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.

  • Article
  • Published:

Enzyme-controlled stereoselective radical cyclization to arenes enabled by metalloredox biocatalysis

Nature Catalysis volume 6, pages 628–636 (2023)Cite this article

Subjects

Abstract

The effective induction of high levels of stereocontrol for free-radical-mediated transformations represents a notorious challenge in asymmetric catalysis. Herein, we describe a metalloredox biocatalysis strategy to repurpose natural cytochromes P450 to catalyse asymmetric radical cyclization to arenes through an unnatural electron transfer mechanism. Directed evolution afforded a series of engineered P450 aromatic radical cyclases with complementary selectivities: P450arc1 and P450arc2 facilitated enantioconvergent transformations of racemic substrates, giving rise to either enantiomer of the product with excellent total turnover numbers (up to 12,000). In addition to these enantioconvergent variants, another engineered radical cyclase, P450arc3, permitted efficient kinetic resolution of racemic chloride substrates (S factor = 18). Furthermore, computational studies revealed a proton-coupled electron transfer mechanism for the radical–polar crossover step, suggesting the potential role of the haem carboxylate as a base catalyst. Collectively, the excellent tunability of this metalloenzyme family provides an exciting platform for harnessing free radical intermediates for asymmetric catalysis.

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: A metalloenzyme platform for stereoselective radical cyclization.
Fig. 2: Discovery and engineering of enantioconvergent P450 radical cyclases.
Fig. 3: Substrate scope of P450arc-catalysed enantioconvergent radical cyclization.
Fig. 4: P450arc-catalysed stereoselective transformations of α-chloro substrate 3a.
Fig. 5: Time course of P450arc-catalysed enantioconvergent radical cyclization processes.
Fig. 6: Reaction energy profile of biocatalytic radical cyclization to arenes using a model system for serine-ligated P450arc.

Similar content being viewed by others

Data availability

All data are available in the main text and the Supplementary Information or available from the authors upon reasonable request. X-ray crystal structures of 2i and (R)-3a are available free of charge from the Cambridge Crystallographic Data Centre under reference numbers CCDC 2184585 and 2184586. Plasmids encoding P450arcs reported in this study are available for research purposes from Y.Y. under a material transfer agreement with the University of California Santa Barbara.

References

  1. Bornscheuer, U. T. et al. Engineering the third wave of biocatalysis. Nature 485, 185–194 (2012).

    CAS  PubMed  Google Scholar 

  2. Reetz, M. T. Laboratory evolution of stereoselective enzymes: a prolific source of catalysts for asymmetric reactions. Angew. Chem. Int. Ed. 50, 138–174 (2011).

    CAS  Google Scholar 

  3. Devine, P. N. et al. Extending the application of biocatalysis to meet the challenges of drug development. Nat. Rev. Chem. 2, 409–421 (2018).

    Google Scholar 

  4. Zetzsche, L. E., Chakrabarty, S. & Narayan, A. R. H. The transformative power of biocatalysis in convergent synthesis. J. Am. Chem. Soc. 144, 5214–5225 (2022).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Zetzsche, L. E. & Narayan, A. R. H. Broadening the scope of biocatalytic C–C bond formation. Nat. Rev. Chem. 4, 334–346 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. 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).

    CAS  Google Scholar 

  7. Leveson-Gower, R. B., Mayer, C. & Roelfes, G. The importance of catalytic promiscuity for enzyme design and evolution. Nat. Rev. Chem. 3, 687–705 (2019).

    CAS  Google Scholar 

  8. Chen, K. & Arnold, F. H. Engineering new catalytic activities in enzymes. Nat. Catal. 3, 203–213 (2020).

    CAS  Google Scholar 

  9. Miller, D. C., Athavale, S. V. & Arnold, F. H. Combining chemistry and protein engineering for new-to-nature biocatalysis. Nat. Synth. 1, 18–23 (2022).

    PubMed  PubMed Central  Google Scholar 

  10. Klaus, C. & Hammer, S. C. New catalytic reactions by enzyme engineering. Trends Chem. 4, 363–366 (2022).

    CAS  Google Scholar 

  11. Jäger, C. M. & Croft, A. K. Anaerobic radical enzymes for biotechnology. ChemBioEng Rev. 5, 143–162 (2018).

    Google Scholar 

  12. Broderick, J. B., Duffus, B. R., Duschene, K. S. & Shepard, E. M. Radical S-adenosylmethionine enzymes. Chem. Rev. 114, 4229–4317 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Sandoval, B. A. & Hyster, T. K. Emerging strategies for expanding the toolbox of enzymes in biocatalysis. Curr. Opin. Chem. Biol. 55, 45–51 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Harrison, W., Huang, X. & Zhao, H. Photobiocatalysis for abiological transformations. Acc. Chem. Res. 55, 1087–1096 (2022).

    CAS  PubMed  Google Scholar 

  15. Emmanuel, M. A., Greenberg, N. R., Oblinsky, D. G. & Hyster, T. K. Accessing non-natural reactivity by irradiating nicotinamide-dependent enzymes with light. Nature 540, 414–417 (2016).

    CAS  PubMed  Google Scholar 

  16. Sandoval, B. A., Meichan, A. J. & Hyster, T. K. Enantioselective hydrogen atom transfer: discovery of catalytic promiscuity in flavin-dependent ‘ene’-reductases. J. Am. Chem. Soc. 139, 11313–11316 (2017).

    CAS  PubMed  Google Scholar 

  17. Biegasiewicz, K. F. et al. Photoexcitation of flavoenzymes enables a stereoselective radical cyclization. Science 364, 1166 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Black, M. J. et al. Asymmetric redox-neutral radical cyclization catalysed by flavin-dependent ‘ene’-reductases. Nat. Chem. 12, 71–75 (2020).

    PubMed  Google Scholar 

  19. Page, C. G. et al. Quaternary charge-transfer complex enables photoenzymatic intermolecular hydroalkylation of olefins. J. Am. Chem. Soc. 143, 97–102 (2021).

    CAS  PubMed  Google Scholar 

  20. Fu, H. et al. An asymmetric sp3–sp3 cross-electrophile coupling using ‘ene’-reductases. Nature 610, 302–307 (2022).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Huang, X. et al. Photoenzymatic enantioselective intermolecular radical hydroalkylation. Nature 584, 69–74 (2020).

    CAS  PubMed  Google Scholar 

  22. Huang, X. et al. Photoinduced chemomimetic biocatalysis for enantioselective intermolecular radical conjugate addition. Nat. Catal. 5, 586–593 (2022).

    CAS  Google Scholar 

  23. Zhou, Q., Chin, M., Fu, Y., Liu, P. & Yang, Y. Stereodivergent atom-transfer radical cyclization by engineered cytochromes P450. Science 374, 1612–1616 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Fu, Y. et al. Engineered P450 atom-transfer radical cyclases are bifunctional biocatalysts: reaction mechanism and origin of enantioselectivity. J. Am. Chem. Soc. 144, 13344–13355 (2022).

    CAS  PubMed  Google Scholar 

  25. Rui, J. et al. Directed evolution of nonheme iron enzymes to access abiological radical-relay C(sp3)−H azidation. Science 376, 869–874 (2022).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Proctor, R. S. J., Colgan, A. C. & Phipps, R. J. Exploiting attractive non-covalent interactions for the enantioselective catalysis of reactions involving radical intermediates. Nat. Chem. 12, 990–1004 (2020).

    PubMed  Google Scholar 

  27. Mondal, S. et al. Enantioselective radical reactions using chiral catalysts. Chem. Rev. 122, 5842–5976 (2022).

    CAS  PubMed  Google Scholar 

  28. Sibi, M. P., Manyem, S. & Zimmerman, J. Enantioselective radical processes. Chem. Rev. 103, 3263–3296 (2003).

    CAS  PubMed  Google Scholar 

  29. Clark, A. J. Copper catalyzed atom transfer radical cyclization reactions. Eur. J. Org. Chem. 2016, 2231–2243 (2016).

    CAS  Google Scholar 

  30. Bhat, V., Welin, E. R., Guo, X. & Stoltz, B. M. Advances in stereoconvergent catalysis from 2005 to 2015: transition-metal-mediated stereoablative reactions, dynamic kinetic resolutions, and dynamic kinetic asymmetric transformations. Chem. Rev. 117, 4528–4561 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Keith, J. M., Larrow, J. F. & Jacobsen, E. N. Practical considerations in kinetic resolution reactions. Adv. Synth. Catal. 343, 5–26 (2001).

    CAS  Google Scholar 

  32. Vedejs, E. & Jure, M. Efficiency in nonenzymatic kinetic resolution. Angew. Chem. Int. Ed. 44, 3974–4001 (2005).

    CAS  Google Scholar 

  33. Zheng, Y., Zhang, S., Low, K.-H., Zi, W. & Huang, Z. A unified and desymmetric approach to chiral tertiary alkyl halides. J. Am. Chem. Soc. 144, 1951–1961 (2022).

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Phillips, I. R., Shephard, E. A. & Ortiz de Montellano, P. R. Cytochrome P450 Protocols (Humana, 2013).

  36. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Yang, Y. & Arnold, F. H. Navigating the unnatural reaction space: directed evolution of heme proteins for selective carbene and nitrene transfer. Acc. Chem. Res. 54, 1209–1225 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Zhou, F., Liu, Y.-L. & Zhou, J. Catalytic asymmetric synthesis of oxindoles bearing a tetrasubstituted stereocenter at the C-3 position. Adv. Synth. Catal. 352, 1381–1407 (2010).

    CAS  Google Scholar 

  39. Whitehouse, C. J. C., Bell, S. G. & Wong, L.-L. P450BM3 (CYP102A1): connecting the dots. Chem. Soc. Rev. 41, 1218–1260 (2012).

    CAS  PubMed  Google Scholar 

  40. Yang, Y., Cho, I., Qi, X., Liu, P. & Arnold, F. H. An enzymatic platform for the asymmetric amination of primary, secondary and tertiary C(sp3)–H bonds. Nat. Chem. 11, 987–993 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Mai, B. K., Neris, N. M., Yang, Y. & Liu, P. C–N bond forming radical rebound is the enantioselectivity-determining step in P411-catalyzed enantioselective C(sp3)–H amination: a combined computational and experimental investigation. J. Am. Chem. Soc. 144, 11215–11225 (2022).

    CAS  PubMed  Google Scholar 

  42. Acevedo-Rocha, C. G., Hoebenreich, S. & Reetz, M. T. In Directed Evolution Library Creation: Methods and Protocols (eds. Gillam, E. M. J. et al.) 103–128 (Springer, 2014).

  43. Weinberg, D. R. et al. Proton-coupled electron transfer. Chem. Rev. 112, 4016–4093 (2012).

    CAS  PubMed  Google Scholar 

  44. Warren, J. J. & Mayer, J. M. Proton-coupled electron transfer reactions at a heme-propionate in an iron-protoporphyrin-IX model compound. J. Am. Chem. Soc. 133, 8544–8551 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This research is supported by the NIH (R35GM147387 to Y.Y. and R35GM128779 to P.L.) and the University of California Santa Barbara (Y.Y.). We acknowledge the BioPACIFIC MIP (NSF Materials Innovation Platform, DMR-1933487) and the NSF MRSEC Program (DMR-1720256) for access to instrumentation. DFT calculations were performed at the Center for Research Computing of the University of Pittsburgh and the Extreme Science and Engineering Discovery Environment supported by the National Science Foundation grant number ACI-1548562. Y.F. is an Andrew W. Mellon Predoctoral Fellow. We thank L. Zhang (University of California Santa Barbara) and Y. Wang (University of Pittsburgh) for critical reading of this manuscript.

Author information

Authors and Affiliations

  1. Department of Chemistry and Biochemistry, University of California Santa Barbara, Santa Barbara, CA, USA

    Wenzhen Fu, Natalia M. Neris, Yunlong Zhao, Benjamin Krohn-Hansen & Yang Yang

  2. Department of Chemistry, University of Pittsburgh, Pittsburgh, PA, USA

    Yue Fu & Peng Liu

  3. Biomolecular Science and Engineering Program, University of California Santa Barbara, Santa Barbara, CA, USA

    Yang Yang

Authors
  1. Wenzhen Fu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Natalia M. Neris
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yue Fu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yunlong Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Benjamin Krohn-Hansen
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Peng Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Yang Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Y.Y. conceived and directed the project. W.F., N.M.N., Y.Z. and B.K.-H. designed and performed the experiments. Y.F. carried out the computational studies with P.L. providing guidance. Y.Y., Y.F. and P.L. wrote the manuscript with the input of all other authors.

Corresponding authors

Correspondence to Peng Liu or Yang Yang.

Ethics declarations

Competing interests

Y.Y., W.F., N.M.N. and Y.Z. are inventors on a patent application (US provisional patent no. 63/477,081) submitted by the University of California Santa Barbara that covers stereoselective biocatalytic radical addition to arenes. The remaining authors declare no competing interests.

Peer review

Peer review information

Nature Catalysis thanks Gonzalo Jiménez-Osé and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Additional information

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

Supplementary information

Supplementary Information

Supplementary Methods, Tables 1–15, Figs. 1–26, Notes 1–12, Discussion and References.

Reporting Summary

Supplementary Data 1

Crystallographic data for compound 2i.

Supplementary Data 2

Crystallographic data for compound 3a.

Supplementary Data 3

Atomic coordinates of the optimized computational models from molecular dynamics simulations.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Fu, W., Neris, N.M., Fu, Y. et al. Enzyme-controlled stereoselective radical cyclization to arenes enabled by metalloredox biocatalysis. Nat Catal 6, 628–636 (2023). https://doi.org/10.1038/s41929-023-00986-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41929-023-00986-5

This article is cited by

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