Fluorescent dye compounds, conjugates and uses thereof

1. A compound having a structure of one of the following formulae:

wherein:

each of R 1 -R 3 and R 6 -R 16 is independently selected from the group consisting of —H, halogen, aryl, substituted aryl, heteroaryl, —CO 2 H, —CO 2 R, —SO 3 H, —SO 3 R, —CH 2 CO 2 H, —CH 2 CO 2 R, —CH 2 SO 3 H, —CH 2 SO 3 R, —CH 2 NH 2 , —CH 2 NHR, —NO 2 , C 1 -C 6 alkyl, substituted C 1 -C 6 alkyl, C 1 -C 6 alkoxy, substituted C 1 -C 6 alkoxy, C 1 -C 6 alkoxyaryl, substituted C 1 -C 6 alkoxyaryl, phenyl, substituted phenyl, biphenyl, substituted biphenyl, benzyl, substituted benzyl, benzoyl, and substituted benzoyl, a bond and a linking group,

wherein R is selected from the group consisting of trifluoroacetyl,

a bond and a linking group, wherein X is succinimide; and

each of R 4 and R 5 taken separately is selected from the group consisting of C 1 -C 6 alkyl and C 1 -C 6 substituted alkyl, R 4 and R 5 taken together are selected from the group consisting of C 3 -C 7 cycloalkyl, C 4 -C 7 unsaturated cycloalkyl, C 3 -C 7 substituted cycloalkyl and C 4 -C 7 substituted unsaturated cycloalkyl,

wherein at least one of R 1 -R 3 and R 6 -R 16 is —SO 3 H,

with the proviso that if the compound has a structure of formula (I), then at least one of R 1 , R 2 , R 3 or R 8 is not hydrogen.

2. The compound of claim 1 having the structure:

3. The compound of claim 1 having the structure:

4. The compound of claim 1 having the structure:

5. An energy transfer dye comprising a donor dye covalently attached to an acceptor dye, wherein the donor dye is capable of absorbing light at a first wavelength and emitting excitation energy in response, the acceptor dye is capable of absorbing the excitation energy emitted by the donor dye and fluorescing at a second wavelength in response, wherein the donor dye is a compound having a structure of one of the following formulae:

wherein:

each of R 1 -R 3 and R 6 -R 16 is independently selected from the group consisting of —H, halogen, aryl, substituted aryl, heteroaryl, —CO 2 H, —CO 2 R, —SO 3 H, —SO 3 R, —CH 2 CO 2 H, —CH 2 CO 2 R, —CH 2 SO 3 H, —CH 2 SO 3 R, —CH 2 NH 2 , —CH 2 NHR, —NO 2 , C 1 -C 6 alkyl, substituted C 1 -C 6 alkyl, C 1 -C 6 alkoxy, substituted C 1 -C 6 alkoxy, C 1 - C 6 alkoxyaryl, substituted C 1 -C 6 alkoxyaryl, phenyl, substituted phenyl, biphenyl, substituted biphenyl, benzyl, substituted benzyl, benzoyl, and substituted benzoyl, a bond and a linking group,

wherein R is selected from the group consisting of trifluoroacetyl,

a bond and a linking group, wherein X is succinimide; and

each of R 4 and R 5 taken separately is selected from the group consisting of C 1 -C 6 alkyl and C 1 -C 6 substituted alkyl, R 4 and R 5 taken together are selected from the group consisting of C 3 -C 7 cycloalkyl, C 4 -C 7 unsaturated cycloalkyl, C 3 -C 7 substituted cycloalkyl and C 4 -C 7 substituted unsaturated cycloalkyl,

wherein at least one of R 1 -R 3 and R 6 -R 16 is —SO 3 H,

with the proviso that if the compound has a structure of formula (I), then at least one of R 1 , R 2 , R 3 or R 8 is not hydrogen.

6. The energy transfer dye of claim 5 , wherein the donor dye is covalently attached to the acceptor dye through a linker.

7. The energy transfer dye of claim 5 , wherein the linker is non-nucleotidic.

8. The energy transfer dye of claim 5 , wherein the linker is nucleotidic.

9. An energy transfer dye comprising a donor dye covalently attached to an acceptor dye, wherein the donor dye is capable of absorbing light at a first wavelength and emitting excitation energy in response, the acceptor dye is capable of absorbing the excitation energy emitted by the donor dye and fluorescing at a second wavelength in response, wherein the acceptor dye is a compound having a structure of one of the following formulae:

wherein:

each of R 1 -R 3 and R 6 -R 16 is independently selected from the group consisting of —H, halogen, aryl, substituted aryl, heteroaryl, —CO 2 H, —CO 2 R, —SO 3 H, —SO 3 R, —CH 2 CO 2 H, —CH 2 CO 2 R, —CH 2 SO 3 H, —CH 2 SO 3 R, —CH 2 NH 2 , —CH 2 NHR, —NO 2 , C 1 -C 6 alkyl, substituted C 1 -C 6 alkyl, C 1 -C 6 alkoxy, substituted C 1 -C 6 alkoxy, C 1 -C 6 alkoxyaryl, substituted C 1 -C 6 alkoxyaryl, phenyl, substituted phenyl, biphenyl, substituted biphenyl, benzyl, substituted benzyl, benzoyl, and substituted benzoyl, a bond and a linking group,

wherein R is selected from the group consisting of trifluoroacetyl,

a bond and a linking group, wherein X is succinimide; and

each of R 4 and R 5 taken separately is selected from the group consisting of C 1 -C 6 alkyl and C 1 -C 6 substituted alkyl, R 4 and R 5 taken together are selected from the group consisting of C 3 -C 7 cycloalkyl, C 4 -C 7 unsaturated cycloalkyl, C 3 -C 7 substituted cycloalkyl and C 4 -C 7 substituted unsaturated cycloalkyl,

wherein at least one of R 1 -R 3 and R 6 -R 16 is —SO 3 H,

with the proviso that if the compound has a structure of formula (I), then at least one of R 1 R 2 , R 1 or R 8 is not hydrogen.

10. The energy transfer dye of claim 9 , wherein the donor dye is selected from the group consisting of 5-carboxyfluorescein (5-FAM), 6-carboxyfluorescein (6-FAM), rhodamine green (R110), 5-carboxyrhodamine, 6-carboxyrhodamine, N,N′-diethyl-2′,7′-dimethyl-5-carboxy-rhodamine (5-R6G), N,N′-diethyl-2′, 7′-dimethyl-6-carboxyrhodamine (6—R6G), N,N,N′,N′-tetramethyl-5-carboxyrhodamine (5-TAMRA), Cy3, N,N,N′,N′-tetramethyl-5-carboxyrhodamine (6-TAMRA), 5-carboxy-X-rhodamine (5-ROX), 6-carboxy-X-rhodamine (6-ROX), 5-carboxy-2′,4′,5′,7′,4,7-hexachlorofluorescein, 6-carboxy-2′, 4′,5,7′,4,7-hexachlorofluorescein, 5-carboxy-2′,7′-dicarboxy-4′,5′-dichloro-fluorescein, 6-carboxy-1′,2′-dicarboxy-4′,5′-dichlorofluorescein, 5-carboxy-2′,4′,5′,7′-tetrachlorofluorescein, 1′,2′-benzo-4′-fluoro-7′,4,7-trichloro-5-carboxyfluorescein, 1′,2′-benzo-4′-fluoro-7′,4,7-trichloro-6-carboxyfluorescein and 1′,2′,7′,8′-dibenzo-4,7-dichloro-5-carboxyfluorescein.

11. The compound of claim 1 , having a structure of formula (I):

12. The compound of claim 1 , wherein R 2 is selected from —SO 3 H, —SO 3 R, —CH 2 NH 2 , —CH 2 NHR, and —NO 2 .

13. The compound of claim 1 , wherein R 6 and R 7 are each independently selected from fluorine, chlorine and bromine.

14. The energy transfer dye of claim 5 , wherein the donor dye has a structure of formula (I):

15. The energy transfer dye of claim 5 , wherein R 2 of the donor dye is selected from —SO 3 H, —SO 3 R, —CH 2 NH 2 , —CH 2 NHR, and —NO 2 .

16. The energy transfer dye of claim 5 , wherein R 6 and R 7 of the donor dye are each independently selected from fluorine, chlorine and bromine.

17. The energy transfer dye of claim 5 , wherein the donor dye has the structure:

18. The energy transfer dye of claim 5 , wherein the donor dye has the structure:

19. The energy transfer dye of claim 5 , wherein the donor dye has the structure:

20. The energy transfer dye of claim 9 , wherein the acceptor dye has a structure of formula (I):

21. The energy transfer dye of claim 9 , wherein R 2 of the acceptor dye is selected from —SO 3 H, —SO 3 R, —CH 2 NH 2 , —CH 2 NHR, and —NO 2 .

22. The energy transfer dye of claim 9 , wherein R 6 and R 7 of the acceptor dye are each independently selected from fluorine, chlorine and bromine.

23. The energy transfer dye of claim 9 , wherein the acceptor dye has the structure:

24. The energy transfer dye of claim 9 , wherein the acceptor dye has the structure:

25. The energy transfer dye of claim 5 , wherein the acceptor dye has the structure:

26. A comnaund having a cfnirture of formula (I):

wherein:

each of R 1 -R 3 and R 6 -R 8 is independently selected from the group consisting of —H, halogen, aryl, substituted aryl, heteroaryl, —CO 2 H, —CO 2 R, —SO 3 H, —SO 3 R, —CH 2 CO 2 H, —CH 2 CO 2 R, —CH 2 SO 3 H, —CH 2 SO 3 R, —CH 2 NH 2 , —CH 2 NHR, —NO 2 , C 1 -C 6 alkyl, substituted C 1 -C 6 alkyl, C 1 -C 6 alkoxy, substituted C 1 -C 6 alkoxy, C 1 -C 6 alkoxyaryl, substituted C 1 -C 6 alkoxyaryl, phenyl, substituted phenyl, biphenyl, substituted biphenyl, benzyl, substituted benzyl, benzoyl, and substituted benzoyl, a bond and a linking group,

wherein R is selected from the group consisting of trifluoroacetyl,

a bond and a linking group, wherein X is succinimide; and

each of R 4 and R 5 taken separately is selected from the group consisting of C 1 -C 6 alkyl and C 1 -C 6 substituted alkyl, R 4 and R 5 taken together are selected from the group consisting of C 3 -C 7 cycloalkyl, C 4 -C 7 unsaturated cycloalkyl, C 3 -C 7 substituted cycloalkyl and C 4 -C 7 substituted unsaturated cycloalkyl,

wherein at least one of R 1 -R 3 and R 6 -R 8 is —SO 3 H,

with the proviso that at least one of R 1 , R 2 , R 3 or R 8 is not hydrogen.

27. The compound of claim 26 , wherein R 2 is selected from —SO 3 H, —SO 3 R, —CH 2 NH 2 , —CH 2 NHR, and —NO 2 .

28. The compound of claim 26 , wherein R 6 and R 7 are each independently selected from fluorine, chlorine and bromine.

29. The compound of 26 having the structure:

30. The compound of 26 having the structure:

31. The compound of 26 having the structure:

32. The compound of 26 having the structure:

33. An energy transfer dye comprising a donor dye covalently attached to an acceptor dye, wherein the donor dye is capable of absorbing light at a first wavelength and emitting excitation energy in response, the acceptor dye is capable of absorbing the excitation energy emitted by the donor dye and fluorescing at a second wavelength in response, wherein the donor or the acceptor dye is a compound having a structure of the formula (I):

wherein:

each of R 1 -R 3 and R 6 -R 8 is independently selected from the group consisting of —H, halogen, aryl, substituted aryl, heteroaryl, —CO 2 H, —CO 2 R, —SO 3 H, —SO 3 R, —CH 2 CO 2 H, —CH 2 CO 2 R, —CH 2 SO 3 H, —CH 2 SO 3 R, —CH 2 NH 2 , —CH 2 NHR, —NO 2 , C 1 -C 6 alkyl, substituted C 1 -C 6 alkyl, C 1 -C 6 alkoxy, substituted C 1 -C 6 alkoxy, C 1 -C 6 alkoxyaryl, substituted C 1 -C 6 alkoxyaryl, phenyl, substituted phenyl, biphenyl, substituted biphenyl, benzyl, substituted benzyl, benzoyl, and substituted benzoyl, a bond and a linking group,

wherein R is selected from the group consisting of trifluoroacetyl,

a bond and a linking group, wherein X is succinimide; and

each of R 4 and R 5 taken separately is selected from the group consisting of C 1 -C 6 alkyl and C 1 -C 6 substituted alkyl, R 4 and R 5 taken together are selected from the group consisting of C 3 -C 7 cycloalkyl, C 4 -C 7 unsaturated cycloalkyl, C 3 -C 7 substituted cycloalkyl and C 4 -C 7 substituted unsaturated cycloalkyl,

wherein at least one of R 1 -R 3 and R 6 -R 8 is —SO 3 H,

with the proviso that at least one of R 1 , R 2 , R 3 or R 8 is not hydrogen.

34. The energy transfer dye of claim 33 , wherein the donor dye is covalently attached to the acceptor dye through a linker.

35. The energy transfer dye of claim 33 , wherein the linker is non-nucleotidic.

36. The energy transfer dye of claim 33 , wherein the linker is nucleotidic.

37. The energy transfer dye of claim 33 , wherein when the acceptor dye is the compound of formula (I), then the donor dye is selected from the group consisting of 5-carboxyfluorescein (5-FAM), 6-carboxyfluorescein (6-FAM), rhodamine green (R110), 5-carboxyrhodamine, 6-carboxyrhodamine, N,N′-diethyl-2′,7′-dimethyl-5-carboxy-rhodamine (5-R6G), N,N′-diethyl-2′,7′-dimethyl-6-carboxyrhodamine (6-R6G), N,N,N′,N′-tetramethyl-5-carboxyrhodamine (5-TAMRA), Cy3, N,N,N′,N′-tetramethyl-5-carboxyrhodamine (6-TAMRA), 5-carboxy-X-rhodamine (5-ROX), 6-carboxy-X-rhodamine (6-ROX), 5-carboxy-2′,4′,5′,7′,4,7-hexachlorofluorescein, 6-carboxy-2′, 4′,5,7′,4,7-hexachlorofluorescein, 5-carboxy-2′,7′-dicarboxy-4′,5′-dichloro-fluorescein, 6-carboxy-1′,2′-dicarboxy-4′,5′-dichlorofluorescein, 5-carboxy-2′,4′,5′,7′-tetrachlorofluorescein, 1′,2′-benzo-4′-fluoro-7′,4,7-trichloro-5-carboxyfluorescein, 1′,2′-benzo-4′-fluoro-7′,4,7-trichloro-6-carboxyfluorescein and 1′,2′,7′,8′-dibenzo-4,7-dichloro-5-carboxyfluorescein.

38. The energy transfer dye of claim 33 , wherein R 2 of the compound of formula (I) is selected from —SO 3 H, —SO 3 R, —CH 2 NH 2 , —CH 2 NHR, and —NO 2 .

39. The energy transfer dye of claim 33 , wherein R 6 and R 7 of the compound of formula (I) are each independently selected from fluorine, chlorine and bromine.

40. The energy transfer dye of claim 33 , wherein the compound of formula (I) has the structure:

41. The energy transfer dye of claim 33 , wherein the compound of formula (I) has the structure:

42. The energy transfer dye of claim 33 , wherein the compound of formula (I) has the structure: