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Direct and two-step activity-based profiling of proteases and glycosidases

Promotores: Prof.dr. H.S. Overkleeft, Prof.dr. G.A. van der Marel

L.I. Willems
24 juni 2014
Thesis in Leiden Repository

Activity-based protein profiling provides a powerful approach for the monitoring of active enzyme populations in complex biological samples by making use of activity-based probes (ABPs), chemical probes that are designed to bind specifically to the active site of an enzyme (family). The research described in this thesis concerns two main topics. First, new techniques are developed for the two-step labeling of enzymatic activity, a strategy that involves the targeting of enzymes with an ABP followed by introduction of the desired reporter entity via a bioorthogonal ligation reaction. In these approaches, both regular and inverse-electron-demand Diels-Alder ligations are applied. The latter of these proved superior in terms of efficiency and selectivity and enables the labeling of enzymatic activity in living cells. Furthermore, two tandem ligation strategies are presented that allow the simultaneous monitoring of multiple biomolecules in a single experiment. The second topic involves the synthesis and biological evaluation of novel activity-based probes for two classes of glycosidases, the retaining α- and β-galactosidases. A fluorescently labeled α-galactopyranose-configured aziridine is demonstrated to enable the labeling of endogenous levels of human retaining α-galactosidase activity in cell extracts. Finally, a number of epoxide-based β-galactopyranose-configured probes is synthesized that target the human retaining β-galactosidase galactocerebrosidase.