CHEMICAL-ENZYMATIC SYNTHESIS AND CONFORMATIONAL-ANALYSIS OF SIALYL LEWIS-X AND DERIVATIVES

Sialyl Lewis x and derivatives have been synthesized using beta-1,4-galactosyltransferase and recombinant alpha-2,3-sialyltransferase and alpha-1,3-fucosyltransferase. The enzymatic glycosylations have been achieved on preparative scales with in situ regeneration of UDP-galactose, CMP-N-acetylneuraminic acid, and GDP-fucose. Additionally, galactosyltransferase and fucosyltransferases have been studied with respect to their substrate specificity and inhibition. The enzymatic procedures have also been used in the synthesis of 2-deoxy-LacNAc, 2'-amino-2-deoxy-LacNAc, 2-azido-Lac, Lewis x, the Lewis x analog with GlcNAc replaced with 5-thioglucose, [Gal-1-C-13]-LacNAc, [Gal-1-C-13]-sialyl Lewis x, and the corresponding terminal glycal. The synthesized C-13-labeled sialyl Lewis x and intermediates (including Lewis x and sialyl LacNAc) were used for conformational study using NMR techniques combined with calculations based on GESA and MM2 programs. GESA calculation of sialyl Lewis x gave four minimum-energy conformers, and the two (A and B) consistent with NMR results were further refined with MM2 calculation. The one (A') with lower energy was picked as the preferred conformer which had all internuclear distances and glycosidic torsional angles consistent with the NMR analysis. The glycosidic torsional angle psi of Gal-GlcNAc, for example, was determined to be 18-degrees on the basis of the coupling between Gal-1-C-13 and GlcNAc, while the predicted value was 15-degrees. The tetrasaccharide appears to form a well-defined hydrophilic surface along NeuAc-Gal-Fuc, and a hydrophobic face underneath NeuAc-GlcNAc. Comparing the conformation of sialyl Lewis x to sialyl Lewis a indicates that the recognition domain of sialyl Lewis x mainly comes from the sialic acid-galactose-fucose residues.