Enantioselective synthesis of a novel trans double bond ceramide analogue via catalytic asymmetric dihydroxylation of an enyne. The role of the transdouble bond of ceramide in the fusion of semliki forest virus with target membranes

Intensive interest is currently focused on the role of ceramide (1), a key lipid molecule that functions as a second messenger. The first asymmetric s ynthesis of a D-erythro-ceramide analogue that contains a C(5)-C(6) trans d ouble bond (3), together with its biological evaluation in a viral-liposome fusion system, is described. Sharpless asymmetric dihydroxylation of 4'-me thoxyphenyl trans-5-octadecyn-2-enyl ether (enyne 8a), prepared by the coup ling reaction of 1-[(E)-(4'-bromo-2'-butenyl)oxy]-4-methoxybenzene (7) with Lithium tetradecyne, generated yne-diol 9 in 96% yield with the desired st ereochemistry at the C(2)and (3) positions and high enantiomeric purity (95 % enantiomeric excess). Birch reduction (Li/EtNH2) of yne-diol 9 furnished (2R,3R,5E)-octadecene-1,2,3-triol (10) stereospecifically. The latter was c onverted to 2 azido derivative 13 in three steps (via a 2-O-triflate-1,3-O- benzylidene intermediate) and 55% overall yield. Reduction of azide 13 and in situ N-acylation with p-nitrophenyl cis-hexadec-4-enoate provided D-eryt hro-Delta(5)-trans-ceramide (3) in 91% yield. The role of the trans double bond of ceramide in mediating fusion of an alphavirus (Semliki Forest virus ) was assessed in a liposomal model system, using target phospholipid/chole sterol vesicles containing either D-erythro-ceramide 1 or 3. The kinetics o f virus fusion, as monitored by a change in pyrene excimer fluorescence ove r a period of 60 s, showed that Delta(5)-trans-ceramide 3 was completely in active, indicating that there is an absolute requirement for the trans doub le bond to be located between C(4) and C(5). These data indicate that the m olecular determinants on the viral envelope glycoprotein are highly specifi c for recognition of the unsaturated site in the ceramide molecule.