A series of fluorescent N-acylsphingosines: synthesis, physical properties, and studies in cultured cells

We have previously shown that when cultured fibroblasts are briefly incubated at 2 degrees C with a fluorescent (NBD) analogue of ceramide, N-[N-(7-nitro-2,1,3-benzoxadiazol-4-yl)-epsilon-aminohexanoyl]-D-e rythro- sphingosine, fluorescent labeling of the mitochondria, endoplasmic reticulum, and nuclear envelope occurs. During further incubation at 37 degrees C, the Golgi apparatus and later the plasma membrane become intensely fluorescent. Concomitantly, the fluorescent ceramide is metabolized to fluorescent analogues of sphingomyelin and glucosylceramide [Lipsky, N. G., & Pagano, R. E. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 2608-2612]. In the present study we synthesized fluorescent N-acylsphingosine analogues using various long-chain bases (D-erythro-sphingosine, L-erythro-sphingosine, D-threo-sphingosine, L-threo-sphingosine, D-erythro-dihydrosphingosine, L-threo-dihydrosphingosine, phytosphingosine, and 3-ketosphingosine) and fluorescent fatty acids (epsilon-NBD-aminohexanoic acid; D- or L-alpha-OH-epsilon-NBD-aminohexanoic acid; D- or L-alpha-NBD-aminohexanoic acid). Using previously described resonance energy transfer assays, we examined the rates of spontaneous transfer of these compounds between liposomes and their ability to undergo transbilayer movement. The fluorescent N-acylsphingosine analogues had half-times for spontaneous transfer of 0.3-4.0 min at 25 degrees C, and all were capable of transbilayer movement in lipid vesicles. The metabolism and intracellular distribution of analogues in cultured fibroblasts were also studied. While most of the fluorescent N-acylsphingosines were significantly metabolized to the corresponding sphingomyelin analogues, metabolism to glucosylceramide was strongly dependent on the long-chain base and the stereochemistry of the fluorescent fatty acid moiety.