Substrate specificity of the epoxidation reaction in sex pheromone biosynthesis of the Japanese giant looper (Lepidoptera: Geometridae)

Female moths of the Japanese giant looper (Ascotis selenaria cretacea, Lepidoptera: Geometridae) secrete (Z,Z)-6,9-cis-3,4-epoxynonadecadiene as a sex pheromone component. To the pheromone glands of the decapitated females, [19,19,19-D₃](Z,Z,Z)-3,6,9-nonadecatriene was applied after an injection of pheromone biosynthesis activating neuropeptide. GC-MS analysis of the gland extract showed its specific conversion into the pheromonal cis-3,4-epoxide indicating that the C₁₉ triene which had been identified in the gland was a precursor of the pheromone. In order to examine the substrate specificity of the enzyme catalyzing this epoxidation step, several unsaturated hydrocarbons not occurring in the gland were applied to it. Not only (Z,Z,Z)-3,6,9-trienes with varying chain lengths (C₁₇, C₁₈ and C₂₀ to C₂₂) but (Z,Z)-3,6-dienes (C₁₇, C₁₉ and C₂₀) were converted into the corresponding cis-3,4-epoxides in a rather good yield, while no 6,7- and 9,10-epoxides could be detected. (Z)-3-Nonadecene was also changed to the cis-epoxide, but (E)-3-, (Z)-2- and (Z)-4-double bonds in the C₁₉ chain were not oxidized. These in vivo experiments revealed that the monooxygenase regiospecifically attacked the (Z)-3-double bond of straight chain hydrocarbons regardless of their length and degree of unsaturation.     

Prenyltransferases from the flavedo of Citrus sinensis

A prenyltransferase activity (EC 2.5.1.1) has been partially purified from the flavedo of Citrus sinensis with 30–40-fold purification and 35–60 % yield. The enzyme catalyses the condensation of IPP with DMAPP or GPP. The products are neryl and geranyl pyrophosphate as well as (2E,6E)- and (2Z,6E)-farnesyl pyrophosphate. The two C₁₅-products are predominant. The E- and Z-synthetase activities are partially dissociated during the purification procedure, as well as by heat or ageing. Preparations devoid of Z-synthetase were obtained. Mg^{2 +} is required for full activity. Mn^{2 +} or Co^{2 +} can replace Mg^{2 +}. The ratio of E/Z-products formed is different for each cation. Mg^{2 +} complexes of allylic substrates or of products protect the enzyme against heat-inactivation and against inactivation by DTNB. The results are interpreted in terms of two or more prenyltransferases stereoselective for the synthesis of E- and Z-products.     

LTA4-derived 5-oxo-eicosatetraenoic acid: pH-dependent formation and interaction with the LTB4 receptor of human polymorphonuclear leukocytes

5-Oxo-(7E,9E,11Z,14Z)-eicosatetraenoic acid (5-oxo-ETE) has been identified as a non-enzymatic hydrolysis product of leukotriene A₄ (LTA₄) in addition to 5,12-dihydroxy-(6E,8E,10E,14Z)-eicosatetraenoic acids (5,12-diHETEs) and 5,6-dihydroxy-(7E,9E,11Z,14Z)-eicosatetraenoic acids (5,6-diHETEs). The amount of 5-oxo-ETE detected in the mixture of the hydrolysis products of LTA₄ was found to be pH-dependent. After incubation of LTA₄ in aqueous medium, the ratio of 5-oxo-ETE to 5,12-diHETE was 1:6 at pH 7.5, and 1:1 at pH 9.5. 5-Oxo-ETE was isolated from the alkaline hydrolysis products of LTA₄ in order to evaluate its effects on human polymorphonuclear (PMN) leukocytes. 5-Oxo-ETE induced a rapid and dose-dependent mobilization of calcium in PMN leukocytes with an EC₅₀ of 250 nM, as compared to values of 3.5 nM for leukotriene B₄ (LTB₄) and >500 nM for 5(S)-hydroxy-(6E,8Z,11Z,14Z)-eicosatetraenoic acid (5-HETE). Pretreatment of the cells with LTB₄ totally abolished the calcium response induced by 5-oxo-ETE. In contrast, the preincubation with 5-oxo-ETE did not affect the calcium mobilization induced by LTB₄. The calcium response induced by 5-oxo-ETE was totally inhibited by the specific LTB₄ receptor antagonist LY223982. These data demonstrate that 5-oxo-ETE can induce calcium mobilization in PMN leukocyte via the LTB₄ receptor in contrast to the closely related analog 5-oxo-(6E,8Z,11Z,14Z)-eicosatetraenoic acid which is known to activate human neutrophils by a mechanism independent of the receptor for LTB₄.     

Oxygenated fatty acids from Lemna trisulca

(12S)-Hydroxyhexadeca-8Z,10E,14Z-trienoic acid and a prostaglandin-like C₁₆ fatty acid have been isolated from the acidic fraction of Lemna trisulca together with several other unsaturated fatty acids.     

ω-ethyl, ω-ethenyl and ω-ethynyl-α-alkylidene-γ-lactones from Clinostemon mahuba

The trunk wood of Clinostemon mahuba contains eight (3R)-2-alkylidene-3-hydroxy-4-methylenebutanolides, seven (3R,4S)-2-alkylidene-3-hydroxy-4-methylbutanolides and seven (3S,4S)-2-alkylidene-3-hydroxy-4-methylbutanolides distinguished by the alkylidene side chains with respect to their E- or Z-geometry, ethenyl, ethynyl or ethyl terminals and lengths (C₁₆ or C₁₈).     

Acetogenins from the aquatic plant Elodea canadensis

13-(2-furyl)-Tridec-12E-en-1-yne and (7S)-hydroxyhexadeca-8E,10Z,13Z-trienoic acid have been isolated from Elodea canadensis in addition to the already known 13-(2-furyl)-tridec-1-yne, hexadec-11Z-enoic, hexadeca-7Z,10Z,13Z-trienoic and (10R)-hydroxyhexadeca-7Z,11E,13Z-trienoic acids.     

An amide from salmea scandens

Costunolide and the isobutylamides of 2E,4E,8Z,10Z- and 2E,4E,8Z,10E-dodeca-2,4,8,10-tetraenoic acid were isolated from the roots of Salmea scandens.     

Conversion of (2Z,4E)-5-(1′,2′-epoxy-2′,6′,6′-trimethylcyclohexyl)-3-methyl-2,4-pentadienoic acid to xanthoxin acid by Cercospora cruenta, a fungus producing (+)-abscisic acid

(±)-(2Z,4E)-5-(1′,2′-epoxy-2′,6′,6′-trimethylcyclohexyl)-3-methyl-2,4-pentadienoic acid was metabolized by Cercospora cruenta, which has the ability to produce (+)-abscisic acid (ABA), to give (±)-(2Z,4E)-xanthoxin acid, (±)-(2Z,4E)-5′-hydroxy-1′,2′-epoxy-1′,2′-dihydro-β-ionylideneacetic acid, (±)-1′,2′-epoxy-1′,2′-dihydro-β-ionone and trace amounts of ABA.     

The Influences of Jasmonic Acid Methyl Ester on Microtubules in Potato Cells and Formation of Potato Tubers

Amides in a CH₂Cl₂ extract from the fruits of Piper retrofractum were detected by HPLC/APCI-MS. Seven new unsaturated amides, together with six known ones, were isolated, and their structures were determined to be N-isobutyl-2E,4E,12Z-octadecatrienamide (1), N-isobutyl-2E,4E,14Z-eicosatrienamide (2), 1-(octadeca-2E,4E,12Z-trienoyl)piperidine (3), 1-(eicosa-2E,4E,14Z-trienoyl)piperidine (4), 1-(octadeca-2E,4E-dienoyl)piperidine (5), 1-(eicosa-2E,4E-dienoyl)piperidine (6), and 1-(eicosa-2E,14Z-dienoyl)piperidine (7) on the basis of chemical and spectroscopic evidence.     

Biosynthetic pathways of the sex pheromone components and substrate selectivity of the oxidation enzymes working in pheromone glands of the fall webworm, Hyphantria cunea

The fall webworm, Hyphantria cunea Drury (Lepidoptera: Arctiidae), is a harmful polyphagous defoliator. Female moths produce the following four pheromone components in a ratio of about 5:4:10:2; (9Z,12Z)-9,12-octadecadienal (I), (9Z,12Z,15Z)-9,12,15-octadecatrienal (II), cis-9,10-epoxy-(3Z,6Z)-3,6-henicosadiene (III), and cis-9,10-epoxy-(3Z,6Z)-1,3,6-henicosatriene (IV). Although ¹³C-labeled linolenic acid was not converted into trienal II at the pheromone glands of H. cunea females, GC-MS analysis of an extract of the pheromone gland treated topically with ¹³C-labeled linolenyl alcohol showed the aldehyde incorporating the isotope. Other C₁₈ and C₁₉ fatty alcohols were also oxidized to the corresponding aldehydes in the pheromone gland, indicating a biosynthetic pathway of IIvia linolenyl alcohol and low substrate selectivity of the alcohol oxidase in the pheromone gland. On the other hand, epoxydiene III was expected to be produced by specific 9,10-epoxidation of the corresponding C₂₁ trienyl hydrocarbon, which might be biosynthesized from dietary linolenic acid in oenocytes and transported to the pheromone gland. The final biosynthetic step in the pheromone gland was confirmed by an experiment using deuterated C₂₁ triene, which was synthesized by the chain elongation of linolenic acid and LiAlD₄ reduction as key reactions. When the labeled triene was administered to the female by topical application at the pheromone gland or injection into the abdomen, deuterated III was detected in a pheromone extract by GC-MS analysis. Furthermore, the substrate selectivity of epoxidase and selective incorporation by the pheromone glands were examined by treatments with mixtures of the deuterated precursor and other hydrocarbons such as C₁₉-C₂₃ trienyl, C₂₁ dienyl, and C₂₁ monoenyl hydrocarbons. The 9,10-epoxy derivative of each alkene was produced, while the epoxidation of the C₂₁ monoene was poorer than those of the trienes and diene. The low selectivity indicated that the species-specific pheromone of the H. cunea female was mainly due to the critical formation of the precursor of each component.     

Synthesis and biological activity of hydroxylated derivatives of linoleic acid and conjugated linoleic acids

Allylic hydroxylated derivatives of the C18 unsaturated fatty acids were prepared from linoleic acid (LA) and conjugated linoleic acids (CLAs). The reaction of LA methyl ester with selenium dioxide (SeO₂) gave mono-hydroxylated derivatives, 13-hydroxy-9Z,11E-octadecadienoic acid, 13-hydroxy-9E,11E-octadecadienoic acid, 9-hydroxy-10E,12Z-octadecadienoic acid and 9-hydroxy-10E,12E-octadecadienoic acid methyl esters. In contrast, the reaction of CLA methyl ester with SeO₂ gave di-hydroxylated derivatives as novel products including, erythro-12,13-dihydroxy-10E-octadecenoic acid, erythro-11,12-dihydroxy-9E-octadecenoic acid, erythro-10,11-dihydroxy-12E-octadecenoic acid and erythro-9,10-dihydroxy-11E-octadecenoic acid methyl esters. These products were purified by normal-phase short column vacuum chromatography followed by high-performance liquid chromatography (HPLC). Their chemical structures were characterized by liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance spectroscopy (NMR). The allylic hydroxylated derivatives of LA and CLA exhibited moderate in vitro cytotoxicity against a panel of human cancer cell lines including chronic myelogenous leukemia K562, myeloma RPMI8226, hepatocellular carcinoma HepG2 and breast adenocarcinoma MCF-7 cells (IC₅₀ 10-75 μM). The allylic hydroxylated derivatives of LA and CLA also showed toxicity to brine shrimp with LD₅₀ values in the range of 2.30-13.8 μM. However these compounds showed insignificant toxicity to honeybee at doses up to 100 μg/bee.     

An acyclic diterpene from Viguiera deltoidea

A new geranylgeraniol type diterpene, named viguieric acid, was isolated from the dichloromethane extracts of V. deltoidea. Its structure was assigned to be (2Z,6Z,10E)-3,15-dimethyl-7-carboxy-11-formyl-2,6,10,14-hexadecatetraen-1-ol.     

Synthesis of “Reversed” Methylenecyclopropane Analogues of Antiviral Phosphonates

Synthesis of “reversed” methylenecyclopropane analogues of nucleoside phosphonates 6a,7a, 6b, and 7b is described. 1-Bromo-1-bromomethylcyclopropane 8 was converted to the bromocyclopropyl phosphonate 9 by Michaelis-Arbuzov reaction with triisopropyl phosphite. Base-catalyzed β-elimination and deacetylation gave the key Z- and E-hydroxymethylcyclopropyl phosphonates 10 and 11 separated by chromatography. The Mitsunobu type of alkylation of 10 or 11 with adenine or 2-amino-6-chloropurine afforded phosphonates 12a, 12b, 13a, and 13b. Acid hydrolysis furnished the adenine and guanine analogues 6a, 7a, 6b, and 7b. The E and Z configuration was assigned on the basis of NOE experiments with phosphonates 6b and 7b. All Z- and E-isomers were also distinguished by different chemical shifts of CH₂O or CH₂N (H₄ or H_4′). Significant differences of the chemical shifts of the cyclopropane C_3(3’) carbons and coupling constants ³J_P,C2(2’) or ³J_P,C3(3’) selective for the Z- or E-isomers were also noted. Phosphonates 6a, 7a, 6b, and 7b are devoid of significant antiviral activity.     

The mechanism of acid-catalyzed conversion of ginsenoside Rf and two new 25-hydroxylated ginsenosides

Ginsenoside Rf is known to have higher chemical stability than other ginsenosides and until lately, the constituents in which it would convert were not known. Only in recent times, it was found that ginsenoside Rf converted to (20E)-Rg₉, (20Z)-Rg₉, Rg₁₀, and 20(R)-Rf. During my continued studies to update the chemical profile of red ginseng, two new ginsenosides converted from ginsenoside Rf, 25-hydroxylated ginsenosides, were discovered. These two new converted ginsenosides, namely (20E),25(OH)-ginsenoside Rg₉ (1), and (20Z),25(OH)-ginsenoside Rg₉ (2), together with ginsenosides (20E)-Rg₉ (3), (20Z)-Rg₉ (4), Rg₁₀ (5), and 20(R)-Rf (6) were isolated from a reaction mixture of ginsenoside Rf in an acid-catalyzed reaction. Their chemical structures (1 and 2) were elucidated by NMR and Mass spectral methods. Compounds 1 and 2 were presumably generated by hydration of (20E)-, and (20Z)-ginsenoside Rg₉. The presence of these six converted ginsenosides was confirmed by UPLC/TOF-MS method in red ginseng. On the basis of these results, I deduced the overall conversion mechanism of ginsenoside Rf and evaluated the significance of ginsenoside Rf as a characteristic mark substance of Panax ginseng.     

Cytotoxic trans-oriented iminoether platinum complexes - Kinetics of binding to DNA oligonucleotides determined by N NMR spectroscopy

The kinetics of reactions between cytotoxic trans-oriented iminoether platinum complexes and DNA oligonucleotides have been studied by 1D and 2D [¹H, ¹⁵N] HMQC NMR spectroscopy. The results for the two isomers of the mono-iminoether compound trans-[PtCl₂(NH₃){E/Z-HNC(OMe)Me}] (trans-E and trans-Z) are compared with those of the bis-iminoether derivative trans-[PtCl₂{E-HNC(OMe)Me}₂] (trans-EE). Earlier we have shown that quite unexpectedly, trans-EE is practically inert towards a central GG residue in a 12-mer double-helical duplex. We now show that the less bulky trans-E and trans-Z compounds do bind to the interior of the duplex [5′-d(G₁G₂T₃A₄C₅C₆G₇G₈ T₉A₁₀C₁₁C₁₂)]₂ which contains terminal and central “hot” GG site. The platination by trans-E and trans-Z is as expected most pronounced for the solvent exposed, terminal GG-step but significantly, competitive binding is also observed for the central GG-step. The rate of platination of the terminal G-sites is almost an order of magnitude larger for the oligomer than for the monomer GMP which was studied for comparison. The role of trans-platinum carrier ligands in influencing the type and rate of formation of adducts with DNA and other relevant biomolecules is discussed.     

Chiroptical properties of diastereomeric five-coordinate Pt(II)–olefin complexes. Molecular structure of [PtCl2{(S,S)-(E)-CNCHCHCN}(R,R)-{C6H5CH(CH3)N(CH3)CH2}2]·C6H6

The chiroptical properties of five-coordinate diastereomeric complexes of general formula [PtCl₂(R,R)-{C₆H₅CH(CH₃)N(CH₃)CH₂}₂{olefin}], with olefin ligands having electron-withdrawing substituents, have been investigated. The sign of CD bands in the 28 000–30 000 cm⁻¹ region appears to be correlated to the absolute configuration of the prochiral coordinated alkene. Single-crystal X-ray diffraction structure determination has been performed on the single diastereomer [PtCl₂(E-but-2-enedinitrile)(R,R)-{C₆H₅CH(CH₃)N(CH₃)CH₂}₂]· C₆H₆. The compound crystallizes in the monoclinic space group C2 with a = 17.842(2), b = 8.466(1), c = 10.464(1) Å, β = 109.34(1)°, Z = 2. The number of observed reflections was 1943 and the final R and R_w values were 0.020 and 0.028 respectively. Trigonal-bipyramidal geometry is observed around the Pt atom, with the two Cl atoms in axial positions. The unsaturated ligand lies in the equatorial plane disclosing S,S absolute configuration.     

Synthesis of 1-C-(tetra-O-acetyl-β-d-galactopyranosyl)-2,3-diiodo-1-propene and its reaction with primary amines

Iodination of 1-C-(tetra-O-acetyl-β-d-galactopyranosyl)allene affords an E/Z-mixture of 1-C-(tetra-O-acetyl-β-d-galactopyranosyl)-2,3-diiodo-1-propene. S_N2 displacement of the allylic iodide with primary amines affords an E/Z-mixture of allylic amines under a variety of conditions. Due to its experimental simplicity and low cost of reagents, this procedure may find wide use in the laboratory for functionalization of sugar allenes.     

Biochemical Characterization of the Oxygenation of Unsaturated Fatty Acids by the Dioxygenase and Hydroperoxide Isomerase of Pseudomonas aeruginosa 42A2

We have studied oxygenation of fatty acids by cell extract of Pseudomonas aeruginosa 42A2. Oleic acid ((9Z)-18:1) was transformed to (10S)-hydroperoxy-(8E)-octadecenoic acid ((10S)-HPOME) and to (7S,10S)-dihydroxy-(8E)-octadecenoic acid (7,10-DiHOME). Experiments under oxygen-18 showed that 7,10-DiHOME contained oxygen from air and was formed sequentially from (10S)-HPOME by isomerization. (10R)-HPOME was not isomerized. The (10S)-dioxygenase and hydroperoxide isomerase activities co-eluted on ion exchange chromatography and on gel filtration with an apparent molecular size of ∼50 kDa. 16:1n-7, 18:2n-6, and 20:1n-11 were also oxygenated to 7,10-dihydroxy fatty acids, and (8Z)-18:1 was oxygenated to 6,9-dihydroxy-(7E)-octadecenoic acid. A series of fatty acids with the double bond positioned closer to ((6Z)-18:1, (5Z,9Z)-18:2) or more distant from the carboxyl group ((11Z)-, (13Z)-, and (15Z)-18:1) were poor substrates. The oxygenation mechanism was studied with [7S-²H]18:1n-9, [7R-²H]18:2n-6, and [8R-²H]18:2n-6 as substrates. The pro-R hydrogen at C-8 was lost in the biosynthesis of (10S)-HPODE, whereas the pro-S hydrogen was lost and the pro-R hydrogen was retained at C-7 during biosynthesis of the 7,10-dihydroxy metabolites. Analysis of the fatty acid composition of P. aeruginosa revealed relatively large amounts of (9E/Z)-16:1 and (11E/Z)-18:1 and only traces of 18:1n-9. We found that (11Z)-18:1 (vaccenic acid) was transformed to (11S,14S)-dihydroxy-(12E)-octadecenoic acid and to a mixture of 11- and 12-HPOME, possibly due to reverse orientation of (11Z)-18:1 at the active site compared with oleic acid. The reaction mechanism of the hydroperoxide isomerase suggests catalytic similarities to cytochrome P450.     

Payne rearrangement during analysis of epoxyalcohols of linoleic and alpha-linolenic acids by normal phase liquid chromatography with tandem mass spectrometry

Hydroperoxides of polyunsaturated fatty acids can be transformed to epoxyalcohols and keto fatty acids by metal enzymes, hematin, and various catalysts. In the current study, we used hematin to transform 9-hydroperoxy-10E,12Z-octadecadienoic acid and 13-hydroperoxy-9Z,11E-octadecadienoic acid to epoxyalcohols (with trans epoxide configuration) and to keto fatty acids. The products were separated by normal phase high-performance liquid chromatography (NP-HPLC) and analyzed using postcolumn addition of isopropanol/water and online negative ion electrospray ionization mass spectrometry (MS). The tandem MS (MS/MS) spectra were studied using analogs prepared from [9,10,12,13-²H₄]linoleic acid (18:2n−6) and from α-linolenic acid (18:3n−3). We also studied the MS/MS spectra of epoxyalcohols formed from 11-hydroperoxy- and 8-hydroperoxy-9Z,12Z-octadecadienoic acids. Results were confirmed by MS/MS analysis of a series of authentic standards. MS/MS ions of 9-keto-10E,12Z-octadecadienoic acid and 13-keto-9Z,11E-octadecadienoic acid could be explained by keto-enol tautomerism. MS/MS spectra of regioisomeric allylic epoxyalcohols differed in relative intensities of characteristic ions. The MS/MS spectra of the epoxyalcohols with 1-hydroxy-2,3-epoxy-4Z-pentene or 3-hydroxy-1,2-epoxy-4Z-pentene elements were virtually identical and showed two characteristic ions that differed by 30 in m/z values (CH(OH)). The results suggested that epoxide migration (Payne rearrangement) occurred during collision-induced dissociation. We conclude that regioisomeric allylic epoxyalcohols can be identified by their MS/MS spectra, whereas regioisomeric epoxyalcohols can be identified by MS/MS in combination with their retention times on NP-HPLC.     

C20-trifluoro-5-oxo-ETE: a metabolically stable 5-oxo-ETE derivative

The total synthesis of C₂₀-trifluoro-6(E),8(Z),11(Z),14(Z) 5-oxo-ETE is reported. This compound was designed as an ω-oxidation-resistant analog of 5-oxo-ETE that would be resistant to metabolism. The trifluoro derivative of 5-oxo-ETE stimulated calcium mobilization in neutrophils and desensitized these cells to subsequent exposure to 5-oxo-ETE.