JPS6143182A - Novel antibiotic ss19508d and preparation thereof - Google Patents

Abstract

NEW MATERIAL:An antibiotic SS19508D. Elementary analysis; C, 55.95; H, 3.50 (experimental values); C, 55,98; H.3.59 (theoretical values). Mass spectrum (EI70eV); M<+>m/z364. Molecular formula; C17H13O7Cl. Melting point: 193-196 deg.C. Specific rotary power {alpha}D<25>=+145 deg. (C=0.30, acetone), etc. USE:An antibiotic having improved antimicrobial activity against Grampositive bacteria and part of Gram-negative bacteria and trichophytons. PREPARATION:A strain having the ability to produce antibiotic SS19508D, e.g. Aspergillus terreus S19508 (FERM-P No. 7636) and a variant strain thereof or natural variant strain, is aerobically cultivated in a culture medium containing a nutrient source by the usual mold cultivation method to give the aimed antibiotic SS19508D having the structure expressed by, e.g. the formula.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a novel antibiotic SS19508D and a method for producing the same.

[Conventional technology]

Conventionally, various antibiotics produced by microorganisms have been mentioned.

[Problem that the invention seeks to solve]

The object of the present invention is to obtain a novel antibiotic useful for medical use.

[Means for solving problems]

The present inventors isolated a large number of microorganisms from natural soil and conducted various studies on their products in order to find useful antibiotics. As a result, Aspergillus terreus, a microorganism belonging to the Aspergillus genus, was isolated from the soil of Mizuhara-cho, Kitakanbara-gun, Niigata Prefecture.
terreus) 819508 (Microtechnical Laboratory No. 7
636) in a culture medium, it was discovered that antibiotic 5819508B, which has excellent antibacterial activity against Durum-positive bacteria, some Durum-negative bacteria, and Shirogensi bacteria, was produced in the culture solution, and the patent was granted earlier. I filed an application (patent application 117-1982).
No. 255).

As a result of further research, the present inventors found that antibiotic 88 was added to the culture solution of Aspergillus terreus 819508.
SS195 is a new antibiotic with a different structure from 19508B.
The inventors discovered that 5819508D exists and completed the present invention. That is, the present invention provides a novel antibiotic 5819508D and a method for producing the same.

Aspergillus terreus strain 819508, which produces the antibiotic SS19508D of the present invention, has the following mycological properties.

(a) Characteristics of the colony) Is it normal for each culture medium at 25°C? The colony diameters on tetodextrose agar and Czapek agar were 2.2 Crn and 2.0 Crn, respectively.
cn1, the growth is suppressed, and the colony diameter on cornmeal agar medium is 3.1 cm, and the growth is rather rapid.

At 37℃? The colony diameters on tetodextrose agar and Czapek agar were 1 and 96Ra 2.4, respectively.
cm, and the colony diameter on cornmeal agar medium is 1.1 cm, which is extremely inhibiting growth.

(II) Growth Q-turn■? On tetodextrose agar medium at 25°C, the basal mycelial layer is rather thin and has shallow radial grooves. Pale brown to brown and velvety. Many conidial heads are formed and the secreted liquid is pale amber in color. The back side of the village is yellow and colored, 3
At 7°C, the basal hyphae +d rises high into a mountainous shape and has shallow radial grooves. Brown and woolly in color. A few conidial heads are formed and the secreted liquid is dark amber. The back side of the village is dark reddish-brown.

■ At 25°C on Czapek agar medium, the basal mycelial layer is somewhat thin and has a shallow radial pattern. Pale yellow to pale and velvety in color, conidial head formation is somewhat lacking, and the secreted fluid is amber in color. The surface of the fruit drop is yellow and colored. 37℃
In this case, the basal 11 filaments are flat with radial grooves, 8 pale yellow to pale brown, and conidial head formation is somewhat lacking.

The back of the collection v4 is yellow and colored.

■ On cornmeal agar medium at 25°C, the basal mycelial layer is extremely thin and flat, mostly colorless, with colored conidial heads scattered here and there, and the underside of the colony is also colorless.

(2) Physiological and ecological properties ■ Optimum growth temperature 24-35°C ■ Growth range In the above medium, p) l 2. It grows at a pH of 0 to 12.0, but the preferred growth range is 4 to 9.

Φ) Microscopic properties (conidial generation) ■ Conidial head cylindrical to oblong cylindrical, reddish-brown to bluish-purple, 50-150 x 40-60 μm in slide culture.

■ Conidiophore ioo~300X4~6μm Most of it is slightly bent, smooth surface,
Colorless, the tip enlarges to form an apical capsule.

■ The top has a hemispherical shape with a diameter of 12 to 16 μm, with metres appearing from the upper half, and those in the middle of growth have several metres and do not reach a hemispherical shape.

■ Metre Approximately 5-12 x 2-3 μm, colorless.

■ Fiallide 6-12 x 2-3 μm, colorless. One or two phialides are produced from metre.

■ Conidia phialide base conidia, long chains, 2-3 cotton diameter, spherical, smooth surface.

Based on the above-mentioned colonies and microscopic properties, this strain was identified as Aspergillus terreus.
) The strain identified as Aspergillus terreus 819508 (As
pergHlus terreus S 19508
) and has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as FEBMP-7636.

The scientific characteristics of Aspergillus terreus can be found in the following documents: Shun Ude-1 Keisuke Tsubaki et al.: Illustrated Encyclopedia of Mycology, Volume 2, pp. 1039-1040, 1978f- (Gdansha Suientific), C. Thorns M, B, Ch
urch: American Journal of
Botany, vol. 5, p. 85, 1918.

The antibiotic SS19508D of the present invention is produced by inoculating the above box strain into a nutrient-containing medium and culturing it aerobically. Antibiotic 5s19508D is the top 6 [
Not only the 5819508 strain, but also its artificial mutants, natural foods, and different strains can be used with the antibiotic SS19508D.
Any vehicle that has the steering force to produce can be used in the present invention. The artificial mutant strain 819508 can be easily turned blue by, for example, ultraviolet rays, cobalt-60 irradiation, or chemical mutagenic agents, like other molds.

Next, the cultivation of the Sengoku strain in the preparation method for the antibiotic SS19508D will be explained. That is, the culture aK of the antibiotic SS19508D fresh strain belonging to Aspergillus silk is prepared using a conventional mold culture method.

As the nutrient medium, any of limited extracts, synthetic media, semi-synthetic media, or natural media containing suitable carbon sources, nitrogen sources, inorganic substances, etc. that can be depleted can be used. As the carbon source, for example, glucose, dextrin, starch, lactose, sucrose, chrycerin, molasses, etc. are used alone or in combination. Furthermore, depending on the assimilation ability of the bacteria, hydrocarbons, alcohols, organic acids, etc. may also be used.
Nitrogen sources include inorganic or organic nitrogen compounds, such as ammonium chloride, ammonium sulfate, ammonium nitrate, urea, monosodium glutamate, etc., or natural products, such as soybean extract, defatted soybean, cottonseed meal, cornstap liquor, rice bran, etc. Yeast extract, pezoton, soyton, etc. may be used alone or in combination. Antibiotic SS1
When producing antibiotic 5819508p by culturing 9508D-producing bacteria, inorganic salts and metal salts are added if necessary. Antibiotic S helps the growth of other 819508 strains
A substance that promotes the production of S19508D or a general antifoaming agent such as silicone oil or Adekanol (trade name) can also be added to the medium as appropriate.

As the cultivation method, methods used in the production of general antibiotics are adopted, but commonly used aerobic culture methods, such as solid culture method, shaking culture method, and aerated agitation culture method, are preferably used. The aerated agitation culture method is most suitable. Cultivation is carried out under aerobic conditions, and the appropriate temperature for cultivation is 24-35°C.
However, it is generally preferable to culture at around 27°C.

The production amount of antibiotic 8819508D reaches its maximum after 3 to 5 days of culture in both shaking culture and aeration agitation culture. When the accumulated amount of antibiotic S 819508 D in the culture solution reaches a drop, the culture is stopped, and the target substance is isolated and purified from the culture solution. Antibiotic S from culture solution
Isolation of S19508D is carried out by using various methods alone or in appropriate combinations, taking into account the physicochemical properties of this antibiotic, as shown in Examples below.

That is, since the antibiotic SS19508D is mainly present in the normal culture solution F, after removing the bacterial cells from the culture solution by centrifugation or filtration, the normal separation means such as extraction treatment using various solvents, various ion exchange methods, etc. Antibiotic S can be extracted from the culture solution by appropriately combining chromatography methods, dull filtration methods using various carriers, absorption layer or partition column chromatography methods, dialysis methods, precipitation methods, etc., and by repeatedly using them.
Separate and purify S 19508D.

- Examples of preferred separation and purification methods include the following. After fermentation, the culture solution is centrifuged, and an appropriate solvent such as ethyl acetate is added to the supernatant for extraction.

The obtained extract is dehydrated using sodium sulfate or the like and distilled off under reduced pressure. The residue is washed with an insoluble solvent such as petroleum ether and subjected to a 4Jp silica glu column chromatography. Next, after elution with an appropriate eluent such as chloroform, the active fractions are collected, and the residue is recrystallized with an appropriate solvent such as Oka-chill after distillation under reduced pressure.
8D pale yellow crystals are obtained.

The antibiotic SS19508D obtained as described above has the following physicochemical properties.

<Physical and chemical properties> ■ Elemental analysis CH Experimental value (%) 55.95 3.50 Theoretical value (%) 5
5.98 3.59 ■ Mass spectrum (EI70eV) M+ m/z 364 ■ Molecular formula % Formula % [α] B-+145° (C = 0.30. Acetone) Infrared λ absorption spectrum (KBr method) Fig. 2 IH-B spectrum (90 MHz) Fig. 3 TMs in heavy chloroform bath solution was measured as a reference material.

(2) "C-1'4R spectrum (22.5 MHz) Measured using TMS in deuterium chloroform solution as a reference substance.

δ(pprn): 193.4 (8), 185.0
(8), 170.0 (s), 168.6 (g), 163
．． 3(B), 151.1(s), 149.5(s), 1
37.6(s), 137.2(d), 113.6(s)
, 108.4 (s), 106.0 (d), 10
4.2(d), 84.6(8), 56.8(q),
5z, 9(q), 22.0 ((1) 6.91 Solubility Soluble in chloroform, dimethyl sulfoxide, ethyl acetate. Insoluble in petroleum ether, n-hexane, water.

■ Distinguishing between basic, acidic, and neutral Weakly acidic @ Color and properties of the substance Slightly yellow crystals (recrystallized from ether) 0 Positive in color reaction ferric chloride reaction 0 Thin layer chromatography carrier Nijiri gel plate F254 (Merck (+'wa Structural formula From the above physical and chemical measurements, the antibiotic SS19 of the present invention
508D was determined to have the following structural formula.

[For production]

Biological Activity> Table 1 shows the minimum inhibitory concentration (MIC") of the anti-pressure substance SS19508D against various microorganisms.

Table 1 Next, an example will be given and explained.

Example potato starch 2.0%, Pharmamedia 2.0%,
Corn staple liquor 1.0%, calcium carbonate 0.
A liquid medium with a composition of 3% was adjusted to pH 7.0 and 500
m7! Dispense 100 microliters into each Sakaguchi flask and sterilize. This was inoculated with Aspergillus terreus strain 819508 (*Koken Bacteria No. 7636) and cultured with shaking at 27°C for 2 days to prepare a seed culture. A liquid medium 1.31 consisting of the same medium composition was placed in a 2.64-liter Zoya fermenter, inoculated with the above seed culture solution 13-, and the culture temperature was increased to 27.
℃, stirring number 35 Orpm, aeration rate 1.31! Culture is carried out for 96 hours under the condition of /min. Next, rotate the culture solution at 3000 rpm.
After centrifugation for 10 minutes at 'n', the resulting supernatant (900 yd) was extracted with 1.8 mm of ethyl acetate at 4°C, and this ethyl acetate layer was dehydrated with sodium sulfate, concentrated and evaporated to dryness. did. After washing this concentrate with a small amount of petroleum ether, it was washed with silica dal (Kiesel G manufactured by Merck & Co.).
el 60.230-400 mesh) Sprinkle on 5f,
A silica gel column (40f!tX500ml) which had been pre-blocked with chloroform was loaded and eluted with chloroform. First, antibiotic 8819508B was eluted, and it was well separated from antibiotic 581950B.
8D was eluted.

The antibiotic SS19508D fractions were collected, concentrated to dryness, and then dissolved in a small amount of ether and recrystallized to obtain antibiotic SS19508D as pale yellow crystals.
I got Mg.

[Brief explanation of the drawing]

Fig. 1 shows the ultraviolet absorption spectrum of the antibiotic SS19508B of the present invention, Fig. 2 shows its infrared absorption spectrum, and Fig. 3 shows the infrared absorption spectrum of the antibiotic SS19508B of the present invention.
Is the diagram the same IH-NVII? , is a spectrum. that's all

Claims (1)

[Claims] 1. Antibiotic SS1950 having the following physicochemical properties
8D. (1) Elemental analysis C H Experimental value (%) 55.95 3.50 Theoretical value (%) 55.98 3.59 (2) Mass spectrum (EI70eV) M^+m/z 364 (3) Molecular formula C_1_7H_1_3O_7Cl (4 ) Melting point: 193-196℃ (5) Specific optical rotation [α]^2^5_D=+145° (C=0.30, acetone) (6) Ultraviolet absorption spectrum Figure 1 λ^M^e^O^H_m_a_x ( ε) 283 nm (
16900) (7) Infrared absorption spectrum (KBr method) Figure 2 (8)^1H-NMR spectrum (90 MHz) Figure 3 Measurements were made using TMS in deuterium chloroform solution as a reference substance. (9)^1^3C-NMR spectrum (22.5MHz
) TMS in deuterated chloroform solution was used as a reference substance. δ (ppm): 193.4 (s), 185.0 (s),
170.0(s), 168.6(s), 163.3(s
), 151.1 (s), 149.5 (s), 137.6
(s), 137.2(d), 113.6(s), 108
．． 4(s), 106.0(d), 104.2(d), 8
4.6(s), 56.8(q), 52.9(q), 22
．． 0(q)(10) Solubility Soluble in chloroform, dimethyl sulfoxide, ethyl acetate. Insoluble in petroleum ether, n-hexane, and water. (11) Distinguishing between basic, acidic, and neutral Weakly acidic (12) Yellow crystals imitating the color and properties of substances (recrystallized from ether) (13) Positive in color reaction ferric chloride reaction (14) Thin layer chromatography Graphic carrier: Silica gel plate F_2_5_4 (manufactured by Merck & Co.) ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ 2. Claim 1 whose structure is represented by the following formula ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ Novel antibiotic SS19508D described in Section 1. 3. SS195, a novel antibiotic belonging to the Aspergillus genus
08D-producing bacteria were cultured, and antibiotic SS1 was obtained from the culture.
Novel antibiotic S characterized by collecting 9508D
Manufacturing method of S19508D. 4. The production method according to claim 3, wherein the SS19508D substance-producing bacterium is Aspergillus terreus S19508 strain.