Active-site directed probes are powerful in studies of enzymatic function. In the Journal of the American Chemical Society Ekkebus et al. report activity-based probes based on a new warhead so far considered unreactive: propargyl amide (PA). By replacing the C-terminal carboxylate of ubiquitin (Ub) with an alkyne functionality, a selective reaction with the active-site cysteine residue of de-ubiquitinating enzymes was observed. The resulting product was shown to be a quaternary vinyl thioether, as determined by X-ray crystallography. Proteomic analysis of proteins bound to an immobilized probe confirmed the selectivity toward deubiquitinating enzymes (DUBs). 

Figure 1 – propargyl amide as a new warhead for DUB activity-based probes (Ekkebus et al. J Am Chem Soc 2013, 135, 2867).

• UbiQ-057 :  Ub-PA
• UbiQ-058 :  TAMRA-Ub-PA
• UbiQ-072 :  Cy5-Ub-PA
• UbiQ-076 :  Biotin-Ahx-Ub-PA 
• UbiQ-077:   Biotin-ANP-Ub-PA
• UbiQ-078 :  HA-Ahx-Ahx-Ub-PA 
• UbiQ-168  : Biotin-Ahx-SUMO2-PA