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Two‐Step Labeling of Endogenous Enzymatic Activities by Diels–Alder Ligation
Author(s) -
Willems Lianne I.,
Verdoes Martijn,
Florea Bogdan I.,
van der Marel Gijsbert A.,
Overkleeft Herman S.
Publication year - 2010
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201000280
Subject(s) - proteases , cathepsin , chemistry , cysteine , enzyme , cycloaddition , biochemistry , chemical ligation , native chemical ligation , combinatorial chemistry , peptide , catalysis
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.