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Repurposing haemoproteins for asymmetric metal-catalysed H atom transfer

Nature volume 644, pages 381–390 (2025)Cite this article

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Abstract

Transition metal–hydrides have been widely exploited in catalysis for the hydrofunctionalization of unsaturated moieties, including carbonyls, alkenes and alkynes1. To complement heterolytic metal–hydride bond cleavage, metal–hydride hydrogen atom transfer (MHAT) has recently gained attention, as a promising strategy for radical hydrofunctionalization of unactivated alkenes2, thus enabling late-stage diversification of complex molecules3,4. However, owing to the weak interactions between the prochiral organic radical and the enantiopure catalyst5, asymmetric MHAT6 remains challenging. Here we show that cytochrome P450 enzymes (CYPs) can be repurposed to catalyse asymmetric MHAT, a new-to-nature reaction. Directed evolution of P450BM3 yielded a triple mutant that catalyses MHAT radical cyclization of unactivated alkenes, producing diverse cyclic compounds—including pyrrolidines and piperidines—with up to 98:2 enantiomeric ratio under aerobic whole-cell conditions. Apart from electron-deficient alkenes, alternative radical acceptors—including hydrazones, oximes and nitriles—were converted by repurposed P450BM3 to enantioenriched cyclization products. Mechanistic investigations support an MHAT mechanism proceeding by homolytic cleavage of a fleeting iron(III)–hydride species2,6. Starting from CYP119, directed evolution afforded a stereocomplementary MHATase, highlighting the potential of repurposed CYPs for MHAT biocatalysis. Our study highlights the prospect of integrating homolytic metal–hydride reactivity into metalloenzymes, thus expanding the scope of asymmetric radical biocatalysis.

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Fig. 1: Catalytic reactions that involve metal–hydrides.
Fig. 2: Discovery and directed evolution of the MHATase catalysing the conversion of diene 1 to enantioenriched pyrrolidine 2.
Fig. 3: Substrate scope for the whole-cell asymmetric radical cyclization by a Giese reaction catalysed by evolved P450BM3.
Fig. 4: Substrate scope for the MHATase-catalysed asymmetric radical cyclization using alternative radical acceptors.
Fig. 5: Mechanistic studies on the radical cyclization catalysed by P450BM3_LQQ with PhSiH3.
Fig. 6: Scalability and access to the opposite pyrrolidine enantiomers with MHATase.

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Data availability

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

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Acknowledgements

T.R.W. thanks the NCCR Catalysis (grant no. 180544), a National Centre of Competence in Research funded by the Swiss National Science Foundation. Additional funding was provided by the NCCR Molecular Systems Engineering (grant no. 200021_178760). We acknowledge T. Weymuth and M. Reiher for help with the computational studies. We acknowledge J. Stropp and D. Klose for their assistance with preliminary electron paramagnetic resonance studies. We acknowledge T. Kardashliev and Z. Zou for providing original plasmids of various haemoproteins. We acknowledge J. Zurflüh for the help with the GC-MS analysis.

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Author notes

  1. These authors contributed equally: Xiang Zhang, Dongping Chen

Authors and Affiliations

  1. Department of Chemistry, University of Basel, Basel, Switzerland

    Xiang Zhang, Dongping Chen, María Álvarez & Thomas R. Ward

  2. National Center of Competence in Research ‘Molecular Systems Engineering’, Basel, Switzerland

    Xiang Zhang, Dongping Chen & Thomas R. Ward

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  1. Xiang Zhang
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Contributions

T.R.W., X.Z. and D.C. conceived and designed the study. X.Z. and D.C. contributed equally to the synthesis of the substrates and products in Fig. 3, directed evolution of both P450BM3 and CYP119, and the mechanistic study in Fig. 5 and Supplementary Information. X.Z. and D.C. performed the catalytic experiment; X.Z. recorded the data. X.Z. and M.Á. contributed to the synthesis of substrates and products in Fig. 4 as well as the double-blind experiments. T.R.W., X.Z. and D.C. analysed the data. T.R.W., X.Z. and D.C. wrote the paper. All authors gave approval for the final version of the paper.

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Correspondence to Thomas R. Ward.

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Extended data figures and tables

Extended Data Fig. 1

Schematic representations of active sites in natural hydrogenases (a) and selected examples illustrating the application of MHAT in total synthesis (b).

Extended Data Fig. 2 Mechanistic studies.

a, UV-Vis spectra of purified P450BM3_LQQ (black), purified P450BM3_LQQ with Na2S2O4 before (red) and after purging with CO (blue), and purified P450BM3_LQQ with PhSiH3 before (green) and after purging with CO (purple). (Inset) Expanded view of the region between 450 and 700 nm. All spectra were collected under anaerobic conditions. b, Investigation of the involvement of P450-FeII in the radical cyclization of diene 1. (Upper) Standard reaction conditions with addition of excess Na2S2O4 did not yield any pyrrolidine 2, in contrast to standard conditions without the additive. (Lower) Additional experiments were carried out in a glove box without any additive or with air or K3Fe(CN)6 as the additive. The clock icon was created with BioRender.com.

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Zhang, X., Chen, D., Álvarez, M. et al. Repurposing haemoproteins for asymmetric metal-catalysed H atom transfer. Nature 644, 381–390 (2025). https://doi.org/10.1038/s41586-025-09308-0

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