# US 2014/0154675 A1 · Flash and Glow 1,2-Dioxetanes

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- Number: US 2014/0154675 A1
- Document ID: US20140154675A1
- Status: Published application
- Kind: A1
- Application number: 14/114,975
- Filed: 2012-04-30
- Published: 2014-06-05
- Earliest claimed: 2011-05-03
- Assignees: Life Technologies Corporation
- Inventors: Albana Mihali; Brooks Edwards; Zhixian Wang
- Web page: https://patentgrep.com/patent/US20140154675A1

## CPC

- C12Q 1/42: involving phosphatase
- C07D 321/00: Heterocyclic compounds containing rings having two oxygen atoms as the only ring hetero atoms, not provided for by groups C07D317/00 - C07D319/00
- C07D 407/12: linked by a chain containing hetero atoms as chain links
- G01N 33/573: for enzymes or isoenzymes
- H10P 14/3241: {being conductive materials}
- 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
- C07D: HETEROCYCLIC COMPOUNDS (macromolecular compounds C08)
- 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)
- H10P: GENERIC PROCESSES OR APPARATUS FOR THE MANUFACTURE OR TREATMENT OF DEVICES COVERED BY CLASS H10

## Abstract

Compounds having chemiluminescent flash and glow properties. Also disclosed are methods using the compounds to generate light, detect and/or quantify enzymes, antigens, and/or nucleic acids. Also disclosed are kits relating to these compounds.

## Claims

### Claim 1

A compound having the structure
wherein,
A and B are independently selected from the group consisting of straight chain alkyl comprising 1 to 20 carbon atoms, straight chain alkenyl comprising 2 to 20 carbon atoms, branched alkyl comprising 3 to 20 carbon atoms, branched alkenyl comprising 3 to 20 carbon atoms, cycloalkyl comprising 3 to 20 carbon atoms, cycloalkenyl comprising 3 to 20 carbon atoms, cycloheteroalkyl comprising 3 to 20 carbon atoms, cycloheteroalkenyl comprising 3 to 20 carbon atoms, polycycloalkyl comprising 4 to 60 carbon atoms, polycycloalkenyl comprising 4 to 60 carbon atoms, polycycloheteroalkyl comprising 4 to 60 carbon atoms and polycycloheteroalkenyl comprising 4 to 60 carbon atoms, any one of which can be unsubstituted or substituted with one or more electron-active groups, solubilizing groups, or light-enhancing groups, and
where A and B together form the cycloalkyl, cycloalkenyl, cycloheteroalkyl, cycloheteroalkenyl, polycycloalkenyl, polycycloheteroalkyl and polycycloheteroalkenyl, the formed group can be unsubstituted or substituted with one or more electron-active groups, solubilizing groups, or light-enhancing groups, or A and B together form the polycycloalkyl that is substituted with a halogen;
R1 is straight chain alkyl comprising 1 to 20 carbon atoms which may be optionally substituted with one or more halogen atoms or branched chain alkyl comprising 3 to 20 carbon atoms which may be optionally substituted with one or more halogen atoms;
R2 is an enzyme-cleavable group that comprises a bond cleavable by an enzyme moiety to yield an oxygen anion on T; and
R3 is hydrogen or an electron-donating group; with the proviso that when R1 is unsubstituted, R3 is an electron-donating group;
wherein, when

### Claim 2 (depends on 1)

The compound of claim 1, wherein at least one of A or B is

### Claim 3 (depends on 1)

The compound of claim 1, wherein A and B together is

### Claim 4 (depends on 1)

The compound of claim 1, wherein R1 is a straight chain alkyl comprising 1 to 5 carbon atoms which may be optionally substituted with one or more halogens atoms.

### Claim 5 (depends on 1)

The compound of claim 1, wherein R1 is a straight chain alkyl comprising 1 to 2 carbon atoms or straight chain trifluoalkyl comprising 1 to 2 carbon atoms.

### Claim 6 (depends on 5)

The compound of claim 5, wherein R1 is methyl or 1,1,1-trifluoroethyl.

### Claim 7 (depends on 1)

The compound of claim 1, wherein

### Claim 8 (depends on 1)

The compound of claim 1, wherein

### Claim 9 (depends on 1)

The compound of claim 1, wherein OR2 is phosphate, acetate, 1-phospho-2,3-diacylglyceride, adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, adenosine, α-D-galactoside, β-D-galactoside, α-D-glucoside, β-D-glucoside, α-D-mannoside, β-D-mannoside, β-fructofuranoside, β-D-glucuronide, or
wherein, B1, B2 and B3 are each independently H, a straight alkyl (branched or straight chain) comprising 1-4 carbon atoms, or a branched chain alkyl comprising 3-6 carbon atoms.

### Claim 10 (depends on 9)

The compound of claim 9, wherein R2 is

### Claim 11 (depends on 1)

The compound of claim 1, wherein R2 is E-L-Nuc-Z, wherein E is a group comprising an electrophilic atom, which atom upon the enzymatic cleavage of the Z group is attacked by the electron pair of the Nuc group and by anchimeric assistance releases the compound anion; L is a linking group;
Nuc is nucleophilic atom; and Z is an enzymatically cleavable group; wherein
E is carboxyl, carbonyl, methylene substituted by a leaving group, phosphate, carbonate, xanthate, sulfite, sulfonate, bisulfite or bisulfide;
L is selected from the group consisting of methylene or polymethylene containing 1 to 4 carbon atoms, —(CH2)m—O—(CH2)n, —(CH2)m—S—(CH2)n—, or —(CH2)m—NR6—(CH2)n—, wherein m and n are 0 to 3 and m+n is 2 or 3, wherein R6 is alkyl containing 1 to 10 carbon atoms and the linking group may be substituted by alkyl containing 1 to 24 carbon atoms, alkenyl containing 2 to 24 carbon atoms, alkyl containing 1 to 24 carbon atoms and mono- or di-substituted with acyloxy containing 1 to 24 carbon atoms, alkenyl containing 2 to 24 carbon atoms and mono- or disubstituted with acyloxy containing 1 to 24 carbon atoms, aryl containing 6 to 10 carbons, alkyl containing 1 to 24 carbon atoms and substituted with phenyl, hydroxyphenyl, indolyl, mercapto, alkylthio containing 1 to 4 carbon atoms, hydroxy, carboxy, amino, guanidino, imidazole or carbamyl, or alkenyl containing 2 to 24 carbon atoms and substituted with phenyl, hydroxyphenyl, indolyl, mercapto, alkylthio containing 1 to 4 carbon atoms, hydroxy, carboxy, amino, guanidino, imidazole, or carbamyl;
Nuc is an oxygen atom or sulfur atom; and
Z is phosphoryl, acetyl, 1-phospho-2,3-diacylglycerosyl, adenosine triphosphoryl, adenosine diphosphoryl, adenosine monophosphoryl, adenosyl, α-D-galactosyl, β-D-galactosyl, α-D-glucosyl, β-D-glucosyl, α-D-mannosyl, β-D-mannosyl, β-fructofuranosyl, β-D-glucosiduransyl, or
wherein, B1, B2 and B3 are each independently H, a straight alkyl comprising 1-4 carbon atoms, or a branched chain alkyl comprising 3-6 carbon atoms.

### Claim 12 (depends on 11)

The compound of claim 11, wherein Z is

### Claim 13 (depends on 1)

The compound of claim 1, wherein the electron-donating group is a straight chain alkyl comprising 1 to 20 carbon atoms, a branched alkyl comprising 3 to 20 carbon atoms, a straight chain alkoxy comprising 1 to 20 carbon atoms, or a branched alkoxy comprising 3 to 20 carbon atoms.

### Claim 14 (depends on 1)

The compound of claim 1, wherein the electron-donating group is a straight chain alkyl comprising 1 to 20 carbon atoms or straight chain alkoxy comprising 1 to 20 carbon atoms.

### Claim 15 (depends on 1)

The compound of claim 1, wherein the electron-donating group is a straight chain alkyl comprising 1 to 5 carbon atoms or straight chain alkoxy comprising 1 to 5 carbon atoms.

### Claim 16 (depends on 1)

The compound of claim 1, wherein the electron-donating group is selected from the group consisting of methyl, ethyl, propyl, butyl, methoxy, ethoxy, propyloxy and butyloxy.

### Claim 17 (depends on 1)

The compound of claim 1, wherein the cleavage of the bond cleavable by an enzyme moiety results in the generation of light at 25° C. which reaches a maximum in less than about 15 minutes.

### Claim 18 (depends on 17)

The compound of claim 17, wherein the generation of light reaches a maximum in less than about 10 minutes.

### Claim 19 (depends on 17)

The compound of claim 17, wherein the generation of light reaches a maximum in less than about 5 minutes.

### Claim 20 (depends on 1)

The compound of claim 1, wherein the cleavage of the bond cleavable by an enzyme moiety results in the generation of light at 37° C. which reaches a maximum in less than about 15 minutes.

### Claim 21 (depends on 20)

The compound of claim 20, wherein the generation of light reaches a maximum in less than about 10 minutes.

### Claim 22 (depends on 20)

The compound of claim 20, wherein the generation of light reaches a maximum in less than about 5 minutes.

### Claim 23 (depends on 1)

The compound of claim 1 which is

### Claim 24 (depends on 1)

A method for generating light, comprising the steps of:
(a) providing the compound of claim 1;
(b) providing an enzyme complex comprising an enzyme moiety which is capable of cleaving the compound;
(c) contacting the enzyme complex with the compound to form a reaction mixture; and,
(d) allowing the reaction mixture to generate light.

### Claim at position 25

-85. (canceled)

### Claim 86 (depends on 1)

An assay method for determining the presence and/or amount of an enzyme in a sample, comprising the steps of:
(a) providing the compound of claim 1;
(b) providing a sample suspected of comprising the enzyme which is capable of cleaving the compound so that it decomposes and generates light;
(c) contacting the sample with the compound to form a reaction mixture; and,
(d) detecting the light generated by the reaction mixture after addition of the compound, wherein the generation of light is indicative of the presence of the enzyme and the amount of light generated can be correlated to the amount of the enzyme present in the sample, and wherein the generation of light at 25° C. to 37° C. reaches a maximum in less than about 15 minutes.

### Claim 87 (depends on 1)

An assay method for determining the presence and/or amount of an antigen in a sample, comprising the steps of:
(a) providing the compound of claim 1;
(b) providing a sample suspected of comprising the antigen;
(c) providing an enzyme-linked antigen comprising the antigen and an enzyme capable of cleaving the compound so that it decomposes and generates light;
(d) providing a solid phase comprising an antibody capable of binding to the antigen;
(e) contacting the sample and enzyme-linked antigen with the solid phase to form an enzyme complex;
(f) contacting the enzyme complex with the compound to form a reaction mixture; and,
(g) detecting the light generated by the reaction mixture after addition of the compound, wherein the generation of light is indicative of the presence of the antigen and the amount of light generated can be correlated to the amount of the antigen present in the sample, and wherein the generation of light at 25° C. to 37° C. reaches a maximum in less than about 15 minutes.

### Claim 88 (depends on 1)

An assay method for determining the presence and/or amount of a nucleic acid in a sample, comprising the steps of:
(a) providing the compound of claim 1;
(b) providing a sample suspected of comprising the nucleic acid;
(c) immobilizing the nucleic acid to a solid phase;
(d) providing an enzyme-linked oligonucleotide comprising an oligonucleotide capable of hydridizing to the nucleic acid and an enzyme capable of cleaving the compound so that it decomposes and generates light;
(e) contacting the immobilized nucleic acid and enzyme-linked oligonucleotide to form an enzyme complex;
(f) contacting the enzyme complex with the compound to form a reaction mixture; and,
(g) detecting the light generated by the reaction mixture after addition of the compound, wherein the generation of light is indicative of the presence of the nucleic acid and the amount of light generated can be correlated to the amount of the nucleic acid present in the sample, and wherein the generation of light at 25° C. to 37° C. reaches a maximum in less than about 15 minutes.

### Claim 89 (depends on 1)

A kit for detecting the presence and/or amount of an analyte in a sample comprising:
(a) the compound of claim 1; and
(b) a buffer.

### Claim at position 30

-93. (canceled)