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Unmasking the reverse catalytic activity of ‘ene’-reductases for asymmetric carbonyl desaturation

Nature Chemistry volume 17, pages 74–82 (2025)Cite this article

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Abstract

Carbonyl desaturation is a fundamental reaction widely practised in organic synthesis. While numerous methods have been developed to expand the scope of this important transformation, most of them necessitate multi-step protocols or suffer from the use of high loadings of metal or strong oxidizing conditions. Moreover, approaches that can achieve precise stereochemical control of the desaturation process are extremely rare. Here we report a biocatalytic platform for desymmetrizing desaturation of cyclohexanones to generate diverse cyclohexenones bearing a remote quaternary stereogenic centre, by reengineering ‘ene’-reductases to efficiently mediate dehydrogenation, the reverse process of their native activity. This ‘ene’-reductase-based desaturation system operates under mild conditions with air as the terminal oxidant, tolerates oxidation-sensitive or metal-incompatible functional groups and, more importantly, exhibits unparalleled stereoselectivity compared with those achieved with small-molecule catalysts. Mechanistic investigations suggest that the reaction proceeded through α-deprotonation followed by a rate-determining β-hydride transfer.

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Fig. 1: Carbonyl desaturation reactions.
Fig. 2: Reaction discovery and directed evolution of CrS and GkOYE enzyme for desymmetrizing dehydrogenation.
Fig. 3: Scope of desymmetrizing dehydrogenation on 4-alkyl-4-aryl cyclohexanones.
Fig. 4: Scope of desymmetrizing dehydrogenation on other classes of cyclohexanones.
Fig. 5: Preparative scale synthesis and product derivatization.
Fig. 6: Mechanistic studies.

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Data availability

Full experimental details are available in Supplementary Information. Crystallographic data for compound 35 reported in this Article have been deposited at the Cambridge Crystallographic Data Centre under deposition number CCDC 2293360. Copies of the data can be obtained free of charge from the Cambridge Crystallographic Data Centre via https://www.ccdc.cam.ac.uk/structures/.

References

  1. Patai, S. & Rappoport, Z. The Chemistry of Enones (John Wiley & Sons, 1989).

  2. Stahl, S. S. & Diao, T. Comprehensive Organic Synthesis 2nd edn (Elsevier, 2014).

  3. Ito, Y., Hirao, T. & Saegusa, T. Synthesis of α,β-unsaturated carbonyl compounds by palladium (II)-catalyzed dehydrosilylation of silyl enol ethers. J. Org. Chem. 43, 1011–1013 (1978).

    CAS  Google Scholar 

  4. Back, T. G. Encyclopedia of Inorganic and Bioinorganic Chemistry (John Wiley & Sons, 2011).

  5. Nicolaou, K. C., Zhong, Y. L. & Baran, P. S. A new method saturated alcohols and carbonyl compounds. J. Am. Chem. Soc. 122, 7596–7597 (2000).

    CAS  Google Scholar 

  6. Diao, T. & Stahl, S. S. Synthesis of cyclic enones via direct palladium catalyzed aerobic dehydrogenation of ketones. J. Am. Chem. Soc. 133, 14566–14569 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Chen, Y., Romaire, J. P. & Newhouse, T. R. Synthesis of cyclic enones by allyl-palladium-catalyzed α, β-dehydrogenation of esters and nitriles. J. Am. Chem. Soc. 137, 5875–5878 (2015).

    CAS  PubMed  Google Scholar 

  8. Wang, Z. et al. Ligand-controlled divergent dehydrogenative reactions of carboxylic acids via C–H activation. Science 374, 1281–1285 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Jie, X., Shang, Y., Zhang, X. & Su, W. Cu-catalyzed sequential dehydrogenation-conjugate addition for β-functionalization of saturated ketones: scope and mechanism. J. Am. Chem. Soc. 138, 5623–5633 (2016).

    CAS  PubMed  Google Scholar 

  10. Chen, M., Rago, A. J. & Dong, G. Platinum-catalyzed desaturation of lactams, ketones, and lactones. Angew. Chem. Int. Ed. 57, 16205–16209 (2018).

    CAS  Google Scholar 

  11. Wang, Z., He, Z., Zhang, L. & Huang, Y. Iridium-catalyzed aerobic α,β-dehydrogenation of γ,δ-unsaturated amides and acids: activation of both α- and β-C–H bonds through an allyl-iridium intermediates. J. Am. Chem. Soc. 140, 735–740 (2018).

    CAS  PubMed  Google Scholar 

  12. Huang, D., Szewczyk, S. M., Zhang, P. & Newhouse, T. R. Allyl-nickel catalysis enables carbonyl dehydrogenation and oxidative cycloalkenylation of ketones. J. Am. Chem. Soc. 141, 5669–5674 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Gnaim, S. et al. Electrochemically driven desaturation of carbonyl compounds. Nat. Chem. 13, 367–372 (2021).

    CAS  PubMed  Google Scholar 

  14. Gnaim, S., Vantourout, J. C., Serpier, F., Echeverria, P.-G. & Baran, P. S. Carbonyl desaturation: where does catalysis stand. ACS Catal. 11, 883–892 (2021).

    CAS  Google Scholar 

  15. Huang, D. & Newhouse, T. R. Dehydrogenative Pd and Ni catalysis for total synthesis. Acc. Chem. Res. 54, 1118–1130 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Zhu, L., Zhang, L. & Luo, S. Catalytic desymmetrizing dehydrogenation of 4-substituted cyclohexanones through enamine oxidation. Angew. Chem. Int. Ed. 57, 2253–2258 (2018).

    CAS  Google Scholar 

  17. Bornscheuer, U. T. The fourth wave of biocatalysis is approaching. Phil. Trans. R. Soc. A 376, 20170063 (2018).

    PubMed  Google Scholar 

  18. Romero, E. O. et al. Enabling broader adoption of biocatalysis in organic chemistry. JACS Au 3, 2073–2085 (2023).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Arnold, F. H. Directed evolution: bringing new chemistry to life. Angew. Chem. Int. Ed. 57, 4143–4148 (2018).

    CAS  Google Scholar 

  20. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Ghisla, S. & Thorpe, C. Acyl-CoA dehydrogenases—a mechanistic overview. Eur. J. Biochem. 271, 494–508 (2004).

    CAS  PubMed  Google Scholar 

  24. Buist, P. H. Fatty acid desaturases: selecting the dehydrogenation channel. Nat. Prod. Rep. 21, 249–262 (2004).

    CAS  PubMed  Google Scholar 

  25. Cerone, M. & Smith, T. K. Desaturases: structural and mechanistic insights into the biosynthesis of unsaturated fatty acids. IUBMB Life 74, 1036–1051 (2022).

    CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Winkler, C. K., Faber, K. & Hall, M. Biocatalytic reduction of activated C=C-bonds and beyond: emerging trends. Curr. Opin. Chem. Biol. 43, 97–105 (2018).

    CAS  PubMed  Google Scholar 

  28. Durchschein, K., Hall, M. & Faber, K. Unusual reactions mediated by FMN-dependent ene- and nitro-reductases. Green Chem. 15, 1764–1772 (2013).

    CAS  Google Scholar 

  29. Hall, M. Enzymatic strategies for asymmetric synthesis. RSC Chem. Biol. 2, 958–989 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Roy, T. K., Sreedharan, R., Ghosh, P., Gandhi, T. & Maiti, D. Ene-reductase: a multifaceted biocatalyst in organic synthesis. Chem. Eur. J. 28, e202103949 (2022).

    Google Scholar 

  31. Murthy, Y. V. S. N., Meah, Y. & Massey, V. Conversion of a flavoprotein reductase to a desaturase by manipulation of the flavin redox potential. J. Am. Chem. Soc. 121, 5344–5345 (1999).

    CAS  Google Scholar 

  32. Jiang, G. et al. Ene reductase enabled intramolecular β-C–H functionalization of substituted cyclohexanones for efficient synthesis of bridged bicyclic nitrogen scaffolds. Angew. Chem. Int. Ed. 62, e202302125 (2023).

    CAS  Google Scholar 

  33. Schittmayer, M. et al. Old yellow enzyme-catalyzed dehydrogenation of saturated ketones. Adv. Synth. Catal. 353, 268–274 (2011).

    CAS  Google Scholar 

  34. Rogers, D. W., Zhao, Y., Traetteberg, M., Hulce, M. & Liebman, J. Enthalpies of hydrogenation and formation of enones. resonance energies of 2-cyclopentenone and 2-cyclohexenone. J. Chem. Thermodyn. 30, 1393–1400 (1998).

    CAS  Google Scholar 

  35. Vaz, A. D. N., Chakraborty, S. & Massey, V. Old yellow enzyme: aromatization of cyclic enones and the mechanism of a novel dismutation reaction. Biochemistry 34, 4246–4256 (1995).

    CAS  PubMed  Google Scholar 

  36. Stueckler, C., Reiter, T. C., Baudendistel, N. & Faber, K. Nicotinamide-independent asymmetric bioreduction of C=C-bonds via disproportionation of enones catalyzed by enoate reductases. Tetrahedron 66, 663–667 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Winker, C. K., Clay, D., Entner, M., Plank, M. & Faber, K. NAD(P)H-independent asymmetric C=C bond reduction catalyzed by ene reductases by using artificial co-substrates as the hydrogen donor. Chem. Eur. J. 20, 1403–1409 (2014).

    Google Scholar 

  38. Kelly, P. P. et al. Ene reductase enzymes for the aromatisation of tetralones and cyclohexenones to napththols and phenols. Adv. Synth. Catal. 358, 731–736 (2016).

    CAS  Google Scholar 

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

    CAS  Google Scholar 

  40. Christoffers, J. & Baro, A. Quaternary Stereocenters: Challenges and Solutions for Organic Synthesis (Wiley, 2005).

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

    CAS  PubMed  Google Scholar 

  42. Krstenansky, J. L. Mesembrin alkaloids: review of their occurrence, chemistry, and pharmacology. J. Ethnopharmacol. 195, 10–19 (2017).

    CAS  PubMed  Google Scholar 

  43. Fringuelli, F., Pizzo, F., Taticchi, A., Halls, T. D. J. & Wenkert, E. Diels–Alder reactions of cycloalkenones. 1. Preparation and structure of the adducts. J. Org. Chem. 47, 5056–5065 (1982).

    CAS  Google Scholar 

  44. Schanze, K., Mattox, F. & Whitten, D. G. Solvent effects upon the thermal cis–trans isomerization and charge-transfer absorption of 4-(diethylamino)-4′-nitroazobenzene. J. Org. Chem. 48, 2808–2813 (1983).

    CAS  Google Scholar 

  45. Lonsdale, R. & Reetz, M. T. Reduction of α,β-unsaturated ketones by old yellow enzymes: mechanistic insights from quantum mechanics/molecular mechanics calculations. J. Am. Chem. Soc. 137, 14733–14742 (2015).

    CAS  PubMed  Google Scholar 

  46. Lai, M.-T., Li, D., Oh, E. & Liu, H.-W. Inactivation of medium-chain acyl-CoA dehydrogenase by a metabolite of hypoglycin: characterization of the major turnover product and evidence suggesting an alternative flavin modification pathway. J. Am. Chem. Soc. 115, 1619–1628 (1993).

    CAS  Google Scholar 

  47. Li, D. et al. Spiropentylacetyl-CoA, a mechanism-based inactivator of acyl-CoA dehydrogenases. J. Am. Chem. Soc. 120, 2008–2017 (1998).

    CAS  Google Scholar 

  48. Kohli, R. M. & Massey, V. The oxidative half-reaction of old yellow enzyme. The role of tyrosine 196. J. Biol. Chem. 273, 32763–32770 (1998).

    CAS  PubMed  Google Scholar 

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Acknowledgements

This work was supported by the National Key R&D Program of China (no. 2022YFA1505600 to Y.Y.). We thank the Zhejiang Provincial Key Laboratory Construction Project and Research Center for Industries of the Future at Westlake University and the Zhejiang Provincial National Science Foundation of China (XHD24B0101 to Y.Y.) for partially supporting this work. We thank Westlake University Instrumentation and Service Center for Molecular Sciences for the facility support and technical assistance. We thank X. Lu, Z. Chen and D. Gu from Westlake University Instrumentation and Service Center for Molecular Sciences for the assistance with the stopped-flow experiment. We thank X. Cui and B. Zhang from CAP for Solar Fuels at Westlake University for the assistance in measuring the oxygen concentrations. We also thank X. Huang (Johns Hopkins University) for the suggestions on the manuscript and T. Hyster (Princeton University), Y. Yang (UCSB), H. Fu (CAMS and PUMS) and P. Hu (Westlake University) for helpful discussions.

Author information

Author notes

  1. These authors contributed equally: Hui Wang, Bin Gao.

Authors and Affiliations

  1. Key Laboratory of Precise Synthesis of Functional Molecules of Zhejiang Province, Department of Chemistry, School of Science and Research Center for Industries of the Future, Westlake University, Hangzhou, China

    Hui Wang, Bin Gao, Heli Cheng, Shixuan Cao, Xinyi Ma & Yuxuan Ye

  2. Key Laboratory of Precise Synthesis of Functional Molecules of Zhejiang Province, Instrumentation and Service Center for Molecular Sciences, Westlake University, Hangzhou, China

    Yinjuan Chen

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Contributions

Y.Y. conceived and directed the project. H.W. performed the protein engineering, substrate scope evaluation and mechanistic study. B.G., H.C., S.C. and X.M. synthesized the substrates and analysed spectroscopy data. Y.C. conducted the deuterium-labelling analysis. All authors discussed the results and commented on the manuscript.

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Correspondence to Yuxuan Ye.

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Nature Chemistry thanks Scott France, Maciej Szaleniec 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 protocols, discussions, figures, NMR spectra and HPLC traces.

Reporting Summary

Supplementary Data 1

Raw data on the Clark experiment to study the kinetics of substrate desaturation.

Supplementary Data 2

Raw data on the stopped-flow experiment to study the kinetics of substrate reduction.

Supplementary Data 3

Raw data on the stopped-flow experiment to study the kinetics of enzyme regeneration by oxygen.

Supplementary Data 4

Primers used in the directed evolution.

Supplementary Data 5

Crystallographic data for compound 35, CCDC reference 2293360.

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Wang, H., Gao, B., Cheng, H. et al. Unmasking the reverse catalytic activity of ‘ene’-reductases for asymmetric carbonyl desaturation. Nat. Chem. 17, 74–82 (2025). https://doi.org/10.1038/s41557-024-01671-1

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