Method of making 1,2-dioxetanes

Abstract

The present invention relates to a method of making a 1,2-dioxetane by oxidizing an enol ether with a peroxomolybdate or peroxotungstate to give the 1,2-dioxetane. The method is particularly useful for making chemiluminescent 1,2-dioxetanes probes for use in a broad range of research and diagnostic applications, including the detection of pathogens and enzymes and chemiluminescent in vitro and in vivo imaging.

Description

December 12, 2025120592P1126PC- 1 -METHOD OF MAKING 1,2-DIOXETANESFIELD OF THE INVENTION

[0001] The present invention relates to a method of making a 1 ,2-dioxetane by oxidizing an enol ether with a peroxomolybdate or peroxotungstate to give the 1 ,2- dioxetane. The method is particularly useful for making chemiluminescent 1 ,2- dioxetanes probes for use in a broad range of research and diagnostic applications, including the detection of pathogens and enzymes and chemiluminescent in vitro and in vivo imaging.BACKGROUND OF THE INVENTION

[0002] 1 ,2-Dioxetanes have been widely explored in the past due to their chemiluminescent properties. The decomposition of the 1 ,2-dioxetane moiety, a ring of two adjacent oxygen atoms and two adjacent carbon atoms, gives an excited-state carbonyl compound that generates chemiluminescence emission upon radiative deactivation of the excited-state carbonyl compound. Chemiluminescent 1 ,2-dioxetane compounds are employed for in vivo and in vitro detection and imaging of various analytes and enzymes and find use in a wide variety of applications, such as in research and diagnostic applications, chemiluminescent microscopy and chemiluminescent in vivo imaging. 1 ,2-Dioxetane-based chemiluminescence probes are described, e.g., in WO 2017 / 130191 .

[0003] Commercially available chemiluminescence probes are, e.g., known under the trademark AquaSpark™ and used for various applications, such as singlet oxygen detection, enzyme detection and pathogen detection (e.g., of Salmonella spp. and Listeria spp.). AquaSpark™ chemiluminescent probes are based on compounds comprising a 1 ,2-dioxetane moiety derived from an adamantyl enol ether and a phenolate moiety comprising a protecting group and an electron withdrawing group. In the AquaSpark™ probes, the emission of chemiluminescence is initiated because of enzymatic cleavage of the protecting group (PG) from the phenolate moiety connectedDecember 12, 2025120592P1126PC- 2 - to the adamantane-based 1 ,2-dioxetane moiety, followed by the release of adamantanone and the subsequent formation of a high energy phenolate (characterized by *), as shown in the following scheme (EWG is an electron withdrawing group):

[0004] The high-energy phenolate readily undergoes decomposition to give an excited-state carbonyl compound as product. Chemiluminescence emission is generated during radiative deactivation of this excited-state carbonyl compound. The process of electron transfer from the phenolate group to the 0-0 IT* orbital of the 1 ,2- dioxetane is called a chemically initiated electron exchange luminescence process (CIEEL). Hence, if there is no bacteria or enzyme present, which can cleave the PG- phenolate bond, no light emission can be observed.

[0005] Over the past years, several processes for the synthesis of 1 ,2-dioxetanes have been developed. In one approach, 1 ,2-dioxetanes are produced by photooxygenation involving reacting a corresponding enol ether moiety with oxygen in a photoreactor as schematically shown below:

[0006] The photoreactor requires a photosensitizer, which transfers oxygen in the ground state (3O2) into its excited state (1O2) in the presence of light. The excited state of molecular oxygen is the reactive species that reacts with the enol ether via a [2+2] cycloaddition to generate the 1 ,2-dioxetane moiety.December 12, 2025120592P1126PC

[0007] In addition, various synthetic routes for the preparation of 1 ,2-dioxetanes from enol ethers involving electron-transfer oxygenation and other mechanisms have been reported. Among others, CN 112851709 A discloses the synthesis of AMPPD (3-(2'- spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy)phenyl-1 ,2-dioxetane, which is suitable for use as a chemiluminescent substrate for alkaline phosphatase (ALP). This synthesis process comprises reacting a suitable enol ether with molybdate in the presence of hydrogen peroxide in an organic solvent overnight to form the product. The organic solvent used is a solvent in which the intermediate and the product are soluble or easily soluble, but the molybdate is only slightly soluble. The process requires dissolving the enol ether in the solvent and then adding the molybdate and aqueous hydrogen peroxide. According to the example, per one mole equivalent of the enol ether, two mole equivalents molybdate and 1.49 mole equivalents H2O2 (in the form of 30 % aqueous H2O2) are used.

[0008] However, peroxomolybdates formed by the reaction of a molybdate with hydrogen peroxide is also known as a reagent for oxidizing olefins to epoxides. This reaction and other reactions are typically competing reactions in the oxidation of enol ethers to dioxetanes. Specifically, the known use of peroxomolybdates in an organic solvent, in which the intermediate and the product are soluble or easily soluble, but the molybdate is only slightly soluble, results in the following products:

[0009] As can be seen, the reaction not only leads to the desired 1 ,2-dioxetane but also results in epoxide formation, ketone formation and in decomposition reactions of the starting enol ether, resulting in various undesirable side-products.December 12, 2025120592P1126PC- 4 -

[0010] Despite the efforts made in the art, the known synthesis methods for 1 ,2- dioxetanes are not entirely satisfactory and suffer from one or more drawbacks, including low yield and / or purity, complex synthesis steps, tedious synthesis procedure, and the use of expensive equipment and / or chemicals.

[0011] Thus, there is still room for improvement in the synthesis of 1 ,2-dioxetanes, such as in the synthesis of 1 ,2-dioxetane-based chemiluminescence probes.OBJECT OF THE INVENTION

[0012] The object of the present invent is therefore the provision of a method of making a 1 ,2-dioxetane, which is simple and convenient to use and provides 1 ,2- dioxetanes in high yield and purity.SUMMARY OF THE INVENTION

[0013] This object is solved by a method involving the use of a peroxomolybdate or peroxotungstate for generating singlet oxygen (1O2) that reacts with the double bond of an enol ether to form a 1 ,2-dioxetane. The peroxomolybdate or peroxotungstate used in the reaction can be obtained by peroxidation of a suitable tungstate or molybdate by singlet oxygen, wherein the singlet oxygen is generated by catalytic disproportionation of hydrogen peroxide, preferably hydrogen peroxide in water, and singlet oxygen catalyzed by the molybdate or tungstate.

[0014] The method of the present invention avoids the use of a photoreactor and can be run at much higher concentrations (at least 50 mg / ml) than those attainable with a photoreactor (approx. 5 mg / mL only). Further, it avoids or at least minimizes the formation of undesirable by-products including epoxides, ketone and degradation products.

[0015] In addition, the method of the present invention can be performed under mild reaction conditions (e.g. at ambient temperature) and under aerobic conditions, using basic laboratory glassware. Also, it is characterized by relatively short reaction times, where singlet oxygen will be reversibly trapped by a molybdate or tungstate compoundDecember 12, 2025120592P1126PC- 5 - to generate oxo molybdate or oxo tungstate, acting as a secondary source of1O2 used for the peroxidation of the double bond of an enol ether. Moreover, the reactions of the method of the present invention can be easily monitored by1H-NMR analysis and analytical HPLC.

[0016] The conversion of an enol ether to a 1 ,2-dioxetane in accordance with the present invention is exemplary shown in the following scheme, showing the peroxidation of enol ether 1 to produce chemiluminescent probe 2:

[0017] In a first aspect, the invention relates to a method of making a 1 ,2-dioxetane comprising the reaction of a peroxomolybdate or peroxotungstate with the double bond of an enol ether to form the 1 ,2-dioxetane, the reaction comprising steps (A) and (B):(A) reacting a molybdate or tungstate with hydrogen peroxide or aqueous hydrogen peroxide to form the peroxomolybdate or peroxotungstate;(B) reacting an enol ether with the peroxomolybdate or peroxotungstate formed in step (A) to form the 1 ,2-dioxetane; wherein the reaction in step (B) is performed in a reaction solvent comprising a water miscible protic solvent, preferably a C1-5 alcohol and water, preferably wherein the reaction is carried out in homogeneous liquid phase.

[0018] Preferably, the molybdate or tungstate is an ammonia or alkali metal salt. Accordingly, in a preferred embodiment, the formed peroxomolybdate or peroxotungstate is the ammonia or alkali metal salt of the peroxomolybdate or peroxotungstate.

[0019] In a preferred embodiment, the invention relates to a method of making a chemiluminescent 1 ,2-dioxetane of formulaDecember 12, 2025120592P1126PC- 6 -wherein PG is a protecting group, R1is an electron withdrawing group, R2is H or a halogen, preferably Cl, R3is C1-18 alkyl or C1-7 cycloalkyl.

[0020] In a second aspect, the invention relates to a composition comprising an ammonium or alkali metal salt of a peroxomolybdate or peroxotungstate, an enol ether, a C1-5 alcohol, water, a base selected from a hydrogen carbonate or dihydrogen phosphate, mono hydrogen phosphate or phosphate such that the pH is adjusted to a range of from 6 to 9, preferably wherein the ingredients are in homogeneous liquid phase.DETAILED DESCRIPTION OF THE INVENTION

[0021] The invention relates to a method of making a 1 ,2-dioxetane, wherein the method comprises the reaction of a peroxomolybdate or peroxotungstate with the double bond of an enol ether to form the 1 ,2-dioxetane, comprising steps (A) and (B)(A) reacting a molybdate or tungstate with hydrogen peroxide or aqueous hydrogen peroxide to form the ammonium or alkali metal salt of a peroxomolybdate or peroxotungstate;(B) reacting an enol ether with the peroxomolybdate or peroxotungstate formed in step (A); wherein the reaction in step (B) is performed in a reaction solvent comprising water miscible protic solvent, preferably a C1-5 alcohol and water, preferably wherein the reaction is carried out in homogeneous liquid phase.

[0022] Preferably, the peroxomolybdate or peroxotungstate is an ammonium or alkali metal salt of a peroxomolybdate or peroxotungstate.December 12, 2025120592P1126PC- 7 -

[0023] Accordingly, in one embodiment, the invention relates to a method of making a 1 ,2-dioxetane, wherein the method comprises the reaction of an ammonium or alkali metal salt of a peroxomolybdate or peroxotungstate with the double bond of an enol ether to form the 1 ,2-dioxetane, comprising steps (A) and (B)(A) reacting an ammonium or alkali metal salt of a molybdate or tungstate with hydrogen peroxide or aqueous hydrogen peroxide to form the ammonium or alkali metal salt of a peroxomolybdate or peroxotungstate;(B) reacting an enol ether with the ammonium or alkali metal salt of the peroxomolybdate or peroxotungstate formed in step (A); wherein the reaction in step (B) is performed in a reaction solvent comprising water miscible protic solvent, preferably a C1-5 alcohol and water, preferably wherein the reaction is carried out in homogeneous liquid phase.

[0024] As used herein, the term “dioxetane” means a 1 ,2-dioxetane. The terms “dioxetane”, “1 ,2-dioxetane” and “chemiluminescent 1 ,2-dioxetane” or “chemiluminescent dioxetane” are used synonymously in this disclosure.

[0025] The term “enol ether” as used herein means an ether of an enol having the general structure RaRbC=CRc-O-Rd.

[0026] While Ra, Rb, and Rcare not particularly limited, Rdis selected from C1-18 alkyl or C1-7 cycloalkyl.

[0027] In one embodiment, Ra, Rb, and Rcare independently selected from H, Ce-14 aryl, C1-18 alkyl and C1-7 cycloalkyl, optionally substituted, respectively. Suitable substituents are, e.g., halogen, cyano, amido, C1-4 alkyl, C1-4 alkoxy, and phenyl.

[0028] In one embodiment, Raand Rbtaken together, may also form a monocyclic, bicyclic or a tricyclic hydrocarbon moiety or another polycyclic moiety.

[0029] In one embodiment, Raand Rctaken together, may also form a monocyclic, bicyclic or a tricyclic hydrocarbon moiety or another polycyclic moiety.December 12, 2025120592P1126PC- 8 -

[0030] In one embodiment, Ra, Rb, and Rcare independently selected from Ce-uaryl, C1-18 alkyl and C1-7 cycloalkyl, the Ra, Rb, and Rcbeing substituted with, preferably, halogen, cyano, amido, C1-4 alkyl, C1-4 alkoxy, and phenyl, or wherein Raand Rbtaken together, form a unsubstituted or substituted monocyclic, bicyclic, tricyclic or another polycyclic hydrocarbon moiety, the substituted bicyclic or tricyclic hydrocarbon moiety being substituted with, preferably, halogen, cyano, amido, C1-4 alkyl, C1-4 alkoxy, and phenyl, and Rcis selected from H, Ce-uaryl, C1-18 alkyl and C1-7 cycloalkyl, the Rcbeing unsubstituted or substituted with, preferably, halogen, cyano, amido, C1-4 alkyl, C1-4 alkoxy, and phenyl.

[0031] A monocyclic hydrocarbon moiety is, e.g., a cyclohexyl moiety.

[0032] In one embodiment, the term “monocyclic hydrocarbon moiety” encompasses a moiety having an oxygen atom or a sulfur atom in the backbone.

[0033] A bicyclic hydrocarbon moiety is, e.g., a norbornyl moiety such as a 7- norbornyl moiety.

[0034] A tricyclic hydrocarbon moiety is, e.g., an adamantyl moiety.

[0035] A polycyclic hydrocarbon moiety is, e.g., a Cuban or a homocuban moiety.

[0036] Enol ethers are termed as electron-rich alkenes by virtue of the electrondonation from the oxygen atom via iT-bonding. It is believed that this property is responsible for good reactivity in cycloaddition reactions such as the [2 + 2] cycloaddition with singlet oxygen1O2.

[0037] Step (A) requires reacting a molybdate or tungstate or an ammonia or alkali metal salt of molybdate or tungstate with hydrogen peroxide or aqueous hydrogen peroxide to form the peroxomolybdate or peroxotungstate or the ammonium or alkali metal salt of a peroxomolybdate or peroxotungstate.

[0038] In one embodiment, the use of an aqueous hydrogen peroxide is preferred.

[0039] The term “alkali metal” as used herein preferably encompasses lithium, sodium, and potassium.December 12, 2025120592P1126PC- 9 -

[0040] The terms “molybdate” or “tungstate” as used herein mean molybdenum and tungsten in their highest oxidation state, namely +VI, and coordinated by oxygen. The terms encompass mono-, di-, tri-, tetra-, penta-, hexa-, hepta-, octamolybdates and - tungstates, etc. The terms also encompass solvates and hydrates thereof.

[0041] The terms “peroxomolybdate” and “peroxotungstate” as used herein mean that the molybdates and tungstates contain one or more units of O2 wherein both oxygen atoms are bound to one metal. The terms may synonymously used with the terms “oxo molybdate” and “oxo tungstate”.

[0042] Preferably, said molybdate used in step (A) is ammonium heptamolybdate tetrahydrate, (NH^eMozC^FhO (CAS 12054-85-2).

[0043] The term “aqueous hydrogen peroxide” encompasses the solution of hydrogen peroxide in water in any concentration. Preferably, the concentration of hydrogen peroxide in the aqueous hydrogen peroxide used in step (A) is at least 1 % or at least 5%, more preferably 1 % to 80%, still more preferably 5 % to 50 %, based on the total weight of hydrogen peroxide and water. Further particularly preferred ranges are from 4 % to 40 % and from 5 % to 35 %, based on the total weight of hydrogen peroxide and water.

[0044] In a further preferred embodiment, hydrogen peroxide is free of water, i.e. , it is 100 % pure hydrogen peroxide.

[0045] In one embodiment, at least 5 mole equivalents of H2O2 are used per 0.1 mole equivalent of the molybdate or tungstate or wherein at least 0.1 mole equivalent of the molybdate or tungstate is used per 1 mole equivalent of the enol ether, or both.

[0046] In another embodiment, at least 5 mole equivalents of H2O2 are used per 0.1 up to less than 2 mole equivalents of the molybdate or tungstate or wherein at least 0.1 mole equivalent up to less than 2 mole equivalents of the molybdate or tungstate is used per 1 mole equivalent of the enol ether, or both.

[0047] In still another embodiment, at least 5 mole equivalents of H2O2 are used per 0.1 up to less than 1.5 mole equivalents of the molybdate or tungstate or wherein atDecember 12, 2025120592P1126PC- 10 - least 0.1 mole equivalent up to less than 2 mole equivalents of the molybdate or tungstate is used per 1 mole equivalent of the enol ether, or both.

[0048] The amount of H2O2 is not limited. For practical reasons, in one embodiment, the amount ranges from 5 to 50 mole equivalents, or 5 to 40 mole equivalents, or 5 to 30 mole equivalents, or 5 to 20 mole equivalents, or 5 to 10 mole equivalents, per 1 mole equivalent of the enol ether.

[0049] According to the invention, the reacting in step (B) is performed in a reaction solvent comprising a water miscible protic solvent, preferably a C1-5 alcohol and water, in homogeneous liquid phase. This means that, in one embodiment, the reaction according to step (A) is performed in the water-miscible protic solvent containing water prior to oxidation of the enol ether.

[0050] However, in another preferred embodiment, the reaction solvent consists of a water miscible protic solvent. Then, the water contained therein stems from water generated by the decomposition of hydrogen peroxide caused by the oxidation of molybdate or tungstate during step (A).

[0051] In a preferred embodiment, the water miscible protic solvent comprises or is a C1-5 alcohol.

[0052] Further preferably, the reactions in step (A) and in step (B) are performed in a C1-5 alcohol and in homogeneous liquid phase.

[0053] The term “C1-5 alcohol” as used herein means alcohols selected from methanol, ethanol, propanol, butanol and pentanol including all possible isomers.

[0054] Preferred alcohols are C1-4 alcohols.

[0055] In a further preferred embodiment, the alcohol used is methanol or t-butyl alcohol.

[0056] According to the invention, the reaction is carried out in homogeneous liquid phase. The term “homogeneous liquid phase” as used herein means that the mixture formed in step (B) is in a unitary phase, i.e. that by inspection with the eye no differentDecember 12, 2025120592P1126PC- 11 - liquid phases are detectable or that no solids are detectable or that no different liquid phases and no solids are detectable.

[0057] This can be achieved by selection of an appropriate amount of water and alcohol.

[0058] Accordingly, additional water and / or C1-5 alcohol may be added in step (A) and / or step (B) to ensure a homogeneous liquid phase.

[0059] In a preferred embodiment, the reaction is carried out in presence of a base such that the pH is adjusted to a range of from 6 to 9, preferably 6 to 8.

[0060] Preferably, the base is selected from a hydrogen carbonate or dihydrogen phosphate, mono hydrogen phosphate or phosphate.

[0061] In a further preferred embodiment, the base is disodium mono hydrogen phosphate.

[0062] However, in another embodiment, it is also possible to use an amine as base.

[0063] The peroxidation preferably is carried out at a temperature, which is high enough to trigger the [2+2] cycloaddition but low enough to prevent the degradation of the formed 1 ,2-dioxetane. Suitably, the reaction temperature is from -10 °C to 50 °C, preferably -10 °C to 40 °C, further preferably -10 °C to 30 °C, and further preferred from -10 °C to room temperature (23 °C).

[0064] In a preferred embodiment, the reaction is carried out in a dark environment.

[0065] If desired, the formed 1 ,2-dioxetane may be isolated. Accordingly, the method according to the invention may comprise step (C):(C) isolating the formed 1 ,2-dioxetane.

[0066] Suitable isolation methods encompass the extraction of the formed 1 ,2- dioxetane from the liquid phase with a suitable organic solvent such as methylene chloride. Subsequently, the solvent may be removed, and the product may be isolated.December 12, 2025120592P1126PC

[0067] In a specific embodiment, the invention relates to a method of making a chemiluminescent 1 ,2-dioxetane of formula IIthe method comprising: reacting an enol ether of formula Ito yield the 1 ,2-dioxetane of formula II, wherein the method comprises the reaction of an ammonium or alkali metal salt of a peroxomolybdate or peroxotungstate with the double bond of the enol ether I to form the 1 ,2-dioxetane II, comprising steps (A) and (B):(A) reacting an ammonia or alkali metal salt of molybdate or tungstate with hydrogen peroxide and aqueous hydrogen peroxide to form the ammonium or alkali metal salt of a peroxomolybdate or peroxotungstate;(B) reacting enol ether of formula I with the ammonium or alkali metal salt of peroxomolybdate or peroxotungstate formed in step (A); wherein the reaction in step (B) is performed in a C1-5 alcohol and wherein the reaction is carried out in homogeneous liquid phase.

[0068] In a preferred embodiment, in the enol ether I and the 1 ,2-dioxetane II, R1is an electron withdrawing group.December 12, 2025 120592P1126PC - 13 -

[0069] The term “electron-withdrawing” us as used herein denotes any group which is known to be electron withdrawing.

[0070] In preferred embodiments, R1is selected from -CH=CH-COOH, -CH=CH- COOR, and -CH=CH-CN, wherein R is C1-10 alkyl.

[0071] In a preferred embodiment, in the enol ether I and the 1 ,2-dioxetane II, R2is H or a halogen. Halogen is preferred. Further preferably, the halogen is Cl.

[0072] In a preferred embodiment, in the enol ether I and the 1 ,2-dioxetane II, R3is Ci -1 s alkyl or C1-7 cycloalkyl. This means that the CC double bond of the enol ether may be regarded as an electron-rich double bond.

[0073] In a preferred embodiment, R3is C1-4 alkyl, further preferably R3is methyl.

[0074] The protecting group PG in the enol ether I and the 1 ,2-dioxetane III can be any protecting group that is known in the art which can be attached to the oxy group of a phenolate group, and wherein the PG-0 group can be cleaved from the phenolate oxygen by means of a bacterium, respectively an enzyme.

[0075] In one embodiment, the protecting group PG of the enol ether is a group R4- L.

[0076] In one embodiment, R4is selected from H, C1-4 alkyl, and C6H5-CH2.

[0077] In a preferred embodiment, R4is selected from a glycoside

[0078] Preferably, the glycoside is a glucoside, a mannoside, a galactoside, a fucoside or an arabinoside.

[0079] In another preferred embodiment, R4is selected from glucuronideDecember 12, 2025120592P1126PC

[0080] In another embodiment, R4is a phosphonate P(=O)(OH)2 or an anion thereof.

[0081] In a further embodiment, R4is a sulfonate S(=O)2(OH) or an anion thereof.

[0082] In another embodiment, R4is a silyl group R5R6R7Si, wherein R5’ R6, R7are independently selected from C1-4 alkyl and phenyl. A preferred silyl group is t-butyl dimethyl silyl ((t-CH3)3C)(CH3)2Si.

[0083] In still other embodiments, R4is selected from 2,4-dinitrobenzenesulfonate4-azidobenzyloxy carbonylwherein pep is a peptide moiety consisting of at least two amino acid residues and linked via a carboxylic group thereof.

[0084] In one embodiment, L is absent.

[0085] In another embodiment, L is a linker. In a preferred embodiment, L is O-(CeH4) or is O-C1-4 alkylene.

[0086] The enol ethers of formula I defined above are known or may be prepared according to known methods. Suitable methods are disclosed in WO 2017 / 130191.

[0087] In preferred embodiments, the chemiluminescent 1 ,2-dioxetanes prepared according to the method of the invention are defined by the following formulae (the yield is also shown for information):December 12, 2025120592P1126PC% 55%December 12, 2025120592P1126PC- 16 -65% 80%.

[0088] As described above, in one embodiment, the chemiluminescent probes are based on compounds comprising a 1 ,2-dioxetane moiety derived from an adamantyl enol ether and a phenolate moiety comprising a protecting group and an electron withdrawing group, i.e. the ortho-substituted phenoxy-adamantyl-1 ,2-dioxetane luminophore of, e.g., formula I (a spiro-adamantyl-dioxetane).

[0089] In a further embodiment, it is contemplated that the above-described method is expanded to enol ethers other than an adamantyl enol ether.

[0090] In one embodiment, it is possible to substitute the spiro-adamantyl-dioxetane unit in an ortho-substituted phenoxy-adamantyl-1 ,2-dioxetane luminophore with a spiro-cyclo-butyl unit. Respective compounds are, e.g., known from R. Tannous et al., “Spirostrain-Accelerated Chemiexcitation of Dioxetanes Yields Unprecedented Detection Sensitivity in Chemiluminescence Bioassays”, ACS Cent. Sci. 2024, 10, 28- 42.

[0091] In a further embodiment, it is possible to substitute the spiro-adamantyl- dioxetane unit in an ortho-substituted phenoxy-adamantyl-1 ,2-dioxetane luminophore with a spiro-7-norbornyl or spiro-homocubanyl unit. Respective compounds are, e.g., known from S. Gutkin et al, “Boosting Chemiexcitation of Phenoxy-1-2-dioxetanes through 7-Norbornyl and Homocubanyl Spirofusion”, JACS, Au 2024, 4, 3558-3566.

[0092] In a further embodiment, it is possible to substitute the spiro-adamantyl- dioxetane unit in the ortho-substituted phenoxy-adamantyl-1 ,2-dioxetane luminophore with a spiro-cyclo-oxetan-3-yl unit. Respective compounds are, e.g., known from O. Shelef et al, “Biocompatible Flash Chemiluminscent Assay Enabled by stericallyDecember 12, 2025120592P1126PC- 17 -Hindered Spiro-Strained-Oxetanyl-1 ,2-Dioxetane”, Chem.Eur. J. 2024, 30, e202402981 .

[0093] In a further embodiment, it is possible to substitute the spiro-adamantyl- dioxetane unit in the ortho-substituted phenoxy-adamantyl-1 ,2-dioxetane luminophore compound with a spiro-fused six-member ring such as a cyclohexyl ring. Respective compounds are, e.g., known from M. David et al, “Chemiexcitation Acceleration of 1 ,2- Dioxetanes by Spiro-Fused Six-Member Rings with Electron-Withdrawing Motifs”, Angew. Chem. Int. Ed. Engl. 2024, 63(46);e202410057.

[0094] By means of the above-defined spiro compounds, the glow-type chemiluminescent reaction often observed with the spiro-adamantyl-dioxetane unit may be modified to a flash luminescent reaction, i.e. faster 0-0 cleavage and accelerated chemiexcitation.

[0095] According to a second aspect, the invention relates to a composition comprising an ammonium or alkali metal salt of a peroxomolybdate or peroxotungstate, an enol ether, a C1-5 alcohol, water, a base selected from a hydrogen carbonate or dihydrogen phosphate, mono hydrogen phosphate or phosphate such that the pH is adjusted to a range of from 6 to 9, preferably 6 to 8, preferably wherein the ingredients are in homogeneous liquid phase.

[0096] In a preferred embodiment, the composition further comprises hydrogen peroxide and a 1 ,2-dioxetane.EXAMPLESExample 1

[0097] Generally applicable method

[0098] 0.1 eq of ammonium heptamolybdate tetrahydrate, 2 eq of disodium hydrogen phosphate and aqueous solution of H2O2 (10% concentration, 3 mL per 0.1 mmol) are stirred in dark for 10 min to form a reddish-brown solution. Next, the selected enol ether, preferably the enol ether of formula I, dissolved in f-BuOH (5mL perDecember 12, 2025120592P1126PC- 18 -0.1 mmol) is added. The resulting solution is then stirred at 25 °C in dark until the reaction is completed (typically after 18 h; may be controlled by HPLC).

[0099] The reaction mixture obtained is partitioned between water and dichloromethane (5x original reaction volume), wherein the aqueous phase is twice extracted with dichloromethane. The combined organic phases are washed with 10 % Na2S20s until a peroxide test is negative, then with brine, dried over MgSCk and concentrated under reduced pressure at 25 °C.

[0100] The obtained residue is then purified by preparative HPLC, using a commercial C18 column and acetonitrile / 0.1 % ammonium carbonate or alternatively acetonitrile / 0.1 % acetic acid as eluent.

[0101] Alternatively, the product is crystallized from an appropriate solvent or no further purification is used. Alternatively, a HILIC chromatography (hydrophilic interaction liquid chromatography) on normal polarity silica is used, using a mixture of halogenated hydrocarbon with aliphatic alcohol and organic acid as eluent. HILIC chromatography is known in the art.Example 2

[0102] In a specific example, the following AquaSpark™ beta-D-galactoside enhanced probe precursorwas oxidized according to the generally applicable method to yield the following dioxetane (yield 70 %):December 12, 2025120592P1126PC- 19 -

[0103] 1H-NMR analysis (400 MHz, DMSO-d6; 5 ppm) confirmed full conversion of the enol ether into the 1 ,2-dioxetane, wherein the spectrum of the product was in good accordance with a reference spectrum:

[0104] 1.10 (s, 4H) 1 .17 (t, J = 7.2 Hz, 1 H) 1 .27 - 1 .38 (m, 1 H) 1 .47 (ddd, J = 11 .8 Hz, J = 2.3 Hz, J = 1 .2 Hz, 1 H) 1 .52 - 1 .75 (m, 8H) 1 .85 - 1 .95 (m, 2H) 1 .98 (s, 1 H) 2.29 - 2.33 (m, 1 H) 2.79 - 2.98 (m, 2H) 3.07 (s, 1 H) 3.40 (br dd, J = 9.5 Hz, J = 3.30 Hz, 2H) 3.49 (dt, J = 9.0 Hz, J = 4.5 Hz, 2H) 3.52 - 3.60 (m, 4H) 3.70 (d, J = 3.2 Hz, 1 H) 4.02 (q, J = 7.1 Hz, 1 H) 4.72 - 4.93 (m, 3H) 6.64 (d, J = 16.0 Hz, 1 H) 6.99 - 7.08 (m, 2H) 7.26 - 7.50 (m, 2H) 7.68 - 7.84 (m, 2H) 7.92 (d, J = 8.4 Hz, 1 H).Example 3

[0105] In a specific example, the following AquaSpark™ alpha-D-mannoside methylester enhanced probe precursorwas oxidized according to the generally applicable method to yield the following dioxetane (yield 89 %):December 12, 2025120592P1126PC

[0106] 1H-NMR analysis (400 MHz, MeOH-d4; 5 ppm) confirmed full conversion of the enol ether into the 1 ,2-dioxetane, wherein the spectrum of the product was in good accordance with a reference spectrum:

[0107] 1.29 (s, 1 H) 1.40 (br s, 2H) 1.50 (br d, J = 12.4 Hz, 1 H) 1.60 - 1.90 (m, 8H) 1 .9 - 2.2 (m, 2H) 2.40 (br d, J = 12.5 Hz, 1 H) 2.97 (br s, 1 H) 3.19 (s, 3H) 3.35 (s, 1 H) 3.60 (ddd, J = 7.3 Hz, J = 4.9 Hz, J = 2.4 Hz, 1 H) 3.70 - 3.78 (m, 3H) 3.80 (s, 3H) 3.89 - 3.93 (m, 1 H) 4.00 (br s, 1 H) 4.94 (s, 2H) 5.48 (s, 1 H) 6.51 (d, J = 16.0 Hz, 1 H) 7.10 (br d, J = 7.6 Hz, 2H) 7.30 (dd, J = 8.6 Hz, J = 2.4 Hz, 2H) 7.70 - 7.85 (m, 2H) 7.88 (br d, J = 8.3 Hz, 1 H).

[0108] LCMS analysis of the latter compound produced peak, which under negative ESI-APCI multimode ionization produced ions with m / z 689.2 (80% intensity, M-H) 556.2 (100% intensity).Example 4

[0109] In a specific example, the following AquaSpark™ alpha-D-mannoside free acid enhanced probe precursorwas oxidized according to the generally applicable method to yield the following dioxetane (yield 87 %):December 12, 2025120592P1126PC- 21 -

[0110] 1H-NMR analysis (400 MHz, MeOH-d4; 5 ppm) confirmed full conversion of the enol ether into the 1 ,2-dioxetane, wherein the spectrum of the product was in good accordance with a reference spectrum:

[0111] 1.29 (s, 1 H) 1.39 (s, 2H) 1.52 (dd, J = 12Hz, J = 2Hz, 1 H) 1.6-1 .9 (m, 8H) 2.01 (bs, 3H) 2.39 (d, J = 8Hz, 1 H) 2.96 (s, 1 H) 3.19 (s, 3H) 3.57-3.62 (m, 1 H), 3.70- 3.79 (m, 3H) 3.89 (dd, J = 6Hz J = 1.2Hz) 4.10 (dd, J = J = 8Hz) 4.93 (s, 2H) 5.49 (s, 1 H) 6.5 (d, J = 15.5Hz 1 H) 7.10 (d, J = 8Hz, 2H) 7.35 (d, J = 8Hz, 2H) 7.73 (d, J = 8Hz, 1 H) 7.78 (d, J = 15.5Hz, 1 H) 7.87 (d, J = 8Hz, 1 H)

[0112] LCMS analysis of the latter compound produced peak, which under negative ESI-APCI multimode ionization produced ions with m / z 673.1 (15% intensity, M-H) 523.1 (100% intensity).

[0113] The latter compound can be further purified, using HILIC mode chromatography on 60 A silica, preferably with 10-40 pm particle size. Acidic eluent, comprising halogenated hydrocarbon, aliphatic alcohol and organic acid, preferably dichloromethane with isopropanol and acetic acid was used, either in negative (filtration) or positive (column) pressure mode.Example 5

[0114] In a specific example, the following AquaSpark™ D-myoinositol phosphate enhanced probe precursorDecember 12, 2025120592P1126PC- 22 -was oxidized according to the generally applicable method to yield the following dioxetane (yield 65 %):

[0115] 1H-NMR analysis (400 MHz, DMSO-d6; 5 ppm) confirmed full conversion of the enol ether into the 1 ,2-dioxetane, wherein the spectrum of the product was in good accordance with a reference spectrum:

[0116] 1.23 (d, J = 10.4 Hz, 2H) 1.34 (d, J = 12.7 Hz, 1 H) 1.46 (d, J = 11.3 Hz, 1 H) 1 .91 (s, 1 H), 1 .77 - 1 .51 (m, 9H) 2.25 (d, J = 11 .0 Hz, 1 H) 2.88 (s, 1 H) 2.93 (t, J = 9.0 Hz, 1 H) 3.07 (s, 1 H) 3.12 (s, 3H) 3.35 (t, J = 9.3 Hz, 1 H) 3.57 (t, J = 9.2 Hz, 1 H) 3.78 - 3.67 (m, 2H) 4.83 (s, 2H) 6.51 (d, J = 16.1 Hz, 1 H) 7.16 (d, J = 8.4 Hz, 2H) 7.28 (d, J = 8.5 Hz, 2H) 7.56 (d, J = 16.0 Hz, 1 H) 7.74 (d, J = 8.4 Hz, 1 H) 7.83 (d, J = 8.4 Hz, 1 H)

[0117] LCMS analysis of the latter compound produced peak, which under negative ESI-APCI multimode ionization produced ions with m / z 753.2 (4% intensity, M-H) 603.1 (100% intensity).Example 6

[0118] In a specific example, the following AquaSpark™ alpha-L-fucoside methylester enhanced probe precursorDecember 12, 2025120592P1126PC- 23 -was oxidized according to the generally applicable method to yield the following dioxetane (yield 75 %):

[0119] 1H-NMR analysis (400 MHz, MeOH-d4; 5 ppm) confirmed full conversion of the enol ether into the 1 ,2-dioxetane, wherein the spectrum of the product was in good accordance with a reference spectrum:

[0120] 1.12 - 1.22 (m, 3H) 1.37 (bs, 2H) 1.48 (bd, J = 12.6 Hz, 1 H) 1.57 - 1.88 (m, 8H) 1.96 (bs 1 H) 2.36 (bd, J = 12.6 Hz, 1 H), 2.95 (s, 1 H), 3.16 (s, 3H), 3.73 (bs, 1 H) 3.79 (s, 3H) 3.88 - 4.07 (m, 3H), 4.92 (s, 2H) 5.47 (s, 1 H) 6.53 (dd, J = 16.0 Hz, J =3.5 Hz, 1 H) 7.10 (d, J = 7.0 Hz, 2H) 7.31 (dd, J = 8.5 Hz, J = 4.5 Hz, 2H) 7.72 (d, J =8.5 Hz, 1 H) 7.81 (dd, J = 16.0 Hz, J = 4.5 Hz, 1 H), 7.85 (d, J = 8.5 Hz, 1 H).

[0121] LCMS analysis of the latter compound produced peak, which under negative ESI-APCI multimode ionization produced ions with m / z 671.2 (100% intensity, M-H) 521.1 (80% intensity).Example 7

[0122] In a specific example, the following AquaSpark™ alpha-L-fucoside free acid enhanced probe precursorDecember 12, 2025120592P1126PC- 24 -was oxidized according to the generally applicable method to yield the following dioxetane (yield 69 %):

[0123] 1H-NMR analysis (400 MHz, MeOH-d4; 5 ppm) confirmed full conversion of the enol ether into the 1 ,2-dioxetane, wherein the spectrum of the product was in good accordance with a reference spectrum:

[0124] 1.16 - 1.22 (m, 3H) 1.37 (bs, 2H) 1.5 (bd, J = 12.4 Hz, 1 H) 1.59 - 1.90 (m, 8H) 2.37 (bd, J = 12.4 Hz, 1 H), 2.95 (s, 1 H), 3.16 (s, 3H), 3.73 (bs, 1 H) 3.88 - 4.07 (m, 3H), 4.92 (s, 2H) 5.48 (s, 1 H) 6.53 (dd, J = 16.0 Hz, J = 3.5 Hz, 1 H) 7.09 (d, J = 8.3 Hz, 2H) 7.35 (dd, J = 8.3 Hz, J = 4.6 Hz, 2H) 7.73 (d, J = 8.3 Hz, 1 H) 7.82 (dd, J = 16.0 Hz, J = 4 Hz, 1 H), 7.85 (d, J = 8.3 Hz, 1 H).

[0125] LCMS analysis of the latter compound produced peak, which under negative ESI-APCI multimode ionization produced ions with m / z 657.2 (8% intensity, M-H) 507.1 (100% intensity).

[0126] The latter compound could be further purified, using HILIC mode chromatography on 60 A silica, preferably with 10-40 pm particle size. Acidic eluent, comprising halogenated hydrocarbon, aliphatic alcohol and organic acid, preferably dichloromethane with isopropanol and acetic acid was used, either in negative (filtration) or positive (column) pressure mode.December 12, 2025120592P1126PC- 25 -Example 8

[0127] In a specific example, the following AquaSpark™ calibration probe precursorwas oxidized according to the generally applicable method to yield the following dioxetane (yield 69 %):

[0128] 1H-NMR analysis (400 MHz, CDCh; 5 ppm) confirmed full conversion of the enol ether into the 1 ,2-dioxetane, wherein the spectrum of the product was in good accordance with a reference spectrum:

[0129] 1.34 - 1.90 (m, 12H) 2.06 (bs, 1 H) 2.23 (d, J = 12.0 Hz, 1 H) 3.03 (bs, 1 H) 3.24 (s, 3H) 6.53 (bs, 1 H) 6.70 (d, J = 16.0 Hz, 1 H) 7.56 (d, J = 8.0 Hz, 1 H) 7.71 (d, J = 8.0 Hz, 1 H) 8.06 (d, J = 16.0 Hz, 1 H).

[0130] LCMS analysis of the latter compound produced peak, which under negative ESI-APCI multimode ionization produced ions with m / z 405.1 (100% intensity, M-H) 255.0 (70% intensity).Example 9

[0131] In a specific example, the following AMPPD alkaline phosphatase probe precursorDecember 12, 2025120592P1126PC- 26 -was oxidized according to the generally applicable method. Further treatment of crude material with bi-stoichiometric amount of Na2COs in deionized water, lyophylization and trituration with cold methanol yielded the following dioxetane (yield 80 %):

[0132] 1H-NMR analysis (400 MHz, D2O; 6 ppm) confirmed full conversion of the enol ether into the 1 ,2-dioxetane disodium salt, wherein the spectrum of the product was in good accordance with a reference spectrum:

[0133] 0.99 (d, J = 10Hz, 1 H) 1.28 (d, J = 13.1 Hz, 1 H) 1.90-1.50 (m, 10H) 2.28 (bs, 1 H) 2.89 (s, 1 H) 3.24 (s, 3H) 7.40-7.15 (m, 4H).

[0134] LCMS analysis of the latter compound produced peak, which under negative ESI-APCI multimode ionization produced ions with m / z 380.1 (100% intensity, M-H) 231.1 (65% intensity).Example 10

[0135] In a specific example, the following AquaSpark™ beta-D-glucuronide free acid enhanced probe precursorDecember 12, 2025120592P1126PC- 27 - was oxidized according to the generally applicable method to yield the following dioxetane (yield 55 %):

[0136] 1H-NMR analysis (400 MHz, MeOH-d4; 5 ppm) confirmed full conversion of the enol ether into the 1 ,2-dioxetane, wherein the spectrum of the product was in good accordance with a reference spectrum:

[0137] 1.41 (br s, 2H) 1.53 (br d, J = 12.6 Hz, 1 H) 1.61 - 1.90 (m, 9H) 2.00 (br s, 1 H) 2.40 (br d, J = 12.6 Hz, 1 H) 2.97 (br s, 1 H) 3.17 - 3.21 (m, 3H) 3.47 - 3.55 (m, 3H) 3.80 (d, J = 9.0 Hz, 1 H) 4.88 - 4.99 (m, 4H) 6.41 (br dd, J = 16.0, 5.0 Hz, 1 H) 7.07 (m, J = 7.2 Hz, 2H) 7.29 (m, J = 7.2 Hz, 2H) 7.66 - 7.71 (m, 1 H) 7.85 (br d, J = 8.0 Hz, 1 H).

[0138] LCMS analysis of the latter compound produced peak, which under negative ESI-APCI multimode ionization produced ions with m / z 687.2 (70% intensity, M-H) 537.1 (100% intensity).

[0139] The latter compound could be further purified, using HILIC mode chromatography on 60 A silica, preferably with 10-40 pm particle size. Acidic eluent, comprising halogenated hydrocarbon, aliphatic alcohol and organic acid, preferably dichloromethane with isopropanol and acetic acid was used, either in negative (filtration) or positive (column) pressure mode.

Claims

December 12, 2025120592P1126PC- 28 -CLAIMS1. A method of making a 1 ,2-dioxetane comprising the reaction of a peroxomolybdate or a peroxotungstate, preferably the reaction of an ammonium or alkali metal salt of a peroxomolybdate or peroxotungstate, with the double bond of an enol ether to form the 1 ,2-dioxetane, the reaction comprising steps (A) and (B):(A) reacting a molybdate or tungstate, preferably an ammonia or alkali metal salt of molybdate or tungstate, with hydrogen peroxide or aqueous hydrogen peroxide to form the peroxomolybdate or peroxotungstate,(B) reacting an enol ether with the peroxomolybdate or peroxotungstate formed in step (A) to form the 1 ,2-dioxetane, wherein the reaction in step (B) is performed in a reaction solvent comprising water miscible protic solvent, preferably a C1-5 alcohol and water, preferably wherein the reaction is carried out in homogeneous liquid phase.

2. The method of claim 1 , wherein step (A) comprises dissolving the molybdate or the tungstate in aqueous hydrogen peroxide to form a solution, and step (B) comprises contacting the solution formed in step (A) with the enol ether and the reaction solvent, preferably wherein the enol ether is pre-dissolved in the reaction solvent.

3. The method of claim 1 or 2, wherein at least 5 mole equivalents of H2O2 are used per 0.1 mole equivalent of the molybdate or tungstate or wherein at least 0.1 mole equivalent of the molybdate or tungstate is used per 1 mole equivalent of the enol ether, or both.

4. The method of any one of claims 1 to 3, wherein the molybdate used in step (A) is ammonium heptamolybdate tetrahydrate.December 12, 2025120592P1126PC- 29 -5. The method of any one of claims 1 to 4, wherein hydrogen peroxide used in step (A) is an aqueous hydrogen peroxide, preferably having a concentration from 5 wt.% to 99.9 wt.% of hydrogen peroxide based on the sum of water and hydrogen peroxide, or wherein the hydrogen peroxide is 100 wt.%, and / or wherein the C1-5 alcohol used in step (B) is f-butyl alcohol.

6. The method of any one of claims 1 to 5, wherein the reaction is carried out in the presence of a base such that the pH is adjusted to a range of from 5.0 to 9.0, preferably 6.0 to 8.0, and wherein the base is preferably selected from the group consisting of hydrogen carbonate, dihydrogen phosphate, monohydrogen phosphate, phosphate, and mixtures thereof, preferably wherein the base is disodium monohydrogen phosphate.

7. The method of any one of claims 1 to 6, wherein the reaction is carried out in the absence of light and / or wherein the reaction is carried out in a temperature range of from -10 °C to 50 °C.

8. The method of any one of claims 1 to 7, wherein the reaction further comprises, after steps (A) and (B), step (C):(C) isolating the formed 1 ,2-dioxetane.

9. The method of any one of claims 1 to 8, wherein the enol ether has the general structure RaRbC=CRc-O-Rd, wherein Ra, Rb, and Rcare independently of each other selected from H, unsubstitued or substituted Ce-u aryl, C1-18 alkyl and C1-7 cycloalkyl, and Rdis selected from Ci -1 s alkyl or C1-7 cycloalkyl.

10. The method of claim 9, wherein the Ra, Rb, and Rcare independently selected from Ce-u aryl, C1-18 alkyl and C1-7 cycloalkyl, the Ra, Rb, and Rcbeing substituted with, preferably, halogen, cyano, amido, C1-4 alkyl, C1-4 alkoxy, and phenyl, or wherein Raand Rbtaken together, form an unsubstituted or substituted monocyclic, bicyclic or tricyclic or another polycyclic hydrocarbon moiety, the substituted monocyclic, bicyclic or tricyclic or another polycyclic hydrocarbonDecember 12, 2025120592P1126PC- 30 - moiety being substituted with, preferably, halogen, cyano, amido, C1-4 alkyl, C1-4 alkoxy, and phenyl, and Rcis selected from H, Ce-14 aryl, C1-18 alkyl and C1-7 cycloalkyl, the Rcbeing unsubstituted or substituted with, preferably, halogen, cyano, amido, C1-4 alkyl, C1-4 alkoxy, and phenyl.11 . The method of any one of claims 1 to 10, wherein the enol ether is of formula Iand the formed 1 ,2-dioxetane is of formulaII whereinPG is a protecting group,R1is an electron withdrawing group, preferably R1is CH=CH-COOH, CH=CH-COOR, or CH=CH-CN, wherein R is C1-8 alkyl,R2is H or a halogen, preferably Cl,R3is C1-18 alkyl or C1-7 cycloalkyl.

12. The method of claim 10 or 11 , wherein the protecting group PG is a group R4-L, wherein R4is selected from:H, C1-4 alkyl, and C6H5-CH2; a glycosideDecember 12, 2025120592P1126PC- 31 -preferably the glycoside is a glucoside, a mannoside, a galactoside, a fucoside or an arabinoside; glucuronidephosphonate P(=O)(OH)2 or an anion thereof; sulfonate S(=O)2(OH) or an anion thereof; silyl R5R6R7Si, wherein R5’ R6, R7are independently selected from C1-4 alkyl and phenyl, preferably t-butyl dimethyl silyl ((t-CH3)3C)(CH3)2Si;two amino acid residues and linked via a carboxylic group thereof; and wherein L is absent or is present, and when present is O-(CeH4) or O-C1-4 alkylene.

13. The method of any one of the claims 1 to 12, wherein the formed 1 ,2-dioxetane is one of the following compounds:December 12, 2025120592P1126PC- 32 -14. The method of any one of claims 1 to 13, wherein in step (A) and / or step (B) additional water and / or a C1-5 alcohol is added to ensure a homogeneous liquid phase.December 12, 2025120592P1126PC- 33 -15. A composition comprising an ammonium or alkali metal salt of a peroxomolybdate or peroxotungstate, an enol ether, a C1-5 alcohol, water, a base selected from a hydrogen carbonate or dihydrogen phosphate, mono hydrogen phosphate or phosphate such that the pH is adjusted to a range of from 6 to 7, preferably wherein the ingredients are in homogeneous liquid phase, and wherein the composition preferably further comprises hydrogen peroxide and a 1 ,2- dioxetane.

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