A method of synthesizing vitamin k2 or a derivative thereof

Abstract

The application belongs to the technical field of organic synthesis, and relates to a synthesis method of vitamin K2 or a derivative thereof. The synthesis method comprises the following steps: obtaining a compound shown in formula I according to a reaction route shown in the following compound 1 and compound 2; wherein the compound 1 and the compound 2 are subjected to enzyme catalysis reaction (enzymatic reaction) to obtain a compound 3; the enzyme used in the enzyme catalysis reaction is one or more of isopentenyl transferase, geranylgeranyl transferase and heptaprenyl pyrophosphate transferase; and n is a natural number from 0 to 7. The synthesis method has the advantages of shortening a synthesis route, simplifying post-treatment operation, less pollution, mild conditions, high reaction efficiency, low production cost, high product purity and the like.

Description

Technical Field

[0001] This invention belongs to the field of organic synthesis technology and relates to a method for synthesizing vitamin K2 or its derivatives. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Vitamin K2 is an important fat-soluble vitamin that helps with blood clotting and is a rare source of fats and oils. Due to its extremely low content in food, it is often called the "golden vitamin" and has many important physiological functions, including preventing and treating osteoporosis, arterial calcification, cardiovascular disease, tumors, and Parkinson's disease. Vitamin K2 can be used to treat and prevent osteoporosis, increase bone density, prevent fractures, prevent cirrhosis from developing into liver cancer, treat vitamin K2 deficiency bleeding, promote prothrombin formation, accelerate blood clotting, and maintain normal clotting time. It also has diuretic properties, strengthens the liver's detoxification function, and can lower blood pressure. Vitamin K2 is a collective term for a series of compounds, usually pale yellow crystals or oily liquids, denoted by MK-n, where n represents the number of isoprene units on the side chain. MK-4 and MK-7 are the most active. MK-4 and MK-7 are fat-soluble vitamins with important physiological activities. MK-4 is mainly found in animal-derived foods, while MK-7 is mostly derived from fermented foods (such as natto). The core function of both is as coenzymes for γ-glutamyl carboxylase, promoting the carboxylation of coagulation factors II, VII, IX, and X, thereby maintaining normal coagulation function. More importantly, they can carboxylate osteocalcin and matrix Gla protein. The former can enhance calcium deposition in bones, increase bone density, and reduce the risk of osteoporosis, while the latter can inhibit vascular smooth muscle calcification, reducing the risk of atherosclerosis and cardiovascular disease. In addition, MK-7 has a longer half-life and higher bioavailability, resulting in a more sustained effect on improving bone and cardiovascular health, while MK-4 plays a role in short-term coagulation regulation and rapid regulation of bone metabolism. Together, they contribute to the dual health maintenance of the skeletal and cardiovascular systems.

[0004] The synthesis of vitamin K2 is generally carried out through chemical synthesis, which suffers from drawbacks such as low product yield, low purity, significant pollution, and complex operation. To address these issues, researchers have employed a bioenzymatic method to prepare vitamin MK-4. This method uses menadione and geranyyl pyrophosphate as starting materials and employs geranyyl transferase catalysis to prepare vitamin MK-4. However, this study found that this method involves a complex reaction process. Because the menadione nucleus has a quinone structure, it exhibits a strong electron-withdrawing effect, leading to a decrease in the electron cloud density of the nucleus fragment and low reactivity. Therefore, a high reaction temperature is required, reaching up to 100 °C. However, under these conditions, the enzyme is prone to inactivation, significantly reducing catalytic efficiency. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a method for synthesizing vitamin K2 or its derivatives. The synthesis method provided by the present invention significantly shortens the synthesis route, simplifies post-processing operations, and has advantages such as less pollution, mild conditions, high reaction efficiency, low production cost, and high product purity.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows:

[0007] A method for synthesizing vitamin K2 or a derivative thereof, comprising the steps of obtaining the compound shown in Formula I by reacting compound 1 (1,4-dimethoxy-2-methylnaphthalene) and compound 2 (long-chain terpene pyrophosphate) according to the following reaction route;

[0008] Compound 1 and Compound 2 are reacted by an enzyme-catalyzed reaction (enzyme-catalyzed reaction) to obtain Compound 3; the enzyme used in the enzyme-catalyzed reaction is one or more of isopentenyltransferase, geranylgeranyltransferase, and heptapentenylpyrophosphate transferase; n is a natural number from 0 to 7.

[0009] When n is 3, the synthesized compound of formula I is vitamin MK-4; when n is 6, the synthesized compound of formula I is vitamin MK-7.

[0010] Specifically, the enzyme-catalyzed reaction is carried out in a buffer system with a pH of 7.0 to 7.5.

[0011] Specifically, the temperature for enzyme-catalyzed reactions is 20~40℃.

[0012] The beneficial effects of this invention are as follows: 1. The synthesis method provided by this invention has the advantages of mild reaction conditions and high efficiency. This invention uses isopentenyltransferase, geranylgeranyltransferase, and heptaisopentenylpyrophosphate transferase to carry out an enzymatic reaction of 1,4-dimethoxy-2-methylnaphthalene and long-chain terpene pyrophosphate to obtain intermediate 3, which yields vitamin K2 or its derivatives through further oxidation. This enzymatic reaction is carried out in a near-neutral buffer solution at pH 7.0-7.5 under normal temperature and pressure conditions, eliminating the need for high temperature and high pressure, significantly reducing the risk of enzyme inactivation and improving catalytic efficiency and stability. Compared with existing technologies that require high temperatures (e.g., 100°C), this invention has a lower reaction temperature, milder conditions, lower energy consumption, and is safer and simpler to operate.

[0013] 2. The synthetic method provided by this invention has the advantages of wide substrate applicability and high selectivity. This invention uses 1,4-dimethoxy-2-methylnaphthalene as the parent core structure, whose electron-donating groups enhance the reactivity, enabling efficient enzyme catalysis at room temperature. This method has a wide selectivity range for the length of the isoprene side chain (n=0~7), allowing for directional linkage between the side chain and the parent core, resulting in fewer side reactions and high reaction selectivity.

[0014] 3. The synthesis method provided by this invention simplifies the synthesis steps and improves the purity of the product: no complex conditions need to be controlled during the reaction process, and the intermediate can be converted into a high-purity target product (such as vitamins MK-4, MK-7, etc.) simply by oxidation, which greatly simplifies the post-processing process and improves the overall purity and yield of the product.

[0015] 4. The synthesis method provided by this invention is characterized by its environmental friendliness and economic efficiency. Compared with traditional chemical synthesis methods, this invention avoids the use of high temperatures and highly corrosive reagents, reducing pollutant emissions and conforming to the concept of green synthesis. At the same time, the mild reaction conditions reduce equipment requirements and operational risks, resulting in lower overall production costs and making it more suitable for large-scale production. Attached Figure Description

[0016] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0017] Figure 1 The 1H NMR spectrum of vitamin MK-4 synthesized in Example 1 of this invention; Figure 2 The FT-IR spectrum of vitamin MK-4 synthesized in Example 1 of this invention; Figure 3 The 1H NMR spectrum of vitamin MK-7 synthesized in Example 4 of this invention; Figure 4The image shows the FT-IR spectrum of vitamin MK-7 synthesized in Example 4 of this invention. Detailed Implementation

[0018] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0019] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0020] In view of the problems of complex operation, high reaction temperature, low yield and low reaction selectivity in the synthesis of vitamin K2, the present invention proposes a method for synthesizing vitamin K2 or its derivatives.

[0021] A typical embodiment of the present invention provides a method for synthesizing vitamin K2 or its derivatives, comprising the steps of obtaining the compound shown in Formula I by reacting compound 1 (1,4-dimethoxy-2-methylnaphthalene) and compound 2 (long-chain terpene pyrophosphate) according to the following reaction route;

[0022] Compound 1 and Compound 2 are reacted by an enzyme-catalyzed reaction (enzyme-catalyzed reaction) to obtain Compound 3; the enzyme used in the enzyme-catalyzed reaction is one or more of isopentenyltransferase, geranylgeranyltransferase, and heptapentenylpyrophosphate transferase; n is a natural number from 0 to 7.

[0023] This invention has discovered that using 1,4-dimethoxy-2-methylnaphthalene as a reaction raw material, with a dimethoxy-substituted naphthalene structure as its core, provides electron-donating groups and exhibits high reactivity. Enzymatic catalysis using one or more of isopentenyltransferase, geranylgeranyltransferase, and heptapentenylpyrophosphate transferase can be carried out at room temperature, efficiently achieving directional linkage between the side chain and the core. This results in advantages such as mild reaction conditions, high reaction efficiency, and high selectivity. Subsequent simple oxidation is all that is needed to obtain the high-purity target product.

[0024] In some embodiments, n is 0, 3, or 6. When n is 0, compound 2 (long-chain terpene pyrophosphate) is isopentenyl pyrophosphate. When n is 3, compound 2 (long-chain terpene pyrophosphate) is geranylgeranyl pyrophosphate. When n is 6, compound 2 (long-chain terpene pyrophosphate) is heptaisopentenyl pyrophosphate.

[0025] In some embodiments, the enzyme-catalyzed reaction is carried out in a buffer system with a pH of 7.0–7.5. The isopentenyltransferase, geranylgeranyltransferase, and heptapentenylpyrophosphate transferase used in this invention exhibit higher activity and better reaction results in this reaction system. Specifically, the buffer system is selected from one or more of phosphate buffer solution, Tris-HCl buffer solution, carbonate buffer solution, and HEPES buffer solution. Specifically, the concentration of the buffer salt in the buffer system is 50–100 mmol / L.

[0026] In some embodiments, a co-solvent is added to the enzyme-catalyzed reaction system. Adding a co-solvent facilitates thorough mixing of compound 1 and compound 2, thereby improving reaction efficiency. Specifically, the co-solvent may be dimethyl sulfoxide.

[0027] In some embodiments, the temperature of the enzyme-catalyzed reaction is 20–40 °C. The isopentenyltransferase, geranylgeranyltransferase, and heptapentenylpyrophosphate transferase used in this invention exhibit higher activity and better reaction efficiency under the specified reaction temperature conditions. Specifically, the reaction time is 12–48 h. The highest reaction efficiency is achieved when the reaction temperature is 35–40 °C and the reaction time is 24–36 h.

[0028] In some embodiments, the molar ratio of compound 1 to compound 2 is 1:1 to 1.5. This condition is beneficial for improving the conversion rate of 1,4-dimethoxy-2-methylnaphthalene and reducing costs.

[0029] In some embodiments, in the reaction system of the enzyme-catalyzed reaction, the concentration of compound 1 is 0.2–0.5 mmol / L, and the concentration of compound 2 is 0.3–0.6 mmol / L. Studies have shown that these concentration conditions are more conducive to the exertion of enzyme activity and improve the efficiency of enzyme-catalyzed reaction.

[0030] In some embodiments, the enzyme added in the enzyme-catalyzed reaction is 1% to 10% of the mass of compound 1. Studies have shown that this condition is beneficial for improving the efficiency of the enzyme-catalyzed reaction.

[0031] In some embodiments, isopropanol is added after the enzyme-catalyzed reaction to induce crystallization and obtain compound 3.

[0032] In some embodiments, compound 3 is obtained by an oxidation reaction to obtain the compound shown in Formula I.

[0033] Specifically, the mass ratio of compound 3 to oxidant is 1:2 to 5, preferably 1:3.5 to 4.0.

[0034] Specifically, in the oxidation reaction, the oxidant used is cerium ammonium nitrate. Preferably, the concentration of cerium ammonium nitrate in the oxidation reaction system is 0.2~1.5 mmol / L. Under these conditions, the reaction is more favorable.

[0035] Specifically, the solvent for the oxidation reaction is dichloromethane.

[0036] Specifically, the oxidation reaction takes 5 to 8 hours.

[0037] Specifically, extraction is performed after the oxidation reaction.

[0038] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0039] The routes for synthesizing vitamin K2 (MK-4) in Examples 1-3 are shown below:

[0040] Example 1: Synthesis of Vitamin K2 (MK-4) 1,4-Dimethoxy-2-methylnaphthalene (2.02 g, 10 mmol) and geranylgeranyl pyrophosphate (4.40 g, 10 mmol) were added as substrates to a reaction flask, followed by 10 mL of phosphate buffer solution (pH 7.5), 0.2 g of 10% geranylgeranyltransferase, and 5 mL of dimethyl sulfoxide as a cosolvent. The reaction was carried out at atmospheric pressure and 35 °C for 12 h. 50 mL of isopropanol was added to induce crystallization, and the resulting intermediate (4.65 g, yield: 98%) was obtained by filtration. 25 mL of dichloromethane and 18 g of cerium ammonium nitrate (CAN) were added to the intermediate for oxidation for 5 h. After the reaction was complete, 20 mL of water was added for extraction, and the concentrate was used to obtain a yellow oily substance. This was purified by crystallization with ethanol to obtain 4.23 g of vitamin K2 (MK-4). Figures 1-2 As shown, the yield was 96%.

[0041] Example 2: Synthesis of Vitamin K2 (MK-4) 1,4-Dimethoxy-2-methylnaphthalene (2.02 g, 10 mmol) and geranylgeranyl pyrophosphate (6.17 g, 14 mmol) were added as substrates to a reaction flask, followed by 10 mL of Tris-HCl buffer solution (pH 7.5), 0.2 g of 10% geranylgeranyltransferase, and 5 mL of dimethyl sulfoxide as a solubilizer. The reaction was carried out at atmospheric pressure and 40 °C for 18 h. 50 mL of isopropanol was added to induce crystallization, and the resulting intermediate (4.71 g, yield: 99%) was obtained by filtration. 25 mL of dichloromethane and 18 g of cerium ammonium nitrate (CAN) were added to the intermediate for oxidation for 5 h. After the reaction was complete, 20 mL of water was added for extraction, and the concentrate was used to obtain a yellow oil. After crystallization and purification, 4.32 g of vitamin K2 (MK-4) was obtained, with a yield of 98%.

[0042] Example 3: Synthesis of Vitamin K2 (MK-4) 1,4-Dimethoxy-2-methylnaphthalene (2.02 g, 10 mmol) and geranylgeranyl pyrophosphate (4.85 g, 11 mmol) were added as substrates to a reaction flask, followed by 10 mL of carbonate buffer solution (pH 7.5), 0.2 g of 10% geranylgeranyltransferase, and 5 mL of dimethyl sulfoxide as a cosolvent. The reaction was carried out at atmospheric pressure and 35 °C for 30 h. 50 mL of isopropanol was added to induce crystallization, and the resulting intermediate (4.55 g, yield: 96%) was obtained by filtration. 24 mL of dichloromethane and 16 g of cerium ammonium nitrate (CAN) were added to the intermediate for oxidation for 5 h. After the reaction was complete, 20 mL of water was added for extraction, and the concentrate was used to obtain a yellow oil. After crystallization and purification, 4.14 g of vitamin K2 (MK-4) was obtained, with a yield of 94%.

[0043] The routes for synthesizing vitamin K2 (MK-7) in Examples 4-6 are shown below:

[0044] Example 4: Synthesis of Vitamin K2 (MK-7) 1,4-Dimethoxy-2-methylnaphthalene (2.02 g, 10 mmol) and heptapentyl pyrophosphate (8.07 g, 12 mmol) were added as substrates to a reaction flask, followed by 10 mL of phosphate buffer solution (pH 7.5), 0.2 g of 10% heptapentyl pyrophosphate transferase, and 5 mL of dimethyl sulfoxide as a co-solvent. The reaction was carried out at atmospheric pressure and 35 °C for 24 h. 50 mL of isopropanol was added to induce crystallization, and the product was filtered to obtain an intermediate (6.68 g, yield: 98%). 35 mL of dichloromethane and 26 g of cerium ammonium nitrate (CAN) were added to the intermediate for oxidation for 5 h. After the reaction was complete, the product was extracted with 20 mL of water and concentrated to obtain a yellow oil. After crystallization and purification, 6.15 g of vitamin K2 (MK-7) was obtained. Figures 3-4 As shown, the yield was 96%.

[0045] Example 5: Synthesis of Vitamin K2 (MK-7) 1,4-Dimethoxy-2-methylnaphthalene (2.02 g, 10 mmol) and heptapentyl pyrophosphate (10.09 g, 15 mmol) were added as substrates to a reaction flask, followed by 10 mL of Tris-HCl buffer solution (pH 7.5), 0.2 g of 10% heptapentyl pyrophosphate transferase, and 5 mL of dimethyl sulfoxide as a co-solvent. The reaction was carried out at atmospheric pressure and 40 °C for 36 h. 50 mL of isopropanol was added to induce crystallization, and the product was filtered to obtain an intermediate (6.75 g, yield: 99%). 35 mL of dichloromethane and 26 g of cerium ammonium nitrate (CAN) were added to the intermediate for oxidation reaction for 5 h. After the reaction was complete, the product was extracted with 20 mL of water and concentrated to obtain a yellow oil. After crystallization and purification, 6.28 g of vitamin K2 (MK-7) was obtained, with a yield of 98%.

[0046] Example 6: Synthesis of Vitamin K2 (MK-7) 1,4-Dimethoxy-2-methylnaphthalene (2.02 g, 10 mmol) and heptapentyl pyrophosphate (8.75 g, 13 mmol) were added as substrates to a reaction flask, followed by 50 mL of carbonate buffer solution at pH 7.5, 0.2 g of 10% heptapentyl pyrophosphate transferase, and 5 mL of dimethyl sulfoxide as a co-solvent. The reaction was carried out at atmospheric pressure and 20 °C for 48 h. 50 mL of isopropanol was added to induce crystallization, and the crystals were filtered to obtain an intermediate (6.55 g, yield: 96%). 33 mL of dichloromethane and 24 g of cerium ammonium nitrate (CAN) were added to the intermediate for oxidation for 5 h. After the reaction was complete, the mixture was extracted with 20 mL of water and concentrated to obtain a yellow oil. After crystallization and purification, 6.03 g of vitamin K2 (MK-7) was obtained, with a yield of 94%.

[0047] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for synthesizing vitamin K2 or a derivative thereof, characterized in that, The steps include obtaining the compound shown in Formula I by reacting compounds 1 and 2 according to the following reaction route; Compound 1 and Compound 2 are reacted by an enzyme to obtain Compound 3; the enzyme used in the enzyme catalysis reaction is one or more of isopentenyltransferase, geranylgeranyltransferase, and heptapentenylpyrophosphate transferase; n is a natural number from 0 to 7.

2. The synthesis method as described in claim 1, characterized in that, n is 0, 3, or 6.

3. The synthesis method as described in claim 1, characterized in that, The enzyme-catalyzed reaction was carried out in a buffer system with a pH of 7.0–7.5; Alternatively, the temperature for enzyme-catalyzed reactions is 20~40℃.

4. The synthesis method as described in claim 1, characterized in that, Add a co-solvent to the enzyme-catalyzed reaction system.

5. The synthesis method as described in claim 1, characterized in that, The molar ratio of compound 1 to compound 2 is 1:1 to 1.

5.

6. The synthesis method according to claim 1, characterized in that the enzyme... In the reaction system of the catalytic reaction, the concentration of compound 1 is 0.2~0.5 mmol / L and the concentration of compound 2 is 0.3~0.6 mmol / L.

7. The synthesis method as described in claim 1, characterized in that, In the enzyme-catalyzed reaction, the added mass of the enzyme is 1% to 10% of the mass of compound 1.

8. The synthesis method as described in claim 1, characterized in that, After the enzyme-catalyzed reaction, isopropanol was added to induce crystallization, yielding compound 3.

9. The synthesis method as described in claim 1, characterized in that, Compound 3 was oxidized to obtain the compound shown in Formula I.

10. The synthesis method as described in claim 9, characterized in that, The mass ratio of compound 3 to oxidant is 1:2~5, preferably 1:3.5~4.0; Alternatively, in the oxidation reaction, the oxidant used is cerium ammonium nitrate; preferably, in the oxidation reaction system, the concentration of cerium ammonium nitrate is 0.2~1.5 mmol / L; Alternatively, the solvent for the oxidation reaction is dichloromethane; Alternatively, extraction can be performed after an oxidation reaction.

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