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

To date, a variety of covalent cysteine-reactive chemical probes have been reported. The most common ones include maleimide for bioconjugation and iodoacetamide alkyne (IAA) for chemoproteomics analyses. Their applicability, however, is limited due to, e.g., the hydrolytic lability of maleimide adducts and the slow reaction kinetics as well as the inability to record the entirety of the cysteinome with IAA. This is compounded by the missing potential to fine-tune their reactivity tailored to advanced applications. To generate a high-reactivity, cysteine-selective chemical probe with broad utility, we have performed an in-depth investigation into the acrylophenone scaffold. The aryl group connected to the vinyl ketone chemotype can be readily substituted, which provides the potential to fine-tune the reactivity and install a bioorthogonal handle. We took advantage of this feature by modifying acrylophenone-alkyne (APA) with two ortho chlorine groups to generate <i>ortho</i>-dichloroacrylophenone-alkyne (CAPA), which increased the stability of the probe and the yield of its cysteine adducts. To showcase the reactivity, we performed reaction rate analyses with model reagents. The selectivity was demonstrated by specifically labeling cysteine residues within two peptides under physiological conditions. To investigate its utility toward bioconjugation reactions, we performed the stoichiometric labeling of two proteins. Remarkably, CAPA was successfully implemented as a high-reactivity cysteine-selective chemical probe into activity-based protein profiling (ABPP) experiments using both in-gel fluorescence (in-gel ABPP) and mass spectrometry analyses. The chemoproteomics workflow, named isoDTB-ABPP, allowed us to highlight that CAPA provides a complementary approach to IAA in expanding the coverage of the cysteinome.