Chemoselective fluorgenic molecular linkers and methods for their preparation and use

• US 7,745,229 B2
• Filed: 04/15/2005
• Issued: 06/29/2010
• Est. Priority Date: 04/16/2004
• Status: Active Grant

• Current Assignee: University of South Carolina
• Original Assignee: University of South Carolina
• Inventors: Sivakumar, Krishnamoorthy, Wang, Qian
• Primary Examiner(s): Haq; Shafiqul
• Application Number:
  US11/578,565
• Publication Number:
  US 20070224695A1
• Time in Patent Office: 1,901 Days
• Field of Search: None
• US Class Current: 436/546
• 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: C07D 491/14 Ortho-condensed systems
• CPC Class Codes: C07K 1/13 Labelling of peptides
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: G01N 33/533 with fluorescent label
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine
• CPC Class Codes: Y10T 436/13 Tracers or tags

Abstract

The present invention describes a bioconjugation strategy and compounds that are useful therein in which a fluorescent signal is produced when two molecular or supramolecular entities are linked by chemoselective combination of one linker having an azido or halide substituent group with another linker having a cyano or an alkyne substituent group. A kit is also provided.

Claims

1. A method of forming a linkage complex having a fluorescent signal, the method comprising:

reacting a fluorophore with a linkage compound to form a linkage complex having a fluorescent signal that is different than a fluorescent signal of the fluorophore;

wherein the fluorophore comprises coumarin substituted at position 3 or 4 with at least one azide group (—

N₃); and

wherein the linkage compound has the structure

2. The method according to claim 1, wherein the linkage complex comprises a triazole.

3. The method according to claim 1, wherein the linkage complex comprises 1,2,3-triazole.

4. The method according to claim 1, wherein the fluorophore comprises a coumarin compound having the structure:

5. The method according to claim 4, wherein:

each R¹ is the same or different and is selected from hydrogen, hydroxyl, alkyl, alkenyl, alkynyl, halo, carboxyl, carbonyl, alkyloxyl, alkoxyl, alkoxyalkyl, amino, alkylamino, azido, hydroxyalkyl, sulfonyl, sulfinyl, phospho, phosphino, or optionally two or more R¹ groups join to form one or more substituted or unsubstituted rings.

6. The method according to claim 4, wherein R² comprises —

N₃, and R³ comprises any R¹ group.

7. The method according to claim 1, wherein the fluorophore comprises 3-azidocoumarin.

8. The method according to claim 1, wherein the fluorophore comprises 3-azido-7-dimethylaminocoumarin.

9. The method according to claim 1, wherein the fluorophore comprises 3-azido-7-hydroxycoumarin.

10. The method according to claim 1, wherein the fluorophore comprises 3-azido-7-methoxycoumarin.

11. The method according to claim 1, wherein the fluorophore comprises 7-acetic-3-azidocoumarin.

12. The method according to claim 1, wherein the fluorophore comprises 3-azido-7-diethylaminocoumarin.

13. The method according to claim 1, wherein the fluorophore comprises 3-azido-6-bromocoumarin.

14. The method according to claim 1, wherein the fluorophore comprises 4-azidocoumarin.

15. The method according to claim 1, wherein the fluorophore is chemically bonded to a second biomaterial.

16. The method according to claim 15, wherein the fluorophore is chemically bonded to the biomaterial according to the structure:

17. The method according to claim 16, wherein L^A is an organic substituent group having at least one reactive group that is selected from carboxylic acid esters, sulfonyl halide, acyl halide, aldehydes, reactive ketones, aromatic halides, bromoacetamides, idoacetamides, maleimides, disulfides, hydroxyl, amines, diazonium compounds, active alkylating reagents, active acylating reagents, or diketones.

18. The method according to claim 16, wherein L^A is an organic substituent group that is selected from branched or unbranched C₁-C₁₂ alky, C₁-C₁₂ alkenyl, C₁-C₁₂ alkynyl, or aryl, heteroaryl, or heterocyclyl, and which has at least one substituent reactive group that is selected from carboxylic acid esters, sulfonyl halide, acyl halide, aldehydes, reactive ketones, aromatic halides, bromoacetamides, idoacetamides, maleimides, disulfides, hydroxyl, amines, diazonium compounds, active alkylating reagents, active acylating reagents, or diketones.

19. The method according to claim 15, wherein the second biomaterial is selected from small molecules, drugs, ligands, catalysts, peptides, proteins, nucleic acids, oligonucleotides, saccharides, viruses, cells, organic polymers, inorganic polymers, nanoparticles, or solid surfaces.

20. The method according to claim 1, wherein the biomaterial is selected from small molecules, drugs, ligands, catalysts, peptides, proteins, nucleic acids, oligonucleotides, saccharides, viruses, cells, organic polymers, inorganic polymers, nanoparticles, or solid surfaces.

21. The method according to claim 1 further comprising

measuring the fluorescent signal of the linkage complex.

40. The method according to claim 1 wherein reacting the fluorophore with the linkage compound to form the linkage complex is catalyzed with copper(I).

22. A kit for forming a linkage complex having a fluorescent signal, the kit comprising

a fluorophore comprising coumarin substituted at position 3 or 4 with at least one azide group (—

N₃); and

a linkage compound, wherein the linkage compound has the structure

23. The kit according to claim 22, wherein the fluorophore comprises a coumarin compound having the structure:

24. The kit according to claim 23, wherein:

each R¹ is the same or different and is selected from hydrogen, hydroxyl, alkyl, alkenyl, alkynyl, halo, carboxyl, carbonyl, alkyloxyl, alkoxyl, alkoxyalkyl, amino, alkylamino, azido, hydroxyalkyl, sulfonyl, sulfinyl, phospho, phosphino, or optionally two or more R¹ groups join to form one or more substituted or unsubstituted rings.

25. The kit according to claim 23, wherein R² comprises —

N₃, and R³ comprises any R¹ group.

26. The kit according to claim 22, wherein the fluorophore comprises 3-azidocoumarin.

27. The kit according to claim 22, wherein the fluorophore comprises 3-azido-7-dimethylaminocoumarin.

28. The kit according to claim 22, wherein the fluorophore comprises 3-azido-7-hydroxycoumarin.

29. The kit according to claim 22, wherein the fluorophore comprises 3-azido-7-methoxycoumarin.

30. The kit according to claim 22, wherein the fluorophore comprises 7-acetic-3-azidocoumarin.

31. The kit according to claim 22, wherein the fluorophore comprises 3-azido-7-diethylaminocoumarin.

32. The kit according to claim 22, wherein the fluorophore comprises 3-azido-6-bromocoumarin.

33. The kit according to claim 22, wherein the fluorophore comprises 4-azidocoumarin.

34. The kit according to claim 22, wherein the fluorophore is chemically bonded to a biomaterial.

35. The kit according to claim 22, wherein the fluorophore is chemically bonded to a second biomaterial.

36. The kit according to claim 35, wherein the fluorophore comprises a compound having the structure:

37. The kit according to claim 35, wherein the second biomaterial is selected from small molecules, drugs, ligands, catalysts, peptides, proteins, nucleic acids, oligonucleotides, saccharides, viruses, cells, organic polymers, inorganic polymers, nanoparticles, or solid surfaces.

38. The kit according to claim 22, wherein the biomaterial is selected from small molecules, drugs, ligands, catalysts, peptides, proteins, nucleic acids, oligonucleotides, saccharides, viruses, cells, organic polymers, inorganic polymers, nanoparticles, or solid surfaces.

39. The kit according to claim 22 further comprising a catalyst, wherein the catalyst comprises copper(I).