Tuning activity-based probe selectivity for serine proteases by on-resin ‘click’ construction of peptidediphenyl phosphonates

Abstract

Activity-based probes (ABPs) are powerful tools for functional proteomics studies. Their selectivity can be influenced by modification of a recognition element that interacts with pockets near the active site. For serine proteases there are a limited number of simple and efficient synthetic procedures for the development of selective probes. Here we describe a new synthetic route combining solid and solution phase chemistries to generate a small library of diphenyl phosphonate probes. Building blocks carrying a P1 recognition element and an electrophilic phosphonate warhead were prepared in solution and ‘clicked’ on-resin onto a tripeptide. We show the ability to modulate the activity and selectivity of diphenyl phosphonate ABPs and demonstrate activity-dependent labeling of endogenous proteases within a tissue proteome. The herein described synthetic approach therefore serves as a valuable method for rapid diversification of serine protease ABPs.