Semisynthesis of Ubiquitin and SUMO-Rhodamine 110-Glycine through Aminolysis of Boc-Protected Thioester Counterparts

Ubiquitin (Ub)-based fluorescent reagents are crucial to explore the activity of deubiquitinases (DUBs). Ub-Rho110-G is one of the preferred tools, whereas the current synthetic route is time-consuming. Here, we report a new semisynthetic strategy to produce Ub-Rho110-G through direct aminolysis of Boc-protected Ub-Mesna using bisglycyl-rhodamine 110. We also applied this strategy to synthesize active SUMO2-Rho110-G for the first time. Biochemical analysis demonstrated that semisynthetic Ub or SUMO-Rho110-G can be effectively used for the detection of the activity of DUBs or SUMO-specific enzymes.