Preparation of 2-(4-hydroxyphenoxy)propionic acid by fermentation

Abstract

A process for the oxidation of 2-phenoxypropionic acid to 2-(4-hydrophenoxy)propionic acid using microorganisms is described.

Description

The present invention relates to a process for the preparation of 2-(4-hydroxyphenoxy)propionic acid.

2-(4-Hydroxyphenoxy)propionic acid is a valuable intermediate, in particular for the preparation of herbicides.

Chemical processes for the preparation of racemic and of pure enantiomeric forms of 2-(4-hydroxyphenoxy)propionic acid have been disclosed. These processes start from hydroquinone and have the disadvantage that they give by-products whose removal, which is necessary, is laborious. This makes economic preparation impossible.

It has been disclosed that microorganisms are able to hydroxylate aromatic compounds. This usually produces dihydroxylated compounds or mixtures of various regioisomers (R. J. W. Byrde, D. Woodcook, Biochem. J. 65 (1957) 682).

The regioselective oxidation of 2-phenoxypropionic acid to 2-(4-hydroxyphenoxy)propionic acid by microorganisms has not hitherto been disclosed.

We have now found, surprisingly, that various microorganisms oxidize the valuable 2-phenoxypropionic acid to 2-(4-hydroxyphenoxy)propionic acid with high regioselectivity.

The present invention relates to a process for the preparation of 2-(4-hydroxyphenoxy)propionic acid by fermentation, which comprises oxidizing 2-phenoxypropionic acid or salts thereof under aerobic conditions in the presence of a microorganism.

A large number of microorganisms are suitable for the process, in particular fungi and bacteria. Utilizable fungi can be isolated, for example, from soil samples, in particular humus-containing soil samples. Particularly suitable for the reaction are Aspergillus niger strains. Also suitable are many fungi from the genera Beauveria, Paecilomyces, Sclerotium and Coprinus, which can be obtained, for example, from collections of strains and whose suitability for the hydroxylation can be determined in a straightforward preliminary test. Suitable bacteria can also be isolated from soil samples, inter alia. Particularly suitable bacteria are Streptomyces strains.

To carry out the process according to the invention, suitable strains are transferred to a nutrient medium containing 2-phenoxypropionic acid and incubated aerobically therein under conditions favorable for growth and production by the particular microorganism. The fermentation is carried out continuously or batchwise for 1 to 10 days.

The cells of the microorganism, which can also be used in the form of dormant, non-growing cells, are allowed to act directly on the substrate. All conventional incubation processes can be employed, but particularly preferred fermenters are in the form of deep, aerated and agitated tanks. Very good results are obtained by incubation of a liquid nutrient medium.

Suitable nutrient media contain sources of carbon and of nitrogen, and inorganic salts and, where appropriate, small amounts of trace elements and vitamins. Sources of nitrogen which can be used are inorganic or organic nitrogen compounds or materials which contain these compounds. Examples are ammonium salts, nitrates, corn steep liquor, brewer's yeast autolysate, soybean meal, wheat gluten, yeast extract, yeast, urea and potato protein. Examples of sources of carbon which can be used are sugars such as glucose, polyols such as glycerol, or fats such as soybean oil.

Examples of inorganic salts are the salts of calcium, magnesium, manganese,, potassium, zinc, copper, iron and other metals. A particularly suitable anion in the salts is the phosphate ion. Growth factors may be added to the nutrient medium, such as biotin, riboflavin or other vitamins.

The ratios of the said nutrients in the mixture depends on the type of fermentation and will be determined in the individual case.

Generally suitable for carrying out the process according to the invention are phenoxypropionic acid concentrations of about 1 to 100 g/l, preferably about 5 to 50 g/l.

The culture conditions are chosen so that the best possible yields are achieved. Cultivation is preferably carried out at from 20.degree. to 40.degree. C., particularly advantageously from 25.degree. to 30.degree. C. The pH is preferably maintained in the range 3 to 9. The pH is particularly advantageously from 4 to 7. An incubation time of from 15 to 100 hours generally suffices. The maximum amount of the desired product accumulates in the medium during this time. The acid is preferably added in the form of a salt, e.g. the sodium salt. The acid can be added to the nutrient medium all at once at the start, after growth has taken place or in several portions or continuously during the cultivation.

The use of 2-phenoxypropionic acid is preferred, but it is also possible to use salts thereof. Preferred salts are alkali metal and alkaline earth metal salts, for example the Na, K and Li salts.

The novel process is suitable for the oxidation both of racemic 2-(4-hydroxyphenoxy)propionic acid and of antipodes thereof. The center of asymmetry remains untouched in the process according to the invention. If, for example, R-2-phenoxypropionic acid is used, the configuration is retained and the product is R-2-(4-hydroxyphenoxy)propionic acid. No other hydroxylation products are found in the reaction. Thus the novel process represents a straightforward and more economic method for the selective preparation of 2-(4-hydroxyphenoxy)propionic acid.

The examples which follow illustrate the invention:

EXAMPLE 1

Two liquid nutrient media were used to examine various strains of bacteria:

______________________________________Medium A______________________________________20 g/l glucose5 g/l yeast extract5 g/l ammonium sulfate0.5 g/l magnesium sulfate 7-hydrate0.05 g/l manganese sulfate 1-hydrate1.5 g/l potassium dihydrogen phosphate3.6 g/l dipotassium hydrogen phosphate1 g/l (R)-2-phenoxypropionic acid2 mg/l iron(II) sulfate 1-hydrate100 .mu.g/l zinc(II) sulfate 4-hydrate300 .mu.g/l boric acid200 .mu.g/l cobalt(II) chloride 6-hydrate10 .mu.g/l copper(II) chloride 2-hydrate20 .mu.g/l nickel(II) chloride 6-hydrate30 .mu.g/l sodium molybdate 2-hydrate______________________________________

The pH was adjusted to 6.8 with 5N sodium hydroxide solution. Glucose and phosphates were each autoclaved separately at 121.degree. C. for 10 minutes. The (R)-2-phenoxypropionic acid was dissolved in water, with a little 5N sodium hydroxide solution, and sterilized by filtration. The remaining medium was autoclaved at 121.degree. C. for 10 minutes.

______________________________________Medium B______________________________________20 g/l glucose40 g/l corn steep liquor1.5 g/l potassium dihydrogen phosphate3.6 g/l dipotassium hydrogen phosphate1 g/l (R)-2-phenoxypropionic acid______________________________________

The pH was adjusted to 6.8 with 5N sodium hydroxide solution. Glucose and phosphates were each autoclaved separately at 121.degree. C. for 10 minutes. The (R)-2-phenoxypropionic acid was dissolved in water, with a little 5N sodium hydroxide solution, and sterilized by filtration. The remaining medium was autoclaved at 121.degree. C. for 45 minutes.

20 ml portions of the sterile medium were introduced into sterile 100 ml Erlenmeyer flasks, which were provided with a sterile cotton plug. One loop of the strain of bacteria to be tested was used to inoculate each flask. The flasks were then incubated at 28.degree. C., shaking at 250 rpm, for 3 to 7 days. Then 1000 .mu.l of fermentation broth were removed, 100 .mu.l of 1N HCl and 800 .mu.l of ethyl acetate were added, and the mixture was vigorously mixed for 15 seconds. 700 .mu.l of the organic phase were carefully removed and evaporated in a test tube under a gentle stream of nitrogen at 50.degree. C. The residue was dissolved in 70 .mu.l of ethyl acetate and transferred quantitatively into a test tube for the gas chromatography. To this were added 30 .mu.l of N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA). The samples were then examined by gas chromatography. The external standard used was an authentic sample of (R,S)-2-(4 -hydroxyphenoxy)propionic acid which had been chemically synthesized.

The table which follows summarizes the results:

______________________________________ Collection/ ConversionGenus/species Number (%) Medium______________________________________Streptomyces DSM 40725 11 AantibioticusStreptomyces ATCC 11891 10 AantibioticusStreptomyces ATCC 13850 18 AflocculusStreptomyces ATCC 19040 97 BhygroscopicusStreptomyces ATCC 21705 98 AhygroscopicusStreptomyces ATCC 15715 4 AkasugaensisStreptomyces ATCC 15714 23 BkasugaensisStreptomyces ATCC 13279 13 AmediocidicusStreptomyces ATCC 19793 23 AniveusStreptomyces ATCC 31055 2 ApanayensisStreptomyces ATCC 21895 4 AroseochromogenesStreptomyces ATCC 11989 24 Aviridifaciens______________________________________

EXAMPLE 2

About 400 different novel Streptomycetes strains were isolated from various soil samples by the method of DREWS (Mikrobiologisches Praktikum, 3rd edition, Springer Verlag, pages 47-48, 1976). These strains were tested by the method of Example 1. The table which follows summarizes the conversion by the strains identified as positive.

______________________________________Strain number Conversion (%) Medium______________________________________1034 8 A1045 28 A1053 22 A1057 7 A1071 11 A2476 13 A3279 4 A3292 33 B3388 26 B3520 42 B3523 80 A3549 80 B3558 24 A3559 32 A3560 98 A3561 90 A3565 4 A3567 72 B3574 41 A3575 24 B3782 44 A3925 24 B4041 14 A5431 19 A5432 99 A5607 24 A5608 26 A5613 33 A5619 51 A5632 18 A5635 15 A5636 5 A5637 39 A5638 38 A5648 25 A5654 59 A______________________________________

EXAMPLE 3

A liquid nutrient medium was prepared with the following ingredients:

______________________________________20 g/l glucose5 g/l yeast extract5 g/l ammonium sulfate0.5 g/l magnesium sulfate 7-hydrate0.05 g/l manganese sulfate 1-hydrate1.5 g/l potassium dihydrogen phosphate3.6 g/l dipotassium hydrogen phosphate3 g/l Carbopol .RTM. 946 (Carboxyvinyl polymer with an extremely high molecular weight)3 g/l (R)-2-phenoxypropionic acid2 mg/l iron(II) sulfate 1-hydrate100 .mu.g/l zinc(II) sulfate 4-hydrate300 .mu.g/l boric acid200 .mu.g/l cobalt(II) chloride 6-hydrate10 .mu.g/l copper(II) chloride 2-hydrate20 .mu.g/l nickel(II) chloride 6-hydrate30 .mu.g/l sodium molybdate 2-hydrate______________________________________

The pH was adjusted to 6.8 with 5N sodium hydroxide solution. Glucose and phosphates were each autoclaved separately at 121.degree. C. for 10 minutes. The (R)-2-phenoxypropionic acid was dissolved in water with a little 5N sodium hydroxide solution and sterilized by filtration. The remaining medium was autoclaved at 121.degree. C. for 10 minutes. 20 ml portions of the sterile medium were introduced into sterile 100 ml Erlenmeyer flasks, which were provided with a sterile cotton plug. One loop of spores of the Aspergillus niger ATCC 11394 fungus strain was used to inoculate one flask. The flask was then incubated at 28.degree. C., shaking at 250 rpm, for three days. Then 1000 .mu.l of fermentation broth were removed, 100 .mu.l of 1N HCl and 800 .mu.l of ethyl acetate were added, and the mixture was vigorously mixed for 15 seconds. 700 .mu.l of the organic phase were carefully removed and evaporated in a test tube under a gentle stream of nitrogen at 50.degree. C. The residue was dissolved in 70 .mu.l of ethyl acetate and transferred quantitatively into a test tube for the gas chromatography. To this were added 30 .mu.l of N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA). The sample was then examined by gas chromatography. An authentic sample of 2-)(4-hydroxyphenoxy)-propionic acid was sued as reference.

EXAMPLE 4

A liquid nutrient medium was prepared with the following ingredients:

______________________________________40 g/l glucose10 g/l yeast extract0.5 g/l magnesium sulfate 7-hydrate0.05 g/l manganese sulfate 1-hydrate1.5 g/l potassium dihydrogen phosphate3.6 g/l dipotassium hydrogen phosphate20 g/l (R)-2-phenoxypropionic acid2 mg/l iron(II) sulfate 1-hydrate100 .mu.g/l zinc(II) sulfate 4-hydrate300 .mu.g/l boric acid200 .mu.g/l cobalt(II) chloride 6-hydrate10 .mu.g/l copper(II) chloride 2-hydrate20 .mu.g/l nickel(II) chloride 6-hydrate30 .mu.g/l sodium molybdate 2-hydrate______________________________________

The pH was adjusted to 6.8 with 5N sodium hydroxide solution. Glucose and phosphates were each autoclaved separately at 121.degree. C. for 10 minutes. The (R)-2-phenoxypropionic acid was dissolved in water with a little 5N sodium hydroxide solution and sterilized by filtration. The remaining medium was autoclaved at 121.degree. C. for 10 minutes. 50 ml of the sterile medium were introduced into a sterile 100 ml Erlenmeyer flask, which was provided with a sterile cotton plug.

After inoculation wit 2.5 ml of a 72 h-old preculture of Beauveria bassiana ATCC 7159 (cultured in the same medium but in the presence of only 5g/l (.eta.)-2-phenoxypropionic acid), the mixture was incubated at 28.degree. C., shaking at 200 rpm, for 4-5 days. The subsequent analysis by gas chromatography revealed that the acid had been 98% hydroxylated.

98% of the (R)-2-phenoxypropionic acid had been oxidized to (R)-2-(4-hydroxyphenoxy)propionic acid. The result was the same when the S enantiomer or the racemate of 2-phenoxypropionic acid was employed.

EXAMPLE 5

A novel strain of Aspergillus niger was isolated from a humus-containing soil sample by the method of DREWS (Mikrobiologisches Praktikum, 3rd edition, Springer Verlag, pages 51-53 (1976)). This strain (No. 1777) was tested by the method of Example 3. 99.8% of the acid had been oxidized after 3 days.

EXAMPLE 6

Further strains of the species Aspergillus niger were tested as described in Example 3 and analyzed after fermentation for 3 and 7 days. The results are summarized in the table which follows:

______________________________________ Collection/ ConversionGenus/species Number (%) Days______________________________________Aspergillus niger ATCC 9142 34 3 ATCC 11394 98 3Aspergillus niger ATCC 26693 100 3Aspergillus niger ATCC 1015 83 3Aspergillus niger ATCC 10577 70 3Aspergillus niger ATCC 13794 99 3Aspergillus niger ATCC 11414 79 3Aspergillus niger ATCC 26550 79 3Aspergillus niger ATCC 9029 71 3Aspergillus niger ATCC 9642 41 3Aspergillus niger ATCC 32656 27 7Aspergillus niger ATCC 6275 11 3Aspergillus niger ATCC 16404 97 3Aspergillus niger ATCC 10575 53 7Aspergillus niger ATCC 10581 85 3Aspergillus niger ATCC 10549 27 7Aspergillus niger ATCC 1027 51 7Aspergillus niger ATCC 1040 97 7Aspergillus niger ATCC 10553 14 7Aspergillus niger ATCC 10864 65 7Aspergillus niger ATCC 1004 79 7Aspergillus niger ATCC 16880 99 7Aspergillus niger ATCC 16888 54 7Aspergillus niger ATCC 6273 89 7Aspergillus niger ATCC 7797 79 7Aspergillus niger ATCC 7983 90 7Aspergillus niger ATCC 10574 100 7Aspergillus niger ATCC 10578 100 7______________________________________

EXAMPLE 7

Further fungi were tested as in Example 3 and analyzed after fermentation for 3 and 7 days.

______________________________________ Collection/ ConversionGenus/species Number (%) Days______________________________________Aspergillus ATCC 8740 7 7carbonariusAspergillus ATCC 6276 5 7carbonariusAspergillus ATCC 10254 62 7foetidusAspergillus ATCC 26850 7 3parasiticusAspergillus DSM 63357 5 3sclerotiorumAspergillus CMI 112328 17 3sclerotiorumAspergillus CMI 116935 15 3sclerotiorumBeauveria bassiana ATCC 7159 99 3Paecilomyces ATCC 26853 63 7farinosusSclerotium rolfsii ATCC 15206 85 7Sclerotium rolfsii ATCC 15204 19 7Sclerotium rolfsii ATCC 15203 89 7Sclerotium rolfsii ATCC 15201 81 7Sclerotium rolfsii ATCC 15195 85 7Sclerotium rolfsii ATCC 26326 4 7Sclerotium ATCC 15196 1 7delphiniiCoprinus cinereus ATCC 20120 2 7______________________________________

Claims

1. A process for the preparation of 2-(4-hydroxyphenoxy)propionic acid by fermentation, wherein hydroxylation takes place substantially only in the p-position, which process comprises oxidizing 2-phenoxypropionic acid of salts thereof under aerobic conditions in the presence of a 2-(4-hydroxyphenoxy) propionic acid activity-producing, soil-inhabiting microorganism selected from the group consisting of a Streptomycetes bacteria and a fungus.

2. The process of claim 1 wherein the microorganism is an Aspergillus niger fungus.

3. The process of claim 1 wherein the microorganism is a Streptomycetes bacterium.

4. A process as defined in claim 1 wherein the enantiomers of 2-phenoxypropionic acid are employed.

5. The process of claim 1, wherein the microorganism is a fungus selected from the group consisting of Beauveria bassiana, Paecilomyces farinosus, Sclerotium folfsii, Sclerotium delphinii, and Coprinus cinereus.