Semi-synthesis of biotin-bearing activity-based ubiquitin probes through sequential enzymatic ligation,N-S acyl transfer and aminolysis reaction

Activity-based Ubiquitin probes(Ub-ABPs)carrying a reporter group have emerged as effective tools for the investigation of deubiquitinating enzymes(DUBs),such as studying the molecular mechanism of DUBs,profiling new DUBs.But so far,the synthesis of commonly used biotin-bearing Ub-ABPs is a technical challenge.Here,we report a one-pot semi-synthetic strategy for the acquiring of Ub-ABPs carrying a biotin tag through sequential enzymatic ligation,N-S acyl transfer and aminolysis reaction without any purification steps.These probes enable to capture the different family of DUBs for enrichment and immunoblotting using the attached biotin tag.