Synthesis and utilization of multivalent sialyl-Lewis X N-linked oligosaccharides in probing selectin valency

This dissertation examined the utility of N-linked oligosaccharides as glycoconjugates to probe and characterize the native oligomeric state of the selectins (an inflammation-induced lectin). This work was segmented into three phases: (i) development of a library of enzymatically modified N-linked oligosaccharides, (ii) defining the in vivo carbohydrate-binding specificity of a well-characterized trivalent liver lectin, the asialoglycoprotein receptor (ASGP-R), and (iii) determine the carbohydrate-binding specificity and localization of the selectins in two animal models of inflammation.; Tyrosinamide-oligosaccharides served as synthons to develop a N-linked oligosaccharide library using a host of glycosidases and glycosyltransferases. Oligosaccharides were purified by RP-HPLC and characterized by proton NMR and MS. Biantennary and triantennary oligosaccharides possessing LewisX (Lex) determinants were used to study the binding specificity of the ASGP-R in mice. Results verified that lectin valency could be determined in vivo by demonstrating Lex triantennary oligosaccharide targeting (66%; % of injected dose in target organ) was a more effective ligand than Lex biantennary (18%) for the trivalent ASGP-R.; Selectin valency was studied using a panel of N-linked oligosaccharides containing a clustered array of one to four sialyl-Lewisx (sLex) determinants. A lipopolysaccharide mouse model of acute inflammation was use to determine E-selectin valency as indicated by the percent targeting of each ligand. E-selectin expression was most prevalent in the kidney. SLex biantennary and triantennary oligosaccharides distributed to the kidney with targeting efficiencies of 20% and 18%, respectively. Conversely, monovalent sLex and tetraantennary sLex oligosaccharides demonstrated no active targeting. These results suggest that E-selectin induced in the kidney is primarily a bivalent receptor.; P-selectin valency was studied in a cobra venom factor (CVF) rat lung injury model. The protective effect afforded by the sLex oligosaccharides was determined by the reduction in lung permeability. The greatest protective effect was seen with sLex tetraantennary oligosaccharide with a 58% reduction in lung permeability, followed by monovalent sLex (47%) and triantennary sLex (36%). Unlike the LPS mouse model, biantennary sLex was ineffective in this model. These results concluded that P-selectin preferentially binds to sLex tetraantennary, indicating a tetravalent arrangement in the CVF model.