The proton nuclear magnetic resonance spectra of gangliosides GM4 (6), GM3 (7), GM2 (8), and GM1 (9) and their asialo derivatives (3–5) have been obtained at 500 MHz and 30 °C in dimethyl-d6 sulfoxide-deuterium oxide (98:2 v/v). Through chemical shift analysis of the upfield (0.6–3.0 ppm), anomeric (4.0–5.0 ppm), and olefinic (5.2–5.6 ppm) regions, well resolved in the one-dimensional spectra, a quantitative determination of NeuAc/GalNAc/Gal/Glc/ceramide ratios is obtained within the series 3–9. Through the use of homonuclear two-dimensional spin-echo J-correlated spectroscopy (2-D-SECSY), the J connectivities of 4–9 are revealed, allowing allocation of all resonances to subspectra. Each subspectrum is assigned to a specific residue through consideration of the number, J-connected pattern, and chemical shifts of its component protons and the magnitude and sequence of their vicinal coupling constants. Thus the monosaccharide composition, anomeric configurations, and aglycon structures of gangliosides and their derivatives may be obtained rapidly and nondestructively through high-field, proton 2-D-SECSY NMR, independent of congener series or other data. Formation of NeuAcα2→3Gal glycosidic linkages causes predictable 0.2–0.6 ppm deshielding of the proton directly attached to the glycosidation site. However, formation of GalNAcβ1→4Gal and Galβ1→3GalNAc glycosidic linkages causes irregular effects on the protons directly involved. The lack of chemical shift additivity in the latter cases indicates that glycosidation shift data can be unreliable in assignment of glycosidic linkage sites. Large, reciprocal long-range glycosidation shifts experienced by resonances of the GalNAc and NeuAc residues of 8 and 9 indicate through-space interactions exist between these residues, as they are disposed in these branched-chain gangliosides.
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