FR2531714A1 - S-adenosyl-L-methionine solutions purification - Google Patents

Abstract

In the purificn. of S-adenosyl-L-methionine (SAM), a crude SAM-contg. liquid is fed into a serial purification process consisting of (A) one or more treatments with a weakly acidic cation-exchange resin of the H-type and (B) one or more treatments with a porous synthetic resin adsorbent, in any desired sequence. In a pref. procedure, step (A) comprises contacting crude SAM-contg. liquid of pH 3.5-6.5 with a cation exchange resin and subsequently fractionally eluting the adsorbed SAM using an aqueous solution of an acid of pH 3.0 or less. Step (B) pref. comprises contacting crude SAM-contg. liquid of pH 6.5 or less with a synthetic resin adsorbent either to selectively adsorb impurities or to adsorb both SAM and impurities, SAM, in the latter case being fractionally eluted with an aqueous solution of an acid of pH 3.5 or less. Steps (A) and (B) are pref. performed in that order. SAM is involved in the in vivo metabolism of fats, proteins and carbohydrates, and exerts a therapeutic effect in fatty liver, lipaemia, arteriosclerosis, depressions and sleeplessness. Simple process which uses easily regenerated materials to give very highly pure SAM.

Description

 The present invention relates to a method for purifying S-adenosyl-L-methionine (which will be abbreviated hereinafter by SAM), and more particularly, to a method for effectively isolating SAM of high purity. liquid containing raw SAM.

 SAM is an important substance which participates, in vivo, in the metabolism of fats, proteins and carbohydrates. Recently, SAM has been found to have a therapeutic effect on heart fat, lipemia, arteriosclerosis, depression and insomnia, and it is desirable to produce SAM in large quantities.

 Many methods have hitherto been known for the purification of SAM. Particular examples include: (1) a method of combining a step of treating a liquid containing SAM with a strongly acidic cation exchange resin and a step of treating this liquid with activated carbon (Japanese patent published under No. 13680/1971) -, (2) a process which consists in treating a liquid containing raw SAM with a weakly acidic cation exchange resin (Enzymologia, Vol. 29, page 283), (3) a process consisting to combine a step of treating a liquid containing raw SAM with a weakly acidic cation exchange resin of the H + type and a step of treating this liquid with activated carbon (Japanese patent application subject to public inspection N "14299/1981), (4) a process which consists in treating a liquid containing raw SAM with a resin of the chelate type (Japanese patent published under N" 20998/1978), and (5) a process passing through a salt of SAM with picric acid or picrolonic acid (patents and United States of America Nos. 3,707,536 and Nos. 3,954,726).

 Method (2) is simpler from the point of view of the processing steps and more economical than the method as), (4) or (5), but it has the disadvantage of an incomplete separation of SAM from impurities and foreign matter and it does not allow SAM to be obtained with sufficiently high purity for use as a medicament. Regarding process (3), which is an improvement of process (1), the purity of SAM is increased but1 since the adsorption of SAM on the activated carbon is high, the percentage of recovery in SAM is reduced . If one seeks to increase the recovery percentage by increasing the amount of organic solvent in an eluent, unfortunately this results in an insufficient separation of SAM from impurities and foreign matter.

 The present invention therefore proposes to provide a process by which one can effectively isolate SAM to a high degree of purity.

The Applicant has carried out intensive research to achieve this objective, and has found that
SAM could be effectively isolated and purified from a liquid containing raw SAM by combining a step of treatment of this liquid with a weakly acidic cation exchange resin-of the H + type with a step of treatment of this liquid with a porous adsorbent synthetic resin.

 Thus, according to the present invention, there is a method for purifying SAM which consists in subjecting a liquid containing raw SAM to a purification process in series which comprises, in any desired order, (A) at least one treatment step liquid containing raw SAM with a weakly acidic cation exchange resin of the H + type and (B) at least one step of treatment of this liquid with a porous synthetic adsorbent resin.

There are no particular limitations with regard to the process for producing the liquid containing
Raw SAM used in the present invention. For example, this liquid is produced by the culture of a microorganism belonging to the genus Saccharomyces, Candida 'or Mucor capable of producing SAM, in a culture medium containing methionine to produce and accumulate SAM inside and / or outside the microbial cells, and extracting the culture broth with an extraction agent such as perchloric acid, hydrochloric acid, sulfuric acid or formic acid; or by enzymatic reaction of adenosine triphosphate and methionine in the presence of methionine adenosyl transferase.

 The essence of the process of the present invention is to purify such a crude SAM-containing liquid by subjecting it to a serial purification process which comprises, in any desired order, (A) at least one step of processing the SAM-containing liquid crude with a weakly acidic cation exchange resin of type H and (B) at least one step of treatment of this liquid with a porous synthetic adsorbent resin.

 The processing step (A) is carried out in the following manner.

 First of all, the pH of the liquid containing raw SAM is generally adjusted to 3, 5--6, 5, preferably to 4-6.5.

When the pH of the liquid containing raw SAN is too low,
SAM is hardly adsorbed on the ion exchange resin.

If it is too high, SAM is susceptible to decomposition.

The process for increasing -pH is not particularly limited. Preferably, the adjustment of the pH is carried out using a combination of an acid and an alkaline substance capable of forming a readily soluble or insoluble precipitate in water, or an anion exchange resin (of the OH type).

By bringing the liquid containing raw SAM, the pH of which has been adjusted in the above-mentioned manner, into contact with the weakly acidic cation exchange resin of the H + type
SAM of positive charge is selectively adsorbed on the cation exchange resin. Impurities with neutral and negative charges in the liquid are not adsorbed on the resin.

 The weakly acidic cation exchange resin used can be any cation exchange resin having carboxylic acid groups as ion exchange groups. Particular examples are Amberlite IRC-50 and IRC-80 (products of Rohm & Haas Co.) and Diaion WK20 (a product of Mitsubishi-Chemical Co., Ltd.). The contacting can be carried out by a batch process or a column process. The latter method is preferred because of its satisfactory operation and its ease of removing impurities.

 SAM adsorbed on the ion exchange resin is then separated by fractional elution of the resin with an aqueous solution of an inorganic or organic acid having a pH generally not exceeding 3.0, preferably between 0.2 and 2.0. The acid used is not particularly limited and it is possible to use, for example, hydrochloric acid, sulfuric acid, acetic acid and p-toluenesulfonic acid. If necessary before fractional elution of SAM, the ion exchange resin can be washed with water or with a dilute aqueous solution of an acid (e.g. pH 3.5 or higher) to remove traces, impurities present.

 The processing step (B) is carried out in the following manner.

 First, the raw SAM-containing liquid is adjusted to pH 6.5 or less, and then brought into contact with the porous adsorbent synthetic resin. The adjustment of the pH and the contacting can be carried out in the same way as in the treatment step (A). Contact with the adsorbent results in selective adsorption of amines, methylthioadenosine (the decomposition product of SAM) and colored matter which is the foreign matter contained in the liquid. By a choice of conditions F as described above, SAN and the foreign matter can be adsorbed together on the adsorbent. In this case, SAM can be selectively separated by fractional elution of the adsorbent with an aqueous solution of an inorganic or organic acid having a pH not exceeding 3.5, preferably between 0.2 and 2.0.

 The inorganic and organic acids which can be used can be all those which fall into the category used in the treatment stage (A). An organic solvent can be used in conjunction with inorganic or organic acid provided that its proportion can provide a uniform solution. Examples of organic solvent are methanol, methanol; n-propanol, iso propanol, acetone, methyl ethyl ketone, methyl formate, ethyl acetate, dioxane and toluene.

If desired, in the same manner as in treatment step (A), the adsorbent can be washed with water or with a dilute aqueous solution of an acid before fractional elution in order to remove the traces of impurities present.

The porous adsorbent synthetic resin used in the present invention is insoluble in water and has a giant network structure. Particular examples include non-polar adsorbents based on a styrene / divinylbenzene copolymer as a matrix such as Amberlite XAD-2 and XAD-4 (products of Rohm & Haas Co.) and Diaion HP-10, HP-20, HP-30, HP-40 and HP-50 (products of
Mitsubishi Chemical Co., Ltd.), and medium polar adsorbents based on a polymer of an acrylic acid ester and / or a methacrylic acid ester or a copolymer of such a monomer with a non-polar monomer such as styrene and divinylbenzene as matrix, for example Amberlite XAD-7 and XAD-8 (products of Rohm & Haas
Co.) and Diaion HP-ZMG (product of Mitsubishi Chemical Co.,
Ltd.). If desired, these adsorbents can be used in combinations.

 These adsorbents have in common a selective adsorption of foreign materials present in the liquid containing raw SAM, for example amines and colored materials. However, they have a selective adsorbability with respect to SAM according to their type. In particular, non-polar adsorbents adsorb foreign matters but not SAM, and moderately polar adsorbents adsorb SAM as well as foreign matters in conditions of relatively low acidity, but adsorb only foreign matter and not SAM under relatively high acidity conditions.

 In the method of the present invention, the processing of step (Aj and the processing of step (B) are each carried out at least once. The sequence of processing steps is optional. Preferred examples of sequence are (1) (A) then (B); (2) (B) then (A); (3) (A), (B) then (A); and (4) (B), (A) then (B). Depending on the needs, the treatment step (A) and / or the treatment step (B) can be further linked to these examples. As the number of treatment steps increases, the costs involved in the purification process are naturally slightly increased.

 When the treatment step (B) must be carried out before the treatment step (A), it is more operational to place oneself in conditions in which only the impurities are selectively adsorbed on the synthetic adsorbent resin without adsorption of SAM. A particular technique for establishing such conditions consists, for example, of using a non-polar adsorbent or a moderately polar adsorbent and bringing it into contact under acidic conditions at a pH not exceeding 3.5, preferably 0.2 to 3.0.

 When the final step of the serial purification process is step (B), it is possible to use a process consisting in first adsorbing SAM on the synthetic adsorbent resin, then in separating it, or a process which consists in passing SAM through the synthetic adsorbent resin without adsorption.

 The SAM solution eluted from the serial purification step described above is concentrated under reduced pressure, as required. Then, it is brought into contact with an organic solvent such as methanol, ethanol, n-pr-opanol, isopropanol, n-butanol, isobutanol, methoxyethanol, acetone, methyl ethyl ketone , methyl formate, ethyl formate, methyl acetate, ethyl acetate, butyl acetate and dioxane. It is thus possible to obtain a precipitate of a salt formed between SAM and an inorganic or organic acid.

Alternatively, without contacting the SAM solution with an organic solvent, a powder of a salt formed between SAM and an inorganic or organic acid can be obtained by removing excess acid from the SAM solution using an anion exchange resin (of the OH type) or an alkaline substance capable of forming a salt insoluble in water by reaction with the acid used in the fractionated elution of SAM, then evaporation of the solvent from the solution under reduced pressure to dryness
The weakly acidic ion exchange resin of the H + type is regenerated at a stage when SB has been eliminated therefrom. It is therefore not always necessary to subject it to a particular regeneration treatment. It can be used repeatedly after only washing with water. Furthermore, the synthetic adsorbent resin can be easily regenerated, for example by washing with a 50% aqueous solution of methanol, then with water.

 Thus, according to the present invention, SAM can be efficiently obtained with a very high degree of purity by simple operations using substances which are easy to regenerate.

 The following examples illustrate the present invention in more detail.

EXAMPLE 1
Saccharomyces cerevisiae (IFO 204) is cultivated in the culture medium of F. Schlenk et al. Journal of Biological Chemistry, Vol. 229, page 1037 (1957)] to form microbial cells in which SAM is accumulated. 210 g of the resulting cells are suspended in 1000 ml of 1.5N perchloric acid and extracted by stirring at room temperature for 1 hour. Then, the cell residues are removed by separating them by centrifugation. Potassium hydrogen carbonate is added to the extract to adjust its pH to 5.0.

The resulting precipitate of potassium perchlorate is removed by suction filter filtration to obtain 1080 ml of an extract containing 1.15 g of SAM.

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

The pH of the resulting SAM solution is adjusted to 4.5 with a weakly basic anion exchange resin,
Amberlite IRA-45 (type OH) (trademark of a product of Rohm & Haas Co.), then passed through a column packed with 1.5 liters of Amberlite XAD-7 (trademark of a synthetic adsorbent resin of the acrylate type manufactured by Rohm & Haas Co.) to adsorb SAM.

The column is washed with 2 liters of 0.0001N acetic acid - then it is subjected to a fractional elution with 0.1N sulfuric acid to obtain 1440 ml of a SAM solution.

 The SAM solution is concentrated under reduced pressure until its total volume is 200 ml.

800 ml of acetone are then added to obtain a precipitate of SAM sulfate. The precipitate is collected by separation by centrifugation, dissolved in a small amount of water and then lyophilized to obtain 1.74 g of SAM sulfate in the form of a white powder. This product has a single stain in paper chromatography and thin layer chromatography on silica gel.

 The SAM yield and its degree of purity are shown in Table 1.

EXAMPLE 2
An extract containing 1.06 g of SAM, obtained in the same manner as in Example 1, is passed through a column packed with 200 ml of Amberlite IRC-84 (type H +) (trademark of a resin of weak acid cation exchange made by Rohm & Haas Co.) to adsorb
SAT. The column is washed with 400 ml of 0.0001N hydrochloric acid and then subjected to fractional elution with hydrochloric acid to obtain 640 ml of a SAM solution.

The SAM solution is adjusted to pH 4.8 with
Amberlite IRA-45 (typeçOH), a weakly basic ion exchange resin, then it is passed through a column packed with 1.5 liters of Amberlite XAD-7 (trademark of a synthetic adsorbent resin manufactured by
Rohm & Haas Co.) to adsorb SAM. The column is washed with 2 liters of 0.0001N acetic acid and then subjected to fractional elution by passing a mixed solvent composed of 0.1N hydrochloric acid and acetone (at a volume ratio of 1: 0., 1) to obtain 1120 ml of a SAM solution.

 The SAM solution is concentrated under reduced pressure until its total volume is 200 ml, then Amberlite IRA-45 (OH type), a weakly basic anion exchange resin, is added to adjust the pH of the solution at 2.0. The resin is separated by suction filtration, and the filtrate is concentrated under reduced pressure.

The concentrate is lyophilized to obtain 1.19 g of SAM hydrochloride in the form of a white powder. This product has a single stain in paper chromatography and thin layer chromatography on silica gel.

 The SAM yield and its degree of purity are indicated in Table 1.

EXAMPLE 3
An extract containing 0.98 g of SAM, obtained in the same manner as in Example 1, is passed through a column packed with 200 ml of Amberlite XAD-2 (trademark of a styrene-type adsorbent / divinylbenzene manufactured by Rohm & Haas Co.) to adsorb foreign matter. Potassium hydrogen carbonate is added to the SAM solution from which foreign matter has been removed as above, to adjust its pH to 5.0.

The resulting precipitate of potassium perchlorate is separated by suction filtration.

The SAM solution is then passed through a column packed with 200 ml of Amberlite RC-50 (type
H +), a weakly acidic cation exchange resin, to adsorb SAM. The column is washed with 400 ml of 0.0001N acetic acid and subjected to fractional elution with 0.1N sulfuric acid to obtain 630 ml of a SAM solution.

 The resulting SAM solution is concentrated under reduced pressure until its total volume is 200 ml, then 800 ml of acetone is added to obtain a SAM sulfate precipitate. off collects the precipitate in, separating it by centrifugation, dissolving it in a small amount of water, and lyophilizing it to obtain 1.50 g of SAM sulfate in the form of a white powder.

This product has a single stain in paper chromatography and thin layer chromatography on silica gel.

 The SAM yield and its degree of purity are shown in Table 1.

EXAMPLE 4
We produce microbial cells containing
SAM accumulated in the same manner as in Example 1. 200 g of the microbial cells are suspended in 1000 ml of 0.1N formic acid. The suspension is heated to 60 ° C. for 10 minutes, then immediately cooled. The cells are separated by centrifugation to obtain 1020 ml of an extract containing 0.91 g of SAM.

 The extract is passed through a column packed with 200 ml of Amberlite XAD-8 (trademark of an adsorbent acrylate manufactured by Rohm & Haas Co.) to adsorb foreign matter.

The SAM solution freed of foreign matter as above is adjusted to pu .5.0 with Amberlite
IRA-45 (OH- type), a weakly basic anion exchange resin, then pass it through a column packed with 200 ml of Amberlite IRC-84 (H + type) 9 cation exchange resin weakly acid, to adsorb SAM.

The column is washed with 400 ml of 0.0001N hydrochloric acid, then it is subjected to a fractional elution with 0.2N hydrochloric acid to obtain 710 ml of a solution of-SAM.

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

 The SAM yield and its degree of 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 cation exchange resin type H, is passed through a column in the same manner as in Example 3. filled with 200 ml of an adsorbent of the acrylate type, Amberlite XAD-7, to adsorb foreign matter.

 The SAM solution from which the foreign materials have been removed as above is concentrated under reduced pressure until its total volume is 200m1.

800 ml of acetone are then added to form a precipitate of SAM sulfate. The precipitate is collected in it. separating by centrifugation, it is dissolved in a small amount of water, and lyophilized to obtain 1.46 g of
SAM in the form of a white powder. This product has a single stain in paper chromatography and thin layer chromatography on silica gel.

 The SAM yield and its degree of purity are shown in Table 1.

Comparative Example 1
The pH of 950 g of an extract containing Q; 98 g of SAM, obtained in the same manner as in Example 1, is adjusted to 5.0 with potassium hydrogen carbonate.

The resulting precipitate of potassium perchlorate is separated by suction filtration to obtain a liquid containing SAM.

 The liquid containing SAM is passed through a column packed with 200 ml of Amberlite IRC-50 (type H), a weakly acidic cation exchange resin, to adsorb SAM. The column is washed with 400 ml of 0.0001N acetic acid, then it is subjected to a fractional elution with 0.1N sulfuric acid to obtain 610 ml of a SAM solution.

 The S M solution is passed through a column packed with 200 ml of chromatographic active carbon.

The column is washed with 600 ml of 0.2N sulfuric acid, then it is subjected to a fractional elution with a mixed solvent consisting of sulfuric acid 1, GN and methanol (1: 1 by volume) to obtain a solution of SAN.

 The SAM solution is concentrated under reduced pressure until its total volume is 200 ml.

800 ml of acetone are added to obtain a precipitate of SAM sulfate. The precipitate is collected by separation by centrifugation, dissolved in a small amount of water, and lyophilized to obtain 1.28 g of
SAM in the form of a white powder. This product has a single stain in paper chromatography and thin layer chromatography on silica gel.

 The SAM yield and its degree of purity are shown in Table 1.

Comparative Example 2
Example 1 is repeated except that only the treatment with Amberlite IRC-50 (type H +) is carried out as a purification treatment.

 The results are shown in Table 1.

Table 1
Purity Reaction Yield Quantity
from SAM to m.thylthio- in SAM
(7) adenosine ninhydrin (%)
(* 1) with (%) (* 3)
materials
other than
SAT (* 2)
Ex. 1 98.1 - 0.08 89
Ex. 2 98.0. - 0.1 90
Ex. 3 98.0 - 0.1 90
Ex. 4 98.1 - 0.08 90
Ex. 5 98.5 - å 0.05 88
at 0.05
Ex. Comp. 1 97.6 - 0.4 77
Ex. Comp. 2 56.7 ++ 1.8 90
"Ex." means Example, and "Comp. Ex." mean
Comparative example.

 (* 1): The purity of SAM is measured by two-dimensional chromatography on paper.

(* 2): The reaction to ninhydrin with materials other than SAM is determined as follows
A sample is developed by two-dimensional thin layer cellulose chromatography, and the presence of spots other than a SAM spot which are detected by the formation of ninhydrin color is measured and estimated according to the following method with three graduations.

-: None
+: Mild presence
++: Considerable presence
(* 3): The quantity of methylthioadenosine is measured by two-dimensional chromatography on paper.

The above results clearly show that by the process of the present invention, it is possible to obtain
SAM of greater purity in a higher yield than in known methods.

Claims (10)

 1. Purification process for S-adenosyl-L-methionine characterized in that it consists in subjecting a liquid containing the crude S-adenosyl-L-methionine to a purification process, this process being a serial purification process comprising , in any desired order, (A) at least one step of treating said liquid with a weakly acidic cation exchange resin of the H + type and (B) at least one step of treating said liquid with a synthetic adsorbent binder  2. Method according to claim 1, characterized in that step (A) comprises contacting the liquid containing the crude S-adenosyl-L methionine and having a pH of 3.5 to 6.5 with said tesin d exchange of cations to adsorb on the resin the S-adenosy1-L-methionine that the liquid contains, then to subject the S-adenosyl L-methionine at fractional elution to separate it from said cation exchange resin using an aqueous solution of an acid having a pH not exceeding 3.0.  3. Method according to claim 18 characterized in that step (B) comprises bringing the containing liquid into contact. crude S-adenosyl-L-methionine and having a pH not exceeding 6.5, with said synthetic adsorbent resin so as to adsorb foreign matter contained in the liquid on the adsorbent.,  4. Method according to claim 1, characterized in that step (B) comprises bringing the liquid containing crude S-adenosyl-L-methionine into contact and having a pH not exceeding 6.5 with said synthetic resin- adsorbent so as to adsorb both S-adenosyl L-methionine and the foreign matter of the liquid on the adsorbent, then to subject to a fractional elution S-adenosyl-L-methionine to separate it from the synthetic adsorbent resin using an aqueous solution of an acid having a pH not exceeding 3.5.  5. Method according to claim 1, characterized in that the purification process comprises step (A> and step (B), in this order.  6. Method according to claim 1, characterized in that the purification process comprises step (A), step (B) and step (A), in this order.  7. Method according to claim 1, characterized in that the purification process comprises step (B) and step (A), in this order.  8. Method according to claim 1, characterized in that the purification process comprises step (B), - step (A) and step (B) 1 in this order.  9. Method according to claim 7, characterized in that step (B) comprises contacting the liquid containing crude S-adenosyl-L-methionine and having a pH not exceeding 6.5 with the synthetic adsorbent resin so as to only absorb foreign matter from the liquid on the adsorbent.  10 Method according to claim 8, characterized in that the first step (B) comprises contacting the liquid containing the crude S-adenosyl-L-methionine and having an rH not exceeding 6.5 with the synthetic adsorbent resin of so as to adsorb on the adsorbent only foreign matter contained in the liquid.