Fluoromethyl product as well as preparation method and application thereof

Abstract

The invention discloses a fluoromethyl product as well as a preparation method and application thereof, and belongs to the technical field of biological catalysis. According to the method, a cascade reaction system and method formed by halide methyltransferase (HMT) and B12-dependent SAM free radical methyltransferase (B12RSMTs) are utilized for the first time, fluoroalkyl modification on inactive carbon is successfully achieved, a fluoromethyl product is successfully prepared, and the provided preparation method is high in specificity, mild in reaction condition and environmentally friendly; the invention provides a new fluoromethyl B12 reaction intermediate, provides a new thought for developing more fluoroalkyl compounds, and lays a foundation for introducing other fluoroalkyl modifications on inactive carbon.

Description

Technical Field

[0001] The present invention relates to the technical field of fluoromethyl product preparation, and in particular to a fluoromethyl product and a preparation method and application thereof. Background Art

[0002] Fluorine is a special element that has the smallest atomic radius and the largest electronegativity except for hydrogen. Therefore, replacing hydrogen in alkanes with fluorine will not cause a big change in space, but can change the physical and chemical properties of the substance, such as pKa and dipole moment, and can also improve the bioavailability of the substance. Due to the special properties that fluorine gives to molecules, it has become an important element in drug molecules and agricultural chemical products. According to statistics, about 20% of drugs and 30-40% of agricultural chemicals contain fluorine. Among them, fluoromethyl drugs have good application effects in anti-cancer, anti-tumor and as inhibitors.

[0003] The use of chemical methods to introduce fluoroalkyl groups into molecules has the disadvantages of high energy consumption, low yield, poor synthetic selectivity and environmental unfriendliness. In comparison, biocatalysis has the advantages of strong specificity, mild reaction conditions and environmental friendliness. With the rapid development of enzyme catalysis, replacing chemical synthesis with biocatalysis will become a future development trend. Halide methyltransferase (HMT) is an enzyme that can catalyze the reversible conversion of S-adenosyl-L-methionine (SAM) and S-adenosylhomocysteine ​​(SAH). Inspired by its reversible reaction, it was found that it can also catalyze the reaction of SAH with halogenated fluoroalkyl reagents to produce non-natural fluoroalkyl SAM analogues. However, some fluoroalkyl SAM analogues catalyzed by HMT are very unstable. For example, the half-life of fluoromethyl SAM (F-SAM) is less than one hour under pH 8.0, so it is difficult to prepare and store, which limits its application. The strategy of multi-enzyme cascade reaction can effectively solve this problem.

[0004] Fluoroalkylation modification on active atoms such as oxygen, nitrogen, and sulfur can be achieved through nucleophilic reactions, while the participation of free radicals is required to catalyze the alkylation reaction of inactive carbon. Vitamin B12-dependent SAM radical methyltransferases (B12-RSMTs) are a type of enzyme that uses iron-sulfur clusters and vitamin B12 as cofactors and SAM as a methyl donor to catalyze the methylation reaction on inactive carbon initiated by free radicals. The use of this type of enzyme to catalyze fluoromethylation has not been reported so far, and achieving fluoroalkylation on inactive carbon is of great significance for enriching fluoroalkylation products. Summary of the invention

[0005] The purpose of the present invention is to provide a fluoromethyl product and a preparation method and application thereof, so as to provide a method for modifying inactive carbon with fluoroalkyl groups, which has simple reaction, mild conditions and is environmentally friendly.

[0006] To achieve the above object, the present invention provides a method for preparing a fluoromethyl product, the steps of which are as follows:

[0007] S1. preparing a cascade reaction system, the components of which include: S-adenosylhomocysteine, fluoroiodomethane, titanium citrate, HMT protein, substrate and another protein; the other protein is one of QCMT protein, CysS protein and GenD1 protein;

[0008] S2. Mix the reaction system and place it in a metal bath for anaerobic reaction to obtain a reaction mixture, add a reagent to the reaction mixture to quench the reaction or continue the reaction, purify it, and freeze-dry it in a vacuum to obtain a fluoromethyl product.

[0009] Preferably, in S1, when the cascade reaction system contains both HMT protein and QCMT protein, the substrate is polypeptide substrate PS24, and the sequence of polypeptide substrate PS24 is shown in SEQ ID NO.1; when the cascade reaction system contains both HMT protein and CysS protein, the substrate is (R)-S-panthenthiovinyl-4-amino-3-methoxythiobenzoate; when the cascade reaction system contains both HMT protein and GenD1 protein, the substrate is gentamicin A, and threose dithiol needs to be added, and the molar ratio of gentamicin A:threose dithiol is 1:20.

[0010] Preferably, when the cascade reaction system contains both HMT protein and QCMT protein, the final concentration molar ratio of S-adenosylhomocysteine:fluoroiodomethane:titanium citrate:substrate:HMT protein:QCMT protein is 2.5:1438:25:1:1:1; when the cascade reaction system contains both HMT protein and CysS protein, the final concentration molar ratio of S-adenosylhomocysteine:fluoroiodomethane:titanium citrate:substrate:HMT protein:CysS protein is 2.5:1438:50:2.5:1:1; when the cascade reaction system contains both HMT protein and GenD1 protein, the final concentration molar ratio of S-adenosylhomocysteine:fluoroiodomethane:titanium citrate:substrate:HMT protein:GenD1 protein is 5:2876:100:5:1:2.

[0011] Preferably, the conditions for the anaerobic reaction of the reaction system after mixing in S2 in a metal bath are: when the cascade reaction system contains both HMT protein and QCMT protein, the reaction conditions are anaerobic reaction at 37°C for 4 hours; when the cascade reaction system contains both HMT protein and CysS protein / GenD1 protein, the reaction conditions are anaerobic reaction at 28°C overnight.

[0012] Preferably, in S2, when the cascade reaction system contains both HMT protein and QCMT protein, an equal volume of methanol is added to the reaction mixture to quench the reaction; when the cascade reaction system contains both HMT protein and CysS protein, an equal volume of 10% trifluoroacetic acid is added to the reaction mixture to quench the reaction; when the cascade reaction system contains both HMT protein and GenD1 protein, an equal volume of 10% trifluoroacetic acid is added to the reaction mixture to quench the reaction, followed by freeze-vacuum drying, and the lyophilized material is reconstituted with a 250 mM NaOH solution with half the volume of the reaction mixture, and then 7.96 M 2,4-dinitrofluorobenzene and methanol are added, and the reaction is carried out at 40°C for 90 minutes.

[0013] Preferably, when the cascade reaction system contains both HMT protein and GenD1 protein, the volume ratio of 250 mM NaOH solution: 7.96 M 2,4-dinitrofluorobenzene: methanol is 50:1:150.

[0014] A fluoromethyl product prepared by the method for preparing a fluoromethyl product as described above.

[0015] An application of the fluoromethyl product as described above in the preparation of drugs, wherein the drugs are anticancer, antitumor and inhibitor drugs.

[0016] A new fluoromethyl B12 reaction intermediate for preparing the above-mentioned fluoromethyl product. The preparation method of the new fluoromethyl B12 reaction intermediate comprises: preparing a cascade reaction system for preparing the fluoromethyl product, placing the reaction system in a metal bath, adopting anaerobic reaction conditions for preparing the fluoromethyl product to prepare a reaction mixture, adding an equal volume of 10% trifluoroacetic acid to quench the reaction, and centrifuging to obtain the supernatant for separation and purification.

[0017] Preferably, the reaction is quenched and then centrifuged at 12000 rpm for 30 min.

[0018] Therefore, the present invention provides a fluoromethyl product and its preparation method and application, and its specific technical effects are as follows:

[0019] (1) The present invention uses for the first time a cascade reaction system and method consisting of HMT and B12RSMTs to successfully achieve fluoroalkyl modification on inactive carbon and successfully prepare fluoromethyl products. The preparation method provided has strong specificity, mild reaction conditions, and is environmentally friendly;

[0020] (2) The present invention provides a new fluoromethyl B12 reaction intermediate, which provides a new idea for the development of more fluoroalkyl compounds, lays a foundation for the study of other fluoroalkyl modifications on inactive carbon, and also provides a theoretical basis for expanding the study of the mechanism of SAM free radical enzymes. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative labor.

[0022] Figure 1 is the HPLC test result of the supernatant obtained after quenching the reaction with 10% trifluoroacetic acid in Example 1 of the present invention;

[0023] Figure 2 This is the result of LC-MS detection after quenching the reaction with methanol in Example 1 of the present invention;

[0024] Figure 3 is a schematic diagram of the cascade reaction in Example 1 of the present invention;

[0025] Figure 4 is the HPLC test result of the supernatant obtained after quenching the reaction with 10% trifluoroacetic acid in Example 2 of the present invention;

[0026] Figure 5 This is the LC-MS analysis result of the new substance prepared in Example 2 of the present invention;

[0027] Figure 6 This is a schematic diagram of the cascade reaction in Example 2 of the present invention;

[0028] Figure 7 is the HPLC test result of the supernatant obtained after quenching the reaction with 10% trifluoroacetic acid in Example 3 of the present invention;

[0029] Figure 8 The LC-MS and MS / MS test results of the product obtained by continuing the reaction of the supernatant obtained after quenching the reaction with 10% trifluoroacetic acid in Example 3 of the present invention;

[0030] Fig. 9 It is a schematic diagram of the cascade reaction in Example 3 of the present invention. DETAILED DESCRIPTION

[0031] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0032] In order to make the purpose, technical scheme and advantages of the present application clearer, more thorough and more complete, the technical scheme of the present invention is clearly and completely described below through the accompanying drawings and examples. The following detailed descriptions are all descriptions of the embodiments, and are intended to provide further detailed descriptions of the present invention. Unless otherwise specified, all technical terms used in the present invention have the same meaning as those generally understood by those skilled in the art to which the present application belongs.

[0033] The instruments, equipment, reagents and materials used in the examples were all obtained through commercial channels.

[0034] Among them, the gene hmt comes from Aspergillus clavatu (GenBank: EAW10780.1), the gene qcmt comes from Methanoculleus thermophilus DSM2373 (Gen Bank: WP_066957658.1), the gene cysS comes from Cystobacter sp.Cbv34 (GenBank: AKP45384.1), and the gene genD1 comes from Micromonosporaechinosp ora ATCC15835 (GenBank: CAF31434.1). After optimizing these genes to codons preferred by Escherichia coli, they were constructed on pET28a, pET21a, pET28b and pET28a vectors respectively. The expression and purification of proteins HMT, QCM T, CysS and GenD1 encoded by hmt, qcmt, cysS and genD1 genes were consistent with the literature. The references are (Angew. Chem. Int. Ed. 2021, 60, 5554-5560), (Angew. Chem. Int. Ed. 2022, 61, e202204198), (J. Am. Chem. Soc. 2017, 139, 5, 1742-1745), (Chembiol. 2015, 22(2): 251-261.). 1.453mM QCMT is prepared by dissolving 77.7mg of QCMT in 1mL of desalting buffer; 0.966mM CysS is prepared by dissolving 72.6mg of CysS in 1mL of desalting buffer; 0.847mM GenD1 is prepared by dissolving 65.8mg of GenD1 in 1mL of desalting buffer; the desalting buffer is: 50mM Tris, 300mM NaCl, pH7.5.

[0035] Embodiment 1

[0036] HMT and QCMT cascade catalysis produces fluoromethyl peptide CH2F-PS24 and fluoromethyl B12 intermediates. The specific steps are as follows:

[0037] S11. Prepare a cascade reaction system of HMT and QCMT with a total volume of 80 μL: including 4 μL of S-adenosylhomocysteine ​​(SAH), 1.6 μL of fluoroiodomethane, 13.6 μL of HMT, 11 μL of QCMT, 4 μL of titanium citrate and 1.1 μL of polypeptide substrate PS24. The sequence of polypeptide substrate PS24 is shown in SEQ ID NO.1. The concentrations of SAH, fluoroiodomethane, HMT, QCMT, titanium citrate and PS24 are 10 mM, 14.38 M, 1.176 mM, 1.453 mM, 100 mM and 15 mM respectively. Among them, except for 14.38 M fluoroiodomethane which is a commercial raw material and PS24 which needs to be dissolved in degassed dimethyl sulfoxide, the rest are dissolved in degassed protein buffer. Protein buffer is: 50 mM Tris, 300 mM NaCl, pH 7.5.

[0038] SEQ ID NO.1:PTMMEDHFGGSQRAGVIAAASGLS

[0039] S12, after the reaction system is mixed evenly, it is placed in a metal bath at 37°C for anaerobic reaction for 4 hours to obtain a reaction mixture. Then, 30 μL of the reaction mixture is taken out and 30 μL of 10% trifluoroacetic acid is added to quench the reaction, and the mixture is centrifuged at 12000 rpm for 30 minutes, and the supernatant is taken and detected by analytical HPLC at 254 nm and 361 nm.

[0040] The results are as follows Figure 1 As shown, the product 5'-deoxyadenosine (5'-dA) was detected at 254 nm and the fluoromethyl B12 intermediate was detected at 361 nm.

[0041] S13, add 50 μL of methanol to the remaining 50 μL of reaction mixture in S12 to quench the reaction, freeze-dry in vacuum, reconstitute with 50 μL of deionized water, and then perform LC-MS detection. The results are as follows: Figure 2 As shown, mass peaks of 808.3828 and 1212.0715 were detected, which are consistent with the theoretical molecular weights of the fluoromethyl peptide CH2F-PS24 with three charges and two charges, respectively.

[0042] S14. Prepare 300 μL of reaction mixture using the system and method of steps S11 and S12. After the reaction is completed, add 300 μL of methanol to quench the reaction. Centrifuge at 12000 rpm for 30 min. Transfer the supernatant to a 0.5 kDa dialysis bag. Seal it and dialyze it in 5 L of deionized water for 8 hours to remove small molecule salts. After freeze-vacuum drying, add 50 μL of deionized water to re-dissolve it.

[0043] To the obtained solution, 30 μL of trypsin (concentration of 100 μg / mL, 100 μg of trypsin was dissolved in 1 mL of 50 mM acetic acid solution) was added, mixed evenly, incubated overnight at 37°C, quenched by adding an equal volume of methanol, centrifuged at 12000 rpm for 30 min, and the supernatant was taken for secondary mass spectrometry MS / MS detection, which was recorded as the reaction group. The control group was a group in which no iodomethane was added to the components, and other components and contents were the same as those of the reaction group.

[0044] The results are shown in Table 1. The ion fragments y1, y2, y3, ..., y9, y10, and y11 obtained in the reaction group are 175.1183, 335.1808, 422.2145, 479.2360, 536.2571, 683.3251, 820.3841, 935.4103, 1064.4430, 1195.4860, and 1326.5400, respectively. The corresponding ion fragments in the control group are 175.1191, 303.1785, 390.2100, 447.2305, 504.2526, 651.3209, 788.3804, 903.4084, 1032.4480, 1163.4900, and 1294.5310, respectively.

[0045] Table 1

[0046]

[0047]

[0048] The results showed that the cascade reaction was a fluoromethyl modification of glutamine Q in the peptide substrate PS24. The schematic diagram of the cascade reaction is shown in Figure 3 As shown, the yield of the fluoromethyl product is 8.49%.

[0049] Embodiment 2

[0050] HMT and CysS cascade catalysis produces fluoromethyl products and fluoromethyl B12 intermediates. The specific steps are as follows:

[0051] S21. Prepare 50 μL of the cascade reaction system of HMT and CysS: including 2.5 μL of SAH, 1 μL of iodofluoromethane, 8.5 μL of HMT, 10.4 μL of CysS, 5 μL of titanium citrate, and 2.5 μL of (R)-S-panthenyl-4-amino-3-methoxythiobenzoate. The concentrations of SAH, iodofluoromethane, HMT, CysS, titanium citrate, and (R)-S-panthenyl-4-amino-3-methoxythiobenzoate are 10 mM, 14.38 M, 1.176 mM, 0.966 mM, 100 mM, and 10 mM, respectively. Among them, except for 14.38 M iodofluoromethane which is a commercial raw material, the rest are dissolved in degassed 100 mM pH 7.6 PB buffer.

[0052] S22, after mixing the prepared system, react anaerobically overnight in a metal bath at 28°C to obtain a reaction mixture. Add 50 μL of 10% trifluoroacetic acid to the reaction mixture to quench the reaction, centrifuge at 12000 rpm for 30 min. Take the supernatant and detect it at 254 nm and 361 nm by analytical HPLC.

[0053] The results are as follows Figure 4 As shown, the product 5'-deoxyadenosine (5'-dA) and new substances were detected at 254 nm, and the fluoromethyl B12 intermediate was detected at 361 nm.

[0054] S23, the new substance was separated and prepared by analytical HPLC and then analyzed by LC-MS. The results are as follows Figure 5 As shown, a mass peak of 460.1957 was obtained, which is consistent with the molecular weight of the standard product of fluoromethyl (R)-S-panthenthiovinyl-4-amino-3-fluoroethoxythiobenzoate. This indicates that the cascade reaction can catalyze the fluoromethylation reaction of the substrate. The schematic diagram of the cascade reaction is shown in Figure 6 As shown, the yield of the fluoromethyl product is 39.7%.

[0055] Embodiment 3

[0056] HMT and GenD1 cascade catalysis produces fluoromethyl products and fluoromethyl B12 intermediates. The specific steps are as follows:

[0057] S31. Prepare a cascade reaction system of HMT and GenD1 with a total volume of 50 μL: including 2.5 μL SAH, 1 μL fluoroiodomethane, 4.3 μL HMT, 11.8 μL GenD1, 5 μL titanium citrate, 1 μL threose dithiol, and 2.5 μL gentamicin A. The concentrations of SAH, fluoroiodomethane, HMT, GenD1, titanium citrate, threose dithiol, and gentamicin A are 10 mM, 14.38 M, 1.176 mM, 0.847 mM, 100 mM, 500 mM, and 10 mM, respectively. Among them, except for 14.38 M fluoroiodomethane which is a commercial raw material, the rest are dissolved in degassed 100 mM pH 7.6 PB buffer.

[0058] S32, after the reaction system is mixed evenly, it is placed in a metal bath at 28°C for anaerobically overnight reaction to obtain a reaction mixture. 50 μL of 10% trifluoroacetic acid is added to the reaction mixture to quench the reaction, and the mixture is centrifuged at 12000 rpm for 30 min. 40 μL of the supernatant is taken and detected by analytical HPLC at 254 nm and 361 nm.

[0059] The results are as follows Figure 7 As shown, the product 5'-deoxyadenosine (5'-dA) was detected at 254 nm and the fluoromethyl B12 intermediate was detected at 361 nm.

[0060] S33. After freeze-vacuum drying, the remaining 60 μL supernatant in step S32 was re-dissolved by adding 30 μL of 250 mM NaOH solution, and then 0.6 μL of 7.96 M 2,4-dinitrofluorobenzene (DNFB) and 90 μL of methanol were added. After mixing, the mixture was reacted at 40°C for 90 minutes, and the supernatant was collected by centrifugation at 12000 rpm for 30 minutes for LC-MS and MS / MS detection.

[0061] The results are as follows Figure 8 As shown, LC-MS detected a mass peak of 999.2599, and its corresponding MS / MS secondary fragment was 178.0875, which is consistent with the theoretical primary molecular weight and secondary fragment of fluoromethyl gentamicin A. This indicates that the cascade reaction can catalyze the fluoromethylation reaction of gentamicin A. The schematic diagram of the cascade reaction is shown in Fig. 9 As shown, the yield of the fluoromethyl product is 5.68%.

[0062] Therefore, the present invention utilizes the cascade reaction system and method composed of HMT and B12RSMTs for the first time, successfully realizes the fluoroalkyl modification on the inactive carbon, and successfully prepares the fluoromethyl product. The preparation method provided has strong specificity, mild reaction conditions, and is environmentally friendly. The present invention provides a new fluoromethyl B12 reaction intermediate, which provides new ideas for the development of more fluoroalkyl compounds, lays a foundation for the study of other fluoroalkyl modifications on inactive carbon, and also provides a theoretical basis for expanding the study of the mechanism of SAM free radical enzymes.

[0063] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. A method for preparing a fluoromethyl product, characterized in that: Here are the steps: S1. preparing a cascade reaction system, the components of which include: S-adenosylhomocysteine, fluoroiodomethane, titanium citrate, HMT protein, substrate and another protein; the other protein is one of QCMT protein, CysS protein and GenD1 protein; S2. Mix the reaction system and place it in a metal bath for anaerobic reaction to obtain a reaction mixture, add a reagent to the reaction mixture to quench the reaction or continue the reaction, purify it, and freeze-dry it in a vacuum to obtain a fluoromethyl product.

2. The method for preparing a fluoromethyl product according to claim 1, characterized in that: In S1, when the cascade reaction system contains both HMT protein and QCMT protein, the substrate is polypeptide substrate PS24, and the sequence of polypeptide substrate PS24 is shown in SEQ ID NO.1; when the cascade reaction system contains both HMT protein and CysS protein, the substrate is (R)-S-panthenthiovinyl-4-amino-3-methoxythiobenzoate; when the cascade reaction system contains both HMT protein and GenD1 protein, the substrate is gentamicin A, and threose dithiol needs to be added, and the molar ratio of gentamicin A:threose dithiol is 1:

20.

3. The method for preparing a fluoromethyl product according to claim 2, characterized in that: When the cascade reaction system contains both HMT protein and QCMT protein, the final concentration molar ratio of S-adenosylhomocysteine:fluoroiodomethane:titanium citrate:substrate:HMT protein:QCMT protein is 2.5:1438:25:1:1:1; when the cascade reaction system contains both HMT protein and CysS protein, the final concentration molar ratio of S-adenosylhomocysteine:fluoroiodomethane:titanium citrate:substrate:HMT protein:CysS protein is 2.5:1438:50:2.5:1:1; when the cascade reaction system contains both HMT protein and GenD1 protein, the final concentration molar ratio of S-adenosylhomocysteine:fluoroiodomethane:titanium citrate:substrate:HMT protein:GenD1 protein is 5:2876:100:5:1:

2.

4. The method for preparing a fluoromethyl product according to claim 1, characterized in that: The reaction system mixed in S2 is placed in a metal bath for anaerobic reaction under the following conditions: when the cascade reaction system contains both HMT protein and QCMT protein, the reaction condition is anaerobic reaction at 37°C for 4 hours; when the cascade reaction system contains both HMT protein and CysS protein / GenD1 protein, the reaction condition is anaerobic reaction at 28°C overnight.

5. The method for preparing a fluoromethyl product according to claim 1, characterized in that: In S2, when the cascade reaction system contains both HMT protein and QCMT protein, an equal volume of methanol is added to the reaction mixture to quench the reaction; when the cascade reaction system contains both HMT protein and CysS protein, an equal volume of 10% trifluoroacetic acid is added to the reaction mixture to quench the reaction; when the cascade reaction system contains both HMT protein and GenD1 protein, an equal volume of 10% trifluoroacetic acid is added to the reaction mixture to quench the reaction, then freeze-dry in a vacuum, and the lyophilized material is reconstituted with a 250 mM NaOH solution with half the volume of the reaction mixture, and then 7.96 M 2,4-dinitrofluorobenzene and methanol are added, and the reaction is carried out at 40°C for 90 minutes.

6. The method for preparing a fluoromethyl product according to claim 5, characterized in that: When the cascade reaction system contains both HMT protein and GenD1 protein, the volume ratio of 250 mM NaOH solution: 7.96 M 2,4-dinitrofluorobenzene: methanol is 50:1:

150.

7. A fluoromethyl product prepared by the method for preparing a fluoromethyl product as claimed in any one of claims 1 to 6.

8. Use of the fluoromethyl product as claimed in claim 7 in the preparation of medicines, characterized in that: The drug is an anticancer, antitumor, or inhibitor drug.

9. A novel fluoromethyl B12 reaction intermediate for preparing the fluoromethyl product as claimed in claim 7, characterized in that: The preparation method of the novel fluoromethyl B12 reaction intermediate comprises: preparing a cascade reaction system for preparing a fluoromethyl product, placing the reaction system in a metal bath, preparing a reaction mixture under anaerobic reaction conditions for preparing a fluoromethyl product, adding an equal volume of 10% trifluoroacetic acid to quench the reaction, and centrifuging to obtain a supernatant for separation and purification.

10. A novel fluoromethyl B12 reaction intermediate for preparing fluoromethyl products according to claim 9, characterized in that: After quenching the reaction, centrifuge at 12000 rpm for 30 min.

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