Covalent probes serve as valuable tools for global investigation of protein function and ligand binding capacity. Despite efforts to expand coverage of residues available for chemical proteomics (e.g. cysteine and lysine), a large fraction of the proteome remains inaccessible with current activity-based probes. Here, we introduce sulfur-triazole exchange (SuTEx) chemistry as a tunable platform for developing covalent probes with broad applications for chemical proteomics. We show modifications to the triazole leaving group can furnish sulfonyl probes with ~5-fold enhanced chemoselectivity for tyrosines over other nucleophilic amino acids to investigate, for the first time, more than 10,000 tyrosine sites in lysates and live cells. We discover that tyrosines with enhanced nucleophilicity are enriched in enzymatic, protein-protein interaction, and nucleotide recognition domains. We apply SuTEx as a chemical phosphoproteomics strategy to monitor activation of phosphotyrosine sites. Collectively, we describe SuTEx as a biocompatible chemistry for chemical biology investigations of the human proteome. Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

Sulfonyl‐triazoles have emerged as a new reactive group for covalent modification of tyrosine sites on proteins through sulfur‐triazole exchange (SuTEx) chemistry. The extent to which this sulfur electrophile can be tuned for developing ligands with cellular activity remains largely underexplored. Here, we performed fragment‐based ligand discovery in live cells to identify SuTEx compounds capable of liganding tyrosine sites on diverse protein targets. We verified our quantitative chemical proteomic findings by demonstrating concentration‐dependent activity of SuTEx ligands, but not inactive counterparts, against recombinant protein targets directly in live cells. Our structure‐activity relationship studies identified the SuTEx ligand HHS‐0701 as a cell‐active inhibitor capable of blocking prostaglandin reductase 2 (PTGR2) biochemical activity.

Chemoproteomic probes serve as valuable tools to study and perturb protein function on a global scale. Despite advances in methodologies for cysteine and lysine profiling, a large fraction of the human proteome remains inaccessible with current activity-based probes. Here, we describe development and application of sulfur-triazole exchange (SuTEx) chemistry for developing covalent probes with broad applications for chemical proteomics. We show modifications to the triazole leaving group can produce sulfonyl probes with high chemoselectivity for tyrosines over other nucleophilic amino acids to investigate thousands of tyrosine sites in lysates and live cells. We discover hyper-reactive tyrosines are enriched in enzymatic, protein–protein interaction and nucleotide recognition domains. We apply SuTEx as a chemical phosphoproteomics strategy to monitor activation of phosphotyrosine sites. Our findings illustrate the broad potential for deploying SuTEx to globally investigate tyrosine reactivity, function and post-translational modification state in complex biological systems.