Reduced dye probes for the detection of radical oxygen species

Abstract

Reduced dyes, such as hydrocyanines, deuterocyanines, and/or other deuterated dyes capable of detecting one or more reactive oxygen species are described herein. The reduced dyes exhibit little or no fluorescence due to the disrupted π conjugation. However, upon reaction with ROS, the reduced dyes are oxidized, regenerating the extended π conjugation and causing a substantial increase in fluorescence intensity. In many case, the oxidized dye is generally membrane impermeable. However, upon reduction, many of the reduced dyes are membrane permeable. Thus, reduced dyes can accumulate in cells and/or tissue to amplify the signal. Once inside the cell or tissue, the reduced dye is reoxidized upon reaction with ROS, and the oxidized dye again becomes membrane impermeable, trapping the dye within the cell. The reduced dyes can be used to image ROS, such as hydroxide radical and superoxide, in serum, cell cultures, tissue explants, and in vivo.

1 - 24 . (canceled) 
     
     
         25 . A method of detecting reactive oxygen species, the method comprising contacting cells, tissue, biological fluids, or combinations thereof, with an effective amount of one or more reduced dyes and determining if the reduced dye has been oxidized. 
     
     
         26 . The method of  claim 25 , wherein oxidation of the reduced dye is detected by fluorescence spectroscopy. 
     
     
         27 . The method of  claim 25 , wherein the oxidation of the reduced dye is detected by fluorescence microscopy. 
     
     
         28 . The method of  claim 25 , wherein the oxidation of the reduced dye is detected by confocal laser scanning microscopy. 
     
     
         29 . The method of  claim 25 , wherein the oxidation of the reduced dye is detected by total internal reflection fluorescence microscopy. 
     
     
         30 . (canceled) 
     
     
         31 . A kit comprising one or more reduced dyes and one or more containers. 
     
     
         32 . The kit of  claim 31 , wherein the kit further comprises instructions for performing an assay for detecting one or more reactive oxygen species. 
     
     
         33 . The kit of  claim 32 , wherein the assay is performed in vivo, in vitro, or ex vivo. 
     
     
         34 . The kit of  claim 31 , wherein the kit further comprises one or more instruments for performing the assay. 
     
     
         35 . The kit of  claim 31 , wherein the one or more instruments are selected from the group consisting of syringes, pipettes, pipette bulbs, spatulas, vials, syringe needles, and combinations thereof. 
     
     
         36 - 40 . (canceled) 
     
     
         41 . The kit of  claim 31 , wherein the one or more reduced dyes are selected from the group consisting of hydrocyanines, deuterocyanines, and combinations thereof. 
     
     
         42 . The kit of  claim 41 , wherein the hydrocyanines, deuterocyanines, or combinations thereof are selected from the group consisting of 
       
         
           
           
               
               
           
         
         wherein X is hydrogen; halogen; C 1-20  alkyl; aryl; —OR, where R is C 1-20  alkyl or aryl; hydroxyl; —NR 5 R 6 , where R 5  and R 6  are independently hydrogen, C 1-20  alkyl or aryl; —CN, —SH; —NO 2 ; —S—C 1-20  alkyl; —S-aryl; aryl, —COOH; —COH; —COR or —COOR, where R is defined above; or —CONR 13 R 14 , wherein R 13  and R 14  are independently hydrogen or R as defined above; 
         R 1 -R 4  are independently hydrogen, C 1-20  alkyl, C 1-20  alkyl sulfate, 
       
       C 1-20  alkyl carboxylate; C 1-20  alkyl amino; aryl, benzyl, oligoethylene glycol, or polyethylene glycol;
 R 5  and R 6  are independently hydrogen; hydroxyl; —OR 8 ; —NH 2 ; —NHR 9 ; 
 
       —NR 10 R 11 , C 1-20  alkyl, C 1-20  alkyl sulfonate, C 1-20  alkyl carboxylic acid or carboxylate; C 1-20  alkyl amino or quaternized amino; —COOH; —CO 2   −  or —SO 3   − ; wherein R 8 -R 11  are the same as R; oligoethylene glycol, or polyethylene glycol;
 n is an integer from 1-5; 
 R 12  is H or D; and 
 the benzene ring represented by dotted lines is optional; 
 
       
         
           
           
               
               
           
         
       
       wherein X is hydrogen; halogen; C 1-20  alkyl; aryl; —OR, where R is C 1-20  alkyl or aryl; hydroxyl; —NR 5 R 6 , where R 5  and R 6  are independently hydrogen, C 1-20  alkyl or aryl; —CN, —SH; —NO 2 ; —S—C 1-20  alkyl; —S-aryl; aryl, —COOH; —COH; —COR or —COOR, where R is defined above; or —CONR 12 R 13 , wherein R 12  and R 13  are independently hydrogen or R as defined above;
 R 1  and R 2  are independently hydrogen, C 1-20  alkyl, C 1-20  alkyl sulfonate, C 1-20  alkyl carboxylic acid or carboxylate; C 1-20  alkyl amino or quaternized amino; aryl, benzyl, oligoethylene glycol, or polyethylene glycol; 
 R 3  and R 4  are independently hydrogen; hydroxyl; —OR 7 ; —NH 2 ; —NHR 8 ; 
 
       —NR 9 R 10 , C 1-20  alkyl, C 1-20  alkyl sulfate, C 1-20  alkyl carboxylate; C 1-20  alkyl amino; —COOH; —CO 2   −  or —SO 3   − ; wherein R 7 -R 10  are the same as R; oligoethylene glycol, or polyethylene glycol;
 n is an integer from 1-5; 
 R 11  is H or D, and 
 the benzene ring represented by dotted lines is optional; 
 
       
         
           
           
               
               
           
         
       
       wherein X is hydrogen; halogen; C 1-20  alkyl; aryl; —OR, where R is C 1-20  alkyl or aryl; hydroxyl; —NR 5 R 6 , where R 5  and R 6  are independently hydrogen, C 1-20  alkyl or aryl; —CN, —SH; —NO 2 ; —S—C 1-20  alkyl; —S-aryl; aryl, —COOH; —COH; —COR or —COOR, where R is defined above; or —CONR 12 R 13 , wherein R 12  and R 13  are independently hydrogen or R as defined above;
 R 1  and R 2  are independently hydrogen, C 1-20  alkyl, C 1-20  alkyl sulfonate, C 1-20  alkyl carboxylic acid or carboxylate; C 1-20  alkyl amino or quaternized amino; aryl, benzyl, oligoethylene glycol, or polyethylene glycol; 
 R 3  and R 4  are independently hydrogen; hydroxyl; —OR 7 ; —NH 2 ; —NHR 8 ; 
 
       —NR 9 R 10 , C 1-20  alkyl, C 1-20  alkyl sulfate, C 1-20  alkyl carboxylate; C 1-20  alkyl amino; —COOH; —CO 2  or —SO 3   − ; wherein R 7 -R 10  are the same as R; oligoethylene glycol, or polyethylene glycol;
 n is an integer from 1-5; 
 R 11  is H or D; and 
 the benzene ring represented by dotted lines is optional; 
 
       
         
           
           
               
               
           
         
         wherein X is hydrogen; halogen; C 1-20  alkyl; aryl; —OR, where R is C 1-20  alkyl or aryl; hydroxyl; —NR 5 R 6 , where R 5  and R 6  are independently hydrogen, C 1-20  alkyl or aryl; —CN, —SH; —NO 2 ; —S—C 1-20  alkyl; —S-aryl; aryl, —COOH; —COH; —COR or —COOR, where R is defined above; or —CONR 17 R 18 , wherein R 17  and R 18  are independently hydrogen or R as defined above; 
         R 1  and R 2  are independently hydrogen, C 1-20  alkyl, C 1-20  alkyl sulfonate, C 1-20  alkyl carboxylic acid or carboxylate; C 1-20  alkyl amino or quaternized amino; aryl, benzyl, oligoethylene glycol, or polyethylene glycol; 
         R 3  and R 4  are independently hydrogen; hydroxyl; —OR 12 ; —NHR 13 ; 
       
       —NR 14 R 15 , C 1-20  alkyl, C 1-20  alkyl sulfate, C 1-20  alkyl carboxylate; C 1-20  alkyl amino; —COOH; —CO 2   −  or —SO 3   − ; wherein R 12 -R 15  are the same as R; oligoethylene glycol, or polyethylene glycol;
 R 5 -R 8  are independently hydrogen or C 1-20  alkyl; 
 n is an integer from 1-5; 
 R 16  is H or D; and 
 the benzene ring represented by dotted lines is optional, 
 wherein if the benzene ring is not present, R 16  is D; 
 
       
         
           
           
               
               
           
         
         wherein X is hydrogen; halogen; C 1-20  alkyl; aryl; —OR, where R is C 1-20  alkyl or aryl; hydroxyl; —NR 5 R 6 , where R 5  and R 6  are independently hydrogen, C 1-20  alkyl or aryl; —CN, —SH; —NO 2 ; —S—C 1-20  alkyl; —S-aryl; aryl, —COOH; —COH; —COR or —COOR, where R is defined above; or —CONR 16 R 17 , wherein R 16  and R 17  are independently hydrogen or R as defined above; 
         R 1  and R 2  are independently hydrogen, C 1-20  alkyl, C 1-20  alkyl sulfonate, C 1-20  alkyl carboxylic acid or carboxylate; C 1-20  alkyl amino or quaternized amino; aryl, benzyl, oligoethylene glycol, or polyethylene glycol; 
         R 3  and R 4  are independently hydrogen; hydroxyl; —OR 11 ; —NHR 12 ; —NR 13 R 14 , C 1-20  alkyl, C 1-20  alkyl sulfate, C 1-20  alkyl carboxylate; C 1-20  alkyl amino; —COOH; —CO 2   −  or —SO 3   − ; wherein R 11 -R 14  are the same as R; oligoethylene glycol, or polyethylene glycol; 
         R 5 -R 8  are independently hydrogen or C 1-20  alkyl; 
         R 15  is H or D; and 
         n is an integer from 1-5; 
       
       
         
           
           
               
               
           
         
         wherein X is hydrogen; halogen; C 1-20  alkyl; aryl; —OR, where R is C 1-20  alkyl or aryl; hydroxyl; —NR 5 R 6 , where R 5  and R 6  are independently hydrogen, C 1-20  alkyl or aryl; —CN, —SH; —NO 2 ; —S—C 1-20  alkyl; —S-aryl; aryl, —COOH; —COH; —COR or —COOR, where R is defined above; or —CONR 12 R 13 , wherein R 12  and R 13  are independently hydrogen or R as defined above; 
         R 1  and R 2  are independently hydrogen, C 1-20  alkyl, C 1-20  alkyl sulfonate, C 1-20  alkyl carboxylic acid or carboxylate; C 1-20  alkyl amino or quaternized amino; aryl, benzyl, oligoethylene glycol, or polyethylene glycol; 
         R 3  and R 4  are independently hydrogen; hydroxyl; —OR 7 ; —NHR 8 ; 
       
       —NR 9 R 10 , C 1-20  alkyl, C 1-20  alkyl sulfate, C 1-20  alkyl carboxylate; C 1-20  alkyl amino; oligoethylene glycol, or polyethylene glycol; 
       —COOH; —CO 2   −  or —SO 3   − ; wherein R 7 -R 10  are the same as R;
 R 11  is H or D; and 
 n is an integer from 1-5; 
 
       
         
           
           
               
               
           
         
         wherein X is hydrogen; halogen; C 1-20  alkyl; aryl; —OR, where R is C 1-20  alkyl or aryl; hydroxyl; —NR 5 R 6 , where R 5  and R 6  are independently hydrogen, C 1-20  alkyl or aryl; —CN, —SH; —NO 2 ; —S—C 1-20  alkyl; —S-aryl; aryl, —COOH; —COH; —COR or —COOR, where R is defined above; or —CONR 12 R 13 , wherein R 12  and R 13  are independently hydrogen or R as defined above; 
         R 1  and R 2  are independently hydrogen, C 1-20  alkyl, C 1-20  alkyl sulfonate, C 1-20  alkyl carboxylic acid or carboxylate; C 1-20  alkyl amino or quaternized amino; aryl, benzyl, oligoethylene glycol, or polyethylene glycol; and 
         R 3  and R 4  are independently hydrogen; hydroxyl; —OR 7 ; —NHR 8 ; 
       
       —NR 9 R 10 , C 1-20  alkyl, C 1-20  alkyl sulfate, C 1-20  alkyl carboxylate; C 1-20  alkyl amino; —COOH; —CO 2   −  or —SO 3   − ; wherein R 7 -R 10  are the same as R; oligoethylene glycol, or polyethylene glycol;
 n is an integer from 1-5; and 
 q is 0, 1, or 2; 
 
       
         
           
           
               
               
           
         
         wherein X is hydrogen; halogen; C 1-20  alkyl; aryl; —OR, where R is C 1-20  alkyl or aryl; hydroxyl; —NR 5 R 6 , where R 5  and R 6  are independently hydrogen, C 1-20  alkyl or aryl; —CN, —SH; —NO 2 ; —S—C 1-20  alkyl; —S-aryl; aryl, —COOH; —COH; —COR or —COOR, where R is defined above; or —CONR 12 R 13 , wherein R 12  and R 13  are independently hydrogen or R as defined above; 
         R 1  and R 2  are independently hydrogen, C 1-20  alkyl, C 1-20  alkyl sulfonate, C 1-20  alkyl carboxylic acid or carboxylate; C 1-20  alkyl amino or quaternized amino; aryl, benzyl, oligoethylene glycol, or polyethylene glycol; and 
         R 3  and R 4  are independently hydrogen; hydroxyl; —OR 7 ; —NHR 8 ; 
       
       —NR 9 R 10 , C 1-20  alkyl, C 1-20  alkyl sulfate, C 1-20  alkyl carboxylate; C 1-20  alkyl amino; —COOH; —CO 2  or —SO 3   − ; wherein R 7 -R 10  are the same as R; oligoethylene glycol, or polyethylene glycol;
 R 11  is D; and 
 n is an integer from 1-5; 
 
       
         
           
           
               
               
           
         
         wherein X is hydrogen; halogen; C 1-20  alkyl; aryl; —OR, where R is C 1-20  alkyl or aryl; hydroxyl; —NR 5 R 6 , where R 5  and R 6  are independently hydrogen, C 1-20  alkyl or aryl; —CN, —SH; —NO 2 ; —S—C 1-20  alkyl; —S-aryl; aryl, —COOH; —COH; —COR or —COOR, where R is defined above; or —CONR 13 R 14 , wherein R 13  and R 14  are independently hydrogen or R as defined above; 
         R 1- R 4  are independently hydrogen, C 1-20  alkyl; C 1-20  alkyl sulfonate, C 1-20  alkyl carboxylic acid or carboxylate; C 1-20  alkyl amino or quaternized amino; aryl, benzyl, oligoethylene glycol, or polyethylene glycol; 
         R 5  and R 6  are independently hydrogen; hydroxyl; —OR 5 ; —NH 2 ; —NHR 6 ; 
       
       —NR 7 R 8 , C 1-20  alkyl; C 1-20  alkyl sulfate; C 1-20  alkyl carboxylate; C 1-20  alkyl amino; —COOH; —CO 2   − ; or —SO 3   − ; wherein R 5 -R 8  are the same as R; oligoethylene glycol, or polyethylene glycol;
 Y and Z are independently carbon, nitrogen or sulfur, wherein if Y and/or Z is carbon, the carbon is tetravalent having two substituents as defined above; if Y and/or Z is sulfur; the sulfur is divalent; and if Y and/or Z is nitrogen, the nitrogen is trivalent, having one substituent as defined above; 
 R 12  is H or D; and 
 the benzene ring represented by dotted lines is optional; 
 
       
         
           
           
               
               
           
         
         wherein X is hydrogen; halogen; C 1-20  alkyl; aryl; —OR, where R is C 1-20  alkyl or aryl; hydroxyl; —NR 5 R 6 , where R 5  and R 6  are independently hydrogen, C 1-20  alkyl or aryl; —CN, —SH; —NO 2 ; —S—C 1-20  alkyl; —S-aryl; —COOH; —COH; —COR or —COOR, where R is defined above; or —CONR 13 R 14 , wherein R 13  and R 14  are independently hydrogen or R as defined above; 
         R 1  and R 2  are independently hydrogen, C 1-20  alkyl; C 1-20  alkyl sulfonate, C 1-20  alkyl carboxylic acid or carboxylate; C 1-20  alkyl amino or quaternized amino; aryl, benzyl, oligoethylene glycol, or polyethylene glycol; 
         R 3  and R 4  are independently hydrogen; hydroxyl; —OR 5 ; —NH 2 ; —NHR 6 ; 
       
       —NR 7 R 8 , C 1-20  alkyl; C 1-20  alkyl sulfonate, C 1-20  alkyl carboxylic acid or carboxylate; C 1-20  alkyl amino or quaternized amino; —COOH; —CO 2   − ; or —SO 3   − ; wherein R 5 -R 8  are independent selected from the group consisting of C 1-20  alkyl or aryl; oligoethylene glycol, or polyethylene glycol;
 Z is carbon, nitrogen, sulfur, or oxygen, wherein if Y and/or Z is carbon, the carbon is tetravalent having two substituents as defined above; if Y and/or Z is sulfur and/or oxygen; the sulfur and/or oxygen is divalent; and if Y and/or Z is nitrogen, the nitrogen is trivalent, having one substituent as defined above; 
 R 12  is H or D; and 
 the benzene ring represented by dotted lines is optional. 
 
     
     
         43 . The kit of  claim 31 , wherein the reduced dye is selected from the group consisting of deuterhydroethydium (D-DHE), dideuterorhodamine, carboxy-D 2 DCFDA, dideuterocalcein AM, dideuterorhodamine 6G, D 2 DCFDA, deuterolucigenin, deuteroRedoxSensor Red CC-1,5-chloromethyl-2′7′-dichlorodideutero-fluorescein diacetate, 6-chloromethyl-2′7′-dichlorodideuterofluorescein diacetate. 
     
     
         44 . The kit of  claim 43 , wherein the reduced dye is D 2 DCFDA or carboxy-D 2 DCFDA. 
     
     
         45 . The method of  claim 25 , wherein the reduced dye is selected from the group consisting of deuterhydroethydium (D-DHE), dideuterorhodamine, carboxy-D 2 DCFDA, dideuterocalcein AM, dideuterorhodamine 6G, D 2 DCFDA, deuterolucigenin, deuteroRedoxSensor Red CC-1,5-chloromethyl-2′7′-dichlorodideutero-fluorescein diacetate, 6-chloromethyl-2′7′-dichlorodideuterofluorescein diacetate. 
     
     
         46 . The method of  claim 45 , wherein the reduced dye is D 2 DCFDA or carboxy-D 2 DCFDA. 
     
     
         47 . The method of  claim 25 , wherein the cells, tissue, biological fluids, or combinations thereof are contacted with the one or more reduced dyes in vitro. 
     
     
         48 . The method of  claim 25 , wherein the cells, tissue, biological fluids, or combinations thereof are contacted with the one or more reduced dyes in vivo.