Organic compounds -- part of the class 532-570 series – Organic compounds – Phosphorus esters
Reexamination Certificate
1997-06-10
2001-05-15
Ambrose, Michael G. (Department: 1626)
Organic compounds -- part of the class 532-570 series
Organic compounds
Phosphorus esters
C558S161000, C558S179000, C558S180000, C558S186000
Reexamination Certificate
active
06232486
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention is concerned with certain structural and stereochemical analogues of the phosphoinositide group of cellular lipids, novel approaches for their preparation by synthesis and key starting materials and intermediates of these approaches. The phosphoinositides comprising 1
D
-1-(1′,2′-di-O-fattyacyl-sn-glycero-3′-phospho)-myo-inositols or phosphatidylinositol (PtdIns) and its mono- and poly-phosphate derivatives are key participants in the intracellular signaling cascade which is generated in response to stimulation of certain cell surface receptors by many agonists. Biosynthetic and metabolic transformations of the phosphoinositides are implicated in initiating, sustaining and regulating this signal cascade in an agonist and tissue specific manner. These lipid transformations are catalyzed by several families of enzymes including the phosphatidylinositol-specific phospholipase C (PI-PLC) and the phosphatidylinositol 3-kinase (PI 3-kinase). Stimulated hydrolysis of phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P
2
), the substrate preferred by the mammalian PI-PLC, is representative. This hydrolysis rapidly and simultaneously generates inositol-1,4,5-trisphosphate (IP
3
) and sn-1,2-diacylglycerol (DAG). Both IP
3
and DAG are second messengers respectively inducing Ca
++
mobilization from intracellular stores and protein kinase C (PKC) activation and are implicated in many physiological responses including mitogenesis (Berridge, 1984; Nishizuka, 1983). Specific PI-PLC enzymes function also in releasing membrane-anchored proteins using glycosyl-PtdIns as the anchoring ligand. PI 3-kinase specifically associates with and is phosphorylated by activated growth factor receptors and oncoproteins which manifest protein-tyrosine kinase activity (Whitman et al, 1988; Auger et al, 1989). It phosphorylates PtdIns(4,5)P
2
specifically at the D-3 hydroxyl to produce phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P
3
) which is the putative novel and critical second messenger of growth signals (Auger et al, 1989; Carpenter and Cantley, 1990; Coughlin et al, 1989; Majerus, 1992). A complex role for PI 3-kinase and its products, the 3-phosphorylated phosphoinositides (3PPI), is emerging in the control of cell division and growth (Carpenter and Cantley, 1996). Additionally a role is seen for PI 3-kinase in transient actin polymerization and association between actin and cellular cytoskeletal elements, and a possible connection between this and the role in cell growth. Thus there is tremendous current interest in elucidating the structure, biochemical behavior, and physiological roles of the various isoforms of these key enzymes and in tracing the downstream targets of the products of their action on the phosphoinsoitides. Probes and modulators incorporating the core PtdIns(4,5)
2
structure as provided by the present invention are required for these multifarious ongoing research investigations.
Endogenous IP
3
is dephosphorylated and is reutilized with DAG for the resynthesis of PtdIns, the PtdIns is rephosphorylated to PtdIns-phosphates, and the latter are converted back to PtdIns by the action of PtdIns-phosphate phosphatases, in the overall PtdIns metabolic cycle. Exogenous inositols and PtdIns, including structurally modified analogues such as those disclosed in Kozikowski (U.S. Pat. No. 5,053,399), Kozikowski et al (U.S. Pat. No. 5,227,508), and, Yang et al (U.S. Pat. No. 4,515,722) are incorporated into the PtdIns cycle and ostensibly into the PtdIns-phosphate pool. The characterization of the biosynthetic PtdIns-phosphates produced from the aforementioned modified exogenous inositols and PtdIns derivatives requires the corresponding modified-PtdIns(4,5)P
2
and related derivatives as reference reagents. These reference reagents are provided by the present invention.
In the prior art (Yang et al, U.S. Pat. No. 4,515,722) synthesized 2-modified analogues of PtdIns and found these to be useful antiinflammatory/analgesic agents. These analogues all incorporated DL-inositol moieties and the preferred lipid moiety was 1-(3′,4′-acyloxybutylphosphonyl. The same biological activity was also claimed for unspecified PtdIns-phosphate derivatives but no application as a research reagent was disclosed.
Several phosphoinositide analogues are known in the prior art relevant to the present invention. The fluorescent 1-pyrenebutyl myo-inositol-1-phosphate and the chromogenic 4-nitrophenyl myo-inositol-1-phosphate have been described as reagents for the assay of bacterial PI-PLC (Hendrickson et al, 1992; Shashidhar et al, 1991) but are poor substrates and considered to be inadequate reagents (Bruzik and Tsai, 1994). The preparation of a nanomolar quantity of a pyrene-labelled PtdIns(4,5)P
2
from the corresponding pyrene-labelled PtdIns by successive phosphorylations at 4-O and 5-O by partially purified PtdIns 4-kinase and PI 5-kinase has been reported also (Gadella et al, 1990) but the required enzyme reagents and method of preparation are not easily accessible. Synthetic PtdIns(4,5)P
2
labelled with photoactive p-benzoyldihydrocinnamoyl and related reporter groups covalently attached to either the 1′-acyloxy or the 1-phosphate have been reported recently (Gu and Prestwich, 1996; Chen et al, 1996). These analogues are broadly similar, but attachment of the reporter group at 1-phosphate creates a 1-phosphotriester analogue and thereby destroys the core 1-phosphodiester function which is an essential structural feature of PtdIns(4,5)P
2
and all phosphoinositide substrate of PI-PLC.
It is considered that a sufficient range of appropriate biochemical probes and modulators of these enzymes are not available (Bruzik and Tsai, 1994). Therefore, an objective of the present invention is to provide substrate analogues as structure/mechanism-based probes and modulators suitable for research studies on PI-PLC, PI 3-kinase and related enzyme families. Additional objectives are to provide novel approaches for their preparation by synthesis and key starting materials and intermediates of these approaches.
SUMMARY OF THE INVENTION
This invention comprises several synthetic analogues of the preferred phosphoinositide substrates of the mammalian PI-PLC and PI 3-kinase enzyme families, exemplified by PtdIns(4,5)P
2
, which retain the core structural requirements for efficient bonding and catalysis, but in which the 2-OH is rendered non-nucleophilic by derivatization or replacement exemplified by 2-OAc and 2-deoxyfluoro respectively, and, which may additionally contain photoaffinity, fluorescent, spin, other reporter groups, and conjugands for linking to polymer, chromatographic matrix, or gold surfaces are incorporated in the fatty acyl or inositol residues as shown in structure. Thus, the invention provides substrate analogues as structure/mechanism-based probes and modulators suitable for research studies on PI-PLC, PI 3-kinase and related enzyme families. Additionally, it provides novel approaches for their preparation by synthesis, and key starting materials and intermediates of these approaches.
REFERENCES:
patent: 4515722 (1985-05-01), Yang et al.
patent: 5053399 (1991-10-01), Kozikowski
patent: 5227508 (1993-07-01), Kozikowski et al.
Elie, C.J.J. et al. Tetrahedron 45(11), 3477-3486 (1989).*
Lewis, K.A. et al. Biochemistry 32, 8836-8841 (1993).*
Chen, J. et al. Journal of Organic Chemistry 1996, 61(1), 393-397.*
Bruzik & Tsai, “Toward the Mechanism of Phosphoinositide-Specific Phospholipases C,”Bioorganic&Medicinal Chemistry,vol. 2, No. 2, pp. 49-72, 1994.
Chen et al., Synthesis of Photoactivatable 1,2-0-Diacyl-sn-glycerol Derivatives of 1-L-Phosphatidyl-D-myo-inositol 4,5-Bisphosphate (PtdInsP2) and 3,45-Trisphosphate (PtdInsP3),J. Org. Chem.,vol. 61, pp. 6305-6312, 1996.
Gadella et al., “Enzymatic Synthesis of Pyrene-Labeled Polyphosphoinositides and Their Behavior in Organic Solvents and Phosphatidylcholine Bilayers,”Biochemistry,vol. 29, pp. 3389-3395, 1990.
Gu & Prestwich, Synthesis of Phosphotriester Analog
Ambrose Michael G.
Nutrimed Biotech
Williams Morgan & Amerson
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