A natural form of (6S)-5-methyltetrahydrofolate and a method for its preparation

Abstract

The present application relates to a kind of natural (6S) -5-methyl tetrahydrofolate and its preparation method, wherein the natural (6S) -5-methyl tetrahydrofolate is prepared by reducing process with folic acid as raw material, and no heavy metal such as platinum or lead, and formaldehyde, benzene sulfonic acid and its ester and other raw materials are used in production process;The content of the natural (6S) -5-methyl tetrahydrofolate is not less than 98%, the amount of JK12A is not higher than 0.1%, methyl tetrahydropterin is not detected, benzene sulfonic acid and its ester are not detected, and enzyme protein residue is not higher than 1 ng / g;The present application has the advantages of convenient post-processing, green environmental protection, high product purity and the like due to the use of green biological production process.

Description

Technical Field

[0001] This invention relates to the field of synthetic technology, specifically to a naturalized (6S)-5-methyltetrahydrofolate and its preparation method. Background Technology

[0002] Folic acid is one of the most important vitamin supplements: it plays a vital role and cannot be synthesized by the human body. The earliest folic acid discovered was found in nature, falling into the category of natural folic acid. Natural folic acid refers to folic acid that exists naturally, and it has been found in yeast extracts, animal livers, and other sources. In 1941, American scholar Mitchell discovered the same component in spinach leaves and named it folic acid, marking the earliest recognition of its existence and its prototype. Nearly a century has passed, but natural folic acid still cannot be supplied on a large scale commercially. Due to its high activity, or in other words, instability, extraction, application, and maintaining a sufficient shelf life remain challenging tasks. Currently, the folic acid added to food and pharmaceuticals is only artificially synthesized. In Asian countries, the primary form is inactive synthetic folic acid, namely pteridine glutamate (FA), which requires multiple enzymatic reactions within the body before it can be utilized. In Europe and America, since the 1960s, directly absorbable folic acid, or active folic acid, has been available. Although bioactive folic acid has undergone a long and continuous improvement process, its advantages are obvious: it no longer needs to go through the conversion process required for inactive folic acid, and can be directly absorbed and utilized. This bypasses the differences in human metabolic enzyme activity, i.e., genotypic differences, and also avoids the various problems caused by unmetabolized folic acid. Of course, the existence of these problems is also the main driving force behind the emergence of artificially synthesized bioactive folic acid.

[0003] Therefore, folic acid should refer to natural folic acid, while synthetic folic acid is a substitute for natural folic acid. Artificially synthesized active folic acid is an upgraded version of natural folic acid substitute.

[0004] Synthetic active folic acid has significantly improved the performance of folic acid supplements. However, due to the complexity of the technology, active folic acid has undergone two stages of development: the first stage was from the racemic methyltetrahydrofolate calcium to 6S-5-methyltetrahydrofolate calcium. The latter possesses the same spatial structure as natural folic acid, thus exhibiting the same biological activity; the second stage was from amorphous 6S-5-methyltetrahydrofolate calcium to crystalline 6S-5-methyltetrahydrofolate calcium. Scientists discovered that by forming stable crystals, 6S-5-methyltetrahydrofolate can resist oxidative degradation under dry conditions. This made active folic acid truly suitable for large-scale use. These stages of development have been achieved over time, consistent with the progress of human civilization.

[0005] Crystallized active folic acid is a milestone in the history of folic acid development. However, products at this stage still have significant problems. While they meet the needs of the general population, they neglect the safety needs of the most important group: the fetus. The problems include two aspects: 1. The production process uses raw materials with high safety risks: heavy metal ions, platinum or lead; formaldehyde, p-toluenesulfonic acid, and other substances with direct and indirect genotoxicity. Some substances, such as p-toluenesulfonic acid and its esters, are not yet included in the detection and control items. Controlling the residues of these substances to a certain extent can meet the safety needs of the general population, but it is still insufficient for the fetus. We all know that life in the single-cell or few-cell stage is very fragile. A single harmful molecule can cause developmental abnormalities, which can be amplified and transmitted as development progresses. Therefore, these harmful substances should not only be below a certain limit or undetectable, but should not be present at all. The Doha theory suggests that the embryonic development period is affected by adverse environments or malnutrition. 2. Poor control of process impurities. There is a lack of systematic safety assessment and control of process impurities.

[0006] The aforementioned issues spurred the emergence of new natural folic acid products.

[0007] Naturalized folic acid has two main characteristics: it possesses the same chemical and spatial structure as natural folic acid, thus exhibiting the same physiological activity; and it has comparable safety characteristics to natural folic acid. Specifically, this means using advanced technology to improve the synthesis process and avoid harmful raw materials; and controlling harmful impurities JK12A and methylpteroic acid to the highest standards currently achievable with technology.

[0008] Naturalized folic acid not only possesses the excellent activity of natural folic acid, but also meets the safety requirements of embryos that begin as single cells. Compared with natural folic acid, the outstanding advantage of naturalized folic acid is its breakthrough improvement in stability.

[0009] Improving folic acid production by using natural folic acid is not simply about making it structurally similar to or identical to natural folic acid. Rather, it involves using green chemistry principles in the production and synthesis process for more environmentally friendly production. Furthermore, the synthesis of 6S-5-methyltetrahydrofolic acid raw materials must not involve the use of any solvents or catalysts that may pose a safety risk to the human body. The goal is to achieve a folic acid product that is non-natural but surpasses the safety requirements of natural folic acid.

[0010] Currently, the large-scale synthesis of 6S-5-methyltetrahydrofolate mainly involves using folic acid as a raw material for reduction to prepare dihydrofolate, followed by further reduction to obtain tetrahydrofolate. During the reduction process, the conversion of the SP2 carbon atom to the SP3 carbon atom on the pteridine ring of dihydrofolate is accompanied by the generation of a chiral center. Therefore, 5-methyltetrahydrofolate in chemical synthesis contains two asymmetric centers. Typically, when hydrogen is added to the double bond at the 5,6 positions, an optically active carbon atom is formed at the 6 position, existing in a racemic (6R,S) form. The (6S) form is the only naturally occurring form of 5-methyltetrahydrofolate and is the active component in the human body. In existing technologies, the reduction of dihydrofolate to tetrahydrofolate usually requires the use of borohydride reduction. Commonly used reducing agents are sodium borohydride and potassium borohydride. Patent US201300409561A1 describes a method of methylation by reduction with sodium borohydride, and patent CN103214487A describes a method of reduction with potassium borohydride. The above-mentioned chemical reduction synthesis process inevitably uses resolving agents and catalysts. Since chiral compounds need to be separated after reduction, such as 6R-tetrahydrofolate and 6S-tetrahydrofolate, benzenesulfonic acid must be used as a resolving agent. However, benzenesulfonic acid esters pose a risk of genotoxicity.

[0011] Green chemistry is a global trend in scientific research and production. It fully utilizes resources and energy, employs non-toxic and harmless raw materials, reduces waste emissions, improves atom utilization, and produces environmentally friendly products that benefit environmental protection, community safety, and human health. This trend is inevitable. It can be seen that the process of preparing 6S-5-methyltetrahydrofolate using organic synthesis methods requires the use of various raw materials that may negatively impact embryonic development. While this process is the mainstream method, its drawbacks are obvious: the product may contain impurities that pose a certain risk to human health, and some waste entering nature will pollute the environment. Therefore, the direction of chemical synthesis is gradually shifting towards green biosynthesis. Patent US20100151533A1 describes a method for producing 6S-5-methyltetrahydrofolate using dihydrofolate reductase, which uses glucose and glucose dehydrogenase as a method for NADPH recovery during synthesis.

[0012] Generally, the first step in current enzymatic synthesis is to obtain dihydrofolate. This is achieved by reducing synthetic folic acid to dihydrofolate using a zinc powder alkaline solvent, with hydrogen gas as a byproduct. Then, dihydrofolate reductase is used, employing glucose and glucose dehydrogenase as a NADPH regeneration system to reduce dihydrofolate to obtain chiral tetrahydrofolate. The key to production using dihydrofolate reductase lies in the regeneration of nicotinamide coenzyme. Since NADPH, the reducing coenzyme, is expensive, it cannot be added in the correct dosage according to the reaction equation. This process inevitably uses a large amount of glucose, and further separation of tetrahydrofolate from the high-concentration glucose solution inevitably leads to reduced costs and product purity. Furthermore, this process requires a large dose of glucose dehydrogenase, making it difficult to control enzyme protein residues in the final product. Therefore, although enzymatic synthesis can basically achieve large-scale production of 6S-5-methyltetrahydrofolate, few companies currently use this process for 6S-5-methyltetrahydrofolate production due to cost and other factors. Summary of the Invention

[0013] The purpose of this invention is to overcome the shortcomings of the prior art by providing a naturalized (6S)-5-methyltetrahydrofolate and its preparation method, which has the advantages of convenient post-processing, green and environmentally friendly, and high product purity.

[0014] To achieve the above objectives, the technical solution adopted by the present invention is: a naturalized (6S)-5-methyltetrahydrofolate, wherein the naturalized (6S)-5-methyltetrahydrofolate is obtained from folic acid through reduction and other processes, and no heavy metals such as platinum or lead, or raw materials such as formaldehyde, benzenesulfonic acid and its esters are used in the production process; the content of the naturalized (6S)-5-methyltetrahydrofolate is not less than 98%, the amount of JK12A is not more than 0.1%, methyltetrahydropteroic acid is not detectable, benzenesulfonic acid and its esters are not detectable, and enzyme protein residue is not more than 1 ng / g.

[0015] This invention also discloses a method for preparing naturally occurring (6S)-5-methyltetrahydrofolate, characterized by comprising the following steps:

[0016] Step 1: Folic acid is dissolved in an alkaline solution and reduced with zinc powder under inert gas protection to obtain dihydrofolic acid. Then, the pH value is adjusted to approximately neutral with acid.

[0017] Step 2: The hydrogenase solution is incubated with hydrogen gas;

[0018] Step 3: In the presence of NADP+ or NADPH, hydrogenase, and hydrogen, dihydrofolate is stereoselectively reduced by dihydrofolate reductase to obtain (6S)-tetrahydrofolate.

[0019] Step 4: After converting (6S)-tetrahydrofolate into a related derivative using formic acid, it is reduced to naturalized (6S)-5-methyltetrahydrofolate.

[0020] The (6S)-5-methyltetrahydrofolate is selected from the acid addition salt of (6S)-5-methyltetrahydrofolate, preferably (6S)-5-methyltetrahydrofolate salt, (6S)-5-methyltetrahydrofolate sulfate or (6S)-5-methyltetrahydrofolate phosphate.

[0021] In another approach, (6S)-5-methyltetrahydrofolate is reacted with a metal salt to obtain a basic salt of (6S)-5-methyltetrahydrofolate; the metal salt may be calcium chloride, magnesium chloride, sodium carbonate, etc., and the basic salt of (6S)-5-methyltetrahydrofolate is preferably calcium salt of (6S)-5-methyltetrahydrofolate, magnesium salt of (6S)-5-methyltetrahydrofolate, or sodium salt of (6S)-5-methyltetrahydrofolate.

[0022] More specifically, in the first step, sodium folic acid is dissolved in a solution of folic acid, sodium hydroxide, or sodium carbonate (pH approximately 13-13.5) with stirring in a reaction vessel of appropriate size to prepare sodium folic acid. Then, zinc powder and phosphoric acid are added to reduce the sodium folic acid to dihydrofolic acid. It is important to note that all reactions in the first step should be carried out under an inert gas atmosphere (e.g., nitrogen or argon).

[0023] In the second step of the method described in this invention, the hydrogen gas generated in step 1 is collected. A schematic diagram of the main reaction in step 1 is shown in the appendix to the specification. Figure 1 The raw materials for the reaction in step 1 are folic acid, phosphoric acid, water, sodium hydroxide, and zinc, and the products are sodium dihydrofolate, water, disodium hydrogen phosphate, sodium dihydrogen phosphate, zinc phosphate, and hydrogen gas.

[0024] In this reaction, sodium folic acid is reduced to dihydrofolic acid. Byproducts of the reduction include zinc phosphate and hydrogen gas. The reaction produces a significant amount of hydrogen gas, which is a flammable and explosive hazardous gas and is often discharged as waste gas in other (6S)5-methyltetrahydrofolic acid synthesis processes. However, in this invention, the hydrogen gas produced in the synthesis route is used as one of the raw materials for the next reaction step. In step 1 of this invention, a mixture of hydrogen gas and an inert gas is saturated and incubated with a hydrogenase solution at a pressure of p = 1.1-1.3 par.

[0025] The hydrogenase of this invention requires an oxygen-free environment to exhibit high activity. However, in step 1 of this invention, due to the instability of dihydrofolate, the reaction itself is carried out under inert gas protection. Therefore, during the reaction, the gas in the reaction vessel is mainly an oxygen-free inert gas, and the proportion of hydrogen gradually increases. It can be directly transferred through a pipeline to a container containing the hydrogenase solution for incubation. Afterwards, sodium dithionate can be added to the hydrogenase solution to remove residual oxygen.

[0026] In step 3 of this invention, while maintaining an inert gas atmosphere, a series of reactants from step 1 are added to the reaction vessel, including, but not limited to, the following reactants: dihydrofolate reductase, NADP+, or NADPH. Simultaneously, the hydrogenase solution mixture from step 2 is added, and the mixture is stirred at 40°C until the dihydrofolate is substantially converted. The reaction solution is then filtered. A schematic diagram of the reaction is attached. Figure 2 .

[0027] In step 4 of this invention, (6S)-tetrahydrofolate is converted to (6S)-5-methyltetrahydrofolate. Conventional methods generally use an appropriate amount of formaldehyde to convert it to 5-10-methylenetetrahydrofolate, which is then reduced by borohydride to obtain (6S)-5-methyltetrahydrofolate. In this invention, formic acid is preferred as the cyclization solvent. First, (6S)-5-methyltetrahydrofolate is converted to (6R)-5-10-methylenetetrahydrofolate, and then (6S)-5-methyltetrahydrofolate is finally obtained by catalytic reaction using the method described in CN201711459340.0, with the aid of Pd / CNTs or Pd / AC.

[0028] The hydrogenase described in this invention is derived from the soluble hydrogenase of the thermophilic archaea Pyrococcus furiosus, a classic hydrogenase. Based on the understanding of this invention, the hydrogenase can also be selected from the hydrogenase of Desulfovibrio vulgaris, the hydrogenase of Ralstonia eutropha H16, and other hydrogenases that can provide hydrogen ions. The hydrogenase described in this invention is an anaerobic enzyme, and its activity will be lost due to oxygen. In step (2) of this invention, the hydrogenase solution needs to be degassed under vacuum and protected with an inert gas before hydrogen is introduced. The volume fraction ψi of the inert gas in the byproduct hydrogen is less than 40%, preferably less than 30%, and most preferably less than 20%. Sodium dithionite is added to the hydrogenase solution before hydrogen is introduced, and the concentration of sodium dithionite is 2-10 μM, preferably 5 μM.

[0029] In step (2) of this invention, the incubation temperature of hydrogen gas and hydrogenase is 50-85℃, preferably 60-80℃, more preferably 70℃, the incubation pressure should be greater than 1.1 bar, preferably 1.3 bar, the hydrogenase concentration is 1-5 g / L, and the incubation time is not less than 10 min. Experiments have shown that a certain pressure must be provided when incubating the hydrogenase solution with hydrogen gas to facilitate the dissolution of hydrogen molecules in the solution; however, excessive pressure can also affect the equipment and the enzyme activity. The incubation time is temperature-dependent; the lower the temperature, the longer the incubation time required. Since the activity of hydrogenase cannot be repeatedly measured, the concentration of hydrogenase in this invention is given as a mass fraction, not as enzyme activity.

[0030] In step (3) of this invention, the preferred reaction concentrations are: NADPH 5-30 mM, preferably 10 mM; hydrogenase concentration 1-100 mg / L, preferably 2.5 mg / L; Tris hydrochloride concentration 50-400 mM, preferably 200 mM; and dihydrofolate reductase concentration 2000 U-60000 U / L, preferably 3000-10000 U / L. The pH is maintained between 7.3 and 8.0 during the reaction, preferably pH 7.5.

[0031] In step (4) of the present invention, 6S-tetrahydrofolate is converted into 6S-5-methyltetrahydrofolate. The conventional method is to use formaldehyde to convert it into a derivative of 5-methyltetrahydrofolate. The present invention uses an alternative method, formic acid, to convert 6S-tetrahydrofolate into a cyclized compound of (6R)-5,10-methylenetetrahydrofolate, and obtains (6S)-5-methyltetrahydrofolate by reduction ring-opening.

[0032] A method for producing (6S)-5-methyltetrahydrofolate using dihydrofolate reductase has been reported, involving the selective stereoreduction of dihydrofolate by dihydrofolate reductase in the presence of glucose and glucose dehydrogenase. This reaction has low atom utilization efficiency, requires glucose additions almost equivalent to dihydrofolate, and the purification of the resulting 6S-tetrahydrofolate is complex. The inventors discovered that using hydrogen gas with the hydrogenase not only results in high reaction efficiency but also produces almost no byproducts, yielding high-purity 6S-tetrahydrofolate. Furthermore, since the preceding process also requires inert gas protection, the byproduct hydrogen gas can be used without complex treatment, meeting the requirements of recycling and pollution-free green chemistry.

[0033] Due to the application of the above technical solution, the present invention has the following advantages compared with the prior art:

[0034] 1. The naturalized (6S)-5-methyltetrahydrofolate of the present invention avoids the use of resolving agents that may produce gene impurities due to the use of green biological production processes, and also avoids the use of formaldehyde. The naturalized folic acid is not detectable for chiral resolving agents benzoic acid and its esters used in conventional methods, and is not detectable for formaldehyde.

[0035] 2. This invention is the first to use hydrogen as a coenzyme regeneration system. The byproducts of hydrogen are only protons or water. As is well known, these byproducts are very environmentally friendly. Moreover, the efficiency of this reaction is higher than that of glucose / glucose dehydrogenase. The mass of hydrogen added is only one-thousandth of that of glucose. Furthermore, this invention also makes full use of hydrogen, a byproduct of upstream translation, to achieve a high level of atom utilization.

[0036] 3. The present invention uses a very small amount of hydrogenase, and the enzyme residue of the produced natural (6S)-5-methyltetrahydrofolate is very low, which further demonstrates the superiority of the present invention. Attached Figure Description

[0037] The technical solution of the present invention will be further described below with reference to the accompanying drawings:

[0038] Appendix Figure 1 This is a schematic diagram of the folic acid to dihydrofolate reaction in step 1 of the present invention;

[0039] Appendix Figure 2 This is a schematic diagram of the dihydrofolate reduction reaction in step 2 of the present invention;

[0040] Appendix Figure 3 The figure shows the effect of the existing JK12A on the heart rate of zebrafish.

[0041] Appendix Figure 4 The figure shows the impact of existing JK12A zebrafish embryonic development.

[0042] Appendix Figure 5 The image shows the results of 5-methyltetrahydropteridine-induced developmental abnormalities in zebrafish.

[0043] Appendix Figure 6 The figure shows the effect of existing 5-methyltetrahydropteroic acid on zebrafish embryonic development. Detailed Implementation

[0044] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0045] HPLC detection method:

[0046] The purity described in this invention is detected using high-performance liquid chromatography (HPLC), under the following specific conditions:

[0047] Detection wavelength: 280nm;

[0048] Column packing material: octadecylsilane-bonded silica gel (250×4.6mm, 5μm);

[0049] Buffer salt solution: NaH2PO4 solution;

[0050] Mobile phase A: Adjust the pH of the buffer salt solution to 6.5 using 32% NaOH solution;

[0051] Mobile phase B: methanol and buffer salt solution (35:65), pH adjusted to 8.0 with 32% NaOH solution;

[0052] The flow rate was 1.0 ml / min;

[0053] The injection volume was 10 μl;

[0054] Elution gradient;

[0055] T(min) A％ B％ 0 100 0 14 45 55 17 0 100 24 0 100 24.01 100 0 33 100 0

[0056] Example 1: Preparation of naturalized (6S)-5-methyltetrahydrofolate

[0057] Select 10g of commercially available synthetic folic acid, add it to a reaction flask, add 60ml of pure water and 2.2g of sodium hydroxide, and stir until the folic acid is completely dissolved. Then add 5g of zinc powder. Stir the solution under nitrogen protection for 3 hours, and then add phosphoric acid to adjust the pH value to 7.0.

[0058] Dissolve 5 mg of hydrogenase (P. furiosus hydrogenase I, purity 23%) in 5 ml of 50 mM Tris hydrochloride buffer. The buffer is repeatedly treated with vacuum and nitrogen to remove oxygen. Finally, add 20 μl of 1 M sodium dithionate. Saturate the hydrogenase buffer with hydrogen at 1.2 bar and incubate at 80°C for 1 hour.

[0059] While maintaining nitrogen protection, the dihydrofolate reaction solution was transferred to the reaction vessel, and 2g of ascorbic acid, 10ml of dihydrofolate reductase solution (enzyme activity 65000U / L), 0.1g of NADPH, and 40μl of hydrogenase buffer after hydrogen incubation were added. A mixture of inert gas and hydrogen (V:V = 1:1) was introduced into the reaction flask, and 50ml of 100mM Tris hydrochloride buffer was added. The reaction was maintained at 40℃ and stirred for 3 hours.

[0060] After the reaction was complete, 40g of formic acid (30% concentration) was added to the reaction solution, and the mixture was stirred for 1 hour. Then, 1g of Pd / CNTs was added to the reaction solution, and hydrogen gas was introduced to maintain a pressure of 0.8 MPa. The reaction temperature was 60℃, and the reaction was carried out for 3 hours. The mixture was filtered, and sodium hydroxide solution was added dropwise until neutral. Then, hydrochloric acid solution was slowly added dropwise to adjust the pH to 4, and a solid precipitated. After stirring for 1 hour, the mixture was filtered, washed with water, and dried under vacuum at 50-60℃ to obtain (6S)-5-methyltetrahydrofolate.

[0061] The crude (6S)-5-methyltetrahydrofolate salt was added to calcium chloride, and 20% ascorbic acid was added. The mixture was recrystallized to obtain (6S)-5-methyltetrahydrofolate calcium with a purity of 99.7%.

[0062] Example 2: Preparation of naturalized (6S)-5-methyltetrahydrofolate

[0063] Select 10g of commercially available synthetic folic acid, add it to a reaction flask, add 60ml of pure water and 2.2g of sodium hydroxide, and stir until the folic acid is completely dissolved. Then add 5g of zinc powder. Stir the solution under nitrogen protection for 3 hours, and then add phosphoric acid to adjust the pH value to 7.0.

[0064] Take 5 mg of hydrogenase (P. furiosus hydrogenase I, purity 30%), transfer it to an autoclave, dissolve it in 5 ml of Tris hydrochloride buffer, and repeatedly treat the buffer with vacuum and argon to remove oxygen. Finally, add 20 μl of 1M sodium dithionate. Saturate the hydrogenase buffer with hydrogen at 1.3 bar and incubate at 70°C for 2 hours.

[0065] While maintaining nitrogen protection, the dihydrofolate reaction solution was transferred to the reaction vessel, and 2g of ascorbic acid, 10ml of dihydrofolate reductase solution (enzyme activity 65000U / L), 0.1g of NADPH, and 40μl of hydrogenase buffer after hydrogen incubation were added. A mixture of inert gas and hydrogen (V:V = 1:1) was introduced into the reaction flask, and 50ml of 200mM Tris hydrochloride buffer was added. The reaction was maintained at 37°C and stirred for 4 hours.

[0066] After the reaction was complete, 40g of formic acid (30% concentration) was added to the reaction solution, and the mixture was stirred for 1 hour. Then, 1g of Pd / CNTs was added to the reaction solution, and hydrogen gas was introduced to maintain a pressure of 0.8 MPa. The reaction temperature was 60℃, and the reaction was carried out for 3 hours. The mixture was filtered, and sodium hydroxide solution was added dropwise until neutral. Then, hydrochloric acid solution was slowly added dropwise to adjust the pH to 4, and a solid precipitated. After stirring for 1 hour, the mixture was filtered, washed with water, and dried under vacuum at 50-60℃ to obtain (6S)-5-methyltetrahydrofolate.

[0067] Example 3: Preparation of naturalized (6S)-5-methyltetrahydrofolate

[0068] A 500L reactor was selected, and 50kg of folic acid (96% purity) was added as reactant. Then, 150kg of sodium hydroxide and 280L of water were added, and the mixture was stirred until the folic acid was completely dissolved. Next, 25kg of zinc powder was added, and the mixture was stirred for 2 hours under an inert argon atmosphere. Phosphoric acid was then added to generate zinc phosphate and remove unreacted zinc powder. Because the density of hydrogen is much lower than that of argon, the hydrogen collected in the pipe above the reactor had a high purity, yielding approximately 500L of a mixed hydrogen and argon gas, with hydrogen comprising 79% (by volume).

[0069] Under argon protection, 5 g of hydrogenase (P. furiosus hydrogenase I, 30% purity) was transferred to a high-pressure reactor and dissolved in 5 L of 100 mM Tris hydrochloride buffer, with 0.8 g of sodium dithionate added. Hydrogen gas was compressed and continuously passed into the hydrogenase buffer, and the mixture was incubated at 80°C with stirring for 2 hours at a pressure of 1.3 bar.

[0070] Under argon protection, 28 L of dihydrofolate reductase enzyme solution (65000 U / L enzyme activity), 0.6 kg of coenzyme NADP+, and Tris hydrochloride buffer were added to the dihydrofolate reaction vessel to a concentration of 100 mM. The incubated hydrogenase buffer solution and hydrogen gas were drawn from the high-pressure reactor into the reaction vessel. The reaction vessel was stirred for 3 hours.

[0071] After the reaction was complete, 12 kg of formic acid (30% concentration) was added to the reaction solution, and the mixture was stirred for 1 hour. Then, 500 g of Pd / CNTs was added to the reaction solution, and hydrogen gas was introduced to maintain a pressure of 0.8 MPa. The reaction temperature was 60℃, and the reaction was carried out for 3 hours. The mixture was filtered, and sodium hydroxide solution was added dropwise until neutral. Then, hydrochloric acid solution was slowly added dropwise to adjust the pH to 4, and a solid precipitated. After stirring for 1 hour, the mixture was filtered, washed with water, and dried under vacuum at 50-60℃ to obtain (6S)-5-methyltetrahydrofolate.

[0072] The crude (6S)-5-methyltetrahydrofolate salt was added to calcium chloride, and 20% ascorbic acid was added. The mixture was recrystallized to obtain (6S)-5-methyltetrahydrofolate calcium with a purity of 99.7%.

[0073] Example 4: Preparation of naturalized (6S)-5-methyltetrahydrofolate

[0074] Select 10g of commercially available synthetic folic acid, add it to a reaction flask, add 60ml of pure water and 2.2g of sodium hydroxide, and stir until the folic acid is completely dissolved. Then add 5g of zinc powder. Stir the solution under nitrogen protection for 3 hours, and then add phosphoric acid to adjust the pH value to 7.0.

[0075] Take 25 mg of hydrogenase (P. furiosus hydrogenase I, purity 30%), transfer it to an autoclave, dissolve it in 5 ml of Tris hydrochloride buffer, and repeatedly treat the buffer with vacuum and argon to remove oxygen. Finally, add 20 μl of 1 M sodium dithionate. Saturate the hydrogenase buffer with hydrogen at 1.5 bar and incubate at 80°C for 2 hours.

[0076] While maintaining nitrogen protection, the dihydrofolate reaction solution was transferred to the reaction vessel, and 2g of ascorbic acid, 10ml of dihydrofolate reductase solution (enzyme activity 65000U / L), 0.1g of NADPH, and 40μl of hydrogenase buffer after hydrogen incubation were added. A mixture of inert gas and hydrogen (V:V = 1:1) was introduced into the reaction flask, and 50ml of 100mM Tris hydrochloride buffer was added. The reaction was maintained at 37°C and stirred for 4 hours.

[0077] After the reaction was complete, 40g of formic acid (30% concentration) was added to the reaction solution, and the mixture was stirred for 1 hour. Then, 1g of Pd / CNTs was added to the reaction solution, and hydrogen gas was introduced to maintain a pressure of 0.8 MPa. The reaction temperature was 60℃, and the reaction was carried out for 3 hours. The mixture was filtered, and sodium hydroxide solution was added dropwise until neutral. Then, hydrochloric acid solution was slowly added dropwise to adjust the pH to 4, and a solid precipitated. After stirring for 1 hour, the mixture was filtered, washed with water, and dried under vacuum at 50-60℃ to obtain (6S)-5-methyltetrahydrofolate.

[0078] Example 5: Preparation of naturalized (6S)-5-methyltetrahydrofolate

[0079] A 5000L high-pressure reactor was selected, and 525kg of folic acid, 1508kg of sodium hydroxide, and 2950L of water were added. The mixture was stirred for 5 minutes until the folic acid was completely dissolved. Then, 90kg of zinc powder was added, and the mixture was stirred for 2 hours under an inert argon atmosphere. Next, 780L of phosphoric acid was added to generate zinc phosphate and remove unreacted zinc powder. The reaction byproduct hydrogen gas was collected and stored. The mixture was filtered, and the filtrate was transferred to the next process to produce tetrahydrofolic acid.

[0080] Under argon protection, 25 g of hydrogenase (P. furiosus hydrogenase I, 30% purity) was transferred to an autoclave and dissolved in 5 L of 200 mM Tris hydrochloride buffer, with 0.8 g of sodium dithionate added. Hydrogen gas was compressed and continuously bubbled into the hydrogenase buffer, and the mixture was incubated at 60°C with stirring for 5 hours at a pressure of 1.1 bar.

[0081] Under argon protection, the dihydrofolic acid mixture was transferred into a 10000L reaction vessel. 52.5 kg of sodium ascorbate was added as the raw material, 288 L of dihydrofolate reductase solution (enzyme activity 65000 U / L) was added, 6.4 Kg of coenzyme NADP+ was added, and Tris hydrochloride buffer solution was added to make its concentration reach 200 mM. Then the hydrogenase buffer solution was transferred to the dihydrofolic acid mixture. Stirring was carried out at 40 °C, and during this period, a mixed gas of hydrogen and argon was used for protection. Stirring was carried out for 3 hours. Filtration was carried out, and the filtrate was taken for the next process.

[0082] 120 kg of formic acid was added to the filtrate, and stirring was carried out for 1 hour. 1000 g of Pd / CNTs was added to the reaction solution, and hydrogen was passed through to maintain a pressure of 0.8 Mpa; the reaction temperature was 60 °C, and the reaction was carried out for 3 hours. Filtration was carried out, sodium hydroxide solution was added dropwise until neutral, and then hydrochloric acid solution was slowly added dropwise to adjust the pH to 4. A solid was precipitated. After stirring for 1 hour, filtration was carried out, and the solid was washed with water and dried under vacuum at 50 - 60 °C to obtain (6S)-5-methyltetrahydrofolic acid hydrochloride.

[0083] Calcium chloride was added to the crude product of (6S)-5-methyltetrahydrofolic acid hydrochloride, and 20% ascorbic acid was added. Recrystallization was carried out to obtain calcium (6S)-5-methyltetrahydrofolate, with a total of 540 kg obtained and a purity of 99.3%.

[0084] Example 6

[0085] Acute toxicity test on rats

[0086] SD rats, SPF grade, 20 animals, 10 males and 10 females, with body weights ranging from 180 - 213 g, were provided by Shanghai Xipuer-Bikai Experimental Animal Co., Ltd. Production license number: SCXK (Shanghai) 2013-0016. The temperature in the breeding room was 20 - 25 °C, and the relative humidity was 40 - 70%. The animal feed was provided by Suzhou Shuangshi Experimental Animal Feed Technology Service Co., Ltd. Sample name: Calcium (6S)-5-methyltetrahydrofolate (the new naturalized folic acid described in this invention). 5 g of the sample was weighed, and distilled water was added to make 20 ml, and then it was fully mixed. According to this ratio, the maximum gavage suspension was prepared. After the animals were fasted (water not prohibited) for 16 hours, 10 male and 10 female rats were selected according to the body weight requirements and placed in cages respectively. According to the principle of the maximum tolerated dose test, the corresponding dose of the test solution was given to each group of experimental animals by oral gavage. The animals were weighed one by one, and the gavage volume was calculated according to 20 ml / kg.BW. Gavage was carried out in 3 times within 24 hours, with a time interval of 4 hours. After exposure, the general state, body weight change, poisoning symptoms, and death situation of the animals were observed, and the observation period was one week.

[0087] As a result, during the test period, the animals in each group were active normally, with good hair gloss, and no signs of poisoning or death were observed; when the animals were sacrificed at the end of the period, the organs were observed by gross anatomy, and no abnormalities were found.

[0088] Summary: The maximum tolerated dose (MTD) of the sample in the oral toxicity test for male and female rats was greater than 15 g / kg, belonging to the non-toxic level.

[0089] Acute toxicity test in mice

[0090] KM mice, SPF grade, with 20 animals (10 males and 10 females), weighing 19 - 22 g. They were provided by Shanghai Jiesijie Experimental Animal Co., Ltd., with the production license number: SCCK (Shanghai) 2016 - 0006. The temperature in the breeding room was 20 - 25°C, and the relative humidity was 40 - 70%. Sample name: 6S - 5 - methyltetrahydrofolate calcium (the naturalized folic acid described in this invention). Weigh 5 g of the sample, add distilled water to 20 ml, and mix well. Prepare the maximum gavage suspension according to this ratio. After the animals were fasted (but not water - deprived) for 16 hours, 10 male and 10 female animals were selected according to the weight requirement and placed in cages by gender. The experimental animals were exposed to the sample by oral gavage. Each animal was weighed individually; the gavage volume was calculated as 20 ml / kg.Bw. The gavage was carried out in 3 times within 24 hours, with a time interval of 4 hours. After exposure, the general state, weight change, poisoning symptoms, and death of the animals were observed. The observation period was one week. Each group of animals was weighed before and after the test. Autopsies were performed on the dead animals and the animals sacrificed at the end of the experiment, and gross pathological changes were observed macroscopically.

[0091] Results: During the test, the animals in each group were active normally, with good hair gloss, and no signs of poisoning or death were observed. At the end of the experiment, the animals were sacrificed, and no abnormalities were observed in the organs during gross dissection.

[0092] The maximum tolerated dose (MTD) of the sample in the acute oral toxicity test for male and female mice was greater than 15 g / kg, belonging to the non - toxic level.

[0093] Example 7

[0094] Embryotoxicity test of JK12A

[0095] The transgenic zebrafish (fli - 1:EGFP) was from the Model Animal Research Institute of Nanjing University. The fish used in the experiment were adult zebrafish less than 1 year old. The pH value of the breeding water was 7 ± 0.2, the temperature was about 28°C, and the light - dark time ratio was 14h:10h. The brine shrimp eggs were fed twice a day. The night before, one female and two male zebrafish were placed in the spawning box, and the fish eggs were collected the next morning. The fish eggs were placed in embryo culture medium (prepared with 0.2 g / L sea salt) and cultured at 28°C.

[0096] JK12A was first attempted to be dissolved in water, but the dissolution was found to be poor. Based on previous experimental records, 20 mM NaHCO3 was chosen for dissolution, and it was found that JK12A completely dissolved at 14.08 mM. The dissolved JK12A was incubated in a water bath at a constant temperature of 50℃ for 4 hours before being taken out for use. The maximum concentration set in this experiment was 14.08 mM. In addition, 7.04 mM, 3.52 mM, 1.76 mM, and 20 mM NaHCO3 groups and a control group were also set up for experiments.

[0097] Test methods

[0098] Zebrafish embryos were cultured in 24-well plates starting at 2 hpf, with 10 embryos per well. 1 mL of JK12A solution of various concentrations was added and the treatment continued until 72 hpf. Dead embryos were removed promptly. Each group had 3 replicates, with 10 embryos per well. At 8 hpf, developmental delays were observed. At 24 hpf, developmental abnormalities were observed. At 48 hpf, pigment development, cardiac edema, heart rate, and intersegmental vessels were observed. At 72 hpf, intestinal reticulum vessel development and body length were measured.

[0099] Results: At 8 hpf, no significant embryonic developmental delay was observed in any of the drug-treated groups, and the survival rates were comparable across groups. At 24 hpf, zebrafish embryos had developed head and tail structures, and tail wagging was observed. Except for the 20 mM NaHCO3 group, the survival rates of zebrafish in all other groups were decreased, with the 7.04 mM group showing the largest decrease. At 48 hpf, the heart rate of zebrafish embryos was inhibited by JK12A, and this inhibition showed a clear concentration-dependent effect (see instruction manual appendix). Figure 3 Meanwhile, the survival rate remained unchanged compared to the 24 hpf stage. Furthermore, intersegmental development was normal, and no pericardial edema was observed. At 72 hpf, the zebrafish embryo's body length was inhibited by JK12A, but the inhibition was not significant. The survival rate decreased slightly compared to the previous stage, with an overall survival rate between 0.8 and 1. In all experimental groups, the intestinal reticulum developed normally, and no malformations occurred (see instruction manual appendix). Figure 4 In conclusion, JK12A has a negative impact on the growth and development of zebrafish embryos, with heart rate being a prominent indicator, suggesting that JK12A may have some influence on embryonic heart development.

[0100] Embryotoxicity of pteridine

[0101] The transgenic zebrafish (fli-1:EGFP) was obtained from the Institute of Model Animals, Nanjing University. The fish used in the experiment were adult zebrafish less than one year old. The pH of the rearing water was 7±0.2, the temperature was around 28℃, and the light-to-dark ratio was 14h:10h. They were fed Artemia eggs twice daily. One female and two male zebrafish were placed in a spawning box the night before, and the eggs were collected the following morning. The eggs were placed in embryo culture medium (prepared with 0.2g / L sea salt) and cultured at 28℃.

[0102] Using 20 mM NaHCO3 as the solvent, the maximum solubility of impurity C was observed at a concentration of 7.84 mM. Based on this, experiments were conducted at five concentrations: 0.49 mM, 0.98 mM, 1.96 mM, and 3.92 mM. The dissolved 5-methyltetrahydropteric acid (impurity C) mother liquor was incubated in a 50°C water bath for 4 hours, then diluted for subsequent experiments. 20 mM NaHCO3 was used as a solvent control.

[0103] Zebrafish embryos were cultured in 24-well plates starting at 2 hpf, with 10 embryos per well. 1 mL of each of the above-mentioned concentrations of Impurity C solution and a control were added, and the culture was continued until 72 hpf. Dead embryos were promptly removed. Each group had 3 replicates, with 10 embryos per well. At 8 hpf, developmental delays were observed; at 24 hpf, developmental abnormalities were observed; at 48 hpf, pigment development, cardiac edema, heart rate, and intersegmental vascularity were observed; and at 72 hpf, intestinal reticulum vascular development and body length were measured.

[0104] Test results

[0105] At 8 hpf, the survival rate of zebrafish embryos in all groups was slightly reduced, but development was normal without delay. At 24 hpf, the zebrafish embryos had developed head and tail, and the tail showed wagging behavior; the survival rate remained unchanged compared to 8 hpf. At 48 hpf, the heart rate of the zebrafish embryos was not suppressed by impurity-C, the survival rate did not change significantly compared to 24 hpf, and there was no pericardial edema; intersegmental development was normal. At 72 hpf, a small number of malformations appeared in the 7.84 mM impurity-C group, mainly manifested as trunk curvature. In addition, the survival rate was slightly reduced only in the control group and the 3.92 mM group compared to the previous two days; the overall survival rate was above 0.8. The embryo body length also decreased with increasing impurity-C concentration, and the intestinal medulla developed normally (see instruction manual appendix). Figure 5 ,6).

[0106] 5-Methyltetrahydropteranoic acid (5-MTEPA) has certain negative effects on the growth and development of zebrafish. Due to solubility limitations, the highest concentration tested in this study was 7.8 mM. Further investigation is needed to determine if other negative effects exist due to the small sample size. Teratogenicity was observed at a concentration of approximately 0.49 mM. Given the small sample size in each group of this experiment, the actual incidence of malformations needs to be confirmed; statistically, the actual incidence may be higher.

[0107] Example 7

[0108] Dihydrofolate reductase-glucose dehydrogenase process

[0109] Select a 500L reactor, add 52kg of folic acid, 150kg of sodium hydroxide, and 295L of water, stir for 5 minutes until the folic acid is completely dissolved, then add 9kg of zinc powder, stir for 2 hours under inert gas protection, then add 78L of phosphoric acid, filter, and transfer the filtrate to the next process to produce tetrahydrofolic acid.

[0110] Under inert gas protection, the dihydrofolate mixture was transferred to an enzyme-catalyzed reduction reactor. 5 kg of sodium ascorbate was added, along with 29 L of dihydrofolate reductase solution (65000 U / L), 0.65 kg of NADP+ coenzyme, 29 L of glucose dehydrogenase solution (72000 U / L), and 25 kg of glucose. The mixture was stirred for 3 hours. After filtration, the filtrate was used for the next step.

[0111] Add 12 kg of formic acid to the filtrate and stir for 1 hour. Add 1000 g of Pd / CNTs to the reaction solution, purge with hydrogen gas, and maintain a pressure of 0.8 MPa. React at 60°C for 3 hours. Filter, add sodium hydroxide solution dropwise until neutral, then slowly add hydrochloric acid solution to adjust the pH to 4. A solid precipitates, and after stirring for 1 hour, filter, wash with water, and dry under vacuum at 50–60°C to obtain (6S)-5-methyltetrahydrofolate.

[0112] The crude (6S)-5-methyltetrahydrofolate salt was added to calcium chloride and 20% ascorbic acid, and recrystallized to obtain (6S)-5-methyltetrahydrofolate calcium, yielding a total of 48.9 kg with a purity of 99.2%.

[0113] Example 8

[0114] Seven batches of 6S-5-methyltetrahydrofolate products were tested for genotoxic impurities.

[0115] As described in the background section of this invention, the organic synthesis process of 6S-5-methyltetrahydrofolate calcium uses benzenesulfonic acid, and the solvent used is ethanol or isopropanol, therefore it may contain ethyl benzenesulfonate or isopropyl benzenesulfonate. Seven batches of the product were tested using liquid chromatography-mass spectrometry (LC-MS).

[0116] Instrument: Agilent 1290-ABSCIEX4500 LC-MS / MS; Column: Agilent C18 column (50mm*2.1mm). Detection conditions: Mobile phase A, 5mM ammonium formate; Mobile phase B, methanol; Elution gradient: 70% B phase isocratic elution for 7 min; Injection volume: 1 μL; Ion source temperature: 350℃ (EI); Acquisition mode: SIM selected ion detection (ion selection parameters are as follows):

[0117]

[0118] The standard substances, ethyl benzenesulfonate, isopropyl benzenesulfonate, and benzenesulfonic acid, were dissolved in ethyl acetate and diluted to a series of concentrations.

[0119] Three batches of raw materials were purchased: two batches of glucose dehydrogenase process raw materials and two batches of the naturalized folic acid described in this invention. The above seven batches of 6S-5-methyltetrahydrofolate calcium were tested.

[0120] The results are as follows:

[0121] Table 1: Detection results of ethyl benzenesulfonate in 7 batches of samples

[0122]

[0123] Table 2: Detection results of isopropyl benzenesulfonate in 7 batches of samples

[0124]

[0125] The results showed that using reductase for processing avoided the introduction of benzenesulfonic acid in the process, and neither ethyl benzenesulfonate nor isopropyl benzenesulfonate of naturalized folic acid were detected.

[0126] Example 9

[0127] Detection of enzyme protein residues in 9 batches of enzymatic process

[0128] Research Project: Content of dihydrofolate reductase protein, oxidized coenzyme, glucose dehydrogenase, and hydrogenase. Methods: Gel chromatography was used to determine the content of dihydrofolate reductase protein, oxidized coenzyme, glucose dehydrogenase, and hydrogenase. Chromatographic Conditions: A TSK GEL column (300 mm * 7.8 mm) was used, with a mobile phase of 0.06 mol / L Tris-HCl solution (pH 9.0) containing 0.15 mol / L sodium chloride, and a flow rate of 0.5 mL / min.

[0129] The reference standard is the original protein.

[0130] The results are as follows:

[0131]

[0132] The results showed that the enzyme protein residue of naturalized folic acid produced using the new process was reduced by at least 70% compared with that produced by the conventional enzymatic process, and the relevant protein residue did not exceed 1 ng / L.

[0133] The above are merely specific application examples of the present invention and do not constitute any limitation on the scope of protection of the present invention. All technical solutions formed by equivalent transformations or equivalent substitutions fall within the scope of protection of the present invention.

Claims

1. A method for preparing synthetically naturalized (6S)-5-methyltetrahydrofolate, characterized in that: Includes the following steps: Step 1: Folic acid is dissolved in an alkaline solution and reduced with zinc powder under inert gas protection to obtain dihydrofolic acid. Then, the pH value is adjusted to neutral with acid. Step 2: Collect the byproduct hydrogen gas from Step 1 and incubate it together with the hydrogenase solution. The hydrogenase content is 1-5 g / L, the incubation time is not less than 10 min, and the volume fraction ψi of inert gas in the hydrogen gas is less than 40%. Sodium dithionate is added to the hydrogenase solution before the hydrogen gas is introduced. The concentration of sodium dithionate is 2-10 μM. The hydrogenase is derived from one of the following: soluble hydrogenase from thermophilic archaea, hydrogenase from desulfovibrio, or hydrogenase from Alcaligenes. Step 3: In the presence of NADP+ or NADPH, hydrogenase, and hydrogen, dihydrofolate is stereoselectively reduced with dihydrofolate reductase to obtain (6S)-tetrahydrofolate. The concentration of NADPH or NPDP+ is 5-30 mM, the reaction concentration of hydrogenase is 1-100 mg / L, the concentration of Tris hydrochloride is 50-400 mM, and the concentration of dihydrofolate reductase is 2000-60000 U / L. Step 4: After converting (6S)-tetrahydrofolate to 6R-5-10-methylenetetrahydrofolate and its salt using formic acid, it is reduced to natural (6S)-5-methyltetrahydrofolate.

2. The method for preparing synthetic naturalized (6S)-5-methyltetrahydrofolate according to claim 1, characterized in that: In step 1, the acid is phosphoric acid.

3. The method for preparing synthetic naturalized (6S)-5-methyltetrahydrofolate according to claim 2, characterized in that: In step 2, the incubation temperature of the hydrogenase solution is 50-85℃, and the incubation pressure should be greater than 1.1 bar.

4. The method for preparing synthetic naturalized (6S)-5-methyltetrahydrofolate according to any one of claims 1-3, characterized in that: Step 3 also includes a pH buffer salt.

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