Synthesis of D-3 phosphorylated phosphoinositides and analogues

Abstract

Disclosed are unique starting materials, reaction sequences and intermediate compounds for the preparation of D-3-phosphorylated phosphoinositides (3-PPI) of unambiguous structure and absolute stereochemistry. The enantiomerically pure D-3-phosphorylated phosphoinositides also provided have many uses, including the development of diagnostics and therapeutics based on the roles of 3-PPI in intracellular signaling.

What is claimed is:  
     
       1. Enantiomerically pure 1,2:4,5-di-O-cyclohexylidene-3-O-allyl-myo-inositol. 
     
     
       2. The enantiomerically pure compound of  claim 1 , wherein said compound is 1D-1,2:4,5-di-O-cyclohexylidene-3-O-allyl-myo-inositol. 
     
     
       3. The enantiomerically pure compound of  claim 1 , wherein said compound is 1L-1,2:4,5-di-O-cyclohexylidene-3-O-allyl-myo-inositol. 
     
     
       4. The enantiomerically pure compound of  claim 1 , wherein said compound is prepared by a method comprising esterifying (±)-1,2:4,5-di-O-cyclohexylidene-3-O-allyl-myo-inositol with an optically active carboxylic acid derivative and a tertiary amine; separating the diastereomeric esters by MPLC on silica or by conducting crystallization; and then performing alkali catalyzed hydrolysis of the separated individual esters. 
     
     
       5. The enantiomerically pure compound of  claim 4 , wherein said optically active carboxylic acid derivative is (1S)-(−)-camphanic acid chloride and wherein said tertiary amine is NEt 3 . 
     
     
       6. A method for synthesizing D-3 phosphorylated phosphoinositides, comprising the steps of: 
       (a) preparing the enantiomerically pure starting material:                    
        wherein R is H (hydroxyl group), 1D-series;  
       (b) protecting the hydroxyl group to prepare the 6-O-benzyl derivative:                    
        wherein R is benzyl;  
       (c) selectively removing the 4,5-O-cyclohexylidene protecting group to prepare the 4,5-diol compound:                    
        wherein R 1  and R 2  are H;  
       (d) protecting the 4- or 5-hydroxyls by partial allylation, or protecting the 4- and 5-hydroxyls by complete allylation, and separating to obtain:                    
        wherein R 1  is allyl and R 2  is H, or R 1  is H and R 2  is allyl, and there is one free hydroxyl in each derivative formed by the partial allylation reaction; or wherein both R 1  and R 2  are allyl and there is no free hydroxyl for the derivative formed by the complete allylation reaction;  
       (e) further protecting the products of any partial allylation reaction, or the unallylated 4,5-diol derivative, by benzylation of the free hydroxyl to obtain:                    
        wherein R 1  is allyl and R 2  is benzyl, or R 1  is benzyl and R 2  is allyl, or both R 1  and R 2  are benzyl, and there is no free hydroxyl in any derivative formed;  
       (f) removing the 1,2-O-cyclohexylidene protecting group from the allylated or benzylated derivatives containing no free hydroxyl to prepare the 1,2-diol compounds:                    
        wherein both R 1  and R 2  are allyl, or R 1  is allyl and R 2  is benzyl, or R 1  is benzyl and R 2  is allyl, or both R 1  and R 2  are benzyl;  
       (g) selectively protecting the 1-hydroxyl with 4-methoxybenzyl to prepare:                    
        wherein PMB is 4-methoxybenzyl, and both R 1  and R 2  are allyl, or R 1  is allyl and R 2  is benzyl, or R 1  is benzyl and R 2  is allyl, or both R 1  and R 2  are benzyl;  
       (h) protecting the free hydroxyl to prepare the 2-benzyl derivatives:                    
        wherein PMB is 4-methoxybenzyl, and both R 1  and R 2  are allyl, or R 1  is allyl and R 2  is benzyl, or R 1  is benzyl and R 2  is allyl, or both R 1  and R 2  are benzyl;  
       (i) deprotecting the hydroxyls by removing the allyl group or groups to produce:                    
        wherein PMB is 4-methoxybenzyl, and both R 1  and R 2  are H, or R 1  is H and R 2  is benzyl, or R 1  is benzyl and R 2  is H, or both R 1  and R 2  are benzyl;  
       (j) phosphorylating the deprotected compound to prepare the dibenzylphosphate derivatives:                    
        wherein PMB is 4-methoxybenzyl, and both R 1  and R 2  are dibenzylphosphate, or R 1  is dibenzylphosphate and R 2  is benzyl, or R 1  is benzyl and R 2  is dibenzylphosphate, or both R 1  and R 2  are benzyl;  
       (k) selectively deprotecting by removing the 4-methoxybenzyl group to produce the 1-hydroxyl derivatives:                    
        wherein both R 1  and R 2  are dibenzylphosphate, or R 1  is dibenzylphosphate and R 2  is benzyl, or R 1  is benzyl and R 2  is dibenzylphosphate, or both R 1  and R 2  are benzyl;  
       (l) condensing the 1-hydroxy derivatives formed by selective deprotection with an sn-3-phosphatidic acid to prepare:                    
        wherein both R 1  and R 2  are dibenzylphosphate, or R 1  is dibenzylphosphate and R 2  is benzyl, or R 1  is benzyl and R 2  is dibenzylphosphate, or both R 1  and R 2  are benzyl; and wherein R 3  and R 4  are acyl groups; and  
       (m) completely deprotecting to remove the benzyl ether and benzyl (phosphate)ester groups to prepare the D-3 phosphorylated phosphoinositides:                    
        wherein both R 1  and R 2  are phosphate, or R 1  is phosphate and R 2  is H, or R 1  is H and R 2  is phosphate, or both R 1  and R 2  are H; and wherein R 3  and R 4  are acyl groups.  
     
     
       7. The method of  claim 6 , wherein R 3  and R 4  in the compounds produced by step (l) and step (m) are the same acyl group. 
     
     
       8. The method of  claim 6 , wherein R 3  or R 4  in the compounds produced by step (l) and step (m) is a short-chain fattyacyl group. 
     
     
       9. The method of  claim 6 , wherein R 3  or R 4  in the compounds produced by step (l) and step (m) is a long-chain fattyacyl group. 
     
     
       10. The method of  claim 6 , wherein R 3  or R 4  in the compounds produced by step (l) and step (m) is a hexadecanoyl group. 
     
     
       11. The method of clam  6 , wherein R 3  and R 4  in the compounds produced by step (l) and step (m) is a saturated fattyacyl group. 
     
     
       12. The method of  claim 6 , wherein R 3  or R 4  in the compounds produced by step (l) and step (m) is an unsaturated fattyacyl group. 
     
     
       13. The method of  claim 6 , wherein R 3  or R 4  in the compounds produced by step (l) and step (m) is a polyunsaturated fattyacyl group. 
     
     
       14. The compound 1D-1-(1′,2′-difattyacyl-sn-glycero-3′phospho)-2,6-dibenzyl-myo-inositol-3,4,5-tris (dibenzylphosphate), 1D-1-(1′,2′-difattyacyl-sn-glycero-3′-phospho)-2,5,6-tribenzyl-myo-inositol-3,4-bis (dibenzylphosphate), 1D-1-(1′,2′-difattyacyl-sn-glycero-3′-phospho)-2,4,6-tribenzyl-myo-inositol-3,5-bis (dibenzylphosphate) or 1D-1-(′,2′-difattyacyl-sn-glycero-3′-phospho)-2,4,5,6-tetrabenzyl-myo-inositol-3-dibenzylphosphate, as in the structure:                    
       wherein both R 1  and R 2  are dibenzylphosphate, or R 1  is dibenzylphosphate and R 2  is benzyl, or R 1  is benzyl and R 2  is dibenzylphosphate, or both R 1  and R 2  are benzyl; and R 3  and R 4  comprise short-chain, long-chain, saturated, unsaturated or polyunsaturated fattyacyl or alkyl residues.  
     
     
       15. The compound of  claim 14 , wherein said compound is 1D-1-(1′,2′-dihexadecanoyl-sn-glycero-3′-phospho)-2,6-dibenzyl-myo-inositol-3,4,5-tris(dibenzylphosphate), 1D-1-(1′,2′-dihexadecanoyl-sn-glycero-3′-phospho)-2,5,6-tribenzyl-myo-inositol-3,4-bis(dibenzylphosphate), 1D-1-(1′,2′-dihexadecanoyl-sn-glycero-3′-phospho)-2,4,6-tribenzyl-myo-inositol-3,5-bis(dibenzylphosphate) or 1D-1-(1′,2′-dihexadecanoyl-sn-glycero-3′-phospho)-2,4,5,6-tetrabenzyl-myo-inositol-3-dibenzylphosphate, as in the structure:                    
       wherein both R 1  and R 2  are dibenzylphosphate, or R 1  is dibenzylphosphate and R 2  is benzyl, or R 1  is benzyl and R 2  is dibenzylphosphate, or both R 1  and R 2  are benzyl; and R 3  and R 4  are each hexadecanoyl.  
     
     
       16. A D-   3   - phosphorylated phosphoinositide of the structure:                      
       
         wherein  
       
       
         RCO are each fattyacyl, and at least one RCO is hexanoyl or a shorter chain fattyacyl;  
       
         R   1   =H or P ( O )( OH ) 2   ; and    
         R   2   =H or P ( O )( OH ) 2 . 
     
     
       17. The D-   3   - phosphorylated phosphoinositide of    claim 16   , wherein one RCO is hexanoyl or a shorter chain fattyacyl.   
     
     
       18. The D-   3   - phosphorylated phosphoinositide of    claim 17   , wherein the RCO residue is hexanoyl.   
     
     
       19. The D-   3   - phosphorylated phosphoinositide of    claim 16   , wherein both RCO residues are hexanoyl or a shorter chain fattyacyl fattyacyl.   
     
     
       20. The D-   3   - phosphorylated phosphoinositide of    claim 19   , wherein both RCO residues are hexanoyl.   
     
     
       21. A D-   3   - phosphorylated phosphoinositide of the structure:                      
       
         wherein  
       
         RCO are each fattyacyl, and at least one RCO is an ω - aminofattyacyl;    
         R   1   =H or P ( O )( OH ) 2   ; and    
         R   2   =H or P ( O )( OH ) 2 . 
     
     
       22. The D-   3   - phosphorylated phosphoinositide of    claim 21   , wherein one RCO is an ω - aminofattyacyl conjugated to a reporter group.   
     
     
       23. The D-   3   - phosphorylated phosphoinositide of    claim 22   , wherein the reporter group is a fluorescent reporter group.   
     
     
       24. A D-   3   - phosphorylated phosphoinositide of the structure:                      
       
         wherein  
       
       
         RCO are each fattyacyl, and at least one RCO is an unsaturated or polyunsaturated fattyacyl;  
       
         R   1   =H or P ( O )( OH ) 2   ; and    
         R   2   =H or P ( O )( OH ) 2 . 
     
     
       25. The D-   3   - phosphorylated phosphoinositide of    claim 24   , wherein one RCO is an unsaturated fattyacyl.   
     
     
       26. The D-   3   - phosphorylated phosphoinositide of    claim 24   , wherein both RCO residues are unsaturated fattyacyls.   
     
     
       27. The D-   3   - phosphorylated phosphoinositide of    claim 24   , wherein one RCO is a polyunsaturated fattyacyl.   
     
     
       28. The D-   3   - phosphorylated phosphoinositide of    claim 24   , wherein both RCO residues are polyunsaturated fattyacyls.   
     
     
       29. A D-   3   - phosphorylated phosphoinositide obtained by a process comprising the regioselective  3   - phosphorylation of preformed phosphatidylinositol or derived phosphates lacking the D -   3   - phosphate as the starting materials, wherein said starting materials lacking the D -   3   - phosphate are from natural plant or animal cell sources and contain  ( poly ) unsaturated fattyacyls, or wherein said starting materials lacking the D -   3   - phosphate are synthetic phosphatidyl - inositols with unsaturated fattyacyls, and of the structure:                      
       
         wherein  
       
         R   1   =H or P ( O )( OH ) 2   ; and    
         R   2   =H or P ( O )( OH ) 2 . 
     
     
       30. A synthetic D-   3   - phosphorylated phosphoinositide library comprising a mixture of D -   3   phosphorylated phosphoinositides in accordance with the structure:                    
       
         wherein  
       
       
         RCO are each fattyacyl;  
       
         R   1   =H or P ( O )( OH ) 2   ; and    
         R   2   =H or P ( O )( OH ) 2 .