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US 6,664,047 B1Granted
Aza-benzazolium containing cyanine dyes
AssigneeMolecular Probes, Inc.
InventorsRichard P. Haugland, Stephen T. 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.
Claims55
A compound having formula
- wherein A represents the atoms necessary to form one to two fused aromatic rings having 6 atoms in each ring selected from the group consisting of —C—, CH or N(R2)β, provided at least one of said ring atoms is N(R2)β, wherein aromatic carbon atoms are optionally substituted one or more times by substituents selected from the group consisting of hydrogen, alkyl having from 1-6 carbons, alkoxy having from 1-6 carbons, trifluoromethyl, halogen, methylthio, —L—Rx and —L—Sc;
- X is selected from the group consisting of O and S,
- α is 0 or 1 and β is 0 or 1 provided that α+all β=1;
- 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, or amino;
- n=0, 1 or 2;
- Y is —CR3═CR4—;
- 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, an alkyl that is saturated or unsaturated, linear or branched, having 1-6 carbons, a halogen, a CYCLIC SUBSTITUENT, —OR8, —SR8, —(NR8R9), TAIL, BRIDGE, —L—Rx and —L—Sc wherein R8 and R9 are independently alkyl groups having 1-6 carbons or 1-2 alicyclic or aromatic rings;
- or R8 and R9 taken in combination are —(CH2)2—W—(CH2)2— where W is a single bond, —O—, —CH2—, or —NR10—, where R10 is H or an alkyl having 1-6 H carbons;
- or R6 and R7 taken in combination form a fused 6-membered aromatic ring wherein ring substituents R11, R12, R13, and R14 are independently selected from the group consisting of hydrogen, halogen, —OR8, —SR8, —(NR8R9) a CYCLIC SUBSTITUENT, TAIL, BRIDGE, —L—Rx, —L—Sc and a saturated or unsaturated alkyl having 1-6 carbons that is linear or branched;
- R5 is selected from the group consisting of CYCLIC SUBSTITUENT, TAIL, BRIDGE, —L—Rx, —L—Sc, a pair of electrons, sulfoalkyl and a saturated or unsaturated alkyl having 1-6 carbons that is linear or branched;
- R30, R31, and R32 are independently selected from the group consisting of hydrogen, alkyl having 1-6 carbons, cycloalkyl having 3-10 carbons, aryl, and heteroaryl;
- and,
- when present, BRIDGE is attached to a DYE compound provided that no more than one of R2, R3, R4, R5, R6, R7, R11, R12, R13, R14 and R15 is BRIDGE;
- wherein
- L and BRIDGE are independently a single covalent bond or a covalent linkage having 1-20 nonhydrogen atoms selected from the group consisting of C, N, O and S;
- Rx is a reactive group;
- Sc is a conjugated substance;
- TAIL is a heteroatom-containing moiety,
- DYE is a compound having formula
- wherein A, X, R2, α, n, Ym, Yp, R5, R6, R7, R30, R31 and R32 are as defined above provided that BRIDGE not be any of R2, R3, R4, R5, R6, R7, R11, R12, R12, R13, R14 and R15.
18 more elements
A complex comprising:
- a) a compound having formula
- wherein A represents the atoms necessary to form one to two fused aromatic rings having 6 atoms in each ring selected from the group consisting of —C—, CH or N(R2)β, provided at least one of said ring atoms is N(R2)β wherein aromatic carbons are optionally substituted one or more times by substituents selected from the group consisting of hydrogen, alkyl having from 1-6 carbons, alkoxy having from 1-6 carbons, trifluoromethyl, halogen, methylthio, —L—Rx and —L—Sc;
- X is selected from the group consisting of O and;
- α is 0 or 1 and β is 0 or 1 provided that α+all β=1;
- 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, or amino;
- n=0, 1 or 2;
- Y is —CR3═CR4—;
- 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, an alkyl that is saturated or unsaturated, linear or branched, having 1-6 carbons, a halogen, a CYCLIC SUBSTITUENT, —OR8, —SR8, —(NR8R9), TAIL, BRIDGE, —L—Rx and —L—Sc wherein R8 and R9 are independently alkyl groups having 1-6 carbons or 1-2 alicyclic or aromatic rings;
- or R8 and R9 taken in combination are —(CH2)2—W—(CH2)2— where W is a single bond, —O—, —CH2—, or —NR10—, where R10 is H or an alkyl having 1-6 carbons;
- or R6 and R7 taken in combination form a fused 6-membered aromatic ring wherein ling substituents R11, R12, R13, and R14 are independently selected from the group consisting of hydrogen, halogen, —OR8, —SR8, —(NR8R9) a CYCLIC SUBSTITUENT, TAIL, BRIDGE, —L—Rx, —L—Sc, and a saturated or unsaturated alkyl having 1-6 carbons that is linear or branched;
- R5 is selected from the group consisting of CYCLIC SUBSTITUENT, TAIL, BRIDGE, —L—Rx, —L—Sc, a pair of electrons, sulfoalkyl and a saturated or unsaturated alkyl having 1-6 carbons that is linear or branched;
- R30, R31, and R32 are independently selected from the group consisting of hydrogen alkyl having 1-6 carbons, cycloalkyl having 3-10 carbons, aryl, and heteroaryl;
- and,
- when present, BRIDGE is attached to a DYE compound provided that no more than one of R2, R3, R4, R5, R6, R7, R11, R12, R13, R14 and R15 is BRIDGE;
- wherein
- L and BRIDGE are independently A single covalent bond or a covalent linkage having 1-20 nonhydrogen atoms selected from the group consisting of C, N, O and S;
- Rx is a reactive group;
- Sc is a conjugated substance;
- TAIL is a heteroatom-containing moiety;
- DYE is a compound having formula
- wherein A, X, R2, α, n, Ym, Yp, R5, R6, R7, R30, R31 and R32 are as defined above provided that BRIDGE not be any of R2, R3, R4, R5, R6, R7, R11, R12, R13, R14 and R15;
- and
- b) a nucleic acid polymer.
21 more elements
A complex comprising:
- a) a compound having formula:
- wherein A represents the atoms necessary to form one to two fused aromatic rings having 6 atoms in each ring selected from the group consisting of —C—, CH or N(R2)β, provided at least one of said ring atoms is N(R2)β wherein aromatic carbons are optionally substituted one or more times by substituents selected from the group consisting of hydrogen, alkyl having from 1-6 carbons, alkoxy having from 1-6 carbons, trifluoromethyl, halogen, methylthio, —L—Rx and —L—Sc;
- X is selected from the group consisting of O and S;
- α is 0 or 1 and β is 0 or 1 provided that α+all β=1;
- 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, or amino;
- n=0, 1 or 2;
- Y is —CR3═CR4—;
- 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, an alkyl that is saturated or unsaturated, linear or branched, having 1-6 carbons, a halogen, a CYCLIC SUBSTITUENT, —OR8, —SR8, —(NR8R9), TAIL, BRIDGE, —L—Rx and —L—Sc wherein R8 and R9 are independently alkyl groups having 1-6 carbons or 1-2 alicyclic or aromatic rings;
- or R8 and R9 taken in combination are —(CH2)2—W—(CH2)2— where W is a single bond, —O—, —CH2—, or —NR10—, where R10 is H or an alkyl having 1-6 carbons;
- or R6 and R7 taken in combination form a fused 6-membered aromatic ring wherein ring substituents R11, R12, R13, and R14 are independently selected from the group consisting of hydrogen, halogen, —OR8, —SR8, —(NR8R9) a CYCLIC SUBSTITUENT, TAIL, BRIDGE, —L—Rx, —L—Sc and a saturated or unsaturated alkyl having 1-6 carbons that is linear or branched;
- R5 is selected from the group consisting of CYCLIC SUBSTITUENT, TAIL, BRIDGE, —L—Rx, —L—Sc, a pair of electrons, sulfoalkyl and a saturated or unsaturated alkyl having 1-6 carbons that is linear or branched;
- R30, R31, and R32 are independently selected from the group consisting of hydrogen, alkyl having 1-6 carbons, cycloalkyl having 3-10 carbons, aryl, and heteroaryl;
- and,
- when present, BRIDGE is attached to a DYE compound provided that no more than one of R2, R3, R4, R5, R6, R7, R11, R12, R13, R14 and R15 is BRIDGE;
- wherein
- L and BRIDGE are independently a single covalent bond or a covalent linkage having 1-20 nonhydrogen atoms selected from the group consisting of C, N, O and S;
- Rx is a reactive group;
- Sc is a conjugated substance;
- TAIL is a heteroatom-containing moiety;
- DYE is a compound having formula
- wherein A, X, R2, α, n, Ym, Yp, R5, R6, R7, R30, R31 and R32 are as defined above provided that BRIDGE not be any of R2, R3, R4, R5, R6, R7, R11, R12, R13, R14 and R15;
- and,
- b) a poly(amino acid).
21 more elements
A kit comprising:
- a) a stock solution comprising;
- i) one or more compounds individually having formula:
- wherein A represents the atoms necessary to form one to two fused aromatic rings having 6 atoms in each ring selected from the group consisting of —C—, CH or N(R2)β, provided at least one of said ring atoms is N(R2)β wherein aromatic carbons are optionally substituted one or more times by substituents selected from the group consisting of hydrogen, alkyl having from 1-6 carbons, alkoxy having from 1-6 carbons, trifluoromethyl, halogen, methylthio, —L—Rx and —L—Sc;
- X is selected from the group consisting of O and S;
- α is 0 or 1 and β is 0 or 1 provided that α+all β=1;
- 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, or amino;
- n=0, 1 or 2;
- Y is —CR3═CR4—;
- p and m=0 or 1, such that p+m1;
- R3, R4, R6, and R7 are independently selected from the group consisting of hydrogen, an alkyl that is saturated or unsaturated, linear or branched, having 1-6 carbons, a halogen, a CYCLIC SUBSTITUENT, —OR8, —SR8, —(NR8R9), TAIL, BRIDGE, —L—Rx and —L—Sc wherein R8 and R9 are independently alkyl groups having 1-6 carbons or 1-2 alicyclic or aromatic rings;
- or R8 and R9 taken in combination are —(CH2)2—W—(CH2)2— where W is a single bond, —O—, —CH2—, or —NR10—, where R10 is H or an alkyl having 1-6 carbons;
- or R6 and R7 taken in combination form a fused 6-membered aromatic ring wherein ring substituents R11, R12, R13, and R14 are independently selected from the group consisting of hydrogen, halogen, —OR8, —SR8, —(NR8R9) a CYCLIC SUBSTITUENT, TAIL, BRIDGE, —L—Rx, —L—Sc and a saturated or unsaturated alkyl having 1-6 carbons that is linear or branched;
- R5 is selected from the group consisting of CYCLIC SUBSTITUENT, TAIL, BRIDGE, —L—Rx, —L—Sc, a pair of electrons, sulfoalkyl and a saturated or unsaturated alkyl having 1-6 carbons that is linear or branched;
- R30, R31, and R32 are independently selected from the group consisting of H, alkyl having 1-6 carbons, cycloalkyl having 3-10 carbons, aryl, and heteroaryl;
- and,
- when present, BRIDGE is attached to a DYE compound provided that no more than one of R2, R3, R4, R5, R6, R7, R11, R12, R13, R14 and R15 is BRIDGE;
- wherein
- L and BRIDGE are independently a single covalent bond or a covalent linkage having 1-20 nonhydrogen atoms selected from the group consisting of C, N, O and S;
- Rx is a reactive group;
- Sc is a conjugated substance;
- TAIL is a heteroatom-containing moiety;
- DYE is a compound having formula
- wherein A, X, R2, α, n, Ym, Yp, R5, R6, R7, R30, R31 and R32 are as defined above provided that BRIDGE not be any of R2, R3, R4, R5, R6, R7, R11, R12, R13, R14 and R15;
- ii) an organic solvent;
- and,
- b) a buffer suitable for dilution of said stock solution.
23 more elements
A compound having formula:
- wherein R2 is an alkyl having 1-6 carbon atoms and wherein meta and ortho positions to R2 are independently and optionally substituted with halogen or an alkyl having 1-6 carbons atoms;
- X is selected from the group consisting of O and S;
- n=0, 1 or 2;
- α is 0 or 1 and β is 0 or 1 provided that α+all β=1;
- Y is —CR3═CR4—;
- 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, an alkyl having 1-6 carbons, a halogen, a CYCLIC SUBSTITUENT, —OR8, —SR8, —(NFR8R9), TAIL and BRIDGE wherein R8 and R9 are independently alkyl groups having 1-6 carbons or 1-2 alicyclic or aromatic rings;
- or R8 and R9 taken in combination are —(CH2)2—W—(CH2)2— where W is a single bond, —O—, —CH2—, or —NR10—, where R10 is H or an alkyl having 1-6 carbons;
- or R6 and R7 taken in combination form a fused 6-membered aromatic ring wherein ring substituents R11, R12, R13, and R14 are independently selected from the group consisting of hydrogen, halogen, —OR8, —SR8, —(NR8R9), CYCLIC SUBSTITUENT, TAIL, BRIDGE,
- and an alkyl having 1-6 carbons;
- R5 is selected from the group consisting of CYCLIC SUBSTITUENT, TAIL BRIDGE, a pair of electrons, sulfoalkyl and an alkyl having 1-6 carbons;
- R30, R31, and R32 are independently selected from the group consisting of H, alkyl having 1-6 carbons, cycloalkyl having 3-10 carbons, aryl, and heteroaryl;
- and,
- when present, BRIDGE is attached to a DYE compound provided that no more than one of R3, R4, R5, R6, R7, R11, R12, R13 and R14 is BRIDGE;
- wherein;
- BRIDGE is independently a single covalent bond or a covalent linkage having 1-20 nonhydrogen atoms selected from the group consisting of C, N, O and S;
- TAIL is a heteroatom-containing moiety;
- DYE is a compound of the formula
- wherein X, R2, α, β, n, Ym, Yp, R5, R6, R7, R30, R31, and R32 are as defined above provided that BRIDGE not be any of R3, R4, R5, R6, R7, R11, R12, R13 and R14.
16 more elements
A compound having formula:
- wherein R2 is an alkyl having 1-6 carbon atoms and fused 6-membered aromatic ring is optionally substituted at a ring carbon by methylthio;
- X is selected from the group consisting of O and S;
- n=0, 1 or 2;
- α is 0 or 1 and β is 0 or 1 provided that α+all β=1;
- Y is —CR3═CR4—;
- 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, an alkyl having 1-6 carbons, a halogen, a CYCLIC SUBSTITUENT, —OR8, —SR8, —(NR8R9), TAIL and BRIDGE wherein R8 and R9 are independently alkyl groups having 1-6 carbons or 1-2 alicyclic or aromatic rings;
- or R8 and R9 taken in combination are —(CH2)2—W—(CH2)2— where W is a single bond, —O—, —CH2—, or —NR10—, where R10 is H or an alkyl having 1-6 carbons;
- or R6 and R7 taken in combination form a fused 6-membered aromatic ring wherein ring substituents R11, R12, R13, and R14 are independently selected from the group consisting of hydrogen, halogen, —OR8, —SR8, —(NR8R9), CYCLIC SUBSTITUENT, TAIL, BRIDGE, and an alkyl having 1-6 carbons;
- R5 is selected from the group consisting of CYCLIC SUBSTITUENT, TAIL, BRIDGE, a pair of electrons, sulfoalkyl and an alkyl having 1-6 carbons;
- R30, R31, and R32 are independently selected from the group consisting of H, alkyl having 1-6 carbons, cycloalkyl having 3-10 carbons, aryl, and heteroaryl;
- and,
- when present, BRIDGE is attached to a DYE compound provided that no more than one of R3, R4, R5, R6, R7, R11, R12, R13 and R14 is BRIDGE;
- wherein;
- BRIDGE is independently a single covalent bond or a covalent linkage having 1-20 nonhydrogen atoms selected from the group consisting of C, N, O and S;
- TAIL is a heteroatom-containing moiety;
- DYE is a compound having formula
- wherein X, R2, α, β, n, Ym, Yp, R5, R6, R7, R30, R31 and R32 are as defined above provided that BRIDGE not be any of R3, R4, R5, R6, R7, R11, R12, R13 and R14.
15 more elements
A solution for staining nucleic acid polymers or poly(amino acids) wherein said solution comprises:
- a) one or more compounds having formula
- wherein A represents the atoms necessary to form one to two fused aromatic rings having 6 atoms in each ring selected from the group consisting of —C—, CH or N(R2)β, provided at least one of said ring atoms is N(R2)β wherein aromatic carbons are optionally substituted one or more times by substituents selected from the group consisting of hydrogen, alkyl having from 1-6 carbons, alkoxy having from 1-6 carbons, trifluoromethyl, halogen, methylthio, —L—Rx and —L—Sc;
- X is selected from the group consisting of O and S;
- α is 0 or 1 and β is 0 or 1 provided that α+all β=1;
- 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, or amino;
- n=0,1 or 2;
- Y is —CR3═CR4—;
- p and m=0 or 1, such that p+m1;
- R3, R4, R6, and R7 are independently selected from the group consisting of hydrogen, an alkyl that is saturated or unsaturated, linear or branched, having 1-6 carbons, a halogen, a CYCLIC SUBSTITUENT, —OR5, —SR8, —(NR8R9), TAIL, BRIDGE, —L—Rx and —L—Sc wherein R8 and R9 are independently alkyl groups having 1-6 carbons or 1-2 alicyclic or aromatic rings;
- or R8 and R9 taken in combination are —(CH2)2—W—(CH2)2— where W is a single bond, —O—, —CH2—, or —NR10—, where R10 is H or an alkyl having 1-6 carbons;
- or R6 and R7 taken in combination form a fused 6-membered aromatic ring wherein ring substituents R11, R12, R13, and R14 are independently selected from the group consisting of hydrogen, halogen, —OR8, —SR8, —(NR8R9), a CYCLIC SUBSTITUENT, TAIL, BRIDGE, —L—Rx, —L—Sc and a saturated or unsaturated alkyl having 1-6 carbons that is linear or branched;
- R5 is selected from the group consisting of CYCLIC SUBSTITUENT, TAIL, BRIDGE, —L—Rx, —L—Sc, a pair of electrons, sulfoalkyl and a saturated or unsaturated alkyl having 1-6 carbons that is linear or branched;
- R30, R31, and R32 are independently selected from the group consisting of hydrogen, alkyl having 1-6 carbons, cycloalkyl having 3-10 carbons, awl, and heteroaryl;
- and,
- when present BRIDGE is attached to a DYE compound provided that no more than one of R2, R3, R4, R5, R6, R7, R11, R12, R13, R14 and R15 is BRIDGE;
- wherein
- L and BRIDGE are independently a single covalent bond or a covalent linkage having 1-20 nonhydrogen atoms selected from the group consisting of C, N, O and S;
- Rx is a reactive group;
- Sc is a conjugated substance;
- TAIL is a heteroatom-containing moiety;
- DYE is a compound having formula
- wherein A, X, R7, α, n, Ym, Yp, R5, R6, R7, R30, R31 and R32 are as defined above provided that BRIDGE not be any of R2, R3, R4, R5, R6, R7, R11, R12, R13, R14 and R15;
- and;
- b) an organic solvent.
21 more elements
Dependencies are inferred from the claim text.