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
Glycoside hydrolases (glycosidases/GHs) are widely abundant enzymes in all kingdoms of life and are important biocatalysts that catalyze the hydrolysis of glycosidic linkages in oligo... Show moreGlycoside hydrolases (glycosidases/GHs) are widely abundant enzymes in all kingdoms of life and are important biocatalysts that catalyze the hydrolysis of glycosidic linkages in oligo/polysaccharides, glycoproteins and glycolipids with tremendous efficiency. Abnormal glycosidase activity is intimately associated with a variety of human diseases. Overexpression of heparanase, for example, is implicated in almost all cancers examined, and correlates with increased tumor size, tumor angiogenesis, enhanced metastasis and poor prognosis. Specific inhibitors of glycosidases are of great value, not only because they can serve as useful biological tools to study the catalytic machinery, mechanism and itinerary of target enzymes by crystal structure analysis of (covalent) inhibitor-enzyme complexes, but also because they may act as starting points for the development of therapeutic drugs for the treatment of glycosidase-mediated diseases. Additionally, covalent mechanism-based inhibitors have been used as scaffolds for the development of activity-based probes (ABPs) which allow profiling of glycosidases in complex biological systems. The research described in this dissertation focus on the development and biochemical evaluation of covalent inhibitors and ABPs for retaining endo- and exo-glycosidases including starch-degrading enzymes and human lysosomal β-glucocerebrosidase (GBA), as well as the synthesis of a panel of uronic acid-type 1-N-iminosugars as potential competitive heparanase inhibitors. Show less
