# US 2010/0167333 A1 · Heavy Metal Binding Compounds and Their Method of Use

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- Number: US 2010/0167333 A1
- Document ID: US20100167333A1
- Status: Published application
- Kind: A1
- Application number: 12/636,687
- Filed: 2009-12-11
- Published: 2010-07-01
- Earliest claimed: 2006-01-12
- Assignees: Life Technologies Corporation
- Inventors: Kyle R. Gee; Jolene Bradford; Vladimir V. Martin
- Web page: https://patentgrep.com/patent/US20100167333A1

## CPC

- C07D 413/04: directly linked by a ring-member-to-ring-member bond
- C07D 209/14: Radicals substituted by nitrogen atoms, not forming part of a nitro radical
- C07D 335/12: Thioxanthenes
- C09B 11/28: Pyronines {; Xanthon, thioxanthon, selenoxanthan, telluroxanthon dyes}
- C07D: HETEROCYCLIC COMPOUNDS (macromolecular compounds C08)
- C09B: ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES {, e.g. PIGMENTS}; MORDANTS; LAKES (fermentation or enzyme using processes to synthesise a desired chemical compound C12P)

## Abstract

The present invention provides a metal chelator and methods that facilitate binding, detecting, monitoring and quantitating of heavy metal ions in a sample. This metal chelating moiety is a —N,N,O-triacetic acid analog of BAPTA and has the following formula

## Claims

### Claim at position 1

-66. (canceled)

### Claim 67

A kit for detecting metal ions in a sample, wherein the kit comprises a compound having the formula:
wherein
RX is —CH2CO2R;
RY is —CH2CO2R;
RX′ is —CH2CO2R;
wherein R is H, a salt ion or —CH2OC(O)(CH2)—CH3 and n is 0 to 6;
R1 is hydrogen;
R2 is hydrogen;
R3 is hydrogen, halogen, alkyl, substituted alkyl, alkoxy, substituted alkoxy, hydroxyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, amino, substituted amino, alkylamino, substituted alkylamino, aldehyde, carboxyl, azido, nitro, nitroso, cyano, thioether, sulfoalkyl, carboxyalkyl, aminoalkyl, reporter molecule, reactive group, carrier molecule or solid support;
R4 is hydrogen;
R5 is hydrogen;
R6 is hydrogen, halogen, alkyl, substituted alkyl, alkoxy, substituted alkoxy, hydroxyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, amino, substituted amino, alkylamino, substituted alkylamino, aldehyde, carboxyl, azido, nitro, nitroso, cyano, thioether, sulfoalkyl, carboxyalkyl, aminoalkyl, reporter molecule, reactive group, carrier molecule or solid support;
R7 is hydrogen, halogen, alkyl, substituted alkyl, alkoxy, substituted alkoxy, hydroxyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, amino, substituted amino, alkylamino, substituted alkylamino, aldehyde, carboxyl, azido, nitro, nitroso, cyano, thioether, sulfoalkyl, carboxyalkyl, aminoalkyl, reporter molecule, reactive group, carrier molecule or solid support;
R8 is hydrogen;
R9 is hydrogen;
R10 is hydrogen;
R11 is hydrogen; and
R12 is hydrogen.

### Claim 68 (depends on 67)

The kit according to claim 67, further comprising instructions for detecting the presence or absence of lead, mercury, nickel, lanthanum or cadmium ions in a sample.

### Claim 69 (depends on 67)

The kit according to claim 67, further comprising instructions for detecting the presence or absence of lead, mercury, nickel, lanthanum or cadmium ions in a sample using flow cytometry.

### Claim 70 (depends on 67)

The kit according to claim 67, further comprising one or more of a sample preparation reagent, a buffering agent, aqueous metal ion reporter molecule dilution buffer, an additional detection reagent, a metal ion calibration reagent, a positive control, a metal ion indicator other than for lead, mercury, nickel, lanthanum or cadmium ions, an antibody or fragment thereof or a reference dye standard.

### Claim 71 (depends on 67)

The kit according to claim 67, wherein R is H, a salt ion or CH2OC(O)CH3.

### Claim 72 (depends on 67)

The kit according to claim 67, wherein said R is hydrogen or a salt ion.

### Claim 73 (depends on 67)

The kit according to claim 67, wherein said R is CH2OC(O)CH3.

### Claim 74 (depends on 67)

The kit according to claim 67, wherein at least one of R3, R6 and R7 is a reporter molecule.

### Claim 75 (depends on 67)

The kit according to claim 67, wherein R3 or R6 is a reporter molecule.

### Claim 76 (depends on 67)

The kit according to claim 67, wherein the reporter molecule is a chromophore, fluorophore, fluorescent protein, phosphorescent dye or a tandem dye.

### Claim 77 (depends on 67)

The kit according to claim 67, wherein the reporter molecule is a xanthene, indole, cyanine, oxazole, dansyl, borapolyazaindacene, benzofuran, quinazolinone, benzazole, oxazine, pyrene, naphthalene, coumarin, biotin, enzyme substrate or fluorescent protein.

### Claim 78 (depends on 77)

The kit according to claim 77, wherein the xanthene is a fluorescein or derivative thereof, rhodamine or derivative thereof, rhodol or derivative thereof or rosamine or a derivative thereof

### Claim 79 (depends on 77)

The kit according to claim 77, wherein the reporter molecule is independently substituted by hydrogen, halogen, amino, substituted amino, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkoxy, sulfo, solid support, reactive group or carrier molecule.

### Claim 80 (depends on 77)

The kit according to claim 77, wherein the reporter molecule is independently substituted by a lipophilic group.

### Claim 81 (depends on 80)

The kit according to claim 80, wherein the lipophilic group is an AM or acetate ester.

### Claim 82 (depends on 67)

The kit according to claim 67, wherein the reporter group, reactive group, solid support and carrier molecule comprise a linker that is a single covalent bond, or a covalent linkage that is linear or branched, cyclic or heterocyclic, saturated or unsaturated, having 1-20 nonhydrogen atoms selected from the group consisting of C, N, P, O and S; and are composed of any combination of ether, thioether, amine, ester, carboxamide, sulfonamide, hydrazide bonds and aromatic or heteroaromatic bonds.

### Claim 83 (depends on 67)

The kit according to claim 67, wherein the reactive group is an acrylamide, an activated ester of a carboxylic acid, a carboxylic ester, an acyl azide, an acyl nitrile, an aldehyde, an alkyl halide, an anhydride, an aniline, an amine, an aryl halide, an azide, an aziridine, a boronate, a diazoalkane, a haloacetamide, a haloalkyl, a halotriazine, a hydrazine, an imido ester, an isocyanate, an isothiocyanate, a maleimide, a phosphoramidite, a reactive platinum complex, a silyl halide, a sulfonyl halide, a thiol or a photoactivatable group.

### Claim 84 (depends on 67)

The kit according to claim 67, wherein the reactive group is carboxylic acid, succinimidyl ester of a carboxylic acid, hydrazide, amine or a maleimide.

### Claim 85 (depends on 67)

The kit according to claim 67, wherein the carrier molecule is an amino acid, a peptide, a protein, a polysaccharide, a nucleoside, a nucleotide, an oligonucleotide, a nucleic acid polymer, a hapten, a psoralen, a drug, a hormone, a lipid, a lipid assembly, a synthetic polymer, a polymeric microparticle, a biological cell or a virus.

### Claim 86 (depends on 67)

The kit according to claim 67, wherein the carrier molecule is an antibody or fragment thereof, an avidin or streptavidin, a biotin, a blood component protein, a dextran, an enzyme, an enzyme inhibitor, a hormone, an IgG binding protein, a fluorescent protein, a growth factor, a lectin, a lipopolysaccharide, a microorganism, a metal binding protein, a metal chelating moiety, a non-biological microparticle, a peptide toxin, a phosphotidylserine-binding protein, a structural protein, a small-molecule drug, or a tyramide.

### Claim 87 (depends on 67)

The kit according to claim 67, wherein the solid support is a microfluidic chip, a silicon chip, a microscope slide, a microplate well, silica gels, polymeric membranes, particles, derivatized plastic films, glass beads, cotton, plastic beads, alumina gels, polysaccharides, polyvinylchloride, polypropylene, polyethylene, nylon, latex bead, magnetic bead, paramagnetic bead, or superparamagnetic bead.

### Claim 88 (depends on 67)

The kit according to claim 67, wherein the solid support is sepharose, poly(acrylate), polystyrene, poly(acrylamide), polyol, agarose, agar, cellulose, dextran, starch, FICOLL, heparin, glycogen, amylopectin, mannan, inulin, nitrocellulose, diazocellulose or starch.

### Claim 89 (depends on 67)

The kit according to claim 67, wherein the compound is
and a biologically compatible counterion.