WO2024256370A1 - Synthèse de glufosinate à l'aide d'un processus à base d'amidase - Google Patents

Synthèse de glufosinate à l'aide d'un processus à base d'amidase Download PDF

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WO2024256370A1
WO2024256370A1 PCT/EP2024/066028 EP2024066028W WO2024256370A1 WO 2024256370 A1 WO2024256370 A1 WO 2024256370A1 EP 2024066028 W EP2024066028 W EP 2024066028W WO 2024256370 A1 WO2024256370 A1 WO 2024256370A1
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glufosinate
alkyl
salt
formula
carbamoyl
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Gunther Zimmermann
Moritz Stefan POTT
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BASF SE
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BASF SE
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Priority to CN202480039400.5A priority Critical patent/CN121335908A/zh
Priority to EP24732871.9A priority patent/EP4727951A1/fr
Priority to KR1020257041323A priority patent/KR20260026015A/ko
Priority to IL325207A priority patent/IL325207A/en
Priority to MX2025015004A priority patent/MX2025015004A/es
Publication of WO2024256370A1 publication Critical patent/WO2024256370A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/28—Phosphorus compounds with one or more P—C bonds
    • C07F9/30—Phosphinic acids [R2P(=O)(OH)]; Thiophosphinic acids ; [R2P(=X1)(X2H) (X1, X2 are each independently O, S or Se)]
    • C07F9/301—Acyclic saturated acids which can have further substituents on alkyl
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N57/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic phosphorus compounds
    • A01N57/18—Biocides, pest repellants or attractants, or plant growth regulators containing organic phosphorus compounds having phosphorus-to-carbon bonds
    • A01N57/20—Biocides, pest repellants or attractants, or plant growth regulators containing organic phosphorus compounds having phosphorus-to-carbon bonds containing acyclic or cycloaliphatic radicals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
    • A01P13/00—Herbicides; Algicides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/28—Phosphorus compounds with one or more P—C bonds
    • C07F9/30—Phosphinic acids [R2P(=O)(OH)]; Thiophosphinic acids ; [R2P(=X1)(X2H) (X1, X2 are each independently O, S or Se)]
    • C07F9/32—Esters thereof
    • C07F9/3205—Esters thereof the acid moiety containing a substituent or a structure which is considered as characteristic
    • C07F9/3211—Esters of acyclic saturated acids which can have further substituents on alkyl
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/06—Alanine; Leucine; Isoleucine; Serine; Homoserine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
    • C12P41/006—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by reactions involving C-N bonds, e.g. nitriles, amides, hydantoins, carbamates, lactames, transamination reactions, or keto group formation from racemic mixtures

Definitions

  • the present invention relates to a method of preparing glufosinate, comprising the steps of hydrolysing an N-carbamoyl glufosinate amide with an Amidase enzyme to form an N- carbamoyl amino acid compound followed by cleaving off the carbamoyl moiety of said N- carbamoyl amino acid compound.
  • the herbicide glufosinate is a non-selective, foliarly-applied herbicide considered to be one of the safest herbicides from a toxicological or environmental standpoint.
  • Current commercial chemical synthesis methods for glufosinate yield a racemic mixture of L- and D-glufosinate (Duke et al. 2010 Toxins 2:1943-1962).
  • L-glufosinate also known as phosphinothricin or (S)-2-amino-4- (hydroxy(methyl) phosphonoyl)butanoic acid
  • D-glufosinate also known as phosphinothricin or (S)-2-amino-4- (hydroxy(methyl) phosphonoyl)butanoic acid
  • the present invention therefore relates to a method of preparing glufosinate and/or a salt thereof or a glufosinate alkyl ester and/or a salt thereof having the formula (I) wherein R is H or C r C 8 alkyl, preferably H or C r C 6 alkyl, more preferably H or C 2 -C 4 alkyl, even more preferably ethyl or butyl, and most preferably ethyl, comprising the steps of: a) hydrolysing an N-carbamoyl glufosinate amide having the formula (II) wherein R is H or C r C 8 alkyl, preferably H or C r C 6 alkyl, more preferably H or C 2 -C 4 alkyl, even more preferably ethyl or butyl, and most preferably ethyl, by an Amidase enzyme to form an N- carbamoyl amino acid having the formula (III)
  • the cleaving step b) provides the glufosinate and/or a salt thereof or a glufosinate alkyl ester and/or a salt thereof having the formula (I) in form of a racemic mixture or in form of an enantiomeric excess of L-glufosinate and/or a salt thereof or a glufosinate alkyl ester and/or a salt thereof having the formula (la) wherein R is H or C r C 8 alkyl, preferably H or C r C 6 alkyl, more preferably H or C 2 -C 4 alkyl, even more preferably ethyl or butyl, and most preferably ethyl; preferably in form of an enantiomeric excess of L-glufosinate and/or a salt thereof or a glufosinate alkyl ester and/or a salt thereof having the formula (la) and the Amidase enzyme is an L
  • 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 the formula (II) is converted to L-glufosinate and/or a salt thereof or a glufosinate alkyl ester and/or a salt thereof having the formula (la), wherein formula (la) is as defined in preferred embodiment A2.
  • the cleaving step b) is performed under enzymatic conditions, preferably using an N-Carbamoyl amino acid hydrolase enzyme, more preferably an L-N-Carbamoyl amino acid hydrolase enzyme or wherein the cleaving step b) is performed under chemical conditions, preferably using sodium nitrite and/or hydrogen chloride.
  • R in formulae (II) and (III) is H or C r C 6 alkyl, preferably H or C 2 -C 4 alkyl, more preferably ethyl or butyl, and most preferably ethyl.
  • the hydrolysing step a) is performed at a pH of 6 to 11, preferably of 6.5 to 10, more preferably of 7 to 9.5 and in particular of 7.5 to 9 and/or at a temperature of 20 to 50 °C, preferably of 25 to 45 °C, more preferably of 30 to 42 °C, and in particular of 32 to 40 °C.
  • R in formulae (II) and (III) is C r C 8 alkyl, preferably C r C 6 alkyl, more preferably C 2 -C 4 alkyl, even more preferably ethyl or butyl, and most preferably ethyl, and the method further comprises the step of c) deprotecting under acidic conditions, preferably using hydrochloric acid or sulfuric acid.
  • the method further comprises the addition of an N-carbamoyl glufosinate amide Racemase enzyme and/or an N-Carbamoyl amino acid racemase enzyme.
  • step a) and step b) are performed in a single container, preferably wherein all reagents are substantially added at the start of the reaction or wherein the reagents for step a) and the reagents for step b) are added to the single container at different times.
  • the method further comprises the step of separating off an N-carbamoyl glufosinate amide having the formula (lib) (lib), wherein R is H or C r C 8 alkyl, preferably H or C r C 6 alkyl, more preferably H or C 2 -C 4 alkyl, even more preferably ethyl or butyl, and most preferably ethyl, which is obtained in hydrolysing step a), preferably using reversed phase chromatography.
  • R is H or C r C 8 alkyl, preferably H or C r C 6 alkyl, more preferably H or C 2 -C 4 alkyl, even more preferably ethyl or butyl, and most preferably ethyl, which is obtained in hydrolysing step a), preferably using reversed phase chromatography.
  • the present invention relates to a composition
  • a composition comprising an N-carbamoyl glufosinate amide having the formula (lib) wherein R is H or C r C 8 alkyl, preferably H or C r C 6 alkyl, more preferably H or C 2 -C 4 alkyl, even more preferably ethyl or butyl, and most preferably ethyl.
  • the composition consists of an N- carbamoyl glufosinate amide having the formula (lib) wherein R is H or C r C 8 alkyl, preferably H or C r C 6 alkyl, more preferably H or C 2 -C 4 alkyl, even more preferably ethyl or butyl, and most preferably ethyl.
  • the present invention relates to a method for selectively controlling weeds in an area, preferably containing a crop of planted seeds or crops that are resistant to glufosinate, comprising: applying an effective amount of a composition comprising L-glufosinate and/or a salt thereof at an enantiomeric proportion of at least 50%, preferably in an enantiomeric excess of greater than 70%, over D-glufosinate and/or a salt thereof and and more than 0.01 wt.-% to less than 10 wt.-%, based on the total amount of the composition, of an N-carbamoyl amino amide having the formula (II) wherein R is H or C r C 8 alkyl, preferably H or C r C 6 alkyl, more preferably H or C 2 -C 4 alkyl, even more preferably ethyl or butyl, and most preferably ethyl, and/or more than 0.01 wt.-% to less than
  • the compounds according to the invention may have one or more stereocenters.
  • the invention preferably encompasses all stereoisomers, i.e., pure enantiomers, pure diastereomers, of the compounds according to the invention, and their mixtures, including racemic mixtures.
  • the present invention relates in one aspect to a method of preparing a glufosinate and/or a salt thereof or a glufosinate alkyl ester and/or a salt thereof having the formula (I) wherein R is H or C C 8 alkyl, preferably H or C C 6 alkyl, more preferably H or C 2 -C 4 alkyl, even more preferably ethyl or butyl, and most preferably ethyl, comprising the steps of: a) hydrolysing an N-carbamoyl glufosinate amide having the formula (II) wherein R is H or C r C 8 alkyl, preferably H or C r C 6 alkyl, more preferably H or C 2 -C 4 alkyl, even more preferably ethyl or butyl, and most preferably ethyl, by an Amidase enzyme to form an N- carbamoyl amino acid having the formula (III) wherein
  • the N-carbamoyl glufosinate amide having the formula (II) can be obtained via any suitable method of preparing.
  • the N-carbamoyl glufosinate amide is a byproduct of the Bucherer-Bergs reaction.
  • the N-carbamoyl glufosinate amide can be produced from the related cyanhydrine.
  • Cyanhydrine or cyanhydrine derivatives can be obtained, e.g., via a process as described inter alia in US 4,521,348 B1, DE 3047024, US 4,599,207 B1 US 6,359,162 B1, CN 102372739 A, and CN 102399240 A.
  • any suitable Amidase enzyme preferably a linear Amidase enzyme, may be used. More preferably, the Amidase enzyme is an enzyme, which hydrolyses an 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 from whether this hydrolysis is the natural or a promiscuous functionality of the enzyme.
  • preferred Amidase enzymes may be selected from the list consisting of peptidases, proteases, linear amidases, or cyclic amidases.
  • Especially preferred Amidase enzymes are selected from the list consisting of Papain (CAS 9001-73-4), Bromelain (CAS 37189-34-7), and Bacterial Proteinase (Proteinase from Bacillus licheniformis, CAS 9014-01-1).
  • the Amidase enzyme is an L-Amidase enzyme.
  • the Amidase enzyme is a D-Amidase enzyme, more preferably a protease, and most preferably a cysteine protease.
  • R in formulae (II) and (III) is H or C r C 6 alkyl, preferably H or C 2 -C 4 alkyl, more preferably ethyl or butyl, and most preferably ethyl.
  • N-carbamoyl glufosinate amide having the formula (lib) may be treated with an N-carbamoyl glufosinate amide Racemase enzyme.
  • the components other than L-glufosinate can be removed from the biotransformation mixture, the mixture optionally concentrated, and then the mixture can be used directly (and/or with the addition of various adjuvants) for the prevention or control of weeds.
  • the biotransformation mixture in some instances, can be used directly (and/or with the addition of various adjuvants) for the prevention or control of weeds.
  • L-glufosinate it is not necessary to produce a solid product after purification. This may be advantageous if the formulation of L-glufosinate is to occur at the same site used for L-glufosinate production.
  • L- glufosinate and many of its salts are readily soluble in water, and water is a convenient liquid to use for formulating products.
  • the amine donor is isolated by filtration and the resulting filtrate is concentrated by distillation.
  • the pH of the filtrate may be adjusted to a desirable value and the resulting solution may be used as is or blended with formulation ingredients.
  • a slurry of L-glufosinate or one of its salts may be prepared as described above and isolated by filtration. The solid could be dissolved directly on the filter by adding water or a suitable solvent to obtain a solution of L-glufosinate.
  • the invention further relates in another preferred embodiment of the second aspect to a composition
  • a composition comprising an N-carbamoyl glufosinate amide having the formula (lib) wherein R is H or C r C 8 alkyl, preferably H or C r C 6 alkyl, more preferably H or C 2 -C 4 alkyl, even more preferably ethyl or butyl, and most preferably ethyl, an N-carbamoyl amino acid having the formula (Illa) wherein R is H or C r C 8 alkyl, preferably H or C r C 6 alkyl, more preferably H or C 2 -C 4 alkyl, even more preferably ethyl or butyl, and most preferably ethyl, and L-glufosinate and/or a salt thereof.
  • R is H or C r C 8 alkyl, preferably H or C r C 6 alkyl, more preferably H or C 2 -C
  • the amount of L-glufosinate and/or a salt thereof is 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 in particular at least 50 wt.-% or at least 60 wt.-%, based on the total amount of the N-carbamoyl glufosinate amide having the formula (lib), the N-carbamoyl amino acid having the formula (Illa), and L-glufosinate and/or a salt thereof.
  • the composition can further comprise the N-carbamoyl glufosinate amide having the formula preferably in an amount of up to 30 wt.-%, preferably up to 20 wt.-%, more preferably up to 10 wt.-%, even more preferably up to 5 wt.-%, still more preferably up to 2.5 wt.-%, and in particular up to 1 wt.-%, based on the total amount of the N-carbamoyl glufosinate amide having the formula (Ila), the N-carbamoyl glufosinate amide having the formula (lib), the N-carbamoyl amino acid having the formula (Illa), and L-glufosinate and/or a salt thereof.
  • the N-carbamoyl glufosinate amide having the formula preferably in an amount of up to 30 wt.-%, preferably up to 20 wt.-%, more preferably up to 10 w
  • the composition can further comprise the N-carbamoyl glufosinate amide having the formula (Ila) in an amount of 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 in particular 0.5 to 1 wt.-%, based on the total amount of the N-carbamoyl glufosinate amide having the formula (Ila), the N-carbamoyl glufosinate amide having the formula (lib), the N-carbamoyl amino acid having the formula (Illa), and L-glufosinate and/or a salt thereof.
  • the N-carbamoyl glufosinate amide having the formula (Ila) in an amount of 0.001 to 30 wt.-%, preferably 0.005 to
  • the herein described composition may be used directly as a herbicidal compositions or as an ingredient in a formulated herbicidal product.
  • compositions described herein are useful for application to a field of crop plants for the prevention or control of weeds.
  • the composition may be formulated as a liquid for spraying on a field.
  • the glufosinate preferably the L-glufosinate, is provided in the composition in effective amounts.
  • effective amount means from about 10 grams active ingredient per hectare to about 1,500 grams active ingredient per hectare, e.g., from about 50 grams to about 400 grams or from about 100 grams to about 350 grams.
  • the active ingredient is L-glufosinate.
  • the concentration of the active ingredients are the same. It is recognized that the herbicidal compositions can be used in combination with other herbicides.
  • the herbicidal compositions of the present invention are often applied in conjunction with one or more other herbicides to control a wider variety of undesirable vegetation.
  • the presently claimed compounds can be formulated with the other herbicide or herbicides, tank mixed with the other herbicide or herbicides or applied sequentially with the other herbicide or herbicides.
  • Some of the herbicides that can be employed in conjunction with the compounds of the present invention include: amide herbicides such as allidochlor, beflubutamid, benzadox, benzipram, bromobutide, cafenstrole, CDEA, chlorthiamid, cyprazole, dimethenamid, dimethenamid-P, diphenamid, epronaz, etnipromid, fentrazamide, flupoxam, fomesafen, halosafen, isocarbamid, isoxaben, napropamide, naptalam, pethoxamid, propyzamide, quinonamid and tebutam; anilide herbicides such as chloranocryl, cisanilide, clomeprop, cypromid, diflufenican, etobenzanid, fenasulam, flufenacet, flufenican, mefenacet, mefluid
  • compositions of the present invention can, further, be used in conjunction with glyphosate or 2,4-D on glyphosate-tolerant or 2,4-D-tolerant crops. It is generally preferred to use the compositions of the invention in combination with herbicides that are selective for the crop being treated and which complement the spectrum of weeds controlled by these compositions at the application rate employed. It is further generally preferred to apply the compositions of the invention and other complementary herbicides at the same time, either as a combination formulation or as a tank mix.
  • the invention further relates in a third aspect to a method for selectively controlling weeds in an area, preferably containing a crop of planted seeds or crops that are resistant to glufosinate, comprising: applying an effective amount of a composition comprising L-glufosinate and/or a salt thereof at an enantiomeric proportion of at least 50%, preferably in an enantiomeric excess of greater than 70%, over D-glufosinate and/or a salt thereof and more than 0.01 wt.-% to less than 10 wt.-%, based on the total amount of the composition, of an N-carbamoyl amino amide having the formula (II) wherein R is H or C r C 8 alkyl, preferably H or C r C 6 alkyl, more preferably H or C 2 -C 4 alkyl, even more preferably ethyl or butyl, and most preferably ethyl, and/or more than 0.01 wt.-% to
  • the composition comprises L-glufosinate and/or a salt thereof at an enantiomeric proportion of 50 to 99%, preferably in an enantiomeric proportion of 60 to 98%, more preferably of 70 to 95%, and in particular of 80 to 90%, over D- glufosinate and/or a salt thereof.
  • composition may comprise the same adjuvants and/or other herbicides as described in more detail above.
  • compositions described herein are useful for application to a field of crop plants for the prevention or control of weeds.
  • the composition may be formulated as a liquid for spraying on a field.
  • the L-glufosinate is provided in the composition in effective amounts.
  • effective amount means from about 10 grams active ingredient per hectare to about 1,500 grams active ingredient per hectare, e.g., from about 50 grams to about 400 grams or from about 100 grams to about 350 grams.
  • the active ingredient is L-glufosinate.
  • the amount of L-glufosinate in the composition can be about 10 grams, about 50 grams, about 100 grams, about 150 grams, about 200 grams, about 250 grams, about 300 grams, about 350 grams, about 400 grams, about 500 grams, about 550 grams, about 600 grams, about 650 grams, about 700 grams, about 750 grams, about 800 grams, about 850 grams, about 900 grams, about 950 grams, about 1,000 grams, about 1,050 grams, about 1,100 grams, about 1,150 grams, about 1,200 grams, about 1,250 grams, about 1,300 grams, about 1,350 grams, about 1,400 grams, about 1,450 grams, or about 1,500 grams L-glufosinate per hectare.
  • £ co//TG10 carrying the recombinant plasmid of the enzyme was used to inoculate 2 ml LB medium (Bertani, G., J Bacteriol, 1951, 62, 293) supplemented with 100 pg/ml ampicillin, 100 pg/ml spectinomycin, 20 pg/ml chloramphenicol and the resulting pre-culture was incubated for 5 h at 37 °C at an agitation of 250 rpm.
  • 1 ml of the pre-culture was used to inoculate 100 ml LB medium supplemented with 100 pg/ml ampicillin, 100 pg/ml spectinomycin, 20 pg/ml chloramphenicol, 1 mM MnCI2, 0.1 mM isopropyl-B-D-thiogalactopyranosid, and 0.5 g/l rhamnose in a 500 ml baffled Erlenmeyer-flask.
  • the culture was incubated at 37 °C for 18 h under shaking conditions. Subsequently, the biomass was harvested by centrifugation at 3220 xg for 10 min at 8 °C.
  • the supernatant was discarded, and the cell pellet resuspended in 8 ml HEPES buffer at a concentration of 100 mM and pH 8.2 supplemented with 1 mM MnCI2.
  • the cell suspension was used without any further preparation for synthesis in case whole cell biotransformation were carried out.
  • 5 ml of the cell suspension were distributed into 5 reaction tubes containing lysing matrix B (0.7 ml quartz-beads at 0 0.1 mm, MP Biomedicals), the tubes chilled on ice, and cells subsequently broken in a homogenizer (Peqlab Precellys24, VWR) for two 30 second cycles. In between cycles samples were chilled on ice.
  • Phosphoric acid 20% used to adjust pH 6.6
  • Vitamin solution Ultrapure water 100 g
  • Vitamin B12 0.5 g
  • preculture medium parts 1 1, 2, and 3 are combined and 2.0 ml of vitamin solution added. Furthermore, the medium was supplemented with 100 pg/ml ampicillin, 100 pg/ml spectinomycin, and 20 pg/ml chloramphenicol.
  • Several transformants were scraped of the LB agar plate and used to inoculated 2x 100 g of preculture media in 1 I baffled Erlenmeyer flasks. These precultures were incubated at 37 °C and 150 rpm. When an OD600 of 12 was reached the precultures were used in their entirety to inoculate the main culture.
  • Diammonium hydrogen phosphate 52.8 g Mangesium sulfate heptahydrate 15.1 g Calcium chloride dihydrate 0.7 g
  • Part 4 was sterilized at 125 °C for 45 min.
  • Part 5 was sterilized by sterile filtration using a filter unit with a pore size of 0.1 pm
  • Glycerol, and antifoam solution were sterilized at 121 °C for 30 min.
  • Thiamine and inductor solution are sterilized by filtration using a filter with a pore size of 0.2 pm.
  • Parts 4 and 5 were combined in the sterilized fermentation vessel (Techfors, Infers HT) and inoculated with the preculture.
  • the vessel was kept at a temperature of 37 °C, a pressure of 0.2 bar, and at a pH of 6.6 by dosing with base solution over the course of fermentation.
  • the pO2 level was kept at 20-40% by adjusting the stirrer speed (commonly 500 rpm) and aeration rate (commonly 6 l/min).
  • Antifoam solution was added as needed. Glycerol and thiamine solutions were combined yielding the feed solution. After inoculation the feed solution was dosed at a rate of 10 g/h.
  • the resulting supernatant was discarded, and cells resuspended in 3850 g of 50 mM potassium dihydrogen phosphate buffer at pH 7.0.
  • the cell suspension was frozen at -80 °C before being lyophilized.
  • the lyophilizer was kept at -50 °C and a pressure of 0.25 mbar. Lyophilized cells were stored at 4 °C.
  • Lyophilized cells were resuspended in ultrapure water at 100 g/l.
  • the cell suspension was cooled on ice before cells were disrupted by three passages through a pressure homogenizer (Panda Plus 2000, GEA) which was set to 800 bar. Pressures of the three passages were commonly between 1000 to 1400 bar.
  • the resulting mixture was cleared from debris by centrifugation at 10000 rpm at 10 °C for 15 min.
  • the resulting pellet was discarded and the concentration of protein in the supernatant analyzed by Bradford assay.
  • the supernatant was frozen at -80 °C and subsequently lyophilized at -50 °C and a pressure of 0.25 mbar.
  • ACM-H has been prepared according to example 2 of WO 2015/173146 A1. d) Preparation of N-carbamoyi glufosinate amide from A CM-H (Ex 4)
  • Ammonium hydrogen carbonate (27.05 g, 342.1 mmol) and diammoniumcarbonate (32.87 g, 342.1 mmol) were dissolved under stirring in 135 ml distilled water and the reaction mixture was heated to 50°C.
  • the concentrations of the N-Carbamoyl amino aicd were determined by chiral HPLC-MS using a Supelco Chirobiotic T2 (Gradient 90% ACN/Water to 60% ACN/Water in 19 minutes, 0.1% Formic acid). Temp: 20°C, flow rate 0.8 mL/min. Retention times of N-Carbamoyl amino acids: L-configured Diastereoisomers (8.6 min); D-configured (10.7 and 11.2 min).
  • reaction mixture was shaken for 24 h at room temperature. After this period of time 1.5 pl MnCI 2 solution (2M in water) was added, followed by N-Carbmoyl amino acid hydrolase (10 mg, lyophilized cell free extract, A0A535Y1H2, SEQ ID NO:2 ).
  • the liquid enzyme formulation used was Sustine® 220 from Novozymes.
  • the reaction product (P-Butyl-ester of Glufosinate) was obtained in a yield >0.01% and detected by LC/MS.
  • the liquid enzyme formulation used was Protease from Bacillus Licheniformis (CAS 9014- 01-1, aqueous, 94 mg/mL Protein).
  • the reaction product P-Butyl-ester of Glufosinate was obtained in a yield >0.01% and detected by LC/MS.
  • SEQ ID NO:1 (A0A3E0C996 from Paraburkar/deria sp. BL6669N2)
  • SEQ ID NO:2 (A0A535Y1H2 from Chloroflexi bacterium)
  • SEQ ID NO:4 (A0A1Y4GC62 from Cloacibacillus sp. An23)

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Abstract

La présente invention concerne un procédé de préparation de glufosinate, comprenant les étapes consistant à hydrolyser un amide de N-carbamoyle glufosinate avec une enzyme amidase pour former un composé d'acide aminé N-carbamoyle suivie par le clivage de la fraction carbamoyle dudit composé d'acide aminé N-carbamoyle.
PCT/EP2024/066028 2023-06-13 2024-06-11 Synthèse de glufosinate à l'aide d'un processus à base d'amidase Ceased WO2024256370A1 (fr)

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CN202480039400.5A CN121335908A (zh) 2023-06-13 2024-06-11 使用基于酰胺酶的工艺的草铵膦合成
EP24732871.9A EP4727951A1 (fr) 2023-06-13 2024-06-11 Synthèse de glufosinate à l'aide d'un processus à base d'amidase
KR1020257041323A KR20260026015A (ko) 2023-06-13 2024-06-11 아미다제-기반 공정을 사용하는 글루포시네이트의 합성
IL325207A IL325207A (en) 2023-06-13 2024-06-11 Synthesis of glufosinate using an amidase-based process
MX2025015004A MX2025015004A (es) 2023-06-13 2024-06-11 Sintesis de glufosinato mediante un proceso basado en amidasa

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CN102372739A (zh) 2011-12-05 2012-03-14 韩扶军 一种草铵膦的合成方法
CN102399240A (zh) 2011-12-27 2012-04-04 江苏优士化学有限公司 一种改进的草铵膦及类似物的合成方法
WO2015173146A1 (fr) 2014-05-13 2015-11-19 Bayer Cropscience Ag Procédé de préparation de cyanhydrines contenant du phosphore
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DE3047024A1 (de) 1978-11-11 1982-07-22 Hoechst Ag, 6000 Frankfurt Verfahren zur herstellung phosphorhaltiger cyanhydrine
US4521348A (en) 1978-11-11 1985-06-04 Hoechst Aktiengesellschaft Phosphorus-containing cyanohydrine derivatives
US4599207A (en) 1983-06-01 1986-07-08 Hoechst Aktiengesellschaft Process for the preparation of phosphorus-containing cyanohydrin derivatives
EP0249188A2 (fr) 1986-06-09 1987-12-16 Meiji Seika Kaisha Ltd. Procédé de production de l'acide L-2-amino-4-(hydroxyméthyl-phosphinyl)-butyrique
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WO2004050877A1 (fr) 2002-12-02 2004-06-17 Basf Aktiengesellschaft Systemes d'expression pouvant etre induits par le l-rhamnose
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CN102399240A (zh) 2011-12-27 2012-04-04 江苏优士化学有限公司 一种改进的草铵膦及类似物的合成方法
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