Improved method for the synthesis of 1- or 3-acyl-sn-glycerols

Optically active 1- or 3-acyl-sn-glycerols were synthesized from 2,3- or 1,2-isopropylidene-sn-glycerols, respectively. The 2,3- or 1,2-isopropylidene-sn-glycerols were condensed with appropriate long saturated or unsaturated fatty acids and the resulting acyl isopropylidene compounds were treated with dimethylboronbromide at - 50 degrees C to give the title compounds. The ketal cleavage of acyl isopropylidene-sn-glycerols by dimethylboronbromide to produce the long 1- or 3-acyl-sn-glycerols was effective and gave good yields (70-90%). The reaction conditions were mild and there was no acyl migration, as shown by optical rotation of the monoacyl-sn-glycerols. The synthesis of 2,3-isopropylidene-sn-glycerol was improved to give an overall yield of 40% from L-arabinose. L-Arabinose was first converted to its 1,1'-diethylmercapto derivative and then condensed with 2-methoxypropene to yield 1,1'-diethyl-mercapto-4,5-isopropylidene-L-arabinose. Oxidation of this compound with sodium periodate followed by reduction with sodium borohydride under alkaline conditions yielded 2,3-isopropylidene-sn-glycerol [alpha]22D = -14.90 degrees, neat (Lit. 8 [alpha]22D = -14.5 degrees, neat; 14 [alpha]25D = -10.8 degrees; methanol C, 16.9). The optical purity of isopropylidene-sn-glycerols was determined as benzoyl derivatives on a high performance liquid chromatographic column packed with a chiral stationary phase.     

Enzymic synthesis of 1-alkyl-2-acyl-sn-glycero-3-phosphorylethanolamines by the CDP-ethanolamine: 1-radyl-2-acyl-sn-glycerol ethanolaminephosphotransferase from microsomal fraction of rat brain

The incorporation of radioactivity from cytidine-5'-phosphate-[(32)P]phosphorylethanolamine into 1-alkyl-2-acyl-sn-glycero-3-phosphorylethanolamines and 1,2-diacyl-sn-glycero-3-phosphorylethanolamines was stimulated more than fourfold by 1-alkyl-2-acyl-sn-glycerols and 1,2-diacyl-sn-glycerols, respectively, with an ethanolaminephosphotransferase (EC 2.7.8.1) present in the microsomal fraction from brains of mature rats. The K(m) values, 0.28 mm for CDP-ethanolamine and 1.9 mm for 1-alkyl-2-acyl-sn-glycerols, were similar to those obtained by other investigators with other 1-radyl-2-acyl-sn-glycerols. The formation of 1,2-diacyl-sn-glycero-3-phosphorylethanolamines from endogenous 1,2-diacyl-sn-glycerols was inhibited by 1-alkyl-2-acyl-sn-glycerols. These properties indicate that the ethanolaminephosphotransferase lacks specificity for the type of group at the 1-position of the lipid substrate. The synthesis of 1-alkyl-2-acyl-sn-glycero-3-phosphorylethanolamines from 1-alkyl-2-acyl-sn-glycerols and CDP-ethanolamine by an enzyme from rat brain supports the inclusion of this reaction in the metabolic pathway for the synthesis of 1-alk-1'-enyl-2-acyl-sn-glycero-3-phosphorylethanolamines.     

Synthesis of ethanolamine phosphoglycerides by human platelets.

Platelet homogenates contain an ethanolaminephosphotransferase (EC 2.7.8.1) that catalyzes the synthesis of ethanolamine phosphoglycerides from cytidine-5'-diphosphate ethanolamine and 1-radyl-2-acyl-sn-glycerols. The enzyme is particulate-bound and requires Mn2+ and bile salts for optimal activity. The apparent Km of the enzyme for cytidine-5'-diphosphate ethanolamine is 1.6 X 10(-5) M when the concentration of 1,2-diacyl-sn-glycerols is 8.8 X 10(-4) M. The pH optimum is 8.5 in Tris-HCl or glycine-NaOH buffer. The activity of the enzyme in platelets from normal subjects is 0.24-0.34 nmole/min/mg of protein.     

Versatile methods for the synthesis of mixed-acid 1,2-diacylglycerols

Two methods for synthesizing mixed-acid 1,2-diacylglycerols starting from 2,3-epoxy-1-propanol (glycidol) or 3-chloro-1,2-propanediol have been described. This first method involves fatty acid addition to a protected glycidol derivative and solid-state isomerization. The second approach exploits the specificity of the trityl group for primary alcohols and the nucleophilic replacement of chlorine by a carboxylate ion in an aprotic solvent. The second method proves to be more general: with 3-chloro-1-O-trityl-1,2-propanediol as an intermediate compound apparently all types of mixed-acid, saturated and unsaturated, chiral and racemic 1,2-diacylglycerols can be prepared in good yields. In the first method tritylglycidol is a good starting compound. The use of this method, however, is restricted because only the 2-position of glycerol can be occupied with an unsaturated fatty acid. For de-blocking protected 1,2-diacylglycerols, the trityl group and other protecting groups were exchanged for the trifluoroacetyl group, which group could then be removed without any detectable acyl migration (< 1%). To this end, the 1,2-diacyl-3-trifluoroacetyl-glycerols were dissolved at room temperature in methanol containing pyridine, whereby the trifluoroacetyl group was split off, giving the 1,2-diacylglycerol.     

Regulation of selectivity of CDPcholine: 1,2-diacyl-sn-glycerol cholinephosphotransferase in rat liver microsomes towards different molecular species of 1,2-diacyl-sn-glycerols.

The suitability of monoenoic, dienoic, tetraenoic, and hexaenoic molecular species of 1,2-diacyl-sn-glycerols as substrates for the CDPcholine: 1,2-diacyl-sn-glycerol cholinephosphotransferase (EC 2.7.8.2) was studied in rat liver microsomes. No statistically significant difference in the rates of phosphatidylcholine synthesis with the various diacylglycerols was found at 0.40 mM, although a moderate discrimination against hexaenoic species relative to monoenoic and dienoic species was observed at 0.25 mM. The addition of palmitoyl-CoA (7.5 micron) significantly enhanced cholinephosphotransferase activity when tetraenoic diacylglycerols were added at 0.25 or 0.40 mM. CDPethanolamine at 24.4 micron was found to inhibit the rates of phophatidylcholine biosynthesis by 54 and 39% with hexaenoic and monoenoic 1,2-diacyl-sn-glycerols, respectively, whereas no significant effects were observed in the case of dienoic and tetraenoic species. These latter findings may partially explain why 1-saturated 2-docosahexaenoyl diacylglycerols are used to a greater extent for phosphatidylethanolamine than for phosphatidylcholine synthesis in rat liver in vivo. The present results also suggest that the selectivity of the cholinephosphotransferase for certain molecular species of 1,2-diacyl-sn-glycerols is a function of diacylglycerol concentration and may be mediated under physiological conditions by substrates for enzymes which compete for common diacylglycerol precursors.     

Selective utilization of endogenous unsaturated phosphatidylcholines and diacylglycerols by cholinephosphotransferase of mouse lung microsomes.

In the presence of CMP, cholinephosphotransferase of mouse lung microsomes catalyzes the conversion of endogenous phosphatidylcholines into 1,2-diacyl-sn-glycerols and CDPcholine. 2. In this conversion cholinephosphotransferase shows a distinct preference for those molecular species of phosphatidylcholine which contain an unsaturated fatty acid. The enzyme hardly utilizes endogenous depalmitoylglycerophosphocholine as a substrate. 3. Membrane-bound 1,2-diacyl-sn-glycerols were also prepared by treatment of mouse lung microsomes with a pure phospholipase C from Bacillus cereus. These 1,2-diacyl-sn-glycerols were subsequently utilized as substrate by cholinephosphotransferase in the formation of phosphatidylcholine. In the latter reaction, cholinephosphotransferase exhibited a pronounced preference for unsaturated 1,2-diacyl-sn-glycerols and hardly utilized the endogenous 1,2-depalmitoyl-sn-glycerol. 4. The low affinity of cholinephosphotransferase for either dipalmitoylglycerophosphocholine or 1,2-dip     

A new synthesis of bis(diacylglycero)phosphate

The chemical synthesis of bis(diacylglycero)phosphate previously named bisphosphatidic acid, starting with a diacylglycerol and phosphatidic acid, is described. The phosphodiester bond formation is catalyzed by triisopropylbenzenesulfonylchloride. This simple approach allows the preparation of saturated as well as unsaturated bis(diacylglycero)phosphate species in one step without the use of any protecting group. The methods used until now yield only mono-acid species, or mixed-acid unsaturated species after many steps involving the introduction and the removal of protecting groups. The synthetic products have been characterized by component analysis and NMR-techniques.     

Structure and polymorphism of saturated monoacid 1,2-diacyl-sn-glycerols

The 1,2-diacyl-sn-glycerols (1,2-DGs) are the predominant naturally occurring isomer found in cell membranes, lipid droplets, and lipoproteins. They are involved in the metabolism of monoacylglycerols, triacylglycerols, and phospholipids. The 1,2-DGs participate in the activation of protein kinase C, in phosphorylation of target proteins, and in transduction of extracellular signals into the cell. We have undertaken a study of the physical properties of a homologous series of synthetic optically active diacylglycerols. Stereospecific 1,2-diacyl-sn-glycerols were synthesized with saturated fatty acyl chains of 12, 16, 18, 22, and 24 carbons in length. Their polymorphic behavior was examined by differential scanning calorimetry and X-ray powder diffraction. The solvent-crystallized form for all the 1,2-DGs packs in the orthorhombic perpendicular subcell (beta') and melts with a single sharp endotherm to an isotropic liquid. On quenching, the C12, C16 and C18 compounds pack in a hexagonal subcell (alpha), whereas the C22 and C24 pack in a pseudohexagonal subcell (sub-alpha). The sub-alpha phase reversibly converts to the alpha phase. The long spacings of these compounds in both the alpha and beta' phases increase with chain length. In the alpha and beta' phases, the acyl chain tilts were found to be 90 degrees and 62 degrees from the basal methyl plane. The polymorphic behavior of 1,2-diacyl-sn-glycerol is quite different from that of the corresponding monoacid saturated 1,3-diacylglycerols which form two beta phases with triclinic parallel subcells.     

Radioassay of the stereospecificity of 2-monoacylglycerol acyltransferase

The 2-monoacylglycerol acyltransferase (EC 2.3.1.22, acylglycerol palmitoyl transferase) catalyzes the synthesis of X-1,2-diacylglycerols from 2-monoacylglycerol and acyl CoA with an apparently variable stereochemical specificity. A microassay for determining the ratio of sn-1,2- and sn-2,3-diacylglycerols formed by the acylation of radioactive 2-monoacylglycerol in intact cells or in cell-free systems in the presence of free fatty acids and cofactors has been developed. The diacylglycerols are isolated by thin-layer chromatography using nonradioactive racemic diacylglycerols as carriers. The enantiomer content is determined following a chemical synthesis of X-1,2-diacylphosphatidylcholines and a stereospecific stepwise release of the sn-1,2- and sn-2,3-diacylglycerols by phospholipase C. By using thin-layer chromatography for the isolation of the hydrolysis products, known samples ranging in enantiomer ratios from 0.05 to 20 and containing 5000 to 200,000 cpm can be assayed to within 1% of the major and within 10% of the minor enatiomer content. The method is applicable to the determination of the enantiomer content of X-1,2-diacylglycerols generated via other acyltransferases and via lipolysis of triacylglycerols and diacylglycerolphospholipids in other biological systems.     

Incorporation of (2-3H)glycerol into rat brain 1,2-diacyl-sn-glycero-3-phosphorylcholine and 1,2-diacyl-sn-glycerol molecular species in vivo

Rat brain 1,2-diacyl-sn-glycerols (diglycerides) and 1,2-diacyl-sn-glycerols obtained from 1,2-diacyl-sn-glycero-3-phosphorylcholine after treatment with phospholipase C differ markedly in carbon number distribution. 70% of the 1,2-diacyl-sn-glycerols had a total of 38 fatty acid carbon atoms, and there was no detectable change in the 1,2-diacyl-sn-glycerol mass pattern between 7 and 23 days of age. In contrast, 1,2-diacyl-sn-glycero-3-phosphorylcholine contained at most 10% of this molecular species in the brains of rats of comparable age. A small increase in the C(36) species of 1,2-diacyl-sn-glycero-3-phosphorylcholine, which is associated with myelination, was noted between 10 and 17 days. The incorporation of intracranially injected [2-(3)H]glycerol into 1,2-diacyl-sn-glycero-3-phosphoryl-choline species with polyunsaturated fatty acids containing 20 or 22 carbon atoms was greater than into the species containing only saturated and/or monoenoic fatty acids between 30 min and 24 hr. The 1,2-diacyl-sn-glycerol fractions containing polyunsaturated fatty acids had the lowest specific activity at 30 min. The specific activity of the particular 1,2-diacyl-sn-glycerol fraction containing the stearate-arachidonate pair is the lowest for 4 hr after intracranial injection of the isotope. Thus, molecular species of 1,2-diacyl-sn-glycerol and 1,2-diacyl-sn-glycero-3-phosphorylcholine differed considerably in their labeling patterns, and a direct precursor-product relationship could not be demonstrated during the time period studied.     

The acylation of lipophilic alcohols by lysosomal phospholipase A2

A novel lysosomal phospholipase A(2) (LPLA2) with specificity toward phosphatidylethanolamine and phosphatidylcholine was previously purified and cloned. LPLA2 transfers sn-1 or sn-2 acyl groups of phospholipids to the C1 hydroxyl of the short-chain ceramide N-acetylsphingosine (NAS) under acidic conditions. The common features of lipophilic alcohols serving as acceptor molecules in the transacylase reaction were examined. 1-O-Hexadecyl-2-acetyl-sn-glycerol (HAG) was acylated by LPLA2 similar to NAS. HAG competed with NAS and inhibited NAS acylation. The transacylation of 1-O-hexadecyl-glycerol (HG), 1-O-palmityl-2-O-methyl-sn-glycerol (PMG), and monoacylglycerols was also investigated. HG, PMG, 1- or 3-palmitoyl-sn-glycerol, and 2-palmitoylglycerol were converted to 1,3-alkylacylglycerol, 1,2-dialkyl-3-acylglycerol, 1,3-diacylglycerol, and 1,2- or 2,3-diacylglycerol, respectively. HG and monoacylglycerol inhibited the acylation of NAS by the enzyme with IC(50) values of 35 and 45 microM, respectively. Additionally, the enzyme acylated glycerol to produce 1- or 3-acyl-sn-glycerol but not 2-acylglycerol. Therefore, the preferred acceptor molecules for LPLA2 are primary alcohols with one long carbon chain and one small nonpolar residue linked to the C2 position of ethanol. The enzyme acylated other natural lipophilic alcohols, including anandamide and oleoylethanolamide. Thus, LPLA2 may function to remodel acyl groups and modulate the biological and pharmacological activities of some lipophilic alcohols.     

Relative acylglycerol acyltransferase activities in homogenates of enzymically dispersed rat jejunal villus and crypt cells

The relative acyltransferase activities were compared in homogenates of rat jejunal villus and crypt cells isolated by differential scraping and hyaluronidase dispersion. The contributions of the monoacylglycerol and phosphatidic acid pathways to the higher acylglycerol and phospholipid biosynthesis were assessed using 2-oleoyl-sn-[3H] glycerol and [I-14C] palmitic acid as tracers. The stereochemical course of the diacylglycerol biosynthesis was determined by stereospecific analysis. Using 2-oleoyl-sn-glycerol as a tracer, the villus cells exhibited for times higher diacylglycerol and 19 times higher triacylglycerol biosynthesis than crypt cells on an equivalent protein basis. Furthermore, while the villus cell homogenates yielded a preponderance (75%) of the 1, 2-diacyl-sn-glycerols, the crypt cell homogenates formed essentially racemic proportions of 1, 2- and 2,3-diacyl-sn-glycerols. Both villus and crypt cell homogenates exhibited comparable acyl acceptor and acyl donor concentration dependence and the same cofactor requirements. It is unlikely that these acyltransferase activities in the crypt cell preparation are due to contamination with the villus cells, because then more comparable proportions of the enantiomeric diacylglycerols and triacylglycerols would have been anticipated. It is concluded that the crypt cells possess intrinsic monoacylglycerol and to a much lesser extent diacylglycerol acyltransferase activities, which are acquired prior to the development of a distinct brush border and which probably do not require dietary stimulus for induction.     

Determination of molecular species of enantiomeric diacylglycerols by chiral phase high performance liquid chromatography and polar capillary gas-liquid chromatography

A simple method is described for the determination of molecular species of enantiomeric sn-1,2- and sn-2,3-diacylglycerols derived from natural triacylglycerols by Grignard degradation. The method is based on a preparative separation of the enantiomeric diacylglycerols as 3,5-dinitrophenylurethane (DNPU) derivatives by high performance liquid chromatography (HPLC) on a chiral column (25 cm x 4.6 mm ID) containing R-(+)-1-(1-naphthyl)ethylamine as a stationary phase. This is followed by polar capillary gas-liquid chromatography (GLC) of the trimethylsilyl (TMS) ether derivatives of the enantiomeric diacylglycerols derived from the DNPU derivatives using trichlorosilane, which does not cause acyl migration and racemization during the reaction. The cleavage is better than 94% complete. The method was standardized with synthetic sn-1,2- and sn-2,3-dipalmitoyl- and rac-1,2-dioleoylglycerols and was applied to the identification and quantitation of individual molecular species of enantiomeric diacylglycerols generated by Grignard degradation of the triacylglycerols from corn oil, cocoa butter, and lard.     

Synthesis of 5,9-hexacosadienoic acid phospholipids. 11. Phospholipid studies of marine organisms

The synthesis of phosphatidylcholines (PC), phosphatidylethanolamines (PE) and phosphatidylserines (PS) containing two acyl chains of the naturally occurring sponge fatty acid (5Z,9Z)-5,9-hexacosadienoic acid as well as its hitherto unknown geometrical isomers is described. The PCs were prepared by deacylation of natural lecithins, followed by reacylation with fatty acid anhydrides. The synthesis of mixed-acid PCs is also reported: a diacyl product was converted to the lyso-PC by treatment with phospholipase A2 and subsequent acylation of the secondary hydroxyl group to give the desired mixed-acid PCs. The PEs and the PSs were prepared from the corresponding PCs by enzymatic transphosphatidylation catalyzed by phospholipase D. Structural assignments of the compounds were confirmed by spectroscopy (1H-NMR and MS). Ammonia chemical ionization mass spectrometry provided molecular ion and significant fragment peaks for PCs and PEs.     

Enzymic synthesis of ether types of choline and ethanolamine phosphoglycerides by microsomal fractions from rat brain and liver.

The formation of product by ethanolamine phosphotransferases (EC 2.7.8.1) and cholinephosphotransferases (EC 2.7.8.2) in microsomal fractions from brains and livers of mature rats is increased several fold by 1,2-diacyl-sn-glycerols. With the addition of 1-alkyl-2-acyl-sn-glycerols, we have found an 11-fold increase with brain microsomes and a 20-fold increase with lvier microsomes in the synthesis of choline ether lipids (1-alkyl-2-acyl- and 1-alk-1'-enyl-2-acyl-sn-glycero-3-phosphorylcholines). For the synthesis of ethanolamine ether lipids (1-alkyl-2-acyl and 1-alk-1'-enyl-2-acyl-sn-glycero-3-phosphorylethanolamines), the stimulation of alkylacylglycerols was 7-fold for brain microsomes and 18-fold for liver microsomes. The alkylacyl glycerols (8 mM) also inhibited the synthesis of diacyl phosphoglycerides by 44 to 65%, indicating that the same ethanolaminephosphotransferases and cholinephosphotransferases are utilized for the synthesis of alkylacyl phosphoglycerides and diacyl phosphoglycerides. A desaturation of the alkyl groups may take place in the same reaction mixture. The rate of incorporation of phosphorylcholine into alkenylacyl glycerophosphorylcholines (choline plasmalogens) with alkylacylglycerols, cytidine diphosphate choline, and liver microsomes was 15 nmoles per mg protein per hour. The in vitro synthesis of choline plasmalogens with alkylacylglycerols had not been observed previously. The corresponding rate of incorporation of phosphorylethanolamine into ethanolamine plasmalogens was 10 nmoles per mg protein per hour, a value greater than any of the previously reported values for ethanolamine plasmalogen formation from alkylacyl glycerophosphorylethanolamines.     

Mixed-chain phosphatidylcholine analogues modified in the choline moiety: preparation of isomerically pure phospholipids with bulky head groups and one acyl chain twice as long as the other

Diacylphosphatidylcholines were synthesized with widely different acyl chain lengths and bulky head groups. Lysophosphatidylcholine was acylated at room temperature within 6 h with a 10-fold molar excess of fatty acid anhydride in dry, alcohol-free chloroform in the presence of 1.2 equivalents of 4-pyrrolidinopyridine as a catalyst, affording the mixed-acid phosphatidylcholines with widely different chain lengths in more than 90% yield and with less than 1% acyl migration. The syntheses of isomerically pure 1-stearoyl-2-decanoyl- and 1-stearoyl-2-undecenoyl(delta 10)-sn-glycero-3-phosphocholines C(18:0)/C(10:0)-PC and C(18:0)/C(11:1 delta 10)-PC, respectively), followed by conversion to various head-group analogues, are illustrated here. The transition peak widths at half-height of the endotherms obtained by differential scanning calorimetry are consistent with very high isomeric purity. Phospholipase D from Streptomyces chromofuscus was used as a catalyst in the hydrolysis of C(18:0)/C(10:0-PC to give the corresponding phosphatidic acid in quantitative yield. The latter compound was condensed with 10 molar equivalents of various N,N,N-trialkylammonium alkanols (as their p-toluenesulfonate or tetraphenylborate salt) in the presence of trichloroacetonitrile in dry pyridine under nitrogen atmosphere to yield the C(18:0)/C(10:0) phospholipids bearing modified head groups, which were purified by flash chromatography.     

Stereospecific analysis of triacylglycerols via racemic phosphatidylcholines and phospholipase C.

A method of simultaneous determination of stereospecific distribution and molecular association of acyl groups in triacylglycerols has been developed. The analysis is based on a random generation of rac-1,2-diacylglycerols by Grignard degradation, synthesis of rac-phosphatidylcholines, and a stereospecific stepwise release of 1,2-sn- and 2,3-sn-diacylglycerols by phospholipase C. The exact structure of the original triacylglycerols is reconstituted on the basis of complete analysis of the molecular species of the 1,2-sn- and 2,3-sn-diacylglycerols as the tertiary-butyldimethylsilyl ethers by gas chromatography with mass spectrometry. The validity of the method is demonstrated by analyses of synthetic triacylglycerols of known structure. A practical application is illustrated by determination of the fatty acid distribution in lard.     

The mechanism-based inactivation of 2,3-dihydroxybiphenyl 1,2-dioxygenase by catecholic substrates.

2,3-Dihydroxybiphenyl 1,2-dioxygenase (EC ), the extradiol dioxygenase of the biphenyl biodegradation pathway, is subject to inactivation during the steady-state cleavage of catechols. Detailed analysis revealed that this inactivation was similar to the O(2)-dependent inactivation of the enzyme in the absence of catecholic substrate, resulting in oxidation of the active site Fe(II) to Fe(III). Interestingly, the catecholic substrate not only increased the reactivity of the enzyme with O(2) to promote ring cleavage but also increased the rate of O(2)-dependent inactivation. Thus, in air-saturated buffer, the apparent rate constant of inactivation of the free enzyme was (0.7 +/- 0.1) x 10(-3) s(-1) versus (3.7 +/- 0.4) x 10(-3) s(-1) for 2,3-dihydroxybiphenyl, the preferred catecholic substrate of the enzyme, and (501 +/- 19) x 10(-3) s(-1) for 3-chlorocatechol, a potent inactivator of 2,3-dihydroxybiphenyl 1,2-dioxygenase (partition coefficient = 8 +/- 2, K(m)(app) = 4.8 +/- 0.7 microm). The 2,3-dihydroxybiphenyl 1,2-dioxygenase-catalyzed cleavage of 3-chlorocatechol yielded predominantly 2-pyrone-6-carboxylic acid and 2-hydroxymuconic acid, consistent with the transient formation of an acyl chloride. However, the enzyme was not covalently modified by this acyl chloride in vitro or in vivo. The study suggests a general mechanism for the inactivation of extradiol dioxygenases during catalytic turnover involving the dissociation of superoxide from the enzyme-catecholic-dioxygen ternary complex and is consistent with the catalytic mechanism.     

Synthesis and polymorphism of 3-acyl-sn-glycerols

3-Acyl-sn-glycerols with even-numbered saturated fatty acyl chains from decanoate to lignocerate were synthesized. Successful hydrolysis of the long acyl chain intermediate 1,2-isopropylidene-3-acyl-sn-glycerols from stearate to lignocerate was accomplished by applying the compounds to silica gel and exposing them to hydrogen chloride gas at -75 degrees C. The purity of the compounds was checked by boric acid impregnated thin-layer chromatography, 13C NMR, and reverse-phase high-pressure liquid chromatography. Differential scanning calorimetry and X-ray diffraction techniques were used to study the polymorphism of the compounds. In the beta phase obtained from solvent of crystallization, the acyl chain packing was in a two-dimensional oblique lattice with specific chain-chain interactions with a tilt angle of 55.4 degrees from the bilayer plane. The thickness of the region containing two glycerol head groups was 12.7 A. The phase transition enthalpy of melting for the beta phase was 1.06 kcal/mol of CH2. On being cooled these compounds crystallized reversibly to an unstable alpha phase, which on being further cooled underwent a second crystallization to a beta or beta' phase. The thermodynamic parameters and long spacings of these compounds in both beta and alpha phases were linear, indicating isostructural packing in each phase. The enthalpy of the melting transition of the alpha phase was 0.69 kcal/mol of CH2. In this phase, the chains were packed in a hexagonal lattice with nonspecific chain-chain interactions. The thickness of the head-group region (12.2 A) and the tilt angle (55 degrees) of the acyl chains in the alpha phase were very similar to those in the beta phase.     

The biosynthesis of gram-negative endotoxin. Formation of lipid A precursors from UDP-GlcNAc in extracts of Escherichia coli

The Gram-negative bacterium Escherichia coli has previously been shown to utilize two unique glucosamine (GlcN)-derived phospholipids in the biosynthesis of lipid A disaccharides (Bulawa, C.E., and Raetz, C. R.H. (1984) J. Biol. Chem. 259, 4846-4851; Ray, B. L., Painter, G.L., and Raetz, C.R.H. (1984) J. Biol. Chem. 259, 4852-4859. We now present evidence that these compounds, UDP-2,3-diacyl-GlcN and 2,3-diacyl-GlcN-1-phosphate (2,3-diacyl-GlcN-1-P), are generated in extracts of E. coli by fatty acylation of UDP-GlcNAc. The initial reaction is an O-acylation of the glucosamine ring, presumably of the 3-OH group, with (R)-beta-hydroxymyristate, followed by removal of the acetyl moiety, and further fatty acylation of the N atom with (R)-beta-hydroxymyristate to yield UDP-2,3-diacyl-GlcN. Hydrolysis of the pyrophosphate bridge in this molecule gives 2,3-diacyl-GlcN-1-P + UMP. In vivo pulse labeling with 32Pi supports this postulated pathway, since UDP-2,3-diacyl-GlcN is labeled prior to 2,3-diacyl-GlcN-1-P. UDP-glucosamine is inactive as a substrate in the initial acylation reaction. These acylations show an absolute specificity for fatty acyl moieties activated with acyl carrier protein. No reaction is detected with fatty acyl-CoA or free fatty acid. The fatty acylation of sugar nucleotides has not been reported previously in E. coli or any other organism.