Chemistry and Physics of Lipids

Published by Elsevier
Print ISSN: 0009-3084
We have synthesized a homologous series of saturated 1,2-di-O-n-acyl-3-O-(beta-D-galactopyranosyl)-sn-glycerols with odd- and even-numbered hydrocarbon chains ranging in length from 10 to 20 carbon atoms, and have investigated their physical properties using differential scanning calorimetry (DSC), X-ray diffraction (XRD) and Fourier-transform infrared (FTIR) spectroscopy. The DSC results show a complex pattern of phase behaviour, which in a typical preheated sample consists of a lower temperature, moderately energetic lamellar gel/lamellar liquid-crystalline (L(beta)/L(alpha)) phase transition and a higher temperature, weakly energetic lamellar/nonlamellar phase transition. On annealing at a suitable temperature below the L(beta)/L(alpha) phase transition, the L(beta) phase converts to a lamellar crystalline (L(c1)) phase which may undergo a highly energetic L(c1)/L(alpha) or L(c1)/inverted hexagonal (H(II)) phase transition at very high temperatures on subsequent heating or convert to a second L(c2) phase in certain long chain compounds on storage at or below 4 degrees C. The transition temperatures and phase assignments for these galactolipids are supported by our XRD and FTIR spectroscopic measurements. The phase transition temperatures of all of these events are higher than those of the comparable phase transitions exhibited by the corresponding diacyl alpha- and beta-D-glucosyl glycerols. In contrast, the L(beta)/L(alpha) and lamellar/nonlamellar phase transition temperatures of the beta-D-galactosyl glycerols are lower than those of the corresponding diacyl phosphatidylethanolamines (PEs) and these glycolipids form inverted cubic phases at temperatures between the lamellar and H(II) phase regions. Our FTIR measurements indicate that in the L(beta) phase, the hydrocarbon chains form a hexagonally packed structure in which the headgroup and interfacial region are undergoing rapid motion, whereas the L(c) phase consists of a more highly ordered, hydrogen-bonded phase, in which the chains are packed in an orthorhombic subcell similar to that reported for the diacyl-beta-D-glucosyl-sn-glycerols. A comparison of the DSC data presented here with our earlier studies of other diacyl glycolipids shows that the rate of conversion from the L(beta) to the L(c) phase in the beta-D-galactosyl glycerols is slightly faster than that seen in the alpha-D-glucosyl glycerols and much faster than that seen in the corresponding beta-D-glucosyl glycerols. The similarities between the FTIR spectra and the first-order spacings for the lamellar phases in both the beta-D-glucosyl and galactosyl glycerols suggest that the headgroup orientations may be similar in both beta-anomers in all of their lamellar phases. Thus, the differences in their L(beta)/L(c) conversion kinetics and the lamellar/nonlamellar phase properties of these lipids probably arise from subtly different hydration and H-bonding interactions in the headgroup and interfacial regions of these phases. In the latter case, such differences would be expected to alter the ability of the polar headgroup to counterbalance the volume of the hydrocarbon chains. This perspective is discussed in the context of the mechanism for the L(alpha)/H(II) phase transition which we recently proposed, based on our X-ray diffraction measurements of a series of PEs.
The condensation of N-tritylaziridine with phosphatidic acid is facile and gives a good yield of N-trityl phosphatidylethanolamine which is detritylated to a phosphatidylethanolamine. This novel route is illustrated by the synthesis of 0-(1,2-dioctadecanoyl-sn-glycero-3-phosphoryl)-ethanolamine.
A novel method of deprotecting primary alcohols protected with either benzyl or trityl groups by using bromodimethylborane under mild reaction conditions (dichloromethane, -20 to 5 degrees C) has been applied to the synthesis of optically pure mono-acid or mixed-acid 1,2- or 2,3-diacyl-sn-glycerols. This method was particularly useful for the synthesis of long saturated acyl (C12 to C24) as well as unsaturated diacyl-sn-glycerols since little or no acyl migration occurred during deprotection. Diacylation of 3-benzyl-sn-glycerol or 1-benzyl-sn-glycerol followed by bromodimethylborane debenzylation gave mono-acid 1,2- or 2,3-diacyl-sn-glycerols, respectively. The mixed-acid 1,2- or 2,3-diacyl-sn-glycerols were prepared from 1-acyl-sn-glycerols or 3-acyl-sn-glycerols, respectively, by tritylation, acylation with a different fatty acid, followed by detritylation with bromodimethylborane.
Spin lattice relaxation times in both the lab and rotating frame, have been measured for deuterons (2H) in a number of unsonicated dispersions of 1,2 dipalmitoyl phosphatidylcholine in D2O over a range of resonant frequencies from 13 MHz to 1 MHz for temperatures from −20°C to 65°C.The proton (1H) spin lattice relaxation time for the lecithin was measured for resonant frequencies of 8.5 MHz, and 40 MHz over a similar range of temperatures.The results agree with broadline measurements by Salsbury et al. [1], and for the liquid crystal phase are consistent with an anisotropic tumbling model of the water molecules bound to the lecithin headgroup. This tumbling occurs with correlation times of ≤10−10 sec and ≈ 10−6 sec about axes parallel to and perpendicular to the bisector of the D-O-D angle within a D2O molecule, hydrogen bonded to the negatively charged phosphate headgroup.
The acyl migration of 1,2-dipalmitoyl-sn-glycerol (1,2-DPG) to 1,3-dipalmitoylglycerol (1,3-DPG) in different states, neat, in the presence of egg yolk lecithin (sonicated and unsonicated) and on silica gel was studied. The isomerization was quantitated by scanning densitometry of charred TLC plates, at different temperatures and for varying periods of time. At equilibrium the amount of 1,3-DPG was found to be 56%. The rates of initial isomerization, and the time required to isomerize to half the equilibrium quantity (i.e., t1/2 eq. = 1,3-DPG 28%) under the above conditions was estimated. In the case of neat melt at 74 degrees C and in an organic solvent the time required to t1/2 eq. is 18 h and a few days, respectively. However, at 62 degrees C in the presence of a polar solvent (sodium phosphate buffer at pH 7.0) the t1/2 eq. is 1-2 h. On dry silica gel (TLC plate) at 24 degrees C the t1/2 eq. is reached in less than 1 h.
A study has been made of the semi-synthesis of 1,3-diols (anacardic alcohols) from natural phenolic lipid resources from Anacardium occidentale and Anacardium giganteum which have given C15 and C11 derivatives, respectively. An isomeric 1,3-diol (isoanacardic alcohol) has been obtained from cardanol separated from technical cashew nut-shell liquid. Homologous 1,3-diols have been synthesised from a range of synthetic 2-alkyl-, 3-alkyl- and 4-alkylphenols and from 6-alkylsalicylic acids. The natural 1,2-diol, urushiol, from Rhus vernicifera has been purified. All these lipidic compounds have been studied for their complexation and the potential recovery of boron as boric acid.
The major marine sponge phospholipids 1,2-di-(5Z,9Z)-5,9-hexacosadienoyl phosphatidylcholine (PC) and phosphatidyl-ethanolamine (PE) hardly incorporate cholesterol into their liposomal bilayers, as reported earlier. Our previous studies indicated that their synthetic short chain (C18-C24) analogs with the same double bond pattern readily incorporated cholesterol, thus demonstrating the importance of the chain length. In order to investigate the possible role of the unusual delta 5,9 diunsaturation 1,2-di-(6Z,9Z)-6,9-hexacosadienoyl phosphatidylcholine and phosphatidylethanolamine were synthesized and their thermotropic behavior studied. Both analogs shows a transition endoterm at 45 degrees C, while the natural 1,2-di-(5Z,9Z)-5,9-hexacosadienoyl PC and its PE counterpart exhibited it at 42 degrees C. A partial incorporation of cholesterol into liposomal bilayers of 1,2-di-(6Z,9Z)-6,9-hexacosadienoyl PC was observed. Our results suggest that while the chain length is the predominant factor in the interactions of these phospholipids with sterols, the double bond location may also play a contributing role.
Hydrated multibilayers of 1-palmitoyl-2-monobromopalmitoyl-sn-glycero-3-phosphorylcholine (BrDPPC), where the 2-chain is brominated at either the C-9 or C-10 position, have been studied by low and wide angle X-ray diffraction methods. Oriented and unoriented samples were investigated. The long spacing was observed over the temperature interval -15 degrees C to 80 degrees C. A monotonic increase from approx. 50 A to approx. 62 A (28 wt. % H2O) occurred with decreasing temperature. The BrDPPC showed no evidence of a sharp gel-to-liquid crystal phase transition. Wide angle scattering showed a diffuse peak corresponding to (4.5 A)-1. Differential scanning calorimetry measurements for hydrated liposomes (50 wt. % H2O) also showed no evidence for a phase transition (-40 less than or equal to T less than or equal to 60 degrees C). These results suggest a low temperature amorphous (glass) state for the acyl side chains of BrDPPC. Monolayer film properties of monobrominated stearic acid also reflect a chain disordering effect occurring upon midchain substitution.
Optical and electron microscopy were employed to characterize microstructures formed by thermal mechanical treatment of glycol suspensions of various pure and binary mixtures of the brain-derived galactosphingolipids hydroxy fatty acid cerebroside (HFA-Cer), non-hydroxy fatty acid cerebroside (NFA-Cer) and sulfatide (S-Cer). Negative staining indicated some new features of the neutral cerebroside suspensions in glycol. HFA-Cer formed a small fraction of both unilamellar cylinders (ULCs) (lumina ca. 27 nm) and giant multilamellar cochleates in addition to the typical nonhelical multilamellar cylinders (MLCs) (lumina ca. 10-30 nm). NFA-Cer formed a gel composed of a significant fraction of very long ULCs (lumina ca. 17 nm) without helical substructure, in addition to multilamellar helical structures such as ribbons and cylinders (lumina ca. 70 nm). Anisotropic lamellar micelle-shards of NFA-Cer were also detected by negative staining. S-Cer formed short ULCs (lumina ca. 44 nm) with no obvious helical substructure. Complex mixture data are thought to result from thermodynamic and kinetic factors. HFA-Cer is highly insoluble and promotes a network of rigid intralamellar hydrogen bonding that tends to exclude other lipids. NFA-Cer stabilizes helical defects in the lamellae, and S-Cer enhances disorder or micellization. The processes of microstructure nucleation and lipid phase separation were affected by mixtures such that metastable microstructures were trapped or the length of lamellar cylinders was altered.
The interactions between two membrane lipids, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) and cholesterol (CHOL), were studied in Langmuir films using surface pressure isotherms and Brewster angle microscopy. The DPPE/CHOL interactions were probed for chosen monolayer and subphase (Na(+), Ca(2+)) composition at 20, 25, and 30 degrees C. The results obtained show that DPPE and CHOL are miscible for the cholesterol mol fractions x(CHOL)=0.3-0.5. Cholesterol induces condensation of the DPPE monolayers. The most significant condensation of the DPPE/CHOL monolayers was observed in the presence of Ca(2+) ions in the subphase at x(CHOL)=0.4. The negative deviation of the molecular surface area (MMA) additivity from the ideal behavior together with negative values of excess free enthalpy of mixing in the monolayers were interpreted in terms of attractive interactions between lipid molecules.
The 13C NMR spectra of all sixteen 1,2-dioctade-cis-enoyl-sn-glycero-3-phosphorylcholines have been obtained. Resonance lines of the olefinic, methylene, methyl and carboxyl carbon nuclei are sufficiently characteristic to permit unequivocal designation of double bond position for each isomer. Two resonances of the sn-glycero-3-phosphorylcholine structure have been reassigned.
Ion binding and lipid ionization of the acidic phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) in monolayers was studied by measuring the lateral pressure Pi as a function of the molecular area A at the air/water interface at different temperatures. The pH of the subphase (pH 2 and 7) and the ionic strength (NaCl) was varied. In addition, different divalent cations (1mM MgCl2, CaCl2 and SrCl2, pH 7) were added. DMPG is partly protonated on pure water at pH 7. An increase in the NaCl concentration in the subphase leads to film expansion. This effect is caused by an ionization of the headgroup of DMPG, i.e. a shift of the apparent pK. More condensed films are obtained on pure water at pH 2, due to the reduction of electrostatic repulsion by headgroup protonation and the possibility for the formation of a hydrogen bonding network. The divalent cations Mg2+, Ca2+ and Sr2+ interact differently with a DMPG monolayer in pure water at pH 7. In the presence of 1mM CaCl2 a condensation of the DMPG film is induced, whereas an expansion of the monolayer is observed in the presence of Mg2+ and Sr2+. Two counteracting effects are operative: (a) ionization of the headgroup due to electrostatic screening leads to film expansion and (b) binding of the divalent cations to the lipid headgroups leads to condensation. The latter effect is more pronounced in the case of Ca2+, whereas the binding of Mg2+ and Sr2+ to DMPG is weaker. Site-specific cation binding has to be assumed in addition to electrostatic effects.
Aqueous dispersions of 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine, on cooling below the chain melting temperature, form hollow cylindrical structures known as 'tubules'. We have studied the formation of tubules in methanol/water, ethanol/water and n-propanol/water. For each alcohol, there is a defined window of alcohol/water ratios in which the lipid precipitates with the tubule morphology. As the chain length of alcohol is increased, the window shifts towards lower alcohol fraction. Light scattering studies show that at very low lipid concentrations the tubules self-assemble directly from the isotropic phase where as for lipid concentrations greater than 4 mg/ml an intermediate L alpha phase is observed. These results indicate that the mechanism of tubule formation may be dependent on lipid concentration.
A novel diacyl glycerol-based lipid with a polyphenolic head group has been synthesized and characterized. X-ray diffraction experiments show that this lipid, 1,2-dipalmitoylgalloylglycerol (DPGG), hydrates to form gel phase bilayers at 20 degrees C with extremely narrow interbilayer fluid separations, indicating that apposing DPGG bilayers strongly adhere to each other. Differential scanning calorimetry shows that fully hydrated DPGG exhibits a pretransition exotherm (3.7 kcal/mol) at 52 degrees C and a high enthalpy (11.3 kcal/mol) main endothermic transition at 69 degrees C. These thermal properties are similar to those of galactosylceramides with similar hydrocarbon chain compositions. The adhesive and thermal properties of DPGG are likely due to both intermolecular hydrogen-bonding and hydrophobic interactions between the aromatic rings on the gallic acids.
The mass spectra of 1,2-dipropyl-, 1,2-dipentyl-, 1,2-dihexyl-, 1,2-diheptyl-, and 1,2-dioctyl-cyclopropene, methyl malvalate, methyl sterculate, malvalyl alcohol, 1,2-dipropyl-, 1,2-dipentyl-, and 1,2-dihexylcyclopropene-3-carboxylic acid, and methyl-9,10-(carbethoxymethano)-9-octadecenoate are presented. A noticable feature of the 1,2-disubstituted cyclopropene spectra is the total absence of a cyclopropenium ion. The cyclopropenes with a carboxyl group in the 3-position yield cyclopropenium ions in the mass spectra. β-Cleavage to a allylic ion appears to be important.
The effect of 1,2-dioleoyglycerol (1,2-DOG) on the promotion of Ca(2+)-induced fusion of phosphatidylserine/phosphatidylcholine (PS/PC) vesicles was studied. 1,2-DOG is able to induce the mixing of membrane lipids at concentrations of 10 mol% without mixing of vesicular contents. At concentrations of 20 mol% or higher, 1,2-DOG promotes fusion, lipid and content mixing, of LUV composed of an equimolar mixture of PS and PC, which otherwise are unable to fuse in the presence of Ca2+. Fusion was demonstrated by fluorescence assays monitoring mixing of aqueous vesicular contents and mixing of membrane lipids. Studies by Fourier transform infrared spectroscopy provided evidence for a fusion mechanism different to that of Ca(2+)-induced fusion of pure PS vesicles. Final equilibrium structures were characterized by 31P-NMR and freeze-fracture electron microscopy. Ca(2+)-induced fusion of 1,2-DOG containing vesicles is accompanied by the formation of isotropic structures which are shown to correspond to structures with lipidic particle morphology. The possible fusion mechanisms and implications are discussed.
The syntheses of four head group labeled analogs of 1,2-di-O-palmitoyl-sn-glycero-3-phosphocholine (DPPC) (6) by a general method from 1,2-di-O-palmitoyl-sn-glycero-3-phosphatidic acid (5) have been performed. The syntheses of 1,2-di-O-palmitoyl-sn-glycero-3-phospho[alpha-13C]choline (6a) and 1,2-di-O-palmitoyl-sn-glycero-3-phospho[beta-13C]choline (6b) were performed from labeled [1-13C]glycine (1a) in 52% overall yield and from [2-13C]glycine (1b) in 56% overall yield, respectively. 1,2-Di-O-palmitoyl-sn-glycero-3-phospho[N(C2H3)3]choline (9) was prepared from 2-aminoethanol in 39% overall yield. 1,2-Di-O-palmitoyl-sn-glycero-3-phospho[alpha-C2H2]choline (12) was prepared from N,N-dimethylglycine ethyl ester in 50% overall yield.
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.
Monomolecular films of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) and 1-palmitoyl-2-[10-(pyren-1-yl)decanoyl]-sn-glycero-3-phosphatidylc holine (PPDPC) were transferred from an air/water interface onto a germanium attenuated total reflection crystal by the Langmuir-Blodgett (LB) technique. The assemblies were thereafter investigated by Fourier transform infrared-attenuated total reflection (FTIR-ATR) spectroscopy. To determine the molecular organization in the deposited layers we monitored the CH2 and C = O stretching and the CH2 bending regions of the infrared spectra of these lipids in detail. Using Fourier self-deconvolution technique, the carbonyl stretching mode was resolved into two models corresponding to the conformational differences in the ester linkages of the phospholipid sn-1 and sn-2 acyl chains. By varying the temperature of the subphase and using different surface pressures, we were able to transfer different conformational states of DPPC onto a germanium ATR crystal. Deposition of DPPC at 40 mN m-1 and at 15 degrees C or at 20 mN m-1 and at 35 degrees C results in LB-assemblies in ordered or disordered states, respectively, as judged by the IR spectra. These structures in LB films correspond to the state of DPPC in liposomes below and above the temperature of the order-disorder phase transition. Irrespective of the surface pressure and subphase temperature used during the deposition, an ordering process was found in DPPC films when the number of the transferred layers was increased from one to five. The pyrene-labelled phosphatidylcholine analogue, PPDPC, behaved differently from DPPC. In the case where one to three layers of PPDPC transferred at 35 mN m-1 and at 20 degrees C only conformational structures resembling those in fully hydrated liposomes above the main transition temperature were observed.
A novel series of N,N'-diacyl-1,2-diaminopropyl-3-carbamoyl-(dimethylaminoethane) cationic derivatives was synthesized and screened for in vitro transfection activity at different charge ratios in the presence and absence of the helper lipids DOPE and cholesterol. Physicochemical properties of lipid-DNA complexes were studied by gel electrophoresis, fluorescence spectroscopy and dynamic light scattering. The interfacial properties of the lipids in isolation were studied using the Langmuir film balance technique at 23 degrees C. It was found that only lipoplexes formulated with the dioleoyl derivative, 1,2lmt[5], mediated significant in vitro transfection activity. Optimum activity was obtained with 1,2lmt[5]/DOPE mixture at a +/-charge ratio of 2. In agreement with the transfection results, 1,2lmt[5] was the only lipid found to complex and retard DNA migration as verified by gel electrophoresis. Despite the efficient complexation, no significant condensation of plasmid DNA was observed as indicated by fluorescence spectroscopy measurements. Monolayer studies showed that the dioleoyl derivative 1,2lmt[5] was the only lipid that existed in an all liquid-expanded state with a collapse area and collapse pressure of 59.5 A2 and 38.7 mN/m, respectively. This lipid was also found to have the highest elasticity with a compressibility modulus at monolayer collapse of 80.4 mN/m. In conclusion, increased acyl chain fluidity and high molecular elasticity of cationic lipids were found to correlate with improved transfection activity.
1,2-Ethylene-di-N-n-propylcarbamate (1) is characterized as an essential activator of Pseudomonas species lipase while 1,2-ethylene-di-N-n-butyl-, t-butyl-, n-heptyl-, and n-octyl-carbamates (2-5) are characterized as the pseudo substrate inhibitors of the enzyme in the presence of the detergent taurocholate or triton X-100. The inhibition and activation reactions are more sensitive in taurocholate than in triton X-100. From CD studies, the enzyme changes conformations in the presence of the detergent and further alters conformations by addition of the carbamate activator or inhibitor into the enzyme-detergent adduct. Therefore, this study suggests that the conformational change of lipase during interfacial activation is a continuous process to expose the active site of the enzyme to substrate. From 600 MHz (1)H NMR studies, the conformations of the alpha- and beta-methylene moieties of the activator 1,2-ethylene-di-N-n-propylcarbamate in the presence of substrate change after adding taurocholate into the mixture, and the conformations of the beta-methylene moieties of the inhibitor 1,2-ethylene-di-N-n-butylcarbamate in the presence of substrate alter after adding taurocholate into the mixture.
Quantitative crystallographic structure analyses are carried out for two polymorphic forms of 1,2-dipalmitoyl-sn-glycerol. A single crystal X-ray determination on the higher melting beta'L-form reveals that the hairpin conformer structure is essentially identical to that of the dilauroyl homolog reported earlier (I. Pascher, S. Sundell and H. Hauser (1981) J. Mol. Biol. 153, 791-806) with inclined acyl chain packing in the O perpendicular methylene subcell. Lamellar electron diffraction intensity data from epitaxially crystallized samples were used to determine the structure of the lower melting alpha L-form. The chains pack in the hexagonal subcell and are perpendicular to the lamellar surface. An appropriately oriented molecular model based on the beta'L-polymorph does not lead to a satisfactory structure solution but models based on the conformationally different 1,2-diglyceride moiety of several phospholipid structures does lead to a closer match to the observed diffraction data. In this proposed packing model for the alpha L-form, the hydroxyl oxygens are somewhat farther away from the unit cell origin than in the beta'L-form crystal structure, and, in combination with the different molecular conformation, this might explain the observed stability of this crystal polymorph against acyl shifts.
This report presents the first X-ray diffraction data on diacetylenic phospholipids. The tubule-forming polymerizable lipid, 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine (DC8,9PC), was studied by low angle X-ray diffraction from partially dehydrated oriented multibilayers in both polymerized and unpolymerized form. Bilayers of this material were found to be highly ordered, yielding as many as 16 orders of lamellar diffraction, in both the polymerized and unpolymerized states. The unit cell dimension was very small for a lipid of this size. In addition to the features usually observed in the electron density profile structure of phospholipid bilayers, the electron-dense diacetylenic portions of the fatty acyl chain produced electron density maxima at two well-defined levels on each side of the bilayer approximately 15 A and 9 A from the bilayer midplane. A model molecular conformation deduced from the one-dimensional electron density map features all-trans acyl chains tilted at approximately 28 degrees from the bilayer normal that are interdigitated with chains of the opposing monolayer by approximately two carbons at the bilayer center. The linear diacetylene moieties on beta- and gamma-chains appear at different positions along the bilayer normal axis and are roughly parallel to the bilayer surface. This model is discussed in terms of a polymerization mechanism.
Computational procedures have been developed by which the total energy of a lipid multibilayer can be calculated and minimized. The energy is expressed as a sum of non-bonded, electrostatic, hydrogen bonded and torsional energy terms and includes intramolecular and intermolecular components. Calculations were carried out on three lipid crystals for which structural data are available from X-ray diffraction analysis. For each crystal, the energy was minimized as a function of all bond rotations, molecular rotations and translations and the lattice constants. The minimized structures differed by only small amounts from the experimental structures, which confirms the validity of the current set of energy functions and parameters for use with lipids. The intermolecular energy of each crystal is analyzed in terms of lateral interactions, interactions between the two monolayers of the same bilayer and interactions between bilayers. The intermolecular non-bonded energy per CH2 or CH3 group in the acyl chains is also given.
The binary phase behavior of pure 1,3-dimyristoyl-2-stearoyl-sn-glycerol (MSM) and 1,2-dimyristoyl-3-stearoyl-sn-glycerol (MMS) was investigated in terms of polymorphism, melting and crystallization behavior, SFC, hardness and microstructure. Samples were crystallized at cooling rates of 3.0 and 0.1 degrees C/min. The asymmetric TAG demonstrated lower melting and crystallization points at both cooling rates. All samples crystallized in the beta' polymorph when cooled at 0.1 degrees C/min and in the alpha polymorph when cooled at 3.0 degrees C/min. The experimentally determined kinetic phase diagram of MSM-MMS was monotectic for both cooling rates. This data was well described by a thermodynamic model using the Bragg-Williams approximation for non-ideality of mixing and suggested that in both the solid and liquid states, like pair interactions (MSM-MSM and MMS-MMS) were favored over MSM-MMS interaction. A strong tendency to phase separation in the solid phase was also observed. For both cooling rates, the fit of the SFC (%)-time curves to a modified form of the Avrami model indicated that crystallization occurred in two distinct kinetic steps. Depressions seen in SFC did not correspond to depressions in hardness or melting temperatures.
We present a steady-state and time-resolved fluorescence emission spectra analysis of the membrane probe 1-myristoyl-2-[12-[(5-dimethylamino-1-naphthalenesulfonyl)amino]dodecanoyl]-sn-glycero-3-phosphocholine (DANSYL) in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and cholesterol multi-lamellar vesicles (MLV) prepared by modified rapid solvent exchange. We report that the dose-dependent cholesterol-induced blue shifts in the steady-state fluorescence emission spectra observed in DMPC MLV are due to complex solvent effects that include time-dependent dipolar relaxation and the formation of internal charge transfer (ICT) states. A key finding of this investigation is identification of two distinguishable DANSYL populations existing at both shallow and deep locations in the membrane; these two DANSYL populations are evidence of laterally phase-separated domains at cholesterol compositions between X(chol) = 0.30 and 0.60 at 30 degrees C in DMPC MLV.
The effect of 2,4-dichlorophenol (DCP) on the structures and phase transitions of fully hydrated 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) liposomes was studied using FT-Raman spectroscopy. Whereas the Raman frequency shifts of the most frequently investigated bands of C-C and C-H stretching regions only indicate the main phase transition (P(beta')-L(alpha)) of the pure DPPC/water system, the Raman shift of C-H scissoring vibration at 1440 cm(-1) was found to be able to reveal the pretransition (L(beta')-P(beta')) as well. Analyzing the spectral parameters of the trans band at 1128 cm(-1), which does not overlap with DCP vibrational modes, a continuous decrease of trans conformations was found with increasing DCP concentration at 26 degrees C accompanying the phase transitions L(beta')-P(beta') and P(beta')-L(alpha). The intensity ratio of the symmetrical and asymmetrical methylene stretching bands (at 2850 cm(-1) and 2880 cm(-1)), defined as the disorder parameter by Levin [Levin, I.W., 1985. Two types of hydrocarbon chain interdigitation in sphingomielin bilayers. Biochemistry 24, 6282-6286], indicated that in the interdigitated phase (L(I)) the order is markedly high and comparable with that of L(beta). Both the phase transition P(beta')-L(alpha) in the DCP/DPPC molar ratio range of 10/100-50/100 and the phase transition L(I)-L(alpha) led to a significant increase of disordered chains and the presence of DCP molecules induced a more disordered chain region than that observed in the L(alpha) phase of DPPC. Nevertheless, it was found that the L(alpha) phase with DCP contains approximately the same amount of trans conformers than that without DCP.
1,2-diacyl-P-O-ethylphosphatidylcholines are synthetic cationic lipids that show some promising properties as nonviral DNA transfection agents. To gain further insight in the effects of the additional ethyl group in the headgroup region on the bilayer structure we systematically investigated a homologous series of fully hydrated ethylphosphatidylcholines with linear saturated chains (C14:0, C16:0, and C18:0) by small- and wide-angle X-ray diffraction. Our data show that all of them form multilamellar vesicles with chain interdigitated gel phases. Paying regard to the very importance of the liquid-crystalline phase in gene transfection, we applied the novel MCG method on high resolution X-ray diffraction data of the C16:0 derivative to be able to gain structural information on this phase. Comparison of this ethylphosphatidylcholine with its parent compound, the unesterified phosphatidylcholine, revealed that the major difference in the liquid-crystalline phase is the significantly reduced water layer between the bilayers for the cationic lipid. This may be one factor that contributes to the comparatively good DNA transfection efficiency of ethylphosphatidylcholines.
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 A(2) (PLA(2)) 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 (PLA(2)s 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.
1,2-Dioleoyl-sn-[3-3H]glycero-3-phospho(1-rac-glycerol) was synthesized from 1,2-dioleoyl-sn-glycerol using a new radiosynthetic procedure. 1,2-Dioleoyl-sn-glycerol was oxidized to the corresponding aldehyde using pyridinium dichromate and pyridine. The aldehyde was reduced to the radiolabeled alcohol using tritiated sodium borohydride and crown ether. This material was then converted to the phosphocholine derivative using 2-chloro-2-oxo-1,3,2-dioxaphospholane, followed by displacement with trimethylamine. In the last step, the 1,2-dioleoyl-sn-[3-3H]glycero-3-phosphocholine was converted to 1,2-dioleoyl-sn-[3-3H]glycero-3-phospho-(1-rac-glycerol) via a classic transphosphatidylation reaction using glycerol and cabbage phospholipase D. A theoretical explanation of unusual chemical behavior of the primary alcohol of diglycerides is also given, based on semi-empirical calculations.
We present results of studies on the effect of pressure on phase transitions in 1,2-dimyristoyl-phosphatidylcholine (DMPC) and 1,2-dipalmitoyl-phosphatidylcholine (DPPC) dispersed in excess water. The P-T diagram of hydrated DMPC shows a Gel III-Gel II-Gel I triple point at 3.5 kbar, 41 degrees C, the Gel III phase being obtained by annealing the sample at high pressure for several hours. In the case of DPPC, a pressure induced phase (X) appears between the Gel II and Gel I phases at approximately 0.93 kbar. With increasing pressure the temperature range of the X phase increases at the expense of that of the Gel I phase until finally at 2.87 kbar, the latter is completely suppressed. The P-T diagram of water-rich DPPC thus has 2 triple points, the Gel II-X-Gel I triple point at 0.93 kbar, 42.5 degrees C and the X-Gel I-liquid crystal triple point at 2.87 kbar, 98.5 degrees C. A pressure induced Gel III-Gel II transition is also observed in DPPC in the pressure range of 1.7-3 kbar.
A method is described for the preparation of 1,2-dialkyl glycerol ethers starting from 3-tetrahydropyranyl-glycerol and alkyl methanesulfonates.By reaction of an excess of 1,2-dialkyl glycerol ethers and (1-14C)-alkyl methanesulfonates 14C-labelled trialkyl glycerol ethers are obtained in good yields.
High-pressure investigations of phospholipids at low degrees of hydration are highly important due to the continuous interest in hydration related phenomena. This is for instance the case when discussing differences between the molecular behaviour under hydrostatic or hydration pressure, e.g. to characterise the structural and thermodynamic conditions that hold for the different theoretical approaches proposed in the literature. The stability of phospholipid aggregates in aqueous solutions is determined by a balance of attractive end repulsive forces as well as entropic and energetic interactions. The pressure-dependent variation of the frequency of molecular vibrations determined by FTIR spectroscopy might be an appropriate tool to study the nature of these interactions as well as of conformational changes for various degrees of hydrations. The paper reports preliminary results for the stretching and bending vibration of the CH2 groups that are mainly situated in the hydrocarbon chains. In a hydration range of about 2 water molecules per lipid in the case of DOPC and 6 water molecules for POPC, the pressure-dependent vibrations in the liquid crystalline phase change between red shift and blue shift. A further interesting parameter is the onset pressure for the correlation field splitting. It increases with dehydration, and it is assumed that the correlation field splitting does not only depend on the disorder related to the gauche conformer population in the hydrocarbon chains but also on the chain tilt in the gel phase.
The synthesis of C2-methyl-1,2-di-O-hexadecylglycerophosphocholine from methylallyl alcohol is described and the 13C-NMR assignments of all glycerol backbone and headgroup resonances are provided. Bond rotations in the glycerol backbone of this diether phospholipid derivative are impeded by steric hindrance exerted by the methyl group. We discuss the utility of this conformationally restricted C2-alkyl phospholipid analog for study of the influence of conformational changes at the lipid-water interface of bilayers on the interactions between lipids and other membrane components.
The glycerol-related racemic derivatives 1a-15a and 1b-15b have been resolved by hydrolysis and transesterifications using lipase B from Candida antarctica. Influence of substrate structure for the enantiomeric ratio E has been explained on the basis of previous molecular modelling. E values and equilibrium constants Keq have been calculated on the basis of ees and eep measured throughout the reaction, and ping-pong bi-bi mechanism using the computer program E&K calculator. Medium effects have been studied for hydrolysis with organic co-solvents and transesterifications in different solvents with varying water activity, aw. For some substrates addition of 30% acetone to the hydrolysis reaction gave a large increase of E. High water activity gave for some substrates an increase of E, but was unfavourable for the reversibility. However, this did not significantly influence the ee of product fraction. The influence of the alkoxy part of various butanates as acyl donors has been studied with respect to E and Keq. It was generally found that 2-chloroethyl butanate gave higher E than vinyl butanate; however, the latter gave irreversible conditions.
A series of long-chain methyl esters with vicinal oxo groups (1,2-diones; 1,2-diketones) were synthesized by potassium permanganate-based oxidation of methyl esters of mono-unsaturated fatty acids. The presence of two additional carbonyl groups may facilitate the synthesis of other derivatives. The starting materials were selected in such a fashion to give the 1,2-dioxo moiety in consecutive positions from the methyl ester group. The compounds were characterized by mass spectrometry and nuclear magnetic resonance spectroscopy. In mass spectrometry, both electron and chemical ionization (methane as reagent gas) were investigated. The position of the dioxo moiety can be determined in both ionization modes, however, in electron ionization mode the corresponding fragment ions are considerably stronger. In electron ionization mode, a fragmentation mechanism depending on the position of the 1,2-dioxo moiety occurs while the spectra derived from chemical ionization mode are mainly characterized by peaks around the molecular ion with both ionization modes appearing suitable.
The drawbacks of the presently used photosensitizers include their relatively low selectivity toward cancer cells, and long-lasting accumulation in healthy tissues. Our recent results indicate that conjugating a photosensitizer with folic acid both enhances the active uptake by cells, and decreases the accumulation in healthy tissue. Here, the interaction between 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) monolayers used as model membranes, and three different photosensitizers were studied; the derivatives were the non-conjugated meta-tetrahydroxyphenylchlorin (m-THPC, CHL1) and tris(3-hydroxyphenyl)-4-carboxyphenylchlorin (CHL2), as well as a folic acid-conjugated m-THPC-like molecule (CHL3). The results obtained indicate that the folate moiety present in the conjugated derivative CHL3 is involved in the interaction with the phospholipid polar heads. This interaction may be responsible for a better miscibility of CHL3 with the DPPC films compared to CHL1 and CHL2, while elimination of CHL3 from the tissue may be due rather to specific, biological processes and not to its polarity.
At 123 K, the crystal structure of 1,2-dipalmitoyl-sn-glycerol (C35H68O5, M(r) = 568.9) is monoclinic with space group P2(1), a = 5.480(1), b = 7.301(1), c = 43.145(7) A, beta = 92.91(1) degrees, V = 1724 A3, Dc = 1.0960 g cm-3 and Z = 2. Integrated X-ray intensities for 3574 independent reflections were measured with Ni-filtered Cu-K alpha radiation (lambda = 1.5418 A, mu = 0.56 mm-1), and these were all used in a full-matrix least-squares refinement which gave R(F2) = 0.142. Bond lengths and angles have e.s.d.s less than 0.01 A and 0.1 degrees for non-hydrogen atoms. Comparison with a previously reported study of the room temperature structure shows a very similar orthorhombic mode of chain packing but significant conformational differences in the polar headgroup. These changes may occur in conjunction with small changes in enthalpy (less than 1 kJ/mol) observed at 202, 193 and 136 K and with a discontinuity in the unit cell volume observed in the range 200-190 K. Analysis of the atomic mean square displacements at 123 K indicates the presence of internal torsional vibrations in the headgroup region that involve the carbonyl and hydroxyl O-atoms.
Analytically pure 1,2-dipalmitoyl-sn-glycero-3-phosphoric acid was prepared in gram amounts, using a simplified version of a previous procedure. The main step, enzymatic cleavage of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine with freshly extracted phospholipase D, was performed in the presence of chloroform and the crude phosphatidic acid was purified by silica gel column chromatography. The interest of the method was illustrated by the synthesis of two dipalmitoyl phosphatidylcholines selectively deuterated on the polar headgroup.
The binary phase behavior of purified 1,3-dilauroyl-2-stearoyl-sn-glycerol (LSL) and 1,2-dilauroyl-3-stearoyl-sn-glycerol (LLS) was investigated at a slow (0.1 degrees C/min) and a relatively fast (3.0 degrees C/min) cooling rate in terms of melting and crystallization, polymorphism, solid fat content (SFC), hardness and microstructure. Much of the behavior of the system is explained by its polymorphism and the influence of thermal processing. The alpha-form and the beta'-form of a double chain length structure were detected in the mixtures cooled at 3.0 degrees C/min, whereas only the beta'-form was detected in those cooled at 0.1 degrees C/min. X-ray diffraction data as well as thermodynamic data propose that the most stable phases are promoted by the symmetrical LSL. The measured trends in structural characteristics, thermal properties, SFC, relative hardness and microstructure delimit three groups of mixtures which imply a competition between the stabilizing effect of LSL and disordering introduced by kinetic effects: (a) LLS-rich mixtures with LSL molar fractions (X(LSL)) less than 0.3, (b) mixtures with X(LSL) clustered around 0.5 and (c) LSL-rich mixtures with X(LSL)>or=0.7. The balance between ordering and kinetic effects determines the polymorphism of the mixtures, which in turn determines the behavior of the LSL/LLS system. The kinetic phase diagram of the LSL/LLS binary system constructed using heating differential scanning calorimetry thermograms displayed a singularity at the 0.5(LSL) molar fraction which delimits two distinct behaviors: eutectic behavior in one region and monotectic behavior in the other. The molecular interactions, as depicted by a non-ideality parameter of mixing obtained from a thermodynamic model based on the Hildebrand equation, suggests an almost ideal mixing behavior and a moderate tendency to the formation of unlike-pairs in the liquid state.
The polymorphic behavior of three series of tris-homoacyl (C14:0, C16:0 and C18:0) cyclopentane-1,2,3-triol analogs of the natural saturated triglycerides has been studied using differential thermal analysis, Fourier transform infrared spectroscopy, and X-ray diffraction. It was found that the triglyceride analogs derived from the 1,2,3/0 and 1,2/3 cyclopentanetriols exhibit different polymorphic behavior than that of the natural triglycerides. The analogs derived from 1,3/2 cyclopentanetriol, however, were found to parallel the polymorphic behavior of the natural triglycerides quite closely. This polymorphic behavior is discussed in terms of the different configurations which the chains assume in each of the triglyceride analogs.
Reported herein is the synthesis of (+)- and (-)-(1,3/2)-1-O-(phosphocholine)-2,3-O-dihexanoylcyclopentane-1,2, 3-triol. These are the enantiomers of a contrained analogue of dihexanoylphosphatidylcholine in which the glycerol backbone is replaced by all-trans cyclopentane-1,2,3-triol. Evidence is presented to demonstrate that the (-)-enantiomer is a substrate for phospholipase A2 (PLA2) (Crotalus atrox) while the (+)-enantiomer is not. This strict enantiomeric (and positional) specificity was exploited in conjunction with a novel application of DEAE-cellulose column chromatography, to achieve racemic resolution with an excellent yield. The constrained backbone geometry, and the experimentally accessible critical micellar concentration (CMC) of these analogues should render them useful probes for assessing the contribution of substrate conformation and flexibility to the catalytic efficiency of PLA2.
The effects of the neurotoxin MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) and its toxic metabolites MPDP+ (1-methyl-4-phenyl-2,3-dihydropyridinium) and MPP+ (1-methyl-4-phenylpyridinium) on liposomal membrane were assessed using fluorescence-polarization and carboxyfluorescein leakage studies as well as in biological membrane preparations. Of the three compounds, MPTP was found to cause the greatest perturbation of membrane followed by MPDP+ and then MPP+. The ability of the three toxins to inhibit cytochrome P-450 enzyme activity (a microsomal membrane-bound enzyme system) was also studied and their relative potency was again found to be MPTP > MPDP+ > MPP+. The changes in the physicochemical property of the liposomal membrane can be related to the ability of the neurotoxin's ability to inhibit cytochrome P-450 activity.
The steady-state and dynamic fluorescence properties of the membrane potential-sensitive bis-oxonol dye Dibac4(3) were characterized in vitro using model ligand systems and in vivo in A10 smooth muscle cells by fluorescence microscopy in conjunction with the ACAS imaging system. In the latter system the dye responds to potassium ion-induced jumps in membrane potential with changes in its fluorescence intensity, which follow pseudo-first-order kinetics. The relationship between the magnitude of the changes and the corresponding rate constants excludes the possibility that a simple, one-step equilibrium between extracellular and cytoplasmic dye would be sufficient to account for this phenomenon. The necessity of invoking an additional step suggested that the redistribution of the free dye between the cytoplasm and the exocellular medium is rapid and that the slow step associated with the fluorescence changes may be the interaction of the dye with proteins in the cytoplasm, along the lines proposed by Bräuner et al. (Biochim. Biophys. Acta 771 (1984), 208, 216). The interaction of the dye with BSA and with egg lecithin SUVs was studied as a model for the in vivo phenomenon. The dependence of fluorescence intensity changes on the concentrations of the reagents shows the formation of a reversible dye/albumin complex with a 2/1-stoichiometry, with Kd = 0.03 +/- 0.01 microM and a reversible adsorption to the SUVs with Kd 0.45 +/- 0.08 microM. The fluorescence lifetime of the dye in solution, < 100 ps, results in a high solution steady-state anisotropy. The latter decreases considerably upon binding to BSA, SUVs and A10 cells concomitant with a large increase in the lifetime. With such a short lifetime of the free dye, selective gating of the excitation source or the photodetector should eliminate the high background of the unbound dye and thereby enhance the sensitivity of the system.
Semi-preparative HPLC on a chiral stationary phase (Chiracel OD) was utilized in the course of this synthesis to separate the four possible diastereomers [cis-(2R,4S)-2a, trans-(2S,4S)-2b, cis-(2S,4R)-2a', and trans-(2R,4R)-2b'] of a 2,4-disubstituted-1,3-dioxolane into optically pure forms (100% de, 100% ee). The syntheses of phosphodiester head group derivatives from each of these four conformationally constrained diastereomeric dioxolanes gave phospholipids which are monocyclic ether lipid analogs. First, the series of four [[(2-pentadecyl-1,3-dioxolan-4-yl)methyl]oxy]phosphocholines 5 were synthesized to give optically pure conformationally constrained analogues of ET-16-OCH(3). A head group variation was also demonstrated by the syntheses of the four diastereomeric [[(2-pentadecyl-1,3-dioxolan-4-yl)-methyl]oxy]phospho-beta-(N-methylmorpholino)ethanols 6.
Lipids that are covalently labeled with the 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) group are widely used as fluorescent analogues of native lipids in model and biological membranes to study a variety of processes. The fluorescent NBD group may be attached either to the polar or the apolar regions of a wide variety of lipid molecules. Synthetic routes for preparing the lipids, and spectroscopic and ionization properties of these probes are reviewed in this report. The orientation of various NBD-labeled lipids in membranes, as indicated by the location of the NBD group, is also discussed. The NBD group is uncharged at neutral pH in membranes, but loops up to the surface if attached to acyl chains of phospholipids. These lipids find applications in a variety of membrane-related studies which include membrane fusion, lipid motion and dynamics, organization of lipids and proteins in membranes, intracellular lipid transfer, and bilayer to hexagonal phase transition in liposomes. Use of NBD-labeled lipids as analogues of natural lipids is critically evaluated.
Transbilayer lipid distribution of small unilamellar vesicles (SUVs) and large unilamellar vesicles (LUVs) was measured using 31P-nuclear magnetic resonance (NMR) spectroscopy, chemical modification with 2,4,6-trinitrobenzene sulfonic acid (TNBS) and dithionite reduction of N-(7-nitrobenz-2-oxa-1,3-diazole-4-yl)-labeled lipid (NBD-lipid). The dithionite assay was the most reproducible of the three assays, with 1.2% error for SUVs and 3.9% error for LUVs. The dithionite assay also agreed best with theoretical inner:outer leaflet ratios, based on vesicle diameters determined by electron microscopy (Thomas et al. (1989) Biochem. Biophys. Acta 978, 85-90). Dithionite assay measurements were within 2.7% of theoretical ratios for SUVs and 2.3% for LUVs, while the NMR assay for SUVs was 14% lower than theoretical ratios and 23% lower for LUVs. The accuracy of NBD-lipids as markers for total transbilayer lipid was investigated. NBD-labeled phosphatidylserine, phosphatidylcholine and phosphatidylglycerol were accurate markers for total transbilayer lipid distribution, as their distributions were in close agreement with theoretical ratios. However, NBD-labeled phosphatidylethanolamine displayed a slight preference for the inner leaflet at low mole fractions of phosphatidylethanolamine, while native phosphatidylethanolamine showed a preference for the outer leaflet at the same concentration. NBD-labeled phosphatidic acid also showed a slight preference for the inner leaflet. We conclude that although dithionite-based assessment of NBD-labeled lipids across membrane bilayers can be a powerful analytical tool, caution must be used in the interpretation of results.
A novel series of synthetic glycolipids, 1,3-di-O-alkyl-2-O-(beta-glycosyl)glycerols, and their efficient synthetic route were proposed. These glyceroglycolipids were synthesized in good overall yields and stereoselectivity in five steps via trichloroacetimidate glycosylation with 1,3-di-O-alkylglycerols. This route was applied to prepare the glycolipids bearing a cello- or malto-oligosaccharide with a definite number of sugar residues from one to six. Thin-layer chromatography, elemental analysis, nuclear magnetic resonance spectroscopy and infrared absorption spectroscopy confirmed that these glycolipids were chemically pure compounds.
A flexible method for synthesising 1,30diflycerides and triglycerides is described. Glycidol esters, prepared by a known route from epichlorohydrin and the sodium salt of a fatty acid, were heated with another or with the same fatty acid and a quaternary ammonium salt. This resulted in a fast, mild reaction and higher yields and greater purity of the diglycerides than hitherto obtained in this synthesis. The mixture of 1,3- and 1,2-diglycerides obtained was isomerised by heating while still in the solid phase to 1,3-diglycerides. Triglycerides were prepared from the diglycerides by acylation using a fatty acid chloride and pyridine in hexane.
A novel series of symmetric double-chained primary and tertiary 1,3-dialkoylamido monovalent cationic lipids were synthesized and evaluated for their transfection activities. In the absence of the helper lipid DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), only the primary and tertiary dioleoyl derivatives 1,3lmp5 and 1,3lmt5, respectively elicited transfection activity. This is a striking difference between symmetrical 1,2-diacyl glycerol-based monovalent cationic lipids that always found both dioleoyl and dimyristoyl analogues being efficient transfection reagents. In the presence of helper lipid, all cationic derivatives induced marker gene expression, except the dilauroyl analogues 1,3lmp1 and 1,3lmt1 that elicited no transfection activity. Combining electrophoretic mobility data of the lipoplexes at different charge ratios with transfection activity suggested two requirements for high transfection activity with monovalent double-chained cationic lipids, that is, binding/association of the lipid to the plasmid DNA and membrane fusion properties of the lipid layers surrounding the DNA.
Top-cited authors
Frank D. Gunstone
Paavo KJ Kinnunen
  • Aalto University
Pieter Cullis
  • University of British Columbia - Vancouver
Yechezkel Barenholz
  • Hebrew University of Jerusalem
Barry R Lentz
  • University of North Carolina at Chapel Hill