JPS5952000B2 - Novel physiologically active substance 3-deoxyafuidicorin and its production method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel physiologically active substance 3-deoxyaphidicolin having specific inhibitory activity on eukaryotic DNA polymerase α and a method for producing the same.

3-deoxyaphidicolin, which is the target substance of this invention, is represented by the following structural formula (I).

(I) The microorganism used to produce 3-deoxyaphuideicolin is a microorganism commonly known as sugar beet and soybean fungus, Phomabetae.
Examples include strains belonging to.

The representative strain is Homa Bethe Frank PS-1
3 (PhomabetaeFrankPS-13) and has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology (Feibokuken Bibori No. 6556). The present invention relates to a strain of microorganisms belonging to Homa Bethe isolated from the natural world and having the ability to produce 3-deoxyaphuideicolin,
Irradiation treatment with ultraviolet rays, X-rays, radiation, etc., N-methyl-N'
-Nitro N-Nitronguanidine (NTG), 2-aminopurine, and other chemical agents are used for mutation treatment, conjugation, and other commonly used mutation treatment methods to increase the production capacity are included. The production of 3-deoxyaphuideicolin, which is carried out by culturing 3-deoxyaphidicolin-producing bacteria belonging to Homa Bethe in a nutrient medium, is carried out in principle according to the culture method of general microorganisms, but usually is cultured in a liquid medium.

As the medium used for culture, a natural medium, a semi-synthetic medium, or a synthetic medium is used. The medium composition is 1
Potato decoction, starch, dextrin, glucose, shaken sugar, glycerin, etc. are used as carbon sources.
Nitrogen sources include peptone, meat scratches, yeast scratches, casein hydrolyzate, corn steep liquor, gnoretenmeal, cornmeal, soybean flour, cottonseed meal, dried yeast, ammonium sulfate, ammonium nitrate, urea, etc. used. In addition, calcium carbonate, metal salts,
Hydrogen dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium chloride, etc. are added as appropriate. Culture temperature is 20-30℃
If the before or after is appropriate and the culture volume is large, a method is used in which a seed culture is performed as appropriate and then transplanted to the main culture.

The culture period for main culture varies depending on the medium conditions, culture format, etc., and for example, 7 to 15 days for static culture, and 50 days for aerated agitation culture. The culture is carried out for approximately 200 hours, and importantly, until a substantial amount of the desired 3-deoxyaphidicolin is produced in the medium. The culture conditions described above are applied by selecting optimal conditions depending on the characteristics of the bacterial strain used.

Since 3-deoxyaphidicolin accumulated in the culture by the above method is mainly contained in the filtration process, after removing the bacterial cells from the culture solution, the filtrate is subjected to known separation and purification methods as appropriate. They are isolated and collected in selected combinations, an example of which is as follows. That is, after adding glacial acetic acid to the culture filtrate to make the width 5.5 to 6.5, it is concentrated under reduced pressure, and an organic solvent such as ethyl acetate that dissolves the target substance 3-deoxyaphidicolin is added thereto. Extract. The extract (ethyl acetate layer) obtained here is concentrated under reduced pressure and subjected to chromatography using an adsorbent such as silica gel. When silica gel is used as the adsorbent, when elution is performed using a mixed solvent system of chloroform and methanol as the elution solvent, the target substance is eluted in the fraction portion of the elution solvent. In this way, the target substance 3-deoxyaphidicolin is isolated and collected from the culture solution. Detection and quantification of the produced 3-deoxyafuidicorin was carried out using silica gel thin layer chromatography (silica gel 60F25 manufactured by Talc, thickness 0.2 mm, developing solvent: chloroformrimethanol, 85:15, V/ V, color former; 2,4-DNP reagent,
heating).

Regarding the target substance 3-deoxyafuidicorin Shape of the substance and white needle-like crystals (2) Analysis values (from FD-mass spectrum and millimas) C2OH34O3 (3) Molecular weight (FD-mass spectrum) (4) Melting point 138. 0 to 140.5°C (5) Infrared absorption spectrum IR: ゛Rcm-” 3350, 2930, 2850,
1440, 1220, 1030, 750 (6) Nuclear magnetic resonance spectrum ▲ ゜r, .噌. , -K^8 ^轡一 /HaTT
^, Engineering↓! (7) Solubility Soluble in methanol, ethanol, chloroform, ethyl acetate, insoluble in n-hexane and water.

(8) Color reaction Represents pick color on silica gel thin layer chromatography due to anisaldehyde.

Based on the above-mentioned physical and chemical properties and the results of separate research, the structure of the target substance 3-deoxyaphidicolin is as follows.

D
The inhibitory activity of NA polymerase α is also being recognized, and although the mechanism of inhibition has not been elucidated in detail, it is regarded as important as a new physiologically active substance.

Next, examples of the present invention will be shown.

Example 1 Homa Bethe Franta strain PS-13 (PhOmab
etaeFrankPS-13; Microtechnical Research Institute No. 6556
No.) was inoculated into the preculture medium, and then cultured at 25°C for 10 days.

The preculture medium used here is as follows. Cut 200 g of peeled potatoes into 1 cm cubes, add about 11 g of tap water, heat treat in an autoclave (1 kg/Cm2, lO min), strain the decoction through 4 pieces of gauze,
get.

Add 20g of shootalose and 20g of powdered agar to this,
The mixture is further heated in an autotable for 5 minutes to dissolve it. Next, 150 ml of the precultured inoculum was inoculated into a sterilized main culture medium per 500 ml flask using a platinum loop, and static culture was performed at 25° C. for 15 days.

The main culture medium used here is a medium obtained by removing powdered agar from the pre-culture medium. After completing the culture, filter and collect the sap 13.
51 was collected and the IfI was adjusted to 5.5-6.5 by adding glacial acetic acid.

This adjusted solution was concentrated to about 1/6 under reduced pressure at a temperature of 40° C. or lower. To this concentrated solution, 1.51 ml of ethyl acetate was added and extracted with stirring, and the ethyl acetate layer was separated. The ethyl acetate layer thus obtained was concentrated to dryness under reduced pressure to obtain 4.5 g of crude material. 4.5 g of this crude material was placed on a silica gel column (Wako Gel C-200 manufactured by Wako Pure Chemical Industries, Ltd., 250 g) packed with chloroform and developed with taloloform, and then chloroformrimethanol (
Developed with a mixed solvent of 98:2, VN), fractionated the eluate into 100ml portions per fraction, and performed appropriate silica gel thin layer chromatography (developed with the solvent chloroformrimethanol (85:15, /V)) Rf value 0.55 at
The fraction (Cz) containing the target substance detected and confirmed at the position of ~0.60 was collected and concentrated to dryness under reduced pressure to obtain powder 211 containing 3-deoxyaphidicoline.
mg was obtained.

Claims (1)

[Claims] 1. 3-deoxyaphidicoline represented by the structural formula (I) ▲ Numerical formulas, chemical formulas, tables, etc. are available▼. 2. A method for producing 3-deoxyaphidicolin, which comprises culturing a 3-deoxyaphidicolin-producing strain of Homa Bethe in a nutrient medium, and separating and collecting 3-deoxyaphidicolin from the resulting culture. . 3. The method according to claim 2, wherein Homa Bethe is designated as Kaikoken Bibori No. 6556.