Spin-label assay for phospholipase A2

A rapid and continuous method for measuring phospholipase A2 activity using electron spin resonance spectroscopy and a spin-labeled phospholipid as a substrate has been developed. The substrate, 1-palmitoyl-2-(4-doxylpentanoyl)glycerophosphocholine, gives rise principally to a broad ESR line in aqueous solution due to strong spin-spin interactions, probably resulting from its micellar formation. Upon addition of bee venom phospholipase A2, the water-soluble product, 4-doxylpentanoic acid, is released which brings about a sharp three-line spectrum. Thus, the kinetics of phospholipase A2 activity can be followed by monitoring the increase in the ESR signal amplitude of the three-line spectrum, which is linearly proportional to the amount of 4-doxylpentanoic acid produced; no separation of the product from the substrate is needed during the measurement. The rate of hydrolysis of 1 nmol min-1 can be accurately measured within a 5-min period of time in a sample volume of 100 microliters. This new method should be useful for assaying phospholipase A2 activities in various biological systems.     

Electron spin resonance studies on the lipid-protein interaction between cardiolipin and anti-cardiolipin antibodies.

Electron spin resonance measurements were performed in order to investigate the influence of anti-cardiolipin antibodies on cardiolipin-containing liposomes. The physical state of the lipid structures and the alterations caused by the interaction with specific antibody were determined by measuring the freedom of motion of spin-labeled stearic acid derivatives incorporated into the lipid structures. The interaction of the cardiolipin-containing liposomes with the anti-cardiolipin antibodies reduced the mobility of the spin-labeled stearic acid probe I (12, 3), whose nitroxide group is assumed to be located near the polar region of the lipid bilayer. The restricted mobility, which qualitatively resembles the interaction of cardiolipin liposomes with calcium ions, is probably the result of a tighter packing of the polar groups in their crystalline array. The binding sites of the cardiolipin structures for anti-cardiolipin antibodies and Ca2 ions seem to be identical. As indicated by the spin-labeled stearic acid probe I (1, 14), the apolar region of the lipid bilayer is not affected by the interaction of the cardiolipin-containing liposomes with the anti-cardiolipin antibodies.     

Influence of the calcium-induced gel phase on the behavior of small molecules in phosphatidylserine and phosphatidylserine-phosphatidylcholine multilamellar vesicles

The behavior of fluorescent and spin-label probes is examined in several fluid and gel phospholipid phases, with particular focus on the Ca2+-induced gel phase in phosphatidylserine (PS). These probes have behavior characteristic of the type of probe and of the type of lipid environment. Anthroyloxy- and doxyl-labeled PS [12-AS-PS and (7,6)PS, respectively] exhibit greatly restricted and/or slow probe motion in Ca(PS)2, even compared to thermotropic gel-phase lipid at the same temperature. In contrast, anthroyloxy- and doxyl-labeled phosphatidylcholine (PC), as well as fluorescent-labeled and spin-labeled fatty acid derivatives, show no apparent change in probe motion in Ca(PS)2 compared to fluid lamellar lipid. Doxyl-labeled phosphatidic acid, phosphatidylethanolamine, and phosphatidylglycerol show restricted motion in Ca(PS)2 relative to fluid-phase lipid, but the electron paramagnetic resonance (EPR) spectra could not be interpreted in terms of simple models for probe ordering. The fluorescent probes diphenylhexatriene (DPH) and trans-parinaric acid methyl ester (tPNA-Me) show motional behavior in Ca(PS)2 that is intermediate between that observed in fluid and in thermotropic gel-phase lipid. When Ca(PS)2 and fluid PS/PC phases coexist, probe molecules distribute between the two phases. Experiments using fluorescence quenching by spin-labeled PC in PS/PC in excess Ca2+ yield the distribution of several fluorophore probes between fluid liquid-crystal and Ca(PS)2 gel phases, expressed as a concentration ratio, RLC/G. The value of RLC/G = 100 in favor of the fluid phase is obtained for 12-AS-PC, 18 for 12-AS-Me, 12 for DPH, 3 for tPnA-Me, and 1 for 12-AS-PS.(ABSTRACT TRUNCATED AT 250 WORDS)     

Backbone dynamics determined by electron paramagnetic resonance to optimize solid-phase peptide synthesis of TOAC-labeled phospholamban

Electron paramagnetic resonance (EPR) was used to optimize the solid-phase peptide synthesis of a membrane-bound peptide labeled with TOAC (2,2,6,6-tetramethyl-piperidine-1-oxyl-4-amino-4-carboxylic acid). The incorporation of this paramagnetic amino acid results in a nitroxide spin label coupled rigidly to the alpha-carbon, providing direct detection of peptide backbone dynamics by EPR. We applied this approach to phospholamban, which regulates cardiac calcium transport. The synthesis of this amphipathic 52-amino-acid membrane peptide including TOAC is a challenge, especially in the addition of TOAC and the next several amino acids. Therefore, EPR of synthetic intermediates, reconstituted into lipid bilayers, was used to ensure complete coupling and 9-fluorenylmethoxycarbonyl (Fmoc) deprotection. The attachment of Fmoc-TOAC-OH leads to strong immobilization of the spin label, whereas Fmoc deprotection dramatically mobilizes it, producing an EPR spectral peak that is completely resolved from that observed before deprotection. Similarly, coupling of the next amino acid (Ser) restores the spin label to strong immobilization, giving a peak that is completely resolved from that of the preceding step. For several subsequent steps, the effect of coupling and deprotection is similar but less dramatic. Thus, the sensitivity and resolution of EPR provides a quantitative monitor of completion at each of these critical steps in peptide synthesis. Mass spectrometry, circular dichroism, and Edman degradation were used in concert with EPR to verify the chemistry and characterize the secondary structure. In conclusion, the application of conventional analytical methods in combination with EPR offers an improved approach to optimize the accurate synthesis of TOAC spin-labeled membrane peptides.     

Effect of fluorophore linkage position of n-(9-anthroyloxy) fatty acids on probe distribution between coexisting gel and fluid phospholipid phases

The quenching of probe fluorescence by spin-labeled phospholipid has been used to determine the distribution of a series of n-(9-anthroyloxy) fatty acids between coexisting gel and fluid liquid-crystal phases in multilamellar phospholipid vesicles. The phase distribution ratio in every case is found to favor the fluid lipid phase, but is much greater between fluid and Ca2+-induced gel than between fluid and thermal gel. For a given gel type, n-(9-anthroyloxy)stearic acids with n = 3, 6, 9 or 12 as well as 11-(9-anthroyloxy)undecanoic acid all exhibit similar behavior, favoring the fluid phase by about a factor of 4 over thermally-induced lipid gel phase and by 18 over Ca2+-induced gel phase. 16-(9-Anthroyloxy)palmitic acid, with the bulky probe at the terminus of the 16-carbon chain, favors the fluid phase less strongly, by a factor of 1.5 or 11 over thermally-induced or Ca2+-induced gel phase, respectively, indicating better packing of this probe in phospholipid gel phases.     

Solid-phase synthesis of peptides containing the spin-labeled 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC).

2,2,6,6-Tetramethylpiperidine-1-oxyl-4-amino4-carboxylic acid (TOAC) is a nitroxide spin-labeled, achiral Calpha-tetrasubstituted amino acid recently shown to be not only an effective beta-turn and 3(10)/alpha-helix promoter in peptides, but also an excellent rigid electron paramagnetic resonance probe and fluorescence quencher. Here, we demonstrate that TOAC can be effectively incorporated into internal positions of peptide sequences using Fmoc chemistry and solid-phase synthesis in an automated apparatus.     

Initial membrane reaction in the biosynthesis of peptidoglycan. Spin-labeled intermediates as receptors for vancomycin and ristocetin.

Phospho-N-acetylmuramyl-pentapeptide translocase (UDP-MurNAc-Ala-DGlu-Lys-DAla-DAla:undecaprenyl phosphate, phospho-MurNAc-pentapeptide transferase) catalyzes the initial membrane reaction in the biosynthesis of peptidoglycan. The spin-labeled nucleotide, UDP-MurNAc-Ala-DGlu-Lys (Nepsilon-2,2,5,5-tetramethyl-N-oxyl-pyrroline-3-carbonyl)-DAla-DAla, was used as a substrate by this enzyme for the synthesis of membrane-associated undecaprenyl-diphosphate-MurNAc-Ala-DGlu-Lys(Nepsilon-Tempyo)-DAla-DAla. The spin-labeled substrate and product complex with the antibiotics vancomycin and ristocetin. The association constants for the spin-labeled nucleotide are 6.2 times 10(5) and 6.2 times 10(4) M-1 for vancomycin and ristocetin, respectively. The association constants for the spin-labeled lipid intermediate are 3.0 times 10(4) and 2.1 times 10(4) M-1 for vancomycin and ristocetin, respectively. These results indicate that the acyl-DAla termini of membranes-associated spin-labeled undecaprenyl-diphosphate-MurNAc-pentapeptide are accessible to vancomycin and ristocetin and that the association constants are smaller than those determined for the corresponding antibiotic spin-labeled UDP-MurNAc-pentapeptide complexes.     

The outer membrane of Proteus mirabilis. II. The extractable lipid fraction and electron-paramagnetic resonance analysis of the outer and cytoplasmic membranes.

1. The lipid fraction extracted from the outer and cytoplasmic membranes of Proteus mirabilis with chloroform/methanol consisted almost entirely of phosphatidylethanolamine, diphosphatidylglycerol and phosphatidylglycerol. 2. The phospholipid content of the cytoplasmic membrane was more than twice that of the outer membrane (38% as against 18% of the total dry weight) and the proportions of the three phospholipids differed somewhat in the two membranes. Yet, the fatty acid composition of the extractable lipids was essentially the same in both membranes. 3. The freedom of motion of spin-labeled fatty acids in the outer membrane of P. mirabilis depended markedly on temperature and on the position of the nitroxide group on the hydrocarbon chain of the probe, suggesting that the local environment of the probe is an associate lipid structure with the properties of a bilayer. Nevertheless, the mobility of the probe was more restricted in the outer membrane than in the cytoplasmic membrane, indicating a higher viscosity of the outer membrane. 4. Chloroform/methanol completely removed the phospholipids from the outer membrane, leaving the lipopolysaccharide moiety intact. The motion of spin-labeled fatty acids in the extracted membranes was, however, highly restricted, suggesting that, in the native outer membrane, the local environment of the probe is composed of phospholipids rather than lipopolysaccharide. Aqueous acetone extraction removed only 75-80% of the phospholipids of the outer membrane. Nevertheless, the mobility of the spin-labeled fatty acid remained highly restricted, suggesting the existence of two phospholipid environments in the outer membrane differing in the nature of their association with the lipopolysaccharide and protein moieties.     

Synthesis of the spin-labeled derivative of an ether-linked phospholipid possessing high antineoplastic activity.

We report here the complete synthesis of the spin-labeled derivative of an antitumor ether phospholipid, 1-O-octadecyl-2-O-(4'-doxylpentyl)-rac-glycerol-3-phosphocholine. This also represents the first time that the synthesis of a nitroxide spin-labeled diether phospholipid is described. In vitro experiments showed that at micromolar concentrations, this new analog is readily incorporated into the plasma membranes of human HL60 and mouse E8/AK.D1 leukemic cells, and subsequently kills the cells. The availability of this new probe should permit the electron spin resonance spectroscopic approach to investigate ways by which anti-tumor ether phospholipids selectively destroy the tumor cells.     

First synthesis of a fully active spin-labeled peptide hormone

For the first time in the electron spin resonance (ESR) and peptide synthesis fields, a fully active spin-labeled peptide hormone was reported. The ESR spectra of this alpha-melanocyte stimulating hormone (alpha-MSH) analogue (acetyl-Toac0-alpha-MSH) where Toac is the paramagnetic amino acid probe 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid, suggested a pH-independent conformation and a more restricted movement comparatively to the free Toac. Owing to its equivalent biological potency in a skin pigmentation assay as compared to the native alpha-MSH and its unique characteristic (paramagnetic, naturally fluorescent and fully active), this analogue is of great potential for investigation of relevant physiological roles reported for alpha-MSH.     

Location and aggregation of the spin-labeled peptide trichogin GA IV in a phospholipid membrane as revealed by pulsed EPR

The lipopeptaibol trichogin GA IV is a 10 amino acid-long residue and alpha-aminoisobutyric acid-rich antibiotic peptide of fungal origin. TOAC (2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) spin-labeled analogs of this membrane active peptide were investigated in hydrated bilayers of dipalmitoylphosphatidylcholine by electron spin echo envelope modulation (ESEEM) spectroscopy and pulsed electron-electron double resonance (PELDOR). Since, the ESEEM of the spin label appears to be strongly dependent on the presence of water molecules penetrated into the membrane, this phenomenon was used to study the location of this peptide in the membrane. This was achieved by comparing the ESEEM spectra for peptides labeled at different positions along the amino acid sequence with spectra known for lipids with spin labels at different positions along the hydrocarbon chain. To increase the ESEEM amplitude and to distinguish the hydrogen nuclei of water from lipid protons, membranes were hydrated with deuterated water. The PELDOR spectroscopy technique was chosen to study peptide aggregation and to determine the mutual distance distribution of the spin-labeled peptides in the membrane. The location of the peptide in the membrane and its aggregation state were found to be dependent on the peptide concentration. At a low peptide/lipid molar ratio (less than 1:100) the nonaggregated peptide chain of the trichogin molecules lie parallel to the membrane surface, with TOAC at the 4th residue located near the 9th-11th carbon positions of the sn-2 lipid chain. Increasing this ratio up to 1:20 leads to a change in peptide orientation, with the N-terminus of the peptide buried deeper into membrane. Under these conditions peptide aggregates are formed with a mean aggregate number of about N = 2. The aggregates are further characterized by a broad range of intermolecular distances (1.5-4 nm) between the labels at the N-terminal residues. The major population exhibits a distance of approximately 2.5 nm, which is of the same order as the length of the helical peptide. We suggest that the constituting monomers of the dimer are antiparallel oriented.     

Membrane assembly of the 16-kDa proteolipid channel from Nephrops norvegicus studied by relaxation enhancements in spin-label ESR

The 16-kDa proteolipid from the hepatopancreas of Nephrops norvegicus belongs to the class of channel proteins that includes the proton-translocation subunit of the vacuolar ATPases. The membranous 16-kDa protein from Nephrops was covalently spin-labeled on the unique cysteine Cys54, with a nitroxyl maleimide, or on the functionally essential glutamate Glu140, with a nitroxyl analogue of dicyclohexylcarbodiimide (DCCD). The intensities of the saturation transfer ESR spectra are a sensitive indicator of spin-spin interactions that were used to probe the intramembranous structure and assembly of the spin-labeled 16-kDa protein. Spin-lattice relaxation enhancements by aqueous Ni(2+) ions revealed that the spin label on Glu140 is located deeper within the membrane (around C9-C10 of the lipid chains) than is that on Cys54 (located around C5-C6). In double labeling experiments, alleviation of saturation by spin-spin interactions with spin-labeled lipids indicates that spin labels both on Cys54 and on Glu140 are at least partially exposed to the lipid chains. The decrease in saturation transfer ESR intensity observed with increasing spin-labeling level is evidence of oligomeric assembly of the 16-kDa monomers and is consistent with a protein hexamer. These results determine the locations and orientations of transmembrane segments 2 and 4 of the 16-kDa putative 4-helix bundle and put constraints on molecular models for the hexameric assembly in the membrane. In particular, the crucial DCCD-binding site that is essential for proton translocation appears to contact lipid.     

Lipid requirement for cell cycling : The effect of selective inhibition of lipid synthesis

Tissue culture cells require lipid which must be provided exogenously or synthesized via endogenous pathways. The exogenous supplies can be largely removed by growing cells in medium containing delipidized serum. Pathways for synthesis of lipid can then be blocked at three steps: (1) fatty acids by removal of biotin, an essential coenzyme; (2) phosphatidylcholine and sphingomyelin by deleting choline from the growth medium; and (3) cholesterol by inhibiting HMG-CoA reductase with 25-hydroxycholesterol. Sustained proliferation is prevented when lipid synthesis is blocked at any one of these steps. Cell proliferation resumes upon restoring synthesis with biotin, choline, or mevalonate (the product of the HMG-CoA reductase reaction) or by providing the lipid end products oleic acid or cholesterol. Using a combined cytophotometric-autoradiographic analysis to determine cell cycle distributions we have demonstrated that prereplicative (G1) cell cycle arrests develop in parallel with the proliferative inhibition. Each of the G1 arrests can be reversed by restoring the synthetic pathways or their lipid products. These observations suggest a causal relationship between the supply of lipids and passage through G1.     

Membrane lysis by the antibacterial peptides cecropins B1 and B3: A spin-label electron spin resonance study on phospholipid bilayers

Custom antibacterial peptides, cecropins B1 (CB1) and B3 (CB3), were synthesized. These peptides have particular sequence characteristics, with CB1 having two amphipathic alpha-helical segments and CB3 having two hydrophobic alpha-helical segments. These differences were exploited for a study of their efficacy in breaking up liposomes, which had different combinations of phosphatidic acid (PA) and phosphatidylcholine (PC), and a study of their lipid binding ability. Binding and nonbinding lysis actions of CB1 and CB3 on liposomes were examined further by electron spin resonance (ESR). The spin-labeled lipids 5'SL-PC, 7'SL-PC, 10'SL-PC, 12'SL-PC, and 16'SL-PC were used as probes. The ESR spectra revealed larger outer hyperfine splittings (2A(max)) for CB1 when the interactions of CB1 and CB3 with liposomes were compared. These observations indicate a larger restriction of the motion of the spin-labeled chains in the presence of CB1. Plots of the effective order parameter at the various probe positions (chain flexibility gradient) versus the peptide-lipid ratio further suggested that the lysis action of CB1 is related to its capacity to bind to the lipid bilayers. In contrast, there is no evidence of binding for CB3. To augment these findings, four spin-labeled peptides, C8SL-CB1, C32SL-CB1, C5SL-CB3, and C30SL-CB3, were also examined for their binding to and their state of aggregation within the lipid bilayers. Association isotherms of the peptides were measured for liposomes containing two molar fractions of PA (0.25 and 0.75). The membrane binding of the CB1 peptides exhibited a cooperative behavior, whereas the association isotherm of CB3 revealed binding to the lipid only for beta = 0.75 liposomes. To further identify the location of CB1 in the lipid bilayers, measurements of the collision rate with chromium oxalate in solution were conducted. Results from ESR power saturation measurements suggested that the NH(2)-terminal alpha-helix of CB1 is located on the surface of the lipid bilayers, whereas the COOH-terminal alpha-helix of CB1 is embedded below the surface of the lipid bilayers. These conclusions were further supported by the observed relationship between the partition distribution of peptides bound to liposomes at different PA/PC ratios and the amounts of free peptides. Based on the above observations, possible mechanisms of the bilayer lysis induced by CB1 and CB3 on liposomes of different composition are discussed.     

TOAC spin-labeled peptides tailored for DNP-NMR studies in lipid membrane environments

The benefit of combining in-cell solid-state dynamic nuclear polarization (DNP) NMR and cryogenic temperatures is providing sufficient signal/noise and preservation of bacterial integrity via cryoprotection to enable in situ biophysical studies of antimicrobial peptides. The radical source required for DNP was delivered into cells by adding a nitroxide-tagged peptide based on the antimicrobial peptide maculatin 1.1 (Mac1). In this study, the structure, localization, and signal enhancement properties of a single (T-MacW) and double (T-T-MacW) TOAC (2,2,6,6-tetramethylpiperidine-N-oxyl-4-amino-4-carboxylic acid) spin-labeled Mac1 analogs were determined within micelles or lipid vesicles. The solution NMR and circular dichroism results showed that the spin-labeled peptides adopted helical structures in contact with micelles. The peptides behaved as an isolated radical source in the presence of multilamellar vesicles, and the electron paramagnetic resonance (EPR) electron-electron distance for the doubly spin-labeled peptide was ∼1 nm. The strongest paramagnetic relaxation enhancement (PRE) was observed for the lipid NMR signals near the glycerol-carbonyl backbone and was stronger for the doubly spin-labeled peptide. Molecular dynamics simulation of the T-T-MacW radical source in phospholipid bilayers supported the EPR and PRE observations while providing further structural insights. Overall, the T-T-MacW peptide achieved better ¹³C and ¹⁵N signal NMR enhancements and ¹H spin-lattice T₁ relaxation than T-MacW.     

Rapid transbilayer movement of spin-labeled steroids in human erythrocytes and in liposomes

The transbilayer movement and distribution of spin-labeled analogs of the steroids androstane (SLA) and cholestane (SLC) were investigated in the human erythrocyte and in liposomes. Membranes were labeled with SLA or SLC, and the analogs in the outer leaflet were selectively reduced at 4C using 6-O-phenylascorbic acid. As shown previously, 6-O-phenylascorbic acid reduces rapidly nitroxides exposed on the outer leaflet, but its permeation of membranes is comparatively slow and thus does not interfere with the assay. From the reduction kinetics, we infer that transbilayer movement of SLA in erythrocytes is rapid at 4C with a half-time of approximately 4.3 min and that the probe distributes almost symmetrically between both halves of the plasma membrane. We have no indication that a protein-mediated transport is involved in the rapid transbilayer movement of SLA because 1) pretreatment of erythrocytes with N-ethyl maleimide affected neither flip-flop nor transbilayer distribution of SLA and 2) flip-flop of SLA was also rapid in pure lipid membranes. The transbilayer dynamics of SLC in erythrocyte membranes could not be resolved by our assay. Thus, the rate of SLC flip-flop must be on the order of, or even faster than, that of probe reduction rate on the exoplasmic leaflet (half-time approximately 0.5 min). The results are discussed with regard to the transbilayer dynamics of cholesterol.     

Fluorescence quenching in model membranes An analysis of the local phospholipid environments of diphenylhexatriene and gramicidin A′

Interactions between the fluorophors diphenylhexatriene or gramicidin A′ and lipids are examined using a spin-labeled phosphatidylcholine as a fluorescence quenching probe. It is found that in phospholipid vesicles of mixed lipid composition at temperatures where phospholipids are completely in the liquid crystal phase, several different species of phosphatidylcholines are randomly distributed around the fluorophors. In vesicles of mixed lipid composition which can undergo thermally induced phase separations, the fluorescence quenching observed at lower temperatures reflects a non-random distribution of lipids around each fluorophor. This observation is explained in terms of the partition of fluorophor between a spin-labeled lipid-rich liquid crystal phase, and a spin-labeled lipiddepleted gel phase. Gramicidin A′ strongly favors partition into the liquid crystal phase, whereas diphenylhexatriene partitions about equally between the two lipid phases. A method is described utilizing fluorescence quenching for the calculation of the partition coefficient for a fluorophor. The partition coefficients so calculated are shown to be in good agreement with previously reported values derived from other methods. It is also shown that Ca²⁺-induced lipid phase separations can be monitored by fluorescence quenching.     

Electron spin resonance and steady-state fluorescence polarization studies of lipid bilayers containing integral proteins

We derive equations that describe changes in the steady-state fluorescence polarization of the probe 1,6-diphenyl-1,3,5-hexatriene (DPH) or in the spectrum of electron spin resonance (ESR) nitroxide spin-labeled lipid probes as a function of the intrinsic molecule concentration in lipid bilayer membranes. We make use of an assumption used by us in an earlier paper. The equations are independent of any membrane model. They are valid when a DPH probe or a spin-labeled chain is equivalent to an unlabeled lipid hydrocarbon chain only as far as their general space-filling properties are concerned. We consider cases where the bilayer is either in a single homogeneous phase or in a two-phase region. We apply our equations to analyze ESR data from delipidated sarcoplasmic reticulum membranes and from egg yolk phosphatidylcholine bilayers containing Ca2+-ATPase, and DPH data from dipalmitoylphosphatidylcholine (DPPC) bilayers containing Ca2+-ATPase, both for T greater than Tc. The following conclusions were derived: (i) Ca2+-ATPase oligomers are "randomly" distributed, for the concentrations studied, in the fluid phase. (ii) There is no fixed stoichiometric ratio of "boundary" lipids and oligomers. (iii) Between 24k and 28k lipid molecules are able to surround each isolated oligomer composed of k Ca2+-ATPase monomers. Finally, we apply our equations to analyze DPH studies on DPPC bilayers containing Ca2+-ATPase for T less than Tc. We find that the results reported are in accord with the predictions of the model. In the Appendix, we show that an analytical expression for probabilities used by us is in very good agreement with the results of computer simulation.     

Stearoyl paratoluenesulfonate. A powerful acylating agent for lipid synthesis

The utility of the mixed carboxylic-sulfonic acid anhydride stearoyl p-toluenesulfonate as a powerful, mild acylating agent for lipid synthesis is shown by the synthesis of rac 1,2-distearoyl-3-iodopropane, lecithin and a spin-labeled choline derivative from the corresponding alcohols. The method constitutes a significant improvement of earlier acylating methods.