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Sulfur(VI) fluorides (SFs) have emerged as valuable electrophiles for the design of "beyond-cysteine" covalent inhibitors and offer potential for expansion of the liganded proteome. Since SFs target a broad range of nucleophilic amino acids, they deliver an approach for the covalent modification of proteins without requirement for a proximal cysteine residue. Further to this, libraries of reactive fragments present an innovative approach for the discovery of ligands and tools for proteins of interest by leveraging a breadth of mass spectrometry analytical approaches. Herein, we report a screening approach that exploits the unique properties of SFs for this purpose. Libraries of SF-containing reactive fragments were synthesized, and a direct-to-biology workflow was taken to efficiently identify hit compounds for CAII and BCL6. The most promising hits were further characterized to establish the site(s) of covalent modification, modification kinetics, and target engagement in cells. Crystallography was used to gain a detailed molecular understanding of how these reactive fragments bind to their target. It is anticipated that this screening protocol can be used for the accelerated discovery of "beyond-cysteine" covalent inhibitors.

Here, we report a comprehensive profiling of sulfur(VI) fluorides (S VI -Fs) as reactive groups for chemical biology applications. S VI -Fs are reactive functionalities that modify lysine, tyrosine, histidine, and serine sidechains. A panel of S VI -Fs were studied with respect to hydrolytic stability and reactivity with nucleophilic amino acid sidechains. The use of S VI -Fs to covalently modify carbonic anhydrase II (CAII) and a range of kinases was then investigated. Finally, the S VI -F panel was used in live cell chemoproteomic workflows, identifying novel protein targets based on the type of S VI -F used. This work highlights how S VI -F reactivity can be used as a tool to expand the liganded proteome.