Synthesis of carbamyl and ether analogs of phosphatidylcholines

A complete synthesis of 1-O-hexadecyl-2-O-N-(heptadec-8-cis-enyl)carbamyl-sn-glycero-3-Phosphocholine, a novel analog of phosphatidylcholine, has been described. Each step is simple to perform and gives the desired products in high yield. Also, some of the intermediates formed during the synthesis have been efficiently utilized to prepare 1-O-hexadecyl-2-O-oleyl-sn-glycero-3-phosphocholine, 1-O-hexadecyl-2-oleoyl-sn-glycero-3-phosphochloine and 3-O-hexadecyl-2-oeloyl-sn-glycero-1-phosphocholine. These phosphatidylcholine (PC) analogs are useful for studying the possible role of phospholipases in the capture and lyses of liposomes in vivo.     

Synthesis of azide and amide analogs of platelet-activating factor and related derivatives

2-Azido-2-deoxy-1-O-hexadecyl-sn-glycero-3-phosphorylcholine was prepared in good yield from D-mannitol via 3-O-hexadecyl-2-O-methanesulfonyl-1-O-triphenylmethyl-sn-glycerol. Nucleophilic displacement of the 2-methanesulfonate function by benzoate or azide ion proceeded with inversion of configuration (Sn2) without racemization. Hydrogenation of the azidophospholipid gave 2-amino-2-deoxy-1-O-hexadecyl-sn-glycero-3-phosphorylcholine which is a versatile intermediate for the preparation of amide analogs of platelet-activating factor and related derivatives. The synthesis of 2-deoxy-2-fluoro-1-O-hexadecyl-sn-glycero-3-phosphorylcholine was also described.     

Metabolic interrelations of hydroxy-substituted ether-linked glycerolipids in the pink portion of the rabbit harderian gland.

The pink portion of the rabbit harderian gland is known to contain a preponderance of ether-linked glycerolipids consisting primarily of 1-(O-acyl)hydroxyalkyl-2,3-diacyl-sn-glycerols and smaller amounts of 1-alkyl-2,3-diacyl-sn-glycerols. In the present study, we have used a combination of chemical, enzymatic, and chromatographic techniques to identify two minor lipid components in the gland as 1-hydroxyalkyl-2-acyl-sn-glycerols and 1-hydroxyalkyl-2,3-diacyl-sn-glycerols. The long-chain acyl groups occurring in the 1-hydroxyalkyl-2-acyl-sn-glycerols and 1-hydroxyalkyl-2,3-diacyl-sn-glycerols are almost exclusively hexadecanoic acid, whereas the 1-(O-acyl)hydroxyalkyl-2,3-diacyl-sn-glycerols have a ratio of hexadecanoic acid to octadecanoic acid of $\text{2}\text{1}$. The 1-(O-acyl) hydroxyalkyl-2,3-diacyl-sn-glycerols and the 1-hydroxyalkyl-2,3-diacyl-sn-glycerols also contain a short-chain acyl moiety identified as 3-methylbutanoic acid (isovaleric acid). This acid was found to occupy the 3-position of the glycerol backbone in these lipid classes.Metabolic experiments demonstrate that 3-methylbutanoic acid in the lipids of the gland is derived from the catabolism of l-leucine. Pulse-chase data show a precursor-product relation between the 1-hydroxyalkyl-2,3-diacyl-sn-glycerols and 1-(O-acyl-hydroxyalkyl-2,3-diacyl-sn-glycerols and rule out direct hydroxylation of 1-alkyl-2,3-diacyl-sn-glycerols as a possible biosynthetic route to the 1-(O-acyl)hydroxyalkyl-2,3-diacyl-sn-glycerols.Characterization of the alkyl and acyl groups and the positional distributions of the acyl moieties in combination with the metabolic information indicated the acylation sequence involved in the formation of 1-(O-acyl)hydroxyalkyl-2,3-diacyl-sn-glycerol is 1-hydroxyalkyl-2-acyl-sn-glycerols → 1-hydroxyalkyl-2,3-diacyl-sn-glycerols → 1-(O-acyl)hydroxyalkyl-2,3-diacyl-sn-glycerols. The data also suggest that hydroxylation of the alkyl side-chain occurs before or at the alkylacylglycerol stage.     

Susceptibility of plasmenyl glycerophosphoethanolamine lipids containing arachidonate to oxidative degradation

Plasmenyl phospholipids (1-alk-1′-enyl-2-acyl-3-glycerophospholipids, plasmalogens) are a structurally unique class of lipids that contain an α-unsaturated ether substituent at the sn-1 position of the glycerol backbone. Several studies have supported the hypothesis that plasmalogens may be antioxidant molecules that protect cells from oxidative stress. Because the molecular mechanisms responsible for the antioxidant properties of plasmenyl phospholipids are not fully understood, the oxidation of plasmalogens in natural mixtures of phospholipids was studied using electrospray tandem mass spectrometry. Glycerophosphoethanolamine (GPE) lipids from bovine brain were found to contain six major molecular species (16:0p/18:1-, 18:1p/18:1-, 18:0p/20:4-, 16:0p/20:4, 18:0a/20:4-, and 18:0a/22:6-GPE). Oxidation of GPE yielded lyso phospholipid products derived from plasmalogen species containing only monounsaturated sn-2 substituents and diacyl-GPE with oxidized polyunsaturated fatty acyl substituents at sn-2. The only plasmalogen species remaining intact following oxidation contained monounsaturated fatty acyl groups esterified at sn-2. The mechanism responsible for the rapid and specific destruction of plasmalogen GPE may likely involve unique reactivity imparted by a polyunsaturated fatty acyl group esterified at sn-2. This structural feature may play a central role determining the antioxidant properties ascribed to this class of phospholipids.     

Oxidatively modified fatty acyl chain determines physicochemical properties of aggregates of oxidized phospholipids

In vivo oxidation of glycerophospholipid generates a variety of products including truncated oxidized phospholipids (tOx-PLs). The fatty acyl chains at the sn-2 position of tOx-PLs are shorter in length than the parent non-oxidized phospholipids and contain a polar functional group(s) at the end. The effect of oxidatively modified sn-2 fatty acyl chain on the physicochemical properties of tOx-PLs aggregates has not been addressed in detail, although there are few reports that modified fatty acyl chain primarily determines the biological activities of tOx-PLs. In this study we have compared the properties of four closely related tOx-PLs which differ only in the type of modified fatty acyl chain present at the sn-2 position: 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (PazePC), 1-palmitoyl-2-(9′-oxo-nonanoyl)-sn-glycero-3-phosphocholine (PoxnoPC), 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC), and 1-palmitoyl-2-(5′-oxo-valeroyl)-sn-glycero-3-phosphocholine (POVPC). Aggregates of individual tOx-PL in aqueous solution were characterized by fluorescence spectroscopy, size exclusion chromatography, native polyacrylamide and agarose gel electrophoresis. The data suggest that aggregates of four closely related tOx-PLs form micelle-like particles of considerably different properties. Our result provides first direct evidence that because of the specific chemical composition of the sn-2 fatty acyl chain aggregates of particular tOx-PL possess a distinctive set of physicochemical properties.     

Hydrolysis of novel diacylglycerol and phorbol diesters by serum lipase

Rat serum, active in the hydrolysis of the tumor-promoting phorbol diester, 12-O-tetradecanoylphorbol-13-acetate (TPA), was examined with regard to lipid interferences of [³H]TPA hydrolysis and enzyme substrate specificity. The enzymatic hydrolysis of TPA could be enhanced 8-fold, ever crude serum, by using a lipid-free acetone powder of rat serum. Addition of lipid to the lipid-free acetone powder produced potent inhibition of TPA hydrolysis. The inclusion of multilamallar liposomes resulted in similar inhibition, and isolation of liposomes by high-speed centrifugation showed that 95% of the radiolabeled TPA was associated with the fatty pellet. Substrate specificity studies demonstrated that the serum activity hydrolyzes the long-chain ester of TPA and the long-chain primary acyl group of diacylglycerols. TPA was hydrolyzed at approximately twice the rate of dioleoylglycerol; however, the most reactive substrates were those synthetic analogs of diacylglycerol containing a short-chain ester group at the sn-2 position. Palmitic acid was liberated from [1-¹⁴C]palmitoyl-2-acetyl-sn-glycerol and [1-¹⁴C]palmitoyl-2-butyryl-sn-glycerol at 120- and 33-tinies the rate of TPA hydrolysis, respectively. Lipase resistant 1-hexadecyl-2-[³H]acetylglycerol was also used as substrate, but the sn-2 ester moiety showed poor lability. The diacylglycerol analogs are new lipase substrates and, in view of their similarities to the fatty acyl portion of TPA, it is thought that these compounds could serve as protein kinase C activators.     

A Comparison of the Membrane Binding Properties of C1B Domains of PKCγ, PKCδ, and PKC?

The C1 domains of classical and novel PKCs mediate their diacylglycerol-dependent translocation. Using fluorescence resonance energy transfer, we studied the contribution of different negatively charged phospholipids and diacylglycerols to membrane binding. Three different C1B domains of PKCs were studied (the classical γ, and the novel δ and ?), together with different lipid mixtures containing three types of acidic phospholipids and three types of activating diacylglycerols. The results show that C1Bγ and C1B? exhibit a higher affinity to bind to vesicles containing 1-palmitoyl-2-oleoyl-sn-phosphatidic acid, 1-palmitoyl-2-oleoyl-sn-phoshatidylserine, or 1-palmitoyl-2-oleoyl-sn-phosphatidylglycerol, with C1B? being the most relevant case because its affinity for POPA-containing vesicles increased by almost two orders of magnitude. When the effect of the diacylglycerol fatty acid composition on membrane binding was studied, the C1B? domain showed the highest binding affinity to membranes containing 1-stearoyl-oleoyl-sn-glycerol or 1,2-sn-dioleoylglycerol with POPA as the acidic phospholipid. Of the three diacylglycerols used in this study, 1,2-sn-dioleoylglycerol and 1-stearoyl-oleoyl-sn-glycerol showed the highest affinities for each isoenzyme, whereas 1,2-sn-dipalmitoylglycerol; showed the lowest affinity. DSC experiments showed this to be a consequence of the nonfluid conditions of 1,2-sn-dipalmitoylglycerol;-containing systems.     

Molecular and mechanistic characterization of platelet-activating factor-like bioactivity produced upon LDL oxidation

Oxidation of LDL is thought to be involved in both initiating and sustaining atherogenesis through the formation of proinflammatory lipids and the covalent modification of LDL particles. Platelet-activating factor (PAF; 1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a potent phospholipid mediator involved in inflammation. Upon oxidation of LDL, oxidized phospholipids with PAF-like structure are generated, and some of them may act via the PAF receptor. We evaluated the contribution of 1-0-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (C16:0 PAF) and of other PAF analogs on the PAF-like bioactivity formed upon Cu2+-initiated oxidation of LDL. Reverse-phase HPLC purification and electrospray ionization-MS analyses showed that upon oxidation of LDL with inactivated PAF-acetylhydrolase (PAF-AH), C16:0 PAF accounted for >30% of PAF-like biological activity and its sn-2 butenoyl analog accounted for >50%. However, upon LDL oxidation in the presence of exogenous 1-0-alkyl-sn-glycero-3-phosphocholine (lyso-PAF) without PAF-AH inactivation, C16:0 PAF formation accounted for >90% of the biological activity recovered. We suggest that the C16:0 PAF, despite being a minor constituent of the LDL peroxidation products, may contribute substantially to the bioactivity formed in oxidized LDL. The higher bioactivity of C16:0 PAF, and the higher selectivity of the LDL-attached lyso-PAF transacetylase toward very short acyl chains [acetate (C2) vs. butanate (C4)], may explain the contribution described above.     

Miscibility of Sphingomyelins and Phosphatidylcholines in Unsaturated Phosphatidylcholine Bilayers

Polyunsaturated phospholipids are common in biological membranes and affect the lateral structure of bilayers. We have examined how saturated sphingomyelin (SM; palmitoyl and stearoyl SM (PSM and SSM, respectively)) and phosphatidylcholine (PC; dipalmitoyl PC and 1-palmitoyl-2-stearoyl PC (DPPC and PSPC, respectively)) segregate laterally to form ordered gel phases in increasingly unsaturated PC bilayers (sn-1: 16:0 and sn-2: 18:1...22:6; or sn-1 and sn-2: 18:1…22:6). The formation of gel phases was determined from the lifetime analysis of trans-parinaric acid. Using calorimetry, we also determined gel phase formation by PSM and DPPC in unsaturated PC mixed bilayers. Comparing PSM with DPPC, we observed that PSM formed a gel phase with less order than DPPC at comparable bilayer concentrations. The same was true when SSM was compared with PSPC. Furthermore, we observed that at equal saturated phospholipid concentration, the gel phases formed were less ordered in unsaturated PCs having 16:0 in sn-1, as compared to PCs having unsaturated acyl chains in both sn-1 and sn-2. The gel phases formed by the saturated phospholipids in unsaturated PC bilayers did not appear to achieve properties similar to pure saturated phospholipid bilayers, suggesting that complete lateral phase separation did not occur. Based on scanning calorimetry analysis, the melting of the gel phases formed by PSM and DPPC in unsaturated PC mixed bilayers (at 45 mol % saturated phospholipid) had low cooperativity and hence most likely were of mixed composition, in good agreement with trans-parinaric acid lifetime data. We conclude that both interfacial properties of the saturated phospholipids and their chain length, as well as the presence of 16:0 in sn-1 of the unsaturated PCs and the total number of cis unsaturations and acyl chain length (18 to 22) of the unsaturated PCs, all affected the formation of gel phases enriched in saturated phospholipids, under the conditions used.     

The effects of oxidised phospholipids and cholesterol on the biophysical properties of POPC bilayers

Oxidation of unsaturated membrane phospholipids by oxidative stress is associated with inflammation, infection, numerous diseases and neurodegenerative disorders. Lipid oxidation is observed in experimental samples when the parent lipid is exposed to oxidative stressors. The effect of phospholipid oxidation on the properties of biological membranes are still being explored, while low concentrations (0.1–2.0?mol%) of oxidised phospholipids are associated with disease states [1]. Previous computational studies have focused on the effect of high concentrations (~50?mol%) of oxidised phospholipids on binary lipid bilayers. This work systematically characterises the effect of lower concentrations (~10?mol%) of two oxidised lipid species, PoxnoPC (1-palmitoyl-2-(9′-oxo-nonanoyl)-sn-glycero-3-phosphocholine) or PazePC (1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine), on POPC/cholesterol and pure POPC bilayers. During μs atomistic simulations in pure POPC bilayers, PoxnoPC and PazePC reoriented their oxidised sn-2 acyl chains towards the solution, and PazePC adopted an extended conformation. The addition of 20?mol% cholesterol not only modulated the fluidity of the bilayers; it also modulated the flexibility of the PoxnoPC oxidised sn-2 tail, reducing bilayer disorder. In contrast, the addition of cholesterol had little effect on bilayers containing PazePC. Our studies show that the effect of oxidised lipids on the biophysical properties of a multicomponent bilayer cannot be intuitively extrapolated from a binary lipid system.     

Oxidized phospholipids as potential molecular targets for antimicrobial peptides

The effects of oxidatively modified phospholipids on the association with model biomembranes of four antimicrobial peptides (AMPs), temporin B and L, indolicidin, and LL-37(F27W) were studied by Langmuir balance and fluorescence spectroscopy. In keeping with previous reports the negatively charged phospholipid phosphatidylglycerol (PG) enhanced the intercalation of all four peptides into lipid monolayers and liposomal bilayers under low ionic strength conditions. Interestingly, similar effect was observed for 1-palmitoyl-2-(9′-oxo-nonanoyl)-sn-glycero-3-phosphocholine (PoxnoPC), a zwitterionic oxidized phospholipid bearing an aldehyde function at the end of its truncated sn-2 acyl chain. Instead, the structurally similar 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (PazePC) containing a carboxylic moiety was less efficient in promoting the membrane association of these peptides. Physiological saline reduced the binding of the above peptides to membranes containing PG, whereas interactions with PoxnoPC were found to be insensitive to ionic strength. Notably, membrane intercalation of temporin L, the most surface active of the above peptides could be into PoxnoPC containing monolayers was strongly attenuated by methoxyamine, suggesting the importance of Schiff base formation between peptide amino groups and the lipid aldehyde function. PoxnoPC and similar aldehyde bearing oxidatively modified phospholipids could represent novel molecular targets for AMPs.     

Two new phospholipids,hydroxyarchaetidylglycerol and hydroxyarchaetidylethanolamine,from the Archaea Methanosarcina barkeri

The structures of two new ether phospholipids of the methanogenic Archaea, Methanosarcina barkeri, were determined as hydroxyarchaetidylglycerol and hydroxyarchaetidylethanolamine by means of chemical, chromatographic and enzymatic analyses, and fast atom bombardment-mass spectrometry. These lipids are hydroxy diether analogs of phosphatidylglycerol and phosphatidylethanolamine, respectively, with β-hydroxyarchaeol (2-O-(3′-hydroxy)phytanyl-3-O-phytanyl-sn-glycerol) as a core lipid. In addition, two other ether phospholipids, usual archaetidylglycerol and archaetidylethanolamine, were also identified in the organism. The stereochemical structure of the unalkylated glycerophosphate of hydroxyarchaetidylglycerol and archaetidylglycerol was determined as sn-glycerol-3-phosphate by use of sn-glycerol-3-phosphate dehydrogenase. The stereochemical configuration of the glycerophosphoglycerol backbone of these lipids was a mirror image of that of diacylphosphatidylglycerol from the organisms of the domains Bacteria and Eucarya, and it was shared with extremely halophilic Archaea. These four phospholipids, in addition to five lipids that had already been reported, accounted for 88% of the total polar lipids of this organism.     

Structural Basis for the Recognition of Oxidized Phospholipids in Oxidized Low Density Lipoproteins by Class B Scavenger Receptors CD36 and SR-BI

Specific oxidized phospholipids (oxPC_CD36) accumulate in vivo at sites of oxidative stress and serve as high affinity ligands for scavenger receptors class B (CD36 and SR-BI). Recognition of oxPC_CD36 by scavenger receptors plays a role in several pathophysiological processes. The structural basis for the recognition of oxPC_CD36 by CD36 and SR-BI is poorly understood. A characteristic feature of oxPC_CD36 is an sn-2 acyl group that incorporates a terminal γ-hydroxy (or oxo)-α,β-unsaturated carbonyl. In the present study, a series of model oxidized phospholipids were designed, synthesized, and tested for their ability to serve as ligands for CD36 and SR-BI. We demonstrated that intact the sn-1 hydrophobic chain, the sn-3 hydrophilic phosphocholine or phosphatidic acid group, and the polar sn-2 tail are absolutely essential for high affinity binding. We further found that a terminal negatively charged carboxylate at the sn-2 position suffices to generate high binding affinity to class B scavenger receptors. In addition, factors such as polarity, rigidity, optimal chain length of sn-2, and sn-3 positions and negative charge at the sn-3 position of phospholipids further modulate the binding affinity. We conclude that all three positions of oxidized phospholipids are essential for the effective recognition by scavenger receptors class B. Furthermore, the structure of residues in these positions controls the affinity of the binding. The present studies suggest that, in addition to oxPC_CD36, other oxidized phospholipids observed in vivo may represent novel ligands for scavenger receptors class B.     

Structural identification of phosphatidylcholines having an oxidatively shortened linoleate residue generated through its oxygenation with soybean or rabbit reticulocyte lipoxygenase

Phosphatidylcholines (PCs) with platelet-activating factor (PAF)-like biological activities are known to be generated by fragmentation of the sn-2-esterified polyunsaturated fatty acyl group. The reaction is free radical-mediated and triggered by oxidants such as metal ions, oxyhemoglobin, and organic hydroperoxides. In this study, we characterized the PAF-like phospholipids produced on reaction of PC having a linoleate group with lipoxygenase enzymes at low oxygen concentrations. When the oxidized PCs were analyzed by gas chromatography-mass spectrometry, two types of oxidatively fragmented PC were detected. One PC had an sn-2-short chain saturated or unsaturated acyl group (C(8)-C(13)) with an aldehydic terminal; the abundant species were PCs with C(9) and C(13). The other PC had a short chain saturated acyl group (C(6)-C(9)) with a methyl terminal, and the most predominant species was PC with C(8). When the extracts of oxidation products were subjected to catalytic hydrogenation, PCs having saturated acyl groups (C(6)-C(14)) were detected; the most abundant was C(12) species. The less regiospecific formation of PAF-like lipids suggests that they were generated by oxidative fragmentation of PC hydroperoxides formed by non-stereoselective oxygenation of the alkyl radical of esterified linoleate that escaped from the active centers of lipoxygenases. One of the PAF-like PC with an aldehydic terminal was found to be bioactive; it inhibited the production of nitric oxide induced by lipopolysaccharide and interferon-gamma in vascular smooth muscle cells from rat aorta.     

Serum choline plasmalogens,particularly those with oleic acid in sn-2, are associated with proatherogenic state

Serum plasmalogens (Pls) (1-O-alk-1’-enyl-2-acyl glycerophospholipids) are of particular interest for studies on metabolic disorders associated with oxidative stress and chronic inflammation. Serum levels of Pls are known to correlate positively with HDL-cholesterol (HDL-C); however, few studies have examined serum Pls molecular species in association with pathophysiological conditions and their clinical significance. To clarify these, we determined serum levels of individual ether glycerophospholipids in Japanese asymptomatic cohorts (n = 428; 362 male and 66 female subjects) by LC/MS/MS, and examined their correlations with clinical parameters. We found that the proportion of choline Pls (PlsCho) among total serum phospholipids was significantly lower in the male group over 40 years old and was associated with multiple risk parameters more strongly than HDL-C. The abundance of serum PlsCho with oleic acid (18:1) in sn-2 exhibited the strongest positive correlation with serum concentrations of adiponectin and HDL-C, while being inversely associated with waist circumference and the serum levels of TG and small dense LDL-cholesterol. The characterization of serum ether glycerophospholipids verified the specificity of PlsCho, particularly the ones with 18:1 in sn-2, as a sensitive biomarker for the atherogenic state.     

Closely related oxidized phospholipids differentially modulate the physicochemical properties of lipid particles

Oxidation of glycerophospholipids results in the formation of large variety of oxidized phospholipid products that differs significantly in their chemical compositions and molecular structures. Biological activities of these oxidized products also differ considerably. Here we report the comparisons of the physicochemical properties of non-oxidized phospholipid particle containing two closely related tOx-PLs: 1-palmitoyl-2-(5-keto-6-octendioyl)-sn-glycero-3-phosphocholine (KOdiA-PC) and 1-palmitoyl-2-(9-keto-10-dodecendioyl)-sn-glycero-3-phosphocholine (KDdiA-PC). DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) was used as a model membrane non-oxidized phospholipid. Physicochemical properties of the lipid particles were characterized by using fluorescence spectroscopy, native polyacrylamide gel and agarose gel electrophoresis. Our result shows that the presence of closely related tOx-PLs, which differ only in the chemical composition of the oxidized fatty acyl chains at the sn-2 position, exerts considerably different effect on the physicochemical properties of non-oxidized phospholipid particles containing them.     

The chemical synthesis of glucosaminylphosphatidylglycerol. Comparison with a new phospholipid isolated from Bacillus megaterium

1. We describe the synthesis of a glucosamine derivative of phosphatidylglycerol having the same structure as that of the natural compound isolated from Bacillus megaterium. 2. 2-O-(3,4,6-Tri-O-acetyl-2-deoxy-2-phthalimido-d-glucopyranosyl)-3-O-benzyl-1-iodo-sn-glycerol was prepared by a Königs–Knorr condensation between 3-O-benzyl-1-toluene-p-sulphonyl-sn-glycerol and 3,4,6-tri-O-acetyl-1-bromo-2-deoxy-2-phthalimido-d-glucopyranose followed by replacement of the toluene-p-sulphonyl group with iodine. The iodide was treated with the silver salt of 2-isolauroyl-1-oleoyl-sn-glycerol 3-(monobenzyl hydrogen phosphate) to form the fully protected phosphoglycolipid. 3. Removal of benzyl protecting groups by catalytic hydrogenolysis, phthaloyl group with hydrazine and acetyl groups with pH10 buffer furnished 2-O-(2-amino-2-deoxy-d-glucopyranosyl)-1-(2-isolauroyl-1-stearoyl-sn-glycero-3-phosphoryl)-sn-glycerol. 4. The synthetic and natural compounds appeared identical when compared by chromatography and by identification of hydrolysis products from chemical and enzymic degradations.     

Synthesis of two purple-membrane glycolipids and the glycolipid sulfate O-(β-d-glucopyranosyl 3-sulfate)-(1→6)-O-α-d-mannopyranosyl-(1→2)-O-α-d-glucopyranosyl-(1→1)-2, 3-di-O-phytanyl-sn-glycerol

The two purple-membrane glycolipids O-β-d-glucopyranosyl- and O-β-d-galactopyranosyl-(1→6)-O-α-d-mannopyranosyl-(1→2)-O-α-d-glucopyranosyl-(1→1)-2, 3-di-O-phytanyl-sn-glycerol were prepared by coupling O-(2,3,4-tri-O-acetyl-α-d-mannopyranosyl)-(1→2)-O-(3,4,6-tri-O-acetyl-α-d-glucopyranosyl)-(1→1)-2, 3-di-O-phytanyl-sn-glycerol (9) with 2,3,4,6-tetra-O-acetyl-α-d-glucopyranosyl bromide or 2,3,4,6-tetra-O-acetyl-α-d-mannopyranosyl bromide, respectively, followed by deacetylation. The glycolipid sulfate O-(β-d-glucopyranosyl 3-sulfate)-(1→6)-O-α-d-mannopyranosyl-(1→2)-O-α-d-glucopyranosyl-(1→1)-2,3-di-O-phytanyl-sn-glycerol was prepared by coupling of 9 with 2,4,6-tri-O-acetyl-3-O-trichloroethyloxycarbonyl-α-d-glucopyranosyl bromide in the presence of Hg(CN)₂/HgBr₂ followed by selective removal of the 3?-trichloroethyloxycarbonyl group, sulfation of HO-3?, and deacetylation. The suitably protected key-intermediate 9 could be prepared by two distinct approaches.     

Synthesis and in vitro activities of a new antiviral duplex drug linking Zidovudine (AZT) and Foscarnet (PFA) via an octadecylglycerol residue

To prepare a new antiviral duplex drug linking Zidovudine (AZT) and Foscarnet (PFA) via a lipophilic octadecylglycerol residue we condensed 1-O-4-monomethoxytrityl-3-O-octadecyl-sn-glycerol-2-hydrogenphosphonate obtained from 3-O-octadecyl-sn-glycerol with AZT by the phosphonate method. The purified condensation product was de-tritylated resulting in 3′-azido-3′-deoxythymidylyl-(5′ → 2-O)-3-O-octadecyl-sn-glycerol, followed by treatment with (ethoxycarbonyl)phosphoric dichloride. The resulting 3′-azido-3′-deoxy-thymidylyl-(5′ → 2)-3-O-octadecyl-sn-glycerol-1-O-(ethoxycarbonyl)phosphonate was purified by preparative RP-18 column chromatography. The antiviral duplex drug 3′-azido-3′-deoxythymidylyl-(5′ → 2-O)-3-O-octadecyl-sn-glycerol-1-O-phosphonoformate trisodium salt (AZT–lipid–PFA) was obtained after alkaline cleavage of the phosphonoformate ethylester residue. The overall yield of the five step synthesis performed at gram scale was about 30%. According to a supposed pathway AZT–lipid–PFA could be cleaved to yield a mixture of different antiviral compounds such as AZT, AZT-5′-monophosphate, octadecylglycerol–AZT, PFA and octadecylglycerol–PFA, possibly producing additive and/or synergistic antiviral effects. In vitro studies showed that the duplex drug exhibits antiviral activities against HIV and especially against drug-resistant strains and clinical isolates of HSV and HCMV. The E₅₀ values of AZT–lipid–PFA against HIV ranged between 170 and 200 nM. The half-maximal inhibitory doses (IC₅₀) against highly acyclovir (ACV)-resistant HSV isolates determined by a plaque reduction assay ranged between 1.87 and 4.59 μM. Using ganciclovir (GCV)-sensitive, GCV resistant and drug cross-resistant HCMV strains the IC₅₀-values of AZT–lipid–PFA were between 2.78 and 1.18 μM. With regard to PFA, the IC₅₀-value of AZT–lipid–PFA determined on a multi-drug-resistant HCMV strain was about 90-fold lower than that of PFA, demonstrating the superior antiviral effect of the duplex-drug.