Beyond the catalytic serine: selective protease engagement with covalent macrocyclic activity-based probes

Activity-based probes (ABPs) are widely used to profile serine protease activity - enzymes central to diverse physiological and pathological processes - but most rely on covalent modification of the conserved catalytic serine residue, often resulting in poor selectivity across related proteases. Here, we introduce covalent macrocyclic activity-based probes (cmABPs) that selectively target non-catalytic residues within serine protease active sites. By combining phage display with systematic electrophile scanning, we identify macrocyclic scaffolds that position sulfur(vi) fluoride (SuFEx) electrophiles to covalently engage alternative nucleophiles such as lysine and tyrosine. Applied to plasma kallikrein, this approach yielded a macrocyclic scaffold that was converted into covalent probes via fluorosulfate scanning. Remarkably, small changes in electrophile structure produced large, tuneable differences in covalent kinetics, with benzenesulfonyl fluoride derivative 23 achieving rapid and complete protein modification. Biochemical and mass spectrometry analyses confirmed selective modification of an active-site lysine by 23, along with robust performance in complex biological samples. Extension to urokinase plasminogen activator further demonstrates the generality of this strategy. More broadly, this work establishes electrophile scanning within macrocyclic scaffolds as a general approach for tuning covalent reactivity and provides a blueprint for designing selective probes that move beyond catalytic-residue targeting.