Classifications

• • 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
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
  • C07C51/347—Preparation of carboxylic acids or their salts, halides or anhydrides by reactions not involving formation of carboxyl groups
  • C07C51/373—Preparation of carboxylic acids or their salts, halides or anhydrides by reactions not involving formation of carboxyl groups by introduction of functional groups containing oxygen only in doubly bound form
• • 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
  • C07F9/38—Phosphonic acids [RP(=O)(OH)2]; Thiophosphonic acids ; [RP(=X1)(X2H)2(X1, X2 are each independently O, S or Se)]
  • C07F9/3804—Phosphonic acids [RP(=O)(OH)2]; Thiophosphonic acids ; [RP(=X1)(X2H)2(X1, X2 are each independently O, S or Se)] not used, see subgroups
  • C07F9/3808—Acyclic saturated acids which can have further substituents on alkyl
  • C07F9/3813—N-Phosphonomethylglycine; Salts or complexes thereof
• • 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
  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic 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
  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
  • C12P7/42—Hydroxy-carboxylic acids

Definitions

• This invention relates to the preparation of N-(phosphonomethyl)- glycine by the hydrogenation of mixtures produced by the reaction of glycohc acid and oxygen in an aqueous solution containing aminomethylphosphonic acid (AMPA) and the enzymes glycolate oxidase ((S)-2-hydroxy-acid oxidase, EC 1.1.3.15) and catalase (EC 1.11.1.6).
• AMPA aminomethylphosphonic acid
• N-(p__osphonomethyl)glyc_ne is a broad-spectrum, postemergent herbicide useful in controlling the growth of a wide variety of plants.
• Glycolate oxidase an enzyme commonly found in leafy green plants and mammalian cells, catalyzes the oxidation of glycolic acid to glyoxylic acid, with the concomitant production of hydrogen peroxide:
• Oxalic acid (100 mM) was reported to inhibit the catalytic action of the glycolate oxidase.
• TIS tris(hydroxymethyl)amino- methane
• N-(phosphono- methyl)glycine Numerous methods are known for preparing N-(phosphono- methyl)glycine from aminomethylphosphonic acid and glyoxylic acid.
• One such method described in Rogers et al., European Patent Application 186,648, involves condensation of glyoxylic acid or a salt thereof with aminomethylphosphonic acid or a salt thereof to form an intermediate product, generally regarded as an aldimine (Schiff base), which without isolation is reduced, as by catalytic hydrogenation, to N-(phosphonomethyl)glycine.
• a second method described in Gaertner, U.S.
• Patent 4,094,928 isolates these same intermediate carbonylalctiminomethanephosphonates by the reaction of glyoxyhc acid esters with aminomethylphosphonate esters in a non-aqueous solvent; after azeotropic distillation of water and removal of the solvent, the carbonylaldiminomethanephosphonate ester is reduced and the ester groups hydrolyzed to produce N-(phosphonomethyl)glycine.
• glyoxyhc acid is a rather costly starting material, and other less expensive routes to the desired material are practiced.
• Existing methods for the preparation of glyoxylic acid such as hydrolysis of a dihaloacetic acid, electrolytic reduction of oxalic acid, oxidation of glyoxal, catalytic oxidation of ethylene or acetaldehyde, and ozonolysis of maleic acid, its esters or anhydride, present one or more difficulties in practice, e.g. costly separation/purification steps, low yields, or large waste streams.
• the method described in Gaertner is also disadvantageous in that it requires several additional steps (with corresponding losses in yield), and the unnecessary isolation of an intermediate.
• N-(phosphonomethyl)glyc_ne Another method for the synthesis of N-(phosphonomethyl)glyc_ne, disclosed in Kleiner, U.S. Pat. 4,670,191, comprises the reaction of aminomethylphosphonic acid or a salt thereof with about two molar equivalents of glyoxyhc acid in aqueous medium.
• the excess glyoxyhc acid evidently functions as a reducing agent, converting an intermediate glyoxyhc acid-aminomethylphosphonic acid reaction product to the desired N-(phosphonomethyl)glycine, and is itself oxidized to one or more by-products, including CC .
• Fields et al. in U.S. Pat.
• N-(phosphonomethyl)glycine by heating an N-acylaminomethylphosphonic acid with glyoxyhc acid or a derivative thereof.
• the mole ratio of the glyoxyhc to the N-acylamino component is preferably 2 to 1; otherwise at smaller ratios the yield suffers.
• the Kleiner and Fields et al. processes entail the disadvantages of not only employing relatively expensive glyoxyhc acid but of employing it as a sacrificial reductant (ca. one mole of glyoxylate employed as reductant for every mole of N-(phosphonomethyl)glycine produced) as well as the condensing agent for the __mino-(or N-acylamino) methylphosphonic acid.
• the process for preparing N-(phosphonomethyl)glycine according to the present invention involves hydrogenating a mixture, wherein the mixture is enzymatically produced by reacting glycolic acid and oxygen in an aqueous solution containing aminomethylphosphonic acid (AMPA) and the enzymes glycolate oxidase and catalase.
• AMPA aminomethylphosphonic acid
• the mixtures produced by enzymatic oxidation of glycohc acid in the presence aminomethylphosphonic acid inherently result in a distribution of oxidation by-products in addition to the desired glyoxylic acid component (including by way of example but not limited thereto, oxalate, formate, and carbon dioxide).
• the present invention further provides for the removal and recovery of the enzymes from the solution produced as a result of enzymatic oxidation as well as the optional removal of FMN prior to the hydrogenation step.
• the present invention provides an improved process for preparing N-(phosphonomethyl)g_ycine comprising the step of reducing a mixture of glyoxylic acid and aminomethylphosphonic acid by hydrogenation; said mixture being enzymatically generated in situ in an aqueous solution by incorporating into the aqueous solution glycolic acid, a first catalyst adapted to catalyze the oxidation of glycolic acid with oxygen to gly ⁇ xylic acid and hydrogen peroxide, and a second catalyst adapted to catalyze the decomposition of hydrogen peroxide, adjusting the pH of the solution to between 6 and about 10, contacting the solution with a source of oxygen at an effective temperature and sufficient time to convert at least a portion of the glycohc component to the glyoxyhc component in the presence of aminomethylphosphonic acid, and ceasing contacting the solution with oxygen prior to the reducing step.
• the catalysts are enzymatic; more preferably the first enzyme is glycolate oxidase ((S)-2-hydroxy-acid oxidase, EC 1.13.15) and the second enzyme is catalase (EC 1.11.1.6).
• the catalysts/enzymes are removed, as by filtration or centrifugation, before the solution is subjected to reducing conditions for the production of N-(phosphonomethyl)glycine.
• the present invention provides for a more efficient and economic process for the production of N-(phosphonomethyl)glycine.
• the improved process for the production of N- (phosphonomethyl)glycine involves the reduction of a mixture containing glyoxyhc acid (or a suitable derivative thereof) with aminomethylphosphonic acid (AMPA) (or a suitable derivative thereof).
• AMPA aminomethylphosphonic acid
• the mixture is prepared by catalytically o-ridizing a glycohc acid component or a suitable salt thereof by contacting the glycohc by contacting the glycohc acid component with a source of molecular oxygen in the presence of AMPA and a catalyst effective to catalyze the reaction of glycohc acid with 0 2 to form glyoxyhc acid.
• glycolate oxidase EC 1.13.15
• glycohc acid oxidase is a natur_dly-occrurring enzyme glycolate oxidase (EC 1.13.15), also known as glycohc acid oxidase, which is capable of catalyzing the reaction to produce glyoxyhc acid in high yields at high glycohc acid conversions in aqueous media under mild conditions of pH and temperature, i.e.,
• the catalytic oxidation of glycohc acid or a suitable salt thereof is conveniently carried out by contacting the glycohc acid with a source of molecular oxygen in the presence of an enzyme catalyst which catalyzes the reaction of glycohc acid with ⁇ 2 to form glyoxyhc acid.
• an enzyme catalyst which catalyzes the reaction of glycohc acid with ⁇ 2 to form glyoxyhc acid.
• One such catalyst is the enzyme glycolate oxidase (EC 1.13.15), also known as glycohc add oxidase.
• Glycolate oxidase may be isolated from numerous sources well-known to the art.
• the glycolate oxidase used in the reaction should be present in an effective concentration, usually a concentration of about 0.01 to about 1000 IU/mL, preferably about 0.1 to about 4 IU/mL.
• An IU International Unit
• An IU International Unit is defined as the amount of enzyme that will catalyze the transformation of one micromole of substrate per minute.
• a procedure for the assay of this enzyme is found in I. Zelitch and S. Ochoa, J. Biol. Chem.. Vol. 201, 707-718 (1953).
• This method is also used to assay the activity of recovered or recycled glycolate oxidase.
• the enzyme-catalyzed reaction of glycohc add with oxygen is well known, high selectivities to glyoxylic add have not been previously obtained, and there are no previous reports of performing the enzymatic oxidation of glycohc add in the presence of aminomethylphosphonic add (AMPA).
• AMPA aminomethylphosphonic add
• glycolate oxidase as a catalyst for the oxidative conversion of glycohc add to glyoxyhc add are obtained by incorporating into the reaction solution a catalyst for the decomposition of hydrogen peroxide.
• a peroxide-destroying catalyst which is effective in combination with glycolate oxidase is the enzyme catalase (E.C. 1.11.1.6).
• Catalase catalyzes the decomposition of hydrogen peroxide to water and oxygen, and it is believed to improve yields of glyoxyhc add in the present process by accelerating the decomposition of the hydrogen peroxide produced along with glyoxyhc add in the glycolate oxidase-catalyzed reaction of glycohc add with ⁇ 2.
• the concentration of catalase should be 50 to 50,000 IU/mI_, preferably 500 to 15,000 IU/mL. It is preferred that the catalase and glycolate oxidase concentrations be adjusted within the above ranges so that the ratio (measured in IU for each enzyme) of catalase to glycolate oxidase is at least about 250:1.
• FMN flavin mononucleotide
• concentration of added FMN is in addition to any FMN present with the enzyme, because FMN is often also added to the enzyme during the preparation of the enzyme.
• the structure of FMN and a method for its analysis is found in K. Yagai, Methods of Biochemical Analysis. Vol.
• Glycohc add (2-hydroxyacetic add) is available commerdaJly.
• its initial concentration is in the range of 0.10 M to 2.0 M, preferably between 0_25 M and 1.0 M. It can be used as such or as a compatible salt thereof, that is, a salt that is water-soluble and whose cation does not interfere with the desired conversion of glycohc add to glyoxylic add, or the subsequent reaction of the glyoxyhc add product with the ⁇ tmino-methylphosphonic add to form N-(phosphonomethyl)gJycine.
• Suitable and compatible salt-forming catio ⁇ ic groups are readily determined by trial.
• Representative of such salts are the alkali metal, alkaline earth metal, ammonium, substituted ammonium, phosphonium, and substituted phosphonium salts.
• the conversion of glycohc add to glyoxylic add is conveniently and preferably conducted in aqueous media.
• Aminomethylphosphonic add (AMPA), or a suitable salt thereof is added to produce a molar ratio of AMPA/glycohc add (starting amount) in the range of from 0.01/1.0 to 3.0/1.0, preferably from 0.25/1.0 to 1.05/1.0.
• the pH of the resulting mixture is adjusted to a value between 6 and 10, preferably between 7.0 and 9.0. Within this pH range, the exact value may be adjusted to obtain the desired pH by adding any compatible, non-interfering base, including alkali metal hydroxides, carbonates, bicarbonates and phosphates.
• the pH of the reaction mixture decreases slightly as the reaction proceeds, so it is often useful to start the reaction near the high end of the maximum enzyme activity pH range, about 9.0 - 8.5, and allow it to drop during the reaction.
• the pH can optionally be maintained by the separate addition of a non-interfering inorganic or organic buffer, since enzyme activity varies with pH.
• glycohc and glyoxyhc adds are highly dissodated in water, and at pH of between 6 and 10 are largely if not substantially entirely present as glycolate and glyoxylate ions.
• glyoxyhc add (and its conjugate base, the glyoxylate anion) may also be present as the hydrate, e.g. (HO)2CHCOOH and/or as the hemiacetal, HOOCCH(OH)OCH(OH)COOH, which compositions and their anionic counterparts are equivalent to glyoxyhc add and its anion for the present purpose of being suitable reactants for N-(phosphonomethyl)glycine formation.
• Oxygen ( ⁇ 2) the oxidant for the conversion of the glycohc add to glyoxylic add, may be added as a gas to the reaction by agitation of the liquid at the gas-liquid interface or through a membrane permeable to oxygen. It is beheved that under most conditions, the reaction rate is at least partially controlled by the rate at which oxygen can be dissolved into the aqueous medium.
• oxygen can be added to the reaction as air, it is preferred to use a relatively pure form of oxygen, and even use elevated pressures. Although no upper limit of oxygen pressure is known, oxygen pressures up to 50 atmospheres may be used, and an upper limit of 15 atmospheres is preferred.
• Agitation is important to maintaining a high oxygen dissolution (hence reaction) rate. Any convenient form of agitation is useful, such as stirring. On the other hand, as is well known to those skilled in the enzyme art, high shear agitation or agitation that produces foam may decrease the activity of the enzyme(s), and should be avoided.
• the reaction temperature is an important variable, in that it affects reaction rate and the stability of the enzymes.
• a reaction temperature of 0°C to 40°C may be used, but the preferred reaction temperature range is from 5°C to 15°C. Operating in the preferred temperature range maximizes recovered enzyme activity at the end of the reaction.
• the temperature should not be so low that the aqueous solution starts to freeze.
• Temperature can be controlled by ordinary methods, such as, but not limited to, by using a jacketed reaction vessel and passing liquid of the appropriate temperature through the jacket.
• the reaction vessel may be constructed of any material that is inert to the reaction ingredients.
• the enzymes may be removed by filtration or centrifugation and reused. Alternatively, they can be denatured and predpitated by heating, e.g. to 70°C for 5 minutes, and/or can be allowed to remain in the reaction mixture if their presence in the subsequent steps of converting the glyoxylic add-aminomethylphosphonic add mixture to N- (phospho ⁇ omethyl)glydne, and of recovering N-(phosphono-methyl) glycine from the reaction mixture, is not objectionable. Following the cessation of contacting the reaction solution with
• flavin mononucleotide may optionally be removed by contacting the solution with activated carbon.
• the solution containing glyoxyhc add and aminomethyl-phosphonic add (which are beheved to be in equilibrium with the corresponding i ine), is reduced, producing N- (phosphonomethyl)glydne.
• Catalytic hydrogenation is a preferred method for preparing N-(phosphonomethyl)glycine from a mixture of glyoxylic add and aminomethylphosphonic add.
• Catalysts suitable for this purpose include (but are not limited to) the various platinum metals, such as indium, osmium, rhodium, ruthenium, platinum, and palladium; also various other transition metals such as cobalt, copper, nickel and zinc.
• the catalyst may be unsupported, for example as Raney nickel or platinum oxide; or it may be supported, for example as platinum on carbon, palladium on alumina, or nickel on kieselguhr. Palladium on carbon, nickel on kieselguhr and Raney nickel are preferred.
• the hydrogenation can be performed at a pH of from 4 to 11, preferably from 5 to 10. Within this pH range, the exact value may be adjusted to obtain the desired pH by adding any compatible, non-interfering base or add.
• Suitable bases include, but are not limited to, alkali metal hydroxides, carbonates, bicarbonates and phosphates, while suitable adds include, but are not limited to, hydrochloric, sulfuric, or phosphoric add.
• the hydrogenation temperature and pressure can vary widely.
• the temperature may generally be in the range of 0°C to 150°C, preferably from 20°C to 90°C, while the EL pressure is generally in the range of from about atmospheric to about 100 atmospheres, preferably from 1 to 10 atmospheres.
• the hydrogenation catalyst is employed at a minimum concentration suffident to obtain the desired reaction rate and total conversion of starting materials under -li ⁇ the chosen reaction conditions; this concentration is easily determined by trial.
• the catalyst may be used in amounts of from 0.001 to 20 or more parts by weight of catalyst per 100 parts of combined weight of the glyoxyhc add and AMPA employed in the reaction.
• N-(Phosphonomethyl)glycine useful as a post-emergent herbidde, may be recovered from the reduced solution, whatever the reducing method employed, by any of the recovery methods known to the art, including those disclosed in the U.S. Patents 4,851,159 and 4,670,191 and in European Patent Applications 186648 and 413 672.
• the yields of glyoxylate, formate and oxalate, and the recovered yield of glycolate are percentages based on the total amount of glycohc add present at the beginning of the reaction. Analyses of reaction mixtures were performed using high pressure liquid chromatography.
• Example 1 Into a 3 oz. Fischer-Porter glass aerosol reaction vessel was placed a magnetic stirring bar and 10 mL of an aqueous solution cont ⁇ ning glycohc add (0.25 M), aminomethylphosphonic add (AMPA, 0.263 M), FMN (0.01 mM), propionic add (HPLC internal standard, 0.125 M), glycolate oxidase (from spinach, 1.0 IU/mL), and catalase (from Asperg ⁇ llus niger, 1,400 IU/mL) at pH 8.5. The reaction vessel was sealed and the reaction mixture was cooled to 15°C, then the vessel was flushed with oxygen by pressurizing to 70 psig and venting to atmospheric pressure five times with stirring.
• AMPA aminomethylphosphonic add
• FMN 0.01 mM
• propionic add HPLC internal standard, 0.125 M
• glycolate oxidase from spinach, 1.0 IU/mL
• catalase from Asperg ⁇ llus niger, 1,400
• Example 2 (Comparative) The reaction in Example 1 was repeated, using 0.33 M K HPO4 in place of 0.265 M AMPA.
• the HPLC yields of glyoxylate, formate, and oxalate were 34.1 %, 11.1 %, and 0.2 %, respectively, and 58.7 % glycolate remained.
• the HPLC yields of glyoxylate, formate, and oxalate were 39.4 %, 44.7 %, and 15.34 %, respectively, and no glycolate remained.
• the remaining activity of glycolate oxidase and catalase were 85 % and 87 %, respectively, of their initial values.
• Example 1 The reaction in Example 1 was repeated, using 0.263 M bicine buffer in place of 0.265 M AMPA. After 5 h, the HPLC yields of glyoxylate, formate, and oxalate were 42.5 %, 49.6 %, and 10.1 %, respectively, and 0.2 % glycolate remained. The remaining activity of glycolate oxidase and catalase were 47 % and 100 %, respectively, of their initial values.
• Example 1 The reaction in Example 1 was repeated using 5,600 IU/mL catalase from.AspergUlus niger. After 6 h, the HPLC yields of glyoxylate, formate, and oxalate were 85.5 %, 7.6 %, and 3.3 %, respectively, and 2.5 % glycolate remained. The remaining activity of glycolate oxidase and catalase were 36 % and 100 %, respectively, of their initial values.
• Example 5 The reaction in Example 1 was repeated using 14,000 IU/mL catalase from Asperg ⁇ lus niger. After 6 h, the HPLC yields of glyoxylate, formate, and oxalate were 88.0 %, 3.3 %, and 3.0 %, respectively, and 3.4 % glycolate remained. The remaining activity of glycolate oxidase and catalase were 28 % and 96 %, respectively, of their initial values.
• Example 7 The reaction in Example 1 was repeated using 56,000 IU/mL catalase from Aspeigillus niger. After 6 h, the HPLC yields of glyoxylate, formate, and oxalate were 84.0 %, 0.4 %, and 2.5 %, respectively, and 8.4 % glycolate remained. The remaining activity of glycolate oxidase and catalase were 16 % and 76 %, respectively, of their initial values.
• Example 7 The remaining activity of glycolate oxidase and catalase were 16 % and 76 %, respectively, of their initial values.
• reaction vessel 1.0 IU/mL
• catalase from Aspergillus niger, 14,000 IU/mL
• the reaction vessel was sealed and the reaction mixture was cooled to 5°C, then the vessel was flushed with oxygen by pressurizing to 70 psig and venting to atmospheric pressure five times with stirring. The vessel was then pressurized to 70 psig of oxygen and the mixture stirred at 5°C. Ahquots (0.10 mL) were removed by syringe through a sampling port (without loss of pressure in the vessel) at regular intervals for analysis by HPLC to monitor the progress of the reaction.
• the HPLC yields of glyoxylate, formate, and oxalate were 92.3 %, 4.36 %, and 5.5 %, respectively, and no glycolate remained.
• the remaining activity of glycolate oxidase and catalase were 87 % and 88 %, respectively, of their initial values.
• the final pH of the reaction mixture was 6.7.
• the resulting mixture of glyoxyhc add (0.23 M) and AMPA (0.20 M) was filtered using an Amicon Centriprep 10 concentrator (10,000 molecular weight cutoff) to remove the soluble enzymes, then the filtrate was placed in a 3-oz. Fischer-Porter bottle equipped with a magnetic stirrer bar. To the bottle was then added 0.100 g of 10% Pd/C and the bottle sealed, flushed with nitrogen gas, then pressurized to 50 psi with hydrogen and stirred at 25°C. After 17 h, the concentration of N-(phosphonomethyl)-glycine (determined by HPLC) was 0.13 M (66% yield based on AMPA).
• Example 8 Into a 3 oz. Fischer-Porter glass aerosol reaction vessel was placed a magnetic stirring bar and 10 mL of an aqueous solution containing glycohc add (0.50 M), aminomethylphosphonic add (AMPA, 0.40 M), FMN (0.01 mM), butyric add (HPLC internal standard, 0.10 M), glycolate oxidase (from spinach, 1.0 IU/mL), and catalase (from Aspergillus niger, 14,000 IU/mL) at pH 8.5. The reaction vessel was sealed and the reaction mixture was cooled to 5°C (instead of 15°C as described in previous examples), then the vessel was flushed with oxygen by pressurizing to 70 psig and venting to atmospheric pressure five times with stirring.
• glycohc add (0.50 M
• AMPA aminomethylphosphonic add
• FMN 0.01 mM
• butyric add HPLC internal standard, 0.10 M
• glycolate oxidase from spinach, 1.0 IU/mL
• the vessel was then pressurized to 70 psig of oxygen and the mixture stirred at 5°C. Ahquots (0.10 mL) were removed by syringe through a sampling port (without loss of pressure in the vessel) at regular intervals for analysis by HPLC to monitor the progress of the reaction. After 17-5 h, the HPLC yields of glyoxylate, formate, and oxalate were 91.0%, 2.9%, and 2.9%, respectively, and 4.1% glycolate remained. The final pH of the reaction mixture was 6.7. The remaining activity of glycolate oxidase was 63% and 91%, respectively, of their initial value.
• the resulting mixture of glyoxylic add (0.46 M) and AMPA (0.40 M) was filtered using an Amicon Centriprep 10 concentrator (10,000 molecular weight cutoff) to remove the soluble enzymes, then the filtrate was placed in a 3-oz. Fischer-Porter bottle equipped with a magnetic stirrer bar. To the bottle was then added 0.100 g of 10% Pd/C and the bottle sealed, flushed with nitrogen gas, then pressurized to 50 psi with hydrogen and stirred at 25°C. After 17 h, the concentration of N-(phosphonomethyl)glyc_ne (determined by HPLC) was 0.29 M (72% yield based on AMPA).
• Example 10 The enzymatic oxidation of glycohc add in Example 8 was repeated, using 10 mL of an aqueous solution containing glycohc add (1.0 M), aminomethylphosphonic add (AMPA, 0.80 M), FMN (0.01 mM), butyric add (HPLC internal standard, 0.10 M), glycolate oxidase (from spinach, 2.0 IU/mL), and catalase (from Aspergilhis niger, 14,000 IU/mL) at pH 8.5. After 66 h, the HPLC yields of glyoxylate, formate, and oxalate were 78.9%, 2.2%, and 12.1%, respectively, and 2.0% glycolate remained. The final pH of the reaction mixture was 6.9. The remaining activity of glycolate oxidase and catalase were 64 % and 87 %, respectively, of their initial values.
• the resulting mixture of glyoxyhc add (0.79 M) and AMPA (0.80 M) was filtered using an Amicon Centriprep 10 concentrator (10,000 molecular weight cutoff) to remove the soluble enzymes, then the filtrate was placed in a 3-oz. Fischer-Porter bottle equipped with a magnetic stirrer bar. To the bottle was then added 0.100 g of 10% Pd/C and the bottle sealed, flushed with nitrogen gas, then pressurized to 50 psi with hydrogen and stirred at 25°C. After 23 h, the concentration of N-(phosphonomethyl)glyc_ne (determined by HPLC) was 0.51 M (65% yield based on glyoxyhc add).
• Example 8 The reaction in Example 8 was repeated at pH 8.0. After 17.5 h, the HPLC yields of glyoxylate, formate, and oxalate were 87.0 %, 2.2 %, and 1.9
• glycolate oxidase and catalase were 44 % and 97 %, respectively, of their initial values.
• Example 12 The reaction in Example 8 was repeated at pH 7. After 17.5 h, the HPLC yields of glyoxylate, formate, and oxalate were 88.0 %, 1.4 %, and 1.9 %, respectively, and 8.2 % glycolate remained. The remaining activity of glycolate oxidase and catalase were 44 % and 93 %, respectively, of their initial values.
• Example 13 Into a 3 oz. Fischer-Porter glass aerosol reaction vessel was placed a magnetic stirring bar and 10 mL of an aqueous solution containing glycohc add (0.50 M), FMN (0.01 mM), isobutyric acid (HPLC internal standard, 0.10 M), glycolate oxidase (from spinach, 1.0 IU/mL), and catalase (from Aspergilhis niger, 14,000 IU/mL) at pH 8.5. The reaction vessel was sealed and the reaction mixture was cooled to 5°C, then the vessel was flushed with oxygen by pressurizing to 70 psig and venting to atmospheric pressure five times with stirring. The vessel was then pressurized to 70 psig of oxygen and the mixture stirred at 5°C.
• glycohc add 0.50 M
• FMN 0.01 mM
• isobutyric acid HPLC internal standard, 0.10 M
• glycolate oxidase from spinach, 1.0 IU/mL
• catalase from Asperg
• glycohc add in Example 8 was repeated, using 10 mL of an aqueous solution containing glycohc add (025 M), aminomethylphosphonic add (AMPA, 0.263 M), FMN (0.01 mM), butyric add (HPLC internal standard, 0.25 M), glycolate oxidase (from spinach, 1.0 IU/mL), and catalase (fromAspergUlus niger, 14,000 IU/mL) at pH 7.0 and 15°C. After 8 h, the HPLC yields of glyoxylate, formate, and oxalate were 82.8%, 0.9%, and 2.1%, respectively, and 13.9% glycolate remained. The final pH of the reaction mixture was 6.6.
• Example 15 The enzymatic oxidation of glycohc add in Example 14 was repeated at pH 8. After 8 h, the HPLC yields of glyoxylate, formate, and oxalate were 86.7%, 1.8%, and 4.1%, respectively, and 13.2% glycolate remained. The final pH of the reaction mixture was 6.7.
• Example 16 The enzymatic oxidation of glycohc add in Example 14 was repeated at pH 9. After 7 h, the HPLC yields of glyoxylate, formate, and oxalate were 70.0%, 5.6%, and 11.1%, respectively, and no glycolate remained. The final pH of the reaction mixture was 6.8.
• Example 17 The enzymatic oxidation of glycolic add in Example 14 was repeated at pH 8.5, and using initial concentrations of glycolic add and AMPA of 0.50 M and 0.40 M, respectively. After 16.5 h, the HPLC yields of glyoxylate, formate, and oxalate were 85.4%, 3.5%, and 6.3%, respectively, and 1.4% glycolate remained. The final pH of the reaction mixture was 7.0. This mixture of glyoxyhc add (0.43 M) and AMPA (0.40 M) was hydrogenated at 1000 psi using the same procedure as described in Example 5. After 4 h, the concentration of N-(phosphonomethyl)glycine (determined by HPLC) was 030 M (75% yield based on AMPA).
• the resulting ⁇ iixture of glyoxylic add (0.435 M) and AMPA (0.375 M) was filtered using an Amicon Centriprep 10 concentrator (10,000 molecular weight cutoff) to remove the soluble enzymes, then the filtrate was mixed with 50 mg of decolorizing carbon (to remove FMN) and again filtered.
• the resulting filtrate was placed in a 3-oz. Fischer-Porter bottle equipped with a magnetic stirrer bar. To the bottle was then added 0.100 g of 10% Pd/C and the bottle sealed, flushed with nitrogen gas, then pressurized to 50 psi with hydrogen and stirred at 25°C. After 17 h, the concentration of N-(phosphonomethyl)- glycine (determined by HPLC) was 0.372 M (99% yield based on AMPA).

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• 1992
  • 1992-11-03 CA CA002123081A patent/CA2123081C/en not_active Expired - Fee Related
  • 1992-11-03 ES ES92310063T patent/ES2087460T3/es not_active Expired - Lifetime
  • 1992-11-03 CZ CZ941114A patent/CZ111494A3/cs unknown
  • 1992-11-03 WO PCT/US1992/009420 patent/WO1993009243A1/en not_active Application Discontinuation
  • 1992-11-03 JP JP5508669A patent/JPH07501940A/ja active Pending
  • 1992-11-03 AU AU30617/92A patent/AU658956B2/en not_active Ceased
  • 1992-11-03 CZ CZ941113A patent/CZ111394A3/cs unknown
  • 1992-11-03 EP EP92924228A patent/EP0611399A1/de active Pending
  • 1992-11-03 DE DE69210771T patent/DE69210771T2/de not_active Expired - Fee Related
  • 1992-11-03 HU HU9401416A patent/HUT71955A/hu unknown
  • 1992-11-03 BR BR9206882A patent/BR9206882A/pt not_active Application Discontinuation
  • 1992-11-03 JP JP50866893A patent/JP3315119B2/ja not_active Expired - Fee Related
  • 1992-11-03 EP EP92310063A patent/EP0545553B1/de not_active Expired - Lifetime
  • 1992-11-03 EP EP92924227A patent/EP0611398A1/de active Pending
  • 1992-11-03 WO PCT/US1992/009419 patent/WO1993009242A1/en not_active Application Discontinuation
  • 1992-11-03 CA CA002123079A patent/CA2123079C/en not_active Expired - Fee Related
  • 1992-11-03 BR BR9206884A patent/BR9206884A/pt not_active Application Discontinuation
  • 1992-11-03 AU AU30616/92A patent/AU673146B2/en not_active Expired - Fee Related
  • 1992-11-03 HU HU9401419A patent/HUT71271A/hu unknown
  • 1992-11-03 EP EP92310064A patent/EP0541333A1/de not_active Withdrawn
  • 1992-11-04 NZ NZ245011A patent/NZ245011A/en not_active IP Right Cessation
  • 1992-11-04 NZ NZ245012A patent/NZ245012A/en not_active IP Right Cessation
  • 1992-11-04 MY MYPI92002001A patent/MY130099A/en unknown
  • 1992-11-05 MX MX9206363A patent/MX9206363A/es not_active Application Discontinuation
  • 1992-11-05 MX MX9206362A patent/MX9206362A/es unknown
  • 1992-11-05 IL IL10364692A patent/IL103646A/en not_active IP Right Cessation
  • 1992-11-05 IL IL10364592A patent/IL103645A/en not_active IP Right Cessation
  • 1992-11-06 CN CN92114383A patent/CN1073482A/zh active Pending
  • 1992-11-06 CN CN92114382A patent/CN1073481A/zh active Pending
  • 1992-11-06 ID IDP497492A patent/ID1002B/id unknown

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