Transphosphatidylation reaction of phosphatidylcholine to 4-methoxyphenol in water-immiscible organic solvents with immobilized phospholipase D

Immobilized phospholipase D (PLD) from Streptomyces sp. catalyzed the transfer reaction of the dipalmitoylphosphatidyl residue from 1,2-dipalmitoyl-3-sn-phosphatidylcholine (DPPC) to an aromatic hydroxy group on 4-methoxyphenol in water-immiscible organic solvents, to afford 1,2-dipalmitoyl-3-sn-phosphatidyl-4-methoxyphenol (DPP-PMP) with a 45% yield, accompanied by a trace amount of 1,2-dipalmitoyl-3-sn-phosphatidic acid sodium salt (DPPA-Na). The formation of DPP-PMP was affected by organic solvents used in the reaction. Benzene, toluene, and methylene chloride gave DPP-PMP with moderate yields but use of diethyl ether resulted in a low yield of DPP-PMP. In both ethyl acetate and water-miscible organic solvents, the transfer reaction did not take place. Immobilization of PLD was carried out by adding a 1 % volume of PLD solution to a suspension of a cation-exchange resin (Amberlite IRC-50, 5% w/v) in benzene with stirring and sonication. In a repeated batch reaction for DPP-PMP synthesis with immobilized PLD, after ten batch cycles the enzyme retained 74% of its initial activity.     

Enzymatic Synthesis of Novel Phosphatidylkojic Acid and Phosphatidylarbutin,and Their Inhibitory Effects on Tyrosinase Activity

Phospholipase D (EC 3.1.4.4) from Streptomyces sp. catalyzed the transfer reaction of the dipalmitoylphosphatidyl residue from l,2-dipalmitoyl-3-sn-phosphatidylcholine (DPPC) to both arbutin and kojic acid in a biphasic system, to afford 1,2-dipalmitoyl-3-sn-phosphatidylarbutin (DPP-arbutin) and l,2-dipalmitoyl-3-sn-phosphatidylkojic acid (DPP-kojic acid), respectively. The transfer reaction of DPPC was accompanied by hydrolysis to phosphatidic acid, and the ratio was significantly affected with organic solvents used in the biphasic system. DPP-arbutin and DPP-kojic acid showed almost the same inhibitory activity to tyrosinase (EC 1.14.18.1) from mushroom as arbutin and kojic acid, respectively.     

31P NMR study of head group behavior in sonicated phosphatidylcholine liposomes in the gel and in the liquid state

We measured the ³¹P[¹H] Nuclear Overhauser Effect (NOE) as a function of temperature and of ¹H irradiation frequency, the linewidth $\text{Δν}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ as a function of temperature and the relaxation time T₁ above and below the thermal transition temperature, of the ³¹P-NMR signal in sonicated liposomes of 1,2-dimiristoyl-3-sn-phosphatidylcholine (DMPC), 1,2-dipalmitoyl-3-sn-phosphatidylcholine (DPPC) and 1,2-dimiristoyl-3-sn-phosphatidylcholine (DSPC). The same measurements were repeated in the presence of high molecular weight dextrans. They strongly reduce the NOE and produce longer relaxation times T₁. According to the current models, we were able to evaluate, in the different situations, the correlation time of the internal motion τ_G and the distance r between interacting groups in the region of the polar head groups. While the first parameter changes abruptly through the phase transition and under the effect of dextrans, the latter does not appear modified in any case. These results are discussed in terms of a conformational change of the phosphocholine head groups.     

First total synthesis of 1,2-dipalmitoyl-3-(N-palmitoyl-6′-amino-6′-deoxy-α-d-glucosyl)-sn-glycerol—a glycoglycerolipid of a marine alga with a high inhibitor activity against human Myt1-kinase

The first total synthesis of 1,2-dipalmitoyl-3-(N-palmitoyl-6′-amino-6′-deoxy-α-d-glucosyl)-sn-glycerol, a glycoglycerolipid isolated from a marine alga extract, is described. Starting from α-methylglucopyranoside the multistep strategy allows the stereoselective synthesis of the final compound using various protective group procedures as well as derivatization of partial molecule domains. The latter offers the development of lead structures for inhibitors of human Myt1-kinase.     

Nucleophilic substitution in glycerol derivatives. Part V. Formation of 1,3-diacylglycerol-2-phosphates in the reaction of 1,2-diacyl-3-iodoeoxy-rac-glycerol with silver dibenzyl phosphate

The reaction of 1,2-dipalmitoyl-3-iododeoxy-rac-glycerol with silver dibenzyl phosphate gave 1,2-dipalmitoyl-rac-glycerol-3-(dibenzyl phosphate) as the major product, contamined with ca. 2.0% of 1,3-dipalmitoyl-rac-glycerol-2-(dibenzyl phosphate). This contamination is acceptable in most preparations; in particular it cannot account for the low optical rotation values which have been recorded in the literature for sn-glycerol-3-phosphates prepared from sn-3-iododeoxy derivatives. The reaction thus remains a valuable key step in the total synthesis of rac- and sn-glycero-3-phosphoric acid esters. Factors controlling regioselectivity in such reactions are discussed.     

Perturbation of DPPC bilayers by high concentrations of pulmonary surfactant protein SP-B

Deuterium (²H) NMR has been used to observe perturbation of dipalmitoylphosphatidylcholine (DPPC) bilayers by the pulmonary surfactant protein B (SP-B) at concentrations up to 17% (w/w). Previous ²H NMR studies of DPPC/dipalmitoylphosphatidylglycerol (DPPG) (7:3) bilayers containing up to 11% (w/w) SP-B and DPPC bilayers containing up to 11% (w/w) synthetic SP-B indicated a slight effect on bilayer chain order and a more substantial effect on motions that contribute to decay of quadrupole echoes obtained from bilayers of deuterated DPPC. This is consistent with the perturbation of headgroup-deuterated DPPC reported here for bilayers containing 6 and 9% (w/w) SP-B. For the higher concentrations of SP-B investigated in the present work, ²H NMR spectra of DPPC deuterated in both the headgroup and chain display a prominent narrow component consistent with fast, large amplitude reorientation of some labeled lipid. Similar spectral perturbations have been reported for bilayers in the presence of the antibiotic polypeptide nisin. The observation of large amplitude lipid reorientation at high SP-B concentration could indicate that SP-B can induce regions of high bilayer curvature and thus provides some insight into local interaction of SP-B with DPPC. Such local interactions may be relevant to the formation, in vitro and in vivo, of tubular myelin, a unique structure found in extracellular pulmonary surfactant, and to the delivery of surfactant material to films at the air–water interface.Abbreviations DPPC 1,2-dipalmitoyl-sn-glycero-3-phosphocholine - DPPG 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol - DPPC-d₆₂ 1,2-perdeuterodipalmitoyl-sn-glycero-3-phosphocholine - DPPC-d₄ 1,2-dipalmitoyl-sn-glycero-3-phospho-(, perdeutero)-choline     

The glycoglycerolipid 1,2-dipalmitoyl-3-(N-palmitoyl-6′-amino-6′-deoxy-α-d-glucosyl)-sn-glycerol is no inhibitor of the human Myt1 kinase

Abstract

Previously, a glycoglycerolipid isolated from marine algae was reported to be a potent and selective inhibitor of the human Myt1 kinase, an enzyme involved in cell cycle regulation with great potential as an anti-cancer target. Based on that report, a lot of research effort has been invested by several working groups to synthesize and derivatize this compound. However, reliable assay data confirming the inhibitory potential and the mechanism of action of these glycoglycerolipids are missing so far. Here, based on experimental data and theoretical considerations, we show that the aforesaid glycoglycerolipid 1,2-dipalmitoyl-3-(N-palmitoyl-6′-amino-6′-deoxy-α-d-glucosyl)-sn-glycerol is not an inhibitor of the human Myt1 kinase.     

Diacyl glycerol arginine-based surfactants: biological and physicochemical properties of catanionic formulations

In this paper, we report on a catanionic vesicles-based strategy to reduce the cytotoxicity of the diacyl glycerol arginine-based synthetic surfactants 1,2-dimyristoyl-rac-glycero-3-O-(N ^α-acetyl-l-arginine) hydrochloride (1414RAc) and 1,2-dilauroyl-rac-glycero-3-O-(N ^α-acetyl-l-arginine) hydrochloride (1212RAc). The behavior of these surfactants was studied either as pure components or after their formulation as pseudo-tetra-chain catanionic mixtures with phosphatidylglycerol (PG) and as cationic mixtures with 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) used as control. The antimicrobial activity of the negatively charged formulations against Acinetobacter baumannii was maintained with respect to the surfactant alone, while a significant improvement of the antimicrobial activity against Staphylococcus aureus was observed, together with a strong decrease of hemolytic activity. The influence of the net charge of the catanionic vesicles on membrane selectivity was studied using model membranes. The dynamics of surface tension changes induced by the addition of 1414RAc/PG aqueous dispersions into phospholipid monolayers composed of zwitterionic DPPC as model system for mammalian membranes and of negatively charged PG mimicking cytoplasmic membrane of Gram-positive bacteria was followed by tensiometry. Our results constitute a proof of principle that tuning formulation can reduce the cytotoxicity of many surfactants, opening their possible biological applications.     

First total synthesis of 1,2-dipalmitoyl-3-(N-palmitoyl-6′-amino-6′-deoxy-α-d-glucosyl)-sn-glycerol—a glycoglycerolipid of a marine alga with a high inhibitor activity against human Myt1-kinase

The first total synthesis of 1,2-dipalmitoyl-3-(N-palmitoyl-6′-amino-6′-deoxy-α-d-glucosyl)-sn-glycerol, a glycoglycerolipid isolated from a marine alga extract, is described. Starting from α-methylglucopyranoside the multistep strategy allows the stereoselective synthesis of the final compound using various protective group procedures as well as derivatization of partial molecule domains. The latter offers the development of lead structures for inhibitors of human Myt1-kinase.     

Influence of lipid membrane rigidity on properties of supporting polymer

Temperature-sensitive hydrogel polymers are utilized as responsive layers in various applications. Although the polymer's native characteristics have been studied extensively, details concerning its properties during interaction with biorelated structures are lacking. This work investigates the interaction between a thermoresponsive polymer cushion and different lipid membrane capping layers probed by neutron reflectometry. N-isopropylacrylamide copolymerized with methacroylbenzophenone first supported a lipid bilayer composed of 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) and subsequently 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). The polymer-membrane systems were investigated above and below the polymer transition temperature (37 and 25°C). Although the same cushion supported each lipid membrane, the polymer hydration profile and thickness were markedly different for DPPE and DPPC systems. Because DPPE and DPPC have different bending rigidities, these results establish that the polymer-membrane interaction is critically mediated by the mechanics of the membrane, providing better insight into cell-hydrogel interactions.     

The enzymatic acyl exchange of phospholipids with lipases

The acyl exchange of phospholipids with lipases was investigated. The lipase from Rhizopus delemar catalyzed the acyl exchange reaction between various phospholipids and fatty acids. When we incubated 1,2-dipalmitoyl-sn-glycero-3-phosphatidyl choline (DPPC) and oleic acid with lipase from R. delemar, the yield of diacyl phosphatidyl choline (PC) was 25% and the fatty acid composition of the converted PC was an oleic acid content of 25% and a palmitic acid content of 75%. This reaction exhibited 1-positional specificity. Three industrial lipases from Rhizopus sp., Mucor javanicus, and Candida cylindracea had the activity of the acyl exchange of phosphatidyl choline. The lipase from R. sp. gave the best result.     

Effect of stereoconfiguration on ripple phases (Pβ′) of dipalmitoylphosphatidylcholine

Mixtures of sn-1 ( ) and sn-3 ( ) enantiomers of fully hydrated dipalmitoylphosphatidylcholine (DPPC) were studied with differential scanning calorimetry and freeze-fracture microscopy. The pretransition temperature of racemic mixtures of DPPC was 1.8 C° below that of either pure sn-1 or sn-3 enantiomers, which had similar pretransition temperatures. The main transition temperature of racemic mixtures was also depressed, but to a lesser extent, 0.8 C°. Freeze-fracture images of liposomes of sn-1, sn-3, and racemic mixtures of DPPC frozen from the P_β′ phase showed well-defined ripples of wavelength 13 nm. Lipid stereoconfiguration had no effect on ripple wavelength, configuration or amplitude, or on the number and nature of surface defects.     

In vitro stereoselective hydrolysis of diacylglycerols by hormone-sensitive lipase

Hormone-sensitive lipase (HSL) contributes importantly to the mobilization of fatty acids in adipocytes and shows a substrate preference for the diacylglycerols (DAGs) originating from triacylglycerols. To determine whether HSL shows any stereopreference during the hydrolysis of diacylglycerols, racemic 1,2(2,3)-sn-diolein was used as a substrate and the enantiomeric excess (ee%) of residual 1,2-sn-diolein over 2,3-sn-diolein was measured as a function of DAG hydrolysis. Enantiomeric DAGs were separated by performing chiral-stationary-phase HPLC after direct derivatization from lipolysis product extracts. The fact that the ee% of 1,2-sn-diolein over 2,3-sn-diolein increased with the level of hydrolysis indicated that HSL has a preference for 2,3-sn-diolein as a substrate and therefore a stereopreference for the sn-3 position of dioleoylglycerol. The ee% of 1,2-sn-diolein reached a maximum value of 36% at 42% hydrolysis. Among the various mammalian lipases tested so far, HSL is the only lipolytic carboxylester hydrolase found to have a pronounced stereospecificity for the sn-3 position of dioleoylglycerol.     

Acyl migration during deacylation of phospholipids rich in docosahexaenoic acid (DHA): an enzymatic approach for evidence and study

Non-enzymatic acyl migration could be counter-productive for the preparation of structured phospholipids with docosahexaenoic acid (DHA) at a designated position. Therefore enzymatic approaches have been developed to investigate acyl migration. First, acyl migration from sn-2 to sn-1 position has been set into relief by a three step enzymatic method using a typo-selective lipase, a phospholipase A2 and a non-selective lipase. The effect of reaction temperature on acyl migration from sn-2 to sn-1 was monitored: lowering the reaction temperature from 40 to 30°C allowed a reduction of DHA migration rate of 40%. Secondly, acyl migration from sn-1 to sn-2 position was negligible. This last result was obtained through the study of structured phosphatidylcholine selective deacylation using a phospholipase A2.     

Label-free,chronological and selective detection of aggregation and fibrillization of amyloid β protein in serum by microcantilever sensor immobilizing cholesterol-incorporated liposome

To facilitate the early diagnosis of Alzheimer's disease and mild cognitive impairment patients, we developed a cantilever-based microsensor that immobilized liposomes of various phospholipids to detect a trace amount of amyloid β (Aβ) protein, and investigated its aggregation and fibrillization on model cell membranes in human serum. Three species of liposomes composed of different phospholipids of 1,2-dipalmtoyl-sn-glycero-3-phosphocholine (DPPC), DPPC/phosphatidyl ethanolamine and 1,2-dipalmitoyl-sn-glycero-3-phosphorylglycerol having varied hydrophilic groups were applied, which showed different chronological interactions with Aβ(1–40) protein and varied sensitivities of the cantilever sensor, depending on their specific electrostatic charged conditions, hydrophilicity, and membrane fluidity. 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) having short hydrophobic carbon chains confirmed to show a large interaction with Aβ(1–40) and a high sensitivity. Furthermore, the incorporation of cholesterol into DMPC was effective to selectively detect Aβ(1–40) in human serum, which effect was also checked by quartz crystal microbalance. Finally, Aβ detection of 100-pM order was expected selectively in the serum by using the developed biosensor.     

Understanding the cytotoxic effects of new isovanillin derivatives through phospholipid Langmuir monolayers

Twenty-one isovanillin derivatives were prepared in order to evaluate their cytotoxic properties against the cancer cell lines B16F10-Nex2, HL-60, MCF-7, A2058 and HeLa. Among them, seven derivatives exhibited cytotoxic activity. We observed that for obtaining smaller IC₅₀ values and for increasing the index of selectivity, two structural features are very important when compared with isovanillin (1); a hydroxymethyl group at C-1 and the replacement of the hydroxyl group at C-3 by different alkyl groups. As the lipophilicity of the compounds was changed, we decided to investigate the interaction of the cytotoxic isovallinin derivatives on cell membrane models through Langmuir monolayers by employing the lipids DPPC (1,2-diplamitoyl-sn-glycero-3-phosphocoline) and DPPS (1,2-diplamitoyl-sn-glycero-3-phosphoserine). The structural changes on the scaffold of the compounds modulated the interaction with the phospholipids at the air-water interface. These results were very important to understand the biophysical aspects related to the interaction of the cytotoxic compounds with the cancer cell membranes.     

Chemical and physicochemical studies of lysylphosphatidylglycerol derivatives. Occurrence of a2' yields 3' lysyl migration

Syntheses of 1,2-didodecanoyl-sn-glycero-3-phosphoryl-1′-(3′-O-L-lysyl)-sn-glycerol (IV) and 1,2-didodecanoyl-sn-glycero-3-phosphoryl-1′-(2′-O-L-lysyl)-sn-glycerol (VIII) as well as 1,2-didodecanoyl-sn-glycerol-3-phosphoryl-1′-sn-glycerol (XII) are described. 2′- and 3′-lysylphosphatidylglycerol are obtained as pure isomers and can be distinguished spectroscopically (infrared, 100 and 300 MHZ NMR). By these criteria a migration of the lysyl group from the 2′ to the 3′ position of the glycerol occurs in the presence of a strong acid catalyst such as HCl. On the other hand, a weak acid such as acetic acid appears ineffective in inducing lysyl migration, even at very high concentrations.Spectroscopic analysis furthermore demonstrated that lysylphosphatidylglycerol extracted from the Staphylococcus aureus membrane, is a 3′-isomer.     

Discrimination between the regioisomeric 1,2- and 1,3-diacylglycerophosphocholines by phospholipases

The artificial 1,3-diacyl-glycero-2-phosphocholines (1,3-PCs), which form similar aggregate structures as the naturally occurring 1,2-diacyl-sn-glycero-3-phosphocholines (1,2-PCs), were tested as substrates for different classes of phospholipases such as phospholipase A₂ (PLA₂) from porcine pancreas, bee and snake venom, and Arabidopsis thaliana, phospholipase C (PLC) from Bacillus cereus, and phospholipase D (PLD) from cabbage and Streptomyces species. The regioisomers of the natural phospholipids were shown to bind to all investigated phospholipases with an affinity similar to the corresponding naturally occurring phospholipids, however their hydrolysis was reduced to different degrees (PLA₂s and PLC) or even abolished (PLDs belonging to the PLD superfamily). The results are in accordance with binding models obtained by docking the substrates to the crystal structures or homology models of the phospholipases.     

Role of Cholesterol, DOTAP, and DPPC in Prostasome/Spermatozoa Interaction and Fusion

Prostasomes are membranous vesicles present in ejaculated human semen. They are very rich in cholesterol and can interact with spermatozoa. Their physiological roles are still under study. Prostasomes were mixed with liposomes prepared from various lipids, such as N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium (DOTAP), DOTAP/1,2-dipalmytoyl-sn-glycero-3-phosphorylcholine (DPPC, 4:1 molar ratio) and DOTAP/cholesterol (4:1, molar ratio) at different pH values (5–8). The mixing of the lipid phases (fusion) was determined by the relief of octadecyl rhodamine B chloride (R₁₈) self-quenching and the radii of the vesicles, by light scattering measurements. The mixing of lipids and the radii of prostasomes were both influenced by the addition of liposome, although in a different manner. The ability of prostasomes (modified by previous treatment with liposomes) to transfer lipid to spermatozoa was also measured. Pretreatment with DOTAP decreased the phenomenon and addition of DPPC abolished it. On the other hand, pretreatment of prostasomes with DOTAP/cholesterol liposomes did not affect the transfer of lipid between prostasome and spermatozoa. Therefore, the ability of vesicles to fuse (or, at least, to exchange the lipid component) was affected by the enrichment in either natural or artificial lipid. This may open new possibilities for the modulation of spermatozoa capacitation and acrosome reaction.     

Thiol activation of a model O-aryl diazeniumdiolate prodrug in phospholipid vesicle media

Thiolysis of the model diazeniumdiolate prodrug, O²-(2,4-dinitrophenyl) 1-(N,N-diethylamino)diazen-1-ium-1,2-diolate (DNP-DEA/NO, 1), by glutathione (GSH), cysteine (CYSH) and 1-heptanethiol (heptylmercaptan, HM) has been examined in anionic (DOPG), neutral (DPPC, DOPE) and cationic (DOTAP) vesicle media and in glycine buffered aqueous solutions. DOTAP vesicles accelerate the bimolecular reaction with glutathione, cysteine and 1-heptanethiol by factors of 81, 8.2 and 4630, respectively, while reaction is inhibited 5- to 10-fold in the presence of neutral and anionic vesicles. The intrinsic nucleophilicity of the thiols has been compared through the second-order rate constants, 22.9, 5.24 and 43.1 M⁻¹ s⁻¹, for nucleophilic attack on 1 by GS⁻, CYS⁻ and M⁻, respectively, obtained in buffered aqueous media. Analysis of the catalysis by DOTAP vesicles, using pseudophase ion-exchange formalism, suggests that the rate increase is due to reactant concentration in the bilayer and interfacial region coupled with enhanced dissociation of the thiol at the vesicle surface. Some contribution from enhanced nucleophilic reactivity at the vesicle interface may also contribute to the greater catalysis by HM. Inhibition of the thiolysis reaction by phospholipid liposomes is attributed to repulsion of the thiolate anions by the negatively charged acyl phosphate of the lipid head group. DOPG = 1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)], DPPC = 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, DOPE = 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, DOTAP = 1,2-dioleoyl-3-trimethylammonium-propane.