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.

Nanoparticulate tablet dosage form of lisofylline-linoleic acid conjugate for type 1 diabetes: in situ single-pass intestinal perfusion (SPIP) studies and pharmacokinetics in rat.

Lisofylline (LSF) is an anti-inflammatory molecule with high aqueous solubility and rapid metabolic interconversion to its parent drug, pentoxifylline (PTX) resulting in very poor pharmacokinetic (PK) parameters, necessitating high dose and dosing frequency. In the present study, we resolved the physicochemical and pharmacokinetic limitations associated with LSF and designed its oral dosage form as a tablet for effective treatment in type 1 diabetes (T1D). Self-assembling polymeric micelles of LSF (lisofylline-linoleic acid polymeric micelles (LSF-LA PLM)) were optimized for scale-up (6 g batch size) and lyophilized followed by compression into tablets. Powder blend and tablets were evaluated as per USP. LSF-LA PLM tablet so formed was evaluated for in vitro release in simulated biological fluids (with enzymes) and for cell viability in MIN-6 cells. LSF-LA PLM in tablet formulation was further evaluated for intestinal permeability (in situ) along with LSF and LSF-LA self-assembled micelles (SM) as controls in a rat model using single-pass intestinal perfusion (SPIP) study. SPIP studies revealed 1.8-fold higher oral absorption of LSF-LA from LSF-LA PLM as compared to LSF-LA SM and ~5.9-fold higher than LSF (alone) solution. Pharmacokinetic studies of LSF-LA PLM tablet showed greater Cmax than LSF, LSF-LA, and LSF-LA PLM. Designed facile LSF-LA PLM tablet dosage form has potential for an immediate decrease in the postprandial glucose levels in patients of T1D.

Human metabolite-derived alkylsuccinate/dilinoleate copolymers: from synthesis to application.

The advances in polymer chemistry have allowed the preparation of biomedical polymers using human metabolites as monomers that can hold unique properties beyond the required biodegradability and biocompatibility. Herein, we demonstrate the use of endogenous human metabolites (succinic and dilinoleic acids) as monomeric building blocks to develop a new series of renewable resource-based biodegradable and biocompatible copolyesters. The novel copolyesters were characterized in detail employing several standard techniques, namely 1H NMR, 13C NMR, and FTIR spectroscopy and SEC, followed by an in-depth thermomechanical and surface characterization of their resulting thin films (DSC, TGA, DMTA, tensile tests, AFM, and contact angle measurements). Also, their anti-fungal biofilm properties were assessed via an anti-fungal biofilm assay and the biological properties were evaluated in vitro using relevant human-derived cells (human mesenchymal stem cells and normal human dermal fibroblasts). These novel highly biocompatible polymers are simple and cheap to prepare, and their synthesis can be easily scaled-up. They presented good mechanical, thermal and anti-fungal biofilm properties while also promoting cell attachment and proliferation, outperforming well-known polymers used for biomedical applications (e.g. PVC, PLGA, and PCL). Moreover, they induced morphological changes in the cells, which were dependent on the structural characteristics of the polymers. In addition, the obtained physicochemical and biological properties can be design-tuned by the synthesis of homo- and -copolymers through the selection of the diol moiety (ES, PS, or BS) and by the addition of a co-monomer, DLA. Consequently, the copolyesters presented herein have high application potential as renewable and cost-effective biopolymers for various biomedical applications.

Hybrid micelles containing methotrexate-conjugated polymer and co-loaded with microRNA-124 for rheumatoid arthritis therapy.

Purpose: Methotrexate (MTX) is a first-line drug for rheumatoid arthritis (RA)therapy. However, MTX monotherapy often results in irreversible joint damage due to its slow onset of action and long duration. microRNA-124 (miR-124) has shown direct bone protection activity against RA. A co-delivery system for MTX and microRNA combination may provide therapeutic synergy. Methods: Methotrexate-conjugated polymer hybrid micelles (M-PHMs) were prepared by self-assembly of two functional amphiphilic polymers (MTX-PEI-LA and mPEG-LA) at an optimized weight ratio. Incorporation of microRNA was achieved through electrostatic interactions between microRNA and cationic polymer MTX-PEI-LA. Cellular uptake, endosome escape, biodistribution, and therapeutic efficacy of M-PHMs/miR-124 complexes were investigated and evaluated in RAW264.7 cells and a rat adjuvant-induced arthritis (AIA) model. Results: M-PHMs/miR-124 complexes exhibited folate receptor-mediated uptake in activated RAW264.7 cells. miR-124 was able to escape from the endosome and down-regulate nuclear factor of activated T cells cytoplasmic1 (NFATc1). M-PHMs/miR-124 complexes accumulated in inflamed joints of AIA rats and showed superior therapeutic efficacy through both anti-inflammatory effect and direct bone protective effect. Combination of miR-124 and MTX in these micelles induced disease remission. Conclusions: M-PHMs/miR-124 was highly effective against RA through therapeutic synergy. Additional studies are warranted to further investigate its therapeutic potential and delineate its mechanisms of action.

Self-aggregated nanoparticles of linoleic acid-modified glycol chitosan conjugate as delivery vehicles for paclitaxel: preparation, characterization and evaluation.

A series of linoleic acid-modified glycol chitosan (LAGC) conjugates were synthesized and characterized by FTIR and (1)H NMR. The effect of the amount of linoleic acid (LA) on the physicochemical properties of LAGC conjugates was investigated. The mean diameters of three LAGC nanoparticles determined by dynamic light scattering ranged from 204 to 289 nm. The critical aggregation concentration values of LAGC conjugates in aqueous solution were 0.0148, 0.0348, and 0.0807 mg/ml, respectively. Paclitaxel (PTX) was physically loaded into the LAGC nanoparticles by a dialysis method. The drug loading content and encapsulation efficiency of PTX-loaded LAGC (PTX-LAGC) nanoparticles increased with an increasing ratio of the hydrophobic LA to hydrophilic glycol chitosan in the conjugates. PTX-LAGC nanoparticles were almost spherical in shape observed by transmission electron microscopy. In vitro release revealed that PTX release from the nanoparticles was reduced as the LA substitution degree of LAGC conjugates increased. Compared with the commercial formulation Taxol, PTX-LAGC-1 nanoparticles exhibited comparable cellular uptake and cytotoxicity against HepG2 cells in vitro. Importantly, PTX-LAGC-1 nanoparticles demonstrated the stronger antitumor efficacy against hepatic H22 tumor-bearing mice than Taxol (p < 0.05). Therefore, glycolipid-like LAGC nanoparticles had a potential as delivery vehicles for tumor therapy.

Vegetable oil thermosets reinforced by tannin-lipid formulations.

Totally bio-based thermosetting polymers which are comparable to synthetic polyester thermosets have been prepared from copolymerization of condensed tannin-fatty acid esters with vegetable oils. Oxidative copolymerization of tannin linoleate/acetate mixed esters with linseed oil and tung oil produced polymer films ranging from soft rubbers to rigid thermosets. Tannin incorporation into the formulations was essential for the final product to achieve necessary mechanical strength. Films had ambient modulus values between 0.12 and 1.6 GPa, with glass transition temperatures ranging from 32 to 72 °C and calculated crosslink densities of 1020-57,700 mol m⁻³. Film stiffness, T(g) and crosslink density increase with greater tannin linoeate/acetate content due mainly to this tannin component providing rigidity through polyphenolic aromatic rings and unsaturated chains as crosslinking sites.

Synthesis and characterization of novel PUFA esters exhibiting potential anticancer activities: an in vitro study.

Polyunsaturated fatty acids (PUFAs) have been reported to play a regulatory role in tumour growth progression. In the present study, we have synthesized ester derivatives of two important PUFA viz., linoleic acid (LA) and arachidonic acid (AA) with propofol, a widely used general anaesthetic-sedative agent. The novel propofol ester analogues have been found to inhibit various cancer cell lines in a dose-dependent manner. Moreover, the compounds have been found to induce apoptotic cell death by enhancing the release of cytochrome c and expression of caspase-3. The data of the present study suggest that novel propofol-PUFA esters have strong potential to emerge as effective anticancer agents.

Stereochemistry of hydrogen removal during oxygenation of linoleic acid by singlet oxygen and synthesis of 11(S)-deuterium-labeled linoleic acid.

Exposure of unsaturated fatty acids to singlet oxygen results in the formation of hydroperoxides. In this process, each double bond in the acyl chain produces two regioisomeric hydroperoxides having an (E)-configured double bond. Although such compounds are racemic, the hydrogen removal associated with the oxygenation may, a priori, take place antarafacially, suprafacially or stereorandomly. The present study describes the preparation of [11(S)-²H]linoleic acid by two independent methods and the use of this stereospecifically labeled fatty acid to reveal the hidden stereospecificity in singlet oxygenations of polyunsaturated fatty acids. It was found that linoleic acid 9(R)- and 13(S)-hydroperoxides formed from [11(S)-²H]linoleic acid both retained the deuterium label whereas the 9(S)- and 13(R)-hydroperoxides were essentially devoid of deuterium. It is concluded that polyunsaturated fatty acid hydroperoxides produced in the presence of singlet oxygen in e.g., plant leaves are formed by a reaction involving addition of oxygen and removal of hydrogen taking place with suprafacial stereochemistry. This result confirms and extends previous mechanistic studies of singlet oxygen-dependent oxygenations.

Dual response surface-optimized synthesis of L-menthyl conjugated linoleate in solvent-free system by Candida rugosa lipase.

Lipase-catalyzed synthesis of L-menthyl conjugated linoleate in solvent-free system was studied in this paper. Duel response surface methodology was employed to explore the factors which would influence the reaction conversion by a range of independent experiments. The conditions of reaction temperature, reaction time, enzyme amount, substrate molar ratio and water content were symmetrically investigated. When the substrates were 1 mmol CLA and 1 mmol L-menthol, the maximum conversion (79.1+/-0.8%) was obtained at 30 degrees C, Candida rugosa lipase of 33.7% (w/w by weight of L-menthol), water content of 32% (w/w by weight of L-menthol), reaction time of 43 h. The product isomers (9Z,11E-/10E,12Z-=63/37) were analyzed by GC/MS. The similarity between the oleic acid and 9Z,11E-CLA conformations which were obtained by molecular modeling could account for the specific catalyzed by C. rugosa lipase.

Biosynthesis of a linoleic acid allylic epoxide: mechanistic comparison with its chemical synthesis and leukotriene A biosynthesis.

Biosynthesis of the leukotriene A (LTA) class of epoxide is a lipoxygenase-catalyzed transformation requiring a fatty acid hydroperoxide substrate containing at least three double bonds. Here, we report on biosynthesis of a dienoic analog of LTA epoxides via a different enzymatic mechanism. Beginning with homolytic cleavage of the hydroperoxide moiety, a catalase/peroxidase-related hemoprotein from Anabaena PCC 7120, which occurs in a fusion protein with a linoleic acid 9R-lipoxygenase, dehydrates 9R-hydroperoxylinoleate to a highly unstable epoxide. Using methods we developed for isolating extremely labile compounds, we prepared and purified the epoxide and characterized its structure as 9R,10R-epoxy-octadeca-11E,13E-dienoate. This epoxide hydrolyzes to stable 9,14-diols that were reported before in linoleate autoxidation (Hamberg, M. 1983. Autoxidation of linoleic acid: Isolation and structure of four dihydroxy octadecadienoic acids. Biochim. Biophys. Acta 752: 353-356) and in incubations with the Anabaena enzyme (Lang, I., C. Göbel, A. Porzel, I. Heilmann, and I. Feussner. 2008. A lipoxygenase with linoleate diol synthase activity from Nostoc sp. PCC 7120. Biochem. J. 410: 347-357). We also prepared an equivalent epoxide from 13S-hydroperoxylinoleate using a "biomimetic" chemical method originally described for LTA(4) synthesis and showed that like LTA(4), the C18.2 epoxide conjugates readily with glutathione, a potential metabolic fate in vivo. We compare and contrast the mechanisms of LTA-type allylic epoxide synthesis by lipoxygenase, catalase/peroxidase, and chemical transformations. These findings provide new insights into the reactions of linoleic acid hydroperoxides and extend the known range of catalytic activities of catalase-related hemoproteins.

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 (SeO(2)) 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 SeO(2) 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 (IC(50) 10-75 microM). The allylic hydroxylated derivatives of LA and CLA also showed toxicity to brine shrimp with LD(50) values in the range of 2.30-13.8 microM. However these compounds showed insignificant toxicity to honeybee at doses up to 100 microg/bee.

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.

Red cell membrane and plasma linoleic acid nitration products: synthesis, clinical identification, and quantitation.

Nitric oxide (*NO) and its reactive metabolites mediate the oxidation, nitration, and nitrosation of DNA bases, amino acids, and lipids. Here, we report the structural characterization and quantitation of two allylic nitro derivatives of linoleic acid (LNO(2)), present as both free and esterified species in human red cell membranes and plasma lipids. The LNO(2) isomers 10-nitro-9-cis, 12-cis-octadecadienoic acid and 12-nitro-9-cis, 12-cis-octadecadienoic acid were synthesized and compared with red cell and plasma LNO(2) species based on chromatographic elution and mass spectral properties. Collision-induced dissociation fragmentation patterns from synthetic LNO(2) isomers were identical to those of the two most prevalent LNO(2) positional isomers found in red cells and plasma. By using [(13)C]LNO(2) as an internal standard, red cell free and esterified LNO(2) content was 50 +/- 17 and 249 +/- 104 nM, respectively. The free and esterified LNO(2) content of plasma was 79 +/- 35 and 550 +/- 275 nM, respectively. Nitrated fatty acids, thus, represent the single largest pool of bioactive oxides of nitrogen in the vasculature, with a net LNO(2) concentration of 477 +/- 128 nM, excluding buffy coat cells. These observations affirm that basal oxidative and nitrating conditions occur in healthy humans to an extent that is sufficient to induce abundant membrane and lipoprotein-fatty acid nitration. Given that LNO(2) is capable of mediating cGMP and non-cGMP-dependent signaling reactions, fatty acid nitration products are species representing the convergence of ()NO and oxygenated lipid cell-signaling pathways.

Sequential substitution of 1,2-dichloro-ethene: a convenient stereoselective route to (9Z,11E)-, (10E,12Z)- and (10Z,12Z)-.

Conjugated linoleic acid (CLA) isomers are present in human foods derived from milk or ruminant meat. To study their metabolism, (9Z,11E)-, (10E,12Z)- and (10Z,12Z)-[1-(14)C]-octadecadienoic acids with high radiochemical and isomeric purities (>98%) were prepared by stereoselective multi-step syntheses involving sequential substitution of 1,2-dichloro-ethene. In the case of the (9Z,11E) isomer, a first metal-catalyzed cross-coupling reaction between (E)-1,2-dichloro-ethene and 2-non-8-ynyloxy-tetrahydro-pyran, obtained from 7-bromo-heptan-1-ol, gave a conjugated chloroenyne. A second coupling reaction with hexylmagnesium bromide provided a heptadecenynyl derivative. Stereoselective reduction of the triple bond and bromination afforded (7E,9Z)-17-bromo-heptadeca-7,9-diene. Formation of the Grignard reagent and carbonation with 14CO(2) gave (9Z,11E)-[1-(14)C]-octadeca-9,11-dienoic acid (overall yield from 7-bromo-heptan-1-ol, 14.4%). (10E,12Z)- and (10Z,12Z)-[1-(14)C]-octadeca-10,12-dienoic acids were synthesized by the same methodology using 1-heptyne, 8-bromo-octan-1-ol and, respectively, (E)-1,2-dichloro-ethene and its (Z) isomer (overall yield from 8-bromo-octan-1-ol, 13.1% (10E,12Z); 17.2% (10Z,12Z)). Impurities (<2% if present) were identified as being (E,E) CLA isomers and were removed by RP-HPLC. Metabolism studies in animal are in progress.

Large-scale preparation of (9Z,12E)-[1-(13)C]-octadeca-9,12-dienoic acid, (9Z,12Z,15E)-[1-(13)C]-octadeca-9,12,15-trienoic acid and their [1-(13)C] all-cis isomers.

Several grams of labelled trans linoleic and linolenic acids with high chemical and isomeric purities (>97%) have been prepared for human metabolism studies. A total of 12.5 g of (9Z, 12E)-[1-(13)C]-octadeca-9,12-dienoic acid and 6.3 g of (9Z,12Z, 15E)-[1-(13)C]-octadeca-9,12,15-trienoic acid were obtained in, respectively, seven steps (7.8% overall yield) and 11 steps (7% overall yield) from 7-bromo-heptan-1-ol. The trans bromo precursors used for the labelling were synthesised by using copper-catalysed couplings. The trans fatty acids were then obtained via the nitrile derivatives. A total of 23.5 g of (9Z,12Z)-[1-(13)C]-octadeca-9, 12-dienoic acid and 10.4 g of (9Z,12Z,15Z)-[1-(13)C]-octadeca-9,12, 15-trienoic acid were prepared in five steps in, respectively, 32 and 18% overall yield. Large quantities of bromo and chloro precursors were synthesised from the commercially available acid according to Barton's procedure. In all cases, the main impurities (>0.5%) of each labelled fatty acid have been characterised.

Synthesis and nuclear magnetic resonance properties of all geometrical isomers of conjugated linoleic acids.

Pure geometric isomers of conjugated linoleic acid were prepared from castor oil as the primary starting material. Methyl octadeca-9Z,11E-dienoate (2) and methyl octadeca-9Z,11Z-dienoate (4) were obtained by zinc reduction of methyl santalbate (1, methyl octadec-11E-en-9-ynoate) and methyl octadec-11Z-en-9-ynoate (3), respectively, as the key intermediates. Methyl octadeca-9E,11E-dienoate (8) and methyl octadeca-9E,11Z-dienoate (9) were prepared by demesylation of the mesyloxy derivative of methyl ricinelaidate (6, methyl 12-hydroxy-octadec-9E-enoate). A study of the nuclear magnetic resonance spectral properties was carried-out, and the shifts of the olefinic carbon atoms of 18:2(9Z,11E) (2) and 18:2(9E,11Z) (9) were readily identified by a combination of incredible natural abundance double quantum transfer experiment, heteronuclear multiple bond correlation, and 1H-13C correlation spectroscopy correlation techniques. Doubts remain in the absolute identification of the individual olefinic carbon atoms of the 18:2(9Z,11Z) (4) and 18:2(9E,11E) (8), except the fact that the shifts of the "inner" (C-10 and C-11) and "outer" (C-9 and C-12) positioned olefinic carbon atoms of the conjugated diene system are distinguishable.