Chemiluminescent 1,2-dioxetanes

BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention pertains to chemiluminescent 1,2-dioxetane derivatives which can be enzymatically activated to decompose and, through decomposition, release light. The dioxetanes are particularly characterized by the presence of an aromatic (phenyl or naphthyl) ring bonded to the dioxetane, which ring bears a meta-substituted or disjoint enzymatically cleavable group, which when cleaved, leaves the phenoxyanion or naphthyloxyanion of the dioxetane, and, at the four or preferably the five position, an electron donating or electron withdrawing group. By selecting the identity of the substituent at the four or five position (the Z moiety) particular aspects of the chemiluminescent properties of the dioxetane, including half life, quantum yield, SIN ratio, etc., can be altered.
2. Background of the Invention
1,2-dioxetane enzyme substrates have been well established as highly efficient chemiluminescent reporter molecules for use in enzyme immunoassays of a wide variety of types. These assays provide a preferred alternative to conventional assays that rely on radioisotopes, fluorophores, complicated color shifting, secondary reactions and the like. Dioxetanes developed for this purpose include those disclosed in U.S. Pat. No. 4,978,614 as well as U.S. Pat. No. 5,112,960. U.S. Pat. No. 4,978,614 discloses, among others, 3-(2'-spiroadamantane)4-methoxy-4-(3"-phosphoryloxy)phenyl-1,2-dioxetane, which has received world-wide attention, and is commercially available under the trade name AMPPD. U.S. Pat. No. 5,112,960, discloses similar compounds, wherein the adamantyl stabilizing ring is substituted, at either bridgehead position, with a variety of substituents, including hydroxy, halogen, and the like, which convert the otherwise static or passive adamantyl stabilizing group into an active group involved in the kinetics of decomposition of the dioxetane ring. Compounds of this type have similarly received international attention, giving a faster and stronger signal than AMPPD in many applications. CSPD corresponds to AMPPD with a chlorine substituent on the adamantyl group, and, like AMPPD, is available from Tropix, Inc. of Bedford, Mass.
Compounds of this type have been particularly developed for enhanced sensitivity in assays for the presence of analytes in concentrations as low as 10.sup.-12 M and lower. In certain applications, compounds of this type are used in conjunction with enhancers to detect analytes in concentration of 10.sup.-12 M or lower. These enhancement agents, which include natural and synthetic water-soluble macromolecules, are disclosed in detail in U.S. Pat. No. 5,145,772. Preferred enhancement agents include water-soluble polymeric quaternary ammonium salts, such as poly(vinylbenzyltrimethylammonium chloride) (TMQ), poly(vinylbenzyltributylammonium chloride) (TBQ) and poly(vinylbenzyldimethylbenzylammonium chloride) (BDMQ).
These enhancement agents improve the chemiluminescent signal of the dioxetane reporter molecules, apparently by providing a hydrophobic environment in which the dioxetane is sequestered. Water, an unavoidable aspect of most assays, due to the use of body fluids, is a natural "quencher" of the dioxetane chemiluminescence. The enhancement molecules apparently exclude water from the microenvironment in which the dioxetane molecules, or at least the excited state emitter species reside, resulting in enhanced chemiluminescence. Other effects associated with the enhancer-dioxetane interaction could also contribute to the chemiluminescence enhancement.
Additional advantages can be secured by the use of selected membranes, including nylon membranes and treated nitrocellulose, providing a similarly hydrophobic surface for membrane-based assays, and other membranes coated with the enhancer-type polymers described.
Nonetheless, it remains a general goal of the industry to improve the performance of these stabilized, chemiluminescent dioxetane reporter molecules, to improve the machine readability, sensitivity, and performance aspects of the immunoassays, dependent on the chemiluminescent signal released by the dioxetanes.
By way of background, and as disclosed in all the patents referenced above, the enzymatically-activated dioxetanes are used as reporter molecules, as substrates for enzymes which cleave the enzyme-labile group bonded to an aromatic substituent on the dioxetane ring. Thus, the enzyme, e.g., alkaline phosphatase is covalently linked or otherwise complexed with either an antigen or antibody, in conventional antigen/antibody ligand binding assays, or a nucleic acid probe in nucleic acid assays. The enzyme-bearing antigen or antibody, or nucleic acid probe, is then admixed with the analyte suspected of containing the target antigen, or nucleic acid sequence, under conditions which permit complexing or hybridization between the antigen/antibody or probe/nucleic acid sequence. After washing away or separating off all noncomplexed or nonhybridized material, the dioxetane substrate is added. If the suspected analyte is present, the enzyme will cleave the enzyme-labile group on the aromatic substituent on the dioxetane, e.g., phenyl or naphthyl, yielding the phenoxy or naphthyloxy anion intermediate. This anion decomposes, by electron transfer through the aromatic ring, cleaving the dioxetane ring, and yielding two carbonyl-based products. The cleavage/decomposition event is the light-releasing event.
To automate clinical assays, and to provide for substantial throughput, continued reductions in the halflife, or T.sub.1/2 of the dioxetane, as well as a reduction in the amount of time required to reach the maximum emission of light of the reporter molecule, is desirable. At the same time, to detect analytes in extremely low concentrations, below, e.g., about 10.sup.-12 M, it is desirable to improve the intensity of the signal of the dioxetane reporter molecule, and simultaneously desirable to avoid increasing the background noise due to nonenzymatically-induced light release, so as to improve the overall sensitivity of the assay. Thus, further improvements in chemiluminescent dioxetane reporter molecules are sought.
SUMMARY OF THE INVENTION
The above goals, and others, are met by a new class of dioxetanes, particularly characterized by a substituent on the aromatic ring bonded to the dioxetane, in addition to the meta-substituted enzyme-labile group. Thus, the novel dioxetanes of this invention have the generalized structure I, II or III below. ##STR1## wherein R is C1-12 alkyl, aralkyl, or aryl, preferably C1-6 alkyl, X is an enzyme labile group cleavable by a specific enzyme which recognizes that group to leave the phenoxy or naphthoxy anion, and is preferably a phosphate or galactoside, Y.sup.1 and Y.sup.2 are independently hydrogen, or an electron donating or withdrawing group, and are preferably hydrogen, methoxy, carboxy or halogen, and most preferably one of Y.sup.1 and Y.sup.2 is hydrogen while the other is chlorine, and Z is an electron-active group, most preferably chlorine, alkoxy, alkyl or amido. When Z is on a phenyl ring, Z is in the four or five position, preferably the five position. When OX and Z are substituted on a naphthyl group, OX is substituted such that the substitution is disjoint, that is the total number of ring atoms between the point of attachment to the dioxetane ring and the point of substitution, including the point of attachment and substitution, is an odd number, as disclosed in U.S. Pat. No. 4,952,707. Substituent Z may be substituted on the naphthyl ring at any position other than those adjacent the one position, or the point of attachment to the dioxetane ring.
By selecting the particular identity of Z, as an electron-withdrawing or an electron-donating group, specific characteristics of the chemiluminescent behavior of the dioxetane, including its T.sub.1/2, time to maximum emission, maximum emission wavelength, and chemiluminescent signal intensity can be affected.
DETAILED DESCRIPTION OF THE INVENTION
The dioxetanes of this invention are critically characterized by the substituents on the aromatic ring attached to the dioxetanes, which ring determines the electron transfer in the aryloxy anion, leading to decomposition and chemiluminescence. Thus, dioxetanes of the invention have the following and generalized structure (I). ##STR2##
Thus, the adamantyl-stabilized dioxetanes of the claimed invention bear two substituents on the phenyl ring, as well as 0, 1 or 2 non-hydrogen substituents on the adamantyl ring. These substituents critically characterize the electronic characteristics of the dioxetane, the oxyanion, and its decomposition behavior. The identities of each substituent are set forth below.
R may be alkyl, aralkyl, cycloalkyl, or aryl, having 1-12 carbon atoms. R is preferably C1-C3 alkyl, most preferably, methyl. The identity of R may be optimized with regard to solubility concerns, where unusual analytes, or buffers, may pose particular problems. Each of Y.sup.1 and Y.sup.2 represent, individually, hydrogen, a hydroxyl group, a halo substituent, a hydroxy lower alkyl group, a halo lower alkyl group, a phenyl group, a halophenyl group, an alkoxy phenyl group, an alkoxy phenoxy group, a hydroxyalkoxy group, a cyano group, an amide group, a carboxyl group or substituted carboxyl group, an alkoxy group and other similar electron-active species. Preferred identities for Y.sup.1 and Y.sup.2 are chlorine, hydroxy, and methoxy.
X is an enzyme-cleavable moiety. Thus, upon proper contact with a suitable enzyme, X is cleaved from the molecule, leaving the oxygen attached to the phenyl ring, and thus, the phenoxy anion. X is ideally phosphate, galactoside, acetate, 1-phospho-2,3-diacylglyceride, 1-thio-D-glucoside, adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, adenosine, .alpha.-D-glucoside, .beta.-D-glucoside, .beta.-D- glucuronide, .alpha.-D-mannoside, .beta.-D-mannoside, .beta.-D-fructofuranoside, .beta.-glucosiduronate, P-toluenesulfonyl-L-arginine ester, and P-toluenesulfonyl-L-arginine amide. X is preferably phosphate or galactoside, most preferably phosphate. It is important to note that when substituted on the phenyl ring, OX is meta with respect to the point of attachment to the dioxetane ring, that is, it occupies the three position.
Z may occupy either the four or five position, most preferably the five position. Z is an electron-active substituent, the character of the electron-active species (electron-donating or electron-withdrawing), optimizing various aspects of the dioxetane moiety. As an example, an electron-donating group, such as a methoxy group, may enhance the dioxetane phenoxy anion decomposition process, by facilitating the transferability of the free electrons from the aromatic ring O.sup.-- donor group, to the dioxetane ring. In contrast, an electron-withdrawing group would reduce or impair the ability to transfer the free electrons to the dioxetane, thus slowing the decomposition reaction and light emission, although ultimately giving a light signal of greater intensity. This should be contrasted with the impact of the electron-withdrawing substituent on the adamantyl group, such as chlorine, which substantially accelerates light emission, sharply reducing T.sub.1/2. Of surprising significance is the fact that substitution in the six position is particularly undesirable. Such six-substituted phenyl dioxetanes exhibit extraordinarily fast decomposition kinetics, and nearly no light emission. While Applicants do not wish to be restricted to this theory, it is believed that this behavior is due to steric considerations, that is, the ortho substituent "turns" the phenyl ring such that it destabilizes the dioxetane ring (destabilization through steric forces, not electron transfer) and a substituent at the six position, e.g., methoxy, does not participate in electron transfer. As discussed below, experiments involving 6-substituted phenyl dioxetanes give essentially no signal.
The phenyl substituent on the dioxetane ring may instead be naphthyl (structures II and III) as ##STR3## In the naphthyl dioxetane, identities for R, Y.sup.1 and Y.sup.2, X and Z remain the same. Instead of being restricted to the "meta" position, OX may occupy corresponding positions in the naphthyl ring, that is, non-conjugated positions, or positions such that the number of carbon atoms between the point of substitution and the point of attachment to the dioxetane ring, including the carbons at both point of attachment and point of substitution, are odd, as set forth in U.S. Pat. No. 4,952,707. Phenyl meta-substituted dioxetanes, and naphthyl dioxetanes substituted according to the pattern described above, may generally be expected to give higher quantum yields than the corresponding para and conjugated systems.
As noted above, Z can be any electron-active substituent that does not interfere with the chemiluminescent behavior of the dioxetane, and thus can be selected from a wide variety of identities. Preferred electron-active substituents include chloro, alkoxy (--OR), aryloxy (--OAr), trialkylammonium (--NR.sub.3 +), alkylamido (--NHCOR, --NRCOR'), arylamido (--NHCOAr, --NRCOAr, --NArCOAr), arylcarbamoyl (--NHCOOAr, --NRCOOAr), alkylcarbamoyl (--NHCOOR, --NRCOOR'), cyano (--CN), nitro (--NO.sub.2), ester (--COOR, --COOAr), alkyl- or arylsulfonamido (--NHSO.sub.2 R, --NHSO.sub.2 Ar), trifluoromethyl (--CF.sub.3), aryl (--Ar), alkyl (--R), trialkyl-, triaryl-, or alkylarylsilyl (--SiR.sub.3, SiAr.sub.3, --SiArR.sub.2), alkyl- or arylamidosulfonyl (--SO.sub.2 NHCOR, --SO.sub.2 NHCOAr), alkyl or aryl sulfonyl (--SO.sub.2 R, SO.sub.2 Ar) alkyl- or arylthioethers (--SR, SAr). The size of the Z substituent is generally limited only by solubility concerns. Where reference is made to alkyl or R, R', etc., the alkyl moiety should have 1-12 carbon atoms. Suitable aryl moieties include phenyl and naphthyl as exemplary moieties. Particularly preferred species include chloro and alkoxy.
Dioxetanes of the type described above, without the inclusion of the Z substituent, as previously noted, are disclosed in patents commonly assigned herewith. Patents addressing dioxetanes of this type without the inclusion of the Y and Z substituents have also been assigned to Wayne State University, such as U.S. Pat. No. 4,962,192. Substitution of the Z substituent on the dioxetanes required development of the synthesis of trisubstituted phenyl phosphonates which is described below, under the title Novel Tri-substituted Phenyl 1,2-Dioxetane Phosphates. The same general synthesis route can be employed for naphthyl dioxetanes embraced herein, bearing in mind the substitution patterns required, as discussed above. The synthesis of these compounds through the route described below involves the preparation of novel tri-substituted benzenes. Thus, as described below, an exemplary compound involved in the synthesis of the dioxetanes of this class includes 3-chloro-5-methoxybenzaldehyde. These tri-substituted compounds constitute key intermediates in a variety of synthetic pathways, the 1,3,5 substitution pattern being a generally preferred and widely applicable pattern. It is Applicants' belief that these intermediates have never previously been prepared, and are marked, in the synthesis route described below, with an asterisk.
NOVEL TRI-SUBSTITUTED PHENYL 1,2-DIOXETANE PHOSPHATES
__________________________________________________________________________Synthesis__________________________________________________________________________General. Commercial reagents were used as obtained without furtherpurification. Baker silica gels (60-200 mesh for gram scale, and 230-400meshreported in parts per million relative to a phosphoric acid standard.Highresolution meass spectral analyses were run by J. L. Kachinski at JohnsHopkinsUniversity. Synthesis of dioxetanes 3 and 4 were carried out followingtheprocedure described below for dioxetanes 1 and 2 respectively. Yields,meltingpoints (uncorrected) and spectral data are summarized for isolatedintermediates. ##STR4##3-Chloro-5-methoxy-4-trifluoromethanesulfonyloxy benzaldehyde (5). Asolutionof 5-Cl-vanillin.sup.1 (13.0 g, 70 mmol), chloroform (4 ml) and pyridine(16 ml) wasstirred at 0.degree. C. Addition of trifluoromethanesulfonic anhydride(12.4 ml, 75 mmol)at 0.degree. C. over 30 min gave clean formation of the triflate. Thereaction mixture waspartitioned between EtOAc and 3N HCl, washed with dilute brine, driedoverNa.sub.2 SO.sub.4, and evaporated under reduced pressure. Purification ofthe resultingyellow oil by silica gel chromatography (30% EtOAc/hexanes) yielded 18.5(83%) triflate 5 as yellow crystals.IR (CHCl.sub.3, cm.sup.-1): 1705, 1590, 1461, 1425, 1225, 1205, 1132,1049, 875, 624.sup.1 H NMR (ppm): 3.99(3H, s), 7.44(1H, d, J=1.6Hz), 7.57(1H, d,J=1.7Hz),9.92(1H, s) ##STR5##3-Chloro-5-methoxybenzaldehyde (6). Triflate 5 (9 g, 28 mmol),palladium(II)acetate (120 mg, 0.5 mmol), 1,1'-bis(diphenylphosphino)ferrocene (620mg,1 mmol) and hplc grade CH.sub.3 CN (10 ml) were mixed well in ateflon-linedstainless steel bomb. After adding freshly made, pulverized protonspongeformate.sup.2 (7.84 g, 30 mmol), the bomb was sealed and heated at90.degree. C. for 4 h.The cooled reaction was then filtered to remove proton sponge crystals,partitioned between EtOAc and 3N HCl, washed once each with dilute brineanddilute NaHCO.sub.3, dried over Na.sub.2 SO.sub.4, and evaporated. Silicagel chromatography(15% EtOAc/hexanes) yielded 4.25 g (88.5%) of chloromethoxybenzaldehyde6,mp 45.degree. C.IR (CHCl.sub.3, cm.sup.-1): 2835, 1700(CO), 1590, 1576, 1461, 1425, 1380,1320, 1280,1265, 1144, 1050, 850, 695.sup.1 H NMR (ppm): 3.84(3H, s), 7.13(1H, m), 7.26(1H, m), 7.41(1H, m),9.89(1H, s)Mass spectrum (El, 70 eV): exact mass calcd for C.sub.8 H.sub.7 ClO.sub.2170.0135, found170.0134. ##STR6##3-Chloro-5-methoxybenzaldehyde dimethyl acetal (7). A methanol solution(20 ml) of benzaldehyde 6 (8.76 g, 51 mmol) was cleanly converted todimethylacetal 7 in the presence of trimethyl orthoformate (5.62 ml, 51 mmol) andacatalytic amount of p-toluenesulfonic acid. The reaction was quenchedwithtriethylamine to pH 7, evaporated to a small volume and partitionedbetweenEtOAc and NaHCO.sub.3. The organic layer was dried, evaporated underreducedpressure and purified by silica gel chromatography (10% EtOAc/hexanes) togive10.68 g (96%) of acetal 7 as a light yellow oil.IR (CHCl.sub.3, cm.sup.-1): 2960, 2938, 2830, 1596, 1578, 1458, 1270,1104, 1050, 989,872, 865, 840.sup.1 H NMR (ppm): 3.31(6H, s), 3.79(3H, s), 5.31(1H, s), 6.85(1H, s),6.88(1H, s), 7.04(1H, s) ##STR7##Diethyl 1-methoxy-1-(3-chloro-5-methoxyphenyl)methane phosphonate 8.Triethyl phosphte (3.2 ml, 19 mmol) was added dropwise to a solution ofacetal 7(4.0 g, 18.5 mmol), boron trifluoride etherate (2.3 ml, 19 mmol) andCH.sub.2 Cl.sub.2(20 ml) at 0.degree. C. After slowly warming the reaction to roomtemperature (30 min),the solution was partitioned with dilute NaHCO.sub.3, dried over Na.sub.2SO.sub.4, evaporatedand purified on silica gel (40%-100% EtOAc/hexanes) to give 4.6 g (77.5%)ofphosphonate 8 as a light yellow oil.IR (CHCl.sub.3, cm.sup.-1): 2990, 1591, 1573, 1458, 1254(PO), 1050(PO),1025(PO),969, 870, 687.sup.1 H NMR (ppm): 1.24(3H, t, J=7Hz), 1.26(3H, t, J=7Hz), 3.37(3H, s),3.78(3H, s), 4.01-4.09(4H, m), 4.40(1H, d, J=16Hz), 6.83(1H, t, J=2Hz),6.88(1H, qt, J=2Hz), 6.98(1H, qt, J=2Hz) ##STR8##3-Chloro-5-methoxy-1-(methoxytricyclo�3.3.1.1.sup.3.7 !dec-2-ylidenemethyl)benzene (9).Phosphonate 8 (4.62 g, 14 mmol) and 2-adamantanone (2.58 g, 17 mmol)were dissolved in anhydrous THF (35 ml) under argon and cooled to-68.degree. C.Dropwise addition of lithium diisopropylamide (18.6 mmol) in anhydrousTHF(20 ml) at -68.degree. C. generated the ylid, followed by subsequentolefination of theketone. The reaction was slowly warmed to room temperature over 2 h andthenstirred at 75.degree. C. for 1 h. The solution was partitioned betweenEtOAc/NH.sub.4 Cl, driedover Na.sub.2 SO.sub.4, evaporated and purified by silica gelchromatography(2% EtOAc/hexanes), yielding 2.5 g (55%) of enol ether 9 as an oil..sup.1 H NMR (ppm): 1.55-1.95(12H, m), 2.61(1H, brs), 3.21(1H, brs),3.28(3H, s),3.78(3H, s), 6.74(1H, s), 6.80(1H, s), 6.87(1H, s) ##STR9##3-Chloro-5-hydroxy-1-(methoxytricyclo�3.3.1.1.sup.3.7 !dec-2-ylidene-methyl)benzene (10).Demethylation to enol ether phenol 10 proceeded cleanly upon heatingenol ether 9 (2.5 g, 7.8 mmol) in DMF (14 ml), at 155.degree. C. in thepresence of sodiumethane thiolate (11.7 mmol). Upon cooling, the mixture was partitionedbetweenEtOAc and NH.sub.4 Cl, dried over Na.sub.2 SO.sub.4 and evaporated underhigh vacuum toremove residual DMF. Chromatographic purification (silica gel,20% EtOAc/hexanes) produced 2.3 g (96%) of phenol 10 as an oil whichcrystallized upon standing. Trituration of the solid with 5%EtOAc/hexanes gavewhite crystals, mp 133.degree. C.IR (CHCl.sub.3, cm.sup.-1): 3854(OH), 3300(OH), 2910, 1590, 1310, 1285,1163, 1096,1080, 1011, 900, 840.sup.1 H NMR (ppm): 1.73-1.96(12H, m), 2.62(1H, brs), 3.20(1H, brs),3.32(3H, s),5.65(1H, brs), 6.73(1H, s), 6.79(1H, m), 6.85(1H, s) ##STR10##Pyridinium 3-chloro-5-(methoxytricyclo�3.3.1.1.sup.3.7 !dec-2-ylidenemethyl)-1-phenylphosphate (11). Triethylamine (450 .mu.l, 3.2 mmol) was added under anargonatmosphere to enol ether 10 (709 mg, 2.3 mmol) dissolved in anhydrousTHF(10 ml). The solution was cooled to 0.degree. C., at which time2-chloro-2-oxo-1,3,2-dioxaphospholane (Fluka, 285 .mu.l, 3.0 mmol) wasaddeddropwise. The reaction was warmed to room temperature, quickly passedthrough an argon-flushed column under inert atmosphere to removetriethylammonium hydrochloride crystals. After rinsing the crystal cakeonce withTHF, the solution was evaporated and pumped dry to give crude phospholane11a.Opening the phospholane ring upon reaction of 11a with NaCN (vacuumdried, 179 mg, 3.65 mmol) in anhydrous DMF (6 ml) under argon, producedthedesired .beta.-cyanoethyl diester pohspate 11b, as well as regeneratingenol etherphenol 10. Removal of DMF under high vacuum while warming the flask to55.degree. C.,left a mixture of compounds 10 and 11b as a yellow-orange oil.The above mixture was dissolved in methanol (8 ml) and stirred at40.degree. C. inthe presence of NaOMe (1 ml of 4.25M NaOMe/MeOH, 6.4 mmol), effecting.beta.-elimination of the cyanoethyl group to give enol ether phosphate11 as thedisodium salt. After evaporating the methanol, the solid was dissolved inwaterand partitioned with minimal EtOAc to recover phenol 10 (333 mg).Purificationof the aqueous phase by preparative HPLC, using a CH.sub.3 CN/H.sub.2 Ogradientthrough a polystyrene column (PLRS-S, Polymer Laboratories), followed byionexchange with pyridinium toluenesulfonate (Amberlyst-IR 120+ resin) andlyophilization, yielded 448 mg (78% over 3 steps, accounting forrecoveredphenol) of enol ether phosphate 11 as a fluffy, off-white powder.IR (CHCl.sub.3, cm.sup.-1): 2910, 1590, 1567, 1278, 1160, 1095, 945.sup.1 H NMR (ppm): 1.73-1.96(12H, m), 2.63(1H, brs), 3.20(1H, brs),3.32(3H, s),5.89(1H, s), 6.72(1H, m), 6.79(1H, t, J=2Hz), 6.85(1H, d, J=2Hz).sup.31 P NMR (ppm): 54(1P) ##STR11##Disodium 3-chloro-5-(methoxyspiro�1,2-dioxetane-3,2'-tricyclo�3.3.1.1.sup.3.7 !-decan!-4-yl)-1-phenyl phosphate (1). A solution of enol ether phosphate 11 and5,10,15,20-tetraphenyl-21H,23H-porphine (TPP, 0.5 ml of a 2% solution in CHCl.sub.3by weight)in CHCl.sub.3 (8 ml) was irradiated with a 250 W, high pressure sodiumlamp at 10.degree. C.while passing a stream of oxygen through the solution. A 5-mil piece ofKaptonpolyimide film (DuPont) placed between the lamp and the reaction mixturefilteredout unwanted UV radiation. Analytical HPLC (UV detector at 270 nm) showedcompletedioxetane formation upon irradiating 5 min. After evaporation of thechloroform at0.degree. C., the residue was dissolved in ice water in the presence ofNa.sub.2 CO.sub.3(27 mg, 0.25 mmol) and purified by preparative HPLC as described above.The fractionswere frozen and lyophilized at 0.degree. C., yielding 65.3 mg (90%) ofdioxetane 1 as a fluffywhite powder. TLC of the dioxetane exhibited blue chemiluminescence bythermaldecomposition upon heating. Enzymatic cleavage of the phosphate alsoindicedchemiluninescent decomposition in aqueous solutions..sup.1 H NMR (D.sub.2 O, ppm): 0.93(1H, d, J=13Hz), 1.21(1H, d, J=13Hz),1.44-1.69(10H, m), 2.16(1H, brs), 2.79(1H, brs), 3.14(3H, s), 7.20(2H,brs),7.30(1H, s).sup.31 P NMR (D.sub.2 O, ppm): 24(1P) ##STR12##3-Chloro-5-hydroxy benzaldehyde dimethyl acetal (12). 5-Chloro-3-methoxybenzaldehyde dimethyl acetal (7, 3.21 g, 14.8 mmol) was demethylatedwithsodium ethane thiolate (19 mmol) in DMF (14 ml) while heating at150.degree. C. Theresultant phenol 12 was cooled, partitioned between EtOAc and NH.sub.4Cl, driedover Na.sub.2 SO.sub.4, evaporated and pumped to dryness on high vacuumto removeresidual DMF. Chromatographic purification (silica gel, 20%EtOAc/hexanes)afforded 2.75 g (92%) of phenol 12 as a yellow oil. An analytical sampleof the oilcrystallized upon further purification, mp 153.degree. C.IR (CHCl.sub.3, cm.sup.-1): 3580(OH), 3325(OH), 2940, 2830, 1599, 1585,1449, 1350,1155, 1105, 1055, 894, 845.sup.1 H NMR (ppm): 3.32(6H, s), 5.30(1H, s), 5.73(1H, brs), 6.81(2H, m),7.01(1H, s) ##STR13##3-Chloro-5-pivaloyloxybenzaldehyde dimethyl acetal (13). Phenol 12 (2.7g,13.3 mmol) and triethylamine (2.8 ml, 20 mmol) in CH.sub.2 Cl.sub.2 (20ml) were stirred at0.degree. C. Addition of trimethylacetyl chloride (1.64 ml, 13.3 mmol)cleanly yielded thepivaloyl ester. Standard workup provided crude pivaloate 13 as an oilwhich wascarried on to the next reaction without purification; no weight wastaken. A smallsamples was purified by prep TLC for spectral characterization.IR (CHCl.sub.3, cm.sup.-1): 2980, 2940, 1749(CO), 1585, 1448, 1349, 1250,1150, 1109,1056, 898.sup.1 H NMR (ppm): 1.34(9H, s), 3.31(6H, s), 5.36(1H, s), 7.06(2H,brs),7.31(1H, s) ##STR14## ##STR15##Diethyl 1-methoxy-1-(3-chloro-5-pivaloyloxyphenyl)methane phosphonate(14).A solution of acetal 13, boron trifluoride etherate (2.6 ml, 21 mmol) andCH.sub.2 Cl.sub.2(10 ml) was stirred at -78.degree. C. Addition of triethyl phosphite (3.0ml, 17.5 mmol)converted the acetal to phosphonate 14. Workup and purification (silicagel,10% EtOAc/hexanes) yielded 2.43 g oil (47% over 2 steps).IR (CHCl.sub.3, cm.sup.-1): 2995, 2980, 1750(CO), 1600, 1581, 1442,1247(P),1110, 1028(PO), 975, 890.sup.1 H NMR (ppm): 1.22-1.26(6H, d of t, J=2Hz, 7Hz), 1.31(9H, s),3.39(3H, s),4.02-4.08(4H, m), 4.44(1H, d, J=16Hz), 7.04(2H, m), 7.27(1H, brs) ##STR16##3-Chloro-5-pivaloyloxy-1-(methoxy-5-chloro-tricyclo�3.3.1.1.sup.3.7 !-dec-2-ylidenemethylbenzene (15). Phosphonate 14 (2.4 g, 6.1 mmol) wasdissolved in anhydrous THF (10 ml) under argon and cooled to -68.degree.C. Dropwiseaddition of lithium diisopropylamide (6.6 mmol) in anhydrous THF (7 ml)at lowtemperature generated the ylid, evident by deep coloration. After 5 min,a THFsolution of 5-chloro-2-adamantanone (941 mg, 5 mmol) was added and thereaction was slowly warmed to room temperature over 40 min, followed byheating at 75.degree. for 1 h to complete olefination. The solution waspartitionedbetween EtOAc/NH.sub.4 Cl, dried over Na.sub.2 SO.sub.4 and evaporated togive a crudemixture of enol ether pivaloate 15 and the corresponding enol etherphenol 16.The crude oil was used without purification in the following hydrolysis.A smallsample was purified by prep TLC for spectral characterization.IR (CHCl.sub.3, cm.sup.-1): 2935, 1750(CO), 1595, 1571, 1450, 1520, 1397,1275, 1160,1110, 1024, 918, 906, 887, 829.sup.1 H NMR (ppm); 1.34(9H, s), 1.68-1.78(4H, m), 2.14-2.25(7H, m),2.77(1H, brs), 3.30(3H, s), 3.42(1H, brs), 6.88(1H, d, J=1.5Hz), 7.04(1H,m),7.11(1H, d, J=1.5Hz) ##STR17##3-Chloro-5-hydroxy-1-(methoxy-5-chloro-tricyclo�3.3.1.1.sup.3.7 !-dec-2-ylidenemethyl)benzene (16). Crude pivaloate 15 was hydrolyzed atroomtemperature with K.sub.2 CO.sub.3 (1.45 g, 10.5 mmol) in 10 ml methanol.Evaporation ofmethanol, followed by standard workup and purification (silica gel,30% EtOAc/hexanes) afforded 1.095 g (63% over 2 steps) of a slightlyyellow oilwhich solidified upon standing. Trituration of the solid produced whitecrystallineenol ether phenol 16, mp 130.degree. C.IR (CHCl.sub.3, cm.sup.-1): 3590(OH), 3300(OH), 2935, 1595, 1163, 1100,1082, 1030,911.sup.1 H NMR (ppm): 1.69-1.83(4H, m), 2.14-2.27(7H, m), 2.77(1H, brs),3.30(3H, s), 3.41(1H, brs), 5.21(1H, brs), 6.67(1H, d, J=1.5Hz), 6.81(1H,m),6.84(1H, d) ##STR18##Disodium 3-chloro-5-(methoxyspiro�1,2-dioxetane-3,2'-(5-chloro-)tricylco-�3.3.1.1.sup.3.7 !-decan!-4-yl)-1-phenyl phosphate (2). Triethylamine(230 .mu.l,1.65 mmol) was added under an argon atmosphere to enol ether 16 (356 mg,1.05 mmol) dissolved in anhydrous THF (5 ml). The solution was cooled to0.degree. C.,at which time 2-chloro-2-oxo-1,3,2-dioxasphospholane (Fluka, 143 .mu.l,1.55 mmol)was added dropwise. The reaction was warmed to room temperature andquicklypassed through an argon-flushed column under inert atmosphere to removetriethylammonium hydrochloride crystals. After rinsing the crystal cakeonce withTHF, the solution was evaporated and pumped dry to give crude phospholane17a.Opening the phospholane ring upon reaction with NaCN (vacuum dried,69 mg, 1.4 mmol) in anhydrous DMF (5 ml) under argon, produced thedesired.beta.-cyanoethyl diester phosphate 17b. Removal of DMF under high vacuumwhilewarming the flask to 55.degree. C. left the crude diester phosphate as anorange oil.A solution of cyanoethyl phosphate 17b and 5,10,15,20-tetraphenyl-21H,23H-porphine (TPP, 1.5 ml of a 2% solution in CHCl.sub.3 by weight)in CHCl.sub.3(10 ml) was irradiated with a 250W, high pressure sodium lamp at10.degree. C. whilepassing a stream of oxygen through the solution. A 5-mil piece of Kaptonpolyimide film (DuPont) placed between the lamp and the reaction mixturefilteredout unwanted UV radiation. Analytical HPLC (UV detector at 270 nm)showedcomplete dioxetane formation upon irradiating 15 min. After evaporationof thechloroform at 0.degree. C., the residue was dissolved in methanol anddeprotected to thedisodium phosphate dioxetane with NaOMe (0.5 ml of 4.25M NaOMe/MeOH,2 mmol). Upon .beta.-elimination of the cyanoethyl group, the solventwasevaporated at 0.degree. and the residue dissolved in ice water.Purification bypreparative HPLC, as described above, followed by lyophilization at0.degree. C., yielded289 mg (60% over 4 steps) of dioxetane 2 as a fluffy white powder..sup.1 H NMR (D.sub.2 O, ppm, mixture of syn/anti isomers): 0.86(1H, d),1.13(1H, d, J=14Hz), 1.30(1H, d), 1.37(1H, d), 1.45-2.07(18H, m),2.27(1H, brs), 2.32(1H, brs), 2.95(2H, brs), 3.09(3H, s), 3.11(3H, s),7.0-7.3(4H, brs), 7.25(1H, s), 7.28(1H, s) ##STR19##3,5-Dmethoxybenzaldehyde dimethyl acetal (18).IR (CHCl.sub.3, cm.sup.-1): 2958, 2935, 1598, 1460, 1426, 1357, 1190,1154, 1101, 1053, 840.sup.1 H NMR (ppm): 3.32(6H, s), 3.78(6H, s), 5.28(1H, s), 6.41(1H, m),6.60(2H, m) ##STR20##3-Hydroxy-5-methoxybenzaldehyde dimethyl acetal (19).IR (CHCl.sub.3 cm.sup.-1): 3590(OH), 3345(OH), 2940, 2830, 1600, 1462,1432, 1355,1190, 1150, 1110, 1055, 841.sup.1 H NMR (ppm): 3.32(6H, s), 3.77(3H, s), 5.28(1H, s), 6.37(1H, d,J=2Hz),6.53(1H, brs), 6.58(1H, brs) ##STR21##3-Methoxy-5-pivaloyloxybenzaldehyde dimethyl acetal (20). (73% over 3steps, oil)IR (CHCl.sub.3, cm.sup.-1): 2960, 2935, 1741(CO), 1608, 1597, 1462, 1350,1273, 1190,1139, 1115, 1056, 999, 902, 848.sup.1 H NMR (ppm): 1.34(9H, s), 3.31(6H, s), 3.80(3H, s), 5.35(1H, s),6.57(1H, d, J=2Hz), 6.75(1H, brs), 6.87(1H, brs) ##STR22##Diethyl 1-methoxy-1-(3-methoxy-5-pivaloyloxyphenyl)methane phosphonate(21). (40%, oil)IR (CHCl.sub.3, cm.sup.-1): 2990, 2980, 1742(CO), 1606, 1590, 1463, 1272,1240, 1136,1110, 1100, 1055, 1023, 970.sup.1 H NMR (ppm): 1.21(3H, t, J=3Hz), 1.23(3H, t), 1.32(9H, s),3.39(3H, s),3.78(3H, s), 4.06(4H, m), 4.44(1H, d, J=16Hz), 6.56(1H, m), 6.72(1H, m),6.85(1H, m) ##STR23##3-Methoxy-5-pivaloyloxy-1-(methoxytricyclo�3.3.1.1.sup.3.7 !dec-2-ylidenemethyl)-benzene (22a).IR (CHCl.sub.3, cm.sup.-1): 2910, 1740(CO), 1600, 1580, 1460, 1325, 1272,1140, 1114,1097, 1079, 1055.sup.1 H NMR (ppm): 1.35(9H, s), 1.56-1.96(12H, m), 2.68(1H, brs),3.23(1H, brs),3.31(3H, s), 3.80(3H, s), 6.53(1H, t, J=2Hz), 6.61(1H, brs), 6.72(1H, m) ##STR24##3-Hydroxy-5-methoxy-1-(methoxytricyclo�3.3.1.1.sup.3.7 !dec-2-ylidenemethyl)-benzene (22). (64%, white crystals, mp 159.degree. C.)IR (CHCl.sub.3, cm.sup.-1): 3590(OH), 3320(OH), 2910, 1591, 1342, 1150,1098.sup.1 H NMR (ppm): 1.78-1.97(12H, m), 2.68(1H, brs), 3.23(1H, brs),3.33(3H, s),3.78(3H, s), 5.49(1H, s), 6.37(1H, m), 6.45(2H, m) ##STR25##Pyridinium 5-methoxy-3-(methoxytricyclo�3.3.1.1.sup.3.7 !dec-2-ylidenemethyl)-1-phenyl phosphate (23). (62%, off-white fluffy powder)IR (CHCl.sub.3, cm.sup.-1): 2911, 1584, 1448, 1425, 1328, 1149, 1099,960, 870.sup.1 H NMR (ppm): 1.68-1.92(12H, m), 2.63(1H, brs), 3.17(1H, brs),3.23(3H, s),3.68(3H, s), 6.55(1H, brs), 6.72(1H, brs), 6.76(1H, brs), 6.98(1H, brs) ##STR26##Disodium 5-methoxy-3-(methoxyspiro�1,2-dioxetane-3,2'-tricyclo�3.3.1.1.sup.3.7 !-decan!-4-yl)-1-phenyl phosphate (3). (85%, white fluffy powder).sup.1 H NMR (D.sub.2 O, ppm): 0.98(1H, brd), 1.22(1H, brd),1.46-1.76(10H, m),2.20(1H, brs), 2.78(1H, brs), 3.14(3H, s), 3.74(3H, s), 6.91(1H, brs),6.68-6.97(2H, very broad signal).sup.31 P NMR (D.sub.2 O, ppm): 44.8(1P) ##STR27##3-Hydroxy-5-methoxy-1-(methoxy-5-chloro-tricyclo�3.3.1.1.sup.3.7 !-dec-2-yldenemethyl)benzene (24). (63%, white crystals, mp 134.degree.C.)IR (CHCl.sub.3, cm.sup.-1): 3590(OH), 3330(OH), 2930, 1610, 1591, 1450,1430, 1341,1150, 1100, 1080, 1056, 1028, 829.sup.1 H NMR (ppm): 1.58-2.40(11H, m), 2.82(1H, brs), 3.31(3H, s),3.42(1H, brs),3.78(3H, s), 6.37-6.41(3H, m) ##STR28##Disodium 5-methoxy-3-(methoxyspiro�1,2-dioxetane-3,2'-(5-chloro-)tricyclo-�3.3.1.1.sup.3.7 !-decan!-4-yl)-1-phenyl phosphate (4). (57% over 4steps, white fluffypowder).sup.1 H NMR (D.sub.2 O, ppm, mixture of syn/anti isomers): 0.94(1H,brd),1.19(1H, brd), 1.42(1H, brd), 1.50(1H, brs), 1.58(1H, brd),1.67-2.16(17H, m), 2.38(1H, brs), 2.40(1H, brs), 3.00(2H, brs), 3.15(3H,s),3.16(3H, s), 3.73(3H, s), 3.74(3H, s), 6.90(1H, brs), 6.93(1H, brs),6.65-7.00(4H, very broad signal).sup.31 P NMR (D.sub.2 O, ppm, mixture of syn/anti isomers): 44.8(2P)References 5-Chlorovanillin was synthesized as described by Hann and Spencer (J. Am. Chem. Soc., 1927, 49:535-537), mp 163.degree. C. Proton sponge formate (N,N,N',N',-tetramethyl-1,8-naphthalenediammonium formate): Formic acid (98%, 1.2 ml, 31 mmol) was added to a solution of proton sponge (6.8 g, 32 mmol) and CH.sub.2 Cl.sub.2 (8 ml) at 0.degree. C. After warming to room temperature, the solvent was evaporated and the proton sponge formate crystallized as white crystals while drying on high vacuum with minimal warming. Proton sponge formate crystals (mp 79.degree. C.) must be used soon after preparation since formic acid will evaporate upon standing, leaving proton sponge (mp 50.degree. C.). ##STR29##3-Methoxy-5-nitro-4-hydroxy benzaldehyde dimethyl acetal (25). Amethanolsolution (30 ml) of 5-nitrovanillin (5.0 g, 97%, 18.4 mmol) was cleanlyconvertedto dimethyl acetal 25 in the presence of trimethyl orthoformate (2.8 ml,25 mmol)and a catalytic amount of p-toluenesulfonic acid. The reaction wasquenchedwith triethylamine to pH 8, evaporated to a small volume and partitionedbetweenEtOAc and NaHCO.sub.3. The aqueous layer was washed once with EtOAc. Theorganic layers were dried over Na.sub.2 SO.sub.4, decanted and evaporatedto anorange-red oil that crystallized upon pumping. Recrystallization from50% EtOAc/hexanes gave 5.55 g (93%) acetal 25 as red-orange crystals,mp 58-59.degree. C.IR (CHCl.sub.3, cm.sup.-1): 3300, 3010, 2930, 2820, 1620, 1543, 1460,1445, 1392, 1341,1320, 1254, 1132, 1101, 1058, 990, 865.sup.1 H NMR (ppm): 3.31(6H, s), 3.94(3H, s), 5.31(1H, s), 7.22(1H, d,J=1.7 Hz),7.78(1H, d) ##STR30##3-Methoxy-5-nitro-4-trifluoromethanesulfonyloxy benzaldehyde dimethylacetal(26). A solution of dimethyl acetal 25 (5.0 g, 20.6 mmol), chloroform (3ml) andpyridine (8 ml) was stirred at 0.degree. C. under argon. Addition oftrifluoromethanesulfonic anhydride (4.0 ml, 23.8 mmol) at 0.degree. C.,over 10 min,followed by stirring at room temperature overnight gave clean formationof thetriflate. The solvents were evaporated under high vacuum while warmingthe oilto 45.degree. C. and traces of pyridine were chased with 4 ml toluene.The resulting oilwas pumped well under high vacuum, taken up in 50% EtOAc/hexanes andtriturated with 50% EtOAc/hexanes to separate the desired triflate (insolution)from the fine pyridinium triflate crystals. Evaporation of thetrituration solution,followed by purification of the oil on a silica gel column, eluting with30%EtOAc/hexanes, yielded 6.43 g (84%) of triflate 26 as a light yellowoil.IR (CHCl.sub.3, cm.sup.-1):.sup.1 H NMR (ppm): 3.35(6H, s), 4.00(3H, s), 5.42(1H, s), 7.43(1H, d,J=1.6Hz),7.73(1H, d) ##STR31##*3-Methoxy-5-nitro-benzaldehyde dimethyl acetal (27). 5-Nitrophenyltriflate 26(7 g, 18.7 mmol), palladium (11) acetate (88 mg, 0.39 mmol),1,1'-bis(diphenyl-phosphino)ferrocene (430 mg, 0.78 mmol) and hpic grade CH.sub.3 CN (10ml) weremixed well in a teflon-lined stainless steel bomb. After adding freshlymade,pulverized proton sponge formate (5.1 g, 19.6 mmol), the bomb was sealedandheated at 90.degree. C. for 2 h. The reaction mixture was taken up inEtOAc, passedthrough a silica gel plug, and then purified on a silica gel column,eluting with0-30% EtOAc/hexanes to yield 1.5 g (35%) methoxynitrobenzaldehyde acetalIR (CHCl.sub.3, cm.sup.-1): 3005, 2960, 2935, 2835, 1532(NO.sub.2), 1463,1450, 1343(NO.sub.2),1280, 1190, 1158, 1104, 1055, 990, 871.sup.1 H NMR (ppm): 3.33(6H, s), 3.89(3H, s), 5.41(1H, s), 7.33(1H, s),7.68(1H, s),7.92(1H, s) ##STR32##Diethyl 1-methoxy-1-(3-methoxy-5-nitrophenyl)methane phosphonate (28).Triethyl phosphite (0.98 ml, 5.7 mmol) was added dropwise to a solutionofdimethyl acetal 27 (1.08 g, 4.7 mmol), boron trifluoride etherate (1.2ml,9.8 mmol) and CH.sub.2 Cl.sub.2 (10 ml) at 0.degree. C. After warming thereaction to roomtemperature overnight, the solution was partitioned with 3N HCl and theaqueouslayer was washed with CH.sub.2 Cl.sub.2 twice. The organic layers werewashed withdilute NaHCO.sub.3, dried over Na.sub.2 SO.sub.4, decanted andevaporated. The cruderesidue was purified on a silica gel column, eluting with 0-80%EtOAc/hexanes, togive 1.36 g (86%) phosphonate 28 as a slightly yellow oil.IR (CHCl.sub.3, cm.sup.-1): 2995, 1532(NO.sub.2), 1350(NO.sub.2), 1280,1258, 1253, 1096,1053, 1025, 973, 721.sup.1 H NMR (ppm): 1.28(6H, t, J=7.1Hz), 3.44(3H, s), 3.90(3H, s),4.08-4.15(4H, m), 4.55(1H, d, J=16Hz), 7.34(1H, d), 7.69(1H, d,J=2.1Hz),7.87(1H, d, J=1.6Hz) ##STR33##Diethyl 1-methoxy-1-(3-amino-5-methoxyphenyl)methane phosphonate (29).Nitro phosphonate 28 is dissolved in methylene chloride and added to a1MNaOH solution containing nBu.sub.4 NBr and sodium hydrosulfite. Thebiphasicsolution is stirred vigorously, with warming if necessary, untilreduction of thenitro substituent to aniline 29 is complete. The cooled solution ispartitionedbetween CH.sub.2 Cl.sub.2 and minimal water, and the aqueous layer iswashed withCH.sub.2 Cl.sub.2 as needed to obtain the crude aniline. The combinedorganic layers aredried, decanted and evaporated. The residue is then passed through ashortsilica gel plug to give aniline 29.IR (CHCl.sub.3, cm.sup.-1):.sup.1 H NMR (ppm):(References for other reduction conditions are appended to the synthesissummary.) ##STR34##Diethyl 1-methoxy-1-(3-methoxy-5-trifluoroacetamidophenyl)methanephosphonate (30). Phosphonate 29 is quantitatively acetylated by additionoftrifluoroacetic anhydride (1 eq) and triethylamine (1.3 eq) in 10 mlCH.sub.2 Cl.sub.2 at 0.degree. C.Evaporation of solvents, followed by silical gel column purificationyieldstrifluoroacetamide 30.IR (CHCl.sub.3, cm.sup.-1):.sup.1 H NMR (ppm): ##STR35##3-Methoxy-5-trifluoroacetamido-1-(methoxytricyclo�3.3.1.1.sup.3.7!dec-2-ylidene-methyl)benzene (31). Phosphonate 30, dissolved in anhydrous THF, iscooled to-68.degree. C. under an argon atmosphere. Similarly, 2-adamantanone (1.1eq) isdissolved in anhydrous THF and cooled to -68.degree. C. under argon in aseparate flask.To the phosphonate solution is added 2.5M nBuLi at -68.degree. C. underargon until thered color of the ylid persists. At this point, 1.2 eq nBuLi is added tocomplete theylid formation and the resulting colored solution is stirred at-68.degree. C. for 5 min.While maintaining the low temperature, 2-adamantanone in THF is slowlyaddedto the ylid over an hour. After the final addition of ketone, thereaction mixture isstirred for 2 h while warming to room temperture. The reaction is thenheated atreflux for 1 h, cooled and quenched by partitioning with EtOAc andsaturatedNH.sub.4 Cl. The organic layer is dried over Na.sub.2 SO.sub.4 andchromatographed withEtOAc/hexanes on a silica gel column to give enol ether 31.IR (CHCl.sub.3, cm.sup.-1):.sup.1 H NMR (ppm): ##STR36##3-Amino-5-methoxy-1-(methoxytricyclo�3.3.1.1.sup.3.7 !dec-2-ylidenemethyl)benzene(32). Trifluoroacetamide enol ether 31 is hydrolyzed at 60.degree. C.with finely groundK.sub.2 CO.sub.2 (3 eq) in MeOH containing trace water. Work up bypartitioning themixture with EtOAc/H.sub.2 O, followed by silica gel chromatographyprovides enolether aniline 32.IR (CHCl.sub.3, cm.sup.-1):.sup.1 H NMR (ppm): ##STR37##3-Carbamoyl-5-methoxy Derivatives (3-NHCO.sub.2 X):3-para-Methoxyphenylcarbamoyl-5-methoxy-1-(methoxytricyclo�3.3.1.1.sup.3.7 !dec-2-ylidenemethyl)benzene (33). Enol ether aniline 32 in methylene chlorideiscarboxylated with 4-methoxyphenyl chloroformate (1.1 eq) in the presenceoftriethylamine (2.0 eq) at 0.degree. C. The reaction mixture ispartitioned withCH.sub.2 Cl.sub.2 /H.sub.2 O, washed with dilute NaHCO.sub.3, dried overNa.sub.2 SO.sub.4, evaporated andchromatographed on silica gel to yield enol ether p-methoxyphenylcarbamate 33.IR (CHCl.sub.3, cm.sup.-1):.sup.1 H NMR (ppm): ##STR38##3-tert-Butylcarbamoyl-5-methoxy-1-(methoxytricyclo�3.3.1.1.sup.3.7!dec-2-ylidenemethyl)benzene (34). A methylene chloride solution of enol etheraniline32, triethylamine (1.5 eq) and BOCON (1.3 eq) is stirred at 55.degree. C.in a tightlycapped Kimax tube to effect t-butyl carbamate formation. The solution iscooled,evaporated to a small volume and, upon addition of MeOH to the residue,thedesired carbamate 34 precipitates.IR (CHCl.sub.3, cm.sup.-1):.sup.1 H NMR (ppm): ##STR39##3-N-Sulfonamido-5-methoxy Derivatives (3-NHSO.sub.2 X:)3-N-Toluenesulfonamido-5-methoxy-1-(methoxytricycl�3.3.1.1..sup.3.7!dec-2-ylidenemethyl)benzene (35). A methylene chloride solution of enol etheraniline32 is sulfonylated with tosyl chloride (1.1 eq) in te presence oftriethylamine(2.0 eq) at 0.degree. C. The reaction mixture is partiontioned withCH.sub.2 Cl.sub.2 /H.sub.2 O, washedwith dilute NaHCO.sub.3, dried over Na.sub.2 SO.sub.4, evaporated andchromatographed onsilica gel to yield N-toluenesulfonamido enol ether 35.IR (CHCl.sub.3, cm.sup.-1):.sup.1 H NMR (ppm): ##STR40##3-N-Trifluoromethylsulfonamido-5-methoxy-1-(methoxytricyclo�3.3.1.1.sup.3.7 !dec-2-ylidenemethyl)benzene (36). A methylene chloride solution of enol etheraniline32 is sulfonylated with trifluoromethylsulfonic anhydride (1.1 eq) at0.degree. C. Thereaction mixture is partioned with CH.sub.2 Cl.sub.2 /H.sub.2 O, driedover Na.sub.2 SO.sub.4, evaporatedand chromatographed on silica gel to yield N-trifluoromethylsulfonamidoenolether 36.IR (CHCl.sub.3, cm.sup.-1):.sup.1 H NMR (pm): ##STR41##3-Amido-5-methoxy Derivatives (3-NHCOX):3-N-Benzamide-5-methoxy-1-(methoxytricyclo�3.3.1.1.sup.3.7 !dec-2-ylidenemethyl)benzene (37). A pyridine solution of enol ether aniline 32isreacted with benzoyl chloride (1.1 eq) at 0.degree. C. The solvent isevaporated andpumped well to yield a crude oil, which is partioned between CH.sub.2Cl.sub.2 /H.sub.2 O, driedand evaporated. Chromatography on silica gel yields benzamido enol etherIR (CHCl.sub.3, cm.sup.-1):.sup.1 H NMR (ppm):The 3-nitrogen-substituted phenyl enol ethers (compounds 33-37) aredemethylated with sodium ethane thiolate, and then phosphorylated andphotooxygenated as described for dioxetanes 1 and 2 to obtain theanalogousdioxetanes.__________________________________________________________________________

EXAMPLES
Various dioxetanes within the scope of this invention have been prepared and tested for essential properties, such as quantum yield (performed by an independent laboratory according to the procedure listed below), T.sub.1/2 and the emission wavelength maxima. These dioxetanes are identified by number, and in the tables following after the number, the identity of the substituent on the adamantyl ring, if any followed by the identity of the Z substituent is given. In the compounds tested, X is phosphate. Values for quantum yield and T.sub.1/2 are obtained both for the dioxetane alone in 0.1 molar DEA, and in the presence of an ehancement agent, Sapphire II.
Protocol for Ouantum Yields Determination
500 .mu.L of 3.2.times.10.sup.-4 M solution of a dioxetane in 0.1M Na.sub.2 CO.sub.3, pH 9.5 was placed in a 12.times.75 mm tube, at 20.degree. C. The solution was equilibrated to 20.degree. C. in a refrigerated water bath for 10 minutes. 2 .mu.L of alkaline phosphatase suspension was added to the tube containing dioxetane and immediately vortexed for 1 sec and placed in the 20.degree. C. water bath. The tube was then placed in MGM Optocomp.RTM. I luminometer and the light signal was measured at 1 sec integration times. After the light signal was measured, the tube was placed back into the 20.degree. C. water bath and the measurement was repeated. The total counts for the dioxetane were determined from the intensity data. Total counts observed for a given concentration of dioxetane is the product of Photon Detection Efficiency (PDE) of the luminometer, the quantum yield of dioxetane and the number of molecules capable of emitting light (concentration of dephosphorylated dioxetanes). PDE for the MGM Optocomp I luminometer was determined to be 2.56.times.10.sup.-3, measured with a Biolink.RTM. absolute standard and utilizing the known spectral response of the luminometer's PMT and the known emission spectrum of the dioxetanes. The quantum yield is calculated by dividing the total counts measured by the PDE and the concentration of the dioxetane.
Calculation of Half Life or Half Time to Steady State Light Emission
From the Turner luminometer readout, the maximum signal was measured. The maximum signal minus the Turner light unit readings at 30, 150, 300, or 600 second intervals was calculated and graphed vs. time in seconds. From the graphs, an exponential equation was calculated to determine the half life.
The half lives of the dioxetanes were also determined directly from the Turner luminometer printouts.
Emission Maxima
To 2 ml of a pH 10 solution of 0.4 mM dioxetane, 0.1M diethanolamine, 1 mM MgCl.sub.2 was added 9.9.times.10.sup.-11 M alkaline phosphatase. The solution was equilibrated 5 minutes in a Spex Fluorolog Fluorimeter and then scanned 5 times at 0.5 sec/nm for chemiluminescent emission. The chemiluminescence emission wavelength maximum was recorded.
Chemiluminescent DNA Sequencing
DNA sequencing with chemiluminescent detection was performed as described in the Tropix SEQ-Light.TM. protocol. Briefly, DNA sequencing reactions were initiated with biotinylated primers using M13 single stranded phage DNA as a template. The reactions were separated by 8M urea denaturing PAGE, transferred horizontally to Tropilon-Plus nylon membrane by capillary action, and cross-linked to the membrane by exposure to UV light using a Spectronics SpectroLinker XL-1500 at 200 mJ/cm.sup.2. The membranes were incubated with blocking buffer (0.2% I-Block.TM., 0.5% sodium, dodecyl sulphate/SDS, in phosphate buffered saline/PBS �20 mM sodium phosphate, pH 7.2, 150 mM NaCl!) for 10 minutes, incubated with a 1/5000 dilution of Avidx-AP streptavidin-alkaline phosphatase in blocking buffer for 20 minutes, washed for 5 minutes in blocking buffer, washed 3.times.5 minutes with wash buffer (0.5% SDS, PBS), washed 2.times.5 minutes with assay buffer (0.1M diethanolamine, 1 mM MgCl.sub.2 pH 10), and then incubated with dioxetane solution (either CSPD, 140-17 or 128-87 diluted to 0.25 mM in assay buffer) for 5 minutes. The membranes were drained, sealed in a plastic folder and exposed to Kodak XAR-5 X-ray film. For the dioxetane 128-87, the exposure time was 70 minutes and for 140-17, 80 minutes, both 65 minutes after substrate addition. For the comparison of dioxetane 128-87 versus CSPD, the membrane exposure time was 5 minutes after a 24 hour incubation with substrate. The details of this type of protocol are reflected in Tropix SEQ-Light.TM. DNA sequencing system, commercially available from Tropix, Inc.
0.1M DEA, pH, 25.degree. C.
Dioxetane concentration 3.7.times.10.sup.-7 M to 6.times.10.sup.-6 M
______________________________________Compound Quantum Yield T 1/2 (min) .mu.L em______________________________________128-70 (H, 5-Cl) 1.4 .+-. 10.sup.-4 35.55 471128-87 (Cl, 5-Cl) 1.2 .+-. 10.sup.-4 9.03 470140-20 (H, 5-OMe) 1.5 .+-. 10.sup.-5 1.55 476140-17 (Cl, 5-OMe) 2.3 .+-. 10.sup.-5 1.09 475140-62 (H, 6-OMe) 1.1 .+-. 10.sup.-6 2.4 490140-73 (Cl, 6-OMe) 6.8 .+-. 10.sup.-7 2.0 487AMPPD 1.5 .+-. 10.sup.-5 2.1 477CSPD 5.2 .+-. 10.sup.-5 1.6 475______________________________________
0.09M DEA+0.1% Sapphire II, pH 9.95, 25.degree. C.
Dioxetane concentration 1.8.times.10.sup.-7 M to 6.1.times.10.sup.-9 M
______________________________________Compound Quantum Yield T 1/2 (min)______________________________________128-70 (H, 5-Cl) 5.2 .+-. 10.sup.-2 172128-87 (Cl, 5-Cl) 3.5 .+-. 10.sup.-2 70.6140-20 (H, 5-OMe) 2.4 .+-. 10.sup.-3 4.34140-17 (Cl, 5-OMe) 1.9 .+-. 10.sup.-3 1.1140-62 (H, 6-OMe) 3.8 .+-. 10.sup.-5 6.49140-73 (Cl, 6-OMe) 5.5 .+-. 10.sup.-5 2.22AMPPD 6.4 .+-. 10.sup.-4 8.2CSPD 6 .+-. 10.sup.-3 4.5______________________________________
To demonstrate positively the interaction of the dioxetane, or at least the excited-state emitter, with enhancement agents of the type known for use in connection with dioxetanes, the wavelength for the emission maximum was detected in the absence of any enhancement agent, in the presence of BDMQ, and on a nylon membrane. The data are set forth in the following table.
______________________________________ Emission Max, nmDioxetane No Addition +BDMQ On Nylon______________________________________128-70 471 463 461128-87 470 464 459140-20 476 466 461140-17 475 464 463140-62 490 482 477140-73 487 479 481______________________________________
Dot Blot Assays
As noted above, the dioxetanes of this invention are suitable for use in dot blot assays. The dioxetanes synthesized according to the synthesis route described above were employed in dot blot assays. In confirmation of the absence of chemiluminescence of the dioxetanes bearing a Z substituent at the six position, it should be noted that Compound 140-62 gave a consistent absence of signal, or, under optimum conditions, a barely detectable signal. Similarly, the dioxetane with the methoxy substituent at the six position with a chlorine substituent on the adamantyl ring, 140-73, gave no signal in dot blot assay, again confirming the lack of chemiluminescent activity in six-substituted metaphosphate phenyl dioxetanes.
Nitrocellulose and nylon membrances were spotted with a biotinylated 35 base oligonucleotide probe. The probe was diluted in 1.times.SSC to yield a starting dilution of 210 pg. Successive 1:2 dilutions of the starting dilution were spotted on the membranes, 12 spots total. The membranes were dried, subjected to optimum U.V. crosslinking (120 mJ/cm.sup.2), blocked for 30 minutes in blocking buffer (nitrocellulose: 0.2% I-Block, 0.1% Tween-20, 1.times.PBS; nylon:0.2% I-Block, 0.5% SDS, 1.times.PBS), incubated 20 minutes in a 1/5000 dilution of streptavidin-aklaline phosphatase conjugate diluted in blocking buffer, and washed as follows: 1.times.5 minutes in blocking buffer; 3.times.5 minutes in 1.times.PBS, 0.3% Tween-20 (nitrocellulose) or 3.times.5 minutes in 1.times.PBS, 0.5% SDS (nylon); 2.times.5 minutes in substrate buffer (0.1M diethanolamine, 0.1 mM MgCl.sub.2, pH 10); 1.times.5 minutes in a 1/20 dilution of Nitro-Block (Tropix, Inc. Bedford, Mass.) diluted in substrate buffer (Nitrocellulose Experiment Only); and 2.times.5 minutes in substrate buffer (Nitrocellulose Experiment Only). The membranes were incubated with 0.25 mM dioxetane diluted in substrate buffer for 5 minutes. Several membranes in both the nitrocellulose and nylon experiments were incubated with 0.25 mg/ml Calfax DB-45, Calfax 10L-45 or Calsoft T-60 (Pilot Chemical Company, Los Angeles, Calif.), 1.0 mg/ml Tween-20, 1.0 mg/ml Nitro-Block, and 0.25 mM dioxetane diluted in substrate buffer for 5 minutes. These membranes were not subjected to a 1/20 dilution of Nitro-Block. The membranese were then exposed to x-ray film and developed.
Thus, as can be seen from the results above, electron withdrawing groups added to the aromatic ring of the dioxetane slow the kinetics of light emissions while tending to increase the chemiluminescent signal. In contrast, electron-donating groups accelerate T.sub.1/2 apparently by facilitating electron transfer from the oxygen, through the aromatic group, to the dioxetane. Thus, by proper selection of the nature and ability of the electron-donating or electron-withdrawing Z substituent, and simultaneous selection of the appropriate substituent for the adamantyl ring, if desired, dioxetanes of specific characteristics, including optimized signal intensity, optimized speed, specific emission wavelength, and the like, can be obtained.
These dioxetanes can be used for assays of all types in which an enzyme capable of cleaving the dioxetane can be attached to one element of the ultimate complex which the analyte, if present, will form. Conventional assay formats are known to those of skill in the art, and are described in the patents set forth above in the Background of the Invention. Exemplary disclosure of suitable assays appears in U.S. Pat. No. 5,112,960, and the same is incorporated herein by reference. The assay format, per se, save for the enhanced performance therein by the dioxetanes of this invention, does not constitute an aspect of the invention.
The dioxetanes of this invention, as well as the intermediates therefore, have been disclosed by reference to both generic description and specific embodiment. Additionally, dioxetane performance has been described generally, and exemplified. The examples are not intended as limiting, and should not be construed as such. Variations in substituent pattern, identity, and the like, consistent with the disclosure will occur to those of ordinary skill in the art. Such variations and modifications remain within the scope of the invention, save as excluded by the positive limitations set forth in the claims below.

Number	Name	Date
4931223	Bronstein et al.	Jun 1990
4952707	Edwards et al.	Aug 1990
4956477	Bronstein et al.	Sep 1990
4978614	Bronstein	Dec 1990
5089630	Bronstein et al.	Feb 1992
5112960	Bronstein et al.	May 1992
5145772	Voyta et al.	Sep 1992
5220005	Bronstein	Jun 1993
5326882	Bronstein et al.	Jul 1994
5330900	Bronstein et al.	Jul 1994
5336596	Bronstein et al.	Aug 1994
5538847	Bronstein et al.	Jul 1996

	Number	Date	Country
Parent	574786	Aug 1990
Parent	367772	Jul 1989

	Number	Date	Country
Parent	806928	Dec 1991
Parent	559152	Jul 1990

Chemiluminescent 1,2-dioxetanes

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