| Summary: | β-1,4-Galactosyltransferases (β-1,4-GalTs) catalyse the transfer of D-galactose from a uridine diphosphate galactose (UDP-Gal) donor to an N-acetylglucosamine (N-GlcNAc) or glucose (Glc) acceptor, forming a β-1,4-glycosidic linkage. β-1,4-GalTs are required for the formation of important glycan epitopes, such as terminal tetrasaccharide Sialyl Lewis X (sLex), which is present in P-selectin glycoprotein ligand 1 (PSGL-1) and other cell adhesion molecules. Therefore, small molecular β-1,4-GalT inhibitors are of great interest as chemical tool compounds to study sLex- and PSGL-1-dependent processes. A UDP-Gal derivative, 5-(5-formylthien-2-yl) UDP-galactose (5-FT UDP-Gal), has previously been described as a potent, broad-spectrum GalT inhibitor; however, the application of 5-FT UDP-Gal in cell assays is compromised by its limited stability and membrane permeability, due to the presence of pyrophosphate and sugar moieties. Therefore, the main aim of this thesis was to develop uncharged β-1,4-GalT inhibitors based on 5-FT UDP-Gal, but with more suitable properties for cellular applications. Several approaches were explored to achieve this goal. In chapter 2, attempts to apply the pro-drug concept using phosphate esters of 5-FT UDP-Gal are described. A series of 5-substituted nucleoside derivatives derived from 5-FT UDP-Gal was also prepared. The inhibitory activities of these derivatives against β-1,4-GalT were assessed in biochemical assays. Direct comparison with the corresponding complete UDP-sugar derivatives allowed the identification of structural factors that determine activity. The effects of the most active nucleoside derivative and its ester prodrug were also investigated in a PSGL-1 expression assay. Attempts to overcome the relative loss of activity from the absence of pyrophosphate and sugar moieties in nucleoside inhibitors using dynamic combinatorial chemistry are described in Chapter 3. A hydrazone dynamic combinatorial library (DCL) was generated from the most potent nucleoside fragment and a series of hydrazides to identify mimics of pyrophosphate and sugar moieties to develop potent inhibitors. A suitable hydrazide was identified from the library and the corresponding nucleoside derivatives were generated and evaluated in the biochemical assay as well as the PSGL-1 expression assay. A known, substrate-based -1,4-GalT inhibitor was prepared as a positive control in the DCL experiments. However, this N-GlcNAc derivative unexpectedly behaved as an acceptor substrate rather than an inhibitor in our phosphatase-coupled assay. These unexpected finding, including attempts to rationalise the discrepancy between these results and reported in previous literature describing this compound as a β-1,4-GalT inhibitor, are described in Chapter 4.
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