Synthesis of multivalent oligosaccharides and their application as substrates for glycosidases

Abstract

Multivalent oligosaccharides often display increased apparent affinities for their protein receptors compared to that of the corresponding monovalent ligands. These increased affinities have been exploited in the design and synthesis of inhibitors of bacterial toxins and selectins, and for blocking the binding of viruses to host cells. However, multivalent carbohydrates will only achieve their potential as therapeutics if they have favorable metabolic stabilities and are not rapidly degraded or modified by carbohydrate processing enzymes. To address these issues this dissertation describes the synthesis of monovalent and multivalent oligosaccharides related to SLeX, followed by an investigation into their susceptibility towards hydrolysis by two different neuraminidases. A tetrasaccharide, á- D-Neu5Ac(2-3)â-D-Gal(1-4)[á-L-Fuc(1-3)]â-D-GlcNAc (43), and a related trisaccharide á-D-Neu5Ac(2-3)â-D-Gal(1-4)â-D-GlcNAc (44), each containing N-acetylneuraminic acid, were chosen as synthetic targets. These oligosaccharides were subsequently linked covalently to both a dendrimeric and a polymeric scaffold, generating two multivalent trisaccharides (113 and 115, respectively) and two multivalent tetrasaccharides (114 and 116, respectively). Preparation of the glycopolymers was achieved by condensation of oligosaccharides 43 and 44, via aminopropyl linkers, to a pre-activated polymer. On the other hand, the glycodendrimers were synthesized by attachment of thioacetylated oligosaccharides 109 and 110 onto a pre-formed dendritic core. To ensure that a direct comparison between the rates of hydrolysis of the monomeric and multimeric substrates could be made, the aminopropyl linkers of the monovalent oligosaccharides, 43 and 44, were acetylated to give 112 and 111. In this way, the linker of all compounds is derivatized as an amide. The multivalent trisaccharides 113 and 115 were subjected to enzymatic hydrolysis by two different neuraminidases, and the apparent kinetic parameters compared with those of the corresponding monovalent counterpart 111. Steady state kinetic studies showed that polyvalent trisaccharide 115 was hydrolyzed with much greater enzymatic efficiency than either the corresponding monovalent 111 or dendrimeric 113 derivatives. The Michaelis constant (Km) was significantly smaller for the polyvalent substrate in comparison to the dendrimeric and monomeric substrates. On the other hand, both the glycopolymer and glycodendrimer substrates displayed virtually identical maximum velocities (Vmax), relative to the monovalent substrate.