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.     

Synthesis of long chain n-3 and n-6 fatty acids having a photoactive conjugated tetraene group

Fatty acids of the n-3 and n-6 families containing a photoactive conjugated tetraene group near the carboxylate were prepared from several naturally occurring fatty acids by sequential iodolactonization and treatment with excess 1,8-diazabicyclo[5.4.0]undec-7-ene. The new conjugated fatty acids include 5E,7E,9E,11Z,14Z- and 5E,7E,9E,11E,14Z-eicosapentaenoic acids derived from arachidonic acid; 5E,7E,9E,11Z,14Z,17Z- and 5E,7E,9E,11E,14Z,17Z-eicosahexaenoic acids from eicosapentaenoic acid; and 4E,6E,8E,10Z,13Z,16Z,19Z- and 4E,6E,8E,10E,13Z,16Z,19Z-docosaheptaenoic acids from docosahexaenoic acid. All of the newly synthesized fatty acids were characterized by UV, ¹H NMR and mass spectroscopy. These new products represent the first examples of directed conjugation of methylene interrupted double bond systems. The products can be synthesized in gram quantities and in high yields (>50%). Interestingly, it did not prove possible to synthesize fatty acids having a triene group near the carboxyl group even using mild conditions and different synthetic approaches. Once initiated, the isomerization always continued until a tetraene group was formed. Because of the sensitivity of the tetraene group to light, these fatty acids have the potential for being used in tracking fatty acid movements in cells employing fluorescence techniques and in UV light-induced cross linking to membrane proteins.     

Formation of cyclopentenones from all-(E) hydroperoxides of linoleic acid via allene oxides. New insight into the mechanism of cyclization

Conversions of (Z,E)- and (E,E)-isomers of linoleic acid 13- and 9-hydroperoxides with flax and maize allene oxide synthase were studied. All-(E) but not (Z,E) hydroperoxides readily undergo cyclization via allene oxides into trans-cyclopentenones. These results suggest that double bond geometry dramatically affects the formation of pericyclic pentadienyl cation intermediate and thus the capability of 18:2-allene oxides to undergo electrocyclization into cyclopentenones.     

Isolation and Characterization of Two Geometric Allene Oxide Isomers Synthesized from 9S-Hydroperoxylinoleic Acid by Cytochrome P450 CYP74C3: STEREOCHEMICAL ASSIGNMENT OF NATURAL FATTY ACID ALLENE OXIDES*

Specialized cytochromes P450 or catalase-related hemoproteins transform fatty acid hydroperoxides to allene oxides, highly reactive epoxides leading to cyclopentenones and other products. The stereochemistry of the natural allene oxides is incompletely defined, as are the structural features required for their cyclization. We investigated the transformation of 9S-hydroperoxylinoleic acid with the allene oxide synthase CYP74C3, a reported reaction that unexpectedly produces an allene oxide-derived cyclopentenone. Using biphasic reaction conditions at 0 °C, we isolated the initial products and separated two allene oxide isomers by HPLC at −15 °C. One matched previously described allene oxides in its UV spectrum (λ_max 236 nm) and NMR spectrum (defining a 9,10-epoxy-octadec-10,12Z-dienoate). The second was a novel stereoisomer (UV λ_max 239 nm) with distinctive NMR chemical shifts. Comparison of NOE interactions of the epoxy proton at C9 in the two allene oxides (and the equivalent NOE experiment in 12,13-epoxy allene oxides) allowed assignment at the isomeric C10 epoxy-ene carbon as Z in the new isomer and the E configuration in all previously characterized allene oxides. The novel 10Z isomer spontaneously formed a cis-cyclopentenone at room temperature in hexane. These results explain the origin of the cyclopentenone, provide insights into the mechanisms of allene oxide cyclization, and define the double bond geometry in naturally occurring allene oxides.     

Oxidation of [1-C]linoleic acid in isolated microsomes from pea leaves

The oxidation of [1-¹⁴C]linoleate in isolated microsomes from pea leaves was found to be stimulated by NADPH addition. The formation of one of the main metabolites, 12-hydroxy-9(Z)-dodecenoic acid is particularly NADPH-dependent. The predominant products in the absence of NADPH were hydroperoxides and in the presence of NADPH, 12-hydroxy-9(Z)-dodecenoic acid. Exogenous [1-¹⁴C]-13-hydroperoxy-9(Z), 11(E)-octadecadieoic acid and [1-¹⁴C]-12-oxo-9(Z)-dodecenoic acidwere the efficient precursors of 12-hydroxy9(Z)-dodecenoic acid. It was concluded that 12-hydroxy-9(Z)-dodecenoic acid is formed by NADPH-dependent enzymatic reduction of 12oxo-9(Z)-dodecenoic acid. The observed inhibition of linoleate oxidation in isolated microsomes by CO and metryapone suggests the involvement of cytochrome P-450 in the reaction. The relative contribution of lipoxygenase and monooxygenase activity to linoleate oxidation in microsomes is discussed.     

Allene oxide cyclase: a new enzyme in plant lipid metabolism

The mechanism of the biosynthesis of 12-oxo-10,15(Z)-phytodienoic acid (12-oxo-PDA) from 13(S)-hydroperoxy-9(Z),11(E),15(Z)-octadecatrienoic acid in preparations of corn (Zea mays L.) was studied. In the initial reaction the hydroperoxide was converted into an unstable allene oxide, 12,13(S)-epoxy-9(Z),11,15(Z)-octadecatrienoic acid, by action of a particle-bound hydroperoxide dehydrase. A new enzyme, allene oxide cyclase, catalyzed subsequent cyclization of allene oxide into 9(S),13(S)-12-oxo-PDA. In addition, because of its chemical instability, the allene oxide underwent competing nonenzymatic reactions such as hydrolysis into alpha- and gamma-ketol derivatives as well as spontaneous cyclization into racemic 12-oxo-PDA. (+/-)-cis-12,13-Epoxy-9(Z)-octadecenoic acid and (+/-)-cis-12,13-epoxy-9(Z),15(Z)-octadecadienoic acid, in which the epoxy group was located in the same position as in the allene oxide substrate, served as potent inhibitors of corn allene oxide cyclase. On the other hand, the isomeric (+/-)-cis-9,10-epoxy-12(Z)-octadecenoic acid had little inhibitory effect. Allene oxide cyclase was present in the soluble fraction of corn homogenate and had a molecular weight of about 45,000 as judged by gel filtration. The enzyme activity was detected in several plant tissues, the highest levels being observed in potato tubers and in leaves of spinach and white cabbage.     

Biosynthesis of 12-oxo-10,15(Z)-phytodienoic acid: identification of an allene oxide cyclase

Incubation of 13(S)-hydroperoxy-9(Z),11(E),15(Z)-octadecatrienoic acid with corn (Zea mays L.) hydroperoxide dehydrase led to the formation of an unstable allene oxide derivative, 12,13(S)-epoxy-9(Z),11,15(Z)-octadecatrienoic acid. Further conversion of the allene oxide yielded two major products, i.e. alpha-ketol 12-oxo-13-hydroxy-9(Z),15(Z)-octadecadienoic acid, and 12-oxo-10,15(Z)-phytodienoic acid (12-oxo-PDA). 12-Oxo-PDA was formed from allene oxide by two different pathways, i.e. spontaneous chemical cyclization, leading to racemic 12-oxo-PDA, and enzyme-catalyzed cyclization, leading to optically pure 12-oxo-PDA. The allene oxide cyclase, a novel enzyme in the metabolism of oxygenated fatty acids, was partially characterized and found to be a soluble protein with an apparent molecular weight of about 45,000 that specifically catalyzed conversion of allene oxide into 9(S),13(S)-12-oxo-PDA.     

Solanesyl diphosphate synthase reaction with artificial substrates. Formation of R and S-enantiomers of 4- and 8- methyl derivatives of geranylgeranyl diphosphate

(E)- and (Z-3-Methyl-3-pentenyl diphosphates acted as artificial substrates in the reaction with geranyl diphosphate catalyzed by solanesyl diphosphate synthase of Micrococcus luteus. The reactions of the E- and Z-isomers proceeded in the same stereochemical manner as that with the natural substrate but stopped at the stage of two steps of condensation, forming C₁₆- and C₂₂-prenyl diphosphates having extra one and two methyl groups at 4- and 4,8-positions, respectively.     

Biocatalytic production of 2(E)-hexenal from hydrolysed linseed oil

Natural 2(E)-hexenal was produced in two steps from hydrolysed linseed oil, which contains the most linolenic acid among the available natural sources. In the first step 13-hydroperoxy-9(Z),11(E),15(Z)-octadecatrienoic acid (13-HPOT) was formed from linolenic acid (100 mM) by soybean lipoxygenase-1 (Lox-1) isoenzyme with oxygen as co-substrate. The reaction resulted in 57 mM 13-HPOT with a yield of 62%. In the second step 13-HPOT (20 mM) was cleaved by green bell pepper hydroperoxide lyase resulting in 1.6 mM 2(E)-hexenal and 5.9 mM 3(Z)-hexenal (37% yield for the hexenal isomers together). Hexenals were isolated from the reaction mixture by repeated steam distillations. During distillations the 2(E)-hexenal:3(Z)-hexenal isomer ratio was changed from 0.27 to 7.86 as a consequence of heat.     

Expression and evolution of delta9 and delta11 desaturase genes in the moth Spodoptera littoralis

Desaturation of fatty acids is a key reaction in the biosynthesis of moth sex pheromones. The main component of Spodoptera littoralis sex pheromone blend is produced by the action of Δ¹¹ and Δ⁹ desaturases. In this article, we report on the cloning of four desaturase-like genes in this species: one from the fat body (Sls-FL1) and three (Sls-FL2, Sls-FL3 and Sls-FL4) from the pheromone gland. By means of a computational/phylogenetic method, as well as functional assays, the desaturase gene products have been characterized. The fat body gene expressed a Δ⁹ desaturase that produced (Z)-9-hexadecenoic and (Z)-9-octadecenoic acids in a (1:4.5) ratio, whereas the pheromone gland Sls-FL2 expressed a Δ⁹ desaturase that produced (Z)-9-hexadecenoic and (Z)-9-octadecenoic acids in a (1.5:1) ratio. Although both Δ⁹ desaturases produced (Z)-9-tetradecenoic acid from myristic acid, transformed yeast grown in the presence of a mixture of myristic and (E)-11-tetradecenoic acids produced (Z,E)-9,11-tetradecadienoic acid, but not (Z)-9-tetradecenoic acid. The Sls-FL3 gene expressed a protein that produced a mixture of (E)-11-tetradecenoic, (Z)-11-tetradecenoic, (Z)-11-hexadecenoic and (Z)-11-octadecenoic acids in a 5:4:60:31 ratio. Despite having all the characteristics of a desaturase gene, no function could be found for Sls-FL4.     

Diterpenes from Croton salutaris

The twigs of Croton salutaris afforded three new acyclic diterpenes and a new tricyclic diterpene as well as a known compound, sonderianol. The structures of three acyclic diterpenes [(10E)-3,12-dihydroxy-3,7,11,15-tetramethyl-1,10,14-hexadecatrien-5,13-dione, (6E,10E)-3,12-dihydroxy-3,7,11,15-tetramethyl-1,6,10,14-hexadecatetraen-5,13-dione and (6Z,10E)-3,12-dihydroxy-3,7,11,15-tetramethyl-1,6,10,14-hexadecatetraen-5,13-dione] and a tricyclic diterpene [12-hydroxy-13-methylpodocarpa-9,11,13-trien-3-one] were determined by spectral methods.     

Epoxyalcohol synthase activity of the CYP74B enzymes of higher plants

The CYP74B subfamily of fatty acid hydroperoxide transforming cytochromes P450 includes the most common plant enzymes. All CYP74Bs studied yet except the CYP74B16 (flax divinyl ether synthase, LuDES) and the CYP74B33 (carrot allene oxide synthase, DcAOS) are 13-hydroperoxide lyases (HPLs, synonym: hemiacetal synthases). The results of present work demonstrate that additional products (except the HPL products) of fatty acid hydroperoxides conversion by the recombinant StHPL (CYP74B3, Solanum tuberosum), MsHPL (CYP74B4v1, Medicago sativa), and CsHPL (CYP74B6, Cucumis sativus) are epoxyalcohols. MsHPL, StHPL, and CsHPL converted the 13-hydroperoxides of linoleic (13-HPOD) and α-linolenic acids (13-HPOT) primarily to the chain cleavage products. The minor by-products of 13-HPOD and 13-HPOT conversions by these enzymes were the oxiranyl carbinols, 11-hydroxy-12,13-epoxy-9-octadecenoic and 11-hydroxy-12,13-epoxy-9,15-octadecadienoic acid. At the same time, all enzymes studied converted 9-hydroperoxides into corresponding oxiranyl carbinols with HPL by-products. Thus, the results showed the additional epoxyalcohol synthase activity of studied CYP74B enzymes. The 13-HPOD conversion reliably resulted in smaller yields of the HPL products and bigger yields of the epoxyalcohols compared to the 13-HPOT transformation. Overall, the results show the dualistic HPL/EAS behaviour of studied CYP74B enzymes, depending on hydroperoxide isomerism and unsaturation.     

Cucujolide biosynthesis in the merchant and rusty grain beetles

Most known aggregation pheromones of cucujid grain beetles are macrolides called “cucujolides”. It has recently been shown by us that cucujolide I[4(E),8(E)-4,8-dimethyldecadien-10-olide] is of terpenoid origin, and that cucujolide II[3(Z)-dodecen-11-olide] is of fatty acid. The objectives of this study were to determine if farnesol could serve as a precursor of cucujolide I in vivo; to study the conversion of fatty acids to cucujolide II; and to study the stereochemistry of the lactonization reactions leading to cucujolides I and II. Experiments were performed through application of stable isotope-labeling techniques, using the merchant grain beetle, Oryzaephilus mercator (Fauvel), and/or the rusty grain beetle, Cryptolestes ferrugineus (Stephens), as study insects. Exogenous (E,E)-farnesol was converted to cucujolide I. Dual-labeling studies with D and ¹⁸O indicate that this conversion proceeded with retention of the hydroxyl oxygen. Lauric and 11-hydroxydodecanoic acids were not effective precursors of cucujolide II, whereas 3(Z)-dodecenoic acid and 11-hydroxy-3(Z)-dodecenoic acid were effective precursors of cucujolide II. These data support the hypothesis that the biosynthetic route from fatty acids to cucujolide II involves chain shortening through β-oxidation to a 3(Z)-dodecenoic acid intermediate and oxidation at carbon-11 to form a 11-hydroxy-3(Z)-dodecenoic acid intermediate, followed by cylization. Dual-labeling studies with D and ¹⁸O indicate that this cyclization proceeded with retention of the C-11 hydroxyl of the 11-hydroxy-3(Z)-dodecenoic acid intermediate.     

Synthesis of 1,1-difluoro-5-(1H-9-purinyl)-2-pentenylphosphonic acids and the related methano analogues. Remarkable effect of the nucleobases and the cyclopropane rings on inhibitory activity toward purine nucleoside phosphorylase

A series of 1,1-difluoro-5-(1H-9-purinyl)-2-pentenylphosphonic acids, (E)-2a,b and (Z)-2a,b, as well as the related methano analogues (±)-3a,b and (±)-4a,b were prepared for evaluation of their PNP inhibitory activities. The cyclopopane ring and the hypoxanthine residue were found to increase the profile of inhibitory activity. The IC₅₀ and K_i values of difluoro{(1R*,2S*)-2-[2-(6-oxo-6,9-dihydro-1H9-purinyl)ethyl]cyclopropyl}methylphosphonic acid (±)-3b toward PNP purified from Cellulomonas sp. were determined to be 70 nM and 8.8nM, respectively.     

Molecular mechanism of enzymatic allene oxide cyclization in plants.

Jasmonates, a collective term combining both jasmonic acid (JA) and related derivatives, are ubiquitously distributed in the plant kingdom. They are characterized as lipid-derived signal molecules which mediate a plethora of physiological functions, in particular stress responses, male fertility, and a multitude of developmental processes. In the course of JA biosynthesis, the first oxylipin with signal character, cis-(+)-12-oxo-phytodienoic acid (OPDA), is produced in a cyclization reaction catalyzed by allene oxide cyclase (AOC). This enzyme-catalyzed ring closure is of particular importance, as it warrants the enantiomeric structure at the cyclopentenone ring which in the end results in the only bioactive JA enantiomer, cis-(+)-JA. In this review, we focus on the structural and molecular mechanisms underlying the above mentioned cyclization reaction. In this context, we will discuss the crystal structure of AOC2 of Arabidopsis thaliana with respect to putative binding sites of the instable substrate, 12,13-epoxy-9(Z),11,15(Z)-octadecatrienoic acid (12,13-EOT), as well as possible intermolecular rearrangements during the cyclization reaction.     

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.     

Design and synthesis of novel imidazole derivatives as potent inhibitors of allene oxide synthase(CYP74)

Allene oxide synthase (AOS) is a key enzyme in the oxylipin pathway in plants leading to jasmonic acid and other jasmonates (JAs), important signal mediators of defense signal networks in plants. AOS uses hydroperoxylinolenic acid as an oxygen donor as well as the substrate, thus the biochemical conversion of 13(S)-hydroperoxylinolenic acid to allene oxide can proceed in the absence of oxygen and NADPH. We have designed the synthesized of a series of novel imidazole derivatives and tested them in a bioassay as AOS inhibitors using a purified recombinant AOS enzyme isolated from Arabidopsis and expressed in E. coli. Among the derivatives prepared, heptyl 8-[1-(2,4-dichlorophenyl)-2-imidazolylethoxy]octanoate (k) was found to be the most potent inhibitor, with an IC₅₀ of 10±5 nM, which is 250,000-fold and 1,000,000-fold more potent than the known AOS inhibitors, acetylsalicyclic acid (2.5 mM) and ketoconazole (10 mM), respectively.     

Fatty acid allene oxides. II. Formation of two macrolactones from 12,13(S)-epoxy-9(Z),11-octadecadienoic acid

An unstable fatty acid allene oxide, 12,13(S)-epoxy-9(Z),11-octadecadienoic acid, was recently identified as the product formed from 13(S)-hydroperoxy-9(Z), 11(E)-octadecadienoic acid in the presence of corn (Zea mays L.) hydroperoxide dehydrase (M. Hamberg (1987) Biochim. Biophys. Acta 920, 76-84). The present paper is concerned with the spontaneous decomposition of 12,13(S)-epoxy-9(Z),11-octadecadienoic acid in acetonitrile solution. Two major products were isolated and characterized, i.e. macrolactones 12-keto-9(Z)-octadecen-11-olide and 12-keto-9(Z)-octadecen-13-olide.     

Redistribution of electric charge accompanying photo-synthetic electron transport in Chromatium

The isoionic pH of Chromatium chromatophores is 5.2±0.1. At pH 7.7, the net charge on the chromatophore is approx. −1·10⁴. If a change in this charge accompanies the oxidation of an electron carrier, the midpoint redox potential (E_m) of that carrier should be a function of the solution ionic strength (I). of that carrier should be a function of the solution ionic strength (I).

The E_m values of P870 and cytochrome c-555 increase strongly with increasing I at low values of I. The E_m of cytochrome c-552 also increases with increasing I, though not so strongly. These effects probably cannot be attributed to an influence of I on the activity coefficient of a dissociable ion. We conclude that, when either P870 or cytochrome c-555 loses an electron, no specific ions (including protons) are bound or released in significant amounts, and the absolute value of the charge on the chromatophore decreases.

The E_m values of the primary and secondary electron acceptors, X and Y, do not depend on I. Because these E_m values have been shown previously to depend on pH, we conclude that the uptake of a proton keeps the charge on the chromatophore constant when either X or Y accepts an electron. This means that the primary and secondary electron transfer reactions in Chromatium result in a net decrease in the charge on the photosynthetic membrane. They do not result in the translocation of protons across the membrane.

The E_m of the soluble flavocytochrome c-552 from Chromatium depends only weakly on I, but depends strongly on the pH. The uptake of a proton appears to keep the net charge on this cytochrome constant upon reduction.