JPH01165390A - Method for purifying dideoxyinosine - Google Patents

Abstract

PURPOSE:To simply and efficiently separate and purify dideoxyinosine at a low cost, by treating 2',3'-dideoxyinosine produced by action of a microorganism or enzyme with a nonpolar porous resin. CONSTITUTION:A solution containing unpurified 2',3'-dideoxyinosine produced by action of a microorganism or enzyme is subjected to, e.g., sterilization, deproteinization and decoloring treatment, and then brought into contact with a nonpolar porous resin, e.g., styrene-divinylbenzene based copolymer or a derivative thereof, to adsorb the 2',3'-dideoxyinosine on the above-mentioned resin. An aqueous solution of a lower aliphatic alcohol is subsequently used as an eluent to elute the 2',3'-dideoxyinosine from the afore-mentioned resin and the eluate is preferably concentrated to crystallize the aimed 2',3'-dideoxyinosine.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to
This article relates to a new method for purifying 3/-dideoxyinosine (hereinafter abbreviated as DDI).

DDI is used as a medicine such as an antiviral agent.

[Conventional technology]

Conventional methods for producing DDI include two types of nucleosides:
Deoxygenation reaction at the ’ or 3’ position is taking place (C
hem, Pharm, Bull, , 22.128 (
(1974)), but there are few reported cases for the following reasons.

■ Protecting groups must be introduced prior to the reaction.

■ The 2' and 3' positions are highly sterically hindered, making it difficult for reactions to occur.

Also, 2',3'-dideoxyadenosine (hereinafter referred to as DD
A deaminating method using enzymes using A) as a raw material has also been reported (Bioahim).

Biophs, Acts, 566(2), 2
59 (1979)), however, this DDA is also difficult to manufacture due to the above-mentioned reasons, and there are few reports of methods using deamination.

For this reason, with regard to isolation and purification methods, only repeated preparative purifications using liquid or thin layer chromatography have been carried out at the laboratory level, and purification methods that can be used industrially have not yet been established. There wasn't.

The enzyme reaction solution produced by the action of microorganisms or enzymes using 2',3'-dideoxyuridine (hereinafter abbreviated as DDU) or 2,3-dideoxyligase-1-phosphate as a substrate contains the desired substance. In addition to the product DDI, there are unreacted substrates DDU and hypoxanthine (hereinafter abbreviated as H7p).

), uracil (hereinafter abbreviated as Ura), a substrate decomposition product, and some by-product nucleic acids.

In order to efficiently obtain DDI of high purity from this reaction solution, treatment using only generally known means such as concentration crystallization is inadequate. The reason for this is that during concentration crystallization, impurities such as Ura + Hyp are not absorbed by the target substance DD.
This is because both I and I have low solubility, so they are mixed into DDI crystals as impurities, making purification difficult.

In addition, DDI undergoes hydrolysis under dispersive or neutral conditions, and the 2,3-dideoxy IJ, y-su residue and hypoxanthine residue are easily cleaved, so ion exchange that requires acid is difficult. Separation and purification by resin treatment was also difficult.

[Problem that the invention seeks to solve]

It has been desired to develop an industrially superior purification method for DI that eliminates the above-mentioned drawbacks, that is, a simple and low-cost purification method.

[Means for solving problems]

In order to solve the above-mentioned problems, the present inventors have conducted intensive studies and found that a solution containing unpurified DDI is treated with a non-polar porous resin after, for example, sterilization, protein removal, and decolorization treatment. In particular, we have discovered that 15DDI can be separated from impurities such as Ura and Hyp, and preferably from impurities such as fi DDU by combining a crystallization separation process, and based on these discoveries, we have completed the present invention. reached.

That is, the present invention provides, when purifying unpurified DDI produced by or derived from the action of microorganisms or enzymes, D
D, characterized by a treatment method for adsorbing DI to a non-polar porous resin, preferably combining this with a crystallization method.
This is a method for purifying DI.

The starting material of the present invention may have any degree of purity as long as it is unpurified DDI. For example, a reaction solution in which a 2,3-dideoxyligase residue and a hypoxanthine residue are combined by the action of a microorganism or an enzyme, or an intermediate treatment product thereof is employed.

Microorganisms include Escherichia genus, Flavobacterium genus, Serratia genus, Enteropa l#-R, Erwinia genus,
Any member of the genus Citrobacter, Corynebacterium, Hafnia, Kluichella, Salmonella, or Xanthomonas that can produce DDI may be used. Further, the enzyme is not particularly limited as long as it is possessed by the above-mentioned microorganisms or has the same function.

The DDI reaction solution used in the present invention contains DDU, which is an impurity.
e Hyp + Ura *It may contain any of some by-product nucleic acids, and the DDIII degree of this solution is not limited as long as it is below the solubility of DDI.

Next, the non-polar porous resin used here may be made of a styrene-diverbenzene copolymer or
Any of its derivatives, such as halogenated polymers with high specific gravity, can be used. For example, Diaion BP series, SP series (and above,
Mitsubishi Chemical Industries).

XAD-4 (o-mu-7:Yde Haas), 0C1
031 (Bayer) etc., but any other non-polar porous resin may be used as long as it has equivalent properties. Especially high specific gravity 5P207
(Mitsubishi Chemical Industries, Ltd.) is suitable because the resin does not float when a DDI-containing solution is fed and it has good operability.

There are two methods for contacting the non-polar porous resin with the DDI-containing solution: a patch method and a column method, but the column method is preferred because it is easier to operate.

There is no particular restriction on the flow rate of liquid to the column, and usually SV=
0.5 to 4.0, preferably about 5v=1 to 2! stomach.

The volumetric loading of the DDI-containing solution fed to the column is:
It varies depending on the concentration of the DDI-containing solution, and at the same time, the resin loading amount of DDI (11#-R) is 5 to 401/l-n, preferably 10 to 30 I! /iR is suitable in terms of separation and economy.

The temperature of the liquid in contact with the column is not particularly limited as long as it is 10 to 50°C. At this temperature, there is almost no difference in the separation between DDI and the impurity H)'p+ TJra in the solution.

In addition, from the viewpoint of stability of DDI, the feed liquid to the resin is
is preferably on the alkaline side, preferably p) 18.0 to 10.
0 is good. In addition, a temperature of 50° C. or lower is suitable from the viewpoint of stability.

Next, a method of elution of DDI from the column will be described.

As the eluent, a lower aliphatic alcohol aqueous solution is suitable.

For example, methyl alcohol, ethyl alcohol, iso7
It is an aqueous solution such as 'o-pyl alcohol. The elution rate is usually about 5V=1 to 2.

The purification operation using a non-polar porous resin may be carried out as follows. That is, the column K D filled with the resin
After feeding a certain amount of DI-containing solution, send it to Suishin.
n and) Iyp. DDI and DDU are then eluted using an aqueous alcohol solution.

Next, the fraction containing the DDI and DDU is concentrated to
By cooling K after crystallizing I, DDU and DDI can be separated and highly purified DDI can be obtained.

- of the concentrated solution during crystallization is on the alkaline side, preferably pH 8
If the ratio is maintained at 10 to 10, decomposition of DDI can be suppressed and the crystallization rate can be improved.

(9) If necessary, in addition to the above-mentioned treatment methods, a solvent extraction method or a liquid chromatography treatment may be performed.

〔Example〕 Hereinafter, the present invention will be explained in detail with reference to Examples.

Example 1 Yeast extract 5001R9/dl, peptone 1000 instant/
dt, Agony extract 10001119/dl and NaCl
Medium containing 200119/dl (pH 7,0) 501
The mixture was dispensed into nlt-500, d shoulder flasks and sterilized.

One platinum loop of Escherichia coli ATCC 10798, which had been previously cultured on a bouillon agar medium at 30°C for 16 hours, was inoculated into this medium, and cultured with shaking at 30°C for 16 hours. After separating the obtained culture solution and bacterial cells by centrifugation,
．． By washing with 05M phosphate buffer (pH 7.0) and further centrifugation), the washed bacterial cells were made into mM.

The washed bacterial cells of Escherichia coli ATCC 10798 were washed with
mM phosphate buffer - (P) (= 7.0) 1
1 to 14, and reacted at 50°C for 24 hours. As a result, a DDI of 'l0m9/di was generated.

(Recovery rate: 15) α This solution was centrifuged (7000G, 40 minutes to sterilize it,
The disinfectant solution was filtered with activated carbon (white cedar charcoal, Ota Pharmaceutical Co., Ltd.) (pore size 0.45 μm filter). After adjusting the pH of the ν solution to 8 using 1N NaOH, it was concentrated to 100% and added with 65 ml of non-polar porous adsorption resin S, P2O7 (Mitsubishi Chemical Industries).
(Force 9 A $XL = 20m x 210m) K 5V
After feeding with =1, I went to Suishin for 300m (sv=2)
, (referred to as fraction -1). Next, it was eluted with 204 ethyl alcohol aqueous solution 2604 to give 7'j (SV=2), (fraction -
2) The temperature during the resin treatment was all 30°C.

When each fraction was measured by liquid chromatography analysis, Ura and Hyp were detected in fraction-1, each with a recovery rate of 99.984, and DDI and DDU were detected in fraction-2, each with a recovery rate of 98.984. Sorry, it was included with 95 regrets.

Both fractions -2 were adjusted to -=8 with IN NaOH, concentrated, and crystallized to obtain υ highly pure DDE crystals of 410η. The elemental analysis values of the obtained DDI are as shown in Table 1.

Table-1 Elemental analysis value (壬) CHN Theoretical value 50.84 5.12 23.72 Elemental analysis value 50.86 5.12 23.91 Example 2 100 d of DDI concentrate obtained by processing in the same manner as Example 1
(DDI700 da/dt) with 5p of non-polar porous adsorption resin
207 (Mitsubishi Chemical Industries) 65m (column φXL=20t
After feeding 5V=1 to mx210+u), set Suijin to 25
04 was carried out (SV=2), (set as fraction -1). next,
It was eluted with a 200% aqueous solution of isopropyl alcohol (SV=2) (referred to as fraction-2). The temperature during all resin treatments was 30°C.

When each fraction was measured by liquid chromatography analysis, Ura and H3'p were detected in fraction-1, with a recovery rate of 99.9796, respectively, and DDI and DDU were detected in fraction-2. The recovery rate for each was 98.

It contained 95qb.

This fraction -2 was adjusted to -=8 with IN NaOH and concentrated.
440m9 of highly pure DDI crystals were obtained by crystallization.

The elemental analysis values of the obtained DDI are shown in Table 2.

Table 2 C)l N Theoretical value 50.84 5.12 23.72 [Effect of the invention] As stated above, according to the present invention, a non-polar porous resin treatment method, preferably a combination of this and a crystallization method As a result, DDE can be efficiently separated and purified, and there are great expectations that it will lead to industrialization.

Claims (5)

[Claims] (1) 2', 3 produced by the action of microorganisms or enzymes
When purifying ′-dideoxyinosine, 2′, 3′
- A method for purifying 2',3'-dideoxyinosine, which comprises adsorbing dideoxyinosine onto a non-polar porous resin. (2) The method according to claim (1), further comprising a crystallization step. (3) The production reaction of 2',3'-dideoxyinosine is 2
, 3-dideoxyribose residue and hypoxanthine residue are bonded by the action of a microorganism or an enzyme, the method according to claim (1). (4) Claim (1) characterized in that the product to be purified contains at least one of 2',3'-dideoxyuridine, hypoxanthine, and uracil as impurities.
The method described in section. (5) The method according to claim (1), wherein the non-polar porous resin contains a styrene-diverbenzene copolymer or a derivative thereof.