Glufosinate synthesis using amidase-based processes

Abstract

The present invention relates to a process for the preparation of glufosinate comprising the steps of hydrolyzing an N-carbamoyl glufosinate amide with an amidase to form an N-carbamoyl amino acid compound followed by cleavage of the carbamoyl moiety of the N-carbamoyl amino acid compound.

Description

TECHNICAL FIELD

[0001] The present invention relates to a process for the preparation of glufosinate comprising the steps of hydrolyzing N-carbamoyl glufosinate amide with an amidase to form an N-carbamoyl amino acid compound, followed by cleaving the carbamoyl moiety of said N-carbamoyl amino acid compound. BACKGROUND

[0002] The herbicide glufosinate is a non-selective foliar-applied herbicide that is considered one of the safest herbicides from a toxicological or environmental perspective. Current commercial chemical synthesis methods for glufosinate produce a racemic mixture of L- and D- glufosinate (Duke et al. 2010 Toxins 2: 1943-1962).

[0003] L-glufosinate (also known as phosphinothricin or (S)-2-amino-4-(hydroxy(methyl)phosphinoxy)butanoic acid) is known to be more effective than D-glufosinate (Ruhland et al. (2002) Environ. Biosafety Res. 1 : 29-37). Therefore, a process that produces the more active L-glufosinate form in excess is of interest. SUMMARY

[0004] In the above context, it is an object of the present invention to provide a mild process for the preparation of glufosinate.

[0005] It is a further object of the present invention to provide a safe process for the preparation of glufosinate.

[0006] It is a further object of the present invention to provide a mild process for the preparation of L-glufosinate in enantiomeric excess.

[0007] It is a further object of the present invention to provide a composition comprising L-glufosinate.

[0008] It is a further object of the present invention to provide a method for the selective control of weeds using a composition obtained using the process according to the present invention.

[0009] The inventors of the present invention surprisingly found that at least one of the above objects can be obtained by the herein described process based on N-carbamoyl glufosinate amide. The inventors of the present invention further found that the claimed process provides a composition comprising glufosinate in an amount sufficient for use as a herbicide.

[0010] Thus, in a first aspect, the present invention relates to a process for the preparation of glufosinate of formula (I) and / or its salts or glufosinate alkyl esters and / or its salts

[0011] (I),

[0012] Wherein R is H or C1-C8 alkyl, preferably H or C1-C6 alkyl, more preferably H or C2-C4 alkyl, even more preferably ethyl or butyl, and most preferably ethyl, the method comprising the following steps:

[0013] a) Hydrolysis of N-carbamoyl glufosinate-ammonium amide with formula (II) by amidase

[0014] (II),

[0015] Wherein R is H or C1-C8 alkyl, preferably H or C1-C6 alkyl, more preferably H or C2-C4 alkyl, even more preferably ethyl or butyl, and most preferably ethyl, to form an N-carbamoyl amino acid having formula (III).

[0016] (III),

[0017] Wherein R is H or C1-C8 alkyl, preferably H or C1-C6 alkyl, more preferably H or C2-C4 alkyl, even more preferably ethyl or butyl, and most preferably ethyl.

[0018] b) Cut the carbamoyl moiety of an N-carbamoyl amino acid having formula (III).

[0019] The components of the preparation method, the composition for selective weed control, and preferred embodiments of the method are described in further detail below. It should be understood that each preferred embodiment is relevant on its own and in combination with other preferred embodiments.

[0020] In the preferred embodiment A1 of the first aspect, the cutting step b) provides glufosinate having formula (I) and / or its salt or glufosinate alkyl ester and / or its salt.

[0021] (I),

[0022] Wherein R is H or C1-C8 alkyl, preferably H or C1-C6 alkyl, more preferably H or C2-C4 alkyl, even more preferably ethyl or butyl, and most preferably ethyl.

[0023] In the preferred embodiment A2 of the first aspect, the cutting step b) provides either a racemic mixture or an enantiomeric excess of L-glufosinate of formula (Ia) and / or its salt or glufosinate alkyl ester and / or its salt, or glufosinate alkyl ester and / or its salt, in the form of L-glufosinate of formula (Ia) and / or its salt or glufosinate alkyl ester and / or its salt.

[0024] (Ia),

[0025] Wherein R is H or C1-C8 alkyl, preferably H or C1-C6 alkyl, more preferably H or C2-C4 alkyl, even more preferably ethyl or butyl, and most preferably ethyl; preferably in the form of an enantiomeric excess of L-glufosinate of formula (Ia) and / or its salt or glufosinate alkyl ester and / or its salt, and the amidase is L-amidase.

[0026] In a preferred embodiment A3 of the first aspect, at least 5%, preferably at least 10%, more preferably at least 20%, even more preferably at least 40%, and most preferably at least 50% of N-carbamoyl glufosinate having formula (II) is converted into L-glufosinate and / or its salt or glufosinate alkyl ester and / or its salt having formula (Ia), wherein formula (Ia) is as defined in preferred embodiment A2.

[0027] In the preferred embodiment A4 of the first aspect, the cutting step b) is carried out under enzymatic conditions, preferably using N-carbamoyl amino acid hydrolase, more preferably LN-carbamoyl amino acid hydrolase, or wherein the cutting step b) is carried out under chemical conditions, preferably using sodium nitrite and / or hydrogen chloride.

[0028] In the preferred embodiment A4a of the first aspect, the cutting step b) is carried out under enzymatic conditions, preferably using N-carbamoyl amino acid hydrolase, more preferably LN-carbamoyl amino acid hydrolase, or wherein the cutting step b) is carried out under chemical conditions, preferably using sodium nitrite and / or hydrogen chloride, and steps a) and b) are carried out in a one-pot process.

[0029] In the preferred embodiment A5 of the first aspect, R in formulas (II) and (III) is H or C1-C6 alkyl, preferably H or C2-C4 alkyl, more preferably ethyl or butyl, and most preferably ethyl.

[0030] In the preferred embodiment A6 of the first aspect, the hydrolysis step a) is carried out at a pH of 6 to 11, preferably 6.5 to 10, more preferably 7 to 9.5, and particularly 7.5 to 9, and / or at a temperature of 20°C to 50°C, preferably 25°C to 45°C, more preferably 30°C to 42°C, and particularly 32°C to 40°C.

[0031] In the preferred embodiment A7 of the first aspect, R in formulas (II) and (III) is a C1-C8 alkyl, preferably a C1-C6 alkyl, more preferably a C2-C4 alkyl, even more preferably ethyl or butyl, and most preferably ethyl, and the method further includes the following steps

[0032] c) Deprotection is carried out under acidic conditions, preferably using hydrochloric acid or sulfuric acid.

[0033] In a preferred embodiment A8 of the first aspect, the method further includes adding N-carbamoyl glufosinate-phosphonamide racemic enzyme and / or N-carbamoyl amino acid racemic enzyme.

[0034] In the preferred embodiment A9 of the first aspect, steps a) and b) are carried out in a single container, preferably wherein all reagents are added substantially at the start of the reaction, or wherein the reagents of step a) and step b) are added to the single container at different times.

[0035] In a preferred embodiment A10 of the first aspect, the method further includes the step of isolating N-carbamoyl glufosinate-ammonium having formula (IIb).

[0036] (IIb),

[0037] Wherein R is H or C1-C8 alkyl, preferably H or C1-C6 alkyl, more preferably H or C2-C4 alkyl, even more preferably ethyl or butyl, and most preferably ethyl, which is obtained in hydrolysis step a), preferably using reversed-phase chromatography.

[0038] In a second aspect, the present invention relates to a composition comprising N-carbamoyl glufosinate-ammonium amide having formula (IIb).

[0039] (IIb),

[0040] Wherein R is H or C1-C8 alkyl, preferably H or C1-C6 alkyl, more preferably H or C2-C4 alkyl, even more preferably ethyl or butyl, and most preferably ethyl.

[0041] In the preferred embodiment B1 of the second aspect, the composition consists of N-carbamoyl glufosinate-ammonium amide having formula (IIb).

[0042] (IIb),

[0043] Wherein R is H or C1-C8 alkyl, preferably H or C1-C6 alkyl, more preferably H or C2-C4 alkyl, even more preferably ethyl or butyl, and most preferably ethyl.

[0044] In another preferred embodiment B2 of the second aspect, the composition comprises N-carbamoyl glufosinate-ammonium amide having formula (IIb).

[0045] (IIb),

[0046] Wherein R is H or C1-C8 alkyl, preferably H or C1-C6 alkyl, more preferably H or C2-C4 alkyl, even more preferably ethyl or butyl, and most preferably ethyl.

[0047] and optionally N-carbamoyl amino acids of formula (IIIa)

[0048] (IIIa),

[0049] Wherein R is H or C1-C8 alkyl, preferably H or C1-C6 alkyl, more preferably H or C2-C4 alkyl, even more preferably ethyl or butyl, and most preferably ethyl, as well as L-glufosinate and / or its salts.

[0050] In the preferred embodiment B3 of the second aspect, the amount of L-glufosinate and / or its salt is based on at least 5 wt.-%, preferably at least 10 wt.-%, even more preferably at least 20 wt.-%, still more preferably at least 30 wt.-%, and most preferably at least 50 wt.-%.

[0051] In the preferred embodiment B4 of the second aspect, R in formulas (IIIa) and (IIb) is H or C1-C6 alkyl, preferably H or C2-C4 alkyl, more preferably ethyl or butyl, and most preferably ethyl.

[0052] In a third aspect, the present invention relates to a method for selectively controlling weeds in an area, preferably containing a batch of planted glufosinate-resistant seeds or crops, the method comprising:

[0053] Apply an effective amount of the composition to the area, the composition comprising an enantiomeric excess of L-glufosinate and / or its salt relative to D-glufosinate and / or its salt in a ratio of at least 50%, preferably greater than 70%, of enantiomers, and

[0054] Based on the total amount of the composition, greater than 0.01 wt.% to less than 10 wt.% of an N-carbamoylamino amide having formula (II)

[0055] (II),

[0056] Wherein R is H or C1-C8 alkyl, preferably H or C1-C6 alkyl, more preferably H or C2-C4 alkyl, even more preferably ethyl or butyl, and most preferably ethyl.

[0057] And / or

[0058] Based on the total amount of the composition, greater than 0.01 wt.% to less than 10 wt.% of N-carbamoyl amino acids having formula (III)

[0059] (III),

[0060] Wherein R is H or C1-C8 alkyl, preferably H or C1-C6 alkyl, more preferably H or C2-C4 alkyl, even more preferably ethyl or butyl, and most preferably ethyl. Detailed Implementation

[0061] Before describing in detail exemplary embodiments of the present invention, definitions important for understanding the present invention are given.

[0062] As used in this specification and the appended claims, unless the context clearly specifies otherwise, the singular form “a / an” also includes the corresponding plural. In the context of this invention, the terms “about” and “approximately” indicate a range of precision that, as will be understood by those skilled in the art, still ensures the technical effect of the features discussed. This term typically indicates a deviation from the indicated numerical value by ±20%, preferably ±15%, more preferably ±10%, and even more preferably ±5%. It should be understood that the term “comprising” is not limiting. For the purposes of this invention, the term “consisting of” is considered a preferred embodiment of the term “comprises from”. If a group is defined below as including at least a certain number of embodiments, this means that it also includes a group preferably consisting only of those embodiments. Furthermore, the terms “first,” “second,” “third,” or “(a),” “(b),” “(c),” “(d),” etc., in the specification and claims are used to distinguish similar elements and are not necessarily used to describe a sequential or chronological order. It should be understood that the terms thus used are interchangeable where appropriate, and the embodiments of the invention described herein can operate in orders other than those described or shown herein. In cases where the terms “first,” “second,” “third,” or “(a),” “(b),” “(c),” “(d),” “i,” “ii,” etc., refer to steps of a method, use, or experiment, there is no temporal or time interval coherence between these steps; that is, these steps may be performed simultaneously or there may be time intervals of seconds, minutes, hours, days, weeks, months, or even years between such steps, unless otherwise indicated in this application as stated above or below. It should be understood that the invention is not limited to the specific methods, schemes, reagents, etc., described herein, as these can vary. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to limit the scope of the invention, which will be limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0063] As used throughout this article, the term "wt.-%" stands for "weight percentage".

[0064] As used herein, the term "alkyl" in each case refers to a straight-chain or branched alkyl group that typically has 1 to 20 carbon atoms, preferably 1 to 8 carbon atoms, often 1 to 6 carbon atoms, more preferably 1 to 4 carbon atoms, such as 2 or 4 carbon atoms. Examples of alkyl groups are methyl, ethyl, n-propyl, isopropyl, n-butyl, 2-butyl, isobutyl, tert-butyl, n-pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, 2,2-dimethylpropyl, 1-ethylpropyl, and n-hexyl.

[0065] Depending on the substitution mode, compounds according to the invention may have one or more stereocenters. Unless otherwise explicitly indicated (e.g., via chemical formula), the invention preferably covers all stereoisomers of compounds according to the invention (i.e., pure enantiomers, pure diastereomers), and mixtures thereof (including racemic mixtures).

[0066] The following describes in detail preferred embodiments of a method for preparing glufosinate and / or its salts or glufosinate alkyl esters and / or their salts having formula (I), comprising compositions of N-carbamoyl glufosinate amide having formula (IIb), N-carbamoyl amino acids having formula (IIIa), and L-glufosinate and / or their salts, and a method for selectively controlling weeds. It should be understood that preferred embodiments of the invention are preferred individually or in combination with each other.

[0067] As indicated above, in one aspect, the present invention relates to a method for preparing glufosinate and / or its salts or glufosinate alkyl esters and / or their salts having formula (I).

[0068] (I),

[0069] Wherein R is H or C1-C8 alkyl, preferably H or C1-C6 alkyl, more preferably H or C2-C4 alkyl, even more preferably ethyl or butyl, and most preferably ethyl, the method comprising the following steps:

[0070] a) Hydrolysis of N-carbamoyl glufosinate-ammonium amide with formula (II) by amidase

[0071] (II),

[0072] Wherein R is H or C1-C8 alkyl, preferably H or C1-C6 alkyl, more preferably H or C2-C4 alkyl, even more preferably ethyl or butyl, and most preferably ethyl, to form an N-carbamoyl amino acid having formula (III).

[0073] (III),

[0074] Wherein R is H or C1-C8 alkyl, preferably H or C1-C6 alkyl, more preferably H or C2-C4 alkyl, even more preferably ethyl or butyl, and most preferably ethyl.

[0075] b) Cut the carbamoyl moiety of an N-carbamoyl amino acid having formula (III).

[0076] It should be understood that glufosinate and / or its salts or glufosinate alkyl esters and / or their salts having formula (I)

[0077] (I)

[0078] Suitable salts of all stereoisomers, the corresponding glufosinate or its alkyl esters, are included. Further, the corresponding zwitterions are covered by formula (I). Suitable salts are exemplarily hydrochloride, ammonium salts, and isopropylammonium salts. In this respect, compounds having formula (I) particularly encompass two stereocenters, one located at the phosphorus atom and the other at the α-carbon atom. Compounds having formula (I) particularly encompass all stereoisomers derived from the stereocenter at the phosphorus atom.

[0079] N-Carbamoyl glufosinate-ammonium having formula (II) can be obtained by any suitable preparative method. For example, N-carbamoyl glufosinate-ammonium is a byproduct of the Bucherer-Bergs reaction. Therefore, N-carbamoyl glufosinate-ammonium can be generated from related cyanohydrins. Cyanohydrins or cyanohydrin derivatives can be obtained, for example, via processes described in particular as those described in US 4,521,348 B1, DE3047024, US 4,599,207 B1, US 6,359,162 B1, CN 102372739 A and CN 102399240 A.

[0080] In a preferred embodiment of the invention, the hydrolysis step a) is carried out at a pH of 6 to 11, preferably 6.5 to 10, more preferably 7 to 9.5, and particularly 7.5 to 9. Preferably, an alkaline hydroxide, more preferably sodium hydroxide or potassium hydroxide, and particularly potassium hydroxide, is used to adjust the pH.

[0081] In a preferred embodiment of the invention, the hydrolysis step a) is carried out at a temperature of 20°C to 50°C, preferably 25°C to 45°C, more preferably 30°C to 42°C, and particularly 32°C to 40°C.

[0082] In a preferred embodiment of the invention, the hydrolysis step a) is carried out under aqueous conditions, preferably in a degassed aqueous phosphate buffer, and more preferably in a degassed aqueous potassium phosphate buffer.

[0083] In a preferred embodiment of the invention, the hydrolysis step a) is preferably carried out at 50 to 1000 rpm, more preferably 100 to 800 rpm, even more preferably 150 to 600 rpm, still more preferably 180 to 400 rpm, and particularly at 200 to 300 rpm during stirring.

[0084] Any suitable amidase can be used, preferably a linear amidase. More preferably, the amidase is an enzyme that hydrolyzes the amide bond (EC 3.4, EC 3.5.1, EC 3.5.2). Therefore, it is preferred that the hydrolysis of the amide bond occurs independently of whether such hydrolysis is a natural or heterofunctional reaction of the enzyme. Thus, preferred amidases can be selected from a list consisting of free peptidases, proteases, linear amidases, or cyclic amidases. Particularly preferred amidases are selected from a list consisting of free papain (CAS 9001-73-4), bromelain (CAS 37189-34-7), and bacterial proteases (proteases from Bacillus licheniformis, CAS 9014-01-1).

[0085] In a preferred embodiment of the present invention, the amidase is an L-amidase.

[0086] In a preferred embodiment of the invention, the amidase is a D-amidase, more preferably a protease, and most preferably a cysteine ​​protease.

[0087] In a preferred embodiment of the invention, R in formulas (II) and (III) is H or C1-C6 alkyl, preferably H or C2-C4 alkyl, more preferably ethyl or butyl, and most preferably ethyl.

[0088] In a preferred embodiment of the invention, cutting step b) provides glufosinate having formula (I) and / or its salt or glufosinate alkyl ester and / or its salt.

[0089] (I),

[0090] Wherein R is H or C1-C8 alkyl, preferably H or C1-C6 alkyl, more preferably H or C2-C4 alkyl, even more preferably ethyl or butyl, and most preferably ethyl.

[0091] In a preferred embodiment of the invention, cutting step b) provides glufosinate of formula (I) and / or its salts or glufosinate alkyl esters and / or their salts in the form of a racemic mixture.

[0092] In another preferred embodiment of the invention, the cutting step b) provides an enantiomeric excess of L-glufosinate of formula (I) and / or its salt or glufosinate alkyl ester and / or its salt, in the form of L-glufosinate of formula (Ia) and / or its salt or glufosinate alkyl ester and / or its salt.

[0093] (Ia),

[0094] Wherein R is H or C1-C8 alkyl, preferably H or C1-C6 alkyl, more preferably H or C2-C4 alkyl, even more preferably ethyl or butyl, and most preferably ethyl. Preferably, an enantiomeric excess of L-glufosinate of formula (Ia) and / or its salt or glufosinate alkyl ester and / or its salt is formed, and the amidase is L-amidase.

[0095] In another preferred embodiment of the invention, the cutting step b) provides an enantiomeric excess of D-glufosinate of formula (I) and / or its salt or glufosinate alkyl ester and / or its salt, in the form of glufosinate of formula (Ib) and / or its salt or glufosinate alkyl ester and / or its salt.

[0096] (Ib),

[0097] Wherein R is H or C1-C8 alkyl, preferably H or C1-C6 alkyl, more preferably H or C2-C4 alkyl, even more preferably ethyl or butyl, and most preferably ethyl. In this regard, it is preferred that the amidase is a D-amidase.

[0098] In a preferred embodiment of the invention, the cutting step b) is carried out under enzymatic conditions, preferably using an N-carbamoyl amino acid hydrolase, more preferably an LN-carbamoyl amino acid hydrolase. Suitable N-carbamoyl amino acid hydrolases 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, EC 3.5.1.77 N-carbamoyl-D-amino acid hydrolases, and mixtures thereof. Suitable N-carbamoyl amino acid hydrolases that can be used in this method include those selected from the group consisting of: A0A7Y0T4N7 and its variants, Q88FQ3 and its variants, Q88Q81 and its variants, A0A126S6J4 and its variants, Q8VUL6 and its variants, H9B8T5 and its variants, Q9FB05 and its variants, C0ZCM8 and its variants, C0Z7R5 and its variants, A0A0K9YX84 and its variants, E3H UL6 and its variants, A0A1V9BSS3 and its variants, A0A1V9BSS3 and its variants, Q9F464 and its variants, A0A4D7Q548 and its variants, Q9F464 and its variants, A0A2S9D976 and its variants, A0A1I6VZZ4 and its variants, A0A1L6RE91 and its variants, A0A3E0C996 and its variants, A0A3M7BGJ4 and its variants, A0A2D7YQN7 and its variants, A 0A535Y1H2 and its variants, A0A223E4I5 and its variants, M2VSE9 and its variants, A0A3T0K6C0 and its variants, A0A416FGE1 and its variants, D1P143 and its variants, A0A6P2ISL4 and its variants, A0A3S6Z2M9 and its variants, A0A0C1US49 and its variants, A0A1Y4GC62 and its variants, A0A3D3VMN7 and its variants, A0A2K8L549 The following are examples of the following: A0A1G0MC89 and its variants, A0A1M6WYS1 and its variants, A0A2K2BYI3 and its variants, A0A510DYR5 and its variants, A0A5Y3XFN7 and its variants, A0A381IB54 and its variants, A0A2V3IQW6 and its variants, and mixtures thereof, wherein a variant is defined as a polypeptide sequence having at least 80%, preferably 90%, and most preferably 95% sequence identity with the corresponding polypeptide sequence.Most preferably, the N-carbamoyl amino acid hydrolase is selected from the group consisting of: A0A3E0C996 (SEQ ID NO: 1) and its variants, A0A535Y1H2 (SEQ ID NO: 2) and its variants, A0A6P2ISL4 (SEQ ID NO: 3) and its variants, A0A1Y4GC62 (SEQ ID NO: 4) and its variants, wherein a variant is defined as a polypeptide sequence having at least 80%, preferably 90%, and most preferably 95% sequence identity with the corresponding polypeptide sequence. It should be understood that the N-carbamoyl amino acid hydrolases outlined above are based on the Uniprot database. www.UniProt.org The database identifier's naming convention indicates the status as of March 19, 2023.

[0099] In this regard, the cutting step b) is preferably carried out at a temperature of 20°C to 50°C, preferably 25°C to 45°C, more preferably 30°C to 42°C, and particularly 32°C to 40°C. Additionally, the reaction pressure is preferably ambient temperature pressure. Preferably, the reaction pressure ranges from 0.995 to 1.030 mbar, more preferably 1.005 to 1.020 mbar, and particularly about 1.013 mbar. In a preferred embodiment of the invention, the cutting step b) is carried out at a pH of 5 to 10, preferably 6 to 9, and particularly about 7.

[0100] In a preferred embodiment of the invention, the cutting step b) is preferably performed during stirring at 50 to 1000 rpm, more preferably at 100 to 800 rpm, even more preferably at 150 to 600 rpm, still more preferably at 180 to 400 rpm, and particularly at 200 to 300 rpm.

[0101] In another preferred embodiment of the invention, the cleavage step b) is performed under chemical conditions. It should be understood that the terms "chemical conditions" or "chemical cleavage" refer to a cleavage step performed without enzymatic catalysis. Any suitable chemical method is possible. Cleavage can be performed, exemplarily, using sodium nitrite and / or hydrogen chloride. N-carbamoyl amino acids having formula (III) can be exemplarily treated with concentrated hydrogen chloride at elevated temperatures.

[0102] (III),

[0103] Wherein R is H or C1-C8 alkyl, preferably H or C1-C6 alkyl, more preferably H or C2-C4 alkyl, even more preferably ethyl or butyl, and most preferably ethyl. Alternatively, N-carbamoyl amino acids having formula (III) as defined above can be treated with sodium nitrite and hydrogen chloride under aqueous conditions. In this regard, the cleavage step b) is preferably carried out at a temperature of 25°C to 120°C, more preferably 50°C to 110°C, and particularly 60°C to 105°C. In addition, the reaction pressure is preferably ambient temperature pressure. Preferably, the reaction pressure ranges from 0.995 to 1.030 mbar, more preferably 1.005 to 1.020 mbar, and particularly about 1.013 mbar. In a preferred embodiment of the invention, the cleavage step b) is carried out at a pH of 0 to 5, preferably 0 to 3. The reaction mixture can be post-treated under standard procedures (i.e., washing and purification).

[0104] In a particularly preferred embodiment of the invention, the cutting step b) is performed under enzymatic conditions.

[0105] In a preferred embodiment of the invention, R in formulas (II) and (III) is a C1-C8 alkyl group, preferably a C1-C6 alkyl group, more preferably a C2-C4 alkyl group, even more preferably an ethyl or butyl group, and most preferably an ethyl group, and the method further includes the following steps:

[0106] c) Deprotection under acidic conditions. Any suitable acid is possible in this regard. Hydrochloric acid or sulfuric acid is preferred.

[0107] In a preferred embodiment of the invention, the method further comprises adding an N-carbamoyl glufosinate racemic enzyme. Any suitable N-carbamoyl glufosinate racemic enzyme is possible, which racemates the N-carbamoyl glufosinate at the α-carbon atom.

[0108] In a preferred embodiment of the invention, the method further includes the addition of an N-carbamoyl amino acid racemase. Any suitable N-carbamoyl amino acid racemase may be used.

[0109] In a preferred embodiment of the present invention, the method further includes adding N-carbamoyl glufosinate-phosphonamide racemic enzyme and N-carbamoyl amino acid racemic enzyme.

[0110] In a preferred embodiment of the invention, steps a) and b) are carried out in a single container, wherein step b) is carried out under enzymatic conditions. In this regard, all reagents are preferably added substantially at the start of the reaction. Alternatively, the reagents for step a) and step b) are preferably added to the single container at different times.

[0111] In a preferred embodiment of the invention, the method further includes the step of isolating N-carbamoyl glufosinate-ammonium amide having formula (IIb).

[0112] (IIb),

[0113] Wherein R is H or C1-C8 alkyl, preferably H or C1-C6 alkyl, more preferably H or C2-C4 alkyl, even more preferably ethyl or butyl, and most preferably ethyl, which is obtained in hydrolysis step a). The separation of N-carbamoyl glufosinate having formula (IIb) is preferably achieved using reversed-phase chromatography. Alternatively, separation can be achieved using ion exchange, extraction, salt formation, crystallization, and filtration.

[0114] N-carbamoyl glufosinate having formula (IIb) can be chemically racemized and reused in hydrolysis step a). To racemize N-carbamoyl glufosinate having formula (IIb), it can 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, and particularly 8.5 to 10. Preferably, the racemization is carried out under aqueous conditions.

[0115] Alternatively, N-carbamoyl glufosinate with formula (IIb) can be treated with N-carbamoyl glufosinate racemic enzyme.

[0116] In a preferred embodiment of the invention, at least 5%, preferably at least 10%, more preferably at least 20%, even more preferably at least 30%, still even more preferably at least 40%, still even more preferably at least 50%, and most preferably at least 60% of N-carbamoyl glufosinate amide of formula (II) is converted into L-glufosinate and / or its salt or glufosinate alkyl ester and / or its salt of formula (Ia).

[0117] (Ia),

[0118] Wherein R is H or C1-C8 alkyl, preferably H or C1-C6 alkyl, more preferably H or C2-C4 alkyl, even more preferably ethyl or butyl, and most preferably ethyl.

[0119] In a preferred embodiment of the invention, 5% to 99%, preferably 10% to 98%, more preferably 20% to 97%, even more preferably 30% to 96%, and particularly 40% to 95% of N-carbamoyl glufosinate amide of formula (II) is converted into L-glufosinate and / or its salt or glufosinate alkyl ester and / or its salt of formula (Ia).

[0120] (Ia),

[0121] Wherein R is H or C1-C8 alkyl, preferably H or C1-C6 alkyl, more preferably H or C2-C4 alkyl, even more preferably ethyl or butyl, and most preferably ethyl.

[0122] In another preferred embodiment of the invention, at least 5%, preferably at least 10%, more preferably at least 20%, even more preferably at least 30%, still even more preferably at least 40%, still even more preferably at least 50%, and most preferably at least 60% of the N-carbamoyl glufosinate amide of formula (II) is converted into L-glufosinate and / or its salt or glufosinate alkyl ester and / or its salt of formula (Ib).

[0123] (Ib),

[0124] Wherein R is H or C1-C8 alkyl, preferably H or C1-C6 alkyl, more preferably H or C2-C4 alkyl, even more preferably ethyl or butyl, and most preferably ethyl.

[0125] In another preferred embodiment of the invention, 5% to 99%, preferably 10% to 98%, more preferably 20% to 96%, even more preferably 30% to 95%, and particularly 40% to 90% of the N-carbamoyl glufosinate of formula (II) is converted into L-glufosinate and / or its salt or glufosinate alkyl ester and / or its salt of formula (Ib).

[0126] (Ib),

[0127] Wherein R is H or C1-C8 alkyl, preferably H or C1-C6 alkyl, more preferably H or C2-C4 alkyl, even more preferably ethyl or butyl, and most preferably ethyl.

[0128] Enantiomer excess of L-glufosinate is preferred.

[0129] In a preferred embodiment of the invention, the method includes the addition of amidase, N-carbamoyl glufosinate-ammonium racemicase and N-carbamoyl amino acid hydrolase, wherein all reaction steps are carried out in a single container (also known as a "one-pot" condition), preferably wherein all reagents are added substantially at the start of the reaction, or wherein the reagents are added to the single container at different times.

[0130] In another preferred embodiment of the invention, the method includes the addition of amidase, N-carbamoyl glufosinate-phosphonamide racemicase and N-carbamoyl amino acid racemicase, wherein all reaction steps are carried out in a single container (also known as a "one-pot" condition), preferably wherein all reagents are added substantially at the start of the reaction, or wherein the reagents are added to the single container at different times.

[0131] In yet another preferred embodiment of the invention, the method includes the addition of an amidase and an N-carbamoyl amino acid hydrolase, wherein all reaction steps are carried out in a single vessel (also known as a "one-pot" condition), preferably wherein all reagents are added substantially at the start of the reaction, or wherein reagents are added to the single vessel at different times. In this regard, it is preferred that the pH of the reaction mixture is 8 or higher.

[0132] The applied enzyme can be administered by any suitable method known in the art.

[0133] In a preferred embodiment of the invention, the applied enzyme is administered as a clarified cell lysate, whole cell, or immobilized enzyme.

[0134] Alternatively, some or all of the components, except L-glufosinate, may be removed from the bioconversion mixture (optionally a concentrated mixture), which can then be used directly (and / or with the addition of multiple adjuvants) for weed prevention or control. In some instances, the bioconversion mixture may be used directly (and / or with the addition of multiple adjuvants) for weed prevention or control.

[0135] Additional steps can be added to further purify L-glufosinate. Such additional purification and separation methods include ion exchange, extraction, salt formation, crystallization, and filtration; each method can be used multiple times or in a suitable combination. If effective, the enzyme can be removed by simple filtration, or if the enzyme is free in solution, it can be removed by ultrafiltration, the use of an absorbent (such as diatomaceous earth, cellulose, or carbon), or denaturation by various techniques known to those skilled in the art.

[0136] Ion exchange processes achieve separation by selectively adsorbing solutes onto a resin chosen for this purpose. Because the product and impurities must be dissolved in a single solution prior to adsorption, the purified product stream is typically concentrated by evaporation or distillation before separation. Schultz et al. and EP 0 249 188 A2 describe examples of purification using ion exchange.

[0137] Purification can be achieved by adding a suitable acid (including hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, acetic acid, etc.) to form an insoluble salt of L-glufosinate. Similarly, purification can be achieved by adding 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 nitrogen-containing bases can be used, including ammonia, hydroxylamine, isopropylamine, triethylamine, tributylamine, pyridine, 2-methylpyridine, 3-methylpyridine, 4-methylpyridine, 2,4-dimethylpyridine, 2,6-dimethylpyridine, morpholine, N-methylmorpholine, 1,8-diazabicyclo[5.4.0]undec-7-ene, and dimethylethanolamine. Concentrating the mixture or adding a solvent (or both) may be advantageous to maximize the yield and optimize the purity of the desired salt. Suitable solvents for this purpose include those with very low solubility of the desired salt (such solvents are often referred to as "antisolvents"). The salts of L-glufosinate can be converted into a form suitable for formulation using standard methods known to those skilled in the art. Alternatively, L-glufosinate can be separated as a zwitterion.

[0138] US 9,255,115 B2 describes how to convert the hydrochloride of L-glufosinate to a zwitterionic form using a base (e.g., sodium hydroxide or sodium methoxide), followed by crystallization from an aqueous alcoholic solvent to provide L-glufosinate of relatively high purity. An advantage of this method is that the resulting crystalline L-glufosinate is non-hygroscopic, thus maintaining higher purity when exposed to humidity over time compared to amorphous L-glufosinate.

[0139] Other salts of L-glufosinate are known in the art. US 5,767,309 and US 5,869,668 teach the use of chiral alkaloids to form diastereomeric salts with racemic glufosinate. Purification is achieved because the amount of L-glufosinate salt precipitated from solution is much greater than the amount of the corresponding D-glufosinate salt precipitated from solution. Therefore, if desired, the method can be used in conjunction with the present invention to obtain L-glufosinate with a high enantiomeric excess.

[0140] 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 previously described. This is advantageous if the unreacted amine donor can be partially or completely isolated and used in subsequent reactions. Similarly, partially or completely isolated, unreacted N-carbamoyl amino acids of formula (III) can be recycled for subsequent reactions.

[0141] Extraction can be used to purify the product. DE 3920570 C2 describes a process in which excess glutamic acid (used as an amine donor) is precipitated by adjusting the pH of the solution to 3.7 to 4.2 with sulfuric acid. After filtration of the glutamic acid, the pH of the filtrate is lowered to 1-2, and other impurities are then extracted into the solvent. After extraction and concentration, ammonia is added to the aqueous solution to a pH of 5-7, and then ammonium sulfate is precipitated. The ammonium sulfate is removed by filtration, and the resulting filtrate is concentrated to give the ammonium salt of L-glufosinate.

[0142] For example, to transport solids to preparation or use sites, it may be necessary to separate L-glufosinate or its salts. Typical industrial separation methods, such as filtration and centrifugation, can be used. The separated product typically needs to be dewatered, free of volatile impurities and solvents (if present), and typical industrial drying equipment can be used for this purpose. Examples of such equipment include ovens, drum dryers, stirred dryers, etc. In some cases, the use of spray dryers may be advantageous.

[0143] No solid product is required after purification. This may be advantageous if the formulation of L-glufosinate occurs at the same location used for L-glufosinate production. 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 separated by filtration, and the resulting filtrate is concentrated by distillation. The pH of the filtrate can be adjusted to the desired value, and the resulting solution can be used as is or mixed with formulation components. In another example, a slurry of L-glufosinate or one of its salts can be prepared as described above and separated by filtration. The solid can be dissolved directly on the filter by adding water or a suitable solvent to obtain an L-glufosinate solution.

[0144] As described above, the invention further relates in a second aspect to a composition comprising N-carbamoyl glufosinate-ammonium amide having formula (IIb).

[0145] (IIb),

[0146] Wherein R is H or C1-C8 alkyl, preferably H or C1-C6 alkyl, more preferably H or C2-C4 alkyl, even more preferably ethyl or butyl, and most preferably ethyl.

[0147] In a preferred embodiment of the second aspect, the composition comprises N-carbamoyl glufosinate-ammonium amide having formula (IIb).

[0148] (IIb),

[0149] Wherein R is H or C1-C8 alkyl, preferably H or C1-C6 alkyl, more preferably H or C2-C4 alkyl, even more preferably ethyl or butyl, and most preferably ethyl.

[0150] In another preferred embodiment of the second aspect, the invention further relates to a composition comprising N-carbamoyl glufosinate-ammonium amide having formula (IIb).

[0151] (IIb),

[0152] Wherein R is H or C1-C8 alkyl, preferably H or C1-C6 alkyl, more preferably H or C2-C4 alkyl, even more preferably ethyl or butyl, and most preferably ethyl; an N-carbamoyl amino acid having formula (IIIa).

[0153] (IIIa),

[0154] Wherein R is H or C1-C8 alkyl, preferably H or C1-C6 alkyl, more preferably H or C2-C4 alkyl, even more preferably ethyl or butyl, and most preferably ethyl; and L-glufosinate and / or its salt.

[0155] Suitable salts are hydrochloride, ammonium salts, and isopropylammonium salts. It should also be understood that the corresponding zwitterions of L-glufosinate are also included.

[0156] In a preferred embodiment of the invention, the amount of L-glufosinate and / or its salt is based on at least 5 wt.-%, preferably at least 10 wt.-%, more preferably at least 20 wt.-%, even more preferably at least 30 wt.-%, still more preferably at least 40 wt.-%, and particularly at least 50 wt.-% or at least 60 wt.-%.

[0157] In a preferred embodiment of the invention, the amount of L-glufosinate and / or its salt is 5 to 99 wt.-%, preferably 10 to 98 wt.-%, more preferably 20 to 96 wt.-%, even more preferably 30 to 95 wt.-%, still more preferably 40 to 94 wt.-%, and particularly at least 50 to 90 wt.-% or at least 60 to 90 wt.-%.

[0158] The composition may contain an N-carbamoyl amino acid having formula (IIIa).

[0159] (IIIa)

[0160] The amount of the N-carbamoyl amino acid having formula (IIIa) is up to 40 wt.-%, preferably up to 20 wt.-%, more preferably up to 10 wt.-%, even more preferably up to 5 wt.-%, still more preferably up to 4 wt.-%, and particularly up to 2 wt.-%. The composition may contain the N-carbamoyl amino acid having formula (IIIa).

[0161] (IIIa)

[0162] The amount of the N-carbamoyl amino acid of formula (IIIa) is based on 0.001 to 40 wt.-%, preferably 0.005 to 20 wt.-%, more preferably 0.01 to 10 wt.-%, even more preferably 0.05 to 5 wt.-%, still more preferably 0.1 to 4 wt.-%, and particularly 0.5 to 2 wt.-%.

[0163] The composition may further comprise an N-carbamoyl amino acid having formula (IIIb).

[0164] (IIIb),

[0165] The amount of the N-carbamoyl amino acid having formula (IIIb) is preferably up to 40 wt.-%, preferably up to 20 wt.-%, more preferably up to 10 wt.-%, even more preferably up to 5 wt.-%, still more preferably up to 4 wt.-%, and particularly up to 2 wt.-%. The composition may further comprise the N-carbamoyl amino acid having formula (IIIb).

[0166] (IIIb),

[0167] The amount of the N-carbamoyl amino acid of formula (IIIb) is 0.001 to 40 wt.-%, preferably 0.005 to 20 wt.-%, more preferably 0.01 to 10 wt.-%, even more preferably 0.05 to 5 wt.-%, still more preferably 0.1 to 4 wt.-%, and particularly 0.5 to 2 wt.-%.

[0168] The composition may contain N-carbamoyl glufosinate-ammonium amide having formula (IIb).

[0169] (IIb)

[0170] The amount of the N-carbamoyl glufosinate-ammonium amide having formula (IIb) is up to 30 wt.-%, preferably up to 20 wt.-%, more preferably up to 10 wt.-%, even more preferably up to 5 wt.-%, still more preferably up to 2.5 wt.-%, and particularly up to 1 wt.-%. The composition may contain N-carbamoyl glufosinate-ammonium amide having formula (IIb).

[0171] (IIb)

[0172] The amount of the N-carbamoyl glufosinate-ammonium amide having formula (IIb) is 0.001 to 30 wt.-%, preferably 0.005 to 20 wt.-%, more preferably 0.01 to 10 wt.-%, even more preferably 0.05 to 5 wt.-%, still more preferably 0.1 to 2.5 wt.-%, and particularly 0.5 to 1 wt.-%.

[0173] The composition may further comprise N-carbamoyl glufosinate-ammonium amide having formula (IIa).

[0174] (IIa),

[0175] The amount of the N-carbamoyl glufosinate having formula (IIa) is preferably up to 30 wt.-%, preferably up to 20 wt.-%, more preferably up to 10 wt.-%, even more preferably up to 5 wt.-%, still more preferably up to 2.5 wt.-%, and particularly up to 1 wt.-%. The composition may further comprise N-carbamoyl glufosinate having formula (IIa).

[0176] (IIa),

[0177] The amount of the N-carbamoyl glufosinate having formula (IIa) is based on 0.001 to 30 wt.-%, preferably 0.005 to 20 wt.-%, more preferably 0.01 to 10 wt.-%, even more preferably 0.05 to 5 wt.-%, still more preferably 0.1 to 2.5 wt.-%, and particularly 0.5 to 1 wt.-%.

[0178] In a preferred embodiment of the invention, R in formulas (IIIa) and (IIb) is H or a C1-C6 alkyl group, preferably H or a C2-C4 alkyl group, more preferably ethyl or butyl, and most preferably ethyl. In this regard, it should be understood that R in formulas (IIIb) and (IIa) is also preferably H or a C1-C6 alkyl group, preferably H or a C2-C4 alkyl group, more preferably ethyl or butyl, and most preferably ethyl (if present).

[0179] In a preferred embodiment of the invention, the composition described herein can be used directly as a herbicidal composition or as an ingredient in a formulated herbicidal product.

[0180] The compositions described herein can be used for application to crop plants in the field for the prevention or control of weeds. The compositions can be formulated as liquids for field spraying. Glufosinate (preferably L-glufosinate) is provided in the composition in an effective amount. As used herein, an effective amount means from about 10 grams of active ingredient per hectare to about 1,500 grams of active ingredient per hectare, for example, from about 50 grams to about 400 grams or from 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 may be about 10 g, about 50 g, about 100 g, about 150 g, about 200 g, about 250 g, about 300 g, about 350 g, about 400 g, about 500 g, about 550 g, about 600 g, about 650 g, about 700 g, about 750 g, about 800 g, about 850 g, about 900 g, about 950 g, about 1,000 g, about 1,050 g, about 1,100 g, about 1,150 g, about 1,200 g, about 1,250 g, about 1,300 g, about 1,350 g, about 1,400 g, about 1,450 g, or about 1,500 g of L-glufosinate per hectare.

[0181] The herbicidal compositions described herein (including concentrates that require dilution before application to plants) contain L-glufosinate (i.e., the active ingredient), optionally with some residual N-carbamoyl glufosinate amide of formula (IIb).

[0182] (IIb) and / or N-carbamoyl amino acids having formula (IIIa)

[0183] (IIIa), and one or more auxiliary components in liquid or solid form.

[0184] The composition is prepared by mixing the active ingredient with one or more adjuvants (e.g., diluents, extenders, carriers, surfactants, organic solvents, wetting agents, or regulators) to provide a composition in the form of finely dispersed particulate solids, pellets, solutions, dispersions, or emulsions. Therefore, the active ingredient can be used with adjuvants (e.g., finely dispersed solids, organically derived liquids, water, wetting agents, dispersants, emulsifiers, or any suitable combination thereof). From an economic and convenience standpoint, water is the preferred diluent. However, not all compounds are resistant to hydrolysis, and in some cases, this may require the use of non-aqueous solvent media, as understood by those skilled in the art.

[0185] Optionally, one or more additional components may be added to the composition to produce a formulated herbicidal composition. Such a formulated composition may include L-glufosinate, a carrier (e.g., a diluent and / or solvent), and other components. The formulated composition contains an effective amount of L-glufosinate.

[0186] Diluents may also be included in the formulated composition. Suitable diluents include water and other aqueous components. Optionally, the diluent is present in the amount required to produce a composition ready for packaging or use.

[0187] The herbicidal compositions described herein (particularly liquids and soluble powders) may contain one or more surfactants as additional adjuvant components, in an amount sufficient to readily disperse the given composition in water or oil. The incorporation of surfactants into the composition greatly enhances its efficacy. As used herein, surfactants include wetting agents, dispersants, suspending agents, and emulsifiers. Anionic, cationic, and nonionic agents can all be used with equal ease.

[0188] Suitable wetting agents include alkylbenzene and alkylnaphthalene sulfonates, sulfated fatty alcohols, amines or amides, long-chain esters of sodium isosulfate, esters of sodium sulfosuccinate, sulfated or sulfonated fatty acid ester petroleum sulfonates, sulfonated vegetable oils, ditert-tert-acetylenide, polyoxyethylene derivatives of alkylphenols (especially isooctylphenol and nonylphenol), and polyoxyethylene derivatives of mono-higher fatty acid esters of hexyl anhydrides (e.g., sorbitan). Exemplary dispersants include methylcellulose, polyvinyl alcohol, sodium lignin sulfonate, polymeric alkylnaphthalene sulfonate, sodium naphthalene sulfonate, polymethylene bisnaphthalene sulfonate, and sodium N-methyl-N-(long-chain acid) laurate.

[0189] A water-dispersible powder composition containing one or more active ingredients, an inert solids expander, and one or more wetting agents and dispersants can be prepared. The inert solids expander is typically mineral-derived, such as natural clay, diatomaceous earth, and synthetic minerals derived from silica. Examples of such expanders include kaolinite, attapulgite clay, and synthetic magnesium silicate. The water-dispersible powder described herein may optionally contain about 5 to about 95 parts by weight of the active ingredient (e.g., about 15 to 30 parts by weight of the active ingredient), about 0.25 to 25 parts by weight of the wetting agent, about 0.25 to 25 parts by weight of the dispersant, and about 4.5 to about 94.5 parts by weight of the inert solids expander, all parts by weight of the total composition. When desired, about 0.1 to 2.0 parts by weight of the solid inert expander may be replaced by a corrosion inhibitor or an antifoaming agent, or both.

[0190] Aqueous suspensions can be prepared by dissolving or mixing water-insoluble active ingredients in an aqueous slurry in the presence of a dispersant and then grinding them to obtain a concentrated slurry with very finely dispersed particles. The resulting concentrated aqueous suspension is characterized by its extremely small particle size, resulting in very uniform coverage when diluted and sprayed.

[0191] Emulsifiable oils are typically solutions of an active ingredient and a surfactant in a solvent that is immiscible with or partially immiscible with water. Suitable solvents for the active ingredient described herein include hydrocarbons and water-immiscible ethers, esters, or ketones. Emulsifiable oil compositions typically contain about 5 to 95 parts of the active ingredient, about 1 to 50 parts of the surfactant, and about 4 to 94 parts of the solvent, all parts by weight of the total emulsifiable oil.

[0192] The compositions described herein may also contain other additives used as adjuvants or in combination with any of the aforementioned adjuvants, such as fertilizers, plant toxins and plant growth regulators, pest control agents, etc. The compositions described herein may also be mixed with other materials (e.g., fertilizers, other plant toxins, etc.) and applied in a single application.

[0193] In each type of formulation described herein (e.g., liquid and solid formulations), the concentration of the active ingredient is the same.

[0194] It should be recognized that the weeding compositions can be used in combination with other herbicides. The weeding compositions of the present invention are typically applied in combination with one or more other herbicides to control a wider range of unwanted vegetation. When used in combination with other herbicides, the claimed compounds of the present invention can be formulated with one or more other herbicides, mixed in a tank with one or more other herbicides, or applied sequentially with one or more other herbicides. Some herbicides that can be used in combination with the compounds of this invention include: amide herbicides, such as dipropionylamine, flubutyroxynil, bensulfuron-methyl, benzoylamine, brobutyroxynil, benzoyl ketone, CDEA, chlorpyrifos, tricyclosporine, dimethomorph, succinylmethrin, bispyribac-methyl, triazolylsulfuron, acetamiprid, flumetsulam, flufenoxuron, flunitrazepam, succinylmethrin, isoxaflutole, dichlorvos, nattochlor, clethodim, fenpyroxylchlor, chlorpyrifos, and chlorpyrifos; aniline herbicides, such as butyroxynil, lorfuran, chlorpyrifos, cyclosporine, pyrifluquinazon, ethoxybenzamide, etc. Acetylsulfuron, fluthiamethoxam, flubendioxonil, benzylsulfuron, flusulfanilamide, oxazolidinylsulfuron, heptamethrin, naproxen, metolachlor, flupyrfluthrin, and propargite; arylaloacetic acid herbicides, such as acetochlor, metolachlor, and metolachlor-M; chlorpyrifos-methyl herbicides, such as acetochlor, metolachlor, butachlor, butachlor, isobutachlor, acetochlor, metolachlor, pyrazochlor, isopropachlor, S-metolachlor, propachlor, chlorpyrifos, propachlor, propargite, terbutachlor, thifensulfuron, and sulfadiazine; sulfonylsulfuron-methyl herbicides, such as ethoxysulfuron, chlorpyrifos, pyriproxysulfuron, and fluazinam. Amines; sulfonamide herbicides, such as sulfadiazine, carbaryl, sulfadiazine, and fenvalerate; antibiotic herbicides, such as phosmet; benzoic acid herbicides, such as dicamba, dicamba, 2,3,6-TBA, and chlorpyrifos; pyrimidinoxybenzoic acid herbicides, such as bispyribac-sodium and pyrimethanil; pyrimidinylthiobenzoic acid herbicides, such as pyrimethanil; phthalic acid herbicides, such as chlorpyrifos; pyridinecarboxylic acid herbicides, such as chlorpyrifos, dichloropyridine, and chlorpyrifos; quinoline carboxylic acid herbicides, such as dichloroquinoline and chloroquinoline; arsenic herbicides, such as dimethylarsine, CMA, DSMA, and hexamethylarsine. Fluoroarsenates, MAA, MAMA, MSMA, potassium arsenite, and sodium arsenite; benzoylcyclohexanedione herbicides, such as mesotrione, sulfadiazine, furazolidone, and cyclosulfonyl ketone; benzofuranylalkyl sulfonate herbicides, such as furazolidone and ethoxyfuran; carbamate herbicides, such as sulfadiazine, tebuconazole, chlorpyrifos, benzylsulfuron, sulfadiazine, and sulfadiazine; phenyl carbamate herbicides, such as abamectin, BCPC, carbaryl, carbaryl, CEPC, chlorpyrifos, chlorfenapyr, CPPC, betaine, betaine, ethyl betaine, acetamiprid, and metribuzin;Cyclohexene oxime herbicides, such as quizalofop-P-ethyl, butoxycycline, clethodim, cyclobutenyl ketamine, thiamethoxam, cyclobenzanil, clethodim, dexamethasone, and styraxone; cyclopropylisoxazole herbicides, such as isoxaflutole and isoxaflutole; dicarboximide herbicides, such as pyrimidinone, indole-methyl, fluphenazine, flufenoxuron, propyzoxystrobin, and clodinafop-propargyl; dinitroaniline herbicides, such as flutamide, diflubenzuron, diflubenzuron, chlorfluazol, isopropoxyl, flufenoxuron, sulfadiazine, ammonium sulfadiazine, pendimethalin, aminopropoxyl, cyclopropoxyl, and trifluralin; dinitrophenol herbicides, such as diflubenzuron, nitropropoxyl, pendimethalin, diflubenzuron, terlipine, DNOC, nitropropoxyl, and diflubenzuron; diphenyl ether herbicides... Herbicides, such as fluroxypyr; nitrophenyl ether herbicides, such as trifluralin, bensulfuron, methyl methoxypyr, methoxypyr, fensulfuron, fensulfuron, fensulfuron, trifluralin, ethoxypyr, fluorinated oxychloride, flufenoxuron, furoxypyr, flufenoxuron, quizalofop-p-ethyl, fensulfuron, trifluralin, and ethoxypyr; dithiocarbamate herbicides, such as dazomet and fensulfuron; halogenated aliphatic herbicides, such as arolactam, trichloropropionic acid, cyhalofop-p-ethyl, tetrafluoropropionic acid, hexachloroacetone, iodomethane, methyl bromide, monochloroacetic acid, SMA, and TCA; imidazolinone herbicides, such as imazalil, methoxypromethazine, imazalil, metribuzin, metribuzin, and imazalil; inorganic herbicides, such as ammonium aminosulfonate, borax, calcium chlorate, etc. Copper sulfate, ferrous sulfate, potassium azide, potassium cyanate, sodium azide, sodium chlorate, and sulfuric acid; nitrile herbicides, such as bromofenozide, bromobenzonitrile, chlorobenzonitrile, diflubenzuron, iodofenozide, iodofenozide, and bispyribac-methyl; organophosphorus herbicides, such as methyl parathion, chlorpyrifos, dimethoate, phosmet, 2,4-DEP, DMPA, EBEP, phosmet, glyphosate, and piperazine; phenoxy herbicides, such as bromophenol oxime, chlorpyrifos, 2,4-DEB, 2,4-DEP, quizalofop-p-ethyl, cyhalothrin, chlorpyrifos, acetamiprid, chlorpyrifos, chlorophenoxyethanol, and quizalofop-p-ethyl; phenoxyacetic acid herbicides, such as 4-CPA, 2,4-D, 3,4-DA, MCPA, and MCPA-thiophene. Phenoethyl 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 hydroxyethyl, 4-CPP, 2,4-D propionic acid, 2,4-D propionic acid, 3,4-DP, 2,4,5-dichloropropionic acid, 2-methyl-4-chloropropionic acid and 2-methyl-4-chloropropionic acid; aryloxyphenoxypropionic acid herbicides, such as quizalofop-p-ethyl, quizalofop-p-ethyl, chlorpyrifos, cyhalofop-p-ethyl, quizalofop-p-ethyl, quizalofop-p-ethyl, quizalofop-p-ethyl, quizalofop-p-ethyl, quizalofop-p-ethyl, quizalofop-p-ethyl, quizalofop-p-ethyl, quizalofop-p-ethyl, isoxaflutole, oxadiazon, quizalofop-p-ethyl, quizalofop-p-ethyl, quizalofop-p-ethyl, and trifluorobutylic acid;Phenylenediamine herbicides, such as dichlorvos and aminopropoxyfen; pyrazolyl herbicides, such as imidacloprid, pyrazosulfuron, sulfadiazine, benzalkonium chloride, pyrrolizumab, and pyrazosulfuron-methyl; pyrazolylphenyl herbicides, such as isopyrazosulfuron and imidacloprid; pyridazine herbicides, such as chlorpyrifos, chlorfenapyr, and pyrazosulfuron; pyridazinone herbicides, such as bromopyrazosulfuron, chlorpyrifos, pyrazosulfuron-methyl, flupyrazosulfuron, flupyrazosulfuron, pyrazosulfuron-methyl, pyrazosulfuron-methyl, and pyridazine; pyridine herbicides, such as chlorpyrifos, iodopyridazine, dichloropyridine, fluthion, flupyrazosulfuron, flupyrazosulfuron, flupyrazosulfuron, flupyrazosulfuron, thiamethoxam, and chlorpyrifos; pyrimidinediamine herbicides, such as isochlorpyrifos and pyrazosulfuron; quaternary ammonium herbicides, such as styrax. Herbicides include: diethyl sulfadiazine, fenvalerate, diquat, fenvalerate, and paraquat; thiocarbamate herbicides, such as butyraz, fenvalerate, EPTC, quizalofop-p-ethyl, chlorpyrifos, isopropyl quizalofop-p-ethyl, chlorpyrifos ... Simazine, terbutaline, and glyphosate; methoxytriazine herbicides, such as atrazine, fenproxate, propiconazole, styraxate, terbutaline, simazine, and terbutaline; methylthiotriazine herbicides, such as atrazine, azidophos, cypermethrin, diquat, isoamyl ethyl, glyphosate, propiconazole, fenproxate, and terbutaline; triazinone herbicides, such as acetamiprid, terbutaline, cycloazinone, cimetidine, benzoxazinone, and cypermethrin; triazole herbicides, such as fenproxate, benzoxazinone, cycloxazinone, cycloxazinone, benzoxazinone, and cycloxazinone; triazole herbicides, such as fenproxate, fenproxate, fenproxate, flufenproxate, propiconazole, metsulfuron-methyl, and thiamethoxam-methyl; triazolopyrimidine herbicides, such as chlorpyrifos, dichlorvos, diflubenzuron, and pyrazosulfuron-methyl. Herbicides such as sulfadiazine, penoxsulam, and pyrazosulfuron; uracil herbicides such as flupropargyl, chlorpyrifos, flupropargyl, isosulfuron, cyclopyr, and terbuprofen; 3-phenyluracil; urea herbicides such as thiamethoxam, bensulfuron, cyclopyr, chlorpyrifos, flupyridine, isosulfuron, ethazin, toluazol, tebuconazole, and pyrazosulfuron; phenylurea herbicides such as succinyluron, clodinafop-methyl, chlorothalonil, ethoxysulfuron, chlorpyrifos, fenpyroxon, chlorpyrifos, oxazol, diuron, felfluroxyfen, fluroxypyr, flusulfuron, isoproturon, flusulfuron, metribuzin, methyl chlorpyrifos, pyranol, bromonazine, methoxysulfuron, chlorpyrifos, metribuzin, chlorpyrifos, paraflurium, cyprodinium, cyclopyr, tetraflurium, and thiamethoxam.Pyrimidinylsulfonylurea herbicides, such as pyrimisulfuron, tetrazolium-sulfuron, bensulfuron-sulfuron, chlorimuron-sulfuron, cypromethazine-sulfuron, ethoxysulfuron, pyrimisulfuron, flupyrsulfuron, flupyrsulfuron, formamide-sulfuron, chlorpyrifos-sulfuron, azoxysulfuron, mesosulfuron-methyl, nicosulfuron, pyrimisulfuron-sulfuron, epoxysulfuron, flupyrsulfuron-sulfuron, pyrimisulfuron-sulfuron-sulfuric acid, sulfonylsulfuron-sulfuron, mesosulfuron-sulfuric acid, sulfonylsulfuron-sulfuron, and triflumsulfuron-sulfuron; triazineylsulfonylurea herbicides, such as chlorsulfuron, ethersulfuron-sulfuron, azoxysulfuron-sulfuric acid, iodosulfuron-sulfuron, mesosulfuron-sulfuron, flusulfuron-sulfuron, thifensulfuron, ethersulfuron-sulfuron, benzylsulfuron-sulfuron, flumethinon-sulfuron, and triflumsulfuron-sulfuron; thiamethoxam-sulfuron-urea herbicides, such as butyrazuron, sulfothiamethoxam, butyrazuron-sulfuron, thiamethoxam, and thiamethoxam; and unclassified herbicides. Herbicides, such as acrolein, allyl alcohol, chlorpyrifos, pyrazosulfuron, glyphosate, bentazon, dicyclosulfuron, butiamethoxam, calcium cyanamide, chlorpyrifos, chlorpyrifos, alfalfa, fluazinam, chlorofluorocarbons, cyclohexane, isoxaflutole, CPMF, mixed methylphenol, o-dichlorobenzene, piperazine, cypermethrin, chlorpyrifos, flupyrfluthrin, fluroxypyr, furazolidone, etc. The herbicides include glyphosate, cypermethrin, indoxacarb, metribuzin, methyl isothiocyanate, fluchlorfon, OCH, propyzoxystrobin, oxadiazon, oxadiazon, pentachlorophenol, pentacycloxadiazon, phenylmercuric acetate, cyclophosphamide, thiamethoxam, pyrimimethoxam, cyclomethoxyfenozide, cyprodinil, thiamethoxam, azoxystrobin, thiamethoxam, thiamethoxam, thiamethoxam, metribuzin, isouron, tripropanol, and glyphosate. The herbicidal compositions of the present invention can also be combined with glyphosate or 2,4-D for use in glyphosate-tolerant or 2,4-D-tolerant crops. It is generally preferred to use the compositions of the present invention in combination with herbicides that are selective for the treated crop and supplement the weed spectrum controlled by these compositions at the applied rate. It is generally further preferred to simultaneously apply the compositions of the present invention with other complementary herbicides, as a combined formulation or as a tank mix.

[0195] As mentioned above, in a third aspect, the invention further relates to a method for selectively controlling weeds in an area, preferably containing a batch of planted glufosinate-resistant seeds or crops, the method comprising:

[0196] Apply an effective amount of the composition to the region, the composition comprising an enantiomeric excess of L-glufosinate and / or its salt at a ratio of at least 50% enantiomerically, preferably greater than 70%, relative to D-glufosinate and / or its salt, and greater than 0.01 wt.-% to less than 10 wt.-% of an N-carbamoylaminoamide of formula (II) based on the total amount of the composition.

[0197] (II),

[0198] Wherein R is H or C1-C8 alkyl, preferably H or C1-C6 alkyl, more preferably H or C2-C4 alkyl, even more preferably ethyl or butyl, and most preferably ethyl.

[0199] And / or

[0200] Based on the total amount of the composition, greater than 0.01 wt.% to less than 10 wt.% of N-carbamoyl amino acids having formula (III)

[0201] (III),

[0202] Wherein R is H or C1-C8 alkyl, preferably H or C1-C6 alkyl, more preferably H or C2-C4 alkyl, even more preferably ethyl or butyl, and most preferably ethyl.

[0203] In a preferred embodiment of the invention, the composition comprises L-glufosinate and / or its salt in an enantiomer ratio of 50% to 99%, preferably 60% to 98%, more preferably 70% to 95%, and particularly 80% to 90% relative to D-glufosinate and / or its salt.

[0204] In a preferred embodiment of the invention, the composition comprises 0.02 to 8 wt.-%, preferably 0.03 to 5 wt.-%, more preferably 0.05 to 3 wt.-%, and particularly 0.1 to 2 wt.-% of an N-carbamoyl amino acid having formula (III) based on the total amount of the composition.

[0205] (III),

[0206] Wherein R is H or C1-C8 alkyl, preferably H or C1-C6 alkyl, more preferably H or C2-C4 alkyl, and most preferably H.

[0207] It should be understood that the composition may contain the same adjuvants and / or other herbicides as described in more detail above.

[0208] The compositions described herein can be used for application to crop plants in the field for the prevention or control of weeds. The compositions can be formulated as liquids for field spraying. L-Glufosinate is provided in the composition in an effective amount. As used herein, an effective amount means from about 10 grams of active ingredient per hectare to about 1,500 grams of active ingredient per hectare, for example, from about 50 grams to about 400 grams or from 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 may be about 10 g, about 50 g, about 100 g, about 150 g, about 200 g, about 250 g, about 300 g, about 350 g, about 400 g, about 500 g, about 550 g, about 600 g, about 650 g, about 700 g, about 750 g, about 800 g, about 850 g, about 900 g, about 950 g, about 1,000 g, about 1,050 g, about 1,100 g, about 1,150 g, about 1,200 g, about 1,250 g, about 1,300 g, about 1,350 g, about 1,400 g, about 1,450 g, or about 1,500 g of L-glufosinate per hectare.

[0209] The invention is further illustrated by the following examples. Example

[0210] Preparation of enzymes

[0211] a) Cloning of enzyme genes (Example 1)

[0212] The amino acid sequences of the corresponding enzymes were identified from public databases (UniProt, https: / / www.uniprot.org; NCBI Protein Database, https: / / www.ncbi.nlm.nih.gov / protein). Sequences from NCBI were marked with "" at the beginning of the corresponding database identifier. "Instructions. The corresponding DNA sequence was derived using the standard codon usage of *Escherichia coli*. The DNA sequence was synthesized (BioCat GmbH) and cloned into plasmid pDHE19.2 (Ress-Loeschke, M. et al., DE 19848129, 1998, BASF AG). The resulting plasmid was used to transform competent cells of *E. coli* strain (TG10, pAgro, pHSG575) (*E. coli* TG10 (Kesseler, M. et al., WO 2004050877A1, 2004, BASF AG)) (Chung, CT et al., Proc Natl Acad Sci USA, 1989, 86, 2172): using pHSG575 (Takeshita, S. et al., Gene, 1987, rhaA-derived compounds of Escherichia coli TG1 were transformed with pAgro4 (pBB541 in Tomoyasu, T. et al., Mol. Microbiol. [Molecular Microbiology], 2001, 40, 397) and pAgro4 (pBB541 in Tomoyasu, T. et al., Mol. Microbiol. [Molecular Microbiology], 2001, 40, 397).

[0213] b) Production of enzymes through recombination (Example 2)

[0214] Preparation of biocatalysts in shake flasks

[0215] E. coli TG10 carrying the enzyme was inoculated into 2 ml LB medium supplemented with 100 µg / ml ampicillin, 100 µg / ml spectinomycin, and 20 µg / ml chloramphenicol (Bertani, G., J Bacteriol [Journal of Bacteriology], 1951, 62, 293), and the resulting preculture was incubated at 37°C with stirring at 250 rpm for 5 h. 1 ml of the preculture was then inoculated into 100 ml 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-thiopyranoside, and 0.5 g / L rhamnose in a 500 ml Erlenmeyer-flask with a baffle. The culture was incubated at 37°C with shaking for 18 h. Biomass was then harvested by centrifugation at 3220 xg for 10 min at 8°C. The supernatant was discarded, and the cell pellet was resuspended in 8 ml of 100 mM HEPES buffer supplemented with 1 mM MnCl2 at pH 8.2. If whole-cell biotransformation is to be performed, the cell suspension can be used without any further synthetic preparation. If using clarified cell lysate, 5 ml of cell suspension was dispensed into five reaction tubes containing lysis matrix B (0.7 ml quartz beads (Ø 0.1 mm), MPBiomedicals), the tubes were cooled on ice, and the cells were then lysed in a homogenizer (Peqlab Precellys 24, VWR) for two 30-second cycles. Between cycles, the sample was cooled on ice. The resulting cell-free lysate was clarified by centrifugation at 20817 xg for 10 min at 8°C. The supernatants were separated and fractions from the same batch (= clarified cell lysate) were combined.

[0216] Fermentative whole-cell biocatalyst production

[0217] E. coli TG10 containing plasmids pAgro4 and pHSG575 were transformed with the pDHE plasmid encoding the target protein. The transformants were cultured on LB agar plates supplemented with 100 µg / ml ampicillin, 100 µg / ml spectinomycin, and 20 µg / ml chloramphenicol.

[0218] Pre-culture medium:

[0219] EcoK12 solution

[0220]

[0221] Sterilization was performed using a filter with a pore size of 0.2 µm.

[0222] Part 1

[0223]

[0224] Part 2

[0225]

[0226] Part 3

[0227]

[0228] All three parts were sterilized at 121°C for 30 minutes.

[0229] Vitamin solution

[0230]

[0231] Sterilization by filtration using filters with a pore size of 0.2 pm

[0232] To prepare the final pre-medium, portions 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 from LB agar plates and used to inoculate 2 x 100 g of pre-medium into 1 L baffled Erlenmeyer flasks. These pre-cultures were incubated at 37°C and 150 rpm. When the OD600 reached 12, the entire pre-culture was used to inoculate the master culture.

[0233] Main culture medium:

[0234] Part 4

[0235]

[0236] Part 4 is sterilized at 125°C for 45 min.

[0237] Part 5

[0238]

[0239] Part 5 uses a filter unit with a pore size of 0.1 µm for sterilization via aseptic filtration.

[0240] Glycerin solution

[0241]

[0242] Thiamine solution

[0243]

[0244] Glycerin and defoaming solution were sterilized at 121°C for 30 min. Thiamine and induction solution were sterilized by filtration through a 0.2 µm filter.

[0245] Parts 4 and 5 were combined in a sterile fermentation vessel (Techfors, Infors HT) and inoculated with a pre-culture. The vessel was maintained at 37°C, 0.2 bar, and pH 6.6 by administering an alkaline solution during fermentation. The pO2 level was maintained at 20%–40% by adjusting the stirrer speed (typically 500 rpm) and aeration rate (typically 6 l / min). Antifoaming solution was added as needed. A feed solution was prepared by combining glycerol and thiamine solutions. After inoculation, the feed solution was administered at a rate of 10 g / h. After 7 h, feed solution administration was switched to a "stop-and-observe" mode, where the feed was activated at a rate of 10 g / h as the pO2 level increased. After consuming 14 h or 330 g of feed solution, the feed rate was increased to 80–100 g / h. Gene expression was induced by adding an induction solution at an oxygen transfer rate of 80 mmol / L / h or, alternatively, at an OD600 of 12. Fermentation was stopped 36 h after induction 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 precipitate 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 and then lyophilized. At this point, the lyophilizer was maintained at -50°C and a pressure of 0.25 mbar. The lyophilized cells were stored at 4°C.

[0246] Production of freeze-dried cell-free extract

[0247] The lyophilized cells were resuspended in 100 g / L ultrapure water. The cell suspension was cooled on ice and then homogenized through three channels using a homogenizer (Panda Plus 2000, GEA) set to 800 bar. The pressure in the three channels was typically between 1000 and 1400 bar. Debris in the resulting mixture was removed by centrifugation at 10000 rpm for 15 min at 10°C. The resulting precipitate was discarded, and the protein concentration in the supernatant was analyzed using a Bradford assay. The supernatant was frozen at -80°C and subsequently lyophilized at -50°C and 0.25 mbar.

[0248] Preparation of starting materials and intermediates

[0249] c) Synthesis of n-Butyl(3-cyano-3-hydroxypropyl)methylphosphonate (ACM-H) (Example 3)

[0250] ACM-H has been prepared according to Example 2 of WO 2015 / 173146 A1.

[0251] d) Preparation of N-carbamoyl glufosinate-ammonium amide from ACM-H (Example 4)

[0252]

[0253] Ammonium bicarbonate (27.05 g, 342.1 mmol) and diammonium carbonate (32.87 g, 342.1 mmol) were dissolved in 135 mL of distilled water with stirring, and the reaction mixture was heated to 50°C. After a 2-hour period, a solution of 15 g of n-butyl(3-cyano-3-hydroxypropyl)methylphosphonate (68.4 mmol, “ACM-H”) in water (15 mL) prepared according to Example 2 was added to the reaction mixture. The reaction was cooled to room temperature and stirred for another 3 days at room temperature. The reaction mixture was concentrated under vacuum. The crude product was purified by column chromatography using a gradient of methanol in dichloromethane (1:9 methanol / dichloromethane to 2:1) to yield the product (10%). ¹H NMR (500 MHz, DMSO-d⁶) δ 7.46 (s, ¹H), 7.08 (s, ¹H), 6.20 (dd, J = 8.2, 3.7 Hz, ¹H), 5.63 (s, 2H), 4.17 – 4.03 (m, ¹H), 3.85 (m, 2H), 1.93 – 1.19 (m, ¹¹H), 0.89 (t, J = 7.4 Hz, 3H). LC-MS (measured): 280.2

[0254] e) Chemical synthesis of N-carbamoyl amino acids from glufosinate (Example 5)

[0255]

[0256] After a 30-minute period, a solution of potassium cyanate (11.8 g, 145 mmol) in water (30 mL) was added to a stirred solution of racemic glufosinate-ammonium salt (50% in water, 39.6 g, 99.9 mmol) at 50°C under vacuum (200 mbar). The reaction mixture was stirred for another 1 h at 50°C under vacuum (200 mbar) and then cooled to room temperature. The reaction mixture was subjected to ion-exchange chromatography (Dowex-50 WX 8 200-400 (H), 220 mL) and the product was eluted with water (1 L). The eluted product was concentrated under reduced pressure to yield a carbamoyl acid product (7.9 g). The remaining carbamoyl acid was separated from the column as a potassium salt. ¹H 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-enantiomers and L-enantiomers were synthesized from commercially available D-glufosinate and L-glufosinate in a similar manner. The specific rotation of the L-enantiomer [α] = +27.5 (c = 1 H₂O, measured as potassium salt). HPLC-MS retention times were obtained using Supelco Chirobiotic T2 (elution buffer: 40% water in acetonitrile, 0.1% formic acid). Temperature: 20°C, flow rate: 0.8 mL / min. Retention times: L-carbamoyl amino acid (7.4 min); D-carbamoyl amino acid (9.2 min).

[0257] Preparation of L-glufosinate P-butyl ester

[0258] f) Preparation of N-carbamoyl glufosinate from N-carbamoyl glufosinate amide (Example 6)

[0259]

[0260] Add 5 mg of enzyme (papain CAS 9001-73-4, powder) to 500 μL of a 10 mg / mL N-carbamoyl glufosinate-ammonium solution in phosphate buffer (potassium dihydrogen phosphate, pH 8.0, 50 mM). Shake the resulting solution at room temperature for 2 days. Subsequently, analysis of the reaction mixture by chiral HPLC-MS showed a 14% conversion to the corresponding L-carbamoyl amino acid (er L:D, > 99 : 1). The concentration of N-carbamoyl amino acids was determined by chiral HPLC-MS using Supelco Chirobiotic T2 (gradient 90% ACN / water to 60% ACN / water, over 19 min, 0.1% formic acid). Temperature: 20°C, flow rate: 0.8 mL / min. Retention times of N-carbamoyl amino acids: L-configuration diastereomer (8.6 min); D-configuration (10.7 and 11.2 min).

[0261] Other enzymes tested showed conversion rates greater than 0.1%.

[0262] -Bromelain (CAS 37189-34-7)

[0263] - Bacterial protease (protease from Bacillus licheniformis, CAS 9014-01-1)

[0264] g) Chemical synthesis of glufosinate from N-carbamoyl amino acids (Example 7)

[0265]

[0266] 100 mg of 4-[butoxy(methyl)phosphoryl]-2-ureo-butyric acid (synthesized using amidase (papain CAS9001-73-4, see Example 5)) 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 another 2 hours. Then concentrated HCl (36%, 7.5 mL) in water was added, and the reaction mixture was heated to 100°C and stirred overnight at this temperature. The reaction mixture was cooled to room temperature and extracted twice with dichloromethane (2 x 10 mL). The aqueous phase was concentrated under reduced pressure to obtain glufosinate hydrochloride. ¹H NMR (500 MHz, 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 = 13.5 Hz, 3H).

[0267] Reaction to glufosinate using an enzymatic carbamoyl cleavage step

[0268] h) Enzymatic two-pot synthesis of glufosinate from N-carbamoyl amino acids (Example 8, SEQ ID NO: 1)

[0269]

[0270] To a solution of 2-(carbamoylamino)-4-[hydroxy(methyl)phosphoryl]butyric acid (0.6 g, 2.5 mmol, produced according to Example 5) in degassed aqueous potassium phosphate buffer (5.4 mL, 0.496 M, pH 8.0), KOH (3 M in water) was added to adjust the pH to 8.0. To a reaction mixture (6.1 mL), potassium phosphate buffer (19.2 mL, 0.100 M, pH 8.0) and N-carbamoyl amino acid hydrolase (A0A1Y4GC62, SEQ ID NO: 4, clarified cell lysate, 1.5 mL, 12.9 mg / mL total protein concentration, protein produced in a shake flask) and MnCl2 solution (1 M in water, 20 μL) were added. The reaction mixture was stirred at 37°C (250 rpm) for 24 h. NMR and HPLC analysis showed a 31% conversion to glufosinate. The enantiomer ratio of glufosinate was analyzed by chiral HPLC. Chiral HPLC: > 99% L-glufosinate / < 1% D-glufosinate; Analytical method: Chirex (D)-penicillamine 250 × 4, 6 mm column from Phenomenex; isocratic elution with 10 mM copper(II) sulfate; UV detection at 245 nm.

[0271] i) One-pot synthesis of p-butyl glufosinate

[0272] 5 mg of N-carbamoyl glufosinate was dissolved in 250 μL of phosphate buffer (potassium dihydrogen phosphate, pH 8.0, 100 mM), and 250 μL of liquid enzyme preparation was added to this solution. The reaction mixture was shaken at room temperature for 24 h. After this period, 1.5 μL of MnCl2 solution (2 M in water) was added, followed by N-carbamoyl amino acid hydrolase (10 mg, lyophilized cell-free extract, A0A535Y1H2, SEQ ID NO: 2).

[0273] (a) The liquid enzyme formulation used was Sustine® 220 from Novozymes. The reaction product (p-butyl glufosinate) was obtained in a yield of >0.01% and detected by LC / MS.

[0274] (b) The liquid enzyme formulation used was a protease from Bacillus licheniformis (CAS 9014-01-1, aqueous, 94 mg / mL protein). The reaction product (p-butyl glufosinate) was obtained in a yield of >0.01% and detected by LC / MS.

[0275] Analytical method: p-butyl glufosinate was analyzed by LC / MS on a Kinetex C18 100 × 2, 1 mm column. Elution was performed isocratically at 40°C, with a flow rate of 0.5 mL / min, using 80% water / 20% acetonitrile + 0.1% formic acid, and a retention time of 1.8 min.

[0276] SEQ ID NO: 1 (A0A3E0C996, from Paraburkholderia sp. BL6669N2)

[0277] MVRIDPDRLLSDLKQLRSFGATGPGVVRLALSPVDLASREWLAGRMTEAGLDAVIDGVGTVFGRSRKSGPALVIGSHTDTQPTGGWLDGAMGVIYGLEIARALAENEATRHLAVDVASWIDEEGTFSGLLGSRSFVGDNVDETIRDATNRQGQRLEDVLAAAGLAGRPRARFEPGRQVAYLEPHIEQGGRLEAAGKSIGVVTTIVG LRELRLRFTGQRNHAGTTPMAIRRDAGAALVAFIPQMNEAFTQLADADTVWTVGRIDLDPGSLSVVPGAAEMYLQFRDANAARLQAMEDRLAELVRDFNARGSVSVELTTIDEPMQPVTMHAALADHLARAAEAVAPGQWIRMPSGAAHDAQVIARCMPACMMFVPSIGGVSHDFIEDTAEAHIVLGCQVAATAAAAMLEEQWAKRS

[0278] SEQ ID NO: 2 (A0A535Y1H2 from Chloroflexi bacteria)

[0279] MTDAARLERRIHELAQIGRTDDPAREIYATAVSRLGLSAEEQRARDLVTSWCAPHGATARRDPAANLYLRFPGADPHAPVVLVGSHLDSVPMGGRFDGALGVCCAVEAVVSLLESGARFARPVEVVGWADEEGARFGYGLFGSAAAFGRLRVDPERVRDKGGTSIAEALRALGESGDLAGAMRDPKGIRAYLELHIEQGPRLERAGAPLGVVSDIVGIFHGLVMVRGEQNHAGATVMGERHDALVAASHMIIALERIASSVPDAVATVGEITVKPGAKNVIPGECTFSLDIRAPKQESIDLVLERFKAEANEIFRKSLREWGLRPLQSVAVTPLDEDLRDLLWKSAMSVGVNAPTLVSGAGHDAQNPSLAGVPTGMIFVRSTGGSHTPTEFAATADAALGAKALEIAIRELATA

[0280] SEQ ID NO: 3 (A0A6P2ISL4 from Burkholderia lata DSM 23089)

[0281] MNPTDFPFPPLNAERLNARVEQLARFTRPDVPWTRRAFSPLFTEARAWLAAQFAEAGLAVSMDAGGNLIGRREGSGRCTKPLVTGSHCDTVVGGGRFDGIIGVLAGIEVAHTLNEQGIVLDHPFEVIDFLSEEPSDYGISCVGSRALSGVLDAGMLRATNAEGETLAEALRRIGGNPDALREPLRAPGSTAAFVELHIEQGPVLETRGLPIGVVTNIVGIRRVLITVTGQPDHAGTTPMDIRRDALVGAAHLIEAAHARASALSGNPHYVVATIGRIAMTPNVPNAVPGQVELMLEVRSDSDAVLDAFPEALLAGAAARLDALRLSARAEHVSRARPTDCQPLVMDAVEQAATQLGYPSMRLPSGAGHDAVYVAPTGPIGMIFIPCLGGRSHCPEEWIEPQQLLDGTRVLYQTLVALDRSLAGAA

[0282] SEQ ID NO: 4 (from A0A1Y4GC62 of Cloacibacillus sp. An23)

[0283] MNCVNDILRSIGKAGRNEDGSYTRACYSAEYFAAVDITEKLMREYGMETSRDAAGNLHGVLPGTEPGLKSIIIGSHLDTVPEGGLFDGAYGVAGGLEVVRRLKEEGRRPRHTIELYGFNAEESSPLGGTFGSRAVTGLVSPEQPGLAEALKSYGHTVEEIMGCRRDFSDAKCYLELHIEQGDYLFSEGQKIGVVSGIVGVIRYKVTALGHSNHAGTTMMKNRRDAMVAMARLITEADRRCRAIDDRLVLTVGTIKCWPGSENVIPGKVECSFEMRHMDKAKTDELIREIREIAENIATVEFEIVNMIDKGAVSCDAHLMDVICEAAEEAGESHVVMPSGAGHDANPMAHRVPIGMIFVPSKDGMSHCPEEWTDSEETAAGAEVLYRTVLALDAED。

Claims

1. A method for preparing glufosinate and / or its salts or glufosinate alkyl esters and / or their salts, wherein the glufosinate or the glufosinate alkyl ester has a molecular structure according to formula (I). (I)， Wherein R is H or C1-C8 alkyl, preferably H or C1-C6 alkyl, more preferably H or C2-C4 alkyl, even more preferably ethyl or butyl, and most preferably ethyl. The process includes the following steps: a) Hydrolysis of N-carbamoyl glufosinate-ammonium amide with formula (II) by amidase (II), Wherein R is H or C1-C8 alkyl, preferably H or C1-C6 alkyl, more preferably H or C2-C4 alkyl, even more preferably ethyl or butyl, and most preferably ethyl. The amidase is preferably selected from a list of free peptidases, proteases, linear amidases, or cyclic amidases, thereby producing an N-carbamoyl amino acid having formula (III). (III), Wherein R is H or C1-C8 alkyl, preferably H or C1-C6 alkyl, more preferably H or C2-C4 alkyl, even more preferably ethyl or butyl, and most preferably ethyl. as well as b) Cut the carbamoyl moiety of the N-carbamoyl amino acid having formula (III).

2. The method according to claim 1, wherein the cutting step b) provides glufosinate having formula (I) and / or its salt or glufosinate alkyl ester and / or its salt. (I)， Wherein R is H or C1-C8 alkyl, preferably H or C1-C6 alkyl, more preferably H or C2-C4 alkyl, even more preferably ethyl or butyl, and especially ethyl.

3. The method according to claim 2, wherein the cutting step b) provides either a racemic mixture or an enantiomeric excess of L-glufosinate of formula (Ia) and / or its salt or L-glufosinate alkyl ester and / or its salt, or L-glufosinate alkyl ester and / or its salt. (I), Wherein R is H or C1-C8 alkyl, preferably H or C1-C6 alkyl, more preferably H or C2-C4 alkyl, even more preferably ethyl or butyl, and most preferably ethyl. Preferably, it is in the form of an enantiomeric excess of L-glufosinate and / or its salt or L-glufosinate alkyl ester and / or its salt having formula (Ia), and the amidase is an L-amidase.

4. The method according to any one of claims 1 to 3, wherein at least 5%, preferably at least 10%, more preferably at least 30%, even more preferably at least 40%, and most preferably at least 50% of the N-carbamoyl glufosinate having formula (II) is converted into L-glufosinate and / or its salt or L-glufosinate alkyl ester and / or its salt having formula (Ia), wherein formula (Ia) is as defined in claim 3.

5. The method according to any one of claims 1 to 4, wherein the cutting step b) is carried out under enzymatic conditions, preferably using N-carbamoyl amino acid hydrolase, more preferably LN-carbamoyl amino acid hydrolase, or wherein the cutting step b) is carried out under chemical conditions, preferably using sodium nitrite and / or hydrogen chloride.

6. The method according to any one of claims 1 to 5, wherein the hydrolysis step a) is carried out at a pH of 6 to 11, preferably 6.5 to 10, more preferably 7 to 9.5, and particularly 7.5 to 9, and / or The experiment is carried out at a temperature of 20°C to 50°C, preferably 25°C to 45°C, more preferably 30°C to 42°C, and particularly 32°C to 40°C.

7. The method according to any one of claims 1 to 6, wherein R in formulas (II) and (III) is a C1-C8 alkyl, preferably a C1-C6 alkyl, more preferably a C2-C4 alkyl, even more preferably ethyl or butyl, and most preferably ethyl, and the method further comprises the following steps: c) Deprotection is carried out under acidic conditions, preferably using hydrochloric acid or sulfuric acid.

8. The method according to any one of claims 1 to 7, wherein the method further comprises adding N-carbamoyl glufosinate racemic enzyme and / or N-carbamoyl amino acid racemic enzyme, the N-carbamoyl glufosinate racemic enzyme causing the N-carbamoyl glufosinate to racemic at the α-carbon atom.

9. The method according to any one of claims 1 to 8, wherein steps a) and b) are carried out in a single container, preferably wherein all reagents are added substantially at the start of the reaction, or wherein the reagents of step a) and step b) are added to the single container at different times.

10. The method according to any one of claims 1 to 8, wherein the method further comprises the step of isolating N-carbamoyl glufosinate having formula (IIb). (IIb), Wherein R is H or C1-C8 alkyl, preferably H or C1-C6 alkyl, more preferably H or C2-C4 alkyl, even more preferably ethyl or butyl, and most preferably ethyl, which is obtained in hydrolysis step a), preferably using reversed-phase chromatography.

11. The method according to any one of the preceding claims, wherein the amidase is selected from the group consisting of: papain (CAS 9001-73-4) and variants thereof, bromelain (CAS 37189-34-7) and variants thereof, and protease from Bacillus licheniformis (CAS 9014-01-1) and variants thereof, wherein a variant is defined as a polypeptide sequence having at least 80%, preferably 90%, and most preferably 95% sequence identity with the corresponding polypeptide sequence.

12. The method according to any one of claims 5 to 11, wherein the N-carbamoyl amino acid hydrolase is selected from the group consisting of enzymes identified by their Uniprot IDs: A0A0K9YX84 and its variants, E3HUL6 and its variants, Q9F464 and its variants, A0A4D7Q548 and its variants, A0A2S9D976 and its variants, A0A3E0C996 (SEQ ID NO:1) and its variants, A0A535Y1H2 (SEQ ID NO:2) and its variants, A0A6P2ISL4 (SEQ ID NO:3) and its variants, A0A1Y4GC62 (SEQ ID NO:4) and its variants, wherein a variant is defined as a polypeptide sequence having at least 80%, preferably 90%, and most preferably 95% sequence identity with the corresponding polypeptide sequence, preferably selected from the group consisting of enzymes identified by their Uniprot IDs: A0A3E0C996 (SEQ ID NO:1) and its variants. (SEQ ID NO:1) and its variants, A0A535Y1H2 (SEQ ID NO:2) and its variants, A0A6P2ISL4 (SEQ ID NO:3) and its variants, A0A1Y4GC62 (SEQ ID NO:4), wherein a variant is defined as a polypeptide sequence having at least 80%, preferably 90%, and most preferably 95% sequence identity with the corresponding polypeptide sequence.

13. A composition comprising N-carbamoyl glufosinate-ammonium amide having formula (II) (II), Wherein R is H or C1-C8 alkyl, preferably H or C1-C6 alkyl, more preferably H or C2-C4 alkyl, even more preferably ethyl or butyl, and most preferably ethyl.

14. The composition according to claim 13, wherein the composition comprises N-carbamoyl glufosinate-ammonium amide having formula (IIb). (IIb), Wherein R is H or C1-C8 alkyl, preferably H or C1-C6 alkyl, more preferably H or C2-C4 alkyl, even more preferably ethyl or butyl, and most preferably ethyl. and optional N-carbamoyl amino acids of formula (IIIa) (IIIa), Wherein R is H or C1-C8 alkyl, preferably H or C1-C6 alkyl, more preferably H or C2-C4 alkyl, even more preferably ethyl or butyl, and most preferably ethyl. And L-glufosinate and / or its salts or L-glufosinate alkyl esters and / or their salts.

15. The composition according to claim 14, wherein the amount of L-glufosinate and / or its salt or L-glufosinate alkyl ester and / or its salt is based on at least 5 wt.-%, preferably at least 10 wt.-%, more preferably at least 20 wt.-%, even more preferably at least 30 wt.-%, and most preferably at least 40 wt.-%.

16. A method for selectively controlling weeds in an area, preferably containing a batch of planted glufosinate-resistant seeds or crops, the method comprising the steps of: Apply an effective amount of the composition to the region, the composition comprising, in an enantiomer ratio of at least 50% and preferably greater than 70% enantiomer excess of L-glufosinate and / or its salt or L-glufosinate alkyl ester and / or its salt relative to D-glufosinate and / or its salt or D-glufosinate alkyl ester and / or its salt, and greater than 0.01 wt.-% to less than 10 wt.-% of an N-carbamoylaminoamide of formula (II) based on the total amount of the composition. (II), Wherein R is H or C1-C8 alkyl, preferably H or C1-C6 alkyl, more preferably H or C2-C4 alkyl, even more preferably ethyl or butyl, and most preferably ethyl. And / or Based on the total amount of the composition, greater than 0.01 wt.% to less than 10 wt.% of N-carbamoyl amino acids having formula (II) (III), Wherein R is H or C1-C8 alkyl, preferably H or C1-C6 alkyl, more preferably H or C2-C4 alkyl, even more preferably ethyl or butyl, and most preferably ethyl.

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