US1976175A - Process of preparing adenosine phosphoric acid - Google Patents

Description

Patented Get. 9, 1934 unitsv srArss PATEL? PRGICESS OF PREPARING ADENOSINE PHOSPHORIC AGED No Drawing.

Application February 18, 1932,

Serial No. 593,924. In Germany February 23,

2 Claims.

with hydrolyzing agents at ordinary or slightly elevated temperature, that is to say at a temperature of about C. to about 50 C., and treating the hydrolysates thus obtained with precipitating agents, a muscle adenosine phosphoric acid is obtained which is different from the yeast 39 adenylic acid, but identical with the muscle adenylic acid. The muscle adenosine phosphoric acid has the following formula:

(I) ran H H H HzN-O=N O=PO-CCl-UC H Hid til.

As starting material there may be utilized the 40 adenosine triphosphoric acid obtained by extracting yeast with aqueous trichloroacetic acid (about 5 to 10%) which has been described by Lohmann (Naturwissenschaften, 1928, page 298) There can also be used adenosine triphosphoric acid which, for example, can be obtained by treating with water plasniolysates obtained from yeast at a low temperature (by destroying the cell membrane of yeast at a low temperature with the aid of an organic solvent as, for instance, acetic ester), eliminating the undissolved constituents, treating the solution with absorbing agents and precipitating the remaining solution, after the adsorbing agents containing ballast substances have been removed, with organic solvents. The

product thus obtained is subjected to the process (Cl. 260-43) I described hereafter. Furthermore there can be used crude extracts containing adenosine triphosphoric acid which can be obtained from yeast, by extracting the latter, for instance, with trichloracetic acid before or after the plasmolysis; this crude extract is then treated as herein described.

The process of the invention is carried out by dissolving in water adenosine triphosphoric acid or a preparation or crude extract containing this acid and hydrolyzing while adding hydrolyzing agents, such as barium hydroxide, caustic soda solution or mineral acids. The hydrolysis is preferably carried out at an alkaline reaction. During this pr cess the character of the adenylic acid which is comparatively instable must be taken into consideration by applying conditions of reaction which are not too violent. Therefore the conditions of temperature are chosen so that the hydrolysis is carried out at ordinary or slightly elevated temperature, that is to say at a temperature between about 10 C. and about 50 C. After hydrolysis the solution is filtered and from the remaining filtered solution the adenosine phosphoric acid is isolated by adding a precipitant and separating the adenosine phosphoric acid from the precipitant.

The adenosine phosphoric acid thus obtained is intended for use in the therapy of circulation of the blood.

The following examples illustrate the invention.

(1) Adenosine triphosphoric acid is dissolved in Water, the solution is mixed with barium hydroxide until the reaction to phenolphthaleine is alkaline and the whole is allowed to stand over night in the incubator. The separated product is centrifuged and the clear solution is mixed with lead acetate and ammonia. The precipitate obtained is filtered by suction, washed and decomposed in an aqueous suspension by means of hydrogen sulfide. The filtrate of the lead sulfide is concentrated to a small volume and precipitated by means of acetone. By allowing the mass to stand in the cold the adenosine phosphoric acid is completely separated. The product obtained can directly be utilized and may, if required, be further purified.

(2) Instead of adenosine triphcsphoric acid a preparation containing adenosine uriphosphoric acid can be subjected to the process described in Example 1. follows:

2 kilos of pressed yeast are finely ground and plasmolyzed at a low tem erature (not above 15 C.) by addition of acetic acid ethyl ester. The thin paste obtained is mixed, while stirring, for

This preparation can be prepared as half an hour at room temperature, with 3 liters of distilled water and the clear solution is separated by centrifuging. 25 grams of bentonite are introduced into the solution, while stirring, and the whole is then centrifuged. The clear solution is neutralized, concentrated in a vacuum to 209 cc. and introduced, while stirring, into 1200 cc. of pure methyl alcohol. The precipitate obtained is filtered by suction, washed with methyl alcohol and ether and dried in a vacuum.

(3) 2 kilos of brewers yeast are plasmolyzed by addition of acetic acid ethyl ester. The plasmolyzed product is introduced into 3 liters of boiling water, the mixture is heated to boiling temperature by the introduction of steam and then quickly cooled. The clear solution obtained by centrifuging is mixed with caustic soda solution until there is a distinct basic reaction to phenolphthaleine and the solution is heated during the night at 50 C. It is filtered, the filtrate is feebly acidified, a copper sulfate solution is added to it and the whole is then mixed with a suspension of calcium hydroxide until the reaction is strongly alkaline. A precipitate is obtained which contains the adenosine phosphoric acid being identical with the muscle adenylic acid. The precipitate is decomposed in known manner and the solution obtained is worked up into the adenosine phosphoric acid.

(4) 2 kilos of yeast are plasmolyzed and mixed, while stirring, with 3 liters of trichloracetic acid of 5% strength. The clear solution obtained by centrifuging is adjusted by means of hydrochloric acid to a content of 1% of hydrochloric acid and the whole is allowed to stand over night at about 10 C.-15 C. in a cooled room. After filtration the solution is worked up according to the process described by Drury and Szent-Gyorgyi (Journal of PhysioL", volume 68, page 216), by precipitating by means of lead acetate, decomposing the precipitate obtained with sulfuric acid, precipitating the solution thus obtained with copper sulfate, decomposing the precipitate with hydrogen sulfide and further precipitating with zinc acetate and decomposing the zinc salt obtained with hydrogen sulfide. The final product is the pure crystallized adenosine phosphoric acid.

In the following claims the process using the adenosine triphosphoric acid obtained from yeast as starting material is equivalent to the process using as starting material preparations from yeast or crude extracts from yeast containing adenosine triphosphoric acid. It has been shown in the examples that instead of adenosine triphosphoric acid those preparations and crude extracts can be treated in the manner described in order to obtain the adenosine phosphoric acid.

We claim:

1. The process of producing a muscle adenosine phosphoric acid which comprises subjecting adenosine triphosphoric acid obtained from yeast in an aqueous solution and in the presence of a hydrolyzing agent of the group consisting of diluted alkali hydroxides, alkaline earth hydroxides and mineral acids to a hydrolysis at a temperature of about 10 C. to about 50 0., isolating the adenosine phosphoric acid from the filtered solution by means of a heavy metal salt as a precipitant and separating the adenosine phosphoric acid from the precipitant.

2. The process of producing a muscle adenosine phosphoric acid which comprises subjecting adenosine triphosphoric acid obtained from yeast in an aqueous solution and in the presence of diluted barium hydroxide to a hydrolysis at a temperature of about 10 C. to about 50 C., isolating the adenosine phosphoric acid from the filtered solution by means of lead acetate as precipitant and separating the adenosine phosphoric acid from the precipitant.

CARL LUDWIG LAUTENSCHLAGER. FRITZ LINDNER.