JPH1121285A - Chemiluminescent 1,2-dioxetane compound - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new compound cleavable by an enzyme, and short in time necessary for completing the assay when used as a reporter molecule in an assay, in particular, biological assay. SOLUTION: This new compound is shown by formula I [R1 is O(CH2 )n CH3 ((n) is 0-19), pref. methoxy; R2 is a group of formula II or formula III (Z is a residue selected from phosphate, galactoside, acetate, 1-phospho-2,3- diacylglyceride, adenosine triphosphate, etc.), pref. of formula IV (M is an alkali metal or ammonium)], e.g. diethyl 1-methoxy-1-(3-pivaloyloxyphenyl) methanephosphate. The compound of formula I is obtained, for example, by using a compound of the formula HC(=O)-ϕ-OR9 (R9 is a lower alkyl, etc.), as the starting material and in accordance with the process described in the USP #279,176 by Edwards, et. al (Sept. 6, 1989).

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to 1,2-dioxetane compounds having improved chemiluminescence. More particularly, the present invention relates to improved, enzymatically cleavable, chemiluminescent 1,2-dioxetane compounds having an enzymatically removable labile group. Such labile groups remain until the appropriate enzyme is added to remove the labile group.
Prevents molecules from breaking down to produce light that can be detected by suitable equipment.

[0002] One enzyme molecule catalyzes the removal of its corresponding labile group from an enzymatically cleavable chemiluminescent 1,2-dioxetane molecule. This is in sharp contrast to the case of chemically cleavable chemiluminescent 1,2-dioxetanes, which require one molecule of a chemical cleavage agent to remove the corresponding labile group from each dioxetane molecule. For example, 3- (2'-spiroadamantane) -4-
Methoxy-4- (3 "-hydroxy) phenyl-1,2
1 mole of sodium hydroxide is required to cleave 1 mole of hydrogen ions from the hydroxy substituent on the phenyl group of dioxetane, while 1,000 to 1000
5,000 mol of 3- (2'-spiroadamantane)-
Only one mole of alkaline phosphatase (AP) is required to cleave the phosphoryloxy group of 4-methoxy-4- (3 "-phosphoryloxy) phenyl-1,2-dioxetane disodium salt; Jablonski, "DNA Probes for Infectious Diseases" (Boca R
aton, Fla .: CRC Press, 1989, p22).

[0003] Light-producing 1, cleavable enzymes
The 2-dioxetane compounds usually have a stabilizing group, for example an adamantylidene group spiro-bonded to the 3-carbon atom of the dioxetane ring, so that an unstable group cleavable by an enzyme is present in the remainder of the molecule. Prior to deliberate cleavage of the bound bond, it helps prevent the dioxetane compound from substantially decomposing at room temperature (about 25 ° C.). The idea of using a spiroadamantyl group for stabilization is described in Tetrahedron Letters , 169 by Wierynga et al.
(1972) and J. Chem. Comm.
Introduced into 1,2-dioxetane chemistry by Soc. Chem. Comm , 944 (1977). Thus, these stabilizing groups can be used in such dioxetanes at temperatures as high as about 4 to about 30 ° C., without substantial degradation, for an acceptable long period of time prior to use, eg, about 12 months to about 12 years. Between storages.

[0004] The present invention further relates to the use of dioxetane molecules in immunological, chemical and nucleic acid probe assays known in the art and to various macromolecules, synthetic polymers, proteins, nucleic acids, Enables the identification or quantification of chemical or biological substances that measure the presence, quantity or structure of analytes, using these as direct chemical / physical probes to study the molecular structure and microstructure of catalytic antibodies, etc. About doing.

[0005]

2. Description of the Related Art In recent years, in particular, Bronstein
U.S. Patent Application No. 889,8, filed July 24, 1986.
No. 23; Bronstein et al., U.S. Patent Application No. 140,035, issued December 31, 1987;
rds), JP-A-2-724 and Edwards et al., 1988.
Since the advent of enzymatically cleavable chemiluminescent 1,2-dioxetanes, described in U.S. Patent Application No. 213,672, filed June 30, 2016, chemiluminescent 1,2-dioxetanes have become increasingly important. It has become a compound.

In sharp contrast to the enzymatically cleavable 1,2-dioxetanes, a variety of chemically cleavable, previously known 1,2-dioxetanes have been identified as reporter molecules in some analytical methods. If any, they were only marginal and certainly not used in bioassays. The reason is that the known chemically cleavable compounds are mostly water-insoluble-certain acetoxy-substituted 1, which are somewhat soluble in water and organic solvents.
Other than 2-dioxetane, and thus modified by adding groups or substituents capable of binding to biological components such as antibodies to form such chemically bound cleavable 1,2-dioxetane. Is not useful for biological assays unless it can be used as a chemically activated chemiluminescent label.

Representative chemiluminescent 1,2 cleavable by enzymes that decompose with luminescence in the presence of a suitable enzyme.
-Dioxetane-e.g. 1,2-dioxetane cleavable by adamantyl-added enzymes, e.g.
(4-methoxyspiro [1,2-dioxetane-3,
2'-tricyclo [3.3.1.1 ^3,7 ] decane] -4
-Yl) phenyl phosphate and its salts (e.g. the sodium salt) are water-soluble and are therefore very suitable for use as reporters in various analytical methods performed in aqueous media, in particular biological assays. These compounds are hereinafter abbreviated as follows: Adamantylidene-methoxyphenoxyphosphorylated dioxetane (AMPP
D), and 3- (4-methoxyspiro [1,2-dioxetane-3,2'-tricyclo [[3.3.1.1
^3,7 ] decane] -4-yl) phenyloxy-3 ″ -β
-D-galactopyranoside and its salts (AMPGD).

AMPPD can be used in aqueous solutions and / or in chemiluminescence enhancers such as poly [vinylbenzyl (benzyldimethylammonium chloride)] (BDMQ) and other heteropolar polymers [Voyta et al. It has been observed that in the presence of quaternary ammonium, phosphonium or sulfonium salts of polymers such as U.S. Pat. “T _1/2 ” is _{１ ／} of the maximum chemiluminescence intensity at a constant steady-state luminescence level.
(The luminescence half-life depends on the stability of the dioxetane oxyanion in various environments).

Statistically, it takes about 7t _1/2 to reach steady state emission characteristics. In the presence of BDMQ, the t _1/2 of AMPPD at a concentration above 2 × 10 ⁻⁵ M in an aqueous solution of pH 9.5 was found to be 7.5 minutes. At 4 × 10 ⁻³ M in the absence of BDMQ, t _1/2 is about 30-60 minutes, while 2 × 10 ⁻⁵ M in aqueous solution
It was found that t _1/2 of AMPPD was 2.5 minutes.

The chemiluminescent 1,2 cleavable by enzymes
-For rapid biological assays using dioxetane as reporter molecule, it is preferred to reach steady-state luminescence properties as fast as possible to detect the endpoint in the assay. Also, chemiluminescence intensity can be measured before reaching steady state, but if you want to get accurate data before steady state emission characteristics, you should use the latest thermal controlled luminescence measurement equipment. Must.

In addition, BDMQ produces stronger thermal and other non-enzymatically activated luminescence, ie, noise luminescence, in buffered aqueous solutions in the presence and absence of a chemiluminescence enhancer. Show. Such noise is due to the emission from the excited state of adamantanone and the emission of the methyl m-oxybenzoate anion derived from the aromatic portion of the AMPPD molecule. Since the measured noise level of AMPPD is about two orders of magnitude higher than the dark current in a standard luminometer, this noise limits the detection level,
Therefore, it is difficult to express the final sensitivity.

AMPP using alkaline phosphatase
D enzymatic cleavage yields anionic dephosphorylated AM
PPD-adamantylidene-methoxymethylphenolate dioxetane, also AMP ^- D-, is formed. The phenolate anion may also be formed in small amounts by hydrolysis, causing a background chemiluminescent signal. This includes organized molecular assemblies such as micelles, liposomes, lamellar layers, thin films, lipid bilayers, liposome vesicles, reverse micelles, microemulsions,
On a microgel, latex, membrane or polymer surface
And in a hydrophobic environment created by a chemiluminescence enhancer such as BDMQ, resulting in strong enhanced levels of luminescence, thus resulting in a high background signal;
The dynamic range of the signal resulting from enzymatic hydrolysis of AMPPD will be significantly reduced.

Based on the above observations, we hypothesized the following mechanism. In the presence of an accelerating polymer such as BDMQ:

[0014]

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1) "CL" represents chemiluminescence. 2) In the aqueous solution to which the buffer of AMPPD was added, 1,2-dioxetane (AMPH) subjected to dephosphorylation and hydrolysis was used.
D) is contained in a small amount. The pH of the solution is sufficiently high (approximately 9.
Above 5), the dephosphorylated dioxetane may be present in an anionic state as AMP ^- D. 3) “CL ₁ ” and “CL ₂ ” represent background chemiluminescence. 4) “A *” represents adamantanone in an excited energy state.

In the absence of the accelerating polymer, AMPPD is present in the aqueous solution as an aggregate:

[0017]

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In the above mechanism, n >>m; n and m are in the presence or absence of the accelerating polymer and A
Depends on MPPD concentration.

The excited state of the adamantanone singlet (n or m> 1) in the form of aggregates produces more signal than the excited energy state of unaggregated adamantanone, and here, in particular, the BDMQ Even if stabilized by the presence of such a chemiluminescence enhancer, more light is emitted. This is probably due to less intersystem crossing because the former singlet state is lower than the latter,
Or because the speed of the intersystem crossing is slower, or because of other factors not yet known. Luminometers are generally designed to detect all emitted photons, regardless of their energy, i.e., their wavelength, so that 415 nm and 47
Both the 7 nm chemiluminescence is detected as background noise emission. Similarly, when recording chemiluminescence using photographs or X-ray films, discrimination between different wavelengths of emission cannot be easily made, and thus the detection sensitivity is limited by background noise.

Finally, the AMP observed under the above conditions
Aggregation of PD involves AMPPD or its phenolate anion and molecules such as:

[0021]

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[0022] It depends on the amphipathic nature of

[0023]

Accordingly, it is an object of the present invention to reduce the time required for analysis, particularly for biological assays using a chemiluminescent 1,2-dioxetane cleavable by an enzyme as a reporter molecule. It is.

It is also an object of the present invention to provide a new and improved enzyme-cleavable chemiluminescent enzyme which, when used as a reporter molecule in an assay, particularly a biological assay, requires less time to complete the assay. 1,2-
To provide dioxetane.

Another object of the present invention is to provide a new and improved, enzymatically cleavable, chemiluminescent 1,2-dioxetane for use as a substrate in enzyme-based assays, particularly biological assays. In essence, this compound provides an improved signal over background and, therefore, an improved level of detection.

[0026] These and other objects and properties, scopes and uses of the present invention are set forth in the following description and appended claims.
It will be readily apparent to those skilled in the art.

[0027]

According to the present invention, there is provided an aqueous medium comprising:
For example, in a sample obtained by adding a biological fluid to a solution, or on a solid surface, for example, on a membrane surface such as a nylon membrane, the enzyme can be reacted with an enzyme or an enzyme denatured with a specific binding pair, and detected optically. A new class of stable, enzymatically cleavable chemiluminescent 3-capable of releasing energy
(Substituted adamant-2'-ylidene) -1,2-dioxetane compounds.

Analysis can be carried out with these modified adamantylidene dioxetanes in an aqueous medium, in which the compounds are used as reporter molecules,
It can be performed faster and with better sensitivity than the analysis performed so far using AMPPD.

Although we do not wish to explain this unexpectedly superior effect in any mechanism or theory, the presence of substituents of the above type on or in the adamantylidene moiety indicates that the dioxetane molecule It may prevent it from working efficiently, and thus they may prevent the formation of micellar or other aggregated stabilized organized assemblies. Some of these substituents also hydrogen bond to other substances in an aqueous environment, including water itself, thereby preventing further formation of aggregates. Also, the electronic and dipole effects may contribute to this phenomenon, as can be seen from shorter t _{1/2 and} lower background noise.

The novel chemiluminescent 1,2-dioxetane compound of the present invention has the general formula (I):

[0031]

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[0032] wherein, R ₁ is a _{-O (CH 2) n CH 3} , n is an integer from 0 to 19; R ₂ has the formula:

[0033]

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(Wherein Z is phosphate, galactoside, acetate, 1-phospho-2,3-diacylglyceride, 1-thio-D-glucoside, adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, adenosine, α -D-glucoside, β-
D-glucoside, α-D-mannoside, β-D-mannoside, β-D-fructofuranoside, β-D-glucoside uronate, p-toluenesulfonyl-L-arginine ester and p-toluenesulfonyl-L- Which is a residue selected from the group consisting of argininamide).

R ₁ is particularly preferably a methoxy group. R ₂ is some fluorophore-forming fluorescent chromophore that emits light. The group Z which can be removed by the enzyme of the substituent OZ of the phenyl or naphthyl group of R ₂ is preferably a phosphate group, in particular of the general formula (III):

[0036]

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Wherein M ⁺ is a cation such as an alkali metal such as sodium or potassium, ammonium or C ₁ -C ₇ alkyl, aralkyl or aromatic quaternary ammonium cation, N (R ₇ ) ₄ ⁺ (each R ₇ is alkyl, such as methyl or ethyl, aralkyl, such as benzyl or where heterocyclic ring system, e.g., to form a pyridinium) a phosphate ester group represented by. disodium salt is particularly Such phosphate ester groups are preferably cleaved with an enzyme such as alkaline phosphatase to yield a group substituted with an oxygen anion, thus destabilizing dioxetane and breaking its oxygen-oxygen bond. The quaternary ammonium cations of such phosphate ester groups may also emit one of their quaternary groups. The polymer main chain by, that the following formula (IV):

[0038]

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(Where n is greater than 1) or can be part of a quaternary ammonium salt polymer, ie, an ionene polymer.

Another preferred enzyme removable group is the β-D-galactoside group, which is the enzyme β-galactoside.
Cleavage with D-galactosidase yields the conjugate acid of dioxetane phenolate, which emits chemiluminescence under pressure.

Enzyme-cleavable substituents which can be used also include enzyme-cleavable alkanoyloxy groups such as acetate ester groups, enzyme-cleavable oxacarboxylate groups, 1-phospho-2,3-.
Diacylglyceride group, 1-thio-D-glucoside group,
Adenosine triphosphates, adenosine diphosphates, adenosine monophosphates, adenosines, α-D-galactoside, β-D-galactoside, α-D-glucoside, β-D-glucoside, α-D-mannoside group, β-D-mannoside group, β
-D-fructofuranoside group, β-D-glucoside uronate group, p-toluenesulfonyl-L-arginine ester group or p-toluenesulfonyl-L-arginineamide group. R ₁ is —O (CH ₂ ) _n CH ₃ (n = 0
To 19, preferably n = 0 to 5).

The overall synthesis of these 3- (substituted adamant-2'-ylidene) 1,2-dioxetanes is:
The aforementioned Bronstein and Edwards application and Edwards et al., U.S. Patent Application Serial No. 27, September 6, 1989.
It can be carried out by the method described in No. 9,176. Thus, for example, R ₁ is a methoxy group, and R ₂
1,2-dioxetane, which is a phenyl group substituted with a phosphoryloxy group, preferably a phenyl group substituted with a meta-phosphoryloxy salt, is disclosed in U.S. Pat.
According to the method described in No. 9,176, it can be synthesized by the reaction steps shown in the following figures.

[0043]

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As illustrated in detail below, if necessary, the HC (（O) -φ-OR ₉ starting material is reacted with an orthoformate, for example, trimethyl orthoformate, methanol and p-toluenesulfonic acid, Intermediate:

[0045]

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Can be obtained. This intermediate (R ₈
O) When reacted with ₃ P and a Lewis acid, the phosphonate ester intermediate:

[0047]

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Is obtained.

In the above reaction step, R ₈ is a lower alkyl group such as methyl, ethyl or butyl. R ₉ is an acyl group having 2 to 14 carbon atoms, for example, acetyl, propionyl, mesitoyl or pivaloyl; Q is a halogen, for example, chloro or bromo, or OR ₈ ;
And M is individually a proton, a metal cation such as Na
⁺ Or K ⁺ , or ammonium, substituted ammonium, quaternary ammonium or (H ⁺ ) pyridinium cation. Thiolate cleavage described in U.S. Patent Application Serial No. 213,197 of Edwards, R ₉ is lower alkyl, lower alkenyl or aralkyl group, such as methyl, allyl or benzyl, instead of base cleavage of the OR ₉ in the reaction step 5 Can be used. The product of base or thiolate cleavage has hydrogen or an alkali metal cation, such as lithium, sodium or potassium, in place of R ₉ .

The above intermediate represented by Ad = O is a known compound.

Major Dissertation, University of Groningen, The Netherlands (1982); Newman et al., J. Or.
g. Chem. , 43, 2232 (1978);
J. Chem. Soc., Chem. Comm. , 3906 (1971) provides access to 4-methyleneadamantan-2-one as a starting material for reaction step 4 above and subsequent reaction with a suitable phosphonate-stabilized carbanion. A method is described. Due to the difference in reactivity of singlet oxygen to enol ether instead of exomethylene function, 3- (4-methoxyspiro [1,2-dioxetane-3,2 '-(4'-methylene) tricyclo [3.3. 1.1 ^3,7 ] -4-yl) phenyl disodium phosphate can be reliably obtained as a photooxygenation product.

If a pivaloyloxyaryl enol ether capable of selective cleavage is obtained in any reaction just prior to adding an enzyme-removable group such as a phosphate ester group, the hydroxyaryl enol ether It is more convenient to avoid separation. This can be done by directly separating the pivaloyl ester with one equivalent of sodium methoxide in methanol and separating the sodium aryloxide component as a dry solid by removing all volatile components at the end of the reaction. . In such a case, reaction step 6 is performed without using a Lewis base, but with this preformed salt in a dry polar aprotic solvent such as dimethylformamide, and the inorganic salt by-product is removed from step 7 or step 8 Removed during processing.

The 2-cyanoethyl phosphate diester product of Step 7 is beta-eliminated to the phosphate monoester of Step 8. In step 8, the diester derivative is combined with a volatile amine such as ammonia or "DBU"
(1,8-diazabicyclo [5.4.0] undec-7
It is preferred to react with an organic amine soluble in a solvent such as -ene) in an alcoholic solvent such as methanol. It is particularly advantageous to use ammonia at a pressure above atmospheric pressure and ambient temperature, since the excess base is easily volatilized at the end of the reaction in vacuo.

The oxidation of the enol ether phosphate in the reaction step 9 is carried out by photochemical reaction with singlet oxygen ( ¹ O ₂ ) in a halogenated solvent, for example, a halogenated hydrocarbon such as chloroform, as described above. Can be done. The solvent may also contain an auxiliary solvent, for example, a lower alkanol such as methanol. Singlet oxygen can be polymer bound Rose Bengal (Polysciences), methylene blue or 5,10,15,20.
-Tetraphenyl-21H, 23H-porphine (TP
It can be generated using a photosensitizer such as P).

Alternatively, the crude 2 obtained in the reaction step 7
The cyanoethyl phosphate diesters can also be oxidized with singlet oxygen to their 1,2-dioxetanes. Chemical methods of 1,2-dioxetane, including methods using electronic oxidation, mediated by triethylsilyl hydrotrioxide, phosphate ozonide or triarylamine radical cation in the presence of triplet oxygen, can also be used. .

As noted above, the present invention also provides for the use of chemiluminescent, cleavable substituted 1,2-dioxetanes in assays known in the art, including assays for detecting enzymes in a sample. And kits for such assays, and methods and means for their use.

For example, when the present invention is used to detect an enzyme in a sample, the sample is contacted with a dioxetane having a group cleavable by the enzyme to be detected. The enzyme cleaves the dioxetane enzymatically cleavable group to form a negatively charged substituent (eg, an oxygen anion) attached to the dioxetane. This negatively charged substituent in turn destabilizes dioxetane and degrades dioxetane to form a fluorescent chromophore group that emits light energy. It is this chromophore group that is detected as indicative of the presence of the enzyme. By measuring the luminescence intensity, the concentration of the enzyme in the sample can also be measured.

There are a variety of different analytical methods which measure the presence or concentration of a particular substance in a sample using a visually detectable means. The above dioxetanes can be used in any of these assays. Examples of such assays include antibodies or antigens such as δ- or β-hCG
An enzyme assay; for example, a chemical assay for detecting potassium or sodium ions; for example, a virus (eg, HTLV VIII or cytomegalovirus), or a bacteria (eg, E. coli) .
coli and nucleic acid assays to detect specific cellular functions (eg, receptor binding sites).

When the substance to be detected is an antibody, an antigen or a nucleic acid, an enzyme capable of cleaving a group that can be cleaved by the enzyme of dioxetane is converted to a substance having a specific affinity for the substance to be detected (that is, specific to the substance to be detected). Substances that bind together),
For example, it is preferable to bind to an antigen, an antibody or a nucleic acid probe. In a general method, for example, carbodiimide coupling, the enzyme is used by being bound to a substance having a specific affinity; preferably, the binding is through an amide bond.

Generally, the analysis is performed as follows. The sample suspected of containing the detection substance is contacted with a solution containing a buffer containing an enzyme bound to a substance having a specific affinity for the detection substance. The resulting solution is incubated to allow the detection substance to bind to the specific affinity portion of the enzyme compound having specific affinity. Then, the excess,
The enzyme compound having the specific affinity is washed away, and dioxetane having a group that can be cleaved by the enzyme portion of the enzyme compound having the specific affinity is added. The enzyme cleaves a group cleavable by the enzyme to break down dioxetane into two carbonyl compounds (eg, esters, ketones or aldehydes). This causes the chromophore to which the group cleavable by the enzyme is attached to excite and emit light. Luminescence is detected (eg, using a cuvette, or a photosensitive film of a camera luminometer, or a photovoltaic cell or photomultiplier) as an indication of the presence of a detection substance in the sample. The emission intensity is measured to determine the concentration of the substance.

A specific analysis example is as follows.

A. Human IgG assay Sheep anti-human Ig in a 96-well microtiter plate
G (F (ab) specific fragment) is applied. next,
Serum samples containing human IgG are added to the wells and incubated for 1 hour at room temperature. After the incubation period, the serum samples were removed from the wells and the wells were filled with 0.15 M NaCl,
Wash four times with an aqueous buffer solution (pH 7.4) containing 0.01 M phosphate and 0.1% bovine serum albumin. Alkaline phosphatase conjugated to anti-human IgG is added to each well and incubated for 1 hour. The wells are then washed four times with the above buffer and the phosphate-containing dioxetane buffer of the invention is added. The luminescence obtained by the enzymatic degradation of dioxetane is detected using a luminometer or a photographic film of a camera luminometer.

B. hCG Assay Rabbit anti-α-hCG is absorbed onto a nylon-mesh membrane. A sample solution containing hCG, for example, urine of a pregnant woman, is sucked through the membrane, and then the membrane is washed with 0.15 M
-Wash with 1 ml of a buffer (pH; 7.4) containing NaCl, 0.01 M phosphate and 0.1% bovine serum albumin. Alkaline phosphatase labeled anti-p-hCG is added to the membrane and the membrane is washed again with 2 ml of the above buffer. The membrane is then placed in a luminometer cuvette or camera luminometer and contacted with the phosphate-containing dioxetane of the present invention. Next, the luminescence resulting from the enzymatic degradation of dioxetane is detected.

C. Assay of serum alkaline phosphatase
The aqueous buffer 2.7ml containing 2-methyl-2-aminopropanol Lee 0.8M placed in Pyrex test tubes 12 × 75 mm, adding serum sample 0.1ml containing alkaline phosphatase. The solution is then brought to an equilibrium of 30 ° C. The phosphate-containing dioxetane 0.2 of the present invention
Add ml and place the tube immediately in the luminometer and record the resulting luminescence. The luminescence level is proportional to the alkaline phosphatase activity rate.

D. Nucleic Acid Hybridization Assay Cerebrospinal Fluid (CS
F) Collect the sample and place it on a membrane such as a nylon or nitrocellulose membrane. Next, the sample is chemically treated with urea or guanidinium isothiocyanate to break the cell wall,
Degrades all cellular components except viral DNA. The thus obtained strand of the viral DNA is separated,
Bound to nitrocellulose filter. Virus D
A DNA probe specific for NA and labeled with alkaline phosphatase is provided to the filter; the probe is hybridized with a complementary viral DNA strand. After hybridization, the filter was placed on a 0.
Wash with an aqueous buffer (pH = 8.10) containing 2M-NaCl and 0.1 mM Tris-HCl to remove excess probe molecules. The phosphate-containing dioxetane of the present invention is added and the luminescence resulting from the enzymatic degradation of dioxetane is measured with a luminometer or detected with a photographic film.

E. Assays for galactosidase In the assays described above and in the Examples below, α- or β
Α-D- or β cleavable by galactosidase
Dioxetanes, each containing a -D-galactoside (galactopyranoside) group, may be added and the luminescence resulting from enzymatic cleavage of the sugar moiety from the chromophore is measured with a luminometer or detected with a photographic film.

F. Electrophoresis Electrophoresis separates a complex mixture of proteins and nucleic acids on a gel support in an electric field according to their molecular size and structure. The method can also be used to separate protein fragments after protein hydrolysis or nucleic acid fragments after cleavage with restriction endonucleases (as in DNA sequencing). After electrophoretic degradation of the components in the gel, or after transfer of the separated components from the gel to the membrane, binding is examined with an enzyme attached to the ligand. For example, peptide fragments are probed with an antibody covalently linked to alkaline phosphatase. In another example, in DNA sequencing, alkaline phosphatase / avidin is linked to a biotinylated nucleotide base. Thereafter, the AMPPD analog of the present invention is added to the gel or membrane filter. After a short incubation, dioxetane is activated by the enzyme to form a luminescent component, resulting in luminescence. Luminescence is detected by X-ray or instant photographic film or by luminometer. A further improvement is achieved by using a multiplex assay that examines two or more fragments simultaneously.

G. Solid State Assays In solid state assays, blocking non-specific binding to the matrix by pretreating the non-specific binding sites with a non-specific protein such as bovine serum albumin (BSA) or gelatin Is preferred. BS
It has been found that some of the commercial formulations of A contain small amounts of phosphatase activity that produces undesirable background chemiluminescence from AMPPD. However, it has also been found that certain water-soluble synthetic macromolecules are sufficient blockers of non-specific binding in solid state assays using dioxetane. Preferred among such materials are water soluble polymer quaternary ammonium salts such as BDMQ, poly [vinylbenzyl (trimethylammonium chloride)] (TMQ) and poly [vinylbenzyl (tributylammonium chloride)] (T
BQ).

H. Nucleotidase Assay The enzyme ATPase assay is performed in two steps. In the first step, the enzyme is reacted at an optimal pH (generally pH 7.4) with a substance consisting of ATP covalently linked to the 1,2-dioxetane substituted by a chromophore via a terminal phosphoester bond, resulting in phosphoryl-chromogenic formation. A group-substituted 1,2-dioxetane is obtained. In the second step, the product of the first step is decomposed by adding an acid to bring the pH to less than 6, preferably 2 to 4,
The resulting light is measured with a luminometer or detected with a chromatographic film. In a similar two-step process, ADP
Assays for ase were performed using the ADP derivative of 1,2-dioxetane substituted with a chromophore of the invention as a substrate, and assays for 5'-nucleotidase were substituted with a chromophore of the invention as a substrate. This is performed using an adenylic acid derivative of 1,2-dioxetane. The second step is also
This can be done by adding the enzyme alkaline phosphatase to degrade the phosphoryl-chromophore-substituted 1,2-dioxetane.

I. Nucleic acid sequencing DNA or RNA fragments produced by sequencing can be detected after electrophoretic separation using the chemiluminescent 1,2-dioxetane of the present invention. DN
A sequencing was performed by the dideoxy chain termination reaction [Sanger, F. et al.
Nat. Acad. Sci. (USA) , 74: 5463 (1977)]
Can be done by Briefly, in each of the four sequencing reactions, single-stranded template DNA is mixed with dideoxynucleotide and biotinylated primer strand DNA. After annealing, Klenow enzyme and deoxyadenosine triphosphate are incubated with each of the four sequencing reaction mixtures, chasing deoxynucleotide triphosphate is added, and the incubation is continued. Thereafter, the DNA fragments in the reaction mixture are separated by polyacrylamide gel electrophoresis (PAGE). The fragments are transferred to a membrane, preferably a nylon membrane, and the fragments are cross-linked to the membrane, preferably by irradiation with short wavelength UV light.

After blocking the non-specific binding sites with a polymer, for example heparin, casein or serum albumin, the DNA fragments on the membrane are used for the individual 1,2 of the invention used.
Contacting avidin or streptavidin covalently linked to an enzyme specific for an enzyme cleavable group of the 2-dioxetane substrate. Since avidin or streptavidin binds biotin greedily, the biotinylated DNA fragment is enzymatically labeled. Avidin or streptavidin binds to phosphatase or galactosidase and the like. DNA fragment-biotin-
The avidin (or streptavidin) -enzyme complex is combined with the appropriate 1,2-dioxetane and an alkaline pH value,
After luminescence, for example by contacting at about pH 8.5, the DNA fragments are visualized on a light-sensitive film, such as X-ray or instant film, or with a photoluminometer device.

The detection method abbreviated above is described by Church et al. [Church, G .; M. Nat. Acad. Sci. (USA) , 81:
1991 (1984)]. DN cleaved chemically and separated by electrophoresis
A [Maxam, A. M. Nat. Acad. Sci. (US
A) , 74: 560 (1977)] to a membrane, preferably a nylon membrane, crosslink the ladder to the membrane with UV light, and
The sequence comprises a biotinylated oligonucleotide as a hybridization probe; avidin or streptavidin covalently linked to an enzyme specific for a chemiluminescent 1,2-dioxetane cleavable by an enzyme of the invention; It is detected by sequentially adding 2-dioxetane. An image of the sequence ladder (produced by PAGE) is obtained as described above.

Continuous reprobing of sequence ladder (repr
obing) firstly cleans the membrane with a heated solution of a cleaning agent, for example about 0.
5 to about 5% sodium dodecyl sulfate (SDS) for about 80
Hybridization probes and chemiluminescent material are first stripped from the membrane by contacting with water in 〜90 ° C., cooled to about 50-70 ° C., and naked DNA fragments are subjected to another biotinylation. Hybridization with an oligonucleotide probe,
This can be done by obtaining a different array and then generating an image chemiluminescence as described above.

Similar detection methods can be applied to RNA fragments generated by RNA sequencing.

The following examples are provided so that those skilled in the art can better understand the present invention. These embodiments are provided for illustrative purposes only. All parts and percentages are by weight / volume unless otherwise specified, except that the TLC solvent mixture is by volume / volume.

Example I 200 g (1.64 mol) of 3-hydroxybenzaldehyde and 270 ml (1.93 mol) of triethylamine were placed in a flask containing 1 liter of methylene chloride in an ice bath. The resulting brown solution was stirred mechanically,
2 ml (1.72 mol) of trimethylacetyl chloride were tricklely added from the dropping funnel over 15 minutes. The resulting slurry was stirred for another 15 minutes, the ice bath was removed and the reaction was allowed to proceed for another 2 hours. TLC (K5F; 25% acetone-
Hexane) showed no starting material and no single high R _f product. The reaction mixture was transferred to a separatory funnel and mixed with 250 ml of 1M hydrochloric acid. Then the organic phase was extracted with water (2 × 400 ml) and finally dried over sodium sulfate. The dry solution was passed through a silica gel plug, rotary evaporated and then pumped under vacuum (1.0 mm Hg) to give 348 g of a greenish brown oil: 3-pivaloyloxybenzaldehyde. This was placed under an argon atmosphere.

Example II 400 mg of p-toluenesulfonic acid dissolved in 25 ml of methanol were added with stirring to the 3-pivaloyloxybenzaldehyde of Example I. Next, trimethyl orthoformate (224 ml; 2.05 mol) was added dropwise. The mixture was allowed to stir for 1 hour with a slight exotherm. 1/2 g sodium bicarbonate was added and the flask was placed in a rotary evaporator (bath temperature 40 ° C.) to remove any volatile components. The resulting oil was passed through a short silica gel column under nitrogen pressure to give an orange-brown oil, which was suctioned under vacuum (1.0 mmHg) with stirring to 426 g.
The crude 3-pivaloyloxybenzaldehyde dimethyl acetal was obtained. No infrared aldehyde absorption (1,695 cm ^-1 ) was observed in the infrared assay.

Example III The crude 3-pivaloyloxybenzaldehyde dimethyl acetal of Example II was dissolved in 1 liter of freshly distilled methylene chloride from P ₂ O ₅ in a 3 liter flask under an argon atmosphere. Next, 347 ml (2.03
mol) of triethyl phosphite were added all at once. The flask was fitted with a filter inlet adapter and cooled in a dry ice / acetone bath under slight argon pressure.
Boron trifluoride ether complex (249 ml; 2.03
mol) was added in several portions with a syringe while stirring vigorously. The resulting reaction mixture was stirred at -55 ° C for 2 hours,
It was then stored in a freezer at -20 ° C overnight. next,
The flask was warmed to room temperature and the contents were stirred for 4 hours.
This orange-brown solution was added to a vigorously stirred slurry of 170 g of sodium bicarbonate in 800 ml of water.
Carefully poured at such a rate that it did not foam violently. After vigorously stirring the biphasic mixture for 1 hour, the layers were separated in a separatory funnel and the aqueous layer was re-methylene chloride (2 x 250m
Extracted in l). The combined organic extracts were dried over sodium sulfate, concentrated, and distilled in vacuo to provide 535 g of diethyl 1-methoxy-1- (3-pivaloyloxyphenyl).
Methane phosphate is a clear, pale yellow oil (0.25 mmHg
With a boiling point of 158 to 161 ° C). This is shown in Example I-II
The yield was 91% for all steps of I. ¹ H NMR (400 MHz; CDCl ₃ ): δ 1.21 and 1.25 (6H, two t,
_{7Hz, OCH 2 C H 3)} ; 3.37 (3H, s, ArCHOC H 3); 3.80 (3H, s,
ArOC H ₃ ); 3.9-4.10 (4H, m, OC H ₂ CH ₃ ); 4.46 (1H, d, 1
5.6Hz, ArC H PO); 6.85 (1H, m); 7.00 (2H, m); 7.26 (1H,
m). IR (neat): 2974, 1596, 1582, 1480, 1255 (P = O), 1
098, 1050, 1020, 965 cm ^-1 .

Example IV The increase in chemiluminescence (compared to the emission from AMPPD) obtained with the compounds according to the invention in the presence of the following accelerating polymers has been demonstrated in the following manner.
Four 1,2-dioxetanes to be compared in 0.1 M diethanolamine (pH 10.0, substrate buffer) containing 1 mM magnesium chloride, 0.02% sodium azide and 0.1% promoter polymer. 0 of one of the
Four sets of three tubes each containing 450 μl of a 4 mM aqueous solution were prepared, and the background signal from each tube was measured with a Berthold LB 952T luminometer (Berthold luminometer).
Instruments; Wildbad, Germany). 1 mM magnesium chloride, 0.0
50 μl of an aqueous solution containing 2.83 × 10 ⁻¹² M alkaline phosphatase in 0.1 M diethanolamine (pH 10.0) containing 2% sodium azide (final enzyme concentration 2.
83 × 10 ^-13 M) was added to each tube, and the chemiluminescence signal was 5
And at 20 minutes with a luminometer.

The accelerating polymers used are as follows: Symbol accelerating polymer SAPPHIRE BDMQ TMQ poly [vinylbenzyl (trimethylammonium chloride)] S / TMQ styrene / TMQ copolymer DAA / TMQ diacetone acrylamide / TMQ copolymer Polymer DMQ / TEQ Poly [vinylbenzyl (dodecyldimethylammonium chloride)] / TEQ Copolymer TEQ Poly [vinylbenzyl (triethylammonium chloride)] TBQ Poly [vinylbenzyl (tributylammonium chloride)] MPB Poly [vinylbenzyl ( N-methylpiperidinium chloride)] BAEDM poly [vinylbenzyl ((2-benzoylaminoethyl) dimethylammonium chloride)] BZ benzal mordant DMEB poly [vinylbenzyl (dimethylethylammonium chloride)] DME (OH) B poly [ Vinyl benzyl (dimethi (2-hydroxyethyl) A Nmo chloride)] EMERALD sapphire and fluorescein TBQ / FLUOR TBQ and fluorescein

The preferred embodiments and examples of the present invention have been mainly described above. Those skilled in the art can easily modify the present invention without departing from the spirit and scope of the present invention described in the claims.

────────────────────────────────────────────────── (5) Int.Cl. ⁶ Identification symbol FI C12Q 1/68 C12Q 1/68 A G01N 33/533 G01N 33/533 (72) Inventor Edwards, Brooks 02138, Mass., Cambridge, USA Huron Avenue 269 (72) Inventor Juo, Lo-Lon 28134, Holston Street, Oston, Massachusetts, USA 28

Claims (2)

[Claims] 1. A compound of formula (I): Wherein R ₁ is —O (CH ₂ ) _n CH ₃ , wherein n is an integer from 0 to 19; R ₂ is represented by the following formula: Wherein Z is phosphate, galactoside, acetate, 1-phospho-2,3-diacylglyceride, 1-thio-D-glucoside, adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, adenosine, α-D- Glucoside, β-D-glucoside, α-D-mannoside, β-D-mannoside, β-
D-fructofuranoside, β-D-glucoside uronate, p-toluenesulfonyl-L-arginine ester and p-toluenesulfonyl-L-argininamide). An unstable substituent, cleavable by an enzyme, is attached to the molecule and is substantially stable at room temperature before intentionally cleaving the bond, and may generate light energy upon decomposition. Chemiluminescent 1,2-dioxetane compound that can be cleaved by the 2. R ₁ is a methoxy group and R ₂ is 2. The 1,2-dioxetane compound according to claim 1, wherein M represents an alkali metal or ammonium group.