A method for the synthesis of l-phosphinothricin from l-threonine

Abstract

The present application relates to a kind of L-threonine synthesis L-glufosinate method, belong to the technical field of biosynthesis chemistry and organic synthesis combination, specifically with L-threonine as substrate by chemical synthesis enzyme catalysis preparation L-glufosinate.L-threonine is added threonine deaminase, and enzyme catalysis deamination reaction is carried out, after amino is completely oxidized into ketone, 3-hydroxybutyroic acid reaction liquid is obtained, catalyst is added, and dehydration reaction is carried out by heating, 1-vinylbutyroic acid reaction liquid is obtained, L-threonine and L-threonine transaminase are added, and enzyme transamination is obtained 1-vinyl amino acid reaction liquid, methyl phosphite monomethyl ester is added, and nucleophilic addition reaction is carried out, and then L-glufosinate parent drug is obtained by acidification, and high purity L-glufosinate original powder is obtained by crystallization, centrifugation, drying.The purpose is to develop a kind of raw material, easy, economic, efficient and green new process.

Description

Technical Field

[0001] This invention belongs to the technical field of the herbicide L-glufosinate, and relates to a method for synthesizing L-glufosinate from L-threonine. Background Technology

[0002] Glufosinate, also known as glufosinate-ammonium, with the chemical name 4-[hydroxy(methyl)phosphono]-DL-homoalanine, is a highly effective, broad-spectrum, low-toxicity, non-selective herbicide developed by Hoechst AG in Germany. Currently, most commercially available glufosinate is racemic, but only L-glufosinate possesses herbicidal activity. While the production processes for DL-glufosinate are mature both domestically and internationally, research on L-glufosinate remains in the laboratory stage, and industrial production has not yet been achieved. Therefore, researching and developing a production process for L-glufosinate is of great significance.

[0003] Currently, the reported methods for preparing L-glufosinate both domestically and internationally mainly include chemical and biological methods. Chemical methods use natural amino acids (L-aspartic acid, L-homoserine, and L-lysine) as chiral sources or construct chiral centers through asymmetric catalysis, but most suffer from drawbacks such as high cost and low optical purity. Biological methods mainly include bioresolution and bioasymmetric synthesis. Bioresolution uses DL-glufosinate or its derivatives as substrates, achieving resolution through microbial degradation of a single configuration. Bioasymmetric synthesis uses 2-carbonyl-4-(hydroxymethylphosphono)butyric acid as a substrate, catalyzed by an enzyme system to obtain L-glufosinate. While these routes can all yield refined glufosinate, they generally suffer from long routes, cumbersome operations, low yields, and high costs, making them unsuitable for industrial production.

[0004] To overcome the above difficulties, a novel process for synthesizing L-glufosinate using L-threonine as a substrate was developed by reviewing relevant literature and research experience.

[0005] L-Threonine is an essential amino acid, primarily used in pharmaceuticals, chemical reagents, food fortifiers, and feed additives. In recent years, international demand for threonine has seen steady and continuous growth, making it one of the fastest-growing amino acids. Its usage has been particularly rapid in chemical and biochemical applications, food additives, and feed additives, making it the third fastest-growing amino acid after lysine and methionine. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a method for synthesizing L-glufosinate from L-threonine, which has a high yield and low cost.

[0007] The technical solution adopted in this invention is:

[0008] L-Threonine, a readily available and relatively inexpensive amino acid in China, was selected as the starting substrate. The amino group at the 2-position was removed using threonine deaminase. After complete removal of the amino group, a 3-hydroxybutyric acid (THA) reaction solution was obtained. This THA reaction solution was preheated to 80-100°C, and an appropriate amount of nanomaterial catalyst (nano-silica supported alumina) was added for dehydration to obtain a 1-vinylketo acid (LVA) reaction solution. After cooling, L-Threonine and L-Threonine transaminase were added for transamination. Then, monomethyl methyl phosphite was added to the reaction solution for nucleophilic addition. Following acid hydrolysis, crystallization, purification, and centrifugal drying, L-glufosinate technical powder was obtained. The specific route is as follows:

[0009]

[0010] A method for synthesizing L-glufosinate from L-threonine, the method comprising the following steps:

[0011] (1) Enzyme-catalyzed deamination: Threonine deaminase was added dropwise to L-threonine (a) to carry out enzyme-catalyzed deamination reaction. After the amino group was completely oxidized to ketone, 3-hydroxybutyric acid reaction solution (b) was obtained.

[0012] (2) Hydroxyl dehydration: Add a catalyst to the reaction solution (b) obtained in step (1), heat, and carry out a dehydration reaction to obtain 1-vinylbutyric acid reaction solution (c).

[0013] (3) Enzymatic transamination: Add appropriate amounts of L-threonine and L-threonine transaminase to step (2), control the temperature at 20-30℃ and pH = 6-9, and react for 6-8 hours to obtain 1-vinylglycine reaction solution (d).

[0014] (4) Nucleophilic addition: The reaction solution (d) obtained in step (3) is added to monomethyl methyl phosphite to carry out a nucleophilic addition reaction, and then acid hydrolysis is performed to obtain L-glufosinate technical material (e).

[0015] (5) Centrifugal drying: The reaction solution (e) obtained in step (4) is crystallized, centrifuged and dried to obtain high-purity L-glufosinate technical powder.

[0016] Preferably, in step (1), the amount of threonine deaminase added is 1%-10% of the reaction substrate mass, and the conditions for enzyme-catalyzed deamination reaction are a temperature of 20-30℃, a pH of 6-9, and a reaction time of 3-5h.

[0017] Preferably, in step (2), the catalyst is nano-silica supported alumina, the amount of catalyst is 0.1%-1% of the total reaction system, and the dehydration reaction is carried out by heating to 80-100℃.

[0018] The preparation process of the above catalyst is as follows: A supported alumina nano-silica catalyst is prepared using a template method. A calcium carbonate template and sodium silicate solution are added to a reaction vessel to obtain a reaction solution. The reaction solution and a 40% sodium hydroxide alkaline solution are then pumped separately into a high-gravity reactor. The pH is adjusted to 8-9, and the mixture is centrifuged for 2 hours. Solid nano-silica spheres are obtained from the outer cavity. These solid spheres are further acidified with 30% hydrochloric acid to remove adhering calcium carbonate, and then washed with 99% ethanol to remove organic impurities. Finally, the mixture is filtered, washed with water, dried at 110℃-140℃, and ground to obtain hollow nano-silica. Figure 1 As shown, the mass ratio of calcium carbonate:sodium silicate:30% hydrochloric acid is 3:1:1.5. The resulting product was further examined using electron microscopy. After passing the examination, hollow silica, ethanol, alkyl sulfonic acid, and alumina were added to a reaction vessel, with a silica:alumina mass ratio of 3:1. The mixture was soaked, washed, and dried at room temperature and pressure to obtain a nano-silica-supported alumina catalyst.

[0019] Preferably, in step (3), the amount of L-threonine added is 0.5-1.5 times the amount of the reaction substrate, the amount of L-threonine transaminase added is 1%-10% of the amount of the reaction substrate, and the conditions for the enzymatic transaminase reaction are a temperature of 20-30℃, pH = 6-9, and a reaction time of 6-8h.

[0020] Preferably, the hydroxyl group is dehydrogenated to obtain 1-vinyl, and the carbon-carbon double bond continuously attacks the lone pair electrons of the trivalent phosphine, causing the trivalent phosphine to capture a proton to form a new pentavalent phosphorus-oxygen double bond, thus completing the nucleophilic addition. In step (4), the nucleophilic addition reaction conditions are: pH = 5-8, temperature -5℃ to 5℃, and reaction time 3-5h.

[0021] Preferably, the purity of L-glufosinate synthesized by the method is ≥95%, and the ee value is ≥99%.

[0022] The beneficial effects of this invention are:

[0023] 1. Compared with traditional chemical synthesis methods, this method reduces the number of synthesis steps, wastewater, and waste salt generated (traditional chemical processes require continuous acid-base neutralization and filtration, generating 30-50 tons of wastewater and 3-5 tons of waste salt per ton of glufosinate; this invention uses enzyme catalysis, generating virtually no waste salt, and produces only 3-5 tons of wastewater per ton of glufosinate); the biosynthesis method for high-serine has readily available and inexpensive raw materials (L-threonine 23,000 RMB / ton (industrialized, stable supply), L-homoserine 60,000 RMB / ton (not yet scaled up, supply limited), with raw material consumption of 1 ton / ton and 1.3 ton / ton respectively, resulting in a raw material saving of approximately 50,000 RMB / ton); L-threonine transaminase has high stereoselectivity, high yield, and high ee value.

[0024] 2. Nanocatalysts possess a large surface area, ensuring sufficient material contact and homogeneous reaction. They also feature a slow-release function, releasing the catalyst according to the reaction progress, which helps control the intensity of the reaction and ensures a smooth and stable reaction. Furthermore, alumina is used as a catalyst to replace traditional concentrated sulfuric acid. Nanocatalysts can be dried and regenerated for recycling, while concentrated sulfuric acid requires concentration and purification, resulting in higher costs and a concentration level that can only reach about 70%, making recycling impossible. Attached Figure Description

[0025] Figure 1 Hollow nano-silica structure diagram. Detailed Implementation

[0026] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention.

[0027] Example 1

[0028] A method for synthesizing L-glufosinate from L-threonine includes the following steps:

[0029] 1) The material in the L-threonine storage tank is pumped into the deamination vessel by the L-threonine feed pump. The L-threonine deaminase is fed into the deamination vessel by gravity from the high-level tank through the flow meter. The amount of threonine deaminase added is 5% of the mass of L-threonine. After stirring and mixing, the temperature is controlled at 25℃, the pH is controlled at 7, and the reaction time is 4h. After the amino group is completely removed, the 3-hydroxybutyric acid reaction solution is obtained.

[0030] 2) The reaction solution is pumped into the dehydration vessel via a 3-hydroxybutanone feed pump. A catalyst is added to the vessel, the temperature is raised to 90°C, and the dehydration reaction is carried out for 3 hours to obtain a 1-vinylketo acid reaction solution.

[0031] The catalyst is nano-silica supported alumina, and the amount of catalyst used is 5% of the total reaction system.

[0032] The preparation process of the above catalyst is as follows: fabrication of nano-hollow shells, embedding and supporting the catalyst using supergravity technology, and centrifugal drying to obtain nano-alumina catalyst.

[0033] The preparation process of the above catalyst is as follows:

[0034] The preparation process of the above catalyst is as follows: A supported alumina nano-silica catalyst is prepared using a template method. A calcium carbonate template and sodium silicate solution are added to a reaction vessel to obtain a reaction solution. The reaction solution and a 40% sodium hydroxide alkaline solution are then pumped separately into a high-gravity reactor. The pH is adjusted to 8-9, and the mixture is centrifuged for 2 hours. Solid nano-silica spheres are obtained in the outer cavity. These solid spheres are further acidified with 30% hydrochloric acid to remove adhering calcium carbonate, and then washed with 99% ethanol to remove organic impurities. Finally, the mixture is filtered, washed with water, dried at 110℃-140℃, and ground to obtain hollow nano-silica. The mass ratio of calcium carbonate:sodium silicate:30% hydrochloric acid is 3:1:1.5. The finished product is further examined using electron microscopy. After passing the examination, hollow silica, ethanol, alkyl sulfonic acid, and alumina are added to a reaction vessel, with a silica:alumina mass ratio of 3:1. The mixture is soaked, washed, and dried at room temperature and pressure to obtain the nano-silica supported alumina catalyst.

[0035] 3) The cooled 1-vinylketo acid reaction solution is pumped into the transamination reactor and thoroughly mixed with L-threonine and L-threonine transaminase from the high-level tank. The amount of L-threonine added is 1 times the mass of the reaction substrate (1-vinylketo acid), and the amount of L-threonine transaminase added is 3% of the mass of the reaction substrate (1-vinylketo acid). The temperature is controlled at 25℃, the pH is controlled at 7, and the reaction time is 4h to obtain 1-vinyl amino acids.

[0036] 4) A 1-vinyl amino acid feed pump pumps the reaction solution into an addition reactor where it reacts with monomethyl methyl phosphite from a storage tank. The temperature is controlled at -5℃, the pH at 6, and the reaction time at 4 hours. Purification is performed (washing with ethanol at room temperature and pressure). The washing mother liquor is then purified by resin adsorption to remove organic matter. The permeate is then distilled under reduced pressure to recover the solvent. The evaporated residue is subjected to electrodialysis to remove salts, and the permeate is fed into the biochemical system. The purified and crystallized solid is centrifuged to obtain a wet powder, which is then dried by cyclone airflow (temperature controlled at 100-105℃). After passing inspection, L-glufosinate technical powder is obtained, with a content ≥95% and an ee value ≥99%.

[0037] Example 2

[0038] Based on Example 1, the yield of L-glufosinate technical powder was obtained by changing the amount of catalyst added in step (2) and the pH conditions in step (2). The results are shown in Table 1 below.

[0039] Table 1. Effects of catalyst addition and reaction pH on total yield

[0040] Serial Number Catalyst addition ratio (%) pH Yield (%) 1 3 6 85.6 2 3 7 84.7 3 3 8 84..4 4 3 9 83.6 5 5 6 86.7 6 5 7 85.4 7 5 8 84.9 8 5 9 84.7 9 7 6 88.2 10 7 7 87.9 11 7 8 86.7 12 7 9 85.8 13 10 6 87.9 14 10 7 86.8 15 10 8 86.1 16 10 9 85.9

[0041] Table 1 shows that when the amount of catalyst exceeds 6%, the catalytic effect is less affected by pH value, and the amount of catalyst added has virtually no impact on the catalytic effect. It is recommended to control the catalyst addition at 7% and pH = 6.

[0042] Example 3

[0043] Based on Example 1, the difference lies in the addition of L-threonine and the corresponding transaminase in step (3). The rest is the same as in Example 1. The results are shown in Table 2.

[0044] Table 2 Effect of L-threonine and transaminase addition on reaction yield

[0045] Serial Number L-threonine equivalent L-threonine transaminase addition ratio (%) Yield (%) 1 0.5 10 46.3 2 0.6 10 55.1 3 0.7 10 64.6 4 0.8 10 72.4 5 0.9 10 83.1 6 1.0 10 87.4 7 1.1 10 87.3 8 1.2 10 87.5 9 1.3 10 87.5 10 1.4 10 87.6 11 1.5 10 87.8 12 1 9 87.6 13 1 8 87.4 14 1 7 87.5 15 1 6 87.4 16 1 5 87.3 17 1 4 87.4 18 1 3 87.4 19 1 2 86.0 20 1 1 73.6

[0046] Analysis of the experimental results showed that the greatest economic benefit was achieved when the L-threonine dosage was 1 times the mass of the substrate 1-vinylketoacid, with an L-threonine transaminase addition of 10% (Table No. 6). Although increasing the L-threonine dosage improved the product yield (Table Nos. 7-11), the improvement efficiency was far less than the increase in dosage. Therefore, a mass ratio of 1:1 was selected as the optimal dosage. Subsequently, the dosage of L-threonine transaminase was changed based on this. The results showed that a dosage of 3% could achieve efficient synthesis of the target product (Table No. 18). Therefore, the dosage of L-threonine and its transaminase should be 1 times and 3% of the mass of the substrate 1-vinylketoacid, respectively.

[0047] Example 4

[0048] Based on Example 1, the difference lies in the temperature of nucleophilic addition in step (4). Other results are the same as in Example 1, as shown in Table 3.

[0049] The nucleophilic addition reaction of dimethyl phosphite is exothermic, requiring timely cooling to remove heat and ensure the reaction proceeds in the forward direction, thereby increasing product yield. Experiments were conducted at different temperatures.

[0050] Table 3 Effect of different reaction temperatures on reaction yield

[0051]

[0052]

[0053] As shown in Table 3, the yield increases with decreasing temperature. This is because nucleophilic addition reactions are exothermic; if heat cannot be removed promptly, the reaction rate will be slow, the reaction will be incomplete, and more byproducts will be produced. Exothermic reactions require timely removal of heat from the external environment to facilitate the forward reaction. Therefore, based on actual production considerations, controlling the reaction temperature at -5℃ yields the best results.

[0054] In summary, this invention reports a highly efficient biological method for synthesizing glufosinate-ammonium phosphate based on L-threonine as a substrate. Compared with traditional biological methods, it has advantages such as mild conditions, readily available raw materials, low reaction cost, environmental friendliness, and high product purity, and has potential for industrial application.

[0055] The specific embodiments described herein are merely illustrative of the spirit of the invention. Those skilled in the art to which this invention pertains may make various modifications or additions to the described specific embodiments or use similar methods to substitute them, without departing from the spirit of the invention or exceeding the scope defined by the appended claims.

Claims

1. A method for synthesizing L-glufosinate from L-threonine, characterized in that, The method includes the following steps: (1) Enzyme-catalyzed deamination: Threonine deaminase was added dropwise to L-threonine (a). The amount of threonine deaminase added was 1%-10% of the mass of the reaction substrate. The enzyme-catalyzed deamination reaction was carried out under the following conditions: temperature 20-30℃, pH = 6-9, reaction time 3-5h. After the amino group was completely oxidized to ketone, 3-hydroxybutyric acid reaction solution (b) was obtained. (2) Hydroxyl dehydration: Add catalyst nano silica supported alumina to the reaction solution (b) obtained in step (1). The amount of catalyst is 1%-10% of the total mass of the reaction system. Heat to carry out dehydration reaction to obtain 3-vinylbutyric acid reaction solution (c). (3) Enzymatic transamination: Add L-threonine and L-threonine transaminase to the reaction solution (c) in step (2) to obtain 3-vinylglycine reaction solution (d) by enzymatic transamination. The amount of L-threonine added is 0.5-1.5 times the amount of the reaction substrate, and the amount of L-threonine transaminase added is 1%-10% of the amount of the reaction substrate. The reaction conditions for enzymatic transamination are temperature 20-30℃, pH=6-9, and reaction time 6-8h. (4) Nucleophilic addition: The reaction solution (d) obtained in step (3) is added to monomethyl methyl methyl phosphite to carry out a nucleophilic addition reaction, and then acidified to obtain L-glufosinate technical material reaction solution (e). (5) Centrifugal drying: The reaction solution (e) obtained in step (4) is crystallized, centrifuged and dried to obtain high-purity L-glufosinate technical powder.

2. The method for synthesizing L-glufosinate from L-threonine according to claim 1, characterized in that, In step (2), the temperature is heated to 80-100℃ to carry out a dehydration reaction.

3. The method for synthesizing L-glufosinate from L-threonine according to claim 1, characterized in that, In step (4), the nucleophilic addition reaction conditions are: pH=5-8, temperature-5℃~5℃, and reaction time-5 hours.

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