Synthetic substrates for enzymes that catalyze reactions of modified cysteines and related methods

Synthetic probes for detecting the activity of enzymes that catalyze reactions of post-translationally modified cysteine residues are described. The probes include "turn-on" probes that include a carbamate linkage that is cleaved via an intramolecular reaction with a free thiol produced by an enzyme catalyzed activity. The probes also include ratiometric, Michael addition-based probes that respond to enzymatic activity by a change in structure that results in a change in fluorescence properties. Methods of using the probes to detect enzymatic activity and disease are described. For example, the probes can be used to detect enzymatic activity in a variety of samples, including live cells and heterogeneous tissues. In addition, prodrugs that can be activated by enzymes that catalyze reactions of post-translationally modified cysteine residues and methods of using the prodrugs to treat disease are described.