Facile syntheses for (5Z,9Z)-5,9-hexadecadienoic acid, (5Z,9Z)-5,9-nonadecadienoic acid, and (5Z,9Z)-5,9-eicosadienoic acid through a common synthetic route

The delta 5,9 fatty acids (5Z,9Z)-5,9-hexadecadienoic acid, (5Z,9Z)-5,9-nonadecadienoic acid, and (5Z,9Z)-5,9-eicosadienoic acid were synthesized for the first time in four steps (9-12% overall yield) starting from commercially available 2-(2-bromoethyl)-1,3-dioxolane. The synthetic approach provided enough material to corroborate the structure and stereochemistry of (5Z,9Z)-5,9-nonadecadienoic acid which was recently identified in the flowers of Malvaviscus arboreus (Malvaceae). The novel phospholipids 1-hexadecanoyl-2-[(5Z,9Z)-5,9-eicosadienoyl]-sn-glycer o-3-phosphocholine and 1-octadecanoyl-2-[(5Z,9Z)-5,9-eicosadienoyl]-sn- glycero-3-phosphocholine were also synthesized from commercially available L-alpha-phosphatidylcholine (egg yolk) and characterized by positive ion electrospray mass spectrometry. These are the first examples of unsymmetrical phospholipids with saturated fatty acids at the sn-1 position and delta 5,9 fatty acids at the sn-2 position.     

Metabolism of unusual membrane phospholipids in the marine sponge Microciona prolifera

Sponges are unique in regard to membrane phospholipid composition. Features virtually without parallel in other organisms are the predominance of the C26-C30 polyenoic acids (demospongic acids) in the phosphatidylethanolamines (PE) and the attachment of identical acyl groups to the glycerol moiety. The biosynthesis and disposition of these unusual phospholipids were followed in the marine sponge Microciona prolifera where PE ( delta 5,9-26:2, delta 5,9-26:2) is a major molecular species. Incorporation experiments with radiolabeled fatty acids, bases, and intact phospholipids revealed the de novo biosynthesis of the two major phosphatides, phosphatidylethanolamines (PE) and phosphatidylcholines (PC), via the cytidine pathway as in higher animals, with ethanolamine selectively incorporated into PE( delta 5,9-26:2, delta 5,9-26:2). Methylation of PE and random acyl chain migration across different phospholipid classes were marginal, but the exchange of PC for PE, apparently mediated by the action of phospholipase, was indicated after uptake of the unnatural PC( delta 9-27:1, delta 9-26:1). The present study demonstrates in the most primitive multicellular animals a phospholipid metabolic pattern similar to that in higher organisms, with unique acyl and phosphoethanolamine transferases apparently involved in the biosynthesis of the (demospongic) di-C26-acyl-PE molecular species.     

Marine natural products from the Turkish sponge Agelas oroides that inhibit the enoyl reductases from Plasmodium falciparum, Mycobacterium tuberculosis and Escherichia coli

The type II fatty acid pathway (FAS-II) is a validated target for antimicrobial drug discovery. An activity-guided isolation procedure based on Plasmodium falciparum enoyl-ACP reductase (PfFabI) enzyme inhibition assay on the n-hexane-, the CHCl(3-) and the aq MeOH extracts of the Turkish marine sponge Agelas oroides yielded six pure metabolites [24-ethyl-cholest-5alpha-7-en-3-beta-ol (1), 4,5-dibromopyrrole-2-carboxylic acid methyl ester (2), 4,5-dibromopyrrole-2-carboxylic acid (3), (E)-oroidin (4), 3-amino-1-(2-aminoimidazoyl)-prop-1-ene (5), taurine (6)] and some minor, complex fatty acid mixtures (FAMA-FAMG). FAMA, consisting of a 1:2 mixture of (5Z,9Z)-5,9-tricosadienoic (7) and (5Z,9Z)-5,9-tetracosadienoic (8) acids, and FAMB composed of 8, (5Z,9Z)-5,9-pentacosadienoic (9) and (5Z,9Z)-5,9-hexacosadienoic (10) acids in approximately 3:3:2 ratio were the most active PfFabI inhibitory principles of the hexane extract (IC(50) values 0.35 microg/ml). (E)-Oroidin isolated as free base (4a) was identified as the active component of the CHCl(3) extract. Compound 4a was a more potent PfFabI inhibitor (IC(50) 0.30 microg/ml=0.77 microM) than the (E)-oroidin TFA salt (4b), the active and major component of the aq MeOH extract (IC(50) 5.0 microg/ml). Enzyme kinetic studies showed 4a to be an uncompetitive PfFabI inhibitor (K(i): 0.4+/-0.2 and 0.8+/-0.2 microM with respect to substrate and cofactor). In addition, FAMA and FAMD (mainly consisting of methyl-branched fatty acids) inhibited FabI of Mycobacterium tuberculosis (MtFabI, IC(50)s 9.4 and 8.2 microg/ml, respectively) and Escherichia coli (EcFabI, IC(50)s 0.5 and 0.07 microg/ml, respectively). The majority of the compounds exhibited in vitro antiplasmodial, as well as trypanocidal and leishmanicidal activities without cytotoxicity towards mammalian cells. This study represents the first marine metabolites that inhibit FabI, a clinically relevant enzyme target from the FAS-II pathway of several pathogenic microorganisms.     

Mass spectral behavior and HPLC of some unusual molecular phospholipid species

The molecular species of the major phospholipids from the marine sponges Parasperella psila and Microciona prolifera were studied using chemical hydrolysis, enzymatic degradation and capillary gas chromatography (GC), high performance liquid chromatography (HPLC), desorption chemical ionization (DCI), fast atom bombardment (FAB) combined with collisionally activated decomposition (CAD) mass spectrometry. Two new solvent systems were developed for the isolation of these species from the sponges. Our investigations indicated the existence of unusual symmetrical phospholipids as major components. 1,2-Di-(5Z,9Z)-5,9-hexacosadienoyl-sn-glycero-3-phosph oethanolamine was found in both organisms, while 1,2-di(5Z,9Z,19Z)-5,9,19-hexacosatrienoyl-sn-gly cero-3-phosphoethanolamine was present in M. prolifera, 1,2-Di-(4Z,7Z,10Z,13Z,16Z,19Z)-4,7,1 0,13,16,19-docosahexaenoyl-sn-glycero-3- phosphocholine was the major molecular species in the PC fraction of M. prolifera.     

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.     

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.     

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.     

Fatty acids from the sponge Halichondria panicea from the Sea of Japan

The fatty acid (FA) composition of total lipids isolated from the marine sponge Halichondria panicea inhabiting Peter the Great Bay of Sea of Japan was studied. GC and GC-MS techniques helped identify 63 FAs, with the main attention being paid to FAs with 14-22 carbon atoms. 4, 8, 12-Trimethyl-13:0 FA was for the first time identified as the main saturated FA along with the branched FAs br-25:1, br-27:1, and br-27:2. The contents of arachidonic, eicosapentaenoic, docosapentaenoic, and the major demospongic acids [26:3(5, 9, 19), 26:3(5, 9, 17), 27:3(5, 9, 20), and 28:3(5, 9, 21)] considerably differed from those previously found for H. panicea, which may be due to seasonal changes in the species composition of organisms consumed by the sponge.     

The Composition of fatty acids and aldehydes of the marine bryozoans <Emphasis Type="Italic">Berenicea meandrina</Emphasis> and <Emphasis Type="Italic">Dendrobeania flustroides</Emphasis> (Bryozoa: Gymnolaemata)

This study examines the composition of lipids, fatty acids, and fatty aldehydes in two marine bryozoan species, Berenicea meandrina and Dendrobeania flustroides, from the Sea of Okhotsk. The share of neutral lipids was up to 57.3% in D. flustroides and 54.9% in B. meandrina; the share of polar lipids was 33.2 and 40.4%, respectively. In all, 57 fatty acids (FA) and 9 aldehydes were identified in total lipids. The main FAs were 16:0, 18:0, 22:6n-3, and 20:5n-3. The content of branched saturated FA in bryozoans was on the average 6.4%. Three isomers of 16:1 (n-9, n-7, and n-5), five isomers of 18:1 (n-13, n-11, n-9, n-7, and n-5), four isomers of 20:1 (n-13, n-11, n-9, and n-7), as well as 22:1n-9 and 22:1n-13 were found; the presence of 7-methyl-6-hexadienoic acid (on the average, 3.0% of total FAs) was demonstrated. Non-methylene-inter-rupted FAs contributed 8.9 and 1.6% of the total FAs in D. flustroides and B. meandrina, respectively, and were identified as 20:2(5,11), 20:2(7,13), 20:3(5,11,14), 22:2(7,13), and 22:2(7,15). In B. meandrina, minor amounts of 24:0, 24:1, 25:0, 26:0, 24:4n-3, 26:3(5,9,19), and 28:3(5,9,19) were found, suggesting sponge biofouling on some bryozoan colonies. Aldehydes (branched saturated and unsaturated C_16–19 homologues) did not exceed 10.3 and 1.9% of the total FAs in D. flustroides and B. meandrina, respectively. The presence of the FA markers that are characteristic of microalgae, protozoans, and detritus in bryozoan lipids agrees well with data on polytrophic feeding of these bryozoans.     

Fatty Acids from the Sea of Japan Sponge Halichondria panicea

The fatty acid (FA) composition of total lipids isolated from the marine sponge Halichondria panicea inhabiting Peter the Great Bay of the Sea of Japan was studied. GC and GC-MS techniques were used in identification of 63 FAs, with the main attention being paid to FAs with 14–22 carbon atoms. 4,8,12-Trimethyl-13:0 FA was for the first time identified as a main saturated FA, along with the branched FAs br-25:1, br-27:1, and br-27:2. The contents of arachidonic, eicosapentaenoic, docosapentaenoic, and the major demospongic acids [26:3(5,9,19), 26:3(5,9,17), 27:3(5,9,20), and 28:3(5,9,21)] considerably differed from those previously found for H. panicea, which may be due to seasonal changes in the species composition of organisms consumed by the sponge.     

Comparative study of the endemic freshwater fauna of Lake Baikal—VI. Unusual fatty acid and lipid composition of the endemic sponge Lubomirskia baicalensis and its amphipod crustacean parasite Brandtia (Spinacanthus) parasitica

Lipids and phospholipids (both plasmalogen and alkyl forms) of the freshwater sponge Lubomirskia baicalensis and the sponge's gammarid parasite Brandtia (Spinacanthus) parasitica were examined. Composition of alkenyl-acyl (plasmalogen), alkylacyl and diacyl forms of major phospholipid classes, phosphatidylethanolamine and phosphatidylcholine were determined. One hundred and eighty-three fatty acids were identified by GC-MS: 46 saturated, 55 monoenoic, 35 dienoic, 25 trienoic and 22 tetra-, penta- and hexaenoic. The freshwater sponges, belonging to the family Lubomirskiidae, were shown to contain unusual long-chain fatty acids: anteiso-5, 9–28:2, branched-5, 9–29:2, 5,9,23–29:3, 5,9,23–30:3, 15,18,21,24–30:4 and 15,18,21,24,27–30:5. Some from these fatty acids were found in lipids of the amphipod parasite.     

Biophysical properties of unusual phospholipids and sterols from marine invertebrates

Liposomes composed of 1,2-di-(5Z,9Z)-5-9-hexacosadienoyl-sn-glycero-3-pho sph ocholine underwent an endothermic phase transition at 42 degrees C. Cholesterol or the marine sterols studied did not affect this transition to an appreciable extent, but rather were excluded from the phospholipid bilayers. Membranes composed of 1,2-di-(5Z,9Z)-5,9-hexacosadienoyl-sn-glycero-3-pho sph ocholine displayed very similar phase properties. Effects of the marine sterols on dipalmitoylphosphatidylcholine bilayers were also investigated.     

Epicuticular hydrocarbons of Drosophila pseudoobscura (Diptera; Drosophilidae) Identification of unusual alkadiene and alkatriene positional isomers

Epicuticular hydrocarbons of Drosophila pseudoobscura were analyzed by gas chromatography and mass spectrometry. Methyl-branched alkanes and alkadienes were the predominant hydrocarbons, with lesser amounts of monoenes (14%) and trienes (9%) also present. Alkanes (49%) were mostly odd carbon number 2-methylalkanes (C₂₅–C₃₁). Alkadienes (27%) were odd carbon number components (C₂₅–C₃₃), with the (Z,Z)-5,9-isomer predominating. Monounsaturated hydrocarbons were a mixture of 5-, 7-, 9-, 11-, 13-, 14- and 15-isomers containing 25–33 carbon atoms. The major alkatriene components contained 29–31 carbon atoms and were either 5,19,17- or 5,9,19-isomers. Sodium[1-¹⁴C]acetate was incorporated into each class of hydrocarbon and into each of the major alkadienes.     

Moth desaturase characterized that produces both Z and E isomers of delta 11-tetradecenoic acids

The redbanded leafroller moth, Argyrotaenia velutinana (Lepidoptera: Tortricidae) uses a 92:8 mixture of (Z)-11- and (E)-11-tetradecenyl acetate in its pheromone blend. These are produced in the abdominal pheromone gland from the corresponding acids, which are biosynthesized in the gland in a 3:2 Z/E ratio by desaturation of myristoyl CoA. The delta 11 desaturase involved in this reaction exhibits unusual substrate and stereospecificities in specifically producing Z11 and E11 isomers of tetradecenoic acid, and exhibiting no activity with C16 and C18 precursor acids. This report describes the cloning and expression of the redbanded leafroller moth delta 11 desaturase, and compares its amino-acid sequence to those of other known insect Z9, Z10, Z11, and E11 desaturases. The metabolic Z9 desaturase from fat body tissue also was cloned and expressed, and found mainly to produce Z9-16:Acid and Z9-18:Acid. The open reading frame of the delta 11 desaturase encodes a protein with 329 amino acids, whereas the open reading frame of the Z9 desaturase encodes a protein with 351 amino acids. Addition of this new delta 11 desaturase with its different substrate and regiospecificites to the databank of characterized integral-membrane desaturases will be key in efforts to determine amino-acid mutations responsible for the wide array of unsaturated fatty-acid products.     

Synthesis of 5,9-hexacosadienoic acid phospholipids. 11. Phospholipid studies of marine organisms

The synthesis of phosphatidylcholines (PC), phosphatidylethanolamines (PE) and phosphatidylserines (PS) containing two acyl chains of the naturally occurring sponge fatty acid (5Z,9Z)-5,9-hexacosadienoic acid as well as its hitherto unknown geometrical isomers is described. The PCs were prepared by deacylation of natural lecithins, followed by reacylation with fatty acid anhydrides. The synthesis of mixed-acid PCs is also reported: a diacyl product was converted to the lyso-PC by treatment with phospholipase A2 and subsequent acylation of the secondary hydroxyl group to give the desired mixed-acid PCs. The PEs and the PSs were prepared from the corresponding PCs by enzymatic transphosphatidylation catalyzed by phospholipase D. Structural assignments of the compounds were confirmed by spectroscopy (1H-NMR and MS). Ammonia chemical ionization mass spectrometry provided molecular ion and significant fragment peaks for PCs and PEs.     

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.     

Fatty acid composition of four microsporidian species compared to that of their host fishes

ABSTRACT. The fatty acid composition of four microsporidian species (Glugea atherinae, Spraguea lophii, Glugea americanus , and Pleistophora mirandellae) and their host fishes has been determined using gas chromatography. Twenty-four fatty acids were identified with differences in relative abundance of fatty acids among the four parasites. Certain even-saturated fatty acids were found in a very high proportion: palmitic acid (16:0) represented one-third of total fatty acids in Pleistophora mirandellae. The level of docosahexaenoic acid (22:6ω3) attained 26–28% in Glugea atherinae, Spraguea lophii , and Glugea americanus , but only 8–9% in P. mirandellae. With respect to fatty acid compositions of host organs, some significant differences were evident between marine and freshwater fishes. Palmitic acid was prevalent in the marine fishes, Atherinae boyeri and Lophius piscatorius , and oleic acid (18:1ω9) in the freshwater fish Leuciscus cephalus. The proportion of docosahexaenoic acid in marine fishes was two or three times as great as in freshwater fish Leuciscus. The high polyunsaturated fatty acid content in both parasites and host fishes may be related to the scavenging of these fatty acids by the parasites rather than a microsporidia-specific fatty acid biosynthesis pathway.     

Pheromone biosynthetic pathways in the moths Helicoverpa zea and Helicoverpa assulta

Sex pheromones of many Lepidopteran species have relatively simple structures consisting of a hydrocarbon chain with a functional group and usually one to several double bonds. The sex pheromones are usually derived from fatty acids through a specific biosynthetic pathway. We investigated the incorporation of deuterium-labeled palmitic and stearic acid precursors into pheromone components of Helicoverpa zea and Helicoverpa assulta. The major pheromone component for H. zea is (Z)11-hexadecenal (Z11-16:Ald) while H. assulta utilizes (Z)9-hexadecenal (Z9-16:Ald). We found that H. zea uses palmitic acid to form Z11-16:Ald via delta 11 desaturation and reduction, but also requires stearic acid to biosynthesize the minor pheromone components Z9-16:Ald and Z7-16:Ald. The Z9-16:Ald is produced by delta 11 desaturation of stearic acid followed by one round of chain-shortening and reduction to the aldehyde. The Z7-16:Ald is produced by delta 9 desaturation of stearic acid followed by one round of chain-shortening and reduction to the aldehyde. H. assulta uses palmitic acid as a substrate to form Z9-16:Ald, Z11-16:Ald and 16:Ald. The amount of labeling indicated that the delta 9 desaturase is the major desaturase present in the pheromone gland cells of H. assulta; whereas, the delta 11 desaturase is the major desaturase in pheromone glands of H. zea. It also appears that H. assulta lacks chain-shortening enzymes since stearic acid did not label any of the 16-carbon aldehydes.     

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.     

Regulation of sialic acid O-acetylation in human colon mucosa

The expression of O-acetylated sialic acids in human colonic mucins is developmentally regulated, and a reduction of O-acetylation has been found to be associated with the early stages of colorectal cancer. Despite this, however, little is known about the enzymatic process of sialic acid O-acetylation in human colonic mucosa. Recently, we have reported on a human colon sialate-7(9)-O-acetyltransferase capable of incorporating acetyl groups into sialic acids at the nucleotide-sugar level [Shen et al., Biol. Chem. 383 (2002), 307-317]. In this report, we show that the CMP-N-acetyl-neuraminic acid (CMP-Neu5Ac) and acetyl-CoA (AcCoA) transporters are critical components for the O-acetylation of CMP-Neu5Ac in Golgi lumen, with specific inhibition of either transporter leading to a reduction in the formation of CMP-5-N-acetyl-9-O-acetyl-neuraminic acid (CMP-Neu5,9Ac2). Moreover, the finding that 5-N-acetyl-9-O-acetyl-neuraminic acid (Neu5,9Ac2 could be transferred from neo-synthesised CMP-Neu5,9Ac2 to endogenous glycoproteins in the same Golgi vesicles, together with the observation that asialofetuin and asialo-human colon mucin are much better acceptors for Neu5,9Ac2 than asialo-bovine submandibular gland mucin, suggests that a sialyltransferase exists that preferentially utilises CMP-Neu5,9Ac2 as the donor substrate, transferring Neu5,9Ac2 to terminal Galbeta1,3(4)R- residues.