Analogues Of Lysophosphatidylcholine Research Articles

Lysolipid containing liposomes for transendothelial drug delivery

BackgroundDesigning efficient 'vectors', to deliver therapeutics across endothelial barriers, in a controlled manner, remains one of the key goals of drug development. Recently, transcytosis of liposome encapsulated fluorescence marker calcein across a tight cell barrier was studied. The most efficient liposomes were found to be liposomes containing sufficient amount of alkyl phospholipid (APL) perifosine. APLs have similar structure as lysophosphatidyl choline (LPC), since APLs were synthesized as metabolically stable analogues of LPC, which increases endothelial permeability directly by inducing endothelial cell contraction, resulting in formation of gaps between endothelial cells. Since one of the unique properties of lysolipid, containing liposomal formulations is dynamic equilibrium of lysolipids, which are distributed among liposomes, micelles, and free form, such liposomes represent a reservoir of free lysolipids. On the other hand lysolipid containing liposomes also represent a reservoir of an encapsulated hydrophilic drug.Presentation of the hypothesisWe hypothesize that free lysolipids, with highest concentration in vicinity of drug carrying liposomes, compromise endothelial integrity, primarily where concentrations of liposomes is the highest, in a similar manner as LPC, by formation of gaps between endothelial cells. Liposome encapsulated drug, which leaks from liposomes, due to liposome destabilization, caused by lysolipid depletion, can therefore be efficiently transported across the locally compromised endothelial barrier.Testing the hypothesisThis hypothesis could be verified: by measuring binding of perifosine and other lysolipids to albumin and to lysophospholipid receptor (LPL-R) group; formation of stress fibers and subsequent cell contraction; activation of RhoA, and endothelial barrier dysfunction; by a synthesis of other LPC analogues with high critical micellar concentration and measuring their effect on transendothelial permeability in presence and absence of albumin.Implications of the hypothesisWe propose that lysolipid containing liposomal formulations might be used as nonspecific transendothelial transport vector, since leakage of liposome encapsulated active drug occurs simultaneously with the release of the lysolipids. The concentration of the active drug is therefore expected to be the highest at the site of compromised endothelial barrier. By appropriate choice of the lysolipids an endothelial barrier would stay open only for a short time. Use of such liposomes would potentially maximize the delivery of the drug while limiting the passage of toxic substances and pathogens across the endothelial barrier. Combining lysolipid containing liposomes with superparamagnetic iron oxide nanoparticles or a targeting ligand might be required to efficiently localize drug delivery to a disease affected tissue and to avoid endothelial disruption over the entire body.

Cholera toxin inhibits human hepatocarcinoma cell proliferation in vitro via suppressing ATX/LPA axis

To investigate the antitumor effect of cholera toxin (CT) in hepatocellular carcinoma (HCC) in vitro and the mechanisms underlying the effect. Human hepatocellular carcinoma cell lines Hep3B and Huh7, which expressed moderate and high level of autotaxin (ATX), respectively, were used. Cytokine level in the cells was evaluated using ELISA assay, and cell proliferation was investigated using MTT assay. ATX expression was determined using Western blot. ATX/lyso-PLD activity in the conditioned medium was measured using FS-3, a fluorescent lysophosphatidylcholine (LPC) analogue, as substrate. Exposure to CT (7.5 and 10 ng/mL) significantly inhibited the cell growth, decreased secretion of proinflammatory cytokine TNF-α and promoted secretion of anti-inflammatory cytokines IL-4 and IL-10. CT at 10 ng/mL markedly suppressed ATX expression in Hep3B and Huh7 cells. Furthermore, ATX and lysophosphatidic acid (LPA) were found to be crucial for growth of the cancer cells. CT could inhibit TNF-α-induced expression and secretion of ATX that led to decreased activity of lysophospholipase D, thus decreasing the conversion of LPC to LPA. CT inhibits hepatocellular carcinoma cell growth in vitro via regulating the ATX-LPA pathway.

Synthesis of Lysophosphatidylcholine Analogues Using D-Mannitol as a Chiral Template and Their Biological Activity for Sepsis

LPC analogues including natural and unnatural LPC, 3-L-2-PC, acetylated LPC and ethylene glycol derivative are prepared from D-mannitol using in convenient procedures by only changing the synthetic sequences, and their protective activities against cecal ligation and puncture (CLP)-induced severe sepsis are compared. The chirality at C2 position in LPC is found to be required as (S)-configuration for sepsis inhibition, comparing from the protection activity between LPC 6 and unnatural LPC 8. The hydroxyl functionality is also very important and required at C2 or C3 position as shown in the protection activities of ethylene glycol analogue 11 and 3-L-2-PC 9.

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

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

ET-18-OCH3 (Edelfosine): A Selective Antitumour Lipid Targeting Apoptosis Through Intracellular Activation of Fas / CD95 Death Receptor

Synthetic ether-linked analogues of phosphatidylcholine and lysophosphatidylcholine, collectively named as antitumour lipids (ATLs), were initially synthesized in the late 60s, but have attracted a renewed interest since the finding that the ether lipid 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3, edelfosine), a synthetic analogue of 2-lysophosphatidylcholine considered the ATL prototype, induces a selective apoptotic response in tumour cells, sparing normal cells. Unlike most chemotherapeutic agents currently used, ET-18-OCH3 does not interact with DNA, but act at the cell membrane, and thereby its effects seem to be independent of the proliferative state of target cells. Each part of the molecular structure of ET-18-OCH3 is important for its optimal proapoptotic activity. Recent progress has unveiled the molecular mechanism underlying the apoptotic action of ET-18-OCH3, involving membrane rafts and Fas/CD95 death receptor, and has led to the proposal of a two-step model for the ET-18-OCH3 selective action on cancer cells, namely: a) ET-18-OCH3 uptake into the tumour cell, but not in normal cells; b) intracellular activation of Fas/CD95 through its translocation and capping into membrane rafts. ET-18-OCH3 constitutes the first antitumour drug acting through the intracellular activation of the Fas/CD95 death receptor. Computational docking studies have allowed us to propose a molecular model for the putative interaction of ET-18-OCH3 with the intracellular Fas/CD95 death domain. This novel mechanism of action represents a new way to target tumour cells in cancer chemotherapy and can be of interest as a new framework in designing novel and more selective proapoptotic antitumour drugs.

Synthesis of novel lysophosphatidylcholine analogues using serine as chiral template.

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

Biological Activities, Mechanisms of Action and Biomedical Prospect of the Antitumor Ether Phospholipid ET-18-OCH3 (Edelfosine), A Proapoptotic Agent in Tumor Cells

The antitumor ether lipid ET-18-OCH(3) (edelfosine) is the prototype of a new class of antineoplastic agents, synthetic analogues of lysophosphatidylcholine, that shows a high metabolic stability, does not interact with DNA and shows a selective apoptotic response in tumor cells, sparing normal cells. Unlike currently used antitumor drugs, ET-18-OCH(3) does not act directly on the formation and function of the replication machinery, and thereby its effects are independent of the proliferative state of target cells. Because of its capacity to modulate cellular regulatory and signaling events, including those failing in cancer cells, like defective apoptosis, ET-18-OCH(3), beyond its putative clinical importance, is an interesting model compound for the development of more selective drugs for cancer therapy. Although ET-18-OCH(3) enhances host defense mechanisms against tumors, its major antitumor action lies in a direct effect on cancer cells, inhibiting phosphatidylcholine biosynthesis and inducing apoptosis in tumor cells. Recent progress has allowed unraveling the molecular mechanism underlying the apoptotic action of ET-18-OCH(3), leading to the notion that ET-18-OCH(3) is selectively incorporated into tumor cells and induces cell death by intracellular activation of the cell death receptor Fas/CD95. This intracellular Fas/CD95 activation is a novel mechanism of action for an antitumor drug and represents a new way to target tumor cells in cancer chemotherapy that can be of interest as a new framework in designing novel antitumor drugs. ET-18-OCH(3) and some analogues are pleiotropic agents that affect additional biomedical important diseases, including parasitic and autoimmune diseases, suggesting new therapeutic indications for these compounds.

Synthesis of azidophospholipids and labeling of lysophosphatidylcholine acyltransferase from developing soybean cotyledons

A photoreactive substrate analog of lysophosphatidylcholine (LPC), 1-[(4-azidosalicyl)-12-amino]dodecanoyl- sn-glycerol-3-phosphocholine (azido-LPC) was synthesized. Fast atom bombardment mass spectrometry was employed to confirm the structures of azido-LPC and its intermediates. Azido-LPC was used to label putative acyl-CoA:LPC acyltransferase from microsomal membranes of developing soybean cotyledons. The synthesized substrate analog acts as a substrate for the target acyltransferases and phospholipases in the dark. When the microsomal membranes were incubated with the acyl acceptor analog and immediately photolyzed, LPC acyltransferase was irreversibly inhibited. Photoinactivation of the enzyme by the photoprobe decreased in the presence of LPC. Microsomal membranes were photolyzed with 125I-labeled azido-LPC and analyzed by SDS-PAGE followed by autoradiography. These revealed that the analog preferentially labeled 54- and 114-kDa polypeptides. Substrate protected the labeling of both the polypeptides. In our earlier report, the same polypeptides were also labeled with photoreactive acyl-CoA analogs, suggesting that these polypeptides could be putative LPC acyltransferase(s). These results demonstrated that the photoreactive phospholipid analog could be a powerful tool to label acyltransferases involved in lipid biosynthesis.

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

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

Multiple enzymatic activities of the human cytosolic 85-kDa phospholipase A2: hydrolytic reactions and acyl transfer to glycerol.

The recombinant human 85-kDa cytosolic phospholipase A2 (cPLA2), when assayed in the presence of glycerol, catalyzes the transfer of acyl chains of radiolabeled phosphatidylcholine and para-substituted phenyl esters of fatty acids to glycerol, in addition to hydrolyzing these substrates. The product of the transacylation reaction is monoacylglycerol (MAG), and the acyl chain is predominantly esterified (> or = 95%) to a primary hydroxyl group of glycerol (sn-1/3); the stereochemistry is not known. Increasing concentrations of glycerol accelerate enzyme turnover both by providing an additional mechanistic pathway for the enzyme-substrate complex to form products and by increasing the intrinsic hydrolytic and transacylation activities of the enzyme. Significant enzymatic hydrolysis of sn-1/3-arachidonylmonoacylglycerol was measured, while sn-1/3-alpha-linolenoyl- and sn-2-arachidonylmonoacylglycerols were not detectably hydrolyzed. 1,3-Propanediol also serves as an acyl acceptor for the enzyme. cPLA2 hydrolyzes analog of lysophosphatidylcholine that lacks the sn-2 hydroxyl group. The enzyme will hydrolyze sn-1-acyl chains of rac-1-(arachidonyl, alpha-linolenoyl, palmitoyl)-2-O-hexadecyl-glycero-3-phosphocholine lipids and transfer the acyl chain to glycerol. Thus, cPLA2 has phospholipase A1 activity but only if an ether linkage rather than an ester linkage is present at the sn-2 position, and it is shown that the sn-1 acyl chains of both enantiomers of phosphatidylcholine are hydrolyzed. Phenyl [14C]-alpha-linolenate and five para-substituted phenyl esters of [3H]-alpha-linolenic acid with pKa values ranging from 7.2 to 10.2 for the phenol leaving groups were incorporated into 1,2-ditetradecyl-sn-glycero-3-phosphomethanol/Triton X-100 mixed micelles as substrates for the transacylation/hydrolysis reactions of the enzyme. Average product ratios, which are defined as the amount of monoacylglycerol formed to phenyl ester hydrolyzed, were 2.1 +/- 0.1 (n = 5) for the para-substituted phenyl esters and 2.0 +/- 0.3 (n = 7) for phenyl alpha-linolenate. The similarity of the ratios, despite the range of pKa values for the leaving groups, is consistent with the formation of a common enzyme intermediate that partitions to give either fatty acid or MAG. That intermediate may be a covalent acyl enzyme. Finally, the acyl chain specificity of cPLA2 was investigated to better understand the preference of the enzyme for phospholipids with sn-2-arachidonyl chains.

Isomeric sn‐1‐octadecenyl and sn‐2‐octadecenyl analogues of lysophosphatidylcholine as substrates for acylation and desaturation by plant microsomal membranes

To provide supporting and independent evidence for lipid-linked desaturation of acyl groups in plant microsomal membranes, ether-analogous substrates were synthesized and used for in-vitro desaturation studies. The substrates included 1-O-(9-cis-octadecenyl)-sn-glycero-3-phosphocholine and 2-O-(9-cis-octadecenyl)-sn-glycero-3-phosphocholine as well as labelled 1-O-(9-cis-[9,10-3H2]octadecenyl)-sn-glycero-3-phosphocholine. In experiments with microsomal membranes from developing fruits of sunflower, it was shown that both isomeric alkenyl ether phospholipids were acylated with [14C]oleoyl-CoA and [14C]palmitoyl-CoA. In the presence of O2 and NADH, the oleoyl groups incorporated into both compounds, i.e. at the sn-1 and sn-2 positions of the glycerol backbone of the substrate, were desaturated to linoleoyl residues in similar proportions. Under the same conditions, an additional double bond, but not an enol-ether double bond, was introduced into the ether-linked side chain of acylated 1-O-(9-cis-[9,10-3H2]octadecenyl)-sn-glycero-3-phosphocholine. This represents the first demonstration of this type of desaturation with an alkenyl ether phospholipid and confirms previous conclusions that plants introduce second and further double bonds into lipid-linked acyl groups.

Inhibition of theClostridium perfringens phospholipase C hydrolysis of a thiophosphate analog of lysophosphatidylcholine by micelle‐bound ammonium and sulfonium cations

Cetyltrimethylammonium and n-octadecyldimethylsulfonium bromides inhibit the Clostridium perfringens phospholipase C-catalyzed hydrolysis of 1-S-phosphocholine-2-O-hexadecanoyl-1-mercapto-2-ethanol (1) at pH 7.5, 37 degrees C, mu = 0.15 with KCl. Mixed micelles containing 1 and either inhibitor are substrates for the enzyme and the fraction of activity remaining is a monotonic, but non-linear function of the mole fraction of inhibitor. Simple saturation kinetics are observed as the concentration of 1 is increased in mixed micelles containing a constant mole fraction of inhibitor. Inhibition constants for cetyltrimethylammonium and n-octadecyldimethylsulfonium bromides are 0.66 +/- 0.04 and 0.25 +/- 0.02 mM, respectively. The data suggest that the significant inhibition previously observed for soluble alkyldisulfonium salts (K50 for dodecamethylene-bis(dimethylsulfonium) bromide, 27 microM) is dependent upon bifunctional cationic interactions rather than hydrophobic binding.

Structural requirements of lysophospholipid-regulated mitochondrial Ca 2+ transport

Analogues of lysophosphatidylcholine, including PAF (platelet-activating-factor) and HePC (an experimental anticancer drug), were studied for their influence on mitochondrial Ca 2+ transport and membrane potential. Lysophospholipids released Ca 2+ from mitochondria and reduced the maximal Ca 2+ uptake. The structure-activity relations indicate that deprotonated head groups like phosphocholines yield active compounds while partially protonated head groups like phosphoethanolamines are essentially inactive. Structural requirements for the apolar part of the molecules were acyl or alkyl chain lengths of less than 18 carbon atoms at the C 1-position of the glycerol backbone and residues of small size and/or low polarity at the C 2-position. Choline lysophospholipids, but not ethanolamine lysophospholipids, may therefore induce mitochondrial Ca 2+ efflux and become mediators of ischaemic tissue damage where dysregulated phospholiphase A 2 activity and an impairment of mitochondrial function are supposed to play a crucial role.

Differential effects of ether lipids on the activity and secretion of interleukin-1 and interleukin-2.

Alkyl lysophospholipids (ALP) are synthetic analogues of lysophosphatidylcholine and represent a new generation of antitumor drugs currently being tested in phase-I trials in patients with cancer. The present study reports the differential modulation of human immunocompetent cells in vitro by ALP. Serum-bound ALP effectively blocked the response of growth factor-dependent cells to interleukin-2 (IL-2), inhibited the cellular production and release of IL-2 and suppressed the comitogenic effect of interleukin-1 (IL-1) on mouse thymocytes. In contrast, ALP-primed, monocyte-derived macrophages (MO) lost their ability to release IL-1 in response to stimuli like lipopolysaccharides (LPS) during terminal maturation from monocytes. Supernatants from ALP-primed, LPS-induced MO possessed costimulatory as well as direct mitogenic activity. Neither ALP alone nor ALP-conditioned MO supernatants stimulated mouse thymocytes. Priming of MO by ET-18-OH, an ALP molecule not substituted in the sn-2 position, occurred at concentrations 4- to 16-fold higher than the most active compounds ET-18-OCH3 and the thioether analogue BM 41.440. ALP also primed MO for subsequent activation of tumor cytotoxicity by LPS and interferon-gamma. IL-1 has multiple biological activities in common with ALP, and it may mediate antitumor activity and other ALP effects in vivo. The ability of ALP to induce differential immunomodulation, as demonstrated in this study, may make ALP worthy of study for the therapy of both autoimmune and neoplastic disease.

Clonogenicity of normal and malignant hematopoietic progenitor cells after exposure to synthetic alkyl-lymphospholipids.

Alkyl-lysophospholipids (ALP) are synthetic analogues of natural lysophosphatidylcholine and represent a new class of anti-tumor agents. They are cytotoxic in vitro with a high selectivity for neoplastic cells which, in contrast to normal cells, lack an alkyl-cleavage enzyme to degrade the adsorbed ALP molecules. As ALP accumulates, it interferes with normal membrane phospholipid turnover and eventually causes disruption of membrane integrity. To evaluate the potential value of ALP in eliminating leukemic cells from remission marrows prior to autologous transplantation, we tested the effect of various ALPs on the clongenicity of normal human marrow cells and on promyelocytic leukemia HL-60. A remarkable difference in the dose response to ALP of normal marrow cells an HL-60 was observed. After an incubation period of 24 h, the same inhibition of clonogenicity in HL-60 occurred at ALP concentrations 4 times lower than in normal marrow cells. Reducing the exposure time to 6 h enhanced the selectivity further: whereas HL-60 colonies were nearly completely inhibited at 16 micrograms ALP/ml, more than 50% of normal CFU-c and BFU-E were recovered after incubation with 48 micrograms/ml. No further increase in selectivity was achieved by changing the incubation temperature. Both thioether- and alkyl-analogues were active and no difference was observed between methoxy- and acylamino-substituted ALPs. We conclude that this selective cytotoxicity makes ALP compounds worth further study as purging agents in autologous bone marrow transplantation programs.

Molecular interactions in the hydrogen belts of membranes glucose-6-phosphatase, lysophosphatidylcholine, and cholesterol

Microsomal glucose-6-phosphatase from rat liver is activated by phosphatidylcholine but inhibited by lysophosphatidylcholine. Inhibition occurs not by membrane lysis but in an intact bilayer; it is reversible; and it is overcome by addition of cholesterol but not if the cholesterol-hydroxyl group is blocked. An analog of lysophosphatidylcholine deprived of hydrogen bonding sites, 1-ether-2-deoxylysophosphatidylcholine, is a partial activator, and its effect on the enzyme in a phosphatidylcholine bilayer is not modulated by cholesterol. It appears to be one of the functions of cholesterol to buffer the lysophospholipids in membranes by complexing with them through hydrogen bonding in the hydrogen belt region. Lysophosphatidylcholine/cholesterol association is favored over phosphatidylcholine/cholesterol association.

Independent expression of the surface markers 5'-nucleotidase and cALLA on leukemic cells.

High levels of the ectozyme 5'-nucleotidase (5'-N) and the common ALL-antigen (cALLA) are coexpressed on leukemic blast cells in common ALL, in the lymphoid blast crisis of CML and also on the lymphoblastoid cell-line Nalm-1. Clinically this coexpression can help to subclassify leukemias and may be of diagnostic and prognostic significance. In an attempt to study the mechanism underlying this simultaneous expression plasmamembrane subfractionation was undertaken on Nalm-1. When membrane-shedding from intact cells is induced by sublytic concentrations of the lysophosphatidyl-choline analogue ET-12-H, membrane subfractions are obtained which contain 30-40% of total cellular 5'-N, which is most of the enzyme carried on the cell surface, in a highly enriched form. Under these conditions only a very low release of intracellular enzymes is observed. On the other hand cALLA is not accumulated in these membrane fractions to any appreciable extent. The predominant part of this antigen is still on the intact cells remaining after the shedding procedure. It is concluded that the simultaneous expression of 5'-N and cALLA on Nalm-1 and leukemic blasts is not regulated by a physical association or a close neighborhood of these antigens on the membrane level.

Release of 1-O-alkylglyceryl 3-phosphorylcholine, O-deacetyl platelet-activating factor, from leukocytes: chemical ionization mass spectrometry of phospholipids.

Evidence is presented for the simultaneous release of platelet-activating factor (PAF-acether) and of its deacetylated derivative (lyso-PAF-acether) from hog leukocytes. On the basis of spectroscopy and chemical reactions, the structure of O-deacetyl-PAF is shown to be 1-O-alkylglyceryl 3-phosphorylcholine, an alkyl ether analog of lyso-phosphatidylcholine. Acetylation of lyso-PAF yields a compound with biological activity and chromatographical behavior indistinguishable from those of native PAF. Lyso-PAF may be considered to be either the precursor or the enzymatic degradation product of PAF. The usefulness of chemical ionization mass spectrometry for structural determination of phospholipids is also demonstrated.

Conformation of phospholipids: Crystal structure of a lysophosphatidylcholine analogue

The conformation and molecular packing of deoxylysophosphatidylcholine monohydrate (3-dodecanoyl-propandiol-1-phosphorylcholine · H 2O) has been determined by single-crystal analysis. The lipid crystallizes in the monoclinic space group P2 1 c with unit cell dimensions: a = 24.83, b = 9.53, c = 10.94 A ̊ and β = 99.66 ° . The unit cell contains four symmetry-related molecules arranged in pairs of conformational enantiomers. The lipid molecules pack head-to-tail forming a common hydrocarbon layer which is bordered on each side by a region of polar groups. Two hydrocarbon chains are thus accommodated per polar group. The profile of this layer arrangement resembles that of a bilayer except that the thickness of the hydrocarbon region corresponds to the chain length of one fatty acid rather than two. The overall orientation of the phosphorylcholine groups is parallel to the layer surface. The torsion angles of the polar group are very similar to those of phosphatidylethanolamine and glycerylphosphorylcholine. Apparently this preferred polar group conformation is determined by intramolecular forces. The water molecules of hydration link adjacent phosphate groups within the polar group layer to hydrogen-bonded ribbons. Each molecule occupies an area of 52 Å 2 in the layer surface. The hydrocarbon chains pack according to the monoclinic M ∥ packing mode with all their chain planes parallel and with a cross-section/chain of 19.7 Å 2. The chains accommodate to the large molecular area by a tilt of 41 ° with respect to the layer normal. The conformational features of the compound are compared with structure data of other membrane lipids. A close resemblance to the gel state of lysophosphatidylcholine and diacylphosphatidylcholine is found which now allows a detailed interpretation of the molecular conformation and packing principles in the fully hydrated lamellar structures of these important membrane lipids.

The polar group conformation of a lysophosphatidylcholine analogue in solution: A high-resolution nuclear magnetic resonance study

The 1H and 13C resonances of 3-lauroyl-propandiol-1-phosphorylcholine in C 2H 3O 2H and 2H 2O have been assigned. LPPC ‡ ‡ Abbreviations used: LPPC, 3-lauroyl-propandiol-1-phosphorylcholine; n.m.r., nuclear magnetic resonance; p.p.m., parts per million. is present as monomers in methanol whereas it forms small micelles in water consisting of about 65 molecules. The vicinal 1H 1H, 1H 13C and 1H 31P spin coupling constants of the polar group resonances were derived from computer simulations of the 1H, 13C and 31P high-resolution nuclear magnetic resonance spectra. From an analysis of these vicinal coupling constants rotamer populations for the CC and CO bonds of the propandiol-3-phosphorylcholine moiety of LPPC were computed using a Karplus treatment (Becker, 1969). In both solvents used there is a preferred conformation in the phosphorylcholine fragment of LPPC whereas the propandiol part is flexible, as is evident from nearly equally populated rotamers around the two CC bonds of propandiol. The motionally averaged conformation of the phosphorylcholine group is characterized by an almost exclusively synclinal conformation of the choline residue (torsion angle α 5, OCCN) and by predominantly antiperiplanar conformations about the CCOP bond (torsion angle α 1) and the POCC bond (torsion angle α 4). Within the error of our conformational analysis there is good agreement between the motionally averaged conformation in solution and the crystal structure of LPPC. The average conformation in solution is independent of the solvent used and of the state of aggregation suggesting that it is mainly determined by intramolecular forces. Electrostatic interaction between the positively charged nitrogen and the anionic phosphate oxygen is responsible for stabilizing the ± synclinal conformation of the choline group. In contrast to the preferred conformation of the phosphorylcholine group, the number of possible conformations in the propandiol group is not restricted. Since, for both torsion angles θ 1 and θ 3 the three staggered conformations are equally populated, there are essentially nine possible conformational combinations for the propandiol moiety of LPPC. Since the interconversion between these different conformations is rapid on the n.m.r. time scale the LPPC molecule must have considerable flexibility about the two CC bonds of the propandiol fragment. This result indicates that there cannot be any stringent requirements for the packing of the propandiol group or the hydrocarbon chains in LPPC micelles imposing any serious constraints on the segmental motion of that group. In this respect LPPC differs markedly from diacyl phospholipids. Under comparable experimental conditions the latter class of lipids has been reported to have preferred conformations about the two CC bonds of the glycerol (torsion angles θ 1 and θ 3, Hauser et al., 1978 a, 1980). The conformational preference in that part of the molecule is a consequence of the parallel alignment of the two hydrocarbon chains optimizing hydrophobic interactions both intra- and intermolecularly. Apparently, intermolecular chain-stacking in the LPPC micelle does not restrict the number of possible rotamers in the propandiol part of the LPPC molecule.