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US 6,004,536 AGranted
Lipophilic cyanine dyes with enchanced aqueous solubilty
AssigneeMolecular Probes, Inc.
InventorsWai-Yee Leung, Richard P. Haugland, Fei Mao
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Abstract
The present invention relates to a family of cyanine dyes possessing lipophilic alkyl chains and either one or more reactive functional groups, bromo or chloro, or phenyl, sulfophenyl or polysulfophenyl substituents or combinations thereof. The dyes of the invention are useful for staining membranes in cells or isolated from cells, and are well-retained therein. Additionally, the reactive dyes of the invention are useful for preparing dye-conjugates, thereby conferring the membrane staining ability of the subject dye onto the resulting dye-conjugate.
Claims28
A compound having the formula ##STR57## wherein R.sup.1 and R.sup.2 are independently linear or branched hydrocarbons having 7-30 carbons, where each hydrocarbon group optionally incorporates a 5- or 6-membered unsaturated hydrocarbon ring, and each hydrocarbon is independently and optionally substituted one or more times by F, Cl, or alkoxy having 1-6 carbons;
- X and Y are independently O, S or CR.sup.3 R.sup.4, where R.sup.3 and R.sup.4, which may be the same or different, are independently alkyl groups having 1-6 carbons, or R.sup.3 and R.sup.4 taken in combination complete a five or six membered saturated ring;
- n=0-3, yielding a methine bridge that is unsubstituted or is optionally substituted by F, Cl or alkyl having 1-6 carbons;
- or any double bond in the methine bridge is optionally incorporated into a 5- or 6- membered hydrocarbons ring that is unsubstituted or is optionally substituted one or more times by alkyl having 1-6 carbons;
- R.sup.5, R.sup.6, R.sup.7 R.sup.8, R.sup.9, R.sup.10, R.sup.11 and R.sup.12 are independently hydrogen, F, Cl, Br, I, CN, sulfo, phenyl, sulfophenyl, polysulfophenyl, carboxy, amino, ammonium, or an alkyl group having 1-22 carbons that is optionally and independently substituted by one or more of F, Cl, or --OR.sup.13 where R.sup.13 is H or an alkyl group having 1-6 carbons;
- or any two adjacent substituents of R.sup.5, R.sup.6, R.sup.7, R.sup.8, R.sup.9, R.sup.10, R.sup.11 and R.sup.12, when taken in combination, form a single fused benzo substituent;
- or any of R.sup.5, R.sup.6, R.sup.7, R.sup.8, R.sup.9, R.sup.10, R.sup.11 and R.sup.12 is a methylbenzamido, chloromethylbenzamido, maleimidyl benzamido, maleimidyl alkylamido, azidobenzamido or azidoperfluorobenzamido;
- .PSI. is a biologically compatible counterion, if necessary;
- provided that at least one of R.sup.5, R.sup.6, R.sup.7, R.sup.8, R.sup.9, R.sup.10, R.sup.11 or R.sup.12 is a methylbenzamido, chloromethylbenzamido, maleimidyl benzamido, maleimidyl alkylamido, azidobenzamido or azidoperfluorobenzamido, and no more than one of R.sup.5, R.sup.6, R.sup.7 and R.sup.8, and no more than one of R.sup.9, R.sup.10, R.sup.11 or R.sup.12 is a methylbenzamido, chloromethylbenzamido, maleimidyl benzamido, maleimidyl alkylamido, azidobenzamido or azidoperfluorobenzamido.
5 more elements
A compound having the formula ##STR60## wherein R.sup.1 and R.sup.2 are independently linear or branched hydrocarbons having 7-30 carbons, where each hydrocarbon group optionally incorporates a 5- or 6-membered unsaturated hydrocarbon ring, and each hydrocarbon is independently and optionally substituted one or more times by F, Cl, or alkoxy having 1-6 carbons;
- X and Y are independently O, S or CR.sup.3 R.sup.4, where R.sup.3 and R.sup.4, which may be the same or different, are independently alkyl groups having 1-6 carbons, or R.sup.3 and R.sup.4 taken in combination complete a five or six membered saturated ring;
- n=0-3, yielding a methine bridge that is unsubstituted or is optionally substituted by F, Cl or alkyl having 1-6 carbons;
- or any double bond in the methine bridge is optionally incorporated into a 5- or 6- membered hydrocarbons ring that is unsubstituted or is optionally substituted one or more times by alkyl having 1-6 carbons;
- R.sup.5, R.sup.6, R.sup.7 R.sup.8, R.sup.9, R.sup.10, R.sup.11 and R.sup.12 are independently hydrogen, F, Cl, Br, I, CN, sulfo, phenyl, sulfophenyl, polysulfophenyl, carboxy, amino, ammonium, methylbenzamido, or an alkyl group having 1-22 carbons that is optionally and independently substituted by one or more of F, Cl, or --OR.sup.13 where R.sup.13 is H or an alkyl group having 1-6 carbons;
- or any two adjacent substituents of R.sup.5, R.sup.6, R.sup.7, R.sup.8, R.sup.9, R.sup.10, R.sup.11 and R.sup.12, when taken in combination, form a single fused benzo substituent;
- or any of R.sup.5, R.sup.6, R.sup.7, R.sup.8, R.sup.9, R.sup.10, R.sup.11 and R.sup.12 is an --L--A;
- wherein
- L is a linking moiety that is a single covalent bond, or is a covalent linkage having 1-20 nonhydrogen atoms selected from the group consisting of C, N, O and S;
- such that the linkage contains any combination of bonds selected from the group consisting of ether, thioether, amine, ester, carboxamide, sulfonamide and hydrazide bonds;
- single, double, triple and aromatic carbon-carbon bonds;
- aromatic and heteroaromatic bonds;
- and
- A is a reactive group;
- .PSI. is a biologically compatible counterion, if necessary;
- provided that at least one of R.sup.5, R.sup.6, R.sup.7 R.sup.8, R.sup.9, R.sup.10, R.sup.11 or R.sup.12 is a phenyl, sulfo, sulfophenyl, or polysulfophenyl.
12 more elements
A method of staining lipophilic structures in cells, comprising:
- combining cells with a cyanine compound of the formula ##STR64## wherein R.sup.1 and R.sup.2 are independently linear or branched hydrocarbons having 7-30 carbons, where each hydrocarbon group optionally incorporates a 5- or 6-membered unsaturated hydrocarbon ring, and each hydrocarbon is independently and optionally substituted one or more times by F, Cl, or alkoxy having 1-6 carbons;
- X and Y are independently O, S or CR.sup.3 R.sup.4, where R.sup.3 and R.sup.4, which may be the same or different, are independently alkyl groups having 1-6 carbons, or R.sup.3 and R.sup.4 taken in combination complete a five or six membered saturated ring;
- n=0-3, yielding a methine bridge that is unsubstituted or is optionally substituted by F, Cl or alkyl having 1-6 carbons;
- or any double bond in the methine bridge is optionally incorporated into a 5- or 6- membered hydrocarbons ring that is unsubstituted or is optionally substituted one or more times by alkyl having 1-6 carbons;
- R.sup.5, R.sup.6, R.sup.7 R.sup.8, R.sup.9, R.sup.10, R.sup.11 and R.sup.12 are independently hydrogen, F, Cl, Br, I, CN, sulfo, phenyl, sulfophenyl, polysulfophenyl, carboxy, amino, ammonium, methylbenzamido, or an alkyl group having 1-22 carbons that is optionally and independently substituted by one or more of F, Cl, or --OR.sup.13 where R.sup.13 is H or an alkyl group having 1-6 carbons;
- or any two adjacent substituents of R.sup.5, R.sup.6, R.sup.7, R.sup.8, R.sup.9, R.sup.10, R.sup.11 and R.sup.12, when taken in combination, form a single fused benzo substituent;
- or any of R.sup.5, R.sup.6, R.sup.7, R.sup.8, R.sup.9, R.sup.10, R.sup.11 and R.sup.12 is a methylbenzamido, chloromethylbenzamido, maleimidyl benzamido, maleimidyl alkylamido, azidobenzamido, azidoperfluorobenzamido, phenyl, sulfo, sulfophenyl, or polysulfophenyl;
- .PSI. is a biologically compatible counterion, if necessary;
- provided that at least one of R.sup.5, R.sup.6, R.sup.7, R.sup.8, R.sup.9, R.sup.10, R.sup.11 or R.sup.12 is a methylbenzamido, chloromethylbenzamido, maleimidyl benzamido, maleimidyl alkylamido, azidobenzamido, azidoperfluorobenzamido, phenyl, sulfo, sulfophenyl, or polysulfophenyl;
- and no more than one of R.sup.5, R.sup.6, R.sup.7 and R.sup.8, and no more than one of R.sup.9, R.sup.10, R.sup.11 or R.sup.12 is a methylbenzamido, chloromethylbenzamido, maleimidyl benzamido, maleimidyl alkylamido, azidobenzamido, or azidoperfluorobenzamido;
- where said compound is present in an amount sufficient to selectively accumulate in said lipophilic structures and for a time sufficient for the cyanine compound to selectively accumulate in said lipophilic structures.
8 more elements
Dependencies are inferred from the claim text.