JPS59167599A - Chemical phosphatization of inosine and guanosine - Google Patents

Abstract

PURPOSE:To prepare 5'-monophosphate of guanosine and inosine useful as a chemical seasoning, economically, in high yield, suppressing the by-production of diphosphate, by treating a mixed crystal of guanosine and inosine with phosphorus oxyhalide or its hydrate in a polar organic solvent. CONSTITUTION:1mol of mixed crystal of guanosine and inosine is treated with preferably about 1.5-4mol of phosphorus oxyhalide or its hydrate in a polar organic solvent (e.g. nitrile, ethylene glycol dialkyl ether, etc.) preferably at -10-+30 deg.C to obtain a mixture of guanosine-5'-phosphate and inosine-5'-phosphate. EFFECT:The 5'-OH can be directly phosphatized without protecting the 2'- and 3'-OH of the sugar group.

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 (lipose), and the 5'-position hydroxyl group (primary hydroxyl group) A mixed crystal of guanosine and inosine is used for direct phosphorylation of guanosine and inosine (hydroxyl groups), and its purpose is to improve the yield of guar/sheer 5'-phosphoric acid and to Guanosine-2'(3
'), to suppress the by-product of guanosine diphosphates such as 5'-diphosphate.

Conventionally, in the production of 5'-nucleosides, liponucleosides are protected at the 2't3'-position of the ribofuranosyl group with an acyl group or alkylidene group, and then treated with various phosphorylating agents in the presence of various reaction solvents. A known method is to phosphorylate under various conditions using
This phosphorylation method has drawbacks such as (1) the reaction requires multiple steps, (2) the amount of phosphorylating agent is large, and (2) the yield is not always good.

In addition, in order to solve these drawbacks, conventionally known methods for chemically phosphorylating unprotected nucleosides involve reacting nucleosides with a phosphorylating agent in the presence of a specific polar organic solvent. There is. Such phosphorylation methods include (A) a method using a Nido 1Jiv compound (Japanese Patent Publication No. 42-20316), (B) a method using an organic acid ester (Japanese Patent Publication No. 42-21351), (
c) Method using phosphoric acid trialkyl ester (Tet
rahedron Letters Nch 50.
pp, 5065.1967iBulletin o
f the Chemical 5ociety
of Japan Vol, 4'2.3505
(1969) I Japanese Patent Publication No. 42-1i071), (D) Method using phosphoric acid tri(ampkoxyalkyl/I/) ester (Japanese Unexamined Patent Publication No. 51-86.482), (E) Method using ethylene glycol dialkyl ether (F) a method using a polar organic solvent and an organic amine or an inorganic acid salt of an organic amine (B)
uiletin of the Chemical
5ociety of Japan, Vol.

48.2084 (1975) and others are known.

However, when these unprotected phosphorylation methods are applied to the phosphorylation of guanosine, the conversion rate to the target guanosine-5'-phosphate is at most 90%.
In contrast, in the case of inosine, the conversion rate to inosine-5'-phosphate is as high as 95%.
This is not industrially satisfactory.

In addition, guanosine-5'-diphosphate, which is difficult to separate from guanosine-5'-phosphate, and guanosine-2'(3')
The disadvantage is that about 10% or more of diphosphates such as 5'-diphosphoric acid are produced as by-products.

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 phosphorylation method.
As a result of extensive research to establish a method for producing 5'-phosphoric acid,
By using a mixed crystal of guanosine and inosine as a raw material, not only inosine but also guanosine can be converted into 5'-monophosphate in an extremely high yield, and the by-product of diphosphates is also significantly reduced. This invention was discovered and further studied to complete the present invention.

That is, the present invention provides guanosine-5'-, which is characterized in that phosphorus oxyhalide or its hydrate is allowed to act on a mixed crystal of guanosine and inosine in a polar organic solvent.
This is a method for producing a mixture of phosphoric acid and inosine-5'-phosphoric 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 means of precipitating the solute is used (see Japanese Patent Publication No. 47-38199).

In this case, methods of crystallization include, for example, cooling, concentration (removal of water), addition of seed crystals, addition of a hydrophilic solvent (e.g. acetone) that does not dissolve guanosine and inosine, pH adjustment (solubility of guanosine and inosine is big pH3
From the following acidic ranges or alkaline ranges of pH 9 or higher
Adjustment to a range of H3 to H9) and combinations thereof can be employed as appropriate. After precipitating a mixed crystal of guanosine and inosine from a solution containing guanosine and inosine in this manner, the mixed crystal is separated by a commonly used method such as suction or pressure filtration, centrifugation, or centrifugal sedimentation. ,
For example, it is desirable to remove the solvent such as water by heating and drying under reduced pressure, and then subjecting 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 ground, passed through a sieve, and sized to, for example, 100 meshes 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 to increase the conversion rate of diphosphates. It may be difficult to achieve the effect of the present invention of suppressing by-products. Usually, the ratio of inosine to guanosine is about 0.1 to 10 (Mo/L//Mo/
It is advantageous to use mixed crystals of about 0.2 to 4 (mol)v/7 l).

The quantitative ratio of inosine and guanosine in the mixed crystal can be arbitrarily adjusted by appropriately selecting the concentration ratio of these in the crystallization stock solution or (and) the crystallization method in the above-mentioned method. .

Examples of the polar organic solvent used in the production method of the present invention include acetonitrile, nitrile compounds such as 10 bionitrile μ,
Organic acid esters such as ethyl acetate, methyl acetate, and methyl propionate; tri-lower alkyl μ esters such as trimethyl phosphate and triethyl phosphate; tri(methoxyethyl/L/) phosphate; tri(ethoxyethyl/L/) phosphate; L/
), phosphoric acid tri(alkoxyalkyl/L/) esters such as ethylene glycol dimethyl ether, ethylene glycol diethyl ether, etc., cyclic ethers/L' such as tetrahydrofuran, 1,4-dioxane, etc. , dichloromethane, chlorophorem, 1,2-dichloroethane, and other halogenated hydrocarbons; 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 benzene,
As long as it does not adversely affect the reaction, such as aromatic hydrogenated hydrogen compounds such as toluene and ethers such as ethyl ether, it can be used in combination with these solvents.

The amount of polar organic solvent used varies depending on the type, but is usually about 5 to about 30% of the amount of guanosine and inosine.
The amount is appropriately selected within the range of twice the amount, preferably about 10 to 20 times the amount.

In addition, pyridine, γ-picoline, N, N-
-, F Methy μm Tertiary amines such as aniline or their inorganic acid salts are added to the amount of guanosine and inocune from about 1 to
Appropriate addition of about 5 times the mole often brings about favorable effects such as further reaction acceleration and yield improvement.

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 amount of guanosine and inosine, preferably about 16
It is 5 to 4 times the molar amount. Its usage is small.

If the amount is too low, guanosine and inosine will remain unreacted, and if it is used in an excessive amount, the by-product of diphosphates will increase and at the same time the yield of the desired 5'-monophosphate will decrease, which is not preferred.

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 production of 5'-monophosphate, and to produce 5'-monophosphate. This is preferable because the amount of by-products of diphosphates is reduced.

The method for obtaining a hydrate of phosphorus oxyhalide is to add phosphorus oxyhalide to the above reaction solvent and dissolve it, and then add a small amount of water or an alcohol such as methanol, ethanol-μ, tertiary pucnol, or The reaction may be carried out by adding organic acids such as formic acid and acetic acid, and the generated 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 is usually about 0.1 to phosphorus oxyhalide. ~0.7
The amount is 0.3 to 0.6 times, preferably 0.3 to 0.6 times
It is the amount of L'.

However, among the above polar organic solvents used as reaction solvents in the production method of the present invention, nitri μ, ethylene glycol shea p-kylete μ, cyclic ethers, halogenated hydrogenated hydrogen, or nitro compounds When using phosphorus oxyhalide and adding an inorganic acid salt such as pyridine, γ-picoline, N,N-dimethylaniline, etc. to the reaction, the phosphorus oxyhalide does not need to be partially hydrated, and the t-reaction Even when used as a partial hydrate, good effects can be obtained as in the case of using a partial hydrate.

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/l's or organic acid) is added thereto. furthermore, a tertiary amine (or its inorganic acid salt)
is added and dissolved, then a mixed crystal of guanosine and inosine is added, and the reaction is carried out under stirring and cooling.

The reaction temperature is practically in the range of -30' to +50°C,
Among these, -10° to +3 (l) is 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, the reaction is completed in a shorter time than in the conventional method of phosphorylating guanosine alone.

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, and 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 hydrolysis solution is adjusted to about 1.5 with sodium hydroxide;
2), the reaction solvent is extracted and separated using another organic solvent, neutralized with an alkali such as sodium hydroxide, and then treated with an adsorption resin or crystallized. 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, it can be obtained as a mixed crystal of diguanosine-5'-disodium phosphate and inosine-57-disodium phosphate by a conventional method.

If necessary, guanosine-5'-phosphoric acid and inosine-5'-phosphoric acid are separated by a fractional elution method using an activated carbon column, and then each is purified as a desired alkali metal salt. It is also possible to obtain it as a single crystal. These 5'-nucleotides or mixtures thereof are rich in taste and are useful substances as chemical seasonings.

By applying the production method of the present invention, the 5′ of guanosine
- The conversion rate to monophosphate is improved, and the amount of diphosphate by-products is also significantly reduced. The conversion rate is almost the same as the conversion rate.

Hereinafter, the present invention will be explained in more detail with reference to Reference Examples and Examples.

Reference Example-1 67.5 liters of phosphorus oxychloride was added dropwise to a solution of 37.9 g of acetonitrile/'100 m/pyridine under cooling with water,
Next, 4.5 g of water was slowly added. The resulting mixture was kept at 0-2C and 28% of guanosine was added to it while stirring.
3F/ was added. After reacting by stirring continuously at this temperature for 6 hours, the reaction solution was mixed with ice water at 700 f. This aqueous solution was analyzed by high performance liquid chromatography, and the results shown in Table 1 were obtained.

Guanosine-5'-diphosphate 0.53
1.2 Guanosine, 5'-diphosphate
1.24 2.8 Reference Example-2 ↓ Tsukushi was added. The resulting mixture was maintained at 10° C. and 28.31 liters 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 f/ice water. This aqueous solution was analyzed by high performance liquid chromatography, and the results shown in Table 2 were obtained.

Table 2 Guanosine-5'-diphosphate 0.58
1.3 Guanosine-2',5'-diphosphate
1.37 3.1 Reference Example-3 Acetonitrile 200ml. 67.5 f of phosphorus oxychloride was added dropwise to a solution of 75.19 g of pyridine while cooling with water, and then 8.9 g of water was slowly added. The obtained mixture was heated to 0°~
28.3 g of guanosine was added to this while stirring at 2°C.
Subsequently, 26.31 inosine was added.

After reacting by stirring continuously at this temperature for 6 hours, the reaction solution was
4. Mixed with ice water. This aqueous solution was analyzed by high performance liquid chromatography, and the results shown in Table 3 were obtained.

Guanosine-5'-phosphate 31.3 8
6.2 guar/singoniphosphoric acid 0.51
1.1 Guanosine-2',5'-nilinic acid 1
．． 22 2.7 Guanosine-3',5'-niline e 2.58 5.8 Reference example-4 Triethyl phosphate)'700ml, phosphorus oxychloride++5
．． 0F! Add 4.5y of water dropwise to the mixed solution under water cooling,
Next, this was followed by 28.3 g of guanosine, followed by 4 g of inosine.
0.2f was added and the mixture was continuously stirred at 5C for 6 hours to react. The reaction solution was mixed with 1.2 ky of ice water, and the aqueous solution was analyzed by high performance liquid chromatography, and the results shown in Table 4 were obtained.

(hereinafter referred to as light/white) Table 4 Guanosine-5'-phosphoric acid 30.8 8
4.8 Guanosine-5'-diphosphate 0.55
1.2 Guanosine-2',5'-diphosphate 1
．． 32 3.0 Guanosine-3',5'-diphosphoric acid 2.90 6.5 Inosine-5'-phosphoric acid 49.7 95.2 Example-1 Acetonitrile/I/120 resistant, pyridine 46.7 g Add 83% phosphorus oxychloride to a solution of 83% under water cooling. Of was added dropwise, and then 5.5 y of water was slowly added. The obtained solution is 0 to 2
℃ and add 28.3 f of guanosine to this while stirring.
and mixed crystals containing 6.2 g of inosine were added. After reacting by continuously stirring at this temperature for 5 hours, the reaction solution was heated to 900 w
t of ice water. This aqueous solution was analyzed by high performance liquid chromatography, and the results shown in Table 5 were obtained.

Table 5 Guanosine-5'-diphosphate 0.18 0
．． 4 guanosine-2', 5'-diphosphate 0.4
2 0.9 Guanosine-3',5'-diphosphoric acid 1.02 2.3 Example-2 76.1 g of pyridine hydrochloride in 140 ml of nitromethane
was added, and 52.5 f of phosphorus oxychloride was added dropwise thereto under water cooling. The resulting solution was maintained at 2° to 3° C., and a mixed crystal containing 28.3f of guanosine and 9.911 inosine was added thereto while stirring. 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 6 G1 Anosine-5'-phosphoric acid 34,0 9
3.6 Guanosine-5'-diphosphate 0.20
0.5 Guanosine-2',5'-diphosphate 0
．． 39 0.9 Gudensine-3',5'-diphosphoric acid 1.03 2.3 Inosine-57-phosphoric acid 12.1 94.0 Example-3 Jig 1: I/l/methane 200gt, r -Picoline 44
．． 133.61 liters of phosphorus oxychloride was added dropwise to the 2V mixed solution under ice cooling, and then 5.2 f of methanol/l'+ was slowly added. The resulting solution was kept at 2°-3°C and 28.3 f of guanosine and 26.3 f of inosine were added to it while stirring.
Added mixed crystals containing. After reacting by stirring continuously at this temperature for 5 hours, the reaction solution was mixed with 1400 g of ice water. The resulting liquid was separated into a dichloroμmethane layer and an aqueous layer, and the aqueous layer was analyzed by high performance liquid chromatography to obtain the results shown in Table 7.

Table 7 Name of produced substance Amount produced [) Production rate [%] Guanosine-5'-phosphoric acid 33.7 92.
8 Guanosine-5'-diphosphate 0.2.1
0.5 Guanosine-2',5'-diphosphate 0.
41 0.9 Guanosine-3',5'-diphosphoric acid 0.98 2.2 Inosine-5'-phosphoric acid 32.1 94゜1 Example-4 Acetonitrile/l/320 Go, N, N-dimethyl Add 213.2 phosphorus oxychloride to a mixture of aniline 91.8 f under ice cooling.
9 was added dropwise, and then 29.1 g of formic acid was slowly added.

The resulting solution was maintained at 2 DEG -3 DEG C., and a mixed crystal containing 28.31 f of guanosine and 57.9 f of inosine was added thereto while stirring. After continuously stirring and reacting at this temperature for 4 hours, the reaction solution was mixed with 2.2 kq of ice water. This aqueous solution was analyzed by high performance liquid chromatography, and the results shown in Table 8 were obtained.

Name of produced substance Production amount CI] Production rate [%]
Guanosine-5-diphosphate 0.17 0.4
Guanosine-2', 5'-diphosphate 0.45
1.0 guanosine-3',5'-diphosphate
? , 00 2.3 Example-5 Triethyl phosphate) L/400txl, phosphorus oxychloride 5
2.31 of water was added dropwise to the mixture of 9 and 7f under ice cooling, and then guanosine 2B, 3f and inosine 8.31 were added to this mixture.
A mixed crystal containing Oy was added, and the mixture was stirred continuously at 10° C. for 4 hours to react. The reaction solution was mixed with ice water at 600 f, and the aqueous solution was analyzed by high performance liquid chromatography to obtain the results shown in Table 9.

Table 9 Guanosine-5'-diphosphate 0.13 0
．． 3 Guanosine-2',5'-diphosphate 0.29
0.7 guanosine-3',5'-diphosphate
0.71 1.6 Example-6 3.2 g of water was added dropwise to a mixed solution of 500 ml of trimethyl phosphate/l' and 82.9 F of phosphorus oxychloride under water cooling, and then 3 g of guanosine 2B and 3 g of guanosine 2B and Inosine 21
．． A mixed crystal containing 5F was added, and the mixture was stirred continuously at 10° C. for 3 hours to react. The reaction solution was mixed with 900 g of ice water, and the aqueous solution was analyzed by high performance liquid chromatography to obtain the results shown in Table 10.

This aqueous solution was extracted with 1,2-dichloroethane to remove trimethyl phosphate, and the raffinate was passed through a charcoal column to adsorb nucleotides such as guanosine-5'-phosphate, thoroughly washed with water, and then % 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 while hot, then Megno 7X/all was added to crystallize, resulting in disodium guanosine-5-phosphate. and mixed crystals of disodium inosine-5-phosphate (hydrated salt,
Purity: 98%)

Guanosine-5'-diphosphate 0.11 0.
3 guanosine, 5'-diphosphate 0.26
0.6 guanosine-3',5'-diphosphate
0.68 1.5 Example-7 Triethyl phosphate)'700w1. Phosphorus oxychloride 115
．． Add 4.5 f of water dropwise to 0 g of the mixed solution under water cooling, and then add 28.31 g of guanosine! and inosine 40.
2F! A mixed crystal containing . The reaction solution was mixed with 1.2 ky 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 was extracted with toluene to remove triethyl phosphate, and the raffinate was passed through a charcoal tower to adsorb nucleotides such as guanosine-5'-diphosphate, thoroughly washed with water, and then 1.8. Inosine-5'-phosphoric acid was eluted (first elution) through a 5% aqueous ammonium chloride solution with aqueous ammonia added to pH 10.0, followed by 2% hydroxylated Fl.
Guanosine-5'-phosphoric acid and the like were eluted through the LAM solution (second elution).

The first eluate was concentrated by adding II hydroxide, crystallized while standing to cool, and crystals of inosine-5'-disodium phosphate (8
water salt, purity 99%) was obtained.

Guanosine-5'-diphosphate 0.12 0
．． 3 Guanosine-2',5'-diphosphate 0.28
0.6 guanosine-3',5'-diphosphate
0.70 1.6 inosine-5'-phosphate
49.6 95.0 Also, the second eluate was purified with hydrochloric acid.
After adjusting to H8.5, it was concentrated and crystallized while standing to cool to obtain crystals of guanosine-5'-disodium phosphate (heptahydrate, purity 98%).

Example-8 Trimethyphosphate/1/1. 12, phosphorus oxychloride 184.91 was added dropwise to a mixture of 7.2 g of water under water cooling, and then guanosine 2B, 3f and inosine 81. A mixed crystal containing OF was added and the mixture was continuously stirred at 5° C. for 4 hours to react. The reaction solution was mixed with 2# ice water, and the aqueous solution was analyzed by high performance liquid chromatography, and the results shown in Table 12 were obtained.

Claims (1)

[Claims] Guanosine-5'-phosphoric acid and inosine-5, which is characterized in that guanosine-5'-phosphoric acid and inosine-5 are treated with phosphorus oxyhalide or its hydrate on a mixed crystal of guanosine, inosine, and phosphorus in a polar organic solvent.
′-Process for producing a mixture with phosphoric acid.