Lysosomal glycosidases are acid hydrolases that fragment glycoconjugates in lysosomes. Their inherited deficiency in human is the cause of a number of lysosomal storage disorders (LSDs),... Show moreLysosomal glycosidases are acid hydrolases that fragment glycoconjugates in lysosomes. Their inherited deficiency in human is the cause of a number of lysosomal storage disorders (LSDs), showing characteristic lysosomal accumulation of undegraded glycoconjugates. In the past, activity-based probes (ABPs) based on cyclophellitol or cyclophellitol aziridine scaffold have emerged as powerful tools enabling sensitive quantification of a number of lysosomal glycosidases in extracts of cells and tissue, as well as in intact cells. This thesis describes the characterization of several novel ABP classes targeting α-glucosidase, β-glucuronidase, α-L-iduronidase, α-mannosidase, β-mannosidase, and β-galactosidase, as well as a broad scale of applications for ABPs in LSD research. Novel glucocerebrosidase inhibitors based on the cyclophellitol scaffold are also described, which are brain-permeable, selective, and potently inactivate the enzyme in adult zebrafish. Additionally, a protocol for gel-based and microscopy-based detection of glucocerebrosidase is 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
Glycosidases are essential in fundamental biological processes and are responsible for the degradation of most (oligo)saccharides, glycolipids and glycoproteins. Malfunctioning of glycosidases... Show moreGlycosidases are essential in fundamental biological processes and are responsible for the degradation of most (oligo)saccharides, glycolipids and glycoproteins. Malfunctioning of glycosidases causes various complex pathologies in man, for example, lysosomal storage disorders such as Fabry disease and Krabbe disease. Activity-Based Protein Profiling (ABPP) is a powerful technique to selectively analyze functional proteins in their physiological surroundings. Potent and selective activity-based glycosidase probes (ABPs) could help to understand the pathological processes connected with these enzymes. The first part of this Thesis describes the design, synthesis and application of a set of ABPs for retaining α-galactosidases, β-galactosidases, α-mannosidases and β-mannosidases.Several reversible, competitive glycosidase inhibitors have been developed in the past and are employed for therapeutic applications. The second part of this Thesis focuses on the design of functionalized bicycle [4.1.0] heptanes, carba-cyclophellitols, as new potential competitive inhibitors of glycosidases (α-galactosidases, β-galactosidases, α-glucosidases and β-glucosidases) and glycosyltransferases (both galactosyltransferases and glucosyltransferases). Herein a specific substituted carba-cyclophellitol turned out to be highly potent towards Thermotoga maritima TmGH1 β-glucosidase and therefore conformational strain induced through a cyclopropyl unit may be added to the armory of tight-binding inhibitor designs. Show less
