阅读说明：本技术 L-草铵膦的制备方法 (Preparation method of L-glufosinate-ammonium ) 是由 胡磊 曾超 奚超群 于 2019-03-29 设计创作，主要内容包括：本发明公开了一种式I化合物的制备方法,由式V化合物反应得到,反应式如下：<Image he="314" wi="700" file="DDA0002011329780000011.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image> R<Sub>1</Sub>选自烃基、芳基、杂芳基、取代烃基、取代芳基、取代杂芳基；R<Sub>2</Sub>选自氨基或-OR<Sub>0</Sub>；R<Sub>0</Sub>选自烃基、硅烷基、苄基、烷基甲基、烷基取代烷氧基、3-8元脂环族基、芳基、杂芳基、取代芳基、取代杂芳基、Ar(CH<Sub>2</Sub>)<Sub>n</Sub>O-基团中的一种,Ar代表芳基、杂芳基,n取1-6。本发明工艺流程简单,对设备无特殊要求,适用于工业化生产,对环境友好,极大地降低了成本。(The invention discloses a preparation method of a compound shown in formula I, which is obtained by reacting a compound shown in formula V, wherein the reaction formula is as follows: R 1 selected from the group consisting of alkyl, aryl, heteroaryl, substituted alkyl, substituted aryl, substituted heteroaryl; r 2 Selected from amino OR-OR 0 ；R 0 Selected from the group consisting of hydrocarbyl, silyl, benzyl, alkyl methyl, alkyl-substituted alkoxy, 3-8 membered cycloaliphatic, aryl, heteroaryl, substituted aryl, substituted heteroaryl, Ar (CH) 2 ) n One of O-groups, Ar represents aryl or heteroaryl, and n is 1-6. The method has the advantages of simple process flow, no special requirements on equipment, suitability for industrial production, environmental friendliness and great reduction of cost.)

1. A process for the preparation of a compound of formula I, characterized by reacting a compound of formula V, wherein the reaction is as follows:

wherein R is₁Selected from alkyl, arylA group, heteroaryl, substituted alkyl, substituted aryl, substituted heteroaryl;

R₂selected from amino OR-OR₀；R₀Selected from the group consisting of hydrocarbyl, silyl, benzyl, alkyl methyl, alkyl-substituted alkoxy, 3-8 membered cycloaliphatic, aryl, heteroaryl, substituted aryl, substituted heteroaryl, Ar (CH)₂)_nOne of O-groups, Ar represents aryl or heteroaryl, and n is 1-6.

2. A preparation method of L-glufosinate-ammonium is characterized in that a compound shown in a formula II is prepared by reacting a compound shown in a formula IV, the reaction formula is as follows, protecting groups are removed to obtain L-glufosinate-ammonium,

wherein R is₃Selected from the group consisting of hydrogen, alkyl, silyl ether protecting groups, benzyl ether protecting groups, alkoxymethyl, alkoxy-substituted methyl, C2-C10 linear or branched alkenylalkyl, C2-C10 linear or branched alkynylalkyl, 3-8 membered cycloaliphatic, aryl, heteroaryl, amino, substituted amino, Ar (CH)₂)_a-one of the groups, Ar represents an aryl, heteroaryl group, a takes 1 to 6.

3. The process of claim 1, wherein the compound of formula V is reacted with enzyme E1 and enzyme E2 to form the compound of formula I:

said E1 is an enzyme capable of hydrolyzing a compound of formula V to a compound of formula I;

said E2 is an enzyme capable of racemizing a compound of formula VI;

coenzyme is needed to be added in the reaction process.

4. A process according to claim 3, wherein the L-glufosinate-ammonium is prepared by reacting a compound of formula IV with an enzyme E1 and an enzyme E2 to form a compound of formula II, deprotecting the groups to form L-glufosinate-ammonium,

5. the process according to claim 2, wherein R3 is hydrogen, R2 is ethyl, propyl or isopropyl, the pH of the reaction system is controlled to 5 to 8.5, and the coenzyme is pyridoxal phosphate.

6. The process according to claim 3, wherein the enzyme E1 is derived from Streptomyces ramulosus, Pseudomonas azotoformans, Mycobacterium tubericus, pork liver, Candidaantartica, Pseudomonas wadesweinhlerensis, Pseudomonas sp, Brevundimonas diminuta.

7. The process according to claim 3, wherein the enzyme E2 is derived from a gene selected from the group consisting of Bacillus, Trypanosoma cruzi, Oenococcus oeni, Amycolatopsis orientalis, Streptomyces lavendale, Arthrobacter nicotinianae, Ochrobactum antrrophi, Achromobacter obae.

8. The method according to claim 3, wherein the enzymes E1 and E2 are genetically engineered bacteria whole cells, broken enzyme solution, freeze-dried powder, immobilized enzymes or immobilized cells.

9. The process according to claim 3, wherein the enzyme E1 is a polypeptide having the amino acid sequence of SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4. or SEQ ID NO: 9, or the protein shown in SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4. or SEQ ID NO: 9 is a protein having the activity of hydrolyzing the compound of formula V to a compound of formula I after substitution, deletion or addition of one or more amino acid residues, or is a protein of SEQ id no: 1. SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4. or SEQ ID NO: 9 has more than 80% homology and is a protein having the activity of hydrolyzing the compound of the formula V into the compound of the formula I.

10. The process according to claim 3, wherein the enzyme E2 is a polypeptide having the amino acid sequence of SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO: 7. SEQ ID NO: 8 or SEQ ID NO: 10, or a protein represented by seq id NO: 5. SEQ ID NO: 6. SEQ ID NO: 7. SEQ ID NO: 8 or SEQ ID NO: 10, or a protein having activity of racemizing the compound of formula VI after substitution, deletion or addition of one or more amino acid residues, or a protein having activity of racemizing the compound of formula VI with SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO: 7. SEQ ID NO: 8 or SEQ ID NO: 10 and a protein having 80% or more homology of the amino acid sequence shown in the formula VI and having racemization activity to the compound of the formula VI.

Technical Field

The invention relates to a preparation method of a pesticide, in particular to a preparation method of L-glufosinate-ammonium.

Background

Glufosinate, chemical name 4- [ hydroxy (methyl) phosphono ] -DL-homoalanine, developed and produced by hester (bayer, germany) in the last 80 th century, is a phosphonic acid herbicide, is a glutamine synthesis inhibitor, a non-selective contact herbicide, which was registered for use as a herbicide in 1984. Glufosinate technical material registration is only available in 2004 and product registration is only available in 2005.

Since the wide application of glyphosate, glyphosate-resistant weeds are increasing and the harm is gradually aggravated. Paraquat is a strong weed-killing herbicide and has strong toxic effect on human and livestock. 7, 1 month in 2014, China cancels paraquat water registration and production permission and stops production; the water aqua is sold and used in China after 2016, 7 months and 1 day. Glufosinate is a world wide large tonnage pesticide variety and is also a herbicide tolerant for the second largest transgenic crop in the world. The glufosinate-ammonium has low toxicity, is relatively safe, is easy to degrade in soil, is safe to crops, is not easy to drift, has a wide weeding spectrum, is high in activity, small in dosage, small in environmental pressure, can quickly kill more than 100 gramineous weeds and broadleaf weeds, can use water as a base agent, is safe and convenient to use, and has the characteristic that the product is superior to other herbicides, so that the glufosinate-ammonium can still be sold after a plurality of high-efficiency and super-high-efficiency products appear.

At present, the synthetic technical routes of glufosinate-ammonium at home and abroad are more, and the review reports are made on Yanghai Chang et al (pesticides, 2002, 41(9), 46-48), but the problems of more reaction steps and high production cost exist in each route generally. Bayer, U.S. Pat. Nos. 4,193,521 and 6359162, disclose a process for synthesizing glufosinate-ammonium by a series of reactions from dichloromethylphosphine and methyl phosphite. The method has high yield and low cost, but the starting route has active physicochemical properties of the raw materials and products for synthesizing the dichloromethylphosphine, and is flammable and explosive. The synthesis process is controlled at 500-600 ℃, and if the control is unstable, yellow phosphorus and phosphine which are very easy to be natural are easily generated, so that the danger is very high. In addition, the materials have strong corrosivity and have strict requirements on material selection of reaction equipment and processing and manufacturing processes of the reaction equipment, and the current domestic processing and manufacturing level is difficult to meet the production requirements.

These methods are all methods for preparing glufosinate-ammonium, which is an L/D mixed type, wherein L-type is mainly used; l-glufosinate-ammonium can be degraded by microorganisms in soil, while D-glufosinate-ammonium is difficult to degrade, and finally soil hardening can be caused. Therefore, the L-glufosinate-ammonium is more efficient, lower in cost and safer than the traditional glufosinate-ammonium.

Therefore, it is necessary to develop a biosynthetic preparation method of L-glufosinate-ammonium, which can improve the utilization rate of raw materials, reduce the production cost and avoid the danger in the industrial process.

Disclosure of Invention

The invention aims to overcome the defects of the background technology and provide a preparation method of L-glufosinate-ammonium, which can improve the utilization rate of raw materials and reduce the production cost and is suitable for industrial production.

The invention provides a preparation method of a compound shown in a formula I, which is obtained by reacting a compound shown in a formula V, wherein the reaction formula is as follows:

R₂selected from amino OR-OR₀；

R₀Selected from the group consisting of hydrogen, hydrocarbyl, silyl, benzyl, alkyl methyl, alkyl-substituted alkoxy, 3-8 membered cycloaliphatic, aryl, heteroaryl, substituted aryl, substituted heteroaryl, Ar (CH)₂)_nOne of O-groups, Ar represents aryl or heteroaryl, and n is 1-6;

R₁selected from the group consisting of alkyl, aryl, heteroaryl, substituted alkyl, substituted aryl, substituted heteroaryl.

Further, the preparation method of the L-glufosinate-ammonium comprises the step of preparing a compound shown in a formula II by reacting a compound shown in a formula IV, wherein the compound shown in the formula II is obtained by removing protecting groups,

wherein R is₃Selected from the group consisting of hydrogen, alkyl, silyl ether protecting groups, benzyl ether protecting groups, alkoxymethyl, alkoxy-substituted methyl, C2-C10 linear or branched alkenylalkyl, C2-C10 linear or branched alkynylalkyl, 3-8 membered cycloaliphatic, aryl, heteroaryl, amino, substituted amino, Ar (CH)₂)_a-one of the groups, Ar represents an aryl, heteroaryl group, a takes 1 to 6;

further, the compound of formula V is reacted with an enzyme E1 and an enzyme E2 to produce a compound of formula I, wherein the reaction formula is as follows:

said E1 is an enzyme capable of hydrolyzing a compound of formula V to a compound of formula I;

said E2 is an enzyme capable of racemizing a compound of formula VI;

coenzyme is needed to be added in the reaction process.

Further, the preparation method of the L-glufosinate-ammonium comprises the steps of reacting the compound shown in the formula IV under the action of enzyme E1 and enzyme E2 to prepare a compound shown in the formula II, deprotecting the group to obtain the L-glufosinate-ammonium,

further, in the preparation method of the L-glufosinate-ammonium, R3 is hydrogen, R2 is ethyl or propyl or isopropyl, the pH of the reaction system is controlled to be 5-8.5, and the coenzyme is pyridoxal phosphate.

Further, the enzyme E1 gene source includes Streptomyces ramulosus, Pseudomonas azotoformans, Mycobacterium tuberculosis, pork liver, Candida antartica, Pseudomonas wadanswerensis, Pseudomonas sp, Brevundimonas diminuta.

Further, in the preparation method of L-glufosinate-ammonium, the enzyme E2 gene source includes Bacillus, tryptanosomatruczi, Ocnococcus oeni, Amycolatopsis orientalis, Streptomyces lavendale, Arthrobacter nicotinianae, Ochrobactum antrrophi, Achromobacter obae.

Further, in the preparation method of the L-glufosinate-ammonium, the enzyme E1 and the enzyme E2 are whole cells, broken enzyme liquid, freeze-dried powder or immobilized enzyme or immobilized cells of the genetically engineered bacteria.

Further, in the preparation method of the L-glufosinate-ammonium, the enzyme E1 has an amino acid sequence of SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4. or SEQ ID NO: 9, or a protein of SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4. or SEQ ID NO: 9 is a protein having the activity of hydrolyzing the compound of formula V to the compound of formula I after substitution, deletion or addition of one or more amino acid residues, or is SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4. or SEQ ID NO: 9 has more than 80% homology and is a protein having the activity of hydrolyzing the compound of the formula V into the compound of the formula I.

Further, in the preparation method of the L-glufosinate-ammonium, the enzyme E2 has an amino acid sequence of SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO: 7. SEQ ID NO: 8 or SEQ ID NO: 10, or a protein of SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO: 7. SEQ ID NO: 8 or SEQ ID NO: 10, or a protein having activity of racemizing the compound of formula VI after substitution, deletion or addition of one or more amino acid residues, or a protein having activity of racemizing the compound of formula VI with SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO: 7. SEQ ID NO: 8 or SEQ ID NO: 10 and a protein having 80% or more homology of the amino acid sequence shown in the formula VI and having racemization activity to the compound of the formula VI.

The advantages of the invention are mainly embodied in the following aspects:

firstly, the process flow is simple, no special requirements are required on equipment, and the method is suitable for industrial production;

secondly, the invention has high utilization rate of the substrate, mild reaction condition and environmental protection;

thirdly, the preparation process of the invention is an upgrade and update of the traditional chemical synthesis method, thus greatly reducing the cost.

Detailed Description

The present invention is described in further detail below with reference to specific examples, which should not be construed as limiting the invention.