Fluorogenic probes for reactive oxygen species

• US 7,842,823 B2
• Filed: 10/27/2006
• Issued: 11/30/2010
• Est. Priority Date: 10/27/2005
• Status: Active Grant

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Chang, Christopher J.
• Primary Examiner(s): HAVLIN, ROBERT H
• Application Number:
  US11/588,785
• Publication Number:
  US 20070141658A1
• Time in Patent Office: 1,495 Days
• Field of Search: 549/213, 544/69, 436/68
• US Class Current: 549/213
• CPC Class Codes: C07F 5/025 Boronic and borinic acid compounds
• CPC Class Codes: G01N 33/533 with fluorescent label
• CPC Class Codes: G01N 33/84 involving inorganic compounds or pH

Abstract

The present invention provides a novel class of fluorogenic probes for reactive oxygen species. Exemplary probes of the invention utilize a boronate deprotection mechanism to provide high selectivity and optical dynamic range for detecting H₂O₂ in aqueous solution over similar reactive oxygen species (ROS) including superoxide, nitric oxide, tert-butyl hydroperoxide, and hydroxyl radical; Peroxyresorufin-1 (PR1), Peroxyfluor-1 (PF1), and Peroxyxanthone-1 (PX1) are first-generation probes that respond to H₂O₂ by an increase in red, green, and blue fluorescence, respectively. The boronate dyes are cell-permeable and can detect micromolar changes in H₂O₂ concentrations in living cells, including hippocampal neurons, using confocal and two-photon microscopy. The unique combination of ROS selectivity, membrane permeability, and a range of available excitation/emission colors establishes the potential value of PR1, PF1, PX1, and related probes for interrogating the physiology and pathology of cellular H₂O₂.

Claims

1. A pro-fluorescent compound having the formula:

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

3. The compound according to claim 2, having the formula:

4. The compound according to claim 1, having the formula:

5. The compound according to claim 1, wherein said compound is covalently attached to a carrier species selected from the group consisting of synthetic polymers and biomolecules.

6. The compound according to claim 5, wherein said biomolecule is a member selected from the group consisting of antibodies, antigens, peptides, nucleic acids, enzymes, haptens, carbohydrates and pharmacologically active agents.

7. A method for assaying a sample for a selected analyte, said method comprising:

(a) contacting said sample with a pro-fluorescent compound according to claim 1, under conditions appropriate to convert said compound to a fluorescent compound;

(b) exciting said fluorescent compound; and

(c) determining a fluorescence property of said sample, wherein the presence of said analyte in said sample results in a change in said fluorescence property.

8. The method according to claim 7, wherein said analyte converts said pro-fluorescent compound into said fluorescent compound.

9. A method for determining whether a compound alters an activity of an enzyme, said method comprising:

(a) contacting a sample comprising said enzyme with a profluorescent compound according to claim 1 under conditions appropriate for said enzyme to convert said pro-fluorescent compound into a fluorescent compound;

(b) exciting said compound; and

(c) determining a fluorescence property of said sample, wherein said activity of said enzyme in said sample results in a change in said fluorescence property.

10. The method according to claim 9, wherein said enzyme generates a product that converts said profluorescent compound into said fluorescent compound.

11. A microarray comprising a compound according to claim 1, said compound being conjugated directly to a solid support, conjugated through a linker, or conjugated to a carrier species attached to said solid support.

12. The microarray according to claim 11, wherein said carrier species is a member selected from a biomolecule, a synthetic polymer and combinations thereof.

13. The microarray according to claim 11, wherein said solid support is divided into a first region and a second region, said first region having attached thereto a first said compound and said second region having attached thereto a second said compound.

16. The compound of claim 1 having the formula:

17. The compound of claim 1 wherein Z is selected from C(O), NR⁵ and substituted or unsubstituted heterocycloalkyl.

18. The compound of claim 1 wherein Z is selected from C(O) and NR⁵.

14. A compound having a formula selected from:

15. A compound having the formula: