JP2534807B2 - Method for producing nucleoside compound - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method capable of producing a nucleoside compound in a high yield.

[0002]

2. Description of the Related Art As a method for producing a nucleoside compound using a base exchange reaction, N. Hori, M. Watanabe, Y.
Yamazaki and Y. Mikami, Agric. Biol. Chem., 53, 197
(1989). This method uses purine nucleoside phosphorylase and pyrimidine nucleoside phosphorylase in the presence of phosphoric acid to react thymine with inosine to carry out a base exchange reaction,
5-methyluridine is produced using ribose-1-phosphoric acid as an intermediate.

The above reaction consists of the following two reactions.

[0004]

Embedded image Therefore, as a whole reaction,

[0005]

Embedded image Becomes

[0006]

The equilibrium constant K1 (K1 = [5-methyluridine] [hypoxanthine] / [inosin] [thymine]) of the base exchange reaction (3) shown in Chemical formula 2 above is 0.21 (40 C)) and the same initial concentration of inosine and thymine, the yield is defined by the equilibrium constant, so the maximum yield of 5-methyluridine is only 31%. This is the same when other bases other than thymine are used, and the yield of 5-methyluridine due to the base exchange reaction remains low.

The present invention utilizes a base exchange reaction,
It is an object to provide a method for producing a nucleoside compound with high yield.

[Means for Solving the Problems] In view of the above circumstances, the inventors of the present invention have conducted diligent research in order to further increase the yield of the nucleoside compound. As a result, they have found a method for obtaining a nucleoside compound in a higher yield by removing hypoxanthine produced by base exchange from the system.

That is, the method for producing a nucleoside compound of the present invention is to convert adenosine or deoxyadenosine into inosine or deoxyinosine by adenosine deaminase, and to convert this inosine or deoxyinosine with a pyrimidine base or a purine base into phosphate. Alternatively, in an aqueous solution in the presence of phosphate, purine nucleoside phosphorylase and pyrimidine nucleoside phosphorylase when the base is a pyrimidine base, and purine nucleoside phosphorylase when the base is a purine base. And a hypoxanthine produced by this base exchange reaction is converted to uric acid by xanthine oxidase.

Here, as the pyrimidine base, uracil, cytosine, thymine, 5-fluorouracil, 5-chlorouracil, 5-bromouracil, 5-iodouracil, 5-
Ethyluracil, 5-trifluoromethyluracil, 5-carboxyuracil and the like, as the purine base 2-chloropurine, 6-chloropurine, 2,6-dichloropurine, 2-amino-6-chloropurine, 2, Examples thereof include 6-diaminopurine, 6-mercaptopurine, 6-methylthiopurine and 2-aminopurine.

The method for producing the nucleoside compound of the present invention will be described below in detail with the pyrimidine base as the base that undergoes a base exchange reaction with the nucleoside.

First, by using adenosine deaminase, adenosine or deoxyadenosine and H
₂ O and inosine or deoxyinosine respectively
NH ₃ and produced. Although this reaction is a reversible reaction, since a large amount of H ₂ O is usually present in the reaction system, the equilibrium is largely inclined toward the side of inosine or deoxyinosine generation. That is, it can be considered that the initial concentration of adenosine or deoxyadenosine as the starting material is equal to the concentration of inosine produced. The deoxyinosine obtained here is a compound that is not industrially produced and is difficult to obtain, but by this method, it is easily produced from deoxyadenosine that is industrially produced in a large amount and is easily available. be able to.

Then, from the produced inosine or deoxyinosine and phosphoric acid or phosphate, hypoxanthine and ribose-1-phosphoric acid or deoxyribose-1-phosphoric acid are obtained by purine nucleoside phosphorylase. To generate.

Next, the formed ribose-1-phosphate or deoxyribo--1-phosphate is subjected to a substitution reaction with a pyrimidine base by a pyrimidine nucleoside phosphorylase. This produces a pyrimidine nucleoside from the reaction of ribose-1-phosphate with a pyrimidine base,
The reaction of deoxyribose-1-phosphoric acid with a pyrimidine base produces a pyrimidine deoxynucleoside.

At the same time, due to the pyrimidine nucleoside phosphorylase present in the reaction solution, as a reverse reaction, these nucleosides and phosphoric acid or phosphate produced by the base exchange reaction are again reacted with pyrimidine base and ribose. -1-Phosphoric acid or deoxyribose-1-decomposes with phosphoric acid to reach an equilibrium state as a whole system.

However, in the method of the present invention, xanthine oxidase is also present in the reaction solution. As a result, hypoxanthine produced by the decomposition of the above inosine or deoxyinosine is irreversibly oxidized by oxygen and xanthine oxidase, and becomes xanthine to uric acid. The produced uric acid is not a substrate for purine nucleoside phosphorylase and pyrimidine nucleoside phosphorylase, and therefore does not participate in the base exchange reaction and is excluded from the system. Therefore, stoichiometrically, all inosine becomes ribose-1-phosphate and uric acid, and all deoxyinosine becomes deoxyribo--1-phosphate and uric acid.

Therefore, the amount of nucleoside produced depends only on the equilibrium constant of the following equation.

[0018]

Embedded image Here, considering the case where adenosine is used as the starting material and thymine is used as the base, the final product, 5-methyluridine, is produced according to the above formula (2). Constant (K = [ribose-1-phosphate] [thymine] / [5-methyluridine] [phosphate]) = 0.062.
Adenosine is completely converted to inosine by adenosine deaminase, and further, the produced inosine becomes ribose-1-phosphate and uric acid by purine nucleoside phosphorylase and xanthine oxidase. Therefore,
When the initial concentrations of adenosine and thymine are equal, the concentrations T and M of thymine and 5-methyluridine at the end of the reaction can be calculated by the formulas (4) and (5) shown in the following chemical formula 4. The initial concentrations of adenosine and phosphoric acid in the formula are A and P.

[0019]

Embedded image When the base that undergoes the exchange reaction with the nucleoside is a purine base, ribose-1-phosphate or deoxyribose-1-generated from adenosine or deoxyadenosine is used.
The substitution reaction between phosphoric acid and purine base occurs by purine nucleoside phosphorylase. As a result,
Purine nucleosides are produced from the reaction between su-1-phosphate and purine bases, and purine deoxynucleosides are produced from the reaction between deoxyribo-s-1-phosphate and purine bases. The reverse reaction, purine nucleoside or purine deoxynucleoside and phosphoric acid or phosphate, to produce purine base and ribo--1-phosphoric acid or deoxyribo--1-phosphoric acid is also a purine nucleoside phosphonate. Caused by a lyase. Therefore, the pyrimidine nucleoside phosphorylase is not necessary when the base that undergoes an exchange reaction with the nucleoside is a purine base.

Adenosine deaminator used in the present invention
Ze, xanthine oxidase, purine nucleoside phosphorylase, and pyrimidine nucleoside phosphorylase can be of any origin. The adenosine deaminase and xanthine oxidase used in the present invention are derived from milk manufactured by Behringer Mannheim Yamanouchi. The purine nucleoside phosphorylase and pyrimidine nucleoside phosphorylase used in the present invention are Bacillus stearosa mosfilus JTS85 which simultaneously produces these two enzymes.
9 was prepared.

This strain has been deposited at the Institute for Microbial Industry, Institute of Industrial Technology, Ministry of International Trade and Industry, and the deposit number is Microindustrial Research Institute No. 9666 (FERM P-9666).

The mycological properties of this strain were determined by
The results of an examination based on the second volume of the manual are shown below. 1. Morphology Bacillus: 5.4-6.5 μm × 0.7-0.9 μm Elliptical spore formation: 2.0-2.3 μm × 1.1 × 1.2 μm, 1 per cell, position is terminal 2. Culture properties NB medium: 62 ° C, 2 days culture Plate: White, slightly yellowish, glossy, opaque, no swelling Colony shape is circular wavy slant: White, slightly yellowish, lustrous, opaque, no swelling Growth Is moderate 3. Biochemical properties (1) Gram stain positive (2) Anaerobic culture No growth (3) Motile and pericardial (4) Oxidase positive (5) Catalase positive (6) Liquefaction and liquefaction ability of gelatin Yes (7) Coagulate litmus milk (8) OF test Fermentation type (9) VP test Negative (10) Evolution of gas from glucose No production (11) Production of acid from glucose Produced (12) Production of acid from arabinose No production (13) Production of acid from mannitol Produced (14) Production of acid from xylol No production (15) Growth on Sabouraud medium Slant grown Liquid grown (16) 0.001% Growth under lysozyme No growth (17) 0.02% Growth under azide No growth (18) 7% Growth under NaCl No growth (2% (19) Casein hydrolysis with resolution (20) Starch hydrolysis with resolution (21) Dihydroxyacetone production No generation (22) Egg york test No growth (23) Use of citric acid Negative (24) Negative production of indol (25) Positive urease activity (26) Negative deamination of phenylalanine (27) Arginine dehydrolase activity positive (28) Tyrosine degradation negative (29) Levan production negative (30) Nitrate reduction positive (31) Sodium nitrate denitrification negative (32) Hydrogen sulfide production positive (33) Inorganic Utilization of nitrogen source Growing with NO ₃ as the sole nitrogen source and with NH ₄ as the sole nitrogen source (34) GC content 47.3% (35) Growing at a growth temperature of 40 to 71 ° C Optimal 60 to 68 ° C (36) pH range pH 5.7 to 8.5 Optimum pH 6.0 to 7.0 In the method of the present invention, the reaction solution is adenosine or a base such as deoxyadenosine, uracil and thymine, a phosphate such as phosphate or potassium phosphate, and adenosine deaminase. , Purine nucleoside phosphorylase, pyrimidine Creo Sid phosphoryl La - Ze, and xanthine oxy da - may include zero. The concentration of adenosine or deoxyadenosine in this reaction solution is 5 to 100 mM,
The base concentration is preferably 5 to 100 mM, and the phosphoric acid or phosphate concentration is preferably 5 to 20 mM. Adenosine or deoxyadenosine and base have the same concentration,
The yield of the nucleoside compound increases as the concentration ratio of adenosine or dedoxyadenosine to phosphoric acid or phosphate increases. The pH of the reaction solution is 7.0 to 9.0, preferably 7.0 to 8.0. The reaction temperature is 35 ℃ to 42 ℃,
It is preferably 37 ° C to 40 ° C.

Collection of the product from the reaction solution can be suitably carried out by reverse layer chromatography of octadecyl group.

[0024]

【Example】

Production Example 1: Preparation of crude enzyme solution of purine nucleoside phosphorylase and pyrimidine nucleoside phosphorylase First, Bacillus stearosa mophilus JTS859 was prepared as follows.
The cells were cultured according to the following procedure.

For culturing the bacterium, a medium of pH 6.0 consisting of 20 g of peptone, 10 g of yeast extract, 3 g of glucose and 1 liter of water was used. 3.2 liters of Bacillus stearosa-Mophilus JTS859 spores were added to 2 liters of this medium.
Add 10 ⁷ pieces and use a jar fermenter having a stirring blade (diameter 60 mm, upper and lower 6 pieces each), and set the stirring blade at 680 rpm.
Aeration rate 1.5 vvm, culture temperature 65 ℃, p
The cells were cultured at H5.9 to 6.2 for 8 hours.

After completion of the culture, the cells were centrifuged (10,000 ×
g, 4 ° C, 15 minutes). Then, a crude enzyme solution was prepared as follows.

160 g of the wet bacterium thus obtained was suspended in a 500 mM potassium phosphate solution (pH 7.0, hereinafter simply referred to as "buffer solution"), and the microbial cells were destroyed with a Dymomill. Then, add buffer to make the whole 1400 ml, then centrifuge (8300 x
g, 20 minutes) to obtain 1300 ml of supernatant. The precipitate was suspended in 500 ml of buffer solution to make 1030 ml, and then centrifuged (8300 xg, 20 minutes) to obtain 990 m of supernatant.
1 was obtained. 900 ml of this supernatant was combined with 1300 ml of the above supernatant and gently stirred at 63 ° C for 1 hour. Then, centrifugation (8300 × g, 40 minutes) was performed to obtain 2130 ml of supernatant.

After heat treating 2130 ml of this supernatant, -10 ° C
A mixed solution of 200 ml of chilled acetone and 200 ml of a buffer solution was added. In addition, acetone cooled to -10 ° C
After adding 1.8 liters and stirring at 5-10 ° C for 15 minutes,
Centrifugation (9000 xg, 5 minutes) was performed. To the obtained supernatant, 2.5 liter of acetone at -10 ° C was added, stirred for 15 minutes, and centrifuged (9000 g, 5 minutes) to obtain a precipitate. This precipitate was suspended in 800 ml of buffer solution to obtain 850 ml of acetone-treated crude enzyme solution. Example 1 1 ml containing 5 mM adenosine, 5 mM thymine, 5 mM potassium phosphate, 0.5 mg adenosine deaminase, 2 U xanthine oxidase, and 250 μl of crude enzyme solution (obtained in Production Example 1) 40 reaction mixture (pH 7.0)
The reaction was carried out at 13 ° C for 13 hours and the concentration of each component contained in the reaction solution was measured. The results are shown in Table 1 below and FIG.

Further, the reaction was carried out under the same conditions as in the above procedure except that adenosine deaminase and xanthine oxidase were not added, and the concentration of each component after the reaction was measured. The results are shown in Table 2 below and FIG.

[0030]

[Table 1]

[0031]

[Table 2] 1 and 2, the vertical axis represents the concentration of each component expressed in mM, and the horizontal axis represents the reaction time. -▽-is adenosine, downward black triangle is adenine,-○-is inosine,-●
-Is hypoxanthine,-□-is 5-methyluridine,-■
-Represents thymine, upward black triangle represents uric acid, and-△-represents xanthine.

As is apparent from FIGS. 1 and 2, when adenosine deaminase and xanthine oxidase are present (FIG. 1), the product is 5- The content of methyluridine is very high. In particular, in the presence of xanthine oxidase, xanthine and uric acid become visible with hypoxanthine as the inosine content decreases,
It has been shown that in equilibrium, the contents of inosine, hypoxanthine and xanthine other than uric acid are almost zero. This indicates that all hypoxanthine was oxidized by xanthine oxidase.

Specifically, when adenosine deaminase and xanthine oxidase were present in the reaction system, 3.6 mM 5-methyluridine was produced. When it was not present, the amount of 5-methyluridine produced was 0.09 mM.

Thus, 5-methyluridine could be obtained in higher yield in the presence of adenosine deaminase and xanthine oxidase. Example 2 1-15 mM thymine, adenosine at the same concentration as thymine,
5 mM potassium phosphate, adenosine deaminase 0.5
Reaction solution containing 1 mg of xanthine oxidase (2 U) and 250 μl of crude enzyme solution (obtained in Production Example 1)
1 was reacted at 40 ° C., and the amount of 5-methyluridine produced when inosine disappeared was measured. The result is shown in Figure 3.
-Indicated. This yield was in very good agreement with the theoretical curves calculated by the formulas (4) and (5) shown in the chemical formula 4 and represented by the broken line. Example 3 5 mM adenosine, 5 mM various bases shown in Table 3 below, 5 mM potassium phosphate, adenosine deaminase
0.5m containing 0.20 mg, 0.8 U of xanthine oxidase, and 100 μl of crude enzyme solution (obtained in Production Example 1)
1 of the reaction solution (pH 7.0) was reacted at 40 ° C. for 10 hours, and the concentration of the produced nucleoside contained in the reaction solution was measured. The results are also shown in Table 3.

[0035]

[Table 3] Example 4 The same operation as in Example 3 was carried out except that 5 mM 2′-deoxyadenosine was used instead of adenosine, and the concentration of the produced nucleoside contained in the reaction solution was measured.
The results are shown in Table 4.

[0036]

[Table 4]

[0037]

INDUSTRIAL APPLICABILITY As described above, the method for producing a nucleoside compound of the present invention makes it possible to obtain a nucleoside compound easily and in high yield by utilizing a base exchange reaction.

[Brief description of drawings]

FIG. 1 is a graph showing the changes over time in the concentration of each component in the reaction liquid in Example 1 of the present invention.

FIG. 2 is a graph showing the change with time of the concentration of each component in the reaction solution under the same conditions as the graph shown in FIG. 1 except that adenosine deaminase and xanthine oxidase were not added.

FIG. 3 is a graph showing the relationship between thymine concentration and 5-methyluridine production in Example 2.

Claims (2)

(57) [Claims] 1. Adenosine or deoxyadenosine is converted to inosine or deoxyinosine by adenosine deaminase, and the obtained inosine or deoxyinosine and pyrimidine base are dissolved in an aqueous solution in the presence of phosphoric acid or phosphate, Purine nucleoside phosphorylase and pyrimidine nucleoside phosphorylase are subjected to base exchange reaction, and further hypoxanthine produced by this base exchange reaction is converted to uric acid by xanthine oxidase. . 2. Adenosine or deoxyadenosine is converted to inosine or deoxyinosine by adenosine deaminase, and the obtained inosine or deoxyinosine and purine base are dissolved in an aqueous solution in the presence of phosphoric acid or phosphate, Purine nucleoside phosphoryla
The base exchange reaction is carried out with zeze, and the hypoxanthine produced by this base exchange reaction is converted into xanthine oxidase.
A method for producing a nucleoside compound, which comprises converting uric acid with azetase.