JPH0987268A - Purification of zopfielin - Google Patents

Abstract

PROBLEM TO BE SOLVED: To purify zopfielin useful as a germicide or an antithrombotic agent, etc., by making the compound basic and absorbing it into an anion exchange resin. SOLUTION: A solution containing (A) a compound represented by the formula or (B) a compound having a structure of a dicarboxylic acid or tetracarboxylic acid obtained by opening one or two rings of diketone furan structure which the component A has is made basic and absorbed into an anionic exchange resin and the component A or the component B is eluted with a solution containing an acid or inorganic salt from the resin and the component A is deposited by making the eluate acidic and purified. Furthermore, a culture medium, etc., obtained by culturing a microorganism belonging to the genus zophielin, e.g. Zopfiella curvata (FERM P-13067) is preferably used as the solution containing the component A or the component B.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for purifying zofierin useful as a bactericidal agent or an antithrombotic agent.

[0002]

2. Description of the Related Art Formula (I):

Embedded image Compound represented by (Zofierin) is Zofiera (Zo
It is a substance produced by a microorganism belonging to the genus pfiella ) and is known to have antibacterial activity and activity as an antithrombotic agent (JP-A-6-184157).

As a method for isolating zofierin, a culture solution obtained by culturing a microorganism belonging to the genus Zophieella is filtered, the obtained culture filtrate is acidified and then extracted with ethyl acetate, and the extract is subjected to silica gel chromatography. And a method of purifying using gel filtration chromatography and further preparative purification using high performance liquid chromatography is known.

However, this method is industrially disadvantageous because it uses multi-step chromatography including silica gel chromatography and uses a large amount of organic solvents such as ethyl acetate and benzene. Also,
Zophieline was only obtained as a white powder by this method, and a sufficient purity has not been secured so far.

On the other hand, zofierin has a diketofuran structure of 1 or 2 depending on the extraction conditions.
It is known that a compound having a ring-opened dicarboxylic acid or tetracarboxylic acid structure can be obtained (JP-A-6-184157).

However, it is not known under what conditions the ring-opened product can become zofierin.

[0007]

An object of the present invention is to provide a method for efficiently purifying zofierin, which is useful as a bactericide or antithrombotic agent, on an industrial scale.

[0008]

According to the present invention, by utilizing the fact that zofierin is easily converted into a ring-opened product under basic conditions and the ring-opened product is converted to zofierin under acidic conditions, respectively, A method for purifying zofierin using an anion exchange resin is provided.

That is, according to the present invention, a solution containing zofierin or a ring-opened product is made basic and adsorbed on an anion exchange resin, and the zofierin or ring-opened product is eluted from the resin with an aqueous solution containing an acid or an inorganic salt and eluted. Provided is a method for purifying zofierin, which comprises precipitating zofierin by acidifying a liquid.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION As a solution containing zofierin or a ring-opened product used in the present invention, a microorganism belonging to the genus Zophieella, for example, Zopfiella carba
curvata ) No.37-3 strain (FERM P-13067)
A culture solution (Japanese Patent Laid-Open No. 184157/1994) obtained by cultivating the above is mentioned.

When the solution used in the present invention is a culture solution obtained by culturing a microorganism belonging to the genus Zophieella, it may be used as it is, but the culture solution is subjected to centrifugation or filtration to cultivate the bacterial cells. You may separate it into a filtrate and use each. When using bacterial cells,
Alternatively, the cells may be crushed with an ultrasonic wave, a ball mill, a homogenizer or the like, and further, an organic solvent such as methanol, ethanol, propanol, butanol, acetone, ethyl acetate, acetonitrile, chloroform (hereinafter, also simply referred to as an organic solvent. ), And may be used as a solvent extract obtained by extraction. When using the culture filtrate,
The culture filtrate may be used as it is or after being concentrated.

The solution used in the present invention is adjusted to be basic, preferably at a pH of 8 to 14 with sodium hydroxide, potassium hydroxide, aqueous ammonia, etc., and then converted to an anion exchange resin, preferably a strongly basic anion exchange resin. Adsorb. The conversion reaction from zofierin to a ring-opened product is usually completed in 4 hours in an aqueous solution of pH 8 at a reaction temperature of 30 ° C.

As the anion exchange resin, commercially available products such as DIAION PA304, PA308,
PA412, (Made by Mitsubishi Chemical Corporation), AMBERLI
TEIRA-400, IRA-904, A-27 (above, manufactured by Rohm and Haas), DOWEX 1,
2, 11 (above, Dow Chemical Co.), DUOLITE
ES-109, A-101D (above, manufactured by Duolite) and the like are used.

In order to adsorb the ring-opened compound contained in the solution adjusted to be basic to the anion exchange resin, the solution may be simply mixed with the anion exchange resin, or the anion exchange resin may be filled. SV0.1 ~ 3
The liquid may be passed at a flow rate of.

Without washing the anion exchange resin adsorbing the ring-opened product, or water or an organic solvent containing water (hereinafter,
After washing with water containing organic solvent), water containing an acid or an inorganic salt in the anion exchange resin or a water containing organic solvent containing an acid or an inorganic salt (hereinafter referred to as an eluate) is SV.
The solution is passed through at a flow rate of 0.1 to 3 to elute zofierin or the ring-opened product.

As the acid, hydrochloric acid, sulfuric acid, nitric acid or the like is used, and it is added to the eluate so as to be acidic, preferably pH 1-4.

As the inorganic salt, sodium, potassium,
Hydrochlorides, sulfates, nitrates, carbonates, bicarbonates and the like composed of calcium and ammonium are used, and the salt concentration is 1 to 5
It is added to the eluate so that it becomes mol / L.

The eluate containing zofierin or the ring-opened product is used as it is, or after the organic solvent contained in the eluate is distilled off, the pH is adjusted to acidic, preferably pH 1 to 4, so that zofierin is precipitated as crystals or precipitates. Come on. In addition, the conversion reaction from the ring-opened form to zofierin is
When the reaction temperature is 30 ° C in an aqueous solution of H2, the reaction is usually completed in 2 hours.

The precipitated zofierin can be obtained as crystals or powder by usual operations such as centrifugation, filtration, washing and drying.

[0020]

【Example】

Example 1 A liquid medium (pH) containing Zofierella carbata No. 37-3 strain containing a potato dextrose medium (manufactured by Difco)
7.0), and cultured at 25 ° C. for 5 days. The obtained culture broth was added to potato dextrose medium to obtain polypeptone (0.1%).
1%, yeast extract 0.1%, tomato juice (added salt, manufactured by Kagome Co., Ltd.) 10% (W / W) was added to the medium (pH 7.0) and inoculated to the medium (140 rpm, 25 ° C, 12).
Shaking culture was performed for one day.

After the completion of the culture, the same amount of acetone was added to 14 L of the culture solution, and the mixture was stirred for 1 hour and filtered to obtain a filtrate. The filtrate is 350 ml of adsorption resin SP-207 (manufactured by Mitsubishi Chemical Corporation).
It was adsorbed on and washed with 1050 ml of 60% acetone, and then eluted with 70% acetone. After the eluate was evaporated to dryness, 100 ml of methanol was added and dissolved. 40 ml of water was added to the solution, and the solution was stirred at room temperature for 4 hours, collected by a separator and dried to obtain high performance liquid chromatography (hereinafter, referred to as HPLC) purity 90.
5.7 g of% zofierin powder was obtained.

5 g of the obtained zofierin was added to 500 ml of water.
After suspension in, adjust to pH 10 with sodium hydroxide,
Stir for 4 hours. The obtained solution was analyzed by HPLC
And thin layer chromatography (hereinafter referred to as TLC)
When analyzed with the following conditions, only the ring-opened product was detected in the solution. HPLC column: GL-pack nucleosil 5C18 ID 4.6mm × 150mm
(GL Science) Mobile phase: 0.05 M phosphate buffer (pH 6) / acetonitrile = 4/6 Column temperature: 35 ° C. Flow rate: 1.0 ml / L Detection: UV254 nm Retention time: Zophieline; 9 minutes, ring opening Body; 1.7 minutes TLC plate: Merck HPTLC F ₂₅₄ developing phase: toluene: acetone: acetic acid = 36: 1: 1 Rf value: Zophieline; 0.5, ring-opened body; 0.1 Next, the solution was treated with DIAION. A column (φ3 cm × 18 cm) packed with PA412 was passed at a flow rate of 125 ml / h for adsorption, 250 ml of water was passed therethrough to remove unadsorbed substances, and then 1M sodium sulfate was passed for elution.
625 ml of the eluate was obtained as a ring-opened body fraction. This fraction was adjusted to pH 2 with 12N hydrochloric acid, and the resulting crystals were collected with a basket separator and dried to give HP.
4.5 g of crystals having an LC purity of 95% were obtained. The obtained crystals were analyzed by ¹ H-NMR and ¹³ C-NMR.

¹ H-NMR (400 MHz, CDCl ₃ ) δ; 0.93 (3 H,
t, J = 7.1Hz), 0.96 (3H, t, J = 7.1Hz), 1.36-1.44 (8H, m),
1.65-1.70 (3H, m), 1.74 (1H, d, J = 4.1Hz), 1.83 (1H, m),
2.88-3.10 (4H, m), 3.13 (1H, dd, J = 15.4,14.9Hz), 3.29 (1
H, t, J = 13.0Hz), 3.99 (1H, m) ¹³ C-NMR (100MHz, CDCl ₃ ) σ; 13.8, 14.0, 18.5, 22.
4, 22.5, 25.0, 28.2, 28.5, 34.2, 34.9, 36.6, 41.7,
71.7, 141.0, 143.4, 145.1, 147.3, 165.0, 165.1, 1
65.4, 165.5 From the above results, it was confirmed that the obtained crystal was zofierin.

Example 2 10 L of the culture broth obtained in the same manner as in Example 1 was adjusted to pH 8 with sodium hydroxide and stirred for 4 hours.
To this culture solution, 250 ml of DIAION PA412 was added, and after stirring for 5 hours, only DIAION PA412 was collected at a recovery rate of 95%, and the column (φ3.9 cm × 20 c
m). 500 ml of water was passed through the column to remove unadsorbed substances, and then 1N hydrochloric acid-methanol was passed for elution. Eluate (1.5 L) was obtained as a zofierin fraction, and an equal amount of water was added to this fraction. After collecting the generated crystals with a basket separator, by drying,
4.5 g of zofierin crystals with an HPLC purity of 95% were obtained. The recovery rate was 81%.

Example 3 10 L of the culture solution obtained by the same method as in Example 1 was used to separate the cells, and the cells were dried. 160g of this dry cell
Is suspended in water and the suspension is adjusted to pH with sodium hydroxide.
Adjusted to 10. The solution obtained by filtering the obtained solution was used as a column packed with DIAIONPA412 (φ3 cm
(* 18 cm) at a flow rate of 125 ml / h to adsorb the ring-opened product. After 250 ml of water was passed through the column to remove unadsorbed substances, 1 M sodium chloride was passed for elution to obtain 875 ml of a ring-opened body fraction. This fraction 12
After adjusting the pH to 2 with N hydrochloric acid and collecting the resulting crystals with a basket separator, drying the crystals
5 g of crystals of zofierin with a purity of 95% were obtained. The recovery rate was 88%.

Example 4 1000 L of the culture solution obtained in the same manner as in Example 1 was adjusted to pH 2 with sulfuric acid, stirred at 70 ° C. for 1 hour, and then collected by filtration. Suspend the collected cells in 300 L of water, adjust the suspension to pH 8 with sodium hydroxide,
Stir for 4 hours. This suspension was filtered to obtain 500 L of the filtrate containing the ring-opened product. The obtained filtrate is treated with DIAION.
Column packed with PA412 (φ22cm × 53cm)
The ring-opened product was adsorbed by passing the solution at a flow rate of 20 L / h. After passing 140 L of water through the column to remove unadsorbed substances, 1 M
Elution was carried out by passing ammonium chloride, and 140 L of a ring-opened product fraction was obtained. This fraction was adjusted to pH 2 with 12N hydrochloric acid.
The resulting crystals were collected by a basket separator and dried to obtain 489 g of crystals of zofierin having an HPLC purity of 95%. The recovery rate was 78%.

[0027]

Industrial Applicability According to the present invention, an efficient purification method of zofierin on an industrial scale is provided.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location A61K 31/34 ADZ A61K 31/34 ADZ C12P 17/18 C12P 17/18 D (C12P 17/18 C12R 1: 645) (72) Inventor Satoshi Kinoshita 2-2-306 Kyowa Town, Hofu City, Yamaguchi Prefecture

Claims (1)

[Claims] 1. Formula (I): A solution containing a compound represented by (hereinafter, referred to as zofierin) or a compound having a dicarboxylic acid or tetracarboxylic acid structure in which one or two diketofuran structures of zofierin are ring-opened (hereinafter, referred to as ring-opened body) A method for purifying zofierin, which comprises adsorbing a basic substance on an anion exchange resin, eluting zofierin or a ring-opened product from the resin with an aqueous solution containing an acid or an inorganic salt, and acidifying the eluate to precipitate zofierin.