Tunable Heteroaromatic Sulfones Enhance in-Cell Cysteine Profiling

Heteroaromatic sulfones react with cysteine via nucleophilic aromatic substitution, providing a mechanistically selective and irreversible scaffold for cysteine conjugation. Here we evaluate a library of heteroaromatic sulfides with different oxidation states, heteroatom substitutions, and a series of electron-donating and electron-withdrawing substituents. Select substitutions profoundly influence reactivity and stability compared to conventional cysteine conjugation reagents, increasing the reaction rate by >3 orders of magnitude. The findings establish a series of synthetically accessible electrophilic scaffolds tunable across multiple centers. New electrophiles and their corresponding alkyne conjugates were profiled directly in cultured cells, achieving thiol saturation in a few minutes at submillimolar concentrations. Direct addition of desthiobiotin-functionalized probes to cultured cells simplified enrichment and elution to enable the mass spectrometry discovery of >3000 reactive and/or accessible ...