D-erythro-Sphingosine (synthetic)

Use of D-erythro-sphingosine as a pharmacological inhibitor of protein kinase C in human platelets

Sphingosine is a naturally occurring long-chain amino diol with potent inhibitory activity against protein kinase C in vitro and in cell systems. The use of sphingosine as a pharmacological tool to probe the activity of protein kinase C has been hampered by its amphiphilicity, possible contamination of its commercial preparations, and the existence of other targets for its action. To address these problems, high-purity D-erythro-sphingosine was prepared and employed to develop an approach for the use of sphingosine as a pharmacological agent. The addition of synthetic D-erythro-sphingosine to intact human platelets resulted in quick uptake and preferential partitioning into the particulate fraction. It was rapidly metabolized by intact platelets, 60% being degraded within 1 min after addition. Sphingosine was found to be a potent inhibitor of gamma-thrombin-induced aggregation and secretion of washed human platelets. Multiple criteria indicated that this effect is probably mediated through the inhibition of protein kinase C: (1) sphingosine inhibited protein kinase C activity in intact platelets with a similar dose/response to its inhibition of platelet aggregation and secretion; (2) sphingosine inhibited phorbol binding to intact platelets under identical conditions and with a similar dose-dependence; (3) exogenous dioctanoylglycerol overcame sphingosine's inhibition of platelet activation. The effectiveness of sphingosine in inhibiting platelet activation was primarily determined by the ratio of sphingosine to total number of platelets. These data are discussed in relation to a general approach for the use of sphingosine and other parameters for determining biological activities of protein kinase C.

Efficient synthesis of D-erythro-sphingosine and D-erythro-azidosphingosine from D-ribo-phytosphingosine via a cyclic sulfate intermediate

The synthesis of naturally occurring d-erythro-sphingosine and synthetically useful D-erythro-2-azidosphingosine from commercially available d-ribo-phytosphingosine is described. An important feature of this synthesis is the selective transformation of the 3,4-vicinal diol of phytosphingosine into the characteristic E-allylic alcohol of sphingosine via a cyclic sulfate intermediate.

Protein kinase C and platelet inhibition by D-erythro-sphingosine: comparison with N,N-dimethylsphingosine and commercial preparation

Sphingosine has been shown to be a potent and specific inhibitor of protein kinase C in vitro and in cell systems including human platelets. Questions have been raised as to the validity of commercial sphingosine as a protein kinase C inhibitor and whether sphingosine or N,N-dimethylsphingosine is the active species. In the present study, we compared the effects of synthetic D-erythro-sphingosine, N,N-dimethylsphingosine and commercial sphingosine on purified protein kinase C in vitro and washed human platelets. These three compounds were found to be of high purity and well-defined structure based on [1H]NMR, FAB-mass Spectrometry, and TLC analysis. Both synthetic D-erythro-sphingosine and commercial sphingosine inhibited protein kinase C in vitro using vesicle as well as mixed micellar assays. N,N-dimethylsphingosine also significantly inhibited purified protein kinase C in vitro. Both preparations of sphingosine inhibited phosphorylation for 40 kD protein, a known substrate of protein kinase C in platelets. Similarly both sphingosine preparations inhibited aggregation and secretion of human platelets induced by 8 nM gamma-thrombin. These results indicate that sphingosine from commercial source, synthetic sphingosine and N,N-dimethylsphingosine are equipotent in inhibiting protein kinase C. These studies also validate the utility of sphingosine as a phamarcologic inhibitor of protein kinase C in vitro and in cell systems.

New approaches to synthesis of stereospecific sphingomyelin

Enormous progress in the asymmetric synthesis of stereochemically and chemically pure D-erythro-sphingosine and ceramides led to the development of a practical, efficient, easily scaleable process to provide industrial quantities of chiral sphingosine and ceramides. This established a new platform of chiral starting materials which facilitate the synthesis of complex sphingolipids. Utilizing stereochemically homogeneous, fully synthetic ceramides, two efficient synthetic methods were developed for the preparation of ultra pure stereochemically homogeneous sphingomyelins. The first method adapted highly efficient phosphoramidite technology from oligonucleotide chemistry. This method allows selective insertion of a phosphocholine moiety into 3-O-protected ceramide through phosphitylation, followed by choline attachment, phosphite oxidation and deprotection. This route provides stereochemically homogeneous sphingomyelin in 35-79% yield. The second route is based on the reaction of selectively protected ceramides with cyclic chlorophosphate followed by treatment with trimethylamine to give the desired sphingomyelins in 50% yield. Multigram quantities of 14C-labeled N-palmitoyl-D-erythro-sphingomyelin were produced with specific activity > 1000 dpm/nmol.

Divergent synthesis of D-erythro-sphingosine, L-threo-sphingosine, and their regioisomers

Starting from a vinyl epoxide, a divergent synthesis of four sphingosine isomers is described. The remaining four isomers can easily be synthesized using the same methodology. Although numerous syntheses of sphingosine have been published, this is the first general route leading to all eight isomers in this important compound class. The synthetic strategy relies on regioselective opening of a vinyl epoxide and a vinylaziridine in the allylic position.