US2018299456A1PendingUtilityA1
In Situ Chemiluminescent Substrates and Assays
Est. expiryJul 8, 2030(~3.9 yrs left)· nominal 20-yr term from priority
G01N 33/581C07D 321/00C07F 9/12C07F 9/4056C12Q 1/6816C07F 9/65583C12Q 1/34C07F 9/6541C12Q 1/42C07H 15/203C07F 9/65512
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Claims
Abstract
Methods for generating a chemiluminescent enzyme substrate in situ, in aqueous or other assay conditions. Also disclosed are methods to use the substrates to generate light, detect and/or quantify enzymes, antigens, and/or nucleic acids. Kits relating to these methods are also disclosed.
Claims
exact text as granted — not AI-modifiedWe claim:
1 . A method for generating light, comprising the steps of:
(a) providing an oxidant; (b) providing an enol ether having the structure:
wherein,
A and B are independently selected from the group consisting of straight chain alkyl containing 1 to 20 carbon atoms, straight chain alkenyl containing 2 to 20 carbon atoms, branched alkyl containing 3 to 20 carbon atoms, branched alkenyl containing 3 to 20 carbon atoms, cycloalkyl containing 3 to 20 carbon atoms, cycloalkenyl containing 3 to 20 carbon atoms, cycloheteroalkyl containing 3 to 20 carbon atoms, cycloheteroalkenyl containing 3 to 20 carbon atoms, polycycloalkyl containing 4 to 60 carbon atoms, polycycloalkenyl containing 4 to 60 carbon atoms, polycycloheteroalkyl containing 4 to 60 carbon atoms and polycycloheteroalkenyl containing 4 to 60 carbon atoms, any 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, polycycloalkyl or polycycloalkenyl, one of the carbon atoms of the cycloalkyl, cycloalkenyl, polycycloalkyl, or polycycloalkenyl is one of two carbon atoms forming the double bond of the enol ether,
R 1 is alkyl containing 1 to 20 carbon atoms, aryl containing 6 to 14 carbon atoms, aralkyl containing 7 to 15 carbon atoms, heteroaryl containing 4 to 20 carbon atoms, or heteroaralkyl containing 5 to 20 carbons,
T is an aryl or heteroaryl ring capable of emitting light, and
R 2 is an enzyme-cleavable group that contains a bond cleavable by an enzyme moiety to yield an oxygen anion on T;
(c) combining an aqueous solution, the oxidant, and the enol ether to form an aqueous solution comprising a 1,2-dioxetane enzyme substrate;
(d) providing an enzyme complex comprising an enzyme moiety which is capable of cleaving the 1,2-dioxetane enzyme substrate;
(e) contacting the enzyme complex with the aqueous solution comprising the 1,2-dioxetane enzyme substrate to form a reaction mixture; and,
(f) allowing the reaction mixture to generate light.
2 . The method of claim 1 , wherein the oxidant is selected from hydrogen peroxide, sodium molybdate, hydrogen peroxide and sodium molybdate, hypochlorite, hypochlorite and hydrogen peroxide, aryl endoperoxide, calcium peroxide peroxyhydrate, and combinations thereof.
3 . The method of claim 2 , wherein the oxidant is hydrogen peroxide, and hydrogen peroxide and sodium molybdate.
4 . The method of claim 1 , wherein at least one of A or B is
5 . The method of claim 1 , wherein A and B together is
6 . The method of claim 1 , wherein R 1 is alkyl containing 1 to 2 carbon atoms or trifluoalkyl containing 1 to 2 carbon atoms.
7 . The method of claim 1 , wherein T is
wherein,
R 3 , R 4 , and R 5 , are independently selected from the group consisting of H, F, Cl, Br, I, cyano, nitro, sulfonate, sulfate, trifluomethyl, trifluroethyl, straight chain alkyl containing 1 to 20 carbon atoms, branched alkyl containing 3 to 20 carbon atoms, straight chain alkenyl containing 2 to 20 carbon atoms, branched alkenyl containing 3 to 20 carbon atoms, cycloalkyl containing 3 to 20 carbon atoms, cycloalkenyl containing 3 to 20 carbon atoms, cycloheteroalkyl containing 3 to 20 carbon atoms, cycloheteroalkenyl containing 3 to 20 carbon atoms, polycycloalkyl containing 4 to 60 carbon atoms, polycycloalkenyl containing 4 to 60 carbon atoms, polycycloheteroalkyl containing 4 to 60 carbon atoms, polycycloheteroalkenyl containing 4 to 60 carbon atoms, alkoxy containing 1 to 20 carbon atoms, aryl containing 6 to 14 carbon atoms, aryloxy containing 6 to 14 carbon atoms, ester containing 2 to 21 carbon atoms, trialkylammonium containing 3 to 30 carbon atoms, trialkylphosphonium containing 3 to 30 carbon atoms, alkylamido containing 2 to 21 carbon atoms, arylamido containing 7 to 15 carbon atoms, alkylcarbamoyl containing 2 to 21 carbon atoms, arylcarbamoyl containing 7 to 15 carbon atoms, alkylsulfonamido containing 1 to 20 carbon atoms, arylsulfonamido containing 6 to 14 carbon atoms, trialkylsilyl containing 3 to 60 carbon atoms, triarylsilyl containing 18 to 42 carbon atoms, alkylarylsilyl containing 7 to 32 carbon atoms, alkylamidosulfonyl containing 1 to 20 carbon atoms, arylamidosulfonyl containing 6 to 14 carbon atoms, alkylsulfonyl containing 1 to 20 carbon atoms, arylsulfonyl containing 6 to 14 carbon atoms, alkylthio containing 2 to 20 carbon atoms and arylthio containing 6 to 14 carbon atoms, and
X is a sulfur atom, oxygen atom, or nitrogen atom.
8 . The method of claim 1 , wherein OR 2 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, B 1 , B 2 and B 3 are each independently H or an alkyl (branched or straight chain) of 1-4 carbon atoms.
9 . The method of claim 8 , wherein R 2 is
10 . The method of claim 1 , wherein R 2 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 1,2-dioxetane enzyme substrate anion; L is a linking group; Nuc is nucleophic 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, —(CH 2 ) m —O—(CH 2 ) n , —(CH 2 ) m —S—(CH 2 ) n —, or —(CH 2 ) m —NR 6 —(CH 2 ) n —, wherein m and n are 0 to 3 and m+n is 2 or 3, wherein
R 6 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, B 1 , B 2 and B 3 are each independently H or an alkyl (branched or straight chain) of 1-4 carbon atoms.
11 . The method of claim 10 , wherein Z is
12 . The method of claim 1 , wherein the enzyme moiety comprises a hydrolytic enzyme.
13 . The method of claim 12 , wherein the hydrolyic enzyme is alkaline phosphatase, β-galactosidase, β-glucosidase, β-glucuronidase, or neuraminidase.
14 . The method of claim 13 , wherein the enzyme moiety is an enzyme.
15 . The method of claim 14 , further comprising the step of detecting the light emitted from the reaction mixture after addition of the aqueous solution of the 1,2-dioxetane enzyme substrate, wherein the emission of light is indicative of the presence of the enzyme, and the amount of light emitted can be correlated to the amount of the enzyme present in the sample.
16 . The method of claim 13 , wherein the enzyme moiety is an enzyme-linked antibody comprising a first antibody capable of binding to an antigen and an enzyme capable of cleaving the 1,2-dioxetane enzyme substrate so that the substrate decomposes and generates light.
17 . The method of claim 16 , wherein the first antibody is covalently or non-covalently linked to the enzyme.
18 . The method of claim 17 , wherein the first antibody is covalently linked to a label and the enzyme is covalently linked to a molecule capable of non-covalent binding to the label.
19 . The method of claim 18 , wherein the label is biotin, or a biotin derivative, and the molecule is avidin or strepavidin.
20 . The method of claim 18 , wherein the label is a hapten and the molecule is an antibody capable of binding to the hapten.
21 . The method of claim 16 , further comprising the steps of:
(a) providing a sample suspected of comprising an antigen; (b) providing a solid phase comprising a second antibody capable of binding to the antigen; (c) contacting the sample and enzyme-linked antibody with the solid phase to form the enzyme complex; and, (d) detecting the light emitted from the reaction mixture after addition of the aqueous solution of the 1,2-dioxetane enzyme substrate, wherein the emission of light is indicative of the presence of the antigen, and the amount of light emitted can be correlated to the amount of the antigen present in the sample.
22 . The method of claim 21 , further comprising the step of removing any unbound enzyme-linked antibody from the enzyme complex.
23 . The method of claim 13 , wherein the enzyme moiety is an enzyme-linked antigen comprising an antigen and an enzyme capable of cleaving the 1,2-dioxetane enzyme substrate so that the substrate decomposes and generates light.
24 . The method of claim 23 , wherein the antigen is covalently or non-covalently linked to the enzyme.
25 . The method of claim 24 , wherein antigen is covalently linked to a label and the enzyme is covalently linked to a molecule capable of non-covalent binding to the label.
26 . The method of claim 25 , wherein the label is biotin, or a biotin derivative, and the molecule is avidin or strepavidin.
27 . The method of claim 25 , wherein the label is a hapten and the molecule is an antibody capable of binding to the hapten.
28 . The method of claim 23 , further comprising the steps of:
(a) providing a sample suspected of comprising an antigen; (b) providing a solid phase comprising an antibody capable of binding to the antigen; (c) contacting the sample and enzyme-linked antigen with the solid phase to form an enzyme complex; and, (d) detecting the light emitted from the reaction mixture after addition of the aqueous solution of the 1,2-dioxetane enzyme substrate, wherein the amount of light emitted can be correlated to the amount of the antigen present in the sample.
29 . The method of claim 28 , further comprising the step of removing any unbound enzyme-linked antigen from the enzyme complex.
30 . The method of claim 13 , wherein the enzyme moiety is an enzyme-linked oligonucleotide comprising an oligonucleotide capable of hydridizing to a nucleic acid and an enzyme capable of cleaving the 1,2-dioxetane enzyme substrate so that the substrate decomposes and generates light.
31 . The method of claim 30 , wherein the oligonucleotide is covalently or non-covalently linked to the enzyme.
32 . The method of claim 31 , wherein oligonucleotide is covalently linked to a label and the enzyme is covalently linked to a molecule capable of non-covalent binding to the label.
33 . The method of claim 32 , wherein the label is biotin, or a biotin derivative, and the molecule is avidin or strepavidin.
34 . The method of claim 32 , wherein the label is a hapten and the molecule is an antibody capable of binding to the hapten.
35 . The method of claim 30 , further comprising the steps of:
(a) providing a sample suspected of comprising a nucleic acid; (b) immobilizing the nucleic acid to a solid phase, (c) contacting the immobilzed nucleic acid and the enzyme-linked oligonucleotide to form an enzyme complex; and, (d) detecting the light emitted from the reaction mixture after addition of the aqueous solution of the 1,2-dioxetane enzyme substrate, wherein the emission of light is indicative of the presence of the nucleic acid, and the amount of light emitted can be correlated to the amount of the nucleic acid present in the sample.
36 . The method of claim 35 , further comprising the step of removing any unbound enzyme-linked oligonucleotide from the enzyme complex.
37 . The method of claim 1 , wherein the reaction mixture further comprises an enhancer.
38 . The method of claim 37 , wherein the enhancer comprises a polymeric quaternary ammonium salt, polymeric quaternary phosphonium salt, or a combination thereof.
39 . The method of claim 38 , wherein the enhancer further comprises an acceptor dye.
40 . The method of claim 39 , wherein the acceptor dye is fluorescein.
41 . The method of claim 38 , wherein the polymeric quaternary ammonium salt is poly(vinylbenzyltrimethylammonium chloride), poly[vinylbenzyl(benzyldimethylammonium chloride)], poly[vinyl(benzyltributylammonium chloride)], poly[vinyl(benzyltripentylammonium chloride)], or a combination thereof.
42 . The method of claim 38 , wherein the polymeric quaternary phosphonium salt is poly(vinylbenzyltrimethylphosphonium chloride), poly(vinylbenzyltributylphosphonium chloride), poly(vinylbenzyltrioctylphosphonium chloride), a copolymer comprising poly(vinylbenzyltributylphosphonium chloride) and poly(vinylbenzyltrioctylphosphonium chloride), or a combination thereof.
43 . The method of claim 1 , wherein the enol ether is
44 . An assay method for determining the presence or amount of an enzyme in a sample, comprising the steps of:
(a) providing an oxidant; (b) providing an enol ether having the structure:
wherein,
A and B are independently selected from the group consisting of straight chain alkyl containing 1 to 20 carbon atoms, straight chain alkenyl containing 2 to 20 carbon atoms, branched alkyl containing 3 to 20 carbon atoms, branched alkenyl containing 3 to 20 carbon atoms, cycloalkyl containing 3 to 20 carbon atoms, cycloalkenyl containing 3 to 20 carbon atoms, cycloheteroalkyl containing 3 to 20 carbon atoms, cycloheteroalkenyl containing 3 to 20 carbon atoms, polycycloalkyl containing 4 to 60 carbon atoms, polycycloalkenyl containing 4 to 60 carbon atoms, polycycloheteroalkyl containing 4 to 60 carbon atoms and polycycloheteroalkenyl containing 4 to 60 carbon atoms, any 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, polycycloalkyl or polycycloalkenyl, one of the carbon atoms of the cycloalkyl, cycloalkenyl, polycycloalkyl or polycycloalkenyl is one of two carbon atoms forming the double bond of the enol ether,
R 1 is alkyl containing 1 to 20 carbon atoms, aryl containing 6 to 14 carbon atoms, aralkyl containing 7 to 15 carbon atoms, heteroaryl containing 4 to 20 carbon atoms, or heteroaralkyl containing 5 to 20 carbons,
T is an aryl or heteroaryl ring capable of emitting light, and
R 2 is an enzyme-cleavable group that contains a bond cleavable by an enzyme moiety to yield an oxygen anion on T;
(c) combining an aqueous solution, the oxidant, and the enol ether to form an aqueous solution comprising a 1,2-dioxetane enzyme substrate;
(d) providing a sample suspected of comprising the enzyme which is capable of cleaving the 1,2-dioxetane enzyme substrate so that the substrate decomposes and generates light;
(e) contacting the sample with the aqueous solution comprising the 1,2-dioxetane enzyme substrate to form a reaction mixture; and,
(f) detecting the light emitted from the reaction mixture after addition of the aqueous solution of the 1,2-dioxetane enzyme substrate, wherein the emission of light is indicative of the presence of the enzyme, and the amount of light emitted can be correlated to the amount of the enzyme present in the sample.
45 . An assay method for determining the presence or amount of an antigen in a sample, comprising the steps of:
(a) providing an oxidant; (b) providing an enol ether having the structure:
wherein,
A and B are independently selected from the group consisting of straight chain alkyl containing 1 to 20 carbon atoms, straight chain alkenyl containing 2 to 20 carbon atoms, branched alkyl containing 3 to 20 carbon atoms, branched alkenyl containing 3 to 20 carbon atoms, cycloalkyl containing 3 to 20 carbon atoms, cycloalkenyl containing 3 to 20 carbon atoms, cycloheteroalkyl containing 3 to 20 carbon atoms, cycloheteroalkenyl containing 3 to 20 carbon atoms, polycycloalkyl containing 4 to 60 carbon atoms, polycycloalkenyl containing 4 to 60 carbon atoms, polycycloheteroalkyl containing 4 to 60 carbon atoms and polycycloheteroalkenyl containing 4 to 60 carbon atoms, any 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, polycycloalkyl or polycycloalkenyl, one of the carbon atoms of the cycloalkyl, cycloalkenyl, polycycloalkyl, or polycycloalkenyl is one of two carbon atoms forming the double bond of the enol ether,
R 1 is alkyl containing 1 to 20 carbon atoms, aryl containing 6 to 14 carbon atoms, aralkyl containing 7 to 15 carbon atoms, heteroaryl containing 4 to 20 carbon atoms, or heteroaralkyl containing 5 to 20 carbons,
T is an aryl or heteroaryl ring capable of emitting light, and
R 2 is an enzyme-cleavable group that contains a bond cleavable by an enzyme moiety to yield an oxygen anion on T;
(c) combining an aqueous solution, the oxidant, and the enol ether to form an aqueous solution comprising a 1,2-dioxetane enzyme substrate;
(d) providing a sample suspected of comprising the antigen;
(e) providing an enzyme-linked antibody comprising a first antibody capable of binding to the antigen and an enzyme capable of cleaving the 1,2-dioxetane enzyme substrate so that the substrate decomposes and generates light;
(f) providing a solid phase comprising a second antibody capable of binding to the antigen;
(g) contacting the sample and enzyme-linked antibody with the solid phase to form an enzyme complex;
(h) contacting the enzyme complex with the aqueous solution comprising the 1,2-dioxetane enzyme substrate to form a reaction mixture; and,
(i) detecting the light emitted from the reaction mixture after addition of the aqueous solution of the 1,2-dioxetane enzyme substrate, wherein the emission of light is indicative of the presence of the antigen, and the amount of light emitted can be correlated to the amount of the antigen present in the sample.
46 . An assay method for determining the presence or amount of an antigen in a sample, comprising the steps of:
(a) providing an oxidant; (b) providing an enol ether having the structure:
wherein,
A and B are independently selected from the group consisting of straight chain alkyl containing 1 to 20 carbon atoms, straight chain alkenyl containing 2 to 20 carbon atoms, branched alkyl containing 3 to 20 carbon atoms, branched alkenyl containing 3 to 20 carbon atoms, cycloalkyl containing 3 to 20 carbon atoms, cycloalkenyl containing 3 to 20 carbon atoms, cycloheteroalkyl containing 3 to 20 carbon atoms, cycloheteroalkenyl containing 3 to 20 carbon atoms, polycycloalkyl containing 4 to 60 carbon atoms, polycycloalkenyl containing 4 to 60 carbon atoms, polycycloheteroalkyl containing 4 to 60 carbon atoms and polycycloheteroalkenyl containing 4 to 60 carbon atoms, any 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, polycycloalkyl or polycycloalkenyl, one of the carbon atoms of the cycloalkyl, cycloalkenyl, polycycloalkyl, or polycycloalkenyl is one of two carbon atoms forming the double bond of the enol ether,
R 1 is alkyl containing 1 to 20 carbon atoms, aryl containing 6 to 14 carbon atoms, aralkyl containing 7 to 15 carbon atoms, heteroaryl containing 4 to 20 carbon atoms, or heteroaralkyl containing 5 to 20 carbons,
T is an aryl or heteroaryl ring capable of emitting light, and
R 2 is an enzyme-cleavable group that contains a bond cleavable by an enzyme moiety to yield an oxygen anion on T;
(c) combining an aqueous solution, the oxidant and the enol ether to form an aqueous solution comprising a 1,2-dioxetane enzyme substrate;
(d) providing a sample suspected of comprising the antigen;
(e) providing an enzyme-linked antigen comprising the antigen and an enzyme capable of cleaving the 1,2-dioxetane enzyme substrate so that the substrate decomposes and generates light;
(f) providing a solid phase comprising an antibody capable of binding to the antigen;
(g) contacting the sample and enzyme-linked antigen with the solid phase to form an enzyme complex;
(h) contacting the enzyme complex with the aqueous solution comprising the 1,2-dioxetane enzyme substrate to form a reaction mixture; and,
(i) detecting the light emitted from the reaction mixture after addition of the aqueous solution of the 1,2-dioxetane enzyme substrate, wherein the amount of light emitted can be correlated to the amount of the antigen present in the sample.
47 . An assay method for determining the presence and/or amount of a nucleic acid in a sample, comprising the steps of:
(a) providing an oxidant; (b) providing an enol ether having the structure:
wherein,
A and B are independently selected from the group consisting of straight chain alkyl containing 1 to 20 carbon atoms, straight chain alkenyl containing 2 to 20 carbon atoms, branched alkyl containing 3 to 20 carbon atoms, branched alkenyl containing 3 to 20 carbon atoms, cycloalkyl containing 3 to 20 carbon atoms, cycloalkenyl containing 3 to 20 carbon atoms, cycloheteroalkyl containing 3 to 20 carbon atoms, cycloheteroalkenyl containing 3 to 20 carbon atoms, polycycloalkyl containing 4 to 60 carbon atoms, polycycloalkenyl containing 4 to 60 carbon atoms, polycycloheteroalkyl containing 4 to 60 carbon atoms and polycycloheteroalkenyl containing 4 to 60 carbon atoms, any 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, polycycloalkyl or polycycloalkenyl, one of the carbon atoms of the cycloalkyl, cycloalkenyl, polycycloalkyl, or polycycloalkenyl is one of two carbon atoms forming the double bond of the enol ether,
R 1 is alkyl containing 1 to 20 carbon atoms, aryl containing 6 to 14 carbon atoms, aralkyl containing 7 to 15 carbon atoms, heteroaryl containing 4 to 20 carbon atoms, or heteroaralkyl containing 5 to 20 carbons,
T is an aryl or heteroaryl ring capable of emitting light, and
R 2 is an enzyme-cleavable group that contains a bond cleavable by an enzyme moiety to yield an oxygen anion on T;
(c) combining an aqueous solution, the oxidant, and the enol ether to form an aqueous solution comprising a 1,2-dioxetane enzyme substrate;
(d) providing a sample suspected of comprising the nucleic acid;
(e) immobilizing the nucleic acid to a solid phase;
(f) providing an enzyme-linked oligonucleotide comprising an oligonucleotide capable of hydridizing to the nucleic acid and an enzyme capable of cleaving the 1,2-dioxetane enzyme substrate so that the substrate decomposes and generates light;
(g) contacting the immobilized and enzyme-linked oligonucleotide to form an enzyme complex;
(h) contacting the enzyme complex with the aqueous solution comprising the 1,2-dioxetane enzyme substrate to form a reaction mixture; and,
(i) detecting the light emitted from the reaction mixture after addition of the aqueous solution of the 1,2-dioxetane enzyme substrate, wherein the emission of light is indicative of the presence of the nucleic acid, and the amount of light emitted can be correlated to the amount of the nucleic acid present in the sample.
48 . A kit for detecting the presence or amount of an analyte in a sample comprising:
(a) an oxidant, and (b) an enol ether having the structure:
wherein,
A and B are independently selected from the group consisting of straight chain alkyl containing 1 to 20 carbon atoms, straight chain alkenyl containing 2 to 20 carbon atoms, branched alkyl containing 3 to 20 carbon atoms, branched alkenyl containing 3 to 20 carbon atoms, cycloalkyl containing 3 to 20 carbon atoms, cycloalkenyl containing 3 to 20 carbon atoms, cycloheteroalkyl containing 3 to 20 carbon atoms, cycloheteroalkenyl containing 3 to 20 carbon atoms, polycycloalkyl containing 4 to 60 carbon atoms, polycycloalkenyl containing 4 to 60 carbon atoms, polycycloheteroalkyl containing 4 to 60 carbon atoms and polycycloheteroalkenyl containing 4 to 60 carbon atoms, any 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, polycycloalkyl or polycycloalkenyl, one of the carbon atoms of the cycloalkyl, cycloalkenyl, polycycloalkyl, or polycycloalkenyl is one of two carbon atoms forming the double bond of the enol ether,
R 1 is alkyl containing 1 to 20 carbon atoms, aryl containing 6 to 14 carbon atoms, aralkyl containing 7 to 15 carbon atoms, heteroaryl containing 4 to 20 carbon atoms, or heteroaralkyl containing 5 to 20 carbons,
T is an aryl or heteroaryl ring capable of emitting light, and
R 2 is an enzyme-cleavable group that contains a bond cleavable by an enzyme moiety to yield an oxygen anion on T.
49 . The kit of claim 48 , wherein the oxidant is selected from hydrogen peroxide, sodium molybdate, hydrogen peroxide and sodium molybdate, hypochlorite, hypochlorite and hydrogen peroxide, aryl endoperoxide, calcium peroxide peroxyhydrate, and combinations thereof.
50 . The kit of claim 49 , wherein the oxidant is hydrogen peroxide, or hydrogen peroxide and sodium molybdate.
51 . The kit of claim 48 , wherein at least one of A or B is
52 . The kit of claim 48 , wherein A and B together is
53 . The kit of claim 48 , wherein R 1 is alkyl containing 1 to 2 carbon atoms or trifluoalkyl containing 1 to 2 carbon atoms.
54 . The kit of claim 48 , wherein T is
wherein,
R 3 , R 4 , and R 5 , are independently selected from the group consisting of H,
F, Cl, Br, I, cyano, nitro, sulfonate, sulfate, trifluomethyl, trifluroethyl, straight chain alkyl containing 1 to 20 carbon atoms, branched alkyl containing 3 to 20 carbon atoms, straight chain alkenyl containing 2 to 20 carbon atoms, branched alkenyl containing 3 to 20 carbon atoms, cycloalkyl containing 3 to 20 carbon atoms, cycloalkenyl containing 3 to 20 carbon atoms, cycloheteroalkyl containing 3 to 20 carbon atoms, cycloheteroalkenyl containing 3 to 20 carbon atoms, polycycloalkyl containing 4 to 60 carbon atoms, polycycloalkenyl containing 4 to 60 carbon atoms, polycycloheteroalkyl containing 4 to 60 carbon atoms, polycycloheteroalkenyl containing 4 to 60 carbon atoms, alkoxy containing 1 to 20 carbon atoms, aryl containing 6 to 14 carbon atoms, aryloxy containing 6 to 14 carbon atoms, ester containing 2 to 21 carbon atoms, trialkylammonium containing 3 to 30 carbon atoms, trialkylphosphonium containing 3 to 30 carbon atoms, alkylamido containing 2 to 21 carbon atoms, arylamido containing 7 to 15 carbon atoms, alkylcarbamoyl containing 2 to 21 carbon atoms, arylcarbamoyl containing 7 to 15 carbon atoms, alkylsulfonamido containing 1 to 20 carbon atoms, arylsulfonamido containing 6 to 14 carbon atoms, trialkylsilyl containing 3 to 60 carbon atoms, triarylsilyl containing 18 to 42 carbon atoms, alkylarylsilyl containing 7 to 32 carbon atoms, alkylamidosulfonyl containing 1 to 20 carbon atoms, arylamidosulfonyl containing 6 to 14 carbon atoms, alkylsulfonyl containing 1 to 20 carbon atoms, arylsulfonyl containing 6 to 14 carbon atoms, alkylthio containing 2 to 20 carbon atoms and arylthio containing 6 to 14 carbon atoms, and
X is a sulfur atom, oxygen atom, or nitrogen atom.
55 . The kit of claim 48 , wherein OR 2 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, B 1 , B 2 and B 3 are each independently H or an alkyl (branched or straight chain) of 1-4 carbon atoms.
56 . The kit of claim 55 , wherein R 2 is
57 . The kit of claim 48 , wherein R 2 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 1,2-dioxetane enzyme substrate anion; L is a linking group; Nuc is nucleophic 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, —(CH 2 ) m —O—(CH 2 ) n , —(CH 2 ) m —S—(CH 2 ) n —, or —(CH 2 ) m —NR 6 —(CH 2 ) n —, wherein m and n are 0 to 3 and m+n is 2 or 3, wherein
R 6 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, B 1 , B 2 and B 3 are each independently H or an alkyl (branched or straight chain) of 1-4 carbon atoms.
58 . The kit of claim 57 , wherein Z is
59 . The kit of claim 48 , further comprising an enhancer.
60 . The kit of claim 59 , wherein the enhancer comprises a polymeric quaternary ammonium salt, polymeric quaternary phosphium salt, or a combination thereof.
61 . The kit of claim 60 , wherein the enhancer further comprises an acceptor dye.
62 . The kit of claim 61 , wherein the acceptor dye is fluorescein.
63 . The kit of claim 60 , wherein the polymeric quaternary ammonium salt is poly(vinylbenzyltrimethylammonium chloride), poly[vinylbenzyl(benzyldimethylammonium chloride)], poly[vinyl(benzyltributylammonium chloride)], poly[vinyl(benzyltripentylammonium chloride)], or a combination thereof.
64 . The kit of claim 60 , wherein the polymeric quaternary phosphonium salt is poly(vinylbenzyltrimethylphosphonium chloride), poly(vinylbenzyltributylphosphonium chloride), poly(vinylbenzyltrioctylphosphonium chloride), a copolymer comprising poly(vinylbenzyltributylphosphonium chloride) and poly(vinylbenzyltrioctylphosphonium chloride); or a combination thereof.
65 . The kit of claim 48 , wherein the enol ether is
66 . A method for making a 1,2-dioxetane enzyme substrate, comprising the steps of:
(a) providing an oxidant; (b) providing an enol ether having the structure:
wherein,
A and B are independently selected from the group consisting of straight chain alkyl containing 1 to 20 carbon atoms, straight chain alkenyl containing 2 to 20 carbon atoms, branched alkyl containing 3 to 20 carbon atoms, branched alkenyl containing 3 to 20 carbon atoms, cycloalkyl containing 3 to 20 carbon atoms, cycloalkenyl containing 3 to 20 carbon atoms, cycloheteroalkyl containing 3 to 20 carbon atoms, cycloheteroalkenyl containing 3 to 20 carbon atoms, polycycloalkyl containing 4 to 60 carbon atoms, polycycloalkenyl containing 4 to 60 carbon atoms, polycycloheteroalkyl containing 4 to 60 carbon atoms and polycycloheteroalkenyl containing 4 to 60 carbon atoms, any 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, polycycloalkyl or polycycloalkenyl, one of the carbon atoms of the cycloalkyl, cycloalkenyl, polycycloalkyl, or polycycloalkenyl is one of two carbon atoms forming the double bond of the enol ether,
R 1 is alkyl containing 1 to 20 carbon atoms, aryl containing 6 to 14 carbon atoms, aralkyl containing 7 to 15 carbon atoms, heteroaryl containing 4 to 20 carbon atoms, or heteroaralkyl containing 5 to 20 carbons,
T is an aryl or heteroaryl ring capable of emitting light, and
R 2 is an enzyme-cleavable group that contains a bond cleavable by an enzyme moiety to yield an oxygen anion on T; and
(c) combining an aqueous solution, the oxidant, and the enol ether to form an aqueous solution comprising a 1,2-dioxetane enzyme substrate.Join the waitlist — get patent alerts
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