Fluorescent pyrene compounds

• US 8,658,434 B2
• Filed: 10/28/2009
• Issued: 02/25/2014
• Est. Priority Date: 10/28/2009
• Status: Active Grant

• Current Assignee: Biotium, Inc.
• Original Assignee: Biotium, Inc.
• Inventors: Mao, Fei, Leung, Wai-Yee, Cheung, Ching-Ying
• Primary Examiner(s): Haq, Shafiqul
• Assistant Examiner(s): YAKOVLEVA, GALINA M
• Application Number:
  US12/607,915
• Publication Number:
  US 20110097735A1
• Time in Patent Office: 1,581 Days
• Field of Search: None
• US Class Current: 436/544
• CPC Class Codes: C07C 2603/50 Pyrenes; Hydrogenated pyrenes
• CPC Class Codes: C07C 309/47 having at least one of the sulfo groups bound to a carbon atom of a six-membered aromatic ring being part of a condensed ring system
• CPC Class Codes: C07D 207/46 with hetero atoms directly attached to the ring nitrogen atom
• CPC Class Codes: C07D 401/12 linked by a chain containing hetero atoms as chain links
• CPC Class Codes: C07K 1/13 Labelling of peptides
• CPC Class Codes: C08H 1/00 Macromolecular products derived from proteins food proteins A23; glue, gelatine C09H
• CPC Class Codes: G01N 2333/475 Assays involving growth factors
• CPC Class Codes: G01N 33/533 with fluorescent label
• CPC Class Codes: G01N 33/57484 involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites

Abstract

The present invention relates to fluorescent pyrene 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 a is 5.

3. The compound of claim 1, wherein L is a bond.

4. The compound of claim 1, wherein L comprises a water-soluble moiety.

5. The compound of claim 1, wherein L comprises a polyethylene glycol.

6. The compound of claim 1, wherein R_x is a reactive group capable of forming a covalent bond by reacting with an amine group.

7. The compound of claim 1, wherein R_x is an activated ester.

8. The compound of claim 1, wherein R_x has the formula —

CO-(Lg), wherein Lg is a leaving group.

9. The compound of claim 1, wherein R_x is an N-hydroxysuccinimide ester.

10. The compound of claim 1, wherein R_x is an aminooxy.

11. 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.

12. A biomolecule conjugated to the compound of Formula I of claim 1 through the reactive group R_x of the compound of Formula I.

13. The biomolecule of claim 12 wherein the biomolecule comprises a polynucleotide.

14. The biomolecule of claim 12 wherein the biomolecule comprises a polypeptide.

15. The biomolecule of claim 14, wherein the polypeptide further comprises an antigen binding site.

16. The biomolecule of claim 14, wherein the polypeptide is a whole immunoglobulin.

17. The biomolecule of claim 14, wherein the polypeptide is a Fab fragment.

18. An immunoglobin conjugated to the compound of Formula I of claim 1 through the reactive group R_x of the compound of Formula I, wherein the immunoglobin is an antibody that binds specifically to an antigen on a cancer cell.

19. The immunoglobulin of claim 18, wherein the antibody binds to erb2.

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

21. The method of claim 20, wherein the substrate biomolecule is a polypeptide, a polynucleotide, a carbohydrate, a lipid or a combination thereof.

22. The method of claim 20, wherein the substrate biomolecule is a polynucleotide.

23. 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 12, 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.

24. The method of claim 23, further comprises 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.

25. The method of claim 23, wherein the labeling takes place in vitro.

26. The method of claim 23, wherein the labeling takes place in vivo.

27. A method of labeling a polypeptide comprising:

forming a complex that comprises the polypeptide and a binding agent, wherein the binding agent is conjugated to the compound of Formula I of claim 1 through the reactive group R_x of the compound of Formula I.

28. The method of claim 27, wherein the binding agent is an antibody.

29. The method of claim 27, 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.

30. The method of claim 27, wherein the labeling occurs on a solid substrate.

31. The method of claim 27, wherein the labeling occurs intracellularly.

32. The method of claim 27, wherein the complex yields a signal to noise ratio greater than about 100.

33. The method of claim 27, 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).

34. The method of claim 27, 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).

35. The method of claim 27, 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).

36. A compound having the structure

37. A compound having the structure:

38. A compound selected from the group consisting of

39. A kit comprising:

i) the compound of claim 36 or 37;

ii) a buffer;

iii) materials or devices for purifying conjugation products; and

iv) instructions instructing the use of the compound.

40. A biomolecule conjugated to the compound of claim 36 or 37.

41. The biomolecule of claim 40, wherein the biomolecule comprises a polynucleotide.

42. The biomolecule of claim 40, wherein the biomolecule comprises a polypeptide.

43. A method of preparing a labeled biomolecule comprising reacting a compound according to claim 36 or 37 and a substrate biomolecule under conditions sufficient to effect crosslinking between the compound and the substrate biomolecule.