Propargyl substituted nucleoside compounds and methods

• US 20050170388A1
• Filed: 12/17/2004
• Published: 08/04/2005
• Est. Priority Date: 12/23/2003
• Status: Active Grant

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applera Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Jeon, Geun-sook, Khan, Shaheer H., Rosenblum, Barnett B.
• Granted Patent: US 7,998,706 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C07H 19/00 Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
• CPC Class Codes: C07H 19/06 Pyrimidine radicals
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 21/02 with ribosyl as saccharide radical
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical

Abstract

The present teachings relate to nucleobase, nucleoside and nucleotide compounds, methods of synthesis, and uses thereof. The present teachings provide compounds, such as nucleobase, nucleoside and/or nucleotide compounds including a propargyl linker, and methods for making or using such compounds.

Claims

1. A nucleotide comprising a nucleobase disposed between a sugar or sugar analog and a propargylic moiety, wherein the nucleobase comprises a pyrimidine nucleobase or pyrimidine nucleobase analog, the propargylic moiety is covalently attached to the nucleobase at the C-5 position of the nucleobase, the sugar or sugar analog is covalently attached to the nucleobase at the N-1 position of the nucleobase, the sugar or sugar analog comprises a 5′

-monophosphate group, 5′

-diphosphate group, or 5′

-triphosphate group, wherein the propargylic moiety comprises

(i) a propargyl group covalently attached by its methylene carbon to the nucleobase,

(ii) a detectable label, and

(iii) a linker covalently linking the acetylene carbon of the propargyl group to the detectable label.

2. The nucleotide of claim 1 wherein the nucleobase comprises a pyrimidine base.

3. The nucleotide of claim 2 wherein the pyrimidine base is uridine.

4. The nucleotide of claim 3 wherein the sugar comprises ribose, 2′

-deoxyribose or 2′

,3′

-dideoxyribose.

5. The nucleotide of claim 4 wherein the sugar is 2′

,3′

-dideoxyribose.

6. The nucleotide of claim 1 wherein the sugar or sugar analog comprises a compound of the structure:

7. The nucleotide of claim 1 wherein the linker comprises from 1-50 atoms selected from C, H, N, O, S, P and Si.

8. The nucleotide of claim 1 wherein the linker comprises at least one anionic moiety.

9. The nucleotide of claim 1 wherein the linker comprises at least one cationic moiety.

10. The nucleotide of claim 1 wherein the linker comprises at least one amide linkage.

11. The nucleotide of claim 10 wherein the linker comprises at least one diradical selected from

12. The nucleotide of claim 10 wherein the linker comprises at least one diradical selected from

13. The nucleotide of claim 12 wherein Z is unsubstituted amine.

14. The nucleotide of claim 12 wherein Z is substituted amine.

15. The nucleotide of claim 1 wherein the detectable label comprises at least one of a fluorescent dye, an energy transfer dye, a quencher and biotin.

16. The nucleotide of claim 1 wherein the detectable label comprises a fluorescent dye comprising a rhodamine dye, a fluorescein dye, a rhodol dye, an energy transfer dye, a cyanine dye, a phthalocyanine or a squaraine.

17. The nucleotide of claim 15 wherein the fluorescent dye comprises a fluorescein dye, a rhodamine dye or an energy transfer dye.

18. The nucleotide of claim 1 wherein the detectable label comprises an energy transfer dye comprising a donor dye covalently attached to an acceptor dye, wherein either the donor dye or the acceptor dye is covalently attached to the linker, and the acceptor dye is capable of absorbing light emitted by the donor dye.

19. The nucleotide of claim 18 wherein the donor dye is covalently attached to the linker.

20. The nucleotide of claim 18 wherein the donor dye comprises a fluorescein dye or a rhodamine dye.

21. The nucleotide of claim 20 wherein the acceptor dye comprises one of a rhodamine dye, a fluorescein dye, a cyanine dye or a non-fluorescent quencher.

22. The nucleotide of claim 18 wherein the donor dye is covalently linked to the acceptor dye through a linker-moiety that is different from the first said linker.

23. The nucleotide of claim 18 wherein the donor dye comprises a fluorescein or a rhodamine dye and the acceptor dye is a non-fluorescent quencher.

102. The method of claim 97 wherein said extending is performed in the presence of four different nucleotide triphosphates and a compound of claim 1.

24. A labeled nucleoside comprising a nucleobase disposed between a sugar or a sugar analog and a propargylic moiety, wherein the nucleobase is selected from a pyrimidine base or a pyrimidine base analog, the propargylic moiety is covalently attached to the nucleobase at the C-5 position of the nucleobase, the sugar or sugar analog is covalently attached to the nucleobase at the N-1 position of the nucleobase, and wherein the propargylic moiety comprises

(i) a propargyl group covalently attached by its methylene carbon to the nucleobase,

(ii) a detectable label, and

(iii) a linker covalently linking the acetylene carbon of the propargyl group to the detectable label.

25. The nucleoside of claim 24 wherein the nucleobase comprises a pyrimidine base.

26. The nucleoside of claim 24 wherein the pyrimidine base is uridine.

27. The nucleoside of claim 24 wherein the sugar comprises ribose, 2′

-deoxyribose or 2′

,3′

-dideoxyribose.

28. The nucleoside of claim 27 wherein the sugar is 2′

,3′

-dideoxyribose.

29. The nucleoside of claim 24 wherein the sugar or sugar analog comprises a compound of the structure:

30. The nucleoside of claim 24 wherein the linker comprises from 1-50 atoms selected from C, H, N, O, S, P and Si.

31. The nucleoside of claim 24 wherein the linker comprises at least one anionic moiety.

32. The nucleoside of claim 24 wherein the linker comprises at least one cationic moiety.

33. The nucleoside of claim 24 wherein the linker comprises at least one amide linkage.

34. The nucleoside of claim 33 wherein the linker comprises at least one diradical selected from

35. The nucleoside of claim 33 wherein the linker comprises at least one diradical selected from

36. The nucleoside of claim 35 wherein Z is unsubstituted amine.

37. The nucleoside of claim 35 wherein Z is substituted amine.

38. The nucleoside of claim 24 wherein the detectable label comprises at least one of a fluorescent dye, an energy transfer dye, a quencher or biotin.

39. The nucleoside of claim 24 wherein the detectable label comprises a rhodamine dye, a fluorescein dye, a rhodol dye, an energy transfer dye, a cyanine dye, a phthalocyanine and a squaraine.

40. The nucleoside of claim 24 wherein the detectable label comprises a fluorescein dye, a rhodamine dye or an energy transfer dye.

41. The nucleoside of claim 24 wherein the detectable label comprises an energy transfer dye comprising a donor dye covalently attached to an acceptor dye, wherein either the donor dye or the acceptor dye is covalently attached to the linker, and the acceptor dye is capable of absorbing the light emitted by the donor dye.

42. The nucleoside of claim 41 wherein the donor dye is covalently attached to the linker.

43. The nucleoside of claim 41 wherein the donor dye comprises a fluorescein dye or a rhodamine dye.

44. The nucleoside of claim 43 wherein the acceptor dye comprises one of a rhodamine dye, a fluorescein dye, a cyanine dye or a non-fluorescent quencher.

45. The nucleoside of claim 44 wherein the donor dye is covalently linked to the acceptor dye through a linker-moiety.

46. The nucleoside of claim 41 wherein the donor dye comprises a fluorescein or a rhodamine dye and the acceptor dye is a non-fluorescent quencher.

47. A polynucleotide comprising at least one nucleobase comprising the structure

48. The polynucleotide of claim 47 wherein NUC comprises a pyrimidine nucleobase.

49. The polynucleotide of claim 47 wherein the pyrimidine nucleobase is uridine.

50. The polynucleotide of claim 47 wherein the sugar comprises ribose, 2′

-deoxyribose or 2′

,3 ′

-dideoxyribose.

51. The polynucleotide of claim 50 wherein the sugar is 2′

,3′

-dideoxyribose.

52. The polynucleotide of claim 47 wherein the sugar or sugar analog comprises a compound of the structure:

53. The polynucleotide of claim 47 wherein the linker comprises from 1-50 atoms selected from C, H, N, O, S, P and Si.

54. The polynucleotide of claim 47 wherein the linker comprises at least one anionic moiety.

55. The polynucleotide of claim 47 wherein the linker comprises at least one cationic moiety.

56. The polynucleotide of claim 47 wherein the linker comprises at least one amide linkage.

57. The polynucleotide of claim 56 wherein the linker comprises at least one diradical selected from

58. The polynucleotide of claim 56 wherein the linker comprises at least one diradical selected from

59. The polynucleotide of claim 58 wherein Z is unsubstituted amine.

60. The polynucleotide of claim 58 wherein Z is substituted amine.

61. The polynucleotide of claim 47 wherein the detectable label comprises at least one of a fluorescent dye, an energy transfer dye, a quencher or biotin.

62. The polynucleotide of claim 47 wherein the detectable label comprises a fluorescent dye selected from a rhodamine dye, a fluorescein dye, a rhodol dye, an energy transfer dye, a cyanine dye, a phthalocyanine and a squaraine.

63. The polynucleotide of claim 61 wherein the fluorescent dye comprises a fluorescein dye, a rhodamine dye or an energy transfer dye.

64. The polynucleotide of claim 47 wherein the detectable label comprises an energy transfer dye comprising a donor dye covalently attached to an acceptor dye, wherein either the donor dye or the acceptor dye is covalently attached to the linker, and the acceptor dye is capable of absorbing the light emitted by the donor dye.

65. The polynucleotide of claim 64 wherein the donor dye is covalently attached to the linker.

66. The polynucleotide of claim 64 wherein the donor dye comprises a fluorescein dye or a rhodamine dye.

67. The polynucleotide of claim 66 wherein the acceptor dye comprises one of a rhodamine dye, a fluorescein dye, a cyanine dye or a non-fluorescent quencher.

68. The polynucleotide of claim 67 wherein the donor dye is covalently linked to the acceptor dye through a linker-moiety that is different from the first said linker.

69. The polynucleotide of claim 64 wherein the donor dye comprises a fluorescein or a rhodamine dye and the acceptor dye is a non-fluorescent quencher.

70. A nucleoside comprising a nucleobase disposed between a sugar or a sugar analog and a propargylic moiety, wherein the nucleobase is selected from a pyrimidine base or a pyrimidine base analog, the propargylic moiety is covalently attached to the nucleobase at the C-5 position of the nucleobase, the sugar or sugar analog is covalently attached to the nucleobase at the N-1 position of the nucleobase, and wherein the propargylic moiety comprises a propargyl group covalently attached to the nucleobase at the methylene carbon of the propargyl group.

71. The nucleoside of claim 70 wherein the nucleobase comprises a pyrimidine base.

72. The nucleoside of claim 70 wherein the pyrimidine base is uridine.

73. The nucleoside of claim 70 wherein the sugar comprises ribose, 2′

-deoxyribose or 2′

,3′

-dideoxyribose.

74. The nucleoside of claim 70 wherein the sugar is 2′

,3′

-dideoxyribose.

75. The nucleoside of claim 70 wherein the sugar or sugar analog comprises a compound of the structure:

76. The nucleoside of claim 70 wherein the linker comprises from 1-50 atoms selected from C, H, N, O, S, P and Si.

77. The nucleoside of claim 70 wherein the linker comprises at least one anionic moiety.

78. The nucleoside of claim 70 wherein the linker comprises at least one cationic moiety.

79. The nucleoside of claim 70 wherein the linker comprises at least one amide linkage.

80. The nucleoside of claim 79 wherein the linker comprises at least one diradical selected from

81. The nucleoside of claim 79 wherein the linker comprises at least one diradical selected from

82. The nucleoside of claim 81 wherein Z is unsubstituted amine.

83. The nucleoside of claim 81 wherein Z is substituted amine.

84. A nucleotide comprising a nucleobase disposed between a sugar or a sugar analog and a propargylic moiety, wherein the nucleobase is selected from a pyrimidine base or a pyrimidine base analog, the propargylic moiety is covalently attached to the nucleobase at the C-5 position of the nucleobase, the sugar or sugar analog is covalently attached to the nucleobase at the N-1 position of the nucleobase, the sugar or sugar analog comprises a 5′

-monophosphate group, 5′

-diphosphate group or 5′

-triphosphate group and wherein the propargylic moiety comprises a propargyl group covalently attached to the nucleobase at the methylene carbon of the propargyl group.

85. The nucleotide of claim 84 wherein the nucleobase comprises a pyrimidine base.

86. The nucleotide of claim 85 wherein the pyrimidine base is uridine.

87. The nucleotide of claim 84 wherein the sugar comprises ribose, 2′

-deoxyribose or 2′

,3′

-dideoxyribose.

88. The nucleotide of claim 84 wherein the sugar is 2′

,3′

-dideoxyribose.

89. The nucleotide of claim 84 wherein the sugar or sugar analog comprises a compound of the structure:

90. The nucleotide of claim 84 wherein the linker comprises from 1-50 atoms selected from C, H, N, O, S, P and Si.

91. The nucleotide of claim 84 wherein the linker comprises at least one anionic moiety.

92. The nucleotide of claim 84 wherein the linker comprises at least one cationic moiety.

93. The nucleotide of claim 84 wherein the linker comprises at least one amide linkage.

94. The nucleotide of claim 92 wherein the diradical comprises at least one diradical selected from

95. The nucleotide of claim 93 comprising at least one diradical selected from

96. The nucleotide of claim 95 wherein Z is unsubstituted amine.

97. A method of extending a polynucleotide comprising

forming a hybridization complex between a 3′

-extendable polynucleotide and a complementary sequence in a polynucleotide, and

extending the 3′

-extendable polynucleotide in the presence of a nucleotide 5′

-triphosphate and a template-dependent nucleic acid polymerase under conditions effective to append the nucleotide 5′

triphosphate to the 3′

-end of the extendable polynucleotide, wherein the nucleotide 5′

-triphosphate comprises nucleobase disposed between a sugar or sugar analog and a propargylic moiety, the nucleobase comprises a pyrimidine nucleobase or pyrimidine nucleobase analog, the propargylic moiety is covalently attached to the nucleobase at the C-5 position of the nucleobase, the sugar or sugar analog is covalently attached to the nucleobase at the N-1 position of the nucleobase, the sugar or sugar analog comprises a 5′

-monophosphate group, a 5′

-diphosphate group, or a 5′

-triphosphate group, and the propargylic moiety comprises (i) a propargyl group covalently attached by its methylene carbon to the nucleobase, (ii) a detectable label, and (iii) a linker covalently linking the acetylene carbon of the propargyl group to the detectable label.

98. The method of claim 97 wherein the extendable polynucleotide comprises an extended primer 10-500 nucleotides in length.

99. The method of claim 97 wherein the nucleotide 5′

triphosphate is non-extendable.

100. The method of claim 97 wherein the nucleotide 5′

triphosphate comprises a dideoxyribose moiety.

101. The method of claim 97 wherein the nucleic acid polymerase comprises a DNA polymerase.

103. A method for making a thymidine analog comprising:

contacting a protected or unprotected 5-halomethyl uridine nucleotide with a copper acetylide to form an adduct comprising a 5-propargyl uridine.

104. The method of claim 103 wherein the copper acetylide comprises a mixture comprising a copper reagent, a base and an alkyne.

105. A compound having the formula:

NUC—

CH₂—

C═

C—

X-LABEL

wherein

NUC comprises a nucleoside comprising a sugar moiety and a nucleobase selected from a pyrimidine nucleobase or a pyrimidine nucleobase analog,

the CH₂ moiety is covalently attached to the 5-atom of the nucleobase,

X is bond or a linker having from 1 to 50 chain atoms each selected from C, H, N, O, S, P and Si, and

the sugar moiety is covalently attached to the N-1 position of the nucleobase.

106. The compound of claim 105 wherein NUC comprises a 5′

-triphosphate group.

107. The compound of claim 105 wherein X comprises a linker having from 1 to 25 chain atoms.

108. A compound comprising the structure