<i>In Cellulo</i> Cysteine Umpolung for Protein Structure Probing

Proteome-wide intramolecular cross-linking of amino acids allows for protein structure probing and is often achieved by reactive intermediates generated <i>in situ</i>. For example, carbene insertion chemistry forms versatile products but relies on two-step protocols that first introduce a photoreactive group and then generate a reactive intermediate via UV light irradiation. Alternatively, direct cross-linking with bifunctional electrophilic reagents enables modification of natural amino acids in a single transformation but proceeds via intermediates with a sufficiently long half-life in solution to capture unfavorable protein conformations. Herein, we report the use of commercially available vinyl thianthrenium tetrafluoroborate (<b>VTT</b>) for a single-step cross-linking strategy based on the umpolung of native cysteinyl thiols to electrophilic episulfonium ions in cells. We demonstrate that the umpolung reaction proceeds within minutes in various cell types. Even when competing with cysteine-reactive maleimide or iodoacetamide reagents, the fast cellular uptake of <b>VTT</b> enables efficient intracellular labeling and ensures the formation of products with a short and stable ethylene linker between cysteine and nucleophilic natural amino acids. We anticipate that the fast generation of episulfonium ions, which enable cross-linking without exogenous activation, will assist researchers in predicting protein structures based on quantitative proteome-wide information generated <i>in cellulo</i>.