CA2121049A1 - The preparation of halogenated hydroxyphenylacetic acids - Google Patents

Abstract

The invention concerns a new method of producing, by fermentation, compounds of the general formula (Ia) and (Ib), in which R1 is a hydrogen, fluorine, chlorine or bromine atom and R2 is a fluorine or chlorine atom.

Description

- ~21~i-19 O.Z. 0050/42777 The preparation of haloqenated hydroxyphen~lacetic acid~
The present invention relate~ to a novel proce~
for the preparation, by fermentation, of compound~ of the S formulae Ia and Ib R2 ~ `., Rl ~ CH2COOH Ia ~:

OH

OH

Rl ~ -CHzCOOH Ib where Rl is hydrogen, fluorine, chlorine or bromine, and R2 is fluorine or chlorine.
Co~pounds of th~ formulae Ia and Ib are valuable intermediates in the preparation of dye~ and phar~aceutical~.
It i~ known that microorgani~m~ are able to hydroxylat~ aro~atic compounds. The hydroxylation of th~
aromatic ring in many ca~e3 represent~ the first step in a 3equence of reaction which lead~ to ~he breakdown of the relevant ~ub~tanc~.
Yoshizako et al. (Agric. Biol. Chem. 49 (3), 1985, 877-879) di~cloge that the breakdown of phenyl~
acetic acid in variou~ fungi takes place via the inter-mediate 2,5-dihydroxyphenylacstic acid (homo~entisic acid). Theq~ fungi include, for example, tho3e of the genera A~pergillus, Fu~ariu~, Gibberella, Mucor, Pellicularia, Penicillium, Phellinus- and Rhizopus.
However, only very small amount~ of homogentisic acid are produced a~ metabolite of phenylacetic acid because it is 212 I tJLl~

- 2 - O.Z. 0050/42777 usually further broken down by cleavage of the aromatic ;~
ring.
It is an object of the present invention to provide a fermentation process for preparing compounds of ;~
the formulae Ia and Ib which starts from easily obtain-able starting compounds and provides good yields.
We have found that this object is achieved by the process, defined in the first paragraph, for the prepara- -tion, by fermentation, of compoundR of the formulae Ia and Ib c~2COO~ Ia OH
R1 ~ CH2COOH Ib R2 ~ ~
-where Rl is hydrogen, fluorine, chlorine or bromine, and R2 i~ fluorine or chlorine, which provide~ particularly good yieldR when compounds of the formula II

Rl~CHz C II
\R3 where Rl and R2 have the abovementioned meanings, and R3 is hydroxyl, methoxy or amino, are hydroxylated in the pre~ence of a microorganism which is able to hydroxylate, but not utilize as C source, 2 ~ 2 ~ ~,3 ~;c .' - 3 - O.Z. 0050/42777 compounds of th~ formula II.
The starting compounds of the formula II required for the proce~3 according to the invention are known.
They can be prepared, for example, by processes described in Houben-Weyl, Methoden der organischen Chemie, Volume 8.
The conversion of the abovementioned starting compounds II into compound~ of the formulae Ia and Ib by microorganisms compri~es hydroxylation in the position para to R2. If R2 i~ in po~ition 2 of the starting com-pound, the proeess according to the invention results in a 2-halo-5-hydroxy derivative of the formula Ia.
If R2 is in position 3 of the starting compound, ' the result i~ a 5-halo-2-~ydroxy derivative of the formula Ib.
Radical~ in po~ition 4 of the aromatic nueleu~, ~such as hydrogen or halogen, are unchanged in the proce~
according to the in~ention. Starting compounds which are preferably u~ed have hydrogen i~ position 4.
8e~ides hydroxylation on the aromatic ring, the proce~s according to tho invention may al80 result in the modification of the carboxyl. Thu~, a methyl ester or an ~m; de i-Q converted in~o the free acid or its salt. If a free acid or it~ salt i8 u~ed as ~tarting compound, this group is not alt~red by th~ proce~O
Th~ m~croorganism~ suitable for the process according to the invention on the on~ hand mu~t have the ability to hydroxylat~ th~ ~tarting compound~ on the aromatic nucleu~, but on th~ other hand they must not utilize the ~tarting compound a~ carbon source.
~icroorganism3 of this typ~ are ~xpediently obtained by mutation of wil~-type ~trains which have the ability to hydroxylate aromatic ring~.
Preferably u~ed as microorgani~m~ are fungi, in particular Deuteromycete~. Tho~Q of the ~enu3 Beauveria are particularly prefe~red, e~pecially of the species ~eauveria bas~iana (DSM 665Q).
, ,~ .

~21~
_ 4 _ O.Z. 0050/42777 It is easy to e~tablish whether a microorganl~m i3 3uitable for hydroxylating the starting compound in the required manner on the aromatic nucleu~ with the aid of analytical method~, eg. gas chromatography, using the nutrient medium.
The starting compound to be hydroxylated is expediently added to the nutrient medium and, during the course of cultivatlon, it is examined to find whether the starting compound decrease~ and the required hydroxylated compound occur3 as meta~olite. Metabolites can be identi-fied by conventional methods such as resting cell experi-ments, use of inhibitors or isotope labeling.
Microorgani3ms suitable for the proce~ according ~ to the invention can be found by examining the ability to hydroxylate aromatic compounds not on the required starting compound itself but on a compound which is ~tructurally related thereto. For example, the convercion of phenylacetic acid into 2,5-dihydroxyphenylacetic acid (homogentisic acid) can be used a~ a criterion for the hydroxylation by ~uitable microorgani3m~.
The mutant3 generated from the ~uitable micro-organism3 are expediently no longer able to use the ~tarting compound a~ carbon source.
Known microbiological technique~ can be employed to generate such mutant~. ~11 conven~ional methQd~ can be used to induce mutation~, ~uch as the u3e of mutagenic sub~tance~, eg. nitro~oguanidine r ethyl methane~ulfonate, ~odium nitrite, or expo~ure to electromagnetic radiation such a~ W, gamms or X-rayR. It i8 al90 pos~ible to u~e transposable genetic elements for the mutagene~is.
Various propertie~ can ~e utilized to i~olate the mutants, such a~ the inability to grow on phenylacetic acid a~ sole C ~ource or the vi~ually evident brown coloration resulting from the homogentisic acid produced.
It is po~sible at thi~ point, if nece~ary, also to carry out an enrichment of the mutants which are sought.
The proce~s according to the invention is carried - ~ .4~ 3 4 ~`3 _ 5 _ o.z. 0050/42777 out with suitable microorganisms which are cultivated in a nutrient medium which contain~ the starting compound in a concentration of from l to 20 g/l, preferably from 5 to 15 g/l.
s The cultivation time depends on the starting compound and the microorganism; as a rule it is a few days. The cultivation i3 expediently continued until there i3 virtually quantitative conversion of the st~rt-ing compound.
The cultivation can be carried out in a con~
tinuous or batchwise procs3s; however, a batchwise procedure i3 preferred.
The halogenated hydroxyphenylacetic acid can be , i~olated and purified from the nutrient medium by conven-tional methods. It is expedient to separa~e the solid biomass from the nutrient medium, to extract the required product, eg. with an organic 801vent ~ and to i~olate it from the extracted phase, if nece3sary after further purification, eg. by cry3tallization.
The invention i3 further explained by the example~ which follow.

Preparation of a mutant (Lu 6577~ of Beauveria ba~iana The fungu Beauveria bas~iana DSM 6650 was mutated u~ing N-methyl-N'-nitro-N-nitro~oguanidine (MNNG). A su3pen~ion of fungal ~pores in 0.1 M pho phate buffer pH 7.0, 0~1% by weight polyethoxysorbitan oleate (Tween~ 80) wa~ adju~ted to a titer of 107 spore~ per ml.
10 ml of thi~ ~pore ~u~pen~ion were adju~ted to a con-centration of 0.2 mg/ml MNNG by addition of a itock solution of S mg/ml MNNG (dissolved in dimethylform-amide~, and incubated at 30C, shaking gently, for 15 min. The spore were then harve~ted by centrifugation (5,000 rpm for 5 minute~) and wa~hed twice with 10 ml Tween buffer. The spores were taken up in Tween buffer, diluted and plated out on complex medium. Compari~on with untreated ~pores regularly ~howed in ~everal experiments ~.

- 6 - O.Z. 0050/42777 a survival rate of from 1 to 2% for the mutated spores.
The mutated spore~ were plated out on agar plate~
containing complex medium of the following composition and incubated at 30C for 5 day~:
g/l D-glucose g/l yeast extract 3.6 g/l K~PO4 1.5 g/l XH2PO~
0.5 g/l MgSO~ x 7 H2O
0 . 05 g/l MnSO4 x H20 2 ml/l trace element solution 20 g/l agar Trace element ~olution:
~ 200 mg/l iron~II) sulfate monohydrate 10 mg/l zinc(II) ~ulfate tetrahydrate 3 mg/l man~anese chloride tetrahydrate 30 mg/l ~oric acid 20 mg/l ~obalt(II) chloride hexahydrate 1 mg/l copper(II) chlorid~ dihydrata 2 mg/l nickel(II) chloride hexahydrate 3 mg/l sodium molybdate dihydrate 500 mg/l ethylenediaminetetraacetic acid (EDTA) Toothpicks were u~ed to inoculate a small piece of mycelium from the re~ulting 3ingle colonie~ into te~t ~ube~ each containing 2 ml of the following minimal medium with ph~nylacetic acid a~ ~ole carbon source:
5 g/l phe~ylacstic acid 5 g/l (N~)zSO4 3.6 g/l K~HPO~
1.5 g/l KH2PO~
0.5 g/l ~gS~4 x 7 ~2 o.05 g/l MnS04 X ~2~
2 ml/l trace element ~olution The mixture~ were shaken (180 rpm) at 30C for ~-7 day~. Clone~ which had not grown densely on the medium during this period were further characterized. The proportions of clones which did not grow ranged from 3 to 2 ~ v~

- 7 - O.Z. 0050/42777 8% in various experiments.
The clones to be characterized further were inoculated from the retained sample into 2 ml of complex medium containing 2 g/l phenylacetic acid in each case, and cultivated 3haking at 30C for 10 days.
The culture3 were then centrifuged and a photo-metric assay was carried out on the culture supernatant:
0-5 ml of 1-6~ NaNO2 and 0.2 ml of 1 M ~2S4 were added to 0.5 ml of culture ~upernatant and incubated for 10 min.
Then 0.25 ml of 0.2% by weight ~DTA in 2.3 M NaOH was added and the samples were measured at 450 nm with the blank a~ reference. For the blank, water in place of any NaNO2 solution was added, otherwise the procedure was the ~ same. ~
Clone~ which had an extinction A450 > 1.8 in the photometric as~ay (1-5% of the assayed clone~) were further investigated in ~haken flask experiment~. For thi in each ca~e a preculture of the clone~ was made up in complex medium containing 2.5 g/l phenylacetic acid ~30 ml in 250 ml Erlenm~yer flasks3. After ~haking at 3 0 C f or 3 day3, 5 ml portion~ of the preculture were used to inoculate a mai~ ~ulture containing 10 Gy/l phenylacetic acid, which wa~ ~haken at 309C. A sample waR
taken after 3 and after 7 days and analyzed for the content of phenylacetic acid and it~ derivative~ by ga~
chromatography.
1 ml of the culture wa~ removed, 100 ~,1 of 5 M
~Cl and 800 ~1 of ethyl acetate wer~ added, and the mixture was mixed for 15 ~ and centrifuged at 12, 000 g for 2 min. 50 ~1 of the organic phase were remo~red and 50 ~l of N-m¢thyl-N-(trLm~thylsilyl)trifluoroacetamide (MSTFA) were added.
The samples were ~ubjected to ga~ chromatography ( 165~C i~oth~rmal, column: methyl~ilicon~ 12.5 m, Hewlett-Packard, 1 ~1 injected~. In the chromatogram, phenylacetic acid appeared after 1.7 min and homogenti~ic acid after 7.8 min.

- 2 ~ ç~
- 8 - O.~ 0050/42777 In this assay, the mutant Lu 6577 showed complete conver~ion of phenylacetic acid to homogenti~ic acid after 7 days.

Preparation of 2-chloro-5-hydroxyphenyla~etic acid from 2-chlorophenylacetic acid The mutant Lu 6577 was inoculated in 5 x 330 ml of the foll~wing medium in 1 1 Erlenmeyer flasks and incubated aerobically, shaking at 180 rpm, at 30C for 3 days:
0.5 g/l 2-chlorophenylacetic acid g/l D-gluco~e g/l yea~t extract ~ 0.5 g/l MgSO4 x 7 H2O
1.5 g/l KH2PO4 3.6 g/l R2HPO4 2 ml/l trac~ element solution Thi~ preculture was used tc inoculate a 10 1 fermenter containing the 3am~ medium and a 2-chloro-phenylacetic acid concentration of 5 g/l. The fe~menter was stirred at 600 rpm and 1 volum~ of air per volume of the reactor wa~ pa~sed through per minute. Whenever the reaction had progre~ed until the 2-chlorophenylacetic acid concentration had fallen to 0.5-2.5 g/l, each time 50 g of 2-chlorophenylacetic acid w~re metered in a~ a ~:
20% ~trength ~tock solution in water. A to~al of 500 g cf 2-chlorophenylacstic acid wa~ reacted in thi~ way. After this amount wa~ reached, the culture wa~ incubated further without metering in 2-chlorophenylac~tic acid.
When the reaction wa~ complete, the cell3 were removed by centrifugation. The culture ~upernatant wa~ adjusted to p~ 2 with HCl, and the resulting 2-chloro-5-hydroxy~
phenylacetic acid was obtained by extraction with ethyl acetate. -2~ Ol~9 _ g - o.Z. 0050/42777 Preparation of 5-chloro-2-hydroxyphenyl acetic acid from ~-3-chloroacetic acid The conversion wa~ carried out as described in Example 2. 3-Chlorophenylacetic acid wa~ employed as ;~
starting compound. The result i~ shown in Table 1.

Preparation of 2-fluoro-5-hydroxyphenylacetic acid from 2-fluorophenylacetic acid The conversion wa~ carried out as described in Example 2. 2-Fluorophenylacetic acid was employed as starting compound in a concentration of O.5 g/l (pre-culture) and 3 g/l (main culture). The result is shown in ' Table 1. ~

Conversion of halogenated phenylacetic acids with Lu 6577 _ . ~
Ex . Precur~or Product Amount Fer - -:
f isol menta^
. ~ rod. ttion . ~ .

~CI}2-COO~ ¦R ~3CHz-CoC~ g ~ Y ¦

_ __ _ ~' 2 Rl - H Rl - H 50 11 Ra-~ Cl R2 . ~1 R3 -~ ~ ~.3 - OH
. .
3 . Rl ~- H Rl - H 5 20 3 0 Ra _ H R~ - OH
R3 Cl R3 -- Cl _ _ l 4 Rl -- H Rl -- H 15 10 ; .-Ra _ F RZ _ F
_ R3 - H R3 - OH I ~::
., - ~'.

Claims (4)

We claim

1. A process for the preparation, by fermentation, of compounds of the formulae Ia and Ib Ia Ib where R1 is hydrogen, fluorine, chlorine or bromine, and R2 is fluorine or chlorine, which comprises hydroxylating compounds of the formula II

II

where R1 and R2 have the abovementioned meanings, and R3 is hydroxyl, methoxy or amino, in the presence of a microorganism which is able to hydroxylate, but not utilize as C source, compounds of the formula II.

2. A process as claimed in claim 1, wherein a fungus is used as microorganism.

3. A process as claimed in claim 2, wherein a representative of the Deuteromycetes is used as fungus.

4. A process as claimed in claim 2, wherein a fungus of the genus Beauveria is used as microorganism.