On terminal alkynes that can react with active-site cysteine nucleophiles in proteases

J Am Chem Soc. 2013 Feb 27;135(8):2867-70. doi: 10.1021/ja309802n. Epub 2013 Feb 15.


Active-site directed probes are powerful in studies of enzymatic function. We report an active-site directed probe based on a warhead so far considered unreactive. 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 Ub alkyne probe confirmed the selectivity toward de-ubiquitinating enzymes. The observed reactivity is not just restricted to propargylated Ub, as highlighted by the selective reaction between caspase-1 (interleukin converting enzyme) and a propargylated peptide derived from IL-1β, a caspase-1 substrate.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alkynes / chemistry*
  • Catalytic Domain
  • Cysteine / chemistry*
  • Peptide Hydrolases / metabolism*


  • Alkynes
  • Peptide Hydrolases
  • Cysteine