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 ortho-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.