Tunable Heteroaromatic Sulfones Enhance in-Cell Cysteine Profiling

Area of Science:

• Chemical Biology
• Proteomics
• Organic Chemistry

Background:

• Cysteine conjugation is crucial for chemical biology and proteomics.
• Existing cysteine alkylation reagents have limitations in reaction rate and stability.

Purpose of the Study:

• To evaluate a library of heteroaromatic sulfones as novel cysteine conjugation reagents.
• To assess the impact of structural modifications on reactivity and stability.
• To profile these probes in cultured cells for proteome-wide thiol analysis.

Main Methods:

• Synthesis and evaluation of diverse heteroaromatic sulfone libraries.
• Kinetic analysis of cysteine conjugation reactions.
• Cell-based profiling using desthiobiotin-functionalized probes.
• Mass spectrometry-based thiol identification and quantification.

Main Results:

• Select heteroaromatic sulfone substitutions increased reaction rates by over 3 orders of magnitude.
• Probes achieved rapid thiol saturation in cultured cells at submillimolar concentrations.
• Mass spectrometry identified over 3000 reactive/accessible thiols in native cellular environments.
• Complementary cysteine detection was observed between different probe types.

Conclusions:

• Heteroaromatic sulfones provide tunable, stable, and highly reactive scaffolds for cysteine conjugation.
• These probes offer significant advantages over conventional reagents for in-cell proteome profiling.
• The developed methodology enables efficient covalent ligand discovery and in-depth proteome analysis.