Fluorinated resorufin compounds and their application

• US 9,212,385 B2
• Filed: 09/12/2012
• Issued: 12/15/2015
• Est. Priority Date: 10/31/2003
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Ge, Yue, Johnson, Iain, Leung, Wai-Yee, Liu, Jixiang, Patch, Brian, Smalley, Peter, Steinberg, Thomas, Gee, Kyle, Batchelor, Robert H.
• Primary Examiner(s): Jones, Dameron Levest
• Application Number:
  US13/612,681
• Publication Number:
  US 20130102014A1
• Time in Patent Office: 1,189 Days
• Field of Search: 424/1.11, 424/1.49, 424/1.65, 424/1.69, 424/1.73, 424/9.1, 424/9.2, 424/9.3, 424/9.4, 424/9.5, 424/9.6, 544/1, 544/3, 544/63, 544/46, 544/99, 544/101, 544/102, 544/347, 530/375, 530/391.3, 435/5, 435/6.16, 435/7.1, 435/7.5, 435/7.92, 435/25, 435/27, 435/28, 435/29, 514/224.8, 514/229.8, 514/250, 206/223, 206/569, 206/570
• US Class Current: 1/1
• CPC Class Codes: C07D 265/38 [b, e]-condensed with two six-membered rings
• CPC Class Codes: C07D 413/12 linked by a chain containing hetero atoms as chain links
• CPC Class Codes: C07D 495/04 Ortho-condensed systems
• CPC Class Codes: C09B 19/00 Oxazine dyes
• CPC Class Codes: C12Q 1/28 involving peroxidase

Abstract

The invention provides novel fluorinated resorufin compounds that are of use in a variety of assay formats. Also provided are methods of using the compounds and kits that include a compound of the invention and instructions detailing the use of the compound in one or more assay formats.

Claims

1. A compound having the formula:

2. The compound according to claim 1, wherein at least one of R³, R⁴, R⁵, and R⁶ is fluorine.

3. The compound according to claim 1, wherein A is OR⁸, E is OR⁸, and R⁸ is hydrogen.

4. The compound according to claim 1, wherein R³ and R⁶ are fluorine.

5. The compound according to claim 1, wherein R⁴ and R⁵ are each fluorine.

6. The compound according to claim 1, wherein R³, R⁴, R⁵ and R⁶ are each fluorine.

7. The compound according to claim 1, wherein R³ and R⁶ are each fluorine and R², R⁴, R⁵ and R⁷ are hydrogen, substituted alkyl, unsubstituted alkyl, substituted alkoxy, unsubstituted alkoxy, substituted alkylthio, unsubstituted alkylthio, substituted aryl, unsubstituted aryl, substituted heteroaryl, or unsubstituted heteroaryl.

8. The compound according to claim 1, wherein R³ and R⁶ are each fluorine and R², R⁴, R⁵ and R⁷ are each hydrogen.

9. The compound according to claim 1, wherein R⁴ and R⁵ are each fluorine and R², R³, R⁶ and R⁷ are hydrogen, substituted alkyl, unsubstituted alkyl, substituted alkoxy, unsubstituted alkoxy, substituted alkylthio, unsubstituted alkylthio, substituted aryl, unsubstituted aryl, substituted heteroaryl, or unsubstituted heteroaryl.

10. The compound according to claim 1, wherein R⁴ and R⁵ are each fluorine and R², R³, R⁶ and R⁷ are each hydrogen.

11. The compound according to claim 1, wherein R¹ is hydrogen, substituted alkyl or unsubstituted alkyl.

12. The compound according to claim 1, wherein R¹ is methyl.

13. The compound according to claim 1, wherein the solid support and carrier molecule independently comprise a linker that is a single covalent bond, or a covalent linkage that is linear or branched, cyclic or heterocyclic, saturated or unsaturated, having 1-20 nonhydrogen atoms selected from the group consisting of C, N, P, O and S;

and are composed of any combination of ether, thioether, amine, ester, carboxamide, sulfonamide, hydrazide bonds and aromatic or heteroaromatic bonds.

14. The compound according to claim 1, wherein the carrier molecule is an amino acid, a peptide, a protein, a polysaccharide, a nucleoside, a nucleotide, an oligonucleotide, a nucleic acid polymer, a hapten, a biotin-binding protein, a psoralen, a drug, a hormone, a lipid, a lipid assembly, a synthetic polymer, a polymeric microparticle, a biological cell or a virus.

15. The compound according to claim 1, wherein the carrier molecule is an antibody or fragment thereof, an avidin or streptavidin, a biotin, a blood component protein, a dextran, an enzyme, an enzyme inhibitor, a hormone, an IgG binding protein, a fluorescent protein, a growth factor, a lectin, a lipopolysaccharide, a microorganism, a metal binding protein, a metal chelating moiety, a non-biological microparticle, a peptide toxin, a phosphotidylserine-binding protein, a structural protein, molecular weight a drug, or a tyramide.

16. The compound according to claim 1, wherein the solid support is a microfluidic chip, a silicon chip, a microscope slide, a microplate well, a silica gel, a polymeric membrane, a particle, a derivatized plastic film, a glass bead, cotton, a plastic bead, an alumina gel, a polysaccharide, polyvinylchloride, polypropylene, polyethylene, nylon, latex bead, magnetic bead, paramagnetic bead, or superparamagnetic bead.

17. The compound according to claim 1, wherein the solid support is Sepharose, poly(acrylate), polystyrene, poly(acrylamide), polyol, agarose, agar, cellulose, dextran, starch, FICOLL, heparin, glycogen, amylopectin, mannan, inulin, nitrocellulose, diazocellulose or starch.

18. A kit for the determination of peroxide in a sample, wherein kit comprises:

a) a compound having the formula

19. The kit according to claim 18, wherein the enzyme is an oxidase or peroxidase.

20. The kit according to claim 18, further comprising a catalyst, a reaction buffer, an enzyme substrate, a peroxide, a stop solution, or a positive control.

21. The kit according to claim 20, wherein the positive control is an oxazine or a thiazine dye.

22. The kit according to claim 19, wherein the enzyme is horseradish peroxidase or myeloperoxidase.

23. The kit according to claim 20, wherein the peroxide is hydrogen peroxide.

24. A kit for determining the presence or absence of an analyte in a sample, wherein the kit comprises:

a) a peroxidase enzyme covalently attached to a carrier molecule; and

b) a fluorogenic compound, wherein the compound has the formula

25. The kit according to claim 24, wherein the peroxidase is horseradish peroxidase.

26. The kit according to claim 24, wherein the carrier molecule is an amino acid, a peptide, a protein, a polysaccharide, a nucleoside, a nucleotide, an oligonucleotide, a nucleic acid polymer, a hapten, a biotin-binding protein, a psoralen, a drug, a hormone, a lipid, a lipid assembly, a synthetic polymer, a polymeric microparticle, a biological cell or a virus.

27. The kit according to claim 24, wherein the carrier molecule is an antibody or fragment thereof, an avidin or streptavidin, a biotin, a blood component protein, a dextran, an IgG binding protein, a fluorescent protein, a growth factor, a lectin, a lipopolysaccharide, a microorganism, a metal binding protein, a metal chelating moiety, a non-biological microparticle, a peptide toxin, a phosphotidylserine-binding protein, a drug, or a tyramide.

28. The kit according to claim 24, wherein the carrier molecule comprises a specific binding pair member that specifically binds with the analyte.

29. The kit according to claim 24, wherein the peroxidase is horseradish peroxidase and the carrier molecule is anti-IgG, anti-IgE or anti-IgA.