AU724148B2

AU724148B2 - Alkane precursors of chemiluminescent dialkyl-substituted 1,2-dioxetane compounds, methods of synthesis and use

Info

Publication number: AU724148B2
Application number: AU36771/97A
Authority: AU
Inventors: Hashem Akhavan-Tafti; Arthur P. Schaap
Original assignee: Wayne State University
Current assignee: Wayne State University; Lumigen Inc
Priority date: 1994-11-23
Filing date: 1997-09-02
Publication date: 2000-09-14
Anticipated expiration: 2015-11-02
Also published as: AU3677197A

Description

(17t, Our Ref: 655257 P/00/011 Regulation 3:2

AUSTRALIA

Patents Act 1990

ORIGINAL

COMPLETE SPECIFICATION STANDARD PATENT 4.

4 b S W A 04o 0RTO Applicant(s): r r Lumigen, Inc. f0 Riverwood Research Center 24485 W Ten Mile Road Southfield Michigan 48034 UNITED STATES OF AMERICA DAVIES COLLISON CAVE Patent Trade Mark Attorneys Level 10, 10 Barrack Street SYDNEY NSW 2000 eebprezr b (OVRI>C~ 9 IC Tc D

.FP~~

Address for Service: Invention Title: Alkane precursors of chemiluminescent dialkyl-substituted 1,2-dioxetane compounds, methods of synthesis and use The following statement is a full description of this invention, including the best method of performing it known to me:- 5020 I AT.AJIE PRECUJRSORS OF CHIMXLUMINRSCN DXLLKYL-SUB8TXTUTUD 1, 2-DIOZANU COMPOUNDS, NNTRODS OF ybYW'IZBS AND V8Z RAC RTNQ OF H NETO FIELnlO THE INVMMTON The present invention relates to chemiluminescent 1. 2-dioxetane compounds that can be triggered by-reagents including enzymes and other chemicals to generate light. Irn particular, the present invention relates to stable aryl group-substituted 1.2-dioxetaries that contain a triggerable x-oxy group (OX) which is a substituent of the aryl group, in which the stable 1,2 -dioxetane forms an unstable dioxetane compound by removal of X, which decomposes to produce light and two -carbonryl compounds.

=ECRIPTION OF RL=iEfl A Prpnaratiori of 1-2-ioxotanera. Kopecky and Mumford *do. reported the first synthesis of a dioxetane (3,3.4-tnimethyl-i, 2-dioxetane by the base-catalyzed cyclization of a ia-bromohydroperoxide, which, irn turn, is prepared from the corresponding alkene R. Kopecky and C. Mumford, Can. J.

*.of*Chem., 47, 709 (1969)). Although this method has been used to produce a variety of alkyl and aryl-substituted 25 1,2-dioxetanes. it can not be used f or the preparation of dioxetanes derived from vinyl ethers, vinyl sul f ides and .enamines.

An alternate synthetic route to 1,2-dioxetanes, especially those, derived from vinyl ethers, vinyl sulf ides and enamines was independently reported by Bartlett and Schaap D. Bartlett and A. P. Schaap, J. Am. Chem. Soc., 92, 3223 (1970)) and Mazur and Foote Mazur and C. S.

Foote, J. Am. Chem. Soc., 92, 3225 (1970)). Photochemical addition of a molecule of oxygen to the appropriate alkene compound in the presence of a photosensitizer produces 1,2-dioxetanes in high yield. This method has been used to produce a large number of dioxetane compounds R.

Kopecky in Chemical and Biological Generation of Excited States, W. Adam and G. Cilento, Academic Press, New York, p. 85, 1982).

Two limitations of this method have been reported.

Certain alkenes with aromatic substituents were found to produce six membered ring peroxides known as endoperoxides on photooxygenation P. Schaap, P. A. Burns and K. A.

Zaklika, J. Am. Chem. Soc., 99, 1270 (1977)). Alkenes with reactive allylic hydrogens frequently undergo an alternate reaction, the "ene reaction, producing an allylic hydroperoxide instead of a dioxetane Baumstark in 20 Advances In Oxygenated Processes, JAI Press, Greenwich, CT, o 1988; Vol.1, pp 31-84).

b. Thermally Stable Dioxetanes from Sterically Hindered SAikenes. The dioxetane derived from the hindered alkene adamantylideneadamantane which was discovered by Wynberg H. Wieringa, J. Strating, H. Wynberg and W. Adam, S. Tetrahedron Lett., 169 (1972) was shown to have an activation energy for decomposition of 37 kcal/mol and a half life 2 at 25 °C of several years J. Turro, G.

4 0 Schuster, H. C. Steinmetzer, G. R. Paler and A. P. Schaap, J. Amer. Chem. Soc., 97, 7110 (1975)). Others have shown that a spiro-fused polycyclic gro .up such as the adazuantyl group can help to increase the stability of dioxetanes derived from amino-substituted alkenes mccapra, 1.

Beheshti, A. Burford,.R. A. Hann and A. Zaklika, J.* Ch~em. Soc.., Chem. Comm., 944. (1977) vinyl ethers (W.

Adam, L. A. Encarnacion and K. Zinner. ,Chem. Ber., 116, 839 (1983)) and vinryl sulfides G. Geller, C. S. Foote and D. 13. Pechman, Tetrahedron Lett., 673 (1983); W. Adam, L.A.

Arias and D. Schuetzow, Tetrahedron Lett., 2835 (1982-))which would be unstable without 'this group.

c. Chemical Trioaerin of Dioxetanan-. The first example.

in the literature is described in relation to the hydxoxysubstituted dioxetane derived from the 2,3-diaryl- 1,4-dioxene P. Schaap and S. Gagnon, J. Amer. Chem.

Soc., 104, 3504 (1982)). However, the hydroxy-substituted dioxetane and any other examples of the dioxetanes derived from the diaryl-1, 4-dioxenes are relatively unstable having e half-lives at 250C of only a few hours. Further, these non-stabilized dioxetanes are destroyed by small quantities of amines Wilson, mnt..Rev. Scl.: Chemi., Ser. Two. 9, 265 (1976)) and metal ions Wilson, M. E. Landis, A. L.

Baumstark, and P. D. Bartlett, Amer. Chem. Soc., 4765 (1973); P. ID. Bartlett, A. L. Baumstark, and M. E.

Landis, J. Amer. Chem. Soc., 96, 5557 (1974)), both comnponents used in the aqueous buffers f or biological assays.

Examples of the chemical triggering of adamantyl- -4stabilized dioxetanes were f irst reported in U. s. patent application P. Schaap, patent application serial No.

887,139*, filed July, 17, 1986) and a paper P. Schaap, T. S. Chen, R. Handley, R. DeSilva, and B. R. Giri, Tetrahedron Lett., 1155 (1987)). These dioxetanes exhibit thermal half-lives of years but can be triggered to produce efficient chemiluminescence on demand. Moderately- stable benzofuranyl dioxetanes substituted with trialkylsilyl and acetyl -protected phenolic groups which produce weak chemiluminescenice have also beep reported- Adam, R. Fell.

N.H. Schulz, Tetrahedron, 49(11), 2227-38 (1993); W. Adam, N.H. Schulz, Chem. Ber.. 125. 2455-61 (1992)). The stabilizing effect of other rigid polycyclic groups has also been reported D. Bartlett and M4. Ho. J. Am. Chem.

Soc., 6 67 (1975); P. Lechtken. Chem. Ber., 109, 2862 (1976)). A PCT application. WO 94/10258 discloses chemical triggering of dioxetanes bearing various rigid polycyclic subs tituents.

d- Enzvnatir Trigapirina of Ad iatyl Diaxeetanos- 20 Dioxetanes which can be triggered by an enzyme to undergo chemiluminescent decomposition are-disclosed in U. S.

-patent application P. Schaap, pa tent application.

No. EP 254051 and a series of papers P. Schaap, R. S.

Handley, and B. P. Giri. Tetrahedron Lett., 935 (19867); A.

P. Schaap, Sandison, and Rt. S. Handley, Tetrahedron Lett., 1159 (1987) and A. P. Schaap, Photochem. Photobiol..

A2.L 50S (1988)).,The highly st able adamantyl-substituted dioxetanes bearing a protected aryloxide substituent are triggered to decompose with emission of light by the action of an enzyme in an aqueous buffer to give a strongly electron-donating aryloxide anion which dramatically increases the rate of decomposition of the dioxetane. As a result, chemiluminescence is emitted at intensities several orders of magnitude above that resulting from slow thermal decomposition of the protected form of the dioxetane. U.S.

Patent No. 5,068,339 to Schaap discloses enzymatically triggerable dioxetanes with covalently linked fluorescer groups. Decomposition of these dioxetanes results in enhanced and red-shifted chemiluminescence through intramolecular energy transfer to the fluorescer. U.S. Patent No. 4,952,707 to Edwards discloses enzymatically triggerable dioxetanes bearing an adamantyl group and 2,5- or 2,7-disubstituted naphthyl groups. U.S. Patent Nos.

5,112,960, 5,220,005, 5,326,882 and a PCT application (88 00695) to Bronstein disclose triggerable dioxetanes bearing adamantyl groups substituted with various groups including chlorine, bromine carboxyl, hydroxyl, methoxy and methylene 20 groups. A publication Ryan, J.C. Huang, O.H. Griffith, J.F. Keana, J.J. Volwerk, Anal. Biochem., 214(2), 548-56 (1993)) discloses a phosphodiester-substituted dioxetane which is triggered by the enzyme phospholipase. U.S. Patent 5,132,204 to Urdea discloses dioxetanes which require two 25 different enzymes to sequentially remove two linked protecting groups in order to trigger the chemiluminescent decomposition. U.S. Patent 5,248,618 to Haces discloses dioxetanes which are enzymatically or chemically triggered -6to unmask a first protecting group generating an intermediate which spontaneously undergoes an intramolecular reaction to split off a second protecting group in order to trigger the chemiluminescent decomposition.

e. Enhanced Chemiluminescence from Dioxetanes in the Presence of Surfactants. Enhancement of chemiluminescence from the enzyme-triggered decomposition of a stable 1,2-dioxetane in the presence of water-soluble substances including an ammonium surfactant and a fluorescer has been reported P. Schaap, H. Akhavan and L. J. Romano, Clin.

Chem., 35(9), 1863 (1989)). Fluorescent micelles consisting of cetyltrimethylammonium bromide (CTAB) and anoyl)aminofluorescein capture the intermediate hydroxysubstituted dioxetane and lead to a 400-fold increase in the chemiluminescence quantum yield by virtue of an efficient transfer of energy from the anionic form of the excited state ester to the fluorescein compound within the •hydrophobic environment of the micelle.

U. S. Patents 4,959,182 and 5,004,565 to Schaap describe 20 additional examples of enhancement of chemiluminescence from chemical and enzymatic triggering of stable dioxetanes in the presence of the quaternary ammonium surfactant CTAB and fluorescers. Fluorescent micelles formed from CTAB and either the fluorescein surfactant described above or 25 1-hexadecyl-6-hydroxybenzothiazamide enhance chemiluminescence from the base-triggered decomposition of hydroxyand acetoxy-substituted dioxetanes. It was also reported that CTAB itself can enhance the chemiluminescence of a phosphate-substituted dioxetane.

U.S. Patent No. 5,145,772 to Voyta discloses enhancement of enzymatically generated cheumiluminesceflce from 1, 2-dioxetazles in the presence of polymers with pendant quaternary amumoniuma groups alone or admixed with fluorescein. other substances reported to enhance chemilumiflesceflce include globular proteins such as bovine albumin and quaternlary azrmtoniumf surfactants. Other cationic polymer compounds were of modest effectiveness as chemilumfiflescence enhancers; nonionic polymeric compounds were generally ineffective and the only anionic polymersignificantly decreased light emission. A PCT application :0 WO 94/21821 discloses enhancement from the combination of a polymeric aummonium salt surf actant, and an enhancement additive. European -Patent Application wo. 0561033 to Aihavan-Tafti published on-Sept. 22, 1993 discloses enhancement of enzymat ical ly g enerated chemi luminescence from 1.2-dioxetales in the presence of polyvinyl phosphonium salts anid polyvinyl phosphonium salts to which ~20 fluorescent energy acceptors are covalently attached.

0U.S., Patent No. 5,451,347 to gooAkhavaf-Tafti filed June 24, 1993 discloses enhancement of 00 enzymatically generated chemi luminlescence from 1 .2-dioxetanes in the presence of dicationic phosphoniufl salts.

Triggerable stabilized dioxetanes known in the art.

incorporate a rigid spiro-fused polycycli'c substituerit or a substituted spiroadamfantyl substituent. The ketone starting -amaterials from which these dioxetanes are prepared are relatively expensive and are of limited availability or must be prepared from costly precursors. No examples of stable triggerable dioxetaries bearing two alkyl grouips in place of rigid spiro-fused polycyclic organic groups are known. Such tr iggerable stabilized dioxetanes can be prepared from inexpensive, readily available starting materials and will therefore provide cost advantages facilitating their commercial potential.

SUMMARY OF THE INVENTION The present invention as claimed relates to compounds having the formula: 02 R, anis selected idenetl from alrancdhed grup ayladccolygrpscontaining to 12 carbon atoms, rakyiyoy an a nld diinlsbttet.wherein R is anoraigruOh2slt -9- R-D-galactosidoxy and R-D-glUcuroflidyloXy groups.

IN THE~ DR AWINS Figure'1 is a spectrum of the chemiluminescence emitted from a solution of dioxetane 2c in dimethyl suif oxide (mKoo) when triggered by addition of a solution of potassium hydroxide in a mixture of methanol and dimethyl suif oxide. The spectrum is corrected for the decay in 6gS 15 chemilumiflescence intensity occurring during the scan.

Figure 2 is a graph of chemiluminescence intensity as a function of time produced by triggering a 10 IlL aliquot of a 10-6 z4 solution of dioxetane 2g with 50 IlL of 1 M4 tetranbutylamUonium f luoride in VSO.

20 Figure 3 is a graph showing a comparison of the time profile of the chemilullinescence intensity emitted by 100 pL of solutions containing either dioxetane 2f of the present invention or 2k CLUMIGEN PPD, Lumigen. Inc., **:Southfield, MI) triggered at 31 OC by addition of 1.12 x 10-17- mol of AP. The reagents consist of 1) a 0.33

M

solution of dioxetane 2f in 0.2 14 2-amino-2-methyl- 1-propanol buffer-, pH 9.6. and 2) a 0.33 mm solution- of dioxetane 2k in 0.2 14 2.amino-2-methyl-1-propalol buffer, pH 9.6. Use of dioxetale 2f of the present invention advantageously achieves a higher maximum intensity compared to dioxetane 2k.

Figure 4 is a graph showing a comparison of the time profile of the chemil umi lCSeflcBn intensity emitted by 100.

pL of'solutions, containing either dioxetane 2f or 2k triggered at 37 *C by addition of 1.12 x 10-~ .7Mol Of AP.

The reagents consist of 1) a 0.33 MM solution of dioxetane 2f in 0.2 M4 2-amino-2-methyl-4-propalol buffer, pH 9.6 containing 1.0 mg/mL of the enhancer l-(tri-n-octylphosphoniunifethyl) (tri -n-butylphosphoniummethyl) benzene dichloride (Enhancer and 2) a 0.33 inK solution 9f dioxetane 2k in 0.2 14 2-amino-2-methyll-propalol buffer, pH 9.6 containing 1.0 mg/uL of the same enhancer. use of dioxetane 2f of the present invention advantageously achieves higher light intensities at all time points compared to dioxetane 2k.

Figure 5 is a graph showing a comparison of the time profile of the chemiluminescence intensity emitted by 100 pL of another pair of s olutions containing either dioxetane 2f or 2k triggered at 37 *C by addition of 1.12 x 10-'7 mol of AP. The reagents consist of 1) a 0.33 mM solution of dioxetane 2f in 0.2 M4 2-amino-2-etl- 1-propanol buffer, pH 9.6 containing 0.5 mg/rnL of polyvinylbenzyltributylphosphonium chloride (EnhancersB) and 2) a 0.33 MM solution of dioxetafie 2k in 0.2 M4 2-amino-2-methyl-lipropanol -11buffer, pH 9.6 containing 0.5 mg/mL of the same enhancer.

The preparation of Enhancer B is described in European Patent Application 561,033 published September 22, 1993.

Use of dioxetane 2f of the present invention advantageously achieves higher light intensities at all time points compared to dioxetane 2k.

Figure 6 is a graph showing a comparison of the time profile of the chemiluminescence intensity emitted by 100 pL of another pair of solutions containing either dioxetane 2f or 2k triggered at 37 °C by addition of 1.12 x 10-17 mol of AP. The reagents consist of 1) a 0.33 mM solution of dioxetane 2f in 0.2 M 2-amino-2-methyl- 1-propanol buffer, pH 9.6 containing 0.5 mg/mL of polyvinylbenzyltributylphosphonium chloride co-polyvinylbenzyltrioctylphosphonium chloride (containing a 3:1 ratio of tributyl:trioctyl groups) (Enhancer C) and 2) a 0.33 mM solution of dioxetane 2k in 0.2 M 2-amino-2-methyl-l-propanol buffer, pH 9.6 containing 0.5 mg/mL of the same enhancer. The preparation of Enhancer C is described in European Patent Application 20 561,033. Use of dioxetane 2f of the present invention o* S" advantageously achieves higher light intensities at all time points compared to dioxetane 2k.

Figure 7 is a graph relating the maximum chemiluminescence intensity emitted by 100 pL of a reagent containing 25 dioxetane 2f triggered at 37 °C to the amount of AP.

Chemiluminescence emission was initiated at 37 °C by addition of 3 pL of solutions of AP containing between 3.36 -12x 10 16 mol and 3.36 x 10- 22 of enzyme to 100 pL of a 0.33 mM solution of dioxetane 2f in 2-amino-2-methyl-l-propanol buffer, 0.2 M (pH 9.6) containing 1.0 mg/mL of Enhancer A.

The term S-B refers to the chemiluminescence signal in Relative Light Units (RLU) in the presence of AP corrected for background chemiluminescence in the absence of AP.

The graph shows the linear detection of alkaline phosphatase. The calculated detection limit (twice the standard deviation of the background) was determined to be 1.4 x 10 22 mol or less than 100 molecules of alkaline phosphatase under these conditions.

Figure 8 is a digitally scanned image of an X-ray film from an experiment detecting alkaline phosphatase on a membrane with chemiluminescence. Solutions of alkaline phosphatase in water containing from 1.1 x 10-15 to 1.1 x 18 mol were applied to identical nylon membranes (Micron Separations Inc., Westboro, MA). The membranes were air dried for 5 min and soaked briefly with a reagent .containing 1 mg/mL of Enhancer A in 0.2 M 2-amino-2-methyl- 1-propanol buffer, pH 9.6 containing 0.88 mM MgCl and either 0.33 mM dioxetane 2f or 0.33 mM dioxetane 2k. The membrane was placed between transparent plastic sheets and exposed to X-ray film (Kodak X-OMAT AR, Rochester, NY). In a comparison of the two reagents, the light produced using dioxetane 2f of the present invention led equivalent images and detection sensitivity. These results illustrate the performance of dioxetane 2f which is to be expected in -13- Western blotting, Southern blotting. DNA fingerprinting and other blotting applications.

D-fSCRPTTON OF THE PREPERRID EMBODIMENTS The present invention relates to compositions containing a stable 1.2-dioxetane which can be triggered by reagents, including enzymes and other chemicals, to generate chemiluminescence. Stable dioxetanes useful in practicing the present invention may be of the formula: 0--0 0, R ?>LjORl 3^ R 1

R

4

R

2 0-X wherein R 3 and R 4 are nonspiro-fused organic groups, wherein R, is an organic group which may be combined with R 2 and wherein R 2 represents an aryl group substituted with an X-oxy group which forms an unstable oxide intermediate dioxetane compound when triggered to remove a chemically labile group X by a reagent, including enzymes and other e. .oO .chemicals. The unstable oxide intermediate dioxetane a decomposes and releases electronic energy to form light and 20 two carbonyl containing compounds of the formula ano OR, R" a n d OMMI R20 R2

O

R

4 25 A preferred method of practicing the present invention uses ~a stable dioxetane of the formula: *e -14- OR 0OR R; R 2 0-X wherein Ri is selected from alkyl, cycloalkyl and aryl groups containing 1 to 12 carbon atoms which may additionally contain heteroatoms,

R

3 and R4 are selected from branched chain alkyl and cycloalkyl groups containing 3 to 8 carbon atoms and may additionally contain heteroatoms and which provide thermal stability, and wherein R 2 is selected from aryl, biaryl, heteroaryl, fused ring polycyclic aryl or heteroaryl groups which can be substituted or unsubstituted and wherein OX is an X-oxy group which forms an unstable oxide intermediate dioxetane compound when triggered to remove a chemically labile group X by a reagent including enzymes and other chemicals.

15 The stable 1,2-dioxetane compounds have relatively long half-lives at room temperature (20-30 even though they can be triggered by chemical reagents. Previous examples of d Sstable, triggerable 1,2-dioxetanes all made use of rigid spiro-fused polycyclic alkyl groups such as adamantyl and substituted adamantyl to confer thermal stability. It has now been discovered that 1,2-dioxetanes bearing a wider range of substituents corresponding to R 3 and R4 in the structure above also exhibit substantial thermal stability at room temperature. Dioxetane compounds substituted with alkyl groups containing as few as 3 carbons (as substituents R 3 and R 4 in the structure above) have half-lives of approximately one year at room temperature and several years at 4 R 3 and R 4 groups whose carbon atom attached to the dioxetane ring carbon is substituted with zero or one hydrogen atoms isopropyl, sec-butyl, t-butyl, cycloalkyl) provide enough thermal stability to the dioxetane compounds to render them useful for practical applications.

R

3 and R 4 groups which are linked to the dioxetane ring through a CH2 group but which are otherwise bulky, for example a neo-pentyl group, are considered to be within the scope of the invention. Further, these dioxetanes can be triggered by the removal of an X group to decompose with emission of light. The degree of rate enhancement upon triggering depends on such factors as the lability of the X group, the amount of the triggering 15 reagent, choice of solvent, pH and temperature. By selecting appropriate conditions, a factor of 106 or greater rate enhancement can be achieved.

The present invention relates to a process using readily available or inexpensive starting materials for preparing a 20 stable 1,2-dioxetane of the formula: O--0

OR

R

3 |L -1 4

R

2 0-X a* wherein

R

3 and R 4 are nonspiro-fused organic groups, wherein Ri is an organic group which may be combined with R, and wherein

R

2 represents an aryl group substituted with an X-oxy group by addition of oxygen to the appropriate -16alkene. An unexpected finding of the present invention is that the alkenes reported here readily undergo photochemical addition of a molecule of oxygen (as singlet oxygen 102) to produce the corresponding 1,2-dioxetane. It is well known in the literature that alkenes bearing allylic hydrogens may preferentially undergo addition of singlet oxygen by a different reaction path to produce an allylichydro-peroxide, dioxetane formation is a minor process at most.

3

OOH

R3

R

1 02 light R3 Ri ene reaction

R

4

R

2 sensitizer R4 R2 hydroperoxide

R

3 R, 02 light 3 1 R -a s2 2 cycloaddition

R

4 ^sensitizer R4 8 2 4 2 dioxetane The requisite alkene compounds are synthesized through coupling arylcarboylate esters substituted with an X-oxy

S*OR

o0 and 0 in the presence of lithium aluminum hydride, other metal 25 hydride, zinc metal or zinc-copper couple in a polar aprotic organic solvent, preferably tetrahydrofuran, with a transition metal halide salt, preferably a titanium chloride compound, and a tertiary amine base. The reaction is -17generally conducted in refluxing tetrahydrofuran and usually goes to completion in about 2 to 24 hours. A significant advantage of the present process is the ability to conduct the reaction on a large scale due to the availability of the ketone starting materials in large quantity.

Triggerable dioxetanes in commercial use are prepared from adamantanone or a substituted adamantanone compound.

Adamantanone is relatively costly. Substituted adamantanones are even more expensive and of more limited supply. In comparison to the preparation of adamantanone, which involves a laborious procedure involving large quantities of dangerous oxidizing materials, alkyl and cycloalkyl ketones are readily prepared in large quantities by standard techniques. Another advantage is the reduced cost of certain of the ketone starting materials. Diisopropyl ketone, for example, is between 15 and 20 times less expensive than adamantanone on a molar basis.

The triggering reagent may be a chemical which requires 1 equivalent or a catalyst such as an enzyme wherein 20 only a small amount is used. Electron donors, organic and •inorganic bases, nucleophilic reagents and reducing agents can be used to remove X.The triggering reagent may also be an enzyme selected from but not limited to phosphatase enzymes, esterase enzymes, cholinesterase enzymes, hydro- 25 lytic enzymes such as a- and 0-galactosidase, a- and P-glucosidase, glucuronidase, trypsin and chymotrypsin.

The OX group may include, without limitation, hydroxyl, -18- OOCR, wherein R 6 is an alkyl or aryl group containing 2 to carbon atoms either of which may contain heteroatoms,.

trialkylsilyloxy, triarylsilyloxy, aryldialkylsilyloxy, OPO3-2 salt, OS03- salt, S-D-galactosidoxy and f-D-glucuronidyloxy groups.

The present invention relates to a method for generating light which comprises providing a chemical reagent and a stable 1,2-dioxetane of the formula: O-0 OR

R

4

R

2 0-X wherein Rs and R 4 are organic groups which are selected from lower alkyl or cycloalkyl containing 3 to 8 carbon atoms and which provide thermal stability, wherein R, is an organic group which may be combined with R, and wherein R, represents an aryl group substituted with an X-oxy group which forms an unstable oxide intermediate dioxetane compound when triggered to remove a chemically labile group *.fl 20 X by a reagent including enzymes and other chemicals wherein the unstable oxide intermediate dioxetane decomposes and releases electronic energy to form light and two carbonyl containing compounds of the formula: R3/° 0 and 0o O R R The present invention also relates to a method for -19detecting triggering reagents selected from chemical reagents including enzymes. In this instance the dioxetane is used as the reagent.

Further the present invention relates to a method and compositions for the detection of enzymes, in immunoassays, e. g. ELISA and the detection of enzyme-linked DNA or RNA probes. Detection of the light emitted may be readily performed using a luminometer, X-ray film or with a camera and photographic film.

EXAMPLES

Nuclear magnetic resonance (NMR) spectra were obtained on a GE QE300 or a Varian Gemini 300 spectrometer as solutions in CDC13 with tetramethylsilane as internal standard 15 or as solutions in CD 3 OD or D 2 0. Mass spectra were obtained on an AEI MS-90" spectrometer.

Examne1 Synthsis_ of J-3-t-Btldimethylsilv nhenvl) -2.2-diiso rounvl-l-methoxvethene (1a).

OCHS

OSi(CH 3 2 t-Bu A three neck flask was purged with argon and charged with 100 mL of anhydrous tetrahydrofuran (THF). The flask was cooled in an ice bath and titanium trichloride (18 g) was added with stirring. Lithium aluminum hydride (2.2 g) was added in small portions causing a brief exothermic reaction. After all of the lithium aluminum hydride was added the cooling bath was removed and triethylamine (16 ml) was added. The black mixture was refluxed for one hour under argon. A solution of 2,4-dimethyl-3-propanone (3.86 g) and methyl 3-t-butyl-dimethylsilyloxybenzoate (3.00 g) in 10 mL of dry THF was added dropwise over 2 hours.

Reaction progress was monitored by TLC on silica plates eluting with 4% ethyl acetate/hexane. The crude reaction mixture was cooled to room temperature and diluted with hexane and decanted. The residue was washed several times using a total of ca. 700 mL of hexane. The combined hexane solutions were filtered and evaporated leaving an oil which was purified by column chromatography on silica gel, eluting with hexane yielding 2.12 g (54 of la: 'H NMR (CDC13) 8 7.3-6.7 4H), 3.18 3H), 2.45 (sept, 1H, J=7.2 Hz), 2.31 (sept, 1H, J=7.2 Hz), 1.24 6H, J=7.2 Hz), 0.99 3H), 0.91 6H, J=7.2 Hz), 0.19 3H); 20 13 C NMR (CDC1 3 6 128.76, 122.84, 121.46, 119.28, 56.06, 30.32, 26.54, 25.56, 21.91, 20.86, -4.58; Mass spectrum 348, 333, 306; exact mass, calc'd. 348.2484, found 348.2479.

*o~ -21- Examnle 2. Synthesis of 2.2-Diisnrnvl-l-(3-hvdroxynphnvl) -l-methoxvethene (Ib

OCH

3

OH

To a solution of 0.97 g (2.78 mmol) of alkene la in ml of dry THF was added 0.81 g (1.1 eq.) of tetra-n-butylammonium fluoride. After stirring for one hour TLC (silica, ethyl acetate/hexane) showed complete conversion of starting material to a new compound. The THF was evaporated and the residue dissolved in ethyl acetate. The ethyl acetate solution was extracted four times with water and dried. Silica gel (2 g) was added and the solvent evaporated. The material was purified by column chromatography on silica gel, eluting with 10 20 ethyl acetate/hexane yielding 0.568 g (87 of lb: H NMR (CDC1 3 8 7.5-6.5 (m, 4H), 4.91 1H), 3.20 3H), 2.47 (sept, 1H), 2.33 (sept. 1H). 1.25 6H), 0.92 6H); "C NMR (CDC1 3 8 129.25, 124.98, 122.60, 116.54, 114.98, 114.56, 56.38, 30.52, 26.80, 22.11, 21.08; Mass spectrum 234, 219, 191; exact mass, calc'd. 234.1620, found 234.1620.

-22- Examnle synthpnin of 1-(3-Acetoxflhnl)-2.2-diisornroov1- 1~methoxvt-hofe (1c).

OCH

3 oCOCH 3 Alkene lb (200 mg. 0.85 nunol) was dissolved in 20 rnL of dry methylene chloride with 0.31liL of anhydrous pyr idine.

The f lask was purged with argon and cooled in an ice bath.

Acetyl chloride (0.115 g, 1.47 nunol) in 5 mL of dry' methylene chloride was added dropwise over one hour. TLC analysis (silica, 20 ethyl acetate/hexane) indicated the reaction to be complete after 2.5 hours of stirring at 0 OC. The solvents were evaporated and the residue dissolved in ethyl acetate. The solution was washed four times with dried over M4gSO, and evaporated. The residue was .22 purified by column chromatography on silica gel, eluting with 10-20 ethyl acetate/hexale yielding 220 mg (93 of lc: 1H NMR (CDCl 3 8 7.37-6.99 4H), 3.19 3H), 2.47 (sept, 1H, J=6.9 Hz), 2.33 (Sept, 1H, J=6.9 Hz), 2.29 (s, 3H), 1.24 6 H, J=6.9 Hz), 0.93 6 H, J=6.9 Hz); 13

C

Nm (cDCl 3 8 169.46, 150.62, 14.9.03, 139.02, 133.64.

128.95, 127.24, 122.89, 120.72, 56.50, 30.49, 26.98, 22.06, 21.25, 21.05.

-23- Exanmi 4. synthpnin of 1-13 -Benzavlovnhanvl) 2 -diinnr~rcmvl I imethnxv~thPne (1d).

OCHa OCOPh Alkene lb (4.5 g, 1.9 mmol) was dissolved in 50 rob of dry CH 2 Cl 2 With 5.3 mL of anhydrous triethylamine. The flask was purged with argon and cooled in an ice bath.

Benzoyl chloride (4.05 g, 2.9 no1) was added dropwise. The cooling bath was removed and stirring continued for 1 hour at room temperature. The mixture was filtered and the solution was washed with water, dried over MgSO& and evaporated. The residue was suspended in hexane, the solid filtered away and the solution evaporated. The residue was purified by column chromatography on silica gel, eluting .:..with 1% ethyl acetate in hexane yielding 3.7 g of dioxetane id: 1H NW (CDCl 3 8.25-7.05 (in, 9H), 3.25 1H), 2.54 (sept, JR, j=6.9 Hz), 2.40 (sept, 1H, J=6.9 Hz), 2.29 (s, 3H), 1.26 6 H, J=6.9 Hz), 0.95 6 H, J=6.9 Hz).

-24ixainns 5. nthonin of i-(3-Pivalovloxvinhenv1)-2.2-iiiAo- OCOC (013 Alkene lb (2 9, 8.6 mmol) was dissolved in 50, mL of dry

CH

2 Cl 2 with 2.4 niL of anhydrous triethylamine. The flask was purged with argon and cooled in an ice bath. Pivaloyl chloride (1.6 g. 2 eq.) was added dropwise over one hour.

The cooling bath was removed and stirring continued for 3 hours at room temperature. The solution was washed with aq.

15 K 2 C0 3 and then water, dried over MgSO. and evaporated. The residue was purified by colunn chromatography on silica eluting with 5% triethylamine in hexane yielding.1.95 g of dioxetane le: 1H NMR (CDC1 3 8 7.34-6.98 (in, 4H) 3.198 0 3H), 2.47 (sept, 1H), 2.33 (sept, 1H), 1.36 9H), 1.24 6 H. J=6.9 Hz), 0.92 6 H, J=6.9 Hz).

Exainli 6. Synthesin of -22-iiR0Dron1'1-m&LhoV-1 (3-nhosTnhnrylnxvohenryl) thene. dinodijim salt (iflo .*25

OCH

0 1

*(S

A solution of 9 mL of dry CHC1 2 and 0.7 mL of anhydrous pyridine (8.7 mmol) was purged with argon and cooled in an ice bath. Phosphorus oxychloride (0.40 g, 2.6 rmmol) was added followed after 5 min by a solution of alkene lb (209 mg, 0.87 mmol) in 0.4 mL of pyridine. The solution was stirred at room temperature for 1 hour. TLC analysis (silica, 30 ethyl acetate/hexane) indicated the reaction to be complete. The solvents were evaporated and the residue taken on to the nest step.

The product from step was dissolved in CH 2 C1 2 and 0.7 mL of pyridine added. The solution was cooled in an ice bath and treated with 618 ag of 2-cyanoethanol (8.7 mmol). The ice bath was removed and stirring continued at room temperature for two hours. The mixture was then concentrated and the residue was purified by column chromatography on silica gel, eluting with a gradient of ethyl acetate in hexane to 100% ethyl acetate yielding of the bis(cyanoethyl phosphate) H NMR (CDC13) 8 0.934 (d, 6H, J=9 Hz), 1.235 6H, J=9 Hz), 2.28-2.45 2H), 20 2.76-2.82 4H), 3.18 1H), 4.31-4.47 4H), 7.11-7.38 4H).

The bis(cyanoethyl phosphate) alkene (420 ag) was dissolved in 4 mL of acetone. Sodium hydroxide (65 mg) was dissolved in 1 mL of water and added to the acetone solution which was then stirred over night. The precipitate was collected and dried to a white powder. 1H NMR (D20) 8 0.907 3H), 0.929 3H), 1.20 3H), 1.22 3H), 2.35- 2.46 2H), 3.23 1H), 6.96-7.37 4H); C NMR (D 2 0) -26- 8 155.15 149.56, 137.84, 136.08, 129.71, 124.55, 122.33 120.48, 57.16, 31.27, 27.21, 22.59, 21.10; 3p nMR (D 2 0) (rel. To ext. H 3

PO

4 6 0.345.

xamnle 7. Svnthesis of 1-(3-t-Butvldimethvl1ilvloxvnhPnvl) -22-dicvclorouvl- 1-methoxvethene (la).

OCH

2 OSi (CH3) 2 t-Bu A three neck flask was purged with argon and charged with 50 mL of anhydrous THF. The flask was cooled in an ice 15 bath and titanium trichloride (11.6 g) was added with stirring. Lithium aluminum hydride (1.4 g) was added in small portions causing a brief exothermic reaction. while the lithium aluminum hydride was being added, an additional mL portion of anhydrous THF was added to aid stirring.

20 The cooling bath was removed when the addition was complete and the black mixture was brought to reflux. Triethylamine (10.5 ml) was added and the black mixture was refluxed for one hour under argon. A solution of dicyclopropyl ketone (2.61 g) and methyl 3-t-butyldimethylsilyloxybenzoate (2.00 g) in 20 mL of dry THF was added dropwise over 75 min. The reaction was judged complete after an additional 1 hour reflux period as monitored by TLC on silica plates eluting with 5% ethyl acetate/hexane. The crude reaction mixture -27 was cooled to room temperature and extracted with four 400 mL portions of hexane. The combined hexane solutions were filtered and evaporated leaving 1.42 g of a yellow oil which was purified by column chromatography on silica gel, eluting first with hexane and then with 20 ethyl acetate/hexane to elute the product alkene la. Further purification was achieved by preparative TLC on silica eluting with 5% ethyl acetate /heae. 1 HNMR (CDCl 3 6 7.3-6.7 (in, 4H), 3.36 3H), 1.80 1H), 1.13 1H), 0.988 Cs, 9H), 0.78-0.67 4H), 0.43-0.37 Cm, 2H). 0.19 6H), 0.11-0.05 2H).

Example Svrthgin of 1-3i-Tirvdmty-iyoy rhenvl) 2-dicvr1ohexv1- 1-mthgyvetheng I 1h).

OSi(CH 3 2 t-BU A mixture of 4.3 g of methyl 3-t-butyldiinethylsilyloxybenzoate and 9.5 g of dicyclohexyl ketone in dry THF were coupled according to the procedure of Example 5 using the Ti reagent made from 25 g of TiCl 3 3 3.0 g of LiAlH 4 and 16.4 g of triethylamine in 150 niL of dry TEF. The crude product mixture (10 g) obtained af ter hexane extraction was purified by column chromatography-on silica gel, eluting -28f irst with hexane, followed by 1 ethyl acetate/hexane and then with 3 ethyl acetate/ hexane. The yield was 3.5 g -(51 of alkene is: 1H lOR (cC1C 3 8 7..22-7.16 1H1), 6.85-6.72 311), 3.155 311), 2.05-0.86 (in, 22H1), 0.995 9H) 0 .21 6H) Exahmla 9- Svrnthanin of 2. 2-Di C~ h@x1-1- (3-hvdrOyVnhiPvl) -l-methnxyethene (1i) .is1 To a solution of 0.7 g of alkene if in dry THF was added 0.62 g (1.2 eq.) of tetra-n-butylamnmiumf fluoride *dropwise. Af ter stirring for one hour TLC (silica, ethyl acetate/hexale) showed complete conversion of starting material to a new compound. The THF was evaporated and 2>20 the residue dissolved in ethyl acetate. The ethyl acetate .solution was extracted with water and dried over MgSO 4 The material was purified by colun chromatography on silica gel, eluting with 0 -10 %ethyl acetate/hexale yielding 0.46 g (92 of If. The alkene was further purified by crystallization in benzeie/hexarle at 4 1H NMR (CDCl 3 8 7.20-6.72 411), 4.72 1H1), 3.174 Cs, 311), 2.06-1.04 22H1); 13~ CI (CDCl 3 8 155.19, 138.20, 131.97, 129.05, 122.50, 116.36, 114.39, 56.48, 41.51, -29- 39.34, 31.40, 30.92, 27.50, 26.37, 26.25, 25.99; Mass spectrum 314, 231, 121; exact mass, calc'd.

314.2246, found 314.2246.

TahlP 1. ninxetafle rnmnarnds 2a 2b 2c 2d 2e 2f 2g 2h 2i 2j 2k

CH(CR

3

CHCH)

CH (CH 3 2 CH (CHO) 2 CH (CH 3 2 CH (CHO 2 CH (CH 3 2 CH (CHO) 2 CH (CHO) 2 CH (CHO) 2 CH (CH 3 2 CH (CH 3 2

C-C

3 Hs c-C 3

H

5 c-C6HIl c-C 6

HII

c-C6Hln CC6I adamantYl adamantYl Si (CH 3 2 t-DU

H

COCH

3 COPh COC WCH 3

PO

3 Na 2 Si (CHO) 2 t-BU Si (CHO 2 t-BU

H

Si (CHO) 2 t-BU P0 3 Na 2 Examnle 10. synthesis of 1.2-Dinxetanes Photooxygenation procedure. Method A. Typically a 100 mg sample of the alkene was dissolved in 20 mL of a 1:1 mixture of methanol and methylene chloride in a photooxygenation tube. Approximately 200 mg of polystyrene-bound Rose Bengal was added and an oxygen bubbler connected.

Oxygen was passed slowly through the apparatus while immersed in a half-silvered Dewar flask containing either Dry Ice/2-propanol or ice water. The sample was irradiated with a 400 w sodium lamp (GE Lucalox) through a film of mil Kapton (DuPont, Wilmington, DE) as UV cutoff filter while continuously bubbling oxygen. Progress of the reaction was monitored by TLC or H NMR. The dioxetane compound was isolated by filtering off the polymer-bound sensitizer 15 and evaporating the solvent at room temperature. Further purification could be achieved by column chromatography on silica gel or crystallization from a suitable solvent as necessary.

1 Method B. Alternatively, methylene blue was used in some 20 cases as photosensitizer. Approximately 100 mg was dissolved in 10 mL of the reaction solvent and irradiation proceeded as described above. The dioxetanes prepared in this manner were purified by column chromatography on silica gel.

-31- EXAHMr 11. Synthanis of 4-(3-t-RitvldimorthV1Rilvloxv- 0- 0Si(CH 3 2 t-BU A 102.8 mg sample of the alkene was photooxygenated for a total of 9 hours by method B at -78 OC. The solvent was evaporated and the mixture purified by preparative

TLC

using 4 ethyl acetate/hexafle to elute the plate.-The' yield of dioxetane 2a was 55.9 mg (50 1H iOR (CDCl 3 )6 7.6-6.7 4H1), 3.14 Cs, 3H), 2.61 (sept. 1H), 2.46 (sept, 111), 1.30 1H1). 1.18 Cd, iN), 1.00 3H),0.92 1H1), 0.46 1H), 0.20 3H1); 13 C NOR (CDCl 3 8 155.88, 137.07, 129.41, 114.526, 98.57, 49.46, 33.51, 29.24. 25.79, 19.43, 18.51, 17.29. 16.69, -4.32.

Rxample 127. Synthanis of Di imorropv1- 4 -hydroxy bhnj--~hx-.-ixtn MIb.

0-

OH

Alkene lb (83.2 mg) was photooxygeflated for a total of 3 hours by method B at -78 The solvent was evaporated.

the residue dissolved in ethyl acetate and the mixture -32purified by preparative TLC using 20 ethyl acetate/hexane to elute the plate. The yield of dioxetane 2b was 79 mg -(-84 1 H NOR (CDCl 3 8 7.4-6.8 4H1), 3.2 3H), 2.62 (sept, 1H), 2.48 (sept, 1Hf), 2.08 (801H), 1.30 3H1), 1.17 3H), 0. 90 3H), 0 .47 3H) 1 3 C IOH (CDCl 3 )8 156.00, 137.21, 129.70, 116.41, 114.61, 98.97, 49.58, 33.55, 29.35, 19.46. 18.56, 17.31, 16.65.

Examnio 11- synthorais of 4-C 3-Acertaxvnhonfli-3. 3-dun-s nrorgnv14-m-thoxV-l1 2-ixtane (2c).

150 .OCOCH 3 A 63 mig sample of the alkene was photooxygenated f or a total of 6.5 hours by method B at -78 OC. The solvent was evaporated, the residue dissolved in ethyl acetate and the mixture purified by preparative TLC using 20 ethyl acetate/hexafle to elute the plate. The yield of dioxetane 2c was 56 mig (80 1 H NM4 (CDCl 3 8 7.37-6.99 4H1), 3.14 3M), 2.59-2.42 CM 2H), 2.32 3H), 1.30 3M, J=7.2 Hz), 1.17 311, J=7.2 Hz) 0.91 3H, J=7.2 Hz), 0.46 3H, J=7.2 Hz) 1 3C NM (CDC1 3 8 150.89, 137.34, 129.39, 12V.73, 114.07, 98.34, 49.60, 33.54, 29.3-1, 21.22, 19.44, 18.53, 17.17, 16.59.

-33- Exainne 14- Syntbta-gir of 4- 13-8enzov~oxynh~nv1) 1-diinnprony1-4-MathoCV&2 dinxptano (2dL.

0-

OCH

3 ocoPh A 3.7 g sample of the alkene was photooxygenated for a total of 19 hours by method B at -78 *C using 500 niL of a 1:1 mixture of acetone and CH 2 Cl 2 and 100 ug-of methylene blue. Progress of the reaction was monitored by 111 NkR.- The solvent was evaporated, the residue dissolved in ethyl acetate and the mixture purified by column chromatography using hexane as eluent. 1H1 NHR (CDCl 3 8 8.22-7.0 Cmt, 911), 3.184 Cs. 3H1), 2.62-2.46 2H1), 1.3.0 3H, J=7.2 Hz), 1.20 3H,. J=7.2 Hz), 0.94 3H, J=7.2 0.52 (d, 3H,. J=7.2 Hz); 13 C HMI (CDC1 3 8 151.09, 137.32, 133.72, 130.18, 129.33, 128.61, 122.66. 98.24, 49.48. 33.44. 29.22.

19.32, 18.42. 17.11, 16.48.

Examnio is;. syntbhe3io of 4(3-PivaOvloxflhn1W13.3diiso *riy--oh~y12Aooai 2a).

0- OCH3 250 OCOC (C1 3 3 A 1.95 g sample of the alkene was photooxygeflated for a -34total of 2.5 hours by method B at 4 0 *C using 300 IIL of a 1:1 mixture of acetone and CH 2 C1 2 Progress of the react-ion was monitored by 1 H HR The solvent was evaporated, the residue dissolved in ethyl acetate and the mixture Purified by column chromatography using hexane as eluent. 1 H NN (C=C13) 8 7.43-7.07 (in, 4H), 3.14 3H), 2.59-2.42 (m, 2H), 1.3.7 1.31 3H, J=6,.9 Hz), 1.17 3H, J=6.9 Hz), 0.92 Cd, 3H. J=6.9 Hz), 0.47 C(d, -3H, J=6.9 Hz).

Examplde 16-. Svt~hni of 4(3-Phnoqnhorv~nxyflhenlj- 3 .3- Aiinonronl-4-mepthoxyv-1-2-dioxet~ari. di~odium saltj (fi.

0-

OCH

3 150

OP)N&

2 A 64 mg sample of the alkerie was photooxygellated for a total of 1. 5 hours in 3 mL of D) 2 0 at 0 OC according to Method B. The solution was stored at 4 OC to induce crys- .20 tallization. The white crystals were filtered, washed with acetone and dried. 1 H NNR (D 2 0) 8 7.43-7.14 4H), 3.132 2.63-2.53 Cm, 2H), 1.225 Cd, 3H, J=7.5 Hz), 1.123 Cd, 3H1, J=7.5 Hz), 0.892 Cd, 3H, J=6.6 Hz), 0.475 3H, J=6.6 Hz); 31 P NHR (D 2 0) (rel. To ext. H 3 P04) 8 0.248.

it should be noted that all other solvent systems used including

D

2 0/p-dioxafle, methanol, methanol/CH2Cl2 required reaction times of several hours and led to significant quantities of decomposition products.

pVxmnm~ 17. synthanis of 4-(3-t-uvldimethV1Ril1=XnhenVi) -j Aic 1oInronv1-4-m~thoxyV-1.-2-dioxetane p OSi (CHO 3 2 t-Bu A 25 mg sample of the alkene was photooxygenated f or a total of 1 hour by method A at -78 6C. 1 H NMR indicated the solution to contain a 3:1 mixture of dioxetane to alkene and a small amount of the ester decomposition product.

Irradiation was stopped at this point and the sensitizer filtered away. The solvent was evaporated and the mixture used as a solution in Xylene for kinetic measurements. 1H NM(R CCDCl 3 peaks due to dioxetane: 8 7.6-6.7 4H), 3.14 3H), 1.80 Wm 3.H) 1.2-1.0 9H), 0.991 9H), 0.221 611).

ExAmnl@ IA-. SvnbPcia of 4-3rAtiiehliyoy prvl)-3. 3-dicvelohQ1V14-Mthgcxv-2-tdiotAn@ (2h).

OCH3 A 2.0 g sample of alkene if was photooxygezlated for a total of 8.5 hours by method B at -78 0C. The solvent was -36evaporated, the residue dissolved in hexane and filtered.

The organic solution was evaporated and the solid residue was purified by column chromatography. The yield of product was 2.0 g (93 1H NMR (CDC13) 8 7.26-6.85 4H), 3.143 3H), 2.3-0.5 22H) 0.995 9H), 0.205 6H); 13

C

NMR (CDC1 3 8 155.57, 136.82, 129.10, 122-121(several unresolved), 114.56, 104.39, 97.31, 49.49, 45.18, 41.79, 28.71, 28.07, 27.80, 27.17, 26.95, 26.83, 26.74, 26.30, 25.68, 18.24, -4.38.

xamnle 19. Synthesis of 3.3-Dicvclohexvl-4-(3-hvdroxvhenv1) -4-methoxv-1. 2-dioxetane (2iH.

-15

OCH

3 20 total of 1.5 hours by method B at -78 OC. The solvent was

OH

A 150 mg sample of alkene ig was photooxygenated for a o 20 total of 1.5 hours by method B at -78 The solvent was evaporated, the residue dissolved in hexane and filtered.

The precipitate was washed with 10 ml of 20 ethyl acetate/hexane and the organic solution evaporated. The solid residue was purified by preparative TLC using 20 ethyl acetate/hexane to elute the plate. The yield of product was 120 mg (72 H NHR (CDC13) 8 7.34-6.93 4H), 5.30 (s, 1H), 3.163 3H), 2.23-0.56 22H); 13 C NMR (CDC1 3 8 155.55, 137.02, 129.42, 116.23, 116.12, 114.62, 104.36, -37- 97.88, 49.60, 45.28, 41.78, 28.70, 28.09, 27.75, 27.14, 26.90, 26.86, 26.72, 26.37.

Examnle 20. Synthesis of 1-(tri-n-octv1nhosnhniummthvl)- 4- (tri-n-butvInhosbhaniummathl benzene dichioride.

Enhancer A 1 ts PBu 3 C1l A mixture of tri-n-butylphosphine (7 g, 34.6 mmol) in toluene (50 mL) was added dropwise to a mixture of :0*si ,a'-dichloro-p-xylene (12.1 g, 69.2 mmol, 2 eq.) in toluene (200 mL) under argon. The reaction mixture was stirred for 12 hours at room temperature under argon, after which time 4-(chloromethyl)benzyl-tri-n-butylphosphonium chloride had crystallized out of solution. The crystals were filtered and washed with toluene and hexane and air dried: 'H NMR (CDC1,) 0.92 1.44 12H), 2.39 (m, 6H), 4.35-4.40 2H), 4.56 2H), 7.36-7.39 2H), 7.47-7.51 (dd, 2H).

To a mixture of 4-(chloromethyl)benzyl-tri-n-butylphosphonium chloride (3 g, 7.9 mol) in fMF at room temperature, under argon was added tri-n-octylphosphine (4.39 g, 12 mmol). The reaction mixture was allowed to stir for several days, after which time TLC examination showed the reaction to be complete. The DMFW was removed under reduced pressure, the residue washed with hexanes and toluene -38several times and then dried .to give 1- (tri-n-octylphosphoniummethyl) 4- (tri -n-butylphosphoniummethyl) benzene dichloride as white crystals: 1H NHR (CDC1 3 8 0.84 91), 0.89 9H), 1.22 (br s, 24H), 1.41 (m,24HU, 2.34 (m, 12H), 4.35-4.40 4H), 7.58 4H); 13 C IOIR (CDCl 3 8 13.34, '13.94, 18.33, 18.62, 18.92, 19.21. 21.76, 21.-81, 2 3.5 8, 23.64, 23.78, 23.98, 26.10, 26.65. 28.86, 30.68, 30.88, 31.53, 129.22, 131.22; 31p NM WD 2 0).8 31.10. 31.94.

RxSAMrIl 21. Measuremont of CheIilumip !o KinetiC.

Chemilwinescence intensities and rate measurements were performed using either a Turner Designs (Sunnyvale, CA) model TD-20e iwainometer or a luminometer built in Jouse (Black Box) which uses -a photon counting photomultiplier.

Temperature control of samples analyzed in the luminometers was achieved by means of a circulating bath connected to the instrument. Quantitative measurement of light intensi- *ties on the Turner luminometer was extended beyond the 104 linear range of the detector by a neutral density filter.

20 Data collection was controlled by an Apple MacIntosh computer using the LUMISOT data reduction program (Lumigen, inc., Southfield, MIV.

Activation energies for thermal decomposition of dioxetanes 2c, h and i were determined'by measuring the first order rate constant kC for decay of chemiluminescence of dilute solutions in xylene at several temperatures.

-39- TabIL- 2. -Thermal 9tabilitv of stabilized nioxetanes W-xpan La loaaL A v 5* 124O (kcal/mol) 2c 28.1 12.85 1. 2 yr 43.9 yr 2h 29.4 13.7 1. 7 yr 72.3 yr 2i 28.5 13.2 1. 1 yr 43. 4 yr Exanmlz 12- Cbemiltiminge~c~le and Fluorescenice Snectra Chemi luminescence and fluorescence spectra were measured using a Fluorolog II fluorimeter (Spex Ind., Edison,. NJ) with 1 cm quartz cuvettes. All measurements were performed at ambient temperature. The spectrum was either scanned when the light intensity reached a constant level or correction was made for the decay of light intensity during 15 the scan. Figure 1 shows a typical chemiluminescence spectrum from the decomposition of dioxetane 2c in triggered by addition of a small volume of a solution of KOH in 1:1 methanol/rMSO. The emission arises from the excited state of the anion of methyl 3-hydroxybenzoate.

Triggered decomposition of each dioxetane of the present invention in DMO0 generates this excited state.

Exa nlne 231. ChemiCal Triggering of the Chemiiminescent D-cnposition of Dioxetaflen 2c.a.i.

stock solutions of dioxetanes 2c, 2e, 2g and for comparison, 4- (3 -t-butyldimethylsilyloxyphenyl) -4-methoxyspiro[1,2-dioxetane-3,21tricyclo[3.3..

3 7 ]decane] (2j), (preparation described in U.S. Patent No. 4,96.2,192) were made to a concentration of 10 6 M in DMSO. Serial dilutions in DMSO were made as required. Ten pL aliquots were triggered in 7 x 50 mm polypropylene tubes in a Turner Designs luminometer by injection of 50 ILL of a solution of tetra-n-butylammonium fluoride (TBAF) in DMSO (11M 104' M) in the appropriate solvent, typically DMSO. Light intensity was attenuated when needed by a neutral density filter. All experiments were conducted at ambient temperature. Peak light intensity and decay rate diminished as the fluoride concentration was decreased. At the lowest concentration of fluoride, decay kinetics were not cleanly first order.

Other triggering reagents found to produce chemiluminescence from dioxetanes 2a-e and 2g-i in DESO or EMF include hydrazine, potassium and tetraalkylammonium hydroxides, alkali metal and tetraalkylammonium alkoxides and sodium azide. Small amounts of a protic co-solvent such as methanol, ethanol or water could be used to dissolve the triggering agent in DMSO.

The duration and intensity of chemiluminescence may be 20 altered by the choice of solvent, triggering agent and ratio of dioxetane/triggering agent. Suitable solvents for practicing the present invention include any aprotic solvent in which the reactants are soluble, especially polar solvents such as EMSO, dimethylformamide, acetonitrile, p-dioxane and the like. The reaction can also be conducted in, for example, a hydrocarbon solvent where only one of the reactants is dissolved and the other is supplied in the medium undissolved. In this case, light is emitted -41f rom the surface of the undissolved reactant.

Examne a24..- Rates mf Trigaged feenmnonition of Dijoxetaies Figure 2 shows a typical chemiluminescence intensity profile upon triggering a 10 ILL aliquot of a 10-6 14 solution of dioxetane 2h with 50 ILL of 1 M4 TEAF in tEISO. Triggering of serial ten-fold dilutions of the dioxetane solution showed that a 10' 9M solution provided a signal 1. 5 times that of background. All chemiluminescence decay curves showed pseudo-first order kinetics. The half lives'for decay were essentially independent-of dioxetane concentration.

Table Rates and Chemiluminoeence Tntengity from Fluor-iAP-triffargd Decomposition of Dioxetane 2h as a unction nf Concentration.

ffinxatang 2h1 JLF] I .1 sc Total intensity 10-6M I M 7.2 1.9 x 1 4

T

107 M4 I 6.2 2.0Ox 10 3

TLU

10-8 14 I1 6.2 2.1 x1 2

TLU

*10'19 M1I4 6.7 1.5 x 101 TLU The rates of fluoride -triggered decomposition of.

dioxetanes 2c, h, i and j were compared in M4SO under identical conditions, i.e. 10 pL aliquot of a 10-'4M solution of dioxetane with 50 pt. of 1 M4 TEAP in rMO. All four dioxetanes were found to undergo reaction at essentially the same rate under these conditions.

-42- Trable rormArison of Thermal a- d Fluoride- triccrproad ruar-mmositiofl Ratios.

nioYAxpaflft t Z.LLLLL, thL1/2 t~ Rat& aerelpTyAtjn 2c 7 sec 1.2 yr 5.5 X 106 2h 7 sec 1.7 yr 7.0 x 106 2i 6 sec 1.1 yr 5.7 x 106 2j 7 sec 3.8 yr 1.4 Examnle 25. Measurement of Relative Chemihimin aceice Oantum Yields The total chemiluminescence intensity generated -by f luoride-triggering of dioxetanes 2c, g, h and i were compared in 04SO under identical conditions, i. e. 10 ;LL aliquot of a 10'6 M solution of dioxetane with 50 pL of 1 m TEAF in 1NS0. Precise values were difficult to reproduce; however, all four dioxetanes were found to generate the same chemi luminescence output within a factor of two under .~.these conditions. Based on the reported chemi luminescence ef ficiency of 25 for dioxetane 2h P. Schaap, T. -S.

Chen,, R. Handley, R. DeSi2lva and B. P. Gin., Tetrahedron Lett.,. 1155 (1987)) the dioxetanes of the present invention are found to produce chemiluminescence with high efficiency upon triggering in DKSO.

ExamnIP 26- Comnarisgn of Chem*iminekeenca Tntenkitrion- Kinetic profile of Rolutions Containina fioxetarie 2f or 2k.

in order to demonstrate the unexpected advantage of the phosphate dioxetane 2f of the present invention, a compari- -43son was made of the time course of chemiluminescence f rom this dioxetane induced by alkaline phosphatase (AP) i.nalkaline buf fer solutions to the commercially available dioxetane 4-methoxy-4- (3-phosphoryloxyphenyl) Spiro 2-dioxetane-3.V 0-tricyclo [3.3 1.1 3 ,7]decane], disodium salt, (LUMIGEN PPD. Lumigen, Inc., Southfield, Xi), dioxetane 2k. Figure 3 illustrates the time profile and relative chemiluminescence intensities at 37 *C from two compositions, one containing 0.33 mm dioxetane 2d of the present invention and the other containing 0.33 m dioxetane 2k in the same buffer. Light emission was Initiated by addition of 1.12 x 10-17 moles of AP to 100 ;LL of the dioxetane solution. The reagent containing dioxetane 2f of the present invention reaches a significantly higher maximuim intensity.

Examnnle 27. Caimarlson of Chamilirmingecenep TntgnsitieR-q xislatte Profile of S61utiana Containina Dinxetarie 2f or 2k.

Figure 4 illustrates the time profile and relative chemiluminescence intensities at 37 *C from two compositions, one containing 0.33 mM dioxetane 2f of the present invention and 1.0 mg/mL. of 1-(tri-n-octylphosphoniumniethyl) -4 (tri-n-butylphosphoniuuuethyl benzene dichloride a a: (Enhancer A) and the other containing 0.33 mM dioxetane 2k and 1.0 mg/mL of the same enhancer. Light emission was initiated by addition of 1.12 X 10-17 moles of AP to 100 ILL.

of the dioxetane solution. The reagent containing dioxetane 2f of the present invention reaches achieves higher light -44intensities at all time points.

Zxamnie 2A.- Conmaringn of Cheri PnncnP !nten-aitime- Kinea-tir Profile of Aolioins Containing fixetane 2f or2k Figure 5 illustrates the time prof ile and relative chemiluminescence intensities at 37 OC from two compositions, one containing 0.33 mM dioxetane 2f of the present invention and 0.5 mg/mL of polyVinrylbenzyltributylphosphonium chloride (Enhancer B) and the other containing 0.33 mm dioxetane 2k and 1.0 mg/mL of the same enhancer. Light emission was initiated by addition of 1.12 x 10-17 moles of AP to 100 pLL of the dioxetane solution. The reagent containing dioxetane 2f of the present invention reaches achieves higher light intensities at all time points.

rwamzilp 29-. Comnari-gon of Cherilurinescant-P Tntonitipeq- Kinetic Profilp of Slutions Cnainina ioxetane 2f or 2k, Figure 6 illustrates the time profile and relative chemi luminescenlce intensities at 37 *C from two compositions, one containing 0.33 mH dioxetane 2f of the presen t invention and 0.5 mg/niL of polyvinylbenzyltributylphosphonium chloride co-po lyvinylbenzyltrioctylphosphoflium chloride (containing a 3:1 ratio of tributyl:trioctyl groups) (Enhancer C) and the other containing 0.33 mM dioxetane 2k and 0.5 mg/JuL of the same enhancer. Light emission was initiated by addition of 1.12 X 1i-17 moles of AP to 100 paL of the dioxetane solution. The reagent containing dioxetane 2f of the present invention reaches achieves higher light intensities at all time points.

RxamnlA 10-. Linparitv and son itivitv of De&tpcrion of Alkaline Pho~nnhata3.b wit-h finxetang 2f.

The linearity of detection of AP using a reagent composition of the present invention containing dioxetane .2f was, determined. To each of 48 veils in a 96-well micropla .te was added 100 IlL of a 0.33 mM solution of 2f in 0.2 M4 2-methyl-2-amino-l-propanol buffer, pH 9.6 containing 0.88 MM Mg* 2 and 1. 0 Mg/M L of Enhancer A. The plate was incubated at 37 *C and chemiluminescence emission initiat'ed by addition of 3 ILL of solutions of AP containing between 3.36 x 10-16 mol and 3.36 x 10-22 aol of enzyme. Light intensities were measured at 10 min. Figure 7 shows the linear detection of alkaline phosphatase. The term S-B refers to the chemiluminescence signal in RLU in the presence of alkaline phosphatase (AIP) corrected for background chemiluminescence MB in the absence of AP. The calculated detection limit (twice the standard deviation of the background) was determined to be 2.0 x 10-22 aol, or 120 molecules of AP under these conditions.

Examnie 1. Comnarison of Chemiltriincence Ouantum.

The relative chemiluininescence quantum yields of dioxetanes 2f and 2k were determined in solutions containing 1 mg/mL of Enhancer C in 0.2 M4 2-amino-2-methYl- 1-propanol buffer, pH 9.6 containing 0. 88 mm* Mg 2 and -46selected enhancers as described in Table 4. A 100 tIL aliquot of each reagent was completely dephosphorylated -by addition of 3.36 X 1O-13 Mol of alkaline phosphatase. The total amount of light emitted in Relative Light Units (RLU) was integrated until light emission ceased. A similar comparison was also made with 500 PIL portions of formulations without any enhancer using either 0.2 31 or 0.75 m1 2-amino- 2-methyl-1-propanol buffer, PH 9.6 containing 0.88 M 1g4 2 Dioxetane 2f produces more light than dioxetane 2k in buf fer alone and in the presence of Enhancers A and C.

Table 5. Total Light Intensity from Phosnhate Dioxetanes rnhanar 0inxtan@2f Dioxatane 2k None 2 1) 2.82 x 10' 1.55 x None (0.75 M1) 5.55 x 104 4.41 x 104 Enhancer A (1 mg/mL) 1.19 X 10 7 9.0 x -Enhancer B (0.5 mg/mL) 1.65 x16 2.09 x Enhancer C (0.5 mg/mL) 4.15 X 10 7 3.65 Exanmie 17. _qtahilitv of ioxptan@ 2f in Amienus Solution&_ The thermal and hydrolytic stability of a 0.33 mH solution of dioxetane 2f containing 1 mg/mL of Enhancer A in 0.2 31 2 -amino- 2 -methyll 1-propalol buffer, pH 9.6 and 0.88 :MM 31g 2 was determined at 37 OC. Solutions of the dioxetane 25 were maintained at room temperature and 37 OC for 5 days.

To each of 12 wells in a 96-well microplate was added 100 IlL of each solution. The plate was incubated at 37 *C and chemi luminescence emission initiated by addition of 10 ILL -47of solutions containing 1.1 x 10-1 mol of AP. Light intensities were integrated for 2.5 hours. Stability of the dioxetane was assessed by comparing the average light yield of the sample incubated at 37 °C to the solution held at room temperature. A decrease in the amount of light emitted indicates decomposition of the dioxetane during the incubation period. The solution maintained at 37 OC was identical to the room temperature solution indicating the dioxetane to be stable under these conditions.

Examole 33. Chemiluminescent Detection of Alkaline Phosnhatase on Membrane.

The utility of a composition of the present invention for the chemiluminescent detection of enzymes on the surface of blotting membranes is demonstrated in the following example. Solutions of alkaline phosphatase in water containing from 1.1 fmol to 1.1 amol were applied to identical nylon membranes (Micron Separations Inc., Westboro, MA). The membranes were air dried for 5 min and 20 soaked briefly with a reagent containing 1 mg/mL of Enhancer A in 0.2 M 2-amino-2-methyl-l-propanol buffer, pH 9.6 containing 0.88 mM MgC1 and either 0.33 mM dioxetane 2f or 0.33 mM dioxetane 2k. The membranes were placed between transparency sheets and exposed to X-ray film (Kodak X-OMAT AR, Rochester, NY). Figure 8 shows that the light produced using the two dioxetanes led to equivalent images and detection sensitivity. These results illustrate the performance of dioxetane 2f which is to be expected in Western -48blotting, Southern blotting, MNA fingerprinting and otherblotting applications.

The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in Australia.

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Claims

3. The compound of Claim I isopropyl groups. wherein R 3 and R 4 are each
4. The compound of Claim iwherein R 3 and R 4 are each cyclohexyl groups. The compound of Claim IL wherein R 3 and R 4 are each cyclopropyl groups.
6. The compound of Claim -3 wherein X is a hydrogen atom.
7. The compound of Claim 3 wherein X is a t-butyldi- methylsilyl group.
8. The compound of Claim 3 wherein X is an acetyl group.
9- The compound of Claim 3 wherein X is a benzoyl group. 9* S *SSC C CC.. C C. S. *S 51 The compound of claim 3 wherein X is a pivaloyl qroup.
11. A compound of claim 1 substantially as hereinbefore described especially with reference to the Examples. DAT ED this twenty first day of June 2000 Lumigen Inc. AND The Board of Governors of Wayne State University by Their Patent Attorneys DAVIES COLLISON CAVE