FLUORINATED RESORUFIN COMPOUNDS AND THEIR APPLICATION

• US 20080113341A1
• Filed: 01/16/2008
• Published: 05/15/2008
• Est. Priority Date: 10/31/2003
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Invitrogen Corp. (Thermo Fisher Scientific Incorporated)
• Inventors: STEINBERG, Thomas, JOHNSON, Iain, BATCHELOR, Robert, SMALLEY, Peter, PATCH, Brian, LIU, Jixiang, LEUNG, Wai-Yee, GE, Yue
• Granted Patent: US 7,875,261 B2
• Time in Patent Office: Days
• US Class Current: 435/5
• 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 method for determining the presence or absence of peroxide 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;

wherein

A is OR⁸ or NR⁹R¹⁰;

E is OR⁸ or NR⁹R¹⁰

wherein

R⁸ is hydrogen, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, substituted heterocycloalkyl, unsubstituted heterocycloalkyl, substituted carboxyalkyl, unsubstituted carboxyalkyl, substituted sulfoalkyl, unsubstituted sulfoalkyl, substituted acyl, unsubstituted acyl, substituted haloalkyl, unsubstituted haloalkyl, substituted alkoxy, unsubstituted alkoxy, a reactive group, substituted reactive group, carrier molecule, substituted carrier molecule, solid support or substituted solid support;

R⁹ is hydrogen, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, substituted heterocycloalkyl, unsubstituted heterocycloalkyl, substituted carboxyalkyl, unsubstituted carboxyalkyl, substituted sulfoalkyl, unsubstituted sulfoalkyl, substituted acyl, unsubstituted acyl, substituted haloalkyl, unsubstituted haloalkyl, substituted alkoxy, unsubstituted alkoxy, a reactive group, substituted reactive group, carrier molecule, substituted carrier molecule, solid support or substituted solid support;

R¹⁰ is hydrogen, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, substituted heterocycloalkyl, unsubstituted heterocycloalkyl, substituted carboxyalkyl, unsubstituted carboxyalkyl, substituted sulfoalkyl, unsubstituted sulfoalkyl, substituted acyl, unsubstituted acyl, substituted haloalkyl, unsubstituted haloalkyl, substituted alkoxy, unsubstituted alkoxy, a reactive group, substituted reactive group, carrier molecule, substituted carrier molecule, solid support or substituted solid support;

or

a member independently selected from

R⁹ in combination with R¹⁰;

R⁹ in combination with R³;

R⁹ in combination with R⁶;

R¹⁰ in combination with R⁴; and

R¹⁰ in combination with R⁵

together with the atoms to which they are joined, form a ring which is a 5-, 6- or 7-membered heterocycloalkyl, a substituted 5-, 6- or 7-membered heterocycloalkyl, a 5-, 6- or 7-membered heteroaryl, or a substituted 5-, 6- or 7-membered heteroaryl;

R¹ is substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, substituted heterocycloalkyl, unsubstituted heterocycloalkyl, OR⁸ or NR⁹R¹⁰;

X¹ is oxygen or sulfur;

X is oxygen or sulfur;

R² is hydrogen, halogen, substituted alkyl, unsubstituted alkyl, substituted alkoxy, unsubstituted alkoxy, substituted alkylthio, unsubstituted alkylthio, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, sulfo, nitro, carboxyl, hydroxyl, a reactive group, substituted reactive group, a carrier molecule, substituted carrier molecule, a solid support or substituted solid support;

R³ is hydrogen, halogen, substituted alkyl, unsubstituted alkyl, substituted alkoxy, unsubstituted alkoxy, substituted alkylthio, unsubstituted alkylthio, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, sulfo, nitro, carboxyl, hydroxyl, a reactive group, substituted reactive group, a carrier molecule, substituted carrier molecule, a solid support or substituted solid support;

R⁴ is hydrogen, halogen, substituted alkyl, unsubstituted alkyl, substituted alkoxy, unsubstituted alkoxy, substituted alkylthio, unsubstituted alkylthio, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, sulfo, nitro, carboxyl, hydroxyl, a reactive group, substituted reactive group, a carrier molecule, substituted carrier molecule, a solid support or substituted solid support;

R⁵ is hydrogen, halogen, substituted alkyl, unsubstituted alkyl, substituted alkoxy, unsubstituted alkoxy, substituted alkylthio, unsubstituted alkylthio, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, sulfo, nitro, carboxyl, hydroxyl, a reactive group, substituted reactive group, a carrier molecule, substituted carrier molecule, a solid support or substituted solid support;

R⁶ is hydrogen, halogen, substituted alkyl, unsubstituted alkyl, substituted alkoxy, unsubstituted alkoxy, substituted alkylthio, unsubstituted alkylthio, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, sulfo, nitro, carboxyl, hydroxyl, a reactive group, substituted reactive group, a carrier molecule, substituted carrier molecule, a solid support or substituted solid support;

R⁷ is hydrogen, halogen, substituted alkyl, unsubstituted alkyl, substituted alkoxy, unsubstituted alkoxy, substituted alkylthio, unsubstituted alkylthio, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, sulfo, nitro, carboxyl, hydroxyl, a reactive group, substituted reactive group, a carrier molecule, substituted carrier molecule, a solid support or substituted solid support;

or

a member independently selected from

R² in combination with R³; and

R⁶ in combination with R⁷

together with the atoms to which they are joined, form a ring which is a 5-, 6- or 7-membered cycloalkyl, a substituted 5-, 6- or 7-membered cycloalkyl, a 5-, 6- or 7-membered heterocycloalkyl, a substituted 5-, 6- or 7-membered heterocycloalkyl, a 5-, 6- or 7-membered aryl, a substituted 5-, 6- or 7-membered aryl, a 5-, 6- or 7-membered heteroaryl, or a substituted 5-, 6- or 7-membered heteroaryl;

with the proviso that at least one member selected from R², R³, R⁴, R⁵, R⁶ and R⁷ is fluorine;

b) incubating the labeled sample for a sufficient amount of time to prepare an incubated sample, wherein the peroxide reacts with the compound in the presence of a peroxidase to produce a fluorescent product;

c) illuminating the incubated sample with an appropriate wavelength to prepare an illuminated sample; and

d) observing the illuminated sample whereby the presence or absence of the peroxide in the sample is determined.

2. ) The method according to claim 1, wherein the peroxidase is horseradish peroxidase, myeloperoxidase or cyclooxygenase.

3. ) The method according to claim 1, wherein the peroxidase is covalently attached to a carrier molecule.

4. ) The method 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.

5. ) 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 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.

6. ) The method according to claim 1, wherein the peroxide is hydrogen peroxide.

7. ) The method according to claim 1, wherein the peroxide is a product formed by an enzymatic reaction.

8. ) The method according to claim 7, wherein the enzymatic reaction is the oxidation of a substrate using an oxidase.

9. ) The method according to claim 8, wherein the oxidase is selected from the group consisting of glutamate oxidase, amine oxidase, choline oxidase, cholesterol oxidase, galactose oxidase, xanthine oxidase, uricase oxidase, pyruvate oxidase, glycerin-3-phosphate oxidase, acyl Co A oxidase, glycerol oxidase and glucose oxidase.

10. ) The method according to claim 8, wherein the enzymatic reaction and the reaction of the peroxide and the peroxidase with the compound are conducted simultaneously.

11. ) The method according to claim 10, wherein the peroxidase is horseradish peroxidase.

12. ) A method for determining the presence or absence of peroxide in a sample, wherein the method comprises:

a) generating peroxide from an enzymatic reaction wherein the enzymatic reaction is the oxidation of a substrate by an oxidase to prepare a peroxide containing sample;

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

wherein

A is OR⁸ or NR⁹R¹⁰;

E is OR⁸ or NR⁹R¹⁰

wherein

R⁸ is hydrogen, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, substituted heterocycloalkyl, unsubstituted heterocycloalkyl, substituted carboxyalkyl, unsubstituted carboxyalkyl, substituted sulfoalkyl, unsubstituted sulfoalkyl, substituted acyl, unsubstituted acyl, substituted haloalkyl, unsubstituted haloalkyl, substituted alkoxy, unsubstituted alkoxy, a reactive group, substituted reactive group, carrier molecule, substituted carrier molecule, solid support or substituted solid support;

R⁹ is hydrogen, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, substituted heterocycloalkyl, unsubstituted heterocycloalkyl, substituted carboxyalkyl, unsubstituted carboxyalkyl, substituted sulfoalkyl, unsubstituted sulfoalkyl, substituted acyl, unsubstituted acyl, substituted haloalkyl, unsubstituted haloalkyl, substituted alkoxy, unsubstituted alkoxy, a reactive group, substituted reactive group, carrier molecule, substituted carrier molecule, solid support or substituted solid support;

R¹⁰ is hydrogen, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, substituted heterocycloalkyl, unsubstituted heterocycloalkyl, substituted carboxyalkyl, unsubstituted carboxyalkyl, substituted sulfoalkyl, unsubstituted sulfoalkyl, substituted acyl, unsubstituted acyl, substituted haloalkyl, unsubstituted haloalkyl, substituted alkoxy, unsubstituted alkoxy, a reactive group, substituted reactive group, carrier molecule, substituted carrier molecule, solid support or substituted solid support;

or

a member independently selected from

R⁹ in combination with R¹⁰;

R⁹ in combination with R³;

R⁹ in combination with R⁶;

R¹⁰ in combination with R⁴; and

R¹⁰ in combination with R⁵

together with the atoms to which they are joined, form a ring which is a 5-, 6- or 7-membered heterocycloalkyl, a substituted 5-, 6- or 7-membered heterocycloalkyl, a 5-, 6- or 7-membered heteroaryl, or a substituted 5-, 6- or 7-membered heteroaryl;

R¹ is substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, substituted heterocycloalkyl, unsubstituted heterocycloalkyl, OR⁸ or NR⁹R¹⁰;

X¹ is oxygen or sulfur;

X is oxygen or sulfur;

R² is hydrogen, halogen, substituted alkyl, unsubstituted alkyl, substituted alkoxy, unsubstituted alkoxy, substituted alkylthio, unsubstituted alkylthio, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, sulfo, nitro, carboxyl, hydroxyl, a reactive group, substituted reactive group, a carrier molecule, substituted carrier molecule, a solid support or substituted solid support;

R³ is hydrogen, halogen, substituted alkyl, unsubstituted alkyl, substituted alkoxy, unsubstituted alkoxy, substituted alkylthio, unsubstituted alkylthio, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, sulfo, nitro, carboxyl, hydroxyl, a reactive group, substituted reactive group, a carrier molecule, substituted carrier molecule, a solid support or substituted solid support;

R⁴ is hydrogen, halogen, substituted alkyl, unsubstituted alkyl, substituted alkoxy, unsubstituted alkoxy, substituted alkylthio, unsubstituted alkylthio, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, sulfo, nitro, carboxyl, hydroxyl, a reactive group, substituted reactive group, a carrier molecule, substituted carrier molecule, a solid support or substituted solid support;

R⁵ is hydrogen, halogen, substituted alkyl, unsubstituted alkyl, substituted alkoxy, unsubstituted alkoxy, substituted alkylthio, unsubstituted alkylthio, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, sulfo, nitro, carboxyl, hydroxyl, a reactive group, substituted reactive group, a carrier molecule, substituted carrier molecule, a solid support or substituted solid support;

R⁶ is hydrogen, halogen, substituted alkyl, unsubstituted alkyl, substituted alkoxy, unsubstituted alkoxy, substituted alkylthio, unsubstituted alkylthio, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, sulfo, nitro, carboxyl, hydroxyl, a reactive group, substituted reactive group, a carrier molecule, substituted carrier molecule, a solid support or substituted solid support;

R⁷ is hydrogen, halogen, substituted alkyl, unsubstituted alkyl, substituted alkoxy, unsubstituted alkoxy, substituted alkylthio, unsubstituted alkylthio, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, sulfo, nitro, carboxyl, hydroxyl, a reactive group, substituted reactive group, a carrier molecule, substituted carrier molecule, a solid support or substituted solid support;

or

a member independently selected from

R² in combination with R³; and

R⁶ in combination with R⁷

together with the atoms to which they are joined, form a ring which is a 5-, 6- or 7-membered cycloalkyl, a substituted 5-, 6- or 7-membered cycloalkyl, a 5-, 6- or 7-membered heterocycloalkyl, a substituted 5-, 6- or 7-membered heterocycloalkyl, a 5-, 6- or 7-membered aryl, a substituted 5-, 6- or 7-membered aryl, a 5-, 6- or 7-membered heteroaryl, or a substituted 5-, 6- or 7-membered heteroaryl;

with the proviso that at least one member selected from R², R³, R⁴, R⁵, R⁶ and R⁷ is fluorine;

c) incubating the labeled sample for a sufficient amount of time to prepare an incubated sample, wherein the peroxide reacts with the compound in the presence of a peroxidase to produce a fluorescent product;

d) illuminating the incubated sample with an appropriate wavelength to prepare an illuminated sample; and

e) observing the illuminated sample whereby the presence or absence of peroxide in the sample is determine.

13. The method according to claim 12, wherein the peroxidase is horseradish peroxidase or myeloperoxidase.

14. The method according to claim 13, wherein the peroxide is hydrogen peroxide.

15. The method according to claim 12, wherein the peroxide is hydrogen peroxide.

16. The method according to claim 12, wherein the oxidase is selected from the group consisting of glutamate oxidase, amine oxidase, choline oxidase, cholesterol oxidase, galactose oxidase, xanthine oxidase, uricase oxidase, pyruvate oxidase, glycerin-3-phosphate oxidase, acyl Co A oxidase, glycerol oxidase and glucose oxidase.

17. The method according to claim 12, wherein the enzymatic reaction and the reaction of the hydrogen peroxide and the horseradish peroxidase with the compound are conducted simultaneously.

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

a) contacting the sample with a peroxidase enzyme covalently attached to a carrier molecule to prepare a contacted sample, wherein the carrier molecule directly or indirectly associates with the analyte;

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

wherein

A is OR⁸ or NR⁹R¹⁰;

E is OR⁸ or NR⁹R¹⁰

wherein

R⁸ is hydrogen, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, substituted heterocycloalkyl, unsubstituted heterocycloalkyl, substituted carboxyalkyl, unsubstituted carboxyalkyl, substituted sulfoalkyl, unsubstituted sulfoalkyl, substituted acyl, unsubstituted acyl, substituted haloalkyl, unsubstituted haloalkyl, substituted alkoxy, unsubstituted alkoxy, a reactive group, substituted reactive group, carrier molecule, substituted carrier molecule, solid support or substituted solid support;

R⁹ is hydrogen, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, substituted heterocycloalkyl, unsubstituted heterocycloalkyl, substituted carboxyalkyl, unsubstituted carboxyalkyl, substituted sulfoalkyl, unsubstituted sulfoalkyl, substituted acyl, unsubstituted acyl, substituted haloalkyl, unsubstituted haloalkyl, substituted alkoxy, unsubstituted alkoxy, a reactive group, substituted reactive group, carrier molecule, substituted carrier molecule, solid support or substituted solid support;

R¹⁰ is hydrogen, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, substituted heterocycloalkyl, unsubstituted heterocycloalkyl, substituted carboxyalkyl, unsubstituted carboxyalkyl, substituted sulfoalkyl, unsubstituted sulfoalkyl, substituted acyl, unsubstituted acyl, substituted haloalkyl, unsubstituted haloalkyl, substituted alkoxy, unsubstituted alkoxy, a reactive group, substituted reactive group, carrier molecule, substituted carrier molecule, solid support or substituted solid support;

or

a member independently selected from

R⁹ in combination with R¹⁰;

R⁹ in combination with R³;

R⁹ in combination with R⁶;

R¹⁰ in combination with R⁴; and

R¹⁰ in combination with R⁵

together with the atoms to which they are joined, form a ring which is a 5-, 6- or 7-membered heterocycloalkyl, a substituted 5-, 6- or 7-membered heterocycloalkyl, a 5-, 6- or 7-membered heteroaryl, or a substituted 5-, 6- or 7-membered heteroaryl;

R¹ is substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, substituted heterocycloalkyl, unsubstituted heterocycloalkyl, OR⁸ or NR⁹R¹⁰;

X¹ is oxygen or sulfur;

X is oxygen or sulfur;

R² is hydrogen, halogen, substituted alkyl, unsubstituted alkyl, substituted alkoxy, unsubstituted alkoxy, substituted alkylthio, unsubstituted alkylthio, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, sulfo, nitro, carboxyl, hydroxyl, a reactive group, substituted reactive group, a carrier molecule, substituted carrier molecule, a solid support or substituted solid support;

R³ is hydrogen, halogen, substituted alkyl, unsubstituted alkyl, substituted alkoxy, unsubstituted alkoxy, substituted alkylthio, unsubstituted alkylthio, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, sulfo, nitro, carboxyl, hydroxyl, a reactive group, substituted reactive group, a carrier molecule, substituted carrier molecule, a solid support or substituted solid support;

R⁴ is hydrogen, halogen, substituted alkyl, unsubstituted alkyl, substituted alkoxy, unsubstituted alkoxy, substituted alkylthio, unsubstituted alkylthio, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, sulfo, nitro, carboxyl, hydroxyl, a reactive group, substituted reactive group, a carrier molecule, substituted carrier molecule, a solid support or substituted solid support;

R⁵ is hydrogen, halogen, substituted alkyl, unsubstituted alkyl, substituted alkoxy, unsubstituted alkoxy, substituted alkylthio, unsubstituted alkylthio, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, sulfo, nitro, carboxyl, hydroxyl, a reactive group, substituted reactive group, a carrier molecule, substituted carrier molecule, a solid support or substituted solid support;

R⁶ is hydrogen, halogen, substituted alkyl, unsubstituted alkyl, substituted alkoxy, unsubstituted alkoxy, substituted alkylthio, unsubstituted alkylthio, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, sulfo, nitro, carboxyl, hydroxyl, a reactive group, substituted reactive group, a carrier molecule, substituted carrier molecule, a solid support or substituted solid support;

R⁷ is hydrogen, halogen, substituted alkyl, unsubstituted alkyl, substituted alkoxy, unsubstituted alkoxy, substituted alkylthio, unsubstituted alkylthio, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, sulfo, nitro, carboxyl, hydroxyl, a reactive group, substituted reactive group, a carrier molecule, substituted carrier molecule, a solid support or substituted solid support;

or

a member independently selected from

R² in combination with R³; and

R⁶ in combination with R⁷

together with the atoms to which they are joined, form a ring which is a 5-, 6- or 7-membered cycloalkyl, a substituted 5-, 6- or 7-membered cycloalkyl, a 5-, 6- or 7-membered heterocycloalkyl, a substituted 5-, 6- or 7-membered heterocycloalkyl, a 5-, 6- or 7-membered aryl, a substituted 5-, 6- or 7-membered aryl, a 5-, 6- or 7-membered heteroaryl, or a substituted 5-, 6- or 7-membered heteroaryl;

with the proviso that at least one member selected from R², R³, R⁴, R⁵, R⁶ and R⁷ is fluorine;

c) incubating the labeled sample for a sufficient amount of time to prepare an incubated sample, wherein the peroxide reacts with the compound in the presence of a peroxidase to produce a fluorescent product;

d) illuminating the incubated sample with an appropriate wavelength to prepare an illuminated sample; and

e) observing the illuminated sample whereby the presence or absence of the analyte in the sample is determined.

19. The method according to claim 18, wherein the peroxidase is horseradish peroxidase or myeloperoxidase.

20. The method according to claim 18, 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.

21. The method according to claim 18, 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 structural protein, a small-molecule drug, or a tyramide.

22. The method according to claim 18, wherein the carrier molecule specifically associates with the analyte.

23. The method according to claim 18, wherein the peroxidase is horseradish peroxidase and the carrier molecule is anti-IgG, anti-IgE or anti-IgA.

24. The method according to claim 18, wherein the peroxide is hydrogen peroxide.

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

b) a compound having the formula

wherein

A is OR⁸ or NR⁹R¹⁰;

E is OR⁸ or NR⁹R¹⁰

wherein

R⁸ is hydrogen, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, substituted heterocycloalkyl, unsubstituted heterocycloalkyl, substituted carboxyalkyl, unsubstituted carboxyalkyl, substituted sulfoalkyl, unsubstituted sulfoalkyl, substituted acyl, unsubstituted acyl, substituted haloalkyl, unsubstituted haloalkyl, substituted alkoxy, unsubstituted alkoxy, a reactive group, substituted reactive group, carrier molecule, substituted carrier molecule, solid support or substituted solid support;

R⁹ is hydrogen, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, substituted heterocycloalkyl, unsubstituted heterocycloalkyl, substituted carboxyalkyl, unsubstituted carboxyalkyl, substituted sulfoalkyl, unsubstituted sulfoalkyl, substituted acyl, unsubstituted acyl, substituted haloalkyl, unsubstituted haloalkyl, substituted alkoxy, unsubstituted alkoxy, a reactive group, substituted reactive group, carrier molecule, substituted carrier molecule, solid support or substituted solid support;

R¹⁰ is hydrogen, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, substituted heterocycloalkyl, unsubstituted heterocycloalkyl, substituted carboxyalkyl, unsubstituted carboxyalkyl, substituted sulfoalkyl, unsubstituted sulfoalkyl, substituted acyl, unsubstituted acyl, substituted haloalkyl, unsubstituted haloalkyl, substituted alkoxy, unsubstituted alkoxy, a reactive group, substituted reactive group, carrier molecule, substituted carrier molecule, solid support or substituted solid support;

or

a member independently selected from

R⁹ in combination with R¹⁰;

R⁹ in combination with R³;

R⁹ in combination with R⁶;

R¹⁰ in combination with R⁴; and

R¹⁰ in combination with R⁵

together with the atoms to which they are joined, form a ring which is a 5-, 6- or 7-membered heterocycloalkyl, a substituted 5-, 6- or 7-membered heterocycloalkyl, a 5-, 6- or 7-membered heteroaryl, or a substituted 5-, 6- or 7-membered heteroaryl;

R¹ is substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, substituted heterocycloalkyl, unsubstituted heterocycloalkyl, OR⁸ or NR⁹R¹⁰;

X¹ is oxygen or sulfur;

X is oxygen or sulfur;

R² is hydrogen, halogen, substituted alkyl, unsubstituted alkyl, substituted alkoxy, unsubstituted alkoxy, substituted alkylthio, unsubstituted alkylthio, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, sulfo, nitro, carboxyl, hydroxyl, a reactive group, substituted reactive group, a carrier molecule, substituted carrier molecule, a solid support or substituted solid support;

R³ is hydrogen, halogen, substituted alkyl, unsubstituted alkyl, substituted alkoxy, unsubstituted alkoxy, substituted alkylthio, unsubstituted alkylthio, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, sulfo, nitro, carboxyl, hydroxyl, a reactive group, substituted reactive group, a carrier molecule, substituted carrier molecule, a solid support or substituted solid support;

R⁴ is hydrogen, halogen, substituted alkyl, unsubstituted alkyl, substituted alkoxy, unsubstituted alkoxy, substituted alkylthio, unsubstituted alkylthio, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, sulfo, nitro, carboxyl, hydroxyl, a reactive group, substituted reactive group, a carrier molecule, substituted carrier molecule, a solid support or substituted solid support;

R⁵ is hydrogen, halogen, substituted alkyl, unsubstituted alkyl, substituted alkoxy, unsubstituted alkoxy, substituted alkylthio, unsubstituted alkylthio, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, sulfo, nitro, carboxyl, hydroxyl, a reactive group, substituted reactive group, a carrier molecule, substituted carrier molecule, a solid support or substituted solid support;

R⁶ is hydrogen, halogen, substituted alkyl, unsubstituted alkyl, substituted alkoxy, unsubstituted alkoxy, substituted alkylthio, unsubstituted alkylthio, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, sulfo, nitro, carboxyl, hydroxyl, a reactive group, substituted reactive group, a carrier molecule, substituted carrier molecule, a solid support or substituted solid support;

R⁷ is hydrogen, halogen, substituted alkyl, unsubstituted alkyl, substituted alkoxy, unsubstituted alkoxy, substituted alkylthio, unsubstituted alkylthio, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, sulfo, nitro, carboxyl, hydroxyl, a reactive group, substituted reactive group, a carrier molecule, substituted carrier molecule, a solid support or substituted solid support;

or

a member independently selected from

R² in combination with R³; and

R⁶ in combination with R⁷

together with the atoms to which they are joined, form a ring which is a 5-, 6- or 7-membered cycloalkyl, a substituted 5-, 6- or 7-membered cycloalkyl, a 5-, 6- or 7-membered heterocycloalkyl, a substituted 5-, 6- or 7-membered heterocycloalkyl, a 5-, 6- or 7-membered aryl, a substituted 5-, 6- or 7-membered aryl, a 5-, 6- or 7-membered heteroaryl, or a substituted 5-, 6- or 7-membered heteroaryl;

with the proviso that at least one member selected from R², R³, R⁴, R⁵, R⁶ and R⁷ is fluorine; and

b) an enzyme.

26. The kit according to claim 25, wherein the enzyme has oxidase or peroxidase activity.

27. The kit according to claim 25, further comprising a catalyst, a reaction buffer, an enzyme substrate, a peroxide, a stop solution, or a positive control.

28. The kit according to claim 27, wherein the positive control is an oxazine or a thiazine dye.

29. The kit according to claim 25, wherein the enzyme is horseradish peroxidase or myeloperoxidase.

30. The kit according to claim 27, wherein the peroxide is hydrogen peroxide.

31. 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

wherein

A is OR⁸ or NR⁹R¹⁰;

E is OR⁸ or NR⁹R¹⁰

wherein

R⁸ is hydrogen, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, substituted heterocycloalkyl, unsubstituted heterocycloalkyl, substituted carboxyalkyl, unsubstituted carboxyalkyl, substituted sulfoalkyl, unsubstituted sulfoalkyl, substituted acyl, unsubstituted acyl, substituted haloalkyl, unsubstituted haloalkyl, substituted alkoxy, unsubstituted alkoxy, a reactive group, substituted reactive group, carrier molecule, substituted carrier molecule, solid support or substituted solid support;

R⁹ is hydrogen, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, substituted heterocycloalkyl, unsubstituted heterocycloalkyl, substituted carboxyalkyl, unsubstituted carboxyalkyl, substituted sulfoalkyl, unsubstituted sulfoalkyl, substituted acyl, unsubstituted acyl, substituted haloalkyl, unsubstituted haloalkyl, substituted alkoxy, unsubstituted alkoxy, a reactive group, substituted reactive group, carrier molecule, substituted carrier molecule, solid support or substituted solid support;

R¹⁰ is hydrogen, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, substituted heterocycloalkyl, unsubstituted heterocycloalkyl, substituted carboxyalkyl, unsubstituted carboxyalkyl, substituted sulfoalkyl, unsubstituted sulfoalkyl, substituted acyl, unsubstituted acyl, substituted haloalkyl, unsubstituted haloalkyl, substituted alkoxy, unsubstituted alkoxy, a reactive group, substituted reactive group, carrier molecule, substituted carrier molecule, solid support or substituted solid support;

or

a member independently selected from

R⁹ in combination with R¹⁰;

R⁹ in combination with R³;

R⁹ in combination with R⁶;

R¹⁰ in combination with R⁴; and

R¹⁰ in combination with R⁵

together with the atoms to which they are joined, form a ring which is a 5-, 6- or 7-membered heterocycloalkyl, a substituted 5-, 6- or 7-membered heterocycloalkyl, a 5-, 6- or 7-membered heteroaryl, or a substituted 5-, 6- or 7-membered heteroaryl;

R¹ is substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, substituted heterocycloalkyl, unsubstituted heterocycloalkyl, OR⁸ or NR⁹R¹⁰;

X¹ is oxygen or sulfur;

X is oxygen or sulfur;

R² is hydrogen, halogen, substituted alkyl, unsubstituted alkyl, substituted alkoxy, unsubstituted alkoxy, substituted alkylthio, unsubstituted alkylthio, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, sulfo, nitro, carboxyl, hydroxyl, a reactive group, substituted reactive group, a carrier molecule, substituted carrier molecule, a solid support or substituted solid support;

R³ is hydrogen, halogen, substituted alkyl, unsubstituted alkyl, substituted alkoxy, unsubstituted alkoxy, substituted alkylthio, unsubstituted alkylthio, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, sulfo, nitro, carboxyl, hydroxyl, a reactive group, substituted reactive group, a carrier molecule, substituted carrier molecule, a solid support or substituted solid support;

R⁴ is hydrogen, halogen, substituted alkyl, unsubstituted alkyl, substituted alkoxy, unsubstituted alkoxy, substituted alkylthio, unsubstituted alkylthio, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, sulfo, nitro, carboxyl, hydroxyl, a reactive group, substituted reactive group, a carrier molecule, substituted carrier molecule, a solid support or substituted solid support;

R⁵ is hydrogen, halogen, substituted alkyl, unsubstituted alkyl, substituted alkoxy, unsubstituted alkoxy, substituted alkylthio, unsubstituted alkylthio, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, sulfo, nitro, carboxyl, hydroxyl, a reactive group, substituted reactive group, a carrier molecule, substituted carrier molecule, a solid support or substituted solid support;

R⁶ is hydrogen, halogen, substituted alkyl, unsubstituted alkyl, substituted alkoxy, unsubstituted alkoxy, substituted alkylthio, unsubstituted alkylthio, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, sulfo, nitro, carboxyl, hydroxyl, a reactive group, substituted reactive group, a carrier molecule, substituted carrier molecule, a solid support or substituted solid support;

R⁷ is hydrogen, halogen, substituted alkyl, unsubstituted alkyl, substituted alkoxy, unsubstituted alkoxy, substituted alkylthio, unsubstituted alkylthio, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, sulfo, nitro, carboxyl, hydroxyl, a reactive group, substituted reactive group, a carrier molecule, substituted carrier molecule, a solid support or substituted solid support;

or

a member independently selected from

R² in combination with R³; and

R⁶ in combination with R⁷

together with the atoms to which they are joined, form a ring which is a 5-, 6- or 7-membered cycloalkyl, a substituted 5-, 6- or 7-membered cycloalkyl, a 5-, 6- or 7-membered heterocycloalkyl, a substituted 5-, 6- or 7-membered heterocycloalkyl, a 5-, 6- or 7-membered aryl, a substituted 5-, 6- or 7-membered aryl, a 5-, 6- or 7-membered heteroaryl, or a substituted 5-, 6- or 7-membered heteroaryl;

with the proviso that at least one member selected from R², R³, R⁴, R⁵, R⁶ and R⁷ is fluorine.

32. The kit according to claim 31, wherein the peroxidase is horseradish peroxidase.

33. The kit according to claim 31, 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.

34. The kit according to claim 31, 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 structural protein, a small-molecule drug, or a tyramide.

35. The kit according to claim 31, wherein the carrier molecule specifically associates with the analyte.

36. The kit according to claim 31, wherein the peroxidase is horseradish peroxidase and the carrier molecule is anti-IgG, anti-IgE or anti-IgA.

37. A composition for the quantitative determination of peroxide in a sample, wherein the composition comprises;

a) a sample comprising peroxidase;

b) a fluorogenic compound that reacts with the peroxidase in the presence of the peroxide to form a fluorescent oxazine or chromogenic thiazine product proportional to the amount of peroxide in the sample, wherein the fluorogenic compound has the formula;

wherein

A is OR⁸ or NR⁹R¹⁰;

E is OR⁸ or NR⁹R¹⁰

wherein

R⁸ is hydrogen, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, substituted heterocycloalkyl, unsubstituted heterocycloalkyl, substituted carboxyalkyl, unsubstituted carboxyalkyl, substituted sulfoalkyl, unsubstituted sulfoalkyl, substituted acyl, unsubstituted acyl, substituted haloalkyl, unsubstituted haloalkyl, substituted alkoxy, unsubstituted alkoxy, a reactive group, substituted reactive group, carrier molecule, substituted carrier molecule, solid support or substituted solid support;

R⁹ is hydrogen, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, substituted heterocycloalkyl, unsubstituted heterocycloalkyl, substituted carboxyalkyl, unsubstituted carboxyalkyl, substituted sulfoalkyl, unsubstituted sulfoalkyl, substituted acyl, unsubstituted acyl, substituted haloalkyl, unsubstituted haloalkyl, substituted alkoxy, unsubstituted alkoxy, a reactive group, substituted reactive group, carrier molecule, substituted carrier molecule, solid support or substituted solid support;

R¹⁰ is hydrogen, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, substituted heterocycloalkyl, unsubstituted heterocycloalkyl, substituted carboxyalkyl, unsubstituted carboxyalkyl, substituted sulfoalkyl, unsubstituted sulfoalkyl, substituted acyl, unsubstituted acyl, substituted haloalkyl, unsubstituted haloalkyl, substituted alkoxy, unsubstituted alkoxy, a reactive group, substituted reactive group, carrier molecule, substituted carrier molecule, solid support or substituted solid support;

or

a member independently selected from

R⁹ in combination with R¹⁰;

R⁹ in combination with R³;

R⁹ in combination with R⁶;

R¹⁰ in combination with R⁴; and

R¹⁰ in combination with R⁵

together with the atoms to which they are joined, form a ring which is a 5-, 6- or 7-membered heterocycloalkyl, a substituted 5-, 6- or 7-membered heterocycloalkyl, a 5-, 6- or 7-membered heteroaryl, or a substituted 5-, 6- or 7-membered heteroaryl;

R¹ is substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, substituted heterocycloalkyl, unsubstituted heterocycloalkyl, OR⁸ or NR⁹R¹⁰;

X¹ is oxygen or sulfur;

X is oxygen or sulfur;

R² is hydrogen, halogen, substituted alkyl, unsubstituted alkyl, substituted alkoxy, unsubstituted alkoxy, substituted alkylthio, unsubstituted alkylthio, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, sulfo, nitro, carboxyl, hydroxyl, a reactive group, substituted reactive group, a carrier molecule, substituted carrier molecule, a solid support or substituted solid support;

R³ is hydrogen, halogen, substituted alkyl, unsubstituted alkyl, substituted alkoxy, unsubstituted alkoxy, substituted alkylthio, unsubstituted alkylthio, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, sulfo, nitro, carboxyl, hydroxyl, a reactive group, substituted reactive group, a carrier molecule, substituted carrier molecule, a solid support or substituted solid support;

R⁴ is hydrogen, halogen, substituted alkyl, unsubstituted alkyl, substituted alkoxy, unsubstituted alkoxy, substituted alkylthio, unsubstituted alkylthio, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, sulfo, nitro, carboxyl, hydroxyl, a reactive group, substituted reactive group, a carrier molecule, substituted carrier molecule, a solid support or substituted solid support;

R⁵ is hydrogen, halogen, substituted alkyl, unsubstituted alkyl, substituted alkoxy, unsubstituted alkoxy, substituted alkylthio, unsubstituted alkylthio, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, sulfo, nitro, carboxyl, hydroxyl, a reactive group, substituted reactive group, a carrier molecule, substituted carrier molecule, a solid support or substituted solid support;

R⁶ is hydrogen, halogen, substituted alkyl, unsubstituted alkyl, substituted alkoxy, unsubstituted alkoxy, substituted alkylthio, unsubstituted alkylthio, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, sulfo, nitro, carboxyl, hydroxyl, a reactive group, substituted reactive group, a carrier molecule, substituted carrier molecule, a solid support or substituted solid support;

R⁷ is hydrogen, halogen, substituted alkyl, unsubstituted alkyl, substituted alkoxy, unsubstituted alkoxy, substituted alkylthio, unsubstituted alkylthio, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, sulfo, nitro, carboxyl, hydroxyl, a reactive group, substituted reactive group, a carrier molecule, substituted carrier molecule, a solid support or substituted solid support;

or

a member independently selected from

R² in combination with R³; and

R⁶ in combination with R⁷

together with the atoms to which they are joined, form a ring which is a 5-, 6- or 7-membered cycloalkyl, a substituted 5-, 6- or 7-membered cycloalkyl, a 5-, 6- or 7-membered heterocycloalkyl, a substituted 5-, 6- or 7-membered heterocycloalkyl, a 5-, 6- or 7-membered aryl, a substituted 5-, 6- or 7-membered aryl, a 5-, 6- or 7-membered heteroaryl, or a substituted 5-, 6- or 7-membered heteroaryl;

with the proviso that at least one member selected from R², R³, R⁴, R⁵, R⁶ and R⁷ is fluorine.

38. The composition according to claim 37, wherein the peroxidase is horseradish peroxidase or myeloperoxidase.

39. The composition according to claim 37, further comprising an enzymatic peroxide producing system in an amount sufficient to produce hydrogen peroxide.

40. The composition according to claim 39, wherein the enzymatic peroxide producing system comprises an oxidase enzyme.

41. The composition according to claim 40, wherein the oxidase enzyme is glutamate oxidase, amine oxidase, choline oxidase, cholesterol oxidase, galactose oxidase, xanthine oxidase, uricase oxidase, pyruvate oxidase, glycerin-3-phosphate oxidase, acyl Co A oxidase, glycerol oxidase or glucose oxidase.

42. A method for detecting metabolic activity in cells in a sample, wherein the method comprises:

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

wherein

A is OR⁸ or NR⁹R¹⁰;

Z is or N⁺R⁹R¹⁰;

wherein

R⁸ is hydrogen, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, substituted heterocycloalkyl, unsubstituted heterocycloalkyl, substituted carboxyalkyl, unsubstituted carboxyalkyl, substituted sulfoalkyl, unsubstituted sulfoalkyl, substituted acyl, unsubstituted acyl, substituted haloalkyl, unsubstituted haloalkyl, substituted alkoxy, unsubstituted alkoxy, a reactive group, substituted reactive group, carrier molecule, substituted carrier molecule, solid support or substituted solid support;

R⁹ is hydrogen, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, substituted heterocycloalkyl, unsubstituted heterocycloalkyl, substituted carboxyalkyl, unsubstituted carboxyalkyl, substituted sulfoalkyl, unsubstituted sulfoalkyl, substituted acyl, unsubstituted acyl, substituted haloalkyl, unsubstituted haloalkyl, substituted alkoxy, unsubstituted alkoxy, a reactive group, substituted reactive group, carrier molecule, substituted carrier molecule, solid support or substituted solid support;

R¹⁰ is hydrogen, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, substituted heterocycloalkyl, unsubstituted heterocycloalkyl, substituted carboxyalkyl, unsubstituted carboxyalkyl, substituted sulfoalkyl, unsubstituted sulfoalkyl, substituted acyl, unsubstituted acyl, substituted haloalkyl, unsubstituted haloalkyl, substituted alkoxy, unsubstituted alkoxy, a reactive group, substituted reactive group, carrier molecule, substituted carrier molecule, solid support or substituted solid support;

or

a member independently selected from

R⁹ in combination with R¹⁰;

R⁹ in combination with R³;

R⁹ in combination with R⁶;

R¹⁰ in combination with R⁴; and

R¹⁰ in combination with R⁵

together with the atoms to which they are joined, form a ring which is a 5-, 6- or 7-membered heterocycloalkyl, a substituted 5-, 6- or 7-membered heterocycloalkyl, a 5-, 6- or 7-membered heteroaryl, or a substituted 5-, 6- or 7-membered heteroaryl;

X is oxygen or sulfur; and

R² is hydrogen, halogen, substituted alkyl, unsubstituted alkyl, substituted alkoxy, unsubstituted alkoxy, substituted alkylthio, unsubstituted alkylthio, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, sulfo, nitro, carboxyl, hydroxyl, a reactive group, substituted reactive group, a carrier molecule, substituted carrier molecule, a solid support or substituted solid support;

R³ is hydrogen, halogen, substituted alkyl, unsubstituted alkyl, substituted alkoxy, unsubstituted alkoxy, substituted alkylthio, unsubstituted alkylthio, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, sulfo, nitro, carboxyl, hydroxyl, a reactive group, substituted reactive group, a carrier molecule, substituted carrier molecule, a solid support or substituted solid support;

R⁴ is hydrogen, halogen, substituted alkyl, unsubstituted alkyl, substituted alkoxy, unsubstituted alkoxy, substituted alkylthio, unsubstituted alkylthio, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, sulfo, nitro, carboxyl, hydroxyl, a reactive group, substituted reactive group, a carrier molecule, substituted carrier molecule, a solid support or substituted solid support;

R⁵ is hydrogen, halogen, substituted alkyl, unsubstituted alkyl, substituted alkoxy, unsubstituted alkoxy, substituted alkylthio, unsubstituted alkylthio, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, sulfo, nitro, carboxyl, hydroxyl, a reactive group, substituted reactive group, a carrier molecule, substituted carrier molecule, a solid support or substituted solid support;

R⁶ is hydrogen, halogen, substituted alkyl, unsubstituted alkyl, substituted alkoxy, unsubstituted alkoxy, substituted alkylthio, unsubstituted alkylthio, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, sulfo, nitro, carboxyl, hydroxyl, a reactive group, substituted reactive group, a carrier molecule, substituted carrier molecule, a solid support or substituted solid support;

R⁷ is hydrogen, halogen, substituted alkyl, unsubstituted alkyl, substituted alkoxy, unsubstituted alkoxy, substituted alkylthio, unsubstituted alkylthio, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, sulfo, nitro, carboxyl, hydroxyl, a reactive group, substituted reactive group, a carrier molecule, substituted carrier molecule, a solid support or substituted solid support;

or

a member independently selected from

R² in combination with R³; and

R⁶ in combination with R⁷

together with the atoms to which they are joined, form a ring which is a 5-, 6- or 7-membered cycloalkyl, a substituted 5-, 6- or 7-membered cycloalkyl, a 5-, 6- or 7-membered heterocycloalkyl, a substituted 5-, 6- or 7-membered heterocycloalkyl, a 5-, 6- or 7-membered aryl, a substituted 5-, 6- or 7-membered aryl, a 5-, 6- or 7-membered heteroaryl, or a substituted 5-, 6- or 7-membered heteroaryl;

with the proviso that at least one member selected from R², R³, R⁴, R⁵, R⁶ and R⁷ is fluorine

b) incubating the labeled sample for a sufficient amount of time to prepare an incubated sample, wherein the compound is capable of entering cells and being reduced to produce a fluorescent product;

c) illuminating the incubated sample with an appropriate wavelength; and

d) observing the illuminated sample whereby the metabolic activity is detected and the resulting signal is proportional to the number of viable cells present in the sample.

43. ) A method for staining a sample, wherein the method comprises:

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

wherein

A is OR⁸ or NR⁹R¹⁰;

Z is O or N⁺R⁹R¹⁰

wherein

R⁸ is hydrogen, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, substituted heterocycloalkyl, unsubstituted heterocycloalkyl, substituted carboxyalkyl, unsubstituted carboxyalkyl, substituted sulfoalkyl, unsubstituted sulfoalkyl, substituted acyl, unsubstituted acyl, substituted haloalkyl, unsubstituted haloalkyl, substituted alkoxy, unsubstituted alkoxy, a reactive group, substituted reactive group, carrier molecule, substituted carrier molecule, solid support or substituted solid support;

R⁹ is hydrogen, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, substituted heterocycloalkyl, unsubstituted heterocycloalkyl, substituted carboxyalkyl, unsubstituted carboxyalkyl, substituted sulfoalkyl, unsubstituted sulfoalkyl, substituted acyl, unsubstituted acyl, substituted haloalkyl, unsubstituted haloalkyl, substituted alkoxy, unsubstituted alkoxy, a reactive group, substituted reactive group, carrier molecule, substituted carrier molecule, solid support or substituted solid support;

R¹⁰ is hydrogen, substituted alkyl, unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl, substituted heterocycloalkyl, unsubstituted heterocycloalkyl, substituted carboxyalkyl, unsubstituted carboxyalkyl, substituted sulfoalkyl, unsubstituted sulfoalkyl, substituted acyl, unsubstituted acyl, substituted haloalkyl, unsubstituted haloalkyl, substituted alkoxy, unsubstituted alkoxy, a reactive group, substituted reactive group, carrier molecule, substituted carrier molecule, solid support or substituted solid support;

or

a member independently selected from

R⁹ in combination with R¹⁰;

R⁹ in combination with R³;

R⁹ in combination with R⁶;

R¹⁰ in combination with R⁴; and

R¹⁰ in combination with R⁵

together with the atoms to which they are joined, form a ring which is a 5-, 6- or 7-membered heterocycloalkyl, a substituted 5-, 6- or 7-membered heterocycloalkyl, a 5-, 6- or 7-membered heteroaryl, or a substituted 5-, 6- or 7-membered heteroaryl;

X is oxygen or sulfur;

R² is hydrogen, halogen, substituted alkyl, unsubstituted alkyl, substituted alkoxy, unsubstituted alkoxy, substituted alkylthio, unsubstituted alkylthio, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, sulfo, nitro, carboxyl, hydroxyl, a reactive group, substituted reactive group, a carrier molecule, substituted carrier molecule, a solid support or substituted solid support;

R³ is hydrogen, halogen, substituted alkyl, unsubstituted alkyl, substituted alkoxy, unsubstituted alkoxy, substituted alkylthio, unsubstituted alkylthio, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, sulfo, nitro, carboxyl, hydroxyl, a reactive group, substituted reactive group, a carrier molecule, substituted carrier molecule, a solid support or substituted solid support;

R⁴ is hydrogen, halogen, substituted alkyl, unsubstituted alkyl, substituted alkoxy, unsubstituted alkoxy, substituted alkylthio, unsubstituted alkylthio, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, sulfo, nitro, carboxyl, hydroxyl, a reactive group, substituted reactive group, a carrier molecule, substituted carrier molecule, a solid support or substituted solid support;

R⁵ is hydrogen, halogen, substituted alkyl, unsubstituted alkyl, substituted alkoxy, unsubstituted alkoxy, substituted alkylthio, unsubstituted alkylthio, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, sulfo, nitro, carboxyl, hydroxyl, a reactive group, substituted reactive group, a carrier molecule, substituted carrier molecule, a solid support or substituted solid support;

R⁶ is hydrogen, halogen, substituted alkyl, unsubstituted alkyl, substituted alkoxy, unsubstituted alkoxy, substituted alkylthio, unsubstituted alkylthio, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, sulfo, nitro, carboxyl, hydroxyl, a reactive group, substituted reactive group, a carrier molecule, substituted carrier molecule, a solid support or substituted solid support;

R⁷ is hydrogen, halogen, substituted alkyl, unsubstituted alkyl, substituted alkoxy, unsubstituted alkoxy, substituted alkylthio, unsubstituted alkylthio, substituted aryl, unsubstituted aryl, substituted heteroaryl, unsubstituted heteroaryl, sulfo, nitro, carboxyl, hydroxyl, a reactive group, substituted reactive group, a carrier molecule, substituted carrier molecule, a solid support or substituted solid support;

or

a member independently selected from

 

R² in combination with R³; and

 

R⁶ in combination with R⁷

together with the atoms to which they are joined, form a ring which is a 5-, 6- or 7-membered cycloalkyl, a substituted 5-, 6- or 7-membered cycloalkyl, a 5-, 6- or 7-membered heterocycloalkyl, a substituted 5-, 6- or 7-membered heterocycloalkyl, a 5-, 6- or 7-membered aryl, a substituted 5-, 6- or 7-membered aryl, a 5-, 6- or 7-membered heteroaryl, or a substituted 5-, 6- or 7-membered heteroaryl;

with the proviso that at least one member selected from R², R³, R⁴, R⁵, R⁶ and R⁷ is fluorine;

b) incubating the labeled sample for a sufficient amount of time to prepare an incubated sample;

c) illuminating the incubated sample with an appropriate wavelength; and

d) observing the illuminated sample whereby the sample is stained.