Structural analysis of leech galactocerebrosides using 1D and 2D NMR spectroscopy, gas chromatography–mass spectrometry, and FAB mass spectrometry 1

Abstract

Cerebrosides were isolated from the leech species, Hirudo medicinalis, and purified to homogeneity by silicic acid chromatography, followed by preparative thin-layer chromatography. Their structure was determined by spectroscopic and chemical methods. 1D and 2D 1H NMR spectroscopy, DQF–COSY and HMQC indicated that the head group consists of a single galactose residue in the β configuration. The galacto configuration was determined by the characteristic chemical shift, the spin–spin splitting and the multiplicity of the characteristic resonance of its equatorial H-4 proton, as well as by the splittings of the other ring protons. GC, GC–MS and fast-atom-bombardment mass spectrometry studies indicated that C 24:0 and C 22:0 are the major saturated fatty acid species. Unsaturated fatty acids present were C 25:2, C 27:2, C 27:3, C 28:3, C 29:3, C 30:3, C 33:3. GC–MS indicated the presence of hydroxylated C 27:2 and one other unidentified hydroxylated fatty acid. The cerebroside contained an unusual polyunsaturated sphingosine analogue, namely 2-amino-1,3-dihydroxydocosatriene.