Environmental Sensitive Fluorogenic Compounds and Their Application for Singlet Oxygen and Protein Detection

• US 20080160627A1
• Filed: 02/29/2008
• Published: 07/03/2008
• Est. Priority Date: 11/19/2003
• Status: Active Grant

• Current Assignee: Invitrogen Corp. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Invitrogen Corp. (Thermo Fisher Scientific Incorporated)
• Inventors: Leung, Wai-Yee, Liu, Jixiang, JOHNSON, Iain, Patch, Brian Richard
• Granted Patent: US 8,173,698 B2
• Time in Patent Office: Days
• US Class Current: 436/127
• CPC Class Codes: C09B 1/00 Dyes with anthracene nucleus not condensed with any other ring
• CPC Class Codes: C09B 11/08 Phthaleins; Phenolphthaleins; Fluorescein
• 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 19/00 Oxazine dyes
• CPC Class Codes: C09B 57/02 Coumarine dyes
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine
• CPC Class Codes: Y10T 436/20 Oxygen containing

Abstract

The present invention provides fluorogenic probes and corresponding fluorescent compounds, methods of using the probes, compounds and kits that include the probes.

Claims

1. A compound having a formula:

2. The compound according to claim 1, wherein the anthracene moiety has a structure according to the following formula:

3. The compound according to claim 2, wherein the compound is:

4. The compound according to claim 1, wherein R², R³, R⁵, and R⁶ are H.

5. The compound according to claim 1, wherein the compound is:

6. The compound according to claim 2, wherein at least one of R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰, R²¹, R²², R²³, R²⁴, R²⁵, R²⁶ or R²⁷ is a reactive group, a carrier molecule or solid support.

7. The compound according to claim 1, wherein the reactive group, 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.

8. The compound according to claim 1, wherein the reactive group is an acrylamide, an activated ester of a carboxylic acid, a carboxylic ester, an acyl azide, an acyl nitrile, an aldehyde, an alkyl halide, an anhydride, an aniline, an amine, an aryl halide, an azide, an aziridine, a boronate, a diazoalkane, a haloacetamide, a haloalkyl, a halotriazine, a hydrazine, an imido ester, an isocyanate, an isothiocyanate, a maleimide, a phosphoramidite, a reactive platinum complex, a silyl halide, a sulfonyl halide, a thiol or a photoactivatable group.

9. The compound according to claim 1, wherein the reactive group is carboxylic acid, succinimidyl ester of a carboxylic acid, hydrazide, amine or a maleimide.

10. The compound according to claim 1, wherein the reactive group reacts with an amine, an alcohol, a cysteine, a haloalkyl, an aldehyde, a ketone, a sulfo group or a thiol.

11. 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.

12. 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, a small-molecule drug, or a tyramide.

13. 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.

14. 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.

15. The compound according to claim 1, wherein the anthracene moiety has a structure according to the following formula:

16. The compound according to claim 15, wherein the compound is:

17. The compound according to claim 15, wherein R², R³, R⁵, and R⁶ are H.

18. The compound according to claim 1, wherein the compound is:

19. The compound according to claim 15, wherein at least one of R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰, R²¹, R²², R²³, R²⁴, R²⁵, R²⁶ or R²⁷ is a reactive group, a solid support or a carrier molecule.

20. A method for the determination of the presence or absence of singlet oxygen in a sample, wherein the method comprises:

a) contacting the sample with a fluorogenic compound to prepare a labeled sample, wherein the fluorogenic compound has the formula;

21. The method according to claim 20, wherein the singlet oxygen is a product formed by a photosensitizer and ground state oxygen in the presence of visible light, hypochlorites and hydrogen peroxide, benzyl cyanide and hydrogen peroxide, oxalyl chloride and hydrogen peroxide, sodium molybdate and hydrogen peroxide, ozone and phosphines, ozone and phosphites, electrodeless discharge of ground state oxygen, hydrogen peroxide in an aqueous solution with Fe (II), (III), or Ce (IV) ions, or pyrolysis of aromatic endoperoxides.

22. The method according to claim 20, wherein the singlet oxygen is a product formed by the reaction of a photosensitizer and ground state oxygen in the presence of visible light.

23. The method according to claim 21, wherein the photosensitizer is an aromatic carbonyl compound, condensed aromatic compound, acridine dye, coumarin dye, crystal violet, fluorene derivative, naphthalocyanine, porphyrin derivative, chlorin, phthalocyanine, thiazine dye, thioketone, xanthene dye, platinum (II) terpyridyl acetylide complex, or combinations thereof.

24. The method according to claim 21, wherein the photosensitizer is an acetonapthone, acetophenone, benzophenone, acridine orange, eosin, fluorene, fluorenone, fluorescein, methylene blue, naphthalocyanine, phthalocyanine, rose bengal, 5,10,15,20-tetrakis(m-hydroxyphenyl)chlorin, thiocoumarin, toluidine blue, methylene blue, zinc tetraphenylporphyrin tetrasulfonate, or combinations thereof.

25. The method according to claim 21, wherein the photosensitizer is methylene blue.

26. The method according to claim 20, wherein the singlet oxygen is a product formed by the reaction of sodium molybdate and hydrogen peroxide.

27. A kit for the determination of singlet oxygen in a sample, wherein the kit comprises:

a) a reaction buffer; and

,

b) a compound according to the formula;

28. The kit according to claim 27, further comprising instructions for use of the kit.

29. The kit according to claim 27, further comprising a singlet oxygen generator.

30. The kit according to claim 27, further comprising methylene blue, sodium molybdate or hydrogen peroxide.

31. A composition comprising:

a) a sample; and

b) a compound according to the formula

32. The composition according to claim 31, further comprising a singlet oxygen generator in an amount sufficient to produce singlet oxygen.

33. The composition according to claim 31, further comprising methylene blue, sodium molybdate or hydrogen peroxide.