Improved method for the synthesis of phosphatidylcholines

An improved method for the synthesis of phosphatidylcholines from phosphatidic acid and choline is described. The technique utilizes the tetraphenylborate salt of choline together with the condensing agent 2,4,6-triisopropylbenzenesulfonyl chloride. The yields in the reaction are consistently in the range 70-75%.     

Lateral diffusion of saturated phosphatidylcholines in cholesterol-containing bilayers

A pulsed field gradient NMR was used to study lateral diffusion in the cholesterol-containing oriented bilayers of saturated (dipalmitoyl- and dimyristoyl-) phosphatidylcholines, upon their limiting hydration. Similar dependences of lateral diffusion coefficients on temperature and cholesterol concentration were observed, which agree with phase diagram showing the presence of the regions of disordered and ordered liquid-crystalline phases and a two-phase region. Under the same conditions, the lateral diffusion coefficient of dipalmitoylphosphatidylcholine is lower, which agrees qualitatively with its larger molecular weight. The comparison of data for dipalmitoylphosphatidylcholine with previous results for dipalmitoylsphingomyelin-cholesterol bilayers under the same conditions, in spite of similarity of phase diagrams, shows large (two–three times) differences in the lateral diffusion coefficient and a different profile of its dependence on cholesterol concentration. The comparison of data for dimyristoylphosphatidylcholine with previous results shows that the values of lateral diffusion coefficient and the shape of its dependence on cholesterol concentration coincide at high concentrations (>15 mol%) but differ at lower concentrations The revealed disagreement may be caused by the fact that the measurements were carried out at different water content in the system. At limiting hydration (more than 35% of water), the lateral diffusion coefficient for lipids decreases when cholesterol concentration rises, while at water content about 25% (as a result of equilibrium hydration from vapors) the lateral diffusion coefficient of phosphatidylcholine may be independent of cholesterol concentration. This is the consequence of the denser packing of molecules in the bilayer at reduced water content, an effect that competes with the ordering effect of cholesterol.     

Lateral diffusion of saturated phosphatidylcholines in cholesterol-containing bilayers

Lateral diffusion in oriented bilayers of saturated cholesterol-containing phosphatidylcholines, dipalmitoylphosphatidylcholine and dimyrilstoylphosphatidylcholine upon their limiting hydration has been studied by NMR with impulse gradient of magnetic field. For both systems, similar dependences of the coefficient of lateral diffusion on temperature and cholesterol concentration were observed, which agree with the phase diagram showing the presence of regions of ordered and unordered liquid-crystalline phases and a two-phase region. Under similar conditions, the coefficient of lateral diffusion for dipalmytoylphosphatidylcholine has lower values, which is in qualitative agreement with its greater molecular mass. A comparison of data for dipalmytoylphosphatidylcholine with the results obtained earlier for dipalmytoylsphyngomyelin/cholesterol under the same conditions shows, despite a similarity in phase diagrams, greater (two- to threefold) differences in the values of the coefficient of lateral diffusion and a different mode of dependence of the coefficient on cholesterol concentration. A comparison of data for dimyrilstoylphosphatidylcholine with the results obtained previously shows that the values of the coefficient of lateral diffusion and the mode of its dependence on cholesterol concentration coincide in the region of higher concentrations (more than 15 mole %) and differ in the region of lower concentrations (below 15 mole %). The discrepancies may be explained by different contents of water in the systems during the measurements. At a limiting hydration (more than 35%) of water, the coefficient of lateral diffusion decreases with increasing cholesterol concentration. If the content of water is about 25% (as a result of equilibrium hydration from vapors), the coefficient of lateral diffusion of phosphatidylcholine is probably independent of cholesterol concentration. This results from a denser packing of molecules in the bilayer at a lower water concentration, an effect that competes with the ordering effect of cholesterol.     

The metastability of saturated phosphatidylcholines depends on the acyl chain length

We report the peak temperatures and enthalpies of the main (gel to liquid crystal) transitions and 's-transitions' of symmetric saturated PC's (CnPC) with n varying from 12 to 22, where s-transition is defined as the transition between metastable and stable phases. The line for s-transition peak temperature versus n (t = 1.98n -16.0 degrees C) crosses the corresponding pre- and main-transition curves between n = 12 and 14. We also find that the metastability pattern for lipids of n less than 14 is different to that for n greater than 14 (and is similar to that of the phosphatidylethanolamine analogs). For n greater than 14 the enthalpy of the s-transition decreases with increasing n and extrapolates to zero above n = 20, implying that the s-(orthorhombic) and gel (quasi-hexagonal) phases become identical. That simultaneous gel-liquid crystal transition and metastability is restricted to the range n = 12 to 20 may be correlated with the predominance in natural lipids of these acyl chain lengths.     

An efficient synthesis of phosphatidylcholines

This article deals with two of the major steps involved in phospholipid synthesis: the preparation of the optically pure precursors, sn-glycero-3-phosphocholine (GPC) and -ethanolamine, from a convenient lipid source such as egg yolk, and acylation of hydroxyl groups present in those precursors involving an acid to yield the corresponding phospholipid. Phosphatidylcholines and phosphatidylethanolamines were separated from lipids extracted from egg yolk using low-pressure column chromatography. The advantages of this method include the use of smaller volumes of solvents and silica gel and reuse of adsorbent. Acylation of GPC is aided by ultrasound from a common laboratory bath cleaner. Ultrasound-assisted base-catalyzed esterification of GPC is accomplished between 2-6 hours providing a phospholipid in more than 80% yield. This scheme is particularly valuable in the synthesis of polymerizable phospholipids.     

A facile synthesis of ceramides.

Lateral diffusion of phosphatide molecules in liquid crystalline bilayers has been analysed as a case of co-operative lattice diffusion. The potential energy of interaction between two molecules is assumed to arise from Van der Waals interactions of the hydrocarbon chains, and to have the form suggested by Salem [6]. From the observed values of the self-diffusion constant (of the order of 10^?8 cm² sec^?1) the depth of the potential “well” for two molecules at the equilibrium separation was estimated to have a lower limit of 1.95 kcal per mole, and the energy barrier to lateral motion was estimated to have an upper limit of 7.21 kcal per mole.     

A facile synthesis of sugar-pyrazole derivatives

A facile synthesis of sugar-pyrazole derivatives has been accomplished by condensation of sugar-chalcone with hydrazine hydrate under neutral conditions resulting in yields of 70–85%. The products are characterized by FTIR and NMR spectroscopy and by elemental analysis. The β-anomeric forms for these derivatives were assigned by NMR spectroscopy.     

Effect of saturated phosphatidylcholines on the functional properties of reconstituted cytochrome oxidase

Cytochrome oxidase was incorporated into liposomes, at various protein/lipid ratios, composed of either a phosphatidylcholine of varying chain length and symmetry or asolectin. Catalytic activity and respiratory control were assayed at two temperatures. All preparations showed higher activity at low protein/lipid ratios, but only asolectin showed respiratory control. A spectroscopic determination of the vectorial orientation of oxidase molecules showed that, for proteoliposomes with saturated lipids, 100% of oxidase molecules could be reduced by external substrate as compared with 75% for asolectin proteoliposomes. Freeze-fracture electron microscopy confirmed that oxidase was incorporated into these proteoliposomes and differential scanning calorimetry indicated that the protein induces significant disruption in the long range packing of the saturated phospholipids. We propose that the oxidase molecules in proteoliposomes formed from saturated phosphatidylcholines do not display respiratory control because they are unable to assume the transmembrane orientation necessary for full vectorial activity.     

A convenient procedure for the synthesis of ceramides

A procedure for the preparation of ceramides by direct coupling of long-chain bases and fatty acids in the presence of a mixed carbodiimide is described. This method has been used to prepare ceramides containing sphing-4-enine or sphinganine and various saturated and unsaturated fatty acids as well as saturated 2-hydroxy acids. Ceramides containing 4-hydroxy sphinganine and saturated nonhydroxy acids have also been prepared. The yields were 60-75%. The characterization of these compounds by gas-liquid chromatography-mass spectrometry as trimethylsilyl derivatives has been previously reported. Some of the ceramides are further characterized in this report by infrared spectroscopy and one compound, in addition, by elementary analysis. Use of racemic constituents for 2-hydroxy acid ceramide syntheses leads to the formation of diastereoisomers which separate by thin-layer chromatography. These were characterized by gas-liquid chromatography-mass spectrometry as the trimethylsilyl derivatives and by infrared spectroscopy. Their configurations were established by syntheses with optically active constituents.     

Empirical estimation of the gel to liquid-crystalline phase transition temperatures for fully hydrated saturated phosphatidylcholines

Phospholipids are a major component of biological membranes. In excess water, phospholipids may self-assemble into fully hydrated lamellae which, upon heating, may undergo the gel to liquid-crystalline phase transition at the characteristic temperature, Tm. Our present knowledge about the Tm values for various phospholipids is far from complete, although it is necessary to know the Tm value for preparing liposomes. In this study, we have derived empirically a general expression of Tm = 154.2 + 2.0(delta C) - 142.8(delta C/CL) - 1512.5(1/CL) in which two apparent structural parameters, delta C and CL, of a phosphatidylcholine molecule and their ratio, delta C/CL, are applied to estimate the Tm value of the phosphatidylcholine bilayer in excess water. The parameter delta C is the effective chain-length difference, in C-C bond lengths, between the two acyl chains for the phosphatidylcholine molecule in the gel-state bilayer, and CL is the effective length of the longer of the two acyl chains, also in C-C bond lengths. A figure containing 163 calculated Tm values is presented, and this information will be useful as a guide for designing experiments.     

A facile stereoselective synthesis of alpha-glycosyl ureas

Alpha-glycosyl ureas can be synthesised directly from tetra-O-benzyl glycosyl azides and isocyanates, using a one-pot procedure that is simple and general in scope. The benzyl protecting groups are easily removed from the urea products by catalytic hydrogenation. The synthesised alpha-glycosyl ureas represent a new class of neo-glycoconjugates with the potential of being resistant towards carbohydrate processing enzymes.     

A facile method for the direct synthesis of lanthionine containing cyclic peptides

A series of disulfide bridged peptides were designed as potential inhibitors of protein-protein interactions. Following solid phase synthesis, completely deprotected linear peptides were first oxidized to their disulfide analogs and then transformed into their lanthionine equivalents via a base-assisted reaction in water. Peptides consisting of cystine bridges of length i, i+3, with and without discrimination of the chiral centers, were studied for this transformation. Lanthionine peptides were also obtained directly from the reduced linear peptides under mild alkaline treatment, and the reaction proceeded via disulfide bond formation. The extent of conversion of a disulfide bridge into its lanthionine counterpart varied according to the primary sequence. Product characterization revealed diastereomeric lanthionine formation. The presence of D-amino acids, peptide conformation, and/or position of the cystine bridge are among the factors determining the facility of this reaction. Elimination of the backbone proton beta to the sulfur atom followed by intramolecular thiol Michael addition is the most likely mechanism for this transformation.     

A facile method for the direct synthesis of lanthionine containing cyclic peptides

Summary A series of disulfide bridged peptides were designed as potential inhibitors of protein-protein interactions. Following solid phase synthesis, completely deprotected linear peptides were first oxidized to their disulfide analogs and then transformed into their lanthionine equivalents via a base-assisted reaction in water. Peptides consisting of cystine bridges of lengthi, i+3, with and without discrimination of the chiral centers, were studied for this transformation. Lanthionine peptides were also obtained directly from the reduced linear peptides under mild alkaline treatment, and the reaction proceeded via disulfide bond formation. The extent of conversion of a disulfide bridge into its lanthionine counterpart varied according to the primary sequence. Product characterization revealed diastereomeric lanthionine formation. The presence of D-amino acids, peptide conformation, and/or position of the cystine bridge are among the factors determining the facility of this reaction. Elimination of the backbone proton beta to the sulfur atom followed by intramolecular thiol Michael addition is the most likely mechanism for this transformation.     

A facile procedure for efficient plantlet regeneration from self-incompatible hybrids in Antirrhinum

A rapid and efficient regeneration system has been developed for interspecific self-incompatible hybrids between Antirrhinum majus×A. hispanicum. Multiple shoots were induced from hypocotyls explants on Murashige and Skoog medium or Loblolly pine medium supplemented with a low concentration of 3-indoleacetic acid (0.05 mg l^–1) and a high concentration of 6-benzyladenine (1 mg l^–1). Rooting took place in 1/2 Murashige and Skoog medium without growth regulators. By using this procedure, it takes about 5–6 weeks from seed to plantlet. The hypocotyls also had a potential to expand and form callus, providing a source for a continuous supply of multiple shoots from the calli at periodic intervals.