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 - 10 . (canceled) 
     
     
         11 . A combination, which comprises:
 a) 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): 
 
       
       
         
           
           
               
               
           
         
       
       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 a). 
       
     
     
         12 . The combination of  claim 11 , which further comprises an oxidizing reagent that is capable of oxidizing the aryl alcohol molecule or unsaturated aliphatic alcohol molecule produced by the cleavage reaction catalyzed by the hydrolytic enzyme. 
     
     
         13 . The combination of  claim 12 , wherein the oxidizing reagent is an aryl alcohol oxidase or an aliphatic alcohol oxidase that is capable of oxidizing the aryl alcohol molecule or unsaturated aliphatic alcohol molecule in the presence of oxygen to produce an aryl aldehyde molecule or unsaturated aliphatic aldehyde molecule and H 2 O 2 . 
     
     
         14 . The combination of  claim 12 , wherein the oxidizing reagent is an aryl alcohol dehydrogenase or an alcohol dehydrogenase that is capable of oxidizing the aryl alcohol molecule or unsaturated aliphatic alcohol molecule in the presence of NAD +  or NADP +  to produce NADH or NADPH. 
     
     
         15 . The combination of  claim 13 , which further comprises NADH or NADPH and an aryl alcohol dehydrogenase or an alcohol dehydrogenase that is capable of reducing the aryl aldehyde molecule or unsaturated aliphatic aldehyde molecule in the presence of NADH or NADPH. 
     
     
         16 . The combination of  claim 11 , 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. 
     
     
         17 - 34 . (canceled) 
     
     
         35 . The combination of  claim 11 , wherein A in the hydrolytic enzyme substrate is a substituted aromatic or heteroaromatic group. 
     
     
         36 . The combination of  claim 11 , wherein A in the hydrolytic enzyme substrate is a 1-alkene of the formula (IV): 
       
         
           
           
               
               
           
         
         wherein the wavy line indicates the point of attachment of A to —[CH(R)] n —O—X in Formula (I), and each G, G′ and G″ is independently H or an optionally substituted group selected from the group consisting of C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C3-C8 cycloalkyl, C3-C8 heterocycloalkyl, aryl, and heteroaryl. 
       
     
     
         37 . The combination of  claim 11 , wherein A in the hydrolytic enzyme substrate is a 1-alkyne of the formula (V): 
       
         
           
           
               
               
           
         
         wherein the wavy line indicates the point of attachment of A to —[CH(R)] n —O—X in Formula (I), and G is H or an optionally substituted member selected from the group consisting of C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C3-C8 cycloalkyl, C3-C8 heterocycloalkyl, aryl, and heteroaryl. 
       
     
     
         38 . The combination of  claim 11 , wherein X in the hydrolytic enzyme substrate comprises a saccharide. 
     
     
         39 . The combination of  claim 38 , wherein the compound is of the formula (VIa) or (VIb): 
       
         
           
           
               
               
           
         
       
       wherein R 2  is H or —CH 2 OQ, and each Q is independently H or a monosaccharide, disaccharide or oligosaccharide, and A, R and n are as defined in  claim 11 . 
     
     
         40 . The combination of  claim 11 , wherein the compound is an ester of formula (VII): 
       
         
           
           
               
               
           
         
         wherein R 3  is H or an optionally substituted aryl, heteroaryl, C1-C8 alkyl, C3-C8 cycloalkyl, or C3-C8 heterocyclyl group, 
         and A, R and n are as defined in  claim 11 . 
       
     
     
         41 . The combination of  claim 11 , wherein the compound is of the formula (VIII): 
       
         
           
           
               
               
           
         
         wherein Z is N, S, S═O, P, or P—OH, and R 4  is O, hydroxy, C1-C4 alkoxy, C1-C4 alkyl, or aryl. 
       
     
     
         42 . The combination of  claim 11 , wherein X in the hydrolytic enzyme substrate comprises a phosphate group. 
     
     
         43 . The combination of  claim 11 , wherein A in the hydrolytic enzyme substrate is a group of the formula (X): 
       
         
           
           
               
               
           
         
         wherein the wavy line indicates the point of attachment of A to —[CH(R)] n —O—X in Formula (I), 
         and each G is independently H or an optionally substituted group selected from the group consisting of C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C3-C8 cycloalkyl, C3-C8 heterocycloalkyl, aryl, and heteroaryl. 
       
     
     
         44 . The combination of  claim 16 , wherein the hydrolytic enzyme is a β-glycosidase. 
     
     
         45 . The combination of  claim 16 , wherein the hydrolytic enzyme is a beta-galactosidase. 
     
     
         46 . The combination of  claim 11 , which further comprises a reagent for assaying, isolating and/or producing a target. 
     
     
         47 . The combination of  claim 11 , which further comprises a reagent for immunoassay, protein sequencing, nucleic acid amplification, hybridization or sequencing.