Two 2-nitroimidazole-1,8-naphthalimide conjugates, 1 and 2, have been synthesised as fluorescence probes for the detection of reductive stress in HeLa cells. The 4-substituted derivative 1 was shown to act as a highly sensitive and selective substrate for nitroreductase where it exhibited a clear blue to green ratiometric fluorescence response visible to the naked eye. Moreover, biological studies demonstrated 1 could be activated in cellulo where the impact of reductive stress was easily monitored using confocal microscopy and flow cytommetry.

Latent activity-based probes have been developed for deubiquitinating enzymes using a thiol–ene strategy, labelling following a specific binding interaction.

The regulation of ubiquitination status in the cell is controlled by ubiquitin ligases acting in tandem with deubiquitinating enzymes. Ubiquitination controls many key processes in the cell from division to death making its tight regulation key to optimal cell function. Activity based protein profiling has emerged as a powerful technique to study these important enzymes. With around 100 deubiquitinating enzymes and 600 ubiquitin ligases in the human genome targeting a subclass of these enzymes or even a single enzyme is a compelling strategy to unpick this complex system. In this review we will discuss different approaches adopted, including activity-based probes centered around ubiquitin-protein, ubiquitin-peptide and mutated ubiquitin scaffolds. We examine challenges faced and opportunities presented to increase specificity in activity-based protein profiling of the ubiquitin conjugation/deconjugation machinery.