SAM analogue and application thereof

Abstract

The invention relates to the technical field of biological catalysis, and particularly discloses an SAM analogue and application thereof. The SAM analogue has a structure as shown in a formula I, and an active molecule can be subjected to a fluoroethyl transfer reaction by utilizing MTases. Wherein FEt-SeAM can effectively avoid the problem of fluoroethyl elimination of FEt-SAM, and is more suitable for fluoroethylation of an acceptor substrate. Compared with a wild type, the methyltransferase mutant provided by the invention has the advantage that the catalytic activity of fluoroethyl transfer is effectively improved. According to the method, an HMT-MTase cyclase cascade reaction system based on SeAH / SAH is further constructed, fluoroethylation of nucleophilic reagents such as O-, N-, S-and C-is successfully carried out, compared with an existing chemical synthesis technology, the method has the advantages that operation is easy and convenient, the reaction condition is mild and environmentally friendly, meanwhile, the fluoroethyl donor FEt-SeAM / FEt-SAM can be recycled, and the atom economy and the reaction efficiency are remarkably improved.

Description

[0001] This application is a divisional application of "the application date is March 7, 2024, the application number is 202410263906.6, and the invention name is SAM analogs, SAM-dependent methyltransferase mutants and their applications". Technical Field

[0002] The invention relates to the technical field of biocatalysis, in particular to SAM analogs and applications thereof. Background Art

[0003] Hydroxylation reactions, represented by methylation, can improve the biological activity or selectivity of the modified molecules, thereby improving the targeting, safety and even physiological effects of these compounds. This is a widely used strategy in the pharmaceutical and agricultural industries to improve the pharmacological properties of candidate drugs and natural products by adjusting their binding affinity, solubility and metabolic profile. At present, hydroxylation is increasingly becoming one of the important strategies for drug structure modification and transformation.

[0004] Fluorine is a unique chemical element with the highest electronegativity and the smallest atomic radius except for hydrogen. Replacing hydrogen in a hydrocarbon group with fluorine will hardly affect its size and shape, so it will not bring spatial load to the catalytic system, but it will greatly affect the physical and chemical properties of the molecule, such as polarity, acidity and alkalinity, dipole moment, cell permeability, lipophilicity and stability, etc. Because fluorine atoms give molecules unique properties, they play an important role in pharmaceutical chemistry, agricultural chemistry and material chemistry. In particular, the introduction of fluorine elements in drugs can improve bioavailability, increase selectivity for target organs, and reduce the dosage of drugs. These special properties have promoted the rapid development of fluorine-containing drugs. In recent years, research on fluorine chemistry has also increased. Fluorine exists in 20-30% of drugs and about 30% of agricultural chemicals. Currently, nearly half of high-end special materials are fluorine-containing materials. Therefore, the design and development of various compounds containing fluorine atoms or fluorine-containing functional groups has very important research value. The selective introduction of fluorine atoms or fluorine-containing functional groups at specific positions in compound molecules is the key to the synthesis of fluorine-containing drugs and fluorine-containing active compounds.

[0005] In recent years, on the basis of traditional hydrocarbyl modification, the research on fluorohydrocarbylation has been further strengthened. Traditional synthetic hydrocarbylation methods have several disadvantages. Their reaction substrates are not environmentally friendly (such as hydrocarbyl halides), and they lack strong regioselectivity, chemical selectivity or stereoselectivity. They often have high energy consumption and low yields during the synthesis process. These inherent defects in chemical synthesis have brought challenges to further research on a wide range of hydrocarbylation, especially fluorohydrocarbylation reactions. At the same time, the lack of fluorination reagents has also brought limitations to the development of fluorohydrocarbylation research.

[0006] Compared with chemical reactions, biocatalytic reactions have the advantages of high selectivity, environmental friendliness, and mild reaction conditions. With the continuous discovery of biocatalysts and the rapid development of enzyme catalysis, the use of enzyme catalysis to replace traditional chemical synthesis has gradually become a significant trend in the pharmaceutical field. The research on enzyme-catalyzed alkylation is inspired by the natural methylation reaction of organisms themselves, that is, using the cofactor S-adenosyl-L-methionine (SAM) as a methyl donor, SAM-dependent methyltransferases (MTases) use SAM to methylate many molecules, including small molecule metabolites, fats, proteins, DNA, RNA, etc. in the body. The reaction mechanism involves the polarity of the nucleophilic reagent on the electrophilic methyl substituent of SAM. N 2 attacks and generates S-adenosylhomocysteine ​​(SAH) as a byproduct. SAM and SAM-dependent MTases are the most widely used biocatalytic systems in nature. MTases are ubiquitous in nature and have been shown to have a wide range of substrates in vitro. In recent years, the study of SAM analogs (such as replacing methyl with ethyl, propyl, aryl or other long-chain hydrocarbon groups) has broadened the substrate breadth and functional diversity of this biocatalytic system, and also brought opportunities and challenges to the use of this strategy for fluoroalkylation reactions.

[0007]

[0008] SAM and its analogs are inherently chemically unstable due to their special sulfonium structure. They degrade in three different ways at pH 7.5-10.0 and 37°C. It undergoes racemization under the action of sulfur to form diastereomers (R, S). At pH values ​​above 3, it undergoes intramolecular cyclization to generate 5'-deoxy-5'-methylthioadenosine (MTA) and L-homoserine lactone, which is the main decomposition pathway for SAM and its analogs. When the pH is above 7, it undergoes deprotonation at C5', resulting in the elimination of adenine and the production of S-ribosylmethionine.

[0009]

[0010] Halide methyltransferase (HMT) is a class of enzymes that can directly catalyze the production of SAM and its analogs from halogenated alkane reagents and SAH. HMTs from different sources have different abilities to catalyze the transfer of hydrocarbon groups, especially for non-natural functional groups that are larger than methyl groups. Chen et al. studied the structure of AclHMT from the Aspergillus fungus species (Chen Q, et al. Mol. Catal. 2023, 550, 113533.), and constructed three mutants (W27F, W27F / P8L, W27F / P8L / V265W) to enhance its catalytic activity for the formation of S-adenosyl-L-ethionine (SAE). The activities of these three mutants are higher than those of wild-type AclHMT, especially W27F / P8L / V265W, which has a 38.7-fold higher activity in catalyzing the formation of SAE than the wild-type. Seebeck et al. found that HMT can generate S-adenosyl-S-(fluoromethyl)-L-homocysteine ​​(F-SAM) from fluoromethyl iodide (FMeI) and SAH (Seebeck FP, et al. Angew. Chem. Int. Ed. 2021, 60, 27178–27183), but it was not characterized and identified. Because under physiological conditions, F-SAM is very unstable and quickly hydrolyzes into L-homoserine lactone and 5'-deoxy-5'-fluoromethylthioadenosine (F-MTA), experiments have found that F-MTA is still unstable and will continue to decompose into 5'-deoxy-5'-thioadenosine, formaldehyde and fluoride. This is not exactly the same as the decomposition pattern of natural SAM. It hydrolyzes to MTA faster and further decomposes on the basis of hydrolysis to MTA, which may be caused by the introduction of fluorine atoms. In order to overcome the instability of F-SAM and achieve the purpose of fluoromethylation of nucleophiles, they innovatively adopted the HMT-MTases cascade reaction system, using secondary methyltransferases to transfer the fluoromethyl groups of F-SAM generated in situ to nucleophiles such as N-, C-, and O-. This reaction strategy has been reported in their previous studies (Seebeck FP, et al. Nat. Catal. 2019, 2, 696-701), which demonstrated that C-, N- and O-specific MTases can be combined with HMT to form an enzyme cascade, requiring only catalytic concentrations of SAM and using methyl iodide (MeI) as a stoichiometric methyl donor. This successfully overcomes the inherent instability of SAM and improves atom economy and reaction efficiency.

[0011] In addition to the fluoromethyl group, other SAM analogs containing fluorinated functional groups also have important research value. Liu et al. used the corresponding SAM analogs to transfer 3,3-difluoroalkenyl and several fluorinated benzyl groups to vancomycin (Liu W, et al. Org. Lett. 2023, 25, 5650-5655). These analogs were prepared by coupling engineered human methionine adenosyltransferase (hMAT2A) with MTase. They also tried to use the same strategy to synthesize fluoroethyl SAM (FEt-SAM) and perform fluoroethyl transfer, but no fluoroethylated products were detected, and only trace amounts of hydroxyethylated vancomycin were detected, indicating that the use of SAM analogs to achieve fluoroethylation is relatively challenging. However, the fluoroethyl group is an important pharmacodynamic functional group, and fluoroethyl-containing drugs are important drugs with multiple activities such as anti-sensitivity and anti-inflammatory activities, which are used to treat rhinitis, bronchitis, asthma and other diseases. Many fluoroethyl-containing drugs are diagnostic drugs for PET-CT. In addition, fluoroethyl-containing 19 F probes play an important role in NMR studies of the structural dynamics of large membrane proteins. Therefore, the study of fluoroethylation reactions is of great research significance. However, there are few studies on the enzymatic synthesis of active drug analogs containing fluorine functional groups. As an important fluorine-containing functional group, there is currently no research on the enzymatic synthesis of related analogs of fluoroethyl. Summary of the invention

[0012] In view of this, the present invention provides a SAM analog and a SAM-dependent methyltransferase mutant, and constructs a HMT-MTase cyclase cascade reaction system based on a fluoroalkyl SAM analog. The FEt-SeAM and FEt-SAM provided by the present invention can utilize MTases to perform a fluoroethyl transfer reaction on active molecules. Among them, FEt-SeAM can successfully avoid the problem of fluoroethyl elimination, and is more suitable for use as a fluoroethyl donor for a variety of MTases enzymatic fluoroethylation reactions. Further, the present invention generates FEt-SeAM in situ through a cyclase cascade reaction, thereby reducing its own decomposition and improving the reaction efficiency. Furthermore, the use of a SAM-dependent methyltransferase mutant for fluoroethylation modification of active molecules significantly improves the yield of the fluoroethylation reaction product compared to wild-type methyltransferase and other transferase mutants.

[0013] In order to achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0014] A mutant of a SAM-dependent methyltransferase, comprising one or more mutation sites among I6V, L160A, L160V, L160I, and L160S, and its wild-type amino acid sequence is shown in GenBank: AAA26712.1 or GenBank: AAF67508.2.

[0015] In some embodiments, the mutant is a carminomycin 4-O-methyltransferase DnrK mutant, whose mutation site is L160A, L160V, L160I or L160S, and its wild-type amino acid sequence is shown in GenBank: AAA26712.1.

[0016] In some embodiments, the mutant is a coumarin C-methyltransferase NovO mutant, the mutation site of which is I6V, and the wild-type amino acid sequence is shown in GenBank: AAF67508.2.

[0017] Experiments show that compared with the wild-type methyltransferase, the mutant provided by the present invention significantly improves the yield of the fluoroethylation reaction product.

[0018] The present invention also provides a biomaterial comprising:

[0019] 1) A nucleic acid encoding the mutant of the present invention;

[0020] 2) an expression cassette comprising the nucleic acid described in 1);

[0021] 3) a recombinant vector comprising 1) the nucleic acid or 2) the expression cassette;

[0022] 4) Transfecting or transforming the host of the recombinant vector described in 3).

[0023] The mutant of the present invention can be obtained by chemical synthesis or biosynthesis, wherein the biosynthesis method comprises: constructing the host shown in 4) of the present invention, culturing, and obtaining the mutant.

[0024] The present invention also provides a fluorocarbon-based SAM analogue having a structure shown in Formula I:

[0025]

[0026] Wherein, X is sulfur or selenium;

[0027] R is a fluorine-substituted C2~C 10 Saturated or unsaturated straight chain or branched hydrocarbon group.

[0028] In some embodiments, the C2-C 10 Saturated or unsaturated straight or branched chain hydrocarbon groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, and the like.

[0029] In some specific embodiments, the SAM analog has any of the following structures:

[0030]

[0031] The present invention synthesizes two new SAM analogs, FEt-SAM and FEt-SeAM. Both compounds can be synthesized and separated by chemical methods to obtain pure products, and can also be obtained by HMT enzymatic reaction. However, under the conditions of the enzyme reaction system, different degrees of decomposition occur.

[0032] The present invention demonstrates the ability of HMT to generate FEt-SAM and FEt-SeAM, creates a SeAH-based HMT-MTase cyclase cascade reaction by using HMT to in situ generate FEt-SeAM, and successfully achieves enzymatic fluoroethylation of O-, N-, S-, and C-nucleophiles.

[0033] The present invention also provides the use of SAM analogs as donors for fluoroalkylation of acceptor substrates.

[0034] The receptor substrate is a common active molecular drug in the art, including but not limited to protein, nucleic acid or small molecule compound, etc. In the specific embodiment of the present invention, it is carminomycin and coumarin.

[0035] The present invention also provides a method for enzymatically fluoroalkylating a receptor substrate, wherein a SAM analog and a receptor substrate are reacted under the catalytic action of the mutant of the present invention to obtain a fluoroalkylated receptor product.

[0036] Furthermore, the SAM analog is the SAM analog represented by Formula I of the present invention.

[0037] The present invention also provides a fluoroalkylation cyclase cascade reaction, in which steps 1 to 2) are carried out simultaneously in the same system:

[0038] 1) catalyzing the reaction of the SAH analogue of formula II with a fluoroalkyl iodine reagent by halogen methyltransferase to produce the SAM analogue of formula I;

[0039]

[0040] 2) the SAM analogue shown in formula I reacts with a receptor substrate under the action of a methyltransferase to obtain a fluoroalkylated receptor product and a SAH analogue shown in formula II;

[0041] Wherein, the SAH analogue shown in formula II obtained in step 2) is recycled for the reaction in step 1);

[0042] The hydrocarbon group in the fluoroalkyl iodination reagent is a straight chain or branched hydrocarbon group of C2 to C10.

[0043] In some preferred embodiments, the SAM-dependent methyltransferase is a mutant described in the present invention.

[0044] In the present invention, the halogen methyltransferase is preferably a halogen methyltransferase mutant. In some specific embodiments, it is specifically an AclHMT (W27F / P8L or W27F / P8L / V265W) mutant.

[0045] The present invention transforms MTases based on protein structure analysis, obtains five SAM-dependent methyltransferase mutants DnrKL160A, DnrKL160V, DnrKL160I, DnrKL160S, and NovO I6V, and designs and synthesizes new fluoroethyl donors, SAM analogs (such as FEt-SAM and FEt-SeAM) shown in Formula I. Experimental results show that the mutants provided by the present invention can participate in the HMT-MTase cyclase cascade reaction for fluoroethylation modification of active molecules, significantly improving the yield of fluoroethylation products, and the SAM analog FEt-SeAM provided by the present invention successfully avoids the fluoroethyl elimination problem of FEt-SAM, has high stability, and meets the conditions for using MTases to perform fluoroethylation transfer reactions. The results show that the SAM analogs and SAM-dependent methyltransferases provided by the present invention are suitable for fluoroethylation modification of active molecules. Compared with the existing chemical synthesis method, the enzymatic reaction is simple to operate, the reaction conditions are mild, and the environment is environmentally friendly. At the same time, the cyclase cascade reaction greatly improves the atom economy and reaction efficiency by cyclically regenerating the fluoroethyl donor FEt-SeAM or FEt-SAM. This provides a new way to prepare drug fluoroethyl analogs. DETAILED DESCRIPTION

[0046] To further illustrate the present invention, the fluoroalkyl SAM analogs, SAM-dependent methyltransferase mutants and the SeAH-based HMT-Mtase cyclase cascade reaction involved in the present invention are described in detail below in conjunction with the examples.

[0047] The technical scheme involved in the present invention includes the synthesis of fluoroethyl SAM analogs, HMT-MTase cyclase cascade reaction based on SeAH, fluoroethylation structural modification of active molecules, and design of enzyme mutants based on protein structure models to enable them to accept and perform fluoroethyl transfer reactions.

[0048] 1. Chemical and enzymatic synthesis of FEt-SAM and FEt-SeAM

[0049] 1. Chemical synthesis scheme

[0050] SAH / SeAH (1 equivalent) was dissolved in acetic acid (200 μL) and formic acid (200 μL). FEtI (30 equivalents) was added at 0°C and wrapped in aluminum foil to protect it from light. AgClO4 (10 equivalents) was dissolved in 100 μL acetic acid and 100 μL formic acid and added to the above solution at 0°C. The mixture was stirred at 0°C for 30 minutes and then transferred to room temperature. After reacting at room temperature for 12 hours, the mixture was cooled to 0°C in an ice bath, and then the same volume of FEtI (30 equivalents) as in the initial reaction was added, and AgClO4 (10 equivalents) was added in the same manner. After stirring at 0°C for 30 minutes, the reaction was continued at room temperature for 12 hours, and FEtI and AgClO4 were added again. FEtI and AgClO4 were added three times in total. The reaction solution was quenched with a pre-cooled 0.1% formic acid aqueous solution and the AgI precipitate was removed by centrifugation. The reaction solution was extracted with an equal volume of precooled anhydrous ether to remove excess FEtI in the reaction system. After removing a small amount of anhydrous ether miscible with the aqueous solution by rotary evaporation, the product was purified by preparative HPLC in CH3CN / H2O, and the purified sample was collected and freeze-dried to obtain a white powdery solid.

[0051] The reaction formula is as follows:

[0052]

[0053] 2. Enzymatic synthesis scheme

[0054] The present invention also constructs AclHMT (W27F / P8L, W27F / P8L / V265W) mutants. After experimental comparison, the activity of these two mutants in catalyzing fluoroethyl is significantly higher than that of wild-type AclHMT, especially W27F / P8L / V265W. Purified AclHMT (W27F / P8L / V265W) (final concentration 200 μM), SAH / SeAH (final concentration 1.0 mM), FEtI (final concentration 2.5 mM) are fully mixed in a 50 mM PBS (pH 7.6) solution, and reacted in a 30° C. water bath for 1 hour. After the reaction is completed, an equal volume of 10% TFA is added to the reaction solution to quench the reaction. The quenched reaction sample is centrifuged at 4° C. and 12,000 rpm for 30 minutes using a high-speed refrigerated centrifuge, and the supernatant is filtered using a pinhole filter. The filtrate is analyzed and detected in CH3CN / H2O by analytical HPLC.

[0055] The reaction formula is as follows:

[0056]

[0057] 2. SeAH-based HMT-MTase cyclase cascade reaction

[0058] Since the enzymatically synthesized FEt-SAM and FEt-SeAM decompose to varying degrees in the reaction system, the introduction of Se atoms successfully avoids the elimination of the fluoroethyl group, and since the Se-C bond is weak, the selenium analogue of SAM (SeAM) is a better hydrocarbon donor than SAM, so FEt-SeAM has stronger reaction activity. Therefore, the HMT-MTase cyclase cascade reaction based on SeAH was used to achieve the purpose of fluoroethylation structure modification of the active molecule.

[0059] Purified AclHMT (W27F / P8L / V265W) (final concentration 40 μM), MTases (final concentration 80 μM), SeAH (final concentration 100 μM), FEtI (final concentration 2.5 mM), receptor substrate (final concentration 1.0 mM) were reacted in a 30°C water bath in a 50 mM PBS (pH 7.6) or other buffer solution. After the reaction, an equal volume of 10% TFA was added to the reaction solution to quench the reaction. The quenched reaction sample was centrifuged at 4°C and 12000 rpm for 30 min using a high-speed refrigerated centrifuge, and the supernatant was analyzed and detected by analytical HPLC in CH3CN / H2O.

[0060] The general formula of the MT-MTase cyclase cascade reaction is as follows:

[0061]

[0062] 3. Designing enzyme mutants based on protein structure models

[0063] The present invention searches for the MTases to be found in the PDB protein structure database, and the cofactor SAH or SAM is generally included in the structure, which can assist in finding the position of the active pocket. The key amino acid residues that may affect the reaction activity around the active pocket are analyzed, especially the residues near the SAM methyl group are mutated into residues with a smaller structure than the natural amino acid, such as alanine. The mutated protein structure is molecularly docked with FEt-SeAM using Autodock-Vina to verify that the mutation has a better spatial structure for introducing a larger fluoroethyl group than the wild type. The above-mentioned HMT-MTase cyclase cascade reaction scheme is used to verify the reaction activity of the mutant.

[0064] In a preferred embodiment, AclHMT is preferably used for HMT, and MppJ, DnrK, and NovO are preferably used for MTases. The aclhmt gene is from Aspergillus clavatus, the mppj gene is from Streptomyces hygroscopicus, the dnrk gene is from Streptomyces peucetius, and the novo gene is from Streptomyces niveus. The aclhmt, mppj, dnrk, and novo genes are synthesized by a biological company and constructed on a pET28a(+) vector with a Kanamycin resistance gene after BL21(DE3) codon optimization, and finally pET28a(+)-AclHMT, pET28a(+)-MppJ, pET28a(+)-DnrK, and pET28a(+)-NovO plasmids are obtained. DnrK uses its best mutant L160A, and NovO uses its best mutant I6V. The expression and purification of AclHMT refer to the literature (Hammer SC, et al. Angew. Chem. Int. Ed. 2021, 60, 5554--5560). The expression and purification of MppJ refer to the literature (Li TL, et al. Chembiochem. 2009, 10, 2480-2487). The expression and purification of DnrK (L160A) refer to the literature (Thorson JS, et al. ACS Chem. Biol. 2016, 11, 2484-2491). The expression and purification of NovO (I6V) refer to the literature (Burley GA, et al. Angew. Chem. Int. Ed. 2019, 58, 17583-17588).

[0065] In order to study and successfully carry out the fluoroethylation reaction in depth, the present invention synthesizes two fluoroethyl SAM analogs, FEt-SAM and Se-substituted FEt-SAM (FEt-SeAM) by chemical method, and explores their stability to verify whether the conditions for the fluoroethylation transfer reaction using MTases are met. The study found that the introduction of Se atoms well solves the problem of the co-elimination of F atoms and deprotonated H due to the electronegativity of S atoms. At the same time, the present invention proves that HMT can catalyze the fluoroethyl iodination reagent (FEtI) and SAH or Se-substituted SAH (SeAH) to produce FEt-SAM and FEt-SeAM. At the same time, the present invention also develops a HMT-MTase cyclase cascade reaction based on SeAH, which can realize the cyclic regeneration of FEt-SeAM analogs and the efficient fluoroethylation reaction of active molecules in the presence of a catalytic amount of raw material SeAH. In addition, the present invention makes more MTases applicable to the cyclase cascade reaction through rational design and modification of MTases, develops a widely applicable fluoroethylation reaction scheme, and provides a new approach for preparing drug fluoroethyl analogs.

[0066] The present invention will be further described below in conjunction with embodiments:

[0067] Example 1: Chemical synthesis of FEt-SAM

[0068] Weigh SAH (10 mg, 26 μmol) in a 10 ml round-bottom flask, add acetic acid (200 μL) and formic acid (200 μL) to stir and dissolve, seal, stir and degas in an ice bath and replace with nitrogen (N2). Add FEtI (68 μL, 780 μmol) dropwise with a syringe at 0°C, and wrap the reaction bottle with aluminum foil to protect it from light. Weigh AgClO4 (54 mg, 260 μmol) and dissolve it in 100 μL acetic acid and 100 μL formic acid, and add it dropwise to the above solution at 0°C with a syringe. The mixture is stirred at 0°C for 30 minutes and then transferred to room temperature. After reacting at room temperature for 12 hours, the mixture is cooled to 0°C in an ice bath, and then the same volume of FEtI (68 μL, 780 μmol) as in the initial reaction is added, and AgClO4 (54 mg, 260 μmol) is added in the same manner as before. After stirring at 0℃ for 30min, the mixture was transferred to room temperature and continued to react for 12h. FEtI and AgClO4 were added again. FEtI and AgClO4 were added three times in total. After each addition, the reaction was continued at room temperature for 12h. After the reaction was completed, the reaction solution was quenched with a pre-cooled 0.1% formic acid aqueous solution, and the AgI precipitate was removed by centrifugal filtration. The reaction solution was extracted three times with an equal volume of pre-cooled anhydrous ether to remove excess FEtI in the reaction system. After removing a small amount of anhydrous ether miscible with the aqueous solution by rotary evaporation, the mixture was purified by preparative HPLC using a reverse phase C18 column chromatography SHIMADZUC18 (250mm×4.6mm, 5μm). The mobile phase A was H2O+1‰TFA, and the mobile phase B was CH3CN+1‰TFA. The elution method was gradient elution, the flow rate was 3 mL / min, the injection volume was 5 mL, the detection wavelength was 254 nm and 215 nm dual wavelength detection, and 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 collected and freeze-dried to obtain a white powdery solid (3.1 mg, yield 28%). Target product 1 H NMR(600MHz,D2O)δ8.39(d,J=1.2Hz,2H),6.12(s,1H),4.93(s,1H),4.85(s,2H),4.63–4.4 8(m,2H),4.15–-3.98(m,3H),3.88(d,J=27.1Hz,2H),3.76–3.61(m,2H),2.52–2.30(m,2H). 19 F NMR(564MHz,D2O)δ-217.15-–-217.54(m).LCMS(ESI)calcd.for C 16 H 24 FN6O5S+ [M] + 431.1507,obsd.431.1436.

[0069] The reaction formula is as follows:

[0070]

[0071] Example 2: Chemical synthesis of FEt-SeAM

[0072] According to the same method and steps of chemical synthesis of FEt-SAM, FEt-SeAM was synthesized with SeAH. It was purified by preparative HPLC through reverse phase C18 column chromatography SHIMADZU C18 (250mm×4.6mm, 5μm), mobile phase A was H2O+1‰TFA, and mobile phase B was CH3CN+1‰TFA. The elution method was gradient elution, the flow rate was 3mL / min, the injection volume was 5mL, the detection wavelength was 254nm and 215nm dual wavelength detection, and the elution program was 0-6min 0%B, 6-30min 0-48%B, 30-33min48-90%B, 33-39min 90%B, 39-39.01min 90%-0%B, 39.01-45min 0%B. The purified sample was collected and freeze-dried in a vacuum to obtain a white powder solid (2.3mg, yield 19%). LCMS (ESI) calculation for C 16 H 24 FN6O5Se + [M] + 479.0952,obsd.479.1049.

[0073] The reaction formula is as follows:

[0074]

[0075] Example 3: AclHMT (W27F / P8L / V265W) and MppJ cascade catalysis to produce fluoroethylated phenylpyruvate (FEt-Ppy)

[0076] Purified AclHMT (W27F / P8L / V265W) (final concentration 40 μM), phenylpyruvate C-methyltransferase MppJ (final concentration 80 μM), SeAH (final concentration 100 μM), FEtI (final concentration 2.5 mM), MgCl2 (final concentration 4 mM), DTT (final concentration 2 mM), phenylpyruvate (Ppy, final concentration 1.0 mM) were reacted in a 100 μl solution containing 50 mM HEPES (pH 8.0) in a 30°C water bath. After the reaction was completed, an equal volume of 10% TFA was added to the reaction solution to quench the reaction. The quenched reaction sample was centrifuged at 4°C and 12000 rpm for 30 min using a high-speed refrigerated centrifuge, and the supernatant was analyzed by analytical HPLC using a reverse phase C18 column SHIMADZU C18 (150 mm×4.6 mm, 2.5 μm). The same reaction system was scaled up to prepare the target product, and the product was purified by preparative HPLC through reverse phase C18 column chromatography SHIMADZU C18 (250 mm × 4.6 mm, 5 μm), mobile phase A was H2O + 1‰ TFA, mobile phase B was CH3CN + 1‰ TFA. The elution method was gradient elution, the flow rate was 3 mL / min, the injection volume was 5 mL, the detection wavelength was 254 nm and 280 nm dual wavelength detection, and the elution program was 0-3min 0% B, 3-21min 0-15% B, 21-35min 15-35% B, 35-50min 75-90% B, 50-50.01min 90-100% B, 50.01-53min 100% B, 53-53.01min 100-0% B, 53.01-60min 0% B. The purified sample was collected and freeze-dried to obtain the product (5 mg, yield 60%). 1 H NMR (600MHz, CD3OD) δ7.38--7.16(m,5H),4.64(t,J=7.5Hz,1H),4.35--4.00(m,1H),3.28--3.16(m,1H),2.59--2.33(m,1H),2.25--1.83(m,1H). 19 F NMR (564MHz, CD3OD) δ-221.35 (tdd, J=47.9, 32.5, 15.0Hz). 19 F NMR(564MHz,CD3OD)δ-222.04(tdd,J=47.8,30.3,19.8Hz).LCMS(ESI)calcd.for C 11 H 11 FO3[MH] 1- 209.0619, obsd.209.0722. 19F NMR showed that the obtained product was a mixture of two isomers with a ratio of 2:1, indicating that the stereoselectivity of MppJ for accepting fluoroethyl to generate the product was 67%.

[0077] The reaction formula is as follows:

[0078]

[0079] Example 4 AclHMT (W27F / P8L / V265W) and DnrK mutant cascade catalysis to produce fluoroethylated carminomycin

[0080] In the SAM-bound crystal structure of Dnrk (PDB: 7phd), there is a conserved key amino acid L160 near the SAM methyl group, and the distance between L160 and the methyl group is Since the fluoroethyl group is larger than the methyl group, the space of the active pocket must be increased to accommodate the fluoroethyl group. Therefore, four mutants, L160A / V / I / S, were constructed in this example and their activities were tested. All four mutants had higher activity than the wild-type DnrK, and the L160A mutant showed the best activity.

[0081] Purified AclHMT (W27F / P8L / V265W) (final concentration 40 μM), carminomycin 4-O-methyltransferase variant DnrK (L160A) (final concentration 80 μM), SeAH (final concentration 100 μM), FEtI (final concentration 2.5 mM), carminomycin (final concentration 1.0 mM) were reacted in 100 μl of a solution containing 50 mM Tris-HCl (pH 8.0) in a 30°C water bath. After the reaction was completed, an equal volume of 10% TFA was added to the reaction solution to quench the reaction. The quenched reaction sample was centrifuged at 4°C and 12000 rpm for 30 min using a high-speed refrigerated centrifuge, and the supernatant was analyzed by analytical HPLC on a reverse phase C18 column SHIMADZU C18 (150 mm×4.6 mm, 2.5 μm). The same reaction system was scaled up to prepare the target product, and the product was purified by preparative HPLC through reverse phase C18 column chromatography SHIMADZU C18 (250 mm × 4.6 mm, 5 μm), mobile phase A was H2O + 1‰ TFA, and mobile phase B was CH3CN + 1‰ TFA. The elution method was gradient elution, the flow rate was 3 mL / min, the injection volume was 5 mL, the detection wavelength was 215 nm and 254 nm dual wavelength detection, and the elution program was 0-3 min 0% B, 3-20 min 0-14% B, 20-29 min 14-56% B, 29-48 min 56-75% B, 48-48.01 min 75%-95% B, 48.01-51 min 95% B, 51-51.01 min 95-0% B, 51.01-59 min 0% B. The purified sample was collected and freeze-dried in a vacuum to obtain a magenta solid product (7.1 mg, yield 84%). 1 H NMR(400MHz,D2O)δ7.58(s,1H),7.33(s,2H),5.40(s,1H),4.93(s,1H),4.72 (s,1H),4.35(d,J=30.0Hz,2H),4.23(q,J=6.6Hz,1H),3.81(d,J=2.8Hz,1H), 3.71–-3.49(m,2H),2.80(d,J=18.1Hz,1H),2.61(d,J=18.2Hz,1H),2.41(s, 3H), 2.19 (d, J = 14.6Hz, 1H), 2.06 (s, 1H), 1.96 (s, 2H), 1.27 (d, J = 6.4Hz, 3H). 19 F NMR (564MHz, D2O) δ-221.81 (tt, J = 47.4, 30.0Hz). 13C NMR(100MHz,D2O)δ215.05,185.75,162.79,159.33,155.50,153.92,136.42 ,133.82,133.65,120.52,119.73,119.15,110.42,110.30,98.89,82.22(d, 1 J CF =166.0Hz),75.94,71.99,68.71(d,J CF =17.1Hz),66.85,66.15,62.40,46.64,34.78,31.33,27.55,24.18,15.59.LCMS(ESI)calcd.for C 28 H 30 FNO 10 [M+H] 1+ 560.1927,obsd.560.1903.

[0082] The reaction formula is as follows:

[0083]

[0084] According to the above method, three mutants of carminomycin 4-O-methyltransferase variants DnrKL160V, L160I and L160S were used for reaction, and the yields of the obtained magenta solid product (fluoroethylated carminomycin) were 20%, 7% and 45%, respectively.

[0085] Example 5: Cascade catalysis of AclHMT (W27F / P8L / V265W) and NovO (I6V) to produce fluoroethylated coumarin

[0086] When the wild-type coumarin C-methyltransferase NovO undergoes a fluoroethyl transfer reaction with the HMT cascade, only a small amount is converted into the fluoroethylated product and the vinyl byproduct obtained by the elimination of F and proton.

[0087] Although there is no crystal structure of NovO bound to SAM, a conserved isoleucine residue (I6) was found in the active site of the structure of NovO bound to SAH (PDB: 5mgz.1). I6 of NovO corresponds to the Leu residue in Dnrk, which may interact with the methyl group of SAM. Therefore, the isoleucine residue can be replaced with a smaller amino acid, and three NovO mutants I6A / V / S were constructed. The results showed that although both I6A and I6S mutants lost the activity of accepting fluoroethyl to generate products and no target product was generated, the I6V mutant significantly improved the activity of the fluoroethylation reaction.

[0088] Purified AclHMT (W27F / P8L / V265W) (final concentration 40 μM), coumarin C-methyltransferase NovO variant NovO (I6V) (final concentration 80 μM), SeAH (final concentration 100 μM), FEtI (final concentration 2.5 mM), coumarin (final concentration 1.0 mM) were reacted in 100 μl of a solution containing 50 mM PBS (pH 6.5) in a water bath at 30°C. After the reaction was completed, an equal volume of 10% TFA was added to the reaction solution to quench the reaction, and the quenched reaction solution was vacuum freeze-dried and then reconstituted with a 20% DMSO solution. The sample was centrifuged at 4°C and 12000 rpm for 30 minutes using a high-speed refrigerated centrifuge, and the supernatant was analyzed by analytical HPLC on a reversed-phase C18 column SHIMADZU C18 (150 mm×4.6 mm, 2.5 μm). The same reaction system was scaled up to prepare the target product, and the product was purified by preparative HPLC through reverse phase C18 column chromatography SHIMADZU C18 (250 mm × 4.6 mm, 5 μm), mobile phase A was H2O + 1‰ TFA, mobile phase B was CH3CN + 1‰ TFA. The elution method was gradient elution, the flow rate was 3 mL / min, the injection volume was 5 mL, the detection wavelength was 254 nm and 310 nm dual wavelength detection, and the elution program was 0-3 min 0% B, 3-21 min 0-15% B, 21-35 min 15-35% B, 35-50 min 75-90% B, 50-50.01 min 90-100% B, 50.01-53 min 100% B, 53-53.01 min 100-0% B, 53.01-60 min 0% B. The purified sample was collected and freeze-dried to obtain a white solid product (3.8 mg, yield 28%). 1 HNMR (600MHz, DMSO-d6) δ11.79(s,1H),10.69(s,1H),9.43(s,1H),8.01(d,J=7.0Hz,2H),7.66(d,J=8.7Hz,1H),7.59(d,J=14.8 Hz,1H),7.52(t,J=7.7Hz,2H),6.92(d,J=8.7Hz,1H),4.64(t,J=6.8Hz,1H),4.56(t,J=6.8Hz,1H),3.15(dt,J=20.4,6.8Hz,2H). 19 F NMR(564MHz,DMSO-d6)δ-213.90(tt,J=47.7,31.6Hz).LCMS(ESI)calcd.for C 18 H 14 FNO5[M+H] 1+344.0929, obsd.344.0902.

[0089] The reaction formula is as follows:

[0090]

[0091] Example 6 Using SAH as a substrate, AclHMT (W27F / P8L / V265W) and nicotinamide N-methyltransferase NNMT cascade catalyze the production of fluoroethylated nicotinamide

[0092] Purified AclHMT (W27F / P8L / V265W) (final concentration 40μM), nicotinamide N-methyltransferase NNMT (final concentration 80μM), SAH (final concentration 100μM), FEtI (final concentration 2.5mM), nicotinamide (NCA, final concentration 1.0mM) were reacted in 100μl of a solution containing 100mM Tris-HCl (pH 8.0) in a water bath at 30°C. After the reaction was completed, an equal volume of 10% TFA was added to the reaction solution to quench the reaction. The quenched reaction sample was centrifuged at 4°C and 12000rpm for 30min using a high-speed refrigerated centrifuge, and the supernatant was analyzed by analytical HPLC on a reverse phase C18 column SHIMADZU C18 (150mm×4.6mm, 2.5μm). The results showed that 18% of the substrate was converted into fluoroethylated nicotinamide product under this cascade reaction system. 1 H NMR (600MHz, D2O) δ9.40 (s, 1H), 9.11 (d, J = 5.8Hz, 1H), 9.00 (d, J = 8.2Hz, 1H), 8.29 (t, J = 7.3Hz, 1H), 5.16--4.97 (m, 4H). 19 FNMR(564MHz,D2O)δ-223.69(tt,J=46.3,26.9Hz). 13 CNMR(100MHz,D2O)δ165.67,147.08,144.94,144.66,134.03,128.51,81.59(d, 1 J CF =170.3Hz),61.93(d,J CF =18.9Hz).LCMS(ESI)calcd.for C8H 10 FN2O + [M] + 169.0772,obsd.169.0838.

[0094] The reaction formula is as follows:

[0095]

[0096] Comparative Example 1

[0097] According to the method of Example 4, wild-type carminomycin 4-O-methyltransferase DnrK was used to carry out a cyclase cascade reaction. The results showed that the activity of catalyzing the production of fluoroethylated products was very weak, and almost no obvious product peak was observed in the cyclase cascade reaction HPLC. The yield of fluoroethylated carminomycin was less than 1%.

[0098] Comparative Example 2

[0099] According to the method of Example 5, the wild-type coumarin C-methyltransferase NovO was used to carry out the cyclase cascade reaction. It was found that the yield of the white solid product, ie, fluoroethylated coumarin, was only 8%.

[0100] The above results show that the SeAH / SAH-based HMT-MTase cyclase cascade reaction provided by the present invention can realize the cyclic regeneration of FEt-SeAM / FEt-SAM analogs and the fluoroethylation reaction of active molecules in the presence of a catalytic amount of raw material SeAH / SAH, wherein the FEt-SeAM obtained by using Se instead of S in the present invention well avoids the problem of fluoroethyl elimination of FEt-SAM to produce vinyl SAM, so FEt-SeAM shows better activity in the fluoroethylation reaction of the substrate. Furthermore, compared with the wild-type methyltransferase and other mutants, the methyltransferase mutant provided by the present invention can significantly improve the yield of the product and more efficiently realize the fluoroethylation reaction.

[0101] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A fluorocarbon-based SAM analogue, characterized in that: It has the structure shown in Formula I: Wherein, X is sulfur or selenium; R is a fluorine-substituted C2~C 10 Saturated or unsaturated straight chain or branched hydrocarbon group.

2. The fluorocarbon-based SAM analogue according to claim 1, characterized in that C2~C 10 The saturated or unsaturated straight-chain or branched hydrocarbon group is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl.

3. The fluorocarbon-based SAM analogue according to claim 1, characterized in that Has any of the following structures:

4. Use of the SAM analogue according to any one of claims 1 to 3 as a donor for fluoroalkylation of an acceptor substrate.

5. A fluoroalkylation cyclase cascade reaction, characterized in that: Steps 1) to 2) are performed simultaneously in the same system: 1) catalyzing the reaction of the SAH analogue of formula II with a fluorocarbon-based reagent by halogen methyltransferase to generate the fluorocarbon-based SAM analogue of formula I; Wherein, X is sulfur or selenium; 2) the fluoroalkyl SAM analogue represented by formula I reacts with a receptor substrate under the action of a SAM-dependent methyltransferase to obtain a fluoroalkylated receptor product and a SAH analogue represented by formula II; wherein the fluorocarbon SAH analogue represented by formula II obtained in step 2) is recycled for the reaction in step 1); The hydrocarbon group in the fluorocarbon-based reagent is a C2-C10 straight-chain or branched hydrocarbon group.

6. The cyclase cascade reaction according to claim 5, characterized in that The SAM-dependent methyltransferase in step 2) includes any one of the following: Wild-type phenylpyruvate C-methyltransferase MppJ, wild-type coumarin C-methyltransferase NovO, wild-type nicotinamide N-methyltransferase NNMT.

7. The cyclase cascade reaction according to claim 5, characterized in that The halogen methyltransferase in step 1) includes one of the following mutants of the wild-type AclHMT enzyme: W27F / P8L mutant, W27F / P8L / V265W mutant.

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