Two-step labeling of endogenous enzymatic activities by Diels-Alder ligation

Lianne I. Willems, Martijn Verdoes, Bogdan I. Florea, Gijsbert A. Van Der Marel, Herman S. Overkleeft

Research output: Contribution to journalArticlepeer-review


A ligation strategy based on the Diels-Alder [4+2] cycloaddition for the two-step activity-based labeling of endogenously expressed enzymes in complex biological samples has been developed. A panel of four diene-derivatized proteasome probes was synthesized, along with a dienophile-functionalized BODIPY(TMR) tag. These probes were applied in a Diels-Alder labeling procedure that enabled us to label active proteasome β-subunits selectively in cellular extracts and in living cells. We were also able to label the activity of cysteine proteases in cell extracts by utilizing a diene-derivatized cathepsin probe. Importantly, the Diels-Alder strategy described here is fully orthogonal with respect to the Staudinger-Bertozzi ligation, as demonstrated by the independent labeling of different proteolytic activities by the two methods in a single experiment.

Original languageEnglish
Pages (from-to)1769-1781
Number of pages13
Issue number12
Publication statusPublished - 16 Aug 2010


  • Activity-based protein profiling
  • Bioorthogonal chemistry
  • Cycloaddition
  • Hydrolases
  • Proteomics

Cite this