Method for producing L-glufosinate-ammonium from cyanohydrin or cyanohydrin derivatives

Abstract

Disclosed is a process for the preparation of L-glufosinate-ammonium and / or a salt thereof or an alkyl ester of L-glufosinate-ammonium and / or a salt thereof wherein the L-glufosinate-ammonium or the alkyl ester of L-glufosinate-ammonium has a molecular structure according to formula (I) wherein R1 is H or a C1-C8 alkyl group; wherein the process comprises the step of reacting in at least one reaction step: (1) a cyanohydrin or a cyanohydrin derivative according to formula (II) wherein R1 is H or C1-C8 alkyl, and R2 is H, C1-C8 alkyl, C6-C10 aryl, C7-C10 aralkyl, C4-C10 cycloalkyl, or C1-C10 acyl; (2) an ammonia source; (3) a carbon dioxide source; and (4) at least two enzymes. # imgabs0 # (I) # imgabs1 # (II)

Description

Technical Field

[0001] The present invention relates to the preparation of L-glufosinate-ammonium, in particular to the preparation of L-glufosinate-ammonium from cyanohydrin or cyanohydrin derivatives. Background Art

[0002] US Pat. No. 4,168,963 describes various phosphorus-containing herbicidal active compounds, among which phosphinothricin (2-amino-4-[hydroxy(methyl)phosphonyl]-butyric acid: glufosinate) or its salts have gained commercial importance in the field of agrochemistry. This herbicide, glufosinate, is a non-selective, foliar-applied herbicide and is considered one of the safest herbicides from a toxicological and environmental perspective.

[0003] Processes for the preparation of intermediates for the synthesis of such phosphorus-containing herbicidally active compounds, in particular glufosinate-ammonium, are described, for example, in US Pat. Nos. 4,521,348, 4,599,207 and 6,359,162.

[0004] WO 2015 / 173146 A1 describes the preparation of glufosinate starting from n-butyl (3-cyano-3-hydroxypropyl) methylphosphonate (ACM-H). Similarly, WO 2017 / 037012 A1 describes the preparation of glufosinate starting from n-butyl (3-cyano-3-acetoxypropyl) methylphosphonate (ACM). Summary of the Invention

[0005] Phosphorus-containing cyanohydrin is a valuable intermediate in various fields, particularly in the production of bioactive substances that can be used for pharmaceutical or agricultural chemical fields. ACM and ACM-H are easily obtained from known intermediates in bulk chemicals and racemic glufosinate-ammonium synthesis. However, there is no technology at present directly synthesizing L-glufosinate-ammonium from these intermediates. Therefore, aforesaid method also has the following shortcoming: the glufosinate-ammonium produced by it does not show any enantiomeric excess, particularly for L-glufosinate-ammonium.

[0006] Other commercial chemical synthesis methods for glufosinate also currently produce only a racemic mixture of L- and D-glufosinate (Duke et al. 2010 Toxins 2:1943-1962). However, L-glufosinate is known to be more effective than D-glufosinate in terms of herbicidal efficacy (Ruhland et al. (2002) Environ. Biosafety Res. 1:29-37).

[0007] Therefore, in view of the prior art listed above, it was an object of the present invention to provide a mild process for preparing L-glufosinate-ammonium.

[0008] Another object of the present invention is to provide a safe process for preparing L-glufosinate-ammonium.

[0009] Furthermore, it was an object of the present invention to provide a process for preparing L-glufosinate in enantiomeric excess.

[0010] Finally, it is an object of the present invention to provide a process for the preparation of L-glufosinate from readily available source materials.

[0011] It has surprisingly been found that at least one of the above-mentioned objects can be achieved by the method described herein.

[0012] Therefore, in a first aspect, the present invention relates to a process for preparing L-phosphinothricin and / or its salts or L-phosphinothricin alkyl esters and / or its salts, wherein the L-phosphinothricin or the L-phosphinothricin alkyl esters have a molecular structure according to formula (I):

[0013] (I),

[0014] where R 1 is H or C1-C8 alkyl,

[0015] The method comprises reacting the following components in at least one reaction step:

[0016] (1) Cyanohydrin or cyanohydrin derivative according to formula (II)

[0017] (II),

[0018] in

[0019] R 1 is H or C1-C8 alkyl, and

[0020] R 2 It is H, C1-C8 alkyl, C6-C 10 Aryl, C7-C 10 Aralkyl, C4-C 10 Cycloalkyl, or C1-C 10 acyl group;

[0021] (2) ammonia source;

[0022] (3) a source of carbon dioxide; and

[0023] (4) At least two enzymes.

[0024] In the following, preferred embodiments of the components of the method for preparation are described in further detail. It should be understood that each preferred embodiment is relevant both in itself and in combination with other preferred embodiments.

[0025] In a first preferred embodiment A1 of the first aspect of the present invention, component (4) comprises, preferably consists of: (4a) at least one amidohydrolase (EC 3.5.2) acting on cyclic amides and (4b) at least one L-amidohydrolase (EC 3.5.1) acting on linear amides.

[0026] In a second preferred embodiment A2 of the first aspect of the invention, components (1), (2) and (3) are contacted first, followed by the addition of component (4), preferably component (4a) first and component (4b) last.

[0027] 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, preferably the reaction is carried out as a one-pot reaction.

[0028] In a fourth preferred embodiment A4 of the first aspect of the present invention, the cyanohydrin is prepared by reacting an aldehyde with a cyanide (preferably hydrogen cyanide or potassium cyanide), and wherein the aldehyde has a molecular structure according to formula (III):

[0029] (III)

[0030] where R 1 is 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.

[0031] In a fifth preferred embodiment A5 of the first aspect of the present invention, R of 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.

[0032] In a sixth preferred embodiment A6 of the first aspect of the present invention, the ammonia source is selected from the list consisting of gaseous ammonia, dissolved ammonia, ammonium salts, or mixtures thereof.

[0033] In a seventh preferred embodiment A7 of the first aspect of the present invention, the carbon dioxide source is gaseous carbon dioxide, dissolved carbon dioxide, carbonates, or mixtures thereof.

[0034] In an eighth preferred embodiment A8 of the first aspect of the present invention, the cyanohydrin or cyanohydrin derivative is a cyanohydrin derivative according to formula (IV):

[0035] (IV),

[0036] where R3 It is a C1-C8 alkyl group, preferably a C1-C4 alkyl group, more preferably a C1-C3 alkyl group, and most preferably a methyl group.

[0037] In a ninth preferred embodiment A9 of the first aspect of the present invention, the amidohydrolase (EC 3.5.2) acting on cyclic amides is an L-amidohydrolase (EC 3.5.2) acting on cyclic amides.

[0038] In a second aspect, the present invention further relates to a composition comprising a cyanohydrin or a cyanohydrin derivative according to formula (II)

[0039] (II),

[0040] in

[0041] R 1 is H or C1-C8 alkyl, and

[0042] R 2 It is H, C1-C8 alkyl, C6-C 10 Aryl, C7-C 10 Aralkyl, C4-C 10 Cycloalkyl, or C1-C 10 acyl groups,

[0043] and L-glufosinate and / or its salts.

[0044] In a third aspect, the present invention further relates to a method for selectively controlling weeds in an area, preferably containing a planted batch of seeds or crops resistant to glufosinate, the method comprising:

[0045] Applying to the area an effective amount of a composition comprising L-phosphinothricin and / or its salt obtained by the process of the invention and more than 0.01 wt.-% to less than 10 wt.-% of a cyanohydrin or a cyanohydrin derivative according to formula (II), based on the total amount of the composition, wherein the enantiomeric ratio of L-phosphinothricin and / or its salt relative to D-phosphinothricin and / or its salt is at least 50%, preferably an enantiomeric excess of more than 70%.

[0046] (II),

[0047] in

[0048] R 1 is H or C1-C8 alkyl, and

[0049] R 2 It is H, C1-C8 alkyl, C6-C 10 Aryl, C7-C 10 Aralkyl, C4-C10 Cycloalkyl, or C1-C 10 acyl group. DETAILED DESCRIPTION

[0050] Before describing exemplary embodiments of the present invention in detail, definitions important to understanding the present invention are given.

[0051] As used in this specification and the appended claims, unless the context clearly dictates otherwise, the singular forms "a" and "an" also include the corresponding plural forms. In the context of the present invention, the terms "about" and "approximately" indicate a range of precision that a person skilled in the art would understand still ensure the technical effect of the discussed feature. The terms typically indicate a deviation of ±20%, preferably ±15%, more preferably ±10%, and even more preferably ±5% from the indicated value. It should be understood that the term "comprising" is not limiting. For the purposes of the present invention, the term "consisting of" is considered a preferred embodiment of the term "composed of." If a group is defined below as including at least a certain number of embodiments, this is meant to also include a group that preferably consists only of those embodiments. Furthermore, the terms "first," "second," "third," or "(a)," "(b)," "(c)," "(d)," etc., in the specification and claims are used to distinguish similar elements and are not necessarily intended to describe a sequential or chronological order. It should be understood that the terms so used are interchangeable where appropriate, and that the embodiments of the present invention described herein are capable of operation in sequences other than those described or illustrated herein. Where the terms "first", "second", "third" or "(a)", "(b)", "(c)", "(d)", "i", "ii" etc. refer to steps of a method or use or assay, there is no temporal or temporal time interval coherence between these steps, i.e., the steps may be performed simultaneously or there may be a time interval of seconds, minutes, hours, days, weeks, months or even years between such steps, unless otherwise indicated in this application as described above or below. It is to be understood that this invention is not limited to the particular methodology, protocols, reagents etc. described herein, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention, which will be limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0052] The term "wt.-%" as used throughout this document stands for "percent by weight".

[0053] As used herein, the term "alkyl" denotes in each case a straight-chain or branched alkyl group having generally 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 are 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.

[0054] Depending on the substitution pattern, the compounds according to the invention may have one or more stereocenters. Unless otherwise expressly indicated (e.g. via a chemical formula), the present invention preferably encompasses all stereoisomers (i.e., pure enantiomers, pure diastereomers) of the compounds according to the invention, as well as mixtures thereof (including racemic mixtures).

[0055] The preferred embodiments of the method for preparing L-phosphinothricin and / or its salt or L-phosphinothricin alkyl ester and / or its salt are described in detail below. It should be understood that the preferred embodiments of the present invention are preferred individually or in combination with each other.

[0056] As mentioned above, in one aspect, the present invention relates to a process for preparing L-phosphinothricin and / or its salts, or L-phosphinothricin alkyl esters and / or its salts, wherein the L-phosphinothricin or the L-phosphinothricin alkyl esters have a molecular structure according to formula (I):

[0057] (I),

[0058] where R 1 is H or C1-C8 alkyl,

[0059] The method comprises reacting the following components in at least one reaction step:

[0060] (1) Cyanohydrin or cyanohydrin derivative according to formula (II)

[0061] (II),

[0062] in

[0063] R 1 is H or C1-C8 alkyl, and

[0064] R 2 It is H, C1-C8 alkyl, C6-C 10 Aryl, C7-C 10 Aralkyl, C4-C 10 Cycloalkyl, or C1-C 10 acyl group;

[0065] (2) ammonia source;

[0066] (3) a source of carbon dioxide; and

[0067] (4) At least two enzymes.

[0068] Preferably, component (4) comprises, preferably consists of: (4a) at least one amidohydrolase (EC 3.5.2) acting on cyclic amides and (4b) at least one L-amidohydrolase (EC 3.5.1) acting on linear amides.

[0069] It should be understood that the preparation of L- glufosinate and / or its salt or L- glufosinate alkyl ester and / or its salt encompasses all stereoisomers, suitable salts of the corresponding L- glufosinate or its alkyl ester. Preferably, the alkyl ester of L- glufosinate represents the alkyl-P-ester of L- glufosinate. Further, the corresponding zwitterion is encompassed by formula (I). Suitable salts are illustratively hydrochlorides, ammonium salts and isopropylammonium salts. In this respect, compounds with formula (I) particularly encompass two stereo centers, wherein one stereo center is located at the phosphorus atom and one stereo center is located at the α carbon atom. Compounds with formula (I) particularly encompass all stereoisomers derived from the stereo center at the phosphorus atom.

[0070] Cyanohydrins or cyanohydrin derivatives can be obtained via any suitable preparation process. Suitable processes are described in, inter alia, US 4,521,348 B1, DE 3047024, US 4,599,207 B1, US 6,359,162 B1, CN 102372739 A, and CN 102399240 A.

[0071] In a preferred embodiment, the cyanohydrin is prepared by reacting an aldehyde with a cyanide (preferably hydrogen cyanide or potassium cyanide), wherein the aldehyde has a molecular structure according to formula (III):

[0072] (III)

[0073] where R 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.

[0074] Such a reaction has the advantage that the cyanohydrin is prepared in situ and the solution can be used directly to carry out the process according to the invention.

[0075] Preferably, R of formula (II) and / or (III) 1is H or C1-C8 alkyl. Therefore, preferably, the cyanohydrin or cyanohydrin derivative is a precursor of L-phosphinothricin or a C1-C8 protected alkyl ester of L-phosphinothricin (preferably a C1-C8 protected alkyl-P-ester of L-phosphinothricin).

[0076] Likewise, preferably, R of formula (II) 2 It is H, C1-C8 alkyl, C6-C 10 Aryl, C7-C 10 Aralkyl, C4-C 10 Cycloalkyl, or C1-C 10 More preferably, R 2 Is H or C1-C 10 acyl, and most preferably, R of formula (II) 2 It is H or acetyl.

[0077] Therefore, in the first most preferred embodiment of the present invention, the cyanohydrin is n-butyl (3-cyano-3-hydroxypropyl) methylphosphonate (ACM-H)

[0078] Preferably, the cyanohydrin derivative is a cyanohydrin derivative according to formula (IV):

[0079] (IV),

[0080] where R 3 It is a C1-C8 alkyl group, preferably a C1-C4 alkyl group, more preferably a C1-C3 alkyl group, and most preferably a methyl group.

[0081] Therefore, in the second most preferred embodiment of the present invention, the cyanohydrin derivative is n-butyl (3-cyano-3-acetoxypropyl) methylphosphonate (ACM).

[0082] The method can be carried out in several steps. Preferably, if the method is carried out in several steps, components (1) to (3) are added first, followed by component (4), preferably component (4a) is added first and component (4b) is added last. If the method is carried out in more than one step, it is preferably carried out in two steps, wherein the first step comprises the reaction of components (1) to (3) and the second step comprises the addition of components (4), preferably (4a) and (4b). In another preferred embodiment, the method is carried out in one step, i.e., as a one-pot process.

[0083] Where the process is carried out in two steps, it is preferred that the first step be 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°C to 100°C, more preferably 50°C to 90°C, and most preferably 75°C to 85°C. It has been surprisingly found that the higher the temperature of the first step, the higher the overall conversion. Preferably, the first step is carried out for at least half an hour, more preferably at least one hour. Typically, the first step is complete after one hour.

[0084] In the case of a two-step process, the second step is preferably carried out at a temperature suitable for the enzymes according to component (4), preferably (4a) and (4b), whereby "suitable" means firstly that the enzymes are stable within this temperature range and secondly that the reaction catalyzed by these enzymes preferably occurs with optimal conversion. Preferably, the temperature of the second step is as listed below for the one-pot reaction.

[0085] In case the process is carried out in one step (i.e. as a one-pot process), it is carried out at a temperature in the range of 20°C to 50°C, preferably in the range of 25°C to 45°C, more preferably in the range of 30°C to 42°C, and most preferably in the range of 32°C to 40°C.

[0086] In a preferred embodiment 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 alkaline hydroxide, more preferably sodium hydroxide or potassium hydroxide, and particularly potassium hydroxide. However, most preferably, the pH is adjusted by an ammonia source and / or a carbon dioxide source. In particular, the pH can be controlled by the amount of ammonium salt or carbonate (preferably ammonium carbonate or ammonium bicarbonate) added to the solution, or by the amount of gaseous ammonia and / or carbon dioxide passing through the solution, or by both.

[0087] In a preferred embodiment 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 carbon dioxide. In particular, the pH can be controlled by the amount of ammonium salt or carbonate (preferably ammonium carbonate or ammonium bicarbonate) added to the solution, or by the amount of gaseous ammonia and / or carbon dioxide passing through the solution, or by both.

[0088] 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, still more preferably at 180 to 400 rpm, and most preferably at 200 to 300 rpm.

[0089] Preferably, in the method of the present invention, any suitable amidohydrolase (EC 3.5.2) acting on cyclic amides may be used.

[0090] Such amide hydrolases acting on cyclic amides (EC 3.5.2) that can be used in the methods of the invention include those from Defluviimonas alba, Rhodococcus erythropolis, Streptomyces coelicolor, Brevibacillus agri, Paenarthrobacter aurescens, Arthrobacter crystallopoietes, Bacillus sp. TS-23, Bacillus fordii, Jannaschia sp., Pseudomonas putida, Geobacillus stearothermophilus, Thermus sp., Dictyostelium discoideum), Rhizobium meliloti, Pseudomonas aeruginosa, Rhizobium radiobacter, Pseudomonas fluorescens, Glycine max, Robinia pseudoacacia, Bacillus licheniformis, Aedes aegypti, Agrobacterium fabrum, Arthrobacter sp., etc., preferably Bacillus albus.

[0091] Suitable amidohydrolases acting on cyclic amides (EC 3.5.2) can be selected from the group consisting of: Q8RSQ2 and variants thereof, O69809 and variants thereof, Q846U5_9BACL and variants thereof, P81006 and variants thereof, Q84FR6_9MICC and variants thereof, Q56S49_9BACI and variants thereof, A1E351_9BAC and variants thereof, Q28SA7 and variants thereof, Q59699 and variants thereof, Q45515 and variants thereof, A0A399DRQ3_9DEIN and variants thereof, Q55DL0 and variants thereof, F7X5M8_SINMM and variants thereof, Q9I676 and variants thereof, Q44184 and variants thereof, B5L363 and variants thereof, I1MEH3 and variants thereof , Q6S4R9 and variants thereof, Q65LN0 and variants thereof, Q171F8 and variants thereof, Q8U8Z6 and variants thereof, P42084 and variants thereof, Q88NW7 and variants thereof, P25995 and variants thereof, Q3Z354 and variants thereof, B1XEG2 and variants thereof, Q9F465_PAEAU and variants thereof, Q01262.1 and variants thereof, A0A250DXG4_GEOSE and variants thereof, A1SPN2 and variants thereof, Q9WYH0 and variants thereof, P58329 and variants thereof, A1SGT4 and variants thereof, E3JD18 and variants thereof, HUTI_BDEBA and variants thereof, A0A161KD37_9CHLR and variants thereof, I0 GL27_CALEA and variants, A0A068WGW0_ECHGR and variants, A0A1J4XHR4_9BACT and variants, A0A1C4QIY5_9ACTN and variants, A0A0K2UMP4_LEPSM and variants, A0A0F5Q0A2_9RHIZ and variants, A0A024KHS5_9RHIZ and variants, A0A060UM69_9PROT and variants, A3DKS9_STAMF and variants, W2EWT0_9ACTN and variants, A0A0B1T9I4_OESDE and variants, A0A0A7LM60_9BACT and variants, A0A08 7M7T5_9RHIZ and variants thereof, C0C180_9FIRM and variants thereof, A0A159Z531_9RHOB and variants thereof, R5JTP2_9CLOT and variants thereof, A0A010RM85_9PEZI and variants thereof, E1R8C9_SEDSS and variants thereof, A0A010YEH8_9BACT and variants thereof, A0A031LV69_9CREN and variants thereof, A0A1F9QT17_9BACT and variants thereof, ALLB_BACVZ and variants thereof, HUTI_FLAPJ and variants thereof, A0A073J5J1_9BACT and variants thereof, A0A034W2Q8_BACDO and variants thereof,A0A0D8IVV8_9FIRM and variants thereof, A0A0B5QKE4_CLOBE and variants thereof, A0A098B7X6_DESHA and variants thereof, A0A0B5H4M8_9EURY and variants thereof, A0A0C1YDP1_9ACTN and variants thereof, Q981H2_RHILO and variants thereof, T1EEH7_HELRO and variants thereof, A0A060DTG8_AZOBR and variants thereof, A0A011MGZ5_MANHA and variants thereof, A0A060LYB6_9BACI and variants thereof, S0F3L7_CHOCR and variants thereof, A0A133VNR0_9EURY and variants thereof, A0A13 3U7U9_9EURY and variants thereof, A0A0U2XD52_ECOLX and variants thereof, M1YZY6_NITG3 and variants thereof, T0N9X6_9EURY and variants thereof, T0LMU2_9EURY and variants thereof, A0A0N1GBZ8_9ACTN and variants thereof, HUTI_ANASK and variants thereof, A0A031JUP0_9SPHN and variants thereof, A0A061N9L2_9BACL and variants thereof, A0A017T4D2_9DELT and variants thereof, A0A174ADZ3_9FIRM and variants thereof, A0A021X7D5_9RHIZ and variants thereof, A0A021XAC5_9RHIZ and variants thereof its variants, 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 variants thereof, A0A0C2FCG7_9ACTN and variants thereof, A0A0S8CI48_9CHLR and variants thereof, A0A1F9CZ74_9DELT and variants thereof, A0A0A3YKD1_9ENTR and variants thereof, A0A084R4T2_STACH and variants thereof, A0A070A1Z0_9PROT and variants thereof, A0A1J4J4Y8_9EUKA and variants thereof, R1BR72_EMIHU and variants thereof, R1DD72_EMIHU and variants thereof, A0A1L0FIA0_9ASCO and variants thereof, F7DRE9_ORNAN and variants thereof, A0LK75_SYNFM and variants thereof,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_9FIRM and its variants, A0A0D5NFS5_9BACL and its variants, A0A0D5NNJ7_9BACL and its variants, A0A1H2AV66_9BACL and its variants, A0A0Q4RXY0_9BACL and its variants, A 0A0Q7SB75_9BACL and variants, A0A015NM92_9BACL and variants, A0A100VRN2_PAEAM and variants, W4BDJ0_9BACL and variants, A0A147K2G0_9EURY and variants, A0A0W8FVM4_9ZZZZ and variants, A0A147JXR0_9EURY and variants, E8R8J7_DESM0 and variants, D5U113_THEAM and variants, A0A1F8T9J2_9CHLR and variants, G3C952_9ARCH and variants, Q6YNI0_9MICC and variants, A0A1G0YIQ9_9BAC T and variants thereof, A0A1J5EHQ6_9DELT and variants thereof, A0A1J5E082_9DELT and variants thereof, A0A1C4PKD1_9ACTN and variants thereof, H8GX25_DEIGI and variants thereof, A0A1H5ZFN3_9BACT and variants thereof, A0A0M9Z5S1_9ACTN and variants thereof, A0A1B2HNC5_9PSEU and variants thereof, A0A1B2GNI8_STRNR and variants thereof, A0A1F8LBZ3_9CHLR and variants thereof, A0A1F8NMM2_9CHLR and variants thereof, A0A1F8SDV1_9CHLR and variants thereof, A0A1H1PLX0_9B ACT 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_042803791.1 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,

[0092] 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 the corresponding polypeptide sequence.

[0093] Further preferably, the amidohydrolase (EC 3.5.2) acting on cyclic amides is selected from the group consisting of: O69809 and variants thereof, Q846U5_9BACL and variants thereof, P81006 and variants thereof, Q84FR6_9MICC and variants thereof, Q56S49_9BACI and variants thereof, A1E351_9BACI and variants thereof, Q28SA7 and variants thereof, Q45515 and variants thereof, A0A399DRQ3_9DEIN and variants thereof, Q55DL0 and variants thereof, F7X5M8_SINMM and variants thereof, Q9I676 and variants thereof, Q44184 and variants thereof, B5L363 and variants thereof, P42084 and variants thereof, P25995 and variants thereof, Q 3Z354 and variants thereof, B1XEG2 and variants thereof, Q9F465_PAEAU and variants thereof, A0A161KD37_9CHLR and variants thereof, A0A1J4XHR4_9BACT and variants thereof, A0A1C4QIY5_9ACTN and variants thereof, A0A0K2UMP4_LEPSM and variants thereof, A0A159Z531_9RHOB and variants thereof, E1R8C9_SEDSS and variants thereof, A0A1F9QT17_9BACT and variants thereof, A0A0D8IVV8_9FIRM and variants thereof, A0A0B5QKE4_CLOBE and variants thereof, 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, A0A0D5NNJ7_9BACL and its variants, A0A1H2AV66_9BACL and its variants, A0A0Q4RXY0_9BACL and its variants, A0A0Q7SB75_9BACL and its variants, A 0A100VRN2_PAEAM and variants, W4BDJ0_9BACL and variants, A0A1J5E082_9DELT and variants, A0A1H5ZFN3_9BACT and variants, A0A1F8NMM2_9CHLR and variants, A0A1F8SDV1_9CHLR and variants, A0A1H1PLX0_9BACT and variants, A0A0Q5I8X4_9DEIO and variants, *WP_046170519.1 and variants, *WP_023514195.1 and variants, *WP_023516147.1 and variants, and *ANZ15483.1, 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 the corresponding polypeptide sequence.

[0094] In addition, suitable amide hydrolases acting on cyclic amides (EC 3.5.2) can be selected from the group consisting of: Q846U5_9BACL and variants thereof, P81006 and variants thereof, Q84FR6_9MICC and variants thereof, Q56S49_9BACI and variants thereof, Q45515 and variants thereof, A0A399DRQ3_9DEIN and variants thereof, Q55DL0 and variants thereof, F7X5M8_SINMM and variants thereof, Q9I676 and variants thereof, Q44184 and variants thereof, B1XEG2 and variants thereof, A0A161KD37_9CHLR and variants thereof , A0A159Z531_9RHOB and variants thereof, E1R8C9_SEDSS and variants thereof, A0A1F9QT17_9BACT and variants thereof, A0A0B5QKE4_CLOBE and variants thereof, A0A0N1GBZ8_9ACTN and variants thereof, BAD75708.1 and variants thereof, A0A064AFD7_9FUSO and variants thereof, 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 the corresponding polypeptide sequence.

[0095] In a preferred embodiment, the amidohydrolase (EC 3.5.2) acting on cyclic amides is selected from the group consisting of Q846U5_9BACL and variants thereof, P81006 and variants thereof, Q84FR6_9MICC and variants thereof, A0A399DRQ3_9DEIN and variants thereof, B1XEG2 and variants thereof, A0A161KD37_9CHLR and variants thereof, A0A159Z531_9RHOB and variants thereof, E1R8C9_SEDSS and variants thereof, A0A1F9QT17_9BACT and variants thereof, A0A0B5QKE4_CLOBE and variants thereof, A0A0N1GBZ8_9ACTN and variants thereof, BAD75708.1 and variants thereof, A0A064AFD7_9FUSO, and mixtures thereof, wherein a variant is defined as a polypeptide sequence having at least 80%, preferably 90%, and most preferably 95% sequence identity to the corresponding polypeptide sequence.

[0096] Most preferably, the amidohydrolase (EC 3.5.2) acting on cyclic amides is selected from the group consisting of Q45515, Q44184 and variants thereof, A0A1C4QIY5_9ACTN and variants thereof, A0A0K2UMP4_LEPSM and variants thereof, *WP_046170519.1 and variants thereof, and E1R8C9_SEDSS and variants thereof, A0A159Z531_9RHOB and variants thereof, and mixtures thereof, wherein a variant is defined as a polypeptide sequence having at least 80%, preferably 90%, and most preferably 95% sequence identity to the corresponding polypeptide sequence.

[0097] It will be appreciated that the amidohydrolases acting on cyclic amides (EC 3.5.2) outlined above are indicated in the nomenclature according to the database identifiers of Uniprot (www.uniprot.org) or the NCBI protein database (www.ncbi.nlm.nih.gov / protein), wherein sequences from NCBI are indicated with a "*" at the beginning of the respective database identifiers.

[0098] In a preferred embodiment of the present invention, the amidohydrolase (EC 3.5.2) acting on cyclic amides is an L-amidohydrolase (EC 3.5.2) acting on cyclic amides.

[0099] In a preferred embodiment of the present invention, R in formula (I), (II), (III) and (IV) 1 is H or C1-C6 alkyl, preferably H or C2-C4 alkyl, more preferably H, ethyl or butyl, and most preferably ethyl.

[0100] Suitable L-amide hydrolases (EC 3.5.1) acting on linear amides are preferably selected from the group consisting of EC 3.5.1 hydrolases acting on linear amides, EC 3.5.1.87 N-carbamoyl-L-amino-acid hydrolases, 3.5.1.77 N-carbamoyl-D-amino-acid hydrolases, and mixtures thereof.Suitable L-amidohydrolases (EC 3.5.1) acting on linear amides that can be used in the methods include those selected from the group consisting of A0A7Y0T4N7_9RHIZ and variants thereof, Q88FQ3_PSEPK and variants thereof, Q88Q81_PSEPK and variants thereof, A0A126S6J4_PSEPU and variants thereof, Q8VUL6_9PSED and variants thereof, H9B8T5_9PSED and variants thereof, Q9FB05_9PSED and variants thereof, C0ZCM8_BREBN and variants thereof, C0Z7R5_BREB and variants thereof, A0A0K9YX84_9BACL and variants thereof, E3HUL6_ACHXA and variants thereof, A0A1V9BSS3_9BACI and variants thereof, A0A1V9BSS3_9BACI and variants thereof, Q9F464 and variants thereof, A0A4D7Q548_GEOKU and variants thereof, Q9F464 and variants thereof, A0A2S9D976_9MICC and variants thereof, A0A1I6VZZ4_9RHIZ and variants thereof, A0A1L6RE91_9LACT and variants thereof, A0A3E0C996_9BURK and variants thereof, A0A3M7BGJ4_HORWE and variants thereof, A0A2D7YQN7_9GAMM and variants thereof, A0A535Y1 H2_UNCCH and variants, A0A223E4I5_9BACI and variants, M2VSE9_GALSU and variants, A0A3T0K6C0_9GAMM and variants, A0A416FGE1_9CLOT and variants, D1P143_9GAMM and variants, A0A6P2ISL4_BURL3 and variants, A0A3S6Z2M9_9FIRM and variants, A0A0C1US49_9BACT and variants, A0A1Y4GC62_9BACT and variants, A0A3D3VMN7_9BACT and variants, A0A 2K8L549_9PROT and variants thereof, A0A1G0MC89_9BACT and variants thereof, A0A1M6WYS1_SELRU and variants thereof, A0A2K2BYI3_POPTR and variants thereof, A0A510DYR5_9CREN and variants thereof, A0A5Y3XFN7_SALER and variants thereof, A0A381IB54_CLODI and variants thereof, A0A2V3IQW6_9FLOR and variants thereof, 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 the corresponding polypeptide sequence.Most preferably, the L-amidohydrolase (EC 3.5.1) acting on linear amides is selected from the group consisting of: A0A3E0C996_9BURK and variants thereof, A0A535Y1H2_UNCCH() and variants thereof, A0A6P2ISL4_BURL3() and variants thereof, A0A1Y4GC62_9BACT() and variants thereof, wherein a variant is defined as a polypeptide sequence having at least 80%, preferably 90%, and most preferably 95% sequence identity with the corresponding polypeptide sequence. It will be understood that the L-amidohydrolase (EC 3.5.1) acting on linear amides outlined above is indicated in the nomenclature of the database identifier according to the Uniprot database (www.uniprot.org).

[0101] In a preferred embodiment of the present invention, R in formula (I) 1 is C1-C8 alkyl, preferably C1-C6 alkyl, more preferably C2-C4 alkyl, even more preferably ethyl or butyl, and most preferably ethyl, and the method of the present invention further comprises a step of deprotection under acidic conditions. In this regard, any suitable acid is possible. Preferably, hydrochloric acid or sulfuric acid is used.

[0102] In a preferred embodiment of the present invention, the method further comprises adding a racemase. Any suitable racemase may be possible. Suitable racemases are selected from the group consisting of EC 5.1 racemases, EC 5.1.1 racemases acting on amino acids and derivatives, EC 5.1.99.5 racemases, and mixtures thereof.Suitable racemases that can be used in the methods include those selected from the group consisting of: Q9RYA6_DEIRA and variants thereof, Q9F466 and variants thereof, Q9F466 and variants thereof, A0A7L5BQP9_9RHIZ and variants thereof, Q00924 and variants thereof, F7X6X4_SINMM and variants thereof, A0A6V7ACK5_RHIRD and variants thereof, A0A7Y0XLH3_9RHIZ and variants thereof, A0A5B8XR30_9DELT and variants thereof, A0A533QH78_9PROT and variants thereof, A0A3M9Z0A0_9CYAN and variants thereof, A0A3A0A4T5_9CHLR and variants thereof, A0 A1F6C9P8_HANXR and variants thereof, A0A4S0NM85_9RHIZ and variants thereof, A0A1V5I086_9SPIR and variants thereof, A0A6P0NEY4_9CYAN and variants thereof, A0A2K0YBY8_9SPHN and variants thereof, A0A1H5NHN7_9RHIZ and variants thereof, A0A317KUZ3_9ACTN and variants thereof, A0A430VJ34_THESC and variants thereof, A0A1J5KHA5_9PROT and variants thereof, A0A535LIJ4_9CHLR and variants thereof, A0A2T6KHH4_9RHOB and variants thereof, A0A3G8JSD5_9ACTN and variants thereof its variants, A0A3A9JRT3_9THEO and its variants, A0A2N7WBP6_9BURK and its variants, A0A1A2N8C4_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, A0A434WJY9_9RHI Z and variants thereof, A0A4R7C3Y1_9RHIZ and variants thereof, A0A2T4IRF7_9RHIZ and variants thereof, A0A2E8B427_9PLAN and variants thereof, A0A538D678_9ACTN and variants thereof, A0A1W6Z0D5_9BORD and variants thereof, A0A3P1UKI1_9RHIZ and variants thereof, U2S1Q0_9FIRM and variants thereof, A0A3D5IHC5_AGRSP and variants thereof, A0A3D5JEU3_9DELT and variants thereof, 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 the corresponding polypeptide sequence.It will be appreciated that the racemases outlined above are indicated in the nomenclature according to the database identifiers of the Uniprot database (www.uniprot.org). Most preferably, the racemase is selected from the group consisting of A0A6V7ACK5_RHIRD and variants thereof, A0A2T6KHH4_9RHOB and variants thereof, wherein a variant is defined as a polypeptide sequence having at least 80%, preferably 90%, and most preferably 95% sequence identity with the corresponding polypeptide sequence.

[0103] In a preferred embodiment of the present invention, the method further comprises adding an N-carbamoylamino acid racemase.Any suitable N-carbamoylamino acid racemase may be possible.

[0104] In a preferred embodiment of the present invention, the method further comprises adding a racemase as outlined above, and an N-carbamoyl amino acid racemase.

[0105] In a preferred embodiment of the present invention, all steps of the method are carried out in a single vessel. In this regard, all components are preferably added substantially at the beginning of the reaction.

[0106] 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 in particular at least 70% of the cyanohydrin or cyanohydrin derivative according to formula (II) is converted into L-phosphinothricin and / or its salt, or L-phosphinothricin alkyl ester and / or its salt.

[0107] In the case where an L-phosphinothricin alkyl ester or salt thereof has been produced, the method of the present invention may involve a final step of deprotecting the L-phosphinothricin alkyl ester or salt thereof to produce its L-phosphinothricin chloride, which can be further converted into L-phosphinothricin by increasing the pH value of the solution. A corresponding detailed description of the process steps can be found in EP 0 508 296 A1.

[0108] Preferably, in the process according to the first aspect of the invention, L-phosphinothricin and / or its salts or L-phosphinothricin alkyl esters and / or its salts are prepared in enantiomeric excess, preferably in an enantiomeric excess of greater than 85%, more preferably greater than 90%, even more preferably greater than 95%, and most preferably greater than 99%.

[0109] The administered enzyme may be administered by any suitable means known in the art. In a preferred embodiment of the present invention, the administered enzyme is administered as clarified cell lysate, whole cells or immobilized enzyme.

[0110] Alternatively, some or all components except L-glufosinate can be removed from the bioconversion mixture (optionally the concentrated mixture), and the mixture can then be used directly (and / or with the addition of various adjuvants) to prevent or control weeds. In some examples, the bioconversion mixture can be used directly (and / or with the addition of various adjuvants) to prevent or control weeds.

[0111] Additional steps for further purification of L-phosphinothricin can be added. Such additional purification and separation methods include ion exchange, extraction, salt formation, crystallization, and filtration; each method can be used multiple times or in suitable combinations. If it works, the enzyme can be removed by simple filtration, or if the enzyme is free in solution, it can be removed by using ultrafiltration, using an absorbent (such as diatomaceous earth, cellulose, or carbon), or denaturing using various techniques known to those skilled in the art.

[0112] Ion exchange processes achieve separations by selectively adsorbing solutes onto resins selected for this purpose. Because the product and impurities must be dissolved in a single solution prior to adsorption, the purified product stream is typically concentrated by evaporation or distillation prior to separation. Examples of purification using ion exchange are described in Schultz et al. and EP 0 249 188 (A2).

[0113] Purification can be achieved by adding a suitable acid (including hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, acetic acid, etc.) to form an insoluble salt of L-phosphinothion. Similarly, purification can be achieved by adding a suitable base to form an insoluble salt. Useful bases include hydroxides, carbonates, sulfates and phosphates of alkaline earth metals or hydroxides, carbonates, sulfates and phosphates of alkaline earth metals. Other bases containing nitrogen can be used, including ammonia, hydroxylamine, isopropylamine, triethylamine, tributylamine, pyridine, 2-picoline, 3-picoline, 4-picoline, 2,4-lutidine, 2,6-lutidine, morpholine, N-methylmorpholine, 1,8-diazabicyclo[5.4.0]undec-7-ene and dimethylethanolamine. It may be advantageous to concentrate the mixture or add a solvent (or both) to maximize the yield and optimize the purity of the desired salt. Solvents suitable for this purpose include those with very low solubility of the desired salt (such solvents are generally referred to as "anti-solvents"). Salts of L-glufosinate can be converted into a form of glufosinate suitable for formulation by standard methods known to those skilled in the art. Alternatively, L-glufosinate can be isolated as a zwitterion.

[0114] US 9,255,115 B2 describes how the hydrochloride salt of L-glufosinate can be converted to a zwitterionic form using a base (e.g., sodium hydroxide or sodium methoxide), which can then be crystallized from an aqueous alcohol solvent to provide relatively high-purity L-glufosinate. This method has the advantage that the resulting crystalline L-glufosinate is non-hygroscopic and, therefore, can maintain a higher purity when exposed to humidity over time compared to amorphous L-glufosinate.

[0115] Other salts of L-glufosinate are known in the art. US 5,767,309 and US 5,869,668 have taught the use of chiral alkaloids and racemic glufosinate to form diastereomeric salts. Realization purification is because the amount that the salt of L-glufosinate precipitates out from solution is much greater than the amount that the salt of corresponding D-glufosinate precipitates out from solution. Therefore, if desired, method can be used together with the present invention to obtain the L-glufosinate with high enantiomeric excess.

[0116] Optionally, purification can be achieved by: first making one or more impurity crystals, by filtering impurity removal, then by forming a salt as previously described further purified L-phosphine-ammonium from the gained filtrate.If unreacted amine donor can be partially or completely separated and used in subsequent reaction, then this is favourable.Similarly, partially or completely separated, unreacted cyanohydrin or cyanohydrin derivatives according to formula (II) can be recycled for subsequent reaction.

[0117] Extraction can be used to purify the product. DE 3920570 C2 describes a process in which excess glutamic acid (used as an amine donor) is precipitated by adjusting the solution pH to 3.7 to 4.2 with sulfuric acid. After filtering the glutamic acid, the filtrate pH is lowered to 1-2, and other impurities are then extracted into a solvent. After extraction and concentration, ammonia is added to the aqueous solution to a pH of 5-7, and ammonium sulfate is then precipitated. The ammonium sulfate is removed via filtration, and the resulting filtrate is concentrated to obtain the ammonium salt of L-phosphinothricin.

[0118] In some embodiments, the present invention provides the method for separating L-phosphine-ammonium or its salt.For example, in order that solid is transported to preparation site or use site, may need to separate L-phosphine-ammonium or its salt.Can use typical industrial separation method, for example filtering, centrifugal etc.The product of separation needs to remove water, volatile impurity and solvent (if existing) and typical industrial drying equipment can be used for this purpose usually.The example of such equipment comprises baking oven, rotary drum dryer, stirring dryer etc.In some cases, use spray dryer may be favourable.

[0119] After purification, there is no need to produce a solid product. If the preparation of L-glufosinate occurs at the same location for L-glufosinate production, this may be advantageous. L-glufosinate and its many salts are soluble in water, and water is a convenient liquid for preparing products. For example, the amine donor is separated via filtration, and the resulting filtrate is concentrated by distillation. The pH of the filtrate can be adjusted to the desired value, and the resulting solution can be used as is or blended with the formulation ingredients. In another example, the slurry of L-glufosinate or a salt thereof can be prepared as described above and separated via filtration. Solid can be dissolved directly on the filter by adding water or a suitable solvent to obtain L-glufosinate solution.

[0120] As mentioned above, in a second aspect, the present invention further relates to a composition comprising a cyanohydrin or a cyanohydrin derivative according to formula (II)

[0121] (II),

[0122] in

[0123] R 1 is H or C1-C8 alkyl, and

[0124] R 2 It is H, C1-C8 alkyl, C6-C 10 Aryl, C7-C 10 Aralkyl, C4-C 10 Cycloalkyl, or C1-C 10 acyl groups,

[0125] and L-glufosinate and / or its salts.

[0126] Suitable salts are the hydrochloride, ammonium and isopropylammonium salts. It will also be understood that the corresponding zwitterion of L-glufosinate is also encompassed.

[0127] In a preferred embodiment 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.-%, and in particular 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.

[0128] In a preferred embodiment 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.-%, still more preferably 60 to 94 wt.-%, and in particular at least 70 to 90 wt.-% or at least 80 to 90 wt.-%, based on the total amount of cyanohydrin or cyanohydrin derivative according to formula (II), and L-glufosinate and / or its salt.

[0129] The composition may comprise the cyanohydrin or cyanohydrin derivative according to formula (II) in an amount of 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 in particular up to 1 wt.-%, based on the total amount of the cyanohydrin or cyanohydrin derivative according to formula (II) and L-phosphinothricin and / or its salt.

[0130] In a preferred embodiment of the present invention, the composition described herein can be used directly as a herbicidal composition or as an ingredient in a formulated herbicidal product.

[0131] The compositions described herein can be used to prevent or control weeds in fields applied to crop plants. The compositions can be formulated into liquids for spraying in fields. Glufosinate (preferably L-glufosinate) is provided in the composition in an effective amount. As used herein, an effective amount means about 10 grams of active ingredient per hectare to about 1,500 grams of active ingredient per hectare, for example, about 50 grams to about 400 grams or about 100 grams to about 350 grams. In certain embodiments, the active ingredient is L-glufosinate. For example, the amount of L-glufosinate in the composition can be about 10 grams, about 50 grams, about 100 grams, about 150 grams, about 200 grams, about 250 grams, about 300 grams, about 350 grams, about 400 grams, about 500 grams, about 550 grams, about 600 grams, about 650 grams, about 700 grams, about 750 grams, about 800 grams, about 850 grams, about 900 grams, about 950 grams, about 1,000 grams, about 1,050 grams, about 1,100 grams, about 1,150 grams, about 1,200 grams, about 1,250 grams, about 1,300 grams, about 1,350 grams, about 1,400 grams, about 1,450 grams, or about 1,500 grams of L-glufosinate per hectare.

[0132] The herbicidal compositions described herein (including concentrates that require dilution before application to plants) contain L-glufosinate (i.e., the active ingredient), optionally some residual cyanohydrin or cyanohydrin derivative according to formula (II), and one or more adjuvant components in liquid or solid form.

[0133] The composition is prepared by mixing the active ingredient with one or more adjuvants (e.g., diluents, extenders, carriers, surfactants, organic solvents, wetting agents, or conditioning agents) to provide a composition in the form of a finely dispersed particulate solid, micropellets, solutions, dispersions, or emulsions. Thus, the active ingredient can be used with an adjuvant (e.g., a finely dispersed solid, a liquid of organic origin, water, a wetting agent, a dispersant, an emulsifier, or any suitable combination of these). From an economic and convenient perspective, water is a preferred diluent. However, not all compounds are resistant to hydrolysis, and in some cases, this may require the use of a non-aqueous solvent medium, as will be appreciated by those skilled in the art.

[0134] Optionally, one or more other components can be added to the composition to produce the herbicidal composition of preparation. The composition of such preparation can include L-glufosinate-ammonium, carrier (for example, diluent and / or solvent) and other components. The composition of preparation includes the L-glufosinate-ammonium of effective dose.

[0135] A diluent may also be included in the formulated composition. Suitable diluents include water and other aqueous components. Optionally, the diluent is present in an amount required to produce a composition ready for packaging or use.

[0136] The herbicidal compositions described herein (particularly liquids and soluble powders) may contain one or more surfactants as additional adjuvant components in an amount sufficient to make the given composition readily dispersible in water or oil. Incorporating surfactants into the composition greatly enhances the efficacy of the composition. As used herein, surfactants include wetting agents, dispersants, suspending agents, and emulsifiers. Anionic, cationic, and nonionic agents can be used with equal ease.

[0137] Suitable wetting agents include alkylbenzene and alkylnaphthalene sulfonates, sulfated fatty alcohols, amines or amides, long-chain acid esters of sodium isosulfate, esters of sodium sulfosuccinate, sulfated or sulfonated fatty acid ester petroleum sulfonates, sulfonated vegetable oils, ditertiary acetylenic diols, polyoxyethylene derivatives of alkylphenols (particularly isooctylphenol and nonylphenol), and polyoxyethylene derivatives of mono-higher fatty acid esters of hexitol anhydrides (e.g., sorbitan). Exemplary dispersants include methylcellulose, polyvinyl alcohol, sodium lignin sulfonate, polymerized alkylnaphthalene sulfonates, sodium naphthalene sulfonate, polymethylene bis-naphthalene sulfonate, and sodium N-methyl-N-(long-chain acid) laurate.

[0138] Water-dispersible powder compositions containing one or more active ingredients, an inert solid extender, and one or more wetting agents and dispersants can be prepared. Inert solid extenders are typically of mineral origin, such as natural clays, diatomaceous earth, and synthetic minerals derived from silicon dioxide. Examples of such extenders include kaolinite, attapulgite clay, and synthetic magnesium silicate. The water-dispersible powders described herein may optionally contain from about 5 to about 95 parts by weight of the active ingredient (e.g., from about 15 to 30 parts by weight of the active ingredient), from about 0.25 to 25 parts by weight of the wetting agent, from about 0.25 to 25 parts by weight of the dispersant, and from 4.5 to about 94.5 parts by weight of the inert solid extender, all parts being by weight of the total composition. When desired, from about 0.1 to 2.0 parts by weight of the solid inert extender may be replaced by a corrosion inhibitor or a defoamer, or both.

[0139] Aqueous suspensions can be prepared by dissolving or mixing together and grinding an aqueous slurry of the water-insoluble active ingredient in the presence of a dispersant to obtain a concentrated slurry of very finely divided particles. The resulting concentrated aqueous suspension is characterized by its extremely small particle size, resulting in very uniform coverage when diluted and sprayed.

[0140] Emulsifiable oils are typically solutions of the 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. Emulsifiable oil compositions typically contain about 5 to 95 parts active ingredient, about 1 to 50 parts surfactant, and about 4 to 94 parts solvent, all parts by weight based on the total weight of the emulsifiable oil.

[0141] The compositions described herein may also contain other additives used as adjuvants or in combination with any of the above adjuvants, such as fertilizers, plant poisons and plant growth regulators, pesticides, etc. The compositions described herein may also be mixed with other materials (e.g., fertilizers, other plant poisons, etc.) and applied in a single application.

[0142] In each formulation type described herein (eg, liquid and solid formulations), the concentration of the active ingredient is the same.

[0143] It will be appreciated that the herbicidal compositions may be used in combination with other herbicides. The herbicidal compositions of the present invention are typically applied in combination with one or more other herbicides to control a wider range of undesirable vegetation. When used in combination with other herbicides, the presently claimed compounds may be formulated with the other herbicide(s), mixed in a tank with the other herbicide(s), or applied sequentially with the other herbicide(s). Some herbicides that can be used in combination with the compounds of the present invention include: amide herbicides, such as dipropylene glycol, fluazifop, benoxydoprop, benzylamine, bromobutyric acid, benzophenone azole, CDEA, chlorsulfuron, tricyclam, dimethenamid, dimethenamid, bisbencarb, triazole sulfone, aniprofen, tetrazolam, fluazifop, flufenacet, flunitrazepam, dimethomorph ... Acesulfame, flufenacet, flufenacet, mefenacet, flusulfamide, oxadiazine, heptamidine, naproxen, mechlorethamine, flupyrad, and propanil; arylalanine herbicides such as Xinyanling, mechlorethamine, and mechlorethamine-M; chloroacetanilide herbicides such as acetochlor, methylchlor, butachlor, butachlor, isobutachlor, acetochlor, dimethochlor, metazachlor, isopropyl metolachlor, S-isopropyl metolachlor, pretilachlor, pyraclostrobin, isopropyl metolachlor, propargyl phthalate, terbutachlor, dimethenamid, and dimethenamid; sulfonylanilide herbicides such as flufenacet-butyl, pyriproxyfen, and flufenacet-M Amines; sulfonamide herbicides such as sulfamethoxazole, carbamate, acefonsulfuron and chlorpyrifos; antibiotic herbicides such as bialaphos; benzoic acid herbicides such as chlorpyrifos, dicamba, 2,3,6-TBA and chlorpyrifos; pyrimidinyloxybenzoic acid herbicides such as bispyribac-butyl and pyrimidinylsulfuron; pyrimidinylthiobenzoic acid herbicides such as pyrimidinylsulfuron; phthalic acid herbicides such as chlorthalid; pyridinecarboxylic acid herbicides such as aminopyralid, clopyralid and picloram; quinoline carboxylic acid herbicides such as quinclorac and clomeclorac; arsenic herbicides such as dimethylarsinate, CMA, DSMA, hexamethasone, Fluoroarsenates, MAA, MAMA, MSMA, potassium arsenite and sodium arsenite; benzoylcyclohexanedione herbicides, such as mesotrione, sulcotrione, furasulfotrione and tembotrione; benzofuranyl alkyl sulfonate herbicides, such as furosulfuron and ethoxyfurosulfuron; carbamate herbicides, such as sulfamethoxazole, teroxazolidinone, thiamethoxazole, benzylamine, benzosulfuron, teroxazolidinone and teroxazolidinone; phenylcarbamate herbicides, such as avenacin, BCPC, carbamate, carbamate, CEPC, chlorpropham, chlorpropham, CPPC, betaine, cypermethrin, ethyl betaine, betaine and chlorpyrifos;Cyclohexene oxime herbicides, such as fenpropimorph, butoxycycline, clethodim, cyclobutene, cycloxydim, cyclohexadione, sethoxydim, dapoxetine and trimethylol; cyclopropyl isoxazole herbicides, such as isoxaclorac and isoxaclorac; dicarboximide herbicides, such as fluazifop, indolesulfuron, fluphenazine, flufenac, fluazifop-butyl and clodinafop-butyl; dinitroaniline herbicides, such as fluazifop, dimethoate, dimethoate, butyralid, chlorpyrifos, isopropylalin, flufenacet, sulfalyl, amoxifen, pendimethalin, aminopropylalin, cyprofluorfen and trifluralin; dinitrophenol herbicides, such as difeno, nitropropenol, pentotropol, diphenylol, terephthalate, DNOC, nitrosoquinol and dioxil; diphenyl ether herbicides Herbicides such as fluazifop, nitrophenyl ether herbicides such as acifluorfen, beniflox, fenpyroxen, methoxyfenpyroxen, fenpyroxen, aniloxam, anisulfoxam, penflufenacil, fluazifop-butyl, fluazifop-butyl, fluazifop-butyl, fluazifop-butyl, fluazifop-butyl and oxyfluorfenacil; dithiocarbamate herbicides such as dazomet and methamphetamine; halogenated aliphatic herbicides such as arolac, trichloropropionic acid, dalapon, tetrafluoropropionic acid, hexachloroacetone, iodomethane, methyl bromide, monochloroacetic acid, SMA and TCA; imidazolinone herbicides such as imazapic, imazapic, imazapic, imazaquin and imazapic; inorganic herbicides such as ammonium sulfamate, borax, calcium chlorate, Copper sulfate, ferrous sulfate, potassium azide, potassium cyanate, sodium azide, sodium chlorate and sulfuric acid; nitrile herbicides such as furobromoxanil, bromoxynil, chlorobenzonil, dichlorvos, iodoxanil, ioxadone and bispyribac; organophosphorus herbicides such as methylaminophosphine, cypermethrin, dithiophos, bialaphos, cypermethrin, 2,4-DEP, DMPA, EBEP, cypermethrin, glyphosate and cypermethrin; phenoxy herbicides such as bromophenoxime, chlorfenapyr, 2,4-DEB, 2,4-DEP, pentofenac, cypermethrin, cypermethrin, ethamethamide, chlorophenoxyethanol and trifloxyfen; phenoxyacetic acid herbicides such as 4-CPA, 2,4-D, 3,4-DA, MCPA, 2-methyl-4-chloroethylthioate (MCPA-thioester) ioethyl) and 2,4,5-T; phenoxybutyric acid herbicides, such as 4-CPB, 2,4-DB, 3,4-DB, MCPB and 2,4,5-TB; phenoxypropionic acid herbicides, such as acetic acid, 4-CPP, 2,4-dichlorprop, 2,4-dichlorprop, 3,4-DP, 2,4,5-tetrachloroprop, 2-methyl-4-chloroprop and 2-methyl-4-chloroprop; aryloxyphenoxypropionic acid herbicides, such as clodinafop-butyl, clodinafop-butyl, chlorbutazone, cyhalofop-butyl, diclofop-butyl, fenoxaprop-butyl, famfop-butyl, thiazolin-butyl, fluazifop-butyl, fluazifop-butyl, fluazifop-butyl, isothiazolin, oxadiazol-butyl, cypermethrin, quizalofop-butyl, quizalofop-butyl and trifloxylic acid;Phenyldiamine herbicides, such as dimethoate and aminopropyrin; pyrazolyl herbicides, such as pyraclostrobin, pyrazol, pyrasulfuron, pyrazosulfuron, and pyraclostrobin; pyrazoylphenyl herbicides, such as isopropylpyraclostrobin and pyraclostrobin; pyridazine herbicides, such as chlorpyrifos, chlorpyrifos, and chlorpyrifos; pyridazinone herbicides, such as bromomycin, chlorpyrifos, chlorpyrifos, fluazifop-butyl ... , diethyl chlorpyrifos, wild fenquat, diquat, quat and paraquat; thiocarbamate herbicides such as butaqua, chlorpyrifos, avena cava, EPTC, cypermethrin, isopropyl chlorpyrifos, methiocarb, cypermethrin, pyralid, cypermethrin, cypermethrin, cypermethrin, cypermethrin, cypermethrin and vemolate; thiocarbonate herbicides such as dimethoate, EXD and cypermethrin; thiourea herbicides such as cypermethrin; triazine herbicides such as isopropylamine, triazine fluazifop and cyanuric acid; chlorotriazine herbicides such as atrazine, clonazine, cyanoacetonitrile, cyclothiazine, glycyrrhizin, cypermethrin, cyproconazole, cyproconazole, cyproconazole, chlorpyrifos ... Simazine, terbuthylazine and thiabendazole; methoxytriazine herbicides such as atraton, terbuthone, promethazine, sec-butylton, simazine and terbuthone; methylthiotriazine herbicides such as ametryn, azidopropynil, cyanamide, dichlorvos, isopentamycin, chlorfenapyr, promethazine, simetropyr and terbuthion; triazinon herbicides such as fenthiocarb, terbuthiocarb, hexazinone, cymetrazone, metamitron and metribuzin; triazole herbicides such as cyproconazole, benzophenone, yellow triazole and fluamidazole; triazolone herbicides such as amicarbazone, benzylpyridinol, foramsulfuron, flucarbazone, propoxysulfuron, mesotrione and thiamethoxam-methyl; triazolopyrimidine herbicides such as clorac-sulam, bisclosulam, bisfloxacin, chlorpyrifos ... sulfamethoxam, sulfamethoxam, penoxsulam and pyrasulfuron; uracil herbicides, such as fluazifop-butyl, bromocriptine, fluazifop-butyl, isothiocarb, lenacil and teracil; 3-phenyluracil; urea herbicides, such as thiamethoxam, benzylpyridinium, cypermethrin, chloranil, fluazifop-butyl, isothiocarb, axilamide, xylazine, teroxazolamide and thiamethoxam; phenylurea herbicides, such as thiamethoxam, clodinuron, chlorbromide, ethoxyconazole, chlorotoluron, cypermethrin, cypermethrin, oxazolamide, diuron, fenuron, fluazifop-butyl, flusulfuron, isoproturon, flusulfuron, mefenamuron, methylthiamethoxam, pyranuron, bromothiocarb, methoxythiocarb, chloranil, mefenamuron, chlorpromazine, paraflumuron, acyluron, lenacil, tetraflumuron and thiadipyrone;Pyrimidinylsulfonylurea herbicides, such as amidosulfuron-methyl, tetrazosulfuron-methyl, bensulfuron-methyl, chlorimuron-methyl, cyprosulfuron-methyl, ethoxysulfuron-methyl, flazasulfuron-methyl, flutosulfuron-methyl, flutosulfuron-methyl, chlorimuron-methyl, cyprosulfuron-methyl, ethoxysulfuron-methyl, flazasulfuron-methyl, flutosulfuron-methyl, chlorim ... Herbicides such as acrolein, acrolein, chlorpyrifos, chlorpyrifos, chlorpyrifos, bentazone, bicyclam, buthidone, calcium cyanamide, chlorpyrifos, fluazifop, oat acid, fluazifop, chlorpyrifos, cypermethrin, clomazone, CPMF, mixed methylphenols, o-dichlorobenzene, piperocarb, endosulfan, chlorpyrifos, fluazifop, fluazifop, furazolidone, fluazifop, chlorpyrifos ... The herbicidal compositions of the present invention can also be combined with glyphosate or 2,4-D for use on glyphosate-tolerant or 2,4-D-tolerant crops. It is generally preferred to use the compositions of the present invention in combination with herbicides that are selective for the crops being treated and that complement the spectrum of weeds controlled by the compositions at the application rates employed. It is further generally preferred to apply the compositions of the present invention and other complementary herbicides simultaneously, either as a combined formulation or as a tank mix.

[0144] As mentioned above, in a third aspect, the present invention further relates to a method for selectively controlling weeds in an area, preferably containing a planted batch of seeds or crops resistant to glufosinate, the method comprising:

[0145] Applying to the area an effective amount of a composition comprising L-phosphinothricin and / or its salt obtained by the process of the invention and more than 0.01 wt.-% to less than 10 wt.-% of a cyanohydrin or a cyanohydrin derivative according to formula (II), based on the total amount of the composition, wherein the enantiomeric ratio of L-phosphinothricin and / or its salt relative to D-phosphinothricin and / or its salt is at least 50%, preferably an enantiomeric excess of more than 70%.

[0146] (II),

[0147] in

[0148] R 1 is H or C1-C8 alkyl, and

[0149] R 2 It is H, C1-C8 alkyl, C6-C 10 Aryl, C7-C 10 Aralkyl, C4-C 10 Cycloalkyl, or C1-C 10 acyl group.

[0150] In a preferred embodiment of the present invention, the composition comprises L-glufosinate and / or its salt in an enantiomeric ratio of 50% to 99%, preferably an enantiomeric ratio of 60% to 98%, more preferably 70% to 95%, and in particular 80% to 90% relative to D-glufosinate and / or its salt.

[0151] In a preferred embodiment of the present invention, the composition comprises 0.02 to 8 wt.-%, preferably 0.03 to 5 wt.-%, more preferably 0.05 to 3 wt.-%, and in particular 0.1 to 2 wt.-% of cyanohydrin or a cyanohydrin derivative according to formula (II), based on the total amount of the composition.

[0152] It will be appreciated that the compositions may contain the same adjuvants and / or other herbicides as described in more detail above.

[0153] Composition as herein described can be used for being applied to the field of crop plants for preventing or controlling weeds.Composition can be formulated into the liquid for spraying in the field.L-glufosinate-ammonium is provided in the composition with an effective amount.As used herein, effective amount means about 10 grams of active ingredients per hectare to about 1,500 grams of active ingredients per hectare, for example, about 50 grams to about 400 grams or about 100 grams to about 350 grams.In certain embodiments, active ingredient is L-glufosinate-ammonium. For example, the amount of L-glufosinate in the composition can be about 10 grams, about 50 grams, about 100 grams, about 150 grams, about 200 grams, about 250 grams, about 300 grams, about 350 grams, about 400 grams, about 500 grams, about 550 grams, about 600 grams, about 650 grams, about 700 grams, about 750 grams, about 800 grams, about 850 grams, about 900 grams, about 950 grams, about 1,000 grams, about 1,050 grams, about 1,100 grams, about 1,150 grams, about 1,200 grams, about 1,250 grams, about 1,300 grams, about 1,350 grams, about 1,400 grams, about 1,450 grams, or about 1,500 grams of L-glufosinate per hectare.

[0154] The present invention is further illustrated by the following examples.

[0155] Examples

[0156] Enzyme preparation

[0157] a) Cloning of enzyme genes (Example 1)

[0158] The amino acid sequences of the corresponding enzymes were identified from public databases (UniProt, https: / / www.uniprot.org; NCBI Protein Database, https: / / www.ncbi.nlm.nih.gov / protein). Sequences from NCBI are indicated by an "*" at the beginning of the corresponding database identifier. The corresponding DNA sequences were derived using standard codon usage for Escherichia coli. The DNA sequences were synthesized (BioCat GmbH) and cloned into the plasmid pDHE19.2 (Ress-Loeschke, M. et al., DE 19848129, 1998, (BASF AG)). The resulting plasmid was used to transform competent cells of E. coli strains (TG10, pAgro, pHSG575) (E. coli TG10 (Kesseler, M. et al., WO 2004050877A1, 2004, (BASF AG))) (Chung, CT et al., Proc Natl Acad Sci USA, 1989, 86, 2172): rhaA-derivatives of E. coli TG1 were transformed with pHSG575 (Takeshita, S. et al., Gene, 1987, 61, 63) and pAgro4 (pBB541 in Tomoyasu, T. et al., Mol. Microbiol., 2001, 40, 397).

[0159] b) Recombinant production of enzymes (Example 2)

[0160] Preparation of biocatalysts in shake flasks

[0161] E. coli TG10 cells carrying the recombinant plasmid for the enzyme were inoculated with 2 ml of LB medium supplemented with 100 µg / ml ampicillin, 100 µg / ml spectinomycin, and 20 µg / ml chloramphenicol (Bertani, G., J Bacteriol, 1951, 62, 293). The resulting preculture was incubated at 37°C with agitation at 250 rpm for 5 hours. One ml of the preculture was used to inoculate 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-ß-D-thiogalactopyranoside, and 0.5 g / l rhamnose in a 500 ml baffled Erlenmeyer flask. The culture was incubated at 37°C with shaking for 18 hours. The biomass was then harvested by centrifugation at 3220 x g for 10 min at 8°C. The supernatant was discarded, and the cell pellet was resuspended in 8 ml of 100 mM HEPES buffer (pH 8.2) supplemented with 1 mM MnCl₂. If whole-cell biotransformation is being performed, the cell suspension can be used without any further preparation for synthesis. If using clarified cell lysate, 5 ml of the cell suspension was distributed into five reaction tubes containing Lysis Matrix B (0.7 ml of quartz beads (Ø 0.1 mm), MP Biomedicals). The tubes were cooled on ice, and the cells were then disrupted using a homogenizer (Peqlab Precellys24, VWR) for two 30-second cycles. The samples were cooled on ice between cycles. The resulting cell-free lysate was clarified by centrifugation at 20817 x g for 10 min at 8°C. The supernatant was separated, and fractions from the same batch (clarified cell lysate) were pooled.

[0162] Fermentation-derived whole-cell biocatalyst production

[0163] Escherichia coli TG10 cells harboring plasmids pAgro4 and pHSG575 were transformed with the pDHE plasmid encoding the target protein. Transformants were cultured on LB agar plates supplemented with 100 µg / ml ampicillin, 100 µg / ml spectinomycin, and 20 µg / ml chloramphenicol.

[0164] Pre-culture medium:

[0165] EcoK12 solution

[0166] Ultrapure water 1.0 kg

[0167] Citric acid monohydrate 40.0 g

[0168] Zinc sulfate heptahydrate 11.0 g

[0169] Diammonium ferric sulfate hexahydrate 8.6 g

[0170] Manganese sulfate monohydrate 3.0 g

[0171] Copper sulfate pentahydrate 0.8 g

[0172] Cobalt sulfate heptahydrate 0.09 g

[0173] Sterilize by filtration using a 0.2 µm pore size filter.

[0174] Part 1

[0175] Ultrapure water 1.0 kg

[0176] Citric acid monohydrate 3.4 g

[0177] Magnesium sulfate heptahydrate 2.4 g

[0178] Calcium chloride dihydrate 0.1 g

[0179] EcoK12 solution 20 g

[0180] Sodium hydroxide solution 25% was used to adjust the pH to 6.6

[0181] Part 2

[0182] Ultrapure water 500 g

[0183] Potassium dihydrogen phosphate 26.6 g

[0184] Diammonium hydrogen phosphate 8.0 g

[0185] Sodium hydroxide solution 25% was used to adjust the pH to 6.4

[0186] Part 3

[0187] Ultrapure water 500 g

[0188] Glycerol 99% 36.0 g

[0189] Sodium gluconate 24.0 g

[0190] Phosphoric acid 20% is used to adjust the pH to 6.6

[0191] All 3 parts were sterilized at 121°C for 30 minutes.

[0192] Vitamin solution

[0193] Ultrapure water 100 g

[0194] Thiamine hydrochloride 1.0 g

[0195] Vitamin B 12 0.5 g

[0196] Sterilize by filtration using a 0.2 µm pore size filter

[0197] To form the final preculture, fractions 1, 2, and 3 were combined and 2.0 ml of the vitamin solution was added. Furthermore, the medium was supplemented with 100 µg / ml ampicillin, 100 µg / ml spectinomycin, and 20 µg / ml chloramphenicol. Several transformants were scraped from LB agar plates and used to inoculate 2 x 100 g of preculture in 1 L baffled Erlenmeyer flasks. These precultures were incubated at 37°C and 150 rpm. When the OD600 reached 12, the entire preculture was used to inoculate the main culture.

[0198] Main culture medium:

[0199] Part 4

[0200] 9.6 kg of ultrapure water

[0201] Citric acid monohydrate 21.1 g

[0202] Potassium dihydrogen phosphate 173.6 g

[0203] Diammonium hydrogen phosphate 52.8 g

[0204] Magnesium sulfate heptahydrate 15.1 g

[0205] Calcium chloride dihydrate 0.7 g

[0206] EcoK12 solution 123 g

[0207] Sodium hydroxide solution 25% to adjust pH to 6.4

[0208] Pluriol P 20001 ml

[0209] Part 4 was sterilized at 125°C for 45 min.

[0210] Part 5

[0211] 300 g ultrapure water

[0212] Thiamine hydrochloride 151 mg

[0213] Vitamin B12 30.2 mg

[0214] Ampicillin sodium 1000 mg

[0215] Spectinomycin hydrochloride 500 mg

[0216] Chloramphenicol 200 mg

[0217] Part 5 Sterilize by sterile filtration using a filter unit with a pore size of 0.1 µm

[0218] Glycerol solution

[0219] Ultrapure water 804 g

[0220] Citric acid monohydrate 29.1 g

[0221] Sodium sulfate 58.1 g

[0222] Diammonium ferric sulfate hexahydrate 4.5 g

[0223] Glycerol 99% 3370 g

[0224] Thiamine solution

[0225] 40 g ultrapure water

[0226] Thiamine hydrochloride 55 mg

[0227] Defoaming solution

[0228] Pluriol P 2000350 g

[0229] alkaline solution

[0230] 25% ammonia 1500 ml

[0231] Induction solution

[0232] 150 g ultrapure water

[0233] Rhamnose monohydrate 100 g

[0234] IPTG 238 mg

[0235] Sterilize the glycerol and antifoaming solution at 121°C for 30 min. Sterilize the thiamine and induction solutions by filtering through a 0.2 µm filter.

[0236] Portions 4 and 5 were combined in a sterilized fermentation vessel (Techfors, Infors HT) and inoculated with the preculture. The vessel was maintained at a temperature of 37°C, a pressure of 0.2 bar, and a pH of 6.6 by dosing alkaline solution during fermentation. The pO2 level was maintained at 20%–40% by adjusting the agitator speed (typically 500 rpm) and the aeration rate (typically 6 l / min). Antifoam solution was added as needed. The glycerol and thiamine solutions were combined to create the feed solution. After inoculation, the feed solution was dosed at a rate of 10 g / h. After 7 h, the feed solution was switched to a "stop and observe" mode, where the feed was activated at a rate of 10 g / h when the pO2 level increased. After 14 h or 330 g of feed solution had been consumed, the feed rate was increased to 80–100 g / h. Gene expression was induced by adding induction solution at an oxygen transfer rate of 80 mmol / l / h, or alternatively, at an OD600 of 12. Fermentation was stopped 36 hours after induction by lowering the temperature to 15°C. The cooled fermentation broth was drained from the fermentor and centrifuged at 4700 rpm and 10°C to pellet the cells. The resulting supernatant was discarded, and the cells were resuspended in 50 mM potassium dihydrogen phosphate buffer (pH 7.0) at 3850 g. The cell suspension was frozen at -80°C and then lyophilized. At this point, the lyophilizer was maintained at -50°C and a pressure of 0.25 mbar. The lyophilized cells were stored at 4°C.

[0237] Generation of lyophilized cell-free extracts

[0238] The lyophilized cells were resuspended in 100 g / L ultrapure water. The cell suspension was cooled on ice and then disrupted by three passages of a pressure homogenizer (Panda Plus 2000, GEA) set to 800 bar. The pressure for the three passages was typically between 1000 and 1400 bar. The resulting mixture was cleared of debris by centrifugation at 10,000 rpm for 15 min at 10°C. 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 subsequently lyophilized at -50°C and a pressure of 0.25 mbar.

[0239] Preparation of starting materials

[0240] c) Synthesis of n-butyl (3-cyano-3-hydroxypropyl) methylphosphonate (ACM-H) (Example 3)

[0241] ACM-H has been prepared according to Example 2 of WO 2015 / 173146 A1.

[0242] d) Synthesis of n-butyl (3-cyano-3-acetoxypropyl) methylphosphonate (ACM) (Example 4)

[0243] ACM has been prepared according to Example 1 of WO 2017 / 037012 A1.

[0244] Preparation of L-phosphinothricin P-butyl ester

[0245] e) Preparation of L-phosphinothricin-butyl ester (IE 5) from ACM-H

[0246]

[0247] 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) methylphosphonate (18.2 mmol, 1 equivalent, "ACM-H") prepared according to Example 3 was added with stirring. The resulting solution was stirred at 37°C. 1 ml of 2 M aqueous MnCl2 was added, followed by 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 produce a 357 mM "ACM-H" solution. The solution was stirred at 37°C.

[0248] After stirring for 3.5 hours, an additional 0.5 g of enzyme (A0A535Y1H2_UNCCH, SEQ ID NO: 2, 500 mg of 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 of lyophilized cell-free extract). After a total reaction time of 99 hours, additional enzyme (A0A535Y1H2_UNCCH, SEQ ID NO: 2, 500 mg of lyophilized cell-free extract) was added, and the reaction was stopped after 116 hours.

[0249] As measured by HPLC, the ultimate concentration of butyl-glufosinate is 56 mmol, which is equivalent to the conversion of 15 mol% ACM-H. The enantiomeric ratio is 92% L: 8% D. After the reaction is completed, the crude product reaction mixture is heated to 80°C for 30min and filtered to remove the cell lysate. The filtrate is concentrated under reduced pressure. L-glufosinate butyl ester is eluted with ammonia (1 M aqueous solution) on Dowex-50 WX 8200-400 (H) and separated. Use Supelco Chirobiotic T2 (gradient 90% ACN / water to 60% ACN / water, 0.1% formic acid in 19 minutes) to determine the concentration of L and D-glufosinate butyl ester by HPLC-MS. Temperature: 20°C, flow velocity: 0.8 mL / min. Retention times of glufosinate-butyl ester: L-configuration diastereomer (13.3 + 13.7 min); D-configuration (14.5 and 16.8 min).

[0250] f) Preparation of L-phosphinothricin-butyl ester (IE 6) from ACM

[0251]

[0252] Diammonium carbonate (9.6 g) was dissolved in water (100 mL) and the pH was adjusted to 8.5 with HCl (37% aqueous solution). 20 mL of the resulting buffer was used to dissolve 3.25 g of n-butyl (3-cyano-3-acetoxypropyl) methylphosphonate (11.2 mmol, 1 equivalent, "ACM," 90%), prepared according to Example 4. The resulting reaction mixture was stirred at 30°C. 0.5 ml of a 2 M aqueous MnCl solution was added, followed by the enzyme (Uniprot ID: A0A159Z531_9RHOB, SEQ ID NO: 1, 2 g of lyophilized cell-free extract and A0A535Y1H2_UNCCH, SEQ ID NO: 2, 1 g of lyophilized cell-free extract) to produce a 546 mM "ACM" solution. The solution was stirred at 30°C for 94 h.

[0253] After this time, the final concentration of butyl-glufosinate was 55 mmol as measured by HPLC, which corresponds to a conversion of 10 mol% of ACM. The enantiomeric ratio was >99% L : <1% D.

[0254] g) Preparation of L-glufosinate from ACM using a heating step (IE 6)

[0255]

[0256] 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) methylphosphonate ("ACM-H") prepared according to Example 3 was added with stirring. The resulting solution was stirred at 80°C for 1 h under microwave heating. The solution was then cooled to 37°C, and 250 μl of 2 M aqueous MnCl2 was added, followed by two amidases (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). The solution was further stirred at 37°C. After 4.5 h, the amidase (A0A535Y1H2_UNCCH, SEQ ID NO: 2, 250 mg of lyophilized cell-free extract) was added. The 4-amino-1-oxo-2-oxo-4-oxo-5-nitropropene was added into the 4-amino-1-oxo-2-oxo-3-oxo-5-nitropropene.After 46 h, pH was readjusted to 8.5 and two kinds of amidases (Uniprot ID: A0A159Z531_9RHOB, SEQ ID NO:1, the cell-free extract of 500 mg freeze-drying) and (A0A535Y1H2_UNCCH, SEQ ID NO:2, the cell-free extract of 250 mg freeze-drying) were added again. After 52 h, HPLC showed that the conversion to butyl-phosphinothricin was 15 mol%. Enantiomeric ratio was 95% L: 5% D. After the reaction was completed, the crude reaction mixture was heated to 80 ℃ and continued for 30 min and filtered to remove cell lysate, and the filtrate was under reduced pressure concentrated. L-phosphinothricin butyl ester was separated and further carried out reverse phase chromatography (acetonitrile-water gradient with 0.1% trifluoroacetic acid) at Dowex-50 WX 8 200-400 (H) with ammoniacal liquor (1M aqueous solution) wash-out. An analytical sample of L-phosphinothricin butyl ester (50 mg) was stirred with aqueous HCl (18% wt) at 100°C for 5 h. The enantiomeric ratio was determined by chiral HPLC (92% L-phosphinothricin: 8% D-phosphinothricin).

[0257] h) Preparation of L-phosphinothricin-butyl ester from ACM using a heating step (IE 7)

[0258]

[0259] Under stirring, 1.6 g of ammonium bicarbonate was dissolved in 12.5 ml of distilled water. Under stirring, 2 g of n-butyl (3-cyano-3-acetoxypropyl) methylphosphonate ("ACM", 90%) prepared according to Example 4 was added. The resulting solution was stirred at 80°C for 3 h under microwave heating. Subsequently, the solution was cooled to 40°C and the pH was adjusted to 8.5 with aqueous ammonia. 375 μl of 2 M MnCl2 aqueous solution was added, followed by two amidases (Uniprot ID: A0A159Z531_9RHOB, SEQ ID NO: 1, 500 mg of freeze-dried cell-free extract) and (A0A535Y1H2_UNCCH, SEQ ID NO: 2, 250 mg of freeze-dried cell-free extract). The solution was stirred at 40°C for 93 h. After this, HPLC showed that 22 mol% of ACM had been converted into butyl-phosphinothricin. The enantiomeric ratio was determined by chiral HPLC (> 99% L < 1% D).

[0260] SEQ ID NO: 1 (from Septicomonas albus)

[0261] MTLIVTNGRVVSPEGVALRDVVVEGETIAAVLPAGEAVKACPGAEVIDATGRIVIPGGVDPHVHLLVGFMGQRSVYDFASGGIAALRGGTTAIVDFALQRRGGSMLKGLAHRRKQ ADANVTLDYGLHLIVTDVTADTLAELPALRAAGVTTLKVYTVYEEDGLKVEDGALFALMQGAARHGLSVVLHAENAGIVERLRAEAVARGDTHPRHHALTRPPIVEIEAVSRAIAF SRATGCGVHILHLVAADAIALVAAARAEGLPVTAETCSHYLALTDEALERPNGHEFILSPPLRDKANQDRLWKGLETAALSLVASDEVSYSAAAKAMGLPSFATVANGITGIEARL PLLYTLGVDQGRIGLQRFVKLFSTWPAEIFGFAGKGRIAPGFDADLVLIDPDGRRVISTDSDYGDIGYTPYAGMELTGFATETIYRGRLVVRDGVFLGTEGQGRFIERVAPRRPAP

[0262] SEQ ID NO: 2 (from Chloroflexi bacterium)

[0263] MTDAARLERRIHELAQIGRTDDPAREIYATAVSRLGLSAEEQRARDLVTSWCAPHGATARRDPAANLYLRFPGADPHAPVVLVGSHLDSVPMGGRFDGALGVCCAVEAVVSLLESGARFARPVEVVGWADEEGARFGYGLFGSAAAFGRLRVDPERVRDKGGTSIAEALRALGESGDLAGAMRDPKGIRAYLELHIEQGPRLERAGAPLGVVSDIVGIFHGLVMVRGEQNHAGATVMGERHDALVAASHMIIALERIASSVPDAVATVGEITVKPGAKNVIPGECTFSLDIRAPKQESIDLVLERFKAEANEIFRKSLREWGLRPLQSVAVTPLDEDLRDLLWKSAMSVGVNAPTLVSGAGHDAQNPSLAGVPTGMIFVRSTGGSHTPTEFAATADAALGAKALEIAIRELATA

Claims

1. A method for preparing L-phosphinothricin and / or its salt or L-phosphinothricin alkyl ester and / or its salt, wherein the L-phosphinothricin or the L-phosphinothricin alkyl ester has a molecular structure according to formula (I): (I)， where R 1 is H or C1-C8 alkyl, The method comprises reacting the following components in at least one reaction step: (1) Cyanohydrin or cyanohydrin derivative according to formula (II) (II), in R 1 is H or C1-C8 alkyl, and R 2 It is H, C1-C8 alkyl, C6-C 10 Aryl, C7-C 10 Aralkyl, C4-C 10 Cycloalkyl, or C1-C 10 acyl group; (2) ammonia source; (3) a source of carbon dioxide; and (4) At least two enzymes.

2. The method according to claim 1, wherein component (4) comprises, preferably consists of: (4a) at least one amidohydrolase (EC 3.5.2) that acts on cyclic amides and (4b) at least one L-amidohydrolase (EC 3.5.1) that acts on linear amides.

3. The process according to claim 1 or 2, wherein components (1), (2) and (3) are contacted first and component (4) is subsequently added, preferably component (4a) is added first and component (4b) is added last.

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

5. The process according to any one of the preceding claims, wherein the cyanohydrin is prepared by reaction of an aldehyde with a cyanide, preferably hydrogen cyanide or potassium cyanide, and wherein the aldehyde has a molecular structure according to formula (III): (III) where R 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.

6. A method according to any one of the preceding claims, wherein R 1 It is a C1-C8 alkyl group, preferably a C1-C6 alkyl group, more preferably a C2-C4 alkyl group, even more preferably an ethyl group or a butyl group, and most preferably an ethyl group.

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

8. The method according to any one of the preceding claims, wherein the carbon dioxide source is gaseous carbon dioxide, dissolved carbon dioxide, carbonates, or mixtures thereof.

9. The process according to any one of the preceding claims, wherein the cyanohydrin or cyanohydrin derivative is a cyanohydrin derivative according to formula (IV): (IV), where R 3 It is a C1-C8 alkyl group, preferably a C1-C4 alkyl group, more preferably a C1-C3 alkyl group, and most preferably a methyl group.

10. The process according to any one of the preceding claims, wherein the L-phosphinothricin and / or its salt or the L-phosphinothricin alkyl ester and / or its salt is prepared in enantiomeric excess, preferably in an enantiomeric excess of greater than 85%, more preferably greater than 90%, even more preferably greater than 95%, and most preferably greater than 99%.

11. The method according to any one of the preceding claims, wherein the amidohydrolase (EC 3.5.2) acting on cyclic amides is an L-amidohydrolase (EC 3.5.2) acting on cyclic amides.

12. The method according to any of the preceding claims, wherein the L-amide hydrolase (EC 3.5.2) acting on cyclic amides is chosen from the group of enzymes identified by their Uniprot ID or NCBI ID (the latter indicated by a "*" at the beginning of the ID) consisting of: O69809 and variants thereof, Q846U5_9BACL and variants thereof, P81006 and variants thereof, Q84FR6_9MICC and variants thereof, Q56S49_9BACI and variants thereof, A1E351_9BACI and variants thereof, Q28SA7 and variants thereof, Q45515 and variants thereof, A0A399DRQ3_9DEIN and variants thereof, Q55DL0 and variants thereof, F7X5M8_SINMM and variants thereof, Q9I676 and variants thereof, Q44184 and variants thereof, B5L363 and variants thereof, P42084 and variants thereof, P2 5995 and variants thereof, Q3Z354 and variants thereof, B1XEG2 and variants thereof, Q9F465_PAEAU and variants thereof, A0A161KD37_9CHLR and variants thereof, A0A1J4XHR4_9BACT and variants thereof, A0A1C4QIY5_9ACTN and variants thereof, A0A0K2UMP4_LEPSM and variants thereof, A0A159Z531_9RHOB and variants thereof, E1R8C9_SEDSS and variants thereof, A0A1F9QT17_9BACT and variants thereof, A0A0D8IVV8_9FIRM and variants thereof, A0A0B5QKE4_CLOBE and variants thereof A0A0N1GBZ8_9ACTN and variants thereof, A0A174ADZ3_9FIRM and variants thereof, U7V9Q6_9FUSO and variants thereof, A0A0J1FAI4_9FIRM and variants thereof, PHYDA_ECOK1 and variants thereof, A0A0S8H576_9BACT and variants thereof, A0A1J4J4Y8_9EUKA and variants thereof, A0A0D5NFS5_9BACL and variants thereof, A0A0D5NNJ7_9BACL and variants thereof, A0A1H2AV66_9BACL and variants thereof, A0A0Q4RXY0_9BACL and variants thereof, A0A0Q7S B75_9BACL and variants, A0A100VRN2_PAEAM and variants, W4BDJ0_9BACL and variants, A0A1J5E082_9DELT and variants, A0A1H5ZFN3_9BACT and variants, A0A1F8NMM2_9CHLR and variants, A0A1F8SDV1_9CHLR and variants, A0A1H1PLX0_9BACT and variants, A0A0Q5I8X4_9DEIO and variants, *WP_046170519.1 and variants, *WP_023514195.1 and variants, *WP_023516147.1 and variants thereof, and *ANZ15483.1, wherein a variant is defined as a polypeptide sequence having at least 80%, preferably 90%, and most preferably 95% sequence identity with the corresponding polypeptide sequence, preferably selected from the group of enzymes identified by their Uniprot ID or NCBI ID (the latter indicated by an "*" at the beginning of the ID) consisting of: Q45515 and variants thereof, Q44184 and variants thereof, P81006 and variants thereof, A0A1C4QIY5_9ACTN and variants thereof, A0A0K2UMP4_LEPSM and variants thereof, *WP_046170519.1 and variants thereof, A0A159Z531_9RHOB and variants thereof, and E1R8C9_SEDSS and variants thereof, wherein a variant is defined as a polypeptide sequence having at least 80%, preferably 90%, and most preferably 95% sequence identity with the corresponding polypeptide sequence.

13. The method according to any of the preceding claims, wherein the L-amidohydrolase (EC 3.5.1) acting on linear amides is chosen from the group of enzymes identified by their Uniprot ID consisting of: A0A0K9YX84_9BACL and variants thereof, E3HUL6_ACHXA and variants thereof, A0A4D7Q548_GEOKU and variants thereof, Q9F464 and variants thereof, A0A2S9D976_9MICC and variants thereof, A0A3E0C996_9BURK and variants thereof, A0A535Y1H2_UNCCH and variants thereof, A0A6P2ISL4_BURL3 and variants thereof, A0A1Y4GC62_9BACT and variants thereof, wherein a variant is defined as a polypeptide sequence having at least 80%, preferably 90%, and most preferably 95% sequence identity with the corresponding polypeptide sequence, preferably chosen from the group consisting of: The group of enzymes identified by ID: A0A3E0C996_9BURK and variants thereof, A0A535Y1H2_UNCCH and variants thereof, A0A6P2ISL4_BURL3 and variants thereof, A0A1Y4GC62_9BACT, wherein a variant is defined as a polypeptide sequence having at least 80%, preferably 90%, and most preferably 95% sequence identity with the corresponding polypeptide sequence.

14. A composition comprising a cyanohydrin or a cyanohydrin derivative according to formula (II): (II), in R 1 is H or C1-C8 alkyl, and R 2 It is H, C1-C8 alkyl, C6-C 10 Aryl, C7-C 10 Aralkyl, C4-C 10 Cycloalkyl, or C1-C 10 acyl groups, and L-glufosinate and / or its salts.

15. A method for selectively controlling weeds in an area, preferably containing a planted batch of seeds or crops resistant to glufosinate, the method comprising: An effective amount of a composition is applied to the area, the composition comprising L-phosphinothricin and / or its salts, the enantiomeric ratio of L-phosphinothricin and / or its salts relative to D-phosphinothricin and / or its salts being at least 50%, preferably an enantiomeric excess of more than 70%, and more than 0.01 wt.-% to less than 10 wt.-% of a cyanohydrin or a cyanohydrin derivative according to formula (II), based on the total amount of the composition: (II), in R 1 is H or C1-C8 alkyl, and R 2 It is H, C1-C8 alkyl, C6-C 10 Aryl, C7-C 10 Aralkyl, C4-C 10 Cycloalkyl, or C1-C 10 acyl group.

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