Insight into C35 terpene Biosyntheses by Nonpathogenic Mycobacterium Species: Functional Analyses of Three Z‐Prenyltransferases and Identification of Dehydroheptaprenylcyclines

Nonpathogenic Mycobacterium species produce rare cyclic C₃₅ terpenes that are biosynthesized by cyclization of Z‐type C₃₅ polyprenyl diphosphate. To provide deeper insight into the biosynthesis of C₃₅ terpenes, we carried out functional analyses of three Z‐prenyltransferase homologues in M. vanbaalenii identified by genomic analysis. Mvan_3822, a novel bifunctional Z‐prenyltransferase, biosynthesizes C₃₅‐heptaprenyl diphosphate as a main product from (E,E)‐farnesyl diphosphate (E,E‐FPP) and (E,E,E)‐geranylgeranyl diphosphate (E,E,E‐GGPP), but produces a C₅₀‐decaprenyl diphosphate from geranyl diphosphate. Mvan_1705 is a novel Z,E,E‐GGPP synthase. In addition, novel cyclic C₃₅ terpenes, (14E)‐ and (14Z)‐dehydroheptaprenylcycline, were identified as minor metabolites in nonpathogenic Mycobacterium cells. C₃₅ terpenes could be biosynthesized by two routes, in which E and Z geometric isomers of heptaprenyl diphosphate are produced from E,E‐FPP and E,E,E‐GGPP, and the prenylreductase responsible for the biosynthesis of C₃₅ terpenes could reduce both E and Z prenyl residues.     

Diterpene Synthase-Catalyzed Biosynthesis of Distinct Clerodane Stereoisomers

The diterpene synthase clerodienyl diphosphate synthase 1 (PvCPS1) from the crop plant switchgrass (Panicum virgatum) stereoselectively converts (E,E,E)-geranylgeranyl diphosphate (GGPP) into the clerodane natural product, cis-trans-clerodienyl diphosphate (CLPP, 1 ). Structure-guided point mutations of PvCPS1 redirected product stereoselectivity toward the formation of a rare cis-clerodane diastereomer, cis-cis-CLPP ( 2 ). Additionally, an alternative cis-clerodane diastereomer, (5S,8S,9R,10R)-13Z-CLPP ( 3 ), was produced when treating PvCPS1 and select variants thereof with the cis-prenyl substrate (Z,Z,Z)-nerylneryl diphosphate (NNPP). These results support the hypothesis that substrate configuration and minor active-site alterations impact precatalysis substrate folding in the stereoselective biosynthesis of clerodane diterpenoid scaffolds, and can be employed to provide enzymatic access to a broader range of bioactive clerodane natural products.     

An artificial substrate for undecaprenyl diphosphate synthase from Micrococcus luteus B-P 26

In order to investigate the reactivity of the homologs of homoallylic substrates, we examined the enzymatic reactions of E- or Z-3-methylhex-3-enyl diphosphate with farnesyl diphosphate using the undecaprenyl diphosphate synthase (UPS) of Micrococcus luteus B-P 26. E-3-methylhex-3-enyl diphosphate reacted with farnesyl diphosphate to give a chiral (4S)-(2Z,6E,10E,14E)-4-ethyl-3,7,11,15-tetramethylhexadeca-2,6,10,14-tetraen-1-ol. However, the Z-isomer is not accepted as a substrate for the bacterial UPS at all.     

Characterization of the Rv3377c Gene Product,a Type‐B Diterpene Cyclase,from the Mycobacterium tuberculosis H37 Genome

The Rv3377c gene from the Mycobacterium tuberculosis H37 genome is specifically limited to those Mycobacterium species that cause tuberculosis. We have demonstrated that the gene product of Rv3377c is a diterpene cyclase that catalyzes the formation of tuberculosinol from geranylgeranyl diphosphate (GGPP). However, the characteristics of this enzyme had not previously been studied in detail with homogeneously purified enzyme. The purified enzyme catalyzed the synthesis of tuberculosinyl diphosphate from GGPP, but it did not bring about the synthesis of tuberculosinol. Optimal conditions for the highest activity were found to be as follows: pH 7.5, 30 °C, Mg^II (0.1 mM ), and Triton X‐100 (0.1 %). Under these conditions, the kinetic values of K_M and k_cat were determined to be 11.7±1.9 μM for GGPP and 12.7±0.7 min^?1, respectively, whereas the specific activity was 186 nmol min^?1 mg^?1. The enzyme activity was inhibited at substrate concentrations higher than 50 μM . The catalytic activity was strongly inhibited by 15‐aza‐dihydrogeranylgeraniol and 5‐isopropyl‐N,N,N,2‐tetramethyl‐4‐(piperidine‐1‐carbonyloxy)benzenaminium chloride (Amo‐1618). The DXDTT_293–297 motif, corresponding to the DXDDTA motif conserved among terpene cyclases, was mutated in order to investigate its function. The middle D295 was found to be the most crucial entity for the catalysis. D293 and two threonine residues function synergistically to enhance the acidity of D295, possibly through hydrogen‐bonding networks. The Rv3377c enzyme could also react with (14R/S)‐14,15‐oxidoGGPP to generate 3α‐ and 3β‐hydroxytuberculosinyl diphosphate. Conformational analyses were carried out with deuterium‐labeled GGPP and oxidoGGPP. We found that GGPP and (14R)‐oxidoGGPP adopted a chair/chair conformation, but (14S)‐oxidoGGPP adopted a boat/chair conformation. Interestingly, the conformations of oxidoGGPP for the A‐ring formation are the opposite of those of oxidosqualene when it is used as a substrate by squalene cyclases for the biosynthesis of hopene and tetrahymanol. (3R)‐Oxidosqualene is folded in a boat conformation, whereas (3S)‐2,3‐oxidosqualene folds into a chair conformation, for the formation of the A‐rings of the hopene and tetrahymanol skeletons, respectively.     

Chemical protection of pheromones containing an internal conjugated diene system from isomerization and oxidation

Conjugated diene systems are common in natural products, including pheromones. The systems are sensitive to heat, light, and oxygen, among other things. They can be protected by antioxidants and UV absorbers, which slow downcis-trans isomerization and oxidation. Three sex pheromones (one as an analog) containingZ,E, E,Z, andE,E units were studied: (Z,E)-9,11-C₁₄OAc, (E,Z)-7,9-C₁₂OAc, and (E,E)-10,12-C₁₆OAc. The UV absorber 2-hydroxy-4-methoxybenzophenone and the antioxidants BHT and BHA were found to be effective in solution. The protective effect of the UV absorber against photoisomerization on paper carriers was not as good as that in solution. Preliminary studies on the utilization of formulations containing these compounds and (Z,E)-9,11-C₁₄OAc in the mass trapping of Egyptian cotton leafworm male in cotton fields showed the new combinations to be as good as a previously used formulation with UOP 688, a compound which is unpleasant to handle.     

Structure-activity relationship of unsaturated fatty acids as mosquito ovipositional repellents

Various straight-chain unsaturated fatty acids from C₁₄ to C₂₄ were evaluated for their ovipositional repellency against gravid females of the southern house mosquitoCulex quinquefasciatus Say, and the relationship between the structures of the fatty acids and their ovipositional repellency was determined. A double bond withZ configuration was prerequisite for an unsaturated fatty acid to be highly repellent;E isomers were less active or even inactive. No relationship was found between the repellency and the number of double bonds in the unsaturated fatty acids. In C₁₈ monounsaturated fatty acids, (Z)-9 acid was more active than (Z)-11 and (Z)-6 acids, indicating that a double bond at the 9 position rendered an acid highly repellent. Among (Z)-9-alkenoic acids of different chain lengths, the most repellent was C₁₈ acid which was also more active than (Z)-11-C₂₀, (Z)-13-C₂₂, and (Z)-15-C₂₄ acids. Oleic[(Z)-9-octadecenoic]acid, which met all these criteria, was the most ovipositionally repellent among the unsaturated fatty acids tested.Diptera: Culicidae.     

Aggregation of unsaturated long-chain fatty alcohols in nonaqueous systems

Aggregation and related phenomena in nonaqueous binary and ternary solutions containing unsaturated long-chain fatty alcohol amphiphiles have been studied. Six C₁₈ fatty alcohols were studied—oleyl alcohol (9Z-octadecen-1-ol), elaidyl alcohol (9E-octadecen-1-ol), linoleyl alcohol (9Z, 12Z-octadecadien-1-ol), elaidolinoleyl alcohol (9E, 12E-octadecadien-1-ol), linolenyl alcohol (9Z, 12Z, 15Z-octadecatrien-1-ol) and elaidolinolenyl alcohol (9E, 12E, 15E-octadecatrien-1-ol). Equivalent conductivity and photon correlation spectroscopy confirmed that unsaturated long-chain fatty alcohols form large and polydisperse aggregates in methanol. Critical micelle concentration (CMC) results showed that the degree of unsaturation and configuration of the double bonds in the fatty alcohol significantly influences aggregation. Aggregation of oleyl alcohol in a series of straight and branched medium-chainlength (C₃-C₈) alkanol solvents was studied. For shorter-chained alkanols (C₁-C₄), decreasing solvent dielectric constant decreases the CMC; however, for longer-chained alkanols (C₄-C₈), no significant effects occurred on the CMC. The effect of solubilized soybean oil on the viscosity of long-chain fatty alcohol/methanol solutions was also analyzed. Relative viscosity results were consistent with those expected for microemulsions. Although preliminary in nature, these results generally support the notion that soybean oil is solubilized by incorporation into large soybean oil-in-fatty alcohol aggregates in methanol solvent, resembling a nonaqueous detergentless microemulsion. Presented at the 67th Colloid and Surface Science Symposium, Toronto, Canada, June 20–23, 1993.     

Modulation of prostaglandin H synthase activity by conjugated linoleic acid (CLA) and specific CLA isomers

Conjugated linoleic acid (CLA) has been shown to inhibit tumorigenesis in animal models and is cytostatic to numerous cell lines in vitro. However, the mechanism of action is unknown. In the current study, we determined the effects of CLA and specific isomers of CLA on the rate of oxygenation of arachidonic acid by prostaglandin H synthase (PGHS) in ram seminal vesicle microsomes. The enzyme was incubated with 0.1 to 100 μM CLA or specific isomers of CLA for 2 min prior to the addition of 44 to 176 μM arachidonate. The isomers tested were 9(E),11(E) CLA; 9(Z),11(E) CLA; 9(Z),11(Z) CLA, and 10(E),12(Z) CLA. For a positive inhibitor control, flurbiprofen was used at 0.75 to 2.50 μM. Enzyme activity was assessed by measuring the rate of oxygen consumption. Inclusion of CLA or specific isomers of CLA in the incubation mixtures inhibits PGHS. The efficacy differs for each isomer, with the 9(Z),11(E) CLA isomer being the most effective and the 9(Z),11(Z) CLA isomer being the least effective inhibitor among the four CLA isomers tested. The K _i values obtained by Dixon replots range from 18.7 μM for the most effective isomer, 9(Z),11(Z) CLA, to 105.3 μM for the least effective isomer, 9(Z),11(Z) CLA. The K _i value for flurbiprofen with ram seminal vesicle microsomes was 0.33 μM. As the concentration of arachidonate was increased, the CLA-dependent inhibition of PGHS decreased, suggesting competitive inhibition. The results of this study demonstrate the potential of CLA and specific isomers of CLA to modulate prostaglandin biosynthesis.     

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.     

(Z,Z)-5,27-Tritriacontadiene: Major sex pheromone ofDrosophila pallidosa (Diptera; Drosophilidae)

A crude cuticular extract from both sexes of 3660 fruit flies (Drosophila pallidosa) was subjected to SiO₂ and AgNO₃/SiO₂ column chromatography, accompanied by bioassay for the sex pheromone activity. After three chromatographic steps, the active fraction was obtained. The main component of the active fraction was determined to be (Z,Z)-5,27-tritriacontadiene [(Z,Z)-5,27-C_33:2, on the basis of gas-liquid chromatographic analysis, chemical derivatization and gas chromatography-mass spectrometry. Synthetic (Z,Z)-5,27-C_33:2 at 5 female equivalents (FE) elicited a clear courtship response with a high courtship index amongD. pallidosa males. Therefore it was concluded that (Z,Z)-5,27-C_33:2 was a major sex pheromone component in this species.     

Biosynthesis of Sesterterpenes,Head‐to‐Tail Triterpenes,and Sesquarterpenes in Bacillus clausii: Identification of Multifunctional Enzymes and Analysis of Isoprenoid Metabolites

We performed functional analysis of recombinant enzymes and analysis of isoprenoid metabolites in Bacillus clausii to gain insights into the biosynthesis of rare terpenoid groups of sesterterpenes, head‐to‐tail triterpenes, and sesquarterpenes. We have identified an (all‐E)‐isoprenyl diphosphate synthase (E‐IDS) homologue as a trifunctional geranylfarnesyl diphosphate (GFPP)/hexaprenyl diphosphate (HexPP)/heptaprenyl diphosphate (HepPP) synthase. In addition, we have redefined the function of a tetraprenyl‐β‐curcumene synthase homologue as that of a trifunctional sesterterpene/triterpene/sesquarterpene synthase. This study has revealed that GFPP, HexPP, and HepPP, intermediates of two isoprenoid pathways (acyclic terpenes and menaquinones), are biosynthesized by one trifunctional E‐IDS. In addition, GFPP/HexPP and HepPP are the primary substrates for the biosynthesis of acyclic terpenes and menaquinone‐7, respectively.     

Mycobacterium tuberculosis Rv1916 is an Acetyl-CoA-Binding Protein

Isocitrate lyase (ICL) isoform 2 is an essential enzyme for some clinical Mycobacterium tuberculosis (Mtb) strains during infection. In the laboratory Mtb strain H37Rv, the icl2 gene encodes two distinct gene products – Rv1915 and Rv1916 – due to a frameshift mutation. This study aims to characterise these two gene products to understand their structure and function. While we were unable to produce Rv1915 recombinantly, soluble Rv1916 was obtained with sufficient yield for characterisation. Kinetic studies using UV-visible spectrophotometry and ¹H-NMR spectroscopy showed that recombinant Rv1916 does not possess isocitrate lyase activity, while waterLOGSY binding experiments demonstrated that it could bind acetyl-CoA. Finally, X-ray crystallography revealed structural similarities between Rv1916 and the C-terminal domain of ICL2. Considering the probable differences between full-length ICL2 and the gene products Rv1915 and Rv1916, care must be taken when using Mtb H37Rv as a model organism to study central carbon metabolism.     

Cuticular hydrocarbons of Drosophila birchii and D. serrata: identification and role in mate choice in D. serrata

The cuticular hydrocarbon compositions of two sympatric species of Australian Drosophila in the montium subgroup of the melanogaster group that use cuticular hydrocarbons in mate recognition have been characterized. Drosophila birchii has 34 components in greater than trace amounts, with a carbon number range of C₂₀ to C₃₃. Drosophila serrata has 21 components above trace level and a carbon number range of C₂₄ to C₃₁. These two species share eight hydrocarbon components, with all but two of them being monoenes. For both species, the (Z)-9-monoenes are the predominant positional isomer. The hydrocarbons of D. birchii are n-alkanes, n-alkenes (Z)-5-, (Z)-7-, (Z)-9-, and (Z)-11-), low to trace levels of homologous (Z,Z)-7,11- and (Z,Z)-9,13-dienes; and trace amounts of (Z,Z)-5,9-C_25:2, a major component of D. serrata. Only one methyl branched hydrocarbon was detected (2-methyl C₂₈), and it occurred at very low levels. The hydrocarbons of D. serrata are dominated by a homologous series of (Z,Z)-5,9-dienes, and notably, are characterized by the apparent absence of n-alkanes. Homologous series of (Z)-5-, (Z)-7-, and (Z)-9-alkenes are also present in D. serrata as well as 2-methyl alkanes. Drosophila serrata females display strong directional mate choice based on male cuticular hydrocarbons and prefer D. serrata males with higher relative abundances of the 2-methyl alkanes, but lower relative abundances of (Z,Z)-5,9-C_24:2 and (Z)-9-C_25:1.     

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.     

Biosynthesis of Oxylipins by Rhizoctonia solani with Allene Oxide and Oleate 8S,9S‐Diol Synthase Activities

Oxylipin biosynthesis by fungi is catalyzed by both the lipoxygenase (LOX) family and the linoleate diol synthase (LDS) family of the peroxidase‐cyclooxygenase superfamily. Rhizoctonia solani, a pathogenic fungus, infects staple crops such as potato and rice. The genome predicts three genes with 9–13 introns, which code for tentative dioxygenase (DOX)–cytochrome P450 fusion enzymes of the LDS family, and one gene, which might code for a 13‐LOX. The objective was to determine whether mycelia or nitrogen powder of mycelia oxidized unsaturated C₁₈ fatty acids to LDS‐ or LOX‐related metabolites. Mycelia converted 18:2n‐6 to 8R‐hydroxy‐9Z,12Z‐octadecadienoic acid and to an α‐ketol, 9S‐hydroxy‐10‐oxo‐12Z‐octadecenoic acid. In addition to these metabolites, nitrogen powder of mycelia oxidized 18:2n‐6 to 9S‐hydroperoxy‐10E, 12Z‐octadecadienoic, and 13S‐hydroperoxy‐9Z,11E‐octadecadienoic acids; the latter was likely formed by the predicted 13‐LOX. 18:1n‐9 was transformed into 8S‐hydroperoxy‐9Z‐octadecenoic and into 8S,9S‐dihydroxy‐10E‐octadecenoic acids, indicating the expression of 8,9‐diol synthase. The allene oxide, 9S(10)epoxy‐10,12Z‐octadecadienoic acid, is unstable and decomposes rapidly to the α‐ketol above, indicating biosynthesis by 9S‐DOX‐allene oxide synthase. This allene oxide and α‐ketol are also formed by potato stolons, which illustrates catalytic similarities between the plant host and fungal pathogen.     

Alkadienes and Alkenes, Sex Pheromone Components of the Almond Seed Wasp Eurytoma amygdali

Whole body extracts of virgin Eurytoma amygdali females were attractive to males in laboratory bioassays. Extracts of various body parts of the female wasp elicited different responses to males, with the thorax extract being the most active. Preparative fractionation of the crude hydrocarbon extract on a silver nitrate impregnated silica gel column (alkanes, alkenes, and alkadienes) revealed that the highest male response was elicited by alkadienes and the lowest by alkenes, with the alkane fraction being inactive. The identification of alkenes and alkadienes was based on gas chromatographic, mass spectrometric, and gas-phase infrared data. Laboratory bioassays suggested that the two alkadienes, (Z,Z)-6,9-tricosadiene [(Z,Z)-6,9-C_23:2], and (Z,Z)-6,9-pentacosadiene [(Z,Z)-6,9-C_25:2], and to a lesser extent alkenes, identified in the female extract of E. amygdali were male attractants.     

Sex pheromone biosynthesis in the red-banded leafroller moth,studied by mass-labeling with stable isotopes and analysis with mass spectrometry

A technique for mass-labeling was developed to study sex pheromone biosynthesis in the red-banded leafroller moth,Argyrotaenia velutinana. With this technique, the pheromone components and all fatty acyl groups in the pheromone gland were analyzed for incorporation of label in the same analytic ran with gas chromatography-mass spectrometry, using chemical ionization and selected ion monitoring (GC-SIM-CI-MS). Sex pheromone glands were incubated with fatty acids or triacylglycerols labeled with at least three deuterium atoms or carbon-13 atoms. The results of these incubations support an interpretation in which hexadecanoate is chain shortened to tetradecanoate, which is desaturated to produce (E)- and (Z)-11-tetradecenoate precursors for the sex pheromone components (E)- and (Z)-11-tetradecen-1-yl acetate. Labeled (E)- and (Z)-11-tetradecenoyl groups in synthetic triacylglycerols were not incorporated into the sex pheromone components, perhaps indicating that this lipid class is not a donor of the immediate fatty acyl precursors in sex pheromone biosynthesis.     

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.     

Biosynthesis of 14,15-Hepoxilins in Human L1236 Hodgkin Lymphoma Cells and Eosinophils

Hepoxilins are epoxy alcohols synthesized through the 12-lipoxygenase (12-LO) pathway in animal cells. The epidermis is the principal source of hepoxilins in humans. Here we report on the formation of novel hepoxilin regioisomers formed by the 15-LO pathway in human cells. The Hodgkin lymphoma cell line L1236 possesses high 15-lipoxygenase-1 (15-LO-1) activity and incubation of L1236 cells with arachidonic acid led to the formation of 11(S)-hydroxy-14(S),15(S)-epoxy 5(Z),8(Z),12(E) eicosatrienoic acid (14,15-HxA₃ 11(S)) and 13(R)-hydroxy-14(S),15(S)-epoxy 5(Z),8(Z),11(Z) eicosatrienoic acid (14,15-HxB₃ 13 (R)). In addition, two hitherto unidentified products were detected and these products were collected and analyzed by positive ion electrospray tandem mass spectrometry. These metabolites were identified as 11(S),15(S)-dihydroxy-14(R)-glutathionyl-5(Z),8(Z),12(E)-eicosatrienoic acid (14,15-HxA₃-C) and 11(S),15(S)-dihydroxy-14(R)-cysteinyl-glycyl-5(Z),8(Z),12(E)-eicosatrienoic acid (14,15-HxA₃-D). Incubation of L1236 cells with synthetic 14,15-HxA₃ 11(S) also led to the formation of 14,15-HxA₃-C and 14,15-HxA₃-D. Several soluble glutathione transferases, in particular GST M1-1 and GST P1-1, were found to catalyze the conversion of 14,15-HxA₃ to 14,15-HxA₃-C. L1236 cells produced approximately twice as much eoxins as cysteinyl-containing hepoxilins upon stimulation with arachidonic acid. Human eosinophils, nasal polyps and dendritic cells selectively formed 14,15-HxA₃ 11(S) and 14,15-HxB₃ 13(R) stereoisomers, but not cysteinyl-containing hepoxilins, after stimulation with arachidonic acid. Furthermore, purified recombinant 15-LO-1 alone catalyzed the conversion of arachidonic acid to 14,15-HxA₃ 11(S) and 14,15-HxB₃ 13(R), showing that human 15-LO-1 possesses intrinsic 14,15-hepoxilin synthase activity.     

Epoxidation of a conjugated linoleic acid isomer

Epoxidation of methyl (9Z, 11E)‐octadecadienoate ( 1 ) with various epoxidizing agents viz. m‐chloroperoxybenzoic acid, dimethyl dioxirane, methyltrioxorhenium/hydrogen peroxide, potassium peroxomonosulfate (Oxone, 2KHSO₅ · KHSO₄ · K₂SO₄)/tetrahydrothiopyran‐4‐one, and Novozyme 435/hydrogen peroxide is described. The reactions furnished the corresponding mono‐epoxy [methyl (11, 12E)‐epoxy‐(9Z)‐octadecenoate ( 2 ) and methyl (9, 10Z)‐epoxy‐(11E)‐octadecenoate ( 3 )] and a mixture of diastereomers of syn‐ and anti‐diepoxy‐stearate [methyl (9, 10Z;11, 12E)‐diepoxystearate ( 4a‐4d )], which were identified by a combination of spectroscopic and spectrometric analyses.