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

Abstract

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.