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:

Using enzymes to tame nitrogen-centred radicals for enantioselective hydroamination

Nature Chemistry volume 15, pages 206–212 (2023)Cite this article

Subjects

Abstract

The formation of C–N bonds—of great importance to the pharmaceutical industry—can be facilitated enzymatically using nucleophilic and nitrene transfer mechanisms. However, neither natural nor engineered enzymes are known to generate and control nitrogen-centred radicals, which serve as valuable species for C–N bond formation. Here we use flavin-dependent ‘ene’-reductases with an exogenous photoredox catalyst to selectively generate amidyl radicals within the protein active site. These enzymes are engineered through directed evolution to catalyse 5-exo, 6-endo, 7-endo, 8-endo, and intermolecular hydroamination reactions with high levels of enantioselectivity. Mechanistic studies suggest that radical initiation occurs via an enzyme-gated mechanism, where the protein thermodynamically activates the substrate for reduction by the photocatalyst. Molecular dynamics studies indicate that the enzymes bind substrates using non-canonical binding interactions, which may serve as a handle to further manipulate reactivity. This approach demonstrates the versatility of these enzymes for controlling the reactivity of high-energy radical intermediates and highlights the opportunity for synergistic catalyst strategies to unlock previously inaccessible enzymatic functions.

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: Biocatalytic C–N bond-forming reactions.
Fig. 2: Reaction discovery and directed evolution of YqjM enzyme for intramolecular hydroamination reaction.
Fig. 3: Mechanistic studies.

Similar content being viewed by others

Data availability

The data that support the findings in this study are available in this article and the Supplementary Information. Source data are provided with this paper.

References

  1. Breuer, M. et al. Industrial methods for the production of optically active intermediates. Angew. Chem. Int. Ed. 43, 788–824 (2004).

    Article  CAS  Google Scholar 

  2. Roughley, S. D. & Jordan, A. M. The medicinal chemist’s toolbox: an analysis of reactions used in the pursuit of drug candidates. J. Med. Chem. 54, 3451–3479 (2011).

    Article  CAS  Google Scholar 

  3. Kerru, N., Gummidi, L., Maddila, S., Gangu, K. K. & Jonnalagadda, S. B. A review on recent advances in nitrogen-containing molecules and their biological applications. Molecules 25, 1909 (2020).

    Article  CAS  Google Scholar 

  4. Slabu, I. S., Galman, J. L., Lloyd, R. C. & Turner, N. J. Discovery, engineering, and synthetic application of transaminase biocatalysts. ACS Catal. 7, 8263–8284 (2017).

    Article  CAS  Google Scholar 

  5. Aleku, G. A. et al. A reductive aminase from Aspergillus oryzae. Nat. Chem. 9, 961–969 (2017).

    Article  CAS  Google Scholar 

  6. Marshall, J. R. et al. Screening and characterization of a diverse panel of metagenomic imine reductases for biocatalytic reductive amination. Nat. Chem. 13, 140–148 (2021).

    Article  CAS  Google Scholar 

  7. Parmeggiani, F., Weise, N. J., Ahmed, S. T. & Turner, N. J. Synthetic and therapeutic applications of ammonia-lyases and aminomutases. Chem. Rev. 118, 73–118 (2018).

    Article  CAS  Google Scholar 

  8. Raj, H. et al. Engineering methylaspartate ammonia lyase for the asymmetric synthesis of unnatural amino acids. Nat. Chem. 4, 478–484 (2012).

    Article  CAS  Google Scholar 

  9. Fu, H. et al. Chemoenzymatic asymmetric synthesis of the metallo-β-lactamase inhibitor aspergillomarasmine A and related aminocarboxylic acids. Nat. Chem. 1, 186–191 (2018).

    CAS  Google Scholar 

  10. Peatzold, J. & Bäckvall, J. E. Chemoenzymatic dynamic kinetic resolution of primary amines. J. Am. Chem. Soc. 127, 17620–17621 (2005).

    Article  Google Scholar 

  11. Ghislieri, D. & Turner, N. J. Biocatalytic approaches to the synthesis of enantiomerically pure chiral amines. Top. Catal. 57, 284–300 (2014).

    Article  CAS  Google Scholar 

  12. Athavale, S. V. et al. Biocatalytic, intermolecular C–H bond functionalization for the synthesis of enantioenriched amides. Angew. Chem. Int. Ed. 60, 24864–24869 (2021).

    Article  CAS  Google Scholar 

  13. Farwell, C. C., Zhang, R. K., McIntosh, J. A., Hyster, T. K. & Arnold, F. H. Enantioselective enzyme-catalyzed aziridination enabled by active-site evolution of a cytochrome P450. ACS Cent. Sci. 1, 89–93 (2015).

    Article  CAS  Google Scholar 

  14. 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, 1209–1224 (2021).

    Article  CAS  Google Scholar 

  15. Zard, S. Z. Recent progress in the generation and use of nitrogen-centered radicals. Chem. Soc. Rev. 37, 1603–1618 (2008).

    Article  CAS  Google Scholar 

  16. Kwon, K., Simons, R. T., Nandakumar, M. & Roizen, J. L. Strategies to generate nitrogen-centered radicals that may rely on photoredox catalysis: development in reaction methodology and applications in organic synthesis. Chem. Rev. 122, 2353–2428 (2022).

    Article  CAS  Google Scholar 

  17. Studer, A. & Curran, D. P. Catalysis of radical reactions: a radical chemistry perspective. Angew. Chem. Int. Ed. 55, 58–102 (2016).

    Article  CAS  Google Scholar 

  18. Horner, J. H., Musa, O. M., Bouvier, A. & Newcomb, M. Absolute kinetics of amidyl radical reactions. J. Am. Chem. Soc. 120, 7738–7748 (1998).

    Article  CAS  Google Scholar 

  19. Rittle, J. & Green, M. T. Cytochrome P450 compound I: capture, characterization, and C–H bond activation kinetics. Science 330, 933–937 (2010).

    Article  CAS  Google Scholar 

  20. 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 

  21. Ye, Y., Fu, H. & Hyster, T. K. Activation modes in biocatalytic radical cyclization reactions. J. Ind. Microbiol. Biotechnol. 48, kuab021 (2021).

    Article  CAS  Google Scholar 

  22. Toogood, H. S. & Scrutton, N. S. Discovery, characterization, engineering, and applications of ene-reductases for industrial biocatalysis. ACS Catal. 8, 3532–3549 (2018).

    Article  CAS  Google Scholar 

  23. Biegasiewicz, K. F. et al. Photoexcitation of flavoenzymes enables a stereoselective radical cyclization. Science 364, 1166–1169 (2019).

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

  25. Clayman, P. D. & Hyster, T. K. Photoenzymatic generation of unstabilized alkyl radicals: an asymmetric reductive cyclization. J. Am. Chem. Soc. 142, 15673–15677 (2020).

    Article  CAS  Google Scholar 

  26. Fu, H. et al. Ground-state electron transfer as an initiation mechanism for biocatalytic C–C bond forming reactions. J. Am. Chem. Soc. 143, 9622–9629 (2021).

    Article  CAS  Google Scholar 

  27. Page, C. G. et al. Quaternary charge-transfer complex enables photoenzymatic intermolecular hydroalkylation of olefins. J. Am. Chem. Soc. 143, 97–102 (2020).

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  29. Grosheva, D. & Hyster, T. K. in Flavin-Based Catalysis: Principles and Applications (eds Cibulka, R. & Fraaije, M.) 291–313 (Wiley, 2021).

  30. Davies, J., Morcillo, S. P., Douglas, J. J. & Leonori, D. Hydroxylamine derivatives as nitrogen-radical precursors in visible-light photochemistry. Chem. Eur. J. 24, 12154–12163 (2018).

    Article  CAS  Google Scholar 

  31. Keum, H., Jung, H., Jeong, J., Kim, D. & Chang, S. Visible-light induced C(sp2)–H amidation with an aryl–alkyl σ-bond relocation via redox-neutral radical–polar crossover. Angew. Chem. Int. Ed. 60, 25235–25240 (2021).

    Article  CAS  Google Scholar 

  32. Warren, J. J., Ener, M. E., Vlček, A., Winkler, J. R. & Gray, H. B. Electron hopping through proteins. Coord. Chem. Rev. 256, 2478–2487 (2012).

    Article  CAS  Google Scholar 

  33. Sandoval, B. A. et al. Photoenzymatic reductions enabled by direct excitation of flavin-dependent ‘ene’-reductases. J. Am. Chem. Soc. 143, 1735–1739 (2021).

    Article  CAS  Google Scholar 

  34. Biegasiewicz, K. F., Cooper, S. J., Emmanuel, M. A., Miller, D. C. & Hyster, T. K. Catalytic promiscuity enabled by photoredox catalysis in nicotinamide-dependent oxidoreductases. Nat. Chem. 10, 770–775 (2018).

    Article  CAS  Google Scholar 

  35. Sandoval, B. A., Kurtoic, S. I., Chung, M. M., Biegasiewicz, K. F. & Hyster, T. K. Photoenzymatic catalysis enables radical-mediated ketone reduction in ene-reductases. Angew. Chem. Int. Ed. 58, 8714–8718 (2019).

    Article  CAS  Google Scholar 

  36. Nakano, Y. et al. Photoenzymatic hydrogenation of heteroaromatic olefins using ‘ene’-reductases with photoredox catalysts. Angew. Chem. Int. Ed. 59, 10484–10488 (2020).

    Article  CAS  Google Scholar 

  37. Prier, C. K., Rankic, D. A. & Macmillan, D. W. C. Visible light photoredox catalysis with transition metal complexes: applications in organic synthesis. Chem. Rev. 113, 5322–5363 (2013).

    Article  CAS  Google Scholar 

  38. Abramovitz, A. S. & Massey, V. Interaction of phenols with old yellow enzyme. Physical evidence for charge-transfer complexes. J. Biol. Chem. 251, 5327–5336 (1976).

    Article  CAS  Google Scholar 

  39. Marshall, S. J., Krause, D., Blencowe, D. K. & White, G. F. Characterization of glycerol trinitrate reductase (NerA) and the catalytic role of active-site residues. J. Bacteriol. 186, 1802–1810 (2004).

    Article  CAS  Google Scholar 

  40. Steward, R. C. & Massey, V. Potentiometric studies of native and flavin-substituted old yellow enzyme. J. Biol. Chem. 260, 13639–13647 (1985).

    Article  Google Scholar 

  41. Reetz, M. T. & Carballeira, J. D. Iterative saturation mutagenesis (ISM) for rapid directed evolution of functional enzymes. Nat. Protoc. 2, 891–903 (2007).

    Article  CAS  Google Scholar 

  42. Nicholls, B. et al. Engineering a non-natural photoenzyme for improved photon efficiency. Angew. Chem. Int. Ed. https://doi.org/10.1002/anie.202113842 (2022).

  43. Bougioukou, D. J., Kille, S., Taglieber, A. & Reetz, M. T. Directed evolution of an enantioselective enoate-reductase: testing the utility of iterative saturation mutagenesis. Adv. Synth. Catal. 351, 3287–3305 (2009).

    Article  CAS  Google Scholar 

  44. Sim, B. A., Griller, D. & Wayner, D. D. M. Reduction potentials for substituted benzyl radicals: pKa values for the corresponding toluenes. J. Am. Chem. Soc. 111, 754–755 (1989).

    Article  CAS  Google Scholar 

  45. Sorigué, D. et al. Mechanism and dynamics of fatty acid photodecarboxylase. Science https://doi.org/10.1126/science.abd5687 (2021).

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

    Article  CAS  Google Scholar 

  47. Kato, Y., Nagao, R. & Noguchi, T. Redox potential of the terminal quinone electron acceptor QB in photosystem II reveals the mechanism of electron transfer regulation. Proc. Natl Acad. Sci. USA 113, 620–625 (2016).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The research reported here was supported by the National Institutes of Health National Institute of General Medical Sciences (R01 GM127703). Transient absorption spectroscopy studies were supported by the Division of Chemical Sciences, Geosciences, and Biosciences, Office of Basic Energy Sciences of the US Department of Energy (DOE) through grant DE-SC0019370. We thank the Princeton Catalysis Initiative and Merck Sharp & Dohme LLC, a subsidiary of Merck & Co., Inc., Rahway, NJ, USA, for financial support. This work made use of the Cornell University NMR Facility, which is supported, in part, by the NSF though MRI Award CHE-1531632. D.G.O. acknowledges support from the Postgraduate Scholarships Doctoral Program of the Natural Sciences and Engineering Research Council of Canada. We thank K. Meihaus, P. Clayman, K. Biegasiewicz and Y. Liu for assistance in preparing this manuscript, and T. Qiao for assistance with density functional theory calculations.

Author information

Author notes

  1. These authors contributed equally: Yuxuan Ye, Jingzhe Cao.

Authors and Affiliations

  1. Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY, USA

    Yuxuan Ye, Jingzhe Cao & Todd K. Hyster

  2. Department of Chemistry, Princeton University, Princeton, NJ, USA

    Jingzhe Cao, Daniel G. Oblinsky, Gregory D. Scholes & Todd K. Hyster

  3. Computational and Structural Chemistry, Merck & Co., Inc., Rahway, NJ, USA

    Deeptak Verma

  4. Department of Process Research and Development, Merck & Co., Inc., Rahway, NJ, USA

    Christopher K. Prier

Authors
  1. Yuxuan Ye
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jingzhe Cao
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Daniel G. Oblinsky
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Deeptak Verma
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Christopher K. Prier
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Gregory D. Scholes
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Todd K. Hyster
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

T.K.H. conceived and directed the project. Y.Y. and J.C. performed and analysed the experiments. D.G.O. performed the transient absorption spectroscopy experiments. D.G.O. and G.D.S. analysed and interpreted the spectroscopy results. D.V. performed the MD simulations. D.V. and C.K.P. analysed and interpreted the MD simulations. All authors discussed the results and commented on the manuscript.

Corresponding author

Correspondence to Todd K. Hyster.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Chemistry thanks Fabio Parmeggiani 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 Parts I–XVI (Figs. 1–32, Tables 1–24, NMR spectra and HPLC traces).

Source data

Source Data Fig. 2

Statistical source data for Fig. 2c.

Source Data Fig. 3

Statistical source data for Fig. 3a,c.

Source Data Fig. 3

Statistical source data.

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

Ye, Y., Cao, J., Oblinsky, D.G. et al. Using enzymes to tame nitrogen-centred radicals for enantioselective hydroamination. Nat. Chem. 15, 206–212 (2023). https://doi.org/10.1038/s41557-022-01083-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41557-022-01083-z

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