Isolation of brominated long-chain fatty acids from the phospholipids of the tropical marine spongeAmphimedon terpenensis

Preliminary investigation of the phospholipid fatty acid composition of the tropical marine spongeAmphimedon terpenensis by gas chromatography/mass spectrometry revealed the presence of some novel brominated fatty acids. Two new brominated fatty acids, (5E, 9Z)-6-bromo-5,9-tetracosadienoic acid (2a) and (5E, 9Z)-6-bromo-5,9-pentacosadienoic acid (3a) were subsequently isolated from a chloroform/methanol (3∶1, vol/vol) extract of the sponge and characterized as their methyl esters 2b and 3b. The known brominated fatty acid (5E, 9Z)-6-bromo-5,9-hexacosadienoic acid (4a) was also isolated. The new fatty acid methyl esters were confirmed as brominated δ5,9 acid derivatives by chemical ionization mass spectrometry. The position of the bromine substituent was determined to be C-6 by nuclear magnetic resonance techniques while the stereochemistry of the two double bonds was deduced by nuclear Overhauser enhancement difference spectroscopy. The biosynthetic implications of the co-occurrence of the three brominated acids are discussed.     

Novel brominated phospholipid fatty acids from the Caribbean spongeAgelas sp.

The very long-chain fatty acids, (5E,9Z)-6-bromo-5,9-tetracosadienoic, (5E,9Z)-6-bromo-23-methyl-5,9-tetracosadienoic, (5E,9Z)-6-bromo-5,9-pentacosadienoic and (5E,9Z)-6-bromo-24-methyl-5,9-pentacosadienoic acids, were identified in the phospholipids (mainly phosphatidylethanolamine) of the spongeAgelas sp. Structure elucidation was accomplished by means of mass spectrometry and chemical transformations, including deuteration with Wilkinson's catalyst. All of the sterols from the sponge had the Δ5,7 nucleus, with 24-methylcholesta-5,7,22-trien-3β-ol (ergosterol) and 24-ethylcholesta-5,7,22-trien-3β-ol being the most abundant.     

Sponge fatty acids. 3. Occurrence of series of n−7 monoenoic andiso-5,9 dienoic long-chain fatty acids in the phospholipids of the marine spongeCinachyrella aff.schulzei keller

The fatty acid composition of phospholipids from the New Caledonian spongeCinachyrella aff.schulzei Keller was studied. More than 60 fatty acids were identified as methyl esters andN-acyl pyrrolidides by gas chromatography and gas chromatography/mass spectrometry. Two isoprenoid fatty acids also were shown to be present, namely 4,8,12-trimethyltridecanoic and 5,9,13-trimethyltetradecanoic acids. The unusual 6-tetradecenoic, 6-pentadecenoic, 12-nonadecenoic and 26-methylheptacosanoic (iso-28∶0) acids were found for the first time in sponge phospholipids. A series of six n−7 monoenoic long-chain fatty acids (C₂₃ to C₂₈) were identified, including the rare 16-tricosenoic, 18-pentacosenoic and 21-octacosenoic acids. Fifteen fatty acids possessing the typical 5,9 dienoic moiety accounted for 30% of the total fatty acid mixture. Two new fatty acids were identified, namely 5(Z)-octacosenoic and 27-methyl-5(Z),9(Z)-octacosadienoic (iso-5,9-29∶2). Based on gas chromatography/Fourier transform infrared experiments, the double bonds were assigned the (Z) configuration. For part 2 of this series, see Reference 1.     

Identification of Novel α-Methoxylated Phospholipid Fatty Acids in the Caribbean Sponge <Emphasis Type="Italic">Erylus goffrilleri</Emphasis>

The phospholipid fatty acid composition of the Caribbean sponge Erylus goffrilleri is described for the first time. A total of 70 fatty acids with chain lengths between 13 and 29 carbons were identified in the sponge. Methyl-branched fatty acids predominated in E. goffrilleri suggesting the presence of a considerable number of bacterial symbionts. The novel fatty acids (5Z,9Z)-2-methoxy-5,9-hexadecadienoic acid, (5Z,9Z)-2-methoxy-5,9-octadecadienoic acid, (5Z,9Z)-2-methoxy-5,9-nonadecadienoic acid, and (5Z,9Z)-2-methoxy-5,9-eicosadienoic acid are described for the first time in the literature. In addition, the iso-methyl-branched fatty acids (9Z)-2-methoxy-15-methyl-9-hexadecenoic acid and (5Z,9Z)-2-methoxy-15-methyl-5,9-hexadecadienoic acid, also identified in E. goffrilleri, were identified for the first time in nature. Based on the identified metabolites it is proposed that the unprecedented biosynthetic sequence: i-17:1Δ9 → 2-OMe-i-17:1Δ9 → 2-OMe-i-17:2Δ5,9 might be responsible for the biosynthesis of the novel iso-α-methoxylated fatty acids in E. goffrilleri.     

Novel Cyclopropane Fatty Acids from the Phospholipids of the Caribbean Sponge <Emphasis Type="Italic">Pseudospongosorites suberitoides</Emphasis>

The cyclopropane fatty acids 17-methyl-trans-4,5-methyleneoctadecanoic acid, 18-methyl-trans-4,5-methylenenonadecanoic acid, and 17-methyl-trans-4,5-methylenenonadecanoic acid were characterized for the first time in nature in the phospholipids (mainly PE, PG and PS) of the hermit-crab sponge Pseudospongosorites suberitoides. Pyrrolidine derivatization was the key in identifying the position of the cyclopropyl and methyl groups in the acyl chains and ¹H NMR was used to determine the trans stereochemistry of the cyclopropane ring. The phospholipids from the sponge also contained an interesting series of iso-anteiso Δ^5,9 fatty acids with chain-lengths between 17 and 21 carbons, with the fatty acids (5Z,9Z)-18-methyl-5,9-nonadecadienoic acid and the (5Z,9Z)-17-methyl-5,9-nonadecadienoic acid being described for the first time in sponges. The anteiso α-methoxylated fatty acid 2-methoxy-12-methyltetradecanoic acid was also identified for the first time in nature in the phospholipids of this interesting marine sponge. The novel cyclopropyl fatty acids could have originated from the phospholipids of a cyanobacterium living in symbiosis with the sponge.     

2-Hydroxy fatty acids from marine sponges 2. The phospholipid fatty acids of the caribbean spongesVerongula gigantea andAplysina archeri

The α-hydroxy fatty acids 2-hydroxy-eicosanoic (1) acid, 2-hydroxyheneicosanoic (2) acid, 2-hydroxydocosanoic (3) acid, 2-hydroxytetracosanoic (4) acid, 2-hydroxy-23-methyl-tetracosanoic acid and 2-hydroxypentacosanoic (5) acid were isolated from the Caribbean spongesVerongula gigantea andAplysina archeri. The very long chain fatty acids 5,9-nonacosadienoic acid (29∶2) and 5,9,23-tricontatrienoic acid (30∶3) were also identified together with theiso-prenoid fatty acid 3,7,11,15-tetramethylhexadecanoic (phytanic) acid that seems to be common in the Aplysinidae.A. archeri contained an extremely long chain fatty acid tentatively characterized as dotricontaenoic (32∶1) acid. These acids were found to occur in phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, phosphatidylcholine and traces of phosphatidylglycerol.     

Identification of novel nonmethylene-interrupted fatty acids, 7E,13E-20∶2, 7E,13E,17Z-20∶3, 9E,15E,19Z-22∶3, and 4Z,9E,15E,19Z-22∶4, in ophiuroidea (Brittle star) lipids

Fatty acids of Ophiuroidea (brittle star) Ophiura sarsi have been investigated by gas-liquid chromatography (GLC). About 2–13% of four unidentified fatty acids were found in total fatty acids from a sample caught at a depth of 1,100 m. Structural analyses were undertaken after partial hydrogenation of their concentrates with hydrazine hydrate and subsequent isolation of the monoenoate products by argentation thin-layer chromatography. The structures of the unidentified fatty acids were determined as 7E,13E-eicosadienoic (20∶2), 7E,13E,17Z-eicosatrienoic (20∶3), 9E,15E,19Z-docosatrienoic (22∶3), and 4Z,9E,15E,19Z-docosatetraenoic (22∶4) acids by gas chromatography-mass spectrometry of dimethyl disulfide adducts and GLC of the monoenoates on a polar column. These fatty acids belong to a family of nonmethylene-interrupted (NMI) polyunsaturated fatty acids frequently observed in marine invertebrates and conifer seeds. As far as the authors know, however, these NMI fatty acid types with mixed geometry of ethylenic bonds have not been reported previously.     

Identification of the new 23-methyl-5,9-pentacosadienoic acid in the spongeCribrochalina vasculum

The phospholipid fatty acids from the spongeCribrochalina vasculum were studied, revealing the presence of the new 23-methyl-5,9-pentacosadienoic acid (26∶2) which completes theiso-anteiso 26∶2 (Δ5,9) series. Other phospholipid fatty acids isolated include 26-methyl-5,9-heptacosadienoic (28∶2) and 25- methyl-5,9-heptacosadienoic (28∶2), as well as the branched acids 8-methylhexadecanoic and 11-methyloctadecanoic. The fatty acids described in this work were found in phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol. The sterol composition ofC. vasculum consisted of petrosterol, and interesting cyclopropane containing sterol, and the more common sitosterol. The phospholipid fatty acid composition of the demospongeIrcinia strobilina is also reported upon.     

Total synthesis of 2-methoxy-14-methylpentadecanoic acid and the novel 2-methoxy-14-methylhexadecanoic acid identified in the sponge <Emphasis Type="Italic">agelas dispar</Emphasis>

The phospholipid FA composition of the Carbbean sponge Agelas dispar was revisited and 40 different FA were identified. Among these a novel 2-methoxylated FA, namely, the anteiso methyl-branched 2-methoxy-14-methylhexadecanoic acid, was identified together with the recently discovered iso methyl-branched 2-methoxy-14-methylpentadecanoic acid and the normal-chain 2-methoxytetradecanoic acid. The structures of the iso and anteiso methyl-branched 2-methoxylated FA were confirmed by total syntheses, which were accomplished in seven steps and in 45–48% overall yields. Other phospholipid FA identified in A. dispar include the unusual methyl-branched 10, 130dimethyltetradecanoic acid, 3,7,11,15-tetramethylhexadecanoic (phytanic) acid, and the 11-methyloctadecanoic acid. In addition, the Δ^5,9 FA (5Z, 9Z)-15-methyl-5,9-hexadecadienoic acid and (5Z, 9Z)-5,9-octadecadienoic acid were characterized. These findings establish alternative FA biosynthetic possibilities for these marine organisms.     

Structure and biosynthesis of novel conjugated polyene fatty acids from the marine green algaAnadyomene stellata

Novel polyunsaturated fatty acids with four conjugated double bonds were found in extracts of the green macroalga,Anadyomene stellata. The isolation of five of these with different chain lengths and varying degrees of unsaturation −16∶5, 18∶4, 20∶5, 20∶6, and 22∶7—was accomplished by organic extraction followed by a combination of vaccum and high-performarce liquid chromatography. One of these that was a novel substance (22∶7) was characterized as 4ZZ,7Z,9E,13Z,16Z,19Z-docosaheptaenoic acid and assigned the trivial name stellaheptaenoic acid. The structure of this new compound, isolated as its methyl ester derivative, was deduced from detailed nuclear magnetic resonance, gas chromatography/mass spectrometry (GC/MS), and other spectroscopic methods. Incubation of a chloroplast preparation, isolated from a crude algal homogenate by differential centrifugation, with six unsaturated fatty acids (palmitoleic, 6Z,9Z,12Z,15Z-octadecatetraenoic acid, arachidonic acid, eicosapentaenoic acid, 7Z,10Z,13Z,16Z-docosatetraenoic acid, and 4Z,7Z,10Z,13Z,16Z,19Z-docosahexaenoic acid) resulted in substantially increased synthesis of unique tetraene compouds as detected by ultraviolet spectrophotometry and tentatively identified by GC/MS.     

6-methyl-8-hexadecenoic acid: A novel fatty acid from the marine spongeDesmapsama anchorata

The novel fatty acid 7-methyl-8-hexadecenoic (1) was identified in the marine spongeDesmapsama anchorata. Other interesting fatty acids identified were 14-methyl-8-hexadecenoic (2), better known through its methyl ester as one of the components of the sex attractant of the female dermestid beetle, and the saturated fatty acid 3-methylheptadecanoic (3), known to possess larvicidal activity. The main phospholipid fatty acids encountered inD. anchorata were palmitic (16∶0), behenic (22∶0) and 5,9-hexacosadienoic acid (26∶2), which together accounted for 50% of the total phospholipid fatty acid mixture.     

On the isolation of 2-hydroxydocosanoic and 2-hydroxytricosanoic acids from the marine spongeAmphimedon compressa

The first α-hydroxy fatty acids from a marine sponge, namely 2-hydroxydocosanoic and 2-hydroxytricosanoic, were identified in the Caribbean spongeAmphimedon compressa. These acids were found to occur in phosphatidylethanolamine and phosphatidylserine and constituted 52% of the total fatty acid mixture of this sponge. The long chain fatty acids 5,9,23-nonacosatrienoic (29∶3) and 5,9,23-tricontatrienoic (30∶3), as well as a new tetratricontatetraenoic (34∶4) acid, were also found inA. compressa.     

A new cytotoxic fatty acid (5<Emphasis Type="Italic">Z</Emphasis>,9<Emphasis Type="Italic">Z</Emphasis>)-22-methyl-5,9-tetracosadienoic acid and the sterols from the far Eastern sponge <Emphasis Type="Italic">Geodinella robusta</Emphasis>

A new fatty acid, (5Z,9Z)-22-methyl-5,9-tetracosadienoic acid (1a), and a rare fatty acid, (5Z,9Z)-23-methyl-5,9-tetracosadienoic acid (2a), the predominant constituents of the free fatty acid fraction from the lipids of the sponge Geodinella robusta, were isolated and partly separated by reversed phase high-performance liquid chromatography, followed by multifold crystallization from MeOH to give 1a and 2a in 70% and 60% purity, respectively. These fatty acids were identified as (5Z,9Z)-22-and (5Z,9Z)-23-methyl-5,9-tetracosadienoic acids by nuclear magnetic resonance techniques, including distortionless enhancement by polarization transfer, heteronuclear multiple quantum connectivity, and correlation spectroscopy experiments, as well as from mass-spectrometric data for their methyl esters, the methyl esters of their perhydro derivatives, and their pyrrolidides. Mixtures of 1a and 2a showed cytotoxic activity against mouse Ehrlich carcinoma cells and a hemolytic effect on mouse erythrocytes. The sterol fraction from the same sponge was analyzed by gas liquid chromatography mass spectrometry, and 24-methylenecholesterol was identified as a main constituent of this fraction. The implications of the co-occurrence of membranolytic long-chain fatty acids and 24-methylenecholesterol as a main membrane sterol are discussed in terms of the phenomenon of biochemical coordination.     

New 17-Methyl-13-Octadecenoic and 3,16-Docosadienoic Acids from the Sponge <Emphasis Type="Italic">Polymastia penicillus</Emphasis>

The phospholipid fatty acid composition of the North-East Atlantic sponge Polymastia penicillus (South Brittany, France) was investigated. Sixty fatty acids (FA) were identified as methyl esters (FAME) and N-acyl pyrrolidides (NAP) by gas chromatography–mass spectrometry (GC/MS), including eight Δ5,9 unsaturated FA and three long-chain 2-hydroxylated FA. The major phospholipid FA were palmitic (14.3% of the total FA mixture), vaccenic (12.7%), 15(Z)-docosenoic (13.4%) and 5(Z),9(Z)-hexacosadienoic (13.3%) acids. In addition to the iso- and anteiso-branched saturated FA, several unusual short-chain branched saturated FA were identified. In addition to the known Δ5,9 FA, and interestingly regarding their identification by GC–MS as N-acyl pyrrolidides, was the co-occurrence of unusual FA possessing a Δ3, Δ4 and Δ5 double bond such as iso-4-pentadecenoic, iso-5-heptadecenoic, anteiso-5-heptadecenoic and two new compounds, not hitherto found in nature, namely 17-methyl-13-octadecenoic (0.8%) and 3,16-docosadienoic (1.1%) acids.     

Occurrence of 5,9,19-octacosatrienoic, 5,9-hexacosadienoic and 17-hexacosenoic acids in the marine spongeXestospongia halichondroides

The marine spongeXestospongia halichondroides contains 13% 28∶3Δ5,9,19, 27% 26∶2Δ5,9, and 21% 26∶1Δ17 in its total fatty acids. Thecis-5,cis-9,cis-19-octacosatrienoic acid structure is unknown in other living organisms.     

New phospholipid fatty acids from the Caribbean spongeEctyoplasia ferox

The phospholipid fatty acids from the Caribbean spongeEctyoplasia ferox were studied. The novel fatty acids 25-methyl-5,9-heptacosadienoic (1) and 26-methyl-5,9-heptacosadienoic (2) were identified in 3.4 and 2.0% abundance, respectively, representing the longest set of Δ5,9iso andanteiso acids yet isolated from a marine sponge. The new acid 10,13-dimethyltetradecanoic (3), the unusual acid 15-methyl-11-hexadecenoic (4) and the also novel acid 9-methyl-11-hexadecenoic (5) were also identified inE. ferox. The principal sterols isolated fromE. ferox were 24-ethylcholest-5-en-3β-ol (46%) and 24(R)-methylcholesta-5,22-dien-3β-ol (14%).     

Extracting long-chain fatty acids from a fermentation medium

Several solvents were evaluated for extracting free long-chain FA (LCFA) from a fermentation medium. Chloroform, chloroform/methanol (1∶1), hexane, and hexane/methyl tert-butyl ether (MTBE) (1∶1) were evaluated as alternative extraction solvents. Parameters considered for optimizing LCFA recoveries included pH and ionic strength. Maximal LCFA recoveries were obtained by adding 2 mL of the hexane/MTBE (1∶1) solvent mixture, 80 μL of 50% H₂SO₄, and 0.05 g NaCl to 1 mL of the aqueous sample and mixing for 15 min at 200 rpm. This method quantified saturated LCFA [capric acid (C_10∶0) to stearic acid (C_18∶0)] and unsaturated LCFA with 18 carbons [linoleic acid (C_18∶2) and oleic acid (C_18∶1)] with a 98 to 100% recovery. Caproic (C_6∶0) and caprylic (C_8∶0) acids were characterized by 27 and 76% recoveries, respectively.     

cis-5-olefinic unusual fatty acids in seed lipids of gymnospermae and their distribution in triacylglycerols

Open-tubular gas chromatographic analysis of fatty acids in the lipids from the seeds of 20 species of Gymnospermae showed that they all contained nonmethylene-interrupted polyenoic (NMIP) acids as minor components and palmitic, oleic, linoleic and α-linolenic acids as major components. The NMIP acids have an additional 5,6-ethylenic bond in ordinary plant unsaturated fatty acids and the following C₂ elongation acids:cis-5,cis-9-octadecadienoic acid (5,9–18∶2) (I); 5,9,12–18∶3 (II); 5,9,12,15–18∶4, 5,11–20∶2, 5,11,14–20∶3 (III); and 5,11,14,17–20∶4 (IV). The main NMIP acids found in neutral lipids are I in two species ofTaxus, II in seven species of Pinaceae, III in two species of Podocarpaceae,Torreya nucifera, Cycas revoluta, andGinkgo biloba, and III and IV in each of three species of Taxodiaceae, and Cupressaceae. The polar lipids constitute the minor fraction of seed lipids in general. The content and composition of NMIP acids in these lipids differe considerably from those in neutral lipids. Analysis of the partial cleavage products of triacylglycerols showed that the NMIP acids distribute mainly in the 1,3-position.     

Cell membrane localization of long chain C24−C30 fatty acids in two marine demosponges

Subcellular fractionation by differential centrifugation was performed on two previously unstudied marine sponges (Reniera sp. andPseudaxinyssa sp.) that represent both major subclasses of the Demospongiae. Long chain fatty acids (LCFA) with 24–30 carbon units were found as major constituents of cell membrane isolates of both sponges. Most LCFA were polyunsaturated and were constituents of the phospholipids, which are typical membrane lipids, and in particular the amino-phospholipids. The LCFA composition of phospholipids from whole sponge tissue was shown to provide a reliable indication of the LCFA composition of cell membrane phospholipids in the sponges studied. An unusual triply branched C₁₆ isoprenoid fatty acid, 4,8,12-trimethyltridecanoic acid, also was identified as a cell membrane acid in the spongePseudaxinyssa sp. Part 16 of “Phospholipid Studies of Marine Organisms.” For Part 15 in this series see Dasgupta, A., Ayanoglu, E., Tomer, K.B., and Djerassi, C. (1987)Chem. Phys. Lipids 43, 101–111.     

On the isolation of the new fatty acid 6,11-eicosadienoic (20∶2) and related 6,11-dienoic acids from the spongeEuryspongia rosea

The phospholipid fatty acids from the spongeEuryspongia rosea were studied revealing the presence of the new 6,11-eicosadienoic (20∶2) acid and the rare 6,11-octadecadienoic acid (18∶2). The isolation of these 6,11-dienoic acids reveals the presence of new biosynthetic possibilities in sponges and suggests the presence of an active Δ6 desaturase inE. rosea. Other acids isolated fromE. rosea include 12-eicosenoic (20∶1) and 17-tetracosenoic acid (24∶1). The major phospholipids encountered in the sponge were phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidylcholine (PC).