New Approach for Characterization of Lysosulfatide by TLC, Fast Atom Bombardment Mass Spectrometry and NMR Spectroscopy

Abstract

Thin layer chromatography of lysosulfatide showed anomalous R_f- values in contrast with such lysosphingolipids as glucopsychosine and galactopsychosine with neutral, acidic, and alkaline developing solvents. This was thought to be due to the presence of oppositely charged sulfate and amino groups in the lysosulfatide. In the negative mode of fast atom bombardment mass spectrometry, the lysosulfatide showed the pseudo molecular ion (M—H)˜ peak at m/z 540 and sulfate ion peak at m/z 97, whereas in the positive mode, it showed not only the pseudo molecular ion (M + H)⁺ peak at m/z 542, but also the major peaks of protonated psychosine at m/z 462 and fragment ions of dehydrated sphingosine at m/z 282 and 264.13C-NMR signals of all carbons of lysosulfatide were determined by using distortionless enhancement by polarization transfer. The difference in chemical shifts of ring carbons of galactose residue between lysosulfatide and galactopsychosine was largest at C-3 (downfield shift), thereby indicating the location of the sulfate group to be at C-3 of galactose. This conclusion is supported by the ¹H-NMR spectra of the lysosulfatide and galactopsychosine. Thus, the chemical structure of lysosulfatide was confirmed by fast atom bombardment mass spectrometry and ¹³C- and ¹H-NMR spectroscopy. Furthermore, ¹³C-NMR signals of C-l to C-5 of the sphingosine moiety showed significantly different chemical shifts between the lysosulfatide and galactopsychosine. These differences suggested that C-l to C-5 of sphingosine might be influenced by intramolecular or inter-molecular interaction between the sulfate group of the galactose residue and the amino group of sphingosine. In particular, the order of the absolute value of difference in chemical shift (| Δδ |) being C-l > C-3> C-5 in the sphingosine between lysosulfatide and galactopsychosine suggested that the sulfate group might influence the carbons at C-l, -3, and -5, inducing a zigzag conformation of sphingosine in lysosulfatide. Also, the downfield shift of protons at C-l, -3, and -5 of sphingosine in lysosulfatide relative to sulfatide indicated that the removal of fatty acid from sulfatide might similarly induce a zigzag conformation of sphingosine in lysosulfatide. The interaction between the sulfate and amino groups was also suggested by the ¹H-NMR spectra of the lysosulfatide and galactopsychosine, because the former showed no amino proton signal.