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US 5,686,261 AGranted
Xanthylium dyes that are well retained in mitochondria
AssigneeMolecular Probes, Inc.
InventorsYu-Zhong Zhang, Richard P. Haugland
Life of this patent
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Abstract
The present invention describes a method of staining mitochondria, and analyzing mitochondrial function, using a class of fluorescent substituted 3',6'-diaminoxanthenes and their reduced analogs 3',6'-diaminodihydroxanthenes, which are oxidized to the fluorescent form of the dye in situ. In their oxidized form, the dyes selectively localize within mitochondria. The dyes of the invention are substituted by an alkylating group that allows their retention in mitochondria even after cell death, fixation, and permeabilization.
Claims20
A method of staining mitochondria with a fluorescent label, comprising:
- a) preparing an aqueous labeling solution containing a compound of the formula ##STR13## or of the formula ##STR14## wherein a fused-ring system of the compound has ring substituents R.sup.1, R.sup.4, R.sup.5, and R.sup.8 that are independently H, Cl, Br, I, (CH.sub.2).sub.n CH.sub.3, or (CH.sub.2).sub.n CO.sub.2 R.sup.13, where n is an integer from 0 to 15 and R.sup.13 is an alkyl with 1-6 carbons;
- and amino substituents R.sup.2, R.sup.3, R.sup.6, and R.sup.7 that are independently H or C.sub.1 -C.sub.6 alkyl;
- or optionally any ring substituent in combination with an adjacent amino substituent independently forms an additional fused ring such that R.sup.1 in combination with R.sup.2, R.sup.3 in combination with R.sup.4, R.sup.5 in combination with R.sup.6, and R.sup.7 in combination with R.sup.8 is (CH.sub.2).sub.3 or (CH.sub.2).sub.2 ;
- .OMEGA. is a covalent linkage that is a single covalent bond, or .OMEGA. is --Z--Y-- where Z is attached to the fused ring system, and
- Z is a covalent bond;
- or Z is (CH.sub.2).sub.I where I=1-6;
- or Z is ##STR15## where R.sup.11 is a hydrocarbon chain with 1-6 carbons that is branched, unbranched, saturated or unsaturated;
- Y is a covalent bond or Y has the formula --OCH.sub.2 R.sup.12 --, --OR.sup.13 --, --SR.sup.12 --, --SR.sup.13 --, --NHCOCH.sub.2 R.sup.12 --, --NHCOR.sup.13 --, --CONHCH.sub.2 R.sup.12 --, --CONHR.sup.13 --, NHSO.sub.2 R.sup.12 --, --NHSO.sub.2 R.sup.13 --, --NHCONHCH.sub.2 R.sub.12 --, --NHCONHR.sup.13 --, --NHCSNHCH.sub.2 R.sub.12 --, or --NHCSNHR.sup.13 --, where R.sup.12 is (CH.sub.2).sub.m and m=1-18, and R.sup.13 is phenylene (--C.sub.6 H.sub.4 --);
- such that Z and Y are not both covalent bonds;
- A is an alkylating group that is
- --CR.sup.9 R.sup.10 X, where R.sup.9 and R.sup.10 are independently H or CH.sub.3, and X is Cl, Br, I, or --OSO.sub.2 R.sup.16, where R.sup.16 is C.sub.1 -C.sub.6 alkyl;
- C.sub.1 -C.sub.6 perfluoroalkyl;
- or R.sup.16 is phenyl, or phenyl substituted one or more times by C.sub.1 -C.sub.6 alkyl, C.sub.1 -C.sub.6 perfluoroalkyl, Cl, Br, I, NO.sub.2, or CN;
- or
- the alkylating group A is maleimidyl (--NC.sub.4 H.sub.2 O.sub.2) or haloacetamido (--NH(C.dbd.O)--CH.sub.2 II) where II is Cl, Br or I;
- .PSI. is a biologically compatible counterion,
- where said compound is greater than 0 nM and less than about 500 nM in concentration;
- and
- b) incubating a sample, where the sample is isolated mitochondria or a cell containing intracellular mitochondria, in the labeling solution for a time sufficient to produce fluorescent labeled mitochondria.
16 more elements
A method of analyzing mitochondrial function in a living eukaryotic cell or cells comprising
- a) preparing an aqueous labeling solution containing a compound of the formula ##STR23## or of the formula ##STR24## wherein a fused-ring system of the compound has ring substituents R.sup.1, R.sup.4, R.sup.5, and R.sup.8 that are independently H, Cl, Br, I, (CH.sub.2).sub.n CH.sub.3, or (CH.sub.2).sub.n CO.sub.2 R.sup.13, where n is an integer from 0 to 15 and R.sup.13 is an alkyl with 1-6 carbons;
- and amino substituents R.sup.2, R.sup.3, R.sup.6, and R.sup.7 that are independently H or C.sub.1 -C.sub.6 alkyl;
- or optionally any ring substituent in combination with an adjacent amino substituent independently forms an additional fused ring such that R.sup.1 in combination with R.sup.2, R.sup.3 in combination with R.sup.4, R.sup.5 in combination with R.sup.6, and R.sup.7 in combination with R.sup.8 is (CH.sub.2).sub.3 or (CH.sub.2).sub.2 ;
- and
- .OMEGA. is a covalent linkage that is a single covalent bond, or .OMEGA. is --Z--Y-- where Z is attached to the fused ring system, and
- Z is a covalent bond;
- or Z is (CH.sub.2).sub.I where I=1-6;
- or Z is ##STR25## where R.sup.11 is a hydrocarbon chain with 1-6 carbons that is branched, unbranched, saturated or unsaturated;
- Y is a covalent bond or Y has the formula --OCH.sub.2 R.sup.12 --, --OR.sup.13 --, --SR.sup.12 --, --SR.sup.13 --, --NHCOCH.sub.2 R.sub.12 --, --NHCOR.sub.13 --, --CONHCH.sub.2 R.sub.12 --, --CONHR.sub.13 --, NHSO.sub.2 R.sub.12 --, --NHSO.sub.2 R.sup.13 --, --NHCONHCH.sub.2 R.sup.12 --, --NHCONHR.sup.13 --, --NHCSNHCH.sub.2 R.sup.12 --, or --NHCSNHR.sup.13 --, where R.sup.12 is (CH.sub.2).sub.m and m=1-18, and R.sup.13 is phenylene (--C.sub.6 H.sub.4 --);
- such that Z and Y are not both covalent bonds;
- and
- A is an alkylating group that is
- CR.sup.9 R.sup.10 X, where R.sup.9 and R.sup.10 are independently H or CH.sub.3, and X is Cl, Br, I, or --OSO.sub.2 R.sup.16, where R.sup.16 is C.sub.1 -C.sub.6 alkyl;
- C.sub.1 -C.sub.6 perfluoroalkyl;
- or R.sup.16 is phenyl, or phenyl substituted one or more times by C.sub.1 -C.sub.6 alkyl, C.sub.1 -C.sub.6 perfluoroalkyl, Cl, Br, I, NO.sub.2, or CN;
- or
- the alkylating group A is maleimidyl (--NC.sub.4 H.sub.2 O.sub.2) or haloacetamido (--NH(C.dbd.O)--CH.sub.2 II) where II is Cl, Br or I;
- .OMEGA. is a biologically compatible counterion,
- where said compound is greater than 0 nM and less than about 500 nM in concentration;
- b) incubating the eukaryotic cell or cells in the labeling solution for a time sufficient to produce fluorescent labeled mitochondria;
- c) preparing the cell or cells for observation;
- d) observing the cell or cells with means for detecting the fluorescent labeled mitochondria;
- and
- e) analyzing the function of said mitochondria, based on the presence or absence of mitochondrial fluorescence.
21 more elements
A method of determining foreign gene expression, comprising
- a) preparing an aqueous labeling solution containing a compound of the formula ##STR29## or of the formula ##STR30## wherein a fused-ring system of the compound has ring substituents R.sup.1, R.sup.4, R.sup.5, and R.sup.8 that are independently H, Cl, Br, I, (CH.sub.2).sub.n CH.sub.3, or (CH.sub.2).sub.n CO.sub.2 R.sup.13, where n is an integer from 0 to 15 and R.sup.13 is an alkyl with 1-6 carbons;
- and amino substituents R.sup.2, R.sup.3, R.sup.6, and R.sup.7 that are independently H or C.sub.1 -C.sub.6 alkyl;
- or optionally any ring substituent in combination with an adjacent amino substituent independently forms an additional fused ring such that R.sup.1 in combination with R.sup.2, R.sup.3 in combination with R.sup.4, R.sup.5 in combination with R.sup.6, and R.sup.7 in combination with R.sup.8 is (CH.sub.2).sub.3 or (CH.sub.2).sub.2 ;
- and
- .OMEGA. is a covalent linkage that is a single covalent bond, or .OMEGA. is --Z--Y-- where Z is attached to the fused ring system, and
- Z is a covalent bond;
- or Z is (CH.sub.2).sub.I where I=1-6;
- or Z is ##STR31## where R.sup.11 is a hydrocarbon chain with 1-6 carbons that is branched, unbranched, saturated or unsaturated;
- Y is a covalent bond or Y has the formula --OCH.sub.2 R.sup.12 --, --OR.sup.13 --, --SR.sup.12 --, --SR.sup.13 --, --NHCOCH.sub.2 R.sup.12 --, --NHCOR.sup.13 --, --CONHCH.sub.2 R.sup.12 --, --CONHR.sub.13 --, NHSO.sub.2 R.sup.12 --, --NHSO.sub.2 R.sup.13 --, --NHCONHCH.sub.2 R.sup.12 --, --NHCONHR.sup.13 --, --NHCSNHCH.sub.2 R.sup.12 --, or --NHCSNHR.sup.13 --, where R.sup.12 is (CH.sub.2).sub.m and m=1-18, and R.sup.13 is phenylene (--C.sub.6 H.sub.4 --);
- such that Z and Y are not both covalent bonds;
- and
- A is an alkylating group that is
- CR.sup.9 R.sup.10 X, where R.sup.9 and R.sup.10 are independently H or CH.sub.3, and X is Cl, Br, I, or --OSO.sub.2 R.sup.16, where R.sup.16 is C.sub.1 -C.sub.6 alkyl;
- C.sub.1 -C.sub.6 perfluoroalkyl;
- or R.sup.16 is phenyl, or phenyl substituted one or more times by C.sub.1 -C.sub.6 alkyl, C.sub.1 -C.sub.6 perfluoroalkyl, Cl, Br, I, NO.sub.2, or CN;
- or
- the alkylating group A is maleimidyl (--NC.sub.4 H.sub.2 O.sub.2) or haloacetamido (--NH(C.dbd.O)--CH.sub.2 II) where II is Cl, Br or I;
- .OMEGA. is a biologically compatible counterion,
- where said compound is greater than 0 nM and less than about 500 nM in concentration;
- b) incubating the eukaryotic cell or cells in the labeling solution for a time sufficient to produce fluorescent labeled mitochondria;
- c) washing, fixing, and permeabilizing the cell or cells, and adding an additional detection reagent to the cell or cells, wherein said additional detection reagent fluorescently labels a protein that is present in the cell as a result of expression of a transfected gene, or said additional detection reagent fluorescently labels an antibody against said protein;
- and
- d) observing the cell or cells with a microscope.
20 more elements
Dependencies are inferred from the claim text.