Quantitative multivalent carbohydrate-protein interactions from novel glycodendrimers.

Abstract

The syntheses and {\it in vitro\/} biological evaluation of three series of glycodendrimers is presented herein. This dissertation marks three ``firsts'' in the field of glycobiology. Chemically well defined hypervalent neoglycoconjugates, termed glycodendrimers, have been designed and prepared. These concepts were extended to include the enzymatic elongation of a glycoside on a dendritic core. Furthermore, the first direct correlation of the number of carbohydrate residues and the multivalent effect has been reported. To further the understanding of multivalency and its role in carbohydrate-protein interactions, glycoconjugates with differing carbohydrate densities, conformations, and interglycosidic spacings must be prepared. The design and synthesis of glycodendrimers addresses these issues. Solid phase synthesis on Wang resin was used to construct dendritic $\alpha$-thiosialosides. The design of these new, hyperbranched clusters was based on the rational scaffolding of L-lysine core structures using established 9-fluorenylmethoxycarbonyl (Fmoc) protecting group and activated benzotriazolyl ester (HOBt) coupling procedures. Chain extension of the lysyl amino groups with chloroacetylglycylglycine active ester allowed introduction of the required functionality necessary for the coupling to an $\alpha$-thiosialoside derivative prepared under phase transfer catalysed conditions. Well defined di-, tetra-, octa-, and hexadeca-valent dendritic $\alpha$-thiosialosides were thus prepared. The synthesis of structurally similar divergent and spherical dendrimers, with even valencies of between two and sixteen and ending with equidistant $\alpha$-thiosialoside residues is also described. The synthesis of the dendritic core was based on the regioselective protection of the primary amines of 3,3$\sp\prime$-iminobis(propylamine) using benzylcyanoformate. The resulting secondary amine was monoalkylated with {\it tert\/}-butyl bromoacetate to provide a divalent core structure orthogonally protected with carbobenzyloxy (Cbz) protected amines and a {\it tert\/}-butyl ester. Selective deprotection {\it via\/} hydrogenation or trifluoroacetolysis afforded amine and acid key precursors, respectively. These were conjugated using standard HOBt/DIC strategy to give divergent, Cbz-protected dendrimers with valencies between two and sixteen in the first through fourth generations. Tethering of the dimer and tetramer to both hexamethylenediamine and tris-(2-aminoethyl)amine provided spherical, hyperbranched dendritic structures with valencies between four and twelve. All Cbz-protected dendrimers were transformed into N-chloroacetylated dendrimers which were coupled to a thiolated sialic acid derivative. Thiourelene {\it p\/}-phenyl $\alpha$-thiosialoside containing dendrimers scaffolded on Starburst$\sp\circler$ PAMAM dendrimers were prepared using an isothiocyanate conjugation strategy. PAMAM cores, generation G0 to G3, were coupled to a sialic acid isothiocyanate derivative to give spherical, hypervalent thiourea derivatives containing four to thirty-two surface sialic acid residues. The above concepts were easily extended to include the preparation of dendrimers with covalently attached N-acetylglucosamine, lactose, N-acetyllactosamine, mannose, Gal$\beta$-(1,3)-GalNAc$\alpha$ (T-antigen), and 3$\sp\prime$-sulfo-Lewis$\sp{\rm X}$-(Glc) residues. Furthermore, glycodendrimers were shown to be amenable to both chemical and enzymatic transformations {\it via\/} the 9-O-acetylation of octavalent $\alpha$-thiosialodendrimers and the enzymatic galactosylation of dendritic N-acetylglucosamine. The preparation of a custom designed heterobifunctional dendrimer containing biotin and four sialic acid residues is also presented. The synthesis was based on the tethering of a tetravalent, Cbz-protected, 3,3$\sp\prime$-iminobis(propylamine)-based core to an amine functionalized biotin derivative using established HOBt/DIC coupling chemistry. Cbz deprotection {\it via\/} hydrogenation afforded the biotin-containing tetraamine which was conjugated to a sialic acid {\it p\/}-isothiocyanatophenyl derivative to provide the desired bi-directional glycodendrimer. Binding studies {\it via\/} double immunodiffusion and/or turbidimetric analysis confirmed the ability of the glycodendrimers to cross-link and precipitate appropriate model lectins. In addition, when used in competitive enzyme linked lectin assays, all glycodendrimers showed improved inhibitory potentials.