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

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 beta 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 C24:0 and C22:0 are the major saturated fatty acid species. Unsaturated fatty acids present were C25:2, C27:2, C27:3, C28:3, C29:3, C30:3, C33:3. GC-MS indicated the presence of hydroxylated C27:2 and one other unidentified hydroxylated fatty acid. The cerebroside contained an unusual polyunsaturated sphingosine analogue, namely 2-amino-1,3-dihydroxydocsatriene.