Fluorogenic probes for reactive oxygen species

• US 20070141658A1
• Filed: 10/27/2006
• Published: 06/21/2007
• 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
• Granted Patent: US 7,842,823 B2
• Time in Patent Office: Days
• US Class Current: 435/23
• 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 has a formula other than:

6. The compound according to claim 1, wherein at least one of A and E is substituted with a member selected from substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl.

7. The compound according to claim 6, wherein said member selected from substituted or unsubstituted alkyl and substituted or unsubstituted heteroalky bears a reactive functional group at the ω

-position.

8. The compound according to claim 6, wherein said substituted or unsubstituted heteroalkyl comprises a polyether.

9. The compound according to claim 8, wherein said polyether is a member selected from ethylene glycol, ethylene glycol oligomers and combinations thereof, wherein said polyether has a molecular weight of from about 60 daltons to about 10,000 daltons.

10. The compound according to claim 6, wherein said heteroalkyl is a member selected from ω

-carboxyl alkyl groups, ω

-carboxyl substituted alkyl groups and combinations thereof.

11. The compound according to claim 6, wherein said substituted alkyl or substituted heteroalkyl is an active ester of a member selected from said ω

-carboxyl alkyl groups, ω

-carboxyl substituted alkyl groups and combinations thereof.

12. The compound according to claim 10, wherein said ω

-carboxyl substituted alkyl group has the formula;

13. The compound according to claim 1, wherein said compound is covalently attached to a carrier species.

14. The compound according to claim 13, wherein said carrier species is a member selected from the group consisting of synthetic polymers and biomolecules.

15. The compound according to claim 14, wherein said biomolecule is a member selected from the group consisting of antibodies, antigens, peptides, nucleic acids, enzymes, haptens, carbohydrates and pharmacologically active agents.

16. 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.

18. 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.

19. The method according to claim 18, wherein said enzyme generates a product that converts said profluorsecent compound into said fluorescent compound.

20. 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.

21. The microarray according to claim 20, wherein said carrier species is a member selected from a biomolecule, a synthetic polymer and combinations thereof.

22. The microarray according to claim 20, 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.

17. The method according to claim wherein said analyte converts said pro-fluorescent compound into said fluorescent compound.