NMR AND CONFORMATIONAL-ANALYSIS OF GANGLIOSIDE-GD1A

The hexasaccharide fragment GD1aOS of the glycolipid ganglioside GD1a [alpha-DNeuAc(2-3)beta-DGal(1-3)beta-DGalNAc(1-4){alpha-DNeuAc(2-3)}beta-DGal(1-4)beta-DGlc-O-ceramide] was released by ozonolysis followed by mild base treatment and isolated in pure form by ion-exchange chromatography. The compound was analyzed extensively by 500- and 600-MHz NMR spectroscopy. One-dimensional TOCSY and 1D NOESY experiments using Gaussian-shaped pulses as well as 2D NOESY and 2D ROESY measurements were carried out to obtain informations for primary and secondary structural characterizations. These techniques combined with C-13-H-1 correlation measurements were essential for the complete assignment of hydrogen and carbon chemical shifts. Proton and carbon spin-lattice relaxation time measurements were carried out to probe the dynamic behavior of the hexasaccharide. The interresidual hydrogen distances calculated from the 1D NOESY values were found to be in good accord with the lowest energy model suggested by the HSEA calculations, including those for the glycosidic linkages containing the internal sialic acid residue. Two-dimensional NOESY and ROESY have also been carried out on GD1aOS and the interresidual hydrogen distances calculated from these two methods are remarkably similar. The lowest energy conformer of GD1aOS shows that the internal, branched sialic acid is stacked underneath the GalNAc residue, establishing a hydrogen bond between the carboxylate group of the sialic acid and the acetamido group of the GalNAc unit. Both sialic acid residues are oriented almost perpendicular to the plane of the neutral tetrasaccharide core as the CPK models indicate.