S-adenosine homocysteine analogue, S-adenosine-L-methionine analogue and application of S-adenosine homocysteine analogue and S-adenosine-L-methionine analogue

Abstract

The invention provides an S-adenosine homocysteine (SAH) analogue, an S-adenosine-L-methionine (SAM) analogue and application thereof. The preparation method comprises the following steps: synthesis of the SAH analogue and a fluoroalkyl SAM analogue, fluoromethylation modification of bioactive molecules by an HMT-MT cyclase cascade reaction system based on the fluoroalkyl SAM analogue, and construction of a fluoro derivative of a clinical drug synthesized by a one-pot enzymatic method. The SAH analogue adopted by the invention can effectively avoid spontaneous degradation of the corresponding SAM analogue, and is used for constructing a cascade reaction system based on HMT-MT cyclase, so that the yield of a fluoromethylation product is increased; it is confirmed that the fluoro-intermediate generated by MT can be accepted by downstream biosynthetase, so that a fluoro-derivative of a clinical drug synthesized by a one-pot enzymic method is constructed.

Description

Technical Field

[0001] This invention relates to the field of biocatalysis technology, and in particular to S-adenosine homocysteine ​​analogs, S-adenosine-L-methionine analogs, and their applications. Background Technology

[0002] Fluorine's unique van der Waals radius, electronegativity, and nonpolarity distinguish it from other halogens. The substitution of carbon-hydrogen bonds for carbon-fluorine bonds subtly affects the dipole moment, lipophilicity, metabolic stability, membrane permeability, and bioavailability of organic molecules. Organofluorine compounds constitute a significant proportion of the materials, pharmaceutical, and agrochemical industries, with new fluorine-containing molecules being approved for market each year. The importance and potential uses of fluorine compounds have generated a strong demand for fluorination reaction methods, greatly promoting the development of organofluorine chemistry, and leading to an increasing number of chemical synthesis methods being applied to prepare fluorine-containing molecules.

[0003] Because the selective direct construction of carbon-fluorine bonds is difficult, fluoroalkylation is used in synthetic chemistry as an alternative to introduce fluorine atoms into molecular structures. Currently, most fluoroalkylating reagents and precursors focus on trifluoroalkyl, difluoroalkyl, and fluoroalkyl groups to mimic motifs such as methyl and hydroxymethyl commonly found in bioactive molecules and drugs. Despite significant progress, most reagents can only transfer fluoroalkyl groups to heteroatoms (N-, O-, S-, P-) of substrate molecules. Some transition metal-catalyzed reactions can achieve carbon-carbon fluoroalkylation, but with low efficiency. These chemical synthesis methods still have limitations in chemoselectivity, regioselectivity, and stereoselectivity. Furthermore, most fluoroalkylating reagents are derived from haloalkanes, and the environmental pressures from their production cannot be ignored.

[0004] S-adenosyl-L-methionine (SAM) is a widely distributed methyl donor in organisms. As a cofactor of SAM-dependent methyltransferases (MTs), it performs various methylation modifications related to life activities. Receptors include small molecules, nucleic acids, lipids, and proteins. The typical reaction mechanism is as follows: a substrate molecule with a nucleophilic group attacks the side-chain methyl group of SAM to undergo S-methylation. N 2. Nucleophilic reaction yields methylated products and S-adenosylhomocysteine ​​(SAH). Biocatalytic fluorination reactions offer advantages such as high efficiency, high selectivity, and high atom economy, but are rarely reported. Recent studies have explored the use of non-natural SAM analogs in engineered MT catalysis for fluoroalkyl transfer, providing a novel biocatalytic technology for the production of fluorinated derivatives of natural products.

[0005] In 2021, Seebeck et al. used halide methyltransferase (HMT) to catalyze the reaction of SAH and fluoroiodomethane (FCH2I) to generate S-adenosyl-L-(fluoromethyl)homocysteine ​​(F-SAM) (Seebeck F P, et al. Angew. Chem. Int. Ed. 2021, 60, 27178–27183). However, due to the instability of this SAM analog under physiological conditions, its structure was only deduced from the degradation products. Building on this, they constructed a cyclic enzyme cascade system by coupling MT and HMT, using the in-situ generated F-SAM as a fluoromethyl donor to fluoromethylate N-, O-, C-containing substrates. Subsequently, HMT recovered SAH and regenerated F-SAM, thus overcoming the cofactor loss caused by the spontaneous degradation of F-SAM.

[0006] Prior patent CN119709915A discloses a method for preparing fluoromethyl products, using HMT and a B12-dependent SAM radical methyltransferase to form a cascade reaction system, similarly achieving fluoroalkyl modification on the inactive carbon through in-situ generation of F-SAM via SAH and fluoroiodomethane. This method also discloses a novel fluoromethyl B12 intermediate.

[0007] Prior patent CN118048338A discloses SAM analogs containing fluoroethyl groups, which include fluoroethyl side chains and thionium or selenium centers. Among them, fluoroethyl selenized SAM (FEt-SeAM) can avoid the elimination problem of fluoroethyl groups, and combines with... Aspergillus clavatus A cyclic enzyme cascade system constructed from the AclHMT mutant and the MT mutant efficiently achieves the fluoroethylation of nucleophiles such as O-, N-, S-, and C-. Its claims also include mutants of SAM-dependent methyltransferases.

[0008] All the above schemes employ an HMT-MT cascade strategy to achieve the cyclic regeneration of SAM analogs. The catalytic efficiency of this system is limited by the kinetic characteristics of HMT and MT, but the key factor limiting the number of cycles is the stability of the SAM analogs. SAM exhibits inherent chemical instability (a half-life of approximately 16 hours at pH 8), primarily degrading spontaneously through two parallel pathways: intramolecular cyclization leads to the formation of 5'-deoxy-5'-methylthioadenosine (MTA) and homoserine lactone (HSL), while depurination leads to the generation of adenine and S-ribosylmethionine. The introduction of fluorine atoms into F-SAM accelerates the decomposition process, and the resulting 5'-deoxy-5'-fluoromethylthioadenosine (F-MTA) further decomposes into 5'-deoxy-5'-mercaptoadenosine, formaldehyde, and fluorides. Therefore, the key to improving the efficiency of fluoroalkyl transfer reactions lies in obtaining stable fluoroalkyl SAM analogs.

[0009] To improve the stability of SAM analogs, functional group substitution based on isosteric groups has been widely adopted, effectively or even completely suppressing spontaneous degradation (Thorson, J. S, et al. ACS Chem. Biol. 2016, 11 (9), 2484-2491; Thorson, J. S, et al. ACS Chem. Biol. 2020, 15 (3), 695-705; Höbartner, C., et al. Nature Chem. 2023, 15 (11), 1523-1531). In 2023, Booker et al. synthesized FMeTeSAM, an F-SAM analog with thionium replaced by tellurium (Booker, SJ, et al. ACS Cent.Sci. 2023, 9 (5), 905-914). The decay of this analog was not mentioned, and it is compatible with various MTs to complete the enzymatic fluoromethyl transfer reaction. However, the enzyme kinetics of some reactions showed that the Te atom might interfere with the binding of cofactors to enzymes. Furthermore, the lengthy chemical synthesis steps reduced the atom economy of this approach. In the same year, Dong et al. designed the decarboxylated F-SAM analog F-dcSAM, which can be synthesized from the decarboxylated SAH analog (dcSAH) via chemical and enzymatic methods (Dong M., et al. ACS Catal. 2023, 13 (20), 13729-13734). This SAM analog exhibited stronger stability than F-SAM and could be integrated into the HMT-MT cascade reaction system to achieve high fluoroalkyl transfer efficiency. However, the absence of the carboxyl group might interfere with the binding conformation of F-dcSAM, causing unexpected chemoselectivity switching in some enzyme reactions with strict substrate recognition.

[0010] In summary, enzyme-catalyzed fluoroalkyl transfer reactions are usually located in the later stages of the biosynthetic pathway, and it remains unclear whether fluorinated intermediates of bioactive molecules can be recognized by downstream post-modifying enzymes. Summary of the Invention

[0011] The purpose of this invention is to provide S-adenosylhomocysteine ​​analogs, S-adenosyl-L-methionine analogs, and their applications. It involves structurally modifying fluoroalkyl SAM analogs to overcome spontaneous decomposition pathways, exhibiting superior chemical stability compared to F-SAM and F-dcSAM, thereby supporting their high efficiency in MT-dependent fluoroalkyl transfer reactions. This invention provides an SAH analog for the synthesis of fluoroalkyl SAM analogs, enabling them to be recognized by MT and maintaining correct regioselectivity and stereoselectivity for the fluoromethylation of clinical drug biosynthetic precursors. The SAH analog provided by this invention can be used to construct an HMT-MT cyclic enzyme cascade reaction system based on fluoroalkyl SAM analogs, thereby further reducing the decomposition of fluoroalkyl SAM analogs and improving the yield of fluoromethylated products. This invention requires confirmation that the fluorinated intermediates generated by MT can be accepted by downstream biosynthetic enzymes, optimizing reaction conditions, and thus constructing a one-pot enzymatic synthesis of fluorinated derivatives for clinical drugs.

[0012] To achieve the above objectives, the present invention provides S-adenosine-L-methionine analogs, which have the structure shown in Formula I: ; Formula I R1 is either a terminal amide or a tetrazolium; R2 is one of the fluorine-substituted C1~C10 saturated or unsaturated straight-chain or branched alkanes; X is NH, O, S, or CH; Y represents N and C; Z represents N and C.

[0013] Preferably, the fluorinated C1-C10 saturated or unsaturated straight-chain or branched alkanes are methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl, etc.

[0014] Preferably, the SAM analogue has any of the following structures: .

[0015] S-adenosylhomocysteine ​​analogues, SAH analogues have the structure shown in Formula II: ; Formula II R1 is either a terminal amide or a tetrazolium; X is NH, O, S, or CH; Y represents N and C; Z represents N and C; n is 1 or 2.

[0016] Preferably, the SAH analogue has any of the following structures: .

[0017] The chemical synthesis method of S-adenosylhomocysteine ​​includes the following steps: Steps for preparing the intermediate (compound B) from the compound shown in Formula A; Steps for preparing intermediates from the compound shown in Formula B: the compound shown in Formula C; Steps for preparing intermediates from the compound shown in formula C: the compound shown in formula D; Steps for preparing intermediates from the compound shown in formula D: the compound shown in formula E; The steps for preparing intermediates from compounds of formula F: compounds of formula G; The steps for preparing the compound of formula II from the compound shown in formula C and the compound shown in formula G; The steps for preparing the compound of formula II from the compound shown in formula E and the compound shown in formula G; .

[0018] Since MT typically only recognizes (S)-SAM analogs as substrates, the fluoromethyl SAM analogs F-amSAM, F-tSAM, and F-7dz-tSAM provided in this embodiment of the invention are synthesized enzymatically to control the enantiomeric specificity of the thionium center. Specifically, the synthesis involves using the corresponding SAH analogs to synthesize and purify fluoroiodomethane via AclHMT catalysis.

[0019] The specific enzymatic synthesis scheme is as follows: Purified AclHMT (200 μM), SAH analogue (1 mM), and fluoroiodomethane (10 mM) were thoroughly mixed in a buffer solution and reacted at 90 rpm for 1 hour in a shaker at 30 °C. For reactions using amide-SAH and tet-SAH, the buffer solution was 100 mM sodium phosphate (pH 6.5). For reactions using 7dz-tSAH, the buffer solution was 100 mM Tris-HCl (pH 8.0). After the reaction was complete, an equal volume of 10% (v / v) trifluoroacetic acid (TFA) was added to quench the reaction. The quenched sample was centrifuged at 4 °C and 10,000 rpm for 10 minutes using a cryogenic centrifuge. The supernatant was filtered through a PES needle filter, and the filtrate was prepared by preparative high-performance liquid chromatography (HPLC) using acetonitrile / water as the mobile phase. The collected target fraction was freeze-dried under vacuum to obtain an oily product.

[0020] The reaction formula is as follows: This invention also provides a cyclic enzyme cascade reaction based on the SAM analog shown in Formula I, in which steps (1) to (2) are performed simultaneously in the same system: (1) The SAH analog of formula II is reacted with fluoroalkyl iodide by halogen methyltransferase to generate the SAM analog of formula I. (2) The SAM analog shown in Formula I is fluoroalkylated with the receptor substrate under the action of methyltransferase to obtain the fluoroalkylated receptor product and the SAH analog shown in Formula II. The SAH analogue of formula II obtained in step (2) is used in the reaction of step (1).

[0021] Preferably, the receptor substrate is a bioactive molecule, including but not limited to small molecule compounds, nucleic acids, lipids or proteins, specifically luteolin and N-acetyl-O-methylaminoacyl-5-hydroxytryptamine; Halogen methyltransferases are derived from Aspergillus clavatus AclHMT, including but not limited to those from Arabidopsis thaliana , Paraburkholderia xenovorans、Vibri parhaemolyticus、 Ustilago maydis、Kordia algicida、Synechococcus elongates Homologous to.

[0022] The methyltransferases are CiCOMT10 and PsmD, including but not limited to O-methyltransferase, N-methyltransferase, S-methyltransferase, and C-methyltransferase.

[0023] The specific cascade reaction scheme is as follows: The reaction system components include: purified AclHMT, 7dz-tSAH, receptor substrate, fluoroiodomethane, buffer solution, and another protein; the other protein is either CiCOMT10 or PsmD. When the other protein was 50 μM CiCOMT10, the concentrations were: AclHMT 50 μM, 7dz-tSAH 50 μM, fluoromethyl iodide 10 mM, substrate 500 μM luteolin, buffer 50 mM Tris-HCl (pH 7.5), with the addition of 10 mM magnesium chloride and 10% (v / v) dimethyl sulfoxide; when the other protein was 50 μM PsmD, the concentrations were: AclHMT 25 μM, 7dz-tSAH 50 μM, fluoromethyl iodide 15 mM, substrate 1 mM N-acetyl-O-methylaminoacyl-5-hydroxytryptamine, buffer 100 mM potassium phosphate (pH 7.5).

[0024] The cascade reactions were carried out in a 30°C water bath for 16 hours. After the reactions were completed, the reaction containing CiCOMT10 was quenched by adding an equal volume of methanol to the solution, and the reaction containing PsmD was quenched by adding an equal volume of 10% (v / v) trifluoroacetic acid to the solution. The quenched samples were centrifuged at 4°C and 12,000 rpm for 30 minutes using a cryogenic high-speed centrifuge, and the supernatant was analyzed by high-performance liquid chromatography.

[0025] A one-pot enzymatic method for preparing diosmin fluorinated derivatives is provided, which is based on the scale-up of the HMT-CiCOMT10 cyclic enzyme cascade of SAH analogs shown in Formula II to prepare fluorinated intermediate 1. Using fluorinated intermediate 1 as the acceptor substrate, in stepwise addition of CiUGT11 and CiRhaT-GD 4X In the cascade system, rutinylation modification is carried out sequentially to generate fluorinated diosmin.

[0026] The specific reaction plan is as follows: The HMT-CiCOMT10 cyclic enzyme cascade reaction was scaled up to 20 mL. After the reaction, an equal volume of methanol was added to quench the reaction. The quenched sample was centrifuged at 4°C and 10,000 rpm for 10 minutes using a low-temperature high-speed centrifuge. The supernatant was collected and filtered through a Nylon 66 needle filter. The filtrate was then subjected to preparative high-performance liquid chromatography (HPLC) with acetonitrile / water as the mobile phase. The collected target fraction was freeze-dried under vacuum to obtain fluorinated intermediate 1. This fluorinated intermediate (1 mM) was added to a buffer solution containing CiUGT11 (44 μM), UDP-glucose (2 mM), magnesium chloride (10 mM), and DL-dithiothreitol (10 mM). After thorough mixing, the mixture was incubated at 37°C for 1.5 hours. Further, CiRhaT-GD was added to the reaction solution. 4X (Final concentration 40 μM) and UDP-rhamnose (final concentration 1 mM) were thoroughly mixed and incubated at 37°C for 4.5 hours. After the reaction was complete, an equal volume of methanol was added to quench the reaction. The quenched sample was then subjected to the above post-processing and purification procedures to obtain fluorodiosmin.

[0027] A one-pot enzymatic method for preparing fluorinated derivatives of physostigmine is based on the scale-up of the HMT-PsmD cyclic enzyme cascade reaction of the SAH analog shown in Formula II to prepare fluorinated intermediate 2. Using fluorinated intermediate 2 as the acceptor substrate, stepwise methylation and deacetylation were carried out in an enzyme cascade system of stepwise addition of PsmC and PsmB to generate fluorophysostigmine.

[0028] The specific reaction plan is as follows: The HMT-PsmD cyclic enzyme cascade reaction was scaled up to 20 mL. After the reaction, an equal volume of 10% (v / v) trifluoroacetic acid was added to quench the reaction. The quenched sample was centrifuged at 4°C and 10,000 rpm for 10 minutes using a cryogenic centrifuge. The supernatant was filtered through a PES syringe filter, and the filtrate was prepared by preparative high-performance liquid chromatography using acetonitrile / water as the mobile phase. The collected target fraction was freeze-dried under vacuum to obtain fluorinated intermediate 2. This fluorinated intermediate (2 mM) was added to a buffer solution containing 4 mM SAM, 75 μM PsmC, and 5% (v / v) glycerol, and the reaction was allowed to stand at 37°C for 6 hours. Subsequently, SAM (final concentration 2 mM) and PsmB (final concentration 50 μM) were added, and the reaction was continued for another 8 hours. After the reaction, trifluoroacetic acid was added to the solution to a final concentration of 5% (v / v) to quench the reaction. The quenched sample was subjected to the above post-processing and purification to obtain fluorophysostigmine.

[0029] Therefore, the present invention utilizes the above-mentioned S-adenosylhomocysteine ​​analogue and S-adenosyl-L-methionine analogue in enzymatic fluoromethylation reaction, with the following technical effects: For SAM analogs with fluorinated alkyl side chains, the fluorine atom promotes their degradation. This invention weakens the degradation caused by intramolecular cyclization of fluorinated alkyl SAM analogs by replacing the carboxyl group with a weakly nucleophilic bioisosteric group, such as a primary amide or tetrazolium. Bioisosteric substitution retains hydrogen bond acceptor atoms similar to the carboxyl group, reducing the impact of functional group substitution on enzyme adaptability. Furthermore, some adenine analogs form more stable glycosidic bonds with ribose, effectively reducing degradation caused by depurination.

[0030] The stability of the fluoromethyl SAM analog described in Example 2 was tested, and the results showed that it had better stability than F-dcSAM under the preferred enzyme reaction conditions, specifically as follows: Figure 1 As shown, F-7dz-tSAM has a significantly extended half-life of 713 min, making it a preferred stable fluoromethyl SAM analog.

[0031] Specific implementation results demonstrate that F-7dz-tSAM provided by this invention can serve as a stable fluoromethyl donor, enabling the fluoromethylation of clinical drug biosynthetic precursors using MT while maintaining regioselectivity and stereoselectivity consistent with the natural reaction. Furthermore, the HMT-MT cyclic enzyme cascade reaction system based on 7dz-tSAH can regenerate F-7dz-tSAM in situ. Using a catalytic amount of SAH analogue can improve the yield of fluoromethyl products and enhance the atom economy of the reaction. Compared with existing chemical synthesis methods, this enzymatic reaction is milder and simpler to operate, providing a new route for the preparation of drug fluoromethyl derivatives.

[0032] Fluorinated intermediates generated by cyclic enzyme cascade reactions can be accepted and modified by downstream enzymes in the biosynthetic pathway to produce fluorinated derivatives of clinical drugs (such as diosmin and physostigmine). This invention provides a one-pot enzymatic method for the production of fluorinated diosmin and fluorinated physostigmine, which can simulate the native environment of biosynthesis. By completing multiple steps in the same reaction system through a multi-enzyme cascade, it avoids cumbersome intermediate separation steps, reduces waste generation, and is characterized by high efficiency, greenness, and sustainability.

[0033] In vitro activity assays showed that fluorinated intermediate 2 had an IC50 value for butyrylcholinesterase (BChE). 50 The value was 0.864 μM, compared to the IC50 of the positive reference physostigmine. 50 A concentration of 6.36 μM offers a significant advantage and represents a potential BChE inhibitor. Specific results are as follows... Figure 2 As shown. Attached Figure Description

[0034] Figure 1 This is a graph showing the stability results of the fluoromethyl SAM analog in Example 2 in 100 mM Tris-HCl buffer at 30°C and pH=8. Figure 2 This is a graph showing the results of fluorinated intermediate 2 and physostigmine inhibiting AChE and BChE in Example 7; Figure 2 (A) is a graph showing the results of fluorinated intermediate 2 and physostigmine inhibiting AChE; Figure 2 (B) is a graph showing the results of fluorinated intermediate 2 and physostigmine inhibiting BChE. Detailed Implementation

[0035] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0036] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0037] In a specific implementation, hmm Genes originate from Aspergillus clavatus (GenBank:EAW10780.1), comt10 Genes originate from Chrysanthemum indica (GenBank: WIF29799.1), ugt11 Genes originate from Chrysanthemum indica (GenBank: WIF29794.1), rhat Genes originate from Chrysanthemum indica (GenBank: UYE98987.1), psmb Genes originate from Streptomyces gray-brown (GenBank: AHL44340.1), psmc Genes originate from Streptomyces griseofuscus (GenBank: AHL44341.1), psmd Genes originate from Streptomyces griseofuscus (GenBank:AHL44342.1). comt10, ugt11, rhat The genes encode the proteins CiCOMT10, CiUGT11, and CiRhaT-GD, respectively. 4X It was constructed on the pMAL-c5x vector, and the construction method, protein expression and purification were consistent with the literature (Ma, D., et al. Cell Rep. 2024, 43 (2), 113725). hmt, psmb, psmc, psmd The genes encode proteins AclHMT, PsmC, PsmB, and PsmD, respectively. After codon optimization, they were synthesized by the supplier and constructed into pET-28a(+) and pET-21a(+) vectors, ultimately yielding plasmids pET-28a(+)-AclHMT, pET-28a(+)-PsmB, pET-21a(+)-PsmC, and pET-21a(+)-PsmD. Expression and purification of AclHMT were performed according to the literature (Hammer SC, et al. Angew. Chem. Int. Ed. 2021, 60, 5554–5560). Expression and purification of PsmB and PsmC were performed according to the literature (Zhang, W., et al. Angew. Chem. Int. Ed. 2013, 53 (1), 136-139). The expression and purification of PsmD were performed according to the literature (Pietruszka, J., et al. Angew. Chem. Int. Ed. 2021, 60 (43), 23412-23418).

[0038] Example 1: Synthesis of SAH analogs (1) Synthesis of N-Fmoc-homocysteine ​​(compound B-1) In a 250 mL round-bottom flask, 60 mL of water, sodium carbonate (1.6 g, 10 mmol), and L-homocysteine ​​(compound A-1, 2.8 g, 10 mmol) were added. Under vigorous stirring, 60 mL of dioxane and 9-fluorenylmethyl-N-succinimide carbonate (6.74 g, 20 mmol) were added. The reaction mixture was stirred overnight at room temperature until a clear liquid was formed. Most of the dioxane was removed by rotary evaporation. The aqueous phase was washed twice with 60 mL of anhydrous diethyl ether, and the pH was adjusted to 4 with citric acid. The solid-liquid mixture was extracted with ethyl acetate (3 × 60 mL). The combined organic phases were washed successively with water and saturated NaCl, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation to give 6.1 g of white solid product B-1, in 86% yield.

[0039] 1 H NMR (500 MHz, DMSO- d6 ) δ 7.88 (d, J = 7.5 Hz, 4H), 7.73 –7.64 (m,6H), 7.42 (t, J = 7.5 Hz, 4H), 7.33 (t, J = 7.5 Hz, 4H), 4.32 (dd, J = 7.2, 2.3 Hz, 4H), 4.24 (t, J = 7.0 Hz, 2H), 4.15 –4.11 (m, 2H), 2.81 –2.73 (m, 4H), 2.19 –2.12 (m, 2H), 2.02 –1.96 (m, 2H). 13 C NMR (126 MHz, DMSO- d6 ) δ 173.67, 156.37, 144.01, 143.95, 140.93,140.91, 127.83, 127.25, 125.44, 125.41, 120.27, 120.25, 65.80, 52.67, 46.87,34.32, 30.46. ESI-MS: m / z 711.2 [MH] - . (2) Synthesis of compound C-1 In a 25 mL round-bottom flask, 8 mL of pyridine, compound B-1 (710 mg, 1 mmol), and ammonium bicarbonate (390 mg, 5 mmol) were added. The flask was sealed with a flip-top stopper, and di-tert-butyl dicarbonate (590 mg, 2.7 mmol) dissolved in 2 mL of pyridine was added using a syringe while stirring. The mixture was stirred at room temperature for 5 hours under sealed conditions, and the conversion of the starting material was monitored by TLC. The mixture was then diluted with ethyl acetate. The organic phase was washed successively with 5% H₂SO₄ and saturated NaCl, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation to obtain an oily crude product. The crude product was slurried with a dichloromethane / petroleum ether mixture, filtered, and the filter cake was washed with petroleum ether and dried under vacuum to give 590 mg of white solid product C-1, with a yield of 83%.

[0040] 1 H NMR (500 MHz, DMSO- d6 ) δ 7.88 (d, J = 7.5 Hz, 4H), 7.75 –7.69 (m,4H), 7.51 (d, J = 8.4 Hz, 2H), 7.41 (t, J = 7.5 Hz, 4H), 7.34 –7.31 (m, 6H), 7.08 (s, 2H), 4.33 –4.21 (m, 6H), 4.03 (q, J = 8.7 Hz, 2H), 2.71 (q, J = 9.1 Hz, 4H),2.08 –1.86 (m, 4H). 13 C NMR (126 MHz, DMSO- d6 ) δ 173.32, 155.97, 143.78, 140.73, 127.65,127.08, 125.33, 120.11, 65.61, 53.53, 46.70, 34.38, 31.65. ESI-MS: m / z 711.2 [M+H] + . (3) Synthesis of compound D-1 5 mL of N,N-dimethylformamide and compound C-1 (710 mg, 1 mmol) were added to a 25 mL round-bottom flask and dissolved. The solution was then pre-cooled at 0 °C for 15 min. Trifluoroacetic anhydride (840 mg, 4 mmol) and 560 mL of pyridine were dissolved in 5 mL of N,N-dimethylformamide and added dropwise to the reaction mixture at 0 °C using a dropping funnel. The reaction was then carried out at room temperature for 12 h. The conversion of the starting material was monitored by TLC. The reaction was quenched by diluting with ethyl acetate and then adding an equal volume of saturated NaHCO3 solution. The aqueous phase was extracted with ethyl acetate (3 × 50 mL). The combined organic phases were washed successively with 1N HCl and saturated NaCl, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation to obtain the crude product. The crude product was separated by column chromatography (petroleum ether:dichloromethane:ethyl acetate = 4:2:1, v / v) to give 412 mg of white solid product D-1, with a yield of 61%.

[0041] 1 H NMR (500 MHz, CDCl3) δ 7.75 (d, J = 7.5 Hz, 4H), 7.57 –7.50 (m, 4H),7.39 (t, J = 7.5 Hz, 4H), 7.30 (t, J = 7.5 Hz, 4H), 4.78 (q, J = 8.7 Hz, 2H), 4.46(s, 4H), 4.16 –4.10 (m, 2H), 2.75 (t, J = 7.0 Hz, 4H), 2.21 (d, J = 7.1 Hz, 4H). 13 C NMR (126 MHz, CDCl3) δ 155.28, 143.43, 141.48, 141.46, 128.03,127.29, 124.99, 120.23, 120.21, 117.94, 67.58, 47.15, 41.68, 33.65, 32.77. HRMS calculated value C 38 H 36 N4NaO5S2 + : 715.2019 [M+H2O+Na] + Experimental value: 715.1945.

[0042] (4) Synthesis of compound E-1 In a 50 mL single-necked flask, 20 mL of ethylene glycol dimethyl ether, compound D-1 (610 mg, 0.9 mmol), dibutyltin oxide (45 mg, 0.18 mmol), and azidotrimethylsilane (414 mg, 3.6 mmol) were added sequentially, and the mixture was heated under reflux for 24 hours. The conversion of the starting material was monitored by TLC. After cooling to room temperature, the reaction was quenched with saturated NaHCO3 solution. The aqueous phase was washed three times with ethyl acetate and then acidified to pH 2 with 6N HCl. The aqueous phase was extracted again with ethyl acetate (3 × 50 mL). The organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation to give 548 mg of yellow solid product E-1, in 85% yield.

[0043] 1 H NMR (500 MHz, DMSO- d 6 )) δ 8.12 (d, J = 8.0 Hz, 2H), 7.88 (d, J = 7.5Hz, 4H), 7.70 (dd, J = 10.3, 7.5 Hz, 4H), 7.41 (t, J = 7.4 Hz, 4H), 7.32 (q, J =7.3 Hz, 2H), 5.06 (td, J = 8.5, 5.4 Hz, 2H), 4.39 (dd, J = 10.5, 7.0 Hz, 2H), 4.29 (dd, J = 10.4, 6.8 Hz, 2H), 4.23 (t, J = 6.9 Hz, 2H), 2.77 – 2.72 (m, 4H), 2.36 – 2.19 (m, 4H). 13 C NMR (126 MHz, DMSO- d 6 ) δ 172.12, 155.90, 143.72, 140.79, 127.72,127.11, 125.31, 125.26, 120.19, 120.16, 65.78, 46.73, 44.97, 33.61, 32.32. ESI-MS: m / z 759.2 [MH] - . (5) Synthesis of 5'-deoxy-5'-iodoadenosine (compound G-1) In a 50 mL round-bottom flask, 15 mL of anhydrous pyridine, adenosine (compound F-1, 160 mg, 0.6 mmol), and triphenylphosphine (314 mg, 1.2 mmol) were added sequentially. Under nitrogen protection, the mixture was reacted at room temperature for 30 min. Then, iodine (230 mg, 0.9 mmol) dissolved in 1 mL of anhydrous pyridine was added. The reaction was carried out in the dark for 20 h, and the conversion of the starting material was monitored by TLC. After the reaction was completed, the mixture was quenched with 10% Na₂S₂O₃ solution, and most of the pyridine was removed by rotary evaporation. The residue was then dissolved in water. The aqueous phase was washed twice with diethyl ether and then evaporated under vacuum to remove water. Anhydrous ethanol was added several times during evaporation to remove residual water through azeotropic extraction. The crude product was purified by column chromatography (dichloromethane:methanol = 5:1, v / v) to give 166 mg of white solid product G-1, with a yield of 74%.

[0044] 1 H NMR (800 MHz, DMSO- d 6 ) δ 8.35 (s, 1H), 8.13 (s, 1H), 7.35 (s, 2H), 5.87 (d, J = 6.2 Hz, 1H), 5.46 – 5.40 (m, 2H), 5.18 (s, 1H), 4.61 (q, J = 5.8 Hz, 1H), 4.14 (d, J = 3.5 Hz, 1H), 3.96 (q, J = 3.5 Hz, 1H), 3.67 (dd, J = 12.0, 3.6Hz, 1H), 3.57 – 3.52 (m, 1H). 13 C NMR (201 MHz, DMSO- d 6 ) δ 156.15, 152.35, 149.05, 139.93, 119.36, 87.89, 85.89, 73.42, 70.66, 61.67. ESI-MS: m / z 378.0 [M+H] + . (6) Synthesis of 5'-deoxy-5'-iodo-7-deadenine (compound G-2) The synthesis method of this compound is similar to that of G-1, using tuberculin (compound F-2) as a raw material, with a yield of 71%.

[0045] 1 H NMR (800 MHz, DMSO- d 6 ) δ 8.40 (s, 1H), 7.69 (d, J = 3.7 Hz, 1H), 6.99(d, J = 3.7 Hz, 1H), 6.13 (d, J = 6.1 Hz, 1H), 4.52 (t, J = 5.7 Hz, 1H), 4.07 –4.06 (m, 1H), 3.97 – 3.95 (m, 1H), 3.60 (dd, J = 10.5, 5.8 Hz, 1H), 3.43 (dd, J =10.5, 6.5 Hz, 1H). 13 C NMR (201 MHz, DMSO- d 6 ) δ 151.75, 147.87, 143.67, 124.76, 102.65,102.16, 87.17, 83.63, 73.63, 73.16, 8.04. ESI-MS: m / z 377.0 [M+H] + . (7) Synthesis of amide-SAH In a 10 mL round-bottom flask, 2 mL of isopropanol, compound C-1 (36 mg, 0.05 mmol), and sodium borohydride (30 mg, 0.8 mmol) were added sequentially. The mixture was reacted at room temperature for 1 h under a nitrogen atmosphere. Then, compound G-1 (30 mg, 0.08 mmol) dissolved in 1 mL of isopropanol was added to the reaction system, and the mixture was heated to 70 °C and reacted in the dark under nitrogen protection for 2 h. The conversion of the starting material was monitored by TLC, and the reaction was quenched by adding an equal volume of water. The solution was acidified to pH 3 with 1N HCl. The solvent was removed by rotary evaporation, and the residue was reconstituted with water and filtered through a PES syringe filter. The filtrate was purified by preparative high-performance liquid chromatography (HPLC) using a Shim-pack GIST C18 column (20 × 250 mm, 5 μm). Mobile phase A was H₂O + 1‰ trifluoroacetic acid, and mobile phase B was CH₃CN + 1‰ trifluoroacetic acid. The flow rate was 10 mL / min, the single injection volume was 5 mL, the detection wavelengths were 254 mm and 215 mm, and the elution program was 0 min, 0% B; 5 min, 0% B; 31 min, 19% B; 33 min, 90% B; 37 min, 90% B; 37.01 min, 0% B; 44 min, 0% B. The combined fractions were freeze-dried under vacuum to give 11 mg of white solid, with a yield of 37%.

[0046] 1 H NMR (800 MHz, D2O) δ 8.51 (s, 1H), 8.43 (s, 1H), 6.14 (d, J = 4.7 Hz, 1H), 4.85 (t, J = 4.9 Hz, 1H), 4.44 (t, J = 5.1 Hz, 1H), 4.34 (dt, J = 7.2, 4.7 Hz, 1H), 4.11 (t, J = 6.6 Hz, 1H), 3.08 (dd, J = 14.3, 4.7 Hz, 1H), 3.01 (dd, J = 14.3,7.0 Hz, 1H), 2.73 – 2.66 (m, 2H), 2.20 – 2.12 (m, 2H). 13C NMR (201 MHz, D2O) δ 171.40, 150.34, 148.34, 145.05, 142.66,118.93, 88.37, 83.45, 73.55, 72.27, 51.98, 33.43, 30.58, 26.87. HRMS calculated value C 14 H 22 N7O4S + 384.1448 [M+H] + Experimental value: 384.1439.

[0047] (8) Synthesis of tet-SAH The synthesis of this compound was similar to that of amide-SAH, using compounds E-1 and G-1 as starting materials. The reaction solvent was replaced with isopropanol / water (2:1, v / v), yielding 10 mg of a white solid in 45% yield. The preparative high-performance liquid chromatography (HPLC) elution program was as follows: 0 min, 0% B; 6 min, 0% B; 31 min, 30% B; 33 min, 90% B; 37 min, 90% B; 37.01 min, 0% B; 44 min, 0% B.

[0048] 1 H NMR (800 MHz, D2O) δ 8.45 (s, 1H), 8.42 (s, 1H), 6.08 (d, J = 4.7 Hz,1H), 5.03 – 4.99 (m, 1H), 4.39 (t, J = 5.2 Hz, 1H), 4.29 – 4.25 (m, 1H), 3.01(dd, J = 14.4, 4.4 Hz, 1H), 2.93 (dd, J = 14.4, 6.9 Hz, 1H), 2.61 – 2.52 (m, 2H), 2.45 – 2.33 (m, 1H). 13 C NMR (201 MHz, D2O) δ 156.42, 149.94, 148.22, 144.47, 142.81,118.89, 88.39, 83.73, 73.47, 72.14, 45.00, 33.19, 31.56, 27.03. HRMS calculated value C 14 H 22 N7O4S + : 409.1513 [M+H] + Experimental value: 409.1526.

[0049] (9) Synthesis of 7dz-tSAH The synthesis of this compound was similar to that of tet-SAH, using compounds E-1 and G-2 as starting materials. 10 mg of solid was obtained, with a yield of 36%.

[0050] 1 H NMR (600 MHz, D2O) δ 8.27 (s, 1H), 7.47 (d, J = 3.8 Hz, 1H), 6.84 (d, J = 4.2 Hz, 1H), 6.19 (d, J = 5.4 Hz, 2H), 4.99 (dd, J = 8.5, 6.0 Hz, 1H), 4.65(t, J = 5.5 Hz, 1H), 4.34 (t, J = 5.4 Hz, 1H), 4.24 (dt, J = 6.5, 4.6 Hz, 1H), 3.00(dd, J = 14.5, 4.5 Hz, 1H), 2.89 (dd, J = 14.5, 6.5 Hz, 1H), 2.62 – 2.52 (m, 2H), 2.44 – 2.32 (m, 2H). 13 C NMR (151 MHz, D2O) δ 156.50, 150.99, 147.48, 142.26, 124.73,102.91, 102.69, 87.26, 83.36, 73.62, 72.19, 45.07, 33.47, 31.61, 27.30. HRMS calculated value C 15 H 22 N9O3S + 408.1561 [M+H] + Experimental value: 408.1561.

[0051] Example 2: Synthesis of fluoromethyl SAM analogs (1) Synthesis of F-amSAM like Figure 1 As shown, the stability of fluoromethyl SAM analogs in 100 mM Tris-HCl buffer at 30 °C and pH=8 was investigated, and the following experiments were conducted based on this.

[0052] Purified AclHMT (200 μM), amide-SAH (1 mM), and fluoroiodomethane (10 mM) were thoroughly mixed in 100 mM sodium phosphate (pH 6.5) buffer, with a total volume of 8.4 mL. The reaction mixture was reacted in a shaker at 30 °C and 90 rpm for 45 min. 420 μL of trifluoroacetic acid was added to bring the final concentration to 5% (v / v) to quench the reaction. The quenched sample was centrifuged at 4 °C and 10,000 rpm for 10 min using a cryogenic centrifuge. The supernatant was filtered through a PES syringe filter, and the filtrate was purified by preparative high-performance liquid chromatography (HPLC) using an Innoval ODS-2 C18 column (10 × 250 mm, 5 μm). Mobile phase A was H₂O + 1‰ trifluoroacetic acid, and mobile phase B was CH₃CN + 1‰ trifluoroacetic acid. The flow rate was 3 mL / min, the single injection volume was 5 mL, the detection wavelengths were 260 mm and 215 mm, and the elution program was 0 min, 0% B; 5 min, 0% B; 27 min, 16.5% B; 29 min, 90% B; 33 min, 90% B; 33.01 min, 0% B; 40 min, 0% B. The collected target fraction was freeze-dried under vacuum to obtain 1.9 μmol of oily product.

[0053] To reduce the decomposition of F-amSAM, the preparation reaction was carried out at pH 6.5. However, the catalytic efficiency of HMT was low at pH 6.5, resulting in a small amount of product. Only the product was characterized by HRMS.

[0054] HRMS calculated value C 15 H 23 FN7O4S + : 416.1511 [M] + Found: 416.1487. (2) Synthesis of F-tSAM The synthesis of this compound was similar to that of F-amSAM, using tet-SAH as the starting material, with a total reaction volume of 13.4 mL. Purification was performed using a Shim-pack GIST C18 column (20 × 250 mm, 5 μm) at a mobile phase flow rate of 10 mL / min. The elution program was: 0 min, 0% B; 6 min, 0% B; 31 min, 30% B; 33 min, 90% B; 37 min, 90% B; 37.01 min, 0% B; 44 min, 0% B. 8.5 μmol of oily product was obtained.

[0055] 1 H NMR (600 MHz, D2O) δ 8.41 (d, J = 8.1 Hz, 1H), 8.35 (d, J = 18.0 Hz,1H), 6.27 – 6.11 (m, 2H), 6.09 – 5.99 (m, 1H), 4.99 – 4.96 (m, 1H), 4.62 –4.61 (m, 1H), 4.58 – 4.50 (m, 1H), 4.17- 4.08 (m, 1H), 3.65 (dt, J = 14.2, 2.6Hz, 2H), 3.57 – 3.54 (m, 2H), 2.73 – 2.70 (m, 2H). 19 F NMR (753 MHz, D2O) δ -215.51 (dt, J = 130.9, 45.9 Hz). 13 C NMR (151 MHz, D2O) δ 157.28, 157.01, 150.00, 149.95, 147.91,147.74, 144.37, 143.51, 143.40, 119.45, 119.30, 90.12, 90.08, 85.13 (dd, J CF =238.0, 67.1 Hz), 79.03, 78.79, 73.09, 73.05, 72.78, 72.70, 62.44, 46.63,44.75 (d, J CF= 4.6 Hz), 40.76, 39.63, 33.72, 33.43, 26.81, 26.46. HRMS calculated value C 15 H 22 FN 10 O3S + : 441.1576 [M] + Experimental value: 441.1565.

[0056] (3) Synthesis of F-7dz-tSAM The synthesis method of this compound is similar to that of F-tSAM, using 7dz-tSAH as the starting material, 100 mM Tris-HCl (pH 8) as the buffer solution, a total reaction volume of 11 mL, and a reaction time of 1 h. 20.2 μmol of oily product was obtained.

[0057] 1 H NMR (800 MHz, D2O) δ 8.27 (d, J = 13.8 Hz, 1H), 7.45 (d, J = 3.7 Hz, 1H), 7.37 (d, J = 3.7 Hz, 1H), 6.86 (dd, J = 30.9, 3.7 Hz, 1H), 6.25 – 6.15 (m,2H), 6.14 – 6.00 (m, 1H), 4.84 (dd, J = 9.6, 5.3 Hz, 1H), 4.73 (t, J = 5.0 Hz, 1H), 4.68 (t, J = 5.3 Hz, 1H), 4.58 – 4.51 (m, 1H), 4.16 – 4.09 (m, 1H), 4.03 –3.98 (m, 1H), 3.75 – 3.53 (m, 2H), 2.70 (q, J = 7.1 Hz, 2H). 19 F NMR (753 MHz, D2O) δ -215.53 (dt, J = 72.9, 45.7 Hz). 13C NMR (201 MHz, D2O) δ 157.36, 156.91, 151.02, 147.19, 146.97,142.14, 142.05, 125.34, 103.22, 103.05, 102.81, 89.31, 85.32 (dd, J CF = 237.5,38.4 Hz), 78.56, 78.44, 73.05, 72.69, 62.44, 45.08 (d, J CF = 4.5 Hz), 41.32, 39.74, 34.26, 33.96, 26.82, 26.70. HRMS calculated value C 16 H 23 FN9O3S + : 440.1623 [M] + Experimental value: 440.1623.

[0058] Example 3: Catalytic production of fluorinated geraniol (fluorinated intermediate 1) by cascade catalysis of AclHMT and CiCOMT10. Purified AclHMT (50 μM), CiCOMT10 (50 μM), 7dz-SAH (50 μM), fluoromethyl iodide (16 mM), luteolin (0.5 mM), and magnesium chloride (10 mM) were thoroughly mixed in 50 mM Tris-HCl (pH 7.5) buffer solution. 10% (v / v) dimethyl sulfoxide was added as a co-solvent, with a total volume of 100 mL. The reaction mixture was reacted in a water bath at 30 °C for 16 h. After the reaction was complete, an equal volume of methanol was added to quench the reaction mixture. The mixture was centrifuged at 12,000 rpm for 30 min at 4 °C, and the supernatant was analyzed by high-performance liquid chromatography (HPLC) using a Shim-pack GIST C18 column (4.6 × 150 mm, 5 μm). The reaction mixture was scaled up to a preparative scale with a total volume of 30 mL. The reaction mixture was reacted in a shaker at 90 rpm at 30 °C for 16 h. The reaction was quenched with an equal volume of methanol, followed by centrifugation at 4°C and 10,000 rpm for 10 minutes using a low-temperature high-speed centrifuge. The supernatant was collected and filtered through a Nylon 66 needle filter. The filtrate was purified by preparative high-performance liquid chromatography (HPLC) using an InnovalODS-2 C18 column (10×250 mm, 5 μm). Mobile phase A was H₂O + 1‰ trifluoroacetic acid, and mobile phase B was CH₃CN + 1‰ trifluoroacetic acid. The flow rate was 3 mL / min, the single injection volume was 5 mL, and the detection wavelengths were 330 nm and 280 nm. The elution program was: 0 min, 10% B; 4 min, 10% B; 42 min, 70% B; 42.01 min, 100% B; 47 min, 100% B; 47.01 min, 10% B; 54 min, 10% B. The collected target fraction was freeze-dried under vacuum to obtain 2 mg of pale yellow solid, with a yield of 42%.

[0059] 1 H NMR (800 MHz, DMSO- d 6 ) δ 12.86 (s, 1H), 10.91 (s, 1H), 9.94 (s,1H), 7.55 (dd, J = 8.5, 2.2 Hz, 1H), 7.52 (d, J = 2.3 Hz, 1H), 7.25 (d, J = 8.5 Hz,1H), 6.81 (s, 1H), 6.47 (d, J = 2.1 Hz, 1H), 6.21 (d, J= 2.0 Hz, 1H), 5.89 (d, J =54.1 Hz, 2H). 19 F NMR (753 MHz, DMSO- d 6 ) δ -150.22 (t, J = 53.6 Hz). 13 C NMR (201 MHz, DMSO- d 6 ) δ 181.75, 164.36, 162.93, 161.48, 157.36,147.68, 147.11, 126.31, 118.34, 117.00, 114.30, 104.47, 103.85, 100.84 (d, J CF =216.7 Hz), 98.99, 93.96. HRMS calculated value C 16 H 12 FO6 + 319.0612 [M+H] + Experimental value: 319.0616.

[0060] Example 4: One-pot enzymatic preparation of fluorodiosmin The fluorinated intermediate 1 (1 mM) described in Example 3 was added to a 50 mM Tris-HCl (pH 7.5) buffer solution containing CiUGT11 (44 μM), UDP-glucose (2 mM), magnesium chloride (10 mM), and DL-dithiothreitol (10 mM), for a total volume of 8 mL. After thorough mixing, the mixture was incubated at 37°C for 1.5 hours. Further, 4 mL of a solution containing CiRhaT-GD was added to the above reaction solution. 4X A mixture of 120 μM UDP-rhamnose (3 mM) and 120 μM UDP-rhamnose (3 mM) was thoroughly mixed and incubated at 37°C for 4.5 hours. After the reaction, an equal volume of methanol was added to quench the reaction. The quenched sample underwent a similar post-processing and purification process as in Example 3, with the following HPLC elution program: 0 min, 5% B; 4 min, 5% B; 30 min, 38% B; 33 min, 95% B; 37 min, 95% B; 37.01 min, 5% B; 44 min, 5% B. 2 mg of fluorodiosmin was finally obtained, with a separation yield of 40%.

[0061] 1 H NMR (800 MHz, DMSO- d 6 ) δ 12.85 (s, 1H), 9.91 (s, 1H), 7.57 (dd, J =8.5, 2.3 Hz, 1H), 7.53 (d, J = 2.2 Hz, 1H), 7.29 (d, J = 8.5 Hz, 1H), 6.87 (s,1H), 6.76 (d, J = 2.1 Hz, 1H), 6.48 (d, J = 2.1 Hz, 1H), 5.90 (d, J = 54.1 Hz, 2H),5.08 (d, J = 7.6 Hz, 1H), 4.54 (d, J = 1.6 Hz, 1H), 3.85 (dd, J = 11.4, 1.8 Hz,1H), 3.66 (dd, J = 3.5, 1.6 Hz, 1H), 3.61 – 3.59 (m, 1H), 3.48 – 3.45 (m, 2H),3.42 – 3.40 (m, 2H), 3.27 (dd, J = 9.0, 7.6 Hz, 1H), 3.17 – 3.14 (m, 2H), 1.07(d, J = 6.2 Hz, 3H). 19 F NMR (753 MHz, DMSO- d 6 ) δ -150.26 (t, J = 54.2 Hz). 13 C NMR (201 MHz, DMSO- d 6 ) δ 182.05, 173.01, 163.66, 163.08, 161.23,157.03, 147.67, 147.30, 126.23, 118.58, 117.01, 114.54, 105.56, 104.82,100.81 (d, J CF= 216.3 Hz), 100.55, 99.90, 99.72, 94.86, 76.26, 75.63, 73.11,72.04, 70.74, 70.33, 69.60, 69.31, 68.36, 66.06, 17.83. HRMS calculated value C 28 H 32 FO 15 + 627.1720 [M+H] + Experimental value: 627.1723.

[0062] Example 5: Catalytic cascade formation of fluorinated intermediate 2 from AclHMT and PsmD Purified AclHMT (25 μM), PsmD (50 μM), 7dz-SAH (50 μM), fluoroiodomethane (16 mM), and N-acetyl-O-methylaminoacyl-5-hydroxytryptamine (1 mM) were thoroughly mixed in 100 mM potassium phosphate (pH 7.5) buffer solution, with a total volume of 100 mL. The mixture was reacted in a water bath at 30 °C for 16 h. After the reaction was complete, an equal volume of 10% TFA was added to quench the reaction mixture. The mixture was centrifuged at 12,000 rpm for 30 min at 4 °C, and the supernatant was analyzed by high-performance liquid chromatography (HPLC) using a Shim-pack GIST C18 column (4.6 × 150 mm, 5 μm). The analysis showed that almost quantitative conversion occurred. The reaction system was scaled up to a preparative scale with a total volume of 20 mL. The reaction mixture was reacted in a shaker at 90 rpm at 30 °C for 16 h. The reaction was quenched by adding TFA (final concentration 5%, v / v). The quenched sample was centrifuged at 4°C and 10,000 rpm for 10 minutes using a low-temperature high-speed centrifuge. The supernatant was collected and filtered through a PES syringe filter. The filtrate was purified by preparative high-performance liquid chromatography using an Innoval ODS-2 C18 column (10×250 mm, 5 μm). Mobile phase A was H2O + 1‰ trifluoroacetic acid, and mobile phase B was CH3CN + 1‰ trifluoroacetic acid. The flow rate was 3 mL / min, the single injection volume was 5 mL, the detection wavelengths were 254 mm and 215 mm, and the elution program was: 0 min, 0% B; 4 min, 0% B; 15 min, 31.5% B; 33 min, 35.5% B; 33.01 min, 90% B; 38 min, 90% B; 38.01 min, 0% B; 46 min, 0% B. The collected target fraction was freeze-dried under vacuum to obtain 5.2 mg of white solid, with a separation yield of 85%.

[0063] 1 H NMR (800 MHz, CDCl3) δ 6.89 – 6.86 (m, 2H), 6.62 (d, J = 8.4 Hz, 1H), 5.52 (s, 1H), 4.49 – 4.37 (m, 2H), 3.39 (td, J = 10.9, 6.6 Hz, 1H), 2.94 (dd, J =65.2, 4.7 Hz, 3H), 2.55 – 2.51 (m, 1H), 2.28 (dd, J = 11.7, 7.7 Hz, 1H), 2.12(s, 3H). 19 F NMR (376 MHz, CDCl3) δ -221.73 (t, J = 47.5 Hz). 13 C NMR (201 MHz, CDCl3) δ 172.43, 159.16, 146.63, 144.14, 127.28,123.04, 117.74, 110.18, 83.40 (d, J CF = 179.8 Hz), 78.18, 57.49, 47.29, 31.14,27.92, 21.94. HRMS calculated value C 15 H 19 FN3O3 + 308.1405 [M+H] + Experimental value: 308.1431.

[0064] [α]D 20 = –147.05±0.29 ( c 0.2, CHCl3) Comparative Example 1 Purified F-7dz-tSAM (2 mM) was used as the fluoromethyl donor. Purified AclHMT (25 μM), PsmD (50 μM), and N-acetyl-O-methylaminoacyl-5-hydroxytryptamine (1 mM) were added and thoroughly mixed in 100 mM potassium phosphate (pH 7.5) buffer solution, with a total volume of 100 mL. The reaction was carried out in a water bath at 30°C for 16 h. Using the same chromatographic analysis method as described above, the conversion rate was 77%.

[0065] Example 6: One-pot enzymatic preparation of fluorophysostigmine The fluorinated intermediate 2 (2 mM) described in Example 5 was added to a 100 mM potassium phosphate (pH 7.0) buffer solution containing 4 mM SAM, 75 μM PsmC, and 5% (v / v) glycerol, for a total volume of 20 mL. After thorough mixing, the mixture was incubated at 37°C for 6 hours. Subsequently, 5 mL of a mixture containing SAM (6 mM) and PsmB (150 μM) was added, and the reaction was continued for 8 hours. After the reaction was completed, trifluoroacetic acid was added to the solution to a final concentration of 5% (v / v) to quench the reaction. The quenched sample underwent a post-processing and purification process similar to that in Example 5, with the following high-performance liquid chromatography elution program: 0 min, 0% B; 4 min, 0% B; 10 min, 10% B; 32 min, 25% B; 35 min, 90% B, hold for 5 min; 40 min, 0% B, equilibrate for 10 min. The final yield was 4.9 mg of fluorophysostigmine, with a separation yield of 42%.

[0066] 1 H NMR (800 MHz, CDCl3) δ 7.02 (d, J = 8.5 Hz, 1H), 6.93 (s, 1H), 6.63(d, J = 8.7 Hz, 1H), 5.37 (s, 1H), 5.02 (d, J = 7.4 Hz, 1H), 4.61 – 4.50 (m, 2H), 3.66 (s, 1H), 3.10 (s, 3H), 2.88 (s, 3H), 2.80 (s, 3H), 2.52 (s, 1H), 2.20(dd, J = 13.9, 5.5 Hz, 1H). 19 F NMR (470 MHz, CDCl3) δ -220.26 (t, J = 46.5 Hz). 13 C NMR (201 MHz, CDCl3) δ 155.74, 148.70, 145.68, 129.46, 123.50,117.37, 109.68, 91.64, 84.58 (d, J CF= 179.0 Hz), 58.42, 52.21, 38.37, 34.07,32.97, 27.88. HRMS calculated value C 15 H 21 FN3O2 + : 294.1612 [M+H] + Experimental value: 294.1617.

[0067] [α]D 20 = –36.17±0.01 ( c 1.0, CHCl3). Example 7: In vitro activity of fluorinated intermediate 2 and fluorophysostigmine Fluorinated intermediate 2, fluorophysostigmine, and physostigmine were prepared into a 50 mM stock solution using dimethyl sulfoxide (DMSO). This stock solution was then diluted proportionally with 100 mM potassium phosphate (pH 8.0) buffer to 12 concentrations ranging from 25 mM to 0.512 nM. The inhibitory activities of these compounds on the hydrolysis of thioacetylcholine and thiobutyrylcholine by AChE and BChE, respectively, were determined in 96-well plates using the Ellman assay and a microplate reader. Dose-response curves were plotted, and the IC50 values ​​of each compound for AChE and BChE were calculated. 50 Values ​​are shown in Table 1. See Table 1 for detailed results. Figure 2 As shown.

[0068] Table 1. IC50 values ​​of physostigmine, fluorinated intermediate 2, and fluorinated physostigmine for AChE and BChE. 50 value

[0069] Therefore, this invention utilizes the aforementioned S-adenosylhomocysteine ​​analogs, S-adenosyl-L-methionine analogs, and their applications. This invention designs and synthesizes three novel SAM analogs: F-amSAM, F-tSAM, and F-7dz-tSAM. By replacing the carboxyl and base groups with bioisosteric electrons, the two main decomposition pathways of SAM analogs are suppressed, improving their chemical stability. Among them, the preferred F-7dz-tSAM has a half-life greater than 12 hours, which can effectively reduce its degradation side reactions as a fluoromethyl donor in enzymatic reactions.

[0070] This invention demonstrates the ability of AclHMT to recognize SAH analogs and synthesize fluoromethyl SAM analogs, as well as the ability of CiCOMT10 and PsmD to perform enzymatic regioselective and stereoselective fluoromethyl modification of clinical drug biosynthetic precursors using F-7dz-tSAM as a fluoromethyl donor.

[0071] The HMT-MT cyclic enzyme cascade reaction based on 7dz-tSAH constructed in this invention can realize in-situ regeneration of F-7dz-tSAM and complete the enzymatic fluoromethylation reaction with a catalytic amount of 7dz-tSAH, thereby improving reaction efficiency and atom economy.

[0072] The fluorinated intermediates provided by this invention can be accepted by downstream enzymes in the biosynthetic pathway, enabling the one-pot enzymatic production of fluorinated diosmin and fluorinated physostigmine, avoiding complex intermediate purification steps and reducing waste generation and energy consumption.

[0073] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. An S-adenosyl-L-methionine analogue, characterized in that, SAM analogues have the structure shown in Formula I: ； Formula I R1 is either a terminal amide or a tetrazolium; R2 is one of the fluorine-substituted C1~C10 saturated or unsaturated straight-chain or branched alkanes; X is NH, O, S, or CH; Y represents N and C; Z represents N and C.

2. The S-adenosine-L-methionine analogue according to claim 1, characterized in that, Fluorine-substituted C1-C10 saturated or unsaturated straight-chain or branched alkanes include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl, etc.

3. The S-adenosine-L-methionine analogue according to claim 1, characterized in that, SAM analogues have any of the following structures: 。 4. S-adenosylhomocysteine ​​analogue, characterized in that, SAH analogues have the structure shown in Formula II: ； Formula II R1 is either a terminal amide or a tetrazolium; X is NH, O, S, or CH; Y represents N and C; Z represents N and C; n is 1 or 2.

5. The S-adenosylhomocysteine ​​analogue according to claim 4, characterized in that, SAH analogs have any of the following structures: 。 6. The application of SAM analogs and SAH analogs, characterized in that, The cyclic enzyme cascade reaction of the SAM analog shown in Formula I and the SAH analog shown in Formula II was carried out simultaneously in the same system in steps (1) to (2): (1) The SAH analog of formula II is reacted with fluoroalkyl iodide by halogen methyltransferase to generate the SAM analog of formula I. (2) The SAM analog shown in Formula I is fluoroalkylated with the receptor substrate under the action of methyltransferase to obtain the fluoroalkylated receptor product and the SAH analog shown in Formula II. In this process, the SAH analogue of formula II obtained in step (2) is recycled for the reaction in step (1); The alkyl group in the fluoroalkyl iodide is a C1-C10 straight-chain or straight-chain alkyl group.

7. The application of the SAM analogue and SAH analogue according to claim 6, characterized in that, The receptor substrate is a bioactive molecule, including but not limited to small molecule compounds, nucleic acids, lipids or proteins, specifically luteolin and N-acetyl-O-methylaminoacyl-5-hydroxytryptamine; Halogen methyltransferases are derived from Aspergillus clavatus AclHMT, including but not limited to those from Arabidopsis thaliana , Paraburkholderia xenovorans, Vibri parhaemolyticus, Ustilago maydis, Kordia algicida, Synechococcus elongates Homologous to; The methyltransferases are CiCOMT10 and PsmD, including but not limited to O-methyltransferase, N-methyltransferase, S-methyltransferase, and C-methyltransferase.

8. A method for synthesizing fluorinated intermediate 1, characterized in that, Based on the HMT-CiCOMT10 cyclic enzyme cascade reaction of the SAH analog shown in Formula II, the fluorinated intermediate 1 was prepared by scaling up the reaction.

9. A method for synthesizing fluorinated intermediate 2, characterized in that, Based on the scale-up of the HMT-PsmD cyclic enzyme cascade of the SAH analog shown in Formula II, fluorinated intermediate 2 was prepared.

10. A method for one-pot enzymatic preparation of diosmin fluorinated derivatives, characterized in that, Using fluorinated intermediate 1 as the acceptor substrate, in stepwise addition of CiUGT11 and CiRhaT-GD 4X In the cascade system, rutinylation modification is carried out sequentially to generate fluorinated diosmin.

11. A method for one-pot enzymatic preparation of physostigmine fluorinated derivatives, characterized in that, Using fluorinated intermediate 2 as the acceptor substrate, stepwise methylation and deacetylation were carried out in an enzyme cascade system of stepwise addition of PsmC and PsmB to generate fluorophysostigmine.

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