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Engineering non-haem iron enzymes for enantioselective C(sp3)–F bond formation via radical fluorine transfer

Nature Synthesis (2024)Cite this article

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A Publisher Correction to this article was published on 26 April 2024

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Abstract

In recent years there has been a surge in the development of methods for the synthesis of organofluorine compounds. However, enzymatic methods for C–F bond formation have been limited to nucleophilic fluoride substitution. Here we report the incorporation of iron-catalysed radical fluorine transfer, a reaction mechanism that is not used in naturally occurring enzymes, into enzymatic catalysis for the development of biocatalytic enantioselective C(sp3)–F bond formation. Using this strategy, we repurposed (S)-2-hydroxypropylphosphonate epoxidase from Streptomyces viridochromogenes (SvHppE) to catalyse an N-fluoroamide-directed C(sp3)–H fluorination. Directed evolution has enabled SvHppE to be optimized, forming diverse chiral benzylic fluoride products with turnover numbers of up to 180 and with excellent enantiocontrol (up to 94% enantiomeric excess). Mechanistic investigations showed that the N–F bond activation is the rate-determining step, and the strong preference for fluorination in the presence of excess NaN3 can be attributed to the spatial proximity of the carbon-centred radical to the iron-bound fluoride.

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Fig. 1: Overview of enzymatic halogenation.
Fig. 2: Optimization of fluorination activity of SvHPPE via directed evolution.
Fig. 3: Reaction scope and determination of absolute configuration of products.
Fig. 4: Mechanistic studies.
Fig. 5: MD simulations.

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Data availability

All data needed to evaluate the conclusions in this study are present in the main paper or Supplementary Information. The crystal structure of 1F is available from the Cambridge Crystallographic Data Centre under reference number CCDC 2267386.

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Acknowledgements

We thank M. Greenberg for helpful discussion and comments on the manuscript. We thank T. Yuan from H. Xiao’s group at Rice University for help on obtaining optical rotation data. We thank M. A. Siegler and JHU X-ray Crystallography Facility for analytical support. Financial support was provided by the Johns Hopkins University and National Institute for General Medical Sciences R00GM129419 and R35GM147639 (to X.H.). This work was also supported by the Spanish MICINN (Ministerio de Ciencia e Innovación) PID2019-111300GA-I00, PID2022-141676NB-I00 and TED2021-130173B-C42 projects (to M.G.-B.), and the Ramón y Cajal programme via the RYC 2020-028628-I fellowship (to M.G.-B.), the Generalitat de Catalunya (2021SGR00623 project to M.G.-B.), the Spanish MIU (Ministerio de Universidades) predoctoral fellowship FPU18/02380 (to J.S.), the National Natural Science Foundation of China (21978272) (to Y.Y.) and the Fundamental Research Funds for the Provincial Universities of Zhejiang (RF-C2022006) (to Y.Y.).

Author information

Author notes

  1. Qun Zhao

    Present address: School of Biotechnology, Jiangnan University, Wuxi, P. R. China

  2. These authors contributed equally: Qun Zhao, Zhenhong Chen, Jordi Soler.

Authors and Affiliations

  1. Department of Chemistry, Johns Hopkins University, Baltimore, MD, USA

    Qun Zhao, Zhenhong Chen, Jinyan Rui, Nathan Tianlin Ji, Qinglan E. Yu & Xiongyi Huang

  2. Institut de Química Computacional i Catàlisi and Departament de Química, Universitat de Girona, Catalonia, Spain

    Jordi Soler & Marc Garcia-Borràs

  3. State Key Laboratory Breeding Base of Green Chemistry-Synthesis Technology, Key Laboratory of Green Chemistry-Synthesis Technology of Zhejiang Province, College of Chemical Engineering, Zhejiang University of Technology, Hangzhou, China

    Xiahe Chen & Yunfang Yang

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Contributions

X.H. conceived and directed the project. X.H., Z.C. and Q.Z. designed the experiments. Q.Z. and Z.C. performed screening of initial enzyme activity. Q.Z. and Z.C. performed directed evolution and results analysis. Q.Z., Z.C., J.R., Q.E.Y. and N.T.J. performed substrate scope study. M.G.-B. conceived and directed the computational modelling studies. J.S. and X.C. performed DFT calculations under the guidance of M.G.-B. and Y.Y. J.S. performed MD simulations under the guidance of M.G.-B. X.H. and Q.Z. wrote the manuscript with input from all other authors; Y.Y. and M.G.-B. wrote the computational sections.

Corresponding authors

Correspondence to Yunfang Yang, Marc Garcia-Borràs or Xiongyi Huang.

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Competing interests

A provisional patent application (no. PCT/US2023/022431) covering enantioselective biocatalytic C–F bond formation has been filed through the Johns Hopkins University with Q.Z., X.H., Z.C. and J.R. as inventors. Authors J.S., N.T.J., Q.E.Y., X.C., Y.Y. and M.G.-B. declare no competing interests.

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Nature Synthesis thanks Kyle Biegasiewicz, Anna Fryszkowska, Hans Senn 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 1–9, Figs. 1–16, discussion, and further experimental and computational details.

Reporting Summary

Supplementary Data 1

Crystallographic data for compound 1F, CCDC 2267386.

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Zhao, Q., Chen, Z., Soler, J. et al. Engineering non-haem iron enzymes for enantioselective C(sp3)–F bond formation via radical fluorine transfer. Nat. Synth (2024). https://doi.org/10.1038/s44160-024-00507-7

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