Fatty acid allene oxides. III. Albumin-induced cyclization of 12,13(S)-epoxy-9(Z),11-octadecadienoic acid

Recently, corn (Zea mays L.) hydroperoxide dehydrase was found to catalyze the conversion of 13(S)-hydroperoxy-9(Z),11(E)-octadecadienoic acid into an unstable fatty acid allene oxide, 12,13(S)-epoxy-9(Z),11-octadecadienoic acid. This study is concerned with the chemistry of 12,13(S)-epoxy-9(Z),11-octadecadienoic acid in the presence of vertebrate serum albumins. Albumins were found to greatly enhance the aqueous half-life of the allene oxide, i.e. 14.1±1.8 min, 11.6±1.2 min and 4.8±0.5 min at 0 C in the presence of 15 mg/ml of bovine, human and equine serum albumins, respectively, as compared with ca. 33 sec in the absence of albumin. Degradation of allene oxide in the presence of bovine serum albumin led to the formation of a novel cyclization product, i.e. 3-oxo-2-pentyl-cyclopent-4-en-1-octanoic acid (12-oxo-10-phytoenoic acid, in which the relative configuration of the side chains attached to the five-membered ring istrans). Steric analysis of the cyclic derivative showed that the compound was largely racemic (ratio between enantiomers, 58∶42). 12-Oxo-10,15(Z)-phytodienoic acid, needed for reference purposes, was prepared by incubation of 13(S)-hydroperoxy-9(Z),11(E),15(Z)-octadecatrienoic acid with corn hydroperoxide dehydrase. Steric analysis showed that the 12-oxo-10,15(Z)-phytodienoic acid thus obtained was not optically pure but a mixture of enantiomers in a ratio of 82∶18. The first paper in this series is Reference 1.     

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.     

Efficient and Specific Conversion of 9-Lipoxygenase Hydroperoxides in the Beetroot. Formation of Pinellic Acid

The linoleate 9-lipoxygenase product 9(S)-hydroperoxy-10(E),12(Z)-octadecadienoic acid was stirred with a crude enzyme preparation from the beetroot (Beta vulgaris ssp. vulgaris var. vulgaris) to afford a product consisting of 95% of 9(S),12(S),13(S)-trihydroxy-10(E)-octadecenoic acid (pinellic acid). The linolenic acid-derived hydroperoxide 9(S)-hydroperoxy-10(E),12(Z),15(Z)-octadecatrienoic acid was converted in an analogous way into 9(S),12(S),13(S)-trihydroxy-10(E),15(Z)-octadecadienoic acid (fulgidic acid). On the other hand, the 13-lipoxygenase-generated hydroperoxides of linoleic or linolenic acids failed to produce significant amounts of trihydroxy acids. Short-time incubation of 9(S)-hydroperoxy-10(E),12(Z)-octadecadienoic acid afforded the epoxy alcohol 12(R),13(S)-epoxy-9(S)-hydroxy-10(E)-octadecenoic acid as the main product indicating the sequence 9-hydroperoxide → epoxy alcohol → trihydroxy acid catalyzed by epoxy alcohol synthase and epoxide hydrolase activities, respectively. The high capacity of the enzyme system detected in beetroot combined with a simple isolation protocol made possible by the low amounts of endogenous lipids in the enzyme preparation offered an easy access to pinellic and fulgidic acids for use in biological and medical studies.     

Positional specificity of γ-ketol formation from linoleic acid hydroperoxides by a corn germ enzyme

We have shown unequivocally that the positional specificity of γ-ketol formation by a corn germ enzyme was different from that observed previously by others with an alfalfa seedling enzyme. When the pure positional isomers of linoleic acid hydroperoxide served as substrates, the corn germ enzyme formed one of two γ-ketols: 12-oxo-9-hydroxy-trans-10-octadecenoic acid from 13-hydroperoxy-cis-9,trans-11-octadecadienoic acid (99+% pure) and 10-oxo-13-hydroxy-trans-11-octadecenoic acid from 9-hydroperoxy-trans-10,cis-12-octadecadienoic acid (96% pure). Also isolated from these reactions was one of two α-ketols commonly found as a result of catalysis by linoleic acid hydroperoxide isomerase: 12-oxo-13-hydroxy-cis-9-octadecenoic acid from the 13-hydroperoxide and 10-oxo-9-hydroxy-cis-12-octadecenoic acid from the 9-hydroperoxide. Evidence is offered that γ-ketol formation is catalyzed by linoleic acid hydroperoxide isomerase, the same enzyme responsible for α-ketol production. Presented at the AOCS Spring Meeting, Dallas, Texas, April, 1975.     

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.     

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.     

Absolute configuration of 12-Oxo-10,15(Z)-phytodienoic acid

12-Oxo-10,15(Z)-phytodienoic acid biosynthesized from 13(S)-hydroperoxy-9(Z),11(E),15(Z)-octadecatrienoic acid using a preparation of corn (Zea mays L) hydroperoxide dehydrase recently was found to be a mixture of enantiomers in a ratio of 82∶18 (Hamberg, M., and Hughes, M.A. (1988)Lipids 23, 469–475). In this work, 12-oxophytodienoic acid and (+)-7-iso-jasmonic acid were converted into a common derivative, methyl 3-hydroxy-2-pentyl-cyclopentane-1-octanoate. From gas liquid chromatographic analysis of the (−)-menthoxycarbonyl derivative of methyl 3-hydroxy-2-pentyl-cyclopentane-1-octanoates prepared from 12-oxophytodienoic acid and (+)-7-iso-jasmonic acid, it could be deduced that the major enantiomer of 12-oxophytodienoic acid had the 9(S),13(S) configuration. Therefore, in the major enantiomer of 12-oxophytodienoic acid, the configurations of the side chainbearing carbons are identical to the configurations of the corresponding carbons of (+)-7-iso-jasmonic acid, thus giving support to previous studies indicating that 12-oxophytodienoic acid serves as the precursor of (+)-7-iso-jasmonic acid in plant tissue. When absolute configurations of C-9 and C-13 are not specifically indicated, phytonoic acid is used to denote 2-pentyl-cyclopentane-1-octanoic acid in which the two side chains have thecis relationship, whereas phytonoic acid (trans isomer) denotes 2-pentyl-cyclopentane-1-octanoic acid in which the two side chains have thetrans relationship.     

Method to produce 9(S)-hydroperoxides of linoleic and linolenic acids by maize lipoxygenase

Seed from maize (corn) Zea mays provides a ready source of 9-lipoxygenase that oxidizes linoleic acid and linolenic acid into 9(S)-hydroperoxy-10(F), 12(Z)-octadecadienoic acid and 9(S)-hydroperoxy-10(E), 12(Z), 15(Z)-octadecatrienoic acid, respectively. Corn seed has a very active hydro-peroxide-decomposing enzyme, allene oxide synthase (AOS), which must be removed prior to oxidizing the fatty acid. A simple pH 4.5 treatment followed by centrifugation removes most of the AOS activity. Subsequent purification by ammonium sulfate fractional precipitation results in negligible improvement in 9-hydroperoxide formation. This facile alternative method of preparing 9-hydroperoxides has advantages over other commonly used plant lipoxygenases.     

Biosynthesis of Jasmonates from Linoleic Acid by the Fungus Fusarium oxysporum. Evidence for a Novel Allene Oxide Cyclase

Fusarium oxysporum f. sp. tulipae (FOT) secretes (+)-7-iso-jasmonoyl-(S)-isoleucine ((+)-JA-Ile) to the growth medium together with about 10 times less 9,10-dihydro-(+)-7-iso-JA-Ile. Plants and fungi form (+)-JA-Ile from 18:3n-3 via 12-oxophytodienoic acid (12-OPDA), which is formed sequentially by 13S-lipoxygenase, allene oxide synthase (AOS), and allene oxide cyclase (AOC). Plant AOC does not accept linoleic acid (18:2n-6)-derived allene oxides and dihydrojasmonates are not commonly found in plants. This raises the question whether 18:2n-6 serves as the precursor of 9,10-dihydro-JA-Ile in Fusarium, or whether the latter arises by a putative reductase activity operating on the n-3 double bond of (+)-JA-Ile or one of its precursors. Incubation of pentadeuterated (d₅) 18:3n-3 with mycelia led to the formation of d₅-(+)-JA-Ile whereas d₅-9,10-dihydro-JA-Ile was not detectable. In contrast, d₅-9,10-dihydro-(+)-JA-Ile was produced following incubation of [17,17,18,18,18-²H₅]linoleic acid (d₅-18:2n-6). Furthermore, 9(S),13(S)-12-oxophytoenoic acid, the 15,16-dihydro analog of 12-OPDA, was formed upon incubation of unlabeled or d₅-18:2n-6. Appearance of the α-ketol, 12-oxo-13-hydroxy-9-octadecenoic acid following incubation of unlabeled or [¹³C₁₈]-labeled 13(S)-hydroperoxy-9(Z),11(E)-octadecadienoic acid confirmed the involvement of AOS and the biosynthesis of the allene oxide 12,13(S)-epoxy-9,11-octadecadienoic acid. The lack of conversion of this allene oxide by AOC in higher plants necessitates the conclusion that the fungal AOC is distinct from the corresponding plant enzyme.     

Detection of the First Epoxyalcohol Synthase/Allene Oxide Synthase (CYP74 Clan) in the Lancelet (Branchiostoma belcheri,Chordata)

The CYP74 clan cytochromes (P450) are key enzymes of oxidative metabolism of polyunsaturated fatty acids in plants, some Proteobacteria, brown and green algae, and Metazoa. The CYP74 enzymes, including the allene oxide synthases (AOSs), hydroperoxide lyases, divinyl ether synthases, and epoxyalcohol synthases (EASs) transform the fatty acid hydroperoxides to bioactive oxylipins. A novel CYP74 clan enzyme CYP440A18 of the Asian (Belcher’s) lancelet (Branchiostoma belcheri, Chordata) was biochemically characterized in the present work. The recombinant CYP440A18 enzyme was active towards all substrates used: linoleate and α-linolenate 9- and 13-hydroperoxides, as well as with eicosatetraenoate and eicosapentaenoate 15-hydroperoxides. The enzyme specifically converted α-linolenate 13-hydroperoxide (13-HPOT) to the oxiranyl carbinol (9Z,11R,12R,13S,15Z)-11-hydroxy-12,13-epoxy-9,15-octadecadienoic acid (EAS product), α-ketol, 12-oxo-13-hydroxy-9,15-octadecadienoic acid (AOS product), and cis-12-oxo-10,15-phytodienoic acid (AOS product) at a ratio of around 35:5:1. Other hydroperoxides were converted by this enzyme to the analogous products. In contrast to other substrates, the 13-HPOT and 15-HPEPE yielded higher proportions of α-ketols, as well as the small amounts of cyclopentenones, cis-12-oxo-10,15-phytodienoic acid and its higher homologue, dihomo-cis-12-oxo-3,6,10,15-phytotetraenoic acid, respectively. Thus, the CYP440A18 enzyme exhibited dual EAS/AOS activity. The obtained results allowed us to ascribe a name “B. belcheri EAS/AOS” (BbEAS/AOS) to this enzyme. BbEAS/AOS is a first CYP74 clan enzyme of Chordata species possessing AOS activity.     

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.     

Catalytic properties of allene oxide synthase from flaxseed (Linum usitatissimum L.)

We investigated the catalytic and kinetic properties of allene oxide synthase (AOS; E.C. 3.2.1.92) from flaxseed (Linum usitatissimum L.). Both Michaelis constant and maximal initial velocity for the conversion of 9(S)-and 13(S)-hydroper-oxides of linoleic and linolenic acid were determined by a photometric assay, 13(S)-Hydroperoxy-9(Z), 11(E)-octadecadienoic acid [13(S)-HPOD] as the most effective substrate was converted at 116.9±5.8 nkat/mg protein by the flax enzyme extract. The enzyme was also incubated with a series of variable conjugated hydroperoxy dienyladipates. Substrates with a shape similar to the natural hydroperoxides showed the best reactivity. Monoenoic substrates as oleic acid hydroperoxides were not converted by the enzyme. In contrast, 12-hydroperoxy-9(Z), 13(E)-octadecadienoic acid was a strong competitive inhibitor for AOS catalyzed degradation of 13(S)-HPOD. The inhibitor constant was determined to be 0.09 μM. Based on these results, we concluded that allene oxide synthase requires conjugated diene hydroperoxides for successful catalysis. Studying the enantiomeric preference of the enzyme, we found that AOS was also able to metabolize (R)-configurated fatty acid hydroperoxides. Conversion of these substrates into labile allene oxides was confirmed by steric analysis of the stable α-ketol hydrolysis products.     

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.     

A pathway for biosynthesis of divinyl ether fatty acids in green leaves

[1-¹⁴C]α-Linolenic acid was incubated with a particulate fraction of homogenate of leaves of the meadow buttercup (Ranunculus acris L.). The main product was a divinyl ether fatty acid, which was identified as 12-[1′(Z),3′(Z)-hexadienyloxy]-9(Z), 11(E)-dodecadienoic acid. Addition of glutathione peroxidase and reduced glutathione to incubations of α-linolenic acid almost completely suppressed formation of the divinyl ether acid and resulted in the appearance of 13(S)-hydroxy-9(Z), 11(E), 15(Z)-octadecatrienoic acid as the main product. This result, together with the finding that 13(S)-hydroperoxy-9(Z), 11(E), 15(Z)-octadecatrienoic acid served as an efficient precursor of the divinyl ether fatty acid, indicated that divinyl ether biosynthesis in leaves of R. acris occurred by a two-step pathway involving an ω6-lipoxygenase and a divinyl ether synthase. Incubations of isomeric hydroperoxides derived from α-linolenic and linoleic acids with the enzyme preparation from R. acris showed that 13(S)-hydroperoxy-9(Z), 11(E)-octadecadienoic acid was transformed into the divinyl ether 12-[1′(Z)-hexenyloxy]-9(Z), 11(E)-dodecadienoic acid. In contrast, neither the 9(S)-hydroperoxides of linoleic or α-linolenic acids nor the 13(R)-hydroperoxide of α-linolenic acid served as precursors of divinyl ethers.     

9-Hydroxy-traumatin, a new metabolite of the lipoxygenase pathway

9-Hydroxy-traumatin, 9-hydroxy-12-oxo-10E-dodecenoic acid, was isolated as a product of 13S-hydroperoxy-9Z, 11E-octadecadienoic acid as catalyzed by enzyme preparations of both soybean and alfalfa seedlings. This suggested that 9Z-traumatin, 12-oxo-9Z-dodecenoic acid, was being converted into 9-hydroxy-traumatin in an analogous manner to the previously identified enzymic conversion of 3Z-nonenal and 3Z-hexenal into 4-hydroxy-2E-nonenal and 4-hydroxy-2E-hexenal, respectively. Other metabolites of 13S-hydroperoxy-9Z,11E-octadecadienoic acid were similar for both soybean and alfalfa seedling preparations, and they are briefly described.     

Formation of γ-ketols from 13- and 9-hydroperoxides of linolenic acid by flaxseed hydroperoxide isomerase

A reexamination of the flaxseed hydroperoxide isomerase reaction showed that a minor enzymic product (ca. 5%), identified as a γ-ketol, was present. The substrates were the 13- or 9-hydroperoxides of linolenic acid, which were converted to 9-hydroxy-12-oxo-cis-15-trans-11-octadecadienoic acid, respectively. These compounds were formed in addition to the major products reported earlier: a 12,13-α-ketol and 12-oxo-cis-10,15-phytodienoic acid from the 13-isomer, and a 9,10-α-ketol from the 9-isomer.     

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.     

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.     

Lack of regioselectivity in formation of oxohydroxyoctadecenoic acids from the 9- or 13-hydroperoxide of linoleic acid

Either 9-hydroperoxy-trans-10,cis-12-octadecadienoic acid or 13-hydroperoxy-cis-9,trans-11-octadecadienoic acid was treated with the catalyst, cysteine-FeCl₃, in the presence of oxygen. Oxohydroxyoctadecenoic acids were among the many products formed as a result of hydroperoxide decomposition. A mixture of 9(13)-oxo-13(9)-hydroxy-trans-11(10)-octadecenoic acids (δ-ketols) was produced from either isomeric hydroperoxide. The formation of isomeric δ-ketols from 9-hydroxy-trans-12,13-epoxy-trans-10-octadecenoic acid (epoxyol), a known product of 13-hydroperoxy-cis-9,trans-11-octadecadienoic acid decomposition, implies that the epoxyol is an intermediate. The mechanism was elucidated by the facile conversion of the epoxyol (methyl ester_ to methyl 9(13)-oxo-13(9)-hydroxy-trans-11(10)-octadecenoates with a Lewis acid, BF₃-etherate. Presented at the 14th World Congress, International Society for Fat Research, Brighton, U.K., September 17–22, 1978. The mention of firm names or trade products does not imply that they are endorsed or recomended by the U.S. Department of Agriculture over other firms or similar products not mentioned.     

Production of hydroxy fatty acids from unsaturated fatty acids byFlavobacterium sp. DS5 hydratase,a C-10 positional- andcis unsaturation-specific enzyme

A new microbial isolate,Flavobacterium sp. DS5, converted oleic and linoleic acids to their corresponding 10-keto-and 10-hydroxy fatty acids. The hydration enzyme seems to be specific to the C-10 position. Conversion products from α- and γ-linolenic acids were identified by gas chromatography/mass spectrometry, Fourier transform infrared, and nuclear magnetic resonance as 10-hydroxy-12(Z),15(Z)-octadecadienoic and 10-hydroxy-6(Z),12(Z)-octadecadienoic acids, respectively. Products from other 9(Z)-unsaturated fatty acids also were identified as their corresponding 10-hydroxy- and 10-keto-fatty acids.Trans unsaturated fatty acid was not converted. From these results, it is concluded that strain DS5 hydratase is indeed a C-10 positional-specific andcis-specific enzyme. DS5 hydratase prefers an 18-carbon monounsaturated fatty acid. Among the C₁₈ unsaturated fatty acids, an additional double bond at either side of the 9,10-position lowers the enzyme hydration activity. Because hydratases from other microbes also convert 9(Z)-unsaturated fatty acids to 10-hydroxy fatty acids, the C-10 positional specificity of microbial hydratases may be universal.