# US 8,540,521 B2 · Benzoxazole-based fluorescent metal ion indicators

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- Number: US 8,540,521 B2
- Document ID: US8540521B2
- Status: Granted
- Kind: B2
- Application number: 13/142,584
- Filed: 2009-12-29
- Published: 2011-12-15
- Granted: 2013-09-24
- Earliest claimed: 2008-12-29
- Assignees: Life Technologies Corporation
- Applicants: Kyle Gee; Vladimir Martin
- Inventors: Kyle Gee; Vladimir Martin
- Web page: https://patentgrep.com/patent/US8540521B2

## CPC

- G01N 21/75: Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated (systems in which material is burnt in a flame or plasma G01N21/72, G01N21/73)
- C07D 263/56: with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached in position 2
- C07D 263/57: Aryl or substituted aryl radicals
- C07D 401/04: directly linked by a ring-member-to-ring-member bond
- C07D 413/04: directly linked by a ring-member-to-ring-member bond
- C07D 417/04: directly linked by a ring-member-to-ring-member bond
- C07D 471/06: Peri-condensed systems
- C07D 491/16: Peri-condensed systems
- C09B 57/00: Other synthetic dyes of known constitution
- C09B 57/02: Coumarine dyes
- C09K 11/06: containing organic luminescent materials
- G01N 33/582: {with fluorescent label}
- G01N 33/84: involving inorganic compounds or pH
- C09K 2211/1007: Non-condensed systems
- C09K 2211/1014: bridged by heteroatoms, e.g. N, P, Si or B
- C09K 2211/1029: containing one nitrogen atom as the heteroatom
- C09K 2211/1033: with oxygen
- C09K 2211/1037: with sulfur
- C09K 2211/1048: with oxygen
- C09K 2211/1088: containing oxygen as the only heteroatom
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- 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)
- C09K: MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE

## Abstract

Disclosed are benzoxazole-based compounds, kits, and methods of producing and using the described compounds in fluorescence-based detection of analytes (e.g., metal ions). Also disclosed are uses of benzoxazole-based compounds as ratiometric metal ion indicators.

## Claims

### Claim 1

A compound of the formula:
or a salt thereof,
wherein M is —NCH2-Py or —N(CH2COOR5)2, wherein R5 is H, an alkyl having 1-6 carbons, —CH2OCOCH3, or a counterion;
Y is —CH3, —CH2COOH, —CH2COOPy, or
X is —N(CH2COOR5)2, —NCH2Py, —OCH2Py, or —OCH2COOR5;
R7, R8, R9, and R10 are independently selected from the group consisting of an alkyl having 1-6 carbons, halogen, amino, nitro, cyano, trifluoromethyl, sulfo, and sulfonamide;
R1 and R2 are independently H, an alkyl having 1-6 carbons, halogen, or sulfo;
R4 is a 5 or 6-membered aromatic ring or a fused ring system comprising at least one 6-membered aromatic ring;
L is an alkylene having 2-6 carbons; and
p is 0 or 1.

### Claim 2

A compound of the formula:
or a salt thereof,
wherein M is —NR3—, wherein R3 is —CH2-Py, —CH2Py, —CH2CH2OR5, or —CH2COOR5, wherein R5 is H, an alkyl having 1-6 carbons, —CH2OCOCH3, or a counterion;
Z is
wherein n is 1, 2, or 3;
R6 is —CH2COOR6, —CH2CH2OR5, —CH2Py, or, when taken in combination with M, forms a structure
wherein m is 1, 2, or 3;
R7, R8, R9, and R10 are independently selected from the group consisting of an alkyl having 1-6 carbons, halogen, amino, nitro, cyano, trifluoromethyl, sulfo, and sulfonamide;
R1 and R2 are independently H, an alkyl having 1-6 carbons, halogen, or sulfo;
R4 is a 5 or 6-membered aromatic ring or a fused ring system comprising at least one 6-membered aromatic ring;
L is an alkylene having 2-6 carbons; and
p is 0 or 1.

### Claim 3 (depends on 1, 2)

The compound of claim 1 or 2 wherein R4 is substituted with an alkyl having 1-6 carbons, an alkoxy having 1-6 carbons, —OH, —COOH, COO−, oxygen, halogen, —SO2NH2, or —N—R10R11, wherein R10 and R11 are independently H or an alkyl group having 1-6 carbon atoms.

### Claim 4 (depends on 1, 2)

The compound of claim 1 or 2 wherein R10 and R11 are independently methyl or ethyl.

### Claim 5 (depends on 1, 2)

The compound of claim 1 or 2 wherein R4 comprises a heteroatom selected from O, N, and S.

### Claim 6 (depends on 1, 2)

The compound of claim 1 or 2 wherein R4 is phenyl or a substituted phenyl moiety.

### Claim 7 (depends on 1, 2)

The compound of claim 1 or 2 wherein R4 is selected from p-H2NSO2—C6H4—, p-KOOC—C6H4—, p-CH3O—C6H4—, p-(CH3)2N—C6H4—,
wherein K is H or a counterion.

### Claim 8 (depends on 1, 2)

The compound of claim 1 or 2 wherein R4 is selected from the group consisting of

### Claim 9 (depends on 1, 2)

The compound of claim 1 or 2 wherein R4 is

### Claim 10 (depends on 1, 2)

The compound of claim 1 or 2 wherein R4 is selected from Ph-CH═CH—, p-CH3O—C6H4—CH═CH—,

### Claim 11 (depends on 1, 2)

The compound of claim 1 or 2 wherein R4 is selected from Ph-CH═CH—, p-CH3O—C6H4—CH═CH—, p-(CH3)2N—C6H4—CH═CH—, and

### Claim 12 (depends on 1, 2)

The compound of claim 1 or 2 wherein R4 is

### Claim 13 (depends on 1, 2)

The compound of claim 1 or 2 wherein M is —N(CH2COOH)2 or —N(CH2OCOCH3)2.

### Claim 14 (depends on 2)

The compound of claim 2 having the formula

### Claim 15

A compound of the formula
or a salt thereof,
wherein M is —NCH2-Py, —N(CH2COOR5)2, wherein R5 is H, an alkyl having 1-6 carbons, —CH2OCOCH3, or a counterion;
X is —N(CH2COOR5)2, —NCH2Py, —OCH2Py, or —OCH2COOR5;
R7, R8, R9, and R10 are independently selected from the group consisting of an alkyl having 1-6 carbons, halogen, amino, nitro, cyano, trifluoromethyl, sulfo, and sulfonamide;
R1 and R2 are independently H, an alkyl having 1-6 carbons, halogen, or sulfo;
R4 is a 5 or 6-membered aromatic ring or a fused ring system comprising at least one 6-membered aromatic ring;
L is an alkylene having 2-6 carbons; and
p is 0 or 1.

### Claim 16 (depends on 1, 2)

The compound of claim 1 or 2, further comprising a counterion selected from Na+, Li+, K+, Tl+, trialkylammonium and tetraalkylammonium.

### Claim 17 (depends on 1, 2)

A complex comprising a compound of claim 1 or 2 and a polycationic metal ion.

### Claim 18 (depends on 17)

The complex of claim 17 wherein the polycationic metal ion is a divalent metal ion.

### Claim 19 (depends on 17)

The complex of claim 17 wherein the polycationic metal ion is Ca2+, Zn2+, Mg2+, Pb2+, Hg2+, or Pb2+.

### Claim 20 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16)

A method of measuring the concentration of a polycationic metal ion in a sample, comprising:
a) combining a compound of any one of claims 1-16 with a sample in an amount sufficient to generate a detectable fluorescent response to the metal ion;
b) illuminating the sample to generate a fluorescence excitation or emission response; and
c) observing the absorbance or emission response.

### Claim 21 (depends on 20)

The method of claim 20 wherein a fluorometer, fluorescence microscope, laser scanner, flow cytometer is used to observe the absorbance or emission response.

### Claim 22 (depends on 20)

The method of claim 20 wherein the polycationic metal ion is Ca2+.

### Claim 23 (depends on 20)

The method of claim 20 wherein the sample comprises living cells or biological fluids.

### Claim 24 (depends on 20)

The method of claim 20, further comprising quantifying the fluorescence excitation or emission response.

### Claim 25 (depends on 20)

The method of claim 20 wherein the compound exhibits a change in emission wavelength maximum upon binding to the polycationic metal ion.

### Claim 26 (depends on 20)

The method of claim 20 wherein the compound exhibits a change in excitation wavelength maximum upon binding to the polycationic metal ion.

### Claim 27 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16)

A kit for measuring the concentration of a polycationic metal ion in a sample comprising a compound of any one of claims 1-16.

### Claim 28 (depends on 27)

The kit of claim 27, further comprising a component selected from a salt solution having biological osmolarity, a buffer, and a metal ion solution.