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

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

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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.

Change history


  1. Purser, S., Moore, P. R., Swallow, S. & Gouverneur, V. Fluorine in medicinal chemistry. Chem. Soc. Rev. 37, 320–330 (2008).

    Article  CAS  PubMed  Google Scholar 

  2. O’Hagan, D. Understanding organofluorine chemistry. An introduction to the C–F bond. Chem. Soc. Rev. 37, 308–319 (2008).

    Article  PubMed  Google Scholar 

  3. Inoue, M., Sumii, Y. & Shibata, N. Contribution of organofluorine compounds to pharmaceuticals. ACS Omega 5, 10633–10640 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Benedetto Tiz, D. et al. New halogen-containing drugs approved by FDA in 2021: an overview on their syntheses and pharmaceutical use. Molecules 27, 1643 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Britton, R. et al. Contemporary synthetic strategies in organofluorine chemistry. Nat. Rev. Methods Primers 1, 47 (2021).

    Article  CAS  Google Scholar 

  6. Furuya, T., Kamlet, A. S. & Ritter, T. Catalysis for fluorination and trifluoromethylation. Nature 473, 470–477 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Yang, X. Y., Wu, T., Phipps, R. J. & Toste, F. D. Advances in catalytic enantioselective fluorination, mono-, di-, and trifluoromethylation, and trifluoromethylthiolation reactions. Chem. Rev. 115, 826–870 (2015).

    Article  CAS  PubMed  Google Scholar 

  8. Caron, S. Where does the fluorine come from? A review on the challenges associated with the synthesis of organofluorine compounds. Org. Process Res. Dev. 24, 470–480 (2020).

    Article  CAS  Google Scholar 

  9. Walker, M. C. & Chang, M. C. Y. Natural and engineered biosynthesis of fluorinated natural products. Chem. Soc. Rev. 43, 6527–6536 (2014).

    Article  CAS  PubMed  Google Scholar 

  10. O’Hagan, D. & Deng, H. Enzymatic fluorination and biotechnological developments of the fluorinase. Chem. Rev. 115, 634–649 (2015).

    Article  PubMed  Google Scholar 

  11. Vaillancourt, F. H., Yeh, E., Vosburg, D. A., Garneau-Tsodikova, S. & Walsh, C. T. Nature’s inventory of halogenation catalysts: oxidative strategies predominate. Chem. Rev. 106, 3364–3378 (2006).

    Article  CAS  PubMed  Google Scholar 

  12. Agarwal, V. et al. Enzymatic halogenation and dehalogenation reactions: pervasive and mechanistically diverse. Chem. Rev. 117, 5619–5674 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Papadopoulou, A., Meyer, F. & Buller, R. M. Engineering Fe(II)/α-ketoglutarate-dependent halogenases and desaturases. Biochemistry 62, 229–240 (2023).

    Article  CAS  PubMed  Google Scholar 

  14. Dong, C. et al. Crystal structure and mechanism of a bacterial fluorinating enzyme. Nature 427, 561–565 (2004).

    Article  CAS  PubMed  Google Scholar 

  15. O’Hagan, D., Schaffrath, C., Cobb, S. L., Hamilton, J. T. G. & Murphy, C. D. Biosynthesis of an organofluorine molecule—a fluorinase enzyme has been discovered that catalyses carbon–fluorine bond formation. Nature 416, 279 (2002).

    PubMed  Google Scholar 

  16. Zechel, D. L. et al. Enzymatic synthesis of carbon–fluorine bonds. J. Am. Chem. Soc. 123, 4350–4351 (2001).

    Article  CAS  PubMed  Google Scholar 

  17. Cros, A., Alfaro-Espinoza, G., De Maria, A., Wirth, N. T. & Nikel, P. I. Synthetic metabolism for biohalogenation. Curr. Opin. Biotechnol. 74, 180–193 (2022).

    Article  CAS  PubMed  Google Scholar 

  18. Cheng, X. & Ma, L. Enzymatic synthesis of fluorinated compounds. Appl. Microbiol. Biotechnol. 105, 8033–8058 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Eustáquio, A. S., O’Hagan, D. & Moore, B. S. Engineering fluorometabolite production: fluorinase expression in salinispora tropica yields fluorosalinosporamide. J. Nat. Prod. 73, 378–382 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  20. Liu, W. & Groves, J. T. Manganese catalyzed C–H halogenation. Acc. Chem. Res. 48, 1727–1735 (2015).

    Article  CAS  PubMed  Google Scholar 

  21. Panda, C., Anny-Nzekwue, O., Doyle, L. M., Gericke, R. & McDonald, A. R. Evidence for a high-valent iron-fluoride that mediates oxidative C(sp3)–H fluorination. JACS Au 3, 919–928 (2023).

  22. Farley, G. W., Siegler, M. A. & Goldberg, D. P. Halogen transfer to carbon radicals by high-valent iron chloride and iron fluoride corroles. Inorg. Chem. 60, 17288–17302 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Bower, J. K., Cypcar, A. D., Henriquez, B., Stieber, S. C. E. & Zhang, S. C(sp3)–H fluorination with a copper(II)/(III) redox couple. J. Am. Chem. Soc. 142, 8514–8521 (2020).

  24. Huang, X., Liu, W., Hooker, J. M. & Groves, J. T. Targeted fluorination with the fluoride ion by manganese-catalyzed decarboxylation. Angew. Chem. Int. Ed. 54, 5241–5245 (2015).

    Article  CAS  Google Scholar 

  25. Huang, X. et al. Late stage benzylic C–H fluorination with [18F]fluoride for PET imaging. J. Am. Chem. Soc. 136, 6842–6845 (2014).

    Article  CAS  PubMed  Google Scholar 

  26. Liu, W. & Groves, J. T. Manganese-catalyzed oxidative benzylic C–H fluorination by fluoride ions. Angew. Chem. Int. Ed. 52, 6024–6027 (2013).

    Article  CAS  Google Scholar 

  27. Liu, W. et al. Oxidative aliphatic C–H fluorination with fluoride ion catalyzed by a manganese porphyrin. Science 337, 1322–1325 (2012).

    Article  CAS  PubMed  Google Scholar 

  28. Pinter, E. N., Bingham, J. E., AbuSalim, D. I. & Cook, S. P. N-directed fluorination of unactivated Csp3–H bonds. Chem. Sci. 11, 1102–1106 (2020).

    Article  CAS  Google Scholar 

  29. Groendyke, B. J., AbuSalim, D. I. & Cook, S. P. Iron-catalyzed, fluoroamide-directed C–H fluorination. J. Am. Chem. Soc. 138, 12771–12774 (2016).

    Article  CAS  PubMed  Google Scholar 

  30. Hintz, H. et al. Copper-catalyzed electrochemical C–H fluorination. Chem Catal. 3, 100491 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Rui, J. et al. Directed evolution of nonheme iron enzymes to access abiological radical-relay C(sp3)–H azidation. Science 376, 869–874 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Liu, P. et al. Protein purification and function assignment of the epoxidase catalyzing the formation of fosfomycin. J. Am. Chem. Soc. 123, 4619–4620 (2001).

    Article  CAS  PubMed  Google Scholar 

  33. Wang, C. et al. Evidence that the fosfomycin-producing epoxidase, HppE, is a non-heme-iron peroxidase. Science 342, 991–995 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Mitchell, A. J. et al. Structure-guided reprogramming of a hydroxylase to halogenate its small molecule substrate. Biochemistry 56, 441–444 (2017).

    Article  CAS  PubMed  Google Scholar 

  35. Gomez, C. A., Mondal, D., Du, Q., Chan, N. & Lewis, J. C. Directed evolution of an iron(II)- and α-ketoglutarate-dependent dioxygenase for site-selective azidation of unactivated aliphatic C–H bonds. Angew. Chem. Int. Ed. 62, e202301370 (2023).

    Article  CAS  Google Scholar 

  36. Neugebauer, M. E. et al. Reaction pathway engineering converts a radical hydroxylase into a halogenase. Nat. Chem. Biol. 18, 171–179 (2022).

    Article  CAS  PubMed  Google Scholar 

  37. Papadopoulou, A. et al. Re-programming and optimization of a l-proline cis-4-hydroxylase for the cis-3-halogenation of its native substrate. ChemCatChem 13, 3914–3919 (2021).

    Article  CAS  Google Scholar 

  38. Olivares, P., Ulrich, E. C., Chekan, J. R., van der Donk, W. A. & Nair, S. K. Characterization of two late-stage enzymes involved in fosfomycin biosynthesis in pseudomonads. ACS Chem. Biol. 12, 456–463 (2017).

    Article  CAS  PubMed  Google Scholar 

  39. Yun, D. et al. Structural basis of regiospecificity of a mononuclear iron enzyme in antibiotic fosfomycin biosynthesis. J. Am. Chem. Soc. 133, 11262–11269 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Higgins, L. J., Yan, F., Liu, P. H., Liu, H. W. & Drennan, C. L. Structural insight into antibiotic fosfomycin biosynthesis by a mononuclear iron enzyme. Nature 437, 838–844 (2005).

    Article  CAS  PubMed  Google Scholar 

  41. Tarantino, G. & Hammond, C. Catalytic C(sp3)–F bond formation: recent achievements and pertaining challenges. Green Chem. 22, 5195–5209 (2020).

    Article  CAS  Google Scholar 

  42. Matthews, M. L. et al. Substrate positioning controls the partition between halogenation and hydroxylation in the aliphatic halogenase, SyrB2. Proc. Natl Acad. Sci. USA 106, 17723–17728 (2009).

  43. Kulik, H. J. & Drennan, C. L. Substrate placement influences reactivity in non-heme Fe(II) halogenases and hydroxylases. J. Biol. Chem. 288, 11233–11241 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Kissman, E. N. et al. Biocatalytic control of site-selectivity and chain length-selectivity in radical amino acid halogenases. Proc. Natl Acad. Sci. USA 120, e2214512120 (2023).

  45. Chan, N. H. et al. Non-native anionic ligand binding and reactivity in engineered variants of the Fe(II)- and α-ketoglutarate-dependent oxygenase, SadA. Inorg. Chem. 61, 14477–14485 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Dai, L. et al. Structural and functional insights into a nonheme iron- and α-ketoglutarate-dependent halogenase that catalyzes chlorination of nucleotide substrates. Appl. Environ. Microbiol. 88, e02497–02421 (2022).

    Article  PubMed  PubMed Central  Google Scholar 

  47. Kastner, D. W., Nandy, A., Mehmood, R. & Kulik, H. J. Mechanistic insights into substrate positioning that distinguish non-heme Fe(II)/α-ketoglutarate-dependent halogenases and hydroxylases. ACS Catal. 13, 2489–2501 (2023).

    Article  CAS  Google Scholar 

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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

Authors and Affiliations



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).

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