Methods for labeling nucleotides, labeled nucleotides and useful intermediates

• US 7,217,813 B2
• Filed: 09/27/2004
• Issued: 05/15/2007
• Est. Priority Date: 10/08/1996
• Status: Expired due to Fees

• 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:
  US10/950,985
• Publication Number:
  US 20050118621A1
• Time in Patent Office: 960 Days
• Field of Search: 536/26.6, 536/25.3, 536/25.32, 435/6, 435/7.1, 435/7.2
• US Class Current: 536/25.3
• 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 nucleoside having formula:

where X represents 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 carbon atoms and at least one oxygen atom in the chain. The invention further provides a method for labeling a nucleoside, kits for labeling a nucleoside, and kits for producing a labeled nucleic acid.

Claims

1. A labeled nucleoside having formula:

2. The labeled nucleoside according to claim 1, wherein the aliphatic diamine has 2–

6 carbon atoms.

3. The labeled nucleoside 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 nucleoside according to claim 1, wherein X represents ethylenediamine.

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

,N′

-tetramethylethylenediamine.

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

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

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

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

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

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

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

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

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

15. The labeled nucleoside according to claim 1, wherein the nucleoside is adenosine, deoxythymidine, cytodine, guanosine, or uridine.

16. The labeled nucleoside according to claim 1, wherein the nucleoside is adenosine, deoxythymidine, cytodine, and either guanosine or uridine.

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

18. A method for labeling a nucleoside comprising:

providing a nucleoside;

providing a labeling substance having formula VII,

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 nucleoside is adenosine, deoxythymidine, cytodine, guanosine, or uridine.

38. The method according to claim 18, wherein the nucleoside is adenosine, deoxythymidine, cytodine, and either guanosine or uridine, or guanosine and uridine.

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

40. A kit for labeling a nucleoside comprising:

a nucleoside; and

a labeling substance having formula VII,

41. The kit according to claim 40, wherein X represents an aliphatic diamine having 2–

6 carbon atoms.

42. The kit according to claim 40, 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.

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

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

,N′

-tetramethylethylenediamine.

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

, SO₃⁻

, Cl⁻

, I⁻

, other halogen or Me₂SO.

46. The kit according to claim 40, wherein A represents NO₃⁻

.

47. The kit according to claim 40, wherein the spacer comprises no more than twenty carbon atoms.

48. The kit according to claim 47, wherein the carbon atoms are non-branched.

49. The kit according to claim 40, wherein the spacer is 1,8-diamino-3,6-dioxaoctane.

50. The kit according to claim 40, wherein the electron donating moiety is an amino group or a thiolate group.

51. The kit according to claim 50, wherein the amino group is an aromatic amino group.

52. The kit according to claim 50, wherein the amino group is an imidazole or purine group.

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

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

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

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

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

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

59. The kit according to claim 40, wherein the nucleoside is adenosine, deoxythymidine, cytodine, guanosine, or uridine.

60. The kit according to claim 40, wherein the nucleoside is a mixture of adenosine, deoxythymidine, cytodine, and either guanosine or uridine, or guanosine and uridine.

61. The kit according to claim 40, wherein the nucleoside is a purine.

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

a labeled nucleoside having formula;

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 G₂NCH₂CH₂NG₂, 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 the spacer comprises no more than twenty carbon atoms.

68. The kit according to claim 67, wherein the carbon atoms are non-branched.

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

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

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

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

73. The kit according to claim 72, wherein the specific binding pair is biotin and either avidin or streptavidin.

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

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

76. The kit according to claim 62, wherein the labeled nucleoside is labeled adenosine, deoxythymidine, cytodine, guanosine, uridine, or combinations thereof.

77. The kit according to claim 62, wherein the unlabeled nucleoside is adenosine, deoxythymidine, cytodine, guanosine, uridine, or combinations thereof.

78. The kit according to claim 62, wherein the labeled nucleoside is a purine.

79. A method for labeling a nucleoside comprising:

providing a label;

providing a spacer comprising a spacer reactive moiety at one end and an electron donating moiety at the other end of the spacer, the spacer comprising a chain having at least four carbon atoms and at least one oxygen atom in the chain, wherein the spacer reactive moiety is capable of coupling the spacer to a label when the spacer reactive moiety is reacter with the label;

providing a nucleoside;

providing a linker having formula I,