Derivatives of amphotericin inhibit infection with human immunodeficiency virus in vitro by different modes of action.

Three water-soluble derivatives of amphotericin B were tested for inhibition of HIV infection in vitro. The compounds amphotericin B methyl ester (AME) and N-(N'-(2-(4'-methylmorpholinio)ethyl)N"-cyclohexyl guanyl) amphotericin B methyl ester (MCG) inhibited HIV infection by 50% at 1 microgram/ml; N-(N'-(3-dimethylaminopropyl)N"-ethyl guanyl) amphotericin B (DAPEG) did so at 5-11 micrograms/ml. While the virus-inhibitory effect of AME was due to an interaction with target lymphocytes, the effect of MCG was due to a direct anti-viral action. AME increased the potential of infected cells to fuse with uninfected cells, but MCG had no significant effect on cell fusion. All compounds had a lower cellular toxicity than amphotericin B and were not toxic at concentrations below 20 micrograms/ml.

Conversion of methylallyl alcohol into C2-methyl-1,2-di-O-hexadecylglycerophosphocholine, a conformationally restricted phospholipid.

The synthesis of C2-methyl-1,2-di-O-hexadecylglycerophosphocholine from methylallyl alcohol is described and the 13C-NMR assignments of all glycerol backbone and headgroup resonances are provided. Bond rotations in the glycerol backbone of this diether phospholipid derivative are impeded by steric hindrance exerted by the methyl group. We discuss the utility of this conformationally restricted C2-alkyl phospholipid analog for study of the influence of conformational changes at the lipid-water interface of bilayers on the interactions between lipids and other membrane components.

Synthesis and biochemical studies of analogs of platelet-activating factor bearing a methyl group at C2 of the glycerol backbone.

Two platelet-activating factor (PAF) analogs containing a methyl group at C2 of the glycerol moiety were synthesized, and some of their biochemical properties were investigated. 1-O-Hexadecyl-2-C,O-dimethyl-rac-glycero-3-phosphocholine (2-methyl-2-methoxy PAF) was prepared in a synthetic scheme beginning with the etherification of 2-methylpropen-1-ol. A reaction sequence involving hydroxylation, tritylation, alkylation, and detritylation afforded 1-O-hexadecyl-2-C,O-dimethyl-rac-glycerol, which was converted into the phosphocholine. A 2-lyso derivative of this PAF analog (2-methyl-lyso PAF) was synthesized from 1-O-hexadecyl-2-C-methyl-3-O-trityl-rac-glycerol. Benzylation followed by detritylation gave 1-O-hexadecyl-2-C-methyl-2-O-benzyl-rac-glycerol, which was converted into the phosphocholine compound. Hydrogenolysis afforded 1-O-hexadecyl-2-C-methyl-rac-glycero-3-phospholine (2-methyl-lyso PAF). The 2-methyl-lyso PAF analog served as a substrate for the acetyl-CoA-dependent acetyltransferase that acetylates 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine. However, 2-methyl-lyso PAF did not have a significant effect on the activities of a CoA-independent transacylase or of the acetylhydrolase that inactivates PAF, and thus does not appear to be a substrate or an inhibitor, respectively, for these enzymes. In addition, this analog exhibited only one-half of the antitumor activity of rac-1-O-alkyl-2-methoxy-rac-glycero-3-phosphocholine in human leukemic (HL-60) cells, and elicited no hypotensive response in rats and no platelet-activating activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Structure and thermotropic properties of hydrated 1-eicosyl-2-dodecyl-rac-glycero-3-phosphocholine and 1-dodecyl-2-eicosyl-rac-glycero-3-phosphocholine bilayer membranes.

The ether-linked phosphatidylcholines 1-eicosyl-2-dodecyl-rac-glycero-3-phosphocholine (EDPC) and 1-dodecyl-2-eicosyl-rac-glycero-3-phosphocholine (DEPC) have been investigated by differential scanning calorimetry (DSC) and X-ray diffraction. DSC of hydrated EDPC shows a single endothermic transition at 34.8 degrees C (delta H = 11.2 kcal/mol) after storage at -4 degrees C while DEPC shows three endothermic transitions at 7.7 and approximately 9.0 degrees C (combined delta H approximately 0.4 kcal/mol) and at 25.2 degrees C (delta H = 4.7 kcal/mol). Both the single transition of EDPC and the two higher temperature transitions of DEPC are reversible, while the approximately 7.7 degrees C transition of DEPC increases in enthalpy on low-temperature incubation. At 23 degrees C, X-ray diffraction of hydrated EDPC shows a sharp reflection at 4.2 A together with lamellar reflections corresponding to a bilayer periodicity, d = 56.2 A. Electron density profiles derived from swelling experiments show a phosphate-phosphate intrabilayer distance, dp-p, of 36 A at all hydrations. This, together with calculated lipid thickness and molecular area considerations, suggests an interdigitated, three chains per head group, bilayer gel phase, L beta*, with no hydrocarbon chain tilt. This is structurally analogous to the bilayer gel phase of hydrated 18:0/10:0 ester PC [McIntosh, T. J., Simon, S. A., Ellington, J. C., Jr., & Porter, N. A. (1984) Biochemistry 23, 4038]. In contrast, DEPC at -4 degrees C shows an L beta' bilayer gel phase with tilted hydrocarbon chains (d = 61.1 A). However, this transforms above 9 degrees C to an interdigitated, triple-chain, L beta* bilayer gel phase (identical with that of EDPC) with d = 56.6 A and a phosphate-phosphate distance of 36 A. Above their respective chain melting transitions, Tm, EDPC and DEPC exhibit liquid-crystalline L alpha bilayer phases with d = 64.5 and 65.0 A at 55 and 45 degrees C, respectively. The ability of both EDPC and DEPC to form triple-chain interdigitated gel-state bilayers suggests that the conformational inequivalence at the sn-1 and sn-2 positions is less pronounced in the ether-linked PCs compared to the ester-linked PCs, where only one of the positional isomers, e.g., 18:0/10:0 PC but not 10:0/18:0 PC, forms the triple-chain structure (J. Mattai, unpublished results). Thus, a different conformation around the glycerol is predicted for ether-linked PC compared to ester-linked PC.

Convenient synthesis of racemic mixed-chain ether glycerophosphocholines from fatty alkyl allyl ethers: useful analogs for biophysical studies.

Diether glycerophosphocholines containing one long and one short chain were synthesized in moderate yield. The synthesis of a diether glycerophosphocholine containing a branched chain and a straight chain is also described. Alkylation of fatty alcohol with allyl bromide and dimethyl sulfoxide anion gave fatty alkyl allyl ether, which was hydroxylated and tritylated. By procedures described previously, 1-O-alkyl-3-O-triphenyl-methylglycerol was alkylated, detritylated, and converted into the phosphocholine product. This method is of general utility for the preparation of a variety of mixed-chain racemic glycerophosphocholines whose physical properties are of interest in studies of biological membranes.

Synthesis of phosphatidylcholine analogs with an alkyl group at C1 or C3 of the glycerol moiety.

The syntheses of 1,2-di-(O-hexadecyl)-rac-glycero-3-phosphocholines containing a methyl group at either C1 or C3 of the glycerol moiety are described. The methyl group was introduced at C1 in a synthetic scheme beginning with the hydroxylation of methyl vinyl ketone. The primary hydroxyl was protected by tritylation and the carbonyl group was reduced with sodium borohydride. Di-O-alkylation with 1-bromohexadecane was accomplished using finely powdered potassium hydroxide in refluxing toluene. Detritylation afforded two diastereomers of 2,3-di-(1-hexadecyloxy)butanol. Reaction with the simple phosphorylating agent, dimethylphosphoryl chloride (Bittman, R., A. F. Rosenthal, and L. A. Vargas. 1984. Chem. Phys. Lipids. 34: 201-205), followed by conversion to the phosphatidic acid and condensation with choline tosylate in the presence of trichloroacetonitrile afforded the diastereomers of 1-methyl-1,2-di-(O-hexadecyl)-rac-glycero-3-phosphocholine. The analog bearing a methyl group at C3 was prepared in a synthetic scheme beginning with the hydroxylation of acrolein dimethyl acetal. After di-O-alkylation with 1-bromohexadecane and sodium hydride in dimethyl sulfoxide/toluene, the acetal was converted to the aldehyde. Reaction with methylmagnesium bromide afforded the diastereomers of 1,2-di-(1-hexadecyloxy)-3-butanol, which were converted to the phosphocholine derivatives. These diether phosphatidylcholine analogs may be useful for investigating the effect of steric bulk at C1 and C3 of the glycerol moiety on the interactions with membrane components.

Kinetics of cholesterol and phospholipid exchange from membranes containing cross-linked proteins or cross-linked phosphatidylethanolamines.

Mono- and dipalmitoylphosphatidylethanolamine derivatives have been synthesized and used to evaluate the role of cross-links between the amino groups of two phospholipid molecules in the rate of cholesterol movement between membranes. Incorporation of the cross-linked phospholipids into small unilamellar vesicles (the donor species) decreased the rate of spontaneous cholesterol exchange with acceptor membranes (small unilamellar vesicles or Mycoplasma gallisepticum cells). These results suggest that the cross-linking of aminophospholipids by reactive intermediates, which may be one of the degenerative transformations associated with peroxidation of unsaturated lipids and cellular aging, can inhibit cholesterol exchangeability in biological membranes. The rates of spontaneous [14C]cholesterol and protein-mediated 14C-labeled phospholipid exchange from diamide-treated mycoplasma and erythrocyte membranes have also been measured. The formation of extensive disulfide bonds in the membrane proteins of M. gallisepticum enhanced the 14C-labeled phospholipid exchange rate but did not affect the rate of [14C]cholesterol exchange. The rates of radiolabeled cholesterol and phospholipid exchange between erythrocyte ghosts and vesicles were both enhanced (but to different extents) when ghosts were treated with diamide. These observations suggest that diamide-induced oxidative cross-linking of sulfhydryl groups in membrane proteins does not lead to random defects in the lipid domain.

Dissociation kinetics and equilibrium binding properties of polyene antibiotic complexes with phosphatidylcholine/sterol vesicles.

The interactions of sonicated vesicles with the polyene antibiotics amphotericin B, candicidin, mediocidin , and a water-soluble, guanidine derivative of amphotericin B were examined by UV-visible spectroscopy at concentrations below which the polyenes become self-associated. The association constants, Kapp, and the numbers of binding sites per sterol or phospholipid molecule (n) were determined at 30 degrees C and pH 7.4. A single class of binding sites was found, with no evidence of cooperativity. For the binding of mediocidin , amphotericin B, and the guanidine derivative with phosphatidylcholine (PC), PC/cholesterol, and PC/ergosterol vesicles, Kapp was in the range of (1.0-3.0) X 10(6) M-1; Kapp was higher for candicidin-vesicle interaction, reaching 9.0 X 10(6) M-1 with PC/ergosterol vesicles. Binding of the guanidine derivative of amphotericin B to PC vesicles lacking sterol was extensive (n = 0.46); since the other polyenes, which have low aqueous solubilities, had n less than 0.05, positive charges in the mycosamine moiety appear to enhance the extent of polyene antibiotic interaction with the glycerophospholipid head group. Higher values of n (and, therefore, of nKapp ) were found with sterol-containing than with sterol-free vesicles, suggestive of penetration of the polyenes toward the interior of the bilayer when sterol is present. For binding to PC/sterol vesicles, nKapp followed the order of candicidin greater than guanidine derivative of amphotericin B greater than amphotericin B much greater than mediocidin . The values of n and nKapp were appreciably higher for amphotericin B-ergosterol than for amphotericin B-cholesterol interaction in vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)