Akin to sulfonyl-fluorides 20– 22 and fluorosulfates 23– 29 (i.e. We recently introduced sulfur-triazole exchange (SuTEx) chemistry as a new class of electrophiles for chemical proteomic applications 19. New chemoselective reactions, therefore, are important for advancing chemical probes used for basic and therapeutic discovery. Beyond liganding sites on proteins 4– 5, 9, 15, covalent probes can be adapted to study post-translational modifications (PTM) including crotonolyation 16, methylation 17, deimination 18, and phosphorylation 19. Development of cysteine- 5– 8 and lysine-reactive chemistry 9– 12, for example, are creating new opportunities for perturbing and degrading proteins based on enzymatic and non-catalytic functions 13– 14. Electrophilic or photoreactive groups embedded in fragments or high molecular weight binders have been used, in combination with proteomic technologies, to uncover ligand sites that can be exploited for pharmacological control 3– 5. Collectively, we describe SuTEx as a versatile covalent chemistry with broad applications for chemical proteomics and protein ligand discovery.Ĭovalent small molecules are enabling tools for investigating protein function in biology 1 and represent an important class of drug molecules 2. Our proteomic studies identified non-catalytic tyrosine- and phosphotyrosine-sites that can be liganded by SuTEx fragments, with site specificity, in lysates and live cells to disrupt protein function. We applied these reactivity principles to identify a large fraction of tyrosine sites (~30%) on proteins (~44%) that can be liganded across >1500 probe-modified sites quantified by chemical proteomics. We discovered the sulfur electrophile is highly sensitive to the position of modification (AG versus LG), which enabled both coarse and fine adjustments in solution and proteome activity. Here, we probed tunability of SuTEx for fragment-based ligand discovery by modifying the adduct group (AG) and LG with functional groups of differing electron-donating and -withdrawing properties. We recently introduced sulfur-triazole exchange (SuTEx) chemistry to demonstrate the triazole as an effective LG for activating nucleophilic substitution reactions on tyrosine sites of proteins. To date, activation of the sulfur electrophile for protein modification has been ascribed principally to stabilization of a fluoride leaving group (LG) in covalent reactions of sulfonyl fluorides and arylfluorosulfates. Tuning reactivity of sulfur electrophiles is key for advancing click chemistry and chemical probe discovery.

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