SYNTHESIS OF A DIVALENT SIALYL-LEWIS-X O-GLYCAN, A POTENT INHIBITOR OF LYMPHOCYTE-ENDOTHELIUM ADHESION - EVIDENCE THAT MULTIVALENCY ENHANCES THE SACCHARIDE BINDING TO L-SELECTIN

The recognition of cell-surface L-selectin by its carbohydrate ligands causes lymphocytes to roll on capillary endothelium at sites of infla mmation, As this primary contact is a prerequisite for extravasation o f the leukocytes to the tissue, its inhibition by free oligosaccharide s capable of competing with the natural L-selectin ligands is an attra ctive therapeutic possibility. The exact structures of the biological ligands of L-selectin are not vet known, but the principal carbohydrat e epitopes share some structural features: they are O-glycosidically l inked mucin-type oligosaccharides with N-acetyllactosamine backbone, w hich is 3'-sialylated or 3'-sulfated, 3-fucosylated and sometimes 6- o r 6'-sulfated at the distal N-acetyllactosamine termini. Multivalency of the ligand, which is believed to enhance the binding, is achieved b y a branched polylactosamine backbone ol by a clustered array of O-gly cans. We report here enzymic synthesis of a large oligosaccharide fulf illing several of the features characteristic to the L-selectin ligand s: it is a dodecameric O-glycosidic core-2-type oligosaccharide aldito l with a branched polylactosamine backbone carrying two distal alpha-2 ,3'-sialylated and alpha-1,3-fucosylated N-acetyllactosamine groups (s ialyl Lewis x, sialyl Le(x)). The structure of each saccharide on the synthesis route from disaccharide Gal beta 1-3GalNAc to the dodecasacc haride alditol was established by several methods including one- and t wo-dimensional H-1-NMR spectroscopy. The last step of. the synthesis, the alpha-1,3-fucosylation of the tr-linked arm proceeded sluggishly, and was associated with a noticeable shift in H1 resonance of the GlcN Ac residue of the branch-bearing N-acrtyllactosamine unit. The final s ynthesis product and its analogs lacking one or both of the fucose res idues were tested as inhibitors of L-selectin-mediated lymphocyte-endo thelium interaction in vitro in rejecting rat kidney transplant, While the non-fucosylated O-glycosidic oligosaccharide alditol did not poss ess any inhibitory activity, the mono-fucosylated one (i.e. monovalent sialyl Le(x)) prevented the binding significantly and the difucosylat ed dodecasaccharide alditol (i,e, divalent sialyl Le(x)) was a very po tent inhibitor (IC50, inhibitory concentration preventing 50% of bindi ng = 0.15 mu M) Besides the multivalency also the Gal beta 1-3GalNAc-o l sequence of the O-glycosidic core appeared to increase the affinity of the glycan to L-selectin, This was indicated by parallel inhibition experiments, where a disialylated and difucosylated branched polylact osamine decasaccharide, similar to the divalent dodecasaccharide aldit ol, but lacking the reduced O-glycosidic core, was a less effective in hibitor (IC50 = 0.5 mu M) than the O-glycosidic dodecasaccharide aldit ol.