Two-stage Enzymatic Hydrolysis of Soybean Concentrated Phospholipid to Prepare Glycerylphosphorylcholine: Optimized by Response Surface Methodology.

A two-stage enzymatic hydrolysis method, in which phospholipase A1 (PLA1) was added after phospholipase A2 (PLA2) was added for a certain time, was successfully carried out to prepare glycerylphosphorylcholine (GPC) from soybean concentrated phospholipid. Effects of reaction variables on hydrolysis reaction were optimized using response surface methodology, and the optimal conditions were as follows: PLA2 load of 1.25%, PLA1 load of 0.70%, substrate concentration of 13%, reaction temperature of 41°C, and stirring rate of 680 rpm. Under the optimal conditions, the GPC yield reached 83.07%, which is close to the predicted value by the fitted model. This paper not only provides an efficient and low-cost method to prepare GPC, but also improves the high-value utilization of soybean concentrated phospholipid.

Asymmetric Total Synthesis Enables Discovery of Antibacterial Activity of Siladenoserinols A and H.

Siladenoserinols A and H were found to show moderate inhibitory activity toward p53-Hdm2 interactions. Our total synthesis allowed us to further examine their bioactivities, which revealed that (i) siladenoserinols A and H were not cytotoxic against cancer cell lines and (ii) siladenoserinol A and its desulfamate analogue exhibited significant antibacterial activity against Gram-positive bacteria including MRSA. Our studies demonstrate that siladenoserinols are a promising new class of bactericidal Gram-positive antibiotics without hemolytic activity.

Total Synthesis and Biological Evaluation of Siladenoserinol†A and its Analogues.

The total synthesis of siladenoserinol A, an inhibitor of the p53-Hdm2 interaction, has been achieved. AuCl3 -catalyzed hydroalkoxylation of an alkynoate derivative smoothly and regioselectively proceeded to afford a bicycloketal in excellent yield. A glycerophosphocholine moiety was successfully introduced through the Horner-Wadsworth-Emmons reaction using an originally developed phosphonoacetate derivative. Finally, removal of the acid-labile protecting groups, followed by regioselective sulfamate formation of the serinol moiety afforded the desired siladenoserinol A, and benzoyl and desulfamated analogues were also successfully synthesized. Biological evaluation showed that the sulfamate is essential for biological activity, and modification of the acyl group on the bicycloketal can improve the inhibitory activity against the p53-Hdm2 interaction.

Synthesis, characterization and inclusion into liposomes of a new cationic pyrenyl amphiphile.

The aggregation properties of a new cationic fluorescent amphiphile tagged on the hydrophobic tail with a pyrene moiety and bearing two hydroxyethyl functionalities on the polar headgroup were investigated by fluorescence experiments as pure components or in mixed liposomes containing an unsaturated phospholipid, 1,2-dioleoyl-sn-glycero-3-phosphocholine, at different molar ratios. The obtained results put in evidence that the conformation and the miscibility of the lipids in the aggregates strongly influence the excimer/monomer ratio. Mixed monolayers at the same composition were investigated by Langmuir compression isotherms to deepen the understanding of lipid organization and miscibility, both in the polar and in the hydrophobic regions. The presence of two hydroxyethyl functionalities on the polar headgroup of the newly synthesized amphiphile exerts a shielding effect of the charge of the amphiphile increasing the compressibility of lipid components in contrast with the disturbing effect of the unsaturated acyl chains of the phospholipid.

Synthesis of deuterated [D32 ]oleic acid and its phospholipid derivative [D64 ]dioleoyl-sn-glycero-3-phosphocholine.

Oleic acid and its phospholipid derivatives are fundamental to the structure and function of cellular membranes. As a result, there has been increasing interest in the availability of their deuterated forms for many nuclear magnetic resonance, infrared, mass spectroscopy and neutron scattering studies. Here, we present for the first time a straightforward, large-scale (gram quantities) synthesis of highly deuterated [D32 ]oleic acid by using multiple, yet simple and high yielding reactions. The precursors for the synthesis of [D32 ]oleic acid are [D14 ]azelaic acid and [D17 ]nonanoic acid, which were obtained by complete deuteration (>98% D) of their (1) H forms by using metal catalysed hydrothermal H/D exchange reactions. The oleic acid was produced with ca. 94% D isotopic purity and with no contamination by the trans-isomer (elaidic acid). The subsequent synthesis of [D64 ]dioleoyl-sn-glycero-3-phosphocholine from [D32 ]oleic acid is also described.

HOCl-mediated glycerophosphocholine and glycerophosphoethanolamine generation from plasmalogens in phospholipid mixtures.

Many mammalian tissues and cells contain, in addition to (diacyl) phospholipids, considerable amounts of plasmalogens, which may function as important antioxidants. Apart from the "scavenger" function mediated by the high sensitivity of the vinyl-ether bond, the functional role of plasmalogens is so far widely unknown. Furthermore, there is increasing evidence that plasmalogen degradation products have harmful effects in inflammatory processes. In a previous investigation glycerophosphocholine (GPC) formation was verified as a novel plasmalogen degradation pathway upon oxidation with hypochlorous acid (HOCl), however these investigations were performed in simple model systems. Herein, we examine plasmalogen degradation in a more complex system in order to evaluate if GPC generation is also a major pathway in the presence of other highly unsaturated glycerophospholipids (GPL) representing an additional reaction site of HOCl targets. Using MALDI-TOF mass spectrometry and (31)P NMR spectroscopy, we confirmed that the first step of the HOCl-induced degradation of GPL mixtures containing plasmalogens is the attack of the vinyl-ether bond resulting in the generation of 1-lysophosphatidylcholine (lysoPtdCho) or 1-lysophosphatidylethanolamine. In the second step HOCl reacts with the fatty acyl residue in the sn-2 position of 1-lysoPtdCho. This reaction is about three times faster in comparison to comparable diacyl-GPL. Thus, the generation of GPC and glycerophosphoethanolamine (GPE) from plasmalogens are relevant products formed from HOCl attack on the vinyl-ether bond of plasmalogens under pathological conditions.

A practical selective synthesis of mixed short/long chains glycerophosphocholines.

Glycerophosphorylcholine (GPC) is transformed into the cyclic stannylene derivatives, which are selectively acylated to 1-acyl-2-lyso-glycerophosphocholines. The reaction is effective using C-2 to C-16 acid chlorides in 2-propanol. After solvent replacement the lyso-phospholipid (lyso-PL) is subjected to a second acylation using acid anhydrides in methylene chloride. A series of 1(2)-short-2(1)-long-diacyl-glycerophosphocholines are obtained in high yields and selectivity. No diacylation product was detected. In order to detect mixed-chain lipids with inverted disposition of acyl chains, the long chain was introduced first and the thus resulting isomeric compounds compared by NMR. An NMR method was developed in order to determine the positional purity of the isomeric compounds.

A role for neutral sphingomyelinase activation in the inhibition of LPS action by phospholipid oxidation products.

Previous studies from our laboratory and others presented evidence that oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphatidylcholine (OxPAPC) and oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphatidylethanolamine can inhibit lipopolysaccharide (LPS)-mediated induction of interleukin-8 (IL-8) in endothelial cells. Using synthetic derivatives of phosphatidylethanolamine, we now demonstrate that phospholipid oxidation products containing alpha,beta-unsaturated carboxylic acids are the most active inhibitors we examined. 5-Keto-6-octendioic acid ester of 2-phosphatidylcholine (KOdiA-PC) was 500-fold more inhibitory than OxPAPC, being active in the nanomolar range. Our studies in human aortic endothelial cells identify one important mechanism of the inhibitory response as involving the activation of neutral sphingomyelinase. There is evidence that Toll-like receptor-4 and other members of the LPS receptor complex must be colocalized to the caveolar/lipid raft region of the cell, where sphingomyelin is enriched, for effective LPS signaling. Previous work from our laboratory suggested that OxPAPC could disrupt this caveolar fraction. These studies present evidence that OxPAPC activates sphingomyelinase, increasing the levels of 16:0, 22:0, and 24:0 ceramide and that the neutral sphingomyelinase inhibitor GW4869 reduces the inhibitory effect of OxPAPC and KOdiA-PC. We also show that cell-permeant C6 ceramide, like OxPAPC, causes the inhibition of LPS-induced IL-8 synthesis and alters caveolin distribution similar to OxPAPC. Together, these data identify a new pathway by which oxidized phospholipids inhibit LPS action involving the activation of neutral sphingomyelinase, resulting in a change in caveolin distribution. Furthermore, we identify specific oxidized phospholipids responsible for this inhibition.

Dephosphorylation and benzoylation reactions occurring in the benzoolysis of diacyl-, alkylacyl-, alk-1-enylacyl-, and acyl-lyso-glycerophosphocholines.

Series of benzoolysis experiments with 1,2-dipalmitoyl glycerophosphocholine (GPC), 1-palmitoyl-2-lyso GPC, and 1-O-hexadecenyl-2-oleoyl GPC, as well as a few experiments with 1-oleoyl-2-acetyl GPC and 1-O-hexadecyl-2-acetyl GPC are reported. The results are used to present a consistent picture of the various dephosphorylation and benzoylation reactions occurring in molten benzoic anhydride.

Influence of alkyl chain lengths in dialkylglycerophosphocholines towards phospholipase A2 inhibition in macrophages.

Rat peritoneal macrophages were cultured with a specific and potent phospholipase A2 activator A 23187, with 1-stearoyl-2-[3H]arachidonoyl-sn-GPC as source of [3H] arachidonic acid, and with a dialkyl-GPC, at 2, 10 or 20 microM. Four dialkyl-GPCs were prepared by chemical synthesis. Position 2 of rac-glycerol was alkylated with an alkane chain of 8 carbons and position 1 was alkylated with various alkane chains (8, 10, 12, or 16 carbons). [3H] arachidonic acid was split, then recovered with cell nonesterified fatty acids and nonphosphorous glycerolipids after endocellular phospholipase A2 activity. It was also recovered with fatty acids and eicosanoids isolated from culture medium. Inhibition of fatty acid release and eicosanoid synthesis depended on mixed chain dialkyl-GPC structures. The highest inhibitory effect on arachidonic acid release was reached with 1-decyl-2octyl-GPC and was practically as high in culture medium (IC50 at 5 microM) as in cells (IC50 at 4 microM). 1,2-di-octyl-GPC and 1-dodecyl-2-octyl-GPC had weaker inhibitory effects (but higher in culture medium than in cells). The asymmetrical 1-hexadecyl-2-octyl-GPC poorly affected enzyme activity.

Synthesis and antineoplastic properties of an ether glycerophosphonocholine, and analog of ET-18-OCH3-GPC.

A glycerophosphonocholine analog of the ether-linked lipid, rac-1-O-octadecyl-2-O-methyl-glycero-3-phosphocholine (ET-18-OCH3-GPC), was synthesized in which the head group is nonhydrolyzable by phospholipase C. The phosphonate analog used in this study is rac-3-octadecyloxy-2-methoxy-propyl-phosphonocholine, C18H37OCH2CH(OCH3)CH2P(O)(O)OCH2CH2N+(CH3)3. The activity of the synthetic phosphonate was tested in the human leukemic cell line, HL-60, and the human undifferentiated cervical carcinoma, C-41. The glycerophosphonocholine inhibited [3H]thymidine uptake by HL-60 cells with an EC50 value of 5-7 microM. The glycerophosphate ET-18-OCH3-GPC had an EC50 value of approximately 2 microM against HL-60 cells. The EC50 values estimated from cell viability experiments were similar to that for [3H]thymidine uptake. The EC50 value for C-41 cells was about 10-15 microM. The data demonstrate that the glycerophosphonocholine is a promising anti-cancer drug for the treatment of both leukemia and solid tumors. Furthermore, the data demonstrate that phospholipase C-catalyzed hydrolysis of ET-18-OCH3-GPC does not play an important role in the cytotoxic action of the ether-linked glycerolipids.

Synthesis of sn-glycero-1-phosphocholine.

The preparation of sn-glycero-1-phosphocholine is described. 2,3-O-Isopropylidene-sn-glycerol is condensed first with the 1,2-dimethylethenylene phosphorochloridate in the presence of triethylamine to yield the cyclic phosphotriester derivative. Choline p-toluenesulfonate, as the second alcohol, is condensed with this intermediate to yield the phosphotriester bearing the 1-methyl-acetonyl blocking group. This is removed under basic conditions to afford the 2,3-O-isopropylidene-sn-glycerol-1-phosphocholine in 57% yield following silicic acid column chromatography. The acetone blocking group is then removed with aqueous HCl (pH 2.3) to give pure sn-glycero-1-phosphocholine.

1-Hydroxy-2-tert-butyldimethylsilyl-sn-glycero-3-phosphorylcholine. A useful intermediate in the synthesis of short acyl chain 1-acyl-sn-glycero-3-phosphorylcholines.

The synthesis of 1-acyl-sn-glycero-3-phosphorylcholines in particular those containing short fatty acyl chains are described. The method involves the use of 1-acyl-2-tert-butyl-dimethylsilyl-sn-glycero-3-phosphorylcholines which can be readily prepared by reacting hens' egg yolk 1-acyl-sn-glycero-3-phosphorylcholines with tert-butyldimethylchlorosilane with imidazole as catalyst and dimethylformamide as solvent. Deacylation of the 1-acyl-2-tert-butyldimethylsilyl-sn-glycero-3-phosphorylcholines with saturated anhydrous potassium carbonate in methanol yields the 2-tert-butyldimethylsilyl-sn-glycero-3-phosphorylcholine. Reacylation of the 2-tert-butyldimethylsilyl-sn-glycero-3-phosphorylcholine with fatty acyl anhydride in the presence of 4-dimethylaminopyridine in anhydrous chloroform followed by removal of the tert-butyldimethylsilyl protecting group by treatment with dry hydrogen chloride gas in anhydrous chloroform at 0 degrees yields the desired 1-acyl-sn-glycero-3-phosphorylcholine. Various facets of the reactions involved in developing the synthetic procedures in this study are discussed.

Preparation of labeled alkyl acyl glycerophosphorylcholine.

A method for the preparation of 1-alkyl 2-acyl glycero-3-phosphorylcholine, labeled in the 2-position with oleic acid-1-(14)C is described. This method, based on the acylation of a 1-alkyl glycero-3-phosphorylcholine-CdCl(2) complex which is, in turn, formed from 1-alk-1-enyl glycero-3-phosphorylcholine by hydrogenation and subsequent reaction with cadmium chloride, offers a relatively simple means of preparing a labeled saturated ether phospholipid suitable for use in metabolic studies.