CH653341A5 - METHOD FOR PURIFYING S-ADENOSYL-L-METHIONINE. - Google Patents

Description

The present invention relates to a process for the purification of S-adenosyl-L-methionine, hereinafter abbreviated as “SAM”, in particular to a process for the effective separation of high purity SAM from a liquid containing crude SAM.

SAM is an important substance that participates in in vivo metabolism in fats, proteins and carbohydrates. Recently, SAM has been found to have therapeutic effects on Jecur adiposum, lipemia, arteriosclerosis, depression and insomnia, and it is therefore desirable to produce SAM in large quantities.

So far, many methods for cleaning SAM are known. Specific examples are (1) a method comprising a combination of a step of treating a liquid containing SAM with a strongly acidic cation exchange resin and a step of treating the liquid with activated carbon as described in JP-AS 13680/1971; (2) a method comprising treating a crude SAM containing liquid with a weakly acidic cation exchange resin as described in "Enzymologia", vol. 29, p. 283; (3) a method comprising a combination of a step of treating a raw SAM-containing liquid with a weakly acidic cation exchange resin of the H + type and a step of treating the liquid with activated carbon as described in JP-OS 145299/1982; (4) a method comprising treating a raw SAM-containing liquid with a chelate resin as described in JP-AS 20998/1978; and (5) a method of salting SAM with picric or picrolic acid as described in U.S. Patent Nos. 3,707,536 and 3,954,726.

The above-mentioned process (2) is simpler and more economical in terms of process steps than processes (1), (4) and (5), but shows the disadvantage that the separation of SAM from impurities and foreign materials is incomplete and no SAM is for the high purity required as a drug can be obtained.

According to the method (3), which is an improvement of the method (1), SAM of higher purity is obtained, but since the adsorption of SAM on activated carbon is strong, the recovery of SAM is reduced. The effort to improve the recovery ratio of SAM by increasing the proportion of organic solvents in an eluent unfortunately leads to insufficient separation of SAM from impurities and foreign materials.

It is therefore an object of the present invention to provide a method by which high purity SAM can be effectively isolated.

This object is achieved by the method according to the invention and defined in claim 1.

The method for producing a raw SAM-containing liquid used as starting material in the method according to the invention is not subject to any particular restrictions. This production can be carried out, for example, by culturing a microorganism of the genus Saccharomyces, Candida or Mucor capable of producing SAM in a culture medium containing methionine to form and accumulate SAM within and / or without the microbial cells and extracting the culture broth with an extract. traction agents such as perchloric, hydrochloric, sulfuric or formic acid; or by enzymatic reaction of adenosine triphosphate and methionine in the presence of methionine / adenosyl transferase.

In the process according to the invention, it is essential that a raw SAM-containing liquid is cleaned by subjecting it to a multi-step cleaning process which, in any order, involves at least one step (A) of treating the liquid with a weakly acidic cation exchange resin of H + type and at least one step (B) treating the liquid with a porous synthetic resin adsorbent.

Treatment (A) can be carried out as follows:

First, the pH of the crude SAM-containing liquid is usually adjusted to 3.5-6.5, preferably 4-6.5. If this pH is too low, SAM will be difficult to adsorb on the cation exchange resin. If this pH is too high, SAM can be decomposed. The method of adjusting the pH is not subject to any particular restrictions. The pH is preferably adjusted

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653 341

Value using a combination of an acid and one to form a poorly or insoluble precipitate in water, or an OH-type anion exchange resin.

When the crude SAM-containing liquid, the pH of which has been adjusted as described above, is contacted with the weakly acidic cation exchange resin of the H + type, the SAM with positive charge is selectively adsorbed on the cation exchange resin. The impurities in the liquid with neutral and negative charge are not adsorbed on the resin.

Any such resin having carboxylic acid groups as ion exchange groups can be used as the weakly acidic cation exchange resin. Specific such resins are, for example, “Amberlite” IRC-50 and IRC-80 from Rohm & Haas Co., and “Diaion” KW20 from Mitsubishi Chemical Co., Ltd. The contact can be made batchwise or by column treatment. Column treatment is preferred because it is easy to carry out and the impurities are easily removed.

The SAM adsorbed on the ion exchange resin is then separated by fractionally eluting the resin with an aqueous solution of an inorganic or organic acid with a pH of usually not more than 3.0, preferably 0.2-2.0. The acid used is not particularly restricted and can be, for example, hydrochloric, sulfuric, acetic or p-toluenesulfonic acid. If necessary, the ion exchange resin may be washed with water or a dilute aqueous solution of an acid, e.g. pH 3.5 or more, prior to fractional elution of SAM to remove traces of contaminants therein.

Treatment stage (B) can be carried out as follows:

First, the raw SAM-containing liquid is adjusted to a pH of 6.5 or less, and then brought into contact with the porous, synthetic resin adsorbent. The adjustment of the pH value and the contacting can be carried out in the same way as described above for treatment step (A). Contacting the adsorbent leads to selective adsorption of amines, the decomposition product methylthioadenosine from SAM, and of colorations that are foreign materials in the liquid. By selecting conditions described below, SAM and the foreign materials can be adsorbed together on the adsorbent. In this case, SAM can be selectively separated by fractionally eluting the adsorbent with an aqueous solution of an inorganic or organic acid with a pH of not more than 3.5, preferably 0.2-2.0.

Any of the inorganic and organic acids mentioned above for treatment step (A) can be used for this. Organic solvent can be used together with the inorganic or organic acid, provided that its proportion enables a uniform solution. Suitable organic solvents are, for example, methanol, ethanol, n-propanol, isopropanol, acetone, methyl ethyl ketone, methyl formate, ethyl acetate, dioxane, toluene. If desired, as in treatment step (A), the adsorbent can be washed with water or a dilute aqueous solution of an acid before the fractional elution in order to remove traces of impurities contained therein.

The porous, synthetic resin adsorbent used in the process according to the invention is water-insoluble and shows a giant network structure. Specific examples of this are non-polar adsorbents based on a styrene / divinylbenzene copolymer as a matrix, such as “Amberlite” XAD-2

and XAD-4 from Rohm & Haas Co., and "Diaion" HP-10, HP-20 HP-30, HP-40 and HP-50 from Mitsubishi Chemical Co., Ldt .; and moderately polar adsorbents based on a polymer of an acrylic acid ester and / or a methacrylic acid ester or a copolymer of such monomers with a non-polar monomer, such as styrene and divinylbenzene as a matrix, such as “Amberlite” XAD-7 and XAD-8 from Rohm & Haas Co ., and "Diaion" HP-ZMG, from Mitsubishi Chemical Co., Ldt. If desired, such adsorbents can be used in combination.

Such adsorbents have in common that they selectively adsorb foreign materials, such as amines and colorations, present in the raw SAM-containing liquid. However, depending on the type, they show selective adsorption capacity for SAM. Specifically, non-polar adsorbents adsorb foreign materials, but not SAM, and moderately polar adsorbents adsorb SAM together with the foreign materials in the relatively weakly acidic range, but only the foreign materials in the relatively strongly acidic range, without adsorbing SAM.

According to the method according to the invention, treatment stages (A) and (B) are carried out at least once each, the order of which is free. Preferred examples of the order are (1) first (A) and then (B); (2) first (B) and then (A); (3) first (A), then (B) and then (A); and (4) first (B), then (A) and then (B). In addition to these examples, treatment stage (A) and / or treatment stage (B) can also be added. With an increasing number of treatment stages, the economy of the cleaning process is of course increasingly reduced.

If treatment stage (B) is carried out before treatment stage (A), it is procedurally advantageous to apply conditions such that the impurities are only selectively adsorbed on the synthetic resin adsorbent, but not SAM. Specific conditions of this type are, for example, the use of a non-polar adsorbent or the use of a moderately polar adsorbent in a pH range of not more than 3.5, preferably 0.2-3.0.

If the last treatment step is step (B), it is possible to adsorb and then separate SAM on the synthetic resin adsorbent first, or not to adsorb the SAM in this step on the synthetic resin adsorbent.

If desired, the solution of SAM eluted by the process according to the invention as described above can be concentrated under reduced pressure and then with an organic solvent such as methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, methoxyethanol, acetone, methyl ethyl ketone , Methyl or ethyl formate or acetate, butyl acetate, dioxane. Here, precipitation of a salt of SAM and an inorganic or organic acid can be obtained. Alternatively, without contacting the solution of SAM with an organic solvent, a powder or a salt of SAM and an inorganic or organic acid can be obtained by removing the excess acid from the solution of SAM using an OH-type anion exchange resin or one for formation a water-insoluble salt is removed by reaction with the alkali which is used in the fractional elution of SAM and then the solvent is removed from the solution to dryness under reduced pressure.

The weakly acidic ion exchange resin of the H + type is regenerated at the time when the SAM is eluted therefrom. It is therefore not always necessary to subject this resin to a special regeneration treatment. It can only be used repeatedly after washing with water. On the other hand

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653 341

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the synthetic resin adsorbent can be easily regenerated, for example by washing with a 50% aqueous solution of methanol and then with water.

According to the method according to the invention, SAM of very high purity can thus be effectively obtained by simple treatment steps using easily regenerable substances.

example 1

Saccharomyces cerevisiae, IFO 2044, was used to form microbial cells with the accumulated SAM in the method described by F. Schlenk et al in the "Journal of Biologica! Chemistry », vol. 229, p. 10377 (1957). 210 g of the cells obtained were suspended in 1000 ml of 1.5N perchloric acid and extracted with shaking for 1 h at room temperature. Then the cell residues were separated by centrifugation. 5.0 potassium hydrogen carbonate was added to the extract to adjust the pH. The resultant precipitation of potassium perchlorate was filtered off with suction, whereby 1080 ml of an extract containing 1.15 g of SAM were obtained.

The extract obtained was passed through a column filled with 200 ml of the weakly acidic cation exchange resin “Amberlite” IRC-50 of the H + type for adsorption of SAM. The column was washed with 400 ml of 0.0001N acetic acid and fractionally eluted with 0.1N sulfuric acid to give 630 ml of a solution of SAM.

The resulting solution of SAM was adjusted to pH 4.5 with the weakly basic anion exchange resin “Amberlite” IRA-45 OH-45 and then with 1.5 1 of the synthetic resin adsorbent “Amberlite” XAD-7 filled column passed to adsorb SAM. The column was washed with 2 1 0.0001N acetic acid and then fractionally eluted with 0.1N sulfuric acid to obtain 1440 ml of a solution of SAM.

The solution of SAM was concentrated to a total volume of 200 ml under reduced pressure. Then 800 ml of acetone was added, whereby a precipitation of SAM sulfate was obtained. The precipitate was separated by centrifugation, dissolved in a little water and then lyophilized, whereby 174 g of SAM sulfate was obtained in the form of a white powder. The product obtained showed a single spot in paper chromatography and silica gel thin layer chromatography.

The recovery ratio of SAM and its purity are shown in Table 1.

Example 2

An extract containing 1.06 g of SAM prepared as described in Example 1 was passed through a column filled with 200 ml of the weakly acidic cation exchange resin “Amberlite” IRC-84 of the H + type in order to adsorb SAM. The column was washed with 400 ml of 0.0001 N hydrochloric acid and then fractionally eluted with hydrochloric acid to obtain 640 ml of a solution of SAM.

The solution of SAM obtained was adjusted to 4.8 using the weakly basic anion exchange resin "Amberlite" IRA-45 OH-45 and then through a column filled with 1.5 l of the synthetic resin adsorbent "Amberlite" XAD-7 directed to adsorb SAM. The column was washed with 2 liters of 0.0001N acetic acid and then fractionally eluted by passing a mixed solvent of 0.1N hydrochloric acid and acetone in a volume ratio of 1: 0.1 to give 1120 ml of a solution of SAM. The resulting solution of SAM was concentrated under reduced pressure to a total volume of 200 mml and then to the pH by adding the weakly basic anion exchange resin «Amberlite» IRA-45 of the OH type

2.0 posed. The resin was suction filtered and the filtrate was concentrated under reduced pressure. The concentrate was lyophilized to give 119 g of SAM hydrochloride as a white powder. The product obtained showed a single spot in paper chromatography and silica gel thin-layer chromatography.

The ratio of the recovery of SAM and its purity are shown in Table 1.

Example 3

An extract containing 0.98 g of SAM, prepared as described in Example 1, was passed through a column filled with 200 ml of the adsorbent “Amberlite” XAD-2 of the styrene / divinylbenzene type in order to adsorb foreign materials. After removing the foreign materials, the solution of SAM was adjusted to pH 5.0 by adding potassium hydrogen carbonate. The potassium perchlorate precipitate formed was filtered off with suction.

The obtained solution of SAM was passed through a column with 200 ml of the weakly acidic cation exchange resin «Amberlite» RC-50 of the H + type to adsorb SAM. The column was washed with 400 ml of 0.0001N acetic acid and fractionally eluted with 0.1N sulfuric acid to give 630 ml of a solution of SAM.

The resulting solution of SAM was concentrated to a total volume of 200 ml under reduced pressure, and then 800 ml of acetone was added, whereby SAM sulfate was precipitated. The precipitate was separated by centrifugation, dissolved in a little water and lyophilized, whereby 150 g of SAM sulfate were obtained in the form of a white powder. The product obtained showed a single spot in paper chromatography and silica gel thin layer chromatography.

The recovery ratio of SAM and its purity are shown in Table 1.

Example 4

According to the procedure described in Example 1, microbial cells with SAM accumulated therein were produced. Of the microbial cells obtained, 200 g were suspended in 1000 ml of 0.1N formic acid. The suspension obtained was heated to 60 ° C. over 10 minutes and cooled immediately thereafter. The cells were separated by centrifugation to obtain 1020 ml of an extract containing 0.91 g of SAM.

The extract obtained was passed through a column with 200 ml of the acrylate adsorbent "Amberlite" XAD-8 in order to adsorb foreign materials.

After removing the foreign materials, the solution of SAM was adjusted to pH 5.0 with the weakly basic anion exchange resin "Amberlite" IRA-45 of the OH type and then through a column with 200 ml of the weakly acidic cation exchange resin "Amberlite" IRC-84 H + -type directed to adsorb SAM. The column was washed with 400 ml of 0.0001N hydrochloric acid and then fractionally eluted with 0.2N hydrochloric acid to obtain 710 ml of a solution of SAM.

The solution of SAM obtained was adjusted to pH 2.0 with the weakly basic anion exchange resin “Amberlite” IRA-45 of the OH type. The resin was suction filtered and the filtrate was concentrated under reduced pressure and lyophilized to give 1.01 g of SAM hydrochloride as a white powder. The product obtained showed a single spot in paper chromatography and silica gel thin layer chromatography.

The recovery ratio of SAM and its purity are shown in Table 1.

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Example 5

From the solution of SAM obtained according to Example 3 by treatment with the styrene-divinylbenzene adsorbent and the weakly acidic cation exchange resin of the H + type, 630 ml were passed through a column filled with 200 ml of the adsorbent “Amberlite” XAD-7 of the acrylate type directed to adsorb foreign materials.

After removing the foreign materials, the solution of SAM was concentrated to a total volume of 200 ml under reduced pressure. Then 800 ml of acetone was added to form a precipitate of SAM sulfate. The precipitate was separated by centrifugation and dissolved in a little water and lyophilized, whereby 146 g of SAM sulfate was obtained in the form of a white powder. The product obtained showed a single spot in paper chromatography and silica gel thin layer chromatography.

The recovery ratio of SAM and its purity are shown in Table 1.

Comparative experiment A

The pH of 950 g of the extract containing 0.98 g of SAM prepared according to Example 1 was adjusted to 5.0 using potassium hydrogen carbonate. The potassium perchlorate precipitate formed was suction filtered to obtain a liquid containing SAM.

The obtained SAM-containing liquid was passed through a column filled with 200 ml of the weakly acidic cation exchange resin "Amberlite" IRC-50 of the H + type to adsorb SAM. The column was washed with 400 ml of 0.0001N acetic acid and then fractionally eluted with 0.1N sulfate to obtain 610 ml of a solution of SAM.

The resulting solution of SAM was passed through a column filled with 200 ml of chromatographically activated carbon. The column was washed with 600 ml of 0.2N sulfuric acid and then fractionally eluted with a mixed solvent of 1.0N sulfuric acid and methanol in a volume ratio of 1: 1, whereby a solution of SAM was obtained.

The resulting solution of SAM was concentrated under reduced pressure to a total volume of 200 ml. Then 800 ml of acetone was added to precipitate SAM sulfate. The precipitate formed was separated by centrifugation, dissolved in a little water and lyophilized, whereby 1.28 g of SAM sulfate was obtained in the form of a white powder. The product obtained showed a single spot in paper chromatography and silica gel thin layer chromatography.

The recovery ratio of SAM and its purity are shown in Table 1.

653 341

Comparative Experiment B Example 1 was repeated with the exception that only the treatment with “Amberlite” IRC-50 of the H + type was carried out for the cleaning treatment.

The results obtained are shown in Table 1.

Table 1

example

purity

Ninhydrin

amounts

Extraction or

from SAM,

reaction proportion ratio

Vergi.

% *

with others

Methylthio by SAM,

Verse.

Materials adenosine,

% By weight

as SAM **

% By weight ***

1

98.1

-

0.08

89

2nd

98.0

-

0.1

90

3rd

98.0

-

0.1

90

4th

98.1

-

0.08

90

5

98.5

-

below

88

0.05

A

97.6

-

0.4

77

B

56.7

+ +

1.8

90

*: The purity of SAM was determined using two-dimensional paper chromatography.

**: The ninhydrin reaction with materials other than SAM was determined as follows:

A test sample was developed by means of two-dimensional cellulose thin-layer chromatography and the spots other than a spot of SAM indicated by ninhy-drin color formation were determined and evaluated on the following three-step scale:

-: no

+: slightly present + +: considerably present

***: The amount of methylthioadenosine was determined by means of two-dimensional paper chromatography.

The table clearly shows that the method according to the invention allows SAM of higher purity to be obtained in a higher recovery ratio than according to known methods.

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Claims (10)

653 341 2nd PATENT CLAIMS 1. A process for the purification of S-adenosyl-L-methionine by introducing a crude liquid containing S-adenosyl-L-methionine into a cleaning process, characterized in that the cleaning is carried out in a multi-stage process, the liquid being carried out in any order in treated in at least one stage (A) with a weakly acidic cation exchange resin of the H + type and in at least one stage (B) with a porous, synthetic resin adsorbent. 2. The method according to claiml, characterized in that that in step (A) the liquid with a pH of 3.5-6.5 for the adsorption of S-adenosyl-L-methionine on the resin is brought into contact with the cation exchange resin and then out of the cation exchange resin by means of an aqueous Solution of an acid with a pH of not more than 3.0 fractionally eluted S-adenosyl-L-methionine. 3. The method according to claim 1, characterized in that the liquid in step (B) at a pH of not more than 6.5 in contact with the synthetic resin adsorbent to remove foreign materials from the liquid adsorb on the resin. 4. The method according to claim 1, characterized in that in step (B) the liquid with a pH of not more than 6.5 is brought into contact with the synthetic resin adsorbent in order to both S-adenosyl L-methionine as well as foreign materials from the liquid to be adsorbed, and that afterwards from the synthetic resin adsorbent using an aqueous solution of an acid with a pH of not more than 3.5 fractionally S-adenosyl-L-methionine eluted. 5. The method according to claim 1, characterized in that the cleaning is carried out in the order of steps (A) and (B). 6. The method according to claim 1, characterized in that one carries out the cleaning in the order of steps (A), (B) and (A). 7. The method according to claim 1, characterized in that one carries out the cleaning in the order of steps (B) and (A). 8. The method according to claim 1, characterized in that one carries out the cleaning in the order of steps (B), (A) and (B). 9. The method according to claim 7, characterized in that in step (B) the liquid with a pH value of not more than 6.5 is brought into contact with the synthetic resin adsorbent in order to only remove foreign materials from the liquid adsorb. 10. The method according to claim 8, characterized in that in the first stage (B) the liquid with a pH of not more than 6.5 is brought into contact with the synthetic resin adsorbent in order to remove only foreign materials from the liquid to adsorb.