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AssigneesAndrew Bruce Holmes, Ze-Yi Lim, Johannes Wilhelmus John Fitzgerald
InventorsLen Stephens, Phillip Thomas Hawkins, Andrew Bruce Holmes, Maria Manifava, Ze-Yi Lim, Nicholas Ktistakis, Johannes Wilhelmus John Fitzgerald Thuring
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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.
A probe having any of the following formulae:
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:
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.
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).
A probe according to any preceding claim wherein the solid support is an organic polymeric support.
A probe according to any preceding claim wherein the solid support is one which swells in water, such as agarose, sepharose, PEG based resin.
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.
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.
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.
A compound having a formula according to any of formulae I′, II′, III′, or IV′.
A compound having a formula according to any V′, VI′, VII′, or VIII′.
A compound having a formula according to any of formulae I″, II″, III″, or IV″.
A compound having a formula according to any of formulae V″, VI″, VII″, or VIII″.
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.
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.
A method of making a compound of Formula I″, II″, III″, or VI″;
A method of making a compound of Formula V″, VI″, VII″, or VIII″;
Dependencies are inferred from the claim text.