Synthesis of 1,2-dioxetanes and intermediates therefor

Abstract: Compounds having formula (I), wherein T is a polycycloalkylidene group (e.g., adamant-2-ylidene); R is a C₁₋₂₀ alkyl, aralkyl or cycloalkyl group; and Y is a fluorescent chromophore (e.g., m-phenylene), produced by reacting a compound having formula (II) with an R-ylating agent (e.g., R₂SO₄) in the presence of an alkali metal alkoxide in a polar aprotic solvent. Also, compounds having formula (III) are produced by reacting a compound having formula (IV) with formula (V), wherein X is an electronegative leaving group (e.g., a halogen anion such as chloride ion) in the presence of a Lewis base (e.g., a trialkyl-amine) dissolved in an aprotic organic solvent (e.g., benzene or toluene). Also, compounds having formula (VI) are produced by reacting a compound of formula (VII) with a tetra-O-acylated-O-hexopyranoside halide, then hydrolysing off the protective acyl groups. The aforementioned compounds and procedures are useful in the synthesis of enzyme-cleavable 1,2-dioxetane ring systems that can serve as members of a binding pair employed, for example, in chemiluminescent immunoassays, DNA probe assays, and direct assays for an enzyme.

Description: BACKGROUND OF THE INVENTION Field of the Invention The invention relates to the production of 1,2-dioxetanes and their intermediates useable in assay methods in which a member of a specific binding pair can be detected and quantified by means of an optically detectable reaction brought about by the enzymolysis of an enzyme-cleavable group in a 1,2-dioxetane molecule. Description of Related Art 1,2-Dioxetanes, cyclic organic peroxides whose central structure is a four-membered ring containing a pair of contiguous carbon atoms and a pair of contiguous oxygen atoms (a peroxide linkage), are a known, but heretofore seldom utilized, class of compounds. Because of their inherent chemical instability, some 1,2-dioxetanes exhibit chemiluminescent decomposition under certain conditions, e.g., by the action of enzymes, as described in copending, commonly-assigned Bronstein, U.S. patent application Serial No. 889,823 entitled "Method of Detecting a Substance Using Enzymatically-Induced Decomposition of Dioxetanes", and in copending, commonly assigned Bronstein, et al., U.S. patent application Serial No. 140,035 entitled "Dioxetanes for Use in Assays", the disclosures of which are incorporated herein by reference. The amount of light emitted during such chemiluminescence is a measure of the concentration of a luminescent substance which, in turn, is a measure of the concentration of its precursor 1,2-dioxetane. Thus, by measuring the intensity and duration of luminescence, the concentration of the 1,2-dioxetane (and hence the concentration of the substance being assayed, i.e., the species bound to the 1,2-dioxetane member of the specific binding pair) can he determined. The appropriate choice of substituents on the 1,2-dioxetane ring allows for the adjustment of the chemical stability of the molecule which, in turn, affords a means of controlling the onset of chemiluminescence, thereby enhancing the usefulness of the chemiluminescent behavior of such compounds for practical purposes, e.g., in chemiluminescence immunoassays and DNA probe assays. The preparation of 1,2-dioxetanes by photooxidation of olefinic double bonds is known. However, a need exists for a convenient, general synthesis of substituted 1,2-dioxetanes from olefinically unsaturated precursors derived from readily available or obtainable starting materials through tractable intermediates. In this connection, a particular need exists for a commercially useful method for producing substituted 1,2-dioxetanes of the formula: from enol ether-type precursors: wherein T, R, Y and Z are defined herein below, and Z represents hydrogen (in which case the dioxetane can be thermally cleaved via rupture of the oxygen-oxygen bond), a chemically cleavable group such as a hydroxyl group, an alkanoyl or aroyl ester group, or an alkyl or aryl silyloxy group, or an enzyme-cleavable group containing a bond cleavable by an enzyme to yield an electron-rich moiety bonded to chromophore Y, e.g., a bond which, when cleaved, yields an oxygen anion, a sulfur anion, an amine, or a nitrogen anion, and particularly an amido anion such as a sulfonamido anion. Enol ethers can be prepared by several classical methods, for example, by acid-catalyzed elimination of alcohol from acetals [R.A. Whol, "Synthesis", p. 38 (1974)], by Peterson or Wittig reactions of alkoxymethylene silanes or phosphoranes with aldehydes or ketones in basic media [Magnus, P. et al., Organometallics , 1 , 553 (1982)], and by reactions of alkoxyacetic acid dianions with ketones followed by propiolactone formation and elimination of CO 2 [Caron, G., et al., Can. J. Chem. , 51 , 981 (1973)]. The O-alkylation of ketone enolate anions is less often used as a general preparative method due to the variable amounts of concomitantly formed alphaalkylated ketones, the extent of which depends on the solvent, base, alkylating agent and ketone structure (see, H.O. House, "Modern Synthetic Reactions" pp. 163-215 (Benjamin, 1965); and J.D. Roberts and M.C. Caserio, "Basic Principles of Organic Chemistry" (Benjamin, 1964)). With the use of hexamethyl phosphoramide (HMPA), a known carcinogenic solvent, it is, at best, possible to obtain yields of the O-alkylation product which are no higher than 70%. Moreover, the separation of enol ether from the C-alkylated ketone is quite tedious. Tetrahedron Letters, vol. 28, no. 9, 1987 , pages 935-938 relates to chemical and enzymatic triggering of 1,2-dioxetanes, particularly to aryl esterase-catalysed chemiluminescence from a naphthyl acetate-substituted dioxetane. EP-A-0 254 051 relates to light producing 1,2-dioxetanes described of the formula wherein ArOX is an aryl ring substituted with an X oxy group and A are passive organic groups which allow the 1,2-dioxetane to produce light when triggered by removing X. X is a chemically labile group which is removed by an activating agent. The 1,2-dioxetane compounds can be triggered to produce light at room temperatures. Tetrahedron Letters, vol. 28, no. 11, 1987 , pages 1159-1162 also relates to chemical and enzymatic triggering of 1,2-dioxetanes, particularly to alkaline phosphatase-catalysed chemiluminescence from an aryl phosphate-substituted dioxetane. Tetrahedron Letters, vol. 28, no. 44, 1987 , pages 5319-5322 relates to the synthesis of 1,2-dioxetanes via electron-transfer oxygenation. This reference reports synthesis of thermally stable 1,2-dioxetanes by the reaction of enol-ethers with tris p-bromophenylammoniumyl hexachloroantimonate via a chain oxygenation reaction involving formation of enol-ether cation radicals. Tetrahedron Letters, vol. 28, no. 11, 1987 , pages 1155-1158 relates to fluoride-induced chemiluminescence from tert-butyldimethylsilyloxy-substituted dioxetanes. In particular, this reference describes the preparation of thermally stable 1,2-dioxetanes bearing tert -butyl-dimethylsilyloxyaryl groups. SUMMARY OF THE INVENTION Adamant-2-yl aryl ketones have been known since the late 1960's (Chem. Abst. 71 :P80812V). No attempts to O-alkylate them, however, have been found in the literature. It has now been discovered that reaction of these ketones, as enolates, with reactive alkylating agents containing "hard" leaving groups [see, Fleming, I., "Frontier Orbitals and Organic Chemical Reactions", p. 40 (Wiley, 1976)],if carried out in a polar aprotic solvent such as dimethyl sulfoxide, dimethylformamide, 1,3-dimethyl-2-imidazolidinone, N-methyl-2-pyrrolidinone, and the like, or a mixture of such solvents, results exclusively in O-alkylation. The enol ethers thus obtained can be used as convenient intermediates in the synthesis of water-soluble or water compatible 1,2-dioxetanes. Such intermediates can be used to prepare substrates which react with singlet oxygen (generated chemically or photochemically) to yield 1,2-dioxetanes of sufficient stability to be useful in subsequent assay techniques based on chemiluminescent dioxetane decomposition: This O-alkylation process is general and therefore extendable to other cycloalkyl aryl ketone substrates, which can be synthesized by the reaction of the appropriate secondary cycloalkyl aldehyde with an aryl Grignard reagent, followed by oxidation of the resulting secondary alcohol with Jones reagent. Preferably, the Grignard reagent is reacted with a secondary cycloalkyl nitrile, followed by acid hydrolysis to form a ketone via an imine salt. In all cases, starting materials and products contain a functional group attached to a secondary carbon atom of the cycloalkyl system, which in the case of fused polycycloalkyl (e.g., adamantyl) systems is flanked on either side by a bridgehead carbon atom. It is, thus, an object of this invention to provide novel synthetic routes to enzyme-cleavable 1,2-dioxetane derivatives. It is a further object of this invention to provide processes for the preparation of novel chemical intermediates in the synthesis of 1,2-dioxetanes. Yet another object of this invention is to provide novel compositions of matter, such as trisubstituted enolether phosphates, useful as synthetic precursors of 1,2-dioxetanes which dioxetanes decompose enzymatically in an optically-detectable reaction. These and other objects of the invention, as well as a fuller understanding of the advantages thereof, can be had by reference to the following description and claims. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Among the 1,2-dioxetanes that can be prepared in accordance with the present invention are those having the formula: In this formula T represents a stabilizing group that prevents the dioxetane compound from decomposing before the bond in the labile ring substituent attached to Y is intentionally cleaved. The stabilizing group is a fused polycycloalkylidene group bonded to the 3-carbon atom of the dioxetane ring through a carbon-carbon or a spiro linkage and having two or more fused rings, each having from 3 to 12 carbon atoms, inclusive, e.g., an adamant-2-ylidene or an adamant-2-yl group, which may additionally contain unsaturated bonds or 1,2 fused aromatic rings, or a substituted or unsubstituted alkyl group having from 1 to 12 carbon atoms, inclusive, such as tertiary butyl or 2-cyanoethyl, or an aryl or substituted aryl group such as carboxyphenyl, or a halogen group such as chloro, or a heteroatom group which can be a hydroxyl group or a substituted or unsubstituted alkoxy or aryloxy group having from 1 to 12 carbon atoms, inclusive, such as an ethoxy, hydroxyethoxy, methoxyethoxy, carboxymethoxy, or polyethyleneoxy group. The symbol Y represents a light-emitting fluorophore-forming fluorescent chromophore group capable of absorbing energy to form an excited energy state from which it emits optically detectable energy to return to its original energy state. Any carbon position in Y can be attached to the dioxetane ring. Examples of suitable Y chromophores include: 1) phenylene and phenylene derivatives, e.g., hydroxyphenyl, hydroxybiphenyl, hydroxy-9,10-dihydrophenanthrene; 2) naphthalene and naphthalene derivatives, e.g., 5-dimethylamino naphthalene-1-sulfonic acid, hydroxy naphthalene, naphthalimides or hydroxy naphthalimides; 3) anthracene and anthracene derivatives, e.g., 9,10-diphenylanthracene, 9-methylanthracene, 9-anthracene carboxaldehyde, hydroxyanthracenes and 9-phenylanthracene; 4) rhodamine and rhodamine derivatives, e.g., rhodols, tetraethyl rhodamine, tetraethyl rhodamine, diphenyldimethyl rhodamine, diphenyldiethyl rhodamine, and dinaphthyl rhodamine; 5) fluorescein and fluorescein derivatives, e.g., 4- or 7-hydroxyfluorescein, 6-iodoacetamido fluorescein, and fluorescein-5-maleimide; 6) eosin and eosin derivatives, e.g., hydroxy eosins, eosin-5-iodoacetamide, and eosin-5-maleimide; 7) coumarin and coumarin derivatives, e.g., 7-dialkylamino-4-methylcoumarin, 4-cyano-7-hydroxy coumarin, and 4-bromomethyl-7-hydroxycoumarin; 8) erythrosin and erythrosin derivatives, e.g., hydroxy erythrosins, erythrosin-5-iodoacetamide and erythrosin-5-maleimide; 9) benzheteroazoles and derivatives, e.g., 2-phenylbenzoxazole, hydroxy-2-phenylbenzoxazoles, hydroxy-2-phenylbenzthiazole and hydroxybenzotriazoles; 10) pyrene and pyrene derivatives, e.g., N-(1-pyrene) iodoacetamide, hydroxypyrenes, and 1-pyrenemethyl iodoacetate: 11) stilbene and stilbene derivatives, e.g., 6,6'-dibromostilbene and hydroxy stilbenes, hydroxydibenzosuberene; 12) nitrobenzoxadiazoles and nitrobenzoxadiazole derivatives, e.g., hydroxy nitrobenzoxadiazoles, 4-chloro-7-nitrobenz-2-oxa-1,3-diazol, 2-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) methylaminoacetaldehyde, and 6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)aminohexanoic acid; 13) quinoline and quinoline derivatives, e.g., 6-hydroxyquinoline and 6-aminoquinoline; 14) acridine and acridine derivatives, e.g., N-methylacridine, N-phenylacridine, hyydroxyacridines, and N-methylhydroxyacridine; 15) acidoacridine and acidoacridine derivatives, e.g., 9-methylacidoacridine and hydroxy-9-methylacidoacridine; 16) carbazole and carbazole derivatives, e.g., N-methylcarbazole and hydroxy-N-methylcarbazole: 17) fluorescent cyanines, e.g., DCM (a laser dye), hydroxy cyanines, 1,6-diphenyl-1,3,5-hexatriene, 1-(4-dimethyl aminophenyl)-6-phenylhexatriene, the corresponding 1,3-butadienes, or any hydroxy derivative of the dienes or trienes; 18) carbocyanine and carbocyanine derivatives, e.g., phenylcarbocyanine and hydroxy carbocyanines; 19) pyridinium salts, e.g., 4(4-dialkylamino styryl) N-methyl pyridinium salts and hydroxy-substituted pyridinium salts; 20) oxonols; and 21) resorofins and hydroxy resorofins. The most suitable Y chromophores are derivatives of benzene or naphthalene: M + is an alkali metal, ammonium, or a C 1 -C 7 alkyl, aralkyl, or aromatic quaternary ammonium cation, (NR 4 ) + wherein R 4 can be any or all of an alkyl, aralkyl, or form part of a heterocyclic ring system. The symbol R represents a C 1 -C 20 unbranched or branched, substituted or unsubstituted, saturated or unsaturated alkyl group, e.g., methyl, allyl or isobutyl: a heteroaralkyl or aralkyl (including ethylenically unsaturated aralkyl) group, e.g., benzyl or vinylbenzyl; a polynuclear (fused ring) or heteropolynuclear aralkyl group which may be further substituted, e.g., naphthylmethyl or 2-(benzothiazol-2-yl)ethyl; a saturated or unsaturated cycloalkyl group, e.g., cyclohexyl or cyclohexenyl; a N, O, or S heteroatom containing group, e.g., 4-hydroxybutyl, methoxyethyl, or polyalkyleneoxyalkyl; an aryl group; or on enzyme labile group containing a bond cleavable by an enzyme to yield an electron rich moiety bonded to the dioxetane ring. Preferably, R is a methyl or ethyl group. One or more of the formula components T, R or Y can also include a substituent which enhances the water solubility of the 1,2-dioxetane such as a carboxylic acid, sulfonic acid or their salts, or a quaternary amino salt group. R may be bonded to Y to form a fused ring fluorophore-forming group which is in turn bonded to the 4-carbon atom of the dioxetane through a Spiro linkage and which therefore results in an excited lactone fragment upon chemical or enzymatic dioxetane decomposition. The required enol ethers are obtained by intramolecular O-alkylation of fused polycycloalkyl aryl ketone enolates by another substituent, e.g., a toluenesulfonyloxyethyl group, in accordance with the methodology presented herein. Y may also be further substituted with one or more electron withdrawing groups, e.g., perfluoroalkyl having from 1 to 7 carbon atoms such as trifluoromethyl; alkyl or arylsulfonyl such as methylsulfonyl; halogen such as fluoro or chloro; cyano; nitro; alkoxycarbonyl such as -COOEt; alkanoyl such as -COCH 3 ; amidosulfonyl such as -SO 2 NHAr; or with one or more electron donating groups such as a branched or unbranched alkyl group having from 1 to 7 carbon atoms; an alkoxy or aralkoxy group having from 1 to 30 carbon atoms which may contain fused aromatic or fused heteroaromatic rings which are further substituted with heteroatom containing moieties, e.g., 2-(5-fluoresceinyl)-ethoxy; an aryloxy group having 1 or 2 rings and which may be further substituted, e.g., phenoxy; a branched or straight chain C 1 -C 7 hydroxyalkyl group, e.g., hydroxymethyl or hydroxyethyl; an aryl group containing one or more hydroxy substituents or alkoxy substituents having 1 to 7 carbon atoms, e.g., 3,5-diethoxyphenyl; or a heteroaryl group having 1 or 2 rings, e.g., benzoxazole, benzthiazole, benzimidazole or benzotriazole. Furthermore, by suitably modifying T, R, and Y groups of 1,2-dioxetanes, the stability of the 1,2-dioxetanes and the rate of decomposition of the 1,2-dioxetanes can be varied. For example, 1,2-dioxetanes can be attached to various molecules (e.g., proteins or haptens) or immobilizing supports (e.g., polymer membranes); they can also constitute side chain groups of homopolymers or copolymers. More particularly, the method for producing 1,2-dioxetanes according to the present invention comprises the following reaction sequence: wherein R 1 can be independently any of the substituents as defined above for R; W is an acid anion such as halide (e.g., chloride); and X and the "R-ylating agent" are as defined below. Step 1, which involves the slow attack of an aromatic Grignard reagent on a nitrile, may be run at reflux in several ethereal solvents such as diethyl ether (34°), THF (67°), or ethylene glycol dimethyl ether (85°). Thus, although the reaction can be run conveniently over the temperature range of 30°-85°C, the use of THF at reflux provides optimum performance with yields above 90%. As will be understood by one skilled in the art, the use of the analogous organolithium compound to replace the Grignard reagent is possible in the above scheme, however, it is known that THF and organolithium compounds (especially n-butyllithium, the metal-halogen exchange reagent) can be incompatible at higher temperatures. Therefore, one can have recourse to the methodology described in Edwards, et al., co-pending and commonly-assigned U.S. Serial No. 213,672, filed June 30, 1988. The reaction of a fused polycycloaldehyde with an aromatic organolitium moiety allows a similar bond construction to be accomplished in diethyl ether over a temperature range of -60° to 0°C. This process then provides a convenient low temperature counterpart to the nitrile reaction, which requires only a facile, ancillary oxidation to arrive at the same ketonic product. Step III is best accomplished in the solvents listed using sodium or potassium hydride or potassium tertbutoxide as the base. This step utilizes reactive alkylating agents to give a kinetic product and can be run conveniently over a temperature range of 0° to 60°C, depending on the "R"-ylating reagent. Dimethyl or diethyl sulfate are particularly useful and inexpensive reagents which display optimum performance between 25° and 60°C. In Step IV, the phenolic ether cleavage with sodium thioethoxide may be accomplished with soft nucleophiles such as with lithium iodide in refluxing pyridine, sodium cyanide in refluxing DMSO, or sodium sulfide in refluxing N-methyl-2-pyrrolidinone are identical in spirit while having other drawbacks from a commercial point of view. Steps V, VI and VII, as indicated herein, may be performed separately or in one operation. The cyclic phosphorichloridate is utilized not only because of its monofunctionality, chemoselectivity, and enol ether-compatible deprotection mode, but also because, by virtue of pseudo-rotation, it is 10 6 times more reactive than acyclic versions. Thus, in cases where an aromatic hydroxyl group is hindered (e.g., a peri position in a polycyclic, aromatic ring system), or if other substituents lower the pKb or nucleophilicity of the enol ether oxyanion, reasonable reaction rates and yields are possible. In benzene, THF, diethyl ether, or DMF, phosphate triester formation with a Lewis base, or with a preformed alkali metal salt can be effected with all of the phosphorochloridates listed over a temperature range of -30° to 50°C. Subsequently, if a pure monosodium cyanoethyl phosphate ester is desired the ring cleavage with alkali cyanide in DMF or DMSO, should be run in a narrow temperature range between 15° and 30°C. In a one pot or in situ mode this is not important and the range widens to 60°C on the high end. It may be apparent that one can employ phase transfer techniques under catalysis by quaternary ammonium ions or crown ethers to generate an even more reactive "naked" cyanide and thus to utilize organic solvents of higher volatility (e.g., CH 2 Cl 2 ), facilitating work-up. Alternatively, the direct use of pure, quaternary ammonium cyanides or sulfinates gives immediate access to phosphate intermediates or products which contain associated gegenions useful in modifying physical properties such as solubility. Such modifications are within the scope of the process parameters disclosed herein. Beta-elimination processes brought to bear on the cyanoethyl substituted phosphate diester may occur under the influence of a wide range of bases. However, aqueous ammonium hydroxide can be used in vast excess due to its ease of removal at the end of the process. The cleavage can be accomplished over a temperature range of 25° to 100°C. At higher temperatures, however, provisions must be made to avoid losses of gaseous ammonia, and thus, a high-pressure vessel or bomb is required. The preferred temperature range is 35° to 55°C, where the phosphate monoester product is quite stable, and where simple glassware outfitted with wired septa can be used as a closed system. Use of alkali metal or quaternary ammonium hydroxides in this step requires close attention to stoichiometry, but as stated above, can provide a variety of mixed gegenion phosphate salts. While chemical methods of dioxetane formation, e.g, triethylsilyl hydrotrioxide, or phosphite ozonide sources of singlet oxygen and triarylamine radical cation mediated one-electron oxidation in the presence of triplet oxygen are known, sensitized photooxygenation is a particularly convenient and forgiving process when reactive olefins are used as substrates. A variety of sensitizing dyes may be used to advantage, with chlorinated hydrocarbons comprising a preferred class of solvents. Reactions are rapid over a temperature range of -78° to 25°C. Low temperatures are not required however for these relatively stable dioxetanes, and in the case of certain phosphate salts, solubility will be reduced. The ability to manipulate gegenions directly via the synthetic methodology disclosed or in subsequent ion exchange steps permits flexibility. The preferred temperature range for all photoxygenation steps is thus 0° to 10°C. The foregoing sequences of reactions can be carried out step-by-step with isolation of the product of each reaction. However, step VI (alkyl cleavage with a nucleophilic acidifying anion such as CN - or organic sulfinate ion) and step VII (deprotection via a beta-elimination reaction) can be performed advantageously without isolation of the intermediate phosphate ester salt; such intermediate need be isolated only when it is desired to confirm its existence. In steps VI and VII of the foregoing reaction sequence, the cation, M + , in the salt used in step VI and the cation, M + , in the base used in Step VII can be an alkali metal (e.g., Na + ), ammonium, or a C 1 - C 7 alkyl, aralkyl, or aromatic quaternary ammonium cation, (NR 4 ) + (wherein R 4 can be any or all of an alkyl, e.g., ethyl, aralkyl, e.g., benzyl, or form part of a heterocyclic ring system, e.g., pyridinium), so that the products of steps VII and VIII would be as follows: In addition, the quaternary ammonium cation can be connected through one of its quaternizing groups to a polymeric backbone, as follows: or can itself be part of a polyquaternary ammonium salt. M + can also be a fluorescent onium moiety such as a substituted benzopyrillium or 2-[4-dimethylaminostyryl]-N-methylpyridinium counterion. Within the framework of the foregoing synthesis, the present invention comprises a process for producing a compound having the formula: wherein T =, R, R 1 and Y are defined above, by reacting a compound having the formula: wherein T is spiro bound at a carbon atom alpha to the carbonyl group, with an alkylating agent (or in more general terms consistent with the definition of R, an "R-ylating agent") selected from the group including R-sulfate, toluenesulfonate ("Tosylate"), methanesulfonate ("mesylate"), trifluoromethanesulfonate ("triflate"), and chloromethyl ethers and trialkyloxonium salts, in a basic, polar, aprotic medium, for example, an alkali metal alkoxide in dimethyl sulfoxide. The invention further provides a process for producing a compound having the formula: wherein T, R, and Y are as defined above, comprising reacting a compound having the formula: with wherein X is an electronegative leaving group such as halogen (e.g., chloro), in the presence of a Lewis base such as a tertiary amine (e.g., triethylamine) dissolved in an aprotic organic solvent, such as an aromatic liquid (e.g., benzene, toluene), and ether (e.g., glyme, diglyme) or a cyclic ether (e.g., tetrahydrofuran ("THF")). In a one-pot process, where synthesis of a phosphate triester and subsequent deprotection to a monoester are done in situ , it is advantageous to preform an alkali metal salt of the aforementioned Y-OH compound in a polar, aprotic solvent such as dimethylformamide, using NaH as the base (see Example 8 below). Addition of the phosphorochloridate affords a solution of the triester which can be directly converted ( - CN, NH 4 OH) to the monoester in the same reaction medium. As an alternative to the use of halophosphate, the analogous halophosphites, i.e., XPO 2 (CH 2 ) 2 , can be used with subsequent oxidation and irradiation to form the dioxetane directly. In another aspect, the invention provides a process for producing compounds having the formulas: wherein T, R and Y are as defined above, R 5 can be independent of any of the substituents described above for R, and R 2 and R 3 are each independently cyano, ortho- or para-nitrophenyl, ortho, para- or ortho, ortho'-dinitrophenyl, comprising reacting a compound having the formula: with wherein X is as defined above, in the presence of a Lewis base such as a tertiary amine (e.g., a trialkylamine) in an aprotic organic solvent such as an aromatic liquid (e.g., benzene or toluene), an ether (e.g., glyme, diglyme) or a cyclic ether (e.g., THF). As an alternative to the use of halophosphates, the analogous nor-oxy compounds (i.e., halophosphites) can be used, followed by oxidation at the phosphorous, deprotection and photooxidation to the dioxetane. In the case of the cyclic phosphite, dioxetane formation and oxidation at the phosphorous can occur simultaneously in the presence of 3 O 2 / 1 O 2 mixtures found in the photooxidation reaction. Preferably, the oxidation described above is effected photochemically by treating the olefin with singlet oxygen ( 1 O 2 ) in the presence of light. 1 O 2 adds across the double bond to form the dioxetane. The reaction is preferably carried out at or below 0°C in a halogenated solvent, e.g., methylene chloride. 1 O 2 can be generated using a photosensitizer. As photosensitizers, 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) or TPP (see Example 9 below) can be used. The dioxetanes of the invention provide a method for generation of light in an optically detectable assay method to determine the presence or concentration of a particular substance in a sample. Examples of such assays include immunoassays to detect antibodies or antigens (e.g., hormones such as α or β-hCG, TSH, LH, etc., cancer-associated antigens such as AFP and CEA) (enzyme-immunoassay); enzyme assays (e.g., alkaline phosphatases and α- or β-D-galactosidases); chemical assays to detect cations, e.g., potassium or sodium ions; and nucleotide probe assays to detect, e.g., viruses (e.g., HSVI, HTLV III, hepatitis virus, 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 (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 (e.g., antigen) is contacted with a buffered solution containing an enzyme bonded to a substance having a specific affinity for the detectable substance (e.g., antibody). The resulting solution is contacted with a solid phase, e.g., antibody-binding beads, to which another substance having the specific affinity, e.g., antibody, is bound. After incubation for a certain period, excess enzyme which is bound to be substance with specific affinity is then washed away, and a 1,2-dioxetane (substrate) having a group Z that is cleavable by the enzyme portion is added. The enzyme cleaves group Z, causing the dioxetane to decompose into ketone and ester moieties; chromophore Y bonded to the ester 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 (e.g., antibody) is not necessary. Instead, 1,2-dioxetanes having a Z group that is cleavable by the enzyme being detected is used. Therefore, an assay for the enzyme involves adding 1,2-dioxetanes to the enzyme-containing sample, and detecting the resulting luminescence as an indication of the presence and the concentration of the enzyme. The following examples are intended to illustrate the invention in detail, but they are in no way to be taken as limiting, and the present invention is intended to encompass modifications and variations of these examples within the framework of their contents. EXAMPLE 1 3-Methoxyphenyl adamant-2-yl ketone Magnesium turnings (1.64 g, 0.067 mol) were placed in a flame-dried flask under argon. A small crystal of iodine and 7 ml of dry tetrahydrofuran ("THF") (freshly distilled over lithium aluminum hydride) was added. A quantity (7 ml, 0.055 mol) of 3-bromoanisole was added by syringe to the slightly agitated suspension of the metal. An exothermic reaction began after brief heating to 50°C. The flask was placed in a water bath at room temperature while THF (33 ml) was added in a thin stream from an addition funnel. After the exothermic reaction had subsided, the mixture was refluxed for 45 minutes. A solution of 2-cyanoadamantane (8.7 g, 0.054 mol; see, "Organic Syntheses", 57 , 8 (Wiley, 1977) or van Leusen, A.M. et al., J. Org. Chem. , 42 , 3114 (1977)) in 50 ml of dry THF was added dropwise over 1.5 hours to the refluxing Grignard reagent. After heating the reaction mixture at reflux temperature overnight, a yellow suspension was obtained. Ether (50 ml) was added, while the flask and its contents were cooled in an ice bath. Concentrated hydrochloric acid (8 ml, 0.096 mol HCl) was added dropwise with vigorous stirring over a period of 20 minutes. The precipitate was separated by filtration, washed with ether, and dried to obtain 29 g of the ketenimine salt as a light, buff-colored, non-hygroscopic powder containing some residual magnesium. The salt was suspended in a mixture of 90 ml of ethanol and 90 ml of concentrated hydrochloric acid and refluxed for 3 hours, during which time the mixture became considerably thinner. After cooling in an ice bath, the resulting solid was broken up, separated by filtration, washed to neutrality and dried to obtain 13.65 9 (93% yield based on 2-cyanoadamantane) of the light gray ketone (m.p. 111-114°C). Thin layer chromatography ("TLC") indicated that the product was sufficiently pure for subsequent manipulation (R f 0.45; Whatman K5F CH 2 Cl 2 : hexanes, 50:50). Recrystallization from hexanes yielded the captioned compound as prismatic crystals (m.p. 113-115°C). I.R. (CH 2 Cl 2 ) : 2900 cm -1 , 1670 cm -1 (C=O), 1590 cm -1 , 1575 cm -1 . 1 H-NMR (400 MHz, CDCl 3 ): δ 1.55-2.05 (m, 12H); 2.30 (s, 2H); 3.41 (s, 1H); 3.84 (s, 3H); 7.03-7.40 (m, 4H). These data confirmed the following structure: EXAMPLE 2 Methoxy(3-methoxyphenyl)methylene adamantane A quantity (11.3 g, 0.042 mol) of 3-methoxyphenyl adamant-2-yl ketone obtained according to Example 1 was suspended in 90 ml of molecular sieve-dried (3 Å) dimethylsulfoxide (DMSO). Heat was applied to dissolve the suspended solid. Upon cooling to room temperature with stirring, a fine suspension was formed. Potassium tertbutoxide (8.5 g, 0.070 mol) was added under an argon atmosphere. After 5 minutes, a nearly homogenous orange solution resulted, which was placed in a water bath at 50°C. Dimethyl sulfate (4 ml, 0.042 mol) was added dropwise by syringe over a period of 10 minutes. After 15 minutes of further stirring, an additional 3.3 ml of dimethyl sulfate (0.034 mol) was added in the same fashion. Subsequently, the colorless solution was stirred overnight at room temperature. After cooling in an ice bath, 0.5 g of K 2 CO 3 and 125 ml of ice water added and the mixture extracted with three 50 ml portions of ethyl acetate. The combined organic fractions were washed with three portions of water, once with 50 ml of saturated aqueous NaCl solution, and dried over K 2 CO 3 . The solvent was removed in vacuo to yield an oil. The oil was dissolved in hexane, and the resulting solution filtered through Celite and concentrated in vacuo to provide 11.5 g (96% yield) of a viscous, straw-colored oily substance. TLC indicated a clean conversion to an enol ether (R f 0.68; E. Merck Al 2 O 3 -CH 2 Cl 2 : hexanes - 50:50) with a trace of the ketone starting material. The oil was distilled from K 2 CO 3 (b.p. 148-150°C, 0.25 mm Hg). Under these conditions, slight yellowing occurred in the still head. I.R. analysis of this distillate revealed a small ketone absorption band at 1670 cm -1 . I.R. (CH 2 Cl 2 ): 2900 cm -1 , 1670 cm -1 (weak),1600 cm -1 , 1590 cm -1 , 1580 cm -1 , 1570 cm -1 , 1095 cm -1 , 1080 cm -1 ; 1 H-NMR 60 MHz (CDCl 3 ): δ 1.5-2.0 (m, 12H), δ 2.55 (s, 1H), δ 3.2 (s, 1H), δ 3.25 (s, 3H), δ 3.75 (s, 3H), and δ 6.7-7.3 (m, 4H). These data confirmed that the structure of the product was: EXAMPLE 3 Methoxy(3-hydroxyphenyl)methylene adamantane A solution of methoxy(3-methoxyphenyl)methylene adamantane (14 g, 0.049 mol), obtained according to Example 2, in 70 ml of molecular sieve-dried (3 Å) dimethylformamide (DMF) was added under an argon atmosphere to a solution of sodium thioethoxide (7.4g, 0.88 mol) in the same solvent. The mixture was refluxed for 3 hours. After cooling in an ice bath with stirring, the reaction was quenched with 62 g of NH 4 Cl in 200 ml of water. Ethyl acetate (120 ml) and a small amount of ice water were added. The aqueous layer was separated and extracted with 75 ml of ethyl acetate. The organic extract was washed with four 100 ml portions of water, then with saturated NaCl solution (100 ml), and quickly dried over Na 2 SO 4 . The solution was filtered and concentrated to an oily substance which was then triturated with 50 ml of hexanes. Upon removal of the solvent on a rotary evaporator, a solid separated, which was then triturated with cold hexanes, filtered and washed with hexanes. The crude, off-white phenolic product (13 g) was recrystallized from 5% MeOH in CH 3 CN to yield 10 g of colorless prismatic crystals (m.p. 131-133°C). I.R. (CH 2 Cl 2 ): 3580 cm -1 , 3320 cm -1 , 2910 cm -1 , 1590 cm -1 , 1580 cm -1 , 1440 cm-l. 1 H-NMR (400 MHz, CDCl 3 ): δ 1.70-1.91 (m, 12H); 2.58 (s, 1H); 3.18 (s, 1 H); 3.26 (s, 3H): 5.25 (s, 1H); 6.70-7.20 (m, 4H). These data confirm the following structure: EXAMPLE 4 Ammonium sodium 3-(adamantylidenemethoxymethyl) phenyl phosphate A quantity (1.1 g, 0.004 mole) of methoxy(3-hydroxyphenyl)methylene adamantane, obtained according to Example 3, was dissolved in 15 ml of molecular sieve-dried (3 Å) benzene under argon. Triethylamine (0.57 ml, 0.004 mole) was added via syringe. The stirred solution was cooled to 0°C in an ice bath for dropwise addition of 2-chloro-2-oxo-1,3,2-dioxaphospholane (0.37 ml, 0.004 mole). After 10 minutes in the cold bath, the viscous mixture was slowly warmed to room temperature and stirred for 3.5 hours. The benzene was removed in vacuo , and 60 ml of ether was added under argon. The suspension was filtered under an inert atmosphere, and the resulting solid-washed with three 20 ml portions of ether. The filtrate was removed in vacuo to yield 1.6 g of the phosphate triester as a colorless, viscous oily substance which was moisture sensitive. I.R. (CH 2 Cl 2 ): 2900 cm -1 , 1600 cm -1 , 1575 cm -1 , 1300 cm -1 (P=O). No phenolic OH stretching or C=O (1670 cm -1 ) absorption was present in the I.R. spectrum. TLC showed the absence of the starting material. These data are consistent with the following structure of 3-(adamantylidenemethoxymethyl) phenyl ethylene phosphate: The oily substance obtained above was dissolved in 7 ml of DMF, sodium cyanide (0.21 g, 0.004 mole) was added, and the mixture stirred for 24 hours at room temperature. The solvent of the resulting yellow solution was distilled off at 50°C in vacuo and further removed by chasing several times with 2 ml portions of xylene. The residue was triturated with ether to produce a gum, which was mixed with CH 2 Cl 2 , filtered and stripped in vacuo to yield 1.5 g of a light yellow, amorphous foam. I.R. (CH 2 Cl 2 ): 2240 cm -1 (weak, CN), 1595 cm -1 , 1570 cm -1 , 1475 cm -1 , 1275 cm -1 (P=O), 1235 cm -1 , 1100 cm -1 . These data are consistent with the following expected structure of sodium 3-(adamantylidenemethoxymethyl)phenyl-2'-cyanoethyl phosphate: This salt (1.5 g, 0.0035 mole) was dissolved in 5 ml of water. Concentrated ammonium hydroxide (5 ml) was then added dropwise. The solution was stirred overnight at room temperature. The resulting white slurry was cooled in an ice bath and treated with 30 ml of acetonitrile. Filtration and washing with two 15 ml portions of cold acetonitrile afforded 0.95 g of a hygroscopic, white solid (sintered at 115°C, melted at 130-133°C) after brief drying under vacuum. HPLC (reverse phase C18 - .1% ammonium acetate/CH 3 CN) gradient) showed one major peak. I.R. (Nujol): 1595 cm -1 , 1575 cm -1 , 1245 cm -1 , 1200 cm -1 , 1095 cm -1 , 1080 cm -1 , 890 cm -1 . U.V. (20% MeOH-dioxane) max 260/nm.; ε = 10,000. 1 H-NMR (400 MHz, D20): δ 1.60-1.80 (m, 12H); 2.44 (s, 1H); 2.97 (s, 1H); 3.22 (s, 1H); 4.65 (s, HOD); 6.88-7.20 (m, 4H). These data confirmed that the structure of the product was: EXAMPLE 5 3-(2'-spiroadamantane)-4-methoxy-4-(3''-phosphoryloxy)phenyl-1,2 dioxetane, sodium ammonium salt In a large culture tube, 0.065 g (0.00017 mole) of the enolether phosphate salt, obtained according to Example 4, was dissolved in 25 ml of CHCl 3 . A quantity (0.210 g) of methylene blue on silica gel (0.0026 g dye/g SiO 2 ) was added as a sensitizer. The tube was placed in a silvered Dewar flask containing a 250 watt, high-pressure sodium lamp inside a water-cooled immersion well. A piece of 5 mil Kapton® (Dupont) was placed inside the well as a U.V. filter. Ice water was pumped through the apparatus to maintain the sample temperature below 10°C. A continuous stream of dry oxygen was passed into the reaction vessel through a capillary tube. The gas flow was adjusted so as to just maintain a uniform suspension of the solid-phase sensitizer. After 25 minutes of irradiation time, the U.V. (260 nm) absorption of the starting material disappeared. The light yellow solution was filtered, evaporated, and reconstituted with 10 ml water. The aqueous sample was filtered through a 0.45 micron nylon filter and chromatographed on a reverse phase, C18 preparative HPLC column using a water/acetonitrile gradient. The fractions showing weak U.V. absorption at 277 nm were combined and lyophilized to provide the dioxetane as a white, cotton-like, hygroscopic solid. AMPPD Na + NH 4 + salt did not exhibit a melting point. Instead, subliming vaporization occurred between 145° - 150°C. A solid residue remained which partially decomposes but did not melt below 270°C. 1 H N.M.R. (D 2 O, ppm): .89-1.85 (m, 12H); 2.10 (s, 1H); 2.75 (s, 1H); 3.15 (s, 3H); 4.65 (s, HOD-NH 4 + ); 7.10-7.36 (m, 4H). I.R. (Nujol mull, cm -1 ): 3120, 1970-1790 (weak, broad-NH 4 + ), 1640 (broad), 1600 (weak), 1580, 1284, 1273, 1122, 980, 895. The structure of the product was thus confirmed as being: EXAMPLE 6 3-(2'-spiroadamantane)-4-methoxy-4-(3''-phosphoryloxy)pheny1-1,2-dioxetane, disodium salt Methoxy (3-phosphoryloxyphenyl)methylene adamantane sodium ammonium salt (3.3g) was dissolved in 15 ml of water containing a drop of pyridine. The solution was slowly run over a 3 cm x 25 cm column of Amberlite IR 120 (plus) ion exchange resin in the pyridinium salt form (Aldrich Chemical Co.). Upon elution with distilled water, the fractions showing absorbance at 260 nm. were combined and lyophilized. A portion of the resulting mono pyridinium salt (1 g, 2.3 mmol) was dissolved in 100 ml of CHCl 3 (dried over Al 2 O 3 ). The resulting solution was placed in a large cylindrical tube and treated with 5, 10, 15, 20-tetraphenyl-21H, 23H-porphine (2 mg. in 1 ml of CHCl 3 ). The homogeneous green solution was cooled to 0° and pre-saturated with oxygen gas via a sparger tube. The mixture was irradiated under constant O 2 flow in a silvered Dewar flask which also contained a cooled immersion well surrounding a 250 watt sodium vapor lamp which was filtered by a single sheet (5 mil) of DuPont Kapton® polyimide film. The temperature in the Dewar remained at 0° - 5°C during a 12 minute irradiation. The solvent was removed in vacuo followed by the addition of 100 ml of distilled water containing 500 mg of NaHCO 3 . The resulting light pink solution was cooled and filtered through a 0.45 micron Teflon® membrane. The resulting aqueous solution of dioxetane was subjected to a CH 3 CN-H 2 O gradient on a polystyrene chromatography column, followed by a second pass with a CH 3 CN - H 2 O gradient. The resulting solution, which was free of inorganic salts, was lyophilized to produce 800 mg of a granular, faintly yellow, white solid. This solid did not exhibit a melting point. Instead, decomposition to give adamantanone as a subliming vapor occurred between 145° - 150°C. A solid residue remained which partially decomposed, but did not melt below 270°C. 1 H N.M.R. (D 2 O): δ .85-δ 1.75 (m, 12H includes 2 doublets at 0.85, 1H and 1.13, 1H); δ 2.15 (s, 1H); δ 2.75 (s, 1H); δ 3.10 (s, 3H); δ 7.10-δ 7.35 (m; 4H). 31 P N.M.R. (D 2 O; p.p.m. vs H 3 PO 4 ) δ 1.53, singlet. 13 C-NMR (400 MHz, D 2 O, p.p.m.): 25.52, 25.68, 31.13, 31.55, 32.13, 32.61, 32.98, 34.20, 35.68, 50.31, 98.49 (dioxetane), 113.61 (dioxetane), 120.95 (broad, low intensity), 121.54, 122.10 (broad, low intensity), 129.37, 134.56, 154.29. When the experiment was repeated (300 MHz in D 2 O at 30°C), the broadened lines sharpened and became more intense relative to the line appearing between them. The sharpened resonances appeared at 120.65 and 121.99 p.p.m. This behavior is a clear indication of restricted rotation of the aromatic group. I.R. (Nujol mull): 1600 cm -1 (weak), 1580 cm -1 , 1285 cm -1 , 1275 cm -1, 1120 cm -1 (broad), 980 cm -1 , 895 cm -1 . EXAMPLE 7 6-Methoxynaphth-2-yl adamant-2'-yl ketone Magnesium turnings (1.1 g, 0.045 mol) were placed in a flame-dried flask under argon together with a crystal of iodine and 5 ml of dry THF. The suspension was heated to 45°C, while a solution of 2-bromo-6-methoxynapthalene (7.13 g, 0.03 mol) in 25 ml of dry THF was added dropwise. When the exothermic reaction began, the flask was submerged in a water bath at room temperature. After the addition was completed, the reaction mixture was refluxed for 30 minutes. A solution of 2-cyanoadamantane (4.85 g, 0.03 mol) was added dropwise over a period of 30 minutes. The resulting golden brown solution was refluxed overnight, cooled in an ice bath, and diluted with 30 ml of ether. Concentrated HCl (5 ml, 0.06 mol) was then added dropwise with stirring. The resulting precipitate was separated by filtration, washed with ether, dried, suspended in a mixture of 35 ml of methoxyethanol and 30 ml of concentrated hydrochloric acid, and refluxed for 5 hours. The solid was collected by filtration while the suspension was still warm, then washed with water. The crude ketone (7.3 g) was obtained as an off-white powder. TIC (R f 0.39; Whatman K5F, CH 2 Cl 2 : hexanes, 40:60) indicated one major product. Recrystallization from 150 ml of ethyl acetate yielded 5 g of buff-colored needles (m.p. 173-75°C). I.R. (CH 2 Cl 2 ): 2900 cm -1 , 1665 cm -1 (C=0), 1620 cm -1 , 1475 cm -1 , 1190 cm -1 , 1165 cm -1 . 1 H-NMR (400 MHz, CDCl 3 ) δ 1.56-2.11 (m, 12H); 2.36 (s, 2H); 3.58 (s, 1H); 3.94 (s, 3H); 7.10-8.26 (m, 6H). The structure of the product was confirmed as: EXAMPLE 8 Disodium methoxy[(7-phosphoryloxy-)naphth-2-yl]methylene adamantane Sodium hydride (50% in mineral oil, 240 mg, 6 mmol) was added under an argon atmosphere to methoxy[(7-hydroxy)naphth-2-yl]methylene adamantane (1.45 g, 4.5 mmol) dissolved in sieve-dried DMF (15 ml). The starting compound is from Edwards, et al., co pending and commonly-assigned U.S. Serial No. 213,672, filed June 30, 1988, Example I. The solution was stirred for 10 min. at room temperature to allow complete sodium naphthoxide formation and then cooled to 0°C, at which time 540 µl (5.87 mmol) of 2-chloro-2-oxo-1,3,2-dioxaphospholane (Fluka) were added dropwise to the suspension. The reaction mixture was slowly warmed to room temperature over 15 min. to ensure formation of methoxy[7-(2-oxo-1,3,2-dioxaphospholan-2-oxy)naphth-2-yl]methylene adamantane. Vacuum-dried sodium cyanide (648 mg, 13.2 mmol) was added as a powder, under argon, followed by stirring at room temperature for 1 hour to effect in situ ring opening of the cyclic phosphate ester. Upon completion of the reaction by TLC analysis (silica gel, 20% EtOAc/hexanes and 30% MeOH/EtOAc) of the reaction products, the solvent was stripped in vacuo while warming gently. The crude monosodium methoxy[(7-[2-cyanoethyl]phosphoryloxy)naphth-2-yl]methylene adamantane was dissolved in 7M NH 4 OH (10 ml) and stirred for 15 hours at 40°C. As the reaction proceeded, the product precipitated as a light yellow gum. The aqueous solution, still containing desired naphthyl phosphate, was drawn off and lyophilized to a brown powder after adding 564 mg (6.7 mmol) of NaHCO 3 . The freeze-dried powder and the gummy precipitate were dissolved together in minimal MeOH and then precipitated as flocculent, tan crystals upon addition of acetonitrile. The precipitate was collected in a Buchner funnel, washed with acetonitrile and dried. Evaporation of the filtrate to a small volume followed by addition of CH 3 CN precipitated more naphthyl phosphate, which was collected and washed as described above. This procedure was repeated two times to remove all of the crude phosphate from the filtrate. The dried crystal cake was purified by preparative HPLC, using an CH 3 CN/H 2 O gradient through a polystyrene column (PLRP-S, Polymer Laboratories). The product fractions were combined and lyophilized to yield 572 mg (28%) of disodium methoxy[(7-phosphoryloxy)naphth-2-yl]methylene adamantane as a white, fluffy powder. 1 H-NMR (D 2 O, p.p.m.): 1.60-1.83 (12H, m); 2.46 (1H, d, J = 0.97 Hz); 3.02 (1H, br. s); 3.22 (3H, s); 7.20 (1H, d, J = 8.43 Hz); 7.29 (1H, d, J = 9.28 Hz); 7.51 (1H, s); 7.65 (1H, s); 7.72 (2H, m). 31 P NMR (D 2 O, 85% H 3 PO 4 std., p.p.m.): 0.99 (1P). EXAMPLE 9 Disodium 3-(2'-spiroadamantane)-4-methoxy-4-(7''-phosphoryloxy)naphth-2''-yl-1,2-dioxetane. A solution of disodium methoxy[(7-phosphoryloxy)naphth-2-yl]methylene adamantane (18.8 mg, 0.042 mmol) and 5,10,15,20-tetraphenyl-21H,23H-porphine (TPP, 20 µl of a 2% solution in CHCl 3 by weight) in 2% MeOH/CHCl 3 (10 ml) was irradiated with a 250 W, high pressure sodium lamp at 10°C while passing a stream of oxygen through the solution. A 5 mil. thick piece of Kapton® polyimide film (DuPont) placed between the lamp and the reaction mixture filtered out unwanted UV radiation. Analytical HPLC (UV detector at 254 nm) showed complete dioxetane formation after irradiating 10.5 minutes. The reaction was also followed by UV spectroscopy with absorption at 255nm due to the conjugated vinyl group disappearing upon photoxygenation. The dioxetane showed one major UV absorption at 230nm. After evaporating the chloroform at 0°C, the residue was dissolved in ice water, passed through a 0.46 µ filter, and separated by preparative HPLC on a polystyrene column with an acetonitrile/water gradient. The fractions were frozen and lyophilized at 0°C, yielding 12.1 mg (60%) of the disodium phosphate dioxetane as a white, fluffy powder. 1 H-NMR (D 2 O, p.p.m.): 0.69 (1H, d); 0.98 (1H, d); 1.34 -1.80 (10H, m); 2.11 (1H, d, J = 1.35 Hz); 2.77 (1H, d, J = 1.96 Hz); 3.08 (3H, s); 7.31-7.98 (6H, m). These data confirm the structure to be as follows: 9.5 µl of a 0.4 mM solution of the above dioxetane in a pH 9 carbonate buffer (0.05M) was added to 490 µl of the same buffer in a glass tube. This solution was treated with 4 x 10 -14 moles of dialyzed alkaline phosphatase (Biozyme; ALPI-11G) in 5 µl of deionized water. The tube was placed in a luminometer (Turner 20E) at 29°C to reveal constant green light emission for over 60 minutes.

Claims: A process for the production of a compound of the formula wherein T is a polycycloalkylidene group; R is a C 1 -C 20 alkyl, aralkyl or cycloalkyl group; Y is a fluorescent chromophore and M + is an alkali metal, ammonium, or a C 1 -C 7 alkyl, aralkyl, or aromatic quaternary ammonium cation, (NR 4 ) + wherein R 4 can be any or all of an alkyl, aralkyl, or form part of a heterocyclic ring system comprising the following steps: (i) reacting a compound of the formula T-CN, wherein T is as defined above with a compound of the formula BrMg-Y-OR 1 wherein R 1 is a C 1 -C 20 alkyl, aralkyl or cycloalkyl group, followed by treatment with an acid HW to obtain a compound of the formula wherein T, Y and R 1 are as defined above and W - is a halide; (ii) subjecting the compound obtained in step (i) above to oxidation to obtain a compound of the formula wherein T, Y and R 1 are as defined above; (iii) reacting the compound obtained in step (ii) above with an R-ylating agent in a basic, polar, aprotic medium in the presence of alkali metal oxide to obtain a compound of the formula wherein T, R and R 1 are as defined above; (iv) subjecting the compound obtained in step (iii) above to a phenolic ether cleavage with sodium thioethoxide, lithium iodide, sodium cyanide or sodium sulfide to obtain a compound of the formula wherein T, Y and R are as defined above; (v) reacting the compound obtained in step (iv) above with wherein X is halogen, in the presence of a Lewis base in an aprotic organic solvent to obtain a compound having the formula wherein T, Y and R are as defined above; (vi) reacting the compound obtained in step (v) above with MCN wherein M is defined as above to obtain a compound having the formula (vii) reacting the compound obtained in step (vi) above with NH + / 4 OH - to obtain a compound having the formula wherein T, Y, R and N are as defined above and (viii) oxygenating the compound obtained in step (vii) above to obtain the compound having the formula A process for the production of a compound of the formula wherein T is a polycycloalkylidene group; R is a C 1 -C 20 alkyl, aralkyl or cycloalkyl group; Y is a fluorescent chromophore and M + is an alkali metal, ammonium, or a C 1 -C 7 alkyl, aralkyl, or aromatic quaternary ammonium cation, (NR 4 ) + wherein R 4 can be any or all of an alkyl, aralkyl, or form part of a heterocyclic ring system comprising the following steps: (i) reacting a compound of the formula T-CN, wherein T is as defined above with a compound of the formula BrMg-Y-OR 1 wherein R 1 is a C 1 -C 20 alkyl, aralkyl or cycloalkyl group, followed by treatment with an acid HW to obtain a compound of the formula wherein T, Y and R 1 are as defined above and W - is a halide; (ii) subjecting the compound obtained in step (i) above to oxidation to obtain a compound of the formula wherein T, Y and R 1 are as defined above; (iii) reacting the compound obtained in step (ii) above with an R-ylating agent in a basic, polar, aprotic medium in the presence of alkali metal oxide to obtain a compound of the formula wherein T, R and R 1 are as defined above; (iv) subjecting the compound obtained in step (iii) above to a phenolic ether cleavage with sodium thioethoxide, lithium iodide, sodium cyanide or sodium sulfide to obtain a compound of the formula wherein T, Y and R are as defined above; (v) reacting the compound obtained in (iv) above with a compound of the formula wherein X denotes a halogen atom to obtain a compound of the formula wherein T, Y and R are as defined above and subjecting said compound to oxidation to obtain a compound having the formula wherein T, Y and R are as defined above; (vi) reacting the compound obtained in step (v) above with MCN wherein M is as defined above to obtain a compound having the formula wherein T, Y, R and M + are as defined above; (vii) reacting the compound obtained in step (vi) above with NH + / 4 OH - to obtain a compound having the formula wherein T, Y, R and M + are as defined above and (viii) oxygenating the compound obtained in step (vii) above to obtain the compound having the formula A process for the production of a compound of the formula wherein T is a polycycloalkylidene group; R is a C 1 -C 20 alkyl, aralkyl or cycloalkyl group; Y is a flourescent chromophore and M + is an alkali metal, ammonium, or a C 1 -C 7 alkyl, aralkyl, or aromatic quaternary ammonium cation, (NR 4 ) + wherein R 4 can be any or all of an alkyl, aralkyl, or form part of a heterocyclic ring system which comprises steps (i) to (vii) as defined in claims 1 and 2 and additionally (viii) subjecting a compound of the formula wherein T, Y, R and M + are as defined above to oxygenation followed by an ion-exchange of NH + / 4 for M + ; or subjecting the compound of the formula to ion-exchange to replace NH + / 4 with M + to obtain a compound having the formula followed by oxygenation. A process for the production of a compound of the formula wherein T, Y, R and M + are as defined in claim 1 which comprises the following steps: (i) reacting a compound of the formula T-CN, wherein T is as defined above with a compound of the formula BrMg-Y-OR 1 wherein R 1 is a C 1 -C 20 alkyl, aralkyl or cycloalkyl group, followed by treatment with an acid HW to obtain a compound of the formula wherein T, Y and R 1 are as defined above and W - is a halide; (ii) subjecting the compound obtained in step (i) above to oxidation to obtain a compound of the formula wherein T, Y and R 1 are as defined above; (iii) reacting the compound obtained in step (ii) above with an R-ylating agent in a basic, polar, aprotic medium in the presence of alkali metal oxide to obtain a compound of the formula wherein T, R and R 1 are as defined above; (iv) subjecting the compound obtained in step (iii) above to a phenolic ether cleavage with sodium thioethoxide, lithium iodide, sodium cyanide or sodium sulfide to obtain a compound of the formula wherein T, Y and R are as defined above; (v) reacting the compound obtained in step (iv) above with a compound of the formula wherein X is a halogen atom and R 2 and R 3 are each independently cyano, nitrophenyl, dinitrophenyl, arylsulfonyl or alkyl sulfonyl to obtain a compound of the formula wherein T, Y, R, R 2 and R 3 are as defined above; (vi) treating the compound obtained in step (v) with M + OH - wherein M + is as defined above to obtain a compound of the formula wherein T, Y, R and M + are as defined above and (vii) oxygenating the compound obtained in step (vi) above to obtain the compound of the formula A process for the production of a compound of the formula wherein T, Y, R and M + are as defined in claim 1 which comprises the following steps: (i) reacting a compound of the formula T-CN, wherein T is as defined above with a compound of the formula BrMg-Y-OR 1 wherein R 1 is a C 1 -C 20 alkyl, aralkyl or cycloalkyl group, followed by treatment with an acid HW to obtain a compound of the formula wherein T, Y and R 1 are as defined above and W - is a halide; (ii) subjecting the compound obtained in step (i) above to oxidation to obtain a compound of the formula wherein T, Y and R 1 are as defined above; (iii) reacting the compound obtained in step (ii) above with an R-ylating agent in a basic, polar, aprotic medium in the presence of alkali metal oxide to obtain a compound of the formula wherein T, R and R 1 are as defined above; (iv) subjecting the compound obtained in step (iii) above to a phenolic ether cleavage with sodium thioethoxide, lithium iodide, sodium cyanide or sodium sulfide to obtain a compound of the formula wherein T, Y and R are as defined above; (v) reacting the compound obtained in step (iv) above with wherein X is a halogen atom and R 5 can be independently any of the substituents as defined above for R to obtain a compound of the formula wherein T, Y, R and R 5 are as defined above; (vi) oxygenating the compound obtained in step (v) above to obtain a compound of the formula wherein T, Y, R and R 5 are as defined above and (vii) hydrolysing the compound obtained in step (vi) above in the presence of M + OH - to obtain the compound of the formula A process according to claims 1 to 5 wherein T= is adamant-2-ylidene, T- is adamant-2-yl, R is methyl or ethyl, YOR 1 is the R-ylating agent is dimethyl or diethyl sulfate, the alkali metal alkoxide is potassium tert-butoxide, and the polar aprotic solvent is dimethyl sulfoxide. A process according to claims 1 to 6 wherein R 1 is methyl. A process according to claims 1 to 6 wherein T= is adamant-2-ylidene, R is methyl or ethyl, and YOH is the Lewis base is a tertiary amine, and the aprotic organic solvent is an aromatic liquid. A process according to claim 8 wherein the Lewis base is a trialkylamine and the aprotic organic solvent is benzene. A process according to claims 1 to 9 further comprising the use as the polycycloalkylidene compound a compound having the formula wherein M + is an alkali metal cation, in the absence of a Lewis base, in a polar aprotic solvent such as dimethylformamide or dimethylsulfoxide. A compound having the formula: wherein T- is a polycycloalkyl group; Y is a fluorescent chromophore; W - is a halide and R 1 is a C 1 - C 20 alkyl, aralkyl or cycloalkyl group. A compound according to claim 11 wherein T is adamant-2-yl, W - is a halogen anion, and YOR 1 is A compound according to claim 12 wherein W - is a chloride ion and R 1 is methyl. A compound having the formula: wherein T- is a polycycloalkyl group; Y is a fluorescent chromophore, and R 1 is a C 1 - C 20 alkyl, aralkyl or cycloalkyl group. A compound according to claim 14 wherein T is adamant-2-yl, and YOR 1 is A compound according to claim 15 wherein R 1 is methyl. A compound having the formula: wherein T= is a polycycloalkylidene group; Y is a fluorescent chromophore; and R is a C 1 - C 20 alkyl, aralkyl or cycloalkyl group. A compound according to claim 17 wherein T= is adamant-2-ylidene, R is methyl or ethyl, and Y is A compound having the formula: wherein T= is a polycycloalkylidene group; Y is a fluorescent chromophore; R is a C 1 - C 12 unbranched or branched, unsubstituted or substituted, alkyl, aralkyl or cycloalkyl group; R 4 is and M + is an alkali metal, ammonium or quaternary ammonium cation. A compound according to claim 17 wherein T= is adamant-2-ylidene, R is methyl or ethyl, M + is Na + , and Y is A compound having the formula: wherein T= is a polycycloalkylidene group, R is a C 1 - C 20 alkyl, aralkyl or cycloalkyl group, and Y is a fluorescent chromophore. A compound according to claim 21 wherein T= is adamant-2-ylidene, R is methyl or ethyl, and Y is A compound having the formula: wherein T= is a polycycloalkylidene group. R is a C 1 -C 20 alkyl, aralkyl, or cycloalkyl group; Y is a fluorescent chromophore; and R 2 and R 3 are each independently cyano, nitrophenyl, dinitrophenyl, alkylsulfonyl or arylsulfonyl. A compound according to claim 23 wherein T is adamant-2-ylidene, R is methyl and R 2 and R 3 are cyano.

Inventors: BRONSTEIN IRENA Y, EDWARDS BROOKS, BRONSTEIN, IRENA Y., EDWARDS, BROOKS
Applicants: TROPIX INC, TROPIX, INC.
Application year: 1989
Main classification: C07C41
IPC codes: C07C43, C07C41, C07F9, C07H15, C07D321, G01N33, G01N21, C12Q1, C07C69