JPS6256487A - Novel antibiotic substance 5-deoxyenterocin - Google Patents

Abstract

NEW MATERIAL:A compound, expressed by the formula and having the follow ing physico-chemical properties; Elementary analysis (%); C, 61.71; H, 4.64. Molecular weight; 428 [measured by the fast atom bomberdment (FAB) mass spectrometry]. Molecular formula; C22H20O9. Melting point; 222-225 deg.C. [alpha]D<20>=-59.4 deg. (c=0.6, methanol). Soluble in methanol, acetone, butanol, ethyl acetate, dimethyl sulfoxide and pyridine and slightly soluble in water, hexane and petroleum ether. Positive to the KMnO4 decoloring reaction, Fehling reaction and Tollens reaction. Negative to Molisch reaction and FeCl3 reaction. Neutral, colorless prismatic crystals, etc. USE:An antimicrobial agent agent Gram-positive and Gram-negative bacteria and microorganisms of the genus Mycoplasma. PREPARATION:A strain belonging to the genus Streptomyces, e.g. Streptomyces species L11-1 (FERM-P No. 7917), is cultivated, preferably under aerobic conditions at 28 deg.C and 7pH for 2-5 days.

Description

[Detailed Description of the Invention] Industrial Application Field The present invention is a novel antibiotic! 5-deoxyenterosine (3
;-Deoxyenmerocin) and its production method.

BACKGROUND ART Enterocin, represented by the formula, is known as an antibiotic produced by actinomycetes and having several hydroxyl groups, a ≠-methoxy-2-oxopyrone ring, and a benzoyl group (J, Antibio also ics, Vol.
, ;lde1. 3.227-23 (/ri 7 Otsu); Publicly known literature/, same magazine, Vol. 29, 10. 11
/4f, ~11/Otsu (7974)). This enterocin has antibacterial activity against Durum-positive and Durum-negative bacteria, and has antibacterial activity against Durum-positive bacteria and Durum-negative bacteria.
s var, enterosLaticusws-
, s”orii strain or 5trep also myces v
iri-docb, romogenes var, M
/ Produced by 27 strains, initially the antibiotic WS-
It was named Zaoritsu substance (Tokuko Sho 17-/3;
7! ;O, known literature/). Then Streptomy
ees hygroscopicus NoA-4,29
11- Vulgamycin produced by
It has been reported that c i n ) is the same antibiotic as enterocin ( Tetrahedron
Lemo ters & lAg + '73 Otsu 7-1.3
70 (/def Otsu)].

Problems to be Solved by the Invention The object of the present invention is to obtain a novel antibiotic useful for medical use.

The present inventors isolated many microorganisms from nature in order to search for new antibiotics, and investigated the biological properties of their products. We found that microorganisms isolated from soil produce substances that have antibacterial activity against Gram-positive bacteria, Gram-negative bacteria, and mycoplasma.

As a result of separating and purifying this antibacterial active substance from the culture and examining its physical and chemical properties, we determined that this substance is a new substance because it has various properties different from those of previously known substances as described below. Based on the results of structural analysis of this antibiotic, this antibiotic was named 5-deoxyenterosin.

The present invention was completed based on such new findings, and provides a novel antibiotic S-deoxyenterosin and a method for producing the same.

The strain isolated by the present inventors that produces the antibiotic 5-deoxyenterosin has the following mycological properties.

(1) Morphological characteristics The L11-/ strain was grown on Nutachi inorganic salt agar medium
! ! 71) CInter, J, System
, Bacteriol.

Vol./l-, 373-3'AOC/ri6o)] was cultured at 2°C for 10-/g days, and the observed findings were as follows. In addition, almost the same morphology was observed in ISP medium 2.3 and j top.

The substratum hyphae are curved and elongate with branching, and the diameter is 0≠~0.
5 μ, the hyphae do not divide and do not attach spores. Aerial hyphae generated from substratum hyphae are curved and elongate with simple branches, and have a diameter of OII to 06μ, forming numerous chained spores at the tip of the branch.

Chains of spores are usually sown 2 to <2 times tightly to form a spiral. The spores are ellipsoidal or short cylindrical in shape, and the size is 06~0, g x oza ~ x2μ, and when observed with a transmission electron microscope, the surface of the spores has a thorn-like shape, and it is a scanning electron microscope. When observed with an electron microscope, it appears wrinkled.
e). Flagellated spores and spore pastes are not observed.

(2) Growth status on each culture medium The cells were cultured on various media at 2°C for 1q days, and the observed findings are shown in Table 1. In addition, the color display is Co1or
Harmony Manual No. 795g (Co
ntainer Corporation of Am
erica) according to the color classification.

(3) Physiological properties■ Growth temperature range', 20-45℃ (optimal temperature range: around 2EC) ■ Growth pH range: O~g (optimum pH range = around 7) ■ Liquefaction of gelatin: positive■ Hydrolysis of starch: Positive ■ Coagulation of skim milk: Negative Peptonization of skim milk: Positive ■ Production of melanin-like pigments: Negative in ISP medium O and 7 (4) Assimilable carbon source L-7 rabinose p-b Tsukudose , D-glucose, inositol, D-mannitol, raffinose,
L-rhamnose, sucrose and hy-D-xylose were all utilized.

(5) Bacterial cell composition, the method of Becker et al. [Appl, Microbi
ol, , / J+t2/~1423 (/ri Otsu4'
)) LL-type of diaminopimelic acid was detected.

The above mycological properties, especially the characteristic properties such as forming aerial hyphae with more chains of spores than true basal hyphae, having LL-type diaminopimelic acid, and not forming flagellated spores or sporangia. It is clear that this strain belongs to the genus Streptomyces.

Therefore, in order to distinguish this strain from known strains, Streptomyces sp.
yces sp.
No. 7 (FERMP-7 de/7).

For example, by culturing the antibiotic 5-deoxyenterocyta of the present invention and a bacterium producing the antibiotic 5-deoxyenterocin belonging to the genus Streptomyces, an antibiotic can be produced from the culture!
-Produced by harvesting deoxyenterosine.

The antibiotic bacterium used in the present invention includes the aforementioned Streptomyces subiciis L.
11-/, but as a general property of actinomycetes, the dental properties are extremely easy to mutate (it is not constant, and is caused by natural or regular ultraviolet irradiation, irradiation, or mutation-inducing agents, for example, Bacterial strains capable of producing deoxyenterosine can be mutated by artificial mutagenic means using N-methyl-N-nitro-N-nitroguanidine, ethyl methanesulfonate, etc.
All can be used in the present invention.

In the method of the present invention, culturing can be carried out under conditions generally used for culturing actinomycetes.

As the culture medium, a nutrient medium containing a carbon source that can be assimilated by microorganisms, a nitrogen source that can be digested, and, if necessary, an inorganic salt is used. As assimilable carbon sources, glucose, fructose, malactone, glycerin, molasses, ~S, \1.~X, etc. are used alone or in combination. As digestible nitrogen sources, corn steep liquor, ammonium sulfate, peptone, yeast, soy flour, meat extract, amino acids, etc. are used alone or in combination. Other sodium salts, potassium salts, magnesium salts, as needed.
Inorganic salts such as phosphates and their low heavy metal salts are added.

Cultivation is usually carried out under aerobic conditions such as shaking or aerated agitation culture, and industrially, deep aeration agitation culture is preferred. The pI of the medium (is preferably around 7 to g1, especially around 7).

The culture temperature is preferably 20 to q5°C, particularly around 2g°C. In the case of liquid culture, the culture time is usually 2 to 50 minutes, but the amount of antibiotic w, L11−/ accumulated in the culture is about 3 hours.
Since it reaches its maximum within a few days, it is preferable to terminate the culture at this point. These medium composition, medium liquid properties, culture temperature,
Culture conditions such as agitation speed and ventilation depend on the type of bacterial strain used and external conditions to obtain favorable results. Needless to say, this is adjusted and selected as appropriate. If foaming occurs during liquid culture, use silicone oil, vegetable oil,
Antifoaming agents such as surfactants are used as appropriate.

Since the antibiotic S-deoxyenterocin thus obtained is mainly contained in the culture P solution, the culture should be filtered with a filter aid such as Celite, Perlite, or High Flow Parcel. It is advantageous to separate the culture solution and the bacterial cells by filtration or centrifugation, and then collect the antibiotic 5-deoxyenterocin from the culture solution.

In order to separate and purify the antibiotic jl-deoxyenterocin from the culture solution, the filtrate is treated with a non-hydrophilic organic solvent such as ethyl acetate, butyl acetate, butanol, chlorophosate, etc.
All you have to do is extract it with rem, methyl isodetyl ketone, etc., and use the extract as an antibiotic. 5-deoxyenterosine is obtained. Further
To purify this, use silica gel, activated alumina, activated carbon,
Examples of adsorption resins Roformoacetone, ethyl acetate-methanol, ethyl acetate-acetone, aqueous acetone, aqueous acetone,!
Examples include mixed solvents such as Recol. Elution can be checked by the paper disk method using Vibrio vercorans or by silica gel thin layer chromatography. The purified antibiotic N,5'-deoxyenterosin can be obtained by combining the eluate obtained in this manner with fractions containing the same components and concentrating them.

The antibiotic 5-deoxyenterocin obtained as described above has the following physicochemical properties.

■Elemental analysis value [as molecular formula C22H2009] C%
H% Measured value Otsu/7/ Theoretical value Otsu/Otsuza 447/ ■Molecular weight t12g (according to FAB mass spectrum) ■Molecular formula% Formula% ■Specific optical rotation [α] 2.0 s 5r 11a c C= o B,
Methanol)■Ultraviolet absorption spectrum l-/L/75μ? The ultraviolet absorption spectrum of a 7tnl methanol solution is shown in Figure 1, k MeOH (nm): 203 (E 7%-10
ft) max 7cm -230
(E, l'!=3119>mu"I(E",=23≠) ■Infrared absorption spectrum Optical external absorption spectrum by KBr method) / is as shown in Figure 2.

3da00, /7g0, /Otsugo, /OtsuI10,15Otsu0
1/≠Otsu0. /g00, l3KO, /335, /2ri5
,/2! ;0. /220, 117! ;, 1130, l0
It has a characteristic absorption band at 0,1000 cm.

(13C-Nuclear Magnetic Resonance Spec)/JjMHz (internal standard TMS; 艷Q, (17 ppm)) The chemical shifts and multiplicities measured in DMSOda are as follows.

/de3;,0(8),77v0(8),/7
07(8), /Otsu3.11<8>, /Otsu/, '7
(s), / 39 j (s), /327 (d), /
, 2g1d) , / , 2g4(d) , /J79(
d), /271d), 104.5(d), g7ri(d), 7ri7(8), 77/(8), 7Sde(
8), 7.2 Otsu (d), Otsu 1 / (d), S Otsu 3 (
q), 5441a(d), 3rd party (1),
3 f(t) (ppm) (However, s; single also, d; double
leL.

Also; triplet, q; q
uarto too) ■ Solubility in solvents Soluble in methanol, acetone, butanol, ethyl acetate, dimethyl sulfoxide, pyridine, water, hexane,
Poorly soluble in petroleum ether ■ Color reaction Potassium permanganate decolorization reaction, Fehling reaction and Tollens reaction are positive, Molisch reaction and ferric chloride reaction are negative ■ Distinguish between acidic, basic and neutral ■ Neutral ■ Detection color Color and shape Colorless prismatic crystals [Phase] Silica gel (Silica gel f manufactured by Tokyo Kasei Co., Ltd.) Thin layer chromatography Developing solvent Rf value Ethyl acetate-methanol-/v (100:/) 0≠/Chloroform-methanol (,!0: /) O, l# chloroform-acetone (, IO', 3') 0.3
! ;0Chemical structureThe position number in the above Mizozo formula is Te t rahedr
onLetters &llI, 'A3A3-1
It was granted based on the description in 1370 (/ri 7 Otsu).

Antibacterial properties: 500μV/ml of antibiotic S-deoxyenterocin
A paper disk with a diameter of g was immersed in the 'ffJ solution, dried, and placed on a broth agar medium containing the test bacteria.The diameter of the growth inhibition circle (diameter) after culturing was measured as follows.

5arcina 1utea ATCCq 34t
/ 2/, 08taphyloccus
aureus A T CC Otsu! ;3gp
1g7Bacillus 5u
btilis AT CC6 Otsu33 0Escheri
chia coli NI HJ-J C20K
lebsiella pneumoniae ATCC1
003/ 23.2 Vibr i
o percolans AT CCgtt/
/ Oto Otsu Pseudomon
as aeruginosaIAMlori50 Candida albicans AT CC7
It 9 / 0 pile biological material 0. Mycopla-sma gallisept at μr/m/noa degree
The growth of icumKP-/3 was completely inhibited.

<Acute Toxicity> No abnormality was observed even after intraperitoneal injection of G00η/9 to mice.

Effects of the Invention As described above, the antibiotic 5-deoxyenterocin of the present invention is effective against Gram-positive bacteria, Gram-negative bacteria, and mycoplasma, and has low toxicity, and can be administered orally to humans, livestock, poultry, etc. Alternatively, when administered parenterally, it is useful as a therapeutic or preventive agent for various diseases caused by these microorganisms.

Example Next, the present invention will be specifically explained with reference to Examples.

Example / Seven platinum loops were collected from a slanted agar culture medium of Streptomyces spiens L11-/ strain (Feikoken Bacillus No. 7-77).
00M seed medium (glucose co%, soluble starch 2%, soy flour type 2, Naei O, 23%, yeast extract, c O,
, 5%, CaCO30,33'N, pH Otsu S]. A 5'oomt Erlenmeyer flask was inoculated and 2g was inoculated at 3'°C.
The seed mother was cultured with shaking for days. This seed mother is glycerin 3
%, soluble starch 05%, C3L 0.3%, meat extract 05
%, polypeptone 0.5%, NaC10,! ;%, Co
Cl 2 ・A H2OQ, Oo/% (pH 4,5
, before sterilization) containing 700 ml of a medium consisting of
A 3% Erlenmeyer flask was inoculated and cultured with shaking at 2 g''C for 3 days. 10 t of the culture solution thus obtained was centrifuged to remove the bacterial cells to obtain a culture filtrate g't.

When this was adjusted to pH 70 and extracted twice with ethyl acetate v21, 5-deoxyenterosine was quantitatively transferred to the ethyl acetate layer. This ethyl acetate layer was diluted with anhydrous sulfuric acid) IJ
After dehydration with aluminum, the residue was concentrated under reduced pressure to obtain about 31 brown oily substances. This oil 'R31 was dissolved in a small amount of ethyl acetate, and a silica gel column filled with ethyl acetate (
/deQml) and chromatography was performed using ethyl acetate. 72? Fractionate Vibrio
A bioassay using Percolans as a test tooth shows that fractions with grains of 33 to 33 are collected and concentrated under reduced pressure to yield a pale yellow powder of 5-deoxyenterosin with a purity of approximately gO%≠t
1. I got the result.

Example Co The pale yellow powder of 5-deoxyenterosine with a purity of about g'0% obtained in Example ≠1Oη was dissolved in a small amount of chloroform, and the silica gel was filled with a mixed solvent of chloroform-acetone <70:3). A column (/10"ml) was charged and chromatography was performed using the same mixed solvent. After fractionation into 20" fractions, the fractions with 79 to 37 wood grains were subjected to silica gel thin layer chromatography (chloro-θform; acetone = 70%). :!;) #Here, a single spot of 5'-oxyenterocin was shown. These fractions were collected, concentrated under reduced pressure, and allowed to stand at low temperature to yield colorless prismatic crystals. The crystals were collected by F and dried to yield S-deoxyenterosine 30%! I got r~.

[Brief explanation of the drawing]

Figure 1 shows the ultraviolet absorption spectrum of the antibiotic 5-deoxyenterocin of the present invention, and Figure 2 shows the external absorption spectrum of the same agency.
300 320 nm C9 drawing of caries surface (contents changed, No. 2r37 procedural amendment filed on July 7, 1985, Patent Application No. 195334, 1985 2, Title of Invention: New Antibiotic 5-Deoquin Entero Shin 3, Relationship with the case of the person making the amendment Patent applicant address: 632 Mifuku, Ohito-cho, Tagata-gun, Shizuoka Prefecture 1 ``Densely sown'' on page 5, line 12 of the specification Correct.
Ige (3ge>]j as ``Pain (Be ige)
(3ge)) Correct it as j. "Yellow Maple" in the 5th line of the soluble dye column in Table 1 on page 7 is corrected to "Yellow Maple (3ng) J.""Toubazu" in the 8th and 9th lines of the soluble dye column in Table 1 on page 7 is corrected to "Toubazu (3ne) J.""S-deoxy J on page 9, line 18 is corrected. Correct it to [5-deoxy]. Correct “object” in the first line of page 16 to “substance.” rstaphyloccus J on page 17, line 9.
Corrected to Staphylococcus J. Procedural amendment (Bandai) September 1985 Yuhi 1985 Patent Application No. 195334 2 Title of the invention New antibiotic 5-deoquinoenteronone 3 Relationship with the case of the person making the amendment Patent applicant address Shizuoka Prefecture 632-1 Nifuku, Ohito-cho, Tagata-gun, Showa 61
August 60, 2017 Regarding Figure 2, the characters used in the figure are written in the appropriate font, and the drawing is a handwritten copy, attached as an attachment. 7. List of attached documents

Claims (1)

[Claims] 1) 5-deoxyenterosine represented by the formula ▲ Numerical formula, chemical formula, table, etc. ▼. 2) A method of producing the antibiotic 5-deoxyenterosin, which is characterized by culturing a bacteria producing the antibiotic 5-deoxyenterosin belonging to the genus Streptomyces in a medium and collecting the antibiotic 5-deoxyenterosin from the culture. Manufacturing method. 3) The production method according to claim 2, wherein the antibiotic 5-deoxyenterosine-producing bacterium is Streptomyces sp.