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Biocatalytic enantioselective C(sp3)–H fluorination enabled by directed evolution of non-haem iron enzymes

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Abstract

Due to the scarcity of C–F bond-forming enzymatic reactions in nature and the contrasting prevalence of organofluorine moieties in bioactive compounds, developing biocatalytic fluorination reactions represents a pre-eminent challenge in enzymology, biocatalysis and synthetic biology. Additionally, catalytic enantioselective C(sp3)–H fluorination remains a challenging problem facing synthetic chemists. Although many non-haem iron halogenases have been discovered to promote C(sp3)–H halogenation reactions, efforts to convert these iron halogenases to fluorinases have remained unsuccessful. Here we report the development of an enantioselective C(sp3)–H fluorination reaction, catalysed by a plant-derived non-haem enzyme 1-aminocyclopropane-1-carboxylic acid oxidase (ACCO), which is repurposed for radical rebound fluorination. Directed evolution afforded a C(sp3)–H fluorinating enzyme ACCOCHF displaying 200-fold higher activity, substantially improved chemoselectivity and excellent enantioselectivity, converting a range of substrates into enantioenriched organofluorine products. Notably, almost all the beneficial mutations were found to be distal to the iron centre, underscoring the importance of substrate tunnel engineering in non-haem iron biocatalysis. Computational studies reveal that the radical rebound step with the Fe(III)–F intermediate has a low activation barrier of 3.4 kcal mol−1 and is kinetically facile.

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Fig. 1: Enzymatic fluorination: an overview.
Fig. 2: Development of chemo- and enantioselective non-haem iron enzyme ACCOCHF for new-to-nature C(sp3)–H fluorination: enzyme mining and engineering.
Fig. 3: Substrate scope of ACCOCHF-catalysed asymmetric C(sp3)–H fluorination.
Fig. 4: The most populated structures of the enzyme–substrate complexes from MD simulations.
Fig. 5: Computational studies using a truncated active site model.

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Data availability

All data are available in the main text and Supplementary Information. Plasmids encoding evolved ACCO variants reported in this study are available for research purposes from Y.Y. under a material transfer agreement with the University of California, Santa Barbara. The solid-state structure of 2a is available free of charge from the Cambridge Crystallographic Data Centre under reference number CCDC 2294341. All the protein structures used are available from the Protein Data Bank using their accession numbers.

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Acknowledgements

This research is supported by the NIH (R35GM147387 to Y.Y.), NSF (CHE-2247505 to P.L.) and Boehringer Ingelheim’s IU More Green Grant (BI number 763955 to Y.Y.). We acknowledge the NSF BioPACIFIC MIP (DMR-1933487) and NSF MRSEC at the University of California, Santa Barbara (DMR-2308708) for access to instrumentation. Computational studies were carried out at the University of Pittsburgh Center for Research Computing and the Advanced Cyberinfrastructure Coordination Ecosystem: Services & Support (ACCESS) program, supported by NSF award numbers OAC-2117681, OAC-1928147 and OAC-1928224. We thank Y. Wang (University of Pittsburgh) for critical reading of this paper.

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Authors

Contributions

Y.Y. conceived and directed the project. L.-P.Z. performed all the enzyme engineering, Michaelis–Menten kinetics, substrate synthesis and substrate scope studies. Y.Y., L.C. and Y. Zhao performed enzyme mining. L.-P.Z. and L.C. performed initial enzyme evaluation. F.G. and R.G. provided some substrates. B.K.M. carried out the computational studies with P.L. providing guidance. H.W. and Y. Zhang participated in discussions and provided suggestions. Y.Y., L.-P.Z., P.L. and B.K.M. wrote the paper with the input of all other authors.

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Correspondence to Peng Liu or Yang Yang.

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Nature Synthesis thanks Kyle Biegasiewicz, 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 Figs. 1–20, Tables 1–16, experimental details, X-ray crystallographic analysis details, computational details, NMR spectra and HPLC analysis.

Reporting Summary

Supplementary Data 1

Crystallographic data for compound 2a, CCDC 2294341.

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Zhao, LP., Mai, B.K., Cheng, L. et al. Biocatalytic enantioselective C(sp3)–H fluorination enabled by directed evolution of non-haem iron enzymes. Nat. Synth (2024). https://doi.org/10.1038/s44160-024-00536-2

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