Aza-benzazolium containing cyanine dyes

A compound having a structure according to Formula I

• wherein A represents the atoms necessary to form one fused 6-membered aromatic ring, at least one member of which is a nitrogen atom, said ring being optionally further substituted one or more times by C1-C6 alkyl, C1-C6 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 or S;
• R2 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 —CR3═CR4— wherein p and m=0 or 1, such that p+m=1;
• R3, R4, R6, and R7 are independently selected from the group consisting of hydrogen, a C1-C6 alkyl, a halogen, a CYCLIC SUBSTITUENT, -0R8, —SR8, —(NR8R9), TAIL;
• BRIDGE, -L-Rx and -L-Sc;
• where R8 and R9 are independently a C1-C6 alkyl group, 1-2 alicyclic or aromatic rings;
• or R8 and R9 taken in combination are —(CH2)2—V—(CH2)2— where V is a single bond, -0-, —CH2—, or —NR10—, where R10 is H or an alkyl having 1-6 carbons;
• wherein CYCLIC SUBSTITUENT is a substituted or unsubstituted aryl, heteroaryl or C3-C10 cycloalkyl;
• or R6 and R7 form a fused aromatic ring —R11═R12—R13═R14— wherein R11, R12, R13, and R14 are independently selected from the group consisting of hydrogen, C1-C6 alkyl group, -0R8, —SR8, —(NR8R9), a CYCLIC SUBSTITUENT, TAIL, BRIDGE, -L-Rx and -L-Sc;
• R5 is independently selected from the group consisting of a C1-C6 alkyl group, a CYCLIC SUBSTITUENT, TAIL, BRIDGE, -L-Rx and -L-Sc;
• or R5 is absent;
• R30, R31, and R32 are independently selected from the group consisting of hydrogen, C1-C6 alkyl, C3-C10 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.

A method for the detection of nucleic acids in a sample, wherein said method comprises:

• a) providing a sample, wherein the sample comprises a biological structure;
• b) combining said sample with a staining solution, wherein said staining solution comprises one or more dyes having a structure according to Formula I
• wherein A represents the atoms necessary to form one fused 6-membered aromatic ring, at least one member of which is a nitrogen atom, said ring or rings being optionally further substituted one or more times by C1-C6 alkyl, C1-C6 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 or S;
• R2 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 —CR3═CR4— wherein p and m=0 or 1, such that p+m=1;
• R3, R4, R6, and R7 are independently selected from the group consisting of hydrogen, a C1-C6 alkyl, a halogen, a CYCLIC SUBSTITUENT, -0R8, —SR8, —(NR8R9), TAIL;
• BRIDGE, -L-Rx and -L-Sc;
• where R8 and R9 are independently a C1-C6 alkyl group, 1-2 alicyclic or aromatic rings;
• or R8 and R9 taken in combination are —(CH2)2—V—(CH2)2— where V is a single bond, -0-, —CH2—, or —NR10—, where R10 is H or an alkyl having 1-6 carbons;
• wherein CYCLIC SUBSTITUENT is a substituted or unsubstituted aryl, heteroaryl or C3-C10 cycloalkyl;
• or R6 and R7 form a fused aromatic ring —R11═R12—R13═R14— wherein R11, R12, R13, and R14 are independently selected from the group consisting of hydrogen, C1-C6 alkyl group, -0R8, —SR8, —(NR8R9), a CYCLIC SUBSTITUENT, TAIL, BRIDGE, -L-Rx and -L-Sc;
• R5 is independently selected from the group consisting of a C1-C6 alkyl group, a CYCLIC SUBSTITUENT, TAIL, BRIDGE, -L-Rx and -L-Sc;
• or R5 is absent;
• R30, R31, and R32 are independently selected from the group consisting of hydrogen, C1-C6 alkyl, C3-C10 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 for the dye to associate with a nucleic acid molecule to form a dye-nucleic acid complex;
• d) illuminating said sample with an appropriate wavelength of light whereby the presence of said nucleic acid is determined.