JPH0631237B2 - Method for preserving and stabilizing 5,6,7,8-tetrahydrofolic acid and its derivatives - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for preserving and stabilizing 5,6,7,8-tetrahydrofolic acid and its derivatives.

5,6,7,8-Tetrahydrofolic acid (hereinafter abbreviated as tetrahydrofolic acid) is a coenzyme represented by the following structural formula (I), and is a useful compound that becomes various useful derivatives in the enzymatic reaction.

For example, an N ⁵ , N ¹⁰ -formyl compound bound to formic acid is a formyl group donor, and an N ⁵ -N ¹⁰ -methylene compound bound to formaldehyde is represented by the following structural formula (II). Serine is produced from glycine as a donor, and the N ⁵ -methyl derivative of the reduced product thereof produces methionine from homocysteine, which plays an important role in the biosynthetic reaction.

These tetrahydrofolic acid and its derivatives are unstable compounds, which decompose over time due to oxygen, heat, light, etc.,
This produces dihydrofolic acid, folic acid, p-aminobenzoic acid and other complex compounds. Therefore, when the degree of deterioration is significant, the enzymatic reaction is sometimes hindered. Therefore, a stabilizing method that prevents deterioration of tetrahydrofolic acid and its derivatives during storage is extremely important.

Conventional Technology Tetrahydrofolic acid is usually prepared in the presence of a noble metal catalyst in an alkaline aqueous solvent of an inorganic base such as ammonium hydroxide.
Obtained by catalytic hydrogenation of folic acid. Further, the operation of taking out tetrahydrofolic acid from the obtained hydrogenation reaction liquid is carried out in order to prevent deterioration of tetrahydrofolic acid due to oxidation, for example, in an inert gas atmosphere such as a nitrogen box in which air is replaced with nitrogen, L-ascorbic acid, Tetrahydrofolic acid powder containing a stabilizer is obtained by neutralizing with hydrochloric acid containing a stabilizer such as mercaptoethanol to precipitate tetrahydrofolic acid, filtering, washing, drying, and recrystallizing.

The tetrahydrofolic acid thus obtained is enclosed in an ampoule filled with an inert gas such as argon gas in the case of powder, and dissolved in an aqueous solution of 1 mol / mercaptoethanol in the case of solution, Normally 0.5g / 3m
It is commercially available by a method in which the solution concentration is adjusted to a certain level, the ampoule is filled with an inert gas as in the case of powder, and stored at low temperature.

Problems to be Solved by the Invention Although such conventional storage methods have some effects, they are not always satisfactory methods.

That is, even if the stabilizer is contained, it has a large temperature dependency when stored as a solution, and deterioration is observed even at a relatively low temperature near 0 ° C., and the stabilizer does not act effectively, and it is particularly abrupt at around room temperature. There was a deterioration in quality. Also, powder is more stable than storing it as a solution, but for example, if you want to re-storing the leftover material taken out from the ampoule, or if you self-made and put it in a container for self-consumption, It is difficult to completely replace the internal air with an inert gas, and deterioration in quality with time is unavoidable.

Means for Solving the Problems The inventors of the present invention have conducted extensive studies on a method of stably storing tetrahydrofolic acid and its derivatives with an advantageous and simple operation over time.

As a result, depending on the usage form of tetrahydrofolic acid and its derivative, the isolation of tetrahydrofolic acid obtained by the hydrogenation reaction of folic acid, and further isolation from the reaction mass that was subsequently reacted to obtain its derivative, that is, powdery It was found that the reaction solution can be used as it is without being necessarily used, so that it can be stably stored for a long period of time by a simple method.

That is, the present invention is a method for preserving and stabilizing tetrahydrofolic acid and its derivatives, characterized by freezing an alkaline aqueous solution of tetrahydrofolic acid and its derivatives and shielding them at low temperature in the presence of an inert gas by blocking outside air. .

In the method of the present invention, as tetrahydrofolic acid and its derivative, it is of course applicable that tetrahydrofolic acid is produced, and the powder isolated according to a conventional method is dissolved in an alkaline aqueous solution such as ammonium hydroxide or caustic soda. Can be, for example, represented by the above structural formula (II),
N ⁵ -, N ¹⁰ - it can be directly applied to the solution obtained by preparing a derivative of methylene body. N ^5, N ¹⁰ - methylene body, L
-Being used as a solution in the synthesis of serine,
Hydrogenation reaction of folic acid After separating the catalyst from the alkaline solution, the reaction solution is reacted with formalin, and the obtained methylene reaction solution is stored as it is according to the method of the present invention, and the reaction is carried out in a usual inert gas seal. It also eliminates the need for complicated equipment for powder separation.

When stored as a solution, the reaction mass after reaction is usually an aqueous solution of tetrahydrofolic acid and its derivative in a concentration of 5 to 10%, and the solution after catalyst separation is sealed in a container in the presence of an inert gas. It can be frozen and stored. There is no effect even if the frozen stock is thawed and used for the enzymatic reaction of L-serine synthesis, for example.

Examples will be shown below.

Example-1 100 g of folic acid (purity 90%) was dissolved in an equivalent amount of dilute aqueous ammonia to a concentration of 10.0% by weight, and 8.02 g of 3% platinum-activated carbon catalyst (50% hydrous product) was added to the solution in the neutral region by a conventional method. The hydrogenation reaction was carried out at room temperature and atmospheric pressure.

After completion of the reaction, hydrogen in the system was continuously replaced with nitrogen gas, and 21.4 g of L-ascorbic acid, 21.0 g of 43.8% formaldehyde aqueous solution and 100 cc of water were charged with stirring. 30 at room temperature
After stirring for 1 minute, filter the catalyst under a nitrogen atmosphere,
It was washed with 0 cc to obtain an aqueous solution of an N ⁵ , N ¹⁰ -methylene derivative [the above structural formula (II)] ammonium salt.

N ⁵ , N ^10- containing 1.72% of the obtained L-ascorbic acid
Aqueous methylene ammonium salt solution (methylene concentration 6.64
%) 150 cc capacity 250 cc capacity glass brown container was charged, the air in the container was replaced with N ₂ gas, and the container was sealed.

Then, -20 ° C. (freezing) and, 0 to 5 ° C., and stored in a thermostatic chamber of 25 ℃, N ^5, N ¹⁰ in the solution by high performance liquid chromatography - examined the change in concentration of methylene body. The results are shown in Table 1, and it was clear that the quality was stabilized by freeze-solidifying and storing the methylene body solution.

Example-2 100 g of folic acid (purity 90%) was dissolved in an equivalent amount of dilute aqueous ammonia to a concentration of 10.0% by weight, and 8.02 g of 3% platinum-activated carbon catalyst (50% hydrous product) was added to the solution in the neutral region by a conventional method. The hydrogenation reaction was carried out at room temperature and atmospheric pressure.

After completion of the reaction, the hydrogen in the system was continuously replaced with nitrogen gas, 21.4 g of L-ascorbic acid was added with stirring, and the mixture was dissolved with stirring at room temperature for 30 minutes. Then, the catalyst was filtered under a nitrogen atmosphere, and water 100c was added.
It was washed with a volume of c to obtain an aqueous solution of ammonium tetrahydrofolate.

Using this solution, the storage stability was examined in the same manner as in Example-1.

The results are shown in Table 2, and it is clear that the quality is stabilized by freeze-solidifying and storing the tetrahydrofolic acid aqueous solution.

Claims (1)

[Claims] 1. An alkaline aqueous solution of 5,6,7,8-tetrahydrofolic acid and its derivative is frozen and solidified, and stored in the presence of an inert gas at low temperature by blocking the outside air. A method for preserving and stabilizing 6,7,8-tetrahydrofolic acid and its derivatives.