Background of the Invention This invention relates to using dioxetanes to detect a substance in a sample.

Dioxetanes are compounds having a 4-membered ring in which 2 of the members are oxygen atoms bonded to each other. Dioxetanes can be thermally or photochemically decomposed to form carbonyl products, i.e., ketones or aldehydes. Release of energy in the form of light (i.e., luminescence) accompanies the decompositions.

Summary of the Invention In, general, the invention features in a first aspect an improvement in an assay method in which a member of a specific binding pair (i.e., two substances which bind specifically to each other) is- detected by- means of an optically detectable reaction. The improvement includes the reaction, with an enzyme, of a dioxetane having the formula

where T is a substituted (i.e., containing one or more C _^ -C- alkyl groups or heteroatom groups, e.g., carbonyl groups) or unsubstituted cycloalkyl (having between 6 and 12 carbon atoms, inclusive, in the ring) or a polycycloalkyl (having 2 or more fused rings, each ring independently having between 5 and 12 carbon

atoms, inclusive) group bonded to the 4-membered ring portion of the dioxetane by a spiro linkage; V is H or an enzyme-cleavable group; Y is a fluorescent chromophore, (i.e., Y is capable of absorbing energy to form an excited, i.e., higher energy, state, from which it emits light to return to its original energy state); X is H, a straight or branched chain alkyl group (having between 1 and 7 carbon atoms, inclusive, e.g., methyl), straight chain or branched heteroalkyl (having between 1 and 7 carbon atoms, inclusive e.g., methoxy, hydroxyethyl, or hydroxypropyl), aryl (having at least 1 ring, e.g., phenyl) , heteroaryl (having at least 1 ring, e.g., pyrrolyl or pyrazolyl) , cycloalkyl (having between 3 and 7 carbon atoms, inclusive, in the ring, e.g., cyclohexyl) , cycloheteroalkyl (having between 2 and 7 carbon atoms, inclusive, in the ring, e.g., dioxane) ,• aralkyl (having at l*east 1 ring, e.g., benzyl), or alkaryl (having at least 1-ring, e.g., tolyl) , or an enzyme-cleavable group, i.e-. , a group having a bond which can be cleaved by an enzyme to yield an electron-rich moiety bonded to the dioxetane, e.g., phosphate, where a phosphorus-oxygen bond can be cleaved by an enzyme, e.g., acid phosphatase or alkaline phosphatase to yield a negatively charged oxygen bonded to the dioxetane; and Z is H, OH, or an enzyme-cleavable group (as defined above), provided that at least one of V, X, or Z must be an enzyme-cleavable group, so that the enzyme cleaves the enzyme-cleavable group to form a negatively charged substituent (e.g., an oxygen anion) bonded to the dioxetane, the negatively charged substituent causing the dioxetane to decompose to form a luminescent substance (i.e., a substance that emits energy in the

form of light) that includes group Y. The luminescent substance is detected as an indication of the presence of the first substance. By measuring the intensity of luminescence, the concentration of the first substance can be determined.

In preferred embodiments, one or more of groups T, X, or Y further include a solubilizing substituent, e.g., carboxylic acid, sulfonic acid, or quaternary amino salt; group T of the dioxetane is a polycycloalkyl group, preferably adamantyl; the enzyme-cleavable group includes phosphate; and the enzyme includes phosphatase.

The invention also features a kit for detecting a first substance in a sample. in a second aspect, the invention features a method of detecting ^* an enzyme in a s ^' ample. The method involves contacting the sample with the above-described dioxetane in which group V, X, or Z is capable of being cleaved by the enzyme being detected. The enzyme cleaves the group to form a negatively charged substituent (e.g., an oxygen anion) bonded to the dioxetane. This substituent destabilizes the dioxetane, thereby causing the dioxetane to decompose to form a luminescent substance that includes group Y of the dioxetane. The luminescent substance is detected as an indication of the presence of the enzyme. By measuring the intensity of luminescence, the concentration of the enzyme can also be determined. The invention provides a simple, very sensitive method for detecting substances in samples, e.g., biological samples, and is ^' particularly useful for substances present in low concentrations. Because dioxetane decomposition serves as the excitation energy source for chromophore Y, an external excitation energy

source, e.g., light, is not necessary. In addition, because the dioxetane molecules are already in the proper oxidation state for decomposition, it is not necessary to add external oxidants, e.g., H_0_ or O Enzyme-triggered decomposition allows for high sensitivity because one enzyme molecule can cause many dioxetane molecules to luminesce, thus creating an amplification effect. Moreover, the wavelength (or energy) of emission and the quantum yields of luminescence can be varied according to the choice of the ^' Y substituent of the dioxetane (as used ^herein, "quantum yield" refers to the number of photons emitted from the luminescent product per number of moles of dioxetane decomposed) . In addition, through appropriate modifications of the T, X, and Y groups of the dioxet.ane, the solubility of the dioxetane and the kinetics of dioxetane decomposition can be varied. The dioxetanes can also be attached to a variety of molecules, e.g., proteins or haptens, σr immobilization substrates, e.g., polymer membranes, or included as a side group in a homopolymer or copolymer.

Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims. Description of the Preferred Embodiments

We now describe the structure, synthesis, and use of preferred embodiments of the invention. Structure

The invention employs dioxetanes having the structure recited in the Summary of the Invention, above. The purpose of -group T is to stabilize the dioxetane, i.e., to prevent the dioxetane from decomposing before the enzyme-cleavable group Z is cleaved. Large, bulky, sterically hindered molecules,

e.g., fused polycyclic molecules, are the most effective stabilizers. In addition, T preferably contains only C-C and C-H single bonds. The most preferred molecule is an adamantyl group consisting of 3 fused cyclohexyl rings. The adamantyl group is bonded to the 4-membered ring portion of the dioxetane through a spiro linkage.

Group Y is a fluorescent chromophore bonded to group Z. Y becomes luminescent when an enzyme cleaves group V, X, or Z, thereby creating an electron-rich moiety which destabilizes the dioxetane, causing the dioxetane to decompose. Decomposition produces 2 individual ketones, one of which contains group T, and the other of which contains groups X, Y, and Z; the energy released from dioxetane decomposition causes the

Y group of the latter ketone to luminesce (if group X is H, an aldehyde is produced) .

The excited state energy of chromophore Y (i.e., the energy chromophore Y must possess in order to emit light) is preferably less than the excited state energy of the ketone containing group T in order to confine luminescence to group Y. For example, when T is adamantyl, the excited state energy of chromophore

Y is preferably less than the excited state energy of spiroadamantanone.

Any chromophore Y can be used according to the invention. In general, it is desirable to use a chromophore which maximizes the quantum yield in order to increase sensitivity. Examples of suitable chromophores include the following:

I) anthracene and anthracene derivatives, e.g., 9, 10-diphenylanthracene, 9-methylanthracene, 9-anthracene σarboxaldehyde, anthrylalcohols and 9-ρhenylanthracene; 2) rhodamine and rhodamine derivatives, e.g., rhodols, tetramethyl rhodamine, tetraethyl rhodamine, diphenyldimethyl rhodamine, diphenyldiethyl rhodamine, and dinaphthyl rhodamine;

3) fluorescein and fluorescein derivatives, e.g., 5-iodoacetamido fluorescein, 6-iodoacetamido fluorescein, and fluorescein-5-maleimide;

4) eosin and eosin derivatives, e.g., hydroxy eosins, eosin-5-iodoacetamide _Λ and eosin-5-maleimide;

5) coumarin and coumarin derivatives, e.g., 7-dialkylamino-4-methylcoumarin,

- 4-bromomethyl-7-methoxycoumarin, and , 4-brornomethyl-7—hydroxy coumarin;

6) erythrosin and erythrosin derivatives, e.g., hydroxy erythrosins, erythrosin-5-iodoacetamide and erythrosin-5-malimide;

7) aciridine and aciridine derivatives, e.g., hydroxy aciridines and 9-methyl aciridine;

8) pyrene and pyrene derivatives, e.g., N-(l-ρyrene) iodoacetamide, hydroxy pyrenes, and 1-ρyrenemethyl iodoacetate;

9) stilbene and stilbene derivatives, e.g., 6,6'-dibromostilbene and hydroxy stilbenes;

10) naphthalene and naphthalene derivatives, e.g., 5-dimethylamino naphthalene-1-sulfonic acid and hydroxy naphthalene;

II) nitrobenzoxadiazoles and nitrobenzoxadiazole derivatives, e.g., hydroxy nit obenzoxadiazoles, 4-chloro-7-nitrobenz-2-oxa-l, 3-diazole, 2-(7-nitrobenz-2-oxa-l,3-diazol-4-yl)

methylaminoacetaldehyde, and 6-(7-nitrobenz-2-oxa-l,3- diazol-4-yl-aminohexanoic acid;

12) quinoline and quinoline derivatives, e.g., 6-hydroxyquinoline and 6-aminoquinoline; 13) acridine and acridine derivatives, e.g.,

N-methylacridine and N-phenylacridine;

14) acidoacridine and acidoacridine derivatives, e.g., 9-methylacidoacridine and hydroxy-9-methylacidoacridine; 15) carbazole and carbazole derivatives, e.g., N-methylcarbazole and hydroxy-N-methylcarbazole;

16) fluorescent cyanines, e.g., DCM (a laser dye), hydroxy cyanines, 1, 6-diphenyl-l,3,-5-hexatriene, 1_(4-dimethyl aminophenyl)-6-phenylhexatriene, and the corresponding 1,3-butadienes;

17) carbocyanine and carbocyanine derivatives, e.g., phenylcarbocyanine and hydroxy carbocyanines;

18) pyridinium salts, e.g., 4(4-dialkyl diamino styryl) N-methyl pyridinium iodate and hydroxy-substituted pyridinium salts;

19) oxonols; and

20) resoro ins and hydroxy resorofins.

The most preferred chromophores are hydroxy derivatives of anthracene or naphthalene; the hydroxy group facilitates bonding to group Z.

Group Z is preferably bonded to chromophore Y through an enzyme-cleavable bond. Contact with the appropriate enzyme cleaves the enzyme-cleavable bond, yielding an electron-rich moiety bonded to chromophore Y; this moiety initiates the decomposition of the dioxetane into 2 individual ketones, or into a ketone and an aldehyde if group X is H. Examples of electron-rich moieties include oxygen, sulfur, and

amine or amido anions . The most preferred moiety is an oxygen anion. Examples of suitable enzyme-cleavable groups, and the enzymes specific to these groups, are given below in Table 1; an arrow denotes the enzyme-cleavable bond. The most preferred ^" group is a phosphate ester, which is cleaved by alkaline or acid phosphatase enzymes.

Table 1 Enzyme-Cleavable Group Enzyme

1) alkaline and acid phosphatases

0°

phosphate ester

2) esterases

acetate ester

3) deca box lases

carboxyl

phospholipase D

l-ρhospho-2,3-diacyl glycerides

5) β-xylosidase

β-D-xyloside

6) β-D-fucosidase

β-D-fucoside

7) thioglucosidase

l-thio-D-glucoside

8) ATPase

adenosine triphosphate analogs

9) ADPase

adenosine diphosphate analogs

10) nucleotidase

MHa

AMP analogs

11) β-D-galactosidase

β-D-galactoside

12) ^α -D-galactosidase

^α -D-galactoside

13) ^α -D-glucosidase

^α -D-glucoside

14) β-D-glucosidase

β-D-glucoside

15) ^α -D-mannosidase

^α -D-mannoside

16) β-D-mannosidase

β-D-mannoside

17) β-D-fructofuranosidase

β-D-fructofuranoside

18 ) β-D-glucosiduronase

β-D-glucosiduronate

19 ) trypsin

p-toluenesulfonyl-L-arginine dye ester

20) trypsin

ΓNJ-

p-toluenesulfonyl-L- arginine dye amide

Suitable X groups are described in the Summary of the Invention, above. Preferably, X contains one or more solubilizing substituents, i.e., substituents which enhance the solubility of the dioxetane in aqueous solution. Examples of solubilizing substituents include carboxylic acids, e.g., acetic acid; sulfonic acids, e.g., methanesulfonic acid; and quaternary amino salts, e.g., ammonium bromide; the most preferred solubilizing substituent is methane-or ethanesulfonic acid.

Preferably, the enzyme which cleaves group V, X, or Z is covalently bonded to a substance having a specific affinity for the substance being detected. Examples of specific affinity substances include antibodies, e.g., anti-hCG, where the substance being detected is an antigen, e.g., hCG; antigens, e.g., hCG, where the substance being detected is an antibody, e.g., anti-hCG; or a probe capable of binding to all or

a portion of a nucleic acid, e.g., DNA or RNA, being detected. Bonding is preferably through an amide bond. Synthesis

In general, the dioxetanes of the invention are synthesized in two steps. The first step involves synthesizing an appropriately substituted olefin having the formula

X

/

where T, X, Y, and Z are as described above. These olefins are preferably synthesized using the Wittig reaction, in which a ketone containing the T group is reacted with a phosphorus ylide (preferably based on triphenylphosphine) containing the X, Y, and Z groups, as follows:

The reaction is preferably carried out at -78°C in an ethereal solvent, e.g., tetrahydrofuran (THF) .

The phosphorus ylide is prepared by reacting triphenyl phosphine with a halogenated compound containing the X, Y, and Z groups in the presence of base; examples of preferred bases include n-butyllithium, sodium amide, sodium hydride, and sodium alkoxide; the most preferred base is n-butyllithium. The reaction sequence is as follows:

where Q is a halogen, e.g., Cl, Br, or I. The preferred halogen is Br. The reaction is preferably carried out at -78°C in THF.

The olefin where T is adamantyl (Ad), X is 5 methoxy (OCH-), Y is anthracene (An), and Z is phosphate (PO.) can be synthesized as follows.

Br-CH-OCH_ is phosphorylated by treating it An-OH with the product of phosphorus acid reacted in the. 10 _. presence of HgCl- with N-methylimidazole; the net result is to replace the hydroxyl group of An with a phosphate group. The phosphorylated product is then reacted with triphenylphosphine at -78°C in THF to form the phosphorus ylide having the formula OCH ₃

& }& ^X Pθ ₄ 5 The reaction is conducted in a dry Ar atmosphere.

Spiroadamantanone (Ad=0) is then added to the solution containing the ylide, while maintaining the temperature at -78°C, to form the olefin having the formula

id An-?0 .

The olefin is then purified using conventional chromatography methods.

The second step in the synthesis of the dioxetanes involves converting the olefin described above to the dioxetane. Preferably, the conversion is effected photochemically by treating the olefin with singlet oxygen ( 0_) in the presence of light.

0_ adds across the double bond to form the dioxetane as follows:

The reaction is preferably carried out at -78°C in a halogenated solvent, e.g., methylene chloride. 0_ is generated using a photosensitizer. Examples of photosensitizers include polymer-bound Rose Bengal (commercially known as Sensitox I and available from Hydron Laboratories, New Brunswick, N.J.) and methylene blue (a well-known dye and pH indicator) . The most preferred sensitizer is Rose Bengal.

The synthesis of the dioxetane having the formula

fo llows

The olefin having the formula

/ An- O

is dissolved in methylene chloride, and the solution is placed in a 2-cm 2 pyrex tube equipped with a glass paddle; the paddle is driven from above by an attached, glass enclosed, bar magnet. The solution is cooled to -78°C and lg of polymer-bound Rose Bengal is added with stirring. Oxygen is then passed over the surface of the agitated solution while the reaction tube is exposed to light from a 500W tungsten-halogen lamp (GE 500 Cl) equipped with a UV-cut off filter (Corning 3060: transmission at 365nm = 0.5%). Thin layer chromatography (tic) is used to monitor the disappearance of the olefin and the concurrent appearance of the dioxetane. After the reaction is complete (as indicated by tic), the solvent is removed and the dioxetane is isolated. Use

A wide variety of assays exist which use visually detectable means to determine the presence or concentration of a particular substance in a sample. The above-described dioxetanes can be used in any of these assays. Examples of such assays include immunoassays to detect antibodies or antigens, e.g., or β-hCG; enzyme assays; chemical assays to detect, e.g., potassium or sodium ions; and nucleic acid assays to detect, e.g., viruses (e.g., HTLV III or cytomegalovirus, or bacteria (e.g., E. Coli)) .

When the detectable substance is an antibody, antigen, or nucleic acid, the enzyme capable of cleaving group Z of the dioxetane is preferably bonded to a substance having a specific affinity for the detectable substance (i.e., a substance that binds specifically to the detectable substance), e.g., an antigen, antibody, or nucleic acid probe, respectively. Conventional methods, e.g., carbodiimide

coupling, are used to bond the enzyme to the specific affinity substance; bonding is preferably through an amide linkage.

In general, assays are performed as follows. A sample suspected of containing a detectable substance is contacted with a buffered solution containing an enzyme bonded to a substance having a specific affinity for the detectable substance. The resulting solution is incubated to allow the detectable substance to bind to the specific affinity portion of the specific affinity-enzyme compound. Excess specific affinity-enzyme compound is then washed away, and a dioxetane having a group Z that is cleavable by the enzyme portion of the specific affinity-enzyme compound is added. The enzyme cleaves group Z, causing the dioxetane to decompose into 2 ketones (or an aldehyde and a ketone when group X is H) ; chromphore Y bonded to one of the ketones is thus excited and luminesces. Luminescence is detected using, e.g., a cuvette or camera luminometer, as an indication of the presence of the detectable substance in the sample. Luminescence intensity is measured to determine the concentration of the substance.

When the detectable substance is an enzyme, a specific affinity substance is not necessary. Instead, a dioxetane having a Z group that is cleavable by the enzyme being detected is used. Therefore, an assay for the enzyme involves adding the dioxetane to the enzyme-containing sample, and detecting the resulting luminescence as an indication of the presence and the concentration of the enzyme.

Examples of specific assays follow.

A. Assay for Human IgG

A 96-well microtiter plate is coated with sheep anti-human IgG (F(ab) ₂ fragment specific). A serum sample containing human IgG is then added to the wells, and the wells are incubated for 1 hr. at room temperature. Following the incubation period, the serum sample is removed from the wells, and the wells are washed four times with an aqueous buffer solution containing 0.15M NaCl, 0.01M phosphate, and 0.1% bovine serum albumin (pH 7.4).

Alkaline phosphatase bonded to anti-human IgG is added to each well, and the wells are incubated for 1 hr. The wells are then washed four times with the above buffer solution, and a buffer solution of a phosphate-containing dioxetane is added. The resulting luminescence caused by enzymatic degradation of the dioxetane is detected in a luminometer, or with photographic film in a camera luminometer.

B. Assay for hCG Rabbit anti- ^α hCG is adsorbed onto a nylon-mesh membrane. A sample solution containing hCG, e.g., urine from a pregnant woman, is blotted through the membrane, after which the membrane is washed with lml of a buffer solution containing 0.15M NaCl, 0.01M phosphate, and 0.1% bovine serum albumin (pH 7.4).

Alkaline phosphatase-labelled anti-β-hCG is added to the membrane, and the membrane is washed again with 2ml of the above buffer solution. The membrane is then placed in the cuvette of a luminometer or into a camera luminometer, and contacted with a phosphate-containing dioxetane. The luminescence resulting from enzymatic degradation of the dioxetane is then detected.

C. Assay for Serum Alkaline Phosphatase

2.7ml of an aqueous buffer solution containing 0.84M 2-methyl-2-aminopropanol is placed in a 12x75mm pyrex test tube, and 0.1ml of a serum sample containing alkaline phosphatase added. The solution is then equilibrated to 30°C. 0.2.nl of a phosphate-containing dioxetane is added, and the test tube immediately placed in a luminometer to record the resulting luminescence. The level of light emission will be proportional to the rate of alkaline phosphatase activity.

D. Nucleic Acid Hybridization Assay

A sample of cerebrospinal fluid (CSF) suspected of containing cytomegalovirus is collected and placed on a nitrocellulose membrane. The sample is then chemically treated with urea or guanidinium isothiocyanate to break the cell walls and to degrade all cellular components except the viral DNA. The strands of the viral DNA thus produced are separated and attached to the nitrocellulose filter. A DNA probe specific to the viral DNA and labelled with alkaline phosphatase is then applied to the filter; the probe hybridizes with the complementary viral DNA strands. After hybridization, the filter is washed with an aqueous buffer solution containing 0.2 M NaCl and .ImM Tris-HCl (ρH=8.0) to remove excess probe molecules. A phosphate-containing dioxetane is added and the resulting luminescence from the enzymatic degradation of the dioxetane is measu -ed in a luminometer or detected with photographic film.

Other embodiments are within the following claims.

For example, the enzyme-cleavable group Z can be bonded to group X of the dioxetane, instead of group

Y. The specific affinity substance can be bonded to the dioxetane through groups X, Y, or T (preferably group X), instead of the enzyme. In this case, the group to which the specific affinity substance is bonded is provided with, e.g., a carboxylic acid, amino, or maleimide substituent to facilitate bonding.

Groups X, Y, or T of the dioxetane can be bonded to a polymerizable group, e.g., a vinyl group, which can be polymerized to form a homopolymer or copolymer.

Groups X, Y, or T of the dioxetane can be bonded to, e.g., membranes, films, beads, or polymers for use in immuno- or nucleic acid assays. The groups are provided with, e.g., carboxylic acid, amino, or maleimide substituents to facilitate bonding.

Groups X, Y, or. T of the dioxetane can contain substituents which ^' enhance the kinetics of the dioxetane enzymatic degradation, e.g., electron-rich moieties (e.g., methoxy) . Groups Y and T of the dioxetane, as well as group X, can contain solubilizing substituents.

Appropriately substituted dioxetanes can be synthesized chemically, as well as photochemically. For example, the olefin prepared from the ittig reaction can be epoxidized using a peracid, e.g., p-nitroperbenzoic acid. The epoxidized olefin can then be converted to the dioxetane by treatment with an ammonium salt, e.g., tetramethylammonium hydroxide. Another example of a chemical synthesis involves converting the olefin prepared from the Wittig reaction to a 1,2 bromohydroperoxide by reacting the olefin with H„0_ and dibromantin (1,3-dibromo-5,5-dimethyl hydantoin) . Treatment of the

1,2-bromohydroperoxide with base, e.g., OH or silver salts, e.g., silver bromide, forms the dioxetane.

Olefin precursors for the dioxetane can be synthesized by reacting a ketone with a perester in the presence of TiCl_ and lithium aluminum hydride

(LAH) . For example, to synthesize an olefin where T is adamantyl (Ad), X is methoxy (OCH_), Y is anthracene (An), and Z is phosphate (PO.), the following reaction sequence is used:

To phosohorylate chromophore Y, e.g., anthracene, a hydroxyl derivative of the chromophore, e.g., hydroxy anthracene, can be reacted with a cyclic acyl phosphate hav-ing the following formula:

H

The reaction product is then hydrolyzed with water to yield the phosphorylated chromophore. The cyclic acyl phosphate is prepared by reacting 2,2,2-trimethoxy-4,5- dimethyl-1,3-dioxaphospholene with phosgene at 0°C, following by heating at 120°C for 2 hr.