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
Glycosidases (GHs) are enzymes responsible for the degradation of carbohydrates and play many roles in human health and pathophysiology. Often, abnormal levels of glycosidase activity are markedly... Show moreGlycosidases (GHs) are enzymes responsible for the degradation of carbohydrates and play many roles in human health and pathophysiology. Often, abnormal levels of glycosidase activity are markedly linked to human pathologies. Example of this is the overexpression of heparanase (HPSE) in several cancer tissues. To date, the biomedical relevance of HPSE mostly pertains cancer treatment. This dissertation reports on the design, synthesis and biochemical evaluation of covalent mechanism-based inhibitors for heparanase, as well as the development of broad-spectrum activity-based probes (ABPs) for retaining exo- and endo-β-D-glucuronidases alike, including HPSE. The design of the novel inhibitors is supported by computational simulations, and the inhibition and selectivity of the newly synthesised compounds towards HPSE is demonstrated in vitro with the use of the newly developed ABPs. The thesis further builds on the concept of ABP-based profiling of GHs and discusses the development of inhibitors and probes targeting retaining β-L-arabinofuranosidases, a group of non-canonical glycosidases predominantly expressed by microorganisms of the human gut microbiome (HGM). As retaining β-L-arabinofuranosidases possess a contentious enzymatic mechanism, the novel chemical tools developed and presented in this thesis were decisive for unraveling β-L-arabinofuranosidases’ mechanism, and might serve in the future as probing tools for studies of the HGM. Show less
Analytical assay development, particularly pertaining to glycomics, is an exciting amalgam of biology, chemistry and engineering. Besides academic research in natural and medical sciences,... Show moreAnalytical assay development, particularly pertaining to glycomics, is an exciting amalgam of biology, chemistry and engineering. Besides academic research in natural and medical sciences, glycomics assays have immense importance in industrial applications such as in quality control and quality assurance of glycoproteins. An up-coming industrial and clinical application is the high-throughput glycan profiling of clinical samples, such as plasma, for identifying disease associations. These glycomics assays are often based on chromatographic and mass spectrometric instrumentation. Thus, they create a requirement of instrumentation infrastructure as well as technical skills which are both not always readily available. This creates a demand in industry for the development of glycomics assays that have a low infrastructure cost as well as minimal training requirements and that are user-friendly. With these objectives in focus, this thesis develops novel exoglycosidase-based high-throughput glycomics assays for use in industrial glycan profiling. In doing so, this thesis also contributes to the development of potential products, such as glycomics kits. Show less
The studies described in this thesis deal with glycosidases, in particular alpha-galactosidases. Activity-based probes are a versatile research tool in many of the investigations on glycosidases... Show moreThe studies described in this thesis deal with glycosidases, in particular alpha-galactosidases. Activity-based probes are a versatile research tool in many of the investigations on glycosidases and are examined regarding diagnostic application. A specific element of the thesis investigations is the focus on plants, either as source of endogenous glycosidases as well as production platform for therapeutic human enzymes. Show less
Glycosidases are important enzymes in the turnover of polysaccharides and glycoconjugates, and are involved in a range of human pathologies including genetic disorders such as Gaucher and Pompe... Show moreGlycosidases are important enzymes in the turnover of polysaccharides and glycoconjugates, and are involved in a range of human pathologies including genetic disorders such as Gaucher and Pompe disease, but also in various cancers. The discovery of potent and selective glycosidase inhibitors for fundamental glycobiology studies and as leads for drug discovery requires access to suitable (glycomimetic) compound libraries, as well as easily applicable assays and screening formats. The research described in this Thesis was aimed to explore how covalent and irreversible glycosidase inhibitors can be applied in the screening of focused compound libraries on various glycosidases using fluorescence polarization activity-based protein profiling (FluoPol-ABPP). 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
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
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... Show moreActivity-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 _- and _-galactosidases. A fluorescently labeled _-galactopyranose-configured aziridine is demonstrated to enable the labeling of endogenous levels of human retaining _-galactosidase activity in cell extracts. Finally, a number of epoxide-based _-galactopyranose-configured probes is synthesized that target the human retaining _-galactosidase galactocerebrosidase. Show less
