Related Experiment Video
Updated: Jun 11, 2026

Visualizing Lignification Dynamics in Plants with Click Chemistry: Dual Labeling is BLISS!
Published on: January 26, 2018
Two-step labeling of endogenous enzymatic activities by Diels-Alder ligation
Lianne I Willems1, Martijn Verdoes, Bogdan I Florea
1Leiden Institute of Chemistry and Netherlands Proteomics Centre, Gorlaeus Laboratories, Einsteinweg 55, 2333 CC Leiden, The Netherlands.
Abstract:
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 beta-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.
Related Concept Videos
[4+2] Cycloaddition of Conjugated Dienes: Diels–Alder Reaction
Diels–Alder Reaction Forming Cyclic Products: Stereochemistry
Diels–Alder Reaction: Characteristics of Dienes
Characteristics of the diene
Conformation
The simplest example of a diene is 1,3-butadiene, an acyclic conjugated π system. At room temperature, the molecule exists as a mixture of s-cis and s-trans conformers by virtue of rotation around the carbon–carbon single bond. Although the s-trans isomer is more stable, the...
Diels–Alder Reaction Forming Bridged Bicyclic Products: Stereochemistry
Labeling DNA Probes
Radioisotopes, fluorophores, or small molecule binding partners like biotin or digoxigenin, are the most widely used reporter tags for labeling DNA probes. These labels can be attached to the probe DNA molecule via...
Diels–Alder Reaction: Characteristics of Dienophiles
Characteristics of Dienophiles
Generally, the best dienophiles are alkenes containing electron-withdrawing substituents such as carbonyl, nitrile, and nitro groups. The feasibility of a Diels–Alder reaction depends on...

