MULTI-COLOR TIME RESOLVED FLUOROPHORES BASED ON MACROCYCLIC LANTHANIDE COMPLEXES

• US 20080213780A1
• Filed: 01/25/2008
• Published: 09/04/2008
• Est. Priority Date: 01/25/2007
• Status: Abandoned Application

• Current Assignee: Lumiphore Inc.
• Original Assignee: Lumiphore Inc.
• Inventors: Butlin, Nathaniel G., Xu, Jide, Corneillie, Todd M.
• Application Number:
  US12/020,470
• Publication Number:
  US 20080213780A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: A61K 49/0043 Fluorescein, used in vivo
• CPC Class Codes: A61K 49/0052 Small organic molecules oligomers, polymers, dendrimers A61K49/0054
• CPC Class Codes: C07D 487/18 Bridged systems
• 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 57/10 Metal complexes of organic compounds not being dyes in uncomplexed form
• CPC Class Codes: C09K 11/06 containing organic luminescent materials
• CPC Class Codes: C09K 2211/145 containing oxygen as the only heteroatom
• CPC Class Codes: C09K 2211/1466 containing nitrogen as the only heteroatom
• CPC Class Codes: C09K 2211/182 of the rare earth metals, i.e. Sc, Y or lanthanide
• CPC Class Codes: G01N 33/533 with fluorescent label

Abstract

The present invention provides a novel class of macrocyclic compounds as well as complexes formed between a metal (e.g., lanthanide) ion and the compounds of the invention. Preferred complexes exhibit high stability as well as high quantum yields of lanthanide ion luminescence in aqueous media without the need for secondary activating agents. Preferred compounds incorporate hydroxy-isophthalamide moieties within their macrocyclic structure and are characterized by surprisingly low, non-specific binding to a variety of polypeptides such as antibodies and proteins as well as high kinetic stability. These characteristics distinguish them from known, open-structured ligands.

Claims

1. A compound having a structure according to Formula I:

2. The compound according to claim 1, having the structure:

3. The compound according to claim 1, having a structure, which is a member selected from:

4. The compound according to claim 1, wherein said linker moieties L¹, L², L³, L⁴, L⁵, L⁶, L⁷, L⁸, L⁹ and L¹⁰ are members independently selected from substituted or unsubstituted C₁ to C₆ alkyl.

5. The compound according to claim 4, wherein said linker moieties L¹, L², L³, L⁴, L⁵, L⁶, L⁷, L⁸, L⁹ and L¹⁰ are members independently selected from substituted or unsubstituted ethyl.

6. A luminescent complex formed between at least one metal ion and a compound according to claim 1.

7. The complex according to claim 6, wherein said metal ion is a lanthanide ion.

8. The complex according to claim 7, wherein said lanthanide is a member selected from neodynium (Nd), samarium (Sm), europium (Eu), terbium (Tb), dysprosium (Dy) and ytterbium (Yb).

9. A method of detecting an analyte in a sample, said method comprising:

(a) contacting said analyte with a compound according to claim 6, wherein an energy transfer pair is formed between said compound and a member selected from said analyte, a fluorophore bound to said analyte and a combination thereof,

(b) exciting said compound such that said compound transfers excitation energy to a member selected from said analyte, said fluorophore bound to said analyte; and

(c) detecting energy emitted by a member selected from said analyte, said fluorophore bound to said analyte and a combination thereof, thereby detecting said analyte.

10. The method according to claim 9 wherein said analyte is a nucleic acid.

11. The method of claim 9 wherein the method is performed in vivo.

12. The method of claim 9 wherein the sample contains a living cell.

13. The method of claim 10 wherein said nucleic acid comprises a genetic point mutation, deletion or insertion relative to a control nucleic acid.

14. The method of claim 10 wherein the detection of said nucleic acid indicates the presence of a cancer in the sample.

15. The method of claim 10 wherein the detection of said nucleic acid indicates an alteration of the expression pattern of said nucleic acid in response to an external stimulus.

16. The method of claim 9 wherein the detecting is performed with single- or multiple-photon microscopy, time-resolved fluorescence microscopy or fluorescence endoscopy.

17. A mixture comprising:

(i) a first compound having a structure according to Formula I;

18. The mixture according to claim 17 wherein said first compound is a luminescent metal ion complex, and said first compound and said second compound interact such that an energy exchange pair is formed wherein excitation energy from said first compound is transferred to a member selected from said fluorophore of said second compound and said quencher of said second compound.

19. The mixture according to claim 18, wherein metal ion is a lanthanide ion which is a member selected from neodynium (Nd), samarium (Sm), europium (Eu), terbium (Tb), dysprosium (Dy) and ytterbium (Yb).

20. The mixture according to claim 19 wherein said first compound is covalently attached through a functional moiety to a first nucleic acid.

21. The mixture according to claim 20 wherein said second compound is a nucleic acid.

22. The mixture according to claim 21 wherein said first nucleic acid and said second nucleic acid independently comprise from about 5 to about 50 nucleotides.

23. The mixture according to claim 22 wherein said first nucleic acid and said second nucleic acid independently comprise from 10 to 40 nucleotides.

24. The mixture according to claim 23, wherein said first nucleic acid and said second nucleic acid independently comprise from 15 to 30 nucleotides.

25. The mixture according to 21 wherein one member selected from said first nucleic acid, said second nucleic acid and a combination thereof participates in both stem-loop formation and hybridization to the target sequence nucleic acid.