A lipoxygenase-divinyl ether synthase pathway in flax (Linum usitatissimum L.) leaves

Incubation of linoleic acid with an enzyme preparation from leaves of flax (Linum usitatissimum L.) led to the formation of a divinyl ether fatty acid, i.e. (9Z,11E,1'Z)-12-(1'-hexenyloxy)-9,11-dodecadienoic [(omega5Z)-etheroleic] acid, as well as smaller amounts of 13-hydroxy-9(Z),11(E)-octadecadienoic acid. The 13-hydroperoxide of linoleic acid afforded the same set of products, whereas incubations of alpha-linolenic acid and its 13-hydroperoxide afforded the divinyl ether (9Z,11E,1'Z,3'Z)-12-(1',3'-hexadienyloxy)-9,11-dodecadienoic [(omega5Z)-etherolenic] as the main product. Identification of both divinyl ethers was substantiated by their UV, mass-, (1)H NMR and COSY spectral data. In addition to the 13-lipoxygenase and divinyl ether synthase activities demonstrated by these results, flax leaves also contained allene oxide synthase activity as judged by the presence of endogenously formed (15Z)-cis-12-oxo-10,15-phytodienoic acid in all incubations.     

Cyclization of natural allene oxide in aprotic solvent: formation of the novel oxylipin methyl cis-12-oxo-10-phytoenoate

Allene oxide, (9Z,11E)-12,13-epoxy-9,11-octadecadienoic acid (12,13-EOD), was prepared by incubation of linoleic acid (13S)-hydroperoxide with flaxseed allene oxide synthase (AOS) and purified (as methyl ester) by low temperature HPLC. Identification of pure 12,13-EOD was substantiated by its UV and (1)H NMR spectra and by GC-MS data for its methanol trapping product. The methyl ester of 12,13-EOD (but not the free carboxylic acid) is slowly cyclized in hexane solution, affording a novel cyclopentenone cis-12-oxo-10-phytoenoic acid. Free carboxylic form of 12,13-EOD does not cyclize due to the exceeding formation of macrolactone (9Z)-12-oxo-9-octadecen-11-olide. The spontaneous cyclization of pure natural allene oxide (12,13-EOD) into cis-cyclopentenone have been observed first time.     

Biomimetic nitration of the linoleic acid metabolite 13-hydroxyoctadecadienoic acid: isolation and spectral characterization of novel chain-rearranged epoxy nitro derivatives

Nitration of unsaturated fatty acids is a (patho)physiologically important pathway of lipid modification induced by nitric oxide-derived species. We report herein on the unexpected chain rearrangement undergone by (13S,9Z,11E)-13-hydroxyoctadeca-9,11-dienoic acid (1), a linoleic acid metabolite, when exposed to nitrating agents of biological relevance. At pH 7.4 and at room temperature, reaction of 1 with peroxynitrite (ONOO-) as well as Fe2+-EDTA/H2O2/NO2- and horseradish peroxidase/H2O2/NO2- led to the formation of two nitration products, which could be isolated as the methyl esters and were identified as diastereoisomeric methyl (12S)-10,11-epoxy-12-hydroxy-9-nitromethylheptadecanoates by extensive 1H, 13C, 15N NMR and MS analysis.     

Detection of divinyl ether synthase in Lily-of-the-Valley (Convallaria majalis) roots

Incubations of linoleic acid with cell-free preparations from Lily-of-the-Valley (Convallaria majalis L., Ruscaceae) roots revealed the presence of 13-lipoxygenase and divinyl ether synthase (DES) activities. Exogenous linoleic acid was metabolized predominantly into (9Z,11E,1′E)-12-(1′-hexenyloxy)-9,11-dodecadienoic (etheroleic) acid. Its identification was confirmed by the data of ultraviolet spectroscopy, mass spectra, ¹H NMR, COSY, catalytic hydrogenation. The isomeric divinyl ether (8E,1′E,3′Z)-12-(1′,3′-nonadienyloxy)-8-nonenoic (colneleic) acid was detected as a minor product. Incubations with linoleic acid hydroperoxides revealed that 13-hydroperoxide was a preferential substrate, while the 9-hydroperoxide was utilized with lesser efficiency.     

(10E,12Z,15Z)-9-hydroxy-10,12,15-octadecatrienoic acid methyl ester as an anti-inflammatory compound from Ehretia dicksonii

The methanol extract of Ehretia dicksonii provided (10E, 12Z, 15Z)-9-hydroxy-10,12,15-octadecatrienoic acid methyl ester (1) which was isolated as an anti-inflammatory compound. Compound 1 suppressed 12-Otetradecanoyl-phorbol-13-acetate (TPA)-induced inflammation on mouse ears at a dose of 500 microg (the inhibitory effect (IE) was 43%). Linolenic acid methyl ester did not inhibit this inflammation at the same dose. However, the related compounds of 1, (9Z,11E)-13hydroxy-9,11-octadecadienoic acid (5) and (9Z,llE)13-oxo-9,11-octadecadienoic acid (6), showed potent activity (IE500 microg of 63% and 79%, respectively). Compounds 1, 4 ((9Z, 12Z, 14E)-16-hydroxy-9,12,14-octadecatrienoic acid), 5 and 6 also showed inhibitory activity toward soybean lipoxygenase at a concentration of 10 microg/ml.     

Acid-catalyzed transformation of 13(S)-hydroperoxylinoleic acid into epoxyhydroxyoctadecenoic and trihydroxyoctadecenoic acids

Acid treatment of (13S)-(9Z,11E)-13-hydroperoxy-9,11-octadecadienoic acid in tetrahydrofuran-water solvent afforded mainly (11R,12R,13S)-(Z)-12,13-epoxy-11-hydroxy-9-octadecenoic acid, diastereomeric (Z)-11,12,13-trihydroxy-9-octadecenoic acids and four isomers of (E)-9,12,13(9,10,13)-trihydroxy-10(11)-octadecenoic acid. Other minor products were oxooctadecadienoic, (E)-9(13)-hydroxy-13(9)-oxo-10(11)-octadecenoic and (E)-12-oxo-10-dodecenoic acids. A heterolytic mechanism for acid catalysis was indicated, even though most of the products characterized also have been observed as a result of homolytic decomposition of the hydroperoxide via an oxy radical. Most of the products found in this study have been observed as metabolites of (13S)-(9Z,11E)-13-hydroperoxy-9,11-octadecadenoic acid in biological systems, and analogous compounds have been reported as metabolites of (12S)-(5Z,8Z,10E, 14Z)-12-hydroperoxy-5,8,10,14-hydroperoxy-5,8,10,14-eicosatetraenoic acid in either blood platelets or lung tissue.     

Free radical oxidation of 15-(S)-hydroxyeicosatetraenoic acid with the Fenton reagent: characterization of an epoxy-alcohol and cytotoxic 4-hydroxy-2E-nonenal from the heptatrienyl radical pathway

The oxidation of (5Z,8Z,11Z,13E,15S)-15-hydroxy-5,8,11,13-eicosatetraenoic acid (15-(S)-HETE, 1a) with the Fenton reagent (Fe2+/EDTA/H2O2) was investigated. In phosphate buffer, pH 7.4, the reaction proceeded with 75% substrate consumption after 1 h to give a mixture of products, one of which was identified as (2E,4S)-4-hydroxy-2-nonenal (3a, 18% yield). Methylation of the mixture with diazomethane allowed isolation of another main product which could be identified as methyl (5Z,8Z,13E)-11,12-trans-epoxy-15-hydroxy-5,8,13-eicosatrienoate (2a methyl ester, 8% yield). A similar oxidation carried out on (15-(2)H)-15-HETE (1b) indicated complete retention of the label in 2b methyl ester and 3b, consistent with an oxidation pathway involving as the primary event H-atom abstraction at C-10. Overall, these results support the recently proposed role of 1a as a potential precursor of the cytotoxic gamma-hydroxyalkenal 3a and disclose a hitherto unrecognized interconnection between 1a and the epoxy-alcohol 2a, previously implicated only in the metabolic transformations of the 15-hydroperoxy derivative of arachidonic acid.     

Detection of an enol intermediate in the hydroperoxide lyase chain cleavage reaction

Guava (Psidium guajava) hydroperoxide lyase (HPL) preparations were incubated with [1-(14)C](9Z,11E,13S,15Z)-13-hydroperoxy-9,11,15-octadecatrienoic acid for 1 min at 0 degrees C, followed by rapid extraction/trimethylsilylation. Analysis of the trimethylsilylated products by gas chromatography-mass spectrometry and radio-high-performance liquid chromatography revealed a single predominant (14)C-labelled compound, identified by its (1)H-nuclear magnetic resonance, ultraviolet and mass spectra as the trimethylsilyl ether/ester of (9Z,11E)-12-hydroxy-9,11-dodecadienoic acid. Longer time incubations afford smaller yield of this enol due to its partial tautomerization into (9Z)-12-oxo-9-dodecenoic acid. The data obtained demonstrate that formation of (9Z)-12-oxo-9-dodecenoic acid in the HPL reaction is preceded by unstable enol oxylipin, and further suggest that hemiacetals are the true products of HPL catalysis.     

Identification of an amino acid determinant of pH regiospecificity in a seed lipoxygenase from Momordica charantia

Lipoxygenases (LOX) form a heterogeneous family of lipid peroxidizing enzymes, which catalyze specific dioxygenation of polyunsaturated fatty acids. According to their positional specificity of linoleic acid oxygenation plant LOX have been classified into linoleate 9- and linoleate 13-LOX and recent reports identified a critical valine at the active site of 9-LOX. In contrast, more bulky phenylalanine or histidine residues were found at this position in 13-LOX. We have recently cloned a LOX-isoform from Momordica charantia and multiple amino acid alignments indicated the existence of a glutamine (Gln599) at the position were 13-LOX usually carry histidine or phenylalanine residues. Analyzing the pH-dependence of the positional specificity of linoleic acid oxygenation we observed that at pH-values higher than 7.5 this enzyme constitutes a linoleate 13-LOX whereas at lower pH, 9-H(P)ODE was the major reaction product. Site-directed mutagenesis of glutamine 599 to histidine (Gln599His) converted the enzyme to a pure 13-LOX. These data confirm previous observation suggesting that reaction specificity of certain LOX-isoforms is not an absolute enzyme property but may be impacted by reaction conditions such as pH of the reaction mixture. We extended this concept by identifying glutamine 599 as sequence determinant for such pH-dependence of the reaction specificity. Although the biological relevance for this alteration switch remains to be investigated it is of particular interest that it occurs at near physiological conditions in the pH-range between 7 and 8.     

First total syntheses of (Z)-15-methyl-10-hexadecenoic acid and the (Z)-13-methyl-8-tetradecenoic acid

The first total syntheses for the (Z)-15-methyl-10-hexadecenoic acid and the (Z)-13-methyl-8-tetradecenoic acid were accomplished in seven steps and in 31-32% overall yields. The (trimethylsilyl)acetylene was the key reagent in both syntheses. It is proposed that the best synthetic strategy towards monounsaturated iso methyl-branched fatty acids with double bonds close to the omega end of the acyl chain is first acetylide coupling of (trimethylsilyl)acetylene to a long-chain bifunctional bromoalkane followed by a second acetylide coupling to a short-chain iso bromoalkane, since higher yields are thus obtained. Spectral data is also presented for the first time for these two unusual fatty acids with potential as biomarkers and as topoisomerase I inhibitors.     

Biosynthetic intermediates and stereochemical aspects of aldehyde biosynthesis in the marine diatom Thalassiosira rotula

Intermediates of the aldehyde biosynthesis in Thalassiosira rotula are investigated. Use of labeled precursors and cell preparations proves production of 2E,4Z-octadienal from 6Z,9Z,12Z-hexadecatrienoic acid (C16:3 omega-4) through the lipoxygenase-dependent intermediate (9S)-9-hydroperoxyhexadeca-6,10,12-trienoic acid. On the contrary, synthesis of 2E,4Z,7Z-decatrienal involves mainly EPA (C20:5 omega-3) by a 11R-lipoxygenase, as suggested by identification of chiral 11R-HEPE (12% e.e.) in the diatom extracts. Consistently with the necessity to have a rapid transport and metabolization of the intermediate hydroperoxides, we show that lipoxygenase and lyase activities are both found in the same subcellular fraction of the microalga.     

Evidence for the formation of Michael adducts from reactions of (E,E)-muconaldehyde with glutathione and other thiols

Glutathione induces the rapid isomerization of (Z,Z)-muconaldehyde to (E,E)-muconaldehyde via (E,Z)-muconaldehyde, probably via reversible Michael addition of the thiol to one of the enal moieties of the muconaldehyde. Reactions of (E,E)-muconaldehyde with glutathione (in the presence and absence of equine glutathione S-transferase), phenylmethanethiol, N-acetyl-l-cysteine, and N-acetyl-l-cysteine methyl ester were investigated using mass spectrometric techniques. In each case, evidence was obtained for the formation of Michael adducts, e.g., reaction between (E,E)-muconaldehyde and glutathione gave 4-glutathionyl-hex-2-enedial and 3,4-bis-glutathionyl-hexanedial. These experiments suggest that (Z,Z)-muconaldehyde, a putative metabolite of benzene, could lead to the long established urinary metabolite of benzene, (E,E)-muconic acid, via glutathione-mediated isomerization to (E,E)-muconaldehyde.     

Constituents from Miliusa balansae (Annonaceae)

The styryl derivatives 3,4-dimethoxy-6-styryl-pyran-2-one and (2E,5E)-2-methoxy-4-oxo-6-phenyl-hexa-2,5-dienoic acid methyl ester were isolated from leaves and branches of Miliusa balansae (Annonaceae). In addition, the geranylated homogentisic acid derivative miliusate, four flavanones and two dihydrochalcones were identified. Their structures were elucidated by spectroscopic means.     

Syntheses and reactions of methyl (Z)-9,10-epoxy-13-oxo-(E)-11-octadecenoate and methyl (E)-9,10-epoxy-13-oxo-(E)-11-octadecenoate

The syntheses and reactions of two epoxyketoacids (methyl (Z)-9,10-epoxy-13-oxo-(E)-11-octadecenoate (IV) and methyl (E)-9,10-epoxy-13-oxo-(E)-11-octadecenoate (V)) are described. The synthetic method is based on the stereoselective oxidation of linoleic acid by soybean lipoxygenase to produce the corresponding 13-hydroperoxide. Reduction of the hydroperoxide with sodium borohydride followed by oxidation, esterification and epoxidation yielded the compounds IV and V with a global yield of 14% and 3%, respectively, referred to the diasteromerically pure isolated compounds. Confirmation of the structures was carried out by reduction of the ketone group with sodium borohydride and by the opening of the oxirane ring with methanolic boron trifluoride. The reduction of compounds IV and V with hydrogen mainly yielded the tetrahydrofuranoid fatty acid, methyl 10,13-epoxyoctadecanoate. This reaction may be considered a new procedure to obtain tetrahydrofuranoid fatty acids.     

The "heterolytic hydroperoxide lyase" is an isomerase producing a short-lived fatty acid hemiacetal

To elucidate the reaction mechanism of hydroperoxide lyase (HPL), the enzyme from guava (Psidium guajava) fruits, was incubated for 10-60 s at 0 degrees C with 13-HPOT. The products were rapidly extracted and derivatized by trimethylsilylation. Two trapping products, namely the trimethylsilyl ether/ester derivatives of the hemiacetal 12-(1'-hydroxy-3'-hexenyloxy)-9,11-dodecadienoic acid and the enol (9Z,11E)-12-hydroxy-9,11-dodecadienoic acid, were detected by gas chromatography-mass spectrometry (GC-MS) analyses. The structural assignments were supported by mass spectra recorded for (a) hydrogenated products; (b) products biosynthesized from [9,10,12,13,15,16] 13-HPOT or [(18)O(2)]13-HPOT; (c) chemically prepared reference compounds. Kinetic experiments showed that the hemiacetal and enol were both unstable and transiently appearing compounds (half-lives, ca. 20 s and 2 min, respectively). Hemiacetal and enol biosynthesized from [(18)O(2)]13-HPOT retained two and one (18)O atoms, respectively, whereas no (18)O was incorporated from [(18)O]water. The data demonstrated that: (1) the true enzymatic product formed from 13-HPOT in the presence of HPL is a short-lived hemiacetal; (2) the hemiacetal spontaneously dissociates into (3Z)-hexenal and the unstable enol form of (9Z)-12-oxo-9-dodecenoic acid; (3) the enzymatic isomerization of 13-HPOT into the hemiacetal occurs homolytically.     

(11Z)-hexadec-11-enal enhances the attractiveness of Diatraea saccharalis main pheromone component in wind tunnel experiments

Abstract:  GC-EAD and GC-MS analysis of pheromone gland extracts of sugarcane borer, Diatraea saccharalis , revealed two antennally active compounds, (9 Z ,11 E )-hexadeca-9,1-dienal and (11 Z )-hexadec-11-enal, in approximately 10 : 1 ratio. Various doses of identified compounds were investigated in wind tunnel experiments individually and in a 10 : 1 ratio. At all tested doses (9 Z ,11 E )-hexadeca-9,1-dienal alone elicited upwind orientation and source location only in a minority of tested males. An admixture of (11 Z )-hex-11-enal enhanced the attractiveness of (9 Z ,11 E )-hexadeca-9,11-dienal significantly. This two-component blend (100 pg) was as attractive as natural pheromone extracted from three female pheromone glands. The data suggest that (11 Z )-hexadec-11-enal is a part of the D. saccharalis sex pheromone.     

(10E,12Z,15Z)-9-Hydroxy-10,12,15-octadecatrienoic Acid Methyl Ester as an Anti-inflammatory Compound from Ehretia dicksonii

The methanol extract of Ehretia dicksonii provided (10E,12Z,15Z)-9-hydroxy-10,12,15-octadecatrienoic acid methyl ester (1) which was isolated as an anti-inflammatory compound. Compound 1 suppressed 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-induced inflammation on mouse ears at a dose of 500 μg (the inhibitory effect (IE) was 43%). Linolenic acid methyl ester did not inhibit this inflammation at the same dose. However, the related compounds of 1, (9Z,11E)-13-hydroxy-9,11-octadecadienoic acid (5) and (9Z,11E)- 13-oxo-9,11-octadecadienoic acid (6), showed potent activity (IE_{500 μg} of 63% and 79%, respectively). Compounds 1, 4 ((9Z,12Z,14E)-16-hydroxy-9,12,14-octadecatrienoic acid), 5 and 6 also showed inhibitory activity toward soybean lipoxygenase at a concentration of 10 μg/ml.     

Identification of (S)-11-cycloheptyl-4-methylundecanoic acid in acylphosphatidylglycerol from Alicyclobacillus acidoterrestris

A method is described for the identification of molecular species of acylphosphatidylglycerols containing a branched cyclo fatty acid ((S)-11-cycloheptyl-4-methylundecanoic acid; brc19:0) from Alicyclobacillus acidoterrestris and its identification as picolinyl ester by means of GC-MS. The combination of TLC, negative RP-HPLC-ESI-MS/MS, enzymatic hydrolysis, and GC-MS was used to identify unusual molecular species of acylphosphatidylglycerols with cyclic and branched FA. The acid, brc19:0, was also synthesized to unambiguously confirm its structure. According to feeding experiments with ¹³C-labeled propionate, the C₃ internal unit (branched methyl) of brc19:0 is assembled from propionate and not from methionine.