Pteridine nucleotide analogs as fluorescent DNA probes

• US 5,525,711 A
• Filed: 05/18/1994
• Issued: 06/11/1996
• Est. Priority Date: 05/18/1994
• Status: Expired due to Term

• Current Assignee: United States Department Of Health And Human Services
• Original Assignee: United States Department Of Health And Human Services
• Inventors: Davis, Michael D., Pfleiderer, Wolfgang, Balis, Frank, Hawkins, Mary E.
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): Marschel, Ardin H.
• Application Number:
  US08/245,923
• Time in Patent Office: 755 Days
• Field of Search: 536/22.1, 536/23.1, 536/25.3, 536/25.31, 536/25.32, 536/25.33, 536/25.34, 536/26.1, 536/26.2, 536/26.7, 536/27.1, 536/27.13, 536/27.2, 536/28.1 , 536/28.4, 536/55, 536/84, 435/6, 436/501, 935/77.78
• US Class Current: 536/22.1
• CPC Class Codes: C07H 19/22 Pteridine radicals
• 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

Abstract

The invention provides novel pteridine nucleotides which are highly fluorescent under physiological conditions and which may be used in the chemical synthesis of fluorescent oligonucleotidcs. The invention further provides for fluorescent oligonucleotides comprising one or more pteridine nucleotides. In addition the invention provides for pteridine nucleotide triphosphates which may be used as the constituent monomers in DNA amplification procedures.

Claims

1. A compound having the formula shown below, with ring vertices 1 through 8 as shown:

##STR25## in which;

R¹¹ is combined with R¹² to form a single oxo oxygen joined by a double bond to ring vertex 4, or with R¹³ to form a double bond between ring vertices 3 and 4;

R¹² when not combined with R¹¹ is a member selected from the group consisting of NH₂, and NH₂ either mono- or disubstituted with a protecting group;

R¹³ when not combined with R¹¹ is lower alkyl or H;

R¹⁴ is a member selected from the group consisting of H, lower alkyl and phenyl;

R¹⁵ is combined with R¹⁶ to form a single oxo oxygen joined by a double bond to ring vertex 2, or with R¹⁷ to form a double bond between ring vertices 1 and 2, such that ring vertices 2 and 4 collectively bear at most one oxo oxygen;

R¹⁶ when not combined with R¹⁵ is a member selected from the group consisting of H, phenyl, NH₂, and NH₂ mono- or disubstituted with a protecting group;

when R¹⁵ is not combined with R¹⁶, R¹⁸ is combined with R¹⁹ to form a single oxo oxygen joined by a double bond to ring vertex 7;

when R¹⁵ is combined with R¹⁶, R¹⁸ is combined with R²⁰ to form a double bond between ring vertices 7 and 8, and R¹⁹ is a member selected from the group consisting of H and lower alkyl; and

R¹⁷ when not combined with R¹⁵, and R²⁰ when not combined with R¹⁸, are ##STR26## in which;

R²¹ is a member selected from the group consisting of H, a triphosphate, and protecting groups;

R²² is a member selected from the group consisting of H, OH and OH substituted with a protecting group; and

R²³ is a member selected from the group consisting of H, a phosphoramidite, an H-phosphonate, a methyl phosphonate, a phosphorothioate, a phosphotriester, a hemisuccinate, a hemisuccinate covalently bound to a solid support, a dicyclohexylcarbodiimide, and a dicyclohexylcarbodiimide covalently bound to a solid support;

when R¹³ is H and R²³ is H, R²¹ is a tfiphosphate; and

when R¹¹ is combined with R¹³ to form a double bond between ring vertices 3 and 4 and R²³ is H, R²¹ is a triphosphate.

2. A compound in accordance with claim 1 in which R¹⁴ is a member selected from the group consisting of H, CH₃ and phenyl.

3. A compound in accordance with claim 1 in which R¹⁴ is a member selected from the group consisting of H and CH₃.

4. A compound in accordance with claim 1 in which R¹⁶, when not combined with R¹⁵, is a member selected from the group consisting of H, phenyl, NH₂, and NH₂ disubstituted with a protecting group.

5. A compound in accordance with claim 1 in which R¹⁶, when not combined with R¹⁵, is a member selected from the group consisting of H and phenyl.

6. A compound in accordance with claim 1 in which, when R¹⁸ is combined with R²⁰, R¹⁹ is a member selected from the group consisting of H and CH₃.

7. A compound in accordance with claim 1 in which R¹⁴ is a member selected from the group consisting of H, CH₃ and phenyl;

R¹⁶, when not combined with R¹⁵, is a member selected from the group consisting of H, phenyl and NH₂ ; and

, when R¹⁸ is combined with R²⁰, R¹⁹ is a member selected from the group consisting of H and CH₃.

8. A compound in accordance with claim 1 in which R¹² is NH₂ either mono- or disubstituted by a protecting group selected from the group consisting of benzoyl, isobutyryl, phthaloyl, di-n-butylaminomethylidene, dimethylaminomethylidene, p-nitrophenylethoxycarbonyl and dimethylaminomethylenamino.

9. A compound in accordance with claim 1 in which R¹² is NH₂ monosubstituted by a protecting group selected from the group consisting of di-n-butylaminomethylidene, p-nitrophenylethoxycarbonyl, and dimethylaminomethylenamino.

10. A compound in accordance with claim 1 in which R¹⁶ is NH₂ either mono- or disubstituted by a protecting group selected from the group consisting of benzoyl, isobutyryl, phthaloyl, di-n-butylaminomethylidene, dimethylaminomethylidene, p-nitrophenylethoxycarbonyl and dimethylaminomethylenamino.

11. A compound in accordance with claim 1 in which R¹⁶ is NH₂ monosubstituted by a protecting group selected from the group consisting of di-n-butylaminomethylidene, p-nitrophenylethoxycarbonyl, and dimethylaminomethylenamino.

12. A compound in accordance with claim 1 in which R²¹ is a member selected from the group consisting of H, trityl, monomethoxytrityl, dimethoxytrityl, phthaloyl, di-n-butylaminomethylene, and dimethylaminomethylidene.

13. A compound in accordance with claim 1 in which R²¹ is a member selected from the group consisting of dimethoxytrityl, di-n-butylaminomethylene, and dimethylaminomethylidene.

14. A compound in accordance with claim 1 in which R²² is a member selected from the group consisting of H, OH and OH substituted with a member selected fro m the group consisting of trityl, monomethoxytrityl, dimethoxytrityl, tetrahydropyran- 1-yl, 4-methoxytetrahydropyran-4-yl, 1-(2-chloro-4-methyl)phenyl-4-methoxypiperidin-4-yl, t-butyldimethylsilyl, p-nitrophenylethylsulfonyl, tetrahydropyranyl, 4-methoxytetrahydropyranyl, 2-nitrobenzyl, 9-phenylxanthen-9-yl and p-nitrophenylethyl.

15. A compound in accordance with claim 1 in which R²² is a member selected from the group consisting of H and OH substituted with a member selected from the group consisting of dimethoxytrityl, tetrahydropyran-1-yl, t-butyldimethylsilyl, 2-nitrobenzyl, and p-nitrophenylethyl.

16. A compound in accordance with claim 1 in which:

R¹¹ is combined with R¹³ to form a double bond between ring vertices 3 and 4;

R¹² is selected from the group consisting of NH₂, and NH₂ mono- or disubstituted with a protecting group;

;

R¹⁴ is H;

R¹⁵ is combined with R¹⁷ to form a double bond between ring vertices 1 and 2;

R¹⁶ is phenyl;

R¹⁸ is combined with R¹⁹ to form a single oxo oxygen joined by a double bond to ring vertex 7; and

R²⁰ is ##STR27##

17. A compound in accordance with claim 1 in which:

R¹¹ is combined with R¹³ to form a double bond between ring vertices 3 and 4;

R¹² is selected from the group consisting of NH₂, and NH₂ mono- or disubstituted with a protecting group;

R¹⁴ is phenyl;

R¹⁵ is combined with R¹⁷ to form a double bond between ring vertices 1 and 2;

R¹⁶ is H;

R¹⁸ is combined with R¹⁹ to form a single oxo oxygen joined by a double bond to ring vertex 7; and

R²⁰ is ##STR28##

18. A compound in accordance with claim 1 in which:

R¹¹ is combined with R¹² to form a single oxo oxygen joined by a double bond to ring vertex 4;

R¹³ is CH₃ ;

R₁₄ is H;

R¹⁵ is combined with R¹⁷ to form a double bond between ring vertices 1 and 2;

R¹⁶ is NH₂ ;

R¹⁸ is combined with R¹⁹ to form a single oxo oxygen joined by a double bond to ring vertex 7; and

R²⁰ is ##STR29##

19. A compound in accordance with claim 1 in which:

R¹¹ is combined with R¹² to form a single oxo oxygen joined by a double bond to ring vertex 4;

R¹³ is H;

R¹⁴ is H;

R¹⁵ is combined with R¹⁷ to form a double bond between ring vertices 1 and 2;

R¹⁶ is selected from the group consisting of NH₂ and NH₂ mono- or disubstituted with a protecting group;

R¹⁸ is combined with R¹⁹ to form a single oxo oxygen joined by a double bond to ring vertex 7; and

R²⁰ is ##STR30##

20. A compound in accordance with claim 1 in which:

R¹¹ is combined with R¹² to form a single oxo oxygen joined by a double bond to ring vertex 4;

R¹³ is H;

R¹⁴ is CH₃.

R¹⁵ is combined with R¹⁷ to form a double bond between ring vertices 1 and 2;

R¹⁶ is selected from the group consisting of NH₂ and NH₂ mono- or disubstituted with a protecting group;

R¹⁸ is combined with R¹⁹ to form a single oxo oxygen joined by a double bond to ring vertex 7; and

R²⁰ is ##STR31##

21. A compound in accordance with claim 1 in which:

R¹¹ is combined with R¹³ to form a double bond between ring vertices 3 and 4;

R¹² is selected from the group consisting of NH₂ and NH₂ mono- or di-substituted with a protecting group;

R¹⁴ is CH₃ ;

R¹⁵ is combined with R¹⁶ to form a single oxo oxygen joined by a double bond to ring vertex 2;

R¹⁷ is ##STR32## R¹⁸ is combined with R²⁰ to form a double bond between ring vertices 7 and 8; and

R¹⁹ is CH₃.

22. A compound in accordance with claim 1 in which:

R¹¹ is combined with R¹³ to form a double bond between ring vertices 3 and 4;

R¹² is selected from the group consisting of NH₂ and NH₂ mono- or di-substituted with a protecting group;

;

R¹⁴ is H;

R¹⁵ is combined with R¹⁶ to form a single oxo oxygen joined by a double bond to ring vertex 2;

R¹⁷ is ##STR33## R¹⁸ is combined with R²⁰ to form a double bond between ring vertices 7 and 8; and

R¹⁹ is CH₃.

23. A compound in accordance with claim 1 in which:

R¹¹ is combined with R¹³ to form a double bond between ring vertices 3 and 4;

R¹² is selected from the group consisting of NH₂ and NH₂ mono- or di-substituted with a protecting group;

R¹⁴ is CH₃ ;

R¹⁵ is combined with R¹⁶ to form a single oxo oxygen joined by a double bond to ring vertex 2;

R¹⁷ is ##STR34## R¹⁸ is combined with R²⁰ to form a double bond between ring vertices 7 and 8; and

R¹⁹ is H.

24. A compound in accordance with claim 1 in which:

R¹¹ is combined with R¹³ to form a double bond between ring vertices 3 and 4;

R¹² is selected from the group consisting of NH₂ and NH₂ mono- or di-substituted with a protecting group;

R¹⁴ is H;

R¹⁵ is combined with R¹⁶ to form a single oxo oxygen joined by a double bond to ring vertex 2;

R¹⁷ is ##STR35## R¹⁸ is combined with R²⁰ to form a double bond between ring vertices 7 and 8; and

R¹⁹ is H.

25. A compound in accordance with claim 16 in which R¹² is NH₂.

26. A compound in accordance with claim 16 in which:

R¹² is mono- or di-substituted with a protecting group; and

R²³ is a member selected from the group consisting of H-phosphonate, phosphoramidite, hemisuccinate, and hemisuccinate covalently bound to a solid support.

27. A compound in accordance with claim 26 in which:

R²¹ is dimethoxytrityl;

R²² is H; and

R²³ is a β

-cyanoethyl, N-diisopropyl phosphoramidite.

28. A compound in accordance with claim 27 in which:

R¹² is dimethylaminomethylenamino.

29. A compound in accordance with claim 26 in which:

R²¹ is dimethoxytrityl;

R²² is H; and

R²³ is a hemisuccinate covalently bound to controlled pore glass.

30. A compound in accordance with claim 29 in which:

R¹² is dimethylaminomethylenamino.

31. A compound in accordance with claim 17 in which R¹² is NH₂.

32. A compound in accordance with claim 17 in which:

R¹² is mono- or di-substituted with a protecting group; and

R²³ is a member selected from the group consisting of H-phosphonate, phosphoramidite, hemisuccinate, and hemisuccinate covalently bound to a solid support.

33. A compound in accordance with claim 32 in which:

R²¹ is dimethoxytrityl;

R²² is H; and

R²³ is a β

-cyanoethyl, N-diisopropyl phosphoramidite.

34. A compound in accordance with claim 33 in which R¹² is dimethylaminomethylenamino.

35. A compound in accordance with claim 32 in which:

R²¹ is dimethoxytrityl;

R²² is H; and

R²³ is a hemisuccinate covalently bound to controlled pore glass.

36. A compound in accordance with claim 35 in which R¹² is dimethylaminomethylenamino.

37. A compound in accordance with claim 18 in which R²³ is a member selected from the group consisting of H, H-phosphonate, phosphoramidite, hemisuccinate, and hemisuccinate covalently bound to a solid support.

38. A compound in accordance with claim 37 in which:

R²¹ is H;

R²² is H; and

R²³ is H.

39. A compound in accordance with claim 37 in which:

R²¹ is dimethoxytrityl;

R²² is H; and

R²³ is a β

-cyanoethyl, N-diisopropyl phosphoramidite.

40. A compound in accordance with claim 37 in which:

R²¹ is dimethoxytrityl;

R²² is H; and

R²³ is a hemisuccinate covalently bound to controlled pore glass.

41. A compound in accordance with claim 19 in which R¹⁶ is NH₂.

42. A compound in accordance with claim 19 in which:

R¹⁶ is NH₂ mono- or di-substituted with a protecting group; and

R²³ is a member selected from the group consisting of H-phosphonate, phosphoramidite, hemisuccinate, and hemisuccinate covalently bound to a solid support.

43. A compound in accordance with claim 42 in which:

R²¹ is dimethoxytrityl;

R²² is H; and

R¹³ is a β

-cyanoethyl, N-diisopropyl phosphoramidite.

44. A compound in accordance with claim 43 in which R¹⁶ is dimethylaminomethylenamino.

45. A compound in accordance with claim 42 in which:

R²¹ is dimethoxytrityl;

R²² is H; and

R²³ is a hemisuccinate covalenfly bound to controlled pore glass.

46. A compound in accordance with claim 45 in which R¹⁶ is dimethylaminomethylenamino.

47. A compound in accordance with claim 20 in which R¹⁶ is NH₂.

48. A compound in accordance with claim 20 in which:

R¹⁶ is NH₂ mono- or di-substituted with a protecting group; and

R²³ is a member selected from the group consisting of H-phosphonate, phosphoramidite, hemisuccinate, and hemisuccinate covalently bound to a solid support.

49. A compound in accordance with claim 48 in which:

R²¹ is dimethoxytrityl;

R²² is H; and

R²³ is a β

-cyanoethyl, N-diisopropyl phosphoramidite.

50. A compound in accordance with claim 49 in which R¹⁶ is dimethylaminomethylenamino.

51. A compound in accordance with claim 48 in which:

R²¹ is dimethoxytrityl;

R²² is H; and

R²³ is a hemisuccinate covalently bound to controlled pore glass.

52. A compound in accordance with claim 51 in which R¹⁶ is dimethylaminomethylenamino.

53. A compound in accordance with claim 21 in which R¹² is NH₂.

54. A compound in accordance with claim 21 in which:

R¹² is NH₂ mono- or di-substituted with a protecting group; and

R²³ is a member selected from the group consisting of H-phosphonate, phosphoramidite, hemisuccinate, and hemisuccinate covalently bound to a solid support.

55. A compound in accordance with claim 54 in which:

R²¹ is dimethoxytrityl;

R²² is H; and

R²³ is a β

-cyanoethyl, N-diisopropyl phosphoramidite.

56. A compound in accordance with claim 55 in which R¹² is p-nitrophenylethoxycarbonyl.

57. A compound in accordance with claim 54 in which:

R²¹ is dimethoxytrityl;

R²² is H; and

R²³ is a hemisuccinate covalently bound to controlled pore glass.

58. A compound in accordance with claim 57 in which R¹² is p-nitrophenylethoxycarbonyl.

59. A compound in accordance with claim 22 in which R¹² is NH₂.

60. A compound in accordance with claim 22 in which:

R¹² is NH₂ mono- or di-substituted with a protecting group; and

R²³ is a member selected from the group consisting of H-phosphonate, phosphoramidite, hemisuccinate, and hemisuccinate covalently bound to a solid support.

61. A compound in accordance with claim 60 in which:

R²¹ is dimethoxytrityl;

R²² is H; and

R²³ is a β

-cyanoethyl, N-diisopropyl phosphoramidite.

62. A compound in accordance with claim 61 in which R¹² is p-nitrophenylethoxycarbonyl.

63. A compound in accordance with claim 60 in which:

R²¹ is dimethoxytrityl;

R²² is H; and

R²³ is a hemisuccinate covalently bound to controlled pore glass.

64. A compound in accordance with claim 63 in which R¹² is p-nitrophenylethoxycarbonyl.

65. A compound in accordance with claim 23 in which R¹² is NH₂.

66. A compound in accordance with claim 23 in which:

R¹² is NH₂ mono- or di-substituted with a protecting group; and

R²³ is a member selected from the group consisting of H-phosphonate, phosphoramidite, hemisuccinate, and hemisuccinate covalently bound to a solid support.

67. A compound in accordance with claim 66 in which:

R²¹ is dimethoxytrityl;

R²² is H; and

R²³ is a β

-cyanoethyl, N-diisopropyl phosphoramidite.

68. A compound in accordance with claim 67 in which R ¹² is p-nitrophenylethoxycarbonyl.

69. A compound in accordance with claim 66 in which:

R²¹ is dimethoxytrityl;

R²² is H; and

R²³ is a hemisuccinate covalently bound to controlled pore glass.

70. A compound in accordance with claim 69 in which R¹² is p-nitrophenylethoxycarbonyl.

71. A compound in accordance with claim 24 in which R¹² is NH₂.

72. A compound in accordance with claim 24 in which:

R¹² is NH₂ mono- or di-substituted with a protecting group; and

R²³ is a member selected from the group consisting of H-phosphonate, phosphoramidite, hemisuccinate, and hemisuccinate covalently bound to a solid support.

73. A compound in accordance with claim 72 in which:

R²¹ is dimethoxytrityl;

R²² is H; and

R²³ is a β

-cyanoethyl, N-diisopropyl phosphoramidite.

74. A compound in accordance with claim 73 in which:

R¹² is p-nitrophenylethoxycarbonyl.

75. A compound in accordance with claim 72 in which:

R²¹ is dimethoxytrityl;

R²² is H; and

R²³ is a hemisuccinate covalently bound to controlled pore glass.

76. A compound in accordance with claim 75 in which:

R¹² is p-nitrophenylethoxycarbonyl.

77. A compound in accordance with claim 16 in which:

R²¹ is a triphosphate;

R²² is H; and

R²³ is H.

78. A compound in accordance with claim 17 in which:

R²¹ is a triphosphate;

R²² is H; and

R²³ is H.

79. A compound in accordance with claim 18 in which:

R²¹ is a triphosphate;

R²² is H; and

R²³ is H.

80. A compound in accordance with claim 19 in which:

R²¹ is a triphosphate;

R²² is H; and

R²³ is H.

81. A compound in accordance with claim 20 in which:

R²¹ is a triphosphate;

R²² is H; and

R²³ is H.

82. A compound in accordance with claim 21 in which:

R²¹ is a triphosphate;

R²² is H; and

R²³ is H.

83. A compound in accordance with claim 22 in which:

R²¹ is a triphosphate;

R²² is H; and

R²³ is H.

84. A compound in accordance with claim 23 in which:

R²¹ is a triphosphate;

R²² is H; and

R²³ is H.

85. A compound in accordance with claim 24 in which:

R²¹ is a triphosphate;

R²² is H; and

R²³ is H.