronic acid. J. Am. Chem. Soc. 131, 438–439 (2009)

    Article  CAS  Google Scholar 

  26. Kim, J. & Bertozzi, C. R. A bioorthogonal reaction of N-oxide and boron reagents. Angew. Chem. Int. Ed. 54, 15777–15781 (2015)

    Article  CAS  Google Scholar 

  27. Li, X. & Curran, D. P. Insertion of reactive rhodium carbenes into boron–hydrogen bonds of stable N-heterocyclic carbene boranes. J. Am. Chem. Soc. 135, 12076–12081 (2013)

    Article  CAS  Google Scholar 

  28. Curran, D. P. et al. Synthesis and reactions of N-heterocyclic carbene boranes. Angew. Chem. Int. Ed. 50, 10294–10317 (2011)

    Article  CAS  MathSciNet  Google Scholar 

  29. Würtemberger-Pietsch, S., Radius, U. & Marder, T. B. 25 years of N-heterocyclic carbenes: activation of both main-group element–element bonds and NHCs themselves. Dalton Trans. 45, 5880–5895 (2016)

    Article  Google Scholar 

  30. Arslan, E ., Schulz, H ., Zufferey, R ., Künzler, P & Thöny-Meyer, L. Overproduction of the Bradyrhizobium japonicum c-type cytochrome subunits of the cbb3 oxidase in Escherichia coli. Biochem. Biophys. Res. Commun. 251, 744–747 (1998)

    Article  CAS  Google Scholar 

  31. Kille, S. et al. Reducing codon redundancy and screening effort of combinatorial protein libraries created by saturation mutagenesis. ACS Synth. Biol. 2, 83–92 (2013)

    Article  CAS  Google Scholar 

  32. Mara, M. W. et al. Metalloprotein entatic control of ligand–metal bonds quantified by ultrafast X-ray spectroscopy. Science 356, 1276–1280 (2017)

    Article  CAS  ADS  Google Scholar 

  33. Renata, H. et al. Identification of mechanism-based inactivation in P450-catalyzed cyclopropanation facilitates engineering of improved enzymes. J. Am. Chem. Soc. 138, 12527–12533 (2016)

    Article  CAS  Google Scholar 

  34. Hernandez, K. E. et al. Highly stereoselective biocatalytic synthesis of key cyclopropane intermediate to ticagrelor. ACS Catal. 6, 7810–7813 (2016)

    Article  CAS  Google Scholar 

  35. Argintaru, O. A., Ryu, D., Aron, I. & Molander, G. A. Synthesis and applications of α-trifluoromethylated alkylboron compounds. Angew. Chem. Int. Ed. 52, 13656–13660 (2013)

    Article  CAS  Google Scholar 

  36. Jiang, Q., Guo, T. & Yu, Z. Copper-catalyzed asymmetric borylation: Construction of a stereogenic carbon center bearing both CF3 and organoboron functional groups. J. Org. Chem. 82, 1951–1960 (2017)

    Article  CAS  Google Scholar 

  37. Kanouni, T., Stafford, J. A., Veal, J. M. & Wallace, M. B. Histone demethylase inhibitors. WO 2014/151106 A1 (2014)

  38. Scopes, D. Pyrrolo [3,2-E] [1,2,4] triazolo [1,5-A] pyrimidines derivatives as inhibitors of microglia activation. US patent 2012/0289523 A1 (2012)

  39. Gibson, D. G. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6, 343–345 (2009)

    CAS  Google Scholar 

  40. Sambrook, J ., Frisch, E. & Maniatis, T. Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1989)

  41. Berry, E. A. & Trumpower, B. L. Simultaneous determination of hemes a, b, and c from pyridine hemochrome spectra. Anal. Biochem. 161, 1–15 (1987)

    Article  CAS  Google Scholar 

  42. Yang, J.-M. et al. Catalytic B−H bond insertion reactions using alkynes as carbene precursors. J. Am. Chem. Soc. 139, 3784–3789 (2017)

    Article  CAS  Google Scholar 

  43. Allen, T. H., Kawamoto, T., Gardner, S., Geib, S. J. & Curran, D. P. N-heterocyclic carbene boryl iodides catalyze insertion reactions of N-heterocyclic carbene boranes and diazoesters. Org. Lett. 19, 3680–3683 (2017)

    Article  CAS  Google Scholar 

  44. Wang, Z. J. et al. Improved cyclopropanation activity of histidine-ligated cytochrome P450 enables the enantioselective formal synthesis of levomilnacipran. Angew. Chem. Int. Ed. 53, 6810–6813 (2014)

    Article  CAS  Google Scholar 

Download references

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.

Author information

Authors and Affiliations

Authors

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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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
Extended Data Table 2 Biosynthesis of organoboranes 3 and 9 via serial substrate addition
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

Supplementary information

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Supplementary Information

This file contains information on borylation with additional figures. (PDF 10741 kb)

Supplementary Data

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Supplementary Data

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Supplementary Data

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Supplementary Data

This file contains cif files for structures CCDC1572198, CCDC1572200 and CCDC1572201. (ZIP 457 kb)

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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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