JPH04197193A - Production of nucleoside compound - Google Patents

Abstract

PURPOSE:To obtain a nucleoside compound in high yield by subjecting (deoxy) inosinnucleoside to base exchange reaction with a pyridine base using a specific enzyme and further converting the produced hypoxanthine to uric acid. CONSTITUTION:(A) A nucleoside consisting of (deoxy)inosine is subjected to base exchange reaction with (B) a pyrimidine base in the presence of an aqueous solution of phosphoric acid (salt) by (D) (i) purinnucleoside phosphorylase and (ii) pyrimidinnucleosidephosphorylase. Further, the produced hypoxanthine is converted to uric acid by (E) xanthinoxidase to provide the objective nucleoside compound.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] This invention relates to a method for producing nucleoside compounds with high yield.

[Conventional technology]

A method for producing a nucleoside compound using a base exchange reaction is described by N., Horl, M., Watanabe.
, Y, Yaaazakl and Y, Mikaa+
1. Agrlc, Blol, Chem,, 53゜1
97 (1989). This method uses purine nucleoside phosphorylase and pyrimidine nucleoside phosphorylase, and in the presence of phosphoric acid, thymine and inosine are reacted to perform a base exchange reaction, using ribose-1-phosphate as an intermediate.
It produces methyluridine.

The above reaction consists of the following two reactions.

-> Inosine + phosphoric acid <- Hypoxanthine in ribose-1-phosphate (1) Ribose-1-phosphate + thymine; = = 5-methyluridine phosphoric acid (2) Therefore, the overall reaction is +Chimi7←-1-5-methyluracil+hypoxanthine (3).

[Problem to be solved by the invention]

The equilibrium constant Kl (Kl-[
5-methyluridine] [hypoxanthine]/[inosine] [thymine]) is 0.21 (at 40°C), and if the initial concentrations of boar and thymine are the same, the yield is determined by the equilibrium constant. , the maximum yield of 5-methyluridine amounts to only 31%. This is the same when other bases than thymine are used, and the yield of nucleoside compounds due to base exchange reaction remains at a low value.

An object of the present invention is to provide a method for producing a nucleoside compound in high yield using a base exchange reaction.

[Means to solve the problem]

In view of the above circumstances, the present inventors conducted extensive research in order to further increase the yield of nucleoside compounds. As a result, we discovered a method for obtaining nucleoside compounds in higher yields by excluding hypoxanthine produced by base exchange from the system.

That is, the method for producing a nucleoside compound of the present invention comprises adding a nucleoside selected from the group consisting of inosine and deoxyinosine and a base selected from the group consisting of pyrimidine bases and purine bases to an aqueous solution in the presence of phosphoric acid or a phosphate salt. Among these, when the base is a pyrimidine base, a base exchange reaction is carried out using purine nucleoside phosphorylase and pyrease, and furthermore, hypoxanthine produced by this base exchange reaction is converted into uric acid by xanthine oxidase.

Here, the pyrimidine bases include uracil, cytosine, thymine, 5-fluorouracil, 5-chlorouracil, 5-bromouracil, 5-iodouracil, 5-ethyluracil, 5-trifluoromethyluracil, 5-
Carboxyuracil, etc., and the purine bases include adenine, guanine, 2-chloropurine, 6-chloropurine, 2.6-dichloropurine, 2-amino-6-chloropurine, 2.6-diamitpurine, 6-mercaptopurine. , 6-methylthiopurine, 2-7 minoprine, and the like.

Hereinafter, the method for producing a nucleoside compound of the present invention will be explained in detail using a pyrimidine base as a base that performs a base exchange reaction with a nucleoside.

First, purine nucleoside phosphorylase converts the starting materials inosine or deoxyinosine and phosphoric acid or phosphate into hypoxanthine and ribose-1.
- Phosphoric acid or deoxyribose-1-phosphate is produced.

Next, the generated ribose-1-phosphate or deoxyribose-1-phosphate undergoes a substitution reaction with a pyrimidine base by pyrimidine nucleoside phosphorylase. As a result, pyrimidine nucleosides are produced from the reaction between ribose-1-phosphate and the pyrimidine base, and pyrimidine deoxynucleosides are produced from the reaction between deoxyribose-1-phosphate and the rimidine base.

At the same time, pyrimidine nucleoside phosphorylase present in the reaction solution causes a reverse reaction in which these nucleosides and phosphoric acid or phosphate produced by the base exchange reaction are again converted into pyrimidine bases and ribose-
It decomposes into 1-phosphoric acid or deoxyribose-1-phosphate, and the system as a whole reaches an equilibrium state.

However, in the method of the present invention, xanthine oxidase is further present in the reaction solution. As a result, hypoxanthine produced by the decomposition of inosine or deoxyinosine is irreversibly oxidized by oxygen and xanthine oxidase to become uric acid via xanthine.

Since the generated uric acid is not a substrate for purine nucleoside phosphorylase and pyrimidine nucleoside phosphorylase, it does not participate in the base exchange reaction and is excluded from the system. Therefore, stoichiometrically all inosine is ribose-
It becomes 1-phosphate and uric acid, and all deoxyinosine becomes deoxyribose-1-phosphate and uric acid. Therefore, when the initial concentrations of inosine or deoxyinosine and the base are equal, the resulting concentrations of ribose-1-phosphate or deoxyribose-1-phosphate and the base are equal. Therefore, the amount of nucleoside produced depends only on the equilibrium constant of the following formula.

Ribose-1-phosphate or the raw base deoxyribose-1-phosphate nucleoside-〉〈- or + Phosphate deoxynucleoside Here, considering the case where inosine is used as the starting material and thymine as the base, the final product is Since a certain 5-methyluridine is produced according to the above formula (2), its yield is determined by the equilibrium constant (K-[ribose-1-phosphate co[thymine]/[5-methyluridine co[ Phosphoric acid] ) 0.082. That is, let the initial concentrations of inosine and phosphoric acid be l and P, respectively,
When the initial concentrations of inosine 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 following equations (4) and (5).

15.12 T2 + (1+P) T-P-1-0(
4) M -1-T (5
) When the base that performs the exchange reaction with the nucleoside is a purine base, ribose-1-phosphate or deoxyribose-1 generated from inosine or deoxyinosine
- The substitution reaction between phosphate and purine bases occurs by purine nucleoside phosphorylase. As a result, purine nucleosides are generated from the reaction between ribose-1-phosphate and purine bases, and purine deoxynucleosides are generated from the reaction between deoxyribose-1-phosphate and purine bases. Further, the reverse reaction of this reaction, in which purine base and ribose-1-phosphate or deoxyribose-1-phosphate are produced from purine nucleoside or purine deoxynucleoside and phosphoric acid or phosphate, also occurs by purine nucleoside phosphorylase. Therefore, when the base that undergoes the exchange reaction with the nucleoside is a purine base, pyrimidine nucleoside phosphorylase is not required.

The xanthine oxidase, purine nucleo"cytophosphorylase, and pyrimidine nucleoside phosphorylase used in the present invention may be of any origin. The xanthine oxidase used in the present invention is derived from milk manufactured by Boehringer Mannheim Yamanouchi. .

Purine nucleoside phosphorylase and pyrimidine nucleoside phosphorylase used in this invention were prepared from Bacillus stearothermosophilus JTS859, which simultaneously produces these two enzymes.

This strain has been deposited with the Institute of Microbiology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, and its deposit number is 9666.
No. (FERM P-9666).

The mycological properties of this strain were investigated in accordance with Purge Aids Manual Volume 2, and the results are shown below.

1, Bacillus morphology: 5.4-B, 5, cnX Q, 7-0.9p
Oval sporulation: 2.0~2.3ρ×1.1X1.2
gm.

1 per cell, located at terminal 2, culture properties NB medium = 62°C, 2 days culture on plate: white, slightly yellow, glossy, opaque, not raised Colony shape: circular wavy slant: white, slightly Glossy with yellow color, opaque, non-heavy growth, moderate growth (3), biochemical properties (1) Durham stain positive (2) anaerobic culture, no growth (3) motility
Yes, pericilla (4) oxidase
Positive (5) Catalase Positive (6) Liquefaction of gelatin Possible to liquefy (7) Lidomus milk coagulate (8) OF test
Fermentation type (9) V-P test
Negative (10) Production of gas from glucose Not produced (11) Production of acid from glucose Produced (12) Production of acid from arabinose No production (13) Production of acid from mannitol Produced (14) ) Acid production from Kijirole Production (15) Growth in Sabouraud medium Slant Growth Liquid Growth (1B) Growth under 0.001% lysozyme No growth (17) Under 0.02% azide Growth No growth (1g) Growth under 7% NaCI No growth (
(Grows up to 2%) (I9) Hydrolysis of casein Degradation ability (20
) Hydrolysis of starch Decomposition ability (21) Production of dihydroxyacetone Not produced (22) Elaguyoke test No growth (23)
Utilization of citric acid Negative (24) Production of indole Negative (25) Urease activity
Positive (26) Deamination of phenylalanine Negative (27) Arginine dehydrolase activity Positive (28) Degradation of tyrosine Negative (29) Production of levan Negative (30) Reduction of nitrate
Positive (31) Denitrifying ability of sodium nitrate Negative (32) Production of hydrogen sulfide Positive (33) Use of inorganic nitrogen source Grown with NO as the sole nitrogen source Grown with NH4 as the sole nitrogen source (34) GC Content 47.3% (35
) Growth temperature: Optimum growth at 40-71”C 6
0~68℃ (3B) pH range pH5,7~8.
5 Optimum pH 6.0 to 7.0 In the method of the present invention, the reaction solution contains a base selected from the group consisting of inosine or deoxyinosine, adenine, uracil and thymine, a phosphate such as phosphoric acid or potassium phosphate, and a purine. nucleoside phosphorylase,
Pyrimidine nucleoside phosphorylase and xanthine oxidase may be included. In this reaction solution,
The concentration of inosine or deoxyboar is 5-too
sM, the concentration of base is preferably 5-100 mM, and the concentration of phosphoric acid or phosphate is 5-20 sM. The concentration of inosine or deoxyinosine and the base is the same, and the higher the concentration ratio of inosine or deoxyinosine and phosphoric acid or phosphate, the higher the yield of the nucleoside compound. The pH of the reaction solution is 7.0~
9.0, preferably 7.0 to 8.0. The reaction temperature is 35°C to 42°C, preferably 37°C to 40°C.

The product can be suitably collected from the reaction solution by reverse layer chromatography using octadecyl groups.

〔Example〕

Production Example 1 Preparation of crude enzyme solutions of purine nucleoside phosphorylase and pyrimidine nucleoside phosphorylase First, Bacillus stearothermophilus JTS859
was cultured according to the following procedure.

For culturing bacteria, 20g peptone 10g yeast extract
A pH 8.0 medium consisting of 3 g, 3 g of glucose, and 1 g of water was used. Add 3.2 x 107 spores of Bacillus stearothermophilus JTS859 to 2 g of this medium, and use a Jarfer Mentor with stirring blades (diameter 80+ am, 6 upper and lower blades each) to adjust the stirring blades to 88 rpm.
While rotating at a ventilation rate of 1.5 VV1% culture temperature of 65
The cells were cultured for 8 hours at a temperature of 5.9 to 6.2.

After culturing, the bacterial cells were centrifuged (10,000 g, 4°C, 1
5 minutes) to collect bacteria. Next, a crude enzyme solution was prepared as follows.

160 g of the obtained wet bacteria were suspended in a 500 M potassium phosphate solution (pH 7.0, hereinafter simply referred to as "buffer"), and the bacterial cells were disrupted with Dymomill. Next, a buffer solution was added to bring the whole to 1400 alt, and then centrifugation (83
00g for 20 minutes) to obtain a supernatant of 1300g.

The precipitate was further suspended in 500@I buffer and the whole was diluted to 1.
After adjusting to 030-, centrifugation (8300g, 20 minutes)
A supernatant 990- was obtained. This supernatant 900- was combined with the previous supernatant 1300- and gently stirred at 63° C. for 1 hour. Then, centrifugation (8300 g, 40 minutes) was performed to obtain supernatant 2130-.

After heat-treating this supernatant 2130-, a mixed solution of acetone 200- and 200 all buffer solutions cooled to -10°C was added. Furthermore, acetone I cooled to -10°C
. 2.5 g of acetone at -10° C. was added to the obtained supernatant, stirred for 15 minutes, and centrifuged (9000 g, 5 minutes) to obtain a precipitate. This precipitate was suspended in 800-buffer to obtain an acetone-treated crude enzyme solution 850-.

Example 1 1 containing 5+ nM inosine, 5 thymine, 5 IIIM potassium phosphate, IU xanthine oxidase, and 25 oρ of crude enzyme solution (obtained in Production Example 1)
- reaction solution (pH 7.0) was reacted at 40° C. for 11 hours, and the concentration of each component contained in the reaction solution was measured. The results are shown in FIG.

In addition, the reaction was carried out under the same conditions as the above procedure except that xanthine oxidase was not added, and the concentration of each component after the reaction was measured. The results are shown in FIG. 2; in these figures, the vertical axis shows the concentration of each component in mM, and the horizontal axis shows the reaction time. Also, -0- is inosine, -
・- is hypoxanthine, -rho is 5-methyluridine,
-■- represents thymine, -mu- represents uric acid, and -Δ- represents xanthine, respectively.

As is clear from Figures 1 and 2, when xanthine oxidase is present (Figure 1), the content of the product 5-methyluridine is lower than when it is absent (Figure 2). It's very expensive. In particular, when xanthine oxidase is present, as the inosine content decreases, xanthine and uric acid are found together with hypoxanthine, and at equilibrium the content of inosine, hypoxanthine, and xanthine other than uric acid becomes almost 0. It is shown. This indicates that almost all inosine was oxidized by xanthine oxidase.

Specifically, when xanthine oxidase was present in the reaction system, 3.8mM of 5-methyluridine was produced. In its absence, the amount of 5-methyluridine produced was 1.4 EvM.

Thus, in the presence of xanthine oxidase, 5-methyluridine could be obtained in higher yield.

Example 2 Thymine at 1-20mM, inosine at the same concentration as thymine,
5ff1M potassium phosphate, 4U of xanthine oxidase, and crude enzyme solution (obtained in Production Example 1)
Reaction solution 1- containing 250d was reacted at 40°C, and the amount of 5-methyluridine present when 7-inosine disappeared was measured. The results are shown in Figure 3. This yield is
A very good agreement was found compared with the theoretical curves calculated by dividing by equations (4) and (5), shown by the broken line.

Examples 3 and 4 5mM inosine, 5iM salt layer 5d as shown in Table 1 below
of potassium phosphate, xanthine oxidase 1υ, and crude enzyme solution (250d obtained in Production Example 1).
- reaction solution (pH 7,0) was reacted at 40°C for 8 hours,
The concentration of the produced nucleoside contained in the reaction solution was measured.

In addition, the reaction was carried out under the same conditions as in the above procedure except that xanthine oxidase was not added, and the concentration of the produced nucleoside after the reaction was measured.The results are also shown in Table 1 below.

(Table 1) 5''M 2-deoxyinosine, 51X base shown in Table 2 below, 5sM potassium phosphate, IU xanthine oxidase, and crude enzyme solution (obtained in Production Example 1) ) A 1 wJ reaction solution (pH 7.0) containing 250 tlJ was reacted at 40° C. for 8 hours, and the concentration of the produced nucleoside contained in the reaction solution was measured.

In addition, the reaction was carried out under the same conditions as in the above procedure except that xanthine oxidase was not added, and the concentration of the produced nucleoside after the reaction was measured.

These results are also listed in Table 2 below.

Table jfi2 [Effects of the Invention] As described above, the method for producing a nucleoside compound of the present invention makes it possible to easily obtain a nucleoside compound in high yield by utilizing a base exchange reaction.

[Brief explanation of the drawing]

FIG. 1 is a graph showing changes over time in the concentration of each component in the reaction solution in Example 1 of the present invention, and FIG. 2 is a reaction solution under the same conditions as the graph shown in FIG. 1 except that xanthine oxidase was not added. FIG. 3 is a graph showing the relationship between the thymine concentration and the amount of 5-methyluridine produced in Example 2. Figure 1 Figure 2

Claims (2)

[Claims] (1) A nucleoside selected from the group consisting of inosine and deoxyinosine and a pyrimidine base are subjected to a base exchange reaction with purine nucleoside phosphorylase and pyrimidine nucleoside phosphorylase in an aqueous solution in the presence of phosphoric acid or a phosphate salt, and then the base is A method for producing a nucleoside compound, which comprises converting hypoxanthine produced by an exchange reaction into uric acid using xanthine oxidase. (2) A nucleoside selected from the group consisting of inosine and deoxyinosine and a purine base are subjected to a base exchange reaction with purine nucleoside phosphorylase in an aqueous solution in the presence of phosphoric acid or a phosphate salt, and further,
A method for producing a nucleoside compound, which comprises converting hypoxanthine produced by this base exchange reaction into uric acid using xanthine oxidase.