Synthesis of Inositol-Containing ... - ACS Publications

Chapter 16

Synthesis of Inositol-Containing Glycophospholipids with Natural and Modified Structure Alexander E. Stepanov and Vitaly I. Shvets

Downloaded by GEORGETOWN UNIV on August 16, 2015 | http://pubs.acs.org Publication Date: December 15, 1998 | doi: 10.1021/bk-1999-0718.ch016

Department of Biotechnology, State Academy of Fine Chemical Technology, Moscow, 117571, Russia

Total syntheses of myo-inositol containing glycolipids related to the family of glycosylphosphatidylinositols anchors of membrane integral proteins have been developed. Directed protection-deprotection strategy of hydroxyl functions was used for the preparation of asymmetrically substituted myo-inositol intermediates. The enantiomerically pure myo-inositol derivatives were obtained by optical resolution of racemates via formation of diastereomers with monosaccharides or with chiral organic acids. The phosphorylated myo-inositol and sphingosine intermediates were prepared by employing phosphite chemistry. Formation of glycosidic bonds of target glycophospholipids was performed by using oxazoline and glycosyl fluoride methods of glycosylation. The described approaches resulted in the syntheses of phosphatidylinositol β-glucosaminides, phosphatidylinositol glucoside, ceramide inositol phosphate and the corresponding phosphorothionate, and the first total synthesis of inositol-containing glycophosphosphingolipid.

The structure, properties and biological function of myo-inositol-containing glycophospholipids have been a subject of intensive studies in recent years, owing to the recognition of the important role of these natural substances in functioning of a living cell. Thus, it was found that glycosylphosphatidylinositols (GPIs) are involved in fundamental biochemical processes such as "anchoring" of many proteins on the surface of the plasma membrane, modulation of physiological state and immunological status of cells (7). GPIs are also recognized as precursors of inositol glycans, which have been considered as second messengers in insulin signaling (2). Investigation of the biological function of GPIs is restricted by their minute content in natural sources. Therefore, development of synthetic methods leading to GPIs with natural structure, as well as their analogues, has become a vital part of broad interdisciplinary studies in our Laboratory. A l l natural GPIs have a common structural fragment of 6-0-(2-amino-2-deoxyp-D-glucopyranosyl)-D-my0-inositol-l -phosphate. This fragment has been reported to possess insulinomimetic activity (7,2). In some natural sources (yeast, fungi, bacteria, plants), numerous inositol-containing phospho- and glycophospholipids 244

©1999 American Chemical Society

In Phosphoinositides; Bruzik, K.; ACS Symposium Series; American Chemical Society: Washington, DC, 1998.


245 (phytoglycolipids) were found. These molecules feature long chain amino alcohols (sphingosine, phytosphingosine, dmydrophytosphingosine) as a structural fragment (3,4). In these lipids, the ceramide moiety is represented by the N-acylated 4hydroxysphinganine; the inositol part of molecule is usually linked with branched oligosaccharide chains, and ceramide and inositol portions are joined together by means of asymmetric phosphoric acid diester. All phytoglycolipids have the common structural core of myo-inositol-phosphoryl-ceramide unit. As compared to intensive synthetic studies of glycerophosphate-based inositol lipids, the synthetic chemistry of phytoglycolipids is much less developed. Meanwhile, in recent years it was discovered that sphingolipids are inhibitors of protein kinase C, and could be involved in control and regulation of the intracellular signal transduction pathways. This fact constituted the reason for our special interest in developing synthetic methods toward the series of inositol containing glycosphingophospholipids. This study included several main synthetic stages: (i) synthesis of asymmetrically substituted myoinositol precursors and sphingosine derivatives in the racemic or chiral forms, with the protection pattern allowing the subsequent introduction of phosphate and carbohydrate moieties at predetenriined positions; (ii) formation of phosphodiesters using trivalent phosphorus reagents; (iii) glycosylation of myoinositol intermediates using glycosyl fluoride, thioglycoside and oxazoline methods. Syntheses of Phosphatidylinositol β-D-Glucosaminides Acylation of 2,3:5,6-di-0-isopropylidene-myoinositol (1) [prepared according to a modified method of Gigg (5)] with levulinic acid in the presence of dicyclohexylcarbodiirnide gave a mixture of mono- and diacyl derivatives 2 and 3 (Scheme 1), respectively, which were separated by column chromatography on silica gel. The monohydroxyl derivative 2 was further used as a suitable intermediate for the introduction of a carbohydrate residue at the C4-position of the cyclitol ring. In the natural GPIs the carbohydrate portion linked to myoinositol is represented by the glucosamine with a free amino group. In the previous reports, formation of myoinositol β-glucosaminides was achieved by using the Koenigs-Knorr (6)> glycosyl fluoride (7) and tricWoroacetirnidate (8) methods, as well as npentenylglycosyl donors (9). These methods gave high stereospecifity and satisfactory yields of aminoglycosyl-myoinositols. In order to prepare some βglycosaminide analogues of natural GPIs we have developed new approach involving the oxazoline method of glycosylation. This method was originally used for the synthesis of oligosaccharides (10). Thus, 2-methyl-(3,4,6-tri-O-acetyl-1,2-dideoxya-D-glycopyranosyl)[2,l-d]-2-oxazoline (4) (77) was used as a glycosyl donor for coupling with the derivative 2. The glycosylation was carried out using equimolar oxazoline-alcohol ratio in the presence of otoluenesulfonic acid (ca. 1 hour in the refluxing mixture of nitromethane-toluene 1:1), and the resulting β-glucosaminide 5 was isolated by column chromatography on silica gel. The glycosylation procedure was found to be stereospecific, forming exclusively the 1,2-fraws-glycosidic product 5. The structure and anomeric configuration of 5 were confirmed by H - and C N M R spectroscopy. Selective removal of levulinoyl protecting group from 5 gave the monohydroxy derivative 6, suitable for coupling with the phosphatidyl portion. For this purpose we chose the methods of trivalent phosphorus chemistry, which showed major advantages when applied to the synthesis of phospholipids (72). The high reactivity of trivalent phosphorus derivatives makes it possible to form phosphoester linkages under mild reaction conditions with both primary and secondary hydroxyl groups. As a more convenient approach to myoinositol phosphoesters, we have decided to use the H-phosphonate method. This methodology is attractive due to !


1 3


246 experimental simplicity and easy availability of the reagents involved. Thus, the βglycosarninide 6 reacted with triethylammonium salt of 1,2-dipalmitoyl-rac-glycero3H-phosphonate (7) (73), and the resulting crude glycero-H-phosphonate was immediately oxidized into the corresponding protected phosphoinositide 8 (Scheme 1). The subsequent stepwise removal of the isopropylidene and acetyl groups gave the desired l-0-(rac-l,2-dipalmitoylglycerophospto pyranosyl)-myoinositol (9) after purification by column chromatography on silica gel. The structure of glycophospholipid 9 was verified by standard spectroscopic methods, and was also confirmed by partial acidic hydrolysis. Refluxing 9 with 0.5 N HC1 in methanol yielded a mixture of D-glucosamine and phosphatidylinositol that were chromatographically identified by comparing with authentic specimens. General considerations suggest that myoinositol phosphodiesters could be synthesized via two alternative reaction sequences: (i) condensation of diacylglycerol H-phosphonate with myoinositol derivative, as has been demonstrated in Scheme 1; and (ii) coupling of myoinositol H-phosphonate with the diacylglycerol component. In order to study the second way we have synthesized l-0-(2-amino-2-deoxy-p-Dglucopyranosyl)^)-(rac-l,2-dpalmitoylglycerophospho)-myoinositol (16), which constitutes both an anomeric and structural isomer of natural GPIs because of the reversed location of the carbohydrate and phosphatidyl moieties at the 1- and 4positions of the cyclitol ring. The starting l-0-p-benzoylpropionyl-2,3;5,6-di-0-isopropylidene-D-myo inositol (10, Scheme 2) was prepared from the diketal 1 and β-benzoylpropionic acid in a similar way to the preparation of 2. The pentasubstituted derivative 10 was phosphitylated at -10°C with a freshly prepared triimidazolylphosphite followed by aqueous work-up to give myoinositol H-phosphonate 11 after chromatography, as an amorphous triethylammonium salt. The P - N M R spectrum of 11 confirmed full regioselectivity of phosphitylation, and the absence of by-products. The subsequent coupling of phosphonate 11 with 1,2-dipalmitoyl-rac-glycerol (12) in the presence of 2,4,6-triisopropylbenzenesulfonyl chloride or pivaloyl chloride gave the corresponding H-phosphonate diester which was immediately oxidized with iodine in aqueous pyridine to afford the phosphate diester 13. We found that pivaloyl chloride was the most convenient activating reagent due to very short reaction time and the absence of side processes. The next synthetic step required the selective removal of the β-benzoylpropionyl protecting group from 13, with retention of the fatty acid residues, followed by introduction of an aminosugar portion at the 1-position of the cyclitol. The selective removal of the β-benzoylpropionyl group from 13 by means of hydrazine hydrate in ethanol gave the protected phosphatidylinositol 14, having only one free hydroxyl group at the 1-position. By employing the oxazoline derivative 4, and using condition analogous to those reported for synthesis of 5, we obtained the product 15 which was converted into the final glycolipid 16 after the removal of the protecting groups. The above described syntheses of 9 and 16 demonstrate that combination of efficient phosphorylation using the trivalent phosphorus reagents and glycosylation using oxazoline methodology is a promising approach toward synthesis of complex myoinositol containing glycophospholipids. 31

Synthesis of Phosphatidylinositol Glucoside. Phospholipid antigens isolated from pathogenic bacteria Mycobacterium were identified as phosphatidylinositol mannosides, and are used as components of diagnostic reagents for tuberculosis and leprosy (14-16). Continuing our program aimed at synthesis of novel biologically active myoinositol derivatives, we took advantage of some recently developed methods in the chemistry of carbohydrates and lipids to accomplish the total synthesis of 1 -0-( 1,2-dipalmitoyl-rαc-glyœrophospho)-4-0-β-D-glycopyranosyl-myoinositol (22) as a structural analog of the bacterial phosphatidylinositol mannoside and the



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Synthesis of Inositol-Containing ... - ACS Publications

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