Fluorescent compounds

• US 9,791,450 B2
• Filed: 06/08/2015
• Issued: 10/17/2017
• Est. Priority Date: 12/14/2007
• Status: Active Grant

• Current Assignee: Biotium, Inc.
• Original Assignee: Biotium, Inc.
• Inventors: Mao, Fei, Leung, Wai-Yee, Cheung, Ching-Ying, Hoover, Hye Eun
• Primary Examiner(s): Otton, Alicia L
• Application Number:
  US14/733,764
• Publication Number:
  US 20160033520A1
• Time in Patent Office: 862 Days
• Field of Search: None
• 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. 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. In one aspect, the invention provides a compound having a maximal fluorescence excitation wavelength, wherein the compound has a structure of Formula II:

wherein Z⁻ is a counterion, Y is a bridge unit permitting electron delocalization between F and Ψ, and F is a moiety having the structure:

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

2. The compound of claim 1, wherein when the two adjacent R₁ and/or the two adjacent R₂ form a fused 6-membered ring, the ring so formed is aromatic.

3. The compound of claim 1, wherein Y is:

4. The compound of claim 1, wherein at least one R of R₁ and R₂ is a sulfonate group.

5. The compound of claim 1, wherein at least one R of R₁ and R₂ comprises a sulfonate group or a phosphonate group.

6. The compound of claim 1, wherein each R of R₁ and R₂ comprises a sulfonate group or a phosphonate group.

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

8. The compound of claim 1, wherein two adjacent (R₁)_a and the atoms in ring A to which it is attached are combined to form a carbocyclic ring.

9. The compound of claim 8, wherein the carbocyclic ring is aromatic.

10. The compound of claim 1, wherein two adjacent (R₂)_b and the atoms in ring B to which it is attached are combined to form a carbocyclic ring.

11. The compound of claim 10, wherein the carbocyclic ring is aromatic.

12. The compound of claim 1, wherein the compound has the formula:

13. The compound of claim 1, wherein the compound has the formula:

14. The compound of claim 1, wherein the compound has the formula:

15. The compound of claim 1, wherein the reactive group is selected from the group consisting of an activated esters, a maleimide, an azide, an alkyne, an acyl azide, an isothiocyanate, a halotriazine, a haloacetamide, a sulfonate ester, a phosphoramidite, an alkylhalide, a hydrazide, an acrylamide, and an epoxide.

16. The compound of claim 1, wherein at least two R of the (R)_p-(L)_q- of the compound is a water-soluble polymer which is a polyalkylene oxide radical.

17. The compound of claim 1, wherein with the molecular weight of the water-soluble polymer ranges from 800 to 3000 daltons.

18. A kit comprising:

i) the compound of claim 1;

ii) a buffer;

iii) materials or devices for purifying conjugation products; and

iv) instructions instructing the use of the compound.

19. A biomolecule having an amine, a thiol, a hydroxyl, an azide, an alkyne, an activated ester, or an aldehyde functional group, wherein the biomolecule comprises a compound of claim 1, wherein the at least one reactive group of the compound has undergone a reaction which attaches the compound to the biomolecule.

20. The biomolecule of claim 19, wherein the biomolecule comprises a polynucleotide or a polypeptide.

21. The biomolecule of claim 20, wherein the polypeptide further comprises an antigen binding site.

22. The biomolecule of claim 20, wherein the polypeptide is a whole immunoglobulin or a Fab fragment.

23. An immunoglobin having an amine, a thiol, a hydroxyl, an azide, an alkyne, an activated ester, or an aldehyde functional group, wherein the immunoglobin comprises a compound of claim 1, wherein the at least one reactive group of the compound has undergone a reaction which attaches the compound to the immunoglobin, wherein the immunoglobin is an antibody that binds specifically to an antigen on a cancer cell.

24. An immunoglobulin having an amine, a thiol, a hydroxyl, an azide, an alkyne, an activated ester, or an aldehyde functional group, wherein the immunoglobin is 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, wherein the fluorescent compound is a compound of claim 1.

25. The immunoglobulin of claim 24, wherein the immunoglobulin retains binding specificity to a target upon conjugation to the fluorescent compound.

26. The immunoglobulin of claim 25, wherein the immunoglobin is an antibody that binds specifically to an antigen on a cancer cell.

27. A polypeptide having an amine, a thiol, a hydroxyl, an azide, an alkyne, an activated ester, or an aldehyde functional group, wherein the polypeptide is 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.

28. A method of preparing a labeled biomolecule comprising reacting a compound of claim 1 and a substrate biomolecule under conditions sufficient to effect crosslinking between the compound and the substrate biomolecule.

29. The method of claim 28, wherein the substrate biomolecule is a polypeptide, a polynucleotide, a carbohydrate, a lipid or a combination thereof.

30. 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 19, 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.

31. The method of claim 30, 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.

32. 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 of claim 1, wherein the at least one reactive moiety of the Formula has undergone a reaction which attaches the label to the binding agent.

33. The method of claim 32, wherein the binding agent is an antibody.

34. The method of claim 33, 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.

35. The method of claim 34, 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).