Interdigitated lamellar phases in the frozen state: Spin-label CW- and FT-EPR.

Interdigitated lamellar phases composed of dipalmitoylphosphatidylcholine (DPPC) and equimolar content of lyso-palmitoylphosphatidylcholine (Lyso-PPC) or DPPC hydrated in ethanol containing water (60% v/v) have been studied in the frozen state. Electron paramagnetic resonance spectra of labeled lipids at C5 or C16 carbon atom positions along the chain are indicative of segmental librational motion over the temperature range 120-260 K. For any dispersion, the mean-square-angular amplitudes of the librations are comparable for both label positions but are larger in DPPC/etOH than in DPPC/Lyso-PPC interdigitated sample. The temperature dependences of the librational amplitudes of the labels in the lipid matrices show a rapid increase at the dynamical transition at Td ≈ 220 K with an activation energy of 20-30 kJ/mol. Three-pulse electron spin echo envelope modulation by D2O revealed comparable solvent accessibility and fractions of singly and doubly hydrogen-bonded nitroxides to deuterons for both positional isomers in the interdigitated lamellae at 77 K. The overall EPR results indicate that the interdigitated DPPC/etOH sample is more loosened packed compared to DPPC/Lyso-PPC sample. The findings of the present work obtained at cryogenic temperatures point out dynamic and molecular properties of interdigitated lamellae that contribute to the biophysical characterization of membrane model systems.

Lipase-catalyzed Synthesis of Oleoyl-lysophosphatidylcholine by Direct Esterification in Solvent-free Medium without Water Removal.

In this work, the synthesis of oleoyl-lysophosphatidylcholine by lipase-catalyzed esterification of glycerophosphocholine (GPC) and free oleic acid in a reaction medium without solvent is presented. The complete solubilisation of GPC, which is a crucial issue in non-polar liquids such as melted free fatty acids, was reached by heating the GPC/oleic acid mixture at high temperature during a short time. The immobilized lipase from Rhizomucor miehei (Lipozyme RM-IM) was shown to catalyze the reaction more efficiently than the immobilized lipases from Thermomyces lanuginosus (Lipozyme TL-IM) and Candida antarctica (Novozym 435). The condition reactions leading to the highest yield were as follows: substrate ratio: 1/20 (GPC/oleic acid); amount of catalyst: 10% (w/w of substrates); temperature: 50°C. Under these conditions, a yield of 75% of oleoyl-lysophosphatidylcholine was achieved in 24 h under stirring and almost no dioleoyl-lysophosphatidylcholine was produced. Unlike other studies dealing with the esterification of GPC with free fatty acids, the removal of the water produced while the reaction proceeds was not necessary to reach high yields.

Lysophosphatidylcholine synthesis by lipase-catalyzed ethanolysis.

Lysophosphatidylcholine (LPC) is amphiphilic substance, and possesses excellent physiological functions. In this study, LPC was prepared through ethanolysis of phosphatidylcholine (PC) in n-hexane or solvent free media catalyzed by Novozym 435 (from Candida antarctica), Lipozyme TLIM (from Thermomcyces lanuginosus) and Lipozyme RMIM (from Rhizomucor miehei). The results showed that three immobilized lipases from Candida Antarctica, Thermomcyces lanuginosus and Rhizomucor miehei could catalyze ethanolysis of PC efficiently. In n-hexane, the LPC conversions of ethanolysis of PC catalyzed by Novozyme 435, Lipozyme TLIM and Lipozyme RMIM could reach to 98.5 ± 1.6%, 94.6 ± 1.4% and 93.7 ± 1.8%, respectively. In solvent free media, the highest LPC conversions of ethanolysis of PC catalyzed by Novozyme 435, Lipozyme TL IM and Lipozyme RM IM were 97.7 ± 1.7%, 93.5 ± 1.2% and 93.8 ± 1.9%, respectively. The catalytic efficiencies of the three lipases were in the order of Novozyme 435 > Lipozyme TLIM > Lipozyme RMIM. Furthermore, their catalytic efficiencies in n-hexane were better than those in solvent free media.

Production of structured phosphatidylcholine with high content of DHA/EPA by immobilized phospholipase A1-catalyzed transesterification.

This paper presents the synthesis of structured phosphatidylcholine (PC) enriched with docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) by transesterification of DHA/EPA-rich ethyl esters with PC using immobilized phospholipsase A1 (PLA1) in solvent-free medium. Firstly, liquid PLA1 was immobilized on resin D380, and it was found that a pH of 5 and a support/PLA1 ratio (w/v) of 1:3 were the best conditions for the adsorption. Secondly, the immobilized PLA1 was used to catalyze transesterification of PC and DHA/EPA-rich ethyl esters. The maximal incorporation of DHA and EPA achieved was 30.7% for 24 h of reaction at 55 °C using a substrate mass ratio (PC/ethyl esters) of 1:6, an immobilized PLA1 loading of 15% and water dosage of 1.25%. Then the reaction mixture was analyzed by 31P nuclear magnetic resonance (NMR). The composition of reaction product included 16.5% PC, 26.3% 2-diacyl-sn-glycero-3-lysophosphatidylcholine (1-LPC), 31.4% 1-diacyl-sn-glycero-3-lysophosphatidylcholine (2-LPC), and 25.8% sn-glycerol-3-phosphatidylcholine (GPC).

The chemical synthesis and cytotoxicity of new sulfur analogues of 2-methoxy-lysophosphatidylcholine.

The chemical synthesis of phosphorothioate/phosphorodithioate analogues of 2-methoxy-lysophosphatidylcholine has been described. For the preparation of new sulfur derivatives of lysophosphatidylcholine both oxathiaphospholane and dithiaphospholane approaches have been employed. Each lysophospholipid analogue was synthesized as a series of five compounds, bearing different fatty acid residues both saturated (12:0, 14:0, 16:0, 18:0) and unsaturated (18:1). The methylation of glycerol 2-hydroxyl function was applied in order to increase the stability of prepared analogues by preventing 1 → 2 acyl migration. The cellular toxicity of newly synthesized 2-methoxy-lysophosphatidylcholine derivatives was measured using MTT viability assay and lactate dehydrogenase release method.

A new synthesis of lysophosphatidylcholines and related derivatives. Use of p-toluenesulfonate for hydroxyl group protection.

A new stereoselective synthesis of lysophosphatidylcholines is reported. The synthesis is based upon (1) the use of 3-p-toluenesulfonyl-sn-glycerol to provide the stereocenter for construction of the optically active lysophospholipid molecule, (2) tetrahydropyranylation of the secondary alcohol function to achieve orthogonal protection of the sn-2- and sn-3-glycerol positions, and (3) elaboration of the phosphodiester headgroup using a 2-chloro-1,3,2-dioxaphospholane/trimethylamine sequence. In the course of developing the synthesis it has been discovered that methoxyacetate displacement of the sn-3-p-toluenesulfonate yields a reactive methoxyacetyl ester, which in turn can be selectively cleaved with methanol/tert-butylamine, while the ester group at the sn-1-position remains unaffected. The sequence has been shown to be suitable for preparation of spectroscopically labeled lysophosphatidylcholines. One of these compounds was readily converted to a double-labeled mixed-chain phosphatidylcholine applicable for real-time fluorescence resonance energy transfer (FRET) assay of lipolytic enzymes. In addition, the work led to new synthetic strategies based on chemoselective manipulation of the tosyl group in the presence of other base-labile groups such as FMOC derivatives that are often used for the protection of amino and hydroxyl groups in syntheses.

Selective plasmenylcholine oxidation by hypochlorous acid: formation of lysophosphatidylcholine chlorohydrins.

The plasmalogen sn-1 vinyl ether bond is targeted by hypochlorous acid (HOCl) produced by activated phagocytes. In the present study, the attack of the plasmalogen sn-1 vinyl ether bond by HOCl is shown to be preferred compared to the attack of double bonds present in the sn-2 position aliphatic chain (sn-2 alkenes) of both plasmenylcholine and phosphatidylcholine. Lysophosphatidylcholine (LPC) is a product from the initial HOCl attack of plasmenylcholine and the sn-2 alkene bonds present in this LPC product are secondary targets of HOCl leading to the production of LPC-chlorohydrins (ClOH). The aliphatic ClOH was demonstrated in both the positive and negative ion mode using collisionally-activated dissociation (CAD) of the molecular ion of LPC-ClOH. Furthermore, HOCl treatment of endothelial cells led to the preferential attack of plasmalogens in comparison to that of diacyl choline glycerophospholipids. Taken together, plasmenylcholine is oxidized preferentially over phosphatidylcholine and leads to the production of LPC-ClOH.

Synthesis and evaluation of fluorogenic substrates for phospholipase D and phospholipase C.

Fluorogenic analogues of phosphatidylcholine and lysophosphatidylcholine, DDPB and lysoDDPB, were synthesized by an enzyme-assisted strategy. The analogues were evaluated as substrates for phospholipases C and D and lysophospholipase D. DDPB was cleaved by bacterial and plant phospholipase D (PLD) enzymes and represents the first direct fluorogenic substrate for real-time measurement of PLD activity. Both fluorogenic substrates have potential in screening for PLD and PC-PLC inhibitors and for monitoring spatiotemporal changes in PLD activity in cells. [structure: see text]

Synthesis of novel lysophosphatidylcholine analogues using serine as chiral template.

Four novel lysophosphatidylcholine (lysoPC) analogues, (S)-N-stearoyl-O-phosphocholineserine methyl ester [(S)-1a], (R)-1-lyso-2-stearoylamino-2-deoxy-sn-glycero-3-phosphatidylcholine [(R)-2a], (R)-N-stearoyl-O-phosphocholineserine methyl ester [(R)-1b], and (S)-1-lyso-2-stearoylamino-2-deoxy-sn-glycero-3-phosphatidylcholine [(S)-2b], were synthesized starting from serine as a chiral template. These synthetic compounds exhibited greatly enhanced hyphal transition inhibitory activity in Candida as compared to the natural lysoPC.

Structural determination of hexadecanoic lysophosphatidylcholine regioisomers by fast atom bombardment tandem mass spectrometry.

The structural determination of sn-1 and sn-2 hexadecanoic lysophosphatidylcholine (LPC) regioisomers was carried out using fast atom bombardment tandem mass spectrometry (FAB-MS/MS). The collision-induced dissociation (CID) of protonated and sodiated molecules produced diverse product ions due mainly to charge remote fragmentations. Based on the information obtained from the CID spectra of protonated and sodiated molecules, sn-1 and sn-2 hexadecanoic LPC isomers could be discriminated. Especially, the abundance ratio of the diagnostic ion pair [m/z 224/226] in the CID spectra of [M + H](+) ions was shown to be greatly different. Moreover, the CID-MS/MS spectra of sodium-adducted molecules for hexadecanoic LPC isomers showed characteristic product ions such as [M + Na - 103](+), [M + Na - 85](+), and [M + Na - 59](+), by which their regio-specificity can be differentiated.

Novel bioactive phospholipids: practical total syntheses of products from the oxidation of arachidonic and linoleic esters of 2-lysophosphatidylcholine(1).

Total syntheses of nine novel phospholipids were accomplished to facilitate the identification and biological testing of compounds that are generated upon oxidative cleavage of arachidonate and linoleate esters of 2-lysophosphatidylcholine, the two most abundant polyunsaturated phospholipids in low-density lipoprotein. An efficient general synthesis exploiting 2-lithiofuran as a 4-oxo-2-butenoyl carbanion equivalent provided phospholipids containing gamma-keto-alpha,beta-unsaturated carbonyl functional arrays. By exploiting facile cis-trans isomerizations, two commercially available cis alkenes, (2Z)-2-butene-1,4-diol and 2,5-dihydrofuran, could be employed as starting materials for preparing the Horner-Wadsworth-Emmons reagent 4-(diethoxyphosphoryl)-2E-butenal, a valuable building block for the synthesis of 2,4-dienals. The reagent was exploited in a total synthesis of 13-oxotridec-9E,11E-dienoic acid, confirming the identity of this product that is generated upon autoxidation of linoleic acid and by decomposition of 13-hydroperoxy-9,11-octadecadienoate (13-HPODE), especially in the presence of redox active transition metal ions, cytochrome p-450, or hydroperoxide lyase.

Synthesis of phosphatidylcholine with defined fatty acid in the sn-1 position by lipase-catalyzed esterification and transesterification reaction.

The incorporation of caproic acid in the sn-1 position of phosphatidylcholine (PC) catalyzed by lipase from Rhizopus oryzae was investigated in a water activity-controlled organic medium. The reaction was carried out either as esterification or transesterification. A comparison between these two reaction modes was made with regard to product yield, product purity, reaction time, and byproduct formation as a consequence of acyl migration. The yield in the esterification and transesterification reaction was the same under identical conditions. The highest yield (78%) was obtained at a water activity (a(w)) of 0.11 and a caproic acid concentration of 0.8 M. The reaction time was shorter in the esterification reaction than in the transesterification reaction. The difference in reaction time was especially pronounced at low water activities and high fatty acid concentrations. The loss in yield due to acyl migration and consequent enzymatic side reactions was around 16% under a wide range of conditions. The incorporation of a fatty acid in the sn-1 position of PC proved to be thermodynamically much more favorable than the incorporation of a fatty acid in the sn-2 position.

High-level secretory production of phospholipase A1 by Saccharomyces cerevisiae and Aspergillus oryzae.

Phospholipase A1 (PLA1) is a hydrolytic enzyme that catalyzes the removal of the acyl group from position 1 of lecithin to form lysolecithin. The PLA1 gene, which had been cloned from Aspergillus oryzae, was expressed in Saccharomyces cerevisiae and A. oryzae. Through the modification of the medium composition and the feeding conditions of substrate, the production level of PLA1 by S. cerevisiae was increased to a level fivefold higher than that indicated in a previous report. In the case of A. oryzae, introduction of multicopies of PLA1 expression units, and the morphological change from the pellet form to the filamentous form were effective for the enhancement of PLA1 production. We succeeded in producing 3,500 U/ml of PLA1 using an industrial-scale fermentor.

Synthesis of a novel lysophosphatidylcholine.

The novel lysophosphatidylcholine (1), which naturally occurs in the marine sponge Spirastrella abata and was reported to inhibit cholesterol biosynthesis in the Chang liver cell, has been synthesized in four steps from methyl cis-11-octadecenoate (2).

1,3-Diacylglycero-2-phosphocholines--synthesis, aggregation behaviour and properties as inhibitors of phospholipase D.

A series of 1,3-diacylglycero-2-phosphocholines (1,3-PCs) with acyl chain lengths of C8-C18 were synthesised by chemical introduction of the phosphocholine moiety into the regioisomerically pure 1,3-diacylglycerols, which were obtained from glycerol and the vinyl esters of fatty acid by means of lipase from Rhizomucor mihei. The 1,3-PCs being regioisomers of the natural glycerophospholipids were studied with respect to their aggregation behaviour in the absence and in the presence of sodium dodecylsulfate (SDS) as well as their properties as substrates and inhibitors of phospholipase D (PLD) from cabbage. While the main structures of the pure 1,3-PCs were micelles (C8), liposomes (C10, C12) or planar bilayers (C14, C16, C18), the addition of SDS resulted in the formation of mixed micelles (C8, C10) and mixed liposomes (C12, C14, C16, C18). None of the 1,3-PCs was found to be hydrolysed by PLD, whereas all of them showed inhibitory properties in the standard assay for PLD. The inhibitory power was strongest with 1,3-didecanoylglycero-2-phosphocholine (IC50 = 43 microM).

New fluorescent lysolipids: preparation and selective labeling of inner liposome leaflet.

Two new fluorescent lysophosphatidylcholine probes have been synthesized for use as a donor-acceptor pair in fluorescence resonance energy transfer (FRET): 9-anthrylvinyl (LAPC) as donor and 3-perylenoyl (LPPC) as acceptor. The partition coefficients between membrane and aqueous phases were 8.3 x 10(5) and 10.5 x 10(5) for LAPC and LPPC, respectively. The inner leaflets of unilamellar lipid vesicles were labeled with these probes to assess conservation of membrane sidedness after membrane fusion. After medium-sized unilamellar vesicles (MUV) were prepared with a probe in both leaflets, probe in the outer leaflet was removed by repeatedly washing with an excess of unlabeled giant unilamellar vesicles (GUV). MUV and GUV were separated by centrifugation. The probes did not flip-flop across bilayers at 25 degrees C for at least 12 h. MUV containing the ganglioside GT1b were labeled with the LAPC/LPPC pair in the inner leaflet and incubated for 30 min at neutral pH with influenza virus. Fusion was triggered by acidification to pH 5.0 and was monitored by an increase in donor fluorescence in a FRET assay. When the inner leaflets of MUV were labeled by LAPC only, its fluorescence did not change after fusion. However, the fluorescence decreased by 60% when the LAPC was removed from the outer leaflets of the fused membranes by repeated washings with GUV. We conclude that the lipids of the inner and outer leaflets of the fused MUV/virus complexes intermixed.

A simple chemical synthesis of the ether analog of lysophosphatidylcholine and platelet-activating factor.

A simple chemical procedure for synthesis of 1-O-alkyl-(rac or sn)-glycero-3-phosphocholine (alkyl analog of lysophosphatidylcholine, II) and platelet activating factor (PAF), 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (III) has been described. The key step of the method is the decomposition of 1-O-hexadecyl-3-diazohydroxyacetone (A. K. Hajra, T. V. Saraswathi and A. K. Das, 1983. Chem. Phys. Lipids. 33: 179-193) with phosphocholine to synthesize 1-O-hexadecyl dihydroxyacetone-3-phosphocholine (I). Conditions for this phosphorolysis were studied with respect to the reaction medium, temperature, and optimum proportion of the reactants. Compound (I) was quantitatively reduced with NaBH4 to synthesize (II) which was acetylated to prepare compound (III). Phospholipase A2 hydrolysis of compound (III) followed by separation of the products afforded the unreacted sn-3-hexadecyl isomer (III) and sn-1-hexadecyl isomer (II) which was acetylated to PAF. The structures of the compounds were verified by NMR and FAB-MS spectra, and their biological activities were determined by measuring the release of serotonin from rabbit blood platelets in response to different doses of these compounds. The suitability of the method as a general technique for synthesis of different ether phosphoglycerides is discussed.

The acquisition of lysophosphatidylcholine by African trypanosomes.

Bloodstream forms of the African trypanosome, Trypanosoma brucei, can acquire substantial amounts of exogenous lysophospholipid. Lysophosphatidylcholine uptake is through a pathway consisting of three enzymes, phospholipase A1, acyl-CoA ligase, and lysophosphatidylcholine:acyl-CoA acyltransferase. The pathway enables the organism to acquire fatty acids and phospholipid head groups such as choline. Radiolabeling and 13C NMR studies show that two molecules of lysophosphatidylcholine are used to generate one molecule of cellular phosphatidylcholine. The three enzymes are associated with the trypanosomal plasma membrane and are accessible to exogenous substrates. The first enzyme, phospholipase A1, generates free fatty acid from exogenous lysophospholipid, which the second enzyme, a ligase, uses to form acyl-CoA. The fatty acyl-CoA formed by this route is in a separate pool from that derived from exogenous free fatty acid and is used by the third enzyme, acyltransferase, to acylate a second molecule of exogenous lysophospholipid. Acyltransferase is accessible to exogenous and endogenous acyl-CoA. The high activity of this pathway in bloodstream forms, compared with procyclic culture form trypanosomes, suggests that it may play a role in the acquisition of fatty acids for synthesis of the membrane form of the variant surface glycoprotein. Extracellular myristoyllysophosphatidylcholine can be used by trypanosomes as a source of myristate in remodeling the lipid anchor of the variant surface glycoprotein.

Simplified methods for the synthesis of 2-hexadecanoylthio-1-ethylphosphorylcholine and for the determination of phospholipase A2 activity.

A simple and straight forward method was developed for the synthesis of 2-hexadecanoylthio-1-ethyl phosphorylcholine (HEPC). The new procedure, which used p-toulenesulfonate instead of 2-bromoethyl phosphorylcholine, not only reduced the reaction time but also allowed the reaction to proceed under mild conditions. Using HEPC as a substrate, we have also developed a microplate assay for measuring phospholipase A2 activity which is rapid and will be useful for analyzing a large number of samples in a very short time. The applicability of this assay method for assessing phospholipases A2 from two different sources and determining their kinetic constants is also demonstrated. This method can also be extended for measuring lipases and lysophospholipases using a suitable thioester. Thus, both synthesis and assay methods will be useful in basic and applied research on phospholipases and related enzymes.