Synthesis of 3-oxalinolenic acid and β-oxidation-resistant 3-oxa-oxylipins

3-Oxalinolenic acid (3-oxa-9(Z), 12(Z), 15(Z)-octadecatrienoic acid or (6(Z), 9(Z), 12(Z)-pentadecatrienyloxy)acetic acid) was synthesized from 5(Z), 8(Z), 11(Z), 14(Z), 17(Z)-eicosapentaenoic acid by a sequence involving the C15 aldehyde 3(Z), 6(Z), 9(Z), 12(Z)-pentadecatetraenal as a key intermediate. Conversion of the aldehyde by isomerization and two steps of reduction afforded 6(Z), 9(Z), 12(Z)-pentadecatrienol, which was coupled to bromoacetate to afford after purification by HPLC >99%-pure 3-oxalinolenic acid in 10–15% overall yield. 3-Oxalinolenic acid was efficiently oxygenated by soybean lipoxygenase-1 into 3-oxa-13(S)-hydroperoxy-9(Z), 11(E), 15(Z)-octadecatrienoic acid, and this hydroperoxide could be further converted chemically into 3-oxa-13(S)-hydroxy-9(Z), 11(E), 15(Z)-octadecatrienoic acid and 3-oxa-13-oxo-9(Z), 11(E), 15(Z)-octadecatrienoic acid. The 3-oxa-hydroperoxide also served as the substrate for the plant enzymes allene oxide synthase, divinyl ether synthase, and hydroperoxide lyase to produce 3-oxa-12-oxo-10, 15(Z)-phytodienoic acid and other 3-oxa-oxylipins that were characterized by MS, 3-Oxalinolenic acid was not oxygenated by 9-lipoxygenase from tomato but was converted at a slow rate into 3-oxa-9(S)-hydroperoxy-10(E), 12(Z), 15(Z)-octadecatrienoic acid by recombinant maize 9-lipoxygenase. Recombinant α-dioxygenase-1 from Arabidopsis thaliana catalyzed the conversion of 3-oxalinolenic acid into a 2-hydroperoxide, which underwent spontaneous degradation into a mixture of 6,9,12-pentadecatrienol and 6,9,12-pentadecatrienyl formate. A novel α-dioxygenase from the moss Physcomitrella patens was cloned and expressed and was found to display the same activity with 3-oxalinolenic acid as Arabidopsis thaliana α-dioxygenase-1. Lipoxygenase-generated 3-oxa-oxylipins are resistant toward β-oxidation and have the potential for displaying enhanced biological activity in situations where activity is limited by metabolic degradation.     

Synthesis of 9,12-Dioxo-10(Z)-dodecenoic acid, a new fatty acid metabolite derived from 9-hydroperoxy-10,12-octadecadienoic acid in lentil seed (Lens culinaris medik.)

The previously unknown linoleic acid peroxidation product 9,12-dioxo-10(Z)-decenoic acid (Z5) was detected in lentil seed fluor (Lens culinaris Medik.) by electron impact mass spectrometry (El-MS) after derivatization with pentafluorobenzyl-hydroxylamine-hydrochloride, methylation of acidic groups with diazomethane, and protection of hydroxylic groups with N-methyl-N-trimethylsilyl-trifluoroacetamide. The structure of the natural product was confirmed by synthesis of Z5, 9,12-dioxo-10(E)-decenoic acid, and derivatives. EI-MS, nuclear magnetic resonance and gas chromatographic data of these compounds and synthetic intermediates are discussed.     

Reinvestigation of the antioxidant properties of conjugated linoleic acid

Despite repeated suggestions that antioxidant activity of conjugated linoleic acid (CLA), a collective of conjugated dienoic isomers of linoleic acid, underlies its reported anticarcinogenic and antiatherosclerotic effects, the antioxidant properties of CLA remain ill-defined. Therefore, this study was undertaken to gain more insight into the mechanism of potential CLA antioxidant activity. It was tested whether CLA could protect membranes composed of 1-palmitoyl-2-linoleoyl phosphatidylcholine (PLPC) from oxidative modification under conditions of metal ion-dependent or-independent oxidative stress. Progress of oxidation was determined by direct spectrophotometric measurement of conjugated diene formation and by gas chromatographic/mass spectrometric analysis of fatty acids. The oxidative susceptibility of CLA was higher than that of linoleic acid, and comparable to arachidonic acid. When oxidation of PLPC (1.0 mM) was initiated using the lipid-soluble 2,2′-azobis(2,4-dimethylvaleronitrile) or the water-soluble 2,2′-azobis(2-amidinopropane) hydrochloride, the radical scavengers vitamin E and butylated hydroxytoluene (BHT) at 0.75 μM efficiently inhibited PLPC oxidation, as evident from a clear lagphase. In contrast, 0.75 μM CLA did not have any significant effect on PLPC oxidation. Inhibition of PLPC oxidation by higher concentrations of CLA appeared due to competition, not to an antioxidant effect. When oxidation of PLPC was initiated by hydrogen peroxide/Fe²⁺ (500 μM/0.05–20 μM), both vitamin E (1 μM) and ethylene glycol-bis(aminoethyl ether) tetraacetic acid (50 μM) efficiently inhibited PLPC oxidation. However, CLA (1–50 μM) did not show a clear protective effect under any of the conditions tested. We conclude that CLA, under these test conditions, does not act as an efficient radical scavenger in any way comparable to vitamin E or BHT. CLA also does not appear to be converted into a metal chelator under metal-ion dependent oxidative stress, as had previously been suggested. On the basis of our observations, a role for CLA as an antioxidant does not seem plausible.     

Stereospecific synthesis of (Z,Z)-11,13-hexadecadienal,a female sex pheromone of the navel orangeworm,Amyelois transitella (Lepidoptera: Pyralidae)

A synthesis of (Z,Z)-11,13-hexadecadienal, a compound previously identified as the major component of the sex pheromone of the female navel orangeworm,Amyelois transitella (Walker), is described. The key step is the reduction of an unsymmetrical conjugated diyne with dicyclohexylborane. This reduction produced theZ,Z isomer with less than 0.5% total of other isomers.Mention of a commercial or proprietary product in this paper does not constitute endorsement of that product by the USDA.     

Structure and intraglyceride distribution of coriolic acid

Coriolic [(R)-13-hydroxy-cis-9,trans-11-octadecadienoic] acid (III, R=Z=H) was isolated as the methyl ester from twoCoriaria seed oils in 66 and 68% yields. The double bonds and hydroxyl group were located by periodate-permanganate oxidation before, and chromic acid oxidation after, hydrogenation of the double bonds. Alternatively the positions of the functional groups were indicated by a convenient micro-ozonolysis-gas-liquid chromatographic procedure. Determination of products from partial hydrolysis of theCoriaria oils with pancreatic lipase (EC 3.1.1.3) revealed a preference of the corioloyl group for the 1,3-positions in triglyceride molecules. The possible significance of coriolic acid as an intermediate in the biogenetic conversion of linoleic acid to conjugated trienoic acids is discussed. Presented in part at the 154th American Chemical Society meeting, Chicago, Ill., September 1967. No. Utiliz. Res. Dev. Div., ARS, USDA.     

Two new icosapentaenoic acids from the temperate red seaweedPtilota filicina J. Agardh

Two new fatty acid metabolites, 5(Z),7(E),9(E),14(Z),17(Z)-icosapentaenoic acid and 5(E),7(E),9(E),14(Z),17(Z)-icosapentaenoic acid, have been isolated from the temperate red marine alga,Ptilota filicina (Ceramiales, Rhodophyta). The structures of these new compounds, isolated as their methyl ester derivatives, have been deduced from detailed¹H nuclear magnetic resonance (NMR),¹³C NMR and 2D-NMR analyses as well as comparisons to known compounds.     

Formation of 12-[18O]Oxo-cis-10,cis-15-phytodienoic acid from 13-[18O]hydroperoxylinolenic acid by hydroperoxide cyclase

13-[¹⁸O] Hydroperoxylinolenic acid was permitted to react with an extract of flaxseed acetone powder containing hydroperoxide cyclase activity. The resulting product, 12-oxo-cis-10,cis-15-phytodienoic acid (12-oxo-PDA), contained¹⁸O in the carbonyl oxygen at carbon 12, suggesting that an epoxide was an intermediate in the hyderoperoxide cyclase reaction. A substrate specificity study showed that acis double bond β,γ to the conjugated hydroperoxide group was essential for the substrate to be converted to a cyclic product by hydroperoxide cyclase.     

Production of polyhydroxy fatty acids from linoleic acid by Clavibacter sp. ALA2

Hydroxy fatty acids are important industrial materials. We isolated a microbial culture, Clavibacter sp. ALA2, which converts linoleic acid to many polyhydroxy fatty acids. Structures of the products were determined as: 12,13,17-trihydroxy-9(Z)-octadecenoic (THOA, main product), 12-[5-ethyl-2-tetrahydrofuranyl]-7,12-dihydroxy-9(Z)-dodecenoic (ETDDA), and 12-[5-ethyl-2-tetrahydrofuranyl]-12-hydroxy-9(Z)-dodecenoic (ETHDA) acid. The yield of THOA was 25% and the relative amount of the products were THOA/ETDDA/ETHDA =9:1.3:1. The structures of the hydroxy unsaturated fatty acids resemble those of plant self-defense substances.     

Syntheses of conjugated octadecadienoic acids

Three approaches for the synthesis of octadecadienoic acids with conjugated double bond systems are presented: synthesis of (10Z, 12Z)-octadecadienoic acid via an enyne-substructure; the use of an educt with a conjugated double bond system for the synthesis of (10E, 12E-octadecadienoic acid; and the Suzuki cross coupling for the synthesis of (7E,9Z)-octadecadienoic acid.     

Conjugated linoleic acid and oxidative stress

At the present time, conjugated linoleic acid (CLA) is the subject of a growing number of studies since it has been demonstrated to possess anticarcinogenic and antiatherogenic activities in experimental animal models and to increase in some pathological states in humans. In both situations, CLA has been claimed to be involved in oxidative stress, as an antioxidant in the first case and as a primary product of a free-radical attack on polyunsaturated fatty acids (PUFA) in the other. The controversial results are due mostly to a lack of a suitable methodology because the presence of conjugated dienes (CD) in lipid moiety has been taken for years as evidence of lipid peroxidation due to the occurrence of this structure in fatty acid hydroperoxides. We have recently developed a new methodology that consists of the extraction of fatty acids, including CD fatty acid hydroperoxides, by mild saponification and their separation and identification by high-performance liquid chromatography with diode array detector. Fatty acid analyses of liver homogenate, oxidized in vitro either with Fe-ADP or t-butyl hydroperoxide (t-ButylHP), of lamb and rats fed CLA at levels known to prevent carcinogenesis, showed that CLA and its metabolites steadily decreased during oxidative stress and that they are more prone to oxidation than their corresponding methylene-interrupted fatty acids. No significant antioxidant effect of CLA was detected in any model tested. However, CD fatty acid hydroperoxides increased in the t-ButylHP model but not in the Fe-ADP model, owing probably to the degradation of CD fatty acid hydroperoxides induced by this oxidative agent. In conclusion, CLA and its metabolites seem to behave, under oxidative stress, as regular PUFA. Thus, it is highly unlikely that the peculiar effects of CLA are directly related to interference in lipoperoxidative processes.     

Production of 3R-hydroxy-polyenoic fatty acids by the yeast Dipodascopsis uninucleata

Various fatty acids were fed to the yeast Dipodascopsis uninucleata UOFS Y 128, and the extracted samples were analyzed for the accumulation of 3-hydroxy metabolites with the help of electron impact gas chromatography-mass spectrometry. Fatty acids containing a 5Z,8Z-diene system (5Z,8Z,11Z-eicosatrienoic, 5Z,8Z,11Z,14Z-eicosatetraenoic, and 5Z,8Z,11Z,14Z,17Z-eicosapentaenoic acids) yielded the corresponding 3-hydroxy-all-Z-eicosapolyenoic acids. Moreover, linoleic acid (9Z,12Z-octadecadienoic acid) and 11Z,14Z,17Z-eicosatrienoic acid were converted to the 3-hydroxylated metabolites of shorter chain length, e.g., 3-hydroxy-5Z,8Z-tetradecadienoic acid and 3-hydroxy-5Z,8Z,11Z-tetradecatrienoic acid, respectively. In contrast, no accumulation of a 3-hydroxy metabolite was observed with oleic acid (9Z-octadecenoic acid), linolelaidic acid (9E,12E-octadecadienoic acid), γ-linolenic acid (6Z,9Z,12Z-octadecatrienoic acid), and eicosanoic acid as substrate. These findings pinpoint that the 3-hydroxylation of a fatty acid in Dipodascopsis uninucleata requires a 5Z,8Z-diene system either directly or following initial incomplete β-oxidation. Following analysis of the enantiomer composition, the arachidonic acid metabolite was identified as 3R-hydroxy-5Z,8Z,11Z,14Z-eicosatetraenoic acid, which rules out a normal β-oxidation as biosynthetic route to this new class of oxylipins.     

Heat treatment of vegetable oils. II. GC-MS and GC-FTIR spectra of some isolated cyclic fatty acid monomers

Gas liquid chromatography coupled with mass spectrometry (GC-MS) showed that the cyclic fatty acid monomers (CFAM) isolated from a heated linseed oil have two ethylenic bonds, while the CFAM isolated from heated sunflower oils were saturated and monoethylenic isomers. GC-MS studies also showed the presence of cyclohexenic derivatives in the case of linseed oil. GLC coupled with Fourier transform infrared spectrometry (GC-FTIR) studies indicated that the CFAM isolated from linseed oil were ofcis (Z),trans (E) structures except two components which werecis,cis (Z,Z) dienoic acids. The unsaturated CFAM isolated from sunflower oils werecis (Z) andtrans (E) monoethylenic isomers. For sunflower oils, the major CFAM were isomers having acis (Z) ethylenic bond. The saturated CFAM isolated from a heated sunflower oil had molecular weights of 296 and 294. The latter could correspond to some bicyclic isomers.     

Three new and bioactive icosanoids from the temperate red marine algaFarlowia mollis

Three new dihydroxyicosanoids, 12(R),13(R)-dihydroxyicosa-5(Z), 8(Z),10(E),14(Z)-tetraenoic acid, 12(R),13(R)-dihydroxyicosa-5(Z), 8(Z),10(E),10(Z),17(Z)-pentaenoic acid and 10(R^*),11(R^*)-dihydroxyoctadeca-6(Z),8(E),12(Z)-trienoic acid, have been isolated from a previously unstudied temperate red marine alga,Farlowia mollis (Cryptonemiales, Rhodophyta). The structures of these new metabolites have been deduced from detailed nuclear magnetic resonance and mass spectrometry analyses on stabilized diacetate-methyl esters and stereochemistry deduced by¹H NMR couplings and CD analysis of a dibenzoate derivative. Collectively, these new natural products modulate fMLP-induced superoxide anion generation in human neutrophils, inhibit the conversion of arachidonic acid to lipoxygenase products by human neutrophils, and inhibit the functioning of the dog kidney Na⁺/K⁺ ATPase.     

Metabolism of arachidonic acid in leukocytes: Isolation of a 5,15-dihydroxy-eicosatetraenoic acid

A novel metabolite of arachidonic acid was isolated from incubations of peripheral blood leukocytes with the fatty acid and the ionophore A23187. The compound was purified by high performance liquid chromatography and identified by ultraviolet photometry and gas chromatography-mass spectrometry as a 5,15-dihydroxy-6,8,11,13-eicosatetraenoic acid. The compound was also isolated from incubations of human leukocytes with the 15S-hydroperoxy-5,8,11,13(Z,Z,Z,E)-eicosatetraenoic acid, suggesting a double dioxygenation mechanism in the formation of this new metabolite of arachidonic acid. A 5S,15S-dihydroxy-6,8,11,13(E,Z,Z,E)-eicosatetraenoic acid was obtained from incubations of 5S-hydroxy-6,8,11,14(E,Z,Z,Z)-eicosatetraenoic acid with the soybean lipoxygenase and reduction with stannous chloride, and was used as reference compound. See ref. 16 for a preliminary report.     

Enzymatic oxidations of linoleic acid and glycerol-1-monolinoleate in doughs and flour-water suspensions

Enzymatic oxidations of linoleic acid and glycerol-1-monolinoleate, and the products formed by these oxidations in flour-water suspensions and doughs were studied. Oxidation of linoleic acid leads through simultaneous reactions to two isomeric hydroperoxy-octadecadienoic acids and two isomeric hydroxy-epoxy-octadecenoic acids. Reduction of the former leads to hydroxyoctadecadienoic acid, while hydrolysis of the latter yields trihydroxy-octadecenoic acids. The hydroperoxy acids are formed by the enzyme lipoxygenase (E.C. 1.13.1.13), whereas the hydroxy-epoxy acids are formed by combined action of lipoxygenase and an unknown factor Y. This factor Y is localized in the gluten fraction. Oxidation of glycerol-1-monolinoleate gives a product having a hydroperoxy group and acis,trans conjugated diene bond. The oxidation of glycerol-1-monolinoleate is probably a lipoxygenase reaction.     

Effects of conjugated linolenic acid and conjugated linoleic acid on lipid metabolism in mice

The effects of two isomers of conjugated linolenic acid (CLnA), α‐eleostearic acid (α‐ESA) and punicic acid (PA), on body fat and lipid metabolism were investigated, compared with a conjugated linoleic acid (CLA) mixture (primarily cis9,trans11‐ and trans10,cis12‐18:2) and α‐linolenic acid (ALA), a non‐conjugated octadecatrienoic acid, in the present study. ICR mice were fed either a control diet or one of four experimental diets supplemented with 1% α‐ESA, 1% PA, 1% CLA mixture and 1% ALA in the form of triacylglycerols (TAG) for 6 weeks. The weights of perirenal and epididymal adipose tissues were significantly decreased while the liver weight was significantly increased in mice fed CLA, compared with the control. In contrast to CLA, the tissue weights in α—ESA‐, PA‐ and ALA‐fed mice were not affected. No significant differences were observed in TAG, total cholesterol, high‐density lipoprotein and low‐density lipoprotein cholesterol levels among the five groups. The liver TAG level was significantly decreased in mice fed α‐ESA and PA while it was significantly increased in mice fed the CLA mixture. These results indicate that CLnA and CLA have differential effects on body fat mass and liver TAG levels in mice.     

Synthesis of the conjugated trienes 5E,7E,9E,14Z,17Z-eicosapentaenoic acid and 5Z,7E,9E,14Z,17Z-eicosapentaenoic acid, and their induction of apoptosis in DLD-1 colorectal adenocarcinoma cells

During the course of our recent study on the antitumor effect of conjugated eicosapentaenoic acids (CEPA), we found that acid mixtures prepared by treating EPA with KOH in ethylene glycol induced potent apoptotic cell death in human tumor cells via membrane phospholipid peroxidation. Interestingly, the KOH-treated CEPA mixtures were more cytotoxic than EPA and CLA and had no effect on normal human fibroblast cells. To identify the specific cytotoxic FA in the CEPA mixture, we synthesized possible candidates for the active species. Here, we report the synthesis of (5E,7E,9E,14Z,17Z)-5,7,9,14,17-eicosapentaenoic acid (E-CEPA) and its 5-(Z) isomer (Z-CEPA), both of which are conjugated trienes that exist naturally in red algae (Ptilota filicina J. Agardh). E-CEPA and Z-CEPA were synthesized from methyl 5-oxopentanoate in six steps, using three types of Wittig reactions as the key steps. Next, we examined the cytotoxicity of E-CEPA and Z-CEPA in human tumor cells and confirmed their bioactivity. Both E-CEPA and Z-CEPA had a strong cytotoxic reaction in tumor cells, and this effect occurred through induction of apoptosis.     

Monooxygenase system of <Emphasis Type="Italic">Bacillus megaterium</Emphasis> ALA2: Studies on palmitic acid oxidation products

We identified many novel oxygenated FA produced from linoleic acid by microbial strain ALA2: 12,13,17-trihydroxy-9(Z)-octadecenoic acid (12,13,17-THOA); 12,13,16-trihydroxy-9(Z)-octadecenoic acid (12,13,16-THOA); 12-hydroxy-13,16-epoxy-9(Z)-octadecenoic acid; and 12,17;13,17-diepoxy-16-hydroxy-9(Z)-octadecenoic acid. 12,13,17-THOA, the main product, inhibits the growth of some plant pathogenic fungi. Recently, we reclassified strain ALA2 as Bacillus megaterium ALA2 NRRL B-21660 and opened a possible link with the well-studied catalytically self-sufficient P450 monooxygenase of Bacillus megaterium ATCC 14581 (NRRL B-3712) and B. subtilis strain 168 (NRRL B-4219). Now we have found that strain ALA2 also oxidizes palmitic acid into three oxygenated products: 13-, 14-, and 15-hydroxy palmitic acids. This indicates that strain ALA2 also possesses a monooxygenase system similar to the abovementioned well-known strains. These data facilitate studies on the oxygenase system of strain ALA2.     

Improvement in the synthesis of 2-(5-amino-1,2,4-thiadiazol-3-yl)-2-(Z)-methoxyiminoacetic acid 2-benzothiazolyl thioester

2-(5-Amino-1,2,4-thiadiazol-3-yl)-2-(Z)-methoxyiminoacetic acid 2-benzothiazolyl thioester(III), an important intermediate of the fourth generation cephalosporins, was efficiently synthesized by reacting 2-(5-amino-1,2,4-thiadiazol-3-yl)-2-(Z)-methoxyiminoacetic acid (I) with 2,2′-dibenzothiazole disulfide (II) in the presence of triphenylphosphine. Effects of reaction time, temperature, solvents, catalysts and feeding molar ratio on the yield and quality of products were investigated, and an improved procedure suitable for industrial production was established. Using 1,2-dichloroethane as solvent, triphenylphosphine as reducer, and triethylamine as catalyst, n(I): n(II): n(triphenylphosphine)51.0: 1.0: 1.0, the product was obtained at room temperature in 98.1% yield. The purity of the product without further purification is 98.7% determined by HPLC method. This procedure could be a suitable alternative to the traditional processes because of its easy handling, high yield and low cost. __________ Translated from Fine Chemicals, 2007, 24(8): 816–819 [译自:精细化工]     

Drosophila Fed ARA and EPA Yields Eicosanoids, 15S-Hydroxy-5Z,8Z, 11Z, 13E-Eicosatetraenoic Acid,and 15S-Hydroxy-5Z,8Z,11Z,13E,17Z-Eicosapentaenoic Acid

Drosophila melanogaster has been a widely used as a model system for its powerful genetic tools. However, it remains to be illustrated if Drosophila can be used to examine the biochemical and physiological metabolism of eicosanoids. Thus, the analysis on the metabolism of C20 polyunsaturated fatty acids (PUFA) in Drosophila was implemented with high performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS). Fatty acid (FA) analysis of the whole body, head, and thorax‐abdomen in Drosophila showed C20 PUFA could only be found in Drosophila fed diets supplemented with eicosapentaenoic acid (EPA) and arachidonic acid (ARA), but not in Drosophila fed base diets. The C20 PUFA were found in abundance in the head. Drosophila fed ARA‐ and EPA‐supplemented diets yielded 15S‐hydroxy‐5Z,8Z,11Z,13E‐eicosatetraenoic acid [15(S)‐HETE] and 15S‐hydroxy‐5Z,8Z,11Z,13E,17Z‐eicosapentaenoic acid [15(S)‐HEPE], respectively, while other sampled eicosanoids could not be detected. Similar results were obtained by incubating fly tissue supplemented with ARA or EPA. Furthermore, a genome sequence scan indicated that no gene encoding the key enzymes synthesizing eicosanoids were found in Drosophila. These findings demonstrate that Drosophila may possess a special lipid metabolic system, which is different from mammals.