JPS61100583A - Production of 5,6,7,8-tetrahydrofolic acid - Google Patents

Abstract

PURPOSE:The catalytic reduction of folic acid or dihydrofolic acid is carried out in an aqueous inorganic base at a specific pH in the presence of a noble metal catalyst to enable economic and advantageous production of the compound of high quality, which is used as a coenzyme, through simple operations in high yield. CONSTITUTION:Folic acid or dihydrofolic acid is dissolved or suspended in an aqueous solution containing an almost equivalent amount, preferably 0.8-1 equivalent amount of an inorganic base such as ammonium hydroxide based on the acid, to keep the pH 5-9, preferably 6-8 and the catalytic hydrogenation is effected in the presence of a noble metal catalyst at a temperature over 0 deg.C, preferably 0-80 deg.C. The amount of the catalyst used in the reaction is 0.15-3.0wt% calculated as metallic element, based on folic acid or 0.15-2wt% based on dihydrofolic acid.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention is directed to 5. e, 'tp Concerning an improved industrial production method of 8-tetrahydrofolic acid.

In the enzymatic reaction, for example, the ♂-1-〇-formyl compound bound to phoric acid acts as a formyl group donor, and the N3-1N10-methylene compound bound to formaldehyde acts as a hydroxymethyl donor from glycine. It produces serine, and its reduced product, the N5-methyl form, is a useful compound that plays an important role in biosynthetic reactions such as producing methionine from homocysteine.

Conventional technology The conventional method for producing tetrahydrofolic acid is to dissolve folic acid in an aqueous sodium hydroxide solution and reduce it with sodium hydrosulfide to obtain dihydrofolic acid, which is then further reduced with sodium borohydride to obtain tetrahydrofolic acid. How to obtain folic acid (Helvetic force Acta He1v.

Chim, Acta, 1980.63(8) 25
54) and the method of reducing folic acid with NacNBH4 in acetic acid (Analytical Biochemistry Analytical Biochemistry)
l, Biochem, 1980.103(2)
255), but these methods not only require a large amount of expensive reducing agent but also have the drawbacks of complicated manufacturing processes, making them unsuitable for production on an industrial scale. do not have.

On the other hand, a method for producing tetrahydrofolic acid by hydrogenating folic acid in the presence of a noble metal catalyst is also known.

For example, USP, 2717250 (1955) and U.S.P.
SP, 2790802. (1957) describes a method in which folic acid is suspended in glacial acetic acid and reduced in the presence of a platinum oxide catalyst; In addition, a large amount of glacial acetic acid, which is a reaction solvent, is required, and the removal of the product is complicated, so it is not an economically advantageous method. Moreover, the yield is low at about 48 cm.

Also, Journal on American Chemical Society (J, Am, Chem, SOC, 69,
250 (1947)) contains folic acid in an approximately 19 molar ratio (3
0.0 for every 6 ■ of folic acid. IN NaOHl 5cc)
There are examples of hydrogenation using a large amount of platinum oxide catalyst, which is 70% by weight based on folic acid, dissolved in a large amount of aqueous sodium hydroxide solution in strong alkaline conditions, but dihydrofolic acid was not obtained in low yield. Although a large amount of catalyst is used, the hydrogenation reaction stops at the dihydrofolic acid stage, and tetrahydrofolic acid is not obtained.

Problems that Ming is trying to solve As described above, in the conventionally known methods, by-products such as hydrogen separation are generated during the hydrogenation reaction, and unreacted folic acid and dihydro compounds remain in the reaction mass. As a result, the yield of the desired product was low, and this method was not satisfactory for production on an industrial scale.

Means for Solving Problems Between 9 and 9 The present inventors have conducted extensive research into an economically advantageous method for producing tetrahydrofolic acid with a high yield using simplified operations. near,
That is, in the case of a monoacid base, use an inorganic base in an amount of about 2 times the mole of folic acid or dihydrofolic acid, dissolve or suspend it in an aqueous solution, and add platinum or rhodium while maintaining the pH of the reaction solution at 5 to 9. The present invention was completed based on the discovery that the objective can be achieved by hydrogenation in the presence of a noble metal catalyst.

According to the method of the present invention, the manufacturing cost is extremely low because an inexpensive inorganic base aqueous solution or liquid is used as the solvent, and the raw materials are hydrogenated while dissolved or almost dissolved in the inorganic base aqueous solution or in a suspended state. The reaction completes in a relatively short time. Therefore, an extremely small amount of catalyst is sufficient. Further, post-treatment of the reaction solution is also simplified. That is, the catalyst is separated from the reaction mass, discharged and neutralized in an acid solution containing a stabilizer such as L-ascorbic acid, and the precipitate of the liberated target product is separated from F and washed.
By drying under reduced pressure, high-quality tetrahydrofolic acid, which can be satisfactorily used in reactions such as biosynthesis of L-serine, can be obtained in good yield without any special purification.

In the method of the present invention, any inorganic base can be used as long as it dissolves folic acid or dihydrofolic acid and does not adversely affect the hydrogenation reaction. For example,
Sodium hydroxide, potassium hydroxide, sodium carbonate,
Examples include inorganic bases such as potassium carbonate, sodium hydrogen carbonate, potassium hydrogen carbonate, and ammonium hydroxide, with ammonium hydroxide being particularly preferred.

In use 1 of the base, the base is used in an equivalent amount to the carboxyl group of the raw material folic acid or dihydrofolic acid, preferably in an equivalent ratio of 0.8 to 1, and the pH of the reaction solution is maintained at 5 to 9, preferably 6 to 8. It is necessary to implement it.

If the pH of the reaction solution is lower than 5, the solubility of the raw materials decreases, leaving a large amount of unreacted substances. If the pH of the reaction solution is higher than 9, the activity of the catalyst decreases, so the amount of hydrogen absorbed becomes much lower than the theoretical amount, the reaction stops, and a large amount of unreacted substances remain.

Usually, an inorganic base can be used in an equivalent ratio of 0.5 to 1.5 to the carboxyl group of folic acid or dihydrofolic acid as raw materials, and the amount used depends on the type of base and the pH of the reaction mass. , shall be enacted as appropriate.

In addition, in the present invention, as a method of carrying out the reaction, the entire amount of base in the vicinity of the equivalent amount may be charged from the start of the reaction, but at the start of the reaction, a part of the base is charged, and as the hydrogenation reaction progresses, the remaining amount is added. A more preferable method is to dropwise charge an aqueous base solution into the reaction system because it can suppress the production of by-products due to decomposition.

The noble metal catalyst used in the method of the present invention is preferably platinum or rhodium, and these catalysts are usually used as supported catalysts on activated carbon, silica, alumina, etc.
It may also be used as an oxide such as platinum oxide.

The amount of catalyst used is 0.1% of the precious metal based on the raw material folic acid.
5-3.0% by weight, 0 if dihydrofolic acid is used as raw material
．． 15-2.0% by weight is used. For example, 5-chi platinum-
When using an activated carbon catalyst, it usually contains 3 to 2
0% by weight is suitable. When folic acid is used as a raw material, if the amount of 5-stage platinum-activated carbon catalyst used is less than 3 parts, the reaction will stop midway, leaving unreacted substances and reaction intermediates, and the amount of catalyst will be more than 20 parts. In some cases, the reaction completes quickly, but no water is added. Therefore, the preferred amount used is 5 to 15% by weight. Further, the catalyst used in the reaction is separated by filtration or the like, washed with water, and then used again in the next reaction. At this time, since the activity of the catalyst has decreased, add a new catalyst if necessary. 〇 Normal pressure is sufficient for the hydrogen pressure during hydrogenation, but the reaction can also be carried out under increased pressure if necessary.

The reaction temperature for hydrogenation is suitably 0°C or higher, and preferably 0 to 80°C when the reaction is carried out under normal pressure. A particularly preferable temperature is 20 to 50°C. Since the reaction rate of the hydrogenation reaction varies greatly depending on the stirring speed of the reaction solution, it is preferable to stir vigorously to bring the reaction solution into sufficient contact with hydrogen.

The operation for extracting tetrahydrofolic acid from the reaction solution is usually performed under an inert gas atmosphere to prevent deterioration of tetrahydrofolic acid due to oxidation.

That is, the reaction solution is separated from the catalyst by a method such as filtration in a nitrogen cylinder in which the air has been replaced with nitrogen, and then neutralized with hydrochloric acid in the presence of a stabilizer such as L-ascorbic acid or mercaptoethanol if necessary to obtain tetrahydrofolic acid. precipitate. At this time, the neutralization pH is preferably around 3.5.

Next, the cooled neutralized liquid is separated by a method such as filtration,
If necessary, remove salts with deaerated cold water and then dry.

In order to dry in a short time at a low temperature, it is preferable to wash with a solvent having a low boiling point and soluble in water, such as acetone or methanol, and then drying.

The tetrahydrofolic acid obtained in this way is a white to off-white crystal, and usually has a purity of 80% or more (HLC analysis).
Shows sufficient activity as a coenzyme in enzyme reactions without special purification.

Hereinafter, the method of the present invention will be specifically explained with reference to Examples. Note that the folic acid used in the examples has a purity of 91.3%, and the number of moles is the value in terms of purity. ◎Example-1 15.01211% of folic acid,
xosxxo mol) and 10.8 ml of N/2 ammonium hydroxide aqueous solution (5.40 x 10 mol) were added and stirred to dissolve most of it. (The pH of the solution was 6.6.) Next, a suspension of 3% platinum-activated carbon catalyst 2.025# (0.44% noble metal/folic acid) in 1601d of distilled water was added to the system. After purging the inside with nitrogen, a hydrogenation reaction was carried out at normal pressure while vigorously stirring in a conventional manner. (Hydrogen absorption rate 100.4%, reaction time 360 minutes)' After the reaction, N/2 ammonium hydroxide aqueous solution 18.6 mA under nitrogen stream! was added to dissolve the very small amount of insoluble matter present. Thereafter, all separation and drying of the reaction products were performed under a nitrogen atmosphere.

That is, the reaction-completed liquid was filtered with a suction filter to separate the catalyst, and then washed with distilled water for 30 minutes. Next, the furnace liquid was discharged into a solution in which 3.31 l of L-ascorbic acid was dissolved in 117.4 mj of N/2 hydrochloric acid, and the solution was neutralized and the pH was adjusted to 1. The deposited precipitate was suction filtered, washed with 30 ml of distilled water at 5°C, and then washed with 20 ml of acetone at 5°C.
Washed with 0ml. The wet cake was then dried under reduced pressure in a vacuum dryer containing a desiccant at 25-30° C. for 8 hours at 15 il Hg to obtain 5.6.7.8-tetrahydrofolic acid. Yield 10.714. ! 1F (yield 77.5%) The purity of the obtained tetrahydrofolic acid by high performance liquid chromatography was 85.0%, and an enzymatic reaction was attempted without purification by conventional column chromatography. , showed activity equivalent to that of commercially available reagents.

4.310 # (8,91 x 10 mol) and N/2 ammonium hydroxide aqueous solution 32,2 # (16,1 x 1 mmol) were added and stirred to dissolve. (The pH of the solution was 6.6.) Next, platinum oxide O, O6SRLC, 1.5%/
A suspension of folic acid (folic acid) in 50 ml of distilled water was added, and a hydrogenation reaction was carried out in the same manner as in Example-1, yielding 2.973!
i (yield 74.9) of 5.6.7.8-tetrahydrofolic acid was obtained.

Example-3 Folic acid 2.296 in a 300 mjt baffled round bottom flask
3J (4,75 x 10 mol) and 16.5 t/(8,25 x 10 mol) of N/2 ammonium hydroxide aqueous solution were added and stirred to dissolve. (The pH of the solution was 6.5.) Next, a suspension of 5% rhodium activated carbon catalyst (0.55% as precious metals/folic acid) in 25 m/distilled water was added and the process was carried out. A hydrogenation reaction was carried out in the same manner as in Example 1 to obtain 5.6.7.8-tetrahydrofolic acid (1.593FC, yield 75.3%).

Example-4 4.384J of folic acid in a 300ml round bottom flask
i' (9,07 x 10 mol) and N/2 aqueous sodium hydroxide solution 3 s, 7y (17,85 x 10'' mol)
was added and stirred to dissolve. (The pH of the solution was 7.3.) Next, a suspension of 0.399N of 5% platinum-activated carbon catalyst in 50-distilled water was added and a hydrogenation reaction was carried out in the same manner as in Example-1. went. (Hydrogen absorption rate: 101.8%, reaction time: 515 minutes) After separating the catalyst by filtration, the p liquid was discharged into a solution consisting of 34d of N/2 hydrochloric acid and 1.0261j of L-ascorbic acid, and neutralized to a pH of 3. Adjusted to 5.

Next, separate and dry according to Example 1, and collect! 2.9
5.6.7.8-tetrahydrofolic acid was obtained with a yield of 26 Jl (72.4 qIJ).

Example 5 2.179!9 of 7,8 dihydrofolic acid and 17.5 ml of N/2 ammonium hydroxide aqueous solution were added to a 300-gopatsuflu round bottom flask and dissolved with stirring. (The pH of the solution was 6.7.2) Next, 5T platinum-activated carbon catalyst 0.112.9 (0.26V dihydrofolic acid as noble metal) was added and a hydrogenation reaction was carried out in the same manner as in Example-1. 1.439 # (yield 65.7) of 5.6.7.8-tetrahydrofolic acid was obtained.

Example-6 N/2 ammonium hydroxide 10.0+1d (0.5 equivalent) was added to folic acid z, 4179 (s, ox1o mol), and p
H6,3 and then 5 Ch platinum-activated carbon catalyst 0.20
2J7. A hydrogenation reaction was started by adding 25-liters of distilled water.

Next, from the point at which the hydrogen absorption rate slows down, 9.8 N/
The reaction was carried out while gradually adding a small amount of ammonium dihydroxide aqueous solution dropwise as the reaction progressed. (The final pH was 6.8.) Thereafter, the same treatment as in Example 1 was carried out, and 5,6,7.8-tetrahydrofolic acid was 1.785.
1 yield of 80.2%) was obtained.

Claims (5)

[Claims] (1) In a method for producing 5,6,7,8-tetrahydrofolic acid by subjecting folic acid or dihydrofolic acid to a catalytic hydrogenation reaction in the presence of a noble metal and a catalyst, an inorganic base in an amount approximately equivalent to the raw material folic acid or dihydrofolic acid is used. A method for producing 5,6,7,8-tetrahydrofolic acid, which comprises dissolving or suspending it in an aqueous solution thereof and carrying out a hydrogenation reaction while maintaining the pH at 5 to 9. (2) The method according to claim (1), in which the hydrogenation reaction is carried out while maintaining the pH at 6 to 8. (3) The method according to claim (1), wherein the inorganic base is used in an equivalent ratio of 0.8 to 1 with respect to the raw material folic acid or dihydrofolic acid. (4) The method according to claim (1), which is carried out while cumulatively adding an inorganic base. (5) The method according to claim (1), wherein the noble metal catalyst is platinum or rhodium.