High-Resolution Proton NMR Studies of Gangliosides. 2. Use of Two-Dimensional Nuclear Overhauser Effect Spectroscopy and Sialylation Shifts for Determination of Oligosaccharide Sequence and Linkage Sites

Homonuclear two-dimensional proton nuclear Overhauser effect (2-D-NOE) spectra have been obtained for asialo-G_M2 (4) and gangliosides G_M2 (8) and G_M1 (9) at 500 MHz and 40 °C in Me₂SO-d₆-D₂O (98:2 v/v). The anomeric protons of each oligosaccharide residue of 4, 8, and 9 are observed to NOE couple via intraresidue 1,3- and 1,5-diaxial interactions and interresidue interactions across the glycosidic linkages. The former couplings are used to confirm the H-3 and H-5 assignments for each residue. From the latter couplings the sequence and glycosidic linkage sites of all oligosaccharide residues, except the sialic acid residues, are revealed. Sialic acid attachment sites are determined for the monosialogangliosides (6–9) through consideration of sialylation-induced glycosidation shifts. Combination of the above sequence and linkage-site data with the monosaccharide composition, anomeric configurations, and characterization of the aglycon (information obtained via two-dimensional spin-echo J-correlated spectroscopy or 2-D-SECSY) allows complete assignment of monosialoganglioside primary structure, independent of other methods of structural analysis. Compared to conventional chemical and enzymatic methods, high-resolution two-dimensional proton nuclear magnetic resonance spectroscopy has the advantages of speed, sensitivity, and sample preservation.