Chemical basis of fluorogenic substrate probe design: revisiting the past to discover the new in biochemistry

Enzyme activity, rather than mere protein abundance, defines the functional state of the proteome. Fluorogenic probes are molecules that are designed to directly report the "activity" of enzymes by fluorescence changes. In this review, we present a comprehensive overview of chemical design strategies for enzyme-activatable fluorogenic substrate probes targeting various classes of enzymes. We discuss the chemical principles required to convert natural substrate reactions into functional molecular sensors that respond to target enzymes with high precision. We also examine how recent advances in these design strategies, together with their integration into innovative analytical platforms, are expanding the impact of enzyme-responsive probes in chemical, biological, and clinical research.