Aza-benzazolium Containing Cyanine Dyes

Abstract

Unsymmetrical cyanine dyes that incorporate an aza-benzazolium ring moiety are described, including cyanine dyes substituted by a cationic side chain, monomeric and dimeric cyanine dyes, chemically reactive cyanine dyes, and conjugates of cyanine dyes. The subject dyes are virtually non-fluorescent when diluted in aqueous solution, but exhibit bright fluorescence when associated with nucleic acid polymers such as DNA or RNA, or when associated with detergent-complexed proteins. A variety of applications are described for detection and quantitation of nucleic acids and detergent-complexed proteins in a variety of samples, including solutions, electrophoretic gels, cells, and microorganisms.

1 . A method for determining the presence of an analyte of interest in a sample, wherein said method comprises:
 a) combining said sample with a staining solution, wherein said staining solution comprises one or more dyes having formula   
       
         
           
           
               
               
           
         
         wherein A represents the atoms necessary to form one to two fused aromatic rings having 6 atoms in each ring, at least one of which is a nitrogen atom, said ring or rings being optionally further substituted one or more times by C 1 -C 6  alkyl, C 1 -C 6  alkoxy trifluoromethyl, halogen, BRIDGE, -L-Rx or -L-Sc;
 wherein Rx is a reactive group; Sc is a conjugated substance; and L and BRIDGE are independently a single covalent bond, or a covalent linkage; 
 
         X is O, S, Se, NR 15 , or CR 16 R 17 , where R 15  is H or an alkyl group having 1-6 carbons; and R 16  and R 17 , which may be the same or different, are independently alkyl groups having 1-6 carbons, or R 16  and R 17  taken in combination complete a five or six membered saturated ring; 
         R 2  is selected from the group consisting of -L-Rx, -L-Sc, TAIL, BRIDGE and an alkyl group having 1-6 carbons that is optionally substituted by sulk), carboxy, amino, substituted amino or substituted ammonium, wherein a is 0 or 1; and TAIL is a heteroatom-containing moiety; 
         Y is —CR 3 ═CR 4 — wherein p and m=0 or 1, such that p m=1; 
         R 3 , R 4 , R 6 , and R 7  are independently selected from the group consisting of hydrogen, a C 1 -C 6  alkyl, a halogen, a CYCLIC SUBSTITUENT, —OR 8 , —SR 8 , —(NR 8 R 9 ), TAIL; BRIDGE, -L-Rx and -L-Sc; where R 8  and R 9  are independently a C 1 -C 6  alkyl group, 1-2 alicyclic or aromatic rings; or R 8  and R 9  taken in combination are —(CH 2 ) 2 —V—(CH 2 ) 2 — where V is a single bond, —O—, —CH 2 —, or —NR 10 —, where R 10  is H or an alkyl having 1-6 carbons;
 wherein CYCLIC SUBSTITUENT is a substituted or unsubstituted aryl, heteroaryl or C 3 -C 10  cycloalkyl; 
 
         or R 6  and R 7  form a fused aromatic ring —R 11 ═R 12 —R 13 ═R 14 — wherein R 11 , R 12 , R 13 , and R 14  are independently selected from the group consisting of hydrogen, C 1 -C 8  alkyl group, —OR 8 , —(NR 8 R 9 ), a CYCLIC SUBSTITUENT, TAIL, BRIDGE, -L-Rx and -L-Sc; 
         R 5  is independently selected from the group consisting of a C 1 -C 6  alkyl group, a CYCLIC SUBSTITUENT, TAIL, BRIDGE, -L-Rx and -L-Sc; or R 5  is absent; 
         R 30 , R 31 , and R 32  are independently selected from the group consisting of hydrogen, C 1 -C 6  alkyl, C 3 -C 10  cycloalkyl, aryl, and heteroaryl, wherein n=0, 1 or 2; 
         wherein, BRIDGE, when present, is bound to a compound having formula I or another unsymmetrical cyanine dye 
         b) incubating said sample and said staining solution for a sufficient amount of time to form a dye-analyte complex; 
         c) illuminating said complex with an appropriate wavelength whereby the presence of said analyte is determined. 
       
     
     
         2 . The method according to  claim 1 , wherein said analyte of Interest is a poly(amino acid) or a nucleic acid. 
     
     
         3 . The method according to  claim 2 , wherein said poly(amino acid) is in solution or immobilized on a solid or semi solid matrix. 
     
     
         4 . The method according to  claim 3 , wherein said matrix is a polymeric gel, an array, a polymeric microparticle or a membrane. 
     
     
         5 . The method according to  claim 4 , wherein said gel is a polyacrylamide gel. 
     
     
         6 . The method according to  claim 3 , wherein said method further comprises heating said sample prior to combining with said staining solution, or healing combined sample and staining solution mixture. 
     
     
         7 . The method according to  claim 3 , wherein said method further comprises removing, destroying, or dispersing below the critical micelle concentration any biological membranes that are present in said sample. 
     
     
         8 . The method according to  claim 3 , wherein said method further comprises adding an anionic detergent to the sample, staining solution or combined sample and staining solution mixture. 
     
     
         9 . The method according to  claim 2 , wherein said nucleic acid is in solution, immobilized on a solid or semi-solid matrix or present in a biological structure. 
     
     
         10 . The method according to  claim 9 , wherein said matrix is a polymeric gel, an array, a glass slide, a polymeric microparticle or a membrane. 
     
     
         11 . The method according to  claim 10 , wherein said gel is an agarose gel. 
     
     
         12 . The method according to  claim 9 , wherein said biological structure is a biological cell, a virus particle or a tissue section. 
     
     
         13 . A method for the detection of immobilized poly(amino acids), wherein method comprises:
 a) immobilizing said poly(amino acids) on a solid or semi-solid matrix;   b) combining said sample with a staining solution prior, during or after immobilization of said poly(amino acid), wherein said staining solution comprises a compound having formula   
       
         
           
           
               
               
           
         
         wherein A represents the atoms necessary to form one to two fused aromatic rings having 6 atoms in each ring, at least one of which is a nitrogen atom, said ring or rings being optionally further substituted one or more times by C 1 -C 6  alkyl, C 1 -C 6  alkoxy trifluoromethyl, halogen, BRIDGE, -L-Rx or -L-Sc;
 wherein Rx is a reactive group; Sc is a conjugated substance; and L and BRIDGE are independently a single covalent bond, or a covalent linkage; 
 
         X is O, S, Se, NR 15 , or CR 16 R 17 , where R 15  is H or an alkyl group having 1-6 carbons; and R 16  and R 17 , which may be the same or different, are independently alkyl groups having 1-6 carbons, or R 16  and R 17  taken in combination complete a five or six membered saturated ring; 
         R 2  is selected from the group consisting of -L-Rx, -L-Sc, TAIL, BRIDGE and an alkyl group having 1-6 carbons that is optionally substituted by sulfo, carboxy, amino, substituted amino or substituted ammonium, wherein α is 0 or 1; and TAIL is a heteroatom-containing moiety; 
         Y is —CR 3 ═CR 4 — wherein p and m=0 or 1, such that p+m=1; 
         R 3 , R 4 , R 6 , and R 7  are independently selected from the group consisting of hydrogen, a C 1 -C 6  alkyl, a halogen, a CYCLIC SUBSTITUENT, —OR 8 , —SR 8 , —(NR 8 R 9 ), TAIL; BRIDGE, -L-Rx and -L-Sc; where R 8  and R 9  are independently a C 1 -C 6  alkyl group, 1-2 alicyclic or aromatic rings; or R 8  and R 9  taken in combination are —(CH 2 ) 2 —V—(CH 2 ) 2 — where V is a single bond, —O—, —CH 2 —, or —NR 10 —, where R 10  is H or an alkyl having 1-6 carbons;
 wherein CYCLIC SUBSTITUENT is a substituted or unsubstituted aryl, heteroaryl or C 3 -C 10  cycloalkyl; 
 
         or R 6  and R 7  form a fused aromatic ring —R 11 ═R 12 —R 13 ═R 14 — wherein R 11 , R 12 , R 13 , and R 14  are independently selected from the group consisting of hydrogen, C 1 -C 6  alkyl group, —OR 8 , —SR 8 , —(NR 8 R 9 ), a CYCLIC SUBSTITUENT, TAIL, BRIDGE, -L-Rx and -L-Sc; 
         R 5  is independently selected from the group consisting of a C 1 -C 6  alkyl group, a CYCLIC SUBSTITUENT, TAIL, BRIDGE, -L-Rx and -L-Sc; or R 5  is absent; 
         R 30 , R 31 , and R 32  are independently selected from the group consisting of hydrogen, C 1 -C 6  alkyl, C 3 -C 10  cycloalkyl, aryl, and heteroaryl, wherein n=0, 1 or 2; 
         wherein, BRIDGE, when present, is bound to a compound having formula I or another unsymmetrical cyanine dye 
         c) incubating said sample and said staining solution for a sufficient amount of time to form a dye-poly(amino acid) complex; 
         d) illuminating said complex with an appropriate wavelength whereby the presence of said poly(amino acid) is determined. 
       
     
     
         14 . The method according to  claim 13 , wherein said compound is overall neutral in charge. 
     
     
         15 . The method according to  claim 14 , wherein said R 2  is a C 2 -C 6  alkyl group substituted by a sulfo group or R 5  is absent. 
     
     
         16 . The method according to  claim 13 , wherein said X is O or S, n is 0 or 1, R 6  and R 7  form a fused aromatic ring —R 11 ═R 12 —R 13 ═R 14 — wherein R 11 , R 12 , R 13 , and R 14  are independently selected from the group consisting of hydrogen, C 1 -C 6  alkyl group, —OR 8 ; R 4  and R 5  are independently a C 1 -C 6  alkyl group or a CYCLIC SUBSTITUENT. 
     
     
         17 . The method according to  claim 13 , wherein said method further comprises heating said sample prior to combining with said staining solution, or heating combined sample and staining solution mixture. 
     
     
         18 . The method according to  claim 13 , wherein said method further comprises removing, destroying, or dispersing below the critical micelle concentration any biological membranes that are present in said sample. 
     
     
         19 . The method according to  claim 13 , wherein said method further comprises adding an anionic detergent to the sample, staining solution or combined sample and staining solution mixture. 
     
     
         20 . The method according to  claim 13 , wherein said method further comprising electrophoretically separating said sample before, after, or while it is combined with said staining solution. 
     
     
         21 . The method according to  claim 20 , wherein said matrix is a polymeric gel. 
     
     
         22 . The method according to  claim 21 , wherein said gel is a polyacrylamide gel. 
     
     
         23 . The method according to  claim 22 , wherein said method further comprises adding an additional detection reagent to said sample, said staining solution, or combined sample and staining solution mixture. 
     
     
         24 . The method according to  claim 20 , wherein said method further comprises transferring said sample to a solid or semi-solid matrix before or after combining with said staining solution. 
     
     
         25 . A method for the detection of nucleic acids in a sample, wherein said method comprises:
 a) combining said sample with a staining solution, wherein said staining solution comprises one or more dyes having formula   
       
         
           
           
               
               
           
         
         wherein A represents the atoms necessary to form one to two fused aromatic rings having 6 atoms in each ring, at least one of which is a nitrogen atom, said ring or rings being optionally further substituted one or more times by C 1 -C 6  alkyl, C 1 -C 6  alkoxy trifluoromethyl, halogen, BRIDGE, -L-Rx or -L-Sc;
 wherein Rx is a reactive group; Sc is a conjugated substance; and L and BRIDGE are independently a single covalent bond, or a covalent linkage; 
 
         X is O, S, Se, NR 15 , or CR 16 R 17 , where R 15  is Nor an alkyl group having 1-6 carbons; and R 16  and R 17 , which may be the same or different, are independently alkyl groups having 1-6 carbons, or R 16  and R 17  taken in combination complete a five or six membered saturated ring; 
         R 2  is selected from the group consisting of -L-Rx, -L-Sc, TAIL, BRIDGE and an alkyl group having 1-6 carbons that is optionally substituted by sulfo, carboxy, amino, substituted amino or substituted ammonium, wherein a is 0 or 1; and TAIL is a heteroatom-containing moiety; 
         Y is —CR 3 ═CR 4 — wherein p and m=0 or 1, such that p m=1; 
         R 3 , R 4 , R 6 , and R 7  are independently selected from the group consisting of hydrogen, a C 1 -C 6  alkyl, a halogen, a CYCLIC SUBSTITUENT, —OR 8 , —SR 8 , —(NR 8 R 9 ), TAIL; BRIDGE, -L-Rx and -L-Sc; where R 8  and R 9  are independently a C 1 -C 6  alkyl group, 1-2 alicyclic or aromatic rings; or R 8  and R 9  taken in combination are —(CH 2 ) 2 —V—(CH 2 ) 2 — where V is a single bond, —O—, —CH 2 —, or —NR 10 —, where R 10  is H or an alkyl having 1-6 carbons;
 wherein CYCLIC SUBSTITUENT is a substituted or unsubstituted aryl, heteroaryl or C 3 -C 10  cycloalkyl; 
 
         or R 6  and form a fused aromatic ring —R 11 ═R 12 —R 13 ═R 14 — wherein R 11 , R 12 , R 13 , and R 14  are independently selected from the group consisting of hydrogen, C 1 -C 8  alkyl group, —OR 8 , —SR 8 , —(NR 8 R 9 ), a CYCLIC SUBSTITUENT, TAIL, BRIDGE, -L-Rx and -L-Sc; 
         R 5  is independently selected from the group consisting of a C 1 -C 6  alkyl group, a CYCLIC SUBSTITUENT, TAIL, BRIDGE, -L-Rx and -L-Sc; or R 5  is absent; 
         R 30 , R 31 , and R 32  are independently selected from the group consisting of hydrogen, C 1 -C 6  alkyl, C 3 -C 10  cycloalkyl, aryl, and heteroaryl, wherein n=0, 1 or 2; 
         wherein, BRIDGE, when present, is bound to a compound having formula I or another unsymmetrical cyanine dye; 
         b) incubating said sample and said staining solution for a sufficient amount of time to form a dye-nucleic acid complex; 
         illuminating said complex with an appropriate wavelength whereby the presence of said nucleic acid is determined. 
       
     
     
         26 . The method according to  claim 25 , wherein said X is O, n is O, R 2  is a methyl group and R 6  and R 7  form a fused aromatic ring —R 11 ═R 12 —R 13 ═R 14 — wherein R 11 , R 12 , R 13 , and R 14  are independently selected from the group consisting of hydrogen, C 1 -C 8  alkyl group, —OR 8 . 
     
     
         27 . The method according to  claim 26 , wherein said R 5  is a CYCLIC SUBSTITUENT that is an aryl group and R 4  is an alkyl group. 
     
     
         28 . The method according to  claim 26 , wherein said R 5  is an alkyl group and R 4  is a CYCLIC SUBSTITUENT that is an aryl group. 
     
     
         29 . The method according to  claim 26 , wherein said compound is substituted by at least one TAIL substituent. 
     
     
         30 . The method according to  claim 29 , wherein said TAIL is according to formula LINK-SPACER-CAP;
 wherein LINK is single covalent bond, an either linkage (—O—), a thioether linkage (—S—) or an amine linkage (—NR 20 —); where R 20  is hydrogen, C 1 -C 8  alkyl or SPACER-CAP;   SPACER is a covalent linkage; and,   CAP is —O—R 21 , —S—R 21 , —NR 21 R 22  or —NR 21 R 22 R 23 ; where R 21 , R 22 , and R 23  are independently selected from the group consisting of hydrogen, C 1 -C 8  alkyl and a C 1 -C 8  cycloalkyl wherein said alkyl or cycloalkyl are optionally substituted by one or more substituents selected from the group consisting of halogen, hydroxyl, C 1 -C 8  alkoxy, amino, carboxy, sulfo and phenyl where said phenyl is optionally substituted by one or more substituents selected from the group consisting halogen, hydroxyl, C 1 -C 8  alkoxy, amino, C 1 -C 8  aminoalkyl, C 1 -C 8  sulfoalkyl and C 1 -C 8  carboxyalkyl; or one or more R 21 , R 22 , and R 23 , taken in combination with R 20  and SPACER, or with SPACER alone, forms a 5- or 6-membered ring.   
     
     
         31 . The method according to  claim 30 , wherein said R 4  or R 5  is independently a TAIL or a CYCLIC SUBSTITUENT substituted by TAIL. 
     
     
         32 . The method according to  claim 31 , wherein said R 4  is a TAIL and R 5  is an alkyl group. 
     
     
         33 . The method according to  claim 32 , wherein said CAP is —NR 21 R 22  where R 21  and R 22  are independently C 1 -C 6 alkyl groups. 
     
     
         34 . The method according to  claim 32 , wherein said CAP is —NR 21 R 22 R 23  where R 21 , R 22  and R 23  are independently C 1 -C 6  alkyl groups. 
     
     
         35 . The method according to  claim 31 , wherein said R 5  is a CYCLIC SUBSTITUENT that is an aryl or heteroaryl and said R 4  is a TAIL where CAP is —O—R 21  or —S—R 21 . 
     
     
         36 . The method according to  claim 31 , wherein said R 5  is a TAIL. 
     
     
         37 . The method according to  claim 36 , wherein said CAP is —NR 21 R 22 R 23  where R 21 , R 22  and R 23  are independently C 1 -C 6  alkyl groups. 
     
     
         38 . The method according to  claim 26 , wherein said nucleic acid is in solution, immobilized on a solid or semi-solid matrix, or present in a biological structure. 
     
     
         39 . The method according to  claim 38 , wherein said solid or semi-solid matrix is a polymeric gel, an array, a glass slide, a polymeric microparticle or a membrane. 
     
     
         40 . The method according to  claim 39 , wherein said gel is an electrophoretic gel that is an agarose gel. 
     
     
         41 . The method according to  claim 38 , wherein said biological structure is a biological cell or portion thereof, virus particle or tissue section. 
     
     
         42 . The method according to  claim 41 , wherein said biological structure is a cell or portion thereof. 
     
     
         43 . The method according to  claim 38 , wherein said nucleic acid is in solution that is free from cells or portions thereof.