This thesis describes the use of an activity-based proteomics method to study the endocannabinoid system. A protocol for label-free chemical proteomics to measure serine hydrolase activity in mouse... Show moreThis thesis describes the use of an activity-based proteomics method to study the endocannabinoid system. A protocol for label-free chemical proteomics to measure serine hydrolase activity in mouse tissue is described. This method is used to compare a Niemann-Pick Type C mouse model to healthy mice. Additionally, several novel activity-based probes for the enzymes diacylglycerol lipase alpha and a/b-hydrolase domain containing protein 6 are described. Show less
To this day, all cyclophellitol-based inhibitors and ABPs have been close analogues of their natural substrate counterparts. As a result, these probes showed high selectivity towards their... Show moreTo this day, all cyclophellitol-based inhibitors and ABPs have been close analogues of their natural substrate counterparts. As a result, these probes showed high selectivity towards their target glycosidases. While such probes are of high value for studying these specific enzyme classes, they impede the simultaneous profiling of glycosidases that process different substrate configurations with a single probe. This Thesis focusses on structural derivatization of cyclophellitol-based probes with the aim of enabling inter-class labelling of glycosidases. The synthesis of cyclophellitol-based inhibitors and probes is described, which either lack the hydroxymethylene functionality at C2, C4 and C5, or are furanose-based cyclitol aziridines. Their labelling is biochemically evaluated on complex biological samples. The synthesis of a novel reversible inhibitor, gluco-1H-imidazole, is described in Chapter 5. In the last Chapters of this Thesis, synthetic methodologies for the construction of endo-glycosidase probes are described. Chapter 6 reports on the introduction of a spiro-epoxide warhead onto a disaccharide moiety for the purpose of constructing probes for GH99 endo-α-mannosidases, while the construction of xylobiose-cyclophellitol probes from xylo-cyclophellitol acceptors via direct glycosylation is described in Chapter 7. Finally, Chapter 8 provides a summary of this Thesis, followed by future prospects. Show less
