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Biocatalytic enantioselective γ-C–H lactonization of aliphatic carboxylic acids

Nature Synthesis volume 3, pages 123–130 (2024)Cite this article

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Abstract

Enantioselective γ-lactonization of unactivated C(sp3)–H bonds of aliphatic carboxylic acids is a synthetic challenge. Without using directing groups, chemical methods largely capitalize on the innate bias of dissimilar C–H bonds and are therefore challenging for flexible linear substrates with identical methylenes. Here we report the use of a fatty acid hydroxylase, P450BSβ, repurposed through directed evolution, for the catalytic oxidative γ-lactonization of aliphatic carboxylic acids in an enantioselective fashion. The biocatalytic reaction uses inexpensive and eco-friendly oxidant, H2O2, at room temperature and forms γ-lactones in 2 h with excellent atom and step economy. This scalable process is tolerant of a range of functional groups, including (hetero)aryl substituents and aliphatic substrates with differing chain lengths. The introduced mutations result in a substrate conformation that prefers γ-regioselectivity with pro-(S)-H abstraction, which is supported by X-ray crystallographic analysis of the evolved hydroxylase variant in complex with palmitoleic acid and further computational studies.

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Fig. 1: Biosynthesis of γ-lactones and their synthetic strategies via direct C–H lactonization.
Fig. 2: Directed evolution of P450BSβ for γ-lactonization.
Fig. 3: Product applications.
Fig. 4: Structural basis of the regio- and enantioselectivity of the evolved enzyme.

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Data availability

All data supporting the findings of this study are available within the paper and its Supplementary Information. The crystal structure of P450BSβ-L78G/Q85F/F173S/G290I in complex with palmitoleic acid reported in this study has been deposited in the PDB under accession code 8HKD. Previously published crystal structures of wild-type P450BSβ (ref. 28) and P450BSβ-L78I/Q85H/G290I (ref. 27) can be accessed from the PDB with accession codes 1IZO and 7WYG, respectively. Crystallographic data for the structures reported in this article have been deposited at the Cambridge Crystallographic Data Centre, under deposition numbers CCDC 2272143 (22), 2272144 (40) and 2255877 (42). Copies of the data can be obtained free of charge via https://www.ccdc.cam.ac.uk/structures.

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Acknowledgements

We thank the Core Facility Center for Life Sciences, USTC, for assistance during X-ray data collection, and F. Shen (Fudan University) for technical assistance. This work is financially supported by the National Natural Science Foundation of China (grant no. 32171410 to X.W.) and the Natural Science Foundation of Anhui Province (grant no. 2008085 to C.H.).

Author information

Author notes

  1. Wen-Ju Bai

    Present address: Abbvie Inc., North Chicago, IL, USA

  2. These authors contributed equally: Qian Ma, Wenhao Shan, Xuan Chu, Haochen Xu.

Authors and Affiliations

  1. College of Bioscience and Biotechnology, Yangzhou University, Yangzhou, China

    Qian Ma, Wenhao Shan, Ziyi Chen & Xiqing Wang

  2. School of Life Science, Anhui University, Hefei, China

    Xuan Chu, Chao He & Xiqing Wang

  3. Department of Chemical Physics, University of Science and Technology of China, Hefei, China

    Haochen Xu & Jie-Lou Liao

  4. School of Life Sciences, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, China

    Fudong Li

  5. Department of Chemistry, Stanford University, Stanford, CA, USA

    Wen-Ju Bai

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Contributions

X.W. and W.-J.B. conceived and designed the project. Q.M. and W.S. performed directed evolution and enzymatic experiments and collected the data. X.C., F.L. and C.H. obtained protein crystals and solved the crystal structure. Z.C. obtained the small-molecule crystals and solved the crystal structure. H.X. and J.-L.L. performed computational experiments. Q.M., W.S., X.C., H.X., Z.C., F.L., J.-L.L., C.H., W.-J.B. and X.W. analysed data. X.W. and W.-J.B. wrote the manuscript with contributions from all authors.

Corresponding authors

Correspondence to Jie-Lou Liao, Chao He, Wen-Ju Bai or Xiqing Wang.

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Peer review information

Nature Synthesis thanks Elaine O’Reilly 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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Supplementary information

Supplementary Information

Experimental details, Supplementary Figs. 1–17 and Tables 1–8.

Reporting Summary

Supplementary Data 1

Crystallographic data for compound 22, CCDC 2272143.

Supplementary Data 2

Crystallographic data for compound 40, CCDC 2272144.

Supplementary Data 3

Crystallographic data for compound 42, CCDC 2255877.

Supplementary Data 4

Crystallographic data for P450BSβ-L78G/Q85F/F173S/G290I, PDB accession code 8HKD.

Supplementary Data 5

Primers used in this study.

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Ma, Q., Shan, W., Chu, X. et al. Biocatalytic enantioselective γ-C–H lactonization of aliphatic carboxylic acids. Nat. Synth 3, 123–130 (2024). https://doi.org/10.1038/s44160-023-00427-y

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