Fluorescent ion indicators and their applications

Abstract

Fluorescent dyes useful for preparing fluorescent metal ion indicators, the fluorescent indicators themselves, and the use of the fluorescent indicators for the detection, discrimination and quantification of metal cations.

1 . A compound having the formula: 
       
         
           
           
               
               
           
         
         where A and B are independently hydrogen, alkyl, cycloalkyl, or aryl; or A and B taken in combination are cycloalkyl or aryl; 
         R 3 , R 4 , j, k, m, n and V are independently hydrogen, halogen, carboxy, alkoxy, aryloxy, thiol, alkylthiol, arylthiol, azido, nitro, nitroso, cyano, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronic acid, aryl, or heteroaryl; or alkyl, or alkoxy that is itself optionally substituted one or more times by halogen, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronic acid, aryl or heteroaryl; 
         each R 5  is independently hydrogen, alkyl having 1-9 carbons, acetoxymethyl, or a biologically compatible salt; 
         T and U are independently hydrogen, alkyl having 1-12 carbons, alkoxy having 1-12 carbons, aryloxy, amino, halogen, cyano, carboxy, acetoxymethylcarbonyl, carboxyalkyl, carbonyl, sulfonyl, phosphonyl, boronic acid, aryl or heteroaryl; provided that at least one of T and U is not hydrogen. 
       
     
     
         2 . The compound of  claim 1  where at least one of T and U is fluoro, methyl, methoxy, carboxymethyl, cyano, carboxy or boronic acid. 
     
     
         3 . A compound having the formula: 
       
         
           
           
               
               
           
         
         where A and B are independently hydrogen, alkyl, cycloalkyl, or aryl; or A and B taken in combination are cycloalkyl or aryl; 
         R 3 , R 4 , j, k, m, n and V are independently hydrogen, halogen, carboxy, alkoxy, aryloxy, thiol, alkylthiol, arylthiol, azido, nitro, nitroso, cyano, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronic acid, aryl, or heteroaryl; or alkyl, or alkoxy that is itself optionally substituted one or more times by halogen, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronic acid, aryl or heteroaryl; 
         T and U are independently selected from hydrogen, an alkyl having 1-2 carbons, alkoxy having 1-12 carbons, aryloxy, amino, halogen, cyano, carboxy, acetoxymethylcarbonyl, carboxyalkyl, carbonyl, sulfonyl, phosphonyl, boronic acid, aryl or heteroaryl; and 
         Z is —OC(═O)R 5 , OCH 2 OC(═O)R 5 , NH 2 , NHR 5 , NR 5 R 6 , NHCH 2 C(═O)R 5  or N(CH 2 C(═O)R 5 ) 2  where R 5  and R 6  are independently methyl or alkyl of 1-10 carbon atoms. 
       
     
     
         4 . The compound of  claim 3  where at least one of T and U is hydrogen, fluoro, methyl, methoxy, cyano or acetoxymethylcarbonyl. 
     
     
         5 . A compound having the formula: 
       
         
           
           
               
               
           
         
         where A and B are independently hydrogen, alkyl, cycloalkyl, or aryl; or A and B taken in combination are cycloalkyl or aryl; 
         R 1 -R 6 , j, k, m, n and V are independently hydrogen, halogen, carboxy, alkoxy, aryloxy, thiol, alkylthiol, arylthiol, azido, nitro, nitroso, cyano, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronic acid, aryl or heteroaryl; or alkyl, or alkoxy that is itself optionally substituted one or more times by halogen, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronic acid, aryl or heteroaryl; 
         R 7  and R 8  are independently hydrogen or alkyl having 1-12 carbons or carboxyalkyl; and 
         T and U are independently hydrogen, alkyl having 1-12 carbons, alkoxy having 1-12 carbons, aryloxy, amino, halogen, cyano, carboxy, carboxyalkyl, carbonyl, sulfonyl, phosphonyl, boronic acid, aryl or heteroaryl. 
       
     
     
         6 . A compound having the formula: 
       
         
           
           
               
               
           
         
         where A and B are independently hydrogen, alkyl, cycloalkyl, or aryl; or A and B taken in combination are cycloalkyl or aryl; 
         R 1 -R 6 , j, k, m, n and V are independently hydrogen, halogen, carboxy, alkoxy, aryloxy, thiol, alkylthiol, arylthiol, azido, nitro, nitroso, cyano, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronic acid, aryl or heteroaryl; or alkyl, or alkoxy that is itself optionally substituted one or more times by halogen, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronic acid, aryl or heteroaryl; 
         Y is NR 9  or CR 10 R 11 ; X and Z are independently selected from O or NR 12 R 13 ; 
       
       where each of R 9 , R 10 , R 11 , R 12  and R 13  are independently hydrogen or alkyl having 1-12 carbons or carboxyalkyl.
 T and U are independently hydrogen, alkyl having 1-12 carbons, alkoxy having 1-12 carbons, aryloxy, amino, halogen, cyano, carboxy, carboxyalkyl, carbonyl, sulfonyl, phosphonyl, boronic acid, aryl or heteroaryl. 
 
     
     
         7 . The compound of  claim 6 , where Y is dimethylmethylene and both of X and Z are oxygen. 
     
     
         8 . A compound having the formula: 
       
         
           
           
               
               
           
         
         where A and B are independently hydrogen, alkyl, cycloalkyl, or aryl; or A and B taken in combination are cycloalkyl or aryl; 
         Y is O, S, Se, CR 9 R 10 , or NR 11 ; 
         R 1 -R 11 , j, k, m, n and V are independently hydrogen, halogen, carboxy, alkoxy, aryloxy, thiol, alkylthiol, arylthiol, azido, nitro, nitroso, cyano, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronic acid, aryl or heteroaryl; or alkyl, or alkoxy that is itself optionally substituted one or more times by halogen, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronic acid, aryl or heteroaryl; 
         Z is an acetyl, acyl having 1-9 carbons, acetoxymethyl or acyloxymethyl having 1-9 carbons. 
       
     
     
         9 . The compound of  claim 8  where Y is oxygen, and R 2  and R 5  are independently hydrogen, fluoro or chloro. 
     
     
         10 . The compound of  claim 8  where Z is acetyl or acetoxymethyl. 
     
     
         11 . A compound having the formula: 
       
         
           
           
               
               
           
         
         where A and B are independently hydrogen, alkyl, cycloalkyl, or aryl; or A and B taken in combination are cycloalkyl or aryl; 
         X and Y are independently H, halogen, OH, OR 7 , OC(═O)R 7 , NH 2 , NHR 7 , NR 7 R 8 , or X and Y taken in combination are O; 
         R 1 -R 6  are independently hydrogen, halogen, carboxy, alkoxy, aryloxy, thiol, alkylthiol, arylthiol, azido, nitro, nitroso, cyano, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronic acid, aryl or heteroaryl; or alkyl, or alkoxy that is itself optionally substituted one or more times by halogen, amino, hydroxy, phosphonyl, sulfonyl, carbonyl, boronic acid, aryl or heteroaryl; 
         R 7  and R 8  are independently alkyl, aryl or heteroaryl; and 
         Z is hydrogen, an alkyl, an aryl or a salt. 
       
     
     
         12 . The compound of  claim 11  where X and Y are independently hydrogen, halogen, hydroxy, methoxy, ethoxy, t-butoxy or phenylmethoxy. 
     
     
         13 . A method of monitoring intracellular calcium using a compound of any one of  claims 3 - 4  and  8 - 10 , comprising:
 a) adding the compound to a sample containing a cell;   b) incubating the sample for a time sufficient for the compound to be loaded into the cell and an indicator compound to be generated intracellularly;   c) illuminating the sample at a wavelength that generates a fluorescence response from the indicator compound;   d) detecting a fluorescence response from the indicator compound;   e) correlating the fluorescence response with the presence of intracellular calcium.   
     
     
         14 . The method of  claim 13 , further comprising:
 stimulating the cell;   monitoring changes in the intensity of the fluorescence response from the indicator compound; and   correlating the changes in fluorescence intensity with changes in intracellular calcium levels.   
     
     
         15 . The method of  claim 13 , further comprising adding a cell-impermeant and non-fluorescent dye to the sample. 
     
     
         16 . The method of  claim 15 , where the cell-impermeant and non-fluorescent dye is a water-soluble azo dye. 
     
     
         17 . The method of  claim 13 , further comprising adding probenecid or a probenecid derivative to the sample. 
     
     
         18 . A kit for performing a calcium assay, comprising at least one compound according to one of  claims 3 - 4  and  8 - 10  and a non-fluorescent and cell-impermeant quencher dye. 
     
     
         19 . The kit of  claim 18 , where the non-fluorescent and cell-impermeant quencher dye is present in a mixed composition with the compound, or the cell-impermeant quencher dye is provided in a container distinct from the compound. 
     
     
         20 . A kit for performing a calcium assay, comprising at least one compound according to one of  claims 3 - 4  and  8 - 10  and a probenecid or a probenecid derivative.