Methods for labeling nucleotides, labeled nucleotides and useful intermediates

• US 6,797,818 B2
• Filed: 01/14/2002
• Issued: 09/28/2004
• Est. Priority Date: 10/08/1996
• Status: Expired due to Term

• Current Assignee: Leica Biosystems Newcastle Ltd. (Danaher Corporation)
• Original Assignee: Kreatech Biotechnology BV (Danaher Corporation)
• Inventors: Heetebrij, Robert Jochem, Volkers, Herman Hendricus, Jelsma, Tinka, Reedijk, Jan, Houthoff, Hendrick Jan
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US10/047,874
• Publication Number:
  US 20020160396A1
• Time in Patent Office: 988 Days
• Field of Search: 435/6, 435/7.1, 435/7.2, 536/26.6
• US Class Current: 536/26.6
• 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: C07H 23/00 Compounds containing boron, silicon, or a metal, e.g. chelates, vitamin B12 esters with inorganic acids C07H11/00; metal salts, see parent compounds
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

The present invention provides a labeled nucleotide having formula:

where X is an aliphatic diamine, and the labeling moiety comprises a label and a spacer, where the spacer is coupled at one end to a platinum atom and at the other end to the label, which spacer comprises a chain having at least four atoms. The invention further provides a method for labeling a nucleotide, kits for labeling a nucleotide, and kits containing labeled nucleotides.

Claims

1. A labeled nucleotide having formula:

wherein;

X represents an aliphatic diamine; and

the labeling moiety comprises a label and a spacer, wherein the spacer is coupled at one end to the Pt atom and at the other end to the label, the spacer comprising a chain having at least four carbon atoms and at least one oxygen atom in the chain.

2. The labeled nucleotide according to claim 1, wherein the aliphatic diamine has 2-6 carbon atoms.

3. The labeled nucleotide according to claim 1, wherein the aliphatic diamine has the formula G₂NCH₂ CH₂ NG₂, wherein G represents H or an alkyl group of from 1 to 6 carbon atoms.

4. The labeled nucleotide according to claim 1, wherein X represents ethylenediamine.

5. The labeled nucleotide according to claim 1, wherein X represents N,N,N′

,N′

-tetramethylethylenediamine.

6. The labeled nucleotide according to claims 1, wherein the spacer comprises no more than twenty carbon atoms.

7. The labeled nucleotide according to claim 6, wherein the carbon atoms are non-branched.

8. The labeled nucleotide according to claim 1, wherein the spacer is 1,8-diamino-3,6-dioxaoctane.

9. The labeled nucleotide according to claim 1, wherein the label is radioactive.

10. The labeled nucleotide according to claim 1, wherein the label is an enzyme.

11. The labeled nucleotide according to claim 1, wherein the label is a component of a specific binding pair.

12. The labeled nucleotide according to claim 11, wherein the specific binding pair is biotin and either avidin or streptavidin.

13. The labeled nucleotide according to claim 1, wherein the label is a dye, a fluorochrome, or a reducing agent.

14. The labeled nucleotide according to claim 1, wherein the label is digoxygenin.

15. The labeled nucleotide according to claim 1, wherein the nucleotide is adenine, thymidine, cytosine, guanine, or uridine.

16. The labeled nucleotide according to claim 1, wherein the nucleotide is adenine, thymidine, cytosine, and either guanine or uridine.

17. The labeled nucleotide according to claim 1, wherein the nucleotide is a purine.

18. A method for labeling a nucleotide comprising:

providing a nucleotide;

providing a labeling substance having formula VII,

wherein;

X represents an aliphatic diamine;

A represents a reactive moiety capable of reacting with the nucleotide, thereby attaching the nucleotide to the labeling substance when the reactive moiety reacts with the nucleotide;

the labeling moiety comprises a spacer comprising an electron donating moiety bonded to the platinum atom, a chain having at least four carbon atoms and at least one oxygen atom in the chain, the chain attached to the electron donating moiety, and a label attached to the end of the chain distal to the electron donating moiety; and

,

reacting the reactive moiety with the nucleotide, thereby labeling the nucleotide.

19. The method according to claim 18, wherein X represents an aliphatic diamine having 2-6 carbon atoms.

20. The method according to claim 18, wherein X represents an aliphatic diamine having the formula G₂NCH₂ CH₂ NG₂, wherein G represents H or an alkyl group of from 1 to 6 carbon atoms.

21. The method according to claim 18, wherein X represents ethylenediamine.

22. The method according to claim 18, wherein X represents N,N,N′

,N′

-tetramethylethylenediamine.

23. The method according to claim 18, wherein A represents NO₃⁻

, SO₃⁻

, Cl⁻

, I⁻

, other halogen or Me₂SO.

24. The method according to claim 18, wherein A represents NO₃⁻

.

25. The method according to claim 18, wherein the spacer comprises no more than twenty carbon atoms.

26. The method according to claim 25, wherein the carbon atoms are non-branched.

27. The method according to claim 18, wherein the spacer is 1,8-diamino-3,6-dioxaoctane.

28. The method according to claim 18, wherein the electron donating moiety is an amino group or a thiolate group.

29. The method according to claim 28, wherein the amino group is an aromatic amino group.

30. The method according to claim 28, wherein the amino group is an imidazole or purine group.

31. The method according to claim 18, wherein the label is radioactive.

32. The method according to claim 18, wherein the label is an enzyme.

33. The method according to claim 18, wherein the label is a component of a specific binding pair.

34. The method according to claim 33, wherein the specific binding pair is biotin and either avidin or streptavidin.

35. The method according to claim 18, wherein the label is a dye, a fluorochrome, or a reducing agent.

36. The method according to claim 18, wherein the label is digoxygenin.

37. The method according to claim 18, wherein the nucleotide is adenine, thymidine, cytosine, guanine, or uridine.

38. The method according to claim 18, wherein the nucleotide is adenine, thymidine, cytosine, and either guanine or uridine, or guanine and uridine.

39. The method according to claim 18, wherein the nucleotide is a purine.

40. A labeling substance having formula VII:

wherein;

X represents an aliphatic diamine;

A represents a reactive moiety; and

the labeling moiety comprises a label and a spacer, wherein the spacer is coupled at one end to the Pt atom and at the other end to the label, the spacer comprising a chain having at least four carbon atoms and at least one oxygen atom in the chain.

41. The labeling substance according to claim 40, wherein X represents an aliphatic diamine having 2-6 carbon atoms.

42. The labeling substance according to claim 40, wherein X represents an aliphatic diamine having the formula G₂NCH₂ CH_{2 NG2}, wherein G represents H or an alkyl group of from 1 to 6 carbon atoms.

43. The labeling substance according to claim 40, wherein X represents ethylenediamine.

44. The labeling substance according to claim 40, wherein X represents N,N,N′

,N′

-tetramethylethylenediamine.

45. The labeling substance according to claim 40, wherein A represents NO₃⁻

, SO₃⁻

, Cl⁻

, I⁻

, other halogen or Me₂SO.

46. The labeling substance according to claim 40, wherein the spacer comprises no more than twenty carbon atoms.

47. The labeling substance according to claim 46, wherein the carbon atoms are non-branched.

48. The labeling substance according to claim 40, wherein the spacer is 1,8-diamino-3,6-dioxaoctane.

49. The labeling substance according to claim 40, wherein the electron donating moiety is an amino group or a thiolate group.

50. The labeling substance according to claim 49, wherein the amino group is an aromatic amino group.

51. The labeling substance according to claim 49, wherein the amino group is an imidazole or purine group.

52. The labeling substance according to claim 40, wherein the spacer reactive moiety is NH₂.

53. The labeling substance according to claim 40, wherein the label is radioactive.

54. The labeling substance according to claim 40, wherein the label is an enzyme.

55. The labeling substance according to claim 40, wherein the label is a component of a specific binding pair.

56. The labeling substance according to claim 55, wherein the specific binding pair is biotin and either avidin or streptavidin.

57. The labeling substance according to claim 40, wherein the label is a dye, a fluorochrome, or a reducing agent.

58. The labeling substance according to claim 40, wherein the label is digoxygenin.

59. The labeling substance according to claim 40, wherein the nucleotide is adenine, thymidine, cytosine, guanine, or uridine.

60. The labeling substance according to claim 40, wherein the nucleotide is adenine, thymidine, cytosine, and either guanine or uridine.

61. The labeling substance according to claim 40, wherein the nucleotide is a purine.

62. A kit for labeling a nucleotide comprising:

a nucleotide; and

a labeling substance having formula VII,

wherein;

X represents an aliphatic diamine;

A represents a reactive moiety capable of reacting with the nucleotide, thereby attaching the nucleotide to the labeling substance when the reactive moiety reacts with the nucleotide;

the labeling moiety comprises a spacer comprising an electron donating moiety bonded to the platinum atom, a chain having at least four carbon atoms and at least one oxygen atom in the chain, the chain attached to the electron donating moiety, and a label attached to the end of the chain distal to the electron donating moiety.

63. The kit according to claim 62, wherein X represents an aliphatic diamine having 2-6 carbon atoms.

64. The kit according to claim 62, wherein X represents an aliphatic diamine having the formula G₂NCH₂ CH₂ NG₂, wherein G represents H or an alkyl group of from 1 to 6 carbon atoms.

65. The kit according to claim 62, wherein X represents ethylenediamine.

66. The kit according to claim 62, wherein X represents N,N,N′

,N′

-tetramethylethylenediamine.

67. The kit according to claim 62, wherein A represents NO₃⁻

, SO₃⁻

, Cl⁻

, I⁻

, other halogen or Me₂SO.

68. The kit according to claim 62, wherein A represents NO₃⁻

.

69. The kit according to claim 62, wherein the spacer comprises no more than twenty carbon atoms.

70. The kit according to claim 69, wherein the carbon atoms are non-branched.

71. The kit according to claim 62, wherein the spacer is 1,8-diamino-3,6-dioxaoctane.

72. The kit according to claim 62, wherein the electron donating moiety is an amino group or a thiolate group.

73. The kit according to claim 72, wherein the amino group is an aromatic amino group.

74. The kit according to claim 72, wherein the amino group is an imidazole or purine group.

75. The kit according to claim 62, wherein the label is radioactive.

76. The kit according to claim 62, wherein the label is an enzyme.

77. The kit according to claim 62, wherein the label is a component of a specific binding pair.

78. The kit according to claim 77, wherein the specific binding pair is biotin and either avidin or streptavidin.

79. The kit according to claim 62, wherein the label is a dye, a fluorochrome, or a reducing agent.

80. The kit according to claim 62, wherein the label is digoxygenin.

81. The kit according to claim 62, wherein the nucleotide is adenine, thymidine, cytosine, guanine, or uridine.

82. The kit according to claim 62, wherein the nucleotide is a mixture of adenine, thymidine, cytosine, and either guanine or uridine, or guanine and uridine.

83. The kit according to claim 62, wherein the nucleotide is a purine.

84. A kit for producing a labeled nucleic acid comprising:

a labeled nucleotide having formula;

wherein X represents an aliphatic diamine; and

the labeling moiety comprises a label and a spacer, wherein the spacer is coupled at one end to the Pt atom and at the other end to the label, the spacer comprising a chain having at least four carbon atoms and at least one oxygen atom in the chain; and

unlabeled nucleotides.

85. The kit according to claim 84, wherein X represents an aliphatic diamine having 2-6 carbon atoms.

86. The kit according to claim 84, wherein X represents an aliphatic diamine having the formula G₂NCH₂CH₂NG₂, wherein G represents H or an alkyl group of from 1 to 6 carbon atoms.

87. The kit according to claim 84, wherein X represents ethylenediamine.

88. The kit according to claim 84, wherein X represents N,N,N′

,N′

-tetramethylethylenediamine.

89. The kit according to claim 84, wherein the spacer comprises no more than twenty carbon atoms.

90. The kit according to claim 89, wherein the carbon atoms are non-branched.

91. The kit according to claim 84, wherein the spacer is 1,8-diamino-3,6-dioxaoctane.

92. The kit according to claim 84, wherein the label is radioactive.

93. The kit according to claim 84, wherein the label is an enzyme.

94. The kit according to claim 84, wherein the label is a component of a specific binding pair.

95. The kit according to claim 94, wherein the specific binding pair is biotin and either avidin or streptavidin.

96. The kit according to claim 84, wherein the label is a dye, a fluorochrome, or a reducing agent.

97. The kit according to claim 84, wherein the label is digoxygenin.

98. The kit according to claim 84, wherein the labeled nucleotide is labeled adenine, thymidine, cytosine, guanine, uridine, or combinations thereof.

99. The kit according to claim 84, wherein the unlabeled nucleotide is adenine, thymidine, cytosine, guanine, uridine, or combinations thereof.

100. The kit according to claim 84, wherein the labeled nucleotide is a purine.