First total synthesis of (5Z,9Z)-(+/-)-2-methoxy-5,9-octadecadienoic acid, a marine derived methoxylated analog of taxoleic acid

The first total synthesis for the sponge derived (5Z,9Z)-(+/-)-2-methoxy-5,9-octadecadienoic acid, an analog of taxoleic acid, was accomplished in seven steps and in a 10% overall yield. It was again corroborated that the best strategy to prepare these cis,cis dimethylene interrupted double bonds is the double-alkyne bromide coupling reaction of 1,5-hexadiyne, which provides the advantage of achieving a 100% cis stereochemical purity for both double bonds after hydrogenation under Lindlar conditions. The alpha-methoxy functionality was best prepared via the Mukaiyama reaction of (4Z,8Z)-heptadecadienal with trimethylsilyl cyanide and triethylamine followed by acid hydrolysis. Selective methylation of the hydroxyl group of (5Z,9Z)-(+/-)-2-hydroxy-5,9-octadecadienoic acid was achieved with sodium hydride/methyl iodide when tetrahydrofuran was used as solvent. Complete spectral data is presented, for the first time, for this unusual marine alpha-methoxylated fatty acid.     

Large-scale preparation of (9Z,12E)-[1-(13)C]-octadeca-9,12-dienoic acid, (9Z,12Z,15E)-[1-(13)C]-octadeca-9,12,15-trienoic acid and their [1-(13)C] all-cis isomers

Several grams of labelled trans linoleic and linolenic acids with high chemical and isomeric purities (>97%) have been prepared for human metabolism studies. A total of 12.5 g of (9Z, 12E)-[1-(13)C]-octadeca-9,12-dienoic acid and 6.3 g of (9Z,12Z, 15E)-[1-(13)C]-octadeca-9,12,15-trienoic acid were obtained in, respectively, seven steps (7.8% overall yield) and 11 steps (7% overall yield) from 7-bromo-heptan-1-ol. The trans bromo precursors used for the labelling were synthesised by using copper-catalysed couplings. The trans fatty acids were then obtained via the nitrile derivatives. A total of 23.5 g of (9Z,12Z)-[1-(13)C]-octadeca-9, 12-dienoic acid and 10.4 g of (9Z,12Z,15Z)-[1-(13)C]-octadeca-9,12, 15-trienoic acid were prepared in five steps in, respectively, 32 and 18% overall yield. Large quantities of bromo and chloro precursors were synthesised from the commercially available acid according to Barton's procedure. In all cases, the main impurities (>0.5%) of each labelled fatty acid have been characterised.     

Analogs of unusual sponge phospholipids. Synthesis and thermotropic properties of 1,2-di-(6Z,9Z)-6,9-hexacosadienoyl phosphatidylcholine and phosphatidylethanolamine

The major marine sponge phospholipids 1,2-di-(5Z,9Z)-5,9-hexacosadienoyl phosphatidylcholine (PC) and phosphatidyl-ethanolamine (PE) hardly incorporate cholesterol into their liposomal bilayers, as reported earlier. Our previous studies indicated that their synthetic short chain (C18-C24) analogs with the same double bond pattern readily incorporated cholesterol, thus demonstrating the importance of the chain length. In order to investigate the possible role of the unusual delta 5,9 diunsaturation 1,2-di-(6Z,9Z)-6,9-hexacosadienoyl phosphatidylcholine and phosphatidylethanolamine were synthesized and their thermotropic behavior studied. Both analogs shows a transition endoterm at 45 degrees C, while the natural 1,2-di-(5Z,9Z)-5,9-hexacosadienoyl PC and its PE counterpart exhibited it at 42 degrees C. A partial incorporation of cholesterol into liposomal bilayers of 1,2-di-(6Z,9Z)-6,9-hexacosadienoyl PC was observed. Our results suggest that while the chain length is the predominant factor in the interactions of these phospholipids with sterols, the double bond location may also play a contributing role.     

Unusual pattern of fatty acid biosynthesis. Evidence for C-19 desaturase activity in freshwater sponges

The two long chain fatty acids common in marine demosponges, (5Z, 9Z)-5,9-hexacosadienoic (delta 5,9-26:2) and (5Z, 9Z, 19Z)-5,9,19-hexacosatrienoic (delta 5,9,19-26:3) acids), were identified also as the major phospholipid components in the freshwater sponge Ephydatia fluviatilis. Whereas the typical marine sponge Microciona prolifera biosynthesizes the delta 5,9,19-26:3 acid solely by homologation of exogenous palmitoleic acid (delta 9-16:1) and subsequent desaturation at positions 5 and 9, it was found that the freshwater sponge could further desaturate the delta 5,9-26:2 acid to the triene, indicating for the first time the existence of delta 19 -desaturase activity in a living organism.     

Synthesis of Methyl (5Z,9Z,17R)-17-Methylnonadeca-5,9-dienoate,the (R)-Enantiomer of the Structure Proposed for a Metabolite of the Philippine Sponge Plakinastrella sp.

The (R)-enantiomer (1) of methyl (5Z,9Z)-17-methyl-nonadeca-5,9-dienoate, the structure proposed for a metabolite of the Philippine sponge, Plakinastrella sp., was synthesized. The ¹H- and ¹³C-NMR spectra of the synthetic material were different from those reported for the natural product. The proposed structure 1 is therefore incorrect.     

Studies on the mechanism of formation of the 5S,12S-dihydroxy-6,8,10,14(E,Z,E,Z)-icosatetraenoic acid in leukocytes

Incubation of peripheral blood leukocytes with arachidonic acid (and ionophore A23187) led to the formation of leukotriene B₄, Δ⁶-trans-leukotriene B₄, Δ⁶-trans-12-epi-leukotriene B₄, 5-hydroxy-icosatetraenoic acid, 12-hydroxy-icosatetraenoic acid and of 5S,12S-dihydroxy-6,8,10,14-(E,Z,E,Z)-icosatetraenoic acid (5S,12S-DiHETE). Incubation of leukocytes with leukotriene A₄ resulted in the formation of leukotriene B₄ and of its two Δ⁶-trans-isomers but not of the 5S,12S-DiHETE. ¹⁸O₂ labeling experiments have shown that the hydroxyl groups at C5 and C12 in the 5S,12S-DiHETE are derived from molecular oxygen. The tetraacetylenic analog of arachidonic acid was found to be a potent inhibitor of the formation of the 5S,12S-DiHETE whereas it potentiated the synthesis of the 5-hydroxy acid and of leukotriene B₄. Addition of the 12-hydroxy-icosatetraenoic acid to leukocytes, or of the 5-hydroxy-icosatetraenoic acid to a suspension of platelets caused the formation of the 5S,12S-DiHETE. It is concluded that the 5S,12S-DiHETE is not derived from leukotriene A₄ but is a product of the successive reactions of arachidonic acid with two lipoxygenases of different positional specificities.     

Properties of unusual phospholipids. III: Synthesis, monolayer investigations and DSC studies of hydroxy octadeca(e)noic acids and diacylglycerophosphocholines derived therefrom

Diacylglycerophosphocholines containing (R)-3-, (R)-12-, (R)-17-hydroxy octadeca(e)noic acids and the corresponding racemates were synthesized and purified to homogeneity. The influence of the position of the hydroxy group on the monolayer packing properties of these fatty acids and their phosphatidylcholines was studied by Langmuir techniques and 1,2-di-[(R)-12-hydroxy-octadec-cis-9-enyl]-sn-glycero-3-phosphocholine displayed the largest lift-off area (330 Ų/molecule). This result was in line with the thermotropic phase behavior of these phospholipids, as measured by differential scanning calorimetry (DSC): the gel- to liquid-crystalline phase transition temperature (T_m) passed through a minimum of −15.1°C for 1,2-di-[(R)-12-hydroxy-octadec-cis-9-enyl]-sn-glycero-3-phosphocholine.     

The first total synthesis of the marine fatty acid (+/-)-9-methoxypentadecanoic acid: a synthetic route towards mid-chain methoxylated fatty acids

The marine fatty acid (+/-)-9-methoxypentadecanoic acid was synthesized for the first time in seven steps (7.8% overall yield) starting from commercially available 9-decen-1-ol. The key step in the synthesis was the coupling of pentylmagnesium bromide with 1-benzyloxy-9,10-epoxydecane under 1,5-cyclooctadiene copper (I) chloride catalysis. Nuclear magnetic resonance data are provided for the first time for this type of methoxylated fatty acids and the synthetic approach utilized is of general applicability since it can be used in the synthesis of other mid-chain methoxylated fatty acids. This synthetic methodology should afford sufficient quantities of these fatty acids for biological evaluation. The spectral data obtained for the title compound will also be helpful in subsequent characterizations of other mid-chain methoxylated fatty acids using nuclear magnetic resonance spectroscopy.     

Metabolic incorporation of 9-(2-anthryl)-nonanoic acid, a new fluorescent and photoactivable probe, into the membrane lipids of Chinese hamster ovary cells

9-(2-Anthryl)-nonanoic acid is a new fluorescent and photoactivable probe, which has been designed for studying the lateral diffusion rate and the lateral distribution of lipids in biological membranes by means of the anthracene photodimerization reaction. It is shown that this anthracene fatty acid is metabolically incorporated into the glycerophospholipids (phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol) of the eukaryotic Chinese hamster ovary cells in culture. Under our culture conditions (Eagle's minimal essential medium plus delipidized fetal calf serum) this incorporation proceeded with a very good rate (up to 45 mol/100 mol, after two days culture) and could be easily modulated depending on the way the cells were fed with the anthracene fatty acid. It occurred to a similar extent at the sn-1 (55 +/- 5%) or at the sn-2 (45 +/- 5%) position on the phospholipid glycerol backbone, without any degradation or elongation. No double labelling at the sn-1 and sn-2 positions was detected. Although incorporation of the anthracene fatty acid affected the cell growth rate (generation time of 48 h compared to a generation time of 21 h for control cells) it did not bring about cell mortality. This incorporation took place not only into the phospholipids but also into the triglycerides with, as a consequence, the appearance of strongly fluorescent lipid vesicles inside the cells. It affected the whole cell fatty acid composition by slightly increasing the amount of palmitic acid and markedly decreasing the amount of stearic and oleic acids.     

Chemical C2-elongation of polyunsaturated fatty acids

Three fatty acids were synthesized from commercially available alpha-linolenic, stearidonic and eicosapentaenoic acids by C2-elongation using a four step preparative technique. The parent fatty acid methyl esters were reduced to alcohols with LiAlH(4), converted to bromides by treatment with triphenylphosphine dibromide, coupled with a lithiated C2-elongation block--2,4,4-trimethyl-2-oxazoline--to form the corresponding 2,2-dimethyloxazolines of C2-elongated fatty acids, and finally, converted to the target polyunsaturated fatty acids by acidic alcoholysis. Yields of more than 60% were achieved on a gram scale. The resulting 11Z,14Z,17Z-eicosatrienoic, 8Z,11Z,14Z,17Z-eicosatetraenoic and 7Z,10Z,13Z,16Z,19Z-docosapentaenoic acids were obtained as colorless oils with >98% purity and could be used for biochemical investigations without additional purification. The elongated fatty acids were free of byproducts that could result from Z-E isomerization or migration of double bonds.     

Synthesis of methyl (5Z,9Z,17R)-17-methylnonadeca-5,9-dienoate, the (R)-enantiomer of the structure proposed for a metabolite of the Philippine sponge Plakinastrella sp.

The (R)-enantiomer (1) of methyl (5Z,9Z)-17-methylnonadeca-5,9-dienoate, the structure proposed for a metabolite of the Philippine sponge, Plakinastrella sp., was synthesized. The 1H- and 13C-NMR spectra of the synthetic material were different from those reported for the natural product. The proposed structure 1 is therefore incorrect.     

Drug-phospholipid conjugates as potential prodrugs: synthesis, characterization, and degradation by pancreatic phospholipase A(2)

The aim of the present study was the synthesis of phospholipids containing a drug molecule instead of a fatty acid. Valproic acid and ibuprofen served as model compounds. The target molecules were synthesized either starting from sn-glycero-3-phosphocholine (1) or using (S)-2-O-benzyl-1-O-tritylglycerol (11) and (R)-2-O-benzyl-1-O-tert-butyldiphenylsilylglycerol (12), respectively, as key intermediates. With respect to the surface properties and the aggregation behavior, the drug-phospholipid conjugates resembled natural phosopholipids. Upon incubation with porcine pancreatic phospholipase A(2), only compounds with a fatty acid in the sn-2 position of the glycerol backbone were degraded. Derivatives with either ibuprofen in the sn-2 position or displaying the unnatural S-configuration were resistant to enzymatic in vitro hydrolysis.     

20-hydroxyeicosatetraenoic acid (20-HETE) metabolism in coronary endothelial cells

We have investigated the role of endothelial cells in the metabolism of 20-hydroxyeicosatetraenoic acid (20-HETE), a vasoactive mediator synthesized from arachidonic acid by cytochrome P450 omega-oxidases. Porcine coronary artery endothelial cells (PCEC) incorporated 20-[(3)H]HETE primarily into the sn-2 position of phospholipids through a coenzyme A-dependent process. The incorporation was reduced by equimolar amounts of arachidonic, eicosapentaenoic or 8,9-epoxyeicosatrienoic acids, but some uptake persisted even when a 10-fold excess of arachidonic acid was available. The retention of 20-[(3)H]HETE increased substantially when methyl arachidonoyl fluorophosphonate, but not bromoenol lactone, was added, suggesting that a Ca(2+)-dependent cytosolic phospholipase A(2) released the 20-HETE contained in PCEC phospholipids. Addition of calcium ionophore A23187 produced a rapid release of 20-[(3)H]HETE from the PCEC, a finding that also is consistent with a Ca(2+)-dependent mobilization process. PCEC also converted 20-[(3)H]HETE to 20-carboxy-arachidonic acid (20-COOH-AA) and 18-, 16-, and 14-carbon beta-oxidation products. 20-COOH-AA produced vasodilation in porcine coronary arterioles, but 20-HETE was inactive. These results suggest that the incorporation of 20-HETE and its subsequent conversion to 20-COOH-AA in the endothelium may be important in modulating coronary vascular function.     

Stereoselective synthesis of (6Z,10E,12Z)-octadeca-6,10,12-trienoic acid, (8Z,12E,14Z)-eicosa-8,12,14-trienoic acid,and their [1-14C]-radiolabeled analogs

To study the metabolic fate of conjugated linoleic acid isomers, we synthesized, in seven steps, from 1-heptyne, (6Z,10E,12Z)-octadeca-6,10,12-trienoic acid, (8Z,12E,14Z)-eicosa-8,12,14-trienoic acid, and their [1-(14)C]-analogs. In the case of (6Z,10E,12Z)-octadecatrienoic acid, a series of palladium-catalyzed cross-coupling reactions between 1-heptyne and (E)-1,2-dichloro-ethene, a coupling reaction with a Grignard reagent and cleavage of the dioxolane gave (E)-dodec-4-en-6-ynal 3. Stereoselective Wittig reaction between aldehyde 3 and triphenyl-[5-(tetrahydro-pyran-2-yloxy)-pentyl]-phosphonium provided a dienyne. Stereocontrolled reduction of the triple bond and replacement of the tetrahydropyranyl group by a bromine gave (5Z,9E,11Z)-1-bromo-heptadeca-5,9,11-triene 10. Formation of the alkenyl lithium derivative and carbonation with CO(2) furnished (6Z,10E,12Z)-octadecatrienoic acid. (8Z,12E,14Z)-eicosa-8,12,14-trienoic acid was obtained by the same route but using triphenyl-[5-(tetrahydro-pyran-2-yloxy)-heptyl]-phosphonium iodide for the Wittig reaction. [1-(14)C]-analogs were obtained from the bromides by carbonation with (14)CO2. In all cases, chemical or radiochemical purities were found to be better than 95% after purification by flash chromatography on silica gel (>99% after additional purification by RP-HPLC). Metabolism studies in animals are in progress.     

Cholesterol interactions with tetracosenoic acid phospholipids in model cell membranes: role of the double-bond position

The synthesis and thermotropic properties of 1,2-di-(9Z)-9-tetracosenoylphosphatidylcholine [delta 9-PC(24:1,24:1), 1], 1,2-di-(5Z)-5-tetracosenoylphosphatidylcholine [delta 5-PC(24:1,24:1), 2], and 1,2-di-(15Z)-15- tetracosenoylphosphatidylcholine [delta 15-PC(24:1,24:1), 3] are reported. Liposomes prepared from these phospholipids differ from those of the natural sponge phospholipids, 1,2-di-(5Z,9Z)-5,9-hexacosadienoylphosphatidylcholine (4a) and the corresponding ethanolamine (4b), both of which virtually exclude cholesterol from their bilayers. The behavior of 1 and 2 is similar to that of 1,2-di-(6Z,9Z)-6,9-hexacosadienoylphosphatidylcholine (5), which exhibits a partial molecular interaction with cholesterol. In the case of 3, cholesterol appears to interact with the saturated acyl chain regions of this phospholipid in a manner similar to that of its interaction with DPPC acyl chains. This study delineates the effect of the double-bond location in long fatty acyl chains of phospholipids on their interactions with cholesterol.     

Sequential substitution of 1,2-dichloro-ethene: a convenient stereoselective route to (9Z,11E)-, (10E,12Z)- and (10Z,12Z)-

Conjugated linoleic acid (CLA) isomers are present in human foods derived from milk or ruminant meat. To study their metabolism, (9Z,11E)-, (10E,12Z)- and (10Z,12Z)-[1-(14)C]-octadecadienoic acids with high radiochemical and isomeric purities (>98%) were prepared by stereoselective multi-step syntheses involving sequential substitution of 1,2-dichloro-ethene. In the case of the (9Z,11E) isomer, a first metal-catalyzed cross-coupling reaction between (E)-1,2-dichloro-ethene and 2-non-8-ynyloxy-tetrahydro-pyran, obtained from 7-bromo-heptan-1-ol, gave a conjugated chloroenyne. A second coupling reaction with hexylmagnesium bromide provided a heptadecenynyl derivative. Stereoselective reduction of the triple bond and bromination afforded (7E,9Z)-17-bromo-heptadeca-7,9-diene. Formation of the Grignard reagent and carbonation with 14CO(2) gave (9Z,11E)-[1-(14)C]-octadeca-9,11-dienoic acid (overall yield from 7-bromo-heptan-1-ol, 14.4%). (10E,12Z)- and (10Z,12Z)-[1-(14)C]-octadeca-10,12-dienoic acids were synthesized by the same methodology using 1-heptyne, 8-bromo-octan-1-ol and, respectively, (E)-1,2-dichloro-ethene and its (Z) isomer (overall yield from 8-bromo-octan-1-ol, 13.1% (10E,12Z); 17.2% (10Z,12Z)). Impurities (<2% if present) were identified as being (E,E) CLA isomers and were removed by RP-HPLC. Metabolism studies in animal are in progress.     

First total syntheses of (Z)-15-methyl-10-hexadecenoic acid and the (Z)-13-methyl-8-tetradecenoic acid

The first total syntheses for the (Z)-15-methyl-10-hexadecenoic acid and the (Z)-13-methyl-8-tetradecenoic acid were accomplished in seven steps and in 31-32% overall yields. The (trimethylsilyl)acetylene was the key reagent in both syntheses. It is proposed that the best synthetic strategy towards monounsaturated iso methyl-branched fatty acids with double bonds close to the omega end of the acyl chain is first acetylide coupling of (trimethylsilyl)acetylene to a long-chain bifunctional bromoalkane followed by a second acetylide coupling to a short-chain iso bromoalkane, since higher yields are thus obtained. Spectral data is also presented for the first time for these two unusual fatty acids with potential as biomarkers and as topoisomerase I inhibitors.     

LYCAT, a homologue of C. elegans acl-8, acl-9, and acl-10, determines the fatty acid composition of phosphatidylinositol in mice

Mammalian phosphatidylinositol (PI) has a unique fatty acid composition in that 1-stearoyl-2-arachidonoyl species is predominant. This fatty acid composition is formed through fatty acid remodeling by sequential deacylation and reacylation. We recently identified three Caenorhabditis elegans acyltransferases (ACL-8, ACL-9, and ACL-10) that incorporate stearic acid into the sn-1 position of PI. Mammalian LYCAT, which is the closest homolog of ACL-8, ACL-9, and ACL-10, was originally identified as a lysocardiolipin acyltransferase by an in vitro assay and was subsequently reported to possess acyltransferase activity toward various anionic lysophospholipids. However, the in vivo role of mammalian LYCAT in phospholipid fatty acid metabolism has not been well elucidated. In this study, we generated LYCAT-deficient mice and demonstrated that LYCAT determined the fatty acid composition of PI in vivo. LYCAT-deficient mice were outwardly healthy and fertile. In the mice, stearoyl-CoA acyltransferase activity toward the sn-1 position of PI was reduced, and the fatty acid composition of PI, but not those of other major phospholipids, was altered. Furthermore, expression of mouse LYCAT rescued the phenotype of C. elegans acl-8 acl-9 acl-10 triple mutants. Our data indicate that LYCAT is a determinant of PI molecular species and its function is conserved in C. elegans and mammals.     

Apparent relative retention of the phosphatidylethanolamine molecular species 18:0-20:5(n-3), 16:0-22:6(n-3) and the sum 16:0-20:4(n-6) plus 16:0-20:3(n-9) in the liver microsomes of pig on an essential fatty acid deficient diet.

Attempts at a better understanding of the cell membrane organization and functioning need to assess the physical properties which partly depend (i) on the positional distribution of the fatty acids in the membrane phospholipids (PLs) and (ii) on the way by which the PL molecular species are affected by exogenous fatty acids. To do that, the effects of essential (polyunsaturated) fatty acid (EFA) deficiency and enrichment were studied in the liver microsomes of piglets feeding on either an EFA-deficient diet or an EFA-enriched diet containing hydrogenated coconut oil or a mixture of soya + corn oils, respectively. After derivatization, the diacylated forms of choline and ethanolamine PLs were analyzed using a combination of chromatographic techniques and fast-atom bombardment-mass spectrometry. The dinitrobenzoyl-diacylglycerol derivatives corresponding to the molecular species of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were identified. It appears that three factors brought about a marked apparent relative retention: the nature of (i) the base of the polar head, (ii) fatty acids at the sn-1 position and (iii) fatty acids at the sn-2 position. The highest apparent relative retentions were displayed by the 18:0-20:5(n-3)-PE and 16:0-22:6(n-3)-PE. It is noteworthy that the behavior of 20:3 n-9--which is synthesized during the EFA-deficient diet by the same bioconversion system as 20:4 n-6--was very similar to that of 20:4 n-6 during the formation of PC and PE molecular species and that the molecular species of PE containing 20:4(n-6) and 20:3(n-9), gathered together as metabolical homologues, were also apparently retained, particularly in association with 16:0. Present observations are consistent with some others showing retention or preferential distribution of EFA in PE and suggest that specific acyltransferase(s), ethanolamine phosphotransferase and methyltransferase would be mainly involved for PE and PC formation in liver endoplasmic reticulum. Fast-atom bombardment-mass spectrometry of intact phospholipids enables us to show that there is no very long chain dipolyunsaturated phospholipid in liver endoplasmic reticulum.     

Free radical oxidation of coriolic acid (13-(S)-hydroxy-9Z,11E-octadecadienoic acid)

The reaction of (13S,9Z,11E)-13-hydroxy-9,11-octadecadienoic acid (1a), one of the major peroxidation products of linoleic acid and an important physiological mediator, with the Fenton reagent (Fe(2+)/EDTA/H(2)O(2)) was investigated. In phosphate buffer, pH 7.4, the reaction proceeded with >80% substrate consumption after 4h to give a defined pattern of products, the major of which were isolated as methyl esters and were subjected to complete spectral characterization. The less polar product was identified as (9Z,11E)-13-oxo-9,11-octadecadienoate (2) methyl ester (40% yield). Based on 2D NMR analysis the other two major products were formulated as (11E)-9,10-epoxy-13-hydroxy-11-octadecenoate (3) methyl ester (15% yield) and (10E)-9-hydroxy-13-oxo-10-octadecenoate (4) methyl ester (10% yield). Mechanistic experiments, including deuterium labeling, were consistent with a free radical oxidation pathway involving as the primary event H-atom abstraction at C-13, as inferred from loss of the original S configuration in the reaction products. Overall, these results provide the first insight into the products formed by oxidation of 1a with the Fenton reagent, and hint at novel formation pathways of the hydroxyepoxide 3 and hydroxyketone 4 of potential (patho)physiological relevance in settings of oxidative stress.