Inositolphospholipids and analogues

Details Inositolphospholipids and analogues Inositolphospholipids and analogues

2002-02-04

2004-05-18

Carr, Deborah D. (Department: 1621)

Organic compounds -- part of the class 532-570 series

Organic compounds

Fatty compounds having an acid moiety which contains the...

C554S079000, C558S091000

Reexamination Certificate

active

06737536

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
Inositolphospholipids are conjugates of a lipid and a myo-inositol linked by a phosphodiester bridge. This phospholipid group and the derived glycosylates and phosphates occur as minor components of biological cells, and are involved in vital cellular functions including intracellular signaling. Two biochemical parents are the phosphatidyl-myo-inositols (PtdIns) and the ceramide-phosphoinositols (CerPhosIns). The present invention concerns synthetic PtdIns, CerPhosIns and their structural and stereochemical analogues. It is not concerned with their glycosylated or phosphorylated derivatives. The invention specifically provides a novel approach to synthesis of inositolphospholipids which is suitable for laboratory scale preparation as well as for large scale industrial production. The synthetic approach is applicable equally well for the preparation of inositolphospholipids carrying saturated lipid chains, unsaturated lipid chains with one or more double or triple bonds, chains with hydroxyl, amino and other functional groups, or combinations of these. In addition, it provides novel high purity diastereomer molecular species of inositolphospholipids that have unequivocally defined structure and absolute stereochemistry in both the myo-inositol and the glycerol residues and are obtainable only by the present new approach. The invention further provides methods for characterizing and using these high purity diastereomeric compounds. The synthetic products have utility, inter alia, as biochemical reagents in studies on the structure and function of cell membranes and mechanisms of intracellular signaling, as reference compounds for analysis of cellular inositolphospholipids, as substrates in assays and diagnostics kits for enzymes involved in signaling via the inositolphospholipids, as lead compounds for the design and development of novel drugs for the treatment of disorders caused by aberrant signaling including diabetes and some cancers, for biodelivery of specific pharmacodynamic fattyacyls covalently incorporated in the inositolphospholipid structure, as the lipid component in liposomal delivery vehicles for cytotoxic drugs, bioactive peptides, proteins and polynucleotides, and in cosmetics formulations.
2. Description of Related Art
Inositolphospholipids: Structures, Biological Roles, and Utility
Inositolphospholipids constitute an important group of biological small molecules which includes PtdIns and CerPhosIns (Carter et al., 1965). Cellular PtdIns belong to the 1D-1-myo-inositol series and have the 1D-1-(1-fattyacyl′-2-fattyacyl
2
-sn-glycero-3-phospho)-myo-inositol absolute stereochemical structure. In the radyl analogues (Radyl-PtdIns), the 1-fattyacyl residue is replaced by O-alkyl, and in CerPhosIns, the 1,2-diacylglycero residue is replaced by a ceramide moiety. The corresponding lyso-series of inositolphospholipids lack the 2-fattyacyl or, and thus have a free hydroxyl group at the glycero-2 position. The sphingosyl-phosphoinositols (Sphingosyl-PhosIns) lack the amide fattyacyl and have a free 2-amino group which may be derivatized further. Representative structures of the cellular series follow.
As noted above, in cellular PtdIns the inositol moiety is in the 1D-1-myo-inositol and the glycerol moiety is in the sn-glycero-3-phospho configuration. The glycero residue is esterified with mixtures of saturated long carbon chain fattyacyls, and unsaturated and polyunsaturated fattyacyls collectively referred to as (poly)unsaturated fattyacyls. Thus PtdIns in natural phospholipids are complex mixtures of molecular species differing in their fattyacyl composition and distribution between glycero-1 and -2 positions. In the naturally occurring CerPhosIns series, the sphingosine/ceramide residue has the D-erythro stereochemical configuration. CerPhosIns show variable chain length and degree of unsaturation in the two alkyl groups of the ceramide residue, and may carry additional unsaturation, hydroxyl or related functional groups. Synthetic PtdIns analogues are based on the sn-glycero-3-phospho as well as the sn-glycero-1-phospho configurations, and the synthetic analogues of CerPhosIns are based on the D-erythro, L-erythro, D-threo, or L-threo stereochemical configurations in the sphingosine/ceramide residues. Diastereomers are formed by all combinations of the 1D-1- and 1L-1-myo-inositols with the aforementioned glycerol or sphingosine/ceramide configurations.
In eukaryotic cells, PtdIns are quantitatively minor but vital components of membrane lipids with critical structural and metabolic roles. In the structural role, PtdIns function akin to the more abundant phosphatidylcholines and phosphatidylethanolamines in membranes (Small, 1986) but this has not been studied in detail. In its metabolic roles, PtdIns is the parent participant in the vital PtdIns cycle which is responsive directly to various extracellular stimuli acting on the cell (Hokin, 1985). Agonist stimulated metabolism is mediated by combinations of many regulatory protein and enzyme families including the PtdIns transfer protein, PtdIns synthase, and phospholipases, kinases and phosphate-phosphatases specific for the PtdIns group implicated in intracellular signaling. Mono, bis, and trisphosphates of PtdIns are formed and their cellular concentrations are regulated by the actions of kinase and phosphate-phosphatase groups of enzyme families which are specific for the 3-, 4-, or 5-positions. The action of PtdIns specific phospholipase C (Rhee et al., 1989) on PtdIns-4,5-bisphosphate generates the intracellular second messengers inositol-1,4,5-trisphosphate and diacylglycerol which respectively mediate release of intracellular Ca ions (Berridge, 1984, 1987, 1993) and activation of protein kinase C (Nishizuka, 1986) respectively. The 3-phosphate series (Whitman et al., 1988) act as messengers in mitogenic and related signals more directly (Toker et al., 1994; Duckworth and Cantley, 1996). Action of cytosolic phospholipase A
2
liberates arachidonic acid from the sn-glycero-2-O-acyl moiety (Lapetina et al, 1981) which is utilized in the arachidonic acid-eicosanoid messenger cascades. Thus PtdIns is a direct and indirect reservoir of additional signaling molecules which mediate and control vital cellular functions (Bell et al., 1996). PtdIns moiety is the lipid component in glycosyl-phosphatidylinositols which function as membrane anchors of important cellular proteins (Englund, 1993), and as transducers in the insulin messenger cascade (Saltiel et al., 1986). The radyl and sphingo analogues of PtdIns and glycosyl-phosphatidylinositols have similar and additional roles (Ferguson and Williams, 1988).
Synthetic inositolphospholipids and analogues are required as research reagents in multifarious signaling and related biomedical fields some of which are summarized in section “Field of the Invention” and are further discussed below.
PtdIns as the amphiphile in liposomal drug delivery vehicles prevents recognition of vesicle surface by the phagocytic cells of the reticuloendothelial system, the circulating mononuclear phagocytic cells, and those located in liver and spleen, and enhances blood circulation time of the drug formulation (Lee et al., 1992).
PtdIns in tissues are mixtures of molecular species, mostly with saturated fattyacyls at the 1-glycero and polyunsaturated fattyacyls at the 2-glycero positions. In early studies, development of therapeutics was linked to the action of cytosolic phospholipase A
2
and the liberation of arachidonic acid from animal tissue derived PtdIns, and of linoleic and linolenic acids from plant derived PtdIns. These studies could not be pursued to definitive conclusions because methods of synthesis and consequently well defined synthetic PtdIns with selected (poly)unsaturated fatty acids were not available. However, very promising results were obtained using plant versus animal tissue derived PtdIns. Plant PtdIns was reported to have antiviral activity and recommended as a prophylactic for HIV; the identity of the fattya

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