title: Two-step labeling of endogenous enzymatic activities by Diels-Alder ligation.
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.
authors.affiliation: Leiden Institute of Chemistry and Netherlands Proteomics Centre, Gorlaeus Laboratories, Einsteinweg 55, 2333 CC Leiden, The Netherlands.
authors.forename: Lianne I
authors.initials: LI
authors.lastname: Willems
authors.forename: Martijn
authors.initials: M
authors.lastname: Verdoes
authors.forename: Bogdan I
authors.initials: BI
authors.lastname: Florea
authors.forename: Gijsbert A
authors.initials: GA
authors.lastname: van der Marel
authors.forename: Herman S
authors.initials: HS
authors.lastname: Overkleeft
mesh_terms: D000818:Animals; D001896:Boron Compounds / Q000378:metabolism*; D002403:Cathepsins / Q000032:analysis; D002460:Cell Line; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D051379:Mice; D015335:Molecular Probes / Q000138:chemical synthesis* / Q000378:metabolism; D010447:Peptide Hydrolases / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*
chemical_list: C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001896:Boron Compounds; D015335:Molecular Probes; D002403:Cathepsins; D010447:Peptide Hydrolases; D046988:Proteasome Endopeptidase Complex