Aza-Benzazolium Containing Cyanine Dyes

• US 20080044811A1
• Filed: 06/01/2007
• Published: 02/21/2008
• Est. Priority Date: 04/30/1999
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Invitrogen Corp. (Thermo Fisher Scientific Incorporated)
• Inventors: Haugland, Richard, Yue, Stephen
• Granted Patent: US 7,871,773 B2
• Time in Patent Office: Days
• US Class Current: 435/5
• CPC Class Codes: C09B 23/02 the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups
• CPC Class Codes: C09B 23/04 one >CH- group, e.g. cyanines, isocyanines, pseudocyanines
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2563/173 staining/intercalating agent, e.g. ethidium bromide
• CPC Class Codes: G01N 1/30 Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
• CPC Class Codes: G01N 2001/302 Stain compositions
• CPC Class Codes: G01N 27/44726 using specific dyes, markers or binding molecules
• CPC Class Codes: G01N 33/52 Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements immunological elements G01N33/54386; involving labelled immunochemicals G01N33/58; for haemoglobin or occult blood G01N33/72
• CPC Class Codes: G01N 33/5308 for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
• CPC Class Codes: G01N 33/531 Production of immunochemical test materials
• CPC Class Codes: G01N 33/582 with fluorescent label
• CPC Class Codes: G01N 33/68 involving proteins, peptides or amino acids involving lipoproteins G01N33/92
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

Unsymmetrical cyanine dyes that incorporate an aza-benzazolium ring moiety are described, including cyanine dyes substituted by a cationic side chain, monomeric and dimeric cyanine dyes, chemically reactive cyanine dyes, and conjugates of cyanine dyes. The subject dyes are virtually non-fluorescent when diluted in aqueous solution, but exhibit bright fluorescence when associated with nucleic acid polymers such as DNA or RNA, or when associated with detergent-complexed proteins. A variety of applications are described for detection and quantitation of nucleic acids and detergent-complexed proteins in a variety of samples, including solutions, electrophoretic gels, cells, and microorganisms.

Claims

1. A method for determining the presence of an analyte of interest in a sample, wherein said method comprises:

a) combining said sample with a staining solution, wherein said staining solution comprises one or more dyes having formula

wherein A represents the atoms necessary to form one to two fused aromatic rings having 6 atoms in each ring, at least one of which is a nitrogen atom, said ring or rings being optionally further substituted one or more times by C₁-C₆ alkyl, C₁-C₆ alkoxy trifluoromethyl, halogen, BRIDGE, -L-Rx or -L-Sc;

wherein Rx is a reactive group;

Sc is a conjugated substance; and

L and BRIDGE are independently a single covalent bond, or a covalent linkage;

X is O, S, Se, NR¹⁵, or CR¹⁶R¹⁷, where R¹⁵ is H or an alkyl group having 1-6 carbons; and

R¹⁶ and R¹⁷, which may be the same or different, are independently alkyl groups having 1-6 carbons, or R¹⁶ and R¹⁷ taken in combination complete a five or six membered saturated ring;

R² is selected from the group consisting of -L-Rx, -L-Sc, TAIL, BRIDGE and an alkyl group having 1-6 carbons that is optionally substituted by sulfo, carboxy, amino, substituted amino or substituted ammonium, wherein α

is 0 or 1; and

TAIL is a heteroatom-containing moiety;

Y is —

CR³═

CR⁴—

wherein p and m=0 or 1, such that p+m=1;

R³, R⁴, R⁶, and R⁷ are independently selected from the group consisting of hydrogen, a C₁-C₆ alkyl, a halogen, a CYCLIC SUBSTITUENT, —

OR⁸, —

SR⁸, —

(NR⁸R⁹), TAIL;

BRIDGE, -L-Rx and -L-Sc;

where R⁸ and R⁹ are independently a C₁-C₆ alkyl group, 1-2 alicyclic or aromatic rings;

or R⁸ and R⁹ taken in combination are —

(CH₂)₂-V-(CH₂)₂—

where V is a single bond, —

O—

, —

CH₂—

, or —

NR¹⁰—

, where R¹⁰ is H or an alkyl having 1-6 carbons;

wherein CYCLIC SUBSTITUENT is a substituted or unsubstituted aryl, heteroaryl or C₃-C₁₀ cycloalkyl;

or R⁶ and R⁷ form a fused aromatic ring —

R¹¹═

R¹²—

R¹³═

R¹⁴—

wherein R¹¹, R¹², R¹³, and R¹⁴ are independently selected from the group consisting of hydrogen, C₁-C₆ alkyl group, —

OR⁸, —

SR⁸, —

(NR⁸R⁹), a CYCLIC SUBSTITUENT, TAIL, BRIDGE, -L-Rx and -L-Sc;

R⁵ is independently selected from the group consisting of a C₁-C6 alkyl group, a CYCLIC SUBSTITUENT, TAIL, BRIDGE, -L-Rx and -L-Sc;

or R⁵ is absent;

R³⁰, R³¹, and R³² are independently selected from the group consisting of hydrogen, C₁-C₆ alkyl, C₃-C₁₀ cycloalkyl, aryl, and heteroaryl, wherein n=0, 1 or 2;

wherein, BRIDGE, when present, is bound to a compound having formula I or another unsymmetrical cyanine dye

b) incubating said sample and said staining solution for a sufficient amount of time to form a dye-analyte complex;

c) illiminating said complex with an appropriate wavelength whereby the presence of said analyte is determined.

2. The method according to claim 1, wherein said analyte of interest is a poly(amino acid) or a nucleic acid.

3. The method according to claim 2, wherein said poly(amino acid) is in solution or immobilized on a solid or semi solid matrix.

4. The method according to claim 3, wherein said matrix is a polymeric gel, an array, a polymeric microparticle or a membrane.

5. The method according to claim 4, wherein said gel is a polyacrylamide gel.

6. The method according to claim 3, wherein said method further comprises heating said sample prior to combining with said staining solution, or heating combined sample and staining solution mixture.

7. The method according to claim 3, wherein said method further comprises removing, destroying, or dispersing below the critical micelle concentration any biological membranes that are present in said sample.

8. The method according to claim 3, wherein said method further comprises adding an anionic detergent to the sample, staining solution or combined sample and staining solution mixture.

9. The method according to claim 2, wherein said nucleic acid is in solution, immobilized on a solid or semi-solid matrix or present in a biological structure.

10. The method according to claim 9, wherein said matrix is a polymeric gel, an array, a glass slide, a polymeric microparticle or a membrane.

11. The method according to claim 10, wherein said gel is an agarose gel.

12. The method according to claim 9, wherein said biological structure is a biological cell, a virus particle or a tissue section.

13. A method for the detection of immobilized poly(amino acids), wherein method comprises:

a) immobilizing said poly(amino acids) on a solid or semi-solid matrix;

b) combining said said sample with a staining solution prior, during or after immobilization of said poly(amino acid), wherein said staining solution comprises a compound having formula

wherein A represents the atoms necessary to form one to two fused aromatic rings having 6 atoms in each ring, at least one of which is a nitrogen atom, said ring or rings being optionally further substituted one or more times by C₁-C₆ alkyl, C₁-C₆ alkoxy trifluoromethyl, halogen, BRIDGE, -L-Rx or -L-Sc;

wherein Rx is a reactive group;

Sc is a conjugated substance; and

L and BRIDGE are independently a single covalent bond, or a covalent linkage;

X is O, S, Se, NR¹⁵, or CR¹⁶R¹⁷, where R¹⁵ is H or an alkyl group having 1-6 carbons; and

R¹⁶ and R¹⁷, which may be the same or different, are independently alkyl groups having 1-6 carbons, or R¹⁶ and R¹⁷ taken in combination complete a five or six membered saturated ring;

R² is selected from the group consisting of -L-Rx, -L-Sc, TAIL, BRIDGE and an alkyl group having 1-6 carbons that is optionally substituted by sulfo, carboxy, amino, substituted amino or substituted ammonium, wherein α

is 0 or 1; and

TAIL is a heteroatom-containing moiety;

Y is —

CR³═

CR⁴—

wherein p and m=0 or 1, such that p+m=1;

R³, R⁴, R⁶, and R⁷ are independently selected from the group consisting of hydrogen, a C₁-C₆ alkyl, a halogen, a CYCLIC SUBSTITUENT, —

OR⁸, —

SR⁸, —

(NR⁸R⁹), TAIL;

BRIDGE, -L-Rx and -L-Sc;

where R⁸ and R⁹ are independently a C₁-C₆ alkyl group, 1-2 alicyclic or aromatic rings;

or R⁸ and R⁹ taken in combination are —

(CH₂)₂-V-(CH₂)₂—

where V is a single bond, —

O—

, —

CH₂—

, or —

NR¹⁰—

, where R¹⁰ is H or an alkyl having 1-6 carbons;

wherein CYCLIC SUBSTITUENT is a substituted or unsubstituted aryl, heteroaryl or C₃-C₁₀ cycloalkyl;

or R⁶ and R⁷ form a fused aromatic ring —

R¹¹═

R¹²—

R¹³═

R¹⁴—

wherein R¹¹, R¹², R¹³, and R¹⁴ are independently selected from the group consisting of hydrogen, C₁-C₆ alkyl group, —

OR⁸, —

SR⁸, —

(NR⁸R⁹), a CYCLIC SUBSTITUENT, TAIL, BRIDGE, -L-Rx and -L-Sc;

R⁵ is independently selected from the group consisting of a C₁-C₆ alkyl group, a CYCLIC SUBSTITUENT, TAIL, BRIDGE, -L-Rx and -L-Sc;

or R⁵ is absent;

R³⁰, R³¹, and R³² are independently selected from the group consisting of hydrogen, C₁-C₆ alkyl, C₃-C₁₀ cycloalkyl, aryl, and heteroaryl, wherein n=0, 1 or 2;

wherein, BRIDGE, when present, is bound to a compound having formula I or another unsymmetrical cyanine dye

c) incubating said sample and said staining solution for a sufficient amount of time to form a dye-poly(amino acid) complex;

d) illiminating said complex with an appropriate wavelength whereby the presence of said poly(amino acid) is determined.

14. The method according to claim 13, wherein said compound is overall neutral in charge.

15. The method according to claim 14, wherein said R² is a C₂-C₆ alkyl group substituted by a sulfo group or R⁵ is absent.

16. The method according to claim 13, wherein said X is O or S, n is 0 or 1, R⁶ and R⁷ form a fused aromatic ring —

R¹¹═

R¹²—

R¹³═

R¹⁴—

wherein R¹¹, R¹², R¹³, and R¹⁴ are independently selected from the group consisting of hydrogen, C₁-C₆ alkyl group, —

OR⁸;

R⁴ and R⁵ are independently a C₁-C₆ alkyl group or a CYCLIC SUBSTITUENT.

17. The method according to claim 13, wherein said method further comprises heating said sample prior to combining with said staining solution, or heating combined sample and staining solution mixture.

18. The method according to claim 13, wherein said method further comprises removing, destroying, or dispersing below the critical micelle concentration any biological membranes that are present in said sample.

19. The method according to claim 13, wherein said method further comprises adding an anionic detergent to the sample, staining solution or combined sample and staining solution mixture.

20. The method according to claim 13, wherein said method further comprising electrophoretically separating said sample before, after, or while it is combined with said staining solution.

21. The method according to claim 20, wherein said matrix is a polymeric gel.

22. The method according to claim 21, wherein said gel is a polyacrylamide gel.

23. The method according to claim 22, wherein said method further comprises adding an additional detection reagent to said sample, said staining solution, or combined sample and staining solution mixture.

24. The method according to claim 20, wherein said method further comprises transferring said sample to a solid or semi-solid matrix before or after combining with said staining solution.

25. A method for the detection of nucleic acids in a sample, wherein said method comprises:

a) combining said sample with a staining solution, wherein said staining solution comprises one or more dyes having formula

wherein A represents the atoms necessary to form one to two fused aromatic rings having 6 atoms in each ring, at least one of which is a nitrogen atom, said ring or rings being optionally further substituted one or more times by C₁-C₆ alkyl, C₁-C₆ alkoxy trifluoromethyl, halogen, BRIDGE, -L-Rx or -L-Sc;

wherein Rx is a reactive group;

Sc is a conjugated substance; and

L and BRIDGE are independently a single covalent bond, or a covalent linkage;

X is O, S, Se, NR¹⁵, or CR¹⁶R¹⁷, where R¹⁵ is H or an alkyl group having 1-6 carbons; and

R¹⁶ and R¹⁷, which may be the same or different, are independently alkyl groups having 1-6 carbons, or R¹⁶ and R¹⁷ taken in combination complete a five or six membered saturated ring;

R² is selected from the group consisting of -L-Rx, -L-Sc, TAIL, BRIDGE and an alkyl group having 1-6 carbons that is optionally substituted by sulfo, carboxy, amino, substituted amino or substituted ammonium, wherein α

is 0 or 1; and

TAIL is a heteroatom-containing moiety;

Y is —

CR³═

CR⁴—

wherein p and m=0 or 1, such that p+m=1;

R³, R⁴, R⁶, and R⁷ are independently selected from the group consisting of hydrogen, a C₁-C₆ alkyl, a halogen, a CYCLIC SUBSTITUENT, —

OR⁸, —

SR⁸, —

(NR⁸R⁹), TAIL;

BRIDGE, -L-Rx and -L-Sc;

where R⁸ and R⁹ are independently a C₁-C₆ alkyl group, 1-2 alicyclic or aromatic rings;

or R⁸ and R⁹ taken in combination are —

(CH₂)₂-V-(CH₂)₂—

where V is a single bond, —

O—

, —

CH₂—

, or —

NR¹⁰—

, where R¹⁰ is H or an alkyl having 1-6 carbons;

wherein CYCLIC SUBSTITUENT is a substituted or unsubstituted aryl, heteroaryl or C₃-C₁₀ cycloalkyl;

or R⁶ and R⁷ form a fused aromatic ring —

R¹¹═

R¹²—

R¹³═

R¹⁴—

wherein R¹¹, R¹², R¹³, and R¹⁴ are independently selected from the group consisting of hydrogen, C₁-C₆ alkyl group, —

OR⁸, —

SR⁸, —

(NR⁸R⁹), a CYCLIC SUBSTITUENT, TAIL, BRIDGE, -L-Rx and -L-Sc;

R⁵ is independently selected from the group consisting of a C₁-C₆ alkyl group, a CYCLIC SUBSTITUENT, TAIL, BRIDGE, -L-Rx and -L-Sc;

or R⁵ is absent;

R³⁰, R³¹, and R³² are independently selected from the group consisting of hydrogen, C₁-C₆ alkyl, C₃-C₁₀ cycloalkyl, aryl, and heteroaryl, wherein n=0, 1 or 2;

wherein, BRIDGE, when present, is bound to a compound having formula I or another unsymmetrical cyanine dye;

b) incubating said sample and said staining solution for a sufficient amount of time to form a dye-nucleic acid complex;

c) illiminating said complex with an appropriate wavelength whereby the presence of said nucleic acid is determined.

26. The method according to claim 25, wherein said X is O, n is 0, R² is a methyl group and R⁶ and R⁷ form a fused aromatic ring —

R¹¹═

R¹²—

R¹³═

R¹⁴—

wherein R¹¹, R¹², R¹³, and R¹⁴ are independently selected from the group consisting of hydrogen, C₁-C₆ alkyl group, —

OR⁸.

27. The method according to claim 26, wherein said R⁵ is a CYCLIC SUBSTITUENT that is an aryl group and R⁴ is an alkyl group.

28. The method according to claim 26, wherein said R⁵ is an alkyl group and R⁴ is a CYCLIC SUBSTITUENT that is an aryl group.

29. The method according to claim 26, wherein said compound is substituted by at least one TAIL substituent.

30. The method according to claim 29, wherein said TAIL is according to formula LINK-SPACER-CAP;

wherein LINK is single covalent bond, an either linkage (—

O—

), a thioether linkage (—

S—

) or an amine linkage (—

NR²⁰—

);

where R²⁰ is hydrogen, C₁-C₈ alkyl or SPACER-CAP;

SPACER is a covalent linkage; and

,

CAP is —

O—

R²¹, —

S—

R²¹, —

NR²¹R²² or —

NR²¹R²²R²³;

where R²¹, R²², and R²³ are independently selected from the group consisting of hydrogen, C₁-C₈ alkyl and a C₁-C₈ cycloalkyl wherein said alkyl or cycloalkyl are optionally substituted by one or more substituents selected from the group consisting of halogen, hydroxyl, C₁-C₈ alkoxy, amino, carboxy, sulfo and phenyl where said phenyl is optionally substituted by one or more substituents selected from the group consisting halogen, hydroxyl, C₁-C₈ alkoxy, amino, C₁-C₈ aminoalkyl, C₁-C₈ sulfoalkyl and C₁-C₈ carboxyalkyl;

or one or more R²¹, R²², and R²³, taken in combination with R²⁰ and SPACER, or with SPACER alone, forms a 5- or 6-membered ring.

31. The method according to claim 30, wherein said R⁴ or R⁵ is independently a TAIL or a CYCLIC SUBSTITUENT substituted by TAIL.

32. The method according to claim 31, wherein said R⁴ is a TAIL and R⁵ is an alkyl group.

33. The method according to claim 32, wherein said CAP is —

NR²¹R²² where R²¹ and R²² are independently C₁-C₆alkyl groups.

34. The method according to claim 32, wherein said CAP is —

NR²¹R²²R²³ where R²¹, R²² and R²³ are independently C₁-C₆ alkyl groups.

35. The method according to claim 31, wherein said R⁵ is a CYCLIC SUBSTITUENT that is an aryl or heteroaryl and said R⁴ is a TAIL where CAP is —

O—

R²¹ or —

S—

R²¹.

36. The method according to claim 31, wherein said R⁵ is a TAIL.

37. The method according to claim 36, wherein said CAP is —

NR²¹R²²R²³ where R²¹, R²² and R²³ are independently C₁-C₆ alkyl groups.

38. The method according to claim 26, wherein said nucleic acid is in solution, immobilized on a solid or semi-solid matrix, or present in a biological structure.

39. The method according to claim 38, wherein said solid or semi-solid matrix is a polymeric gel, an array, a glass slide, a polymeric microparticle or a membrane.

40. The method according to claim 39, wherein said gel is an electrophoretic gel that is an agarose gel.

41. The method according to claim 38, wherein said biological structure is a biological cell or portion thereof, virus particle or tissue section.

42. The method according to claim 41, wherein said biological structure is a cell or portion thereof.

43. The method according to claim 38, wherein said nucleic acid is in solution that is free from cells or portions thereof.