Stable analogs of 13­hydroxy-9,10-trans-epoxy-(11E)-octadecenoate (13,9-HEL), an oxidized derivative of linoleic acid implicated in the epidermal skin barrier.

Hydroxy-epoxy- and trihydroxy derivatives of linoleic acid are proposed to play an essential function in formation of the mammalian skin permeability barrier, which could account for the essential nature of its precursor, linoleic acid. Recent literature suggests that a specific oxidized enone derivative of LA esterified in ceramides facilitates binding to proteins, potentially serving a structural role in formation of the epidermal skin barrier. However, it is still to be established if other linoleic acid derivatives are also required for skin barrier formation, and whether the essential role is performed exclusively by an esterified, structural lipid or as an unesterified, labile signaling lipid, or by some combination of these derivatives. Progress in this domain is limited by lack of availability of hydroxy­epoxy-and trihydroxy- and octadecenoate derivatives of linoleic acid and related compounds, and challenges in maintaining them in the unesterified lipid pool. Here we describe methods for the total synthesis of hydroxy­epoxy-octadecenoate derivatives of linoleic acid (HEL1), and stable analogs that are designed to be resistant to inactivation by: (a) acylation/esterification (thus trapping these lipids in the free acid pool), (b) dehydrogenation, and (c) analogs combining both modifications. We further provide a total synthesis of corresponding hydroxy­epoxy- derivatives of sebaleic acid (a regioisomer of linoleic acid present in skin), and of small molecule scaffolds containing the allylic and non-allylic epoxide 7-carbon substructures shared by both families of hydroxy­epoxy-and trihydroxy- octadecenoates. Finally, we demonstrate that 2,2-dimethyl analogs of hydroxy­epoxy-and trihydroxy- octadecenoates are resistant to esterification with an in vitro assay and thus provide a novel template for stabilizing labile, bioactive lipids as free acids by preventing acylation/esterification.

Synthesis and molecular targets of N-13-hydroxy-octadienoyl-ethanolamine, a novel endogenous bioactive 15-lipoxygenase-derived metabolite of N-linoleoyl-ethanolamine found in the skin and saliva.

N-Arachidonoyl-ethanolamine (AEA) is an endocannabinoid (eCB) and endogenous lipid mimicking many of the effects of Δ9-tetrahydrocannabinol, notably on brain functions, appetite, pain and inflammation. The eCBs and eCB-like compounds contain fatty acids, the main classes being the monoacylglycerols and the N-acyl-ethanolamines (NAEs). Thus, each long chain fatty acid likely exists under the form of a monoacylglycerol and NAE, as it is the case for arachidonic acid (AA) and linoleic acid (LA). Following their biosynthesis, AA and AEA can be further metabolized into additional eicosanoids, notably by the 15-lipoxygenase pathway. Thus, we postulated that NAEs possessing a 1Z,4Z-pentadiene motif, near their omega end, would be transformed into their 15-lipoxygenase metabolites. As a proof of concept, we investigated N-linoleoyl-ethanolamine (LAE). We successfully synthesized LEA and LEA-d4 as well as their 15-lipoxygenase-derived derivatives, namely 13-hydroxy-9Z,11E-octadecadienoyl-N-ethanolamine (13-HODE-EA) and 13-HODE-EA-d4, using Novozyme 435 immobilized on acrylic resin and soybean lipoxygenase respectively. We also show that both human 15-lipoxygenase-1 and -2 can biosynthesize 13-HODE-EA. Co-incubation of LEA and LA with either human 15-lipoxygenase led to the biosynthesis of 13-HODE-EA and 13-HODE in a ratio equal to or greater than 3:1, indicating that LEA is preferred to LA by these enzymes. Finally, we show that 13-HODE-EA is found in human saliva and skin and is a weak although selective TRPV1 agonist. The full biological importance of 13-HODE-EA remains to be explored.

Enzymatic synthesis of ascorbyl ester derived from linoleic acid.

Antioxidants are substances that defend cells against damage, kidnapping and destroying free radicals. They have been largely used in the food industry due the possibility to control the oxidation process, aimed to increase shelf life. Thus, esterification reaction to obtain ascorbyl linoleate catalyzed by Novozym 435 lipase assisted by ultrasound bath was investigated. In this work, molecular sieve (4 Å) was added to the reaction medium to remove the water formed during the esterification reaction to improve the process performance. According to the results, ascorbyl linoleate production up to 90 % was reached after 1 h of reaction time carried out using ultrasound bath, 1:9 molar ratio of substrates L-ascorbic acid to linoleic acid, 20 mL of tert-butanol as organic solvent, 5 wt% of Novozym 435 lipase, 10 wt% of molecular sieve at 70 °C.

Mitochondrially targeted nitro-linoleate: a new tool for the study of cardioprotection.

BACKGROUND AND PURPOSE: Cardiac ischaemia-reperfusion (IR) injury remains a significant clinical problem with limited treatment options available. We previously showed that cardioprotection against IR injury by nitro-fatty acids, such as nitro-linoleate (LNO2 ), involves covalent modification of mitochondrial adenine nucleotide translocase 1 (ANT1). Thus, it was hypothesized that conjugation of LNO2 to the mitochondriotropic triphenylphosphonium (TPP(+) ) moiety would enhance its protective properties. EXPERIMENTAL APPROACH: TPP(+) -LNO2 was synthesized from aminopropyl-TPP(+) and LNO2 , and characterized by direct infusion MS/MS. Its effects were assayed in primary cultures of cardiomyocytes from adult C57BL/6 mice and in mitochondria from these cells, exposed to simulated IR (SIR) conditions (oxygen and metabolite deprivation for 1h followed by normal conditions for 1h) by measuring viability by LDH release and exclusion of Trypan blue. Nitro-alkylated mitochondrial proteins were also measured by Western blots, using antibodies to TPP(+) . KEY RESULTS: TPP(+) -LNO2 protected cardiomyocytes from SIR injury more potently than the parent compound LNO2 . In addition, TPP(+) -LNO2 modified mitochondrial proteins, including ANT1, in a manner sensitive to the mitochondrial uncoupler carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP) and the ANT1 inhibitor carboxyatractyloside. Similar protein nitro-alkylation was obtained in cells and in isolated mitochondria, indicating the cell membrane was not a significant barrier to TPP(+) -LNO2 . CONCLUSIONS AND IMPLICATIONS: Together, these results emphasize the importance of ANT1 as a target for the protective effects of LNO2 , and suggest that TPP(+) -conjugated electrophilic lipid compounds may yield novel tools for the investigation of cardioprotection.

Synthesis of linoleoyl ethanolamide.

Linoleoyl ethanolamide has been showed to serve as a new and additional class of endogenous signaling molecule and exhibited a variety of biological activities in cells and tissues. Herein, we reported an effective method for the synthesis of linoleoyl ethanolamide. Enzymatic and chemical syntheses of linoleoyl ethanolamide were first compared and then reaction conditions were optimized. When the reaction was conducted at 30°C for 1 h by reacting 0.5 mmol methyl linoleate with 5 mmol ethanolamine in the presence of 15 µL, 5.4 mol/L sodium methoxide in methanol as catalyst, 97.2% linoleoyl ethanolamide was produced in the crude reaction mixture without further purification after the removal of excess ethanolamine. Additionally, the reaction can be conducted on a large scale, which resulted in the formation of 95.9% linoleoyl ethanolamide. Compared to previous studies, the amidation reaction between methyl linoleate and ethanolamine with sodium methoxide as catalyst for the synthesis of linoleoyl ethanolamide is more effective and faster. In addition, the reaction is scalable and reaction conditions are mild. This is the first time to use methyl linoleate to synthesize linoleoyl ethanolamide. Commercial linoleoyl ethanolamide is very expensive. However, the scalability and ease for such synthesis make it possible to study the biological and nutritional functions of the cannabinoid-like linoleoyl ethanolamide in animal or human subjects.

Molecular mechanism of metal-independent decomposition of lipid hydroperoxide 13-HPODE by halogenated quinoid carcinogens.

Halogenated quinones are a class of carcinogenic intermediates and newly identified chlorination disinfection by-products in drinking water. 13-Hydroperoxy-9,11-octadecadienoic acid (13-HPODE) is the most extensively studied endogenous lipid hydroperoxide. Although it is well known that the decomposition of 13-HPODE can be catalyzed by transition metal ions, it is not clear whether halogenated quinones could enhance its decomposition independent of metal ions and, if so, what the unique characteristics and similarities are. Here we show that 2,5-dichloro-1,4-benzoquinone (DCBQ) could markedly enhance the decomposition of 13-HPODE and formation of reactive lipid alkyl radicals such as pentyl and 7-carboxyheptyl radicals, and the genotoxic 4-hydroxy-2-nonenal (HNE), through the complementary application of ESR spin trapping, HPLC-MS, and GC-MS methods. Interestingly, two chloroquinone-lipid alkoxyl conjugates were also detected and identified from the reaction between DCBQ and 13-HPODE. Analogous results were observed with other halogenated quinones. This represents the first report that halogenated quinoid carcinogens can enhance the decomposition of the endogenous lipid hydroperoxide 13-HPODE and formation of reactive lipid alkyl radicals and genotoxic HNE via a novel metal-independent nucleophilic substitution coupled with homolytic decomposition mechanism, which may partly explain their potential genotoxicity and carcinogenicity.

Production of chemoenzymatic catalyzed monoepoxide biolubricant: optimization and physicochemical characteristics.

Linoleic acid (LA) is converted to per-carboxylic acid catalyzed by an immobilized lipase from Candida antarctica (Novozym 435). This per-carboxylic acid is only intermediate and epoxidized itself in good yields and almost without consecutive reactions. Monoepoxide linoleic acid 9(12)-10(13)-monoepoxy 12(9)-octadecanoic acid (MEOA) was optimized using D-optimal design. At optimum conditions, higher yield% (82.14) and medium oxirane oxygen content (OOC) (4.91%) of MEOA were predicted at 15 µL of H(2)O(2), 120 mg of Novozym 435, and 7 h of reaction time. In order to develop better-quality biolubricants, pour point (PP), flash point (FP), viscosity index (VI), and oxidative stability (OT) were determined for LA and MEOA. The results showed that MEOA exhibited good low-temperature behavior with PP of -41(°)C. FP of MEOA increased to 128(°)C comparing with 115(°)C of LA. In a similar fashion, VI for LA was 224 generally several hundred centistokes (cSt) more viscous than MEOA 130.8. The ability of a substance to resist oxidative degradation is another important property for biolubricants. Therefore, LA and MEOA were screened to measure their OT which was observed at 189 and 168(°)C, respectively.

Fragmentation of a linoleate-derived γ-hydroperoxy-α,ß-unsaturated epoxide to γ-hydroxy- and γ-oxo-alkenals involves a unique pseudo-symmetrical diepoxycarbinyl radical.

Many of the pathological effects of lipid peroxidation are mediated by aldehydes generated through fragmentation of lipid peroxides. Among these aldehydes, the γ-hydroxy- and γ-oxo-α,ß-alkenals, e.g., 4-hydroxy-2-nonenal (HNE) and 4-oxo-2-nonenal (ONE), are especially prone to modifying proteins and DNA through covalent adduction. In addition the "mirror image" γ-hydroxy- and γ-oxo-α,ß-alkenal phospholipids can serve as high-affinity ligands for biological receptors triggering pathology. Therefore, the mechanisms by which these aldehydes are generated in vivo are under intense scrutiny. We now report observations supporting the intermediacy of a unique pseudo-symmetrical diepoxycarbinyl radical that accounts for the coproduction of HNE, ONE, and their mirror image analogues 9-hydroxy-12-oxo-10(E)-dodecenoic acid and 9-keto-12-oxo-10-dodecenoic acid upon fragmentation of 13-hydroperoxy-cis-9,10-epoxyoctadeca-11-enoic acid.

Structure-activity relationships in toll-like receptor 2-agonists leading to simplified monoacyl lipopeptides.

Toll-like receptor 2-agonistic lipopeptides typified by S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-R-cysteinyl-S-serine (PAM(2)CS) compounds are potential vaccine adjuvants. In continuation of previously reported structure-activity relationships on this chemotype, we have determined that at least one acyl group of optimal length (C(16)) and an appropriately oriented ester carbonyl group is essential for TLR2-agonistic activity. The spacing between one of the palmitoyl ester carbonyl and the thioether is crucial to allow for an important H-bond, which observed in the crystal structure of the lipopeptide:TLR2 complex; consequently, activity is lost in homologated compounds. Penicillamine-derived analogues are also inactive, likely due to unfavorable steric interactions with the carbonyl of Ser 12 in TLR2. The thioether in this chemotype can be replaced with a selenoether. Importantly, the thioglycerol motif can be dispensed with altogether and can be replaced with a thioethanol bridge. These results have led to a structurally simpler, synthetically more accessible, and water-soluble analogue possessing strong TLR2-agonistic activities in human blood.

Improved synthesis of (E)-12-nitrooctadec-12-enoic acid, a potent PPARγ activator. Development of a "buffer-free" enzymatic method for hydrolysis of methyl esters.

Endogenous nitro-fatty acids, acting as partial agonist of PPARγ, are able to lower the insulin and glucose levels without the side effects associated with common antidiabetic drugs. (E)-12-Nitrooctadec-12-enoic acid, a potent activator of this peroxisome receptor, was synthesized in a very efficient sequence via a Henry-retro-Claisen ring fragmentation, followed by a novel enzymatic cleavage of methyl esters. The latter method was then applied in the last step of the synthesis of a few labile natural products, such as prostaglandins, isoprostanes, and phytoprostanes.

A novel cationic lipophosphoramide with diunsaturated lipid chains: synthesis, physicochemical properties, and transfection activities.

Cationic lipophosphoramidates constitute a class of cationic lipids we have previously reported to be efficient for gene transfection. Here, we synthesized and studied a novel lipophosphoramidate derivative characterized by an arsonium headgroup linked, via a phosphoramidate linker, to an unconventional lipidic moiety consisting of two diunsaturated linoleic chains. Physicochemical studies allowed us to comparatively evaluate the specific fluidity and fusogenicity properties of the liposomes formed. Although corresponding lipoplexes exhibited significant but relatively modest in vitro transfection efficiencies, they showed a remarkably efficient and reproducible ability to transfect mouse lung, with in vivo transfection levels higher than those observed with a monounsaturated analogue previously described. Thus, these results demonstrate that this diunsaturated cationic lipophosphoramidate constitutes an efficient and versatile nonviral vector for gene transfection. They also invite further evaluations of the transfection activity, especially in vivo, of gene delivery systems incorporating the lipid reported herein and/or other lipids bearing polyunsaturated chains.

Bioactivity of nitrolinoleate: effects on adhesion molecules and CD40-CD40L system.

The vascular effects of nitrolinoleate (LNO2), an endogenous product of linoleic acid (LA) nitration by nitric oxide-derived species and a potential nitrosating agent, were investigated on rat endothelial-leukocyte interactions. Confocal microscopy analysis demonstrated that LNO2 was capable to deliver free radical nitric oxide (*NO) into cells, 5 min after its administration to cultured cells, with a peak of liberation at 30 min. THP-1 monocytes incubated with LNO2 for 5 min presented nitrosation of CD40, leading to its inactivation. Other anti-inflammatory actions of LNO2 were observed in vivo by intravital microscopy assays. LNO2 decreased the number of adhered leukocytes in postcapillary venules of the mesentery network. In addition to this, LNO2 reduced mRNA and protein expression of beta2-integrin in circulating leukocytes, as well as VCAM-1 in endothelial cells isolated from postcapillary venules, confirming its antiadhesive effects on both cell types. Moreover, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, a nitric oxide scavenger, partially abolished the inhibitory action of LNO2 on leukocyte-endothelium interaction, suggesting that the antiadhesion effects of LNO2 involve a dual role in leukocyte adhesion, acting as a nitric oxide donor as well as through nitric oxide-independent mechanisms. In conclusion, LNO2 inhibited adhesion molecules expression and promoted *NO inactivation of the CD40-CD40L system, both important processes of the inflammatory response.

Synthesis of 11-thialinoleic acid and 14-thialinoleic acid, inhibitors of soybean and human lipoxygenases.

Lipoxygenases catalyse the oxidation of polyunsaturated fatty acids and have been invoked in many diseases including cancer, atherosclerosis and Alzheimer's disease. Currently, no X-ray structures are available with substrate or substrate analogues bound in a productive conformation. Such structures would be very useful for examining interactions between substrate and active site residues. Reported here are the syntheses of linoleic acid analogues containing a sulfur atom at the 11 or 14 positions. The key steps in the syntheses were the incorporation of sulfur using nucleophilic attack of metallated alkynes on electrophilic sulfur compounds and the subsequent stereospecific tantalum-mediated reduction of the alkynylsulfide to the cis-alkenylsulfide. Kinetic assays performed with soybean lipoxygenase-1 showed that both 11-thialinoleic acid and 14-thialinoleic acid were competitive inhibitors with respect to linoleic acid with K(i) values of 22 and 35 microM, respectively. On the other hand, 11-thialinoleic acid was a noncompetitive inhibitor with respect to arachidonic acid with K(is) and K(ii) values of 48 and 36 microM, respectively. 11-Thialinoleic acid was also a noncompetitive inhibitor of human 15-lipoxygenase-1 with arachidonic acid (K(is) = 11.4 microM, K(ii) = 18.1 microM) or linoleic acid as substrate (K(is) = 20.1 microM, K(ii) = 20.0 microM), and a competitive inhibitor of human 12-lipoxygenase with arachidonic acid as substrate (K(i) = 2.5 microM). The presence of inhibitor did not change the regioselectivity of soybean lipoxygenase-1, human 12- or 15-lipoxygenase-1.

Biological evaluations of fatty acid esters originated during storage of Prasaplai, a Thai traditional medicine.

Three fatty acid esters, (E)-4-(3,4-dimethoxyphenyl)but-3-en-1-yl linoleate (1), (E)-4-(3,4-dimethoxyphenyl)but-3-en-1-yl oleate (2), and (E)-4-(3,4-dimethoxyphenyl)but-3-en-1-yl palmitate (3), originated during storage by the interaction of components in Prasaplai, were synthesized. These three artificial esters were subjected to four biological evaluations. All three compounds were active against Mycobacterium tuberculosis H(37)Ra for which compounds 1 and 3 had inhibitory concentration at 200 microg mL(-1) while compound 2 inhibited at 100 microg mL(-1). When all these compounds were subjected to anti-HSV-1 test, compound 2 showed positive activity at 42.6 microg mL(-1) without any cytotoxic activity against human vero cell line while compound 3 had the cytotoxicity to vero cell at IC(50) 38 microg mL(-1). Compound 1 was inactive for this test.

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.

An exogenous marker: a novel approach for the characterization of oxidative stress.

Oxidative stress (OS) and its consequences which promote alterations in biomolecules, to tissue damage and to the development of pathological conditions, continue to attract many investigators. The identification of reliable biomarker is essential for the characterization of OS and possibly for early discovery of OS-associated diseases. The aim of the present study was to offer a new concept in the development of novel probes for OS, based on the design, synthesis, and utilization of exogenous markers, as alternative to the search for endogenous markers. This article describes: (a) the synthesis of such a marker, linoleoyl tyrosine 2-deoxyguanosyl ester (LTG), constructed from three endogenous subunits: linoleic acid, tyrosine, and 2'-deoxyguanosine, representing the three major groups from which the body is composed, unsaturated fatty acids (USFA), proteins, and DNA, respectively, all bound covalently and (b) the development of analytical tools (LC/MS/MS) to enable the identification of the different LTG oxidized products formed under OS by exposure of LTG to different reactive oxygen species (ROS) such as, copper ions and hypochlorous acid.

NMR and HPLC-MS/MS analysis of synthetically prepared linoleic acid diol glucuronides.

Hydroxylated fatty acids are important mediators of many physiological and pathophysiological processes in a variety of human tissues. Recent evidence shows that in humans many of these are ultimately excreted in the urine as the glucuronide conjugates. In this paper we describe a general approach for the chemical synthesis of glucuronide conjugate derivatives of fatty acids. The synthesis strategy employs three steps (epoxidation, hydrolysis and glucuronidation) using methyl linoleate as a model non-hydroxylated starting compound. Hydroxylated starting compounds would require only the glucuronidation step. NMR and HPLC-MS/MS experiments were used to help determine the structure of the synthesized glucuronide conjugates and to identify fragmentation product ions useful for discriminating positional isomers in biological samples. This synthetic strategy should prove useful for generating analytical standards in order to identify and quantify glucuronide metabolites of hydroxylated fatty acids in humans.

Enzymatic synthesis of L-ascorbyl linoleate in organic media.

A novel L-ascorbyl fatty acid ester, L-ascorbyl linoleate was successfully prepared by enzymatic esterification and transesterification in a non-aqueous medium using immobilized lipase as biocatalyst. Changes in enzymatic activity and product yield were studied for the following variable: the nature of the fatty acid, the fatty acid concentration and water content. The yield of synthesis for the C18 unsaturated fatty acids were higher than for the C18 saturated fatty acid. Initial enzyme concentration does not affect the equilibrium of the reaction. And the product yield (33.5%) in the transesterification was higher than that of the esterification (21.8%) at a high-substrate concentration 0.3 M. The medium water content was found to have a distinct influence on the L-ascorbyl linoleate synthesis.

Furan fatty acids: occurrence, synthesis, and reactions. Are furan fatty acids responsible for the cardioprotective effects of a fish diet?

Furan FA (F-acids) are tri- or tetrasubstituted furan derivatives characterized by either a propyl or pentyl side chain in one of the alpha-positions; the other is substituted by a straight long-chain saturated acid with a carboxylic group at its end. F-acids are generated in large amounts in algae, but they are also produced by plants and microorganisms. Fish and other marine organisms as well as mammals consume F-acids in their food and incorporate them into phospholipids and cholesterol esters. F-acids are catabolized to dibasic urofuran acids, which are excreted in the urine. The biogenetic precursor of the most abundant F-acid, F6, is linoleic acid. Methyl groups in the beta-position are derived from adenosylmethionine. Owing to the different alkyl substituents, synthesis of F-acids requires multistep reactions. F-acids react readily with peroxyl radicals to generate dioxoenes. The radical-scavenging ability of F-acids may contribute to the protective properties of fish and fish oil diets against mortality from heart disease.

Enzymatic synthesis and antioxidant properties of L-ascorbyl oleate and L-ascorbyl linoleate.

L-Ascorbyl oleate and L-ascorbyl linoleate were synthesized by an immobilized lipase from Candida antarctica with yields of 38% and 44%, respectively. L-Ascorbyl oleate was stable in sterile culture medium over 12 h at 37 degrees C but L-ascorbyl linoleate degraded by 17%. Ascorbyl oleate had a better protective effect on human umbilical cord vein endothelial cells treated with H2O2 than of L-ascorbic acid-2-phosphate-6-palmitate (Asc2P6P).