Fluorescent dye compounds, conjugates and uses thereof

1. 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, and the donor dye is a compound selected from the group consisting of structures (I), (II), (III) and (IV)

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, biphenyl, substituted biphenyl, benzyl, substituted benzyl, benzoyl, and substituted benzoyl, wherein R is selected from 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, substituted benzoyl, and a linking group; 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 comprises the structure (I), then at least one of R 1 , R 2 , R 3 or R 8 is independently selected from the group consisting of 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, biphenyl, substituted biphenyl, benzyl, substituted benzyl, benzoyl, and substituted benzoyl, wherein R is selected from the group consisting of 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, substituted benzoyl, and a linking group.

2. The energy transfer dye of claim 1 , wherein the donor dye is covalently attached to the acceptor dye through a linker.

3. The energy transfer dye of claim 2 , wherein the linker is non-nucleotidic.

4. The energy transfer dye of claim 2 , wherein the linker is nucleotidic.

5. The energy transfer dye according to any one of claims 1 - 4 , wherein the acceptor dye is a compound comprising a structure selected from the group consisting of structures (I), (II), (III) and (IV)

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, biphenyl, substituted biphenyl, benzyl, substituted benzyl, benzoyl, and substituted benzoyl, wherein R is selected from the group consisting of 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; 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, and 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 comprises the structure (I), then at least one of R 1 , R 2 , R 3 or R 8 is independently selected from the group consisting of 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, biphenyl, substituted biphenyl, benzyl, substituted benzyl, benzoyl, and substituted benzoyl, wherein R is selected from the group consisting of 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.

6. The energy transfer dye of claim 5 , 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.

7. The compound of claim 1 , wherein each of R 6 and R 7 is independently selected from the group consisting of fluorine, chlorine and bromine.

8. The compound of claim 7 , wherein each of R 6 and R 7 is fluorine.

9. The compound of claim 7 , wherein each of R 6 and R 7 is chlorine.

10. The compound of claim 7 , wherein each of R 6 and R 7 is bromine.

11. The compound of claim 1 , wherein R 6 is —H and R 7 is selected from the group consisting of —H, —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 , and —CH 2 NHR.

12. The compound of claim 1 , wherein R 6 is —H and R 7 is selected from the group consisting of —H, —SO 3 H, —SO 3 R, —CH 2 CO 2 H, —CH 2 CO 2 R, —CH 2 NH 2 , and —CH 2 NHR.

13. The compound of claim 1 , wherein R 3 is —H and R 2 is selected from the group consisting of —H, —SO 3 H, —SO 3 R, —CH 2 CO 2 H, —CH 2 CO 2 R, —CH 2 NH 2 , and —CH 2 NHR.

14. The compound of claim 1 , wherein R is selected from the group consisting of 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, substituted benzoyl, a linking group and trifluoracetyl.