The exploitation of the unusual hydrophobic properties of diamondoid derivatives has been the primary goal of the research described in this Thesis. 1-Adamantaneacetic acid was used as a protective... Show moreThe exploitation of the unusual hydrophobic properties of diamondoid derivatives has been the primary goal of the research described in this Thesis. 1-Adamantaneacetic acid was used as a protective group in the solution-phase synthesis of (phosphorothioate) oligonucleotides. Pioneered by de Koning et al. a Solution-Phase Extraction Method (SPEM) amendable to scale-up was developed for the preparation of DNA and phosphorthioate oligonucleotide fragments. This approach utilizes extractive work-up procedures as the only tool in the isolation of oligonucleotides. Next, the design and synthesis of diamoniod decorated iminosugars as potential inhibitors of glycoside proccesing enzymes are described. Several libraries of natural iminosugars such as deoxynojirimycin and castanospermine decorated with an N-pentyloxy spacer were prepared. These iminosugar derivatives were evaluated as inhibitors of the enzymes involved in glucosylcramide metabolism, namely glucosylceramide synthase, beta-glucocerebrosidase and beta-glucosidase. Show less
The ongoing research in the field of lysosomal storage diseases (LSD), and more specific Gaucher disease is the basis for the research described in this thesis. The progress of Gaucher disease and... Show moreThe ongoing research in the field of lysosomal storage diseases (LSD), and more specific Gaucher disease is the basis for the research described in this thesis. The progress of Gaucher disease and the effect of therapeutic intervention is correlated to the level of chitotriosidase (CHIT1), the first identified human chitinase. Mea- surement of plasma CHIT1 activity in man is done by an assays using a fluorogenic substrate. The ability of CHIT1 to transglycosylate can complicate the enzyme assay, however umbelliferone 4__-deoxychitobioside is not prone to be transglycosylated. And gives a proportional fluorophore to active enzyme ratio read-out. Because of this umbelliferone 4__-deoxychitobioside has become a popular fluorogenic substrate for the measurement of human chitinases, an improved scalable route towards this compound is described in Chapter 2. Chapter 3 describes the synthesis and biological evaluation of three novel fluorogenic substrates, containing substituents of different sizes on the 4__-OH of the non-reducing sugar. The locally elevated activity of CHIT1 allows sitespecific drug delivery via the prodrug approach. Chapter 4 describes the design and synthesis of novel prodrugs in which a chitobiose core, the substrate for CHIT1, is coupled to known inhibitors of GCS which are able to restore the influx/efflux balance of GC in Gaucher cells. It is known that some iminosugars and N-alkylated derivatives thereof have a taste bitter. In Chapter 5 attempts are made to palliated this bitter taste by appending a galactosyl moiety to DNJ. Aside from potentially masking the bitter taste this modification will also help to direct the inhibitors to the colon were they will be processed by lactase. Cholesteryl-_-glucoside and cholesteryl-_ -glucoside, the synthesis of which is described in Chapter 6, will be used as as internal standards to get a better insight in the biosynthesis of the potentially neurotoxic steryl-glucosides, which are potentially linked to a high level of glycosylceramide. Chapter 7 summarizes the research described in chapters 2 to 6 and future prospects based on these results are presented. 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
