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Photobiocatalytic benzylic C–H acylation enabled by the synergy of a thiamine-dependent enzyme, an organophotocatalyst and hydrogen-atom transfer

Nature Synthesis volume 4, pages 1453–1461 (2025)Cite this article

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Abstract

Direct functionalization of abundant C(sp3)–H bonds is highly attractive. Photobiocatalysis offers promise for expanding enzyme reactivity but has been limited to the use of preactivated radical precursors. Key challenges for C(sp3)–H bond activation include the lack of robust activation modes for the inert bonds under biocatalytic conditions and controlling the reactivity and stereochemistry of prochiral radicals. Here we report a triple activation strategy enabling photobiocatalytic C(sp3)–H bond acylation with aldehydes. By combining hydrogen-atom transfer for prochiral radical formation, organic-dye-modulated single-electron transfer and an engineered thiamine-dependent enzyme, we describe a radical acyl transferase for functionalizing C(sp3)–H bonds. This robust radical enzymatic system achieves benzylic C(sp3)–H and aldehyde C(sp2)–H oxidative coupling in an enantioselective manner (up to 97% e.e.).

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Fig. 1: Photobiocatalytic C(sp3)–H bond acylation with aldehydes.
Fig. 2: Catalysis design, development and evolution.
Fig. 3: Substrate promiscuity of the biocatalytic C(sp3)–H bond radical acylation.
Fig. 4: Mechanistic studies.

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Data availability

All data are available in the main text or Supplementary Information. Crystallographic data for the structures reported in this article have been deposited at the Cambridge Crystallographic Data Centre, under deposition numbers CCDC 2355842 (3g), 2358465 (3k) and 2416063 (3q). Copies of the data can be obtained free of charge via https://www.ccdc.cam.ac.uk/structures/.

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Acknowledgements

This work was supported by the Jiangsu Basic Research Center for Synthetic Biology (grant no. BK20233003). We appreciate financial support from the National Key Research and Development Program of China (2022YFA0913000 to X.H.), the National Natural Science Foundation of China (22277053 to X.H.; 22122305 to B.W.; 223B2703 to Y.X.), the Natural Science Foundation of Jiangsu Province (BK20220760 to X.H.), the Fundamental Research Funds for the Central Universities (0205/14380351 and 0205/14380346 to X.H.) and the Excellent Research Program of Nanjing University (ZYJH004 to X.H.).

Author information

Author notes

  1. These authors contributed equally: Xichao Peng, Jianqiang Feng, Fulu Liu, Wen-Zheng Xie.

Authors and Affiliations

  1. State Key Laboratory of Coordination Chemistry, Chemistry and Biomedicine Innovation Center (ChemBIC), School of Chemistry and Chemical Engineering, Frontier Interdisciplinary Science Research Center, Nanjing University, Nanjing, China

    Xichao Peng, Fulu Liu, Wen-Zheng Xie, Hailong Sun, Yuanyuan Xu, Yang Ming, Zhongqiu Xing, Yi-Tao Long & Xiaoqiang Huang

  2. State Key Laboratory of Physical Chemistry of Solid Surfaces, Fujian Provincial Key Laboratory of Theoretical and Computational Chemistry, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, China

    Jianqiang Feng & Binju Wang

  3. Institute of Molecular Engineering Plus, College of Chemistry, Fuzhou University, Fuzhou, China

    Jianqiang Feng

  4. Jiangsu Co-Innovation Center of Efficient Processing and Utilization of Forest Resources, International Innovation Center for Forest Chemicals and Materials, Nanjing Forestry University, Nanjing, China

    Yu Zheng

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Contributions

X.P. developed the catalysis. X.P. and F.L. performed most of the experiments. H.S., Y.M., Y.X., Z.X. and Y.Z. assisted in synthetic experiments. J.F. and B.W. performed theoretical calculations. W.-Z.X. and Y.-T.L. contributed to UV–vis spectroelectrochemical and EPR experiments. X.P. and X.H. wrote the paper with input from all authors. X.H. coordinated and conceived the project.

Corresponding authors

Correspondence to Binju Wang, Yi-Tao Long or Xiaoqiang Huang.

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Nature Synthesis thanks Robert Kourist and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Thomas West, in collaboration with the Nature Synthesis team.

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Extended data

Extended Data Fig. 1 Unsuccessful examples.

Conditions: 1 (0.004 mmol), 2 (0.016 mmol), HAT reagent (0.020 mmol), enzyme (PfBAL_T481L, PfBAL_T481L-A480G or PfBAL_T481L-A480G-Y397A-W163C, 2 mol%), Eosin Y (3 mol%), 20% v/v DMSO in 100 mM MOPS buffer (pH 8.0, containing 2.5 mM MgSO4 and 0.15 mM ThDP) were stirred for 14 h at room temperature under N2 atmosphere with the irradiation of 450–460 nm LEDs; total volume of the reaction is 0.8 ml. The reactions were analysed by GC-MS.

Extended Data Fig. 2 Active site view of the molecular-dynamics-simulated structure of the mutant.

A representative snapshot was selected in the final stage of 200 ns MD trajectory, which resembles the most populated structures from the clustering analysis of the MD trajectory (Supplementary Table 25). MD, Molecular Dynamics.

Supplementary information

Supplementary Information (download PDF )

Supplementary Figs. 1–12, Tables 1–25 and Methods.

Reporting Summary (download PDF )

Supplementary Data 1

X-ray crystallographic data for 3g, CCDC 2355842.

Supplementary Data 2

X-ray crystallographic data for 3k, CCDC 2358465.

Supplementary Data 3

X-ray crystallographic data for 3q, CCDC 2416063.

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Peng, X., Feng, J., Liu, F. et al. Photobiocatalytic benzylic C–H acylation enabled by the synergy of a thiamine-dependent enzyme, an organophotocatalyst and hydrogen-atom transfer. Nat. Synth 4, 1453–1461 (2025). https://doi.org/10.1038/s44160-025-00866-9

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