Gas chromatography-mass spectrometry of cis-9,10-epoxyoctadecanoic acid (cis-EODA). II. Quantitative determination of cis-EODA in human plasma

Cytochrome P450 dependent epoxidation and non-enzymic lipid peroxidation of oleic acid (cis-9-octadecenoic acid) result in the formation of cis-9,10-epoxyoctadecanoic acid (cis-EODA). This oleic acid oxide has been identified indirectly in blood and urine of humans. Reliable concentrations of circulating cis-EODA have not been reported thus far. In the present article, we report on the first GC-tandem MS method for the accurate quantitative determination in human plasma of authentic cis-EODA as its pentafluorobenzyl (PFB) ester. cis-[9,10-2H2]-EODA (cis-d2-EODA) was synthesized by chemical epoxidation of commercially available cis-[9,10-2H2]-9-octadecenoic acid and used as an internal standard for quantification. Endogenous cis-EODA and externally added cis-[9,10-2H2]-EODA were isolated from acidified plasma samples (1 ml; pH 4.5) by solvent or solid-phase extraction, converted into their PFB esters, isolated by HPLC and quantified by selected reaction monitoring. The parent ions [M-PFB]- at mass-to-charge ratio (m/z) 297 for cis-EODA and m/z 299 for (cis-d2-EODA) were subjected to collisionally-activated dissociation and the corresponding characteristic product ions at m/z 171 and 172 were monitored. In plasma of nine healthy humans (5 females, 4 males), cis-EODA was found to be present at 47.6+/-7.4 nM (mean+/-S.D.). Plasma cis-EODA levels were statistically insignificantly different (P=0.10403, t-test) in females (51.1+/-3.4 nM) and males (43.1+/-2.2 nM). cis-EODA was identified as a considerable contamination in laboratory plastic ware and found to contribute to endogenous cis-EODA by approximately 2 nM. The present GC-tandem MS method should be useful in investigating the physiological role(s) of cis-EODA in humans.     

In-silico and In-vitro based studies of Streptomyces peucetius CYP107N3 for oleic acid epoxidation

Certain members of the cytochromes P450 superfamily metabolize polyunsaturated long-chain fatty acids to several classes of oxygenated metabolites. An approach based on in silico analysis predicted that Streptomyces peucetius CYP107N3 might be a fatty acid-metabolizing enzyme, showing high homology with epoxidase enzymes. Homology modeling and docking studies of CYP107N3 showed that oleic acid can fit directly into the active site pocket of the double bond of oleic acid within optimum distance of 4.6 Å from the Fe. In order to confirm the epoxidation activity proposed by in silico analysis, a gene coding CYP107N3 was expressed in Escherichia coli. The purified CYP107N3 was shown to catalyze C₉-C₁₀ epoxidation of oleic acid in vitro to 9,10-epoxy stearic acid confirmed by ESI-MS, HPLC-MS and GC-MS spectral analysis. [BMB Reports 2012; 45(12): 736-741]     

Identification of CYP2C9 as a human liver microsomal linoleic acid epoxygenase

Leukotoxin (9,10-epoxy-12-octadecanoate) and isoleukotoxin (12, 13-epoxy-9-octadecenoate) are monoepoxides of linoleic acid, synthesized by a cytochrome P450 monooxygenase and possibly by an oxidative burst of inflammatory cells. Recent experiments in this laboratory have indicated that the toxicity of leukotoxin and isoleukotoxin is not due to these epoxides, but to the 9,10- and 12, 13-diol metabolites. Leukotoxin and isoleukotoxin are metabolized primarily by the soluble epoxide hydrolase to form leukotoxin diol. Investigations with recombinant cytochrome P450 enzymes have demonstrated that leukotoxin and isoleukotoxin can be formed by these enzymes. This study used a combination of experimental approaches to identify the major cytochrome P450 enzyme in human liver involved in linoleic acid epoxidation. The kinetic paramenters were determined; the K(m) of linoleic acid epoxidation by pooled human liver microsomes was 170 microM and the V(max) was 58 pmol/mg/min. Correlation analysis was performed using individual samples of human liver microsomes, and the best correlation of linoleic acid epoxidation activity was with tolbutamide hydroxylase activity, CYP2C9. Recombinant CYP2C9 was the most active in linoleic acid epoxygenation, and antibody and chemical inhibition also indicated the importance of CYP2C9. This enzyme, therefore, may serve as a therapeutic target in the treatment of inflammation in order to reduce the amount of circulating leukotoxin/isoleukotoxin and their related diols.     

Regio- and enantioselectivity of soybean fatty acid epoxide hydrolase.

Soluble epoxide hydrolase purified from soybean catalyzes trans-addition of water across the oxirane ring of cis-9,10-epoxystearic acid with inversion of configuration at the attacked carbon, yielding threo-9,10-dihydroxystearic acid. Kinetic analyses of the progress curves, obtained at low substrate concentrations (i.e. [S] much less than Km), and determination of the enantiomeric excess of the residual substrate by chiral-phase high-performance liquid chromatography at different reaction times, indicate that the epoxide hydrolase hydrates preferentially cis-9R, 10S-epoxystearic acid (V/Km ratio, approximately 20). Interestingly, this enantiomer is obtained by epoxidation of oleic acid catalyzed by peroxygenase, a hydroperoxide-dependent oxidase, we have previously described in soybean (Blée, E., and Schuber, F. (1990) J.Biol. Chem. 265, 12887-12894). For the epoxide hydrolase to show high enantioselectivity there must be a free carboxylic acid functionality on the substrate which probably influences its positioning within the active site. This selectivity, which in principle can be used for kinetic resolution of the cis-9,10-epoxystearic acid enantiomers, is much reduced with methyl cis-9,10-epoxystearate. 18O-Labeling experiments indicate that water attacks both cis-9,10-epoxystearic acid enantiomers on the oxirane carbon which has the S-chirality. Results show that soybean epoxide hydrolase produces exclusively threo-9R,10R-dihydroxystearic acid, i.e. a naturally occurring metabolite in higher plants. cis-9,10-Epoxy-18-hydroxystearic acid, a cutin monomer, was a poorer substrate of the epoxide hydrolase than 9,10-epoxystearic acid (V/Km ratio for the preferred enantiomers, approximately 19). From a physiological point of view, peroxygenase and this newly described epoxide hydrolase could be responsible, in vivo, for the biosynthesis of a class of oxygenated fatty acid compounds known to be involved in cutin monomers production and in plant defense mechanisms.     

Determination of monoenoic fatty acid double bond position by permanganate-periodate oxidation followed by high-performance liquid chromatography of carboxylic acid phenacyl esters

This investigation was carried out to develop methods for a reverse-phase, high-performance liquid chromatography analysis of the monocarboxylic and dicarboxylic acids produced by permanganate-periodate oxidation of monoenoic fatty acids. Oxidation reactions were performed using [U-14C]oleic acid and [U-14C]oleic acid methyl ester in order to measure reaction yields and product distributions. The 14C-labeled oxidation products consisted of nearly equal amounts of monocarboxylic and dicarboxylic acid (or dicarboxylic acid monomethyl ester), with few side products (yield greater than 98%). Conversion of the carboxylic acids to phenacyl esters proceeded to completion. HPLC of carboxylic acid phenacyl esters was performed using a C18 column with a linear solvent gradient beginning with acetonitrile/water (1/1) and ending with 100% acetonitrile. Excellent resolution was achieved for all components of a mixture of C5 through C12 monocarboxylic acid phenacyl esters and C6 through C11 dicarboxylic acid phenacyl esters. Resolution was also achieved for all components of a mixture of C5 through C12 monocarboxylic acid phenacyl esters and C6 through C11 dicarboxylic acid monomethyl, monophenacyl esters. The resolution obtained by HPLC demonstrates that, for a wide range of monoenoic fatty acids, both products of a permanganate-periodate oxidation can be identified on a single chromatogram. Free fatty acids and fatty acid methyl esters were analyzed with equal success. Neither the oxidation nor the esterification reaction caused detectable hydrolysis of methyl ester. The method is illustrated for free acids and methyl esters of 14:1 (cis-9), 16:1 (cis-9), 18:1 (cis-6), 18:1 (cis-9), and 18:1 (cis-11).     

Oxidized and nitrated oleic acid in biological systems: analysis by GC-MS/MS and LC-MS/MS, and biological significance

Compared to the arachidonic acid (C20:4) cascade, the oleic acid (C18:1) family comprises a handful known metabolites. The pathophysiology of oleic acid and its oxidized and nitrated metabolites, i.e., cis-9,10-epoxyoctadecanoic acid (cis-EpOA) and the two vinylic nitro-oleic acids cis-9-nitro-oleic acid (9-NO(2)-OA) and cis-10-nitro-oleic acid (10-NO(2)-OA), is only little investigated and little understood. cis-EpOA, 9-NO(2)-OA and 10-NO(2)-OA have been detected in plasma of healthy and ill human subjects by means of gas chromatography-tandem mass spectrometry (GC-MS/MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) techniques in their acid and esterified forms. cis-EpOA is formed from oleic acid by the catalytic action of various cytochrome P450 isozymes. In end-stage liver disease, cis-EpOA plasma concentration is lower than in healthy subjects suggesting liver as the main organ responsible for cis-EpOA synthesis. The origin of 9-NO(2)-OA and 10-NO(2)-OA and of other nitrated oleic acid metabolites is unknown. In vitro models, nitro-oleic acid species can be formed non-enzymatically from oleic acid and nitrogen dioxide. Thus, endogenous nitro-oleic acids could serve as biomarkers of fatty acid nitration by reactive nitrogen species. Synthetic 9-NO(2)-OA and 10-NO(2)-OA at concentrations of three orders of magnitude higher than their endogenous counterparts have interesting pharmacological features and are currently intensely investigated. The present article reviews and discusses currently available analytical methods for the quantitative determination of cis-EpOA, 9-NO(2)-OA and 10-NO(2)-OA in biological samples, notably in human plasma, and the potential biological significance of these oleic acid metabolites. Special emphasis is given to GC-MS/MS and LC-MS/MS methods utilizing the stable-isotope dilution technique. The sensitivity and specificity of the MS/MS approach make electron-capture negative ion chemical ionization (ECNICI) GC-MS/MS and negative electrospray ionization (NESI) LC-MS/MS methodologies indispensable in experimental and clinical settings on oxidative and nitrative oleic acid metabolism. These techniques are particularly suited to delineate the oleic acid cascade.     

CYP94A5, a new cytochrome P450 from Nicotiana tabacum is able to catalyze the oxidation of fatty acids to the omega-alcohol and to the corresponding diacid.

A full length cDNA encoding a new cytochrome P450-dependent fatty acid hydroxylase (CYP94A5) was isolated from a tobacco cDNA library. CYP94A5 was expressed in S. cerevisiae strain WAT11 containing a P450 reductase from Arabidopsis thaliana necessary for catalytic activity of cytochrome P450 enzymes. When incubated for 10 min in presence of NADPH with microsomes of recombinant yeast, 9,10-epoxystearic acid was converted into one major metabolite identified by GC/MS as 18-hydroxy-9,10-epoxystearic acid. The kinetic parameters of the reaction were Km,app = 0.9 +/- 0.2 microM and Vmax,app = 27 +/- 1 nmol x min(-1) x nmol(-1) P450. Increasing the incubation time to 1 h led to the formation of a compound identified by GC/MS as 9,10-epoxy-octadecan-1,18-dioic acid. The diacid was also produced in microsomal incubations of 18-hydroxy-9,10-epoxystearic acid. Metabolites were not produced in incubations with microsomes of yeast transformed with a control plasmid lacking CYP94A5 and their production was inhibited by antibodies raised against the P450 reductase, demonstrating the involvement of CYP94A5 in the reactions. The present study describes a cytochrome P450 able to catalyze the complete set of reactions oxidizing a terminal methyl group to the corresponding carboxyl. This new fatty acid hydroxylase is enantioselective: after incubation of a synthetic racemic mixture of 9,10-epoxystearic acid, the chirality of the residual epoxide was 40/60 in favor of 9R,10S enantiomer. CYP94A5 also catalyzed the omega-hydroxylation of saturated and unsaturated fatty acids with aliphatic chain ranging from C12 to C18.     

Chlorohydrin formation from unsaturated fatty acids reacted with hypochlorous acid.

Stimulated neutrophils produce hypochlorous acid (HOCl) via the myeloperoxidase-catalyzed reaction of hydrogen peroxide with chloride. The reactions of HOCl with oleic, linoleic, and arachidonic acids both as free fatty acids or bound in phosphatidylcholine have been studied. The products were identified by gas chromatography-mass spectrometry of the methylated and trimethylsilylated derivatives. Oleic acid was converted to the two 9,10-chlorohydrin isomers in near stoichiometric yield. Linoleic acid, at low HOCl:fatty acid ratios, yielded predominantly a mixture of the four possible monochlorohydrin isomers. Bischlorohydrins were also formed, in increasing amounts at higher HOCl concentrations. Arachidonic acid gave a complex mixture of mono- and bischlorohydrins, the relative proportions depending on the amount of HOCl added. Linoleic acid appears to be slightly more reactive than oleic acid with HOCl. Reactions of oleic and linoleic acids with myeloperoxidase, hydrogen peroxide, and chloride gave chlorohydrin products identical to those with HOCl. Lipid chlorohydrins have received little attention as products of reactions of neutrophil oxidants. They are more polar than the parent fatty acids, and if formed in cell membranes could cause disruption to membrane structure. Since cellular targets for HOCl appear to be membrane constituents, chlorohydrin formation from unsaturated lipids could be significant in neutrophil-mediated cytotoxicity.     

Molecular cloning of CYP76B9, a cytochrome P450 from Petunia hybrida, catalyzing the omega-hydroxylation of capric acid and lauric acid

A cDNA encoding a cytochrome P450 (CYP76B9) was isolated from Petunia hybrida. Northern blot analysis revealed preferential expression of the gene in flowers and leaves. The recombinant yeast microsomes expressing CYP76B9 was allowed to react with capric acid and lauric acid as substrates. One major metabolite was produced from each fatty acid after incubation with yeast microsomes expressing CYP76B9. The metabolites were identified by gas chromatography-mass spectrometry (GC-MS) as omega-hydroxy capric acid and omega-hydroxy lauric acid. The kinetic parameters of the reactions were Km=9.4 microM and Vmax=13.6 mol min(-1) per mol of P450 for capric acid, and Km=5.7 microM and Vmax=19.1 mol min(-1) per mol of P450 for lauric acid. We found that the omega-hydroxy metabolites of capric acid and lauric acid can affect the plant growth of Arabidopsis thaliana. Plants grown in the presence of omega-hydroxy fatty acids exhibited shorter root length than control plants with the corresponding non-hydroxylated fatty acids.     

Isomerization of stable isotopically labeled elaidic acid to cis and trans monoenes by ruminal microbes

A previous study showed that oleic acid was converted by mixed ruminal microbes to stearic acid and also converted to a multitude of trans octadecenoic acid isomers. This study traced the metabolism of one of these trans C18:1 isomers upon its incubation with mixed ruminal microbes. Unlabeled and labeled (18-[13C]trans-9 C18:1) elaidic acid were each added to four in vitro batch cultures with three cultures inoculated with mixed ruminal bacteria and one uninoculated culture. Samples were taken at 0, 12, 24, and 48 h and analyzed for 13C enrichment in component fatty acids by gas chromatography-mass spectrometry. At 0 h of incubation, enrichment was detected only in elaidic acid. By 48 h of incubation, 13C enrichment was 18% (P < 0.01) for stearic acid, 7% to 30% (P < 0.01) for all trans C18:1 isomers having double bonds between carbons six through 16, and 5% to 10% for cis-9 and cis-11 monoenes. After 48 h, 13C enrichment in the uninoculated cultures was only detected in the added elaidic acid. This study shows trans fatty acids exposed to active ruminal cultures are converted to stearic acid but also undergo enzymic isomerization yielding a multitude of positional and geometric isomers.     

Molecular definitions of fatty acid hydroxylases in Arabidopsis thaliana

Towards defining the function of Arabidopsis thaliana fatty acid hydroxylases, five members of the CYP86A subfamily have been heterologously expressed in baculovirus-infected Sf9 cells and tested for their ability to bind a range of fatty acids including unsubstituted (lauric acid (C12:0) and oleic acid (C18:1)) and oxygenated (9,10-epoxystearic acid and 9,10-dihydroxystearic acid). Comparison between these five P450s at constant P450 content over a range of concentrations for individual fatty acids indicates that binding of different fatty acids to CYP86A2 always results in a higher proportion of high spin state heme than binding titrations conducted with CYP86A1 or CYP86A4. In comparison to these three, CYP86A7 and CYP86A8 produce extremely low proportions of high spin state heme even with the most effectively bound fatty acids. In addition to their previously demonstrated lauric acid hydroxylase activities, all CYP86A proteins are capable of hydroxylating oleic acid but not oxygenated 9,10-epoxystearic acid. Homology models have been built for these five enzymes that metabolize unsubstituted fatty acids and sometimes bind oxygenated fatty acids. Comparison of the substrate binding modes and predicted substrate access channels indicate that all use channel pw2a consistent with the crystal structures and models of other fatty acid-metabolizing P450s in bacteria and mammals. Among these P450s, those that bind internally oxygenated fatty acids contain polar residues in their substrate binding cavity that help stabilize these charged/polar groups within their largely hydrophobic catalytic site.     

Epoxidation of 6,7- and 10,11-oxidosqualenes by the squalene epoxidase present in rat liver microsomes

Incubation of 6,7-oxidosqualene (2) or 10,11-oxidosqualene (3) with rat liver microsomes led to the formation of mixtures of the corresponding dioxidosqualenes (4 and 5, or 6 and 7, respectively), resulting from the epoxidation of 2 and 3 at their terminal double bonds. The epoxidation requires the presence of both NADPH and FAD. In addition, the HPLC analysis of the Mosher esters resulting from the controlled hydrolysis of dioxide 5 to give the corresponding epoxydiols 9 followed by derivatization with (R)-MTPA, showed that the epoxidation had been stereoselective. These facts support the hypothesis that these dioxidosqualenes had been generated by the squalene epoxidase present in the incubation medium.     

High sensitivity negative ion GC-MS method for detection of desaturated and chain-elongated products of deuterated linoleic and linolenic acids.

A sensitive negative chemical ionization (NCI) gas chromatography-mass spectrometry (GC-MS) method for the detection of pentafluorobenzyl (PFB) esters of deuterated fatty acids is described. Deuterated linoleic [18:2n-6 2H4-9,10,12,13] and linolenic [18:3n-3 2H5-17,17,18,18,18] acids were converted to chain-elongated and desaturated products during incubations with homogenates prepared from rat liver. The extracted fatty acids were derivatized with pentafluorobenzyl bromide and analyzed in the negative ion mode by GC-MS. The detection limit of the PFB esters in NCI using selected ion monitoring was below 10 femtograms. In general, detection of the PFB derivatives using the negative ion mode was more than three orders of magnitude more sensitive than using a positive chemical ionization (PCI) method with methyl ester derivatives. The PFB esters of the 2H4-18:2n-6 metabolites eluted with their unlabeled analogues, whereas the PFB esters of the 2H5-18:3n-3 metabolites were resolved from the unlabeled compounds on polar capillary FFAP columns. Isotope ratios of the 2H4-18:2n-6 metabolites were used to quantify the deuterated compounds from standard dilution curves generated from the ion abundances of the unlabeled fatty acids. The 2H5-18:3n-3 metabolites were quantified similarly using 18:3n-3. This method is feasible for the study of the in vivo metabolism of deuterated essential fatty acids in whole animals.     

omega-Hydroxylation of epoxy- and hydroxy-fatty acids by CYP94A1: possible involvement in plant defence

The C(18) fatty acid derivatives 9,10-epoxystearic acid and 9,10-dihydroxystearic acid were hydroxylated on the terminal methyl by microsomes of yeast expressing CYP94A1 cloned from Vicia sativa. The reactions did not occur in incubations of microsomes from yeast transformed with a void plasmid or in the absence of NADPH. After incubation of a synthetic racemic mixture of 9,10-epoxystearic acid, the chirality of the residual epoxide was shifted to 66:34 in favour of the 9S,10R enantiomer. Both the 9S,10R and 9R,10S enantiomers were incubated separately. We determined respective K(m) and V(max) values of 1.2+/-0.1 microM and 19.2+/-0.3 nmol/min per nmol of cytochrome P450 for the 9R,10S enantiomer and of 5.9+/-0.1 microM and 20.2+/-1.0 nmol/min per nmol of cytochrome P450 for the 9S,10R enantiomer. This demonstrated that CYP94A1 is enantioselective for the 9R,10S, which is preferentially formed in V. sativa microsomes. Cutin analysis of V. sativa seedlings revealed that it is mainly constituted of derivatives of palmitic acid, a C(16) fatty acid. Our results suggest that CYP94A1 might play a minor role in cutin synthesis and could be involved in plant defence. Indeed, 18-hydroxy-9,10-epoxystearic acid and 9,10,18-trihydroxystearic acid have been described as potential messengers in plant-pathogen interactions.     

Gas chromatography-mass spectrometry determination of metabolites of conjugated cis-9,trans-11,cis-15 18:3 fatty acid

Structural determination of polyunsaturated fatty acids by gas chromatography-mass spectrometry (GC-MS) requires currently the use of nitrogen containing derivatives such as picolinyl esters, 4,4-dimethyloxazoline or pyrrolidides derivatives. The derivatization is required in most cases to obtain low energy fragmentation that allows accurate location of the double bonds. In the present work, the following metabolites of rumelenic (cis-9,trans-11,cis-15 18:3) acid, from rat livers, were identified: cis-8,cis-11,trans-13,cis-17 20:4, cis-5,cis-8,cis-11,trans-13,cis-17 20:5, cis-7,cis-10,cis-13,trans-15,cis-19 22:5, and cis-4,cis-7,cis-10,cis-13,trans-15,cis-19 22:6 acids by GC-MS as their 4,4-dimethyloxazoline and methyl esters derivatives. Specific fragmentation of the methyl ester derivatives revealed some similarity with their corresponding DMOX derivatives. Indeed, intense ion fragments at m/z=M+-69, corresponding to a cleavage at the center of a bis-methylene interrupted double bond system were observed for all identified metabolites. Moreover, intense ion fragments at m/z=M+-136, corresponding to allylic cleavage of the n-12 double bonds were observed for the C20:5, C22:5, C22:6 acid metabolites. For the long chain polyunsaturated fatty acids from the rumelenic metabolism, we showed that single methyl esters derivatives might be used for both usual quantification by GC-FID and identification by GC-MS.     

Synthesis of highly pure oxyphytosterols and (oxy)phytosterol esters. Part II. (Oxy)-sitosterol esters derived from oleic acid and from 9,10-dihydroxystearic acid [1

Several efficient synthetic routes giving readily access to (oxy)-sitosterol esters and (oxy)-cholesterol esters derived respectively from oleic acid and from 9,10-dihydroxystearic acid were developed for the first time. This approach allowed that sufficient amounts of the latter were available in order to carry out further biological studies.     

Oleic acid modulates the post-translational glycosylation of macrophage ApoE to increase its secretion

There has been increasing interest in a potential role for fatty acids in adversely affecting organismal substrate utilization and contributing to the cardiovascular complications in insulin resistance. Fatty acids have already been implicated in regulating the expression of a number of genes in resident cells of the vessel wall. In the current studies, we evaluated a potential role for fatty acids in the regulation of macrophage apoE expression. Incubation in oleic acid increased the synthesis and secretion of apoE by human monocyte-derived macrophages. Part of this stimulation was mediated at a post-translational locus. Oleic acid increased the secretion of apoE from macrophages that constitutively expressed a human apoE3 cDNA. Incubation in palmitic acid decreased apoE secretion from these cells. The effect of oleic acid on apoE secretion could not be accounted for by the known effect of fatty acid on cellular sterol, because incubation in oleic acid did not suppress the degradation of nascent apoE. Incubation in oleic acid for at least 6 h was required to observe an effect on apoE secretion. Oleic acid altered the glycosylation pattern of cellular and secreted apoE, with a loss of the most heavily sialylated isoform. Oleic acid had no effect on the glycosylation of interleukin 6 secreted from macrophages. Elimination of apoE glycosylation, by substitution of threonine 194 with alanine, eliminated oleic acid-mediated stimulation of apoE secretion. These results indicate that oleic acid increases apoE secretion from macrophages at a locus involving post-translational glycosylation.     

Unique reactivity of alpha,beta-unsaturated carboxylic acid imidazolides: catalytic asymmetric synthesis of alpha,beta-epoxy esters and alpha,beta-epoxy carboxylic acid derivatives

Catalytic asymmetric synthesis of alpha,beta-epoxy esters and alpha,beta-epoxy carboxylic acid derivatives is described. Catalytic asymmetric epoxidation of alpha,beta-unsaturated carboxylic acid imidazolides using La-BINOL-Ph(3)As=O complex gave the corresponding alpha,beta-epoxy peroxy tert-butyl esters, which were directly converted to the alpha,beta-epoxy methyl esters by adding methanol to the reaction. This catalytic system had broad generality for epoxidation of various substrates. With the use of 5-10 mol% of the catalyst, both beta-aryl and beta-alkyl-substituted-alpha,beta-epoxy methyl esters were obtained in up to 91% yield and in up to 93% enantiomeric excess. In addition, efficient transformations of alpha,beta-epoxy peroxy tert-butyl esters into the alpha,beta-epoxy amides, alpha,beta-epoxy aldehydes, and gamma,delta-epoxy beta-keto esters are also reported.     

Urinary metabolites of cis-9,10-methylene octadecanoic acid. cis-3,4-Methylene hexanedioic acid and related compounds.

cis-3,4-Methylene hexanedioic acid has been discovered in human urine. It has been isolated and identified by mass spectrometry and synthesis. The daily excretion in nine subjects on a free diet was 88 mumol/day (range, 32 to 144 mumol/day). cis-3,4-Methylene hexanedioic acid was given orally to a rat. About 90% of the dose was recovered unchanged in the urine within 24 h. Intragastric administration of cis-9,10-methylene [9,10-3H2]octadecanoic acid to rats gave four labeled urinary metabolites. The major one was cis-3,4-methylene hexanedioic acid, the others were 2,3-methylene pentanedioic acid and isomers of methylene heptanedioic acid and methylene octanedioic acid. Within 72 h, about 40% of the administered radioactivity could be recovered from the urine and another 40% from the carcass. About 20% of the recovered radioactivity was found to be water. Of the radioactivity administered to rats orally as cis-9,10-methylene [9,10-3H2]octadecanoic acid methyl ester, about 50% could be recovered from the lymph of the thoracic duct within 9 h. Intraperitoneal administration of cis-9,10-methylene octodecanoic acid methyl ester to rats gave the same metabolites. Of the given amount, 50 mol % could be recovered from the urine as cis-3,4-methylene hexanedioic acid and 19 mol % as homologues within 38 days.