Enzymatic synthesis of omega-amino-ceramide: preparation of a sensitive fluorescent substrate for ceramidase.

Sphingolipid ceramide N-deacylase catalyzes the reversible reactions in which the N-acyl linkage of ceramides of various sphingolipids is hydrolyzed or synthesized under different conditions. We report here a new method for preparation of ceramide containing omega-amino-fatty acid by using the condensation reaction of the enzyme. omega-Aminododecanoic acids were efficiently condensed by the enzyme to sphingosine in 25 mM glycine-NaOH buffer, pH 10, containing 0.3% Triton X-100 when the amino residue at the omega position of the fatty acid was blocked with trifluoroacetate. The reaction product was purified sequentially from the reaction mixture on a C18 reversed-phase column and Sep-Pak Plus Silica and Sep-Pak QMA cartridges with an overall yield of 80% and determined to be omega-aminododecanoylsphingosine by thin-layer chromatography and fast atom bombardment-mass spectrometry analyses after removing the block of trifluoroacetate by alkaline treatment. The enzyme can also be applied successfully to the synthesis of various glycosphingolipids and sphingomyelin containing omega-aminododecanoic acids. The 7-nitrobenz-2-oxa-1,3-diazole (NBD)-labeled N-dodecanoylsphingosine was easily prepared from the omega-amino-ceramide by coupling with NBD-fluoride. This fluorescent ceramide was found to be hydrolyzed by ceramidase of B16 melanoma cells much faster than NBD-labeled N-hexanoylsphingosine in vitro as well as in vivo, indicating that the former is an excellent substrate for the assay of ceramidase.