Ceramides are naturally occurring compounds recognized to mediate apoptosis. N-acylsphingosines, containing a double bond at carbons 4 and 5 of their sphingoid backbone, are thought to be the active form, because N-acylsphinganines with completely saturated sphingoid are inactive. In the present study, we synthesized a series of N-acyl-4D-ribo-phytosphingosines (phytoceramides) that contain a hydroxyl group at carbon 4 and investigated structure-cytotoxicity relationship of the presumed functional groups in ceramides. N-Acetylphytoceramide (PCer2) and N-hexanoylphytoceramide (PCer6) were found to be more cytotoxic than ceramides as determined by released lactate dehydrogenase activity and morphological criteria. This was not caused by intracellular conversion of phytoceramides to ceramides, because no N-hexanoylsphingosine was formed after incubation of cell lysate with PCer6. Among phytoceramides having acyl chains two to eight carbons long, the cytotoxicity was highest with five or six carbons. The carbonyl group of the amide bond did not seem to be critical, because substitution of the oxygen with sulfur did not influence the cytotoxicity. The phytoceramide-induced cell death was observed to be apoptotic in nature with the use of terminal deoxynucleotidyl transferase dUTP nick-end labeling and propidium iodide staining. Because phytoceramides can be readily synthesized from yeast sources, they may present a potential and economical alternative to ceramide in future studies and therapies.
To probe for free radical intermediates in the model methylmalonate to succinate rearrangements promoted by vitamin B12., a model series with a pentenyl side chain radical trap has been devised. The control free radical, generated by tri-n-butyltin hydride treatment of bromomethylpentenylmalonate thioester, undergoes rapid cyclization to the six-membered ring, and, as anticipated, no succinate rearrangement product is detected. By contrast when the bromide is treated with vitamin B12., little cyclized product is observed;
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