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.     

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.     

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.     

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.     

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.     

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.     

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.     

Oxygenated fatty acid constituents of soybean phosphatidylcholines

Bitter-tasting phosphatidylcholines from hexane-defatted soybean flakes were chromatographically separable from ordinary soy phosphatidylcholines (SPC). The bitter-tasting SPC contain 32% oxygenated fatty acids in addition to palmitic, stearic, oleic, linoleic, and linolenic acids. Identification of these oxygenated acids was based on infrared, ultraviolet, proton nuclear magnetic resonance, and mass spectral characteristics of methyl ester derivatives which were separated and purified by column and thin layer chromatography. The fatty acid methyl esters identified were (a) 15, 16-epoxy-9, 12-octadecadienoate, (b) 12, 13-epoxy-9-octadecenoate, both with double bonds and epoxide groups predominantly ofcis configuration; (c) 13-oxo-9,11-and 9-oxo-10, 12-octadecadienoates; (d) 13-hydroxy-9, 11- and 9-hydroxy-10, 12-octadecadienoates; (e) 9, 10, 13-trihydroxy-11- and 9,12,13-trihydroxy-10-octadecenoates. In addition, trace amounts of (f) 11-hydroxy-9,10-epoxy-12-and 11-hydroxy-12,13-epoxy-9-octadecenoates; (g) 13-oxo-9-hydroxy-10-and 9-oxo-13-hydroxy-11-octadecenoates; (h) 9,10-dihydroxy-12- and 12, 13-dihydroxy-9-octadecenoates; and (i) 9,12,13-dihydroxyethoxy-10- and 9,10,13-dihydroxyethoxy-11-octadecenoates were indicated by mass spectrometry. Dihydroxyethoxy compounds (i) were possibly formed upon extraction of the SPC from flakes by 80% ethanol. Except for the first two epoxy compounds, labelled a and b, the oxygenated fatty acids are similar to the products formed by homolytic decomposition of linoleic acid hydroperoxide. The first two compounds with predominantlycis configuration may occur by action of fatty acid hydroperoxides on an unsaturated fatty acid. Presented in part at the 13th World Congress of the International Society for Fat Research, Marseille, France, August 31–September 4, 1976.     

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.     

Isolation of unsaturated diols after oxidation of conjugated linoleic acid with peroxygenase

Oat seeds are a rich source of peroxygenase, an iron heme enzyme that participates in oxylipin metabolism in plants. An isomer of CLA, 9(Z), 11(F)-octadecadienoic acid (1), believed to have anticarcinogenic activity, was used as a substrate for peroxygenase in an aqueous medium using t-butyl hydroperoxide as the oxidant. After acidification of the reaction medium, the products were extracted with ethyl ether, converted to their methyl esters, and characterized using HPLC. Major products after reaction for 24 h showed resonances from ¹H NMR spectroscopy that were further downfield than the expected epoxides and were thought to be diol hydrolysis products. However, analyses by HPLC with atmospheric pressure chemical ionization MS (APCI-MS) of the putative allylic diols or their bis-trimethylsilyl ether derivatives gave incorrect M.W. The M.W. of the diols could be obtained by APCI-MS after removal of unsaturation by hydrogenation or by EI-MS after conversion of unsaturation by hydrogenation or by EI-MS after conversion of the allylic 1,2-diols to cyclic methyl boronic esters. Data from MS in conjunction with analyses using ¹H and ¹³C NMR showed that the methylated products from 1 were methyl 9,10(threo)-dihydroxy- 11(E)-octadecenoate, methyl 9,10(erythro)-dihydroxy-11(E)-octadecenoate, methyl 9,12(threo)-dihydroxy-10(E)-octadecenoate. Solid-phase extraction without prior acidification and conversion of the products to methyl esters allowed identification of the following epoxides: methyl 9,10(Z)-epoxy-11(E)-octadecenoate (6M), methyl 9,10(E)-epoxy-11(E)-octadecenoate, and methyl 11,12(E)-epoxy-9(Z)-octadecenoate. At times of up to at least 6h, 6M accounted for approximately 90% of the epoxide product. Product analysis after the hydrolysis of isolated epoxide 6M showed that hydrolysis of epoxide 6 could largely account for the diol products obtained from the acidified reaction mixtures.     

Monooxygenase system of Bacillus megaterium ALA2: Studies on linoleic acid epoxidation products

Bacillus megaterium ALA2 produces many oxygenated FA from linoleic acid: 12,13-dihydroxy-9(Z)-octadecenoic acid; 12,13,17-trihydroxy-9(Z)-octadecenoic acid; 12,13,16-trihydroxy-9(Z)-octadecenoic acid; 12-hydroxy-13,16-epoxy-9 (Z)-octadecenoic acid; and 12,17;13,17-diepoxy-16-hydroxy-9 (Z)-octadecenoic acid. Recently, we studied the monooxygenase system of B. megaterium ALA2 by comparing its palmitic acid oxidation products with those of the well-studied catalytically self-sufficient P450 monooxygenase of B. megaterium ATCC 14581 (NRRL B-3712) and of B. subtilis strain 168 (NRRI B-4219). We found that their oxidation products are identical, indicating that their monooxygenase systems (hydroxylation) are similar. Now, we report that strain ALA2 epoxidizes linoleic acid to 12,13-epoxy-9(Z)-octadecenoic acid and 9,10-epoxy-12 (Z)-octadecenoic acid, the initial products in the linoleic acid oxidation. The epoxidation enzyme did not oxidize specific double bond of the linoleic acid. The epoxidation activity of strain ALA2 was compared with the above-mentioned two Bacillus strains. These two Bacillus strain also produced 12,13-expoxy-9 (Z)-octadecenoic acid and 9,10-epoxy-12(Z)-octadecenoic acid, indicating that their epoxidation enzyme systems might be similar. The ratios of epoxy FA production by these three strains (A1 A2, NRRI B-3712, and NRRI B-4219) were, respectively, 5.56∶0.66∶0.18 for 12,13-epoxy-9(Z)-octadecenoic acid and 2.43∶0.41∶0.57 for 9,10-epoxy-12(Z)-octadecenoic acid per 50 mL medium per 48 h.     

Reaction of mono-epoxidized conjugated linoleic acid ester with boron trifluoride etherate complex

The reaction of methyl 11, 12-E-epoxy-9Z-octadecenoate (1) with boron trifluoride etherate furnished a mixture of methyl 12-oxo-10E-octadecenoate (3a) and methyl 11-oxo-9E-octadecenoate (3b) in 66% yield. Methyl 9, 10-Z-epoxy-11 E-octadecenoate (2) with boron trifluoride etherate furnished a mixture of methyl 9-oxo-10 E-octadecenoate (4a, 45%) and methyl 10-oxo-11 E-octadecenoate (4b, 19%). A plausible mechanism is proposed for these reactions, which involves the attack on the epoxy ring system by BF₃, followed by deprotonation, oxo formation, and double bond migration to give a mixture of two positional α,β-unsaturated C₁₈ enone ester derivatives (3a/3b, 4a/4b). The structures of these C₁₈ enone ester derivatives (3a/3b, 4a/4b) were identified by a combination of NMR spectroscopic and mass spectrometric analyses.     

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.     

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.     

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.     

A study of the structures and reactions of some methyl substituted unsaturated C18 ester intermediates in the synthesis of a racemic mixture of 10-methyloctadecanoic acid

Methyl 10-undecenoate was hydrated to methyl 10-hydroxyundecanoate using mercury (II) acetate in aqueous tetrahydrofuran (THF). Chromic acid oxidation of methyl 10-hydroxyundecanoate gave methyl 10-oxoundecanoate, which was hydrolyzed to 10-oxoundecanoic acid. Reaction of n-octyl magnesium bromide complex in THF with 10-oxoundecanoic acid furnished 10-hydroxy-10-methyloctadecanoic acid after hydrolysis. The latter compound was esterified, and dehydration of methyl 10-hydroxy-10-methyloctadecanoate withp-toluenesulfonic acid in benzene gave a mixture of unsaturated branched fatty ester intermediates:viz. methyl 10-methyl-9-octadecenoate, 10-methyl-10-octadecenoate and 10-octyl-10-undecenoate. Treatment of the mixture of unsaturated branched fatty ester intermediates with mercury (II) acetate in methanol gave exclusively methyl 10-methoxy-10-methyloctadecanoate. Epoxidation of the same mixture of unsaturated fatty esters withm-chloroperbenzoic acid provided a mixture of epoxy derivatives: methyl 9,10-epoxy-10-methyloctadecanoate, 10,11-epoxy-10-methyloctadecanoate and 2-octyl-oxirane-nonanoate. Catalytic hydrogenation of the mixture of unsaturated fatty esters gave a racemic mixture of methyl 10-methyloctadecanoate, which was hydrolyzed to 10-methyloctadecanoic acid. The structures of the mixture of unsaturated branched fatty ester intermediates and their derivatives were characterized by chemical and spectroscopic analyses.     

Preparation of fatty epoxy alcohols using oat seed peroxygenase in nonaqueous media

Peroxygenase is an enzyme of higher plants that is capable of using hydroperoxide and hydrogen peroxide for oxidation of a double bond to an epoxide. A microsomal fraction was prepared from dry oat (Avena sativa) seeds. The peroxygenase activity of this fraction was tested using fatty acid hydroperoxide 2a [13(S)-hydroperoxy-9(Z), 11(E)-octadecadienoic acid] and its methyl ester 2b as sources of peroxygen. These were prepared by the action of soybean lipoxygenase on linoleic acid. A high-performance liquid chromatographic assay was used to differentiate between peroxygen cleavage and peroxygen cleavage with accompanying double-bond oxidation Higher activity was obtained with 2b compared to 2a, and peroxygen cleavage activity was observed in both aqueous and organic solvent media. Double-bond oxidation activity was high only in aqueous media and nonpolar organic solvents. Structural elucidation of the epoxidized product showed it to be the oxylipid, methyl cis-9,10-epoxy-13(S)-hydroxy-11(E)-octade-cenoate 4b, demonstrating specificity for epoxidation of the cis double bond. Trihydroxy product was not detected, demonstrating that the epoxide was not hydrolyzed.     

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.     

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.     

Purification, molecular cloning, and expression of the gene encoding fatty acid 13-hydroperoxide lyase from guava fruit (Psidium guajava)

Guava fruit was identified as a particularly rich source of 13-hydroperoxide lyase activity. The enzyme proved stable to chromatographic procedures and was purified to homogeneity. Based on gel filtration and gel electrophoresis, the native enzyme appears to be a homotetramer with subunits of 55 kD. Starting with primers based on the peptide sequence, the enzyme was cloned by polymerase chain reaction with 3′ and 5′ rapid amplification of cDNA ends. The sequence shows approximately 60–70% identity to known 13-hydroperoxide lyases and is classified in cytochrome P450 74B subfamily as CYP74B5. The cDNA was expressed in Escherichia coli (BL21 cells), with optimal enzyme activity obtained in the absence of isopropyl-β-d-thiogalactopyranoside and σ-aminolevulinic acid. The expressed enzyme metabolized 13(S)-hydroperoxylinolenic acid over 10-fold faster than 13(S)-hydroperoxylinoleic acid and the 9-hydroperoxides of linoleic and linolenic acids. 13(S)-Hydroperoxylinolenic acid was converted to 12-oxododec-9(Z)-enoic acid and 3(Z)-hexenal, as identified by gas chromatography-mass spectrometry. The turnover number with this substrate, with enzyme concentration estimated from the Soret absorbance, was≈2000/s, comparable to values reported for the related allene oxide synthases. Distinctive features of the guava 13-hydroperoxide lyase and related cytochrome P450 are discussed.