Hydrolase enzyme substrates and uses thereof

Abstract

The present invention provides novel methods for determining the presence or amount of a hydrolytic enzyme in a sample, based on novel substrates for the enzymes, and also provides compositions and methods that provide highly sensitive assay methods for such hydrolytic enzymes.

1 . A hydrolytic enzyme substrate, which is a compound of formula (I): 
       
         
           
           
               
               
           
         
         wherein: 
         A is an aromatic or heteroaromatic group, a 1-alkene or a 1-alkyne, each of which is optionally substituted; 
         each R is independently H or an optionally substituted C1-C4 alkyl or aryl; 
         n is an integer from 1-4; 
         and X is a group comprising a substrate moiety,
 wherein the substrate moiety comprises a recognition component of a substrate for the hydrolytic enzyme, and wherein the activity of said hydrolytic enzyme is capable of hydrolyzing said compound of formula (I) to form compounds (II) and (III): 
 
       
       
         
           
           
               
               
           
         
       
     
     
         2 - 10 . (canceled) 
     
     
         11 . A combination, which comprises:
 a) a hydrolytic enzyme substrate of  claim 1 ; and   b) a hydrolytic enzyme that is capable of cleaving said hydrolytic enzyme substrate to produce an aryl alcohol molecule or unsaturated aliphatic alcohol molecule as a product of said cleavage reaction catalyzed by said hydrolytic enzyme, wherein said aryl alcohol molecule or unsaturated aliphatic alcohol molecule has a structure of said formula (II):   
       
         
           
           
               
               
           
         
         wherein A, R and n are as defined in  claim 1 . 
       
     
     
         12 - 16 . (canceled) 
     
     
         17 . A method for assessing activity and/or amount of a hydrolytic enzyme in a sample, which method comprises:
 a) contacting a hydrolytic enzyme substrate of any of the embodiments 1-25 with a sample containing or suspected of containing a hydrolytic enzyme with a hydrolytic enzyme substrate having a structure of formula (I):   
       
         
           
           
               
               
           
         
         under conditions where said hydrolytic enzyme, if present in said sample, cleaves said substrate to produce an aryl alcohol molecule or unsaturated aliphatic alcohol molecule having a structure of formula (II) and a compound having a structure of formula (III): 
       
       
         
           
           
               
               
           
         
         wherein A, R, n and X are as defined in  claim 1 ; and 
         b) assessing the presence and/or amount of said aryl alcohol molecule or unsaturated aliphatic alcohol molecule to assess activity and/or amount of said hydrolytic enzyme in said sample. 
       
     
     
         18 . The method of  claim 17 , wherein the hydrolytic enzyme is an esterase, a beta-galactosidase, or a glycosidase. 
     
     
         19 . The method of  claim 17 , wherein the step of assessing the presence and/or amount of the aryl alcohol molecule or unsaturated aliphatic alcohol molecule comprises oxidizing the aryl alcohol molecule or unsaturated aliphatic alcohol molecule with an oxidizing reagent. 
     
     
         20 . The method of  claim 19 , wherein the presence and/or amount of the aryl alcohol molecule or unsaturated aliphatic alcohol molecule is assessed by oxidizing the aryl alcohol molecule or unsaturated aliphatic alcohol molecule with an aryl alcohol oxidase or an aliphatic alcohol oxidase in the presence of oxygen to produce H 2 O 2  and assessing the presence and/or amount of the H 2 O 2 . 
     
     
         21 . The method of  claim 17 , wherein the presence and/or amount of the aryl alcohol molecule or unsaturated aliphatic alcohol molecule is assessed by oxidizing the aryl alcohol molecule or unsaturated aliphatic alcohol molecule with an aryl alcohol dehydrogenase or an alcohol dehydrogenase in the presence of NAD +  or NADP +  to produce NADH or NADPH, and assessing the presence and/or amount of the NAD+, NADP+, NADH or NADPH. 
     
     
         22 . The method of  claim 17 , wherein the presence and/or amount of the aryl alcohol molecule or unsaturated aliphatic alcohol molecule is assessed by:
 a) oxidizing the aryl alcohol molecule or unsaturated aliphatic alcohol molecule with an aryl alcohol oxidase or an aliphatic alcohol oxidase in the presence of oxygen to produce aryl aldehyde molecule or unsaturated aliphatic aldehyde molecule and H 2 O 2 ;   b) reducing the aryl aldehyde molecule or unsaturated aliphatic aldehyde molecule with an aryl alcohol dehydrogenase or an alcohol dehydrogenase in the presence of NADH or NADPH to form a reaction cycle in which the reduced aryl alcohol molecule or unsaturated aliphatic alcohol molecule is oxidized by the aryl alcohol oxidase or an aliphatic alcohol oxidase in the presence of oxygen to produce additional aryl aldehyde molecule or unsaturated aliphatic aldehyde molecule and H 2 O 2 ; and   c) assessing the presence and/or amount of the H 2 O 2 , or the amount of NADH, NADPH, NAD+, or NADP+.   
     
     
         23 . The method of  claim 17 , wherein the hydrolytic enzyme substrate comprises at least a part of a β-glycosidase substrate molecule, and the hydrolytic enzyme is a β-glycosidase or beta-galactosidase. 
     
     
         24 . The method of  claim 23 , wherein the step of assessing the presence and/or amount of the aryl alcohol molecule or unsaturated aliphatic alcohol molecule comprises oxidizing the aryl alcohol molecule or unsaturated aliphatic alcohol molecule with an oxidizing reagent. 
     
     
         25 . The method of  claim 23 , wherein the presence and/or amount of the aryl alcohol molecule or unsaturated aliphatic alcohol molecule is assessed by oxidizing the aryl alcohol molecule or unsaturated aliphatic alcohol molecule with an aryl alcohol oxidase or an aliphatic alcohol oxidase in the presence of oxygen to produce oxidized aryl alcohol molecule or unsaturated aliphatic alcohol molecule and H 2 O 2  and assessing the presence and/or amount of the H 2 O 2 . 
     
     
         26 . The method of  claim 23 , wherein the presence and/or amount of the aryl alcohol molecule or unsaturated aliphatic alcohol molecule is assessed by oxidizing the aryl alcohol molecule or unsaturated aliphatic alcohol molecule with an aryl alcohol dehydrogenase or an alcohol dehydrogenase in the presence of NAD +  or NADP +  to produce NADH or NADPH, and assessing the presence and/or amount of the NAD+, NADP+, NADH or NADPH. 
     
     
         27 . The method of  claim 23 , wherein the presence and/or amount of the aryl alcohol molecule or unsaturated aliphatic alcohol molecule is assessed by:
 a) oxidizing the aryl alcohol molecule or unsaturated aliphatic alcohol molecule with an aryl alcohol oxidase or an aliphatic alcohol oxidase in the presence of oxygen to produce oxidized aryl alcohol molecule or unsaturated aliphatic alcohol molecule and H 2 O 2 ;   b) reducing the oxidized aryl alcohol molecule or unsaturated aliphatic alcohol molecule with an aryl alcohol dehydrogenase or an alcohol dehydrogenase in the presence of NADH or NADPH to form a reaction cycle in which the reduced aryl alcohol molecule or unsaturated aliphatic alcohol molecule is oxidized by the aryl alcohol oxidase or an aliphatic alcohol oxidase in the presence of oxygen to produce additional oxidized aryl alcohol molecule or unsaturated aliphatic alcohol molecule and H 2 O 2 ; and   c) assessing the presence and/or amount of the H 2 O 2 .   
     
     
         28 . The method of  claim 17 , wherein the hydrolytic enzyme substrate comprises at least a part of an alkaline phosphatase substrate molecule, and the hydrolytic enzyme is an alkaline phosphatase. 
     
     
         29 . The method of  claim 28 , wherein the step of assessing the presence and/or amount of the aryl alcohol molecule or unsaturated aliphatic alcohol molecule comprises oxidizing the aryl alcohol molecule or unsaturated aliphatic alcohol molecule with an oxidizing reagent. 
     
     
         30 . The method of  claim 28 , wherein the presence and/or amount of the aryl alcohol molecule or unsaturated aliphatic alcohol molecule is assessed by oxidizing the aryl alcohol molecule or unsaturated aliphatic alcohol molecule with an aryl alcohol oxidase or an aliphatic alcohol oxidase in the presence of oxygen to produce oxidized aryl alcohol molecule or unsaturated aliphatic alcohol molecule and H 2 O 2 , and assessing the presence and/or amount of the H 2 O 2 . 
     
     
         31 . The method of  claim 28 , wherein the presence and/or amount of the aryl alcohol molecule or unsaturated aliphatic alcohol molecule is assessed by oxidizing the aryl alcohol molecule or unsaturated aliphatic alcohol molecule with an aryl alcohol dehydrogenase or an alcohol dehydrogenase in the presence of NAD +  or NADP +  to produce NADH or NADPH, and assessing the presence and/or amount of the NAD+, NADP+, NADH or NADPH. 
     
     
         32 . The method of  claim 28 , wherein the presence and/or amount of the aryl alcohol molecule or unsaturated aliphatic alcohol molecule is assessed by
 a) oxidizing the aryl alcohol molecule or unsaturated aliphatic alcohol molecule with an aryl alcohol oxidase or an aliphatic alcohol oxidase in the presence of oxygen to produce oxidized aryl alcohol molecule or unsaturated aliphatic alcohol molecule and H 2 O 2 ;   b) reducing the oxidized aryl alcohol molecule or unsaturated aliphatic alcohol molecule with an aryl alcohol dehydrogenase or an alcohol dehydrogenase in the presence of NADH or NADPH to form a reaction cycle in which the reduced aryl alcohol molecule or unsaturated aliphatic alcohol molecule is oxidized by the aryl alcohol oxidase or an aliphatic alcohol oxidase in the presence of oxygen to produce additional oxidized aryl alcohol molecule or unsaturated aliphatic alcohol molecule and H 2 O 2 ; and   c) assessing the presence and/or amount of the H 2 O 2 .   
     
     
         33 . The method of  claim 17 , which is conducted as part of an assay, isolation and/or production of a target. 
     
     
         34 . The method of  claim 17 , which is conducted as part of an immunoassay, protein sequencing, nucleic acid amplification, hybridization or sequencing.