LABELING REAGENTS AND METHODS OF THEIR USE

• US 20090004753A1
• Filed: 01/30/2008
• Published: 01/01/2009
• Est. Priority Date: 01/30/2007
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Invitrogen Corp. (Thermo Fisher Scientific Incorporated)
• Inventors: Rukavishnikov, Aleksey, Witczak, Diane M., Antoulinakis, Evan, Gee, Kyle
• Granted Patent: US 8,586,743 B2
• Time in Patent Office: Days
• US Class Current: 436/166
• CPC Class Codes: C07D 209/14 Radicals substituted by nitrogen atoms, not forming part of a nitro radical
• CPC Class Codes: C07D 209/18 Radicals substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
• CPC Class Codes: C07D 311/18 substituted otherwise than in position 3 or 7
• CPC Class Codes: C07D 311/82 Xanthenes
• CPC Class Codes: C07D 401/10 linked by a carbon chain containing aromatic rings
• CPC Class Codes: C07D 493/14 Ortho-condensed systems
• CPC Class Codes: C07D 495/22 in which the condensed system contains four or more hetero rings
• CPC Class Codes: C07F 5/022 without C-boron linkages
• 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 11/26 Triarylmethane dyes in which at least one of the aromatic nuclei is heterocyclic phthaleins C09B11/24
• CPC Class Codes: C09B 23/04 one >CH- group, e.g. cyanines, isocyanines, pseudocyanines
• CPC Class Codes: C09B 23/08 more than three >CH- groups, e.g. polycarbocyanines
• CPC Class Codes: C09B 45/00 Complex metal compounds of azo dyes
• CPC Class Codes: C09B 56/16 Methine- or polymethine-azo dyes
• CPC Class Codes: G01N 33/532 Production of labelled immunochemicals
• CPC Class Codes: G01N 33/58 involving labelled substances G01N33/53 takes precedence
• CPC Class Codes: G01N 33/582 with fluorescent label

Abstract

The present disclosure is directed to a reactive ester agent capable of conjugating a reporter molecule to a carrier molecule or solid support. The reactive ester agent has the general formula:

wherein the variables are described throughout the application.

Claims

1. A compound of Formula IA or a tautomer or salt thereof:

2. The compound of claim 1, wherein X is an amino, thio, or oxo group.

3. The compound of claim 1, wherein L is a covalent bond, -alkyl-, -substituted alkyl-, -alkenyl-, -substituted alkenyl-, -heterocyclyl-, -substituted heterocyclyl-, -aryl-, -substituted aryl-, -heteroaryl-, -substituted heteroaryl-, -cycloakyl-, -substituted cycloalkyl-, -oxy-, -alkoxy-, -substituted alkoxy-, -thio-, -amino-, or -substituted amino-.

4. The compound of claim 1, wherein R¹ and R² are chloro.

5. The compound of claim 1, wherein R³ is —

COO⁻

, —

SO₃⁻

, substituted azenyl, PEG, phosphate, or bisphosphonate.

6. The compound of claim 1, wherein R³ is —

SO₃⁻

.

7. The compound of claim 1, wherein R^a is a dye.

8. The compound of claim 7, wherein the dye is a xanthene, a cyanine, an indole, a benzofuran, a coumarin, or a borapolyazaindacine.

9. The compound of claim 1, wherein R^a is a chelating moiety, a hapten, an antibody, an enzyme, a radiolabel, a metal ion or metal ion containing substance, a pigment, a chromogen, a phosphor, a fluorogen, a bioluminescent substance, a chemiluminescent substance, or a semiconductor nanocrystal.

10. The compound of claim 1, wherein R^a is avidin, streptavidin or an analog thereof.

11. The compound of claim 1, wherein R^b is a solid support.

12. The compound of claim 1, wherein R^b is a carrier molecule.

13. The compound of claim 12, wherein the carrier molecule is selected from the group consisting of an amino acid, a peptide, a protein, a carbohydrate, a polysaccharide, a nucleoside, a nucleotide, an oligonucleotide, a nucleic acid polymer, a drug, a lipid, and a synthetic polymer.

14. The compound of claim 12, wherein the carrier molecule is a protein.

15. The compound of claim 1, wherein the compound of Formula IA is a salt.

16. The compound of claim 15, wherein the salt comprises a potassium or sodium ion.

17. The compound of claim 1, wherein R^a is:

18. The compound of claim 1, wherein R^a is:

19. The compound of claim 1, wherein R^a is:

20. The compound of claim 1, wherein R^a is:

21. The compound of claim 1, wherein R^a is:

22. A compound of Formula II or a tautomer or salt thereof:

23. A compound of Formula III or a tautomer or salt thereof:

24. A compound selected from the group consisting of:

25. A compound having the following structure or a tautomer or salt thereof:

26. The compound of claim 25, wherein R¹ and R² are chloro.

27. The compound of claim 25, wherein R³ is —

SO₃⁻

.

28. A method of making a compound of Formula I comprising:

contacting a carrier molecule or a solid support comprising a nucleophile with a compound of Formula IA or a tautomer or salt thereof;

29. The method of claim 28, wherein X is an amino, thio, or oxo group.

30. The method of claim 28, wherein L is a covalent bond, -alkyl-, -substituted alkyl-, -alkenyl-, -substituted alkenyl-, -heterocyclyl-, -substituted heterocyclyl-, -aryl-, -substituted aryl-, -heteroaryl-, -substituted heteroaryl-, -cycloakyl-, -substituted cycloalkyl-, -oxy-, -alkoxy-, -substituted alkoxy-, -thio-, -amino-, or -substituted amino-.

31. The method of claim 28, wherein R¹ and R² are chloro.

32. The method of claim 28, wherein R³ is —

COO⁻

, —

SO₃⁻

, substituted azenyl, PEG, phosphate, or bisphosphonate.

33. The method of claim 28, wherein R³ is —

SO₃⁻

.

34. The method of claim 28, wherein R^a is a dye.

35. The method of claim 34, wherein the dye is a xanthene, a cyanine, an indole, a benzofuran, a coumarin, or a borapolyazaindacine.

36. The method of claim 28, wherein R^a is a chelating moiety, a hapten, an antibody, an enzyme, a radiolabel, a metal ion or metal ion containing substance, a pigment, a chromogen, a phosphor, a fluorogen, a bioluminescent substance, a chemiluminescent substance, or a semiconductor nanocrystal.

37. The method of claim 28, wherein R^a is avidin, streptavidin or an analog thereof.

38. The method of claim 28, wherein R^b is a solid support.

39. The method of claim 28, wherein R^b is a carrier molecule.

40. The method of claim 39, wherein the carrier molecule is selected from the group consisting of an amino acid, a peptide, a protein, a carbohydrate, a polysaccharide, a nucleoside, a nucleotide, an oligonucleotide, a nucleic acid polymer, a drug, a lipid, and a synthetic polymer.

41. The method of claim 39, wherein the carrier molecule is a protein.

42. The method of claim 28, wherein the compound of Formula IA is a salt.

43. The method of claim 42, wherein the salt comprises a potassium or sodium ion.

44. The method of claim 28, further comprising incubating the carrier molecule or solid support with the compound of Formula IA after the contacting step.

45. The method of claim 28, wherein the contacting step is done in an aqueous solution.

46. The method of claim 28, wherein the reporter molecule is hydrophobic.

47. The method of claim 28, wherein the compound of Formula IA is soluble in an aqueous solution.

48. The method of claim 28, wherein R^a is:

49. The method of claim 48, wherein R⁸ is the point of attachment to L is through a -phenyl-.

50. The method of claim 28, wherein R^a is:

51. A method of labeling a carrier molecule or solid support comprising:

contacting the carrier molecule or solid support with a compound of Formula IA or a tautomer or salt thereof;

52. The method of claim 51, wherein the labeled carrier molecule or solid support comprises a compound of Formula I:

53. A method of making a compound of Formula IA or a tautomer or salt thereof comprising:

contacting a compound of Formula IB or a tautomer or salt thereof;

54. The method of claim 53, wherein the contacting step is done in the presence of dimethylaminopyridine (DMAP).

55. The method of claim 53, wherein the contacting step is done in an organic solvent.

56. A kit for forming a conjugate with a carrier molecule or solid support and a labeling reagent, wherein the kit comprises:

a) a labeling reagent according to Formula IA or a tautomer or salt thereof;