Quinone methide analog signal amplification

Abstract

Disclosed herein are novel quinone methide analog precursors and embodiments of a method and a kit of using the same for detecting one or more targets in a biological sample. The method of detection comprises contacting the sample with a detection probe, then contacting the sample with a labeling conjugate that comprises an enzyme. The enzyme interacts with a quinone methide analog precursor comprising a detectable label, forming a reactive quinone methide analog, which binds to the biological sample proximally to or directly on the target. The detectable label is then detected. In some embodiments, multiple targets can be detected by multiple quinone methide analog precursors interacting with different enzymes without the need for an enzyme deactivation step.

1 . A compound having Formula (III): 
       
         
           
           
               
               
           
         
         wherein 
         each Q is carbon; 
         each R 7  is independently selected from hydrogen, —Z—R 1 , a moiety comprising a LG group, a moiety comprising a detectable label, a moiety comprising a LG group and a detectable label, a halo group, an aliphatic group, an alkoxy group, or where two adjacent R 7  groups together form an aliphatic ring or aryl ring where the formed aliphatic ring or aryl ring may be substituted with at least one of a moiety comprising LG group and a moiety comprising a detectable label; 
         —Z—R 1  is —OP(O)(OH) 2 , —SP(O)(OH) 2 , —O-sugar or a derivative or analog thereof, —NR a P(O)(OH) 2 , —OC(═O)R a , —N(R a )C(═O)R a , —NO 2 , —NR a —C(═O)—N(R c ) 2 , —OSO 3 H, —OR a , or a β-lactam-containing moiety; 
         LG is a halide, alkoxy, carboxylate, inorganic ester, thiolate, amine, carboxylate, azide, sulfate ester, aryloxy, phenoxide or —N(R b ) 3   +  where each R b  is independently hydrogen or a lower alkyl or two R b  moieties together form a heteroaliphatic ring; 
         m is 1, 
         at least one of the R 7  groups comprises —Z—R 1 ; and 
         the detectable label is selected from the group consisting of a hapten, a chromophore, a dye, and a fluorophore; and 
         wherein the compound of Formula (III) comprises at least one detectable label. 
       
     
     
         2 . The compound of  claim 1 , wherein —Z—R 1  is —O-sugar or a derivative or analog thereof. 
     
     
         3 . The compound of  claim 2 , wherein —O-sugar is —O-alpha-glucose, —O-beta-glucose, —O-alpha-galactose, —O-beta-galactose, —O-alpha-lactose, —O-beta-lactose, —O-alpha-glucuronic acid, and —O-beta-glucuronic acid. 
     
     
         4 . The compound of  claim 1 , wherein the β-lactam-containing moiety is 
       
         
           
           
               
               
           
         
         wherein 
         R is alkyl; and 
         Z is O or S. 
       
     
     
         5 . A quinone methide analog precursor compound having Formula (IV) or a salt or solvate thereof: 
       
         
           
           
               
               
           
         
         wherein 
         R 8  is —C(LG)(R 5 )(R 3 R 4 ), —R 3 R 4  or —C(LG)(R 5 )(R 6 ); 
         R 9 , R 11  and R 12  are each independently hydrogen, halo, cyano, aliphatic, alkoxy, NO 2 , N(R c ) 2 , aryl, haloalkyl, —C(O)alkyl, —C(S)alkyl, —C(O)OH, —C(O)Oalkyl, —C(O)NHR c , —C(O)N(R c ) 2 , —R 3 R 4 , or where R u  and R 12  together form an aliphatic ring or aryl ring; 
         R 10  is hydrogen, halo, cyano, aliphatic, alkoxy, NO 2 , N(R c ) 2 , aryl, haloalkyl, —C(O)alkyl, —C(S)alkyl, —C(O)OH, —C(O)Oalkyl, —C(O)NHR c , —C(O)N(R c ) 2 —R 3 R 4 , —C(LG)(R 5 )(R 6 ), or where R 10  together with one of R 9  or R 11  form an aliphatic ring or aryl ring; 
         LG is a leaving group; 
         Z—R 1  is —OP(O)(OH) 2 , —SP(O)(OH) 2 , —NR a P(O)(OH) 2 , —OC(═O)R a , —N(R a )C(═O)R a , —NO 2 , —NR a —C(═O)—N(R c ) 2 , —OSO 3 H, —OR a , —O-β-lactam-containing moiety, —S-β-lactam-containing moiety, or —O-enzyme-appropriate sugar; 
         R 3  is a bond, —(CH 2 ) n NH—, —O(CH 2 ) n NH—, —N(H)C(O)(CH 2 ) n NH—, —C(O)N(H)(CH 2 ) n NH—, —(CH 2 ) n O—, —O(CH 2 ) n O—, —O(CH 2 CH 2 O) n —, —N(H)C(O)(CH 2 ) n O—, —C(O)N(H)(CH 2 ) n O—, —C(O)N(H)(CH 2 CH 2 O) n —, —(CH 2 ) n S—, —O(CH 2 ) n S—, —N(H)C(O)(CH 2 ) n S—, —C(O)N(H)(CH 2 ) n S—, —(CH 2 ) n NH—, —C(O)N(H)(CH 2 CH 2 O) n CH 2 CH 2 NH, —C(O)(CH 2 CH 2 O) n CH 2 CH 2 NH—, —C(O)N(H)(CH 2 ) n NHC(O)CH(CH 3 )(CH 2 ) n NH— or —N(H)(CH 2 ) n NH—, 
       
       
         
           
           
               
               
           
         
          where each n is independently an integer ranging from 1 to 12; 
         R 4  is a chromogen, a fluorophore, a luminophore, a hapten or a combination thereof; 
         each R 5  is independently hydrogen, halo, cyano, lower alkyl, lower haloalkyl, —C(O)alkyl, —C(S)alkyl, —C(O)OH, —C(O)Oalkyl, —C(O)NHR c  or —C(O)N(R) 2 ; 
         each R 6  is independently hydrogen, halo, cyano, lower alkyl, lower haloalkyl, —C(O)alkyl, —C(S)alkyl, —C(O)OH, —C(O)Oalkyl, —C(O)NHR c  or —C(O)N(R) 2 ; 
         each R a  is hydrogen; 
         each R c  independently is hydrogen, aryl, aliphatic or heteroaliphatic, or two R c  moieties together form a heteroaliphatic ring; and 
         at least one of R 8  and R 10  comprises LG, and at least one of R 8  and R 10  comprises R 3 R 4 ; and 
         if LG is halo, then R 5  and R 6  are not halo. 
       
     
     
         6 . The quinone methide analog precursor compound of  claim 5 , wherein the quinone methide analog precursor compound has a Formula: 
       
         
           
           
               
               
           
         
       
     
     
         7 . The quinone methide analog precursor compound of  claim 5 , wherein the quinone methide analog precursor compound has a Formula: 
       
         
           
           
               
               
           
         
       
     
     
         8 . The quinone methide analog precursor compound of  claim 5 , wherein the quinone methide analog precursor compound has a Formula: 
       
         
           
           
               
               
           
         
       
       where R 13 -R 20  are each independently hydrogen, halo, cyano, aliphatic, alkoxy, —NO 2 , —N(R c ) 2 , aryl, haloalkyl, —C(O)alkyl, —C(S)alkyl, —C(O)OH, —C(O)Oalkyl, —C(O)NHR c , —C(O)N(R c ) 2 , —R 3 R 4  or two adjacent groups together form an aliphatic ring or aryl ring, and at least one of R 1 -R 20  comprises —R 3 R 4 . 
     
     
         9 . The quinone methide analog precursor compound of  claim 5 , wherein Z—R 1  is —OP(O)(OH) 2 , —NO 2 , —NHC(O)R, —OC(O)CH 3 , —OC(O)CH 2 CH 3 , —NHC(O)NH 2 , —OS(O) 2 OH, OCH 3 , or a salt thereof. 
     
     
         10 . The quinone methide analog precursor compound of  claim 5 , wherein the sugar is α-glucose, β-glucose, α-galactoside, β-galactoside, α-glucuronose or β-glucuronose. 
     
     
         11 . The quinone methide analog precursor compound of  claim 5 , wherein LG is fluoride, chloride, azide, acetate, methoxy, ethoxy, isopropoxy, phenoxide, —OS(O) 2 CH 3 , —OS(O) 2 C 6 H 4 CH 3 , —OS(O) 2 C 6 H 5 , —OS(O) 2 C 6 H 4 CX 3 , —OC 6 H 5 , —N 2   + , —NH 3   + , —NC 6 H 5   + , —O-alkyl, —OC(O)alkyl, —OC(O)H, —N(R b ) 3   + , or 1,4-diazabicyclo[2.2.2]octane; wherein each X is independently is fluoro, chloro, bromo or iodo; and wherein each R b  independently is hydrogen or lower alkyl, or two R b  moieties together form a heteroaliphatic ring. 
     
     
         12 . The quinone methide analog precursor compound of  claim 5 , wherein R 4  is 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
       
     
     
         13 . The quinone methide analog precursor compound of  claim 5 , wherein the quinone methide analog precursor compound is selected from the group consisting of: 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
       
     
     
         14 . The quinone methide analog precursor compound of  claim 5 , wherein the quinone methide analog precursor compound is selected from the group consisting of 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
       
     
     
         15 . The quinone methide analog precursor compound of  claim 5 , wherein R 4  is a chromophore. 
     
     
         16 . The quinone methide analog precursor compound of  claim 5 , wherein R 4  is derived from 
       
         
           
           
               
               
           
         
       
     
     
         17 . A compound having Formula (VI): 
       
         
           
           
               
               
           
         
         wherein 
         —Z—R 1  is —O—P(O)(OH) 2  or —O-sugar; 
         LG is a leaving group; 
         R 3  is a bond, —(CH 2 ) n NH—, —O(CH 2 ) n NH—, —N(H)C(O)(CH 2 ) n NH—, —C(O)N(H)(CH 2 ) n NH—, —(CH 2 ) n O—, —O(CH 2 ) n O—, —O(CH 2 CH 2 O) n —, —N(H)C(O)(CH 2 ) n O—, —C(O)N(H)(CH 2 ) n O—, —C(O)N(H)(CH 2 CH 2 O) n —, —(CH 2 ) n S—, —O(CH 2 ) n S—, —N(H)C(O)(CH 2 ) n S—, —C(O)N(H)(CH 2 ) n S—, —(CH 2 ) n NH—, —C(O)N(H)(CH 2 CH 2 O) n CH 2 CH 2 NH, —C(O)(CH 2 CH 2 O) n CH 2 CH 2 NH—, —C(O)N(H)(CH 2 ) n NHC(O)CH(CH 3 )(CH 2 ) n NH— or —N(H)(CH 2 ) n NH—, 
       
       
         
           
           
               
               
           
         
          where each n is independently an integer ranging from 1 to 12; 
         R 4  is a detectable label;
 R 21 , R 22 , R 23 , and R 24  are each independently hydrogen, halo, cyano, aliphatic, alkoxy, NO 2 , N(R c ) 2 , aryl, haloalkyl, —C(O)alkyl, —C(S)alkyl, —C(O)OH, —C(O)Oalkyl, —C(O)NHR c , —C(O)N(R c ) 2 , —R 3 R 4 , or two adjacent groups together form an aliphatic ring or aryl ring; 
 R a  is hydrogen or aliphatic; and 
 
         each R c  independently is hydrogen, aryl, aliphatic or heteroaliphatic. 
       
     
     
         18 . The compound of  claim 15 , wherein R 3 —N(H)C(O)(CH 2 ) n NH—, —C(O)N(H)(CH 2 ) n NH—(CH 2 ) n NH—, or —C(O)N(H)(CH 2 CH 2 O)CH 2 CH 2 NH—, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. 
     
     
         19 . The compound of  claim 15 , wherein the detectable label is a chromophore. 
     
     
         20 . The compound of  claim 15 , wherein LG is halo.