# Patent

## Title
Immobilized phosphatidic acid probe

## Abstract

The invention relates to immobilized phosphatidic acid probes which can be used to identify important proteins for signal transduction, housekeeping and diagnosis.

## Background
This application is a 371 of PCT/GB01/03791 on Aug. 23, 2001, which is hereby incorporated by reference.
FIELD OF THE INVENTION
The invention relates to a solid support reagent to detect the presence or expression level of proteins in biomedical research. The invention relates to probes which can be used to identify important proteins for signal transduction, housekeeping and diagnosis.
BACKGROUND TO THE INVENTION
Phosphatidic acid (PA) synthesised via the glycerol-3-phosphate or the dihydroxyacetone phosphate pathway is an important intermediate in the biosynthesis of glycerophospholipids and triacylglycerols [Athenstaedt and Daum, Eur. J. Biochem. 266: 1-16 (1999)]. In addition, because PA can be produced from hydrolysis of phosphatidylcholine (PC) by phospholipase D (PLD), it is recently attracting considerable interest as a potential second messenger. This hypothesis is based on the observation that many agonists cause PLD-activation (and thus PA formation) in a variety of cell types and tissues to regulate many cellular pathways including secretion, respiratory burst, calcium influx, mitosis, etc [Exton, Biochimica et Biophysica Acta 1439: 121-133 (1999)]. If PA is indeed a second messenger, it would be expected to interact specifically with a certain class of cellular proteins. However, the identification of such proteins has not been achieved to date in a systematic approach, and would be extremely complicated by conventional methods for the following reasons:

    
    
        1. PA is unstable in cells since it is rapidly dephosphorylated to give diacylglycerol [Hodgin et al. TIBS 23: 200-204 (1998)].
        2. Free PA (both synthetic or from biological origin) is difficult to handle due to its poor solublity in aqueous media. Extensive sonication to obtain liposomes is hence required. (Pure naturally-occurring or synthetic PA is difficult to solubilise in vitro, requiring either organic solvents or extensive sonication to produce liposomes).
        3. Even if PA were reconstituted into liposomes, it would be impossible to identify detergent-extracted PA-binding proteins (vide infra) since the detergent used would destroy the integrity of the liposome structure.
    
    


SUMMARY OF THE INVENTION
To overcome the above problems, and to identify the class of proteins which bind PA, we have invented an immobilised PA derivative attached onto a solid support. An example is illustrated in Formula I. The preferred stereochemistry is shown in Formula II. Our invention can become an important research tool in fundamental research and it will provide unique opportunities in the fields of diagnostics and drug discovery. In the following description we use the terms “PA resin” and “PA bead” to refer to the immobilised phosphatidic acid derivative probes of this invention.




A further embodiment is illustrated in Formula III. The preferred stereochemistry is shown in Formula IV.




The invention covers phosphatidic acid functionalised solid supports of the general formula as is depicted in Formulae I, II, III and IV and includes the following characteristics:
The linker consists of aryl, heteroaryl, alkyl with possible heteroatoms and/or unsaturations, preferably chains of (CH2)n, with n=8-20, most preferably n=11. The heteroatom X maybe O, S, or, most preferably NH. The functional group (FG) is a carbonyl from a carboxylate (thiolo)ester, or, most preferably an amide. The R-substituent at the sn2-position or sn-position of the sn-glycerol-3-phosphate derivative carries an aryl, alkyl group, or a combination, preferably R=CmH2m+1, m=8-20, m=16 is optimal. Unsaturations are allowed, such as in an arachidonyl side chain. It is preferred that the diacyl glycerol is based on the sn-glycerol-3-phosphate family, as in the natural series. Alternatively, the diacyl glycerol may be based on the enantiomeric sn-glycerol-1-phosphate family, or a racemic mixture may be used. The phosphate head groups maybe substituted by phosphonic acid or thiono phosphate. The ion M represents any cation, preferably Na+, NH4+. The solid support with the attachment to the functional group is illustrated as:




Any suitable covalent attachment may link the solid support to the functional group. It is to be noted that this symbolic illustration is not to be interpreted as representing solely a —CH2— linkage between the functional group and the solid support. The nature of the solid supports is limited to those which swell in water, such as agarose, sepharose, PEG based resins.
Examples of probes covered by these general formulae are shown in Table 2.
In a further embodiment the PA-functionalised solid support maybe modified to carry photoaffinity labels such as aryl azides, α-halo-carbonyl compounds, diaryl ketones, etc. for mapping the binding pocket. Specifically, a 125I-labeled azido salicylate (Pierce product catalogue, 2000) could be attached through an α-amide linkage at the sn2-position of the sn-glycerol-3-phosphate derivative. After incubation with a specific PA-binding protein, photolysis, hydrolysis of the acyl glycerol bonds, and PAGE analysis of the tryptic digests, information about the radio-labelled binding sequence may be obtained.
Yet another embodiment involves attachment of fluorescent reporter groups in the carboxyl side chain ester attached to the sn2-alkoxy substituent of the sn-glycerol-3-phosphate derivative (Formulae I or II) or to the carboxyl side chain ester attached to the sn-1 position of the sn-glycerol-3-phosphate derivative (Formulae III or IV) to obtain binding affinities.
The specificity of the PA resin for tissue culture extracts may be influenced by the nature of detergent used.
Currently, our preferred surfactant is a non-ionic surfactant NP-40, which is commercially available and is similar to TRITON (RTM) X-100 (which is polyoxyethylene (9.5 average) p-t-octylphenol). We believe that the inclusion of a non-ionic surfactant aids and/or maintains the specificity of the probe for PA.
The linker has the function of affecting the way that the phosphatidic “head group” is presented (e.g. for recognition and binding by a specific protein). The choice of linker may be made so as either to mimic the “natural” presentation of the head group as it would occur at a lipid membrane surface, or alternatively the linker may be chosen deliberately so as to result in a non-natural presentation. Those skilled in the art will recognise that since there are many (about 50) possible different types of PA in a cell, the ability to manipulate the linker of the PA resin of the invention can provide useful benefit.
With regard to the stereocentre of the diacyl glycerol, it is to be noted that the stereochemistry in the natural series is based on sn-glycerol-3-phosphate (Formula II, IV). Under certain circumstances it might be desirable to have the chiral carbon in a non-natural stereochemistry (or enantiomeric, as allowed by Formla I or III, based on sn-glycerol-1-phosphate) as this might give interesting/useful effects on the binding and/or specificity of PA.
With regard to the phosphate head group, it may be desirable to make a substitution (as described, above), for example in order to protect the group from hydrolysis. It is to be noted that substitution by e.g. phosphoric acid or thiono phosphate would render the head group resistant to hydrolysis by endogenous or added hydrolase.
In other embodiments of the invention, other derivatives of phosphatidic acid are attached onto a solid support to provide probes of the invention. Such derivatives include phosphoinositides such as (D)-Ptd Ins (3,4,5) P3 (PIP3), (D)-Ptd Ins (3,4) P2 (PIP2), (D)-Ptd Ins (3,5) P2 (PIP2), (D)-Ptd Ins (4,5) P2 (PIP2), and (D)-Ptd Ins (3) P (PIP) (collectively referred to as PIPn's,—see FIG. 3), or analogues thereof. Examples are shown in Table 3. Immobilised PIPn's or their analogues can be used to identify PIPn binding proteins.
The immobilised PA-derivative or phosphoinositide may be non-covalently or covalently attached onto the solid support. Where the PA-derivative or phosphoinositide is non-covalently attached it is preferred that the strength of the non-covalent attachment is such that it is not disrupted under conditions in which a protein may be bound specifically to the probe. A suitable binding energy is greater than that of thiol for gold (i.e. greater than about 200 KJ/mole).
Rao et al (JBC 1999, 274 (53), 37893-37900) reports avidin-coated beads pre-bound to biotinylated PtdIns-3,4-P2 and PtdIns-3,4,5-P3, as shown in FIG. 1 on page 37894 of that document.
We have appreciated that phosphoinositides other than PtdIns-3,4-P2 and PtdIns-3,4,5-P3 may be non-covalently immobilised onto a solid support to provide probes for use in identifying binding proteins for those phosphoinositides. For example, any of the free phosphoinositides shown in Table 3, or functional analogues thereof may be non covalently attached onto a solid support.
It is preferred, however, that a PA-derivative or phosphoinositide is covalently attached onto the solid support. A disadvantage of non covalent attachment is that the non covalent interaction is disrupted under conditions required to remove proteins bound to the solid support. Thus, the solid support cannot be reused after an analysis of protein binding has been performed. This can add significantly to the cost of performing experiments, particularly where more expensive solid supports are used, such as agarose beads.
Covalent attachment of inositol 1,4,5-trisphosphate (IP3) to Affi-Gel 10 resin has been described by Prestwich et al 1991 (Am. Chem. Soc. 1991, 113, 1822-1825). This document discloses attachment of the 1-O-(3-aminopropyl)ester of inositol 1,4,5-trisphosphate (IP3) to resin (as shown in Scheme I, compound 1c, on page 1823 of the document) and use of the resulting bioaffinity matrix to purity the known IP3 receptor (IP3R). Note that IP3 is not a “phosphoinositide”.
Shirai et al (Biochim. Biophys. Acta 1998, 1402, 292-302) discloses attachment of a PI 3,4,5-P3 analogue (PIP3-APB, as shown in FIG. 1on page 294 of the document) to Affi-Gel 10 beads and their use to purify PIP3 binding proteins.
In one aspect of the invention phosphoinositides other than PIP3-APB may be attached onto a solid support. Preferably the attachment is covalent. Suitable examples are shown in Table 3.
Probes of the invention may be used to identify PA binding proteins or phosphoinositide binding proteins. In order to efficiently identify such proteins, it is advantageous if the probes can bind proteins which are present in relatively low abundance and/or proteins which have relatively low PA or phosphoinositide affinity. We believe that key factors in binding of relatively low abundance and/or low affinity proteins are: the part of the PA-derivative or phosphoinositide which is covalently linked to the solid support; the physical characteristics of the linkage; and the nature of the groups at the sn1-, and sn2-positions of the sn-glycerol-3-phosphate derivative.
In particular, it is thought that attachment of the PA-derivative or phosphoinositide via a long-chain fatty acid side chain of the molecule to the solid support ensures that the head group of the PA-derivative or phosphoinositol is available for binding by a PA-binding or phosphoinositide-binding protein. It is believed that this arrangement mimics cellular PA or phosphoinositide. It is thought that the length of the linkage between the head group and the solid support should not be too short, otherwise the solid support may sterically interfere with binding. The presence of a fatty acid side chain, and especially a long-chain fatty acid, at the sn1-position and/or the sn2-position position of the sn-glycerol-3-phosphate derivative is thought to be required for optimal binding by PA/phosphoinositide binding proteins. Suitable length fatty acid side chains are 8-20 carbon atoms in length, unsaturations being allowed. Fatty acid side chains with 17 carbon atoms (including the ester carbon) are preferred at the sn2-position of the sit-glycerol-3-phosphate derivative, and fatty acid side chains with 12 carbon atoms (including the ester carbon) are preferred at the sn1-position of the sn-glycerol-3-phosphate derivative, as these lengths are thought to best mimic cellular PA/phosphoinositide.
Our results show that probes of the invention with suitable length fatty acid side chains specifically bind several proteins (see for example Table 1). It is believed that such probes can be used to detect many more phosphoinositide binding proteins than the PIP3-APB beads described by Shirai et al.
Examples of embodiments of the invention in which a phosphoinositide is covalently attached onto a solid support are shown in general Formulae V and VI:




where:

    
    
        R=aryl, alkyl group, or a combination, preferably R=CmH2m+1,
        m=8-20, m=16 is optimal.
        R3=P(O)(OM)2; R4=H; R5=H (PI(3)P);
        R3=H; R4=P(O)(OM)2; R5=H (PI(4)P);
        R3=H; R4=H; R5=P(O)(OM)2 (PI(5)P);
        R3=P(O)(OM)2; R4=P(O)(OM)2; R5=H (PI(3,4)P2);
        R3=P(O)(OM)2; R4=H; R5=P(O)(OM)2 (PI(3,5)P2);
        R3=H; R4=P(O)(OM)2; R5=P(O)(OM)2 (PI(4,5)P2); or
        R3=P(O)(OM)2; R4=P(O)(OM)2; R5=P(O)(OM)2 (PI(3,4,5)P3).
        M=any cation, preferably Na+, NH4+
        *Denotes a stereogenic centre. More preferably a stereogenic centre with an R absolute configuration.
        Linker=aryl, heteroaryl, alkyl with possible heteroatoms and/or unsaturations. Preferably chains of (CH2)n with n=8-20, most preferably n=11.
        X=O, S, or, most preferably NH.
        FG=Carbonyl from a carboxylate, thiolo(ester), or, most preferably an amide.
        Unsaturations are allowed, such as in an arachidonyl side chain.
    
    






    
    
         =solid support with attachment to functional group. Any suitable covalent attachment may link the solid support to the functional group. Suitable solid supports are organic polymeric supports. Preferred solid supports are those which swell in water, such as agarose, sepharose, PEG based resins. Affi-Gel 10 and Affi-Gel 15 (Bio-Rad) are preferred examples.
    
    


Examples of probes covered by these general formulae are shown in Table 3.
It is to be noted that one or more of the free OH groups on the inositol ring of formulae V, VI, VII, VIII (and formulae V′, VI′, VII′, VIII′, V′′, VI′′, VII′′, VIII′′, see below) could be O-alkyl, or O-aryl derivatives or heteroatom analogues (examples of heteroatoms being S, N, P). One or more of the phosphate esters in formulae V, VI, VII, VIII (and formulae V′, VI′, VII′, VIII′, V′′, VI′′, VII′′, VIII′′, see below) could be replaced by a thionophosphate, or a phosphinic acid derivative or a phosphonic acid derivative.
It may be that effective protein binding to probes of the invention may be achieved where the optimum length of the linker attached to the sn1-, or sn-2position of the sn-glycerol-3-phosphate derivative is reduced, but a longer attachment to the solid support is used. This is expected to occur in cases where a protein recognises the head group of the PA-derivative or phosphoinositide, substantially independently of the identity of the fatty acid side chains (or other groups attached to the sn1-, and sn2-positions of the sn-glycerol-3-phosphate derivative), but is sterically inhibited from binding unless the head group is at least a certain distance from the solid support. Suitable probes are provided according to General formulae VII and VIII:




Where:

    
    
        R=aryl, alkyl group, or a combination, preferably R=CmH2m+1,
        m=8-20, m=16 is optimal.
        R3=P(O)(OM)2; R4=H; R5=H (PI(3)P);
        R3=H; R4=P(O)(OM)2; R5=H (PI(4)P);
        R3=H; R4=H; R5=P(O)(OM)2 (PI(5)P);
        R3=P(O)(OM)2; R4=P(O)(OM)2; R5=H (PI(3,4)P2);
        R3=P(O)(OM)2; R4=H; R5=P(O)(OM)2 (PI(3,5)P2);
        R3=H; R4=P(O)(OM)2; R5=P(O)(OM)2 (PI(4,5)P2); or
        R3=P(O)(OM)2; R4=P(O)(OM)2; R5=P(O)(OM)2 (PI(3,4,5)P3).
        M=any cation, preferably Na+, NH4+
        *Denotes a stereogenic centre. More preferably a stereogenic centre with an R absolute configuration.
        X=O, S, or, most preferably NH.
        FG A=Carbonyl from a carboxylate, thiolo(ester), or, most preferably an amide.
        Linker A=aryl, heteroaryl, alkyl with possible heteroatoms and/or unsaturations. Preferably chains of (CH2)n.
        Linker B=aryl, heteroaryl, alkyl with possible heteroatoms and/or saturations.
        These could be any atoms, more preferably C, N, O, S, more preferably still methylene groups linked via amide and ester bonds.
        Preferably the total length of linker A and linker B is 8-60 atoms, more preferably 19-31 atoms, most preferably 22 atoms.
        FG B=Amide, thiolo(ester), or, most preferably ester.
        Unsaturations are allowed, such as in an arachidonyl side chain.
    
    






    
    
         =solid support with attachment to functional group.
    
    


It will be appreciated that equivalent structures may be provided for general formulae I-IV.
There is also provided according to the invention use of a probe of the invention to bind a binding partner of the Phosphatidic Acid derivative or phosphoinositide attached onto the solid support. Preferably the binding partner is a protein.
A probe of the invention may be used for testing the PA/PIPn-binding and/or affinity of a protein.
The invention provides an assay method which involves the step of detecting and/or measuring the binding of a probe of the invention when said probe is exposed to a protein in a test sample. Such an assay may involve the steps of identifying and/or isolating said protein by binding to said probe. Said probe may be used to detect/measure/identify and/or isolate more than one type of PA and/or PIPn binding protein from a test sample containing many proteins. More than one type of probe may be used to detect/measure/identifying and/or isolate more than one type of PA and/or PIPn binding protein. The test sample may be a tissue or tissue culture extract, preferably a lysed extract. The test sample may be obtained by lysis of cells in a buffer containing at least one non-ionic surfactant, such as TRITON (RTM) X-100 or NP-40. The probe may be exposed to said test sample in the presence or absence of soluble PA and/or PIPn. Protein-probe binding may be compared between more than one test sample to determine PA-binding protein and/or PIPn binding protein variation between said samples.
There is also provided: use of an assay method of the invention to detect/measure/identify and/or isolate a PA-binding protein and/or a PIPn-binding protein in a test sample; use of an assay method of the invention to detect and/or measure the ability of an agent, applied to said PA-binding protein-containing and/or PIPn-binding protein-containing test sample, to agonise or antagonise protein-probe binding; use of an assay method of the invention to detect and/or measure the ability of an agent, applied to said probe, to agonise or antagonise protein-probe binding.
The invention further provides a PA-binding protein or a PIPn-binding protein detected/measured/identified and/or isolated by an assay method of the invention, and an agent capable of agonising or antagonising protein-probe binding detected and/or measured by use of an assay method of the invention.
In a further aspect of the invention, a probe of the invention coupled to scintillant may be used to identify an agonist or antagonist of the interaction of a PA/PIPn-binding protein with PA or PIPn. Such uses are particularly suited for high throughput screening of candidate agonists/antagonists, especially single step high throughput screening. A radiolabelled protein (radiolabelled for example with tritiated leucine, or 35S-methionine) known to bind PA or PIPn is tested for binding to a probe of the invention coupled to scintillant in the presence and absence of one or more candidate agonists and/or antagonists. The advantage of using probe coupled to scintillant is that the difference in signal obtained between normal binding (i.e. in a control sample without any candidate antagonist or agonist) of PA or PIPn binding protein to the probe and reduced or enhanced binding (i.e. in samples with agonist or antagonist) is much greater than can be obtained without the scintillant. Consequently, agonists and antagonists can be more readily identified. A similar strategy but using fluorescence detection can be envisaged, with the probe and the protein containing fluorophores of different excitation.
There is also provided according to the invention a method of making a probe which comprises attaching a Phosphatidic Acid derivative onto a solid support. A suitable comprises reacting a compound of formula I′, II′, III′, or IV′:




in which:

    
    
        (a) the linker consists of aryl, heteroaryl, alkyl with possible heteroatoms and/or unsaturations, preferably chains of (CH2)n, with n=8-20, most preferably n=11;
        (b) the R-substituent carries an aryl, alkyl group, or a combination, preferably R=CmH2m+1, m=8-20, m=16 is optimal;
        (c) the ion M represents any cation, preferably Na+, NH4+;
        (d) unsaturations are allowed, such as in an arachidonyl side chain;
        
            X=NH,O,S
            RG2=a reactive group capable of reaction with XH, e.g. N-hydroxy-succinimide-activated carboxylate
        
        
    
    






    
    
        
        
             =solid support with attachment to RG2.
        
        
    
    


Such methods may further comprises deprotecting a compound of formula I′′, II′′, III′′, or IV′′ to form the compound of formula I′, II′, III′, or IV′:




where:

    
    
        (a) the linker consists of aryl, heteroaryl, alkyl with possible heteroatoms and/or unsaturations, preferably chains of (CH2)n, with n=8-20, most preferably n=11;
        (b) the heteroatom X maybe O, S, or, most preferably NH;
        (c) the R-substituent carries an aryl, alkyl group, or a combination, preferably R=CmH2m+1, m=8-20, m=16 is optimal;
        (d) unsaturations are allowed, such as in an arachidonyl side chain;
        (e) R′=any suitable protecting group, preferably Bn; trialkyl silyl; CNCH2CH2—
        (f) R′′=any suitable protecting group, preferably Fmoc; CBz, when X is NH
    
    


Examples of compounds of formulae I′ or II′ are shown in Table 2.
There is also provided a method of making a probe which comprises attaching a phosphoinositide onto a solid support. A suitable method comprises reacting a compound of formula V′ or VI′:




where: R=aryl, alkyl group, or a combination, preferably R=CmH2m+1,

    
    
        m=8-20, most preferably m=16 is optimal.
        R3=P(O)(OM)2; R4=H; R5=H (PI(3)P);
        R3=H; R4=P(O)(OM)2; R5=H (PI(4)P);
        R3=H; R4=H; R5=P(O)(OM)2 (PI(5)P);
        R3=P(O)(OM)2; R4=P(O)(OM)2; R5=H (PI(3,4)P2);
        R3=P(O)(OM)2; R4=H; R5=P(O)(OM)2 (PI(3,5)P2);
        R3=H; R4=P(O)(OM)2; R5=P(O)(OM)2 (PI(4,5)P2); or
        R3=P(O)(OM)2; R4=P(O)(OM)2; R5=P(O)(OM)2 (PI(3,4,5)P3).
        M=any cation, preferably Na+, NH4+
        *Denotes a stereogenic centre. More preferably a stereogenic centre with an R absolute configuration.
        Linker=aryl, heteroaryl, alkyl with possible heteroatoms and/or unsaturations. Preferably chains of (CH2)n with n=8-20, most preferably n=11.
        X=NH, O, S
        Unsaturations are allowed, such as in an arachidonyl side chain.
         with
    
    






    
    
         where
    
    






    
    
         =solid support with attachment to RG2.
        RG2=a reactive group capable of reaction with XH, e.g. N-hydroxy-succinimide-activated carboxylate
    
    


A further suitable method comprises reacting a compound of formula VII′ or VIII′:




Where:

    
    
        R=aryl, alkyl group, or a combination, preferably R=CmH2m+1,
        m=8-20, most preferably m=16 is optimal.
        R3=P(O)(OM)2; R4=H; R5=H (PI(3)P);
        R3=H; R4=P(O)(OM)2; R5=H (PI(4)P);
        R3=H; R4=H; R5=P(O)(OM)2 (PI(5)P);
        R3=P(O)(OM)2; R4=P(O)(OM)2; R5=H (PI(3,4)P2);
        R3=P(O)(OM)2; R4=H; R5=P(O)(OM)2 (PI(3,5)P2);
        R3=H; R4=P(O)(OM)2; R5=P(O)(OM)2 (PI(4,5)P2); or
        R3=P(O)(OM)2; R4=P(O)(OM)2; R5=P(O)(OM)2 (PI(3,4,5)P3).
        M=any cation, preferably Na+, NH4+
        *Denotes a stereogenic centre. More preferably a stereogenic centre with an R absolute configuration.
        Linker A=aryl, heteroaryl, alkyl with possible heteroatoms and/or unsaturations. Preferably chains of (CH2)n.
        X=NH, O, S
        Unsaturations are allowed, such as in an arachidonyl side chain.
         with
    
    






    
    
        Linker B=Aryl, heteroaryl, alkyl with possible heteroatoms and/or unsaturations. These could be any atoms, more preferably C, N, O, S, more preferably still methylene groups linked via amide ester bonds.
        Preferably the total length of linker A and linker B is 8-60 atoms, more preferably 19-31 atoms, most preferably 22 atoms.
        FG B=Amide, thiolo(ester), or most preferably ester
    
    






    
    
         =solid support with attachment to FG B
        RG2=a reactive group capable of reaction with XH, e.g. N-hydroxy-succinimide-activated carboxylate
    
    


Such methods may further comprise deprotecting a compound or formula V′′, VI′′, VII′′, or VIII′′ to form the compound of formula V′, VI′, VII′ or VIII′:




where:

    
    
        R=aryl, alkyl group, or a combination, preferably R=CmHm2+1,
        m=8-20, most preferably m=16 is optimal.
        R3=P(O)(OBn)2; R4=H; R5=H (PI(3)P);
        R3=H; R4=P(O)(OBn)2; R5=H (PI(4)P);
        R3=H; R4=H; R5=P(O)(OBn)2 (PI(5)P);
        R3=P(O)(OBn)2; R4=P(O)(OBn)2; R5=H (PI(3,4)P2);
        R3=P(O)(OBn)2; R4=H; R5=P(O)(OBn)2 (PI(3,5)P2);
        R3=H; R4=P(O)(OBn)2; R5=P(O)(OBn)2 (PI(4,5)P2); or
        R3=P(O)(OBn)2; R4=P(O)(OBn)2; R5=P(O)(OBn)2 (PI(3,4,5)P3).
        *Denotes a stereogenic centre. More preferably a stereogenic centre with an R absolute configuration.
        Linker=Aryl, heteroaryl, alkyl with possible heteroatoms and/or unsaturations. Preferably chains of (CH2)n with n=8-20, most preferably n=11.
        X=O, S, or, most preferably NH.
        Unsaturations are allowed, such as in an arachidonyl side chain.
    
    






    
    
        Where:
        R=aryl, alkyl group, or a combination, preferably R=CmH2m+1,
        m=8-20, most preferably m=16 is optimal.
        R3=P(O)(OBn)2; R4=H; R5=H (PI(3)P);
        R3=H; R4=P(O)(OBn)2; R5=H (PI(4)P);
        R3=H; R4=H; R5=P(O)(OBn)2 (PI(5)P);
        R3=P(O)(OBn)2; R4=P(O)(OBn)2; R5=H (PI(3,4)P2);
        R3=P(O)(OBn)2; R4=H; R5=P(O)(OBn)2 (PI(3,5)P2);
        R3=H; R4=P(O)(OBn)2; R5=P(O)(OBn)2 (PI(4,5)P2); or
        R3=P(O)(OBn)2; R4=P(O)(OBn)2; R5=P(O)(OBn)2 (PI(3,4,5)P3).
        *Denotes a stereogenic centre. More preferably a stereogenic centre with an R absolute configuration.
        Linker A=aryl, heteroaryl, alkyl with possible heteroatoms and/or unsaturations. Preferably chains of (CH2)n.
        X=O, S, or, most preferably NH.
        Unsaturations are allowed, such as in an arachidonyl side chain
    
    


Examples of compounds of formula V′, VI′, VII′, or VIII′ are shown in Table 3.
There is further provided according to the invention a compound having a formula according to any of formulae I′, II′, III′, IV′, V′, VI′, VII′, VIII′, I′′, II′′, III′′, IV′′, V′′, VI′′, VII′′, VIII′′, and use of such compounds to make a probe of the invention.
There is also provided according to the invention a method of making a compound of Formula I′, II′, M′, or IV′ which method comprises removal of the protecting groups of a compound of Formula I′′, II′′, III′′, or IV′′, respectively, preferably by reductive debenzylation.
There is further provided according to the invention a method of making a compound of Formula V′, VI′, VII′, or VIII′ which method includes reductive debenzylation of a compound of Formula V′′, VI′′, VII′′, or VIII′′, respectively.
The invention also provides a method of making a compound of Formula I′′, II′′, III′′, or IV′′ by phosphitylation of alcohol:




with (R′′′O)2 PNiPr2 and oxidation of the phosphitylated product, where R′′′=Bn; CNCH2CH2—; trialkyl silyl.

A method of making a compound of Formula V′′, VI′′, VII′′, or VIII′′ is also provided by coupling a first alcohol of formula:




with a second alcohol of formula:





through a phosphodiester linkage.

Preferably the second alcohol is phosphitylated with BnOP(NiPr2)2 to produce a phosphoramidite of formula:




which is then coupled to the first alcohol above to make the compound of Formula V′′, VI′′, VIII′′, or VIII′′.

There is further provided a method of making a compound of Formula I′, II′, III>, or IV′ which comprises making a compound of Formula I′′, II′′, III′′ or IV′′ by the above method followed by removal of the protecting groups of the compound of Formula I′′, II′′, III′, or IV′′, preferably by reductive debenzylation.
There is also provided a method of making a compound of Formula V′, VI′, VII′, or VIII′, which comprises making a compound of Formula V′′, VI′′, VII′′, or VIII′′ by a method above followed by reductive debenzylation of the compound of Formula V′′, VI′′, VII′′, VIII′′.
There is further provided a method of making a probe of the invention which comprises coupling a compound of Formula I′, II′, III′, IV′, V′, VI′, VII′, or VIII′, made by an above method, to the solid support.
The compound may be coupled to an N-hydroxy-succinimide-activated carboxylate of the solid support.
The alcohol of Formula:




may be made from a compound of Formula 44:





for example as described in Example 3.

Further Definition of the Linker of Formulae I-IV, V, VI, I′-IV′, V′, VI′, I′′-IV′′, V′′, VI′′

As an alternative to the linker definitions given above for these formulae, the linker may comprise or consist of carbon chains, for example made up of aryl, heteroaryl, alkyl, or combinations of these. An example of such a linker is —(CH2)n-aryl-. Preferably the linker comprises or consits of chains of (CH2)n, with n=8-20, most preferably n=11. Where the linker comprises a chain of (CH2)n, preferably the chain is attached directly to the ester carbon attached to the linker.
Further Definition of the R-Substituent of Formulae I-IV, V, VI, I′-IV′, V′, VI′, I′′-IV′′, V′′, VI′′
As an alternative to the definitions of the R-substituent given above for these formulae, the R-substituent may comprise or consist of carbon chains, for example made up of aryl, alkyl, or a combination. Preferably the R-substituent comprises or consits of chains of R=CmH2m+1, m=16 is optimal. Where the R-substituent comprises CmH2m+1, preferably this is attached directly to the ester carbon attached to the R-substituent.



DESCRIPTION OF FIGURES
FIG. 1. Identification of PA-binding proteins from brain cytosol. Brain cytosol (lane 1) was mixed with PA resin (lanes 2-4) without (lanes 2, 5) or with (lanes 3, 4) preaddition of soluble C:12 PA at 75 μM. After incubation for 2 hr at 4° C. and three washes, polypeptides bound to the resin were analysed by SDS-PAGE and stained with silver. The molecular weight range of this gel is 200-20 kD. Notice that several bands are reduced in intensity after pretretament with soluble PA.
FIG. 2. Simian COS-7 cells were grown on plastic plates to 80% confluency. Following removal of the culture medium and three washes in PBS, the cells were lysed in buffer containing 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 10 mM EDTA and 0.2% of Na deoxycholate (lanes 1-3), 0.4% NP-40 (lanes 4-6) or 0.4% CHAPS (lanes 7-9). The lysates were centrifuged at 12,000×g for 10 min and insoluble material was discarded. The supernatant from each lysate (shown in lanes 1, 4, 7) was mixed with PA resin (lanes 2, 5, 8) or PIP2 resin (lanes 3, 6, 9) for 2 hr at 4° C. After three washes, polypeptides bound to the resin were resolved by SDS-PAGE followed by electroblotting onto nitrocellulose. The blot was probed with antibodies to coatomer subunit β-cop (upper band) and to PLCó (lower band). Notice that the specificity of the PA resin for coatomer and of the PIP2 resin for PLCó is maintained only for samples lysed with NP-40;
FIG. 3. Phosphoinositides and dipalmitoyl PA;
FIG. 4. Immobilised PA and Ptd Ins (4.5) P2;
FIG. 5. Specificity of PA and Ptd Ins (4,5) P2 beads.
FIG. 6. Phosphoinositide-derivatised beads: structures and protein-binding properties. Structures of the beads. B. Binding of recombinant protein kinase B to PtdIns(3,4,5)P3 beads. Purified N-terminally EE-tagged PKB (100 nM) was incubated with leukocyte cytosol ( ̃4 mg ml−1 protein) or buffer, in the presence of the indicated PtdIns(3,4,5)P3 stereoisomer (20 μM). In some samples, EDTA was replaced with 1 mM MgCl2 and EGTA by a Ca2+/EGTA buffer ([Ca2+]=100 nM). Samples (1 ml) were mixed with PtdIns(3,4,5)P3-derivatised or ethanolamine-derivatised (control) Affigel-10 (10l) for 45 min. Bead-bound PKB was detected after washing by SDS-PAGE and an anti-EE immunoblot. In the absence of PtdIns(3,4,5)P3,  ̃1% of the added PKB was bead-bound. C, D. Examples of the binding of phosphoinositide-binding proteins in total cytosol extracts to the derivatised beads. In C, Pig leukocyte cytosol (4.5 ml; 8 mg ml−1 protein) was incubated with free D- or L-PtdIns(3,4,5)P3, and aliquots were mixed with PtdIns(3,4,5)P3 beads (20 l; see Methods). InD, Rat liver cytosol (1 ml; 7.5 mg ml−1 protein) was, incubated with PtdIns(3,5)P2 beads (10 l), with or without 20 μM free PtdIns(3,5)P2 (see Methods). Proteins that remained bound after washing were separated by SDS-PAGE and silver-stained: the positions of m.w. markers are shown. Arrows identify abundant proteins whose binding was competed effectively by free phosphoinositides (these are labelled according to the identities that were later given to them after ion-exchange separations of similar extracts, see FIGS. 7, 8 and Table1).
FIG. 7. Anion-exchange chromatography of pig leukocyte cytosol proteins that bind PtdIns(3,4,5)P3. Cytosolic protein ( ̃3 g) was fractionated by anion-exchange chromatography (Q-Sepharose HR; see Methods). A. Conductivity and absorbance traces. B. Silver-stained SDS-PAGE gel of proteins that bound to PtdIns(3,4,5)P3 beads, with and without 50 μM PtdInS(3,4,5)P3. Bands A-L, whose binding was inhibited by PtdIns(3,4,5)P3, were isolated from a scaled-up separation (see Methods). Table 1 lists proteins that have been identified.
FIG. 8. Cation-exchange chromatography of pig leukocyte cytosol proteins that bind PtdIns(3,4,5)P3. Pig leukocyte cytosol ( ̃2.4 g protein) was separated by cation-exchange chromatography (S-Sepharose HP; see Methods). A. Conductivity and absorbance traces. B. Silver-stained SDS-PAGE gel showing proteins recovered on PtdIns(3,4,5)P3 beads after incubation with or without 25 μM PtdIns(3,4,5)P3. PtdIns(3,4,5)P3 inhibited the binding of bands V-Y, which were isolated from a scaled-up separation (see Methods). Table 1 identifies some of these proteins.
FIG. 9. Anion-exchange chromatography of NP-40-solubilised pig leukocytemembrane PtdIns(3,4,5)P3 binding proteins. NP-40-solubilised pig leukocyte membranes ( ̃90 mg protein) were separated by anion-exchange chromatography on Q-Sepharose HR (see Methods). A silver-stained SDS-PAGE gel shows the proteins recovered from PtdIns(3,4,5)P3 beads after incubation with or without 27 μM PtdIns(3,4,5)P3. The major bands whose binding was inhibited by PtdIns(3,4,5)P3 are designated M-U and were isolated by scaled-up assays (see Methods). The identities of some of these are in Table 1.
FIG. 10. Domain structures of proteins isolated on phosphoinositide beads. The SMART programme was used to identify domains.
FIG. 11. Protein binding to derivatised beads: inhibition by free phosphoinositides. Recombinant proteins (in Cos cell lysates or expressed and purified) were bound to PtdIns(3,4,5)P3 or PtdIns(3,4)P2 beads in the presence of various free phosphoinositides (for details, see Methods). For PIP3-E, a full length construct was expressed in Cos cells while a truncated version (residues 40-437) was purified from E.Coli. In the examples shown, all lipids were presented to a particular protein at the same concentration—this was chosen as a concentration at which the most effective lipid showed just-maximal competition. Unless otherwise indicated, the first lane was loaded with a sample equivalent to 1% (Cos cell lysates) or 10% (purified recombinant proteins) of the protein sample that was incubated with beads. As appropriate, tagged proteins were detected with antibodies (anti-myc, anti-GFP, anti-HIS, or anti-EE; see Methods) or by silver-staining (PIP3-G/H-EE, EE-myosin 1F, GST-cytohesin-4, DAPP1, PIP3-E, MEG-2 and CDC42-GAP). For each protein, the data shown are representative of information collected in 4 independent experiments.
FIG. 12. Binding of DAPP1 and GST ytohesin4 to phosphoinositide-containing PtdEtn/PtdCho/PtdSer (1:1:1) surfaces on an HPA sensor-chip (see Methods). The data represent the mean±S.E.M. (n=36 independent determination) of the mass of protein binding at equilibrium to the chip surface after flowing 100 nM recombinant DAPP1 or GST-cytohesin-4 over the chip.



EXAMPLE 1
The immobilised PA [1] was prepared by coupling of the ω-amino phosphatidic acid (−) [2], which was synthesised in 7 steps starting from the optically pure protected glycerol derivative (−) [3], with the N-hydroxysuccinimide (NHS) activated ester agarose resin, Affi-Gel 10, as is depicted in Scheme 1. The poor solubility of [2] in a range of solvents neccesitated the use of the solvent combination chloroform-methanol-water (0.8:1:0.2). Excess resin (ca. 5 equiv. of NHS-groups) was used, resulting in a loading of 80% [2] onto the beads, as determined by the quantification of recovered [2] by 1H-NMR in the presence of an internal standard.
When a brain cytosolic extract was treated with the PA-resin [1], both in the presence and absence of soluble PA, and the resin-bound fraction subsequently analysed by SDS-PAGE, a number of bands appeared representing proteins with the desired characteristics. They had relatively high intensity when incubated in the absence of soluble PA, but their appearance was dramatically reduced when soluble PA was present, as is illustrated in FIG. 1.
The Synthesis of the Reagent [1] is as Follows (Numbers in Brackets Refer to the Structures Shown in Scheme 1)
1-O-[12-(N-benzyloxycarbonyl-amino)dodecanoyl]-3-O-(4-methoxybenzyl-sn-glycerol [6]: The diol [5] (2.47 g, 12 mmol) [see Chen, Profit and Prestwich: J. Org. Chem. 61: 6305-6312 (1996)], DCC (2.64 g, 13 mmol) and DMAP (1.56 g, 13 mmol) was added to dry CH2Cl2 (100 mL) under nitrogen and the reaction was stirred for 30 mins at 0° C. 12-(Benzyloxycarbonyl)-amino-dodecanoic acid (2.27 g, 6.5 mmol) in dry CH2Cl2 (5 mL) was transferred via cannula into the reaction mixture under nitrogen and stirred at room temperature overnight. The reaction was quenched by addition of water (50 mL) and the aqueous phase was extracted with CH2Cl2 (3×40 mL). The combined organic extracts were washed with brine (20 mL) and dried over MgSO4. Flash chromatography (50% EtOAc in hexane) of the residue gave alcohol [6] (3.15 g, 5.8 mmol, 89%) as a white solid: m.p. 51-53° C. (from EtOAc); [α]D20=+1.7 (c 0.52 in CHCl3); Rf=0.48 (50% EtOAc/hexane); γmax(CHCl3)/cm−1 3689, 3585, 3450, 3004, 2931, 2856, 1720, 1655, 1612, 1514, 1463, 1454, 1252, 1234, 1174, 1132, 1101, 1035; δH (400 MHz; CDCl3) 7.35-7.28 (5 H, m, Bn), 7.24 (2 H, d, J 8.6, PMB), 6.87 (2 H, d, J 8.6, PMB), 5.08 (2 H, s, OCH2), 4.84 (1 H, bs, NH), 4.50-4.42 (2 H, m, OCH2), 4.16 (1 H, dd, J 11.5, 4.5, CH2CHCH2), 4.11 (1 H, dd, J 11.5, 6.0, CH2CHCH2), 3.99-3.97 (1 H, m, CH2CHCH2), 3.79 (3 H, s, OCH3), 3.51 (1 H, dd, J 9.6, 4.4 CH2CHCH2), 3.44 (1 H, J 9.6, 6.0, CH2CHCH2), 3.16 (2 H, q, J 6.7, CH2NH), 2.70 (1 H, bs, OH), 2.30 (2 H, t, J 7.6, OCOCH2), 1.59 (2 H, qn, J 6.7, CH2CH2NH), 1.47-1.37 (2 H, m, OCOCH2CH2), 1.25 (16 H, bs, C11H22NH); δC (100 MHz; CDCl3) 173.9 (OCO), 159.4 (NHCO), 156.4 (CH3OC), 136.7, 129.8, 129.4, 129.2, 128.4, 128.1, 113.9 (7×Bn and PMB), 73.1 (CH), 70.6, 68.9, 66.5, 65.4 (4×CH2), 55.2 (OCH3), 41.1 (CH2NH), 34.1, 29.9, 29.5, 29.4, 29.3, 29.2, 29.1, 29.0, 26.7, 24.5 (10×CH2); m/z (CI) [Found (M+H)+544.3302. C31H46O7N requires M, 544.3274].
1-O-[12-(N-benzyloxycarbonyl-amino)dodecanoyl]-2-O-hexadecanoyl-3-O-(4-methoxybenzyl)sn-glycerol [7]: To a solution of alcohol [6] (3.14 g, 5.7 mmol) in dry CH2Cl2 (20 mL) under nitrogen was added DMAP (0.035 g, 0.29 mmol). The resulting solution was cooled to 0° C. and dry pyridine (0.68 g, 0.70 mL, 8.68 mmol) was added dropwise. After stirring for 30 mins, palmitoyl chloride (1.75 g, 1.93 mL, 6.37 mmol) was added dropwise under nitrogen and the reaction mixture was stirred overnight. Water (50 mL) was added to quench the reaction. The aqueous phase was extracted with ether (3×50 mL) and the ethereal layers were washed with 2 M HCl (20 mL). The acid phase was back extracted with ether (50 mL) and the combined ethereal layers were washed with brine (20 mL) and dried over MgSO4. Flash chromatography eluting with 50% EtOAc in hexane gave the ester [7] (3.17 g, 4.1 mmol, 70%) as a white solid: (Found: C, 72.6; H, 9.85; N, 1.8. C47H75O8N requires C, 72.2; H, 9.7; N, 1.8%); m.p. 44-47° C. (from EtOAc); [α]D20=+5.5 (c 0.59 in CHCl3); Rf=0.20 (20% EtOAc/hexane); γmax(CHCl3)/cm−1 3451, 2926, 2854, 1728, 1612, 1514, 1465, 1366, 1302, 1252, 1173, 1110; δH (400 MHz; CDCl3) 7.35-7.30 (5 H, m, Bn), 7.23 (2 H, d, J 11.5, PMB), 6.87 (2 H, d, J 8.7, PMB), 5.22 (1 H, qn, J 5.1, CH2CHCH2), 5.09 (2 H, s, OCH2), 4.74 (1 H, bs, NH), 4.48 (1 H, d, J 12.0, OCH2), 4.44 (1 H, d, J 12.0, OCH2), 4.32 (1 H, dd, J 11.8, 3.8, CH2CHCH2), 4.17 (1 H, dd, J 11.8, 6.4, CH2CHCH2), 3.80 (3 H, s, OCH3), 3.55 (2 H, dd, J 5.1, 1.1, CH2CHCH2), 3.18 (2 H, q, J 6.6, CH2NH), 2.31 (2 H, t, J 7.4, OCOCH2), 2.27 (2 H, t, J 8.1, OCOCH2), 1.66-1.56 (6 H, m, OCOCH2CH2, CH2CH2NH), 1.55-1.47 (2 H, m, CH2CH2CH2NH), 1.25 (36 H, bs, COC15H31, C11H22NH), 0.88 (3 H, t, J 6.6 CH3); δC (100 MHz; CDCl3) 173.4, 173.1 (2×OCO), 159.3 (NHCO), 156.4 (CH3OC), 136.7, 129.8, 129.3, 129.0, 128.5, 128.06, 113.81 (7×Bn and PMB), 72.9 (CH2), 70.1 (CH), 70.0, 67.9, 66.5 (3×CH2), 55.8 (OCH3), 41.1 (CH2NH), 34.3, 34.1 31.9, 29.9, 29.7, 29.5, 29.4, 29.3, 29.2, 29.1, 24.9, 24.8, 22.6 (13×CH2), 14.1 (CH3); m/z (CI) [Found (M+H)+ 782.56140. C47H76O8N requires M, 782.55707].
1-O-[12-(N-benzyloxycarbony-amino)dodecanoy]-2-O-hexadecanoyl-sn-glycerol [8]: To a solution of CH2Cl2 (50 mL) and H2O (5 mL) under air was added the diacylglycerol [7] (3.12 g, 4.00 mmol). DDQ (1.81 g, 8.00 mmol) was added and the reaction was stirred overnight at room temperature. The reaction was diluted with CH2Cl2 (50 mL) and washed with saturated NaHCO3 solution (20 mL), brine (20 mL) and dried over MgSO4. Flash chromatography eluting with 40-50% EtOAc in hexane gave the alcohol [8] (2.40 g, 3.62 mmol, 91%) as an off white solid: (Found: C, 70.7; H, 10.1; N, 2.1. C39H67O7N requires C, 70.7; H, 10.2; N, 2.1%); m.p. 56-58° C. [α]D20=−3.2 (c 1.02 in CHCl3); Rf=0.20 (30% EtOAc/hexane); γmax(CHCl3)/cm−1 3450, 2924, 2854, 1724, 1602, 1517, 1465, 1413, 1372, 1251, 1164; δH (400 MHz; CDCl3) 7.34-7.25 (5 H, m, Bn), 5.10-5.05 (3 H, m, CH2CHCH2, OCH2C6H5), 4.81 (1 H, bs, NH), 4.31 (1 H, dd, J 11.9, 4.4, CH2CHCH2), 4.20 (1 H, dd, J 11.9, 5.8, CH2CHCH2), 3.71 (2 H, d, J 5.8, CH2CHCH2), 3.16 (2 H, q, J 6.6, CH2NH), 2.32 (2 H, t, J 7.5, OCOCH2), 2.30 (2 H, t, J 7.8, OCOCH2), 1.63-1.59 (4 H, m, OCOCH2CH2, CH2CH2NH), 1.58-1.45 (2 H, m, CH2CH2CH2NH), 1.25 (36 H, bs, COC15H31, C11H22NH), 0.87 (3 H, t, J 6.6, CH2CH3); δC (63.5 MHz; CDCl3) 173.7, 173.4 (OCO), 156.4 (NHCO), 136.7, 128.5, 128.0, 112.6 (4×Bn), 72.1 (CH), 66.6, 62.1, 61.4 (3×CH2), 41.1 (NHCH2), 34.3, 34.1, 31.9, 30.0, 29.7, 29.6, 29.5, 29.3, 29.2, 29.1, 26.7, 24.9, 22.7 (12×CH2), 14.1 (CH3); m/z (CI) [Found (M+Na)+ 684.4828. C39H67O7NNa requires M, 684.4816].
1-O-[(12-N-benzyloxycarbonyl-amino)dodecanoyl]-2-O-hexadecanoyl-sn-glycer-3-yl bis-O-benzylphosphate [9]: Dry CH2Cl2 (10 mL) was added into a mixture of alcohol [8] (1.00 g, 1.51 mmol), 1H-tetrazole (1.04 g, 3.02 mmol) and bisbenzyl(N,N,-diisopropylamino)phosphine (0.31 g, 4.54 mmol) under nitrogen. The reaction mixture was stirred at room temperature for 2 h until tlc indicated no starting material was left. The reaction mixture was cooled to −78° C. and mCPBA (1.43 g, 8.3 mmol) was added in one portion. The reaction mixture was warmed to room temperature over 2 h and stirred overnight. The reaction mixture was diluted with CH2Cl2 (30 mL) and washed with 10% NaHSO3 solution (50 mL). The aqueous phase was back extracted with CH2Cl2 (2×50 mL). The combined organic extracts were washed with saturated NaHCO3 solution (20 mL), brine (20 mL) and dried over MgSO4. The residue was chromatographed (40% EtOAc in hexane) affording phosphate [9] (1.14 g, 1.24 mmol, 82%) as an off white gum: (Found: C, 69.2; H, 8.8; N, 1.5; P, 3.4. C53H80O10NP requires C, 69.0; H, 8.75; N, 1.5; P, 3.4%); m.p. 40-42° C. (from EtOAc/hexane); [α]D20=+1.8 (c 0.23 in CHCl3); Rf=0.11 (30% EtOAc/hexane); γmax(CHCl3)/cm−1 3542, 3022, 2853, 1733, 1601, 1512, 1433, 1222, 1217, 1032; δP (101.3 MHz; CDCl3) −0.64; δH (400 MHz; CDCl3) 7.37-7.27 (15 H, m, Bn), 5.16 (1 H, qn, J 5.0, CH2CHCH2), 5.08 (H, s, OCH2), 5.05-5.00 (4 H, m, OCH2), 4.85 (1 H, bs, NH), 4.25 (1 H, dd J 11.9, 4.4, CH2CHCH2), 4.13-4.05 (3 H, m, CH2CHCH2), 3.16 (2 H, q, J 6.5, CH2NH), 2.26 (2 H, t, J 7.2, OCOCH2), 2.24 (2 H, t, J 7.6, OCOCH2), 1.58-1.57 (4 H, m, OCOCH2CH2), 1.48-1.46 (2 H, m, CH2CH2NH), 1.25 (39 H, bs, COC15H31, C11H22NH), 0.87 (3 H, t, J 6.6 CH3); δC (100 MHz; CDCl3) 173.1, 172.7 (OCO), 156.4 (NHCO), 136.7, 135.6, 135.5 (3×CH2C), 128.6, 128.5, 128.1, 128.0, 127.9, 127.8, (6×Bn), 69.5 (CH), 69.4, 69.3, 66.5, 65.4, 61.6 (5×CH2), 41.10 (CH2NH), 34.1, 33.9, 31.9, 29.68, 29.7, 29.6, 29.5, 29.4, 29.3, 29.2, 29.1, 29.0, 26.7, 24.8, 22.7 (15×CH2), 14.11 (CH3); m/z (+FAB) [Found (M+Na)+ 944.5417. C53H80O10NPNa requires M, 944.5427].
1-O-(12-Amino)dodecanoyl-2-O-hexadecanoyl-sn-glycer-3-ylphosphate [2]: To a solution of tBuOH (18 mL) and H2O (3 mL) in a steel tube was added the benzyl phosphate [9] (0.1 g, 0.11 mmol). The reaction mixture was placed in an autoclave and the steel tube and autoclave was vented with H2 four and five times respectively. The autoclave was finally pressurised to 15 bar and stirred for 18 h. The pressure was slowly released and the reaction mixture was centrifuged. The tBuOH/H2O layer was discarded and the residue was washed with MeOH/CHCl3 (1:1 v/v). The suspension was centrifuged and the organic layer was collected and passed through a pad of celite. The filtrate was centrifuged to remove traces of celite and the organic layer was collected and concentrated in vacuo affording phosphate [2] (0.052 g, 0.084 mmol, 78%) as a white solid: [α]D20=−8.9 (c 0.09 in CHCl3/H2O 1:1); γmax(KBr)/cm−1 3838, 2918, 2849, 1732, 1684, 1469, 1417, 1382, 1242, 1166, 1044, 937; δp [101 MHz; CD3OD/CDCl3 (1:1)] 1.31; δH [500 MHz; CD3OD/CDCl3 (1:1)] 4.88 (1 H, bs, CH2CHCH2), 4.13 (1 H, dd, J 12.0, 3.0, CCH2CHCH2), 3.83 (1 H, dd, J 12.0, 3.0, CH2CHCH2), 3.66-3.64 (2 H, m, CH2CHCH2), 2.55 (2H, t, J 7.6, CH2NH2), 2.01-1.98 (4 H, m, COCH2), 1.33-1.28 (6 H, m, COCH2CH2, CH2CH2NH2), 0.98-0.92 (38 H, m, C15H31, C11H22), 0.54 (3 H, t, J 6.5 (CH3); ∘H [500 MHz; CD3OD/CDCl3 (1:1)] 173.5, 173.1 (2×OC═O), 70.2, 62.0 (CH and CH2), 39.0, 33.6, 33.6, 33.3, 31.3, 29.0, 28.8, 28.7, 28.5, 27.7, 27.6, 27.4, 27.2, 26.7, 25.3, 24.2, 23.8, 22.0, 13.1 (19×C15H31, C11H22).
1-O-[(12-N-affigel-10-amino)dodecanoyl]-2-O-hexadecanoyl-sn-glycer-3-yl phosphate [1]: Affigel-10 (2 mL slurry, 30.00 μmol) was filtered and washed with CHCl3/MeOH/H2O (0.8:1:0.2, 15 mL). It was transferred to a stirred solution of the acid [2] (4.0 mg, 6.14 μmol) and NaHCO3 (0.025 g, 0.30 mmol) in CHCl3/MeOH/H2O (0.8:1:0.2, 2 mL). The reaction mixture was stirred at room temperature for 18 h. The reaction mixture was filtered and washed with H2O (5 mL), CHCl3/MeOH/H2O (0.8:1:0.2, 10 mL) and H2O (5 mL) again. The gel was stored in H2O at 0° C. The filtrates were combined and 4.83 mmol of the internal standard was added. The solvent was removed in vacuo and the white residue was taken up in CDCl3/CD3OD (1:1 v/v) for proton NMR. The orthoformate:amine ratio was estimated to be 1:0.22 which implied that 5.33 μmol of the amine was loaded on to the beads.
Use of the Invention for Identification of PA Binding Proteins
In this document we disclose that resin-bound PA, illustrated as [1]. in Scheme 1, shows surprisingly a high affinity for a family of proteins which are expected to play important roles in both housekeeping cellular functions and in signal transduction. The observed affinity of the proteins for PA allows important substances in the MW range 60250 kD, and preferably in the range 60-160 kD to be identified.
Using this PA derivative we have identified a family of cytosolic brain proteins that showed strong and specific binding to PA. An unexpected common characteristic among some of the known proteins identified is their involvement in distinct stages of intracellular transport, from coated vesicle formation (coatomer and ADP ribosylation factor, Arf), to fusion (N ethylmaleimide sensitive factor NSF) and vesicle movement along microtubules (kinesin). Surprisingly, the resin-bound PA was much more stable than the cellular counterpart, and could be advantageously re-used a number of times (e.g. 5 times) without noticeable degradation or loss of activity. This is of crucial importance for any practical application.
A general approach for identifying PA binding proteins from tissue extracts is as follows: The tissue is homogenised using standard methods, and two fractions are produced, cysosol and membranes. The cytosol fraction is mixed 1:1 with buffer A (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 10 mM EDTA, 1% NP-40, protease inhibitors) and then incubated with the PA resin equilibrated for 30 min in buffer B (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.1% Tween-20, 0.02% Na azide). The membrane fraction is mixed 1:3 with buffer A but containing 2% NP-40 for 30 min on ice. The sample is then spun at 100,000×g for one hr to produce a soluble membrane extract. This extract is mixed with PA beads equilibrated as described above and processed similarly as above.
The sample is put in a rotator at 4° C. for 2 hr, and then washed three times with buffer B in the cold. These washes are very important since they remove non-specifically bound proteins. To provide an extra level of specificity we do the following modification. To one of duplicate samples excess soluble PA is added before the beads are introduced (the soluble PA solution is made by drying C:12 or C:8 PA dissolved in chloroform, resuspending in buffer A and sonicating for 5 min to make a stock solution of 250 mM). The assumption is that excess soluble PA will compete with the PA on the resin thus reducing the amount of protein that is recovered bound to the resin (see FIG. 1). Bands of interest are excised from the gel and treated with trypsin. The tryptic digests produced from the various bands are analysed by mass spectroscopy.
The catalogue of PA binding proteins obtainable using the invention is not complete. Nevertheless, such proteins are expected to fall into three embodiments:
The First Embodiment
Proteins of known identity and function but whose exact mechanism of action is not well understood. Kinesin, N-ethylmaleimide-sensitive factor (NSF), coatomer and ADP ribosylation factor (Arf) of the PA binding proteins identified so far using the PA resin fall into this category. We will briefly discuss kinesin as an example but similar arguments can be made for the rest of the known proteins. Membrane vesicle and organelle movement in eucaryotic cells is driven along microtubules by motor proteins [Rogers and Gelfand, Current Opinion in Cell Biology 12: 57-62 (2000)]. Kinesins, which are among the best understood of those motor proteins, hydrolyse ATP to generate movement along microtubules [Sablin, Current Opinion in Cell Biology 12: 3541 (2000)]. Part of the kinesin molecule has been crystalised, and mice carrying targeted knockouts for conventional kinesin have been generated. Thus the kinesin field is very advanced. However, the exact mechanism by which kinesins bind the membrane of the organelles or vesicles that they help transport is unknown [Bloom and Goldstein, J. Cell Biology 140: 1277-1280 (1998)]. Our identification of kinesin among the PA binding proteins suggests that PA may play a role in the recognition of the membrane by kinesins and may help solve this long-standing problem. Kinesin, in addition, has been implicated as an important target for the treatment of cancer [Cytokinetics, Inc., Chemistry & Biology 6: R225-R226 (1999)]; any discovery leading to a better understanding of the function of this protein may lead to the discovery of novel, or the design of more rational, drug therapies.
The Second Embodiment
Proteins of known identity but whose function is not understood. Neurochondrininorbin, a PA binding protein identified using the PA resin falls into this category. The gene for this protein was found to be upregulated following treatment of rat hippocampal slices with tetraethylammonium, a compound that induces long-term potentiation-like synaptic enhancement [Shinozaki et al. Biochem. Biophys. Res. Comm. 240: 766-771 (1997)]. Subsequent work has shown that norbin is present in dendrites of neural outgrowth [Shinozaki et al. Molecular Brain Research 71: 364-368 (1999)] whereas independent work based on expression cloning has indicated an additional role for this protein in bone metastasis [Ishizuka et al. Biochim. Biophys. Acta 1450: 92-98 (1999)]. For such a protein and others to be identified, discovery of their PA binding property may provide important clues as to their function and should help to design experiments to elucidate this function. For those that appear to have medical relevance, this approach may also help design strategies that control their function.
The Third Embodiment
Totally Novel Proteins.
Scope of the Invention
The PA resin described above and the PIPn resins described below are general analytical tools for identification of additional PA-binding or PIPn-binding proteins from different tissues and biological fluids. We envisage that the cytosolic and membrane contents of any cell type can be screened for PAIPIPn binding proteins using these resins. (In addition to brain, a partial list includes liver, kidney, heart, pancreas, macrophages, neutrophils.) In all cases, cytosolic or membrane fractions could be subjected to assays as described above. Once a series of proteins, which bind directly to PA or PIPn, have been identified, they could be examined as to which amino acids are involved in the binding, using a photoaffinity labeled PA-analogue (see above) or PIPn analogue. Comparison among those proteins should result in a common motif which may define the PA binding motif or a PIPn binding motif. Once the motif is identified, it can be used as a search tool to identify most proteins, that are expected to bind PA or PIPn and that are described in the databases. Thus the PA and PIPn resins are expected to reveal the majority of the members of the PA and PIPn binding protein families.
We foresee important applications of the PA and PIPn resins in diagnostics, Extracts from healthy or pathological tissues could be compared side by side and their fall complement of PA or PIPn binding proteins may hence be established. Any protein whose amount and/or electrophoretic mobility changes in the pathological tissue in comparison to the healthy tissue could be identified by mass spectroscopy. Such proteins will be candidates both as markers for the disease and as therapeutic targets (see below). Additionally, to identify key PA-binding or PIPn-binding proteins involved in cell differentiation, similar assays may be done in matched samples of tissue culture cells that differ in some important physiological parameter. Examples include cell lines before and after differentiation into a neuronal phenotype (e.g. PC12 cells with and without treatment with nerve growth factor) or endothelial cell lines induced to mimic angiogenesis with vascular permeability factor [Hanahan and Folkman, Cell 86: 353-364 (1996)]. Since this assay involves extracts from whole cells grown on plastic as opposed to tissue sources, we have tested extraction conditions that maintain the specificity of the PA resin. Surprisingly, specificity is maintained with low concentrations of non ionic detergents (FIG. 2).
The approach of identifying candidate proteins by comparing their expression level and pattern between “normal” and “altered” tissues or cell lines has similarity to current proteomics strategies that are in use by many pharmaceutical companies whereby total cellular proteins from such tissues are analysed with a view to identify potentially interesting changes in expression profiles. We point out two essential differences with the approach proposed here: (a) The PA (or PIPn) resin acts as a concentration/enrichment reagent thus allowing small differences, or differences in rare proteins to be more readily detectable. (b) Since a functional requirement is built into the screening process (i.e. PA, or PIPn, binding), the resulting proteins from our approach can be studied with some prior knowledge of their potential function.
We foresee important applications of the PA and PIPn resins in therapeutics. The PA and PIPn resins provide unique tools for identification of small molecule compounds that interfere with or enhance PA (or PIPn) binding of proteins identified using the schemes above since it is essentially a solid phase reagent amenable to automated assays. Following identification of a candidate target protein, specific monoclonal antibodies against this protein could be raised and the protein itself may then be produced in miligram amounts. The preferred binding assay is based on detection by ELISA using the specific antibodies raised. Other configurations of the binding assay include the use of PA or PIPn functionalised with a fluorescent reporter group (detection of binding will be done by fluorometry) or the use of radioactive protein (detection of binding will be done by scintillation counting). Candidate compounds (obtained from commercial sources) can be introduced in the binding assay prior to adding the PA resin. If a compound interferes with binding, detection of the protein is expected to be reduced. If it enhances binding, detection should be higher. Compounds identified using this screen might become interesting drug lead candidates.
An Experimental Protocol for Obtaining Lead Compound
Isogeneic healthy rats are treated with streptozotocin (65 mg/kg i.v.) to render them diabetic. After sacrification of animals at different stages of disease progression (0, 1, 2, 3, 4, 6, 8 week), several organs (liver, kidney, heart, brain) are dissected for further study. Extracts from each organ at various stages of diabetes are assayed for PA-binding proteins (or PIPn-binding proteins) using the PA resin or PIPn resin) described here. Assume that protein Ω is identified to be present at higher levels in the liver as a function of disease progression. Antibodies against Ω are then raised, thus allowing a screen for interfering compounds to be initiated. The antibodies can be used for diagnosis of disease progression. In addition, since Ω was identified by its ability to differentially bind to PA (or PIPn) as the disease progresses, molecules that interfere with this binding maybe of relevance as a therapeutic compound.
One specific example of the use of the PA resin to identify subtle changes in the pattern of protein expression is concerned with the binding of Arf6 to PA. We have found that the active form of Arf6 (i.e. bound to GTP) binds with 50-fold higher affinity to the PA beads in comparison to the inactive form (i.e. bound to GDP). Thus, when cellular samples containing equal amounts of Arf(GDP) or Arf6(GTP) were analysed for PA binding side by side, 50 times more Arf6(GTP) than Arf6(GDP) was bound to the PA resin. The implications for this are significant since, if an altered cell line or tissue contains “activated” Arf6 in comparison to its normal counterpart, analysis of the two samples with the PA beads will detect this potentially important difference.




EXAMPLE 2
Synthesis and Biological Evaluation of a PtdIns(3,4,5)P3 Affinity Matrix
New PtdIns(3,4,5)P3 binding proteins have been identified utilising PtdIns(3,4,5)P3 modified affinity matrix 11 which was synthesised from myo-inositol derivative 12, phosphoramidite 19 and an agarose based solid support.
The role of myo-inositol phospholipids in cell signalling systems is well established.1-4 One such signal transduction mechanism involves the in vivo production of PtdIns(3,4,5)P3 via phosphorylation of PtdIns(4,5)P2 mediated by PI3K.5 Although PtdIns(3,4,5)P3 binding proteins are known,6 many of the cellular processes downstream of PI3K activation do not yet have a defined lipid binding protein mapped above them. For this reason we embarked on the preparation and evaluation of an affinity matrix based on PtdIns(3,4,5)P3.
The phospholipid was attached to an agarose matrix by an amide linkage formed between a carboxylic acid-terminated side chain on the agarose and a 3-(ω-aminoacyl) glycerol derivative on the phospholipid (Scheme 2). The phospholipid 21 was prepared by coupling the alcohols 127 and 13 (from the commercially available (S)-(+)-1,2-O-isopropylideneglycerol 14) through a phosphodiester linkage.




We initially protected the primary alcohol 14 as the t-butyldiphenylsilyl ether, but encountered difficulties in its removal at a later stage of the synthesis. A more efficient process involved 4-methoxybenzylation of the primary alcohol 14 (Scheme 3),8 followed by acetonide removal to give the PMB-ether 15. Selective esterification of the primary alcohol in 15 with the Cbz-protected w-amino acid 16, followed by palmitoylation of the secondary alcohol 17 gave the diester 18. Oxidative removal (CAN) of the PMB protecting group and phosphitylation of 13 with BnOP(NiPr2)29 gave the phosphoramidite 19.†




The lipid side chain, in the form of the phosphoramidite 19, was then coupled with the enantiomerically pure alcohol (−)-12 to afford the perbenzylated compound 20 (Scheme 4). Reductive debenzylation was readily effected using H2 (50 psi) in the presence of Pd-black and NaHCO3 in ButOH—H2O (6:1) as the solvent, to afford the amine 21 in good yield.† This was then coupled with the N-hydroxysuccinimide (NHS) activated ester resin, Affi-Gel 10,§ to afford the PtdIns(3,4,5)P3 modified matrix 11. Excess resin (ca 5 equivalents) was required to ensure the complete consumption of the amine which was determined by a negative Kaiser test.‡




Pilot experiments showed that PKB (25 mM) [a known6,10 PtdIns(3,4,5)P3 binding protein] would bind to the matrix 11 and could be completely displaced by 10 mM D,D-PtdIns(3,4,5)P3†† but not at all by 10 mM LL-PtdIns(3,4,5)P3, thus establishing the potential specificity of the matrix. When applied to a pig neutrophil cytosol a number of proteins have been identified that bind to the resin 11 in a PtdIns(3,4,5)P3 sensitive manner. Several novel proteins were identified and the full biological results are discussed below in Example 4. One of these proteins was subsequently shown to be identical to the recently characterised protein, DAPP1, possessing a Src homology (SH2) domain and a pleckstrin homology (PH) domain. This novel protein had been independently identified from a data base search by comparison of the PH domain sequences with known PtdIns(3,4,5)P3 binding proteins.11 The fact that the ‘functional screen assay’ identified several proteins including DAPP1, which is involved in endosomal trafficking or sorting,12 is noteworthy and exemplifies the strength of the approach. Very recently biotinylated PtdIns(3,4,5)P3 has been used as an affinity ligand for the purification of recombinant PtdIns(3,4,5)P3 binding proteins.13 
In summary we have demonstrated a synthesis of a PtdIns(3,4,5)P3-modified matrix and demonstrated its use as a tool for the identification of proteins binding to PtdIns(3,4,5)P3. The flexible nature of the methodology and the biological success of resin 11 warrants further investigation into the preparation and biological evaluation of other D-3 phosphorylated myo-inositol phospholipid modified matrixes.14 
FOOTNOTES AND REFERENCES FOR EXAMPLE 2
† All new compounds exhibited spectroscopic and analytical data in accord with the assigned structure. Selected data (J values in Hz) for 19:[α]D22+7.0 (c 1.9 in CHCl3); δH (250 MHz, CDCl3), 7.38-7.28 (10 H, m, Ph), 5.20-5.10 (1 H, m), 5.10 (2 H, bs, OCH2Ph), 4.80-4.60 (3 H, m), 4.36 (1 H, m), 4.12 (1 H, m), 3.85-3.55 (4 H, m), 3.18 (2 H, aq, J 6.7, CH2NH), 2.29 (4 H, at, J 7.3), 1.64-1.40 (6 H, m), 1.30-1.20 (38 H, m), 1.17 (12 H, 2xd, J 6.8, 4×Me), 0.97 (3 H, t, J 6.9, Me); δP (101.25 MHz, CDCl3), 149.2, 149.1; m/z (FIB) [Found: (M+Na)+ 921.6022. C52H87N2O8Pna requires 921.6098]. For 21: [α]D22+3.0 (c0.1 in H20); vmax (KBr/cm−1) 3403, 2920, 2850, 1742, 1238, 1094; δH (250 MHz, D2O), 5.25 (1 H, bs), 4.45-3.80 (10H, m), 2.95-2.85 (2 H, m), 2.40-2.25 (4 H, m), 1.65-1.05 (44 H, m), 0.85-0.70 (3 H, m); δP (101.25 MHz, D2O), 5.81, 4.79, 3.55, 0.80; m/z (-ve FAB) 1142 [(M−Na)−, 25%], 1119 (50), 1098 (100), 1076 (90).
§ Affigel 10 was Purchased From BioRad.
‡ The matrix 11 was constructed by reacting 60 mole of N-hydroxysuccinimide activated resin (4 mL) with 12.2 mmole of the amine 21 in the presence of 122 mmole NaHCO3 at 0° C. overnight. †† D,D-PtdIns(3,4,5)P3 refers to the dipalmitoyl analogue of PtdIns(3,4,5)P3 containing the 1(D)-myo-inositol ring stereochemistry and sn-2-diacylglycerol side chain; L,L-PtdIns(3,4,5)P3 refers to the enantiomer.

    1. C. L. Carpenter and L. C. Cantley, Curr. Opin. Cell Biol., 1996, 8, 153.
    2. A. Toker, M. Meyer, K. K. Reddy, J. R. Falck, R. Aneja, S. Aneja, A. Parra, D. J. Burns, L. M. Ballas and L. C. Cantley, J. Biol. Chem., 1994, 269, 32358.
    3. M. J. Berridge, Nature, 1993, 361, 315.
    4. L. R. Stephens, T. R. Jackson and P. T. Hawkins, Biochem. Biophys. Acta, 1993, 1179, 27.
    5. C. P. Downes and A. N. Carter, Cellular Signalling, 1991, 3, 501.
    6. P. R. Shepherd, D. J. Withers and K. Siddle, Biochemical J., 1998, 333, 471.
    7. G. F. Painter, S. J. A. Grove, I. H. Gilbert, A. B. Holmes, P. R. Raithby, M. L. Hill, P. T. Hawkins and L. R. Stephens, J. Chem. Soc., Perkin Trans. 1, 1999, 923.
    8. J. Chen, A. A. Profit and G. D. Prestwich, J. Org. Chem., 1996, 61, 6305.
    9. E. Dreef, C. J. J. Elie, P. Hoogerhout, G. A. van der Marel and J. H. van Boom, Tetrahedron Lett., 1988, 29, 6513.
    10. S. R. James, C. P. Downes, R. Gigg, S. J. A. Grove, A. B. Holmes and D. R. Alessi, Biochemical J., 1996, 315, 709.
    11. S. Dowler, R A. Currie, C. P. Downes and D. R. Alessi, Biochein. J., 1999, 342, 7
    12. K. Anderson, P. Lipp, M. Bootman, S. H. Ridley, J. Coadwell, L. Ronnstrand, J. Lennartsson, A. B. Holmes, G. F. Painter, J. Thuring, Z.-Y. Lim, H. Erdjument-Bromage, A. Grewal, P. Temspt, L. R. Stephens and P. T. Hawkins, Curr. Biol., 2000, 10, 1403.
    13. D. S. Wang, T. T. Ching, J. St Pyrek and C. S. Chen, Anal. Biochem., 2000, 208, 301.
    14. P. T. Hawkins et al., Nature Biotechnol., manuscript in preparation.


EXAMPLE 3
Synthesis and Biological Evaluation of a PtdIns(4,5)P2 and Phosphatidic Acid Affinity Matrix




Ptd Ins(4,5)P2 and dipalmitoyl PA were synthesised. In order to identify the direct downstream effectors of these molecules, dipalmitoyl PA and PtdIns(4,5)P2 were immobilised onto a solid support, Affi Gel-10 giving 31 and 32. Using 31 and 32 as affinity matrices, a number of known proteins as well as a set of novel proteins were found to bind specifically to PA.

Phosphoinositides (PIPn's) (FIG. 3) represent a class of membrane phospholipids, which exhibit a wide range of activities in cell signaling cascades.15 The 3-phosphorylated lipid products of phosphatidylinositol-3-kinase (PI3K), viz. PtdIns(3)P, PtdIns(3,4)P2 and PtdIns(3,4,5)P3, mediate cell functions as diverse as cell movement, division and survival as well as glucose transport and many other functions, upon cell surface receptor stimulation by hormones and growth factors.16-18 The PtdIns(3,4,5)P3 is the product of 3-phosphorylation of the relatively abundant PtdIns(4,5)P2. Another well established function of PtdIns(4,5)P2 is phospholipase C (PLC)-promoted hydrolysis to give diacylglycerol and inositol(1,4,5)P3; the former activates protein kinase C (PKC) where the latter releases Ca2+ from internal stores.19 Recent work has shown that PtdIns(4,5)P2 is a highly versatile signaling molecule in its own right, and is involved in fundamental processes in membrane trafficking and plasma membrane-cytoskeleton linkages.20 As such PtdIns(4,5)P2 serves as an effector of many multiple downstream proteins, many of which remain to be identified. The biosynthesis of PtdIns(4,5)P2 involves mainly 5-phosphorylation of PtdIns(4)P by PI4P-5-kinase,19 which is activated by phosphatidic acid (PA), a product of phospholipase D (PLD)-catalyzed hydrolysis of phosphatidyl choline.21 It is suspected that PA, besides being an important intermediate in the biosynthesis of glycerophospholipids, fulfills crucial roles in lipid based signaling and intracellular trafficking, because PLD activation is associated with the regulation of those processes.22,23 This would imply the existence of PA-interacting proteins that operate downstream of PLD activation.
To identify the direct downstream effectors of PtdIns(4,5)P2 and PA, and hence to gain more insight in their roles in signaling and house-keeping, we embarked on the synthesis of immobilized analogs of PA 31 and PtdIns(4,5)P2 32, having saturated fatty acid chains, as illustrated in FIG. 4.
These affinity reagents were prepared by attaching a terminal fatty acid amino function at the sn-1 position of the glycerol moiety, as in 33 and 34, to an agarose solid support.




The soluble PA analog 33 was prepared by extension of methodology previously developed in our group and by others (Scheme 5).24,25 The commercially available (S)-(+)-1,2-O-isopropylidene glycerol 35 was converted to the alcohol 40 in 5 steps, followed by phosphitylation with BnO2P(NiPr2) and in situ oxidation with mCPBA to give 41. Reductive debenzylation was readily effected using H2 (15 bar) in the presence of Pd-black and NaHCO3 in tBuOH-H2O (6:1) as the solvent, to afford the amine 33, isolated as the sodium salt, in good yield. Alternatively, 40 was phosphitylated with BnOP(NiPr2)2 to give the phosphoramidite 42, which is the lipid synthon in the preparation of the ω-amino PtdIns(4,5)P2 34 (vide infra).25,26




The synthesis of 3427 (Scheme 6) started from the readily available myo-inositol orthoformate 43, that was converted in 6 steps to the optically pure camphor acetal (−)-44, derived from (−)-camphor. The intermediate (−)-44, in which the resolving camphor fragment also served to protect the 3- and 4-positions of the myo-inositol ring, has previously been utilized by us in the synthesis of dipalmitoyl PtdIns(3,4,5)P3.28 Subsequent p-methoxybenzylation followed by acetal deprotection afforded the 3,4-diol
(−)-46.29 Chemoselective benzylation of the 3-position was readily effected via the in situ generated stannane acetal in the presence of tetrabutylammonium bromide and benzyl bromide in refluxing acetonitrile. Using these conditions developed by Gigg et al.,30 the ratio of 3-benzyl:4benzyl was approximately 4:1 as judged by 1H-NMR analysis. It should be noted that preformation of the cyclic stannane acetal, followed by benzylation in the presence of CsF in DMF,31 resulted in a lower yield and selectivity. The required 3-benzylated product (−)-47 was purified by flash chromatography and trituration. Deprotection of the 5-O-allyl ether using Wilknson's catalyst followed by acid treatment furnished the known diol (+)-48.




Definite proof for the observed regioselectivity of the stannylation-benzylation process was obtained by an independent synthesis of the diol (+)-48,32 starting from the known optically pure diol (+)-49.31 A four step sequence (Scheme 7) yielded (+)-48, which had identical optical rotatory and 1H-NMR data as compared with the material prepared via Scheme 6. The diol (+)-48 was phosphorylated and PMB-deprotected under standard conditions to afford the known alcohol (−)-53, which was then coupled with the phosphoramidite 42 in the presence of 1H-tetrazole, followed by in situ mCPBA oxidation to give the fully protected phosphoinositide (−)-54. Global deprotection was carried out using similar conditions to those discussed to give the ω-amino PtdIns(4,5)P2 analog 34.




Finally, the PA amine 33 and PtdIns(4,5)P2 amine 34 were coupled to the N-hydroxysuccinimide (NHS)-activated ester resin, Affi-Gel 10, to afford the corresponding affinity matrices 31 and 32. The PtdIns(4,5)P2 modified matrix was prepared in water using excess NaHCO3, by reacting the activated Affi Gel 10 [resin 60 mmol (4 mL)] with the amine 34 (14.3 mmol) to give a loading of 4.5 mmol as judged from the recovery of the amino phospholipid 34, that was quantified by 500 MHz 1H-NMR spectroscopy in the presence of myo-inositol orthoformate 43 as the internal standard. The poor solubility of the amino-terminated PA 31 necessitated the use of the solvent combination chloroform-methanol-water 4:5:1 to yield material with a loading of 5.3 mmol starting from 30 mmol activated ester resin.
By treating the PA-modified affinity resin 31 with a brain cytosol extract in the presence of a non-ionic detergent, we found that a number of proteins bind specifically to those beads, i.e. their binding was inhibited by soluble dilauroyl PA. Amongst the PA-binding proteins identified were the b-cop subunit of coatomer, ADP ribosylation factor (Arf), N′-ethylmaleimide-sensitive factor (NSF) and kinesin, all of which are involved in intracellular traffic. In addition a set of 5 novel proteins was found. To further emphasize the observed binding specificity and the strength of the affinity reagents PA 31 and PtdIns(4,5)P2 32 to identify (detect) phospholipid-protein interactions, we treated in parallel brain cytosol with PA-resin 31 and various amounts of immobilized PtdIns(4,5)P2 32 (FIG. 5). Cytosol was mixed for 1 h with PA beads (150 nmol) or three different concentration of PtdIns(4,5)P2 beads (120 nmol maximum) as shown. Following washes, the proteins bound to the beads were analysed by SDS-PAGE probed with antibodies to b-cop, Arf, PLCd (a known effector of PtdIns(4,5)P2, vide supra) and PKC (an abundant cytosolic protein that binds acidic phospholipids and diacyl glycerol). We were pleased to see that b-cop and Arf bound strongly to the PA-beads 31, and only weakly to PtdIns(4,5)P2 beads. Conversely, under the same conditions PLCd had a strong interaction with the PtdIns(4,5)P2 beads, wheareas its binding to PA-resin 31 was undetectable. Finally, the negative control PKC did not bind to any of the beads.
In conclusion, we have synthesized a PA-functionalized solid support 31 and the corresponding 4,5-phosphoinositide 32 involving the key synthon 44. The observed binding profiles with several cytosolic proteins and the high degree of specificity warrant the further use of these materials in cellular biology.
We have covalently-coupled several inositol phospholipid (phosphoinositide) species to sepharose beads to provide novel affinity-capture tools. Initial use of these beads to capture proteins in leucocyte, platelet, brain and liver cytosol resulted in the identification of some 21 proteins which specifically bound to the phosphoinositide moeity attached to the beads. 11 of these proteins (4 of which remain undescribed in the literature) possess established phosphoinositide binding domains (9 have one or more PH domains; 2 have one or more FYVE domains) establishing the effectiveness of this approach and pointing to the possibility that novel domains may exist in the other proteins identified. Phosphoinositide binding proteins are known to be key components of intracellular signalling pathways used by growth factor receptors and inflammatory stimuli and also of intracellular vesicle trafficking pathways and thus it is anticipated that high-throughput use of the matrices we have created here could be used as a discovery tool for new proteins involved in these pathways and also as a probe for functional expression of established members of these families.
REFERENCES FOR EXAMPLE 3


    (15) Toker, A; Cantley, L. C. Nature 1997, 387, 673-676.
    (16) Rameh, L. E.; Cantley, L. C. J. Biol. Chem. 1999, 274, 8347-8350.
    (17) Dekker, L. V.; Segal, A. W. Science 2000, 287, 982-985.
    (18) Corvera, S.; Czech, M. P. Trends Cell Biol. 1998, 8, 442-446.
    (19) Toker, A. Curr. Opin. Cell Biol. 1998, 10, 254-261.
    (20) Czech, M. P. Cell 2000, 100, 603-606.
    (21) Frohman, M. A.; Sung, T. C.; Morris, A. J. Biochim. Biophys. Acta 1999, 1439, 175-186.
    (22) Exton, J. H. Biochim. Biophys. Acta 1999, 1439, 121-133.
    (23) Liscovitch, M.; Czarny, M.; Fiucci, G.; Tang, X. Biochem. J. 2000, 345, 401-415.
    (24) Painter, G. F.; Thuring, J. W. J. F.; Lim, Z.-Y.; Holmes, A. B.; Hawkins, P. T.; Stephens, L. R. Chem. Commum., submitted.
    (25) Chen, J.; Profit, A. A.; Prestwich, G. D. J. Org. Chem. 1996, 61, 6305-6312.
    (26) For other syntheses of sn-1-aminoacyl PIPn analogues, see: Prestwich, G. D. Acc. Chem. Res. 1996, 29, 503-513 and refs. cited therein. Falck, J. R.; Krishna, U. M.; Reddy Katipally, K.; Capdevila, J. H.; Ulug, E. T. Tetrahedron Lett. 2000, 41, 4271-4275. Falck, J. R.; Krishna, U. M.; Capdevila, J. H. Bioorg. Med. Chem. Lett., 2000, 10, 1711-1713.
    (27) For some recent syntheses of PtdIns(4,5)P2 analogues see: Gu, Q.-M.; Prestwich, G. D. J. Org. Chem. 1996, 61, 8642-8647. Falck, J. R.; Krishna, U. M.; Capdevila, J. H. Tetrahedron Lett. 1999, 40, 8771-8774.
    (28) Painter, G. F.; Grove, S. J. A.; Gilbert, I. H.; Holmes, A. B.; Raithby, P. R.; Hill, M. L.; Hawkins, P. T.; Stephens, L. R., J. Chem. Soc. Perkin Trans. 1 1999, 923.
    (29) Grove, S. J. A.; Gilbert, I. H.; Holmes, A. B.; Painter, G. F.; Hill, M. L. Chem. Commun. 1997,1633-1634.
    (30) Desai, J.; Gigg, R.; Gigg, R.; Payne, S. Carbohydr. Res. 1992, 225, 209-228.
    (31) Wang, D.-S.; Chen, C.-S. J. Org. Chem. 1996, 61, 5905-5910.
    (32) Desai, J.; Gigg, R.; Gigg, R.; Martin-Zamora, E. Carbohydr. Res. 1994, 262, 59-77.


EXAMPLE 4
Identification of Phosphoinositide-Binding Proteins by Targetted Proteomics Using Selective Affinity Matrices
Summary
Phosphoinositides play a critical role in many cellular regulatory processes and there is a need for a systematic approach to identifying their target proteins. We show that matrices displaying tethered homologues of natural phosphoinositides can be used to capture many phosphoinositide binding proteins in cell and tissue extracts simultaneously and that these proteins can be effectively identified by coupling a simple functional display (based on competition for binding to free phosphoinositides and gel electrophoresis) with mass-spectrometric fingerprinting and/or sequencing. We present the identification of over 20 proteins isolated by this method, mostly from leukocyte extracts: they include known and novel proteins with established phosphoinositide binding domains and also known proteins with surprising and unusual phosphoinositide-binding properties. We also describe the use of these matrices to construct simple and very effective phosphoinositide-binding assays for recombinant proteins expressed in cell lysates or in purified form.
Introduction
Phosphoinositides (PtdIns and its phosphorylated derivatives) are membrane phospholipids that dictate the localisation and function of many intracellular target proteins. These target proteins influence many critical processes in eukaryote cells, including signalling by cell-surface receptors, vesicle trafficking and cytoskeletal assembly and disassembly, and novel phosphoinositide functions continue to emerge (reviewed in Martin, 1998; Toker, 1998; Leevers et al., 1999; Rameh and Cantley, 1999; Cockcroft, 2000). Recent years have seen an increase in the number of known phosphoinositides to eight (PtdIns, PtdIns3P, PtdIns4P, PtdIns5P, PtdIns(3,4)P2, PtdIns(4,5)P2, PtdIns(3,5)P2 and PtdIns(3,4,5)P3), making the analysis of their functions ever more complex.
Some selective interaction between a particular phosphoinositide and a discriminatory phosphoinositide-binding domain(s) in an involved protein is central to most phosphoinositide-regulated events. Some pleckstrin homology (PH) domains selectively bind PtdIns(4,5)P2, some have a particular affinity for PtdIns(3,4,5)P3 and/or PtdIns(3,4)P2, and others bind anionic phospholipids non-selectively (Lemmon and Ferguson, 2000). Some C2B domains bind PtdIns(3,4,5)P3 and PtdIns(4,5)P2 (e.g. Schiavo et al., 1996) and some FYVE domains selectively bind PtdIns3P (e.g. Stenmark and Aasland, 1999; Burd and Emr, 1998).
Once the phosphoinositide-binding properties of particular domain types were recognised, additional phosphoinositide-binding proteins were speedily identified by cloning novel molecules found in genome databases (e.g. Isakoff et al., 1998; Dowler et al., 2000). However, a major limitation of this approach is that it cannot identify phosphoinositide-binding sites that do not belong to one of these recognised domain families. There are now many proteins that have been established to bind phosphoinositides, mainly by various ad hoc strategies, but within which primary sequence determinants have not yet been defined: e.g. several cytoskeletal/focal adhesion proteins (Flanagan and Janmey, 2000), signalling proteins (e.g. the MARCKS-protein, Wang et al., 2000) or proteins involved in vesicle trafficking (e.g. the AP2 adaptor, Gaidarov and Keen, 1999). The problem of identifying protein targets is most severe when studying recently discovered phosphoinositides (e.g. PtdIns5P, PtdIns(3,5)P2; Rameh et al., 1997; Dove et al., 1997) for which there is no paradigmatic information.
An ideal way to get an overview of the constellation of proteins that interact specifically with one or more of the phosphoinositides would be by screening protein mixtures from cells or tissues with an assumption-free proteomic method. ‘Proteomnics’ is often taken to mean mapping of the expression of all proteins (e.g. by 2-D gels followed by high-throughput mass spectrometry) in a manner akin to microarray analysis of a cell's entire mRNA complement—but this cannot yield information on protein interactions with other proteins, with nucleic acids or with small molecules. To learn about these, targetted analyses of macromolecular interactions must be used to define function-critical subsets of the proteome.
Affinity matrices derivatised with synthetic phosphoinositides offer one such approach. Previous studies using matrices carrying Ins(1,3,4,5)P4-like structures [isosteric with the PtdIns(3,4,5)P3 headgroup; Hammonds-Odie et al., 1996; Stricker et al. 1997; Shirai et al. 1998; Tanaka et al. 1997] or linked to biotinylated diC8-PtdIns(3,4,5)P3 (Rao et al. 1999) have identified a few novel PtdIns(3,4,5)P3-binding proteins. This encouraged us to think that we might efficiently identify multiple phosphoinositide-binding proteins from a single tissue by combining protein isolation on new affinity matrices carrying the tethered homologues of natural phosphoinositides with protein identification by state-of-the-art technologies. To avoid false positives, we would target proteins whose binding was competitively inhibited by free phosphoinositides.
In this example, we report such surveys of the phosphoinositide-binding proteins of several tissues, particularly neutrophils. Not only have we isolated many proteins with established phosphoinositide-binding domains, including two novel proteins with PH domains and two novel proteins with FYVE domains, but we also identified members of previously untargetted protein families which reveal unexpected phosphoinositide-binding properties.
Results
The Use of PtdIns(3,4,5)P3-delivatised Beads to Isolate PtdIns(3,4,5)P3-binding Proteins From Leukocytes
FIG. 6a shows the nature of the derivatised beads used in this study. Initial experiments defined conditions for identifying proteins that bound specifically to the PtdIns(3,4,5)P3 moiety on the PtdIns(3,4,5)P3-derivatised beads. In the adopted assay, tissue samples were pre-incubated, with or without a competing phosphoinositide (usually PtdIns(3,4,5)P3), at near-physiological salt concentration (≧0.1 M NaCl) and with a non-ionic detergent (≧0.1% NP40) and reagents likely to inhibit PtdIns(3,4,5)P3 hydrolysis (β-glycerophosphate, F−, orthovanadate, divalent cations chelated). They were then incubated with PtdIns(3,4,5)P3 beads, and proteins that were retained by the beads were identified by SDS-PAGE. FIG. 6b demonstrates that D-PtdIns(3,4,5)P3 stereospecifically inhibited the binding of recombinant protein kinase B (PKB), an established PtdIns(3,4,5)P3 target (e.g. Stephens et al., 1998), to bead-bound D-PtdIns(3,4,5)P3, and that leukocyte cytosol had little effect on its binding. These results suggested that the PtdIns(3,4,5)P3 on the beads was displayed effectively and that the derivatised beads might provide a facile route for isolating phosphoinositide target proteins.
We are currently trying to understand the function of the phosphoinositide 3-kinase signalling system in neutrophils, so much of the work employed pig leukocyte cytosol as an abundant source of relevant PtdIns(3,4,5)P3-binding proteins. The binding of several cytosolic proteins to the PtdIns(3,4,5)P3-beads was inhibited by free PtdIns(3,4,5)P3 (D- or L-isomer, or both), but many other proteins interacted with the beads in a PtdIns(3,4,5)P3-independent way (FIG. 6c), making it difficult to recover proteins of interest in sufficient purity for unambiguous identification. We reduced the protein complexity of the samples applied to the beads by first fractionating them by ion-exchange chromatography. Using this approach we were able to isolate several proteins in sufficient yield and purity to attempt their identification (A-L, FIG. 7; V-Y, FIG. 8). We also screened various detergent and high salt (1 M NaCl) extracts of leukocyte membranes for PtdIns(3,4,5)P3-binding proteins, and proteins M-U were isolated from chromatographically fractionated NP40 extracts (FIG. 9).
Identification of PtdIns(3,4,5)P3-Binding Proteins
Some of the proteins isolated were digested with trypsin and identified by mass fingerprinting and sequencing (see Methods and Table 1). This identified: porcine orthologues of four characterised PtdIns(3,4,5)P3-binding proteins (rasGAPIP4BP, BTK, ETK and centaurin-α); five proteins (C, E, F, G/H, and X) that were novel at the time of isolation; and porcine orthologues of seven proteins that were not known to bind PtdIns(3,4,5)P3-rho/CDC42 GTPase-activating protein (CDC42-GAP), myosin 1F, megakaryocyte protein-tyrosine phosphatase (MEG2), Type II inositol polyphosphate 5-phosphatase and/or ezrin (both present in band D), and the α and β subunits of mitochondrial fatty acid oxidase.
We cloned the human orthologues of proteins C, E, F, G/H, and X: near-identical ORFs have been independently described for F (cytohesin-4; Ogasawara et al., 2000), X (DAPP1, Dowler et al., 1999; also termed PHISH, Rao et al., 1999; or Bam32, Marshall et al., 2000) and C (PLC-L2, Otsuki et al., 1999). Schematic “domain profiles” for these proteins are in FIG. 10.
Proteins Isolated on PtdIns(3,4)P2, PtdIns(3,5)P2 and PtdIns3P Matrices
We also analysed cytosol from pig platelet, sheep brain, sheep liver and rat liver for proteins that bound to beads derivatised with PtdIns(3,4)P2, PtdIns(3,5)P2 or PtdIns3P, particularly focussing on proteins that were not readily displaced by PtdIns(3,4,5)P3 and so might selectively bind other phosphoinositide(s). The PtdIns(3,4)P2 and PtdIns(3,5)P2 beads showed a limited degree of non-phosphoinositide-dependent protein binding (an example of using the PtdIns(3,5)P2 beads to isolate proteins from rat liver cytosol is given in FIG. 6d), but there was a high background of non-specific protein adsorption to PtdIns3P beads, so it was hard to characterise genuine PtdIns3P-dependent binding (not shown).
Several proteins were isolated from the fractionated cytosols (SR1-SR7 and SD1: Table 1 gives their tissue and bead origins). Type C 6-phosphofructokinase (PFK-C) was isolated on PtdIns3P beads, vinculin on PtdIns(3,4)P2 beads and -tocopherol trasfer protein (ATTP) on PtdIns(3,5)P2 and PtdIns(3,4)P2 beads. The PtdIns3P and PtdIns(3,4)P2 beads also yielded two proteins that were novel at the time of isolation (SR1 and SR3; Table 1). The human orthologues of SR1 and SR3 were cloned (FIG. 10) and very recently a near identical ORF has been described for SR3 (DFCP-1; Derubeis et al 2000).
The Phosphoinositide-Binding Specificities of Recombinant Proteins
Several of the proteins were made in recombinant form in Cos-7 cells, E. coli or baculovirus-infected Sf9 cells. We purified CT-EE-tagged PIP3-G/H, NT-EE-SR1, NT-EE-SR3 and NT-EE-myosin IF from Sf9 cells; and GST-CDC42GAP, GST-cytohesin-4, GST-MEG2, GST-DAPP1 and HIS-ATTP from E. coli. We could not purify full-length PIP3-E, but obtained an N-terminal truncation containing its PH domain (residues 40-437).
We investigated the relative abilities of various phosphoinositides to compete with the binding to PtdIns(3,4,5)P3 or PtdIns(3,4)P2 beads of tagged and purified proteins (EE-, GST- or His-; some tags were removed by thrombin cleavage; see Methods) and of proteins heterologously expressed in Cos-7-cell lysates (N-terminally myc- or GFP-tagged). With lysates, assays were run under conditions similar to those used to isolate the proteins—with micellar NP40, physiological salt, EDTA and other reagents to minimise phosphoinositide metabolism (see Methods). Assays on purified proteins used micellar NP40 in PBS, with 1 mM MgCl2 to approximate to the physiological divalent cation environment and minimise the stripping of Zn2+ from FYVE domains.
FIG. 11 shows examples of the results obtained: for each protein the competing phosphoinositides were all at one concentration, chosen to achieve just maximal inhibition by the most effectively competing lipid. Since the surface lipid concentrations on the derivatised beads are unknown, and may vary between batches and during multiple rounds of bead re-use, the results indicate only the relative affinities of the relevant binding sites on the proteins for various phosphoinositides. Several of the relative affinity screens were replicated with pure proteins and Cos-7 cell lysates, so the results are likely to reflect the intrinsic phosphoinositide-binding properties of the proteins. The PH domain-containing proteins all bound to PtdIns(3,4,5)P3 beads and were displaced most effectively by PtdIns(3,4,5)P3 (cytohesin-4, PIP3-G/H, PIP3-E) or equally by PtdIns(3,4,5)P3 and PtdIns(3,4)P2 (DAPP1). By contrast, the FYVE domain-containing proteins (SR1 and SR3) were harvested with PtdIns(3,4)P2 beads, and PtdIns3P displaced them most effectively. SR3 bound better to PtdIns(3,4)P2 beads that were being re-used, maybe because of their partial conversion to PtdIns3P (not shown).
Our isolation of three proteins containing Sec14 domains was unexpected, and they had different and unusual binding specificities. α-tocopherol-transfer protein (ATTP) was displaced most effectively from PtdIns(3,4)P2 beads by PtdIns(3,4)P2, PtdIns4P or PtdIns5P. MEG2 was displaced most effectively from PtdIns(3,4,5)P3 beads by PtdIns(3,4,5)P3 or PtdIns(4,5)P2. CDC42GAP bound weakly to PtdIns(3,4,5)P3 or PtdIns(4,5)P2 beads (data not shown), and was poorly displaced by the phosphoinositides tested (PtdIns(4,5)P2 and PtdIns(3,4,5)P3 were the most effective). Myosin IF bound to PtdIns(3,4,5)P3 beads and was displaced best by PtdIns(3,4,5)P3 or PtdIns(4,5)P2.
Surface Plasmon Resonance Analysis of Binding to Phosphoinositide-doped Lipid Monolayers
We used a surface plasmon resonance (SPR) biosensor to analyse and characterise the binding of some proteins to lipid surfaces of defined composition. FIG. 12 shows the binding of DAPP1 and cytohesin-4 to self-assembled PtdEtn/PtdSer/PtdCho (1:1:1) monolayers on alkane-coated HPA chips. Both proteins bound weakly to this surface, and binding was promoted by small proportions of phosphoinositides (usually 3-10 mole percent). In accord with the specificities from the bead displacement assays, cytohesin-4 bound best to surfaces containing PtdIns(3,4,5)P3 and DAPP1 to surfaces containing PtdIns(3,4,5)P3 or PtdIns(3,4)P2. These results are consistent with the receptor-driven, and phosphoinositide 3-kinase-dependent, recruitment of DAPP1 (Anderson et al., 2000; Marshall et al., 2000) and cytohesin-4 (A. McGregor, data not shown) from the cytosol to the plasma membranes of stimulated cells.
We also examined PIP3-G/H, SR3, ATTP, MEG2 and CDC42GAP, but it was difficult to assess their phosphoinositide affinities because they all bound strongly to the PtdEtn/PtdSer/PtdCho surface.
Discussion
The affinity matrix-based approach that we used to capture, display and identify a substantial number of phosphoinositide-binding proteins makes no prior assumptions about the nature of such binding proteins, such as the possession of a particular domain(s). It rapidly identifies multiple proteins by a three-step protocol—a cell fraction is chromatographically sub-fractionated, fractions are screened for proteins that bind to phosphoinositides, and proteins are identified by mass spectrophotometric fingerprinting and/or sequencing. We re-isolated 14 previously known proteins and identified, cloned and characterised seven new phosphoinositide-binding gene products, the ORFs for three of which remain undefined in the databases (SR1, PIP3-E and PIP3-G/H). The sequences of these ORFs are given below. The retrieval of several, now authenticated PtdIns(3,4,5)P3-binding proteins validates this method ie. BTK, ETK, centaurin-α and DAPP1 (Li et al., 1997; Qiu et al., 1998; Jackson et al., 2000; Anderson et al., 2000; Dowler et al., 1999; Marshall et at., 2000). In addition, we isolated cytohesin-4, which is the newest member of a family of Arf GTP-exchangers with established credentials as PtdIns(3,4,5)P3 effectors (e.g. Jackson et al., 2000).
We also identified several phosphoinositide-binding proteins that earlier screens did not detect. Some are novel (PIP3-E; PIP3-G/H; SR1), whilst others are previously known proteins whose phosphoinositide affinities were not previously recognised (ATTP; MEG2; CDC42GAP; mitochondrial fatty acid oxidase; Type C phosphofructokinase). It was also clear from inspection of the original gels of fractionated cytosol and membrane extracts that many more phosphoinositide-binding proteins were present than we have identified (we estimate more than 30 proteins in leukocyte cytosol alone). We note, for example, that some important PtdIns(3,4,5)P3 targets (e.g. PKB and PDK-1) have not yet emerged from this screen.
We identified many more proteins that interact specifically with acutely regulated phosphoinositide messengers, particularly PtdIns(3,4,5)P3, than with phosphoinositides implicated in intracellular processes such as membrane trafficking (e.g. PtdIns3P). PtdIns(3,4,5)P3 may genuinely have many more target proteins than other phosphoinositides, but it is also possible that our harvest retrieved fewer protein targets of the less highly charged phosphoinositides because these are harder to identify. PtdIns(3,4,5)P3 may tend to have a higher affinity for its effectors than PtdIns3P, making it easy to discriminate between specific and non-specific binding. Moreover, phosphoinositide-binding proteins that are involved in membrane trafficking often require multiple functional interactions with membranes. For instance, EEA1 only associates with early endosomes whose membranes simultaneously contain both PtdIns3P and rab5 (Lawe et al., 2000). In such situations, proteins that recognise phosphoinositides need a fairly low phosphoinositide affinity, so that functional association with a membrane will require both interactions. In this regard, it will be interesting to examine the role of PtdIns3P binding in the localisation of our novel FYVE domain-containing proteins (SR1 and SR3). The sequence of the SR3 ORF is given below.
Our identification of a trio of polyphosphoinositide-binding proteins that contain SEC14-like domains (ATTP, MEG2, and CDC42GAP) was unexpected. Designation of a SEC14 domain relies on sequence homology with the canonical yeast PtdIns transfer protein Sec14p (Bankaitis et al., 1990; Alb et al., 1995). Eukaryote databases currently contain more than one hundred SEC14 domain-containing proteins, but with no consensus on any shared common function. It has been reported that several other SEC14 domain-containing yeast proteins catalyse inter-membrane PtdIns transfer (Li et al., 2000), and that two SEC14 domain-containing proteins from soybean, Ssh1p and Ssh2p, bind PtdIns(4,5)P2 (Ssh2p) and either PtdIns(4,5)P2 or PtdIns(3,5)P2 (Ssh1p) (Kearns et al., 1998).
Even with this background, polyphosphoinositide binding by ATTP, MEG2, and CDC42GAP, which have no obvious links to phosphoinositide function, was a surprise. These proteins are thought to have very different functions: ATTP is involved in intracellular-tocopherol trafficking (Arita et al., 1995), CDC42GAP stimulates GTP hydrolysis by the small GTPase CDC42 (Lancaster et al., 1994; Barfod et al., 1993), and MEG2 is a haemopoietic protein-tyrosine phosphatase (Gu et al., 1992). They show no sequence similarity outside the SEC14 domain, so their SEC14 domains probably bind polyphosphoinositides—but why remains to be determined. These results make it likely that a subset of the many SEC14 domain-containing proteins constitutes an unrecognised class of proteins with a propensity for binding PtdIns and/or phosphorylated PtdIns derivatives. This idea is intriguing, given that SEC14 domains often occur in proteins involved in signalling: for example, additional protein-tyrosine phosphatases; GEFs and GAPs that regulate the guanine nucleotides status of Rho and Ras (including neurofibromin-related protein NF-1); and a diacyglycerol kinase-related protein from Drosophila.
A striking feature was how many of the proteins that we harvested are involved, directly (myosin 1F, ezrin, vinculin) or indirectly (via the control of rho family GTPases; cytohesin-4, α-centaurin, PIP3-G/H), in control of the cytoskeleton. The sequence of the myosin-1F ORF is shown below. It is becoming ever clearer that phosphoinositides have essential roles in coordinating the complex spatial and temporal events underlying cell adhesion and movement (see Martin, 1998; Flanagan and Jamney, 2000), and relating the phosphoinositide-binding properties of individual proteins to an understanding of these processes is a major challenge.
We do not understand why phosphofructokinase type C and mitochondrial fatty acid oxidase were recovered on our phosphoinositide-beads: evidence for any physiological significance of these interactions can only come from further work.
We not only used the derivatised beads to isolate the above proteins, but also showed that they can be used to establish the phosphoinositide-binding selectivities of proteins: we provide data on nine proteins that are either novel or previously uncharacterised in this respect. This method is quick and avoids many of the presentational and practical problems inherent in other methods, such as binding to phosphoinositides displayed on nitrocellulose or on Biacore chips. It would seem relatively straight forward to convert this method to a high-throughput format which could be used to screen for compounds which interfere with phosphoinositide binding to recombinant proteins. The phosphoinositide selectivities that were determined using these beads confirmed the conclusions from earlier studies of the same proteins by other methods. For example, cytohesins are highly selective towards PtdIns(3,4,5)P3 (Klarlund et al., 1998), but DAPP1/PHISH/Bam32 binds PtdIns(3,4,5)P3 or PtdIns(3,4)P2 (Kavran et al., 1998; Dowler et al., 1999). It is therefore likely that PIP3-G/H and PIP3-E, which are newly isolated and showed clear PtdIns(3,4,5)P3 selectivity, are genuine PtdIns(3,4,5)P3 targets.
In summary, we have used a targetted proteomic approach to screen cell and tissue extracts for proteins that bind with high specificity to phosphoinositides displayed on synthetic affinity matrices and to identify these proteins by mass spectrometry. These affinity matrices retrieved phosphoinositide-binding proteins even from quite complex protein mixtures, suggesting it will be possible to use this approach in conjunction with modern 2-D gel/high-throughput mass spectrometry technologies to rapidly screen for variations in the expression of phosphoinositide-binding proteins. In this regard, it is well established that some Bruton's tyrosine kinase PH domain mutations destroy BTK's ability to bind PtdIns(3,4,5)P3 with high affinity and cause X-linked immunodeficiency (Vetrie et al., 1993); and mutations that disrupt ATTP cause a severe and progressive ataxia (Ben Hamida et al., 1993), apparently by disrupting α-tocopherol delivery to the brain. Both would prevent the proteins from being retrieved from the patients' cell extracts, raising the possibility of screening cell extracts from other patients with possible cell regulatory defects for changes in the their profiles of functional phosphoinositide-binding proteins.
Experimental Procedures
Isolation of Cells, Preparation of Cell Fractions and Protein Fractionation
Isolation procedures were at 0-4° C., using buffers adjusted at 4° C. Isolated preparations were used immediately or “snap-frozen” in liquid N2 and stored at −80° C.
Pig Leukocytes
Isolation. Fresh blood was mixed with 0.146 volumes of ACD (80 mM trisodium citrate, 18 mM NaH2PO4.H2O, 15.6 mM citric acid, 161 mM D-glucose). One fifth of a volume of 3% PVP/0.91% (w/v) NaCl was added, erythrocytes were allowed to settle, and the upper layer was centrifuged (500×gav 8 min). The leukocyte pellet was suspended in calcium-free Hanks medium and cells were collected by centrifugation and briefly shocked (20 sec; >20 volumes of ice-cold H2O). The suspension was re-adjusted to the composition of Hanks' medium, and the cells were collected and washed in calcium-free Hanks. 30-40 l blood typically yielded 100-150 ml of packed cells.
Cytosol. Leukocytes were washed with 40 mM Tris-HCl (pH 7.5), 120 mM NaCl, 2.5 mM MgCl2 (500×gav, 10 min) and 1 volume of cells was suspended in 4 volumes of lysis buffer (100 mM NaCl, 40 mM Tris-HCl pH 7.5, 2 mM EGTA, 2.5 mM MgCl2, 1 mM DTT, 0.1 mM PMSF and 10 μg.ml−1 each of protease inhibitors (aprotinin, leupeptin, antipain and pepstatin A: ALAP). Cells were disrupted by sonication (4×12 sec; Heat systems probe sonicator, 1.2 cm tip), and the homogenate was centrifuged (900×gav for 10 min, then 160,000×gav for 60 min). 100 ml cell pellet gave  ̃250 ml cytosol containing  ̃2 g protein.
Anion-exchange chromatography of cytosol. Cytosol (3 g protein) was thawed and 0.1 mM PMSF and a protease inhibitor mixture (ALAP: see above) were added. After centrifugation (160,000×gav; 30 min), the supernatant was filtered (0.2 μm cellulose acetate; glass microfibre prefilter, Whatman), diluted 4-fold with 20 mM Tris/HCl, pH 7.5, 0.04% Tween-20, 0.5 MM EGTA, 0.1 mM EDTA, 1% betaine, 0.01% azide, 1 mM DTT and 0.1 mM PMSF, and pumped at 5 ml min−1 onto a 2.6×16 cm ( ̃75 ml) Pharmacia Q-Sepharose HR column. Elution at 3.5 ml min−1 used a linear NaCl gradient (750 ml, from 0 to 0.5 M; 22×40 ml fractions) in 30 mM Tris-HCl, pH 7.5, 0.5 mM EGTA, 0.1 mM EDTA, 0.03% Tween-20, 1% betaine, 1 mM DTT, 0.01% azide. 0.8 ml samples of the fractions were assayed for proteins binding to 10 μl PtdIns(3,4,5)P3-beads in the presence or absence of 50 μM PtdIns(3,4,5)P3 (see below). Fractions of interest were pooled and proteins A-L (see FIG. 7) were isolated by a scaled up procedure on 0.1-0.2 ml PtdIns(3,4,5)P3-beads (see below).
Cation-exchange chromatography of cytosol. Cytosol (2.4 g protein) was thawed, protease inhibitors (ALAP, see above) were added, the mixture was centrifuged and filtered (see above), adjusted to pH 6.7 (Hepes), diluted 5-fold in 30 mM Hepes, pH 6.7, 0.0625% Tween-20, 1% betaine, 1 mM dithiothreitol and 0.0125% azide, and loaded (3.5 ml. min−1) onto a 2.6×16 cm Pharmacia S-Sepharose HP cation-exchange column. Elution at 3.5 ml min−1 used a linear NaCl gradient (400 ml; 3.5 ml. min−1; 0 to 0.5 M; 15×30 ml fractions) in 30 mM Hepes pH 6.7, 0.5 mM EGTA, 0.05 mM EDTA, 1% betaine, 0.05% Tween-20, 0.01% azide, 1 mM DTT. Fractions were assayed for protein binding to PtdIns(3,4,5)P3-beads, yielding proteins V-Y (see below and FIG. 8).
Membranes. PBS-washed leukocytes ( ̃100 ml) were suspended in lysis buffer (3 volumes of 20 mM Hepes, pH 7.4, 0.1 M sucrose, 0.1 M KCl, 2 mM EGTA, 0.5 mM EDTA, 10 mM β-glycerophosphate, 0.1 mM PMSF and protease inhibitors (ALAP)), sonicated and a ‘post-nuclear supernatant’ obtained by centrifugation (900×gav; 10 min). 15 ml samples were centrifuged (100,000×gav; 60 min) into discontinuous sucrose gradients (12 ml 0.6 M sucrose over 12 ml 1.25 M sucrose, both in lysis buffer) in a swing-out rotor. The 0.6 M/1.25 M sucrose interface was collected, diluted >5-fold with lysis buffer and pelleted (160,000×gav; 30 min). The plasma membrane-enriched pellet was suspended and stored in 20 mM Hepes pH 7.2, 0.125 M NaCl, 5 mM EDTA, 5 mM EGTA, 5 mM β-glycerophosphate, 10 mM NaF and stored. 100 ml cells typically yielded 30-40 mg membrane protein.
Anion-Exchange Chromatography of Detergent-Solubilised Membranes.
Membranes (90 mg) were solubilised on ice for 30 min in 85 ml of 40 mM Tris-HCl, 9 mM Hepes, 0.2 M NaCl, 2.2 mM EDTA, 2.2 mM EGTA, 4.5 mM NaF, 2.2 mM β-glycerophosphate, 1.5% NP40, 1 mM dithiothreitol, 0.1 mM PMSF and protease inhibitors (ALAP, see above) at pH 7.8. After centrifugation (250,000×gav; 60 min), the supernatant was filtered, diluted  ̃7-fold and loaded onto a 1.6×7 cm Pharmacia Q-Sepharose H column: final concentrations were 0.4% NP40, 30 mM NaCl, 30 mM Tris, pH 7.8. Elution was with a linear NaCl gradient (0-0.5 M; 44×6 ml fractions) in 30 mM Tris, pH 7.8, 0.5 mM EGTA, 0.1 mM EDTA, 0.4% NP40, 0.01% azide. Fractions were assayed for proteins binding to PtdIns(3,4,5)P3-beads, yielding proteins M and N (FIG. 9). A larger experiment (600 mg membrane protein) yielded proteins O-U. ps Other Cytosol Preparations
Pig platelets. Blood/ACD ( ̃35 l) was centrifuged (200×gav, 15 min, room temperature). 5 mM EGTA was added to the supernatant, which was centrifuged (1,200×gav; 20 min). The platelet pellets were: suspended in 2 l of 10 mM Hepes-NaOH, pH 7.2, 150 mM NaCl, 4 mM KCl, 3 mM EDTA, 2 mM EGTA; sedimented (1,200×gav; 20 min); suspended in 250 ml ice-cold lysis buffer (30 mM Tris-HCl, pH 7.6, 80 mM NaCl, 2 mM EGTA, 0.5 mM EDTA, 1 mM DTT, 0.5 mM PMSF and protease inhibitors (ALAP)); sonicated (three 15 second bursts, with 30 seconds between bursts); and centrifuged (106×gav; 45 min). Supernatants were stored at −80° C.
Sheep liver and brain. Tissues were quickly frozen and stored at −80° C. Samples were thawed, chopped in ice-cold lysis buffer (see above), disrupted (Polytron) and centrifuged (15,000 gav, 30 min; then 150,000 gav, 60 min). Floating fat was discarded, and the supernatant was used immediately for chromatography or stored frozen.
Rat liver. Livers were frozen in N2 and stored at −80° C. They were homogenised (Waring blender, 4×20 sec, in 25 mM Hepes, 2 mM EDTA, 2 mM EDTA, 5 mM-glycerophosphate, 150 mM NaCl, 10 mM NaCl, 2 mM betaine, 10 mM DTT and protease inhibitors (ALAP; see above)), further disrupted with a tight Dounce homogeniser (10-20 strokes) and centrifuged (4000×gav, 15 min; 100,000×gav, 90 min). Floating fat was removed, and filtered supernatant were stored at −80° C.
Anion exchange chromatography. Cytosols were fractionated by anion-exchange chromatography by procedures analogous to those described above for leukocyte cytosol, leading to the isolation of proteins SR1-SR7 and SD1 (see Table 1).
Synthesis of Phosphoinositides and Phosphoinositide-Coupled Beads
Synthesis of the biological (D-) stereoisomers of the dipalmitoyl forms of PtdIns3P, PtdIns4P, PtdIns5P, PtdIns(3,5)P2, PtdIns(3,4)P2, PtdIns(4,5)P2 and PtdIns(3,4,5)P3, and of the biologically inactive reference compound L-PtdIns(3,4,5)3, are described elsewhere (Painter et al., 1999). Lipid stocks were stored as dry films at −80° C.: the sodium salts of PtdIns(3,4,5)P3, PtdIns(3,4)P2. PtdIns(3,5)P2 and PtdIns(4,5)P2 were adjusted to pH 7.0 and dried under vacuum; PtdIns3P, PtdIns4P and PtdIns5P were converted to the free acids through a CHCl3/MeOH/0.1M HCl (1:1:0.9) phase partition and the lower phases dried. Stocks (4-10 mM) were prepared by bath-sonicating dry lipid in H2O (PtdIns(3,4,5)P3, PtdIns(3,4)P2, PtdIns(3,5)P2, PtdIns(4,5)P2) or DMSO (PtdIns3P, PtdIns4P, PtdIns5P): concentrations were determined by organic phosphorus assay.
Coupling of phosphoinositides to Affigel-10 beads is described in Examples 2 and 3. Derivatised and washed beads were stored at 4° C. in 0.1 M sodium phosphate buffer, pH 7.0, 0.01% azide. They were equilibrated for ≧30 min in assay buffer (typically 20 mM Hepes, pH 7.2 at 4° C., 0.2 M NaCl, 5 mM EDTA, 5 mM EGTA, 5 mM β-glycerophosphate, 10 mM NaF, 0.1% NP40) before use.
Identification of Phosphoinositide-binding Proteins
Before samples were subjected to binding studies, they were generally adjusted to: 20-30 mM Hepes/Na or Tris/HCl, pH 7.2-7.5, 5 mM β-glycerophosphate, ≧0.1 mM EDTA (in excess of the free Mg2+), ≧0.1 M NaCl, 10 mM NaF, 0.1% NP-40, 1 mM sodium orthovanadate. They were kept on ice for 15 min, with or without free phosphoinositides (typically 5-50 μM), and then transferred onto phosphoinositide-derivatised beads [typically 1 ml onto 10 μl beads (analytical); or 30-100 ml onto 0.1-0.3 ml beads (preparative)], mixed and returned to ice (45 min). Beads were washed (3 times; <8 min) with 20 mM Hepes pH 7.2, 0.2 M NaCl, 5 mM EDTA, 5 mM EGTA, 5 mM β-glycerophosphate, 10 mM NaF, 0.1% NP-40, and then once in 5 mM Hepes, pH 7.2. Proteins were eluted with SDS-PAGE sample buffer (95° C. for 5 min), separated by SDS-PAGE and silver-stained (analytical experiments) or transferred to nitrocellulose (preparative). Beads, recycled by washing in SDS-PAGE buffer followed by 0.1 M sodium phosphate buffer, pH 7.0, were used up to five times.
Proteins that showed specific binding to phosphoinositide beads were digested with trypsin and processed for mass spectrometric fingerprinting (Erdjument-Bromage et al., 1998). Briefly, peptide mixtures were partially fractionated on Poros 50 R2 RP micro-tips, and resulting peptide pools were analysed by matrix-assisted laser-desorption/ionisation reflectron time-of-flight mass spectrometry (MALDI-reTOF MS) using a Reflex III instrument (Bruker Franzen; Bremen, Germany). Some samples were also analysed by electrospray ionisation (ESI) tandem MS on an API 300 triple quadrupole instrument (PE-SCIEX; Thornhill, Canada) with a custom-made fine ionization source (Geromanos et al., 2000). Mass values from the MALDI-TOF experiments were used to search a non-redundant protein database (‘NR’, NCBI, Bethesda, Md.), using the PeptideSearch algorithm (Mann et al., 1993). MS/MS spectra from the ESI triple quadrupole analyses were inspected for y′′ ion series, and the output was used as a search string in the SequenceTag (Mann and Wilm, 1994) and PepFrag (Fenyö et al., 1998) programs. Identifications were verified by comparing the computer-generated fragment ion series of predicted tryptic peptides with experimental MS/MS data. Sometimes, peptides were fractionated by reversed-phase HPLC (0.8 mm Vydac C18 column; LC-Packings, San Francisco, Calif.) (Elicone et al., 1994). Peak fractions were analysed by MALDI-TOF MS and automated Edman sequencing (477A; Applied Biosystems, Foster City, Calif.) (Tempst et al., 1994).
Cloning and Expression of Phosphoinositide-Binding Proteins
The resulting sequences revealed that complete mRNAs had not been described for five of the PtdIns(3,4,5)P3-binding proteins (C, E, F, G/H and X) or two PtdIns(3,4)P2-binding proteins (SR1 and SR3), but databases included fragment ESTs for each. Image ESTs were used to prepare 32P-labelled probes to screen cDNA libraries for C, E, G/H, SR1 and SR3. Useful clones were mainly from random-primed and oligodT-primed human U937 cell libraries in the λZAPII vector (Stratagene), and useful G/H clones came from a human spleen oligodT/random-primed library in λgtII (Clontech). SR3 clones were recovered from a mixed oligodT/random-primed rat brain library in λZAPII (Stratagene). Multiple overlapping clones allowed prediction of full-length human ORFs for each protein (rat brain sequence was used to PCR SR3 from human liver and brain cDNA (Clontech)). For F, peptide sequences allowed overlapping ESTs to be compiled into a predicted ORF, and this was obtained by nested PCR from human leukocyte cDNA (Clontech). For X, 5′-RACE-PCR from leukocyte cDNA (Clontech) allowed 5′-extension of EST 684797 to a full-length ORF, and gene-specific primers were used to recover a full-length clone.
Peptide sequences from other proteins indicated that they were porcine orthologues of an unconventional type I myosin (I: myosin 1F; X98411; Crozet et al., 1997), the protein tyrosine phosphatase MEG2 (V: Gu et al., 1992), CDC42GAP (K; Lancaster et al., 1994) and -tocopherol transfer protein (SR2; ATTP, Arita et al., 1995), respectively. Partial clones of human myosin 1F (C. Petit, Institut Pasteur, Paris, France) were used to screen U937 cell libraries: overlapping clones defined the full-length human cDNA. We obtained full-length cDNAs for human MEG2 (P. Majerus, University of Washington, St Louis, Mo., USA), and CDC42GAP (A. Hall, UCL, London, UK). Human ATTP cDNA was obtained by PCR from human liver cDNA (Clontech).
Recombinant Proteins
ORFs were subcloned into expression vectors by standard restriction enzyme cloning and PCR methods. When PCR was used, products were verified by sequencing. pGEX4T (Pharmacia) and pQE (Qiagen) vectors were used for bacterial expression of N-terminally GST- or His-tagged proteins, respectively. GST proteins were purified on glutathione-Sepharose-4B (Pharmacia) and His-tagged proteins on metal affinity resin (Talon, Clontech). Proteins were recovered from the elution buffers by gel filtration on PD10 columns (Pharmacia) in PBS, 1 mM EGTA, 0.01% azide. Thrombin cleavage of GST proteins was carried out on the glutathione-Sepharose beads in PBS, 1 mM DTT for 16 h at 4° C. Where needed, proteins were concentrated to ≧1 mg ml−1 using centrifugal filters (Centricon). They were stored at −80° C. in 50% glycerol.
A modified pAcoGEE (Stephens et al., 1997) vector was used for baculovirus-driven expression of N-terminally EE-tagged SR1, SR3 and myo 1F, and for C-terminally EE-tagged PIP3-G/H proteins. Sf9 cells (ETCC) were grown in suspension culture (0.5-2.0×106 cells ml−1) for up to 10 weeks in TNM-FH medium supplemented with 11% foetal bovine serum (heat-inactivated) and antibodies (penicillin/streptomycin). Transfer vectors were co-transfected with linearised baculovirus DNA (Baculo-Gold, PharMingen) into SF9 cells using cationic liposomes (Insectin, PharMingen). Recombinant viruses were plaque-purified and amplified via 3 cycles of infection to high titre (>108 infectious particles ml−1), and protein production was optimised for each construct. Typically, 2-4 litres of culture were infected at 106 cells ml−1 with 10-40 ml of viral stock and cultured for 1.8-2.5 days, cells were harvested, washed in TNM-FH and snap-frozen. Pellets were thawed and sonicated into lysis buffer [1% (w/v) Triton X-100; 0.15 M NaCl; 40 mM HEPES, pH 7.4, 1 mM dithiothreitol, 0.1 mM PMSF and protease inhibitors (ALAP)] (and, for myosin 1F, 5 mM EGTA). Homogenates containing EE-tagged proteins were centrifuged (100,000×g, 60 min) and mixed with EE-beads (anti-EE monoclonal antibody, dimethyl pimelimidate-crosslinked to Protein G-Sepharose; capacity  ̃2 mg of a 50 kD target protein ml−1 beads). Immunoprecipitation (90 min, end-on-end at 4° C.) was followed by washing (4 times in 0.1% w/v Triton X-100; 0.3 M NaCl; 20 mM HEPES/Na, pH 7.4, 1 mM DTT; and 3× with PBS plus 1 mM DTT: plus, for SR1 and SR3, 10-20% ethylene glycol). Proteins were eluted with peptide EEYMPME (NH2-terminus acetylated: 100 μg ml−1). Loaded beads were incubated, 3-4 times for 20 mins on ice, with an equal volume of elution buffer, pooled supernatants were concentrated to ≦2.5 ml (if necessary, with Centriplus concentrators, Amicon,) and passed through a PD-10 column (Pharmacia) (in elution buffer without peptide), and protein-containing fractions were concentrated and stored frozen. Concentrations were determined by SDS-PAGE followed by Coomasie staining and Bradford protein assay (BioRad) (with BSA standard, correcting for the 1.29-fold greater than average colour yield of BSA).
Use of Phosphoinositide-Deivatised Beads to Investigate the Specificities of Recombinant Proteins
107 Cos-7 cells were transfected by electroporation with 20 μg expression vector (usually Myc- or GFP-tagged), cells were allowed to recover in DMEM containing 10% FBS in 2×15 cm diameter dishes for 36-48 H, and were washed and lysed into 5 ml per dish of 1.0% NP-40, 20 mM Hepes, pH 7.5, 0.12 M NaCl, 5 mM EDTA, 5 mM EGTA, 5 mM β-glycerophosphate, 1 mM orthovanadate, 10 mM NaF. Lysates were centrifuged (190,000×gav; 30 min).
Samples of the supernatants were diluted up to 4-fold in lysis buffer, depending on expression levels, and mixed with indicated concentrations of phosphoinositide in 1 ml (FIG. 11) for 10 min on ice. They were transferred to 5-20 μl phosphoinositide beads in lysis buffer and mixed gently for 45 min. Sedimented beads were washed (4×, ≦15 min) in modified lysis buffer (0.1% NP-40 rather than 1% NP-40). Proteins were eluted with SDS sample buffer, separated by SDS-PAGE and detected by immunoblotting [anti-myc (monoclonal 9E10) or anti-GFP (rabbit polyclonal): Clontech).
Assays with purified recombinant proteins (prepared via expression of EE, GST or His-tagged versions in E. coli or Sf9 cells—see above), were as just described, except that 0.2-2 μM proteins were incubated with indicated concentrations of phosphoinositides in PBS, 1 mM DTT, 1 mM MgCl2, 0.1% NP-40, 0.5 mg ml−1 BSA (omitted when bound protein was assessed by silver-staining). The beads were washed in PBS, 1 mM MgCl2, 0.1% NP-40, 0.1 mg ml−1 BSA (no BSA for silver-staining), and proteins were eluted in sample buffer, separated by SDS-PAGE and detected—either directly by silver-staining or after transfer to Immobilon by immunoblotting (anti-EE mouse ascites from Babco; anti-His-monoclonal from Clontech; anti-GST polyclonal from Amersham-Pharmacia).
Binding of Recombinant Proteins to Phosphoinositide-Containing Biacore Chips
Unilamellar phospholipid vesicles containing 2.5 mM natural phospholipids [PtdCho, PtdEtn and PtdSer (1:1:1); Avanti Polar Lipids Inc., Alabama] were prepared in PBS (pH 7.4), 1 mM MgCl2, 1 mM EGTA, 0.01% azide by extrusion through 100 nm pores in polycarbonate filters. A lipid monolayer was formed on a hydrophobic affinity sensor-chip (HPA, Biacore AB) by first cleaning the chip with an 80 injection of 40 mM octyl D-glucoside (20 1 min−1) then injecting 30 μl of the vesicle solution (2 μl min−1) and washing with 40 μl of 10 mM NaOH (100 μl min−1). Separate flow cells were then injected manually with PtdIns(3,4)P2, PtdIns(3,5)P2, PtdIns(4,5)P2 or PtdIns(3,4,5)P3. Measurements of binding levels at equilibrium were made using proteins at a concentration of 100 nM in a Biacore 3000 instrument.
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TABLE 1





Origin
Name
Size (kDa)
Identity
Acc. No.







PtdIns(3,4,S)P3 beads






Pig leukocyte cytosol
PIP3-A
92
RasGAPIP4BP
TR:Q14644


Pig leukocyte cytosol
PIP3-B
65
Bruton's Tyrosine Kinase (BTK)
SW:P35991


Pig leukocyte cytosol
PIP3-C
145
NOVEL [PLC-L2]
TR:Q9UPR0


Pig leukocyte cytosol
PIP3-D
77
Ezrin/Type II Ins(1,4,5)P3 phosphatase
SW:P31976/P329





(mixed)
9


Pig leukocyte cytosol
PIP3-E
61
NOVEL
EM:XXXXX


Pig leukocyte cytosol
PIP3-F
45
NOVEL [Cytohesin-4]
TR:Q9UIA0


Pig leukocyte cytosol
PIP3-G/H
197/184
NOVEL
EM:XXXXX


Pig leukocyte cytosol
PIP3-I
127
Myosin 1F
EM:XIXXXX


Pig leukocyte cytosol
PIP3-J
73
ETK
SW:P51813


Pig leukocyte cytosol
PIP3-K
51
CDC42-GAP
SW:Q07960


Pig leukocyte cytosol
PIP3-L
200
n.d.



Pig leukocyte memb.
PIP3-M
94
n.d.



Pig leukocyte memb.
PIP3-N
65
n.d.



Pig leukocyte memb.
PIP3-O

n.d. (-subunit mitochondrial fatty acid
SW:Q29554





oxidase?)



Pig leukocyte memb.
PIP3-P

-subunit mitochondrial fatty acid oxidase
SW:046629


Pig leukocyte memb.
PIP3-Q
64
n.d.



Pig leukocyte memb.
PIP3-R
56
n.d.



Pig leukocyte memb.
PIP3-S
28
RasGAPIP4BP (fragment)



Pig leukocyte memb.
PIP3-T
34
n.d.



Pig leukocyte memb.
PIP3-U
62
n.d.



Pig leukocyte cytosol
PIP3-V
59
MEG2
SW:P43378


Pig leukocyte cytosol
PIP3-W
45
PIP3-F
TR:Q9UIA0


Pig leukocyte cytosol
PIP3-X
31
NOVEL [DAPP1]
TR:Q9UN19


Pig leukocyte cytosol
PIP3-Y
42
Centaurin-
TR:O02780


PtdIns(3,4)P2 beads






Sheep liver cytosol
SR1
46
NOVEL
EM:XXXXX


Sheep liver cytosol
SR2

-tocopherol transfer protein (ATTP)
SW:P49638


Sheep liver cytosol
SR3
80
NOVEL [DFCP-1]
EM:XXXXX


Pig platelet cytosol
SR4
128
Vinculin
SW:P18206


Pig platelet cytosol
SR5
46
SR1



PtdIns(3,5)P2 beads






Rat liver cytosol
SD1
30
ATTP



PtdIns3P beads






Pig platelet cytosol
SR6
80
6-phosphofructokinase type C (PFK-C)
SW:K6PP_HUMN


Pig platelet cytosol
SR7
46
SR1





Proteins isolated on phosphoinositide-derivatised beads.


Proteins were excised on nitrocellulose, digested with trypsin and peptides identified by mass fingerprinting and aminoacid sequencing (see Methods).


Proteins that were unknown at the time of identification are described as NOVEL: independent ORFs encoding some of these proteins have since been described and their names are given in square brackets.


EMBL accession numbers are given for our ORFs where they remain undefined in the databases (PIP3-E, PIP3- G/H, SR1), they define new species orthologues (MYO1F) or add significantly to existing entries (SR3).


Protein O was tentatively identified on the basis of its size and co-chromatography with the -subunit of the mitochondrial fatty acid oxidase.


n.d. designates phosphoinositide-binding proteins that have not yet been identified.


SW: Swissprot.


TR: Trembl.


EM: Embl.













TABLE 2





Preferred Compounds and Probes of the Invention













 








 








 








 








 








 


Changes in chain length at the sn-2 position


 








 








 








 








 








 








 








 








 








 








 








 








 


Changes in chain length at the sn-1 position


 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 


C9 at sn-2 position


 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 


C10 at sn-2 position


 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 


C11 at sn-2 position


 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 


C12 at sn-2 position


 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 


C14 at sn-2 position


 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 


C15 at sn-2 position


 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 


C16 at sn-2 position


 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 


C11 at sn-2 position


 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 


C12 at sn-2 position


 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 


C13 at sn-2 position


 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 


C14 at sn-2 position


 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 


C15 at sn-2 position


 








 








 








 








 








 








 








 








 








 








 








 








 








 


C16 at sn-2 position


 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 


Diacylglycol


 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 


Enantiomeric structures


 








 








 








 








 








 








 


Changes in chain length at the sn-2 position


 








 








 








 








 








 








 








 








 








 








 








 








 


Changes in chain at the sn-1 position


 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 


C9 at sn-2 position


 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 


C10 at sn-2 position


 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 


C11 at sn-2 position


 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 


C12 at sn-2 position


 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 


C13 at sn-2 position


 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 


C14 at sn-2 position


 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 


Diacylglycerol


 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 


Changes in chain length at the sn-2 position


 








 








 








 








 








 








 








 








 








 








 








 








 


Changes in chain length at the sn-1 position


 








 








 








 








 








 








 








 








 








 








 








 








 








 








 


C11 at sn-2 position


 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 


C12 at sn-2 position


 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 


C13 at sn-2 position


 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 


C14 at sn-2 position


 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 


C15 at sn-2 position


 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 


C16 at sn-2 position


 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 


Diacylglycerol


 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 






















TABLE 3





Further preferred compounds and probes of the invention







C8


C8H16


 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 


C9


 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 


C10


 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 


C11


 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 


C12


 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 


C13


 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 


C14


 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 


C15


 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 


C16

## Claims

1. A probe having any of the following general formulae:



wherein:
R=aryl or alkyl group;
R=CmH2m+1, where m=8-20;
R3=P(O)(OM)2; R4=H; R5=H (PI(3)P);
R3=H; R4=P(O)(OM)2, R5=H (PI(4)P);
R3=H; R4=H; R5=P(O)(OM)2 (PI(5)P);
R3=P(O)(OM)2; R4=P(O)(OM)2; R5=H (PI(3,4)P2);
R3=P(O)(OM)2; R4=H; R5=P(O)(OM)2 (PI(3,5)P2);
R3=H; R4=P(O)(OM)2; R5=P(O)(OM)2 (PI(4,5)P2); or
R3=P(O)(OM)2; R4=P(O)(OM)2; R5=P(O)(OM)2 (PI(3,4,5)P3);
M=any cation including Na+, NH4+;
*Denotes a stereogenic centre;
Linker=(CH2)n with n=8-20;
X=O, S, or NH;
FG=Carbonyl from a carboxylate, thiolo(ester), or an amide;
unsaturations are allowed, including in an arachidonyl side chain;




 =solid support with attachment to functional group;




wherein:
R=aryl or alkyl group;
R=CmH2m+1, where m=8-20;
R3=P(O)(OM)2; R4=H; R5=H (PI(3)P);
R3=H; R4=P(O)(OM)2; R5=H (PI(4)P);
R3=H; R4=H; R5=P(O)(OM)2 (PI(5)P);
R3=P(O)(OM)2; R4=P(O)(OM)2; R5=H (PI(3,4)P2);
R3=P(O)(OM)2; R4=H; R5=P(O)(OM)2 (PI(3,5)P2);
R3=H; R4=P(O)(OM)2, R5=P(O)(OM)2 (PI(4,5)P2); or
R3=P(O)(OM)2; R4=P(O)(OM)2, R5=P(O)(OM)2 (PI(3,4,5)P3);
M=any cation including Na+ and NH4+;
*Denotes a stereogenic centre;
X=O, S, or NH;
FG A=Carbonyl from a carboxylate, thiolo(ester), or an amide;
Linker A=(CH2)n with n=8-20;
Linker B=aryl, heteroaryl, alkyl with possible heteroatoms and/or saturations;
these could be any atoms, including C, N, O, S, or methylene groups linked via amide and ester bonds;
FG B=Amide, thiolo(ester), or ester;
unsaturations are allowed, including in an arachidonyl side chain;




 =solid support with attachment to functional group.

2. The probe according to claim 1, wherein the total length of linker A and linker B of formula VII or VIII is 8-60 atoms.

3. The probe according to claim 1, wherein the R group is alkyl.

4. The probe according to claim 1, wherein the probe has a formula of one of the compounds as listed below:

5. A probe consisting of a phosphatidic acid functionalised solid support of the general formula as is depicted in Formulae I, II, III or IV:



(a) the linker consists of (CH2)n, with n=8-20;
(b) the heteroatom X is O, S, or NH;
(c) the functional group (FG) is a carbonyl from a carboxylate (thiolo)ester, or an amide;
(d) the R-substituent carries an aryl or alkyl group;
R=CmH2m+1, where m=8-20;
(e) the ion M represents any cation, including Na+ and NH4+;
(f) unsaturations are allowed, including in an arachidonyl side chain; and
(g)




 =solid support with attachment to functional group.

6. The probe according to claim 5, wherein the diacyl glycerol stereocenter is sn2.

7. The probe according to claim 5, wherein the diacyl glycerol stereocenter is the enantiomeric 2(S)-configuration.

8. The probe according to claim 5, wherein the phosphate head groups are substituted by phosphonic acid or thiono phosphate.

9. The probe according to claim 5 which has a formula of one of the compounds as listed below:

10. The probe according to claim 5 which includes attachment of a fluorescent reporter group in the carboxyl side chain ester attached to the sn2-alkoxy substituent of the sn-glycerol-3-phosphate derivative, of formulae I, II, IV, VI, or to the sn1-alkoxy substituent of the sn-glycerol-3-phosphate derivative of formulae III, IV, VII, VIII.

11. The probe according to claim 5 which is capable of binding a protein which itself binds PA, wherein the probe has the following formulae:

12. A probe which is capable of binding a protein which itself binds PI(3,4,5)P3, wherein the probe has the following formulae:

13. A probe which is capable of binding a protein which itself binds PI(4,5)P2, wherein the probe has the following formulae:

14. The probe according to claim 5 which is capable of binding a PA-binding protein with protein molecular weight in the range 60-250 kD, or 60-160 kD.

15. A probe which comprises a phosphoinositide attached onto a solid support by non-covalent binding, wherein the strength of the non-covalent attachment is such that it is not disrupted under conditions in which a protein is bound specifically to the probe with a binding energy greater than about 200 KJ/mole so that the non-covalent attachment is not disrupted under conditions in which a protein is bound specifically to the probe, and wherein the phosphoinositide is PI(3)P, PI(4)P, PI(5)P, PI(3,4)P2, PI(3,5)P2, PI(4,5)P2, or PI(3,4,5)P3.

16. A method of making a probe according to claim 1 which comprises reacting a compound of formula V′ or VI′:



wherein:
R=aryl or alkyl group;
R=CmH2m+1, where m=8-20;
R3=P(O)(OM)2; R4=H; R5=H (PI(3)P);
R3=H; R4=P(O)(OM)2; R5=H (PI(4)P);
R3=H; R4=H; R5=P(O)(OM)2 (PI(5)P);
R3=P(O)(OM)2; R4=P(O)(OM)2; R5=H (PI(3,4)P2);
R3=P(O)(OM)2; R4=H; R5=P(O)(OM)2 (PI(3,5)P2);
R3=H; R4=P(O)(OM)2; R5=P(O)(OM)2 (PI(4,5)P2); or
R3=P(O)(OM)2; R4=P(O)(OM)2; R5=P(O)(OM)2 (PI(3,4,5)P3);
M=any cation, including Na+, NH4+;
*Denotes a stereogenic centre;
Linker=(CH2)n with n=8-20;
X=NH, O, or S;
unsaturations are allowed, including in an arachidonyl side chain;
with




where:




 =solid support with attachment to RG2; and
RG2=a reactive group capable of reaction with XH, including N-hydroxy-succinimide-activated carboxylate.

17. A method of making a probe according to claim 1 which comprises reacting a compound of formula VII′ or VIII′:



where:
R=aryl or alkyl group;
R=CmH2m+1, where m=8-20;
R3=P(O)(OM)2; R4=H; R5=H (PI(3)P);
R3=H; R4=P(O)(OM)2; R5=H (PI(4)P);
R3=H; R4=H; R5=P(O)(OM)2 (PI(5)P);
R3=P(O)(OM)2; R4=P(O)(OM)2; R5=H (PI(3,4)P2);
R3=P(O)(OM)2; R4=H; R5=P(O)(OM)2 (PI(3,5)P2),
R3=H; R4=P(O)(OM)2; R5=P(O)(OM)2 (PI(4,5)P2); or
R3=P(O)(OM)2, R4=P(O)(OM)2; R5=P(O)(OM)2 (PI(3,4,5)P3);
M=any cation, including Na+ and NH4+;
*Denotes a stereogenic centre;
Linker A=(CH2)n with n=8-20;
X=NH, O, or S;
unsaturations are allowed, including in an arachidonyl side chain;
with




Linker B=aryl, heteroaryl, alkyl with possible heteroatoms and/or saturations;
these could be any atoms, including C, N, O, S, or methylene groups linked via amide and ester bonds;
the total length of linker A and linker B is 8-60 atoms;
FG B=Amide, thiolo(ester), or ester




 =solid support with attachment to FG B; and
RG2=a reactive group capable of reaction with XH, including N-hydroxy-succinimide-activated carboxylate.

18. The method according to claim 16 further comprising deprotecting a compound of formula V′′, VI′′, VII′′, or VIII′′ to form a compound of formula V′, VI′, VII′ or VIII′:



wherein,
R=aryl or alkyl group;
R is CmH2m+1, where m is 8-20;
R3 is P(O)(OBn)2; R4=H; R5 is H (PI(3)P),
R3 is H; R4 is P(O)(OBn)2; R5 is H (PI(4)P);
R3 is H; R4 is H; R5 is P(O)(OBn)2 (PI(5)P);
R3 is P(O)(OBn)2; R4 is P(O)(OBn)2; R5 is H (PI(3,4)P2);
R3 is P(O)(OBn)2; R4 is H; R5 is P(O)(OBn)2 (PI(3,5)P2);
R3 is H; R4 is P(O)(OBn)2; R5 is P(O)(OBn)2 (PI(4,5)P2); or
R3 is P(O)(OBn)2; R4 is P(O)(OBn)2; R5 is P(O)(OBn)2 (PI(3,4,5)P3);
*Denotes a stereogenic centre;
Linker is (CH2)n wherein n=8-20;
X is O, S, or, NH;
unsaturations are allowed, including in an arachidonyl side chain;




wherein,
R is aryl or alkyl group;
R is CmH2m+1; where m is 8-20;
R3 is P(O)(OBn)2; R4 is H; R5 is H (PI(3)P);
R3 is H; R4 is P(O)(OBn)2, R5 is H (PI(4)P);
R3 is H; R4 is H; R5 is P(O)(OBn)2 (PI(5)P);
R3 is P(O)(OBn)2; R4 is P(O)(OBn)2; R5 is H (PI(3,4)P2);
R3 is P(O)(OBn)2; R4 is H; R5 is P(O)(OBn)2 (PI(3,5)P2);
R3 is H; R4 is P(O)(OBn)2; R5 is P(O)(OBn)2 (PI(4,5)P2); or
R3 is P(O)(OBn)2; R4 is P(O)(OBn)2; R5 is P(O)(OBn)2 (PI(3,4,5)P3);
*Denotes a stereogenic centre;
Linker A is (CH2)n wherein n=8-20;
X is O, S, or NH;
unsaturations are allowed, including in an arachidonyl side chain.

19. The method of claim 16 wherein the R group is alkyl.

20. The method of claim 16, wherein the compound of formula V′, VI′, VII′, or VIII′ is of one of the compounds as listed below:

21. A method of making a probe according to claim 5 comprising reacting a compound of formula I′, II′, III′, or IV:



wherein,
(a) the linker consists of (CH2)n, wherein n is 8-20;
(b) the R-substituent carries an aryl or alkyl group;
R is CmH2m+1, where m is 8-20;
(c) the ion M represents any cation, including Na+, NH4+;
(d) unsaturations are allowed, including in an arachidonyl side chain;
X is NH, O, or S
RG2 is a reactive group capable of reaction with XH;




 is a solid support with attachment to RG2.

22. The method of claim 21 further comprising deprotecting a compound of formula I′′, II′′, III′′, or IV′′ to form the compound of formula I′, II′, III′, or IV′:



wherein,
(a) the linker consists of (CH2)n, wherein n is 8-20;
(b) the heteroatom X is O, S, or NH;
(c) the R-substituent carries an aryl or alkyl group;
R is CmH2m+1, where m is 8-20;
(d) unsaturations are allowed, including in an arachidonyl side chain;
(e) R′ is any suitable protecting group, including Bn; trialkyl silyl; and CNCH2CH2—;
(f) R′′ is any suitable protecting group, including Fmoc; CBz, when X is NH.

23. The method according to claim 21, wherein the compound of formula I′ or II′ is of one of the compounds as listed below:

24. A method of making a compound of Formula I′, II′, III′, or IV′ which method comprises removal of the protecting groups of a compound of Formula I′′, II′′, III′′, or IV′′, respectively, including by reductive debenzylation



wherein:
(a) the linker consists of (CH2)n, wherein n is 8-20;
(b) the R-substituent carries an aryl or alkyl group;
R is CmH2m+1, where m is 8-20;
(c) the ion M represents any cation, including Na+, NH4+;
(d) unsaturations are allowed, including in an arachidonyl side chain;
X is NH, O, or S;
RG2 is a reactive group capable of reaction with XH;




 is a solid support with attachment to RG2;




wherein,
(a) the linker consists of (CH2)n, wherein n is 8-20;
(b) the heteroatom X is O, S, or NH;
(c) the R-substituent carries an aryl or alkyl group;
R is CmH2m+1, where m is 8-20;
(d) unsaturations are allowed, including in an arachidonyl side chain;
(e) R′ is any suitable protecting group, including Bn; trialkyl silyl; and CNCH2CH2—;
(f) R′′ is any suitable protecting group, including Fmoc; CBz, when X is NH.

25. A method of making a compound of Formula V′, VI′, VII′, or VIII′ which method includes reductive debenzylation of a compound of Formula V′′, VI′′, VII′′, or VIII′′, respectively



wherein:
R=aryl or alkyl group;
R=CmH2m+1, where m=8-20;
R3=P(O)(OM)2; R4=H; R5=H (PI(3)P);
R3=H; R4=P(O)(OM)2; R5=H (PI(4)P);
R3=H; R4=H; R5=P(O)(OM)2 (PI(5)P);
R3=P(O)(OM)2; R4=P(O)(ONA)2, R5=H (PI(3,4)P2);
R3=P(O)(OM)2; R4=H; R5=P(O)(OM)2 (PI(3,5)P2);
R3=H; R4=P(O)(OM)2; R5=P(O)(OM)2 (PI(4,5)P2); or
R3=P(O)(OM)2; R4=P(O)(OM)2; R5=P(O)(OM)2 (PI(3,4,5)P3);
M=any cation, including Na+, NH4+;
*Denotes a stereogenic centre;
Linker=(CH2)n with n=8-20;
X=NH, O, or S
unsaturations are allowed, including in an arachidonyl side chain;
with




where:




 =solid support with attachment to RG2; and
RG2=a reactive group capable of reaction with XH, including N-hydroxy-succinimide-activated carboxylate;




wherein:
R=aryl or alkyl group;
R=CmH2m+, where m=8-20;
R3=P(O)(OM)2; R4=H; R5=H (PI(3)P);
R3=H; R4=P(O)(OM)2; R5=H (PI(4)P);
R3=H; R4=H; R5=P(O)(OM)2 (PI(5)P);
R3=P(O)(OM)2; R4=P(O)(OM)2; R5=H (PI(3,4)P2);
R3=P(O)(OM)2; R4=H; R5=P(O)(OM)2 (PI(3,5)P2);
R3=H; R4=P(O)(OM)2; R5=P(O)(OM)2 (PI(4,5)P2); or
R3=P(O)(OM)2; R4=P(O)(OM)2; R5=P(O)(OM)2 (PI(3,4,5)P3);
M=any cation, including Na+, NH4+;
*Denotes a stereogenic centre;
Linker A=(CH2)n with n=8-20;
X=NH, O, or S;
unsaturations are allowed, including in an arachidonyl side chain;
with




Linker B=aryl, heteroaryl, alkyl with possible heteroatoms and/or saturations: these could be any atoms, including C, N, O, S, or methylene groups linked via amide and ester bonds;
the total length of linker A and linker B is 8-60 atoms
FG B=Amide, thiolo(ester), or ester




 =solid support with attachment to FG B; and
RG2=a reactive group capable of reaction with XH, including N-hydroxy-succinimide-activated carboxylate;




wherein:
R is an aryl or alkyl group;
R is CmH2m+1, where m is 8-20;
R3 is P(O)(OBn)2; R4 is H; R5 is H (PI(3)P);
R3 is H; R4 is P(O)(OBn)2; R5 is H (PI(4)P);
R3 is H; R4 is H; R5 is P(O)(OBn)2 (PI(5)P);
R3 is P(O)(OBn)2; R4 is P(O)(OBn)2; R5 is H (PI(3,4)P2);
R3 is P(O)(OBn)2; R4 is H; R5 is P(O)(OBn)2 (PI(3,5)P2);
R3 is H; R4 is P(O)(OBn)2; R5 is P(O)(OBn)2 (PI(4,5)P2); or
R3 is P(O)(OBn)2; R4 is P(O)(OBn)2; R5 is P(O)(OBn)2 (PI(3,4,5)P3);
*Denotes a stereogenic centre;
Linker is (CH2)n wherein n is 8-20;
X is O, S, or NH;
unsaturations are allowed, including in an arachidonyl side chain;




wherein:
R is an aryl or alkyl group;
R is CmH2m+1, where m is 8-20;
R3 is P(O)(OBn)2; R4 is H; R5 is H (PI(3)P);
R3 is H; R4 is P(O)(OBn)2; R5 is H (PI(4)P);
R3 is H; R4 is H; R5 is P(O)(OBn)2 (PI(5)P);
R3 is P(O)(OBn)2; R4 is P(O)(OBn)2; R5 is H (PI(3,4)P2);
R3 is P(O)(OBn)2; R4 is H; R5 is P(O)(OBn)2 (PI(3,5)P2);
R3 is H; R4 is P(O)(OBn)2; R5 is P(O)(OBn)2 (PI(4,5)P2); or
R3 is P(O)(OBn)2; R4 is P(O)(OBn)2; R5 is P(O)(OBn)2 (PI(3,4,5)P3);
*Denotes a stereogenic centre;
Linker A is (CH2)n wherein n is 8-20;
X is O, S, or NH;
unsaturations are allowed, including in an arachidonyl side chain.

26. A method of making a compound of Formula I′′, II′′, III′′, or IV′′;



wherein,
(a) the linker consists of (CH2)m, wherein n is 8-20;
(b) the heteroatom X is O, S, or NH;
(c) the R-substituent carries an aryl or alkyl group;
R is CmH2m+1, where m is 8-20;
(d) unsaturations are allowed, including in an arachidonyl side chain;
(e) R′ is any suitable protecting group, including Bn; trialkyl silyl; and CNCH2CH2—;
(f) R′′ is any suitable protecting group, including Fmoc; CBz, when X is NH.
By phosphitylation of alcohol:




with (R′′′O)2 PNPr2 and oxidation of the phosphitylated product, where R′′=Bn;
CNCH2CH2—; trialkyl silyl.

27. A method of making a compound of Formula V′′, VI′′, VII′′, or VIII′′;



wherein:
R is an aryl or alkyl group;
R is CmH2m+1, where m is 8-20;
R3 is P(O)(OBn)2; R4 is H; R5 is H (PI(3)P);
R3 is H; R4 is P(O)(OBn)2; R5 is H (PI(4)P);
R3 is H; R4 is H; R5 is P(O)(OBn)2 (PI(5)P);
R3 is P(O)(OBn)2; R4 is P(O)(OBn)2; R5 is H (PI(3,4)P2);
R3 is P(O)(OBn)2; R4 is H; R5 is P(O)(OBn)2 (PI(3,5)P2);
R3 is H; R4 is P(O)(OBn)2; R5 is P(O)(OBn)2 (PI(4,5)P2); or
R3 is P(O)(OBn)2; R4 is P(O)(OBn)2; R5 is P(O)(OBn)2 (PI(3,4,5)P3);
*Denotes a stereogenic centre;
Linker is (CH2)n wherein n is 8-20;
X is O, S, or NH;
unsaturations are allowed, including in an arachidonyl side chain;




wherein:
R is an aryl or alkyl group;
R is CmH2m+1, where m is 8-20;
R3 is P(O)(OBn)2; R4 is H; R5 is H (PI(3)P);
R3 is H; R4 is P(O)(OBn)2; R5 is H (PI(4)P);
R3 is H; R4 is H; R5 is P(O)(OBn)2 (PI(5)P);
R3 is P(O)(OBn)2; R4 is P(O)(OBn)2; R5 is H (PI(3,4)P2);
R3 is P(O)(OBn)2; R4 is H; R5 is P(O)(OBn)2 (PI(3,5)P2);
R3 is H; R4 is P(O)(OBn)2; R5 is P(O)(OBn)2 (PI(4,5)P2); or
R3 is P(O)(OBn)2; R4 is P(O)(OBn)2; R5 is P(O)(OBn)2 (PI(3,4,5)P3);
*Denotes a stereogenic centre;
Linker is (CH2)n wherein n is 8-20;
X is O, S, or NH;
unsaturations are allowed, including in an arachidonyl side chain;
by coupling a first alcohol of formula:




with a second alcohol of formula:




through a phosphodiester linkage.

28. The method according to claim 27, in which the second alcohol is phosphitylated with BnOP(N1Pr2)2 to produce a phosphoramidite of formula:



which is then coupled to the first alcohol of claim 27 to make the compound of Formula V′′, VI′′, VII′′, or VIII′′.




wherein:
R is an aryl or alkyl group;
R is CmH2m+1, where m is 8-20;
R3 is P(O)(OBn)2; R4 is H; R5 is H (PI(3)P);
R3 is H; R4 is P(O)(OBn)2, R5 is H (PI(4)P);
R3 is R4 is H; R5 is P(O)(OBn)2 (PI(5)P);
R3 is P(O)(OBn)2; R4 is P(O)(OBn)2; R5 is H (PI(3,4)P2);
R3 is P(O)(OBn)2; R4 is H; R5 is P(O)(OBn)2 (PI(3,5)P2);
R3 is H; R4 is P(O)(OBn)2; R5 is P(O)(OBn)2 (PI(4,5)P2); or
R3 is P(O)(OBn)2; R4 is P(O)(OBn)2; R5 is P(O)(OBn)2 (PI(3,4,5)P3);
*Denotes a stereogenic centre;
Linker is (CH2)n wherein n is 8-20,
X is O, S, or NH;
unsaturations are allowed, including in an arachidonyl side chain;




wherein:
R is an aryl or alkyl group;
R is CmH2m+1, where m is 8-20;
R3 is P(O)(OBn)2; R4 is H; R5 is H (PI(3)P);
R3 is H; R4 is P(O)(OBn)2; R5 is H (PI(4)P);
R3 is H; R4 is H; R5 is P(O)(OBn)2 (PI(5)P);
R3 is P(O)(OBn)2; R4 is P(O)(OBn)2; R5 is H (PI(3,4)P2);
R3 is P(O)(OBn)2; R4 is H; R5 is P(O)(OBn)2 (PI(3,5)P2);
R3 is H; R4 is P(O)(OBn)2; R5 is P(O)(OBn)2 (PI(4,5)P2); or
R3 is P(O)(OBn)2; R4 is P(O)(OBn)2; R5 is P(O)(OBn)2 (PI(3,4,5)P3);
*Denotes a stereogenic centre;
Linker is (CH2)n wherein n is 8-20;
X is O, S, or NH;
unsaturations are allowed, including in an arachidonyl side chain.

29. A method of making a compound of Formula I′, II′, III′, or IV′ which comprises making a compound of Formula I′′, II′′, III′′ or IV′′ by a method of claim 26 followed by removal of the protecting groups of the compound of Formula I′′, II′′, III′, or IV′′, including by reductive debenzylation



wherein:
(a) the linker consists of (CH2)n, wherein n is 8-20;
(b) the R-substituent carries an aryl or alkyl group;
R is CmH2m+1, where m is 8-20;
(c) the ion M represents any cation, including Na+, NH4+;
(d) unsaturations are allowed, including in an arachidonyl side chain;
X is NH, O, or S;
RG2 is a reactive group capable of reaction with XH;




 is a solid support with attachment to RG2;




wherein,
(a) the linker consists of (CH2)n, wherein n is 8-20;
(b) the heteroatom X is O, S, or NH;
(c) the R-substituent carries an aryl or alkyl group;
R is CmH2m+1, where m is 8-20;
(d) unsaturations are allowed, including in an arachidonyl side chain;
(e) R′ is any suitable protecting group, including Bn; trialkyl silyl; and CNCH2CH2—;
(f) R′′ is any suitable protecting group, including Fmoc; CBz, when X is NH.

30. A method of making a compound of Formula V′, VI′, VII′, or VIII′, which comprises making a compound of Formula V′′, VI′′, VII′′, or VIII′′ by a method of claim 27 followed by reductive debenzylation of the compound of Formula V′′, VI′′, VII′′, VIII′′:



wherein:
R=aryl or alkyl group;
R=CmH2m+1, where m=8-20;
R3=P(O)(OM)2; R4=H; R5=H (PI(3)P);
R3=H; R4=P(O)(OM)2; R5=H (PI(4)P);
R3=H; R4=H; R5=P(O)(OM)2 (PI(5)P);
R3=P(O)(OM)2; R4=P(O)(OM)2; R5=H (PI(3,4)P2);
R3=P(O)(OM)2; R4=H; R5=P(O)(OM)2 (PI(3,5)P2);
R3=H; R4=P(O)(OM)2; R5=P(O)(OM)2 (PI(4,5)P2); or
R3=P(O)(OM)2; R4=P(O)(OM)2; R5=P(O)(OM)2 (PI(3,4,5)P3);
M=any cation, including Na+, NH4+;
*Denotes a stereogenic centre;
Linker=(CH2)n with n=8-20;
X=NH, O, or S
unsaturations are allowed, including in an arachidonyl side chain;
with




where:




 =solid support with attachment to RG2; and
RG2=a reactive group capable of reaction with XH, including N-hydroxy-succinimide-activated carboxylate;




wherein:
R=aryl or alkyl group;
R=CmH2m+1, where m=8-20;
R3=P(O)(OM)2; R4=H; R5=H (PI(3)P);
R3=H; R4=P(O)(OM)2; R5=H (PI(4)P);
R3=H; R4=H; R5=P(O)(OM)2 (PI(5)P),
R3=P(O)(OM)2; R4=P(O)(OM)2; R5=H (PI(3,4)P2);
R3=P(O)(OM)2; R4=H; R5=P(O)(OM)2 (PI(3,5)P2);
R3=H; R4 P(O)(OM)2; R5=P(O)(OM)2 (PI(4,5)P2); or
R3=P(O)(OM)2; R4=P(O)(OM)2; R5=P(O)(OM)2 (PI(3,4,5)P3);
M=any cation, including Na+, NH4+;
*Denotes a stereogenic centre;
Linker A=(CH2)n with n=8-20;
X=NH, O, or S;
unsaturations are allowed, including in an arachidonyl side chain;
with




Linker B=aryl, heteroaryl, alkyl with possible heteroatoms and/or saturations.:these could be any atoms, including C, N, O, S, or methylene groups linked via amide and ester bonds;
the total length of linker A and linker B is 8-60 atoms
FG B=Amide, thiolo(ester), or ester




 =solid support with attachment to FG B; and
RG2=a reactive group capable of reaction with XH, including N-hydroxy-succinimide-activated carboxylate;




wherein:
R is an aryl or alkyl group;
R is where m is 8-20;
R3 is P(O)(OBn)2; R4 is H; R5 is H (PI(3)P);
R3 is H; R4 is P(O)(OBn)2; R5 is H (PI(4)P);
R3 is H; R4 is H; R5 is P(O)(OBn)2 (PI(5)P);
R3 is P(O)(OBn)2; R4 is P(O)(OBn)2, R5 is H (PI(3,4)P2),
R3 is P(O)(OBn)2, R4 is H; R5 is P(O)(OBn)2 (PI(3,5)P2);
R3 is H; R4 is P(O)(OBn)2; R5 is P(O)(OBn)2 (PI(4,5)P2); or
R3 is P(O)(OBn)2; R4 is P(O)(OBn)2; R5 is P(O)(OBn)2 (PI(3,4,5)P3);
*Denotes a stereogenic centre;
Linker is (CH2)n wherein n is 8-20;
X is O, S, or NH;
unsaturations are allowed, including in an arachidonyl side chain;




wherein:
R is an aryl or alkyl group;
R is CmH2m+1, where m is 8-20;
R3 is P(O)(OBn)2; R4 is H; R5 is H (PI(3)P);
R3 is H; R4 is P(O)(OBn)2; R5 is H (PI(4)P);
R3 is H; R4 is H; R5 is P(O)(OBn)2 (PI(5)P);
R3 is P(O)(OBn)2; R4 is P(O)(OBn)2; R5 is H (PI(3,4)P2);
R3 is P(O)(OBn)2; R4 is H; R5 is P(O)(OBn)2 (PI(3,5)P2);
R3 is H; R4 is P(O)(OBn)2; R5 is P(O)(OBn)2 (PI(4,5)P2); or
R3 is P(O)(OBn)2; R4 is P(O)(OBn)2; R5 is P(O)(OBn)2 (PI(3,4,5)P3);
*Denotes a stereogenic centre;
Linker A is (CH2)n wherein n is 8-20;
X is O, S, or NH;
unsaturations are allowed, including in an arachidonyl side chain.

31. A method of making a probe consisting of a phosphatidic acid functionalised solid support of the general formula as is depicted in Formula I, II, III or IV comprising coupling a compound of Formula I′, II′, III′ or IV' made by a method of claim 24 to the solid support



(a) the linker consists of (CH2)n, with n=8-20;
(b) the heteroatom X is O, S, or NH;
(c) the functional group (FG) is a carbonyl from a carboxylate (thiolo)ester, or an amide;
(d) the R-substituent carries an aryl or alkyl group;
R=CmH2m+1, where m=8-20
(e) the ion M represents any cation, including Na+, NH4+;
(f) unsaturations are allowed, including in an arachidonyl side chain; and
(g)




 =solid support with attachment to functional group;




wherein:
(a) the linker consists of (CH2)n, wherein n is 8-20
(b) the R-substituent carries an aryl or alkyl group;
R is CmH2m+1, where m is 8-20;
(c) the ion M represents any cation, including Na+, NH4+;
(d) unsaturations are allowed, including in an arachidonyl side chain;
X is NH, O, or S;
RG2 is a reactive group capable of reaction with XH;




 is a solid support with attachment to RG2.

32. A method of making a probe having any of the general Formulae V, VI, VII or VIII comprising coupling a compound of Formula V′, VI′, VII′ or VIII′ made by a method of claim 25 to the solid support



wherein:
R=aryl or alkyl group;
R=CmH2m+1, where m=8-20;
R3=P(O)(OM)2; R4=H; R5=H (PI(3)P);
R3=H; R4=P(O)(OM)2; R5=H (PI(4)P);
R3=H; R4=H; R5=P(O)(OM)2 (PI(5)P),
R3=P(O)(OM)2; R4=P(O)(OM)2; R5=H (PI(3,4)P2);
R3=P(O)(OM)2; R4=H; R5=P(O)(OM)2 (PI(3,5)P2);
R3=H; R4=P(O)(OM)2; R5=P(O)(OM)2 (PI(4,5)P2); or
R3=P(O)(OM)2; R4=P(O)(OM)2; R5=P(O)(OM)2 (PI(3,4,5)P3);
M=any cation, including Na+, NH4+;
*Denotes a stereogenic centre;
Linker=(CH2)n with n=8-20;
X=O, S, or NH;
FG=Carbonyl from a carboxylate, thiolo(ester), or an amide;
unsaturations are allowed, including in an arachidonyl side chain;




 =solid support with attachment to functional group;




wherein:
R=aryl or alkyl group;
R=CmH2m+1, where m=8-20;
R3=P(O)(OM)2; R4=H; R5=H (PI(3)P);
R3=H; R4=P(O)(OM)2; R5=H (PI(4)P);
R3=H; R4=H; R5=P(O)(OM)2 (PI(5)P);
R3=P(O)(OM)2; R4=P(O)(OM)2; R5=H (PI(3,4)P2);
R3=P(O)(OM)2; R4=H; R5=P(O)(OM)2 (PI(3,5)P2);
R3=H; R4=P(O)(OM)2; R5=P(O)(OM)2 (PI(4,5)P2); or
R3=P(O)(OM)2; R4=P(O)(OM)2; R5=P(O)(OM)2 (PI(3,4,5)P3);
M=any cation, including Na+, NH4+;
*Denotes a stereogenic centre;
X=O , S, or NH
FG A=Carbonyl from a carboxylate, thiolo(ester), or an amide;
Linker A=(CH2)n with n=8-20;
Linker B=aryl, heteroaryl, alkyl with possible heteroatoms and/or saturations:
these could be any atoms, including C, N, O, S, or methylene groups linked via amide and ester bonds;
FG B=Amide, thiolo(ester), or ester;
unsaturations are allowed, including in an arachidonyl side chain;




 =solid support with attachment to functional group.




wherein:
R=aryl or alkyl group;
R=CmH2m+1, where m=8-20;
R3=P(O)(OM)2; R4=H; R5=H (PI(3)P);
R3=H; R4=P(O)(OM)2; R5=H (PI(4)P);
R3=H; R4=H; R5=P(O)(OM)2 (PI(5)P);
R3=P(O)(OM)2; R4=P(O)(OM)2, R5=H (PI(3,4)P2),
R3=P(O)(OM)2; R4=H; R5=P(O)(OM)2 (31(3,5)P2);
R3=H; R4=P(O)(OM)2; R5=P(O)(OM)2 (PI(4,5)P2); or
R3=P(O)(OM)2; R4=P(O)(OM)2; R5=P(O)(OM)2 (PI(3,4,5)P3);
M=any cation, including Na+, NH4+;
*Denotes a stereogenic centre;
Linker=(CH2)n with n=8-20;
X=NH, O, or S
unsaturations are allowed, including in an arachidonyl side chain;
with




where:




 =solid support with attachment to RG2; and
RG2=a reactive group capable of reaction with XH, including N-hydroxy-succinimide-activated carboxylate;




wherein:
R=aryl or alkyl group;
R=CmH2m+1, where m=8-20;
R3=P(O)(OM)2; R4=H; R5=H (PI(3)P);
R3=H; R4=P(O)(OM)2; R5=H (PI(4)P);
R3=H; R4=H; R5=P(O)(OM)2 (PI(5)P);
R3=P(O)(OM)2; R4=P(O)(OM)2; R5=H (PI(3,4)P2);
R3=P(O)(OM)2; R4=H; R5=P(O)(OM)2 (PI(3,5)P2);
R3=H; R4=P(O)(OM)2; R5=P(O)(OM)2 (PI(4,5)P2); or
R3=P(O)(OM)2; R4=P(O)(OM)2; R5=P(O)(OM)2 (PI(3,4,5)P3);
M=any cation, including Na+, NH4+;
*Denotes a stereogenic centre;
Linker A=(CH2)n with n=8-20;
X=NH, O, or S;
unsaturations are allowed, including in an arachidonyl side chain;
with




Linker B=aryl, heteroaryl, alkyl with possible heteroatoms and/or saturations:
these could be any atoms, including C, N, O, S, or methylene groups linked via amide and ester bonds;
the total length of linker A and linker B is 8-60 atoms;
FG B=Amide, thiolo(ester), or ester




 =solid support with attachment to FG B; and
RG2=a reactive group capable of reaction with XH, including N-hydroxy-succinimide-activated carboxylate.

33. A method according to claim 31 in which the compound is coupled to a carboxylic acid group of the solid support.

34. A method according to claim 27 in which the alcohol of Formula:



is made from a compound of Formula 44:

35. The probe according to claim 1 is coupled to a scintillant or a fluorophore.

36. The method of claim 17 further comprising de-protecting a compound of the formula VII′′ or VIII′′ to form a compound of formula VII′ or VIII′.

37. The method according to claim 17, wherein the R group is an alkyl.

38. The method according to claim 17, wherein the compound of formula VII′ or VIII′ is one of the compounds as listed below: