Xanthene dyes and their application as luminescence quenching compounds

Claim 1

A compound, having the formula wherein R 1 is H; R 6 is H; R 2 , R 3 , R 4 and R 5 are independently H, F, Cl, Br, I, CN; or C 1 -C 18 alkyl, or C 1 -C 18 alkoxy, where each alkyl or alkoxy is optionally further substituted by F, Cl, Br, I, a carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C 1 -C 6 alcohol; or —SO 3 X where X is H or a counterion; or R 1 taken in combination with R 2 , or R 6 taken in combination with R 5 is a fused six-membered aromatic ring; R 8 and R 9 are independently H, C 1 -C 6 alkyl, C 1 -C 6 carboxyalkyl, C 1 -C 6 sulfoalkyl, a salt of C 1 -C 6 carboxyalkyl, or a salt of C 1 -C 6 sulfoalkyl, wherein the alkyl portions are optionally substituted by amino, hydroxy, carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C 1 -C 6 alkyl; or —L—R x ; or —L—S c ; or one or more of R 8 and R 9 is a Q moiety; or R 8 taken in combination with R 9 forms a saturated 5- or 6-membered heterocycle that is optionally fused to a Q moiety and said heterocycle is optionally further substituted by methyl, carboxylic acid, a salt of carboxylic acid, a carboxylic acid ester of a C 1 -C 6 alkyl, or by —L—R x , or —L—S c ; wherein each Q moiety is 1-4 aromatic or heteroaromatic rings that is optionally substituted by halogen, cyano, sulfo, alkali or ammonium salt of sulfo, carboxy, alkali or ammonium salt of carboxy, nitro, alkyl, perfluoroalkyl, alkoxy, alkylthio, amino, monoalkylamino, dialkylarnino; alkylamido; or is substituted by —L—R x ; or is substituted by —L—S c ; wherein each heteroaromatic ring in Q is a 5- or 6-membered aromatic heterocycle having 1 to 3 heteroatoms selected from the group consisting of O, N or S in any combination; and when Q is 2-4 rings, said 2-4 rings are fused to each other; and K is O or N + R 18 R 19 ; wherein R 18 and R 19 are independently H, C 1 -C 6 alkyl, C 1 -C 6 carboxyalkyl, C 1 -C 6 sulfoalkyl, a salt of C 1 -C 6 carboxyalkyl, or a salt of C 1 -C 6 sulfoalkyl, wherein the alkyl portions are optionally substituted by amino, hydroxy, carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C 1 -C 6 alkyl; or —L—R x ; or —L—S c ; or one or more of R 18 and R 19 is a Q moiety; or R 18 taken in combination with R 19 forms a saturated heterocyclic ring that is a morpholine, a pyrazine, or a piperazine, that is optionally substituted by methyl, sulfonic acid, a salt of sulfonic acid, carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C 1 -C 6 alkyl; or R 18 taken in combination with R 19 forms a saturated 5- or 6-membered heterocycle that is a piperidine, or a pyrrolidine that is optionally fused to a Q moiety, and the heterocycle is optionally further substituted by methyl, carboxylic acid, a salt of carboxylic acid, a carboxylic acid ester of a C 1 -C 6 alkyl, or by —L—R x , or —L—S c ; R 10 is H, CN, a carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C 1 -C 6 alcohol; or R 10 is a saturated or unsaturated C 1 -C 18 alkyl that is optionally substituted one or more times by F, Cl, Br, carboxylic acid, a salt of carboxylic acid, a carboxylic acid ester of a C 1 -C 6 alcohol, —SO 3 X, amino, alkylamino, or dialkylamino, the alkyl groups of which have 1-6 carbons; or R 10 has the formula where R 12 , R 13 , R 14 , R 15 and R 16 are independently H, F, Cl, Br, I, —SO 3 X, a carboxylic acid, a salt of carboxylic acid, CN, hydroxy, amino, hydrazino; or C 1 -C 18 alkyl, C 1 -C 18 alkoxy, C 1 -C 18 alkylthio, C 1 -C 18 alkanoylamino, C 1 -C 18 alkylaminocarbonyl, C 2 -C 36 dialkylaminocarbonyl, C 1 -C 18 alkyloxycarbonyl, or C 6 -C 18 arylcarboxamido, the alkyl or aryl portions of which are optionally substituted one or more times by F, Cl, Br, I, hydroxy, carboxylic acid, a salt of carboxylic acid, a carboxylic acid ester of a C 1 -C 6 alcohol, —SO 3 X, amino, alkylamino, dialkylamino or alkoxy, the alkyl portions of each having 1-6 carbons; or one pair of adjacent substituents R 13 and R 14 , R 14 and R 15 or R 15 and R 16 , when taken in combination, form a fused 6-membered aromatic ring that is optionally further substituted by carboxylic acid, or a salt of carboxylic acid; or one of R 12 , R 13 , R 14 , R 15 and R 16 is —L—R x or —L—S c ; provided that at least one of R 8 , R 9 , R 18 , and R 19 is, or is fused to, a Q moiety; and further provided that at least one of R 8 , R 9 , R 12 , R 13 , R 14 , R 15 , R 16 , R 18 , or R 19 is —L—R x or —L—S c ; or at least one Q moiety is substituted by —L—R x or —L—S c ; wherein L is a covalent linkage; and R x is a reactive functional group that is a maleimide, isocyanate, isothiocyanate, a phosphoramidite, a reactive platinum complex, perfluorobenzamido, azidoperfluorobenzamido, a succinimidyl ester, a sulfosuccinimidyl ester, an alkali or alkaline earth metal salt of a sulfosuccinimidyl ester, a symmetric anhydride, a mixed anhydride of a chloroformate having 2-8 carbons, a mixed anhydride of a carboxylic acid or perfluorinated carboxylic acid having 2-8 carbons, a mixed anhydride of a sulfonic acid or fluorinated sulfonic acid having 1-8 carbons, or an ester of a phenol or a naphthol that is further substituted one or more times by nitro, sulfo, carboxy, alkali or alkaline earth metal salt of sulfo or carboxy, cyano, fluoro, chloro, or trifluoromethyl; or R x is the adduct of a carboxylic acid and a carbodiimide having 2-14 carbons; and S c is a conjugated substance.

Claim 24

A method of detecting a change in separation distance between one or more luminophore donors and quenching compound acceptors in a sample, wherein at least one quenching compound acceptor has the formula wherein R 1 is H; R 6 is H; R 2 , R 3 , R 4 and R 5 are independently H, F, Cl, Br, I, CN; or C 1 -C 18 alkyl, or C 1 -C 18 alkoxy, where each alkyl or alkoxy is optionally further substituted by F, Cl, Br, I, a carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C 1 -C 6 alcohol; or —SO 3 X where X is H or a counterion; or R 1 taken in combination with R 2 , or R 6 taken in combination with R 5 is a fused six-membered aromatic ring; R 8 and R 9 are independently H, C 1 -C 6 alkyl, C 1 -C 6 carboxyalkyl, C 1 -C 6 sulfoalkyl, a salt of C 1 -C 6 carboxyalkyl, or a salt of C 1 -C 6 sulfoalkyl, wherein the alkyl portions are optionally substituted by amino, hydroxy, carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C 1 -C 6 alkyl; or —L—R x ; or —L—S c ; or one or more of R 8 and R 9 is a Q moiety; or R 8 taken in combination with R 9 forms a saturated 5- or 6-membered heterocycle that is optionally fused to a Q moiety and said heterocycle is optionally further substituted by methyl, carboxylic acid, a salt of carboxylic acid, a carboxylic acid ester of a C 1 -C 6 alkyl, or by —L—R x , or —L—S c ; wherein each Q moiety is 1-4 aromatic or heteroaromatic rings that is optionally substituted by halogen, cyano, sulfo, alkali or ammonium salt of sulfo, carboxy, alkali or ammonium salt of carboxy, nitro, alkyl, perfluoroalkyl, alkoxy, alkylthio, amino, monoalkylamino, dialkylamino; alkylamido; or is substituted by —L—R x ; or is substituted by —L—S c ; wherein each heteroaromatic ring in Q is a 5- or 6-membered aromatic heterocycle having 1 to 3 heteroatoms selected from the group consisting of O, N or S in any combination; and when Q is 2-4 rings, said 2-4 rings are fused to each other; and K is O or N + R 18 R 19 ; wherein R 18 and R 19 are independently H, C 1 -C 6 alkyl, C 1 -C 6 carboxyalkyl, C 1 -C 6 sulfoalkyl, a salt of C 1 -C 6 carboxyalkyl, or a salt of C 1 -C 6 sulfoalkyl, wherein the alkyl portions are optionally substituted by amino, hydroxy, carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C 1 -C 6 alkyl; or —L—R x ; or —L—S c ; or one or more of R 18 and R 19 is a Q moiety; or R 18 taken in combination with R 19 forms a saturated heterocyclic ring that is a morpholine, a pyrazine, or a piperazine, that is optionally substituted by methyl, sulfonic acid, a salt of sulfonic acid, carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C 1 -C 6 alkyl; or R 18 taken in combination with R 19 forms a saturated 5- or 6-membered heterocycle that is a piperidine, or a pyrrolidine that is optionally fused to a Q moiety, and the heterocycle is optionally further substituted by methyl, carboxylic acid, a salt of carboxylic acid, a carboxylic acid ester of a C 1 -C 6 alkyl, or by —L—R x , or —L—S c ; R 10 is H, CN, a carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C 1 -C 6 alcohol; or R 10 is a saturated or unsaturated C 1 -C 18 alkyl that is optionally substituted one or more times by F, Cl, Br, carboxylic acid, a salt of carboxylic acid, a carboxylic acid ester of a C 1 -C 6 alcohol, —SO 3 X, amino, alkylamino, or dialkylamino, the alkyl groups of which have 1-6 carbons; or R 10 has the formula where R 12 , R 13 , R 14 , R 15 and R 16 are independently H, F, Cl, Br, I, —SO 3 X, a carboxylic acid, a salt of carboxylic acid, CN, hydroxy, amino, hydrazino; or C 1 -C 18 alkyl, C 1 -C 18 alkoxy, C 1 -C 18 alkylthio, C 1 -C 18 alkanoylamino, C 1 -C 18 alkylaminocarbonyl, C 2 -C 36 dialkylaminocarbonyl, C 1 -C 18 alkyloxycarbonyl, or C 6 -C 18 arylcarboxamido, the alkyl or aryl portions of which are optionally substituted one or more times by F, Cl, Br, I, hydroxy, carboxylic acid, a salt of carboxylic acid, a carboxylic acid ester of a C 1 -C 6 alcohol, —SO 3 X, amino, alkylamino, dialkylamino or alkoxy, the alkyl portions of each having 1-6 carbons; or one pair of adjacent substituents R 13 and R 14 , R 14 and R 15 or R 15 and R 16 , when taken in combination, form a fused 6-membered aromatic ring that is optionally further substituted by carboxylic acid, or a salt of carboxylic acid, or one of R 12 , R 13 , R 14 , R 15 and R 16 is —L—R x or —L—S c ; provided that at least one of R 8 , R 9 , R 18 , and R 19 is, or is fused to, a Q moiety; and further provided that at least one of R 8 , R 9 , R 12 , R 13 , R 14 , R 15 , R 16 , R 18 , or R 19 is —L—R x or —L—S c ; or at least one Q moiety is substituted by —L—R x or —L—S c ; wherein L is a covalent linkage; and R x is a reactive functional group that is a maleimide, isocyanate, isothiocyanate, a phosphoramidite, a reactive platinum complex, perfluorobenzamido, azidoperfluorobenzamido, a succinimidyl ester, a sulfosuccinimidyl ester, an alkali or alkaline earth metal salt of a sulfosuccinimidyl ester, a symmetric anhydride, a mixed anhydride of a chloroformate having 2-8 carbons, a mixed anhydride of a carboxylic acid or perfluorinated carboxylic acid having 2-8 carbons, a mixed anhydride of a sulfonic acid or fluorinated sulfonic acid having 1-8 carbons, or an ester of a phenol or a naphthol that is further substituted one or more times by nitro, sulfo, carboxy, alkali or alkaline earth metal salt of sulfo or carboxy, cyano, fluoro, chloro, or trifluoromethyl; or R x is the adduct of a carboxylic acid and a carbodiimide having 2-14 carbons; and S c is a conjugated substance; said method comprising the steps of: a) illuminating said sample; b) detecting a first luminescence response of said sample; c) exposing said sample to an environmental condition expected to change said separation distance; d) illuminating said sample; e) detecting a second luminescence response of said sample; and f) comparing said first and second luminescence response to determine a detectable difference in luminescence, where said detectable difference in luminescence correlates with said change in separation distance.

Claim 25

A method of detecting a change in separation distance between one or more luminophore donors and quenching compound acceptors in a sample, wherein at least one quenching compound acceptor has the formula wherein R 1 is H; R 6 is H; R 2 , R 3 , R 4 and R 5 are independently H, F, Cl, Br, I, CN; or C 1 -C 18 alkyl, or C 1 -C 18 alkoxy, where each alkyl or alkoxy is optionally further substituted by F, Cl, Br, I, a carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C 1 -C 6 alcohol; or —SO 3 X where X is H or a counterion; or R 1 taken in combination with R 2 , or R 6 taken in combination with R 5 is a fused six-membered aromatic ring; R 8 and R 9 are independently H, C 1 -C 6 alkyl, C 1 -C 6 carboxyalkyl, C 1 -C 6 sulfoalkyl, a salt of C 1 -C 6 carboxyalkyl, or a salt of C 1 -C 6 sulfoalkyl, wherein the alkyl portions are optionally substituted by amino, hydroxy, carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C 1 -C 6 alkyl; or —L—R x ; or —L—S c ; or one or more of R 8 and R 9 is a Q moiety; or R 8 taken in combination with R 9 forms a saturated 5- or 6-membered heterocycle that is optionally fused to a Q moiety and said heterocycle is optionally further substituted by methyl, carboxylic acid, a salt of carboxylic acid, a carboxylic acid ester of a C 1 -C 6 alkyl, or by —L—R x , or —L—S c ; wherein each Q moiety is 1-4 aromatic or heteroaromatic rings that is optionally substituted by halogen, cyano, sulfo, alkali or ammonium salt of sulfo, carboxy, alkali or ammonium salt of carboxy, nitro, alkyl, perfluoroalkyl, alkoxy, alkylthio, amino, monoalkylamino, dialkylamino; alkylamido; or is substituted by —L—R x ; or is substituted by —L—S c ; wherein each heteroaromatic ring in Q is a 5- or 6-membered aromatic heterocycle having 1 to 3 heteroatoms selected from the group consisting of O, N or S in any combination; and when Q is 2-4 rings, said 2-4 rings are fused to each other; and K is O or N + R 18 R 19 ; wherein R 18 and R 19 are independently H, C 1 -C 6 alkyl, C 1 -C 6 carboxyalkyl, C 1 -C 6 sulfoalkyl, a salt of C 1 -C 6 carboxyalkyl, or a salt of C 1 -C 6 sulfoalkyl, wherein the alkyl portions are optionally substituted by amino, hydroxy, carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C 1 -C 6 alkyl; or —L—R x ; or —L—S c ; or one or more of R 18 and R 19 is a Q moiety; or R 18 taken in combination with R 19 forms a saturated heterocyclic ring that is a morpholine, a pyrazine, or a piperazine, that is optionally substituted by methyl, sulfonic acid, a salt of sulfonic acid, carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C 1 -C 6 alkyl; or R 18 taken in combination with R 19 forms a saturated 5- or 6-membered heterocycle that is a piperidine, or a pyrrolidine that is optionally fused to a Q moiety, and the heterocycle is optionally further substituted by methyl, carboxylic acid, a salt of carboxylic acid, a carboxylic acid ester of a C 1 -C 6 alkyl, or by —L—R x , or —L—S c ; R 10 is H, CN, a carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C 1 -C 6 alcohol; or R 10 is a saturated or unsaturated C 1 -C 18 alkyl that is optionally substituted one or more times by F, Cl, Br, carboxylic acid, a salt of carboxylic acid, a carboxylic acid ester of a C 1 -C 6 alcohol, —SO 3 X, amino, alkylamino, or dialkylamino, the alkyl groups of which have 1-6 carbons; or R 10 has the formula where R 12 , R 13 , R 14 , R 15 and R 16 are independently H, F, Cl, Br, I, —SO 3 X, a carboxylic acid, a salt of carboxylic acid, CN, hydroxy, amino, hydrazino; or C 1 -C 18 alkyl, C 1 -C 18 alkoxy, C 1 -C 18 alkylthio, C 1 -C 18 alkanoylamino, C 1 -C 18 alkylaminocarbonyl, C 2 -C 36 dialkylaminocarbonyl, C 1 -C 18 alkyloxycarbonyl, or C 6 -C 18 arylcarboxamido, the alkyl or aryl portions of which are optionally substituted one or more times by F, Cl, Br, I, hydroxy, carboxylic acid, a salt of carboxylic acid, a carboxylic acid ester of a C 1 -C 6 alcohol, —SO 3 X, amino, alkylamino, dialkylamino or alkoxy, the alkyl portions of each having 1-6 carbons; or one pair of adjacent substituents R 13 and R 14 , R 14 and R 15 or R 15 and R 16 , when taken in combination, form a fused 6-membered aromatic ring that is optionally further substituted by carboxylic acid, or a salt of carboxylic acid, or one of R 12 , R 13 , R 14 , R 15 and R 16 is —L—R x or —L—S c ; provided that at least one of R 8 , R 9 , R 18 , and R 19 is, or is fused to, a Q moiety; and further provided that at least one of R 8 , R 9 , R 12 , R 13 , R 14 , R 15 , R 16 , R 18 , or R 19 is —L—R x or —L—S c ; or at least one Q moiety is substituted by —L—R x or —L—S c ; wherein L is a covalent linkage; and R x is a reactive functional group that is a maleimide, isocyanate, isothiocyanate, a phosphoramidite, a reactive platinum complex, perfluorobenzamido, azidoperfluorobenzamido, a succinimidyl ester, a sulfosuccinimidyl ester, an alkali or alkaline earth metal salt of a sulfosuccinimidyl ester, a symmetric anhydride, a mixed anhydride of a chloroformate having 2-8 carbons, a mixed anhydride of a carboxylic acid or perfluorinated carboxylic acid having 2-8 carbons, a mixed anhydride of a sulfonic acid or fluorinated sulfonic acid having 1-8 carbons, or an ester of a phenol or a naphthol that is further substituted one or more times by nitro, sulfo, carboxy, alkali or alkaline earth metal salt of sulfo or carboxy, cyano, fluoro, chloro, or trifluoromethyl; or R x is the adduct of a carboxylic acid and a carbodiimide having 2-14 carbons; and S c is a conjugated substance; said method comprising the steps of: a) illuminating said sample; b) detecting a first luminescence response of said sample; c) exposing said sample to an environmental condition expected to change said separation distance; d) illuminating said sample; e) detecting a second luminescence response of said sample; and f) comparing said first and second luminescence response to determine a detectable difference in luminescence, where said detectable difference in luminescence correlates with said change in separation distance.