DE1225342B - Process for the production of 6-aminopenicillanic acid from gas solutions - Google Patents

Description

FEDERAL REPUBLIC OF GERMANY

GERMAN

PATENT OFFICE

EDITORIAL

Int. α .:

C12d

German class: 3Oh-6

Number: 1 225 342

File number: M 53620IV a / 30 h

Filing date: July 19, 1962

Opening day: September 22, 1966

The invention relates to a method for isolating pure 6-aminopenicillanic acid in high yield from impure aqueous solutions, such as penicillin fermentation solutions.
. 6-aminopenicillanic acid

H ₂ N-

CH
CO-

CH

N-

CH ₈
CH-COOH

is made by fermenting penicillin-producing microorganism seeds without the addition of a precursor or produced by microbial hydrolysis of benzyl penicillin. After isolation and purification from the Fermentation solution or the solution of the microbial hydrolyzate can use the 6-aminopenicillanic acid with acylating agents to produce new or known penicillins that can be used for treatment bacterial infections. For example, it will be like this 6-aminopenicillanic acid reacted with phenylacetyl chloride to form benzylpenicillin.

It is known that 6-aminopenicillanic acid is produced from fermentation solutions by adsorption on an in the styrene-divinylbenzene-sulfate present in the hydrogen ion form can win on acid - cation exchange resin and eluting at a pH of 7.0 (see Journal of the American Chemical Society, 1960, pp. 3790 to 3791).

It has now been found that the recovery of 6-aminopenicillanic acid is significantly facilitated can be when the adsorbate on such a cation exchange resin with an alkaline aqueous solution at pH values in the range from 8 to 9, as this results in a very sharp eluate cut, containing the 6-aminopenicillanic acid is obtained, so that the acid adsorbed on the resin through a relatively small volume of elution liquid can be eluted.

The inventive method for obtaining 6-aminopenicillanic acid from fermentation solutions by Adsorption of 6-aminopenicillanic acid on a particulate styrene-divinylbenzene-sulfonic acid cation exchange resin, Eluting the adsorbate with an aqueous solution and recovering the 6-aminopenicillanic acid from the eluate is thereby characterized in that the cation exchange resin with an aqueous alkaline solution at pH from 8 to 9 is eluted.

A process for obtaining 6-aminopenicillanic acid is known from the German Auslegeschrift 1 075 799 and the Belgian patent specification 569 728, a process for obtaining
6-aminopenicillanic acid from fermentation solutions

Applicant:

Merck & Co., Incorporated,

Rahway, N.J. (V. St. A.)

Representative:

Dr.-Ing. W. Abitz and Dr. D. Morf,

Patent Attorneys, Munich 27, Pienzenauer Str. 28

Named as inventor:

John William Rothrock, North Plainfield, NJ;
Theodore August Jacob, Westfield, NJ;
Irving Putter, Martinsville, NJ (V. St. A.)

Claimed priority:

V. St. v. America 3 August 1961 (128 981)

which consists in that the fermentation liquid of the mold in which the 6-aminopenicillanic acid is contained, concentrated and then treated with an ion exchange resin, the eluate of the resin concentrated and the 6-aminopenicillanic acid is precipitated in crystalline form therefrom by adding an acid. In this known process, the recovery of 6-aminopenicillanic acid from the fermentation solution is preferred by adsorption on a highly basic anion exchange resin and eluting with dilute Hydrochloric acid obtained. Apart from the fact that this known process has a larger number of process stages concentration, precipitation of precipitates, centrifugation and extraction with organic solvents requires before the 6-aminopenicillanic acid by adsorption on the Ion exchange resin can be obtained, in the known method are also about 33% lower yields of 6-aminopenicillanic acid, based on the fermentation solution originally used, obtained than in the process according to the invention.

A strongly basic anion exchange resin is also used according to French patent 1,256,281 used to obtain 6-aminopenicillanic acid from fermentation solutions.

In the inventive method, the adsorption is carried out by the impure aqueous

609 667/378

3 4

Solution of 6-aminopenicillanic acid at a pH value anion exchange resins, preferably of the quater-

from about 1 to 4 with a particulate styrenic ammonium type. Particularly suitable for this

Divinylbenzene sulfonic acid cation exchange resin is used under the trade name "Dowex-lX-2"

is acting. The adsorption can be obtained by percolating anion exchange resin in which the

the aqueous solution through a column of cationic quaternary nitrogen through a polymeric benzyl radical

exchange resin or by batch adsorption, and three methyl groups is substituted. Other

d. H. Suspending the resin in the aqueous 6-amino resins of this type are available under the trade name

penicillic acid solution available for a period of no more than »Amberlite IRA-400«,

about 30 minutes and filtering off the resin adsorbate ■ ·. .

of the exhausted aqueous solution, carried out io example!

will. To 241 of an aqueous 0.1 molar sodium

The elution step is carried out by adding the phosphate buffer solution with a pH of 7.5

Resin adsorbate with a weakly alkaline aqueous are 200 g of potassium benzylpenicillin and 2500 g

Solution is treated, whereby the 6-amino-Proteus retgerii cells are added; from a vege-

penicillanic acid was obtained from the resin relatively free from the native culture of the organism. That

is eluted by impurities. The pH value mixture is ^{incubated for 4 hours at 37 0} C, whereupon

of the elution solution, should be between 8 and 9. Under- the cells are centrifuged off. ... ',.

'Half the pH value of 8 is the 6-aminopenicillan. The pH of the filtrate is adjusted with hydrochloric acid

acid eluted too slowly, and solutions adjusted to pH 2.5 and the acidified filtrate

pH values higher than 9 cause a decomposition 20 with 81 »Dowex-50X-4« resin in the hydrogen!

of 6-aminopenicillanic acid. ion form stirred for 30 minutes. Then the resin

The 6-aminopenicillanic acid can be separated from the eluate with the adsorbed 6-aminopenicillanic acid.

solution can be obtained by two methods. The filtered and washed with water,

The first of these methods involves the adsorption of the acid is removed from the adsorbate by ten minutes

Eluate solution on an anion exchange resin of qua- 25 suspension in aqueous sodium hydroxide solution at pH 8.0

ternary ammonium type to remove a larger and 5 ° C eluted The resin is from the eluate

Part of the remaining impurities. This filtered off and washed with water. The filtrate

Adsorption becomes with the resin in the salt form, and the washing liquids are combined and the

preferably as chloride, and subjected to freeze-drying with cessation of 105 g

pH of the eluate carried out to about 7. 30 partially purified 6-aminopenicillanic acid is obtained.

After adsorption, the resin adsorbate is mixed with the partially purified 6-aminopenicillanic acid,

dilute acidic solutions eluted. The eluate of this is dissolved in water and the pH of the aqueous

second resin adsorption is concentrated in vacuo, the solution is adjusted to 7.0 using sodium hydroxide solution. the

and the 6-aminopenicillanic acid obtained from the concentrated dark green solution is by filtering

Eluate partially decolorized by adjusting the pH of solution 35 with activated charcoal. The 708 mi

crystallized to about 4.3. This is the preferred making up filtrate is cooled to 5 ° C and using

Method of recovery when the original concentrated hydrochloric acid is acidified to pH 4.3. the

6-aminopenicillanic acid solution a filtered penicillin 6-aminopenicillanic acid crystallizes from the solution

fermentation solution is. the end. The crystals are filtered off from the solution,

The second method of obtaining the 6-amino-40 is washed with ice water and acetone and in vacuo

penicillanic acid from the cathion exchange resin - dried. The dried crystals of the handy

eluate in solutions of 6-aminopenicillanic acid, which is pure by microbial hydrolysis, weigh 51.7 g. Additional

Buffered penicillin solutions produced product borrowed amounts of the material are then from the

applicable. ^: In this case, the eluate of the mother liquor is crystallized.

Cation exchange resin concentrated and the pH value 45. The isolation described above can also be used under

adjusted to 4.3, whereupon practically pure 6-amino use of the resins Dowex-50X-2, Dowex-50X-8

penicillanic acid crystallized from the solutions. or Amberlite IR-120 as an adsorbent

As a cation exchange resin it will become an acidic sulfone. Generally, the Dowex resins have

Acid resin used, which by core sulfonation of a relatively low degree of crosslinking

Styrene-divinylbenzene copolymer grains get 50 higher adsorption capacity for 6-aminopenicillane

will. A known group of such resins is acid.

is available under the trade name "Dowex-50". Example 2
These resins are made from a mixture of styrene and divinylbenzene

polymerisates produced with different levels of hardening depending on the made in the preparation of the 55 by fermentation with a resin used ratio of divinylbenzene to selected strain of Pemcillium chrysogenum May own styrene. A resin with »4% crosslinking« without the addition of an additional penicillin precursor, is one made from grains made from 4% divinylbenzoleinic acid such as phenylacetic acid units and consist of about 96% styrene units. The mycelium is removed from the fermentation solution, manufactured resin. The "Dowex-50" resins are 60 and the filtrates are combined to make 9271 "X" numbers after the name indicating the filtered fermentation solution with a through biological Degree of networking. For example, "Dowex-50X-4" is from a test determined 6-amino-penicillanic acid content of Mixed polymer produced containing 4% divinylbenzene about 520μ / πύ. The test was abstained by. Other resins of this type are under the trademark Phenylacetyl Chloride and Be trade names "Amberlite IR-120" available. 65 Mood of the after that of N. G. Heatley in

The further purification of the penicillin biochem. J., 1944, vol. 38, p. 61

Fermentation solutions contained 6-aminopenicillanic acid using penicillins found using method

The anion exchange resins used are strongly basic carried out by B. Subtilis as the test organism.

The original sample is also tested for penicillin without phenylacetyl chloride treatment. the Difference between the biological determinations was found to be a reliable determination of the existing 6-aminopenicillanic acid. This filtrate is made into 1901 Dowex 50X-4 resin in the hydrogen ion form added. The pH of the filtered fermentation solution is adjusted to 2.1 and on this Maintained value while the mixture of resin and fermentation solution is stirred at 5 ° C for 20 minutes. That Resin is filtered off and washed with water. The 6-aminopenicillanic acid is eluted from the resin, by suspending the resin in water and adjusting the pH to 8.5 with concentrated aqueous ammonia is set. The suspension is stirred for 15 minutes at 5 ° C. and to remove the eluted resin filtered.

The filtrate containing the eluted 6-aminopenicillanic acid is neutralized to pH 7 and passed through a column percolated the 191 Amberlite IRA-401 resin in ao Contains chloride form. The resin with the adsorbed 6-aminopenicillanic acid is washed with water and eluted with 0.05N hydrochloric acid, and the eluate fractions containing 90 g of 6-aminopenicillanic acid, are combined and concentrated in vacuo to 1190 ml. The concentrate becomes more insoluble for removal Impurities filtered. The filtrate is cooled to 8 ° C. and treated with concentrated hydrochloric acid acidified to a pH of 4.3. 65 g of crystalline 6-aminopenicillanic acid separate from the solution off and are filtered off, washed and dried.

Claims (1)

Claim: Process for the production of 6-aminopenicillanic acid from fermentation solutions by adsorption 6-aminopenicillanic acid on a styrene-divinylbenzene-sulfonic acid cation exchange resin in particulate form, Elute the adsorbate with an aqueous solution and recover the 6-aminopenicillanic acid from the Eluate, characterized in that the cation exchange resin with an aqueous alkaline solution at a pH of 8 to 9 is eluted. Considered publications:
German Auslegeschrift No. 1 075 799;
French Patent No. 1256 281;
Belgian patent specification No. 569 728. 609 667/378 9.66 ® Bundesdruckerei Berlin