Method for preparing fluoroisoferulic acid through single-enzyme catalysis and fluoroisoferulic acid

Abstract

The invention provides a method for preparing fluoro-isoferulic acid through single enzyme catalysis and fluoro-isoferulic acid, and belongs to the technical field of enzyme catalysis. The method for preparing fluoroisoferulic acid through single enzyme catalysis comprises the steps that caffeic acid, fluoroiodomethane, a pH buffer solution and halide methyltransferase are mixed, a reaction raw material solution is obtained, and the pH value of the reaction raw material solution is 7.5-7.7; carrying out a dark reaction on the reaction raw material solution to obtain a reaction mixed solution; and sequentially quenching and purifying the reaction mixed solution to obtain the fluoroisoferulic acid. The fluoroisoferulic acid is synthesized in a single-enzyme catalysis mode, the catalysis efficiency is high, and the structure type and the preparation mode of the caffeic acid fluorine-containing medicine can be enriched.

Description

Technical Field

[0001] The present invention relates to the technical field of enzyme catalysis, and particularly relates to a method for preparing fluoroferulic acid by single enzyme catalysis and fluoroferulic acid. Background Art

[0002] In recent years, in the field of drug research and development, on the basis of traditional alkylation modification, the research on fluoroalkylation has been further strengthened. Traditional fluoroalkylation is prepared by chemical synthesis, and the chemical synthesis method usually has the following disadvantages. For example, it lacks strong regioselectivity, chemoselectivity or stereoselectivity, usually has high energy consumption during the synthesis process, but the yield is low.

[0003] Among many fluorine-containing groups, monofluoromethyl, as a bioisosteric unit of a series of functional groups in biological systems, brings advantages such as metabolic stability, high bioavailability, strong lipophilicity and strong membrane permeability to the main chain compound with fluorine substituents. In the pharmaceutical field, the site difference of fluoromethylation can significantly affect the physicochemical properties, biological activity and pharmacokinetic properties of the prepared drugs. Site-selective fluoromethylation is one of the relatively key strategies to improve the performance of drugs.

[0004] However, at present, the selectivity of fluoromethylation of the commonly used chemical fluoromethylation reagent in industry is difficult to control, and there is an urgent need for a new way to improve the selectivity of fluoromethylation. Summary of the Invention

[0005] In view of this, in order to at least partially solve the above-mentioned technical problems, the present invention provides a method for preparing fluoroferulic acid by single enzyme catalysis and fluoroferulic acid.

[0006] According to an embodiment of one aspect of the present invention, a method for preparing fluoroferulic acid by single enzyme catalysis is provided, including: mixing caffeic acid, fluoroiodomethane, pH buffer solution, and halide methyltransferase to obtain a reaction raw material solution, and the pH value of the reaction raw material solution is 7.5 - 7.7; performing a light-shielded reaction on the reaction raw material solution to obtain a reaction mixture; quenching and purifying the reaction mixture in sequence to obtain fluoroferulic acid.

[0007] In some embodiments, the yield of fluoroferulic acid is more than 30%.

[0008] In some embodiments, the halide methyltransferase (HMT) includes at least one of AclHMT, AtHMT, VpaHMT, BxHMT, kalHMT, selHMT, umaHMT.

[0009] In some embodiments, the temperature of the light-shielded reaction is 20 - 40 °C, and the time of the light-shielded reaction is 10 - 15 h.

[0010] In some embodiments, the molar ratio of caffeic acid to halide methyltransferase is (4 - 6):1.

[0011] In some embodiments, the molar ratio of fluoroiodomethane to caffeic acid is (80 - 120):1.

[0012] In some embodiments, the pH buffer is at least one of an alkali metal phosphate solution, an alkali metal borate solution, and an alkali metal carbonate solution.

[0013] In some embodiments, the pH value of the reaction raw material solution is 7.6.

[0014] In some embodiments, the reagent used for quenching is a trifluoroacetic acid solution with the same volume as the reaction raw material solution.

[0015] In some embodiments, purification includes: centrifuging at 11000 - 13000 rpm for 20 - 40 min under the condition of 3 - 5°C.

[0016] According to an embodiment of another aspect of the present invention, there is provided a fluoro - isoferulic acid prepared by the method as described above.

[0017] In some embodiments, the fluoro - isoferulic acid is 4 - fluoromethoxy caffeic acid.

[0018] According to the embodiments of the present invention, the present invention significantly improves the reaction efficiency by using single - enzyme catalysis. Due to the use of single - enzyme catalysis, it has a better catalytic effect compared with the double - enzyme cascade catalysis in the related technology (requiring the co - use of HMT and SAM - dependent methyltransferase), significantly reduces the cost, and provides convenient conditions for large - scale industrial production. The raw materials of the present invention are simple and easy to obtain, have low toxicity, and are environmentally friendly in the preparation process. With the selectivity of halide methyltransferase, fluoro - isoferulic acid is prepared by fluoromethyl substitution at specific substitution sites, providing more diverse products for the pharmaceutical field, enriching the structural types of caffeic acid - containing fluorine drug preparations, and being beneficial for subsequent applications in technical fields such as the preparation of anti - diabetic drugs and anti - tumor drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Shows the process flow chart of the method for preparing fluoro - isoferulic acid by single - enzyme catalysis according to the embodiments of the present invention;

[0020] Figure 2 Shows the nuclear magnetic resonance hydrogen spectrum of the fluoro - isoferulic acid prepared in Example 1 of the present invention;

[0021] Figure 3 Shows the nuclear magnetic resonance carbon spectrum of the fluoro - isoferulic acid prepared in Example 1 of the present invention;

[0022] Figure 4 The nuclear magnetic resonance fluorine spectrum of fluoro - isoferulic acid prepared in Example 1 of the present invention is shown. Detailed implementation manners

[0023] Hereinafter, embodiments of the present invention will be described with reference to the accompanying drawings. However, it should be understood that these descriptions are merely exemplary and are not intended to limit the scope of the present invention. In the following detailed description, for the sake of explanation, many specific details are set forth to provide a comprehensive understanding of the embodiments of the present invention. However, obviously, one or more embodiments can also be implemented without these specific details. In addition, in the following description, descriptions of well - known structures and technologies are omitted to avoid unnecessarily confusing the concepts of the present invention.

[0024] The terms used herein are only for describing specific embodiments and are not intended to limit the present invention. The term "comprising" used herein indicates the presence of features, steps, operations, but does not exclude the presence or addition of one or more other features.

[0025] In the case of using expressions such as "at least one of A, B, and C, etc.", generally, it should be interpreted according to the meaning usually understood by those skilled in the art (for example, "a system having at least one of A, B, and C" should include, but is not limited to, a system having only A, only B, only C, having A and B, having A and C, having B and C, and / or having A, B, and C, etc.). In the case of using expressions such as "at least one of A, B, or C, etc.", generally, it should be interpreted according to the meaning usually understood by those skilled in the art (for example, "a system having at least one of A, B, or C" should include, but is not limited to, a system having only A, only B, only C, having A and B, having A and C, having B and C, and / or having A, B, and C, etc.).

[0026] In the present invention, the term "double - enzyme cascade reaction" refers to a continuous enzymatic reaction process that occurs in vivo or in vitro, involving two different enzymes acting in sequence to gradually convert a substrate into a final product; wherein, the substrate undergoes a specific chemical reaction under the action of the first enzyme to generate an intermediate product; then the intermediate product generated by the reaction serves as the substrate of the second enzyme and further reacts under the catalysis of the second enzyme to generate the final product.

[0027] Currently, about 20% of the drugs on the market contain fluorine atoms or fluoroalkyl groups. In the field of drug research and development, fluorine elements have attracted much attention due to their unique properties. Specifically, fluorine atoms have a small radius and a large electronegativity. Introducing fluorine or fluorine - containing groups into drug molecules can significantly affect various properties of drugs. For example, it can change the conformation of drug molecules, regulate the acidity coefficient (pKa) value of drugs, improve membrane permeability, and can also optimize metabolic pathways and pharmacokinetic properties.

[0028] Among numerous fluorine-containing groups, monofluoromethyl, as a bioisostere of a series of functional groups in biological systems (i.e., groups with similar physical and chemical properties and similar biological activities in biological systems), combined with the enhanced metabolic stability, bioavailability, lipophilicity, and membrane permeability brought by fluorine substituents, provides an effective approach for drug design.

[0029] It can be understood that site-selective fluoromethylation is one of the important strategies for improving drug properties during the process of drug research and development. In drug molecules, the site differences of fluoromethylation can significantly affect the physicochemical properties, biological activities, and pharmacokinetic characteristics of drugs. Biocatalytic reactions, with their advantages of mild reaction conditions, high substrate specificity, and environmental friendliness, have become a potential strategy for selectively introducing fluoromethyl into complex molecules.

[0030] Isoferulic acid is a caffeic acid derivative commonly found in propolis and the traditional Chinese medicine Cimicifuga foetida, with a methyl substituent introduced at the 4-OH site of caffeic acid. Isoferulic acid can be applied in hypoglycemic drugs. Isoferulic acid activates the α-1A adrenergic receptor, triggers the phospholipase C (PLC)-protein kinase C (PKC) signaling pathway, increases the level of glucose transporter 4 (GLUT4) in muscle, thereby stimulating 2 C 12 cells to uptake glucose. Moreover, isoferulic acid can also regulate the expression of genes related to hepatic glucose metabolism. By activating the α-1A adrenergic receptor, it enhances the secretion of endogenous β-endorphin and regulates the mRNA levels of phosphoenolpyruvate carboxykinase (PEPCK) and GLUT4, which are crucial in the gluconeogenesis pathway. Isoferulic acid has inhibitory effects on both α-glucosidase and pancreatic α-amylase, and can inhibit the rise of postprandial blood glucose, which is of positive significance for controlling blood glucose levels. Intestinal glucose is transported into the blood by glucose transporter 2 (GLUT2), and isoferulic acid can significantly inhibit intestinal glucose uptake by interfering with GLUT2. Isoferulic acid can exert hypoglycemic effects through different pathways, and the fluorine derivatives of isoferulic acid are expected to further improve the activity and pharmacokinetics of ferulic acid through the unique properties of fluorine elements, and thus enhance its role in regulating blood glucose and preventing diabetes-related complications.

[0031] Among them, the structures of caffeic acid, ferulic acid, and isoferulic acid are shown in the following formula (I):

[0032] Formula (I).

[0033] In plants, fungi, and bacteria, halide methyltransferase (HMT) is an S-adenosyl-L-methionine (SAM)-dependent methyltransferase. It can catalyze the nucleophilic substitution reaction between a halide ion and S-adenosyl-L-methionine (SAM) to generate halomethane and S-adenosyl-L-homocysteine (SAH), as shown in the following formula (II).

[0034] Formula (II).

[0035] Tobacco caffeic acid-O-methyltransferase (NtCOMT) can specifically catalyze the methylation of the 3-OH site of the substrate caffeic acid to generate ferulic acid using SAM as the methyl donor. In related technologies, a fluorine decarboxylated SAM analogue (F-dcSAM) was synthesized, and the 3-OH site fluoromethylation of caffeic acid was achieved under the catalysis of NtCOMT, as shown in the following formula (III). In the presence of dcSAH, using FCH 2 I as the fluoromethyl donor, the fluorine analogue of ferulic acid was also produced through the HMT-NtCOMT double enzyme cascade reaction, and the fluoromethylation efficiency was 52%. The specific process is shown in the following formula (IV).

[0036] Formula (III);

[0037] Formula (IV).

[0038] However, the above double enzyme cascade reaction or the reaction involving SAM analogues is usually used to achieve the fluoromethylation of the 3-OH site of caffeic acid, and it is difficult to achieve the fluoromethylation of the 4-OH site to generate the fluorinated isoeugenol of the 4-OH site. Moreover, no other efficient method that can replace the double enzyme cascade reaction for synthesizing fluorinated isoeugenol has been found yet, which will limit the development and application of anti-diabetic drugs based on fluorinated isoeugenol.

[0039] In addition, the double enzyme cascade reaction requires the co-participation of HMT, SAM-dependent methyltransferase, and SAH analogues. The reaction system is complex, and the preparation and purification processes of the two enzymes increase the cost, making it difficult to achieve the batch production of fluorine-containing drugs.

[0040] In the process of realizing the concept of the present invention, it was found that under the single enzyme catalysis of halide methyltransferase, caffeic acid and fluoromethane iodide specifically bind to achieve the fluoromethylation of the 4-OH site, and fluorinated isoeugenol is prepared, which enriches the structural types of caffeic acid-containing fluorine drugs, and the present invention provides a new method for single fluorine catalysis.

[0041] Specifically, according to an embodiment of one aspect of the present invention, a method for preparing fluorinated isoeugenol by single enzyme catalysis is provided. Figure 1The flowchart of the method for preparing fluoro - isoferulic acid by single - enzyme catalysis according to an embodiment of the present invention is shown. As Figure 1 shown, the method includes operations S101 - S103.

[0042] In operation S101, caffeic acid, fluoroiodomethane, pH buffer solution, and halide methyltransferase are mixed to obtain a reaction raw material solution, and the pH value of the reaction raw material solution is 7.5 - 7.7.

[0043] In this embodiment, caffeic acid is used as a reaction substrate and belongs to phenolic acid compounds. The phenolic hydroxyl group therein is the target site for fluoro - methylation substitution. Fluoroiodomethane is used as the donor of the fluoromethyl group. Halide methyltransferase (HMT) can specifically catalyze the transfer of the fluoromethyl group from fluoroiodomethane to a specific hydroxyl site of caffeic acid, such as the 3 - OH site and / or the 4 - OH site. Under the catalytic action of halide methyltransferase (HMT), the fluoromethyl group is transferred to a specific hydroxyl group of caffeic acid. Fluoroiodomethane has moderate activity and low toxicity, which is beneficial to environmental protection. Adjusting the pH of the reaction raw material solution to the above - mentioned range enables HMT to have higher activity.

[0044] In operation S102, the reaction raw material solution is subjected to a light - avoiding reaction to obtain a reaction mixture.

[0045] In this embodiment, caffeic acid has a catechol structure and is prone to photo - oxidation reaction under the action of light. Fluoroiodomethane also has a certain degree of photosensitivity; although HMT has low light sensitivity, conformational changes may occur under long - term light irradiation, reducing the catalytic efficiency. Based on the light - avoiding reaction conditions, it helps to maintain the chemical stability of the reaction substrate and fluoroiodomethane, and can maintain the catalytic specificity of HMT.

[0046] In operation S103, the reaction mixture is quenched and purified in sequence to obtain fluoro - isoferulic acid.

[0047] In this embodiment, the enzyme - catalyzed reaction is terminated by quenching to avoid excessive fluoro - methylation reaction and ensure the regioselectivity of the reaction product.

[0048] According to an embodiment of the present invention, when comparing the single-enzyme catalysis of HMT of the present invention with the two-enzyme cascade reaction of HMT and methyltransferase in the prior art, the single-enzyme catalysis of HMT of the present invention has a higher fluoromethylation efficiency compared to the two-enzyme cascade reaction. The two-enzyme cascade reaction has relatively limited selectivity for the reaction site and can only achieve fluoromethylation at the 3-OH position of caffeic acid, which limits its application in the modification of more extensive drug molecules. The present invention uses single-enzyme catalysis of HMT and uses fluoroiodomethane as a fluoromethyl donor to transfer the fluoromethyl group to the 4-OH site of caffeic acid to generate fluoroisoferulic acid with fluorine substitution at the 4-OH site, so that fluoroisoferulic acid of caffeic acid can be prepared by single-enzyme catalysis of HMT. Isoferulic acid can be applied to hypoglycemic drugs and is widely used as a natural antioxidant in the pharmaceutical and food industries. The fluoroisoferulic acid of the present invention is conducive to the subsequent application and promotion in drug research and development. Moreover, the single-enzyme catalysis method of HMT of the present invention can achieve a more excellent fluoromethylation efficiency compared to the two-enzyme cascade reaction and does not require the participation of SAM analogs. Therefore, the single-enzyme catalysis simplifies the reaction system, reduces the costs in aspects such as enzyme purification and preservation, and provides a more practical solution for large-scale industrial applications. The present invention avoids the complex requirements of the two-enzyme cascade reaction. The reaction conditions of the present invention are easier to control and optimize, reducing the operation difficulty and improving the stability and repeatability of the reaction.

[0049] In some embodiments, the yield of fluoroisoferulic acid is more than 30%, preferably more than 50%. For example, when using AclHMT and selHMT as halide methyltransferases, the yield of the prepared fluoroisoferulic acid is relatively higher.

[0050] In some embodiments, the halide methyltransferase (HMT) includes at least one of AclHMT, AtHMT, VpaHMT, BxHMT, kalHMT, selHMT, umaHMT. Specifically, for example, when using AclHMT, the yield of the prepared fluoroisoferulic acid is 92%, indicating that AclHMT has a high selectivity for substitution at the 4-OH site. Using umaHMT can not only obtain the fluoromethyl product at the 3-OH site (yield 30%) and fluoroisoferulic acid (yield 40%) simultaneously, but also detect the fluoromethyl product with substitution at the dihydroxy site (yield 12%). The diverse selectivity of umaHMT provides more structural choices for subsequent drug design, meets the requirements for the diversity of fluoromethylation sites in drug research and development, and enables these diverse caffeic acid fluorine-containing products to have potential new biological activities and application values. Although the yield prepared by selHMT is lower than that of AclHMT, it can still prepare fluoroisoferulic acid with a yield of 57.5%, indicating that the above-mentioned halide methyltransferases all have high regioselectivity and catalytic activity in the single-enzyme catalysis of caffeic acid.

[0051] In some embodiments, the temperature of the light-shielded reaction is 20~40°C, for example, it can be 20°C, 30°C or 40°C, and preferably 30°C. With such a setting, it is possible to ensure that HMT has both high activity and high stability. The reaction at the above temperature can be regulated by a water bath or an incubator. The time of the light-shielded reaction is 10~15h, for example, it can be 10h, 11h, 12h, 13h, 14h or 15h, and preferably 12h. With such a setting, it is possible to ensure the full progress of the reaction and not affect the purity of the product.

[0052] In some embodiments, the molar ratio of caffeic acid to halide methyltransferase is (4~6):1, for example, it can be 4:1, 5:1 or 6:1, and preferably 5:1. With such a setting, it helps to promote the efficient utilization of the enzyme active site, avoid the low conversion rate due to the shortage of reaction substrates or enzyme amount, and ensure the relatively stable reaction rate. If the molar ratio of the two is too low, the reaction rate will decrease due to too little caffeic acid substrate; if the molar ratio of the two is too high, it may lead to the occupation of other binding sites of the enzyme by high-concentration caffeic acid or the change of the enzyme conformation, reducing the catalytic efficiency, and also avoiding the waste of reaction substrates.

[0053] In some embodiments, the molar ratio of fluoroiodomethane to caffeic acid is (80~120):1, for example, it can be 80:1, 90:1, 100:1, 110:1 or 120:1, and preferably 100:1. The reason for adjusting fluoroiodomethane to the above excessive state is that fluoroiodomethane may be hydrolyzed or undergo side reactions and be partially lost. With such a setting, it is ensured that there is sufficient fluoromethyl donor present to maintain the forward progress of the reaction, and thus promote a relatively high fluoromethylation conversion rate of caffeic acid.

[0054] In some embodiments, the pH buffer is at least one of alkali metal phosphate solution, alkali metal borate solution, and alkali metal carbonate solution. Specifically, for example, it can be sodium phosphate solution, sodium nitrate solution, sodium bicarbonate solution, etc., as long as it is a metal salt solution with a certain alkalinity, and the present invention does not make special limitations thereto. The pH buffer is suitable for maintaining a stable pH environment.

[0055] In some embodiments, the pH value of the reaction raw material solution is 7.6. It has been found in the related embodiments of the present invention that this pH is the appropriate pH value for HMT, which can further promote the progress of single-enzyme catalysis and improve the fluoromethylation conversion rate.

[0056] In some embodiments, the reagent used for quenching is trifluoroacetic acid solution with the same volume as the reaction raw material solution. Specifically, for example, it can be trifluoroacetic acid solution with a mass fraction of 10%. Using the above reagent for quenching helps to more accurately control the termination of the reaction, and because trifluoroacetic acid has a relatively high volatility, it is more convenient to remove during the purification process.

[0057] In some embodiments, the purification includes: under the condition of 3 - 5 °C, such as 3 °C, 4 °C or 5 °C, at a rotation speed of 11000 - 13000 rpm, such as 11000 rpm, 12000 rpm or 13000 rpm, centrifuging for 20 - 40 min, such as 20 min, 30 min or 40 min. With such settings, it helps to maintain the stability of the prepared product at low temperature, and can effectively separate products with different densities by centrifugation. Moreover, due to the low temperature, it can be achieved only by simple heating, which simplifies the experimental steps and saves costs.

[0058] The embodiments of the present invention develop a biocatalytic method for realizing the site - specific fluoromethylation at the 4 - OH site of caffeic acid based on HMT single - enzyme catalysis to generate fluoro - isoferulic acid, so as to promote the development of new fluorine - containing drugs with potential pharmacological activities such as preventing diabetes - related cardiovascular diseases. The present invention simplifies the caffeic acid fluoromethylation reaction system, and makes up for the disadvantages of the existing double - enzyme cascade reaction, such as difficult reaction condition control and high reaction cost. The present invention has a higher fluoromethylation efficiency through HMT single - enzyme catalysis compared with the existing double - enzyme cascade reaction, providing a more efficient and economical technical path for the large - scale production of fluorine - containing drugs.

[0059] According to an embodiment of another aspect of the present invention, there is provided a fluoro - isoferulic acid prepared by the method as described above.

[0060] According to the embodiments of the present invention, the present invention synthesizes fluoro - isoferulic acid through the single - enzyme catalysis of HMT. Such fluoro - isoferulic acid helps to enrich the structural types of caffeic acid - containing fluorine - containing drugs and is expected to be applied in the research and development of drugs for inhibiting blood sugar, antibacterial and anti - inflammatory, and anti - tumor in the future.

[0061] The present invention will be further described below through examples, drawings, and related test experiments and their results. In the following detailed description, for the convenience of explanation, many specific details are elaborated to provide a comprehensive understanding of the embodiments of the present invention. However, obviously, one or more embodiments can also be implemented without these specific details. Moreover, without conflict, the details in the following embodiments can be combined arbitrarily into other feasible embodiments.

[0062] It should be noted that the following specific embodiments are only for illustration, and the protection scope of the present invention is not limited thereto. The chemical drugs and raw materials used in the following embodiments are all obtained commercially or prepared by recognized treatment methods.

[0063] It should be noted that the above-mentioned halide methyltransferase of the present invention is preferably selected from AclHMT, AtHMT, VpaHMT, BxHMT, kalHMT, selHMT, umaHMT. The gene source species of aclhmt, athmt, vpahmt, bxhmt, kalhmt, selhmt, umaHMT are Aspergillus clavatus, Arabidopsis thaliana, Vibrio parahaemolyticus, Paraburkholderia xenovorans, Kordia algicida, Synechococcus elongates, Ustilago maydis respectively. The above genes were synthesized by a biological company and constructed on the pET28a(+) vector with a Kanamycin resistance gene after codon optimization by BL21(DE3). The expression and purification of HMT were referred to the literature (Hammer S C, et al. Angew. Chem. Int. Ed. 2021, 60, 5554–5560).

[0064] Specifically, the protein sequences of the halide methyltransferase used are shown as SEQ ID No.1 to SEQ ID No.3 below.

[0065] AclHMT (SEQ ID No.1):

[0066] MSTPSLIPSGVHEVLAKYKDGNYVDGWAELWDKSKGDRLPWDRGFPNPALEDTLIQKRAIIGGPLGQDAQGKTYRKKALVPGCGRGVDVLLLASFGYDAYGLEYSATAVDVCQEEQAKNGDQYPVRDAEIGQGKITFVQGDFFEDTWLEKLNLTRNCFDVIYDYTFFCALNPSMRPQWALRHTQLLADSPRGHLICLEFPRHKDPSVQGPPWGSASEAYRAHLSHPGEEIPYDASRQCQFDSSKAPSAQGLERVAYWQPERTHEVGKNEKGEVQDRVSIWQRPPQSSLLEHHHHHH.

[0067] umaHMT (SEQ ID No.2):

[0068] MTSSLSKDDQIQNLRRLFADSGVPNDPKAWDQAWIDSTTPWDANRPQPALVELLEGAHDADAKVPDVDGNLIPVSQAIPKGDGTAVVPGCGRGYDARVFAERGLTSYGVDISSNAVAAANKWLGDQDLPTELDDKVNFAEADFFTLGTSKSLVLELSKPGQATLAYDYTFLCAIPPSLRTTWAETYTRLLAKHGVLIALVFPIHGDRPGGPPFSISPQLVRELLGSQKNADGSAAWTELVELKPKGPET RPDVERMMVWRRSLEHHHHHH*。

[0069] selHMT (SEQ ID No.3):

[0070] MTNAVNQAQFWEQRYQEGSDRWDLGQAAPVWRSLLAGTNAPAPGRIAVLGCGRGHDARLFAEQGFEVVGFDFAPSAIAAAQALAQGTTAQFLQRDIFALPQEFAGQFDTVLEHTCFCAIDPDRRAEYVEVVRQILKPKGCLLGLFWCHDRPSGPPYGCSLTELRDRFAQGWQEEQLESVTESVEGRRGEEYLGRWRRLDLEHHHHHH*。

[0071] Example 1

[0072] Process of AclHMT catalyzing the fluoromethylation of caffeic acid:

[0073] The reaction raw material solution (100 μL) contains 1 mM caffeic acid, 100 mM CH 2 FI, 50 mM sodium phosphate buffer (the pH value of the buffer is adjusted to 7.6 using hydrochloric acid and sodium hydroxide), and 200 μM halide methyltransferase (AclHMT).

[0074] The reaction raw material solution is reacted in the dark in a water bath at 30 °C for 12 hours. After the reaction is completed, it is quenched with 10 wt% trifluoroacetic acid (TFA) of the same volume as the reaction raw material solution. The quenched reaction mixture is centrifuged at 4 °C and 12,000 rpm for 30 min using a high-speed refrigerated centrifuge to obtain fluoroisoferulic acid, and its reaction process is shown as follows.

[0075]

[0076] Ferulic acid fluoride was analyzed and detected by analytical liquid chromatography (HPLC) using a reverse-phase C18 chromatographic column (150 mm × 4.6 mm, 2.5 μm) of a certain company under ultraviolet detector detection wavelengths of 254 nm and 215 nm. Mobile phase A was H 2 O + trifluoroacetic acid with a mass fraction of 1‰, and mobile phase B was acetonitrile (CH 3 CN) + trifluoroacetic acid with a mass fraction of 1‰. The molecular weight of ferulic acid fluoride was determined by liquid chromatography LC-MS analysis. A large amount of prepared ferulic acid fluoride was also dissolved in 400 μL of deuterated dimethyl sulfoxide (DMSO-d6) and subjected to 1 1H-NMR, 19 19F-NMR, 13 13C-NMR identification by a nuclear magnetic resonance spectrometer of a certain company with a proton resonance frequency of 800 MHz. Figure 2 The nuclear magnetic resonance hydrogen spectrum of ferulic acid fluoride prepared in Example 1 of the present invention is shown; Figure 3 The nuclear magnetic resonance carbon spectrum of ferulic acid fluoride prepared in Example 1 of the present invention is shown; Figure 4 The nuclear magnetic resonance fluorine spectrum of ferulic acid fluoride prepared in Example 1 of the present invention is shown. As Figures 2 to 4 shown, by analyzing the structure of the fluoromethylated product, it can be confirmed that ferulic acid fluoride (i.e., ferulic acid with fluorine substitution at the 4-OH site) was prepared in the present invention. The experimental results show that AclHMT (Aspergillus clavatus HMT) can recognize the substrate caffeic acid of NtCOMT and exhibit excellent fluoromethylation efficiency (93%). Among them, the yield of ferulic acid fluoride reached 87%, and the yield of the difluoromethylated product was 6%. This indicates that AclHMT has high selectivity and efficient catalytic ability for the fluoromethylation of caffeic acid at the 4-OH position. In addition, the fluoromethylation reaction of caffeic acid catalyzed by AclHMT can also be applied to large-scale preparation reactions, which helps to scale up the reaction system proportionally to a 40 mL preparation method.

[0077] Example 2

[0078] The process of umaHMT-catalyzed fluoromethylation of caffeic acid:

[0079] The reaction raw material solution (100 μL) contained 1 mM caffeic acid, 100 mM CH 2 FI, 50 mM sodium phosphate buffer (the pH value of the buffer was adjusted to 7.6 using hydrochloric acid and sodium hydroxide), and 200 μM halide methyltransferase (umaHMT).

[0080] The reaction raw material solution was reacted in a 30 °C water bath in the dark for 12 hours. After the reaction was completed, it was quenched with 10 wt% trifluoroacetic acid (TFA) of the same volume as the reaction raw material solution. The quenched reaction mixture was centrifuged at 4 °C and 12,000 rpm for 30 min using a high-speed refrigerated centrifuge to obtain fluoroferulic acid, and its reaction process is shown in the following formula.

[0081]

[0082] Fluoroferulic acid was analyzed and detected by analytical HPLC on a reversed-phase C18 column (150 mm × 4.6 mm, 2.5 μm) at ultraviolet detector wavelengths of 254 nm and 215 nm. Mobile phase A was H 2 O with 1‰ mass fraction of trifluoroacetic acid, and mobile phase B was CH 3 CN with 1‰ mass fraction of trifluoroacetic acid. The molecular weight of fluoroferulic acid was determined by liquid chromatography LC-MS analysis. A large amount of prepared fluoroferulic acid was also dissolved in 400 μL of deuterated dimethyl sulfoxide (DMSO-d6) and analyzed by a nuclear magnetic resonance spectrometer with a proton resonance frequency of 800 MHz from a certain company for 1 1H-NMR, 19 19F-NMR, 13 13C-NMR identification to confirm the structure of the fluoromethylated product. The experimental results showed that umaHMT (Ustilago maydis HMT) could recognize the substrate caffeic acid of NtCOMT to generate various fluoromethylated products, among which the yield of the 3-fluoromethyl product was 30%, the yield of fluoroferulic acid was 40%, and a difluoromethyl product with fluoromethylation at both the 3-OH site and the 4-OH site was also detected, with a yield of 12%. This indicates that the selectivity of the umaHMT-catalyzed reaction is different from that of AclHMT, and it can generate various caffeic acid fluoromethylated products, providing more diverse structural choices for the synthesis of fluorine-containing drugs.

[0083] Example 3

[0084] The process of selHMT-catalyzed fluoromethylation of caffeic acid:

[0085] The reaction raw material solution (100 μL) contained 1 mM caffeic acid, 100 mM CH 2 FI, 50 mM sodium phosphate buffer (the pH value of the buffer was adjusted to 7.6 using hydrochloric acid and sodium hydroxide), and 200 μM halide methyltransferase (selHMT).

[0086] The reaction raw material solution was reacted for 12 hours in a 30 °C water bath in the dark. After the reaction was completed, it was quenched with 10 wt% trifluoroacetic acid (TFA) of the same volume as the reaction raw material solution. The quenched reaction mixture was centrifuged at 4 °C and 12,000 rpm for 30 min using a high-speed refrigerated centrifuge to obtain fluoroferulic acid.

[0087] The fluoroferulic acid was analyzed and detected on an analytical HPLC using a reversed-phase C18 column (150 mm × 4.6 mm, 2.5 μm) at detection wavelengths of 254 nm and 215 nm by an ultraviolet detector. Mobile phase A was H 2 O + trifluoroacetic acid with a mass fraction of 1‰, and mobile phase B was CH 3 CN + trifluoroacetic acid with a mass fraction of 1‰. The molecular weight of fluoroferulic acid was determined by liquid chromatography LC-MS analysis. The experimental results showed that the fluoromethylation efficiency of selHMT (Synechococcus elongates HMT) was relatively low compared to AclHMT and umaHMT. selHMT could recognize the substrate caffeic acid of NtCOMT to generate various fluoromethylation products. Among them, the yield of the fluoromethyl product at the 3-OH site was 2.5%, the yield of fluoroferulic acid was 57.5%, and the yield of the difluoromethyl product was 9%.

[0088] Comparative Example 1

[0089] The reaction raw material solution (100 μL) contained 3 mM caffeic acid, 100 mM CH 2 FI, 1 mM dcSAH, 6 mM MgCl 2 , 150 μM AclHMT, and 100 mM sodium phosphate buffer (the pH value of the buffer was adjusted to 6.5 by adding hydrochloric acid and sodium hydroxide).

[0090] The reaction raw material solution was incubated at 30 °C for 36 hours in the dark. After the reaction was completed, it was quenched with 10 wt% trifluoroacetic acid (TFA) of the same volume as the reaction raw material solution.

[0091] After identification, a ferulic acid fluoroderivative (caffeic acid 3-OH fluoromethylation product) was obtained, and its reaction yield was 52%. The reaction process is shown in the following formula.

[0092]

[0093] Comparing Example 1 with Comparative Example 1, it can be seen that the yield of the fluoromethylated product obtained in Example 1 is relatively higher, improving the reaction efficiency. Moreover, the product prepared in Example 1 can be widely used in inhibiting blood sugar elevation and anti-tumor drugs, and is widely used as an antioxidant in the pharmaceutical and food industries. Comparing Example 2 with Comparative Example 1, it can be seen that Example 2 can simultaneously prepare multiple fluoromethylated products, providing more structural selectivity for subsequent drug design and meeting the relevant requirements for the diversity of fluoromethylation sites in drug research and development.

[0094] The specific embodiments described above further elaborate on the purpose, technical solutions, and beneficial effects of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not used to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for preparing fluoroisoferulic acid by single enzyme catalysis, characterized in that: include: Mixing caffeic acid, fluoroiodomethane, pH buffer, and halide methyltransferase to obtain a reaction raw material solution, wherein the pH value of the reaction raw material solution is 7.5-7.7; The reaction raw material solution is subjected to light-protected reaction to obtain a reaction mixture; The reaction mixture is sequentially quenched and purified to obtain fluoroisoferulic acid.

2. The method according to claim 1, characterized in that The yield of the fluoroisoferulic acid is more than 30%.

3. The method according to claim 1, characterized in that The halide methyltransferase includes at least one of AclHMT, AtHMT, VpaHMT, BxHMT, kalHMT, selHMT, and umaHMT.

4. The method according to claim 1, characterized in that: The temperature of the light-proof reaction is 20-40° C., and the time of the light-proof reaction is 10-15 hours.

5. The method according to claim 1, characterized in that: The molar ratio of caffeic acid to the halide methyltransferase is (4-6):1; The molar ratio of fluoroiodomethane to caffeic acid is (80~120):

1.

6. The method according to claim 1, characterized in that The pH buffer solution is at least one of an alkali metal phosphate solution, an alkali metal borate solution, and an alkali metal carbonate solution.

7. The method according to claim 1, characterized in that The pH value of the reaction raw material solution is 7.

6.

8. The method according to claim 1, characterized in that: The reagent used for the quenching is a trifluoroacetic acid solution with the same volume as that of the reaction raw material solution.

9. The method according to claim 1, characterized in that: The purification comprises: The centrifuge was performed at 11000-13000 rpm for 20-40 min at 3-5°C.

10. Fluoroisoferulic acid prepared by the method according to any one of claims 1 to 9.

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