ENZYMATIC-SYNTHESIS OF OCTADECAMERIC SACCHARIDES OF MULTIPLY BRANCHEDBLOOD-GROUP I-TYPE, CARRYING 4 DISTAL ALPHA-1,3-GALACTOSE OR BETA-1,3-GLCNAC RESIDUES

Radiolabeled oligosaccharide constructs were prepared to evaluate carb ohydrate determinants involved in gamete adhesion in mice. The octasac charide primer GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3(GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-6)Gal beta 1-4GlcNAc (1) was incubated wi th UDP-GlcNAc and beta 1,6-GlcNAc-transferase of hog gastric microsome s, producing the tetraantennary decasaccharide GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc beta 1-3[GlcNAc beta 1-3(GlcNAc beta 1-6)G al beta 1-4GlcNAc beta 1-6]Gal beta 1-4GlcNAc (2). The decasaccharide was then incubated with UDP-Gal and beta 1,4-galactosyltransferase fro m bovine milk, yielding the tetradecasaccharide Gal beta 1-4GlcNAc bet a 1-3(Gal beta 1-4GlcNAc beta 1-6)Gal beta 1-4GlcNAc beta 1-3[Gal beta 1-4GlcNAc beta 1-3(Gal beta 1-4GlcNAc beta 1-6)Gal beta 1-4GlcNAc bet a 1-6]Gal beta 1-4GlcNAc (3). Incubation of the tetradecasaccharide 3 with UDP-Gal and alpha 1,3-galactosyltransferase from bovine thymus ga ve the octadecameric glycan Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3(G al alpha 1-3Gal beta 1-4GlcNAc beta 1-6)Gal beta 1-4GlcNAc beta 1-3[Ga l alpha 1-3Gal beta 1-4GlcNAc beta 1-3(Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-6)Gal beta 1-4GlcNAc beta 1-6]Gal beta 1-4GlcNAc (4). Incubati on of the tetradecasaccharide 3 with UDP-GlcNAc and beta 1,3-GlcNAc-tr ansferase present in human serum gave the octadecameric saccharide Glc NAc beta 1-3Gal beta 1-4GlcNAc beta 1-3(GlcNAc beta 1-3Gal beta 1-4Glc NAc beta 1-6)Gal beta 1-4GlcNAc beta 1-3[GlcNAc beta 1-3Gal beta 1-4Gl cNAc beta 1-3(GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-6)Gal beta 1-4G lcNAc beta 1-6]Gal beta 1-4GlcNAc (5). From 104 nmol of 1, the octadec amer 4 was obtained in a yield of 53 nmol, enough for characterization by 1D proton NMR, matrix-assisted laser desorption ionization mass sp ectrometry, and degradative experiments. The constructs were studied a s inhibitors of mouse gamete binding in experiments that are reported in detail in the accompanying paper [Litscher, E. S., et al. (1995) Bi ochemistry 34, 4662-4669].