JPS6246156B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing coenzyme Q from reduced coenzyme Q.

Coenzyme Q is a substance that is involved in the electron transport system in vivo and exhibits excellent pharmacological effects against various diseases. Crude coenzyme Q can be obtained by synthesis, fermentation, or natural products, but when purified to high-purity coenzyme Q, it is difficult to obtain coenzyme Q, which is itself a very unstable compound and also contains similar impurities. Therefore, it was extremely difficult. Conventionally, as a method for purifying coenzyme Q, chromatography using inorganic substances such as cica gel, alumina, and florisil is known. Chromatography using this inorganic adsorbent is a highly effective purification method for target substances containing similar impurities, but in an industrial setting, inorganic adsorbents have high specific gravity and are difficult to operate. There are many problems, and there are also problems with repeated use. Methods using polyethylene powder as an adsorbent were also known, but their surface areas were too small to serve as an industrial means of purification.

The present inventors have completed the present invention as a result of various studies on commercially viable methods for producing highly pure coenzyme Q.

That is, the present invention is a method for producing coenzyme Q, in which reduced coenzyme Q, which is a raw material, is previously treated with a porous synthetic resin to remove impurities, and then the treated reduced coenzyme Q is oxidized.

Porous synthetic resins used in the present invention include, for example, styrene-divinylbenzene copolymers [Amberlite XAD-2 (manufactured by Rohm and Haas), Amberlite XAD-4 (manufactured by Rohm and Haas), Non-polar synthetic resins such as high porous polymer HP (manufactured by Mitsubishi Chemical Industries, Ltd.), polyacrylic esters [Amberlite
XAD-7 (manufactured by Rohm and Haas), Amberlite XAD-8 (manufactured by Rohm and Haas)], sulfoxide [Amberlite XAD-
9 (manufactured by Rohm and Haas)], Amide [Amberlight XAD-11 (Rohm & Haas)]
Examples include polar synthetic resins such as A. In addition, these porous synthetic resins are more suitable as the unit surface area of the resin is larger, but usually
It is 100 m ² /g or more, preferably 400 m ² /g or more. The pore size of the porous resin is preferably about 3 times or more the molecular size of the compound to be treated, usually 50 Å or more, and the larger the average pore size, the better the results.

To explain the method of the present invention in more detail, the synthesis, fermentation, and extraction of natural products are reduced as is or by adding general reducing agents such as hydrosulfite soda, sodium boron hydroxide, etc., by conventional methods. After forming coenzyme Q, the porous synthetic resin is used as a carrier for chromatography with common solvents to concentrate the reduced coenzyme Q fraction in the effluent. Next, the obtained reduced coenzyme Q is treated with a mild oxidizing agent in a conventional manner to produce coenzyme Q. Next, the purified coenzyme Q obtained
Among them, those having a melting point above room temperature can be crystallized as a common purification method, for example, by crystallizing in ascent, to obtain highly pure coenzyme Q ₁₀ . In addition, examples of the reduced coenzyme Q according to the present invention include hydroquinone, which is a reduced product of coenzyme Q, as well as hydroquinone monoester, hydroquinone diester, etc. in which the hydroxyl group of the hydroquinone nucleus is acetylated.

When the reduced coenzyme Q is a coenzyme Q mono-ester or di-ester, chromatography is performed on a porous synthetic resin as in the case of a simple reduction of coenzyme Q, followed by saponification followed by an oxidation step. You can attach it to

Extraction and elution solvents used in the present invention include methanol, ethanol, isopropanol, n-propanol, acetone, methyl ethyl ketone, isopropyl ether, tetrahydrofuran, dioxane, methyl cellosolve, ethyl acetate, benzene, toluene, hexane, petroleum ether, petroleum benzine, isopentane, carbon tetrachloride,
Industrially inexpensive polar or non-polar solvents such as chloroform, dimethylformamide, and water can be used alone or as a mixed solvent in various proportions. When using a solvent, for example, for non-polar porous synthetic resins such as styrene-divinylbenzene copolymer, a polar solvent such as methanol is used to adsorb relatively non-polar impurities, and then methanol Add acetone to the solution and develop with a mixed solvent with weakened polarity to elute the target substance.
In contrast, for polar porous synthetic resins such as acrylic acid ester polymers, they are developed and eluted using a non-polar solvent such as hexane. The resin after the elution operation can be reused by washing it with a solvent that does not adsorb any impurities. As the cleaning solvent, relatively nonpolar organic solvents such as acetone, isopropyl ether, and benzene are effective.

A particularly preferred method in the present invention is to use a non-polar synthetic resin as the porous synthetic resin, and use water, alcohols, ketones, dimethyl sulfoxide, N,N-dimethylformamide, etc. as the elution solvent.
A method of developing using a polar solvent such as acetonitrile is preferred. The polar solvents used in this case are generally mixed solvents based on alcohols having 1 to 5 carbon atoms and ketones having 3 to 6 carbon atoms, such as methanol and water, methanol and acetone, methanol and n-hexane, Combinations such as acetone and water are industrially advantageous. It goes without saying that various other combinations or the use of a single solvent are also possible.

The method of the invention is generally carried out in the following order.

A column with a column length/axis ratio of 1.0 or more is filled with water, and a porous synthetic resin with an amount more than three times (volume/weight) of the target product is filled by natural sedimentation. Next, reduced coenzyme Q
After replacing the column with a solvent that does not elute coenzyme Q, the reduced coenzyme Q to be treated is allowed to flow from the top of the column. Chromatography is carried out in the same manner as in general methods, but if crystals of reduced coenzyme Q are precipitated, the column may be heated as necessary. The target substance can be obtained by collecting the effluent in sections and concentrating the eluted fraction containing the target substance. Next, the column is washed with a solvent having a high elution power such as acetone, benzene, ether, or esters to remove adsorbed substances, and the column is replaced with the solvent to perform purification chromatography again.

Furthermore, unlike inorganic adsorbents, ion exchange resins, etc., the porous synthetic resin used in the method of the present invention is chemically inert, so it is possible to elute the adsorbate with a solvent with a high elution power, such as acetone. It is extremely advantageous industrially because it can be easily and completely regenerated to its original state.

The present invention has the following significantly superior effects compared to conventional methods. In other words, the separation selectivity is extremely high, and the amount of impurities adsorbed to the porous synthetic resin is extremely large, and unlike inorganic adsorbents, there is no adsorption point, so decomposition does not occur, and the There is no loss at all, the recovery rate is extremely high, the porous synthetic resin can be used repeatedly, and unlike conventional methods that use inorganic adsorbents and hydrophobic solvents such as hydrocarbons and ethers, there is no loss due to static electricity, etc. It is possible to select a safer solvent with less risk of fire, and highly pure coenzyme Q can be obtained by a simple purification method, which is satisfactory in terms of economic efficiency and practicality.

Next, examples of the present invention will be shown, but the present invention is not limited to the following examples.

Example 1 High porous polymer HP-20 (specific surface area 71.80
m ² /g, pore volume 1.077ml/g, 40 mesh, manufactured by Mitsubishi Chemical Industries, Ltd.) was packed into a glass column (φ45mm x 300mm) and inverted with acetone:methanol (3:7) mixture. After washing, let it stand. This is combined with isodecaprenol and 2,3-dimethoxy-
5-Methyl-hydroquinone and boron trifluoride
2,3 obtained by condensation in the presence of an ether complex catalyst
-Dimethoxy-5-methyl-6-decaprenylhydroquinone (reduced coenzyme Q ₁₀ )-containing 30 g (purity 48%) of an oily substance is stirred and emulsified with 30 ml of the above mixture, and the mixture is made to flow through the packed column. Then drain with the same mixture. The effluent was divided into approximately 100 ml portions, and a minute amount of each fraction was subjected to silica gel thin layer chromatography to collect the fractions containing 2,3-dimethoxy-5-methyl-6-decaprenylhydroquinone, and the solvent is distilled off by vacuum concentration. The obtained treated product is dissolved in 400 ml of isopropyl ether, 20 ml of 5% potassium hydroxide water is added, and air is introduced for 30 minutes at room temperature with stirring to oxidize. Next, the isopropyl ether layer is washed with water, the solvent is dried, and then distilled off under reduced pressure to obtain 16.1 g of a red oil containing coenzyme Q ₁₀ . The obtained red oil was treated again with a porous synthetic resin and then subjected to thin layer chromatography to obtain 13.9 g of coenzyme Q ₁₀ with a purity of 96%.

Example 2 High porous polymer HP-20 (40 mesh,
(manufactured by Mitsubishi Chemical Industries, Ltd.) was packed into a 45 mmφ column and filled with an acetone:methanol (2:8) mixture. Next, 2,3-dimethoxy-5-methyl-6-nonaprenylhydroquinone (reduced coenzyme Q ₉ ) 30g of fats and oils (purity 46)
%) is added to 30 ml of the above mixture, stirred to form an emulsion, and then flowed through a packed column. Then drain with the same mixture. Next, 2,3-dimethoxy-5-
The treated product obtained by concentrating the fraction containing methyl-6-nonaprenylhydroquinone under reduced pressure was dissolved in 400 ml of isopropyl ether, and 36 g of lead dioxide was added.
Stir for an hour. After separating the lead oxide and concentrating under reduced pressure, 16.4 g of a red oil containing coenzyme _Q9 is obtained.

Example 3 Pseudomonas genus bacteria
denitrificans, NRRL B-1665 (Northern
Regional Research Laboratory, Peonria;
Illionis 61604)] and collected by centrifugation, and the obtained wet bacterial cell paste is heated and extracted with a hexane:methanol mixture in the presence of sodium hydroxide and pyrogallol. Next, add 5% hexane layer
Wash with hydrosulfite soda water, further wash with water, dehydrate with Glauber's salt, and concentrate under reduced pressure. Next, the acetone-soluble part of the concentrate was taken, and the acetone was distilled off to obtain 30 g of an oily substance containing 2,3-dimethoxy-5-methyl-6-decaprenylhydroquinone (reducing coenzyme Q ₁₀ ) (purity 55 %) was treated in the same manner as in Example 1 to obtain 19.2 g of a red oil containing coenzyme Q ₁₀ .

Example 4 High porous polymer HP-40 (specific surface area 704.7
m ² /g, pore volume 0.687ml/g, 40 mesh, manufactured by Mitsubishi Chemical Industries, Ltd.) 200ml was poured into a gasla cylinder (φ45mm x 300mm) and inverted with an acetone:methanol (3:7) mixture. Washed and left to stand. Next, on a packed column, 2,3- produced by condensing isodecaprenol and 2,3-dimethoxy-5-methyl-hydroquinone-4-monoacetate using a boron trifluoride/ether complex catalyst.
Dimethoxy-5-methyl-6-decaprenylhydroquinone-4-monoacetate (reduced coenzyme
Stir 20 g of fat (purity 38%) containing ester of Q ₁₀ ) with 20 ml of the above mixture to emulsify it and then add it. Then drain with the same mixture. Effluent is approximately 100ml
The fractions containing 2,3-dimethoxy-5-methyl-6-decaprenylhydroquinone-4-monoacetate were collected by performing silica gel thin layer chromatography on a very small amount of each fraction, and then removing the solvent. is distilled off by vacuum concentration. Dissolve 9.0 g of the obtained oil in 150 ml of isopropyl ether, add 10 ml of methanol containing 10% caustic potassium, let stand for 30 minutes, add 30 ml of water, and oxidize by introducing air at room temperature for 30 minutes while stirring. . Next, the isopropyl ether layer was washed with water and the solvent was distilled off under reduced pressure, resulting in 8.6 g of a red oil containing coenzyme _Q10 .
is obtained.

Claims (1)

[Claims] 1. Treating reduced coenzyme Q with a porous synthetic resin,
A method for producing coenzyme Q, characterized in that the treated reduced coenzyme Q is then oxidized. 2. The method for producing coenzyme Q according to claim 1, which uses a polar solvent when the porous synthetic resin is a non-polar porous synthetic resin. 3. The method for producing coenzyme Q according to claim 1, which uses a non-polar solvent when the porous synthetic resin is a polar porous synthetic resin.