Cyanine compounds and their application as quenching compounds

• US 7,271,265 B2
• Filed: 08/11/2004
• Issued: 09/18/2007
• Est. Priority Date: 08/11/2003
• Status: Active Grant

• Current Assignee: Molecular Probes Incorporated (Thermo Fisher Scientific Incorporated)
• Original Assignee: Invitrogen Corp. (Thermo Fisher Scientific Incorporated)
• Inventors: Cheung, Ching-Ying, Yue, Stephen, Haugland, Rosaria
• Primary Examiner(s): Anderson; Rebecca
• Assistant Examiner(s): Kosack; Joseph R.
• Application Number:
  US10/916,822
• Publication Number:
  US 20050191643A1
• Time in Patent Office: 1,133 Days
• Field of Search: 548/469, 548/159, 548/465, 546/277.4
• US Class Current: 546/277.4
• CPC Class Codes: C07D 209/12 Radicals substituted by oxygen atoms
• CPC Class Codes: C07D 209/14 Radicals substituted by nitrogen atoms, not forming part of a nitro radical
• CPC Class Codes: C07D 209/24 with an alkyl or cycloalkyl radical attached to the ring nitrogen atom
• CPC Class Codes: C07D 401/04 directly linked by a ring-member-to-ring-member bond
• CPC Class Codes: C09B 23/12 the polymethine chain being branched "branched" means that the substituent on the polymethine chain forms a new conjugated system, e.g. most trinuclear cyanine dyes
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes

Abstract

The present invention provides methods and non-fluorescent carbocyanine quencher compounds having the general formula:

Wherein the A moiety is a substituted pyridinium, unsubstituted pyridinium, substituted quinolinium, unsubstituted quinolinium, substituted benzazolium, unsubstituted benzazolium, substituted indolinium, or unsubstituted indolinium. The invention further provides luminescent donor molecule-quencher pairs and luminescent donor molecule-quencher-luminescent acceptor molecule conjugates wherein the quencher is a cyanine compound of the present invention. The energy transfer pairs are used to detect an analyte of interest in a sample.

Claims

1. A compound having formula:

2. The compound according to claim 1, wherein A is

3. The compound according to claim 2, wherein A is

4. The compound according to claim 2, wherein A is

5. The compound according to claim 1, wherein the reactive group, solid support and carrier molecule independently 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.

6. The compound according to claim 5, 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.

7. The compound according to claim 5, wherein the reactive group is carboxylic acid, succinimidyl ester of a carboxylic acid, hydrazide, amine or a maleimide.

8. The compound according to claim 5, 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 biotin-binding protein, a psoralen, a drug, a hormone, a lipid, a lipid assembly, a synthetic polymer, a polymeric microparticle, a biological cell or a virus.

9. The compound according to claim 5, 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.

10. The compound according to claim 5, wherein the solid support is a microfluidic chip, a silicon chip, a microscope slide, a microplate well, a silica gel, a polymeric membrane, a particle, a derivatized plastic film, a glass bead, cotton, a plastic bead, an alumina gel, a polysaccharide, polyvinylchloride, polypropylene, polyethylene, nylon, latex bead, magnetic bead, paramagnetic bead, or superparamagnetic bead.

11. The compound according to claim 5, 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.

12. The compound according to claim 1, wherein the compound has the formula:

13. The compound according to claim 12, wherein at least one of R², R³, or R⁴ is a reactive group, carrier molecule or solid support.

14. A compound having the formula:

15. The compound according to claim 14, wherein the reactive group, solid support and carrier molecule independently 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.

16. The compound according to claim 15, 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.

17. The compound according to claim 15, wherein the reactive group is carboxylic acid, succinimidyl ester of a carboxylic acid, hydrazide, amine or a maleimide.

18. The compound according to claim 15, 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 biotin-binding protein, a hormone, a lipid, a lipid assembly, a synthetic polymer, a polymeric microparticle, a biological cell or a virus.

19. The compound according to claim 15, 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.

20. The compound according to claim 15, wherein the solid support is a microfluidic chip, a silicon chip, a microscope slide, a microplate well, a silica gel, a polymeric membrane, a particle, a derivatized plastic film, a glass bead, cotton, a plastic bead, an alumina gel, a polysaccharide, polyvinylchloride, polypropylene, polyethylene, nylon, latex bead, magnetic bead, paramagnetic bead, or superparamagnetic bead.

21. The compound according to claim 15, 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.

22. The compound according to claim 14, wherein at least one of R², R³, R⁴ or R¹² is a reactive group, carrier molecule or solid support.

23. The compound according to claim 22, wherein the reactive group is an amine- or thiol-reactive group.

24. The compound according to claim 14, wherein:

at least one of R²², R²³, R²⁴, R²⁵ and R²⁶ is —

(C═

O)—

O—

R²⁷;

wherein R²⁷ is hydrogen, amine, substituted amine, alkyl, substituted alkyl.

25. The compound according to claim 24, wherein R²⁷ is alkyl.

26. The compound according to claim 14 wherein R²⁴ is —

(C═

O)—

O—

CH₃.

27. The compound according to claim 14, wherein Z is S or CR³R⁴.

28. A compound that is

29. A composition comprising:

a. a luminescent donor molecule;

b. a luminescent acceptor molecule; and

,

c. a compound that is a non-fluorescent acceptor molecule, having the formula;

30. The composition according to claim 29, further comprising a carrier molecule wherein the luminescent donor molecule, the luminescent acceptor molecule and the non-fluorescent compound are covalently bonded to the carrier molecule.

31. The composition according to claim 30, 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 biotin-binding protein, a polymeric microparticle, a biological cell or a virus.

32. The composition according to claim 30, wherein the carrier molecule is a streptavidin or avidin, a biotin, an antibody, a antibody fragment, a protein A, an oligonucleotide or a nucleotide.

33. The composition according to claim 29, wherein the luminescent donor molecule is a fluorescent dye, a chemiluminescent compound or a fluorescent protein.

34. The composition according to claim 29, wherein the luminescent acceptor molecule is a fluorescent dye, a chemiluminescent compound or a fluorescent protein.

35. The composition according to claim 29, wherein the luminescent donor molecule is a fluorescent protein.

36. The composition according to claim 35, wherein the fluorescent protein is a phycoerythrin protein.

37. The composition according to claim 29, wherein the luminescent acceptor molecule is a fluorescent dye.

38. A composition comprising:

a. a luminescent donor molecule;

b. a luminescent acceptor molecule;

c. a compound that is a non-fluorescent acceptor molecule, wherein the compound has the formula

39. The composition according to claim 38, wherein the carrier molecule is a streptavidin or avidin, a biotin, an antibody, an antibody fragment, a protein A, an oligonucleotide or a nucleotide.

40. The composition according to claim 38, wherein the luminescent donor molecule is a fluorescent protein.

41. The composition according to claim 40, wherein the fluorescent protein is a phycoerythrin protein.

42. The composition according to claim 38, wherein the luminescent acceptor molecule is a fluorescent dye.

43. The composition according to claim 42, wherein the fluorescent dye is a xanthene, cyanine, borapolyazaindacene, oxazole, merocyanine, coumarin, pyrene, or indole.

44. The composition according to claim 38, wherein the luminescent donor molecule is a phycoerythrin fluorescent protein, the luminescent acceptor molecule is a cyanine fluorescent dye and the carrier molecule is an antibody or fragment thereof or a streptavidin or avidin.

45. A composition comprising:

a) a compound that is a non-fluorescent acceptor molecule, wherein the compound has the formula;

46. The composition according to claim 45, 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 biotin-binding protein, a polymeric microparticle, a biological cell or a virus.

47. The composition according to claim 45, wherein the carrier molecule is a streptavidin or avidin, a biotin, an antibody, an antibody fragment, a protein A, an oligonucleotide or a nucleotide.

48. A method for absorbing residual energy from an energy donor molecule during energy transfer between a luminescent donor molecule and a luminescent acceptor molecule, wherein the method comprises:

a) covalently bonding a luminescent donor molecule to a luminescent acceptor molecule to prepare an energy transfer pair;

b) covalently bonding a compound to the energy transfer pair to prepare a quencher labeled energy transfer pair, wherein the compound has the formula

49. The method according to claim 48, wherein the luminescent donor molecule is a fluorescent dye, a chemiluminescent compound or a fluorescent protein.

50. The method according to claim 48, wherein the luminescent donor molecule is a fluorescent protein.

51. The method according to claim 50, wherein the fluorescent protein is a phycoerythrin protein.

52. The method according to claim 48, wherein the luminescent acceptor molecule is a fluorescent dye, a chemiluminescent compound or a fluorescent protein.

53. The method according to claim 48, wherein the luminescent acceptor molecule is a fluorescent dye.

54. The method according to claim 53, wherein the fluorescent dye is a xanthene, cyanine, borapolyazaindacene, oxazole, merocyanine, coumarin, pyrene, or indol.

55. The method according to claim 48, wherein the luminescent donor molecule is a phycoerythrin fluorescent protein, the luminescent acceptor molecule is a cyanine fluorescent dye and the carrier molecule is an antibody or fragment thereof or a streptavidin or avidin.

56. A method for detecting presence or absence of an analyte in a sample, wherein the method comprises:

a. contacting the sample with a composition to prepare a labeled sample, wherein the composition comprises a carrier molecule, a luminescent donor molecule, a luminescent acceptor molecule and a compound having the formula

57. The method according to claim 56, wherein the sample comprises live cells, intracellular fluids, extracellular fluids, biological fluids, biological fermentation media, environmental sample, industrial samples, proteins, peptides, buffer solutions biological fluids or chemical reactors.

58. The method according to claim 56, wherein the sample comprises blood cells, immune cells, cultured cells, muscle tissue, neurons, extracellular vesicles;

vascular tissue, blood fluids, saliva, urine;

water, soil, waste water, sea water;

pharmaceuticals, foodstuffs or beverages.

59. The method according to claim 56, wherein the sample is immobilized on a polymeric membrane, within a polymeric gel, on a microparticle, on a microarray, on a silicon chip, on a glass slide, on a microwell plate, and on a microfluidic chip.

60. The method according to claim 56, wherein the luminescent donor molecule is a fluorescent dye, a chemiluminescent compound or a fluorescent protein.

61. The method according to claim 56, wherein the luminescent donor is a fluorescent protein.

62. The method according to claim 61, wherein the fluorescent protein is a phycoerythrin protein.

63. The method according to claim 56, wherein the luminescent acceptor molecule is a fluorescent dye, a chemiluminescent compound or a fluorescent protein.

64. The method according to claim 56, wherein the luminescent acceptor molecule is a fluorescent dye.

65. The method according to claim 64, wherein the fluorescent dye is a xanthene, cyanine, borapolyazaindacene, oxazole, merocyanine, coumarin, pyrene, or indole.

66. The method according to claim 56, 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 biotin-binding protein, a psoralen, a drug, a hormone, a lipid, a lipid assembly, a synthetic polymer, a polymeric microparticle, a biological cell or a virus.

67. The method according to claim 56, wherein the carrier molecule is a streptavidin or avidin, a biotin, an antibody, an antibody fragment, a protein A, an oligonucleotide or a nucleotide.

68. The method according to claim 56, wherein the luminescent donor molecule is a phycoerythrin fluorescent protein, the luminescent acceptor molecule is a cyanine fluorescent dye and the carrier molecule is an antibody or fragment thereof or a streptavidin or avidin.

69. A method for detecting the presence of an analyte in a sample, wherein the method comprises:

a. contacting the sample with a composition to prepare a labeled sample, wherein the composition comprises a carrier molecule and a compound having the formula

70. The method according to claim 69, wherein the composition further comprises a luminescent donor molecule.

71. The method according to claim 70, wherein the luminescent donor molecule is a fluorescent protein, a fluorescent dye or a nucleic acid stain.

72. The method according to claim 69, 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 biotin-binding protein, a psoralen, a drug, a hormone, a lipid, a lipid assembly, a synthetic polymer, a polymeric microparticle, a biological cell or a virus.

73. The method according to claim 69, wherein the carrier molecule is a streptavidin or avidin, a biotin, an antibody, an antibody fragment, a protein A, an oligonucleotide or a nucleotide.

74. The method according to claim 69, wherein the sample comprises live cells, intracellular fluids, extracellular fluids, biological fluids, biological fermentation media, environmental sample, industrial samples, proteins, peptides, buffer solutions biological fluids or chemical reactors.

75. The method according to claim 69, wherein the sample comprises blood cells, immune cells, cultured cells, muscle tissue, neurons, extracellular vesicles;

vascular tissue, blood fluids, saliva, urine;

water, soil, waste water, sea water;

pharmaceuticals, foodstuffs or beverages.

76. The method according to claim 69, wherein the sample is immobilized on a polymeric membrane, within a polymeric gel, on a microparticle, on a microarray, on a silicon chip, on a glass slide, on a microwell plate, and on a microfluidic chip.

77. A kit for detecting a ligand in a sample, wherein the kit comprises:

a composition comprising a carrier molecule, a luminescent donor molecule, a luminescent acceptor molecule and a non-fluorescent acceptor molecule having the formula

78. The kit according to claim 77, further comprising instructions for detecting the ligand.

79. The kit according to claim 77, 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 biotin-binding protein, a psoralen, a drug, a hormone, a lipid, a lipid assembly, a synthetic polymer, a polymeric microparticle, a biological cell or a virus.

80. The kit according to claim 77, wherein the carrier molecule is a streptavidin or avidin, a biotin, an antibody or fragment thereof, a protein A, an oligonucleotide or a nucleotide.

81. The kit according to claim 77, wherein the luminescent donor molecule is a fluorescent dye, a chemiluminescent compound or a fluorescent protein.

82. The kit according to claim 77, wherein the luminescent donor molecule is a fluorescent protein.

83. The kit according to claim 82, wherein the fluorescent protein is a phycoerythrin protein.

84. The kit according to claim 77, wherein the luminescent acceptor molecule is a fluorescent dye, a chemiluminescent compound or a fluorescent protein.

85. The kit according to claim 84, wherein the luminescent acceptor molecule is a fluorescent dye.

86. The kit according to claim 85, wherein the fluorescent dye is a xanthene, cyanine, borapolyazaindacene, oxazole, merocyanine, coumarin, pyrene, or indole.

87. The kit according to claim 77, wherein the luminescent donor molecule is a phycoerythrin fluorescent protein, the luminescent acceptor molecule is a cyanine fluorescent dye and the carrier molecule is an antibody or fragment thereof or a streptavidin or avidin.

88. The kit according to claim 87, wherein the antibody or antibody fragment is further covalently bonded to a streptavidin or avidin.

89. A kit for labeling a carrier molecule, wherein the kit comprises:

a non-fluorescent compound, wherein the compound has the formula

90. The kit according to claim 89, further comprising instructions for covalently bonding the compound to the carrier molecule.

91. The kit according to claim 89, further comprising one or more of the following:

a buffering agent, a purification medium, a vial of the carrier molecule, an organic solvent, a luminescent acceptor molecule or a luminescent donor molecule.