JP2993033B2 - Production of polyene-based antibiotic - Google Patents

Abstract

PURPOSE:To readily and efficiently obtain the subject antibiotic useful for medical drug, etc., by irradiating light containing ultraviolet rays in long wavelength region to a solution of polyene-based antibiotic having cis-type polyene structure. CONSTITUTION:Light containing ultraviolet rays in long wavelength region is irradiated to a solution of polyene-based antibiotic having cis-type polyene structure to afford the aimed antibiotic. Besides, preferably ultraviolet rays in 300-400nm region are contained in said light and salts are contained in said solution in a concentration of >=5mM.

Description

Description: TECHNICAL FIELD The present invention relates to a method for efficiently producing a trans-polyene antibiotic by irradiation with light including ultraviolet rays. Since the substance obtained by the present production method has antifungal activity, it can be used for medicines, animal drugs and the like.

[Conventional technology]

Partlysine is known as a polyene antibiotic having a cis-polyene structure (J. Antibiotics, 35 , 9).
97 (1982)). Whether the three-dimensional structure of the polyene is trans or cis has already been determined at the stage when these antibiotics are produced in the fermentation broth by the microorganism, and it is very difficult to artificially convert this three-dimensional structure at present. However, it is known that when a dilute solution of a cis polyene antibiotic, partlysine, is irradiated with light, the ultraviolet maximum absorption wavelength shifts to a longer wavelength of 4 nm. This wavelength shift is thought to be due to the change of the cis partlysine to the trans form (US Pat. No. 3,773,925; issued Nov. 20, 1973).

[Problems to be solved by the invention]

Polyene is generally a substance that is very unstable to light irradiation.For example, as described in the above-mentioned literature, simply irradiating light to a partlysine solution does not cause decomposition of a trans-type compound that occurs simultaneously with conversion to a trans-type. It happens and eventually breaks it all down. Therefore, there is no known example in which a trans-type compound obtained by a photoreaction is isolated as a chemically pure substance. There is no example of using this for production as an industrially feasible method by light irradiation. It is desired to develop a method for easily and efficiently converting a cis-polyene antibiotic to a trans-polyene antibiotic.

[Means for solving the problem]

The present inventors have conducted intensive studies to solve the above problems, and as a result, by adding salts to a solution of a cis-polyene antibiotic, and irradiating this solution with ultraviolet light in a long wavelength region, a trans-polyene antibiotic was obtained. We found a way to convert it efficiently and based on this finding, completed the present invention. That is, the present invention provides a solution of a polyene-based antibiotic having a cis-type polyene structure containing salts with a wavelength of 30.
The present invention relates to a method for producing a polyene antibiotic having a trans-polyene structure, which is characterized by irradiating light containing ultraviolet light of 0 nm to 400 nm.

Partlysin B (J. Antibiotics, 35 (8), 997-1012) is a polyene antibiotic having a cis-polyene structure.
(1982), U.S. Pat. No. 3,773,925), and Partlysine B methyl ester (JP-B-55-50960). These antibiotics may contain impurities such as crude products as well as purified products.

Solvents for dissolving polyene antibiotics include hydrophilic organic solvents such as methanol, ethanol, acetonitrile, dimethylformamide, dimethylformamide, and dimethylsulfoxide, and water-containing organic solvents containing 30% or more of these, and contain about 5 to 30% of water. An aqueous alcohol solution, particularly an aqueous methanol solution, is preferred. Further, salts are added to the solution. The kind of the salt may be sodium chloride, sodium acetate, ammonium acetate or the like, and the kind is not limited. The salt concentration may be 5 mM or more, preferably 5 to 1000 mM, particularly preferably 10 mM to 200 mM. The concentration of the polyene antibiotic is suitably about 1 to 1000 mg /. When the solvent is a water-containing solvent, the pH is preferably 10 or less in order to reduce the decomposition of the generated trans-type polyene antibiotic, and particularly, pH 5.5 to 9
Is preferred.

The wavelength of the ultraviolet light used in the present invention is suitably from 300 to 400 nm. Further, since light of the longer wavelength side does not cause isomerization reaction, there is no problem in using it with a wavelength of 300 to 400 nm. On the other hand, light having a wavelength shorter than 300 nm decomposes polyene antibiotics, and is therefore preferably not used. The degradation product is considered to be a substance in which the polyene chromophore in which the polyene bond has been broken has been opened. The quantum yield due to irradiation near 300 nm is about 2 × 10 ^-2 . The quantum yield indicates the ratio of the reactive molecule to the absorbed photon (Photon).

As the light source, various light sources that can be industrially used are used. For example, natural light, daylight fluorescent lamp, cool white fluorescent lamp, near-ultraviolet lamp, long-wavelength ultraviolet lamp and the like can be used. Of these, a long-wavelength ultraviolet lamp that emits a single ultraviolet ray of 365 nm is particularly preferred. The irradiation time is determined so that conversion to the transformer type is sufficiently performed.

The trans-type compound can be purified by reverse-phase chromatography, which is a usual separation and purification method for polyene antibiotics. For example, capsule pack OD
S column (manufactured by Shiseido), LRP-1 (manufactured by Whatman,
Column chromatography using C-18 reverse phase chromatography carrier) or the like is effective.

[Action]

By irradiating the solution of the polyene antibiotic having cis-polyene production with light including ultraviolet rays in a long wavelength region, the cis-polyene antibiotic is excited to be isomerized to the trans-form.

〔Example〕

Example 1 1 kl of a medium having the composition shown in Table 1 was heat-sterilized and placed in a fermentation tank, and the partlysin B-producing bacterium Streptomyces.
Arene (Streptomyces arenae) (FERMP-8099, FERM B
P-1537), and cultured at 28 ° C. for 3 days.

After the culture, the mycelium was collected by centrifugation, and 100 methanol was added thereto. Adjust the pH to 6.0 with acetic acid and allow
Left for hours. Subsequently, the mixture was filtered, and the obtained filtrate 100 was added to 4
It was concentrated under reduced pressure to 10 at 0 ° C. The precipitate was collected by centrifugation, lyophilized, and 200 g of crude partricin B (purity 65
%).

100 g of crude partricin B was purified using reverse-phase partition HPLC (capsule-pack ODS column, manufactured by Shiseido Co., Ltd.) to obtain 80 g of a dried product of purified partricin B (98% purity). Examination of the physicochemical properties of the purified partricin B revealed a molecular weight of 1112.
(Measured by FABMS), thin layer chromatogram [PLKC18F TLC plate manufactured by Whatman, developing solvent: CH ₃ CN-50m
_{M NH 4 OAc (pH9.0) =} 45: in Rf value 0.34 to 55], HPLC
(Column: YMC AM-312 φ4.5 × 150mm, solvent: CH ₃ CN-50mM
NH ₄ OAc (pH 5.5) = 4.5: 5.5, flow rate: 1.00 ml / min], retention time 5.10 minutes, ultraviolet absorption (UV) spectrum (MeOH
1 and the infrared absorption (IR) spectrum were measured by pertricin B (J. Antibiotics,
35, 997, (1982)) and good agreement, gave a single spot by TLC, it gave a single peak by HPLC.

Purified partricin B prepared by the above method was dissolved in a 90% methanol solution (50 mM ammonium acetate was added, and the pH was adjusted to 5.8 with acetic acid) to a concentration of 40 mg /. This solution 1
Put the stainless bat so that the liquid depth is 1cm,
Long wavelength UV lamp (wavelength
Irradiation was performed for 20 minutes from a distance of 12.7 cm using ^two 365 nm, 8 W ultraviolet lamps and an intensity of 410 μw / cm ² . Irradiation liquid under reduced pressure 100m
The resulting precipitate was collected by centrifugation. The precipitate was washed twice with 25 ml of water and dissolved in a small amount of tetrahydrofuran solution. This was loaded on an LRP-1 reverse phase column, and acetonitrile: 0.05 M ammonium acetate (pH 9.0) 4.5 = 5.5
Eluted. The absorbance at 360 nm was measured, and the eluted fraction of the product (hereinafter, referred to as UVP-A) by ultraviolet irradiation was collected, concentrated under reduced pressure, and left in a refrigerator overnight.
The yellow crystals of UVP-A precipitated the next day were collected by centrifugation and dried to obtain 30 mg of a purified UVP-A product. The physicochemical properties of UVP-A are as follows.

(1) Elemental analysis value: C: 62.85%, H: 7.66% N: 2.55% (2) Molecular weight: 1112 (measured by FABMS) (3) Light intensity: ▲ [α] ²⁶ _D ▼: +335゜ (c = 0.1%, dimethyl sulfoxide) (4) UV spectrum: as shown in FIG. 1 (measured in MeOH, indicated by solid line) (5) Rf value: 0.26 [PLKC18F TLC plate manufactured by Whatman, developed Solvent: CH ₃ C
N-50mM, ammonium acetate (pH 9.0) 45:55] (6) HPLC retention time: 8.00 minutes [Column: YMC AM-312 φ4.5 × 150mm, solvent: CH ₃ CN-50mM]
NH ₄ OAc (pH 5.5) = 4.5: 5.5, flow rate: 1 ml / min] Table 2 shows a comparison of the physicochemical properties of pertricin B and UVP-A. As shown in the figure, the molecular weight is the same, the UV spectrum and the degree of light emission are different, indicating that UVP-A is a trans-isomer of partricin B. Comparing the ¹ H-NMR (FIGS. 2 and 3) of the two, there is a good agreement overall. A difference is observed between 5.5 and 6.5 ppm, which indicates a difference in steric structure between the two polyene structures.

As shown in Table 3, UVP-A had a stronger antibacterial activity than Partricin B, and had a minimum inhibitory concentration (MIC) against Candida albicans of 0.005 μg / ml.

Example 2 A purified product of partricin B was prepared in the same manner as in Example 1, and dissolved at a concentration of 40 mg /. Each one of this solution
Was irradiated with light of various wavelengths to isolate UVP-A, and the amount thereof was measured.

Example 3 UVP-A produced as a result of irradiating partlysin B with light using various light sources in the same manner as in Example 2 was as shown in Table 5.

Example 4 A methanol extract 1 of a cultured cell of Streptomyces allene obtained by the same method as in Example 1 (partlysin B5
0 mg) is irradiated with ultraviolet rays in the same manner as in Example 1,
When UVP-A was isolated from the irradiated solution, 20 mg of a purified product was obtained as a dry product.

Example 5 In the same manner as in Examples 1 and 2, 12.7 of a solution of pertoricin B (40 mg / solution) dissolved in various solvents was used by using two long-wavelength ultraviolet lamps (365 nm wavelength, 8 W ultraviolet lamp, intensity 410 μm / cm ² ). UVP-A produced by irradiating ultraviolet rays for 20 minutes from a distance of cm was isolated and its amount was measured.

Example 6 30 g of partricin B prepared in the same manner as in Example 1 was dissolved in 250 ml of dimethylsulfoxide, and 200 ml of a 2% ether solution of diazomethane was added at room temperature with slow stirring. The product was then left overnight in the dark, and ether was added to precipitate the product. The precipitate was collected by filtration, washed with ether and dried under vacuum. The obtained crude partricin B methyl ester was purified by the method described in JP-B-55-50960 to obtain 5 mg of a purified product as a dried product. This compound has the following physicochemical properties, and is characterized by Parthlysin B ethyl ester (JP-B-55).
-50960).

(1) Properties: yellow powder (2) Molecular weight: 1126 (measured by FABMS) (3) Molecular formula: C ₅₉ H ₈₆ N ₂ O ₁₉ (4) UV _max : 401, 378, 359, 340 nm Further infrared absorption spectrum , An absorption at 1715 cm ⁻¹ derived from C ＝ O of the methyl ester was observed, and ¹ H-NM
In R, a characteristic peak of 3.25 ppm of a methyl group due to the presence of an ester group was observed.

Example 7 5 mg of partlysine B methyl ester obtained in the same manner as in Example 6 was dissolved in 1 of a 90% aqueous methanol solution (50 mM ammonium acetate was added, and the pH was adjusted to 6.0 with acetic acid).
According to the method shown in Example 1, ultraviolet light of 365 nm was irradiated using a long wavelength ultraviolet lamp. The irradiated liquid was concentrated, and the resulting precipitate was collected by centrifugation. Next, Novavac C18 (a product of Waters) as a carrier and 65 as a mobile phase.
High-performance liquid chromatography (HPLC, flow rate 1.0 ml / mi) using 10 mM ammonium acetate buffer (pH 8.5) containing 5% acetonitrile and a UV (405 nm) detector as a detector.
Purified by n). The peak eluted at 7.5 minutes was collected and concentrated under vacuum, and the resulting precipitate was collected by centrifugation and dried. 1.5 mg of a dried product was obtained. The purified product (designated as UVP-B) had the following properties.

(1) Properties: yellow powder (2) Molecular weight: 1126 (measured by FABMS) (3) Molecular formula: C ₅₉ H ₈₆ N ₂ O ₁₉ (4) UV _max : 405, 382, 344 nm Part ricin B methyl ester Comparing the physicochemical properties of UVP-B, the molecular weight and molecular formula were the same, the UV peak absorption wavelength was different, and the particin B ethyl ester having a cis-type polyene structure changed to UVP-B having a trans-type polyene structure. Indicates a thing. UVP-B
The minimum inhibitory concentration (MIC) against Candida albicans ATCC 10231 was 0.39 μg / ml, and the activity was stronger than that of partricin B methyl ester (MIC = 0.52).

Example 8 In the same manner as in Examples 1 and 2, a long-wavelength ultraviolet lamp (365 nm wavelength) was added to 1 of 40 mg of partlysin B / 95% ethanol aqueous solution adjusted to pH 8 by adding 50 mM of various salts.
m, 8W UV lamp, intensity 410 μm / cm ² ) 12.
UVP-A generated by irradiating UV rays from a distance of 7 cm for 60 minutes
Was isolated and its amount was measured, as shown in Table 7.

Example 9 In the same manner as in Examples 1 and 2, ammonium acetate 50
95 mg of partlysin B adjusted to pH 8 by adding mM
% Ethanol aqueous solution in 1 ml by fluorescence spectrophotometer, 302,
UVP generated by irradiating light with wavelengths of 320, 365, and 405 nm for 30 minutes
-A was quantified. Quantification is performed by HPLC, and YMC
Use pack AM-312 (manufactured by Yamamura Chemical Laboratory Co., Ltd.) and use acetonitrile-50 mM ammonium acetate (pH
5.5) = 4.5 = 5.5 was used. Table 8 shows the quantum yield. The irradiation was carried out under the condition that partlysin B was converted to 100% UVP-A and the generated UVP-A was not decomposed.

[Effects of the Invention] According to the production method of the present invention, as a drug or animal drug, a transene that can be used as a drug or animal drug by a simple device and operation from a highly toxic polyene antibiotic having a cis-polyene structure. A polyene antibiotic having a polyene structure can be efficiently converted and produced in high yield.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the UV spectrum (solid line) of the trans isomer of partricin B, which is a polyene antibiotic having trans-polyene production obtained by the production method of the present invention. This is shown in comparison with the UV spectrum (broken line) of partlysin B, which is a polyene antibiotic having a polymorphic structure. FIG. 2 is a chart showing ¹ H-NMR of partlysin B. FIG. 3 is a chart showing ¹ H-NMR of UVP-A.

──────────────────────────────────────────────────続 き Continued on the front page (58) Field surveyed (Int.Cl. ⁶ , DB name) C07H 17/08 CAplus (STN)

Claims (5)

(57) [Claims] 1. A polyene antibiotic having a trans-polyene structure, comprising irradiating a solution of a polyene antibiotic having a cis-polyene structure containing salts with light having a wavelength of 300 to 400 nm. How to make a substance 2. The method according to claim 1, wherein the salt concentration of the solution containing salts is 5 mM or more. 3. The method according to claim 1, wherein the salts are ammonium acetate, potassium acetate,
The production method according to claim 1, wherein the method is selected from the group consisting of sodium acetate, sodium chloride and ammonium chloride. 4. A solvent for dissolving a polyene antibiotic, comprising hydrated methanol, hydrated ethanol, dimethylformamide,
2. The process according to claim 1, wherein the process is selected from the group consisting of dimethylacetamide and dimethylsulfoxide. 5. The method according to claim 1, wherein the polyene antibiotic having cis-polyene production is partricin B or partricin B methyl ester.