Novel chemiluminescent dioxetane dimers

Abstract

Described herein are 1,2-dioxetane dimers that are useful as chemiluminescent probes, diagnostic agents, and imaging agents. Also described herein are compositions containing such compounds and methods of using the same.

Description

Attorney Docket No. 772156: DABX-003PCTNOVEL CHEMILUMINESCENT DIOXETANE DIMERSCROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 734,464, filed December 16, 2024, the disclosures of which are hereby incorporated by reference in their entireties.BACKGROUND

[0002] Chemiluminescent dioxetanes are strained cyclic peroxides that can undergo rapid decomposition to generate an excited, transient species that subsequently decays to ground state via emission of light.

[0003] Such compounds are useful as luminescent probes in a range of assays, including enzyme activity assays, immunoassays, and DNA detection assays. Chemiluminescence-based assays can offer excellent sensitivity because, unlike fluorescence and absorption-based assays, no light excitation source is required.

[0004] Dioxetanes can be generated in situ at the time of their use or prepared in advance in stable form and then later activated. When generated in situ via oxidation of a precursor alkene, chemiluminescent dioxetanes can also function as a detection or imaging method for reactive oxygen species (ROS). An example of a stable chemiluminescent dioxetane is 4-methoxy-4-(3-phosphatephenyl)spiro[1 ,2-dioxetane- 3,2'-adamantane], This compound, also known as LUMIGEN® PPD, can be activated upon treatment with an alkaline phosphatase (ALP). ALP is an enzyme that catalyzes the hydrolysis of phosphate groups. Once activated, the resulting compound subsequently undergoes fragmentation of the 1 ,2-dioxatane ring and emits light, thus functioning as a luminescent probe in alkaline phosphatase-labeled assays.

[0005] Because of improvements in assay technologies, there is a continued desire for better and faster probes that are suitable for detection under range of conditions, including both aqueous and non-aqueous media. Many dioxetane compounds suffer from weak emissions in aqueous media and take a long time to reach maximum luminescence after contact with a desired analyte. Current probes may be insufficient for future assay systems. There is a clearly felt unmet need in the diagnostics field for highly chemiluminescent compounds that achieve maximum intensity in very short time periods.Attorney Docket No. 772156: DABX-003PCTSUMMARY

[0006] There is a need for dioxetanes that provide a rapid response to the presence of an analyte including, for example, ALP. There is also a need for dioxetanes that are strongly emissive and suitable for use in both aqueous and non-aqueous environments. Various compounds disclosed herein provide such features.

[0007] The present disclosure provides a compound of Formula I and salts thereof.

[0008] Each of R1and R2is independently an optionally substituted C3-C10 alkyl, or R1and R2taken together with the carbon to which they are attached provide an optionally substituted C5-C10 cycloalkyl ring. R3is C1-C10 alkyl, C6-C10 aryl, or heteroaryl, any of which may be optionally substituted.

[0009] Each phenyl ring A and B has 3 R5groups, each R5located at a ring position of 2-6, wherein each of R5is independently H, Q, X, hydroxy, halogen, amino, thio, nitro, trifluoromethyl, or C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, C1-C10 alkyloxy, C1-C10 alkylamino, C1-C10 trialkylammonium salt, C1-C10 alkylthio, C2-C10 acyl, C1-C10 alkyloxycarbonyl, C1-C10 alkylaminocarbonyl, C1-C10 alkylthiocarbonyl, C2-C10 acyloxy, C2-C10 acylamino, C2-C10 acylthio, C1-C10 alkylcarbonate, C1-C10 alkylcarbamate, C1- C10 carbamido, aryloxy, C1-C10 alkylsulfinyl, C1-C10 alkylsulfonyl, arylthio, arylamino, arylsulfinyl, arylsulfonyl, arylcarbonyl, heteroarylcarbonyl, heteroaryloxy, heteroarylthio, heteroarylamino, heteroarylsulfinyl, heteroarylsulfonyl, cyano, phosphonate, C1-C10 alkylphosphonate, C1-C10 alkylphosphinate, C1-C10 trialkylphosphonium salt, C4-C10 heterocycloamino, C6-C10 aryl, or n-excessive heteroaryl, any of which may be optionally substituted, and wherein at least one R5on one of the rings is Q.Attorney Docket No. 772156: DABX-003PCT

[0010] Q is a n-conjugated electron-donating group.

[0011] X is -OH, -O-G, an -O’ salt, or a boronate group.

[0012] G is an alcohol protecting group, an analyte-responsive protecting group or divalent fragmentable linker having a pendant protecting group.

[0013] The conjugated linker comprises a chemical moiety that is capable of forming a conjugated system with phenyl rings A and B, wherein the conjugated linker is bound at a ring position of 2, 3, 4, 5, or 6 on phenyl ring A and, independently, bound at a ring position 2’, 3’, 4’, 5’, or 6’ on phenyl ring B. In some embodiments, the conjugated linker is bound to the same ring position on both rings A and B. For example, the conjugated linker may be bound at the 4-position on phenyl ring A and the 4’-position on phenyl ring B. In other embodiments, the conjugated linker is bound to a different ring position on both rings A and B, such as the 3-position on phenyl ring A and the 4’- position on phenyl ring B.

[0014] The present disclosure also provides an aqueous composition comprising one or more chemiluminescent dioxetane compounds described here. In an exemplary embodiment, the aqueous composition has a peak luminescent intensity of from 100 photons / sec to 50,000 photons / sec and an emission half-life, T1 / 2, of from 100 microseconds to 30 seconds upon treatment with a pH 9.7 amine-based buffer at 37°C.

[0015] The present disclosure also provides a method for determining the presence of an analyte in a sample, comprising contacting the sample with a compound of Formula I and monitoring the sample for luminescence.

[0016] Advantages, some of which are unexpected, are achieved by various embodiments of the present disclosure. Various compounds described herein can advantageously provide a rapid, high-intensity luminescent signal in non-aqueous media, aqueous media, or both. It is a significant advantage that assays involving such compounds can be performed faster than those with compounds lacking the features of the presently described compounds. Moreover, the compounds of the present disclosure provide increased intensity of luminescence, including in aqueous media. Due to such advantageous properties, various embodiments of the present disclosure can provide a method or kit that can detect an analyte, including, for example, ALP, in an aqueous or non-aqueous sample in under 1 minute, under 30 seconds, or in 100 microseconds to 30 seconds.Attorney Docket No. 772156: DABX-003PCTBRIEF DESCRIPTION OF THE DRAWINGS

[0017] FIG. 1 is a graph showing the time profile of chemiluminescence intensity of the compound of Example 1 , using 50 pL of a 0.0001 mg / mL sample of the compound in methanol, which was triggered with 5 pL of 10mM NaOH at 37°C.

[0018] FIG. 2 is a spectrograph showing the emission spectra of a number of embodied dioxetane dimer compounds of the present disclosure and known examples HPD (3-(4'-methoxyspiro[adamantane-2,3'-[1 ,2]dioxetan]-4'-yl)phenol) and VHPD (5- (4'-methoxyspiro[adamantane-2,3'-[1 ,2]dioxetan]-4'-yl)-2-vinylphenol) in the same conditions. The compounds are structures (3), (7), (9), (11 ), as shown in Examples 3, 7, 9, 11.

[0019] FIG. 3 is a graph showing the kinetic data of the emission of a number of embodied dioxetane dimer compounds of the present disclosure and known examples HPD (3-(4'-methoxyspiro[adamantane-2,3'-[1 ,2]dioxetan]-4'-yl)phenol) and VHPD (5- (4'-methoxyspiro[adamantane-2,3'-[1 ,2]dioxetan]-4'-yl)-2-vinylphenol) in the same conditions. The compounds are structures (3), (11 ), as shown in Examples 3, 11.DESCRIPTION

[0020] Reference will now be made in detail to certain embodiments of the disclosed subject matter, examples of which are illustrated in part in the accompanying drawings. While the disclosed subject matter will be described in conjunction with the enumerated claims, it will be understood that the exemplified subject matter is not intended to limit the claims to the disclosed subject matter.

[0021] The present disclosure describes novel compounds that are useful in chemiluminescent applications. Applicants have extensive experience in the synthesis of chemiluminescent compounds, particularly as labels in bioassays. However, in the process of investigating possible new dioxetane compounds, they unexpectedly synthesized the embodied compound (2) in a side reaction:Attorney Docket No. 772156: DABX-003PCTCompound (2) was initially noticed as an unidentified bright spot that showed up while the desired product, compound (1 ), was being purified via chromatography. Subsequent NMR and MS studies verified the product structure. The discovery that “dimerized” dioxetane compounds could be synthesized, produce light, and are stable led to the synthesis of additional embodied compounds that comprise dioxetane dimers coupled together via a ^-conjugated system.Dioxetane Dimer Compounds

[0022] The present disclosure provides a compound of Formula I, or a salt thereof.

[0023] Each of R1and R2is independently an optionally substituted C3-C10 alkyl, or R1and R2taken together with the carbon to which they are attached provide an optionally substituted C5-C10 cycloalkyl ring, e.g., a monocyclic, bicyclic, or tricyclic ring. In various embodiments, R1and R2are linked such that they provide, together with the carbon to which they are attached, a spirocyclic bridged bicyclo or tricyclo group. For example, R1and R2taken together with the carbon to which they are attached can be a spirocyclic adamantane, norbornane, or bornane.

[0024] Each R3is C1-C10 alkyl, Ce-C-io aryl, or heteroaryl, each of which is optionally substituted. As a non-limiting example, R3can be an unsubstituted C1-C10 alkyl or Ci- C10 alkyl substituted with one or more halogen, hydroxy, amino, thio, alkoxy, alkylamino, alkylthio, sulfate, or carboxylate.

[0025] Each phenyl ring A and B can independently has three R5groups, each R5located at a ring position of 2-6, wherein each R5is independently H, Q, X, hydroxy, halogen, amino, thio, nitro, trifluoromethyl, or C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, C1-C10 alkyloxy, C1-C10 alkylamino, C1-C10 trialkylammonium salt, C1-C10Attorney Docket No. 772156: DABX-003PCT alkylthio, C2-C10 acyl, C1-C10 alkyloxycarbonyl, C1-C10 alkylaminocarbonyl, C1-C10 alkylthiocarbonyl, C2-C10 acyloxy, C2-C10 acylamino, C2-C10 acylthio, C1-C10 alkylcarbonate, C1-C10 alkylcarbamate, C1-C10 carbamido, aryloxy, C1-C10 alkylsulfinyl, C1-C10 alkylsulfonyl, arylthio, arylamino, arylsulfinyl, arylsulfonyl, arylcarbonyl, heteroarylcarbonyl, heteroaryloxy, heteroarylthio, heteroarylamino, heteroarylsulfinyl, heteroarylsulfonyl, cyano, phosphonate, C1-C10 alkylphosphonate, C1-C10 alkylphosphinate, C1-C10 trialkylphosphonium salt, C4-C10 heterocycloamino, C6-C10 aryl, or n-excessive heteroaryl, any of which may be optionally substituted, and wherein at least one R5on one of phenyl rings A and B is Q. In some embodiments, at least one R5on each of phenyl rings A and B is Q.

[0026] In various embodiments, each R5is independently selected from the group H, Q, X, halogen, optionally substituted C1-C10 alkyl, hydroxy, optionally substituted C1- C10 alkyloxy, amino, optionally substituted C1-C10 alkylamino, thio, optionally substituted C1-C10 alkylthio, optionally substituted C2-C10 acyloxy, optionally substituted C2-C10 acylamino, optionally substituted C2-C10 acylthio, optionally substituted C1-C10 alkylcarbonate, optionally substituted C1-C10 alkylcarbamate, optionally substituted C1-C10 carbamido, optionally substituted aryloxy, optionally substituted arylthio, optionally substituted arylamino, optionally substituted heteroaryloxy, optionally substituted heteroarylthio, and optionally substituted heteroarylamino.

[0027] In various further embodiments, at least one R5on each of phenyl rings A and B provides a net electron donating effect on the aromatic ring to which they are attached. For example, the aromatic ring to which X, Q, and R5attach is electron- enriched relative to an identical theoretical compound in which Q and R5is H.

[0028] Q is a n-conjugated group, an electron donating group, or both. Q can be substituted or unsubstituted. In various embodiments, Q is a n-conjugated electron donating group. In various embodiments, Q is an optionally substituted C2-C10 alkenyl, an optionally substituted C2-C10 heterocycloalkenyl, an optionally substituted C6-C10 aryl, or an optionally substituted heteroaryl. In some embodiments, when Q is C2-C10 alkenyl it is substituted with one or more electron-donating groups, it is free of electronwithdrawing groups, or both. In various embodiments, when Q is C2-C10 alkenyl the vinylic positions and allylic positions, if present, are unsubstituted. In some further embodiments. For example, Q can be unsubstituted vinyl. In some embodiments,Attorney Docket No. 772156: DABX-003PCT when Q is Ce-C-io aryl it is substituted with one or more electron-donating groups, it is free of electron-withdrawing groups, or both. For example, Q can be unsubstituted phenyl, phenyl substituted with one or more electron-donating groups, or phenyl substituted with one or more substituents selected from a group consisting of electrondonating substituents. As another example, Q can be vinyl or phenyl substituted with substituents such that the net effect of the substituents is an electron-donating effect. In a further example, Q is a n-excessive heteroaryl such as thiophenyl, furanyl, pyrrolyl, benzothiophenyl, benzofuranyl, or indolyl. In certain embodiments, Q is substituted or unsubstituted thiophen-2-yl or thiophen-3-yl.

[0029] X is -OH, -O-G (as further defined below), an -O’ salt, or a boronate group or a group that generates an oxy anion upon chemical or enzymatic trigger. When X is a boronate group, it has the structure:OR8F OR9. wherein R8and R9of the boronate group are each independently H or an optionally substituted C1-C10 alkyl, or R8and R9taken together with the boronate to which they are attached are an optionally substituted C2-C10 cyclic boronate ester. For example, X can 4,4,5,5-tetramethyl-1 ,3,2-dioxaborolanyl or -B(OH)2.

[0030] In various embodiments where X is -O-G, G is an alcohol protecting group, an analyte-responsive protecting group, or both. For example, G can be trialkylsilyl, alkylarylsilyl, arylsulfonyl, dioxobenzyl, trityl, alkylcarbonate, phosphoryl, dihydropyranyl, tetrahydropyranyl, dihydrofuranyl, tetrahydrofuranyl, pyranosyl, pyranuronyl, furanosyl, acyl, benzoyl, or benzyl. In some embodiments, G is pyranosyl or pyranuronyl such as galactosyl, glucosyl, or glucuronyl. In further such embodiments, G is p-galactosyl, p-glucosyl, or p-glucuronyl. G can also be a phosphorous-containing group, such as a phosphate, phosphonate, and the like. For example, G can be -PO3H2 or a salt or ester thereof. In further embodiments, G is 2,4- dinitrobenzenesulfonyl, 3,4,6-trimehyl-2,5-dioxobenzyl, 4-azidobenzyloxy, tertbutyldimethylsilyl, acetyl, pivaloyl, an enzyme-cleavable moiety. For example, G can be a phosphatase-cleavable moiety or a peptidase-cleavable moiety. G can also comprise a divalent fragmentable linker having a pendant protecting group such that removal of the pendant protecting group triggers fragmentation of the linker and removal of the protecting group, G. Thus, G can contain a divalent fragmentable linkerAttorney Docket No. 772156: DABX-003PCT such as 4-aminobenzyl, 4-(alkylamino)benzyl, 4-oxybenzyl, 4-(oxymethyl)benzyl, oxymethyl, aminomethyl, alkylaminomethyl, and the like, together with a terminal protecting group such as trialkylsilyl, alkylarylsilyl, arylbenzenesulfonyl, dioxobenzyl, trityl, alkylcarbonate, phosphoryl, dihydropyranyl, tetrahydropyranyl, dihydrofuranyl, tetrahydrofuranyl, pyranosyl, pyranuronyl, furanosyl, acyl, benzoyl, benzyl or boronate group.

[0031] Examples of X include the following structures:

[0032] The conjugated linker comprises a chemical moiety that is capable of forming a conjugated system with phenyl rings A and B, wherein the conjugated linker is bound at a ring position of 2, 3, 4, 5, or 6 on phenyl ring A and independently at a ring position 2’, 3’, 4’, 5’, or 6’ on phenyl ring B. Non-limiting examples of conjugated linkers include the following structures:Attorney Docket No. 772156: DABX-003PCTor combinations of thereof.

[0033] Each of R10, R11, and R12is independently hydrogen, hydroxy, halo, or C1-C10 alkyl, C2-C10 alkenyl, or C2-C10 alkynyl, any of which is optionally substituted.

[0034] Each R13is independently N, S, O, Se, or Si(R14)2, wherein each R14is hydrogen or an optionally substituted alkyl.

[0035] Each Y is independently a hydrogen or a halogen.

[0036] n is at least 1 and up to 200.

[0037] The present disclosure also provides a compound of Formula II, or a salt thereof.Attorney Docket No. 772156: DABX-003PCT(Formula II)

[0038] Each of X, R3and R5are as described above for Formula I.

[0039] Each of R14and R15is independently H, halogen, C1-C10 alkyl, C2-C10 alkenyl, C6-C10 aryl, any of which is optionally substituted. In some embodiments, each of R14and R15is independently H or halogen.

[0040] The conjugated linker in Formula II is bound to phenyl rings A and B at the 4- position on phenyl ring A and the 4’-position on phenyl ring B and may be any group as defined in Formula I.

[0041] The present disclosure provides a composition comprising one or more of the compounds described herein, an olefin precursor thereof, or salt thereof. An olefin precursor provides any compound described herein (e.g., a compound of Formula I or Formula II) upon treatment with an analyte, an oxidizing agent, an alkaline phosphatase, or photooxidation conditions.

[0042] In various embodiments, the composition further comprises a buffer solution. The buffer solution can, but need not necessarily be, an alkaline or amine-based buffer solution. An example amine-based buffer is 221 buffer available from Sigma-Aldrich (St. Louis, MO). Likewise, the composition can, but need not necessarily, have a basic pH. For example, composition can have a pH of 4 to 12, 5 to 12, 6 to 12, 7 to 12, 8 to 12, 9 to 12, 10 to 12, 4 to 11 , 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, or 4 to 5. The pH of the composition can be selected based on whether luminescence is intended to trigger immediately upon analyte-triggered removal of a protecting group or substrate, as is typically the case for alkaline pH values, or the pH of the composition can be acidic so as to luminesce upon treatment with base.Attorney Docket No. 772156: DABX-003PCT

[0043] In various embodiments, the composition has a peak luminescent intensity of greater than 1 ,000 photons / sec and an emission half-life, T1 / 2, of 3 minutes or less upon treatment with pH 9.7 buffer at 37°C. In various examples, the peak luminescent intensity can be greater than 2,000 photons per second (photons / sec), greater than 3,000 photons / sec, greater than 4,000 photons / sec, greater than 5,000 photons / sec, greater than 6,000 photons / sec, greater than 7,000 photons / sec, greater than 8,000 photons / sec, greater than 9,000 photons / sec, or greater than 10,000 photons / sec. In various examples, the peak luminescent intensity can be 50,000 photons / sec or less. For example, the peak luminescent intensity can be from 100 to 50,000 photons / sec, from 1000 to 50,000 photons / sec, or from 10,000 to 50,000 photons / sec. Additionally or alternatively, the peak luminescent intensity can be up to 50,000 photons / sec. In some examples, the T1 / 2 can be 3 minutes or less, 2 minutes or less, 1 minute or less, 30 seconds or less, 20 seconds or less, 15 seconds or less, or 10 seconds or less. In some embodiments, the Ti / 2 can be at least 1 millisecond, at least 10 milliseconds, at least 25 milliseconds, at least 50 milliseconds, at least 100 milliseconds, at least 500 milliseconds, at least 1 second, at least 5 seconds, at least 100 microseconds, at least 500 microseconds, at least 1 millisecond, at least 10 milliseconds, at least 25 milliseconds, at least 50 milliseconds, at least 100 milliseconds, at least 500 milliseconds, or at least 1 second. For example, the T 1 / 2 can be from 100 microseconds to 30 seconds, 500 microseconds to 30 seconds, 1 millisecond to 30 seconds, 10 milliseconds to 30 seconds, 25 milliseconds to 30 seconds, 50 milliseconds to 30 seconds, 100 milliseconds to 30 seconds, 500 milliseconds to 30 seconds, 1 second to 30 seconds, 5 seconds to 30 seconds, 100 microseconds to 10 seconds, 500 microseconds to 10 seconds, 1 millisecond to 10 seconds, 10 milliseconds to 10 seconds, 25 milliseconds to 10 seconds, 50 milliseconds to 10 seconds, 100 milliseconds to 10 seconds, 500 milliseconds to 10 seconds, or 1 second to 10 seconds. In one example, the present disclosure provides an aqueous composition comprising one or more dioxetane compounds and having a peak luminescent intensity of greater than 1000 photons / sec and a T1 / 2 of 10 seconds or less upon treatment with pH 9.7 buffer at 37°C.Methods of Using the Compounds

[0044] The present disclosure also provides a method of detecting an analyte in a sample, the method comprising contacting the sample with one or more of theAttorney Docket No. 772156: DABX-003PCT compounds described herein, an olefin precursor thereof, a salt thereof, or a composition comprising the same, and then monitoring the sample for luminescence. In various embodiments, the method involves measuring the intensity of a resulting luminescence and correlating the intensity to the presence of the analyte. The assays described herein can be configured to measure chemiluminescence according to the non-limiting examples described in J.E. Wampler, Instrumentation: Seeing the Light and Measuring It, in Chemi- and Bioluminescence, J.G. Burr, ed., Marcel Dekker, New York, 1-44 (1985), A.K. Campbell, Detection and Quantification ofChemiluminescence, in Chemiluminescence Principles and Applications in Biology and Medicine, Ellis Norwood, Chichester, 68-126 (1988), F. Berthold, Instrumentation for Chemiluminescence Immunoassays, in Luminescence Immunoassay and Molecular Applications, K. Van Dyke and R. Van Dyke, eds., CRC Press, Boca Raton, 11-25 (1990), and T. Nieman, Chemiluminescence: Theory and Instrumentation, Overview, in Encyclopedia of Analytical Science, Academic Press, Orlando, 608-613 (1995).

[0045] In some embodiments, the method of detecting an analyte in a sample is directed to an immunoassay-based method. In such methods, the detected analyte, for example alkaline phosphatase (ALP), is linked to an antibody that selectively binds to the antigen of interest (e.g., biomarker, protein, etc.). For example, the dioxetane molecules described herein may react with an ALP moiety attached to an antibody to produce chemiluminescence as described in Liu et aL, 28 Molecules 6565 (2023).

[0046] Generally, the detected analyte can be any substance which directly or indirectly generates a chemiluminescent reaction from the composition including any compound described herein (e.g., a compound of Formula I or Formula II). For example, in various embodiments, the analyte can be an enzyme like alkaline phosphatase, a peptidase, a glucosidase, an oxidizing agent such as hydrogen peroxide or other reactive oxygen species, glutathione, fluoride, or a base under alkaline conditions.

[0047] The compounds of the present disclosure can be configured as probes to detect variety of different analytes by modifying group X or G. For example, the compounds described here can be configured to detect p-galactosidase by furnishing a glucosyl group at G, configured to detect hydrogen peroxide or other oxidizing agents by furnishing a boronate at X, configured to detect alkaline phosphatase byAttorney Docket No. 772156: DABX-003PCT furnishing a phosphate at X or a phosphoryl group at G, configured to detect glutathione by furnishing a dinitrobenzenesulfonylaminobenzyl group at G, configured to detect fluorine by furnishing a trialkylsilyl group at G, and configured to detect alkaline conditions when X is OH.

[0048] The compounds and compositions described herein can be triggered directly by an analyte so as to produce a signal identifying the presence of the analyte and probe or can be triggered in a two step-process, one step which involves contacting the analyte and another step which involves raising the pH. In some embodiments, the method further involves increasing the pH of the sample. For example, the pH can be adjusted to from 7 to 14, 8 to 14, 9 to 14, 10 to 14, or 11 to 14.

[0049] The rate of luminescence increase, or rise time, can be described according to either the time to the maximum emission (tmax) or the emission half-life (T1 / 2). Chemiluminescence intensity / time profile comprises a period of initial rising emission intensity and a subsequent period of steady-state intensity or decay. Embodiments described herein show a fast rise time suggesting a fast reaction of S' — P* that corresponds to a short initial rising period, as shown in one embodiment in FIG. 1 . For enzymatic chemiluminescent reactions, intensity may remain plateaued at a high level or show a decay, as in the case of a fast-emitting or quenching dioxetane compound. In the case of the current dioxetane dimers, the compounds show a rapid emission that is quenched in under a minute (FIG. 1). The absence of a long (for example, greater than 60 seconds) steady-state intensity indicates either substrate depletion or subsequent inactivation of the enzyme.

[0050] In some applications, it is desirable that the compounds emit all possible light in the briefest possible period of time upon being triggered by the analyte so as to provide the strongest signal possible. When the chemiluminescence is emitted gradually over a period of time, light intensity (photons / sec) is diminished and detection sensitivity can be impaired. An example of the profile of a luminescent signal versus time is shown in Fig. 1. In various embodiments, the analyte is detected in a time range of 100 microseconds to 30 seconds, 500 microseconds to 30 seconds, 1 millisecond to 30 seconds, 10 milliseconds to 30 seconds, 25 milliseconds to 30 seconds, 50 milliseconds to 30 seconds, 100 milliseconds to 30 seconds, 500 milliseconds to 30 seconds, 1 second to 30 seconds, 5 seconds to 30 seconds, 100 microseconds to 10 seconds, 500 microseconds to 10 seconds, 1 millisecond to 10Attorney Docket No. 772156: DABX-003PCT seconds, 10 milliseconds to 10 seconds, 25 milliseconds to 10 seconds, 50 milliseconds to 10 seconds, 100 milliseconds to 10 seconds, 500 milliseconds to 10 seconds, or 1 second to 10 seconds..

[0051] The compounds described herein can be used as an enzyme substrate. For example, various compounds in which G is a phosphorous-containing group can be used as a substrate for alkaline phosphatase (ALP) enzyme, and the like. Without being limited by theory, one example mechanism involves the ALP enzyme hydrolyzing the phosphorous-containing group to provide a phenol which is immediately deprotonated due to the alkaline environment of the solution (e.g., pH 9.7 buffer). Formation of the oxy anion triggers decomposition of the di-1 ,2-dioxetane into three compounds: two 2-adamantanones and a phenyl ester that is in an excited state. The excited phenyl ester then immediately decays to the ground state by releasing light.

[0052] The resulting light intensity is a linear function of the amount of the enzyme. The compounds described herein can thus be used to detect a label enzyme used in an assay. For example, the steps of the chemical process in which the dioxetane dimers described herein provide light can be described according to the following steps: (i) X + S — > X + S' (ii) S' P* and (iii) P* — > P + light. Step (i) represents catalytic turnover of the substrate, wherein X is an enzyme or other component that converts the substrate to its activated form, step (ii) represents degradation of the activated substrate to a transient excited species, and step (iii) represents decay of the excited species to ground state and emission of light, and is extremely short in comparison to the other steps and generally has no meaningful effect on reaction kinetics. Light intensity is the product of the catalytic turnover of substrate in step (i) and the lifetime of the resulting light-producing compound P* in step (ii). Step (ii) is usually first order with a rate constant k and can be characterized by its half-life: T1 / 2 = (In 2) / k. In the case of the embodied compounds, the conjugated dioxetane dimers show very fast luminescence that appears to be driven by the conjugation in combination with the excitation of the first dioxetane driving excitation of the second on the same molecule. Based on these results, the speed and intensity of dioxetane chemiluminescence can be increased by use of conjugated linkers and substituents that are n-conjugated, electron-donating, or both. Moreover, as further described in Example 1 , an example conjugated dioxetane dimer was tested in aqueous conditions. The compounds andAttorney Docket No. 772156: DABX-003PCT compositions of the present disclosure thus represent a significant improvement over commercially-available dioxetanes for certain applications.

[0053] In other embodiments, the present disclosure relates to an assay that uses the compounds described herein as a chemiluminescent probe. The assay can be a homogeneous (non-separation) immunoassay in which bound and unbound ligands do not need to be separated, or the assay can be a heterogeneous assay in which labeled binding pair complexes are separated from unbound labeled reactants. The assay can be configured to be performed manually or it can be automated and performed robotically. The assay can be performed in test tubes, cuvettes, microwells, or a combination thereof. In various embodiments, the test tubes, cuvettes, microwell, or other containers in which the assay is performed are at least partially opaque, fully opaque, black, white, or a combination thereof. The assay can be performed on immobilized proteins in western blots, immobilized nucleic acids in southern or northern blots.

[0054] The present disclosure further provides a kit for determining the presence of an analyte, the kit comprising the compound of any one or more of the compounds described herein, an olefin precursor thereof, a salt thereof, or a composition comprising the same. The kit can contain instructions according to the method described herein.Processes of Making Dioxetane Dimer Compounds

[0055] Compounds of the present disclosure can be prepared, for example, according to the general synthetic approach described in Synthetic Path 1. In some embodiments, one or more of the individual steps shown may be known in the art (See, for example, Jerry March, Michael B. Smith, March's Advanced Organic Chemistry 6th edition, 2007, Wiley Interscience and J. McMurry, Organic Chemistry, 5th Ed. (Brooks / Cole, Pacific Grove, 2000)), but the embodied processes as a whole unexpectedly allow synthesis of novel compounds.Attorney Docket No. 772156: DABX-003PCTSynthetic Path 1

[0056] The resulting olefins can be further modified, e.g., via removing or replacing protecting group G or further functionalizing positions R5on the ring. Additional examples of synthetic methods for producing the compounds of present disclosure are found in Examples 1-13 (below).Additional Disclosure

[0057] The term light “intensity” or luminescence “intensity” as used herein refers to the rate of emission in photons / sec. Intensity can be measured by use of a luminometer. A luminometer is a photodetector in a housing which excludes ambient light. Any suitable luminometer can be used, including photomultiplier tubes andAttorney Docket No. 772156: DABX-003PCT photodiodes. However, a Turner Designs (Sunnyvale, CA) model TD-20e luminometer was used in the Examples provided herein.

[0058] The term “sensitivity” as used herein refers to the lowest level at which a signal for an analyte or product being measured can be reproducibly detected.

[0059] The term “alkyl” as used herein refers to substituted or unsubstituted straight chain, branched or cyclic, saturated mono- or bi-valent groups having from 1 to 20 carbon atoms, 10 to 20 carbon atoms, 12 to 18 carbon atoms, 6 to 10 carbon atoms, 1 to 10 carbons atoms, 1 to 8 carbon atoms, 2 to 8 carbon atoms, 3 to 8 carbon atoms, 4 to 8 carbon atoms, 5 to 8 carbon atoms, 1 to 6 carbon atoms, 2 to 6 carbon atoms, 3 to 6 carbon atoms, or 1 to 3 carbon atoms. Examples of straight chain mono-valent (Ci-C2o)-alkyl groups include those with from 1 to 8 carbon atoms such as methyl (i.e. , CHs), ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl groups. Examples of branched mono-valent (Ci-C2o)-alkyl groups include isopropyl, iso-butyl, sec-butyl, t- butyl, neopentyl, and isopentyl. Examples of straight chain bi-valent (Ci-C2o)alkyl groups include those with from 1 to 6 carbon atoms such as -CH2-, -CH2CH2-, - CH2CH2CH2-, -CH2CH2CH2CH2-, and -CH2CH2CH2CH2CH2-. Examples of branched bi-valent alkyl groups include -CH(CHs)CH2- and -CH2CH(CHS)CH2-. Examples of cyclic alkyl groups include cyclopropyl, cyclobutyl, cyclopently, cyclohexyl, cyclooctyl, bicyclo[1 .1 .1 ]pentyl, bicyclo[2.1.1]hexyl, bicyclo[2.2.1]heptyl, and adamantyl. Cycloalkyl groups further include substituted and unsubstituted polycyclic cycloalkyl groups such as, but not limited to, norbornyl, adamantyl, bornyl, camphenyl, isocamphenyl, and carenyl groups, and fused rings such as, but not limited to, decalinyl, and the like. For example cycloalkyl includes an adamantyl substituted by one, two, three, four, or more substituents, e.g., at the tertiary bridgehead positions at the methylene bridges. In some embodiments, alkyl includes a combination of substituted and unsubstituted alkyl. As an example, alkyl, and also (C-i)alkyl, includes methyl and substituted methyl. As a further example, alkyl can include methyl and substituted (C2-C8)alkyl. Alkyl can also include substituted methyl and unsubstituted (C2-C8)alkyl. In some embodiments, alkyl can be methyl and C2-C8 linear alkyl. In some embodiments, alkyl can be methyl and C2-C8 branched alkyl. The term methyl is understood to be -CH3, which is not substituted. The term methylene is understood to be -CH2-, which is not substituted. For comparison, the term (Ci)alkyl is understood to be a substituted or an unsubstituted -CH3 or a substituted or anAttorney Docket No. 772156: DABX-003PCT unsubstituted -CH2-. Representative substituted alkyl groups can be substituted one or more times with any of the groups listed herein, for example, cycloalkyl, heterocyclyl, aryl, amino, haloalkyl, hydroxy, cyano, carboxy, nitro, thio, alkoxy, and halogen groups. As further example, representative substituted alkyl groups can be substituted one or more fluoro, chloro, bromo, iodo, amino, amido, alkyl, alkoxy, alkylamido, alkenyl, alkynyl, alkoxycarbonyl, acyl, formyl, arylcarbonyl, aryloxycarbonyl, aryloxy, carboxy, haloalkyl, hydroxy, cyano, nitroso, nitro, azido, trifluoromethyl, trifluoromethoxy, thio, alkylthio, arylthiol, alkylsulfonyl, alkylsulfinyl, dialkylaminosulfonyl, sulfonic acid, carboxylic acid, dialkylamino and dialkylamido. In some embodiments, representative substituted alkyl groups can be substituted from a set of groups including amino, hydroxy, cyano, carboxy, nitro, thio and alkoxy, but not including halogen groups. Thus, in some embodiments, alkyl can be substituted with a non-halogen group. For example, representative substituted alkyl groups can be substituted with a fluoro group, substituted with a bromo group, substituted with a halogen other than bromo, or substituted with a halogen other than fluoro. In some embodiments, representative substituted alkyl groups can be substituted with one, two, three or more fluoro groups or they can be substituted with one, two, three or more non-fluoro groups. For example, alkyl can be trifluoromethyl, difluoromethyl, or fluoromethyl, or alkyl can be substituted alkyl other than trifluoromethyl, difluoromethyl or fluoromethyl. Alkyl can be haloalkyl or alkyl can be substituted alkyl other than haloalkyl.

[0060] The term “alkenyl” as used herein refers to substituted or unsubstituted straight chain, branched or cyclic, saturated mono- or bi-valent groups having at least one carbon-carbon double bond and from 2 to 20 carbon atoms, 10 to 20 carbon atoms, 12 to 18 carbon atoms, 6 to 10 carbon atoms, 2 to 10 carbons atoms, 2 to 8 carbon atoms, 3 to 8 carbon atoms, 4 to 8 carbon atoms, 5 to 8 carbon atoms, 2 to 6 carbon atoms, 3 to 6 carbon atoms, 4 to 6 carbon atoms, 2 to 4 carbon atoms, or 2 to 3 carbon atoms. The double bonds can be trans or cis orientation. The double bonds can be terminal or internal. The alkenyl group can be attached via the portion of the alkenyl group containing the double bond, e.g., vinyl, propen-1-yl and buten-1-yl, or the alkenyl group can be attached via a portion of the alkenyl group that does not contain the double bond, e.g., penten-4-yl. Where specified, the parent moiety should be understood to be attached to the alkenyl group at a vinylic position of the double bondAttorney Docket No. 772156: DABX-003PCT rather than a non-vinylic position. For example, where an aromatic ring is substituted with a TT-conjugated alkenyl group, it should be understood to be substituted at the vinyl position rather than a non-vinylic position. As a further example, an aromatic ring substituted with a TT-conjugated propenyl group would be understood to be a propen- 1-yl or a propen-2 -yl group rather than a propen-3-yl group. Examples of mono-valent (C2-C2o)-alkenyl groups include those with from 1 to 8 carbon atoms such as vinyl, propenyl, propen-1-yl, propen-2-yl, butenyl, buten-1-yl, buten-2-yl, sec-buten-1-yl, sec-buten-3-yl, pentenyl, hexenyl, heptenyl and octenyl groups. Examples of branched mono-valent (C2-C2o)-alkenyl groups include isopropenyl, iso-butenyl, sec- butenyl, t-butenyl, neopentenyl, and isopentenyl. Examples of straight chain bi-valent (C2-C2o)alkenyl groups include those with from 2 to 6 carbon atoms such as -CHCH-, -CHCHCH2-, -CHCHCH2CH2-, and -CHCHCH2CH2CH2-. Examples of branched bivalent alkyl groups include -C(CHs)CH- and -CHC(CHs)CH2-. Examples of cyclic alkenyl groups include cyclopentenyl, cyclohexenyl and cyclooctenyl. For example, alkenyl can be vinyl and substituted vinyl. For example, alkenyl can be vinyl and substituted (Cs-CsjalkenyL Alkenyl can also include substituted vinyl and unsubstituted (Cs-CsjalkenyL Representative substituted alkenyl groups can be substituted one or more times with any of the groups listed herein, for example, monoalkylamino, dialkylamino, cyano, acetyl, amido, carboxy, nitro, alkylthio, alkoxy, and halogen groups. As further example, representative substituted alkenyl groups can be substituted one or more fluoro, chloro, bromo, iodo, amino, amido, alkyl, alkoxy, alkylamido, alkenyl, alkynyl, alkoxycarbonyl, acyl, formyl, arylcarbonyl, aryloxycarbonyl, aryloxy, carboxy, haloalkyl, hydroxy, cyano, nitroso, nitro, azido, trifluoromethyl, trifluoromethoxy, thio, alkylthio, arylthiol, alkylsulfonyl, alkylsulfinyl, dialkylaminosulfonyl, sulfonic acid, carboxylic acid, dialkylamino and dialkylamido. In some embodiments, representative substituted alkenyl groups can be substituted from a set of groups including monoalkylamino, dialkylamino, cyano, acetyl, amido, carboxy, nitro, alkylthio and alkoxy, but not including halogen groups. Thus, in some embodiments alkenyl can be substituted with a non-halogen group. In some embodiments, representative substituted alkenyl groups can be substituted with a fluoro group, substituted with a bromo group, substituted with a halogen other than bromo, or substituted with a halogen other than fluoro. For example, alkenyl can be 1- fluorovinyl, 2-fluorovinyl, 1 ,2-difluorovinyl, 1 ,2,2-trifluorovinyl, 2,2-difluorovinyl,Attorney Docket No. 772156: DABX-003PCT trifluoropropen-2-yl, 3,3,3-trifluoropropenyl, 1 -fluoropropenyl, 1 -chlorovinyl, 2- chlorovinyl, 1 ,2-dichlorovinyl, 1 ,2,2-trichlorovinyl or 2,2-dichlorovinyl. In some embodiments, representative substituted alkenyl groups can be substituted with one, two, three or more fluoro groups or they can be substituted with one, two, three or more non-fluoro groups.

[0061] The term “alkynyl” as used herein, refers to substituted or unsubstituted straight and branched chain alkyl groups, except that at least one triple bond exists between two carbon atoms. Thus, alkynyl groups have from 2 to 50 carbon atoms, 2 to 20 carbon atoms, 10 to 20 carbon atoms, 12 to 18 carbon atoms, 6 to 10 carbon atoms, 2 to 10 carbons atoms, 2 to 8 carbon atoms, 3 to 8 carbon atoms, 4 to 8 carbon atoms, 5 to 8 carbon atoms, 2 to 6 carbon atoms, 3 to 6 carbon atoms, 4 to 6 carbon atoms, 2 to 4 carbon atoms, or 2 to 3 carbon atoms. Examples include, but are not limited to ethynyl, propynyl, propyn-1-yl, propyn-2-yl, butynyl, butyn-1-yl, butyn-2-yl, butyn-3-yl, butyn-4-yl, pentynyl, pentyn-1-yl, hexynyl, Examples include, but are not limited to -OCH, -OC(CH3), -C=C(CH2CH3), -CH2C=CH, -CH2C=C(CH3), and -CH2C=C(CH2CH3) among others.

[0062] The term “aryl” as used herein refers to substituted or unsubstituted univalent groups that are derived by removing a hydrogen atom from an arene, which is a cyclic aromatic hydrocarbon, having from 6 to 20 carbon atoms, 10 to 20 carbon atoms, 12 to 20 carbon atoms, 6 to 10 carbon atoms or 6 to 8 carbon atoms. Examples of (Ce- C2o)aryl groups include phenyl, napthalenyl, azulenyl, biphenylyl, indacenyl, fluorenyl, phenanthrenyl, triphenylenyl, pyrenyl, naphthacenyl, chrysenyl, anthracenyl groups. Examples include substituted phenyl, substituted napthalenyl, substituted azulenyl, substituted biphenylyl, substituted indacenyl, substituted fluorenyl, substituted phenanthrenyl, substituted triphenylenyl, substituted pyrenyl, substituted naphthacenyl, substituted chrysenyl, and substituted anthracenyl groups. Examples also include unsubstituted phenyl, unsubstituted napthalenyl, unsubstituted azulenyl, unsubstituted biphenylyl, unsubstituted indacenyl, unsubstituted fluorenyl, unsubstituted phenanthrenyl, unsubstituted triphenylenyl, unsubstituted pyrenyl, unsubstituted naphthacenyl, unsubstituted chrysenyl, and unsubstituted anthracenyl groups. Aryl includes phenyl groups and also non-phenyl aryl groups. From these examples, it is clear that the term (Ce-C2o)aryl encompasses mono- and polycyclic (Ce- C2o)aryl groups, including fused and non-fused polycyclic (Ce-C2o)aryl groups.Attorney Docket No. 772156: DABX-003PCT

[0063] The term “heterocyclyl” as used herein refers to substituted aromatic, unsubstituted aromatic, substituted non-aromatic, and unsubstituted non-aromatic rings containing 3 or more atoms in the ring, of which, one or more is a heteroatom such as, but not limited to, N, O, and S. The term “heteroaryl” is a fully aromatic heterocyclyl and thus a subset of the term heterocyclyl. The term “heterocycloalkenyl” refers to a heterocyclyl group containing an olefin within a non-aromatic ring, such that the olefin is the point of connection to the parent moiety. A heterocyclyl group can thus be a heterocycloalkyl, heterocycloalkenyl, or a heteroaryl, or if polycyclic, any combination thereof. In some embodiments, heterocyclyl groups include 3 to 20 ring members, whereas other such groups have 3 to 15 ring members. In some embodiments, heterocyclyl groups include heterocyclyl groups that include 3 to 8 carbon atoms (Cs-Cs), 3 to 6 carbon atoms (Cs-Ce) or 6 to 8 carbon atoms (Ce-Cs). A heterocyclyl group designated as a C2-heterocyclyl can be a 5-membered ring with two carbon atoms and three heteroatoms, a 6-membered ring with two carbon atoms and four heteroatoms and so forth. Likewise, a C4-heterocyclyl can be a 5-membered ring with one heteroatom, a 6-membered ring with two heteroatoms, and so forth. The number of carbon atoms plus the number of heteroatoms equals the total number of ring atoms. A heterocyclyl ring can also include one or more double bonds. A heteroaryl ring is an embodiment of a heterocyclyl group. The phrase heterocyclyl group includes fused ring species including those that include fused aromatic and non- aromatic groups. Representative heterocyclyl groups include, but are not limited to piperidynyl, pyrrolidinyl, piperazinyl, and morpholinyl. For example, heterocyclyl groups include, without limitation:wherein X1represents H, (Ci-C2o)alkyl, (Ce-C2o)aryl or an amine protecting group (e.g., a t-butyloxycarbonyl group) and wherein the heterocyclyl group can be substituted or unsubstituted. Representative heteroaryl groups include furanyl, pyridinyl, pyrazinyl, pyrimidinyl, triazinyl, thiophenyl, tetrahydrofuranyl, pyrrolyl, oxazolyl, imidazolyl, triazyolyl, tetrazolyl, benzoxazolinyl, and benzimidazolinylAttorney Docket No. 772156: DABX-003PCT groups. In some embodiments, the heteroaryl is a 5-membered heteroaryl. In some embodiments, the heteroaryl is other than pyridine, pyrimidine, pyridazine, pyrazine, or fused derivatives thereof. A n-excessive heteroaryl is a heteroaryl that is electronrich such that it can function as an electron donating group. Examples of n-excessive heteroaryls are furan, thiophene, indole, pyrrole, benzofuran, and benzothiophene.

[0064] The term “alkoxy” as used herein refers to an oxygen atom connected to an alkyl group, including a cycloalkyl group, as are defined herein. Examples of linear alkoxy groups include but are not limited to methoxy, ethoxy, propoxy, butoxy, pentyloxy, hexyloxy, and the like. Examples of branched alkoxy include but are not limited to isopropoxy, sec-butoxy, tert-butoxy, isopentyloxy, isohexyloxy, and the like. Examples of cyclic alkoxy include but are not limited to cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy, and the like. An alkoxy group can include one to 12-20 or 12-40 carbon atoms bonded to the oxygen atom, and can further include double or triple bonds, and can also include heteroatoms. Thus, alkyoxy also includes an oxygen atom connected to an alkyenyl group and oxygen atom connected to an alkynyl group. For example, an allyloxy group is an alkoxy group within the meaning herein. A methoxyethoxy group is also an alkoxy group within the meaning herein, as is a methylenedioxy group in a context where two adjacent atoms of a structure are substituted therewith.

[0065] The terms “aryloxy” and “heteroaryloxy” as used herein refer to an oxygen atom connected to an aryl group or a heteroaryl group, respectively, as are defined herein. The point of substitution to the parent moiety is at the oxygen atom in both cases. The term “arylthio” as used herein refers to a sulfur atom connected to an aryl group as are defined herein. The point of substitution to the parent moiety is at the sulfur atom.

[0066] The term “arylcarbonyl” as used herein refers to a carbonyl (CO) group connected to an aryl group as are defined herein. The point of substitution to the parent moiety is at the carbonyl group.

[0067] The term “heteroarylcarbonyl” as used herein refers to a carbonyl (CO) group connected to a heteroaryl group as are defined herein. The point of substitution to the parent moiety is at the carbonyl group.

[0068] The term and “arylalkyl” as used herein refers to alkyl groups as defined herein in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to anAttorney Docket No. 772156: DABX-003PCT aryl group as defined herein. Representative aralkyl groups include benzyl, biphenylmethyl and phenylethyl groups and fused (cycloal kylaryl)alkyl groups such as 4-ethyl-indanyL Aralkenyl groups are alkenyl groups as defined herein in which a hydrogen or carbon bond of an alkenyl group is replaced with a bond to an aryl group as defined herein. The point of substitution to the parent moiety is at the alkyl group.

[0069] The term “amino” as used herein refers to a substituent of the form -NH2, - NHR, -NR2, -NR3+, wherein each R is independently selected, and protonated forms of each, except for -NR3+, which cannot be protonated. Accordingly, any compound substituted with an amino group can be viewed as an amine. An “amino group” within the meaning herein can be a primary, secondary, tertiary, or quaternary amino group. An “alkylamino” refers to a moiety composed of an alkyl substituent bound to the remainder of the molecule via a nitrogen and includes a monoalkylamino and dialkylamino groups. An “arylamino” refers to a moiety composed of an aryl substituent bound to the remainder of the molecule via a nitrogen. A “heterocycloamino” refers to a moiety composed of heterocycle as defined herein that is bound to the remainder of the molecule via a nitrogen.

[0070] The term “acyl” as used herein refers to a group containing a carbonyl moiety wherein the group is bonded via the carbonyl carbon atom. The carbonyl carbon atom is also bonded to another carbon atom, which can be part of a substituted or unsubstituted alkyl, alkenyl, alkynyl, alkoxy, aryl, cycloalkyl, heterocyclyl, group or the like.

[0071] The term “acyloxy” as used herein refers to a group -OCOR wherein the group is bonded via an oxygen atom, the carbonyl moiety is adjacent the oxygen, and R is a carbon atom, which can be part of a substituted or unsubstituted alkyl, alkenyl, alkynyl, alkoxy, aryl, cycloalkyl, heterocyclyl, group or the like. The term “acylamino” as used herein refers to a group -NCOR wherein the group is bonded via a nitrogen atom, the carbonyl moiety is adjacent the nitrogen, and R is a carbon atom, which can be part of a substituted or unsubstituted alkyl, alkenyl, alkynyl, alkoxy, aryl, cycloalkyl, heterocyclyl, group or the like. The term “acylthio” as used herein refers to a group - SCOR wherein the group is bonded via a sulfur atom, the carbonyl moiety is adjacent the sulfur, and R is a carbon atom, which can be part of a substituted or unsubstituted alkyl, alkenyl, alkynyl, alkoxy, aryl, cycloalkyl, heterocyclyl, group or the like.Attorney Docket No. 772156: DABX-003PCT

[0072] The term “alkoxycarbonyl” as used herein refers to a group containing a carbonyl moiety wherein the group is bonded via the carbonyl carbon atom. The carbonyl carbon atom is also bonded to an oxygen atom which is further bonded to an alkyl group. Alkoxycarbonyl also includes the group where a carbonyl carbon atom is also bonded to an oxygen atom which is further bonded to an alkyenyl group. Alkoxycarbonyl also includes the group where a carbonyl carbon atom is also bonded to an oxygen atom which is further bonded to an alkynyl group. In a further case, which is included in the definition of alkoxycarbonyl as the term is defined herein, and is also included in the term “aryloxycarbonyl,” the carbonyl carbon atom is bonded to an oxygen atom which is bonded to an aryl group instead of an alkyl group.

[0073] The term “alkylamido” as used herein refers to a group containing a carbonyl moiety wherein the group is bonded via the carbonyl carbon atom. The carbonyl carbon atom is also bonded to a nitrogen group which is bonded to one or more alkyl groups. In a further case, which is also an alkylamido as the term is defined herein, the carbonyl carbon atom is bonded to a nitrogen atom which is bonded to one or more aryl group instead of, or in addition to, the one or more alkyl group. In a further case, which is also an alkylamido as the term is defined herein, the carbonyl carbon atom is bonded to a nitrogen atom which is bonded to one or more alkenyl group instead of, or in addition to, the one or more alkyl and or / aryl group. In a further case, which is also an alkylamido as the term is defined herein, the carbonyl carbon atom is bonded to a nitrogen atom which is bonded to one or more alkynyl group instead of, or in addition to, the one or more alkyl, alkenyl and / or aryl group.

[0074] The term “alkylthio” as used herein refers to a sulfur atom connected to an alkyl, alkenyl, or alkynyl group as defined herein. The point of substitution to the parent moiety is at the sulfur atom. The term “alkylthiocarbonyl” as used herein refers to a carbonyl group bound to a sulfur atom connected to an alkyl, alkenyl, or alkynyl group as defined herein. The point of substitution to the parent moiety is at the carbonyl.

[0075] The term “arylthio” as used herein refers to a sulfur atom connected to an aryl group as defined herein. The point of substitution to the parent moiety is at the sulfur atom.

[0076] The term “alkylsulfonyl” as used herein refers to a sulfonyl group connected to an alkyl, alkenyl, or alkynyl group as defined herein. The term “arylsulfonyl” as usedAttorney Docket No. 772156: DABX-003PCT herein refers to a sulfonyl group connected to an aryl group as defined herein. In both cases, the point of substitution to the parent moiety is at the sulfonyl group.

[0077] The term “alkylsulfinyl” as used herein refers to a sulfinyl group connected to an alkyl, alkenyl, or alkynyl group as defined herein. The term “arylsulfinyl” as used herein refers to a sulfinyl group connected to an aryl group as defined herein. In both cases, the point of substitution to the parent moiety is at the sulfinyl group.

[0078] The term “alkylphosphonate” as used herein refers to the group - P(=O)(OR1 )(OR2) where the group is bound to the parent moiety at the phosphorous and at least one of R1and R2is a carbon atom, which can be part of a substituted or unsubstituted alkyl, alkenyl, alkynyl, alkoxy, aryl, cycloalkyl, heterocyclyl, group or the like.

[0079] The term “carboxy” as used herein refers to a group containing a carbonyl moiety wherein the group is bonded via the carbonyl carbon atom and the carbonyl carbon atom is also bonded to a hydroxy group or oxygen anion so as to result in a carboxylic acid or carboxylate. Carboxy also includes both the protonated form of the carboxylic acid and the salt form. For example, a carboxy moiety can be understood as -COOH or -CO2H.

[0080] The term “carbamate” as used herein refers to the group -O-C(=O)-NR1R2where the group is bound to the parent moiety at the oxygen and each of R1and R2is a carbon atom, which can be part of a substituted or unsubstituted alkyl, alkenyl, alkynyl, alkoxy, aryl, cycloalkyl, heterocyclyl, group or the like. The term “alkylcarbamate” as used herein refers to a carbamate moiety wherein at least one of R1and R2is an alkyl.

[0081] The term “carbamido” as used herein refers to the group -O-C(=O)-NH2 where the group is bound to the parent moiety at the oxygen.

[0082] The term “dialkylaminosulfonyl” as used herein refers to a sulfonyl group connected to a nitrogen further connected to two alkyl groups, as defined herein, and which can optionally be linked together to form a ring with the nitrogen. This term also includes the group where the nitrogen is further connected to one or two alkenyl groups in place of the alkyl groups. The point of substitution to the parent moiety is at the sulfonyl group.

[0083] The term “dialkylamino” as used herein refers to an amino group connected to two alkyl groups, as defined herein, and which can optionally be linked together toAttorney Docket No. 772156: DABX-003PCT form a ring with the nitrogen. This term also includes the group where the nitrogen is further connected to one or two alkenyl groups in place of the alkyl groups. The point of substitution to the parent moiety is at the nitrogen atom.

[0084] The term “dialkylamido” as used herein refers to an amido group connected to two alkyl groups, as defined herein, and which can optionally be linked together to form a ring with the nitrogen. This term also includes the group where the nitrogen is further connected to one or two alkenyl groups in place of the alkyl groups. The point of substitution to the parent moiety is at the amido group.

[0085] The term “formyl” as used herein refers to a group containing an aldehyde moiety. The point of substitution to the parent moiety is at the carbonyl group.

[0086] The terms “halo,” “halogen,” or “halide” group, as used herein, by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom.

[0087] Each of the various substituent groups described herein can be substituted or unsubstituted. The term “substituted” as used herein refers to a group that is substituted with one or more groups (substituents) including, but not limited to, the following groups: deuterium (D), halogen (e.g., F, Cl, Br, and I), R, OR, OC(O)N(R)2, CN, NO, NO2, ONO2, azido, CF3, OCF3, methylenedioxy, ethylenedioxy, (C3- C2o)heteroaryl, N(R)2, Si(R)3, SR, SOR, SO2R, SO2N(R)2, SO3R, P(O)(OR)2, OP(O)(OR)2, C(O)R, C(O)C(O)R, C(O)CH2C(O)R, C(S)R, C(O)OR, OC(O)R, C(O)N(R)2, C(O)N(R)OH, OC(O)N(R)2, C(S)N(R)2, (CH2)O-2N(R)C(0)R, (CH2)O- 2N(R)N(R)2, N(R)N(R)C(O)R, N(R)N(R)C(O)OR, N(R)N(R)CON(R)2, N(R)SO2R, N(R)SO2N(R)2, N(R)C(O)OR, N(R)C(O)R, N(R)C(S)R, N(R)C(O)N(R)2, N(R)C(S)N(R)2, N(COR)COR, N(OR)R, C(=NH)N(R)2, C(O)N(OR)R, or C(=NOR)R wherein R can be hydrogen, (Ci-C2o)alkyl or (C6-C2o)aryl. Substituted also includes a group that is substituted with one or more groups including, but not limited to, the following groups: fluoro, chloro, bromo, iodo, amino, amido, alkyl, alkoxy, alkylamido, alkenyl, alkynyl, alkoxycarbonyl, acyl, formyl, arylcarbonyl, aryloxycarbonyl, aryloxy, carboxy, haloalkyl, hydroxy, cyano, nitroso, nitro, azido, trifluoromethyl, trifluoromethoxy, thio, alkylthio, arylthiol, alkylsulfonyl, alkylsulfinyl, dialkylaminosulfonyl, sulfonic acid, carboxylic acid, dialkylamino and dialkylamido.

[0088] In lists of possible chemical moieties that may be optionally substituted, it is understood that whether substitution is possible on a particular moiety and if so, theAttorney Docket No. 772156: DABX-003PCT type of substitution that can be made, is based on a general understanding of synthetic chemistry by one of skill in the art and that embodiments and teachings herein are intended to and encompass all possible structures that comply with the respective laws related to written description and enablement.

[0089] Where there are two or more adjacent substituents, the substituents can be linked to form a carbocyclic or heterocyclic ring. Such adjacent groups can have a vicinal or germinal relationship, or they can be adjacent on a ring in, e.g., an orthoarrangement.

[0090] Each instance of “substituted” is understood to be independent. For example, a substituted aryl can be substituted with bromo and a substituted heterocycle on the same compound can be substituted with alkyl. It is envisaged that a substituted group can be substituted with one or more non-fluoro groups. As another example, a substituted group can be substituted with one or more non-cyano groups. As another example, a substituted group can be substituted with one or more groups other than haloalkyl. As yet another example, a substituted group can be substituted with one or more groups other than tert-butyl. As yet a further example, a substituted group can be substituted with one or more groups other than trifluoromethyl. As yet even further examples, a substituted group can be substituted with one or more groups other than nitro, other than methyl, other than methoxymethyl, other than dialkylaminosulfonyl, other than bromo, other than chloro, other than amido, other than halo, other than benzodioxepinyl, other than polycyclic heterocyclyl, other than polycyclic substituted aryl, other than methoxycarbonyl, other than alkoxycarbonyl, other than thiophenyl, or other than nitrophenyl, or groups meeting a combination of such descriptions. Further, substituted is also understood to include fluoro, cyano, haloalkyl, tert-butyl, trifluoromethyl, nitro, methyl, methoxymethyl, dialkylaminosulfonyl, bromo, chloro, amido, halo, benzodioxepinyl, polycyclic heterocyclyl, polycyclic substituted aryl, methoxycarbonyl, alkoxycarbonyl, thiophenyl, and nitrophenyl groups. In various embodiments, a substituted group may be substituted with a group other than a carbonyl-containing group, nitro, cyano, sulfinyl, sulfonyl, or a halogen-containing group. In various embodiments, a substituted group may be substituted with a group other than an electron-withdrawing group. Some substituted groups in certain embodiments may be substituted solely with one or more electron-donating groups.Attorney Docket No. 772156: DABX-003PCT

[0091] The term “boronate group” as used herein refers to the following structure in which R8and R9are each independently H or C1-C10 alkyl, or R8and R9taken together with the boronate to which they are attached provide a C2-C10 cyclic boronate ester

[0092] As used herein, the term “n-conjugated” group refers to a substituent that has an unhybridized P-orbital that overlaps or aligns with an unhybridized P-orbital in the parent moiety to which the n-conjugated is attached, such that electrons may be shared between the two P-orbitals and a lower energy state is achieved. An example parent moiety is the phenyl group to which X and R5are attached. A substituent that is a n-conjugated group can also have n-bonding electrons that are delocalized through both the substituent and the parent moiety to which it is attached. Examples of n-conjugated groups include substituted or unsubstituted C2-C10 alken-1-yl, C2-C10 alken-2-yl, C2-C10 alkenyn-1-yl, C2-C10 heterocycloalken-1-yl, C2-C10 heterocycloalken-2-yl, C6-C10 aryl, or heteroaryl. Further examples of n-conjugated groups include substituted or unsubstituted vinyl, ethynyl, C6-C10 aryl, or heteroaryl further substituted with a C2-C10 alken-1 -yl, C2-C10 alken-2-yl, C2-C10 alkenyn-1-yl, C2- C10 heterocycloalken-1-yl, C2-C10 heterocycloalken-2-yl, C6-C10 aryl, or heteroaryl. Yet further examples include, for example, substituted or unsubstituted biaryl, biheteroaryl arylvinyl, heteroaryl vinyl, and C2-C10 alken-1-yl aryl, phenyl heteroaryl, and heteroaryl aryl.

[0093] As used herein, the term “electron-donating group” refers to a group that has a net electron donating effect relative to hydrogen. Electron-donating groups are well known in the art. See, for example, Jerry March, Michael B. Smith, March's Advanced Organic Chemistry 6th edition, 2007, Wiley Interscience and J. McMurry, Organic Chemistry, 5th Ed. (Brooks / Cole, Pacific Grove, 2000. Electron-donating groups, sometimes abbreviated EDGs, can be defined according to their Hammett Substituent Constant also known as sigma values (o values). In various embodiments, the electron donating group has a sigma value of 0.3 or lower, 0.2 or lower, 0.1 or lower, or a negative sigma value. In further embodiments, embodiments, the electron donating group is a non-halogen group having a has a sigma value of 0.3 or lower, 0.2 or lower, 0.1 or lower, or a negative sigma value. In cases where the position of the substituent substantially influences its sigma value, the sigma value should be determined relativeAttorney Docket No. 772156: DABX-003PCT to the position of the group X. For example, Ometa values could be provided to determine the sigma value of a substituent at an R5ortho to X and oPara values could be provided to determine the sigma value of a substituent at an R5para to X. Sigma values can be obtained according to published tables or experimentally. See, for example, J.E. Leffler and E. Grunwald, Rates and Equilibria of Organic Reactions, Wiley, 1963 (Dover reprint). Various examples of electron donating groups include oxy anion, hydroxyl, amino, thio, alkylamino, dialkylamino, alkoxy, alkylthio, acylamino, acyloxy, alkyl, alkenyl, vinyl, aryl, electron-rich heteroaryl,

[0094] A further manner of determining whether a particular substituent on a given structure is electron donating is by comparing the pKa of the phenolic group (i.e. , X = OH) of the substituted structure with the pKa of the phenolic of an unsubstituted but otherwise identical structure.

[0095] In various embodiments, R5can be understood to provide a net donating effect if the pKa of a phenolic group at X is 9.0 or greater, 9.5 or greater, 10.0 or greater, 10.5 or greater, or 11.0 or greater. In various embodiments, each non-hydrogen R5present provides a net electron donating effect if the pKa of a phenolic group at X is greater than that of a compound where each R5is H.

[0096] In further embodiments, X is OH or an oxy anion and has a pKa of 9.0 or greater, 9.5 or greater, 10.0 or greater, 10.5 or greater, or 11 .0 or greater.

[0097] An “electron-withdrawing group,” sometimes abbreviated EWG, refers to a group that has a net electron withdrawing effect relative to hydrogen. Electronwithdrawing groups are well known in the art. See, for example, Jerry March, Michael B. Smith, March's Advanced Organic Chemistry 6th edition, 2007, Wiley Interscience and J. McMurry, Organic Chemistry, 5th Ed. (Brooks / Cole, Pacific Grove, 2000). Although it is thought that the presence of an EWG slows the rate of formation of the reactive luminescent intermediate, some embodiments of the present disclosure can contain one or more EWG, provided that the net overall effect of substituents is an electron donating effect. For example, in some embodiments, R5can include one or more electron-withdrawing groups (EWG) provided that said R5has an overall net electron donating effect on the aryl ring to which it is attached. Examples of electron withdrawing groups include acrylate groups, such as alkyl acrylate (e.g., CH3C(O)CH=CH-) and cyanoacrylate (NCCH=CH-) groups.Attorney Docket No. 772156: DABX-003PCT

[0098] As used herein, the term alcohol protecting group refers to a substituent group on an oxy group which renders the oxygen inert to various conditions in which an alcohol would typically react, but which is readily removed when subjected to certain conditions. Alcohol protecting groups as described herein will typically improve the stability of the dioxetane moiety, and upon their removal will promote decomposition of the dioxetane. Thus, alcohol protecting groups include phosphates such as POsNa2, PO3CI2, and PO3H2, glycosyl groups, dinitrobenzenesulfonylaminobenzyl groups, and other groups which can be enzymatically hydrolyzed to provide the unprotected alcohol. Some alcohol protecting groups are described in Theodora W. Greene, Peter G. M. Wuts (1999). Protecting Groups in Organic Synthesis (3 ed.). J. Wiley. Alcohol protecting groups include acetyl, benzoyl, benzyl, methoxyethoxymethyl, dimethyltrityl, methoxylmethyl, methylthiomethyl, pivaloyl, tetrahydropyranyl, tetrahydrofuranyl, trityl, trialkylsilyl, trialkylsiloxymethyl, dialkylarylsilyl, glycosyl, pyranyl, galactosyl, and ethoxyethyl groups. Alcohol protecting groups also include groups in which the alcohol is substituted with a fragmentable linker that is further substituted with a protecting group, wherein upon deprotecting of such protecting group the linker fragments and eliminates from the alcohol. The following compounds are yet further examples of alcohols substituted with an alcohol protecting group:

[0099] In various embodiments, the protecting group G may be an enzyme-cleavable group, wherein removal of said cleavable group by the analyte of interest, e.g., in theAttorney Docket No. 772156: DABX-003PCT presence of an enzyme capable of cleaving said enzyme cleavable group, provides the unstable phenolate-dioxetane species that subsequently decomposes and emits light. For example, G may be a peptide moiety consisting of two or more amino acid residues cleavable by a specific enzyme.

[0100] In some instances, the compounds described herein (e.g., the compounds of the Formulas l-ll can contain chiral centers. All diastereomers of the compounds described herein are contemplated herein, as well as racemates.

[0101] Values expressed in a range format should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range were explicitly recited. For example, a range of “0.1 % to 5%” or “0.1 % to 5%” should be interpreted to include not just 0.1 % to 5%, but also the individual values (e.g., 1 %, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.1 % to 0.5%, 1.1 % to 2.2%, 3.3% to 4.4%) within the indicated range.

[0102] In this document, the terms “a,” “an,” or “the” are used to include one or more than one unless the context clearly dictates otherwise. The term “or” is used to refer to a nonexclusive “or” unless otherwise indicated. In addition, it is to be understood that the phraseology or terminology employed herein, and not otherwise defined, is for the purpose of description only and not of limitation.

[0103] Any use of section headings is intended to aid reading of the document and is not to be interpreted as limiting. Further, information that is relevant to a section heading can occur within or outside of that particular section. Furthermore, all publications, patents, and patent documents referred to in this document are incorporated by reference herein in their entirety, as though individually incorporated by reference. In the event of inconsistent usages between this document and those documents so incorporated by reference, the usage in the incorporated reference should be considered supplementary to that of this document; for irreconcilable inconsistencies, the usage in this document controls.

[0104] In the methods described herein, the steps can be carried out in any order without departing from the principles of the invention, except when a temporal or operational sequence is explicitly recited. Furthermore, specified steps can be carried out concurrently unless explicit claim language recites that they be carried outAttorney Docket No. 772156: DABX-003PCT separately. For example, a claimed step of doing J and a claimed step of doing K can be conducted simultaneously within a single operation, and the resulting process will fall within the literal scope of the claimed process.

[0105] Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” As used herein, in connection with a measured quantity, the term “about” refers to that variation in the measured quantity as would be expected by the skilled artisan making the measurement and exercising a level of care commensurate with the objective of the measurement and the precision of the measuring equipment used. Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0106] Each embodiment described above is envisaged to be applicable in each combination with other embodiments described herein. For example, embodiments corresponding to Formula (I) are equally envisaged as being applicable to Formula (II). As another example, embodiments corresponding to Formula (II) are equally envisaged as being applicable to Formula (I).

[0107] Those skilled in the art will appreciate that many modifications to the embodiments described herein are possible without departing from the spirit and scope of the present disclosure. Thus, the description is not intended and should not be construed to be limited to the examples given but should be granted the full breadth of protection afforded by the appended claims and equivalents thereto. In addition, it is possible to use some of the features of the present disclosure without the corresponding use of other features. Accordingly, the foregoing description of or illustrative embodiments is provided for the purpose of illustrating the principles of the present disclosure and not in limitation thereof and can include modification thereto and permutations thereof.Attorney Docket No. 772156: DABX-003PCTEXAMPLES

[0108] The present disclosure can be better understood by reference to the following examples which are offered by way of illustration. All reagents, starting materials, and solvents used in the following examples were purchased from commercial suppliers (for example, Sigma Aldrich, St. Louis, MO) and were used without further purification unless otherwise indicated.General Methods

[0109] Embodiments of the synthesis described in Synthetic Pathway 1 , along with additional synthetic pathways, are provided in the Example section for representative dioxetane dimer compounds. The example compounds and processes provide one of skill in the art with the ability to make compounds embodied herein without undue experimentation. Various compounds of the present disclosure can be synthesized according to methods including, but not limited to, the synthetic approaches described in PCT International Application WO 1996 / 015122 A1 , U.S. Patent No. 4,962,192, or U.S. Patent No. 5,004,565. Further, in some examples, one or more of the individual steps may be known in the art (See, for example, Jerry March, Michael B. Smith, March's Advanced Organic Chemistry 6th edition, 2007, Wiley Interscience and J. McMurry, Organic Chemistry, 5th Ed. (Brooks / Cole, Pacific Grove, 2000)) but the embodied processes as a whole unexpectedly allow synthesis of the novel dioxetane dimer compounds.

[0110] Chemiluminescence (emission) intensity measurements are done using a Turner Designs (Sunnyvale, CA) model TD-20e luminometer. Spectral data for chemiluminescent emission was obtained on a spectrofluorometer (FP-8650, Jasco, Easton, MD). Kinetic data for chemiluminescence decay recorder on Omega Lumistar plate reader (BMG Labtech, Cary, NC) in 5 mM solutions in methanol (100 pL), triggered by 1 mM NaOH solution (10 pL). Other devices known in the art may also be used for detection purposes, including, for example, a BMG Labtech luminescence plate reader, or a charge-coupled device (CCD) camera luminometer, or any other suitable light intensity measuring devices. Nuclear magnetic resonance (NMR) spectra were obtained using a 500 MHz spectrometer (Bruker BioSpin, Billerica, MA.). Mass spectroscopy data was obtained on SCIEX TQ6500+ mass spectrometer (AB SCIEX, Framingham, MA).

[0111] EXAMPLE 1 :Attorney Docket No. 772156: DABX-003PCTScheme 1

[0112] 140 mL 1 ,4-dioxane was added to a 250 mL round bottom flask, followed by 15.88 g (40 mmol) of iodo-alkene (IPA). 6.7 g (40 mmol) of potassium trifluorovinylborate was subsequently added along with a solution of 13.9 g of potassium carbonate in 70 mL water. After bubbling argon through the mixture for 30 mins, dichloro-1 ,1 ’bis(diphenylphosphino)ferrocene palladium (II) dichloromethane [Pd(dppf)2Cl2 ] (350 mg, 0.4 mmol) was added. The mixture was refluxed under an argon atmosphere for 5 hours. After allowing to cool to room temperature, the resulting products were extracted with ethyl acetate (50 mL x 4), washed with brine (50 mL x 3) and dried over Na2SO4. The final product mixture was purified by column chromatography to obtain product (1 ) in an amount of 10.61 g (90%) and product (2) in an amount of 450 mg (yield 4.5%).

[0113] Nuclear magnetic resonance (NMR) of product (1 ): (CDCh). 7.39 (d, J = 6Hz, 1 H), 7.02-6.87 (m, 1 H), 6.84 (d, J = 5Hz, 1 H), 6.83 (s, 1 H), 5.79 (dd, J1 = 24Hz, J2 =2Hz, 1 H), 5.35 (dd, J1 =24hz, J2 =2Hz, 1 H), 3.33 (s, 3H), 3.26(s, 1 H), 2.71 (s, 1 H), 2.0-1.80 (m, 12H).

[0114] NMR of product (2): (d6-DMSO). 5(ppm), 9.72 (s, 2H), 7.51 (d, J = 12Hz, 2H), 7.39 (s, 2H), 6.80 (s, 2H), 6.73 (d, J = 6Hz, 2H), 3.33 (s, 6H), 3.17 (s, 2H), 2.67 (s, 2H), 2.0-1.80 (m, 24H).

[0115] Mass spectroscopy (MS) of product (2): 564 [M+1]+

[0116] EXAMPLE 2Scheme 2Attorney Docket No. 772156: DABX-003PCT

[0117] Compound (1 ) (280 mg, 1 mmol) was combined with IPA (400 mg, 1 mmol) in 50 mL of CHsCN and bubbled under argon for 15 minutes. Pd(OAc)2 (17 mg) and tri- o-tolyphosphine (15 mg) were added to the solution, followed by 350 pL of triethylamine. The mixture was refluxed and stirred under argon overnight. After allowing to cool to room temperature, the CHsCN was removed under vacuum, and the residue was resuspended in 150 mL of ethyl acetate, washed with brine (50 mL x 3), and dried over Na2SO4. The resulting product was concentrated under vacuum and purified by column chromatography using 10% ethyl acetate in hexane to obtain 231 mg of compound (2).

[0118] NMR: (d6-DMSO). 5(ppm), 9.72 (s, 2H), 7.51 (D, J = 12Hz, 2H), 7.39 (s, 2H), 6.80 (s, 2H), 6.73 (d, J = 6Hz, 2H), 3.33 (s, 6H), 3.17 (s, 2H), 2.67 (s, 2H), 2.0-1.80 (m, 24H).

[0119] MS: 564 [M+1]+

[0120] EXAMPLE 3

[0121] Compound (2) was combined with 5 mL of methanol and 5 mL of dichloromethane, and 250 mg of rose bengal polymer beads were added. The mixture was bubbled with oxygen and run under a 1000 W mercury lamp in an ice bath for 1 hour. The beads were removed via filtration, and the filtrate was concentrated. The residue was purified by preparative TLC using 30% ethyl acetate in hexane to obtain 18 mg of compound (3).

[0122] NMR: (CDC ). 7.70 (s,4H), 7.55-6.80 (br, 4H), 5.86 (br, 2H), 3.31 (s, 6H), 3.08 (s, 2H), 2.31 (s, 2H), 2.0-1.45 (m, 20H), 1.43-1.40 (m, 2H), 1.20-1.08 (m, 2H).

[0123] Light emission wavelength maximum (Em(max))= 575 nm.

[0124] MS:629 [M+1]+

[0125] EXAMPLE 4Attorney Docket No. 772156: DABX-003PCT

[0126] 30 mg (0.2 mmol) of TBDMSCI and 35 mg (0.5 mmol) of imidazole were added to a solution of (2) (56 mg, 0.1 mmol) in 5 mL of DMF. After stirring at room temperature overnight, the mixture was diluted with 100 mL of ethyl acetate, washed with brine (50 mL x 3), dried over Na2SO4 and concentrated in vacuo. The crude mixture was diluted in 2 mL of ethyl acetate, and hexane was added to crystalline out the product. Filtration was done to collect 70 mg of compound (4) (yield 89%). The resulting product purity was sufficient for the subsequent step.

[0127] NMR: (CDCb). 5(ppm), 7.62 (d, J = 12Hz, 2H), 7.47 (s, 2H), 6.97 (d, J-12Hz, 2H), 6.82 (s, 2H), 3.35 (s, 6H), 3.28 (s, 2H), 2.77 (s, 2H), 2.0-1 .80 (m, 24H), 0.89 (s, 18H), 0.12 (s, 12H).

[0128] EXAMPLE 5

[0129] 56 mg of compound (4) in 10 mL of methanol and 10 mL of dichloromethane was combined with 250 mg of rose bengal polymer beads. The mixture was bubbled with oxygen and run under a 1000 W mercury lamp in an ice bath for 1 hour. The beads were removed via filtration, and the filtrate was concentrated. The crude mixture was diluted in 2 mL of ethyl acetate and hexane was added to crystallize out the product. Filtration was done to collect 36 mg of product (5).Attorney Docket No. 772156: DABX-003PCT

[0130] NMR: (CDC ). 5(ppm), 7.70 (s,2H), 7.51 (s, 2H), 7.55-6.80 (br, 5H), 5.86 (br, 2H), 3.29 (s, 6H), 3.05 (s, 2H), 2.30 (s, 2H), 2.0-1.92 (m, 2H), 1.77-1.45 (m, 18H), 1.32-1.28 (m, 2H), 1.20-1.08 (m 2H). 1.10 (s, 18H), 0.26 (s, 6H).

[0131] EXAMPLE 6Scheme 6

[0132] 30 mL 1 ,4-dioxane was combined with 400 mg (1 mmol) of IPA in a 100 mL round bottom flask. The 430 mg (3 mmol) of potassium carbonate in 15 mL water solution was added. Argon was bubbled through the mixture for 30 mins, then 82 mg (0.5 mmol) of benzene-1 ,4-diboronic acid was added, followed by the addition of dichloro-1 ,1 ’bis(diphenylphosphino)ferrocene palladium (II) dichloromethane (24 mg, 0.026 mmol). The mixture was refluxed under argon for 5 hours and subsequently cooled to room temperature. The resulting product was extracted with ethyl acetate (50 mL x 4), washed with brine (50 mL x 3), dried over Na2SO4, and then purified by column to obtain 180 mg of product (6).

[0133] NMR: (d6-DMSO). 5(ppm), 9.62 (s, 2H), 7.62 (s, 4H), 7.32 (d, J = 6Hz, 2H), 6.92 (s, 2H), 6.81 (dd, J1 = 6Hz, J2 = 2 Hz, 2H), 2H), 3.27 (s, 6h), 3.20 (s, 2H), 2.72 (s, 2H), 1.94-1.76 (m, 24H).

[0134] EXAMPLE 7Attorney Docket No. 772156: DABX-003PCT

[0135] Compound (6) (33 mg) was combined with 10 mL of methanol and 10 mL of dichloromethane, and 300 mg of rose bengal polymer beads were added to the mixture. The mixture was bubbled under oxygen and run under a 1000 W mercury lamp in an ice bath for 1 hour. The beads were removed via filtration, and the filtrate concentrated. The residue was purified by preparative TLC using 30% ethyl acetate in hexane to obtain 22 mg of compound (7).

[0136] NMR: (CDC ). 5(ppm), 7.70 (s,4H), 7.55-6.80 (br, 2H), 7.40 (d, J=8Hz, 4H), 5.50 (br, 2H), 3.31 (s, 6H), 3.09 (s, 2H), 2.31 (s, 2H), 1.95-1.45 (m, 18H), 1.43-1.40 9m, 2H), 1.20-1.08 (m, 2H).

[0137] Light emission wavelength maximum (Em(max)) = 480 nm

[0138] EXAMPLE 8

[0139] 30 mL of 1 ,4-dioxane was added to a 100 mL round bottom flask, followed by addition of 500 mg (1 mmol) of iodo-alkene (IPA) and 430 mg (3 mmol) of potassium carbonate in 15 mL water. Argon was bubbled through the mixture for 30 mins, then 80 mg (0.57 mmol) of 4-vinyl benzene-boronic acid was added, followed by the addition of dichloro-1 ,1 ’bis(diphenylphosphino)ferrocene palladium (II)Attorney Docket No. 772156: DABX-003PCT dichloromethane [Pd(dppf)2Cl2.CH2Cl2 54 mg, 0.066 mmol]. The mixture was stirred and refluxed under argon for 5 hours. After refluxing, the mixture was cooled to room temperature, and the product was extracted with ethyl acetate (50 mL x 4), washed with brine (50 mL x 3), and dried over Na2SO4. The mixture was then purified by a silica gel column (20% ethyl acetate on hexane) to obtain 150 mg of the desired product (8).

[0140] NMR: (d6-DMSO). 5(ppm), 9.82 (s, 1 H), 9.63 (s, 1 H), 7.62-7.57 (m, 4H), 7.44 (d, J = 20Hz, 1 H), 7.32-7.25 (m, 2H), 6.90 (s, 1 H), 6.83-6.80 (m, 2H), 6.75 (d, J = 10Hz, 1 H), 3.27 (s, 3H), 3.25 (s, 3H), 3.19 (s, 2H), 2.71 (s, 1 H), 2.68 (s, 1 H), 1.99- 1.75 (m, 24H).

[0141] MS: 641 [M+1]+.

[0142] EXAMPLE 9

[0143] 56 mg of compound (8) was added to a solution of 15 mL of methanol and 15 mL of dichloromethane. 300 mg of rose bengal polymer beads were then added to the mixture. Oxygen was bubbled in while a photoreaction was performed under a 1000 W mercury lamp in an ice bath for 1 hour. The rose bengal polymer beads were removed via filtration, and the filtrate concentrated. The residue was purified by preparative TLC using 25% ethyl acetate in hexane to obtain 32 mg of compound (9), which was identified by NMR.

[0144] NMR: (CDCb). 5(ppm), 7.70 (s, J = 10Hz, 2H), 7.65 (d, J = 5Hz, 1 H), 7.56 (d, J = 5Hz, 2H), 7.49 (d, J = 10Hz, 1 H), 7.36 (d, J = 5Hz, 1 H), 7.40-6.80 (br, 5H), 5.42- 5.37 (br, 2H), 3.31 (s, 3H), 3.28 (s, 3H), 3.07 (s, 2H), 2.31 (s, 1 H), 2.26 (s, 1 H), 1.95- 1.45 (m, 20H), 1.43-1.40 (m, 2H), 1.20-1.08 (m, 2H).

[0145] Light emission wavelength maximum (Em(max)) = 515 nm.

[0146] EXAMPLE 10Attorney Docket No. 772156: DABX-003PCT

[0147] 30 mL of 1 ,4-dioxane was added to a 100 mL round bottom flask, followed by the addition of 800 mg (2 mmol) of iodo-alkene (IPA) and 830 mg (6 mmol) of potassium carbonate in 15 mL water. Argon was bubbled through the mixture for 30 mins, then 175 mg (1.02 mmol) of 2,5-thiophenediyl-bis-boronic acid was added, followed by the addition of dichloro-1 ,1 ’bis(diphenylphosphino)ferrocene palladium (II) dichloromethane [Pd(dppf)2Cl2.CH2Cl2 34 mg, 0.042 mmol]. The mixture was stirred and refluxed under argon for 4 hours. After refluxing, the mixture was cooled to room temperature, and the product extracted with ethyl acetate (50 mL x 3), washed with brine (50 mL x 3), and dried over Na2SO4. The mixture was then purified by a silica gel column (10% ethyl acetate on hexane to 20% ethyl acetate on hexane) to obtain 250 mg of the desired product (10).

[0148] NMR: (CDsOD). 5(ppm), 7.63 (d, J = 10Hz, 2H), 7.61 (s, 2H), 6.90 (s, 2H), 6.83 (s, 2H), 6.83 (d, J = 10Hz, 2H), 3.33 (s, 6H), 3.25 (s, 2H), 2.76 (s, 2H), 1.99-1.75 (m, 24H).

[0149] MS: 621 [M+1]+

[0150] EXAMPLE 11Scheme 11

[0151] 100 mg of compound (10) was added to a solution of 15 mL of methanol and15 mL of dichloromethane. 300 mg of rose bengal polymer beads were then added toAttorney Docket No. 772156: DABX-003PCT the mixture. Oxygen was bubbled in while a photoreaction was performed under a 1000 W mercury lamp in an ice bath for 1 hour. The rose bengal polymer beads were removed via filtration, and the filtrate concentrated. The residue was purified by preparative TLC using 30% ethyl acetate in hexane to obtain 76 mg of compound (11 ), which was identified by NMR.

[0152] NMR: (CDC ). 7.63 (d, J = 10Hz, 2H), 7.53 (s, 2H), 7.53-6.90 (br, 4H), 5.96 (br, 2H), 3.29 (s, 6H), 3.07 (s, 2H), 2.28 (s, 2H), 2.0-1.60 (m, 18H), 1.53 (d, J = 10Hz, 2H), 1.34 (d, J = 10Hz, 2H), 1.16 (d, J = 10Hz, 2H).

[0153] Light emission wavelength maximum (Em(max)) = 520 nm.

[0154] EXAMPLE 12 - A dioxetane compound having the following structure was synthesized using the methods disclosed in the Examples above:

[0155] A graph showing the intensity of light emission over time is provided in FIG. 1 and shows a chemiluminescence half-life of 0.55 seconds.

[0156] EXAMPLE 13 - Additional example embodiments considered herein are shown below.

[0157] Emission studies are done on the example embodiments shown in Example 12 and Example 13, and unexpectedly show that having a second dioxetane moietyAttorney Docket No. 772156: DABX-003PCT that is n-conjugated to the first dioxetane moiety results in a greatly decreased T1 / 2 along with a significantly higher peak intensity of luminescence upon dioxetane fragmentation.

Claims

Attorney Docket No. 772156: DABX-003PCTCLAIMSWhat is claimed is:

1. A compound of Formula Iwherein each of R1and R2is independently an optionally substituted C3-C10 alkyl, or R1and R2taken together with the carbon to which they are attached provide a optionally substituted C5-C10 cycloalkyl ring;R3is C1-C10 alkyl, C6-C10 aryl, or heteroaryl, any of which may be optionally substituted; each phenyl ring A and B has three R5groups, each R5located at a ring position of 2-6, wherein each of R5is independently H, Q, X, hydroxy, halogen, amino, thio, or C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, C1-C10 alkyloxy, C1- C10 alkylamino, C1-C10 trialkylammonium salt, C1-C10 alkylthio, C2-C10 acyl, C1-C10 alkyloxycarbonyl, C1-C10 alkylaminocarbonyl, C1-C10 alkylthiocarbonyl, C2-C10 acyloxy, C2-C10 acylamino, C2-C10 acylthio, C1-C10 alkylcarbonate, C1-C10 alkylcarbamate, C1-C10 carbamido, aryloxy, C1-C10 alkylsulfinyl, C1-C10 alkylsulfonyl, arylthio, arylamino, arylsulfinyl, arylsulfonyl, arylcarbonyl, heteroarylcarbonyl, heteroaryloxy, heteroarylthio, heteroarylamino, heteroarylsulfinyl, heteroarylsulfonyl, cyano, nitro, trifluoromethyl, phosphonate, C1-C10 alkylphosphonate, C1-C10 alkylphosphinate, C1-C10 trialkylphosphonium salt, C4-C10 heterocycloamino, C6-C10 aryl, or n-excessive heteroaryl, any of which is may be optionally substituted, and wherein at least one R5on one of phenyl rings A or B is Q;Q is a n-conjugated electron-donating group;Attorney Docket No. 772156: DABX-003PCTX is -OH, -O-G, an -O’ salt, or a boronate group;G is an alcohol protecting group, an analyte-responsive protecting group or a divalent fragmentable linker having a pendant protecting group; and a conjugated linker comprising a chemical moiety that is capable of forming a conjugated system with phenyl rings A and B, wherein the conjugated linker is bound at a ring position of 2, 3, 4, 5, or 6 on phenyl ring A and independently at a ring position 2’, 3’, 4’, 5’, or 6’ on phenyl ring B.

2. The compound of claim 1 , wherein each R5is independently H, Q, X, halogen, or Ci-Cio alkyl, hydroxy, C1-C10 alkyloxy, amino, C1-C10 alkylamino, thio, C1-C10 alkylthio, C2-C10 acyloxy, C2-C10 acylamino, C2-C10 acylthio, C1-C10 alkylcarbonate, C1-C10 alkylcarbamate, C1-C10 carbamido, aryloxy, arylthio, arylamino, heteroaryloxy, heteroarylthio, or heteroarylamino.

3. The compound of claim 1 , wherein at least one R5provides a net electron donating effect on the aromatic ring to which said R5are attached.

4. The compound of any of claims 1-3, wherein Q is C2-C10 alkenyl, C2-C10 heterocycloalkenyl, C6-C10 aryl, or heteroaryl, any of which is optionally substituted.

5. The compound of any of claims 1-4, wherein R1and R2taken together with the carbon to which they are attached provide a spirocyclic bridged bicyclo or tricyclo group.

6. The compound of claim 5, wherein R1and R2taken together with the carbon to which they are attached provide a spirocyclic adamantane, norbornane, or bornane.

7. The compound of any of claims 1-6, wherein X is -OH.

8. The compound of any of claims 1-6, wherein X is -O-G, or a boron group having the structureAttorney Docket No. 772156: DABX-003PCT wherein R8and R9are each independently H or an optionally substituted C1-C10 alkyl, or R8and R9taken together with the boronate to which they are attached are an optionally substituted C2-C10 cyclic boronate ester.

9. The compound of any of claims 1-8, wherein at least one R5on each of phenyl rings A or B is Q.

10. The compound of claim 8, wherein X is -O-G.

11. The compound of any of claims 1-10, wherein X is a group that generates an oxy anion upon chemical or enzymatic trigger.

12. The compound of any of claims 1-11 , wherein G is an analyte-responsive protecting group.

13. The compound of claim 12, wherein G is trialkylsilyl, alkylarylsilyl, arylsulfonyl, dioxobenzyl, trityl, alkylcarbonate, phosphoryl, dihydropyranyl, tetrahydropyranyl, dihydrofuranyl, tetrahydrofuranyl, pyranosyl, pyranuronyl, furanosyl, acyl, benzoyl, or benzyl.

14. The compound of claim 13, wherein G is pyranosyl or pyranuronyl.

15. The compound of claim 14, wherein G is galactosyl, glucosyl, or glucuronyl.

16. The compound of claim 15, wherein G is p-galactosyl, p-glucosyl, or p- glucuronyl.

17. The compound of claim 16, wherein G is -PO3H2 or a salt or ester thereof.

18. The compound of claim 15, wherein G is 2,4-dinitrobenzenesulfonyl, 3,4,6- trimehyl-2,5-dioxobenzyl, 4-azidobenzyloxy, tert-butyldimethylsilyl, acetyl, pivaloyl, or a peptidase-cleavable moiety.

19. The compound of any of claims 1-12, wherein G comprises a divalent fragmentable linker and a terminal trialkylsilyl, alkylarylsilyl, arylbenzenesulfonyl,Attorney Docket No. 772156: DABX-003PCT dioxobenzyl, trityl, alkylcarbonate, phosphoryl, dihydropyranyl, tetrahydropyranyl, dihydrofuranyl, tetrahydrofuranyl, pyranosyl, pyranuronyl, furanosyl, acyl, benzoyl, benzyl or boronate group.

20. The compound of claim 19, wherein the divalent fragmentable linker is 4- aminobenzyl, 4-(alkylamino)benzyl, 4-oxybenzyl, 4-(oxymethyl)benzyl, oxymethyl, aminomethyl, or alkylaminomethyl.

21. The compound of any of claims 1-20, wherein X has the structure:

22. The compound of any of claim 1-21 , wherein R3is unsubstituted C1-C10 alkyl or C1-C10 alkyl substituted with one or more halogen, hydroxy, amino, thio, alkoxy, alkylamino, alkylthio, sulfate, or carboxylate.

23. The compound of any of the preceding claims, wherein the conjugated linker is:Attorney Docket No. 772156: DABX-003PCTor combinations thereof, wherein each of R10, R11, and R12is independently hydrogen, hydroxy, halo, or C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, any of which is optionally substituted; each R13is independently N, S, O, Se, or Si(R14)2, wherein each R14is hydrogen or an optionally substituted alkyl; each Y is independently a hydrogen or halo; and n is from 1 to 200.

24. The compound of claim 1 having Formula II:Attorney Docket No. 772156: DABX-003PCTwherein each phenyl ring A and B has three R5groups, each R5located at a ring position of 2-6, wherein each of R5is independently H, Q, X, hydroxy, halogen, amino, thio, or C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, C1-C10 alkyloxy, C1-C10 alkylamino, C1-C10 trialkylammonium salt, C1-C10 alkylthio, C2- C10 acyl, C1-C10 alkyloxycarbonyl, C1-C10 alkylaminocarbonyl, C1-C10 alkylthiocarbonyl, C2-C10 acyloxy, C2-C10 acylamino, C2-C10 acylthio, C1-C10 alkylcarbonate, C1-C10 alkylcarbamate, C1-C10 carbamido, aryloxy, C1-C10 alkylsulfinyl, C1-C10 alkylsulfonyl, arylthio, arylamino, arylsulfinyl, arylsulfonyl, arylcarbonyl, heteroarylcarbonyl, heteroaryloxy, heteroarylthio, heteroarylamino, heteroarylsulfinyl, heteroarylsulfonyl, cyano, nitro, trifluoromethyl, phosphonate, C1-C10 alkylphosphonate, C1-C10 alkylphosphinate, C1-C10 trialkylphosphonium salt, C4-C10 heterocycloamino, C6-C10 aryl, or n-excessive heteroaryl, any of which is optionally substituted and wherein at least one R5is Q;Q is a n-conjugated electron-donating group;X is -OH, -O-G, an -O’ salt, or a boronate group;G is an alcohol protecting group; the conjugated linker is bound to phenyl ring A at the 4-position and at phenyl ring B at the 4’-position; and each R14and R15is independently H, halogen, or C1-C10 alkyl, C2-C10 alkenyl, C6-C10 aryl, any of which is optionally substituted.

25. A compound consisting of one of:Attorney Docket No. 772156: DABX-003PCT26. A composition comprising a compound of any one of claims 1-25.

27. The composition of claim 26, further comprising an amine buffer.

28. The composition of claim 27 in the form of an aqueous composition.

29. The composition of claim 28 having a peak luminescent intensity of greater than 1000 photons / sec and a T1 / 2 of 30 seconds or less upon treatment with pH 9.7 buffer at 37°C, and wherein the composition is substantially free of surfactants.Attorney Docket No. 772156: DABX-003PCT30. A method of detecting an analyte in a sample, comprising contacting the sample with a compound of any one of claims 1-25 or the composition of any of claims 26-29 and monitoring the sample for luminescence.

31. The method of claim 30, further comprising measuring the intensity of a resulting luminescence and correlating the intensity to the presence of the analyte.

32. The method of claim 30, wherein correlating the intensity to the presence of the analyte comprises determining the amount of an antigen of interest.

33. The method of claim 31 or 32, wherein the contacted sample is monitored for less than 1 minute.

34. The method of any of claims 30-33, wherein the analyte is alkaline phosphatase, an oxidizing agent, hydrogen peroxide, glutathione, fluoride, or a base.

35. The method of any of claims 30-34, wherein the sample is an aqueous sample having a pH of 4 to 12.

36. The method of claim 35, wherein the sample is an aqueous sample having a pH of 8 to 12.

37. A kit for determining the presence of an analyte, the kit comprising the compound of any one of claims 1-25 or the composition of any one of claims 26-29.

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