Lysophosphatidic acid (LPA) is a lipid mediator that, among several other cellular responses, can stimulate cells to mobilize calcium (Ca2+). LPA is known to activate at least three different subtypes of G protein-coupled receptors. These receptors can then stimulate different kinds of G proteins. In the present study, LPA and LPA analogs were synthesized from (R)- and (S)-glycidol and used to characterize the ability to stimulate Ca2+ mobilization. The cytosolic Ca2+ concentration ([Ca2+]i) was measured in fura-2-acetoxymethylester-loaded human erythroleukemia (HEL) cells. Furthermore, a reverse transcriptase polymerase chain reaction was used to characterize LPA receptor subtypes expressed in HEL cells. The results show that HEL cells mainly express LPA1 and LPA2, although LPA3 might possibly be expressed as well. Moreover, LPA and its analogs concentration-dependently increased [Ca2+]i in HEL cells. The response involved both influx of extracellular Ca2+ and release of Ca2+ from intracellular stores. This is the first time the unnatural (S)-enantiomer of LPA, (S)-3-O-oleoyl-1-O-phosphoryl-glycerol, has been synthesized and studied according to its ability to activate cells. The results indicate that this group of receptors does not discriminate between (R)- and (S)-enantiomers of LPA and its analogs. When comparing ether analogs having different hydrocarbon chain lengths, the tetradecyl analog (14 carbons) was found to be the most effective in increasing [Ca2+]i. Pertussis toxin treatment of the HEL cells resulted in an even more efficient Ca2+ mobilization stimulated by LPA and its analogs. Furthermore, at repeated incubation with the same ligand no further increase in [Ca2+]i was obtained. When combining LPA with the ether analogs no suppression of the new Ca2+ signal occurred. All these findings may be of significance in the process of searching for specific agonists and antagonists of the LPA receptor subtypes.

Short synthetic routes to protected derivatives of 2-amino-2-deoxy-a-D-glucopyranosyl-(1?6)-D-myo-inositol are described. Various 2-azido-2-deoxy-glucosyl donors were synthesized, starting from D-glucal or glucosamine hydrochloride. Derivatives of 1,2- and 2,3- D-myo-inositol-camphanylidene acetals were prepared to function as glycosyl acceptors. The subsequent glycosylations produced useful building blocks for the synthesis of GPI-anchor substances. © 2002 Elsevier Science Ltd. All rights reserved.

New efficient routes to enantiopure phospholipids, starting from (S)-glycidol, are described. Lysophosphatidic acids and phosphatidic acids were obtained in good overall yields from (S)-glycidol, in only three and four steps, respectively. Moreover, the strategy can also be used to produce phosphatidylcholines in three steps. Using dialkylphosphoramidites, (S)-glycidol was phosphorylated to give (R)-1-O-glycidyl dialkyl phosphates. Regiospecific epoxide opening, using hexadecanol or cesium palmitate, followed by phosphate deprotection, provided lysophosphatidic acids. 2-O-Esterification prior to phosphate deprotection provided 1,2-O-diacyl and 1-O-alkyl-2-O-acyl phosphatidic acids. Phosphorylation of (S)-glycidol using phosphorus oxychloride followed by in situ treatment with choline tosylate produced (R)-glycidyl phosphocholine. Subsequent nucleophilic opening of the epoxide using cesium palmitate produced 1-O palmitoyl-sn-glycero-3-phosphocholine, which has been used in syntheses of phosphatidylcholines.

This thesis describes the synthesis of a series of myo-inositol-containing oligosaccharides, and the exploration of using the chiral epoxide (S)-glycidol in short synthetic routes to glycoglycerolipids and glycerophospholipids.

In chapter 2, synthesis of the core heptasaccharyl myo-inositol Galp(α1-6)Galp(α1 -3)Galf(β1-3)[Glcp(α1-P0₄-6)Manp](α1-3)Manp(α1-4)GlcNp(α1-6)Ins-1-P0₄, found in the lipophosphoglycans of Leishmania parasites, is described. This part primarily deals with problematic deprotections of complex carbohydrates. In this context, the stability of anomeric phosphodiesters towards hydrolytic cleavage was studied.

In chapter 3, a short synthetic route to protected derivatives of 2-amino-2-deoxy-α-D-glucopyranosyl-(1➔6)-D-myo-inositol is presented. These derivatives are key building blocks in the synthesis of inositolphosphoglycans (IPGs) and glycosylphosphatidylinositol (GPI)-anchors.The building blocks were subsequently used for synthesis of IPGs, analogous to putative second messengers to insulin, to provide a small targeted library of compounds. A thiol-terminated spacer was introduced by radical elongation of allyl ethers with benzyl mercaptan, for immobilization of the IPGs by coupling to maleimide-functionalized solid phases, proteins or various probes. The aim of this project was to provide IPGs for evaluation of biological activity, isolation of antibodies, and identification of receptors for the second messengers to insulin.

Chapters 4 and 5 describe syntheses of galactoglycerolipids and glycerophospholipids via glycosylation or phosphorylation of the three-carbon synthon (S)-glycidol. Using this approach, three galactoglycerolipids and the corresponding glycerolipid were synthesized and an oxidation-reduction procedure for tritiation of the glycerolipid was developed. Two of the galactolipids have recently been identified in the human colon carcinoma cell line HT29. The synthetic method developed for the galactolipids was subsequently used to explore a new efficient route to enantiomerically pure glycerophospholipids. Lysophosphatidic acids and phosphatidic acids were obtained in good overall yields from (S)-glycidol, in only three and four steps, respectively. Moreover, a strategy for synthesis of lysophosphatidylcholines, in only three steps, is also described.

Synthesis of three galactoglycerolipids (3-O-(ß-D-galactopyranosyl)-1-O-hexadecyl-2-O-palmitoyl-sn-glycerol, 3-O-(a-D-galactopyranosyl-(1?4)-ß-D-galactopyranosyl)-1-O- hexadecyl-2-O-palmitoyl-sn-glycerol, 3-O-(a-D-galactopyranosyl-(1?4)-ß-D-galactopyranosyl)-1-O- hexadecyl-sn-glycerol), and the corresponding glycerolipid (1-O-hexadecyl-2-O-palmitoyl-sn-glycerol) is described. The first two compounds were recently identified in the human colon carcinoma cell line HT29. The three-carbon synthon (S)-glycidol was used for construction of the glycerol moiety. Glycosylation of (S)-glycidol with protected galactosyl and digalactosyl donors produced galactosyl and digalactosyl glycidols. Lewis acid catalyzed opening of the epoxide produced protected galactosyl and digalactosyl glycerolipids. Deprotection, or palmitoylation followed by deprotection, yielded the target compounds. The corresponding glycerolipid was synthesized analogously and an oxidation-reduction procedure for tritiation was developed. The synthesized compounds will be used in studies of the role of galactosyl glycerolipids in differentiation and colon cancer development. © 2002 Elsevier Science Ltd. All rights reserved.

Synthesis of 1 and 2a is described using a block synthetic strategy. Compound 4 was used as precursor for the two mannose derivatives which, coupled together, forms the dimannoside building block. Thioglycoside 7 was coupled to 8 yielding inositol phosphoglycan 9a, which was selectively deprotected and reacted with 2,3,4,6-tetra-O-benzyl-a-D-glucopyranos-1-yl H- phosphonate to form the protected target molecule 12. Deprotection of 12 by acidic deacetalisation/desilylation and subsequent catalytic hydrogenolysis resulted in cleavage of the anomeric phosphodiester to produce 1. Debenzylation with sodium in liquid ammonia followed by acidic deacetalisation/desilylation gave the target compound 2a. (C) 2000 Published by Elsevier Science Ltd.