Cyanine compounds and their application as quenching compounds

Abstract

The present invention provides methods and non-fluorescent carbocyanine quencher compounds having the general formula: Wherein the A moiety is a substituted pyridinium, unsubstituted pyridinium, substituted quinolinium, unsubstituted quinolinium, substituted benzazolium, unsubstituted benzazolium, substituted indolinium, or substituted indolinium. The invention further provides luminescent donor molecule-quencher pairs and luminescent donor molecule-quencher-luminescent acceptor molecule conjugates wherein the quencher is a cyanine compound of the present invention. The energy transfer pairs are used to detect an analyte of interest in a sample.

1 . A compound having formula: 
       
         
           
           
               
               
           
         
         wherein: 
         R 2  is alkyl, substituted alkyl, alkoxy, substituted alkoxy, sulfoalkyl, substituted sulfoalkyl, aminoalkyl, substituted aminoalkyl, reactive group, carrier molecule, or solid support; 
         R 3  and R 4  are each independently alkyl, substituted alkyl, sulfoalkyl, reactive group, carrier molecule, or solid support; or 
         R 3  and R 4  taken together form a 5- or 6-membered saturated ring or a substituted 5- or 6-membered saturated ring; 
         each R 5  is independently hydrogen, amino, substituted amino, sulfo, alkyl, substituted alkyl, alkoxy, substituted alkoxy, fused benzene, substituted fused benzene, trifluoromethyl, sulfoalkyl, substituted sulfoalkyl, aminoalkyl, substituted aminoalkyl, halogen, reactive group, carrier molecule, or solid support; 
         R 22 , R 23 , R 24 , R 25 , and R 26  are each independently hydrogen, alkyl, substituted alkyl, alkoxy, substituted alkoxy, alkoxycarbonyl, substituted alkoxycarbonyl, amino, substituted amino, aminoalkyl, substituted aminoalkyl, hydroxyl, halogen, thioether, carbonyl, substituted carbonyl, sulfo, substituted sulfo, sulfoalkyl, reactive group, carrier molecule, or solid support; or, 
         a member independently selected from
 R 22  in combination with R 23 ; 
 R 23  in combination with R 24 ; 
 R 24  in combination with R 25 ; and 
 R 25  in combination with R 26 ; 
 together with the atoms to which they are joined, form a ring which is a 5-, 6- or 7-membered cycloalkyl, a substituted 5-, 6- or 7-membered cycloalkyl, a 5-, 6- or 7-membered heterocycloalkyl, a substituted 5-, 6- or 7-membered heterocycloalkyl, a 5-, 6- or 7-membered aryl, a substituted 5-, 6- or 7-membered aryl, a 5-, 6- or 7-membered heteroaryl, or a substituted 5-, 6- or 7-membered heteroaryl. 
 
       
     
     
         2 . The compound according to  claim 1 , wherein the reactive group, solid support and carrier molecule independently comprise a linker that is a single covalent bond, or a covalent linkage that is linear or branched, cyclic or heterocyclic, saturated or unsaturated, having 1-20 nonhydrogen atoms selected from the group consisting of C, N, P, O and S; and are composed of any combination of ether, thioether, amine, ester, carboxamide, sulfonamide, hydrazide bonds and aromatic or heteroaromatic bonds. 
     
     
         3 . The compound according to  claim 1 , wherein the reactive group is an acrylamide, an activated ester of a carboxylic acid, a carboxylic ester, an acyl azide, an acyl nitrile, an aldehyde, an alkyl halide, an anhydride, an aniline, an amine, an aryl halide, an azide, an aziridine, a boronate, a diazoalkane, a haloacetamide, a haloalkyl, a halotriazine, a hydrazine, an imido ester, an isocyanate, an isothiocyanate, a maleimide, a phosphoramidite, a reactive platinum complex, a silyl halide, a sulfonyl halide, a thiol or a photoactivatable group. 
     
     
         4 . The compound according to  claim 1 , wherein the reactive group is carboxylic acid, succinimidyl ester of a carboxylic acid, hydrazide, amine or a maleimide. 
     
     
         5 . The compound according to  claim 1 , wherein the carrier molecule is an amino acid, a peptide, a protein, a polysaccharide, a nucleoside, a nucleotide, an oligonucleotide, a nucleic acid polymer, a hapten, a biotin-binding protein, a psoralen, a drug, a hormone, a lipid, a lipid assembly, a synthetic polymer, a polymeric microparticle, a biological cell or a virus. 
     
     
         6 . The compound according to  claim 1 , wherein the carrier molecule is an antibody or fragment thereof, an avidin or streptavidin, a biotin, a blood component protein, a dextran, an enzyme, an enzyme inhibitor, a hormone, an IgG binding protein, a fluorescent protein, a growth factor, a lectin, a lipopolysaccharide, a microorganism, a metal binding protein, a metal chelating moiety, a non-biological microparticle, a peptide toxin, a phosphotidylserine-binding protein, a structural protein, a small-molecule drug, or a tyramide. 
     
     
         7 . The compound according to  claim 1 , wherein the solid support is a microfluidic chip, a silicon chip, a microscope slide, a microplate well, a silica gel, a polymeric membrane, a particle, a derivatized plastic film, a glass bead, cotton, a plastic bead, an alumina gel, a polysaccharide, polyvinylchloride, polypropylene, polyethylene, nylon, latex bead, magnetic bead, paramagnetic bead, or superparamagnetic bead. 
     
     
         8 . The compound according to  claim 1 , wherein the solid support is Sepharose, poly(acrylate), polystyrene, poly(acrylamide), polyol, agarose, agar, cellulose, dextran, starch, FICOLL, heparin, glycogen, amylopectin, mannan, inulin, nitrocellulose, diazocellulose or starch. 
     
     
         9 . The compound according to  claim 1 , having the structure: 
       
         
           
           
               
               
           
         
       
     
     
         10 . The compound according to  claim 1 , having the structure: 
       
         
           
           
               
               
           
         
       
     
     
         11 . The compound according to  claim 1 , having the structure: 
       
         
           
           
               
               
           
         
       
     
     
         12 . The compound according to  claim 1 , having the structure: 
       
         
           
           
               
               
           
         
       
     
     
         13 . The compound according to  claim 1 , wherein:
 R 2  is alkyl, sulfoalkyl, carboxyalkyl, reactive group, carrier molecule, or solid support;   R 3  and R 4  are each independently alkyl or carboxyalkyl;   R 5  is independently hydrogen, amino, sulfo, trifluoromethyl, or halogen; or C 1 -C 6  alkyl, C 1 -C 6  alkoxy, C 1 -C 6  alkylamino, C 2 -C 12  dialkylamino, each of which is optionally further substituted by carboxy, sulfo, amino, or hydroxy; or any two adjacent substituents form a fused benzo ring that is optionally substituted one or more times by amino, sulfo, trifluoromethyl, or halogen; or by C 1 -C 6  alkyl, C 1 -C 6  alkoxy, C 1 -C 6  alkylamino, C 2 -C 12  dialkylamino, each of which is optionally further substituted by carboxy, sulfo, amino, or hydroxy;   R 22 , R 23 , R 25  and R 26  are each hydrogen; and   R 24  is alkoxycarbonyl.   
     
     
         14 . The compound according to  claim 13 , wherein:
 R 2 , R 3  and R 4  are each methyl;   R 5  is independently hydrogen or sulfo; and   R 24  is methoxycarbonyl.