HK1185349A

HK1185349A - In situ chemiluminescent substrates and assays

Info

Publication number: HK1185349A
Application number: HK13112769.9A
Authority: HK
Inventors: Alison Sparks; Zhixian Wang; Melissa Gee
Original assignee: Life Technologies Corporation
Priority date: 2010-07-08
Filing date: 2011-07-06
Publication date: 2014-02-14

Description

In situ chemiluminescent substrates and assays

Technical Field

The present invention relates generally to diagnostic assays and more specifically to chemiluminescent substrates. Methods useful for the generation of in situ chemiluminescent substrates and methods for their use in diagnostic assays are disclosed.

Background

Clinical diagnostic assays currently use chemiluminescence as a preferred state-of-the-art detection technique. Assays containing chemiluminescent readout have the most sensitive detection limit and the widest dynamic range of analyte quantitation. There is an increasing need to design simplified, miniaturized, autonomous, and/or robust diagnostic platforms for such occasions or developing countries: with less standard laboratory equipment, controlled environments, and technical support. Currently commercially available dioxetane substrates have not made good progress, where controlled storage conditions are not readily available. Thus, there is a need for methods of preparing in situ dioxetane substrates using precursors with better thermal stability.

Summary of The Invention

In a first aspect, the present invention provides a method of generating light, the method comprising the steps of: (a) providing an oxidizing agent; (b) providing an enol ether having the structure:

(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 capable of cleaving a 1, 2-dioxetane enzyme substrate; (e) contacting the enzyme complex with an aqueous solution comprising a 1, 2-dioxetane enzyme substrate to form a reaction mixture; and (f) allowing the reaction mixture to produce light.

The oxidizing agent may be selected from: hydrogen peroxide, sodium molybdate, hydrogen peroxide and sodium molybdate, hypochlorite and hydrogen peroxide, aryl endoperoxides, calcium peroxide peroxyhydrate, and combinations thereof. In some embodiments, the oxidizing agent may be hydrogen peroxide, or hydrogen peroxide and sodium molybdate.

In the enol ether [1], a and B may be independently selected from: a linear alkyl group having 1 to 20 carbon atoms, a linear alkenyl group having 2 to 20 carbon atoms, a branched alkyl group having 3 to 20 carbon atoms, a branched alkenyl group having 3 to 20 carbon atoms, a cycloalkyl group having 3 to 20 carbon atoms, a cycloalkenyl group having 3 to 20 carbon atoms, a cycloheteroalkyl group having 3 to 20 carbon atoms, a cycloheteroalkenyl group having 3 to 20 carbon atoms, a polycycloalkyl group having 4 to 60 carbon atoms, a polycyclocycloalkenyl group having 4 to 60 carbon atoms, a polycycloheteroalkyl group having 4 to 60 carbon atoms and a polycycloheteroalkenyl group having 4 to 60 carbon atoms, any of which may be unsubstituted or substituted with one or more electronically active groups, solubilizing groups or photo-enhancing groups, and wherein A and B together form the cycloalkyl group, Cycloalkenyl, polycycloalkyl or polycycloalkenyl, one of the carbon atoms of which is one of the 2 carbon atoms forming the double bond of the enol ether,

R₁may be an alkyl group having 1 to 20 carbon atoms, an aryl group having 6 to 14 carbon atoms, an aralkyl group having 7 to 15 carbon atoms, a heteroaryl group having 4 to 20 carbon atoms, or a heteroaralkyl group having 5 to 20 carbons,

t may be an aryl or heteroaryl ring capable of emitting light, and

R₂may be an enzyme cleavable group containing a bond cleavable by an enzyme moiety to generate an oxygen anion on T.

In some embodiments, the enol ethers may have at least one ofWhile in other embodiments A and B together are

In some embodiments, R₁It may be an alkyl group having 1 to 2 carbon atoms or a trifluoroalkyl group having 1 to 2 carbon atoms.

In some embodiments, T may be

Wherein R is₃、R₄And R₅Independently selected from: H. f, Cl, Br, I, cyano, nitro, sulfonate, sulfate, trifluoromethyl, trifluoroethyl, straight-chain alkyl having 1 to 20 carbon atoms, branched-chain alkyl having 3 to 20 carbon atoms, straight-chain alkenyl having 2 to 20 carbon atoms, branched-chain alkenyl having 3 to 20 carbon atoms, cycloalkyl having 3 to 20 carbon atoms, cycloalkenyl having 3 to 20 carbon atoms, cycloheteroalkyl having 3 to 20 carbon atoms, cycloheteroalkenyl having 3 to 20 carbon atoms, polycycloalkyl having 4 to 60 carbon atoms, polycycloalkenyl having 4 to 60 carbon atoms, polycycloheteroalkyl having 4 to 60 carbon atoms, polycycloheteroalkenyl having 4 to 60 carbon atoms, alkoxy having 1 to 20 carbon atoms, aryl having 6 to 14 carbon atoms, trifluoromethyl, and trifluoromethyl, Aryloxy having 6 to 14 carbon atoms, ester having 2 to 21 carbon atoms, trialkylammonium having 3 to 30 carbon atoms, trialkylphosphonium having 3 to 30 carbon atoms, alkylamido having 2 to 21 carbon atoms, arylamido having 7 to 15 carbon atoms, alkylcarbamoyl having 2 to 21 carbon atoms, arylcarbamoyl having 7 to 15 carbon atoms, alkylsulfonamido having 1 to 20 carbon atoms, arylsulfonamido having 6 to 14 carbon atoms, trialkylsilyl having 3 to 60 carbon atoms, triarylsilyl having 18 to 42 carbon atoms, alkylarylsilyl having 7 to 32 carbon atoms, alkylamidosulfonyl having 1 to 20 carbon atoms, trialkylsilyl having 3 to 60 carbon atoms, trialkylsilyl having 3 to 42 carbon atoms, trialkylsilyl having 1 to 30 carbon atoms, trialkylsilyl having 1 to 20 carbon atoms, trialkylsilyl having a structure which is a linear or branched structure, An arylamidosulfonyl group having 6 to 14 carbon atoms, an alkylsulfonyl group having 1 to 20 carbon atoms, an arylsulfonyl group having 6 to 14 carbon atoms, an alkylthio group having 2 to 20 carbon atoms, and an arylthio group having 6 to 14 carbon atoms, and X is a sulfur atomOxygen atom or nitrogen atom.

In some embodiments, OR₂Can be phosphate, acetate, 1-phospho-2, 3-diacylglycerol, adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, adenosine, alpha-D-galactoside, beta-D-galactoside, alpha-D-glucoside, beta-D-glucoside, alpha-D-mannoside, beta-fructofuranoside, beta-D-glucuronide orWherein B is₁、B₂And B₃Each independently is H or alkyl (branched or straight chain) of 1 to 4 carbon atoms.

In some embodiments, R₂Can be

In some embodiments, R₂Can be E-L-Nuc-Z, wherein R₂Is E-L-Nuc-Z, wherein E is a group comprising an electrophilic atom which, upon enzymatic cleavage of the Z group, is attacked by the electron pair of the Nuc group and releases the 1, 2-dioxetane enzyme substrate anion by vicinal assistance; l is a linking group; nuc is a nucleophilic atom; and Z is an enzyme-cleavable group; wherein E is carboxy, carbonyl, methylene substituted with a leaving group, phosphate, carbonate, xanthate, sulfite, sulfonate, bisulfite or disulfide; l is selected from: methylene or polymethylene radicals containing from 1 to 4 carbon atoms, - (CH)₂)_m-O-(CH₂)_n、-(CH₂)_m-S-(CH₂)_n-or- (CH)₂)_m-NR₆-(CH₂)_n-, where m and n are 0 to 3 and m + n is 2 or 3, where R₆Is an alkyl group having 1 to 10 carbon atoms, and the linking group may be substituted with an alkyl group having 1 to 24 carbon atoms, an alkenyl group having 2 to 24 carbon atoms, an alkyl group having 1 to 24 carbon atomsSubstituted by radicals and mono-or disubstituted by acyloxy having 1 to 24 carbon atoms, alkenyl having 2 to 24 carbon atoms and mono-or disubstituted by acyloxy having 1 to 24 carbon atoms, aryl having 6 to 10 carbon atoms, alkyl having 1 to 24 carbon atoms and substituted by phenyl, hydroxyphenyl, indolyl, mercapto, alkylthio having 1 to 4 carbon atoms, hydroxy, carboxy, amino, guanidino, imidazole or carbamoyl or alkenyl having 2 to 24 carbon atoms and by phenyl, hydroxyphenyl, indolyl, mercapto, alkylthio having 1 to 4 carbon atoms, hydroxy, carboxy, amino, guanidino, imidazole or carbamoyl; nuc is an oxygen atom or a sulfur atom; and Z is phosphoryl, acetyl, 2, 3-diacylglycerolyl (diacylglyceroyl) 1-phosphate, adenosine triphosphatyl, adenosine diphosphate, adenosine monophosphate, adenosine, alpha-D-galactosyl, beta-D-galactosyl, alpha-D-glucosyl, beta-D-glucosyl, alpha-D-mannosyl, beta-fructofuranosyl, beta-D-glucosidursyl orWherein B is₁、B₂And B₃Each independently is H or alkyl (branched or straight chain) of 1 to 4 carbon atoms. In some of these, Z is

The enzyme portion may comprise a hydrolase. In some embodiments, the hydrolase may be an alkaline phosphatase, a β -galactosidase, a β -glucosidase, a β -glucuronidase, or a neuraminidase.

In some embodiments, the enzyme moiety may be an enzyme.

In some embodiments, the method may additionally comprise the steps of: detecting any 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 enzyme present in the sample.

In some embodiments, the enzyme moiety can be an enzyme-linked antibody comprising a first antibody capable of binding to an antigen and an enzyme capable of cleaving a 1, 2-dioxetane enzyme substrate such that the substrate is cleaved and light is generated.

In some embodiments, the first antibody can be covalently or non-covalently linked to the enzyme. In some of these, the first antibody may be covalently linked to a label, and the enzyme may be covalently linked to a molecule that: the molecule is capable of non-covalently binding the label. In some of these, the label may be biotin or a biotin derivative, and the molecule may be avidin or streptavidin. In other embodiments, the label may be a hapten and the molecule may be an antibody capable of binding to the hapten.

In some embodiments, the method may additionally comprise the steps of: (a) providing a sample suspected of containing 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 an enzyme complex; and, (d) detecting 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 antigen present in the sample.

In some embodiments, the method may additionally comprise the steps of: removing any unbound enzyme-linked antibody from the enzyme complex.

In some embodiments, the enzyme moiety can be an enzyme-linked antigen comprising an antigen and an enzyme capable of cleaving a 1, 2-dioxetane enzyme substrate such that the substrate decomposes and generates light.

In some embodiments, the antigen may be covalently or non-covalently linked to the enzyme.

In some embodiments, the antigen may be covalently linked to a label, and the enzyme may be covalently linked to a molecule that: the molecule is capable of non-covalently binding the label. In some of these, the label may be biotin or a biotin derivative, and the molecule may be avidin or streptavidin. In other embodiments, the label may be a hapten and the molecule may be an antibody capable of binding to the hapten.

In some embodiments, the method may additionally comprise the steps of: (a) providing a sample suspected of containing 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 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 antigen present in the sample.

In some embodiments, the method may additionally comprise the steps of: removing any unbound enzyme-linked antigen from the enzyme complex.

In some embodiments, the enzyme moiety can be an enzyme-linked oligonucleotide comprising an oligonucleotide capable of hybridizing to a nucleic acid and an enzyme capable of cleaving a 1, 2-dioxetane enzyme substrate such that the substrate decomposes and generates light.

In some embodiments, the oligonucleotide may be covalently or non-covalently linked to the enzyme.

In some embodiments, the oligonucleotide may be covalently linked to a label, and the enzyme may be covalently linked to a molecule that: the molecule is capable of non-covalently binding the label. In some of these, the label may be biotin or a biotin derivative, and the molecule may be avidin or streptavidin. In other embodiments, the label may be a hapten and the molecule may be an antibody capable of binding to the hapten.

In some embodiments, the method may additionally comprise the steps of: (a) providing a sample suspected of comprising nucleic acids; (b) immobilizing the nucleic acid on a solid phase, (c) contacting the immobilized nucleic acid and the enzyme-linked oligonucleotide to form an enzyme complex; and (d) detecting 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 nucleic acid present in the sample.

In some embodiments, the method may additionally comprise the steps of: removing any unbound enzyme-linked oligonucleotides from the enzyme complex.

In some embodiments, the reaction mixture may additionally comprise a promoter.

In some embodiments, the accelerator may comprise a polymeric quaternary ammonium salt, a polymeric quaternary phosphonium salt, or a combination thereof. In some of these, the enhancer may additionally comprise an acceptor dye. In some of these, the acceptor dye may be fluorescein.

In some embodiments, the polymeric quaternary ammonium salt can be poly (vinylbenzyltrimethylammonium chloride), poly [ vinylbenzyl (benzyldimethylammonium chloride) ], poly [ vinyl (benzyltributylammonium chloride) ], poly [ vinyl (benzyltripentylammonium chloride) ], or combinations thereof.

In other embodiments, the polymeric quaternary phosphonium salt may be poly (vinylbenzyltrimethyl phosphonium chloride), poly (vinylbenzyltributylphosphonium chloride), poly (vinylbenzyltrioctylphosphonium chloride), copolymers comprising poly (vinylbenzyltributylphosphonium chloride) and poly (vinylbenzyltrioctylphosphonium chloride), or combinations thereof.

In some embodiments, the enol ether [1] can be

In another aspect, the invention provides an assay method for determining the presence or amount of an enzyme in a sample, the method comprising the steps of: (a) providing an oxidizing agent; (b) providing an enol ether having structure [1] and having substituents as described above; (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 containing an enzyme capable of cleaving a 1, 2-dioxetane enzyme substrate such that the substrate decomposes and produces light; (e) contacting the sample with an aqueous solution comprising a 1, 2-dioxetane enzyme substrate to form a reaction mixture; and, (f) detecting 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 enzyme present in the sample.

In another aspect, the invention provides an assay method for determining the presence or amount of an antigen in a sample, the method comprising the steps of: (a) providing an oxidizing agent; (b) providing an enol ether having structure [1] and having substituents as described above; (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 containing an antigen; (e) providing an enzyme-linked antibody comprising a first antibody capable of binding to an antigen and an enzyme capable of cleaving a 1, 2-dioxetane enzyme substrate such 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 an aqueous solution comprising a 1, 2-dioxetane enzyme substrate to form a reaction mixture; and, (i) detecting 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 antigen present in the sample.

In another aspect, the invention provides an assay method for determining the presence or amount of an antigen in a sample, the method comprising the steps of: (a) providing an oxidizing agent; (b) providing an enol ether having structure [1] and having substituents as described above; (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 containing an antigen; (e) providing an enzyme-linked antigen comprising an antigen and an enzyme capable of cleaving a 1, 2-dioxetane enzyme substrate such 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 an aqueous solution comprising a 1, 2-dioxetane enzyme substrate to form a reaction mixture; and, (i) detecting 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 antigen present in the sample.

In another aspect, the invention provides an assay method for determining the presence or amount of a nucleic acid in a sample, the method comprising the steps of: (a) providing an oxidizing agent; (b) providing an enol ether having structure [1] and having substituents as described above; (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 a nucleic acid, (e) immobilizing the nucleic acid on a solid phase, (f) providing an enzyme-linked oligonucleotide comprising an oligonucleotide capable of hybridizing to the nucleic acid and an enzyme capable of cleaving a 1, 2-dioxetane enzyme substrate such that the substrate decomposes and generates light; (g) contacting the immobilized and enzyme-linked oligonucleotides to form an enzyme complex; (h) contacting the enzyme complex with an aqueous solution comprising a 1, 2-dioxetane enzyme substrate to form a reaction mixture; and, (i) detecting 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 with the amount of nucleic acid present in the sample.

In another aspect, the invention provides a kit for detecting the presence and/or amount of an analyte in a sample. The kit comprises an oxidant and an enol ether, wherein the enol ether has a structure [1] and has a substituent group as described above. Various embodiments of the enol ethers have structure [1] and its substituents, and oxidizing agents, also as described, are equally applicable to the kits and methods of the present invention.

In some embodiments, the kit may further comprise an enhancer.

In some embodiments, the enol ether having structure [1] can be any of the enol ethers [2] through [18] shown above.

In another aspect, the present invention provides a method for preparing a substrate for a 1, 2-dioxetane enzyme, said method comprising the steps of: (a) providing an oxidizing agent; (b) providing an enol ether having the structure:

and

(c) combining an aqueous solution, the oxidant, and the enol ether to form an aqueous solution comprising a 1, 2-dioxetane enzyme substrate.

Drawings

FIG. 1 shows the use of Na₂MoO₄/H₂O₂Oxidation System in aqueous solution at alkaline pH for conversion of AMPPD enol ether phosphate (AMPPD-EE) to the dioxetane substrate AMPPD^®Reaction scheme (iv).

The HPLC trace of fig. 2 shows the elution peak of the raw material AMPPD enol ether phosphate (AMPPD-EE) control.

The HPLC trace of FIG. 3 shows the expected dioxetane AMPPD^®Elution peaks of control.

FIG. 4 is an HPLC trace showing the mixing of control AMPPD enol ether phosphate (AMPPD-EE) and AMPPD^®The elution peak of (4).

The HPLC trace of FIG. 5 shows complete oxidation of AMPPD enol ether phosphate (AMPPD-EE) to AMPPD^®。

FIG. 6 shows the use of Na₂MoO₄/H₂O₂Oxidation system ADP-StarConversion of enol ether phosphate (ADP-EE) to ADP-Star ^®(ADP-EE-O), reaction scheme for subsequent activation of dioxetanes with alkaline phosphatase.

FIG. 7 shows the ADP-value of alkaline phosphataseStar ^®Activation of (ADP) dioxetane control, 2 ADP-StarEnol ether (ADP-EE-O1 and ADP-EE-O2) oxidation and commercially available CDP-Star^®(CDP) dioxetane-controlled ADP-Star^®An emission curve.

FIG. 8 shows AMPPD^®Thermal stability profile at 55 ℃.

FIG. 9 shows a graph of the thermal stability of AMPPD enol ether phosphate (AMPPD-EE) at 55 ℃.

FIG. 10 shows AMPPD enol ether phosphate (AMPPD-EE) alone (left panel) and AMPPD^®And AMPPD enol ether phosphate (AMPPD-EE) (right panel) at 40 ℃. In the right panel, the bar on the left side is with AMPPD^®The relevant data, while the corresponding bar on the right is the data relating to AMPPD-EE.

FIG. 11 shows the thermal stability of AMPPD enol ether phosphate (AMPPD-EE) in the presence of sodium molybdate at 40 ℃.

Fig. 12 shows method a: the enzyme dephosphorylates phosphate enol ether (AMPPD-EE) followed by aqueous oxidation to dioxetane.

FIG. 13 shows alkaline phosphatase dilution curvesSignal (expressed in relative light units RLU): luminescence (in situ AMPPD prepared by method A) relative to control (AMPPD) in method A^®)。

FIG. 14 shows the signal to noise ratio (S/N) of alkaline phosphatase dilution curves: luminescence (in situ AMPPD prepared by method A) relative to control (AMPPD) in method A^®)。

FIG. 15 shows the effect of hydrogen peroxide and sodium molybdate on enzyme activity.

FIG. 16 shows that decreasing the sodium molybdate concentration restores enzyme activity.

Fig. 17 shows method B: aqueous oxidation of AMPPD enol ether phosphate (AMPPD-EE) to dioxetane (in situ AMPPD) followed by enzymatic dephosphorylation.

FIG. 18 shows the signal (in RLU) of the alkaline phosphatase dilution curve: in situ AMPPD prepared by method B relative to control AMPPD^®。

FIG. 19 shows the signal to noise ratio (S/N) of alkaline phosphatase dilution curves: in situ AMPPD prepared by method B relative to control AMPPD^®。

FIG. 20 shows the signal (in RLU) of the alkaline phosphatase dilution curve: in situ ADP-Star(ADP-Star-EE) relative to CSPD^®、ADP-Star ^®And CDP-Star ^®And (6) comparison.

FIG. 21 shows the signal to noise ratio (S/N) of alkaline phosphatase dilution curves: in situ ADP-Star(ADP-Star-EE) relative to CSPD^®、ADP-Star ^®And CDP-Star ^®And (6) comparison.

FIG. 22 shows a 30-minute IL-6 ELISA sensitivity curve: in situ AMPPD relative to control AMPPD^®。

FIG. 23 shows the time course of in situ AMPPD production as assessed by IL-6 ELISA.

FIG. 24 shows in situ ADP-Star(ADP Star EE) relative to the control ADP-Star ^®、CDP-Star ^®And CSPD^®Light emission curve of (1).

FIG. 25 shows in situ ADP-Star (ADP Star EE) relative to control dioxetane, CSPD^®、ADP-Star ^®And CDP-Star ^®Maximum light signal curve (%) over time.

FIG. 26 shows rhIL-6 ELISA detection curves: in situ ADP-Star(ADP-Star EE) relative to control Dioxolane, CSPD^®、ADP-Star ^®And CDP-Star ^®。

FIG. 27 shows a TBQ promoter (poly [ vinyl (benzyltributylammonium chloride))]) ADP generated in situ in the presence ofStar(ADP) signal to background (S/B) kinetics.

FIG. 28 shows ADP-Star(ADP x) IL-6 ELISA sensitivity comparisons at 30-minute time points.

FIG. 29 shows ADP-Star(ADP) IL-6 ELISA sensitivity comparisons at 60-minute time points.

Detailed Description

Before the present invention is described in detail, it is to be understood that this invention is not limited to the embodiments disclosed herein. Further, when methods and kits are described in terms of "comprising" (interpreted as meaning "including, but not limited to") individual steps or components, the methods and kits can also "consist essentially of or" consist of the individual steps and components, and such terms should be interpreted as defining a substantially closed member set. Finally, it should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

Defining:

acceptor dyes denote molecules that: which can receive energy, in particular light, from other light-emitting molecules and in turn emit detectable energy, also preferably light.

An analyte refers to a substance or chemical component that is determined in an analytical procedure. The term as used herein includes, but is not limited to, antigens or antibodies.

The antigen represents a substance to which an antibody can bind.

Antibodies represent such gamma globulin proteins: it is present in the blood or other body fluids of vertebrates and is used by the immune system to recognize and neutralize foreign bodies. This term as used herein includes, but is not limited to: antibodies or fragments thereof that can then be polyclonal, monoclonal, recombinant to the antigen.

Accelerators mean water-soluble substances such as: which increases the specific light energy production resulting from enzymatic cleavage of the 1, 2-dioxetane enzyme substrate and its subsequent decomposition, wherein this observed light production is higher than that observed in the absence of the promoter.

An enzyme refers to a protein that catalyzes a chemical reaction.

Haptens refer to small molecules that can elicit an immune response only when linked to a large carrier such as a protein.

Hydrolases (hydrolytical enzymes or hydrosases) represent such enzymes: it catalyzes the hydrolysis of chemical bonds and can be classified as EC 3 in the EC numbering classification of enzymes.

Nucleic acid refers to DNA, RNA or fragments thereof.

Oligonucleotides represent short nucleic acid polymers. The term as used herein includes, but is not limited to, nucleic acid polymers containing from 2 to 1000 nucleic acids.

An Oxidant (Oxidant) is a chemical compound that can easily transfer an oxygen atom, or a substance that gains electrons in a redox chemical reaction. The term is used herein interchangeably with the term oxidizing agent or oxidizer.

The present invention provides methods for the in situ preparation of chemiluminescent enzyme substrates under aqueous or other conditions, and/or methods for producing light using such substrates. The generation of light may be used to determine the presence of an enzyme, antigen or nucleic acid in a sample. These methods involve stabilized enol ethers and oxidizing agents that, when combined in an aqueous solution, form a 1, 2-dioxetane enzyme substrate. The invention also provides kits for detecting the presence and/or amount of an analyte in a sample. The kit includes a stabilized enol ether and an oxidizing agent that, when combined in an aqueous solution, form a 1, 2-dioxetane enzyme substrate. These methods, as well as kits, can be used in assays well known in the art.

Viewed from one aspect, the present invention provides a method of generating light. The method comprises the following steps: providing an oxidizing agent; providing an enol ether having the structure shown below:

　　[1]；

combining an aqueous solution, the oxidant, and the enol ether to form an aqueous solution comprising a 1, 2-dioxetane enzyme substrate; providing an enzyme complex comprising an enzyme moiety capable of cleaving a 1, 2-dioxetane enzyme substrate; contacting the enzyme complex with an aqueous solution comprising a 1, 2-dioxetane enzyme substrate to form a reaction mixture; and causing the reaction mixture to generate light.

In one step of these methods, an oxidizing agent is provided. The oxidant is one that can convert an enol ether [1] to its corresponding 1, 2-dioxetane enzyme substrate.

In some embodiments, the oxidizing agent may be selected from: hydrogen peroxide, sodium molybdate, hydrogen peroxide and sodium molybdate, hypochlorite and hydrogen peroxide, aryl endoperoxides, calcium peroxide peroxyhydrate, and combinations thereof. In some of these embodiments, the oxidizing agent may be hydrogen peroxide, or hydrogen peroxide and sodium molybdate.

The oxidizing agent may be provided as a solution or as a powder. Where the oxidizing agent comprises multiple components, one or more (but not all) of these components may or may not be combined with the enol ether.

In another step of these methods, an enol ether [1] is provided.

In some embodiments, the enol ether [1] can have a and B, the a and B being independently selected from the group consisting of: linear alkyl, linear alkenyl, branched alkyl, branched alkenyl, cycloalkyl, cycloalkenyl, cycloheteroalkyl, cycloheteroalkenyl, polycycloalkyl, polycyclocycloalkenyl, polycycloheteroalkyl, and polycycloheteroalkenyl, any of which may be unsubstituted or substituted with one or more electronically active groups, solubilizing groups, or photo-enhancing groups, and wherein 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 the 2 carbon atoms forming the double bond of the alkene alcohol ether.

Examples of electron-reactive groups include: F. cl, Br, I, cyano, nitro, sulfonate, sulfate, trifluoromethyl, trifluoroethyl, straight-chain alkyl having 1 to 20 carbon atoms, branched-chain alkyl having 3 to 20 carbon atoms, straight-chain alkenyl having 2 to 20 carbon atoms, branched-chain alkenyl having 3 to 20 carbon atoms, cycloalkyl having 3 to 20 carbon atoms, cycloalkenyl having 3 to 20 carbon atoms, cycloheteroalkyl having 3 to 20 carbon atoms, cycloheteroalkenyl having 3 to 20 carbon atoms, polycycloalkyl having 4 to 60 carbon atoms, polycyclocycloalkenyl having 4 to 60 carbon atoms, polycycloheteroalkyl having 4 to 60 carbon atoms, polycycloheteroalkenyl having 4 to 60 carbon atoms, alkoxy having 1 to 20 carbon atoms, aryl having 6 to 14 carbon atoms, Aryloxy group having 6 to 14 carbon atoms, ester having 2 to 21 carbon atoms, trialkylammonium having 3 to 30 carbon atoms, trialkylphosphonium having 3 to 30 carbon atoms, alkylamido having 2 to 21 carbon atoms, arylamido having 7 to 15 carbon atoms, alkylcarbamoyl having 2 to 21 carbon atoms, arylcarbamoyl having 7 to 15 carbon atoms, alkylsulfonamido having 1 to 20 carbon atoms, arylsulfonamido having 6 to 14 carbon atoms, trialkylsilyl having 3 to 60 carbon atoms, triarylsilyl having 18 to 42 carbon atoms, alkylarylsilyl having 7 to 32 carbon atoms, alkylamidosulfonyl having 1 to 20 carbon atoms, trialkylsilyl having 3 to 60 carbon atoms, trialkylsilyl having 3 to 42 carbon atoms, trialkylsilyl having 1 to 30 carbon atoms, trialkylsilyl having 1 to 20 carbon atoms, trialkylsilyl having a structure, An arylamidosulfonyl group having 6 to 14 carbon atoms, an alkylsulfonyl group having 1 to 20 carbon atoms, an arylsulfonyl group having 6 to 14 carbon atoms, an alkylthio group having 2 to 20 carbon atoms, and an arylthio group having 6 to 14 carbon atoms.

Examples of solubilizing groups include: carboxylic acid, malonic acid, hydroxyl, sulfate, sulfonate, phosphate, and ammonium groups; poly (ethoxy)_nGroup [ - (O-CH)₂-CH₂-)_n]Wherein n =1-30, terminated with carboxylic acid, malonic acid, hydroxyl, sulfate, sulfonate, phosphate, and ammonium groups; poly [ -O- (CH)₂-)_n]Wherein n =1-30, terminated with carboxylic acid, malonic acid, hydroxyl, sulfate, sulfonate, phosphate, and ammonium groups.

Examples of photo-enhancing groups include: cationic or polycationic moieties such as alkylammonium, alkylphosphonium, alkylsulfonium groups; alkylaryl ammonium, alkylaryl phosphonium, and alkylaryl sulfonium groups; or an aryl ammonium, phosphonium, and sulfonium group; and poly (alkylammonium), poly (alkylphosphonium), poly (alkylsulfonium) groups; poly (alkylarylammonium), poly (alkylarylphosphonium), and polyalkylarylsulfonium groups; or poly (arylammonium), poly (arylphosphonium), and poly (arylsulfonium) groups.

In some embodiments, a and B may be independently selected from: a linear alkyl group having 1 to 20 carbon atoms, a linear alkenyl group having 2 to 20 carbon atoms, a branched alkyl group having 3 to 20 carbon atoms, a branched alkenyl group having 3 to 20 carbon atoms, a cycloalkyl group having 3 to 20 carbon atoms, a cycloalkenyl group having 3 to 20 carbon atoms, a cycloheteroalkyl group having 3 to 20 carbon atoms, a cycloheteroalkenyl group having 3 to 20 carbon atoms, a polycycloalkyl group having 4 to 60 carbon atoms, a polycyclocycloalkenyl group having 4 to 60 carbon atoms, a polycycloheteroalkyl group having 4 to 60 carbon atoms and a polycycloheteroalkenyl group having 4 to 60 carbon atoms, any of which may be unsubstituted or substituted with one or more electronically active groups, solubilizing groups or photo-enhancing groups, and wherein A and B together form the cycloalkyl group, A cycloalkenyl group, a polycycloalkyl group, or a polycycloalkenyl group, one of the carbon atoms of the cycloalkyl, cycloalkenyl, polycycloalkyl, or polycycloalkenyl group being one of the 2 carbon atoms forming the double bond of the enol ether.

In some embodiments, at least one of A or B may be

。

In other embodiments, A and B together may be

。

In some embodiments, the enol ether [1]]Having R₁Said R is₁May be alkyl, aryl, aralkyl, heteroaryl or heteroaralkyl. In some of these, R₁Can be alkyl containing 1-20 carbon atoms, aryl containing 6-14 carbon atoms, or alkyl containing 7-15 carbon atomsAralkyl, heteroaryl having 4 to 20 carbon atoms, or heteroaralkyl having 5 to 20 carbons. In some of these, R₁It may be an alkyl group having 1 to 2 carbon atoms or a trifluoroalkyl group having 1 to 2 carbon atoms.

In some embodiments, the enol ether [1] has a T, which can be an aryl or a fused polycyclic compound including, but not limited to, heteroaryl rings capable of emitting light. T is chosen such that it does not interfere with the generation of light and satisfies the valence of the 4-carbon atom of the dioxetane ring to which it is attached. T represents any of a number of fluorescent chromophore groups that form light emitting fluorophores that allow the corresponding dioxetane to break down the fragment to absorb energy and form excited states from which they emit optically detectable energy to return to their ground state. T is also substituted with an enzyme cleavable group containing the bond: the bond can be cleaved by an enzyme to produce an electron rich moiety, e.g., an oxyanion, sulfide anion, or nitrogen anion, attached to the dioxetane ring either directly or by subsequent adjustment of the pH.

In some embodiments, T may be an aryl group, such as phenyl, which may be substituted with an electron active group, a solubilizing group, or a light enhancing group.

In some embodiments, T may be a fluorophore moiety comprising a fused polycyclic ring having an enzyme cleavable labile ring substituent comprising a bond: the bond, when cleaved by an enzyme, provides an electron rich fused polycyclic moiety to further provide a dioxetane compound that can be cleaved to emit light.

Included within fused polycyclic compounds (residues of which can be used to form such fluorophore moieties) are fused polycyclic aromatic hydrocarbon ring fluorophore compounds containing from 9 to about 30 (inclusive) ring carbon atoms, such as naphthalene:

，

pentalene, azulene, heptatriene-heptalene, asymmetric indacene (asindacene), symmetric indacene (s-indacene), biphenylene, perylene, acenaphthylene, phenanthrene, anthracene, acephenanthrene (acephenanthrylene), acenaphthylene, triphenylene, pyrene, heptatriene, acenaphthylene, phenanthrylene, and phenanthrylene,(chrysene), naphthonaphthalene and the like, as well as derivatives thereof substituted with one or more stable substituents such as branched or straight chain alkyl groups having from 1 to 20 (inclusive) carbon atoms, e.g., methyl, n-butyl or decyl, branched or straight chain heteroalkyl groups having from 1 to 7 (inclusive) carbon atoms, e.g., methoxy, hydroxyethyl or hydroxypropyl; aryl having 1 or 2 rings, for example, phenyl; heteroaryl having 1 or 2 rings, for example, pyrrolyl or pyrazolyl; cycloalkyl groups having 3 to 7 (inclusive) carbon atoms in the ring, for example, cyclohexyl; heterocycloalkyl having 3 to 6 (inclusive) carbon atoms in the ring, e.g., dioxane; aralkyl having 1 or 2 rings, for example, benzyl; alkaryl having 1 or 2 rings, for example, tolyl; electron withdrawing groups such as perfluoroalkyl groups having 1 to 7 (inclusive) carbon atoms, e.g., trifluoromethyl; halogen; CO 2₂H、ZCO₂H、SO₃H、NO₂、ZNO₂CN or ZCN, wherein Z is a branched or straight chain alkyl group having 1 to 7 (inclusive) carbon atoms, e.g. methyl, or an aryl group having 1 or 2 rings, e.g. phenyl; electron-donating groups, e.g. branched or straight-chain C₁-C₇Alkoxy, for example, methoxy or ethoxy: aralkyloxy having 1 or 2 rings, for example, phenoxy; branched or straight chain C₁-C₇Alkoxy, for example, methoxy or ethoxy; aralkyloxy having 1 or 2 rings, for example, phenoxy; branched or straight chain C₁-C₇Hydroxyalkyl, for example, hydroxymethyl or hydroxyethyl; hydroxyaryl having 1 or 2 rings, for example, hydroxyphenyl; branched or straight chain C₁-C₇Alkyl estersGroups, for example, acetates; aryl ester groups having 1 or 2 rings, for example, benzoate; or heteroaryl having 1 or 2 rings, for example, benzoxazole, benzothiazole, benzimidazole or benzotriazole.

Furthermore, the fused polycyclic moiety of the fluorophore moiety represented by T may also be the residue of a fused polycyclic aromatic heterocyclic fluorophore compound such as benzo [ b ] thiophene, naphtho [2,3-b ] thiophene, thianthrene, benzofuran, isobenzofuran, chromene, xanthene (xanthene), phenoxathine, quinoline, isoquinoline, phenanthridine, phenazine, phenoxazine, phenothiazine, phenanthroline, purine, 4H-quinolizine, phthalazine, naphthyridine, indole, indolizine, chroman, isochroman, indoline, isoindoline, and the like, which are unsubstituted or substituted with one or more of the foregoing stable substituents and contain from 9 to about 30 (inclusive) ring atoms, the majority of which are carbon atoms.

In some embodiments, T may be

Wherein R is₃、R₄And R₅Independently selected from: H. f, Cl, Br, I, cyano, nitro, sulfonate, sulfate, trifluoromethyl, trifluoroethyl, straight-chain alkyl, branched-chain alkyl, straight-chain alkenyl, branched-chain alkenyl, cycloalkyl, cycloalkenyl, cycloheteroalkyl, cycloheteroalkenyl, polycycloalkyl, polycycloheteroalkyl, polycycloheteroalkenyl, alkoxy, aryl, aryloxy, ester, trialkylammonium, trialkylphosphonium, alkylamido, arylamido, alkylcarbamoyl, arylcarbamoyl, alkylsulfonamido, arylsulfonamido, trialkylsilyl, triarylsilyl, alkylarylsilyl, alkylamidosulfonyl, arylamidosulfonyl, alkylsulfonyl, arylsulfonyl, alkylthio, and arylthio. In some of these, R₃、R₄And R₅May be independently selected from: H.F. cl, Br, I, cyano, nitro, sulfonate, sulfate, trifluoromethyl, trifluoroethyl, straight-chain alkyl having 1 to 20 carbon atoms, branched-chain alkyl having 3 to 20 carbon atoms, straight-chain alkenyl having 2 to 20 carbon atoms, branched-chain alkenyl having 3 to 20 carbon atoms, cycloalkyl having 3 to 20 carbon atoms, cycloalkenyl having 3 to 20 carbon atoms, cycloheteroalkyl having 3 to 20 carbon atoms, cycloheteroalkenyl having 3 to 20 carbon atoms, polycycloalkyl having 4 to 60 carbon atoms, polycyclocycloalkenyl having 4 to 60 carbon atoms, polycycloheteroalkyl having 4 to 60 carbon atoms, polycycloheteroalkenyl having 4 to 60 carbon atoms, alkoxy having 1 to 20 carbon atoms, aryl having 6 to 14 carbon atoms, Aryloxy having 6 to 14 carbon atoms, ester having 2 to 21 carbon atoms, trialkylammonium having 3 to 30 carbon atoms, trialkylphosphonium having 3 to 30 carbon atoms, alkylamido having 2 to 21 carbon atoms, arylamido having 7 to 15 carbon atoms, alkylcarbamoyl having 2 to 21 carbon atoms, arylcarbamoyl having 7 to 15 carbon atoms, alkylsulfonamido having 1 to 20 carbon atoms, arylsulfonamido having 6 to 14 carbon atoms, trialkylsilyl having 3 to 60 carbon atoms, triarylsilyl having 18 to 42 carbon atoms, alkylarylsilyl having 7 to 32 carbon atoms, alkylamidosulfonyl having 1 to 20 carbon atoms, trialkylsilyl having 3 to 60 carbon atoms, trialkylsilyl having 3 to 42 carbon atoms, trialkylsilyl having 1 to 30 carbon atoms, trialkylsilyl having 1 to 20 carbon atoms, trialkylsilyl having a structure which is a linear or branched structure, An arylamidosulfonyl group having 6 to 14 carbon atoms, an alkylsulfonyl group having 1 to 20 carbon atoms, an arylsulfonyl group having 6 to 14 carbon atoms, an alkylthio group having 2 to 20 carbon atoms, and an arylthio group having 6 to 14 carbon atoms.

In some embodiments, the enol ether [1]]May have OR₂The OR of₂Is phosphate, acetate, 1-phospho-2, 3-diacylglycerol, adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, adenosine, alpha-D-galactoside, beta-D-galactoside, alpha-D-glucoside, beta-D-glucoside, alpha-D-mannoside, beta-fructofuranoside, beta-D-glucuronide orWherein B is₁、B₂And B₃Each independently is H or alkyl (branched or straight chain) of 1 to 4 carbon atoms. In some of these, R₂Is that

。

R₂(i.e., an enzyme-cleavable substituent) may include a phosphate group represented by the following general formula:

wherein M is⁺Represents a cation such as an alkali metal, e.g. sodium or potassium, ammonium or C₁-₇Alkyl, aralkyl or aromatic quaternary ammonium cations, N (DR)₃)₄ ⁺ ^，Wherein each D₃Can be an alkyl group, e.g., methyl or ethyl, an aralkyl group (e.g., benzyl), or form part of a heterocyclic ring system (e.g., pyridinium, specifically, disodium salt). That is, such quaternary ammonium cations may also be linked to the polymeric backbone through one of their quaternized groups.

Where n is greater than 1, or may be part of a polyquaternium (i.e., an ionene polymer).

In some embodiments, R₂Can be E-L-Nuc-Z, wherein E is a group comprising an electrophilic atom which, after enzymatic cleavage of the Z group, is replaced by the electron of the Nuc groupTo attack and release 1, 2-dioxetane enzyme substrate anions through ortho-interactions; l is a linking group; nuc is a nucleophilic atom; and Z is an enzyme-cleavable group; wherein

E may be carboxy, carbonyl, methylene substituted with a leaving group, phosphate, carbonate, xanthate, sulfite, sulfonate, bisulfite or disulfide;

l may be selected from: methylene or polymethylene radicals containing from 1 to 4 carbon atoms, - (CH)₂)_m-O-(CH₂)_n、-(CH₂)_m-S-(CH₂)_n-or- (CH)₂)_m-NR₆-(CH₂)_n-, where m and n are 0 to 3 and m + n is 2 or 3, where R₆Is an alkyl group having 1 to 10 carbon atoms, and the linking group may be substituted with an alkyl group having 1 to 24 carbon atoms, an alkenyl group having 2 to 24 carbon atoms, an alkyl group having 1 to 24 carbon atoms, and mono-or di-substituted with an acyloxy group having 1 to 24 carbon atoms, an alkenyl group having 2 to 24 carbon atoms, and mono-or di-substituted with an acyloxy group having 1 to 24 carbon atoms, an aryl group having 6 to 10 carbon atoms, an alkyl group having 1 to 24 carbon atoms, and substituted with a phenyl group, a hydroxyphenyl group, an indolyl group, a mercapto group, an alkylthio group having 1 to 4 carbon atoms, a hydroxyl group, a carboxyl group, an amino group, a guanidino group, an imidazole or a carbamoyl group, or an alkenyl group having 2 to 24 carbon atoms, and substituted with a phenyl group, a hydroxyphenyl group, an indolyl group, a mercapto group, an alkylthio group having 1 to 4 carbon atoms, Hydroxy, carboxy, amino, guanidino, imidazole, or carbamoyl;

nuc may be an oxygen atom or a sulfur atom; and is

Z may be phosphoryl, acetyl, 2, 3-diacylglyceroyl-1-phosphate, adenosine triphosphatyl, adenosine diphosphoryl, adenosine monophosphoryl, adenosine adenosyl, alpha-D-galactosyl, beta-D-galactosyl, alpha-D-glucosyl, beta-D-glucosyl, alpha-D-mannosyl, beta-fructofuranosyl, beta-D-glucopyranosyl orWherein B is₁、B₂And B₃Each independently is H or alkyl (branched or straight chain) of 1 to 4 carbon atoms.

In some embodiments, Z can be

In some embodiments, the enol ether [1] can be

In another step of these methods, the aqueous solution, the oxidizing agent, and the enol ether are combined to form an aqueous solution comprising a 1, 2-dioxetane enzyme substrate. The aqueous solution may comprise water, one or more buffer components, one or more organic solvents, one or more colorants, one or more preservatives, or a combination thereof. Determination of the concentrations of oxidant and enol ether in the aqueous solution required to provide a substrate for a 1, 2-dioxetane enzyme is within the skill of one of ordinary skill in the diagnostic arts.

In another step of these methods, an enzyme complex is provided comprising an enzyme moiety capable of cleaving a 1, 2-dioxetane enzyme substrate. The enzyme moiety may be an enzyme, an enzyme-linked antibody, an enzyme-linked antigen, or an enzyme-linked oligonucleotide.

The enzyme moiety comprises an enzyme capable of cleaving a 1, 2-dioxetane enzyme substrate. In some embodiments, the enzyme may be a hydrolase. Hydrolytic enzymes include enzymes that: which cleave ester bonds and are classified as EC 3.1, or cleave sugar bonds and are classified as EC 3.2, including, but not limited to, alkaline phosphatase, β -galactosidase, β -glucosidase, β -glucuronidase, or neuraminidase.

The sample to be assayed is suspected to contain an enzyme, an antigen or a nucleic acid. The sample may be of biological or non-biological origin. In case the sample is of biological origin, it may be blood, serum, plasma, urine, stool, saliva, mucus, semen, tissue extract, cell culture medium, cells, cell extract, etc.

In some embodiments, the enzyme moiety may be an enzyme. In these embodiments, the enzymes in the sample will constitute an enzyme complex. Contacting the enzyme complex with a 1, 2-dioxetane enzyme substrate to form a reaction mixture, and allowing the reaction mixture to produce light.

In some embodiments, the emission of light may be detected, and such emission will indicate the presence of the enzyme, and the amount of light emitted may be correlated to the amount of enzyme present in the sample.

In some embodiments, the enzyme moiety can be an enzyme-linked antibody. The enzyme-linked antibody comprises a first antibody capable of binding an antigen and an enzyme capable of cleaving a 1, 2-dioxetane enzyme substrate such that the substrate is cleaved and light is generated. In these embodiments, the enzyme-linked antibody, antigen, and secondary antibody (which is capable of binding to the antigen and immobilized on a solid phase) constitute an enzyme complex. Contacting the enzyme complex with a 1, 2-dioxetane enzyme substrate to form a reaction mixture, and allowing the reaction mixture to produce light.

In some embodiments, a sample suspected of containing an antigen can be contacted with an enzyme-linked antibody (which comprises a first antibody and an enzyme) and a solid phase (which comprises a second antibody), wherein both antibodies are capable of binding to the antigen to provide an enzyme complex that is capable of cleaving a 1, 2-dioxetane enzyme substrate such that the substrate decomposes and generates light. The sample, enzyme-linked antibody, and solid phase can be combined in any order.

In some embodiments, the method may additionally comprise the steps of: any unbound enzyme-linked antibody is removed from the enzyme complex by washing the enzyme complex. This can be achieved by adding and removing buffers compatible with the components of the enzyme complex. Such buffers are well known in the field of diagnostics. Other washing steps of the solid phase may be performed.

The first antibody, the second antibody, or both may be polyclonal, monoclonal, or recombinant.

The solid phase can be a bead, a test tube, a multiwell plate, a microarray, a gel, a membrane, a microparticle, a nanocrystal, a quantum dot, or the like. Materials for preparing these solid phases are known in the field of diagnostics.

The second antibody may be immobilized on the solid phase by non-covalent or covalent attachment of the second antibody to the solid phase by techniques known in the art of diagnostics.

The first antibody may be covalently or non-covalently linked to the enzyme. When non-covalently linked, the first antibody is covalently linked to a label, and the enzyme is covalently linked to a molecule that: the molecule is capable of non-covalently binding the label.

In some embodiments, the label may be biotin or a biotin derivative, and the molecule may be avidin or streptavidin. Biotin derivatives are biotin molecules with substitutions. Biotin molecules, in which a portion of the biotin structure is missing, are also considered biotin derivatives. Biotin derivatives include substituted naturally occurring biotin as well as synthetic biotin.

In some embodiments, the label may be a hapten and the molecule may be an antibody capable of binding to the hapten. The use of digoxigenin (digoxigenin) as a hapten, and the use of anti-digoxigenin as a molecule, are known in the field of diagnostics.

In some embodiments, the enzyme moiety may be an enzyme-linked antigen. The enzyme-linked antigen comprises an antigen and an enzyme capable of cleaving a 1, 2-dioxetane enzyme substrate. In these embodiments, the enzyme complex comprises an antigen linked to an enzyme bound to a solid phase comprising an antibody capable of binding to the antigen. Contacting the enzyme complex with a 1, 2-dioxetane enzyme substrate to form a reaction mixture, and allowing the reaction mixture to produce light.

In some embodiments, a sample suspected of containing an antigen may be contacted with an enzyme-linked antigen and a solid phase comprising an antibody capable of binding to the antigen. The sample, enzyme-linked antigen, and solid phase may be combined in any order.

In some embodiments, the method may additionally comprise the steps of: any unbound enzyme-linked antigen is removed from the enzyme complex by washing the enzyme complex. This can be achieved by adding and removing buffers compatible with the components of the enzyme complex. Such buffers are well known in the field of diagnostics. Other washing steps of the solid phase may be performed.

The antibody may be a polyclonal, monoclonal or recombinant antibody.

The antibody may be immobilized on the solid phase by non-covalent or covalent attachment of the antibody to the solid phase.

The antigen may be covalently or non-covalently linked to the enzyme. When non-covalently linked, the antigen may be covalently linked to a label, and the enzyme may be covalently linked to a molecule that: the molecule is capable of non-covalently binding the label.

In some embodiments, the label may be biotin or a biotin derivative as described above, and the molecule may be avidin or streptavidin.

In some embodiments, the label may be a hapten and the molecule may be an antibody capable of binding to the hapten. The use of digoxigenin as a hapten, and the use of anti-digoxigenin as a molecule, are known in the field of diagnostics.

In some embodiments, the enzyme moiety is an enzymatically linked oligonucleotide. The enzymatically linked oligonucleotide comprises: an oligonucleotide capable of hybridizing to a nucleic acid, and an enzyme capable of cleaving a substrate of a 1, 2-dioxetane enzyme. In these embodiments, the enzyme complex comprises an enzyme-linked oligonucleotide that is hybridized to a solid phase comprising a nucleic acid. Contacting the enzyme complex with a 1, 2-dioxetane enzyme substrate to form a reaction mixture, and allowing the reaction mixture to produce light.

In some embodiments, the method may additionally comprise the steps of: providing a sample suspected of comprising nucleic acids; immobilizing the nucleic acid on a solid phase; contacting the immobilized nucleic acid and the enzyme-linked oligonucleotide to form an enzyme complex; and, after adding the aqueous solution of the 1, 2-dioxetane enzyme substrate, detecting light emitted from the reaction mixture, 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 nucleic acid present in the sample.

In some embodiments, the method may additionally comprise the steps of: any unbound enzyme-linked oligonucleotide is removed from the enzyme complex by washing the enzyme complex. This can be achieved by adding and removing buffers compatible with the components of the enzyme complex. Such buffers are well known in the field of diagnostics. Other washes of the solid phase may be performed.

The oligonucleotide may be covalently or non-covalently linked to the enzyme. When non-covalently linked, the oligonucleotide may be covalently linked to a label, and the enzyme may be covalently linked to a molecule that: the molecule is capable of non-covalently binding the label.

In another step of the method, the sample is contacted with an aqueous solution comprising a substrate of a 1, 2-dioxetane enzyme to form a reaction mixture.

In some embodiments, the sample can be added to an aqueous solution comprising a substrate for a 1, 2-dioxetane enzyme, while in other embodiments, an aqueous solution comprising a substrate for a 1, 2-dioxetane enzyme can be added to the sample.

In some embodiments, the reaction mixture may additionally comprise a promoter. The enhancer may comprise CTAB (cetyltrimethylammonium bromide) and other micelle-forming substances. The accelerator may comprise a polymeric quaternary ammonium salt, a polymeric quaternary phosphonium salt, or a combination thereof. The polymeric quaternary ammonium salt can be poly (vinylbenzyltrimethylammonium chloride), poly [ vinylbenzyl (benzyldimethylammonium chloride) ], poly [ vinyl (benzyltributylammonium chloride) ], poly [ vinyl (benzyltripentylammonium chloride) ], or combinations thereof. The polymeric quaternary phosphonium salt can be poly (vinylbenzyltrimethyl phosphonium chloride), poly (vinylbenzyltributylphosphonium chloride), poly (vinylbenzyltrioctylphosphonium chloride), a copolymer comprising poly (vinylbenzyltributylphosphonium chloride) and poly (vinylbenzyltrioctylphosphonium chloride), or a combination thereof.

In some embodiments, the enhancer may additionally comprise an acceptor dye. In these embodiments, the acceptor dye may be a fluorescent dye. In some of these embodiments, the fluorescent dye may be fluorescein.

In another step of these methods, the reaction mixture is allowed to produce light.

In some embodiments, the light can be observed by eye, or can be measured using X-ray film or an instrument capable of detecting and measuring the light produced. Instruments capable of detecting and measuring the light generated include, but are not limited to, photometers, cameras with film or charge coupled cameras.

Viewed from another aspect the invention provides a kit for detecting the presence or amount of an analyte in a sample, said kit comprising an oxidant and an enol ether having the structure [1], both as defined above.

In some embodiments, the oxidizing agent may be selected from: hydrogen peroxide, sodium molybdate, hydrogen peroxide and sodium molybdate, hypochlorite and hydrogen peroxide, aryl endoperoxides, calcium peroxide peroxyhydrate, and combinations thereof. In some of these embodiments, the oxidizing agent may be hydrogen peroxide or hydrogen peroxide and sodium molybdate.

In some embodiments, the kit may further comprise an enhancer as described above. In some embodiments, the kit may further comprise an acceptor dye as described above.

In some embodiments, the kit may further comprise instructions for using the components thereof.

The following examples are intended to illustrate, but not limit, the invention.

Example 1

General synthesis of enol ether phosphate.

a. Synthesis of enol ether phosphoric triester

A、B、R₁And R₆As defined in the detailed description of the invention.

Phosphorus oxychloride (1.5 eq) was added slowly to pyridine (0.7 ml/mmol phenol enol ether, dried over basic alumina overnight) at 0 ℃ under argon atmosphere. A little white smoke was observed, but no precipitate was formed. To the POCl₃To the solution, a solution of phenol enol ether (30 mmol, 1 eq.) in dry THF (3 ml/mmol phenol enol ether) was added through a dropping funnel over 90 minutes. During the addition, a white pyridine hydrochloride precipitate formed. The storage bottle and dropping funnel were rinsed with additional volumes of THF and added to the reaction mixture. The suspension was stirred at 0 ℃ for 15 minutes and at room temperature for 3 hours. The reaction mixture was then cooled back to 0 ℃ and 3-hydroxypropionitrile (3.95 equivalents) was added slowly as a thin stream via syringe. After stirring at 0 ℃ for 5 minutes, the mixture was stirred at room temperature overnight while more white precipitate formed. The precipitate was removed by filtration and washed with EtOAc in hexanes. Concentrated under reduced pressure and combinedTo give a pale yellow oil.

To the crude product was added saturated NaHCO₃Solution (13 ml/mmol phenol enol ether) and then sufficient water is added to dissolve any salt present. The aqueous solution was extracted 3 times with 60% EtOAc in hexanes. The combined organic solutions were washed sequentially with water and brine over anhydrous Na₂SO₄Dried and concentrated by rotary evaporator. The gummy crude product was triturated with 5% EtOAc in hexanes 3 times (heated, cooled to room temperature, then 0 ℃). TLC showed removal by trituration of most of the residual pyridine, and traces of unreacted starting phenol enol ether, and a small amount of diaryl monocyanoethyl phosphate triester by-product. The product was then aspirated under vacuum to constant weight gum.

b. Synthesis of enol ether phosphate

A、B、R₁And R₆As defined in the detailed description of the invention.

To a solution of phosphotriester enol ether (28 mmol, 1 eq.) in anhydrous MeOH (3 ml/mmol phosphotriester) was added a 4.37M solution of NaOMe in MeOH (2 eq) in a thin stream by syringe at 0 ℃ under argon atmosphere. The mixture was stirred at 0 ℃ for a few minutes and then warmed to room temperature. A large amount of precipitate formed at room temperature with stirring. The flask was tapped occasionally to knock the settled solids back into the stirred solution. The thick suspension was stirred overnight. The reaction mixture was placed on a rotary evaporator to remove about half the MeOH volume and 1.5% water/acetone (7 ml/mmol phosphotriester) was added to the remaining suspension. An additional volume of acetone (7 ml/mmol of phosphotriester) was added to transfer most of the powder to the filter. The filter cake was rinsed with cold acetone (2 ml/mmol phosphotriester) and pumped to dryness in a vacuum desiccator to give a white powder.

The crude powder was further purified as follows: it was dissolved in water (0.35 ml/mmol phosphotriester) and the solution was filtered on a Buchner funnel. The storage vessel and filter funnel were rinsed with an additional volume of water (0.35 ml/mmol phosphotriester) and filtered. The combined filtrates were then transferred to the refrigerator bottle and the filter flask was rinsed with an additional volume of water (0.35 ml/mmol of phosphotriester) and added to the solution. When the combined aqueous solutions were added to acetone (13 ml/mmol phosphotriester), a large amount of precipitate precipitated from the solution. An additional volume of acetone (1.4 ml/mmol phosphotriester) was added to dilute the suspension for easier filtration. The suspension was allowed to stand on the bench for 30 minutes and then a white precipitate was collected by filtration. The filter cake was washed several times with acetone and dried in a vacuum desiccator to a constant weight white powder.

Example 2

And (3) synthesizing AMPPD enol ether phosphate.

The synthesis of AMPPD enol ether phenols is reported in U.S. patent No. 5,177,241.

b. And (3) synthesizing AMPPD enol ether phosphate triester.

Phosphorus oxychloride (1.5 eq) was added slowly to pyridine (0.7 ml/mmol phenol enol ether, dried over basic alumina overnight) at 0 ℃ under argon atmosphere. No precipitate was formed. To the POCl₃To the solution, a solution of AMPDD enol ether phenol (30 mmol, 1 eq.) in dry THF (3 ml/mmol phenol enol ether) was added via dropping funnel over 90 minutes.A white pyridine hydrochloride precipitate formed during the addition. The storage bottle and dropping funnel were rinsed with additional volumes of THF and added to the reaction mixture. The suspension was stirred at 0 ℃ for 15 minutes and at room temperature for 3 hours. The reaction mixture was then cooled back to 0 ℃ and 3-hydroxypropionitrile (3.95 equivalents) was added slowly as a thin stream via syringe. After stirring at 0 ℃ for 5 minutes, the mixture was stirred at room temperature overnight while more white precipitate formed. The precipitate was removed by filtration and washed with EtOAc in hexanes. The combined filtrates were concentrated under reduced pressure to give a pale yellow oil. To the crude product was added saturated NaHCO₃Solution (13 ml/mmol phenol enol ether) and then sufficient water is added to dissolve any salt present. The aqueous solution was extracted 3 times with 60% EtOAc in hexanes. The combined organic solutions were washed sequentially with water and brine over anhydrous Na₂SO₄Dried and concentrated by rotary evaporator. The gummy crude product was triturated with 5% EtOAc in hexanes 3 times (heated, cooled to room temperature, then 0 ℃). TLC showed removal by trituration of most of the residual pyridine, and traces of unreacted starting enol ether phenol, and a small amount of diaryl monocyanoethyl phosphate triester by-product. The product was then aspirated under vacuum to constant weight gum.

c. And (3) synthesizing AMPPD enol ether phosphate.

To a solution of AMPPD enol ether phosphotriester (28 mmol, 1 eq) in anhydrous MeOH (3 ml/mmol phosphotriester) at 0 ℃ under argon atmosphere was added a 4.37M solution of NaOMe in MeOH (2 eq) in a thin stream by syringe. The mixture was stirred at 0 ℃ for a few minutes and then warmed to room temperature. A large amount of precipitate formed at room temperature with stirring. The flask was tapped occasionally to knock the settled solids back into the stirred solution. The thick suspension was stirred overnight. The reaction mixture was placed on a rotary evaporator to remove about half the MeOH volume and 1.5% water/acetone (7 ml/mmol phosphotriester) was added to the remaining suspension. An additional volume of acetone (7 ml/mmol of phosphotriester) was added to transfer most of the powder to the filter. The filter cake was rinsed with cold acetone (2 ml/mmol phosphotriester) and pumped to dryness in a vacuum desiccator to give a white powder.

Example 3

ADP-StarEnol ether phosphate and ADP-Star ^®Synthesis of (2)

Phosphonate synthesis is reported in U.S. patent No. 5,582,980, column 5, lines 18-62.

c. ADP-StarAnd (3) synthesizing methoxy enol ether.

A solution of diethyl 1-methoxy-1- (4-chloro-3-methoxyphenyl) methanephosphonate (21.1 g, 65.4 mmol, 1.1 eq) in 140 ml of anhydrous THF was cooled to-78 ℃ under argon and treated over 20 minutes through the dropping funnel with 41 ml (1.6M, 65.4 mmol, 1.1 eq) of a solution of n-butyllithium in hexane. The resulting orange reaction mixture was stirred at-78 ℃ for 15 minutes, then 2-adamantanone powder (8.93 g, 59.5 mmol) was added in one portion. The reaction mixture was stirred at-78 ℃ for 40 minutes, then warmed to room temperature, and finally heated to reflux for 1.5 hours, and any vigorous butane evolution was noted. The reaction mixture was cooled back to room temperature and kept at this temperature overnight. The next morning, volatiles were removed from the reaction mixture by rotary evaporation. Then in saturated NaHCO₃The residue was partitioned between solution and 5% EtOAc in hexanes. The aqueous solution was extracted 3 times with 5% EtOAc in hexanes (ca. 300 ml). The combined organic solutions were washed with brine, over anhydrous Na₂SO₄Dried and passed through a silica gel plug. After concentrating the filtrate, a yellow oil was obtained. The crude product was crystallized in 30 ml MeOH. After a second recrystallization from 20 ml MeOH, 11.99 g (63.2%) of a pale yellow solid were obtained. The combined mother liquors were further purified by silica gel chromatography (0-4% EtOAc in hexanes) and crystallized 2 times in 8 ml MeOH to give 1.74 g (9.2%) of the second crop of product as a pale yellow solid.

。

d. ADP-StarAnd (3) synthesizing enol ether phenol.

NaH (60% in mineral oil, 1.63) was washed with hexanes (3X15 ml)g, 40.8 mmol, 1.3 eq) 3 times, the resulting wet NaH powder was blown dry with a stream of argon and briefly pumped under vacuum for 5 minutes. To a suspension of NaH powder in anhydrous DMF (35 ml) under argon atmosphere at 0 ℃, EtSH (3.1 ml, 42.3 mmol, 1.35 equiv) was added dropwise by syringe over 10 minutes. During the addition process, severe gas evolution occurs immediately; the resulting clear sodium ethylmercaptide solution was stirred at 0 ℃ for 5 minutes and at room temperature for 25 minutes. The solution was cooled back to 0 ℃ and solid ADP-StarThe sodium ethylmercaptide solution was treated once with methoxy enol ether (10 g, 31.4 mmol). Heating the suspension to 120-125 ℃ for refluxing; a homogeneous mixture was obtained during heating and became cloudy during subsequent refluxing. After 2 hours of reflux, TLC indicated the reaction was complete. The reaction mixture was cooled back to room temperature and saturated NaHCO was used₃The solution was quenched. The aqueous solution was extracted 3 times with 20% EtOAc in hexane (ca. 150 mL). The combined organic solutions were washed with brine, over anhydrous Na₂SO₄Dried and passed through a plug of crude silica gel (40-140 mesh). The filtrate was concentrated by rotary evaporation to give a white powder with a slight off-odour. The crude product was purified by trituration. After heating with 30 ml hexanes, then cooling and storage in the refrigerator overnight, 8.78 g (91.8%) of the product was obtained as a white solid after filtration.

。

e. ADP-StarAnd (3) synthesizing enol ether phosphate triester.

Phosphorus oxychloride (4.2 ml, 46.3 mmol, 1.5 equiv.) was added slowly to pyridine (22 ml, dried over basic alumina overnight) at 0 ℃ under argon. A little white smoke was observed, but no precipitate was formed. To the samePOCl₃In the solution, ADP-ion was added through the dropping funnel over 90 minutesStarA solution of enol ester phenol (9.41 g, 30.87 mmol) in 94 ml of anhydrous THF. During the addition, a white pyridine hydrochloride precipitate formed. The storage bottle and dropping funnel were rinsed with an additional 10 ml of THF and added to the reaction mixture. The suspension was stirred at 0 ℃ for 15 minutes and at room temperature for 2 hours and 40 minutes. The reaction mixture was then cooled back to 0 ℃ and 3-hydroxypropionitrile (8.3 ml, 122 mmol, 3.95 equivalents based on phenol) was added slowly as a thin stream via syringe. After stirring at 0 ℃ for 5 minutes, the mixture was stirred at room temperature overnight (about 15.5 hours) with more white precipitate formed. The precipitate was removed by filtration and washed with 60 ml EtOAc in hexanes. The combined filtrates were concentrated under reduced pressure to give a pale yellow oil.

To the crude product was added 400 ml of saturated NaHCO₃To the solution, sufficient water is then added to dissolve any salts present. The aqueous solution was extracted 3 times with 60% EtOAc in hexane (ca. 400 mL). The combined organic solutions were washed with water and brine (150 ml each) over anhydrous Na₂SO₄Dried and concentrated by rotary evaporation. The gummy crude product was triturated 3 times (heated, cooled to room temperature, then 0 ℃) with 40 ml of a 5% EtOAc in hexanes each time. TLC showed that most of the residual pyridine was removed by these triturations, traces of unreacted starting material ADP-StarEnol ether phenol, and a small amount of diaryl monocyanoethyl phosphotriester byproduct. The product was then aspirated under vacuum to constant weight; 13.4 g (92%) were obtained as a pale yellow gum.

。

f. ADP-StarAnd (3) synthesizing enol ether phosphate.

To ADP-StarTo a solution of enol ether phosphotriester (13.83 g, 28.2 mmol) in 85 ml of anhydrous MeOH was added a 4.37M solution of NaOMe in MeOH (12.9 ml, 56.3 mmol, 2 eq) in thin stream via syringe. The mixture was stirred at 0 ℃ for 2 minutes and then warmed to room temperature. A large amount of precipitate formed at room temperature with stirring. The flask was tapped occasionally to knock the settled solids back into the stirred solution. The thick suspension was stirred overnight for 17.5 hours. Use of acetonitrile/NaHCO₃The gradient was monitored by analytical HPLC, indicating the desired product at 8.3 min; the incomplete reaction intermediate, namely, the mono-cyanoethyl mono-aryl phosphodiester, is 13.6 min; the by-product diaryl phosphate diester is at 16.9 min, and ADP-StarPhenol enol ether was used for 19.6 min. The reaction mixture was placed on a rotary evaporator to remove approximately 45 ml of MeOH, and to the remaining suspension was added 3ml of water, followed by 200 ml of acetone. The solution was filtered on a buchner funnel. The storage vessel and filter funnel were rinsed with an additional 10 ml of water and added to the filtrate. The combined filtrates were then transferred to a 1-liter freeze dryer bottle and the filter flask rinsed with an additional 10 ml of water and added to the solution. When the combined aqueous solutions were added to 360 ml of acetone, a large amount of precipitate precipitated from the solution. An additional 40 ml of acetone was added to dilute the suspension for easier filtration later. The suspension was allowed to stand on the bench for 30 minutes and then a white precipitate was collected by filtration. The filter cake was washed several times with acetone (total 200 ml) and dried in a vacuum desiccator to constant weight to give 10.07 g (83.9%) of a white powder as the first crop.

。

When the filtrate was cooled in a refrigerator, more precipitate precipitated from the solution. Filtration gave 1.388 g (11.6%) of an additional second crop of product. Its proton NMR spectrum was identical to that of the first product. Analytical HPLC peak integration for both batches of product was greater than 99.4%.

g. ADP-Star ^®And (4) synthesizing.

According to CDP reported in U.S. Pat. No. 5,582,980Star ^®In the operation of the compound 5 in columns 5 to 6, 2-adamantanone was used instead of 5-chloro-2-adamantanone to synthesize ADP-Star ^®. All other steps and related to CDP-Star ^®Those reported are the same.

Other examples of enol ether phosphates that can be used as substrates include, but are not limited to:

benzothiazole enol ether phosphates and the like as cited in U.S. patent No. 6,355,441.

(3-Phosphoryloxyphenyl) Methoxymethylene Tricycle [7.3.1.0^2,7]Tridec-2, 7-ene disodium salt. U.S. Pat. No. 6,461,876

[ (3-Phosphoryloxyphenyl) (2,2, 2-trifluoroethoxy) methylene]Tricyclic [7.3.1.0^2,7]Tridec-2, 7-ene disodium salt. U.S. patent No. 6,461,876.

(3-Phosphoryloxy-4-chlorophenyl) methoxymethylene tricyclo [7.3.1.0^2,7]Tridec-2, 7-ene disodium salt. U.S. patent No. 6,461,876.

[ (4-methoxy) -4- (3-phosphoryloxyphenyl)]Spiro [1, 2-dioxetane-3, 13' - (8-n-propyl) tricyclo [7.3.1.0^2,7]Tridec-2, 7-ene]Disodium salt. U.S. patent No. 6,461,876.

(3-Phosphoryloxyphenyl) Methoxymethylene adamantan-4, 5-ene disodium salt. U.S. patent No. 6,461,876.

Example 4

General Synthesis of Enanolether glycosides by phase transfer catalysis

To a vigorously stirred mixture of phenol enol ether (1 eq.) and PTC catalyst, Bu at room temperature₄NBr (1.05 equiv.) in 1N NaOH solution and CH₂Cl₂In a two-phase mixture of (1.5 equivalents) per acyl glycosyl bromide in CH is added as a thin stream₂Cl₂The solution of (1). The mixture was stirred for 60 minutes. Saturated NaHCO₃Adding the solution to the reactant, and using CH₂Cl₂The solution was extracted 3 times. Washing the combined CH with water₂Cl₂Solution in anhydrous Na₂SO₄Dried, concentrated and purified by silica gel chromatography to give the protected glycosylenol ether.

The peracylglycosylenol ether (1 eq) was dissolved in 1:1 THF/MeOH, deprotected by addition of 1N NaOH at 0 deg.C, and the reaction was allowed to warm to room temperature. After complete deprotection with solid NaHCO₃The reaction was neutralized, the solvent was removed by rotary evaporation, and purified by reverse phase chromatography.

Examples of peracyl bromoglycosides are: α -D-glucopyranosyl bromide, 2,3,4, 6-tetraacetate (CAS # 572-09-8); α -D-galactopyranosyl bromide, 2,3,4, 6-tetraacetate (CAS # 3068-32-4); alpha-D-glucopyranosuronic acid-1-bromo-1-deoxy-methyl ester, 2,3, 4-triacetate (CAS # 21085-72-3).

Example 5

Glucon-StarSynthesis of enol ether tetraacetate by phase transfer catalysis

a. And (4) synthesizing Glucon-Star enol ether tetraacetate.

To CDP vigorously stirred at room temperatureStarEnol ether phenol (1.02 g, 3 mmol) and PTC catalyst, tetrabutylammonium bromide (1.02 g, 3.15 mmol) in 1N NaOH (20 ml) and 14 ml CH₂Cl₂To the mixture in (1), 6 ml of CH was added₂Cl₂alpha-D-glucopyranosyl bromide in (1), 2,3,4, 6-tetraacetate (2.47 g, 6 mmol). The reaction was stirred for 30 minutes until TLC indicated very little starting material remained. With saturated NaHCO₃The reaction was quenched with the solution. By CH₂Cl₂The aqueous layer was extracted 3 times and the combined organic layers were washed with brine and over anhydrous Na₂SO₄And drying. 4 drops of triethylamine were added to the solution and the solution was passed through a short silica gel column eluting with 100 ml of 40% EtOAc in hexanes to give an orange gum.

After overnight storage at 4 ℃, the crude product was dissolved in a small amount of CH₂Cl₂And chromatographed on silica gel eluting with 20% to 50% EtOAC/hexanes. The fractions containing the coupling product were collected and concentrated to give a pale yellow gum (2.31 g,>100%)。

b. Glucon-Starand (3) synthesizing enol ether.

Crude Glucon-StarTo a solution of enol ether tetraacetate (1.5 g theoretical yield, 3 mmol) was added 15 drops of a 4.37M solution of NaOMe in MeOH by pipette. The mixture was stirred overnight and a small amount of acetate hydrolysis occurred. An additional 30 drops of 4.37M NaOMe in MeOH was added and the mixture turned from yellow to orange, as confirmed by TLC, and hydrolysis was complete after 4 hours. Ammonium chloride (1 g) was added to quench the reaction, and the solution was stirred for 1 hour. Dichloromethane was added to precipitate the product and 5% MeOH/CH was added₂Cl₂Until no more precipitate precipitated out. The precipitate was collected by filtrationAnd with 5% MeOH/CH₂Cl₂And (6) washing. The crude gum was purified by silica gel chromatography, first eluting with 30% EtOAc in hexanes to recover CDP-StarEnol ether phenol, then with 5-10% MeOH/CH₂Cl₂Washing the column to obtain Glucon-StarEnol ether as a light yellow foam (1.18 g, 79%).

Example 6

General Synthesis of Enanolethlycosides by Schmidt glycosylation

Stirring of peracylglycosyltrichloroacetimidate (1.2-1.5 equivalents) in CH at room temperature under argon₂Cl₂To a solution in (6 ml/equivalent phenol) was added solid phenol enol ether (1 equivalent) in one portion. The mixture was cooled to-23 ℃ and BF was applied over a period of 10 minutes₃•OEt₂(0.2 eq) in CH₂Cl₂The solution in (0.6 ml/eq phenol) was slowly worked up. In BF₃•OEt₂During the addition, the reaction mixture became cloudy and slowly warmed to 0 ℃ over 1.5 hours. With Et₃N (5 equivalents of BF)₃•OEt₂) The reaction was quenched and stirred at 0 ℃ for 10 minutes. In saturated NaHCO₃Solution and CH₂Cl₂The reaction mixture was partitioned, purified by silica gel chromatography, and the over-acylated glycosyl enol ether was collected as a foam.

Peracylglycosyltrichloroacetimidate synthesis is reported in reviews by r.r. Schmidt, adv. carbohydr. chem. biochem., 1994, 50:21 and references cited therein.

Example 7

Synthesis of glucuronoxylene ethers by Schmidt glycosidation

a. Synthesis of peracylglucuronyl AMPPD enol ether.

Peracylglucuronyltrichloroacrylimidate (1.2 equivalents) in CH at room temperature under argon₂Cl₂(24 ml/eq) to the solution was added a solid phenol enol ether (1 eq). The mixture was cooled to-25 ℃ and boron trifluoride diethyl etherate (0.2 eq) in CH was added slowly over 10 min₂Cl₂And (4) solution treatment. The resulting cloudy mixture was stirred at the following temperature gradient: stirring at-23 deg.C to-20 deg.C for 1 hr, at-20 deg.C to-10 deg.C for 30 min, and at-10 deg.C to +5 deg.C for 30 min. Et at +5 ℃₃The reaction was quenched for 15 min with N, followed by addition of saturated NaHCO₃And (3) solution. By CH₂Cl₂The mixture was extracted, washed with water, over anhydrous Na₂SO₄Dried and a small amount of Et is added₃N is added to the organic solution. After rotary evaporation and silica gel chromatography, the crude product was obtained.

b. Synthesis of glucuronyl AMPPD enol ethers.

Dissolving the peracylglucuronyl AMPPD enol ether (1 equivalent) in 1:1 THDeprotection was performed in F/MeOH at 0 deg.C by addition of 1N NaOH, then warmed to room temperature. After complete deprotection with solid NaHCO₃The reaction was neutralized, the solvent was removed by rotary evaporation, and purified by reverse phase chromatography.

Example 8

Conversion of enol ethers to dioxetanes in aqueous solution

The 2 enol ether phosphates were successfully converted to their corresponding 1, 2-dioxetane alkaline phosphatase substrates by oxidation in aqueous alkaline conditions. Preparation of 1, 2-dioxetane, AMPPD, from enol ether precursors^®And ADP-StarAnd subsequently used in an alkaline phosphatase detection assay and an IL-6 ELISA (enzyme-linked immunosorbent assay). The following details the model oxidation conditions and confirmation of dioxetane formation.

a. Oxidation of AMPPD enol ether phosphate to AMPPD^®。

As shown in the reaction scheme depicted in FIG. 1, Na is used₂MoO₄/H₂O₂An oxidation system effective to oxidize AMPPD enol ether phosphate (AMPPD-EE) to the 1, 2-dioxetane substrate AMPPD in an aqueous solution at an alkaline pH^®. The reagents used for this purpose are:

AMP buffer: 0.1M Aminomethylpropanol buffer, pH 9.5.

AMPPD^®(Life Technologies), 1mg/mL in AMP buffer

AMPPD-EE, 1mg/mL (2.3mM) in AMP buffer (2.5 mM).

H₂O₂Stock (c): 10% (3M) aqueous solution (from 50% solution, Aldrich).

Na₂MoO₄Stock (c): 4.8mg/mL (Aldrich), about 20 mM.

Combine 10 μ L AMPPD-EE (0.23 mM), 40 μ L AMP buffer, 40 μ L L H₂O₂Stock (1M) and 20 μ L Na₂MoO₄The stock (3.6mM) was allowed to react at 37 ℃ for 1 hour and then at 55 ℃ for 10 minutes. The progress of the reaction was monitored by HPLC. The HPLC trace of the elution peak of starting material (AMPPD-EE) is shown in fig. 2 as a control trace. The desired 1, 2-dioxetane (AMPPD)^®) The HPLC trace of the elution peak of (a) is shown in fig. 3. The HPLC traces of FIGS. 4 and 5 show that AMPPD-EE (8.1 min) oxidizes to the desired dioxetane substrate, AMPPD^®(7.3 min). The reaction clearly produced a product (AMPPD) in 1 hour 10 minutes^®)。

b. ADP-StarOxidation of enol ether phosphate to ADP-Star ^®。

According to the reaction scheme shown in FIG. 6, in alkaline pH, Na is used₂MoO₄/H₂O₂Oxidation of ADP-Star enol ether phosphate (ADP + EE) to ADP-Star ^®(ADP). The reagents used were:

AMP buffer: 0.1M Aminomethylpropanol buffer, pH 9.5.

0.1M Na₂MoO₄(Aldrich)。

ADP-EE-O stock: 10mg ADP + EE + 50. mu.L 0.1M Na in 2mL AMP buffer₂MoO₄Stock (11.7 mM ADP-EE and 2.5mM Na)₂MoO₄)。

H₂O₂Stock (c): 10% (3M) aqueous solution (from 50% solution, Aldrich).

100 μ L ADP-EE-O stock was pooled with 40 μ L H₂O₂The stock phases were combined and the mixture was allowed to react at 55 ℃ for 1 hour. After this time, the reaction mixture was diluted with 0.5mL of AMP buffer to give a solution containing 1mg/mL (2.3mM) of the resulting 1, 2-dioxetane and 0.5mM Na₂MoO₄The solution of (1). This solution is called ADP x-EE oxidation mixture. The following certificateFrom real ADP EE to ADP-Star ^®Oxidation of (2): the resulting dioxetanes were activated with alkaline phosphatase and the light emission was measured on a Turner photometer. The following reagents were used for this purpose:

0.1M Aminomethylpropanol buffer, pH 9.5 (AMP buffer),

CDP-Star ^®(CDP, Life Technologies: 6.2mg/mL (12.5mM) in AMP buffer,

ADP-Star ^®(ADP. sup.,. Life Technologies): 1mg/mL (2.2mM) in 0.1M AMP buffer,

ADP-EE oxidation mixture 1mg/mL (2.3mM) in 0.1M AMP buffer,

TBQ 10X Sapphire II ™ chemiluminescence promoters (Life Technologies), and

alkaline phosphatase stock (AP): 8ng/mL (from 17.4mg/mL concentrate).

10 μ L ADP or ADP-EE oxidation mixture (0.22 or 0.23mM, respectively), 10 μ L TBQ, 80 μ L AMP buffer and 10 μ L AP were pooled and luminescence from the mixture was measured continuously for 25 minutes at 37 ℃. 1.6 μ L CDP (0.2mM), 10 μ L TBQ, 80 μ L AMP buffer and 10 μ L AP were pooled and luminescence from this mixture was measured continuously for 25 minutes at 37 ℃.

FIG. 7 shows ADP-StarLight emission curves (for ADP-StarDioxetane control and ADP-Star2 products of enol ether phosphate oxidation) show a better performance than commercially available CDP-Star ^®Dioxetane gives higher light emission than did dioxetane.

Example 9

Thermal stability of reagents

Examine AMPPD^®AMPPD enol ether phosphate (AMPPD-EE) and AMPPD-EE aqueous solution in the presence of Na₂MoO₄Thermal stability in the presence. AMPPD^®The thermal half-life at 40 ℃ was 40 days and the half-life at 55 ℃ was reduced to 10 days, as shown in FIGS. 8 and 10. In contrast, AMPPD enol ether phosphate showed almost no thermal decomposition at 40 ℃ for more than 4 months and showed minimal thermal decomposition at 55 ℃ for more than 5 months, as shown in fig. 9 and 10. AMPPD-EE was also examined for Na₂MoO₄Thermal stability at 40 ℃ in the presence of AMPPD enol ether phosphate in the presence of Na was not observed₂MoO₄Thermal decomposition in the presence, as shown in fig. 11.

In addition to the evaluation of thermal stability of enol ether phosphate, Na was also examined₂MoO₄、H₂O₂And urea H₂O₂(H is common in commercial applications₂O₂Alternative) to the reported thermal stability. Sodium molybdate and hydrogen peroxide did not exhibit any significant thermal decomposition as shown in table a.

TABLE A evaluation of the thermal stability of the oxidizing reagents

Dioxobutanes prepared in situ (such as AMPPD) are contemplated^®And ADP-Star ^®) Has a stability similar to that of the dioxetane produced. The dioxetane prepared in situ can be stored for future use under normal restrictions, for example, for 6 months at 4 ℃.

Example 10

Chemiluminescent assay design using enol ether substrates-method A

In order to adapt the in situ 1, 2-dioxetane substrate formation to the assay design, it is possible to integrate the in situ substrate formation in several ways. For example, an assay can be performed in which enzymatic activity or enzymatic labeling produces a 1, 2-dioxyheterocycleThe butane substrate precursor (enol phenolate) as a first step, then oxidized in situ to 1, 2-dioxetane in a second step, which decomposes after formation to produce a chemiluminescent signal readout, thereby producing a detectable signal (method a). In this method, the oxidation step may be considered a "stop" protocol. Alternatively, an assay can be run in which the in situ oxidation of the 1, 2-dioxetane precursor to 1, 2-dioxetane is the first step, and in which enzymatic activity or enzymatic labeling produces 1, 2-dioxetane phenolate as the second step (method B). In part IV (Na)₂MoO₄And H₂O₂Potential quenching effect on alkaline phosphatase activity) describes the second assay, method B, in which the first step is oxidation followed by enzymatic detection.

Method a (enzyme activation followed by oxidation):

1) enzymatic activation of the enol ether precursor of the 1, 2-dioxetane substrate (e.g., by alkaline phosphatase in AMP buffer, resulting in an enol ether phenolate).

2) Oxidation of enol ether phenolates (e.g. by H in buffer pH 10₂O₂/Na₂MoO₄)。

Method a is shown in fig. 12. In this method, in situ oxidation to a substrate following enzyme activation may be suitable for an endpoint assay readout, where the cumulative signal is measured after enzyme activity. The step of oxidation to substrate also functions as an assay stopping protocol.

The reagents used were:

AMP buffer 0.1M aminomethylpropanol buffer, pH 9.8.

TBQ 10X Sapphire II ™ chemiluminescence promoters (Life Technologies),

AP alkaline phosphatase stock at 8, 1.6, 0.32, 0.064ng/mL (from 17.4mg/mL concentrate),

AMPPD-EE 1mg/mL (2.5mM) in AMP buffer,

H₂O₂stock (c): 10% (3M) aqueous solution (from 50% solution, Aldrich),

Na₂MoO₄stock (c): 0.5M Na₂MoO₄ (Aldrich)，

Phosphate 2M K₂HPO₄pH 9.6, and

comparison:

merging 10 mu L AMPPD^®(0.21mM), 10 μ L TBQ, 80 μ L AMP buffer and 10 μ L AP (at different concentrations), luminescence from the mixture was measured continuously for 30 minutes at 37 ℃.

AMPPD-EE In-situ oxidation:

10 μ L AMPPD-EE (0.21mM) + 10 μ L TBQ + [10 μ L AP (at different concentrations) + 10 μ L AMP buffer at 37 ℃ for 1 hour] + [8.5µL Na₂MoO₄(35mM) + 50 μ L phosphate + 25 μ L H₂O₂Stock (0.63M), sonicated for 30 seconds at 37 deg.C]Read for 90 min, read for 5 min (light).

Using method a, AMPPD enol ether phosphate (AMPPD-EE): 1) dephosphorylation, using alkaline phosphatase (cross dilution range) (10 μ L AMPPD-EE (0.21mM) + 10 μ L TBQ + [10 μ L AP (at different concentrations) + 10 μ L AMP buffer at 37 ℃ for 1 hour]) 2) in situ oxidation to AMPPD^®[8.5µL Na₂MoO₄(35mM) + 50 μ L phosphate + 25 μ L H₂O₂Stock (0.63M), sonicated for 30 seconds at 37 deg.C]And 3) comparison from in situ AMPPD^®Relative to AMPPD^®Light emission of control (read every 5 minutes for 90 minutes). From in situ AMPPD^®And control AMPPD^®A very similar linear chemiluminescent readout of the alkaline phosphatase dilution curve was generated, as shown in fig. 13 and 14.

Example 11

Na₂MoO₄And H₂O₂Potential quenching effect on alkaline phosphatase activity

Before developing assay conditions for in situ dioxetane production using method B, we investigated whether reagents from the oxidation system would adversely affect alkaline phosphatase activity. The experimental results show that hydrogen peroxide does not affect alkaline phosphatase activity, but sodium molybdate significantly quenches alkaline phosphatase activity with up to 85% light reduction (see figure 15). Reducing the concentration of sodium molybdate to<1mM, most of the alkaline phosphatase activity was restored (see FIG. 16). Based on these results, method B in situ dioxetane production was developed using much lower sodium molybdate concentrations in the oxidation system. Any quenching effect from the singlet oxygen generation/reaction system may be enzyme specific and must be evaluated for any enzyme/detection substrate pair. For example, Na₂MoO₄The quenching may be specific for alkaline phosphatase and/or related enzyme families, and may not exhibit a quenching effect on other hydrolytic enzymes.

The reagents used were:

AMP buffer: 0.1M Aminomethylpropanol buffer, pH 9.8,

AMPPD^®: 1mg/mL AMPPD (Life Technologies) in AMP buffer

TBQ 10X Sapphire II ™ chemiluminescence promoters (from Life Technologies),

AP alkaline phosphatase stock, 40ng/mL (from 17.4mg/mL concentrate),

H₂O₂stock (c): 10% (3M) aqueous solution (from 50% solution, Aldrich),

Na₂MoO₄stock (c): 0.5M (Aldrich),

the experimental conditions are as follows:

comparison:

merging 10 mu L AMPPD^®(2.3mM), 10 μ L TBQ, 120 μ L AMP buffer and 10 μ L AP (40ng/mL), and luminescence from the mixture was continuously measured at 37 ℃ for 30 minutes.

And (3) testing:

merging 10 mu L AMPPD^®(2.3mM), 10 muL TBQ, 80 muL AMP buffer, 25 muL L H₂O₂Stock (10%) and 10 μ L AP (40ng/mL), and luminescence from this mixture was measured continuously at 37 ℃ for 30 minutes.

Merging 10 mu L AMPPD^®(2.3mM), 10 muL TBQ, 115 muL AMP buffer solution, different amounts of Na₂MoO₄Stock and 10 μ L AP (40ng/mL), and luminescence from this mixture was measured continuously at 37 ℃ for 30 minutes.

Example 12

Chemiluminescent assay design using enol ether substrates-method B

An alternative to integrating the in situ dioxetane substrate production into the assay is to oxidize the enol ether phosphate in a first step, which can be performed in the assay well or in a discrete container from which the substrate is added to the assay well. This step is followed by enzymatic activation (e.g., dephosphorylation in an alkaline phosphatase-based assay) and chemiluminescent readout of the in situ dioxetane substrate. Method B can be used for dynamic mode assays to measure assay signals generated over time. Method B also allows the user to perform a first step (in situ dioxetane generation) prior to the assay if workflow flexibility is required.

Method B (post-oxidation enzyme activation):

1) oxidation of enol ether precursors of 1, 2-dioxetane substrates (e.g., by H in buffer pH 10₂O₂/Na₂MoO₄To yield a 1, 2-dioxetane substrate).

2) Enzymatic activation of the in situ prepared 1, 2-dioxetane substrate (e.g., by alkaline phosphatase in AMP buffer to give 1, 2-dioxetane phenolate).

Method B is shown in fig. 17. In the process, AMPPD enol ether phosphate is oxidized in situ to AMPPD^®Dephosphorylation with alkaline phosphatase (across dilution range) and comparison from in situ AMPPD^®Relative to AMPPD^®Control light emission. From in situ AMPPD^®And control AMPPD^®A very similar linear chemiluminescence readout for alkaline phosphatase dilution curves was also generated (see fig. 18 and 19). The experimental conditions used are as follows.

Oxidation of AMPPD enol ethers to AMPPD as follows^®: combine 200 μ L of 5mg/ml (9mM) AMPPD-EE +5 μ L of 0.1M Na in 0.1M aminomethylpropanol buffer pH 9.8 (AMP buffer)₂MoO₄ (1.8mM) + 75µL 10% H₂O₂(0.8M) and the mixture was incubated at 55 ℃ for 1 hour. AMP buffer was then added to a final volume of 1mL to obtain a solution containing oxidized AMPPD-EE (AMPPD-EE-O) at a concentration of about 1mg/mL (2.5mM) and 0.5mM Na₂MoO₄The solution of (1). The luminescent property of AMPP-EE-O and AMPPD are combined^®The luminescence properties of (a) were compared.

Reagents used for the assay:

AMP buffer 0.1M aminomethylpropanol buffer, pH 9.8,

AMPPD 1mg/mL (2.3mM) AMPPD (Life technologies) in AMP buffer,

AMPPD-EE-O: 1mg/mL (2.5mM) in AMP buffer,

TBQ 10X Sapphire II ™ chemiluminescence promoters (Life Technologies),

alkaline phosphatase at a concentration of 40, 8, 1.6, 0.32, 0.064ng/mL (from 17.4mg/mL concentrate).

Luminoskan Measuring ( Measurements were performed in triplicate )：

20 μ L (0.46mM) AMPPD, 10 μ L TBQ, 60 μ L AMP buffer and 10 μ L AP (4, 0.8, 0.16, 0.032, 0.0064ng/mL) were combined and luminescence from the mixture was continuously measured at room temperature for 2 hours.

20 μ L of AMPPD-EE-O (0.5mM), 10 μ L of TBQ, 60 μ L of AMP buffer and 10 μ L of AP (4, 0.8, 0.16, 0.032, 0.0064ng/mL) were combined and luminescence from the mixture was continuously measured at room temperature for 2 hours.

Also as previously discussed, ADP-Star ^®From ADP-StarIn situ generation of enol ether phosphate. As shown in FIG. 7, from in situ ADP-Star ^®And control ADP-StarThe light emission curves of (a) are overlapping. As described below, method B was modified to accommodate ADP-StarEnol ether phosphate solubility.

Reagents used：

Solution A: 23.3 mM ADP-Star-EE in 30% acetonitrile/70% sodium carbonate solution containing 12.5mM Na₂MoO₄。

Solution B: 10% H₂O₂。

Sodium carbonate solution: 32 g of sodium bicarbonate + 12 g of sodium carbonate in 10 l of water

Combine 1mL of solution A and 0.7mL of solution B and heat at 55 ℃ for 15 minutes (until the color disappears) to give 13.7 mM in situ ADP-StarStock solutions.

And (3) testing:

1.7mL of in situ ADP-StarStock solutions were combined with 56.3mL of 0.1M aminomethylpropanol buffer, pH 9.5, and 1mg/mL of Sapphire II ™ chemiluminescence promoter (Life Technologies)And, to obtain a peptide containing 0.4mM in situ ADP-StarAnd a 1mg/ml solution of a Sapphire II cell line. ADP-generated in situ in alkaline phosphatase dilution curvesStarPerformance of (D) in comparison with dioxetane CSPD, ADP-StarAnd CDP-StarSimilar to control dioxetanes (see fig. 20 and 21).

The principle of in situ dioxetane substrate production can be readily adapted for enzyme assay use as demonstrated using similar enzyme dilution curves generated by 2 different assay/substrate production methods (method a and method B) using in situ generated AMPPD and control AMPPD. In situ ADP-StarAnd control dioxetane CSPD^®(Life Technologies)、ADP-Star ^®And CDP-Star ^®Comparable results were obtained with method B. The use of alkaline phosphatase substrates and assays confirm these substrate generation and assay design principles, but may be universally applied to the assay of dioxetane substrates and other hydrolytic enzymes such as beta-galactosidase, beta-glucuronidase, beta-glucosidase and neuraminidase.

Example 13

Model recombinant human interleukin 6 (rhIL-6) ELISA using enol ether substrates

1. rhIL-6 ELISA using in situ AMPPD detection.

In comparison with in situ AMPPD and control AMPPD^®The model recombinant rhIL-6 detection ELISA was run in parallel to evaluate the performance of in situ AMPPD in immunoassays. Experiments have shown that using "equimolar" amounts of substrate, assuming 100% conversion of enol ether phosphate, in situ generated AMPPD behaves like control AMPPD in the assay^®Equally well (see fig. 22). The in situ AMPPD can be prepared in the ELISA incubation step without adding additional time to the assay and without any further inconvenience compared to the ELISA assay itself. Total assay sensitivity and dynamic range and use of CDP-Star ^®(CDP.) detection of internal rhIL-6 ELISA very equivalent。

rhIL-6 ELISA Plate preparation (96- Microtiter plate well ) ：

1. ELISA plates were coated overnight (w /) with capture antibody (2mg/mL anti-human IL-6).

2. Washing the plate holes.

3. The plate was blocked with 300. mu.L/well of blocking buffer (1 XPBS/0.02% Tween 20/1% BSA).

4. Washing the plate holes.

rhIL-6 And (3) determination:

1. mu.L of sample or standard (3000 pg/. mu.L-0.0003 pg/mL rhIL-6, w/1:6 dilution) was added to the wells and incubated for 30 minutes at room temperature.

2. Washing plate hole

3. Add 100u L12.5 ng/ml biotinylated detection anti IL6 antibody (R & D Systems), and at room temperature 15 minutes, washing plate hole.

4. 100 μ L of alkaline phosphatase-conjugated streptavidin (Jackson Laboratories) was added at a dilution of 1:40,000.

5. Washing the plate holes.

6. 100ul of substrate solution was added to each well and incubated at room temperature for 30 min.

7. RLU was measured for each well using a luminometer at a temperature of 25 ℃ for 30 minutes.

AMPPD In situ dioxetane preparation of：

Solution a was prepared by combining the following solutions to give a 12.7 mM stock solution of AMPPD enol ether phosphate.

5mg/mL AMPPD enol ether phosphate (from example 2; molecular weight 394).

2mL of 0.1M aminomethylpropanol buffer, pH 9.8 (AMP buffer).

50 μL 0.1M Na₂MoO₄ _。

Step 1-preparation of solution B.

100 μ L of solution A

38 μL 10% H₂O₂(preparation of 9.2 mM AMPPD)^®Molecular weight 426)

The solution was heated at 55 ℃ for 1 hour (O elimination)₂Bubbling, color becoming clear)

This is solution B. In this step, a chemiluminescent promoter may also be added.

Step 2 (2.54 mM) — prepare solution C containing 2.54 mM AMPPD.

138 μ L of solution B (with 9.2 mM in situ generated AMPPD)^®)

362 μ L of 0.1M AMP buffer, pH 9.8

This contained 2.54 mM AMPPD^®Solution C of (1).

Step 2 (4 mM) -prepare solution C containing 4mM AMPPD.

138 μ L of solution B (with 9.2 mM in situ generated AMPPD)^®)

149.4 μ L0.1M AMP buffer, pH 9.8

This contained 4mM AMPPD^®Solution C of (1).

Step 3 (0.25 mM) -prepare solution D containing 0.25 mM AMPPD.

1mL of solution C (2.54 mM AMPPD)^®)

1 mL10X Sapphire II (Life Technologies)

8 mL of 0.1M AMP buffer, pH 9.8

This contained 0.25 mM AMPPD^®Solution D of (1).

100 μ L of solution D was added to each well.

Step 3 (0.4 mM) — prepare solution D containing 0.4mM AMPPD.

1ml of solution C (4 mM AMPPD)^®)

1 ml 10X Sapphire II (Life Technologies)

8 ml of 0.1M AMP buffer, pH 9.8

This contained 0.4mM AMPPD^®Solution D of (1)

100 μ L of solution D was added to each well.

This establishes comparative assay conditions as the standard assay test uses 0.4mM 1, 2-dioxetane and 1mg/ml accelerator.

In another set of experiments, the oxidation time to generate in situ AMPPD was varied and then control AMPPD was used^®Substrate performance was assessed by rhIL-6 ELISA. Time course experiments showed that most of the AMPPD enol ether phosphate appeared to be converted to in situ AMPPD before 30 minutes. The use of 15 minutes of in situ generated AMPPD had a very minor impact on immunoassay performance (sensitivity and dynamic range) relative to 60 minutes of in situ generated AMPPD. There was also a very slight difference in the detection signal (RLU) using 30 minutes of in situ generated AMPPD versus 60 minutes of in situ generated AMPPD. Such as T_oThe results indicate that some conversion of AMPPD enol ether phosphate to in situ AMPPD may occur during the assay (see figure 23).

2. rhIL-6 detection ELISA Using in situ ADP-Star detection

The reference detection experiment compares the in-situ ADP-StarRelative to control dioxetane CSPD^®、ADP-Star ^®And CDP-Star ^®Light emission (in RLU) and signal-to-noise ratio (S: N). Maximum light emission as a function of dioxetane substrate, with CSPD^®Luminescence at 30 minutes, CDP-Star ^®Luminescence is achieved in 45 minutes, ADP-Star ^®Luminescence was achieved at 60 minutes (see fig. 24 and 25).

Performing and in-situ ADP-StarAnd control dioxetane CSPD^®、ADP-Star ^®And CDP-Star ^®Parallel modeling of rhIL-6 ELISA to evaluate in situ ADP-StarPerformance in immunoassays. Experiments show that ADP generated in situStarPerformance of (a) was comparable to the control dioxetane substrate. ADP-containing in situ preparationStarThe assay sensitivity (lower limit of detection of IL-6), S/N and dynamic range were similar for all substrates included (see FIG. 26).

In application development, it may be desirable to eliminate one or more of the solution preparation steps a-D described above. In certain applications, it may also be desirable to modify the solution composition. One example of modifying the composition of the solution is to prepare a detection solution in which the enhancer is present in the oxidation step and then diluted with a buffer to assay conditions. Preparation using improved in situ dioxetanes with ADP-Star ^®The alkaline phosphatase detection curves show an in situ ADP-Star(deep blue and yellow emission curves) similar sensitivity (see figure 27). Indeed, in situ ADP-StarThe detection solution produces a more stable light emission after reaching the emission maximum. In addition, improved in situ ADP-StarDioxetane preparation resulted in comparable immunoassay detection sensitivity to control dioxetanes (see fig. 28 and 29).

Generated in situ in the accelerator solution StarADP- ：

a) Use 11.7 mM ADP in AMP buffer-StarEnol ether phosphoric acidSalt (from example 3) and 10 mg/ml Sapphire II (Life Technologies) + Na₂MoO₄And oxidizing. After oxidation, the solution was diluted to 1x with AMP buffer. Final solution concentration: 0.58mM in situ generated ADP-Star/0.5mg/ml Sapphire II.

b) 4mM ADP in AMP buffer was used-StarEnol ether phosphate (from example 3) and 10 mg/ml Sapphire + Na₂MoO₄And oxidizing. After oxidation, the solution was diluted to 1x with AMP buffer. And finally: 0.4mM in situ generated ADP-Star/1mg/ml Sapphire II.

Having described specific embodiments of the present invention with reference to the accompanying drawings, it is to be understood that the invention is not limited to those precise embodiments, and that various changes and modifications may be effected therein by one of ordinary skill in the pertinent art without departing from the scope or spirit of the present invention as defined by the appended claims.

Claims

1. A method for generating light, the method comprising the steps of:

(a) providing an oxidizing agent;

(b) providing an enol ether having the structure:

wherein the content of the first and second substances,

a and B are independently selected from: a linear alkyl group having 1 to 20 carbon atoms, a linear alkenyl group having 2 to 20 carbon atoms, a branched alkyl group having 3 to 20 carbon atoms, a branched alkenyl group having 3 to 20 carbon atoms, a cycloalkyl group having 3 to 20 carbon atoms, a cycloalkenyl group having 3 to 20 carbon atoms, a cycloheteroalkyl group having 3 to 20 carbon atoms, a cycloheteroalkenyl group having 3 to 20 carbon atoms, a polycycloalkyl group having 4 to 60 carbon atoms, a polycyclocycloalkenyl group having 4 to 60 carbon atoms, a polycycloheteroalkyl group having 4 to 60 carbon atoms and a polycycloheteroalkenyl group having 4 to 60 carbon atoms, any of which may be unsubstituted or substituted with one or more electronically active groups, solubilizing groups or photo-enhancing groups, and wherein A and B together form the cycloalkyl group, Cycloalkenyl, polycycloalkyl or polycycloalkenyl, one of the carbon atoms of which is one of the 2 carbon atoms forming the double bond of the enol ether,

R₁is an alkyl group having 1 to 20 carbon atoms, an aryl group having 6 to 14 carbon atoms, an aralkyl group having 7 to 15 carbon atoms, a heteroaryl group having 4 to 20 carbon atoms or a heteroaralkyl group having 5 to 20 carbons,

t is an aryl or heteroaryl ring capable of emitting light, an

R₂Is an enzyme-cleavable group that contains a bond that can be cleaved by an enzyme moiety to generate 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 capable of cleaving the 1, 2-dioxetane enzyme substrate;

(e) contacting the enzyme complex with the aqueous solution comprising a 1, 2-dioxetane enzyme substrate to form a reaction mixture; and the combination of (a) and (b),

(f) causing the reaction mixture to generate light.

2. The method of claim 1, wherein the oxidizing agent is selected from the group consisting of: hydrogen peroxide, sodium molybdate, hydrogen peroxide and sodium molybdate, hypochlorite and hydrogen peroxide, aryl endoperoxides, calcium peroxide peroxyhydrate, and combinations thereof.

3. The method of claim 2, wherein the oxidizing agent 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 are

。

6. The method of claim 1, wherein R₁Is an alkyl group having 1 to 2 carbon atoms or a trifluoroalkyl group having 1 to 2 carbon atoms.

7. The method of claim 1, wherein T is

Wherein the content of the first and second substances,

R₃、R₄and R₅Independently selected from: H. f, Cl, Br, I, cyano, nitro, sulfonate, sulfate, trifluoromethyl, trifluoroethyl, compounds containing from 1 to 20 carbon atomsStraight chain alkyl, branched alkyl having 3 to 20 carbon atoms, straight chain alkenyl having 2 to 20 carbon atoms, branched alkenyl having 3 to 20 carbon atoms, cycloalkyl having 3 to 20 carbon atoms, cycloalkenyl having 3 to 20 carbon atoms, cycloheteroalkyl having 3 to 20 carbon atoms, cycloheteroalkenyl having 3 to 20 carbon atoms, polycycloalkyl having 4 to 60 carbon atoms, polycycloalkenyl having 4 to 60 carbon atoms, polycycloheteroalkyl having 4 to 60 carbon atoms, polycycloheteroalkenyl having 4 to 60 carbon atoms, alkoxy having 1 to 20 carbon atoms, aryl having 6 to 14 carbon atoms, aryloxy having 6 to 14 carbon atoms, ester having 2 to 21 carbon atoms, trialkylammonium having 3 to 30 carbon atoms, Trialkylphosphonium having 3 to 30 carbon atoms, alkylamido having 2 to 21 carbon atoms, arylamido having 7 to 15 carbon atoms, alkylcarbamoyl having 2 to 21 carbon atoms, arylcarbamoyl having 7 to 15 carbon atoms, alkylsulfonamido having 1 to 20 carbon atoms, arylsulfonamido having 6 to 14 carbon atoms, trialkylsilyl having 3 to 60 carbon atoms, triarylsilyl having 18 to 42 carbon atoms, alkylarylsilyl having 7 to 32 carbon atoms, alkylamidosulfonyl having 1 to 20 carbon atoms, arylamidosulfonyl having 6 to 14 carbon atoms, alkylsulfonyl having 1 to 20 carbon atoms, arylsulfonyl having 6 to 14 carbon atoms, Alkylthio having 2 to 20 carbon atoms and arylthio having 6 to 14 carbon atoms, and

x is a sulfur atom, an oxygen atom or a nitrogen atom.

8. The method of claim 1, wherein OR₂Is phosphate, acetate, 1-phospho-2, 3-diacylglycerol, adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, adenosine, alpha-D-galactoside, beta-D-galactoside, alpha-D-glucoside, beta-D-glucoside, alpha-D-mannoside, beta-fructofuranoside, beta-D-glucuronide orWherein B is₁、B₂And B₃Each independently is H or alkyl (branched or straight chain) of 1 to 4 carbon atoms.

9. The method of claim 8, wherein R₂Is that

。

10. The method of claim 1, wherein R₂Is E-L-Nuc-Z, wherein E is a group comprising an electrophilic atom which, upon enzymatic cleavage of the Z group, is attacked by the electron pair of the Nuc group and releases the 1, 2-dioxetane enzyme substrate anion by vicinal assistance; l is a linking group; nuc is a nucleophilic atom; and Z is an enzyme-cleavable group; wherein

E is carboxy, carbonyl, methylene substituted with a leaving group, phosphate, carbonate, xanthate, sulfite, sulfonate, bisulfite or disulfide;

l is selected from: methylene or polymethylene radicals containing from 1 to 4 carbon atoms, - (CH)₂)_m-O-(CH₂)_n、-(CH₂)_m-S-(CH₂)_n-, or- (CH)₂)_m-NR₆-(CH₂)_n-, where m and n are 0 to 3 and m + n is 2 or 3, where

R₆Is an alkyl group having 1 to 10 carbon atoms, and the linking group may be substituted with an alkyl group having 1 to 24 carbon atoms, an alkenyl group having 2 to 24 carbon atoms, an alkyl group having 1 to 24 carbon atoms, and mono-or di-substituted with an acyloxy group having 1 to 24 carbon atoms, an alkenyl group having 2 to 24 carbon atoms, and mono-or di-substituted with an acyloxy group having 1 to 24 carbon atoms, an aryl group having 6 to 10 carbon atoms, an aryl group having 1 to 24 carbon atomsAlkyl of carbon atoms is mono-or di-substituted and is substituted by phenyl, hydroxyphenyl, indolyl, mercapto, alkylthio of 1 to 4 carbon atoms, hydroxy, carboxy, amino, guanidino, imidazole or carbamoyl or alkenyl of 2 to 24 carbon atoms and is substituted by phenyl, hydroxyphenyl, indolyl, mercapto, alkylthio of 1 to 4 carbon atoms, hydroxy, carboxy, amino, guanidino, imidazole or carbamoyl;

nuc is an oxygen atom or a sulfur atom; and is

Z is phosphoryl, acetyl, 2, 3-diacylglyceroyl-1-phosphate, adenosine triphosphatyl, adenosine diphosphoryl, adenosine monophosphoryl, adenosine adenosyl, alpha-D-galactosyl, beta-D-galactosyl, alpha-D-glucosyl, beta-D-glucosyl, alpha-D-mannosyl, beta-fructofuranosyl, beta-D-glucopyranosyl orWherein B is₁、B₂And B₃Each independently is H or alkyl (branched or straight chain) of 1 to 4 carbon atoms.

11. The method of claim 10, wherein Z is

。

12. The method of claim 1, wherein the enzyme moiety comprises a hydrolase.

13. The method of claim 12, wherein the hydrolase is an alkaline phosphatase, a β -galactosidase, a β -glucosidase, a β -glucuronidase, or a neuraminidase.

14. The method of claim 13, wherein the enzyme moiety is an enzyme.

15. The method of claim 14, further comprising the steps of: detecting 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 such 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 that: the molecule is capable of non-covalently binding the label.

19. The method of claim 18, wherein the label is biotin or a biotin derivative and the molecule is avidin or streptavidin.

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 containing 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 the combination of (a) and (b),

(d) detecting 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 steps 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 such 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 the antigen is covalently linked to a label and the enzyme is covalently linked to a molecule that: the molecule is capable of non-covalently binding the label.

26. The method of claim 25, wherein the label is biotin or a biotin derivative and the molecule is avidin or streptavidin.

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 containing 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 the enzyme complex; and the combination of (a) and (b),

(d) detecting 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 steps 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 hybridizing to a nucleic acid and an enzyme capable of cleaving the 1, 2-dioxetane enzyme substrate such 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 the oligonucleotide is covalently linked to a label and the enzyme is covalently linked to a molecule that: the molecule is capable of non-covalently binding the label.

33. The method of claim 32, wherein the label is biotin or a biotin derivative and the molecule is avidin or streptavidin.

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 nucleic acids;

(b) immobilizing the nucleic acid on a solid phase,

(c) contacting the immobilized nucleic acid and the enzyme-linked oligonucleotide to form an enzyme complex; and the combination of (a) and (b),

(d) detecting 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 steps of: removing any unbound enzyme-linked oligonucleotides from the enzyme complex.

37. The process of claim 1, wherein the reaction mixture further comprises a promoter.

38. The method of claim 37, wherein the accelerator comprises a polymeric quaternary ammonium salt, a 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 (vinylbenzyltrimethyl phosphonium 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, the method comprising the steps of:

(a) providing an oxidizing agent;

(b) providing an enol ether having the structure:

wherein the content of the first and second substances,

t is an aryl or heteroaryl ring capable of emitting light, an

(d) providing a sample suspected of comprising an enzyme capable of cleaving the 1, 2-dioxetane enzyme substrate such that the substrate decomposes and produces light;

(e) contacting the sample with the aqueous solution comprising a substrate of a 1, 2-dioxetane enzyme to form a reaction mixture; and the combination of (a) and (b),

(f) detecting 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, the method comprising the steps of:

(a) providing an oxidizing agent;

(b) providing an enol ether having the structure:

wherein the content of the first and second substances,

R₁is alkyl containing 1-20 carbon atoms, aryl containing 6-14 carbon atomsA group, an aralkyl group having 7 to 15 carbon atoms, a heteroaryl group having 4 to 20 carbon atoms or a heteroaralkyl group having 5 to 20 carbons,

t is an aryl or heteroaryl ring capable of emitting light, an

(d) providing a sample suspected of containing 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 such 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 a 1, 2-dioxetane enzyme substrate to form a reaction mixture; and the combination of (a) and (b),

(i) detecting 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, the method comprising the steps of:

(a) providing an oxidizing agent;

(b) providing an enol ether having the structure:

wherein the content of the first and second substances,

t is an aryl or heteroaryl ring capable of emitting light, an

(d) providing a sample suspected of containing the antigen;

(e) providing an enzyme-linked antigen comprising an antigen and an enzyme capable of cleaving the 1, 2-dioxetane enzyme substrate such 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;

(i) detecting 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, the method comprising the steps of:

(a) providing an oxidizing agent;

(b) providing an enol ether having the structure:

wherein the content of the first and second substances,

t is an aryl or heteroaryl ring capable of emitting light, an

(d) providing a sample suspected of comprising said nucleic acid;

(e) immobilizing the nucleic acid to a solid phase;

(f) providing an enzyme-linked oligonucleotide comprising an oligonucleotide capable of hybridizing to the nucleic acid and an enzyme capable of cleaving the 1, 2-dioxetane enzyme substrate such that the substrate decomposes and generates light;

(g) contacting the immobilized and enzyme-linked oligonucleotides to form an enzyme complex;

(i) detecting 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, the kit comprising:

(a) an oxidizing agent, and

(b) an enol ether having the structure:

wherein the content of the first and second substances,

t is an aryl or heteroaryl ring capable of emitting light, an

R₂Is an enzyme cleavable group that contains a bond that can be cleaved by an enzyme moiety to generate an oxygen anion on T.

49. The kit of claim 48, wherein the oxidizing agent is selected from the group consisting of: hydrogen peroxide, sodium molybdate, hydrogen peroxide and sodium molybdate, hypochlorite and hydrogen peroxide, aryl endoperoxides, calcium peroxide peroxyhydrate, and combinations thereof.

50. The kit of claim 49, wherein the oxidizing agent 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 are

。

53. The kit of claim 48, wherein R₁Is an alkyl group having 1 to 2 carbon atoms or a trifluoroalkyl group having 1 to 2 carbon atoms.

54. The kit of claim 48, wherein T is

Wherein the content of the first and second substances,

R₃、R₄and R₅Independently selected from: H. f, Cl, Br, I, cyano, nitro, sulfonate, sulfate, trifluoromethyl, trifluoroethyl, straight-chain alkyl having 1 to 20 carbon atoms, branched-chain alkyl having 3 to 20 carbon atoms, straight-chain alkenyl having 2 to 20 carbon atoms, branched-chain alkenyl having 3 to 20 carbon atoms, cycloalkyl having 3 to 20 carbon atoms, cycloalkenyl having 3 to 20 carbon atoms, cycloheteroalkyl having 3 to 20 carbon atoms, cycloheteroalkenyl having 3 to 20 carbon atoms, polycycloalkyl having 4 to 60 carbon atoms, polycycloalkenyl having 4 to 60 carbon atoms, polycycloheteroalkyl having 4 to 60 carbon atoms, polycycloheteroalkenyl having 4 to 60 carbon atoms, alkoxy having 1 to 20 carbon atoms, aryl having 6 to 14 carbon atoms, trifluoromethyl, and trifluoromethyl, Aryloxy group having 6 to 14 carbon atoms, ester having 2 to 21 carbon atoms, trialkylammonium having 3 to 30 carbon atoms, trialkylphosphonium having 3 to 30 carbon atoms, or trialkylphosphonium having 2 to 21 carbon atomsAlkylamido of atoms, arylamido of 7 to 15 carbon atoms, alkylcarbamoyl of 2 to 21 carbon atoms, arylcarbamoyl of 7 to 15 carbon atoms, alkylsulfonamido of 1 to 20 carbon atoms, arylsulfonamido of 6 to 14 carbon atoms, trialkylsilyl of 3 to 60 carbon atoms, triarylsilyl of 18 to 42 carbon atoms, alkylarylsilyl of 7 to 32 carbon atoms, alkylamidosulfonyl of 1 to 20 carbon atoms, arylamidosulfonyl of 6 to 14 carbon atoms, alkylsulfonyl of 1 to 20 carbon atoms, arylsulfonyl of 6 to 14 carbon atoms, alkylthio of 2 to 20 carbon atoms and arylthio of 6 to 14 carbon atoms, and is

X is a sulfur atom, an oxygen atom or a nitrogen atom.

55. The kit of claim 48, wherein OR₂Is phosphate, acetate, 1-phospho-2, 3-diacylglycerol, adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, adenosine, alpha-D-galactoside, beta-D-galactoside, alpha-D-glucoside, beta-D-glucoside, alpha-D-mannoside, beta-fructofuranoside, beta-D-glucuronide orWherein B is₁、B₂And B₃Each independently is H or alkyl (branched or straight chain) of 1 to 4 carbon atoms.

56. The kit of claim 55, wherein R₂Is that

。

57. The kit of claim 48, wherein R₂Is E-L-Nuc-Z, wherein E is a group comprising an electrophilic atom which, upon enzymatic cleavage of the Z group, is attacked by the electron pair of the Nuc group and releases the 1, 2-dioxetane enzyme substrate anion by vicinal assistance; l is a linking group; nuc is a nucleophilic atom; and Z is an enzyme-cleavable group; wherein

R₆Is an alkyl group having 1 to 10 carbon atoms, and the linking group may be substituted with an alkyl group having 1 to 24 carbon atoms, an alkenyl group having 2 to 24 carbon atoms, an alkyl group having 1 to 24 carbon atoms, and mono-or di-substituted with an acyloxy group having 1 to 24 carbon atoms, an alkenyl group having 2 to 24 carbon atoms, and mono-or di-substituted with an acyloxy group having 1 to 24 carbon atoms, an aryl group having 6 to 10 carbon atoms, an alkyl group having 1 to 24 carbon atoms, and substituted with a phenyl group, a hydroxyphenyl group, an indolyl group, a mercapto group, an alkylthio group having 1 to 4 carbon atoms, a hydroxyl group, a carboxyl group, an amino group, a guanidino group, an imidazole or a carbamoyl group, or an alkenyl group having 2 to 24 carbon atoms, and substituted with a phenyl group, a hydroxyphenyl group, an indolyl group, a mercapto group, an alkylthio group having 1 to 4 carbon atoms, Hydroxy, carboxy, amino, guanidino, imidazole, or carbamoyl;

nuc is an oxygen atom or a sulfur atom; and is

Z is phosphoryl, acetyl, 2, 3-diacylglyceroyl-1-phosphate, adenosine triphosphatyl, adenosine diphosphate, adenosine monophosphate, adenosine, alpha-D-galactosyl, beta-D-galactosyl, alpha-D-glucosyl, beta-D-glucosyl,alpha-D-mannosyl, beta-fructofuranosyl, beta-D-glucopyranosyl orWherein B is₁、B₂And B₃Each independently is H or alkyl (branched or straight chain) of 1 to 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 accelerator comprises a polymeric quaternary ammonium salt, a polymeric quaternary phosphonium 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 (vinylbenzyltrimethyl phosphonium 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 preparing a substrate for a 1, 2-dioxetane enzyme, said method comprising the steps of:

(a) providing an oxidizing agent;

(b) providing an enol ether having the structure:

wherein the content of the first and second substances,

t is an aryl or heteroaryl ring capable of emitting light, an

R₂Is an enzyme-cleavable group that contains a bond that can be cleaved by an enzyme moiety to generate an oxygen anion on T; and