# US 2004/0072244 A1 · Immobilised phosphatidic acid probe

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- Number: US 2004/0072244 A1
- Document ID: US20040072244A1
- Status: Published application
- Kind: A1
- Application number: 10/362,571
- Filed: 2003-08-28
- Published: 2004-04-15
- Earliest claimed: 2000-08-23
- Assignees: Andrew Bruce Holmes; Ze-Yi Lim; Johannes Wilhelmus John Fitzgerald
- Inventors: Len Stephens; Phillip Thomas Hawkins; Andrew Bruce Holmes; Maria Manifava; Ze-Yi Lim; Nicholas Ktistakis; Johannes Wilhelmus John Fitzgerald Thuring
- Web page: https://patentgrep.com/patent/US20040072244A1

## CPC

- G01N 33/54353: {with ligand attached to the carrier via a chemical coupling agent (coatings G01N33/54393)}
- G01N 33/68: involving proteins, peptides or amino acids {(involving lipoproteins G01N33/92)}
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

Probes comprising an immobilised Phosphatidic Acid attached onto a solid support are described, for example, as shown in formula I and V: (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 functional group (FG) is a carbonyl from a carboxylate (thiolo)ester, or, most preferably an amide; (d) the R-substituent carries an aryl, alkyl group, or a combination, preferably R=CmH2m+1, m=8-20, m=16 optimal; (e) the ion M represents any cation, preferably Na+, NH4+; (f) unsaturations are allowed, such as in an arachidonyl side chain and (g) =solid support with attachment to functional group, where: R=aryl, alkyl group, or a combination, preferably R=CmH2m+1, m=8-20, m=16 is optimal. R3 is P(O) (OM)2; 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; R5P=P(O)(OM)2(PI(4,5)P2); or R3=P(O)(OM)2; R4=P(O)(OM)2; R5=H(O)(OM)2(PI(3,4,5)P3). M=any cation, preferably Na+, NH4+ *Denotes a stereogenic center. 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. Use of the probes to identify Phosphatidic Acid binding protein or phosphoinositide binding proteins is also described.

## Claims

### Claim 1

A probe having any of the following formulae:
1252
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(3,4P2);
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=(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,
1253
=solid support with attachment to functional group,
1254
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(3,4P2);
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=(CH2)n with n=8-20,
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,
FG B=Amide, thiolo(ester), or, more preferably ester,
Unsaturations are allowed, such as in an arachidonyl side chain.
1255
=solid support with attachment to functional group.

### Claim 2 (depends on 1)

A probe according to claim 1 wherein the total length of linker A and linker B of formula VII or VIII is 8-60 atoms, more preferably 19-31 atoms, most preferably 22 atoms.

### Claim 3 (depends on 1, 2)

A probe according to claim 1 or 2 wherein the R group consists of alkyl.

### Claim 4 (depends on 1)

A probe according to claim 1 which has a formula as shown in Table 3.

### Claim 5

A probe, comprising or consisting of a phosphatidic acid functionalised solid support of the general formula as is depicted in Formulae I, II, III or IV:
1256
(a) the linker consists of (CH2)n, with n=8-20, most preferably n=11;
(b) the heteroatom X maybe O, S, or, most preferably NH;
(c) the functional group (FG) is a carbonyl from a carboxylate (thiolo)ester, or, most preferably an amide;
(d) the R-substitute carries an aryl, alkyl group, or a combination, preferably R=CmH2m+1, m=8-20, m=16 is optimal;
(e) the ion M represents any cation, preferably Na+, NH4+;
(f) unsaturations are allowed, such as in an arachidonyl side chain; and
(g)=solid support with attachment to functional group.
1257

### Claim 6 (depends on 5)

A probe according to claim 5 wherein the diacyl glycerol stereocenter is sn2.

### Claim 7 (depends on 5)

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

### Claim 8 (depends on 5)

A probe according to claim 5 wherein the phosphate had group maybe substituted by phosphonic acid or thiono phosphate.

### Claim 9 (depends on 5)

A probe according to claim 5 which has a formula as shown in Table 3.

### Claim 10

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

### Claim 11

A probe which comprises or consist of an immobilised Phosphatidic Acid derivative non covalently attached onto a solid support, wherein 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 (suitably with a binding energy greater than about 200 KJ/mole).

### Claim 12 (depends on 11)

A probe according to claim 11 in which the immobilised Phosphatidic Add derivative is non covalently attached onto the sold support by means of a biotin-avidin, or biotin-streptavidin interaction.

### Claim 13 (depends on 11, 12)

A probe according to claims 11 or 12 in which the immobilised Phosphatidic Acid derivative comprises a phosphoinositide.

### Claim 14

A probe according to any preceding claim wherein the solid support is an organic polymeric support.

### Claim 15

A probe according to any preceding claim wherein the solid support is one which swells in water, such as agarose, sepharose, PEG based resin.

### Claim 16

A probe according to any preceding claim wherein the PA-functionalised, PA derivative-functionalised, or phosphoinositide-functionalised solid support is modified to carry a photoaffinity label, such as aryl azide, α-halo-carbonyl compound, diaryl ketone.

### Claim 17 (depends on 16)

A probe according to claim 16 wherein a 125-labeled azido salicylate (Pierce product calalogue, 2000) is attached through an ω-amide linkage at the sn2-position or the sn1-position of the sn-glycerol-3-phosphate derivative.

### Claim 18 (depends on 5, 6, 7, 8)

A probe according to any of claims 5 to 8, or 10 to 17 which is reagent [1] of Scheme 1, or a functional derivative thereof which is capable of binding a protein which itself binds PA.

### Claim 19

A probe which is reagent [11] of Scheme 2, or a functional derivative thereof which is capable of binding a protein which itself binds PI(3,4,5)P3.

### Claim 20

A probe which is reagent [32] of sample 3, or a functional derivative thereof which is capable of binding a protein which itself binds PI(4,5)P2.

### Claim 21 (depends on 5, 11)

A probe according to claim 5 or 11 which is capable of binding a PA-binding protein with protein molecular weight in the range 60-250 kD, preferably 60-160 kD.

### Claim 22 (depends on 13)

A probe according to claim 13 in which die phosphoinositide is PI(3)P, PI(4)P, PI(5P, PI(3,4)P2, PI(3,5)P2, PI(4,5)P2, or PI(3,4,5)P3, or an analogue thereof.

### Claim 23 (depends on 1)

A method of making a probe according to claim 1 which comprises reaction a compound of formula V′ or VI′:
1258
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)2);
R3=P(O)(OM)2; R4=H; R5P(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=(CH2)n, with n=8-20, most preferably n=11, X=NH, O, S
Unsaturations are allowed, such as in an arachidonyl side chain:
1259
=solid support with attachment RG2,
RG2=a reactive group capable of reaction with XH, e.g. N-hydroxy-succinimide-activated carboxylate.

### Claim 24 (depends on 1)

A method of making a probe according to claim 1 which comprises a compound of formula VII′ or VIII′;
1260
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=(CH2)n with n=8-20,
X=NH, O, S
Unsaturations are allowed, such as in an arachidonyl side chain,
with
1261
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 ia 8-60 atoms, more preferably 19-31 atoms, most preferably 22 atoms,
FG B=Amide, thiolo(ester), or most preferably ester
1262
=solid support with attachment to FG B
RG2=a reactive group capable of reaction with XH, e.g. N-hydroxy-succinimide-activated carboxylate.

### Claim 25 (depends on 23, 24)

A method according to claim 23 or 24 which further comprises deprotecting a compound or formula V″, VI″, VII″, or VIII″ to form the compound of formula V′, VI′, VII′ or VIII′:
1263
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=(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=(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:
1264
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=(CH2)n with n=8-20,
X=O, S, or, most preferably NH,
Unsaturations are allowed, such as in an arachidonyl side chain

### Claim 26 (depends on 23, 24, 25)

A method according any of claims 23 to 25 wherein the R group consist of alkyl.

### Claim 27 (depends on 23, 24, 25, 26)

A method according to any of claims 23 to 26 wherein the compound of formula V′, VI′, VII′, or VIII′ is a compound shown in Table 3.

### Claim 28 (depends on 5)

A method according to claim 5 which comprises reacting a compound of formula I′, II′, III′, or IV′:
1265
(a) the linker consist 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=8-10, 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
1266
=solid support with attachment to RG2.

### Claim 29 (depends on 28)

A method according to claim 28 which further comprises deprotecting a compound of formula I″, II″, III″, or IV″ to form the compound of formula I′, II′, III′, or IV′:
1267
where:
(a) the linker consists of (CH2)n, with n=8-20, must 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 protection group, preferably Bn; trialkyl silyl; CNCH2CH2—
(f) R″=any suitable protecting group, preferably Fmoc; CBz, when X is NH

### Claim 30 (depends on 28, 29)

A method according to claim 28 or 29 wherein the compound of formula I′ or II′ is a compound as show in Table 2.

### Claim 31 (depends on 18, 21)

A method of making a probe according to claim 18 or 21 which method is substantially as described in Scheme 1.

### Claim 32 (depends on 1)

A method of making a probe according to claim 1 which method is substantially as described in Example 2.

### Claim 33 (depends on 1)

A method of making a probe according to claim 1 which method is substantially as described in Example 3.

### Claim 34

A compound having a formula according to any of formulae I′, II′, III′, or IV′.

### Claim 35 (depends on 34)

A compound according to claim 34 which has a formula as shown in Table 2.

### Claim 36

A compound having a formula according to any V′, VI′, VII′, or VIII′.

### Claim 37 (depends on 36)

A compound according to claim 36 which has a formula as shown in Table 3.

### Claim 38

A compound having a formula according to any of formulae I″, II″, III″, or IV″.

### Claim 39

A compound having a formula according to any of formulae V″, VI″, VII″, or VIII″.

### Claim 40 (depends on 5, 34, 35)

Use of a compound according to claim 34, 35, or 38 to make a probe according to claim 5.

### Claim 41 (depends on 11, 36, 37)

Use of a compound according to claim 36, 37, or 39 to make a probe according to claim 11.

### Claim 42 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22)

Use of a probe according to any of claims 1 to 22 to bind a binding partner of the Phosphatidic Acid derivative or phosphoinositide attached onto the solid support.

### Claim 43 (depends on 42)

Use of a probe according to claim 42 wherein the binding partner is a protein.

### Claim 44 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22)

Use of a probe according to any of claims 1 to 22 for testing the PA/PIPn-binding and/or affinity of a protein.

### Claim 45 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22)

An assay method which involves the step of detecting and/or measuring the binding of a probe according to any of claims 1 to 22 when said probe is exposed to a protein in test sample.

### Claim 46 (depends on 45)

An assay according to claim 45 which involves the steps of identifying and/or isolating said protein by binding to said probe.

### Claim 47 (depends on 45, 46)

An assay according to claim 45 or 46 wherein said probe is 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.

### Claim 48 (depends on 45, 46, 47)

An assay according to any of claims 45 to 47 wherein more than one type of probe is used to detect/measure/identifying and/or isolate more than one of PA and/or PIPn binding protein.

### Claim 49 (depends on 45, 46, 47, 48)

An assay according to any of claims 45 to 48 wherein said test sample is a tissue or tissue culture extract, preferably a lysed extract.

### Claim 50 (depends on 48)

An assay according to claim 48 wherein said test sample is obtained by lysis of cells in a buffer containing at least one non-ionic surfactant, such as TRITON (RTM) X-100 or NP-40.

### Claim 51 (depends on 45, 46, 47, 48, 49, 50)

An assay according to any of claims 45 to 50 wherein said probe is exposed to said test sample in the presence or absence of soluble PA and/or PIPn.

### Claim 52 (depends on 45, 46, 47, 48, 49, 50, 51)

An assay according to any of claims 45 to 51 wherein protein-probe binding is compared between more than one test sample to determine PA-binding protein and/or PIPn binding protein variation between said samples.

### Claim 53 (depends on 45, 46, 47, 48, 49, 50, 51, 52)

Use of an any assay according to any of claims 45 to 52 to detect/measure/identify and/or isolate a PA-binding protein and/or a PIPn-binding protein in a test sample.

### Claim 54 (depends on 45, 46, 47, 48, 49, 50, 51, 52)

Use of an assay according to any of claims 45 to 52 to detect and/or measure the ability of on agent, applied to said PA-binding protein-containing and/or PIPn-binding protein-containing test sample, to agonise or antagonise protein-probe binding.

### Claim 55 (depends on 45, 46, 47, 48, 49, 50, 51, 52)

Use of an assay according to any of claims 45 to 52 to detect and/or measure the ability of an agent, applied to said probe, to agonise or antagonise protein-probe binding.

### Claim 56 (depends on 45, 46, 47, 48, 49, 50, 51, 52)

A PA-binding protein or a PIPn-binding protein detected/measured/identified and/or isolate by an assay according to any of claims 45 to 52.

### Claim 57 (depends on 54, 55)

An agent capable of agonising or antagonising protein-probe binding detected and/or measured by use of an assay according to claim 54 or 55.

### Claim 58

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, preferably by reductive debenzylation.

### Claim 59

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.

### Claim 60

A method of making a compound of Formula I″, II″, III″, or VI″;
By phosphitylation of alcohol:
1268
With (R′″O)2 PNPr2 and oxidation of the phosphitylated product, where R′″=Bn; CNCH2CH2—; trialkyl silyl.

### Claim 61

A method of making a compound of Formula V″, VI″, VII″, or VIII″; by coupling a first alcohol of formula:
1269
with a second alcohol of formula:
1270
through a phosphodiester linkage.

### Claim 62 (depends on 60, 61)

A method according to claim 61 in which the second alcohol is phosphitylated with BnOP(NiPr2)2 to produce a phosphoramidite of formula:
1271
which is then coupled to the first alcohol of claim 60 to make the compound of Formula V″, VI″, VII″, or VIII″.

### Claim 63 (depends on 60)

A method of making a compound of Formula I′, II′, III′, or IV′ which comprises making a compound of Formula I″, II″, III″, of IV″ by a method of claim 60 followed by removal of the protecting groups of the compound of Formula I″, II″, III″, or IV″, preferably by reductive debenzylation.

### Claim 64 (depends on 61, 62)

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 61 or 62 followed by reductive debenzylation of the compound of Formula V″, VI″, VII″, VIII″.

### Claim 65 (depends on 5, 58, 63)

A method of making a probe according to claim 5 which comprises coupling a compound of Formula I′, II′, III′, or IV′ made by a method of claim 58 or 63 to the solid support.

### Claim 66 (depends on 1, 59, 64)

A method of making a probe according to claim 1 which comprises coupling a compound of Formula V′, VI′, VII′, or VIII′ made by a method of claim 59 or 64 to the solid support.

### Claim 67 (depends on 65, 66)

A method according to claim 65 or 66 in which the compound is coupled to a carboxylic acid group of the solid support.

### Claim 68 (depends on 61, 62, 64)

A method according to claim 61, 62, 64, or 66 in which the alcohol of Formula:
1272
is from a compound of Formula 44:
1273

### Claim 69 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22)

A probe according to any of claims 1 to 22 coupled to a scintillant or a fluorophore.

### Claim 70 (depends on 69)

Use of a probe according to claim 69 in an assay for identifying an agonist or antagonist of the interaction of a PA/PIPn-binding protein with PA or PIPn.

### Claim 71 (depends on 70)

Use according to claim 70 in a high throughput screen of candidate agonists and/or antagonists.

### Claim 72 (depends on 71)

Use according to claim 71 in a single step high throughput screen.

### Claim 73 (depends on 70, 71, 72)

An agonist or antagonist identified by use according to any of claims 70 to 72.