Methods of labelling polynucleotides with dibenzorhodamine dyes

• US 20040072209A1
• Filed: 05/20/2003
• Published: 04/15/2004
• Est. Priority Date: 11/25/1997
• Status: Active Grant

• Current Assignee: Applera Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applera Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Menchen, Steven Micheal, Benson, Scott C., Lam, Joe Y.L.
• Granted Patent: US 6,919,445 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C07D 209/60 Naphtho [b] pyrroles; Hydrogenated naphtho [b] pyrroles
• CPC Class Codes: C07D 221/10 Aza-phenanthrenes
• CPC Class Codes: C07D 311/78 Ring systems having three or more relevant rings
• CPC Class Codes: C07D 491/14 Ortho-condensed systems
• CPC Class Codes: C07D 491/147 the condensed system containing one ring with oxygen as ring hetero atom and two rings with nitrogen as ring hetero atom
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/20 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• 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 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C09B 11/02 derived from diarylmethanes
• CPC Class Codes: C09B 11/24 Phthaleins containing amino groups ; Phthalanes; Fluoranes; Phthalides; Rhodamine dyes; Phthaleins having heterocyclic aryl rings; Lactone or lactame forms of triarylmethane dyes
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

Dibenzorhodamine compounds having the structure

are disclosed, including nitrogen- and aryl-substituted forms thereof. In addition, two intermediates useful for synthesizing such compounds are disclosed, a first intermediate having the structure

including nitrogen- and aryl-substituted forms thereof, and a second intermediate having the structure

including nitrogen- and aryl-substituted forms thereof, wherein substituents at positions C-14 to C18 taken separately are selected from the group consisting of hydrogen, chlorine, fluorine, lower alkyl, carboxylic acid, sulfonic acid, —CH₂OH, alkoxy, phenoxy, linking group, and substituted forms thereof. The invention further includes energy transfer dyes comprising the dibenzorhodamine compounds, nucleosides labeled with the dibenzorhodamine compounds, and nucleic acid analysis methods employing the dibenzorhodamine compounds.

Claims

1. A dibenzorhodamine compound having the structure

2. The compound of claim 1 comprising a first bridging group which when taken together with the C-12-bonded nitrogen and the C-12 and C-13 carbons forms a first ring structure having from 4 to 7 members;

and/or

a second bridging group which when taken together with the C-2-bonded-nitrogen and the C-1 and C-2 carbons forms a second ring structure having from 4 to 7 members.

3. The compound of claim 2 wherein one or both of the first and second ring structures has five members.

4. The compound of claim 3 wherein the five membered ring structure includes one gem disubstituted carbon.

5. The compound of claim 4 wherein the gem substituents are lower alkyl.

6. The compound of claim 5 wherein the gem substituents are methyl.

7. The compound of claim 3 wherein the five membered ring is not aromatic.

8. The compound of claim 3 wherein the five membered ring is substituted with linking group or water-solubilizing group.

9. The compound of claim 2 wherein the first and second ring structures are the same.

10. The compound of claim 2 wherein the first and second ring structures are different.

11. The compound of claim 1 comprising a C-7 substituent selected from the group consisting of acetylene, lower alkyl, lower alkene, cyano, phenyl, heterocyclic aromatic, electron-rich heterocycle, and substituted forms thereof.

12. The compound of claim 11 wherein the C-7 substituent is phenyl having the structure

13. The compound of claim 12 wherein substituents at any one of positions C-14 to C-18 are selected from the group consisting of chlorine, fluorine, lower alkyl, carboxylic acid, sulfonic acid, —

CH₂OH, alkoxy, phenoxy, linking group, water solubilizing group, and substituted forms thereof.

14. The compound of claim 11 wherein a substituent at position C-18 is selected from the group consisting of carboxylic acid and sulfonate.

15. The compound of claim 13 wherein a substituent at position C-18 is carboxylic acid.

16. The compound of claim 12 wherein a substituent at positions C-14 and C-17 is chlorine.

17. The compound of claim 12 wherein a substituent at positions C-14 to C-17 is chlorine.

18. The compound of claim 12 wherein a substituent at positions C-14 to C-17 is fluorine.

19. The compound of claim 12 wherein a substituent at one of positions C-15 and C-16 is linking group and the other is hydrogen, a substituent at positions C-14 and C-17 is chlorine, and a substituent at position C-18 is carboxy.

20. The compound of claim 1 comprising one or more nitrogen substituents selected from the group consisting of lower alkyl, lower alkene, lower alkyne, phenyl, aromatic, electron-rich heterocycle, polycyclic aromatic, water-solubilizing group, linking group, including substituted forms thereof.

21. The compound of claim 20 wherein the nitrogen substituents are selected from the group consisting of lower alkyl, phenyl, and substituted forms thereof.

22. The compound of claim 21 wherein the nitrogen substituents are selected from the group consisting of substituted lower alkyl and substituted phenyl, wherein the substituent is linking group.

23. The compound of claim 21 wherein the nitrogen substituents are selected from the group consisting of substituted lower alkyl and substituted phenyl, wherein the substituent is sulfonate.

24. The compound of claim 20 wherein the nitrogen substituents are water-solubilizing group.

25. The compound of claim 24 wherein the water-solubilizing group is selected from the group consisting of sulfonate, phosphate, quaternary amine, sulfate, polyhydroxyl, and water-soluble polymer.

26. The compound of claim 1 comprising a third bridging group which when taken together with the C-12-bonded nitrogen and the C-11 and C-12 carbons forms a third ring structure having from 5 to 7 members;

and/or

a fourth bridging group which when taken together with the C-2-bonded nitrogen and the C-2 and C-3 carbons forms a fourth ring structure having from 5 to 7 members.

27. The compound of claim 26 wherein one or both of the third and fourth ring structures has six members.

28. The compound of claim 27 wherein the six membered ring structure includes one gem disubstituted carbon.

29. The compound of claim 28 wherein the gem substituents are lower alkyl.

30. The compound of claim 29 wherein the gem substituents are methyl.

31. The compound of claim 27 wherein the six membered ring is not aromatic.

32. The compound of claim 1 comprising one or more substituents at one or more of positions C-1, C-3 to C-6, C-8 to C-11, and C-13, selected from the group consisting of fluorine, chlorine, lower alkyl, lower alkene, lower alkyne, sulfate, sulfonate, sulfone, sulfonamide, sulfoxide, amino, ammonium, amido, nitrile, lower alkoxy, phenoxy, aromatic, phenyl, polycyclic aromatic, electron-rich heterocycle, water-solubilizing group, and linking group, including substituted forms thereof.

33. The compound of claim 32 wherein the one or more substituents are sulfonate.

34. The compound of claim 1 having the structure

35. The compound of claim 1 having the structure

36. The compound of claim 1 having the structure

37. The compound of claim 1 having the structure

38. The compound of claim 1 having the structure

39. The compound of claim 1 having the structure

40. The compound of claim 1 having the structure

95. An energy transfer dye comprising:

a donor dye capable of absorbing light at a first wavelength and emitting excitation energy in response;

an acceptor dye capable of absorbing the excitation energy emitted by the donor dye and fluorescing at a second wavelength in response; and

a linker for linking the donor dye and the acceptor dye, the linker serving to facilitate the efficient transfer of energy between the donor dye and the acceptor dye;

wherein at least one of the donor dye and acceptor dye is a dibenzorhodamine compound of claim 1.

96. The energy transfer dye of claim 95 wherein the first wavelength is greater than about 600 nm.

97. The energy transfer dye of claim 95 wherein the linker has the structure

98. The energy transfer dye of claim 97 wherein R₂₂ is a five or six membered ring selected from the group consisting of cyclopentene, cyclohexene, cyclopentadiene, cyclohexadiene, furan, thiofuran, pyrrole, isopyrole, isoazole, pyrazole, isoimidazole, pyran, pyrone, benzene, pyridine, pyridazine, pyrimidine, pyrazine oxazine, indene, benzofuran, thionaphthene, indole and naphthalene.

99. The energy transfer dye of claim 97 wherein the linker has the structure

100. The energy transfer dye of claim 95 wherein the linker has the structure

101. A labeled nucleoside/tide having the formula:

NUC—

D

wherein

NUC is a nucleoside/tide or nucleoside/tide analog;

D is a dibenzorhodamine dye compound of claim 1, NUC and D being connected by a linkage;

wherein if NUC comprises a purine base, the linkage is attached to the 8-position of the purine, if NUC comprises a 7-deazapurine base, the linkage is attached to the 7-position of the 7-deazapurine, and if NUC comprises a pyrimidine base, the linkage is attached to the 5-position of the pyrimidine.

102. The labeled nucleoside/tide of claim 101 wherein NUC comprises a base selected from the group consisting of uracil, cytosine, deazaadenine, and deazaguanosine.

103. The labeled nucleoside/tide of claim 101 wherein the linkage is

104. A method of polynucleotide sequencing comprising the steps of:

forming a mixture of a first, a second, a third, and a forth class of polynucleotides such that;

each polynucleotide in the first class includes a 3′

-terminal dideoxyadenosine and is labeled with a first dye;

each polynucleotide in the second class includes a 3′

-terminal dideoxycytidine and is labeled with a second dye;

each polynucleotide in the third class includes a 3′

-terminal dideoxyguanosine and is labeled with a third dye; and

each polynucleotide in the forth class includes a 3′

-terminal dideoxythymidine and is labeled with a forth dye;

wherein at least one of the first, second, third, or forth dyes is a dibenzorhodamine compound of claim 1;

the other of the dyes being spectrally resolvable from the dibenzorhodamine dye(s) and from each other;

electrophoretically separating the polynucleotides thereby forming bands of similarly sized polynucleotides;

illuminating the bands with an illumination beam capable of causing the dyes to fluoresce; and

identifying the classes of the polynucleotides in the bands by the fluorescence spectrum of the dyes.

105. A method of fragment analysis comprising:

forming labeled polynucleotide fragments, the fragments being labeled with a dibenzorhodamine compound of claim 1;

subjecting the labeled polynucleotide fragments to a size-dependent separation process; and

detecting the labeled polynucleotide fragments subsequent to the separation process.

106. The method of claim 105 wherein the size-dependent separation process is electrophoresis and the labeled polynucleotide fragments are detected by fluorescence.

41. An intermediate useful for the synthesis of dibenzorhodamine compounds having the structure

42. The compound of claim 41 wherein substituents at any one of positions C-14 to C-18 are selected from the group consisting of chlorine, fluorine, lower alkyl, carboxylic acid, sulfonic acid, —

CH₂OH, alkoxy, phenoxy, linking group, water solubilizing group, and substituted forms thereof.

43. The compound of claim 42 wherein a substituent at position C-18 is selected from the group consisting of carboxylic acid and sulfonate.

44. The compound of claim 43 wherein a substituent at position C-18 is carboxylic acid.

45. The compound of claim 42 wherein a substituent at positions C-14 and C-17 is chlorine.

46. The compound of claim 42 wherein a substituent positions C-14 to C-17 is chlorine.

47. The compound of claim 42 wherein a substituent at positions C-14 to C-17 is fluorine.

48. The compound of claim 42 wherein a substituent at one of positions C-15 and C-16 is linking group and the other is hydrogen, a substituent at positions C-14 and C-17 is chlorine, and a substituent at position C-18 is carboxy.

49. The compound of claim 41 comprising a first bridging group which when taken together with the C-12-bonded nitrogen and the C-12 and C-13 carbons forms a first ring structure having from 4 to 7 members.

50. The compound of claim 49 wherein the ring structure has five members.

51. The compound of claim 50 wherein the five membered ring structure includes one gem disubstituted carbon.

52. The compound of claim 51 wherein the gem substituents are lower alkyl.

53. The compound of claim 52 wherein the gem substituents are methyl.

54. The compound of claim 50 wherein the five membered ring is not aromatic.

55. The compound of claim 50 wherein the five membered ring is substituted with linking group.

56. The compound of claim 41 comprising one or more nitrogen substituents selected from the group consisting of lower alkyl, lower alkene, lower alkyne, phenyl, aromatic, electron-rich heterocycle, polycyclic aromatic, water-solubilizing group, linking group, and substituted forms thereof.

57. The compound of claim 56 wherein the nitrogen substituents are selected from the group consisting of lower alkyl, phenyl, and substituted forms thereof.

58. The compound of claim 57 wherein the nitrogen substituents are selected from the group consisting of substituted lower alkyl and substituted phenyl, wherein the substituent is linking group.

59. The compound of claim 57 wherein the nitrogen substituents are selected from the group consisting of substituted lower alkyl and substituted phenyl, wherein the substituent is sulfonate.

60. The compound of claim 56 wherein the nitrogen substituents are water-solubilizing group.

61. The compound of claim 60 wherein the water-solubilizing group is selected from the group consisting of sulfonate, phosphate, quaternary amine, sulfate, polyhydroxyl, and water-soluble polymer.

62. The compound of claim 41 comprising a bridging group which when taken together with the C-12-bonded nitrogen and the C-11 and C-12 carbons forms a ring structure having from 5 to 7 members.

63. The compound of claim 62 wherein the ring structure has six members.

64. The compound of claim 63 wherein the six membered ring structure includes one gem disubstituted carbon.

65. The compound of claim 64 wherein the gem substituents are lower alkyl.

66. The compound of claim 65 wherein the gem substituents are methyl.

67. The compound of claim 63 wherein the six membered ring is not aromatic.

68. The compound of claim 41 comprising one or more substituents at one or more of positions C-8 to C-11, and C-13, selected from the group consisting of fluorine, chlorine, lower alkyl, lower alkene, lower alkyne, sulfate, sulfonate, sulfone, sulfonamide, sulfoxide, amino, ammonium, amido, nitrile, lower alkoxy, phenoxy, aromatic, phenyl, polycyclic aromatic, water-solubilizing group, electron-rich heterocycle, and linking group, including substituted forms thereof.

69. The compound of claim 68 wherein the one or more substituents are sulfonate.

70. An intermediate useful for the synthesis of dibenzorhodamine compounds having the structure

71. The compound of claim 70 wherein the first ring structure has five members.

72. The compound of claim 71 wherein the five membered ring structure includes one gem disubstituted carbon.

73. The compound of claim 72 wherein the gem substituents are lower alkyl.

74. The compound of claim 73 wherein the gem substituents are methyl.

75. The compound of claim 71 wherein the five membered ring is not aromatic.

76. The compound of claim 71 wherein the five membered ring is substituted with linking group or water-solubilizing groups.

77. The compound of claim 70 wherein the second ring structure has six members.

78. The compound of claim 77 wherein the six membered ring structure includes one gem disubstituted carbon.

79. The compound of claim 78 wherein the gem substituents are lower alkyl.

80. The compound of claim 79 wherein the gem substituents are methyl.

81. The compound of claim 77 wherein the six membered ring is not aromatic.

82. The compound of claim 70 comprising one or more nitrogen substituents selected from the group consisting of lower alkyl, lower alkene, lower alkyne, phenyl, aromatic, electron-rich heterocycle, polycyclic aromatic, water-solubilizing group, and linking group, including substituted forms thereof.

83. The compound of claim 82 wherein the nitrogen substituents are selected from the group consisting of lower alkyl, phenyl, and substituted forms thereof.

84. The compound of claim 83 wherein the nitrogen substituents are selected from the group consisting of substituted lower alkyl and substituted phenyl, wherein the substituent is linking group.

85. The compound of claim 83 wherein the nitrogen substituents are selected from the group consisting of substituted lower alkyl and substituted phenyl, wherein the substituent is sulfonate.

86. The compound of claim 82 wherein the nitrogen substituents are water-solubilizing group.

87. The compound of claim 86 wherein the water-solubilizing group is selected from the group consisting of sulfate, phosphate, quaternary amine, sulfonate, polyhydroxyl, and water-soluble polymer.

88. The compound of claim 70 comprising one or more substituents at one or more of positions C-8 to C-11, and C-13, selected from the group consisting of fluorine, chlorine, lower alkyl, lower alkene, lower alkyne, sulfate, sulfonate, sulfone, sulfonamide, sulfoxide, amino, ammonium, amido, nitrile, lower alkoxy, phenoxy, aromatic, phenyl, polycyclic aromatic, water-solubilizing group, and linking group, including substituted forms thereof.

89. The compound of claim 88 wherein the one or more substituents are sulfonate.

90. The compound of claim 70 having the structure

91. The compound of claim 70 having the structure

92. The compound of claim 70 having the structure

93. The compound of claim 70 having the structure

94. The compound of claim 93 wherein R is selected from the group consisting of methyl or ethyl.