Phosphoramidates as novel activity-based probes for serine proteases

Activity-based probes (ABPs) are small molecules that exclusively form covalent bonds with catalytically active enzymes. In the last decade, they have especially been used in functional proteomics studies of proteases. Here, we present phosphoramidate peptides as a novel type of ABP for serine proteases. These molecules can be made in a straightforward manner by standard Fmoc-based solid-phase peptide synthesis, allowing rapid diversification. The resulting ABPs covalently bind different serine proteases, depending on the amino acid recognition element adjacent to the reactive group. A reporter tag enables downstream gel-based analysis or LC-MS/MS-mediated identification of the targeted proteases. Overall, we believe that these readily accessible probes will provide new avenues for the functional study of serine proteases in complex proteomes. Protease probes: We synthesized serine protease activity-based probes entirely on solid support. They carry a phosphoramidate reactive electrophile for covalent attachment to the protease active site. The selectivity of these probes can be adjusted by changing the residue proximal to the electrophile, and they can be used for detection of proteases in complex proteomes.