Methods of sensing with fluorescent conjugates of metal-chelating nitrogen heterocycles
First Claim
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1. A method of determining the presence of target ions in a sample, comprising:
- a) adding to said sample, in an amount sufficient to generate a detectable fluorescent response to said target ion, a compound containing a metal binding moiety according to the formula ##STR8## wherein pyridyl ring substituents R1, R2, R3, R4, R5, R6, R7, and R8 are independently H, C1 -C6 alkyl, C1 -C6 alkoxy, C1 -6 perfluoroalkyl, halogen, CN, sulfonic acid, salt of sulfonic acid, amino, salt of amino, alkylamino having 1-6 carbons, dialkylamino having 2-12 carbons;
carboxy, carboxyalkyl having 2-7 carbons, carboxyalkoxy having 2-7 carbons, carboxyalkylthio having 2-7 carbons, carboxyalkylamino having 2-7 carbons, or a 1-(acyloxy)alkyl ester of any such carboxy, such ester having 2-7 carbons;
or aryl, or heteroaryl;
or -L-F;
or any two adjacent substituents of R1, R2, R3, R4, R5, R6, R7, and R8, taken in combination, form a fused six-membered aromatic ring, such that no more than one fused ring is bound to each pyridyl ring, and where said fused rings are optionally and independently further substituted by C1 -C6 alkyl, C1 -C6 alkoxy, C1 -C6 perfluoroalkyl, CN, sulfonic acid, salt of sulfonic acid, amino, salt of amino, alkylamino having 1-6 carbons, dialkylamino having 2-12 carbons;
carboxy, carboxyalkyl having 2-7 carbons, carboxyalkoxy having 2-7 carbons, carboxyalkylthio having 2-7 carbons, or a 1(acyloxy)alkyl ester of any such carboxy, such ester having 2-7 carbons;
or -L-F; and
K is a single covalent bond, orK is --(CR112)a --Xb --(CR122)c --,wherein a, b and c are each 0 or 1, provided that when b=1, a+c must equal 0 or 2;
each R11 and R12 is optionally and independently H, or C1 -C6 alkyl;
X is O, S, NR13, or --CR14 R15 --, wherein R13 is H, C1 -C6 alkyl, -L-F, or phenyl that is optionally further substituted by C1 -C6 alkyl, C1 -C6 alkoxy, NO2, C1 -C6 perfluoroalkyl, halogen, CN, sulfonic acid, salt of sulfonic acid, amino, salt of amino, alkylamino having 1-6 carbons, dialkylamino having 2-12 carbons;
carboxy, carboxyalkyl having 2-7 carbons, carboxyalkoxy, having 2-7 carbons, carboxyalkylthio having 2-7 carbons, or carboxyalkylamino having 2-7 carbons, or a 1-(acyloxy)alkyl ester of any such carboxy, such ester having 2-7 carbons;
or -L-F;
R14 and R15 are independently H, C1 -C6 alkyl, C1 -C6 alkoxy, NO2, C1 -C6 perfluoroalkyl, halogen, CN, sulfonic acid, salt of sulfonic acid, amino, salt of amino, alkylamino having 1-6 carbons, dialkylamino having 2-12 carbons, carboxy, carboxyalkyl having 2-7 carbons, carboxyalkoxy having 2-7 carbons, carboxyalkylthio having 2-7 carbons, carboxyalkylamino having 2-7 carbons, or a 1-(acyloxy)alkyl ester of any such carboxy, such ester having 2-7 carbons;
or -L-F;
orK is ##STR9## wherein R16, R17, and R18 are independently H, C1 -C6 alkyl, C1 -C6 alkoxy, NO2,C1 -C6 perfluoroalkyl, halogen, CN, sulfonic acid, salt of sulfonic acid, amino, salt of amino, alkylamino having 1-6 carbons, dialkylamino having 2-12 carbons;
carboxy, carboxyalkyl having 2-7 carbons, carboxyalkoxy having 2-7 carbons, carboxyalkylthio having 2-7 carbons, carboxyalkylamino having 2-7 carbons, or a 1-(acyloxy)alkyl ester of any such carboxy, such ester having 2-7 carbons;
or aryl, or heteroaryl, or -L-F;
orK is a formal single bond, and R4 and R5 when taken in combination are --CR9 ═
CR10 --, forming a phenanthroline moiety, wherein R9 and R10 are independently H, C1 -C6 alkyl, C1 -C6 alkoxy, C1 -C6 perfluoroalkyl, CN, sulfonic acid, salt of sulfonic acid, amino, salt of amino, alkylamino having 1-6 carbons, dialkylamino having 2-12 carbons;
carboxy, carboxyalkyl having 2-7 carbons, carboxyalkoxy having 2-7 carbons, carboxyalkylthio having 2-7 carbons, carboxyalkylamino having 2-7 carbons, or a 1-(acyloxy)alkyl ester of any such carboxy, such ester having 2-7 carbons;
or -L-F; and
the remaining ring substituents R1, R2, R3, R6, R7, and R8 are as defined above;
such that at least one but not more than two substituents on said metal ion-binding moiety is -L-F;
wherein each F is independently a fluorophore that is attached to said metal-binding moiety by L, where each L is independently a single bond or a covalent linkage that is 9 or fewer non-hydrogen atoms in length;
b) illuminating said sample to generate said fluorescence response; and
c) observing said sample with means for detecting said fluorescence response, where the fluorescence response indicates the presence of target ions.
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Abstract
The present invention describes the use of a family of fluorescent indicators for metal cations. The indicators are fluorophore conjugates of pyridyl-based metal ion chelators. The indicators are very sensitive detection as quantification reagents for a variety of metals, in a variety of oxidation states, even in the presence of high concentrations of Ca2+, Na+, or K+ or other ions, such as is found in seawater, making them highly useful for assaying physiological samples, biological samples, or environmental samples.
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Citations
17 Claims
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1. A method of determining the presence of target ions in a sample, comprising:
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a) adding to said sample, in an amount sufficient to generate a detectable fluorescent response to said target ion, a compound containing a metal binding moiety according to the formula ##STR8## wherein pyridyl ring substituents R1, R2, R3, R4, R5, R6, R7, and R8 are independently H, C1 -C6 alkyl, C1 -C6 alkoxy, C1 -6 perfluoroalkyl, halogen, CN, sulfonic acid, salt of sulfonic acid, amino, salt of amino, alkylamino having 1-6 carbons, dialkylamino having 2-12 carbons;
carboxy, carboxyalkyl having 2-7 carbons, carboxyalkoxy having 2-7 carbons, carboxyalkylthio having 2-7 carbons, carboxyalkylamino having 2-7 carbons, or a 1-(acyloxy)alkyl ester of any such carboxy, such ester having 2-7 carbons;
or aryl, or heteroaryl;
or -L-F;or any two adjacent substituents of R1, R2, R3, R4, R5, R6, R7, and R8, taken in combination, form a fused six-membered aromatic ring, such that no more than one fused ring is bound to each pyridyl ring, and where said fused rings are optionally and independently further substituted by C1 -C6 alkyl, C1 -C6 alkoxy, C1 -C6 perfluoroalkyl, CN, sulfonic acid, salt of sulfonic acid, amino, salt of amino, alkylamino having 1-6 carbons, dialkylamino having 2-12 carbons;
carboxy, carboxyalkyl having 2-7 carbons, carboxyalkoxy having 2-7 carbons, carboxyalkylthio having 2-7 carbons, or a 1(acyloxy)alkyl ester of any such carboxy, such ester having 2-7 carbons;
or -L-F; andK is a single covalent bond, or K is --(CR112)a --Xb --(CR122)c --, wherein a, b and c are each 0 or 1, provided that when b=1, a+c must equal 0 or 2; each R11 and R12 is optionally and independently H, or C1 -C6 alkyl; X is O, S, NR13, or --CR14 R15 --, wherein R13 is H, C1 -C6 alkyl, -L-F, or phenyl that is optionally further substituted by C1 -C6 alkyl, C1 -C6 alkoxy, NO2, C1 -C6 perfluoroalkyl, halogen, CN, sulfonic acid, salt of sulfonic acid, amino, salt of amino, alkylamino having 1-6 carbons, dialkylamino having 2-12 carbons;
carboxy, carboxyalkyl having 2-7 carbons, carboxyalkoxy, having 2-7 carbons, carboxyalkylthio having 2-7 carbons, or carboxyalkylamino having 2-7 carbons, or a 1-(acyloxy)alkyl ester of any such carboxy, such ester having 2-7 carbons;
or -L-F;
R14 and R15 are independently H, C1 -C6 alkyl, C1 -C6 alkoxy, NO2, C1 -C6 perfluoroalkyl, halogen, CN, sulfonic acid, salt of sulfonic acid, amino, salt of amino, alkylamino having 1-6 carbons, dialkylamino having 2-12 carbons, carboxy, carboxyalkyl having 2-7 carbons, carboxyalkoxy having 2-7 carbons, carboxyalkylthio having 2-7 carbons, carboxyalkylamino having 2-7 carbons, or a 1-(acyloxy)alkyl ester of any such carboxy, such ester having 2-7 carbons;
or -L-F;
orK is ##STR9## wherein R16, R17, and R18 are independently H, C1 -C6 alkyl, C1 -C6 alkoxy, NO2, C1 -C6 perfluoroalkyl, halogen, CN, sulfonic acid, salt of sulfonic acid, amino, salt of amino, alkylamino having 1-6 carbons, dialkylamino having 2-12 carbons;
carboxy, carboxyalkyl having 2-7 carbons, carboxyalkoxy having 2-7 carbons, carboxyalkylthio having 2-7 carbons, carboxyalkylamino having 2-7 carbons, or a 1-(acyloxy)alkyl ester of any such carboxy, such ester having 2-7 carbons;
or aryl, or heteroaryl, or -L-F;
orK is a formal single bond, and R4 and R5 when taken in combination are --CR9 ═
CR10 --, forming a phenanthroline moiety, wherein R9 and R10 are independently H, C1 -C6 alkyl, C1 -C6 alkoxy, C1 -C6 perfluoroalkyl, CN, sulfonic acid, salt of sulfonic acid, amino, salt of amino, alkylamino having 1-6 carbons, dialkylamino having 2-12 carbons;
carboxy, carboxyalkyl having 2-7 carbons, carboxyalkoxy having 2-7 carbons, carboxyalkylthio having 2-7 carbons, carboxyalkylamino having 2-7 carbons, or a 1-(acyloxy)alkyl ester of any such carboxy, such ester having 2-7 carbons;
or -L-F; and
the remaining ring substituents R1, R2, R3, R6, R7, and R8 are as defined above;such that at least one but not more than two substituents on said metal ion-binding moiety is -L-F;
wherein each F is independently a fluorophore that is attached to said metal-binding moiety by L, where each L is independently a single bond or a covalent linkage that is 9 or fewer non-hydrogen atoms in length;b) illuminating said sample to generate said fluorescence response; and c) observing said sample with means for detecting said fluorescence response, where the fluorescence response indicates the presence of target ions. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17)
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Specification