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

Chembiochem. 2014 May 5;15(7):946-9. doi: 10.1002/cbic.201402012. Epub 2014 Mar 12.


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.

Keywords: activity-based probes; native chemical ligation; protein modifications; solid-phase peptide synthesis; ubiquitin.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Carbocyanines / chemistry
  • Catalytic Domain
  • Cell Line, Tumor
  • Electrophoresis
  • Endopeptidases / chemistry
  • Endopeptidases / metabolism
  • Fluorescent Dyes / chemistry*
  • Humans
  • Mice
  • Substrate Specificity
  • Ubiquitins / chemistry*


  • Carbocyanines
  • Fluorescent Dyes
  • UBD protein, human
  • Ubiquitins
  • cyanine dye 5
  • Endopeptidases
  • OTUD7B protein, human