JPH0710236B2 - Purification method of dideoxyadenosine - Google Patents

Description

TECHNICAL FIELD The present invention relates to a novel method for purifying 2 ′, 3′-didequinadenosine (hereinafter abbreviated as DDA) produced by the action of microorganisms or enzymes. It is a thing.

[Conventional technology]

The conventional method for producing DDA is 2'of nucleosides.
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.

For this reason, 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 fractionation by liquid chromatography is slightly performed at the laboratory level.

2 ', 3'-dideoxyuridine (hereinafter abbreviated as DDU)
Alternatively, in the enzymatic reaction solution produced by the action of the microorganism or the enzyme using 2,3-dideoxyribose-1-phosphate as a substrate, DDA and adenine which are unreacted substrates as impurities in addition to DDA of the target product ( Hereinafter, abbreviated as Ad), uracil (hereinafter abbreviated as U) which is a decomposition product of a substrate, and some other by-produced nucleic acids.

In order to efficiently obtain high-purity DDA from this reaction solution, generally used means such as concentrated crystallization is not suitable. The reason for this is that both Ad and U have low solubilities and can be removed to some extent by concentration, but unreacted DDU and DDA, which is the target product, have high solubilities, making separation and purification difficult.

DDA undergoes hydrolysis under acidic conditions to give 2,3-
Since the dideoxyribose residue and the adenine group are easily cleaved, it was also difficult to separate and purify by treatment with an ion exchange resin which requires an acid.

[Problems to be solved by the invention]

It is desired to develop an industrially excellent purification method for DDA that solves the above-mentioned drawbacks.

[Means for solving problems]

As a result of diligent studies to solve the above problems, the present inventors concentrated the DDA-containing solution after, for example, sterilization, deproteinization, and decolorization, and after passing the solution (hereinafter, abbreviated as DDA concentrated solution). By treating with non-polar porous resin, DDA can be treated with Ad, U, DD
It was found that high-purity DDA can be separated by separating from impurities such as U, and the present invention has been completed based on this discovery.

That is, the present invention is a method for purifying DDA, which comprises adsorbing DDA to a nonpolar porous resin when purifying DDA produced by the action of a microorganism or an enzyme.

The starting material of the present invention may be unpurified DDA 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 an adenine residue are bound to each other or an intermediate treatment product thereof is adopted.

As microorganisms, Escherichia, Flavobacterium, Serratia, Enterobacter, Erwinia,
Any one capable of producing DDA 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 DDA concentrate used in the present invention may contain any of DDU, Ad, U, which are impurities, and some by-produced nucleic acids. Further, the DDA concentration of this solution is not limited as long as it is not higher than the solubility of DDA.

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 DDA concentrate is fed and the operability is good.

The liquid contacting method of the non-polar porous resin and the DDA concentrated liquid includes a batch method and a column method, but the column method 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 volume loading of the DDA concentrate to feed the column is D
DDA resin load (g / l
-R) is suitably 5 to 40 g / l-R, preferably 10 to 30 g / l-R in terms of separability and economy.

The liquid contact temperature to the column is not particularly limited as long as it is 10 to 60 ° C. At this temperature, DDA and the impurities Ad in the concentrate,
There is almost no difference in separability from U and DDU.

Next, the method for eluting DDA 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 column packed with the resin is used for DDA.
After feeding a fixed amount of the concentrated liquid, water is pushed to elute U. Next, DDU and Ad are eluted with an aqueous alcohol solution, and DDA is eluted by further increasing the alcohol concentration. Then, the DDA fraction can be concentrated, crystallized, and then cooled to separate high-purity DDA.

〔Example〕

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

Example 1 Yeast extract 0.5 g / dl, peptone 1.0 g / dl, meat extract 1.0 g / d
50 ml of a medium (pH 7.0) containing 1 and NaCl 0.5 g / dl was dispensed into a 500 ml shoulder flask and sterilized. Escherichia coli pre-incubated in broth agar medium at 30 ° C for 16 hours was added to this medium.
One platinum loop of ATCC10798 was inoculated and shake-cultured at 30 ° C for 16 hours. 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 Ad (pH =
7.0) 1 was added to 1% so as to react at 50 ° C. for 24 hours. As a result, 85 mg / dl of DDA was produced.
(Recovery rate 18%) 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). After concentrating the liquid to 15 ml, excess (No.5C paper)
did. 13 ml of this DDA concentrate (DDA 6.2 g / dl) was added to 65 ml of non-polar porous synthetic adsorption resin SP207 (Mitsubishi Kasei Co., Ltd.) (φ × L = 2).
After feeding SV = 1 to 0 mm × 210 mm), 260 ml of water pressing was carried out (SV = 2) ((fraction-1)). Then, elution was carried out with 390 ml of 10% ethyl alcohol-water solution (SV = 2), (designated as fraction-2). Finally, it was eluted with 390 ml of 20% ethyl alcohol-water solution (SV = 2), (designated as fraction-3).

Each fraction was measured by liquid chromatography analysis. As a result, only U was detected in Fraction-1 with a recovery rate of 99%, and Fraction-2 contained Ad, DDU and some DDA, and the recovery rates of Ad and DDU were 99% and 98%, respectively. , Fraction-3 contained DDA and the recovery rate was 95%.

Fraction -3 was concentrated and DDA was crystallized and then cooled to 10 ° C to obtain 600 mg of highly pure DDA. Table 1 shows the obtained elemental analysis values of DDA.

Example 2 13 ml of DDA concentrate obtained by treating in the same manner as in Example 1 (DDA6.
2 g / dl) was fed to non-polar porous synthetic adsorption resin SP207 (Mitsubishi Kasei Kogyo) 65 ml (φ × L = 20 mm × 210 mm) at SV = 1, and then water pushing was performed at 260 V at SV = 2. (As fraction 1). Next, it was eluted with 520 ml of 20% methyl alcohol-water solution (SV = 2), (designated as fraction-2). Finally, 40%
Elution with 390 ml of methyl alcohol-water solution (SV =
2), (designated as Fraction-3).

Each fraction was measured by liquid chromatography analysis. As a result, only U was detected in fraction-1 and the recovery rate was 98.
%, Fraction-2 contains Ad, DDU and some DDA.
And DDU recoveries of 98% and 98%, respectively.
Was included and the recovery rate was 93%. Fraction -3 was concentrated and DDA was crystallized and then cooled to 10 ° C to obtain 580 mg of high-purity DDA.
I took it. Table 2 shows the elemental analysis values of the obtained DDA.

[Effects of the Invention] As described above, the present invention is capable of efficiently separating and purifying DDA by treatment with a nonpolar resin, and therefore, a road to industrialization is greatly expected.

Claims (4)

[Claims] 1. When purifying 2 ', 3'-dideoxyadenosine produced by the action of a microorganism or an enzyme,
A method for purifying 2 ', 3'-dideoxyadenosine, characterized in that 2', 3'-dideoxyadenosine is adsorbed on a non-polar porous resin. 2. The method according to claim 1, wherein the production reaction of 2 ', 3'-dideoxyadenosine binds the 2,3-dideoxyribose residue and the adenine residue by the action of a microorganism or an enzyme. The method described. 3. The method according to claim 1, wherein the substance to be purified contains at least one of 2 ', 3'-dideoxyuridine, adenine and uracil as impurities. 4. The method according to claim 1, wherein the non-polar porous resin contains a styrene-divinylbenzene copolymer or a derivative thereof.