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A metalloenzyme platform for catalytic asymmetric radical dearomatization

Nature Chemistry volume 16, pages 1999–2008 (2024)Cite this article

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Abstract

Catalytic asymmetric dearomatization represents a powerful means to convert flat aromatic compounds into stereochemically well-defined three-dimensional molecular scaffolds. Using new-to-nature metalloredox biocatalysis, we describe an enzymatic strategy for catalytic asymmetric dearomatization via a challenging radical mechanism that has eluded small-molecule catalysts. Enabled by directed evolution, new-to-nature radical dearomatases P450rad1–P450rad5 facilitated asymmetric dearomatization of a broad spectrum of aromatic substrates, including indoles, pyrroles and phenols, allowing both enantioconvergent and enantiodivergent radical dearomatization reactions to be accomplished with excellent enzymatic control. Computational studies revealed the importance of additional hydrogen bonding interactions between the engineered metalloenzyme and the reactive intermediate in enhancing enzymatic activity and enantiocontrol. Furthermore, designer non-ionic surfactants were found to significantly accelerate this biotransformation, providing an alternative means to promote otherwise sluggish new-to-nature biotransformations. Together, this evolvable metalloenzyme platform opens up new avenues to advance challenging catalytic asymmetric dearomatization processes involving free radical intermediates.

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Fig. 1: Catalytic asymmetric radical dearomatization using metalloredox biocatalysis.
Fig. 2: Discovery, directed evolution and substrate scope of P450rad1, P450rad2 and P450rad3.
Fig. 3: Directed evolution and substrate scope of P450rad4.
Fig. 4: Computational and experimental studies to shed light on the high levels of enantioselectivity observed with radical dearomatase P450rad1.
Fig. 5: Preparative-scale biocatalytic radical dearomatization and derivatization of dearomatized products.

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Data availability

All data are available in the main text and the Supplementary Information. Crystallographic data for compounds 2e, 4a, 6a and 8a reported in this Article have been deposited at the CCDC under deposition numbers 2245164 (2e), 2245162 (4a), 2245161 (6a) and 2245163 (8a). Copies of the data can be obtained free of charge via https://www.ccdc.cam.ac.uk/structures/. The raw data for the docking structures used in the Supplementary Information are also available from the authors upon reasonable request. Plasmids encoding evolved radical dearomatases 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. von Ragué Schleyer, P. Introduction: aromaticity. Chem. Rev. 101, 1115–1118 (2001).

    Google Scholar 

  2. Zheng, C. & You, S.-L. Catalytic asymmetric dearomatization (CADA) reaction-enabled total synthesis of indole-based natural products. Nat. Prod. Rep. 36, 1589–1605 (2019).

    CAS  PubMed  Google Scholar 

  3. Wertjes, W. C., Southgate, E. H. & Sarlah, D. Recent advances in chemical dearomatization of nonactivated arenes. Chem. Soc. Rev. 47, 7996–8017 (2018).

    CAS  PubMed  Google Scholar 

  4. Zhuo, C.-X., Zhang, W. & You, S.-L. Catalytic asymmetric dearomatization reactions. Angew. Chem. Int. Ed. 51, 12662–12686 (2012).

    Google Scholar 

  5. Zheng, C. & You, S.-L. Advances in catalytic asymmetric dearomatization. ACS Cent. Sci. 7, 432–444 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Roche, S. P. & Porco, J. A. Jr. Dearomatization strategies in the synthesis of complex natural products. Angew. Chem. Int. Ed. 50, 4068–4093 (2011).

  7. Wang, Y., Zhang, W.-Y., Yu, Z.-L., Zheng, C. & You, S.-L. SmI2-mediated enantioselective reductive dearomatization of non-activated arenes. Nat. Synth. 1, 401–406 (2022).

    Google Scholar 

  8. Zhang, W.-Y., Wang, H.-C., Wang, Y., Zheng, C. & You, S.-L. Enantioselective dearomatization of indoles via SmI2-mediated intermolecular reductive coupling with ketones. J. Am. Chem. Soc. 145, 10314–10321 (2023).

    CAS  PubMed  Google Scholar 

  9. 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 

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

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  12. Bloom, J. D. & Arnold, F. H. In the light of directed evolution: pathways of adaptive protein evolution. Proc. Natl Acad. Sci. USA 106, 9995–10000 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. 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 

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

    CAS  PubMed  Google Scholar 

  15. Gibson, D. T., Koch, J. R. & Kallio, R. E. Oxidative degradation of aromatic hydrocarbons by microorganisms. I. Enzymic formation of catechol from benzene. Biochemistry 7, 2653–2662 (1968).

    CAS  PubMed  Google Scholar 

  16. Boyd, D. R. & Bugg, T. D. H. Arene cis-dihydrodiol formation: from biology to application. Org. Biomol. Chem. 4, 181–192 (2006).

    CAS  PubMed  Google Scholar 

  17. Johnson, R. A. Microbial arene oxidations. In Organic Reactions (ed. Overman, L. E.) 117–264 (John Wiley & Sons, 2004).

  18. Baker Dockrey, S. A., Lukowski, A. L., Becker, M. R. & Narayan, A. R. H. Biocatalytic site- and enantioselective oxidative dearomatization of phenols. Nat. Chem. 10, 119–125 (2018).

    CAS  PubMed  Google Scholar 

  19. Jacoby, C. et al. Channeling C1 metabolism toward S-adenosylmethionine-dependent conversion of estrogens to androgens in estrogen-degrading bacteria. mBio 11, https://doi.org/10.1128/mbio.01259-20 (2020).

  20. 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 

  21. 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  PubMed Central  Google Scholar 

  22. 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 

  23. Fu, W. et al. Enzyme-controlled stereoselective radical cyclization to arenes enabled by metalloredox biocatalysis. Nat. Catal. 6, 628–636 (2023).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Emmanuel, M. A. et al. Photobiocatalytic strategies for organic synthesis. Chem. Rev. 123, 5459–5520 (2023).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. 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 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  27. 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 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  29. 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 

  30. Gao, X., Turek-Herman, J. R., Choi, Y. J., Cohen, R. D. & Hyster, T. K. Photoenzymatic synthesis of α-tertiary amines by engineered flavin-dependent “ene”-reductases. J. Am. Chem. Soc. 143, 19643–19647 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Nicholls, B. T. et al. Engineering a non-natural photoenzyme for improved photon efficiency. Angew. Chem. Int. Ed. 61, e202113842 (2022).

    CAS  Google Scholar 

  32. 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 

  33. Fu, H., Qiao, T., Carceller, J. M., MacMillan, S. N. & Hyster, T. K. Asymmetric C-alkylation of nitroalkanes via enzymatic photoredox catalysis. J. Am. Chem. Soc. 145, 787–793 (2023).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Page, C. G. et al. Regioselective radical alkylation of arenes using evolved photoenzymes. J. Am. Chem. Soc. 145, 11866–11874 (2023).

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

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

    CAS  Google Scholar 

  38. Cheng, L. et al. Stereoselective amino acid synthesis by synergistic photoredox-pyridoxal radical biocatalysis. Science 381, 444–451 (2023).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Quasdorf, K. W. & Overman, L. E. Catalytic enantioselective synthesis of quaternary carbon stereocentres. Nature 516, 181–191 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Süsse, L. & Stoltz, B. M. Enantioselective formation of quaternary centers by allylic alkylation with first-row transition-metal catalysts. Chem. Rev. 121, 4084–4099 (2021).

    PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

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

  43. 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 

  44. 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 

  45. Mukaiyama, T., Ogata, K., Sato, I. & Hayashi, Y. Asymmetric organocatalyzed Michael addition of nitromethane to a 2-oxoindoline-3-ylidene acetaldehyde and the three one-pot sequential synthesis of (−)-horsfiline and (−)-coerulescine. Chem. Eur. J. 20, 13583–13588 (2014).

    CAS  PubMed  Google Scholar 

  46. Díaz-Marrero, A. R. et al. Carijodienone from the octocoral Carijoa multiflora. A spiropregnane-based steroid. J. Nat. Prod. 74, 292–295 (2011).

    PubMed  Google Scholar 

  47. Bandini, M. & Eichholzer, A. Catalytic functionalization of indoles in a new dimension. Angew. Chem. Int. Ed. 48, 9608–9644 (2009).

    CAS  Google Scholar 

  48. Brandenberg, O. F. et al. Stereoselective enzymatic synthesis of heteroatom-substituted cyclopropanes. ACS Catal. 8, 2629–2634 (2018).

    CAS  Google Scholar 

  49. McIntosh, J. A. et al. Enantioselective intramolecular C—H amination catalyzed by engineered cytochrome P450 enzymes in vitro and in vivo. Angew. Chem. Int. Ed. 52, 9309–9312 (2013).

    CAS  Google Scholar 

  50. Højgaard, C., Sørensen, H. V., Pedersen, J. S., Winther, J. R. & Otzen, D. E. Can a charged surfactant unfold an uncharged protein? Biophys. J. 115, 2081–2086 (2018).

    PubMed  PubMed Central  Google Scholar 

  51. Cortes-Clerget, M. et al. MC-1. A “designer” surfactant engineered for peptide synthesis in water at room temperature. Green Chem. 21, 2610–2614 (2019).

    CAS  Google Scholar 

  52. Lipshutz, B. H. et al. TPGS-750-M: a second-generation amphiphile for metal-catalyzed cross-couplings in water at room temperature. J. Org. Chem. 76, 4379–4391 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. Klumphu, P. & Lipshutz, B. H. “Nok”: a phytosterol-based amphiphile enabling transition-metal-catalyzed couplings in water at room temperature. J. Org. Chem. 79, 888–900 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. Noey, E. L. et al. Origins of stereoselectivity in evolved ketoreductases. Proc. Natl Acad. Sci. USA 112, E7065–E7072 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  55. Xu, R.-Q., Yang, P., Tu, H.-F., Wang, S.-G. & You, S.-L. Palladium(0)-catalyzed intermolecular arylative dearomatization of β-naphthols. Angew. Chem. Int. Ed. 55, 15137–15141 (2016).

    CAS  Google Scholar 

  56. Hu, J. et al. Pd-catalyzed dearomative asymmetric allylic alkylation of naphthols with alkoxyallenes. J. Org. Chem. 85, 7896–7904 (2020).

    CAS  PubMed  Google Scholar 

  57. Mohamed, M. A., Yamada, K. & Tomioka, K. Accessing the amide functionality by the mild and low-cost oxidation of imine. Tetrahedron Lett. 50, 3436–3438 (2009).

    CAS  Google Scholar 

  58. Wang, G., Lu, R., He, C. & Liu, L. Kinetic resolution of indolines by asymmetric hydroxylamine formation. Nat. Commun. 12, 2512 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

This research is supported by the National Institutes of Health (NIH; R35GM147387 to Y.Y. and R35GM128779 to P.L.). We acknowledge the National Science Foundation (NSF) BioPolymers, Automated Cellular Infrastructure, Flow, and Integrated Chemistry Materials Innovation Platform (BioPACIFIC MIP; DMR-1933487) and the NSF Materials Research Science and Engineering Center (MRSEC) at the University of California, Santa Barbara (DMR-2308708) for access to instrumentation. MD simulations were performed at the Center for Research Computing of the University of Pittsburgh and the Advanced Cyberinfrastructure Coordination Ecosystem: Services & Support (ACCESS) programme supported by the NSF, grant numbers OAC-2117681 and OAC-2138259. Y.F. is an Andrew W. Mellon Predoctoral Fellow. We thank Y.-M. Wang (University of Pittsburgh) for critical reading of this manuscript and B. Lipshutz (University of California, Santa Barbara) for the generous donation of surfactants used in this study.

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Author notes

  1. These authors contributed equally: Yue Fu, Yunlong Zhao.

Authors and Affiliations

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

    Wenzhen Fu, Yunlong Zhao, Huanan Wang & Yang Yang

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

    Yue Fu & Peng Liu

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

    Yang Yang

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Contributions

Y.Y. conceived and directed the project. W.F., Y.Z. and H.W. prepared the substrates. W.F. and Y.Z. performed enzyme screening, enzyme engineering and substrate scope studies. Y.F. carried out the computational studies with P.L. providing guidance. Y.Y., P.L., W.F. and Y.F. wrote the manuscript with the input of all other authors.

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Correspondence to Peng Liu or Yang Yang.

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Competing interests

Y.Y., W.F. and Y.Z. are inventors on a patent application (International Patent Application Serial No. PCT/US2023/085941) submitted by the University of California, Santa Barbara that covers compositions, methods and applications of evolved radical dearomatases. The remaining authors declare no competing interests.

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Nature Chemistry thanks Zhongyue Yang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–18, Tables 1–25, Discussion, Methods, analytical data, spectra and references.

Reporting Summary

Supplementary Data 1

Crystallographic data for compound 2e; CCDC reference 2245164.

Supplementary Data 2

Crystallographic data for compound 4a; CCDC reference 2245162.

Supplementary Data 3

Crystallographic data for compound 6a; CCDC reference 2245161.

Supplementary Data 4

Crystallographic data for compound 8a; CCDC reference 2245163.

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Fu, W., Fu, Y., Zhao, Y. et al. A metalloenzyme platform for catalytic asymmetric radical dearomatization. Nat. Chem. 16, 1999–2008 (2024). https://doi.org/10.1038/s41557-024-01608-8

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