Fluorescent dye compounds, conjugates and uses thereof

• US 8,729,267 B2
• Filed: 06/21/2011
• Issued: 05/20/2014
• Est. Priority Date: 09/16/2004
• Status: Active Grant

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Inventors: Graham, Ronald, Zou, Ruiming, Upadhya, Krishna G., Benson, Scott C.
• Primary Examiner(s): Aulakh, Charanjit
• Application Number:
  US13/165,575
• Publication Number:
  US 20110250699A1
• Time in Patent Office: 1,064 Days
• Field of Search: 546/58, 546/61, 546/102, 436/800
• US Class Current: 546/61
• CPC Class Codes: C07D 219/06 Oxygen atoms
• 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: C09B 15/00 Acridine dyes
• CPC Class Codes: C09K 11/06 containing organic luminescent materials
• CPC Class Codes: C09K 2211/1011 Condensed systems
• CPC Class Codes: C09K 2211/1029 containing one nitrogen atom as the heteroatom
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: G01N 33/582 with fluorescent label
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

The present teachings generally relate to fluorescent dyes, linkable forms of fluorescent dyes, energy transfer dyes, reagents labeled with fluorescent dyes and uses thereof.

Claims

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

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:

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

12. The compound of claim 1, wherein R₂ is selected from —

SO₃H, —

SO₃R, —

CH₂NH₂, —

CH₂NHR, and —

NO₂.

13. The compound of claim 1, wherein R₆ and R₇ are each independently selected from fluorine, chlorine and bromine.

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:

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.

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 R₂ of the donor dye is selected from —

SO₃H, —

SO₃R, —

CH₂NH₂, —

CH₂NHR, and —

NO₂.

16. The energy transfer dye of claim 5, wherein R₆ and R₇ 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:

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

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:

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.

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 R₂ of the acceptor dye is selected from —

SO₃H, —

SO₃R, —

CH₂NH₂, —

CH₂NHR, and —

NO₂.

22. The energy transfer dye of claim 9, wherein R₆ and R₇ 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:

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

27. The compound of claim 26, wherein R₂ is selected from —

SO₃H, —

SO₃R, —

CH₂NH₂, —

CH₂NHR, and —

NO₂.

28. The compound of claim 26, wherein R₆ and R₇ 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):

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 R₂ of the compound of formula (I) is selected from —

SO₃H, —

SO₃R, —

CH₂NH₂, —

CH₂NHR, and —

NO₂.

39. The energy transfer dye of claim 33, wherein R₆ and R₇ 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: