Patent ID: 6686145
Filing Date: 2004-02-03
Classification: C07D,C09B,G01N

Abstract:
An assay method which comprises:i) binding one component of a specific binding pair with a second component of said pair, said first component being labeled with a fluorescent donor dye and said second component being labeled with fluorescent (or quenching) acceptor dye to bring about an energy transfer relationship between said first and second components; and ii) detecting the binding of the first and second components by measuring emitted fluorescence; wherein the fluorescent donor dye is a compound of formula: optionally substituted by groups R2-R9, wherein groups R6, R7, R8 and R9 are attached to the rings containing X and Y or, optionally are attached to atoms of the Za and Zb ring structures; R2 to R9 which are the same or different include â€”R10 and â€”Lâ€”R10 where R10 is selected from neutral groups that reduce water solubility, polar groups that increase water solubility, functional groups that can be used in labeling reactions, reactive groups, electron donating and withdrawing groups that shift the absorption and emission wavelengths of the fluorescent molecule, lipid and hydrocarbon solubilising groups, and L is selected from the group consisting of a straight or branched C1-20 alkyl chain, a C2-20 monoether or polyether and a C2-20 atom chain containing up to four secondary amide linkages; R1 is selected from hydrogen, aryl, heteroaryl, cyano, nitro, aldehyde, halogen, hydroxy, amino, quaternary amino, acetal, ketal, phosphoryl, sulphydryl, water-solubilizing groups, and alkyl groups optionally substituted by amino, C1-C4 alkyl-substituted amino, quaternary amino, carbonyl including aldehyde and ketone, acetal, ketal, halo, cyano, aryl, heteroaryl, hydroxyl, sulphonate, sulphate, carboxylate, amide, nitro, and groups reactive with amino, hydroxyl, aldehyde, phosphoryl, or sulphydryl groups; A is selected from O, S and NR11 where R11 is the substituted amino radical: where Râ€² is selected from hydrogen, a C1-4 alkyl and aryl and Râ€³ is selected from C1-8 alkyl, aryl, heteroaryl, an acyl radical having from 2-7 carbon atoms, and a thiocarbamoyl radical; X and Y may be the same or different and are selected from bis-C1-C4 alkyl and C4-C5 spiro alkyl substituted carbon, oxygen, sulphur, selenium, CH&boxH;CH, and Nâ€”W wherein N is nitrogen and W is selected from hydrogen, a group â€”(CH2)nR12 where n is an integer from 1 to 26 and R12 is selected from hydrogen, amino, aldehyde, acetal, ketal, halo, cyano, aryl, heteroaryl, hydroxyl, sulphonate, sulphate, carboxylate, substituted amino, quaternary amino, nitro, primary amide, substituted amide, and groups reactive with amino, hydroxyl, carbonyl, phosphoryl, and sulphydryl groups; Za and Zb each represent the atoms necessary to complete one or two fused aromatic rings each ring having six carbon atoms; provided that when X and Y are other than carbon, at least one of R1-R9 comprises a reactive group for covalent reaction with a functional group on a target material or comprises a functional group for covalent reaction with a reactive group on a target material, or, when X and Y are different and are selected from O and Se, at least one of R1-R9 is other than hydrogen, methyl, phenyl or naphthyl.