β-Fluorovinylsulfonamide as a Highly Reactivity- and Structure-Tunable Electrophile for Covalent Targeting of Proteins.

Covalent targeting of proteins with small molecule probes is a powerful approach for analyzing and manipulating biological functions. Recent advancements in the design of target selective covalent ligands further expand the scope of accessible proteins in this approach. Here we report β-fluorovinylsulfonamide (FVS) as a versatile electrophile for the design of covalent ligands. FVS irreversibly reacts with cysteine residues in proteins to afford stable vinyl sulfide adducts. The reactivity of FVS is maintained with the introduction of various substituents at the β-position, allowing for fine-tuning of the electrophilic reactivity and flexible molecular design of FVS-based covalent ligands. These reaction properties were leveraged in the development of highly selective covalent ligands for RSK2 and BTK. FVS compounds are also able to react with lysine residues in proteins to form enamine-type reversible covalent adducts, which are susceptible to hydrolysis under neutral aqueous conditions. This lysine reactivity of FVS led to the discovery of FVS compound 53 as a reversible covalent ligand for PFKL, which selectively reacts with K677 of PFKL and allosterically upregulates PFKL activity in living cells.