Enzymatic decarbamoylation of glufosinate derivatives

Abstract

The present application relates to a method for producing glufosinate, comprising the step of enzymatically cleaving off the carbamoyl moiety of a carbamoyl amino acid compound.

Description

[Technical field]

[0001] The present application relates to a method for producing glufosinate, comprising the step of enzymatically cleaving off the carbamoyl moiety of an N-carbamoyl amino acid compound. [Background technology]

[0002] The herbicide glufosinate is a non-selective foliar herbicide and is considered to be one of the safest herbicides from a toxicological or environmental protection point of view. Current commercial chemical synthesis of glufosinate results in a racemic mixture of L-glufosinate and D-glufosinate (Duke et al. 2010 Toxins f2:1943-1962). [ka] Scheme 1. Synthesis of hydantoins via their respective aldehydes (wherein R is, for example, H or alkyl).

[0003] It is known that hydantoins can be intermediates in the synthesis of racemic glufosinate. They can be obtained from the respective aldehydes (Scheme 1) by the Bucherer-Bergs reaction. Further synthetic routes are described, for example, in Chinese Patent No. 111662325.

[0004] CN Patent No. 113045604 discloses a method for synthesizing glufosinate starting from hydantoin. The reaction needs to be carried out under high pressure and at temperatures of 130-180 °C. The reaction conditions are therefore quite harsh. However, the use of autoclaves and / or temperatures above 120 °C on an industrial scale also poses safety risks for colleagues. CN Patent No. 111662325 also describes the hydrolysis of hydantoin to obtain glufosinate, but this reaction requires strong acid or strong base and reflux conditions in water.

[0005] L-glufosinate (also known as phosphinothricin or (S)-2-amino-4-(hydroxy(methyl)phosphonoyl)butanoic acid) is known to be more potent than D-glufosinate (Ruhland et al. (2002) Environ. Biosafety Res. 1:29-37). Therefore, methods to produce the more active L-glufosinate in excess have attracted further attention. Summary of the Invention [Problem to be solved by the invention]

[0006] Against the above background, it was an object of the present invention to provide a gentle process for the production of glufosinate.

[0007] Furthermore, it is an object of the present invention to provide a safe method for producing glufosinate.

[0008] It is a further object of the present invention to provide a mild process for the preparation of enantiomerically excess L-glufosinate.

[0009] A further object of the present invention is to provide a composition comprising L-glufosinate.

[0010] It is a further object of the present invention to provide a method for selectively controlling weeds using the composition obtained according to the production method of the present invention. [Means for solving the problem]

[0011] The inventors have surprisingly found that at least one of the above objectives can be obtained by the N-carbamoylamino acid-based process described herein.Furthermore, the inventors have found that the claimed method provides a composition comprising a sufficient amount of glufosinate for use as a herbicide. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] Thus, in a first aspect, the present invention provides a compound of formula (3): [ka] (wherein R is H or C1-C8 alkyl), the method for producing glufosinate, its alkyl ester, or its salt, comprising the steps of: [ka] wherein R is H or C1-C8 alkyl.

[0013] In the following, preferred embodiments of the components of the present method, composition and method for selectively controlling weeds are described in more detail. It should be understood that each preferred embodiment is preferred not only by itself but also in combination with other preferred embodiments.

[0014] In a preferred embodiment A1 of the first aspect, the cleavage step comprises reacting a compound of formula (3) [ka] (wherein R is H or C1-C8 alkyl, preferably H or C1-C6 alkyl, more preferably H or C2-C4 alkyl, further preferably H, ethyl or butyl, particularly preferably H), a glufosinate, an alkyl ester thereof, or a salt thereof.

[0015] In a preferred embodiment A2 of the first aspect, the cleavage step comprises cleaving glufosinate having the formula (3), its alkyl ester or its salt in the form of a racemic mixture or in the form of a cleavage product of the formula (3a): [ka] (wherein R 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, particularly preferably H), preferably in the form of an enantiomerically excess of L-glufosinate, an alkyl ester thereof or a salt thereof having the formula (3a):

[0016] In a preferred embodiment A3 of the first aspect, at least 50%, preferably at least 60%, in particular at least 80% of the N-carbamoylamino acids having formula (2) are converted to L-glufosinate, its alkyl esters or salts thereof having formula (3a), wherein formula (3a) is as defined in embodiment A2.

[0017] In a preferred embodiment A4 of the first aspect, the cleavage is carried out by an N-carbamoyl amino acid hydrolase enzyme, preferably an LN-carbamoyl amino acid hydrolase enzyme.

[0018] In a preferred embodiment A5 of the first aspect, the cleavage is performed by an N-carbamoyl amino acid hydrolase enzyme selected from the group consisting of Uniprot ID: A0A1Y4GC62_9BACT (SEQ ID NO: 2), Uniprot ID: A0A6P2ISL4_BURL3 (SEQ ID NO: 3) and mixtures thereof.

[0019] In a preferred embodiment A6 of the first aspect, R in formulae (2) and (3) is H or C1-C6 alkyl, preferably H or C2-C4 alkyl, more preferably H, ethyl or butyl, especially H.

[0020] In a preferred embodiment A7 of the first aspect, the cleavage step is carried out at a pH of 6 to 11, preferably 6.5 to 10, more preferably 7 to 9.5, in particular 7.5 to 9, and / or at a temperature of 20 to 50°C, preferably 25 to 45°C, more preferably 30 to 42°C, in particular 32 to 40°C.

[0021] In a preferred embodiment A8 of the first aspect, R in formulas (2) and (3) is C1-C8 alkyl, preferably C1-C6 alkyl, more preferably C2-C4 alkyl, even more preferably ethyl or butyl, especially ethyl, and the process further comprises the steps of: c) deprotection under acidic conditions, preferably using hydrochloric acid or sulfuric acid Further includes:

[0022] In a preferred embodiment A9 of the first aspect, the method further comprises the addition of an N-carbamoyl amino acid racemase enzyme.

[0023] In a preferred embodiment A10 of the first aspect, the N-carbamoyl amino acid having the formula (2) is a N-carbamoyl amino acid having the formula (1) [ka] wherein R is H or C1-C8 alkyl, to form an N-carbamoyl amino acid having the formula (2), preferably carried out under enzymatic conditions.

[0024] In a preferred embodiment A11 of the first aspect, an N-carbamoylamino acid having formula (2) is provided by the hydrolysis step described above, and the hydrolysis of the hydantoin having formula (1) is carried out by a hydantoinase enzyme.

[0025] In a preferred embodiment A12 of the first aspect, the method further comprises the addition of a hydantoin racemase enzyme.

[0026] In a preferred embodiment A13 of the first aspect, the hydrolysis step and the cleavage step are carried out in a single vessel, preferably with all reagents being added substantially at the start of the reaction, or with the reagents for the hydrolysis step and the reagents for the cleavage step being added at different times to a single vessel.

[0027] In a second aspect, the present invention provides a compound of formula (2a) [ka] (wherein R is H or C1-C8 alkyl), and L-glufosinate or a salt thereof.

[0028] In a third aspect, the present invention provides a method for selectively controlling weeds in an area containing planted seeds or crops, preferably resistant to glufosinate, comprising: An effective amount of a composition containing L-glufosinate or a salt thereof in an enantiomeric excess of at least 80% over D-glufosinate or a salt thereof and a compound represented by formula (2) in an amount of more than 0.01% by weight and less than 10% by weight based on the total amount of the composition [ka] and an N-carbamoyl amino acid having the formula: (wherein R is H or C1-C8 alkyl),

[0029] Detailed Description Before describing exemplary embodiments of the invention in detail, definitions that are important for understanding the invention are presented.

[0030] As used herein and in the appended claims, the singular forms "a" and "an" also include the respective plural forms, unless the context clearly dictates otherwise. In the context of the present invention, the terms "about" and "approximately" refer to a margin of accuracy that a person skilled in the art would understand to still ensure the technical effect of the feature in question. This term usually indicates a deviation of ±20%, preferably ±15%, more preferably ±10%, and even more preferably ±5% from the indicated numerical value. The term "comprising" should be understood to be non-limiting. The term "consisting of" for the purposes of the present invention is considered to be a preferred embodiment of the term "comprising of". Hereinafter, when a group is defined as comprising at least a certain number of embodiments, this means that the preferred group consisting of only these embodiments is also included. Furthermore, the terms "first", "second", "third" or "(a)", "(b)", "(c)", "(d)" and similar terms in the specification and claims are used to distinguish between similar elements and do not necessarily describe a sequential or chronological order. It is to be understood that terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein can be performed in other orders than those described or illustrated herein. When the terms "first", "second", "third" or "(a)", "(b)", "(c)", "(d)", "i", "ii" and the like refer to steps of a method or use or assay, unless otherwise specified in this application, as described herein above or below, there is no time or consistency between such 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. It is to be understood that the invention is not limited to the specific methods, procedures, reagents, etc. described herein, as these may vary.It should also be understood that the terms used herein are used only for the purpose of describing particular embodiments, and are not intended to limit the scope of the present invention, which is limited only by the scope of 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.

[0031] The term "wt. %" as used throughout this specification stands for "percent by weight."

[0032] The term "alkyl" as used herein in each case denotes a straight-chain or branched alkyl group having usually 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, iso-propyl, n-butyl, 2-butyl, iso-butyl, tert-butyl, n-pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, 2,2-dimethylpropyl, 1-ethylpropyl and n-hexyl.

[0033] Depending on the substitution pattern, the compounds according to the invention may have one or more stereocenters. Unless expressly indicated otherwise (e.g. via a chemical formula), the present invention preferably encompasses all stereoisomers of the compounds according to the invention, i.e. pure enantiomers, pure diastereomers and mixtures thereof, including racemic mixtures.

[0034] Preferred embodiments relating to a method for producing a composition comprising glufosinate having the formula (3), an alkyl ester thereof or a salt thereof, an N-carbamoylamino acid having the formula (2a) and L-glufosinate or a salt thereof, and a method for selectively controlling weeds are described in detail below. It should be understood that the preferred embodiments of the present invention are preferred either alone or in combination with each other.

[0035] As indicated above, the present invention relates in one aspect to a compound represented by formula (3): [ka] (wherein R is H or C1-C8 alkyl), the method for producing glufosinate, its alkyl ester, or its salt, comprising the steps of: [ka] wherein R is H or C1-C8 alkyl, using an enzyme to remove the carbamoyl moiety of the N-carbamoyl amino acid having the formula:

[0036] Formula (3) [ka] It is understood that the glufosinate, its alkyl ester or its salt having the formula (3) includes all stereoisomers of each glufosinate or its alkyl ester, suitable salts. Furthermore, each zwitterion is also included in formula (3). Suitable salts are illustratively hydrochloride, ammonium salt, and isopropylammonium salt. In this regard, in particular, the compound of formula (3) includes two stereocenters, where one stereocenter is located at the phosphorus atom and one stereocenter is located at the α carbon atom. In particular, the compound of formula (3) includes all stereoisomers derived from the stereocenter at the phosphorus atom.

[0037] In a preferred embodiment, the cleavage step is carried out according to formula (3): [ka] (wherein R is H or C1-C8 alkyl, preferably H or C1-C6 alkyl, more preferably H or C2-C4 alkyl, further preferably H, ethyl or butyl, particularly preferably H), a glufosinate, an alkyl ester thereof, or a salt thereof.

[0038] In a preferred embodiment of the present invention, the cleavage step provides glufosinate having formula (3), an alkyl ester thereof or a salt thereof in the form of a racemic mixture.

[0039] In another preferred embodiment of the present invention, the cleavage step comprises converting glufosinate having formula (3), its alkyl ester or its salt into a compound having formula (3a): [ka] (wherein R is H or C1-C8 alkyl, preferably H or C1-C6 alkyl, more preferably H or C2-C4 alkyl, further preferably H, ethyl or butyl, particularly preferably H), in the form of an enantiomerically excess of L-glufosinate, an alkyl ester thereof, or a salt thereof.

[0040] In another preferred embodiment of the present invention, the cleavage step comprises reacting glufosinate having formula (3), its alkyl ester or its salt with a cyclopentadiene having formula (3b): [ka] (wherein R is H or C1-C8 alkyl, preferably H or C1-C6 alkyl, more preferably H or C2-C4 alkyl, further preferably H, ethyl or butyl, particularly preferably H), in the form of an enantiomerically excess of D-glufosinate, an alkyl ester thereof, or a salt thereof.

[0041] In another preferred embodiment of the present invention, at least 50%, preferably at least 60%, more preferably at least 80%, even more preferably at least 90%, especially at least 95% of the N-carbamoylamino acids having formula (2) are L-glufosinate, alkyl esters thereof or non-converted forms thereof having formula (3a), wherein R in formula (3a) 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, especially H.

[0042] In another preferred embodiment of the present invention, 50 to 99.9%, preferably 60 to 99.8%, more preferably 80 to 99.5%, even more preferably 90 to 99.2%, and particularly 95 to 99% of the N-carbamoylamino acid having the formula (2) is L-glufosinate, an alkyl ester thereof or a non-converted form thereof having the formula (3a), wherein R in the formula (3a) is H or a C1 to C8 alkyl, preferably H or a C1 to C6 alkyl, more preferably H or a C2 to C4 alkyl, further preferably H, ethyl or butyl, and particularly H.

[0043] In another preferred embodiment of the invention, at least 50%, preferably at least 60%, more preferably at least 80%, even more preferably at least 90%, especially at least 95% of the N-carbamoylamino acids having formula (2) are [ka] (wherein R 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, in particular H), or an alkyl ester thereof or a salt thereof.

[0044] In another preferred embodiment of the present invention, 50 to 99.9%, preferably 60 to 99.8%, more preferably 80 to 99.5%, even more preferably 90 to 99.2%, and particularly 95 to 99% of the N-carbamoylamino acid having the formula (2) is L-glufosinate, an alkyl ester thereof or a non-converted form thereof having the formula (3b), wherein R in the formula (3b) is H or a C1 to C8 alkyl, preferably H or a C1 to C6 alkyl, more preferably H or a C2 to C4 alkyl, further preferably H, ethyl or butyl, and particularly H.

[0045] An enantiomeric excess of L-glufosinate is preferred.

[0046] In a preferred embodiment of the present invention, the cleavage is performed by an N-carbamoyl amino acid hydrolase enzyme, preferably an LN-carbamoyl amino acid hydrolase enzyme. Alternatively, the cleavage can be performed by a DN-carbamoyl amino acid hydrolase enzyme. Suitable N-carbamoyl amino acid hydrolase enzymes are 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 N-carbamoyl amino acid hydrolase enzymes that can be used in the present method include those from Cloacibacillus sp. An23, Burkholderia lata, and the like. Suitable N-carbamoyl amino acid hydrolase enzymes that can be used in the present method include A0A7Y0T4N7_9RHIZ and mutants thereof, Q88FQ3_PSEPK and mutants thereof, Q88Q81_PSEPK and mutants thereof, A0A126S6J4_PSEPU and mutants thereof, Q8VUL6_9PSED and mutants thereof, H9B8T5_9PSED and mutants thereof, Q9FB05_9PSED and mutants thereof, C0ZCM8_BREBN and mutants thereof, C0Z7R5_BREB and mutants thereof, A0A0K9YX84_9BACL and mutants thereof, E3HUL6_ACHXA and mutants thereof, A0A1V9BSS3_9BACI and mutants thereof, A0A1V9B SS3_9BACI and its mutants, Q9F464 and its mutants, A0A4D7Q548_GEOKU and its mutants, Q9F464 and its mutants, A0A2S9D976_9MICC and its mutants, A0A1I6VZZ4_9RHIZ and its mutants, A0A1L6RE91_9LACT and its mutants, A0A3E0C996_9BURK and its mutants, A0A3M7BGJ4_HORWE and its mutants, A0A2D7YQN7_9GAMM and its mutants, A0A535Y1H2_9CHLR and its mutants, A0A223E4I5_9BACI and its mutants, M2VSE9_GALSU and its mutants, A0A3T0K6C0_9GAMM and its mutants,A0A416FGE1_9CLOT and its mutants, D1P143_9GAMM and its mutants, A0A6P2ISL4_BURL3 (SEQ ID NO: 3) and its mutants, A0A3S6Z2M9_9FIRM and its mutants, A0A0C1US49_9BACT and its mutants, A0A1Y4GC62_9BACT (SEQ ID NO: 2) and its mutants, A0A3D3VMN7_9BACT and its mutants, A0A2K8L549_9PROT and its mutants, A0A1G0MC89_9BACT and its mutants, A0A1M6WYS1_SELRU and its mutants, A0A2K2BYI3_POPTR and its mutants, A0A510 DYR5_9CREN and variants thereof, A0A5Y3XFN7_SALER and variants thereof, A0A381IB54_CLODI and variants thereof, A0A2V3IQW6_9FLOR and variants thereof, *WP_035255878.1 and variants thereof, *WP_012685058.1 and variants thereof, *WP_056530217.1 and variants thereof, *WP_075270569.1 and variants thereof, and mixtures thereof, wherein variants are defined as polypeptide sequences having at least 80%, preferably 90%, and most preferably 95% sequence identity to the respective polypeptide sequences.

[0047] In a preferred embodiment of the invention, the cleavage is performed by an N-carbamoyl amino acid hydrolase enzyme selected from the group consisting of Uniprot ID: A0A1Y4GC62_9BACT (SEQ ID NO: 2) and variants thereof, Uniprot ID: A0A6P2ISL4_BURL3 (SEQ ID NO: 3) and variants thereof, Uniprot ID: A0A535Y1H2_9CHLR and variants thereof, Uniport ID: A0A3E0C996_9BURK and variants thereof, and mixtures thereof, where variants are defined as polypeptide sequences having at least 80%, preferably 90%, most preferably 95% sequence identity to the respective polypeptide sequence.

[0048] It is to be understood that the above N-carbamoyl amino acid hydrolase enzymes are designated by database identifier nomenclature according to the Uniprot database (www.UniProt.org).

[0049] In a preferred embodiment of the invention, R in formulae (2) and (3) is H or C1-C6 alkyl, preferably H or C2-C4 alkyl, more preferably H, ethyl or butyl, especially H.

[0050] In a preferred embodiment of the invention, the cleavage step is carried out at a pH of 6-11, preferably 6.5-10, more preferably 7-9.5, especially 7.5-9.

[0051] In a preferred embodiment of the invention, the cleavage step is carried out at a temperature of 20-50°C, preferably 25-45°C, more preferably 30-42°C, in particular 32-40°C.

[0052] Furthermore, the reaction pressure is preferably ambient pressure. Preferably, the reaction pressure in the cleavage step is in the range of 0.995 to 1.030 mbar, more preferably 1.005 to 1.020 mbar, in particular about 1.013 mbar.

[0053] In a preferred embodiment of the present invention, the cleavage step is carried out during stirring, preferably at 50-1000 rpm, more preferably at 100-800 rpm, even more preferably at 150-600 rpm, still more preferably at 180-400 rpm, especially at 200-300 rpm.

[0054] In a preferred embodiment of the invention, R in formulas (2) and (3) is C1-C8 alkyl, preferably C1-C6 alkyl, more preferably C2-C4 alkyl, even more preferably ethyl or butyl, especially ethyl, and the process comprises: c) deprotection under acidic conditions, preferably using hydrochloric acid or sulfuric acid Further includes:

[0055] In a preferred embodiment of the invention, the method further comprises the addition of an N-carbamoyl amino acid racemase enzyme. Any suitable N-carbamoyl amino acid racemase enzyme may be used.

[0056] The N-carbamoyl amino acid having the formula (2) can be obtained via any suitable manufacturing method.

[0057] In a preferred embodiment, the N-carbamoylamino acid having formula (2) is provided by chemical synthesis. A suitable chemical approach may start with glufosinate (or its alkyl derivative) and may include the addition of a cyanate such as potassium cyanate. Such a reaction preferably involves elevated temperatures in the range of 35-80° C., preferably 40-60° C., and / or reduced pressure in the range of 50-400 mbar, preferably 100-300 mbar.

[0058] In a preferred embodiment, the N-carbamoyl amino acid having the formula (2) is a N-carbamoyl amino acid having the formula (1) [ka] wherein R is H or C1-C8 alkyl to form an N-carbamoyl amino acid having formula (2). The hydrolysis of the hydantoin of formula (1) can be carried out chemically or enzymatically. In a preferred embodiment, the hydrolysis of the hydantoin of formula (1) is carried out under enzymatic conditions.

[0059] In a preferred embodiment, the N-carbamoyl amino acid having formula (2) is provided by the hydrolysis step described above, and the hydrolysis of the hydantoin having formula (1) is carried out by a hydantoinase enzyme.

[0060] Hydantoins having formula (1) can be obtained via any suitable preparation method. DE 3142036 discloses several synthesis methods by way of example.

[0061] Hydantoins having formula (1) can be chemically synthesized starting from alkyl 3-cyano-3-hydroxypropyl(methyl)phosphinate, such as butyl 3-cyano-3-hydroxypropyl(methyl)phosphinate, which can be treated with concentrated sulfuric acid in methanol and then the mixture can be heated to a temperature higher than about 25°C, such as about 40°C. The resulting reaction mixture can be cooled to about 25°C and treated with sodium methoxide and sodium sulfate in methanol. The crude alkyl 3-cyano-3-hydroxypropyl(methyl)phosphinate can be added to a solution of diammonium carbonate, for example, in water, and the reaction mixture can be heated to a temperature of about 70°C. After standard workup, the desired alkyl hydantoin (e.g., butyl hydantoin) can be obtained. In this context, the alkyl can be illustratively methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, or octyl, and is preferably ethyl or butyl.

[0062] Hydantoins having formula (1) may further illustratively be chemically synthesized starting from an aqueous solution of glufosinate ammonium and potassium cyanate. After heating the reaction mixture at about 50° C., the reaction mixture is cooled to about 25° C. and concentrated hydrogen chloride in water is added. After standard workup, the desired hydantoins can be obtained. The obtained hydantoins can further illustratively be alkylated with triethyl orthoacetate to provide the respective alkyl hydantoins (e.g., ethyl hydantoin).

[0063] In a preferred embodiment of the invention the hydrolysis step is carried out at a pH between 6 and 11, preferably between 6.5 and 10, more preferably between 7 and 9.5, in particular between 7.5 and 9. The pH is preferably adjusted using an alkali hydroxide, more preferably sodium hydroxide or potassium hydroxide, in particular potassium hydroxide.

[0064] In a preferred embodiment of the invention, the hydrolysis step is carried out at a temperature of 20-50°C, preferably 25-45°C, more preferably 30-42°C, in particular 32-40°C.

[0065] In a preferred embodiment of the invention, the hydrolysis step is carried out under aqueous conditions, preferably in a degassed aqueous phosphate buffer, more preferably in a degassed aqueous potassium phosphate buffer.

[0066] In a preferred embodiment of the present invention, the hydrolysis step is carried out with stirring, preferably at 50-1000 rpm, more preferably at 100-800 rpm, even more preferably at 150-600 rpm, still more preferably at 180-400 rpm, especially at 200-300 rpm.

[0067] In another preferred embodiment, the N-carbamoyl amino acid having formula (2) is provided by the hydrolysis step described above, wherein the hydrolysis of the hydantoin having formula (1) is carried out chemically.

[0068] The term "chemical" is understood to mean a reaction carried out under chemical conditions, i.e. not under enzymatic conditions. Thus, the chemical hydrolysis step is carried out under chemical conditions, i.e. not under enzymatic conditions. In general, any suitable chemical approach is possible.

[0069] A suitable chemical hydrolysis step may be carried out under alkaline conditions, preferably using sodium hydroxide, potassium hydroxide, and the like.

[0070] Any suitable hydantoinase enzyme may be used.

[0071] Such hydantoinase enzymes that can be used in the present method 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, and the like. 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., and preferably Defluviimonas alba.

[0072] Suitable hydantoinase enzymes are EC 3.5.2 hydrolases that act on cyclic amides.

[0073] Further suitable hydantoinase enzymes include Q8RSQ2 and mutants thereof, O69809 and mutants thereof, Q846U5_9BACL and mutants thereof, P81006 and mutants thereof, Q84FR6_9MICC and mutants thereof, Q56S49_9BACI and mutants thereof, A1E351_9BAC and mutants thereof, Q28SA7 and mutants thereof, Q59699 and mutants thereof, Q45515 and mutants thereof, A0A399DRQ3_9DEIN and mutants thereof, Q55DL0 and mutants thereof, F7X5M8_SINMM and mutants thereof, Q9I676 and mutants thereof, Q44184 and mutants thereof, B5L363 and mutants thereof, I1MEH3 and mutants thereof, Q6S4R9 and mutants thereof, Q65LN0 and mutants thereof, Q171F8 and mutants thereof, Q8U8Z6 and mutants thereof, P42084 and mutants thereof, Q88NW7 and mutants thereof, P25995 and mutants thereof, Q3Z354 and mutants thereof, B1XEG2 and mutants thereof, Q9F465_PAEAU and mutants thereof, Q01262.1 and mutants thereof, A0A250D XG4_GEOSE and its mutants, A1SPN2 and its mutants, Q9WYH0 and its mutants, P58329 and its mutants, A1SGT4 and its mutants, E3JD18 and its mutants, HUTI_BDEBA and its mutants, A0A161KD37_9CHLR and its mutants, I0GL27_CALEA and its mutants, A0A068WGW0_ECHGR and its mutants, A0A1J4XHR4_9BACT and its mutants, A0A1C4QIY5_9ACTN and its mutants, 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 (see also SEQ ID NO: 1) and its variants, R5JTP2_9CLOT and its variants, A0A010RM85_9PEZI and its variants,E1R8C9_SEDSS and its mutants, A0A010YEH8_9BACT and its mutants, A0A031LV69_9CREN and its mutants, A0A1F9QT17_9BACT and its mutants, ALLB_BACVZ and its mutants, HUTI_FLAPJ and its mutants, A0A073J5J1_9BACT and its mutants, A0A034W2Q8_BACDO and its mutants, A0A0D8IVV8_9FIRM and its mutants, A0A0B5QKE4_CLOBE and its mutants, A0A098B7X6_DESHA and its mutants , A0A0B5H4M8_9EURY and its mutants, A0A0C1YDP1_9ACTN and its mutants, Q981H2_RHILO and its mutants, T1EEH7_HELRO and its mutants, A0A060DTG8_AZOBR and its mutants, A0A011MGZ5_MANHA and its mutants, A0A060LYB6_9BACI and its mutants, S0F3L7_CHOCR and its mutants, A0A133VNR0_9EURY and its mutants, A0A133U7U9_9EURY and its mutants, A0A0U2XD52_ECOLX and and its mutants, M1YZY6_NITG3 and its mutants, T0N9X6_9EURY and its mutants, T0LMU2_9EURY and its mutants, A0A0N1GBZ8_9ACTN and its mutants, HUTI_ANASK and its mutants, A0A031JUP0_9SPHN and its mutants, A0A061N9L2_9BACL and its mutants, A0A017T4D2_9DELT and its mutants, A0A174ADZ3_9FIRM and its mutants, A0A021X7D5_9RHIZ and its mutants, A0A021XAC5_9RHIZ and its mutants, mutants thereof, A0A0C2UIW0_9BACL and mutants thereof, A0A1F8NGY1_9CHLR and mutants thereof, D3F1S3_CONWI and mutants thereof, A0A021XG06_9RHIZ and mutants thereof, U7V9Q6_9FUSO and mutants thereof, D6XY37_BACIE and mutants thereof, A0A0J1FAI4_9FIRM and mutants thereof, B5Y9A6_COPPD and mutants thereof, PHYDA_ECOK1 and mutants thereof, A0A0A9X9B7_LYGHE and mutants thereof, A0A0S8H576_9BACT and mutants thereof,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 mutants, A0A1L0FIA0_9ASCO and its mutants, F7DRE9_ORNAN and its mutants, A0LK75_SYNFM and its mutants, A0A0Q1A918_9BACT and its mutants, H2YZ10_CIOSA and its mutants, I4YD99_WALMC and its mutants, A0A077YYH5_TRITR and its mutants, A0A077Y189_9SPHI and its mutants, A0A089K5P4_9BACL and its mutants, A0A0Q7W2T1_9RHIZ and its mutants, A0A174NIK6_9 FIRM and its mutants, A0A0D5NFS5_9BACL and its mutants, A0A0D5NNJ7_9BACL and its mutants, A0A1H2AV66_9BACL and its mutants, A0A0Q4RXY0_9BACL and its mutants, A0A0Q7SB75_9BACL and its mutants, A0A015NM92_9BACL and its mutants, A0A100VRN2_PAEAM and its mutants, W4BDJ0_9BACL and its mutants, A0A147K2G0_9EURY and its mutants, A0A0W8FVM4_9ZZZZ and its mutants body, A0A147JXR0_9EURY and its mutants, E8R8J7_DESM0 and its mutants, D5U113_THEAM and its mutants, A0A1F8T9J2_9CHLR and its mutants, G3C952_9ARCH and its mutants, Q6YNI0_9MICC and its mutants, A0A1G0YIQ9_9BACT and its mutants, A0A1J5EHQ6_9DELT and its mutants, A0A1J5E082_9DELT and its mutants, A0A1C4PKD1_9ACTN and its mutants, H8GX25_DEIGI and its mutants,A0A1H5ZFN3_9BACT and its mutants, A0A0M9Z5S1_9ACTN and its mutants, A0A1B2HNC5_9PSEU and its mutants, A0A1B2GNI8_STRNR and its mutants, A0A1F8LBZ3_9CHLR and its mutants, A0A1F8NMM2_9CHLR and its mutants, A0A1F8SDV1_9CHLR and its mutants, A0A1H1PLX0_9BA CT and its mutants, I0IDC5_PHYMF and its mutants, A0A0Q5I8X4_9DEIO and its mutants, A0A0F4JEH6_9ACTN and its mutants, BAD75708.1, *WP_014453859.1 and its mutants, *WP_046170519.1 and its mutants, *CDP53201.1 and its mutants, *WP_035078314.1 and its mutants, *WP_0 42803791.1 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 variants thereof, *KUO41395.1 and variants thereof, *KOV34818.1 and variants thereof, *ANZ15483.1 and variants thereof, *KJY32595.1 and variants thereof, and mixtures thereof, where a variant is defined as a polypeptide sequence having at least 80%, preferably 90%, and most preferably 95% sequence identity to the respective polypeptide sequence.

[0074] More preferably, the hydantoinase enzyme is O69809 and mutants thereof, Q846U5_9BACL and mutants thereof, P81006 and mutants thereof, Q84FR6_9MICC and mutants thereof, Q56S49_9BACI and mutants thereof, A1E351_9BACI and mutants thereof, Q28SA7 and mutants thereof, Q45515 and mutants thereof, A0A399DRQ3_9DEIN and mutants thereof, Q55DL0 and mutants thereof, F7X5M8_SINMM and mutants thereof, Q9I676 and mutants thereof, Q44184 and mutants thereof, B5L3 63 and its mutants, P42084 and its mutants, P25995 and its mutants, Q3Z354 and its mutants, B1XEG2 and its mutants, Q9F465_PAEAU and its mutants, A0A161KD37_9CHLR and its mutants, A0A1J4XHR4_9BACT and its mutants, A0A1C4QIY5_9ACTN and its mutants, A0A0K2UMP4_LEPSM and its mutants, A0A159Z531_9RHOB and its mutants, E1R8C9_SEDSS and its mutants, A0A1F9QT17_9BACT and its mutants Variants, A0A0D8IVV8_9FIRM and its mutants, A0A0B5QKE4_CLOBE and its mutants, A0A0N1GBZ8_9ACTN and its mutants, A0A174ADZ3_9FIRM and its mutants, U7V9Q6_9FUSO and its mutants, A0A0J1FAI4_9FIRM and its mutants, PHYDA_ECOK1 and its mutants, A0A0S8H576_9BACT and its mutants, A0A1J4J4Y8_9EUKA and its mutants, A0A0D5NFS5_9BACL and its mutants, A0A0D5NNJ7_9B ACL and its mutants, A0A1H2AV66_9BACL and its mutants, A0A0Q4RXY0_9BACL and its mutants, A0A0Q7SB75_9BACL and its mutants, A0A100VRN2_PAEAM and its mutants, W4BDJ0_9BACL and its mutants, A0A1J5E082_9DELT and its mutants, A0A1H5ZFN3_9BACT and its mutants, A0A1F8NMM2_9CHLR and its mutants, A0A1F8SDV1_9CHLR and its mutants, A0A1H1PLX0_9BACT and its mutants,A0A0Q5I8X4_9DEIO and variants thereof, *WP_046170519.1 and variants thereof, *WP_023514195.1 and variants thereof, *WP_023516147.1 and variants thereof, and *ANZ15483.1, and mixtures thereof, wherein variants are defined as polypeptide sequences having at least 80%, preferably 90%, and most preferably 95% sequence identity to the respective polypeptide sequences.

[0075] Further suitable hydantoinase enzymes include Q846U5_9BACL and mutants thereof, P81006 and mutants thereof, Q84FR6_9MICC and mutants thereof, Q56S49_9BACI and mutants thereof, Q45515 and mutants thereof, A0A399DRQ3_9DEIN and mutants thereof, Q55DL0 and mutants thereof, F7X5M8_SINMM and mutants thereof, Q9I676 and mutants thereof, Q44184 and mutants thereof, B1XEG2 and mutants thereof, A0A161KD37_9CHLR and mutants thereof, A0A159Z531_9 The polypeptide may be selected from the group consisting of RHOB 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, where a variant is defined as a polypeptide sequence having at least 80%, preferably 90%, most preferably 95% sequence identity to the respective polypeptide sequence.

[0076] In a preferred embodiment, the hydantoinase enzyme 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 variants are defined as polypeptide sequences having at least 80%, preferably 90%, most preferably 95% sequence identity to the respective polypeptide sequence.

[0077] Most preferably, the hydantoinase 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, E1R8C9_SEDSS and variants thereof, A0A159Z531_9RHOB and variants thereof, and mixtures thereof, where variants are defined as polypeptide sequences having at least 80%, preferably 90%, most preferably 95% sequence identity to the respective polypeptide sequence.

[0078] It is to be understood that the above hydantoinase enzymes are designated by a database identifier nomenclature according to the Uniprot database (www.UniProt.org) or the NCBI protein database (www.ncbi.nlm.nih.gov / protein), where sequences from NCBI are designated with a "*" in front of the respective database identifier.

[0079] In a preferred embodiment, the hydantoinase enzyme has SEQ ID NO:1.

[0080] In a preferred embodiment of the present invention, the hydantoinase enzyme is an L-hydantoinase enzyme.

[0081] In a preferred embodiment of the present invention, the hydantoinase enzyme is a D-hydantoinase enzyme.

[0082] In a preferred embodiment of the invention, R in formulae (1) and (2) is H or C1-C6 alkyl, preferably H or C2-C4 alkyl, more preferably ethyl or butyl, especially ethyl.

[0083] In a preferred embodiment of the present invention, the method further comprises the addition of a hydantoin racemase enzyme. Any suitable hydantoin racemase enzyme may be used. Suitable hydantoin racemase enzymes 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 hydantoin racemases, and mixtures thereof. Suitable hydantoin racemase enzymes that can be used in the method include Q9RYA6_DEIRA and mutants thereof, Q9F466 and mutants thereof, Q9F466 and mutants thereof, A0A7L5BQP9_9RHIZ and mutants thereof, Q00924 and mutants thereof, F7X6X4_SINMM and mutants thereof, A0A6V7ACK5_RHIRD and mutants thereof, A0A7Y0XLH3_9RHIZ and mutants thereof, A0A5B8XR30_9D ELT and its mutants, A0A533QH78_9PROT and its mutants, A0A3M9Z0A0_9CYAN and its mutants, A0A3A0A4T5_9CHLR and its mutants, A0A1F6C9P8_HANXR and its mutants, A0A4S0NM85_9RHIZ and its mutants, A0A1V5I086_9SPIR and its mutants, A0A6P0NEY4_9CYAN and its mutants, A0A2K0YBY8_9SPHN and its mutants , A0A1H5NHN7_9RHIZ and its mutants, A0A317KUZ3_9ACTN and its mutants, A0A430VJ34_THESC and its mutants, A0A1J5KHA5_9PROT and its mutants, A0A535LIJ4_9CHLR and its mutants, A0A2T6KHH4_9RHOB and its mutants, A0A3G8JSD5_9ACTN and its mutants, A0A3A9JRT3_9THEO and its mutants, A0A2N7WBP 6_9BURK and its mutants, A0A1A2N8C4_9MYCO and its mutants, A0A1R3TB43_9RHIZ and its mutants, X1T733_9ZZZZ and its mutants, A0A6P1SX79_9RHOB and its mutants, A0A0Q5VT22_9RHIZ and its mutants, A0A2N1RKS5_9SPIR and its mutants, A0A529XJR5_9RHIZ and its mutants, A0A358TXS4_9FIRM and its mutants,A0A1Q9UJX6_9ACTN and variants thereof, A0A434WJY9_9RHIZ 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, wherein variants are defined as polypeptide sequences having at least 80%, preferably 90%, most preferably 95% sequence identity to the respective polypeptide sequence. It is to be understood that the above hydantoin racemase enzymes are indicated by the database identifier nomenclature according to the Uniprot database (www.UniProt.org). Most preferably, the racemase enzyme is selected from the group consisting of A0A6V7ACK5_RHIRD and variants thereof, A0A2T6KHH4_9RHOB and variants thereof, variants being defined as polypeptide sequences having at least 80%, preferably 90%, most preferably 95% sequence identity to the respective polypeptide sequences.

[0084] In certain embodiments of the invention, the method comprises the addition of a hydantoin racemase enzyme and an N-carbamoyl amino acid racemase enzyme.

[0085] In a preferred embodiment of the invention, the hydrolysis step and the cleavage step are carried out in a single vessel. In this connection, it is preferred that the hydrolysis step is carried out under enzymatic conditions. All reagents are preferably added substantially at the beginning of the reaction. Alternatively, the reagents for the hydrolysis step and the reagents for the cleavage step are preferably added at different times to a single vessel.

[0086] In a preferred embodiment of the present invention, the method comprises the step of hydrolyzing the compound of formula (1b) [ka] wherein R is H or C1-C8 alkyl. Separation of the hydantoin having formula (1b) is preferably accomplished using reverse phase chromatography. Alternatively, separation may be accomplished using ion exchange, extraction, salt formation, crystallization and filtration.

[0087] The hydantoin having formula (1b) may be chemically racemized and reused in the hydrolysis step. To racemize the hydantoin having formula (1b), it may be treated with a suitable base, preferably at a pH of 8 or higher, more preferably 8 to 14, even more preferably 8.5 to 12, especially 8.5 to 10. Preferably, the racemization is carried out under aqueous conditions.

[0088] Alternatively, a hydantoin having formula (1b) may be treated with a hydantoin racemase enzyme.

[0089] In a preferred embodiment of the invention, the method comprises the addition of a hydantoinase enzyme, a hydantoin racemase enzyme, and an N-carbamoyl amino acid hydrolase enzyme, with all reaction steps carried out in a single vessel (also known as "one-pot" conditions), preferably with all reagents added substantially at the beginning of the reaction or with the reagents being added at different times to a single vessel.

[0090] In another preferred embodiment of the invention, the method comprises the addition of a hydantoinase enzyme, a hydantoin racemase enzyme, an N-carbamoyl amino acid racemase enzyme, and an N-carbamoyl amino acid hydrolase enzyme, with all reaction steps carried out in a single vessel (also known as "one-pot" conditions), preferably with all reagents added substantially at the beginning of the reaction or with the reagents being added at different times to a single vessel.

[0091] In another preferred embodiment of the invention, the method comprises the addition of an N-carbamoyl amino acid racemase enzyme, and an N-carbamoyl amino acid hydrolase enzyme, and all reaction steps are carried out in a single vessel (also known as "one-pot" conditions), preferably with all reagents added substantially at the beginning of the reaction or with the reagents being added at different times to a single vessel.

[0092] In yet another preferred embodiment of the present invention, the method comprises the addition of a hydantoinase enzyme, and an N-carbamoyl amino acid hydrolase enzyme, and all reaction steps are carried out in a single vessel (also known as "one-pot" conditions), preferably with all reagents added substantially at the beginning of the reaction or with reagents added at different times to a single vessel. In this regard, it is preferred that the pH of the reaction mixture is between 7 and 9.

[0093] The enzyme applied may be applied in any suitable manner known in the art.

[0094] In a preferred embodiment of the invention, the applied enzyme is applied as a clarified cell lysate, whole cells, or as an immobilized enzyme.

[0095] Alternatively, some or all of the components other than L-glufosinate can be removed from the biotransformation mixture, the mixture can be optionally concentrated, and the mixture can then be used directly (and / or with the addition of various adjuvants) for the prevention or control of weeds. The biotransformation mixture can, in some embodiments, be used directly (and / or with the addition of various adjuvants) for the prevention or control of weeds.

[0096] 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 suitable combinations. The enzyme may be removed by simple filtration if supported, or by ultrafiltration if free in solution, by the use of absorbents such as celite, cellulose, or carbon, or by denaturation by various techniques known to those skilled in the art.

[0097] Ion exchange processes effect separation by selective adsorption of solutes onto resins selected for this purpose. Because the product and impurities must be dissolved in a single solution prior to adsorption, concentration of the purified product stream by evaporation or distillation is usually required prior to isolation. Examples of the use of ion exchange for purification are described by Schultz et al. and in EP 0249188 A2.

[0098] Purification may be accomplished by the formation of an insoluble salt of L-glufosinate by the addition of a suitable acid, including hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, acetic acid, and the like. Similarly, purification may be accomplished by the addition of a suitable base to form an insoluble salt. Useful bases include hydroxides, carbonates, sulfates, and phosphates of alkali metals, or hydroxides, carbonates, sulfates, and phosphates of alkaline earth metals. Other bases containing nitrogen may 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 in which the desired salt has very low solubility (such solvents are often referred to as "anti-solvents"). Salts of L-glufosinate can be converted to 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.

[0099] US Patent No. 9,255,115B2 describes a method for obtaining relatively high purity L-glufosinate by converting the hydrochloride of L-glufosinate to a zwitterionic form using a base such as sodium hydroxide or sodium methoxide, followed by crystallization from an aqueous alcoholic solvent. This method has the advantage that it produces non-hygroscopic crystalline L-glufosinate, which can maintain a high purity compared to amorphous L-glufosinate even when exposed to moisture for a long period of time.

[0100] Other salts of L-glufosinate are known in the art. U.S. Pat. Nos. 5,767,309 and 5,869,668 teach the use of chiral alkaloid bases to form diastereomeric salts with racemic glufosinate. Purification is achieved because salts of L-glufosinate precipitate from solution in much greater amounts than the corresponding salts of D-glufosinate. This method can therefore be used with the present invention to obtain L-glufosinate with high enantiomeric excess, if desired.

[0101] Optionally, purification can be achieved by first crystallizing one or more impurities, removing the impurities by filtration, and then further purifying L-glufosinate from the resulting filtrate by forming a salt as described above. This is advantageous if the unreacted amine donor can be partially or completely isolated and used in a subsequent reaction. Similarly, the partially or completely isolated unreacted N-carbamoylamino acid having formula (2) can be recycled for use in a subsequent reaction.

[0102] Extraction may be used to purify the product. German Patent No. 3920570C2 describes a process in which excess glutamic acid (used as an amine donor) is precipitated by adjusting the pH of the solution to 3.7-4.2 with sulfuric acid. After filtering 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 bring the pH to 5-7, precipitating ammonium sulfate. The ammonium sulfate is removed by filtration, and the resulting filtrate is concentrated to obtain the ammonium salt of L-glufosinate.

[0103] Isolation of L-glufosinate or a salt thereof may be desirable, for example, for purposes of shipping the solid to a site of formulation or use. Typical industrial methods of isolation may be used, such as filtration, centrifugation, etc. The isolated product often requires removal of moisture, volatile impurities, and solvents (if present), and for this purpose typical industrial drying equipment can be used. Examples of such equipment include ovens, rotary drum dryers, agitator dryers, etc. In some cases, it may be advantageous to use a spray dryer.

[0104] It is not necessary to produce a solid product after purification. This can be advantageous if formulation of L-glufosinate is performed at the same location used to manufacture L-glufosinate. L-glufosinate and many of its salts are readily soluble in water, which is a convenient liquid for product formulation. For example, the amine donor is isolated by filtration and the resulting filtrate is concentrated by distillation. The pH of the filtrate is adjusted to a desired value and the resulting solution can be used as is or blended with formulation ingredients. In another example, a slurry of L-glufosinate or one of its salts can be prepared as described above and isolated by filtration. The solids can be dissolved directly on the filter by adding water or a suitable solvent to obtain a solution of L-glufosinate.

[0105] Furthermore, equation (3) [ka] (wherein R is H or C1-C8 alkyl), the method for producing glufosinate, its alkyl ester, or its salt, comprising the steps of: [ka] A method is disclosed that includes the step of chemically cleaving off the carbamoyl moiety of an N-carbamoyl amino acid having the formula: (wherein R is H or C1-C8 alkyl).

[0106] The term "chemically cleaved" should be understood to refer to a cleavage step that is not carried out under enzymatic conditions. Any suitable chemical approach is possible.

[0107] Preferably, the chemical cleavage is carried out under acidic conditions, preferably using hydrogen chloride, sulfuric acid, and mixtures thereof, more preferably hydrogen chloride, especially concentrated hydrogen chloride (i.e., 34-38% hydrogen chloride in water).

[0108] Preferably, the chemical cleavage is carried out by adding sodium nitrite.

[0109] Preferably, the chemical cleavage is carried out at elevated temperatures, more preferably at temperatures between 50 and 130°C, even more preferably at temperatures between 70 and 120°C, and particularly preferably at temperatures between 80 and 110°C.

[0110] Preferably, the reaction pressure is ambient pressure. Preferably, the reaction pressure is in the range of 0.995 to 1.030 mbar, more preferably 1.005 to 1.020 mbar, especially about 1.013 mbar.

[0111] In a preferred embodiment of the invention, the cleavage is carried out at a pH of 0-5, preferably 0-3.

[0112] In a particular embodiment, the chemical cleavage is carried out by i) dissolving the N-carbamoyl amino acid having formula (2) under acidic conditions, preferably at a temperature of −15 to 20° C., more preferably −10 to 10° C., in particular −5 to 5° C., ii) adding sodium nitrite, preferably followed by stirring at 10 to 35° C., preferably 15 to 30° C., in particular 20 to 25° C. or at room temperature, and optionally iii) adding an acid, preferably concentrated hydrogen chloride, and heating the reaction mixture to a temperature of 50 to 130° C., preferably 70 to 120° C., in particular 80 to 110° C. Step iii) is suitable for cleaving off the ester, if present.

[0113] The reaction mixture can be worked up by standard procedures (ie, washing and purification).

[0114] As mentioned above, the present invention further relates in a second aspect to a compound of formula (2a) [ka] (wherein R is H or C1-C8 alkyl), and L-glufosinate or a salt thereof.

[0115] In a preferred embodiment of the invention, R in formula (2a) is H or C1-C6 alkyl, preferably H or C2-C4 alkyl, more preferably H, ethyl or butyl, especially H.

[0116] Suitable salts are the hydrochloride, ammonium, and isopropylammonium salts. Additionally, it is understood that the respective zwitterions of L-glufosinate are also included.

[0117] In a preferred embodiment of the invention, the amount of L-glufosinate or a salt thereof is at least 30% by weight, preferably at least 40% by weight, more preferably at least 50% by weight, even more preferably at least 60% by weight, still more preferably at least 70% by weight, in particular at least 80% by weight or at least 90% by weight, based on the total amount of N-carbamoylamino acid having formula (2a) and L-glufosinate or a salt thereof.

[0118] In a preferred embodiment of the present invention, the amount of L-glufosinate or a salt thereof is 20 to 99.9% by weight, preferably 30 to 99.8% by weight, more preferably 40 to 99.7% by weight, even more preferably 50 to 99.6% by weight, still more preferably 60 to 99.5% by weight, particularly 70 to 99.5% by weight or 80 to 99.5% by weight, based on the total amount of the N-carbamoylamino acid having the formula (2a) and L-glufosinate or a salt thereof.

[0119] In a preferred embodiment of the present invention, the amount of L-glufosinate or a salt thereof is 80 to 99.995% by weight, preferably 90 to 99.99% by weight, more preferably 95 to 99.95% by weight, even more preferably 97 to 99.92% by weight, and particularly 99 to 99.9% by weight, based on the total amount of the N-carbamoylamino acid having the formula (2a) and L-glufosinate or a salt thereof.

[0120] The composition comprises a compound represented by formula (2a) [ka] The composition may contain an N-carbamoylamino acid having the formula (2a) in an amount of up to 70% by weight, preferably up to 60% by weight, more preferably up to 50% by weight, even more preferably up to 40% by weight, even more preferably up to 30% by weight, particularly up to 20% by weight or up to 10% by weight, based on the total amount of the N-carbamoylamino acid having the formula (2a) and L-glufosinate or a salt thereof. The composition may contain an N-carbamoylamino acid having the formula (2a) in an amount of up to 40% by weight, preferably up to 20% by weight, more preferably up to 10% by weight, even more preferably up to 5% by weight, even more preferably up to 3% by weight, particularly up to 1% by weight, based on the total amount of the N-carbamoylamino acid having the formula (2a) and L-glufosinate or a salt thereof.

[0121] The composition comprises a compound represented by formula (2a) [ka] The composition may contain an N-carbamoylamino acid having the formula (2a) in an amount of 0.1 to 80% by weight, preferably 0.2 to 70% by weight, more preferably 0.3 to 60% by weight, even more preferably 0.4 to 50% by weight, still more preferably 0.5 to 40% by weight, particularly 0.5 to 30% by weight or 0.5 to 20% by weight, based on the total amount of the N-carbamoylamino acid having the formula (2a) and L-glufosinate or a salt thereof. The composition may contain an N-carbamoylamino acid having the formula (2a) in an amount of 0.001 to 40% by weight, preferably 0.005 to 20% by weight, more preferably 0.01 to 10% by weight, even more preferably 0.05 to 5% by weight, still more preferably 0.08 to 3% by weight, particularly 0.1 to 1% by weight, based on the total amount of the N-carbamoylamino acid having the formula (2a) and L-glufosinate or a salt thereof.

[0122] The composition comprises a compound represented by formula (2b) [ka] The composition may further comprise an N-carbamoylamino acid having the formula (2a), an N-carbamoylamino acid having the formula (2b), and L-glufosinate or a salt thereof, preferably in an amount of up to 40% by weight, preferably up to 20% by weight, more preferably up to 10% by weight, even more preferably up to 5% by weight, still more preferably up to 3% by weight, in particular up to 1% by weight, based on the total amount of the N-carbamoylamino acid having the formula (2a), the N-carbamoylamino acid having the formula (2b), and L-glufosinate or a salt thereof. [ka] The N-carbamoylamino acid having the formula (2a), the N-carbamoylamino acid having the formula (2b), and L-glufosinate or a salt thereof can be contained in an amount of 0.001 to 40% by weight, preferably 0.005 to 20% by weight, more preferably 0.01 to 10% by weight, even more preferably 0.05 to 5% by weight, still more preferably 0.08 to 3% by weight, and particularly 0.5 to 1% by weight, based on the total amount of the N-carbamoylamino acid having the formula (2a), the N-carbamoylamino acid having the formula (2b), and L-glufosinate or a salt thereof.

[0123] The composition may further comprise a compound represented by the formula (1b): [ka] and / or Formula (1a) [ka] The compound may include a hydantoin having the formula:

[0124] The hydantoin (having formula (1b) and / or (1a)) may be present in an amount of up to 30% by weight, preferably up to 20% by weight, more preferably up to 10% by weight, even more preferably up to 5% by weight, still more preferably up to 2% by weight, and in particular up to 0.5% by weight, based on the total amount of hydantoin (having formula (1b) and / or (1a)), N-carbamoylamino acid having formula (2a), and L-glufosinate or a salt thereof.

[0125] The hydantoin (having formula (1b) and / or (1a)) can be contained in an amount of 0.001 to 30% by weight, preferably 0.005 to 20% by weight, more preferably 0.01 to 10% by weight, even more preferably 0.05 to 5% by weight, still more preferably 0.08 to 2% by weight, and particularly preferably 0.1 to 0.5% by weight, based on the total amount of the hydantoin (having formula (1b) and / or (1a)), the N-carbamoylamino acid having formula (2a), and L-glufosinate or a salt thereof.

[0126] In a preferred embodiment of the present invention, R in formulae (2a) and (1b), if present, is H or C1-C6 alkyl, preferably H or C2-C4 alkyl, more preferably H, ethyl or butyl, in particular H or ethyl. In this context, R in formulae (2b) and (1a) is also understood to be preferably H or C1-C6 alkyl, preferably H or C2-C4 alkyl, more preferably H, ethyl or butyl, in particular H or ethyl. In this context, R in formulae (2b), (1a) and (1b), if present, is also understood to be preferably H or C1-C6 alkyl, preferably H or C2-C4 alkyl, more preferably H, ethyl or butyl, in particular H or ethyl.

[0127] In one preferred embodiment of the present invention, the compositions described herein can be used directly as herbicidal compositions or can be used as ingredients in formulated herbicidal products.

[0128] The compositions described herein are useful for application to fields of crop plants for the prevention or control of weeds. The compositions may be formulated as a liquid for spraying on a field. The 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 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 some 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.

[0129] The herbicidal compositions described herein (including concentrates that require dilution before application to plants) contain L-glufosinate (i.e., the active ingredient), optionally Formula (2a) [ka] and one or more adjuvant components in liquid or solid form.

[0130] The composition may also include a hydantoin of formula (1), as further described herein.

[0131] The composition is prepared by mixing the active ingredient with one or more adjuvants, such as diluents, extenders, carriers, surfactants, organic solvents, moisturizers, or conditioning agents, to provide the composition in the form of fine particulate solids, pellets, solutions, dispersions, or emulsions.Thus, the active ingredient can be used with adjuvants, such as fine particulate solids, organic liquids, water, wetting agents, dispersants, emulsifiers, or any suitable combination thereof.From the viewpoint of economy and convenience, water is the preferred diluent.However, not all compounds are resistant to hydrolysis, and in some cases, the use of non-aqueous solvent vehicles may be indicated, as will be understood by those skilled in the art.

[0132] Optionally, one or more additional ingredients can be added to the composition to produce a formulated herbicidal composition. Such a formulated composition can include L-glufosinate, a carrier (e.g., a diluent and / or a solvent), and other ingredients. The formulated composition includes an effective amount of L-glufosinate.

[0133] Diluents can also be included in the formulated composition. Suitable diluents include water and other aqueous components. Optionally, the diluent is present in an amount necessary to produce a composition that is ready for packaging or use.

[0134] The herbicidal compositions described herein, particularly liquids and soluble powders, can contain one or more surfactants as further adjuvant components in a sufficient amount to make a given composition easily dispersible in water or oil. The incorporation of surfactants into the composition greatly enhances its efficacy. The surfactants used herein include wetting agents, dispersing agents, suspending agents, and emulsifying agents. Anionic agents, cationic agents, and nonionic agents can be used equally well.

[0135] Suitable wetting agents include alkylbenzene and alkylnaphthalene sulfonates, sulfated fatty alcohols, amines or acid amides, long chain acid esters of sodium isothionate, esters of sodium sulfosuccinate, sulfated or sulfonated fatty acid esters petroleum sorbonates, sulfonated vegetable oils, secondary acetylene glycols, polyoxyethylene derivatives of alkylphenols (especially isooctylphenol and nonylphenol), and polyoxyethylene derivatives of mono-higher fatty acid esters of hexitol anhydrides (such as sorbitan). Exemplary dispersants include methylcellulose, polyvinyl alcohol, sodium lignin sulfonate, polymeric alkylnaphthalene sulfonates, sodium naphthalene sulfonate, polymethylene bisnaphthalene sulfonate, and sodium N-methyl-N-(long chain acid) laurate.

[0136] Water-dispersible powder compositions 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 typically a synthetic mineral derived from a mineral, such as natural clay, diatomaceous earth, and silica. Examples of such extenders include kaolinite, attapulgite clay, and synthetic magnesium silicate. The water-dispersible powders described herein can optionally contain about 5 to about 95 parts by weight of the active ingredient (e.g., about 15 to about 30 parts by weight of the active ingredient), about 0.25 to about 25 parts by weight of a wetting agent, about 0.25 to about 25 parts by weight of a dispersing agent, and about 4.5 to about 94.5 parts by weight of an inert solid extender. All parts are by weight of the total composition. If desired, about 0.1 to about 2.0 parts by weight of the solid inert extender can be replaced with a corrosion inhibitor or an antifoaming agent or both.

[0137] Aqueous suspensions can be prepared by dissolving or by mixing and milling an aqueous slurry of a water-insoluble active ingredient in the presence of a dispersing agent to obtain a concentrated slurry of very fine particles. The resulting concentrated aqueous suspension is characterized by its extremely small particle size and therefore, when diluted and sprayed, gives a very uniform coverage.

[0138] Emulsifiable oils are typically water-immiscible or partially water-immiscible solvents in which an active ingredient is dissolved along with a surfactant. Suitable solvents for the active ingredients described herein include hydrocarbons and water-immiscible ethers, esters, or ketones. Emulsifiable oil compositions generally contain about 5-95 parts active ingredient, about 1-50 parts surfactant, and about 4-94 parts solvent, all parts being by weight based on the total weight of the emulsifiable oil.

[0139] The compositions described herein can also contain other additives, such as fertilizers, phytotoxic agents and plant growth regulators, insecticides, etc., used as adjuvants or in combination with any of the above adjuvants. The compositions described herein can also be mixed with other materials, such as fertilizers, other phytotoxic agents, etc., and applied at the same time.

[0140] In each of the formulation types described herein, eg, liquid and solid formulations, the concentration of active ingredient is the same.

[0141] It is recognized that the herbicidal composition can be used in combination with other herbicides. The herbicidal 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 compound of the present invention can be formulated with the other herbicides, tank mixed with the other herbicides, or applied sequentially with the other herbicides. Herbicides that can be used in combination with the compound of the present invention include: amide herbicides, such as alidochlor, beflubutamid, benzadox, benzipram, bromobutide, cafenstrole, CDEA, chlorthiamid, cyprazole, dimethenamid, dimethenamid-P, diphenamid, epronaz, etonipromide, fentrazamide, flupoxam, fomesafen, halosafen, isocarbamide, isoxaben, napromide, naptalam, petoxamide, propyzamide, quinonamide and tebutam; anilide herbicides such as chloranocryl, cisanilide, clomeprop, cypromide, diflufenican, etobenzanide, fenasulam, flufenacet, flufenican, mefenacet, mefluidide, metamifop, monalide, naproanilide, pentanochlor, picolinafen and propanil; arylalanine herbicides such as benzoylprop, flamprop and franprop. Pu-M; chloroacetanilide herbicides, such as acetochlor, alachlor, butachlor, butenachlor, delaclor, diethathyl, dimethachlor, metazachlor, metolachlor, S-metolachlor, pretilachlor, propachlor, propisochlor, purinachlor, terbuchlor, thenylchlor and xylachlor; sulfonanilide herbicides, such as benzofluor, perfluidone, pyrimisulfan and profluazole; sulfonamide herbicides, such as antibiotic herbicides such as viranaphos; benzoic acid herbicides such as chloramben, dicamba, 2,3,6-TBA and tricamba; pyrimidinyloxybenzoic acid herbicides such as bispyribac and pyriminobac; pyrimidinylthiobenzoic acid herbicides such as pyrithiobac; phthalic acid herbicides such as chlorthal; picolinic acid herbicides such as aminopyralid, clopyralid and picloram;Quinoline carboxylic acid herbicides such as quinclorac and quinmerac; arsenical herbicides such as cacodylic acid, CMA, DSMA, hexaflurate, MAA, MAMA, MSMA, potassium arsenite and sodium arsenite; benzoylcyclohexanedione herbicides such as mesotrione, sulcotrione, tefuryltrione and tembotrione; benzofuranyl alkylsulfonate herbicides such as benfuresate and ethofumesate; carbamate herbicides such as asulam, carboxazole chlorprocarb, dichlormate, phenastra carbanilate herbicides, such as balban, BCPC, carbasulam, carbetamide, CEPC, chlorbufam, chlorpropham, CPPC, desmedipham, phenisofam, phenmedipham, phenmedipham-ethyl, propham and swep; cyclohexene oxime herbicides, such as alloxydim, butroxydim, clethodim, cloproxydim, cycloxydim, profoxydim, sethoxydim, tepraloxydim and tralkoxydim; cyclopropylisoxazole herbicides, such as valvan, BCPC, carbasulam, carbetamide, CEPC, chlorbufam, chlorpropham, CPPC, desmedipham, phenisofam, phenmedipham-ethyl, propham and swep; dicarboximide herbicides, such as benzfendizone, cinidon-ethyl, flumezin, flumiclorac, flumioxazin and flumipropin; dinitroaniline herbicides, such as benfluralin, butralin, dinitramine, ethalfluralin, fluchloralin, isoproparin, methalproparin, nitralin, oryzalin, pendimethalin, prodiamine, profluralin and trifluralin; dinitrophenol herbicides, such as dinophenate, dinoprop, dinosam, di Noseb, Dinoterb, DNOC, Ethinofen and Medinoterb; Diphenyl ether herbicides such as Ethoxyfen; Nitrophenyl ether herbicides such as Acifluorfen, Aclonifen, Bifenox, Chlomethoxyfen, Clomitrofen, Etonipromide, Fluorodifen, Fluoroglycofen, Fluoronitrofen, Fomesafen, Furyloxyfen, Halosafen, Lactofen, Nitrofen, Nitrofluorfen and Oxyfluorfen; Dithiocarbamate herbicides such as Dazomet and Metam;Halogenated aliphatic herbicides such as Arorac, Chloropone, Dalapon, Flupropanate, Hexachloroacetone, Iodomethane, Methyl Bromide, Monochloroacetic Acid, SMA and TCA; imidazolinone herbicides such as Imazamethabenz, Imazamox, Imazapic, Imazapyr, Imazaquin and Imazethapyr; 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 Bromobonyl, Bromooxygen, Cinyl, Chloroxynil, Dichlorobenyl, Iodobonyl, Ioxynil and Pyraclonil; Organophosphorus Herbicides such as Amiprophos-methyl, Anilofos, Benzulide, Villanaphos, Butamiphos, 2,4-DEP, DMPA, EBEP, Fosamine, Glyphosate and Piperophos; Phenoxy Herbicides such as Bromofenoxime, Clomeprop, 2,4-DEB, 2,4-DEP, Diphenopentene, Disul, Ervon, Ethonipromide, Fenteracol and Trifopsim; Phenoxyacetic Herbicides such as 4-CPA, 2,4-D, 3,4-DA, MCPA, MCPA-thioethyl 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 cloprop, 4-CPP, dichlorprop, dichlorprop-P, 3,4-DP, fenoprop, mecoprop and mecoprop-P; aryloxyphenoxypropionic acid herbicides such as chloradifop, clodinafop, clofop, cyhalofop, diclofop, fenoxaprop, fenoxaprop-P, fentiaprop phenylenediamine herbicides such as dinitramine and prodiamine; pyrazolyl herbicides such as benzofenap, pyrazolinate, pyrasulfotole, pyrazoxyfen, pyroxasulfone and topramezone; pyrazolylphenyl herbicides such as fluazolate and pyraflufen; pyridazine herbicides such as cledazine, pyridaphor and pyridate;Pyridazinone herbicides such as brompyrazone, chloridazon, dimidazon, flufenpyr, methoflurazone, norflurazone, oxapyrazon and paidanone; pyridine herbicides such as aminopyralid, cliodinate, clopyralid, dithiopyr, fluroxypyr, haloxyzin, picloram, picolinafen, pyrichlor, thiazopyr and triclopyr; pyrimidinediamine herbicides such as iprimidum and thioclorim; quaternary ammonium herbicides such as cyperquat, diethamquat, difenzo Quat, Diquat, Morphamquat and Paraquat; Thiocarbamate herbicides such as butyrate, cycloate, diallate, EPTC, esprocarb, ethiolate, isopolynate, methiobencarb, molinate, orbencarb, pebulate, prosulfocarb, pyributicarb, sulfarate, thiobencarb, thiocarbazyl, triallate and bemorate; Thiocarbonate herbicides such as dimexano, EXD and proxan; Thiourea herbicides such as methiuron; Triazine herbicides , for example, dipropetryn, triaziflam and trihydroxytriazine; chlorotriazine herbicides, for example, atrazine, chlorazine, cyanazine, cyprazine, eglinazine, ipazine, mesoprazine, procyazine, proglinazin, propazine, sebutyrazine, simazine, terbuthylazine and trietazine; methoxytriazine herbicides, for example, atraton, methometon, prometon, secbumeton, simeton and terbumeton; methylthiotriazine herbicides, for example, ametryn, adiprothryn, cyanazine, triazinone herbicides such as ametridione, amivudine, hexazinone, isomethiozine, metamitron and metribuzin; triazole herbicides such as amitrole, cafenstrole, epronaz and flupoxam; triazolone herbicides such as amicarbazone, bencarbazone, carfentrazone, flucarbazone, propoxycarbazone, sulfentrazone and thiencarbazone-methyl;Triazolopyrimidine herbicides, such as cloransulam, diclosulam, florasulam, flumetsulam, metosulam, penoxsulam and piroxsulam; uracil herbicides, such as butafenacil, bromacil, flupropacil, isocyl, lenacil and terbacil; 3-phenyluracil; urea herbicides, such as benzthiazuron, cumyluron, cyclone, dichloralurea, diflufenzopyr, isonoruron, isouron, methabenzthiazuron, molybdenum herbicides, nisouron and noruron; phenylurea herbicides, such as anisuron, buturon, chlorbromuron, chloreturon, chlorotoluron, chloroxuron, dymuron, difenoxuron, dimefuron, diuron, fenuron, fluometuron, fluothiuron, isoproturon, linuron, methiuron, methyldimuron, methobenzuron, metobromuron, metoxuron, monolinuron, monuron, nebulon, parafluron, fenobenzuron, siduron, te trafluron and thidiazuron; pyrimidinylsulfonylurea herbicides, such as amidosulfuron, azimsulfuron, bensulfuron, chlorimuron, cyclosulfamuron, ethoxysulfuron, flazasulfuron, flucetosulfuron, flupyrsulfuron, foramsulfuron, halosulfuron, imazosulfuron, mesosulfuron, nicosulfuron, orthosulfamuron, oxasulfuron, primisulfuron, pyrazosulfuron, rimsulfuron, Sulfometuron, sulfosulfuron and trifloxysulfuron; triazinylsulfonylurea herbicides such as chlorsulfuron, cinosulfuron, ethametsulfuron, iodosulfuron, metsulfuron, prosulfuron, thifensulfuron, triasulfuron, tribenuron, triflusulfuron and tritosulfuron; thiadiazolyl urea herbicides such as buthiuron, ethidimuron, tebuthiuron, thiazafluron and thidiazuron; and Unclassified herbicides, such as acrolein, allyl alcohol, aminocyclopyrachlor, azaphenidine, benazolin, bentazone, benzobicyclon, buthidazole, calcium cyanamide, cambendichlor, chlorfenac, chlorfenprop, chlorflurazole, chlorfenol, cinmethylin, clomazone, CPMF, cresol, orthodichlorobenzene, dimepiperate, endothal, fluoromidine, fluridone, flurochlorine Don, flurtamone, fluthiaset, indanofan, methazole, methyl isothiocyanate, nipiraclofen, OCH, oxadiargyl, oxadiazon, oxaziclomefone, pentachlorophenol, pentoxazone, phenylmercuric acetate, pinoxaden, prosulfarin, pyribenzoxim, pyriftalid, quinoclamine, rodetanil, sulglicapin, thidiadiamine, tridifen, trimeturon, tripropindan and tritac. The herbicidal composition of the present invention can also be used in combination with glyphosate or 2,4-D on crops that are tolerant to glyphosate or 2,4-D. In general, it is preferred to use the composition of the present invention in combination with herbicides that are selective for the crops treated and that complement the spectrum of weeds controlled by these compositions at the application rate employed. It is also generally preferred to apply the composition of the present invention and other complementary herbicides simultaneously, as a combination formulation or as a tank mix.

[0142] As mentioned above, the present invention also relates to a third aspect, a method for selectively controlling weeds in an area, preferably comprising a planted seed crop or a crop tolerant to glufosinate, comprising: An effective amount of a composition containing L-glufosinate or a salt thereof in an enantiomeric ratio of at least 80% relative to D-glufosinate or a salt thereof and a compound represented by formula (2) in an amount of more than 0.01% by weight and less than 10% by weight based on the total amount of the composition [ka] The method includes applying to the area an effective amount of an N-carbamoyl amino acid having the formula:

[0143] In a preferred embodiment of the present invention, the composition contains L-glufosinate or a salt thereof in an enantiomeric proportion of 80 to 99.9% relative to D-glufosinate or a salt thereof, preferably in an enantiomeric proportion of 85 to 99.8%, more preferably in an enantiomeric proportion of 90 to 99.7%, in particular in an enantiomeric proportion of 92 to 99.5%.

[0144] In a preferred embodiment of the present invention, the composition contains from 0.02 to 8% by weight, preferably from 0.03 to 5% by weight, more preferably from 0.05 to 3% by weight, particularly preferably from 0.1 to 2% by weight, of a compound represented by formula (2) based on the total weight of the composition. [ka] (wherein R is H or C1-C8 alkyl, preferably H).

[0145] It is to be understood that the compositions may include the same adjuvants and / or other herbicides as those described in more detail above.

[0146] Preferred dispersants are as described in more detail above.

[0147] The compositions described herein are useful for application to fields of crop plants for the prevention or control of weeds. The compositions may be formulated as a liquid for spraying on a field. L-glufosinate is provided in the composition in an effective amount. As used herein, an effective amount means about 10 grams of active ingredient 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 some 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.

[0148] The present invention is further illustrated by the following examples. EXAMPLES

[0149] Materials and Methods Preparation of enzymes a) Cloning of the enzyme gene (EX1) The amino acid sequence of each enzyme was 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 a "*" at the beginning of the respective database identifier). The respective 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 plasmids were used to transform competent cells of E. coli TG10 strain, pAgro, pHSG575 (E. coli TG10 (Kesseler, M. et al., WO 2004050877A1, 2004, (BASF AG)):pHSG575 (Takeshita, S. et al., Gene, 1987, 61, 63) and rhaA-derivatives of E. coli TG1 transformed with pAgro4 (pBB541 in Tomoyasu, T. et al., Mol. Microbiol., 2001, 40, 397) (Chung, CT et al., Proc Natl Acad Sci USA, 1989, 86, 2172).

[0150] b) Recombinant production of enzymes (EX2) Preparation of biocatalysts in shake flasks E. coli TG10 carrying the recombinant plasmid of the enzyme was used to inoculate 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, and the resulting preliminary culture was cultivated at 37°C and 250 rpm for 5 hours. 1 ml of this preliminary culture 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-β-D-thiogalactopyranoside, and 0.5 g / l rhamnose in a 500 ml baffled Erlenmeyer flask. The culture was incubated at 37°C for 18 hours with shaking. The biomass was then harvested by centrifugation at 3220xg for 10 min at 8 °C. The supernatant was discarded and the cell pellet was suspended in 8 ml of HEPES buffer (concentration 100 mM and pH 8.2, with 1 mM MnCl2). This cell suspension was used without further preparation for synthesis if whole cell biotransformation was performed. If clarified cell lysate was used instead, 5 ml of cell suspension was distributed into 5 reaction tubes containing lysis matrix B (0.7 ml of 0.1 mm quartz beads, MP Biomedicals), the tubes were chilled on ice, and the cells were subsequently disrupted in a homogenizer (Peqlab Precellys24, VWR) for two 30 s cycles. The samples were chilled on ice between cycles. The resulting cell-free lysate was clarified by centrifugation at 20817xg for 10 min at 8 °C. The supernatant was isolated and fractions from the same batch were combined (= clarified cell lysate).

[0151] Fermentative whole cell biocatalytic production E. coli TG10 containing the plasmids pAgro4 and pHSG575 was transformed with the pDHE plasmid encoding the protein of interest, and the transformants were cultured on LB agar medium supplemented with 100 μg / ml ampicillin, 100 μg / ml spectinomycin, and 20 μg / ml chloramphenicol.

[0152] Preculture 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

[0153] Sterilization was achieved by filtration using a filter with a pore size of 0.2 μm.

[0154] Part 1 Ultrapure water 1.0kg Citric acid monohydrate 3.4g Magnesium sulfate heptahydrate 2.4g Calcium chloride dihydrate 0.1g EcoK12 solution 20g Sodium hydroxide solution 25% Used to adjust pH to 6.6

[0155] Part 2 Ultra pure water 500g Potassium dihydrogen phosphate 26.6g Diammonium hydrogen phosphate 8.0g Sodium hydroxide solution 25% Used to adjust pH to 6.4

[0156] Part 3 Ultra pure water 500g Glycerol 99% 36.0g Sodium gluconate 24.0g Phosphoric acid 20% Used to adjust pH to 6.6

[0157] All three parts were sterilized at 121°C for 30 minutes.

[0158] Vitamin Solution Ultra pure water 100g Thiamine hydrochloride 1.0g Vitamin B 12 0.5g

[0159] Sterilization was achieved by filtration using a filter with a pore size of 0.2 μm.

[0160] To make the final preculture medium, parts 1, 2 and 3 were combined and 2.0 ml of vitamin solution was added. Additionally, the medium was supplemented with 100 μg / ml ampicillin, 100 μg / ml spectinomycin and 20 μg / ml chloramphenicol. Several transformants were scraped off the LB agar plates and used to inoculate 100 g of preculture medium twice in 1 L baffled Erlenmeyer flasks. These precultures were grown at 37°C and 150 rpm. When the OD600 reached 12, the precultures were used directly to inoculate the main cultures.

[0161] Main culture medium: Part 4 Ultrapure water 9.6kg Citric acid monohydrate 21.1g Potassium dihydrogen phosphate 173.6g Diammonium hydrogen phosphate 52.8g Mannium sulfate heptahydrate 15.1g Calcium chloride dihydrate 0.7g EcoK12 solution 123g Sodium hydroxide solution 25% Used to adjust pH to 6.4 Pluriol P2000 1ml

[0162] Part 4 was sterilized at 125°C for 45 minutes.

[0163] Part 5 Ultra pure water 300g Thiamine hydrochloride 151mg Vitamin B12 30.2mg Ampicillin sodium salt 1000mg Spectinomycin hydrochloride 500mg Chloramphenicol 200mg

[0164] Part 5 was sterilized by sterile filtration using a 0.1 μm pore size filter unit.

[0165] Glycerol Solution Ultra pure water 804g Citric acid monohydrate 29.1g Sodium sulfate 58.1g Diammonium iron sulfate hexahydrate 4.5g Glycerol 99% 3370g

[0166] Thiamine solution Ultra pure water 40g Thiamine hydrochloride 55mg

[0167] antifoam solution Pluriol P2000 350g

[0168] Base solution Ammonia water 25% 1500ml

[0169] Inducer solution Ultra pure water 150g Rhamnose monohydrate 100g IPTG 238mg

[0170] Glycerol and antifoam solutions were sterilized for 30 minutes at 121° C. Thiamine and inducer solutions were sterilized by filtration through 0.2 μm pore size filters.

[0171] Parts 4 and 5 were combined in a sterile fermentation vessel (Techfors, Infors HT) and inoculated with the preculture. The fermentation vessel was kept at a temperature of 37°C, a pressure of 0.2 bar and a pH of 6.6. The pO2 level was kept at 20-40% by adjusting the agitator speed (usually 500 rpm) and the aeration rate (usually 6 l / min). Antifoam solution was added as required. The glycerol and thiamine solutions were combined to obtain the feeding solution. After inoculation, the feeding solution was administered at a rate of 10 g / h. After 7 h, the feeding solution administration was switched to the "stop-and-sea" mode. Here, the feeding was activated at a rate of 10 g / h when the pO2 level increased. After 14 h or after consuming 330 g of feeding solution, the feeding rate was increased to 80-100 g / h. Gene expression was induced at an oxygen transfer rate of 80 mmol / l / h or at an OD600 of 12 by the addition of inducer solution. Thirty-six hours after induction, fermentation was stopped by lowering the temperature to 15°C. The cooled fermentation broth was drained from the fermenter and centrifuged at 4700 rpm and 10°C to pellet the cells. The resulting supernatant was discarded and the cells were resuspended in 3850 g of 50 mM potassium dihydrogen phosphate buffer, pH 7.0. The cell suspension was frozen at -80°C before lyophilization, with the lyophilizer held at -50°C and 0.25 mbar pressure. The lyophilized cell-free extract was stored at 4°C.

[0172] Preparation of lyophilized cell-free extract Lyophilized cells were suspended at 100 g / l in ultrapure water. After cooling the cell suspension on ice, the cells were disrupted by passing three times through a pressure homogenizer (Panda Plus 2000, GEA) set at 800 bar. The pressure for the three passes was typically 1000-1400 bar. The resulting mixture was cleared of debris by centrifugation at 10000 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 then lyophilized at -50 °C and 0.25 mbar pressure.

[0173] Preparation of Starting Materials and Intermediates c) Chemical synthesis of 5-([2-[butoxy(methyl)phosphoryl]ethyl]imidazolidine-2,4-dione (Ex3) [ka] To a stirred solution of [2-[butoxy(methyl)phosphoryl]-1-cyano-ethyl]acetate (100 g, 90% purity, Cas 167004-78-6) in methanol (400 mL) was added concentrated sulfuric acid (1 g) and the reaction mixture was heated to 40° C. and stirred at this temperature for 15 h. After the reaction mixture was cooled to room temperature, sodium methoxide in methanol (30%, 3.52 g) was added, followed by sodium sulfate (2 g) and stirred at room temperature for 30 min. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure (84.5 g).

[0174] To the crude butyl 3-cyano-3-hydroxypropyl(methyl)phosphinate (84.5 g, 366 mmol) was added a solution of diammonium carbonate (70.4 g, 732 mmol) in water (290 mL). The reaction mixture was heated to 70° C. for 4 h and then evaporated to dryness under reduced pressure. The residue was suspended in hot isopropanol (70° C.), the resulting suspension was filtered, and the filter cake was washed with isopropanol (2×10 mL). The filtrate was concentrated under reduced pressure and filtered through silica (eluted with 1.5 L dichloromethane / methanol 9:1). The filtrate was concentrated under reduced pressure to give 55.5 g of product, and the resulting solid was recrystallized from isopropanol / diisopropyl ether (34% yield). 1 H NMR (500MHz, deuterium oxide) δ 4.42-4.37(m,1H),4.08-4.00(m,2H),2.19-1.77(m,4H),1.70-1.64(m,2H ),1.61(d,J=13.8Hz,3H),1.46-1.34(m,2H),0.92(td,J=7.4,0.8Hz,3H).

[0175] d) Chemical synthesis of 5-([2-[ethoxy(methyl)phosphoryl]ethyl]imidazolidine-2,4-dione from racemic glufosinate (Ex4) [ka] To a stirred solution of glufosinate ammonium (50% in water, 50 g, 126 mmol) under vacuum (200 mbar) was added a solution of potassium cyanate (17 g, 202 mmol) in water (50 ml) at 50° C. over 40 min. The reaction mixture was stirred at 50° C. under vacuum (200 mbar) for a further 1.5 h and then cooled to room temperature. After stirring at room temperature and ambient pressure for a further 14 h, concentrated HCL (125 mL, 36%) was added and the reaction mixture was heated to reflux for 30 min. The reaction mixture was concentrated under reduced pressure. The residue was dissolved in water (50 mL) at 50° C. and filtered. The filtrate was subjected to ion exchange chromatography (Dowex-50 WX 8200-400(H), 500 mL) eluting with water (1 L) to give the product in essentially quantitative yield. 1 H NMR (500MHz, deuterium oxide) δ 4.41-4.36 (m, 1H), 2.15-1.70 (m, 4H), 1.52 (d, J = 13.9Hz, 3H).

[0176] [ka] To a mixture of acetic acid (50 mL) and triethyl orthoacetate (75 mL, 409 mmol) at room temperature was added 2-(2,5-dioxoimidazolidin-4-yl)ethyl-methyl-phosphinic acid (10 g, 48.5 mmol, synthesized as above). The reaction mixture was heated to reflux (110° C., heating bath temperature) for 15 min. The reaction was then concentrated under reduced pressure and purified by column chromatography (dichloromethane / methanol 9:1) to give ethyl 2-(2,5-dioxoimidazolidin-4-yl)ethyl(methyl)phosphinate (4.8 g, 42%). 1 H NMR (500MHz, deuterium oxide) δ 4.41-4.36(m,1H),4.13-4.03(m,2H),2.19-1.74(m,4H),1.60(d,J=13.9Hz,3H),1.35-1.28(m,3H).

[0177] e) Synthesis of N-carbamoylamino acids (under chemical conditions) (Ex5) [ka] To a stirred solution of racemic glufosinate ammonium (50% in water, 39.6 g, 99.9 mmol) under vacuum (200 mbar) was added a solution of potassium cyanate (11.8 g, 145 mmol) in water (30 ml) at 50° C. over 30 min. The reaction mixture was stirred under vacuum (200 mbar) at 50° C. for a further 1 h and then cooled to room temperature. The reaction mixture was subjected to ion exchange chromatography (Dowex-50 WX 8200-400(H), 220 mL) and the product was eluted with water (1 L). The eluted product was concentrated under reduced pressure to give the carbamoyl acid product (7.9 g). The remaining carbamoyl acid was re-isolated from the column as the potassium salt. 1 H NMR (500 MHz, deuterium oxide) δ 4.31-4.25 (m, 1H), 2.19-1.81 (m, 4H), 1.52 (d, J = 14.1 Hz, 3H). D- and L-enantiomers were synthesized in a similar manner from commercially available D- and L-glufosinate. Specific rotation [α] of compound L-enantiomer = 27.5° (c = 1 H2O, measured as potassium salt). HPLC-MS retention times using Supelco Chirobiotic T2 (eluent 40% water in acetonitrile, 0.1% formic acid). Temperature: 20 °C, flow rate 0.8 mL / min. Retention time of L-carbamoyl amino acids (7.4 min); retention time of D-carbamoyl amino acids (9.2 min).

[0178] f) Chemical synthesis of 5-([2-[ethoxy(methyl)phosphoryl]ethyl]imidazolidine-2,4-dione from N-carbamoyl amino acid (Ex6) [ka] (2R)-4-[ethoxy(methyl)phosphoryl]-2-ureido-butanoic acid (i.e., synthesised via Ex11 or Ex12) (164 mg) was dissolved in aqueous HCl (5%, 3 mL). The reaction mixture was shaken at 40° C. for 48 h. NMR showed complete conversion of the N-carbamoyl amino acid to D-hydantoin. The D-hydantoin can be readily racemized according to Example 14 (enzymatic) or by treatment with aqueous ammonia at pH 8.5 (in a manner similar to that described in Example 15).

[0179] Reactions using a chemical carbamoyl cleavage step g) Enzymatic synthesis of butyl-protected N-carbamoyl amino acids (EX7) [ka] To a solution of ([2-[butoxy(methyl)phosphoryl]ethyl]imidazolidine-2,4-dione (7.8 g, 29.7 mmol) in degassed potassium phosphate buffer (60 mL, 0.496 M, pH 8.0) was added KOH (3 M in water, 390 μL) to adjust the pH to 8.0. This mixture was diluted with potassium phosphate buffer (223 mL, 0.100 M, pH 8.0) and hydantoinase enzyme (Uniprot ID: A0A159Z531_9RHOB, SEQ ID NO: 1, clarified cell lysate, 16.5 mL, total protein concentration 8.1 mg / mL) and MnCl2 solution (1 M in water, 240 μL) were added. The reaction mixture was stirred (250 rpm) at 37 °C for 24 h. The crude material was filtered to remove the cell lysate and the filter cake was washed with water (60 mL). The filtrate was concentrated under reduced pressure, redissolved in THF / wet MeOH and filtered through silica (eluent was pure methanol). The crude was then purified by reverse phase chromatography (water / acetonitrile 99:1 to 95:5 gradient) to give 4-[butoxy(methyl)phosphoryl]-2-ureido-butanoic acid (2.05 g, 25%). 1H NMR (500 MHz, deuterium oxide) δ 4.11-4.07(m,1H),4.06-4.00(m,2H),2.09-1.80(m,4H),1.71-1.63(m,2H),1.59(d,J=13.7Hz,3H),1.46-1.34(m,2H),0.92(t,J=7.4Hz,3H).

[0180] h) Enzymatic synthesis of N-carbamoyl amino acids (EX7) [ka] 2-(2,5-Dioxoimidazolidin-4-yl)ethyl-methyl-phosphinic acid (2.47 g) was dissolved in water (2 mL) and NaOH (50% in water, 1.91 g) was added. The reaction was diluted to 6 mL with water and shaken at 50° C. for 28 hours. After this reaction time, NMR revealed that 96% of the hydantoin was converted to the sodium salt of the N-carbamoyl amino acid.

[0181] i) Chemical synthesis of glufosinate (Ex8) [ka] 4-[Butoxy(methyl)phosphoryl]-2-ureido-butanoic acid (100 mg), synthesized using hydantoinase (Uniprot: A0A159Z531_9RHOB), was dissolved in aqueous HCl (3.5 M, 10 mL) and the stirred reaction mixture was cooled to 0° C. A solution of sodium nitrite (26 mg) in water (2 mL) was added and the reaction mixture was warmed to room temperature. The reaction mixture was stirred at room temperature for an additional 2 h. Concentrated HCl in water (36%, 7.5 mL) was then added and the reaction mixture was heated to 100° C. and stirred at this temperature overnight. The reaction mixture was cooled to room temperature and extracted twice with methylene chloride (2×10 mL). The aqueous phase was concentrated under reduced pressure to give the hydrochloride salt of glufosinate. 1 H NMR (500MHz, deuterium oxide) δ 3.84-3.78 (m, 1H), 2.17-2.00 (m, 2H), 1.74-1.54 (m, 2H), 1.27 (d, J = 13.5Hz, 3H).

[0182] j) Enzymatic synthesis of glufosinate (Ex9) [ka] To a solution of 2-(carbamoylamino)-4-[hydroxy(methyl)phosphoryl]butanoic acid (0.6 g, 2.5 mmol) in degassed aqueous potassium phosphate buffer (5.4 mL, 0.496 M, pH 8.0) was added KOH (3 M in water) to adjust the pH to 8.0. To the reaction mixture (6.1 ml) was added potassium phosphate buffer (19.2 mL, 0.100 M, pH 8.0) and N-carbamoylamino acid hydrolase enzyme (Uniprot ID: A0A1Y4GC62_9BACT, SEQ ID NO: 2, clarified cell lysate, 1.5 mL, total protein concentration 12.9 mg / mL, protein produced in shake flask) and MnCl2 solution (1 M in water, 20 μL). The reaction mixture was stirred (250 rpm) at 37° C. for 24 h. NMR and HPLC analysis showed a 31% conversion to glufosinate. The enantiomeric ratio of glufosinate was analyzed by chiral HPLC: >99%-L-glufosinate / <1%-D-glufosinate; analytical method: Chirex(D)-Pencillamine 250×4.6 mm column from Phenomenex; isocratic elution 10 mM copper(II) sulfate; UV detection at 245 nm).

[0183] k) Enzymatic synthesis of glufosinate (Ex10) In parallel, the reaction of Ex9 was carried out under identical conditions using another N-carbamoyl amino acid hydrolase enzyme (Uniprot ID: A0A6P2ISL4_BURL3, SEQ ID NO: 3, clarified cell lysate, 1.5 mL, 10.2 mg / mL total protein concentration, protein produced in shake flask) and also yielded L-glufosinate (21% conversion, chiral HPLC: >99% L-glufosinate / <1% D-glufosinate).

[0184] a) Enzymatic one-pot synthesis of ethyl-glufosinate from the ethyl ester of hydantoin (Ex11, SEQ ID NO:1+4) [ka] Ammonia (25% in water) was added to a solution of 5-[2-[ethoxy(methyl)phosphoryl]ethyl]imidazolidine-2,4-dione (5.6 g, 24 mmol) in water (20 mL) to adjust the pH to 8.5. To this mixture was added MnCl2 solution (2 M in water, 1 mL) and hydantoinase enzyme (Uniprot ID: A0A159Z531_9RHOB, SEQ ID NO: 1, clarified cell lysate, 7.5 mL, total protein concentration 33 mg / mL) and N-carbamoyl amino acid hydrolase enzyme (A0A535Y1H2_9CHLR, SEQ ID NO: 4, clarified cell lysate, 2.8 mL, total protein concentration 44.8 mg / mL). The reaction mixture was stirred at 37° C. for 72 hours. The pH was maintained at 8.5 with ammonia (25% in water) during the 72 hour reaction time. After 7, 27, 30 and 49 hours total reaction time, N-carbamoyl amino acid hydrolase enzyme (A0A535Y1H2_9CHLR, Seq ID:4, 2.8 mL, clarified cell lysate) was added. NMR showed 36% conversion of L-glufosinate to ethyl ester after 24 hours and 43% conversion after 72 hours (enantiomeric ratio L>99%, D>1% by chiral HPLC). After completion of the reaction, the crude reaction mixture was heated to 80° C. for 30 minutes and filtered to remove the cell lysate. The mixture of L-glufosinate ethyl ester and ethyl ester of N-carbamoyl amino acid was separated on Dowex-50 WX 8 200-400(H). N-carbamoyl amino acid was eluted with water and ethyl ester of L-glufosinate was eluted with ammonia (0.5 M in water) to give L-glufosinate ethyl ester. Alternatively, L-glufosinate ethyl ester could be isolated by crystallization. The remaining carbamoyl amino acid could be recycled via Ex6. The concentrations of L-glufosinate and D-glufosinate ethyl esters were determined by HPLC-MS using a Supelco Chirobiotic T2 (eluent 25% water in acetonitrile, 0.1% formic acid). Temperature: 20°C, flow rate 1.0 mL / min. Retention times of glufosinate ethyl ester: L-configuration diastereoisomer (7.6+7.8 min); D-configuration (8.5 and 11.5 min).

[0185] b) Enzymatic one-pot synthesis of ethyl-glufosinate from the ethyl ester of hydantoin (Ex12, SEQ ID NO:1+3) [ka] Ammonia (25% in water) was added to a solution of 5-[2-[ethoxy(methyl)phosphoryl]ethyl]imidazolidine-2,4-dione (5.6 g, 24 mmol) in water (20 mL) to adjust the pH to 8.5. To this mixture was added MnCl2 solution (2 M in water, 1 mL) and hydantoinase enzyme (Uniprot ID: A0A159Z531_9RHOB, SEQ ID NO: 1, clarified cell lysate, 7.5 mL, total protein concentration 33 mg / mL) and N-carbamoyl amino acid hydrolase enzyme (A0A6P2ISL4_BURL3, SEQ ID NO: 3, clarified cell lysate, 2.8 mL, total protein concentration 44 mg / mL). The reaction mixture was stirred at 37° C. for 48 hours. The pH was maintained at 8.5 with ammonia (25% in water) during the 48 hour reaction time. After 7, 27 and 30 hours total reaction time, N-carbamoyl amino acid hydrolase enzyme (SEQ ID NO:3, 2.8 mL, clarified cell lysate) was added. NMR showed 24% conversion of L-glufosinate to ethyl ester (enantiomeric ratio L:D>99:1). Concentrations of L-glufosinate and D-glufosinate ethyl esters were determined by HPLC-MS using a Supelco Chirobiotic T2 (eluent 25% water in acetonitrile, 0.1% formic acid). Temperature: 20°C, flow rate 1.0 mL / min. Retention times of glufosinate ethyl ester: L-configuration diastereoisomer (7.6+7.8 min); D-configuration (8.5 and 11.5 min).

[0186] c) Enzymatic synthesis of N-carbamoyl amino acids (Ex13, SEQ ID NO:1) [ka] 2-(2,5-Dioxoimidazolidin-4-yl)ethyl-methyl-phosphinic acid (25 g) was dissolved in aqueous ammonia (53 mL, 10 M) with heating. The reaction was cooled to 37° C. and then the pH was adjusted to 8.7 using ammonia. MnCl2 solution (2 M in water, 2.5 mL) and hydantoinase enzyme (Uniprot ID: A0A159Z531_9RHOB, SEQ ID NO: 1, lyophilized cell-free extract, 1.28 g) were added and the pH was adjusted to 8.7 using aqueous ammonia. The reaction mixture was stirred at 37° C. for 72 h and the pH was continuously adjusted to 8.7 using 10 M aqueous ammonia. After 24 h, HPLC showed 95% conversion of the hydantoin to carbamoyl acid. HPLC conditions: Conversion of hydantoins to carbamoyl acids was measured by HPLC-MS using a Luna C8 150×3.0 mm column (water + 0.1% formic acid). Temperature: 40° C., flow rate 0.5 mL / min. Retention times: hydantoins 3.4 min, N-carbamoyl amino acids 2.5 min.

[0187] d) Racemization of hydantoins (ethyl esters) using racemase at pH 7.0 (Ex14, racemase A0A6V7ACK5_RHIRD, Seq ID: 5) [ka] (5R)-5-[2-[ethoxy(methyl)phosphoryl]ethyl]imidazolidine-2,4-dione (37.5 mg, 95 / 5 ratio of D- to L-configuration hydantoin) was dissolved in water (75 μL) and the pH was adjusted to 7.0 with ammonia (10 M in water). Hydantoin racemase (Seq ID:5; A0A6V7ACK5_RHIRD, 20.2 mg / ml, 150 μL, clarified cell lysate) was added to this mixture, followed by 1.6 μL of MnCl2 (2 M in water). The reaction mixture was shaken at 37 °C for 24 h. After 4 h total reaction time, the D / L-hydantoin ratio changed from 95 / 5 to 53 / 47. After 20 h, the racemization of hydantoin was nearly complete (ratio 51 / 49). The concentrations of L- and D-hydantoin were determined by HPLC-MS using a Supelco Chirobiotic T2 (eluent 25% water in acetonitrile, 0.1% formic acid). Temperature: 20° C., flow rate 0.8 mL / min. Retention times of the ethyl ester of glufosinate: D-configuration diastereoisomer (5.8+6.1 min); L-configuration (7.2 and 10.5 min).

[0188] e) Racemization of hydantoin at pH 8.5 (Ex15) [ka] 2-[(4S)-2,5-Dioxoimidazolidin-4-yl]ethyl-methyl-phosphinic acid (206 mg, enantiomeric ratio L:D 92:8, determined by chiral HPLC) was dissolved in 900 μL of water and the pH was adjusted to 8.5 with 50 μL of ammonia (10 M in water). 10 μL of 2 M MnCl2 solution was added to the reaction mixture, followed by 30 μL of water. The reaction mixture was shaken at 37 °C for 24 h. After 3 h total reaction time, the enantiomeric ratio was L:D 53:47, and after 24 h total reaction time, it was 50:50. This indicates that hydantoin easily racemizes under concentrated basic conditions in aqueous ammonia. The concentrations of L- and D-hydantoin were determined by HPLC-MS using a Supelco Chirobiotic T2 (eluent 40% water in acetonitrile, 0.1% formic acid). Temperature: 20° C., flow rate 0.8 mL / min. Retention times for L-hydantoin (11.3 min); D-hydantoin (6.6 min).

[0189] f) Racemization of hydantoins using racemase at pH 7.8 (Ex16, racemase A0A2T6KHH4_9RHOB, Seq ID: 6) [ka] 2-[(4S)-2,5-Dioxoimidazolidin-4-yl]ethyl-methyl-phosphinic acid (206 mg, enantiomeric ratio L:D 92:8, determined by chiral HPLC) was dissolved in 500 μL of water and the pH was adjusted to 7.8 with ammonia (10 M in water). Hydantoin racemase (A0A2T6KHH4_9RHOB, Seq ID:6, 100 μL, cell-free extract, total protein concentration 26.1 mg / mL) was added to this mixture, followed by 10 μL of MnCl2 (2 M in water). The reaction was adjusted to 1 mL and the pH was adjusted to 7.8 with ammonia (10 M in water). The reaction mixture was shaken at 37 °C for 24 h. After 2 h total reaction time, the enantiomeric ratio was L:D 50:50.

[0190] g) Enzyme screening for new biocatalysts (Ex17) E. coli TG10 containing the plasmids pAgro4 and pHSG575 was transformed with the pDHE plasmid encoding the protein of interest. The resulting single clone was used to inoculate a well of a 48-well flower-type microtiter plate (m2plabs) with 1 ml of preculture medium (fermentative whole-cell biocatalytic production, see EX2) supplemented with 1 mM MnCl2, 100 μg / ml ampicillin, 100 μg / ml spectinomycin, and 20 μg / ml chloramphenicol. The cultivation was carried out overnight at 37°C and 1000 rpm. For the main cultivation, the preculture medium (fermentative whole-cell biocatalytic production, see EX2) was supplemented with 1 mM MnCl2, 100 μg / ml ampicillin, 100 μg / ml spectinomycin, 20 μg / ml chloramphenicol, 1 mM IPTG, and 1% rhamnose. 1 ml of the resulting medium was dispensed into wells of a 48-well flower-shaped microtiter plate (m2plabs) and inoculated with 10 μl of preculture. The main culture was grown overnight at 37°C and 1000 rpm. The cells were then pelleted by centrifugation at 3750xg and 4°C for 15 min, and the supernatant was discarded. For whole cell screening, the cell pellet was suspended in 500 μl of 50 mM HEPES buffer (pH 8.4, supplemented with 1 mM MnCl2). For clarified cell lysates, the cell pellet was suspended in 500 μl of 50 mM HEPES buffer (pH 8.4) supplemented with 1 mM MnCl2, 1 mg / ml lysozyme, 0.3 mg / ml polymyxin b sulfate, 0.01 mg / ml DNase, and 0.01 mg / ml RNase, and the suspension was grown at room temperature for 1 h at 1000 rpm. The resulting cell lysates were centrifuged at 3750xg for 20 min at 4°C to remove debris. 50 μl of clarified cell lysate or whole cell suspension was used in a 200 μl reaction containing 10 mM of the relevant substrate and 1 mM MnCl2 in 100 mM HEPES at pH 8.4. Reactions were carried out overnight at 37°C and then quenched with TFA at a final concentration of 5%. Precipitates were removed by centrifugation and the supernatants were quantitatively analyzed using HPLC coupled with mass spectrometry detection. HPLC-MS used a Luna C8 150x3.0 mm column (water + 0.1% formic acid).A temperature of 40°C and a flow rate of 0.5 mL / min were used for the detection of N-carbamoyl acids, hydantoins and glufosinate itself, while butyl ester containing molecules were separated on a Kinetex C18 100 x 2.1 mm column (flow rate 0.5 mL / min, 20% acetonitrile in water, 0.1% formic acid).

[0191] 1) Hydantoinases were screened using whole cells and 10 mM racemic 5-([2-[ethoxy(methyl)phosphoryl]ethyl]imidazolidine-2,4-dione (glufosinate hydantoin, synthetic Ex3) or 5-([2-[butoxy(methyl)phosphoryl]ethyl]imidazolidine-2,4-dione (butyl ester of glufosinate hydantoin, synthetic Ex4) as substrates. The production of the respective N-carbamoyl amino acids from the hydantoinases Q45515, Q44184, A0A1C4QIY5_9ACTN, A0A0K2UMP4_LEPSM, *WP_046170519.1, and E1R8C9_SEDSS showed yields of 2-(carbamoylamino)-4-[hydroxy(methyl)phosphoryl]butanoic acid (the N-carbamoyl acid of glufosinate) of >0.1%.Hydantoinase O69809, Q846U5_9BACL, P81006, Q84FR6_9MICC, Q56S49_9BACI, A1E351_9BACI, Q28SA7, Q45515, A0A399DRQ3_ 9DEIN, Q55DL0, F7X5M8_SINMM, Q9I676, Q44184, B5L363, P42084, P25995, Q3Z354, B1XEG2, Q9F465_PAEAU, A0A161KD37_ 9CHLR, A0A1J4XHR4_9BACT, A0A1C4QIY5_9ACTN, A0A0K2UMP4_LEPSM, A0A159Z531_9RHOB, E1R8C9_SEDSS, A0A1F9QT17_9 BACT, A0A0D8IVV8_9FIRM, A0A0B5QKE4_CLOBE, A0A0N1GBZ8_9ACTN, A0A174ADZ3_9FIRM, U7V9Q6_9FUSO, A0A0J1FAI4_9FI RM, PHYDA_ECOK1, A0A0S8H576_9BACT, A0A1J4J4Y8_9EUKA, A0A0D5NFS5_9BACL, A0A0D5NNJ7_9BACL, A0A1H2AV66_9BACL , A0A0Q4RXY0_9BACL, A0A0Q7SB75_9BACL, A0A100VRN2_PAEAM, W4BDJ0_9BACL, A0A1J5E082_9DELT, A0A1H5ZFN3_9BACT, A 0A1F8NMM2_9CHLR, A0A1F8SDV1_9CHLR, A0A1H1PLX0_9BACT, A0A0Q5I8X4_9DEIO, *WP_046170519.1, *WP_023514195.1, *WP_023516147.1 and *ANZ15483.1 showed 4-[butoxy(methyl)phosphoryl]-2-ureido-butanoic acid (the N-carbamoyl acid of glufosinate-butyl ester) yields of >0.1%.

[0192] 2) Carbamoylases were screened using clarified cell lysate and 10 mM 2-(carbamoylamino)-4-[hydroxy(methyl)phosphoryl]butanoic acid (the N-carbamoyl acid of glufosinate) or 4-[butoxy(methyl)phosphoryl]-2-ureido-butanoic acid (the N-carbamoyl acid of glufosinate-butyl ester) as substrate. The production of glufosinate or butyl ester of glufosinate was monitored. Carbamoylases A0A3E0C996_9BURK, A0A535Y1H2_9CHLR, A0A6P2ISL4_BURL3, and A0A1Y4GC62_9BACT showed glufosinate yields of >0.1%. Carbamoylases A0A0K9YX84_9BACL, E3HUL6_ACHXA, Q9F464, A0A4D7Q548_GEOKU, Q9F464, A0A2S9D976_9MICC, A0A3E0C996_9BURK, A0A535Y1H2_9CHLR, A0A6P2ISL4_BURL3 and A0A1Y4GC62_9BACT showed butyl ester yields of glufosinate >0.1%.

[0193] h) Small-scale enzymatic cascade reaction (EX18) [ka] Lyophilized cell-free extracts were dissolved in 1M HEPES buffer, pH 8.4. Reactions were set up in 1M HEPES buffer, pH 8.4, containing 75 mM MnCl2 and 100 mM racemic 5-[2-[ethoxy(methyl)phosphoryl]ethyl]imidazolidine-2,4-dione at a 400 μl scale. The reactions were started by adding hydantoinase Q44184 (SEQ ID 7) and carbamoylase A0A535Y1H2_9CHLR (SEQ ID 4) at final concentrations of 19 mg / ml and 7.3 mg / ml, respectively. The reactions were then incubated at 37°C for 24 hours and then stopped by heating at 95°C for 5 minutes. The precipitate was removed by centrifugation, diluted 100-fold, and the supernatant was quantitatively analyzed using chiral HPLC coupled with mass spectrometry detection. The reaction yield of the ethyl ester of glufosinate was 22.3%, and the enantiomeric ratio was L>99%, D>1%.

[0194] i) Enzymatic 1-pot synthesis of glufosinate from N-carbamoyl amino acids (Ex19, SEQ ID NO:1+3) [ka] To 2-(2,5-dioxoimidazolidin-4-yl)ethyl-methyl-phosphinic acid (7.3 g) in 30 mL aqueous ammonia (2 M) solution was added ammonia (25% in water) to adjust the pH to 8.0. To this mixture was added MnCl2 solution (2 M in water, 1 mL), hydantoinase enzyme (Uniprot ID: A0A159Z531_9RHOB, SEQ ID NO: 1, lyophilized clarified cell lysate, 1.2 g) and N-carbamoyl amino acid hydrolase enzyme (Uniprot ID: A0A6P2ISL4_BURL3, clarified cell lysate, 2.8 mL, total protein concentration 44 mg / mL). The reaction mixture was stirred at 37° C. for 44 hours. N-carbamoylamino acid hydrolase enzyme (A0A6P2ISL4_BURL3, 2.8 mL) was added after 4 h total reaction time and again (A0A6P2ISL4_BURL3, 11.2 mL) after 23 h total reaction time. After 44 h, 83% of the hydantoin was converted to N-carbamoylamino acid and 10% to glufosinate as determined by NMR. Chiral HPLC analysis showed an enantiomeric ratio of L-glufosinate:D-glufosinate of 92:8. The ratio of L-glufosinate to D-glufosinate was determined by HPLC-MS using a Supelco Chirobiotic T2 (eluent 40% water in acetonitrile, 0.1% formic acid). Temperature: 20 °C, flow rate 0.8 mL / min. Retention time of L-glufosinate (6.8 min): retention time of D-glufosinate (7.4 min). The remaining carbamoyl amino acids can be recycled via Ex6.

[0195] SEQ ID NO:1 (from Defluviimonas alba) [ka]

[0196] SEQ ID NO: 2 (from Cloacibacillus sp. An23) [ka]

[0197] SEQ ID NO:3 (from Burkholderia lata) [ka]

[0198] SEQ ID NO:4 (from Chloroflexi bacterium) [ka]

[0199] SEQ ID NO:5 (from Rhizobium radiobacter (Agrobacterium tumefaciens)) [ka]

[0200] SEQ ID NO:6 (from Yoonia sediminilitoris) [ka]

[0201] SEQ ID NO:7 (from Rhizobium radiobacter (Agrobacterium tumefaciens) (Agrobacterium radiobacter)) [ka]

Claims

1. Formula (3) 【Chemistry 1】 (Wherein, R is H or C １ ~C ８ A method for producing glufosinate, its alkyl ester, or a salt thereof, comprising the steps of: 【Chemistry 2】 (Wherein, R is H or C １ ~C ８ enzymatically cleaving and removing the carbamoyl moiety of an N-carbamoyl amino acid having a carboxyl group that is an alkyl group.

2. The cleavage step is carried out by reacting a compound represented by formula (3) 【Chemistry 3】 (Wherein, R is H or C １ ~C ８ Alkyl, preferably H or C １ ~C ６ Alkyl, more preferably H or C ２ ~C ４ The method according to claim 1, wherein the glufosinate, its alkyl ester or its salt is provided with an alkyl group, more preferably H, ethyl or butyl, in particular H.

3. The cleavage step comprises cleaving glufosinate having the formula (3), its alkyl ester or its salt in the form of a racemic mixture or in the form of a cleavage product of the formula (3a): 【Chemistry 4】 (Wherein, R is H or C １ ~C ８ Alkyl, preferably H or C １ ~C ６ Alkyl, more preferably H or C ２ ~C ４ 3. The method according to claim 1 or 2, wherein the compound is provided in the form of an enantiomeric excess of L-glufosinate, its alkyl ester or a salt thereof having the formula (3a):

4. The method according to any one of claims 1 to 3, wherein at least 50%, preferably at least 60%, in particular at least 80% of the N-carbamoylamino acid having the formula (2) is converted to L-glufosinate having the formula (3a) according to claim 3, an alkyl ester thereof or a salt thereof.

5. The method according to any one of claims 1 to 4, wherein the cleavage is carried out by an N-carbamoyl amino acid hydrolase enzyme, preferably an LN-carbamoyl amino acid hydrolase enzyme.

6. The cleavage step is performed by using the Uniprot sequences of A0A0K9YX84_9BACL and mutants thereof, E3HUL6_ACHXA and mutants thereof, Q9F464 and mutants thereof, A0A4D7Q548_GEOKU and mutants thereof, Q9F464 and mutants thereof, A0A2S9D976_9MICC and mutants thereof, A0A3E0C996_9BURK and mutants thereof, A0A535Y1H2_9CHLR and mutants thereof, A0A6P2ISL4_BURL3 (SEQ ID NO: 3) and mutants thereof, and A0A1Y4GC62_9BACT (SEQ ID NO: 2) and mutants thereof. The method is carried out by an N-carbamoyl amino acid hydrolase enzyme selected from the group of enzymes identified by ID, and variants are defined as polypeptide sequences having at least 80%, preferably 90%, most preferably 95% sequence identity to the respective polypeptide sequences, and preferably those Uniprot sequences consisting of A0A3E0C996_9BURK and variants thereof, A0A535Y1H2_9CHLR and variants thereof, A0A6P2ISL4_BURL3 (SEQ ID NO: 3) and variants thereof, A0A1Y4GC62_9BACT (SEQ ID NO: 2), 6. The method according to any one of claims 1 to 5, wherein the method is carried out with an N-carbamoyl amino acid hydrolase enzyme selected from the group of enzymes identified by their Uniprot IDs, wherein variants are defined as polypeptide sequences having at least 80%, preferably 90%, most preferably 95% sequence identity to the respective polypeptide sequence, preferably selected from the group of enzymes identified by their Uniprot IDs consisting of A0A1Y4GC62_9BACT (SEQ ID NO: 2) and variants thereof, A0A6P2ISL4_BURL3 (SEQ ID NO: 3) and variants thereof, and mixtures thereof, wherein variants are defined as polypeptide sequences having at least 80%, preferably 90%, most preferably 95% sequence identity to the respective polypeptide sequence.

7. In the formulas (2) and (3), R is H or C １ ~C ６ Alkyl, preferably H or C ２ ~C ４ The process according to any one of claims 1 to 6, wherein the alkyl is H, ethyl or butyl, more preferably H.

8. and / or the cleavage step is carried out at a pH of from 6 to 11, preferably from 6.5 to 10, more preferably from 7 to 9.5, in particular from 7.5 to 9; and / or The process according to any one of claims 1 to 7, carried out at a temperature of 20 to 50°C, preferably 25 to 45°C, more preferably 30 to 42°C, in particular 32 to 40°C.

9. In the formulas (2) and (3), R is C １ ~C ８ Alkyl, preferably C １ ~C ６ Alkyl, more preferably C ２ ~C ４ alkyl, even more preferably ethyl or butyl, especially ethyl; c) deprotection under acidic conditions, preferably using hydrochloric acid or sulfuric acid The method of any one of claims 1 to 8, further comprising:

10. The method of any one of claims 1 to 9, further comprising the addition of an N-carbamoyl amino acid racemase enzyme.

11. The N-carbamoyl amino acid having the formula (2) is represented by the formula (1): 【Chemistry 5】 (Wherein, R is H or C １ ~C ８ 11. The method according to any one of claims 1 to 10, wherein the method is provided by a step comprising hydrolysing a hydantoin having the formula (I) of formula (I), the formula being N-carbamoyl amino acid having the formula (2), preferably carried out under enzymatic conditions.

12. 12. The method according to any one of claims 1 to 11, wherein the N-carbamoyl amino acid having formula (2) is provided by the hydrolysis step, and the hydrolysis of the hydantoin having formula (1) is carried out by a hydantoinase enzyme, preferably further comprising the addition of a hydantoin racemase enzyme.

13. 13. The method according to claim 11 or 12, wherein the hydrolysis step and the cleavage step are carried out in a single vessel, preferably with all reagents being added substantially at the start of the reaction, or the reagents for the hydrolysis step and the reagents for the cleavage step being added at different times to the single vessel.

14. Formula (2a) 【Chemistry 6】 (Wherein, R is H or C １ ~C ８ A composition comprising an N-carbamoylamino acid having an N-carbamoyl group (wherein the N-carbamoyl group is an alkyl group), and L-glufosinate or a salt thereof.

15. 1. A method for selectively controlling weeds in an area containing planted seeds or crops, preferably resistant to glufosinate, comprising: An effective amount of a composition containing L-glufosinate or a salt thereof in an enantiomeric excess of at least 80% over D-glufosinate or a salt thereof, and a compound represented by the formula (2) of more than 0.01% by weight and less than 10% by weight based on the total amount of the composition 【Chemistry 7】 (Wherein, R is H or C １ ~C ８ The method comprises applying to the area an effective amount of an N-carbamoyl amino acid having a molecular weight of 1,2-diaminopropyl, 1,3-diaminopropyl, 1,4-diaminopropyl, 1,5-diaminopropyl, 1,6 ...

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