Quinazoline-containing kits for labeling aldehyde or ketone moieties

Abstract

Novel fluorescent derivitization reagents are described that are suitable for coupling to biomolecules that contain aldehyde or ketone functional groups. In one embodiment is provided reagents that have the following formula: wherein Q is carbonyl, thiocarbonyl, or sulfonyl, and R 5 is -L-Z; L is arylene, or a C 1-6 perfluoroalkylene, or a single covalent bond; Z is a carbonyl hydrazide, hydrazide, sulfonyl hydrazide, or a thiocarbonyl hydrazide; R 11 -R 14 are independently H, C 1-6 alkyl, C 1-6 alkoxy, C 1-6 perfluoroalkyl, C 1-6 alkylamino, di(C 2-12 -alkyl)amino, amino, carboxy, cyano, halogen, hydroxy, nitro, phenyl, or sulfo; and R 21 -R 24 are independently H, C 1-6 alkyl, C 1-6 alkoxy, C 1-6 perfluoroalkyl, C 1-6 alkylamino, di(C 2-12 -alkyl)amino, amino, carboxy, cyano, halogen, hydroxy, nitro, phenyl, sulfo, or -L-Z. The method of treating a sample with the derivativization reagents is described. The reagents are particularly useful for labeling glycoproteins or glycopeptides, nucleic acids, and lipopolysaccharides in electrophoresis gels.

1 . A method for labeling an aldehyde- and/or ketone containing target substance in a sample, the method comprising steps:
 a) combining a ketone or aldehyde reactive compound with a sample that contains the target substance, wherein the ketone or aldehyde reactive compound has the formula:   
       
         
           
           
               
               
           
         
         
           where the substituents R 1  and R 2 , when taken in combination, form a first aromatic ring system that comprises a 5- or 6-membered aromatic ring that optionally incorporates one or more heteroatoms N, O, or S, and optionally incorporates 1 or 2 additional fused aromatic rings that each optionally incorporate one or more heteroatoms; which first aromatic ring system is optionally substituted by hydrogen, halogen, C 1-6  alkyl, C 1-6  alkoxy, C 1-6  perfluoroalkyl, C 1-6  alkylamino, C 2-12  dialkylamino, sulfo, carboxy, hydroxy, amino, nitro, cyano, aryl, or any combination thereof, or by a covalently bound reactive group -L-Z; and 
           the substituents R 3  and R 4 , when taken in combination, form a second aromatic ring system that comprises a 5- or 6-membered aromatic ring that optionally incorporates 1-3 additional heteroatoms N, O, or S, or oxo, thiooxo, sulfone, or amino groups, and optionally incorporates 1 or 2 additional fused aromatic rings that themselves optionally incorporate one or more heteroatoms, which second aromatic ring system is independently and optionally substituted by hydrogen, halogen, C 1-6 alkyl, C 1-6  alkoxy, C 1-6  perfluoroalkyl, C 1-6  alkylamino, C 2-12  dialkylamino, sulfo, carboxy, hydroxy, amino, nitro, cyano, aryl, or any combination thereof, or by a covalently bound reactive group -L-Z, or 
           R 2  and R 3 , when taken in combination, form an additional 5- or 6-membered bridging moiety that is doubly fused to the first and second aromatic ring systems, which bridging moiety comprises a combination of carbon atoms in saturated or unsaturated carbon-carbon bonds, and optionally includes one of O, N, and S atoms, which carbon and nitrogen atoms of the bridging moiety are optionally substituted by C 1-6  alkyl or by -L-Z; 
           wherein
 each L is independently a single covalent bond, or a covalent linkage that has 1-20 nonhydrogen atoms selected from the group consisting of C, N, O, P, and S; with a combination of stable chemical bonds, optionally including single, aromatic or heteroaromatic bonds that are carbon-carbon bonds, carbon-nitrogen bonds, nitrogen-nitrogen bonds, carbon-oxygen bonds, carbon-sulfur bonds, phosphorus-oxygen bonds, phosphorus-nitrogen bonds, or combinations thereof, 
 each Z is independently a functional group capable of reacting with an aldehyde or ketone to form a covalent bond selected from the group consisting of aliphatic amine, aromatic amine, ethylenediamine, NR 6 —NH 2  (hydrazide), —NR 6 (C═O)NR 7 NH 2  (semicarbazide), —NR 6 (C═S)NR 7 NH 2  (thiosemicarbazide), —(C═O)NR 6 NH 2  (carbonylhydrazide), —(C═S)NR 6 NH 2  (thiocarbonylhydrazide), —(SO 2 )NR 6 NH 2 (sulfonylhydrazide), —NR 6 NR 7 (C═O)NR 8 NH 2  (carbazide), —NR 6 NR 7 (C═S)NR 8 NH 2  (thiocarbazide), and —O—NH 2  (hydroxylamine), where each R 6 , R 7 , and R 8  is independently H, or C 1-6  alkyl; 
 
           X is OH or —NH-Q-R 5 , where
 wherein Q is a stable divalent radical that is an electron-withdrawing linking group; and 
 R 5  is C 1-6 alkyl, C 1-6  alkoxy, C 1-6 alkylamino, or -L-Z; 
 
           with the proviso that the compound is substituted by at least one reactive group, -L-Z; and 
         
         b) incubating the sample and the ketone or aldehyde reactive compound for a sufficient amount of time to form a covalent conjugate with the target substance. 
       
     
     
         2 . The method according to  claim 1 , wherein the compound has the formula: 
       
         
           
           
               
               
           
         
         wherein Q is carbonyl, thiocarbonyl, or sulfonyl, and R 5  is -L-Z;
 L is arylene, or a C 1-6 perfluoroalkylene; and 
 Z is a (C═0)NR 6 NH 2  (carbonyl hydrazide), —NR 6 —NH 2  (hydrazide), —(SO 2 )NR 6 NH 2  (sulfonyl hydrazide) or —(C═S)NR 6 NH 2  (thiocarbonyl hydrazide);
 wherein R 6  is hydrogen or alkyl having 1-6 carbons 
 
 R 11 -R 14  are independently H, C 1-6 alkyl, C 1-6  alkoxy, C 1-6  perfluoroalkyl, C 1-6  alkylamino, di(C 2-12 -alkylamino,amino, carboxy, cyano, halogen, hydroxy, nitro, phenyl, or sulfo; 
 R 21 -R 24  are independently H, C 1-6  alkoxy, C 1-6  perfluoroalkyl, C 1-6  alkylamino, di(C 2-12 -alkyl)amino, amino, carboxy, cyano, halogen, hydroxy, nitro, phenyl, sulfo, or -L-Z. 
 
       
     
     
         3 . The method according to  claim 1 , wherein the target substance is a peptide, a protein, a nucleic acid, or a lipopolysaccharide. 
     
     
         4 . The method according to  claim 1 , wherein the target substance is a glycopeptide, a glycoprotein, or a lipopolysaccharide. 
     
     
         5 . The method according to  claim 4 , further comprising adding an oxidizing agent to the sample. 
     
     
         6 . The method according to  claim 5 , wherein the oxidizing agent is periodate. 
     
     
         7 . The method according to  claim 1 , wherein the target substance is a deoxyribonucleic acid. 
     
     
         8 . The method according to  claim 7 , further comprising adding a depurinating agent to the sample. 
     
     
         9 . The method according to  claim 8 , wherein the depurinating agent is HCl. 
     
     
         10 . The method according to  claim 1 , wherein the target substance is in a solid or semi-solid matrix. 
     
     
         11 . The method according to  claim 10 , wherein the solid or semi-solid matrix is a gel or blot. 
     
     
         12 . The method according to  claim 1 , further comprising detecting the target substance. 
     
     
         13 . The method according to  claim 1 , wherein the compound is: 
       
         
           
           
               
               
           
         
       
     
     
         14 . A kit for labeling an aldehyde- and/or ketone containing target substance in a sample, the kit comprising:
 a) a ketone or aldehyde reactive compound having the formula:   
       
         
           
           
               
               
           
         
         
           where the substituents R 1  and R 2 , when taken in combination, form a first aromatic ring system that comprises a 5- or 6-membered aromatic ring that optionally incorporates one or more heteroatoms N, O, or S, and optionally incorporates 1 or 2 additional fused aromatic rings that each optionally incorporate one or more heteroatoms; which first aromatic ring system is optionally substituted by hydrogen, halogen, C 1-6  alkyl, C 1-6  alkoxy, C 1-6  perfluoroalkyl, C 1-6  alkylamino, C 2-12  dialkylamino, sulfo, carboxy, hydroxy, amino, nitro, cyano, aryl, or any combination thereof, or by a covalently bound reactive group -L-Z; and 
           the substituents R 3  and R 4 , when taken in combination, form a second aromatic ring system that comprises a 5- or 6-membered aromatic ring that optionally incorporates 1-3 additional heteroatoms N, O, or S, or oxo, thiooxo, sulfone, or amino groups, and optionally incorporates 1 or 2 additional fused aromatic rings that themselves optionally incorporate one or more heteroatoms, which second aromatic ring system is independently and optionally substituted by hydrogen, halogen, C 1-6 alkyl, C 1-6  alkoxy, C 1-6  perfluoroalkyl, C 1-6  alkylamino, C 2-12  dialkylamino, sulfo, carboxy, hydroxy, amino, nitro, cyano, aryl, or any combination thereof, or by a covalently bound reactive group -L-Z; or 
           R 2  and R 3 , when taken in combination, form an additional 5- or 6-membered bridging moiety that is doubly fused to the first and second aromatic ring systems, which bridging moiety comprises a combination of carbon atoms in saturated or unsaturated carbon-carbon bonds, and optionally includes one of O, N, and S atoms, which carbon and nitrogen atoms of the bridging moiety are optionally substituted by C 1-6 alkyl or by -L-Z; 
           wherein
 each L is independently a single covalent bond, or a covalent linkage that has 1-20 nonhydrogen atoms selected from the group consisting of C, N, O, P, and S; with a combination of stable chemical bonds, optionally including single, aromatic or heteroaromatic bonds that are carbon-carbon bonds, carbon-nitrogen bonds, nitrogen-nitrogen bonds, carbon-oxygen bonds, carbon-sulfur bonds, phosphorus-oxygen bonds, phosphorus-nitrogen bonds, or combinations thereof, 
 each Z is independently a functional group capable of reacting with an aldehyde or ketone to form a covalent bond selected from the group consisting of aliphatic amine, aromatic amine, ethylenediamine, NR 6 —NH 2  (hydrazide), —NR 6 (C═O)NR 7 NH 2  (semicarbazide), —NR 6 (C═S)NR 7 NH 2  (thiosemicarbazide), —(C═O)NR 6 NH 2  (carbonylhydrazide), —(C═S)NR 6 NH 2  (thiocarbonylhydrazide), —(SO 2 )NR 6 NH 2 (sulfonylhydrazide), —NR 6 NR 7 (C═O)NR 8 NH 2  (carbazide), —NR 6 NR 7 (C═S)NR 8 NH 2  (thiocarbazide), and —O—NH 2  (hydroxylamine), where each R 6 , R 7 , and R 8  is independently H, or C 1-6  alkyl; 
 
           X is OH or —NH-Q-R 5 , where
 wherein Q is a stable divalent radical that is an electron-withdrawing linking group; and 
 R 5  is C 1-6  alkyl, C 1-6  alkoxy, C 1-6  alkylamino, or -L-Z; 
 
           with the proviso that the compound is substituted by at least one reactive group, -L-Z; 
         
         b) a buffer; and, 
         c) instructions for labeling the target substance. 
       
     
     
         15 . The kit according to  claim 14 , wherein the compound has the formula: 
       
         
           
           
               
               
           
         
         wherein Q is carbonyl, thiocarbonyl, or sulfonyl, and R 5  is -L-Z;
 L is arylene, or a C 1-6 perfluoroalkylene; and 
 Z is a (C═O)NR 6 NH 2  (carbonyl hydrazide), —NR 6 —NH 2  (hydrazide), —(SO 2 )NR 6 NH 2  (sulfonyl hydrazide) or —(C═S)NR 6 NH 2  (thiocarbonyl hydrazide);
 wherein R 6  is hydrogen or alkyl having 1-6 carbons 
 
 R 11 -R 14  are independently H, C 1-6  alkyl, C 1-6  alkoxy, C 1-6  perfluoroalkyl, C 1-6  alkylamino, di(C 2-12 -alkyl)amino, amino, carboxy, cyano, halogen, hydroxy, nitro, phenyl, or sulfo; 
 R 21 -R 24  are independently H, C 1-6  alkyl, C 1-6  alkoxy, C 1-6  perfluoroalkyl, C 1-6  alkylamino, di(C 2-12 -alkyl)amino, amino, carboxy, cyano, halogen, hydroxy, nitro, phenyl, sulfo, or -L-Z. 
 
       
     
     
         16 . The kit according to  claim 14 , further comprising an oxidizing agent or a depurinating agent. 
     
     
         17 . The kit according to  claim 16 , wherein the depurinating agent that is HCl. 
     
     
         18 . The kit according to  claim 16 , wherein the oxidizing agent that is periodate. 
     
     
         19 . The kit according to  claim 14 , wherein the kit further comprises and additional detection reagent. 
     
     
         20 . The kit according to  claim 14 , wherein the compound is: 
       
         
           
           
               
               
           
         
       
     
     
         21 . A staining solution for labeling an aldehyde- and/or ketone containing target substance in a sample, the staining solution comprising:
 a) a ketone or aldehyde reactive compound having the formula:   
       
         
           
           
               
               
           
         
         
           where the substituents and R 2 , when taken in combination, form a first aromatic ring system that comprises a 5- or 6-membered aromatic ring that optionally incorporates one or more heteroatoms N, O, or S, and optionally incorporates 1 or 2 additional fused aromatic rings that each optionally incorporate one or more heteroatoms; which first aromatic ring system is optionally substituted by hydrogen, halogen, C 1-6  alkyl, C 1-6  alkoxy, C 1-6  perfluoroalkyl, C 1-6  alkylamino, C 2-12  dialkylamino, sulfo, carboxy, hydroxy, amino, nitro, cyano, aryl, or any combination thereof, or by a covalently bound reactive group -L-Z; and 
           the substituents R 3  and R 4 , when taken in combination, form a second aromatic ring system that comprises a 5- or 6-membered aromatic ring that optionally incorporates 1-3 additional heteroatoms N, O, or S, or oxo, thiooxo, sulfone, or amino groups, and optionally incorporates 1 or 2 additional fused aromatic rings that themselves optionally incorporate one or more heteroatoms, which second aromatic ring system is independently and optionally substituted by hydrogen, halogen, C 1-6  alkyl, C 1-6  alkoxy, C 1-6  perfluoroalkyl, C 1-6  alkylamino, C 2-12  dialkylamino, sulfo, carboxy, hydroxy, amino, nitro, cyano, aryl, or any combination thereof, or by a covalently bound reactive group -L-Z, or 
           R 2  and R 3 , when taken in combination, form an additional 5- or 6-membered bridging moiety that is doubly fused to the first and second aromatic ring systems, which bridging moiety comprises a combination of carbon atoms in saturated or unsaturated carbon-carbon bonds, and optionally includes one of O, N, and S atoms, which carbon and nitrogen atoms of the bridging moiety are optionally substituted by C 1-6  alkyl or by -L-Z; 
           wherein
 each L is independently a single covalent bond, or a covalent linkage that has 1-20 nonhydrogen atoms selected from the group consisting of C, N, O, P, and S; with a combination of stable chemical bonds, optionally including single, aromatic or heteroaromatic bonds that are carbon-carbon bonds, carbon-nitrogen bonds, nitrogen-nitrogen bonds, carbon-oxygen bonds, carbon-sulfur bonds, phosphorus-oxygen bonds, phosphorus-nitrogen bonds, or combinations thereof, 
 each Z is independently a functional group capable of reacting with an aldehyde or ketone to form a covalent bond selected from the group consisting of aliphatic amine, aromatic amine, ethylenediamine, NR 6 —NH 2  (hydrazide), —NR 6 (C═O)NR 7 NH 2  (semicarbazide), —NR 6 (C═S)NR 7 NH 2  (thiosemicarbazide), —(C═O)NR 6 NH 2  (carbonylhydrazide), —(C═S)NR 6 NH 2  (thiocarbonylhydrazide), —(SO 2 )NR 6 NH 2  (sulfonylhydrazide), —NR 6 NR 7 (C═O)NR 8 NH 2  (carbazide), —NR 6 NR 7 (C═S)NR 8 NH 2  (thiocarbazide), and —O—NH 2  (hydroxylamine), where each R 6 , R 7 , and R 8  is independently H, or C 1-6  alkyl; 
 
           X is OH or —NH-Q-R 5 , where
 wherein Q is a stable divalent radical that is an electron-withdrawing linking group; and 
 R 5  is C 1-6  alkyl, C 1-6  alkoxy, C 1-6  alkylamino, or -L-Z; 
 
           with the proviso that the compound is substituted by at least one reactive group, -L-Z; and 
         
         a buffer.