CHEMILUMINESCENT 3-(SUBSTITUTED ADAMANT-2'-YLIDENE) 1,2-DIOXETANES

Abstract: Enzymatically cleavable chemiluminescent 1,2-dioxetane compounds capable of producing light energy when decomposed, substantially stable at room temperature before a bond by which an enzymatically cleavable labile substituent thereof is intentionally cleaved, are disclosed. These compounds can be represented by formula (I) wherein X and X<1> each represent, individually, hydrogen, a hydroxyl group, a halo substituent, an unsubstituted lower alkyl group, a hydroxy (lower) alkyl group, a halo (lower) alkyl group, a phenyl group, a halophenyl group, an alkoxyphenyl group, a hydroxyalkoxy group, a cyano group, a carboxyl or substituted carboxyl group or an amide group, with at least one of X and X<1> being other than hydrogen; and R1 and R2 individually or together, represent an organic substituent that does not interfere with the production of light when the dioxetane compound is enzymatically cleaved and that satisfies the valence of the dioxetane compound's 4-carbon atom, with the provisos that if R1 and R2 represent individual substituents the R2 substituent is aromatic, heteroaromatic, or an unsaturated substituent in conjugation with an aromatic ring, and that at least one of R1 and R2 is, or R1 and R2 taken together are, an enzymatically cleavable labile group-substituted fluorescent chromophore group that produces a luminescent substance when the enzymatically cleavable labile substituent thereof is removed by an enzyme. The corresponding dioxetanes which, instead of being substituted at the 5' or 7', or at the 5' and 7' positions, instead contain a 4' methylene group, are also disclosed, as are intermediates for all these 3-substituted adamant-2'-ylidenedioxetanes, and their use as reporter molecules in assays.

Description: I Description p h-amiiι_ττHτ.escent 3-(Substituted Adamant-2'-Ylidene) 1.2-Dioxetanes This application is a continuation-in-part of copending U.S. patent application Serial No. 559,152, filed July 24, 1990, which in turn is a division of copending U.S. patent application Serial No. 367,772, filed July 17, 1989 (based on PCT application PCT/0589/00016, filed January 3, 1989 in the U.S. Receiving Office based on Japanese patent application No. 185319/88, filed July 25, 1988, and now-abandoned U.S. patent application Serial No. 140,197, filed December 31, 1987) . Technical Field This invention relates to improved chemiluminescent 1,2-dioxetane compounds. More particularly, this invention relates to improved enzymatically cleavable chemiluminescent 1,2-dioxetane compounds that contain enzymatically removable labile groups. Such labile groups prevent the molecule from decomposing to produce light, i.e., visible light or light detectable by appropriate instrumentation, until an appropriate enzyme is added to remove the labile group. One enzyme molecule will effect the removal, through a catalytic cycle, of its complementary labile group from thousands of enzymatically cleavable chemiluminescent 1,2-dioxetane molecules. This is in marked contrast to the situation with chemically cleavable chemiluminescent 1,2-dioxetaneε, where one molecule of chemical cleaving agent is needed to remove the complementary labile group from each dioxetane molecule. For example, one mole of sodium hydroxide is needed to cleave one mole of hydrogen ions from the hydroxyl substituent on the phenyl group in 3-(2 '-spiro-ada antane) -4-methoxy-4-(3"-hydroxy)phenyl- 1, 2-dioxetane, while only a single mole of alkaline phosphatase ("AP") is needed to cleave the phosphoryloxy group in 1,000-5,000 moles of 3-(2 ■ -spiroadamantane) -4- methoxy-4-(3" phosphoryloxy)phenyl-1,2-dioxetane disodium salt per second; see Jablonski, "DNA Probes for Infectious Diseases" (Boca Raton, Fla.:CRC Press, 1989), p. 22. Enzymatically cleavable light-producing 1,2- dioxetane compounds will usually also contain stabilizing groups, such as an adamantylidene group spiro bonded to the dioxetane ring's 3-carbon atom, that will aid in preventing the dioxetane compound from undergoing substantial decomposition at room temperature (about 25°C) before the bond by which the enzymatically cleavable labile group is attached to the remainder of the molecule is intentionally cleaved. The concept of the use of spiroadamantyl group for stability was introduced to 1,2-dioxetane chemistry by ierynga, et al. , Tetrahedron Letters, 169 (1972) and McCapra, et al., J. Chem. Soc.. Chem. Comm. , 944 (1977) . These stabilizing groups thus permit such dioxetanes to be stored for acceptably long periods of time before use, e.g., for from about 12 months to as much as about 12 years at temperatures ranging from about 4 to about as much as 30°C, without undergoing substantial decomposition. This invention further relates to the incorporation of its dioxetane molecules in art-recognized immunoassays, chemical assays and nucleic acid probe assays, and to their use as direct chemical/physical probes for studying the molecular structures or microstructures of various macromolecules, synthetic polymers, proteins, nucleic acids, catalytic antibodies, and the like, to permit an analyte — the chemical or biological substance whose presence, amount or structure is being determined — to be identified or quantified. Background Art Chemiluminescent 1,2-dioxetanes have assumed increasing importance in recent years, particularly with the advent of the enzymatically cleavable chemiluminescent 1,2-dioxetanes disclosed in Bronstein U.S. patent application Serial No. 889,823, filed July 24, 1986 (the "'823 application"); Bronstein, et al. U.S. patent application Serial No. 140,035, filed December 31, 1987; Edwards U.S. patent application Serial No. 140,197, filed December 31, 1987 (the "'197 application") and Edwards, et al. U.S. patent application Serial No. 213,672 ("'672 application"), filed June 30, 1988. Again in marked contrast to enzymatically cleavable 1,2-dioxetanes, the various chemically cleavable chemiluminescent 1,2-dioxetanes known up to now have had little if any utility as reporter molecules in any type of analytical technique, and certainly not in bioassays. This is because the known chemically cleavable compounds are for the most part water insoluble — except for certain acetoxy-substituted 1,2-dioxetanes that are somewhat water-soluble as well as organic solvent-soluble — and thus may not be useful in biological assays unless they could somehow be modified by adding to them groups or substituents that allow conjugation to a biological species, e.g., an antibody, thus permitting such conjugated chemically cleavable 1,2-dicxetanes to be used - ά - as chemically activated che iluminogenic labels. The water solubility of typical enzymatically cleavable chemiluminescent 1,2-dioxetanes, on the other hand — e.g., adamantyl-appende enzymatically cleavable 1,2-dioxetanes that decompose in the presence of a suitable enzyme with light emission, such as 3-(4- methoxyspiro[1,2-dioxetane-3,2 '- tricyclo[3.3.1.l 3,7 ]decan]-4-yl)phenyl phosphate and its salts, e.g., the disodium salt, identified hereinbelow in shorthand fashion as adamantylidenemethoxyphenoxyphosphorylated dioxetane ("AMPPD") , and 3-(4-methoxyspiro[l,2-dioxetane-3,2 '- tricyclo[3.3.1.1 3 ' 7 ]decan]-4-yl)phenyloxy-3"-3-D- galactopyranoside and its salts ("AMPGD") — makes them eminently suitable for use as reporter molecules in many types of analytical techniques carried out in aqueous media, and especially in bioassays. It has been observed that AMPPD in aqueous solution, and also in the presence of chemiluminescence enhancers, e.g., a polymeric ammonium, phosphonium or sulfoniu salt such as poly[vinylbenzyl(benzyldimethylammonium chloride)] ("BDMQ") and other heteropolar polymers (see Voyta, et al. U.S. patent application serial No. 203,263, filed June 1, 1988) , exhibits longer than optimum periods of time to reach constant light emission characteristics ("tV, defined as the time necessary to attain one half of the maximum chemiluminescence intensity at constant, steady-state light emission levels; this emission half- life varies as a function of the stability of the dioxetane oxyanion in various environments) . Statistically, approximately seven t periods are required to reach steady-state light emission kinetics. The t for AMPPD at concentrations above 2 x 10~ 5 M in aqueous solution at pH 9.5 in the presence of BDMQ has been found to be 7.5 minutes. At 4 x 10 ~3 M in the absence of BDMQ the t\ has been found to be approximately 30-60 minutes, while at 2 x 10 "5 M in aqueous solution the t^ for AMPPD has been found to be 2.5 minutes. In rapid bioassays that employ enzymatically cleavable chemiluminescent 1,2-dioxetanes as reporter molecules it is desirable to reach steady-state light emission kinetics as quickly as possible so as to detect an "endpoint" in the assay. And while chemiluminescence intensity can be measured before achieving steady-state kinetics, sophisticated, thermally controlled luminometry instrumentation must be used if one wishes to acquire precise data prior to steady-state emission kinetics. Furthermore, AMPPD, in aqueous buffered solution both in the presence and the absence of chemiluminescence enhancers such as BDMQ, exhibits higher than desirable thermal and otherwise nonenzymatically activated light emission, or "noise". Such noise can be attributed to emissions from the excited states of ada antanone and of the methyl m-oxybenzoate anion derived from the aromatic portion of the AMPPD molecule. This noise can limit the levels of detection, and thus prevent the realization of ultimate sensitivity, as the measured noise level of AMPPD is approximately two orders of magnitude above the dark current in a standard luminometer. Enzymatic cleavage of AMPPD with alkaline phosphatase also generates anionic, dephosphorylated AMPPD — adamantylidenemethoxymethylpher.olate dioxetane, or "AMP ~ D". This phenolate anion can also be formed hydrolytically in small amounts, giving rise tc a background chemiluminescence signal which, in an organized molecular assembly, such as a micelle, liposome, lamellar phase, thin layer, iipid bilayer, liposome vesicle, reversed micelle, microemulsion, microgei, latex, membrane or polymer surface, and ir. a hydrophobic environment such as that produced by a chemiluminescence enhancer, e.g., BDMS, can generate strong, enhanced levels of light emission, thereby creating high background signals and substantially C lowering the dynamic range of the signal resulting from enzymatic hydrolysis of AMPPD. Consistent with the above-described observations, we have postulated the following mechanisms. In the presence of enhancing polymers such as BDMQ: 5 Enzymatic Pathway (AP present . : AP slow [AMPPD ] ■ > [AMPθD ■ > CL at 477nc (t ; = 7 . 5 min. ) Thermal Pathway 'no AP present. : at 477nn "CL" represents chemiluminescence. AMPPD in aqueous buffered solution contains a small amount c: dephosphorylated, hydroxylated 1,2-dioxetane ("AMPHB".. If t : solution pH is sufficiently high (above about 9.5., the dephosphorylated dioxetane may be present in tne anicr . i:: s t a; as AMP β D. "CL^" and "CL-" represent background chemiluminescence. "A*" represents adamantanone in tne excited enerεv s t a t e. SUBSTITUTESHEET Even in the absence of enhancing polymer, AMPPD can exist in aqueous solution as an aggregate: Enzymatic Pathway: AP ster " AMPPD], $> [AMPθ fa »D], -^ CL at 477nm (t = 2.5 min. ) Thermal and Hvdrolvtic Pathway; .AMPPD], AMP® D CL, at 477nm +AMPHD [A*)τn l- 2 at 15nm In the above mechanisms, n>>>m; n and m are a function of the presence cr absence of enhancing polymer, and AMPPD concentration. The excited state of the adamantanone singlet in aggregate form (n cr > 1) may exnibit hignεr yields of signal emission, here again particulariy if "stabilized" to emit more light, as by the presence cf a chemiluminescence enhancer such as BDMQ, than does the excited energy state cf unaggregatea adamantanone. This is perhaps due to the former's having lower singlet states, lower yields cf intersyεtem crossing or slower mtersystem crossing than tne latter, cr to other as yet unknown factors. Since luminometers, generally, are designed to detect ail photons emitted regardless cf their energy, or their wavelength, 415 nm and 477 n chemiluminescence are both detected as background noise emissions. Similarly, when photographic cr X-ray film is used to record chemiluminescence, no discrimination between the different wavelength emissions can easily be made, thus, sensitivity cf detection is limited by background noise. Finally, the observed aggregation of AMPPD under the conditions described above may result from the amphiphilic nature of AMPPD, or its phenolate anion, and like molecules: It is, therefore, an object of this invention to decrease the time necessary to conduct assays, and particularly bioassays, in which enzymatically cleavable chemiluminescent 1,2-dioxetanes are used as reporter molecules. It is also an object of this invention to provide new and improved enzymatically cleavable chemiluminescent 1,2-dioxetanes which, when used as reporter molecules in assays, and particularly bioassays, reduce the time required to complete the assay. A further object of this invention is to provide new and improved enzymatically cleavable chemiluminescent 1,2-dioxetanes for use as substrates for enzyme-based assays, and particularly bioassays, which provide improved signal to background behavior and thus pr vide improved detection levels. A still further object of this invention is to provide novel intermediates useful in synthesizing these improved enzymatically cleavable 1,2-dioxetanes. Another object of this invention is to provide methods of preparing these enzymatically cleavable chemiluminescent 1,2-dioxetanes and intermediates therefor. These and other objects, as well as the nature, scope and utilization of this invention, will become readily apparent to those skilled in the art from the following description and the appended claims. Disclosure of the Invention This invention provides a new class of stable, enzymatically cleavable chemiluminescent 3-(substituted adamant-2'-ylidene)-1,2-dioxetane compounds capable of reacting in aqueous media, e.g. , in a sample of biological fluid in solution or on a solid surface, e.g., a membrane surface such as a nylon membrane, with an enzyme or enzyme modified specific binding pair to release optically detectable energy. In aqueous media these modified adamantylidene dioxetanes enable assays in which they are used as reporter molecules to be conducted faster and with greater sensitivity than hitherto possible using AMPPD. While we do not wish to be bound by any mechanism or theory advanced to explain this unexpectedly superior behavior, it may be that the presence of substituents of the type disclosed herein on or in the adamantylidene moiety prevents the dioxetane molecules from packing efficiently, and thus prevents them from forming "stabilized" organized assemblies, whether in the form of micelles or some other aggregated state. Certain of these substituents may also hydrogen bond to other substances in their aqueous environment, including water itself, thereby further preventing aggregate formation. And, it is also possible that electronic and dipole effects may contribute to this phenomenon, as evidenced by shorter t-^'s and lower background noise. Brief Description of the Drawings FIGS. 1 - 5 show TSH, RLV v. TSH for each of AXPPD and its bromo-, B-hydroxy-, A-hydroxy- and chloroadamant-2'-ylidene analogs, respectively, obtained as described in Example XII below. FIGS. 6 - 10 compare the total luminescence emissions obtained from AMPPD and its A-hydroxy-, B- hydroxy, chloro- and bromoadamant-2 '-ylidene analogs, respectively, obtained as described in Example XIII below. FIG. 11 shows the improved chemiluminescence intensity obtained using the chloroadamant-2 '-ylidene analog of AMPPD as compared to AMPPD itself as the reporter molecule in a nucleic acid assay; see Example XIV below. FIG. 12 shows the kinetics of the light emissions obtained using the chloroadamant-2 '-ylidene analog of AMPPD and AMPPD itself as the reporter molecules in a nucleic acid assay; see Example XVI below. FIG. 13 is the dose response curve at five minutes for alkaline phosphatase dilution with the chloroadamant- 2 *-ylidene analog of AMPPD plus poly[vinyl(benzyldimethylammonium chloride)) ("BDMQ"), compared to the dose response curve at five minutes for AMPPD itself plus BDMQ; see Example XIX below. FIG. 14 is the dose response curves at 20 minutes for the same substances whose dose response curves are shown in FIG. 13. FIG. 15 is the dose response curve at five minutes for alkaline phosphatase dilution with the chloroada ant- 2 '-ylidene analog of AMPPD plus BDMQ-fluorescein ("emerald") , compared to the dose response curve at five minutes for AMPPD itself plus emerald; again see Example XIX below. FIG. 16 is the dose response curves at 20 minutes for the same substances whose dose_response curves are shown in FIG. 15. FIG. 17 shows a TPA sequence's images, obtained as described in Example XX below using (1) AMPPD and. (2) AMPPD's chloroadamant-2 '-ylidene analog. FIG. 18 shows the signals obtained as described in Example XXII using (1) AMPPD and (2) substituted analogues of the invention. FIG. 19 plots the chemiluminescent signal obtained according to Example XXVII obtained with (1) AMPPD and (2) Cl-AMPPD. FIG. 20 reflects the exposure obtained pursuant to Example XXVIX for the detection of pBR322 plasmid DNA, using both (1) AMPPD and (2) Cl-AMPPD. FIG. 21 gives exposures for detection of pBR322 pursuant to Example XXX using (1) AMPPD, (2) Cl-AMPPD and (3) Br-AMPPD. FIG. 22 is a graphic illustration of chemiluminescent signal obtained pursuant to Example XXXI plotted as a function of time for both (1) AMPPD and (2) Cl-AMPPD. FIGS. 23 and 30 reflect the exposures obtained pursuant to Examples XXXII and XXXIV in detection of human transferrin for AMPPD, Cl-AMPPD and Br-AMPPD. FIG. 24 reflects a comparison in signals obtained pursuant to Example XXXV comparing AMPPD, Cl-AMPPD, Br- AMPPD and LumiPhos 530. * - FIGS. 25 and 26 compare images obtained pursuant to Example XXXVI using AMPPD and Cl-AMPPD. FIGS. 27 and 28 reflect exposures obtained pursuant to Examples XXXVIII and XXXIX comparing exposures obtained using AMPPD, Cl-AMPPD and Br-AMPPD. FIG. 29 reflects chemiluminescent detection of human transferrin pursuant to Exmple 40, comparing AMPPD, Cl- AMPPD and Br-AMPPD. Best Mode for carrying out the Invention The novel chemiluminescent 3-(substituted adamant- 2 '-ylidene) 1,2-dioxetanes of this invention can be represented by the general formula: In Formula I, X and X 1 each represent, individually, a substituent at the 5' and 7' positions on the ada ant- 2'-ylidene substituent which can be hydrogen, a hydroxyl group (a εligntly electron withdrawing group when hydrogen-bonded to water), a halo substituent, i.e., fluoro or chloro (electron withdrawing groups) or bromo or iodo (polarizable, mesomeric groups) , an unsubstituted straight or branched chain lower alkyl group, preferably methyl; a substituted straight or branched chain lower alkyl group monosubstituted or having two or more substituents which can be the same or different, e.g., a hydroxyalkyl group such as a hydroxymethyl group, a haloalkyl group such as trifluoromethy1, and the like; an alkoxy group, particularly C x _-. alkoxy (methoxy, ethoxy, propoxy, ...) ; an unsubstituted aryl group, preferably a phenyl group; a substituted aryl group, preferably one whose aryl ring contains six carbon atoms monosubstituted or having two or more substituents which can be the same or different, e.g., a halo substituent, as in p- bromophenyl or p-chlorophenyl, an alkoxy substituent, e.g., p-methoxyphenyl (an electron donating group), a hydroxyalkoxy substituent, e.g., hydroxyethoxy or hydroxypropoxy, a cyano group, or an amide group, e.g., a formamido or acetamido group, a carboxyl or substituted carboxyl group and the like, with the proviso that at least one of X and X 1 is other than hydrogen. When the adamantylidene group is monosubstituted with one of the foregoing substituents other than hydrogen, a mixture of the syn- and anti-isomerε will be obtained when such compounds are synthesized. In certain cases, e.g., for monoiodo-substituted adamantylidene dioxetanes, one isomer will exhibit greater chemiluminescence intensity on decomposition than the other. In other cases, e.g., for monohydroxy-substituted adamantylidene dioxetanes, the two isomers will be equivalent or nearly so in chemiluminescence properties, such as intensity. In either case, the isomers may be separated before being used, e.g., as reporter molecules in bioassays, by techniques such as those disclosed in Edwards, et al. U.S. patent application Serial No. 244,006, filed September 14, 1988, or they may be used as chromatographed, isomeric mixtures without separation. Dioxetanes whose adamantylidene substituents are further substituted with two of the foregoing substituents other than hydrogen (X and X 1 ψ hydrogen) do not exhibit syn/anti isomerism, although, of course, position isomers are possible when two different substituents are present, e.g., 5'-hydroxy-7'-chloro- and 5'-chloro-7'-hydroxy-substituted adamantylidene dioxetaneε. The symbols R λ and R 2 can represent any of the substituents on the 4-carbon atom of the dioxetane ring disclosed in the aforementioned Bronstein; Bronstein, et al.; Edwards; Edwards, et al. and Voyta, et al. applications, so long as when ' R 1 and R 2 represent individual substituents the R 2 substituent is aromatic, heteroaromatic, or an unsaturated substituent in conjugation with an aromatic ring, and at least one of R 2 and R 2 is, or R 2 and R 2 taken together are, an enzymatically cleavable labile group-substituted fluorescent chromophore group that produces a luminescent substance when the enzymatically removable labile substituent thereof is removed by an enzyme. Thus, for example, the symbol 2 can represent hydrogen, or a bond when R 2 represents a substituent bound to the dioxetane ring through a spiro linkage, or an organic substituent that does not interfere with the production of light and that satisfies the valence of the dioxetane ring carbon atom to which it is attached to result in a tetravalent dioxetane ring carbon atom, such as an alkyl, aryl, aralkyl, alkaryl, heteroalkyl, heteroaryl, cycloalkyl or cycloheteroalkyl group, e.g., a straight or branched chain alkyl group having from 1 to 7 carbon atoms, inclusive; a straight pr branched chain hydroxyalkyl group having from 1 to 7 carbon atoms, inclusive, an -OR group in which R is a C 1 -C 20 unbranched or branched, unsubstituted or substituted, saturated or unsaturated alkyl, cycloalkyl, cycloalkenyl, aryl, aralkyl or aralkenyl group, any of which may additionally be fused to R 2 such that the emitting fragment contains a lactone ring or an N, 0 or S heteroato -containing group, or an enzyme cleavable group bonded directly to the 4- carbon atom of the dioxetane ring, or to one of the other aforementioned R 2 groups, that contains a bond cleavable by an enzyme to yield either directly or by subsequent adjustment of pH an electron-rich moiety, e.g., an oxygen anion, a sulfur anion or a nitrogen anion (the latter being, for example, an oxime or an amido anion such as a sulfonamido anion) bonded to the dioxetane ring. Preferably, R χ is an alkoxy group, and especially a methoxy group, when R 2 is singly bonded to the dioxetane's 4-carbon atom. The symbol R 2 can also represent any organic substituent that does not interfere with the production of light and that satisfies the valence of the 4-carbon atom of the dioxetane ring to which it is attached. Preferably, R 2 will represent any of a number of light- emitting fluorophore-forming fluorescent chromophore groups that permit the corresponding dioxetane decomposition fragments to absorb energy and form an excited state from which they emit optically detectable energy to return to their ground state, substituted with an enzyme cleavable group that contains a bond cleavable by an enzyme to yield either directly or by subsequent adjustment of pH an electron-rich moiety, again, for example, an oxygen anion, a sulfur anion or a nitrogen anion, bonded to the dioxetane ring. Thus, for example, the symbol R 2 can represent, alone (or together with the symbol R 2 to give a substituent spiro bonded to the 4-carbon atom of the dioxetane ring) fluorescent chromophore groups such as: -phenyl and phenyl derivatives; -naphthalene and naphthalene derivatives e.g., 5- dimethylaminonaphthalene-1-sulfonic acid and hydroxy naphthalenes; -anthracene and anthracene derivatives, e.g., 9,10- diphenylanthracene, 9-methylanthracene, 9-anthracene carboxaldehyde, anthryl alcohols and 9-phenylanthracene; -rhodamine and rhodamine derivatives, e.g., rhodols, tetramethyl rhodamine, tetraethyl rhodamine, diphenyldimethyl rhodamine, diphenyldiethyl rhodamine and dinaphthyl rhodamine; -fluorescein and fluorescein derivatives, e.g., 5- iodoacetamido fluorescein, 6-iodoacetamido fluorescein and fluorescein-5-maleimide; -cou arin and coumarin derivatives, e.g., 7- dialkylamino-4-methylcoumarin, 4-bromomethyl-7- methoxycoumarin and 4-bromomethy1-7-hydroxy coumarin; -erythrosin and erythrosin derivatives, e.g., hydroxy erythrosins, erythrosin-5-iodoacetamide and erythrosin-5-maleimide; -acridine and acridine derivatives, e.g., hydroxy acridines and 9-methyl acridine; -pyrene and pyrene derivatives., e.g., N-(l-pyrene) iodoaceta ide, hydroxy pyrenes and 1-pyrenemethyl iodoacetate; -stilbene and stilbene derivatives, e.g., 6,6'-dibro ostilbene and hydroxy stilbenes; -nitrobenzoxadiazoles and nitrobenzoxadiazole derivatives, e.g., hydroxy nitrobenzoxadiazoles, 4- chloro-7-nitrobenz-2-oxa-l,3-diazole, 2-(7-nitrobenz-2- oxa-1,3-diazole-4-yl)methylaminoacetaldehyde and 6-(7'- nitrobenz-2-oxa-l,3-diazole-4-yl) minohexanoic acid; -quinoline and quinoline derivatives, e.g., 6- hydroxyquinoline and 6-aminoquinoline; -acridine and acridine derivatives, e.g., N- methylacridine and N-phenylacridine; -acidoacridine and acidoacridine derivatives, e.g., 9-methylacidoacridine and hydroxy-9-methylacidoacridine; -carbazole and carbazole derivatives, e.g., N- methylcarbazole and hydroxy-N-methylcarbazole; -fluorescent cyanines, e.g., DCM (a laser dye), hydroxy cyanines, 1,6-diphenyl-l,3,5-hexatriene, l-(4- dimethyl aminophenyl)-6-phenylhexatriene and the corresponding 1,3-butadienes; -carbocyanines and carbocyanine derivatives, e.g., phenylcarbocyanine and hydrox_. carbocyanines; -pyridiniu salts, e.g., 4 (4-dialkyldiaminostyryl)- N-methyl pyridinium iodate and hydroxy-substituted pyridinium salts; -oxonols; and -resorofins and hydroxy resorofins, The symbol R 2 , together with the symbol R 1 , can also represent a fused fluorescent chromophore group, bonded to the 4-carbon atom of the dioxetane ring through a εpiro linkage, having the general formula: -o-, -S-, cr -NR 6 , where each of R 4 , R 5 and R 6 , independently, is hydrogen, a branched or straight chain alkyl group having 1 to 20 carbon atoms, inclusive, e.g., methyl, n- butyl or decyl, a branched or straight chain heteroalkyl group having 1 to 7 carbon atoms, inclusive, e.g., methoxy, hydroxyethyl or hydroxypropyl; an aryl group having 1 or 2 rings, e.g, phenyl, naphthyl or anthryl; a heteroaryl group having 1 or 2 rings, e.g., pyrrolyl or pyrazolyl; a cycloalkyl group having 3 to 7 carbon atoms, inclusive, in the ring, e.g., cyclohexyl; a heterocycloalkyl group having 3 to 6 carbon atoms, inclusive, in the ring, e.g., dioxane; an aralkyl group having 1 or 2 rings, e.g., benzyl; or an alkaryl group having 1 or 2 rings, e.g., tolyl; and each R,, independently, can be hydrogen; an electron-withdrawing group, such as a perfluoroalkyl group having between 1 and 7 carbon atoms, inclusive, e.g., trifluoromethy1; a halogen; C0 2 H, -ZC0 2 H, -S0 3 H, ZS0 3 H, -N0 2 , ZN0 2 , -C≡N, or -Z- j^ C≡N, where Z is a branched or straight chain alkyl group having 1 to 7 carbon atoms, inclusive, e.g., methyl; an aryl group having 1 or 2 rings, e.g., phenyl; an electron-donating group, e.g., a branched or straight chain C 1 -C 7 alkoxy group, e.g., methoxy or ethoxy; an aralkoxy group having 1 or 2 rings, e.g., phenoxy; a branched or straight chain C 1 -C 7 hydroxyalkyl group, e.g., hydroxymethyl or hydroxyethyl; a hydroxyaryl group having 1 or 2 rings, e.g. , hydroxyphenyl; a branched or straight chain C-^- - j alkyl ester group, e.g., acetate; an aryl ester group having 1 or 2 rings, e.g., benzoate; or a heteroaryl group having 1 or 2 rings, e.g., benzoxazole, benzthiazole, benzimidazole or benztriazole. Furthermore, two or more of the R 3 groups can form a fused ring or rings which can themselveε be unsubstituted or substituted. The symbol R 2 , alone or together with the symbol R^ can likewise represent a particular clasε of fused polycyclic ring-containing fluorophore moieties having a labile ring substituent containing a bond which, when cleaved, renders the fused polycyclic moiety electron- rich to in turn render the dioxetane compound decomposable to emit light. The members of this class are those in which the labile ring substituent's point of attachment to the fused polycyclic ring, in relation to this ring's point(s) of attachment to the dioxetane ring (single bond attachment or, when R l r represents a bond, a spiro linkage) , is such that the total number of ring sp 2 atoms separating these points of attachment, including the sp" ring carbon atoms at the points of attachment, is an odd whole number; see the Edwards, et al. '672 application. Included among the fused polycyclic ring compounds whose residues can be used to form this fluorophore moiety are the fused polycyclic aromatic hydrocarbon ring fluorophoric compounds mentioned above, and particularly ones containing from 9 to about 30 ring carbon atoms, inclusive, such as naphthalene: the substituent bonds indicating a l,6-substitution pattern as in disodium 6-(4-methoxyspiro[1,2-dioxetane- 3,2'-(5'-hydroxy)tricyclo[3.3.1.1 3 ' 7 ]decan]-4-yl)-1- naphthalenyl phosphate, pentalene, azulene, heptalene, as-indacene, s-indacene, biphenylene, perylene, acenaphthylene, phenanthrene, anthracene, acephenanthrylene, aceanthrylene, triphenylene, pyrene, chrysene, naphthacene, and the like, as well as derivatives thereof substituted with one or more non- labile substituents such as those mentioned above as being represented by the symbols R 3 , R 4 and R 5 . The fused polycyclic ring portion of such "odd pattern substituted" fluorophore moieties represented by R 2 alone or together with R-. can also be tne residues of nonaro atic, i.e., less than fully aromatic, fused polycyclic hydrocarbon ring fluorophoric compounds, having a labile ring substituent containing a bond which, when cleaved, renders the fused, less than fully aromatic polycyclic moiety electron-rich to in turn render the dioxetane compound decomposable to emit light, unsubstituted or substituted with one or more cf the aforementioned non-labile subst.ituents, and containing from 10 to about 30 ring carbon atoms, inclusive, such as fluorene, 3,4-dihydro-3,3-dimethylnaphthalene, dibenzosuberene, 9,10-dihydrophenanthrene, indene, indeno [1,2-a] indene, phenalene, fluoroanthrene, and the like. Further, the fused polycyclic ring portion of fluorophore moieties represented by R 2 alone or together with R λ can also be the residue of a fused polycyclic heteroaromatic or less than fully aromatic fused ring heterocyclic fluorophoreforming group, e.g., dibenzothiophene, dibenzofuran, 2,2-dimethyl-2H~chromene, xanthene, piperidine, quinoline, isoquinoline, phenanthridine, carbostyryl, phenoxazine, phenothiazine, phenanthroline, purine, phthalazine, naphthyridine, N- acylindole, chroman, isochroman, N-acylindoline, isoindoline, and the like, unsubstituted or substituted with one or more of the aforementioned non-labile substituents, and containing from 9 to about 30 ring atoms, inclusive, the bulk of which are carbon atoms. A preferred enzymatically removable group with which at least one of R 2 and R 2 is substituted is a phosphate group, particularly a phosphate ester group represented by the general formula: wherein M+ represents a cation such as alkali metal, e.g., sodium or potassium, ammonium, or a C--C-, alkyl, aralkyl cr aromatic quaternary ammonium cation, N(R-,)+ 4 , n which each R can be alkyl, e.g. , methyl or ethyl, aralkyl, e.g., benzyl, or form part of a heterocyclic ring system, e.g. , pyridinium. The disodium salt is particularly preferred. Such phosphate ester groups can be cleaved using an enzyme such as alkaline phosphatase to produce oxygen anion-substituted groups that will, in turn, destabilize the dioxetane with rupture of its oxygen-oxygen bond to produce light. The quaternary ammonium cations in such phosphate ester groups can also be connected through one of their quaternary groups to a tolvmeric backbone, viz. where n is greater than 1, or can be part of a polyquaternary ammonium salt, i.e., an ionene polymer. Another preferred enzymatically removable group is the /3-D-galactoside group, which can be cleaved with the enzyme 0-D-galactosidase to yield the conjugate acid of the dioxetane phenolate, which upon chemilumineεces under pressure. Enzymatically cleavable substituents that can be used also include enzyme-cleavable alkanoyloxy groups, e.g., an acetate ester group, or an enzyme-cleavable oxacarboxylate group, l-phospho-2,3-diacylglyceride group, l-thio-D-glucoside group, adenosine triphosphate analog group, adenosine diphoεphate analog group, adenosine monophosphate analog group, -adenosine analog group, α-D-galactoside group, 3-D-galactoside group, α-D- glucoside group, 3-D-glucoside group, α-D-mannoside group, 3-D-mannoside group, /3-D-fructofuranoside group, 3-D-glucosiduronate group, p-toluenesulfonyl-L-arginine ester group or p-toluenesulfonyl-L-arginine amide group. When R 2 is phenyl, as noted, R 1 may be -0(CH 2 ) n CH 3 , when N = 0-19, preferably n = 0-5. In such compounds, important properties may be further conferred on the molecule, or controlled, by additional substitution on the phenyl ring. Stability, solubility, aggregation, binding ability and decomposition kinetics may be further controlled in compounds of the following structure wherein Z is the enzyme-cleavable group discussed above, the oxygen is ortho, meta or para, and Q is hydrogen, aryl, substituted aryl, aralkyl, heteroary, heteroalkyl of up to 20 carbon atoms, an allyl group, a hydroxy (lower) alkyl, a lower alkyl OSiR 3 (where R 3 is lower alkyl, aryl, alkoxy C^ g , alkoxyalkyl of 12 or fewer carbon atoms, hydroxy (lower) alkyl, amino (lower) alkyl, -OR 4 or -SR 4 (wherein R 4 is alkenyl, substituted (lower) alkenyl, (lower) alkyl or aralkyl of up to 20 carbon atoms) , -S0 R (wherein R 5 is methyl, phenyl or NHC 6 H 5 ) , substituted or unsubstituted (lower alkyl, nitro, cyano, a halogen, hydroxy, carboxyl, trimethylsilyl or phosphoryloxy group. The substituted adamant-2-ylidene moiety spiro bonded to the 3-carbon atom of the dioxetane ring, illustrated in formula I above, can be replaced by other similarly substituted fused polycycloalkylidene groups having two or more fused rings, each ring having from 3 to 12 carbon atoms, inclusive, such as bicyclo[3.3.1.]nonan-9-ylidene, hexacyclo-[5.5. 1.0. 2 ' 6 0. 3 ' 10 0. 4 ' 8 0 9 ' 13 ]tridecan-5-ylidene, pentacyclo[5.4.0.0. 2 ' 6 0. 3,10 0 5 ' 9 ]undecan-4-ylidene, and the like. PCT Application No. W088/00695, published January 28, 1988 based on the Bronstein '823 application, discloses enzymatically cleavable 1,2-dioxetanes which are substituted at the 3-carbon atom with a defined substituent "T-V" and at the 4-carbon atom with defined substituents "X" and "Y-Z". This published application also discloses, at p.3 ,1s.6-12, that: 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 possesses phosphatase activity, at p.22, 1.33-p.23, 1.6 that: 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, and at p.23,Is.11-21 that: 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. The problem solved by the 3-(substituted adamant-2'- ylidene) 1,2-dioxetane compounds disclosed and claimed herein is not addressed in this published PCT application, nor are these compounds themselveε disclosed in this or any other reference of which the inventors are aware. The overall εyntheεiε of these 3-(substituted adamant-2 '-ylidene)-1,2-dioxetanes can be accomplished using methods such as those disclosed in the aforementioned Bronstein and Edwards applications and in Edwards, et al. U.S. patent application Serial No. 279,176 ("'176 application"), filed September 6, 1989. Thus, for example, 1,2-dioxetanes coming within the scope of formula I above in which X represents a hydroxyl group, X 1 represents hydrogen, R χ represents a methoxy group and R 2 represents a phosphoryloxy salt-substituted phenyl group, preferably a meta-phosphoryloxy salt- substituted phenyl group, can be synthesized in accordance with the methods disclosed in the '176 application by a reaction sequence that can be illustrated schematically as follows: H " 7Q, 3R j H 0 = c - jr - OI 1L*5 ++ cC,i^OH 2. Biase Water scavenger * Acid Catalyst 2 CN SUBSTITUTE SHEET H As specifically exemplified below, the O = c - OR c starting material can, if desired, be reacted with an orthoformate such as trimethylorthofcrmate, methanol, and p-toluenesulfonic acid, to give the intermediate: P - ° 9 which, when then reacted with (R s O) 3 P and Lewis acid, gives the phosphonate ester intermediate: In the foregoing reaction sequence R 8 represents a lower alkyl group, e.g., methyl, ethyl or butyl. R g represents an acyl group containing from 2 to about 14 carbon atoms, inclusive, such as acetyl, propionyl, mesitoyl or pivaloyl, Q repreεents a halogen, e.g., chloro or bromo, or OR 8 , and M represents, independently, a proton, a metal cation, e.g., Na + , or K + , or an ammonium, substituted ammonium, quaternary ammonium or (H + ) pyridinium cation. Thiolate cleavage as deεcribed in the Edwardε *197 application can be used in place of base cleavage of the OR 9 group in step 5 of the reaction sequence illustrated above, in which case R 9 can be a lower alkyl, lower alkenyl or aralkyl group, e.g., methyl, allyl or benzyl. The product of base or thiolate cleavage can have, in place of R 9 , hydrogen or an alkali metal cation, e.g., lithium, sodium or potassium. The intermediates represented above by the formula: where only one of X and X 1 is other than hydrogen are known compounds, or are readily synthesizable from known starting materials using art-recognized methods. For example, in the case of the monosubstituted adamantan-2- ones (one of X and X 1 is hydrogen) : -5-hydroxyadamantan-2-one is prepared as described in Geluk, Synthesis. 374 (1972); -5-bromoadamantan-2-one and 5-chloroadamantan-2-one are prepared as described in Geluk, et al., Tetrahedron. Where X or X 1 is fluoro, unsubstituted (lower) alkyl, e.g., t-butyl, substituted (lower) alkyl, e.g., trifluoromethyl, unsubstituted aryl, e.g. , phenyl, or substituted aryl, e.g., p-chlorophenyl, p-methoxyphenyl or p-nitrophenyl, see le Noble, et al. , J. Am. Che . Soc.. 108 1598 (1986) and Walborsky, et al., J. Am. Chem. Soc.. 109. 6719 (1987) (X or X 1 = hydroxymethyl). Simple unit processes allow the conversion of several of the abovementioned X or X 1 substituents, or others known in the art, to 5-X- or 5-X x -adamantane-2- oneε where X or X may be trialkylεilyloxy, iodo or cyano groups. These moieties are stable under the mild conditions used in step 4 of the foregoing reaction sequence. For example, when 5-hydroxyadamantan-2-one is refluxed for 7 hours with 57% hydriodic acid, 5- iodoadamantan-2-one (m.p. 73-76°C) is obtained. 5- Carboxyadamantan-2-one, prepared as described in Lantvoev, J. Obshch. Khim. , 12. 2361 (1976) or Le Noble, et al., J. Orσ. Chem.. 48, 1101 (1983), after saponification of the methyl ester, can be converted to 5-cyanoadamantan-2-one by the three step procedure of Tabushi, et al., J. Orσ. Chem. , 38, 3447 (1973), used for access to the isomeric l-cyanoadamantan-2-one through the intermediacy of a keto amide. 5- Trimethylsilyloxyadamantan-2-one ( " m.p. 34-38°C) , useful as a protected version of 5-hydroxyadamantan-2-one, allows the use of only one equivalent of base in step 4 of the foregoing reaction sequence to prepare the corresponding enol ether, which can then be desilylated using standard techniques. As will be appreciated by one skilled in the art, other X and X 1 groups need not be static during the entire reaction sequence, but may be transformed by reactions which are compatible with other structural considerations at any stage. For example, it has been discovered that when X or X 1 is a chlorine or a bromine atom, enol ether intermediates produced in steps 4 and 5 of the foregoing reaction sequence are subject to facile solvolysis in the presence of molar excesses of diols or liquid ammonia in a bomb at high temperature. The reaction rate with diols such as ethylene glycol or propylene glycol becomes appreciable only at elevated temperatures (105-120°C) in the presence of a proton acceptor such aε potaεεiu carbonate. In general, this reaction is slow, but clean, and avoids the use of silver or heavy metal salts often used to stimulate hydroxyalkyl ether formation. 3-(Methoxy-5-(2-hydroxyethoxy) ricyclo- [3.3.1.l 3 ' 7 ]dec-2-ylidenemethyl)phenol can be esterified with trimethylacetyl chloride and triethylamine to give the corresponding diester, which can then be selectively cleaved, using potassium carbonate in methanol, to give the phenolic monoester. The ensuing phosphorylation step, incorporating simultaneous ^-elimination and saponification of the hindered ester with sodium methoxide in methanol, will furnish the hydroxyethoxy enol ether phosphate. Reaction with liquid ammonia in dioxane, under pressure, to give 3-(methoxy-5- aminotricyclo[3.3.l.i 3 ' 7 ]dec-2-ylidenemethyl)phenol from the corresponding 5-bromo compound is carried out using the procedure deεcribed by Hummelen, Diεsertation, University of Groningen, The Netherlands, p. 60 (1985). Immediate acylation of the thus-obtained amino enol ether phenol, using two equivalents of acetyl chloride or acetic formic anhydride and 4-dimethylaminopyridine as the base, following the procedure of Gawronski, et al., J. Am. Chem. Soc. , 109. 6726 (1987) used for the esterification of (5-hydroxyadamantylidene)ethanol, will give the formamido or acetamido phenolic esters, which can be selectively saponified as described supra and then phosphorylated and photooxygenated as described infra. Meijer, Dissertation. University of Groningen, The Netherlands (1982); Numan, et al., J. Orσ. Chem.. 4J3, 2232 (1978); and Faulkner, et al., J. Chem. Soc.. Chem. Comm. , 3906 (1971) , provide accesε to 4- methyleneadamantan-2-one (X and X 1 hydrogen, methylene at the 4' position in Formula I above) as the starting material for step 4 of the foregoing reaction sequence and subsequent reaction with a suitable phoεphonate- stabilized carbanion. The difference in the reactivity of singlet oxygen toward the enol ether instead of the exomethylene function ensures that disodium 3-(4- methoxyspiro-[l,2-dioxetane-3,2 '-(4'- methylene)tricyclo[3.3.1.l 3 ' 7 ]decan]-4-yl-)phenyl phosphate will be obtained as the photooxygenation product. If a selectively cleavable pivaloyloxyaryl enol ether will be obtained in any of the reactions immediately preceding the addition of an enzyme-removable group such as a phosphate ester group, it will be more convenient to avoid isolation of the hydroxyaryl enol ether. This can be accomplished by directly splitting the pivaloyl ester with one equivalent of sodium methoxide in methanol and isolating the sodium aryloxide species as a dry solid by removing all volatiles at the concluεion of the reaction. In εuch a case, step 6 of the foregoing reaction sequence will be run using thiε preformed salt in a dry, polar aprotic solvent such as dimethylformamide without using a Lewis base, and the inorganic salt by-products will be removed in the work-up phase of step 7 or step 8. The 2-cyanoethyIphosphate diester product of step 7 undergoes beta-elimination to the phosphate monoester in step S. In step 8, the derivatives where X or X 1 = chlorine or bromine are preferably reacted with a volatile amine such as ammonia, or with a solvent-soluble organic amine such as "DBU" (1,8- diazabicyclo[5.4.0]undec-7-ene) in an alcohol solvent, e.g., methanol. The use of ammonia at atmospheric pressure or above and at ambient temperatures iε particularly advantageouε, aε excess baεe is simply volatized in vacuo at the end of the reaction. Oxidation of the enol ether phosphate in step 9 of the foregoing reaction sequence can be carried out photochemically, as indicated, by reaction with singlet oxygen ( 1 0 2 ) in a halogenated solvent, e.g., a halogenated hydrocarbon such as chloroform, which may also contain a cosolvent, e.g., a lower alkanol such as methanol. Singlet oxygen can be generated using a photosensitizer such as polymer-bound Rose Bengal (Polyεciences, Inc.), methylene blue, or 5, 10, 15, 20-tetraphenyl-21H, 23H- porphine ("TPP"). Alternatively, the crude 2-cyanoεthyl phosphate diesterε obtained in step 7 of the foregoing reaction sequence can be oxidized with singlet oxygen to their 1,2-dioxetane counterparts. Subsequent reaction with sodium methoxide in methanol at room temperature, followed by aqueous work-up and preparative reverse phase high pressure liquid chromatography, gives the pure 1,2- dioxetane phosphate monoester salts as mixtures of their εyn- and anti-isomerε. Chemical methods of l,2-dioxetane formation, including ones using triethylεilylhydro- trioxide, phosphate ozonideε or triarylamine radical cation-mediated one electron oxidation in the presence of triplet oxygen, can also be used. Starting materials of the formula: Ad = / \ where both X and X 1 are the same and are other than hydrogen, or intermediates from which such starting materials can be synthesized using art-recognized methods, are also known. For example, the use of symmetrically substituted 5,7-bis-X_,X 1 ,-adamantan-2-ones in step 4 of the foregoing reaction sequence will result in symmetrical 1,2-dioxetanes which contain X and X 1 substituents in both a syn and anti relationship to the four-membered dioxetane ring. The quinone monoacetal- based syntheεiε strategy of Stetter, et al. provides access to 5,7-dihydroxyadamantan-2-one by way of bicyclo[3.3.1]nonan-3,7-dione-9-ethyleneacetal; Stetter, et al. , Liebiσs Ann. Chem.. 1807 (1977); see also Hamill, et al., Tetrahedron. 21_, 4317 (1971). 5,7- Dihydroxyadamantan-2-one can be converted to 5,7- dibromoadamantan-2-one or its 5,7-dichloro and 5,7-diiodo analogs using 47% aqueous hydrobromic acid, thionyl chloride or 57% aqueous hydriodic acid'under the conditions described in Geluk, et al., loc. cit. 5,7- Dialkyladamantan-2-oneε, e.g., 5,7-dimethyl-adamantan-2- one, can be εyntheεized by the method of Kira, et al. , J. Am. Chem. Soc.. 111. 8256 (1989) . Solvolysis of 3- (methoxy-5,7-dibromotricyclo[3.3.3.1 3 ' ]dec-2- ylidenedimethyl)phenol with diols such as ethylene glycol or 1,4-butanediol in the presence of potassium carbonate will also furnish the corresponding symmetrical bis- hydroxyalkoxysubstituted 1,2-dioxetanes upon following the modified route to the monosubstituted derivatives described above. When one wisheε to take advantage of cooperative effectε produced by an X group other than hydrogen that is different from an X 1 group also present that is also other than hydrogen, particularly where advantages can be obtained using εuch enzymatically cleavable 1,2- dioxetanes as isomeric mixtures, unsymmetrical 5,7 (X,X X )adamantan-2-ones will be used in step 4 of the foregoing reaction sequence. 5-Bromo-7- trifluoroadamantan-2-one can be prepared according to the method disclosed in Sorochinskii, et al., Zh. Obshch. Khim.. 17, 2339 (1981) . 5-Chloro-7-hydroxyadamantan-2- one and 5-methyl-7-hydroxyadamantan-2-one have been described by Stetter, et al., loc. cit. , and 5-bromo-7- hydroxyadamantan-2-one can be synthesized from 7- methylenebicyclo[3.3.l]nonane-3,9-dione-9-ethylene acetal following the procedure of Stetter, et al., by dissolving this compound in anhydrous ethanol and saturating the solution at 0°C with gaseous hydrogen bromide instead of the gaseouε hydrogen chloride used to obtain the corresponding chloro derivative. Intermediates and methods for the synthesis of compounds of formula I above in which R-_ is other than lower alkoxy and R 2 is other than phosphoryloxy salt- substituted phenyl are found in the abovementioned Bronstein, Bronstein, et al., Edwards and Edwards, et al., ('672) applications. This invention, as indicated above, is also directed to the use of its chemiluminescent, enzymatically cleavable substituted 1,2-dioxetanes in art-recognized assays, including assays for detecting enzymes in samples, to kits for use in such assays, and to like uses and means for accomplishing such uses. For example, when using this invention to detect an enzyme in a sample, the sample is contacted with a dioxetane bearing a group capable of being cleaved by the enzyme being detected. The enzyme cleaves the dioxetane's enzyme cleavable group to form a negatively charged substituent (e.g., an oxygen anion) bonded to the dioxetane. This negatively charged substituent in turn destabilizes the dioxetane, causing the dioxetane to decompose to form a fluorescent chromophore group that emits light energy. It is thiε chromophore group that is detected as an indication of the presence of the enzyme. By measuring the intensity of luminescence, the concentration of the enzyme in the sample can also be determined. A wide variety of other assayε 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 εuch aεεays include immunoassayε to detect antibodies or antigens, e.g., δ- or 3-hCG; enzyme assayε; chemical assays to detect, e.g., potaεεium or sodium ions; and nucleic acid aεεays to detect, e.g. , viruses (e.g., HTLV III or cytomegalovirus, or bacteria (e.g., E. coli) . and certain cell functions (e.g., receptor binding sites) . When the detectable substance is an antibody, antigen, or nucleic acid, the enzyme capable of cleaving the enzyme cleavable group of the dioxetane iε preferably bonded to a substance having a εpecific affinity for the detectable substance (i.e., a substance that binds specifically to the detectable substance), e.g., an antigen, an antibody, or a nucleic acid probe. Conventional methods, e.g., carbodiimide coupling, are used to bond the enzyme to the εpecific affinity substance; bonding is preferably through an amide linkage. In general, asεayε are performed as follows. A sa ple 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 εubstance 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 cleavable by the enzyme portion of the specific affinity-enzyme compound is added. The enzyme cleaves the enzyme cleavable group, causing the dioxetane to decompose into two carbonyl compounds (e.g., an ester, a ketone or an aldehyde) . The chromophore to which the enzyme cleavable group had been bonded is thus excited and luminesceε. Lu ineεcence is detected (uεing, e.g., a cuvette, or light-sensitive film in a camera luminometer / or a photoelectric cell or photomultiplier tube) , as an indication of the presence of the detectable substance in the sample. Luminescence intensity is measured to determine the concentration of the εubstance. Examples of specific assayε follow. A. Assay for Human IgG A 96-well microtiter plate is coated with sheep antihuman 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 hour at room temperature. Following the incubation period, the serum sample is removed from the wells, and the wellε are waεhed four times with an aqueous buffer solution containing 0.15 M NaCl, 0.01 M phosphate, and 0.1% bovine serum albumin (pH 7.4) . Alkaline phosphatase bonded to a-nti-human IgG is added to each well, and the wellε 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 of this invention iε 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- -hCG is adεorbed 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 1 ml of a buffer εolution containing 0.15 M NaCl, 0.01 M phosphate, and 0.1% bovine serum albumin (pH 7.4). Alkaline phosphatase-labeled anti-p-hCG iε added to the membrane, and the membrane is washed again with 2 ml 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 phoεphate-containing dioxetane of thiε invention. The luminescence reεulting from enzymatic degradation of the dioxetane iε then detected. C. Assay for Serum Alkaline Phosphataεe 2.7 ml of an aqueous buffer solution containing 0.8 M 2-methyl-2-aminopropanol is placed in a 12 X 75 mm pyrex test tube, and 0.1 ml of a εerum sample containing alkaline phoεphataεe added. The εolution is then equilibrated to 30°C. 0.2 ml of a phoεphate-containing dioxetane of thiε invention iε added, and the test tube immediately placed in a luminometer to record the resulting luminescence. The level of light emisεion will be proportional to the rate of alkaline phosphatase activity. D. Nucleic Acid Hybridization Assay A sample of cerebroεpinal fluid (CSF) εuεpected of containing cytomegaloviruε iε collected and placed on a membrane, e.g., a nylon or nitrocellulose membrane. The sample is then chemically treated with urea or guanidinium isothiocyanate to break the cell wallε and the degrade all cellular componentε except the viral DNA. The strands of the viral DNA thuε produced are separated and attached to the nitrocellulose filter. A DNA probe specific to the viral DNA and labeled 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 0.1 mm Tris-HCl (pH = 8.10) to remove excess probe molecules. A phosphate-containing dioxetane of this invention is added and the reεulting luminescence from the enzymatic degradation of the dioxetane is measured in a luminometer or detected with photographic film. E. Assay for Galactosidase In the assays described above and in the working examples to follow dioxetanes containing a- or β- galactosidase-cleavable α-D- or /3-D- galactoside (galactopyranoside) groups, respectively, can be added, and the luminescence resulting from the enzymatic cleavage of the sugar moiety from the chromophore measured in a luminometer or detected with photographic film. F. Electrophoresiε Electrophoresiε allowε one to ^ separate complex ixtureε of proteinε and nucleic acidε according to their molecular εize and εtructure on gel εupports in an electrical field. This technique is also applicable to separate fragments of protein after proteolyεiε, or fragmentε of nucleic acids after scisεion by reεtriction endonucleaseε (aε in DNA εequencing) . After electrophoretic reεolution of εpecies in the gel, or after transfer of the separated species from a gel to a membrane, the bonds are probed with an enzyme bound to a ligand. For example, peptide fragments are probed with an antibody covalently linked to alkaline phosphatase. For another example, in DNA sequencing alkaline phosphatase - avidin bindε to a biotinylated nucleotide baεe. Thereafter, an AMPPD analog of this invention is added to the gel or membrane filter. After short incubation, light is emitted aε the result of enzymatic activation of the dioxetane to form the emitting species. The luminescence is detected by either X-ray or instant photographic film, or scanned by a luminometer. Multichannel analysis further improves the process by allowing one to probe for more than one fragment simultaneously. G. Solid State Assays In solid state asεayε, it is desirable to block nonspecific binding to the matrix by pretreatment of nonspecific binding sites with nonspecific proteins such as bovine serum albumin (BSA) or gelatin. It has been found that some commercial preparations of BSA contain small amounts of substanceε that exhibit phosphatase activity that will produce undeεirable background chemiluminescence from AMPPD. It has also been discovered, however, that certain water-soluble synthetic macromolecular substances are efficient blockers of nonεpecific binding in εolid εtate assays using dioxetanes. Preferred among such substances are water- soluble polymeric quaternary ammonium saltε such as BDMQ, poly(vinylbenzyltrimethylammonium chloride) (TMQ) , and poly(vinylbenzyltributylammonium chloride) (TBQ) . Other such substanceε are diεclosed in the aforementioned Voyta, et al. '263 application and listed in Table III below. H. Asεay for Nucleotidaεe An assay for the enzyme ATPase is performed in two stepε. In the firεt step, the enzyme is reacted at its optimal pH (typically pH 7.4) with a substrate comprising ATP covalently linked via a terminal phosphoester bond to a chromophore-subεtituted 1,2-dioxetane to produce a phosphoryl-chromophore-subεtituted 1,2-dioxetane. In the εecond step, the product of the first step is decomposed by the addition of, acid to bring the pH to below 6, preferably to pH 2-4, and the resulting light measured in a luminometer or detected with chromatographic film. In a similar two-step procedure, ADPase iε assayed using as the substrate an ADP derivative of a chromophore- substituted 1,2-dioxetane of this invention, and 5'- nucleotidase assayed using as the substrate an adenylic acid derivative of a chromophore-subεtituted 1,2- dioxetane of thiε invention. The εecond εtep can also be carried out by adding the enzyme alkaline phosphataεe to decompoεe the phosphoryl-chromophore-εubstituted 1,2- dioxetane. I. Nucleic Acid Sequencing DNA or RNA fragmentε, produced in sequencing protocols, can be detected after electrophoretic separation using the chemiluminescent 1,2-dioxetanes of this invention. DNA sequencing can be performed by a dideoxy chain termination method [Sanger, F. , et al.. Proc. Nat. Acad. Sci. fUSAI . 74:5463 (1977)]. Briefly, for each of the four sequencing reactions, single-stranded template DNA is mixed with dideoxynucleotides and biotinylated primer strand DNA. After annealing, Klenow enzyme and deoxyadenosine triphosphate are incubated with each of the four sequencing reaction mixtures, then chase deoxynucleotide triphosphate iε added and the incubation continued. Subsequently, DNA fragments in reaction mixtures are separated by polyacrylamide gel electrophoresis (PAGE) . The fragments are tranεferred to a membrane, preferably a nylon membrane, and the fragmentε cross-linked to the membrane by exposure to UV light, preferably of short wave length. After blocking non-specific binding sites with a polymer, e.g., heparin, casein or serum albumin, the DNA fragments on the membrane are contacted with avidin or streptavidin covalently linked to an enzyme specific for the enzyme-cleavable group of the particular 1,2- dioxetane substrate of thiε invention being used. As avidin or streptavidin bind avidly to biotin, biotinylated DNA fragments will now be tagged with an enzyme. For example, when the che iluminscent εubεtrate is disodium 3-(4-methoxyεpiro[l,2-dioxetane-3,2 '- (5'- chloro) tricyclo[3.3.1.1 3,7 ]decan]-4-yl)phenyl phosphate dioxetane salt (C1-.AMPPD) , avidin or streptavidin will be conjugated to a phosphatase. Similarly, when the chemiluminescent substrate is disodium 3-(4- ethoxyspiro[1,2-dioxetane-3,2 • -(5'- chloro)tricyclo[3.3.1.1 3 ' 7 ]decan]-4-yl)phenyl β-D- galactopyranose (Cl-AMPGD) , avidin or streptavidin are conjugated with galactosidaεe. Following generation of luminescence by contacting the complex of DNA fragment-biotin-avidin (or streptavidin)-enzyme with the appropriate 1,2-dioxetane at alkaline pH values, e.g., above about pH 8.5, DNA fragments are visualized on light-sensitive film, e.g. , X-ray or instant film, or in a photoelectric luminometer instrument. The detection method outlined above can also be applied to the genomic DNA sequencing protocol of Church et al. [Church, G.M. , et al.. Proc. Nat. Acad. Sci. fUSA.. 81:1991 (1984)]. After transferring chemically cleaved and electrophoretically separated DNA [Maxam, A.M. et al., Proc. Nat. Acad. Sci. (USA. , 74:560 (1977)] to a membrane, preferably a nylon membrane, and cross- linking the ladders to the membrane by UV light, specific DNA sequences may be detected by sequential addition of: biotinylated oligonucleotides as hybridization probes; avidin or streptavidin covalently linked to an enzyme specific for an enzyme-cleavable chemiluminescent 1,2- dioxetane of this invention; and, the appropriate 1,2- dioxetane. Images of sequence ladders (produced by PAGE) may be obtained as described above. Serial reprobing of sequence ladders can be accomplished by first stripping the hybridized probe and che iluminescent material from-a membrane by contacting the membrane - ith a heated εolution of detergent, e.g., from about 0.5 to about 5% sodium dodecylsulfate (SDS) in water at from about 80°C to about 90°_C, cooling to from about 50°C to about 70°C, hybridizing the now-naked DNA fragments with another biotinylated oligonucleotide probe to generate a different sequence, then generating an imaging chemiluminescence as described above. Similar detection methodε can be applied to RNA fragmentε generated by RNA sequencing methods. In order that those skilled in the art can more fully underεtand thiε invention, the following examples are set forth. These examples are given solely for purposeε of illuεtration, and εhould not be conεidered as expressing limitations unlesε so set forth in the appended claims. All parts and percentages are weight by volume, except TLC solvent mixtures, which are volume by volume, or unleεε otherwise stated. The 2 H NMR data given in certain of these examples for enol ether intermediates uses the prime symbol (') to designate aromatic protons, while non-primed numberε refer, in all cases, to substituent adamant-2 '-ylidene ring positions, thus: EXAMPLE I Two hundred grams (1.64 mol) of 3- hydroxybenzaldehyde and 270 ml. (1.93 mol) of triethylamine were charged to a flask containing one liter of methylene chloride in an ice bath. The resulting brown solution was mechanically εtirred, and 212 ml (1.72 mol) of trimethylacetyl chloride waε added in a thin stream from an addition funnel over a 15 minute period. The resulting slurry was stirred for an additional 15 minutes, the ice bath waε removed, and the reaction was then allowed to proceed for an additional two hours. TLC (K5F; 25% acetone-hexaneε) showed the absence of starting material and a single higher R f product. The reaction mixture was transferred to a separatory funnel and admixed with 250 ml of IM hydrochloric acid. The organic phase waε then extracted with water (2 x 400 ml) and finally dried over εodium εulfate. The dried solution waε 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, which was held under an argon atmosphere. EXAMPLE II Four hundred mg of p-toluenesulfonic acid dissolved in 25 ml of methanol was added, with stirring, to the 3- pivaloyloxy-benzaldehyde of Example I. Trimethylorthoformate (224 ml; 2.05 mol) was then added dropwise. The minor exotherm that resulted was allowed to proceed unchecked while the mixture was stirred for 1 hour. One half gram of sodium bicarbonate was added, and the flaεk waε placed on the rotary evaporator (bath temperature 40°C) to remove all volatileε. The reεulting oil waε passed through a εhort εilica gel column under nitrogen pressure to give an orange-brown oil which was pumped under vacuum (1.0 mm Hg) with stirring to yield 426 g of crude 3-pivaloyloxybenzaldehyde dimethyl acetal. Infrared analysis revealed no aldehyde carbonyl absorption (1695 cm -1 ) . EXAMPLE III The crude 3-pivaloyloxybenzaldehyde dimethyl acetal of Example II was dissolved in one liter of methylene chloride, freshly distilled from P 2 0 5 , under an argon atmosphere in, a 3 liter flask. Then, 347 ml (2.03 mol) of triethyl phosphite was added all at once. The flask was fitted with a septum inlet adapter and cooled in a dry ice-acetone bath under slight argon pressure. Boron trifluoride etherate (249 ml; 2.03 mol) was added in several portions by syringe, with vigorous stirring. The reεulting reaction mixture waε εtirred at -55°C for two hours, then stored in a freezer at -20°C overnight. Next, the flask was warmed to room temperature and its contents stirred for 4 hours. The orange-brown solution was then poured carefully into a vigorously stirred slurry of 170 g of sodium bicarbonate in 800 ml of water at a rate such that vigorous foaming was avoided. After vigorously stirring the biphasic mixture for one hour the layers were then separated in a separatory funnel and the aqueous layer was again extracted with methylene chloride (2 x 250 ml) . The combined organic extracts were dried over sodium sulfate, concentrated, and vacuum diεtilled to yield 535 g of diethyl 1-methoxy-l-(3-pivaloyloxyphenyl)methane phoεphonate aε a clear, light yellow oil (b.p. 158-161°C at 0.25 mm Hg) . This represented a 91% yield for the overall procedure of Examples I - III. 1 HNMR (400 MHz; CDCC 3 ) : δ 1.21 and 1.25 (6H, two t, 7HZ, OCH 2 CH 3 ) ; 3.37 (3H, s, ArCHOCH 3 ) ; 3.80 (3H, s, 5 ArOCH 3 ) ; 3.90 - 4.10 (4H, m, OCH 2 CH 3 ) ; 4.46 (IH, d, 15.6 Hz, ArCHPO) ; 6.85 (IH, m) ; 7.00 (2H, m) ; 7.26 (IH, m) . IR (neat): 2974, 1596, 1582, 1480, 1255 (P=0) , 1098, 1050, 1020, 965 cm -1 . EXAMPLE IV C A εolution of diisopropylamine (11.6 ml, 82.8 mmol) in 75 ml of tetrahydrofuran was cooled to -78°C in a dry ice-acetone bath under an argon atmosphere. Thirty ml of a 2.5 M solution of n-butyllithium in hexanes (Aldrich, 75.0 mmol) was added by syringe and, after stirring the resulting lithium diisopropylamide solution for 20 minutes, 13.47 g (37.6 mmol) of diethyl l-methoxy-l-(3- pivaloyloxyphenyl)methane phosphonate in 25 ml of tetrahydrofuran waε added dropwiεe from an addition funnel over a 5 minute period. The resulting red solution was stirred at low temperature for another 30 minutes to ensure complete formation of the phosphonate carbanion. A solution of 4.99 g (30.1 mmol) of 5- hydroxyadamantan-2-one in 25 ml of tetrahydrofuran was then added dropwise. The resulting slightly cloudy mixture waε stirred for 5 inuteε at -78°C and then εlowly warmed to room temperature over 40 minutes. The εolution, now orange in color, was refluxed for 90 minuteε, cooled, diluted with 200 ml of a εaturated sodium bicarbonate εolution, and extracted with ethyl acetate (3 x 75 ml) . The combined organic extracts were washed with a saturated sodium chloride solution, quickly dried over sodium sulfate and concentrated to give 12.49 g of an orange gum, a mixture of phenolic enol ether and its pivaloate ester. 1 HNMR (Pivaloate ester, 400 Hz, in CDC1 3 : δ 7.33 (1 H, m, H-5'), 7.12 (1 H, d, J = 7.7 Hz, ArH) , 6.95 - 7.02 (2H, m, ArH), 3.43 (1 H, br. s, H-l) , 3.28 (3H, ε, OMe) , 2.79 (1 H, br. ε, H-3), 2.23 (1 H, br. ε, H-7), 1.59 - 1.87 (11 H, m) , 1.34 (9 H, s, COC(CH 3 ) 3 ) . IR (in CHC1 3 ) : 3590, 3442 (OH) , 2924, 2848, 1742 (ester C = O) , 1665, 1602, 1578, 1426, 1274, 1152, 1118, 918 cm "1 . This mixture was taken up in 100 ml of methanol and refluxed for 3.5 hours in the presence of 10.7 g of anhydrous potassium carbonate. The methanol was then stripped off and the residue partitioned between water and ethyl acetate. The organic layer was washed with a saturated sodium chloride solution and rotary evaporated to a residue which was recrystallized from chloroform- petroleum ether to give 6.5 g of 3-(methoxy-5-hydroxy- tricyclo[3.3.1.1 3,7 ]dec-2-ylidenemethyl)phenol as an off- white solid (m.p. 171-172°C). An additional 0.80 g was obtained from the mother liquor, giving an overall yield of phenolic enol ether of 79%, based on 5- hγdroxyadamantan-2-one. 1 HNMR (400 MHz, in CDCl 3 ) : δ 1.18 (1 H, dd, J = 8.4 , 7.7 HZ, H-5') , 6.75 - 6.88 (3 H, , ArH) , 6.36 (1 H, br. s, ArOH) , 3.41 (1 H, br. s, H-l) , 2.28 '3 H, s, OMe) , 2.79 (1 H, br. s, H-3), 2.22 (1 H, br. s, H-7), 1.56 - 1.98 (11 H, m) . IR (in CHC1 3 ). : 3586, 3320 (OH), 3000, 2920, 2844, 1665, 1590, 1578, 1445, 1296, 1092, 885 cm "1 . HRMS calc. for C 18 H 22 0 3 (M + ) 286.1573, found 286.1569. EXAMPLE V A solution of 5.04 g (17.6 mmol) of 3-(Methoxy-5- hydroxytricyclo[3.3.1.1 3 ' 7 ]dec-2-ylidenemethyl)phenol in 35 ml of tetrahydrofuran, prepared under argon, was admixed with 3.4 ml (24.6 mmol) of triethylamine and then cooled to 0°C in an ice bath. 2-Chloro-2-oxo-i,3,2- dioxaphospholane (1.95 ml, 21.1 mmol) was added dropwise with stirring. After 5 minutes the ice bath was removed, and stirring waε continued for 45 minutes at room temperature. The reaction mixture was diluted with 30 ml of anhydrous diethyl ether and filtered under argon to exclude moisture. The triethylamine hydrochloride was then washed further with 20 ml of diethyl ether and the filtrate concentrated on the rotary evaporator to give the phosphate triester as a viscous, light orange oil. The triester, dissolved in 30 ml of molecular sieve- dried dimethylformamide under argon, was reacted for 3.5 hours at room temperature with 1.02 g (20.8 mmol) of dry sodium cyanide, added all at once with stirring. The solvent was then removed under vacuum (1.0 mm Hg) witih heating to 50°C. A sample of the reεulting orange-brown residue, when dissolved in water and subjected to reverse phase analytical chromatography [0.1% sodium bicarbonate (water) - acetonitrile gradient] on a PLRP polystyrene column (Polymer Laboratorieε) , evidenced complete reaction to the intermediate cyanoethyl phosphate diester sodium salt. The residue was then taken up in 35 ml of methanol and treated dropwise with 4.85 ml of a 4.37 M solution (21.2 mmol) of sodium methoxide in methanol, with stirring, for 30 minutes at room temperature. Reverse phase analytical HPLC showed ,9-elimination to the phosphate monoester to be complete. The solvent waε removed and the residue triturated with 10% water/acetone to give a gummy solid. Further trituration with 3% water/acetone gave a hard, off-white solid which waε filtered and dried under vacuum (1.0 mm Hg) to give 8.35 g of crude disodium 3-(methoxy-5- hydroxytricyclo[3.3.1.l 3,7 ]dec-2-ylidenemethyl) phenyl phosphate, contaminated with inorganic salts. Reverse phase preparative HPLC using a water- acetonitrile gradient on a PLRP polyεtyrene column (Polymer Laboratories) and lyophilization of the appropriate fractions gave 5.2 g (72%) of purified compound as a white, granular solid, softening at 120°C and melting to a light brown gum at 163-168°C. 1 HNMR (400 MHz, in D 2 0) : δ 7.16 (1 H, dd, J = 8.3, 7.4 Hz, H-5'), 7.05 (1 H, d, J = 8.3, Hz, ArH), 6.93 (1 H, br. ε, H-2'), 6.86 (1 H, d, J = 7.4 Hz, ArH) , 3.2 (3 H, s, OMe), 3.17 (1 H, br. s, H-l), 2.61 (1 H, br. ε, H- 3), 2.06 (1 H, br. s, H-7) 1.42 - 1.73 (10 H, m) . Elemental analysis showed that the phoεphate salt exists as a dihydrate. Anal. Calc. for C 18 H 21 Na 2 0 6 P-2H 2 0:C, 48.44, H, 5.65, P, 6.94. Found: C, 48.37, H, 5.90, P, 6.87. EXAMPLE VI A solution of 0.8g of disodium 3-(methoxy-5- hydroxytricyclo [3.3.1.1 3,7 ] dec-2-ylidenemethyl) phenyl phosphate in 96 ml of 25% anhydrous methanol/chloroform containing 5.35 x 10 ~5 M methylene blue sensitizing dye was divided among three glass tubes. Each tube was then cooled to 5°C in a water bath and saturated with oxygen by passing a stream of the gas through the solution. After 5 minutes, and while continuing to bubble oxygen through the solution, the tubes were irradiated with light from a cooled, 250 watt high pressure sodium vapor lamp while maintaining the temperature at 5°C. A 5 mil thick piece of Kapton polyimide film (duPont) , placed between the sodium vapor lamp and the tubes, filtered out unwanted UV radiation. After 20 minutes of irradiation the solutionε had turned pink. Reverse phase analytical HPLC [0.1% sodium bicarbonate (water) - acetonitrile gradient) on a PLRP polystyrene column (Polymer Laboratories) showed two product peaks: an early eluting, broadened peak (retention time 3.79 minutes) and a sharp, later eluting peak (retention time 5.77 minutes) . The ratio of early eluting product (A) to later eluting product (B) was 1.3:1 in each tube's εolution. The solutions were combined and the solvent removed under vacuum (25.0 mm Hg) on an ice bath. The residue was then dissolved in 70 ml of water and filtered through a 0.45 μm nylon membrane. Reverse phase preparative HPLC (water-acetonitrile gradient) easily separated two products. Combination of the appropriate preparative HPLC fractions, followed by analytical HPLC, εhowed them to be homogeneous. Lyophilization gave 0.32 g of one product (A) and 0.26 g of another (B) , which were shown by 1 HNMR to be isomeric (syn and anti) 1,2-dioxetanes, i.e., syn- disodiu 3-(4-methoxyspiro-[1,2-dioxetane-3 ,2 '-(5'- hydroxy)tricyclo[3.3.1.1 3 ' 7 ]decan]-.4-y1)phenyl phosphate: and its anti-isomer (anti-disodium 3-(4-methoxyspiro- ;i,2-dioxetane-3,2 '-(5 1 - hydroxy)tricyclo[3.3.1.1 3 ' 7 ]decan]-4-yl)phenyl phosphate) Each of these isomers produced chemiluminescence, with unique light vs. time and noise profiles, upon cleavage at pH 10 in aqueous buffer medium with alkaline phosphatase. 1 HNMR (A isomer, 400 MHz, in D 2 0) : δ 6.98 - 7.6 (4H, m, ArH) , 3.11 (3H, s, OMe), 2.97 (IH, br. s, H-l) , 2.34 (IH, br. S, H-3) , 1.79 (IH, br. s, H-7), 1.3 - 1.68 (8H, m) , 1.01 (1 H, d, J 13.5 Hz), 0.8 (IH, d, J = 13Hz) . 1 HNMR (B iso er, 400 MHz, in D 2 0) : δ 6.94 - 7.62 (4H, m, ArH), 3.09 (3H, ε, OMe), 2.91 (IH, br. s, H-l), 2.27 (IH, br. S, H-3), 1.84 (lHf br. s, H-7), 1.21 - 1.75 (8H, m) , 1.02 (IH, d, J = 12.8 Hz), 0.87 (IH, d, J = 12.8 Hz) . Elemental analyεis εhowed that iεomer B exists as a dihydrate. Anal. Calc. for C 18 H 21 Na 2 0 8 P-2H 2 0 (isomer B) ; C, 45.2, H, 5.27, P, 6.47. Found: C, 45.53, H, 5.35, P f 6.11. It was not possible to specifically designate which of the two isomerε obtained waε the syn-isomer and which was the anti-isomer on the basis of ^-HN R data. EXAMPLE VII Following in general the procedure of Example IV above, a solution of 5.35 ml (38.2 mmol) of diisopropylamine in 35 ml of tetrahydrofuran waε cooled to -78°C in an ice bath under an argon atmosphere. Fourteen ml of a 2.5 M solution of n-butyllithium in hexanes (35.0 mmol) waε added by syringe and, after stirring for 20 minuteε, 10.86 g (30.3 mmol) of diethyl l-methoxy-l-(3-pivaloyloxyphenyl)methane phosphonate in 30 ml of tetrahydrofuran was added dropwise over a 10 minute period. The resulting orange solution was stirred at low temperature for 1 hour, then admixed with a solution of 5.21 g (22.75 mmol) of 5-bromoadamantan-2-one in 20 ml of tetrahydrofuran over a 7 minute period, with stirring. Stirring waε continued for an additional 10 minuteε, at which point the cold bath waε removed and the reaction mixture waε allowed to warm slowly to rooir. temperature over a one hour period. The solution was then refluxed for another hour, cooled, diluted with 100 ml of hexaneε, and poured into a separatory funnel containing saturated aqueous sodium bicarbonate solution and extracted with 10% ethyl acetate in hexanes (3 x 50 ml) . The combined organic extracts were washed with an aqueous 15% sodium chloride solution, quickly dried over sodium εulfate, and concentrated to give 11.62 g of a light orange viscous oil. Plug filtration on a short silica gel column, eluting with 10% ethyl acetate in n-hexaneε, gave 11.0 g of a yellowish- green gum. 1 HNMR (Pivaloyl ester, 400 MHz, in CDC1 3 ) : δ 7.34 (IH, t, J = 7.8 Hz, H-5'), 7.1 (IH, d, J = 7.7 Hz, .ArH), 6.95 - 7.02 (2H, , ArH), 3.39 (IH, br. s, H-l), 3.28 (3H, s, OMe), 2.74 (IH, br. ε, H-3), 2.32 - 2.51 (6H, m, H-4, H-6, H-9), 2.17 (IH, br. ε, H-7), 1.67 - 1.92 (4H, m, H-8, H-10) , 1.34 (9H, s, COC(CH 3 ) 3 ). IR (neat) : 2924, 2850, 1745 (ester C=0) , 1654, 1602, 1578, 1478, 1274, 1110, 1018, 808, 758 cm "1 . This gum was taken up in 30 ml of methanol and refluxed for 2 hours with 2.4 g of anhydrous potassium carbonate. The methanol was then removed and the residue partitioned between water and 30% ethyl acetate in hexanes. The organic layer was washed with aqueous saturated sodium chloride solution, dried over sodium sulfate and concentrated to give 9.32 g of a yellow gum. This gum was flash chromatographed on silica gel to give 7.06 g (88% yield based on 5-bromoadamantan-2-one) of a slightly yellow gum that could not be crystallized. IR and NMR indicated, however, that the compound obtained, 3-(methoxy-5-bromotricyclo [3.3.1.l 3,7 ]dec-2- ylidenemethyl)phenol, was pure enough to be uεed for the ensuing phosphorylation reaction. A sample of the pure phenolic compound was obtained from 3-(methoxy-5-bromotricyclo [3.3.l.l 3,7 ]dec-2- ylidenemethyl) phenyl acetate, obtained as follows: The impure phenolic compound (5.57 g; 15.95 mmol) was disεolved in 30 ml of molecular εieve-dried pyridine under an argon atmoεphere. Fifty mg of 4- dimethylaminopyridine was added as catalyst. Next, 1.8 ml (19.15 mmol) of acetic anhydride was added by syringe and the reaction mixture was εtirred at room temperature for 3 hourε. The reaction mixture waε then tranεferred to a εeparatory funnel containing 250 ml of an aqueouε saturated sodium bicarbonate solution, then extracted with 10% ethyl acetate in hexanes (2 x 100 ml) . The combined extracts were washed several times with water, then quickly dried over εodium sulfate. The dried solution was concentrated on a rotary evaporator, and the residue was recryεtallized from n-hexaneε containing a few dropε of ethyl acetate. The thuε-obtained off-white εolid waε again recryεtallized to give 5.21 g (83.5% yield) of the acetate, m.p. 108-110°C. HRMS calc. for C 20 H 23 BrO 3 (M + ) 390.0833, found 390.0831. 1 HNMR (400 MHz, in CDC1 3 ) : δ 7.35 (IH, dd, J = 8, 7.7 HZ, H-5 1 ), 7.13 (IH, dd, J = 7.7, 1Hz, ArH), 7.03 (IH, dd, J = 8, 1Hz, ArH), 5.99 (IH, d, 1Hz, H-2') , 3.39 (IH, br. ε, H-l), 3.27 (3H, ε, OMe), 2.75 (IH, br. ε, H- 3), 2.32 - 2.51 (6H, M, H-4, H-6, H-9), 2.29 (3H, S, OAc), 2.18 (IH, br. s, H-7), 1.69 1.92 (4H, m, H-8, H- 10) . IR (in CHC1 3 ) : 3000, 2930, 2850, 1760 (eεter C 0), 1660, 1602, 1577, 1368, 1192, 1095, 1070, 1016, 804 cm -1 . Treatment of the recrystallized acetate with potassium carbonate in methanol for 15 hours at room temperature gave 3-(methoxy-5- bromotricyclo[3.3.1. l 3, ]dec-2-ylidenemethyl]phenol, m.p. 42-45°C, as a white, crispy foam that could not be recrystallized further. 1 HNMR (400 MHz, in CDCl 3 ) : δ 7.21 (IH, t, J = 7.2 HZ, H-5') , 6.73 - 6.85 (3H, m, ArH) , 5.18 (IH, s, ArOH) , 3.38 (IH, br. s, H-l), 3.28 (3H, s, OMe) , 2.74 (IH, br. ε, H-3), 2.3 - 2.52 (6H, m, H-4 , H-6, H-9) , 2.17 (IH, br. ε, H-7), 1.68 - 1.92 (4H, m, H-8, H-10) . IR (in CHC1 3 ) : 3584, 3320 (OH), 2925, 2850, 1665, 1588, 1578, 1445, 1435, 1092, 1080, 1015, 880, 808 cm "1 . EXAMPLE VIII Again following in general the procedure of Example IV above, a solution of 2.97 ml (21.3 mmol) of diisopropylamine in 21 ml of tetrahydrofuran waε cooled to -78°C in a dry ice-acetone bath under an argon atmosphere, admixed with 8.5 ml of a 2.5 M εolution of n- butyllithium in hexanes (21.3 mmol) , added dropwise by syringe, and stirred for 20 minuteε. Diethyl l-methoxy- l-(3-pivaloyloxyphenyl)methane phosphonate (7.26 g; 20.3 mmol) in 20 ml of tetrahydrofuran was then added dropwiεe by syringe over a 10 minute period, and the solution was stirred at low temperature for 1 hour. A εolution of 2.79 g (15.2 mmol) of 5- chloroadamantan-2-one in 15 ml of tetrahydrofuran was added over a 5 minute period and, after stirring at low temperature for 10 minutes, the cold bath was removed and the mixture warmed to room temperature. The mixture waε then refluxed for 1.5 hours, cooled, diluted with 50 ml of n-hexanes, and poured into a separatory funnel containing 150 ml of an aqueous saturated sodium bicarbonate solution. Extraction with 5% ethyl acetate- n-hexanes was followed by drying the combined organic fractions, concentrating them and pumping them under vacuum (1.0 mm Hg) to yield a reεidue which, when chromatographed on εilica gel, gave 5.15 g of the higher R f 3-(methoxy-5-chlorotricyclo[3.3.1. l >,7 ]dec-2- ylidenemethyl)phenyl trimethylacetate aε a colorless oil (structure confirmed by IR and NMR), and 0.865 g of a later-eluting material composed predominantly of the corresponding phenol. This latter fraction, when reacylated with pivaloyl chloride and triethylamine in methylene chloride (see Example I above) , followed by chromatography, gave an additional 0.35 g of the pivaloyl ester (93% total yield based on 5-chloroadamantan-2-one) . X HNMR (Pivaloyl ester, 400 MHz, in CDC1 3 ) : 6 7.36 (IH, t, J = 7.8 Hz, H-5'), 7.13 (IH, d, J = 7.7 Hz, ArH) , 6.98 - 7.04 (2H, m, ArH), 3.45 (IH, br. s, H-l), 3.3 (3H, s, OMe), 2.8 (IH, br. s, H-3), 2.13 - 2.32 (7H, m, H-4, H-6, H-7, H-9), 1.65 - 1.9 (4H, m, H-8, H-10) , 1.36 (9H, ε, COC(CH 3 ) 3 ) . IR (neat) : 2932, 2835, 1750 (eεter C = O) , 1664, 1602, 1578, 1478, 1274, 1112, 1022, 827, 758 cm "1 . The combined pivaloyl ester fractions (5.5 g, 14.1 mmol) were taken up in 40 ml of methanol and refluxed with 5.37 g of anhydrous potaεεium carbonate for 40 minuteε. The residue remaining after the methanol was stripped off was partitioned between water and 30% ethyl acetate-hexanes, and the organic fractions were concentrated and plug-filtered on a short silica gel column to give 4.07 g (88% yield ba^e on 5- chloroadamantan-2-one) of 3-(methoxy-5- chlorotricyclo[3.3.1.l 3,7 ]dec-2-ylidenemethyl) phenol as a crispy white foam which became slightly tacky upon exposure to air. HRMS calc. for C 18 H 21 C10 2 (M + ) 304.1227, found 304.1230. X HNMR (400 MHZ, in CDC1 3 ) : δ 7.23 (IH, dd, J = 7.7, 7.6 Hz, H-5'), 6.85 (IH, d, J = 7.6 Hz, ArH), 6.77 - 6.83 (2H, m, ArH), 3.44 (IH, br. s, H-l), 3.31 (3H, ε, OMe), 2.8 (IH, br. ε, H-3), 2.1 - 2.31 (7H, m, H-4, H-6, H-7, H-9) , 1.65 - 1.89 (4H, m, H-8, H-10) . IR (in CHC1 3 ) : 3590, 3330 (OH), 2930, 2855, 1655, 1590, 1580, 1440, 1295, 1094, 1080, 1022, 880, 826 cm "1 . EXAMPLE IX 3-(Methoxy-5-bromotricyclo[3.3.l.l 3,7 ]dec-2- ylidene ethyl)phenol (1.49 g, 4.26 mmol) was dissolved in 8.5 ml anhydrous ethylene glycol and then placed, together with 0.28 g of potasεiu carbonate, in a εealed glass tube. The tube was heated in an oil bath at 110°C for seven hours. The contents of the tube were then concentrated in vacuo (1.0 mm Hg) with heating. The residue was partitioned between saturated sodium chloride solution and ethyl acetate. The organic fraction waε then stripped and chromatographed on a short silica gel column to furniεh 1.31 g (92% yield baεed on the phenol εtarting material) of 3-(methoxy-5-(2- hydroxy)ethoxytricyclo[3.3.1. l 3,7 ]dec-2- ylidenemethyl)phenol aε an off-white foam. X HNMR (400 MHz CDC1 3 ) : δ 7.19 (IH, t, J = 7.6 Hz, H- 5 5') , 6.83 (IH, d, J = 7.6 Hz, ArH) , 6.73 - 6.80 (2H, m, ArH) , 5.83 (IH, s, ArOH) , 3.67 (2H, m, OCH 2 CH 2 OH) , 3.51 (2H, t, J = 4.6 Hz, OCH 2 CH 2 OH) , 3.44 (IH, br. ε, H-l), 3.28 (3H, s, OMe) , 2.81 (IH, br. s, H-3), 2.24 (IH, br. ε, H-7), 1.55 - 1.90 (10H, m) . 0 IR (CHC1 3 ) : 3580, 3320 (OH) , 2929, 2842, 1664, 1586, 1575, 1440, 1092, 1078, 885 cm -1 . EXAMPLE X 3-(Methoxy-5-chlorotricyclo[3.3.1.1 3 ' 7 ]dec-2- ylidenemethyl)phenol (1.23 g, 4.0 mmol) waε 5 phosphorylated in the manner described in Example V above with one exception — ammonia in methanol was used for β- elimination. The crude ammonium sodium salt obtained was triturated with acetone, then pumped in vacuo (1.0 mm Hg) to give 1.2 g of an off-white solid. Reverse phase 0 analytical HPLC showed that the phosphorylated enol ether product thus obtained was pure enough for direct photooxygenation to the corresponding 1,2-dioxetane. The 3-(methoxy-5-chlorotricyclo[3.3.1.1 3 ' 7 ] dec-2- ylidenemethyl)phenyl phosphate salt (0.65 g) waε 5 dissolved in 100 ml of anhydrous 10% methanol/chloroform which also contained 5.35 x 10" 5 M methylene blue as a sensitizing dye. The reεulting εolution waε divided among three glass tubes and irradiated aε described in Example VI above. Work-up, in this case, involved C disεolution of the pumped reεidue in 70 ml of water containing 258 mg of sodium bicarbonate. Upon carrying out reverse phase preparative HPLC, the syn- and anti- isomers were collected together, excluding impurities. Analytical HPLC [0.1% NaHC0 3 (H 2 0)-acetonitrile gradient] showed two product peaks (retention timeε of 8.01 and 8.32 minutes). The area percent ratio (270 nm) of the early eluting isomer to the later eluting isomer waε found to be 0.4:1. • ' ■ H NMR confirmed that the lyophilized white solid obtained was a mixture of syn- and anti- disodium 3-(4-methoxyεpiro-[l,2-dioxetane-3,2 '-(5'- chloro)tricyclo[3.3.1.1 3,7 ]decan]-4-y1)phenyl phoεphate. Elemental analyεiε indicated that the product exiεtε in the form of a dihydrate. Anal. Calc. for C 18 H 20 ClNa 2 O 7 P->2H 2 O: C, 43.52; H, 4.87; Cl, 7.14. Found:C, 43.23; H, 4.99; Cl, 7.65. 1 HNMRf400 MHz, in D 2 0, two isomers) : δ 6.97-7.68 (4H, m, ArH), 3.08 and 3.09 (3H, 2s, OMe), 2.95 (IH, br.s, H- 1) , 0.76-2.35 (12H, m) . EXAMPLE XI 3-(Methoxy-5-bromotricyclo[3.3.1.1 3 ' 7 ]dec-2- ylidenemethyl)phenol was phosphorylated and then photooxygenated as described for its 5-chloro analog in Example X above. Work-up in 0.3% (w/v) aqueouε εodium bicarbonate solution gave a filtered, aqueous solution of the crude 1,2-dioxetane phosphate salt, which was then subjected to reverse phase preparative HPLC (water- acetonitrile gradient) to give the syn- and anti-isomers. collected together, for lyophilization. When the lyophilized product, a white, fluffy solid, was subjected to analytical HPLC, two peaks were obtained with retention times of 8.52 and 8.94 minuteε. The area percent ratio (270 nm) of the early eluting isomer to the later eluting isomer was found to be 0.5:1. 1 HNMR confirmed that the product was a mixture of syn- and anti-disodium 3-(4-methoxyεpiro-[1,2-dioxetane-3,2 '-(5'- bromo)tricyclo[3.3.1.1 3 ' 7 ]decan]-4-yl)phenyl phoεphate. 1 HNMR (400 MHz, in D 2 0, two iεomers) : δ 6.99-7.52 (4H, m, ArH), 3.07 and 3.09 (3H, 2ε, OMe), 2.91 (IH, br.s, H- 1) , 0.822.32 (12H, m) . EXAMPLE XII Immunoassayε for TSH were conducted on a series of TSH εtandardε using a Hybritech Tandem-E TSH kit (Hybritech, Inc., San Diego, California) according to the manufacturer's instructionε included with the kit, except that upon completion of the anti-TSH-alkaline phosphatase conjugate incubation step and waεh, the plastic beads were additionally washed with 0.1 M diethanolamine, l mM magnesium chloride, 0.02% sodium azide buffer, pH 10.0, and then briefly stored in 200 μl of the εame buffer. Chemilumineεcent signals from anti-TSH-alkaline phosphatase conjugate bound to the surface of the beads were initiated by adding to the tubes containing beads 300 Al of 0.67 mM buffer solutions containing, respectively, disodium 3-(2'-spiroadamantane)-4-methoxy- 4-(3"-phosphoryloxy)phenyl-1,2-dioxetane ("AMPPD") , disodium 3-(4-methoxyspiro-[1,2-dioxetane-3,2 '-(5•- hydroxy)tricyclo[3.3.l.l 3 ' 7 ]decan]-4-yl)phenyl phosphate (A isomer; "A-OH-AMPPD") , the corresponding disodium B- iεomer ("B-OH-AMPPD") , and diεodium 3-(4-methoxyεpiro- [1,2-dioxetane-3,2 '-(5'-chloro) ricycle 3.3.1. l 3,7 ]decanj- 4-yl)phenyl phoεphate ("Cl-AMPPD"), in 0.1 M diethanolamine, 1 mm magnesium chloride, 0.02% sodium azide, pH 10.0. The intensity ^ of light emission was subsequently recorded at 7, 13, 19, 25, 31, 40, 50 and 60 minutes after the substrate addition, as a 5 second integral at room temperature (about 25°C) , using a Berthold LB952T Luminometer (Berthold Instrument, Wildbad, Federal Republic of Germany) . (Signals measured only not signal-to-noise) . TSH, RLU v. TSH for each of AMPPD, BR-AMPPD, B-OH- AMPPD, A-OH-AMPPD and CL-AMPPD is shown in FIGS. 1, 2, 3, 4 and 5, respectively. EXAMPLE XIII A comparison of the total luminescence emiεεion from AMPPD and from the corresponding 1,2-dioxetanes whose adamant-2'-ylidene groups are monosubstituted with hydroxy (A and B isomers) , chloro and bromo groups was made by carrying out total dephosphorylation experiments on each of these compounds. An aqueous solution of the 1,2-dioxetane (4.0 mm) in 0.05M sodium carbonate/sodium bicarbonate containing 1 mM magnesium chloride was prepared and then equilibrated at 30°C. Ten μl of a 7.64 X 10 M aqueous εolution of alkaline phosphatase (calf intestine; Biozyme) was then added, and the chemiluminescence from the resulting solution was recorded using a Turner Model 20E luminometer (Turner Instruments; Sunnyvale, California). The rates of chemiluminescence decay for each of the five compounds in question, expreεεed in relative light units (RLU's) per minute, are given in the following table. Table I Decay Rate (RLU's) OH-Adamant-2'- -ylidene (B isomer) 1.5 10 Cl-Adamant-2 '- -ylidene 1.2 5.5 15 Br-Adamant-2•- -ylidene 1.2 0.8 40 These total chemiluminescence emisεions are depicted graphically in FIGS. 6, 7, 8, 9 and 10, respectively. EXAMPLE XIV A strip of neutral BIODYNE A nylon membrane (Pall Corporation, Glen Cove, N.Y.) was dotted twice (side-by- side) with the following concentrations of biotinylated pBr 322 35-mer oligonucleotide probe (Synthetic Genetics, San Diego, California) : DNA 1/ Single stranded, unlabeled DNA, 1 ng. Next, the membrane was blocked in 0.2% casein/0.1% Tween 20 detergent in aqueous phoεphate buffered εaline solution (PBS) for 1 hour, following which 1/5000 diluted avidin-alkaline phosphatase conjugate (Sigma, Inc., St. Louis, MO.) in 0.2% casein in PBS was added. The membrane was then incubated for 30 minuteε, waεhed twice (for 5 minutes each time) in 0.2% casein/0.1% Tween 20 detergent in PBS, washed four times (for 5 minutes each time) in 0.3% Tween 20 detergent in PBS, and twice (for 5 minutes each time) in aqueous 0.1 M diethanolamine containing 1 mM magnesium chloride, pH 10.0. The membrane was then cut up the middle to give two εtripε, each bearing one εet of dots. One of the stripε was incubated for 5 minutes in aqueous AMPPD εolution (0.25 mM in 0.1 M diethanolamine containing 1 mM magnesium chloride, pH 10) , the other for 5 minuteε in the corresponding chloroadamant-2'-ylidene compound (0.25 mM in the same buffer) . The two stripε were then placed in camera luminometerε and exposed on Polaroid Type 612 instant black and white film. The improved chemiluminescence intensity obtained using the chloro compound, as compared to AMPPD itself, can be seen by comparing column 2 (Cl-AMPPD) to column 1 (AMPPD) in FIG. 11. EXAMPLE XV pBr 322 plasmid (4700 bp) was subjected to a nick translation process using a Trans-Light kit (Tropix, Inc., Bedford, Mass.) to generate a mixture of biotinylated single stranded polynucleotides of 200-2000 bps in length. This mixture waε dotted onto a dry BIODYNE A membrane aε five parallel colu nε of dots of the following concentrationε: DNA Pair of Dotε No, Concentration fpicoqramε. 1 2 3 4 5 6 7 8 9 The membrane waε εubjected to ultraviolet irradiation (UVP Mineral Light; UVP, San Gabriel, Calif.) for 3 minutes to fix the DNA to the surface of the membrane, then air dried. Next, the membrane was blocked in 0.2% casein/0.1% Tween 20 detergent in PBS for 1 hour, following which 1/5000 diluted avidin-alkaline phosphatase conjugate (Tropix, Inc.) in 0.2% casein in PBS was added. The membrane was then incubated for 30 minuteε, washed three timeε (for 5 minuteε each time) in 0.2% casein/0.1% Tween 20 detergent in PBS and washed once for five minutes in aqueous 0.1 M diethanolamine containing 1 mm magnesium chloride and 0.02% sodium azide, pH 10.C (substrate buffer). Next, the five columns of rows of dotε 1-5 were individually cut from the membrane ("εtrips 1-5") , aε were the five columns of rows of dots 6-10 ("stripε 6- 10") . Stripε 1-5 were waεhed with εubεtrate buffer for 30 minutes. Strips 6-10 were blocked in 0.1% BDMQ in substrate buffer for 30 minuteε. Both εets of stripε were then incubated for 5 minutes in substrate buffer, then individually incubated for five minuteε in aqueous solutionε (0.25 mM) of 1,2-dioxetanes aε indicated below: Strip No. 1.2-Dioxetane 1 AMPPD 2 OH-Adamant-2 '-ylidene (A isomer) 3 OH-Adamane-2 '-ylidene (B isomer) 4 Cl-Adamant-2 '-ylidene 5 Br-Adamant-2 *-ylidene 6 AMPPD 7 OH-Adamant-2 '-ylidene (A isomer) 8 OH-Adamant-2 '-ylidene (B isomer) 9 Cl-Adamant-2 '-ylidene 10 Br-Adamant-2 '-ylidene All strips were then placed in camera luminometers and exposed on Polaroid Type 612 instant black and white film. The improved chemilumineεcence intenεity obtained using the 3-(substituted adamant-2 '-ylidene)1,2- dioxetanes, as compared to AMPPD itself, can be seen from the results shown in Table II below. IΛDLE ALKALINE PIIQSEHΛTΛSE-LΛPE EP P Λ PRQPE PETEC o 1EHDRANE HU1LAHP WITHOUT PPMQ P ?cyi G STEP c/> c Detection (In plco- CD BDMQ limit gra s DNλ) ω MeiΦrane PlPXCtan JBlo ln SJLCB 5t P»lriι e> 1/ 1 l>. ej C 1 AMPPD no 2.500 5.000 H 2 OII-Λdamant-2* -ylldene (A isomer) no 5.000 10.000 m 3 OH-Adamant-2'--ylidene (B isomer) no 5.000 20.000 4 Cl-Adamant-2* ■ -ylidene no 1.250 1.250 I 5 Br-Adamant-2 • ■ -ylidene no 1.250 2.500 I m 6 AMPPD BDMQ 0.625 1.250 σ m 7 Oil-Adamant^ 1 - ylidene (A isomer) BDMQ 0.625 2.500 I H β OH-Adamant-2•- ylidene (B isomer) BDMQ 1.250 5.000 9 Cl-Adamant-2 1 - ylidene BDMQ 0.313 0.313 10 Br-Adamant-2'- ylidene BDMQ 0.313 0.313 1/ Five minute exposure waa performed 40 minutes after dioxetane addition. 2/ One minute exposure was performed 77 minutes after dioxetane addition. EXAMPLE XVI A strip of neutral BIODYNE A nylon membrane was dotted twice (side-by-side) with~l2.5 picograms of biotinylated pBR 322 35-mer oligonucleotide probe (Biogen, Inc., Cambridge, Mass.), dried, and subjected to ultraviolet radiation from an UVP Mineral Light lamp for 5 minutes to fix the DNA to the surface of the membrane. Next, the membrane was wetted with 5 x SSC (0.015 M sodium citrate/0.15 M εodium chloride) and blocked in 0.2% caεein, 0.1% Tween 20 detergent in PBS for 1 hour. The blocked membrane was then incubated with avidin- alkaline phosphatase conjugate ("Avidx" conjugate; Tropix, Inc.), diluted 1-10,000 in 0.2% casein, for 30 minutes. The membrane was then washed twice (for 5 minuteε each time) with aqueous 0.2% casein/0.1% Tween 20 detergent, then twice (for 5 minutes each time) with aqueous 0.1% Tween 20 detergent in PBS, and finally twice (for 5 minutes each time) in aqueous 0.1 M diethanolamine containing 1 mM magnesium chloride, pH 10 (assay buffer) . The washed membrane was cut in half to give two strips, each bearing one dot. The strips were incubated, respectively, in aqueous 0.25 mM solutions of AMPPD and the corresponding chloroadamant-2 '-ylidene compound in 0.1 M diethanolamine containing 1 mM magnesium chloride, pH 10 for five minuteε, then drained and sealed in plastic bags which were taped to the window of a Turner Model 20E luminometer. The chemiluminescence emission from each strip waε integrated for 20 hours. The kinetics of the light emissions obtained are shown in FIG. 12. EXAMPLE XVII. The enhancement of chemiluminescence emission (as compared to the emission from AMPPD) provided by the corresponding hydroxyadamant-2'-ylidene (A and B isomers) and chloroadamant-2'-ylidene compounds, all in the further presence of the enhancer polymers listed below, was demonstrated in the following manner. Four sets of three tubes each, each tube in each set containing 450 μl of a 0.4 mM aqueous solution of one of the four 1,2-dioxetanes being compared in 0.1 M diethanolamine containing 1 mM magnesium chloride, 0.02% sodium azide and 0.1% of the enhancer polymer, pH 10.0 (substrate buffer) were prepared and the background signal from each tube measured using a Berthold LB 952T luminometer (Berthold Instruments; ildbad, Federal Republic of Germany) . Next, 50 μl of an aqueous solution, 2.83 X 10 ~12 M, of alkaline phosphatase in 0.1 M diethanolamine containing l mM magnesium chloride, 0.02% sodium azide, pH 10.0, (final enzyme concentration 2.83 X 10 _13 M) was added to each tube and the chemiluminescent signals were measured in the luminometer at 5 and 20 minuteε. The intenεity of the chemiluminescent signals and the signal: background ratios obtained for each of the four 1,2-dioxetanes in the presence of the enhancer polymers are shown in Table III below. The enhancer polymers used, and the symbols for such polymers used in Table III, were the following: SYMBOL ENHANCER POLYMER SAPPHIRE -BDMQ TMQ poly(vinylbenzyltrimethylammonium chloride) S/TMQ styrene/TMQ copolymer DAA/TMQ diacetone acrylamide/TMQ copolymer DMQ/TEQ poly(vinylbenzyldodecyldimethylammonium chloride) /TEQ copolymer TEQ poly(vinylbenzyltriethylammonium chloride) TBQ poly(vinylbenzyltributylam onium chloride) MPB poly(vinylbenzyl-N-methylpiperidinum chloride) BAEDM poly[vinylbenzyl(2-benzoylamino)ethyl- dimethylammonium chloride] BZ benzal mordant DMEB poly(vinylbenzyldimethylethyl- ammonium chloride) DME(OH)B poly[vinylbenzyldi ethyl(2-hydroxy)eth- ylammonium chloride EMERALD sapphire and fluorescein TBQ/FLUOR TBQ and fluorescein SUBSTITUTE SHEET EXAMPLE XVIII The background signals and t% parameters (as compared to those of AMPPD) obtained for the substituted adamant-2'-ylidene compounds listed below in two different buffers were determined in the following manner. Aqueous 4 X 10 "4 M solutions of the 1,2-dioxetanes in a buffer solution made up of 0.05 M sodium carbonate/sodium bicarbonate containing 1 mM magnesium chloride, pH 9.5, were prepared, aε were aqueous 4 X 10 ~4 M solutionε of the 1,2-dioxetanes in a buffer solution made up of 0.1 M diethanolamine containing 1 mM magnesium chloride and 0.02% sodium azide, pH 10.0. One ml per tube of each of these solutions was placed in a Turner Model 20E luminometer and the backg r ound signals were measured. Next, t values were measured for each sample aε follows. One hundred μl of sample dioxetane in 900 μl of one of the above-described buffer solutions was pipetted into a tube (final dioxetane concentration 4 X 10 "5 M) and equilibrated at 30°C. Ten μl a 1-1,000 dilution of calf intestine alkaline phosphatase in the same buffer (enzyme concentration 7.6 X 10 "10 M) was then added, and the resulting chemiluminescent intensity was recorded, using a Turner Model 20E luminometer, over a 30 minute period. values were then calculated from the decay curves. The results of these determinations are given in Table IV below. TABLE IV 0.05 M Sodium Carbonate/Sodium Bicarbonate, l mM Magnesium Chloride, pH 9.5 DIOXETANE AMPPD A-OH-AMPPD B-OH-AMPPD Cl-AMPPD Br-AMPPD 2. 0.1 M Diethanolamine, 1 mM Magnesium Chloride, 0.02% Sodium Azide, pH 10.0 DIOXETANE AMPPD A-OH-AMPPD B-OH-AMPPD Cl-AMPPD Br-AMPPD 1.13 0.56 EXAMPLE XIX Alkaline phosphatase (calf intestine; Biozyme) was diluted 1-1,000,000 to generate a stock solution, concentration 2.54 X 10 "12 M. A series of tubes was prepared, in duplicate, containing 450 μl of an aqueous 0.1 M diethanolamine solution containing 1 mM magnesium chloride and 0.02% sodium azide, pH 10, pluε 0.1% of one of the enhancer polymers specified below and 4.4 x 10~ 4 M of a 1,2-dioxetane: AMPPD or the corresponding chloroadamant-2 '-ylidene compound. Fifty μl of alkaline phosphatase stock solution was then added to give samples containing the following enzyme concentrations: 2.54 X 10 ~12 M 8.49 X 10 "13 M 2.83 X 10 ~13 M 9.45 X 10 "14 M 3.15 X 10 "14 M 1.05 X 10~ 14 M 3.49 X 10 "15 M 1.16 X 10 "15 M 3.88 X 10 "16 M 1.29 X 10 "16 M 4.31 X 10 "17 M The final concentration of 1,2-dioxetane in each tube was 4 X 10 "4 M; the final concentration of enhancer polymer was 0.09%. Five second integrals were recorded at 5 and 20 minutes following 1,2-dioxetane addition. FIG. 13 show dose response curves for alkaline phosphatase dilution with the chloroadamant-2 '-ylidene 1,2-dioxetane with enhar.—: I and II at 5 minutes after dioxetane addition, aε compared to the dose response curves for AMPPD with the same enhancers exhibited as RLV vs [AP] and signal/noise (S/N vs [AP] . FIG. 14 shows alkaline phosphatase dilutions detected with the chloroadamant-2 '-ylidene 1,2-dioxetane plus BDMQ- fluorescein ("emerald I") and poly(vinylbenzyltributylammonium chloride) ("TBQ")- fluorescein (emerald II) , at 5 and 20 minutes after substrate, addition, as compared to AMPPD pluε the same enhancer polymers. EXAMPLE XX pBR 322 plasmid (Biogen, Inc., Cambridge, Maεε.) containing an inεert of TPA εequence waε digeεted with MSP1 reεtriction enzyme. Chemical cleavageε were performed as described in Maxam, et al., PNAS, 74, 560 (1977) to yield G, AG, AC, TC and C - and one other, T, as described by Rubin, et al.. Nucleic Acids Research, 8 . , 4613 (1980) . One seventh of each reaction tube's contents was loaded per lane onto 0.4 mm TBE-gradient sequencing gel (60 cm in length) . After 4 hours of electrophoresiε, DNA was electrotransferred to a BIODYNE A nylon membrane (0.45 μm) and treated with ultraviolet light to fix the DNA to the membrane'ε εurface. Next, the membrane was dried, prehybridized for 30 minutes at 45°C in aqueouε buffer solution containing 1% BSA, 0.5 M sodium phosphate and 7% SDS, pH 7.2, then hybridized for 2 hours at 45°C with 10 μl of NNB snap direct alkaline phosphatase conjugated probe (Molecular Biosyεtems, Inc., San Diego, California) in 40 ml of the above-described BSA buffer solution. The membrane was then washed twice in aqueous 5 X SSC/1% SDS (for 5 minutes each time) at 45°C, twice in aqueous 1 X SSC/1% SDS (for 5 minutes each time) at 45°C, once in an aqueouε solution containing 125 mM εodium chloride, 50 mM Triε and 1% Triton X-100 detergent, pH 8.0, twice in aqueous IX SSC (for 1 minute each time) at room temperature, and finally twice (for one minute each time) at room temperature in an aqueous solution"containing 0.1 M diethanolamine, 1 mM magnesium chloride and 0.02% sodium azide at pH 10.0. The membrane waε then wetted with aqueous AMPPD solution (0.25 mM) , wrapped in Saran wrap, and exposed to Kodak XAR X-ray film for 40 minutes. A five minute exposure waε then taken 1 hour after AMPPD addition. The sequence images are shown in FIG. 17(1). Repeating this entire procedure using the corresponding chloroadamant-2 '-ylidene 1,2-dioxetane compound gave the sequence images shown in FIG. 17(2). EXAMPLE XXI The thermal background of each dioxetane aε specified in Table 4 was measured in a solution of 0.4 mM dioxetane in 0.1 M diethanolamine, 1 mM MgCl 2 , pH 10.0 at 30°C in a Turner Model 20E luminometer (Sunnyvale, CA) . The average chemiluminescence intensity of each sample in the absence of an enzyme is listed under "Background at 0.4 mM in Turner Light Units (TLU) in Table l. The half-life of the dioxetane anion alεo listed in Table l was measured as follows: A 1 ml solution of 0.04 mM dioxetane (in the same buffer aε above) was completely dephosphorylated by the addition of 0.764 picomoles of alkaline phosphatase at 30°C in a Turner Model 20E luminometer. The half-life of the dioxetane anion was then calculated from the first order decay of the chemiluminescent emisεion. TABLE 4 COMPARISON OF HALF-LIVES -AND THERMAL BACKGROUNDS OF VARIOUS DIOXETANE PHOSPHATES TLU = Turner Light Unitε * Anion produced by the addition of 0.764 picomoleε of alkaline phosphatase to 40 nanomoles of AMPPD and R- AMPPD in 0.1 M DEA, 1 mM MgCl 2 , pH 10.0. EXAMPLE XXII The detection meanε for alkaline phosphatase with different chemiluminescent 1,2-dioxetane substrates were determined as follows: Duplicates of serial dilutions (1 to 3) of alkaline phosphatase were incubated at room temperature with 0.4 mM dioxetane in C.IM diethanolamine, 1 mM MgCl 2 , pH 10.0, containing l mg/ml of either of the polymeric enhancerε Enhancer 1 or Enhancer 2 , in a Berthold LB952T luminometer (Wildbad, Germany) SUBSTITUTESHEET ...Following either a 5 or a 20 minute incubation with a substrate, the chemiluminescence intensity was measured as a 5 second integrated Relative Light Units (RLU) and plotted as in Figure 10. The alkaline phosphatase concentration, which resulted in a. chemiluminescent signal twice the signal obtained without the enzyme, was extrapolated from the graph shown in Figure 18. The • alkaline phosphatase concentrations at twice background were used aε the detection limits n Table 5. TABLE 5 DETECTION LIMITS FOR ALKALINE PHOSPHATASE WITH DIFFERENT CHEMILUMINESCENT 1,2-DIOXETANE SUBSTRATE/ENHANCER SYSTEMS ENHANCER 1 ENHANCER 2 DIOXETANE 5 MIN 20 MIN 5 MIN 20 MIN AMPPD 4.0 1.4 3.5 1.1 HO-AMPPD 3.0 0.8 1.0 0.3 Cl-AMPPD 1.0 0.6 0.8 0.4 Br-AMPPD 2.6 0.5 0.4 0.3 Detection limits expressed in femtomoles/liter Duplicates of serial dilutions (1 to 2) cf alkaline phosphatase were incubated at room temperature with 0.4 M AMPPD cr Cl-AMPPD in 0.1 M diethanolamine, l mM MgCl 2 , SUBSTITUTESHEET pH 10.0, containing 1 mg/ml of sither; of the polymeric enhancers. Enhancer 1 or Enhancer 2, in a Berthold LB952T luminometer. After a 5 minute incubation, the chemiluminescent intensities were measured as a 5 second integrated Relative Light Unit (RLU) and the duplicates were averaged and plotted vs alkaline phosphatase concentration in the upper graph of Figure 18. The lower graph shows the ratio of the chemiluminescent signal to the background (no enzyme) chemiluminescent signal as a function of the enzyme concentration. EXAMPLE XXIV The chemiluminescent signal to noise levels were obtained with 2.83 x 10 ~13 M alkaline phosphataεe and various R-substituted adamantyl 1,2-dioxetane phosphateε in the presence of several enhancerε. All measurements were performed in a Berthold LB952T luminometer at room temperature. First, the background chemiluminescence levels (signal in the absence of the enzyme) of each sample (in triplicate) were measured. Each sample consisted of 0.2 μmoleε of dioxetane in 0.45 ml of 0.1 M diethanolamine, ImM MgCl 2 , pH 10.0, without or with l mg/ml of the indicated enhancer. Tubes were inserted into the luminometer and the luminescence intensity was measured. Next, the tubes were removed from the luminometer and alkaline phosphatase (50 μl containing 1.415 x 10" 16 moles) was added to each tube, and the luminescence intensity was measured at 5 and 20 minutes after substrate addition. The final concentration of dioxetane in each tube was 0.4 mM. Table 6 showε the ratio of the chemilumineεcent εignal obtained in the preεence of alkaline phoεphataεe at 5 ana 20 minuteε to the background εignal (obtained in the absence of the enzyme) . TABLE 6 COMPARISON OF CHEMILUMINESCENT SIGNAL-TO-NOΣ8E LEVELS OF VARIOUS R-SUBSTITUTED AMPPD COMPOUNDS All enhancers used at 1 mg/ml in 0.1 M DEA, pH 10. Alkaline phosphatase concentration = 2.83 x 10" i3 M Measurements performed in a Berthold LB952T Luminometer. E.XAMPLE XXV Table 7 shows the detection limits for alkaline phosphatase detection with the R-substituted dioxetanes determined using procedures deεcribed in Table 5, Example XXII, with the exception of an inεtrument which waε utilized to obtain the chemiluminescence intenεity readings. The luminometer used in this example was SUBSTITUTESHEET -84 - LabSystems Luminoskan microtiter plate reader. TABLE 7 ALKALINE PHOSPHATASE DETECTION LIMITS WITH CHaEMILUMINESCENCE (AT 21 BACKGROUND) 5 MINUTE INCUBATION 20 MINUTE INCUBATION 1) Buffer: 0.1 M diethanolamine, 1 mM MgCl 2 , 0.02% sodium azide, pH 10.0. 2) Signal recorder in a Labsystems Luminoskan microtiter plate reader. SUBSTITUTESHξET -85- EXAMPLE XXVI Table 8 shows the 20 minute incubation data from Table 4, plus measurements performed in a Wilj microtiter plate luminometer. T.ABLE 8 ALKALINE PHOSPHATASE DETECTION LIMITS WITH CHEMILUMINESCENCE (AT 2X BACKGROUND) 20 MINUTE INCUBATION, LABSYSTEMS 23 MINUTE INCUBATION, WILJ HO-AMPPD-A 1 . 5 x 10 ' •15 M 1 . 4 X 10~ 15 M SUBSTITUTE SHEET E.XAMPLE XXVII The performance of AMPPD and Cl-AMPPD on a nylon membrane was compared as follows: 12.5 picograms of biotinylated-pBR322-(35 mer) in 1 μL was spotted onto dry nylon membrane and crosslinked to the membrane with UV light. The membrane subsequently wetted with 1XSSC buffer, incubated with 0.2%- casein, 0.1% Tween-20 in phosphate buffered saline (PBS) for 1 hour at room temperature, washed 4 times with 0.3% Tween-20 in PBS, washed twice in Substrate Buffer (0.1 M diethanolamine, 1 mM MgCl 2 , pH 10.0), and incubated for 5 minutes in 0.25 mM dioxetane in Substrate Buffer. In this fashion, two membranes: one incubated with AMPPD and the εecond with Cl-AMPPD, were generated. The membraneε were then εealed in plastic, attached to the outside of a 12x75mm glasε test tube, and inserted into Turner Model 20-E luminometer equipped with a side-mounted photomultiplier detector and thermally equilibrated to 30°C. The chemiluminescence intensity was then monitored for 20-24 hourε. Aε shown in Figure 19, the chemilumineεcence signal was plotted as a function of time for both AMPPD and Cl-AMPPD. EXAMPLE XXVIII The half-times to the maximum chemiluminescence intensity obtained with AMPPD and Cl-AMPPD on PVDF and nylon membranes were calculated from data collected as described in Example XXVII, for the nylon membrane. For PVDF membrane, the revised protocol included the εecond Substrate buffer waεh followed by the membrane incubation for 5 minuteε at room temperature in the Tropix NitroBlock™ reagent and a εubsequent wash with Substrate Buffer prior to the dioxetane εubεtrateε addition. The half-times to the maximum chemiluminescent signal were calculated from the a plot of the logarithm (maximum chemiluminescent signal minus signal at each time point) as a function of time and is shown in Table 9. TABLE 9 CHEMILUMINESCENT DETECTION OF BIOTINYLATED DNA ON MEMBRANES - HALF-TIME TO MAXIMUM CHEMILUMINESCENCE - * PVDF membrane was treated with NitroBlock prior to addition of substrate. 12.5 pg of biotinylated pBR322-35oer was detected with z Avidix-AP and 0.24 M dioxetane in 0.1 M DEA, pH 10.0. EXAMPLE XXIX The detection of pBR322 plasmid DNA with a biotinylated pBR322 DNA probe using AMPPD and Cl-AMPPD on PVDF and nylon membranes was performed in the following fashion: pBR322 DNA was denatured by boiling, serially diluted samples in 1XSSC buffer (final mass of DNA per slot is indicated in the Figure 20) , applied to ImmobilonP PVDF membrane, Pall Biodyne A nylon membrane, and Amersham Hybond N nylon membrane using a Schleicher SUBSTITUTESHEET and Schuell slot blot apparatus. The DNA was crosslinked to the Biodyne A and Hybond N membranes with UV light. Immobilon P membrane was first blocked and then UV treated. The membranes were then wejtted with 1XSSC buffer, prehybridized at 65°C in hybridization buffer (IM NaCl, 0.2% heparin, 0.5% polyvinyl pyrrolidone, 4% sodium dodecylsulfate, 1 mM ethylenediamineletracetic acid, 5% dextran sulfate, 50 mM Tris-HCl, pH 7.5), and hybridized at 65°C with 12 ng/ml pBR322 probe (biotinylated using a nick translation procedure) in the same buffer, washed once at 65°C in the same buffer minus dextran sulfate, EDTA and tepahn, washed twice for 10 minuteε in 1XSSC/1% SDS at 75°C, twice for 15 minuteε in 0.1XSSC/1% SDS at 75°C, twice for 5 minuteε in 1XSSC at room temperature, blocked for 1 hour in 0.2% caεein, 0.11% Tween -20 in PBS, washed once for 5 minutes in 0.2% casein in PBS, incubated for 30 minutes in a 1-15,000 dilution of streptavidin labeled alkaline phosphatase in 0.2% casein/PBS, then washed 6 times with 0.2% casein, Tween -20 in PBS, waεhed twice in Subεtrate Buffer (0.1 M diethanolamine, 1 mM MgCl 2 , pH 10.0), and then incubated for 5 minutes in 0.25 mM AMPPD and Cl-AMPPD (two separate membranes) in Substrate Buffer. Prior to substrates addition, the PVDF membrane was incubated for 5 minutes in NitroBlock*, and washed twice with Substrate Buffer. The membranes were then wrapped in plastic, and exposed to Polaroid Type 612 film at the times indicated after substrates addition as shown in Figure 20. EXAMPLE XXX The detection of pBR322 plasmid DNA with a biotinylated pBR322 DNA probe using AMPPD, Cl-AMPPD, and Br-AMPPD on PVDF, nylon, and nitrocelluloεe membraneε waε performed uεing protocolε εi ilar to thoεe described in Example XXIX, with one exception in the case of PVDF and nitrocellulose membranes to which the target DNA was fixed by baking the membranes at 80°C for 2 hourε. Also, prior to dioxetane addition, both PVDF and nitrocellulose membranes were treated with Nitroblock. The Polaroid Type 612 images of the chemiluminescent signal from the decomposition of various dioxetane phosphateε catalyzed by the alkaline phosphatase DNA probe conjugates on different membrane supports is shown in Figure 21. EXAMPLE XXXI The kinetics of alkaline phosphatase dependent light emission from AMPPD and Cl-AMPPD on nitrocellulose membranes were compared according to the following procedure: pBR322-(35 mer) which had been previously 3 '-end labeled with biotin-11-dUTP (1 μL containing 12.5 pg) was spotted onto dry nitrocellulosemembrane. The membrane was then wetted with 1XSSC buffer, incubated with 0.2% casein, 0.1% Tween-20 in phosphate buffered saline (PBS) for 1 hour at room temperature, washed 4 times with 0.3% Tween-20 in PBS, washed twice in Substrate Buffer (0.1 M diethanolamine, 1 mM MgCl 2 , pH 10.0), incubated for 5 minutes with NitroBlock™ reagent, washed twice in Substrate Buffer, and subsequently two pieces of the membrane were incubated for 5 minutes each in 0.25 mM AMPPD and Cl-AMPPD in Substrate Buffer. The membranes were then sealed in plastic, attached to the outside of a 12x75mm glass test tube, and inserted into Turner Model 20-E luminometer thermally equilibrated to 30°C equipped with a side photomultiplier detector. The chemiluminescence intensity was monitored for 20-24 hours. As shown in Figure 22, the chemiluminescence intensity signal waε plotted as a function of time for both AMPPD and Cl-AMPPD. EXAMPLE XXXII The half-time to the maximum chemiluminescence signal intensity for a membrane based protein, IgG, detection with alkaline phosphatase conjugated goat anti-mouse antibodies and AMPPD and CL-AMPPD was evaluated using the following protocol. One μl of l μg/ml solution of purified mouse IgG in 20% methanol electrophoresiε transfer buffer was spotted onto dry nitrocellulose and wet PVDF membranes. The membranes were subsequently rinsed in 0.1% Tween-20/PBS, blocked for 1 hour in blocking buffer (0.2% casein, 0.1% Tween-20 in PBS), incubated with a 1-10,000 dilution of alkaline phosphatase conjugated goat anti-mouse antibody in blocking buffer, washed twice for 5 minutes in blocking buffer, washed twice for 5 minutes in 0.1% Tween-20/PBS, washed twice for 5 minutes in substrate buffer (0.1 M diethanolamine, 1 mM MgCl 2 , pH 10.0), separated into two examples and incubated for 5 minutes each in 0.25 mM AMPPD and Cl-AMPPD. The membranes were sealed in plastic, attached to the outside of a 12 x 75 mM glasε tube and inserted into Turner Model 20E luminometer, equipped with a side photomultiplier detector and thermally equilibrated to 30°C. As shown in Table 10, the chemiluminescence intensities were monitored for 8 to 12 hours, and the half-life to maximum signal were calculated as in Example XXVIII. TABLE 10 CHEMILUMINESCENT DETECTION OF MOUSE IgG ON MEMBRANES - HALF-TIME TO MAXIMUM CH.EMILUMINESCENCE - * PVDF membrane was treated with NitroBlock prior to addition of substrate. 1 ng of mouse was detected with goat anti-mouse-AP and 0.24 mM dioxetane in 0.1 M DEA, pH 10.0. EXAMPLE XXXIII Western blotting analysis was performed for the detection of human transferrin on a nylon membrane with chemiluminescence, Serial dilutions of purified human transferrin of 0-5,1,2,4,8,16,32,64,128, and 256 nanograms were separated by SDS polyacrylamide gel electrophoresis and electrophoretically transferred to Pall Biodyne A nylon membrane. The membranes were subsequently washed with phosphate buffered saline (PBS) , blocked for 30 minutes with 0.3% casein in PBS, incubated with a 1-1000 dilution of mouse anti-human transferrin in 0.3% Tween-20/PBS, washed four times for 5 minutes with 0.3% Tween-20/PBS, incubated with a 1-10,000 dilution of alkaline phosphatase labeled goat anti-mouse antibody in 0.3% Tween-20/PBS, washed four times for 5 minutes in 0.3% Tween-20/PBS, washed twice in Substrate Buffer (0.1 SUBSTITUTESHEET M diethanolamine, 1 mM MgCl 2 , pH 10.0), incubated for 5 minutes each in 0.25 mM AMPPD, Cl-AMPPD and Br-AMPPD in Substrate Buffer, wrapped in plastic, and then exposed to Polaroid type 612 instant black and white film for 5 minutes. The results, are shown in 30. EXAMPLE XXXIV The chemiluminescent detection of human tranεferrin on Immobilon-P PVDF membrane was performed according to the protocol described in Example 13, except for the following changes: Immediately prior to dioxetanes addition, two of the four PVDF membranes were incubated for 5 minutes in NitroBlock"' and then waεhed twice in Subεtrate Buffer. 5 minute expoεures for AMPPD and Cl-AMPPD without and with NitroBlock are εhown in Figure 23. E.XAMPLE XXXV Chemiluminescent detection of murine interleukin-4 gene using direct alkaline phoεphataεe labeled oligonucleotide probeε with AMPPD, Cl-AMPPD, Br-AMPPD and LumiPhos 530. A plasmid containing the gene or the murine interleukin-4 (MIL-4) gene was serially diluted in 1XSSC and spotted onto dry Biodyne A membrane at 12.8, 6.4, 3.2, 1.6, .8, .4, .2, .1, .05, .025, .0125, .0063, and .00315 nanograms of DNA. The DNA waε UV fixed to the membrane and subsequently wetted with 1XSSC, prehybridized in hybridization buffer (7% SDS, 1% casein, 0.25 M Na P0 4 , pH 7.2 with phoεphoric acid) for 30 minuteε at 50°C, hybridized with 5 nM alkaline phoεphataεe labeled oligonucleotide probe for 2 hours at 50°C, waεhed aε follows: twice for 5 minutes in 2XSSC, 1%SDS at room temperature; twice for 15 minutes in IX SSC, 1% SDS at 50°C, twice for 5 minutes in 1 XSSC, 1% Triton X-100 at room temperature; twice for 5 minutes in IXSSC; and twice for 5 minutes in Substrate Buffer (0.1 M diethanolamine, 1 mM MgCl 2 , pH 10). Separate samples of membranes were then incubated for 5 minutes each in 0-25 mM AMPPD, Cl-AMPPD Br-AMPPD and LumiPhos 530 (Boehringer Mannheim, Indianapolis, USA), wrapped in plastic, and exposed to Kodak XAR-5 x-ray film for 1 hour after a 1 hour incubation. The resultε of this experiment are shown in Figure 24. EXAMPLE XXXVI Comparison of the intensities of chemiluminescent signal intensitieε obtained with Cl-AMPPD and AMPPD were studied as shown in Figure 9. Sequencing reactions were performed using M13mpl8 DNA with a 5'-biotinylated universal primer with BioRad BST polymerase. Two sets of reactions were εeparated on a 7.67 M urea ionic-gradient polyacrylamide gel and then tranεferred to nylon membrane by pasεive capillary tranεfer. The DNA waε croεεlinked to the membrane by UV irradiation for 5 minutes. The membrane was then incubated at room temperature in blocking buffer (0.2% casein, 0.1% Tween-20 in PBS) for 30 minutes, for 30 minutes with a 1-5000 dilution of streptavidin labeled alkaline phosphatase in 0.2% casein, PBS, washed 5 minutes in blocking buffer, washed twice in 0.3% Tween-20 in PBS, and washed for l minute in substrate buffer (0.1 M diethanolamine, 1 mM MgCl 2 , pH 10) . The membrane was divided into two pieceε and incubated for 5 minutes in 0.25 mM dioxetane (one strip each for AMPPD and Cl-AMPPD) . The membranes were then sealed in plastic and exposed to Kodak XAR-5 x-ray film for 7 minutes beginning 5 minuteε after εubεtrate addition aε shown in Figure 25. EXAMPLE XXXVII Comparison of DNA band resolution using CSPD and CL-AMPPD was performed using sequencing reactions, transfers and chemiluminescent detections were performed under the same conditions as thoεe deεcribed in Example XXXVI uεing AMPPD and Cl-AMPPD. The expoεure εhown in Figure 26 is a 1 minute exposure 24 hours after εubεtrate addition. EXAMPLE XXXVIII Detection of pBR322 plasmid DNA with a biotinylated pBR322 DNA probe uεing AMPPD, Cl-AMPPD and BR-AMPPD on nitrocelluloεe membrane. pBR322 plasmid DNA was serially diluted and spoiled at 512, 256, 128, 64, 32, 16, 8, 4, 2, and 1 picogra of DNA onto 3 εtripε of dry nitrocellulose membrane. The membrane stripε were then baked for 2 hours at 80°C and treated with UV light for 5 minutes, wetted with IXSSC, prehybridized for 60 minuteε in hybridization buffer (IM NacL, 0.2% heparin, 0.5% polyvinylpyrrolidone, 40% sodium dodecylεulfite, l mM ethylenediamine tetracetic acid, 5% dextran sulfate, 50 mM Tris-HCl, pH 7.5) at 651, hybridized overnight with 15 ng/ l pBR322 probe biotinylated by nick translation, washed for 5 minutes in hybridization buffer minus heparin, dextransulfate and EDTA at 65°C, washed twice for 5 minutes with IXSSC/1% SDS at 75°C, twice for 15 minutes with 0.01XSSC/1% SDS at 75°C and once for 5 minutes with IXSSC at room temperature. The membranes were then processed for chemiluminescent detection at room temperature by rinsing twice with 0.2% casein in PBS, blocked for 1 hour with blocking buffer (0.20% casein, 0.1% Tween-20 in PBS), washing for 5 minutes with 0.21% casein in PBS, then for 5 minutes with 0.2% casein in PBS, incubating the membranes for 30 minutes with a 1-15,000 dilution of streptavidin labeled alkaline phosphatase in 0.2% casein/PBS, washing twice for 5 minutes in NitroBlock solution, then waεhing four timeε for 5 minutes in 0.3% Tween-20/PBS, then twice for 5 minutes in substrate buffer (0.1 M diethanolamine, 1 mM MgCl 2 , pH 10) , and incubating one strip in each of the three dioxetane phosphateε: AMPPD, Cl-AMPPD and Br-AMPPD at 0.25 mM dioxetane concentration. Timed expoεures on Polaroid 612 film after various incubation times are shown in Figure 27. EXAMPLE XXXIX Detection of pBR322 DNA with a biotinylated pBR322 DNA probe with streptavidin alkaline phosphatase conjugate and chemiluminescent dioxetane phosphateε, AMPPD, Cl-AMPPD and Br-AMPPD on neutral nylon membrane were performed using a protocol described in Example XXXVIII, except that the 5 minute NitroBlock incubation was omitted. The results are shown in Figure 28. EXAMPLE XL The chemiluminescent detection of human transferrin was performed on Immobilon-P PVDF membrane. The gel electrophoresis, transfer, blocking, and detection steps were performed according to the protocol described in Example XXIV, except that all of the PVDF membranes were incubated for 5 minutes in NitroBlock™ reagent. Figure 29 showε the reεultε of the compariεon of AMPPD, Cl-AMPPD, and Br-AMPPD in the detection cf human tranεferrin on PVDF membraneε. E3CAMPLE XL The thermal background of each dioxetane aε specified in Table 11 was measured in a solution of 0.4 mM dioxetane in 0.1 M diethanolamine, 1 mM MgCl 2 , pH 10.0 at 30°C in a Turner Model 20E lumindmeter (Sunnyvale, CA) . The average chemiluminescence intensity of each sample in the absence of an enzyme is listed "Non- Specific Background" at 0.4 mM in Relative Luminometer Units (TLU) in Table 11. The half-life of the dioxetane anion also listed in Table 1 was measured as follows: A 1 ml solution of 0.04 mM dioxetane (in the same buffer as above) was completely dephosphorylated by the addition of 0.764 picomoles of alkaline phosphatase at 30°C in a Turner Model 20E luminometer. The half-life of the dioxetane anion was then calculated from the first order decay of the chemiluminescent emission. TABLE 11 HALF-LIVES AND NON-SPECIFIC BACKGROUND EMISSION OF DIOXETANES AT 31.5° C The tuDβ contained 2.52 x 10 " 1 3 M ai aime onosDπatase in 0.1 M αiβinanoiamine. 1 mM MgC!2 , C.02% soαium az ce. c-* "l O. The above discussion of thiε invention iε directed primarily to preferred embodiments and practices thereof, It will be readily apparent to thoεe εkilled in the art that further 10 changeε and odificationε in the actual implementation of the concepts deεcribed herein can eaεily be made without departing from the εpirit and εcope of the invention as defined by the following claimε.

Claims: Claims 1. An enzymatically cleavable chemilumineεcent 1,2- dioxetane compound capable of producing light energy when decomposed, substantially stable at room ^ temperature before a bond by which an enzymatically cleavable labile subεtituent thereof iε intentionally cleaved, represented by the formula: wherein: X and X 1 each represent, individually, hydrogen, a hydroxyl group, a halo substituent, an -0(CH 2 ) n CH 3 group where n=0-6, a hydroxy (lower) alkyl group, a halo (lower) alkyl group, a hydroxyalkoxy group, a phenyl group, a halophenyl group, an alkoxyphenyl group, a hydroxalkoxy group, a cyano group, a carboxyl or substituted carboxyl group or an amide group, with at least one of X and X 1 being other than hydorgen; and R j^ and R 2 , individually or together, represent an organic subεtituent that does not interfere with the production of light when the dioxetane compound is enzymatically cleaved and that satisfies the valence of the dioxetane compound's 4-carbon atom, with the provisos that if R and R 2 represent individual SUBSTITUTESHEET substituents the R 2 substituent is aromatic, heteroaromatic, or an unsaturated substituent in conjugation with an aromatic ring, and that at least one of and R 2 is, or R and R 2 taken together are, an enzymatically cleavable labile group- substituted fluorescent chromophore group that becomes luminescent when the enzymatically cleavable labile substituent thereof is removed by an enzyme. 2. A compound of claim 1 wherein X iε hydroxyl and X 1 is hydrogen. 3. A compound of claim 1 wherein X iε chloro and X 1 is hydrogen. 4. A compound of claim 1 wherein X is bromo and X 1 is hydrogen. 5. A compound of any one of claims 2 - 4, inclusive, wherein R. is methoxy. 6. A compound of claim 5 wherein R-, is a meta phosphate- εubεtituted phenoxy group. 7. A compound of claim 6 wherein the phosphate substituent is present as the disodium salt. 8. A compound of claim 5 wherein R 2 is a meta β -D- galactoside-substituted phenoxy group. 9. Disodium 3-(4-methoxyspiro[l,2-dioxetane-3,2'-(5'- hydroxy)trιcyclo[3.3.1.1 3'7 ]decan]-4-yl)phenyl phosphate. SUBSTITUTESHEET 10. Disodium 3-(4-methoxyspiro[l,2-dioxetane-3,2*-(5'- chloro)tricyclo[3.3.1.1 3'7 ]decan]-4-yl)phen l phosphate. 11. Disodium 3-(4-methoxyspiro[l,2-dioxetane-3,2'-(5'- bromo)tricyclo[3.3.1.1 3'7 ]decan]-4-yl)phenyl phosphate. 12. Syn-disodium 3-(4-methoxyspiro[l,2-dioxetane-3,2'-(5'- hyroxy)tricyclo[3.3.1.1 3'7 ]decan]-4-ylphenyl phosphate. 13. Syn-disodium 3-(4-methoxyspiro[l,2-dioxetane-3,2'-(5'- chloro)tricyclo[3.3.1.1 3 ' 7 ]decan]-4-:yl)phenyl phosphate. 14. Syn-disodium 3-(4-methoxyspiro[l,2-dioxetane-3,2'-(5'- bromo)-tricyclo[3.3.l.l 3 ' 7 ]decan]-4-yl)phenyl phosphate. 15. Anti-disodium 3-(4-methoxyspiro[l,2-dioxetane-3,2'- (5 ' -hydroxy) tπcyclo[3.3.1.1 3 '7 ] decan] -4-yl phenyl phoεphate. 16. Anti-diεodium 3-(4-methoxyεpiro[l, 2-dioxetane-3 , 2 ' - (5 '-chloro) tπcyclo[3.3. l.l J 3 | 7 ] decan] -4-yl) phenyl phoεphate. 17. Anti-disodium 3-(4-methoxyεpiro[l,2-dioxetane-3 ,2 '- (5 '-bromo) tricyclo[3.3.1. I 3 ' 7 ] decan] -4-yl) phenyl phoεphate. 18. .An enol ether of the formula: SUBSTITUTE SHEET wherein : X and X 1 each represent, individually, hydrogen, a hydroxyl group, a halo substituent, an unsubstituted lower alkyl group, a hydroxy (lower) alkyl group, a halo (lower) alkyl group, a phenyl group, a halophenyl group, an alkoxyphenyl group, a hydroxyalkoxy group, a cyano group, a carboxyl or subεtituted carboxyl group or an amide group, with at leaεt one of X and X 1 being other than hydrogen; Y represents a lower alkyl group; and Y' represents hydrogen, a lower alkyl group, a lower alkenyl group, an aralkyl group having up to 20 carbon atoms, an alkali metal cation an acyl group having from two to about 14 carbon atomε, inclusive, an alkali metal cation, where M represents, independently, a proton, an alkali metal cation, ammonium, substituted ammonium, quaternary ammonium or an H + pyridinium cation. 19. A compound of claim 18 wherein X is hydroxyl and X 1 is hydrogen. 20. A compound of claim 18 wherein X is chloro and X 1 iε hydrogen. 21. A compound of claim 18 wherein X is bromo and X 1 is hydrogen. 22. A compound of any one of claims 19-21, inclusive, wherein Y is methyl. . - 23. A compound of claim 22 wherein Y 1 iε 24. A compound of claim 23 wherein each M is sodium. 25. Disodium 3-(methoxy-5- hydroxytricyclo[3.3.1.1 3 ' 7 ]dec-2-ylidenemethy1)phenyl phosphate. 26. Disodium 3-(methoxy-5- chlorotricyclo[3.3.1.1 3,7 ]dec-2-ylidenemethy1)phenyl phosphate. 27. Disodium 3-(Methoxy-5- bro otricyclo[3.3.1.1 3 ' 7 )dec-2-ylidene ethyl)phenyl phosphate. 28. In an assay method in which a member of a specific binding pair is detected by means of an optically detectable reaction, the improvement wherein said optically detectable reaction includes the reaction, with an enzyme, of an enzymatically cleavable chemiluminescent 1,2-dioxetane compound capable of producing light energy when decomposed, substantially εtable at room temperature before a bond by which an enzymatically cleavable labile εubstituent thereof is intentionally cleaved, represent"ed-by the formula: wherein: X and X 1 each represent, individually, hydrogen, a hydroxyl group, a halo substituent, an unsubstituted lower alkyl group, a hydroxy (lower) alkyl group, a halo (lower) alkyl group, a phenyl group, a halophenyl group, an alkoxyphenyl group, a hydroxyalkoxy group, a cyano group or an amide group, with at least one of X and X 1 being other than hydrogen; and R and R 2 , individually or together, represent an organic substituent that does not interfere with the production of light when the dioxetane compound is enzymatically cleaved and that satisfies the valence of the dioxetane compound's 4-carbon atom, with the provisos that if R 1 and R 2 represent individual substituents the R 2 substituent is aromatic, heteroaromatic, or an unsaturated substituent in conjugation with an aromatic ring, and that at least one of R-_ and R 2 is, or R 1 and R 2 taken together are, an enzymatically cleavable labile group-subεtituted fluorescent chromophore group that produces a lu ineεcent εubεtance when the enzymatically cleavable labile substituent thereof is removed by an enzyme, so that said enzyme cleaves said enzymatically cleavable labile substituent to form a negatively charged substituent bonded to said 1,2-dioxetane compound, said negatively charged subεtituent causing said 1,2-dioxetane compound to decompose to form a luminescent substance comprising said fluorescent chromophore group. 29. An assay method of claim 28 wherein X is hydroxyl and X 1 is hydrogen. 30. An assay method of claim 28 wherein X is chloro and X 1 is hydrogen. 31. An assay method of claim 28 wherein X is bromo and X 1 iε hydrogen. 32. An assay method of any one of claimε 29 - 31, inclusive, wherein R 1 iε methoxy. 33. An aεsay method of claim 32 wherein R 2 is a meta phosphate-substituted phenoxy group. 34. An assay method of claim 33 wherein the phosphate substituent is present as the disodium salt. 35. An assay method of claim 32 wherein R 2 is a meta 0-D-galactoside-substituted phenoxy group. 36. An assay method of claim 28 wherein the 1,2- dioxetane compound is disodium 3-(4-methoxyspiro[l, 2- dioxetane-3 , 2 '-(5*-hydroxy)tricyclo[3.3.1.1 3 ' 7 ]decan]-4- yl)phenyl phosphate. 37. An asεay method of claim 28 wherein the 1,2- dioxetane compound is disodium 3-(4-methoxyspiro[l,2- dioxetane-3,2 '-(5'-chloro)tricyclo[3.3.1.1 3 ' 7 ]decan]-4- yl)phenyl phosphate. 38. An assay method of claim 28 wherein the 1,2- dioxetane compound is disodium 3-(4-methoxyεpiro[l,2- dioxetane-3,2 '-(5'-bromo)tricyclo[3.3.1.1 3 ' 7 ]decan]-4- yl)phenyl phosphate. 39. An assay method of claim 28 wherein said specific binding pair comprises an antigen and an antibody. 40. An assay method of claim 28 wherein said specific binding pair comprises a nucleic acid and a probe capable of binding to all or a portion of said nucleic acid. 41. An asεay method of claim 28 wherein said specific binding pair comprises an enzyme and a 1,2- dioxetane compound containing a group cleavable by said enzyme. 42. An assay method of claim 41 wherein said enzyme-cleavable group comprises a galactopyranoside, and said enzyme comprises a galactosidase. 43. An assay method of claim 40 wherein the nucleic acid is DNA, RNA or fragment thereof. 44. An asεay method of claim 40 wherein the probe iε a labeled oligonucleotide complementary to the nucleic acid. 45. An aεsay method of claim 44 wherein the oligonucleotide probe is biotinylated. 46. An assay method of claim 44 wherein the DNA, RNA or fragment thereof is produced b a sequencing protocol. 47. An assay method of claim 46 further comprising the steps of (a) contacting the DNA, RNA or fragment thereof with a labeled complementary oligonucleotide probe to form a hybridizing pair, (b) contacting the hybridized pair with a molecule capable of strong binding to the label of the oligonucleotide covalently conjugated with an enzyme capable of cleaving an enzy atically- cleavable 1,2-dioxetane to releaεe light energy, (c) adding εuch a 1,2-dioxetane substrate, and (d) detecting the light produced. 48. An assay method of claim 47 wherein the oligonucleotide label is biotin or a biotin derivative. 49. An assay method of claim 47 wherein the molecule capable of strong interaction with the label of the oligonucleotide iε avidin or streptavidin. 50. An assay of method claim 47 wherein the enzyme is an acid or alkaline phosphatase, χ is methoxy, and R 2 is a meta phosphate-substituted phenoxy group. 51. An assay method of claim 47 wherein the enzyme is a galactosidase, R λ is methoxy, and R 2 is a meta β-O- galactoside-substituted phenoxy group. 52. An assay method of claim 47 wherein light energy is detected by light-sensitive film. 53. An assay method of claim 47 wherein light energy is detected by a photoelectric cell. 54. An assay method of claim 44 wherein said oligonucleotide probe is covalently labeled with an enzyme capable of decomposing said 1,2-dioxetane to emit light energy. 55. An assay method of claim 44 wherein said label on said oligonucleotide probe comprises a covalently bound antigen that is immunochemically bound to an antibody-enzyme conjugate, wherein said antibody is directed to εaid antigen and εaid enzyme iε capable of decompoεing said 1,2-dioxetane compound to emit light energy. 56. An assay method of either claim 54 or 55 wherein said enzyme is an acid or alkaline phoεphataεe, Ra j _ is methoxy, and R 2 is a meta phosphate-εubεtituted phenoxy group. 57. An assay method of either claim 54 or 55 wherein said enzyme is a galactosidase, R 2 is methoxy, and R 2 is a meta 3-D-galactoside-substituted phenoxy grou . 58. An assay method of any one of claims 40, 44, 54 or 55 wherein the binding of said probe to said nucleic acid iε carried out on a nylon membrane. 59. An assay method of any one of claims 47 - 53, inclusive, wherein the hybridizing between said DNA, RNA or fragment thereof and said labeled oligonucleotide probe is conducted on a nylon membrane. 60. An assay method of claim-28, conducted using a solid matrix, wherein nonspecific binding to said matrix is blocked by pretreating said matrix with a polymeric quaternary ammonium salt. 61. An assay method of claim 28, carried out in the further presence of a water-soluble enhancing subεtance that increases specific light energy production above that produced in its absence. 62. An assay method of claim 61, wherein said water-soluble enchancing subεtance iε serum albumin. 63. An assay method of claim 61, wherein said enhancing substance is a polymeric quaternary ammonium salt. 64. An assay method of claim 63, wherein said polymeric quaternary ammonium salt is poly(vinylbenzyltrimethylammonium chloride) , poly[vinylbenzyl(benzyldimethylammonium chloride)] or poly[vinyl(benzyltributylammonium chloride) ] . 65. An assay method of claim 61, wherein said enhancing substance comprises a positively charged polymeric quaternary ammonium salt and fluorescein capable of forming a ternary complex with the negatively charged product of said 1,2-dioxetane compound produced following enzyme-catalyzed decomposition of said 1,2- dioxetane compound, whereby energy transfer occurs between said negatively charged product and fluoreεcein and light energy iε emitted by fluoreεcein. 66. An aεεay method of claim 65, wherein εaid polymeric quaternary ammonium εalt is poly(vinylbenzyltrimethylammonium chloride) , poly[vinylbenzyl(benzyldimethylammonium chloride)] or poly[vinyl(benzyltributylammonium -chloride) ] . 67. A kit for detecting a firεt substance in a sample, comprising an enzymatically cleavable chemiluminescent 1,2-dioxetane compound capable of producing light energy when decomposed, substantially stable at room temperature before a bond by which an enzymatically cleavable labile subεtituent thereof is intentionally cleaved, represented by the formula: - wherein: X and X 1 each represent, individually, hydrogen, a hydroxyl group, a halo substituent, an unsubstituted lower alkyl group, a hydroxy (lower) alkyl group, a halo (lower) alkyl group, a phenyl group, a halophenyl group, an alkoxyphenyl group, a hydroxyalkoxy group, a cyano group or an amide group, with at least one of X and X 1 being other than hydrogen; and R λ and R 2 , individually or together, represent an organic substituent that does not interfere with the production of light when the dioxetane compound is enzymatically cleaved and that satisfies the valence of the dioxetane compound's 4-carbon atom, with the provisos that if and R 2 represent individual substituents the R 2 substituent is aromatic, heteroaromatic, or an unsaturated substituent in conjugation with an aromatic ring, and that at leaεt one of R χ and R 2 is, or R λ and R 2 taken together are, an enzymatically cleavable labile group-subεtituted fluoreεcent chromophore group that produceε a lumineεcent εubstance when the enzymatically cleavable labile substituent thereof is removed by an enzyme; and an enzyme capable of cleaving said enzymatically cleavable labile group of said 1,2- dioxetane compound. 68. The kit of claim 67, wherein R 1 is methoxy, R 2 iε a meta phosphate-subεtituted phenoxy group, and said enzyme is acid or alkaline phosphatase. 69. The kit of claim 67, wherein ± is methoxy, R 2 is a /3-D-galactoside-subεtituted phenoxy group, and εaid enzyme is a galactosidaεe. 70. The kit of any one of claimε 67 - 69, inclusive, further comprising image-reproducing meanε for detecting said light energy. 71. The kit of claim 70, wherein said image- reproducing means is photographic film. 72. A kit for detecting a nucleic acid or fragment thereof in a sample by hybridization of said nucleic acid or fragment to a complementary labeled oligonucleotide probe, comprising an enzymatically cleavable chemiluminescent 1,2-dioxetane compound capable of producing light energy when decomposed, substantially stable at room temperature before a bond by which an enzymatically cleavable labile subεtituent thereof is intentionally cleaved, represented by the formula: wherein: X and X 1 each represent, individually, hydrogen, a hydroxyl group, a halo substituent, an unsubstituted lower alkyl group, a hydroxy (lower) alkyl group, a halo (lower) alkyl group, a phenyl group, a halophenyl group, an alkoxyphenyl group, a hydroxyalkoxy group, a cyano group, a carboxyl or subεtituted carboxyl group or an amide group, with at least one of X and X 1 being other than hydrogen; and 2 and R 2 , individually or together, represent an organic substituent that does not interfere with the production of light when the dioxetane compound is enzymatically cleaved and that satisfieε the valence of the dioxetane compound's 4-carbon atom, with the provisos that if R 1 and R 2 represent individual εubεtituentε the R 2 εubεtituent iε aromatic, heteroaromatic, or an unεaturated substituent in conjugation with an aromatic ring, and that at leaεt one of R, and R 2 iε, or -_ and R 2 taken together are, an enzymatically cleavable labile group-substituted fluorescent chromophore group that produces a luminescent substance when the enzymatically cleavable labile substituent thereof is removed by an enzyme, a covalently enzyme-labeled oligonucleotide probe; and, a nylon membrane upon which εaid nucleic acid-oligonucleotide probe hybridization is conducted. 72. The kit of claim 72 wnerein Rl iε methoxy, R 2 iε a meta pnoεphate-substituted phenoxy group, and said enzyme is acid or alkaline phosphatase. C 74. The kit of claim 72 wherein R : is methoxy, R i a meta β-D-galactoside-substituted phenoxy group, and said enzyme is a galactosidase. 75. The kit of any one of claims 72 - 74, incluεive, further compriεing image-reproducing meanε for 5 detecting said light energy. 76. The kit of claim 75 wherein εaid image- reproducing means is photographic film. 77. A kit for detecting a nucleic acid or fragment thereof in a sample by hybridization of said nucleic aci 0 or fragment to a complementary labeled oligonucleotide probe, comprising an enzymatically cleavable chemiluminescent 1,2-dioxetane compound capable of producing light energy when decomposed, substantially stable at room temperature before a bond by which an 5 enzymatically cleavable labile substituent thereof is intentionally cleaved, represented by the formula: wherein: X and X 1 each represent, individually, hydrogen, a hydroxyl group, a halo substituent, an unsubstituted lower alkyl group, a hydroxy (lower) alkyl group, a halo (lower) alkyl group, a phenyl group, a halophenyl group, an alkoxyphenyl group, a hydroxyalkoxy group, a cyano group or an amide group, with at least one of X and X 1 being other than hydrogen; and R± and R 2 , individually or together, repreεent an organic substituent that does not interfere with the production of light when the dioxetane compound is enzymatically cleaved and that satisfies the valence of the dioxetane compound's 4-carbon atom, with the provisoε that if R 2 and R 2 represent individual substituents the R 2 substituent is aromatic, heteroaromatic, or an unsaturated substituent in conjugation with an aromatic ring, and that at leaεt one of : and R 2 is, or x and R 2 taken together are, an enzymatically cleavable labile group-εubstituted fluorescent chromophore group that produces a luminescent substance when the enzymatically cleavable labile subεtituent thereof is removed by an enzyme, a complementary oligonucleotide probe covalently labeled with biotin or a biotin derivative; avidin or streptavidin covalently bound to an enzyme capable of decomposing said 1,2-dioxetane compound to emit light energy; and, a nylon membrane upon which said nucleic acid or fragment thereof is hybridized to said oligonucleotide probe. 78. The kit of claim 77 wherein R ; is methoxy, R 2 is a meta phosphate-εubεtituted phenoxy group, and said enzyme is acid or alkaline phosphatase. 79. The kit of claim 77, wherein R x is methoxy, R 2 is a meta ,9-D-galactoside-substituted phenoxy group, and said enzyme is a galactosidase. 80. The kit of any one of claims 77 - 79, inclusive, further comprising image-reproducing meanε for detecting εaid light energy. 81. The kit of claim 80 wherein said image- reproducing meanε iε photographic film. 82. A kit for detecting a nucleic acid or fragment thereof in a sample by hybridization of εaid nucleic acid or fragment to a complementary labeled oligonucleotide probe, compriεing an enzymatically cleavable chemiluminescent 1,2-dioxetane compound capable of producing light energy when decomposed, substantially stable at room temperature before a bond by which an enzymatically cleavable labile substituent thereof is intentionally cleaved, represented by the formula: wherein: X and X 1 each represent, iri ividually, hydrogen, a hydroxyl group, a halo substituent, an unsubstituted lower alkyl group, a hydroxy (lower) alkyl group, a halo (lower) alkyl group, a phenyl group, a halophenyl group, an alkoxyphenyl group, a hydroxyalkoxy group, a cyano group, a carboxyl or substituted carboxyl group or an amide group, with at least one of X and X 1 being other than hydrogen; and Ri and R 2 , individually or together, represent an organic εubstituent that does not interfere with the production of light when the dioxetane compound iε enzymatically cleaved and that satisfies the valence of the dioxetane compound's 4-carbon atom, with the provisos that if R 1 and R 2 represent individual substituentε the R 2 substituent is aromatic, heteroaromatic, or an unsaturated substituent in conjugation with an aromatic ring, and that at least one of R 1 and R 2 iε, or R χ and R 2 taken together are, an enzymatically cleavable labile group-substituted fluorescent chromophore group that produces a luminescent subεtance when the enzymatically cleavable labile substituent thereof is removed by an enzyme a complementary oligonucleotide probe covalently labeled with an antigen; an antibody directed to said antigen covalently bound to an enzyme capable of decomposing said 1,2-dioxetane compound to emit light energy; and, a nylon membrane upon which said nucleic acid or fragment thereof is hybridized to said oligonucleotide probe. 83. The kit of claim 82 wherein said enzyme is an acid or alkaline phoεphatase, R 1 is methoxy, and R 2 is a meta phosphate-substituted phenoxy group. 84. The kit of claim 82 wherein said enzyme is a galactosidase, R is methoxy, and R 2 is a meta β-O- galactoside-substituted phenoxy group. 85. The kit of any one of claims 82 - 84, inclusive, further comprising image-reproducing means for detecting said light energy. 86. The kit of claim 85 wherein εaid image- reproducing meanε is photographic film. 87. A kit for detecting a protein in a εample, comprising an enzymatically cleavable chemiluminescent 1,2-dioxetane compound capable of producing light energy when decomposed, subεtantially εtable at room temperature before a bond by which an enzymatically cleavable labile substituent thereof is intentionally cleaved, represented by the formula: wherein: X and X x each represent, individually, hydrogen, a hydroxyl group, a halo substituent, an unsubεtituted lower alkyl group, a hydroxy (lower) alkyl group, a halo (lower) alkyl group, a phenyl group, a halophenyl group, an alkoxyphenyl group, a hydroxyalkoxy group, a cyano group, a carboxyl or substituted carboxyl group or an amide group, with at least one of X and X 1 being other than hydrogen; and R λ and R 2 , individually or together, represent an organic substituent that does not interfere with the production of light when the dioxetane compound is enzymatically cleaved and that satisfieε the valence of the dioxetane compound's 4-carbon atom, with the provisos that if R 1 and R 2 represent individual substituents the R 2 substituent is aromatic, heteroaromatic, or an unsaturated substituent in conjugation with an aromatic ring, and that at leaεt one of R 1 and R 2 is, or R± and R 2 taken together are, an enzymatically cleavable labile group-subεtituted fluoreεcent chromophore group that produceε a luminescent substance when the enzymatically cleavable labile substituent thereof is removed by an enzyme, an antibody directed to said protein covalently bound to an enzyme capable of decomposing said 1,2- dioxetane compound to emit light energy; and, a membrane upon which protein-antibody binding is conducted. 88. The kit of claim 87 wherein said membrane is a nylon, nitrocellulose membrane or PVDF membrane. 89. The kit of claim 87 wherein R is methoxy, R 2 is a meta phosphate-substituted phenoxy group, and said enzyme is acid or alkaline phosphatase. 90. The kit of claim 87 wherein R λ is methoxy, R 2 is a meta )S-D-galactoside-subεtituted phenoxy group, and εaid enzyme is a galactosidase. 91. The kit of any one of claims 87 - 90, inclusive, further compriεing image-reprcducing means for detecting said light energy. 92. The kit of claim 91 wherein said image reproducing means is photographic film. 93. A kit for detecting a protein in a sample comprising an enzymatically cleavable chemiluminescent 1,2-dioxetane compound capable of producing light energy when decomposed, substantially stable at room temperature before a bond by which an enzymatically cleavable labile substituent thereof is intentionally cleaved, represented by the formula: wherein: X and X 1 each represent, individually, hydrogen, a hydroxyl group, a halo substituent, an unsubstituted lower alkyl group, a hydroxy (lower) alkyl group, a halo (lower) alkyl group, a phenyl group, a halophenyl group, an alkoxyphenyl group, a hydroxyalkoxyphenyl group, a cyano group, a carboxyl or substituted carboxyl group or an amide group, with at least one of X and X 1 being other than hydrogen; and R and R 2 individually or together, represent an organic εubεtituent that does not interfere with the production of light when the dioxetane compound is enzymatically cleaved and that satisfies the valence of the dioxetane compound's 4-carbon atom, with the provisos that if R λ and R 2 represent individual substituents the R 2 substituent is aromatic, heteroaromatic, or an unsaturated subεtituent in conjugation with an aromatic ring, and that at leaεt one of R 1 and R 2 iε, or R 1 and R 2 taken together are, an enzymatically cleavable labile group-εubstituted fluorescent chromophore group that produceε a luminescent εubεtance when the enzymatically cleavable labile εubεtituent thereof iε removed by an enzyme, a first antibody directed to said protein; and, a second antibody directed to said first antibody covalently bound to an enzyme capable of decomposing said 1,2-dioxetane compound. 94. The kit of claim 93 wherein R 1 is methoxy, R 2 is a meta phosphate-substituted phenoxy group, and εaid enzyme* is acid or alkaline phosphatase. 95. The kit of claim 93 wherein R 2 iε methoxy, R 2 iε a meta 0-D-galactoside-substituted phenoxy group, and said enzyme is a galactosidase. 96. The kit of any one of claims 67, 72, 77, 82, 87 or 93, further comprising a water-soluble enhancing substance that increases specific light energy production above that produced in its absence. 97. The kit of claim 96 wherein said water-soluble enhancing substance is serum albumin. 98. The kit of claim 96 wherein said enhancing substance is a polymeric quaternary ammonium εalt. 99. The kit of claim 97 wherein said polymeric quaternary ammonium salt iε poly(vinylbenzyltrimethylammonium chloride) , poly[vinylbenzyl(benzyldimethylammonium chloride)] or poly[vinylbenzyl(tributylammoniumchloride) ] . 100. The kit of claim 96 wherein"said enhancing subεtance comprises a positively charged polymeric quaternary ammonium salt and fluorescein capable of forming a ternary complex with the negatively charged product of said 1,2-dioxetane compound produced following enzyme-catalyzed decomposition cf said 1,2-dioxetane compound, whereby energy tranεfer occurε between εaid negatively charged product and fluoreεcein and light energy is emitted by fluoreεcein. 101. The kit cf claim 100 wherein said polymeric quaternary ammonium salt iε poly(vinylbenzyltrimethylammonium chloride) poly[vinylbenzyl(benzyldimethylammonium chloride)] or poly[vinylbenzyl(tributylammoniumchloride) . 102. A method of detecting an enzyme in a sample comprising the steps of: (a) providing an enzymatically cleavable chemiluminescent 1,2-dioxetane compound capable of producing light energy when decomposed, substantially stable at room temperature before a bond by which an enzymatically cleavable labile εubεtituent thereof is intentionally cleaved, represented by the formula: wherein: X and X 1 each represent, individually, hydrogen, a hydroxyl group, a halo substituent, an unsubstituted lower alkyl group, a hydroxy (lower) alkyl group, a halo (lower) alkyl group, a phenyl group, a halophenyl group, an alkoxyphenyl group, a hydroxyalkoxy group, a cyano group, a carboxyl or substituted carboxyl group or an amide group, with at least one of X and X 1 being other than hydrogen; and R 1 and R 2 , individually or together, represent an organic substituent that does not interfere with the production of light when the dioxetane compound is enzymatically cleaved and that satisfies the valence of the dioxetane compound's 4-carbon atom, with the provisos that if R ± and R 2 represent individual substituents the R 2 subεtituent iε aromatic, heteroaromatic, or an unsaturated substituent in conjugation with an aromatic ring, and that at least one of R λ and R 2 iε, or R λ and R 2 taken together are, an enzymatically cleavable labile group-εubstituted, fluorescent chromophore group that produces a lumineεcent subεtance when the enzymatically cleavable labile substituent thereof is removed by an enzyme, (b) contacting said 1,2-dioxetane compound with εaid εample containing εaid enzyme; whereupon said enzyme cleaves said enzymatically cleavable labile substituent from said 1,2-dioxetane compound to form a negatively charged subεtituent bonded to said 1,2-dioxetane compound, εaid negatively charged substituent causing said 1,2-dioxetane compound to decompoεe to form a lumineεcent εubstance comprising said fluorescent chromophore group; and (c) detecting said lumineεcent εubstance aε an indication of the presence of εaid enzyme. 103. A method of claim 102- wherein R ] _ iε methoxy, R 2 iε a meta phoεphate-subεtituted phenoxy group, and said enzyme is acid or alkaline phosphatase. 104. A method of claim 102 wherein R λ iε methoxy, R 2 iε a meta 3-D-galactoside-substituted phenoxy group, and εaid enzyme iε a galactosidase. 105. An enzymatically cleavable chemilumineεcent 1,2-dioxetane compound capable of producing light energy when decomposed, substantially stable at room temperature before a bond by which an enzymatically cleavable labile εubstituent thereof is intentionally cleaved, represented by the formula: wherein x and R 2 , individually or together, represent an organic substituent that does not interfere with the production of light when the dioxetane compound is enzymatically cleaved and that satisfieε the valence of the dioxetane compound'ε 4-carbon atom, with the proviεoε that if R-_ and R 2 repreεent individual εubεtituentε the R 2 substituent is aromatic, heteroaromatic, or an unsaturated substituent in conjugation with an aromatic ring, and that at leaεt one of R and R 2 iε, or R-, and R 2 taken together are, an enzymatically cleavable labile group-substituted fluorescent chromophore group that becomes luminescent when the enzymatically cleavable labile substituent thereof is removed by an enzyme. wherein Z is an enzyme-cleavable group, the oxygen on the phenyl ring is ortho, meta or para, and Q iε hydrogen, aryl, substituted aryl, aralkyl, heteroary, heteroalkyl of up to 20 carbon atoms, an allyl group, a hydroxy (lower) alkyl, a lower alkyl -OSiR 3 (where R 3 iε lower alkyl, aryl, alkoxy C 1 _ 6 , alkoxyalkyl of 12 or fewer carbon atoms, hydroxy (lower) alkyl, amino (lower) alkyl), -OR 4 or -SR 4 (wherein R 4 is alkenyl, subεtituted (lower) alkenyl, (lower) alkyl or aralkyl of up to 20 carbon atoms) ,-S0 2 R 5 (wherein R 5 is methyl, phenyl or NHC 6 H 5 ) , subεtituted or unεubεtituted (lower) alkyl, nitro, cyano, a halogen, hydroxy, carboxy, trimethylsilyl or phosphoryloxy group. 107. The assaying method Claim 60 wherein said assay comprises pretreating a nitrocellulose membrane with BDMQ and TBQ. 108. The assaying method Claim 60 wherein said assay comprises pretreating a PVDj- ' membrane with BDMQ and TBQ.

Inventors: BRONSTEIN IRENA, EDWARDS BROOKS, JUO ROUH-RONG, BRONSTEIN, IRENA, EDWARDS, BROOKS, JUO, ROUH-RONG
Applicants: TROPIX INC, TROPIX, INC.
Application year: 1991
Main classification: C07D321
IPC codes: A61K39, C07C43, C07D321, C07F9, C09K11, C12Q1, G01N21, G01N33