Method for synthesizing l-glufosinate or salt thereof

Abstract

Provided is a method for synthesizing L-glufosinate, comprising the following steps: first making L-homoserine react with acetic acid to form a compound A or a salt thereof; then catalyzing the compound A or the salt thereof by a synthetase to form a compound B; then carrying out chlorination and ring opening on the compound B to form an intermediate compound C; then carrying out an Arbuzov reaction on the intermediate compound C and a phosphine reagent to form a compound D or a salt thereof; and carrying out a hydrolysis reaction on the compound D or the salt thereof regardless of whether the intermediate is separated or not to form L-glufosinate or a salt thereof. The method for synthesizing L-glufosinate combines a chemical synthesis method with an enzyme catalysis method to obtain L-glufosinate having both a high e.e. value and high yield, and has simple preparation steps and controllable cost.

Description

A method for synthesizing L-glufosinate or its salt Technical Field

[0001] The present invention relates to a method for preparing L-glufosinate ammonium by combining bio-enzyme catalysis and chemical synthesis, and particularly relates to a method for synthesizing L-glufosinate ammonium. Background Art

[0002] L-Glufosinate, also known as refined glufosinate, is a fungicide-active tripeptide compound isolated by Bayer from the fermentation broth of Streptomyces hygroscopicus. Bialaphos is composed of two L-alanine molecules and a then-unknown amino acid. L-Glufosinate belongs to the phosphonic acid class of herbicides and shares the same mechanism of action as glufosinate. Glufosinate is a racemate of D / L-Glufosinate, but only L-Glufosinate possesses herbicidal activity. It acts on plant glutamine synthetase, inhibiting L-glutamine synthesis. This leads to the accumulation of cytotoxic ammonium ions, disrupting ammonium metabolism, amino acid depletion, chlorophyll decomposition, and inhibiting photosynthesis, ultimately killing weeds. The glufosinate preparations currently sold on the market are generally racemic D / L-type glufosinate. Therefore, the biological activity of L-glufosinate is twice that of the DL racemate at the same concentration. If glufosinate products can be used in the form of pure chemical isomers with L-configuration, the usage of glufosinate can be reduced by about 50%, which is of great significance for improving economic efficiency, reducing usage costs and alleviating environmental pressure.

[0003] Currently, methods for producing L-glufosinate ammonium both domestically and internationally are primarily chemical synthesis and biosynthesis. In "Research Progress in the Synthesis of Glufosinate Ammonium," Mao Mingzhen et al. reviewed several chemical methods for preparing refined glufosinate ammonium, including asymmetric synthesis, racemate resolution, and chiral auxiliary induction. Industrial large-scale production methods primarily rely on asymmetric synthesis and racemate resolution, but both suffer from multiple process steps, low yields, and expensive chiral raw materials. In "Green Biosynthesis Technology and Application Prospects of Refined Glufosinate Ammonium," Dong Yi reviewed several biosynthetic methods for L-glufosinate ammonium, which offer advantages such as strict stereoselectivity, mild reaction conditions, and high yields, showing potential as alternatives to traditional chemical synthesis. Traditional biosynthesis methods are mainly divided into two types. One is to use L-phosphinothricin derivatives as substrates and obtain them through direct enzymatic hydrolysis, but the precursors are expensive and difficult to obtain; the other is to use racemic glufosinate precursors as substrates and obtain them through enzymatic selective resolution. Patent CN112553285A discloses a method for preparing L-phosphinothricin by using ω-transaminase and bioenzymatic deracemization, but the method has a low theoretical yield and easily causes waste of raw materials.

[0004] Therefore, the present invention provides a L-glufosinate-ammonium synthesis process with simple operation, low cost and good yield, which has very important industrial application significance.

[0005] Summary of the Invention

[0006] In response to the problems existing in the above-mentioned prior art, the present invention provides a method for synthesizing L-glufosinate or its salt, which combines chemical synthesis and enzyme catalysis to obtain L-glufosinate with high ee value and high yield. The preparation steps are simple and the cost is controllable.

[0007] In order to achieve the above object, the present invention adopts the following technical scheme: a method for synthesizing L-phosphinothricin ammonium or its salt, the structural formula of L-phosphinothricin ammonium is The steps of its synthesis are as follows:

[0008] (1) L-homoserine is reacted with acetic acid to form compound A or a salt thereof as shown in structural formula I. Structural formula I is

[0009] (2) Compound A or its salt is catalyzed by a synthase to form compound B as shown in structural formula II. Structural formula II is

[0010] (3) Compound B is subjected to chlorination ring opening to form an intermediate compound C as shown in structural formula III. Structural formula III is

[0011] (4) The intermediate compound C is subjected to an Arbuzov reaction with a phosphine reagent to form a compound D or a salt thereof as shown in structural formula IV. Structural formula IV is

[0012] (5) Subjecting compound D or its salt to a hydrolysis reaction to form L-phosphinothricin ammonium or its salt.

[0013] The method for synthesizing L-glufosinate or its salt of the present invention comprises the following steps:

[0014] (1) L-homoserine is used as a raw material, an acetic acid solution and a protective agent are added, and L-homoserine and acetic acid are reacted at a temperature of 60-100° C. for 3-5 hours, followed by purification to obtain compound A or a salt thereof;

[0015] (2) dissolving compound A or its salt in a buffer solution, heating the solution in a water bath to 20-45°C, adding lactone synthase, reacting for 6-12 hours, and then purifying to obtain compound B;

[0016] (3) Compound B was dissolved in anhydrous ethanol, stirred and cooled to 0°C in an ice bath, and a ring-opening reagent was slowly added. The mixture was reacted at 0°C for 15-60 minutes and then heated to 80°C for 3-5 hours. Compound B disappeared, and then purified to obtain compound C.

[0017] (4) Compound C is placed in an argon atmosphere, a phosphine reagent is added, the mixture is sealed, and the mixture is heated to 135-145° C. and stirred for 46-50 hours. The mixture is then purified to obtain Compound D or a salt thereof;

[0018] (5) Compound D or its salt is dissolved in an inorganic acid solution, stirred and hydrolyzed at 100°C for 12 hours, concentrated and stopped, and then water and powdered activated carbon are added. The mixture is stirred and reacted at 90°C for 30 minutes. The mixture is then filtered and washed while hot, and propylene oxide is added dropwise at 15-25°C. The mixture is stirred and reacted for 3-6 hours to promote uniform distribution of the intermediate product and prevent precipitation. After further concentration, anhydrous ethanol is added and stirred, and the mixture is placed in an ice-water bath and stirred for crystallization to obtain a crystal, i.e., L-phosphinothionyl ammonium.

[0019] Preferably, in step (1), the concentration of the acetic acid solution ranges from 1.0M to 2.0M, and preferably the concentration of the acetic acid solution is 1.4M.

[0020] Preferably, in step (1), the reaction temperature is 75° C. and the reaction time is 3 h.

[0021] Preferably, in step (1), the protecting agent is carbodiimide.

[0022] Preferably, in step (1), the lactone synthase is one or more of homoserine lactonase RhlI, homoserine lactonase LasI, lactone synthase DWF4, lactone synthase GaCPD2, lactone synthase XsTPS3 and lactone synthase ApCPS.

[0023] Preferably, in step (2), the pH range of the buffer solution is 5 to 8; preferably the pH range is 6.8.

[0024] Preferably, in step (2), the buffer solution is a citrate-phosphate buffer solution or a Tris-HCl buffer solution.

[0025] Preferably, in step (2), the temperature is 37° C. and the reaction time is 10 h.

[0026] Preferably, in step (3), the ring-opening reagent is thionyl chloride.

[0027] Preferably, in step (4), the phosphine reagent is diethyl methylphosphite.

[0028] Preferably, in step (4), the reaction temperature is 140° C. and the reaction time is 48 h.

[0029] Preferably, in step (5), the inorganic acid solution is a hydrochloric acid solution.

[0030] Preferably, in step (5), the amount of powdered activated carbon added is 4.5% to 5.5% by weight of compound D, preferably 5%.

[0031] The beneficial effects of the present invention are as follows: combining the advantages of chemical synthesis and enzyme catalysis, using cheap and easily available L-homoserine as a starting material, and preparing L-phosphinothricin with a high enantiomeric excess through a five-step reaction, with an ee value of 95.4%, a purity of 97.7%, and a total yield of 92.5%. Compared with the existing preparation process, the present invention has simple steps, controllable costs, and takes into account high ee value and yield, and has very important industrial application significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] FIG1 is a liquid phase diagram of compound A prepared in Example 1;

[0033] FIG2 is a liquid phase diagram of compound B prepared in Example 1;

[0034] FIG3 is a liquid phase diagram of compound C prepared in Example 1;

[0035] FIG4 is a liquid phase diagram of compound D prepared in Example 1;

[0036] FIG5 is a liquid phase diagram of L-glufosinate prepared in Example 1. DETAILED DESCRIPTION

[0037] In order to explain the present invention more clearly and completely, the present invention is described below with reference to specific embodiments, which however are not intended to limit the present invention.

[0038] The term ee as used in the present invention means "enantiomeric excess" and refers to the percentage of one enantiomer exceeding the other enantiomer in a product, used to express its optical intensity. For example, the percentage of L-glufosinate exceeding D-glufosinate in glufosinate represents the enantiomeric excess of L-glufosinate. The specific calculation formula is:

[0039] Enantiomeric excess (ee) of L-glufosinate = [(content of L-glufosinate - content of D-glufosinate) / (content of L-glufosinate + content of D-glufosinate)] × 100%

[0040] Example 1

[0041] A method for synthesizing L-glufosinate or its salt comprises the following steps:

[0042] (1) Synthesis of Compound A or its salt

[0043] 92 g of the starting material L-homoserine was added to a round-bottom flask I, followed by 1.32 L of a 2.4 M acetic acid solution and 0.1 L of a 0.6 M carbodiimide aqueous solution to dissolve the L-homoserine until it was clear. The mixture was then heated to 75 ° C. to form a reflux reaction for 3 hours. After the reaction was completed, the temperature was lowered and stirred until the L-homoserine disappeared during detection. The reaction solution was then concentrated to a stop, and after adding anhydrous ethanol in an amount twice (w / w) of L-homoserine, compound A or its salt was obtained. The liquid chromatogram in Figure 1 showed that compound A was The structural formula is represented by structural formula I, and the obtained compound A or its salt is directly used in the next reaction;

[0044] The synthesis of compound A or its salt is shown in Reaction Formula I, which is as follows:

[0045] (2) Synthesis of Compound B

[0046] Compound A or its salt obtained in step (1) above was added to round-bottom flask II, and 1.6 L of citric acid-phosphate buffer solution of pH 6.8 was added to dissolve compound A or its salt. The mixture was heated to 37° C. in a water bath, and 1.6 g of homoserine lactone synthase LasI (0.66 U / mg) and 2.2 g of DWF4 (0.27 U / mg) were slowly added under stirring. The mixture was reacted for 10 h until compound A disappeared. The reaction solution was concentrated and removed, and water (10 times the amount (v / w) of L-homoserine) and ethyl acetate (20 times the amount (v / w) of L-homoserine) were added to extract the mixture three times. The ethyl acetate layer was dried over anhydrous sodium sulfate, filtered, and then dried to obtain compound B. The liquid chromatogram of FIG2 showed that compound B was The structural formula is represented as structural formula II;

[0047] The synthesis of compound B is shown in Reaction Formula II, which is as follows:

[0048] Conclusion: Based on 92 g of the starting material L-homoserine, 106.4 g of compound B was obtained. According to the two-step reaction of steps (1) and (2), the yield of compound B was 96.2% and the purity was 98.7%.

[0049] (3) Synthesis of Compound C

[0050] 85.6 g of compound B obtained in step (2) above was added to round-bottom flask III, and 15 ml of anhydrous ethanol was used to dissolve each gram of compound B, with a total amount of anhydrous ethanol of 1284 ml. After stirring and cooling to 0°C in an ice bath, 75 ml of dichlorothione was slowly added. After the addition of dichlorothione was completed, the mixture was reacted at 0°C for 15 minutes, then heated to 80°C and stirred until compound B disappeared. The reaction solution was then slowly poured into a stirred ice-water mixture, and then concentrated to a stop. Ethyl acetate was added in an amount (v / v) 5 times the volume of the concentrated solution for extraction. The extraction was performed twice. The ethyl acetate layer was taken, washed once with a saturated sodium chloride solution, separated, dried over anhydrous sodium sulfate, filtered, and then dried to obtain compound C. The liquid chromatogram in Figure 3 showed that compound C was The structural formula is represented as structural formula III;

[0051] The synthesis reaction formula of compound C is shown in Reaction Formula III, which is as follows:

[0052] Conclusion: Starting from 85.6 g of compound B, 113.3 g of compound C was obtained with a yield of 91.2% and a purity of 91%.

[0053] (4) Synthesis of Compound D or its salt

[0054] Take a 250ml Schlenk reaction flask, add 113.3g of compound C, evacuate and replace with argon three times, then pass argon protection, add 117g of phosphine reagent diethyl methyl phosphite, seal, heat and stir at 140°C for 48h, then cool to room temperature, transfer the reaction solution to a 250ml round-bottom flask, and perform vacuum distillation. First, distill at 55°C under water pump pressure to obtain 11.5g of colorless liquid, then heat to 70°C and continue distillation to obtain 14.1g of colorless liquid, then change the water pump pressure reduction to oil pump pressure reduction, and evaporate some high boiling point liquid at 110°C. Then transfer to a separatory funnel and dissolve with 100ml of dichloromethane, then add 80ml of saturated sodium chloride solution to wash, wash twice, take the dichloromethane layer, first dry and filter with anhydrous sodium sulfate, and concentrate the filtrate to remove dichloromethane to obtain compound D or its salt. The liquid chromatogram of Figure 4 shows that compound D is The structural formula is represented by structural formula IV;

[0055] The synthesis of compound D is shown in Reaction Formula IV, which is as follows:

[0056] Conclusion: Based on 113.3 g of compound C, 129.6 g of compound D or its salt was obtained with a yield of 85.1% and a product purity of 78.0%.

[0057] (5) Synthesis of L-phosphinothricin (TM)

[0058] Take a 2L round-bottom flask, add 129.6g of compound D or its salt, 612ml of 6M hydrochloric acid, and heat and stir to maintain the internal temperature at 100°C for 12h. The reaction solution is slightly cooled and concentrated to a stop, 300ml of water and 1.44g of powdered activated carbon are added, and stirring is continued at 90°C for 30min. The activated carbon is removed by hot filtration, and the filtrate is washed with 150ml of dichloromethane, washed twice, and the aqueous phase is taken. The temperature is controlled at 15-25°C, and 10ml of propylene oxide is added dropwise. The reaction is stirred for 3-6 hours, first concentrated, and then 80ml of anhydrous ethanol is added and stirred. Then, it is placed in an ice-water bath and stirred for crystallization. The solid product is filtered out, and the liquid chromatogram of Figure 4 identifies it as the compound L-glufosinate ammonium, with the structural formula: Indicated by TM;

[0059] The synthesis of L-glufosinate-ammonium is shown in Reaction Formula V, which is as follows:

[0060] Conclusion: Starting from 129.6 g of compound D or its salt, 74.8 g of crystalline compound L-glufosinate ammonium was obtained, and the yield of step (5) was 89.0%. At the same time, the total yield obtained by multiplying the yields of steps (1) and (2), the yield of (3), the yield of (4) and the yield of (5) was 66.48%. The purity of the final product L-glufosinate ammonium was 97.7%, and the ee value was 95.4%.

[0061] Example 2

[0062] Compared with Example 1, this embodiment is different in that:

[0063] (2) Synthesis of Compound B

[0064] Compound A or its salt obtained in the above step (1) was added to a round-bottom flask II (the amount of the initial material L-homoserine added was 73 g), and 1.6 L of a pH 6.8 Tris-HCl buffer solution (0.5 M) was added in a water bath to 37° C., and then 1.6 g of homoserine lactone synthase RhlI (1.02 U / mg) and 2.2 g of ApCPS (0.66 U / mg) were slowly added under stirring. The reaction was allowed to react for 12 h until the starting materials disappeared. The reaction solution was concentrated and extracted according to the process of Example 1, and finally dried to obtain 86 g of compound B. Based on 73 g of the starting material L-homoserine, according to the two-step reaction of steps (1) and (2), the yield of compound B was 98.0% and the purity was 98.5%.

[0065] (3) Synthesis of Compound C

[0066] 86 g of compound B obtained in step (2) above was added to round-bottom flask III, and 15 ml of anhydrous ethanol was used to dissolve each gram of compound B, with a total amount of anhydrous ethanol of 1290 ml. After stirring and cooling to 0° C. in an ice bath, 150 ml of thionyl chloride was slowly added. After the addition of thionyl chloride was completed, the mixture was reacted at 0° C. for 30 min, then heated to 80° C. and stirred until compound B disappeared. The reaction solution was then concentrated and extracted according to the process of Example 1, and finally dried by spin drying to obtain 122.3 g of compound C. Calculated based on 86 g of compound B as the starting material, the yield of compound C was 98.0% and the purity was 90.5%.

[0067] (4) Synthesis of Compound D or its salt

[0068] A 500 ml Schlenk reaction flask was added with 122.3 g of compound C, and the mixture was evacuated and filled with argon to replace the g three times. Then, argon was passed through the flask for protection. 234 g of diethyl methylphosphite, a phosphine reagent, was added, and the mixture was sealed. The reaction mixture was heated with stirring at 140° C. for 48 h, then cooled to room temperature. The reaction solution was distilled and washed under reduced pressure according to the process of Example 1, and finally concentrated to obtain 161.3 g of compound D or its salt. Based on 122.3 g of compound C, the yield of compound D or its salt was 98.9%, and the purity was 96.2%.

[0069] 161.3 g of compound D or its salt was finally hydrolyzed to obtain 101.9 g of L-phosphinothionate, with a yield of 97.4% for L-phosphinothionate. The total yield obtained by multiplying the yields of steps (1) and (2), the yield of (3), the yield of (4) and the yield of (5) was 92.5%, with a purity of 96.4% and an ee value of 92.8%.

[0070] Example 3

[0071] Compared with Example 1, this embodiment is different in that:

[0072] (2) Synthesis of Compound B

[0073] Compound A or its salt obtained in the above step (1) was added to round-bottom flask II (the amount of initial material L-homoserine added was 88 g), and 1.6 L of Tris-HCl buffer solution (0.5 M) with pH 6.8 was added in a water bath to 37° C. Then, 1.6 g of homoserine lactone synthase RhlI (1.02 U / mg), 1.6 g of LasI (0.66 U / mg) and 2.2 g of ApCPS (0.66 U / mg) were slowly added under stirring, and the reaction was carried out for 12 h until the raw materials disappeared. The reaction solution was concentrated and extracted according to the process of Example 1, and finally dried to obtain 101.7 g of compound B. Based on 88 g of the starting material L-homoserine, according to the two-step reaction of steps (1) and (2), the yield was 96.2% and the purity was 92.1%.

[0074] (3) Synthesis of Compound C

[0075] 101.7 g of compound B obtained in step (2) above was added to round-bottom flask III, and 15 ml of anhydrous ethanol was used to dissolve each gram of compound B, with a total amount of anhydrous ethanol of 1917 ml. After stirring and cooling to 0°C in an ice bath, 150 ml of thionyl chloride was slowly added. After the addition of thionyl chloride was completed, the mixture was reacted at 0°C for 60 min, then heated to 80°C and stirred until compound B disappeared. The reaction solution was then concentrated and extracted according to the process of Example 1, and finally dried to obtain 143.6 g of compound C. Calculated based on 101.7 g of compound B, the yield of compound C was 97.3% and the purity was 92.1%.

[0076] (4) Synthesis of Compound D or its salt

[0077] A 500 ml Schlenk reaction flask was added with 86.8 g of compound C, and the mixture was evacuated and replaced with argon three times. Then, argon was introduced for protection. 234 g of diethyl methylphosphite, a phosphine reagent, was added, and the mixture was sealed. The mixture was heated with stirring at 140° C. for 48 h, and then cooled to room temperature. The reaction solution was distilled and washed under reduced pressure according to the process of Example 1, and finally concentrated to obtain compound D or a salt thereof.

[0078] Compound D or its salt is finally hydrolyzed to obtain 72.6 g of L-glufosinate ammonium. Based on 76.8 g of compound C, according to steps (4) and (5), the yield of L-glufosinate ammonium is 96.0%. The total yield of steps (1) and (2), the yield of (3), and the yield of steps (4) and (5) is 89.9%, with a purity of 97.7% and an ee value of 95.4%.

[0079] Finally, it should be noted that the above embodiments are only used to illustrate rather than limit the technical solutions of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the present invention can still be modified or replaced by equivalents. Any modification or partial replacement that does not depart from the spirit and scope of the present invention should be included in the scope of the claims of the present invention.

Claims

1. A method for synthesizing L-phosphinothricin or its salt, characterized in that: The following steps are involved: (1) reacting L-homoserine with acetic acid to form compound A or a salt thereof as shown in structural formula I; (2) subjecting compound A or a salt thereof to a synthetase-catalyzed reaction to form compound B as shown in structural formula II; (3) subjecting compound B to ring opening by chlorination to form an intermediate compound C as shown in structural formula III; (4) subjecting the intermediate compound C to an Arbuzov reaction with a phosphine reagent to form a compound D or a salt thereof as shown in structural formula IV; (5) subjecting compound D or its salt to a hydrolysis reaction to form L-phosphinothricin ammonium or its salt; Among them, structural formula I is Structural formula II is Structural formula III is Structural formula IV is 2. The method for synthesizing L-phosphinothricin or its salt according to claim 1, characterized in that: In step (1), the reaction conditions are: using acetic acid solution as solvent, using carbonyl diamine as protective agent, and reacting at a temperature of 60-100° C. for 3-5 hours; preferably, the reaction temperature is 75° C. and the reaction time is 3 hours.

3. The method for synthesizing L-phosphinothricin or its salt according to claim 2, characterized in that: The concentration of the acetic acid solution is 1.0M to 2.0M, preferably 1.4M.

4. The method for synthesizing L-phosphinothricin or its salt according to claim 1, characterized in that: In step (2), the reaction conditions are: using a buffer solution as the solvent, heating in a water bath to 20-45° C., adding lactone synthase to catalyze the reaction for 6-12 hours; preferably, the reaction temperature is 37° C. and the reaction time is 10 hours.

5. The method for synthesizing L-phosphinothricin or its salt according to claim 4, characterized in that: The lactone synthase is one or more of homoserine lactonase RhlI, homoserine lactonase LasI, lactone synthase DWF4, lactone synthase GaCPD2, lactone synthase XsTPS3 and lactone synthase ApCPS.

6. A method for synthesizing L-phosphinothricin or its salt according to claim 4, characterized in that: The buffer solution is a citric acid-phosphate buffer solution or a Tris-HCl buffer solution; the pH range of the buffer solution is 5 to 8; the preferred pH range is 6.

8.

7. The method for synthesizing L-phosphinothricin or its salt according to claim 1, characterized in that: In step (3), the reaction conditions are: using anhydrous ethanol as the solvent, slowly adding the ring-opening reagent dichlorothionyl in an ice bath, first reacting at 0° C. for 15 to 60 minutes, and then heating to 80° C. for 3 to 5 hours.

8. The method for synthesizing L-phosphinothricin or its salt according to claim 1, characterized in that: In step (4), the reaction conditions are: under argon protection, using diethyl methylphosphite as the phosphine reagent, and reacting at a temperature of 135 to 145° C. for 46 to 50 hours; preferably, the reaction temperature is 140° C. and the reaction time is 48 hours.

9. The method for synthesizing L-phosphinothricin or its salt according to claim 1, characterized in that: In step (5), the reaction conditions are as follows: Compound D or its salt is first subjected to hydrolysis reaction in the presence of hydrochloric acid solution at a reaction temperature of 100° C. for a reaction time of 12 h, then concentrated, and then water and powdered activated carbon are added, and the reaction is continued at 90° C. for 30 min, and then filtered and washed while hot, and then propylene oxide is added dropwise at 15-25° C., and the reaction is stirred for 3-6 h. After concentrating again, anhydrous ethanol is added and then crystallized in an ice bath.

10. The method for synthesizing L-phosphinothricin or its salt according to claim 9, characterized in that: The concentration of the hydrochloric acid solution is 5 to 7 M, preferably 6 M; The added amount of powdered activated carbon is 4.5-5.5% of the mass of compound D; preferably 5%.

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