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US 2015/0031579 A1Published application
Aza-Benzazolium Containing Cyanine Dyes
AssigneeLife Technologies Corporation
InventorsRichard HAUGLAND, Stephen Yue
Life of this patent
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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.
Claims43
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 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, S, Se, NR15, or CR16R17, where R1 is H or an alkyl group having 1-6 carbons;
- and R16 and R17, which may be the same or different, are independently alkyl groups having 1-6 carbons, or R16 and R17 taken in combination complete a five or six membered saturated ring;
- 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, —OR8, —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, —O—, —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 R8 and R 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, —OR8, —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
- 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.
19 more elements
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 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 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, S, Se, NR15, or CR16R17, where R15 is H or an alkyl group having 1-6 carbons;
- and R18 and R17, which may be the same or different, are independently alkyl groups having 1-6 carbons, or R16 and R17 taken in combination complete a five or six membered saturated ring;
- 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, R8, and R7 are independently selected from the group consisting of hydrogen, a C1-C6 alkyl, a halogen, a CYCLIC SUBSTITUENT, —OR8, —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 Re and R9 taken in combination are —(CH2)2—V—(CH2)2— where V is a single bond, —O—, —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, —OR8, —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 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.
20 more elements
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 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, S, Se, NR15, or CR16R17, where R15 is H or an alkyl group having 1-6 carbons;
- and R16 and R17, which may be the same or different, are independently alkyl groups having 1-6 carbons, or R16 and R17 taken in combination complete a five or six membered saturated ring;
- 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, —OR8, —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, —O—, —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-C8 alkyl group, —OR8, —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-C8 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;
- b) incubating said sample and said staining solution for a sufficient amount of time to form a dye-nucleic acid complex;
- c) illuminating said complex with an appropriate wavelength whereby the presence of said nucleic acid is determined.
19 more elements
Dependencies are inferred from the claim text.