DE3329218A1 - Process for the purification of S-adenosyl-L-methionine - Google Patents

Abstract

The described process entails S-adenosyl-L-methionine being purified in such a way that a liquid containing crude S-adenosyl-L-methionine is introduced into a purification process. The purification process consists of, in any desired sequence, A) at least one stage of treatment of the liquid with a weakly acid cation exchange resin of the H<+> type and B) at lest one stage of treatment of the liquid with a porous synthetic resin as adsorbent.

Description

4th
description

The invention relates to a method for purifying S-adenosyl-L-methionine (hereinafter abbreviated as SAM) and in particular, a method for efficiently isolating SAM with high purity from a crude SAM containing solution.

SAM is an important substance that participates in the metabolism of fats, proteins and carbohydrates in vivo is. It has recently been found that SAI-I has a therapeutic effect on Jecur adiposum, Lipemia, atherosclerosis, depression, and insomnia identifies. There is therefore a need to manufacture SAM in larger quantities.

Many methods are already known for cleaning SAM. Specific examples of this are (1) a process comprising a combination of a step of treating the liquid containing SAM with one strongly acidic cation exchange resin and a stage of treatment with activated carbon (JA-AS 13680/1971)} (2) a method comprising treating the crude SAM-containing liquid with a weakly acidic cation exchange resin (Enzymology, Volume 29, page 183); (3) a method comprising a combination of one Step of treating a liquid containing crude SAM with a weakly acidic cation exchange resin of the H type and a stage of treatment with activated carbon (JA-OS 145299/1981); (4) a procedure, comprising treating the crude SAM-containing liquid with a chelating resin (JA-AS 20998/1978); and (5) a method using a salt

by SAM with picric acid or picrolonic acid (U.S. Patents 3,707,536 and 3,954

Process (2) is simpler and more economical in terms of process steps than process (1) _S (4) or (5). However, this process has the disadvantage that the separation of SAM from impurities and foreign materials is incomplete and in this way no SAM can be produced with the purity required for pharmaceuticals., In process (3)? which is an improvement over the method (1) is _9, the purity of the SAM is indeed increased, but due to the Tatsach © _v the adsorption of SAM on the activated carbon is strongly "the yield ratio of SAM vermindertο attempts _9, the yield ratio by increasing the amount An excessive amount of organic solvent in the eluent ₃ disadvantageously leads to an insufficient separation of SAM from the impurities and foreign materials.

The object of the invention therefore © s ₉ to tunnel a method available ,, can be effectively isolated by the SAM with a high purity "

There were extended © investigations in this regard performed _ΰ and © s xtfurde thereby found that SAM can be effectively isolated from a raw © s SAM-containing liquid and purified, if a combination of a stuf © treatment with a weakly acidic cation exchange resin of H -Type and stage of treatment with a porous ^ synthetic resin adsorbent applies «,

The invention therefore relates to a method for purifying SAM ₀ which is characterized in that

"1 you put a liquid containing raw SAM in a Introduces cleaning process, the cleaning process is a series or series cleaning process, which in any desired order from (A) at least one stage of treating the liquid with a weakly acidic cationic exchange resin of the H type and (B) at least one stage of treatment the liquid is composed of a porous synthetic resin adsorbent.

There is no particular limitation on the manufacturing method of the raw SAM-containing liquid. For example, this can be produced by using a microorganism of the genus Saccharomyces, Candida or Mucor, which can produce SAM, are cultured in a culture medium containing methionine is to generate SAM inside and / or outside of the microbial cells, and the culture broth with an extractant, such as perchloric acid, hydrochloric acid, sulfuric acid or formic acid, is extracted, or by taking adenosine triphosphate and methionine in the presence of methionine adenosyl transferase enzymatically implemented with each other.

For the method according to the invention it is essential that that a crude SAM-containing liquid is purified in such a way that it is in a series cleaning process is introduced, in any order from (A) at least one stage of the Treatment of the crude SAM-containing liquid with a weakly acidic cation exchange resin of the H type and (B) at least one step of treatment with a porous synthetic resin adsorbent consists.

The treatment step (A) is carried out as follows "

First, the pH of the raw SM-containing liquid is usually _{adjusted to 3 P} 5 Ms 6 ₉ 5 _S, preferably Ms 6.5 »If the pH of the raw SAM-containing liquid is too low _B then the adsorption of SAM will occur the ion exchange resin. If the pH of the liquid is too high, SAM becomes sensitive to decomposition. . The method of setting the pH is not subject to any particular restrictions. The pH setting is preferably carried out in such a way that a combination of an acid and an alkali is capable of producing a precipitate which is sparingly soluble or insoluble in water to form _ϋ or an anion exchange ^{resin (QH =} = type) used

By contacting the liquid containing crude SAM with the pH value set in the above manner with the weakly acidic cation exchange resin of the H type, SAM _0, which has a positive charge, is selectively adsorbed on the cation exchange resin. The impurities with neutral and negative charges in the liquid are not adsorbed on the resin.

This uses a weakly acidic cation exchange resin any cation exchange resin with carbon

The examples are Amberlit® IRC-50 and IRC-80 (products from Rohm & Haas Co ₀ ) and Diaion WISO (a product from Mitsubishi Chemical Co. ₀₅ LtIo) ₀ The contacting can be carried out by a batch process or a column process _o the column method of contamination is due to its good operability and ease of removal preferred

The SAM adsorbed on the ion exchange resin is then separated off by treating the resin with an aqueous Solution of an inorganic or organic acid having a pH of usually not more than 3.0., Preferably 0.2 to 2.0, is eluted in a fractionated manner. The acid used is not particularly limited. Examples are hydrochloric acid, sulfuric acid, acetic acid and p-toluenesulfonic acid. If necessary, can Before the fractional elution of the SAM, the ion exchange resin with water or a dilute, aqueous Acid solution (e.g. with a pH value of 3.5 or more) can be washed to remove traces of impurities to remove.

The treatment step (B) is carried out as follows.

First, the liquid containing raw SAM is adjusted to pH 6.5 or less and is then contacted with the porous synthetic resin adsorbent. The pH adjustment and the Contacting can take place in the same way as in treatment stage (A). Contacting with the adsorbent leads to a selective adsorption of amines, methylthioadenosine (the decomposition product of SAM) and dyes, the foreign materials are in the liquid. By selecting the conditions in the manner described below, SAM and the foreign materials are adsorbed with each other on the adsorbent. In this case the SAM be selectively separated by treating the adsorbent with an aqueous solution of an inorganic or organic acid with a pH of not more than 3 »5, preferably 0.2 to 2.0, fractionated eluted.

Inorganic and organic acids which can be used herein may be the same as those used in the treatment step (A). An organic solvent may be used together with the inorganic or organic acid as long as it is present in an amount of ₉ which can give a uniform solution. Examples of suitable organic solvents are methanol, ethanol ₉ n-propanol, isopropanol, acetone, methyl ethyl ketone, s, methyl formate, ethyl acetate, dioxane and toluene "If desired, in the same manner as in Behandlungsstuf © (A) the adsorbent with water or a dilute , aqueous acid solution must be washed before the fractional elution in order to remove any traces of impurities that may be present.

The inventively used © _ΰ porous synthetic resin adsorbent is water-insoluble and has a large network structure _o Specific examples are non-polar adsorbent _ΰ based on styrene / divinylbenzene copolymers as matrix, such as Amberlite XAD-2 and XAD-4 (product © by Rohm & Haas Co.) and Diaion HP-10, HP-20, HP-30, HP-40 and HP-50 (products of Mitsubishi Chemical Co _Oi) Ltd.), and moderately polar adsorbents based on a polymer of acrylic acid ester and / or methacrylic acid or a copolymer of such a monomer with a non-polar monomers ₀ as styrene and Bivinyl "benzene .als matrix" Z ₀ B ₀ Arabian Lite ΣΑ0 ~ 7 and XAD-8 (products of Rohm & Haas Co ") and Diaion HP-ZMG ( Product of Mitsubishi Chemical Co. _{O £)} Ltd.). If desired, these adsorbents can be used in combination _{or the like}

What these adsorbents have in common is that they contain foreign materials in the liquid containing raw SM,

such as amines and dyes, selectively adsorb. However, depending on their type, they have selective adsorption capacity for SAM. For example, non-polar adsorbents adsorb foreign materials, but not SAM, while moderately polar adsorbents SAM along with the foreign materials with relatively weak acidity adsorb, but with relatively strong acidity only adsorb the foreign materials and not the SAM adsorb.

In the method according to the invention, the treatment the stage (A) and the treatment of stage (B) are each carried out at least once. The chronological order or the sequence of treatment stages is arbitrary. Preferred Examples of the order are (1) (A) and then (B); (2) (B) and then (A); (3) (A), (B) and then (A); and (4) (B), (A) and then (B). If necessary can be the treatment stage (A) and / or the treatment stage (B) to be added to these examples. As the number of treatment levels increases, will naturally the economy of the cleaning process is slightly reduced.

If the treatment stage (B) before the treatment stage (A) is carried out, it is advantageous from an operational point of view to set such conditions, that only the impurities are selectively adsorbed on the synthetic resin adsorbent, without adsorption of SAM taking place. A special one The technique of establishing these conditions is, for example, to use a non-polar adsorbent to use or to use a moderately polar adsorbent, and contacting at a Acidity of a pH of not more than 3.5, preferably 0.2 to 3.0, to make.

When the Endstuf © d © s number cleaning process istρ the step (B) it is possible _s © j applicable in processes in which one first adsorbs the SAM on the synthetic resin adsorbent and then separating ₉ or a method ;, in which SAM passed through the synthetic resin = adsorbent without adsorption

The SAM "solution has been eluted ₉ di © of the described Reihenreinigungsstuf © _9, if desired under reduced pressure, concentrated Then it is with an organic solvent ₉ as methanol, ethanol _ΰ n-Propanolp isopropanol _g n-butanol ^ isobutanol, Methoxyethanolj, acetone _ΰ Methyl © thy Iketon ₉ Methylformiatj, Ethylformąat _s Methyl acetate _s Ethyl acetate, butyl acetate and dioxane ₉ contacted _o As a result, a precipitate of a salt can be obtained _s formed from SAM and an organic or inorganic acid. Alternatively, without contacting the SAM solution with an organic solvent, a powdery salt from SAM and an inorganic or organic acid can be obtained by removing the excess acid from the SAM solution using an anion exchange resin (0 ° = Τγρ) or an alkali, which is able to ₉ by reaction with the "in the fractional elution of SAM acid used in salt © _s to form and then removed the solvent under reduced pressure from the solution evaporates is ₀ dry until the residue _o

The weakly acidic ion exchange of the H-type becomes regenerated in a stage, where the SAM elutes therefrom Xforden isto It is therefore not always necessary “that To subject the exchange resin to a special regeneration treatment *, it can only be used after washing with water

can be used repeatedly. On the other hand, the synthetic resin adsorbent can be easily regenerated by typing it with a 5C $> igen, for example aqueous methanol solution and then washes with water.

Thus, according to the present invention, SAM with a very high purity can be effectively obtained through simple process steps and using substances that are easy to regenerate.
10

The invention is illustrated in the examples.

example 1

Saccharomyces cerevisiae (IFO 2044) was in the culture medium by F. Schlenk et al. [Journal of Biological Chemistry, Volume 229, Page 1037 (1957)] cultivated to Form microbial cells with SAM accumulated in them. 210 g of the resulting cells were in 1000 ml of 1.5N Perchloric acid suspended and extracted with shaking at room temperature for 1 h. Then the cell debris became separated by centrifugation. Potassium hydrogen carbonate was added to the extract to adjust the pH to 5.0. The resulting precipitate of potassium perchlorate was removed by suction filtration, whereby 1080 ml of an extract containing 1.15 g of SAM was obtained.

The extract was passed through a column filled with 200 ml of weakly acidic cation exchange resin [Amberlite IRC-50 (H ⁺ type), trademark for a product of Rohm & Haas Co.) to adsorb SAM. The column was washed with 400 ml of 0.0001N acetic acid and fractionally eluted with 0.1N sulfuric acid to obtain 630 ml of a SAM solution.

The resulting SAM = solution was adjusted to a pH of 4.5 with a weakly basic anion exchange resin [Ambeplite IRA-45 (QIT type), trademark for a product by Rohm & Haas Co ₀ ] and then passed through a column ₀ filled with 1 _S 5 1 of Amberlite XAD-7 (trademark for © in acrylate type synthetic resin adsorbent ₉ manufactured by Rohm & Haas Co.) ₀ to adsorb SAM. The column was washed with 2 1 O ₅ 0001N Essigsäur © and eluted then fractionated with 0.1 N sulfuric acid ^ whereby 1440 ml of a SAM = solution were obtained.

The SAM solution was concentrated under reduced pressure until the total volume 200 ml fraudulent Then 800 ml Iceton added ^ whereby © in precipitation of SAM sulfate was obtained "The precipitate was ₉ dissolved in a small amount of water and then lyophilized separated by centrifugation _s whereby 1.74 g of SAM sulfate were obtained as a white powder. This product shows a single spot on paper chromatography and thin layer chromatography with silica gel.

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

ILe i s ρ i e 1 2

A extracts containing 1 ₉ O6 g SAMj, obtained as in Example 1 ₉ was passed through a column packed with 200 ml of Amberlite IRC = 84 (H ⁺ type) (trade name for a weakly acidic cation exchange resin manufactured by Rohm & Haas Co which: _o ) Was filled with ₅ to adsorb SAM * The column was _{washed with 400 ml of O 9} O00IN hydrochloric acid and then fractionally eluted with hydrochloric acid, whereby 640 ml of a SAM solution were obtained.

33Z9Z1Ö

The SAM solution was made with Amberlite IRA-45 (OH ~ -type), a weakly basic anion exchange resin, adjusted to a pH value of 4.8 and then through passed a column filled with 1.5 liters of Amberlite XAD-7 (trademark for a synthetic resin adsorbent, manufactured by Rohm & Haas Co.) was filled to adsorb SAM. The column was filled with 2 10 0001N Acetic acid washed and then fractionated eluted by adding a mixed solvent consisting of 0.1N hydrochloric acid. and acetone (in a volume ratio of 1: 0.1) was passed through. In this way 1120 ml a SAM solution.

The SAM solution was concentrated under reduced pressure until its total volume was 200 ml. On that became Amberlite IRA-45 (OH ~ -type), a weakly basic one Anion exchange resin, added to adjust the pH adjust the solution to 2.0. The resin was separated by suction filtration and the filtrate at reduced Focused pressure. The concentrate was lyophilized to give 1.19 g of SAM hydrochloride as White dust. The product showed by paper chromatography and thin layer chromatography on silica gel a single spot.

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

Example 5

An extract containing 0.98 g of SAM obtained as in Example 1 was passed through a column containing 200 ml of Amberlite XAD-2 (trademark for a styrene / divinylbenzene type adsorbent) from Rohm & Haas Co.) was filled to foreign materials

to adsorb "potassium The resulting precipitate of potassium perchlorate was added to the SAM solution from which the impurity © ;, as described above, _s had been removed was added to adjust the pH to 5.0» was removed by suction filtration.

The SAM solution was then passed through a column filled with 200 ml of Amberlite RC-50 (H ⁺ type), a weakly acidic cation exchange resin, in order to adsorb SAM. The column was washed with 400 ml of 0.0001N acetic acid and _{fractionally eluted with O 5} 1N sulfuric acid, whereby 630 ml of a SAM solution were obtained.

The resulting SAM · = · solution was concentrated under reduced pressure until its total volume became 200 ml. Then 800 ml of acetone were added, whereby a precipitate of SAM sulfate was obtained. The precipitate was separated by centrifugation, dissolved in a small amount of water and lyophilized to give 1 ₅ 5O g SAM-sulfate obtained as a white powder. This product shows a in paper chromatography and thin layer chromatography with silica gel

The recovery ratio of SAM "and its purity are compiled in Table 1 «,

At s ji e 1 4

Microbial cells with accumulated therein SM were prepared as in Example 1 ₀ 200 g of the microbial cells were suspended ₃ 1N formic acid in 1000 ml O. The suspension was heated to 60 ° C for 10 min and then cooled immediately.

33292Ί8

ren separated, whereby 1020 ml of an extract with a Content of 0.91 g of SAM were obtained.

The extract was purified by a 200 ml Amberlite XAD-8 (Trademark for an acrylate adsorbent, manufactured by Rohm & Haas Co.) filled column To adsorb foreign materials.

The SAM solution from which the foreign materials had been removed was adjusted to a pH of 5.0 with Amberlite IRA-45 (OH ~ type), a weakly basic anion exchange resin, and then by one with 200 ml of Amberlite IRC -84 (H ⁺ type), a weakly acidic cation exchange resin, was passed to adsorb SAM. The column was washed with 400 ml of 0.0001N hydrochloric acid and then fractionally distilled with 0.2N hydrochloric acid to obtain 710 ml of a SAM solution.

Amberlite IRA-45 (OH ~ -type), a weakly basic anion exchange resin, was added to the SAM solution to adjust its pH to 2.0. The resin was removed by suction filtration and the filtrate concentrated under reduced pressure and lyophilized to give 1.01 g of SAM hydrochloride as a white powder became. This product showed by paper chromatography and thin layer chromatography on silica gel a single spot.

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

Example 5

630 ml of a SAM solution obtained by treatment with the styrene / divinylbenzene adsorbent and the

weakly acidic H-type cation exchange resin as in Example 3 " was passed through a column filled with 200 ml of an acrylate-type adsorbent (AmberIite XAD-7) to adsorb foreign materials.

The SAM solution from which the foreign matters had been removed as described above was concentrated under reduced pressure ₃ until its total volume became 200 ml. Then 800 ml of acetone were added to form a precipitate of SAM ° sulfate. The precipitate was separated by centrifugation, dissolved in a small amount of water and lyophilized, whereby 1.46 g of SAM sulfate was obtained as a white powder. This product showed a single spot by paper chromatography and thin layer chromatography with silica gel.

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

Example I ₁

950 g of an extract containing 0.98 g SAM obtained according to Example 1 ₉ were adjusted with potassium hydrogen carbonate at a pH of 5.0. The resulting precipitate of potassium perchlorate was removed by suction filtration to give a liquid containing SAM.
30th

The liquid containing SAM was passed through a column filled with 200 ml of Amberlite XRC-50 (H ⁺ = TyP) ₅ a weakly acidic cation exchange resin to adsorb SAM. = The column was washed with 400 ml of 0.0001 N acetic acid and then fractionally with 0.1N sulfate

eluted to give 610 ml of a SAM solution.

The SAM solution was filled with 200 ml of activated charcoal * 5 column filled for chromatography passed. the Column was washed with 600 ml of 0.2N sulfuric acid and then with a mixed solvent consisting of 1, ON Sulfuric acid and methanol (1: 1, vol.) »Fractionally eluted, whereby a SAM solution was obtained.

The SAM solution was concentrated under reduced pressure until its total volume became 200 ml. 800 ml of acetone were added was added to give a precipitate of SAM sulfate. The precipitate was removed by centrifugation separated, dissolved in a small amount of water and lyophilized, leaving 1.28 g of SAM sulfate as a white Powders were obtained. This product showed on paper chromatography and thin layer chromatography a single spot on silica gel.

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

Comparative example 2

Example 1 was repeated except that the treatment with the Amberlite IRC-50 (H ⁺ type) was carried out as the cleaning treatment.

The results obtained are shown in Table 1.

10

Table 1

Purity ninhydrin reaction Amount of recovery of SAM with other mate- methylthio- relative. (%) (+1) rials as SAM (+2) adenosine SAM (%) (%) (+3)

Ex.1 98.1 2 98.0 3 98.0 4th 98.1 5 98.5 VgIB.1 97.6 CvJ 56.7

0.08 89 0.1 90 0.1 90 0.08 90 0.05 88 0.4 77 1.8 90

Example stands for example and VgIB. for comparative example. (+1) The purity of SAM was measured by two-dimensional paper chromatography.

(+2) The ninhydrin reaction with materials other than SAM was carried out as follows: A sample was developed by two-dimensional thin layer chromatography on cellulose and the presence of others Stain as a stain of SAM was detected from the color formation with ninhydrin and rated as follows.

unavailable

+ minor presence ++ significant presence.

(+3) The amount of methylthioadenosine was through two-dimensional paper chromatography determined.

The above results clearly show that SAM with higher purity by the method according to the invention and can be obtained with a higher recovery ratio than with known methods «,

End of description »
35

Claims (5)

IAUS "& WESSERT" 332S218 PATENT LAWYERS DR. WALTER KRAUS DIPLOMA CHEMIST DR.-ING. ANNEKÄTE WEISERT DIPL.-ING. SPECIALIZATION CHEMISTRY IRMGARDSTRASSE 15 D-8OO0 MÜNCHEN 71 TELEPHONE OB9 / 797077-797078 TELEX 05-212156 kpatd TELEGRAM CRAUS PATENT 3919 WK / My NIPPON ZEON CO., LTD «Tokyo s Japan Process for the purification of S-adenosyl-L-methionine Claims 1 "J method for the purification of S-adenosyl-L-methionine ^ characterized ₉ that a crude S-adenosyl-L ° methionine-containing liquid is introduced into a cleaning process, the cleaning process being a series cleaning process that occurs in any _The desired sequence consists of (A) at least one step of treating the liquid with a weakly acidic cationic exchange resin of the H type and (B) at least one step of treating the liquid with a porous synthetic resin adsorbent. 2. The method according to claim 1, characterized in that that in step (A) the crude S-adenosyl-L-methionine-containing liquid which has a pH of 3.5 to 6.5 has, contacted with the cation exchange resin to that contained in the liquid S-adenosyl-L-methionine to adsorb on the resin, and that one then fractionates the S-adenosyl-L-methionine of the cation exchange resin using an aqueous solution of an acid having a pH eluted by no more than 3.0. 3. The method according to claim 1, characterized in that that in step (B) the crude S-adenosyl-L-methionine-containing liquid which has a pH of not more than 6.5 has contacted the synthetic resin adsorbent to thereby Adsorb foreign materials in the liquid on the adsorbent. 4. The method according to claim 1, characterized in that in step (B) the crude S-adenosyl-L-methionine containing liquid which has a pH of not more than 6.5 with the synthetic Resin adsorbent contacted, causing both S-adenosyl-L-methionine and foreign materials in the liquid is adsorbed on the adsorbent, and that the S-adenosyl-L-ifethionine is then removed from the synthetic resin adsorbent using an aqueous solution of an acid having a pH of not more than 3.5 fractionated eluted. 5. The method according to claim 1, characterized in that that the cleaning process consists of the step (A) and the step (B) in this order. 35 6 »Method according to claim 1 _9, characterized in that the cleaning process consists of stage (A), stage (B) and stage (A) in this order. 7 »The method according to claim 1, characterized in that that the cleaning process consists of the stage (B) and the stage (A) in this order. 8 _{e 9} The method of claim 1 characterized in ₅ that the cleaning process of step (B), the step (A), and d @ r step (B) in this order, " 9 »Process according to claim 7 _S, characterized in that in stage (B) the crude S-adenosyl-L-methionine containing liquid _g which has a pH value of not more than 6 ₉ 5 _s with the are synthetic resin adsorbent contacted _s whereby only foreign materials in the liquid on the adsorbent adsorbs _o 1Oo method according to claim 8 _B, characterized in ₉ that in a © r © rst @ n stage (B) di © the crude S-adenosyl-L-raethionine © at-containing liquid, which © in © n pH ¥ ®rt of no more than 6j, 5 has _{contacted 9} with the synthetic resin · = adsorbent, whereby only the fr © mdmat © riali © n in the liquid is adsorbed on the adsorbent