Identification of (e)-11-hydroxy-9-octadecenoic acid and (E)-9-hydroxy-10-octadecenoic acid by biotransformation of oleic acid by Pseudomonas sp. 32T3

Pseudomonas sp. 32T3, a newly identified strain originally isolated from a vegetable oil-contaminated soil, produces three monohydroxy acids—(E)-11-hydroxy-9-octadecenoic acid, (E)-10-hydroxy-8-octadecenoic acid, and (E)-9-hydroxy-10-octadecenoic acid—as bioconversion products of oleic acid. The bacterial cells were grown in a mineral medium containing oleic acid as the main carbon substrate. The compounds were identified as the corresponding methyl esters on the basis of their chromatographic and spectroscopic (¹H and ¹³C nuclear magnetic resonance and gas chromatography-mass spectrometry) features.     

Hydroxy fatty acids through hydroxylation of oleic acid with selenium dioxide/tert.-butylhydroperoxide

Oleic acid was hydroxylated in the allylic positions with the selenium dioxide/tert.-butylhydroperoxide system to give 8-hydroxy-9(E)-octadecenoic acid, 11-hydroxy-9(E)-octadecenoic acid and the novel 8,11-dihydroxy-9(E)-octadecenoic acid. This is a viable method for obtaining hydroxy fatty acids. The unsaturated hydroxy acids were hydrogenated with the hydrazine/air system to give the cor-responding saturated products. 8,11-Dihydroxyoctadecanoic acid thus obtained is also a novel compound. The saturated and unsaturated dihydroxy products were obtained aserythro/threo isomers as determined by nuclear magnetic resonance. Presented in part at the 83rd AOCS Annual Meeting, Toronto, Ontario, Canada, 1992.     

Transformation of fatty acid hydroperoxides by alkali and characterization of products

It has previously been determined that (13S,9Z,11E)-13-hydroperoxy-9,11-octadecadienoic acid was mainly converted into (13S,9Z,11E)-13-hydroxy-9,11-octadecadienoic acid by 5 N KHO with preservation of the stereochemistry of the reactant [Simpson, T.D., and Gardner, H.W. (1993)Lipids 28, 325–330]. In addition, about 20–25% of the reactant was converted into several unknown by-products. In the present work it was confirmed that the stereochemistry was conserved during the hydroperoxy-diene to hydroxydiene transformation, but also, novel by-products were identified. It was found that after only 40 min reaction (9Z)-13-oxo-trans-11,12-epoxy-9-octadecenoic acid accumulated to as much as 7% of the total. Later, (9Z)-13-oxo-trans-11,12-epoxy-9-octadecenoic acid began to disappear, and several other compounds continued to increase in yield. Two of these compounds, 2-butyl-3,5-tetradecadienedioic acid and 2-butyl-4-hydroxy-5-tetradecenedioic acid, were shown to originate from (9Z)-13-oxo-trans-11,12-epoxy-9-octadecenoic acid, and they accumulated up to 2–3% each after 4 to 6 h. Some other lesser products included 11-hydroxy-9,12-heptadecadienoic acid, 3-hydroxy-4-tridecenedioic acid, 13-oxo-9,11-octadecadienoic acid and 12,13-epoxy-11-hydroxy-9-octadecenoic acid. Except for the latter two, most or all of the compounds could have originated from Favorskii rearrangement of the early product, (9Z)-13-oxo-trans-11,12-epoxy-9-octadecenoic acid, through a cyclopropanone intermediate.     

Fatty acid hydroperoxide isomerase inSaprolegnia parasitica: Structural studies of epoxy alcohols formed from isomeric hydroperoxyoctadecadienoates

The major part (80%) of the fatty acid hydroperoxide isomerase activity present in homogenates of the fungus,Saprolegnia parasitica, was localized in the particle fraction sedimenting at 105,000×g. 13(S)-Hydroperoxy-9(Z),11(E)-octadecadienoic acid and 9(S)-hydroperoxy-10(E),12(Z)-octadecadienoic acid were both good substrates for the particle-bound hydroperoxide isomerase. The products formed from the 13(S)-hydroperoxide were identified as an α,β- and a γ,δ-epoxy alcohol, i.e., 11(R),12(R)-epoxy-13(S)-hydroxy-9(Z)-octadecenoic acid and 9(S),10(R)-epoxy-13(S)-hydroxy-11(E)-octadecenoic acid, respectively. The 9(S)-hydroperoxide was converted in an analogous way into an α,β-epoxy alcohol, 10(R),11(R)-epoxy-9(S)-hydroxy-12(Z)-octadecenoic acid and a γ,δ-epoxy alcohol, 12(R),13(S)-epoxy-9(S)-hydroxy-10(E)-octadecenoic acid. 9(R,S)-Hydroperoxy-10(E),12(E)-octadecadienoic acid and 13(R,S)-hydroperoxy-9(E),11(E)-octadecadienoic acid were poor substrates for theS. parasitica hydroperoxide isomerase. Experiments with 13(R,S)-hydroperoxy-9(Z),11(E)-octadecadienoic acid showed that the 13(R)-hydroperoxy enantiomer was slowly isomerized by the enzyme. The major product was identified as α,β-epoxy alcohol 11(R),12(R)-epoxy-13(R)-hydroxy-9(Z)-octadecenoic acid.     

A new reaction of unsaturated fatty acid hydroperoxides: Formation of 11-hydroxy-12,13-epoxy-9-octadecenoic acid from 13-hydroperoxy-9,11-octadecadienoic acid

One of the main compounds formed from 13L-hydroperoxy-9cis,11trans-octadecadienoic acid anaerobically at 100 C in aqueous ethanol was found to bethreo-11-hydroxy-12,13-epoxy-9-octadecenoic acid. The major part (ca. 90%) of this compound was formed from the fatty acid hydroperoxide in a reaction involvingcis-addition to the Δ¹¹ double bond of the proximally linked hydroperoxide oxygen and hydroxyl ion or hydroxyl radical from the solvent. A small part (ca. 10%) was formed bycis-addition of the two hydroperoxide oxygens to the Δ¹¹ double bond. 11-Hydroxy-12,13-epoxy-9-octadecenoic acid and its isomer, tentatively identified as 11-hydroxy-9,10-epoxy-12-octadecenoic acid, also were isolated from a sample of autoxidized linoleic acid.     

Linoleic acid metabolism in the red algaLithothamnion corallioides: Biosynthesis of 11(R)-hydroxy-9(Z),12(Z)-octadecadienoic acid

Incubation of [1-¹⁴C]linoleic acid with an enzyme preparation obtained from the red algaLithothamnion corallioides Crouan resulted in the formation of 11-hydroxy-9(Z),12(Z)-octadecadienoic acid as well as smaller amounts of 9-hydroxy-10(E),12(Z)-octadecadienoic acid, 13-hydroxy-9(Z),11(E)-octadecadienoic acid and 11-keto-9(Z),12(Z)-octadecadienoic acid. Steric analysis showed that the 11-hydroxyoctadecadienoic acid had the (R) configuration. The 9- and 13-hydroxyoctadecadienoic acids were not optically pure, but were due to mixtures of 75% (R) and 25% (S) enantiomers (9-hydroxyoctadecadienoate), and 24% (R) and 76% (S) enantiomers (13-hydroxy-octadecadienoate). 11-Hydroxyoctadecadienoic acid was unstable at acidic pH. In acidified water, equal parts of 9(R,S)-hydroxy-10(E),12(Z)-octadecadienoate and 13(R,S)-hydroxy-9(Z),11(E)-octadecadienoate, plus smaller amounts of the corresponding (E),(E) isomers were produced. In aprotic solvents, acid treatment resulted in dehydration and in the formation of equal amounts of 8,10,12- and 9,11,13-octadecatrienoates. The enzymatic conversion of linoleic acid into the hydroxyoctadecadienoic acids and the ketooctadecadienoic acid was oxygen-dependent; however, inhibitor experiments indicated that neither lipoxygenase nor cytochrome P-450 were involved in the conversion. This conclusion was supported by experiments with¹⁸O₂ and H₂ ¹⁸O, which demonstrated that the hydroxyl oxygen of the hydroxy-octadecadienoic acids and the keto oxygen of the 11-ketooctadecadienoic acid were derived from water and not from molecular oxygen. The term “oxylipin” was introduced recently (ref. 1) as an encompassing term for oxygenated compounds which are formed from fatty acids by reaction(s) involving at least one step of mono- or dixoygenase-catalyzed oxygenation.     

Ultrasound-assisted synthesis of santalbic acid and a study of triacylglycerol species inSantalum album (Linn.) seed oil

Methyl ricinoleate (1) was treated with bromine and the dibromo derivative (2) was reacted with ethanolic KOH under ultrasonic irradiation to give 12-hydroxy-octadec-9-ynoic acid upon acidification with dil. HCl. The latter compound was methylated with BF₃/methanol to give methyl 12-hydroxy-octadec-9-ynoate (3). Compound3 was treated with methanesulfonyl chloride in the presence of triethylamine in CH₂Cl₂ to give methyl 12-mesyloxy-octadec-9-ynoate (4). Reaction of methyl 12-mesyloxy-octadec-9-ynoate with aqueous KOH under ultrasonic irradiation (20 kHz) gave (11E)-octadecen-9-ynoic acid (5, santalbic acid, 40%) and (11Z)-octadecen-9-ynoic acid (6, 60%) on acidification with dil. HCl. These isomers were separated by urea fractionation. The¹³C nuclear magnetic resonance (NMR) spectroscopic properties of the methyl ester and the triacylglycerol (TAG) esters of these enynoic fatty acid isomers were studied. The carbon shifts of the unsaturated carbon nuclei of the methyl ester of theE-isomer were unambiguously assigned as 88.547 (C-9), 79.287 (C-10), 109.760 (C-11), and 143.450 (C-12) ppm while the unsaturated carbon shifts of the (Z)-enynoate isomer appeared at 94.277 (C-9), 77.561 (C-10), 109.297 (C-11), and 142.668 (C-12) ppm. In the¹³C NMR spectral analysis of the TAG molecules of type AAA containing either the (Z)-or (E)-enyne fatty acid, the C-1 to C-6 carbon atoms on the α- and β-acyl positions were differentiated. The unsaturated carbon atoms in the α- and β-acyl chains were also resolved into two signals except that of the C-11 olefinic carbon. Sandal (Santalum album) wood seed oil (a source of santalbic acid) was separated by silica chromatography into three fractions. The least polar fraction (7.2 wt%) contained TAG which had a random distribution of saturated and unsaturated fatty acids, of which oleic acid (69%) was the predominant component. The second fraction (3.8 wt%) contained santalbic acid (58%) and oleic acid (28%) together with some other normal fatty acids. Santalbic acid in this fraction was found in both the α- and β-acyl positions of the glycerol “backbone”. The most polar fraction (89 wt%) consisted of TAG containing santalbic acid only. The distribution of the various fatty acids on the glycerol “backbone” was supported by the results from the¹³C NMR spectroscopic analysis.     

Metabolic profile of linoleic acid in stored apples: Formation of 13(R)-hydroxy-9(Z),11(E)-octadecadienoic acid

During our ongoing project on the biosynthesis of R-(+)-octane-1,3-diol the metabolism of linoleic acid was investigated in stored apples after injection of [1-¹⁴C]-, [9,10,12,13-³H]-, ¹³C₁₈- and unlabeled substrates. After different incubation periods the products were analyzed by gas chromatography-mass spectroscopy (MS), high-performance liquid chromatography-MS/MS, and HPLC-radiodetection. Water-soluble compounds and CO₂ were the major products whereas 13(R)-hydroxy- and 13-keto-9(Z),11(E)-octadecadienoic acid, 9(S)-hydroxy-and 9-keto-10(E),12(Z)-octadecadienoic acid, and the stereoisomers of the 9,10,13- and 9,12,13-trihydroxyoctadecenoic acids were identified as the major metabolites found in the diethyl ether extracts. Hydroperoxides were not detected. The ratio of 9/13-hydroxy- and 9/13-keto-octadecadienoic acid was 1∶4 and 1∶10, respectively. Chiral phase HPLC of the methyl ester derivatives showed enantiomeric excesses of 75% (R) and 65% (S) for 13-hydroxy-9(Z),11(E)-octadecadienoic acid and 9-hydroxy-10(E),12(Z)-octadecadienoic acid, respectively. Enzymatically active homogenates from apples were able to convert unlabeled linoleic acid into the metabolites. Radiotracer experiments showed that the transformation products of linoleic acid were converted into (R)-octane-1,3-diol. 13(R)-Hydroxy-9(Z), 11(E)-octadecadienoic acid is probably formed in stored apples from 13-hydroperoxy-9(Z),11(E)-octadecadienoic acid. It is possible that the S-enantiomer of the hydroperoxide is primarily degraded by enzymatic side reactions, resulting in an enrichment of the R-enantiomer and thus leading to the formation of 13(R)-hydroxy-9(Z),11(E)-octadecadienoic acid.     

Biotransformation of linoleic acid by Clavibacter sp. ALA2: Heterocyclic and heterobicyclic fatty acids

Clavibacter sp. ALA2 transformed linoleic acid into a variety of oxylipins. In previous work, three novel fatty acids were identified, (9Z)-12,13,17-trihydroxy-9-octadecenoic acid and two tetrahydrofuran-(di)hydroxy fatty acids. In this report, we confirm the structures of the tetrahydrofuran-(di)hydroxy fatty acids by nuclear magnetic resonance as (9Z)-12-hydroxy-13,16-epoxy-9-octadecenoic acid and (9Z)-7,12-dihydroxy-13,16-epoxy-9-octadecenoic acid. Three other products of the biotransformation were identified as novel heterobicyclic fatty acids, (9Z)-12,17;13,17-diepoxy-9-octadecenoic acid, (9Z)-7-hydroxy-12,17;13,17-diepoxy-9-octadecenoic acid, and (9Z)-12,17;13,17-diepoxy-16-hydroxy-9-octadecenoic acid. Thus, Clavibacter ALA2 effectively oxidized linoleic acid at C-7,-12,-13,-16, and/or-17.     

Fatty acid bioconversions by Pseudomonas aeruginosa PR3

The bioconversions of oleic acid, linoleic acid, and ricinoleic acid by Pseudomonas aeruginosa PR3 were investigated. The conversion of oleic acid to 7,10-dihydroxy-8(E)-octadecenoic acid (DOD) was improved to better than 80% yields by modifying the culture medium and reaction parameters. The microbial cultures were stable and retained the same level of DOD production capacity for up to 6 mon as examined herewith. Strain PR3 did not bioconvert linoleic acid. However, strain PR3 converted ricinoleic acid to a novel compound, 7,10,12-trihydroxy-8(E)-octadecenoic acid (TOD), with a yield of about 35%. The product was further metabolized by strain PR3. TOD was purified by thin-layer chromatography, and its structure was determined by ¹H and ¹³C nuclear magnetic resonance and mass spectrometry. This is the first report on the production of 7,10,12-trihydroxy unsaturated fatty acid by microbial transformation.     

High-performance liquid chromatography and spectroscopic studies on fish oil oxidation products extracted from frozen atlantic mackerel

The formation of stable hydroxy derivatives from hydroperoxides produced during the oxidation of linoleic acid methyl ester and fish oil were studied by reverse-phase high-performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS) and ¹³C nuclear magnetic resonance (NMR) spectroscopy. The oxidation products identified were mixtures of four isomeric hydroxy derivatives: 13-hydroxy-9-cis,11-trans-octadecadienoic, 13-hydroxy-9-trans,11-trans-octadecadienoic, 9-hydroxy-10-trans,12-cis-octadecadienoic, and 9-hydroxy-10-trans,12-trans-octadecadienoic acids. The presence of hydroxy compounds was confirmed by ¹³C NMR, which gave rise to a hydroxy carbon peak at 87 ppm, and by GC-MS, which showed three peaks corresponding to isomeric mixtures of trimethylsilyl ethers of the oxidized linoleic acid methyl ester. The mass spectra scans of the three peaks showed that they represent isomers of molecular weight 382 and are consistent with the molecular formula C₂₂H₄₂O₃Si. In oil extracted from stored frozen mackerel, 13-hydroxy-9-cis,11-trans-octadecadienoic acid was more prominent compared to the model lipid systems. HPLC offered a sensitive means of detection of hydroxy compounds produced both in the initiation and latter stages of oxidation. The effect of antioxidants added to the fish mince prior to storage can also be monitored by HPLC. Thus, the monitoring of lipid oxidation hydroxy derivatives by HPLC is of practical value in the efficient processing and quality control of fish, fish oils, and other fatty foodstuffs in order to enhance the acceptability, nutritional, and safety aspects.     

Biosynthesis of tetrahydrofuranyl fatty acids from linoleic acid by <Emphasis Type="Italic">clavibacter</Emphasis> sp. ALA2

Clavibacter sp. ALA2 converts linoleic acid into many novel oxygenated products including hydroxy FA and tetrahydrofuranyl unsaturated FA (THFA). One of them was tentatively identified by GC-MS as 12,13,16-trihydroxy-9(Z)-octadecenoic acid (12,13,16-THOA) (Hou, C.T., H.W. Gardner, and W. Brown, J Am. Oil Chem. Soc. 78∶1167–1169, 2001). We have separated and purified 12,13,16-THOA from its isomer, 12,13,17-THOA, by silica gel column chromatography and by preparative TLC. Its structure was then confirmed by proton and ¹³C NMR analyses. Purified 12,13,16-THOA was used as a substrate to study the biosynthesis of THFA. Within 24 h of incubation, cells of strain ALA2 converted 12,13,16-THOA to both 12-hydroxy-13,16-epoxy-9(Z)-octadecenoic acid (12-hydroxy-THFA) and 7,12-dihydroxy-13,16-epoxy-9(Z)-octadecenoic acid (7,12-dihydroxy-THFA). The relative abundance of 7,12-dihydroxy-THFA increased with incubation time, whereas that of 12,13,16-THOA and of 12-hydroxy-THFA decreased. Therefore, the biosynthetic pathway of THFA from linoleic acid by strain ALA2 is as follows: linoleic acid→12,13-dihydroxy-9(Z)-octadecenoic acid→12,13,16-THOA→12-hydroxy-THEA→7,12-dihydroxy-THFA.     

Mechanism of linoleic acid hydroperoxide reaction with alkali

Treatment of (13S,9Z,11E)-13-hydroperoxy-9,11-octadecadienoic acid (13S-HPODE) with strong alkali resulted in the formation of about 75% of the corresponding hydroxy acid, (13S,9Z,11E)-13-hydroxyl-9,11-octadecadienoic acid (13S-HPODE), and the remaining 25% of products was a mixture of several oxidized fatty acids, the majority of which was formed from (9Z,11R,S,12S,R)-13-oxo-11, 12-epoxy-9-octadecenoic acid by Favorskii rearrangement (Gardner, H.W.,et al. (1993)Lipids 28, 487–495). In the present work, isotope experiments were completed in order to get further information about the initial steps of the alkali-promoted decomposition of 13S-HPODE.1. Reaction of [hydroperoxy-¹⁸O₂]13S-HPODE with 5 M KOH resulted in the formation of [hydroxy-¹⁸O]13S-HPODE and [epoxy-¹⁸O](9Z,11R,S,12S,R)-13-oxo-11, 12-epoxy-9-octadecenoic acid;2. treatment of a mixture of [U-¹⁴C]13S-HPODE and [hydroperoxy-¹⁸O₂]13S-HPODE with KOH and analysis of the reaction product by radio-TLC showed that 13S-HPODE was stable under the reaction conditions and did not serve as precursor of other products;3. reaction of a mixture of [U-¹⁴C]13-oxo-9,11-octadecadienoic acid (13-OODE) and [hydroperoxy-¹⁸O₂]13S-HPODE with KOH resulted in the formation of [U-¹⁴C-epoxy-¹⁸O](9Z,11R,S,12S,R)-13-oxo-11,12-epoxy-9-octadecenoic acid;4. treatment of a mixture of [hydroperoxy-¹⁸O₂] 13S-HPODE and [carboxyl-¹⁸O₁]13S-HPODE with KOH afforded (9Z,11R,S,12S,R)-13-oxo-11,12-epoxy-9-octadecenoic acid having an¹⁸O-labeling pattern which was in agreement with its formation by intermolecular epoxidation. It was concluded that (9Z,11R,S,12S,R)-13-oxo-11, 12-epoxy-9-octadecenoic acid is formed from 13S-HPODE by a sequence involving initial dehydration into the α,β-unsaturated ketone, 13-OODE, followed by epoxidation of the Δ¹¹ double bond of this compound by the peroxyl anion of a second molecule of 13S-HPODE. Rapid conversion of hydroperoxides by alkali appreared to require the presence of an α,β-unsaturated ketone intermediate as an oxygen acceptor. This was supported by experiments with a saturated hydroperoxide, methyl 12-hydroperoxyoctadecanoate, which was found to be much more resistant to alkali-promoted conversion than 13S-HPODE.     

10(S)-Hydroxy-8(E)-octadecenoic acid, an intermediate in the conversion of oleic acid to 7,10-dihydroxy-8(E)-octadecenoic acid

The new microbial isolate Pseudomonas aeruginosa (PR3) has been reported to produce from oleic acid a new compound, 7,10-dihydroxy-8(E)-octadecenoic acid (DOD), with 10-hydroxy-8-octadecenoic acid (HOD) being a probable intermediate. The production of DOD involves the introduction of two hydroxyl groups at carbon numbers 7 and 10, and a rearrangement of the double bond from carbons 9–10 to 8–9. It has been shown that the 8–9 unsaturation of HOD was possibly in the cis configuration. Now we report that the rearranged double bond of HOD is trans rather than cis, as determined by spectral data. Also, it was found that the 10-hydroxyl was in the S-configuration as determined by gas chromatographic separation of R- and S-isomers after preparation of the (−)-menthoxycarbonyl derivative of the hydroxyl group followed by oxidative cleavage of the double bond and methyl esterification. This latter result coincides with our recent finding that the main final product, DOD, is in the 7(S),10(S)-dihydroxy configuration. In addition, a minor isomer of HOD (about 3%) with the 10(R)-hydroxyl configuration was also detected. From the data obtained herein, we concluded that 10(S)-hydroxy-8(E)-octadecenoic acid is the probable intermediate in the bioconversion of oleic acid to 7(S),10(S)-dihydroxy-8(E)-octadecenoic acid by PR3.     

Cysteine adds to lipid hydroperoxide

Cysteine reacts with linoleic acid hydroperoxide to yield several products, some of which were identified as fatty acid-cysteine adducts. The addition was catalyzed by ferric chloride (10⁻⁵ M) by initiating free radical reactions. When isomerically pure 13-hydroperoxy-cis-9,trans-11-octadecadienoic acid and cysteine were reacted in 80% ethanol under N₂, the major adducts were 9-S-cysteine-13-hydroxy-10-ethoxy-trans-11-octadecenoic acid (I) and 9-S-cysteine-10,13-dihydroxy-trans-11-octadecenoic acid (II). When the reaction included both isomers of the hydroperoxide (13-and 9-hydroperoxide) and air, an adduct of 9-oxononanoic acid and cysteine also was isolated. Additional experiments gave information about possible mechanisms of I and II formation.     

Structure-activity relationship of unsaturated fatty acids as mosquito ovipositional repellents

Various straight-chain unsaturated fatty acids from C₁₄ to C₂₄ were evaluated for their ovipositional repellency against gravid females of the southern house mosquitoCulex quinquefasciatus Say, and the relationship between the structures of the fatty acids and their ovipositional repellency was determined. A double bond withZ configuration was prerequisite for an unsaturated fatty acid to be highly repellent;E isomers were less active or even inactive. No relationship was found between the repellency and the number of double bonds in the unsaturated fatty acids. In C₁₈ monounsaturated fatty acids, (Z)-9 acid was more active than (Z)-11 and (Z)-6 acids, indicating that a double bond at the 9 position rendered an acid highly repellent. Among (Z)-9-alkenoic acids of different chain lengths, the most repellent was C₁₈ acid which was also more active than (Z)-11-C₂₀, (Z)-13-C₂₂, and (Z)-15-C₂₄ acids. Oleic[(Z)-9-octadecenoic]acid, which met all these criteria, was the most ovipositionally repellent among the unsaturated fatty acids tested.Diptera: Culicidae.     

New cyclopentenone fatty acids formed from linoleic and linolenic acids in potato

[1-¹⁴C]Linoleic acid was incubated with a whole homogenate preparation from potato stolons. The reaction product contained four major labeled compounds, i.e., the α-ketol 9-hydroxy-10-oxo-12(Z)-octadecenoic acid (59%), the epoxy alcohol 10(S),11(S)-epoxy-9(S)-hydroxy-12(Z)-octadecenoic acid (19%), the divinyl ether colneleic acid (3%), and a new cyclopentenone (13%). The structure of the last-mentioned compound was determined by chemical and spectral methods to be 2-oxo-5-pentyl-3-cyclopentene-1-octanoic acid (trivial name, 10-oxo-11-phytoenoic acid). Steric analysis demonstrated that the relative configuration of the two side chains attached to the five-membered ring was cis, and that the compound was a racemate comprising equal parts of the 9(R), 13(R) and 9(S), 13(S) enantiomers. Experiments in which specific trapping products of the two intermediates 9(S)-hydroperoxy-10(E), 12(Z)-octadecadienoic acid and 9(S), 10-epoxy-10, 12(Z)-octadecadienoic acid were isolated and characterized demonstrated the presence of 9-lipoxygenase and allene oxide synthase activities in the tissue preparation used. The allene oxide generated from linoleic acid by action of these enzymes was further converted into the cyclopentenone and α-ketol products by cyclization and hydrolysis, respectively. Incubation of [1-¹⁴C]linolenic acid with the preparation of potato stolons afforded 2-oxo-5-[2′(Z)-pentenyl]-3-cyclopentene-1-octanoic acid (trivial name, 10-oxo-11, 15(Z)-phytodienoic acid), i.e., an isomer of the jasmonate precursor 12-oxo-10, 15(Z)-phytodienoic acid. Quantitative determination of 10-oxo-11-phytoenoic acid in linoleic acid-supplied homogenates of different parts of the potato plant showed high levels in roots and stolons, lower levels in developing tubers, and no detectable levels in leaves.     

Homolytic decomposition of linoleic acid hydroperoxide: Identification of fatty acid products

An isomeric mixture of linoleic acid hydroperoxides, 13-hydroperoxy-cis-9,trans-11-octadecadienoic acid (79%) and 9-hydroperoxy-cis-12,trans-10-octadecadienoic acid (21%), was decomposed homolytically by Fe(II) in an ethanol-water solution. In one series of experiments, the hydroperoxides were decomposed by catalytic concentrations of Fe(II). The 10⁻⁵ M Fe(III) used to initiate the decomposition was kept reduced as Fe(II) by a high concentration of cysteine added to the reaction in molar excess of the hydroperoxides. Nine different monomeric (no detectable dimeric) fatty acids were identified from the reaction. Analyses of these fatty acids revealed that they were mixtures of positional isomers identified as follows: (I) 13-oxo-trans,trans-(andcis,trans-) 9,11-octadecadienoic and 9-oxo-trans,trans- (andcis,trans-) 10,12-octadecadienoic acids; (II) 13-oxo-trans-9,10-epoxy-trans-11-octadecenoic and 9-oxo-trans-12, 13-epoxy-trans-10-octadecenoic acids; (III) 13-oxo-cis-9,10-epoxy-trans-11-octadecenoic and 9-oxo-cis-12, 13-epoxy-trans-10-octadecenoic acids; (IV) 13-hydroxy-9,11-octadecadienoic and 9-hydroxy-10,12-octadecadienoic acids; (V) 11-hydroxy-trans-12, 13-epoxy-cis-9-octadecenoic and 11-hydroxy-trans-9, 10-epoxy-cis-12-octadecenoic acids; (VI) 11-hydroxy-trans-12, 13-epoxy-trans-9-octadecenoic and 11-hydroxy-trans-9,10-epoxy-trans-12-octadecenoic acids; (VII) 13-oxo-9-hydroxy-trans-10-octadecenoic acids; (VIII) isomeric mixtures of 9, 12, 13-dihydroxyethoxy-trans-10-octadecenoic and 9, 10, 13-dihydroxyethoxy-trans-11-octadecenoic acids; and (IX) 9, 12, 13-trihydroxy-trans-10-octadecenoic and 9, 10, 13-trihydroxy-trans-11-octadecenoic acids. In another experiment, equimolar amounts of Fe(II) and hydroperoxide were reacted in the absence of cysteine. A large proportion of dimeric fatty acids and a smaller amount of monomeric fatty acids resulted. The monomeric fatty acids were examined by gas liquid chromatography-mass spectroscopy. Spectra indicated that the monomers were largely similar to those produced by the Fe(III)-cysteine reaction. Presented in part at the American Chemical Society Meeting, Los Angeles, March 1974. ARS, USDA.     

Novel oxylipins from the temperate red alga polyneura latissima: Evidence for an arachidonate 9(S)-lipoxygenase

The oxylipin chemistry of the temperate red alga Polyneura latissima has been investigated. The structures of three novel oxylipins, 8-[1′(Z),3′(Z),6′(Z)-dodecatriene-1′-oxyl-5(Z),7(E)-octadienoic acid, 7(S ^*)-hydroxy-8(S ^*),9(S ^*)-epoxy-5(Z), 11(Z),14(Z)-eicosatrienoic acid, 7(R ^*)-hydroxy-8(S ^*), 9(S ^*)-epoxy-5(Z), 11(Z),14(Z)-eicosatrienoic acid, together with two known eicosanoids, 9(S)-hydroxy-5(Z), 7(E), 11(Z), 14(Z)-eicosatetraenoic acid, and 9, 15-dihydroxy-5(Z),7(E),11(Z),13(E)-eicosatetraenoic acid, were elucidated by spectroscopic methods and chemical degradation. The oxygenation pattern of these oxylipins suggests that P. latissima metabolizes polyunsaturated fatty acids via a 9(S)-lipoxygenase.     

Regio- and stereochemical analysis of trihydroxyoctadecenoic acids derived from linoleic acid 9- and 13-hydroperoxides

The methyl esters of 9S,10S,13R-trihydroxy-11E-octadecenoic acid, 9S,10R,13S-trihydroxy-11E-octadecenoic acid, and 9S,10R,13R-trihydroxy-11E-octadecenoic acid were prepared from 9S-hydroperoxy-10E,12Z-octadecadienoic acidvia the epoxy alcohols methyl 10R,11R-epoxy-9S-hydroxy-12Z-octadecenoate and methyl 10S,11S-epoxy-9S-hydroxy-12Z-octadecenoate. The trihydroxyesters, as well as four stereoisomeric methyl 9,12,13-trihydroxy-10E-octadecenoates earlier prepared [Hamberg, M.,Chem. Phys. Lipids 43, 55–67 (1987)], were characterized by thin-layer chromatography, gas-liquid chromatography, mass spectrometry, and by chemical degradation. Availability of these chemically defined trihydroxyoctadecenoates made it possible to design a method for regio- and stereochemical analysis of 9,10,13- and 9,12,13-trihydroxyoctadecenoic acids obtained from various sources. Application of the method revealed that the mixture of 9,10,13- and 9,12,13-trihydroxyoctadecenoic acids formed during autoxidation of linoleic acid in aqueous medium contained comparable amounts of the sixteen possible regio- and stereoisomers. Furthermore, hydrolysis of the allylic epoxy alcohol, methyl 9S,10R-epoxy-13S-hydroxy-11E-octadecenoate, yielded a major trihydroxyoctadecenoate,i.e., methyl 9S,10S,13S-trihydroxyl-11E-octadecenoate, together with smaller amounts of methyl 9S,10R,13S-trihydroxy-11E-octadecenoate, methyl 9S,12R,13S-trihydroxy-10E-octadecenoate, and methyl 9S,12S,13S-trihydroxy-10E-octadecenoate.