JPH0198496A - Purification of dideoxyadenosine - Google Patents

Abstract

PURPOSE:To purify dideoxyadenosine produced by action of a microorganism or enzyme in high purity advantageously on an industrial scale, by absorbing the dideoxyadenosine into a nonpolar and porous resin. CONSTITUTION:2',3'-Dideoxyadenosine produced by action a microorganism or enzyme and containing 2',3'-dioxyuridine, adenine and uracil as impurities is absorbed into a nonpolar and porous resin (preferably styrene-divinylbenzene based copolymer), preferably using a column system. Liquid rate passing through the column, i.e. SV is preferably 1-2 and temperature for bringing the liquid into contact with the column is preferably 10-60 deg.C. As the eluate, an aqueous solution of lower aliphatic alcohol is preferred. As the eluting rate, SV is preferably 1-2.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention provides 7, produced by the action of microorganisms or enzymes.
This invention relates to a new method for purifying 3'-dideoxyadenosine (hereinafter referred to as DDA (!)).

[Conventional technology]

Conventional methods for producing DDA include two nucleoside
Deoxygenation reaction at the ′ or 3′ position is taking place (C
h@m, 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 τ and 3' positions are highly sterically hindered, making it difficult for reactions to occur.

For this reason, as for the isolation and purification method, repeated purification of preparative separation using liquid chromatography has only been carried out at the laboratory level, and an industrially usable method a has not yet been established.

z, 3'-dideoxyuridine (hereinafter abbreviated as DDU) or 2,3-sodeo-siri? In the enzyme reaction solution produced by the action of microorganisms or enzymes using -su-1-phosphate as a substrate, in addition to the target product DDA, there are unreacted substrates DDU and adenium (hereinafter abbreviated as Ad) as impurities.

) and uracil (hereinafter abbreviated as U), which is a decomposition product of the substrate.
, and some other by-product nucleic acids.

In order to efficiently obtain high-purity ODA from this reaction solution, commonly used means such as concentration crystallization are not suitable. The reason for this is that both Ad and U have low solubility and can be removed to some extent by concentration, but unreacted DDU and DDA, the target product, both have high solubility and are difficult to separate and purify.

In addition, DDA undergoes hydrolysis under acidic and alkaline conditions, forming 2,3-dideoxyribose residues and ar2/
Since the groups are easily cleaved, separation and purification by ion exchange resin treatment, which requires acid or alkali, is also difficult.

[Problem that the invention seeks to solve]

It is desired to develop an industrially valuable purification method for DA that eliminates the above-mentioned drawbacks.

[Failure to solve the problem]

As a result of intensive studies to solve the above-mentioned problems, the present inventors concentrated the DDA-containing solution after sterilization, protein removal, and decolorization, filtered it, and produced a P solution (hereinafter abbreviated as DDA concentrated solution).

) was discovered to be able to be separated from impurities such as Ad, U, and DDU by treating it with a non-polar porous resin, and high-purity DDA could be fractionated.Based on this discovery, the present invention was completed. It was.

That is, the present invention is a method for purifying DDA, which is characterized in that when DDA produced by the action of microorganisms or enzymes is purified, DDA is adsorbed onto a non-polar porous resin.

The starting material of the present invention may be unpurified DDA and its degree of purity does not matter. For example, a reaction solution in which a 2,3-dideoxyribose residue and an adenine residue are combined by the action of a microorganism or an enzyme, or an intermediate product thereof, is employed.

Microorganisms include Escherichia, Fladebacterium, Serratia, Enterobacter, Erwinia,
Any species that can produce DDA may be used, such as Citrobacter, Corynebacterium, Hatsuea, Kluihera, Salmonella, or Xanthomonas. In addition, the enzyme is not particularly limited as long as it is possessed by the above-mentioned microorganisms or has other functions. The DDA concentrate used in the present invention is free from DDU, which is an impurity.
, Ad, U, among some by-product nucleic acids,
It may contain either. Also, the DDA of this solution
The a degree is not limited as long as it is below the solubility of DDA.

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

XAD-4 (O-Mu-7y De 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), the resin does not float when feeding DDA liquid.

It is suitable for its ease of operation.

There are two methods of contacting the non-polar porous resin and the DDA 9 condensate: a batch 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 it is usually 5V=0.
．． 5 to 4.0, preferably about 5v=1 to 2.

The volume load of DDA@condensed liquid to be fed to the column varies depending on the amount of DDA a supplied liquid, and is 5 to 40
v/v (%), and at the same time the resin loading amount of DDA (, I
l/1-n) is 5 to 4011/l-n, preferably 10
~31/A-R is suitable from the viewpoint of separability and economy.

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

Next, a method for eluating DDA from the column will be described.

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

For example, methyl alcohol, ethyl 7 A/: + -
/l/, is an aqueous solution such as isopropyl alcohol. The elution rate is usually about 5V=1 to 2.

The actual purification operation using a non-polar porous resin may be carried out as follows. That is, DD is added to the column filled with the resin.
After feeding a certain amount of A concentrate, it is washed with water and U is eluted.

Next, DDU and Ad are eluted using an alcohol aqueous solution, and DDA is further increased by increasing the alcohol concentration.
elute. Then, by concentrating this DDA fraction, cooling it after crystallization, highly pure DDA can be isolated.

〔Example〕

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

Example 1 Yeast extract 0.5 f! /dt, Begton 1. O/
l/dt.

Meat extract 1.0/;l/dt and NaC 10.5g
50 ml of a medium (pH 7.0) containing /dL was dispensed into a soft-shouldered flask and sterilized. One platinum loopful of Escherichia coli ATCC 10798, which had been precultured at 30°C for 116 hours on a bouillon agar medium, was inoculated into this medium.
Shaking culture was performed at °C for 16 hours. After separating the bacterial cells from the obtained culture solution by centrifugation, 0.05 M IJ
Washed bacterial cells were prepared by washing with acid buffer (pH 7.0) and further centrifuging.

The washed bacterial cells of Escherichia coli ATCC 10798 were mixed with 10 μM of DDU and 20 mM of Ad.
The mixture was added to 1 ton of 0-phosphate buffer (pH=7.0) in an amount of 1 t, and reacted for 50'0124 hours.

As a result, DDA of 85η/dt was generated.

(Recovery rate 18%) After sterilizing this solution by centrifugation (7000, 9, 40 minutes), add 500% activated carbon (white sugi charcoal, Shikinichi Yakuhin Kogyo) to the sterilizing solution.
After adding 9, protein removal, and decolorization (50° (:!, 1 hr),
It was filtered (pore size 0.45 μm filter). The two liquids were concentrated to a concentration of 15 and then filtered (No. 5 CP paper).

This DDA concentrate 13 R1 (DDA 6.29/d
t) to non-polar porous synthetic adsorption resin 5P207 (Mitsubishi Chemical Industries) 65d (φxL=20mx210xi+)
After feeding with V=1, I performed Suishin for 260d (SV=2
), referred to as C fraction-1) Next, eluted with 390 ml of 10% ethyl alcohol-aqueous solution (SV = 2), referred to as C fraction-2) Finally, 2 (1 ethyl alcohol-aqueous solution 3901 n/ (SV=2), designated as C fraction-3).

Both portions were 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, with recovery rates of 99% and 98% for Ad and DDU, respectively. ,
Fraction-3 contained DDA, and the recovery rate was 95%.

Fraction-3 was concentrated, DDA was crystallized, and then cooled to 10° C. to obtain highly pure DDA 6009. Obtained DD
The elemental analysis values of A are shown in Table-1.

Table-1 Elemental analysis value CHN O Theoretical value 51.06 5.57 29.77 13.60
Measured value 51.22 5.53 29.78 13.4
7 Example 2 DDA concentrate 13a obtained by processing in the same manner as in Example 1
l (DDA 6.2.9/dt) in 65 ml (φ
×L=201tIIx 210x ) K SV =1
After 74 days, water was run at 8 V = 2 for 260 hours (referred to as fraction-1).Next, it was eluted with 520 mJ of a 20% methyl alcohol-aqueous solution (sv = 2), and the C fraction was designated as fraction-2. Finally, add 390 ml of 40-thimethylalcohol aqueous solution.
9 (SV=2), designated as C fraction-3).

Each fraction was determined by liquid chromatography analysis. As a result, only U was detected in both fractions-1, and the recovery rate was 9.
Fraction-2 contains Ad, DDU, and some DDA, and the recovery rate of Ad and DDU is 98%, respectively.
98%, Ryobu-3 contains DDA, recovery rate 93
It was Chi. After concentrating fraction-3 and crystallizing ODA, 10
It was cooled to ℃ and high-purity DDA5809 was collected. The elemental analysis values of the obtained DDi are shown in Table-2.

Table-2 Elemental analysis value CHN O Theoretical value 51.06 5.57 29.77 13.6
0 measurement value 51.30 5.53 29.80 13.
37 [Effects of the Invention] As described above, the present invention provides DDA by non-polar resin treatment.
Since it can be efficiently separated and purified, there are great expectations that it will lead to industrialization.

Claims (4)

[Claims] (1) 2', 3 produced by the action of microorganisms or enzymes
When purifying '-dideoxyadenosine, 2',3
1. A method for purifying 2',3'-dideoxyadenosine, which comprises adsorbing '-dideoxyadenosine onto a non-polar porous resin. (2) The production reaction of 2',3'-dideoxyadenosine is one in which a 2,3-dideoxyribose residue and an adenine residue are combined by the action of a microorganism or an enzyme. Method. (3) The method according to claim (1), wherein the product 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.