Xanthene dyes and their application as luminescence quenching compounds

• US 6,399,392 B1
• Filed: 04/21/2000
• Issued: 06/04/2002
• Est. Priority Date: 04/23/1999
• Status: Expired due to Term

• Current Assignee: Molecular Probes Incorporated (Thermo Fisher Scientific Incorporated)
• Original Assignee: Molecular Probes Incorporated (Thermo Fisher Scientific Incorporated)
• Inventors: Singer, Victoria L., Yue, Stephen T., Haugland, Richard P.
• Primary Examiner(s): CEPERLEY, MARY
• Application Number:
  US09/556,464
• Time in Patent Office: 774 Days
• Field of Search: 436/546, 436/172, 436/800, 436/805, 530/391.3, 530/402, 435/6, 435/7.5, 435/188, 549/227, 549/394, 548/525, 548/156, 548/184, 548/455, 544/244, 546/173, 546/196, 546/199, 546/201, 546/208, 546/256
• US Class Current: 436/172
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C09B 11/24 Phthaleins containing amino groups ; Phthalanes; Fluoranes; Phthalides; Rhodamine dyes; Phthaleins having heterocyclic aryl rings; Lactone or lactame forms of triarylmethane dyes
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching
• CPC Class Codes: G01N 33/582 with fluorescent label
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine
• CPC Class Codes: Y10S 436/805 Optical property

Abstract

The quenching compounds of the invention are nitrogen-substituted xanthenes that are substituted by one or more aromatic or heteroaromatic quenching moieties. The quenching compounds of the invention exhibit little or no observable fluorescence and efficiently quench a broad spectrum of luminescent compounds. The chemically reactive quenching compounds possess utility for labeling a wide variety of substances, including biomolecules. These labeled substances are highly useful for a variety of energy-transfer assays and applications.

Claims

1. A compound, having the formula

wherein

R¹ is H;

R⁶is H;

R², R³, R⁴ and R⁵ are independently H, F, Cl, Br, I, CN;

or C₁-C₁₈ alkyl, or C₁-C₁₈ 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 C₁-C₆ alcohol;

or —

SO₃X

where X is H or a counterion;

or R¹ taken in combination with R², or R⁶ taken in combination with R⁵ is a fused six-membered aromatic ring;

R⁸ and R⁹ are independently H, C₁-C₆ alkyl, C₁-C₆ carboxyalkyl, C₁-C₆ sulfoalkyl, a salt of C₁-C₆ carboxyalkyl, or a salt of C₁-C₆ 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 C₁-C₆ alkyl;

or —

L—

R_x;

or —

L—

S_c;

or one or more of R⁸ and R⁹ is a Q moiety;

or R⁸ taken in combination with R⁹ 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 C₁-C₆ 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⁺R¹⁸R¹⁹;

wherein R¹⁸ and R¹⁹ are independently H, C₁-C₆ alkyl, C₁-C₆ carboxyalkyl, C₁-C₆ sulfoalkyl, a salt of C₁-C₆ carboxyalkyl, or a salt of C₁-C₆ 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 C₁-C₆ alkyl;

or —

L—

R_x;

or —

L—

S_c;

or one or more of R¹⁸ and R¹⁹ is a Q moiety;

or R¹⁸ taken in combination with R¹⁹ 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 C₁-C₆ alkyl;

or R¹⁸ taken in combination with R¹⁹ 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 C₁-C₆ alkyl, or by —

L—

R_x, or —

L—

S_c;

R¹⁰ is H, CN, a carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C₁-C₆ alcohol;

or R¹⁰ is a saturated or unsaturated C₁-C₁₈ 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 C₁-C₆ alcohol, —

SO₃X, amino, alkylamino, or dialkylamino, the alkyl groups of which have 1-6 carbons;

or R¹⁰ has the formula

 

where R¹², R¹³, R¹⁴, R¹⁵ and R¹⁶ are independently H, F, Cl, Br, I, —

SO₃X, a carboxylic acid, a salt of carboxylic acid, CN, hydroxy, amino, hydrazino;

or C₁-C₁₈ alkyl, C₁-C₁₈ alkoxy, C₁-C₁₈ alkylthio, C₁-C₁₈ alkanoylamino, C₁-C₁₈ alkylaminocarbonyl, C₂-C₃₆ dialkylaminocarbonyl, C₁-C₁₈ alkyloxycarbonyl, or C₆-C₁₈ 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 C₁-C₆ alcohol, —

SO₃X, amino, alkylamino, dialkylamino or alkoxy, the alkyl portions of each having 1-6 carbons;

or one pair of adjacent substituents R¹³ and R¹⁴, R¹⁴ and R¹⁵ or R¹⁵ and R¹⁶, 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 R¹², R¹³, R¹⁴, R¹⁵ and R¹⁶ is —

L—

R_x or —

L—

S_c;

provided that at least one of R⁸, R⁹, R¹⁸, and R¹⁹ is, or is fused to, a Q moiety; and

further provided that at least one of R⁸, R⁹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁸, or R¹⁹ 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.

2. A compound, as claimed in claim 1, wherein the conjugated substance is an antigen, a steroid, a vitamin, a drug, a hapten, a metabolite, a toxin, an environmental pollutant, an amino acid, a peptide, a protein, a nucleotide, an oligonucleotide, a nucleic acid polymer, a carbohydrate, a lipid, an ion-complexing moiety, or a non-biological polymer.

3. A compound as claimed in claim 1, wherein the conjugated substance is an amino acid, a peptide, a protein, a nucleotide, an oligonucleotide, a nucleic acid polymer, a carbohydrate, a lipid, or a drug.

4. A compound, as claimed in claim 1, wherein the conjugated substance is a hapten or a member of a specific binding pair.

5. A compound as claimed in claim 1, wherein the conjugated substance is further conjugated to one or more luminophores, where each luminophore may be the same or different.

6. A compound, as claimed in claim 5, wherein at least one of the luminophores is a fluorophore.

7. A compound, as claimed in claim 6, wherein the fluorophore is a pyrene, an anthracene, a naphthalene, an acridine, a stilbene, an indole, a benzindole, an oxazole, a benzoxazole, a thiazole, a benzothiazole, a 4-amino-7-nitrobenz-2-oxa-1,3-diazole, a cyanine, a carbocyanine, a carbostyryl, a porphyrin, a salicylate, an anthranilate, an azulene, a perylene, a pyridine, a quinoline, a coumarin, a polyazaindacene, a xanthene, an oxazine, a benzoxazine, a carbazine, a phenalenone, or a benzphenalenone.

8. A compound, as claimed in claim 6, wherein the fluorophore is a carbazine, an oxazine, a coumarin, a pyrene, a xanthene, a naphthalene, a phenalenone, or a 4-bora-3a,4a-diaza-s-indacene.

9. A compound, as claimed in claim 6, wherein the fluorophore is a fluorescein, a rhodamine, or a rhodol.

10. A compound as claimed in claim 1, wherein each Q moiety is a substituted or unsubstituted phenyl, naphthyl, anthracenyl, benzothiazole, benzoxazole, or benzimidazole.

11. A compound, as claimed in claim 1, wherein each Q moiety is a phenyl or substituted phenyl.

12. A compound, as claimed in claim 1 wherein K is N+R¹⁸R¹⁹.

13. A compound, as claimed in claim 1, wherein R⁸ taken in combination with R⁹ forms a saturated 5- or 6-membered heterocycle that is fused to a Q moiety, and R¹⁸ taken in combination with R¹⁹ forms a saturated 5- or 6-membered heterocycle that is fused to a Q moiety.

14. A compound, as claimed in claim 1, wherein R⁸═

R¹⁹ and R⁹═

R¹⁸.

15. A compound, as claimed in claim 1, wherein R¹, R², R⁵, and R⁶ are each H.

16. A compound, as claimed in claim 1, wherein R¹⁰ has the formula

17. A compound, as claimed in claim 16, wherein one of R¹²-R¹⁶ is —

L—

R_x or —

L—

S_c.

18. A compound, as claimed in claim 16, wherein R¹² is —

L—

R_x;

wherein L is selected such that —

L—

R_x has the formula

19. A compound, as claimed in claim 1, wherein R_x is a phosphoramidite, a reactive platinum complex, a succinimidyl ester of a carboxylic acid, a haloacetamide, a hydrazine, an isothiocyanate, a maleimide group or an azidoperfluorobenzamido group.

20. A compound, as claimed in claim 1, wherein R_x is a phosphoramidite, a reactive platinum complex, or a succinimidyl ester of a carboxylic acid.

21. A compound, as claimed in claim 1, having the formula

wherein

R⁸═

R¹⁹ and R⁹═

R¹⁸;

R¹² is —

L—

R_x, or —

L—

S_c; and

each Q moiety is a substituted or unsubstituted phenyl, naphthyl, anthracenyl, berzothiazole, benzoxazole, or benzimidazole.

22. A compound, as claimed in claim 1, having a fluorescence quantum yield of less than about 0.05.

23. 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

R¹ is H;

R⁶is H;

R², R³, R⁴ and R⁵ are independently H, F, Cl, Br, I, CN;

or C₁-C₁₈ alkyl, or C₁-C₁₈ 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 C₁-C₆ alcohol;

or —

SO₃X where X is H or a counterion;

or R¹ taken in combination with R², or R⁶ taken in combination with R⁵ is a fused six-membered aromatic ring;

R⁸ and R⁹ are independently H, C₁-C₆ alkyl, C₁-C₆ carboxyalkyl, C₁-C₆ sulfoalkyl, a salt of C₁-C₆ carboxyalkyl, or a salt of C₁-C₆ 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 C₁-C₆ alkyl;

or —

L—

R_x;

or —

L—

S_c;

or one or more of R⁸ and R⁹ is a Q moiety;

or R⁸ taken in combination with R⁹ 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 C₁-C₆ 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⁺R¹⁸R¹⁹;

wherein R¹⁸ and R¹⁹ are independently H, C₁-C₆ alkyl, C₁-C₆ carboxyalkyl, C₁-C₆ sulfoalkyl, a salt of C₁-C₆ carboxyalkyl, or a salt of C₁-C₆ 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 C₁-C₆ alkyl;

or —

L—

R_x;

or —

L—

S_c;

or one or more of R¹⁸ and R¹⁹ is a Q moiety;

or R¹⁸ taken in combination with R¹⁹ 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 C₁-C₆ alkyl;

or R¹⁸ taken in combination with R¹⁹ 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 C₁-C₆ alkyl, or by —

L—

R_x, or —

L—

S_c;

R¹⁰ is H, CN, a carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C₁-C₆ alcohol;

or R¹⁰ is a saturated or unsaturated C₁-C₁₈ 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 C₁-C₆ alcohol, —

SO₃X, amino, alkylamino, or dialkylamino, the alkyl groups of which have 1-6 carbons;

or R¹⁰ has the formula

 

where R¹², R¹³, R¹⁴, R¹⁵ and R¹⁶ are independently H, F, Cl, Br, I, —

SO₃X, a carboxylic acid, a salt of carboxylic acid, CN, hydroxy, amino, hydrazino;

or C₁-C₁₈ alkyl, C₁-C₁₈ alkoxy, C₁-C₁₈ alkylthio, C₁-C₁₈ alkanoylamino, C₁-C₁₈ alkylaminocarbonyl, C₂-C₃₆ dialkylaminocarbonyl, C₁-C₁₈ alkyloxycarbonyl, or C₆-C₁₈ 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 C₁-C₆ alcohol, —

SO₃X, amino, alkylamino, dialkylamino or alkoxy, the alkyl portions of each having 1-6 carbons;

or one pair of adjacent substituents R¹³ and R¹⁴, R¹⁴ and R¹⁵ or R¹⁵ and R¹⁶, 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 R¹²,

R¹³, R¹⁴, R¹⁵ and R¹⁶ is —

L—

R_x or —

L—

S_c;

provided that at least one of R⁸, R⁹, R₁₈, and R¹⁹ is, or is fused to, a Q moiety; and

further provided that at least one of R⁸, R⁹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁸, or R¹⁹ 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.

24. A method, as claimed in claim 23, wherein said detectable difference is an increase in luminescence.

25. A method, as claimed in claim 23, wherein said detectable difference is a decrease in luminescence.

26. A method, as claimed in claim 23, wherein said luminescence is fluorescence.

27. A method, as claimed in claim 23, wherein said quenching compound acceptor is initially covalently bound to a conjugated substance.

28. A method, as claimed in claim 27, wherein said luminophore donor is initially covalently conjugated to the same conjugated substance.

29. A method, as claimed in claim 28, wherein the conjugated substance is cleaved by said environmental condition, resulting in an increase in luminescence.

30. A method, as claimed in claim 28, wherein the conjugated substance changes conformation in response to said environmental condition, resulting in said detectable difference in luminescence.

31. A method, as claimed in claim 27, wherein the conjugated substance is an oligonucleotide, nucleic acid polymer, peptide or protein.

32. A method, as claimed in claim 27, wherein the conjugated substance is a member of a specific binding pair.

33. A kit for labeling a substance, comprising:

a) a quenching compound of the formula

 

wherein

R¹ is H;

R⁶ is H;

R², R³, R⁴ and R⁵ are independently H, F, Cl, Br, I, CN;

or C₁-C₁₈ alkyl, or C₁-C₁₈ 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 C₁-C₆ alcohol;

or —

SO₃X where X is H or a counterion;

or R² taken in combination with R², or R⁶ taken in combination with R⁵ is a fused six-membered aromatic ring;

R⁸ and R⁹ are independently H, C₁-C₆ alkyl, C₁-C₆ carboxyalkyl, C₁-C₆ sulfoalkyl, a salt of C₁-C₆ carboxyalkyl, or a salt of C₁-C₆ 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 C₁-C₆ alkyl;

or —

L—

R_x;

or one or more of R⁸ and R⁹ is a Q moiety;

or R⁸ taken in combination with R⁹ 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 C₁-C₆ alkyl, or by —

L—

R_x,

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;

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⁺R¹⁸R¹⁹;

wherein R¹⁸ and R¹⁹ are independently H, C₁-C₆ alkyl, C₁-C₆ carboxyalkyl, C₁-C₆ sulfoalkyl, a salt of C₁-C₆ carboxyalkyl, or a salt of C₁-C₆ 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 C₁-C₆ alkyl;

or —

L—

R_x;

or one or more of R¹⁸ and R¹⁹ is a Q moiety;

or R¹⁸ taken in combination with R¹⁹ 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 C₁-C₆ alkyl;

or R¹⁸ taken in combination with R¹⁹ 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 C₁-C₆ alkyl, or by —

L—

R_x;

R¹⁰ is H, CN, a carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C₁-C₆ alcohol;

or R¹⁰ is a saturated or unsaturated C₁-C₁₈ 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 C₁-C₆ alcohol, —

SO₃X, amino, alkylamino, or dialkylamino, the alkyl groups of which have 1-6 carbons;

or R¹⁰ has the formula

 

where R¹², R¹³, R¹⁴, R¹⁵ and R¹⁶ are independently H, F, Cl, Br, I, —

SO₃X, a carboxylic acid, a salt of carboxylic acid, CN, hydroxy, amino, hydrazino;

or C₁-C₁₈ alkyl, C₁-C₁₈ alkoxy, C₁-C₁₈ alkylthio, C₁-C₁₈ alkanoylamino, C₁-C₁₈ alkylaminocarbonyl, C₂-C₃₆ dialkylaminocarbonyl, C₁-C₁₈ alkyloxycarbonyl, or C₆-C₁₈ 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 C₁-C₆ alcohol, —

SO₃X, amino, alkylamino, dialkylamino or alkoxy, the alkyl portions of each having 1-6 carbons;

or one pair of adjacent substituents R¹³ and R¹⁴, R¹⁴ and R¹⁵ or R¹⁵ and R¹⁶, 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 R¹², R¹³, R¹⁴, R¹⁵ and R¹⁶ is —

L—

R_x;

provided that at least one of R⁸, R⁹, R¹⁸, and R¹⁹ is, or is fused to, a Q moiety; and

further provided that at least one of R⁸, R⁹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁸, or R¹⁹ is —

L—

R_x;

or at least one Q moiety is substituted by —

L—

R_x;

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

b) instructions for conjugating said quenching compound to said substance; and

c) one or more of the following;

a buffering agent, a purification medium, a sample of said substance, an organic solvent, an enzyme, or an enzyme inhibitor.

34. A kit, as claimed in claim 33, wherein R_x is a phosphoramidite, a reactive platinum complex, or a succinimidyl ester of a carboxylic acid.

35. A kit, as claimed in claim 33, wherein said substance is an oligonucleotide, nucleic acid polymer, peptide or protein.

36. A kit, as claimed in claim 33, wherein said substance is a member of a specific binding pair.

37. A kit, as claimed in claim 33, wherein said quenching compound has the formula

wherein

R¹² is — L—

R_x; and

R_x is a phosphoramidite, a reactive platinum complex, a succinimidyl ester of a carboxylic acid, a haloacetamide, a hydrazine, an isothiocyanate, a maleimide group or an azidoperfluorobenzamido group.

44. A kit, as claimed in claim 33, wherein said quenching compound has the formula

wherein

R¹² is —

L—

S_c; and

S_c is an oligonucleotide, nucleic acid polymer, peptide or protein.

38. A kit, comprising:

a) a quenching compound of the formula

 

wherein

R¹ is H;

R⁶ is H;

R², R³, R⁴ and R⁵ are independently H, F, Cl, Br, I, CN;

or C₁-C₁₈ alkyl, or C₁-C₁₈ 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 C₁-C₆ alcohol;

or —

SO₃X

where X is H or a counterion;

or R¹ taken in combination with R², or R⁶ taken in combination with R⁵ is a fused six-membered aromatic ring;

R⁸ and R⁹ are independently H, C₁-C₆ alkyl, C₁-C₆ carboxyalkyl, C₁-C₆ sulfoalkyl, a salt of C₁-C₆ carboxyalkyl, or a salt of C₁-C₆ 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 C₁-C₆ alkyl;

or —

L—

S_c;

or one or more of R⁸ and R⁹ is a Q moiety;

or R⁸ taken in combination with R⁹ 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 C₁-C₆ alkyl, or by —

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—

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⁺R¹⁸R¹⁹;

wherein R¹⁸ and R¹⁹ are independently H, C₁-C₆ alkyl, C₁-C₆ carboxyalkyl, C₁-C₆ sulfoalkyl, a salt of C₁-C₆ carboxyalkyl, or a salt of C₁-C₆ 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 C₁-C₆ alkyl;

or —

L—

S_c;

or one or more of R¹⁸ and R¹⁹ is a Q moiety;

or R¹⁸ taken in combination with R¹⁹ 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 C₁-C₆ alkyl;

or R¹⁸ taken in combination with R¹⁹ 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 C₁-C₆ alkyl, or by —

L—

S_c;

R¹⁰ is H, CN, a carboxylic acid, a salt of carboxylic acid, or a carboxylic acid ester of a C₁-C₆ alcohol;

or R¹⁰ is a saturated or unsaturated C₁-C₁₈ 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 C₁-C₆ alcohol, —

SO₃X, amino, alkylamino, or dialkylamino, the alkyl groups of which have 1-6 carbons;

or R¹⁰ has the formula

 

where R¹², R¹³, R¹⁴, R¹⁵ and R¹⁶ are independently H, F, Cl, Br, I, —

SO₃X, a carboxylic acid, a salt of carboxylic acid, CN, hydroxy, amino, hydrazino;

or C₁-C₁₈ alkyl, C₁-C₁₈ alkoxy, C₁-C₁₈ alkylthio, C₁-C₁₈ alkanoylamino, C₁-C₁₈ alkylaminocarbonyl, C₂-C₃₆ dialkylaminocarbonyl, C₁-C₁₈ alkyloxycarbonyl, or C₆-C₁₈ 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 C₁-C₆ alcohol, —

SO₃X, amino, alkylamino, dialkylamino or alkoxy, the alkyl portions of each having 1-6 carbons;

or one pair of adjacent substituents R¹³ and R¹⁴, R¹⁴ and R¹⁵ or R¹⁵ and R¹⁶, 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 R¹², R¹³, R¹⁴, R¹⁵ and R¹⁶ is —

L—

S_c;

provided that at least one of R⁸, R⁹, R¹⁸, and R¹⁹ is, or is fused to, a Q moiety; and

further provided that at least one of R⁸, R⁹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁸, or R¹⁹ is —

L—

S_c;

or at least one Q moiety is substituted by —

L—

S_c;

wherein

L is a covalent linkage; and

S_c is a conjugated substance; and

b) a luminophore donor.

39. A kit, as claimed in claim 38, further comprising an additional detection reagent.

40. A kit, as claimed in claim 38, further comprising one or more luminescence standards.

41. A kit, as claimed in claim 38, wherein said quenching compound and said luminophore are covalently bound to the same conjugated substance, S_c.

42. A kit, as claimed in claim 41, wherein said conjugated substance is an oligonucleotide, nucleic acid polymer, peptide or protein.

43. A kit, as claimed in claim 38, wherein S_c is a member of a specific binding pair, and said luminophore donor is covalently bound to the complementary member of said specific binding pair.