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Posttranslational, site-directed photochemical fluorine editing of protein sidechains to probe residue oxidation state via 19F-nuclear magnetic resonance


The fluorination of amino acid residues represents a near-isosteric alteration with the potential to report on biological pathways, yet the site-directed editing of carbon–hydrogen (C–H) bonds in complex biomolecules to carbon–fluorine (C–F) bonds is challenging, resulting in its limited exploitation. Here, we describe a protocol for the posttranslational and site-directed alteration of native γCH2 to γCF2 in protein sidechains. This alteration allows the installation of difluorinated sidechain analogs of proteinogenic amino acids, in both native and modified states. This chemical editing is robust, mild, fast and highly efficient, exploiting photochemical- and radical-mediated C–C bonds grafted onto easy-to-access cysteine-derived dehydroalanine-containing proteins as starting materials. The heteroaryl–sulfonyl reagent required for generating the key carbon-centered C• radicals that install the sidechain can be synthesized in two to six steps from commercially available precursors. This workflow allows the nonexpert to create fluorinated proteins within 24 h, starting from a corresponding purified cysteine-containing protein precursor, without the need for bespoke biological systems. As an example, we readily introduce three γCF2-containing methionines in all three progressive oxidation states (sulfide, sulfoxide and sulfone) as d-/l- forms into histone eH3.1 at site 4 (a relevant lysine to methionine oncomutation site), and each can be detected by 19F-nuclear magnetic resonance of the γCF2 group, as well as the two diastereomers of the sulfoxide, even when found in a complex protein mixture of all three. The site-directed editing of C–H→C–F enables the use of γCF2 as a highly sensitive, ‘zero-size-zero-background’ label in protein sidechains, which may be used to probe biological phenomena, protein structures and/or protein–ligand interactions by 19F-based detection methods.

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Fig. 1: Strategy for the insertion of ‘zero-size-zero-background’ sidechain labels into proteins.
Fig. 2: The range of γCF2-containing sidechains that can be introduced by C–C-bond formation.
Fig. 3: The synthesis of pySOOF reagents for C• radical generation in protein mutagenesis.
Fig. 4: Production of histone eH3.1 with γCF2-tagged Met variants (Met sulfide, Met(O) sulfoxide and Met(O2) sulfone) for 19F-NMR studies.
Fig. 5: 19F-NMR allows the distinction of labeled methionine diastereoisomers in the context of an intact protein.

Data availability

Raw MS and 19F-NMR data have been deposited with the following identifier:


  1. Walsh, C. T., Garneau-Tsodikova, S. & Gatto, G. J. Jr. Protein posttranslational modifications: the chemistry of proteome diversifications. Angew. Chem. Int. Ed. 44, 7342–7372 (2005).

    Article  CAS  Google Scholar 

  2. Strahl, B. D. & Allis, C. D. The language of covalent histone modifications. Nature 403, 41–45 (2000).

    Article  CAS  PubMed  Google Scholar 

  3. Farrelly, L. A. et al. Histone serotonylation is a permissive modification that enhances TFIID binding to H3K4me3. Nature 567, 535–539 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Nadal, S., Raj, R., Mohammed, S. & Davis, B. G. Synthetic post-translational modification of histones. Curr. Opin. Chem. Biol. 45, 35–47 (2018).

    Article  CAS  PubMed  Google Scholar 

  5. Chin, J. W. Expanding and reprogramming the genetic code of cells and animals. Annu. Rev. Biochem. 83, 379–408 (2014).

  6. Chen, H., Venkat, S., McGuire, P., Gan, Q. & Fan, C. Recent development of genetic code expansion for posttranslational modification studies. Molecules 23, 1662 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  7. Nguyen, D. P., Garcia Alai, M. M., Kapadnis, P. B., Neumann, H. & Chin, J. W. Genetically Encoding Nϵ-Methyl-l-lysine in recombinant histones. J. Am. Chem. Soc. 131, 14194–14195 (2009).

    Article  CAS  PubMed  Google Scholar 

  8. Akahoshi, A., Suzue, Y., Kitamatsu, M., Sisido, M. & Ohtsuki, T. Site-specific incorporation of arginine analogs into proteins using arginyl-tRNA synthetase. Biochem. Biophys. Res. Commun. 414, 625–630 (2011).

    Article  CAS  PubMed  Google Scholar 

  9. Chatterjee, C. in Total Chemical Synthesis of Proteins (eds Brik, A. et al.) 489–513 (Wiley-VCH, 2021).

  10. Qi, Y.-K., Ai, H.-S., Li, Y.-M. & Yan, B. Total chemical synthesis of modified histones. Front. Chem. 6, 19–19 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  11. Wright, T. H., Vallée, M. R. J. & Davis, B. G. From chemical mutagenesis to post-expression mutagenesis: A 50 year odyssey. Angew. Chem. Int. Ed. 55, 5896–5903 (2016).

    Article  CAS  Google Scholar 

  12. Dadová, J., Galan, S. R. G. & Davis, B. G. Synthesis of modified proteins via functionalization of dehydroalanine. Curr. Opin. Chem. Biol. 46, 71–81 (2018).

    Article  PubMed  Google Scholar 

  13. Sletten, E. M. & Bertozzi, C. R. Bioorthogonal chemistry: fishing for selectivity in a sea of functionality. Angew. Chem. Int. Ed. 48, 6974–6998 (2009).

    Article  CAS  Google Scholar 

  14. Wright, T. H. & Davis, B. G. Post-translational mutagenesis for installation of natural and unnatural amino acid side chains into recombinant proteins. Nat. Protoc. 12, 2243–2250 (2017).

    Article  PubMed  Google Scholar 

  15. Wright, T. H. et al. Posttranslational mutagenesis: a chemical strategy for exploring protein side-chain diversity. Science (2016).

  16. Yang, A. et al. A chemical biology route to site-specific authentic protein modifications. Science (2016).

  17. Zhang, M., He, P. & Li, Y. Contemporary approaches to α,β-dehydroamino acid chemical modifications. Chem. Res. Chin. Univ. 37, 1044–1054 (2021).

    Article  CAS  Google Scholar 

  18. Josephson, B. et al. Light-driven post-translational installation of reactive protein side chains. Nature 585, 530–537 (2020).

    Article  CAS  PubMed  Google Scholar 

  19. Müller, M. M. & Muir, T. W. Histones: at the crossroads of peptide and protein chemistry. Chem. Rev. 115, 2296–2349 (2015).

    Article  PubMed  Google Scholar 

  20. Bannister, A. J. & Kouzarides, T. Regulation of chromatin by histone modifications. Cell Res. 21, 381–395 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Zheng, Y., Huang, X. & Kelleher, N. L. Epiproteomics: quantitative analysis of histone marks and codes by mass spectrometry. Curr. Opin. Chem. Biol. 33, 142–150 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Rothbart, S. B. et al. An interactive database for the assessment of histone antibody specificity. Mol. Cell. 59, 502–511 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Duan, Q., Chen, H., Costa, M. & Dai, W. Phosphorylation of H3S10 blocks the access of H3K9 by specific antibodies and histone methyltransferase. Implication in regulating chromatin dynamics and epigenetic inheritance during mitosis. J. Biol. Chem. 283, 33585–33590 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Claridge, T. D. W. in High-Resolution NMR Techniques in Organic Chemistry (3rd edn) (ed Claridge, T. D. W.) 421–455 (Elsevier, 2016).

  25. Chalker, J. M. & Davis, B. G. Chemical mutagenesis: selective post-expression interconversion of protein amino acid residues. Curr. Opin. Chem. Biol. 14, 781–789 (2010).

    Article  CAS  PubMed  Google Scholar 

  26. Kim, G., Weiss, S. J. & Levine, R. L. Methionine oxidation and reduction in proteins. Biochim. Biophys. Acta 1840, 901–905 (2014).

    Article  CAS  PubMed  Google Scholar 

  27. Bagert, J. D. et al. Oncohistone mutations enhance chromatin remodeling and alter cell fates. Nat. Chem. Biol. 17, 403–411 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Jang, Y. et al. H3.3K4M destabilizes enhancer H3K4 methyltransferases MLL3/MLL4 and impairs adipose tissue development. Nucleic Acids Res. 47, 607–620 (2019).

    Article  CAS  PubMed  Google Scholar 

  29. Brown, Z. Z. et al. Strategy for “detoxification” of a cancer-derived histone mutant based on mapping its interaction with the methyltransferase PRC2. J. Am. Chem. Soc. 136, 13498–13501 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Hintzen, J. C. J. et al. γ-Difluorolysine as a 19F NMR probe for histone lysine methyltransferases and acetyltransferases. Chem. Comm. 57, 6788–6791 (2021).

    Article  CAS  PubMed  Google Scholar 

  31. Brittain, W. D. G., Lloyd, C. M. & Cobb, S. L. Synthesis of complex unnatural fluorine-containing amino acids. J. Fluor. Chem. 239, 109630 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Papworth, C., Bauer, J. & Braman, J. Site-directed mutagenesis in one day with >80% efficiency. Strategies 9, 3–4 (1996).

    Google Scholar 

  33. Chalker, J. M., Bernardes, G. J. L. & Davis, B. G. A “tag-and-modify” approach to site-selective protein modification. Acc. Chem. Res. 44, 730–741 (2011).

    Article  CAS  PubMed  Google Scholar 

  34. Lai, K.-Y. et al. LanCLs add glutathione to dehydroamino acids generated at phosphorylated sites in the proteome. Cell 184, 2680–2695.e2626 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Chalker, J. M. et al. Methods for converting cysteine to dehydroalanine on peptides and proteins. Chem. Sci. 2, 1666–1676 (2011).

    Article  CAS  Google Scholar 

  36. Dadová, J. et al. Precise probing of residue roles by post-translational β,γ-C,N Aza-Michael mutagenesis in enzyme active sites. ACS Cent. Sci. 3, 1168–1173 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  37. Imiołek, M. et al. Residue-selective protein C-formylation via sequential difluoroalkylation-hydrolysis. ACS Cent. Sci. 7, 145–155 (2021).

    Article  PubMed  PubMed Central  Google Scholar 

  38. Li, B. X. et al. Site-selective tyrosine bioconjugation via photoredox catalysis for native-to-bioorthogonal protein transformation. Nat. Chem. 13, 902–908 (2021).

    Article  CAS  PubMed  Google Scholar 

  39. Mollner, T. A. et al. Post-translational insertion of boron in proteins to probe and modulate function. Nat. Chem. Biol. 17, 1245–1261 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Lin, Y. A. et al. Rapid cross-metathesis for reversible protein modifications via chemical access to Se-Allyl-selenocysteine in proteins. J. Am. Chem. Soc. 135, 12156–12159 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Galan, S. R. G. et al. Post-translational site-selective protein backbone α-deuteration. Nat. Chemi. Biol. 14, 955–963 (2018).

    Article  CAS  Google Scholar 

  42. Stadmiller, S. S., Aguilar, J. S., Waudby, C. A. & Pielak, G. J. Rapid quantification of protein-ligand binding via 19F NMR lineshape analysis. Biophys. J. 118, 2537–2548 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Suzuki, Y. et al. Resolution of oligomeric species during the aggregation of Aβ1–40 using 19F NMR. Biochemistry 52, 1903–1912 (2013).

    Article  CAS  PubMed  Google Scholar 

  44. Danielson, M. A. & Falke, J. J. Use of 19F NMR to probe protein structure and conformational changes. Annu. Rev. Biophys. Biomol. Struct. 25, 163–195 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Hattori, Y. et al. Protein (19)F-labeling using transglutaminase for the NMR study of intermolecular interactions. J. Biomol. Nmr. 68, 271–279 (2017).

    Article  CAS  PubMed  Google Scholar 

  46. Hore, P., Jones, J. & Wimperis, S. NMR: The Toolkit 2nd edn (Oxford Univ. Press, 2015).

  47. Baldwin, A. J. (2022).

  48. Karunanithy, G. et al. Harnessing NMR relaxation interference effects to characterise supramolecular assemblies. Chem. Comm. 52, 7450–7453 (2016).

    Article  CAS  PubMed  Google Scholar 

  49. Wu, J. et al. Synergy of synthesis, computation and NMR reveals correct baulamycin structures. Nature 547, 436–440 (2017).

    Article  CAS  PubMed  Google Scholar 

  50. Gortari, I. D. et al. Time averaging of NMR chemical shifts in the MLF peptide in the solid state. J. Am. Chem. Soc. 132, 5993–6000 (2010).

    Article  PubMed  Google Scholar 

  51. Anil, B., Song, B., Tang, Y. & Raleigh, D. P. Exploiting the right side of the Ramachandran plot: substitution of glycines by D-alanine can significantly increase protein stability. J. Am. Chem. Soc. 126, 13194–13195 (2004).

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This research has received funding from the Swiss National Science Foundation (P2BSP2_178609, P.G.I.), Biotechnology and Biological Sciences Research Council (BBSRC, BB/P026311/1, V.G., B.G.D., P.G.I.), European Research Council (ERC, 101002859), and Oxford Clarendon Scholarship (B.J.). The Next Generation Chemistry theme at the Franklin Institute is supported by the Engineering and Physical Sciences Research Council (EPSRC, V011359/1 (P)).

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Authors and Affiliations



P.G.I. and B.G.D. conceived and designed the experiments. P.G.I. synthesised the pySOOF reagents. B.J. performed the chemical mutagenesis experiments. B.G. performed the NMR experiments. P.G.I., B.J., B.G., M.J.D., V.G., A.J.B. and B.G.D. collected and/or analyzed data. P.G.I., B.J. and B.G.D. wrote the paper. All authors read and commented on the paper.

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Correspondence to Andrew J. Baldwin or Benjamin G. Davis.

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Isenegger, P.G., Josephson, B., Gaunt, B. et al. Posttranslational, site-directed photochemical fluorine editing of protein sidechains to probe residue oxidation state via 19F-nuclear magnetic resonance. Nat Protoc 18, 1543–1562 (2023).

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