JPH0691830B2 - Purification method of dideoxyinosine - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial field of application] The present invention relates to 2 ', 3'-dideoxyinosine produced by the action of microorganisms or enzymes (hereinafter abbreviated as DDI).
The present invention relates to a new purification method.

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

[Prior Art] As a conventional method for producing DDI, 2'of nucleosides is used.
Or 3'-position deoxidation reaction is performed (Chem.P
harm.Bull., 22,128 (1974)), but there are few reports for the following reasons.

Protecting groups must be introduced prior to reaction.

The 2'-position and the 3'-position have large steric hindrance and reaction is difficult to occur.

Also, a method of deamination using an enzyme from 2 ', 3'-dideoxyadenosine (hereinafter abbreviated as DDA) as a raw material has been reported (Biochim.Biophs.Acta., 5).
66 (2), 259 (1979)). However, this DDA is also difficult to manufacture for the above reasons, and there are few reports of methods using deamination.

Therefore, as for the isolation and purification method, the purification method that can be industrially used has not been established yet, because the purification method by liquid or thin layer chromatography is repeatedly performed at the laboratory level. .

2 ', 3'-dideoxyuridine (hereinafter abbreviated as DDU)
Alternatively, in the enzyme reaction solution produced by the action of the microorganism or the enzyme using 2,3-dideoxyribose-1-phosphate as a substrate, in addition to the target product DDI, the unreacted substrate DDU
And hypoxanthine (hereinafter abbreviated as Hyp), a substrate degradation product uracil (hereinafter abbreviated as Ura), and some by-product nucleic acids.

In order to efficiently obtain high-purity DDI from this reaction solution, the generally known treatment such as concentrated crystallization is unsuitable. The reason is that, during concentrated crystallization, etc.
This is because impurities such as Ura and Hyp have a lower solubility than DDI which is the target substance, so that they are mixed as impurities into DDI crystals and make purification difficult.

DDI is hydrolyzed under acidic or neutral conditions, and the 2,3-dideoxyribose residue and hypoxanthine residue are easily cleaved.Therefore, separation and purification by treatment with an ion-exchange resin that requires acid is performed. Was also difficult.

[Problems to be solved by the invention]

It has been desired to develop an industrially excellent purification method for DDI that eliminates the above-mentioned drawbacks, that is, a method that can be purified simply and at low cost.

[Means for solving problems]

The present inventors, in order to solve the above problems, as a result of intensive studies, a solution containing unpurified DDI, for example, sterilization,
After deproteinization and decolorization treatment, nonpolar DDI is adsorbed on the porous resin, and then the adsorbed DDI is eluted to remove DDI.
The inventors have found that they can be separated from impurities such as Ura and Hyp, and are preferably separated from impurities such as DDU by combining crystallization separation steps, and have completed the present invention based on these findings.

That is, the present invention, produced by the action of microorganisms or enzymes, or in purifying unpurified DDI derived therefrom, a method of adsorbing DDI to a non-polar porous resin, and then eluting the adsorbed DDI, preferably this Is a method for purifying DDI, which is characterized by combining the crystallization method with the crystallization method.

The starting material of the present invention may be unpurified DDI and its purity does not matter. By the action of microorganisms or enzymes, for example,
A reaction solution in which a 2,3-dideoxyribose residue and a hypoxanthine residue are bound to each other or an intermediate treatment product thereof is adopted.

As microorganisms, Escherichia, Flavobacterium, Serratia, Enterobacter, Erwinia,
Any genus capable of producing DDI such as Citrobacter, Corynebacterium, Hafnia, Kluyhera, Salmonella, or Xanthomonas can be used. In addition, the enzyme is not particularly limited as long as it has the above-mentioned microorganism and other enzymes having the same function.

The DDI reaction liquid used in the present invention contains impurities DDU, Hyp, Ur
a, any of some by-produced nucleic acids may be contained. Further, the DDI concentration of this solution is not limited as long as it is not higher than the solubility of DDI.

Next, the non-polar porous resin used here, for example, the matrix is a styrene-divinylbenzene-based copolymer or,
Any derivative thereof, for example, a substance which is a polymer obtained by halogenating the derivative and increasing the specific gravity thereof can be used. For example, Diaion HP series, SP series (above, Mitsubishi Kasei), XAD-4 (Rohm and Haas), OC
Although 1031 (Bayer Co., Ltd.) and the like can be used, any other nonpolar porous resin may be used as long as it has equivalent properties. In particular, SP207 (Mitsubishi Kasei), which has a high specific gravity, is suitable because the resin does not float when the DDI-containing solution is fed and the operability is good.

The liquid contacting method of the non-polar porous resin and the DDI-containing solution includes a batch type and a column type, but the column type is preferable in terms of operation and is preferable.

There is no particular restriction on the flow rate through the column, and usually SV = 0.5
˜4.0, preferably about SV = 1˜2.

The constitutional load of the DDI-containing solution fed to the column is DDI.
DDI resin load (g / l-R) is 5-40g / l-R, preferably 10-30g / l
-R is suitable in terms of separability and economy.

The liquid contact temperature to the column is not particularly limited as long as it is 10 to 50 ° C. At this temperature, DDI and impurities Hyp, U in solution
There is almost no difference in separability from ra.

From the viewpoint of DDI stability, the pH of the feed liquid to the resin is preferably on the alkaline side, and more preferably pH 8.0 to 10.0. Also, a temperature of 50 ° C or lower is suitable from the viewpoint of stability.

Next, the method for eluting DDI from the column will be described. A suitable lower aliphatic alcohol aqueous solution is suitable as the eluent. For example, it is an aqueous solution of methyl alcohol, ethyl alcohol, isopropyl alcohol, or the like. The elution rate is
SV = 1-2 is good.

The actual refining operation using a non-polar porous resin may be as follows. That is, the DDI was packed in the column packed with the resin.
After feeding a fixed amount of the solution containing Ura and Hyp, water is pressed to elute Ura and Hyp. Next, DDI and DDU are eluted with an aqueous alcohol solution.

Next, the DDI- and DDU-containing fraction is concentrated, DDI is crystallized, and then cooled to separate DDU and DDI, whereby high-purity DDI can be obtained.

The pH of the concentrated liquid during crystallization is on the alkaline side, preferably pH 8 to 1.
If kept at 0, the decomposition of DDI is suppressed and the crystallization rate can be improved.

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

〔Example〕

 Hereinafter, the present invention will be described in detail with reference to examples.

Example 1 Yeast extract 500 mg / dl, peptone 1000 mg / dl, meat extract 100
Add 50 ml of medium (pH 7.0) containing 0 mg / dl and NaCl 500 mg / dl to 5
It was dispensed into a 00 ml shoulder flask and sterilized. One platinum loop of Escherichia coli ATCC10798, which had been pre-cultured in broth agar medium at 30 ° C. for 16 hours, was inoculated into each of the medium, and cultured at 30 ° C. for 16 hours with shaking. The cells were separated from the obtained culture broth by centrifugation, washed with 0.05 M phosphate buffer (pH 7.0), and further centrifuged to prepare washed cells.

Wash Escherichia coli ATCC 10798 washed cells with 20 mM D
100 mM phosphate buffer containing DU and 20 mM Hyp (pH =
7.0) 1 was added to 1% so as to react at 50 ° C. for 24 hours. As a result, 70 mg / dl of DDI was produced.

(Recovery rate 15%) After sterilizing this solution by centrifugation (7000G, 40 minutes), add 50mg of activated carbon (white heron charcoal, Takeda Pharmaceutical Co., Ltd.) to the sterilization solution to remove protein and decolorize (50 ℃, 1hr) ), Followed by filtration (pore size 0.45 μm filter). The solution was adjusted to pH 8 with 1N NaOH, concentrated to 100 ml, and fed to non-polar porous adsorption resin SP207 (Mitsubishi Kasei) 65 ml (column φ × L = 20 mm × 210 mm) with SV = 1, and then pressed with water. 300 ml was performed (SV = 2), (fraction-1
And). Next, it was eluted with 260 ml of 20% aqueous ethyl alcohol solution (SV = 2), (fraction-2). The temperature during the resin treatment was 30 ° C. in all cases.

When each fraction was measured by liquid chromatography analysis, Ura and Hyp were detected in fraction-1, the recovery rate was 99,98% respectively, and in fraction-2, the DDI and DDU recovery rates were respectively. It was included in 98% and 95%.

This fraction-2 was adjusted to pH = 8 with 1 N NaOH, concentrated, and then crystallized to obtain 410 mg of highly pure DDI crystals. Obtained DDI
Table 1 shows the elemental analysis values of.

Example 2 100 ml of DDI concentrate obtained by treating in the same manner as in Example 1 (DDI7
00 mg / dl) was fed to non-polar porous adsorption resin SP207 (Mitsubishi Kasei) 65 ml (column φ × L = 20 mm × 210 mm) at SV = 1, and then 250 ml of water was pushed (SV = 2), (image) Min-1
And). Next, 10% isopropyl alcohol aqueous solution 20
Elution with 0 ml (SV = 2), (designated fraction-2). The temperature during the resin treatment was 30 ° C. in all cases.

When each fraction was measured by liquid chromatography analysis, Ura and Hyp were detected in fraction-1, the recovery rate was 99,97%, respectively, and fraction-2 contained DDI and DDU, respectively. The recovery rate was 98% and 95%.

This fraction-2 was adjusted to pH = 8 with 1 N NaOH and then concentrated to obtain 440 mg of highly pure DDI crystals by crystallization. Table 2 shows the elemental analysis values of the obtained DDI.

[Effects of the Invention] As described above, according to the present invention, a non-polar porous resin treatment method, preferably by combining this with a crystallization method
Since DDI can be efficiently separated and purified, the road to industrialization is highly expected.

Claims (1)

[Claims] 1. When purifying unpurified 2 ', 3'-dideoxyinosine produced by the action of a microorganism or an enzyme, 2', 3'-dideoxyinosine is adsorbed on a non-polar porous resin and then adsorbed. A method for purifying 2 ', 3'-dideoxyinosine, which comprises eluting 2', 3'-dideoxyinosine as described above and separating it from other impurities.