JP4156678B2 - Method for producing 5'-nucleotide - Google Patents

Description

【表１】

【００１５】
実施例２ イノシンまたはグアノシンの結晶単独のリン酸化反応
イノシン，グアノシンの結晶粉末を各々８５．３ｇ，１０３．６ｇを用いて、実施例１と同様に、室温（約１８℃）でリン酸トリエチルに懸濁させた後、加温処理の有無で両者の反応液をＨＬＣを用いて分析した結果を〔表２〕に示した。
【表２】

【００１６】
実施例３ ＴＥＰ下の加温条件（加温温度）の比較
イノシンとグアノシンの混晶粉末（イノシン，グアノシンが各々８５．３ｇ，１０３．６ｇ）を、室温（約１８℃）でリン酸トリエチル２１０６.２ｇに懸濁させ、加温処理を２５℃，５０℃，１００℃下で各々１５分行ない、約５℃に冷却した後、オキシ塩化リン２９４.３ｇと水１１.３ｇを添加して３.５時間反応させた。
続いて、各々の反応液を３２１１ｇの水（約５℃）に加えて加水分解した。この加水分解液をＨＬＣを用いて分析した結果を〔表３〕に示した。
【表３】

【００１７】
実施例４ ＴＥＰ下の加温条件（加温時間）の比較
実施例３と全く同様にして、リン酸トリエチル下での加熱温度を５０℃として、その処理時間を１５分，３０分，６０分と変化させた場合の反応液のＨＬＣ分析結果を〔表４〕に示した。
【表４】

【００１８】
実施例５ オキシ塩化リンの添加量の比較
イノシンとグアノシンの混晶粉末（イノシン，グアノシンが各々８５．３ｇ，１０３．６ｇ）を、室温（約１８℃）でリン酸トリエチル２１０６．２ｇに懸濁させ、加熱して５０℃とし１５分撹拌した後、約０℃に冷却し、ヌクレオシドに対してオキシ塩化リン１.４，１.６，１.８，２.０，２.７５倍モル量と水をそれぞれ５.４，７.２，７.２，９，１１.３ｇ添加して３．５時間反応させた。
続いて、各々の反応液を３２１１ｇの水（約５℃）に加えて加水分解した。この加水分解液をＨＬＣを用いて分析した結果を〔表５〕に示した。
【表５】

【００１９】
実施例６ リン酸化反応条件の比較
イノシンとグアノシンの混晶粉末（イノシン，グアノシンが各々８５．３ｇ，１０３．６ｇ）を、室温（約１８℃）でリン酸トリエチル２１０６．２ｇに懸濁させ、加熱して５０℃とし１５分撹拌した後、約−５℃，０℃，５℃に冷却し、ヌクレオシドに対してオキシ塩化リン１．８倍モル量と水７．２ｇを添加して３．５時間反応させた。
続いて、各々の反応液を３２１１ｇの水（約５℃）に加えて加水分解した。この加水分解液をＨＬＣを用いて分析した結果を〔表６〕に示した。
【表６】

【００２０】
【発明の効果】
本発明によれば、ヌクレオシドより５′−ヌクレオチドを高純度，高収率で得ることができるとともに、ヌクレオシドリン酸化反応時間が短縮化され、ヌクレオチド精製工程の不純物除去操作がきわめて容易である。また、リン酸化剤であるオキシハロゲン化リンの量も常量より少なくてよい。[0001]
[Industrial application fields]
The present invention relates to a method for improving phosphorylation of nucleosides. More specifically, the present invention relates to a method for phosphorylating a hydroxyl group in an unprotected state after changing the crystal shape of the nucleoside by heating in an organic solvent.
The method of the present invention is used as an industrial and economical method for producing a single product or a mixture of 5'-nucleotide or 5'-nucleotide mixture, particularly inosinic acid, guanylic acid and cytidylic acid, which are useful as seasonings and pharmaceuticals.
[0002]
[Prior art]
Conventionally, for example, the following methods are known as methods for chemically phosphorylating nucleosides.
1) Method of reacting nucleoside with phosphorus oxychloride in phosphoric acid trialkyl ester (Japanese Patent Publication No. 42-11071)
2) A method using a mixed crystal of guanosine and inosine (Japanese Patent Laid-Open No. 59-167599)
3) Mixed phosphorylation method of inosine and guanosine (JP-A-2-115195)
4) Method of using a metallizing agent for phosphorylation (Japanese Patent Laid-Open No. 59-80694)
5) Method of using an aluminizing agent for phosphorylation (Japanese Patent Laid-Open No. 59-163397)
The knowledge about the chemical phosphorylation of nucleosides related to the subject of the present invention is as follows.
When phosphorylation is carried out by adding phosphorus oxychloride under triethyl phosphate, for example, when the nucleoside is a mixed crystal of inosine / guanosine, the rate of phosphorylation of inosine / guanosine is not the same. In comparison, since the phosphorylation rate of guanosine is about 1/3, by-products such as diphosphates, hypoxanthine, and guanine are produced in obtaining a 5'-mononucleotide mixture.
As a method for suppressing the production of this by-product, a method of performing a phosphorylation reaction using a mixed crystal of guanosine and inosine is known as seen in 2) above.
In addition, as seen in 3), the phosphorylation reaction of an inosine or guanosine alkali metal salt is preceded by the latter phosphorylation reaction, and then the former is added to carry out the phosphorylation reaction sequentially. It has been known.
Furthermore, as seen in the above 4) and 5), a method is known in which phosphorylation is performed after activating the hydroxyl group of a nucleoside using a metallizing agent or an aluminizing agent.
However, in the nucleoside phosphorylation process, there is no description of a method in which the organic solvent suspension of nucleoside is kept at a temperature of about 20 ° C. or higher and the resulting nucleoside suspension is subjected to phosphorylation.
[0003]
[Problems to be solved by the invention]
A method for producing nucleotides from nucleosides, for example, a method for producing nucleotides by chemical phosphorylation of a mixture containing inosine and guanosine in an arbitrary ratio, or a mixed crystal of inosine and guanosine, is used as an industrial process. It is not yet fully satisfactory in terms of generation and operation methods. Therefore, it is desired to develop a method for producing 5'-nucleotides that is highly pure, has a high yield, has few by-products, and can shorten the reaction time.
[0004]
[Means for Solving the Problems]
In the chemical phosphorylation of a mixture of inosine and guanosine, the present inventors slurried these nucleoside crystals in an organic solvent (for example, triethyl phosphate, trimethyl phosphate, etc.) and then subjected to a heating treatment to obtain a nucleoside. It has been found that by changing the crystal form or increasing the surface area of the crystal, the phosphorylation rate is increased and the unreacted substances are significantly reduced. As a result of further investigation based on these findings, the inventors have completed a production method for obtaining a mixture of 5'-nucleotides with high purity and high yield which has not been conventionally obtained.
[0005]
That is, the present invention
(1) An organic solvent suspension of nucleosides After Tsu coercive to 20 ° C. or higher, under cooling suspension of the resulting nucleosides, and wherein the subjecting to phosphorylation 5'-nucleotide of the production method,
(2) Suspension of nucleoside in organic solvent 30 ° C ~ The method for producing a 5′-nucleotide according to claim 1, which is kept at 80 ° C.
(3) Nucleoside suspension in organic solvent Over 20 ℃ 10 minutes ~ The method for producing a 5′-nucleotide according to claim 1, which is maintained for 120 minutes,
(4) Phosphorylation reaction -30 ° C ~ The method for producing a 5′-nucleotide according to item 1, performed at a temperature of 10 ° C.,
(5) The method for producing a 5′-nucleotide according to item 1, wherein the organic solvent is a tri-lower (C _1-6 ) alkyl ester of phosphate,
(6) The method for producing a 5′-nucleotide according to item 5, wherein the tri-lower (C _1-6 ) alkyl ester phosphate is triethyl phosphate,
(7) The method for producing a 5′-nucleotide according to item 1, wherein the phosphorylation reaction is performed using phosphorus oxyhalide,
(8) The method for producing a 5′-nucleotide according to item 7, wherein the phosphorus oxyhalide is phosphorus oxychloride,
(9) Phosphorus oxyhalide for nucleosides 1 to The method for producing a 5′-nucleotide according to item 7, which is used in a 5-fold molar amount,
(10) The method for producing a 5′-nucleotide according to item 1, wherein the nucleoside is inosine,
(11) The method for producing a 5′-nucleotide according to item 1, wherein the nucleoside is guanosine,
(12) The method for producing 5′-nucleotide according to Item 1, wherein the nucleoside is a mixed crystal of inosine and guanosine, and (13) the method for producing 5′-nucleotide according to Item 1, wherein the nucleoside is cytidine.
[0006]
In the present invention, nucleoside means a sugar and purine base or pyrimidine base having a glucoside bond, and specifically, ribonucleoside and deoxyribonucleoside are used. Examples of ribonucleosides include inosine, guanosine, cytidine, adenosine, and uridine. Examples of deoxyribonucleosides include deoxyinosine, deoxyguanosine, deoxycytidine, and deoxyuridine. Ribonucleosides are preferably used. Particularly preferably inosine or guanosine is used.
In the present invention, the above nucleosides can be used alone or in the form of a mixture as a raw material nucleoside. Moreover, you may use a nucleoside as a salt. Examples of such salts include inorganic bases (for example, alkali metals such as sodium and potassium, alkaline earth metals such as calcium and magnesium, ammonia and the like) or organic bases (for example, trialkylamines such as trimethylamine and triethylamine, pyridine). Etc.), or inorganic acids (eg hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid etc.) or organic acids (eg acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid) Salts with acid, malic acid, succinic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, etc.).
[0007]
As the raw material nucleoside in the present invention, it is particularly preferable to use a mixed crystal of inosine and guanosine. As the mixed crystal, one obtained by a method known per se can be used. For example, a mixed crystal obtained by crystallization by means of precipitation of a solute from an aqueous solution containing inosine and guanosine is used (see Japanese Patent Publication No. 47-38199). In this case, crystallization methods include, for example, cooling, concentration, addition of seed crystals, addition of a hydrophilic solvent that does not dissolve nucleosides (for example, acetone), pH adjustment (an acidic region having a high nucleoside solubility of pH 3 or lower, or pH 9). The above alkaline range is adjusted to a pH range of 3 to 9), and a combination thereof can be appropriately employed. After the mixed crystal with the nucleoside is precipitated from the solution containing the nucleoside by such a method, the mixed crystal is separated by a commonly used method such as suction or pressure filtration, centrifugation, centrifugal sedimentation, etc. It is desirable to remove a solvent such as water by heating and drying, and then subject to a phosphorylation reaction.
[0008]
In the present invention, any organic solvent may be used as long as it does not adversely influence the reaction. The organic solvent is preferably a polar solvent such as tri-phosphate (C _1-6 ) alkyl esters such as trimethyl phosphate and triethyl phosphate, trimethoxyethyl phosphate and triethoxyethyl phosphate. Examples include phosphoric acid tri-lower (C _1-6 ) alkoxy lower (C _1-6 ) alkyl esters, sulfoxides such as dimethyl sulfoxide, and amides such as dimethylformamide and N-dimethylacetamide. These organic solvents can be used alone or in admixture of two or more. Of these organic solvents, tri-lower (C _1-6 ) alkyl esters of phosphate are preferably used. Particularly preferred examples include triethyl phosphate and trimethyl phosphate.
The amount of the organic solvent to be used varies depending on the kind thereof, but is usually appropriately selected within the range of about 5 to about 20 times the weight of the nucleoside, and preferably about 8 to about 17 times.
[0009]
In the present invention, the phosphorylation reaction is performed as follows. First, nucleoside or a salt thereof is suspended in an organic solvent, and the resulting suspension is maintained at a temperature of about 20 ° C. or higher. In this case, the temperature is preferably about 20 to about 100 ° C, more preferably about 30 to about 80 ° C, and particularly preferably about 40 to about 60 ° C. The time during which the suspension is kept at a temperature of about 20 ° C. or more varies depending on the temperature, the amount of the raw nucleoside, etc., but is preferably about 10 to about 120 minutes, more preferably about 10 to about 60 minutes, and particularly preferably. Is about 10 to about 20 minutes.
In general, since the phosphorylation reaction undergoes a liquid-liquid reaction following dissolution of the nucleoside, the reaction rate varies depending on the particle size and particle shape of the nucleoside crystal used in the reaction. Usually, the finer the nucleoside crystals are, the larger the surface area is, so the reaction rate can be apparently increased. The particle size of the nucleoside crystal is preferably about 1 to about 1000 μm, more preferably about 20 to about 500 μm.
[0010]
However, in the present invention, when the organic solvent suspension of nucleoside is maintained at a temperature of about 20 ° C. or higher, nucleoside crystals having any particle size and particle shape can be used. Due to the heating treatment, the nucleoside crystal apparently changes to an amorphous crystal due to a phenomenon similar to the crystal transition, so that the surface area of the nucleoside crystal increases, resulting in an increase in the reaction rate with the phosphorylating agent.
Subsequently, the resulting nucleoside suspension is subjected to a phosphorylation reaction. The phosphorylation reaction is performed using a phosphorylating agent. In this case, the reaction temperature is preferably about −30 ° C. to about 10 ° C., particularly preferably about 0 ° C. to about 10 ° C.
The phosphorylating agent used in the present invention is a phosphorylating agent usually used for a phosphorylation reaction, preferably phosphorus oxyhalide such as phosphorus oxychloride and phosphorus oxybromide.
[0011]
In the phosphorylation reaction, phosphorous oxyhalide is usually used as a partial hydrate rather than subjected to the reaction as it is, so that the selectivity to 5'-mononucleotide production is usually higher and 2 'or 3'-mono This is preferable because the amount of by-products of phosphate and diphosphate decreases.
The method for obtaining a phosphorus oxyhalide hydrate 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 or tertiary butanol to the reaction. You can do it.
The amount of phosphorus oxyhalide to be used is generally about 1 to about 5 times mol, preferably about 1.5 to about 4 times mol for the nucleoside. If the amount used is too small, the nucleoside remains unreacted, and if the amount used is excessive, the by-product of diphosphates increases and the yield of the target 5'-mononucleotide decreases, which is not preferable. Usually, it is appropriately selected from the above range of use in consideration of the type of phosphorylating agent or solvent.
While the reaction time varies depending on the type of solvent and the presence or absence of an accelerator (eg, a very small amount of sodium hydroxide), it is generally about 30 minutes to about 10 hours. In this reaction method, the reaction is completed in a shorter time than the conventional phosphorylation method.
The reaction product thus obtained is mixed with cold water (eg, about 10 ° C. or less, preferably about 5 ° C. or less) by a conventional method, whereby an unreacted phosphorylating agent and the produced nucleoside phosphohalogenate are obtained. Hydrolyze to obtain a solution (hydrolyzed solution) containing 5'-nucleotide.
[0012]
As a method for purifying the 5′-nucleotide thus obtained, a method known per se, for example,
1) A method in which the pH of the hydrolyzed solution is adjusted to about 1.5 with sodium hydroxide and then treated with activated carbon.
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. Subsequently, after passing through any of these purification methods, it can be obtained as a mixed crystal of nucleoside-5'-disodium phosphate by a conventional method.
[0013]
【Example】
The present invention will be specifically described below with reference to examples and comparative examples.
In Examples, IMPNa ₂ is disodium inosine-5′-monophosphate, GMPNa ₂ is disodium guanosine-5′-monophosphate, IR is inosine, GR is guanosine, and TEP is phosphorus. Triethyl acid and HLC means high performance liquid chromatography.
Example 1 Phosphorylation Reaction of Inosine / Guanosine Mixed Crystal A mixed crystal powder of inosine and guanosine (85.3 g and 103.6 g of inosine and guanosine, respectively) was suspended on 2106.2 g of triethyl phosphate at room temperature (about 18 ° C.). After turbidity and cooling to about 5 ° C., 294.3 g of phosphorus oxychloride and 11.3 g of water were added and reacted for 3.5 hours.
On the other hand, a mixed crystal powder of inosine and guanosine (85.3 g and 103.6 g of inosine and guanosine respectively) was suspended in 2106 g of triethyl phosphate at room temperature (about 18 ° C.) and heated to 50 ° C. in the same manner. After stirring for 15 minutes, 294.3 g of phosphorus oxychloride and 11.3 g of water were added under cooling to about 5 ° C. and reacted for 3.5 hours.
Subsequently, each reaction solution was hydrolyzed by adding it to 3211 g of water (about 5 ° C.). The results of analyzing this hydrolyzed solution using HLC are shown in [Table 1].
The conditions for quantitative analysis by HLC are as follows. This condition is the same in the following examples.
[0014]

[Table 1]

[0015]
Example 2 Phosphorylation reaction of inosine or guanosine crystals alone 85.3 g and 103.6 g of inosine and guanosine crystal powders were used to form triethyl phosphate at room temperature (about 18 ° C.) in the same manner as in Example 1. After suspending, the results of analyzing both reaction solutions with and without heating treatment using HLC are shown in [Table 2].
[Table 2]

[0016]
Example 3 Comparison of heating conditions (heating temperature) under TEP A mixed crystal powder of inosine and guanosine (85.3 g and 103.6 g of inosine and guanosine, respectively), triethyl phosphate 2106 at room temperature (about 18 ° C.) Suspended in 0.2 g, heated for 15 minutes at 25 ° C., 50 ° C., and 100 ° C., cooled to about 5 ° C., then added with 294.3 g of phosphorus oxychloride and 11.3 g of water to add 3 The reaction was allowed for 5 hours.
Subsequently, each reaction solution was hydrolyzed by adding it to 3211 g of water (about 5 ° C.). The results of analysis of this hydrolyzed solution using HLC are shown in [Table 3].
[Table 3]

[0017]
Example 4 Comparison of heating conditions (heating time) under TEP In exactly the same manner as in Example 3, the heating temperature under triethyl phosphate was set to 50 ° C., and the treatment time was 15, 30, and 60 minutes. Table 4 shows the results of HLC analysis of the reaction liquid when changed.
[Table 4]

[0018]
Example 5 Comparison of Addition Amount of Phosphorus Oxychloride A mixed crystal powder of inosine and guanosine (85.3 g and 103.6 g of inosine and guanosine, respectively) was suspended in 2106.2 g of triethyl phosphate at room temperature (about 18 ° C.). And heated to 50 ° C., stirred for 15 minutes, cooled to about 0 ° C., and 1.4, 1.6, 1.8, 2.0, 2.75 times the molar amount of phosphorus oxychloride with respect to the nucleoside. And water were added to each of 5.4, 7.2, 7.2, 9, and 11.3 g and reacted for 3.5 hours.
Subsequently, each reaction solution was hydrolyzed by adding it to 3211 g of water (about 5 ° C.). The results of analysis of this hydrolyzed solution using HLC are shown in [Table 5].
[Table 5]

[0019]
Example 6 Comparison of Phosphorylation Reaction Conditions Mixed crystals of inosine and guanosine (85.3 g and 103.6 g of inosine and guanosine, respectively) were suspended in 2106.2 g of triethyl phosphate at room temperature (about 18 ° C.). 2. Heat to 50 ° C., stir for 15 minutes, cool to about −5 ° C., 0 ° C., 5 ° C., add 1.8 times the molar amount of phosphorus oxychloride and 7.2 g of water to the nucleoside. The reaction was allowed for 5 hours.
Subsequently, each reaction solution was hydrolyzed by adding it to 3211 g of water (about 5 ° C.). The results of analysis of this hydrolyzed solution using HLC are shown in [Table 6].
[Table 6]

[0020]
【The invention's effect】
According to the present invention, 5'-nucleotide can be obtained from nucleoside with high purity and high yield, and the nucleoside phosphorylation reaction time is shortened, so that the impurity removal operation in the nucleotide purification step is very easy. Further, the amount of phosphorous oxyhalide, which is a phosphorylating agent, may be less than the normal amount.

Claims (13)

An organic solvent suspension of nucleosides After maintaining the 20 ° C. or higher, under cooling suspension of the resulting nucleosides, preparation of 5'-nucleotides, wherein the subjecting to phosphorylation. Nucleoside suspension in organic solvent 30 ° C ~ The method for producing a 5'-nucleotide according to claim 1, which is maintained at 80 ° C. Nucleoside suspension in organic solvent Over 20 ℃ 10 minutes ~ The method for producing a 5'-nucleotide according to claim 1, which is maintained for 120 minutes. Phosphorylation reaction -30 ° C ~ The method for producing a 5'-nucleotide according to claim 1, which is carried out at a temperature of 10C. The method for producing 5'-nucleotide according to claim 1, wherein the organic solvent is a tri-lower (C _1-6 ) alkyl ester of phosphate. The method for producing 5'-nucleotide according to claim 5, wherein the tri- (lower) (C _1-6 ) alkyl ester phosphate is triethyl phosphate. The method for producing a 5'-nucleotide according to claim 1, wherein the phosphorylation reaction is carried out using phosphorus oxyhalide. The method for producing 5'-nucleotide according to claim 7, wherein the phosphorus oxyhalide is phosphorus oxychloride. Phosphorus oxyhalide to nucleosides 1 to The method for producing a 5'-nucleotide according to claim 7, which is used in a 5-fold molar amount. The method for producing a 5'-nucleotide according to claim 1, wherein the nucleoside is inosine. The method for producing a 5'-nucleotide according to claim 1, wherein the nucleoside is guanosine. The method for producing a 5'-nucleotide according to claim 1, wherein the nucleoside is a mixed crystal of inosine and guanosine. The method for producing a 5'-nucleotide according to claim 1, wherein the nucleoside is cytidine.