CS223812B2 - Method of making the 6-aminopenicillane acid - Google Patents

Abstract

1348359 Enzymatic preparation of cepnalosporins and penicillins SNAM PROGETTI SpA 27 April 1972 [28 April 1971] 19726/72 Heading C2A [Also in Division C3] The invention comprises a process for the treatment of a substance which contains a ring structure characteristic of penicillins or cephalosporins to convert it to another substance containing the ring structure, which process comprises contacting a reaction medium containing the substance with a filament in which is distributed in finely divided form at least one enzyme capable of causing the desired conversion, the or each enzyme being encased in the filament in such a manner that the or each enzyme can effect conversion and that it or they are substantially retained in the filament during the contacting. The enzyme or enzymes may be incorporated in a filament formed from a nitrated, esterified or etherified cellulosic polymer, a polyolefine, a polymer or copolymer of acrylonitrile, an acrylate, methacrylate, vinyl ester, vinyl chloride, vinylidene chloride or styrene, a polyamide or polyvinylbutyral. Suitable polymers include ethyl cellulose, poly-γ- methylglutamate and cellulose triacetate. Hydrolysis of natural penicillins may be carried out by this process. Such penicillins include those of Formula I wherein R is alkyl of 1 to 10 carbon atoms, with from 3 to 10 carbon atoms, aralkyl, R<SP>1</SP>OCH 2 - or R<SP>1</SP>SCH 2 -, wherein R<SP>1</SP> is alkyl, alkenyl, phenyl or optionally substituted phenyl. The pH of the reaction medium may be buffered at 8. The filament may contain an acylase derived from bacterial cells such as E. coli, B. megaterium, Aerobacter aerogenes or Alcaligenes faecalis cells. The filament may contain an enzyme derived from Actinomyces or fungal cells, e.g. Aspergillus, Penicillum, Botrytis cinerea, Fusanium, Mucor, Alternaria or Streptomyces cells. Synthesis of some penicillins, e.g. ampicillin, may be effected by the process of the invention. The invention also comprises a filament in which is distributed in finely divided form at least one enzyme capable of causing conversion of a substance containing a ring structure characteristic of penicillins or cephalosporins to another substance containing the ring structure, the enzyme being present in the filament in a manner such that it is retained in the filament, when the latter is contacted by a reaction medium containing the substance and that it is capable of effecting the conversion whilst being so retained.

Description

The present invention relates to a process for the preparation of 6-aminopenicillanic acid by the hydrolysis of penicillin G, wherein an aqueous solution of penicillin G at a concentration of 1 to 6% w / v is hydrolyzed at pH 8 in the presence of penicillin acylase embedded in cellulose triacetate fibers.

The invention relates to a process for the production of an acid

6-aminopenicillan by enzymatic cleavage of natural penicillin G.

The method according to the invention is based on the use of enzymatic compositions which are rendered insoluble by depositing in the fiber. This results in a significant improvement in the properties of the end product and a significant simplification of operations and, consequently, a reduction in price.

Zn ^and Mo, that it is ^from several Tet ^6-l aminopenici rope ^and Selina ^{alkyl (which} just ^gives le ^6-APA), a valuable starting material for the production of semisynthetic penicillin.

Preparation of 6-APA by direct fermentation with Penicillium chrysogenum in the absence of a precursor is technically very complex and yields are very low.

Recently they have been proposed for chemical processes, which are relatively expensive, require a ^ ctálrá technology at the ^DU SLE ^DK instability in the starting and final products _ and cause product degradation, associated with considerable losses.

! When used enzymatic processes to ^{M p} ou un ^b IV ^{and EC} in NYC ^h o ^d I ^{h to} roorgamsmů ^to Produce ^p ter ^{é y} enicilinac lasu. Because, ^z is these cells may be used only once at a relatively high concentration (for reducing the time of hydrolysis), it is necessary from time to time by fermentation of cells containing the enzyme produced.

In addition, the extraction of the 6-APA hydrolytic product from the reaction mixture is very complex and the resulting product requires repeated crystallization and very often contains a proteinaceous material which, if not removed, for example by degradation with proteolytic enzymes, causes allergies when using the resulting peniciens.

All this increases the price and makes the production tec ^h nolo ^gi e ^considerable number ^ them that.

The process according to the invention represents a significant improvement in this respect.

Enzymattelte ^at the right ute ^f ENE in the structure of a fiber to maintain its effectiveness TO, THE ^{f p} a very long time. Samples of this type retained their efficacy for almost 12 months on repetitive working cycles.

In addition to the obvious advantages, the extended efficacy of the structures of the invention also has the advantage of simplifying the process, wherein the effect of the cost of the enzyme catalyst on the final product price is very small or almost negligible.

The present process for producing a ^6-h ydro aminopenicinanové carbamic acid ^l ýzou penicillin G, characterized in that an aqueous solution of PEI ^ i ^ i ^ i ^^ Lin G concentration. For 1-6 ^h ^ ^t mo Nost / volume h ^yd ro ^ly pn it observes a pH of 8 in the presence of a penicillin acylase en ute ^{f s} in the ^pickle of NEC ^{H -} celutezy triacetate.

The fiber containing the spinning enzyme is washed prior to use to remove adsorbed impurities and used to catalyze the reaction. Vzhtedem к the protein e ^ ^z e it ^from diffusing DO- reaction medium to give a highly pure product, which among other things contains the protein contaminants and hazardous because alergekých reactions. Catalyst which is a modification of the present invention ^b dy ^p U.S. extended ^{t the company,} the MFE ^p ou ^live discontinuously. The catalyst is contacted with the reaction mixture after completion of its mouth en ^{b i, t} j. ^{L hy} dro YZ ^y oditelí from REA ^{CZK nus} mixture and brought into st ^yk US new ^AR ěerst ^Gd: in ^E mixture.

The same catalyst may also be used ^from i ^{to t} pn ontinu ^AL n ^e m ^b uv caused ^to Oton. The penicillin solution in the column, suitably buffered, is passed through a column containing the enzyme strand. By appropriately adjusting the flow rate and the amount of fiber, it is possible to obtain an effluent with the highest degree of hydrolysis.

The enzyme catalyst is prepared as described in Italian Patent No. '

According to this patent, the enzyme-deposited fibers can be produced by spinning.

frame ^to mites of the ^L and ^{K t} vornýc well of ^poly mers ^hp * in which there are dispersed enzyme preparations are very small droplets of Veli ^{to the} ostium of the same ^{of DOV} j and ^{k are} the APIC ^ky emulsion.

^{"E l} him that" mo ^h ou Vol ^lákň ova ^t dry or wet. The fiber obtained contains small cavities inside which the enzymes are stored. Enzymes are separated from the surroundings very "thin membrane that prevents the escape of the enzymes and their dispersion in rea ^CZK caliber sweep ATE umornuje ^to jejteh was catalytically action.

Cellulose triacetate is used as the fiber forming polymer for the enzyme deposition in the process of the invention.

As the enzymatic material, a crude extract obtained from the cells of the bacteria (E. coli, B. Megaterium, Aerobacter aerogenes, Alcali genera faecalis, etc.) can be used.

During hydrolysis, which has a gain of 6-APA ^from his ev ^y c ^hook zed from a ^{n e} it mites ^to u ^p ou ^ITE penicillin in a concentration between ¹ and ^{from 6% (h} mΌ ^t nose ^{t / v)} ^ ^ o ^ ednos ncentraci 2 ^and 3 ° / o. ^The organic solution ^pH is maintained at - about 8.0 by the automatic addition of base.

When ^{the 1} mm oztizuá operation m ^{live longer} employed will suitably buffered solution of penicillin pro- J toga regulation of pH at various heights of the column is difficult.

The hydrolysis of penicillin G (benz lpemcШzu ^s) may be ^p rov ^Aad pn teprntě ive out of five of ^{d 15} to ⁶⁰ degrees Celsius, preferably from 37 to 40 ° C.

Example 1

While stirring the reactor dissolved 20 g of the triacetate celutezy (Fh-a) in ²⁸⁰ g of ME ^ ^ the ortho (C ^E r and ^{b). In} jrné NATO ^instance synthesized according ^to mites ^s enz ^y násleďujteta way ^of his manner: ES cells Escherichia coli (wet weight 60 g) obtained by the fermentation in a suitable medium in the presence of phenylacetic acid are converted into phosphate buffer 0.01M, pH 7.0. The crude product thus obtained was heated at 50 ° C for three minutes and then precipitated with ammonium sulfate until pH 5.5. The fraction obtained in the range of 25 to 75% of saturation was separated. This fraction was dissolved in 30 ml of pH 8 phosphate buffer containing 30% v / v glycerin. The resulting enzyme solution is added to the polymer solution. Emulsification of both phases is achieved by vigorous stirring for 30 minutes at 0 ° C.

The emulsion is transferred to a small reservoir maintained at -6 ° C and then spun under nitrogen. The coagulation of the fiber is carried out in toluene at room temperature.

The toluene was removed by vacuum drying for several hours.

In a glass column equipped with a thermostated jacket of 70 cm height and 3 cm diameter, 37 g of fibers were placed. The fibers are washed with water and glycerin until the proteins disappear from the wash liquor.

600 ml of a 2% w / v aqueous solution of penicillin potassium potassium (Squibb) was circulated through the column. The pH was maintained at 8.0 by continuous addition of 0.5 N NaOH using a constant pH device. The column is thermostatized to 37 ° C. After 5 hours, when the soda consumption rate decreased to 1/20 of the original value, the reaction mixture was withdrawn, cooled to 3 ° C, acidified with 2 N HCl to pH 3.0 (33.5 mL 2 N HCl 3) and extracted twice 200 ml of butyl acetate.

The first extract contains 298 mg and the second extract 2 mg of penicillin (as determined by hydroxylamine). Since the total amount of penicillin used was 12 g, the conversion was 97%. No decomposition products were detected by chromatography. The pH of the aqueous phase (800 ml), free of residual unreacted penicillin and phenylootic acid, was adjusted to 7.0 by adding 33 ml of 2 N NaOH and then evaporated to a volume of 110 ml under vacuum at 37 ° C.

The concentrated 6-APA solution was cooled to 0 ° C and adjusted to pH 4.2 with 3 N HCl. The 6-APA precipitated, was filtered off and dried under vacuum at 45 ° C to constant weight (6.6 grams).

The content of 6-APA in aqueous mother liquors from crystallization (130 ml) was 0.4% w / w.

The content of 6-APA in the crystalline product as determined by hydroxylamine is 97.5%. Infrared spectrum, chromatography and optical rotation measurement confirm that it is a practically pure product.

Within 6 months, 10 preparations of 6-APA were carried out in exactly the same manner as described above, using the same enzyme-containing fibers each without a marked decrease in productivity.

Example 2

The procedure was carried out under the same conditions as in Example 1, and a fiber with the enzyme preparation deposited was produced from Escherichia Coli cells, purified in the same manner as in Example 1, but fifteen times.

The activity of the same amount of fibers as in the previous example is ten times higher.

The same column as in Example 1 (480 ml volume) was charged with 37 g of fibers. The fibers were washed with water and glycerin until the proteins disappeared from the wash liquors.

Into a 5 L container are initially introduced 3 L of a 2 wt.% Penicillin G potassium salt solution (Squibb).

The solution is continuously pumped into the fiber column through a reservoir. In order to reduce the hydrolysis time, it is necessary to ensure complete homogeneity of the reaction medium, therefore the mixture in the reservoir is vigorously stirred and recirculated at a rate of 500 ml / min.

The pH is maintained at 8.0 by the addition of aqueous 0.5 N NaOH using an automatic pH maintenance device. The temperature of the reaction mixture was automatically maintained at 37 ° C by thermostating the column.

The progress of the hydrolysis can be monitored based on the consumption of the NaOH solution registered on the recorder. After two hours and 10 minutes, the sodium hydroxide consumption rate drops to less than 5% of the original rate. At this point, the reaction is stopped and the mixture is withdrawn from the circulation. The consumption of 0.5 N NaOH is 310 ml. A total of 3300 ml of free solution containing 6-APA, phenylacetic acid formed by hydrolysis and the remainder of unreacted penicillin are withdrawn from the circulation.

Phenylacetic acid and penicillin are removed from the reaction mixture by extraction with butyl acetate. The extraction is carried out twice with 1100 ml of fresh solvent as follows:

The reaction mixture was placed in a 6 L tank equipped with a stirrer ¹ . After cooling to 5 ° C, the pH is adjusted to 3 by addition of aqueous 1 N HCl solution. 1100 ml of butyl acetate are added and stirring is carried out to disperse well between the aqueous and organic phases. While stirring for 15 minutes, the pH is kept constant by the continuous addition of 1 N HCl. The mixture was then allowed to stand at 5 ° C for 20 minutes. At the bottom an aqueous phase is formed which is slightly turbid by the presence of traces of solvent in the form of very fine droplets, the top layer being a clear organic phase. The phases are separated in a separator and a pure aqueous phase is obtained by centrifugation from the butyl acetate emulsion in the aqueous phase.

The entire amount of the aqueous phase is treated once more in the same manner with 1100 ml of fresh solvent and the aqueous phase is isolated in the same manner. During both extractions, 180 ml of an aqueous 1 N HCl solution were used.

In both butyl acetate fractions the presence of penicillin was determined using hydroxylamine. In the first fraction resulting from the first extraction the penicillin content is 3 mmol / l, in the second fraction the penicillin content is not present.

Value. The pH of the aqueous phase obtained in the extraction is adjusted to 6.9 with 165 ml of aqueous 1 N NaOH solution and then the aqueous phase is evaporated in a rotary evaporator under vacuum. The thermostated rotary evaporator bath is maintained at 45 ° C, the condenser coolant temperature is -5 ° C, the suction side pressure is 1 torr. Within 5 hours, the volume of the aqueous phase was reduced to 1.8 of the original volume.

Transfer the residue to a 1 L flask, cool to 2 ° C and then adjust to 4.2 with 6 N HCl. During pH adjustment, 6-APA precipitates in crystalline form. The precipitate was collected by vacuum filtration and then dried under vacuum (0.5 torr at 45 ^° C) to constant weight. 32.4 g of product are obtained. The mother liquors from the crystallization (450 ml) were titrated with hydroxylamine and found to contain 180 mmol / 16-APA corresponding to 0.39% by weight.

The 6-APA obtained is redissolved in water and titrated with hydroxylamine. By comparison with a standard solution of very pure 6-APA, purified by multiple recrystallization, the content of pure 6-APA in the product was found to be 98.5%. From the original. 161 mmol of penicillin are obtained

32.4 x 0.985

216 = 147 mmol of 6-APA.

Assuming the starting product is pure, the overall yield of 6-APA is 91.4% of theory.

In 'continuous' operation, the mother liquors from the crystallization can be partially recycled and 'then' evaporated under vacuum. Thus, the overall yield of the product can be increased up to a limit of 95% of theory.

Claims (3)

Process for the production of 6-amino-oenicillanic acid by hydrolysis of penicillin G, characterized in that an aqueous solution of penicillin G at a concentration of 1 to 6% w / v is hydrolyzed at pH in the region of 8 in the presence of e.nicilinacylase embedded in fibers cellulose triacetate. 2. Process according to claim 1, characterized in that an aqueous solution of 'penicillin G' at a concentration of 2 to 3% w / v is used for the hydrolysis. 3. The process of claim 1 wherein the penicillin G solution is fed to the hydrolysis zone together with a buffer.