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
thiols
labeled in their native cellular environments in a fraction of the
standard analysis time. Surprisingly, only half of the annotated cysteines
were identified by both iodo­acet­amide-des­thio­biotin
and methyl­sulfonyl­benzo­thiazole-des­thio­biotin
in replicate experiments, demonstrating complementary detection by
mass spectrometry analysis. These probes offer advantages over existing
cysteine alkylation reagents, including accelerated reaction rates,
improved stability, and robust ionization for mass spectrometry applications.
Overall, heteroaromatic sulfones provide modular tunability, shifted
chromatographic elution times, and superior in-cell cysteine profiling
for in-depth proteome-wide analysis and covalent ligand discovery.