# US 8,785,652 B2 · Detection of hydrogen peroxide

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- Number: US 8,785,652 B2
- Document ID: US8785652B2
- Status: Granted
- Kind: B2
- Application number: 13/587,387
- Filed: 2012-08-16
- Published: 2013-02-21
- Granted: 2014-07-22
- Earliest claimed: 2011-08-16
- Assignees: Promega Corporation
- Applicants: Dieter Klaubert; John Shultz; James Unch; Michael P. Valley; Hui Wang; Wenhui Zhou
- Inventors: Dieter Klaubert; John Shultz; James Unch; Michael P. Valley; Hui Wang; Wenhui Zhou
- Web page: https://patentgrep.com/patent/US8785652B2

## CPC

- C07F 5/04: Esters of boric acids
- C07D 235/00: Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, condensed with other rings
- C07D 241/54: Nitrogen atoms
- C07D 277/64: with only hydrocarbon or substituted hydrocarbon radicals attached in position 2
- C07F 5/02: Boron compounds
- C12Q 1/66: involving luciferase
- G01N 33/5038: {involving detection of metabolites per se}
- G01N 2500/10: involving cells
- C07F: ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM (metal-containing porphyrins C07D487/22)
- C07D: HETEROCYCLIC COMPOUNDS (macromolecular compounds C08)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- 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)

## Abstract

The present invention provides compounds useful for detection of hydrogen peroxide and methods of using same.

## Claims

### Claim 1

A compound of formula
wherein
R1 is boronic acid or a borate ester;
each R4, R5 and R7 is independently selected from H, halo, methyl, and trifluoromethyl; and
L1 is a linker.

### Claim 2 (depends on 1)

The compound of claim 1, wherein
R1 is —B(OR6)2; and
each R6 is independently selected from H and C1-4 alkyl.

### Claim 3 (depends on 1)

The compound of claim 1, wherein
R1 is
and
each R12 and R13 is independently selected from H, C1-4 alkyl, CF3, phenyl or substituted phenyl; or R12 and R13 together can be an alkyl ring having from 3-7 carbons or can be replaced by a fused 6-membered aromatic ring.

### Claim 4 (depends on 1)

The compound of claim 1, wherein
R1 is
and
each R14, R15 and R16 is independently selected from H, C1-4 alkyl, CF3, phenyl and substituted phenyl; or both R15 together can form an alkyl ring having from 3-7 carbons; R14 and R15 together or R15 and R16 together can be an alkyl ring having from 3-7 carbon atoms or can be replaced by a 6-membered aromatic ring.

### Claim 5 (depends on 1)

The compound of claim 1,
wherein
L1 is
A is —C6(R10)4—, —O—C6(R10)4— or —(CR11═CR11)n—or —S—C6(R10)4— or —NR′—C6(R10)4 or a direct bond;
R′ is H or C1-4 alkyl;
each R3 is independently halo, H, C1-4 alkyl; C1-4 hydroxyalkyl, or C1-4 alkylcarboxylic acid;
each R10 is independently H, halo, CH3, OCH3, or NO2;
each R11 is independently H or CH3;
n is 1 or 2; and
X is a selected from —O—,

### Claim 6 (depends on 1)

A method of detecting hydrogen peroxide in a cell comprising:
(a) contacting cells with a compound according to claim 1;
(b) adding a luciferase reaction mixture to the contacted cells; and
(c) measuring bioluminescence thereby detecting the presence of hydrogen peroxide in the cell.

### Claim 7 (depends on 6)

The method of claim 6, wherein the luciferase reaction mixture comprises D-cysteine.

### Claim 8 (depends on 6)

The method of claim 6, wherein the cells are treated with a test compound or test condition prior to contacting with a compound according to Formula (I)
wherein
R1 is boronic acid or a borate ester;
each R4, R5 and R7 is independently selected from H, halo, methyl, and trifluoromethyl; and
L1 is a linker.

### Claim 9 (depends on 8)

The method of claim 8, wherein the test condition comprises a change in temperature, oxygen tension or ionic strength or an osmotic change.

### Claim 10 (depends on 6)

The method of claim 6, wherein the cell is in a sample.

### Claim 11 (depends on 10)

The method of claim 10, wherein the sample is obtained from an animal.

### Claim 12 (depends on 1)

A method of detecting hydrogen peroxide in a cell comprising:
(a) contacting cells with a compound according to claim 1 in a first reaction vessel to form an incubation mixture;
(b) transferring at least a portion of the incubation mixture to a second reaction vessel;
(c) adding a luciferase reaction mixture to the second reaction vessel; and
(d) measuring bioluminescence thereby detecting the presence of hydrogen peroxide in the cell.

### Claim 13 (depends on 12)

The method of claim 12, wherein the luciferase reaction mixture comprises D-cysteine.

### Claim 14 (depends on 12)

The method of claims 12, wherein the cells are treated with a test compound or test condition prior to contacting with a compound according to Formula (I)
wherein
R1 is boronic acid or a borate ester;
each R4, R5 and R7 is independently selected from H, halo, methyl, and trifluoromethyl; and
L1 is a linker.

### Claim 15 (depends on 14)

The method of claim 14, wherein the test condition comprises a change in temperature, oxygen tension or ionic strength or an osmotic change.

### Claim 16 (depends on 15)

The method of claim 15, wherein the cell is obtained from an animal.

### Claim 17 (depends on 1)

A method of detecting hydrogen peroxide in a sample comprising:
(a) contacting a sample with a compound according to claim 1 to form a first mixture;
b) contacting at least a portion of the first mixture with a luciferase reaction mixture and D-cysteine and
c) measuring bioluminescence thereby detecting the presence of hydrogen peroxide in the sample.

### Claim 18 (depends on 17)

The method of claim 17, wherein the D-cysteine is part of the reaction mixture for a luciferase reaction mixture.

### Claim 19 (depends on 17)

The method of claim 17, wherein the sample is treated with a test compound or test condition prior to contacting with a compound according to Formula (I)
wherein
R1 is boronic acid or a borate ester;
each R4, R5 and R7 is independently selected from H, halo, methyl, and trifluoromethyl; and
L1 is a linker.

### Claim 20 (depends on 19)

The method of claim 19, wherein the test condition comprises a change in temperature, oxygen tension or ionic strength or an osmotic change.

### Claim 21 (depends on 17)

The method of claim 17, wherein the sample comprises a cell or cell medium.

### Claim 22 (depends on 21)

The method of claim 21, wherein the sample comprises a cell or physiological sample from an animal.

### Claim 23 (depends on 1)

A method of determining the effect of a test compound or test condition on the presence or amount of hydrogen peroxide in a sample comprising:
a) treating a sample with a test compound or test condition,
b) contacting the sample from a) with a compound according to claim 1;
c) adding a luciferase reaction mixture to the sample;
d) measuring bioluminescence.

### Claim 24 (depends on 23)

The method of claim 23, wherein the sample comprises a cell or cell medium.

### Claim 25 (depends on 23)

The method of claim 23, wherein the sample comprises a cell or physiological sample from an animal.

### Claim 26 (depends on 23)

The method of claim 23, wherein the test condition comprises a change in temperature, oxygen tension, or ionic strength or an osmotic change.

### Claim 27 (depends on 1)

A kit comprising a compound according to claim 1.