US5648270A — Methods of sensing with fluorescent conjugates of…

Claim 1

Independent Claim 821 words

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 R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , and R 8 are independently H, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 1 - 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 R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , and R 8 , 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 C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 1 -C 6 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, or K is --(CR 11 2 ) a --X b --(CR 12 2 ) c --, wherein a, b and c are each 0 or 1, provided that when b=1, a+c must equal 0 or 2; each R 11 and R 12 is optionally and independently H, or C 1 -C 6 alkyl; X is O, S, NR 13 , or --CR 14 R 15 --, wherein R 13 is H, C 1 -C 6 alkyl, -L-F, or phenyl that is optionally further substituted by C 1 -C 6 alkyl, C 1 -C 6 alkoxy, NO 2 , C 1 -C 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, 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; R 14 and R 15 are independently H, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, NO 2 , C 1 -C 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 -L-F; or K is ##STR9## wherein R 16 , R 17 , and R 18 are independently H, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, NO 2 , C 1 -C 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 K is a formal single bond, and R 4 and R 5 when taken in combination are --CR 9 ═CR 10 --, forming a phenanthroline moiety, wherein R 9 and R 10 are independently H, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 1 -C 6 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 R 1 , R 2 , R 3 , R 6 , R 7 , and R 8 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.

Claim 2

Independent Claim 821 words

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 R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , and R 8 are independently H, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 1 - 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 R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , and R 8 , 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 C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 1 -C 6 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, or K is --(CR 11 2 ) a --X b --(CR 12 2 ) c --, wherein a, b and c are each 0 or 1, provided that when b=1, a+c must equal 0 or 2; each R 11 and R 12 is optionally and independently H, or C 1 -C 6 alkyl; X is O, S, NR 13 , or --CR 14 R 15 --, wherein R 13 is H, C 1 -C 6 alkyl, -L-F, or phenyl that is optionally further substituted by C 1 -C 6 alkyl, C 1 -C 6 alkoxy, NO 2 , C 1 -C 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, 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; R 14 and R 15 are independently H, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, NO 2 , C 1 -C 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 -L-F; or K is ##STR9## wherein R 16 , R 17 , and R 18 are independently H, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, NO 2 , C 1 -C 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 K is a formal single bond, and R 4 and R 5 when taken in combination are --CR 9 ═CR 10 --, forming a phenanthroline moiety, wherein R 9 and R 10 are independently H, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 1 -C 6 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 R 1 , R 2 , R 3 , R 6 , R 7 , and R 8 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.

Claim 3

Dependent Claim 36 words

A method, as claimed in claim 1, further comprising the step of determining the concentration of said target ions, by comparing said fluorescence response with the fluorescence response obtained with known concentrations of said target ions.

Claim 4

Dependent Claim 30 words

A method, as claimed in claim 1, wherein said means for detecting a fluorescence response is a camera, a fluorometer, a fluorescence microscope, a laser scanner, or a flow cytometer.

Claim 5

Dependent Claim 16 words

A method, as claimed in claim 1, wherein said sample comprises living cells or biological fluids.

Claim 6

Dependent Claim 25 words

A method, as claimed in claim 1, wherein said sample is a soil or water sample, or is obtained from a soil or water sample.

Claim 7

Dependent Claim 160 words

A method, as claimed in claim 1, wherein said compound has the formula ##STR10## wherein the substituents R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , and R 10 , when present, are independently hydrogen, sulfonic acid, salt of sulfonic acid, amino, salt of amino, carboxy, alkyl having 1-6 carbons, or alkoxy having 1-6 carbons; provided that no more than three of the substituents R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 are not hydrogen; each fluorophore is independently a fluorescein, a rhodol, a rosamine or a rhodamine fluorophore, that is linked to the metal-binding moiety by a covalent linkage that is a single bond, an alkyl linkage, a phenylene linkage, a sulfonamide, a carboxamide, an alkyl amine, or a thiourea.

Claim 8

Dependent Claim 31 words

A method, as claimed in claim 1, wherein said compound has the formula ##STR11## and said target ion is one of Ni 2+ , Zn 2+ , and Cu 2+ .

Claim 9

Dependent Claim 33 words

A method, as claimed in claim 1, wherein said compound has the formula ##STR12## and said target ion is one of Fe 2+ , Cu 2+ , Cu + and Hg 2+ .

Claim 10

Dependent Claim 22 words

A method, as claimed in claim 1, wherein said compound has the formula ##STR13## and said target ion is Ni 2+ .

Claim 11

Dependent Claim 159 words

A method, as claimed in claim 1, wherein each fluorophore is independently a xanthene, a benzimidazole, a phenoxazine, an ethidium, a propidium, an acridine, a carbocyanine, a merocyanine, a coumarin, a pyrene, a chrysene, a stilbene, an anthracene, a naphthalene, a salicylic acid, a benz-2-oxa-1,3-diazole, a dipyrrometheneboron difluoride, or a dibenzopyrrometheneboron difluoride; each covalent linkage is a single bond, an alkyl linkage, a phenylene linkage, a sulfonamide, a carboxamide, an alkyl amine, or a thiourea linkage that is 9 or fewer non-hydrogen atoms in length; said detectable fluorescence response is a change in fluorescence emission intensity, fluorescence lifetime, excitation wavelength or emission wavelength; said compound is present in a concentration of 100 nM to 20 μM; said target ion is present in a concentration of 5 μM to 1 mM; said illuminating step is accomplished using excitation at greater than 360 nm; and said observing step is accomplished using a fluorometer, fluorescence microscope, a laser scanner, or flow cytometer.

Claim 12

Dependent Claim 19 words

A method, as claimed in claim 1, wherein said compound is present as part of a fiber optic probe.

Claim 13

Dependent Claim 24 words

A method, as claimed in claim 11, wherein said target ion is Cu + , and said additional metal ion is Cu 2+ .

Claim 14

Dependent Claim 37 words

A method, as claimed in claim 1, wherein said sample comprises other metal ions that are not the target ions, and the presence of said target ion is determined in the presence of the other metal ions.

Claim 15

Dependent Claim 36 words

A method, as claimed in claim 13, wherein the presence of said target ion is determined in the presence of one or more of Ca 2+ , Na + , K + or Cl - ions.

Claim 16

Dependent Claim 132 words

A method, as claimed in claim 1, wherein said target ion is selected from the group consisting of Mn 2+ , Fe 2+ , Fe 3+ , Co 2+ , Ni 2+ , Cu 2+ , Cu + , Zn 2+ , Al 3+ , Cd 2+ , Ag + , Au + , Tl + , Pd 2+ , Hg 2+ , Hg + , Sn 2+ , Pb 2+ , Sr 2+ , Ba 2+ , Mo 3+ , Ga 3+ , In 3+ , La 3+ , Eu 3+ , Tb 3+ , Dy 3+ , Ru 3+ , Sc 3+ , As 3+ , Sb 3+ , Cr 3+ , Bi 3+ , Ce 3+ , Ce 4+ , Pd 2+ , Pt 2+ and Pt 4+ .

Claim 17

Dependent Claim 60 words

A method, as claimed in claim 15, wherein said target ion is selected from the group consisting of Mn 2+ , Fe 2+ , Fe 3+ , Ni 2+ , Cu 2+ , Cu + , Zn 2+ , Cd 2+ , Ag + , Au + , Hg 2+ , Pb 2+ , Eu 3+ and Tb 3+ .

Claim 18

Dependent Claim 40 words

A method, as claimed in claim 16, wherein said target ion is selected from the group consisting of Fe 2+ , Fe 3+ , Ni 2+ , Cu 2+ , Cu + , Hg 2+ , or Pb 2+ .

Annuity Stage	Window Opens	Payment Deadline	Surcharge Period Until	Estimated Fee	Status
3.5 Years (Year 4)	2000-07-14	2001-01-13	2001-07-15	$2000.00	Window Closed / Lapsed
7.5 Years (Year 8)	2004-07-14	2005-01-13	2005-07-15	$3760.00	Window Closed / Lapsed
11.5 Years (Year 12)	2008-07-14	2009-01-13	2009-07-15	$7700.00	Window Closed / Lapsed

Methods of sensing with fluorescent conjugates of metal-chelating nitrogen heterocycles

Abstract

Inventors

Bibliographic Details

Claims Analysis

References Cited (Backward Citations)

USPTO Annuity & Maintenance Windows

Assigned Categories & Tags

Assign Category Tag