Electron-deficient nitrogen heterocycle-substituted fluorescein dyes

• US 6,221,604 B1
• Filed: 02/07/2000
• Issued: 04/24/2001
• Est. Priority Date: 02/07/2000
• Status: Expired

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: PE Corp.
• Inventors: Upadhya, Krishna G., Menchen, Steven M., Zhen, Weiguo
• Primary Examiner(s): CEPERLEY, MARY
• Application Number:
  US09/498,702
• Time in Patent Office: 442 Days
• Field of Search: 435/6, 435/546, 435/800, 436/546, 436/800, 530/402, 536/24.32, 544/235, 544/238, 544/283, 544/333, 544/353, 544/405, 546/119, 546/144, 546/173, 546/283.1, 548/146, 548/159, 548/215, 548/217, 548/250, 548/256, 548/266.2, 548/305, 548/311.4, 548/364.4, 935/8
• US Class Current: 435/6.12
• CPC Class Codes: C07D 405/04 directly linked by a ring-member-to-ring-member bond
• CPC Class Codes: C07D 405/14 containing three or more hetero rings
• 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 11/08 Phthaleins; Phenolphthaleins; Fluorescein
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine

Abstract

The invention provides compositions electron-deficient nitrogen heterocycle-substituted fluorescein dyes and methods in which the dyes are conjugated to substrates and used as detection labels in molecular biology experiments. The electron-deficient nitrogen heterocycles include pyridine, quinoline, pyrazine, and the like. Substrates include polynucleotides, nucleosides, nucleotides, peptides, proteins, carbohydrates, and ligands.

Claims

1. A compound having the formula:

wherein;

at least one of R¹, R², R³, R⁴, R⁵, or R⁷ is an electron-deficient nitrogen heterocycle;

R¹, when taken alone, is H, F, Cl, (C₁-C₆) alkyl, (C₁-C₆) substituted alkyl, (C₁-C₆) alkoxy, sulfonate, sulfone, amino, imminium, amido, nitrile, reactive linking group, phenyl, substituted phenyl, aryl, substituted aryl, or heterocycle, or when taken together with R⁷ is benzo or heterocycle;

R², when taken alone, is H, F, Cl, (C₁-C₆) alkyl, (C₁-C₆) substituted alkyl, (C₁-C₆) alkoxy, sulfonate, sulfone, amino, imminium, amido, nitrile, reactive linking group, phenyl, substituted phenyl, aryl, substituted aryl, or heterocycle;

R³, when taken alone, is H, F, Cl, (C₁-C₆) alkyl, (C₁-C₆) substituted alkyl, (C₁-C₆) alkoxy, sulfonate, sulfone, amino, imminium, amido, nitrile, reactive linking group, phenyl, substituted phenyl, aryl, substituted aryl, or heterocycle;

R⁴, when taken alone, is H, F, Cl, (C₁-C₆) alkyl, (C₁-C₆) substituted alkyl, (C₁-C₆) alkoxy, sulfonate, sulfone, amino, imminium, amido, nitrile, reactive linking group, phenyl, substituted phenyl, aryl, substituted aryl, or heterocycle, or when taken together with R⁵ is benzo or heterocycle;

R⁵, when taken alone, is H, F, Cl, (C₁-C₆) alkyl, (C₁-C₆) substituted alkyl, (C₁-C₆) alkoxy, sulfonate, sulfone, amino, imminium, amido, nitrile, reactive linking group, phenyl, substituted phenyl, aryl, substituted aryl, or heterocycle, or when taken together with R⁴ is benzo or heterocycle;

R⁷, when taken alone, is H, F, Cl, (C₁-C₆) alkyl, (C₁-C₆) substituted alkyl, (C-C₆) alkoxy, sulfonate, sulfone, amino, imminium, amido, nitrile, reactive linking group, phenyl, substituted phenyl, aryl, substituted aryl, or heterocycle, or when taken together with R¹ is benzo or heterocycle; and

R⁶ is selected from the group consisting of (C₁-C₆) alkyl, (C₂-C₆) alkene, (C₂-C₆) alkyne, cyano, heterocyclic aromatic, phenyl, and substituted phenyl having the structure;

wherein X¹, X², X³, X⁴ and X⁵ taken separately are H, Cl, F, (C₁-C₆) alkyl, (C₂-C₆) alkene, (C₂-C₆) alkyne, CO₂H, SO₃H, CH₂OH, or reactive linking group.

2. The fluorescein dye of claim 1 wherein the electron-deficient heterocycle is selected from the group consisting of 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-quinolyl, 3-quinolyl, 4-quinolyl, 2-imidazole, 4-imidazole, 3-pyrazole, 4-pyrazole, pyridazine, pyrimidine, pyrazine, cinnoline, pthalazine, quinazoline, quinoxaline, 3-(1,2,4-N)-triazolyl, 5-(1,2,4-N)-triazolyl, 5-tetrazolyl, 4-(1-O, 3-N)-oxazole, 5-(1-O, 3-N)-oxazole, 4-(1-S, 3-N)-thiazole, 5-(1-S, 3-N)-thiazole, 2-benzoxazole, 2-benzothiazole, 4-(1,2,3-N)-benzotriazole, and benzimidazole.

3. The fluorescein dye of claim 1 wherein R⁴ taken together with R⁵ is benzo.

4. The fluorescein dye of claim 1 wherein R¹ taken together with R⁷ is benzo.

5. The fluorescein dye of claim 1 in which R⁶ is a substituted phenyl having the structure:

6. The fluorescein dye of claim 5 wherein one of X³ and X⁴ is carboxyl and the other is hydrogen.

7. The fluorescein dye of claim 1 wherein R¹, R², R³ and R⁴ each taken separately are phenyl or substituted phenyl.

8. The fluorescein dye of claim 1 wherein R¹, R², R³ and R⁴ each taken separately are naphthyl or substituted naphthyl.

9. The fluorescein dye of claim 1 wherein R² and R³ each taken separately are fluoro or chloro.

10. The fluorescein dye of claim 1 wherein R² and R³ each taken separately are 2-pyridyl or 3-pyridyl.

11. The fluorescein dye of claim 1 wherein R² and R³ each taken separately are 2-quinolyl or 3-quinolyl.

12. The fluorescein dye of claim 1 wherein R⁵ and R⁷ are hydrogen.

13. The fluorescein dye of claim 1 in which the reactive linking group is selected from the group consisting of succinimidyl ester, isothiocyanate, sulfonyl chloride, 2,6-dichlorotriazinyl, pentafluorophenyl ester, phosphoramidite, maleimide, haloacetyl, and iodoacetamide.

15. A method of labelling a substrate with a fluorescein dye of claim 1, comprising the step of reacting the substrate with the reactive linking group of the fluorescein dye wherein a substrate-dye conjugate is formed.

16. The method of claim 15 wherein the reactive linking group is N-hydroxysuccinimide.

17. The method of claim 15 wherein the reactive linking group is phosphoramidite.

18. The method of claim 15 wherein the substrate is selected from the group consisting of a polynucleotide, a nucleotide, a nucleoside, a peptide, a protein, a carbohydrate, a ligand, a particle, and a surface.

19. The method of claim 18 wherein the particle is a nanoparticle, a microsphere, a bead, and a liposome.

20. The method of claim 18 wherein the surface is glass.

21. An energy transfer dye compound comprising:

a donor dye capable of absorbing light at a first wavelength and emitting excitation energy in response linked by a linker to an acceptor dye capable of absorbing the excitation energy emitted by the donor dye and fluorescing at a second wavelength in response;

wherein at least one of the donor dye and acceptor dye is a fluorescein dye of claim 1.

22. The energy transfer dye of claim 21 wherein the linker has the structure:

23. The energy transfer dye of claim 21 wherein the linker has the structure:

24. The energy transfer dye of claim 21 in which the linker has the structure:

wherein n is 1 or 2.

25. A labelled nucleoside or nucleotide having the formula:

wherein DYE is a fluorescein dye of claim 1 or an energy transfer dye of claim 23;

B is a nucleobase;

R⁸ is H, monophosphate, diphosphate, triphosphate, thiophosphate, or phosphate analog;

R⁹ and R¹⁰, when taken alone, are each independently H, HO, F, and a moiety which blocks polymerase-mediated target-directed polymerization, or when taken together form 2′

-3′

-didehydroribose; and

L is a linker.

26. The labelled nucleoside or nucleotide of claim 25 wherein B is selected from the group consisting of uracil, thymine, cytosine, adenine, 7-deazaadenine, guanine, and 8-deazaguanosine.

27. The labelled nucleoside or nucleotide of claim 25 in which L is:

wherein n is 0, 1, or 2.

28. The labelled nucleoside or nucleotide of claim 25 which is enzymatically incorporatable.

29. The labelled nucleoside or nucleotide of claim 25 which is a terminator.

30. The terminator nucleotide of claim 29 which has the structure:

wherein DYE is a fluorescein dye;

B is a nucleobase;

R⁸ is triphosphate, α

-thiotriphosphate, or triphosphate analog;

R⁹ and R¹⁰, when taken alone, are each independently H, F, and a moiety which blocks polymerase-mediated target-directed polymerization, or when taken together form 2′

-3′

-didehydroribose; and

L is a linker.

31. The labelled nucleoside or nucleotide of claim 25 which is enzymatically extendable.

32. A labelled oligonucleotide having the formula:

wherein the oligonucleotide comprises 2 to 100 nucleotides;

DYE is a fluorescein dye of claim 1 or an energy transfer dye of claim 21;

B is a nucleobase;

L is a linker;

R¹⁰ is H, OH, halide, azide, amine, alkylamine, alkyl (C₁-C₆), allyl, alkoxy (C₁-C₆), OCH₃, or OCH₂CH═

CH₂;

R¹⁵ is H, phosphate, internucleotide phosphodiester, or internucleotide analog; and

R¹⁶ is H, phosphate, internucleotide phosphodiester, or internucleotide analog.

33. A labelled oligonucleotide having the formula:

wherein the oligonucleotide comprises 2 to 100 nucleotides;

DYE is a fluorescein dye of claim 1;

X is O, NH, or S;

B is a nucleobase;

L is a linker;

R¹⁰ is H, OH, halide, azide, amine, alkylamine, alkyl (C₁-C₆), allyl, alkoxy (C₁-C₆), OCH₃, or OCH₂CH═

CH₂; and

R¹⁵ is internucleotide phosphodiester or internucleotide analog.

34. The labelled oligonucleotide of claim 33 in which L is alkyldiyl (C₁-C₁₂).

35. The labelled oligonucleotide of claim 33 in which L is a mobility-modifier comprising —

(CH₂CH₂O)_n—

, where n is 1 to 100.

36. A phosphoramidite compound having the formula:

wherein DYE is a fluorescein dye of claim 1 or an energy transfer dye of claim 21;

L is a linker;

R¹¹ and R¹² taken separately are selected from the group consisting of alkyl (C₁-C₁₂), alkene, aryl, and cycloalkyl containing up to 10 carbon atoms;

or R¹¹ and R¹² taken together with the nitrogen atom form a saturated nitrogen heterocycle; and

R¹³ is a phosphite ester protecting group.

37. The phosphoramidite compound of claim 36 wherein R¹³ is selected from the group consisting of methyl, 2-cyanoethyl, and 2-(4-nitrophenyl)ethyl.

38. The phosphoramidite compound of claim 36 wherein R¹¹ and R¹² are each isopropyl.

39. The phosphoramidite compound of claim 36 wherein R¹¹ and R¹² taken together is morpholino.

40. The phosphoramidite compound of claim 36 wherein L is alkyldiyl (C₁-C₁₂).

41. The phosphoramidite compound of claim 36 wherein the fluorescein dye is attached at R⁶ by a linker.

42. The phosphoramidite compound of the structure:

wherein DYE is a fluorescein dye of claim 1.

43. A phosphoramidite compound having the formula

wherein DYE is a fluorescein dye of claim 1 or an energy transfer dye of claim 21;

B is a nucleobase;

L is a linker;

R¹¹ and R¹² taken separately are selected from the group consisting of alkyl (C₁-C₆), alkene, aryl, and cycloalkyl containing up to 10 carbon atoms;

or R¹¹ and R¹² taken together with the nitrogen atom form a saturated nitrogen heterocycle;

R¹³ is a phosphite ester protecting group; and

R¹⁴ is an acid-cleavable hydroxyl protecting group.

44. The phosphoramidite compound of claim 43 wherein R¹³ is selected from the group consisting of methyl, 2-cyanoethyl, and 2-(4-nitrophenyl)ethyl.

45. The phosphoramidite compound of claim 43 wherein R¹¹ and R¹² are each isopropyl.

46. The phosphoramidite compound of claim 43 wherein R¹¹ and R¹² taken together is morpholino.

47. The compound of claim 43 wherein L is:

and n ranges from 2 to 10.

48. The compound of claim 43 wherein L is:

and n is0, 1, or 2.

49. The compound of claim 43 wherein L is

and n ranges from 1 to 10.

50. The compound of claim 43 wherein B is selected from the group consisting of uracil, thymine, cytosine, adenine, 7-deazaadenine, guanine, and 8-deazaguanosine.

51. A method of synthesizing a labelled oligonucleotide comprising the step of coupling a phosphoramidite compound of claim 36 to an oligonucleotide on a solid support.

52. A method of synthesizing a labelled oligonucleotide comprising the step of coupling a nucleoside phosphoramidite reagent to a solid support wherein the solid support is labelled with a dye according to claim 1 or an energy transfer compound of claim 21.

53. A method of generating a labelled 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 labelled with a dye according to claim 1 or an energy transfer compound of claim 21.

54. A method of oligonucleotide ligation, comprising the steps of:

annealing two probes to a target sequence and

forming a phosphodiester bond between the 5′

terminus of one probe and the 3′

terminus of the other probe;

wherein one or both probes are labelled with a dye according to claim 1 or an energy transfer compound of claim 21.

55. A method of fragment analysis comprising the steps of:

subjecting polynucleotide fragments, the fragments being labelled with a fluorescein dye of claim 1 or an energy transfer compound of claim 21, to a size-dependent separation process; and

detecting the labelled polynucleotide fragment subsequent to the separation process.

56. The method of claim 55 wherein the fragments are labelled with a mobility-modifying label.

57. The method of claim 55 wherein the fragments are formed by ligation.

58. The method of claim 55 wherein the size-dependent separation process is electrophoresis and the labelled polynucleotide fragment is detected by fluorescence.

59. A method of amplification comprising the steps of:

annealing two or more primers to a target DNA sequence and

extending the primers by polymerase and a mixture of enzymatically-extendable nucleotides;

wherein a primer or a nucleotide is labelled with a dye according to claim 1.

60. A method of amplification comprising the steps of:

annealing two or more primers and a fluorescent dye-quencher probe to a target DNA sequence and

extending the primers by polymerase and a mixture of enzymatically-extendable nucleotides;

wherein the probe is labelled with a dye according to claim 1.

61. A kit for labelling an oligonucleotide, comprising a dye including a reactive linking group according to claim 1 and an oligonucleotide.

62. A kit for labelling an oligonucleotide, comprising a phosphoramidite compound according to claim 36 and an oligonucleotide.

63. A kit for generating a labelled primer extension product, comprising enzymatically-extendable nucleotides capable of supporting continuous primer extension, a terminator and a primer, wherein said primer or said terminator is labelled with a dye according to claim 1.

64. A kit for generating a labelled primer extension product, comprising enzymatically-extendable nucleotides capable of supporting continuous primer extension, a terminator and a primer, wherein said primer or said terminator is labelled with an energy transfer dye of claim 21.

14. Fluorescein dyes having the structures:

65. A kit for generating a labelled primer extension product, comprising enzymatically-extendable nucleotides capable of supporting continuous primer extension, a terminator and a primer, wherein said primer or said terminator is labelled with a dye according to claim 14.

66. The kit of claim 65 in which the terminator is a set of four different terminators, one which terminates at a target A, one which terminates at a target G, one which terminates at a target C and one which terminates at a target T or U.

67. The kit of claim 66 in which the set of four different terminators is a set of mobility-matched terminators.