CN117164655A - Stable decarboxylated S-adenosine-L-methionine analogue and application thereof - Google Patents

Abstract

The invention discloses a stable decarboxylated S-adenosyl-L-methionine analog and its application, and belongs to the field of biocatalytic conversion. The stable decarboxylated S-adenosyl-L-methionine analog has the structure shown in Formula I. The stable decarboxylated S-adenosyl-L-methionine analog can effectively block the intramolecular cyclization reaction of the S-adenosyl-L-methionine analog, has excellent stability, and is used for receptors It has better reaction efficiency when hydrocarbylating the substrate or cyclase cascade reaction.

Description

A stable decarboxylated S-adenosyl-L-methionine analogue and its application

Technical field

The present invention relates to the technical field of biocatalytic conversion, and in particular to a stable decarboxylated S-adenosyl-L-methionine analog and its application.

Background technique

In the process of drug development, various hydrocarbon groups are often introduced to optimize the lipophilicity, metabolic stability and other properties of the drug, thereby improving the drug efficacy. Currently, hydrocarbylation is increasingly becoming one of the important strategies for structural modification and transformation of drugs.

In addition, in medicinal chemistry, fluorine atoms or fluorine-containing functional groups are often introduced into drug molecules to selectively prevent the oxidative metabolism of the drug by protecting the easily oxidized metabolism sites, improve the metabolic stability of the drug molecule, and prolong the drug's shelf life. The action time in the body; the introduction of fluorine atoms or fluorine-containing functional groups can also change the lipophilicity of the drug molecules, enhance the absorption and distribution of the drug in the target tissue, and thereby improve the bioavailability of the drug; the introduction of fluorine atoms or fluorine-containing functional groups can also By affecting the spatial conformation of drug molecules, the selectivity of compounds to target proteins is improved, and the mutual bonding ability of protein-ligands is improved. Therefore, it is of great value to research and develop various compounds containing fluorine atoms or fluorine-containing functional groups, and selectively introducing 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 activities. The key to the compound.

S-adenosyl-L-methionine (SAM) is an important methylation reagent in life. It realizes various methylation reagents in life through various methyltransferases (MTase). Methyl-like transfer reaction, the specific catalytic mechanism is that methyltransferase transfers methyl groups from SAM molecules to acceptor substrates such as proteins, nucleic acids, and metabolic small molecules through a typical S _N 2 mechanism, and generates S-adenosine isotype Cysteine (SAH) as a by-product. In recent years, taking advantage of the versatility and high efficiency of SAM and methyltransferases, a large number of SAM analogs replacing S-methyl substituents have been developed as biomolecular tools for the transfer of various hydrocarbon functional groups. Many methyltransferases have also been found to be highly versatile and can transfer a variety of functional groups from SAM analogs to substrate molecules and generate corresponding alkylation products. Relevant research has been reported on technical solutions in this area.

Patent WO2013029075A1 discloses a method of transferring an alkyl or alkenyl group of 1 to 10 carbon atoms to a small molecule compound with a nucleophilic center. The synthesized product includes an alkylated sulfonium salt or a sulfoxonium salt. The claims also include the use of SAM-dependent methyltransferase to catalyze the transfer of alkyl or alkenyl groups from onium salts to active substrates.

Patent 201510155209.X discloses a method for biocatalytic hydrocarbylation of catechol compounds. The method uses a variety of SAM analogs as hydrocarbon donors, and realizes the hydrocarbylation reaction of catechol compounds under the catalysis of catechol methyltransferase. In addition, this method can realize the recycling and regeneration of the hydrocarbylation donor by adding a solid acid catalyst.

Patent WO2020053196A1 also discloses a biocatalytic alkylation method, which includes two cyclic alkylation steps: the first step uses S-methyltransferase to transfer an alkyl group from an alkyl donor. to a sulfur- or selenium-containing carrier compound to generate an alkylated sulfur-selenium carrier compound. The second step utilizes N, C, O, S or P methyltransferase to convert the selectively substituted alkyl group from the alkylated The carrier compound is transferred to the substrate, thereby producing an alkylated product and a dealkylated carrier compound. The produced dealkylated carrier compound can be continued to be used in step one of the next cycle to regenerate the alkylated carrier compound.

Although enzyme catalysis research using SAM analogs as hydrocarbon donors has been widely welcomed, these SAM analogs reported so far still have certain shortcomings. For example, the sulfonium structure of SAM and its analogs makes them inherently chemically unstable. Under physiological conditions (such as T = 37°C, pH 7.5), SAM and its analogues can easily undergo intramolecular cyclization reactions to generate 5'-deoxy-5'-methylthioadenosine (MTA) and L-homoserine lactone. (This is the main decomposition pathway of SAM and its analogs), or depurination to generate adenine and S-ribomethionine. G et al. studied the stability of various hydrocarbyl-substituted SAM analogs and found that under physiological conditions of pH 7.5, the half-lives of these SAM analogs ranged from 3 minutes to 5 hours (/> G, et al. ACS Chem. Biol. 2013, 8, 1134–1139).

Seebeck et al. used a cyclase cascade reaction to observe the production of the suspected intermediate product fluoromethyl SAM analog (F-SAM) for the first time (Seebeck F P, et al. Angew. Chem. Int. Ed. 2021, 60, 27178 -27183). However, due to the extreme instability of F-SAM, experiments failed to characterize and identify it. It is inferred from the experimental identification of the main decomposition products that F-SAM decomposition still mainly proceeds along the direction of producing 5′-deoxy-5′-fluoromethioadenosine (F-MTA). At the same time, experiments found that the stability of F-MTA produced by decomposition is still extremely poor and will continue to be rapidly degraded over time. Therefore, it can be speculated that the factors that cause F-SAM to be extremely unstable, in addition to the structural reasons of sulfonium described above, are that the strong electron attraction of fluorine atoms increases the instability of the three C-S bonds connected to sulfur atoms, further promoting The decomposition of F-SAM may also become a "common problem" among SAM analogues containing fluorine functional groups. Booker et al. found that the synthetic FMeTeSAM with a Te atom in the sulfonium center showed stronger stability than F-SAM and could be directly used as a fluoromethyl donor to achieve enzymatic fluoromethyl transfer reactions (Booker S J, et al. al. ACS Cent. Sci. 2023, 9, 905–914). However, currently FMeTeSAM can only be synthesized through chemical methods and the synthesis steps are too lengthy and cumbersome, adding a certain complexity to the experiment.

Therefore, it is crucial to research and develop new methods to solve the instability problem currently faced by SAM analogs.

Contents of the invention

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 analogue has good stability and has better reactivity when used for alkylation of acceptor substrates or cyclase cascade reactions.

In order to achieve the above objects, the technical solutions adopted by the present invention are as follows:

The invention provides a stable decarboxylated S-adenosyl-L-methionine analog (dcSAM analog), which has the structure shown in Formula I:

Among them, preferably, M is selected from sulfur or selenium;

Preferably, R is selected from substituted or unsubstituted C ₁ to C ₁₀ saturated or unsaturated linear or branched chain hydrocarbon groups, substituted or unsubstituted C ₁ to C ₁₀ saturated or unsaturated hydrocarbon groups containing N, O, and S heteroatoms. Saturated straight or branched chain hydrocarbon radicals, or

R ₁ is selected from substituted or unsubstituted C ₁ to C ₁₀ saturated or unsaturated linear or branched chain hydrocarbon groups.

Preferably, the substitution includes fluorine substitution.

Fluorine atom substitution can significantly improve the physical and chemical properties, pharmacokinetics, etc. of drug molecules, thereby improving drug efficacy.

The above-mentioned C ₁ to C ₁₀ saturated or unsaturated linear or branched chain hydrocarbon group is preferably a C ₁ to C ₁₀ linear or branched alkyl group, a C ₂ to C ₁₀ linear or branched alkenyl group, or a C ₂ to C ₁₀ linear or branched alkyl group. Straight chain or branched chain alkyne group.

The C ₁ to C ₁₀ linear or branched alkyl group is preferably a C ₁ to C ₆ linear or branched alkyl group, including but not limited to methyl, ethyl, n-propyl, isopropyl, and n-butyl. , isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, etc.

The C ₂ to C ₁₀ linear or branched olefin group is preferably a C ₂ to C ₄ linear or branched olefin group, including but not limited to vinyl, propenyl, allyl, 1-butenyl, cis -2-butenyl, trans-2-butenyl, 2-methyl-1-propenyl, etc.

The C ₂ to C ₁₀ linear or branched alkynyl group is preferably a C ₂ to C ₄ linear or branched alkynyl group, including but not limited to ethynyl, propynyl, 1-butynyl, and 2-butynyl. base, 3-methyl-2-propynyl, etc.

Preferred in the present invention, the substituted or unsubstituted C ₁ to C ₁₀ saturated or unsaturated linear or branched hydrocarbon groups containing N, O, S heteroatoms include but are not limited to amine groups and azide groups. group, ketone group, ester group, amide group, carboxylic acid group, ether group or thioether group.

Preferably, M is selected from sulfur.

Preferably, the R is selected from substituted or unsubstituted C ₁ to C ₁₀ saturated or unsaturated linear or branched chain hydrocarbon groups, or substituted or unsubstituted C ₁ to C ₁₀ containing N, O, S heteroatoms. Saturated or unsaturated straight or branched chain hydrocarbon groups.

The preferred range of the C ₁ to C ₁₀ saturated or unsaturated linear or branched chain hydrocarbon group is the same as above, and will not be repeated here.

Preferably, the substituent of the C ₁ to C ₁₀ saturated or unsaturated linear or branched chain hydrocarbon group is selected from halogen, amino, nitro, hydroxyl, carboxyl, ester group, acyl, C ₃ to C ₁₀ aromatic group. group, heteroaryl group, and one or more of C ₃ to C ₉ cycloalkyl groups.

The halogen is preferably a fluorine atom.

The C ₃ to C ₁₀ aryl group is preferably a C ₆ to C ₁₀ aryl group, specifically including but not limited to phenyl, benzyl, naphthyl, etc.

The C ₃ to C ₁₀ heteroaryl groups specifically include but are not limited to thiazolyl, thienyl, quinolyl, etc.

The C ₃ to C ₉ cycloalkyl group is preferably a C ₃ to C ₆ cycloalkyl group, specifically including but not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, methylcyclopropyl, etc.

Preferably, the substituted C ₁ to C ₁₀ saturated or unsaturated linear or branched chain hydrocarbon group of the present invention is a C ₁ to C ₁₀ saturated or unsaturated linear or branched chain hydrocarbon group containing one or more F atom substituents. Chain hydrocarbon group.

Preferably, the R is selected from -CH ₂ F, -CHF ₂ , -CF ₃ , -CH ₂ CH ₂ F, -CH ₂ CHF ₂ , -CH ₂ CF ₃ , -CH ₂ CH ₂ CH ₂ F, - CH ₂ CH ₂ CHF ₂ , -CH ₂ CH ₂ CF ₃ , 3-chloroallyl, 3-fluoroallyl, 3-aminoallyl, -CH ₂ OCH ₃ , -CH ₂ OC ₄ H ₉ , -CH ₂ OH or -CH ₂ COOH.

The above "-" indicates the connection position.

Preferably, the stable decarboxylated S-adenosyl-L-methionine analog has any of the following structures:

For SAM analogs containing fluorine functional groups, fluorine atoms will exacerbate the decomposition of SAM analogs. The present invention obtains a stable decarboxylated S-adenosyl-L-methionine acid analog through decarboxylation, blocking the main path of unstable decomposition.

The present invention relates to the above-mentioned stable decarboxylated S-adenosyl-L-methionine acid analogues (F-dcSAM) containing fluoromethyl groups and S-adenosyl-L-methionine analogues containing fluoromethyl groups. (F-SAM) was tested for stability, and the results showed that F-dcSAM has better stability.

The stable decarboxylated S-adenosyl-L-methionine analogue of the present invention can be prepared by a chemical method or an enzymatic method. The reaction raw materials are easily available, the conditions are mild, and the operation is simple.

The reaction formula of the chemical method can be specifically:

Chemistry 1

Chemistry 2

The reaction formula of the enzymatic method can be specifically:

Enzymatic method 1

Enzymatic method 2

Enzymatic method 3

The above-mentioned stable decarboxylated S-adenosyl-L-methionine analog directly blocks the intramolecular cyclization reaction of the S-adenosyl-L-methionine analog to produce MTA and L-homoserine through decarboxylation. The main decomposition path of esters, significantly improving stability.

The present invention also provides the use of the above-mentioned stable decarboxylated S-adenosyl-L-methionine analog as an acceptor substrate alkylation donor.

The receptor substrate is preferably a biomolecule.

Preferably, the biological molecules include but are not limited to proteins, nucleic acids or metabolic small molecules.

Preferably, the present invention transfers the hydrocarbon group in the stable decarboxylated S-adenosyl-L-methionine analog to the biomolecule through an enzymatic alkylation reaction, thereby completing the structural modification and property modification of the biomolecule.

The invention also provides a method for enzymatic alkylation using the above-mentioned stable decarboxylated S-adenosyl-L-methionine analog, which includes the following steps:

Under the action of methyltransferase, the stable decarboxylated S-adenosyl-L-methionine analogue shown in Formula I undergoes a nucleophilic substitution reaction with the acceptor substrate to obtain a hydrocarbylated acceptor. Product structure.

Stable decarboxylated S-adenosyl-L-methionine analogues can improve the reaction efficiency of hydrocarbylation of receptor substrates and prepare receptor products containing hydrocarbyl groups more efficiently.

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

The invention also provides the above-mentioned stable decarboxylated S-adenosyl-L-methionine analogue cyclase cascade reaction, which includes the following steps:

1) The halogen methyltransferase catalyzes the reaction between decarboxylated S-adenosyl homocysteine analogs (dcSAH analogs) and R-containing compounds to generate stable decarboxylated S-adenosyl-L-methionine analogs ( dcSAM analogs);

2) Under the action of methyltransferase, the stable decarboxylated S-adenosyl-L-methionine analog undergoes a nucleophilic substitution reaction with the acceptor substrate to prepare the hydrocarbylated acceptor product and decarboxylated S- Adenosyl homocysteine analogs;

3) The decarboxylated S-adenosyl homocysteine analog obtained in step 2) is continued to be used in step 1) of the next cycle to prepare a stable decarboxylated S-adenosyl-L-methionine analog again. ;

R in the R-containing compound is the same as the substituent R in the above-mentioned stable decarboxylated S-adenosyl-L-methionine analog.

In the above-mentioned cyclase cascade reaction, dcSAH analogs and dcSAM analogs can be prepared cyclically, which greatly reduces production costs, improves production efficiency, and is conducive to large-scale production.

Preferably, the molecular formula of the R-containing compound in step 1) is R-X.

Preferably, X in the R-X is selected from halogen, p-toluenesulfonyl or methylsulfonyl.

The above-mentioned cyclase cascade reaction realizes the recycling and regeneration of dcSAM analogues and a more efficient alkylation reaction in the presence of a catalytic amount of raw material dcSAH analogues.

Compared with the prior art, the stable decarboxylated S-adenosyl-L-methionine analog provided by the present invention has the structure shown in Formula I. The stable decarboxylated S-adenosyl-L-methionine analog can effectively block the intramolecular cyclization reaction of the S-adenosyl-L-methionine analog, has excellent stability, and is used for receptors It has better reaction efficiency when hydrocarbylating the substrate or cyclase cascade reaction.

Description of drawings

Figure 1 is a stability test chart of F-dcSAM in Example 2. A) is a stability test of F-dcSAM at pH 1.0; B) is a stability test of F-dcSAM at pH 8.0;

Figure 2 is the stability test chart of F-SAM in Comparative Example 1. A) is the stability test chart of F-SAM at pH 1.0; B) is the stability test chart of F-SAM at pH 8.0; C) This is the LC-MS analysis chart of F-SAM decomposition products.

Detailed ways

In order to further illustrate the present invention, the stable decarboxylated S-adenosyl-L-methionine analog provided by the present invention and its application are described in detail below in conjunction with the examples.

In a preferred embodiment, the halogen methyltransferase is preferably AclHMT, and the source species of the aclhmt gene is Aspergillus clavatus. The aclhmt gene was synthesized by a biological company, and was constructed on the pET28a(+) vector with Kanamycin resistance gene after BL21(DE3) codon optimization, and finally the pET28a(+)-AclHMT plasmid was obtained. The expression and purification of AclHMT is described in the literature (Hammer S C, et al. Angew. Chem. Int. Ed. 2021, 60, 5554-5560).

Example 1

1) Synthesis of dcSAM:

Dissolve 5'-deoxy-5'-methylthioadenosine (MTA) (1 mg, 3.36 μmol) in 100 μL formic acid and 100 μL acetic acid solution at 0°C. After being fully dissolved, continue to add 3-iodopropylcarbamic acid tert-butyl ester (4.8 mg, 16.9 μmol) to the reaction solution at 0°C, and then slowly add AgClO ₄ (3 mg, 26.4 μmol) dropwise, in which 3-iodopropyl carbamate Both tert-butyl carbamate and AgClO ₄ solids must be dissolved in 50 μL formic acid and 50 μL acetic acid solution in advance. The reaction bottle was sealed with a rubber stopper and inserted into a balloon filled with nitrogen (N ₂ ). The reaction was carried out in the dark at 0°C for 20 min and then transferred to room temperature to continue the reaction for 5 h. After the reaction, the reaction was quenched with 1 mL of cold water, and the reaction product was filtered to remove AgI precipitate. Add 1 mL trifluoroacetic acid (TFA) to the filtrate, stir at room temperature for 30 min, and then stop the reaction. Use a vacuum freeze dryer to freeze-dry and evaporate the solvent in the reaction solution. The freeze-dried solid is dissolved in 2 mL H ₂ O, and purified using preparative HPLC through a reversed-phase C18 column SHIMADZU C18 (250 mm × 4.6 mm, 5 μm). Mobile phase A is H ₂ O+1‰TFA, and fluidity B is CH ₃ CN+1‰TFA. The elution method is gradient elution, the flow rate is 3mL/min, the injection volume is 5mL, the detection wavelength is 254nm and 215nm dual wavelength detection, the elution program is 0-6min 0%B, 6-30min 0-48%B, 30-33min 48-90%B, 33-39min 90%B, 39-39.01min 90%-0%B, 39.01-45min 0%B. The purified and collected samples were vacuum freeze-dried to obtain a white powdery solid (yield 30%). Target product ¹ H NMR (600MHz, D ₂ O) 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 N ₆ O ₃ S[M] ⁺ 355.1547,obsd.355.1425.

The reaction formula is as follows:

2) NNMT catalyzes the methylation reaction of dcSAM:

The source species of nnmt gene is Homo sapiens (Human). The nnmt gene was synthesized by a biological company, and was constructed on the pET28a(+) vector with Kanamycin resistance gene after BL21(DE3) codon optimization, and finally the pET28a(+)-NNMT plasmid was obtained. The expression and purification of NNMT was described in the literature (Cravatt B F, et al. Nat Chem Biol 2013, 9, 300-306).

Add the purified nicotinamide N-methyltransferase NNMT (final concentration 100 μM), dcSAM (final concentration 3.0mM), and nicotinamide (final concentration 1.0mM) into 100 μL of a solution containing 100mM Tris-HCl (pH 8.0), React at 30°C for 12 hours. After the reaction, an equal volume (100 μL) of 10% TFA was added to the reaction solution to quench the reaction. After quenching, the reaction sample was centrifuged in a high-speed refrigerated centrifuge at 4°C and 12,000 rpm for 30 minutes. The supernatant was analyzed by analytical HPLC through a reversed-phase C18 column SHIMADZU C18 (150 mm × 4.6 mm, 2.5 μm). The mobile phase A was H ₂ O+1‰TFA, liquidity B is CH ₃ CN+1‰TFA. The elution method is gradient elution, the flow rate is 1mL/min, the injection volume is 50μL, the detection wavelength is 254nm and 215nm dual wavelength detection, the elution program is 0-4min 0% B, 4-20min 0-48%B, 20 -21min 48-90% B, 21-23min 90% B, 23-23.01min 90-0% B, 23.01-28min 0% B. The scale was expanded to prepare the target product under the same reaction system, and the reaction yield was 12%. ¹ H NMR (600MHz, D ₂ O) 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 C ₇ H ₉ N ₂ O ⁺ [M] ⁺ 137.0766,obsd.137.0709.

The reaction formula is as follows:

3) HMT and NNMT cascade catalyze the cyclic enzyme cascade methylation reaction of dcSAH and CH ₃ I:

Add HMT (final concentration 50 μM), NNMT (final concentration 50 μM), dcSAH (final concentration 200 μM), CH ₃ I (final concentration 100 mM), and nicotinamide (final concentration 1.0 mM) in 100 μL containing 100 mM Tris-HCl (pH 8.0). solution, react at 30°C for 1 hour. After the reaction, an equal volume (100 μL) of 10% TFA was added to the reaction solution to quench the reaction. The reaction post-treatment and HPLC analysis methods were consistent with the above step 2).

Example 2

1) Synthesis of fluoromethyl-containing decarboxylated SAM analog (F-dcSAM):

①Synthesis of dcSAH: The synthesis of dcSAH refers to the literature method (Anglin J, et al. J. Med. Chem. 2012, 55, 8066-8074). ②Synthesis of F-dcSAM analogs: Dissolve dcSAH (4.5 mg, 13.2 μmol) in 100 μL formic acid and 100 μL acetic acid solvent at 0°C, stir thoroughly until dissolved, add CH ₂ FI (5 μL, 66 μmol) at 0°C, Continue to slowly add AgClO ₄ (11 mg, 26.4 μmol), react under N ₂ protection at 0°C for 20 min in the dark, and continue the reaction at room temperature for 5 h. After the reaction, 10 mL of cold water was added to quench the reaction, and the reactant was filtered to remove silver iodide precipitate. The filtrate was freeze-dried under vacuum and then dissolved in 2 mL H ₂ O. It was purified using preparative HPLC through a reversed-phase C18 column SHIMADZU C18 (250 mm × 4.6 mm, 5 μm). The mobile phase A was H ₂ O + 1‰TFA. Property B is CH ₃ CN+1‰TFA. Elution method: gradient elution, flow rate is 3mL/min, injection volume is 5mL, detection wavelength is 254nm and 215nm dual wavelength detection, elution program is 0-6min0%B, 6-30min0-48%B, 30-33min 48-90%B, 33-39min 90%B, 39-39.01min 90%-0%B, 39.01-45min 0%B. The purified and collected samples were vacuum freeze-dried to obtain a white powdery solid (yield 30%). Target compound ¹ H NMR (600MHz, D ₂ O) 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–-4.59(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, D ₂ O) δ-215.59 (t, J＝45.7 Hz,0.4F),-216.13(t,J＝45.6Hz,0.6F).LCMS(ESI)calcd.for C ₁₄ H ₂₂ FN ₆ O ₃ S ⁺ [M] ⁺ 373.1295,obsd.373.1453.

The reaction formula is as follows:

2) DnrK catalyzes the fluoromethylation reaction of F-dcSAM:

The source species of dnrk gene is Streptomyces peucetius. The dnrk gene was synthesized by a biological company, and was constructed on the pET28a(+) vector with Kanamycin resistance gene after BL21(DE3) codon optimization, and finally the pET28a(+)-DnrK plasmid was obtained. The expression and purification of DnrK was as described in the literature (Thorson J S, et al. ACS Chem. Biol. 2016, 11, 2484-2491).

The purified erythromycin 4-O-methyltransferase DnrK (final concentration 100 μM), F-dcSAM (final concentration 3.0mM), and erythromycin (final concentration 1.0mM) were placed in 100L containing 100mM Tris-HCl (pH 8.0), react at 30°C for 1 hour. After the reaction, an equal volume (100 μL) of methanol was added to the reaction solution to quench the reaction. After quenching, the reaction sample was centrifuged in a high-speed refrigerated centrifuge at 4°C and 12,000 rpm for 30 minutes. The supernatant was analyzed by analytical HPLC through reversed-phase C18 chromatographic column SHIMADZUC18 (150 mm × 4.6 mm, 2.5 μm). The mobile phase A was H ₂ O+1‰TFA, liquidity B is CH ₃ CN+1‰TFA. The elution method is gradient elution, the flow rate is 1mL/min, the injection volume is 50μL, the detection wavelength is 254nm and 215nm dual wavelength detection, the elution program is 0-2min 0% B, 2-11min 0-14%B, 11 -15min 14-46% B, 15-25min 46-53% B, 25-25.01min 53%-95% B, 25.01-27min 95-0% B, 27.01-31min 0% B.). Under the same reaction system The scale was expanded to prepare the target product, and the reaction yield was 80%. ¹ H NMR (600MHz, D ₂ O) δ7.60 (t, J = 7.8 Hz, 1H), 7.50–7.34 (m, 2H), 5.80 (d, J = 54 Hz, 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.

The reaction formula is as follows:

3) TPMT catalyzes the fluoromethylation reaction of F-dcSAM:

The source species of the tpmt gene is Homo sapiens (Human). The tpmt gene was synthesized by a biological company, and was constructed on the pET28a(+) vector with Kanamycin resistance gene after BL21(DE3) codon optimization, and finally the pET28a(+)-TPMT plasmid was obtained. The expression and purification of TPMT was described in the literature (ZhouZ S, et al. J. Am. Chem. Soc. 2010, 132, 3642-3643).

Add the purified thiopurine 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) in 100 μL containing 100 mM PBS. (pH 6.0) solution, react at 30°C for 12 hours. After the reaction, an equal volume (100 μL) of 10% TFA was added to the reaction solution to quench the reaction. After quenching, the reaction sample was centrifuged in a high-speed refrigerated centrifuge at 4°C and 12,000 rpm for 30 minutes. The supernatant was analyzed by analytical HPLC through a reversed-phase C18 column SHIMADZU C18 (150 mm × 4.6 mm, 2.5 μm). The mobile phase A was H ₂ O+1‰TFA, liquidity B is CH ₃ CN+1‰TFA. The elution method is gradient elution, the flow rate is 1mL/min, the injection volume is 50μL, the detection wavelength is 254nm and 215nm dual wavelength detection, the elution program is 0-4min 0%B, 4-20min 0-48%B, 20 -21min 48-90%B, 21-23min 90%B, 23-23.01min 90-0%B, 23.01-28min 0%B. The scale was expanded to prepare the target product under the same reaction system, and the reaction yield was 80%. ¹ H NMR (600MHz, DMSO-d6) δ 8.78 (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.forC ₆ H ₅ FN ₄ S[M+H] ¹⁺ 185.0240,obsd.185.0292.

The reaction formula is as follows:

4) HMT and DnrK cascade catalyze the cyclic enzyme cascade methylation reaction of dcSAH and CH ₂ FI:

Add HMT (final concentration 50 μM), DnrK (final concentration 50 μM), dcSAH (final concentration 200 μM), CH ₂ FI (final concentration 100 mM), and erythromycin (final concentration 1.0 mM) in 100 μL containing 100 mM Tris-HCl (pH 8.0 ) solution, react at 30°C for 1 hour. After the reaction, an equal volume (100 μL) of methanol was added to the reaction solution to quench the reaction. The reaction post-treatment and HPLC analysis methods were consistent with those described in step 2).

5) HMT and TPMT cascade catalyze the cyclic enzyme cascade methylation reaction of dcSAH and CH ₂ FI:

Add HMT (final concentration 50 μM), TPMT (final concentration 50 μM), dcSAH (final concentration 200 μM), CH ₂ FI (final concentration 100 mM), mercaptopurine (final concentration 1.0 mM), and TCEP (final concentration 2.0 mM) in 100 μL. In a solution of 100mM PBS (pH 6.0), react at 30°C for 12h. After the reaction, an equal volume (100 μL) of 10% TFA was added to the reaction solution to quench the reaction. The reaction post-treatment and HPLC analysis methods were consistent with those described in step 3).

Figure 1 is a stability test chart of F-dcSAM in Example 2. A) is a stability test of F-dcSAM at pH 1.0; B) is a stability test of F-dcSAM at pH 8.0.

Comparative example 1

The synthesis of F-SAM is consistent with the synthesis method of F-dcSAM in Example 2. The raw material dcSAH was replaced with commercially available S-adenosylhomocysteine (SAH), and the dosage was 5 mg (13.2 μmol). The structural formula of F-SAM is as follows:

Figure 2 is the stability test chart of F-SAM in Comparative Example 1. A) is the stability test chart of F-SAM at pH 1.0; B) is the stability test chart of F-SAM at pH 8.0; C ) is the LC-MS analysis chart of F-SAM decomposition products.

The stability test results of fluoromethyl SAM analogues F-SAM and F-dcSAM show that F-dcSAM has better stability than F-SAM regardless of pH 1.0 or pH 8.0. Among them, under the condition of pH 8.0, only 35% of F-SAM remained after 30 minutes, and F-SAM was almost completely degraded after 120 minutes. Compared with F-dcSAM, it only decomposes to produce about 10% adenine after 5.7 hours under pH 8.0 conditions, which has better stability.

In summary, F-dcSAM synthesized through decarboxylation has significantly stronger stability than F-SAM. Therefore, the carboxyl group of SAM and its analogs is removed to obtain a stable decarboxylated S-adenosyl-L-methionine analog, which inhibits the decomposition path of SAM and its analogs to produce MTA or MTA analogs, greatly improving the stability sex.

The description of the above embodiments is only used to help understand the method and its core idea 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 scope of the claims of the present invention.

Claims (10)

1. A stable decarboxylated S-adenosyl-L-methionine analogue, characterized in that it has the structure shown in Formula I: Wherein, M is selected from sulfur or selenium; R is selected from substituted or unsubstituted C ₁ to C ₁₀ saturated or unsaturated linear or branched chain hydrocarbon groups, substituted or unsubstituted C ₁ to C ₁₀ saturated or unsaturated linear hydrocarbon groups containing N, O, and S heteroatoms. chain or branched hydrocarbon radical, or R ₁ is selected from substituted or unsubstituted C ₁ to C ₁₀ saturated or unsaturated linear or branched chain hydrocarbon groups; The substitution includes fluorine substitution. 2. The stable decarboxylated S-adenosyl-L-methionine analog according to claim 1, characterized in that the M is selected from sulfur; The R is selected from substituted or unsubstituted C ₁ to C ₁₀ saturated or unsaturated linear or branched chain hydrocarbon groups, or substituted or unsubstituted C ₁ to C ₁₀ saturated or unsaturated linear or branched hydrocarbon groups containing N, O, and S heteroatoms. Saturated straight or branched chain hydrocarbon group; The substituent of the C ₁ to C ₁₀ saturated or unsaturated linear or branched hydrocarbon group is selected from halogen, amino, nitro, hydroxyl, carboxyl, ester group, acyl, C ₃ to C ₁₀ aryl and heteroaryl , one or more types of C ₃ to C ₉ cycloalkyl groups. 3. The stable decarboxylated S-adenosyl-L-methionine analog according to claim 2, characterized in that the substituted C ₁ to C ₁₀ saturated or unsaturated linear or branched hydrocarbon group It is a C ₁ to C ₁₀ saturated or unsaturated linear or branched chain hydrocarbon group containing one or more F atom substituents. 4. The stable decarboxylated S-adenosyl-L-methionine analog according to claim 1, characterized in that the R is selected from _-CH2F , _-CHF2 , _-CF3 , -CH ₂ CH ₂ F, -CH ₂ CHF ₂ , -CH ₂ CH ₂ CH ₂ F, -CH ₂ CH ₂ CHF ₂ , -CH ₂ CF ₃ , -CH ₂ CH ₂ CF ₃ , 3-chloroallyl, 3 -Fluoroallyl, 3-aminoallyl, -CH ₂ OCH ₃ , -CH ₂ OC ₄ H ₉ , -CH ₂ OH or -CH ₂ COOH. 5. The stable decarboxylated S-adenosyl-L-methionine analog according to claim 1, characterized in that the stable decarboxylated S-adenosyl-L-methionine analog has the following properties: Any of the structures shown: 6. Use of the stable decarboxylated S-adenosyl-L-methionine analog according to any one of claims 1 to 5 as a donor for the alkylation of an acceptor substrate. 7. A method for enzymatic alkylation using the stable decarboxylated S-adenosyl-L-methionine analogue according to any one of claims 1 to 5, characterized in that it includes the following steps: Under the action of methyltransferase, the stable decarboxylated S-adenosyl-L-methionine analogue shown in Formula I undergoes a nucleophilic substitution reaction with the acceptor substrate to obtain a hydrocarbylated acceptor product structure. 8. The method of enzymatic alkylation according to claim 7, wherein the methyltransferase is selected from the group consisting of methyltransferases whose binding center is N, C, O, S or P. 9. The cyclase cascade reaction of the stable decarboxylated S-adenosyl-L-methionine analogue according to any one of claims 1 to 5, characterized in that it includes the following steps: 1) The decarboxylated S-adenosyl homocysteine analogue reacts with an R-containing compound catalyzed by halogen methyltransferase to generate a stable decarboxylated S-adenosyl-L-methionine analogue; 2) Under the action of methyltransferase, the stable decarboxylated S-adenosyl-L-methionine analog undergoes a nucleophilic substitution reaction with the acceptor substrate to prepare the hydrocarbylated acceptor product and decarboxylated S- Adenosyl homocysteine analogs; 3) The decarboxylated S-adenosyl homocysteine analog obtained in step 2) is continued to be used in step 1) of the next cycle to prepare a stable decarboxylated S-adenosyl-L-methionine analog again. ; R in the R-containing compound is the same as the substituent R in the stable decarboxylated S-adenosyl-L-methionine analog according to any one of claims 1 to 5. 10. The cyclase cascade reaction according to claim 9, wherein the molecular formula of the R-containing compound in step 1) is R-X; X in the R-X is selected from halogen, p-toluenesulfonyl or methylsulfonyl.