JPS6118796A - Preparation of adenosine 5'-triphosphate - Google Patents

Abstract

PURPOSE:To obtain the titled compound [adenosine triphosphate (ATP)] industrially and advantageously in high yield with ease, by reacting adenosine 5'-diphosphate (ADP) with beta-cyclodextrin (beta-CD) in the presence of a magnesium salt in an aqueous solution. CONSTITUTION:Adenosine 5'-diphosphate is reacted with beta-cyclodextrin (beta-CD) in a molar amount of preferably 0.1-2 times that of the ADP and if necessary creatine in a molar amount of usually 0.1-10 times that of the ADP in the presence of a magnesium salt, e.g. magnesium chloride, in an aqueous solution, e.g. phosphoric acid buffer solution of 7pH, usually at 20-50 deg.C for 20-120hr to give the aimed ATP.

Description

[Detailed Description of the Invention] [Object of the Invention] The present invention is directed to the production of adenosine 5'-triphosphate in aqueous solution, which is important in biological reactions and industrially. This invention relates to a new manufacturing method.

Prior Art It is well known that azo/syn 5'-triphosphate (ATP) is produced in vivo by the combination of azo/syn 5'-diphosphate (ADP) and a phosphate group. Typical systems include photophosphorylation, oxidative phosphorylation, and the bentose phosphate cycle.

In recent years, one of the most popular methods for synthesizing this high-energy compound, ATP, from ADP using carbamyl phosphate or mercaptoacetate and a bromine oxidizing agent in a non-aqueous solution such as pyridine (Th, W 1eland).

E, B 1iuenlein, A rgeur,
Chem+ 1internal, Edit, 7
89.3 (1968)) and the development of a model system for ATP regeneration using enzymes as part of a bioreactor (Imahori, Kondo, et al.).

Nakajima, Iwasaki, JP-A-58-209990. Murata, Kimura.

Proteins, Nucleic Acids, Enzymes 26 915 (1981))
.

However, methods based on organic synthesis have low yields and require complicated separation and purification steps due to the use of non-aqueous solutions, and enzymatic ATP regeneration systems require strict control of reaction conditions such as temperature and pH. There was a problem of low productivity.

While the invention is intended, the present invention provides an industrially advantageous method for producing ATP from ADP in a simple and high yield in an aqueous solution without requiring conventional non-aqueous solutions or enzymes. The purpose is to

[Structure of the invention] Means for determining the problem β-cyclodextrin (hereinafter referred to as β-CD) has a hydrophobic cavity and the ability to include various compounds in an aqueous solution. Various organic reactions are carried out using this property. The inventors
Focusing on this β-17cD, it was discovered that ATP was generated by reacting it with ADP in the presence of a magnesium salt in an aqueous solution, leading to the present invention.

That is, the method for producing ATP according to the present invention is characterized by allowing ADP to react with β-cyclodextrin in the presence of a magnesium salt in an aqueous solution, and the time required for producing ATP and the production rate are determined by the presence of creatine. facilitated by

As ADP as a raw material in the present invention, commercially available products containing trace amounts of AMT and ATP can also be suitably used. In addition, β-CD requires the presence of the above-mentioned hydrophobic cavity, and for example, when cyclohexanol is added to the reaction system, β-CD
- It immediately forms a complex with CD, inhibiting the reaction associated with the above-mentioned inclusion effect, and no ATP production is observed at all.

Addition of magnesium salts such as magnesium chloride, magnesium bromide, magnesium sulfate, magnesium phosphate, and magnesium perchlorate is important for interaction with ADP in the early stage of the reaction and for stabilizing ATP after generation. That is, in the early stage of the reaction, if there is no magnesium salt in the ADP-β-CD system, ADP, AT
Almost no P is produced, and ATP is produced as a raw acid after the reaction.
yield decreases. On the other hand, creatine is known as a substance that can form high-energy phosphate bonds, and its addition improves the production time and production rate of ATP, but AD
It is estimated that in the range of 0.1 to 10 times the standard addition amount in terms of moles, the production rate is not affected and recycling is catalytically rapid.

In the present invention, to produce ATP from ADP, A
It is sufficient to react with β-CD in a phosphate buffer of DPe pH 7 in the presence of the above magnesium salt at 20 to 50°C for 20 to 120 hours, and the addition of creatine aims to shorten the reaction time and improve the production rate. be able to.

The concentration of ADP is not particularly limited, but is between 0.25 and 250.
A range of mM is suitable. The usage ratio of β-CD based on ADP is in the range of 0.01 to 4 times the mole, preferably 0.1 to 2 times the mole. At concentrations lower than 0.01 times molar, A
The induction period for TP production becomes longer, and rapid increases and decreases in ATP occur.

On the other hand, at a concentration higher than 4 times molar, the production and decrease of ATP are also slow. Also, the concentration of magnesium salt is A
Although it has little effect on the amount of TP produced, as mentioned above,
In order to prevent a decrease in P, it is preferable to add 0.1 to 50 times the mole of ADP.

In order to isolate ATP from the reaction mixture, for example, liquid high performance chromatography, thin layer chromatography (TLC) can be used.
) and stored in an aqueous solution of alkaline salts such as sodium.

In addition, industrially, it is possible to use ordinary ion exchange resins.
I can do that. For example, using Dou+ex IX4 (formic acid type), AMP, ADP, and ATP are separated and purified by increasing the concentration of formic acid and its ammonium salt.

Control -■ The reaction mechanism for ATP production is as follows: 1) 2ADP ≠ A M P + A TP2)
ATP is produced in an aqueous solution by the interaction between β-CD, which has a hydrophobic cavity, and the ADP-Mg conjugate, although it is unclear which process takes place.

Example Next, the present invention will be specifically explained using examples.

Example 1 Creatine, β-CD, MgCl2, and ADP were added to a test tube containing 1715M-+7 phosphate buffer adjusted to pH 7.0 so as to have the concentrations shown in Table 1, respectively.
It was immersed in a 37°C water bath and shaken.

Table 1 Concentration of each reaction reagent Uffi 2i degree (Lr1M) Creatine
2.50 β-CD 2.50 MgCl, 0.25 ADP 2.50 The reaction solution was treated with a 7-ion exchange packing material [TSKIEX-540
-DEAEJ column (4.0 mm l, DX250 m
+n) for separation and purification, elution with 1/10M phosphate buffer (pH 7,0) containing 5% acetonitrile, and each component was detected at UV 26Or+m. FIGS. 1 and 2 show the absorption characteristics after 0 and 40 hours, respectively, from the start of the reaction, and an increase in the ATP peak was confirmed after 40 hours. In addition, imbutyric acid-ammo=
Standard A in Tl-1c using 7-water = 66:1:33
The Rf of the TP peak and the newly generated peak match as shown in Table 2.

Table 2 TLC data 1 digit 1 --- Rf value AMP ...... 0.45
ADP ・・・・・・ 0.28 0.45ATP
O,120,270,44 Formation 0.13 0.25 0.42 = (Color development is due to UV irradiation) Table 3 shows the amount of ATP produced calculated from the relative peak areas in Figures 1 and 2, It can be seen that 13% of ATP was produced based on the amount of ADP in the starting material.

Table 3 ATP yield lifetime 1 1 1 1 1 1 = / l, Akira 0
-ATP 13.7 ADP -29,4 AMP 12.4 [Effects of Gyomei According to the present invention, adenosine 5'-triphosphate is produced in an extremely mild aqueous solution using adenosine 5'-diphosphate as a starting material. It can be produced in one step under suitable reaction conditions and has great industrial value.

[Brief explanation of the drawing]

The drawings show an example of the present invention, and FIGS. 1 and 2 are graphs showing the chromatographic absorption characteristics of the reaction solution 0 and 40 hours after the start of the reaction, respectively.

Claims (1)

[Claims] A method for producing adenosine 5'-triphosphate, which comprises reacting adenosine 5'-diphosphate with β-cyclodextrin in the presence of a magnesium salt and, if necessary, creatine in an aqueous solution.