Water-soluble rhodamine dye conjugates

• US 7,504,496 B2
• Filed: 02/14/2005
• Issued: 03/17/2009
• Est. Priority Date: 11/03/1999
• Status: Expired due to Fees

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Inventors: Lee, Linda G., Graham, Ronald J., Swartzman, Elana, Werner, William E., Lu, Lily
• Primary Examiner(s): Riley; Jezia
• Application Number:
  US11/059,035
• Publication Number:
  US 20050153355A1
• Time in Patent Office: 1,492 Days
• Field of Search: 435/6, 536/4.1, 536/23.1, 536/24.3, 536/25.3, 536/26.6
• US Class Current: 536/26.6
• CPC Class Codes: C07D 311/80 Dibenzopyrans; Hydrogenated dibenzopyrans
• CPC Class Codes: C07D 491/04 Ortho-condensed systems
• CPC Class Codes: C07D 491/06 Peri-condensed systems
• CPC Class Codes: C07H 19/207 the phosphoric or polyphosphoric acids being esterified by a further hydroxylic compound, e.g. flavine adenine dinucleotide or nicotinamide-adenine dinucleotide
• 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 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: G01N 2458/00 Labels used in chemical analysis of biological material
• CPC Class Codes: G01N 33/582 with fluorescent label
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine

Abstract

The present invention provides novel, water-soluble, red-emitting fluorescent rhodamine dyes and red-emitting fluorescent energy-transfer dye pairs, as well as labeled conjugates comprising the same and methods for their use. The dyes, energy-transfer dye pairs and labeled conjugates are useful in a variety of aqueous-based applications, particularly in assays involving staining of cells, protein binding, and/or analysis of nucleic acids, such as hybridization assays and nucleic acid sequencing.

Claims

1. A labeled nucleoside/tide or nucleoside/tide analog comprising a rhodamine dye conjugated by a linker (L′

) to a nucleoside/tide or nucleoside/tide analog (NUC), wherein;

the rhodamine is a rhodamine-type parent xanthene having attached to the xanthene C9 carbon a phenyl group, wherein the phenyl group is further substituted with (i) a carboxy group or a sulfonate group or a salt thereof;

(ii) one to three substituted or unsubstituted aminopyridinium groups; and

(iii) a substituted or unsubstituted alkylthio or arylthio group; and

the nucleoside/tide or nucleoside/tide analog and linker taken together comprise the structure;

2. The labeled nucleoside/tide or nucleoside/tide analog of claim 1 comprising the formula:

3. The labeled nucleoside/tide or nucleoside/tide analog of claim 2 wherein Y comprises the rhodamine-type parent xanthene ring structures:

4. The labeled nucleoside/tide or nucleoside/tide analog of claim 3 wherein R² when taken together with R³ or R^3′

is (C₂-C₆) alkyldiyl.

5. The labeled nucleoside/tide or nucleoside/tide analog of claim 3 wherein:

an alkyldiyl or alkyleno bridge formed by taking R² together with R³ or R^3′

, R⁷ together with R⁶ or R^6′

, or R⁴ together with and R³ or R^3′

, is ethano, propano, 1,1-dimethylethano, 1,1-dimethylpropano or 1,1,3-trimethylpropano;

an aryleno bridge formed by taking R¹ together with R² is benzo or naphtho;

an alkyldiyl or alkyleno bridge formed by taking R³ together with R^3′

, or R⁶ together with R^6′

, is butano;

an alkyldiyl or alkyleno bridge formed by taking R⁵ together with R⁶ or R^6′

is ethano, propano, 1,1-dimethylethano, 1,1-dimethyipropano and 1,1,3-trimethylpropano; and

an aryleno bridge formed by taking R¹⁰, R¹¹, R¹² and R¹³ together, or R¹⁸, R¹⁹, R²⁰ and R²¹ together, is benzo.

6. The labeled nucleoside/tide or nucleoside/tide analog of claim 2 in which Z¹ is phenyldiyl.

7. The labeled nucleoside/tide or nucleoside/tide analog of claim 2 in which L′

is selected from;

8. The labeled nucleoside/tide or nucleoside/tide analog of claim 2 in which L′

is;

9. The labeled nucleoside/tide or nucleoside/tide analog of claim 2 in which R⁹ comprises a phenyl ring and Y is selected from the structures:

10. The labeled nucleoside/tide or nucleoside/tide analog of claim 2 wherein R²², R²³, R²⁵, and R²⁶ are each hydrogen.

11. The labeled nucleoside/tide or nucleoside/tide analog of claim 2 which comprises the structure:

12. The labeled nucleoside/tide or nucleoside/tide analog of claim 11 in which L′

is selected from;

—

C≡

C—

CH₂— and

—

C≡

C—

CH₂—

O—

CH₂—

.

13. The labeled nucleoside/tide or nucleoside/tide analog of claim 1 comprising the formula:

14. The labeled nucleoside/tide or nucleoside/tide analog of claim 13 in which Y is selected from:

15. The labeled nucleoside/tide or nucleoside/tide analog of claim 14 wherein:

an alkyldiyl or alkyleno bridge formed by taking R² together with R³, R⁴ together with R^3′

, R⁵ together with R⁶, or R⁷ together with R^6′

, is ethano, propano, 1,1-dimethylethano, 1,1-dimethyipropano or 1,1,3-trimethylpropano; and

an aryleno bridge formed by taking R¹⁰, R¹¹, R¹² and R¹³ together or R¹⁸, R¹⁹, R²⁰ and R²¹ together is benzo.

16. The labeled nucleoside/tide or nucleoside/tide analog of claim 13 in which L is selected from phenyldiyl and naphthyldiyl.

17. The labeled nucleoside/tide or nucleoside/tide analog of claim 13 in which L is —

(CH₂)_i-φ

- where i is an integer from 1 to 6 and φ

is phenyldiyl or naphthyldiyl.

18. The labeled nucleoside/tide or nucleoside/tide analog of claim 13 in which Z is selected from phenyl, benzyl, naphthyl, pyridyl and purinyl.

19. The labeled nucleoside/tide or nucleoside/tide analog of claim 13 in which L is selected from:

20. The labeled nucleoside/tide or nucleoside/tide analog of claim 13 in which L′

is;

21. The labeled nucleoside/tide or nucleoside/tide analog of claim 13 wherein R²², R²³, R²⁵, and R²⁶ are each hydrogen.

22. The labeled nucleoside/tide or nucleoside/tide analog of claim 13 in which Y is selected from the group consisting of:

23. The labeled nucleoside/tide or nucleoside/tide analog of claim 13 which has the structure:

24. The labeled nucleoside/tide or nucleoside/tide analog of claim 23 in which L′

is selected from;

25. The labeled nucleoside/tide or nucleoside/tide analog of claim 1 used in conjunction with a donor dye or an acceptor dye whereby the rhodamine dye and the donor dye or acceptor dye form an energy-transfer dye pair.

26. The labeled nucleoside/tide or nucleoside/tide analog of claim 25 wherein the donor dye or acceptor dye is a fluorescein, rhodamine, cyanine, phthalocyanine or squaraine.

27. The labeled nucleoside/tide or nucleoside/tide analog of claim 25 wherein the donor dye or acceptor dye is 4′

-aminomethyl-6-carboxyfluorescein and the 4′

-aminomethyl-6-carboxyfluorescein is covalently attached to the rhodamine dye by a linker.

28. The labeled nucleoside/tide or nucleoside/tide analog of claim 27 wherein the 4′

-aminomethyl-6-carboxyfluorescein is further covalently attached by the linker L′

to the nucleobase B of the nucleoside/tide or nucleoside/tide analog.

29. The labeled nucleoside/tide or nucleoside/tide analog of claim 1 which is enzymatically incorporatable.

30. The labeled nucleoside/tide or nucleoside/tide analog of claim 1 which is a terminator.

31. The labeled nucleoside/tide or nucleoside/tide analog of claim 1 which is enzymatically extendable.

32. The labeled nucleoside/tide or nucleoside/tide analog of claim 1 wherein R⁷¹ and R⁷⁰ are hydrogen.

33. The labeled nucleoside/tide or nucleoside/tide analog of claim 1 wherein R⁷¹ and R⁷⁰ are hydroxyl.

34. The labeled nucleoside/tide or nucleoside/tide analog of claim 1 wherein R⁷¹ is hydroxyl, and R⁷⁰ is hydrogen.

35. The labeled nucleoside/tide or nucleoside/tide analog of claim 1 in which nucleobase B is selected from adenine, 7-deazaadenine, cytosine, guanine, 7-deazaguanine, thymine and uracil.

36. A labeled polynucleotide or polynucleotide analog comprising a rhodamine dye conjugated to a nucleoside/tide or nucleoside/tide analog, wherein the rhodamine is a rhodamine-type parent xanthene having attached to the xanthene C9 carbon a phenyl group, wherein the phenyl group is further substituted with (i) a carboxy group or a sulfonate group or a salt thereof;

(ii) one to three substituted or unsubstituted aminopyridinium groups; and

(iii) a substituted or unsubstituted alkylthio or arylthio group.