Method of making chemiluminescent 1,2-dioxetanes

Abstract

The invention relates to a method of making a 1,2-dioxetane, the method comprising reacting a naphthalene endo peroxide (NEPO) with the double bond of an enol ether to form the 1,2-dioxetane. The 1,2-dioxetanes are of great commercial interest for a variety of applications, such as singlet oxygen detection, enzyme detection and pathogen (e.g., Salmonella spp. and Listeria spp.) detection.

Description

August 29, 2025Biosynth AG 119687P1126PCMETHOD OF MAKING CHEMILUMINESCENT 1,2-DIOXETANESFIELD OF THE INVENTION

[0001] The present invention relates to a method of making 1 ,2-dioxetanes by oxidizing an enol ether with a naphthalene endoperoxide (NEPO).BACKGROUND OF THE INVENTION

[0002] Chemiluminescence is a light-emission phenomenon initiated by chemical reactions and is a powerful tool for in vivo and in vitro detection and imaging of various analytes and enzymes. The main advantage of chemiluminescence over fluorescence lies in the fact that irradiation by an external light source is not required and, thus, the background signal is extremely low and the sensitivity is high.

[0003] Most known light-emitting compounds undergo chemiexcitation through spontaneous decomposition of cyclic peroxide moieties. 1 ,2-Dioxetanes have been widely explored in the past as light-emitting functionalities. The decomposition of such 1 ,2-dioxetanes gives an excited-state carbonyl compound that generates chemiluminescence emission upon radiative deactivation of the excited-state carbonyl compound. 1 ,2-Dioxetane-based chemiluminescence probes are described, e.g., in WO 2017 / 130191.

[0004] Commercially available chemiluminescence probes are, e.g., known under the trademark AquaSpark™. Such probes are of great commercial interest for a variety of applications, such as singlet oxygen detection, enzyme detection and pathogen (e.g., Salmonella spp. and Listeria spp.) detection. 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 one or more electron withdrawing group(s). In the AquaSpark™ probes, the emission of chemiluminescence is initiated as a consequence of enzymatic cleavage of the protecting group (PG) from theAugust 29, 2025Biosynth AG 119687P1126PC phenolate moiety connected 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):

[0005] 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.

[0006] However, some of the AquaSpark™ probes are designed for the detection of non-biological material such as common chemical analysts such as fluorides, peroxides and thiols.

[0007] 1 ,2-Dioxetanes may be produced by the reaction of the corresponding enol ether moiety with oxygen in a photoreactor as is schematically shown in the following equation:August 29, 2025Biosynth AG 119687P1126PC

[0008] The photoreactor requires a photosensitizer, which transfers oxygen in the ground state3C>2 into its excited state1O2 in the presence of light. The excited state of molecular oxygen is the reactive species that reacts with the enol ether to generate the 1 ,2-dioxetane. By reaction with oxygen and light in the presence of a photosensitizer, the double bond in the enol ether moiety of the probe and the in situ formed singlet oxygen undergo a [2+2] cycloaddition to generate the 1 ,2-dioxetane moiety.

[0009] The known synthesis methods for 1 ,2-dioxetanes are complex and involve tedious steps and expensive instruments. Thus, there is still room for improvement in the manufacturing process of 1 ,2-dioxetanes.OBJECTS OF THE INVENTION

[0010] Despite these advances, there is a need in the industry to develop a reaction for the manufacture of 1 ,2-dioxetanes, which makes the use of a photoreactor unnecessary, and which is thus easier to handle.SUMMARY OF THE INVENTION

[0011] The present invention relates to a reaction for making a 1 ,2-dioxetane which avoids the use of a photoreactor.

[0012] The invention is based on a method comprising the use of a naphthalene endoperoxide (NEPO) compound for generating singlet oxygen (1O2) used for the peroxidation of the double bond of an enol ether. Herein, the NEPO compound is formed by the peroxidation of a suitable naphthalene compound by singlet oxygen which is generated by catalytic disproportionation of hydrogen peroxide into water and singlet oxygen catalyzed by sodium molybdate.

[0013] The reaction can be performed under mild reaction conditions such as ambient temperature, under aerobic conditions and with very basic laboratory glassware. The reaction can additionally be run at much higher concentrations than the concentrationAugust 29, 2025Biosynth AG 119687P1126PC possible with the photoreactor, which is in the range of approx. 5 mg / mL only, preferably at a concentration of at least 50 mg / ml. The reactions can be easily monitored by1H-NMR analysis and analytical HPLC.

[0014] The NEPO approach is further beneficial compared to a reaction in a photoreactor due to its fairly short reaction times, where singlet oxygen will be reversibly trapped by a naphthalene compound to generate a naphthalene endoperoxide (NEPO). This endoperoxide is considered as a secondary source of1O2 used for the peroxidation of the double bond of an enol ether. The reaction is exemplarily shown in the following scheme showing the peroxidation of enol ether 1 by NEPO 4 to produce AquaSpark™ chemiluminescent probe 2. NEPO compound 4 is made by peroxidation of naphthalene compound 3 by singlet oxygen which is generated by catalytic disproportionation of hydrogen peroxide into water and singlet oxygen catalyzed by sodium molybdate:

[0015] According to a first aspect, the invention relates to a method of making a 1 ,2- dioxetane, the method comprising step (A):August 29, 2025Biosynth AG 119687P1126PC(A) reacting a naphthalene endo peroxide (NEPO) of formula IIwith the double bond of an enol ether to form the 1 ,2-dioxetane, wherein the variables in the NEPO compound II are defined in the appended claims.

[0016] In a preferred embodiment, the invention relates to a method of making a chemiluminescent 1 ,2-dioxetane of formula IIIIII, the method comprising reacting an enol ether of formula IAugust 29, 2025Biosynth AG 119687P1126PC with a naphthalene endoperoxide (NEPO) of formula IIto yield the 1 ,2-dioxetane of formula III, wherein the variables in compounds I, II and III are defined in the appended claims.

[0017] According to a second aspect, the invention relates to the use of a naphthalene endoperoxide (NEPO) compound of formula II for generating singlet oxygen (1O2) used for the peroxidation of the double bond of an enol ether to make a 1 ,2-dioxetane, preferably wherein the enol ether is of formula I, and the 1 ,2-dioxetane is of formula III.

[0018] According to a third aspect, the invention relates to a kit, the kit comprising a naphthalene endoperoxide compound (NEPO) of formula II and an enol ether, preferably comprising a naphthalene endoperoxide (NEPO) of formula II and an enol ether of formula I, as defined in the appended claims.

[0019] According to a fourth aspect, the invention relates to an improved synthesis of NEPO compound 4:August 29, 2025Biosynth AG 119687P1126PCDETAILED DESCRIPTION OF THE INVENTION

[0020] According to the first aspect, the invention relates to a method of making a 1 ,2- dioxetane, the method comprising step (A):(A) reacting a naphthalene endo peroxide (NEPO) of formula IIII with the double bond of an enol ether to form the 1 ,2-dioxetane.

[0021] 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.

[0022] The term “enol ether” as used herein means an ether of an enol having the general structure RaRbC=CRc-O-Rd.

[0023] While Ra, Rb, and Rcare not particularly limited, Rdis selected from C1-18 alkyl or C1-7 cycloalkyl.

[0024] In one embodiment, Ra, Rb, and Rcare independently selected from H, Ce-uaryl, 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.

[0025] In one embodiment, Raand Rbtaken together, may also form a bicyclic or a tricyclic hydrocarbon moiety.August 29, 2025Biosynth AG 119687P1126PC

[0026] In one embodiment, Raand Rctaken together, may also form a bicyclic or a tricyclic hydrocarbon moiety.

[0027] Enol ethers are also termed as electron-rich alkenes by virtue of the electrondonation from the oxygen atom via n-bonding. It is believed that this property is responsible for good reactivity in cycloaddition reactions such as the [2 + 2] cycloaddition with singlet oxygen1O2.

[0028] In one embodiment, in the NEPO compound of formula II, X in 1 -position and Y in 4-position are electron donating groups, respectively, which are the same or which are different from one another.

[0029] In another embodiment, X in 1 -position is an electron donating group, and Y in 4-position is an electron-withdrawing group such as a carboxylic group.

[0030] In one embodiment, R8is H, C1-4 alkyl, COOH, CH2COOH, CH2CH2COOH or CH=CH-COOH. The carboxylic hydrogen may be substituted with a C1-10 alkyl.

[0031] In one embodiment, R9is H or C1-4 alkyl, preferably H.

[0032] In a preferred embodiment, X in 1 -position is C1-4 alkyl, C1-4 alkoxy, CH2-COOH, (CH2)2-COOH. The carboxylic hydrogen may be substituted with a C1-10 alkyl.

[0033] In a particularly preferred embodiment, X in 1 -position is CH3.

[0034] In one embodiment, Y in 4-position is C1-4 alkyl , C1-4 alkoxy, CH2-COOH, (CH2)2- COOH, or CH2CH(COOH)2. The carboxylic hydrogen may be substituted with a C1-10 alkyl.

[0035] In a particularly preferred embodiment, Y is (CH2)2-COOH. The carboxylic hydrogen may be substituted with a C1-10 alkyl.

[0036] In one embodiment, R8is in 2-position and is selected from H, CH3, CH2-COOH, or (CH2)2-COOH. The carboxylic hydrogen may be substituted with a C1-10 alkyl.August 29, 2025Biosynth AG 119687P1126PC

[0037] In another preferred embodiment, X in 1 -position is an electron donating group, Y in 4-position is a carboxylic group, and R8is H, C1-4 alkyl, or CH=CH-COOH, wherein C1-4 alkyl may be substituted with one or two carboxylic groups, and R9is H. The carboxylic hydrogen may be substituted with a C1-10 alkyl to form an ester.

[0038] In a preferred embodiment, NEPO is selected from one of compounds 4, 5, or

[0039] NEPO compounds 4, 5, and 6 and the synthesis thereof are, e.g., known from Negano et al., Chem. Pharm. Bull., 1994, 42, 883 - 887.

[0040] Said endoperoxides may be synthesized according to known methods through the oxidation by a primary source of singlet oxygen. The term “primary source of singlet oxygen” as used herein means the oxidation with singlet oxygen generated in a photoreactor or generated by hydrogen peroxide.

[0041] According to the invention, said endoperoxides have shown to be useful secondary sources of singlet oxygen. The term “secondary source of singlet oxygen” means that the singlet oxygen is generated from a NEPO compound.

[0042] Further NEPO compounds that are suitable in the method according to the invention may be selected from the following compounds:August 29, 2025Biosynth AG 119687P1126PC

[0043] In the method defined in the first aspect, the use of NEPO 4 as a secondary source of singlet oxygen has been proved beneficial. In one embodiment, the use of NEPO 4 is therefore preferred.

[0044] The NEPO compounds used in the present invention may all be synthesized according to known methods, namely by oxidation of a suitable naphthalene compound with singlet oxygen which is generated by hydrogen peroxide in the presence of sodiumAugust 29, 2025Biosynth AG 119687P1126PC molybdate. Preferably, said sodium molybdate is the hydrate Na2MoO4 x 2H2O, which is commercially available.

[0045] Accordingly, the method according to the invention comprises prior to step (A) step (0):(0) reacting a naphthalene compound of formula II’II’ with hydrogen peroxide in water in the presence of sodium molybdate to yield the compound of formula II, wherein the variables in compound II’ have the same meaning as in compound II.

[0046] The naphthalene compounds of formula II are known or may be prepared according to methods which are known to the person skilled in the art.

[0047] Thus, in a preferred embodiment, the method defined in the first aspect comprises the catalytic disproportionation of hydrogen peroxide into water and singlet oxygen by sodium molybdate upon forming a NEPO compound.

[0048] In a specific embodiment, the invention relates to a method of making a chemiluminescent 1 ,2-dioxetane of formula IIIAugust 29, 2025Biosynth AG 119687P1126PCIII the method comprising reacting an enol ether of formula Iwith a naphthalene endoperoxide (NEPO) of formula IIto yield the 1 ,2-dioxetane of formula III.

[0049] In a preferred embodiment, in the enol ether I and the 1 ,2-dioxetane III, R1is an electron withdrawing group.August 29, 2025Biosynth AG 119687P1126PC

[0050] The term “electron-withdrawing” us as used herein denotes any group which is known to be electron withdrawing.

[0051] In preferred embodiments, R1is selected from -CH=CH-COOH, -CH=CH- COOR, and -CH=CH-CN, wherein R is C1-10 alkyl.

[0052] In a preferred embodiment, in the enol ether I and the 1 ,2-dioxetane III, R2is H or a halogen. Halogen is preferred. Further preferably, the halogen is Cl.

[0053] In a preferred embodiment, in the enol ether I and the 1 ,2-dioxetane III, R3is Ci- 18 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.

[0054] In a preferred embodiment, R3is C1-4 alkyl, further preferably R3is methyl.

[0055] 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.

[0056] In one embodiment, the protecting group PG of the enol ether is a group R4-L.

[0057] In one embodiment, R4is selected from H, C1-4 alkyl, and C6H5-CH2.

[0058] In a preferred embodiment, R4is selected from a glycoside

[0059] Preferably, the glycoside is a glucoside, a mannoside, a galactoside, a fucoside or an arabinoside.August 29, 2025Biosynth AG 119687P1126PC

[0060] In another preferred embodiment, R4is selected from glucuronide

[0061] In another embodiment, R4is a phosphonate P(=O)(OH)2 or an anion thereof.

[0062] In a further embodiment, R4is a sulfonate S(=O)2(OH) or an anion thereof.

[0063] 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.

[0064] In still another embodiment, R4is selected from the boron compoundor an anion thereof.

[0065] In still another embodiment, R4is selected from 4,4,5,5-tetramethyl-1 ,3,2-

[0066] In still other embodiments, R4is selected from 2,4-dinitrobenzenesulfonateAugust 29, 2025Biosynth AG 119687P1126PC, 4-azidobenzyloxy carbonylH / “X ,Pop1— N — a — CHj - 5 and \_ / wherein Pep is a peptide moiety consisting of an amino or a peptide residue linked via a carboxylic group thereof.

[0067] In one embodiment, L is absent.

[0068] In another embodiment, L is a linker. In a preferred embodiment, L is O-(CeH4) or is O-C1-4 alkylene.

[0069] 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.

[0070] The peroxidation preferably is carried out in a solvent 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 III. Suitably, the reaction temperature is from - 10 °C to room temperature (23 °C).

[0071] The inventors of the present invention discovered that the solvent may have a crucial impact on the progress of the reaction. Typically, in solvents such as methanol and acetonitrile the conversion is low, respectively no conversion can be observed. A small conversion can typically be observed in deuterated water and tetrachloromethane. Surprisingly, higher conversion can be observed in dichloromethane and chloroform, whereas the highest conversion can be observed in deuterated chloroform.

[0072] Accordingly, the use of deuterated chloroform as solvent for performing the reaction between NEPO compound II and the enol ether I is preferred.

[0073] The use of CHBrs as solvent is also possible.

[0074] The progress of the reaction may be monitored by analytical HPLC.August 29, 2025Biosynth AG 119687P1126PC

[0075] In preferred embodiments, the enol ethers 7, 9, 11, 13, 15 and the corresponding chemiluminescent 1 ,2-dioxetanes 8, 10, 12, 14, 16 are as follows:August 29, 2025Biosynth AG 119687P1126PCAugust 29, 2025Biosynth AG 119687P1126PC

[0076] According to a second aspect, the invention relates to the use of a naphthalene endoperoxide (NEPO) compound for generating singlet oxygen (1O2) used for the peroxidation of the double bond of an enol ether to make a 1 ,2-dioxetane.

[0077] In a preferred embodiment, the invention relates to the use of a naphthalene endoperoxide (NEPO) compound of formula II for generating singlet oxygen (1O2) used for the peroxidation of the double bond of an enol ether of formula I to make a chemiluminescent 1 ,2-dioxetane of formula III.

[0078] According to a third aspect, the invention relates to a kit comprising a naphthalene endoperoxide (NEPO) of formula II and an enol ether of formula I.

[0079] Improved synthesis of NEPO 4 according to fourth aspect of the invention

[0080] The synthesis of NEPO 4 has been reported by J. M. Aubrey et al., J. Org. Chem. 1989, 54, 726-728 by oxidizing naphthalene compound 3 in aqueous solution with sodium molybdate Na2MoO4 x 2 H2O and hydrogen peroxide in a carbonate buffer. For the work-up, phosphoric acid H3PO4 is used.

[0081] As can be seen from the following Table, Entry 1 , the conversion and yield is acceptable, however, the oxidation of compound 3 is performed in a relatively low concentration using a huge excess of catalyst, and a huge excess of hydrogen peroxide. This is not acceptable under industrial conditions:

[0082] The inventors of the present invention developed a method which allows considerably reducing the amount of catalyst Na2MoO4 x 2 H2O and hydrogen peroxide, as is disclosed by entry 2 in the table of Example 2. This is advantageous in view of industrial requirements, e.g., to keep the amount of waste material as low as possible.

[0083] According to the fourth aspect, the invention discloses a method of making a NEPO compound 4August 29, 2025Biosynth AG 119687P1126PCfrom a compound 3the method comprising the oxidation of compound 3 in water with hydrogen peroxide in the presence of sodium molybdate hydrate Na2MoO4 x 2 H2O, wherein the concentration of the compound 3 is in the range of from 0.05 to 0.15 molar, the concentration of sodium molybdate hydrate Na2MoO4 x 2 H2O in terms of equivalents per one equivalent of compound 3 is in the range of from 0.3 to 0.8, and the concentration of hydrogen peroxide in terms of equivalents per one equivalent of compound 3 is in the range of from 15 to 25.

[0084] The term “sodium molybdate” encompasses hydrates such as Na2MoO4 x 2 H2O.

[0085] The method further comprises a step of adding phosphoric acid to the mixture obtained and isolating the precipitated compound 4.

[0086] In another embodiment, the concentration of the compound 3 is in the range of from 0.08 to 0.13 molar, the concentration of sodium molybdate hydrate Na2MoO4 x 2 H2O in terms of equivalents per one equivalent of compound 3 is in the range of from 0.4August 29, 2025Biosynth AG 119687P1126PC to 0.6, and the concentration of hydrogen peroxide in terms of equivalents per one equivalent of compound 3 is in the range of from 19 to 23.EXAMPLESExample 1Synthesis of probe 10 using NEPO 4

[0087] AquaSpark™ beta-D-galactoside enhanced probe precursor 9 (395 mg) was treated with 3.0 equivalents of NEPO 4 in CDCI3. The heterogeneous reaction mixture was allowed to stir vigorously at 23 °C for 24 h. Full conversion into product 10 was proofed by analytical HPLC. Purification was done by silica gel column chromatography after washing the silica gel with EtOAc / CHsOH 1 :1 and then pure EtOAc in order to remove oily material that silica gel may contain. The solution of the crude material in chloroform was directly loaded onto the silica gel. The elution was performed with EtOAc until NEPO precursor 3 contained in traces in NEPO 4 eluted completely. At this point, the polarity was increased stepwise from EtOAc / CHsOH 9:1 to EtOAc / CHsOH 1 :2. Solvent concentration on a rotary evaporator (heating bath at 18° C), subsequent addition of 2 mL MTBE (methyl t-butyl ether) to the residue to obtain a solid, evaporation of the MTBE and drying at 23 °C in the drying cabinet for 20 h afforded product 10 in quantitative yield of > 99%.August 29, 2025Biosynth AG 119687P1126PC

[0088] 1H-NMR analysis (400 MHz, DMSO-d6; 5 ppm) confirmed full conversion of precursor 9 into probe 10 and was in good accordance with a reference spectrum:

[0089] 1.10 (s, 4 H) 1.17 (t, J = 7.15 Hz, 1 H) 1.27 - 1.38 (m, 1 H) 1.47 (ddd, J = 11.77, 2.29, 1 .22 Hz, 1 H) 1 .52 - 1 .75 (m, 8 H) 1.85 - 1 .95 (m, 2 H) 1 .98 (s, 1 H) 2.29 - 2.33 (m, 1 H) 2.79 - 2.98 (m, 2 H) 3.07 (s, 1 H) 3.40 (br dd, J = 9.54, 3.30 Hz, 2 H) 3.49 (dt, J = 8.96, 4.51 Hz, 2 H) 3.52 - 3.60 (m, 4 H) 3.70 (d, J = 3.18 Hz, 1 H) 4.02 (q, J = 7.09 Hz, 1 H) 4.72 - 4.93 (m, 3 H) 6.64 (d, J = 16.02 Hz, 1 H) 6.99 - 7.08 (m, 2 H) 7.26 - 7.50 (m, 2 H) 7.68 - 7.84 (m, 2 H) 7.92 (d, J = 8.44 Hz, 1 H).Example 2:Oxidation of enol ether 3

[0090] Entry 1 in the below table discloses the initial set of conditions reported by Aubry et al. A huge excess of catalyst and hydrogen peroxide was used. However, this is not acceptable under industrial conditions:

[0091] As summarized in the above table, the amount of catalyst was diminished from 4.4 to 0.5 equivalents. Moreover, the amount of hydrogen peroxide was diminished from 53.3 equivalents reported by Aubry et al. to 21.4 equivalents. The best reaction conditions are listed in entry 2. For this reaction, the reaction time could be reduced by performing the reaction under more concentrated conditions. A yield of 63% was achieved with a conversion of 75%. Analysis by1H-NMR showed 25% precursor 3, which might have not reacted, or the product had decomposed.

Claims

August 29, 2025Biosynth AG 119687P1126PCCLAIMS1 . A method of making a 1 ,2-dioxetane, the method comprising step (A):(A) reacting a naphthalene endo peroxide (NEPO) of formula IIII with the double bond of an enol ether to form the 1 ,2-dioxetane, wherein in formula II:X in 1 -position and Y in 4-position are each electron donating groups and are the same or different from one another, orX in 1 -position is an electron donating group, and Y in 4-position is an electronwithdrawing group, preferably a carboxylic group;R8is H, C1-4 alkyl, COOH, CH2COOH, CH2CH2COOH or CH=CH-COOH, wherein the carboxylic hydrogen may be substituted with a C1-10 alkyl; andR9is H or C1-4 alkyl.

2. The method of claim 1 , wherein X in 1 -position is C1-4 alkyl, C1-4 alkoxy, CH2-COOH, (CH2)2-COOH, preferably CH3, wherein the carboxylic hydrogen may be substituted with a C1-10 alkyl; andY in 4-position is C1-4 alkyl, C1-4 alkoxy, CH2-COOH, (CH2)2-COOH, and CH2CH(COOH)2, preferably (CH2)2-COOH, wherein the carboxylic hydrogen may be substituted with a C1-10 alkyl.August 29, 2025Biosynth AG 119687P1126PC3. The method of any one of the preceding claims, wherein R8is in 2-position and is selected from H, CH3, CH2-COOH, or (CH2)2-COOH; wherein the carboxylic hydrogen may be substituted with a C1-10 alkyl.

4. The method of any one of the preceding claims, wherein the naphthalene endo peroxide (NEPO) is selected from one of the following compounds:August 29, 2025Biosynth AG 119687P1126PC5. The method of any one of the preceding claims, wherein the naphthalene endo peroxide (NEPO) is compound 446. The method of any one of the preceding claims, wherein the enol ether has the general structure RaRbC=CRc-O-Rd, wherein Ra, Rb, and Rcare independently of each selected from H, or unsubstitued or substituted Ce- aryl, C1-18 alkyl and C1-7 cycloalkyl, and Rdis selected from C1-18 alkyl or C1-7 cycloalkyl.

7. The method of claim 6, 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 a unsubstituted or substituted bicyclic or a tricyclic 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-u 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.August 29, 2025Biosynth AG 119687P1126PC8. The method of any one of the preceding claims, comprising prior to step (A) step (0):(0) reacting a naphthalene compound II’with hydrogen peroxide in water in the presence of sodium molybdate to yield the compound of formula II, wherein X, Y, R8, and R9are defined as in any one of claims 1 to 3 or wherein the compound of formula II is one of the compounds of claim 4 or 5.

9. The method of any one of the preceding claims, wherein the 1 ,2-dioxetane is a 1 ,2- dioxetane of formula IIIand wherein the method comprises reacting an enol ether of formula IAugust 29, 2025Biosynth AG 119687P1126PCwith a naphthalene endoperoxide (NEPO) of formula IIII to yield the compound of formula III, wherein in the compound of formula I and formula IIIPG is a protecting group,R1is an electron withdrawing group,R2is H or a halogen, preferably Cl,R3is C1-18 alkyl or C1-7 cycloalkyl, and wherein, in the compound of formula II, X, Y, R8, and R9are defined as in any one of claims 1 to 3 or wherein the compound of formula II is one of the compounds of claim 4 or 5.

10. The method of claim 9, wherein R1is CH=CH-COOH, CH=CH-COOR, or CH=CH- CN, wherein R is C1-8 alkyl.August 29, 2025Biosynth AG 119687P1126PC11 . The method of any one of claims 9 or 10, wherein the protecting group PG of the enol ether of formula I is a group R4-L, wherein R4is selected fromH, C1-4 alkyl, and C6H5-CH2; a glycosidepreferably, the glycoside is a glucoside, a mannoside, a galactoside, 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;August 29, 2025Biosynth AG 119687P1126PCmoiety consisting of an amino or a peptide residue linked via a carboxylic group thereof; andL is absent or is present, and when present is O-(CeH4) or O-C1-4 alkylene.

12. The method of any one of the proceeding claims, wherein the reaction of the enol ether with the naphthalene endo peroxide (NEPO) is carried out in CH2CI2, CHCI3, or CDCI3, preferably in CDCI3.

13. The method of any one of the preceding claims, wherein the enol ether of formula I and 1 ,2-dioxetane of formula III are as follows:August 29, 2025Biosynth AG 119687P1126PCAugust 29, 2025Biosynth AG 119687P1126PC14. Use of a naphthalene endoperoxide (NEPO) for generating singlet oxygen (1O2) for the peroxidation of the double bond of an enol ether to make a 1 ,2-dioxetane as defined in any one of claims 1 to 7, preferably wherein the NEPO compound is of formula II, the enol ether is of formula I, and the 1 ,2-dioxetane is of formula III as defined in any one of claims 9 to 13.

15. A kit comprising a naphthalene endoperoxide (NEPO) and an enol ether as defined in any one of claims 1 to 7, preferably comprising a naphthalene endoperoxide (NEPO) of formula II and an enol ether of formula I as defined in any one of claims 9 to 13.

16. A method of making a naphthalene endoperoxide (NEPO) compound 44 from compound 33, wherein the method comprises the oxidation of compound 3 in water with hydrogen peroxide in the presence of sodium molybdate hydrate Na2MoO4 x 2 H2O, whereinAugust 29, 2025Biosynth AG 119687P1126PC the concentration of the compound 3 is in the range of from 0.05 to 0.15 molar, the concentration of sodium molybdate hydrate Na2MoO4 x 2 H2O in terms of equivalents per one equivalent of compound 3 is in the range of from 0.3 to 0.8, and the concentration of hydrogen peroxide in terms of equivalents per one equivalent of compound 3 is in the range of from 15 to 25, wherein the method optionally comprises the further step of adding phosphoric acid to the obtained mixture obtained and isolating the precipitated compound 4.

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