Synthetic methodlogy is described, aiding in the synthetic preparation of putative inhibitors of retaining and inverting glycosidases and glycosyl transferases. All constructs are cyclophellitol... Show moreSynthetic methodlogy is described, aiding in the synthetic preparation of putative inhibitors of retaining and inverting glycosidases and glycosyl transferases. All constructs are cyclophellitol-based cyclitols. Show less
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
Beenakker, T.J.M.; Wander, D.P.A.; Codée, J.D.C.; Aerts, J.M.F.G.; Marel, G.A. van der; Overkleeft, H.S. 2017
Cyclophellitol and cyclophellitol aziridine are potent and irreversible inhibitors of retaining β‐glucosidases. They preferentially adopt a 4H3 half‐chair conformation, thereby mimicking the... Show moreCyclophellitol and cyclophellitol aziridine are potent and irreversible inhibitors of retaining β‐glucosidases. They preferentially adopt a 4H3 half‐chair conformation, thereby mimicking the substrate‐transition‐state conformation characteristic of retaining β‐glucosidases. As a consequence, both compounds bind tightly to the enzyme active site, and attack of the catalytic nucleophile onto the epoxide/aziridine results in enzyme deactivation. Replacement of the epoxide oxygen in cyclophellitol by a (substituted) carbon yielded carba‐cyclophellitols, a conceptually new class of inhibitors of retaining β‐glucosidases, as we demonstrated in a recent communication. In this paper, in‐depth synthetic studies of this class of compounds are described, and the preparation of a comprehensive set of structurally and configurationally new carba‐cyclophellitols is presented. 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
Glycosidases are a large, evolutionary conserved enzyme family that catalyze the hydrolysis of glycosidic linkages of glycopolymers. These enzymes are involved in many (patho)physiological... Show moreGlycosidases are a large, evolutionary conserved enzyme family that catalyze the hydrolysis of glycosidic linkages of glycopolymers. These enzymes are involved in many (patho)physiological processes and they are applied in the glycopolymer assembly and biotechnological industry. The research described in this Thesis aims to develop inhibitors (mechanism-based and competitive) and activity-based probes for glycosidases using the naturally occurring molecule cyclophellitol as template. Different cyclophellitol derivatives have been synthesized and they could be used to study the role of glycosidases in different (patho)physiological and biotechnological processes. Show less
Glycoconjugates (a carbohydrate connected to a lipid, protein or other carbohydrate) play a key role in great variety of biological processes. The synthesis of these constructs is tightly regulated... Show moreGlycoconjugates (a carbohydrate connected to a lipid, protein or other carbohydrate) play a key role in great variety of biological processes. The synthesis of these constructs is tightly regulated by enzymes. Defects in these enzymes may result in an impaired degradation of the glycoconjugate. Consequently, the levels of glycoconjugates are increased and this may eventually lead to storage disorders such as Gaucher disease. The research described in this thesis focuses on the synthesis of chemical tools (activity-based probes, ABPs) to study the enzymes involved in the degradation of glycoconjugates. Using these probes, it was demonstrated that the activity of peptide N-glycanase (the enzyme that is responsible for the hydrolysis of N-linked glycoproteins)inhibitors is determined by its reactive group. Furthermore, the activity-based protein profiling strategy was used to study degradation of glycosy lated proteins. It appeared that deglycosylation of O-GlcNAclated proteins is not a prerequisite for proteasomal degradation. To study beta-glucosidases (enzymes that catalyze the hydrolysis of O-glycosidic linkages), ABPs based on cyclophellitol have been developed. Especially fluorescently labeled probes bind efficiently and selectively to beta-glucosidases. These probes have been used to investigate Gaucher disease. Both wild-type and mutant forms of the enzyme could be labeled in vitro and in living cells which allowed rapid identification activity of this glucosidase. Show less
