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Chemoenzymatic synthesis of C14-functionalized steroids

Nature Synthesis volume 2, pages 729–739 (2023)Cite this article

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Abstract

C14-functionalized steroids display biological activity and have been used as medicines. The lack of synthetic methods to access C14-functionalized steroids has, however, impeded steroidal drug discovery. In the present article, we report a modular chemoenzymatic synthesis of a diverse range of C14-functionalized steroids. This method was enabled through identification of a C14α-hydroxylase (CYP14A) from Cochliobolus lunatus, which displays high catalytic efficiency and substrate promiscuity. Protein engineering of CYP14A generated two variants, I111L–M115K and I111L–V124W, with greatly improved C14-hydroxy regiocontrol. Using this biocatalytic method, a range of C14α-hydroxy steroids with a C17 side chain were prepared in good yields, and were transformed into ∆14 olefins through a superficial elimination. The ∆14 olefin served as a versatile handle to install a variety of functional groups at the C14 position through hydro- or difunctionalization. The utility of this method was further demonstrated through application to concise semisynthesis of cardenolide periplogenin, (+)-digitoxigenin and its three diastereomers.

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Fig. 1: Approaches to access C14-functionalized steroids.
Fig. 2: UPLC analysis of the C14α-OH steroids produced by biocatalysis.
Fig. 3: In silico model of CYP14A with substrate 1b.
Fig. 4: C14α-hydroxy dehydration and ∆14 olefin functionalization.
Fig. 5: Facile synthesis of periplogenin.
Fig. 6: Diversity-oriented synthesis of (+)-digitoxigenin and its three diastereomers.

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Data availability

Crystallographic data for the structure reported in this article have been deposited at the Cambridge Crystallographic Data Centre, under deposition nos. CCDC 2116723 (2b), 2116721 (4cc), 2116722 (4af), 2211826 (4cg), 2116724 (4ch), 2213082 (4ai) and 2213084 (4ci). Copies of the data can be obtained free of charge via https://www.ccdc.cam.ac.uk/structures. All other characterization data and detailed experimental procedures are available in the supplementary materials. The sequence of the P450 genes reported in the present article has been deposited in GenBank under accession nos. ON605617 (CYP14A from CGMCC 3.3589 and JTU2.406) and ON605618 (P450lun, from CGMCC 3.4381).

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Acknowledgements

We thank X.-G. Meng (CCNU) for X-ray crystallographic analysis assistance, L. Shao (Shanghai Institute of Pharmaceutical Industry) for kind gifting of C. lunatus JTU 2.406, H.-G. Cheng, T. Yang, J.-X. Ye, L. Zhou and H. Wei (WHU) for helpful discussions, and L. Cao (WHU) for assistance with the preparation of the manuscript. This work was supported by the National Key R&D Program of China (grant no. 2018YFA0900400 to X.Q.), the Fundamental Research Funds for the Central Universities (grant no. 2042021kf0214 to Q.Z.) and the start-up funding from Wuhan University. We dedicate this paper to Professor Dawei Ma (SIOC) on the occasion of his 60th birthday.

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Author notes

  1. These authors contributed equally: Fuzhen Song, Mengmeng Zheng.

Authors and Affiliations

  1. Institute for Advanced Studies, College of Chemistry and Molecular Sciences, TaiKang Center for Life and Medical Sciences, Wuhan University, Wuhan, China

    Fuzhen Song, Junlin Wang, Hengjiang Cong & Qianghui Zhou

  2. Key Laboratory of Combinatorial Biosynthesis and Drug Discovery Ministry of Education, School of Pharmaceutical Sciences, Wuhan University, Wuhan, China

    Mengmeng Zheng, Huanhuan Liu & Xudong Qu

  3. State Key Laboratory of Microbial Metabolism and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China

    Mengmeng Zheng, Zhi Lin, Benben Liu, Zixin Deng & Xudong Qu

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Contributions

Q.Z. and X.Q. conceived this project. F.S., M.Z., J.W., H.L., Z.L. and B.L. performed the experiment under the supervision of X.Q., Q.Z. and Z.D. H.C. performed the test and analysis of single crystal structures. F.S., M.Z., Q.Z. and X.Q. cowrote the manuscript. All authors discussed the results and commented on the manuscript.

Corresponding authors

Correspondence to Qianghui Zhou or Xudong Qu.

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Peer review information

Nature Synthesis thanks Thomas Bayer, Sebastian Cosgrove, Yang Yang 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

Supplementary Tables 1–6 and Figs. 1–7.

Reporting Summary

Supplementary Data 1

Crystallographic Data for 2b, CCDC2116723.

Supplementary Data 2

Crystallographic Data for 4af, CCDC 2116722.

Supplementary Data 3

Crystallographic Data for 4ai, CCDC 2213082.

Supplementary Data 4

Crystallographic Data for4cc, CCDC 2116721.

Supplementary Data 5

Crystallographic Data for 4cg, CCDC 2211826.

Supplementary Data 6

Crystallographic Data for 4ch, CCDC 2116724.

Supplementary Data 7

Crystallographic Data for 4ci CCDC 2213084.

Supplementary Table 8

Source data for Supplementary Figs. 4a and 5aa–5ma.

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Song, F., Zheng, M., Wang, J. et al. Chemoenzymatic synthesis of C14-functionalized steroids. Nat. Synth 2, 729–739 (2023). https://doi.org/10.1038/s44160-023-00280-z

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