Fluorinated resorufin compounds and their application

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.

1 . A compound having the formula: 
       
         
           
           
               
               
           
         
         wherein
 A is OH; 
 E is OH; 
 R 1  is methyl; 
 X 1  is oxygen; 
 X is oxygen; 
 and either: 
 (i) R 3  and R 6  are each fluorine and R 2 , R 4 , R 5  and R 7  are hydrogen, substituted alkyl, unsubstituted alkyl, substituted alkoxy, unsubstituted alkoxy, substituted alkylthio, unsubstituted alkylthio, substituted aryl, unsubstituted aryl, substituted heteroaryl, or unsubstituted heteroaryl; or 
 (ii) R 4  and R 5  are each fluorine and R 2 , R 3 , R 6  and R 7  are hydrogen, substituted alkyl, unsubstituted alkyl, substituted alkoxy, unsubstituted alkoxy, substituted alkylthio, unsubstituted alkylthio, substituted aryl, unsubstituted aryl, substituted heteroaryl, or unsubstituted heteroaryl; or 
 (iii) R 3 , R 4 , R 5  and R 6  are each fluorine and R 2  and R 7  are hydrogen, substituted alkyl, unsubstituted alkyl, substituted alkoxy, unsubstituted alkoxy, substituted alkylthio, unsubstituted alkylthio, substituted aryl, unsubstituted aryl, substituted heteroaryl, or unsubstituted heteroaryl. 
 
       
     
     
         2 . The compound according to  claim 1 , wherein R 3  and R 6  are each fluorine and R 2 , R 4 , R 5  and R 7  are hydrogen, substituted alkyl, unsubstituted alkyl, substituted alkoxy, unsubstituted alkoxy, substituted alkylthio, unsubstituted alkylthio, substituted aryl, unsubstituted aryl, substituted heteroaryl, or unsubstituted heteroaryl. 
     
     
         3 . The compound of  claim 2 , wherein R 2 , R 4 , R 5  and R 7  are hydrogen. 
     
     
         4 . The compound of  claim 1 , wherein R 4  and R 5  are each fluorine and R 2 , R 3 , R 6  and R 7  are hydrogen, substituted alkyl, unsubstituted alkyl, substituted alkoxy, unsubstituted alkoxy, substituted alkylthio, unsubstituted alkylthio, substituted aryl, unsubstituted aryl, substituted heteroaryl, or unsubstituted heteroaryl. 
     
     
         5 . The compound of  claim 4 , wherein R 2 , R 3 , R 6  and R 7  are hydrogen. 
     
     
         6 . The compound of  claim 1 , wherein R 3 , R 4 , R 5  and R 6  are each fluorine and R 2  and R 7  are hydrogen, substituted alkyl, unsubstituted alkyl, substituted alkoxy, unsubstituted alkoxy, substituted alkylthio, unsubstituted alkylthio, substituted aryl, unsubstituted aryl, substituted heteroaryl, or unsubstituted heteroaryl. 
     
     
         7 . The compound according to  claim 1 , having the formula: 
       
         
           
           
               
               
           
         
       
     
     
         8 . An oxazine or thiazine dye compound having the formula: 
       
         
           
           
               
               
           
         
         wherein
 A is OH; 
 Z is O; 
 X is oxygen or sulfur; 
 R 2  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; 
 R 3  is hydrogen, halogen, 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; 
 R 4  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; 
 R 5  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; 
 R 6  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; 
 R 7  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
 R 2  in combination with R 3 ; and 
 R 6  in combination with R 7    
 
 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 R 2 , R 3 , R 4 , R 5 , R 6  and 
 R 7  is fluorine. 
 
       
     
     
         9 . The compound according to  claim 8 , wherein
 (i) R 3  and R 6  are each fluorine and R 2 , R 4 , R 5  and R 7  are hydrogen, substituted alkyl, unsubstituted alkyl, substituted alkoxy, unsubstituted alkoxy, substituted alkylthio, unsubstituted alkylthio, substituted aryl, unsubstituted aryl, substituted heteroaryl, or unsubstituted heteroaryl; or   (ii) R 4  and R 5  are each fluorine and R 2 , R 3 , R 6  and R 7  are hydrogen, substituted alkyl, unsubstituted alkyl, substituted alkoxy, unsubstituted alkoxy, substituted alkylthio, unsubstituted alkylthio, substituted aryl, unsubstituted aryl, substituted heteroaryl, or unsubstituted heteroaryl; or   (iii) R 3 , R 4 , R 5  and R 6  are each fluorine and R 2  and R 7  are hydrogen, substituted alkyl, unsubstituted alkyl, substituted alkoxy, unsubstituted alkoxy, substituted alkylthio, unsubstituted alkylthio, substituted aryl, unsubstituted aryl, substituted heteroaryl, or unsubstituted heteroaryl.   
     
     
         10 . A method for determining the presence or absence of peroxide in a sample, wherein the method comprises:
 a) acting the sample with a fluorogenic compound according to  claim 1  to prepare a labeled sample;   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.   
     
     
         11 . The method according to  claim 10 , wherein the peroxidase is horseradish peroxidase, myeloperoxidase or cyclooxygenase. 
     
     
         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 according to  claim 1  to prepare a labeled sample,   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 . A kit for the determination of peroxide in a sample, wherein the kit comprises:
 a) compound according to  claim 1 ;   b) an enzyme and;   c) optionally comprising a catalyst, a reaction buffer, an enzyme substrate, a peroxide, a stop solution, or a positive control.   
     
     
         14 . The kit according to  claim 13 , wherein the enzyme has oxidase or peroxidase activity. 
     
     
         15 . The kit according to  claim 13 , wherein the enxyme is horseradish peroxidase or myeloperoxidase. 
     
     
         16 . 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 according to  claim 1 .   
     
     
         17 . The kit according to  claim 16 , wherein the peroxidase is horseradish peroxidase. 
     
     
         18 . The kit according to  claim 16 , wherein the carrier molecule specifically associates with the analyte and is optionally 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, a virus, 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. 
     
     
         19 . The kit according to  claim 16 , wherein the peroxidase is horseradish peroxidase and the carrier molecule is anti-IgG, anti-IgE or anti-IgA. 
     
     
         20 . A composition for the quantitative determination of peroxide in a sample, wherein the composition comprises;
 a) a sample comprising peroxidase; and   b) fluorogenic compound according to  claim 1 , wherein the fluorogenic compound 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.   
     
     
         21 . A compound having formula: 
       
         
           
           
               
               
           
         
         wherein
 A is OR 8  or NR 9 R 10 ; 
 Z is O or N + R 9 R 10 ; 
 wherein
 R 8  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; 
 R 9  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; 
 R 10  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
 R 9  in combination with R 10 ; 
 R 9  in combination with R 3 ; 
 R 9  in combination with R 6 ; 
 R 10  in combination with R 4 ; and 
 R 10  in combination with R 5    
 
 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 
 R 2  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; 
 R 3  is hydrogen, halogen, 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; 
 R 4  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; 
 R 5  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; 
 R 6  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; 
 R 7  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
 R 2  in combination with R 3 ; and 
 R 6  in combination with R 7    
 
 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 R 2 , R 3 , R 4 , R 5 , R 6  and R 7  is fluorine. 
       
     
     
         22 . A method for detecting metabolic activity in cells in a sample, wherein the method comprises:
 a) contacting the sample with a compound according to  claim 21  to prepare a labeled sample;   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.   
     
     
         23 . A method for staining a sample, wherein the method comprises:
 a) contacting the sample with a compound according to  claim 8  to prepare a labeled sample;   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.