Cyanine dye compounds

• US 20050214810A1
• Filed: 12/06/2004
• Published: 09/29/2005
• Est. Priority Date: 12/05/2003
• Status: Active Grant

• Current Assignee: Molecular Probes Incorporated (Thermo Fisher Scientific Incorporated)
• Original Assignee: Molecular Probes Incorporated (Thermo Fisher Scientific Incorporated)
• Inventors: Huang, Shih-Jung, Yue, Stephen, Dallwig, Jason Alfred, Thomas, Gerald Alan, Hagen, David Carl
• Granted Patent: US 7,446,202 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C09B 23/04 one >CH- group, e.g. cyanines, isocyanines, pseudocyanines
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: G01N 33/582 with fluorescent label
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

Cyanine dye compounds having a negatively charged substituent that are nucleic acid stains, particularly for fluorescent staining of DNA, including compounds having the formula wherein W forms one or two fused 5- or 6-membered aromatic rings, a has a value of 0 or 1, n has a value of 0, or 1, X is O, S, or Se, and D is a pyridinium, or quinolinium moiety, provided that the compound has at least one negatively charged substituent.

Claims

1. A compound having the formula:

wherein W represents the atoms necessary to form one or two fused substituted 5- or 6-membered aromatic rings or one or two unsubstituted 5- or 6-membered aromatic rings;

R² is a substituted alkyl, unsubstituted alkyl, substituted arylalkyl, unsubstituted arylalkyl, substituted heteroalkyl, unsubstituted heteroalkyl, alkoxy, carboxy, carboxyalkyl, hydroxy, hydroxyalkyl, sulfo, sulfoalkyl, amino, aminoalkyl, alkylamino, dialkylamino, or trialkylammonium;

α

is 0 or 1;

n is 0 or 1;

X is O, S, or Se;

D is a substituted pyridinium, unsubstituted pyridinium, substituted quinolinium, or unsubstituted quinolinium moiety;

with the proviso that the compound is substituted by at least one negatively charged moiety at a physiological pH.

2. The compound according to claim 1, wherein the compound exhibits a fluorescence enhancement when non-covalently associated with a nucleic acid molecule.

3. The compound according to claim 2, wherein the fluorescence enhancement is greater when the nucleic acid is DNA than when the nucleic acid is RNA.

4. The compound according to claim 1, wherein the negatively charged moiety is sulfo, carboxy, phosphate, phosphonate, an alkyl group substituted by sulfo, an alkyl group substituted by a carboxy, an alkyl group substituted by phosphate, or an alkyl group substituted by phosphonate.

5. The compound acccording to claim 1, wherein W comprises —

C, —

CR¹, or —

N(R2)_β

;

wherein β

is 0 or 1, provided that α

+β

=1; and

wherein each R¹ is independently hydrogen, a reactive group, a carrier molecule, a solid support, carboxy, sulfo, phosphate, phosphonate, amino, hydroxy, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, trifluoromethyl, halogen, substituted alkyl, unsubstituted alkyl, alkoxy, substituted alkylamino, unsubstituted alkylamino, substituted dialkylamino, or unsubstituted dialkylamino.

6. The compound according to claim 1, wherein W includes a fused 6-membered aromatic ring.

7. The compound according to claim 1, wherein n is 0.

8. The compound according to claim 1, wherein D is a substituted pyridinium, unsubstituted pyridinium, substituted quinolinium or unsubstituted quinolinium moiety.

9. A compound according to claim 1, wherein D has the formula

wherein R³ is hydrogen, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, substituted aryl, unsubstituted aryl, substituted arylalkyl, unsubstituted arylalkyl, substituted heteroarylalkyl; unsubstituted heteroarylalkyl, substituted heteroaryl, unsubstituted heteroaryl, substituted cycloalkyl, unsubstituted cycloalkyl, substituted heterocycloalkyl, unsubstituted heterocycloalkyl, halogen, alkoxy, substituted alkylamino, unsubstituted alkylamino, substituted alkylthio, unsubstituted alkylthio, reactive group, solid support, or carrier molecule;

R⁴ is hydrogen, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, substituted aryl, unsubstituted aryl, substituted arylalkyl, unsubstituted arylalkyl, substituted heteroarylalkyl;

unsubstituted heteroarylalkyl, substituted heteroaryl, unsubstituted heteroaryl, substituted cycloalkyl, unsubstituted cycloalkyl, substituted heterocycloalkyl, unsubstituted heterocycloalkyl, halogen, alkoxy, substituted alkylamino, unsubstituted alkylamino, substituted alkylthio, unsubstituted alkylthio, reactive group, solid support, or carrier molecule;

R⁵ is hydrogen, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, substituted aryl, unsubstituted aryl, substituted arylalkyl, unsubstituted arylalkyl, substituted heteroarylalkyl;

unsubstituted heteroarylalkyl, substituted heteroaryl, unsubstituted heteroaryl, substituted cycloalkyl, unsubstituted cycloalkyl, substituted heterocycloalkyl, unsubstituted heterocycloalkyl, halogen, alkoxy, substituted alkylamino, unsubstituted alkylamino, substituted alkylthio, unsubstituted alkylthio, reactive group, solid support, or carrier molecule;

R⁶ is hydrogen, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, substituted aryl, unsubstituted aryl, substituted arylalkyl, unsubstituted arylalkyl, substituted heteroarylalkyl;

unsubstituted heteroarylalkyl, substituted heteroaryl, unsubstituted heteroaryl, substituted cycloalkyl, unsubstituted cycloalkyl, substituted heterocycloalkyl, unsubstituted heterocycloalkyl, halogen, alkoxy, substituted alkylamino, unsubstituted alkylamino, substituted alkylthio, unsubstituted alkylthio, reactive group, solid support, or carrier molecule;

or

a member selected from

R⁵ in combination with R⁶

R⁴ in combination with R⁵;

R⁴ in combination with R³;

R⁴ in combination with R⁶;

R³ in combination with R⁶

together with the atoms to which they are joined, form a ring which is a 5-, 6- or 7-membered heterocycloalkyl, a substituted 5-, 6- or 7-membered heterocycloalkyl, a 5-, 6- or 7-membered cycloalkyl, a substituted 5-, 6- or 7-membered cycloalkyl, a 5-, 6- or 7-membered heteroaryl, a substituted 5-, 6- or 7-membered heteroaryl, a 5-, 6- or 7-membered aryl or a substituted 5-, 6- or 7-membered aryl.

10. The compound according to claim 9, wherein at least one of R³ and R⁴is alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, alkoxy, alkylthio, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl or substituted heteroarylalkyl.

11. The compound according to claim 9, wherein R³is alkyl, substituted alkyl, phenyl, substituted phenyl, benzyl, substituted benzyl, alkylthio, substituted alkylthio, indolyl, imidazolyl, or thiazolyl;

and,

R⁴ is alkyl, substituted alkyl, phenyl, substituted phenyl, benzyl, substituted benzyl, alkylthio, substituted alkylthio, indolyl, imidazolyl, or thiazolyl.

12. The compound according to claim 9, wherein R³is hydrogen, alkyl, substituted alkyl, —

(CH₂)_a-aryl or —

(CH₂)_a-heteroaryl; and

,

R⁴ is hydrogen, alkyl, substituted alkyl, —

(CH₂)_a-aryl or —

(CH₂)_a-heteroaryl;

wherein a is an integer from 0 to about 6.

13. The compound according to claim 1, having the formula

wherein each R^1a is independently hydrogen, carboxy, sulfo, phosphate, phosphonate, amino, hydroxyl, trifluoromethyl, halogen, alkyl, substituted alkyl, alkoxy, alkylamino, substituted alkylamino, dialkylamino, substituted dialkylamino, fused benzene, substituted fused benzene, trifluomethyl, halogen, reactive group, solid support or carrier molecule;

each R^1b is independently hydrogen, carboxy, sulfo, phosphate, phosphonate, amino, hydroxyl, trifluoromethyl, halogen, alkyl, substituted alkyl, alkoxy, alkylamino, substituted alkylamino, dialkylamino, substituted dialkylamino, fused benzene, substituted fused benzene, trifluomethyl, halogen, reactive group, solid support or carrier molecule;

t is an integer from 1 to 4;

R² is a substituted alkyl, unsubstituted alkyl, substituted arylalkyl, unsubstituted arylalkyl, substituted heteroalkyl, unsubstituted heteroalkyl, alkoxy, carboxy, carboxyalkyl, hydroxy, hydroxyalkyl, sulfo, sulfoalkyl, amino, aminoalkyl, alkylamino, dialkylamino, or trialkylammonium;

n=0 or 1;

R³ is hydrogen, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, substituted aryl, unsubstituted aryl, substituted arylalkyl, unsubstituted arylalkyl, substituted heteroarylalkyl;

unsubstituted heteroarylalkyl, substituted heteroaryl, unsubstituted heteroaryl, substituted cycloalkyl, unsubstituted cycloalkyl, substituted heterocycloalkyl, unsubstituted heterocycloalkyl, halogen, alkoxy, substituted alkylamino, unsubstituted alkylamino, substituted alkylthio, unsubstituted alkylthio, reactive group, solid support, or carrier molecule; and

R⁴ is hydrogen, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, substituted aryl, unsubstituted aryl, substituted arylalkyl, unsubstituted arylalkyl, substituted heteroarylalkyl;

unsubstituted heteroarylalkyl, substituted heteroaryl, unsubstituted heteroaryl, substituted cycloalkyl, unsubstituted cycloalkyl, substituted heterocycloalkyl, unsubstituted heterocycloalkyl, halogen, alkoxy, substituted alkylamino, unsubstituted alkylamino, substituted alkylthio, unsubstituted alkylthio, reactive group, solid support, or carrier molecule.

14. The compound according to claim 13, wherein R⁴ is hydrogen, alkyl, —

(CH₂)_a-aryl, or —

(CH₂)_a-heteroaryl, wherein a is an integer from 0 to about 6.

15. The compound according to claim 13, wherein R⁴ is an alkyl, phenyl, benzyl, alkylthio, indolyl, imidazolyl, or thiazolyl.

16. The compound according to claim 13, wherein at least one of R¹ and R² comprises a negatively charged substituent.

17. The compound according to claim 13, wherein at least one R¹ and R² is sulfo, carboxy, phosphate, phosphonate, an alkyl group substituted by sulfo, an alkyl group substituted by a carboxy, an alkyl group substituted by phosphate, or an alkyl group substituted by phosphonate.

18. The compound according to claim 1, having the formula

19. The compound according to claim 1, wherein the reactive group, solid support and carrier molecule comprise a linker that is a single covalent bond, or a covalent linkage that is linear or branched, cyclic or heterocyclic, saturated or unsaturated, having 1-20 nonhydrogen atoms selected from the group consisting of C, N, P, O and S;

and are composed of any combination of ether, thioether, amine, ester, carboxamide, sulfonamide, hydrazide bonds and aromatic or heteroaromatic bonds.

20. The compound according to claim 1, wherein the reactive group is an acrylamide, an activated ester of a carboxylic acid, a carboxylic ester, an acyl azide, an acyl nitrile, an aldehyde, an alkyl halide, an anhydride, an aniline, an amine, an aryl halide, an azide, an aziridine, a boronate, a diazoalkane, a haloacetamide, a haloalkyl, a halotriazine, a hydrazine, an imido ester, an isocyanate, an isothiocyanate, a maleimide, a phosphoramidite, a reactive platinum complex, a silyl halide, a sulfonyl halide, a thiol or a photoactivatable group.

21. The compound according to claim 1, wherein the reactive group is carboxylic acid, succinimidyl ester of a carboxylic acid, hydrazide, amine or a maleimide.

22. The compound according to claim 1, wherein the carrier molecule is an amino acid, a peptide, a protein, a polysaccharide, a nucleoside, a nucleotide, an oligonucleotide, a nucleic acid polymer, a hapten, a psoralen, a drug, a hormone, a lipid, a lipid assembly, a synthetic polymer, a polymeric microparticle, a biological cell or a virus.

23. The compound according to claim 1, wherein the carrier molecule is an antibody or fragment thereof, an avidin or streptavidin, a biotin, a blood component protein, a dextran, an enzyme, an enzyme inhibitor, a hormone, an IgG binding protein, a fluorescent protein, a growth factor, a lectin, a lipopolysaccharide, a microorganism, a metal binding protein, a metal chelating moiety, a non-biological microparticle, a peptide toxin, a phosphotidylserine-binding protein, a structural protein, a small-molecule drug, or a tyramide.

24. The compound according to claim 1, wherein the solid support is a microfluidic chip, a silicon chip, a microscope slide, a microplate well, silica gels, polymeric membranes, particles, derivatized plastic films, glass beads, cotton, plastic beads, alumina gels, polysaccharides, polyvinylchloride, polypropylene, polyethylene, nylon, latex bead, magnetic bead, paramagnetic bead, or superparamagnetic bead.

25. The compound according to claim 1, wherein the solid support is sepharose, poly(acrylate), polystyrene, poly(acrylamide), polyol, agarose, agar, cellulose, dextran, starch, FICOLL, heparin, glycogen, amylopectin, mannan, inulin, nitrocellulose, diazocellulose or starch.

26. A composition of matter comprising a nucleic acid reporter molecule and a sample, wherein the nucleic acid reporter molecule has the formula:

wherein W represents the atoms necessary to form one or two fused substituted 5- or 6-membered aromatic rings or one or two unsubstituted 5- or 6-membered aromatic rings;

R² is a substituted alkyl, unsubstituted alkyl, substituted arylalkyl, unsubstituted arylalkyl, substituted heteroalkyl, unsubstituted heteroalkyl, alkoxy, carboxy, carboxyalkyl, hydroxy, hydroxyalkyl, sulfo, sulfoalkyl, amino, aminoalkyl, alkylamino, dialkylamino, or trialkylammonium;

α

is 0 or 1;

n is 0 or 1;

X is O, S, or Se;

D is a substituted pyridinium, unsubstituted pyridinium, substituted quinolinium, or unsubstituted quinolinium moiety;

with the proviso that the compound is substituted by at least one negatively charged moiety at a physiological pH.

27. The composition according to claim 26, wherein the sample comprises biological fluids, buffer solutions, live cells, fixed cells, eukaryotic cells, prokaryotic cells, nucleic acid polymers, nucleosides, nucleotides, a polymeric gel or tissue sections.

28. The composition according to claim 27, wherein the nucleic acid polymers are single stranded RNA, double stranded RNA, single stranded DNA or double stranded DNA.

33. The complex according to claim 26, wherein the complex is present in an aqueous or aqueous miscible solution.

34. The complex according to claim 26, wherein the complex is present in an electrophoretic matrix or polymeric gel.

29. A complex comprising a nucleic acid reporter molecule non-covalently associated with a nucleic acid molecule, wherein the nucleic acid molecule has the formula:

wherein W represents the atoms necessary to form one or two fused substituted 5- or 6-membered aromatic rings or one or two unsubstituted 5- or 6-membered aromatic rings;

R² is a substituted alkyl, unsubstituted alkyl, substituted arylalkyl, unsubstituted arylalkyl, substituted heteroalkyl, unsubstituted heteroalkyl, alkoxy, carboxy, carboxyalkyl, hydroxy, hydroxyalkyl, sulfo, sulfoalkyl, amino, aminoalkyl, alkylamino, dialkylamino, or trialkylammonium;

α

is 0 or 1;

n is 0 or 1;

X is O, S, or Se;

D is a substituted pyridinium, unsubstituted pyridinium, substituted quinolinium, or unsubstituted quinolinium moiety;

with the proviso that the compound is substituted by at least one negatively charged moiety at a physiological pH.

30. The complex according to claim 29, wherein the nucleic acid molecule is single stranded RNA, double stranded RNA, single stranded DNA or double stranded DNA.

31. The complex according to claim 29, wherein the nucleic acid molecule is an oligonucleotide, a chromosome or a fragment thereof.

32. The complex according to claim 29, wherein the complex is present in a biological sample.

35. A method for detecting the presence or absence of nucleic acid in a sample, wherein the method comprises:

a. combining a nucleic acid reporter molecule with the sample to prepare a labeling mixture, wherein the nucleic acid reporter molecule is according to the formula;

wherein W represents the atoms necessary to form one or two fused substituted 5- or 6-membered aromatic rings or one or two unsubstituted 5- or 6-membered aromatic rings;

R² is a substituted alkyl, unsubstituted alkyl, substituted arylalkyl, unsubstituted arylalkyl, substituted heteroalkyl, unsubstituted heteroalkyl, alkoxy, carboxy, carboxyalkyl, hydroxy, hydroxyalkyl, sulfo, sulfoalkyl, amino, aminoalkyl, alkylamino, dialkylamino, or trialkylammonium;

α

is 0 or 1;

n is 0 or 1;

X is O, S, or Se;

D is a substituted pyridinium, unsubstituted pyridinium, substituted quinolinium, or unsubstituted quinolinium moiety;

with the proviso that the compound is substituted by at least one negatively charged moiety at a physiological pH;

b. incubating the labeling mixture for a sufficient amount of time for the nucleic acid reporter molecule to associate with nucleic acid in the sample to form an incubated mixture;

c. illuminating the incubated sample with an appropriate wavelength to form an illuminated mixture; and

,

d. observing the illuminated mixture whereby the presence or absence of the nucleic acid in a sample is detected.

36. The method according to claim 35, further comprising quantifying the nucleic acid present in the sample.

37. The method according to claim 35, wherein the nucleic acid is DNA.

38. The method according to claim 35, wherein the sample comprises biological fluids, buffer solutions, live cells, fixed cells, eukaryotic cells, prokaryotic cells, nucleic acid polymers, nucleotides, nucleosides, a polymeric gel or tissue sections.

39. The method according to claim 35, wherein the sample comprises cells, tissue, or biological fluids.

40. The method according to claim 35, wherein the sample is present in or on a microarray or a microwell plate.

41. A method for detecting DNA in the presence of RNA, wherein the method comprises the steps:

a. combining a nucleic acid reporter molecule with a sample to prepare a labeling mixture, wherein the nucleic acid reporter molecule has a DNA/RNA ratio of fluorescence enhancement greater than about one wherein the nucleic acid reporter molecule is according to the formula

wherein W represents the atoms necessary to form one or two fused substituted 5- or 6-membered aromatic rings or one or two unsubstituted 5- or 6-membered aromatic rings;

R² is a substituted alkyl, unsubstituted alkyl, substituted arylalkyl, unsubstituted arylalkyl, substituted heteroalkyl, unsubstituted heteroalkyl, alkoxy, carboxy, carboxyalkyl, hydroxy, hydroxyalkyl, sulfo, sulfoalkyl, amino, aminoalkyl, alkylamino, dialkylamino, or trialkylammonium;

α

is 0 or 1;

n is 0 or 1;

X is O, S, or Se;

D is a substituted pyridinium, unsubstituted pyridinium, substituted quinolinium, or unsubstituted quinolinium moiety;

with the proviso that the compound is substituted by at least one negatively charged moiety at a physiological pH;

b. incubating the labeling mixture for a sufficient amount of time for the nucleic acid reporter molecule to associate with DNA in the sample to form an incubated mixture;

c. illuminating the incubated mixture with an appropriate wavelength to form an illuminated mixture; and

,

d. observing the illuminated mixture whereby the DNA is detected in the presence of RNA.

42. The method according to claim 41, further comprising quantifying the DNA present in the sample.

43. The method according to claim 41, wherein the sample comprises biological fluids, buffer solutions, live cells, fixed cells, eukaryotic cells, prokaryotic cells, nucleic acid polymers, nucleotides, nucleosides, a polymeric gel or tissue sections.

44. The method according to claim 41, wherein the sample comprises cells, tissue, or biological fluids.

45. The method according to claim 41, wherein the sample is present in or on a microarray or a microwell plate.

46. A kit for detecting nucleic acid in a sample, wherein the kit comprises a nucleic acid reporter molecule according to the formula:

wherein W represents the atoms necessary to form one or two fused substituted 5- or 6-membered aromatic rings or one or two unsubstituted 5- or 6-membered aromatic rings;

R² is a substituted alkyl, unsubstituted alkyl, substituted arylalkyl, unsubstituted arylalkyl, substituted heteroalkyl, unsubstituted heteroalkyl, alkoxy, carboxy, carboxyalkyl, hydroxy, hydroxyalkyl, sulfo, sulfoalkyl, amino, aminoalkyl, alkylamino, dialkylamino, or trialkylammonium;

α

is 0 or 1;

n is 0 or 1;

X is O, S, or Se;

D is a substituted pyridinium, unsubstituted pyridinium, substituted quinolinium, or unsubstituted quinolinium moiety;

with the proviso that the compound is substituted by at least one negatively charged moiety at a physiological pH.

47. The kit according to claim 46, further comprising instructions for detecting DNA in the presence of RNA.

49. The kit according to claim 46, further comprising one or more of a sample preparation reagent, a buffering agent, aqueous nucleic acid reporter molecule dilution buffer, an organic solvent, nucleic acid control, or an additional detection reagent.

51. The kit according to claim 49, wherein the detergent is present in a final concentration of about 0.01% to about 0.5% (w/v).

52. The kit according to claim 49, wherein the detergent is CHAPS, Tween 20, Trinton-X or SDS.

53. The kit according to claim 46, further comprising a microarray.

54. The kit according to claim 46, further comprising a microwell plate.

56. The staining solution according to claim 54, wherein the detergent is present at a concentration of about 0.01% to about 0.5%.

57. The staining solution according to claim 54, wherein the detergent is CHAPS, Triton-X, SDS or Tween 20.

58. The staining solution according to claim 54, wherein the detergent is CHAPS and is present at a concentration of about 0.2%.

59. The staining solution according to claim 54, further comprising a stabilizer.

48. The kit according to claim 48, further comprising instructions for quantitating DNA from about 1 ng to about 2000 ng.

50. The kit according to claim 48, wherein the nucleic acid reporter molecule dilution buffer contains a detergent.

55. A staining solution comprising a nucleic acid reporter molecule and a detergent, wherein the nucleic acid reporter molecule has the formula:

wherein W represents the atoms necessary to form one or two fused substituted 5- or 6-membered aromatic rings or one or two unsubstituted 5- or 6-membered aromatic rings;

R² is a substituted alkyl, unsubstituted alkyl, substituted arylalkyl, unsubstituted arylalkyl, substituted heteroalkyl, unsubstituted heteroalkyl, alkoxy, carboxy, carboxyalkyl, hydroxy, hydroxyalkyl, sulfo, sulfoalkyl, amino, aminoalkyl, alkylamino, dialkylamino, or trialkylammonium;

α

is 0 or 1;

n is 0 or 1;

X is O, S, or Se;

D is a substituted pyridinium, unsubstituted pyridinium, substituted quinolinium, or unsubstituted quinolinium moiety;

with the proviso that the compound is substituted by at least one negatively charged moiety at a physiological pH.