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

Nature Protocols volume 18, pages 1543–1562 (2023)Cite this article

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Abstract

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.

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Data availability

Raw MS and 19F-NMR data have been deposited with the following identifier: https://doi.org/10.5281/zenodo.6836127.

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Acknowledgements

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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Author notes

  1. These authors contributed equally: Patrick G. Isenegger, Brian Josephson.

Authors and Affiliations

  1. Department of Chemistry, University of Oxford, Oxford, UK

    Patrick G. Isenegger, Brian Josephson, Veronique Gouverneur, Andrew J. Baldwin & Benjamin G. Davis

  2. The Rosalind Franklin Institute, Oxfordshire, UK

    Ben Gaunt, Matthew J. Davy, Andrew J. Baldwin & Benjamin G. Davis

  3. Department of Pharmacology, University of Oxford, Oxford, UK

    Matthew J. Davy & Benjamin G. Davis

  4. Kavli Institute for Nanoscience Discovery, University of Oxford, Oxford, UK

    Andrew J. Baldwin

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Contributions

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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Josephson, B. et al. Nature 585, 530–537 (2020): https://doi.org/10.1038/s41586-020-2733-7

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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). https://doi.org/10.1038/s41596-022-00800-9

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