A native chemical ligation handle that enables the synthesis of advanced activity-based probes: Diubiquitin as a case study

Abstract

We present the development of a native chemical ligation handle that also functions as a masked electrophile that can be liberated during synthesis when required. This handle can thus be used for the synthesis of complex activity-based probes. We describe the use of this handle in the generation of linkage-specific activity-based deubiquitylating enzyme probes that contain substrate context and closely mimic the native ubiquitin isopeptide linkage. We have generated activity-based probes based on all seven isopeptide-linked diubiquitin topoisomers and demonstrated their structural integrity and ability to label DUBs in a linkage-specific manner. Protease profiling: A new native chemical ligation handle that functions as a masked electrophile and allows the total chemical synthesis of complex activity-based probes (ABPs) has been developed. We show the efficiency of our approach by synthesizing deubiquitylating enzyme (DUB) ABPs based on all seven diubiquitin (diUb) isopeptide topoisomers and examining their ability to capture DUBs selectively with distinct target preference. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.