Water-soluble rhodamine dyes conjugates

• US 8,030,096 B2
• Filed: 08/12/2010
• Issued: 10/04/2011
• 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: Swartzman, Elana E., Graham, Ronald, Lee, Linda G., Lu, Lily, Werner, William E.
• Primary Examiner(s): Riley; Jezia
• Application Number:
  US12/855,649
• Publication Number:
  US 20110081688A1
• Time in Patent Office: 418 Days
• Field of Search: 436/800, 530/300, 530/350, 435/6, 435/7.1, 435/7.2, 536/4.1, 536/23.1, 536/24.3, 536/25.3, 536/26.6
• US Class Current: N/A
• 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 rhodamine dye or a salt thereof, comprising a rhodamine-type parent xanthene ring having attached to the xanthene C9 carbon a phenyl group that is further substituted with an ortho carboxy or ortho sulfonate group or a salt thereof, one to three substituted or unsubstituted aminopyridinium groups and a substituted or unsubstituted alkylthio, arylthio or heteroarylthio group, wherein the rhodamine dye comprises the structure:

2. The rhodamine dye of claim 1, wherein Z has the form Z¹-L-R_x, or a salt thereof, wherein:

Z¹ is selected from the group consisting of a (C₁-C₁₂) alkyldiyl, a (C₁-C₁₂) alkyldiyl independently substituted with one or more of the same or different W¹ groups, a (C₅-C₁₄) aryldiyl or heterodiyl and a (C₅-C₁₄) aryldiyl or heterodiyl independently substituted with one or more of the same or different W² groups;

L is selected from the group consisting of a bond and a linker; and

R_x is a reactive functional group.

3. The rhodamine dye of claim 2, in which Y is selected from the group consisting of:

4. The rhodamine dye of claim 3, wherein R², when taken together with R³ or R^3′

is a (C₂-C₆) alkyldiyl or an alkyleno.

5. The rhodamine dye of claim 4, wherein:

alkyl is selected from the group consisting of methyl, ethyl and a propyl;

aryl is selected from the group consisting of phenyl and naphthyl;

arylaryl is biphenyl;

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

, R⁷ together with R⁶ or R^6′

, or R⁴ together with R³ or R^3′

, are selected from the group consisting of ethano, a propano, 1,1-dimethylethano, 1,1-dimethylpropano and 1,1,3-trimethylpropano;

aryleno bridges formed by taking R¹ together with R² are selected from the group consisting of benzo and naphtho;

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

, or R⁶ together with R^6′

, is a butano;

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

are selected from the group consisting of ethano, a propano, 1,1-dimethylethano, 1,1-dimethylpropano and 1,1,3-trimethylpropano; and

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

6. The rhodamine dye of claim 4, wherein L is a bond.

7. The rhodamine dye of claim 2, wherein R_x is selected from the group consisting of carboxyl, carboxylate, an ester and an activated ester.

8. The rhodamine dye of claim 2, wherein Z¹ is selected from the group consisting of a (C₁-C₁₂) alkyleno, a (C₁-C₁₂) alkano, a (C₅-C₁₀) aryldiyl or heteroaryldiyl, phenyldiyl, phena-1,4-diyl, naphthadiyl, naphtha-2,6-diyl, pyridindiyl and purindiyl.

9. The rhodamine dye of claim 2, wherein Y is selected from the group consisting of:

10. The rhodamine dye of claim 2, wherein L is a bond.

11. The rhodamine dye of claim 2 which comprises the structure:

12. The rhodamine dye of claim 11, wherein Y is selected from the group consisting of Y-1, Y-2, Y-3 and Y-4.

13. The rhodamine dye of claim 11, wherein Y is selected from the group consisting of Y-20a, Y-21a, Y-22a, Y-23a, Y-24a, Y-25a, Y-31a, Y-34a, Y-35a, Y-36a, Y-39a, Y-41a, Y-42a, Y-43a, Y-44a, Y-45a and Y-46a.

14. The rhodamine dye of claim 1 having the structure:

15. The rhodamine dye of claim 14, wherein Y¹ is selected from the group consisting of:

16. The rhodamine dye of claim 15, wherein:

alkyl is selected from the group consisting of methanyl, ethanyl and propanyl;

aryl is selected from the group consisting of phenyl and naphthyl;

arylaryl is biphenyl;

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

, R⁷ together with R⁶ or R^6′

, or R⁴ together with R³ or R^3′

, are selected from the group consisting of ethano, a propano, 1,1-dimethylethano, 1,1-dimethylpropano and 1,1,3-trimethylpropano;

aryleno bridges formed by taking R¹⁰, R¹¹, R¹² and R¹³ together or R¹⁸, R¹⁹, R²⁰ and R²¹ together are benzo.

17. The rhodamine dye of claim 14, wherein L is selected from the group consisting of a (C₁-C₆) alkyldiyl, a (C₁-C₆) alkano, a (C₅-C₂₀) aryldiyl, phenyldiyl, phena-1,4-diyl, naphthyldiyl, naphtha-2,6-diyl, naphtha-2,7-diyl, a (C₆-C₂₆) arylalkyldiyl, —

(CH₂)_i-φ

- and —

(CH₂)_i-Ψ

-, where each i is independently an integer having the value between 1 and 6, φ

is selected from the group consisting of a (C₅-C₂₀) aryldiyl, phenyldiyl and phena-1,4-diyl and Ψ

is selected from the group consisting of naphthyldiyl, naphtha-2,6-diyl and naphtha-2,7-diyl.

18. The rhodamine dye of claim 14, wherein R_x is selected from the group consisting of carboxyl, carboxylate, an ester and an activated ester.

19. The rhodamine dye of claim 14, wherein Z is selected from the group consisting of a (C₁-C₁₂) alkyl, a (C₁-C₁₂) alkanyl, a (C₅-C₁₀) aryl or heteroaryl, phenyl, naphthyl, naphth-1-yl, naphth-2-yl, pyridyl and purinyl.

20. The rhodamine dye of claim 14, wherein Y¹ is selected from the group consisting of:

21. The rhodamine dye of claim 14 having the structure:

22. The rhodamine dye of claim 21, wherein Y¹ is selected from the group consisting of Y-1b, Y-2b, Y-3b, Y-4-b, Y-1c, Y-2c, Y-3c and Y-4-c.

23. The rhodamine dye of claim 21, wherein Y¹ is selected from the group consisting of Y-20b, Y-20c, Y-21b, Y-21c, Y-22b, Y-22c, Y-23b, Y-23c, Y-24b, Y-24c, Y-25b, Y-25c, Y-31b, Y-31c, Y-34b, Y-34c, Y-35b, Y-35c, Y-36b, Y-36c, Y-37b, Y-39b, Y-39c, Y-41c, Y-42b, Y-43b, Y-43c, Y-46b and Y-46c.

24. An energy-transfer dye pair comprising a donor dye linked to an acceptor dye, wherein the donor dye or the acceptor dye is a compound according to claim 1 and either or both of said donor and acceptor dyes include an optional linking moiety.

25. The dye pair of claim 24 having the structure:

26. The dye pair of claim 25, wherein Y is selected from the group consisting of Y-1, Y-2, Y-3, Y-4, Y-20a, Y-21a, Y-22a, Y-23a, Y-24a, Y-25a, Y-31a, Y-34a, Y-35a, Y-36a, Y-39a, Y-41a, Y-42a, Y-43a, Y-44a, Y-45a and Y-46a.

27. The dye pair of claim 25, wherein L is a bond.

28. The dye pair of claim 25, wherein R⁴¹ has the formula —

C(O)NR⁴⁵—

, wherein R⁴⁵ is selected from the group consisting of hydrogen and a (C₁-C₆) alkyl.

29. The dye pair of claim 25, wherein Z¹ is selected from the group consisting of a (C₁-C₁₂) alkyleno, a (C₁-C₁₂) alkano, a (C₅-C₁₀) aryldiyl or heteroaryldiyl, phenyldiyl, phena-1,4-diyl, naphthadiyl, naphtha-2,6-diyl, pyridindiyl and purindiyl.

30. The dye pair of claim 25, wherein L″

is —

R⁴³—

Z³—

C(O)—

R⁴⁴—

R⁴⁵—

, wherein R⁴³ is a (C₁-C₆) alkyldiyl, and is bonded to R⁴², wherein R⁴² is selected from the group consisting of O, S and NH;

Z³ is selected from the group consisting of a 5-6 membered cyclic alkenyldiyl or heteroalkenyldiyl and a (C₅-C₁₄) aryldiyl or heteroaryldiyl;

R⁴⁴ is selected from the group consisting of O, S and NH; and

R⁴⁵ is a (C₁-C₆) alkyldiyl.

31. The dye pair of claim 25, wherein DD/AD is a fluorescein dye in which the linking moiety is a reactive functional group, and wherein L″

is attached to the fluorescein dye at the xanthene C4 carbon.

32. The dye pair of claim 25 having the structure:

33. The dye pair of claim 32, wherein Y is selected from the group consisting of Y-1, Y-2, Y-3, Y-4, Y-20a, Y-21a, Y-22a, Y-23a, Y-24a, Y-25a, Y-31a, Y-34a, Y-35a, Y-36a, Y-39a, Y-41a, Y-42a, Y-43a, Y-44a, Y-45a and Y-46a.

34. The dye pair of claim 24 having the structure:

35. The dye pair of claim 34, wherein Y¹ is selected from the group consisting of Y-1b, Y-2b, Y-3b, Y-4-b, Y-1c, Y-2c, Y-3c, Y-4-c, Y-20b, Y-20c, Y-21b, Y-21c, Y-22b, Y-22c, Y-23b, Y-23c, Y-24b, Y-24c, Y-25b, Y-25c, Y-31b, Y-31c, Y-34b, Y-34c, Y-35b, Y-35c, Y-36b, Y-36c, Y-37b, Y-39b, Y-39c, Y-41c, Y-42b, Y-43b, Y-43c, Y-46b and Y-46c.

36. The dye pair of claim 34, wherein L is selected from the group consisting of a (C₁-C₆) alkyldiyl and a (C₁-C₃) alkano.

37. The dye pair of claim 34, wherein R⁴¹ is an amide of the formula —

C(O)NR⁴⁵—

, wherein R⁴⁵ is selected from the group consisting of hydrogen and a (C₁-C₆) alkyl.

38. The dye pair of claim 34, wherein Z is selected from the group consisting of a (C₁-C₁₂) alkyl, a (C₁-C₁₂) alkanyl, a (C₅-C₁₀) aryl or heteroaryl, phenyl, naphthyl, naphth-1yl, naphth-2-yl, pyridyl and purinyl.

39. The dye pair of claim 34, wherein L″

is —

R⁴³—

Z³—

C(O)—

R⁴⁴—

R⁴⁵—

, wherein R⁴³ is a (C₁-C₆) alkyldiyl and is bonded to R⁴², wherein R⁴² is selected from the group consisting of O, S and NH;

Z³ is selected from the group consisting of a 5-6 membered cyclic alkenyldiyl or heteroalkenyldiyl and a (C₅-C₁₄) aryldiyl or heteroaryldiyl;

R⁴⁴ is selected from the group consisting of O, S and NH; and

R⁴⁵ is a (C₁-C₆) alkyldiyl.

40. The dye pair of claim 34, wherein DD/AD is a fluorescein dye in which the linking moiety is a reactive group R_x and wherein L″

is attached to the fluorescein dye at the xanthene C5 carbon.

41. The dye pair of claim 34 having the structure:

42. The dye pair of claim 35, wherein Y¹ is selected from the group consisting of Y-1b, Y-2b, Y-3b, Y-4-b, Y-1c, Y-2c, Y-3c, Y-4-c, Y-20b, Y-20c, Y-21b, Y-21c, Y-22b, Y-22c, Y-23b, Y-23c, Y-24b, Y-24c, Y-25b, Y-25c, Y-31b, Y-31c, Y-34b, Y-34c, Y-35b, Y-35c, Y-36b, Y-36c, Y-37b, Y-39b, Y-39c, Y-41c, Y-42b, Y-43b, Y-43c, Y-46b and Y-46c.

43. A polynucleotide labeled with a rhodamine dye according to claim 2 or an energy-transfer dye pair according to claim 28.

44. A method of generating a labeled 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 labeled with a rhodamine dye according to claim 2 or an energy-transfer dye pair according to claim 28.

45. The method of claim 44, wherein the terminator has the structure:

46. The method of claim 44, wherein the terminator is a mixture of four different terminators, one which terminates at a template A, one which terminates at a template G, one which terminates at a template C and one which terminates at a template T or U.

47. The method of claim 44, wherein each of the four different terminators is labeled with a different, spectrally-resolvable fluorophore.