CS224610B2 - Method for producing semi-synthetic peniciline - Google Patents

Description

The invention relates to a process for the preparation of semisynthetic penicillins by acylation of 6-aminepenicillanic acid with suitable amino acids or amides thereof in the presence of penicillin acylase.

The novel semisynthetic penicillins which are stable to beta-lactamesis with a broader spectrum of activity can be obtained by the process according to the invention.

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.

Enzymatic preparations embedded in a fibrous structure retain their efficacy for a very long time. Specimens of this type have maintained their efficacy for almost 12 months during repetitive duty cycles.

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

SUMMARY OF THE INVENTION The present invention provides a process for the preparation of semisynthetic penicillin by acylating 6-aminopenicillanic acid in an aqueous solution at a concentration of 1-3% w / v at pH 6 with a suitable amino acid or amide thereof in the presence of penicillin acylase. cellulose triacetate.

The fiber containing enzyme produced by the spinning process is washed prior to use to remove adsorbed impurities from the outside and used after washing to catalyze the reaction.

Since the protein cannot diffuse into the environment, a highly pure product is obtained which, besides other things, does not contain proteinaceous impurities, so dangerous due to allergic reactions. The catalyst which is devoid of solubility by the treatment according to the invention can be used discontinuously. The catalyst is contacted with the reaction mixture and, upon termination, separated from the reaction mixture and contacted with a fresh batch of fresh mixture.

The same catalyst can also be used in a continuous column process. 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 conversion.

The enzyme catalyst is prepared as described in Italian patent no. 836,462. According to this patent, enzyme-deposited threads can be prepared by spinning solutions of thread-forming polymers in which the enzyme preparations are dispersed in very small droplets of the same size as the emulsion droplets.

The emulsions may be wet or dry-coated. The fiber obtained contains small cavities inside which enzymes are stored. The enzymes are separated from the environment by a very thin membrane which prevents the enzymes from escaping and dispersing them in the reaction mixture, but allows their catalytic action.

In the process according to the invention, cellulose triacetate is used as the extrudable polymer.

As an enzymatic material, a crude extract derived from cells or fungi or aotinomycetes (Aspergillus, Penioillium, Botrytis cinerea, Fusarium, Mueor, Alternaria, Streptomyces etc.) or a crude extract from bacterial cells (E. coli, B. Megaterium, Aerobacter aerogenes) may be used as enzymatic material. , Alcaligenes faecalis, etc.).

The synthesis may be carried out at a temperature of from 15 to 60 ° C, preferably from 37 to 40 ° C.

Penicillins suitable for synthesis include penicillins which have a greater therapeutic use, such as, for example

6- (alpha-phenoxypropionamines) cilin foam,

6- (alpha-phenoxybutyram do) penicillin

6- (2,6-dimethoxybenzoamido) cilin foam,

6- (5-methyl-3-phenyl-4-isoxazolecarboxyamido) penicillin, 6- (5 6- (5-methyl-3-o-chlorophenyl-4-isoxazolecarboxyamido) penicillin,

5-ie) (-) - alpha-aminophenylacetamidojpenicillin etc.

The starting 6-aminopenicillanic acid (6-APA) can be obtained by enzymatic hydrolysis of natural penicillins such as penicillin O by hydrolyzing a 1 to 6% w / v aqueous solution of penicillin G at pH 8 in the presence of penicillin acylase stored in cellulose triacetate fibers.

He did

Preparation of immobilized enzyme

While stirring, 20 g of cellulose triacetate is dissolved in 280 g of methylene chloride in the reactor.

In another vessel, the enzyme solution is prepared as follows: Escherichia coli cells (60 g wet weight), obtained by fermentation in a suitable medium in the presence of phenylootic acid, are transferred to 0.01 M pH buffer pH 7.0. The crude product thus obtained was heated at 50 ° C for three minutes and then precipitated with ammonium sulfate until a pH of 5.5 was reached. The fractions obtained in the range of 25-75% saturation are separated. This fraction was dissolved in 30 ml of phosphate buffer 0.01 M at pH 8 containing 30% by volume 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 was transferred to a small container held at -6 ° C and then spun under nitrogen. The thread coagulation is carried out in toluene at room temperature.

The toluene was removed by vacuum drying for several hours.

Preparation of an aqueous solution of 6-aminopenicillanic acid

In a glass column equipped with a thermostatic braid 70 cm high and 3 cm diameter, 37 g of fibers were placed. The fibers are washed with water and glycerol until the proteins disappear from the wash liquor.

600 ml of an aqueous solution of 2% (w / v) penicillin G potassium was circulated through the column. The pH was maintained at 8.0 by continuously adding 0.5 N NaOH using a constant pH device. The column is thermostatized to 37 ° C. After 5 hours, when the rate of consumption of sodium hydroxide was reduced to 1/20 of the original value, the reaction mixture was stripped, cooled to 3 ° C, acidified with 2 N HCl to pH 3.0 (33.5 mL 2 N HCl) 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.

Preparation of ampicillin [6- (D - (-) - alpha-aminophenylation melt to) foam

The pH of the aqueous solution of 6-APA containing 8.5 mg / ml of 6-APA was adjusted to 6.0 and D-alpha-aminophenylacetamide hydrobromide (final concentration of 10 mg / ml) was added.

The resulting aqueous solution was circulated through the same column, thermostated at 37 ° C, maintaining the pH at about 6.0. After 6-7 hours of circulation, ampicillin is synthesized in about 90% yield.

Example 2

The procedure described in Example 1 was followed except that DL-mandelic amide at 12 mg / ml was used instead of D-alpha-aminophenylaeetamide hydrobromide and the pH was maintained at about 5.5. After 6 hours, alpha-hydroxybenzylpenicillin was obtained in yield.

Claims (1)

to 90%. PURPOSE OF THE INVENTION A process for preparing semi-synthetic penicillin by acylating 6-aminopenicillanic acid in an aqueous solution at a concentration of 1-3% w / v at pH 6 with a suitable amino acid or amide thereof in the presence of penicillinase, characterized in that penicillin acylase is used.