Stable decarboxylated s-adenosyl-l-methionine analogs and uses thereof

Abstract

The application discloses a stable decarboxylation S-adenosyl-L-methionine analogue and application thereof, and belongs to the field of biological catalytic conversion. The stable decarboxylation S-adenosyl-L-methionine analogue has a structure shown in formula I. The stable decarboxylation S-adenosyl-L-methionine analogue can effectively block intramolecular cyclization reaction of S-adenosyl-L-methionine analogue, has excellent stability, and has better reaction efficiency when being used in hydrocarbylation or cyclase cascade reaction of a receptor substrate.

Description

Technical Field

[0001] This invention relates to the field of biocatalytic conversion technology, and in particular to a stable decarboxylated S-adenosine-L-methionine analog and its applications. Background Technology

[0002] In drug development, various hydrocarbon groups are often introduced to optimize properties such as lipophilicity and metabolic stability, thereby improving drug efficacy. Currently, hydrocarbonation is increasingly becoming one of the important strategies for drug structure modification and transformation.

[0003] In addition, in medicinal chemistry, fluorine atoms or fluorine-containing functional groups are often introduced into drug molecules to selectively prevent oxidative metabolism of drugs by protecting easily oxidized metabolic sites, thereby improving the metabolic stability of drug molecules and prolonging the duration of action of drugs in vivo. The introduction of fluorine atoms or fluorine-containing functional groups can also alter the lipophilicity of drug molecules, enhancing drug absorption and distribution in target tissues, and thus improving drug bioavailability. Furthermore, the introduction of fluorine atoms or fluorine-containing functional groups can affect the spatial conformation of drug molecules, improving the selectivity of compounds for target proteins and enhancing the bonding ability between proteins and ligands. Therefore, the research and development of various compounds containing fluorine atoms or fluorine-containing functional groups is of great value, and the selective introduction of fluorine atoms or fluorine-containing functional groups into specific positions in compound molecules is currently the key to the preparation of fluorine-containing drugs and fluorine-containing active compounds.

[0004] S-adenosyl-L-methionine (SAM) is an important methylating agent in living organisms. It facilitates various methyl transfer reactions within the body through a variety of methyltransferases (MTases). The specific catalytic mechanism involves methyltransferases acting via typical SAM... N The mechanism involves transferring methyl groups from SAM molecules to acceptor substrates such as proteins, nucleic acids, and small metabolic molecules, generating S-adenosyl homocysteine ​​(SAH) as a byproduct. In recent years, leveraging the versatility and efficiency of SAM and methyltransferases, numerous SAM analogs replacing S-methyl substituents have been developed as biomolecular tools for transferring various hydrocarbon functional groups. Many methyltransferases have also been found to possess strong versatility, enabling the transfer of multiple functional groups from SAM analogs to substrate molecules, generating corresponding alkylated products. Related research on this technical approach has been reported.

[0005]

[0006] Patent WO2013029075A1 discloses a method for transferring alkyl or alkenyl groups with 1-10 carbon atoms to small molecule compounds having a nucleophilic center, the synthesized products including alkylated thioonium salts or sulfonium oxyonium salts. Its claims also include the use of SAM-dependent methyltransferases to catalyze the transfer of alkyl or alkenyl groups from ononium salts to the active substrate.

[0007] Patent 201510155209.X discloses a method for the biocatalytic alkylation of catechol compounds. The method utilizes various SAM analogs as alkyl donors to achieve the alkylation reaction of catechol compounds under the catalysis of catechol methyltransferase. Furthermore, the method allows for the recycling and regeneration of the alkylation donors by adding a solid acid catalyst.

[0008] Patent WO2020053196A1 also discloses a method for biocatalytic alkylation, which includes two cyclic alkylation steps: the first step uses S-methyltransferase to transfer alkyl groups from an alkyl donor to a sulfur- or selenium-containing support compound to generate an alkylated sulfur-selenium support compound; the second step uses N, C, O, S, or P methyltransferase to transfer selectively substituted alkyl groups from the alkylated support compound to the substrate, thereby generating an alkylated product and a dealkylated support compound. The generated dealkylated support compound can be used in the next cycle of step one to regenerate the alkylated support compound.

[0009] Although the use of SAM analogs as hydrocarbon donors in enzyme catalysis research has been widely welcomed, these reported SAM analogs still have certain drawbacks. For example, the thionium structure of SAM and its analogs makes them inherently chemically unstable. Under physiological conditions (e.g., T = 37℃, pH 7.5), SAM and its analogs readily undergo intramolecular cyclization reactions to generate 5′-deoxy-5′-methionine (MTA) and L-homoserine lactone (this is the main degradation pathway of SAM and its analogs), or undergo depurination to generate adenine and S-ribosemethionine. G et al. studied the stability of various hydrocarbon-substituted SAM analogs and found that under physiological conditions of pH 7.5, the half-lives of these SAM analogs were all within the range of 3 min to 5 h. G, et al. ACS Chem. Biol. 2013, 8, 1134–1139).

[0010]

[0011] Seebeck et al. first observed the formation of a suspected intermediate fluoromethyl SAM analog (F-SAM) using a cyclic enzyme cascade reaction (Seebeck FP, et al. Angew. Chem. Int. Ed. 2021, 60, 27178-27183). However, due to the extreme instability of F-SAM, the experiment failed to characterize and identify it. Based on the identification of the main decomposition products, it is inferred that the decomposition of F-SAM still mainly proceeds along the direction of producing 5′-deoxy-5′-fluoromethylthioadenosine (F-MTA). Simultaneously, the experiment found that the stability of the decomposed F-MTA remains extremely poor, continuing to degrade rapidly over time. Therefore, it can be inferred that in addition to the thionium structure described above, the strong electron-withdrawing property of the fluorine atom increases the instability of the three CS bonds connected to the sulfur atom, further promoting the decomposition of F-SAM. This may also be a common problem among SAM analogs containing fluorine functional groups. Booker et al. found that FMeTeSAM with a Te atom at its thionium center exhibits stronger stability than F-SAM and can be directly used as a fluoromethyl donor to achieve enzymatic fluoromethyl transfer reactions (Booker SJ, et al. ACS Cent. Sci. 2023, 9, 905–914). However, FMeTeSAM can currently only be synthesized chemically, and the synthesis steps are too lengthy and cumbersome, adding complexity to the experiments.

[0012] Therefore, it is crucial to research and develop new methods to address the instability issues currently faced by SAM analogs. Summary of the Invention

[0013] In view of this, the technical problem to be solved by the present invention is to provide a stable decarboxylated S-adenosyl-L-methionine analog and its application. The stable decarboxylated S-adenosyl-L-methionine analog exhibits good stability and better reactivity when used for the alkylation of acceptor substrates or in cyclic enzyme cascade reactions.

[0014] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0015] This invention provides a stable decarboxylated S-adenosine-L-methionine analog (dcSAM analog) having the structure shown in Formula I:

[0016]

[0017]

[0018] Preferably, M is selected from sulfur or selenium;

[0019] Preferably, R is selected from substituted or unsubstituted C1 to C2.10 Saturated or unsaturated straight-chain or branched hydrocarbon groups, substituted or unsubstituted C1-C1 heteroatoms containing N, O, or S heteroatoms. 10 Saturated or unsaturated straight-chain or branched hydrocarbon groups, or

[0020] R1 is selected from substituted or unsubstituted C1 to C1. 10 Saturated or unsaturated straight-chain or branched hydrocarbon groups.

[0021] Preferably, the substitution includes fluorine substitution.

[0022] Fluorine atom substitution can significantly improve the physicochemical properties and pharmacokinetics of drug molecules, thereby enhancing drug efficacy.

[0023] The above C1~C 10 Saturated or unsaturated straight-chain or branched hydrocarbon groups are preferred, specifically C1 to C2. 10 Straight-chain or branched alkyl groups, C2-C 10 Straight-chain or branched olefinic groups, C2-C 10 Straight-chain or branched alkyne group.

[0024] The C1~C 10 The straight-chain or branched alkyl group is preferably a C1-C6 straight-chain or branched alkyl group, including but not limited to methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, etc.

[0025] The C2~C 10 The straight-chain or branched olefinic group is preferably a C2-C4 straight-chain or branched olefinic group, including but not limited to vinyl, propenyl, allyl, 1-butenyl, cis-2-butenyl, trans-2-butenyl, 2-methyl-1-propenyl, etc.

[0026] The C2~C 10 The straight-chain or branched alkyne group is preferably a C2-C4 straight-chain or branched alkyne group, including but not limited to ethynyl, propynyl, 1-butynyl, 2-butynyl, 3-methyl-2-propynyl, etc.

[0027] Preferably, the C1-C1 heteroatoms containing N, O, or S heteroatoms are substituted or unsubstituted. 10 Saturated or unsaturated straight-chain or branched hydrocarbon groups include, but are not limited to, amine groups, azide groups, ketone groups, ester groups, amide groups, carboxylic acid groups, ether groups, or thioether groups.

[0028] Preferably, in this invention, M is selected from sulfur.

[0029] Preferably, R is selected from substituted or unsubstituted C1 to C2. 10Saturated or unsaturated straight-chain or branched hydrocarbon groups, or substituted or unsubstituted C1-C2 groups containing N, O, or S heteroatoms. 10 Saturated or unsaturated straight-chain or branched hydrocarbon groups.

[0030] The C1~C 10 The preferred range of saturated or unsaturated straight-chain or branched hydrocarbon groups is the same as above, and will not be repeated here.

[0031] Preferably, in this invention, C1 to C 10 The substituents of the saturated or unsaturated straight-chain or branched hydrocarbon groups are selected from halogens, amino groups, nitro groups, hydroxyl groups, carboxyl groups, ester groups, acyl groups, and C3-C6 groups. 10 One or more of aryl and heteroaryl, C3 to C9 cycloalkyl groups.

[0032] The halogen is preferably a fluorine atom.

[0033] The C3~C 10 The aryl group is preferably C6-C6. 10 Aryl groups, including but not limited to phenyl, benzyl, and naphthyl groups.

[0034] The C3~C 10 Heteroaryl groups include, but are not limited to, thiazolyl, thiophene, and quinolinyl groups.

[0035] The C3-C9 cycloalkyl group is preferably a C3-C6 cycloalkyl group, specifically including but not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, methylcyclopropyl, etc.

[0036] Preferably, in this invention, the substituted C1 to C 10 Saturated or unsaturated straight-chain or branched hydrocarbon groups are C1-C6 substituents containing one or more F atoms. 10 Saturated or unsaturated straight-chain or branched hydrocarbon groups.

[0037] Preferably, R is selected from -CH2F, -CHF2, -CF3, -CH2CH2F, -CH2CHF2, -CH2CF3, -CH2CH2CH2F, -CH2CH2CHF2, -CH2CH2CF3, 3-chloroallyl, 3-fluoroallyl, 3-aminoallyl, -CH2OCH3, -CH2OC4H9, -CH2OH, or -CH2COOH.

[0038] The "-" above indicates the connection position.

[0039] Preferably, the stable decarboxylated S-adenosine-L-methionine analogue of the present invention has any of the following structures:

[0040]

[0041] For SAM analogs containing fluorine functional groups, fluorine atoms exacerbate the decomposition of SAM analogs. This invention obtains stable decarboxylated S-adenosine-L-methionine acid analogs through decarboxylation, blocking the main pathway of unstable decomposition.

[0042] The present invention conducted stability tests on the above-mentioned stable decarboxylated S-adenosine-L-methionine analog (F-dcSAM) containing fluoromethyl groups and the S-adenosine-L-methionine analog (F-SAM) containing fluoromethyl groups. The results showed that F-dcSAM has better stability.

[0043] The stable decarboxylated S-adenosine-L-methionine analogues of the present invention can be prepared by chemical or enzymatic methods, with readily available raw materials, mild conditions, and simple operation.

[0044] The specific reaction formula for the chemical method can be:

[0045] Chemical Method 1

[0046]

[0047] Chemical Method 2

[0048]

[0049] The reaction formula for the enzymatic method can be specifically as follows:

[0050] Enzymatic method 1

[0051]

[0052] Enzymatic method 2

[0053]

[0054] Enzymatic method 3

[0055]

[0056] The aforementioned stable decarboxylated S-adenosine-L-methionine analogues significantly improve stability by directly blocking the main decomposition pathway of intramolecular cyclization of S-adenosine-L-methionine analogues to produce MTA and L-homoserine lactone through decarboxylation.

[0057] The present invention also provides the application of the above-mentioned stable decarboxylated S-adenosine-L-methionine analogue as a alkylation donor for an acceptor substrate.

[0058] The receptor substrate is preferably a biomolecule.

[0059] Preferably, the biomolecules include, but are not limited to, proteins, nucleic acids, or small metabolic molecules.

[0060] In a preferred embodiment of the present invention, the hydrocarbon group in a stable decarboxylated S-adenosine-L-methionine analog is transferred to a biomolecule via an enzymatic alkylation reaction, thereby completing the structural modification and property alteration of the biomolecule.

[0061] The present invention also provides a method for enzymatic alkylation using the above-mentioned stable decarboxylated S-adenosine-L-methionine analogue, comprising the following steps:

[0062] Under the action of methyltransferase, the stable decarboxylated S-adenosine-L-methionine analog shown in Formula I undergoes a nucleophilic substitution reaction with the receptor substrate to obtain the alkylated receptor product structure.

[0063]

[0064] Stable decarboxylated S-adenosine-L-methionine analogues can improve the reaction efficiency of receptor substrate alkylation, and more efficiently prepare receptor products containing hydrocarbon groups.

[0065] In the above-mentioned enzymatic alkylation method, the methyltransferase is selected from methyltransferases whose binding center is N, C, O, S or P.

[0066] The present invention also provides the above-mentioned stable decarboxylation S-adenosine-L-methionine analog cyclic enzyme cascade reaction, comprising the following steps:

[0067] 1) A stable decarboxylated S-adenosine homocysteine ​​analog (dcSAH analog) is generated by reacting an R-containing compound with a halogenated methyltransferase-catalyzed decarboxylated S-adenosine-L-methionine analog (dcSAM analog).

[0068] 2) Under the action of methyltransferase, the stable decarboxylated S-adenosine-L-methionine analog undergoes a nucleophilic substitution reaction with the receptor substrate to prepare the alkylated receptor product and the decarboxylated S-adenosine homocysteine ​​analog.

[0069] 3) The decarboxylated S-adenosine homocysteine ​​analog obtained in step 2) is used in the next cycle of step 1) to prepare a stable decarboxylated S-adenosine-L-methionine analog again.

[0070] The R in the R-containing compound is the same as the substituent R in the above-mentioned stable decarboxylated S-adenosine-L-methionine analog.

[0071]

[0072] In the above-mentioned cyclic enzyme cascade reaction, dcSAH and dcSAM analogs can be prepared in a cyclic manner, which greatly reduces production costs, improves production efficiency, and is conducive to large-scale production.

[0073] Preferably, in step 1), the molecular formula of the R-containing compound is RX.

[0074] Preferably, the X in RX is selected from halogen, p-methylbenzenesulfonyl, or methanesulfonyl.

[0075] The above-mentioned cyclic enzyme cascade reaction achieves the recycling and regeneration of dcSAM analogs and a more efficient alkylation reaction in the presence of a catalytic amount of the starting material dcSAH analog.

[0076] Compared with existing technologies, the stable decarboxylated S-adenosyl-L-methionine analog provided by this invention has the structure shown in Formula I. This stable decarboxylated S-adenosyl-L-methionine analog can effectively block intramolecular cyclization reactions of S-adenosyl-L-methionine analogs, exhibits excellent stability, and demonstrates better reaction efficiency when used for the alkylation of acceptor substrates or in cyclase cascade reactions. Attached Figure Description

[0077] Figure 1 The following is a stability test diagram of F-dcSAM in Example 2: A) shows the stability test of F-dcSAM at pH 1.0; B) shows the stability test of F-dcSAM at pH 8.0.

[0078] Figure 2 The following are the stability test results for F-SAM in Comparative Example 1: A) Stability test result for F-SAM at pH 1.0; B) Stability test result for F-SAM at pH 8.0; C) LC-MS analysis results for F-SAM decomposition products. Detailed Implementation

[0079] To further illustrate the present invention, the stable decarboxylated S-adenosine-L-methionine analogues provided by the present invention and their applications are described in detail below with reference to embodiments.

[0080] In a preferred embodiment, the halogen methyltransferase is preferably AclHMT, and the aclhmt gene is derived from the species *Aspergillus clavatus*. The aclhmt gene was synthesized by a biotechnology company and, after codon optimization at BL21(DE3), constructed into a pET28a(+) vector carrying a Kanamycin resistance gene, ultimately yielding the pET28a(+)-AclHMT plasmid. The expression and purification of AclHMT were performed according to the literature (Hammer SC, et al. Angew. Chem. Int. Ed. 2021, 60, 5554–5560).

[0081] Example 1

[0082] 1) Synthesis of dcSAM:

[0083] 5′-Deoxy-5′-methylthioadenosine (MTA) (1 mg, 3.36 μmol) was dissolved in 100 μL of formic acid and 100 μL of acetic acid at 0 °C. After complete dissolution, tert-butyl 3-iodopropylcarbamate (4.8 mg, 16.9 μmol) was added to the reaction solution at 0 °C, followed by the slow dropwise addition of AgClO4 (3 mg, 26.4 μmol). Both tert-butyl 3-iodopropylcarbamate and AgClO4 solids were dissolved in 50 μL of formic acid and 50 μL of acetic acid solution beforehand. The reaction flask was sealed with a rubber stopper and a nitrogen (N2)-filled balloon was inserted. The reaction was carried out at 0 °C in the dark for 20 min, then transferred to room temperature and the reaction was continued for 5 h. After the reaction was completed, the reaction was quenched with 1 mL of cold water, and the reactants were filtered to remove AgI precipitate. 1 mL of trifluoroacetic acid (TFA) was added to the filtrate, and the reaction was stopped after stirring at room temperature for 30 min. The solvent in the reaction solution was lyophilized and evaporated using a vacuum freeze dryer. The lyophilized solid was dissolved in 2 mL of H₂O and purified using preparative HPLC with a reversed-phase C18 column (250 mm × 4.6 mm, 5 μm). Mobile phase A was H₂O + 1‰ TFA, and mobile phase B was CH₃CN + 1‰ TFA. Gradient elution was used at a flow rate of 3 mL / min and an injection volume of 5 mL. Detection was performed at dual wavelengths of 254 nm and 215 nm. The elution program was: 0–6 min 0% B, 6–30 min 0–48% B, 30–33 min 48–90% B, 33–39 min 90% B, 39–39.01 min 90%–0% B, 39.01–45 min 0% B. The purified sample was lyophilized under vacuum to obtain a white powder (yield 30%). Target product 1 H NMR(600MHz,D2O)δ8.27(s,1H),8.26(s,1H),6.02(d,1H),4.77–-4.69(m,1H),4.50–-4.36(m,2H),3.87–3.72(m,2H),3.42 -3.34(m,1H),3.33–-3.25(m,1H),2.95(dt,J=18.9,7.7Hz,2H),2.83(s,s,3H),2.05(p,J=7.6Hz,2H).LCMS(ESI)calcd.for C 14 H 23 N6O3S[M] + 355.1547,obsd.355.1425.

[0084] The reaction formula is as follows:

[0085]

[0086] 2) NNMT-catalyzed methylation of dcSAM:

[0087] The nnmt gene originates from *Homo sapiens* (Human). The nnmt gene was synthesized by a biotechnology company and, after codon optimization (BL21(DE3)), constructed into the pET28a(+) vector carrying the Kanamycin resistance gene, ultimately yielding the pET28a(+)-NNMT plasmid. NNMT expression and purification were performed according to the literature described in (Cravatt BF, et al. *Nat Chem Biol* 2013, 9, 300–306).

[0088] The purified nicotinamide N-methyltransferase NNMT (final concentration 100 μM), dcSAM (final concentration 3.0 mM), and nicotinamide (final concentration 1.0 mM) were added to 100 μL of a solution containing 100 mM Tris-HCl (pH 8.0) and reacted at 30 °C for 12 h. After the reaction, an equal volume (100 μL) of 10% TFA was added to the reaction solution to quench the reaction. The quenched reaction sample was centrifuged at 4 °C and 12,000 rpm for 30 min using a high-speed refrigerated centrifuge. The supernatant was then analyzed by analytical HPLC using a reversed-phase C18 column (150 mm × 4.6 mm, 2.5 μm). Mobile phase A was H2O + 1‰ TFA, and mobile phase B was CH3CN + 1‰ TFA. Gradient elution was used at a flow rate of 1 mL / min and an injection volume of 50 μL. Detection was performed using dual wavelengths of 254 nm and 215 nm. The elution program was: 0-4 min 0% B, 4-20 min 0-48% B, 20-21 min 48-90% B, 21-23 min 90% B, 23-23.01 min 90-0% B, 23.01-28 min 0% B. The same reaction system was scaled up to prepare the target product, with a yield of 12%. 1 H NMR(600MHz,D2O)δ9.79(s,1H),9.46(d,1H),9.17(d,1H),8.76(s,1H),8.41(t,1H),8.23(s,1H),4.44(s,3H).LCMS(ESI)calcd.for C7H9N2O + [M] + 137.0766,obsd.137.0709.

[0089] The reaction formula is as follows:

[0090]

[0091] 3) The HMT and NNMT cascade catalyze the cyclic enzyme methylation reaction of dcSAH and CH3I:

[0092] HMT (final concentration 50 μM), NNMT (final concentration 50 μM), dcSAH (final concentration 200 μM), CH3I (final concentration 100 mM), and nicotinamide (final concentration 1.0 mM) were added to 100 μL of a solution containing 100 mM Tris-HCl (pH 8.0) and reacted at 30 °C for 1 h. After the reaction was completed, an equal volume (100 μL) of 10% TFA was added to the reaction solution to quench the reaction. The post-reaction processing and HPLC analysis methods were the same as in step 2) above.

[0093] Example 2

[0094] 1) Synthesis of fluorinated methyl decarboxylated SAM analogs (F-dcSAM):

[0095] ① Synthesis of dcSAH: The synthesis of dcSAH followed the method described in the literature (Anglin J, et al. J. Med. Chem. 2012, 55, 8066–8074). ② Synthesis of F-dcSAM analogs: dcSAH (4.5 mg, 13.2 μmol) was dissolved in 100 μL of formic acid and 100 μL of acetic acid at 0 °C. After thorough stirring until dissolved, CH2FI (5 μL, 66 μmol) was added at 0 °C, followed by the slow addition of AgClO4 (11 mg, 26.4 μmol). The reaction was carried out under N2 protection at 0 °C in the dark for 20 min, and then continued at room temperature for 5 h. After the reaction was completed, 10 mL of cold water was added to quench the reaction, and the reaction mixture was filtered to remove the silver iodide precipitate. The filtrate was freeze-dried under vacuum and dissolved in 2 mL of H2O. Purification was performed using preparative HPLC with a reversed-phase C18 column (SHIMADZU C18, 250 mm × 4.6 mm, 5 μm). Mobile phase A was H2O + 1‰ TFA, and mobile phase B was CH3CN + 1‰ TFA. Gradient elution was used at a flow rate of 3 mL / min and an injection volume of 5 mL. Detection was performed at dual wavelengths of 254 nm and 215 nm. The elution program was: 0-6 min 0% B, 6-30 min 0-48% B, 30-33 min 48-90% B, 33-39 min 90% B, 39-39.01 min 90%-0% B, 39.01-45 min 0% B. The purified sample was freeze-dried under vacuum to obtain a white powder (30% yield). Target compound. 1H NMR(600MHz,D2O)δ8.48–8.43(m,2H),6.24–6.20(m,1H),6.20–5.99(m,2H),4.89–-4.83(m,1H),4.69–-4.5 9(m,2H),4.25–-4.16(m,1H),4.12–3.98(m,1H),3.72–-3.68(m,1H),3.66–-3.52(m,1H),3.22–3.10(dt,J=6 Hz, 2H), 2.33–-2.24 (h, J = 8.3, 7.9Hz, 2H). 19 F NMR(565MHz,D2O)δ-215.59(t,J=45.7Hz,0.4F),-216.13(t,J=45.6Hz,0.6F).LCMS(ESI)calcd.for C 14 H 22 FN6O3S + [M] + 373.1295,obsd.373.1453.

[0096] The reaction formula is as follows:

[0097]

[0098] 2) DnrK-catalyzed fluoromethylation of F-dcSAM:

[0099] The dnrk gene originates from *Streptomyces peucetius*. The dnrk gene was synthesized by a biotechnology company and, after codon optimization with BL21(DE3), constructed into the pET28a(+) vector carrying the Kanamycin resistance gene, ultimately yielding the pET28a(+)-DnrK plasmid. The expression and purification of DnrK were performed according to the literature (Thorson JS, et al. ACSChem. Biol. 2016, 11, 2484–2491).

[0100] The purified erythromycin 4-O-methyltransferase DnrK (final concentration 100 μM), F-dcSAM (final concentration 3.0 mM), and erythromycin (final concentration 1.0 mM) were added to 100 L of a solution containing 100 mM Tris-HCl (pH 8.0) and reacted at 30 °C for 1 h. After the reaction was completed, an equal volume (100 μL) of methanol was added to the reaction solution to quench the reaction. The quenched reaction sample was centrifuged at 4 °C and 12,000 rpm for 30 min using a high-speed refrigerated centrifuge. The supernatant was then analyzed by analytical HPLC using a reversed-phase C18 column SHIMADZ UC18 (150 mm × 4.6 mm, 2.5 μm). Mobile phase A was H2O + 1‰ TFA, and mobile phase B was CH3CN + 1‰ TFA. The elution method was gradient elution at a flow rate of 1 mL / min and an injection volume of 50 μL. Detection was performed using dual wavelengths of 254 nm and 215 nm. The elution program was as follows: 0-2 min 0% B, 2-11 min 0-14% B, 11-15 min 14-46% B, 15-25 min 46-53% B, 25-25.01 min 53%-95% B, 25.01-27 min 95-0% B, 27.01-31 min 0% B. The reaction was scaled up using the same system to prepare the target product, with a yield of 80%. 1 H NMR(600MHz,D2O)δ7.60(t,J=7.8Hz,1H),7.50–7.34(m,2H),5.80(d,J=54Hz, 2H),5.39(s,1H),4.70(s,1H),4.21(q,J=6.3Hz,1H),3.79(s,1H),3.70–3.64 (m,1H),2.80(d,J=17.8Hz,1H),2.63(d,J=17.8Hz,1H),2.39(s,3H),2.17(d, J=14.4Hz,1H),2.08–2.00(m,1H),2.00–-1.89(m,2H),1.25(d,J=6.5Hz,3H). 19 F NMR(565MHz,D2O)δ-152.39(t,J=55.1Hz,1F).LCMS(ESI)calcd.For C 27 H 28 FNO 10 [M+H] 1+ 546.1731,obsd.546.1770.

[0101] The reaction formula is as follows:

[0102]

[0103] 3) TPMT-catalyzed fluoromethylation of F-dcSAM:

[0104] The tpmt gene originates from the species *Homo sapiens* (Human). The tpmt gene was synthesized by a biotechnology company and, after codon optimization with BL21(DE3), constructed into the pET28a(+) vector carrying the Kanamycin resistance gene, ultimately yielding the pET28a(+)-TPMT plasmid. TPMT expression and purification were performed according to the literature (Zhou Z S, et al. J. Am. Chem. Soc. 2010, 132, 3642–3643).

[0105] The purified mercaptopurine S-methyltransferase TPMT (final concentration 100 μM), F-dcSAM (final concentration 3.0 mM), mercaptopurine (final concentration 1.0 mM), and TCEP (final concentration 2.0 mM) were added to 100 μL of a solution containing 100 mM PBS (pH 6.0) and reacted at 30 °C for 12 h. After the reaction, an equal volume (100 μL) of 10% TFA was added to the reaction solution to quench the reaction. The quenched reaction sample was centrifuged at 4 °C and 12,000 rpm for 30 min using a high-speed refrigerated centrifuge. The supernatant was then analyzed by analytical HPLC using a reversed-phase C18 column (150 mm × 4.6 mm, 2.5 μm). Mobile phase A was H2O + 1‰ TFA, and mobile phase B was CH3CN + 1‰ TFA. The elution method was gradient elution at a flow rate of 1 mL / min and an injection volume of 50 μL. Detection was performed using dual wavelengths of 254 nm and 215 nm. The elution program was: 0–4 min 0% B, 4–20 min 0–48% B, 20–21 min 48–90% B, 21–23 min 90% B, 23–23.01 min 90–0% B, and 23.01–28 min 0% B. The same reaction system was scaled up to prepare the target product, with a yield of 80%. 1 H NMR (600MHz, DMSO-d6) δ8.78 (s, 1H), 8.55 (s, 1H), 6.43 (d, J = 48Hz, 2H). 19 F NMR(565MHz,DMSO-d6)δ-188.89(t,J=55.1Hz,1F).LCMS(ESI)calcd.forC6H5FN4S[M+H] 1+ 185.0240,obsd.185.0292.

[0106] The reaction formula is as follows:

[0107]

[0108] 4) The HMT and DnrK cascade catalyzes the cyclic enzyme methylation reaction of dcSAH and CH2FI:

[0109] HMT (final concentration 50 μM), DnrK (final concentration 50 μM), dcSAH (final concentration 200 μM), CH2FI (final concentration 100 mM), and erythromycin (final concentration 1.0 mM) were added to 100 μL of a solution containing 100 mM Tris-HCl (pH 8.0) and reacted at 30 °C for 1 h. After the reaction was completed, an equal volume (100 μL) of methanol was added to the reaction solution to quench the reaction. The post-reaction processing and HPLC analysis methods were the same as described in step 2).

[0110] 5) The HMT and TPMT cascade catalyzes the cyclic enzyme methylation reaction of dcSAH and CH2FI:

[0111] HMT (final concentration 50 μM), TPMT (final concentration 50 μM), dcSAH (final concentration 200 μM), CH2FI (final concentration 100 mM), mercaptopurine (final concentration 1.0 mM), and TCEP (final concentration 2.0 mM) were added to 100 μL of a solution containing 100 mM PBS (pH 6.0) and reacted at 30 °C for 12 h. After the reaction, an equal volume (100 μL) of 10% TFA was added to the reaction solution to quench the reaction. The post-reaction processing and HPLC analysis methods were the same as described in step 3).

[0112] Figure 1 The following is a stability test diagram of F-dcSAM in Example 2: A) shows the stability test of F-dcSAM at pH 1.0; B) shows the stability test of F-dcSAM at pH 8.0.

[0113] Comparative Example 1

[0114] The synthesis of F-SAM is consistent with the synthesis method of F-dcSAM in Example 2. The starting material dcSAH is replaced with commercially available S-adenosyl homocysteine ​​(SAH), and the amount used is 5 mg (13.2 μmol). The structural formula of F-SAM is as follows:

[0115]

[0116] Figure 2 The following are the stability test results for F-SAM in Comparative Example 1: A) Stability test result for F-SAM at pH 1.0; B) Stability test result for F-SAM at pH 8.0; C) LC-MS analysis results for F-SAM decomposition products.

[0117] Stability tests on the fluoromethyl SAM analogs F-SAM and F-dcSAM showed that F-dcSAM exhibited superior stability compared to F-SAM at both pH 1.0 and pH 8.0. Specifically, F-SAM showed only 35% degradation after 30 minutes at pH 8.0, and almost complete degradation after 120 minutes. In contrast, F-dcSAM only decomposed to produce approximately 10% adenine after 5.7 hours at pH 8.0, demonstrating better stability.

[0118] In summary, F-dcSAM synthesized via decarboxylation exhibits significantly greater stability compared to F-SAM. Therefore, removing the carboxyl group from SAM and its analogues yields stable decarboxylated S-adenosyl-L-methionine analogues, inhibiting the decomposition pathway of SAM and its analogues to produce MTA or MTA analogues, thus greatly improving stability.

[0119] The above description of the embodiments is only for the purpose of helping to understand the method and core ideas of the present invention. It should be noted that those skilled in the art can make several improvements and modifications to the present invention without departing from the principles of the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims

1. A stable decarboxylated S-adenosyl-1-methionine analog, characterized in that, It has the structure shown in Equation I: Formula I; M is selected from sulfur or selenium; R is selected from -CH2F.

2. The stable decarboxylated S-adenosyl-1-methionine analogue according to claim 1, characterized in that, The stable decarboxylated S-adenosine-1-methionine analogue has the following structure: 。 3. The use of the stable decarboxylated S-adenosine-1-methionine analogue according to any one of claims 1 to 2 in the preparation of alkylated donors of acceptor substrates.

4. A method for enzymatic alkylation for non-diagnostic and / or therapeutic purposes using the stable decarboxylated S-adenosyl-1-methionine analogue according to claim 1, characterized in that, Includes the following steps: Under the action of methyltransferase, the stable decarboxylated S-adenosine-1-methionine analog shown in Formula I undergoes a nucleophilic substitution reaction with the receptor substrate to obtain the -CH2F alkylated receptor product structure. Formula I; M is selected from sulfur or selenium; R is selected from -CH2F.

5. The enzymatic alkylation method according to claim 4, characterized in that, The methyltransferase is selected from methyltransferases whose binding center is N, C, O, S or P.

6. The non-diagnostic and / or therapeutic cyclic enzyme cascade of the stable decarboxylated S-adenosyl-1-methionine analog according to any one of claims 1 to 2, characterized in that, Includes the following steps: 1) Stable decarboxylated S-adenosine homocysteine ​​analogues are generated by reacting with R-containing compounds via halogen methyltransferase catalysis. 2) Under the action of methyltransferase, the stable decarboxylated S-adenosine-1-methionine analog undergoes a nucleophilic substitution reaction with the receptor substrate to prepare the -CH2F alkylated receptor product and the decarboxylated S-adenosine homocysteine ​​analog. 3) The decarboxylated S-adenosine homocysteine ​​analog obtained in step 2) is used in the next cycle of step 1) to prepare a stable decarboxylated S-adenosine-1-methionine analog again; The definition of R in the R-containing compound is the same as in claim 4; The structure of the decarboxylated S-adenosine homocysteine ​​analog is as follows: M is selected from sulfur or selenium.

7. The cyclic enzyme cascade reaction according to claim 6, characterized in that, The molecular formula of the R-containing compound in step 1) is RX; The X in RX is selected from halogen, p-methylbenzenesulfonyl, or methanesulfonyl.

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