FLUORESCENT COMPOUNDS

• US 20090305410A1
• Filed: 12/12/2008
• Published: 12/10/2009
• Est. Priority Date: 12/14/2007
• Status: Active Grant

• Current Assignee: Biotium, Inc.
• Original Assignee: Biotium, Inc.
• Inventors: Leung, Wai-Yee, Cheung, Ching-Ying, Mao, Fei, Hoover, Hye Eun
• Granted Patent: US 9,097,667 B2
• Time in Patent Office: Days
• US Class Current: 435/375
• CPC Class Codes: C09B 11/08 Phthaleins; Phenolphthaleins; Fluorescein
• 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: C09B 23/0066 the polymethine chain being part of a carbocyclic ring,(e.g. benzene, naphtalene, cyclohexene, cyclobutenene-quadratic acid)
• CPC Class Codes: C09B 23/06 three >CH- groups, e.g. carbocyanines
• CPC Class Codes: C09B 23/083 five >CH- groups
• CPC Class Codes: C09B 23/086 more than five >CH- groups
• CPC Class Codes: C09B 57/001 Pyrene dyes
• CPC Class Codes: C09B 57/02 Coumarine dyes
• CPC Class Codes: C09B 69/00 Dyes not provided for by a single group of this subclass
• CPC Class Codes: G01N 2021/6439 with indicators, stains, dyes, tags, labels, marks
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 33/582 with fluorescent label

Abstract

The present invention relates to fluorescent dyes in general. The present invention provides a wide range of fluorescent dyes and kits containing the same, which are applicable for labeling a variety of biomolecules, cells and microorganisms. The present invention also provides various methods of using the fluorescent dyes for research and development, forensic identification, environmental studies, diagnosis, prognosis, and/or treatment of disease conditions.

Claims

1. A compound of Formula I:

2. The compound of claim 1, wherein the fluorophore is a xanthene dye, a coumarin dye, a pyrene dye or a cyanine dye.

3. The compound of claim 1, wherein the fluorophore is a coumarin dye.

4. The compound of claim 1, wherein the fluorophore is a pyrene dye.

5. The compound of claim 1, wherein the fluorophore is a rhodamine dye.

6. The compound of claim 1, wherein the water-soluble polymer is a polyalkylene oxide.

7. The compound of claim 1, wherein the water-soluble polymer is a polyethylene oxide.

8. The compound of claim 1, wherein the water-soluble polymer is a carbohydrate.

9. The compound of claim 1, wherein the water-soluble polymer is a polypeptide.

10. The compound of claim 1, wherein the water-soluble polymer has a molecular weight of greater than about 300 Da.

11. The compound of claim 1, wherein the water-soluble polymer has a molecular weight of greater than about 800 Da.

12. The compound of claim 1, wherein the water-soluble polymer has a molecular weight ranging from about 800 Da to about 3000 Da.

13. The compound of claim 1, wherein the reactive group forms a covalent bond with an amino, a sulfhydryl or a hydroxy nucleophile.

14. The compound of claim 1, wherein the reactive group is an isothiocyanate, an isocyanate, a monochlorotriazine, a dichlorotriazine, a halogen-substituted pyridine, a halogen-substituted diazine, a phosphoramidite, a maleimide, an aziridine, a sulfonyl halide, an acid halide, a hydroxysuccinimidyl ester, a hydroxysulfosuccinimidyl ester, a tetrafluorophenol ester, an imido ester, a hydrazine, an azidonitrophenyl, an azide, an alkyne, a 3-(2-pyridyl dithio)-propionamide, a glyoxal or an aldehyde.

15. The compound of claim 1, wherein a maximal fluorescence excitation wavelength of the compound ranges from about 350 to about 1200 nm.

16. The compound of claim 1, wherein a maximal fluorescence emission wavelength of the compound ranges from about 360 to 1250 nm.

17. The compound of claim 1, wherein the fluorophore is a coumarin of the formula:

18. The compound of claim 17, wherein

19. The compound of claim 17, wherein the fluorophore is substituted by one or more sulfonate groups.

20. The compound of claim 1, wherein the fluorophore is a compound of the formula:

21. The compound of claim 20, wherein m is 0 and

22. The compound of claim 21, wherein R₁, comprises a water soluble polymer group and R₂ comprises a reactive group.

23. The compound of claim 22, wherein R₁, comprises a polyethylene glycol.

24. The compound of claim 22, wherein R₂ comprises an N-hydroxysuccinimide group.

25. A compound having a maximal fluorescence excitation wavelength, wherein the compound has a structure of Formula II:

F—

Y═

Ψ

 

 

Formula II

wherein;

F is a moiety having the structure;

26. The compound of claim 25, wherein when at least two adjacent R₁, and/or two adjacent R₂ are present, the two adjacent R₁, and/or the two adjacent R₂ are combinable to form a 6-membered ring which is unsubstituted or substituted by one or more (R)_p-(L)_q-.

27. The compound of claim 26 wherein when the two adjacent R₁, and/or the two adjacent R₂ are combinable to form a 6-membered ring, the ring so formed is aromatic.

28. The compound of claim 25 wherein when Ψ

is Formula 1, and the maximal fluorescence excitation wavelength of the compound is equal to or greater than 660 nm, or Ψ

is other than Formula 1, then R₅ and R₆ are independently (R)_p (L)_q-, or R₅ and R₆ are combinable to form a cyclic moiety which is unsubstituted or substituted by one or more (R)_p-(L)_q-.

29. The compound of claim 25, wherein Y is:

30. The compound of claim 25, wherein at least one R of R₁ and R₂ is a charged moiety.

31. The compound of claim 25, wherein at least one R of R₁ and R₂ comprises a sulfonate group or a phosphonate group.

32. The compound of claim 25, wherein each R of R₁ and R₂ comprises a sulfonate group or a phosphonate group.

33. The compound of claim 25, wherein X₂ and X₃ are independently

34. The compound of claim 25, wherein X₂ and X₃ are independently

35. The compound of claim 25, wherein the water-soluble polymer is a polyalkylene oxide.

36. The compound of claim 25, wherein the water-soluble polymer is a polyethylene oxide.

37. The compound of claim 25, wherein the water-soluble polymer has a molecular weight of greater than about 300.

38. The compound of claim 25, wherein the water-soluble polymer has a molecular weight of greater than about 800.

39. The compound of claim 25, wherein the water-soluble polymer has a molecular weight ranging from about 800 to about 3000.

40. The compound of claim 26, wherein two adjacent (R₁)_a and the atoms in ring A to which it is attached are combined to form a carbocyclic ring.

41. The compound of claim 40, wherein the carbocyclic ring is aromatic.

42. The compound of claim 26, wherein two adjacent (R₂)_b and the atoms in ring B to which it is attached are combined to form a carbocyclic ring.

43. The compound of claim 42, wherein the carbocyclic ring is aromatic.

44. The compound of claim 25, wherein the compound has the formula:

45. The compound of claim 25, wherein the compound has the formula:

46. The compound of claim 25, wherein the compound has the formula:

47. The compound of claim 25, wherein the compound has the formula:

48. The compound of claim 25, wherein the compound has the formula:

49. The compound of claim 25, wherein the compound has the formula:

50. The compound of claim 25, wherein the maximal fluorescence excitation wavelength of the compound ranges from about 350 to about 1200 nm.

51. The compound of claim 25, wherein a maximal fluorescence emission wavelength of the compound ranges from about 360 to about 1250 nm.

52. The compound of claim 25, wherein the water-soluble polymer is a polyalkylene oxide.

53. The compound of claim 25, wherein the water-soluble polymer is a polyethylene oxide.

54. The compound of claim 25, wherein the water-soluble polymer is a carbohydrate.

55. The compound of claim 25, wherein the water-soluble polymer is a polypeptide.

56. The compound of claim 25, wherein the water-soluble polymer has a molecular weight of greater than 300.

57. The compound of claim 25, wherein the water-soluble polymer has a molecular weight of greater than 800.

58. The compound of claim 25, wherein the compound is

59. A substituted cyanine dye comprising a reactive group and one or more water soluble polymer groups, wherein the cyanine dye has a maximal fluorescence excitation wavelength of equal to or greater than about 660 nm.

60. A substituted cyanine dye comprising a reactive group and one or more water soluble polymer groups, wherein the cyanine dye has an absorption maximal wavelength of equal to or greater than about 660 nm.

61. The substituted cyanine dye of claim 59 or 60 wherein the dye is substituted by a non-spiro moiety.

62. A binding agent labeled with a substituted cyanine dye of claim 59 or 60, wherein the binding agent binds selectively to a target polypeptide to form a complex, and wherein formation of the complex yields a signal to noise ratio of fluorescence that is at least 100.

63. The binding agent of claim 62 wherein the substituted cyanine dye is substituted with a non-spiro substituent.

64. The binding agent of claim 62 wherein the binding agent is a polypeptide.

65. The binding agent of claim 63 wherein the polypeptide is an antibody.

66. A compound of Formula III:

67. The compound of claim 66 wherein at least one of R₆, R₇, R₈ or R₉ is combinable with a neighboring R₁ or R₂ and any intervening atoms in a ring to which the neighboring R₁ or R₂ is attached, to form a 5- or 6-membered ring which is unsubstituted or substituted by one or more (R)_p (L)_q-.

68. The compound of claim 67 wherein when the at least one of R₆, R₇, R₈ or R₉ and a neighboring R₁ or R₂ are combinable to form a 5- or 6-membered ring, the ring is unsaturated.

69. The compound of claim 67 wherein when the at least one of R₆, R₇, R₈ or R₉ and a neighboring R₁ or R₂ are combinable to form a 5- or 6-membered ring, the ring is saturated.

70. The compound of claim 66, wherein X₁ is ═

NH₂ .

71. The compound of claim 66, wherein X₁ is ═

O.

72. The compound of claim 66, wherein X₂ is —

OH.

73. The compound of claim 66, wherein X₂ is —

NH₂.

74. The compound of claim 66, wherein a maximal fluorescence excitation wavelength of the compound ranges from about 450 to 750 nm.

75. The compound of claim 66, wherein a maximal fluorescence emission wavelength of the compound ranges from about 470 to 800 nm.

76. A compound of Formula V:

77. A kit comprising:

i) the compound of claim 1, 25, 59, 60, 66, or 76;

ii) a buffer;

iii) materials or devices for purifying conjugation products; and

iv) instructions instructing the use of the compound.

78. A biomolecule comprising a label having a structure of a Formula of claim 1, 25, 59, 60, 66, or 76, wherein the at least one reactive moiety of the Formula has undergone a reaction which attaches the label to the biomolecule.

79. The biomolecule comprising a label of claim 78 wherein the biomolecule comprises a polynucleotide.

80. The biomolecule comprising a label of claim 78 wherein the biomolecule comprises a polypeptide.

81. The biomolecule of claim 80, wherein the polypeptide further comprises an antigen binding site.

82. The biomolecule of claim 80, wherein the polypeptide is a whole immunoglobulin.

83. The biomolecule of claim 80, wherein the polypeptide is a Fab fragment.

84. An immunoglobin comprising a label having a structure of a Formula of claim 1, 25, 59, 60, 66, or 76 wherein the at least one reactive moiety of the Formula has undergone a reaction which attaches the label to the immunoglobin, wherein the immunoglobin is an antibody that binds specifically to an antigen on a cancer cell.

85. The immunoglobin of claim 84 wherein the antibody binds to erb2.

86. A method of preparing a labeled biomolecule comprising reacting a compound of claim 1, 25, 59, 60, 66, or 76 and a substrate biomolecule under conditions sufficient to effect crosslinking between the compound and the substrate biomolecule.

87. The method of claim 86, wherein the substrate biomolecule is a polypeptide, a polynucleotide, a carbohydrate, a lipid or a combination thereof.

88. The method of claim 87, wherein the substrate biomolecule is a polynucleotide.

89. A method for labeling a cell within a population of cells whereby the cell is differentially labeled relative to neighboring cells within the population, the method comprising contacting the cell with a biomolecule of claim 78, wherein the biomolecule comprises a targeting moiety that binds to a binding partner that is indicative of the cell, and thereby differentially labeling the cell relative to neighboring cells within the population.

90. The method of claim 89, further comprising the step of imaging the cell, the imaging step comprising:

i) directing exciting wavelength to the cell; and

ii) detecting emitted fluorescence from the cell.

91. The method of claim 89, wherein the labeling takes place in vitro.

92. The method of claim 89, wherein the labeling takes place in vivo.

93. An immunoglobulin labeled with a fluorescent compound comprising a polyalkylene oxide and a fluorophore that has an absorption maximal wavelength equal to or greater than 685 nm.

94. An immunoglobulin labeled with a fluorescent compound comprising a polyalkylene oxide and a fluorophore that has an absorption maximal wavelength at or greater than 750 nm.

95. The immunoglobulin of claim 93 or 94, wherein the immunoglobulin retains binding specificity to a target upon conjugation to the fluorescent compound.

96. The immunoglobulin of claim 95, wherein the immunoglobin is an antibody that binds specifically to an antigen on a cancer cell.

97. The immunoglobin of claim 96 wherein the antibody binds to erb2.

98. The immunoglobulin of claim 93 or 94, wherein the fluorescent compound is a compound of claim 1, 25, 59, 60, or 66.

99. A polypeptide labeled with a fluorescent compound, the polypeptide exhibiting a serum half-life no shorter than that of a corresponding polypeptide that lacks the fluorescent compound, wherein the fluorescent compound is a compound of claim 1, 25, 59, 60, 66, or 76.

100. A method of labeling a polypeptide comprising:

forming a complex that comprises the polypeptide and a binding agent, wherein the binding agent comprises a fluorescent label having a structure of formula of claim 1, 25, 59, 60, 66, or 76, wherein the at least one reactive moiety of the Formula has undergone a reaction which attaches the label to the binding agent.

101. The method of claim 100 wherein the binding agent is an antibody.

102. The method of claim 101 wherein the complex comprises (a) a primary antibody that binds to the polypeptide, and (b) the binding agent which functions as a secondary antibody exhibiting binding capability to the primary antibody.

103. The method of claim 102, wherein the labeling occurs on a solid substrate.

104. The method of claim 102 that labels a polypeptide intracellularly.

105. The method of claim 102, wherein the complex yields a signal to noise ratio greater than about 100, wherein the signal to noise ratio is calculated by the formula:

(fluorescent signal from a complex comprising the polypeptide bound by a primary antibody which in turn is bound to the binding agent)/(fluorescent signal from a mixture of the polypeptide, an isotype control primary antibody and the binding agent).

106. The method of claim 102, wherein the complex yields a signal to noise ratio greater than about 250, wherein the signal to noise ratio is calculated by the formula:

(fluorescent signal from a complex comprising the polypeptide bound by a primary antibody which in turn is bound to the binding agent)/(fluorescent signal from a mixture of the polypeptide, an isotype control primary antibody and the binding agent).

107. The method of claim 102, wherein the complex yields a signal to noise ratio greater than about 270, wherein the signal to noise ratio is calculated by the formula:

(fluorescent signal from a complex comprising the polypeptide bound by a primary antibody which in turn is bound to the binding agent)/(fluorescent signal from a mixture of the polypeptide, an isotype control primary antibody and the binding agent).

108. The method of claim 102, wherein the complex yields a total fluorescence signal at least 5% greater than that generated by a complex formed with the same primary antibody and with the same secondary antibody that has a comparable degree of labeling with a DyLight 680™

dye.

109. The method of claim 102 wherein the complex yields a total fluorescence signal at least 5% greater than that generated by a complex formed with the same primary antibody and with the same secondary antibody that has a comparable degree of labeling with a Cy5.5®

dye.

110. The method of claim 102 wherein the complex yields a total fluorescence signal at least 5% greater than that generated by a complex formed with the same primary antibody and with the same secondary antibody that has a comparable degree of labeling with an Alexa Fluor 680®

dye.

111. The method of claim 108, 109, or 110 wherein the label having a structure of a Formula of claim 25 is the compound having the structure:

112. The method of claim 111 wherein each complex is excited at 635 nm or 633 nm.

113. The method of claim 111 wherein each complex is present at identical protein concentrations.