Beta-lactamase substrates having phenolic ethers

Abstract

Provided are fluorescent substrates for β-lactamases having the general formula I: in which R is a benzyl, 2-thienylmethyl, or cyanomethyl group; R′ is selected from the group consisting of H, physiologically acceptable salts or metal, ester groups, ammonium cations, —CHR 2 OCO(CH 2 ) n CH 3 , —CHR 2 OCOC(CH 3 ) 3 , acylthiomethyl, acyloxy-alpha-benzyl, deltabutyrolactonyl, methoxycarbonyloxymethyl, phenyl, methylsulphinylmethyl, β-morpholinoethyl, dialkylaminoethyl, and dialkylaminocarbonyloxymethyl, in which R 2 is selected from the group consisting of H and lower alkyl; A is selected from the group consisting of S, O, SO, SO 2 and CH 2 ; and Z is a donor fluorescent moiety. Also provided are methods of use of the compound of general formula I.

1 . A compound having the general formula:  
       
         
           
           
               
               
           
         
       
       in which R is a benzyl, 2-thienylmethyl, or cyanomethyl group; R′ is selected from the group consisting of H, physiologically acceptable salts or metal, ester groups, ammonium cations, —CHR 2 OCO(CH 2 ) n CH 3 , —CHR 2 OCOC(CH 3 ) 3 , acylthiomethyl, acyloxy-alpha-benzyl, deltabutyrolactonyl, methoxycarbonyloxymethyl, phenyl, methylsulphinylmethyl, β-morpholinoethyl, dialkylaminoethyl, and dialkylaminocarbonyloxymethyl, in which R 2  is selected from the group consisting of H and lower alkyl; A is selected from the group consisting of S, O, SO, SO 2  and CH 2 ; and Z is a donor fluorescent moiety.  
     
     
         2 . The compound of  claim 1 , wherein the donor fluorescent moiety is selected from the group consisting of:  
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
       
       R 3  is a linker for the fluorescent donor.  
     
     
         3 . The compound of  claim 2 , wherein the linker is selected from the group consisting of a direct bond to a heteroatom in the fluorescent moiety, —O(CH 2 ) n —, —S(CH 2 ) n —, —NR 2 (CH 2 ) n —, —N + R 2  (CH 2 ) n , —OCONR 2 (CH 2 ) n —, —O 2 C(CH 2 ) n —, —SCSNR 2 (CH 2 ) n —, —SCSO(CH 2 ) n —, —S(CH 2 ) n CONR 2 (CH 2 ) m , —S(CH 2 ) n NR 2 CO(CH 2 ) m , and  
       
         
           
           
               
               
           
         
       
       in which R 2 , n and m are as previously defined; and m is an integer from 0 to 4.  
     
     
         4 . The compound of  claim 1 , wherein the compound has the structure:  
       
         
           
           
               
               
           
         
       
     
     
         5 . A method for detecting the presence of β-lactamase activity in a sample, comprising: 
 contacting the sample with at least one compound of general formula I:                          in which R is a benzyl, 2-thienylmethyl, or cyanomethyl group, or a quencher; R′ is selected from the group consisting of H, physiologically acceptable salts or metal, ester groups, ammonium cations, —CHR 2 OCO(CH 2 ) n CH 3 , —CHR 2 OCOC(CH 3 ) 3 , acylthiomethyl, acyloxy-alpha-benzyl, deltabutyrolactonyl, methoxycarbonyloxymethyl, phenyl, methylsulphinylmethyl, β-morpholinoethyl, dialkylaminoethyl, and dialkylaminocarbonyloxymethyl, in which R 2  is selected from the group consisting of H and lower alkyl; A is selected from the group consisting of S, O, SO, SO 2  and CH 2 ; and Z is a donor fluorescent moiety.    
     
     
         6 . The method of  claim 5 , wherein said sample has a β-lactamase reporter gene.  
     
     
         7 . The method of  claim 6 , wherein said β-lactamase reporter gene is in a mammalian cell.  
     
     
         8 . The method of  claim 5 , wherein samples having β-lactamase activity are separated from samples having no β-lactamase activity by fluorescent-activated cell sorting.  
     
     
         9 . The method of  claim 5 , wherein the β-lactamase activity results from a β-lactamase enzyme that was prepared by mutagenesis of another β-lactamase enzyme.  
     
     
         10 . The method of  claim 5 , wherein said compound is a membrane permeant derivative.  
     
     
         11 . The method of  claim 5 , wherein the donor fluorescent moiety is selected from the group consisting of:  
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
       
       R 3  is a linker for the fluorescent donor.  
     
     
         12 . The method of  claim 11 , wherein the linker is selected from the group consisting of a direct bond to a heteroatom in the fluorescent moiety, —O(CH 2 ) n —, —S(CH 2 ) n —, —NR 2 (CH 2 ) n —, —N + R 2 (CH 2 ) n , —OCONR 2 (CH 2 ) n —, —O 2 C(CH 2 ) n —, —SCSNR 2 (CH 2 ) n —, —SCSO(CH 2 ) n —, —S(CH 2 ) n CONR 2 (CH 2 ) m , —S(CH 2 ) n NR 2 CO(CH 2 ) m , and  
       
         
           
           
               
               
           
         
       
       in which R 2 , n and m are as previously defined; and m is an integer from 0 to 4.  
     
     
         13 . The method of  claim 5 , wherein the compound has the structure:  
       
         
           
           
               
               
           
         
       
     
     
         14 . A method for determining whether a compound of  claim 1  is a substrate for a β-lactamase enzyme, comprising: contacting said compound with a sample containing said β-lactamase enzyme; exciting at the wavelength for the said compound when cleaved; and measuring fluorescence.  
     
     
         15 . The method of  claim 14 , wherein said compound is a membrane permeant derivative.  
     
     
         16 . The method of  claim 14 , wherein said β-lactamase enzyme has been prepared by mutagenesis of another β-lactamase enzyme.