Vibrio cholerae cytolysin (VCC) forms oligomeric pores in lipid bilayers containing cholesterol. Membrane permeabilization is inefficient if the sterol is embedded within bilayers prepared from phosphatidylcholine only but is greatly enhanced if the target membrane also contains ceramide. Although the enhancement of VCC action is stereospecific with respect to cholesterol, we show here that no such specificity applies to the two stereocenters in ceramide; all four stereoisomers of ceramide enhanced VCC activity in cholesterol-containing bilayers. A wide variety of ceramide analogs were as effective as D-erythro-ceramide, as was diacylglycerol, suggesting that the effect of ceramide exemplifies a general trend of lipids with a small headgroup to augment the activity of VCC. Incorporation of these cone-shaped lipids into cholesterol-containing bilayers also gave similar effects with streptolysin O, another cholesterol-specific but structurally unrelated cytolysin. In contrast, the activity of staphylococcal ␣-hemolysin, which does not share with the other toxins the requirement for cholesterol, was far less affected by the presence of lipids with a conical shape. The collective data indicate that sphingolipids and glycerolipids do not interact with the cytolysins specifically. Instead, lipids that have a conical molecular shape appear to effect a change in the energetic state of membrane cholesterol that in turn augments the interaction of the sterol with the cholesterol-specific cytolysins.To bacterial pore-forming cytolysins, cholesterol is a logical choice as a target molecule, because it confers specificity for animal as opposed to bacterial cell membranes. The specificity for cholesterol is shared between Vibrio cholerae cytolysin (VCC) 1 (1) and streptolysin O (SLO) (2). Otherwise, these toxins are not related, and the oligomeric pores they form are very different in size and morphology (1, 3). Although with SLO the sterol is already required in the initial event of membrane binding of the monomeric toxin (4), it only comes into play at the stage of oligomerization in the case of VCC (5, 6). When the sterol is incorporated into phosphatidylcholine (PC) bilayers at physiologically realistic concentrations (i.e. up to 40% by mol), these membranes do not become significantly sensitive to VCC. However, it was previously found that membrane susceptibility toward the cytolysin was greatly enhanced by inclusion of ceramide; free ceramide and monohexosyl ceramides proved similarly effective (7). A combined specificity for cholesterol and sphingolipids has previously been shown for the fusion protein of Semliki Forest virus. In that instance, the interaction with ceramide proved to be highly stereoselective (8 -10). Accordingly, we have examined the structural properties of the ceramide molecule responsible for the sensitization of membranes to VCC. To our surprise, no dependence on stereospecific features of ceramide could be detected. Membrane sensitization was readily achieved with a variety of synthetic ceramides...

A variety of beta-keto phosphonates can be converted to gamma-keto phosphonates through reaction with ethyl(iodomethyl)zinc. The presence of alpha-alkyl substituents, Lewis basic functionality, and modestly acidic NH-protons are accommodated in substrates of this reaction. Chain extension of beta-keto phosphonates that contained olefinic functionality proceeded more quickly than cyclopropanation; however, it was not possible to effect the chain extension to the exclusion of cyclopropane formation. A primary reason for this imperfect chemoselectivity appears to be the slow chain extension of beta-keto phosphonates. Nevertheless, the simplicity, the scope, and efficiency of this method serve to make it an attractive alternative to the established methods for gamma-keto phosphonate formation.

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The identification of two natural products, FR-900848 and U-106305, has stimulated interest concerning the relationship between configurational isomerism, conformational isomerism, and biological activity of polycyclopropanes. Efforts to investigate the relationship between configurational and conformational isomerism through molecular modeling suggest that significantly different three-dimensional structures will result from unique primary structures. Any effort to address these issues demands that stereoselective methods for the preparation of polycyclopropanes be developed. We have investigated the application of zinc-carbenoid cyclopropanation in the presence of chiral dioxaboralanes to the preparation of eight stereochemically unique bicyclopropanes. The trans-vinylcyclopropane starting materials demonstrated very little substrate-induced stereoselectivity, while the cis-vinylcyclopropane demonstrates modest to excellent stereocontrol. A model for the substrate-based stereocontrol is proposed. We also used the spectroscopic data gathered in this investigation to probe the substrate-mediated stereocontrol in the rhodium(II)-catalyzed cyclopropanation of vinylcyclopropanes with ethyl diazoacetate.

The stereoselective preparation of three stereoisomeric tercyclopropanes and a quinquecyclopropane was investigated. Two of the tercyclopropanes were C 2-symmetric and were prepared efficiently through the two-directional application of Charette's reagent-stereocontrolled cyclopropanation methodology. The nonsymmetric tercyclopropane was prepared by an iterative one-directional application of the same reagent-mediated cyclopropanation method. It was shown that the reagent-controlled transformations are far more effective for the stereoselective preparation of the tercyclopropanes than are the reactions which rely upon the influence of the substrate stereocenters. A C 2-symmetric quinquecyclopropane, which possesses the repeating trans-syn stereochemistry, was prepared by iterative application of the two-directional strategy.