JPS5965059A - Novel antibiotic substance neoviridogrisein-cl and its preparation - Google Patents

Abstract

NEW MATERIAL:The antibiotic substance neoviridogrisein-Cl-c and antibiotic substance neoviridogrisein-Cl-t of formula (c and t represent the steric isomers containing the amino acid residue derived from cis-chloroproline and trans- chloroproline, respectively). USE:Useful as a drug, animal drug and feed additive. PROCESS:A microbial strain belonging to Streptomyces genus and separated as a neoviridogrisein-producing strain (FERM-P No.3562 and ATCC 31289) or a strain devoid of prolylhydroxylase activity and obtained by the mutagenetic treatment of said microbial strain (e.g. Streptomyces sp. G-89), is cultured in a nutrient medium containing 4-chloroproline or a 4-chloroproline-containing material, to obtain the antibiotic substance of formula.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a new antibiotic neopyridoglyzein 7-C1 and a method for producing the same, and more particularly to the antibiotic neopyridoglyzein-C1 represented by the formula CH-CH3 1 CHl. -c and the antibiotic neopyridoglyzein-CI-t (c and t each represent a stereoisomer containing amino acid residues derived from cis-chloroproline and trans-chloroproline) and their production methods.

The present inventors first discovered the antibiotic neopyridoglyzein 1 represented by the following general formula (in the formula, R3 represents a methyl group or an ethyl group, and Peng represents a hydrogen atom, a hydroxyl group, or a methyl group). (compounds in which R1 is an ethyl group and horse is a hydrogen atom), 111 (compounds in which R1 is a methyl group and 8 is a hydrogen atom), 111 (compounds in which R is an ethyl group and 8 is a hydroxyl group), (a compound in which R+ is a methyl group and R2 is a hydroxyl group) and -MP (R,
is a methyl group and R2 is a methyl group) and their production methods (for example, Japanese Patent Publication No. 33-333
(See Publication No. 2 and Japanese Unexamined Patent Publication No. 57-6 ≠ Genpei Publication.)

The present inventors have discovered a further useful antibiotic neopyridoglysein (hereinafter simply referred to as "neopyridoglysein").
As a result of conducting research to find analogues, we found that when culturing known neopyridoglysein-producing bacteria, by adding and culturing a substance containing μm chloroproline or goochloroglolin to the nutrient medium, we found that the formula (1) ) The present invention has been completed by successfully producing a new compound that has not been published in any literature.

Neopyridoglysein provided by the present invention=C1
, -C and neopyridoglyzein-C1-t are stereoisomers, respectively, as described below, and both have almost the same antibacterial activity as the neopyridoglyzeins represented by the general formula (11). It is useful as an additive in medicine, veterinary medicine or feed.

Another invention provided by the present application is a method for producing neopyridoglyzein-CI-c and/or neopyridoglyzein-C1-t represented by formula (1). The method for producing the antibiotic involves adding a microorganism belonging to the genus Streptomyces that has the ability to produce the antibiotic to a nutrient medium known per se, and adding a substance containing chlorine or chloroglobulin. This can be carried out by culturing and collecting the antibiotic from the culture.

The microorganisms used in the practice of the present invention can be microorganisms belonging to any genus as long as they have the ability to produce antibiotics, but preferred are microorganisms that have the ability to produce antibiotics. Streptomyces was isolated as a neopyridoglysein producing bacterium.
+nyce++) sp, prgg, r' (Feikoken Bacterial Serial No. 3!t, 2 and ATCC 31 Cor.) or strains lacking cyclic prolyl hydroxylase activity obtained by mutation of said strains. I can do that. In addition, among the mutant strains, the present applicant has specifically identified
1: Ministry of International Trade and Industry, Agency of Industrial Science and Technology, Microorganisms, 1: Fan Technology (1)
) Streptomyces GI) and G-f strains deposited at the Bidoken Co., Ltd. No. 1 III-1 can be mentioned.

1. The orchidological properties of the strain are as follows.

This #;:ri strain III extends short colorless aerial hyphae from well-branched, simple-branched basal hyphae, forming a loose spiral spore chain at the tip. The surface of the spore is smooth. No formation of whorls and r-glue spores is observed.

The culture characteristics of this strain in each J1n medium are as follows.

(1) Growth in sucrose/nitrate agar medium: Slightly growing aerial hyphae Two white aerial hyphae are formed thinly here and there Substratum hyphae:
Colorless to grayish-white Soluble pigment: None Glue: Asparagine agar medium V'-
f・Good Aerobic hyphae: Rarely formed, and when formed, white medium hyphae (pale yellowish white to light yellow (,2d b-, 2rt)) Soluble pigment: None ■ Glycerin/asparagine agar Medium growth in medium: Aerial hyphae: Hardly formed, if formed, white base medium type:
Pale yellow to grayish yellow (2 db to 2 dc to 3 ec) Soluble pigment: None Growth on yeast malt agar medium; Good Hypha in aerobic mode: White to slightly grayish white Hypha in the base: Bright yellow (2 fb) However, in the late stage of cultivation, it becomes brownish gray (Jli).
).

Soluble pigment: absent or sometimes slightly brown.

■ Growth on starch agar medium; Moderate Aerial cocoon filament: Hardly formed, but if formed, white base medium hyphae: pale yellow (2 db), and the middle part of the colony is light grayish yellowish brown (3 ge). Village.

Soluble pigments: None Starch decomposition Slightly separated (C + Rachi "" / N cold culture beef) Growing: Moderate Aerial hyphae: Hardly formed, but scattered white aerial hyphae are occasionally observed .

Hyphae in the base ° gray yellow (3eC) ~ light gray yellow C3gθ) 1
, lJ bath pigment: At the initial stage of culture, a light purple to light reddish brown pigment is produced, but after about 10 days of culture, the color becomes light brown, and then becomes pale (it produces a small amount of melanin pigment).

■ Growth on nutrient agar medium: Good Mycelium in aerobic mode: Substrate mycelium forming thin white aerial hyphae: Pale yellow (2 db) Oj-soluble pigment: None Growth on oatmeal agar medium: Good Mycelia in aerobic mode :White to gray-white base Medium hyphae: Gray-yellow (3ec) to bright gray-red (4'ge)
Soluble pigment: None This strain grows at an optimum temperature range of 2J to 33°C, but can also grow at 20°C or 37'O, although growth is poor. However, it cannot grow at temperatures above 52°C. This strain liquefies gelatin on a glucose-pegtone-gelatin medium and hydrolyzes starch, albeit weakly, on an inorganic salt-Buppun agar medium-L.

This strain peptonizes skim milk. However, no coagulation was observed. This strain does not produce melanin-like pigments in peptone-yeast-iron-agar medium and tryptone-yeast liquid medium, but it sometimes produces melanin-like pigment in tyron-7 agar medium. This strain was tested on D-xylose, D-glucose, and D-
Fructose, L-lano, north, and D-mannit are used, L-arabinose and 1-inositonoperafinose are not used, and a small amount of nyucrose is used.

The strain used in this application has a morphological classification of 5 (Spira-1e
It belongs to section s), grows white to light grayish-white aerial mycelium on yeast malt agar medium, and does not utilize L-arabinose.

This strain produces neopyridoglysein 1v (
The present invention suppresses the production of A.Glysein-C1 to A.Glysein-C and -C'-1.
The selectivity of /1 production can be increased, and this is more suitable for achieving the object of the present invention.

Next, a method for producing the antibiotic of the present invention using the above-mentioned producing bacteria will be described. The above neopyridoglycein-CI-
To produce C and C1-t;
20°C to 33°C, preferably xg'c to 30C, 21
] to /g days, and the culture is extracted, isolated, and purified. As the medium components, known ones used for culturing strains belonging to the genus Streptomyces can be used. A medium containing a suitable carbon source, such as soluble starch, glucose, and/or nitrate, a nitrogen source, such as (I powder, cottonseed meal, dried yeast, etc.), an inorganic salt, such as dibasic potassium diphosphate, salt, etc. Culture is carried out by adding a suitable concentration of chloroproline or ≠-chloroproline to the culture medium, for example, 0./mM or less, preferably 0.5 to 0.5 gmM.

Culture is carried out under atmospheric conditions l-', 20'U to 33°C.
It is desirable to start the culture at 1-24' to 30°C and adjust the pH during the culture to ss to tr, o.

Neobijan grisein-cl-C accumulated in the culture solution
1-C1-t and neopyridoglyzeins containing it can be isolated by known methods for isolating debsipeptide antibiotics and various purification methods based on the physical properties of the substance. In addition, it can be recovered as a mixture of neopyridoglyzeins if necessary.

For example, neopyridoglysein-C present in the culture solution
Neopyridoglyzein containing IC, IR-CI-t, benzene, toluene, ethinoacetate, phthyl acetate,
n-butanol, methylene chloride, chloroform 11
, extracted with a water-repellent organic solvent such as methyl isobutyl ketone. In addition to extraction methods using organic solvents, adsorption/desorption using activated carbon, Amberlite They can be used and recovered by combining them as appropriate by the 1-layer method or the thin-layer method. In case 1, countercurrent distribution θ using a suitable solvent can also be prepared (Jf'III 1 to recovery step I).

Next, neopyridoglysein-C1-C and 1" of the present invention
The physicochemical properties of 1l-C-1,-1 will be described.

Neopyridoglysein-C1-c and neopyridoglysein-C1-c and neopyridoglysein-C1-c are methanol, ethanol, glypanol, rl-butanol, di]gieritz, ethyl acetate, butyl acetate, acetone, methyl ethyl carbon, methyl butyl ketone,
Soluble in benzene, toluene, methylene chloride, chloroform, carbon tetrachloride, N,N-methylforno, amide, dimethylsulfoxide, diethylnial,
It is a white amorphous solid that is sparingly soluble in water and isopropyl ether, and insoluble in petroleum ether and hexane.

Glycein-C,1-c and Glycein-C1--
t is stable for one month at a temperature of 310 or less in an aqueous solution at a pH of 7θ or less. In addition, frying treatment H for 30 minutes at ℃ to
UV absorption maximum measured in 0 methanol and E1 at that wavelength, which is stable in the range 2,3-10! The value (force): within 1) is shown below.

1Jl-glysein-CI-c 305nm
(RIO) Neohi lJ Tokuri L: I7-CI-1,
3θJ-II+11 (RIO)0, IN The ultraviolet absorption maximum in methanolic caustic soda solution and F at that wavelength (translation in parentheses) are shown below.
zag) Neohiritokuri Sein-C Coat 3<zOn
The molecular weight determined by ln(f&) mass spectrometry is shown below.

Neopyridoglysein-C1-C7 6 neopyridoglysein-C1, --- 191.
At the same time, a molecular ion peak appeared in the molecule, and about 4 intensity peaks of the molecular ion peak were observed in the male, indicating that this antibiotic has 4 h of each chlorine source in the molecule. .

Silica gel grade 60F, 2jt/, manufactured by Merck Co., Ltd.
Fl and IJIθ, and then expand the thin layer Y'7 to graph i<r and then small.

/) Solvent thread/ (Ri IJ "1 Porn, : Methanol 2-3 Q : /)'s 1 Isha I・'Biri I・Crysein-C,1,-e R:C
O, 11 fi 1
Rf O, jf/ne (Biri 1 chrysein 11 (control)
urO Hiro/-z) (N medium system! (Benzene ° ethyl acetate ° methanol; acid resistance / water jO: jO: 10:10
:3) neopyridoglysein-(:lc R
f O, A, 2' bili1darizein-Cl -t R
f O,tKne]pyridoglyzein I+ (control)
Rf O, tO neopyridoglysein-C'l-c and Cl-14-7N hydrochloric acid /10'C/ were hydrolyzed for a period of time, concentrated to dryness, /CCI; Thin layer 0
Using Matodarafi (Avicelcel [''-S F' manufactured by Nonacone Pharmaceutical Co., Ltd.), n-butanol'acetic acid, water 4Z:
Solvent system of /:/ or methanol:pyridine water fO:
Developed using a solvent system of 3:20), high 1 Hiro paper electrophoresis (Toyo Roshi 46j/A, manufactured by Toyo Roshi Co., Ltd.), formic acid:acetic acid:water-2jtNia!:riOO pH/Z buffer Electrify the liquid at 1 O'C for 60 minutes at OV/:m)...
and amino acid portion Eve'r Hl'(Ll)'7',
Made 3! ;-40 type)
Leda.

Neobi January Grisein-(:'lt: :Thread(-
0/, 11 I//・t-ri IJ l'J bume phosphorus, ゛lilu I//, B, N-dimethylj-1 yne, -]χ
R-n, noeniril! /N, 3-H I Loki/Vinyl Phosphate Ne, T Bi+Moon Grisei 7-C-1-t: :1. f, -7: rneu//, Hirori [JrozoIJphosphorus, sarco/one, β, N-dimethyl 1neu/no, -no'sil; Hydroquine picolinic acid Among these, 3-human quinbinine monophosphate is
J Madography = (Merck Silica Gel Play 1.t
The Rf value of OF2.9'1 was developed using chloroporm:methanol-,2:l,
Using Avicel Cellulose Sip manufactured by Nonacono Pharmaceutical, r
]-butanol:acetic acid:water-ti:i:When developed with i:i, the Rf values o and to match the Rf values of the standard sample) and its presence was confirmed by mass spectrometry.

Therefore, neopyridoglyzein-(A-c, neopyridoglyzein-C1- as its constituent amino acid,
The presence of 3-hydroquine picolinic acid was confirmed.

In order to determine the stereoisomerism of Hiro-chloroproline, each Goo-chloroproline was separated, converted to N-acetyl methyl ester, and subjected to gas chromatography (column; 3
mrn×/, j ITL, carrier; Chromosolve W,
Stationary phase; o! r%PKOA, carrier gas: N2
・/, 7 kg/r:J, temperature: 17 g°C). Next, the retention time is shown.

Neo-Abi 1-twin blade zein-C1-c medium ll-chlorofus/363 minutes neopyridoglyzein-CI-tlj hiro-
Chloroconb/27] minsu ψ-chloroproline (
Marked substance) 3.65 minutes I. Lance-Goo chloroproline (standard substance) 273 minutes and above were combined to determine the chemical structure of neopyridoglyzein-C1-C and neopyridoglyzein-C1-t. has been reconciled with the above equation (1).

Next, we will demonstrate the antibacterial activity of this antibiotic against various pathogenic bacteria by comparing it with the known substance neoviridoglysein/■ (pyridoglysein) and clarify its usefulness.

The following table shows the minimum inhibitory concentration of neopyridoglysein-C1-c and neopyridoglysein-C1-c against various microorganisms using the same 1v agar dilution method. [ I-↓ Shikaku a) CI-c, C1-t, ■ are each: t4 HiIJ
rchrysei-C1-c, neopyridoglysein-
C1-t, an abbreviation for neopyridoglysein ■.

(b) pc-peninline, TC: tetracycline, E
M: Erythromysi7. LM: Leukomai7no, ()r
() Bacteria resistant to mountain drugs.

(C) Measured using Streptococcus plain...-F infusion medium. Strepto: I Socas Pneumoniae Type I is a medium containing 10% defibrinated horse blood in plain heart infusion medium.

Also, neopyridoglysein-Cl-c and the same Cl-
1 shows a synergistic effect with griseopyridine like other neopyridoglyzeins. This is neopyridoglyzein-Cl-c, neopyridoglyzein-Cl-1,
When Griseopyridin alone was applied at a concentration that did not show an inhibition zone to a paper disk and placed at a certain distance on an agar medium inoculated with test teeth and incubated overnight, no inhibition zone was observed around each disk, and both This is clear from the fact that an inhibition circle is created in the middle of the . Therefore, although the new antibiotics neopyridoglyzein-CI-c and neopyridoglyzein-Cl-t of the present invention are effective antibiotics alone, they are even more effective when mixed with griseopyridine. Becomes an effective antibiotic. In practice, it can be used as a mixture of neopyridoglyzeins and griseoviridein without separating each substance.

Next, examples of the present invention will be given to illustrate the present invention more specifically.

Example 1 A medium jO- consisting of 2.0% soluble starch, 0.2 qlr yeast extract, 02% malt extract, and 0.1% soybean flour (adjusted to pH 1., I) was placed in a 230-containing square flask. , Streptomyces Sp. after sterilization. G-J'ri (Feikoken Joyori No. 1I11) was inoculated and cultured with rotary shaking at λr'c for 72 hours, and this was used as a seed mother. Soluble starch. 2.0%, L-asparagine O. S%.

Salt 0. /%, dipotassium phosphate 0. /%, Magnesium sulfate/umum o. o6%. Zinc a acid '-0'% + chloride 77 Kay 0.00/% (pHAgtic
After sterilization, a 20-liter culture medium consisting of a 100-mL culture medium (preparation) was placed in a 100-mL rectangular flask, inoculated with 0.2-liter seedlings, and cultured with rotary shaking in a JJ"C for .2 hours. Separately sterilized trans-l-chloro -L-Florin or Nosugu Chlorol L
-OJ proline.

/． It was added to each flask at concentrations of 0, 2.0, and ≠OmM, and the rotary shaking culture was continued for an additional 7 and 20 hours. After culturing, 3 ml of the culture solution was extracted with benzene l-, and a certain amount of it was placed on a Merck silica gel plate tOF2μ.
Spotted on.

After development with chloroform:methanol-30:/, quantitative analysis was performed using TLC-chromatography scanner CS-TalO manufactured by Shimadzu Corporation, and the values shown in the table below were obtained.

(Unit: mcg: / al Example + 1J Soluble starch, 20%, yeast extract 02%, malt extract 02% soybean flour 0.7% (adjusted to pH 47)
After sterilizing the medium jQtrl consisting of JQtrl in an A3O-containing square flask, it was inoculated with Streptomyces Sp. . Soluble starch 20%, L-asparagine Osq' + salt 0. /%, dibasic potassium phosphate 0
．． /%, magnesium sulfate o. or%, zinc sulfate o. o
i%.

Manganese chloride o. After sterilization, a medium SO- consisting of oot% (adjusted to #H & .) was placed in a 2304-volume Erlenmeyer flask and inoculated with the above seed mother, followed by rotary shaking culture at xr°C.

After hours, sterilized trans-guchloro-L-proline was added to a concentration of 5 mM, and the culture was continued. After vaccination 6
After culturing for days, the culture solution was filtered to obtain a ≠r− filtrate.

After adjusting the pH of the filtrate to 75, it was extracted twice with JQml ethyl acetate and concentrated to dryness. It was dissolved in 2j-benzene and filtered to remove insoluble matter. The benzene solution was spotted on a Merck & Co. silica gel 60 F, 2jII thin layer plate, and preparative thin layer chromatography was performed.

Development was carried out using chloroform:methanol=30:l. 31s after deployment.

neopyridoglysein-Cl-c by irradiation with ultraviolet rays of λ
I scraped off a part. The scraped silica gel was extracted with 10-methanol and dried. Further, the dried product was dissolved in pure methylene chloride, filtered to remove insoluble materials, and then dried to obtain 22 m of neopyridoglysein-Cl-C single specimen f.

Example 3 Culture was carried out in the same manner as in Example λ, with the addition of cl/l-chloro-L-proline. Example from the culture solution cultivated for 6 days! A single specimen of neopyridoglycein-C'-C was obtained using the recovery and purification method described in 1.

Patent applicant: Sanraku Ocean Co., Ltd.

Claims (1)

[Claims] (+j Formula % Antibiotic neopyridoglyzein-CI-c and antibiotic neopyridoglyzein-C1-t (c and t each represent cis-chloroproline and (2) Belongs to the genus Streptomyces, and the antibiotic neopyridoglyzein-C1-c and the antibiotic neopyridoglyzein-CI- A method for producing the antibiotic, which comprises culturing a microorganism capable of producing T in a nutrient medium, and collecting the antibiotic from the culture. (3) An antibiotic belonging to the genus Streptomyces. Microorganisms that have the ability to produce neopyridoglyzein-C1-c and the antibiotic neopyridoglyzein-C1-t are Streptomyces sp, prtpr (Feikoken Bacterial Serial No. 3JT2) or Streptomyces sp. Sp, G-7
2 (Feikoken Joyori No. 1g≠)
Manufacturing method described in section.