Optically-detectable enzyme substrates and their method of use

Abstract

The present invention relates to compounds that are substrates for an enzyme, and upon reaction with the enzyme provide a detectable response, such as an optically detectable response. In particular, the compounds have utility in detecting the presence of a β-lactamase in a sample. In addition to the compounds, methods are disclosed for analyzing a sample for the presence of a β-lactmase, for example, as an indicator of expression of a nucleic acid sequence including a sequence coding for a β-lactmase. Kits are disclosed that include the disclosed compounds and additional components, for example, cells, antibodies, a β-lactmase or instructions for using the components in an assay.

1 . A compound having the formula:  
       
         
           
           
               
               
           
         
         A is S, O, SO, SO 2  or CH 2 ;  
         X is O, S or NH;  
         L is a linker;  
         R 2  is hydrogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl;  
         R 3  is hydrogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl;  
         R 4  is hydrogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl;  
         R 5  is hydrogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl;  
         R 6  is hydrogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl;  
         R 7  is hydrogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl;  
         R 8  is hydrogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl;  
         R 9  is  
         
           
             
             
                 
                 
             
           
         
         in which W is an alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, aryloxy, substituted aryloxy, heteroaryl, substituted heteroaryl, a dye moiety or CN, and;  
         s is an integer from 0 to 5;  
         W′ and W″ are independently hydrogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, heteroaryl, substituted heteroaryl, (═O), (═NH), OR′°, NHR″, or halogen; 
 wherein R 10  is hydrogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl; or substituted heteroaryl; and,  
 R″ is hydrogen, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl; substituted heteroaryl or OR 12 ; 
 wherein R 12  is hydrogen, substituted or unsubstituted alkyl, and substituted or unsubstituted heteroalkyl or substituted or unsubstituted aryl or heteroaryl; and  
 
 
         Y is a dye moiety or a quencher moiety.  
       
     
     
         2 . The compound according to  claim 1 , wherein the compound is other than a cephalosporin, and at least one of W, W′ or W″ is a quencher of fluorescence emitted by the first dye moiety.  
     
     
         3 . The compound according to  claim 1 , wherein the compound is other than a cephalosporin, at least one of W, W′ and W″ includes a second dye moiety, and the first dye moiety is a quencher of the fluorescence of the second dye moiety.  
     
     
         4 . The compound according to  claim 1 , wherein R 9  is:  
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
       
       where the symbol n is an integer selected from 1 to about 8.  
     
     
         5 . The compound according to  claim 1 , wherein R 9  is other than benzyl, 2-thienylmethyl or cyanomethyl.  
     
     
         6 . The compound according to  claim 1 , wherein the compound has the formula:  
       
         
           
           
               
               
           
         
         where A is S, O, SO, SO 2  or CH 2 ; one of Y and R 9  includes a dye moiety, and the other has the formula:  
         
           
             
             
                 
                 
             
           
         
         where W is substituted or unsubstituted alkyl, substituted or unsubstituted heteroaklyl, substituted or unsubstituted aryl, substituted or unsubstituted aryloxy, substituted or unsubstituted heteroaryl, or CN; the symbol s represents an integer selected from 0 to 5;  
         W′ and W″ are independently H, substituted or unsubstituted alkyl, (═O), (═NH), OR 10 , NHR 11 , or halogen; 
 Wherein W, W′, and W″ are not quenchers of the fluorescence emitted by the dye moiety;  
 R 10  is hydrogen, alkyl, substituted alkyl, heteroalkyl, or substituted heteroalkyl;  
 
         R 11  is hydrogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, or OR 12 ; 
 wherein R 12  is hydrogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl.  
 
       
     
     
         7 . The compound according to  claim 6 , wherein R 9  is other than benzyl, 2-thienylmethyl or cyanomethyl.  
     
     
         8 . The compound according to  claim 6 , wherein Y includes a dye moiety and R 9  is  
       
         
           
           
               
               
           
         
         where W is alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, aryloxy, substituted aryloxy, heteroaryl, substituted heteroaryl, or CN;  
         s is an integer from 0 to about 5; and,  
         W, W′, and W″ are not quenchers of the fluorescence emitted by the dye moiety.  
       
     
     
         9 . The compound according to  claim 6 , wherein R 9  is benzyl, substituted benzyl, 5-membered heteroaryl, substituted 5-membered heteroaryl, or CN.  
     
     
         10 . The compound according to  claim 6 , wherein A is S or SO.  
     
     
         11 . The compound according to  claim 6 , wherein, W′ is (═O) and W″ is (OH).  
     
     
         12 . The compound according to  claim 6 , wherein R 9  is  
       
         
           
           
               
               
           
         
         where R 10  and R 11  are independently H, —OH, —NH 2 , substituted or unsubstituted C 1 -C 18  alkyl, substituted or unsubstituted C 1 -C 18  heteroalkyl, substituted or unsubstituted C 1 -C 18  cycloalkyl, substituted or unsubstituted C 1 -C 18  heterocycloalkyl, substituted or unsubstituted C 1 -C 18  aryl, or substituted or unsubstituted C 1 -C 18  heteroaryl.  
       
     
     
         13 . The compound according to  claim 6 , wherein R 9  is other than benzyl, 2-thienylmethyl or cyanomethyl.  
     
     
         14 . The compound according to  claim 6 , wherein the compound has the formula:  
       
         
           
           
               
               
           
         
         where R 9  and each R a  group is independently H, substituted or unsubstituted akyl or heteroalkyl, or substituted or unsubstituted aryl or heteroaryl.  
       
     
     
         15 . The compound according to  claim 1 , wherein the compound is:  
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
       
     
     
         16 . The compound according to  claim 1 , wherein the first dye moiety is bonded to two substrate moieties, and the two substrate moieties are independently a cephalosporin, a clavulanate, an aceturate, a malonamate, benzofuranone, benzopyranone or a simple β-lactam ring substrate.  
     
     
         17 . The compound according to  claim 16 , wherein at least one of the two substrate moieties bonded to the single dye moiety is other than a cephalosporin substrate moiety.  
     
     
         18 . The compound of  claim 16 , wherein the compound is:  
       
         
           
           
               
               
           
         
       
     
     
         19 . The compound according to  claim 16 , wherein the two substrate moieties are two clavulanates, two aceturates, two malonamates, two benzofuranones, two benzopyranones, a clavulanate and a cephalosporin, a clavulanate and an aceturate, a clavulanate and a malonamate, a clavulanate and a benzofuranone, a clavulanate and a benzopyranone, a clavulanate and a simple β-lactam ring substrate, an aceturate and a cephalosporin, an aceturate and a malonamate, an aceturate and a benzofuranone, an aceturate and a benzopyranone, an aceturate and a simple β-lactam ring substrate, a malonamate and a cephalosporin, a malonamate and a benzofuranone, a malonamate and a benzopyranone, a malonamate and a simple β-lactam ring substrate, a benzofuranone and a cephalosporin, a benzofuranone and a benzopyranone, a benzofuranone and a simple β-lactam ring substrate, a benzopyranone and a cephalosporin, or a benzopyranone and a simple β-lactam ring substrate  
     
     
         20 . The compound according to  claim 1 , wherein the compound is a clavulanic acid derivative (a clavulanate) and has the formula:  
       
         
           
           
               
               
           
         
         where Y is a first dye moiety;  
         L is a linker moiety;  
         A is S, O, SO, SO 2 , or CH 2 ;  
         X is O, S or NH;  
         W′ and W″ are independently hydrogen, alkyl, substituted alkyl, (═O), (═NH), OR 13 , NHR 14 ; 
 wherein R 13  is hydrogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl, or substituted heteroaryl;  
 R 14  is hydrogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, or OR 15 ;  
 wherein R 15  is hydrogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl, or substituted heteroaryl.  
 
       
     
     
         21 . The compound according to  claim 20 , wherein X and A are 0.  
     
     
         22 . The compound according to  claim 21 , wherein W′ is (═O) and W″ is (OH).  
     
     
         23 . The compound according to  claim 21 , wherein the compound has the formula:  
       
         
           
           
               
               
           
         
         where each R a  group is independently hydrogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroaklyl, aryl, substituted aryl, heteroaryl, or substituted heteroaryl.  
       
     
     
         24 . The compound according to  claim 1 , wherein the compound has the formula:  
       
         
           
           
               
               
           
         
       
     
     
         25 . A method for determining the presence or absence of β-lactamase enzyme in a sample, the method comprising: 
 a) contacting the sample with a β-lactamase substrate to form a contacted sample, wherein the β-lactamase substrate has the formula:                          A is S, O, SO, SO 2  or CH 2 ;    X is O, S or NH;    L is a linker;    R 2  is hydrogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl;    R 3  is hydrogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl;    R 4  is hydrogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl;    R 5  is hydrogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl;    R 6  is hydrogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl;    R 7  is hydrogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl;    R 8  is hydrogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl;    R 9  is                          in which W is an alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, aryloxy, substituted aryloxy, heteroaryl, substituted heteroaryl, dye moiety, or CN, and;    s is an integer from 0 to 5;    W′and W″ are independently hydrogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, heteroaryl, substituted heteroaryl, (═O), (═NH), OR 10 , NHR 11 , or halogen; 
 wherein R 10  is hydrogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl; or substituted heteroaryl; and,  
 R 11  is hydrogen, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl; substituted heteroaryl or OR 12 ; 
 wherein R 12  is hydrogen, substituted or unsubstituted alkyl, and substituted or unsubstituted heteroalkyl or substituted or unsubstituted aryl or heteroaryl; and  
 
 Y is a dye moiety or quencher moiety;  
   b) incubating the contacted sample for a sufficient amount of time for the β-lactamase enzyme to to cleave the β-lactamase substrate to form an incubated sample;    c) illuminating the incubated sample with an appropriate wavelength; and    d) observing the illuminated sample whereby the presence or absence of β-lactamase enzyme in the sample is determined.    
     
     
         26 . A method for determining the presence or absence of β-lactamase enzyme in a sample, the method comprising: 
 a) contacting the sample with a β-lactamase substrate to form a contacted sample, wherein the β-lactamase substrate has the formula:                                                                                                                                                        b) incubating the contacted sample for a sufficient amount of time for the β-lactamase enzyme to to cleave the β-lactamase substrate to form an incubated sample;    c) illuminating the incubated sample with an appropriate wavelength; and    d) observing the illuminated sample whereby the presence or absence of β-lactamase enzyme in the sample is determined.    
     
     
         27 . A method of localizing a fluorescent dye product in an environment comprising an aqueous solution and a β-lactamase, the method comprising: 
 a) contacting the environment with a non-fluorescent compound comprising a dye moiety and a β-lactam moiety;    b) incubating the product of step a) for a sufficient amount of time for the β-lactamase to cleave the dye moiety from the β-lactam moiety, thereby producing a fluorescent dye product which is insoluble in the aqueous solution, and thereby localizing the fluorescent dye product in the environment.    
     
     
         28 . The method according to  claim 27 , wherein the non-fluorescent compound has the formula:  
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
       
     
     
         29 . The method according to  claim 27 , wherein the environment is a member selected from a biological cell and a cell-free environment.  
     
     
         30 . The method according to  claim 28 , wherein the biological cell environment is an interior of a cell, an interior of a cell organelle, and an exterior of a cell.  
     
     
         31 . A method for detecting the presence or absence of a target antigen in a sample, the method comprising: 
 a) contacting the sample with an antibody to form a contacted sample, wherein the antibody binds either directly or indirectly to the target antigen, and wherein the antibody comprises a β-lactamase enzyme;    b) adding a non-fluorescent β-lactamase substrate to the incubated sample to form an added sample;    c) incubating the added sample for a sufficient amount of time for the α-lactamase enzyme to cleave the non-fluorescent β-lactamase substrate to form a fluorescent dye moiety;    d) illuminating the fluorescent dye moiety with an appropriate wavelength to form an illuminated sample;    e) observing the illuminated sample whereby the presence or absence of the target antigen is determined.    
     
     
         32 . A method for detecting the presence or absence of a target antigen in a sample, the method comprising: 
 a) contacting the sample with an antibody to form a contacted sample, wherein the antibody binds either directly or indirectly to the target antigen, and wherein the antibody comprises a β-lactamase enzyme;    b) adding a non-fluorescent β-lactamase substrate to the incubated sample to form an added sample, wherein the non-fluorescent β-lactamase substrate has the formula:                          c) incubating the added sample for a sufficient amount of time for the α-lactamase enzyme to cleave the non-fluorescent p-lactamase substrate to form a fluorescent dye moiety;    d) illuminating the fluorescent dye moiety with an appropriate wavelength to form an illuminated sample;    e) observing the illuminated sample whereby the presence or absence of the target antigen is determined.    
     
     
         33 . A kit for determing the presence or absence of β-lactamase in a sample, the kit comprising: 
 a) a compound having the formula:                          A is S, O, SO, SO 2  or CH 2 ;    X is O, S or NH;    L is a linker;    R 2  is hydrogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl;    R 3  is hydrogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl;    R 4  is hydrogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl;    R 5  is hydrogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl;    R 5  is hydrogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl;    R 7  is hydrogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl;    R 8  is hydrogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl;    R 9  is                          in which W is an alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, aryloxy, substituted aryloxy, heteroaryl, substituted heteroaryl, a dye moiety or CN, and;    s is an integer from 0 to 5;    W′ and W″ are independently hydrogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, heteroaryl, substituted heteroaryl, (═O), (═NH), OR′°, NHR″, or halogen; 
 wherein R 10  is hydrogen, alkyl, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl; or substituted heteroaryl; and,  
 R″ is hydrogen, substituted alkyl, heteroalkyl, substituted heteroalkyl, aryl, substituted aryl, heteroaryl; substituted heteroaryl or OR 12 ; 
 wherein R 12  is hydrogen, substituted or unsubstituted alkyl, and substituted or unsubstituted heteroalkyl or substituted or unsubstituted aryl or heteroaryl; and  
 
 Y is a dye moiety or a quencher moiety; and  
   b) instructions on the use of the kit.    
     
     
         34 . The kit according to  claim 33 , further comprising at least one of a reaction buffer, a α-lactamase, a antibody conjugated to a α-lactamase enzyme, a nucleic acid sequence coding for a α-lactamase, and a cell comprising a nucleic acid sequence coding for a β-lactamase.  
     
     
         35 . A kit for determing the presence or absence of β-lactamase in a sample, the kit comprising: 
 a) a compound having the formula:                                                                                                                                      b) instructions on the use of the kit.    
     
     
         37 . The kit according to claim  36 , further comprising at least one of a reaction buffer, a α-lactamase, a antibody conjugated to a β-lactamase enzyme, a nucleic acid sequence coding for a α-lactamase, and a cell comprising a nucleic acid sequence coding for a β-lactamase.