Two Antipodal Click Ceramides Labeled at the Acyl or Sphingosine Chain: Synthesis and Evaluation of Structure by NMR...

Click sphingolipids and their photoactivatable versions have been used for about 10 years to monitor the behaviour of related natural molecules, in particular ceramides, in the study of intracellular and intercellular processes. We have previously synthesized click ceramides to study the catalytic specificity of certain enzymes, to detect transport proteins, and in photocontrolled sphingolipid biosynthesis. One click-ceramid (caCer) was made from artificial click-fatty acid and natural sphingosine. The antipodal click-ceramid (csCer) was made from natural palmitic acid and artificial acetylene-terminated sphingosine, obtained by a cross metathesis. Since the yield of these two ceramide analogues with terminal acetylene unit in the initial schemes was low and limited their availability, we developed new synthetic routes and increased the efficiency of caCer synthesis by 3.6-fold and csCer synthesis by 2.8-fold. We found that acylation of sphingosine was not limited to the formation of a monoacylation product, corresponding to ceramide (N-acylation), but continues with acylation of the primary OH group to form a diacylation product, corresponding to 1-O-acylceramide. ¹H nuclear magnetic resonance (NMR) spectroscopy was successfully used to elucidate the structure of sphingosines with different degrees of acylation, whereas ¹³C NMR spectroscopy has proven to be low informative.