Compositions and methods for determining the presence of active leukocyte cells using an electrochemical assay

Abstract

The present disclosure relates to compositions, methods and test devices for determining the presence of active leukocyte cells, for example, by using novel LE and/or HNE substrates in an electrochemical assay.

What is claimed is: 
     
         1 . A substrate composition with a specificity for leukocyte esterases having a first moiety for participating in a redox reaction and a second moiety comprising an amine blocking group or alcohol blocking group, wherein said substrate has a chemical formula of Formula I: 
       
         
           
           
               
               
           
         
         Wherein “A” comprises oxygen (O) or NR a , where R a  is hydrogen (H) or optionally substituted alkyl, aryl, or aralkyl, “B” has the general structure 
       
       
         
           
           
               
               
           
         
         where X, Y, and L are the 
         same or different and are independently nitrogen (N) or carbon, R 5  and R 6 are the same or different and are independently hydrogen (H), a halogen, (OH) 2 P(O) 2 , optionally substituted alkyl or aryl, halogenated alkyl or halogenated aryl, optionally substituted alkoxy (but not at position 4), OH, amino, optionally substituted alkylamino or arylamino, NO 2 , thio, optionally substituted alkylthio or arylthio, hydroxycarbonyl, formyl, optionally substituted alkoxycarbonyl or aryloxycarbonyl, amido, optionally substituted alkyl or aryl amido, or optionally substituted hydroxyalkyl or hydroxyaryl, and “C” is an amine blocking group or alcohol blocking group. 
       
     
     
         2  The substrate composition of  claim 1 , wherein moiety B is selected from one of the following: 
       
         
           
           
               
               
           
         
         Where X, Y, and Z are the same or different and are independently nitrogen (N) or carbon, R′ is hydrogen (H) or optionally substituted alkyl or aryl, and R 6  is hydrogen (H), a halogen, (OH) 2 P(O) 2 , optionally substituted alkyl or aryl, halogenated alkyl or halogenated aryl, optionally substituted alkoxy (but not at position 4), OH, amino, optionally substituted alkylamino or arylamino, NO 2 , thio, optionally substituted alkylthio or arylthio, hydroxycarbonyl, formyl, optionally substituted alkoxycarbonyl or aryloxycarbonyl, amido, optionally substituted alkyl or aryl amido, or optionally substituted hydroxyalkyl or hydroxyaryl. 
       
     
     
         3 . The substrate composition of  claim 1 , wherein said second moiety C comprises one of the following: acetyl (Ac), benzoyl (Bz), benzyl (Bn), β-methoxyethoxymethyl (MEM), dimethoxytrityl (DMT), methyoxymethyl (MOM), methoxytrityl [(4-methoxyphenyl)diphenylmethyl] (MMT), p-Methoxybenzyl (PMB), methylthiomethyl, pivaloyl (Piv), tetrahydropyranyl (THP), tetrahydrofuran (THF), trityl (Tr), sily (e.g. TMS, TBDMS, TOM, TIPS), methyl, and ethoxyethyl (EE), benzyloxycarbonyl (Cbz); p-methoxybenzylcarbonyl (Moz or MeOZ), tert-butoxycarbonyl (BOC), 9-fluorenylmethyloxycarbonyl (FMOC), 3,4-Dimethoxybenzyl (DMPM), p-methoxyphenyl (PMP), tosyl (Ts), trichloroethoxycarbonyl (Troc), arylsulfonyl, or alkylsulfonyl (e.g.Nosyl and Nps). 
     
     
         4 . The substrate composition of  claim 1 , wherein said second moiety C comprises a sulfonyl group with a substituted or unsubstituted aryl, heteroaryl, or heterocycle 
     
     
         5 . The substrate composition of  claim 1 , wherein said second moiety C is one of Tosyl, pyridine-sulfonyl, methoxypyridine-sulfonyl, or (methoxycarbonyl)pyridine-sulfonyl 
     
     
         6 . A substrate composition for leukocyte esterase with the general structure depicted in Formula IV: 
       
         
           
           
               
               
           
         
         Wherein “A” comprises oxygen (O) or NR a , where R a  is hydrogen (H) or optionally substituted alkyl, aryl, or aralkyl, R 1  and R 2  are either the same or different and are independently hydrogen or optionally substituted alkyl “B” has the 
       
       
         
           
           
               
               
           
         
         general structure 
         where X, Y, and Z are the same or different and are independently nitrogen (N) or carbon, R 5  and R 6 are the same or different and are independently hydrogen (H), a halogen, (OH) 2 P(O) 2 , optionally substituted alkyl or aryl, halogenated alkyl or halogenated aryl, optionally substituted alkoxy (but not at position 4), OH, amino, optionally substituted alkylamino or arylamino, NO 2 , thio, optionally substituted alkylthio or arylthio, hydroxycarbonyl, formyl, optionally substituted alkoxycarbonyl or aryloxycarbonyl, amido, optionally substituted alkyl or aryl amido, or optionally substituted hydroxyalkyl or hydroxyaryl, and R 4  is a substituted or unsubstituted aryl, heteroaryl, or heterocycle. 
       
     
     
         7 . The substrate composition of  claim 6 , wherein moiety B is selected from one of the following: 
       
         
           
           
               
               
           
         
         Where X, Y, and Z are the same or different and are independently nitrogen (N) or carbon, R′ is hydrogen (H) or optionally substituted alkyl or aryl, and R6 is hydrogen (H), a halogen, (OH) 2 P(O) 2 , optionally substituted alkyl or aryl, halogenated alkyl or halogenated aryl, optionally substituted alkoxy (but not at position 4), OH, amino, optionally substituted alkylamino or arylamino, NO 2 , thio, optionally substituted alkylthio or arylthio, hydroxycarbonyl, formyl, optionally substituted alkoxycarbonyl or aryloxycarbonyl, amido, optionally substituted alkyl or aryl amido, or optionally substituted hydroxyalkyl or hydroxyaryl. 
       
     
     
         8 . The substrate composition of  claim 6 , wherein said composition has the structure: 
       
         
           
           
               
               
           
         
         Wherein “A” comprises oxygen (O) or NR a , where R a  is hydrogen (H) or optionally substituted alkyl, aryl, or aralkyl, R 1  and R 2  are either the same or different and are independently hydrogen or optionally substituted alkyl, where W, X, Y, and Z are the same or different and are independently nitrogen (N) or carbon, R 5  and R 6 are the same or different and are independently hydrogen (H), a halogen, (OH) 2 P(O) 2 , optionally substituted alkyl or aryl, halogenated alkyl or halogenated aryl, optionally substituted alkoxy (but not at position 4), OH, amino, optionally substituted alkylamino or arylamino, NO 2 , thio, optionally substituted alkylthio or arylthio, hydroxycarbonyl, formyl, optionally substituted alkoxycarbonyl or aryloxycarbonyl, amido, optionally substituted alkyl or aryl amido, or optionally substituted hydroxyalkyl or hydroxyaryl, and R 7  is hydrogen (H), OH, amino, alkyl, aryl, alkoxy, aryloxy, hydroxycarbonyl, alkoxycarbonyl, or aryloxycarbonyl. 
       
     
     
         9 . The substrate composition of  claim 6 , wherein said composition has the structure: 
       
         
           
           
               
               
           
         
         Wherein “A” comprises oxygen (O) or NR a , where R a  is hydrogen (H) or optionally substituted alkyl, aryl, or aralkyl, R 1  and R 2  are either the same or different and are independently hydrogen or optionally substituted alkyl, where W, X, Y, and Z are the same or different and are independently nitrogen (N) or carbon, R 5  and R 6 are the same or different and are independently hydrogen (H), a halogen, (OH) 2 P(O) 2 , optionally substituted alkyl or aryl, halogenated alkyl or halogenated aryl, optionally substituted alkoxy (but not at position 4), OH, amino, optionally substituted alkylamino or arylamino, NO 2 , thio, optionally substituted alkylthio or arylthio, hydroxycarbonyl, formyl, optionally substituted alkoxycarbonyl or aryloxycarbonyl, amido, optionally substituted alkyl or aryl amido, or optionally substituted hydroxyalkyl or hydroxyaryl, and R 7  is H, CH 3 , OCH 3 , or CO 2 CH 3 . 
       
     
     
         10 . The substrate composition of  claim 8 , wherein said composition has the structure: 
       
         
           
           
               
               
           
         
         Wherein “A” comprises oxygen (O) or NR a , where R a  is hydrogen (H) or optionally substituted alkyl, aryl, or aralkyl, R 1  and R 2  are either the same or different and are independently hydrogen or optionally substituted alkyl, where W, X, Y, and Z are the same or different and are independently nitrogen (N) or carbon, R 6  is hydrogen (H), a halogen, (OH) 2 P(O) 2 , optionally substituted alkyl or aryl, halogenated alkyl or halogenated aryl, optionally substituted alkoxy (but not at position 4), OH, amino, optionally substituted alkylamino or arylamino, NO 2 , thio, optionally substituted alkylthio or arylthio, hydroxycarbonyl, formyl, optionally substituted alkoxycarbonyl or aryloxycarbonyl, amido, optionally substituted alkyl or aryl amido, or optionally substituted hydroxyalkyl or hydroxyaryl, and R 7  is hydrogen (H), OH, amino, alkyl, aryl, alkoxy, aryloxy, hydroxycarbonyl, alkoxycarbonyl, or aryloxycarbonyl. 
       
     
     
         11 . The substrate composition of  claim 8 , wherein said composition has the structure: 
       
         
           
           
               
               
           
         
         Wherein “A” comprises oxygen (O) or NR a , where R a  is hydrogen (H) or optionally substituted alkyl, aryl, or aralkyl, R 1  and R 2  are either the same or different and are independently hydrogen or optionally substituted alkyl, where W, X, Y, and Z are the same or different and are independently nitrogen (N) or carbon, R 6  is hydrogen (H), a halogen, (OH) 2 P(O) 2 , optionally substituted alkyl or aryl, halogenated alkyl or halogenated aryl, optionally substituted alkoxy (but not at position 4), OH, amino, optionally substituted alkylamino or arylamino, NO 2 , thio, optionally substituted alkylthio or arylthio, hydroxycarbonyl, formyl, optionally substituted alkoxycarbonyl or aryloxycarbonyl, amido, optionally substituted alkyl or aryl amido, or optionally substituted hydroxyalkyl or hydroxyaryl, and R 7  is H, CH 3 , OCH 3 , or CO 2 CH 3 . 
       
     
     
         12 . A substrate composition with a specificity for leukocyte esterases having a first moiety for participating in a redox reaction and a second moiety comprising an amine blocking or alcohol blocking group, wherein said substrate has a chemical formula of Formula I: 
       
         
           
           
               
               
           
         
         Wherein “A” comprises oxygen (O) or NR a , where R a  is hydrogen (H) or optionally substituted alkyl, aryl, or aralkyl, “B” has the general structure 
       
       
         
           
           
               
               
           
         
         where X, Y, and Z are the same or different and are independently nitrogen (N) or carbon, R 5  and R 6 are the same or different and are independently hydrogen (H), a halogen, (OH) 2 P(O) 2 , optionally substituted alkyl or aryl, halogenated alkyl or halogenated aryl, optionally substituted alkoxy, OH, amino, optionally substituted alkylamino or arylamino, NO 2 , thio, optionally substituted alkylthio or arylthio, hydroxycarbonyl, formyl, optionally substituted alkoxycarbonyl or aryloxycarbonyl, amido, optionally substituted alkyl or aryl amido, or optionally substituted hydroxyalkyl or hydroxyaryl, and “C” is an amine blocking group or alcohol blocking group. 
       
     
     
         13 . The substrate composition of  claim 12 , wherein moiety B is the following 
       
         
           
           
               
               
           
         
         Wherein X, Y, and Z are the same or different and are independently nitrogen (N) or carbon, R′ is hydrogen (H) or optionally substituted alkyl or aryl, and R 6  is hydrogen (H), a halogen, (OH) 2 P(O) 2 , optionally substituted alkyl or aryl, halogenated alkyl or halogenated aryl, optionally substituted alkoxy, OH, amino, optionally substituted alkylamino or arylamino, NO 2 , thio, optionally substituted alkylthio or arylthio, hydroxycarbonyl, formyl, optionally substituted alkoxycarbonyl or aryloxycarbonyl, amido, optionally substituted alkyl or aryl amido, or optionally substituted hydroxyalkyl or hydroxyaryl. 
       
     
     
         14 . A substrate composition for leukocyte esterase with the general structure depicted in Formula IV: 
       
         
           
           
               
               
           
         
         Wherein “A” comprises oxygen (O) or NR a , where R a  is hydrogen (H) or optionally substituted alkyl, aryl, or aralkyl, R 1  and R 2  are either the same or different and are independently hydrogen or optionally substituted alkyl “B” has 
       
       
         
           
           
               
               
           
         
         the general structure, 
         where X, Y, and Z are the same or different and are independently nitrogen (N) or carbon, R 5  and R 6  are the same or different and are independently hydrogen (H), a halogen, (OH) 2 P(O) 2 , optionally substituted alkyl or aryl, halogenated alkyl or halogenated aryl, optionally substituted alkoxy, OH, amino, optionally substituted alkylamino or arylamino, NO 2 , thio, optionally substituted alkylthio or arylthio, hydroxycarbonyl, formyl, optionally substituted alkoxycarbonyl or aryloxycarbonyl, amido, optionally substituted alkyl or aryl amido, or optionally substituted hydroxyalkyl or hydroxyaryl, and R 4  is a substituted or unsubstituted aryl, heteroaryl, or heterocycle. 
       
     
     
         15 . The substrate composition of  claim 6 , wherein moiety B is the following: 
       
         
           
           
               
               
           
         
         Wherein X, Y, and Z are the same or different and are independently nitrogen (N) or carbon, R′ is hydrogen (H) or optionally substituted alkyl or aryl, and R 6  is hydrogen (H), a halogen, (OH) 2 P(O) 2 , optionally substituted alkyl or aryl, halogenated alkyl or halogenated aryl, optionally substituted alkoxy, OH, amino, optionally substituted alkylamino or arylamino, NO 2 , thio, optionally substituted alkylthio or arylthio, hydroxycarbonyl, formyl, optionally substituted alkoxycarbonyl or aryloxycarbonyl, amido, optionally substituted alkyl or aryl amido, or optionally substituted hydroxyalkyl or hydroxyaryl. 
       
     
     
         16 . A device for detecting leukocyte esterase comprising a substrate that releases one of phenol, a derivative of phenol, or optionally substituted hydroquinone as the electrochemical mediator upon cleavage of an ester linkage by leukocyte esterase. 
     
     
         17 . A method for detecting leukocyte esterase in a biological sample comprising:
 detecting release of one phenol, a derivative of phenol, or optionally substituted hydroquinone as an electrochemical mediator in an electrochemical assay.   
     
     
         18 . A method for diagnosis of infection comprising:
 determining the level of leukocyte esterase in a test sample based on release of one of phenol, a derivative of phenol, or an optionally substituted hydroquinone as the electrochemical mediator in an electrochemical assay.