JPH0375560B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for industrially and inexpensively producing 5'-nucleotides useful as chemical seasonings by chemically phosphorylating nucleosides. More specifically, the present invention provides inosine and guanosine without protecting the 2'- and 3'-position hydroxyl groups (secondary hydroxyl groups) of the sugar moiety (ribose). A mixed crystal of guanosine and inosine is used for direct phosphorylation of guanosine-5'-phosphoric acid (hydroxyl group).
The objective is to suppress the by-product of guanosine diphosphates such as 5'-diphosphoric acid, guanosine-2'(3'), and 5'-diphosphoric acid. Conventionally, the method for producing 5'-nucleosides involves converting ribonucleosides into 2'-,
After protecting the 3'-position with an acyl group or alkylidene group, phosphorylation is performed using various phosphorylating agents in the presence of various reaction solvents under various conditions, and then the protecting group is hydrolyzed and removed. A method for producing 5'-ribonucleotides is known, but this phosphorylation method has drawbacks such as requiring multiple steps for the reaction, requiring a large amount of phosphorylating agent, and not necessarily providing a good yield. In addition, in order to solve these drawbacks, conventional
As a method for chemically phosphorylating unprotected nucleosides, a method is known in which a nucleoside is reacted with a phosphorylating agent in the presence of a specific polar organic solvent. Such phosphorylation methods include (A)
Method using nitrile compounds (Special Publication 1977-
20316), (B) method using organic acid ester (Tokukosho
42-21351), method using (C) phosphoric acid trialkyl ester (Tetrahedron Letters No. 50,
pp.5065, 1967; Bulletin of the Chemical
Society of Japan Vol.42, 3505 (1969); Tokko Akira
42-11071), (D) method using phosphoric acid tri(alkoxyalkyl) ester (Japanese Patent Application Laid-Open No. 51-86482),
(E) A method using ethylene glycol dialkyl ether (Japanese Patent Publication No. 46-31865), and (F) A method using a polar organic solvent and an organic amine or an inorganic acid salt of an organic amine (Bulletin of the Chemical Society
of Japan, Vol. 48, 2084 (1975). However, when these unprotected phosphorylation methods are applied to guanosine phosphorylation, the conversion rate to the desired guanosine-5'-phosphate is at most about 90%, and inosine In this case, the conversion rate to inosine-5'-phosphate is as high as 95%, which is not industrially satisfactory. In addition, guanosine-5'-diphosphate, which is difficult to separate from guanosine-5'-phosphate,
Diphosphates such as 2′(3′), 5′-diphosphoric acid
It has the disadvantage that more than 10% is produced as a by-product. Under these circumstances, the present inventors have overcome the above-mentioned obstacles that occur when applying the conventional unprotected phosphorylation method to guanosine phosphorylation, and have developed an industrially advantageous guanosine-5'-phosphorylation method. As a result of extensive research to establish a method for producing acid, we found that by using a mixed crystal of guanosine and inosine as a raw material, not only inosine but also guanosine could be produced at extremely high yields.
It was discovered that the by-product of diphosphates was also significantly reduced by 5'-monophosphate, and after further investigation, the present invention was completed. That is, the present invention provides guanosine-5'-phosphoric acid and inosine-5'-phosphoric acid, which is characterized in that phosphorus oxyhalide or its hydrate is reacted with a mixed crystal of guanosine and inosine in a polar organic solvent. This is a method for producing a mixture with an acid. In the production method of the present invention, a mixed crystal of guanosine and inosine obtained by a method known per se can be used. For example, a mixed crystal obtained by crystallizing an aqueous solution containing guanosine and inosine by a method of precipitating a solute is used (see Japanese Patent Publication No. 38199/1983). In this case, the crystallization method includes, for example, cooling,
Concentration (removal of water), addition of seed crystals, addition of hydrophilic solvents (e.g. acetone) that do not dissolve guanosine and inosine, pH adjustment (acidic range below PH3 where guanosine and inosine have high solubility, or pH
Adjustment to pH 3 to 9 from an alkaline range of 9 or more) and combinations thereof can be appropriately employed. After precipitating a mixed crystal of guanosine and inosine from a solution containing guanosine and inosine by such a method, the mixed crystal is separated by a commonly used method such as suction or pressurization, centrifugation, centrifugal sedimentation, etc. For example, it is desirable to remove the solvent such as water by heating and drying under reduced pressure, and then to subject it to the phosphorylation reaction. At this time, it is advantageous to wash the separated wet crystals with a hydrophilic solvent such as acetone or methyl ethyl ketone, which does not dissolve guanosine and inosine, since it can be dried at low temperatures and in a short time. Alternatively, the dried mixed crystal may be appropriately pulverized and then passed through a sieve, sized to, for example, 100 mesh or less, and subjected to the reaction. When applying the production method of the present invention, if the inosine content in the mixed crystal of guanosine and inosine is too low or too high, it is necessary to improve the conversion rate of guanosine to 5'-monophosphate and In some cases, it may be difficult to achieve the effect of the present invention of suppressing by-products such as. Usually, the ratio of inosine to guanosine is about 0.1 to 10 (mol/mol), preferably about 0.2
It is advantageous to use mixed crystals of ˜4 (mol/mol). The quantitative ratio of inosine and guanosine in the mixed crystal can be arbitrarily adjusted by appropriately selecting their concentration ratio in the crystallization stock solution and/or the crystallization method in the above-mentioned method. The polar organic solvents used in the production method of the present invention include nitrile compounds such as acetonitrile and propionitrile, organic acid esters such as ethyl acetate, methyl acetate, and methyl propionate, and phosphoric acid esters such as trimethyl phosphate and triethyl phosphate. Acid tri-lower alkyl esters, phosphoric acid tri(alkoxyalkyl) esters such as tri(methoxyethyl) phosphate and tri(ethoxyethyl) phosphate, ethylene glycol dialkyl ethers such as ethylene glycol dimethyl ether and ethylene glycol diethyl ether, Tetrahydrofuran, 1,
Examples include cyclic ethers such as 4-dioxane, halogenated hydrocarbons such as dichloromethane, chloroform, and 1,2-dichloroethane, and nitro compounds such as nitromethane, nitroethane, and nitrobenzene. These polar organic solvents may be used in combination of two or more, and organic solvents other than these may also be used, such as aromatic hydrocarbons such as benzene and toluene,
Any ether such as ethyl ether that does not adversely affect the reaction can be used in combination with these solvents. The amount of the polar organic solvent to be used varies depending on the type thereof, but is usually appropriately selected within the range of about 5 to about 30 times the amount of guanosine and inosine, preferably about 10 to 20 times. In addition, pyridine, γ-picoline,
By appropriately adding tertiary amines such as N,N-dimethyl-aniline or their inorganic acid salts in a molar amount of about 1 to 5 times the amount of guanosine and inosine, it is preferable to further accelerate the reaction and improve the yield. Often effective. The phosphorylating agent used in the production method of the present invention is a phosphorus oxyhalide such as phosphorus oxychloride and phosphorus oxybromide. The amount of phosphorus oxyhalide used is usually about 1 to 5 times the molar amount of guanosine and inosine, preferably about 1.5 to 4 times the molar amount. If the amount used is too small, guanosine and inosine will remain unreacted, and if it is used in an excessive amount, the by-product of diphosphates will increase and the yield of the desired 5'-monophosphate will decrease, which is undesirable. .
Usually, it is appropriately selected from the above-mentioned range of use, taking into consideration the type of phosphorylating agent or solvent. During phosphorylation, rather than subjecting phosphorus oxyhalide to the reaction as it is, it is usually better to use it as a partially hydrated product, which has higher selectivity for the formation of 5'-monophosphate, This is preferable because the amount of by-products of phosphates and diphosphates is reduced. The method for obtaining hydrated phosphorus oxyhalide is
After adding and dissolving the phosphorus oxyhalide in the above reaction solvent, add a small amount of water or methanol,
Alcohols such as ethanol and tertiary butanol,
Alternatively, an organic acid such as formic acid or acetic acid may be added to cause the reaction, and the resulting partial hydrate is subjected to the phosphorylation reaction of the present invention as a solution without being separated. The amount of water, alcohol, or organic acid added at this time varies depending on the ratio of phosphorus oxyhalide to the mixed crystal of guanosine and inosine, but usually approximately
It is 0.1 to 0.7 times the molar amount, preferably 0.3 to 0.6 times the molar amount. However, among the above polar organic solvents used as reaction solvents in the production method of the present invention, nitriles, ethylene glycol dialkyl ethers,
When using cyclic ethers, halogenated hydrocarbons, or nitro compounds and adding inorganic acid salts such as pyridine, γ-picoline, and N,N-dimethylaniline to the reaction, phosphorus oxyhalide is It is not necessary to partially hydrate it, and even if it is used as it is in the reaction, good effects can be obtained as in the case of using a partially hydrated product. In the production method of the present invention, a phosphorylating agent is added to the reaction solvent while cooling to about 10°C or less, and a predetermined amount of water (or alcohol or organic acid) is added to the reaction solvent.
and, if necessary, further add and dissolve a tertiary amine (or its inorganic acid salt), then add a mixed crystal of guanosine and inosine, stir,
The reaction is carried out under cooling. The reaction temperature is practically in the range of -30° to +50°C, with -10° to +30°C being particularly preferred. In this temperature range, the reaction time varies depending on the type of solvent, presence or absence of promoter addition, etc., but is generally 30 minutes to 10 hours. In this reaction method,
Compared to the conventional method of phosphorylating guanosine alone, the reaction completes in a shorter time. By mixing the reaction product thus obtained with cold water in a conventional manner, the unreacted phosphorylating agent and the generated nucleoside phosphohalogenate are hydrolyzed to produce guanosine-5'-phosphate and inosine. A solution (hydrolysis solution) containing -5'-phosphoric acid is obtained. As a method for purifying the guanosine-5'-phosphate and inosine-5'-phosphate obtained in this way, for example, 1. The pH of the hydrolyzed solution is adjusted to about 1.5 with sodium hydroxide.
2. A method in which the reaction solvent is extracted and separated using another organic solvent, neutralized with an alkali such as sodium hydroxide, and then purified by adsorption resin treatment or crystallization. 3. A method in which the reaction solvent is extracted and separated using another organic solvent and then treated with activated carbon. Next, after passing through any of these purification methods, guanosine-5′-
It can be obtained as a mixed crystal of disodium phosphate and inosine-5'-disodium phosphate.
If necessary, guanosine-5'-phosphate and inosine-5'-phosphate are separated by a fractional elution method using an activated carbon column, and then each is converted into a highly pure single salt of the desired alkali metal. It is also possible to obtain it as a crystal. These 5'-nucleotides or mixtures thereof have rich taste and are useful substances as chemical seasonings. By applying the production method of the present invention, the conversion rate of guanosine to 5'-monophosphate is improved, and the amount of diphosphate by-products is also significantly reduced.
The conversion rate of inosine to 5'-monophosphate in this case is almost the same as the conversion rate when inosine is phosphorylated alone under the same conditions. Hereinafter, the present invention will be explained in more detail with reference to Reference Examples and Examples. Reference example 1 67.5 g of phosphorus oxychloride was added dropwise to a solution of 100 ml of acetonitrile and 37.9 g of pyridine under ice cooling, and then water was added.
4.5g was added slowly. Keep the resulting mixture at 0° to 2°C and add guanosine to it while stirring.
Added 28.3g. After reacting with continuous stirring at this temperature for 6 hours, the reaction solution was mixed with 700 g of ice water. This aqueous solution was analyzed by high performance liquid chromatography, and the results shown in Table 1 were obtained.

[Table] Reference Example 2 46.0 g of phosphorus oxychloride was added and dissolved in 300 ml of triethyl phosphate under ice cooling, and then 1.8 g of water was slowly added. The resulting mixture was kept at 10° C. and 28.3 g of guanosine was added thereto while stirring. After reacting by stirring continuously at this temperature for 5 hours, the reaction solution was mixed with 500 g of ice water. This aqueous solution was analyzed by high performance liquid chromatography, and the results shown in Table 2 were obtained.

[Table] Reference Example 3 Add 67.5 g of phosphorus oxychloride dropwise to a solution of 200 ml of acetonitrile and 75.1 g of pyridine under ice cooling, then add water.
8.9g was slowly added. Keep the resulting mixture at 0° to 2°C and add guanosine to it while stirring.
28.3 g followed by 26.3 g of inosine were added. After continuously stirring and reacting at this temperature for 6 hours, 1.4Kg of the reaction solution was
of ice water. This aqueous solution was analyzed by high performance liquid chromatography, and the results shown in Table 3 were obtained.

[Table] Reference example 4 Triethyl phosphate 700ml, phosphorus oxychloride 115.0
4.5 g of water was added dropwise to the mixed solution of 1.5 g under ice cooling, then 28.3 g of guanosine was added, followed by 40.2 g of inosine.
g was added thereto, and the mixture was stirred continuously at 5° C. for 6 hours to react. The reaction solution was mixed with 1.2 kg of ice water, and the aqueous solution was analyzed by high performance liquid chromatography, and the results shown in Table 4 were obtained.

[Table] Example 1 83.0 g of phosphorus oxychloride was added dropwise to a solution of 120 ml of acetonitrile and 46.7 g of pyridine under ice cooling, and then water was added.
5.5g was added slowly. The obtained solution is
Keep at 2℃ and add guanosine 28.3 to this while stirring.
g and a mixed crystal containing 6.2 g of inosine were added. After reacting with continuous stirring at this temperature for 5 hours, the reaction solution was mixed with 900 ml of ice water. This aqueous solution was analyzed by high performance liquid chromatography, and the results shown in Table 5 were obtained.

[Table] Example 2 76.1 g of pyridine hydrochloride was added to 140 ml of nitromethane, and 52.5 g of phosphorus oxychloride was added dropwise thereto under ice cooling. The resulting solution was maintained at 2° to 3°C, and 28.3 g of guanosine and 9.9 g of inosine were added to it while stirring.
A mixed crystal containing g was added. After continuously reacting at this temperature for 5 hours, the reaction solution was mixed with 600 g of ice water. This aqueous solution was extracted with ethyl ether to remove nitromethane, and the raffinate was analyzed by high performance liquid chromatography to obtain the results shown in Table 6.

[Table] Example 3 133.6 g of phosphorus oxychloride was added dropwise to a mixture of 200 ml of dichloromethane and 44.2 g of γ-picoline under ice cooling.
Next, 15.2 g of methanol was slowly added. The resulting solution was maintained at 2 DEG to 3 DEG C., and a mixed crystal containing 28.3 g of guanosine and 26.3 g of inosine was added thereto while stirring. After reacting by continuously stirring at this temperature for 5 hours, the reaction solution was mixed with 1400 g of ice water. The obtained liquid was separated into a dichloromethane layer and an aqueous layer, and the aqueous layer was analyzed by high performance liquid chromatography, and the results shown in Table 7 were obtained.

[Table] Example 4 Add 213.2 g of phosphorus oxychloride to a mixture of 320 ml of acetonitrile and 91.8 g of N,N-dimethylaniline under ice cooling.
g was added dropwise, and then 29.1 g of formic acid was slowly added. The resulting solution was maintained at 2° to 3°C, and 28.3 g of guanosine and 57.9 g of inosine were added to it while stirring.
Added mixed crystals containing. After reacting by continuously stirring at this temperature for 4 hours, the reaction solution was mixed with 2.2 kg of ice water. This aqueous solution was analyzed by high performance liquid chromatography, and the results shown in Table 8 were obtained.

[Table] Example 5 Triethyl phosphate 400ml, phosphorus oxychloride 59.7
2.3g of water was added dropwise to the mixed solution of
A mixed crystal containing . The reaction solution was mixed with 600 g of ice water, and the aqueous solution was analyzed by high performance liquid chromatography to obtain the results shown in Table 9.

[Table] Example 6 Trimethyl phosphate 500ml, phosphorus oxychloride 82.9
3.2g of water was added dropwise to the mixed solution of
A mixed crystal containing . The reaction solution was mixed with 900 g of ice water, and the aqueous solution was analyzed by high performance liquid chromatography, and the results shown in Table 10 were obtained. This aqueous solution was extracted with 1,2-dichloroethane to remove trimethyl phosphate, and the raffinate was passed through a charcoal tower to adsorb nucleotides such as guanosine-5'-phosphate, thoroughly washed with water, and then 2% It was eluted with an aqueous sodium hydroxide solution. The eluate was adjusted to pH 8.5 with hydrochloric acid, water was distilled off under reduced pressure, and the concentrated solution was decolorized and filtered under heat, and then methanol was added to crystallize it to obtain disodium guanosine-5'-phosphate and Mixed crystal of inosine-5'-disodium phosphate (hydrated salt, purity
98%).

[Table] Example 7 Triethyl phosphate 700ml, phosphorus oxychloride 115.0
4.5 g of water was added dropwise to the mixture of g under ice cooling, and then 28.3 g of guanosine and 40.2 g of inosine were added to the mixture.
A mixed crystal containing . The reaction solution was mixed with 1.2 kg of ice water, and the aqueous solution was analyzed by high performance liquid chromatography, and the results shown in Table 11 were obtained. This aqueous solution is extracted with toluene to remove triethyl phosphate, and the raffinate is passed through a charcoal tower to adsorb nucleotides such as guanosine-5'-diphosphate, washed thoroughly with water, and then 1.5% ammonium chloride aqueous solution Inosine-5'-phosphoric acid is eluted through a solution adjusted to pH 10.0 by adding ammonia water (first elution), and then guanosine-5'-phosphoric acid etc. are eluted through a 2% aqueous sodium hydroxide solution (first elution). 2 elution).
Add sodium hydroxide to the first eluate and concentrate,
Crystallization was performed while cooling to obtain crystals of inosine-5'-disodium phosphate (octahydrate, purity 99%).

[Table] In addition, the second eluate was adjusted to pH 8.5 with hydrochloric acid, concentrated, and crystallized while standing to cool to give crystals of guanosine-5'-disodium phosphate (heptahydrate, purity 98%). I got it. Example 8 Trimethyl phosphate 1.1, phosphorus oxychloride 184.9
7.2 g of water was added dropwise to the mixed solution of g under ice cooling, and then 28.3 g of guanosine and 81.0 g of inosine were added to the mixture.
A mixed crystal containing . The reaction solution was mixed with 2 kg of ice water, and the aqueous solution was analyzed by high performance liquid chromatography, and the results shown in Table 12 were obtained.

【table】

Claims (1)

[Claims] 1. Guanosine-5′-, which is characterized by reacting phosphorus oxyhalide or its hydrate with a mixed crystal of guanosine and inosine in a polar organic solvent.
A method for producing a mixture of phosphoric acid and inosine-5'-phosphoric acid.