Proc. Indian Acad. Sci. (Chem. Sci.), Vol. 106, No. 5, October 1994, pp. 1231-1251. Printed in India. Synthesis and applications of phosphatidylinositols and their analogues I M S SHASHIDHAR Division of Organic Chemistry (Synthesis), National Chemical Laboratory, Pune 411008, India Aktraet. There is an upsurge of interest in the chemistry and biochemistry of phosphoino- tides, their analogues and the related enzymes due to their involvement in the inositol phosphates mediated cellular signal transduction pathways. The present review deals with the recent developments in the synthesis and applications of phosphatidylinositol and its derivatives. Keywords. Phospholipid; phosphatidylinositol; inositol phosphates; phospholipase C; signal transduction; second messenger. Introduction Lipids constitute a major class of biologically important molecules. There is an increasing awareness of the active roles played by membrane lipids such as phos- phatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, and phosphati- dylinositol (Ptdlns) in the structure and function of cells. Ptdlns has generated a lot of interest due to its involvement in the myo-inositol mediated cellular signal transduction pathway. This article intends to cover some of the recent developments in the synthesis and applications of Ptdlns and their analogues. Results and discussions Phosphatidylinositols PtdIns exist as a minor component in bio-membranes (5-10% of total phospholipids). The first demonstration of lipid bound myo-inositol in mammals was made by Folch 1NCL commun, no. 6014 Abbreviations: PtdIns, phosphatidylinositol; AMP, adinosinemonophosphate; PtdIns (3) P, phosphatidy- linositol-3-phosphate; PtdIns (4)P, phosphatidylinositol-4-phosphate; PtdIns(3,4)P2, phosphatidylinositol- 3,4-bisphosphate; PtdIns(4,5)P2, phosphatidylinositol-4,5-bisphosphate; PtdIns(3,4,5)Pa, phosphatidy- linositol-3,4,5-triphosphate; Ins(1)P, Myo-inositol-l-phosphate; Ins(1,2-cyc)P, Myo-inositol-l,2-cyclic- phosphate; Ins (1,4, 5) P3, Myo-inositol- 1,4,5-triphosphate; DAG, diacylglycerol; TBDMS, t-butyldimethyl- silyl; MSNT, l-(mesitylene-2-sulphonyl)-3-nitro-l,2,4-triazole; NPCL, 5,5-dimethyl-2-oxo-2-chloro-l,3,2- dioxaphophorinan; PLA2, phospholipase A2; PLD, phospholipase D; PI-PLC, phosphatidylinositol- specific phospholipase C; PI-4-kinase, phosphatidylinositol-4-kinase; P, PO 3 H2; Bn, benzyl; Pro, propenyl; DAG, diacylglycerol; A, allyl; MOM, methoxymethyl; Ac, acetyl; THP, tetrahydropyranyl; PMB, p-methoxybenzyl; nD, D-enantiomer of the compound n; nL, L-enantiomer of the compound n; nHL, racemate n) 1231 1232 M S Shashidhar and Wooley who discovered myo-inositol in brain lipids (Folch and Wooley 1942). Several phosphorylated (on the myo-inositol ring) derivatives of Ptdlns are known, most important of them being Ptdlns (4, 5)P2 (see below). Ptdlns (3)P was identified in transformed fibroblasts (Whitman et al 1988). Other phosphoinositides containing the 3-phosphate, such as, Ptdlns(3,4)P2 and Ptdlns(3,4,5)Pa have been found in many cell types (Majerus et al 1990). Glycosyl phosphatidylinositols which were recently discovered, are a class of glycolipids that anchor proteins, polysaccharides or small oligosaccharides to cell. membranes through covalent linkages. They have been found in a wide variety of cells and tissues. Myo-inositol as well as its di-mannosides are also present (Anderson and Roberto 1930; Anderson et al 1938) in the mycobacterium phospholipids. This lipid consists of a tri-substituted myo~ in which the 2- and 6-hydroxy groups are linked to carbohydrates and the 1-hydroxy group is linked to phosphatidic acid (Lee and Ballou 1965). Phosphatidylinositols: Importance Cellular signal transduction mechanisms translate external signals into internal signals through second messengers. Two major signal transduction pathways are now known, one employs cyclic AMP and the other employs Ins(l,4, 5)Pa and DAG. In the past few years it has been shown that an extracellular signal (such as a drug or a hormone) can activate PI-PLC which hydrolyzes the membrane lipid PtdIns (4, 5) P2 to Ins (1,4, 5)P3 and DAG, both of which serve as second messengers. Ins (1,4, 5)Pa is released into the cytosol, whereas DAG remains in the cell membrane, Consequent to this a series of complex reactions occur, resulting in the mobilization of calcium ions from endoplasmic reticuhim to the cytosol. DAG stimulates protein kinase C which catalyzes the transfer of phosphate groups from ATP to other proteins, which in turn alters the protein function. 1,2-Diacylglycerol also serves as a messenger by providing the substrate for icosanoid production. Both Ins (1,4,5)P3 and DAG are generated rapidly in low concentrations and are quickly removed, properties characteristic of molecules that signal cells to carry out designated functions. The supply of PtdIns (4,5) P2 in the cell membrane is maintained by sequential phosphory- lation of the more abundant PtdIns by specific 4- and 5-kinases. Evidence has also begun to accumulate to show that phosphoinositides might be involved in vesicular traffic in yeast and some mammalian cells (Skinner 1993; Hay and Martin 1993; Cleves et al 1991) However, the exact role played by phosphoinositides in vesicular traffic has not been clearly demonstrated. PI-PLC enzymes which cleave phosphatidylinositols exist in a wide variety of tissues and organisms. Extracellular PI-PLCs have been isolated from the culture media of several microorganisms. Intracellular/membrane bound PI-PLCs are prevalent in mammalian cells. The extracellular PI-PLCs are water-soluble and are relatively specific for Ptdlns and glycosyl Ptdlns. PI-PLCs specific for Ptdlns(4,5)P2 are calcium ion dependent for their activity, whereas bacterial enzymes have no metal ion dependence and do not cleave phosphorylated forms of Ptdlns. Calcium ion indepen- dent PI-PLCs specific for glycosyl Ptdlns have been purified from Trypanasoma brucei and rat liver. The enzyme from rat liver has been implicated in insulin action. The two types of enzymes produce different products when acting on analogous Ptdlns substrates. The mammalian enzyme usually produces a mixture of Ins(1,2- cyc)P and Ins(l)P, depending on the pH, enzyme sub-type and other conditions Phosphatidylinositols and analogues 1233 (Dawson et al 1971; Kim et al 1989) whereas the bacterial enzyme produces Ins (1,2- cyc) P exclusively (Ferguson et al 1985; Volwerk et al 1990). 0 PLA, --~ __OAR 1 o PLD "-~ R2~'-..(~~ .~ H ~176 ~ HO~y~#O -- P-- ~.. .... o. '~HO"'~~H "'"OH 1 -4-KINAS--E--/ PI - PLC PI R, = c~( CH.z)~6CO -- R2= CH3(CH2)4 (CH=CHCH2)4 CH2CO-- Scheme 1. The developments outlined above have revived interest in the chemistry and biochemistry of phosphoinositides and related enzymes. Scheme 1 shows some of the important enzymes for which Ptdlns serves as a substrate. Studies toward the delineation of the roles of various enzymes involved in the myo-inositol mediated signal transduction processes has led to the design and synthesis of a number of myo-inositol phospholipids and their analogues. Such analogues are also of medical interest because of their potential as pharmacological agents. Phosphatidylinositols: Synthesis Myo-inositol, which forms the head group of Ptdlns is a meso isomer of hexahydroxy cyclohexane, In myo-inositol five of the hydroxy groups are equatorial and only one is axial. Thus, for all practical purposes the ring is regarded as rigid (flipping of the ring would result in one equatorial and five axial hydroxy groups). However, heavy substitution on the myo-inositol ring might distort the chair conformation of the cyclohexane ring. Different representations of the myo-inositol ring and the most widely used numbering of the ring carbons are shown in scheme 2 using myo- inositol-l-phosphate (Ins(1)P) as an example (for an account on the implications of the stereochemistry of myo-inositol phosphate see Parthasarthy and Eisenberg 1986). All naturally occurring analogues contain myo-inositol head group substituted at the D-1 position an.d the glycerol moiety acylated at sn-1 and sn-2 positions. No phosphoinositides of the L-series are known to occur in nature. The key step in the chemical synthesis of phospholipids is phosphorylation, leading to the formation of the phosphodiester bond. Phospholipids have been synthesized using various phosphodiesters, phosphotriesters, phosphites and H-phosphonates 1234 M S Shashidhar HO Q 0 OH 0~, 2~ "a/~OH HO OH HO---~\ 1 4 \~OH 4~rl / t HO'@'y '''OH ~ k-"~OHoH5 H OH OH Myo- Inositol D-lns(I)P D-ins (3) P or or L- Cns(3)P L-Cns (I) P Scheme 2. (Lindh and Stawinski 1989). Synthesis of phosphoinositides in addition requires a suitably protected myo-inositol derivative. Several methodologies for the synthesis of enantiomerically pure myo-inositol derivatives have been developed in the recent past (Cosgrove 1980; Billington 1989; Potter 1990; Bruzik and Tsai 1992). The most extensively used hydroxy protecting group during the synthesis of phosphoinositols is the benzyl group, which can be cleaved by hydrogenolysis or using ethanethiol. However, when the use of such conditions are not compatible with.the structure of the desired product, ketals, acetals, esters or orthoesters have been used. Many of the O-alkylated derivatives of myo-inositol are prepared taking advantage of the ease of alkylation of the equatorial 1 (or 3-)-hydroxy group over the axial 2-hydroxy group. Phosphatidylinositols as substrates Dipalmitoyl PtdIns(4,5)P 2 was synthesized (Dreef et al 1988) starting from the optically active 4,5-di-O-allyl-3,6-di-O- benzyl-D-myo-inositol 1 (scheme 3). OH OBn BnO~OH ~ ~ BnO.OH AO'@"~"'O Bn Pro 0": "'O Bn IIAO OPro I 2 3 R = CHs(CH2)t4CO-- Ptd lns (4, 5 ) P2 Scheme 3. Phosphatidylinositols and analogues 1235 The key intermediate 2 was prepared from 1 by temporary protection of the 1-hydroxy group as t-butyldimethylsilyl (TBDMS) ether. The phosphoramidite 3 was then coupled to 2 and the phosphite obtained was oxidized with t-butylhydroperoxide.