A Highly Reactive Cysteine-Targeted Acrylophenone Chemical Probe That Enables Peptide/Protein Bioconjugation and...

Area of Science:

• Chemical Biology
• Proteomics
• Organic Chemistry

Background:

• Existing cysteine-reactive probes like maleimide and iodoacetamide alkyne (IAA) have limitations including hydrolytic instability, slow kinetics, and incomplete cysteinome coverage.
• There is a need for high-reactivity, cysteine-selective chemical probes with tunable properties for advanced applications in chemical biology.

Purpose of the Study:

• To develop a novel, highly reactive, and stable cysteine-selective chemical probe with broad utility.
• To investigate the acrylophenone scaffold for its potential in designing tunable chemical probes.
• To establish a new chemoproteomics workflow for expanded cysteinome analysis.

Main Methods:

• Modification of the acrylophenone-alkyne scaffold to generate ortho-dichloroacrylophenone-alkyne (CAPA).
• Reaction rate analyses with model reagents and selectivity studies on peptides under physiological conditions.
• Application of CAPA in protein labeling for bioconjugation and activity-based protein profiling (ABPP) using in-gel fluorescence and mass spectrometry.

Main Results:

• CAPA demonstrated increased stability and higher yield of cysteine adducts compared to parent compounds.
• Selective labeling of cysteine residues in peptides and stoichiometric labeling of proteins were achieved.
• CAPA proved effective in activity-based protein profiling (ABPP), complementing IAA in expanding cysteinome coverage.

Conclusions:

• Ortho-dichloroacrylophenone-alkyne (CAPA) is a versatile, high-reactivity, cysteine-selective chemical probe.
• CAPA offers enhanced stability and reactivity, overcoming limitations of existing probes.
• The developed isoDTB-ABPP workflow utilizing CAPA expands the scope of chemoproteomics analysis.