Fluorescent substrates for Detecting organophosphatase enzyme activity

Abstract

Disclosed are compounds of the formula (I): wherein R 3 , R 4 , R 5 , R 9 , and R 10 are selected from the group consisting of H and groups or atoms other than H, and R 6 and R 8 are halo or hydrogen; X 1 , X 2 , and X 3 are independently O or S; provided that R 9 and R 10 are not simultaneously H, when all of X 1 , X 2 , and X 3 are O; and of the formula (II) wherein R 11 -R 14 are selected from the group consisting of H and groups or atoms other than H; X 4 -X 9 are independently O or S; n and m are 0 or 1 but m and n cannot be 0 simultaneously; R 15 -R 24 can be H or any substituent so long as the compound of formula II upon hydrolysis provides a fluorescent compound. These compounds are useful as substrates with high specificity for organophosphatase particularly human paraoxonase and bacterial organophosphorus hydrolase. Also disclosed is a method for detecting and/or measuring the paraoxonase activity in a fluid comprising contacting the fluid with a fluorescent substrate and measuring the fluorescence of the fluorescent product formed.

1 - 56 . (canceled) 
     
     
         57 . A compound of the formula II: 
       
         
           
           
               
               
           
         
       
       wherein
 R 11 -R 14  are selected from the group consisting of C 1 -C 8  alkyl, C 5 -C 8  cycloalkyl, C 1 -C 6  haloalkyl, C 1 -C 6  perfluoroalkyl, C 2 -C 6  alkenyl, and C 2 -C 6  alkynyl, and aryl, arylcarbonyl, and heteroaryl, which may be optionally substituted with a substituent selected from the group consisting of hydroxyl, cyano, nitro, halo, amino, amido, azido, acetal, ketal, imido, sulfo, sulfonyl, sulfinyl, thiocyanato, aldehydro, keto, carbamoyl, urethane, ureido, and guanidino; 
 X 4 -X 9  are independently O or S; 
 n and m are 0 or 1 but m and n cannot be 0 simultaneously; and 
 R 15 -R 24  can be H or any substituent so long as the compound of formula II upon hydrolysis provides a fluorescent compound. 
 
     
     
         58 . The compound of  claim 57 , wherein the hydrolysis takes place at the P—X 6  and/or P—X 9  bonds. 
     
     
         59 . The compound of  claim 57 , wherein m and n are 1. 
     
     
         60 . The compound of  claim 57 , wherein R 15 -R 24  are independently selected from the group consisting of H, hydroxyl, cyano, nitro, halo, amino, amido, azido, acetal, ketal, imido, sulfo, sulfonyl, sulfinyl, sulfomethyl, a salt of sulfomethyl, thiocyanato, aldehydro, keto, carbamoyl, urethane, ureido, guanidino, C 1 -C 6  alkylamino, C 1 -C 6  acylamino, C 1 -C 6  alkylamido, C 1 -C 6  alkyl, C 1 -C 6  alkoxy, C 1 -C 6  alkylthio, C 5 -C 8  cycloalkyl, C 1 -C 6  haloalkyl, C 1 -C 6  perfluoroalkyl, formyl, carboxamide of the formula —(C═O)NR 1 R 2  where R 1  and R 2  are independently H, alkyl having 1-6 carbon atoms, an aryl, or R 1  and R 2  taken together form a saturated 5- or 6-membered ring having the formula —(CH 2 ) 2 -M-(CH 2 ) 2 — where the ring moiety M is a single bond, an oxygen atom, a methylene group, or the secondary amine —NR 7 — where R 7  is H or alkyl having 1-6 carbon atoms, an aryl, or R 1  and R 2  taken together form a saturated 5- or 6-membered ring having the formula —(CH 2 ) 2 -M-(CH 2 ) 2 — where the ring moiety M is a single bond, an oxygen atom, a methylene group, or the secondary amine —NR 7 — where R 7  is H or alkyl having 1-6 carbon atoms, C 5 -C 8  halocycloalkyl, C 1 -C 6  hydroxyalkyl, C 5 -C 8  hydroxycycloalkyl, C 1 -C 6  alkoxy C 1 -C 6  alkyl, C 2 -C 6  alkoxycarbonyl, C 2 -C 6  alkoxycarbonyl C 1 -C 6  alkyl, carboxy C 1 -C 6  alkyl, carboxy C 1 -C 6  alkoxy, dicarboxy C 1 -C 6  alkyl, dicarboxy C 1 -C 6  alkoxy, C 2 -C 6  cyanoalkyl, phosphono C 1 -C 6  alkyl, phosphoryl C 1 -C 6  alkyl, mono-, di-, and trisaccharides, nucleic acids, oligonucleotides, amino acids, peptides, and proteins, and C 2 -C 6  alkenyl, C 2 -C 6  alkynyl, aryl, arylcarbonyl, and heteroaryl, which may be optionally substituted with a substituent selected from the group consisting of hydroxyl, cyano, nitro, halo, amino, amido, azido, acetal, ketal, imido, sulfo, sulfonyl, sulfinyl, thiocyanato, aldehydro, keto, carbamoyl, urethane, ureido, and guanidino. 
     
     
         61 . The compound of  claim 57 , wherein R 11 -R 14  are independently selected from the group consisting of C 1 -C 6  alkyl, C 2 -C 6  alkenyl, C 2 -C 6  alkynyl, aryl, and heteroaryl. 
     
     
         62 . The compound of  claim 57 , wherein R 11 -R 14  are independently selected from the group consisting of C 1 -C 6  alkyl, C 2 -C 6  alkenyl, and C 2 -C 6  alkynyl. 
     
     
         63 . The compound of  claim 57 , wherein R 11 -R 14  groups are independently selected from the group consisting of C 1 -C 6  alkyl. 
     
     
         64 . The compound of  claim 57 , wherein R 11 -R 14  is ethyl. 
     
     
         65 . A compound of formula II 
       
         
           
           
               
               
           
         
       
       wherein X 4 -X 9  are O, R 15 -R 24  are H, R 11 -R 14  are ethyl; and m and n are 1. 
     
     
         66 . A compound of formula II: 
       
         
           
           
               
               
           
         
       
       wherein X 4 , X 5 , X 7 , and X 8  are O; X 6  and X 9  are S; R 15 -R 24  are H; R 11 -R 14  are ethyl; and m and n are 1. 
     
     
         67 . A method for specifically and selectively detecting and/or measuring the activity of an organophosphatase enzyme in a fluid, which contains at least organophosphatases and phosphatases, said method comprising: 
       
         
           
           
               
               
           
         
         (a) contacting the fluid with a compound of the formula II: 
       
       wherein R 11 -R 14  are selected from the group consisting of H and groups or atoms other than H, X 4 -X 9  are independently O or S, n and m are 0 or 1 but m and n cannot be 0 simultaneously, and R 15 -R 24  can be H or any substituent so long as the compound of formula II upon hydrolysis provides a fluorescent product;
 (b) collecting the fluorescent product; 
 (c) measuring the fluorescence of a fluorescent product formed during the contacting; and 
 (d) correlating the measured fluorescence with the activity of the organophosphatase enzyme. 
 
     
     
         68 . A method for selectively detecting an organophosphatase enzyme in a sample suspected to contain an organophosphatase and a phosphatase comprising
 (a) contacting the sample with a compound of the formula II:   
       
         
           
           
               
               
           
         
       
       wherein R 11 -R 14  are selected from the group consisting of H and groups or atoms other than H, X 4 -X 9  are independently O or S, n and m are 0 or 1 but m and n cannot be 0 simultaneously, and R 15 -R 24  can be H or any substituent so long as the compound of formula II upon hydrolysis provides a fluorescent product;
 (b) collecting the fluorescent product; 
 (c) measuring the fluorescence of a fluorescent product formed during the contacting; and 
 (d) correlating the measured fluorescence with the activity of the organophosphatase enzyme. 
 
     
     
         69 . A method for specifically and selectively detecting and/or measuring the activity of an organophosphatase enzyme immobilized on a support comprising:
 (a) contacting the support with a compound of the formula II:   
       
         
           
           
               
               
           
         
       
       wherein R 11 -R 14  are selected from the group consisting of H and groups or atoms other than H, X 4 -X 9  are independently O or S, n and m are 0 or 1 but m and n cannot be 0 simultaneously, and R 15 -R 24  can be H or any substituent so long as the compound of formula II upon provides a fluorescent product;
 (b) collecting the fluorescent product; 
 (c) measuring the fluorescence of a fluorescent product formed during the contacting; and 
 (d) correlating the measured fluorescence with the activity of the organophosphatase enzyme.