Detection of biomolecules by sensitizer-linked substrates

• US 20070112180A1
• Filed: 08/29/2006
• Published: 05/17/2007
• Est. Priority Date: 07/19/1999
• Status: Abandoned Application

• Current Assignee: California Institute of Technology
• Original Assignee: California Institute of Technology
• Inventors: Crane, Brian, Dmochowski, Ivan, Winkler, Jay, Dunn, Alexander, Gray, Harry, Wilker, Jonathan
• Application Number:
  US11/512,765
• Publication Number:
  US 20070112180A1
• Time in Patent Office: Days
• US Class Current: 530/391.700
• CPC Class Codes: C12Q 1/26 involving oxidoreductase
• CPC Class Codes: G01N 2333/795 Porphyrin- or corrin-ring-containing peptides
• CPC Class Codes: G01N 2333/80 Cytochromes
• CPC Class Codes: G01N 2333/90245 acting on paired donors with incorporation of molecular oxygen (1.14)
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching
• CPC Class Codes: G01N 33/582 with fluorescent label

Abstract

Methods and compositions for detecting and characterizing target biomolecules using sensitizer-linked substrate molecules are disclosed. High throughput screening assays and therapeutic applications of the inventions are also included.

Claims

1. A sensitizer-linked substrate molecule comprising a substrate and a sensitizer connected by a linker.

2. The molecule of claim 1, wherein said substrate is a binding element of a target biomolecule.

3. The molecule of claim 1, wherein said sensitizer is located less than about 100 Å

from the surface of a target biomolecule when the substrate of the sensitizer-linked substrate molecule is bound to the target biomolecule.

4. The molecule of claim 1, wherein said biomolecule is a metalloprotein.

5. The molecule of claim 4, wherein said metalloprotein is a heme protein.

6. The molecule of claim 4, wherein said biomolecule is Cytochrome P450.

7. The molecule of claim 1, wherein said sensitizer is a photosensitizer.

8. The molecule of claim 7, wherein said photosensitizer is a Ru(bpy)₃²⁺ complex.

9. The molecule of claim 8, wherein said Ru(bpy)₃²⁺ complex is the Δ

or Λ

enantiomer.

10. The molecule of claim 7, wherein said photosensitizer is selected from the group [Ru(phen)₂dppz]²⁺ or [Ru(phen)₂dppa]²⁺.

11. The molecule of claim 7, wherein said photosensitizer is a coumarin molecule.

12. The molecule of claim 1, wherein said linker is a molecule of sufficient length to allow the substrate to bind to the active site of the biomolecule so that upon binding the sensitizer is located at or near the surface of the biomolecule.

13. The molecule of claim 12, wherein said linker is an alkyl chain, (CH₂)_n, in which n is from 1 to 13.

14. The molecule of claim 1, wherein said substrate is a molecule that binds to Cytochrome P450.

15. The molecule of claim 14, wherein, said substrate selected from the group consisting of adamantane (Ad), ethylbenzene (EB), and imidazole (Im).

16. The molecule of claim 1, wherein said sensitizer-linked substrate molecule is selected from the group consisting of [Ru—

C₁₃-EB]²⁺, [Ru—

C₁₂-EB]²⁺, [Ru—

C₁₁-EB]²⁺, [Ru—

C₁₀-EB]²⁺, [Ru—

C₉-EB]²⁺, [Ru—

C₇-EB]²⁺, [Ru—

C₁₁AD]²⁺, [Ru—

C₉-Ad]²⁺, [Ru—

C₁₁]-Im]²⁺, [Ru—

C₁₃-Im]²⁺, Ru-dppa-C₆-Ad, Ru-dppa-gly-Ad, and Ru-dppa-Ad.

17. The molecule of claim 1, wherein said sensitizer-linked substrate molecule is selected from the group consisting of Δ

-[Ru—

C₉-Ad]²⁺ and Λ

-[Ru—

C₉-Ad]²⁺.

18. The molecule of claim 1, wherein the said sensitizer-linked substrate molecule is selected from the group consisting of 4-, 6-, 7- substituted coumarin classes A and B, as shown by structures (I) and (II):

19. The molecule of claim 1, wherein the said sensitizer-linked substrate molecule is selected from the group consisting of classes C and D, as shown by structures (III) and (IV):

20-37. -37. (canceled)