Linoleate hydroperoxides are cleaved heterolytically into aldehydes by a Lewis acid in aprotic solvent

Treatment of isomeric methyl linoleate hydroperoxides with a Lewis acid, BF₃, in anhydrous ether led to a carbon-to-oxygen rearrangement that caused cleavage into shorter-chain aldehydes. Methyl (9Z,11E)-13-hydroperoxy-9,11-octadecadienoate afforded mainly hexanal and methyl (E)-12-oxo-10-dodecenoate, whereas methyl (10E,12Z)-9-hydroperoxy-10,12-octadecadienoate cleaved into 2-nonenal and methyl 9-oxononanoate. The 2 aldehydes obtained from each hydroperoxide isomer were uncharacteristic of the complex volatile profile usually obtained by β-scission of oxy radicals derived from homolysis of the hydroperoxide group. Rather, the reaction resembled the one catalyzed by the plant enzyme, hydroperoxide lyase. Presented in part at the American Oil Chemists' Society Meeting, Chicago, Illinois, May 8–12, 1983. The mention of firm names or trade products does not imply that they are endorsed or recommended by the USDA over other firms or similar products not mentioned.     

Linolenate 9R-Dioxygenase and Allene Oxide Synthase Activities of Lasiodiplodia theobromae

Jasmonic acid (JA) is synthesized from linolenic acid (18:3n-3) by sequential action of 13-lipoxygenase, allene oxide synthase (AOS), and allene oxide cyclase. The fungus Lasiodiplodia theobromae can produce large amounts of JA and was recently reported to form the JA precursor 12-oxophytodienoic acid. The objective of our study was to characterize the fatty acid dioxygenase activities of this fungus. Two strains of L. theobromae with low JA secretion (~0.2 mg/L medium) oxygenated 18:3n-3 to 5,8-dihydroxy-9Z,12Z,15Z-octadecatrienoic acid as well as 9R-hydroperoxy-10E,12Z,15Z-octadecatrienoic acid, which was metabolized by an AOS activity into 9-hydroxy-10-oxo-12Z,15Z-octadecadienoic acid. Analogous conversions were observed with linoleic acid (18:2n-6). Studies using [11S-²H]18:2n-6 revealed that the putative 9R-dioxygenase catalyzed stereospecific removal of the 11R hydrogen followed by suprafacial attack of dioxygen at C-9. Mycelia from these strains of L. theobromae contained 18:2n-6 as the major polyunsaturated acid but lacked 18:3n-3. A third strain with a high secretion of JA (~200 mg/L) contained 18:3n-3 as a major fatty acid and produced 5,8-dihydroxy-9Z,12Z,15Z-octadecatrienoic acid from added 18:3n-3. This strain also lacked the JA biosynthetic enzymes present in higher plants.     

An efficient ultrasound-assisted zinc reduction of fatty esters containing conjugated enynol and conjugated enynone systems

Reduction of methyl 8-hydroxy-11-E/Z-octadecen-9-ynoate (1) with zinc in either aqueous n-propanol or water under concomitant ultrasound irradiation furnished a mixture of methyl 8-hydroxy-9Z,11E-octadecadienoate (3a) and methyl 8-hydroxy-9Z, 11Z-octadecadienoate (3b) (96% yield). Reduction of methyl 8-oxo-11-E/Z-octadecen-9-ynoate (2) under similar conditions gave methyl 8-oxo-10-Z-octadecenoate exclusively (4, 70%). The latter compound was epoxidized and converted to a C₁₈ furanoid fatty ester (6, methyl 8,11-epoxy-8,10-octadecadienoate) in 70% yield.     

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.     

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.     

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.     

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.     

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.     

Enantioselective formation of an α, β-epoxy alcohol by reaction of methyl 13(S)-hydroperoxy-9(Z), 11(E)-octadecadienoate with titanium isopropoxide

Methyl 11(R), 12(R)-epoxy-13(S)-hydroxy-9(Z)-octadecenoate (threo isomer) was generated from linoleic acid by the sequential action of an enzyme and two chemical reagents. Linoleic acid was treated with lipoxygenase to yield its corresponding hydroperoxide [13(S)-hydroperoxy-9(Z), 11(E)-octadecadienoic acid]. After methylation with CH₂N₂, the hydroperoxide was treated with titanium (IV) isopropoxide [Ti(O-i-Pr)₄] at 5°C for 1 h. The products were separated by normal-phase high-performance liquid chromatography and characterized with gas chromatography-mass spectrometry, infrared spectroscopy, and nuclear magnetic resonance spectroscopy. Approximately 30% of the product was methyl 13(S)-hydroxy-9(Z), 11(E)-octadecadienoate. Over 60% of the isolated product was methyl 11(R), 12(R)-epoxy-13(S)-hydroxy-9(Z)-octadecenoate. After quenching Ti(O-i-Pr)₄ with water, the spent catalyst could be removed from the fatty products by partitioning between CH₂Cl₂ and water. These results demonstrate that Ti(O-i-Pr)₄ selectively promotes the formation of an α-epoxide with the threo configuration. It was critically important to start with dry methyl 13(S)-hydroperoxy-9(Z),11(E)-octadecadienoate because the presence of small amounts of water in the reaction medium resulted in the complete hydrolysis of epoxy alcohol to trihydroxy products.     

Enantioselective conversion of linoleate hydroperoxide to an α, β-epoxy alcohol by niobium ethoxide

Niobium (V) ethoxide [Nb(OC₂H₅)₅] catalyzed the rearrangement of methyl 13(S)-hydroperoxy-9(Z),11(E)-octadecadienoate (Me-HPODE) to epoxy hydroxy isomers. At low temperature (5°C) in aprotic solvent, Me-HPODE was converted to the diastereomeric α, β-epoxy alcohols, methyl 11(R,S),12(R,S)-epoxy-13(S)-hydroxy-9(Z)octadecenoate. These products are referred to as oxylipids and structurally resemble those obtained from the vanadium- and epoxygenase-catalyzed rearrangement of Me-HPODE but are distinct from products obtained from ferrous iron-, hematin-, and hemoglobin-catalyzed rearrangements. Because the product of the niobium-catalyzed rearrangement of Me-HPODE was predominantly the erythro diastereomer, the rearrangement is distinguished from that produced by a titanium catalyst, in which the threo diastereomer [methyl 11(R), 12(R)-epoxy-13(S)-hydroxy-9(Z)-octadecenoate] predominates, and from that produced by a vanadium catalyst, in which both diastereomers are produced in equal proportion. The synthesis of alcohol epoxide by Nb(OC₂H₅)₅ was inhibited by traces of water, but inclusion of molecular sieves in the reaction medium did not improve yield, as the alcohol epoxide rearranged to ketonic materials.     

Reaction of α-tocopherol in heated bulk phase in the presence of methyl linoleate (13S)-hydroperoxide or methyl linoleate

α-Tocopherol and methyl (9Z, 11E)-(S)-13-hydroperoxy-9, 11-octadecadienoate (13-MeLOOH) were allowed to stand at 100°C in bulk phase. The products were isolated and identified as methyl 13-hydroxyoctadecadienoate (1), stereoisomers of methyl 9,11,13-octadecatrienoate (2), methyl 13-oxo-9, 11-octadecadienoate (3), epoxy dimers of methyl linoleate with an ether bond (4), a mixture of methyl (E)-12, 13-epoxy-9-(α-tocopheroxy)-10-octadecenoates and methyl (E)-12, 13-epoxy-9-(α-tocopheroxy)-11-(α-tocopheroxy)-9-octadecenoates (5), a mixture of methyl 9-(α-tocopheroxy)-10,12-octadecadienoates and methyl 13-(α-tocopheroxy)-9, 11-octadecadienoates (6), α-tocopherol spirodiene dimer (7), and α-tocopherol trimer (8). α-Tocopherol and 13-MeLOOH were dissolved in methyl myristate, and the thermal decomposition rate and the distributions of reaction products formed from α-tocopherol and 13-MeLOOH were analyzed. α-Tocopherol disappeared during the first 20 min, and the main products of α-tocopherol were 5 and 6 with the accumulation of 1–4 which were the products of 13-MeLOOH. The results indicate that the alkyl and alkoxyl radicals from the thermal decomposition of 13-MeLOOH could be trapped by α-tocopherol to produce 5 and 6. The reaction products of α-tocopherol during the thermal oxidation of methyl linoleate were compounds 6 and 7. Since the radical flux during the autoxidation might be low, the excess α-tocopheroxyl radical reacted with each other to form 7.     

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.     

Synthesis of phenyl substituted C18 furanoid fatty esters

Methyl 9,12-epoxy-10-phenyl-9,11-octadecadienoate was prepared by acid catalyzed cyclization of methyl 9,12-dioxo-10-phenyloctadecanoate, which was derived from the oxidation of methyl 9-hydroxy-12-oxo-10-phenyloctadecanoate. The latter was exclusively obtained from methylcis-9,10-epoxy-12-oxooctadecanoate with phenyllithium in the presence of copper (I) bromide. A mixture of positional isomers, methyl 9,12-epoxy-10(11)-phenyl-9,11-octadecadienoates, was also prepared by another route. The spectroscopic properties of the various intermediates and products were studied. The positional isomers of the phenyl substituted furanoid fatty esters were characterized by¹³C nuclear magnetic resonance spectrometry.     

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.     

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.     

Iron-catalyzed reaction products of α-tocopherol with methyl 13(S)-hydroperoxy-9(Z),11(E)-octadecadienoate

α-Tocopherol was reacted with methyl 13(S)-hydroperoxy-9(Z),11(E)-octadecadienoate in the presence of an iron-chelate, Fe(III)-acetylacetonate, at 37°C in benzene. The reaction was carried out either aerobically or anaerobically. The main products of α-tocopherol under air were isolated and identified as two stereoisomers of 4a,5-epoxy-8a-hydroperoxy-α-tocopherone, four stereoisomers of methyl 9-(8a-dioxy-α-tocopherone)-12,13-epoxy-10(E)-octadecenoate, four stereoisomers of methyl 11-(8a-dioxy-α-tocopherone)-12,13-epoxy-9(Z)-octadecenoate, two stereoisomers of methyl 13(S)-(8a-dioxy-α-tocopherone)-9(Z),11(E)-octadecadinoate, and α-tocopherol dimer. Besides the 8a-(lipid-peroxy)-α-tocopherones, two stereoisomers of methyl 11-(α-tocopheroxy)-12(S),13(S)-epoxy-9(E)-octadecenoate, two stereoisomers of methyl 9-(α-tocopheroxy)-12(S),13(S)-epoxy-10(E)-octadecenoate, and two isomers of methyl (α-tocopheroxy)-octadecadienoate were obtained under nitrogen atmosphere. The results indicate that the peroxyl radicals from lipid hydroperoxides prefer to react with the 8a-carbon radical of α-tocopherol and the carbon-centered radicals react with the phenoxyl radical of α-tocopherol.     

Biosynthesis of new divinyl ether oxylipins in Ranunculus plants

[1-¹⁴C]Linolenic acid was incubated with homogenates of leaves from the aquatic plants Ranunculus lingua (greater spearwort) or R. peltatus (pond water-crowfoot). Analysis by reversed-phase high-performance liquid radiochromatography demonstrated the formation of a new divinyl ether FA, i.e., 12-[1′(E), 3′(Z)-hexadienyloxy]-9(Z), 11(Z)-dodecadienoic acid [11(Z)-etherolenic acid] as well as a smaller proportion of ω5(Z)-etherolenic acid previously identified in terrestrial Ranunculus plants. The same divinyl ethers were formed upon incubation of 13(S)-hydroperoxy-9(Z), 11(E), 15(Z)-octadecatrienoic acid, a lipoxygenase metabolite of linolenic acid, whereas the isomeric hydroperoxide, 9(S)-hydroperoxy-10(E), 12(Z), 15(Z)-octadecatrienoic acid, was not converted into divinyl ethers in R. lingua or R. peltatus. Incubation of [1-¹⁴C]linoleic acid or 13(S)-hydroperoxy-9(Z), 11(E)-octadecadienoic acid produced the divinyl ether 12-[1′(E)-hexenyloxy]-9(Z), 11(Z)-dodecadienoic acid [11(Z)-etheroleic acid] and a smaller amount of ω5(Z)-etheroleic acid. The experiments demonstrated the existence in R. lingua and R. peltatus of a divinyl ether synthase distinct from those previously encountered in higher plants and algae.     

Volatiles from thermal decomposition of isomeric methyl (12S, 13S)-(E)-12,13-epoxy-9-hydroperoxy-10-octadecenoates

Two epimers of methyl (12S,13S)-(E)-12,13-epoxy-9-hydroperoxy-10-octadecenoate were isolated after esterification of a mixture of fatty acids obtained from decomposition of (13S)-(9Z,11E)-13-hydroperoxy-9,11-octadecadienoic acid by an Fe⁺⁺-cysteine catalyst. These epimeric epoxyhydro-peroxyoctadecenoates were decomposed by heat (210 C) in the injection port of a gas chromatograph, and the cleavage fragments were subsequently separated by gas chromatography (GC) and identified by mass spectrometry (MS). Among the scission products obtained, the most prominent in the GC peak profile were methyl octanoate and methyl 9-oxononanoate. Other peaks were identified as pentane, 1-pentanol, hexanal, 2-heptanone, 2-pentylfuran, methyl heptanoate, 2-octenal, 4,5-epoxy-2-decenal, methyl 8-(2-furyl)-octanoate, 11-oxo-9-undecenoate and methyl 13-oxo-9,11-tridecadienoate. In addition, 3,4-epoxynonanal, methyl 8-oxooctanoate, 3-hydroxy-2-pentyl-2,3-dihydrofuran and methyl 10-oxodecanoate were tentatively identified. Except for the furan compounds, the formation of the fragmentation products could be explained by conventional free-radical scission mechanisms. The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned.     

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.     

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.