Method for preparing fluoroisoferulic acid catalyzed by single enzyme and fluoroisoferulic acid

Abstract

The present invention provides a method for preparing fluoroisoferulic acid by single enzyme catalysis and fluoroisoferulic acid, belonging to the technical field of enzyme catalysis. The method for preparing fluoroisoferulic acid by single enzyme catalysis comprises: mixing caffeic acid, fluoroiodomethane, a pH buffer, and a halide methyltransferase to obtain a reaction raw material solution, wherein the pH value of the reaction raw material solution is 7.5 to 7.7; subjecting the reaction raw material solution to a light-proof reaction to obtain a reaction mixture; and sequentially quenching and purifying the reaction mixture to obtain fluoroisoferulic acid. The present invention synthesizes fluoroisoferulic acid by single enzyme catalysis, and has high catalytic efficiency, which helps to enrich the structural types and preparation methods of caffeic acid fluorine-containing drugs.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of enzyme catalysis, and in particular to a method for preparing fluorinated isoferulic acid by single enzyme catalysis and fluorinated isoferulic acid. BACKGROUND

[0002] In recent years, in the field of drug research and development, further research on fluorohydrocarbonylation has been strengthened on the basis of traditional hydrocarbonylation modification. Traditional fluorohydrocarbonylation is prepared by chemical synthesis, which usually has the following disadvantages, such as lack of strong regioselectivity, chemoselectivity or stereoselectivity, and usually has high energy consumption but low yield in the synthesis process.

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

[0004] However, the selectivity of the chemical fluoromethylation reagent commonly used in industry is difficult to control, and a new way to improve the selectivity of fluoromethylation is urgently needed. SUMMARY

[0005] Therefore, in order to at least partially solve the above-mentioned technical problems, the present application provides a method for preparing fluorinated isoferulic acid by single enzyme catalysis and fluorinated isoferulic acid.

[0006] According to an embodiment of one aspect of the present application, a method for preparing fluorinated isoferulic acid by single enzyme catalysis is provided, comprising: mixing caffeic acid, fluoromethyl iodide, a pH buffer and a halide methyltransferase to obtain a reaction raw material solution, the pH value of the reaction raw material solution being 7.5-7.7; performing a light-avoiding reaction on the reaction raw material solution to obtain a reaction mixture; and sequentially performing quenching and purification on the reaction mixture to obtain fluorinated isoferulic acid.

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

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

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

[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 quenching reagent used is trifluoroacetic acid solution of the same volume as the reaction raw material solution.

[0015] In some embodiments, the purification comprises centrifugation at a speed of 11000-13000 rpm for 20-40 min at 3-5℃.

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

[0017] In some embodiments, the fluoroisoferulic acid is 4-fluoromethoxycaffeic acid.

[0018] According to an embodiment of the present application, the present application significantly improves the reaction efficiency by using single enzyme catalysis. Due to the use of single enzyme catalysis, compared with the double enzyme cascade catalysis in the related art (which needs to use HMT and SAM-dependent methyltransferase together), the catalytic effect is better, the cost is significantly reduced, and the large-scale industrial production is facilitated. The raw material of the present application is simple, low toxicity, and more environmentally friendly in the preparation process. With the selectivity of halide methyltransferase, fluoromethyl substitution is carried out at a specific substitution site to prepare fluoroisoferulic acid, which provides more diversified products for the pharmaceutical field, enriches the structure type of caffeic acid fluorine-containing drug preparation, and is beneficial to subsequent application in the technical field of preparing anti-diabetic drugs, anti-tumor drugs, etc. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 A flow chart of a method for preparing fluoroisoferulic acid by single enzyme catalysis according to an embodiment of the present application is shown;

[0020] Figure 2 A nuclear magnetic resonance hydrogen spectrum of fluoroisoferulic acid prepared in Example 1 of the present application is shown;

[0021] Figure 3 A nuclear magnetic resonance carbon spectrum of fluoroisoferulic acid prepared in Example 1 of the present application is shown;

[0022] Figure 4 The nuclear magnetic resonance fluorogram of the fluoroisoferulic acid prepared in Embodiment 1 of the present application is shown. DETAILED DESCRIPTION

[0023] Hereinafter, embodiments of the present application will be described with reference to the accompanying drawings. It is to be understood, however, that the description is merely exemplary and is not intended to limit the scope of the present application. In the following detailed description of the embodiments of the present application, numerous specific details are set forth in order to provide a thorough understanding of the embodiments of the present application. It will be apparent, however, to one skilled in the art that the embodiments of the present application can be practiced without these specific details. In other instances, well-known structures and functions have been omitted or simply referenced in order not to obscure the concept of the present application.

[0024] The terms used herein are merely for the purpose of describing particular embodiments and are not intended to limit the present application. The term "include" used herein indicates the presence of a feature, step, operation, but does not exclude the presence or addition of one or more other features.

[0025] In the case of using an expression similar to "at least one of A, B, and C", it is generally to be interpreted that the expression includes one or more of the items enumerated after the phrase "at least one of", e.g., to include one of A or B or C, A and B, A and C, B and C, or A and B and C, etc. In the case of using an expression similar to "at least one of A, B, or C", it is generally to be interpreted that the expression includes one or more of the items enumerated after the phrase "at least one of", e.g., to include A or B or C, A and B, A and C, B and C, or A and B and C, etc.

[0026] In the present application, the term "double enzyme cascade reaction" refers to a continuous enzymatic reaction process occurring in vivo or in vitro, involving two different enzymes acting in turn 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 a substrate for the second enzyme, which further undergoes a reaction under the catalysis of the second enzyme to generate the final product.

[0027] At present, about 20% of the drugs on the market contain fluorine atoms or fluorine alkyl groups. In the field of drug research and development, fluorine elements are of great concern due to their unique properties. Specifically, fluorine atoms have small radii and high electronegativity. The introduction of fluorine or fluorine-containing groups into drug molecules can significantly affect various properties of the drugs. For example, it can change the conformation of the drug molecule, adjust the acidity coefficient (pKa) value of the drug, improve the membrane permeability, and optimize the metabolic pathway and pharmacokinetic properties.

[0028] Among the many fluorine-containing groups, the monofluoromethyl group serves as a bioisosteric unit (i.e., a group with similar physical and chemical properties and producing similar biological activities in biological systems) for a series of functional groups in biological systems. Combined with the enhanced metabolic stability, bioavailability, lipophilicity and membrane permeability brought by the fluorine substituent, it provides an effective approach for drug design.

[0029] Understandably, site-selective fluoromethylation is a key strategy for improving drug performance during drug development. Differences in the site of fluoromethylation within a drug molecule can significantly impact its physicochemical properties, biological activity, and pharmacokinetic profile. Enzyme catalysis, with its mild reaction conditions, high substrate specificity, and environmental friendliness, has become a promising strategy for selectively introducing fluoromethyl groups into complex molecules.

[0030] Isoferulic acid is a caffeic acid derivative commonly found in propolis and the traditional Chinese medicine Cimicifuga heracleifolia. It incorporates a methyl substituent at the 4-OH site of caffeic acid. Isoferulic acid can be used in hypoglycemic drugs. It activates the α-1A adrenergic receptor, triggering the phospholipase C (PLC)-protein kinase C (PKC) signaling pathway and increasing the level of glucose transporter 4 (GLUT4) in muscle, thereby stimulating C2C. 12 Isoferulic acid also regulates the expression of genes involved in hepatic glucose metabolism. By activating α-1A adrenergic receptors, it enhances endogenous β-endorphin secretion and modulates the mRNA levels of phosphoenolpyruvate carboxykinase (PEPCK) and GLUT4, key enzymes in the hepatic gluconeogenesis pathway. Isoferulic acid inhibits both α-glucosidase and pancreatic α-amylase, suppressing the postprandial rise in blood glucose, which has positive implications for blood glucose control. Intestinal glucose is transported into the bloodstream via glucose transporter 2 (GLUT2), and isoferulic acid can significantly inhibit intestinal glucose uptake by interfering with GLUT2. Isoferulic acid can exert its hypoglycemic effects through various pathways, and fluorinated derivatives of isoferulic acid are expected to further enhance the activity and pharmacokinetics of ferulic acid through the unique properties of fluorine, thereby improving 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 that catalyzes the nucleophilic substitution reaction between halide ions and S-adenosyl-L-methionine (SAM) to produce methyl halides and S-adenosyl-L-homocysteine ​​(SAH). The specific process is shown in the following formula (II).

[0034] Formula (II).

[0035] Tobacco caffeic acid-O-methyltransferase (NtCOMT) can specifically catalyze the methylation of the substrate caffeic acid at the 3-OH site to produce ferulic acid, using SAM as a methyl donor. A fluorodecarboxylation SAM analog (F-dcSAM) was synthesized in related technology. Under the catalysis of NtCOMT, fluoromethylation of the 3-OH site of caffeic acid was achieved, as shown in the following formula (III). Using dcSAH and FCH2I as the fluoromethyl donor, a dual-enzyme cascade reaction (HMT-NtCOMT) also produced the fluoroanalog of ferulic acid, with a fluoromethylation efficiency of 52%. The specific process is shown in the following formula (IV).

[0036] Formula (III);

[0037] Formula (IV).

[0038] However, the above-mentioned dual-enzyme cascade reaction or the reaction involving SAM analogs is usually used to achieve fluoromethylation of the 3-OH site of caffeic acid, but it is difficult to achieve fluoromethylation of the 4-OH site to generate fluoroisoferulic acid at the 4-OH site. At present, no other efficient method that can replace the dual-enzyme cascade reaction to synthesize fluoroisoferulic acid has been found, which will limit the development and application of anti-diabetic drugs based on fluoroisoferulic acid.

[0039] In addition, the dual-enzyme cascade reaction requires the joint participation of HMT, SAM-dependent methyltransferase and SAH analogs. The reaction system is complex, and the preparation and purification process of the two enzymes increases the cost, making it difficult to achieve mass production of fluorinated 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 fluoroiodomethane specifically combined to achieve fluoromethylation at the 4-OH site to prepare fluoroisoferulic acid, enriching the structural types of caffeic acid fluorine-containing drugs, and the present invention provides a new method of single fluorine catalysis.

[0041] Specifically, according to an embodiment of one aspect of the present invention, a method for preparing fluoroisoferulic acid by single enzyme catalysis is provided. Figure 1A flow chart of a method for preparing fluorinated isoferulic acid by single enzyme catalysis is shown in the embodiment of the present application, as shown in Figure 1 The method comprises operations S101-S103.

[0042] In operation S101, caffeic acid, fluoromethyl iodide, a pH buffer, 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 the embodiment, caffeic acid is used as a reaction substrate and belongs to phenolic acid compounds, in which the phenolic hydroxyl group is a target point for fluoromethylation substitution. Fluoromethyl iodide is used as a fluoromethyl donor, and halide methyltransferase (HMT) can specifically catalyze the transfer of fluoromethyl from fluoromethyl iodide to a specific hydroxyl site of caffeic acid, such as a 3-OH site and / or a 4-OH site. Under the catalysis of halide methyltransferase (HMT), fluoromethyl is transferred to a specific hydroxyl group of caffeic acid. Fluoromethyl iodide has moderate activity and low toxicity, which is conducive to environmental protection. Adjusting the pH of the reaction raw material solution to the above range makes HMT have higher activity.

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

[0045] In the embodiment, caffeic acid has a catechol structure and is prone to photooxidation under light irradiation. Fluoromethyl iodide also has a certain photosensitivity. Although HMT has low photosensitivity, long-term light irradiation may cause conformational changes and reduce catalytic efficiency. Based on the light-free reaction condition, the chemical stability of the reaction substrate and fluoromethyl iodide can be maintained, and the catalytic specificity of HMT can be maintained.

[0046] In operation S103, the reaction mixture is subjected to quenching and purification in sequence to obtain fluorinated isoferulic acid.

[0047] In the embodiment, the enzyme catalysis reaction is terminated by quenching to avoid excessive fluoromethylation reaction and ensure the regioselectivity of the reaction product.

[0048] According to the embodiments of the present application, compared with the single-enzyme catalysis of HMT and the double-enzyme cascade reaction of HMT and methyltransferase in the prior art, the present application utilizes the single-enzyme catalysis of HMT, which has higher fluoromethylation efficiency than the double-enzyme cascade reaction. The double-enzyme cascade reaction has 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 a wider range of drug molecules. The present application uses HMT single-enzyme catalysis and fluoromethane as a fluoromethyl donor to transfer fluoromethyl to the 4-OH position of caffeic acid to generate fluorosubstituted fluorinated isoferulic acid at the 4-OH position, so that the fluorinated isoferulic acid of caffeic acid can be produced by HMT single-enzyme catalysis. Isoferulic acid can be applied to hypoglycemic drugs and is widely used as a natural antioxidant in the pharmaceutical and food industries. The fluorinated isoferulic acid of the present application is conducive to subsequent application and promotion in drug research and development. Moreover, the single-enzyme catalysis of HMT in the present application can achieve more excellent fluoromethylation efficiency compared with the double-enzyme cascade reaction, and does not require the participation of SAM analogs. Therefore, single-enzyme catalysis simplifies the reaction system, reduces the cost of enzyme purification, preservation, etc., and provides a more practical solution for large-scale industrial applications. The present application avoids the complex requirements of the double-enzyme cascade reaction, and the reaction conditions of the present application are easier to control and optimize, reducing the operation difficulty, improving the stability and repeatability of the reaction.

[0049] In some embodiments, the yield of fluorinated isoferulic acid is 30% or more, preferably 50% or more. For example, when AclHMT and selHMT are used as halide methyltransferase, the yield of fluorinated isoferulic acid prepared is relatively higher.

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

[0051] In some embodiments, the temperature of the light-avoiding reaction is 20-40℃, for example, it can be 20℃, 30℃ or 40℃, preferably 30℃. In this way, the HMT can have both high activity and high stability. The reaction at the above-mentioned temperature can be regulated by a water bath or a thermostat. The time of the light-avoiding reaction is 10-15h, for example, it can be 10h, 11h, 12h, 13h, 14h or 15h, preferably 12h. In this way, the reaction can be ensured to proceed sufficiently without affecting 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, preferably 5:1. In this way, it is helpful to promote the efficient use of the enzyme active site, avoid the low conversion rate caused by insufficient reaction substrate or enzyme amount, and ensure the stable reaction rate. If the molar ratio is too low, the reaction rate will be reduced due to the insufficient caffeic acid substrate; if the molar ratio is too high, it may lead to high concentration of caffeic acid occupying other binding sites of the enzyme or changing the conformation of the enzyme, reducing the catalytic efficiency, and also avoiding the waste of reaction substrate.

[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, preferably 100:1. The reason for adjusting the fluoroiodomethane to the above-mentioned excessive state is that the fluoroiodomethane may be partially lost by hydrolysis or side reactions. In this way, it is ensured that there is enough fluoromethyl donor to maintain the forward progress of the reaction, thereby promoting the caffeic acid to have a high fluoromethylation conversion rate.

[0054] In some embodiments, the pH buffer is at least one of an alkali metal phosphate solution, an alkali metal borate solution, an alkali metal carbonate solution. For example, it can be a sodium phosphate solution, a sodium nitrate solution, a sodium bicarbonate solution, etc., as long as it is a metal salt solution with certain alkalinity, which is not particularly limited in the present application. 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. In the related embodiments of the present application, it is found that this pH is the suitable pH value of HMT, which can further promote the single enzyme catalysis and improve the fluoromethylation conversion rate.

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

[0057] In some embodiments, purification includes centrifugation at 3-5°C, for example, 3°C, 4°C, or 5°C, and at 11,000-13,000 rpm, for example, 11,000 rpm, 12,000 rpm, or 13,000 rpm, for 20-40 minutes, for example, 20 minutes, 30 minutes, or 40 minutes. This helps maintain the stability of the prepared product at low temperatures, allows for effective separation of products of different densities by centrifugation, and, due to the low temperature, requires only simple heating, simplifying experimental procedures and saving costs.

[0058] The present invention provides a biocatalytic method for site-specific fluoromethylation of caffeic acid 4-OH using HMT single enzyme catalysis to generate fluoroisoferulic acid, thereby promoting the development of novel fluorine-containing drugs with potential pharmacological activities such as preventing diabetes-related cardiovascular diseases. The present invention simplifies the caffeic acid fluoromethylation reaction system, overcoming the shortcomings of the existing dual-enzyme cascade reaction, which is difficult to control reaction conditions and has high reaction costs. Compared with the existing dual-enzyme cascade reaction, the present invention achieves a higher fluoromethylation efficiency through HMT single enzyme catalysis, providing a more efficient and economical technical path for the large-scale production of fluorine-containing drugs.

[0059] According to another embodiment of the present invention, there is provided a fluoroisoferulic acid prepared by the above method.

[0060] According to an embodiment of the present invention, the present invention synthesizes fluoroisoferulic acid through single enzyme catalysis of HMT. Such fluoroisoferulic acid helps to enrich the structural types of caffeic acid fluorine-containing drugs and is expected to be subsequently used in the research and development of drugs such as those for inhibiting blood sugar, antibacterial and anti-inflammatory, and anti-tumor.

[0061] The present invention will be further described below by way of examples, drawings, and related test experiments and results thereof. In the detailed description that follows, for ease of explanation, many specific details are set forth to provide a comprehensive understanding of the embodiments of the present invention. However, it is apparent that one or more embodiments may be implemented without these specific details. Moreover, the details in the following embodiments may be arbitrarily combined into other feasible embodiments, unless conflicting.

[0062] It should be noted that the following specific examples are for illustration only and the scope of protection of the present invention is not limited thereto. The chemicals and raw materials used in the following examples were either commercially available or prepared in-house using recognized processing methods.

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

[0064] Specifically, the protein sequence of the halide methyltransferase used is as shown in SEQ ID No. 1~ SEQ ID No. 3.

[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 catalyzing the fluoromethylation of caffeic acid by AclHMT:

[0073] The reaction solution (100 μΐ^) contained 1 mM caffeic acid, 100 mM CH2FI, 50 mM sodium phosphate buffer (pH 7.6 adjusted by hydrochloric acid and sodium hydroxide) and 200 μΜ halide methyltransferase (AclHMT).

[0074] The reaction solution was incubated in a 30 °C water bath for 12 hours in the dark. After the reaction was completed, the same volume of 10 wt% trifluoroacetic acid (TFA) was added to quench the reaction. The quenched reaction mixture was centrifuged at 4 °C, 12000 rpm for 30 min using a high-speed refrigerated centrifuge to obtain the fluorinated isoferulic acid. The reaction process is shown below.

[0075]

[0076] The fluoroisoferulic acid is analyzed and detected by an analytical liquid chromatography (HPLC) on a reversed-phase C18 column (150 mm x 4.6 mm, 2.5 μm) of a company, under detection wavelengths of 254 nm and 215 nm of an ultraviolet detector, a mobile phase A is H2O + 0.1% (mass fraction) trifluoroacetic acid, and a mobile phase B is acetonitrile (CH3CN) + 0.1% (mass fraction) trifluoroacetic acid. The molecular weight of the fluoroisoferulic acid is determined by liquid chromatography LC-MS analysis. A large amount of prepared fluoroisoferulic acid is dissolved in 400 μL of deuterated dimethyl sulfoxide (DMSO-d6), and a nuclear magnetic resonance spectrometer of a company with a proton resonance frequency of 800 MHz is used to 1 H-NMR, 19 F-NMR, 13 C-NMR identification, Figure 2 Fig. 1 shows a nuclear magnetic resonance hydrogen spectrum of the fluoroisoferulic acid prepared in Example 1 of the present application; Figure 3 Fig. 2 shows a nuclear magnetic resonance carbon spectrum of the fluoroisoferulic acid prepared in Example 1 of the present application; Figure 4 Fig. 3 shows a nuclear magnetic resonance fluorine spectrum of the fluoroisoferulic acid prepared in Example 1 of the present application. As shown in Fig. 3, by analyzing the structure of the fluoromethylation product, it can be confirmed that the fluoroisoferulic acid (i.e., isoferulic acid with fluorine substitution at the 4-OH site) is prepared in the present application. Figures 2-4 The experimental results show that AclHMT (Aspergillus clavatus HMT) can recognize the substrate caffeic acid of NtCOMT and exhibit excellent fluoromethylation efficiency (93%), in which the yield of the generated fluoroisoferulic acid reaches 87%, and the yield of the difluoromethylation product is 6%. This indicates that AclHMT has high selectivity and high catalytic capacity for fluoromethylation of caffeic acid 4-OH. In addition, the AclHMT catalyzed fluoromethylation reaction of caffeic acid can also be applied to a large-scale preparation reaction, which is helpful to scale up the reaction system to a 40 mL preparation mode.

[0077] Example 2

[0078] Process of umaNMT catalyzed fluoromethylation of caffeic acid:

[0079] The reaction raw material solution (100 μL) contains 1 mM caffeic acid, 100 mM CH2FI, 50 mM sodium phosphate buffer (the pH value of the buffer is regulated to 7.6 by using hydrochloric acid and sodium hydroxide), and 200 μM halide methyltransferase (umaNMT).

[0080] The reaction raw material solution was reacted in a 30 °C water bath for 12 hours in the dark. After the reaction was completed, the same volume of 10 wt% trifluoroacetic acid (TFA) as the reaction raw material solution was used for quenching. The quenched reaction mixture was centrifuged at 4 °C and 12000 rpm for 30 min using a high-speed refrigerated centrifuge to obtain fluorinated isoferulic acid, and the reaction process is shown in the following formula.

[0081]

[0082] The fluorinated isoferulic acid was analyzed and detected by analytical HPLC on a reversed-phase C18 chromatographic column (150 mm x 4.6 mm, 2.5 μm) under the detection wavelength of 254 nm and 215 nm of an ultraviolet detector, and the mobile phase A was H2O + 0.01% trifluoroacetic acid, and the mobile phase B was CH3CN + 0.01% trifluoroacetic acid. The molecular weight of fluorinated isoferulic acid was determined by liquid chromatography LC-MS analysis. A large amount of prepared fluorinated isoferulic acid was dissolved in 400 μL of deuterated dimethyl sulfoxide (DMSO-d6), and the structure of the fluoromethylation product was identified by a certain company's proton resonance frequency of 800 MHz nuclear magnetic resonance spectrometer 1 H-NMR, 19 F-NMR, 13 C-NMR identification, and the structure of the fluoromethylated product was confirmed. The experimental results show that umaHMT (Ustilago maydis HMT) can recognize the substrate caffeic acid of NtCOMT to generate various fluoromethylated products, and the yield of 3-fluoromethyl product is 30%, the yield of fluorinated isoferulic acid is 40%, and the yield of double fluoromethyl product with 3-OH and 4-OH sites is 12%. This shows that the selectivity of umaHMT catalytic reaction is different from that of AclHMT, and various caffeic acid fluoromethylated products can be generated, which provides more diversified structural selection for fluorine-containing drug synthesis.

[0083] Example 3

[0084] The process of selHMT catalyzing caffeic acid fluoromethylation is as follows:

[0085] The reaction raw material solution (100 μL) contains 1 mM caffeic acid, 100 mM CH2FI, 50 mM sodium phosphate buffer (the pH value of the buffer is adjusted to 7.6 by using hydrochloric acid and sodium hydroxide), and 200 μM halide methyltransferase (selHMT).

[0086] The reaction raw material solution was reacted in a 30 °C water bath for 12 hours in the dark. After the reaction was completed, the same volume of 10 wt% trifluoroacetic acid (TFA) as the reaction raw material solution was used for quenching. The quenched reaction mixture was centrifuged at 4 °C and 12000 rpm for 30 min using a high-speed refrigerated centrifuge to obtain fluorinated isoferulic acid.

[0087] Fluoroisoferulic acid was analyzed and detected by analytical HPLC with a reversed-phase C18 column (150 mm x 4.6 mm, 2.5 μm) under the detection wavelength of 254 nm and 215 nm of a UV detector, and the mobile phase A was H2O + 0.01% (mass fraction) trifluoroacetic acid, and the mobile phase B was CH3CN + 0.01% (mass fraction) trifluoroacetic acid. The molecular weight of fluoroisoferulic acid was determined by LC-MS analysis. The experimental results show that the fluoromethylation efficiency of selHMT (Synechococcus elongates HMT) is relatively low compared with AclHMT and umaHMT, selHMT can recognize the substrate caffeic acid of NtCOMT to generate multiple fluoromethylated products, the yield of fluoromethylated product at the 3-OH position is 2.5%, the yield of fluoroisoferulic acid is 57.5%, and the yield of difluoromethylated product is 9%.

[0088] Comparative Example 1

[0089] The reaction raw material solution (100 μL) contains 3 mM caffeic acid, 100 mM CH2FI, 1 mM dcSAH, 6 mM MgCl2, 150 μM AclHMT, and 100 mM sodium phosphate buffer (hydrochloric acid and sodium hydroxide are added to adjust the pH value of the buffer to 6.5).

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

[0091] The identified ferulic acid fluorine derivative (caffeic acid 3-OH fluoromethylated product) has a reaction yield of 52%, and the reaction process is as shown in the following formula.

[0092]

[0093] Comparing Example 1 with Comparative Example 1, it is found that the yield of the fluoromethylated product obtained in Example 1 is relatively higher, which improves the efficiency of the reaction, and the product prepared in Example 1 can be widely used in inhibiting blood glucose rise and antitumor drugs, and widely used as an antioxidant in the pharmaceutical and food industries. Comparing Example 2 with Comparative Example 1, it is found that Example 2 can simultaneously prepare multiple fluoromethylated products, which provides more structural selectivity for subsequent drug design and meets the related needs of fluoromethylated site diversity in drug research and development.

[0094] The above-described specific embodiments further illustrate the purpose, technical solutions and beneficial effects of the present application, and it should be understood that the above-described is only a specific embodiment of the present application and is not intended to limit the present application, and any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims

1. A method for preparing fluoroisoferulic acid by single enzyme catalysis, characterized in that: include: mixing caffeic acid, fluoroiodomethane, a pH buffer, and a halide methyltransferase to obtain a reaction raw material solution, wherein the pH value of the reaction raw material solution is 7.5 to 7.7; The reaction raw material solution is subjected to light-proof reaction to obtain a reaction mixture; The reaction mixture is sequentially quenched and purified to obtain fluoroisoferulic acid; The halide methyltransferase is selected from any one of AclHMT of SEQ ID No. 1, umaHMT of SEQ ID No. 2, and selHMT of SEQ ID No.

3.

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 temperature of the light-proof reaction is 20-40° C., and the time of the light-proof reaction is 10-15 hours.

4. The method according to claim 1, wherein 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.

5. The method according to claim 1, wherein 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.

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

6.

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

8. The method according to claim 1, characterized in that The purification comprises: The centrifuge was performed at 11,000 to 13,000 rpm at 3 to 5°C for 20 to 40 minutes.

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