Note

Determination of the position of acetic and sulfuric esters in glycosphingolipids by two-dimensional proton-nuclear magnetic resonance spectroscopy

The full assignment of ¹H and ¹³C NMR signals of galactosylceramide 3-sulfate (galactosyl sulfatide) and ¹H signals of galactosylceramide 6-sulfate was achieved by using ¹H–¹H DQF-COSY and ¹H–¹³C heteronuclear COSY. Analyses were performed on a mixture of galactosyl sulfatides with four representative ceramide types consisting of a combination of non-hydroxy or 2-hydroxy fatty acids and sphingenine or 4D-hydroxysphinganine (trihydroxysphinganine) as the long-chain bases. The ¹H and ¹³C NMR parameters of galactosyl sulfatide with 4-hydroxysphinganine as well as ¹³C signals of complex lipids with 4-hydroxysphinganine were elucidated for the first time. Not only sulfation of the galactosyl residue, but also modification of the aglycon, including hydroxylation of fatty acids and hydration of the double bond in sphingoid bases, altered the chemical shifts substantially. In addition, the unique long-range coupling constants, ⁴J_H,H and ⁵J_H,H, in the galactosyl residue of galactosyl sulfatide could be determined.

To determine O-esterified positions, a rapid and complete acetalization to prepare an intermediate was established using ethyl vinyl ether as a new reagent. The new method was applied to O-esterified glycolipids followed by GC–MS analysis of the monosaccharide derivatives after methylation and methanolysis, revealing the derivatives with correctly substituted positions. This method was superior in terms of its shorter reaction time and complete acetalization, particularly of the N-glycolyl hydroxyl residue, to previously reported methods using methyl vinyl ether.

A novel GM3 O-acetylated at C-4 and at C-9 of N-glycolylneuraminic acid (4,9-di-O-Ac GM3), together with a second GM3 O-acetylated at O-4 of the neuraminic acid and O-6 of d-galactose (4,6′-di-O-Ac GM3) were isolated from equine erythrocytes as a mixture in approximate 1:1 ratio. These two major species were chromatographically inseparable. Their structures, especially the positions of the acetoxy group(s), were determined by means of 1D- and 2D-¹H NMR and fast atom bombardment-MS as well as by gas chromatography-MS of partially O-methylated O-trimethylsilylated monosaccharides derived from the di-O-Ac GM3s. In addition, 4-O-Ac GM3 was chemically mono-O-acetylated with trimethyl orthoacetate under acidic conditions, giving exclusively 4,9-di-O-Ac GM3, the NMR and mass spectra of which were used as references to confirm the 4,9-di-O-acetylated structure of the naturally-occurring GM3.

The structure of Tubeimoside I isolated from the bulb of Bolbostemma paniculatum [Maxim] Franquet (Tu-bei-mu) was determined as 1 mainly by comparative NMR studies of 1 and its degration products. The compound 1 has inter-saccharidechain bridging by a dicrotalic acid to from a unique macrocyclic structure.