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: 
       
         
           
           
               
               
           
         
         where R 1  is H, 
       
       
         
           
           
               
               
           
         
         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 11 , 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 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 heteroalkyl, 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 - 8 . (canceled) 
     
     
         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 . (canceled) 
     
     
         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 alkyl 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, P, SO, SO 2 , or CH 2 ; 
         X is O, S or NH; 
         W′ and W″ are independently hydrogen, alkyl, substituted alkyl, (═O), (═NH), OR″, 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 - 24 . (canceled) 
     
     
         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 is a compound according to  claim 1 ; 
 b) incubating the contacted sample for a sufficient amount of time for the β-lactamase enzyme 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 . (canceled) 
     
     
         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 - 30 . (canceled) 
     
     
         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 β-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 . (canceled) 
     
     
         33 . A kit for determining the presence or absence of β-lactamase in a sample, the kit comprising:
 a) a compound according to  claim 1 ; and 
 b) instructions on the use of the kit. 
 
     
     
         34 - 37 . (canceled)