Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

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

Nature volume 565, pages 67–72 (2019)Cite this article

Subjects

Abstract

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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Enzymatic C–H functionalization systems.
Fig. 2: Haem-protein-catalysed sp3 C–H alkylation.
Fig. 3: Substrate scope for benzylic C–H alkylation with P411-CHF.
Fig. 4: Application of P411 enzymes for sp3 C–H alkylation.

Similar content being viewed by others

Data availability

All relevant data are provided in Supplementary Information. Any additional information is available from the corresponding author on request.

References

  1. Hartwig, J. F. & Larsen, M. A. Undirected, homogeneous C–H bond functionalization: challenges and opportunities. ACS Cent. Sci. 2, 281–292 (2016).

    Article  CAS  Google Scholar 

  2. Saint-Denis, T. G., Zhu, R.-Y., Chen, G., Wu, Q.-F. & Yu, J.-Q. Enantioselective C(sp3)‒H bond activation by chiral transition metal catalysts. Science 359, eaao4798 (2018).

    Article  Google Scholar 

  3. Frey, P. A. & Hegeman, A. D. Enzymatic Reaction Mechanisms Ch. 14 (Oxford Univ. Press, New York, 2007).

  4. Yokoyama, K. & Lilla, E. A. C–C bond forming radical SAM enzymes involved in the construction of carbon skeletons of cofactors and natural products. Nat. Prod. Rep. 35, 660–694 (2018).

    Article  CAS  Google Scholar 

  5. Bauerle, M. R., Schwalm, E. L. & Booker, S. J. Mechanistic diversity of radical S-adenosylmethionine (SAM)-dependent methylation. J. Biol. Chem. 290, 3995–4002 (2015).

    Article  CAS  Google Scholar 

  6. McLaughlin, M. I. & van der Donk, W. A. Stereospecific radical-mediated B12-dependent methyl transfer by the fosfomycin biosynthesis enzyme Fom3. Biochemistry 57, 4967–4971 (2018).

    Article  CAS  Google Scholar 

  7. Shisler, K. A. & Broderick, J. B. Glycyl radical activating enzymes: structure, mechanism, and substrate interactions. Arch. Biochem. Biophys. 546, 64–71 (2014).

    Article  CAS  Google Scholar 

  8. Poulos, T. L. Heme enzyme structure and function. Chem. Rev. 114, 3919–3962 (2014).

    Article  CAS  Google Scholar 

  9. Coelho, P. S., Brustad, E. M., Kannan, A. & Arnold, F. H. Olefin cyclopropanation via carbene transfer catalyzed by engineered cytochrome P450 enzymes. Science 339, 307–310 (2013).

    Article  ADS  CAS  Google Scholar 

  10. 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  ADS  CAS  Google Scholar 

  11. Brandenberg, O. F., Fasan, R. & Arnold, F. H. Exploiting and engineering hemoproteins for abiological carbene and nitrene transfer reactions. Curr. Opin. Biotechnol. 47, 102–111 (2017).

    Article  CAS  Google Scholar 

  12. Liao, K. et al. Site-selective and stereoselective functionalization of non-activated tertiary C–H bonds. Nature 551, 609–613 (2017).

    ADS  CAS  PubMed  Google Scholar 

  13. Weldy, N. M. et al. Iridium(iii)-bis(imidazolinyl)phenyl catalysts for enantioselective C–H functionalization with ethyl diazoacetate. Chem. Sci. 7, 3142–3146 (2016).

    Article  CAS  Google Scholar 

  14. Wang, Y., Wen, X., Cui, X. & Zhang, X. P. Enantioselective radical cyclization for construction of 5-membered ring structures by metalloradical C‒H alkylation. J. Am. Chem. Soc. 140, 4792–4796 (2018).

    Article  CAS  Google Scholar 

  15. Caballero, A. et al. Silver-catalyzed C–C bond formation between methane and ethyl diazoacetate in supercritical CO2. Science 332, 835–838 (2011).

    Article  ADS  CAS  Google Scholar 

  16. Wu, W.-T., Yang, Z.-P. & You, S.-L. in Asymmetric Functionalization of C–H Bonds (ed. You, S.-L.) Ch. 1 (Royal Society of Chemistry, Cambridge, 2015).

  17. Key, H. M., Dydio, P., Clark, D. S. & Hartwig, J. F. Abiological catalysis by artificial haem proteins containing noble metals in place of iron. Nature 534, 534–537 (2016).

    Article  ADS  CAS  Google Scholar 

  18. Mbuvi, H. M. & Woo, L. K. Catalytic C–H insertions using iron(iii) porphyrin complexes. Organometallics 27, 637–645 (2008).

    Article  CAS  Google Scholar 

  19. Li, Y., Huang, J.-S., Zhou, Z.-Y., Che, C.-M. & You, X.-Z. Remarkably stable iron porphyrins bearing nonheteroatom-stabilized carbene or (alkoxycarbonyl)carbenes: isolation, X-ray crystal structures, and carbon atom transfer reactions with hydrocarbons. J. Am. Chem. Soc. 124, 13185–13193 (2002).

    Article  CAS  Google Scholar 

  20. Griffin, J. R., Wendell, C. I., Garwin, J. A. & White, M. C. Catalytic C(sp3)–H alkylation via an iron carbene intermediate. J. Am. Chem. Soc. 139, 13624–13627 (2017).

    Article  CAS  Google Scholar 

  21. Herschlag, D. & Natarajan, A. Fundamental challenges in mechanistic enzymology: progress toward understanding the rate enhancements of enzymes. Biochemistry 52, 2050–2067 (2013).

    Article  CAS  Google Scholar 

  22. 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 

  23. 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 

  24. Zhang, J.-T. et al. Total synthesis of malyngamides K, L, and 5″-epi-C and absolute configuration of malyngamide L. J. Org. Chem. 76, 3946–3959 (2011).

    Article  CAS  Google Scholar 

  25. Edwards, J. T. et al. Decarboxylative alkenylation. Nature 545, 213–218 (2017).

    Article  ADS  CAS  Google Scholar 

  26. He, J., Hamann, L. G., Davies, H. M. L. & Beckwith, R. E. J. Late-stage C–H functionalization of complex alkaloids and drug molecules via intermolecular rhodium-carbenoid insertion. Nat. Commun. 6, 5943 (2015).

    Article  ADS  Google Scholar 

  27. Yang, J.-M., Cai, Y., Zhu, S.-F. & Zhou, Q.-L. Iron-catalyzed arylation of α-aryl-α-diazoesters. Org. Biomol. Chem. 14, 5516–5519 (2016).

    Article  CAS  Google Scholar 

  28. Xu, B., Li, M.-L., Zuo, X.-D., Zhu, S.-F. & Zhou, Q.-L. Catalytic asymmetric arylation of α-aryl-α-diazoacetates with aniline derivatives. J. Am. Chem. Soc. 137, 8700–8703 (2015).

    Article  CAS  Google Scholar 

  29. Cardillo, G., Gentilucci, L., Qasem, A. R., Sgarzi, F. & Spampinato, S. Endomorphin-1 analogues containing β-proline are μ-opioid receptor agonists and display enhanced enzymatic hydrolysis resistance. J. Med. Chem. 45, 2571–2578 (2002).

    Article  CAS  Google Scholar 

  30. Sridharan, V., Suryavanshi, P. A. & Menéndez, J. C. Advances in the chemistry of tetrahydroquinolines. Chem. Rev. 111, 7157–7259 (2011).

    Article  CAS  Google Scholar 

  31. 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).

Download references

Acknowledgements

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.

Reviewer information

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

Author information

Author notes

  1. Lena Wohlschlager

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

  2. Hans Renata

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

Authors and Affiliations

  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

Authors
  1. Ruijie K. Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kai Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Xiongyi Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Lena Wohlschlager
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Hans Renata
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Frances H. Arnold
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

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.

Corresponding author

Correspondence to Frances H. Arnold.

Ethics declarations

Competing interests

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

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

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.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Zhang, R.K., Chen, K., Huang, X. et al. Enzymatic assembly of carbon–carbon bonds via iron-catalysed sp3 C–H functionalization. Nature 565, 67–72 (2019). https://doi.org/10.1038/s41586-018-0808-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41586-018-0808-5

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing