Label target and labeling reagents comprising rigid group backbones

• US 7,863,431 B2
• Filed: 01/22/2004
• Issued: 01/04/2011
• Est. Priority Date: 03/12/2002
• Status: Active Grant

• Current Assignee: Enzo Life Sciences Incorporated (Enzo Biochem Incorporated)
• Original Assignee: Enzo Life Sciences Incorporated (Enzo Biochem Incorporated)
• Inventors: Stavrianopoulos, Jannis G., Rabbani, Elazar
• Primary Examiner(s): Riley; Jezia
• Application Number:
  US10/763,102
• Publication Number:
  US 20090069500A1
• Time in Patent Office: 2,539 Days
• Field of Search: 435/6, 536/23.1, 536/24.3, 536/26.6
• US Class Current: 536/23.1
• 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 23/02 the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups
• CPC Class Codes: C09B 47/00 Porphines; Azaporphines non-dyeing compounds C07D487/22
• CPC Class Codes: C09B 57/00 Other synthetic dyes of known constitution
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine

Abstract

This invention provides for labeling reagents, labeled targets and processes for preparing labeling reagents. The labeling reagents can take the form of cyanine dyes, xanthene dyes, porphyrin dyes, coumarin dyes or composite dyes. These labeling reagents are useful for labeling probes or targets, including nucleic acids and proteins. These reagents can be usefully applied to protein and nucleic acid probe based assays. They are also applicable to real-time detection processes.

Claims

1. A labeled target comprising

T-L-M

wherein T is a target, M is a marker moiety and L is a chemical group covalently linking said M to T, said chemical group L comprising a backbone that comprises at least one rigid group that comprises one or more of:

2. The labeled target of claim 1, wherein said target T comprises a protein, a peptide, a nucleic acid, a nucleotide or a nucleotide analog, a receptor, a natural or synthetic drug, a synthetic oligomer, a synthetic polymer, a hormone, a lymphokine, a cytokine, a toxin, a ligand, an antigen, a hapten, an antibody, a carbohydrate, a sugar or an oligo- or polysaccharide.

3. The labeled target of claim 2, wherein said nucleic acid or nucleotide or nucleotide analog is modified.

4. The labeled target of claim 2, wherein said ligand comprises biotin, iminobiotin, digoxygenin or fluorescein.

5. The labeled target of claim 1, wherein said marker moiety M comprises a ligand that comprises biotin, iminobiotin, digoxygenin or fluorescein.

6. The labeled target of claim 1, wherein said marker moiety M comprises a fluorescent dye.

7. The labeled target of claim 6, wherein said fluorescent dye comprises a xanthene dye, a cyanine dye, a coumarin dye, a porphyrin dye or a composite dye.

8. The labeled target of claim 7, wherein said xanthene dye comprises fluorescein, rhodamine or rhodol, or derivatives thereof.

9. The labeled target of claim 1, wherein said backbone comprises one or more carbon atoms.

10. The labeled target of claim 1, wherein said backbone comprises at least one non-carbon atom.

11. The labeled target of claim 10, wherein said non-carbon atom comprises sulfur, oxygen or nitrogen.

12. The labeled target of claim 1, wherein said chemical group L further comprises at least one additional moiety comprising peptide bonds, amino acids, aliphatic chains from C₁ through C₂₀, alkene groups, alkyne groups, saturated or unsaturated or partially saturated rings, heterocyclic rings and sugars.

13. The labeled target of claim 1, wherein said structure (a) comprises two alkene groups in cis conformation to each other.

14. The labeled target of claim 1, wherein said structure (a) comprises two alkene groups in trans conformation to each other.

15. The labeling reagent of claim 1, wherein one or more hydrogens are substituted by a chemical moiety.

16. A labeling reagent comprising

R-L-M

wherein R is a reactive group, M is a marker moiety and L is a chemical group covalently linking said M to R, said chemical group L comprising a backbone that comprises at least one rigid group that comprises one or more of:

17. The labeling reagent of claim 16, wherein said reactive group R comprises sulfhydryl, hydroxyl, amine, isothiocyanate, isocyanate, monochlorotriazine, dichlorotriazine, mono- or di-halogen substituted pyridine, mono- or di-halogen substituted diazine, maleimide, aziridine, sulfonylhalide, acid halide, hydroxysuccinimide ester, hydroxysulfosuccinimide ester, imidoester, hydrazine, azidonitrophenyl, azide, 3-(2-pyridyl dithio)-proprionamide, glyoxal or aldehyde.

18. The labeling reagent of claim 17, wherein as a reactive group R is capable of forming a carbon-carbon linkage with a target.

19. The labeling reagent of claim 18, wherein said reactive group R comprises an alkene group, an alkyne group, a halogenated compound or a metallo-organic compound.

20. The labeling reagent of claim 19, wherein said metallo-organic compound comprises mercury, zinc, copper or platinum.

21. The labeling reagent of claim 19, wherein said metallo-organic compound comprises an aromatic group, an aromatic heterocyclic group, an alkene group or an alkyne group.

22. The labeling reagent of claim 16, wherein said marker moiety M comprises a ligand that comprises biotin, iminobiotin, digoxygenin or fluorescein.

23. The labeling reagent of claim 16, wherein said marker moiety M comprises a fluorescent dye.

24. The labeling reagent of claim 23, wherein said fluorescent dye comprises a xanthene dye, a cyanine dye, a coumarin dye, a porphyrin dye or a composite dye.

25. The labeling reagent of claim 24, wherein said xanthene dye comprises fluorescein, rhodamine or rhodol, or derivatives thereof.

26. The labeling reagent of claim 16, wherein said backbone comprises one or more carbon atoms.

27. The labeling reagent of claim 16, wherein said backbone comprises at least one non-carbon atom.

28. The labeling reagent of claim 27, wherein said non-carbon atom comprises sulfur, oxygen or nitrogen.

29. The labeling reagent of claim 27, wherein said chemical group L further comprises at least one additional moiety comprising peptide bonds, amino acids, aliphatic chains from C₁ through C₂₀, alkene groups, alkyne groups, saturated or unsaturated or partially saturated rings, heterocyclic rings and sugars.

30. The labeling reagent of claim 16, wherein said structure (a) comprises two alkene groups in cis conformation to each other.

31. The labeling reagent of claim 16, wherein said structure (a) comprises two alkene groups in trans conformation to each other.

32. The labeling reagent of claim 16, wherein one or more hydrogens are substituted by a chemical moiety.

33. A labeled target comprising

T-L-M

wherein T is a target, M is a marker moiety and L is a chemical group covalently linking said M to T, said chemical group L comprising a backbone that comprises at least two consecutive polar rigid units.

34. The labeled target of claim 33, wherein said target T comprises a protein, a peptide, a nucleic acid, a nucleotide or a nucleotide analog, a receptor, a natural or synthetic drug, a synthetic oligomer, a synthetic polymer, a hormone, a lymphokine, a cytokine, a toxin, a ligand, an antigen, a hapten, an antibody, a carbohydrate, a sugar or an oligo- or polysaccharide.

35. The labeled target of claim 34, wherein said nucleic acid or nucleotide or nucleotide analog is modified.

36. The labeled target of claim 34, wherein said ligand comprises biotin, iminobiotin, digoxygenin or fluorescein.

37. The labeled target of claim 33, wherein said marker moiety M comprises a ligand that comprises biotin, iminobiotin, digoxygenin or fluorescein.

38. The labeled target of claim 33, wherein said marker moiety M comprises a fluorescent dye.

39. The labeled target of claim 38, wherein said fluorescent dye comprises a xanthene dye, a cyanine dye, a coumarin dye, a porphyrin dye or a composite dye.

40. The labeled target of claim 39, wherein said xanthene dye comprises fluorescein, rhodamine or rhodol, or derivatives thereof.

41. The labeled target of claim 33, wherein at least one of said two consecutive rigid polar units of said backbone is heteroatomic.

42. The labeled target of claim 41, wherein said heteroatomic backbone comprises a carbon atom that is bonded to a N, S, O, P or halogen atom.

43. The labeled target of claim 41, wherein said heteroatom comprises an —

OH, —

SH, —

SO₃, —

PO₄, —

COOH or —

NH₂ groups.

44. The labeled target of claim 41, wherein at least one of said two consecutive rigid polar units comprises a peptide bond.

45. The labeled target of claim 41, wherein at least one of said two consecutive polar units comprises a ring structure.

46. The labeled target of claim 45, wherein said ring structure further comprises polar or charged functional groups attached to said ring.

47. The labeled target of claim 46, wherein said polar or charged functional groups comprise halide, —

OH, —

SH, —

SO₃, —

PO₄, —

COOH or —

NH₂ groups.

48. The labeled target of claim 45, wherein said ring structure comprises a sugar.

49. The labeled target of claim 45, wherein said ring structure comprises a substituted heterocyclic aromatic compound.

50. The labeled target of claim 33, wherein said backbone comprises one or more carbon atoms.

51. The labeled target of claim 33, wherein said backbone comprises at least one non-carbon atom.

52. The labeled target of claim 51, wherein said non-carbon atom comprises sulfur, oxygen or nitrogen.

53. The labeled target of claim 33, wherein said chemical group L further comprises at least one additional moiety comprising peptide bonds, amino acids, aliphatic chains from C₁ through C₂₀, alkene groups, alkyne groups, saturated or unsaturated or partially saturated rings, heterocyclic rings and sugars.

54. A labeling reagent comprising

R-L-M

wherein R is a reactive group, M is a marker moiety and L is a chemical group covalently linking said M to R, said chemical group L comprising a backbone that comprises at least two consecutive polar rigid units.

55. The labeling reagent of claim 54, wherein said reactive group R comprises sulfhydryl, hydroxyl, amine, isothiocyanate, isocyanate, monochlorotriazine, dichlorotriazine, mono- or di-halogen substituted pyridine, mono- or di-halogen substituted diazine, maleimide, aziridine, sulfonylhalide, acid halide, hydroxysuccinimide ester, hydroxysulfosuccinimide ester, imidoester, hydrazine, azidonitrophenyl, azide, 3-(2-pyridyl dithio)-proprionamide, glyoxal or aldehyde.

56. The labeling reagent of claim 54, wherein as a reactive group R is capable of forming a carbon-carbon linkage with a target.

57. The labeling reagent of claim 54, wherein said reactive group R comprises an alkene group, an alkyne group, a halogenated compound or a metallo-organic compound.

58. The labeling reagent of claim 57, wherein said metallo-organic compound comprises mercury, zinc, copper or platinum.

59. The labeling reagent of claim 57, wherein said metallo-organic compound comprises an aromatic group, an aromatic heterocyclic group, an alkene group or an alkyne group.

60. The labeling reagent of claim 54, wherein said marker moiety M comprises a ligand that comprises biotin, iminobiotin, digoxygenin or fluorescein.

61. The labeling reagent of claim 54, wherein said marker moiety M comprises a fluorescent dye.

62. The labeling reagent of claim 61, wherein said fluorescent dye comprises a xanthene dye, a cyanine dye, a coumarin dye, a porphyrin dye or a composite dye.

63. The labeling reagent of claim 62, wherein said xanthene dye comprises fluorescein, rhodamine or rhodol, or derivatives thereof.

64. The labeled target of claim 54, wherein at least one of said two consecutive rigid polar units is heteroatomic.

65. The labeled target of claim 64, wherein said heteroatomic rigid polar units comprises a carbon atom that is bonded to a N, S, O, P or halogen atom.

66. The labeled target of claim 64, wherein said heteroatom comprises an —

OH, —

SH, —

SO₃, —

PO₄, —

COOH or —

NH₂ groups.

67. The labeled target of claim 54, wherein at least one of said two consecutive rigid polar units comprises a peptide bond.

68. The labeled target of claim 54, wherein at least one of said two consecutive polar units comprises a ring structure.

69. The labeled target of claim 68, wherein said ring structure further comprises polar or charged functional groups attached to said ring.

70. The labeled target of claim 69, wherein said polar or charged functional groups comprise halide, —

OH, —

SH, —

SO₃, —

PO₄, —

COOH or —

NH₂ groups.

71. The labeled target of claim 68, wherein said ring structure comprises a sugar.

72. The labeled target of claim 68, wherein said ring structure comprises a substituted heterocyclic aromatic compound.

73. The labeling reagent of claim 54, wherein said backbone comprises one or more carbon atoms.

74. The labeling reagent of claim 54, wherein said backbone comprises at least one non-carbon atom.

75. The labeling reagent of claim 74, wherein said non-carbon atom comprises sulfur, oxygen or nitrogen.

76. The labeling reagent of claim 54, wherein said chemical group L further comprises at least one additional moiety comprising peptide bonds, amino acids, aliphatic chains from C₁ through C₂₀, alkene groups, alkyne groups, saturated or unsaturated or partially saturated rings, heterocyclic rings and sugars.