JPH0633314B2 - Alkylation of nicotinamide adenine dinucleotide - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a method for alkylating nicotinamide adenine dinucleotide (hereinafter referred to as NAD ⁺ ), and more specifically, alkylating NAD ⁺ with propiolactone. Corresponding N ¹
- (2-carboxyethyl) - relates to a method for producing nicotinamide adenine dinucleotide (hereinafter referred to as N ¹ -CE-NAD ^+).

[Problems to be Solved by Conventional Techniques and Inventions]

A production system that allows a reaction performed in a living body to be carried out in vitro and utilizes the reaction is generally called a bioreactor. You have to solve the problem.
One of the problems to be solved is the development of a method for recovering and reusing enzymes and / or coenzymes. For the purpose of solving this problem, a technique for polymerizing a coenzyme has been developed.
The object of the present invention, N ¹ -CH-NAD ^+, is an intermediate in the method for producing polymerized NAD ⁺ necessary for industrial use of a bioreactor using a redox enzyme that requires a coenzyme NAD ^+. is there.

Conventionally, as a method for polymerizing coenzyme NAD ⁺ , for example, (i) N
After alkylating the N ¹ position of the adenine ring of AD ⁺ , it is transferred to the N ⁶ position of the adenine ring of NAD ⁺ using the transfer reaction of deimulose (for example, Eur. J. Biochem Vol. 49, No. 5).
11 (1974), Eur.J.Biochem. 53, 481.
Page (1975) and Eur. J. Biochem Vol. 54, No. 47.
5 (1975)], (ii) A method for directly acylating the N ⁶ -position of the adenine ring of NAD ⁺ [eg, Eur. J. Biochem.
Vol. 7, p. 511 (1977)] and (iii) synthesize Cl, Br or SH substitution product of carbon at N ^6- position or 8-position of adenine ring of NAD ⁺ , and resubstitute with appropriate modifying group. Methods [eg, Eur. J. Biochem. Vol. 62, 211]
Page (1976)] and the like, and further polymerized NAD ⁺ has been obtained by further binding the intermediate to a carrier or a soluble polymer.

Among these methods for obtaining the intermediate, the most promising method for industrialization is the method (i). In this method, propiolactone was used as an alkylating agent [Eur. J. Biochem,
Volume 80, page 111 (1977)] The method is famous. In this method, the NAD ⁺ aqueous solution was adjusted to pH 6 to 6.5 with LiOH, and propiolactone was added intermittently to carry out an alkylation reaction at room temperature for 2 to 10 days, and then transfer to a daimlose to give N ⁶ −. A general method was to obtain (2-carboxyethyl) -nicotinamide adenine dinucleotide (hereinafter referred to as N ⁶ -CE-NAD ⁺ ) and further polymerize this N ⁶ -CE-NAD ^+. . However, due to the extremely low as about 30% yield in the alkylation for from NAD ⁺ to obtain the N ¹ -CE-NAD ⁺ In this method, N ⁶ -CE-NAD ⁺ yield is low and as a result It was

The present invention provides N ¹ -CE-NA in the alkylation of NAD ⁺ described above.
The purpose is to improve the yield of D ⁺ .

[Means and actions for solving problems]

The inventors have found that the low yield in NAD ⁺ alkylation is due to the consumption of NAD ⁺ as a by-product, which is a by-product of this by-product with repeated low-temperature or short-term reactions, or to prevent alkylation of the hydroxyl group of the sugar moiety of NAD ^+, but the alkylation of NAD ⁺ were line summer in the presence of boric acid in order to prevent the formation of more than connexion byproducts, preventing any generation of by-products at all I couldn't do it. As a result of further research, in the NAD ⁺ alkylation reaction, the reaction solution
The present invention has been completed based on the discovery that pH is the most important requirement for preventing the formation of by-products and improving the yield of N ¹ -CE-NAD ⁺ .

That is, in the present invention, nicotinamide adenine dinucleotide is alkylated with β-propiolactone to give N ^1-
A method for alkylating nicotinamide adenine dinucleotide, characterized in that when producing (2-carboxyethyl) -nicotinamide adenine dinucleotide, the reaction liquid is alkylated while maintaining the pH at 2 to 5.

The source and purity of NAD ⁺ used in the present invention are not particularly limited, but higher purity is preferable.

The alkylation of the present invention is carried out in an aqueous solution, and NAD ⁺ may be dissolved in the reaction solution, and the concentration thereof is not particularly limited, but it is usually 3 mol or less per 1 l of the reaction solution, preferably It is about 0.1 to 1 mol.

The alkylation of the present invention is carried out while keeping the pH of the reaction solution usually 2 to 5, preferably 3 to 4. PH of aqueous solution of NAD ⁺
Can not be unequivocally specified depending on the purity and concentration of NAD ⁺ , but is usually around 2 to 3, so that it may be subjected to alkylation without adjusting the pH. However, in general LiOH, NaOH, KOH and Ca (OH) ₂
The pH is adjusted with an alkaline substance such as to be used for alkylation. Among these alkaline substances, LiOH is most preferable because it is easy to remove the alkaline metal salt or alkaline earth metal salt with acetone or ethanol. Further, these alkaline substances are usually added as an aqueous solution to the reaction solution, but addition to the solid state is not prevented.

When the pH of the reaction solution is less than 2, decomposition of NAD ⁺ itself easily occurs, and the reaction rate is too small to be suitable for practical use. On the other hand, if the pH of the reaction solution is higher than 5, the risk of producing by-products increases.

If the pH of the reaction solution is adjusted to 2 to 5 prior to the alkylation reaction, the pH of the reaction solution remains within 2 to 5 even if the pH of the reaction solution fluctuates during the alkylation reaction. It is not necessary to correct the pH of the reaction solution during the chemical reaction.

The amount of β-propiolactone used in the present invention is the same as that of conventional NA.
The amount is 2 mol or more, preferably about 2-25 mol, relative to 1 mol of D ⁺ . The whole amount of β-propiolactone may be added all at once before the reaction, or may be added continuously or intermittently during the reaction period.

The alkylation in the present invention is carried out at room temperature to room temperature without heating or cooling, preferably 4 to 4
It is set to 0 ° C. It does not prevent heating or cooling.

From the N ¹ -CE-NAD ⁺ thus obtained, N ⁶ -CE-NAD can be obtained by a conventional method as follows.

That is, In the present invention, N ¹ -CE-NAD ⁺ yield results drastically improved in the alkylation, but N ⁶ -CE-NAD ⁺ yield is remarkably improved, the N ¹ -CE-NAD ⁺ N ⁶ -CE-NAD ^+, by adding a slight improvement in the process, N ⁶ -CE-
The yield of NAD ⁺ can be further improved. That is, for example, although N ¹ -CE-NAD ⁺ for separation and recovery is carried out by precipitating the N ¹ -CE-NAD ⁺ was added to the alkylation reaction product liquid in a mixed solvent of acetone and ethanol,
By making the molar ratio of this mixed solvent (acetone / ethanol) larger than in the conventional method (about 1/1)
The yield of N ⁶ -CE-NAD ⁺ can be further improved. In addition, for example, QAE-Sephadex A-25 (trade name, product of Pharmacia) N ⁶ by removing sulfite ion from the daimlose transfer reaction product using anion exchange resin such as
The yield of -CE-NAD ⁺ can be further improved.

〔Example〕

 The present invention will be described more specifically by way of examples.

Example 1 1 g of NAD ⁺ (manufactured by Kojin) was dissolved in 2.5 ml of water, and an aqueous solution of 2M LiOH was added to this aqueous solution (pH 2.5) to adjust the pH to 3.
It was adjusted to 5, 2 ml of β-propiolactone was added thereto, and the alkylation reaction was carried out at room temperature (25 ° C.) for 5 days.

After cooling the reaction product liquid to 0 ° C., the reaction product liquid was mixed with a mixed solvent of 20 times its volume of cold acetone and ethanol (acetone:
Ethanol = 7: 3) was added dropwise to produce a white precipitate. The N ¹ -CE-NAD ⁺ of this white precipitate was centrifuged and dried to obtain 0.82 g.

This N ¹ -CE-NAD ⁺ 0.82 g was added with a 1.3 wt% NaHCO ₃ solution 5
It was dissolved in 0 ml, and 0.5 g of Na ₂ S ₂ O ₄ was added thereto under a nitrogen stream, reduction was carried out at 45 ° C. for 10 minutes, and air was flown for 10 minutes after the reduction was completed. The pH of this reaction product solution was adjusted to 11.0 with a 2M LiOH aqueous solution, and a daimlose transfer reaction was carried out by keeping it at 70 ° C for 2 hours. After removing by the graph, adjust the pH to 7.5 with 1M HCl, add 0.5 ml of 80% acetaldehyde and yeast alcohol dehydrogenase (250 U), and add 1 at 30 ℃.
Enzymatic oxidation reaction was performed while stirring for time. The generated N ⁶ -CE-NAD ⁺ adsorbed eluted separated by DEAE-SephadexA-25 (trade name) column chromatography N ⁶ -CE
From the section containing -NAD ⁺ , purification was performed by performing the same precipitation operation using the mixed solvent of acetone and ethanol as described above. N ⁶
The yield of -CE-NAD ⁺ was 0.68 g.

Comparative Example The same procedure as in Example 1 was carried out except that the alkylation was carried out at pH 6.0, and 0.43 g of N ¹ -CE-from 1 g of NAD ^+.
NAD ⁺ was obtained and from this N ¹ -CE-NAD ^{+ an} additional 0.32 g of N ⁶
-CE-NAD ⁺ was obtained.

〔The invention's effect〕

N ¹ -CE-NAD in by connexion NAD ⁺ alkylation to the present invention ⁺
The yield of NAD ⁺ was significantly improved.
N ⁶ -CE-NAD ⁺ is significantly improved.

Claims (1)

[Claims] 1. When the nicotinamide adenine dinucleotide is alkylated with β-propiolactone to produce N ^1- (2-carboxyethyl) -nicotinamide adenine dinucleotide, the pH of the reaction solution is kept at 2-5. Method for alkylating nicotinamide adenine dinucleotide characterized by alkylating