JPS599159B2 - New substance 477-2h and its manufacturing method - Google Patents

Description

[Detailed description of the invention] The present invention relates to novel substance 477-2.

The present invention also relates to culturing a microorganism belonging to the genus Micromonospora and having the ability to produce 477-2 in a nutrient medium, and culturing until 477-2 is detected in the medium, thereby producing and accumulating 477= 477-2 characterized by collecting 2.
Regarding the manufacturing method. It has been known that antibiotics such as dientamicin can be obtained from cultures of microorganisms of the genus Micromonospora. The present inventors have studied the metabolic products of microorganisms of the genus Micromonospora, and have discovered a compound in a culture of a certain bacterium that has antibacterial properties that are distinct from known substances. As a result of detailed examination of the physicochemical properties of this compound, it was discovered that it is a new substance having the following structural formula, and it was named 477-2. 2E1゜゛゜゛lNHCH.

NH2NH2 According to the present invention, 477-2h belongs to the genus Micromonospora, and a microorganism capable of producing 477-2h is cultured in a medium, the culture is continued until 477-2h is detected, and the microorganism is extracted from the culture. It can be obtained by isolation and purification using an appropriate method.

477-2h is a free basic substance which, by reaction with 1 to 4 equivalents of acid, forms mono-, di-, tri-, and tetra-salts such as hydrochloride, hydrobromide, hydroiodide, sulfate, phosphates, carbonates, nitrates, acetates, fumarates, malates,
It is converted into salts such as citrate, mandelate, ascorbate, tartrate, and succinate.

477-2h and its acid salts are useful as precursors in the production of antibiotics such as sagamycin, and have weak antibacterial activity against a wide range of Gram-positive and Gram-negative bacteria, making them useful for cleaning and sterilizing laboratory equipment. It is useful and can also be used in combination with detergents as a cleaning agent.

The physicochemical properties of the free base of 477-2h related to the present invention are as follows.

(1) Basic white powder (2) Elemental analysis value (3) Molecular weight 630 (4) Molecular formula C24H46N4O,, (5) Ultraviolet absorption Svectori The ultraviolet absorption spectrum of an aqueous solution of this substance is characteristic between 220 and 360 nm. It does not show a typical absorption maximum, but only shows terminal absorption.

(6) This substance is extremely soluble in water, soluble in methanol, and slightly soluble in ethanol and acetone, but organic solvents such as chloroform, benzene, ethyl acetate, butyl acetate, ether, butanol, petroleum ether, and n-hexane It is insoluble in

(7) Color reaction: Ninhydrin reaction: Positive Potassium permanganate reaction: Positive Biuretz reaction: Negative Sakaguchi reaction: Negative (8) The proton nuclear magnetic resonance spectrum of this substance in heavy water (PD9.8) has the following values. shows.

The unit is PpmOl. lO-1.50 (1H,.m), 1
．． 70-2.20 (1H..m), 2.52 (3H..m)
s), 2.60-3.10 (4H), 3.10-4.2
0(17H), 4.94(1H,.d,.J=3.4H
z), 5.03 (1H, .d, .J=3.9Hz), 5
．． 25 (1H,.d,.J=3.4Hz)9) The carbon nuclear magnetic resonance spectrum of this substance in heavy water (PD9.9) shows the following values.

The unit is Ppml lOl. 9, 100.6, 99.0, 88.8, 87.
6, 75.0, 74.6, 73.9, 72.7, 72.
3, 72.2, 70.8, 70.5, 70.4, 68.
3, 67.2, 62.9, 62.7, 61.3, 56.
From the physicochemical properties of 1, 51.5, 50.3, 36.4, and 33.9, the present compound is considered to have the following structural formula.

0) Silica gel thin layer chromatography of this substance using various developing agents [E. Kiesel Gel 6O made by Mark (
Perform at room temperature.

The Rf value of the sample (deployment time: 3 hours)] is shown below. For comparison, dientamicin A was selected and its Rf value is also listed. Both indicate capacity ratio. The microorganism used in the present invention belongs to the genus Micromonospora, and 477-2h
Any microorganism that has the ability to produce can be used.

A preferred species of bacteria is Micromonospora sagamiensis. A specific example of a preferred bacterium is Micromonospora sagamiensis G-477. This bacterium has been deposited at the Institute of Microbial Technology, Agency of Industrial Science and Technology, located in Inage City, Chiba Prefecture, and has been identified as Microbiological Research Institute No. 4459. The mycological properties of the species to which this bacterium belongs are published in Japanese Patent Publication No. 50-39155.
listed in the number. As a method for culturing microorganisms, a conventional method for culturing actinomycetes is used. A medium containing appropriate amounts of carbon source, nitrogen source, and inorganic salts is used as the medium. Carbon sources include glucose, starch, dextrin, mannose,
Flagdose, syrup, molasses, etc. are used alone or in combination. Inorganic and organic nitrogen sources include ammonium chloride, ammonium sulfate, urea, ammonium nitrate, sodium nitrate, etc. Natural nitrogen sources include peptone, meat factory scratches, yeast factory scratches, dried yeast, corn steep liquor, soy flour, Casamino acids, solupulpegitapuru protein, cottonseed gas, etc. are used alone or in combination. In addition, salt as needed,
In addition to appropriately adding inorganic salts such as potassium chloride, calcium carbonate, and phosphates, organic or inorganic substances that promote the growth of the bacteria used and the production of 477-2h can be added as appropriate. As a culture method, a liquid culture method, especially a deep agitation culture method, is suitable.

It is desirable to culture at a culture temperature of 25 to 40°C and a pH near neutral. When culture is carried out in liquid culture for usually 1 to 12 days, 477-2h is accumulated in the culture solution. When the amount of culture fluid produced reaches the maximum, the culture is stopped and the target product is purified and isolated from the culture fluid. 477- from culture solution
The 2-h purification and isolation utilizes separation and purification methods commonly used to isolate microbial metabolites from their culture fluids. Since No. 477-2h is a water-soluble, basic substance as described above, it can be purified by a method commonly used for purifying so-called water-soluble/basic antibiotics.

Namely, adsorption/desorption method using cation exchange resin, cellulose column chromatography, Sephadex LH-20
This can be carried out by appropriately combining methods such as adsorption/desorption using a column, silica gel chromatography, and carbon chromatography. It is also possible to isolate and purify the free base or sulfate salt of this substance. Next, an example of purification and isolation of 477-2h from a culture solution will be shown.

After culturing, solid matter was removed from the culture solution, and the resulting culture solution was adjusted to weak alkalinity. Then, cation exchange resin Amberlite IRC-50 (NH4+ type) (ROHM & Co., Ltd.) was added.
Pass the column through a column packed with HAAS Co., USA), elute with dilute ammonia water, and collect the eluate in an appropriate amount of fractions.

The fractions containing 477-2h were collected and concentrated to dryness under reduced pressure to obtain a crude white powder of 477-2h.

Next, this crude powder is purified by silica gel chromatography. Silica gel is suspended in n-butanol, ethanol, and concentrated aqueous ammonia (8:10:7), packed into a column, the coarse powder is placed on the column, and the column is eluted with a solvent of the same composition. The fractions of 477-2h were collected and concentrated to dryness, and the residue was dissolved in a small amount of water, adjusted to be slightly alkaline, and passed through a column packed with Amberlite IRC-50 (NH4+ type).

After washing with water, elute with 1N aqueous ammonia, collect fractions containing 477-2h, concentrate under reduced pressure, and freeze-dry to obtain purified 477-2h. As an example of using 477-2h in the production of Saga sewing machines, Micromonospora sagamiensis varitus nonreducans ATC
When producing Sagamisin using C2l949 in the same manner as in Example 3 of JP-A No. 51-38490, the yield of Sagamisin is increased by culturing in a fermentation medium containing 250 μy/ml of 477-2h. is twice as large as when 4772h is not used.

EXAMPLES The method for producing 477-2h of the present invention will be specifically explained below using Examples.

Example 1 A. Cultivation of Micromonospora sagamiensis G477: Micromonospora sagamiensis (Mi
crOnOnOspOrasagamiensis)G
-477 (Feikoken Bibori No. 4459) is used.

The 1st, 2nd, and 3rd types of media were Stapyrose K (manufactured by Matsutani Chemical Co., Ltd.) 2% (w/v) (the same applies hereinafter), glucose 0.5%, peptone 0.5%, yeast yeast scratch 0.5 %,
Meat processing scratches 0.3%, calcium carbonate 0.2% (before sterilization P
H8. Use the medium of O).

A platinum loopful of seed culture 1 is inoculated into 10 ml of the above medium in a large test tube, and cultured with shaking at 30°C for 3 days. This seed culture solution 10
m1 is inoculated into a second type medium of 350Tn1 placed in an Erlenmeyer flask with 21 bottles. The second type culture is cultured with shaking at 30°C for 2 days. This seed culture solution 1.5
1 (for 5 flasks) was inoculated into Type 3 medium 151 in a 301-capacity Jarfurmenta 1, and incubated at 34°C for 24 hours using an aeration stirring method (rotation speed 250 rpm, aeration rate 151/M).
Culture is carried out according to m). Finally, this third type culture solution 15.
Fermentation medium 1501 in a 3001 capacity tank is inoculated with the following. The fermentation medium composition was 4% Soucrose, 1% Soybeanmeal, and Pharmanedia (TradersOl).
lmilcOmpa U. S. A. ) 2%, cornmeal 1%, soybean casein 0.5%,
Dipotassium phosphate 0.025%, magnesium sulfate 0.0
5%, calcium phyto 0.2%, corn oil (C
OrnOil) 0.1%, ferrous sulfate 0.015%, L
-Glutamine 0.0001%, L-cystine 0.005
%, β-alanine 0.005%, nicotinic acid 0.000
5%, calcium pantothenate 0.0005%, zinc sulfate (7 hydrate) 0.0005%, potassium alum (24 hydrate) 0.001%, ammonium molybdate (4 hydrate) 0.025% (sterilization) Use a medium with a pH of 8.0.

This fermentation was carried out at 34℃ for 6 days using an aerated agitation culture method (rotation speed: 1
80 rpm, ventilation amount 1501/Mi! t).
B. 477-2h purification and isolation After the completion of the above culture, the pH of the culture solution was adjusted to PH2 with 12N monosulfuric acid.
．． The mixture was heated and stirred at 80°C for 10 minutes.

Thereafter, 2 kg of Radiolite #600 (manufactured by Showa Kagaku Kogyo Co., Ltd.) was added as a filter aid, and the bacterial cells were separated. This filtrate is passed through a column filled with Diaion HPK-25 (NH4+ type) (manufactured by Mitsubishi Kasei) 101, and the effluent is discarded.

After washing the resin with water, it was eluted with 2N aqueous ammonia, and 477
The fractions containing -2h are concentrated to 301 under reduced pressure. The concentrated solution was adjusted to pH 8. with 6N hydrochloric acid. After adjusting to O, it was passed through a column packed with Amberlite IRC-50 (NH4+ type) 41, washed with water, and eluted with 2N aqueous ammonia.
The fraction containing h is concentrated to dryness under reduced pressure. Dissolve the residue in water, pH 8. After adjusting to O, Amberlight CG-5
The column was passed through a column packed with 1000 mt of 0 type I (NH4+ type) (Rohm & Haas USA), washed with water, and eluted with 0.1N aqueous ammonia containing 0.2M ammonium chloride. After a few trace components are eluted first, a section containing 477-2h is eluted. The fractions were collected and adjusted to pH 7.8 with 6N monohydrochloric acid, passed through a column packed with Amberlite IRC-50 (NH4+ type) 200rfL1, washed with water, and eluted with 1N aqueous ammonia. The fraction containing 477-2h was concentrated and freeze-dried to yield 7.5 tons of powder.

40007 silica gel (WakOgelC-200,
Wako Pure Chemical Industries, Ltd.) was suspended in n-butanol, ethanol, and concentrated ammonia water (8:10:7) and packed into a column, and 7.5 f of coarse powder was suspended in a solvent of the same composition. eluted with a solvent of the same composition.

The elution fraction is separated into 500 ml portions at an elution rate of 66.7 ml/Mm. The fractions containing 477-2h were collected and concentrated to dryness. The residue was dissolved in a small amount of water and the pH was adjusted to 7.8, followed by Amberlite IRC-50 (NH4+ type) 100T.
Pass through a column packed with IL1, wash with water, elute with 1N aqueous ammonia, collect fractions containing 477-2h, concentrate under reduced pressure, and freeze-dry to obtain the free base of 477-2h.
3t of purified sample is obtained.

Example 2 477-2h free base obtained in Example 1 1r<10m
Dissolve in 1 water and neutralize with 0.5N sulfuric acid.

Claims (1)

[Claims] 1. A novel substance 477-2h represented by the formula ▲ Numerical formula, chemical formula, table, etc. ▼ and its acid addition salt. 2. A microorganism belonging to the genus Micromonospora and having the ability to produce 477-2h is cultured in a nutrient medium until 477-2h can be substantially detected, 477-2h is accumulated in the medium, and 477-2h is extracted from the culture solution. A method for producing 477-2h, which comprises isolating and recovering 2h.