Process for preparing L-glufosinate from cyanohydrin or cyanohydrin derivatives

Abstract

L-グルホシネート及び / 若しくはその塩又はL-グルホシネートアルキルエステル及び / 若しくはその塩を調製するプロセスであって、L-グルホシネート又はL-グルホシネートアルキルエステルは、式(I):【化1】TIFF2026510249000026.tif18170(式中、R1は、H又はC1~C8アルキルである)による分子構造を有し、そのプロセスは、以下の構成要素:(1)式(II)【化2】TIFF2026510249000027.tif16170(式中、R1は、H又はC1~C8アルキルであり、及びR2は、H、C1~C8アルキル、C6~C10アリール、C7~C10アラルキル、C4~C10シクロアルキル、又はC1~C10アシルである)によるシアンヒドリン又はシアンヒドリン誘導体、(2)アンモニアの供給源、(3)二酸化炭素の供給源、及び(4)少なくとも2種の酵素を少なくとも1つの反応ステップで反応させるステップを含む、プロセス。

Description

[Technical Field]

[0001] The present invention relates to the preparation of L-glufosinate, and more particularly to the preparation of L-glufosinate from cyanohydrin or cyanohydrin derivatives. [Background technology]

[0002] U.S. Patent No. 4,168,963 describes various phosphorus-containing herbicidal compounds, of which phosphinothricin (2-amino-4-[hydroxy(methyl)phosphinoyl]-butanoic acid: glufosinate) or its salts have become commercially important in the field of agricultural chemistry. This herbicide, glufosinate, is a non-selective foliar herbicide that is considered one of the safest herbicides from a toxicological or environmental perspective.

[0003] Methods for preparing intermediates for the synthesis of such phosphorus-containing herbicidal compounds, particularly glufosinates, are described, for example, in U.S. Patent Nos. 4,521,348, 4,599,207, and 6,359,162.

[0004] International Publication No. 2015 / 173146A1 describes the preparation of glufosinate starting from n-butyl(3-cyano-3-hydroxypropyl)methylphosphinat (ACM-H). Similarly, International Publication No. 2017 / 037012A1 describes the preparation of glufosinate starting from n-butyl(3-cyano-3-acetoxypropyl)methylphosphinat (ACM). [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] U.S. Patent No. 4,168,963 [Patent Document 2] U.S. Patent No. 4,521,348 [Patent Document 3] U.S. Patent No. 4,599,207 [Patent Document 4] U.S. Patent No. 6,359,162 [Patent Document 5] International Publication No. 2015 / 173146 A1 [Patent Document 6] International Publication No. 2017 / 037012 A1 [Summary of the Invention] [Problems to be Solved by the Invention]

[0006] Cyanohydrins containing phosphorus are useful intermediates for the production of biologically active substances that can be used in various fields, particularly in the pharmaceutical or agrochemical sectors. ACM and ACM-H are readily available from bulk chemicals and known intermediates in the synthesis of racemic glufosinate. However, there is no existing technology for directly synthesizing L-glufosinate from these intermediates. Therefore, the above process also has the disadvantage that the glufosinate produced therefrom does not exhibit any enantiomeric excess, particularly with respect to L-glufosinate.

[0007] Other current commercial chemical synthesis methods of glufosinate also only yield a racemic mixture of L-glufosinate and D-glufosinate (Duke et al. 2010 Toxins 2:1943 - 1962). However, from the perspective of herbicidal effects, L-glufosinate is known to be more potent than D-glufosinate (Ruhland et al. (2002) Environ. Biosafety Res. 1:29 - 37).

[0008] Therefore, considering the prior art listed above, an object of the present invention is to provide a mild process for preparing L-glufosinate.

[0009] A further object of the present invention is to provide a safe process for preparing L-glufosinate.

[0010] Furthermore, an object of the present invention is to provide a process for preparing L-glufosinate in an enantiomer excess.

[0011] Finally, an object of the present invention is to provide a process for preparing L-glufosinate from readily available raw materials. [Means for solving the problem]

[0012] Surprisingly, it has been found that at least one of the above objectives can be achieved by the process described herein.

[0013] Accordingly, in a first aspect, the present invention relates to a process for preparing L-glufosinate and / or a salt thereof or L-glufosinate alkyl ester and / or a salt thereof, wherein L-glufosinate or L-glufosinate alkyl ester is of formula (I): [ka] (In the formula, R 1 (It is H or C1-C8 alkyl) It has a molecular structure due to, The process consists of the following components: (1) Formula (II) [ka] (In the formula, R 1 is H or C1-C8 alkyl, and R 2 H, C1-C8 alkyl, C6-C 10 Aryl, C7~C 10 Aralkil, C4~C 10 Cycloalkyl, or C1-C 10 (It is an asyl) Cyanide or cyanohydrin derivatives, (2) Sources of ammonia, (3) Sources of carbon dioxide, and (4) At least two enzymes The present invention relates to a process that includes the step of reacting in at least one reaction step.

[0014] Preferred embodiments of the components of the preparation process are described in more detail below. Naturally, each preferred embodiment is suitable on its own or in combination with other preferred embodiments.

[0015] In a first preferred embodiment A1 of the first aspect of the present invention, component (4) comprises, preferably, (4a) at least one amide hydrolase (EC3.5.2) that acts on a cyclic amide, and (4b) at least one L-amide hydrolase (EC3.5.1) that acts on a linear amide.

[0016] In a second preferred embodiment A2 of the first aspect of the present invention, components (1), (2) and (3) are first brought into contact, then component (4) is added, preferably component (4a) first and component (4b) last.

[0017] In a third preferred embodiment A3 of the first aspect of the present invention, components (1) to (4) are added in the same reaction step, and preferably the reaction is carried out as a one-pot reaction.

[0018] In a fourth preferred embodiment A4 of the first aspect of the present invention, cyanohydrin is prepared by the reaction of an aldehyde with a cyanide, preferably hydrogen cyanide or potassium cyanide, wherein the aldehyde is of formula (III): [ka] (In the formula, R 1has a molecular structure by (being H or C1-C8 alkyl, preferably C1-C6 alkyl or H, more preferably C2-C4 alkyl or H, even more preferably ethyl or butyl or H, and most preferably ethyl).

[0019] In the fifth preferred embodiment A5 of the first aspect of the present invention, R in the formula (I), (II), and / or (III) 1 is H or C1-C8 alkyl, preferably H or C1-C6 alkyl, more preferably H or C2-C4 alkyl, even more preferably H, ethyl or butyl, and most preferably ethyl.

[0020] In the sixth preferred embodiment A6 of the first aspect of the present invention, the source of ammonia is selected from the list consisting of gaseous ammonia, dissolved ammonia, ammonium salts, or mixtures thereof.

[0021] In the seventh preferred embodiment A7 of the first aspect of the present invention, the source of carbon dioxide is gaseous carbon dioxide, dissolved carbon dioxide, carbonate, or mixtures thereof.

[0022] In the eighth preferred embodiment A8 of the first aspect of the present invention, the cyanohydrin or cyanohydrin derivative is of the formula (IV):

Chemical formula

[0023] In the ninth preferred embodiment A9 of the first aspect of the present invention, the amidohydrolase (EC3.5.2) acting on the cyclic amide is L-amidohydrolase (EC3.5.2) acting on the cyclic amide.

[0024] In the second aspect of the present invention, the formula (II) [ka] (In the formula, R 1 is H or C1-C8 alkyl, and R 2 H, C1-C8 alkyl, C6-C 10 Aryl, C7~C 10 Aralkil, C4~C 10 Cycloalkyl, or C1-C 10 (It is ashil) Cyanide or cyanohydrin derivatives, The present invention further relates to compositions comprising L-glufosinate and / or salts thereof.

[0025] In a third embodiment, the present invention provides a method for selectively controlling weeds in a region, preferably a region containing planted seed crops or glufosinate-resistant crops, The L-glufosinate and / or salt thereof obtained by the process of the present invention is included with D-glufosinate and / or salt thereof in a proportion of at least 50% enantiomers, preferably in an enantiomer excess of more than 70%, and is present in a proportion of more than 0.01 wt.-% and less than 10 wt.-% of formula (II) based on the total amount of the composition. [ka] (In the formula, R 1 is H or C1-C8 alkyl, and R 2 H, C1-C8 alkyl, C6-C 10 Aryl, C7~C 10 Aralkil, C4~C 10 Cycloalkyl, or C1-C 10 (It is an asyl) The present invention further relates to a method comprising applying an effective amount of a composition containing cyanohydrin or a cyanohydrin derivative to a region. [Modes for carrying out the invention]

[0026] Before describing exemplary embodiments of the present invention in detail, important definitions for understanding the invention are given.

[0027] Where used herein and in the appended claims, the singular forms “a” and “an” also include their respective plural forms unless the context explicitly indicates otherwise. In relation to the present invention, the terms “about” and “approximately” represent a range of accuracy that a person skilled in the art would understand to still ensure the technical effect of the feature. This term typically represents a deviation of ±20%, preferably ±15%, more preferably ±10%, and even more preferably ±5% from the given number. Naturally, the term “including” is not limiting. For the purposes of the present invention, the term “consisting of” is considered a preferred embodiment of the term “comprising of.” Hereinafter, where a group is defined as including at least a certain number of embodiments, it preferably also includes a group consisting only of these embodiments. Furthermore, terms such as “First,” “Second,” “Third,” or “(a),” “(b),” “(c),” “(d),” and similar terms in this specification and the claims are used to distinguish between similar elements and do not necessarily describe an order or chronological sequence. Naturally, terms used in this manner are interchangeable in appropriate contexts, and embodiments of the invention described herein may be performed in an order other than those described or explained herein. Where terms such as “First,” “Second,” “Third,” or “(a),” “(b),” “(c),” “(d),” “i,” “ii,” etc., relate to a method or use or steps of an assay, there is no consistency in time or time intervals between steps. That is, unless otherwise specified in this application as described herein or prior to, those steps may be performed simultaneously, or there may be time intervals of a few seconds, a few minutes, a few hours, a few days, a few weeks, a few months, or even several years between such steps. Naturally, the invention is not limited to the specific methods, protocols, reagents, etc. described herein, as they may vary. Naturally, the terms used herein are for the purpose of describing specific embodiments only and do not limit the scope of the invention, which is limited solely by the appended claims.Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art.

[0028] As used throughout this specification, the term "wt.-%" means "percent by weight".

[0029] As used herein, the term "alkyl" refers to a linear or branched alkyl group having typically 1 to 20 carbon atoms, preferably 1 to 8 carbon atoms, frequently 1 to 6 carbon atoms, more preferably 1 to 4 carbon atoms, for example, 2 or 4 carbon atoms. Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, 2-butyl, isobutyl, tert-butyl, n-pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, 2,2-dimethylpropyl, 1-ethylpropyl, and n-hexyl.

[0030] Depending on the substitution pattern, the compounds according to the present invention may have one or more stereocenters. Unless otherwise expressly indicated (for example by chemical formula), the present invention preferably encompasses all stereoisomers of the compounds according to the present invention, i.e., pure enantiomers, pure diastereomers, and mixtures thereof, including racemic mixtures.

[0031] Preferred embodiments relating to the process for preparing L-glufosinate and / or its salts or L-glufosinate alkyl esters and / or its salts are described in detail below. Naturally, the preferred embodiments of the present invention are preferred individually or in combination with each other.

[0032] As described above, the present invention, in one aspect, is a process for preparing L-glufosinate and / or a salt thereof or L-glufosinate alkyl ester and / or a salt thereof, wherein L-glufosinate or L-glufosinate alkyl ester is of formula (I): [ka] (In the formula, R 1 (It is H or C1-C8 alkyl) It has a molecular structure due to, The process consists of the following components: (1) Formula (II) [ka] (In the formula, R 1 is H or C1-C8 alkyl, and R 2 H, C1-C8 alkyl, C6-C 10 Aryl, C7~C 10 Aralkil, C4~C 10 Cycloalkyl, or C1-C 10 (It is an asyl) Cyanide or cyanohydrin derivatives, (2) Sources of ammonia, (3) Sources of carbon dioxide, and (4) At least two enzymes The present invention relates to a process that includes the step of reacting in at least one reaction step.

[0033] Preferably, component (4) comprises (4a) at least one amide hydrolase (EC3.5.2) that acts on cyclic amides, and (4b) at least one L-amide hydrolase (EC3.5.1) that acts on linear amides, and preferably consists of these.

[0034] Naturally, the preparation of L-glufosinate and / or its salts or L-glufosinate alkyl esters and / or their salts encompasses all stereoisomers and suitable salts of L-glufosinate or its alkyl esters. Preferably, the alkyl ester of L-glufosinate means the alkyl-P ester of L-glufosinate. Furthermore, each zwitterion is encompassed in formula (I). Suitable salts are, exemplary, hydrochloride salts, ammonium salts, and isopropylammonium salts. In this regard, the compounds of formula (I) particularly encompass two stereocenters, one located at the phosphorus atom and the other at the alpha carbon atom. The compounds of formula (I) particularly encompass all stereoisomers derived from the stereocenter at the phosphorus atom.

[0035] Cyanide or cyanohydrin derivatives can be obtained by any suitable preparation process. Suitable processes are described, in particular, in U.S. Patent No. 4,521,348B1, German Patent No. 3047024, U.S. Patent No. 4,599,207B1, U.S. Patent No. 6,359,162B1, Chinese Patent Application Publication No. 102372739A, and Chinese Patent Application Publication No. 102399240A.

[0036] In a preferred embodiment, cyanohydrin is prepared by the reaction of an aldehyde with a cyanide, preferably hydrogen cyanide or potassium cyanide, wherein the aldehyde is of formula (III): [ka] (In the formula, R 1 (The compound is H or C1-C8 alkyl, preferably H or C1-C6 alkyl, more preferably H or C2-C4 alkyl, even more preferably H, ethyl or butyl, most preferably ethyl.) It has a molecular structure due to [the following].

[0037] Such a reaction has the advantage that cyanhydrin is prepared in situ and its solution can be used directly to carry out the process of the present invention.

[0038] Preferably, R of formula (II) and / or (III) 1 The is H or C1-C8 alkyl. Therefore, preferably, the cyanohydrin or cyanohydrin derivative is a precursor of L-glufosinate, or a C1-C8 protected alkyl ester of L-glufosinate, preferably a C1-C8 protected alkyl-P ester of L-glufosinate.

[0039] Similarly, preferably, R of formula (II) 2 H, C1-C8 alkyl, C6-C 10 Aryl, C7~C 10 Aralkil, C4~C 10 Cycloalkyl, or C1-C 10 It is an acyl. More preferably, R of formula (II) 2 H or C1~C 10 It is an acyl, most preferably R of formula (II). 2 is either H or acetyl.

[0040] Therefore, in the first most preferred embodiment of the present invention, the cyanohydrin is n-butyl(3-cyano-3-hydroxypropyl)methylphosphinat (ACM-H).

[0041] Preferably, the cyanohydrin derivative is of formula (IV): [ka] (In the formula, R 3 (The alkyl group is C1-C8 alkyl, preferably C1-C4 alkyl, more preferably C1-C3 alkyl, and most preferably methyl.) It is a cyanohydrin derivative.

[0042] Therefore, in the second most preferred embodiment of the present invention, the cyanohydrin derivative is n-butyl(3-cyano-3-acetoxypropyl)methylphosphinat (ACM).

[0043] This process can be carried out in several steps. Preferably, when the process is carried out in several steps, components (1) to (3) are added first, followed by component (4), preferably component (4a) first and finally component (4b). When the process is carried out in more than one step, it is preferably carried out in two steps, the first step involving the reaction of components (1) to (3), and the second step involving the addition of components (4), preferably (4a) and (4b). In another preferred embodiment, the process is carried out in one step, i.e., it is a one-pot process.

[0044] If this process is carried out in two steps, the first step is preferably carried out at a higher temperature than the second step. Preferably, the first step is carried out at a temperature in the range of 40 to 100°C, more preferably 50 to 90°C, and most preferably 75 to 85°C. Surprisingly, it has been found that the higher the temperature in the first step, the higher the overall conversion rate. Preferably, the first step is carried out for at least half an hour, more preferably at least one hour. Typically, the first step is completed after one hour.

[0045] If the process is carried out in two steps, the second step is preferably carried out at a temperature suitable for the enzymes of component (4), preferably (4a) and (4b), where suitable means, firstly, that the enzymes are stable within this temperature range, and secondly, that the reaction catalyzed by these enzymes preferably occurs with an optimal conversion rate. Preferably, the temperature of the second step is as applied to the one-pot reaction described below.

[0046] If this process is carried out in one step, i.e., a one-pot process, it is carried out at a temperature in the range of 20 to 50°C, preferably 25 to 45°C, more preferably 30 to 42°C, and most preferably 32 to 40°C.

[0047] In preferred embodiments of the present invention, the reaction is carried out at a pH of 6 to 11, preferably 6.5 to 10, more preferably 7 to 9.5, and particularly 7.5 to 9. The pH can be adjusted using an alkali hydroxide, more preferably sodium hydroxide or potassium hydroxide, particularly potassium hydroxide. However, most preferably, the pH is adjusted by a source of ammonia and / or a source of carbon dioxide. In particular, the pH can be controlled by the amount of ammonium salt or carbonate added to the solution, preferably ammonium carbonate or ammonium bicarbonate, or by the amount of gaseous ammonia and / or carbon dioxide introduced through the solution, or both simultaneously.

[0048] In preferred embodiments of the present invention, the reaction is carried out under aqueous conditions, preferably in a degassed aqueous phosphate buffer, more preferably in a degassed aqueous potassium phosphate buffer. However, the buffering effect of the solution can also be adjusted by a source of ammonia and / or a source of carbon dioxide. In particular, the pH can be controlled by the amount of ammonium salt or carbonate added to the solution, preferably ammonium carbonate or ammonium bicarbonate, or by the amount of gaseous ammonia and / or carbon dioxide introduced through the solution, or both simultaneously.

[0049] In a preferred embodiment of the present invention, the process is carried out under stirring, preferably at 50 to 1000 rpm, more preferably at 100 to 800 rpm, even more preferably at 150 to 600 rpm, even more preferably at 180 to 400 rpm, and most preferably at 200 to 300 rpm.

[0050] Preferably, in the process of the present invention, any suitable amide hydrolase (EC3.5.2) that acts on a cyclic amide can be used.

[0051] Examples of amide hydrolases (EC3.5.2) that act on such cyclic amides and can be used in the process of the present invention include: Defluviimonas alba, Rhodococcus erythropolis, Streptomyces coelicolor, Brevibacillus agri, Paenarthrobacter aurescens, Arthrobacter crystallopoietes, Bacillus sp. TS-23, Bacillus fordii, Jannaschia sp., Pseudomonas putida, and Geobacillus stearothermophilus. Examples include *Stearothermophilus*, *Thermus* sp., *Dictyostelium discoideum*, *Rhizobium meliloti*, *Pseudomonas aeruginosa*, *Rhizobium radiobacter*, *Pseudomonas fluorescens*, soybean (*Glycine max*), black locust (*Robinia pseudoacacia*), *Bacillus licheniformis*, *Aedes aegypti*, *Agrobacterium fabrum*, and *Arthrobacter* sp., preferably derived from *Defluviimonas alba*.

[0052] Suitable amide hydrolases (EC3.5.2) that act on cyclic amides include Q8RSQ2 and its variants, O69809 and its variants, Q846U5_9BACL and its variants, P81006 and its variants, Q84FR6_9MICC and its variants, Q56S49_9BACI and its variants, A1E351_9BAC and its variants, Q28SA7 and its variants, Q59699 and its variants, Q45515 and its variants, A0A399DRQ3_9DEIN and its variants, Q55DL0 and its variants, F7X5M8_SINMM and so Variants of Q9I676 and its variants, Q44184 and its variants, B5L363 and its variants, I1MEH3 and its variants, Q6S4R9 and its variants, Q65LN0 and its variants, Q171F8 and its variants, Q8U8Z6 and its variants, P42084 and its variants, Q88NW7 and its variants, P25995 and its variants, Q3Z354 and its variants, B1XEG2 and its variants, Q9F465_PAEAU and its variants, Q01262.1 and its variants, A0A250DXG4_GEOSE and its variants, A1 SPN2 and its variants, Q9WYH0 and its variants, P58329 and its variants, A1SGT4 and its variants, E3JD18 and its variants, HUTI_BDEBA and its variants, A0A161KD37_9CHLR and its variants, I0GL27_CALEA and its variants, A0A068WGW0_ECHGR and its variants, A0A1J4XHR4_9BACT and its variants, A0A1C4QIY5_9ACTN and its variants, A0A0K2UMP4_LEPSM and its variants, A0A0F5Q0A2_9RHIZ and its variants , A0A024KHS5_9RHIZ and its variants, A0A060UM69_9PROT and its variants, A3DKS9_STAMF and its variants, W2EWT0_9ACTN and its variants, A0A0B1T9I4_OESDE and its variants, A0A0A7LM60_9BACT and its variants, A0A087M7T5_9RHIZ and its variants, C0C180_9FIRM and its variants, A0A159Z531_9RHOB and its variants, R5JTP2_9CLOT and its variants, A0A010RM85_9PEZI and its variants,E1R8C9_SEDSS and its variants, A0A010YEH8_9BACT and its variants, A0A031LV69_9CREN and its variants, A0A1F9QT17_9BACT and its variants, ALLB_BACVZ and its variants, HUTI_FLAPJ and its variants, A0A073J5J1_9BACT and its variants, A0A034W2Q8_BACDO and its variants, A0A0D8IVV8_9FIRM and its variants, A0A0B5QKE4_CLOBE and its variants, A0A098B7X6_DESHA and its variants , A0A0B5H4M8_9EURY and its variants, A0A0C1YDP1_9ACTN and its variants, Q981H2_RHILO and its variants, T1EEH7_HELRO and its variants, A0A060DTG8_AZOBR and its variants, A0A011MGZ5_MANHA and its variants, A0A060LYB6_9BACI and its variants, S0F3L7_CHOCR and its variants, A0A133VNR0_9EURY and its variants, A0A133U7U9_9EURY and its variants, A0A0U2XD52_ECOLX and The variants M1YZY6_NITG3 and its variants, T0N9X6_9EURY and its variants, T0LMU2_9EURY and its variants, A0A0N1GBZ8_9ACTN and its variants, HUTI_ANASK and its variants, A0A031JUP0_9SPHN and its variants, A0A061N9L2_9BACL and its variants, A0A017T4D2_9DELT and its variants, A0A174ADZ3_9FIRM and its variants, A0A021X7D5_9RHIZ and its variants, A0A021XAC5_9RHIZ and so The variants of A0A0C2UIW0_9BACL and its variants, A0A1F8NGY1_9CHLR and its variants, D3F1S3_CONWI and its variants, A0A021XG06_9RHIZ and its variants, U7V9Q6_9FUSO and its variants, D6XY37_BACIE and its variants, A0A0J1FAI4_9FIRM and its variants, B5Y9A6_COPPD and its variants, PHYDA_ECOK1 and its variants, A0A0A9X9B7_LYGHE and its variants, A0A0S8H576_9BACT and its variants,A0A151ABI4_9EURY and its variants, A0A064AFD7_9FUSO and its variants, A0A0C2FCG7_9ACTN and its variants, A0A0S8CI48_9CHLR and its variants, A0A1F9CZ74_9DELT and its variants, A0A0A3YKD1_9ENTR and its variants, A0A084R4T2_STACH and its variants, A0A070A1Z0_9PROT and its variants, A0A1J4J4Y8_9EUKA and its variants, R1BR72_EMIHU and its variants, R1DD72_EMI HU and its variants, A0A1L0FIA0_9ASCO and its variants, F7DRE9_ORNAN and its variants, A0LK75_SYNFM and its variants, A0A0Q1A918_9BACT and its variants, H2YZ10_CIOSA and its variants, I4YD99_WALMC and its variants, A0A077YYH5_TRITR and its variants, A0A077Y189_9SPHI and its variants, A0A089K5P4_9BACL and its variants, A0A0Q7W2T1_9RHIZ and its variants, A0A174NIK6_9 FIRM and its variants, A0A0D5NFS5_9BACL and its variants, A0A0D5NNJ7_9BACL and its variants, A0A1H2AV66_9BACL and its variants, A0A0Q4RXY0_9BACL and its variants, A0A0Q7SB75_9BACL and its variants, A0A015NM92_9BACL and its variants, A0A100VRN2_PAEAM and its variants, W4BDJ0_9BACL and its variants, A0A147K2G0_9EURY and its variants, A0A0W8FVM4_9ZZZZ and its variants Body, A0A147JXR0_9EURY and its variants, E8R8J7_DESM0 and its variants, D5U113_THEAM and its variants, A0A1F8T9J2_9CHLR and its variants, G3C952_9ARCH and its variants, Q6YNI0_9MICC and its variants, A0A1G0YIQ9_9BACT and its variants, A0A1J5EHQ6_9DELT and its variants, A0A1J5E082_9DELT and its variants, A0A1C4PKD1_9ACTN and its variants, H8GX25_DEIGI and its variants,A0A1H5ZFN3_9BACT and its variants, A0A0M9Z5S1_9ACTN and its variants, A0A1B2HNC5_9PSEU and its variants, A0A1B2GNI8_STRNR and its variants, A0A1F8LBZ3_9CHLR and its variants, A0A1F8NMM2_9CHLR and its variants, A0A1F8SDV1_9CHLR and its variants, A0A1H1PLX0_9BAC T and its variants, I0IDC5_PHYMF and its variants, A0A0Q5I8X4_9DEIO and its variants, A0A0F4JEH6_9ACTN and its variants, BAD75708.1, *WP_014453859.1 and its variants, *WP_046170519.1 and its variants, *CDP53201.1 and its variants, *WP_035078314.1 and its variants, *WP_042 The following can be selected from the group consisting of 803791.1 and its variants, *EQB70510.1 and its variants, *EQB65904.1 and its variants, *WP_023512514.1 and its variants, *WP_023514195.1 and its variants, *WP_023516147.1 and its variants, *KGT87257.1 and its variants, *WP_045756097.1 and its variants, *WP_056239694.1 and its variants, *KUO41395.1 and its variants, *KOV34818.1 and its variants, *ANZ15483.1 and its variants, *KJY32595.1 and its variants, and mixtures thereof. A variant is defined as a polypeptide sequence having at least 80%, preferably 90%, and most preferably 95% sequence identity with respect to its respective polypeptide sequence.

[0053] More preferably, the amide hydrolase (EC3.5.2) acting on cyclic amides is O69809 and its variants, Q846U5_9BACL and its variants, P81006 and its variants, Q84FR6_9MICC and its variants, Q56S49_9BACI and its variants, A1E351_9BACI and its variants, Q28SA7 and its variants, Q45515 and its variants, A0A399DRQ3_9DEIN and its variants, Q55DL0 and its variants, F7X5M8_SINMM and its variants, Q9I676 and its variants Body, Q44184 and its variants, B5L363 and its variants, P42084 and its variants, P25995 and its variants, Q3Z354 and its variants, B1XEG2 and its variants, Q9F465_PAEAU and its variants, A0A161KD37_9CHLR and its variants, A0A1J4XHR4_9BACT and its variants, A0A1C4QIY5_9ACTN and its variants, A0A0K2UMP4_LEPSM and its variants, A0A159Z531_9RHOB and its variants, E1R8C9_SEDSS and its variants, A0A1F9QT17_9BACT and its variants, A0A0D8IVV8_9FIRM and its variants, A0A0B5QKE4_CLOBE and its variants, A0A0N1GBZ8_9ACTN and its variants, A0A174ADZ3_9FIRM and its variants, U7V9Q6_9FUSO and its variants, A0A0J1FAI4_9FIRM and its variants, PHYDA_ECOK1 and its variants, A0A0S8H576_9BACT and its variants, A0A1J4J4Y8_9EUKA and its variants, A0A0D5NFS5_9BA CL and its variants, A0A0D5NNJ7_9BACL and its variants, A0A1H2AV66_9BACL and its variants, A0A0Q4RXY0_9BACL and its variants, A0A0Q7SB75_9BACL and its variants, A0A100VRN2_PAEAM and its variants, W4BDJ0_9BACL and its variants, A0A1J5E082_9DELT and its variants, A0A1H5ZFN3_9BACT and its variants, A0A1F8NMM2_9CHLR and its variants, A0A1F8SDV1_9CHLR and its variants,A mutant is defined as a polypeptide sequence having at least 80%, preferably 90%, and most preferably 95% sequence identity with respect to its respective polypeptide sequence. The mutant is selected from the group consisting of A0A1H1PLX0_9BACT and its variants, A0A0Q5I8X4_9DEIO and its variants, *WP_046170519.1 and its variants, *WP_023514195.1 and its variants, *WP_023516147.1 and its variants, and *ANZ15483.1, as well as mixtures thereof.

[0054] Furthermore, suitable amide hydrolases (EC3.5.2) that act on cyclic amides include Q846U5_9BACL and its variants, P81006 and its variants, Q84FR6_9MICC and its variants, Q56S49_9BACI and its variants, Q45515 and its variants, A0A399DRQ3_9DEIN and its variants, Q55DL0 and its variants, F7X5M8_SINMM and its variants, Q9I676 and its variants, Q44184 and its variants, B1XEG2 and its variants, A0A161KD37_9CHLR and its variants, and A0A15 The group can be selected from 9Z531_9RHOB and its variants, E1R8C9_SEDSS and its variants, A0A1F9QT17_9BACT and its variants, A0A0B5QKE4_CLOBE and its variants, A0A0N1GBZ8_9ACTN and its variants, BAD75708.1 and its variants, A0A064AFD7_9FUSO and its variants, and mixtures thereof. A variant is defined as a polypeptide sequence having at least 80%, preferably 90%, and most preferably 95% sequence identity with respect to its respective polypeptide sequence.

[0055] In preferred embodiments, the amide hydrolase (EC3.5.2) acting on cyclic amides is Q846U5_9BACL and its variants, P81006 and its variants, Q84FR6_9MICC and its variants, A0A399DRQ3_9DEIN and its variants, B1XEG2 and its variants, A0A161KD37_9CHLR and its variants, A0A159Z531_9RHOB and its variants, E1R8C9_SEDSS and its variants, A A mutant is selected from the group consisting of 0A1F9QT17_9BACT and its variants, A0A0B5QKE4_CLOBE and its variants, A0A0N1GBZ8_9ACTN and its variants, BAD75708.1 and its variants, A0A064AFD7_9FUSO, and mixtures thereof, and the mutant is defined as a polypeptide sequence having at least 80%, preferably 90%, and most preferably 95% sequence identity with respect to the respective polypeptide sequence.

[0056] Most preferably, the amide hydrolase (EC3.5.2) acting on cyclic amides is selected from the group consisting of Q45515, Q44184 and its variants, A0A1C4QIY5_9ACTN and its variants, A0A0K2UMP4_LEPSM and its variants, *WP_046170519.1 and its variants, and E1R8C9_SEDSS and its variants, A0A159Z531_9RHOB and its variants, and mixtures thereof, wherein a variant is defined as a polypeptide sequence having at least 80%, preferably 90%, and most preferably 95% sequence identity with respect to its respective polypeptide sequence.

[0057] Naturally, the amide hydrolases (EC3.5.2) acting on cyclic amides outlined above are indicated using database identifier nomenclature according to Uniprot (www.uniprot.org) or the NCBI protein database (www.ncbi.nlm.nih.gov / protein), with sequences from NCBI indicated by an asterisk (*) preceding the respective database identifier.

[0058] In a preferred embodiment of the present invention, the amide hydrolase (EC3.5.2) acting on cyclic amides is L-amide hydrolase (EC3.5.2) acting on cyclic amides.

[0059] In a preferred embodiment of the present invention, R of formulas (I), (II), (III), and (IV) 1 The compound is H or C1-C6 alkyl, preferably H or C2-C4 alkyl, more preferably H, ethyl or butyl, most preferably ethyl.

[0060] A suitable L-amide hydrolase (EC3.5.1) acting on linear amides is preferably selected from the group consisting of EC3.5.1 hydrolases acting on linear amides, EC3.5.1.87 N-carbamoyl-L-amino-acid hydrolase, 3.5.1.77 N-carbamoyl-D-amino-acid hydrolase, and mixtures thereof. Suitable L-amide hydrolases (EC3.5.1) that can be used in this process include A0A7Y0T4N7_9RHIZ and its variants, Q88FQ3_PSEPK and its variants, Q88Q81_PSEPK and its variants, A0A126S6J4_PSEPU and its variants, Q8VUL6_9PSED and its variants, H9B8T5_9PSED and its variants, Q9FB05_9PSED and its variants, and C0ZCM. 8_BREBN and its variants, C0Z7R5_BREB and its variants, A0A0K9YX84_9BACL and its variants, E3HUL6_ACHXA and its variants, A0A1V9BSS3_9BACI and its variants, A0A1V9BSS3_9BACI and its variants, Q9F464 and its variants, A0A4D7Q548_GEOKU and its variants, Q9F464 and its variants, A0A2S9D976_9MICC and its variants, A0A1I6V ZZ4_9RHIZ and its variants, A0A1L6RE91_9LACT and its variants, A0A3E0C996_9BURK and its variants, A0A3M7BGJ4_HORWE and its variants, A0A2D7YQN7_9GAMM and its variants, A0A535Y1H2_UNCCH and its variants, A0A223E4I5_9BACI and its variants, M2VSE9_GALSU and its variants, A0A3T0K6C0_9GAMM and its variants, A0A 416FGE1_9CLOT and its variants, D1P143_9GAMM and its variants, A0A6P2ISL4_BURL3 and its variants, A0A3S6Z2M9_9FIRM and its variants, A0A0C1US49_9BACT and its variants, A0A1Y4GC62_9BACT and its variants, A0A3D3VMN7_9BACT and its variants, A0A2K8L549_9PROT and its variants, A0A1G0MC89_9BACT and its variants,Examples include selections from the group consisting of A0A1M6WYS1_SELRU and its variants, A0A2K2BYI3_POPTR and its variants, A0A510DYR5_9CREN and its variants, A0A5Y3XFN7_SALER and its variants, A0A381IB54_CLODI and its variants, A0A2V3IQW6_9FLOR and its variants, and mixtures thereof. A variant is defined as a polypeptide sequence having at least 80%, preferably 90%, and most preferably 95% sequence identity with respect to its respective polypeptide sequence. Most preferably, the L-amide hydrolase (EC3.5.1) acting on linear amides is selected from the group consisting of A0A3E0C996_9BURK and its variants, A0A535Y1H2_UNCCH() and its variants, A0A6P2ISL4_BURL3() and its variants, A0A1Y4GC62_9BACT (and its variants), where a variant is defined as a polypeptide sequence having at least 80%, preferably 90%, and most preferably 95% sequence identity with respect to the respective polypeptide sequence. Naturally, the linear amide-acting L-amide hydrolase (EC3.5.1) outlined above is indicated by database identifier nomenclature according to the Uniprot database (www.uniprot.org).

[0061] In a preferred embodiment of the present invention, R of formula (I) 1 The compound is a C1-C8 alkyl, preferably a C1-C6 alkyl, more preferably a C2-C4 alkyl, even more preferably ethyl or butyl, and most preferably ethyl. The process of the present invention further comprises a step of deprotection under acidic conditions. In connection therewith, any suitable acid can be used. Preferably, hydrochloric acid or sulfuric acid is used.

[0062] In a preferred embodiment of the present invention, the process further comprises the addition of a racemase enzyme. Any suitable racemase enzyme may be used. Suitable racemase enzymes are selected from the group consisting of EC5.1 racemase, EC5.1.1 racemase and derivatives acting on amino acids, EC5.1.99.5 racemase, and mixtures thereof. Suitable racemase enzymes that can be used in this process include Q9RYA6_DEIRA and its variants, Q9F466 and its variants, Q9F466 and its variants, A0A7L5BQP9_9RHIZ and its variants, Q00924 and its variants, F7X6X4_SINMM and its variants, A0A6V7ACK5_RHIRD and its variants, A0A7Y0XLH3_9RHIZ and its variants, A0A5B8XR30_9DELT and its variants Body, A0A533QH78_9PROT and its variants, A0A3M9Z0A0_9CYAN and its variants, A0A3A0A4T5_9CHLR and its variants, A0A1F6C9P8_HANXR and its variants, A0A4S0NM85_9RHIZ and its variants, A0A1V5I086_9SPIR and its variants, A0A6P0NEY4_9CYAN and its variants, A0A2K0YBY8_9SPHN and its variants, A0A1H5NHN7_9R HIZ and its variants, A0A317KUZ3_9ACTN and its variants, A0A430VJ34_THESC and its variants, A0A1J5KHA5_9PROT and its variants, A0A535LIJ4_9CHLR and its variants, A0A2T6KHH4_9RHOB and its variants, A0A3G8JSD5_9ACTN and its variants, A0A3A9JRT3_9THEO and its variants, A0A2N7WBP6_9BURK and its variants, A0A1 A2N8C4_9MYCO and its variants, A0A1R3TB43_9RHIZ and its variants, X1T733_9ZZZZ and its variants, A0A6P1SX79_9RHOB and its variants, A0A0Q5VT22_9RHIZ and its variants, A0A2N1RKS5_9SPIR and its variants, A0A529XJR5_9RHIZ and its variants, A0A358TXS4_9FIRM and its variants, A0A1Q9UJX6_9ACTN and its variants,Examples include selections from the group consisting of A0A434WJY9_9RHIZ and its variants, A0A4R7C3Y1_9RHIZ and its variants, A0A2T4IRF7_9RHIZ and its variants, A0A2E8B427_9PLAN and its variants, A0A538D678_9ACTN and its variants, A0A1W6Z0D5_9BORD and its variants, A0A3P1UKI1_9RHIZ and its variants, U2S1Q0_9FIRM and its variants, A0A3D5IHC5_AGRSP and its variants, A0A3D5JEU3_9DELT and its variants, and mixtures thereof. A variant is defined as a polypeptide sequence having at least 80%, preferably 90%, and most preferably 95% sequence identity with respect to its respective polypeptide sequence. Naturally, the racemase enzyme outlined above is represented by the database identifier nomenclature following the Uniprot database (www.uniprot.org). Most preferably, the racemase enzyme is selected from the group consisting of A0A6V7ACK5_RHIRD and its variants, and A0A2T6KHH4_9RHOB and its variants, where a variant is defined as a polypeptide sequence having at least 80%, preferably 90%, and most preferably 95% sequence identity with respect to the respective polypeptide sequence.

[0063] In a preferred embodiment of the present invention, the process further comprises the addition of an N-carbamoyl amino acid racemase enzyme. Any suitable N-carbamoyl amino acid racemase enzyme may be used.

[0064] In a preferred embodiment of the present invention, the process further comprises the addition of the racemase enzyme outlined above and the N-carbamoyl amino acid racemase enzyme.

[0065] In a preferred embodiment of the present invention, all steps of the process are carried out in a single vessel. In connection therewith, all components are preferably added substantially at the start of the reaction.

[0066] In a preferred embodiment of the present invention, at least 15%, preferably at least 20%, more preferably at least 30%, even more preferably at least 50%, and particularly at least 70%, of cyanohydrin or a cyanohydrin derivative according to formula (II) is converted to L-glufosinate and / or its salt or L-glufosinate alkyl ester and / or its salt.

[0067] If an L-glufosinate alkyl ester or a salt thereof is produced, the process of the present invention may include a final step of deprotecting the L-glufosinate alkyl ester or salt thereof to obtain the L-glufosinate chloride, which can be further converted to L-glufosinate by increasing the pH of the solution. A detailed description of each of these process steps can be found in European Patent Application Publication No. 0508296A1.

[0068] Preferably, in the process according to the first aspect of the present invention, L-glufosinate and / or its salt or L-glufosinate alkyl ester and / or its salt is prepared with an enantiomer excess, preferably more than 85%, more preferably more than 90%, even more preferably more than 95%, and most preferably more than 99%.

[0069] The enzyme to be applied can be applied in any suitable manner known in the art. In preferred embodiments of the present invention, the enzyme to be applied is applied as a clarified cell lysate, whole cells, or immobilized enzyme.

[0070] Alternatively, some or all of the components other than L-glufosinate may be removed from the biotransformation mixture, the mixture may be optionally concentrated, and then the mixture may be used directly (and / or with the addition of various adjuvants) for weed prevention or control. The biotransformation mixture may, in some cases, be used directly (and / or with the addition of various adjuvants) for weed prevention or control.

[0071] Additional steps may be added to further purify L-glufosinate. Such further purification and isolation methods include ion exchange, extraction, salt formation, crystallization, and filtration, each of which may be used multiple times or in appropriate combinations. The enzyme, if supported, can be removed by simple filtration, or, if dissolved and free, by ultrafiltration, the use of absorbents such as Celite, cellulose, or carbon, or by denaturation using various techniques known to those skilled in the art.

[0072] The ion exchange process results in separation by selective adsorption of solutes onto a resin selected for this purpose. Since the products and impurities must be dissolved in a single solution before adsorption, concentration by evaporation or distillation of the purified product stream is usually required before isolation. An example of the use of ion exchange for purification is described by Schultz et al. and in European Patent Application Publication No. 0249188(A2).

[0073] Purification may be achieved by the formation of an insoluble salt of L-glufosinate by adding a suitable acid, such as hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, and acetic acid. Similarly, purification may be achieved by the formation of an insoluble salt by adding a suitable base. Useful bases include alkali metal hydroxides, carbonates, sulfates, and phosphates, or alkaline earth metal hydroxides, carbonates, sulfates, and phosphates. Other nitrogen-containing bases may be used, including ammonia, hydroxylamine, isopropylamine, triethylamine, tributylamine, pyridine, 2-picoline, 3-picoline, 4-picoline, 2,4-lutidine, 2,6-lutidine, morpholine, N-methymorpholine, 1,8-diazabicyclo[5.4.0]undeca-7-ene, and dimethylethanolamine. To maximize yield and optimize the purity of the desired salt, it may be advantageous to concentrate the mixture or add a solvent (or both). Suitable solvents for this purpose include those with very low solubility of the desired salt (such solvents are often called "poor solvents"). Salts of L-glufosinate can be converted to glufosinate in a form suitable for formulation by standard methods known to those skilled in the art. Alternatively, L-glufosinate can be isolated as a zwitterion.

[0074] U.S. Patent No. 9,255,115B2 describes a method for obtaining relatively high-purity L-glufosinate by converting the hydrochloride salt of L-glufosinate to a zwitterionic form using a base such as sodium hydroxide or sodium methoxide, and then crystallizing it from an aqueous alcohol solvent. This method produces crystalline L-glufosinate that is not hygroscopic, and therefore has the advantage of maintaining a higher purity compared to amorphous L-glufosinate even when exposed to moisture over time.

[0075] Other salts of L-glufosinate are known in the art. U.S. Patents 5,767,309 and 5,869,668 teach the use of chiral alkaloid bases to form diastereomer salts with racemic glufosinate. Since salts of L-glufosinate precipitate from solution in much larger quantities than the corresponding salts of D-glufosinate, purification is achieved. This method can therefore be used in conjunction with the present invention to obtain L-glufosinate with a high enantiomeric excess, if desired.

[0076] Optionally, purification can be achieved by first crystallizing one or more impurities, removing the impurities by filtration, and then further purifying the L-glufosinate from the resulting filtrate by forming a salt as previously described. This is advantageous if the unreacted amine donor can be partially or completely isolated and used in subsequent reactions. Similarly, partially or completely isolated unreacted cyanohydrin or cyanohydrin derivative according to formula (II) may be reused for use in subsequent reactions.

[0077] Extraction may be used to purify the product. German Patent No. 3920570C2 describes a process for precipitating excess glutamic acid (used as an amine donor) by adjusting the pH of the solution to 3.7-4.2 using sulfuric acid. After filtering out the glutamic acid, the pH of the filtrate is lowered to 1-2, where other impurities are extracted into the solvent. After extraction and concentration, ammonia is added to the aqueous solution to raise the pH to 5-7, and ammonium sulfate is precipitated. The ammonium sulfate is removed by filtration, and the resulting filtrate is concentrated to obtain the ammonium salt of L-glufosinate.

[0078] Isolation of L-glufosinate or its salts may be desirable, for example, for the purpose of shipping the solid to the place of formulation or use. Typical industrial isolation methods, such as filtration and centrifugation, can be used. The isolated product often requires the removal of water, volatile impurities, and solvents (if present), and typical industrial drying equipment may be used for this purpose. Examples of such equipment include ovens, rotary drum dryers, and agitated dryers. In some cases, it may be advantageous to use a spray dryer.

[0079] There is no need to produce a solid product after purification. This can be advantageous if the formulation of L-glufosinate is carried out in the same location used for the production of L-glufosinate. Many L-glufosinate and its salts are readily soluble in water, and water is a convenient liquid to use for product formulation. For example, the amine donor can be isolated by filtration, and the resulting filtrate can be concentrated by distillation. The pH of the filtrate can be adjusted to a desired value, and the resulting solution can be used as is or blended with formulation components. In another example, a slurry of one of L-glufosinate or its salts may be prepared as described above and isolated by filtration. The solid can then be dissolved directly on a filter by adding water or a suitable solvent to obtain a solution of L-glufosinate.

[0080] As described above, in a second embodiment, the present invention relates to formula (II) [ka] (In the formula, R 1 is H or C1-C8 alkyl, and R 2 H, C1-C8 alkyl, C6-C 10 Aryl, C7~C 10 Aralkil, C4~C 10 Cycloalkyl, or C1-C 10 (It is ashil) Cyanide or cyanohydrin derivatives, The present invention further relates to compositions comprising L-glufosinate and / or salts thereof.

[0081] Suitable salts are hydrochloride, ammonium salt, and isopropylammonium salt. Naturally, the zwitterions of each of these salts are also included.

[0082] In preferred embodiments of the present invention, the amount of L-glufosinate and / or its salt is at least 20 wt.-%, preferably at least 30 wt.-%, more preferably at least 40 wt.-%, even more preferably at least 50 wt.-%, still more preferably at least 60 wt.-%, particularly at least 70 wt.-%, or at least 80 wt.-%, based on the total amount of cyanohydrin or cyanohydrin derivative according to formula (II) and L-glufosinate and / or its salt.

[0083] In preferred embodiments of the present invention, the amount of L-glufosinate and / or its salt is in the range of 20 to 99 wt.-%, preferably 30 to 98 wt.-%, more preferably 40 to 96 wt.-%, even more preferably 50 to 95 wt.-%, even more preferably 60 to 94 wt.-%, particularly at least 70 to 90 wt.-%, or at least 80 to 90 wt.-%, based on the total amount of cyanhydrin or cyanhydrin derivative according to formula (II) and L-glufosinate and / or its salt.

[0084] The composition may contain cyanohydrin or a cyanohydrin derivative according to formula (II) in an amount up to 30 wt.-%, preferably up to 20 wt.-%, more preferably up to 10 wt.-%, even more preferably up to 5 wt.-%, still more preferably up to 2.5 wt.-%, and particularly up to 1 wt.-%, based on the total amount of cyanohydrin or a cyanohydrin derivative according to formula (II) and L-glufosinate and / or its salt.

[0085] In one preferred embodiment of the present invention, the compositions described herein can be used directly as herbicides or as components in formulated herbicides.

[0086] The compositions described herein are useful for application to crop fields for the prevention or control of weeds. These compositions can be formulated as liquids for spraying into fields. Glufosinate, preferably L-glufosinate, is supplied in an effective amount in the composition. As used herein, an effective amount means from about 10 grams to about 1,500 grams of the active ingredient per hectare, for example, about 50 grams to about 400 grams, or about 100 grams to about 350 grams. In some embodiments, the active ingredient is L-glufosinate. For example, the amount of L-glufosinate in the composition can be approximately 10 grams, 50 grams, 100 grams, 150 grams, 200 grams, 250 grams, 300 grams, 350 grams, 400 grams, 500 grams, 550 grams, 600 grams, 650 grams, 700 grams, 750 grams, 800 grams, 850 grams, 900 grams, 950 grams, 1,000 grams, 1,050 grams, 1,100 grams, 1,150 grams, 1,200 grams, 1,250 grams, 1,300 grams, 1,350 grams, 1,400 grams, 1,450 grams, or 1,500 grams of L-glufosinate per hectare.

[0087] The herbicidal compositions described herein (including concentrates that require dilution before application to plants) contain L-glufosinate (i.e., the active ingredient), optionally a partially residual cyanohydrin or cyanohydrin derivative of formula (II), and one or more adjuvant components, in liquid or solid form.

[0088] The composition is prepared by mixing the active ingredient with one or more adjuvants, such as diluents, fillers, carriers, surfactants, organic solvents, humectants, or conditioning agents, resulting in a composition in the form of finely divided particulate solids, pellets, solutions, dispersions, or emulsions. Therefore, the active ingredient can be used with adjuvants, such as finely divided solids, organic liquids, water, wetting agents, dispersants, emulsifiers, or any suitable combination thereof. From the standpoint of economy and convenience, water is a preferred diluent. However, not all compounds are resistant to hydrolysis, and in some cases, as will be understood by those skilled in the art, the use of non-aqueous solvent media may be indicated.

[0089] A formulated herbicidal composition can be prepared by optionally adding one or more additional components to the composition. Such a formulated composition may include L-glufosinate, a carrier (e.g., a diluent and / or solvent), and other components. The formulated composition contains an effective amount of L-glufosinate.

[0090] Diluents may also be included in the formulated composition. Suitable diluents include water and other aqueous components. Optionally, the diluent may be present in the amount necessary to produce a packaged or ready-to-use composition.

[0091] The herbicidal compositions described herein, particularly liquids and soluble powders, may contain one or more surfactants as further adjuvant components in an amount sufficient to allow the given composition to disperse easily in water or oil. The incorporation of surfactants into the composition greatly enhances its effectiveness. Surfactants used herein include wetting agents, dispersants, and suspending agents, and emulsifiers are among them. Anionic, cationic, and nonionic agents can also be readily used.

[0092] Suitable wetting agents include alkylbenzenes and alkylnaphthalene sulfonates, sulfated fatty alcohols, amines or acid amides, long-chain esters of sodium isothionate, esters of sodium sulfosuccinate, sulfated or sulfonated fatty acid ester petroleum sulfonates, sulfonated vegetable oils, disterminate acetylene glycols, polyoxyethylene derivatives of alkylphenols (especially isooctylphenol and nonylphenol), and polyoxetylene derivatives of mono-higher fatty acid esters of hexitol anhydride (e.g., sorbitan). Exemplary dispersants include methylcellulose, polyvinyl alcohol, sodium lignin sulfonate, high molecular weight alkylnaphthalene sulfonate, sodium naphthalene sulfonate, polymethylenebisnaphthalene sulfonate, and sodium N-methyl-N-(long-chain acid)laurate.

[0093] A water-dispersible powder composition can be prepared containing one or more active ingredients, an inert solid extender, and one or more wetting and dispersing agents. The inert solid extender is usually of mineral origin, such as natural clay, diatomaceous earth, and synthetic minerals derived from silica. Examples of such extenders include kaolinite, attapulgite clay, and synthetic magnesium silicate. The water-dispersible powders described herein may optionally contain about 5 to about 95 parts by weight of an active ingredient (e.g., about 15 to 30 parts by weight of the active ingredient), about 0.25 to 25 parts by weight of a wetting agent, about 0.25 to 25 parts by weight of a dispersing agent, and 4.5 to about 94.5 parts by weight of an inert solid extender, all parts being by the total weight of the composition. If necessary, about 0.1 to 2.0 parts by weight of the inert solid extender may be replaced with a corrosion inhibitor, an antifoaming agent, or both.

[0094] Aqueous suspensions can be prepared by dissolution, or by mixing and grinding an aqueous slurry of a water-insoluble active ingredient in the presence of a dispersant to obtain a concentrated slurry of extremely finely divided particles. The resulting concentrated aqueous suspension is characterized by its extremely small particle size, and therefore, when diluted and sprayed, it provides an extremely uniform coating.

[0095] Emulsified oils are typically prepared by dissolving an active ingredient together with a surfactant in a water-immiscible or partially water-immiscible solvent. Suitable solvents for the active ingredients described herein include hydrocarbons and water-immiscible ethers, esters, or ketones. Emulsified oil compositions generally contain about 5 to 95 parts of the active ingredient, about 1 to 50 parts of the surfactant, and about 4 to 94 parts of the solvent, all parts being by weight based on the total weight of the emulsified oil.

[0096] The compositions described herein may also contain other additives, such as fertilizers, plant toxicants and plant growth regulators, and insecticides, which are used as adjuvants or in combination with any of the above adjuvants. The compositions described herein may also be mixed with other materials, such as fertilizers and other plant toxicants, and applied in a single application.

[0097] In each of the formulation types described herein, for example, liquid formulations and solid formulations, the concentration of the active ingredient is the same.

[0098] The herbicide composition is recognized as being usable in combination with other herbicides. The herbicide composition of the present invention is often applied in combination with one or more other herbicides to control a wider variety of undesirable vegetation. When used in combination with other herbicides, the currently claimed compound can be formulated together with one or more other herbicides, mixed in a tank with one or more other herbicides, or applied sequentially with one or more other herbicides. Some of the herbicides that can be used in combination with the compound of the present invention are amide herbicides, such as aridochlor, beflubutamide, benzadox, benzipram, bromobutide, cafenstrole, CDEA, chlorthiamide, ciprazole, dimethenamide, dimethenamide P, diphenamide, epronaz, etonipromide, fentrazamide, flupoxam, homesaphen, halosaphen, isocarbamide, isoxaben, nap Lopamide, naptalam, petoxamide, propizamide, quinonamide, and tebutam; anilide herbicides, such as chloranocryl, cisanilide, clomeprop, cypromid, diflufenican, etobenzanide, phenashram, flufenacet, flufenican, mefenacet, mefluidide, metamihop, monalid, naproanilide, pentanocrol, picolinafen, and propanil; arylalanine herbicides Herbicides, e.g., benzoylprop, flancrop, and flancrop M; chloroacetanilide herbicides, e.g., acetochlor, alachlor, butachlor, butenachlor, delaclor, dietatyl, dimethachlor, metazachlor, metrachlor, S-methachlor, pretilachlor, propachlor, propisochlor, prinachlor, terbuchlor, tenylchlor, and xylaclor; sulfone Anilide herbicides, e.g., benzofluor, perfluidone, pyrimisulfan, and profluazole; sulfonamide herbicides, e.g., ashram, carvasram, fenashuram, and oryzarin; antibiotic herbicides, e.g., biranafos; benzoic acid herbicides, e.g., chloramben, dicamba, 2,3,6-TBA, and tricamba; pyrimidinyl oxybenzoic acid herbicides, e.g., bispyribac and pyriminobac;Pyrimidinylthiobenzoate herbicides, e.g., pyrithiobac; phthalate herbicides, e.g., chlortal; picolinic acid herbicides, e.g., aminopyralide, clopyralide, and picloram; quinoline carboxylic acid herbicides, e.g., quinchlorac and kinmelac; arsenic herbicides, e.g., cacodylic acid, CMA, DSMA, hexaflurate, MAA, MAMA, MSMA, potassium arsenite, and sodium arsenite; benzoylcyclohexanedione herbicides, e.g., mesotrione, sulcotrione, Tefuryltrione and tembotrione; benzofuranyl alkyl sulfonate herbicides, e.g., benfresate and etofmesate; carbamate herbicides, e.g., ashuram, carboxazole, chloroprocarb, dichlormate, phenashram, carbtylate, and terbucarb; carbanylate herbicides, e.g., barban, BCPC, carbasram, carbetamide, CEPC, chlorbufame, chlorprofam, CPPC, desmedifam, phenisofa, fenmedifam Herbicides such as fenmedifam ethyl, profam, and swep; cyclohexene oxime herbicides, e.g., alloxidim, butroxidim, cretodym, cloproxidim, cycloxidim, profoxidim, cethoxidim, tepraloxidim, and tralcoxidim; cyclopropyl isoxazole herbicides, e.g., isoxachlortol and isoxaflutol; dicarboxyimide herbicides, e.g., benzfenzizone, synidone ethyl, flumezin, flumimicrolac, flumioxazi , and flumipropine; dinitroaniline herbicides, e.g., benfluralin, butruarin, dinitramine, ethalfluralin, fluroralin, isoproparin, metalproparin, nitralin, oryzarin, pendimethalin, prodiamine, profluralin, and trifluralin; dinitrophenol herbicides, e.g., dinophenate, dinoprop, dinosum, dinoseb, dinoterb, DNOC, ethinofen, and medinoterb; diphenyl ether herbicides, e.g., ethoxyfen;Nitrophenyl ether herbicides, e.g., asifluorfen, acronifen, bifenox, clomethoxyfen, clomitrofen, etonipromide, fluorodiphen, fluoroglycofen, fluoronitrofen, homesafen, fliroxyfen, halosaphen, lactofen, nitrofen, nitrofluorfen, and oxyflufen; dithiocarbamate herbicides, e.g., dazomet and metam; halogenated aliphatic herbicides, For example, Arolac, Chloropone, Darapone, Flupropanate, Hexachloroacetone, Iodomethane, Methyl bromide, Monochloroacetic acid, SMA, and TCA; Imidazolinone herbicides, for example, Imazametabenz, Imazamox, Imazapick, Imazapyr, Imazakine, and Imazetapyr; Inorganic herbicides, for example, Ammonium sulfamate, Borax, Calcium chlorate, Copper sulfate, Ferrous sulfate, Potassium azide, Potassium cyanide, Sodium azide, Sodium chlorate, and Sulfuric acid; Nitrile herbicides, e.g., bromobonyl, bromoxynil, chloroxynil, diclobenil, iodobonyl, ioxynil, and pyraclonil; organophosphate herbicides, e.g., amiprophos-methyl, anirophos, benslid, biranafos, butamiphos, 2,4-DEP, DMPA, EBEP, hosamin, glyphosate, and piperophos; phenoxy herbicides, e.g., bromophenoxime, clomeprop, 2,4-DEB, 2,4-DEP, diphenopenten, disul, ervon, eto Nipromide, fentelacol, and trihopsim; phenoxyacetic acid herbicides, e.g., 4-CPA, 2,4-D, 3,4-DA, MCPA, MCPA-thioethyl, and 2,4,5-T; phenoxybutyric acid herbicides, e.g., 4-CPB, 2,4-DB, 3,4-DB, MCPB, and 2,4,5-TB; phenoxypropionic acid herbicides, e.g., Cloprop, 4-CPP, Dichlorprop, Dichlorprop P, 3,4-DP, Fenoprop, Mecoprop, and Mecoprop P;Aryloxyphenoxypropionic acid herbicides, e.g., chlorajifop, clodinahop, clohop, cyhalofop, diclohop, phenoxaprop, phenoxaprop P, fentiaprop, fluazihop, fluazihop P, haloxyhop, haloxyhop P, isoxapyrifop, metamihop, propaxifop, quizalohop, quizalohop P, and triphop; phenylenediamine herbicides, e.g., dinitramine and prodiamine; pyrazolyl herbicides, e.g., benzofenap, pyrazolinate, pyrazolyl Luphotol, pyrazoxyfen, pyroxasulfone, and topramezone; pyrazolylphenyl herbicides, e.g., fluazolate and pyraflufen; pyridazine herbicides, e.g., credazine, pyridafor, and pyridate; pyridazinon herbicides, e.g., brompyrazone, chloridazone, dimidazone, flufenpyr, metoflurazone, norflurazone, oxapirazone, and pidanone; pyridine herbicides, e.g., aminopyralide, cliodinate, clopyralide, dithiopyr, fluroxypyr, haloxidine, picloram, picolina Fen, pyrichlor, thiazopyr, and triclopyr; pyrimidinediamine herbicides, e.g., iprimidum and thiochlorim; quaternary ammonium herbicides, e.g., cypercoat, dietumcoat, diphenzocoat, diquat, morphamcoat, and paraquat; thiocarbamate herbicides, e.g., butyrate, cycloate, dialate, EPTC, esprocarb, ethiolate, isopolinate, methibencarb, molinate, olbencarb, pebrate, prosulfocarb, pyributicarb, sulfate, thioben Carb, thiocarbasil, trialate, and vemolate; thiocarbonate herbicides, e.g., dimexano, EXD, and proxane; thiourea herbicides, e.g., methionone; triazine herbicides, e.g., dipropetrin, triaziflame, and trihydroxytriazine; chlorotriazine herbicides, e.g., atrazine, chlorazine, cyanazine, siprazine, eglinadin, ipazine, mesoprazine, procyazin, proglinadin, propazine, sebutyrazine, simazine, terbutyrazine, and trietazine;Methoxytriazine herbicides, e.g., atraton, metmeton, prometon, sebumetone, simeton, and terbumeton; methylthiotriazine herbicides, e.g., ametrin, adiprothrin, cyanatrin, desmethrin, dimethametrine, metoprothrin, prometon, simetrine, and terbutrin; triazinon herbicides, e.g., ametridione, amivudine, hexazinone, isomethione, metamitron, and metrivudine; triazole herbicides, e.g., amitrol, cafenstrol, epronaz, and flupoxam; Triazolone herbicides, e.g., amicarbazone, bencarbazone, carfentrazone, flucarbazone, propoxycarbazone, sulfentrazone, and thiencarbazone methyl; triazolopyrimidine herbicides, e.g., chloransram, diclosram, florasram, flumetulam, metosram, penoxsram, and piroxsram; uracil herbicides, e.g., butafenacil, bromacil, flupropacil, isocyl, renacil, and terbacil, 3-phenyluracil; urea herbicides, e.g., benzthiazulon, cumylon, cyclol Dichloral urea, diflufenzopyr, isonorolone, isouron, metabenzthiazulon, monisouron, and norlon; phenylurea herbicides, such as anislon, buturon, chlorbromuron, chloretulon, chlorotoluron, chloroxuron, dimuron, diphenokithron, dimeflon, diuron, fenuron, fluomethuron, fluothiron, isoproturon, linuron, methiron, methyldimuron, metobenzuron, metobromuron, metoxuron, monolinuron, monuron, nebulon, parafluron, phenobendz Ron, Siduron, Tetrafluron, and Thidiazuron; Pyrimidinyl sulfonylurea herbicides, such as Amidosulfuron, Azimsulfuron, Bensulfuron, Chlorimuron, Cyclosulfamuron, Ethoxysulfuron, Flazasulfuron, Flucetosulfuron, Flupirsulfuron, Folamsulfuron, Halosulfuron, Imazosulfuron, Mesosulfuron, Nicosulfuron, Orthosulfamuron, Oxasulfuron, Primisulfuron, Pyrazosulfuron, Rimusulfuron, Sulfometsuron, Sulfosulfuron, and Trifloxysulfuron;Triazinyl sulfonylurea herbicides, for example, chlorsulfuron, cinosulfuron, etamethosulfuron, iodosulfuron, metosulfuron, prosulfuron, thifensulfuron, triasulfuron; Tribenulon, triflusulfuron, and tritosulfuron; thiadiazolylurea herbicides, e.g., butiuron, ethidimylon, tebutiuron, thiazaflurone, and tidiazuron; and unclassified herbicides, e.g., acrolein, allyl alcohol, aminocyclopyrachlor, azaphenidine, benazoline, bentazone, benzobicyclon, butidazole, calcium cyanamide, cambendichlor, chlorfenac, chlorfenprop, chlorflurazole, chlorflurenol, scinmethilin, chromazon, CPMF, cresol, ortho-dic Examples include lolobenzene, dimepiperate, endotar, fluoromidine, flulidone, flurochloridone, flurthamone, fluthiaset, indanophan, mesazole, methyl isothiocyanate, nipiraclofen, OCH, oxaziargyl, oxadiazone, oxadiclomefone, pentachlorophenol, pentoxazone, phenylmercury acetate, pinoxadene, prosulfarin, pyribenzoxime, pyrifthalide, quinoclamin, rhodetanyl, sulglycapine, tidiadimine, tridiphan, trimethulone, tripropindan, and tritac. The herbicides of the present invention can further be used in combination with glyphosate or 2,4-D for glyphosate-resistant or 2,4-D-resistant crops. It is generally preferable to use the compositions of the present invention in combination with herbicides that are selective for the crop being treated and that complement the range of weeds controlled by these compositions at the adopted application ratio. It is more generally preferable to apply the composition of the present invention and other complementary herbicides simultaneously as a combination formulation or as a tank mixture.

[0099] As described above, in a third embodiment, the present invention provides a method for selectively controlling weeds in a region, preferably a region containing planted seed crops or glufosinate-resistant crops, The L-glufosinate and / or salt thereof obtained by the process of the present invention is included with D-glufosinate and / or salt thereof in a proportion of at least 50% enantiomers, preferably in an enantiomer excess of more than 70%, and is present in a proportion of more than 0.01 wt.-% and less than 10 wt.-% of formula (II) based on the total amount of the composition. [ka] (In the formula, R 1 is H or C1-C8 alkyl, and R 2 H, C1-C8 alkyl, C6-C 10 Aryl, C7~C 10 Aralkil, C4~C 10 Cycloalkyl, or C1-C 10 (It is an asyl) The present invention further relates to a method comprising applying an effective amount of a composition containing cyanohydrin or a cyanohydrin derivative to a region.

[0100] In a preferred embodiment of the present invention, the composition comprises L-glufosinate and / or a salt thereof with respect to D-glufosinate and / or a salt thereof in a proportion of 50 to 99% enantiomers, preferably 60 to 98% enantiomers, more preferably 70 to 95% enantiomers, and particularly 80 to 90% enantiomers.

[0101] In preferred embodiments of the present invention, the composition contains 0.02 to 8 wt.-%, preferably 0.03 to 5 wt.-%, more preferably 0.05 to 3 wt.-%, and particularly 0.1 to 2 wt.-%, of cyanohydrin or a cyanohydrin derivative according to formula (II), based on the total amount of the composition.

[0102] Naturally, the composition may also contain the same adjuvants and / or other herbicides as those described in more detail above.

[0103] The compositions described herein are useful for application to crop fields for the prevention or control of weeds. These compositions can be formulated as liquids for field spraying. L-glufosinate is supplied in an effective amount within the composition. As used herein, an effective amount means from about 10 grams of the active ingredient per hectare to about 1,500 grams of the active ingredient per hectare, for example, about 50 grams to about 400 grams, or about 100 grams to about 350 grams. In some embodiments, the active ingredient is L-glufosinate. For example, the amount of L-glufosinate in the composition can be approximately 10 grams, 50 grams, 100 grams, 150 grams, 200 grams, 250 grams, 300 grams, 350 grams, 400 grams, 500 grams, 550 grams, 600 grams, 650 grams, 700 grams, 750 grams, 800 grams, 850 grams, 900 grams, 950 grams, 1,000 grams, 1,050 grams, 1,100 grams, 1,150 grams, 1,200 grams, 1,250 grams, 1,300 grams, 1,350 grams, 1,400 grams, 1,450 grams, or 1,500 grams of L-glufosinate per hectare.

[0104] The present invention will be further explained by the following examples. [Examples]

[0105] Enzyme preparation a) Cloning of enzyme genes (Ex1) The amino acid sequences of each enzyme were identified from publicly available databases (UniProt, https: / / www.uniprot.org; NCBI Protein Database, https: / / www.ncbi.nlm.nih.gov / protein. Sequences from NCBI are indicated with an asterisk (*) at the beginning of their respective database identifiers). Each DNA sequence was then derived using the standard codon usage frequencies of Escherichia coli. The DNA sequences were synthesized (BioCat GmbH) and cloned into plasmid pDHE19.2 (Ress-Loeschke, M. et al., German Patent No. 19848129, 1998, (BASF AG)). Using the obtained plasmid, competent cells were transformed into rhaA--inducible strains of E. coli TG1 transformed with (Chung, CT et al., Proc Natl Acad Sci USA, 1989, 86, 2172), E. coli (E. coli) strains TG10 (Kesseler, M. et al., International Publication No. 2004050877A1 pamphlet, 2004, (BASF AG)), pHSG575 (Takeshita, S. et al., Gene, 1987, 61, 63), and pAgro4 (pBB541 as described in Tomoyasu, T. et al., Mol. Microbiol., 2001, 40, 397).

[0106] b) Recombinant production of enzymes (Ex2) Preparation of biocatalysts in a shaking flask Using Escherichia coli (E. coli) TG10 carrying a recombinant plasmid of the enzyme, 2 ml of LB medium (Bertani, G., J Bacteriol, 1951, 62, 293) supplemented with 100 μg / ml ampicillin, 100 μg / ml spectinomycin, and 20 μg / ml chloramphenicol was inoculated, and the resulting preculture was incubated at 37°C for 5 hours with stirring at 250 rpm. Using 1 ml of the preculture, 100 ml of LB medium supplemented with 100 μg / ml ampicillin, 100 μg / ml spectinomycin, 20 μg / ml chloramphenicol, 1 mM MnCl2, 0.1 mM isopropyl-β-D-thiogalactopyranoside, and 0.5 g / l rhamnose was inoculated in a 500 ml baffled Erlenmeyer flask. This culture was incubated at 37°C for 18 hours under shaking conditions. Next, biomass was recovered by centrifugation at 3220 × g for 10 minutes at 8°C. The supernatant was discarded, and the cell pellet was resuspended in 8 ml of 100 mM, pH 8.2 HEPES buffer with 1 mM MnCl2 added. This cell suspension was used without further preparation for synthesis when performing whole-cell biotransformation. When using clarified cell lysates instead, 5 ml of the cell suspension was distributed into five reaction tubes containing lysis matrix B (0.7 ml of φ0.1 mm quartz beads, MP Biomedicals), the tubes were cooled on ice, and then the cells were disrupted in a homogenizer (Peqlab Precellys24, VWR) for two 30-second cycles. The sample was cooled on ice between cycles. The resulting cell-free lysates were clarified by centrifugation at 20817 × g for 10 minutes at 8°C. The supernatant was isolated, and fractions from the same batch were combined (=clarified cell lysates).

[0107] Production of fermentation-based whole-cell biocatalysts Escherichia coli (E. coli) TG10 containing plasmids pAgro4 and pHSG575 was transformed with the pDHE plasmid encoding the target protein. The transformants were cultured in LB Can template supplemented with 100 μg / ml ampicillin, 100 μg / ml spectinomycin, and 20 μg / ml chloramphenicol.

[0108] Pre-culture medium: EcoK12 solution Ultrapure water 1.0kg Citric acid monohydrate 40.0g Zinc sulfate heptahydrate 11.0g Diammonium iron sulfate hexahydrate 8.6g Manganese sulfate monohydrate 3.0g Copper sulfate pentahydrate 0.8g Cobalt sulfate heptahydrate 0.09g

[0109] Sterilization was performed by filtration using a filter with a pore size of 0.2 μm.

[0110] Part 1 Ultrapure water 1.0kg Citric acid monohydrate 3.4g Magnesium sulfate heptahydrate 2.4g Calcium chloride dihydrate 0.1g EcoK12 solution 20g Used to adjust the pH of a 25% sodium hydroxide solution to 6.6.

[0111] Part 2 Ultra pure water 500g Potassium dihydrogen phosphate 26.6g Diammonium hydrogen phosphate 8.0g Used to adjust the pH of a 25% sodium hydroxide solution to 6.4.

[0112] Part 3 Ultra pure water 500g 99% Glycerol, 36.0g Sodium gluconate 24.0g Used to adjust pH to 6.6 with 20% phosphoric acid.

[0113] All three parts were sterilized at 121°C for 30 minutes.

[0114] Vitamin solution Ultra pure water 100g Thiamine hydrochloride 1.0g Vitamin B 12 0.5g

[0115] Sterilization was performed by filtration using a filter with a pore size of 0.2 μm.

[0116] To prepare the final pre-culture medium, parts 1, 2, and 3 were combined and 2.0 ml of vitamin solution was added. Furthermore, 100 μg / ml ampicillin, 100 μg / ml spectinomycin, and 20 μg / ml chloramphenicol were added to the medium. Several transformants were scraped from the LB can template and used to inoculate two 1 L baffled Erlenmeyer flasks containing 100 g of pre-culture medium. These pre-cultures were incubated at 37°C and 150 rpm. When the OD600 reached 12, the entirety of these pre-cultures was used to inoculate the main culture.

[0117] This culture medium: Part 4 Ultrapure water 9.6kg Citric acid monohydrate 21.1g Potassium dihydrogen phosphate 173.6g Diammonium hydrogen phosphate 52.8g Mangesium sulfate heptahydrate 15.1g Calcium chloride dihydrate 0.7g EcoK12 solution 123g Adjust the pH of the 25% sodium hydroxide solution to 6.4. Pluriol P2000 1ml

[0118] Part 4 was sterilized at 125°C for 45 minutes.

[0119] Part 5 Ultra pure water 300g Thiamine hydrochloride 151 mg Vitamin B12 30.2mg Ampicillin sodium salt 1000 mg Spectinomycin hydrochloride 500mg Chloramphenicol 200mg

[0120] Part 5 was sterilized by sterile filtration using a filter unit with a pore size of 0.1 μm.

[0121] Glycerol solution Ultra pure water 804g Citric acid monohydrate 29.1g Sodium sulfate 58.1g Diammonium iron sulfate hexahydrate 4.5g 99% Glycerol, 3370g

[0122] Thiamine solution Ultra pure water 40g Thiamine hydrochloride 55mg

[0123] antifoam solution Pluriol P2000 350g

[0124] Base solution 25% ammonia solution, 1500ml

[0125] Induced solution Ultra pure water 150g Rhamnose monohydrate 100g IPTG 238mg

[0126] The glycerol and defoaming solution were sterilized at 121°C for 30 minutes. The thiamine and derivate solution were sterilized by filtration using a filter with a pore size of 0.2 μm.

[0127] Parts 4 and 5 were combined in a sterile fermentation vessel (Techfors, Infors HT), and the pre-culture was inoculated. The vessel was maintained at a temperature of 37°C, a pressure of 0.2 bar, and a pH of 6.6 by adding a base solution during fermentation. The pO2 level was maintained at 20-40% by adjusting the stirrer speed (usually 500 rpm) and aeration rate (usually 6 l / min). An antifoaming solution was added as needed. A feed solution was obtained by combining a glycerol solution and a thiamine solution. After inoculation, the feed solution was added at a rate of 10 g / hour. After 7 hours, the feed solution addition was switched to "stop and observe" mode, and the supply was driven at a rate of 10 g / hour when the pO2 level rose. After 14 hours, or after consuming 330 g of feed solution, the supply rate was increased to 80-100 g / hour. Gene expression was induced when the oxygen transfer rate reached 80 mmol / l / hour, or alternatively, when the OD600 reached 12 by adding an inducer solution. After 36 hours, fermentation was stopped by lowering the temperature to 15°C. The cooled fermentation broth was discharged from the fermenter and the cells were pelleted by centrifugation at 4700 rpm and 10°C. The resulting supernatant was discarded, and the cells were resuspended in 3850 g of 50 mM potassium dihydrogen phosphate buffer (pH 7.0). This cell suspension was frozen at -80°C and then lyophilized. During this process, the lyophilizer was maintained at -50°C and a pressure of 0.25 mbar. The lyophilized cells were stored at 4°C.

[0128] Production of freeze-dried cell-free extracts Lyophilized cells were resuspended in ultrapure water at 100 g / l. This cell suspension was cooled on ice and then passed through a pressure homogenizer (Panda Plus2000, GEA) set to 800 bar three times to disrupt the cells. The pressure for all three passes was consistently between 1000 and 1400 bar. The resulting mixture was clarified from the debris by centrifugation at 10000 rpm at 10°C for 15 minutes. The resulting pellet was discarded, and the protein concentration in the supernatant was analyzed by Bradford assay. The supernatant was frozen at -80°C and then lyophilized at -50°C and a pressure of 0.25 mbar.

[0129] Preparation of starting materials c) Synthesis of n-butyl(3-cyano-3-hydroxypropyl)methylphosphinat (ACM-H) (Ex3) ACM-H was prepared according to Example 2 of International Publication No. 2015 / 173146A1.

[0130] d) Synthesis of n-butyl(3-cyano-3-acetoxypropyl)methylphosphinat (ACM) (Ex4) ACM was prepared according to Example 1 of International Publication No. 2017 / 037012A1.

[0131] Preparation of L-glufosinate p-butyl ester e) Preparation of L-glufosinate butyl ester from ACM-H (IE5) [ka] 2.47 g of ammonium bicarbonate (31.2 mmol, 1.7 equivalents) was dissolved in 50 ml of distilled water with stirring. 4 g of n-butyl(3-cyano-3-hydroxypropyl)methylphosphinat (18.2 mmol, 1 equivalent "ACM-H"), prepared according to Ex3, was added with stirring. The resulting solution was stirred at 37°C. 1 ml of 2 M aqueous solution of MnCl2 was added, followed by the enzymes (Uniprot ID: A0A159Z531_9RHOB, SEQ ID NO: 1, 1 g of lyophilized cell-free extract, and A0A535Y1H2_UNCCH, SEQ ID NO: 2, 500 mg of lyophilized cell-free extract) to obtain a 357 mM solution of "ACM-H". This solution was stirred at 37°C.

[0132] After stirring for 3.5 hours, an additional 0.5 g of enzyme (A0A535Y1H2_UNCCH, SEQ ID NO: 2, 500 mg, lyophilized cell-free extract) was added. After stirring for 27 hours, 2.47 g of ammonium bicarbonate (31.2 mmol, 1.7 equivalents) was added in combination with the enzyme (Uniprot ID: A0A159Z531_9RHOB, SEQ ID NO: 1, 1 g of lyophilized cell-free extract, and A0A535Y1H2_UNCCH, SEQ ID NO: 2, 500 mg, lyophilized cell-free extract). After a total reaction time of 99 hours, an additional enzyme ((A0A535Y1H2_UNCCH, SEQ ID NO: 2, 500 mg, lyophilized cell-free extract) was added, and the reaction was stopped after 116 hours.

[0133] The final concentration of butyl glufosinate was 56 mmol, as measured by HPLC. This corresponds to a 15 mol% conversion of ACM-H. The enantiomer ratio was 92% L:8% D. After the reaction was complete, the crude reaction mixture was heated at 80°C for 30 minutes and filtered to remove cell lysates. The filtrate was concentrated under reduced pressure. L-glufosinate butyl ester was separated by elution using Dowex-50WX8 200-400(H) with ammonia (1M aqueous solution). The concentrations of L-glufosinate and D-glufosinate butyl esters were determined by HPLC-MS using a Supelco Chirobiotic T2 (gradient of 90% ACN / water to 60% ACN / water over 19 minutes, 0.1% formic acid). Temperature: 20°C, flow rate: 0.8 mL / min. Retention times of glufosinate butyl esters: L-configuration diastereoisomer (13.3 + 13.7 mins); D-configuration (14.5 and 16.8 mins).

[0134] f) Preparation of L-glufosinate butyl ester from ACM (IE6) [ka] Diammonium carbonate (9.6 g) was dissolved in water (100 mL), and the pH was adjusted to 8.5 using HCl (37% aqueous solution). Using the resulting 20 mL buffer solution, 3.25 g of n-butyl(3-cyano-3-acetoxypropyl)methylphosphinat (11.2 mmol, 1 equivalent "ACM", 90%) prepared according to Ex4 was dissolved. The resulting reaction mixture was stirred at 30°C. 0.5 ml of 2 M aqueous solution of MnCl2 was added, followed by the addition of enzymes (Uniprot ID: A0A159Z531_9RHOB, SEQ ID NO: 1, 2 g lyophilized cell-free extract, and A0A535Y1H2_UNCCH, SEQ ID NO: 2, 1 g lyophilized cell-free extract) to obtain a 546 mM solution of "ACM". This solution was stirred at 30°C for 94 hours.

[0135] After this time, the final concentration of butyl glufosinate was measured by HPLC to 55 mmol. This corresponds to a 10 mol% conversion of ACM. The enantiomer ratio was >99%L:<1%D.

[0136] g) Preparation of L-glufosinate from ACM using a heating step (IE6) [ka] 2.16 g of ammonium bicarbonate was dissolved in 12.5 ml of distilled water with stirring. 2 g of n-butyl(3-cyano-3-hydroxypropyl)methylphosphinate ("ACM-H"), prepared according to Ex3, was added with stirring. The resulting solution was stirred at 80°C under microwave heating for 1 hour. Next, the solution was cooled to 37°C, and 250 μl of 2 M aqueous solution of MnCl2 was added, followed by the addition of two types of amidase (Uniprot ID: A0A159Z531_9RHOB, SEQ ID NO: 1, 500 mg of lyophilized cell-free extract) and (A0A535Y1H2_UNCCH, SEQ ID NO: 2, 250 mg of lyophilized cell-free extract). This solution was further stirred at 37°C. After 4.5 hours, amidase (A0A535Y1H2_UNCCH, SEQ ID NO: 2, 250 mg of lyophilized cell-free extract) was added. After 46 hours, the pH was readjusted to 8.5, and two amidases (Uniprot ID: A0A159Z531_9RHOB, SEQ ID NO: 1, 500 mg lyophilized cell-free extract) and (A0A535Y1H2_UNCCH, SEQ ID NO: 2, 250 mg lyophilized cell-free extract) were added again. After 52 hours, HPLC showed a conversion to 15 mol% butyl glufosinate. The enantiomer ratio was 95% L:5% D. After the reaction was complete, the crude reaction mixture was heated at 80°C for 30 minutes, filtered to remove cell lysates, and the filtrate was concentrated under reduced pressure. L-glufosinate butyl ester was separated by elution with ammonia (1 M aqueous solution) using Dowex-50WX8 200-400(H), and further subjected to reverse-phase chromatography (gradient of acetonitrile in water with 0.1% trifluoroacetic acid). A sample of L-glufosinate butyl ester (50 mg) was stirred with an aqueous solution of HCl (18% wt) at 100°C for 5 hours. The enantiomer ratio was determined by chiral HPLC (92% L-glufosinate:8% D-glufosinate).

[0137] h) Preparation of L-glufosinate butyl ester from ACM using a heating step (IE7) [ka] 1.6 g of ammonium bicarbonate was dissolved in 12.5 ml of distilled water with stirring. 2 g of n-butyl(3-cyano-3-acetoxypropyl)methylphosphinate ("ACM", 90%), prepared according to Ex4, was added with stirring. The resulting solution was stirred at 80°C under microwave heating for 3 hours. Next, the solution was cooled to 40°C and the pH was adjusted to 8.5 using aqueous ammonia. 375 μl of 2 M aqueous solution of MnCl2 was added, followed by the addition of two amidases (Uniprot ID: A0A159Z531_9RHOB, SEQ ID NO: 1, 500 mg lyophilized cell-free extract) and (A0A535Y1H2_UNCCH, SEQ ID NO: 2, 250 mg lyophilized cell-free extract). The solution was stirred at 40°C for 93 hours. After this time, HPLC showed that 22 mol% of ACM had been converted to butyl glufosinate. The enantiomer ratio was determined by chiral HPLC (>99%L<1%D).

[0138] Sequence ID 1 (derived from Defluviimonas alba) [ka] Sequence ID No. 2 (derived from bacteria of the phylum Chloroflexi, or green nonsulfur bacteria) [ka]

Claims

1. A process for preparing L-glufosinate and / or a salt thereof or L-glufosinate alkyl ester and / or a salt thereof, wherein the L-glufosinate or the L-glufosinate alkyl ester is of formula (I): 【Chemistry 1】 (In the formula, R １ is H or C １ ~C ８ (It is alkyl.) It has a molecular structure due to, The aforementioned process consists of the following components: (1) Formula (II) 【Chemistry 2】 (In the formula, R １ is H or C １ ~C ８ It is alkyl, and R ２ is H, C １ -C ８ alkyl, C ６ -C １０ aryl, C ７ -C １０ aralkyl, C ４ -C １０ cycloalkyl, or C １ -C １０ acyl) Cyanide or cyanohydrin derivatives, (2) Ammonia sources, (3) Sources of carbon dioxide, and (4) At least two enzymes A process comprising the step of reacting in at least one reaction step.

2. The process according to claim 1, wherein the component (4) comprises (4a) at least one amide hydrolase (EC3.5.2) that acts on a cyclic amide, and (4b) at least one L-amide hydrolase (EC3.5.1) that acts on a linear amide, preferably consisting of these.

3. The process according to claim 1 or 2, wherein the components (1), (2) and (3) are first brought into contact, then component (4) is added, preferably component (4a) first and component (4b) last.

4. The process according to any one of claims 1 to 3, wherein the components (1) to (4), preferably (1) to (4b), are added in the same reaction step, and preferably the reaction is carried out as a one-pot reaction.

5. The cyanohydrin is prepared by the reaction of an aldehyde with a cyanide, preferably hydrogen cyanide or potassium cyanide, wherein the aldehyde is of formula (III): 【Transformation 3】 (In the formula, R １ is H or C １ ~C ８ Alkyl, preferably H or C １ ~C ６ Alkyl, more preferably H or C ２ ~C ４ Alkyl, more preferably H, ethyl or butyl, most preferably ethyl. Having a molecular structure such as The process according to any one of claims 1 to 4.

6. R １ C １ ~C ８ Alkyl, preferably C １ ~C ６ Alkyl, more preferably C ２ ~C ４ The process according to any one of claims 1 to 5, wherein the alkyl, more preferably ethyl or butyl, and most preferably ethyl.

7. The process according to any one of claims 1 to 6, wherein the ammonia source is selected from the list consisting of gaseous ammonia, dissolved ammonia, ammonium salts, or mixtures thereof.

8. The process according to any one of claims 1 to 7, wherein the source of carbon dioxide is gaseous carbon dioxide, dissolved carbon dioxide, carbonate, or a mixture thereof.

9. The aforementioned cyanohydrin or cyanohydrin derivative is defined by formula (IV): 【Chemistry 4】 (In the formula, R ３ C １ ~C ８ Alkyl, preferably C １ ~C ４ Alkyl, more preferably C １ ~C ３ Cyanide hydrin derivatives are alkyl (most preferably methyl), The process according to any one of claims 1 to 8.

10. The process according to any one of claims 1 to 9, wherein the L-glufosinate and / or the salt thereof or the L-glufosinate alkyl ester and / or the salt thereof is prepared with an enantiomer excess, preferably more than 85%, more preferably more than 90%, even more preferably more than 95%, and most preferably more than 99%.

11. The process according to any one of claims 1 to 10, wherein the amide hydrolase (EC3.5.2) acting on the cyclic amide is an L-amide hydrolase (EC3.5.2) acting on the cyclic amide.

12. L-amide hydrolases (EC3.5.2) that act on the cyclic amides include O69809 and its variants, Q846U5_9BACL and its variants, P81006 and its variants, Q84FR6_9MICC and its variants, Q56S49_9BACI and its variants, A1E351_9BACI and its variants, Q28SA7 and its variants, Q45515 and its variants, A0A399DRQ3_9DEIN and its variants, Q55DL0 and its variants, F7X5M8_SINMM and its variants, Q9I676 and its variants, Q44184 and The variants are B5L363 and its variants, P42084 and its variants, P25995 and its variants, Q3Z354 and its variants, B1XEG2 and its variants, Q9F465_PAEAU and its variants, A0A161KD37_9CHLR and its variants, A0A1J4XHR4_9BACT and its variants, A0A1C4QIY5_9ACTN and its variants, A0A0K2UMP4_LEPSM and its variants, A0A159Z531_9RHOB and its variants, E1R8C9_SEDSS and its variants, A0A1F9QT17_9BA CT and its variants, A0A0D8IVV8_9FIRM and its variants, A0A0B5QKE4_CLOBE and its variants, A0A0N1GBZ8_9ACTN and its variants, A0A174ADZ3_9FIRM and its variants, U7V9Q6_9FUSO and its variants, A0A0J1FAI4_9FIRM and its variants, PHYDA_ECOK1 and its variants, A0A0S8H576_9BACT and its variants, A0A1J4J4Y8_9EUKA and its variants, A0A0D5NFS5_9BACL and its variants, A0A0D5NNJ 7_9BACL and its variants, A0A1H2AV66_9BACL and its variants, A0A0Q4RXY0_9BACL and its variants, A0A0Q7SB75_9BACL and its variants, A0A100VRN2_PAEAM and its variants, W4BDJ0_9BACL and its variants, A0A1J5E082_9DELT and its variants, A0A1H5ZFN3_9BACT and its variants, A0A1F8NMM2_9CHLR and its variants, A0A1F8SDV1_9CHLR and its variants, A0A1H1PLX0_9BACT and its variants,A0A0Q5I8X4_9DEIO and its variants, *WP_046170519.1 and its variants, *WP_023514195.1 and its variants, *WP_023516147.1 and its variants, and *ANZ15483.1, their Uniprot ID or NCBI A group of enzymes identified by ID (the latter indicated by an asterisk "*" at the beginning of the ID) is selected, and a mutant is defined as a polypeptide sequence having at least 80%, preferably 90%, and most preferably 95% sequence identity with respect to the respective polypeptide sequence, and preferably the L-amide hydrolase (EC3.5.2) acting on the cyclic amide is Q45515 and its mutants, Q44184 and its mutants, P81006 and its mutants, A0A1C4QIY5_9ACTN and its mutants, A0A0K2UMP4_LEPSM and its mutants, *WP_046170519.1 and its mutants, A0A159Z531_9RHOB and its mutants, and E1R8C9_SEDSS and its mutants, their Uniprot ID or NCBI The process according to any one of claims 1 to 11, wherein a mutant is defined as a polypeptide sequence having at least 80%, preferably 90%, and most preferably 95% sequence identity with respect to the respective polypeptide sequence, selected from a group of enzymes identified by ID (the latter indicated by an asterisk "*" at the beginning of the ID).

13. The L-amide hydrolase (EC3.5.1) that acts on the linear amide is composed of A0A0K9YX84_9BACL and its variants, E3HUL6_ACHXA and its variants, A0A4D7Q548_GEOKU and its variants, Q9F464 and its variants, A0A2S9D976_9MICC and its variants, A0A3E0C996_9BURK and its variants, A0A535Y1H2_UNCCH and its variants, A0A6P2ISL4_BURL3 and its variants, A0A1Y4GC62_9BACT and its variants, and their Uniprot The process according to any one of claims 1 to 12, wherein a mutant is defined as a polypeptide sequence having at least 80%, preferably 90%, and most preferably 95% sequence identity with respect to its respective polypeptide sequence, and preferably the L-amide hydrolase (EC3.5.1) acting on the linear amide is selected from the group of enzymes identified by their Uniprot IDs, consisting of A0A3E0C996_9BURK and its variants, A0A535Y1H2_UNCCH and its variants, A0A6P2ISL4_BURL3 and its variants, and A0A1Y4GC62_9BACT, and the mutant is defined as a polypeptide sequence having at least 80%, preferably 90%, and most preferably 95% sequence identity with respect to its respective polypeptide sequence.

14. Formula (II): 【Transformation 5】 (In the formula, R １ is H or C １ ~C ８ It is alkyl, and R ２ H, C １ ~C ８ Alkyl, C ６ ~C １０ Ariel, C ７ ~C １０ Aralkil, C ４ ~C １０ Cycloalkyl, or C １ ~C １０ (It is an asylum.) Cyanide or cyanohydrin derivatives, A composition comprising L-glufosinate and / or a salt thereof.

15. A method for selectively controlling weeds in an area, preferably an area containing crops of planted seeds or crops resistant to glufosinate, L-glufosinate and / or its salt is contained with respect to D-glufosinate and / or its salt in a proportion of at least 50% enantiomers, preferably with an enantiomer excess of more than 70%, and based on the total amount of the composition, it is greater than 0.01 wt.-% and less than 10 wt.-% of formula (II) 【Transformation 6】 (In the formula, R １ is H or C １ ~C ８ It is alkyl, and R ２ H, C １ ~C ８ Alkyl, C ６ ~C １０ Ariel, C ７ ~C １０ Aralkil, C ４ ~C １０ Cycloalkyl, or C １ ~C １０ (It is an asylum.) A method comprising applying an effective amount of the composition, which contains cyanohydrin or a cyanohydrin derivative, to the area.

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