Abstract
Original covalent probes with an N‐acyl‐N‐alkyl sulfonamide cleavable linker were developed to target a broad set of human Matrix Metalloproteases (MMPs). The electrophilicity of this cleavable linker was modulated to improve the selectivity of the probes as well as reduce their unspecific reactivity in complex biological matrices. We first demonstrated that targeting the S3 subsite of MMPs enables access to broad‐spectrum affinity‐based probes that exclusively react with the active version of these proteases. The probes were further assessed in proteomes of varying complexity, where human MMP‐13 was artificially introduced at known concentration and the resulting labeled MMP was imaged by in‐gel fluorescence imaging. We showed that the less reactive probe was still able to covalently modify MMP‐13 while exhibiting reduced off‐target unspecific reactivity. This study clearly demonstrated the importance of finely controlling the reactivity of the NASA warhead to improve the selectivity of covalent probes in complex biological systems.