JPH0539257A - Compound and its production - Google Patents

Abstract

PURPOSE:To obtain a new compound from a bacterium belonging to the genus Pseudomonas, useful as a compound for a medicine or agricultural chemical, having antimicrobial activity and antifungal activity. CONSTITUTION:A compound shown by formula I (R is H or SCH3), namely antibiotic T1, 801A shown by formula II and antibiotic T1, 801C shown by formula III. The compound shown by formula I, for example, is obtained by culturing SC-1,801 strain (FERM P-12,327) using various kinds of media containing a carbon source, a nitrogen source, inorganic substances, etc., at 28-33 deg.C at pH6-8 and by collecting from the culture mixture.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a compound and a method for producing the same.

[0002]

2. Description of the Related Art As a compound produced by a microorganism belonging to the genus Pseudomonas, pioluteolin (R. Takeda, J.
Am. Chem. Soc., 80 , 4749, 1958), pyrrole nitrin (K.Arima et al., Agri. Biol. Chem., 28 , 575, 196).
4), Pseudomonic acid (EB Chain et al.,
J. Chem. Soc., Perkin Trans. I, 294, 1977) and the like are known.

[0003]

Presently, as chemotherapeutic agents or pesticides, penicillin G, streptomycin, kanamycin, chloramphenicol, tetracycline, erythromycin, lincomycin, amphotericin B,
Although compounds such as kasugamycin and polyoxin have been used, development of more potent compounds has been demanded due to the problem of efficacy or emergence of resistant bacteria.

[0004]

[Means for Solving the Problems] The inventors of the present invention collected samples of various types of soils and searched for the compounds produced by the microorganisms separated therefrom, and found that the microorganisms belonging to the genus Pseudomonas To produce,
The present invention has been completed by discovering that the compound can be accumulated in the culture by culturing the microorganism in an appropriate nutrient medium and culture conditions. Hereinafter, the present invention will be described in detail. The present invention uses the formula 3

[Chemical 3](In the formula, R is H or SCH₃Represents ) Represented by
Compound (hereinafter, R 180 is a compound represented by H, T1801A,
R is SCH ₃The compound represented by is called T1801C
B), formula 4

[Chemical 4](In the formula, R is H or SCH₃Represents ) Represented by
Compound (hereinafter, R 180 is a compound represented by H, T1801B,
R is SCH ₃The compound represented by is called T1801D
), And the above compounds belonging to the genus Pseudomonas
By culturing the producer in a medium and accumulating the compound in the culture,
Is represented by the formulas 3 and 4 above.
A method for producing the compound is provided.

The microorganisms used in the production of the compounds represented by the chemical formulas 3 and 4 of the present invention are microorganisms belonging to the genus Pseudomonas and having the ability to produce the compounds represented by the chemical formulas 3 and 4. Either can be used.
An example of the microorganism is the bacterial strain SC-1801 isolated by the present inventors from soil in Shizuoka prefecture.

[0006] The SC-1801 strain is a strain belonging to the genus Pseudomonas, and has been deposited with the Institute of Microbial Technology, Institute of Industrial Technology. (Ministry of Microbiology, No. 12327, FERM P-
12327).

In the method of the present invention, the medium for culturing the microorganism producing the compounds represented by Chemical formulas 3 and 4 includes various carbon sources, nitrogen sources, inorganic substances and the like which are commonly used for culturing Pseudomonas microorganisms. Can be used. Carbon sources include glucose, maltose, starch, dextrin, glycerin, molasses and the like. Examples of the nitrogen source include peptone, yeast extract, meat extract, soybean powder, corn steep liquor, cottonseed powder, dry yeast, casamino acid, ammonium chloride, ammonium sulfate, ammonium nitrate, sodium nitrate, urea and the like. As inorganic substances, potassium, sodium, magnesium, iron,
Salts and phosphates such as manganese such as potassium chloride, sodium chloride, magnesium sulfate, ferrous sulfate, manganese sulfate, potassium phosphate, sodium phosphate and the like. Culturing is usually carried out aerobically, and aeration stirring culture is suitable. The culture temperature is 25 to 35 ° C, preferably 28 to 33 ° C, and the pH is preferably 6 to 8. The culturing time varies depending on various conditions, but usually the maximum amount of the compound accumulated in the culture reaches about 15 to 30 hours.

Chemical formula 3 and chemical formula 4 from the culture after the completion of the culture
The compound represented by can be collected by a known means for separating and purifying the compound from the culture of the microorganism, alone or in combination. For example, means for utilizing the difference in solubility with impurities, means for utilizing the difference in adsorption affinity of various adsorbents such as activated carbon, silica gel, alumina, macroporous nonionic resins, etc., means for removing impurities by ion exchange resins Etc. are used individually, in combination, or repeatedly.

The structures of the compounds of the present invention represented by Chemical formulas 3 and 4 were determined based on identification analysis, particularly spectroscopic examination. The physicochemical properties of the substance are as follows. 1. T1801A Formula 5

[Chemical 5] Appearance: Brown oil Molecular formula: C ₉ H ₁₂ O ₂ S 3 Elemental analysis: Actual value (%) C 43.45, H 5.01, S 38.
7 Calculated value (%) C 43.52, H 4.87, S 38.72 Molecular weight: 248 (FD-MS) High resolution mass spectrometry: Actual value (m / z) 247.9992 Calculated value (m / z) 247.9999 UV-visible absorption spectrum (nm, In isopropanol):
212 (sh), 240 (sh) 260 (sh), 333 Infrared absorption spectrum (cm ^-1 , KBr tablet method): 3396, 2924,
1558, 1418 1268, 1204, 974, 852 1H-NMR (δppm, CDCl ₃ ): 2.32 (S 3H), 2.36 (S 3
H), 2.43 (S 3H) 6.64 (S 1H), 6.83 (S 1H), 6.93 (S 1H) ¹³ C-NMR (δppm, CDCl ₃ ): 15.03 (q), 19.73 (q),
20.06 (q) 113.3 (d), 120.4 (s), 124.1 (s) 128.7 (s), 148.
0 (s), 151.5 (s) Color reaction: Positive iodine, ferric chloride-potassium ferricyanide Negative ninhydrin, Dragendrov, Erich, anisaldehyde TLC (silica gel): Rf 0.51 (toluene-ethyl acetate (50: 1)) HPLC: retention time 17.7 minutes (column: YMC-Pack AM-303
(4.6 × 250 mm), eluent: methanol-water (60:40), flow rate: 0.65 ml / min, detection wavelength: 220 nm) 2. T1801B Formulation 6

[Chemical 6] Appearance: Green powder Molecular formula: C ₉ H ₁₀ O ₂ S ₃ Elemental analysis: Actual value (%) C 44.51, H 5.02, S 37.
8 Calculated value (%) C 43.88, H 4.09, S 39.04 Molecular weight: 246 (FD-MS) High resolution mass spectrometry: Measured value (m / z) 245.9838 Calculated value (m / z) 245.9842 UV-visible absorption spectrum (nm, In isopropanol):
230, 377, 521 Infrared absorption spectrum (cm ^-1 , KBr tablet method): 3472, 2928,
1646, 1626 1500, 1264, 1080, 854 ¹ H-NMR (δppm, CDCl ₃ ): 2.29 (S 3H), 2.58 (S 3
H) 2.68 (S 3H), 6.27 (S 1H) 13 C-NMR (δppm, CDCl 3): 13.98 (q), 18.03 (q),
18.53 (q) 125.5 (d), 142.3 (s), 148.1 (s) 155.0 (s), 177.
2 (s), 177.8 (s) Color reaction: positive iodine negative ferric chloride-potassium ferricyanide, ninhydrin, dragendrov, Erich, anisaldehyde TLC (silica gel): Rf 0.41 (toluene-ethyl acetate (50: 1)) HPLC: retention time 34.0 minutes (column: YMC-Pack AM-30
3 (4.6 x 250 mm), eluent: methanol-water (60:40), flow rate: 0.65 ml / min, detection wavelength: 220 nm) 3. T1801C Formulation 7

[Chemical 7] Appearance: Yellow powder Molecular formula: C ₁₀ H ₁₄ O ₂ S ₄ Elemental analysis: Actual value (%) C 43.73, H 5.41, S 46.
3 Calculated value (%) C 40.79, H 4.79, S 43.55 Molecular weight: 294 (FD-MS) High resolution mass spectrometry: Measured value (m / z) 293.9853 Calculated value (m / z) 293.9876 UV-visible absorption spectrum (nm, In isopropanol):
234 (sh), 348 Infrared absorption spectrum (cm ^-1 , KBr tablet method): 3468,3344,
2924, 1380 1278, 1182, 976, 864 ¹ H-NMR (δppm, CDCl ₃ ): 2.44 (S 12H), 7.31 (S
2H) ¹³ C-NMR (δppm, CDCl ₃ ): 19.07 (q), 126.8 (s),
151.7 (s) Color reaction: Positive iodine, ferric chloride-potassium ferricyanide Negative ninhydrin, Dragendrov, Erich, anisaldehyde TLC (silica gel): Rf 0.48 (toluene-ethyl acetate (50: 1)) HPLC: retention time 38.5 Minute (column: YMC-Pack AM-30
3 (4.6 x 250 mm), eluent: methanol-water (60:40), flow rate: 0.65 ml / min, detection wavelength: 220 nm) 4. T1801D Formulation 8

[Chemical 8] Appearance: Yellow powder Molecular formula: C ₁₀ H ₁₂ O ₂ S ₄ Elemental analysis: Actual value (%) C 42.51, H 4.62, S 37.
6 Calculated value (%) C 41.07, H 4.14, S 43.85 Molecular weight: 292 (FD-MS) High resolution mass spectrometry: Measured value (m / z) 291.9724 Calculated value (m / z) 291.9719 UV-visible absorption spectrum (nm, In isopropanol):
392 Infrared absorption spectrum (cm ^-1 , KBr tablet method): 3488,2940,
1652, 1488 1418, 1224, 1128, 726 1H-NMR (δppm, CDCl ₃ ): 2.55 (S 12H) ¹³ C-NMR (δppm, CDCl ₃ ): 17.81 (q), 145.7 (s),
174.2 (s) Color reaction: positive iodine negative ferric chloride-potassium ferricyanide, ninhydrin, dragendrov, Erich, anisaldehyde TLC (silica gel): Rf 0.52 (toluene-ethyl acetate (50: 1)) HPLC: retention time 76.3 Minute (column: YMC-Pack AM-303
(4.6 x 250 mm), eluent: methanol-water (60:40), flow rate: 0.65 ml / min, detection wavelength: 220 nm)

[0010]

EXAMPLES Next, examples of the present invention will be shown, but these examples are merely examples and do not limit the present invention.

Example 1 One platinum loop of Pseudomonas sp. SC-1801 was placed in a large test tube (2.2 x 200 mm) in a liquid medium (peptone 1%, meat extract 0.3%, sodium chloride 0.5%, pH 7.0) 10 ml.
The cells were inoculated and cultured with shaking at 30 ° C for 18 hours. Of this culture
1 ml to liquid medium (Peptone 1 in a 500 ml Sakaguchi flask)
%, Meat extract 0.3%, sodium chloride 0.5%, pH 7.0
) 100 ml was inoculated and cultured at 30 ° C for 8 hours with shaking. 15 L of main culture medium in a 30 L jar fermenter (peptone 1%, yeast extract 0.5%, silicone oil)
KS-66 (Shin-Etsu Chemical) 0.03%, pH 7.0) was inoculated at a ratio of 1%, temperature 30 ° C, aeration rate 7.5 L / min, stirring speed 250rp
The cells were cultured under aeration and stirring for 18 hours under the condition of m 2. The obtained culture solution 14
L was separated into bacterial cells and culture supernatant by centrifugation, the culture supernatant fraction was extracted twice with 4 L of ethyl acetate, and a total of 8 L of the obtained extract was concentrated under reduced pressure. The concentrate was extracted with 100 ml of toluene, and the extract was added to the upper end of a silica gel column (preliminarily equilibrated with toluene). The column was first eluted with toluene then toluene-ethyl acetate (98: 2) and the active fractions were collected to give 75 mg of an oil. This was dissolved in 10 ml of hexane and preliminarily equilibrated with hexane-isopropanol (1000: 1) on a Rover column (LiChrop
repCN, size B, Merck) and eluted with the same solvent, and the active fraction was collected to obtain 40 mg of a crude substance. 25 this
Reversed-phase HPLC column (μBondasphere, 15 μ, 5 × 30 cm, Waters) dissolved in ml of methanol-water (60:40) and equilibrated with methanol-water (60:40) in advance.
And then eluted with the same solvent. At this time, the flow rate of the eluate was 50 ml / min, and the detection was ultraviolet absorption at 220 nm.
Elution fractions with retention times of 35, 68, 79, and 138 were collected, evaporated to dryness under reduced pressure, and T1801A and T1801 in the order of elution.
Purified products of B, T1801C and T1801D are each 5.
0, 1.5, 1.7 and 0.7 mg were obtained. The properties of each of the obtained purified products were in agreement with the physicochemical properties of each substance described above.

Example 2 Antibacterial and antifungal activity The minimum inhibitory concentration (MIC) against various microorganisms was measured by the agar plate dilution method. The results are shown in Table 1.

[Table 1]

[0013]

The compound of the present invention has antibacterial / antifungal activity and is used as a compound for pharmaceuticals or agricultural chemicals.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI technical display location (C12P 11/00 C12R 1:38) (72) Inventor Sannori Kato Kasuga, Konohana-ku, Osaka-shi, Osaka Ichinaka 3-chome No. 98 Sumitomo Chemical Co., Ltd.

Claims (4)

[Claims] 1. Formula 1 (In the formula, R represents H or SCH _3. ) 2. Formula 2 (In the formula, R represents H or SCH _3. ) 3. A microorganism belonging to the genus Pseudomonas and capable of producing the compound according to claim 1 or 2 is cultured, and these compounds are produced and accumulated in the culture,
Formula 1 or Formula 2 characterized by collecting this
A method for producing the compound of. 4. The method according to claim 3, wherein the microorganism capable of producing a compound is Pseudomonas sp. SC-1801.