Synthesis of glufosinate using an amidase-based process

Abstract

The present invention relates to a method for preparing glufosinate, comprising the steps of first hydrolyzing N-carbamoyl glufosinate amide with an amidase enzyme to form an N-carbamoyl amino acid compound, and then cleaving the carbamoyl portion of the N-carbamoyl amino acid compound.

Description

Technical Field

[0001] The present invention relates to a method for preparing glufosinate, which comprises forming an N-carbamoyl amino acid compound by hydrolyzing N-carbamoyl glufosinate amide with an amidase enzyme, and then cleaving the carbamoyl moiety of the N-carbamoyl amino acid compound.

Background Art

[0002] Glufosinate, a herbicide, is a non-selective herbicide for foliar application and is considered one of the safest herbicides from the toxic or environmental viewpoints. In the current commercial chemical synthesis methods of glufosinate, a racemic mixture of L- and D-glufosinate is produced (Duke et al. 2010 Toxins 2:1943-1962).

[0003] L-Glufosinate (also known as phosphinothricin or (S)-2-amino-4-(hydroxy(methyl)phosphonyl)butanoic acid) is known to be more potent than D-glufosinate (Ruhland et al. (2002) Environ. Biosafety Res. 1:29-37). Therefore, there is a further interest in methods that produce a higher amount of the more active L-glufosinate form.

Summary of the Invention

Problems to be Solved by the Invention

[0004] From the above background, an object of the present invention was to provide a mild method for preparing glufosinate.

[0005] A further object of the present invention was to provide a safe method for preparing glufosinate.

[0006] A further object of the present invention was to provide a mild method for preparing L-glufosinate with enantiomeric excess.

[0007] Furthermore, an object of the present invention was to provide a composition containing L-glufosinate.

[0008] Furthermore, an object of the present invention was to provide a method for selectively controlling weeds using a composition obtained according to the preparation method of the present invention. [Means for solving the problem]

[0009] Surprisingly, the inventors have found that at least one of the above-mentioned objectives can be achieved by the N-carbamoyl glufosinate amide-based process described herein. Furthermore, the inventors have found that the claimed method yields a composition containing a sufficient amount of glufosinate for use as a herbicide.

[0010] Therefore, in the first embodiment, the present invention relates to formula (I): [ka] A method for preparing a glufosinate and / or a salt thereof or a glufosinate alkyl ester and / or a salt thereof, having (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, most preferably ethyl): a) Equation (II): [ka] By hydrolyzing an N-carbamoyl glufosinate amide having (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, most preferably ethyl) with an amidase enzyme, formula (III): [ka] The steps of forming an N-carbamoyl amino acid having (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, most preferably ethyl), b) A step of cleaving the carbamoyl portion of an N-carbamoyl amino acid having formula (III), This includes methods.

[0011] The following describes in more detail preferred embodiments of the preparation method, composition, and components of the method for selectively controlling weeds. It should be understood that each preferred embodiment is preferable not only on its own but also in combination with other preferred embodiments.

[0012] In a preferred embodiment A1 of the first aspect, the cutting step b) is performed by formula (I): [ka] A glufosinate and / or a salt thereof or a glufosinate alkyl ester and / or a salt thereof is obtained, having (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, most preferably ethyl).

[0013] In a preferred embodiment A2 of the first aspect, by step b), a glufosinate and / or a salt thereof having formula (I) or a glufosinate alkyl ester and / or a salt thereof is obtained in racemic form, or formula (Ia): [ka] L-glufosinate and / or a salt thereof or glufosinate alkyl ester and / or a salt thereof having (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, most preferably ethyl) is obtained in an enantiomer-rich form, preferably L-glufosinate and / or a salt thereof or glufosinate alkyl ester and / or a salt thereof having formula (Ia) is obtained in an enantiomer-rich form, and the amidase enzyme is L-amidase enzyme.

[0014] In preferred embodiment A3 of the first embodiment, 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 the N-carbamoyl glufosinate amide having formula (II) is converted to L-glufosinate and / or a salt thereof or glufosinate alkyl ester and / or a salt thereof having formula (Ia), where formula (Ia) is as defined in preferred embodiment A2.

[0015] In a preferred embodiment A4 of the first aspect, cleavage step b) is carried out under enzymatic conditions, preferably using N-carbamoyl amino acid hydrolase, more preferably LN-carbamoyl amino acid hydrolase, or cleavage step b) is carried out under chemical conditions, preferably using sodium nitrite and / or hydrogen chloride.

[0016] In a preferred embodiment A4a of the first aspect, cleavage step b) is carried out under enzymatic conditions, preferably using N-carbamoyl amino acid hydrolase, more preferably LN-carbamoyl amino acid hydrolase, or cleavage 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.

[0017] 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.

[0018] In the preferred embodiment A6 of the first aspect, the hydrolysis step a) is carried out at a pH of 6-11, preferably pH 6.5-10, more preferably pH 7-9.5, particularly pH 7.5-9, and / or at a temperature of 20-50 °C, preferably 25-45 °C, more preferably 30-42 °C, particularly 32-40 °C.

[0019] In the preferred embodiment A7 of the first aspect, R in formulas (II) and (III) is C1-C8 alkyl, preferably C1-C6 alkyl, more preferably C2-C4 alkyl, still more preferably ethyl or butyl, and most preferably ethyl, and the method c) further comprises a step of performing deprotection under acidic conditions, preferably using hydrochloric acid or sulfuric acid.

[0020] In the preferred embodiment A8 of the first aspect, the method further comprises adding a racemization enzyme of N-carbamoylglufosinate amide and / or a racemization enzyme of N-carbamoyl amino acid.

[0021] In the preferred embodiment A9 of the first aspect, steps a) and step b) are carried out in a single container, preferably all the reagents are added substantially at the start of the reaction, or the reagents for step a) and the reagents for step b) are added to the single container at different times.

[0022] In the preferred embodiment A10 of the first aspect, the method is the formula (IIb) obtained in the hydrolysis step a):

Chemical formula

[0023] In a second embodiment, the present invention relates to formula (IIb): [ka] The present invention relates to a composition comprising an N-carbamoyl glufosinate amide having (wherein R is H or a C1-C8 alkyl group, preferably H or a C1-C6 alkyl group, more preferably H or a C2-C4 alkyl group, even more preferably ethyl or butyl, and most preferably ethyl).

[0024] In a preferred embodiment B1 of the second aspect, the composition is formula (IIb): [ka] The formula comprises an N-carbamoyl glufosinate amide having (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, most preferably ethyl).

[0025] In another preferred embodiment B2 of the second aspect, the composition is formula (IIb): [ka] N-carbamoyl glufosinate amide having (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, most preferably ethyl), Optionally, equation (IIIa): [ka] The formula comprises an N-carbamoyl amino acid having H or a C1-C8 alkyl group, preferably H or a C1-C6 alkyl group, more preferably H or a C2-C4 alkyl group, even more preferably ethyl or butyl, and most preferably ethyl, and L-glufosinate and / or a salt thereof.

[0026] In preferred embodiment B3 of the second aspect, the amount of L-glufosinate and / or its salt is at least 5% by weight, preferably at least 10% by weight, more preferably at least 20% by weight, even more preferably at least 30% by weight, and most preferably at least 50% by weight, based on the total amount of N-carbamoylglufosinate amide having formula (IIb), N-carbamoyl amino acid having formula (IIIa), and L-glufosinate and / or its salt.

[0027] In 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, most preferably ethyl.

[0028] In a third aspect, the present invention provides a method for selectively controlling weeds in a predetermined area, preferably an area containing a crop of sown seeds or a glufosinate-resistant crop: A composition comprising L-glufosinate and / or a salt thereof, wherein the enantiomer ratio to D-glufosinate and / or a salt thereof is at least 50%, preferably the enantiomer excess ratio is greater than 70%, Formula (II): [ka] An N-carbamoylaminoamide having (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, most preferably ethyl) is added to the composition in an amount of more than 0.01% by weight and less than 10% by weight, based on the total amount of the composition. and / or Formula (III): [ka] An N-carbamoyl amino acid having (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, most preferably ethyl) is added in an amount of more than 0.01% by weight and less than 10% by weight based on the total amount of the composition. The present invention relates to a method comprising applying a composition containing the above to the area in an effective amount. [Modes for carrying out the invention]

[0029] Before describing exemplary embodiments of the present invention in detail, we will provide some important definitions necessary for understanding the invention.

[0030] As used herein and in the appended claims, the singular forms “a” and “an” also encompass their respective plural forms unless the context clearly indicates otherwise. The terms “about” and “approximately” in relation to the present invention refer to a range of precision that a person skilled in the art would recognize as still ensuring the technical effect of the feature in question. This term typically represents a deviation of ±20%, preferably ±15%, more preferably ±10%, and even more preferably ±5% from the given numerical value. The term “comprising” should be understood to be non-limiting. The term “consisting of” in relation to the object of the present invention is considered a preferred embodiment of the term “comprising of.” Hereinafter, where a group is defined as comprising at least a certain number of embodiments, this also means that a preferred group consisting only of these embodiments is also included. Furthermore, the terms “first,” “second,” “third,” or “(a),” “(b),” “(c),” “(d),” and similar terms in this specification and the claims are used to distinguish similar elements and do not necessarily describe a continuous or chronological order. It should be understood that the terms used in this manner are interchangeable under appropriate circumstances, and that embodiments of the present invention described herein may be carried out in an order other than that described or illustrated herein. Where terms such as “first,” “second,” “third,” or “(a),” “(b),” “(c),” “(d),” “i,” “ii,” etc., relate to a method, use, or assay step, unless otherwise specified in this application, there may be no time interval between these steps, or the time interval may be inconsistent, as described or described herein, i.e., these steps may be carried out simultaneously, or there may be time intervals of a few seconds, a few minutes, a few hours, a few days, a few weeks, a few months, or even several years between such steps. It should be understood that the present invention is not limited to the specific methods, procedures, reagents, etc. described herein, as they may be modified.The terms used herein are for the sole purpose of describing specific embodiments and are not intended to limit the scope of the invention, which should be understood to be limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art.

[0031] As used throughout this specification, the term "weight %" refers to "weight percent".

[0032] As used herein, the term "alkyl" refers in each case to a linear or branched alkyl group having typically 1 to 20 carbon atoms, preferably 1 to 8, often 1 to 6, more preferably 1 to 4, for example, 2 or 4 carbon atoms. Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, 2-butyl, isobutyl, tert-butyl, n-pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, 2,2-dimethylpropyl, 1-ethylpropyl, and n-hexyl.

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

[0034] Preferred embodiments of the present invention are described in detail below: a method for preparing glufosinate and / or a salt thereof having formula (I) or glufosinate alkyl ester and / or a salt thereof; a composition comprising N-carbamoyl glufosinate amide having formula (IIb), N-carbamoyl amino acid having formula (IIIa), and L-glufosinate and / or a salt thereof; and a method for selectively controlling weeds. It should be understood that the preferred embodiments of the present invention are preferable individually or in combination with each other.

[0035] As shown above, in one embodiment, the present invention relates to formula (I): [ka] A method for preparing a glufosinate and / or a salt thereof or a glufosinate alkyl ester and / or a salt thereof, having (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, most preferably ethyl): a) Equation (II): [ka] By hydrolyzing an N-carbamoyl glufosinate amide having (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, most preferably ethyl) with an amidase enzyme, formula (III): [ka] The steps of forming an N-carbamoyl amino acid having (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, most preferably ethyl), b) A step of cleaving the carbamoyl portion of an N-carbamoyl amino acid having formula (III), This includes methods.

[0036] Equation (I): [ka] It should be understood that glufosinates and / or salts thereof, or glufosinate alkyl esters and / or salts thereof, encompass all stereoisomers, preferred salts of each glufosinate, or preferred salts of its alkyl ester. Furthermore, each zwitterion is also encompassed in formula (I). Preferred salts include hydrochloride, ammonium salts, and isopropylammonium salts. In this regard, compounds of formula (I) particularly contain two stereocenters, one located at the phosphorus atom and the other at the α-carbon atom. Compounds of formula (I) particularly encompass all stereoisomers derived from the stereocenter of the phosphorus atom.

[0037] N-carbamoyl glufosinate amides having formula (II) can be obtained by any suitable preparation method. For example, N-carbamoyl glufosinate amides are byproducts of the Bucherer-Berks reaction. Thus, N-carbamoyl glufosinate amides can be produced from the relevant cyanohydrin. Cyanogen hydrin or cyanohydrin derivatives can be obtained, for example, by processes described in particular U.S. Patent No. 4,521,348B1, German Patent No. 3047024, U.S. Patent No. 4,599,207B1, U.S. Patent No. 6,359,162B1, Chinese Patent Application Publication No. 102372739A and Chinese Patent Application Publication No. 102399240A.

[0038] In a preferred embodiment of the present invention, 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. The pH is preferably adjusted using an alkali metal hydroxide, more preferably sodium hydroxide or potassium hydroxide, and particularly potassium hydroxide.

[0039] In a preferred embodiment of the present invention, hydrolysis step a) is carried out at a temperature of 20 to 50°C, preferably 25 to 45°C, more preferably 30 to 42°C, and particularly 32 to 40°C.

[0040] In a preferred embodiment of the present invention, 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.

[0041] In a preferred embodiment of the present invention, the hydrolysis step a) is carried out while stirring is performed, preferably at 50 to 1000 rpm, more preferably at 100 to 800 rpm, even more preferably at 150 to 600 rpm, even more preferably at 180 to 400 rpm, and particularly at 200 to 300 rpm.

[0042] Any suitable amidase enzyme, preferably a linear amidase enzyme, can be used. More preferably, the amidase enzyme is an enzyme that hydrolyzes amide bonds (EC 3.4, EC 3.5.1, EC 3.5.2). Therefore, preferably, the hydrolysis of amide bonds occurs regardless of whether this hydrolysis is a natural function or a promiscuous function of the enzyme. Accordingly, preferred amidase enzymes can be selected from the group consisting of peptidases, proteases, linear amidases, or cyclic amide amidases. Particularly preferred amidase enzymes are selected from the group consisting of papain (CAS 9001-73-4), bromelain (CAS 37189-34-7), and bacterial proteinases (proteinases derived from Bacillus licheniformis, CAS 9014-01-1).

[0043] In a preferred embodiment of the present invention, the amidase enzyme is L-amidase enzyme.

[0044] In preferred embodiments of the present invention, the amidase enzyme is D-amidase, more preferably a protease, and most preferably a cysteine ​​protease.

[0045] In preferred embodiments of the present 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.

[0046] In a preferred embodiment of the present invention, step b) cuts formula (I): [ka] A glufosinate and / or a salt thereof or a glufosinate alkyl ester and / or a salt thereof is obtained, having (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, most preferably ethyl).

[0047] In a preferred embodiment of the present invention, step b) cleavage step b) yields a glufosinate and / or a salt thereof having formula (I) or a glufosinate alkyl ester and / or a salt thereof in the form of a racemic mixture.

[0048] In another preferred embodiment of the present invention, by step b), the glufosinate and / or salt thereof having formula (I) or glufosinate alkyl ester and / or salt thereof is obtained by formula (Ia): [ka] L-glufosinate and / or its salt or glufosinate alkyl ester and / or its salt having formula (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, most preferably ethyl) is obtained in a form with an enantiomer excess. Preferably, L-glufosinate and / or its salt or glufosinate alkyl ester and / or its salt having formula (Ia) is formed with an enantiomer excess, and the amidase enzyme is L-amidase enzyme.

[0049] In another preferred embodiment of the present invention, by step b), a glufosinate and / or a salt thereof having formula (I) or a glufosinate alkyl ester and / or a salt thereof is obtained by formula (Ib): [ka] D-glufosinate and / or its salt or glufosinate alkyl ester and / or its salt having (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, most preferably ethyl) is obtained in a form with an enantiomer excess. In this regard, the amidase enzyme is preferably a D-amidase enzyme.

[0050] In a preferred embodiment of the present invention, cleavage step b) is carried out under enzymatic conditions, preferably using N-carbamoyl amino acid hydrolase, more preferably LN-carbamoyl amino acid hydrolase. Suitable N-carbamoyl amino acid hydrolases are selected from the group consisting of linear amide hydrolases EC 3.5.1, N-carbamoyl-L-amino acid hydrolase EC 3.5.1.87, N-carbamoyl-D-amino acid hydrolase EC 3.5.1.77, and mixtures thereof.Suitable N-carbamoyl amino acid hydrolases that can be used in this method include 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, E3HUL6 and its variants, A0 A1V9BSS3 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, A0A535Y1H2 and its variants, A0A 223E4I5 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 and its variants, A0A1G0MC89 and its variants, A0A Examples include selections from the group consisting of 1M6WYS1 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. A variant is defined as a polypeptide sequence having at least 80%, preferably 90%, and most preferably 95% sequence homology to the respective 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, and A0A1Y4GC62 (SEQ ID NO: 4) and its variants, where a variant is defined as a polypeptide sequence having at least 80%, preferably 90%, and most preferably 95% sequence homology to the respective polypeptide sequence. It should be understood that the N-carbamoyl amino acid hydrolases outlined above are represented using database identifier nomenclature according to the Uniprot database (www.UniProt.org) as of March 19, 2023.

[0051] In connection with this, step b) is carried out at a temperature preferably 20 to 50°C, preferably 25 to 45°C, more preferably 30 to 42°C, and particularly 32 to 40°C. Furthermore, the reaction pressure is preferably atmospheric pressure. Preferably, the reaction pressure is in the range of 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 present invention, step b) is carried out at a pH of 5 to 10, preferably 6 to 9, and particularly about 7.

[0052] In a preferred embodiment of the present invention, cutting step b) is carried out while stirring is performed, preferably at 50 to 1000 rpm, more preferably at 100 to 800 rpm, even more preferably at 150 to 600 rpm, even more preferably at 180 to 400 rpm, and especially at 200 to 300 rpm.

[0053] In another preferred embodiment of the present invention, cleavage step b) is carried out under chemical conditions. It should be understood that the terms “chemical conditions” or “chemical cleavage” refer to cleavage steps not carried out under enzymatic conditions. Any suitable chemical method is possible. Cleavage can be carried out, exemplary, using sodium nitrite and / or hydrogen chloride. For example, formula (III): [ka] An N-carbamoyl amino acid having (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, most preferably ethyl) may be treated with concentrated hydrochloric acid at a high temperature. Alternatively, the N-carbamoyl amino acid of formula (III) defined above may be treated with sodium nitrite and hydrogen chloride under aqueous conditions. In this regard, cleavage step b) is preferably carried out at a temperature of 25-120°C, more preferably 50-110°C, and particularly 60-105°C. Furthermore, the reaction pressure is preferably atmospheric pressure. Preferably, the reaction pressure is in the range of 0.995-1.030 mbar, more preferably 1.005-1.020 mbar, and particularly about 1.013 mbar. In a preferred embodiment of the present invention, cleavage step b) is carried out at a pH of 0-5, preferably 0-3. The reaction mixture can be worked up by standard procedures (i.e., washing and purification).

[0054] In a particular preferred embodiment of the present invention, the cleavage step b) is carried out under enzymatic conditions.

[0055] In preferred embodiments of the present invention, 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, most preferably ethyl, and this method is c) Further comprising the step of deprotecting under acidic conditions. In this regard, any suitable acid can be used. Preferably, hydrochloric acid or sulfuric acid is used.

[0056] In a preferred embodiment of the present invention, the method further comprises adding a racemase of N-carbamoylglufosinate amide. Any suitable N-carbamoylglufosinate amide racemase that racems N-carbamoylglufosinate amide at the α-carbon atom may be used.

[0057] In a preferred embodiment of the present invention, the method further comprises adding an N-carbamoyl amino acid racemase. Any suitable N-carbamoyl amino acid racemase may be used.

[0058] In a preferred embodiment of the present invention, the method further comprises adding a racemase of N-carbamoyl glufosinate amide and a racemase of N-carbamoyl amino acids.

[0059] In a preferred embodiment of the present invention, steps a) and b) are carried out in a single container, and step b) is carried out under enzymatic conditions. In connection therewith, preferably, all reagents are added substantially at the start of the reaction. Alternatively, preferably, the reagents of step a) and step b) are added to a single container at different time points.

[0060] In a preferred embodiment of the present invention, the method is to obtain formula (IIb) in hydrolysis step a): [ka] The process further includes the step of separating an N-carbamoyl glufosinate amide having formula (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, most preferably ethyl). Separation of the N-carbamoyl glufosinate amide having formula (IIb) is preferably achieved using reverse-phase chromatography. Alternatively, separation may be achieved using ion exchange, extraction, salt formation, crystallization and filtration.

[0061] N-carbamoyl glufosinate amides having formula (IIb) can be chemically racemized and reused in hydrolysis step a). To racemize N-carbamoyl glufosinate amides having formula (IIb), they can be treated with a suitable base, preferably at a pH of 8 or higher, more preferably 8-14, even more preferably 8.5-12, and particularly 8.5-10. Preferably, the racemization is carried out under aqueous conditions.

[0062] Alternatively, an N-carbamoylglufosinate having formula (IIb) may be treated with a racemizing enzyme for N-carbamoylglufosinate.

[0063] In preferred embodiments of the present invention, at least 5%, preferably at least 10%, more preferably at least 20%, even more preferably at least 30%, even more preferably at least 40%, even more preferably at least 50%, and most preferably at least 60% of N-carbamoyl glufosinate amide having formula (II) is, [ka] The L-glufosinate and / or its salt or glufosinate alkyl ester and / or its salt is converted to an L-glufosinate and / or its salt having (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, most preferably ethyl).

[0064] In preferred embodiments of the present 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 the N-carbamoyl glufosinate amide having formula (II) is, [ka] The L-glufosinate and / or its salt or glufosinate alkyl ester and / or its salt is converted to an L-glufosinate and / or its salt having (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, most preferably ethyl).

[0065] In another preferred embodiment of the present invention, at least 5%, preferably at least 10%, more preferably at least 20%, even more preferably at least 30%, even more preferably at least 40%, even more preferably at least 50%, and most preferably at least 60% of N-carbamoyl glufosinate amide having formula (II) is, [ka] The L-glufosinate and / or its salt or glufosinate alkyl ester and / or its salt is converted to an L-glufosinate and / or its salt having (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, most preferably ethyl).

[0066] In another preferred embodiment of the present 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 amide having formula (II) is, [ka] The L-glufosinate and / or its salt or glufosinate alkyl ester and / or its salt is converted to an L-glufosinate and / or its salt having (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, most preferably ethyl).

[0067] It is preferable that L-glufosinate has an excess of enantiomers.

[0068] In a preferred embodiment of the present invention, the method comprises adding an amidase enzyme, a racemase of N-carbamoyl glufosinate amide, and a hydrolase of N-carbamoyl amino acids, all reaction steps being carried out in a single vessel (also known as "one-pot" conditions), preferably all reagents being added substantially at the start of the reaction, or reagents being added to a single vessel at different time points.

[0069] In another preferred embodiment of the present invention, the method comprises adding an amidase enzyme, a racemase of N-carbamoyl glufosinate amide, and a racemase of N-carbamoyl amino acids, all reaction steps being carried out in a single vessel (also known as "one-pot" conditions), preferably all reagents being added substantially at the start of the reaction, or reagents being added to a single vessel at different time points.

[0070] In further preferred embodiments of the present invention, the method comprises adding an amidase enzyme and an N-carbamoyl amino acid hydrolase, all reaction steps are carried out in a single vessel (also known as "one-pot" conditions), and preferably all reagents are added substantially at the start of the reaction, or the reagents are added to the single vessel at different time points. In this regard, the pH of the reaction mixture is preferably 8 or higher.

[0071] The enzyme to be applied can be applied in any preferred manner known in the art.

[0072] In preferred embodiments of the present invention, the enzyme to be applied is applied as a cleared cell lysate, whole cells, or immobilized enzyme.

[0073] Alternatively, some or all of the components other than L-glufosinate may be removed from the biotransformation mixture, and the mixture may be selectively concentrated and then used as is (and / or with the addition of various auxiliaries) for weed prevention or control. In some cases, this biotransformation reaction mixture may be used as is (and / or with the addition of various auxiliaries) for weed prevention or control.

[0074] Additional steps may be added to further purify L-glufosinate. Such further purification and isolation methods include ion exchange, extraction, salt formation, crystallization, and filtration, each of which may be used multiple times or in appropriate combinations. The enzyme can be removed by simple filtration if supported, or by ultrafiltration if free in solution, using absorbents such as Celite, cellulose, or carbon, or by denaturation using various techniques known to those skilled in the art.

[0075] The ion exchange process achieves separation by selectively adsorbing the solute onto a resin selected for this purpose. Since the product and impurities must be dissolved in a single solution before adsorption, it is usually necessary to concentrate the stream of purified product by evaporation or distillation before isolation. An example of using ion exchange for purification is described by Schultz et al. and in European Patent Application Publication No. 0249188A2.

[0076] Purification can be achieved by adding a suitable acid such as hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, or acetic acid to form an insoluble salt of L-glufosinate. Similarly, purification can also be achieved by adding a suitable base to form an insoluble salt. Useful bases include alkali metal hydroxides, carbonates, sulfates, and phosphates or alkaline earth metal hydroxides, carbonates, sulfates, and phosphates. Other nitrogen-containing bases can also be used, such as 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. To maximize yield and optimize the purity of the desired salt, concentration of the mixture or addition of a solvent (or both) may be advantageous. Suitable solvents for this purpose include those in which the desired salt has very low solubility (such solvents are often called "non-solvents"). Salts of L-glufosinate can be converted to glufosinate in a form suitable for formulation by standard methods known to those skilled in the art. Alternatively, L-glufosinate can be isolated in zwitterionic form.

[0077] U.S. Patent No. 9,255,115B2 describes a method for obtaining relatively high-purity L-glufosinate by converting the hydrochloride salt of L-glufosinate into a zwitterionic form using a base such as sodium hydroxide or sodium methoxide, and then crystallizing it in an aqueous alcoholic solvent. This method has the advantage of producing non-hygroscopic L-glufosinate crystals, which maintain higher purity than amorphous L-glufosinate even when exposed to moisture for a long period of time.

[0078] Other salts of L-glufosinate are also known in the art. U.S. Patents 5,767,309 and 5,869,668 teach the use of a chiral alkaloid base to form diastereomer salts of glufosinate triasemide. Purification is achieved by taking advantage of the fact that the salt of L-glufosinate precipitates from solution in much larger quantities than the salt of the corresponding D-glufosinate. Therefore, if desired, this method can be used in combination with the present invention to obtain L-glufosinate with a high enantiomer excess.

[0079] Optionally, purification can be achieved by first crystallizing one or more impurities, removing these impurities by filtration, and then further purifying the L-glufosinate by forming a salt from the resulting filtrate as described above. This is advantageous when some or all of the unreacted amine donor can be isolated and used in subsequent reactions. Similarly, it may be advantageous to isolate some or all of the unreacted N-carbamoyl amino acids having formula (III) and reuse them in subsequent reactions.

[0080] Extraction can also be used to purify the product. German Patent No. 3920570C2 describes a process for precipitating excess glutamic acid (used as an amine donor) by adjusting the pH of the solution to 3.7-4.2 with sulfuric acid. After filtering out the glutamic acid, the pH of the filtrate is reduced to 1-2, and then other impurities are extracted into the solvent. After extraction and concentration, ammonia is added to the aqueous solution to raise the pH to 5-7, causing ammonium sulfate to precipitate. By filtering out the ammonium sulfate and concentrating the resulting filtrate, the ammonium salt of L-glufosinate is obtained.

[0081] Isolating L-glufosinate or its salts may be desirable, for example, when shipping the solid to the place of formulation or use. Typical industrial methods of isolation, such as filtration or centrifugation, can be used. The isolated product often needs to be dewatered of water, volatile impurities, and solvents (if present), and typical industrial drying equipment can be used for this purpose. Examples of such equipment include ovens, rotary drum dryers, and agitated dryers. In some cases, spray dryers may be advantageous.

[0082] It is not essential to produce a solid product after purification. This can be advantageous if the formulation of L-glufosinate is carried out in the same place as the production of L-glufosinate. Many L-glufosinate and its salts are readily soluble in water, and water is a convenient liquid to use for product formulation. For example, the amine donor can be isolated by filtration, and the resulting filtrate can be concentrated by distillation. The pH of the filtrate may be adjusted to a 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 a salt thereof can be prepared as described above and isolated by filtration. The solid can be directly dissolved by adding water or a suitable solvent on the filter to obtain a solution of L-glufosinate.

[0083] As described above, in a second embodiment, the present invention further includes formula (IIb): [ka] The present invention relates to a composition comprising an N-carbamoyl glufosinate amide having (wherein R is H or a C1-C8 alkyl group, preferably H or a C1-C6 alkyl group, more preferably H or a C2-C4 alkyl group, even more preferably ethyl or butyl, and most preferably ethyl).

[0084] In a preferred embodiment of the second aspect, the composition is formula (IIb): [ka] The formula comprises an N-carbamoyl glufosinate amide having H or a C1-C8 alkyl group, preferably H or a C1-C6 alkyl group, more preferably H or a C2-C4 alkyl group, even more preferably ethyl or butyl, and most preferably ethyl.

[0085] Furthermore, in another preferred embodiment of the second aspect of the present invention, formula (IIb): [ka] N-carbamoyl glufosinate amide having (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, most preferably ethyl) and formula (IIIa): [ka] The present invention relates to a composition comprising an N-carbamoyl amino acid having (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, most preferably ethyl) and L-glufosinate and / or a salt thereof.

[0086] Preferred salts are hydrochloride salts, ammonium salts, and isopropylammonium salts. Furthermore, it should be understood that the zwitterions of each of the L-glufosinate are also included.

[0087] In a preferred embodiment of the present invention, the amount of L-glufosinate and / or its salt is at least 5% by weight, preferably at least 10% by weight, more preferably at least 20% by weight, even more preferably at least 30% by weight, even more preferably at least 40% by weight, particularly at least 50% by weight or at least 60% by weight, based on the total amount of N-carbamoylglufosinate amide having formula (IIb), N-carbamoyl amino acid having formula (IIIa), and L-glufosinate and / or its salt.

[0088] In preferred embodiments of the present invention, the amount of L-glufosinate and / or its salt is in the range of 5 to 99% by weight, preferably 10 to 98% by weight, more preferably 20 to 96% by weight, even more preferably 30 to 95% by weight, even more preferably 40 to 94% by weight, particularly at least 50 to 90% by weight or at least 60 to 90% by weight, based on the total amount of N-carbamoyl glufosinate amide having formula (IIb), N-carbamoyl amino acid having formula (IIIa), and L-glufosinate and / or its salt.

[0089] The composition is formula (IIIa): [ka] The composition may contain an N-carbamoyl amino acid having formula (IIb) 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 4% by weight, and especially up to 2% by weight, based on the total amount of N-carbamoyl glufosinate amide having formula (IIb), N-carbamoyl amino acid having formula (IIIa), and L-glufosinate and / or salt thereof. The composition is formula (IIIa): [ka] The N-carbamoyl amino acid having formula (IIb) can be included 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, even more preferably 0.1 to 4% by weight, and particularly preferably 0.5 to 2% by weight, based on the total amount of N-carbamoyl glufosinate amide having formula (IIb), N-carbamoyl amino acid having formula (IIIa), and L-glufosinate and / or salt thereof.

[0090] Furthermore, the composition is formula (IIIb): [ka] The composition may contain an N-carbamoyl amino acid having formula (IIb), 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, even more preferably up to 4% by weight, and particularly up to 2% by weight, based on the total amount of N-carbamoyl glufosinate amide having formula (IIb), N-carbamoyl amino acid having formula (IIIa), N-carbamoyl amino acid having formula (IIIb), and L-glufosinate and / or salts thereof. Furthermore, the composition may contain formula (IIIb): [ka] The N-carbamoyl amino acid having formula (IIb) can be included 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, even more preferably 0.1 to 4% by weight, and particularly preferably 0.5 to 2% by weight, based on the total amount of N-carbamoyl glufosinate amide having formula (IIb), N-carbamoyl amino acid having formula (IIIa), N-carbamoyl amino acid having formula (IIIb), and L-glufosinate and / or salts thereof.

[0091] The composition is formula (IIb): [ka] The composition may contain N-carbamoyl glufosinate amide having formula (IIb) 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, even more preferably up to 2.5% by weight, and especially up to 1% by weight, based on the total amount of N-carbamoyl glufosinate amide having formula (IIb), N-carbamoyl amino acid having formula (IIIa), and L-glufosinate and / or salt thereof. The composition is formula (IIb): [ka] N-carbamoyl glufosinate amide having formula (IIb) can be included 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, even more preferably 0.1 to 2.5% by weight, and particularly preferably 0.5 to 1% by weight, based on the total amount of N-carbamoyl glufosinate amide having formula (IIb), N-carbamoyl amino acid having formula (IIIa), and L-glufosinate and / or salt thereof.

[0092] Furthermore, the composition is formula (IIa): [ka] N-carbamoyl glufosinate amide having formula (IIa), N-carbamoyl glufosinate amide having formula (IIb), N-carbamoyl amino acid having formula (IIIa), and L-glufosinate and / or salt thereof may be included in an amount preferably 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, even more preferably up to 2.5% by weight, and especially up to 1% by weight, based on the total amount of N-carbamoyl glufosinate amide having formula (IIa), N-carbamoyl glufosinate amide having formula (IIb), N-carbamoyl amino acid having formula (IIIa), and L-glufosinate and / or salt thereof. Furthermore, the composition may include formula (IIa): [ka] N-carbamoyl glufosinate amide having formula (IIa), N-carbamoyl glufosinate amide having formula (IIb), N-carbamoyl amino acid having formula (IIIa), and L-glufosinate and / or salt thereof may be included 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, even more preferably 0.1 to 2.5% by weight, and particularly preferably 0.5 to 1% by weight, based on the total amount of N-carbamoyl glufosinate amide having formula (IIa), N-carbamoyl glufosinate amide having formula (IIb), N-carbamoyl amino acid having formula (IIIa), and L-glufosinate and / or salt thereof.

[0093] In preferred embodiments of the present invention, R in formulas (IIIa) and (IIb) is H or C1-C6 alkyl, preferably H or C2-C4 alkyl, more preferably ethyl or butyl, most preferably ethyl. In connection therewith, it should be understood that when formulas (IIIb) and (IIa) are present, R in the formula is also preferably H or C1-C6 alkyl, preferably H or C2-C4 alkyl, more preferably ethyl or butyl, most preferably ethyl.

[0094] In a preferred embodiment of the present invention, the compositions described herein can be used as herbicides as is, or as components of formulated herbicides.

[0095] The compositions described herein are useful for application to crop fields to prevent or control weeds. The compositions can be formulated as liquids for application to fields. Glufosinate, preferably L-glufosinate, is contained in an effective amount in the composition. As used herein, an effective amount means about 10 grams to about 1,500 grams of the active ingredient per hectare, for example, about 50 grams to about 400 grams or about 100 grams to about 350 grams. In some embodiments, the active ingredient is L-glufosinate. For example, the amount of L-glufosinate in the composition can be such that the amount of L-glufosinate per hectare is approximately 10 grams, 50 grams, 100 grams, 150 grams, 200 grams, 250 grams, 300 grams, 350 grams, 400 grams, 500 grams, 550 grams, 600 grams, 650 grams, 700 grams, 750 grams, 800 grams, 850 grams, 900 grams, 950 grams, 1,000 grams, 1,050 grams, 1,100 grams, 1,150 grams, 1,200 grams, 1,250 grams, 1,300 grams, 1,350 grams, 1,400 grams, 1,450 grams, or 1,500 grams.

[0096] The herbicide compositions described herein (including concentrates that require dilution before application to plants) contain L-glufosinate (i.e., the active ingredient) and optionally, a certain amount of residual herbicide of formula (IIb): [ka] N-carbamoyl glufosinate amide and / or formula (IIIa): [ka] It contains an N-carbamoyl amino acid having the same properties as one or more auxiliary components in liquid or solid form.

[0097] The composition is prepared by mixing the active ingredient with one or more auxiliary agents, such as diluents, fillers, carriers, surfactants, organic solvents, wetting agents, or conditioning agents, to obtain a composition in the form of fine solid particles, pellets, solutions, dispersions, or emulsions. Therefore, the active ingredient can be used with auxiliary agents such as fine solids, organic liquids, water, wetting agents, dispersants, emulsifiers, or any suitable combination thereof. From an economic and convenient standpoint, water is the preferred diluent. However, not all compounds are hydrolyzable, and in some cases, as will be understood by those skilled in the art, the use of non-aqueous solvent media may be necessary.

[0098] Optionally, one or more additional components may be added to the composition to produce a formulated herbicide composition. Such a formulated composition may contain L-glufosinate, a carrier (e.g., a diluent and / or solvent) and other components. The formulated composition contains an effective amount of L-glufosinate.

[0099] Diluents may also be included in the formulated composition. Suitable diluents include water and other aqueous components. Optionally, the diluent may be present in an amount necessary to produce the packaged or ready-to-use composition.

[0100] The herbicide compositions described herein, particularly liquids and soluble powders, may contain, as further auxiliary components, one or more surfactants in sufficient quantities to facilitate the dispersion of a given composition in water or oil. The incorporation of surfactants into a composition significantly enhances its efficacy. Surfactants used herein include wetting agents, dispersants, suspending agents, and emulsifiers. Anionic, cationic, and nonionic agents with similar functions may also be used.

[0101] Suitable wetting agents include alkylbenzenes 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 sulfonates, sulfonated vegetable oils, disterminate acetylene glycols, polyoxyethylene derivatives of alkylphenols (especially isooctylphenol and nonylphenol), and polyoxyethylene derivatives of monovalent higher fatty acid esters of hexitol anhydride (e.g., sorbitan). Exemplary dispersants include methylcellulose, polyvinyl alcohol, sodium ligninsulfonate, alkylnaphthalene sulfonate polymers, sodium naphthalene sulfonate, polymethylenebisnaphthalene sulfonate, and sodium N-methyl-N-(long-chain acid)laurate.

[0102] A water-dispersible powder composition can be prepared to contain one or more active ingredients, an inert solid extender, and one or more wetting and dispersing agents. The inert solid extender is usually of mineral origin, such as synthetic minerals derived from natural clays, diatomaceous earth, and silica. Examples of such extenders include kaolinite, attapulgite clay, and synthetic magnesium silicate. The water-dispersible powders described herein may optionally contain about 5 to about 95 parts by weight of an active ingredient (e.g., about 15 to 30 parts by weight of the active ingredient), about 0.25 to 25 parts by weight of a wetting agent, about 0.25 to 25 parts by weight of a dispersing agent, and about 4.5 to about 94.5 parts by weight of an inert solid extender, all parts being by the total weight of the composition. If necessary, about 0.1 to 2.0 parts by weight of the inert solid extender may be replaced with a corrosion inhibitor, an antifoaming agent, or both.

[0103] Aqueous suspensions can be prepared by dissolving an active ingredient that is insoluble in water, or by grinding an aqueous slurry in the presence of a dispersant, thereby obtaining a concentrated slurry with very fine particles. The resulting concentrated aqueous suspension is characterized by its extremely fine particle size, and therefore, when diluted and sprayed, it spreads very uniformly.

[0104] Emulsifiable oil is typically a liquid preparation in which an active ingredient is dissolved together with a surfactant in a solvent that is immiscible or partially immiscible with water. Suitable solvents for the active ingredients described herein include hydrocarbons and water-immiscible ethers, esters, or ketones. An emulsifiable oil composition typically contains about 5 to 95 parts of the active ingredient, about 1 to 50 parts of the surfactant, and about 4 to 94 parts of the solvent, all parts being based on the total weight of the emulsifiable oil.

[0105] The compositions described herein may also include other additives, such as fertilizers, plant toxic substances, and plant growth regulators used as auxiliary agents, or as a combination with any of the auxiliary agents described above. The compositions described herein may also be mixed with other materials, such as fertilizers, other plant toxic substances, etc., and applied in a single application.

[0106] The concentrations of the active ingredient are the same in each of the formulation types described herein, for example, liquid and solid formulations.

[0107] It is recognized that this herbicide composition can be used in combination with other herbicides. The herbicide composition of the present invention is often applied in combination with one or more other herbicides to control a wider range of undesirable vegetation. When used in combination with other herbicides, the compounds claimed herein can be formulated together with one or more other herbicides, tank-mixed with one or more other herbicides, or applied sequentially with one or more other herbicides. Some of the herbicides that can be used in combination with the compounds of the present invention include: amide herbicides, e.g., aridochlor, beflubutamide, benzadox, benzipram, bromobutide, cafenstrole, CDEA, chlorthiamide, ciprazole, dimethenamide, dimethenamide P, diphenamide, epronaz, etonipromide, fentrazamide, flupoxam, homesaphen, halosaphen, isocal Bamid, isoxaben, napropamide, naptalam, petoxamide, propizamide, quinonamide and tebutam; anilide herbicides, such as chloranocryl, cisanilide, clomeprop, cypromid, diflufenican, etobenzanide, fenashuram, flufenacet, flufenican, mefenacet, mefluidide, metamihop, monalid, naproanilide, pentanocryl Rol, picolinafen and propanil; arylalanine herbicides, e.g., benzoylprop, flancrop and flancrop M; chloroacetanilide herbicides, e.g., acetochlor, alachlor, butachlor, butenachlor, delaclor, dietatil, dimethachlor, metazachlor, metrachlor, S-methachlor, pretilachlor, propachlor, propisochlor, prinachlor, terbuchlor, tenylchlor and xylaclor; sulfonanilide herbicides, e.g., benzofluor, perfluidone, pyrimisulfan and profluazole; sulfonamide herbicides, e.g., ashram, carvasram, phenashram and oryzarin; antibiotic herbicides, e.g., viranaphos; benzoic acid herbicides, e.g., chloramben, dicamba, 2,3,6-TBA and tricamba;Pyrimidinyl oxybenzoic acid herbicides, e.g., bispyribac and pyriminobac; pyrimidinyl thiobenzoic acid herbicides, e.g., pyrithiobac; phthalate herbicides, e.g., chlortal; picolinic acid herbicides, e.g., aminopyralide, clopyralide and picloram; quinoline carboxylic acid herbicides, e.g., quinchlorac and kinmelac; arsenic herbicides, e.g., cacodylic acid, CMA, DSMA, hexaflurate, MAA, MAMA, MSMA, potassium arsenite and sodium arsenite; ben Zoylcyclohexanedione herbicides, e.g., mesotrione, sulcotrione, tefuryltrione, and tembotrione; benzofuranylalkyl sulfonic acid herbicides, e.g., benfresate and etofmesate; carbamate herbicides, e.g., ashuram, carboxazole, chlorprocarb, dichlormate, fenashuram, carbtylate, and terbucarb; carbanylate herbicides, e.g., barban, BCPC, carbaslam, carbetamide, CEPC, chlorbufame, chlorprofam, CPPC, desmedi Fam, phenisophan, fenmedifam, fenmedifam ethyl, profam and suep; cyclohexene oxime herbicides, e.g., alloxidim, butroxidim, cretodym, cloproxidim, cycloxidim, profoxidim, cethoxidim, tepraloxidim and tralcoxidim; cyclopropyl isoxazole herbicides, e.g., isoxachlortol and isoxaflutol; dicarboxyimide herbicides, e.g., benzfenzizone, synidone ethyl, flumezine, flumezine, flumezine, flumezine Oxazine and flumipropine; dinitroaniline herbicides, e.g., benfluralin, butruarin, dinitramine, ethalfluralin, fluroralin, isoproparin, metalproparin, nitralin, oryzarin, pendimethalin, prodiamine, profluralin and trifluralin; dinitrophenol herbicides, e.g., dinophenate, dinoprop, dinosamu, dinoseb, dinoterb, DNOC, ethinofen and medinoterb; diphenyl ether herbicides, e.g., ethoxyfen;Nitrophenyl ether herbicides, e.g., asifluorfen, acronifen, bifenox, clomethoxyfen, chlornitrofen, etonipromide, flulodifen, fluoroglycofen, fluoronitrofen, homesafen, flyloxyfen, halosaphene, lactofen, nitrofen, nitrofluorfen and oxyflufen; dithiocarbamate herbicides, e.g., dazomet and metam; halogenated aliphatic herbicides, e.g., allorac chloropone, darapone, flupropanate, hexachloroacetone, iodomethane, methyl bromide, monochloroacetic acid, SMA and TCA; imidazolinone herbicides, e.g., imazametabenz, imazamox, imazapick, imazapyr, imazakine and imazetapyr; inorganic herbicides, e.g., ammonium sulfamate, borax, calcium chlorate, copper sulfate, iron sulfate, potassium azide, potassium cyanate, sodium azide, sodium chlorate and sulfuric acid; nitrile herbicides Herbicides, such as bromobonyl, bromoxynil, chloroxynil, diclobenil, iodobonyl, ioxynil, and pyraclonil; organophosphate herbicides, such as amiprofos-methyl, anirofos, benslid, biranafos, butamiphos, 2,4-DEP, DMPA, EBEP, fosamine, glyphosate, and piperofos; phenoxy herbicides, such as bromophenoxime, clomeprop, 2,4-DEB, 2,4-DEP, diphenopenten, disul, ervon, etonipro Mido, fentelacol and trihopsim; phenoxyacetic acid herbicides, e.g., 4-CPA, 2,4-D, 3,4-DA, MCPA, MCPA thioethyl and 2,4,5-T; phenoxybutyric acid herbicides, e.g., 4-CPB, 2,4-DB, 3,4-DB, MCPB and 2,4,5-TB; phenoxypropionic acid herbicides, e.g., cloprop, 4-CPP, dichlorprop, dichlorprop-P, 3,4-DP, phenoprop, mecoprop and mecoprop-P;Aryloxyphenoxypropionic acid herbicides, e.g., chlorajifop, clodinahop, clohop, cyhalofop, diclohop, phenoxaprop, phenoxaprop-P, fentiaprop, fluazihop, fluazihop-P, haloxyhop, haloxyhop-P, isoxapyrifop, metamihop, propaxifop, quizalohop, quizalohop-P, and triphop; phenylenediamine herbicides, e.g., dinitramine and prodiamine; pyrazolyl herbicides, e.g., benzofenap, pyrazolinate, pyrazolyl Sulfotol, pyrazoxyfen, pyroxasulfone, and topramazeon; pyrazolylphenyl herbicides, e.g., fluazolate and pyraflufen; pyridazine herbicides, e.g., credazine, pyridafor, and pyridate; pyridazinon herbicides, e.g., brompyrazone, chloridazone, dimidazone, flufenpyr, metoflurazone, norflurazone, oxapirazone, and pidanone; pyridine herbicides, e.g., aminopyralide, cliodinate, clopyralide, dithiopyr, fluroxypyr, haloxidine, picro Lam, picolinafen, pyriclor, thiazopyr, and triclopyr; pyrimidinediamine herbicides, e.g., iprimidum and thiochlorim; quaternary ammonium herbicides, e.g., cypercoat, dietumcoat, diphenzocoat, diquat, morphamcoat, and paraquat; thiocarbamate herbicides, e.g., butyrate, cycloate, dialate, EPTC, esprocarb, etiolate, isopolinate, methibencarb, molinate, olbencarb, pevlate, prosulfocarb, pyributical β, sulfate, thiobencarb, thiocarbasil, triate and vernalate; thiocarbonate herbicides, e.g., dimexano, EXD and proxan; thiourea herbicides, e.g., methionone; triazine herbicides, e.g., dipropetrin, triaziflame and trihydroxytriazine; chlorotriazine herbicides, e.g., atrazine, chlorazine, cyanazine, siprazine, eglinadin, ipazine, mesoprazine, procyazin, proglinadin, propazine, sebutyrazine, simazine, terbutyrazine and trietazine;Methoxytriazine herbicides, e.g., atlaton, metmeton, prometon, sebumetone, simeton and terbumeton; methylthiotriazine herbicides, e.g., ametrin, adiprothrin, cyanatrin, desmethrin, dimethametrine, metoprothrin, prometon, simetrine and terbutrin; triazinon herbicides, e.g., ametridione, amivudine, hexazinone, isomethionine, metamitron and metrivudine; triazole herbicides, e.g., amitrol, cafenstrol, epronaz and flupoxam; Ryazolon herbicides, e.g., amicarbazone, bencarbazone, carfentrazone, flucarbazone, propoxycarbazone, sulfentrazone and thiencarbazone methyl; triazolopyrimidine herbicides, chloransram, diclosram, florasram, flumetulam, metosram, penoxsram and piroxsram; uracil herbicides, e.g., butafenacil, bromacil, flupropacil, isocyl, renacil and tarbacil; 3-phenyluracils; urea herbicides, e.g., benzthiazulon, cumylon, cyclon, di Chloralurea, diflufenzopyr, isonorolone, isouron, metabenzuthiazulon, monisouron and norolone; phenylurea herbicides, e.g., anislon, buturon, chlorbromuron, chloretulon, chlorotoluron, chloroxuron, dimuron, diphenokithron, dimeflon, diuron, fenuron, fluomethuron, fluothiron, isoproturon, linuron, methiron, methyldimuron, metobenzuron, metobromuron, metoxuron, monolinuron, monuron, nebulon, parafluron, phenobenzuron n, siduron, tetraflurone and tidiazuron; pyrimidinyl sulfonylurea herbicides, such as amidesulfuron, azimsulfuron, bensulfuron, chlorimuron, cyclosulfamuron, ethoxysulfuron, flazasulfuron, flucetosulfuron, flupyrsulfuron, horamsulfuron, halosulfuron, imazosulfuron, mesosulfuron, nicosulfuron, orthosulfamuron, oxasulfuron, primisulfuron, pyrazosulfuron, limsulfuron, sulfometsuron, sulfosulfuron and trifloxysulfuron;Triazinyl sulfonylurea herbicides, e.g., chlorsulfuron, cinosulfuron, etamethosulfuron, iodosulfuron, metosulfuron, prosulfuron, thifensulfuron, triasulfuron, trivenuron, triflusulfuron and tritosulfuron; thiadiazolylurea herbicides, e.g., butiuron, ethidimylumon, tebutiuron, thiazaflurone and tidiazuron; and unclassified herbicides, e.g., acrolein, allyl alcohol, aminocyclopyrachlor, azaphenidine, benazoline, bentazone, benzobicyclon, butidazole, calcium cyanamide, cambendichlor, chlorfenac, chlorfenprop, chlorflurazole, chlorflurenol, sinmethilin, chromazon, CPMF, cresol, ortho-dichlorobenzene, dimepiperate, endota; Examples include fluoromidine, flulidone, flurochloridone, flurthamone, fluthiaset, indanophan, metasol, methyl isothiocyanate, nipiraclofen, OCH, oxaziargyl, oxadiazone, oxadiclomefone, pentachlorophenol, pentoxazone, phenylmercury acetate, pinoxadene, prosulfarin, pyribenzoxime, pyrifthalide, quinoclamin, rhodetanyl, sulglycapine, tidiadimine, tridiphan, trimethulone, tripropindan, and tritac. Furthermore, the herbicide compositions of the present invention can be used in combination with glyphosate or 2,4-D against glyphosate-resistant or 2,4-D-resistant crops. Generally, it is preferable to use the compositions of the present invention in combination with herbicides that are selective to the crop being treated and complement the weed spectrum controlled at the application rates in which such compositions are employed. In general, the compositions of the present invention and other complementary herbicides are more preferably applied simultaneously, either as a combination formulation or as a tank mix.

[0108] As described above, in a third embodiment, the present invention provides a method for selectively controlling weeds in a predetermined area, preferably an area containing a crop of sown seeds or a glufosinate-resistant crop: A composition comprising L-glufosinate and / or a salt thereof, wherein the enantiomer ratio to D-glufosinate and / or a salt thereof is at least 50%, preferably with an enantiomer excess of more than 70%, and formula (II): [ka] An N-carbamoylaminoamide having (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, most preferably ethyl) is added to the composition in an amount of more than 0.01% by weight and less than 10% by weight, based on the total amount of the composition. and / or Formula (III): [ka] The present invention relates to a method comprising applying to an area in an effective amount a composition containing an N-carbamoyl amino acid having (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, most preferably ethyl) in an amount of more than 0.01% to less than 10% by weight based on the total amount of the composition.

[0109] In a preferred embodiment of the present invention, the composition comprises L-glufosinate and / or a salt thereof 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 a salt thereof.

[0110] In a preferred embodiment of the present invention, the composition is formula (III): [ka] The composition contains an N-carbamoyl amino acid having (wherein R is H or C1-C8 alkyl, preferably H or C1-C6 alkyl, more preferably H or C2-C4 alkyl, most preferably H) in an amount of 0.02-8% by weight, preferably 0.03-5% by weight, more preferably 0.05-3% by weight, and particularly 0.1-2% by weight, based on the total amount of the composition.

[0111] It should be understood that the composition may include the same auxiliary agents and / or other herbicides as described in more detail above.

[0112] The compositions described herein are useful for application to crop fields to prevent or control weeds. The compositions can be formulated as liquids for application to fields. L-glufosinate is contained in an effective amount in the compositions. As used herein, an effective amount means about 10 grams to about 1,500 grams of the active ingredient per hectare, for example, about 50 grams to about 400 grams or about 100 grams to about 350 grams. In some embodiments, the active ingredient is L-glufosinate. For example, the amount of L-glufosinate in the composition can be such that the amount of L-glufosinate per hectare is approximately 10 grams, 50 grams, 100 grams, 150 grams, 200 grams, 250 grams, 300 grams, 350 grams, 400 grams, 500 grams, 550 grams, 600 grams, 650 grams, 700 grams, 750 grams, 800 grams, 850 grams, 900 grams, 950 grams, 1,000 grams, 1,050 grams, 1,100 grams, 1,150 grams, 1,200 grams, 1,250 grams, 1,300 grams, 1,350 grams, 1,400 grams, 1,450 grams, or 1,500 grams.

[0113] The present invention will be further explained by the following examples. [Examples]

[0114] Enzyme preparation a) Cloning of enzyme genes (Example 1) The amino acid sequences of each enzyme were identified from publicly available databases (UniProt, https: / / www.uniprot.org; NCBI Protein Database, https: / / www.ncbi.nlm.nih.gov / protein. Sequences from NCBI are indicated with an asterisk (*) preceding the respective database identifier). From these, the DNA sequences were derived using the standard codon usage frequencies of Escherichia coli. The DNA sequences were synthesized (BioCat GmbH) and cloned into plasmid pDHE19.2 (Ress-Loeschke, M. et al., German Patent No. 19848129, 1998 (BASF AG)). Competent cells of E. coli strain TG10, pAgro, pHSG575 (Chung, CT et al., Proc Natl Acad Sci USA, 1989, 86, 2172) were transformed using the obtained plasmid. This Escherichia coli (E. coli) strain TG10, pAgro, pHSG575 is an rhaA-deficient derivative of Escherichia coli (E. coli) TG1, transformed with pHSG575 (Takeshita, S. et al., Gene, 1987, 61, 63) and pAgro4 (pBB541 in Tomoyasu, T. et al., Mol. Microbiol., 2001, 40, 397) (E. coli (E. coli) TG10 (Kesseler, M. et al., International Publication No. 2004050877A1, 2004 (BASF AG))).

[0115] b) Recombinant production of the enzyme (Example 2) Preparation of biocatalysts in a shaking flask Using Escherichia coli (E. coli) TG10 containing the recombinant plasmid of the enzyme, 2 ml of LB medium (Bertani, G., J Bacteriol, 1951, 62, 293) supplemented with 100 μg / ml ampicillin, 100 μg / ml spectinomycin, and 20 μg / ml chloramphenicol was inoculated. The resulting pre-culture was incubated at 37°C for 5 hours with shaking at 250 rpm. Using 1 ml of this pre-culture, 100 ml of LB medium supplemented with 100 μg / ml ampicillin, 100 μg / ml spectinomycin, 20 μg / ml chloramphenicol, 1 mM MnCl2, 0.1 mM isopropyl-β-D-thiogalactopyranoside, and 0.5 g / l rhamnose was inoculated in a 500 ml baffled Erlenmeyer flask. The culture was incubated at 37°C for 18 hours under shaking conditions. Next, the biomass was recovered by centrifugation at 3220×g for 10 minutes at 8°C. The supernatant was discarded, and the cell pellet was suspended in 8 ml of 100 mM HEPES buffer at pH 8.2 with 1 mM MnCl2 added. When performing the bioconversion reaction using whole cells, this cell suspension was used for synthesis without further processing. Alternatively, when using cell lysates from which residue had been removed, 5 ml of the cell suspension was divided into 5 reaction tubes containing lysing matrix B (0.7 ml of Φ0.1 mm quartz beads, MP Biomedicals). After cooling the tubes on ice, the cells were lysed using a homogenizer (Peqlab Precellys24, VWR) for two 30-second cycles. The sample was cooled on ice between cycles. The resulting cell-free lysates were centrifuged at 20817×g for 10 minutes at 8°C to remove residue. The supernatant was isolated, and the fractions from the same batch were combined (= cell lysates with residue removed).

[0116] Production of whole-cell biocatalysts by fermentation Escherichia coli (E. coli) TG10 containing plasmids pAgro4 and pHSG575 was transformed with the pDHE plasmid encoding the target protein. The transformants were cultured on LB agar plates supplemented with ampicillin 100 μg / ml, spectinomycin 100 μg / ml, and chloramphenicol 20 μg / ml.

[0117] Pre-culture medium: EcoK12 solution Ultrapure water 1.0kg Citric acid monohydrate 40.0g Zinc sulfate heptahydrate 11.0g Diammonium iron sulfate hexahydrate 8.6g Manganese sulfate monohydrate 3.0g Copper sulfate pentahydrate 0.8g Cobalt sulfate heptahydrate 0.09g Sterilization is performed by filtration using a filter with a pore size of 0.2 μm. Ingredients (part) 1 Ultrapure water 1.0kg Citric acid monohydrate 3.4g Magnesium sulfate heptahydrate 2.4g Calcium chloride dihydrate 0.1g EcoK12 solution 20g 25% sodium hydroxide solution (used to adjust pH to 6.6) Ingredient 2 Ultra pure water 500g Potassium dihydrogen phosphate 26.6g Diammonium hydrogen phosphate 8.0g 25% sodium hydroxide solution (used to adjust pH to 6.4) Ingredient 3 Ultra pure water 500g 99% Glycerol 36.0g Sodium gluconate 24.0g 20% phosphoric acid (used to adjust pH to 6.6) All three components were sterilized at 121°C for 30 minutes. Vitamin solution Ultra pure water 100g Thiamine hydrochloride 1.0g Vitamin B12 0.5g

[0118] Sterilization by filtration using a filter with a pore size of 0.2 μm. To prepare the final pre-culture medium, components 1, 2, and 3 were combined, and 2.0 ml of vitamin solution was added. Furthermore, 100 μg / ml of ampicillin, 100 μg / ml of spectinomycin, and 20 μg / ml of chloramphenicol were added to this medium. Several transformants were scraped from the LB agar plate and inoculated into two 1 L baffled Erlenmeyer flasks containing 100 g of pre-culture medium. These pre-cultures were incubated at 37°C and 150 rpm. When the OD600 reached 12, the entire volume of these pre-cultures was inoculated into the main culture.

[0119] This culture medium: Ingredient 4 Ultrapure water 9.6kg Citric acid monohydrate 21.1g Potassium dihydrogen phosphate 173.6g Diammonium hydrogen phosphate 52.8g Magnesium sulfate heptahydrate 15.1g Calcium chloride dihydrate 0.7g EcoK12 solution 123g Adjust the pH of the 25% sodium hydroxide solution to 6.4. Pluriol P 2000 1ml Component 4 was sterilized at 125°C for 45 minutes. Ingredient 5 Ultra pure water 300g Thiamine hydrochloride 151 mg Vitamin B12 30.2mg Ampicillin sodium salt 1000 mg Spectinomycin hydrochloride 500mg Chloramphenicol 200mg Component 5 was sterilized by sterile filtration using a filter unit with a pore size of 0.1 μm. Glycerol solution Ultra pure water 804g Citric acid monohydrate 29.1g Sodium sulfate 58.1g 4.5g of diammonium iron sulfate hexahydrate 99% Glycerol 3370g Thiamine solution Ultra pure water 40g Thiamine hydrochloride 55mg Antifoaming agent solution Pluriol P 2000 350g Base solution 25% ammonia solution, 1500ml Inducing solution Ultra pure water 150g Rhamnose monohydrate 100g IPTG 238mg

[0120] The glycerol and antifoaming agent solution was sterilized at 121°C for 30 minutes. The thiamine and derivative source solution were sterilized by sterile filtration using a 0.2 μm pore size filter.

[0121] Components 4 and 5 were combined in a sterile fermenter (Techfors, Infors HT), and the preculture was inoculated. Throughout the fermentation period, the fermenter temperature was maintained at 37°C, the pressure at 0.2 bar, and the pH at 6.6 by adding a base solution. The pO2 level was maintained at 20-40% by adjusting the stirrer speed (usually 500 rpm) and aeration rate (usually 6 L / min). An antifoaming agent solution was added as needed. A combination of glycerol solution and thiamine solution was used as the feed solution. After inoculation, the feed solution was added at a rate of 10 g / h. After 7 hours, the feed solution was switched to "stop and see" mode, and once the pO2 level rose, administration was resumed at a rate of 10 g / h. After 14 hours or after 330 g of feed solution had been consumed, the feed rate was increased to 80-100 g / h. Gene expression was induced by adding the induction source solution when the oxygen transport rate reached 80 mmol / L / h or the OD600 reached 12. After 36 hours of induction, fermentation was stopped by lowering the temperature to 15°C. The cooled fermentation broth was discharged from the fermenter, and the cells were pelleted by centrifugation at 4700 rpm and 10°C. The resulting supernatant was discarded, and the cells were suspended in 3850 g of 50 mM potassium dihydrogen phosphate buffer at pH 7.0. The cell suspension was frozen at -80°C and then lyophilized. For this procedure, the lyophilizer was maintained at -50°C and a pressure of 0.25 mbar. The lyophilized cells were stored at 4°C.

[0122] Production of freeze-dried cell-free extracts Lyophilized cells were resuspended in ultrapure water at 100 g / L. After cooling the cell suspension on ice, the cells were disrupted by passing them three times through a high-pressure homogenizer (Panda Plus 2000, GEA) set to 800 bar. The pressure during the three passes was typically 1000-1400 bar. The resulting mixture was centrifuged at 10000 rpm for 15 minutes at 10°C to remove residue. 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 under a pressure of 0.25 mbar.

[0123] Preparation of starting materials and intermediate products c) Synthesis of n-butyl (3-cyano-3-hydroxypropyl)methylphosphinate (ACM-H) (Example 3) ACM-H was prepared according to Example 2 of International Publication No. 2015 / 173146A1.

[0124] d) Preparation of N-carbamoyl glufosinate amide from ACM-H (Example 4) [ka] 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. A solution of 15 g (68.4 mmol, "ACM-H") of n-butyl (3-cyano-3-hydroxypropyl)methylphosphinate, prepared according to Example 2, in 15 mL of water was added to the reaction mixture over 2 hours. The reaction mixture was allowed to cool to room temperature and stirred at room temperature for a further 3 days. The reaction mixture was concentrated under vacuum. The crude product was purified by column chromatography using a methanol gradient in dichloromethane (methanol / dichloromethane 1:9 → 2:1) to obtain the product (10%). 1H NMR(500MHz,DMSO-d6)δ 7.46(s,1H),7.08(s,1H),6.20(dd,J=8.2,3.7Hz,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=7.4Hz,3H).LC-MS(actual value):280.2

[0125] e) Chemical synthesis of N-carbamoyl amino acids from glufosinate (Example 5) [ka] A racemic solution of glufosinate ammonium (50% in water, 39.6 g, 99.9 mmol) was added under reduced pressure (200 mbar) while stirring, using 30 ml of aqueous solution of potassium cyanate (11.8 g, 145 mmol) at 50°C for 30 minutes. The reaction mixture was stirred for a further 1 hour under reduced pressure (200 mbar) at 50°C, and then allowed to cool 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 obtain the carbamoylic acid product (7.9 g). The residual carbamoylic acid was isolated again 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). Starting from commercially available D- and L-glufosinates, D- and L-enantiomers were synthesized by the same method. Specific rotation [α] of the L-enantiomer = +27.5 (measured as c=1, H2O, potassium salt). Retention times by HPLC-MS using Supelco Chirobiotic T2 (eluate: 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).

[0126] Preparation of L-glufosinate p-butyl ester f) Preparation of N-carbamoyl glufosinate acid from N-carbamoyl glufosinate amide (Example 6 of the Invention) [ka] 500 μL of a 10 mg / mL N-carbamoyl glufosinate amide solution in phosphate buffer (potassium dihydrogen phosphate, pH 8.0, 50 mM) was mixed with 5 mg of enzyme (papain in this case: CAS 9001-73-4, powder). The resulting solution was shaken at room temperature for 2 days. Subsequently, the reaction mixture was analyzed by chiral HPLC-MS, showing that 14% was converted to the corresponding L-carbamoyl amino acid (enantiomer ratio L:D, >99:1). The concentration of the N-carbamoyl amino acid was determined by chiral HPLC-MS using Supelco Chirobiotic T2 (gradient from 90% ACN / water to 60% ACN / water, 19 minutes, 0.1% formic acid). Temperature: 20°C, flow rate: 0.8 mL / min. Retention times of N-carbamoyl amino acid: L configuration diastereoisomer (8.6 min); D configuration (10.7 and 11.2 min).

[0127] Of the other enzymes tested, those showing a conversion rate greater than 0.1% are as follows: - Bromelain (CAS 37189-34-7) - Bacterial proteinase (proteinase derived from Bacillus licheniformis, CAS 9014-01-1)

[0128] g) Chemical synthesis of glufosinate from N-carbamoyl amino acids (Example 7 of the invention) [ka] 4-[butoxy(methyl)phosphoryl]-2-ureidobutanoic acid (100 mg), synthesized using amidase (papain, CAS 9001-73-4, see Invention Example 5), was dissolved in aqueous hydrochloric acid (3.5 M, 10 mL), and the reaction mixture was stirred and cooled to 0°C. A solution of sodium nitrite (26 mg) in water (2 mL) was added to the reaction mixture, and the reaction mixture was allowed to stand and warm to room temperature. The reaction mixture was stirred at room temperature for a further 2 hours. Concentrated hydrochloric acid (36%, 7.5 mL) was 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 obtain glufosinate hydrochloride. 1H 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).

[0129] Reaction to obtain glufosinate using an enzymatic carbamoyl cleavage step. h) Enzymatic two-pot synthesis of glufosinate from N-carbamoyl amino acids (Invention Example 8, SEQ ID NO: 1) [ka] 2-(carbamoylamino)-4-[hydroxy(methyl)phosphoryl]butanoic acid (0.6 g, 2.5 mmol, prepared according to Invention Example 5) was dissolved in degassed aqueous potassium phosphate buffer (5.4 mL, 0.496 M, pH 8.0), and KOH (3 M, in water) was added to adjust the pH to 8.0. To the reaction mixture (6.1 ml), potassium phosphate buffer (19.2 mL, 0.100 M, pH 8.0), N-carbamoyl amino acid hydrolase (A0A1Y4GC62, SEQ ID NO: 4, cell lysate with residue removed, 1.5 mL, total protein concentration 12.9 mg / mL, protein produced in a shaking flask), and MnCl2 solution (1 M, 20 μL in water) were added. The reaction mixture was stirred at 37°C for 24 hours (250 rpm). NMR and HPLC analysis showed that the conversion rate of glufosinate was 31%. The enantiomer ratio of glufosinate was analyzed by chiral HPLC. Chiral HPLC: L-glufosinate > 99% / D-glufosinate < 1%; Analytical method: Chirex(D)-Pencillamine 250 × 4.6 mm column from Phenomenex; Isocratic elution, copper(II) sulfate 10 mM, UV detection 245 nm).

[0130] i) One-pot synthesis of glufosinate p-butyl esters 5 mg of N-carbamoyl glufosinate amide 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 hours. After this time, 1.5 μL of MnCl2 solution (in water, 2 M) was added, followed by 10 mg of N-carbamoyl amino acid hydrolase (lyophilized cell-free extract, A0A535Y1H2, SEQ ID NO: 2). (a) Sustine® 220 from Novozymes was used as the liquid enzyme preparation. The reaction product (p-butyl ester of glufosinate) was obtained in yield >0.01% and detected by LC / MS. (b) A protease derived from Bacillus licheniformis (CAS 9014-01-1, aqueous solution, 94 mg / mL protein) was used as the liquid enzyme preparation. The reaction product (p-butyl ester of glufosinate) was obtained in yield >0.01% and detected by LC / MS.

[0131] Analytical method: P-butyl ester of glufosinate was analyzed by LC / MS using a Kinetex C18 100×2.1 mm column. Isocratic elution, 40°C, flow rate 0.5 mL / min, 80% water / 20% acetonitrile + 0.1% formic acid, retention time 1.8 min.

[0132] Sequence ID: 1 (A0A3E0C996, derived from Paraburkholderia sp. BL6669N2) [ka] Sequence ID: 2 (A0A535Y1H2, derived from Chloroflexi bacteria) [ka] Sequence ID: 3 (A0A6P2ISL4, derived from Burkholderia lata DSM 23089) [ka] Sequence ID: 4 (A0A1Y4GC62, derived from Cloacibacillus sp. An23) [ka]

Claims

1. A method for preparing glufosinate and / or a salt thereof or a glufosinate alkyl ester and / or a salt thereof, wherein the glufosinate or the glufosinate alkyl ester is of formula (I): 【Chemistry 1】 (In the formula, 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 Having a molecular structure according to alkyl, more preferably ethyl or butyl, most preferably ethyl, The aforementioned method is: a) Equation (II): 【Chemistry 2】 (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 alkyl, still more preferably ethyl or butyl, most preferably ethyl) having N-carbamoyl glufosinate amide, Preferably, hydrolysis is performed with an amidase enzyme selected from the group consisting of peptidases, proteases, linear amide-acting amidases, or cyclic amide-acting amidases, resulting in formula (III): 【Transformation 3】 (In the formula, 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 A step of producing an N-carbamoyl amino acid having an alkyl group, more preferably ethyl or butyl group, most preferably ethyl group; b) A step of cleaving the carbamoyl portion of an N-carbamoyl amino acid having formula (III), Methods that include...

2. Cutting step b) gives equation (I): 【Chemistry 4】 (In the formula, 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, which yields a glufosinate and / or a salt thereof or a glufosinate alkyl ester and / or a salt thereof having an alkyl (more preferably ethyl or butyl, most preferably ethyl).

3. By step b), a glufosinate and / or a salt thereof having formula (I) or a glufosinate alkyl ester and / or a salt thereof is obtained in racemic mixture form, or formula (Ia): 【Transformation 5】 (In the formula, 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 L-glufosinate and / or its salts or glufosinate alkyl esters and / or its salts having an alkyl (more preferably ethyl or butyl, most preferably ethyl) are obtained in a form with an enantiomer excess. Preferably, the method according to claim 2, wherein an L-glufosinate and / or a salt thereof having formula (Ia) or a glufosinate alkyl ester and / or a salt thereof is obtained in an enantiomer excess, and the amidase enzyme is an L-amidase enzyme.

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 an N-carbamoyl glufosinate amide having formula (II) is converted to an L-glufosinate and / or a salt thereof having formula (Ia) or an L-glufosinate alkyl ester and / or a salt thereof, wherein formula (Ia) is as described in claim 3.

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

6. 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 method according to any one of claims 1 to 5, carried out at a temperature of 20 to 50°C, preferably 25 to 45°C, more preferably 30 to 42°C, and particularly 32 to 40°C.

7. In equations (II) and (III), R is C １ ~C ８ Alkyl, preferably C １ ~C ６ Alkyl, more preferably C ２ ~C ４ Alkyl, more preferably ethyl or butyl, most preferably ethyl, and the above method is c) The method according to any one of claims 1 to 6, further comprising the step of deprotecting under acidic conditions, preferably using hydrochloric acid or sulfuric acid.

8. The method according to any one of claims 1 to 7, further comprising adding an N-carbamoyl glufosinate amide racemase and / or an N-carbamoyl amino acid racemase that racems the N-carbamoyl glufosinate amide at an 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 all reagents are added substantially at the start of the reaction, or the reagents for step a) and step b) are added to the single container at different times.

10. The above method yields formula (IIb) obtained in hydrolysis step a): 【Transformation 6】 (In the formula, 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 any one of claims 1 to 8, further comprising the step of separating an N-carbamoyl glufosinate amide having an alkyl group, more preferably ethyl or butyl, most preferably ethyl, preferably using reverse-phase chromatography.

11. The method according to any one of claims 1 to 10, wherein the amidase enzyme is selected from the group of enzymes consisting of papain (CAS 9001-73-4) and its variants, bromelain (CAS 37189-34-7) and its variants, and proteinase (CAS 9014-01-1) derived from Bacillus licheniformis and its variants, and the variant is defined as a polypeptide sequence having at least 80%, preferably 90%, and most preferably 95% sequence homology to the respective polypeptide sequence.

12. The aforementioned N-carbamoyl amino acid hydrolase consists of 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, and Uniprot. The method according to any one of claims 5 to 11, wherein the mutant is defined as a polypeptide sequence having at least 80%, preferably 90%, and most preferably 95% sequence homology to the respective polypeptide sequence, preferably selected from the group of enzymes defined by Uniprot ID consisting of A0A3E0C996 (SEQ ID NO: 1) and its mutants, A0A535Y1H2 (SEQ ID NO: 2) and its mutants, A0A6P2ISL4 (SEQ ID NO: 3) and its mutants, and A0A1Y4GC62 (SEQ ID NO: 4), and the mutant is defined as a polypeptide sequence having at least 80%, preferably 90%, and most preferably 95% sequence homology to the respective polypeptide sequence.

13. a) Equation (II): 【Transformation 7】 (In the formula, 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 A composition comprising an N-carbamoyl glufosinate amide having an alkyl group, more preferably ethyl or butyl, most preferably ethyl.

14. Equation (IIb): 【Transformation 8】 (In the formula, 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 N-carbamoyl glufosinate amide having an alkyl group, more preferably ethyl or butyl, most preferably ethyl, Optionally, Formula (IIIa): 【Chemistry 9】 (In the formula, 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 An N-carbamoyl amino acid having an alkyl group, more preferably ethyl or butyl, most preferably ethyl, The composition according to claim 13, comprising L-glufosinate and / or a salt thereof, or L-glufosinate alkyl ester and / or a salt thereof.

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 at least 5% by weight, preferably at least 10% by weight, more preferably at least 20% by weight, even more preferably at least 30% by weight, and most preferably at least 40% by weight, based on the total weight of the N-carbamoyl glufosinate amide having formula (II), the N-carbamoyl amino acid having formula (III), and L-glufosinate and / or its salt or L-glufosinate alkyl ester and / or its salt.

16. A method for selectively controlling weeds in a designated area, preferably an area containing a crop of sown seeds or a glufosinate-resistant crop: A composition comprising L-glufosinate and / or its salt or L-glufosinate alkyl ester and / or its salt, wherein the enantiomer ratio is at least 50%, preferably the enantiomer excess is 70%, with respect to D-glufosinate and / or its salt or D-glufosinate alkyl ester and / or its salt, and formula (II): 【Chemistry 10】 (In the formula, 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 An N-carbamoylaminoamide having an alkyl group (more preferably ethyl or butyl, most preferably ethyl) is added in an amount of more than 0.01% by weight and less than 10% by weight based on the total amount of the composition. and / or Formula (III): 【Chemistry 11】 (In the formula, 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 An N-carbamoyl amino acid having an alkyl group (more preferably ethyl or butyl, most preferably ethyl) is added in an amount of more than 0.01% by weight and less than 10% by weight, based on the total amount of the composition. A method comprising the step of applying a composition containing the composition to the area in an effective amount.

Images