Abstract
ABSTRACT
Sulfonyl exchange electrophiles are increasingly used in covalent probe and drug discovery, yet their intrinsic reactivity and plasma stability remain poorly defined, limiting rational warhead selection. Here we establish a systematic LC‐MS framework to quantify the reactivity of sulfonyl imidazole and triazole electrophiles across key biological nucleophiles together with matched plasma stability. Sulfonyl imidazoles display attenuated cysteine reactivity and markedly improved stability while retaining intrinsic tyrosine reactivity, whereas sulfonyl triazoles exhibit high electrophilicity and rapid degradation. These data reveal clear relationships between aryl electronics, leaving group ability, nucleophile selectivity, and
in vivo
‐relevant stability. To demonstrate practical utility, we applied a SuFEx‐click transformation to convert the kinase‐directed probe XO44 into a sulfonyl imidazole analogue, yielding a stable SuDEx probe (JL‐51) that maintains the proteome‐labeling profile of the parent reagent. The corresponding sulfonyl triazole congener was rapidly hydrolyzed. This work provides a mechanistic foundation and practical strategy for designing next‐generation sulfonyl exchange covalent probes.