Regioselective phosphorylation of myo-inositol with BINOL-derived phosphoramidites and its application for protozoan lysophosphatidylinositol.

A regioselective phosphorylation method for myo-inositol was developed by utilizing readily preparable BINOL-derived phosphoramidites. The method also facilitated the complete separation of the diastereomeric products by simple chromatography. Based on this phosphorylation and Ni-catalyzed alkyl-alkyl cross-coupling reaction for long fatty acids, we achieved the first synthesis of a lysophosphatidylinositol, EhPIa having long fatty acid C30:1, as a partial structure of glycosylphosphatidylinositol (GPI) anchor from the cell membrane of a protozoa, Entamoeba histolytica.

Synthesis and evaluation of immunostimulant plasmalogen lysophosphatidylethanolamine and analogues for natural killer T cells.

Plasmalogen lysophosphatidylethanolamine (pLPE) had been identified as a self antigen for natural killer T cells (NKT cells). It is very important in the development, maturation and activation of NKT cells in thymus. Besides, pLPE is a novel type of antigen for NKT cells. To evaluate the structure-activity relationship (SAR) of this new antigen, pLPE and its analogues referred to different aliphatic chains and linkages at the sn-1 position of the glycerol backbone were synthesized, and the biological activities of these analogues was characterized. It is discovered that the linkages between phosphate and lipid moiety are not important for the antigens' activities. The pLPE analogues 1, 3, 4, 7 and 9, which have additional double bonds on lipid parts, were identified as new NKT agonists. Moreover, the analogues 4, 7 and 9 were discovered as potent Th2 activators for NKT cells.

19F/18F exchange synthesis for a novel [18F]S1P3-radiopharmaceutical.

(19)F/(18)F isotope exchange is a useful method to label drug molecules containing (19)F-fluorine with (18)F without modifying the drug molecule itself. Sphingosine-1-phosphate (S1P) is an important cellular mediator that functions by signaling through cell surface receptors. S1P is involved in several cell responses and may be related to many central nervous system disorders, including neural malfunction in Alzheimer's disease. In this study, [(18)F]1-benzyl-N-(3,4-difluorobenzyl)-2-isopropyl-6-(2-methoxyethoxy)-1H-indole-3-carboxamide, a novel (18)F-labeled positron emission tomography tracer for the S1P3 receptor, was successfully synthesized using the (19)F/(18)F isotope exchange reaction. Parameters of the reaction kinetics were studied, and correlations between the initial (18)F-activity, the amount of precursor, radiochemical yield and specific activity (SA) were determined. Contrary to expectations, high initial (18)F-activity decreased the radiochemical yield, and only a minor increase of SA occurred. This is most probably due to the complexity of the molecule and the subsequent susceptibility to radiolytic bond disruption. On the basis of the present results, a convenient condition for the (19)F/(18)F exchange reaction is the use of 2 µmol precursor with 20 GBq of (18)F-activity. This afforded a radiochemical yield of ~10% with an SA of 0.3 GBq/µmol. Results from this study are of interest for new tracer development where high initial (18)F-activity and (19)F/(18)F isotope exchange is used.

The chemical synthesis of metabolically stabilized 2-OMe-LPA analogues and preliminary studies of their inhibitory activity toward autotaxin.

The chemical synthesis of five new metabolically stabilized 2-OMe-LPA analogues (1a-e) possessing different fatty acid residues has been performed by phosphorylation of corresponding 1-O-acyl-2-OMe-glycerols which were prepared by multistep process from racemic glycidol. The now analogues were subjected to biological characterization as autotaxin inhibitors using the FRET-based, synthetic ATX substrate FS-3. Among tested compounds 1-O-oleoyl-2-OMe-LPA (1e) appeared to be the most potent, showing ATX inhibitory activity similar to that of unmodified 1-O-oleoyl-LPA. Parallel testing showed, that similar trend was also observed for corresponding 1-O-acyl-2-OMe-phosphorothioates (2a-e, synthesized as described by us previously). 1-O-oleoyl-2-OMe-LPA (1e) was found to be resistant toward alkaline phosphatase as opposed to unmodified 1-O-oleoyl-LPA.

Switching Lysophosphatidylserine G Protein-Coupled Receptor Agonists to Antagonists by Acylation of the Hydrophilic Serine Amine.

Three human G protein-coupled receptors (GPCRs)-GPR34/LPS1, P2Y10/LPS2, and GPR174/LPS3-are activated specifically by lysophosphatidylserine (LysoPS), an endogenous hydrolysis product of a cell membrane component, phosphatidylserine (PS). LysoPS consists of l-serine, glycerol, and fatty acid moieties connected by phosphodiester and ester linkages. We previously generated potent and selective GPCR agonists by modification of the three modules and the ester linkage. Here, we show that a novel modification of the hydrophilic serine moiety, that is, N-acylations of the serine amine, converted a GPR174 agonist to potent GPR174 antagonists. Structural exploration of the amide functionality provided access to a range of activities from agonist to partial agonist to antagonist. The present study would provide a new strategy for the development of lysophospholipid receptor antagonists.

Lipase-catalyzed Synthesis of Feruloylated Lysophospholipid in Toluene-Ionic Liquids and Its Antioxidant Activity.

In this study, Novozym 435-catalyzed interesterification of ethyl ferulate (EF) with phosphatidylcholine (PC) in a two-phase system consisting of an ionic liquid (IL) and toluene was optimized to prepare feruloylated lysophospholipids (FLPs). Optimum conditions for the interesterification process were found to be [Bmim][Tf2N]/toluene ratio of 1:1 (v/v), solvent volume of 4 mL, molecular sieves (4 Å) concentration of 80 mg/mL, reaction temperature of 55°C, substrate molar ratio of 5:1 (PC/EF), Novozym 435 concentration of 50 mg/mL. Under these conditions, two FLPs products (1-FLP and 2-FLP) with total conversion rate of 50.79% were obtained. Because the formation of 1-FLP was significantly higher than 2-FLP, 1-FLP was purified and characterized by LC-MS and NMR. In addition, 1-FLP showed DPPH scavenging activity comparable with those of EF and BHT. Therefore, this study provides a good method for transformation of ferulic acid to improve its solubility and promote its application as functional ingredient in the food and pharmaceutical industries.

Synthesis of lysophospholipids.

New synthetic methods for the preparation of biologically active phospholipids and lysophospholipids (LPLs) are very important in solving problems of membrane-chemistry and biochemistry. Traditionally considered just as second-messenger molecules regulating intracellular signalling pathways, LPLs have recently shown to be involved in many physiological and pathological processes such as inflammation, reproduction, angiogenesis, tumorogenesis, atherosclerosis and nervous system regulation. Elucidation of the mechanistic details involved in the enzymological, cell-biological and membrane-biophysical roles of LPLs relies obviously on the availability of structurally diverse compounds. A variety of chemical and enzymatic routes have been reported in the literature for the synthesis of LPLs: the enzymatic transformation of natural glycerophospholipids (GPLs) using regiospecific enzymes such as phospholipases A1 (PLA1), A2 (PLA2) phospholipase D (PLD) and different lipases, the coupling of enzymatic processes with chemical transformations, the complete chemical synthesis of LPLs starting from glycerol or derivatives. In this review, chemo-enzymatic procedures leading to 1- and 2-LPLs will be described.

Non-naturally Occurring Regio Isomer of Lysophosphatidylserine Exhibits Potent Agonistic Activity toward G Protein-Coupled Receptors.

Lysophosphatidylserine (LysoPS), an endogenous ligand of G protein-coupled receptors, consists of l-serine, glycerol, and fatty acid moieties connected by phosphodiester and ester linkages, respectively. An ester linkage of phosphatidylserine can be hydrolyzed at the 1-position or at the 2-position to give 2-acyl lysophospholipid or 1-acyl lysophospholipid, respectively. 2-Acyl lysophospholipid is in nonenzymatic equilibrium with 1-acyl lysophospholipid in vivo. On the other hand, 3-acyl lysophospholipid is not found, at least in mammals, raising the question of whether the reason for this might be that the 3-acyl isomer lacks the biological activities of the other isomers. Here, to test this idea, we designed and synthesized a series of new 3-acyl lysophospholipids. Structure-activity relationship studies of more than 100 "glycol surrogate" derivatives led to the identification of potent and selective agonists for LysoPS receptors GPR34 and P2Y10. Thus, the non-natural 3-acyl compounds are indeed active and appear to be biologically orthogonal with respect to the physiologically relevant 1- and 2-acyl lysophospholipids.

Developing a High-Throughput Assay for the Integral Membrane Glycerol 3-Phosphate Acyltransferase.

Phospholipid biosynthesis begins with the acylation of glycerol 3-phosphate (G3P). In most Gram-positive bacteria including many pathogens, a membrane protein called PlsY is the only acyltransferase that catalyzes this essential step, making it a potential target for the development of antibiotics. A convenient enzymatic assay should facilitate such drug discovery activities. Previously, we developed a continuous assay by monitoring phosphate, one of the enzymatic product, using a fluorescently labeled phosphate binding protein in a bilayer environment called lipid cubic phase (LCP). However, some intrinsic characteristics of LCP, such as high viscosity, make the assay incompatible with common high-throughput liquid-handling platforms. Here, we adapted the assay by hosting PlsY in detergent micelles, enabling us to conduct the assay using standard multi-channel pipets in a high-throughput manner. With optimal enzyme loading, the reaction velocity was linear up to 30 min. PlsY showed Michaelis-Menten kinetics behavior in micelles with a Vmax of 57.5 µmol min-1 mg-1, and Kmof 1.14 mM G3P and 6.2 µM acyl phosphate. The inhibitory product lysophosphatidic acid inhibited PlsY with the IC50 of 19 µM. The results principally demonstrated the feasibility of using the assay for high-throughput screening, and the protocol provided an encouraging starting point for further optimization and validation of the assay for automated platforms.

Inhibition of transcellular tumor cell migration and metastasis by novel carba-derivatives of cyclic phosphatidic acid.

Cyclic phosphatidic acid (1-acyl-sn-glycerol-2,3-cyclic phosphate; cPA) is a naturally occurring analog of lysophosphatidic acid (LPA) with a variety of distinctly different biological activities from those of LPA. In contrast to LPA, a potent inducer of tumor cell invasion, palmitoyl-cPA inhibits FBS- and LPA-induced transcellular migration and metastasis. To prevent the conversion of cPA to LPA we synthesized cPA derivatives by stabilizing the cyclic phosphate ring; to prevent the cleavage of the fatty acid we generated alkyl ether analogs of cPA. Both sets of compounds were tested for inhibitory activity on transcellular tumor cell migration. Carba derivatives, in which the phosphate oxygen was replaced with a methylene group at either the sn-2 or the sn-3 position, showed much more potent inhibitory effects on MM1 tumor cell transcellular migration and the pulmonary metastasis of B16-F0 melanoma than the natural pal-cPA. The antimetastatic effect of carba-cPA was accompanied by the inhibition of RhoA activation and was not due to inhibition of the activation of LPA receptors.

Synthesis, pharmacology, and cell biology of sn-2-aminooxy analogues of lysophosphatidic acid.

An efficient enantioselective synthesis of sn-2-aminooxy (AO) analogues of lysophosphatidic acid (LPA) that possess palmitoyl and oleoyl acyl chains is presented. Both sn-2-AO LPA analogues are agonists for the LPA1, LPA2, and LPA4 G-protein-coupled receptors, but antagonists for the LPA3 receptor and inhibitors of autotaxin (ATX). Moreover, both analogues stimulate migration of intestinal epithelial cells in a scratch wound assay.

Lysophosphatidic acids, cyclic phosphatidic acids and autotaxin as promising targets in therapies of cancer and other diseases.

Lysophospholipids have long been recognized as membrane phospholipid metabolites, but only recently lysophosphatidic acids (LPA) have been demonstrated to act on specific G protein-coupled receptors. The widespread expression of LPA receptors and coupling to several classes of G proteins allow LPA-dependent regulation of numerous processes, such as vascular development, neurogenesis, wound healing, immunity, and cancerogenesis. Lysophosphatidic acids have been found to induce many of the hallmarks of cancer including cellular processes such as proliferation, survival, migration, invasion, and neovascularization. Furthermore, autotaxin (ATX), the main enzyme converting lysophosphatidylcholine into LPA was identified as a tumor cell autocrine motility factor. On the other hand, cyclic phosphatidic acids (naturally occurring analogs of LPA generated by ATX) have anti-proliferative activity and inhibit tumor cell invasion and metastasis. Research achievements of the past decade suggest implementation of preclinical and clinical evaluation of LPA and its analogs, LPA receptors, as well as autotaxin as potential therapeutic targets.

An efficient, one-pot synthesis of various ceramide 1-phosphates from sphingosine 1-phosphate.

An efficient, one-pot procedure for the synthesis of ceramide 1-phosphates with varying N-acyl substituents, to serve as tool compounds for analytical and biological investigations, was developed. Sphingosine 1-phosphate was silylated in situ to increase its solubility and to protect the 3-hydroxy functionality and then allowed to react with activated acid derivatives in the presence of diisopropylethylamine. Simultaneous cleavage of the silyl protecting groups and separation from reagents and by-products was achieved by medium pressure chromatography on reversed phase material. Thus, ceramide 1-phosphates with various fatty acid chains and with fluorescent and affinity labels attached to the sphingoid backbone were prepared in good yields.

Examining the effect of regioisomerism on the physico-chemical properties of lysophosphatidylethanolamine-containing liposomes using fluoro probes.

Lysophospholipids (LysoPLs) receive steadily increasing attention in the area of lipid chemistry and biology. However, the physico-chemical properties of individual LysoPL regioisomers have not yet been investigated. Herein, we report the synthesis of fluoro analogues of lysophosphatidylethanolamines (LPEs) and examine the physico-chemical properties of the LPE regioisomers using chemically synthesized fluoro probes.

Sphingosine 1-phosphate (S1P) induces shape change in rat C6 glioma cells through the S1P2 receptor: development of an agonist for S1P receptors.

Treatment with isoprenaline led to a change in the cell morphology of rat C6 glioma cells. This morphological change was reverted by the addition of sphingosine 1-phosphate (S1P). Using this morphological change as a response marker we determined that DS-SG-44 ((2S,3R)-2-amino-3-hydroxy-4-(4-octylphenyl)butyl phosphoric acid) was an agonist of S1P receptors. The DS-SG-44-induced morphological reversion was not observed with such structurally related molecules as DS-SG-45 ((2S,3R)-2-amino-3-hydroxy-4-(3-octylphenyl)butyl phosphoric acid) and DS-SG-12 ((2S,3R)-2-amino-4-(4-octylphenyl)butane-1,3-diol). The S1P- and DS-SG-44-induced shape changes were neither reproduced with the S1P1/S1P3 receptor agonist VPC24191 nor inhibited by the S1P1/S1P3 receptor antagonist, VPC23019. Transfection with small interfering RNA (siRNA) for the S1P2 receptor greatly inhibited the DS-SG-44-induced shape change, and in part an S1P-induced response. In the presence of VPC23019, siRNA transfection for the S1P2 receptor almost completely blocked the S1P- and DS-SG-44-induced shape changes. Our results suggested that DS-SG-44, a newly-synthesized S1P analogue, acted as an S1P receptor agonist and that the S1P-induced shape change in rat C6 glioma cells was mediated mainly through the S1P2 receptor, and cooperatively through the S1P1/S1P3 receptors.

Formation of lysophospholipids from unsaturated phosphatidylcholines under the influence of hypochlorous acid.

The formation of lysophosphatidylcholines from unsaturated phosphatidylcholines upon treatment with hypochlorous acid was evaluated by means of MALDI-TOF mass spectrometry and 31P NMR spectroscopy. With an increasing number of double bonds in a fatty acid residue, the yield of lysophosphatidylcholines with a saturated fatty acid residue increased considerably in comparison to the total amount of higher molecular weight products like chlorohydrins and glycols. High amounts of lysophosphatidylcholines were formed from phospholipids containing arachidonic or docosahexaenoic acid residues. In phospholipids with monounsaturated fatty acid residues, the position of the double bond did not influence the yield of lyso-products. Besides the exclusive formation of chlorohydrin and glycol, hypochlorous acid caused the cleavage of the unsaturated fatty acid residue independent of its location at the first or second position of the glycerol backbone. In contrast, strong alkaline conditions, i.e. saponification led also to a hydrolysis of the saturated fatty acid residue from phosphatidylcholines. It is concluded that both MALDI-TOF mass spectrometry and 31P NMR spectroscopy are able to detect the formation of lysophosphatidylcholines. We conclude also that the formation of lysophospholipids from unsaturated phosphatidylcholines by hypochlorous acid can be relevant in vivo under acute inflammatory conditions.

Alterations in red blood cell sugar transport by nanomolar concentrations of alkyl lysophospholipid.

Acyl lysolipids presented in vitro to red blood cells in amounts comparable to blood serum levels inhibit protein-mediated glucose transport (Naderi, A., Carruthers, A. and Melchior, D.L. (1989) Biochim. Biophys. Acta 985, 173-181). In this study, an alkyl lysolipid (2-O-methyl-1-O-octadecyl-sn-glycero-3- phosphocholine; ALP), was found to be an order of magnitude more effective in inhibiting sugar transport than the most potent acyl lysolipid. Bilayer concentrations of ALP as low as 5 ALP molecules per transporter (0.1 mol% of total membrane lipid) result in a 50% inhibition of transport activity. ALP acts as a competitive inhibitor of exchange L-glucose transport, of CCB binding to the glucose transporter and of D-glucose inhibition of CCB binding to the transporter. Inhibition of zero-trans sugar uptake by ALP is noncompetitive. The two enantiomers of ALP show a different ability to inhibit sugar transport. The action of ALP is consistent with a mechanism in which ALP interacts with a transmembrane portion of the sugar transport molecule resulting in a competitive displacement of D-glucose or cytochalasin B from the cytosolic facing side of the transport molecule. The simplest explanation of our findings is a direct interaction of the ALP molecule with the transport protein.

Structure-activity relationships of fluorinated lysophosphatidic acid analogues.

Lysophosphatidic acid (LPA, 1- or 2-acyl-sn-glycerol 3-phosphate) displays an intriguing cell biology that is mediated via interactions with seven-transmembrane G-protein-coupled receptors (GPCRs) and the nuclear hormone receptor PPARgamma. To identify receptor-selective LPA analogues, we describe a series of fluorinated LPA analogues in which either the sn-1 or sn-2 hydroxyl group was replaced by a fluoro or fluoromethyl substituent. We also describe stabilized phosphonate analogues in which the bridging oxygen of the monophosphate was replaced by an alpha-monofluoromethylene (-CHF-) or alpha-difluoromethylene (-CF(2)-) moiety. The sn-2- and sn-1-fluoro-LPA analogues were unable to undergo acyl migration, effectively "freezing" them in the sn-1-O-acyl or sn-2-O-acyl forms, respectively. We first tested these LPA analogues on insect Sf9 cells induced to express human LPA(1), LPA(2), and LPA(3) receptors. While none of the analogues were found to be more potent than 1-oleoyl-LPA at LPA(1) and LPA(2), several LPA analogues were potent LPA(3)-selective agonists. In contrast, 1-oleoyl-LPA had similar activity at all three receptors. The alpha-fluoromethylene phosphonate analogue 15 activated calcium release in LPA(3)-transfected insect Sf9 cells at a concentration 100-fold lower than that of 1-oleoyl-LPA. This activation was enantioselective, with the (2S)-enantiomer showing 1000-fold more activity than the (2R)-enantiomer. Similar results were found for calcium release in HT-29 and OVCAR8 cells. Analogue 15 was also more effective than 1-oleoyl-LPA in activating MAPK and AKT in cells expressing high levels of LPA(3). The alpha-fluoromethylene phosphonate moiety greatly increased the half-life of 15 in cell culture. Thus, alpha-fluoromethylene LPA analogues are unique new phosphatase-resistant ligands that provide enantiospecific and receptor-specific biological readouts.

Concise synthesis of ether analogues of lysobisphosphatidic acid.

We describe a versatile, efficient method for the preparation of ether analogues of (S,S)-lysobisphosphatidic acid (LBPA) and its enantiomer from (S)-solketal. Phosphorylation of a protected sn-2-O-octadecenyl glyceryl ether with 2-cyanoethyl bis-N,N-diisopropylamino phosphine and subsequent deprotection generated the bisether LBPA analogues. By simply changing the sequence of deprotection steps, we obtained the (R,R)- and (S,S)-enantiomers of 2,2'-bisether LBPA. An ELISA assay with anti-LBPA monoclonal antibodies showed that the bisether LBPAs were recognized with the same affinity as the natural 2,2'-bisoleolyl LBPA. [reaction: see text]