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 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.     

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.     

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.     

The absolute optical configurations of the isomeric 9,10-epoxystearic, 9,10-dihydroxystearic and 9,10,12-trihydroxystearic acids

The absolute optical configuration of (−)-cis-9,10-epoxystearic acid has been verified as being L. Here (−)-erythro-9,10-dihydroxystearic acid, isolated from castor oil, was converted by stereospecific reactions to (+)-cis-9,10-epoxystearic acid and was thereby proved to be D-9,D-10-dihydroxystearic acid. Removal of the D-12-hydroxy group from the higher meltingerythro-9,10,12-trihydroxystearate derived from ricinoleic acid, after protection of the glycol group, gave the L-9,L-10-dihydroxystearate derivative. This proved the high melting diastereoisomer to be L-9,L-10,D-12-trihydroxystearate and directly verified the supposition that the higher melting, arsenite-complexing diastereoisomer of such oxidation pairs has thetrans-10,12-diol grouping. On this basis, the higher meltingthreo-trihydroxystearate from ricinoleate must be D-9,L-10,D-12-trihydroxystearate and removal of the 12-hydroxy group must give D-9,L-10-dihydroxystearate which proved to be the levorotatory enantiomer. The dextrorotatory L-9,D-10-dihydroxystearate was transformed by stereospecific reactions to (+)-trans-9,10-epoxystearic acid, thereby defining the absolute configurations oftrans-9,10-epoxystearic acids. On the basis of these results conclusions may be drawn as to the stereospecificity and site of action of enzymes which hydrate 9,10-epoxystearic acids.     

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.     

Pheromone specificity inEriocrania semipurpurella (Stephens) andE. sangii (Wood) (Lepidoptera: Eriocraniidae) based on chirality of semiochemicals

The fifth abdominal segment of femaleEriocrania semipurpurella (Stephens) andE. sangii (Wood) contains a pair of exocrine glands. Hexane extracts of this segment were prepared from both species and analyzed by gas chromatography with simultaneous flame ionization and electroantennographic detection (EAD). For both species, the EAD active peaks were identified as nonan-2-one, (Z)-6-nonen-2-one, and (Z)-6-nonen-2-ol by means of mass spectrometry and comparison of retention indices with those of synthetic standards. Enantiomeric separation of chiral alcohols from the female extracts was achieved by gas chromatographic analysis on a cyclodextrin column. InE. semipurpurella, a mixture of (2S,6Z)-nonen-2-ol and (2R,6Z)-nonen-2-ol (2: I) was found, whereas inE. sangii (2S,6Z)-nonen-2-ol was the predominant enantiomer and only traces of theR enantiomer were indicated by the antennal response. In field tests, a blend of the three compounds was not attractive to conspecific males. A subtractive assay showed that the alcohol in various enantiomeric mixtures was the only attractive compound, whereas addition of (Z)-6-nonen-2-one to the alcohol completely inhibited the attraction of both species. A trapping experiment including a wide range of ratios between theR andS enantiomers showed that baits containing 95–100% of theS enantiomer were attractive to maleE. sangii, whereas males ofE. semipurpurella were attracted to all tested ratios of the enantiomers. However, the response profiles of maleE. semipurpurella differed between populations from southern Sweden, south Finland, and the Kola Peninsula in Russia. In south Sweden males were maximally attracted to a racemic mixture of the alcohols. At the Kola PeninsulaE. semipurpurella was attracted to baits containing 95–100% of theR enantiomer. In south Finland all tested ratios between 0 and 100%R enantiomer trappedE. semipurpurella, but the trap catches appeared to be bimodally distributed with peaks around 15 and 70%R enantiomer. The trapping results suggest the existence of pheromone races or sibling species among the specimens identified asE. semipurpurella.Dedicated to Prof. H. J. Bestmann on the occasion of his 70th birthday.     

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.     

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.     

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.     

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.     

Novel Cyclopropane Fatty Acids from the Phospholipids of the Caribbean Sponge <Emphasis Type="Italic">Pseudospongosorites suberitoides</Emphasis>

The cyclopropane fatty acids 17-methyl-trans-4,5-methyleneoctadecanoic acid, 18-methyl-trans-4,5-methylenenonadecanoic acid, and 17-methyl-trans-4,5-methylenenonadecanoic acid were characterized for the first time in nature in the phospholipids (mainly PE, PG and PS) of the hermit-crab sponge Pseudospongosorites suberitoides. Pyrrolidine derivatization was the key in identifying the position of the cyclopropyl and methyl groups in the acyl chains and ¹H NMR was used to determine the trans stereochemistry of the cyclopropane ring. The phospholipids from the sponge also contained an interesting series of iso-anteiso Δ^5,9 fatty acids with chain-lengths between 17 and 21 carbons, with the fatty acids (5Z,9Z)-18-methyl-5,9-nonadecadienoic acid and the (5Z,9Z)-17-methyl-5,9-nonadecadienoic acid being described for the first time in sponges. The anteiso α-methoxylated fatty acid 2-methoxy-12-methyltetradecanoic acid was also identified for the first time in nature in the phospholipids of this interesting marine sponge. The novel cyclopropyl fatty acids could have originated from the phospholipids of a cyanobacterium living in symbiosis with the sponge.     

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.     

Identification of Novel α-Methoxylated Phospholipid Fatty Acids in the Caribbean Sponge <Emphasis Type="Italic">Erylus goffrilleri</Emphasis>

The phospholipid fatty acid composition of the Caribbean sponge Erylus goffrilleri is described for the first time. A total of 70 fatty acids with chain lengths between 13 and 29 carbons were identified in the sponge. Methyl-branched fatty acids predominated in E. goffrilleri suggesting the presence of a considerable number of bacterial symbionts. The novel fatty acids (5Z,9Z)-2-methoxy-5,9-hexadecadienoic acid, (5Z,9Z)-2-methoxy-5,9-octadecadienoic acid, (5Z,9Z)-2-methoxy-5,9-nonadecadienoic acid, and (5Z,9Z)-2-methoxy-5,9-eicosadienoic acid are described for the first time in the literature. In addition, the iso-methyl-branched fatty acids (9Z)-2-methoxy-15-methyl-9-hexadecenoic acid and (5Z,9Z)-2-methoxy-15-methyl-5,9-hexadecadienoic acid, also identified in E. goffrilleri, were identified for the first time in nature. Based on the identified metabolites it is proposed that the unprecedented biosynthetic sequence: i-17:1Δ9 → 2-OMe-i-17:1Δ9 → 2-OMe-i-17:2Δ5,9 might be responsible for the biosynthesis of the novel iso-α-methoxylated fatty acids in E. goffrilleri.     

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.     

Semiochemicals via epoxide inversion

A sequence of reactions is presented for inverting the configurations of both epoxide carbons in 1,2-disubstituted epoxides. As examples, (+)-disparlure was converted to its enantiomer, (–)-disparlure, andexo-endo conversion of a cyclohexene oxide was demonstrated.     

Bioconversion of n−3 and n−6 PUFA by <Emphasis Type="Italic">Clavibacter</Emphasis> sp. ALA2

Clavibacter sp. ALA2 oxidized n−3 and n−6 PUFA into a variety of oxylipins. Structures of products converted from EPA and DHA were determined as 15,18-dihydroxy-14,17-epoxy-5(Z),8(Z),11(Z)-eicosatrienoic acid and 17,20-dihydroxy-16,19-epoxy-4(Z),7(Z),10(Z),13(Z)-docosatetraenoic acid by GC-MS and NMR analyses. In contrast, γ-linolenic acid and arachidonic acid were converted to diepoxy bicyclic FA, tetrahydrofuranyl monohydroxy FA, and trihydroxy FA. Thus, the structures of bioconversion products were different between n−3 and n−6 PUFA. Furthermore, strain ALA2 placed hydroxy groups and cyclic structures at the same position from the ω-terminal despite the number of carbons in the chain and the double bonds in the PUFA.     

High performance liquid chromatographic separation of monoacylglycerol enantiomers on a chiral stationary phase

High performance liquid chromatographic separation of monoacylglycerol enantiomers as di-3,5-dinitrophenylurethane derivatives was carried out on a chiral stationary phase, N-(S)-2-(4-chlorophenyl)isovaleroyl-D-phenylglycine chemically bonded tov-aminopropyl silanized silica. Complete separation of the urethane derivatives of racemic monoacylglycerols with saturated acyl groups of C₁₂−C₁₈ was achieved using a stainless steel column (25 cm long) packed with the 5μ particles, an isocratic elution at ambient temperature with a mixture of hexane/ethylene dichloride/ethanol as a mobile phase, and a UV detector. Thesn-1 enantiomers were eluted ahead of the correspondingsn-3 enantiomers. Complete separation of thesn-2 isomers from the corresponding enantiomers and partial separation of the enantiomer homologues differing by two acyl carbons also were observed.     

Biosynthesis and isomerization of 11-hydroperoxylinoleates by manganese- and iron-dependent lipoxygenases

Manganese lipoxygenase (Mn-LO) oxygenates linoleic acid (LA) to a mixture of the hydroperoxides—11(S)-hydroperoxy-9Z,12Z-octadecadienoic acid [11(S)-HPODE] and 13(R)-hydroperoxy-9Z,11E-octadecadienoic acid [13(R)-HPODE]- and also catalyzes the conversion of 11(S)-HPODE to 13(R)-HPODE via oxygen-centered (LOO•) and carbon-centered (L•) radicals [Hamberg, M., Su, C., and Oliw, E. (1998) Manganese Lipoxygenase. Discovery of a Bis-allylic Hydroperoxide as Product and Intermediate in a Lipoxygenase Reaction, J. Biol. Chem. 273, 13080–13088]. The aims of the present work were to investigate whether 11-HPODE can also be produced by iron-dependent lipoxygenases and to determine the enzymatic transformations of stereoisomers of 11-HPODE by lipoxygenases. Rice leaf pathogen-inducible lipoxygenase, but not soybean lipoxygenase-1 (sLO-1), generated a low level of 11-HPODE (0.4%) besides its main hydroperoxide, 13(S)-HPODE, on incubation with LA. Steric analysis revealed that 11-HPODE was enriched with respect to the R enantiomer [74% 11(R)]. In agreement with previous results, 11(S)-HPODE incubated with Mn-LO provided 13(R)-HPODE, and the same conversion also took place with the methyl ester of 11(S)-HPODE. 11(R,S)-HPODE was metabolized biphasically in the presence of Mn-LO, i.e., by a rapid phase during which the 11(S)-enantiomer was converted into 13(R)-HPODE and a slow phase during which the 11(R)-enantiomer was converted into 9(R)-HPODE. sLO-1 catalyzed a slow conversion of 11(S)-HPODE into a mixture of 13(R)-HPODE (75%), 9(S)-HPODE (10%), and 13(S)-HPODE (10%), whereas 11(R,S)-HPODE produced a mixture of nearly racemic 13-HPODE (≈70%) and 9-HPODE (≈30%). The results showed that 11-HPODE can also be produced by an iron-dependent LO and suggested that the previously established mechanism of isomerization of 11(S)-HPODE involving suprafacial migration of O₂ is valid also for the isomerizations of 11(R)-HPODE by Mn-LO and of 11(S)-HPODE by sLO-1.