Mixed Alkyl Aryl Phosphonates as Quenched Activity-Based Probes for Real-Time Imaging of Active Neutrophil Serine Proteases

Activity-based probes have been instrumental in the study of proteases, and quenched fluorescent versions can be utilized in real time imaging. Unfortunately, this application has not yet been reported for serine proteases, which make up the largest mechanistic class of proteases. Here, we describe quenched activity-based probes for detection of serine proteases, specifically the neutrophil serine proteases: neutrophil elastase, proteinase 3, and cathepsin G. We demonstrate that these reagents can selectively label serine proteases in complex proteomes and we illustrate their use in the live cell imaging of activation of primary human neutrophils. We expect that these reagents will find use in real-time imaging of active neutrophil serine proteases and may be further developed for imaging of other serine proteases.