CA1265083A

CA1265083A - Process for the preparation of aromatically substituted l-amino acids

Info

Publication number: CA1265083A
Application number: CA000493202A
Authority: CA
Inventors: Reinhold Keller; Merten Schlingmann
Original assignee: Hoechst AG
Current assignee: Hoechst AG
Priority date: 1984-10-18
Filing date: 1985-10-17
Publication date: 1990-01-30
Anticipated expiration: 2007-01-30
Also published as: ES8609186A1; IL76740A0; DK475585A; PT81320A; ZA857971B; NO854127L; EP0178553B1; ES547901A0; CS738585A2; HUT39414A; JPS61108398A; EP0178553A3; CS258132B2; GR852491B; FI854025L; DE3438189A1; FI854025A7; ATE56472T1; PT81320B; DE3579663D1

Abstract

Abstract of the disclosure Aromatically substituted L-amino acids are ob-tained particularly advantageously by enzymatic cleavage of the racemate of the lower alkyl esters using.alpha.-chymo-trypsin, extraction of the D-ester from the basified reac-tion mixture using an organic solvent, racemization, ex-traction of the D,L-ester using an aqueous acid, and re-turn, after concentration, to the process, and isolation of the L-amino acid from the alkaline residue from extrac-tion after acidification. The enzyme is advantageously immobilized on an inorganic carrier. The racemization is advantageously carried out on the extract, that is to say without isolation of the D-ester.

Description

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- 2 HOE 84/F 248 German Offenlegungsschrift 2,215,853(U.S. 3.813.317) discloses the CQnversion of rac~mic phenylalanines wlth one and tWD
ring substituents in~o the corresponding L amino ac;ds by first esterify;ng the racemate ~ith an alkanol hav;ng up S to 4 carbon atoms, subject;ng the racemic esters to the action of a chymotryps;n at an acid pH, isolating the L~
amino acid by precipitation and where appropriate~
extracting the ester of the D-amino acid from the filtrate and hydrolyzing it. The preferred enzyme is~-chymo-trypsin, and the preferred pH range for the enzymaticcleavage is 5 to 6u The liquid remaining after removal of the L-amino ac;d ;s basif;ed before the extraction of the ester of the D-amino acid~
It has no~ been found that aroma~ically substitu-ted L-amino acids are particularly advantageously access-ible by first basifying the mixture obtained from the enzymatic cleavage, then extracting the ester of the D-amino acid, racemizing, ~here appropriate after dryin~ of the extract, the ester9 and returning the racemate to the process. After extraction of the ester of the D-amino acid, the L-amino acid is isolated from the alkaline res;-due after acidification.
Thus the ir,vention relates to a process for the preparation of aromatically substituted L-amino acids by cleavage of the racemic lo~er alkyl esters using -chymo-trypsin in a ~eakly acidic aqueous medium, which comprises the reaction mixture being made ~eakly alkaline, the ester of the D-amino acid being extracted with an organic sol-vent, the ester bein~ racemized and returned to the pro-cess~ and the L-amino acid bein~ isolated from the aqueous alkaline extract after acidification. Preferred embodi-ments of this invention are illustra~ed in detail belo~
and are defined in the patent claims.
Particularly suitable aromatically substituted amino acids are tryptophan, and especially phenylalanine, tyrosine and 3,4~dihydroxyphenylalanine.
The esterification of the racemic amino acids can . ~ -., , be carried out in accordance ~ith the statements in German OffenLegungsschr;f~ 2,215~853 n Lo~er alkanols having up to 4 carbon atoms ~except tert.-bu~anol~ are preferred~
in p3rticular ~ethanol9 ethanol, n-propanol, isopropanol and n~butanol.
The~ -chymotrypsin is advantageously used in the immobilized form, inorgan;c carri~rs being pref~rred.
This immobilized enzyme is then brought into contact with the aqueous substrate solu~ion in a fixed or flu;di~ed bed. The reaction is preferably carried out at 20 to 45G, advantageously at 30C - 40C, and at a pH in the range 4.5 to 6.5, advantageously at a constant pH ;n the ranQe 5.0 to 600, depending on the ester used. The sub-strate concentration is not critical per se, and 5-~5% by ~eight is advan~ageous, in particular 10 to 15X by weight The aqueous solution of the L-amino acid and o~
the ester of the D-amino acid ~hich is obtained after the enzymatic cleavage is ~ade ~eakly alkaline, advantageously to a pH in the range 7.5 to 8.5, advantageously 8.0, and extracted - advantageously at 20 to 40C, in particular room temperature - using a suitable organic solvent.
The organic solvent ~hich is selected for the ex-traction is advantageously one whose miscibility ~ith uater is lo~, since then the race~izat;on can be carried out in a particularly advantageous manner ~ithout isola~
tion of the ester (the presence of water during the race-mization can give rise to side reactions, such as hydroly-sis). For this reason, a solvent ~hich forms an azeotrope ~ith water and hence allo~s easy drying of the extract is advantageous. However, i~ is also possible, even though not generally advantageous, to remove completely the sol-vent ~hich has been used for the extraction and to race-mize the ester as the solid.
Suitable solvents for the extraction are aliphatic hydrocarbons, such as hexane, and in particular aroma~ic hydrocarbons, such as toluene or xylene, ethers, in par-ticular lower dialkyl ethers~ such ~s d~ethyl ether, di~
isQpropyl ether or di-n-butyl ether9 ketones, in particu-lar louer dialkyl ketones, such as nethyl ethyl ketone or :
,. ,, . : :
~: .. : , :
~,;:: . "..........

_ 4 _ methyl isobutyl ketone9 halogena~ed hydrocarbons, ;n par-ticular chlorinated lower aliphatics~ such as methylene chloride, chloroform or ~etrachloromethaneO as ~ell as esters, in particular lo~er alkyl esters of lo~er alipha-tic carboxylic acids, such as ethyl acetate or butylacetate.
The racemization can be catalyzed in a manner kno~n per se by keto compounds and/or acids. Thus, in the case where the solvent is not a ke~one, addition of alde-hydes or ketones can considerably increase the rate ofracemization. The race~ization of opt;cally active amino acid esters in ketones, ~here appropriate uith the addi-tion of an acid, is disclosed in the published Japanese Patent Application 109 912/1979. It ;s also possible in this kno~n racemi~ation reaction ~o add a solvent, such as toluene, tetrachloromethane or methanol~ The racemiza-tion of free opt;cally active amino acids us;ng aldehydes and acids is disclosed in the publisehd European Patent Application 57,092 and in S~ Yamada et a(., J. Org. Chem.
48 (1983) 843~846. I~ is advantageous for ~hese keto com-pounds to be used in catalyt;c amounts, preferably 0.001 to 0.1 mole per mole of ester. Acetaldehyde and salicyl-aLdehyde are preferred.
The D, L mixture produced in the racemization is then extracted from the racemization mixture using slightly acidified ~ater, during ~hich the ester is con-verted in~o the salt form. This aqueous solution is ad-justed to the desired concentration, advantageously 5 to 15, in particular 10 to 150 X by ~eight, by addition of ~0 new substrate and is returned to the process~
The L-amino acid is isolated from the ~eakly alka-l;ne aqueous residue containing it by acidification. De-pending on the solubility of the acid, it can crystallize immediately or after concentration. The acid can also be isolated in a kno~n manner by extraction using suitable solvents. The purif;cation ;s carried out in a known manner.
The process according to the invention permits aromatically substituted L-amino acids to be obtained in :~
,, , :, .

~- .

; - , : , :, ~', .: . , yields exceeding 90% and in an opticaL purity exceeding 98~. The combina~ion of enzymatic cleavageO extraction of ~he D-es~er~ ;ts racemization and return resul~s in a par~icularly advantageous combina~;on of these operat;ons ~h;ch also allows a cont;nuous embodiment of the process.
The invention is illustrated in detail by the examples ~hich follo~. Unless other~ise stated, percen-tage data relate to ~eight.
~.~
mmobiLi~ation of~-chymotrypsin The enzyme can be immob;lized on aluminum silicate by the 0ethod of Hal~achs et al.O Biotechnology and Bio-engineering XIX t1977) 1667~1677. Ho~everO the following procedure is part;cularly preferred:
138 g of dry silica gel tparticle size 0.1 to 0.3 mm~ are taken up in 1 l of a 3.5X strength solution of 3-aminopropyltrietho~ysilane in acetone~ and the m;x ture is gently stirred at room temperature for 2 hours.
The acetone is then removed in a rotary evaporator, and the silica gel is dried ;n a vacuum drying oven at 100C
for 8 hours. The product contains 0.89 mmol of amino groups per gram of sil;ca gel. This aminated silica gel is t3ken up in ~OQ ml of 25X strength aqueous glutaraldehyde solution and is main~ained under vacuum tabout 130 mbar) at room temperature for 2 hoursO The carrier ;s then fil-tered off and thoroughly washed ~ith de;onized water.
138 9 of the s;lica gel thus activated are intro-duced ;nto a solu~;on of 13~8 g ofo~ochymotrypsin tsup-plied by Novo, 800 S Oral Grade) ;n 300 ml of 0.5 M phos~
phate buffer ~pH 7.5)~ and the mixture is st;rred at room temperature for 4 hours~ The catalyst ;s then fil~ered off and ~ashed w;th 1 l each of deionized water, saturated sodium chlor;de solut;on and deion;zed water. For stor-age, the immobil~zedc~chymotrypsin is taken up in 5DO ml 35 of 0.1 M phosphate buffer solution ~pH 7.$, 0.1 mmol sod;um az;de).
To determ;ne the act;vity, 1 9 of the immob;lized '~chymotrypsin is added to 50 ml of a 10X strength aqueous solut;on of D,L-phenylalan;ne methyl ester hydrochloride, .

. .
,:

and the solution ;s adjusted ~o pH 6.0, equilibrated at 30C and stirredO The pH is ma;ntained constant by addition of 0.1 N sodium hydrox;de solution us;ng an auto-matic burette. The amount of sodium hydroxide solution consun,ed is the measured variable and is directly propor-tional ~o the concen~ration of L-phenylalanine~
Activity: 338 U/g of carrier (dry) = 3~35 kg of L-phenyl-alanine/kg of carrier and h (U = international unit for enzyme activity, 1 U = 1 ~mol substrate minute conversion~

Enzymatic cleavage of racematP
100 9 of D,L-phenylalanine methyl ester hydrochlo-ride tresidual content of D,L~phenylalanine below 0.3%, HPLC detection limit) are placed in a reaction vessel ~hich can be thermostated and is equipped with an internal stirrer, pH electrode and automatic burette, and are dis solved in 1 l of deionized water. The pH of ~he solution is adjusted to 6.0 ~ith 5 N sodium hydroxide solution ~9~5 ml). The solution is equilibrated at 30C and is circulated by pumping at a flo~ rate of 5.0 l per hour through a column ~hich is packed ~ith 150 ml of the enzyme immobilized as ;n Example 1 tdiameter 5.D cm, bed height 8 cm).
~5 The pH of the solution is maintained at 6.0 during the reaction by addition of 5 N sodium hydroxide solution t37.B ml)~ After a reaction time of 25 minutes, according to HPLC analys;s the conversion achieved is 49.2X based on D,L phenylalanine methyl ester. The reaction is then stopped and the enzyme bed is ~ashed with 300 ml of de-ioni2ed ~ater~

Extraction of D-phenylalanine methyl ester and subsequent racemization The aqueous solution of D-phenylalanine methyl ester and L-phenylalanine obtained as in Example 2 is ad-justed to a pH of 8~0 by addition of 5 N sodium hydroxide solution ~36.5 ml), ~ith stirring, and~ at room tempera-ture, is ex~racted by shaking three times ~ith 300 ml each :: . .
'~.''' .: ' ' ' :' ~
, ~' "''' ~ '',,:

~im~ of methyl isobutyl ketone. According to HPLC analy-sis, after the th;rd e~trac~;on there ;s no longer any ester in the aqueous phase ~de~ection limit7 below 0~2X~.
14.D g of glacial acetic acid tOAS mole-equivalent based on D-ester) are added ~o the combined extracts, and the ~ater contained in the extract is removed by azeotropic distillation through a Vigreux column. The dry extract is heated under reflux for a further 2 hours, dur;ng ~hich the degree of racemization is measured by determination of the oDtical ro~ation of the solution using a polarime-ter. The solution is cooled to room temperature and is then extracted by shaking three times ~ith 300 ~l each ti~e of 0.05 N hydrochloric acid~ According to HPLC
analysis, 98.5% of the ester used is contained in the com-bined aqueous phase. This aqueous solution is adjus~edto 10X by addition of D,L-phenylaLanine methyl ester hydrochloride and ;s used for another enzymatic cleavage.
If n-he~ane, di-n-butyL ether, ~oluene or xylene are used as the extractant, then catalytic a~ounts of acetaldehyde, salicylaldehyde or benzaldehyde are added to increase the rate of the reaction~
In place of the glacial acetic acid, i~ is also possible to use hydrochloric acid, benzoic acid, citric acid, p-toluenesulfonic acid or formic acid. Without acids, the racemization takes place very much sLo~er and lasts more than 10 hours. Acid concentrat;ons exceeding 0.5 mole-equivalent br;ng about no further increase in the rate.
~e~
3û Working up and ;solation of L-phenylalanine The solution result;ng after the e~traction with methyl isobutyl ketone as in Example 3, ~hich contains about 3~5% of L-phenylalanine, is adjus~ed to pH 5~5 ~the isoelectric point of phenyLalanir,e~ using concen~rated hydrochloric acid, and is concentrated in a rotary evapo~
rator to a content of about 10% of L-phenylalanine~ The precipitated L-phenylalanine is induced to dissolve by heat;ng to 90C, and the solut;on is f;ltered hot. The clear solut;on ;s allowed to stand at roo~ temperature, ': ' ,:.. : , ' :'~

~5~

whereupon the L~phenylalan;ne separa~es out in large crys~
tals~ The mother liquor is s~ored a~ ~5C overnight, during ~h;ch ~hite crys~als separate out and are f;ltered off and ~ashed ~ith a lit~le cold ~ater. The crystals are dried ln a vacuum drying oven at 105C for 3 hours.
35.3 9 of L-phenylalanine~ or 92X based on 50 9 of l-phe-nylalanine nethyl ester hydrochloride used~ are obtained.
The optical purity is 98~5 to 99.5~.

Enzymatic cleavage of racemate-D,L-Phenylalanine ethyl ester In analogy to Example 2~ 150 9 of D,L phenylala-nine ethyl ester hydrogensulfate are dissolved in 1 l of deionized ~a~er. The pH of the solution is adjusted to 5.5 ~ith 5 N sodium hydroxide solution. The solut;on is equil;brated at 40C and is ~irculated through the column by pumping at a flow rate of 60 l/hourO the pH be;ng main-tained at 5.5 throughout the entire reaction by addit;on of 5 N sodium hydroxide solutionO
Further procPss;ng ls carr;ed out in accordance ~ith Examples 3 and 4~
~e~ .
Enzymatic cleavage of racemate:
D,L-Phenylalanine n-propyl ester D,L-Phenylalanine n-propyl ester hydrogensulfate a e reacted in analogy to Exa~ple 5, but the pH is adjus-ted to and naintained at 5.3. Further ~orking up is car-ried out in analogy to Examples 3 and 4~
It is also possible to use the isopropyl or n-butyl ester of D,L-phenylalanine ~ith equaL success.

L-Tryptophan In analogy to Example 2, 50 9 of D,L-tryptophan methyl ester hydrochloride are dissolved in 1 l of deion-ized ~ater, and the enzymatic clezvage is rarried out asdescribed~ The D-tryptophan ~ethyl ester is extracted as in Example 3 from the aqueous solution thus obtained and, in analogy to Example 4, the re~aining aqueous phase is adjusted to pH 5.9 ~isoelectric point of tryptophan) using :: :
; "

~2~5~13 _ 9 _ concentrated hydrochloric acid, and is concentrated ;n a rotary evaporator to a content of about 5X of L-trypto-phan. The crystals ~hich have separated out are filtered off, washed ~ith cold ~ater and dried at 105C ~or 3 hours.
19.4 ç, or 97X based on 25 ~ of L-tryptophan methyl ester hydrochloride used, are obtained. The optical purity is 98.7%.

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Claims

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A process for the preparation of aromatically sub-stituted L-amino acids by enzymatic cleavage of the race-mate of the lower alkyl esters using .alpha.-chymotrypsin in the weakly acid pH range and extraction of the ester of the D form, which comprises the reaction mixture obtained in the enzymatic cleavage being made weakly alkaline, the ester of the D-amino acid being extracted from this alka-line solution using an organic solvent, and being race-mized, whereupon the racemate is returned to the process, and the extracted alkaline solution is made weakly acid, whereupon the L-amino acid is isolated from it.

2. The process as claimed in claim 1, wherein the .alpha.-chymotrypsin is used in the immobilized form.

3. The process as claimed in claim 2, wherein the -chymotrypsin is immobilized on an inorganic carrier.

4. The process as claimed in claim 1, wherein an organic solvent of low miscibility with water is used for the extraction of the D-ester.

5. The process as claimed in claim 2 or claim 3, wherein an organic solvent of low miscibility with water is used for the extraction of the D-ester.

6. The process as claimed in claim 4, wherein the solvent used forms an azeotrope with water.

7. The process as claimed in claim 1, 2 or 3, wherein the extract, where appropriate dried, of the D-ester is subjected to the racemization.

8. The process as claimed in claim 1, 2 or 3, wherein the racemized ester is extracted under acid conditions and, after concentration, is returned to the process.

9. The process as claimed in claim 1, 2 or 3, wherein the ester is an alkyl ester having 1 to 4 carbon atoms, excepting tert.-butyl.

10. The process as claimed in claim 1, 2 or 3, wherein the ester is a methyl, ethyl, n-propyl, isopropyl or n-butyl ester of phenylalanine or tryptophan.