Electron-deficient nitrogen heterocycle-substituted fluorescein dyes

1. A compound having the formula:

wherein:

at least one of R 1 , R 2 , R 3 , R 4 , R 5 , or R 7 is an electron-deficient nitrogen heterocycle;

R 1 , when taken alone, is H, F, Cl, (C 1 -C 6 ) alkyl, (C 1 -C 6 ) substituted alkyl, (C 1 -C 6 ) alkoxy, sulfonate, sulfone, amino, imminium, amido, nitrile, reactive linking group, phenyl, substituted phenyl, aryl, substituted aryl, or heterocycle, or when taken together with R 7 is benzo or heterocycle;

R 2 , when taken alone, is H, F, Cl, (C 1 -C 6 ) alkyl, (C 1 -C 6 ) substituted alkyl, (C 1 -C 6 ) alkoxy, sulfonate, sulfone, amino, imminium, amido, nitrile, reactive linking group, phenyl, substituted phenyl, aryl, substituted aryl, or heterocycle;

R 3 , when taken alone, is H, F, Cl, (C 1 -C 6 ) alkyl, (C 1 -C 6 ) substituted alkyl, (C 1 -C 6 ) alkoxy, sulfonate, sulfone, amino, imminium, amido, nitrile, reactive linking group, phenyl, substituted phenyl, aryl, substituted aryl, or heterocycle;

R 4 , when taken alone, is H, F, Cl, (C 1 -C 6 ) alkyl, (C 1 -C 6 ) substituted alkyl, (C 1 -C 6 ) alkoxy, sulfonate, sulfone, amino, imminium, amido, nitrile, reactive linking group, phenyl, substituted phenyl, aryl, substituted aryl, or heterocycle, or when taken together with R 5 is benzo or heterocycle;

R 5 , when taken alone, is H, F, Cl, (C 1 -C 6 ) alkyl, (C 1 -C 6 ) substituted alkyl, (C 1 -C 6 ) alkoxy, sulfonate, sulfone, amino, imminium, amido, nitrile, reactive linking group, phenyl, substituted phenyl, aryl, substituted aryl, or heterocycle, or when taken together with R 4 is benzo or heterocycle;

R 7 , when taken alone, is H, F, Cl, (C 1 -C 6 ) alkyl, (C 1 -C 6 ) substituted alkyl, (C 1 -C 6 ) alkoxy, sulfonate, sulfone, amino, imminium, amido, nitrile, reactive linking group, phenyl, substituted phenyl, aryl, substituted aryl, or heterocycle, or when taken together with R 1 is benzo or heterocycle; and

R 6 is selected from the group consisting of (C 1 -C 6 ) alkyl, (C 2 -C 6 ) alkene, (C 2 -C 6 ) alkyne, cyano, heterocyclic aromatic, phenyl, and substituted phenyl having the structure:

wherein X 1 , X 2 , X 3 , X 4 and X 5 taken separately are H, Cl, F, (C 1 -C 6 ) alkyl, (C 2 -C 6 ) alkene, (C 2 -C 6 ) alkyne, CO 2 H, SO 3 H, CH 2 OH, or reactive linking group.

2. The fluorescein dye of claim 1 wherein the electron-deficient heterocycle is selected from the group consisting of 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-quinolyl, 3-quinolyl, 4-quinolyl, 2-imidazole, 4-imidazole, 3-pyrazole, 4-pyrazole, pyridazine, pyrimidine, pyrazine, cinnoline, pthalazine, quinazoline, quinoxaline, 3-(1,2,4-N)-triazolyl, 5-(1,2,4-N)-triazolyl, 5-tetrazolyl, 4-(1-O, 3-N)-oxazole, 5-(1-O, 3-N)-oxazole, 4-(1-S, 3-N)-thiazole, 5-(1-S, 3-N)-thiazole, 2-benzoxazole, 2-benzothiazole, 4-(1,2,3-N)-benzotriazole, and benzimidazole.

3. The fluorescein dye of claim 1 wherein R 4 taken together with R 5 is benzo.

4. The fluorescein dye of claim 1 wherein R 1 taken together with R 7 is benzo.

5. The fluorescein dye of claim 1 in which R 6 is a substituted phenyl having the structure:

6. The fluorescein dye of claim 5 wherein one of X 3 and X 4 is carboxyl and the other is hydrogen.

7. The fluorescein dye of claim 1 wherein R 1 , R 2 , R 3 and R 4 each taken separately are phenyl or substituted phenyl.

8. The fluorescein dye of claim 1 wherein R 1 , R 2 , R 3 and R 4 each taken separately are naphthyl or substituted naphthyl.

9. The fluorescein dye of claim 1 wherein R 2 and R 3 each taken separately are fluoro or chloro.

10. The fluorescein dye of claim 1 wherein R 2 and R 3 each taken separately are 2-pyridyl or 3-pyridyl.

11. The fluorescein dye of claim 1 wherein R 2 and R 3 each taken separately are 2-quinolyl or 3-quinolyl.

12. The fluorescein dye of claim 1 wherein R 5 and R 7 are hydrogen.

13. The fluorescein dye of claim 1 in which the reactive linking group is selected from the group consisting of succinimidyl ester, isothiocyanate, sulfonyl chloride, 2,6-dichlorotriazinyl, pentafluorophenyl ester, phosphoramidite, maleimide, haloacetyl, and iodoacetamide.

14. Fluorescein dyes having the structures:

15. A method of labelling a substrate with a fluorescein dye of claim 1 , comprising the step of reacting the substrate with the reactive linking group of the fluorescein dye wherein a substrate-dye conjugate is formed.

16. The method of claim 15 wherein the reactive linking group is N-hydroxysuccinimide.

17. The method of claim 15 wherein the reactive linking group is phosphoramidite.

18. The method of claim 15 wherein the substrate is selected from the group consisting of a polynucleotide, a nucleotide, a nucleoside, a peptide, a protein, a carbohydrate, a ligand, a particle, and a surface.

19. The method of claim 18 wherein the particle is a nanoparticle, a microsphere, a bead, and a liposome.

20. The method of claim 18 wherein the surface is glass.

21. An energy transfer dye compound comprising:

a donor dye capable of absorbing light at a first wavelength and emitting excitation energy in response linked by a linker to an acceptor dye capable of absorbing the excitation energy emitted by the donor dye and fluorescing at a second wavelength in response;

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

22. The energy transfer dye of claim 21 wherein the linker has the structure:

23. The energy transfer dye of claim 21 wherein the linker has the structure:

24. The energy transfer dye of claim 21 in which the linker has the structure:

wherein n is 1 or 2.

25. A labelled nucleoside or nucleotide having the formula:

wherein DYE is a fluorescein dye of claim 1 or an energy transfer dye of claim 23 ;

B is a nucleobase;

R 8 is H, monophosphate, diphosphate, triphosphate, thiophosphate, or phosphate analog;

R 9 and R 10 , when taken alone, are each independently H, HO, F, and a moiety which blocks polymerase-mediated target-directed polymerization, or when taken together form 2′-3′-didehydroribose; and

L is a linker.

26. The labelled nucleoside or nucleotide of claim 25 wherein B is selected from the group consisting of uracil, thymine, cytosine, adenine, 7-deazaadenine, guanine, and 8-deazaguanosine.

27. The labelled nucleoside or nucleotide of claim 25 in which L is:

wherein n is 0, 1, or 2.

28. The labelled nucleoside or nucleotide of claim 25 which is enzymatically incorporatable.

29. The labelled nucleoside or nucleotide of claim 25 which is a terminator.

30. The terminator nucleotide of claim 29 which has the structure:

wherein DYE is a fluorescein dye;

B is a nucleobase;

R 8 is triphosphate, α-thiotriphosphate, or triphosphate analog;

R 9 and R 10 , when taken alone, are each independently H, F, and a moiety which blocks polymerase-mediated target-directed polymerization, or when taken together form 2′-3′-didehydroribose; and

L is a linker.

31. The labelled nucleoside or nucleotide of claim 25 which is enzymatically extendable.

32. A labelled oligonucleotide having the formula:

wherein the oligonucleotide comprises 2 to 100 nucleotides;

DYE is a fluorescein dye of claim 1 or an energy transfer dye of claim 21 ;

B is a nucleobase;

L is a linker;

R 10 is H, OH, halide, azide, amine, alkylamine, alkyl (C 1 -C 6 ), allyl, alkoxy (C 1 -C 6 ), OCH 3 , or OCH 2 CH═CH 2 ;

R 15 is H, phosphate, internucleotide phosphodiester, or internucleotide analog; and

R 16 is H, phosphate, internucleotide phosphodiester, or internucleotide analog.

33. A labelled oligonucleotide having the formula:

wherein the oligonucleotide comprises 2 to 100 nucleotides;

DYE is a fluorescein dye of claim 1 ;

X is O, NH, or S;

B is a nucleobase;

L is a linker;

R 10 is H, OH, halide, azide, amine, alkylamine, alkyl (C 1 -C 6 ), allyl, alkoxy (C 1 -C 6 ), OCH 3 , or OCH 2 CH═CH 2 ; and

R 15 is internucleotide phosphodiester or internucleotide analog.

34. The labelled oligonucleotide of claim 33 in which L is alkyldiyl (C 1 -C 12 ).

35. The labelled oligonucleotide of claim 33 in which L is a mobility-modifier comprising —(CH 2 CH 2 O) n —, where n is 1 to 100.

36. A phosphoramidite compound having the formula:

wherein DYE is a fluorescein dye of claim 1 or an energy transfer dye of claim 21 ;

L is a linker;

R 11 and R 12 taken separately are selected from the group consisting of alkyl (C 1 -C 12 ), alkene, aryl, and cycloalkyl containing up to 10 carbon atoms; or R 11 and R 12 taken together with the nitrogen atom form a saturated nitrogen heterocycle; and

R 13 is a phosphite ester protecting group.

37. The phosphoramidite compound of claim 36 wherein R 13 is selected from the group consisting of methyl, 2-cyanoethyl, and 2-(4-nitrophenyl)ethyl.

38. The phosphoramidite compound of claim 36 wherein R 11 and R 12 are each isopropyl.

39. The phosphoramidite compound of claim 36 wherein R 11 and R 12 taken together is morpholino.

40. The phosphoramidite compound of claim 36 wherein L is alkyldiyl (C 1 -C 12 ).

41. The phosphoramidite compound of claim 36 wherein the fluorescein dye is attached at R 6 by a linker.

42. The phosphoramidite compound of the structure:

wherein DYE is a fluorescein dye of claim 1 .

43. A phosphoramidite compound having the formula

wherein DYE is a fluorescein dye of claim 1 or an energy transfer dye of claim 21 ;

B is a nucleobase;

L is a linker;

R 11 and R 12 taken separately are selected from the group consisting of alkyl (C 1 -C 6 ), alkene, aryl, and cycloalkyl containing up to 10 carbon atoms; or R 11 and R 12 taken together with the nitrogen atom form a saturated nitrogen heterocycle;

R 13 is a phosphite ester protecting group; and

R 14 is an acid-cleavable hydroxyl protecting group.

44. The phosphoramidite compound of claim 43 wherein R 13 is selected from the group consisting of methyl, 2-cyanoethyl, and 2-(4-nitrophenyl)ethyl.

45. The phosphoramidite compound of claim 43 wherein R 11 and R 12 are each isopropyl.

46. The phosphoramidite compound of claim 43 wherein R 11 and R 12 taken together is morpholino.

47. The compound of claim 43 wherein L is:

and n ranges from 2 to 10.

48. The compound of claim 43 wherein L is:

and n is0, 1, or 2.

49. The compound of claim 43 wherein L is

and n ranges from 1 to 10.

50. The compound of claim 43 wherein B is selected from the group consisting of uracil, thymine, cytosine, adenine, 7-deazaadenine, guanine, and 8-deazaguanosine.

51. A method of synthesizing a labelled oligonucleotide comprising the step of coupling a phosphoramidite compound of claim 36 to an oligonucleotide on a solid support.

52. A method of synthesizing a labelled oligonucleotide comprising the step of coupling a nucleoside phosphoramidite reagent to a solid support wherein the solid support is labelled with a dye according to claim 1 or an energy transfer compound of claim 21 .

53. A method of generating a labelled primer extension product, comprising the step of enzymatically extending a primer-target hybrid in the presence of a mixture of enzymatically-extendable nucleotides capable of supporting continuous primer extension and a terminator, wherein said primer or said terminator is labelled with a dye according to claim 1 or an energy transfer compound of claim 21 .

54. A method of oligonucleotide ligation, comprising the steps of:

annealing two probes to a target sequence and

forming a phosphodiester bond between the 5′ terminus of one probe and the 3′ terminus of the other probe;

wherein one or both probes are labelled with a dye according to claim 1 or an energy transfer compound of claim 21 .

55. A method of fragment analysis comprising the steps of:

subjecting polynucleotide fragments, the fragments being labelled with a fluorescein dye of claim 1 or an energy transfer compound of claim 21 , to a size-dependent separation process; and

detecting the labelled polynucleotide fragment subsequent to the separation process.

56. The method of claim 55 wherein the fragments are labelled with a mobility-modifying label.

57. The method of claim 55 wherein the fragments are formed by ligation.

58. The method of claim 55 wherein the size-dependent separation process of electrophoresis and the labelled polynucleotide fragment is detected by fluorescence.

59. A method of amplification comprising the steps of:

annealing two or more primers to a target DNA sequence and

extending the primers by polymerase and a mixture of enzymatically-extendable nucleotides;

wherein a primer or a nucleotide is labelled with a dye according to claim 1 .

60. A method of amplification comprising the steps of:

annealing two or more primers and a fluorescent dye-quencher probe to a target DNA sequence and

extending the primers by polymerase and a mixture of enzymatically-extendable nucleotides;

wherein the probe is labelled with a dye according to claim 1 .

61. A kit for labelling an oligonucleotide, comprising a dye including a reactive linking group according to claim 1 and an oligonucleotide.

62. A kit for labelling an oligonucleotide, comprising a phosphoramidite compound according to claim 36 and an oligonucleotide.

63. A kit for generating a labelled primer extension product, comprising enzymatically-extendable nucleotides capable of supporting continuous primer extension, a terminator and a primer, wherein said primer or said terminator is labelled with a dye according to claim 1 .

64. A kit for generating a labelled primer extension product, comprising enzymatically-extendable nucleotides capable of supporting continuous primer extension, a terminator and a primer, wherein said primer or said terminator is labelled with an energy transfer dye of claim 21 .

65. A kit for generating a labelled primer extension product, comprising enzymatically-extendable nucleotides capable of supporting continuous primer extension, a terminator and a primer, wherein said primer or said terminator is labelled with a dye according to claim 14 .

66. The kit of claim 65 in which the terminator is a set of four different terminators, one which terminates at a target A, one which terminates at a target G, one which terminates at a target C and one which terminates at a target T or U.

67. The kit of claim 66 in which the set of four different terminators is a set of mobility-matched terminators.

68. A compound having a formula:

wherein

at least one of R 1 , R 2 , R 3 , R 4 , R 5 , or R 7 is an electron - deficient nitrogen heterocycle;

R 1 is H, F, Cl, ( C 1 -C 6 ) alkyl, ( C 1 -C 6 ) substituted alkyl, ( C 1 -C 6 ) alkoxy, sulfonate, sulfone, amino, imminium, amido, nitrile, reactive linking group, phenyl, substituted phenyl, aryl, substituted aryl, or heterocycle, or when taken together with R 7 is benzo or heterocycle;

R 2 is H, F, Cl, ( C 1 -C 6 ) alkyl, ( C 1 -C 6 ) substituted alkyl, ( C 1 -C 6 ) alkoxy, sulfonate, sulfone, amino, imminium, amido, nitrile, reactive linking group, phenyl, substituted phenyl, aryl, substituted aryl, or heterocycle;

R 3 is H, F, Cl, ( C 1 -C 6 ) alkyl, ( C 1 -C 6 ) substituted alkyl, ( C 1 -C 6 ) alkoxy, sulfonate, sulfone, amino, imminium, amido, nitrile, reactive linking group, phenyl, substituted phenyl, aryl, substituted aryl, or heterocycle;

R 4 is H, F, Cl, ( C 1 -C 6 ) alkyl, ( C 1 -C 6 ) substituted alkyl, ( C 1 -C 6 ) alkoxy, sulfonate, sulfone, amino, imminium, amido, nitrile, reactive linking group, phenyl, substituted phenyl, aryl, substituted aryl, or heterocycle, or when taken together with R 5 is benzo or heterocycle;

R 5 is H, F, Cl, ( C 1 -C 6 ) alkyl, ( C 1 -C 6 ) substituted alkyl, ( C 1 -C 6 ) alkoxy, sulfonate, sulfone, amino, imminium, amido, nitrile, reactive linking group, phenyl, substituted phenyl, aryl, substituted aryl, or heterocycle, or when taken together with R 4 is benzo or heterocycle;

R 7 is H, F, Cl, ( C 1 -C 6 ) alkyl, ( C 1 -C 6 ) substituted alkyl, ( C 1 -C 6 ) alkoxy, sulfonate, sulfone, amino, imminium, amido, nitrile, reactive linking group, phenyl, substituted phenyl, aryl, substituted aryl, or heterocycle, or when taken together with R 5 is benzo or heterocycle; and

X 1 , X 2 , X 3 , X 4 and X 5 are individually selected from the group consisting of H, Cl, F, ( C 1 -C 6 ) alkyl, ( C 2 -C 6 ) alkene, ( C 2 -C 6 ) alkyne, CO 2 H, SO 3 H, CH 2 OH, and reactive linking group.

69. The compound of claim 68 wherein at least one of X 1 , X 2 , X 3 , X 4 and X 5 is SO 3 H.

70. The compound claim 69 wherein the electron- deficient nitrogen heterocycle is selected from the group consisting of 2 - pyridyl, 3 - pyridyl, 4 - pyridyl, 2 - quinolyl, 3 - quinolyl, 4 - quinolyl, 2 - imidazole, 4 - imidazole, 3 - pyrazole, 4 - pyrazole, pyridazine, pyrimidine, pyrazine, cinnoline, pthalazine, quinazoline, quinoxaline, 3 -( 1 , 2 , 4 - N )- triazolyl, 5 -( 1 , 2 , 4 - N )- triazolyl, 5 - tetrazolyl, 4 -( 1 - O, 3 - N )- oxazole, 5 -(1- O, 3 - N )- oxazole, 4 -( 1 - S, 3 - N )- thiazole, 5 -( 1 - S, 3 - N )- thiazole, 2 - benzoxazole, 2 - benzothiazole, 4 -( 1 , 2 , 3 - N )- benzotriazole, and benzimidazole.

71. The compound of claim 69 wherein the electron- deficient nitrogen heterocycle is selected from the group consisting of 2 - pyridyl, 3 - pyridyl, 4 - pyridyl, 2 - quinolyl, 3 - quinolyl, and 4 - quinolyl.

72. The compound of claim 69 wherein each of R 1 and R 4 is an electron - deficient nitrogen heterocycle selected from the group consisting of 2 - pyridyl and 3 - pyridyl.

73. The compound of claim 69 wherein each of R 1 and R 4 is 2 - pyridyl.

74. The compound of claim 69 wherein each of R 1 and R 4 is 3 - pyridyl.

75. The compound of claim 69 wherein at least one of R 5 and R 7 is H.

76. The compound of claim 69 wherein:

each of R 1 and R 4 is an electron - deficient nitrogen heterocycle;

each of R 2 and R 3 is H; and

each of R 5 and R 7 is H.

77. An energy transfer dye compound comprising:

a donor dye capable of absorbing light at a first wavelength and emitting excitation energy in response linked by a linker to an acceptor dye capable of absorbing the excitation energy emitted by the donor dye and fluorescing at a second wavelength in response;

wherein at least one of the donor dye and acceptor dye is a compound of claim 69 .

78. The energy transfer dye compound of claim 77 wherein the electron- deficient nitrogen heterocycle is selected from the group consisting of 2 - pyridyl, 3 - pyridyl, 4 - pyridyl, 2 - quinolyl, 3 - quinolyl, 4 - quinolyl, 2 - imidazole, 4 - imidazole, 3 - pyrazole, 4 - pyrazole, pyridazine, pyrimidine, pyrazine, cinnoline, pthalazine, quinazoline, quinoxaline, 3 -( 1 , 2 , 4 - N )- triazolyl, 5 -( 1 , 2 , 4 - N )- triazolyl, 5 - tetrazolyl, 4 -( 1 - O, 3 - N )- oxazole, 5 -(1- O, 3 - N )- oxazole, 4 -( 1 - S, 3 - N )- thiazole, 5 -( 1 - S, 3 - N )- thiazole, 2 - benzoxazole, 2 - benzothiazole, 4 -( 1 , 2 , 3 - N )- benzotriazole, and benzimidazole.

79. The energy transfer dye compound of claim 77 wherein the electron- deficient nitrogen heterocycle is selected from the group consisting of 2 - pyridyl, 3 - pyridyl, 4 - pyridyl, 2 - quinolyl, 3 - quinolyl, and 4 - quinolyl.

80. The energy transfer dye compound of claim 77 wherein each of R 1 and R 4 is an electron - deficient nitrogen heterocycle selected from the group consisting of 2 - pyridyl and 3 - pyridyl.

81. The energy transfer dye compound of claim 77 wherein each of R 1 and R 4 is 2 - pyridyl.

82. The energy transfer dye compound of claim 77 wherein each of R 1 and R 4 is 3 - pyridyl.

83. The energy transfer dye compound of claim 77 wherein at least one of R 5 and R 7 is H.

84. The energy transfer dye compound of claim 77 wherein:

each of R 1 and R 4 is an electron - deficient nitrogen heterocycle;

each of R 2 and R 3 is H; and

each of R 5 and R 7 is H.

85. A labeled nucleoside or nucleotide having a formula:

wherein DYE is a compound of claim 69 or an energy transfer dye compound of claim 77 in which the linker has a structure:

wherein

B is a nucleobase;

R 8 is H, monophosphate, diphosphate, triphosphate, thiophosphate, or phosphate analog;

R 9 and R 10 , when taken alone, are each independently H, HO, F, or a moiety which blocks polymerase - mediated target - directed polymerization, or when taken together, form 2 ′ - 3 ′ - didehydroribose; and

L is a linker.

86. The labeled nucleoside or nucleotide of claim 85 wherein the electron- deficient nitrogen heterocycle is selected from the group consisting of 2 - pyridyl, 3 - pyridyl, 4 - pyridyl, 2 - quinolyl, 3 - quinolyl, 4 - quinolyl, 2 - imidazole, 4 - imidazole, 3 - pyrazole, 4 - pyrazole, pyridazine, pyrimidine, pyrazine, cinnoline, pthalazine, quinazoline, quinoxaline, 3 -( 1 , 2 , 4 - N )- triazolyl, 5 -( 1 , 2 , 4 - N )- triazolyl, 5 - tetrazolyl, 4 -( 1 - O, 3 - N )- oxazole, 5 -( 1 - O, 3 - N )- oxazole, 4 -( 1 - S, 3 - N )- thiazole, 5 -( 1 - S, 3 - N )- thiazole, 2 - benzoxazole, 2 - benzothiazole, 4 -( 1 , 2 , 3 - N )- benzotriazole, and benzimidazole.

87. The labeled nucleoside or nucleotide of claim 85 wherein the electron- deficient nitrogen heterocycle is selected from the group consisting of 2 - pyridyl, 3 - pyridyl, 4 - pyridyl, 2 - quinolyl, 3 - quinolyl, and 4 - quinolyl.

88. The labeled nucleoside or nucleotide of claim 85 wherein each of R 1 and R 4 is an electron - deficient nitrogen heterocycle selected from the group consisting of 2 - pyridyl and 3 - pyridyl.

89. The labeled nucleoside or nucleotide of claim 85 wherein each of R 1 and R 4 is 2 - pyridyl.

90. The labeled nucleoside or nucleotide of claim 85 wherein each of R 1 and R 4 is 3 - pyridyl.

91. The labeled nucleoside or nucleotide of claim 85 wherein at least one of R 5 and R 7 is H.

92. The labeled nucleoside or nucleotide of claim 85 wherein:

each of R 1 and R 4 is an electron - deficient nitrogen heterocycle;

each of R 2 and R 3 is H; and

each of R 5 and R 7 is H.

93. A labelled oligonucleotide having a formula:

wherein the oligonucleotide comprises 2 to 100 nucleotides;

DYE is a compound of claim 69 or an energy transfer dye compound of claim 77 ;

B is a nucleobase;

L is a linker;

R 10 is H, OH, halide, azide, amine, alkylamine, alkyl ( C 1 -C 6 ), allyl, alkoxy ( C 1 -C 6 ), OCH 3 , or OCH 2 CH═CH 2 ;

R 15 is H, phosphate, internucleotide phosphodiester, or internucleotide analog; and

R 16 is H, phosphate, internucleotide phosphodiester, or internucleotide analog.

94. The labelled oligonucleotide of claim 93 wherein the electron- deficient nitrogen heterocycle is selected from the group consisting of 2 - pyridyl, 3 - pyridyl, 4 - pyridyl, 2 - quinolyl, 3 - quinolyl, 4 - quinolyl, 2 - imidazole, 4 - imidazole, 3 - pyrazole, 4 - pyrazole, pyridazine, pyrimidine, pyrazine, cinnoline, pthalazine, quinazoline, quinoxaline, 3 -( 1 , 2 , 4 - N )- triazolyl, 5 -( 1 , 2 , 4 - N )- triazolyl, 5 - tetrazolyl, 4 -( 1 - O, 3 - N )- oxazole, 5 -( 1 - O, 3 - N )- oxazole, 4 -( 1 - S, 3 - N )- thiazole, 5 -( 1 - S, 3 - N )- thiazole, 2 - benzoxazole, 2 - benzothiazole, 4 -( 1 , 2 , 3 - N )- benzotriazole, and benzimidazole.

95. The labelled oligonucleotide of claim 93 wherein the electron- deficient nitrogen heterocycle is selected from the group consisting of 2 - pyridyl, 3 - pyridyl, 4 - pyridyl, 2 - quinolyl, 3 - quinolyl, and 4 - quinolyl.

96. The labelled oligonucleotide of claim 93 wherein each of R 1 and R 4 is an electron - deficient nitrogen heterocycle selected from the group consisting of 2 - pyridyl and 3 - pyridyl.

97. The labelled oligonucleotide of claim 93 wherein each of R 1 and R 4 is 2 - pyridyl.

98. The labelled oligonucleotide of claim 93 wherein each of R 1 and R 4 is 3 - pyridyl.

99. The labelled oligonucleotide of claim 93 wherein at least one of R 5 and R 7 is H.

100. The labelled oligonucleotide of claim 93 wherein:

each of R 1 and R 4 is an electron - deficient nitrogen heterocycle;

each of R 2 and R 3 is H; and

each of R 5 and R 7 is H.

101. A method of generating a labelled primer extension product, comprising:

enzymatically extending a primer - target hybrid in the presence of a mixture of enzymatically - extendable nucleotides capable of supporting continuous primer extension and a terminator, wherein said primer or said terminator is labelled with a compound of claim 69 or an energy transfer dye compound of claim 77 .

102. The method of claim 101 wherein the electron- deficient nitrogen heterocycle is selected from the group consisting of 2 - pyridyl, 3 - pyridyl, 4 - pyridyl, 2 - quinolyl, 3 - quinolyl, 4 - quinolyl, 2 - imidazole, 4 - imidazole, 3 - pyrazole, 4 - pyrazole, pyridazine, pyrimidine, pyrazine, cinnoline, pthalazine, quinazoline, quinoxaline, 3 -( 1 , 2 , 4 - N )- triazolyl, 5 -( 1 , 2 , 4 - N )- triazolyl, 5 - tetrazolyl, 4 -( 1 - O, 3 - N )- oxazole, 5 -( 1 - O, 3 - N )- oxazole, 4 -( 1 - S, 3 - N )- thiazole, 5 -( 1 - S, 3 - N )- thiazole, 2 - benzoxazole, 2 - benzothiazole, 4 -( 1 , 2 , 3 - N )- benzotriazole, and benzimidazole.

103. The method of claim 101 wherein the electron- deficient nitrogen heterocycle is selected from the group consisting of 2 - pyridyl, 3 - pyridyl, 4 - pyridyl, 2 - quinolyl, 3 - quinolyl, and 4 - quinolyl.

104. The method of claim 101 wherein each of R 1 and R 4 is an electron - deficient nitrogen heterocycle selected from the group consisting of 2 - pyridyl and 3 - pyridyl.

105. The method of claim 101 wherein each of R 1 and R 4 is 2 - pyridyl.

106. The method of claim 101 wherein each of R 1 and R 4 is 3 - pyridyl.

107. The method of claim 101 wherein at least one of R 5 and R 7 is H.

108. The method of claim 101 wherein:

each of R 1 and R 4 is an electron - deficient nitrogen heterocycle;

each of R 2 and R 3 is H; and

each of R 5 and R 7 is H.

109. A method of oligonucleotide ligation, comprising:

annealing two probes to a target sequence; and

forming a phosphodiester bond between the 5 ′ terminus of one probe and the 3 ′ terminus of the other probe;

wherein one or both probes are labelled with a compound of claim 69 or an energy transfer dye compound of claim 77 .

110. The method of claim 109 wherein the electron- deficient nitrogen heterocycle is selected from the group consisting of 2 - pyridyl, 3 - pyridyl, 4 - pyridyl, 2 - quinolyl, 3 - quinolyl, 4 - quinolyl, 2 - imidazole, 4 - imidazole, 3 - pyrazole, 4 - pyrazole, pyridazine, pyrimidine, pyrazine, cinnoline, pthalazine, quinazoline, quinoxaline, 3 -( 1 , 2 , 4 - N )- triazolyl, 5 -( 1 , 2 , 4 - N )- triazolyl, 5 - tetrazolyl, 4 -( 1 - O, 3 - N )- oxazole, 5 -( 1 - O, 3 - N )- oxazole, 4 -( 1 - S, 3 - N )- thiazole, 5 -( 1 - S, 3 - N )- thiazole, 2 - benzoxazole, 2 - benzothiazole, 4 -( 1 , 2 , 3 - N )- benzotriazole, and benzimidazole.

111. The method of claim 109 wherein the electron- deficient nitrogen heterocycle is selected from the group consisting of 2 - pyridyl, 3 - pyridyl, 4 - pyridyl, 2 - quinolyl, 3 - quinolyl, and 4 - quinolyl.

112. The method of claim 109 wherein each of R 1 and R 4 is an electron - deficient nitrogen heterocycle selected from the group consisting of 2 - pyridyl and 3 - pyridyl.

113. The method of claim 109 wherein each of R 1 and R 4 is 2 - pyridyl.

114. The method of claim 109 wherein each of R 1 and R 4 is 3 - pyridyl.

115. The method of claim 109 wherein at least one of R 5 and R 7 is H.

116. The method of claim 109 wherein:

each of R 1 and R 4 is an electron - deficient nitrogen heterocycle;

each of R 2 and R 3 is H; and

each of R 5 and R 7 is H.