AU700925B2 - Chemiluminescent dialkyl-substituted 1,2-dioxetane compounds, methods of synthesis and use - Google Patents

Description

Our Ref: 655216 P/00/01 1 Regulation 3:2

AUSTRALIA

Patents Act 1990

ORIGINAL

COMIPLETE SPECIFICATION STAN'DARD PATENT 0 0000 ~0 00.90 :0 *0 0.9 0 0* 0$ 0 t000 0.900 .9.

00 0 Applicant(s): Lurnigen, Inc.

Riverwood Research Center 24485 W Ten Mile Road Southfield Michigan 48034 UNITED STATES OF AMERICA Th eBoard oE ioviernors -0+ FW, ir Deir"' MCy;'!re L4bao2 Address for Service: Invention Title: DAVIES COLLISON CAVE Patent Trade Mark Attorneys Level 10, 10 Barrack Street SYDNEY 14SW 2000 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:- 5090n 4 11$ I V j 4 1 A..

V.

t.

I 4v I CHEMILUNINESCENT DIALKYL-SUESTITUTED 1,2-DIOXETANE COMPOUNDS, METHODS OF SYNTHESIS AND USE BCGROMUND O TTRE INVK=TTON F'TELD QF E nu~Tos The present invention relates to chemiluminescent 1,2-dioxetane compounds that can be triggered by reagents including enzymes and other chemicals to generate light. In particular, the present invention relates to stable aryl group-substituted 1,2-dioxetanes 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 carbonyl. compounds.

ERITO

R

a. Prerpar~lif ~lixtns Kopecky and Mumf-ord reported the first synthesis of a dioxetane (3,3,4-trimethyl-1,2-dioxetale by the base-catalyzed cyclization of a f9-bromohydroperoxide, which, in turn, is prepared from the corresponding alkene R. Kopecky and C. Mumford, Can. j.

Chem., 47, 709 Although this method has been used to produce a variety of alkyl and aryl-substituted 1,2-dioxetanes, it can not be used for the preparation of dioxetanes derived from vinyl ethers, vinyl sulf ides and enamines.

An altenate synthetic route to 1,2-dioxetanes, especially thos^,*derived from vinyl ethers, vinyl sulf ides t b r, c -2and 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 Advances In Oxygenated Processes, JAI Press, Greenwich, CT, 1988; Vol.1, pp 31-84).

b. Thermally Stable Dioxetanes from Stericallv Hindered AIkenes. The dioxetane derived from the hindered alkene adamantylideneadamantane which was discovered by Wynberg H. Wieringa, J. Strating, H. Wynberg and W. Adam, TeCrahedron Lett., 169 (1972) was shown to have an activation energy for decomposition of 37 kcal/mol and a half life (t 1 2 at 25 C of several years J. Turro, G.

r .e, A. o 4tt4 -3- Schuster, H. C. Steinmetzer, G. R. Faler and A. P. Schaap, J. Amer. Chem. Soc., 97, 7110 (1975)). Others have shown t, that a spiro-fused polycyclic group such as the adamantyl group can help to increase the stability of dioxetanes derived from amino-substituted alkenes McCapra, I.

Beheshti, A. Burford, R. A. Hann and K. A. Zaklika, J.

i Chem. Soc., Chem. Comm., 944 (1977)), vinyl ethers (W.

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

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

c. Chemical Triagering of Dioxetanes. The first example in the literature is described in relation to the hydroxysubstituted 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 Sfrom the diaryl-1,4-dioxenes are relatively unstable having half-lives at 25°C of only a few hours. Further, these non-stabilized dioxetanes are destroyed by small quantities of amines Wilson, Int..Rev. Sci.: Chem., Ser. Two, 9, 265 (1976)) and metal ions Wilson, M. E. Landis, A. L.

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

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

Examples of the chemical triggering of adamantyl- -4stabilized dioxetanes were first 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. S. Handley, R. DeSilva, and B. P. 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 chemiluminescence have also been reported Adam, R. Fell, M.H. Schulz, Tetrahedron, 49(11), 2227-38 (1993); W. Adam, M.H. Schulz, Chem. Ber., 125, 2455-61 (1992)). The stabilizing effect of other rigid polycyclic groups has also been reported Bartlett and M. Ho, J. Am. Chem.

Soc., 96, 627 (1975); P. Lechtken, Chem. Ber., 109, 2862 *o (1976)). A PCT application, WO 94/10258 discloses chemical s triggering of dioxetanes bearing various rigid polycyclic .substituents.

d. Enzymatic TriaTerina of Adamantvl Dioxetanes.

Dioxetanes which can be triggered by an enzyme to undergo chemiluminescent decomposition are disclosed in U. S.

patent application P. Schaap, patent application serial No. 887,139) and a series of papers P. Schaap, R. S.

Handley, and B. P. Giri, Tetrahedron Lett., 935 (1987); A.

25 P. Schaap, M. D. Sandison, and R. S. Handley, Tetrahedron Lett., 1159 (1987) and A. P. Schaap, Photochem. Photobiol., SA 50S (1988)). The highly stable adamantyl-substituted dioxetanes bearing a protected aryloxide substituent are -L

V

n 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 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 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 a.

C*

h -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 S. excited state ester to the fluorescein compound within the hydrophobic environment of the micelle.

S: 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 0o" 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 .,r phosphate-substituted dioxetane.

U.S. Patent No. 5,145,772 to Voyta discloses enhancement of enzymatically generated chemiluminescence from 1,2-dioxetanes in the presence of polymers with pendant quaternary ammonium groups alone or admixed with fluorescein. Other substances reported to enhance chemiluminescence include globular proteins such as bovine albumin and quaternary ammonium surfactants. Other cationic polymer compounds were of modest effectiveness as chemiluminescence enhancers; nonionic polymeric compounds were generally ineffective and the only anionic polymer significantly decreased light emission. A PCT application WO 94/21821 discloses enhancement from the combination of a polymeric ammonium salt surfactant and an enhancement additive. European Patent Application No. 92113448.2 to Akhavan-Tafti published on Sept. 22, 1993 discloses enhancement of enzymatically generated chemiluminescence from 1,2-dioxetanes in the presence of polyvinyl phosphonium salts and polyvinyl phosphonium salts to which 20 fluorescent energy acceptors are covalently attached.

Co-pending application U.S. Serial No. 08/082,091 to Akhavan-Tafti filed June 24, 1993 discloses enhancement of enzymatically generated chemiluminescence from 1,2-dioxetanes in the presence of dicationic phosphonium 25 salts.

Triggerable stabilized dioxetanes known in the art incorporate a rigid spiro-fused polycyclic substituent or a substituted spiroadamantyl substituent. The ketone starting #9.

0~ 4Q 9 4 9 .4.9 9 9, 4.4 .4 materials 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 dioxetanes bearing two alkyl groups in place of rigid spiro-fused polycyclic organic groups are known. Such triggerable stabilized dioxetanes can be prepared from inexpensive, readily available starting materials and will therefore provide cost advantages facilitating their commercial potential.

OBJECTS

It is an object of the present invention to provide novel dialkyl and aryl OX-substituted triggerable 1,2-dioxetane compounds which are stable at room temperature over an extended period of time. It is also an object of the present invention to provide such stable 1,2- I""dioxetane compounds which can be triggered to decompose with the generation of chemiluminescence. It is also an object of the present invention to provide such stable 1,2-dioxetane compounds which can be prepared from inexpensive, readily available starting materials. It is an object of the present invention to provide a method and compositions containing a stable 1,2-dioxetane which can be triggered by reagents, including enzymes and other chemicals, to generate cheimiluminescence. Further, it is an object of the present invention to provide a method and compositions for additionally enhancing the chemiluminescence through the use of enhancer substances. Further the

I

-9present invention relates to a method and compositions for the detection of enzymes, and for use in immunoassays and the detection of enzyme-linked nucleic acids, antibodies and antigens such as are generally known in the art.

Further, it is an object of the present invention to provide a method and compositions for chemical lighting applications.

IN THE DRAWINGS -Figure 1 is a spectrum of the chemiluminescencq emitted from a solution of dioxetane 2c in dimethyl sulfoxide (DMSO) when triggered by addition of a solution of potassium hydroxide in a mixture of methanol and dimethyl sulfoxide. The spectrum is corrected for the decay in chemiluminescence intensity occurring during the scan.

Figure 2 is a graph of chemiluminescence intensity as a function of time produced by triggering a 10 pL aliquot of a 10 6 M solution of dioxetane 2g with 50 gL of 1 M tetra-n-butylammonium fluoride in DMSO.

20 Figure 3 is a graph showing a comparison of the time profile of the chemiluminescence intensity emitted by 100 L of solutions containing either dioxetane 2f of the present invention or 2k (LUMIGEN PPD, Lumigen, Inc., Southfield, MI) 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 dioxtane 2f in 0.2 M 2-amino-2-methyl- 1-propanol buffer, pH 9.6, and 2) a 0.33 mM solution of i dioxetane 2k in 0.2 M 2-amino-2-methyl-l-propanol buffer, pH 9.6. Use of dioxetane 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 chemiluminescence intensity emitted by 100 UL of solutions containing either dioxetane 2f or 2k triggered at 37 OC 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-l-propanol buffer, pH 9.6 containing 1.0 mg/mL of the enhancer l-(tri-n-octylphosphoniummethyl)-4- (tri-n-butylphosphoniummethyl) benzene dichloride (Enhancer 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 1.0 mg/mL 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 20 profile of the chemiluminescence intensity emitted by 100 L L 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, 25 pH 9.6 containing 0.5 mg/mL of polyvinylbenzyltributylphosphonium chloride (Enhancer B) and 2) a 0.33 mM solution of dioxetane 2k in 0.2 M 2-amino-2-methyl-l-propanol.

-11- buffer, 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-1-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 561,033. Use of dioxetane 2f of the present invention 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 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

I

-12x 10' 1 6 mol and 3.36 x 10- 22 of enzyme to 100 PL of a 0.33 mM solution o'f 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 Sdeviation 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 A from an experiment detecting alkaline phosphatase on a membrane with chemiluminescence. Solutions of alkaline 15 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 MgC1 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 4 performance of dioxetane 2f which is to be expected in -13- Western blotting, Southern blotting, DNA fingerprinting and other blotting applications.

DESCRIPTION OF THE PREFERRED 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

R

3 IoR

R

4

R

2 0-X wherein R 3 and R 4 are nonspiro-fused organic groups, wherein RI 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 chemicals. The unstable oxide intermediate dioxetane *5 I decomposes and releases electronic energy to form light and 20 two carbonyl containing compounds of the formula

OR

0 and O

RR

2 0 A preferred method of practicing the present invention uses a stable dioxetane of the formula: r -14- 0-0 R>L IORI R4 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 R 4 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 OC) even though. they can be triggered by chemical reagents. Previous examples of stable, triggerable 1,2-dioxetanes all made use of rigid spiro-fused polycyclic alkyl groups such as adamantyl and 20 substituted adamantyl to confer thermal stability. It has now been discovered that 1,2-dioxetanes bearing a wider range of substituents corresponding to R3 and R4 in the structure above also exhibit substantial thermal stability i 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 tha 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 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: S0- 0 OR R R 2 0-X wherein R 3 and R 4 are nonspiro-fused organic groups, wherein Ri 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 by addition of oxygen to the appropriate

I

-C-a -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 allylic' hydro-peroxide, dioxetane formation is a minor process at most.

OOH

R

3 R I 02 light R3 LR Sene reaction

R

4 R2 sensitizer R4 R2 R4 R, hydroperoxide O--0

R

3 R1 02 light R 3

R

sensitizer 2 2 cycloaddition R4 R2 R 4 R2 dioxetane The requisite alkene compounds are synthesized through Ca coupling arylcarboylate esters substituted with an X-oxy group and dialkyl ketones of the formula shown below: -eo

*OR,

O and O

R

4

R

2

X

in the presence of lithium aluminum hydride, other metal 25 hydride, zinc metal or zinc-copper couple in a polar C 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 oft 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 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 P-galactosidase, a- and 9** A-glucosidase, glucuronidase, trypsin and chymotrypsin.

The OX group may include, without limitation, hydroxyl, -18-

OOCR

6 wherein R 6 is an alkyl or aryl group containing 2 to carbon atoms either of which may contain heteroatoms, trialkylsilyloxy, triarylsilyloxy, aryldialkylsilyloxy, OP0 3 2 salt, OSO3" salt, S-D-galactosidoxy and B-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: A 0--O R 0-0 OR

R

4

R

2 0-X wherein R 3 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 1 is an organic group which may be combined with R, 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 chemicals wherein the unstable oxide intermediate dioxetane decomposes and releases electronic energy to form light and two carbonyl containing compounds of the formula: R

OR,

O and O= 25 RR 2 0 4'CR4 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.

Nuclear magnetic resonance (NMR) spectra were obtained on a GE QE300 or a Varian Gemini 300 spectrometer as solutions in CDC1 3 with tetramethyisilane as internal standard or as solutions in CD 3 OD or D 2 0. Mass spectra were obtained on an AEI MS-90" spectrometer.

nhenvl) -22-diisonroDvl-1-methoxvethene (1a).

O* O OSi (CH3) t-Bu It'' with 100 mL of anhydrous tetrahydrofuran (THF). The flask was cooled in an ice bath and titanium trichloride (18 g) r izs isueIsteraet 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 Qbe (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 20 13C NMR (CDC13) 8 128.76, 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.

Ass

I

-21- Examiile 2. Synthei opy 2-Diorrv- 1 -hvdr=

OCH)

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 THY 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 evaporattoo* ed. The material was purified by column chromatography on obo silica gel, eluting with 10 20 ethyl acetate/hexane 1% yielding 0.568 g (87 %)of lb: ~H NMR (CDCl 3 87.5-6.5 (in, 4H), 4.91 (s,1lH), 3.20 3H), 2.47 (sept, IH), 2.33 (sept, 1H), 1.25 6H),.0.92 6H); 13 (CDCl 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- Exainr)lg 3. Svnrhe~sis of 1-C3-A&cetoxvoen1)-2.2-diic~prov.

J-rnethoMvet-hene

OCH

3 0C0CH 3 Alkene lb (200 mg, 0. 85 mmol) was dissolved in 20 mLt of dry methyl.ene chloride with 0.3lmL of anhydrous pyridine.

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

Acetyl chloride (0.115 g, 1.47 mmol) in 5 mL of dry met .hylene 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 15 0 C. The solvents were evaporated and the residue dissolved in ethyl acetate. The solution was washed four times with water, dried over MgSO 4 and evaporated. The residue was purified by columin chromatography on silica gel, eluting with 10-20 ethyl acetate/hexane yielding 220 mg (93 of lc: 1 H NM' (CDCl 3 8 7.37-6.99 (mn, 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) 13C NMR (CDC1 3 8 169.46, 150.62, 149.03, 139.02, 133.64, 128.95, 127.24, 122.69, 120.72, 56.50, 30.49, 26.98, 22.06, 21.25, 21.05.

-23- Zainrje 4. syntlaesis of 1- (3-BenZovloxvrheri1) -2,2-di isoinrotvl-1-metoxethe 1~

OCH

3 OCOPh Alkene lb (4.5 g, 1.9 mmol) was dissolved in 50 rnL 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 mmol) 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 a..,with 1% ethyl acetate in hexane yielding 3.7 g of dioxetane 1d: 1 H NmR (CDCl 3 8 8.25-7.05 (in, 9H), 3.25 1H), 2.54 (sept, lH, j=6.9 Hz), 2.40 (sept, 1H, J=6.9 Hz), 2.29 (s, 90093H),. 1.26 6 H, J=6-9 Hz), 0.95 6 H, J=6.9 Hz) -24- ExaMnile 5.Synthesis of l-(3-Pivadloyloxv)henvl)-2.2-diison~ronvl--methoxvethele

OCH

3 'V OCOC (CH 3 Alkene lb (2 g, 8.6 mmol) was dissolved in 50 rnL of dry

CH

2 Cl 2 with 2.4 mrL of anhydrous triethylamine. The flask was purged with argon and cooled in an ice bath. Pivaloyl chloride (1.6 g, 2 eq.) was addqd 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 4 and evaporated. The residue was purified by column chromatography on silica gel, eluting with 5% triethylamine in hexane yielding 1.95 g of dioxetane le: 1H NMR (CDCl 3 8 7.34-6.98 4H) 3.198 3H), 2.47 (sept. 1H), 2.33 (sept, lH), 1.36 Cs, 9H), 1.24 6 H, J=6.9 Hz), 0.92 6 H, J=6.9 Hz).

Examde 6 Svthes~~ f 2.2-Di* sonrgyjv-1-methoxV-1 (3-Phosphnrvia Vhenvl ethene, disodium Salt (If).

-k- A solution of 9 mL of dry CH 2 Cl1 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 mmol) 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 mg of 2-cyanoethanol (8.7 mmol). The ice bath was removed and stirring continued at room temperature for two hours. The mixture was then 15 concentrated and the residue was purified by column chromatography on silica gel, eluting with a t-adient of ethyl acetate in hexane to 100% ethyl acetate yielding of the bis(cyanoethyl phosphate) H NMR (CDC1 3 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 mg) 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. H NMR (D 2 0) 6 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) 1]R

D

-26- 6 155.15 149.56, 137.84, 136.08, 129.71, 124.55, 122.33 120.48, 57.16, 31.27, 27.21, 22.59, 21.10; 31

P

NMR (D 2 0) (rel. To ext. H 3 P0 4 8 0.345.

Examnle 7. Synthesis of 1-(3-t-Butvldimethvlsilv oxynhenvl)-2.2-dicvcloDronvl- 1-methoxvethene ia) ,OCH3 OSi (CH 3 2t-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 a a 4 small portions causing a brief exothermic reaction. While the lithium aluminum hydride was being added, an additional 20 mL portion of anhydrous THF was added to aid stirring.

20 The cooling bath was removed when the addition was complete a.

and the black mixture was brought to reflux. Triethylamine (10.5 ml) was added and the black mixture was refluxed for a "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 -27was cooled to room temperature and extracted with four 400 mL portions of hexane. The combined hexane so~lutions 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/hexane. 1 NMR (CDCl 3 87.3-6.7 Cm, 4H1), 3.36 Cs, 3H1), 1.80 (in, 1H1), 1.13 (in, 111), 0.988 (s, 9H), 0.78-0.67 Cm. 4H1), 0.43-0.37 2H1), 0.19 6H), 0.11-0.05 Cm, 2H1).

Examnrle S. =vthesis of J-(3-t-DBtXtvdjmethvyIgjvlo2Z:iQhenv1) 2-dirMvclohe2vl- 1-rnetboxvethene (1h).

OSi (CHD) 2 t-Bu .A mixture of 4.3 g of methyl 3-t-butyldimethylsilyloxybenzoate and 9.5 g of dicyclohexyl ketone in dry THF were coupled according to the procedure of Example 5 using the Ti reagent made f rom 25 g of TiC1 3 0 g of LiAlH 4 and 16.4 g of triethylamine in 150 niL of dry.TIHP. The crude product mixture (10 g) obtained after hexane extraction wa s purified by column chromatography. on silica gel, eluting 1, -28first with hexane, followed by 1 ethyl acetate/hexajae and then with 3 ethyl acetate/ hexane. The yield was 3.5 g (51 of alkene le: 1H NNR (CDCl 3 8 7.2 2-7.16 1H), 6.85-6.72 Cm, 3H), 3.155 2.05-0.86 22H)', 0.9959 9H), 0.21 Cs, 6H).

EXamnle 9. Svntlaegig of 2.2-Dicvclohexvyl-1-(3-hvdroxvcnhnl- 1-methoxyetlaene (1i) 0O** to 4e..

0* 09 at** a S 0 0 0* 9 a 0* I) to To a solution of 0.7 g of alkene if in dry THF was added 0.62 g (1.2 eq.) of tetra-n-butylamzmonium fluoride dropwise. 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 20 the residue dissolved in ethyl acetate. The ethyl acetate solution was extracted with water and dried over MgS0 4 The material was purified by column chromatography on silica gel, eluting with 0 -10 %ethyl acetate/hexane yielding 0.46 g (92 of If. The alkene was further purified by crystallization in benzene/hexaie at 4 OC 1H NMR (CDCl 3 8 7.20-6.72 Cm, 4H), 4.72 lH), 3.174 3H), 2.06-1.04 Cm, 22H); 13 C NMR (CDC1 3 8 155.19, 138.20, 131.97, 129.05, 122.50, 116.36, 114.39, 56.48, 41.51,

A

k -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.

Table 1. P DI etafe C omounds, .0CM 3 eq.,

*O

0S** CC

C

C *C

''CC

2a 2b 2c 2d 2e 2f 2g 2h1 2i 2j 2k CH(2 3 2

EH(H

3 CH (CH 3 2 CH (CH 3 2 CH (CH 3 2 CH (CH 3 2 CHi(CH 3 2 CH (CH 3 2 CH (CH 3 2 CH (CH 3 2 CH (CH 3 2 CH (CH 3 2

C-C

3

H

5

C-C

3

H

5 c-C 6 H11 c-C 6

H

1 1

C-C

6

H

1 1 C-C 6 Hll adamantyl adamantyl Si (CH 3 2 t-Bu

H

COCH

3 COPh COC (CH 3 3

PO

3 Na 2 Si (CH 3 2 t-BU Si (CH 3 2 t-BU

H

Si (CH 3 2 t-BU P0 3 Na 2 Example 10- Synthesis of 1.2-Dioxetanes 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 I 15 and evaporating the solvent at room temperature. Further o purification could be achieved by column chromatography on silica gel or crystallization from a suitable solvent as necessary.

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- 11. ~vthesis of 4-(3-t-Biitv1direthv1ilylox"nhgnv1) 3-diisorgror1-4-methoxv-1,.2-dioeanfJj a.

0-

OCH

3 Os! (CHO) 2 t-BU A 1,02.8 mg sample of the alkene was photooxygenated for a total of 9 hours by method B at -78 0 C. The solvent was evaporated and the mixture purified by preparative TLC using 4 ethyl acetate/hexane to elute the plate.-The yield of dioxetane 2a was 55.9 mg (50 1 H NMR (cD~C1 3 )8 7.6-6.7 Cm, 4H), 3.14 3H), 2.61 (sept, 1H), 2.46 (sept, 1H), 1.30 1H), 1.18 1H), 1.00 3H),0.92 1H), 0.46 1H), 0.20 3H); M CDCl1 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.

Examle 1. svrthes of 3.3-Diisonrapvl-4-(3-hvdroxvphenvl) -4 th!2xv-1 .2-dixtn 2) 0-

OH

Alkene lb (83.2 mg) was photooxygenated for a total of 3 hours by method B at -78 0 C. 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 NMR (CDC13) 8 7.4-6.8 4H), 3.2 3H), 2.62 (sept, 1H), 2.48 (sept, 1H), 2.08 1.30 3H), 1.17 3H), 0.90 3H), 0.47 3H); 13 C NMR (CDC1 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.

Examnle 13. Synthesis of 4-(3-AcetoxvPhenv1)-3.3-dii=o- PIICANV1 -d-mB+hcl~V- P a-di.6xetane 12@~ m~~pl-A tU-'hnWr-- 2-urrinxptanp (2n)~ o

SO..

0** II

S

4005 15

T

OCOCH

3 A 63 mg sample of the alkene was photooxygenated for a total of 6.5 hours by method B at -78 OC. The solvent was evaporated, the residue dissolved in ethyl acetate and the 20 mixture purified by preparative TLC using 20 ethyl acetate/hexane to elute the plate. The yield of dioxetane 2c was 56 mg (80 'H NM (CDC13) 6 7.37-6.99 4H), 3.14 3H), 2.59-2.42 2H), 2.32 3H), 1.30 3H, J=7.2 Hz), 1.17 3H, J=7.2 Hz), 0.91 3H, J=7.2 Hz), 0.46 3H, J=7.2 Hz); 13 C NMR (CDC13) 8 150.89, 137.34, 129.39, 122.73, 114.07, 98.34, 49.60, 33.54, 29.31, 21.22, 19.44, 18.53, 17.17, 16.59.

-33- Exampole vthesis of 4-(3-Baenzovyoxv]henv11-3. 3-dimporarorv1-4-methox-1.2-dioxetafne (2d).

0-

OCH

3 q~ OCOPh A 3.7 g sample of the alkene was photooxygenated fora total of 19 hours by method B at -78 OC using 500 mL of a 1:1 mixture of acetone and CH 2 C1 2 and 100 mg-of methylene blue. Progress of the reaction was monitored by H NlR.. The solvent was evaporated, the residue dissolved in ethyl acetate and the mixture purified by column chromatography 0 using hexane as eluent. 1 H NMR (CDC13) 8 8.22-7.0 (in, 9H) 3.184 3H), 2.62-2.46 (mi, 2H), 1.3.0 3H, J=7.2 Hz), .o 1.20 3H, J=7.2 Hz), 0.94 3H, J=7.2 Hz), 0.52 (d, 3H, J=7.2 Hz); 3 C NM (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.

Examnle 15. Svnthesis of 4-(3-Pivalov1oxvnhenv:1)-3,3-diisooronv1-4-methoxV-1.2-dioxetane (2e1.

0o-

OCH

3

OCOC(CH

3 3 A 1.95 g sample of the alkene was photooxygenated for a -34total of 2.5 hours by method B at 4 "C using 300 niL of a 1:1 mixture of acetone and CH 2 Cl 2 Progress of the reaction was monitored by H NMM. The solvent was evaporated, the residue dissolved in ethyl acetate and 'the mixture purified by column chromatography using hexane as eluent. 1H NNR (CDCl 3 8 7.43-7.07 Cm, 4H), 3.14 3H), 2.59-2.42 (in, 2H), 1.37 9H), 1.31 3H. J=6.9 Hz), 1.17 3H, J=6.9 Hz), 0.92 3H, J=6.9 Hz), 0.47 Cd, 3H, J=6.9 Hiz).

1 xamolp 16. Synthesis of 4-(3-phshor~xoev)33 di~otro~v-Met]3oxv-1. 2-dioxetaie. diggdiUrn salt' (2f) 0-

OCH

3 OP0 3 Naz A 64 mg sample of the alkene was photooxygenated for a j~.total of 1.5 hours in 3 MiL Of D 2 0 at 0 *C according to Method B. The solution was stored at 4 @C to induce crystallization. The white crystals were filtered, washed with acetone and dried. 1H NMR (D 2 0) 867.43-7.14 (in, 4H) 3.132 3M), 2.63-2.53 (mn, 2H), 1.225 3H, J=7.5 Hz), 1.123 Cd, 3H, J=7.5 Hz), 0.892 3H, J=6.6 Hz), 0.475 3M1, J=6.6 Hz); 31 P NMR (D 2 0) (rel. To ext. H 3 P0 4 6 0.248.

it should be noted that all other solvent systems used includi.ng D 2 0/P-dioxafle, methanol, methanol/CH 2 Cl 2 required reaction times of several hours and led to significant quantities of decomposition products.

Zxamn~le 17. =vthesig of A-C3-t-Etyl~iiMetbyv1jIyloMZanhenl)-3.3-dicvclonxro=v-4-ethox-1.2-digetale (2g)l 2 t-Bu *0O* 1' t. S S

I

e.S.

*5 0*SS A 25 mg sample of the alkene was photooxygenated for a total of 1 hour by method A at -78 1 HNI indicated the solution to contain a 3:1 mixcture 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 evaporate d and the mixture used as a solution in xylene for kinetic measurements. 1 NMR (CDCl 3 peaks due to dioxetane: 8 7.6-6.7 4H), 3.14 3H), 1.80 1.2-1.0 9H), 0.991 9H), 0.221 6H).

inl ramrilp 18. Synthesis of §-(3-t-ButyjdirnetbvjS 122Mxv.

nhenvll-3 3-dic~vc1ohexv1-4-meth0Xv-1.2-diOXetafle (2hL *0-0 C)A1 OSi (CH3) 2 t-BU A 2.0 g sample of alkene if was photooxygenated for a total of 8.5 hours by method B at -78 OC. The solvent was ii 0000 0* 0,00 00,4.

V.

to ~f0 000* f 04$.

-36evaporated, the residue dissolved in hexane and filtered.

The organic solution was evaporated and the solid residue was purified by columin chromatography. The yield of product was 2. 0 g (93 NMR (CDCl 3 8 7.26-6. 85 (in, 3.143 3H), 2.3-0.5 (mn, 22H) 0.995 9H), 0.205 6H); 13C 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.

ExaMtlle 19. Synthesin of 3.3-Dicvclnhexv]-4-(3-hvdrpxvnDhenvl)-4-Tnethoxv-1.2-dioxetane (2j).

A 150 mg sample of alkene 1g was photooxygenated for a 20 total of 1.5 hours by method B at -78 0 C. The solvent was evapora ted, the residue dissolved in hexane and filtered.

The precipitate was washed with 10 ml of 20 ethyl a cetate/hexafle 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 1H NMR (CD)Cl 3 8 7.34-6.93 4H1), 5.30 (s, 1H), 3.163 3H), 2.23-0.56 22H1); 2 3 C NMR (CDCl 3 )8 155.55, 137.02, 129.42, 116.23, 116.12, 114.62, 104.36, 'MM-7 -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.

Expin~le 20. Synthesis of 1-(tri--oCtvlhonhosnmetylJ- 4- ttri-n-butvlr~ho~honiimmthvl) henzene dichlorCide,

I

4..

Ii 9* a a, *t9~ PCu Cl- A mixture of tri-n-butylphosphine (7 g, 34.6 iimoi) in toluene (50 mL) was added dropwise to a mixture of cz~ec'-dichloro-p-xylene (12.1 g, 69.2 inmol, 2 eq.) in toluene (200 inL) under argon. The reaction mixture was stirred for 12 hours at room temperature under argon, after which time 4- (chloromethyl)benzyl-tri-n-butylphosphoniuma chloride had crystallized out of solution. The crystals were filtered and washed with toluene and hexane and air dried: 'Hi NMR (CDC.

3 8 0.92 1.44 (mn, 12H), 2.39 (mn, 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 9, 7.9 inmol) in DMF at room temperature, under argon was added tri-n-octylphosphine (4.39 g, 12 inmol). The reaction mixture was allowed to stir for several days, after which time TLJC examination showed the reaction to be complete. The DMF was removed under reduced pressure, the residue washed with hexanes and toluene 1_ m_ 14 -38several times and then dried to give 1-(tri-n-octylphosphoniummethyl) -4 (tri-n-butylphosphoniummethyl) benzene dichloride as white crystals: H NMR (CDClI) 80.84 (t,9H), 0.89 9H), 1.22 (br s, 24H), 1.41 (m,24H), 2.34 (m, 12H), 4.35-4.40 4H), 7.58 4H); 13 C NMR (CDC1) 8 13.34, 13.94, 18.33, 18.62, 18.92, 19.21, 21.76, 21.81, 23.58, 23.64, 23.78, 23.98, 26.10, 26.65, 28.86, 30.68, 30.88, 31.53, 129.22, 131.22; P NMR (D 2 0) 6 31.10. 31.94.

Example 21. Measurement of Chemiluminiescence Kinetics.

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

15 Temperature control of samples analyzed in the luminometers was achieved by means of a circulating bath connected to the instrument. Quantitative measurement of light intensities on the Turner luminometer was extended beyond the 10 4 linear range of the detector by a neutral density filter.

Data collection was controlled by an Apple MacIntosh computer using the LUMISOFT data reduction program (Lumigen, Inc., Southfield, MI).

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

.'S

-39- Table 2. Thermal Stability of StabiliZed pioxetanes (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 ajQ'P 22. Che.MihjMinasceane and Flugrescenc- 912rrtra Chemiluminescence and fluorescence spectra were measured using a Fluorolog 11 f luorixneter (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 the scan. Figure I shows a typical chemiluminescehce spectrum from the decomposition of dioxetane 2c in DMSO triggered by addition of a small volume of a solution of oH in 1:1 methanol/DMSO. The emission arises from the excited state of the anion of methyl 3-hydroxybenzoate.

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

Examnle 23. CheMical Triaern oF the Ch~jjns~t Deconositipfl of Digxetanes 2c~g~i.

Stock solutions of dioxetanes 2c, 2e, 2 g and for compari.son, 4- (3-t-butyldimethylsilyloxyphenyl) -4-methoxyspiro 2-dioxetane-3,2 -triclo (3.3 11 7 Idecanl (2j), (preparation described in U.S. Patent No. 4,9M,192) were

JF

made to a concentration of 10-6 M in DMSO. Serial dilutions in DMSO were made as required. Ten JL aliquots were triggered in 7 x 50 mm polypropylene tubes in a Turner Designs luminometer by injection of 50 pL of a solution of tetra-n-butylammonium fluoride (TBAF) in DMSO (1M 10 4

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 DMSO or MF 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 Spracticing the present invention include any aprotic solvent in which the reactants are soluble, especially polar solvents such as DMSO, 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 -41from the surface of the undissolved reactant.

==lamn 24. Rarea of 3:rigagaed Derromro~itonO of Tioxetanen Figure 2 shows a typical chemiluminescence Intensity profile upon triggering a 10 ILL aliquot of a 106Hsolution of dioxetane 2h with 50 IlL of 1 M TEAF in Lr4SO. Triggering of serial ten-fold'dilutions of the dioxetane solution showed that a 10-9 M 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.

Tab2le 3. Rates and Chemiluminscnc Intgn~ity f=o Fluori P-tria Ared Dgegomnoition of Dioxetane. 2h as a Thinction of Concentration.

r~ioetan 2W! jF Total intensit= H 1 H 7.2 19Xi 4 l0"~ M 1 H 6.2 2.0 x i 3

TLU

iO-8 H 6.2 2.1 x 102 mLu 1 9 Hi 6.7 1.5xl101 TLU The rates of fluoride-triggered decomposition of.

dioxetanes 2c, h, i and j were compared in DMSO under identical conditions, i.e. 10 pL aliquot of a 10-6 M solution of dioxetane with 50 IlL of 1 M TEAF in DKSO. All four dioxetanes were found to undergo reaction at essentially the same rate under these conditions.

-42- Table 4. CornarjsOl of Thermal and Fluaride-triaclered Decomnoit ion At.

Pigx==f 2c 2h 2i 2j 71 sec 1./2 yhrma 7 sec 1.7 yr 7 sec 1.1 yr 7 sec 3.8 yr Ea&= acceler-at ion 5.5 X 106 7. 0 x 106 5.7 x 10 6 1.4 0~e~ ~t9.

4 V.

4 rxamnie 25. MeasUrement of Relative Chmuiecne OUanruM YieldS The total chemiluminescence intensity generated 'by fluoride-triggering of dioxetanes 2c, g, h and i were compared in DMSO under identical conditions, i.e. 10 pL aliquot of a 10-6 M solution of dioxetane with 50 JJL of 1 m 15 TEAF in DHSO. P recise 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 chemiluminescence efficiency of 25 for dioxetane 2h P. Schaap, T. -S.

20 Chen, R. S. Handley, R. DeSilva and B. P. Gini, Tetrahedron Lett., 1155 (1987)) the dioxetanes of the present invention are found to produce chemiluminescence with high efficiency upon triggering in r(SO.

EXaM2ie 26. Comparison of ChemiluAMingsCence intensitieawintir~ Prof j1e of Soutions Contain inxetane 2f or 2h

A.

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 from this dioxetane induced by alkaline phosphatase (AP) in alkaline buffer solutions to the commercially available dioxetane 4-methoxy-4- (3 -phosphoryloxyphenyl) spiro E1,2-dioxetane-3,2V-tricycloE3.3 .1.1 3 7 ]decane], disodium sl, (LUMIGEN PPD, Lumigen, Inc., Southfield, MI), 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 pzL of the dioxetane solution. The reagent containing dioxcetane 2 f of the present invention reaches a significantly higher maximum intensity.

Zxa=r1g 27. Comrarjiqnn of Chemi luminescence Intensitieps- Kinetic Profile-of qnlutinn Containing 12ioxetana 2f or 2k.

9..9 Figure 4 illustrates the time profile and relative chemiluminescence intensities at 37 *C from two composi- .***tions, one containing 0.33 mM dioxetane 2f of the present invention and 1.0 mg/mL of 1-(tri-n-octylphosphoniummethyl) (tri-n-butylphosphoniulmethyl benzene dichloride (Enhancer A) and the other containing 0.33 mmN dioxetane 2k and 1.0 mg/mL of the same enhancer. Light emission was initiated by addition of 1.12 x 1.0-17 moles of AP to 100 ptL of the dioxetafle solution. The reagent containing dioxetane 2f of the present invention reaches achieves higher light -44intensities at all time points.

EXamnle 28. Comnaringn of CheiilmneCene intensities- Kinetirc Prof ile, of Solutigns Containling Diaxetane- 2f or 2k Figure 5 illustrates the time profile 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/niL of polyVinylbenzyltributylphosphonium chloride (Enhancer B) and the other containing 0.33 mm dioxetane 2k and 1.0 mg/niL of the same enhancer. Light emission was initiated by addition of 1.12 x1-7moles of AP to 100 ILL of the dioxetane solution. The reagent containing dioxetane 2f of the present invention reaches *a~tachieves higher light intensities at all time points.

*4*ea~nj 29. Coaro of Cheiliuminesgerice Inten~sitiga- Kinetirc profile of Sglutiong Containing Dioxerane 2f or 2kS.

Figure 6 illustrates the time profile and relative I chemiluminescence intensities at 37 0 C from two composi- 1K)20 tions, one containing 0.33 mM dioxetane 2f of the present invention and 0. 5 mg/rL of polyvinrylbenzyltributylphos- ~phonium, chloride co-polyvinylbenzyltrioctylphosphonium 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/mL of the same enhancer. Light emission was initiated by addition of 1. 12 X 10-17 moles of, AP to 100 JJL of the dioxetane solution. The reagent containing dioxetane 2f of the present invention reaches 4 achieves higher light intensities at all time points.

Examnie 30. Linearity and Sensitivitv of Detection of Alkaline Phos2hatase with Dioxetane 2f.

The linearity of detection of AP using a reagent composition of the present invention containing dioxetane 2f was determined. To each of 48 wells in a 6-well microplate was added 100 gL of a 0.33 mM solution of 2f in 0.2 M 2-methyl-2-amino-l-propanol buffer, pH 9.6 containing 0.88 mM Mg+2 and 1.0 mg/mL of Enhancer A. The plate was incubated at 37 *C and chemiluminescence emission initiated by addition of 3 pL of solutions of AP containing between 3.36 x 10' 16 mol and 3.36 x 10 22 mol of enzyme. Light intensities were measured at 10 min. Figure 7 shows the linear detection of alkaline phosphatase. The term S-B refers to l' the chemiluminescence signal in RLU in the presence of alkaline phosphatase (AP) corrected for background chemiluminescence in the absence of AP. The calculated detection limit (twice the standard deviation of the background) 20 was determined to be 2.0 x 10"22 mol, or 120 molecules of AP under these conditions.

Examnle 31. Comparison of Chemiluminescence Ouantum.

Yields.

The relative chemiluminescence quantum yields of dioxetanes 2f and 2k were determined in solutions containing 1 mg/mL of Enhancer C in 0.2 M 2-amino-2-methyl- 1-propanol buffer, pH 9.6 containing 0.88 mM Mg 2 and -0 -46selected enhancers as described in Table 4. A 100 rL aliquot of each reagent was completely dephosphorylated by addition of 3.36 x 10 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 pL portions of formulations without any enhancer using either 0.2 M or 0.75 M 2-amino- 2-methyl-l-propanol buffer, pH 9.6 containing 0.88 mM Mg 2 Dioxetane 2f produces more light than dioxetane 2k in buffer alone and in the presence of Enhancers A and C.

Table 5. Total Light Intensity from Phosphate Dioxetane= Enhancr Dioxetane 2f Dioxetane 2k None (0.2 M) 2.82 x 105 1.55 x 10 15 None (0.75 M) 5.55 x 104 4.41 x 10 4 Enhancer A (1 mg/mL) 1.19 x 10' 9.0 x 106 Enhancer B (0.5 mg/mL) 1.65 x 106 2.09 x 106 Enhancer C (0.5 mg/mL) 4.15 x 10' 3.65 x 10 7 i*l 20 Examlqe 12. Stability of Dioxetane 2f in Euheous Solutions.

The thermal and hydrolytic stability of a 0.33 mM solution of dioxetane 2f containing 1 mg/mL of Enhancer A in 0.2 M 2-amino-2-methyl-1-propanol buffer, pH 9.6 and 0.88 mM Mg* 2 was determined at 37 Solutions of the dioxetane were maintained at room temperature and 37 OC for 5 days.

To each of 12 wells in a 96-well microplate was added 100 -L of each solution. The plate was incubated at 37 OC and chemiluminescence emission initiated by addition of -47of solutions containing 1.1 x 10- 15 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 *C was identical to the room temperature solution indicating the dioxetane to be stable under these conditions.

Example 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 15 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, DNA fingerprinting and otherblotting applicat ions.

Lii I *1I~

~J~S

Claims (2)

1. 2- salt, S-D-galactosidoxy and S-D-glucuronidyloxy groups. -3- The dioxetane of Claim 1 wherein R 3 and R, are independently selected from branched chain alkyl and cycloalkyl groups containing 3 to 8 carbon atoms.
2. 4- The dioxetane of Claim 1 wherein R 2 is a meta-phenyl group substituted by the OX group in the position meta to the dioxetane ring and which can contain additional substituents on the phenyl group. I-+I The dioxetane of Claim 3 wherein R 3 and R 4 are each isopropyl groups. -6- The dioxetane of Claim 3 wherein R 3 and R 4 are each cyclohexyl groups. I 51 -7- The dioxetane of Claim 3 wherein R2 and R 4 are each cyclopropyl groups. -8- The dioxetane of Claim 2 having the formula: (CH 3 -9- 4 I. "S 1~ 4 I 444 r 4 4 S. 44 fr 3444 The dioxetane of Claim 2 having the formula: The dioxetafle of Claim 2 having the formula: 52 -11- A dioxetane compound having the formula: (CH 3 2 CH 0 0 CH 3 CCH 3 2 C1 OH -12- A dioxetarie compound having the formula: (CH 3 2 CH 0-0 OCH 3 (CH 3 2 CHQ Osi (CH.1) 2 t-BU 90*4)-13- A dioxetane compound having the formula: (CH 3 2 CH 0-0 OCH3 (CH 3 2 CH OCO (CC-ji. 53 A dioxetane compound having the formula: (CH 3 2 CH (CH 3 2 CH' OCOPh -16- A dioxetane compound having the formula: (CHO) 2 CH. (CHO 2 CH' OPO (CH 2 CH 2 CN) 2 ~09~ 'pa. a. 0*a. *0 V *S *9 a a flat '.59 a a *9 U boa. a ta a .aaa A ,C U a ta 'a. -17- A dioxetane compound having the formula: (CH3) 2CH OP03Na 2 A dioxetane compound having the formula: -19- A dioxetane compound having the formula: OSi (CH 3 2 t-Bu A dioxetane compound having the formula: S.0 a 0 0 0 00*00 OSi (CH 3 2 t -Bu Lumigen, Inc. 4. Thne cc~qr6 e- C 0 CtAwO('5 by Their Patent Attorneys DAVIES COLLISON CAVE So's. DATED this 29th day of August, 1997 ABSTRACT A chemiluminescent assay method and compositions are described which use a dialkyl-substituted dioxetane which is deprotected to trigger a chemiluminescent reaction. Chemiluminescent 1,2- dioxetane compounds substituted on the dioxetane ring with two nonspirofused alkyl groups which can be triggered by a reagent to generate light are disclosed. Dialkyl-substituted dioxetanes are useful for the detection of triggering agents including enzymes. The enzyme may be present. alone or linked to a member of a specific binding pair in an immunoassay, DNA probe assay or other assay where the enzyme is bound to a reporter molecule. SO9.. 1 t. Q