JPS60259190A - New antibiotic 446-s3-1 and production thereof - Google Patents

Abstract

NEW MATERIAL:An antibiotic 446-S3-1 having the following physicochemical properties; Elementary analysis (%); C, 48.50; H, 5.49; O, 33.71; N, 12.30. Molecular weight; (M+H)<+> observed at 570 mass number and (M+Na)<+> at 592 mass number and (M+K)<+> at 608 mass number and 569mol.wt. Melting point 173- 176 deg.C. Solubility; Readily soluble in water and methanol, soluble in ethanol and acetone and fairly soluble in CHCl3 and insolbule in ethyl acetate, benzene and n-hexane. Color reactions; Positive to KMNO4 reaction and the Molisch reaction and negative to the anthrone reaction and FeCl3 reaction, etc. Color; White, etc. USE:An antimicrobial agent. PREPARATION:A strain, e.g. Streptomyces griseus 446-S3 (FERM-P No.7416), is aerobically cultivated preferably at 7pH and 28+ or -1 deg.C for 4-6 days.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a new antibiotic 446-33-1 and a method for producing the same.

In the process of searching for antibiotics produced by natural microorganisms, the present inventors discovered that an actinomycete strain 446-33 isolated from soil produced a new antibiotic, investigated the mycological properties of this strain, and The present invention was also completed by isolating the antibiotic and investigating its physicochemical and biological properties.

Antibiotic 446-33-1 obtained by the present invention has the following physicochemical properties.

(1) Elemental analysis values: C, 48,50%, H5, 49%, 033.71%, N12.30% (2) Molecular weight: In secondary ion mass spectrum (SI-MS) analysis, mass number 570 to (M+H)", 592 to (M+
From the observation of (M+K)' at 608 and 608, the molecular weight is concluded to be 569.

(3) Melting point: 17:1176°C (4) Ultraviolet absorption spectrum: Absorption maximum in methanol (λM@OHnm(,,)a
x is 214 (16200), 257 (shoulder 9800)
shows. As shown in Figure 1. Also, aqueous solution, 0.1
The ultraviolet absorption spectrum in normal hydrochloric acid and 0.1N aqueous sodium hydroxide solution does not show a characteristic absorption maximum, but only shows terminal absorption.

(5) Infrared absorption spectrum: The infrared absorption spectrum measured with KBr is as shown in FIG. The wavelengths showing maximum absorption are as follows.

The unit is c'.3400.2930.1680.151
5.1270.1100° (6) Solubility in solvents: Very soluble in water and methanol, ethanol,
Soluble in acetone, slightly soluble in chloroform, and insoluble in ethyl acetate, benzene, and n-hexane.

(7) Color reaction: (Positive) Potassium permanganate reaction, Molisch reaction (Negative) Anthrone reaction, Aniline hydrogen phthalate reaction, Ferric chloride reaction, Ninhydrin reaction (8) Basic, neutral, acidic Classification: Color of basic (9) substance: White Color QOl Rf value Nijiri gel thin layer chromatography (Merck TLC)
The Rf value when using a plate, silica gel (60hs thick, 0.25mm thick) in the usual manner is as follows.

(a) Chloroform:methanol (2:1) Rf value 0.43 (θ Benzene:methanol (2:1) Rf value 0.
．． 21 (→ n-butanol:acetic acid:water (3:1:1)
Rfli 0. 43 11) Acid hydrolyzate - Uracil and lysine present in it.

12) 'HNMR(400MI(z,n,o)δ
(ppm): 1.37 (IH), 1.64 (I
H), 1.78 (IH), 1.84 (LH), 1
．． 93 (IH), 2.00 (IH), 3.3 (
IH), 3.32 (3H,s), 3.77 (IH
, t) , 4.23 (IH), 4.4 (IH, t)
, 4.50 (IHX t), 4.53 (LH)
, 4.65 (IH), 4.80 (IH), 5.4
(IH, d), 5.78 (IHld), 5.88
(IH,d), 6.04 (IH), 7.78 (I
I-1, d) 13) 13CNMR (100MHz,
20) δ (ppm): 28.38 (t), 2
8.61 (t), 31.33 (t), 42.33
(t), 53.10 (d), 58.70 (q)
, 62.72 (d), 65.72 (d), 72.
80 (d), 76.42 (d), 78.97 (
d), 82.39 (d), 90.71 (d),
100.00 (d), 102.48 (d), 109
．． 92 (d), 141.45 (d), 142.0
6 (s), ljl, 62 (s), 161.74
(s), 166.30 (s), 173.20 (s
), 176.47 (s) 14) Based on the above physical and chemical properties, antibiotics 446-33
The structural formula of -1 is estimated as follows.

The biological properties of antibiotic 446-83-1 are as follows.

(1) Method established by the Japanese Antibacterial Chemotherapy Society [Minimum Inhibitory Concentration (MI)]
C.) Regarding the revision of the measurement code (chemotherapy, 29.7
6-79.1981)), the minimum inhibitory concentration (M I
C) is as shown in Table 1.

Table 1 Bacillus subtilis 〉100 (Bacillus 5ubtilis) ATCC6
633 Micrococcus Iuteus 〉■00 (Micrococcus Iuteus) ATCC
9341 Staphylococcus ara 〉100 Staphylococcusaureus
FDA209 P, JC-1 Streptococcus fue >to.

Calis (5treptococcus fa@calis) Streptococcus new 12.5 Moniae (St
reptococcus pneumoniae) I
ID 553 “Streptococcus new 1
2.5 Streptococcus spneu
moniae) 11 D 554"Streptococcus pyogenesCo" (5treptococcus pyogenesCo
ok ) Streptococcus pyogenes 100 Neisseria gonorrhoeae 115
D844” Neisseria meningitide >100 chairs (Ne1
sseria, meningitidis) IID854” Haemophilus influenzae >100 the()laem
OP 1us influenzae) I I D 985 ”Pseudomonas aeruginosa N CT C0490 Citrobacter freunde >100i (C4tr
976 Enterobacter freundi 100
era a (3rogenes) ATCC13048 Enterobacter cloacae 〉100 works (Enter
obacter cloacae ) 963 Escherichia coli 〉100 (Escherichia colt ) NIHJ J
C-2 Klebsiella pneumonia 〉100 pieces (Klebs
iella pneu+aoniae) PCI-602 Proteus vulgaris 〉100 (Proteus vulgaris) X-19 Salmonella enteritide 〉100 is (Salm
onella enteritidis ) G 14 Serratia marcescens 〉100 (5erratia marcescens ) IAM
1184 Mycobacterium smegma 3.13 Theis (Myc
obacterium sn+egmatis) ATCC607° (Note) Chocolate agar medium was used for test bacteria marked with *, and ordinary agar medium supplemented with 1% glucose was used for test bacteria marked with **.

In addition, Aspergillus oryzae
soryzae) I F O5239, Penicillium chrysogenum (Penicillium chry
sogenum) A 'l' CC10002, Candida albicans
) 3147 and Satucharomyces cerevisiae (Sa
ccharomyces cerevisiae)
The minimum inhibitory concentration against fungi such as IF 00205 is all 100 mcg/m6 or higher.

(2) When physiological saline solution is administered intravenously to toxic mice, 1,
000 μ/kg showed no toxicity at all.

As described above, antibiotic 446-33-1 is mainly effective against streptococci and acid-fast bacteria, and has extremely low toxicity, so it is expected to be used as a therapeutic agent for infectious diseases in humans, animals, etc., and for other uses.

As a result of comparing the above-mentioned physicochemical properties and biological properties with those of known antibiotics, no corresponding substance was found, so antibiotic 446-83-1 was determined to be a new antibiotic.

Furthermore, 'H-NMRS'3C-NMR and amino acid analysis of the acid hydrolyzate revealed that antibiotic 446-33-1 is a so-called nucleoside antibiotic containing a uracil group and a lysine unit residue. was confirmed.

On the other hand, since there are no substances containing lysine among known nucleoside antibiotics, 446-33-1 can be determined to be a novel antibiotic.

An example of a bacterium producing the antibiotic 446-33-1 of the present invention is Streptomyces, an actinomycete that was newly isolated from the soil of Oaza Yamadera, Yamagata City, Yamagata Prefecture in August 1982 by a conventional method.・Griseus・446-33 (Strep
tomyces griseus 446-33). The mycological properties of this 446-33 strain are as follows.

Unless otherwise specified, the composition of the culture medium used in the following experiments is based on the identification of actinomycetes in the Applied Microorganism Industry (2nd revised edition, August 1981), edited by the Patent Office, industry-specific examination standards, pp. 28-33. The composition of the medium used was followed.

Mycological properties of strain 446-33 1) Form When strain 446-33 was cultured for two weeks at 27°C on starch/inorganic salt agar medium and yeast/malt agar medium, pale green-yellow aerial mycelia were abundant. When observed under a microscope after epiphytic growth, the aerial hyphae are simply branched, gently curved, and partially straight, and show tufted branches. No axle-like branches or spiral spore-bearing hyphae are observed.

When observed under an electron microscope, the tip of the aerial hyphae forms a chain of 10 or more cylindrical spores, and the size of the spores is 0.5 in diameter.
~1.0 pm, length approximately 0.9-1.8 μm, and the surface of the spore is smooth. Flagella, sporangia, and sclerotia are not observed.

2) Growth state experiments and observations on various media were conducted by E. B. Scherling and D. Gottlieb (Int. J. 5yst).

Bacteriol, Vol. 16, p. 313, 1966). When describing colors, please refer to Nihon Shikiken Color Name Book 3rd edition (1974) published by Nihon Shikiken Business Co., Ltd.
JIS symbol is shown in parentheses. The following are the results of 27°C culture and oral observation for 2 weeks, unless otherwise specified.

(11 Growth on sucrose/nitrate agar medium was good;
Spread and develop. The attachment of aerial mycelia is poor and powdery.
White (N9.0) or very pale green-yellow (10Y)
9/1.5). The color of the underside of the basal hyphae is pale yellow (5,5Y 9/6) and bright green-yellow (IOY 8
．． 5/9) of soluble dye.

(2) Growth on glucose-asparagine agar medium is somewhat poor and flat. The attachment of aerial hyphae is also poor, and it appears as a pale green-yellow (10Y9/1.5) powder, and small water droplets are sometimes observed on the aerial hyphae. The color of the back surface of the substrate mycelium is very pale green-yellow (IOY 9/1.5), and it produces a very pale green-yellow (10Y 9/1.5) soluble pigment.

(3) Glycerin-asparagine agar medium (ISP-5
Medium) It grows and grows well. Aerial mycelial growth is moderate, very pale reddish yellow (2,5Y 9/2)
Very pale yellow (5,5Y 9
/1.5) powdery parts are scattered. The color of the back surface of the substrate mycelium is gray-yellow (5,5Y 8/3), pale green-yellow (IO
produces a soluble dye of Y 915).

(4) Starch/inorganic salt agar medium (ISP-4 medium) Growth is good and spreads. The settlement of aerial mycelia is very good, and the central part of the colony is powdery, and the peripheral part is powdery with unevenness, and is very pale yellow-green (SGY 8.5/1.5).
exhibits. The back side of the base mycelium is bright yellow (5,5Y 1
15) to produce a pale green-yellow (10Y 915) soluble dye.

(5) Tyrosine agar medium It shows extremely good growth and flourishes.

Aerial mycelium is also well established, with grayish-white (5G 910.5) velvet-like parts and pale yellow-green (5GY 8.
5/1.5>, uneven powdery parts are mixed.

The color of the back surface of the substrate mycelium is yellowish brown (9YR4/4), with some parts showing dark yellowish brown (9YR3/3), and producing a very pale yellow (5°5Y9/3) soluble pigment.

(6) Growth on nutrient agar medium is poor, with a small amount of white (N9.0) aerial mycelia growing on it. The color of the back side of the base mycelium is very bright yellow (5,5Y
9/3), no formation of soluble dye was observed.

(7) Yeast/malt agar medium (ISP-2 medium) Shows very good growth and has some unevenness. Aerial mycelium grows very well in powder form, and its color is very pale greenish-yellow (IOY9/1.5>. , 5Y 8.5/9).

(8) Oatmeal agar medium (ISP-3 medium) Growth is somewhat poor and flat. The attachment of aerial mycelium is also poor, and it is scattered in the form of a pale greenish-yellow (IOY9/1.5) powder. The color of the back side of the base mycelium is very pale yellow (5,5Y 9/3)
The color is gray to yellow (5,5Y 8/3), and the back side of the periphery of the village is deep yellow (2,5Y 7/10). Produces a very pale yellow (5,5Y 9/3) soluble dye.

3) Physiological properties (1) Growth temperature range As a result of 27 experiments at 15°C and 20°C using starch/inorganic salt agar medium and yeast/malt agar medium, 15°C to 3
It grows at each temperature of 7°C, but does not grow at 40°C and 45°C.

The optimum temperature for growth is around 27°C.

(2) Liquefaction of gelatin (puncture culture using glucose peptone gelatin medium at 20°C) is positive.

That is, liquefaction is not observed on the 7th day of culture, and liquefaction begins around the 14th day and progresses very gradually.

(3) Hydrolysis of starch (cultivated on a starch/inorganic salt agar medium at 27°C and determined according to a conventional method) The hydrolytic power of starch is relatively strong.

(4) Coagulation and peptonization of skim milk (2 in skim milk medium)
The cells were cultured at 7°C and 37°C. ) Coagulation of skim milk is 27
It is not observed at either ℃ or 37℃. Peptonization is
It starts on the 4th day at 27°C and around the 10th day at 37°C, and progresses gradually until it is almost completed in the mouth for about 3 weeks at either temperature.

(5) Production of melanin-like pigments (tyrosine agar, peptone yeast iron agar, tryptone yeast
Broth: All were cultured at 27°C. ) No production of melanin-like pigments was observed in any of the above media.

4) Utilization of carbon source Bridham Gottlieb agar medium (1' SP-9
As a result of culturing at 27°C using D
-xylose, D-glucose, D-fructose, D
- utilizes mannitol, D-galactose and tyrosine, the availability of sucrose and inositol is questionable, and does not utilize L-arabinose, L-rhamnose, raffinose and cellulose.

5) Analysis of diaminopimelic acid in cell walls Diaminopimelic acid, one of the constituent amino acids of cell walls, was analyzed by T. Hasegawa et al.: Journal of the General Endo.
Applied Microbiology (T, Haseg)
awa, et al, *J, Gen, ^pp1. M
icrobiol, ) 29, 319-322 (
The result of analysis according to the method of (1983) was that it was LL-type.

To summarize the above results, the aerial hyphae of 446-33 are simply branched, most of them are gently curved, the epiphytic part of the conidia shows tufted branches, and whorls and spiral formation are not observed. The surface of the spore is smooth. Flagella, spore mass, and sclerotia are not observed. On various media, substrate hyphae are formed that are very pale yellow, very pale green-yellow to yellow-brown, and dark yellow-brown to gray-yellow, and aerial hyphae are very pale green-yellow,
It exhibits a very pale yellow-green color, a very pale yellow color, or a very pale reddish yellow color. In addition, a very faint green-yellow color,
Produces a very pale yellow, pale green-yellow, or dull yellow soluble pigment. As a result of testing from 15℃ to 45℃,
It grows between 15°C and 37°C, and does not grow above 40°C. It liquefies gelatin and peptonizes skim milk without curdling. It thaws starch well and does not produce melanin-like pigments. , D-xylose, D-glucose, D-fructose, D-mannite, D-galactose and tyrosine, the use of sucrose and inositol is questionable, and the use of L-arabinose, L-rhamnose, raffinose and cellulose is not used. . It is clear that the cell wall is diaminopic and belongs to the yellow series in the Pridham and Tresner classification. Therefore, a comparison was made using the 8th edition of ``Birshi's Manual of Determinative Bacteriology'' and the International Journal of Systematic Bacteriology, Vol. 18, p. 332, and as shown in Table 2, Streptomyces It matches well with Griseus' description. However, the new antibiotic 446-33-
Since there was a clear difference in the production of Streptomyces griseus, we concluded that it is appropriate to treat strain 446-83 as a new strain belonging to Streptomyces griseus (SLreptomye-I griseus).
iseus) 446-33.

This strain is Streptomyces sp. 446-
33 (Streptomyces sp, 446-
33), and the accession number is FERMP-
7416).

In carrying out the method for producing antibiotic 446-33-1 of the present invention, antibiotic 446-33-1, which belongs to the genus Streptomyces,
Strains capable of producing 33-1, such as Streptomyces sp.
No. 16) in a nutrient-containing medium and grown aerobically, a culture containing antibiotic 446-33-1 is obtained. As nutrients, those known as nutritional sources for actinomycetes can be used. For example, commercially available carbohydrates such as starch, dextrin, maltose, sucrose, glucose, glycerin, and molasses, organic acids, carbon sources such as fats and oils, and defatted soybean flour, soybean flour, cottonseed flour, NZ-
Nitrogen sources such as amines, corn steep liquor, casein melt, distilled soda solubles, fishmeal, peptone, meat extract, yeast extract, ammonia sulfate, sodium nitrate, ammonia nitrate, ammonia, phosphates, magnesium salts, zinc salts, Inorganic salts such as potassium salts, sodium salts, calcium salts, and other trace amounts of metal salts can also be added as necessary. These substances may be used by the producing bacteria and useful for the production of antibiotic 446-33-1.
All known culture materials for actinomycetes can be used.

Liquid culture is preferable for efficient production of antibiotic 446-33-1, and the culture temperature is such that the producing bacteria grow and produce antibiotic 446.
-33-1 as long as it is within the range of production. Cultivation is normally continued until sufficient accumulation of antibiotic 446-33-1 occurs. For example, 446 cells were cultured on an agar slant in a liquid medium (pH 7.0) consisting of 1.8% glycerin, 0.6% polypeptone, 085% meat extract, and 0.3% salt.
-33 strain is inoculated and cultured aerobically with rotary shaking at 28 ± 1°C. Accumulation of the target antibiotic is observed from the second day of culture, reaching a maximum on the fourth to sixth day. . For quantitative determination of antibiotic 446-33-1, Mycobacterium smegmatis was used as a test bacterium.
A conventional cylindrical plate method or paper disk plate method using an AT CC607 (smegmatis) may be used.

In the culture thus obtained, antibiotic 446
Most of -33-1 is present in the culture filtrate or centrifugation supernatant. Various methods are used to collect antibiotic 446-33-1 from these solutions. That is, various methods such as adsorption and desorption using adsorbents or purification methods using various ion exchange resins can be implemented.

For example, if Diaion HP20 (manufactured by Mitsubishi Chemical Industries, Ltd., trade name), which is a porous nonionic adsorption resin, is used as an adsorbent, antibiotic 446-33-1
and is eluted with acetone water, methanol water, etc. In addition, it is adsorbed in the hydrogen form of Amberlite IRC-50 (manufactured by Rohm & Co., Ltd., USA, trade name), which is a weakly acidic cation exchange resin, and is adsorbed in the hydrogen form of a dilute acid, such as 0.
Eluted with 05N hydrochloric acid. The crude powder of antibiotic 446-33-1 obtained by this method is applied to a silica gel column using a mixed solvent system consisting of an appropriate combination of solvents such as chloroform, methanol, benzene, ethyl acetate, acetone, etc. It can be purified by chromatographic methods. In addition, weakly acidic cation exchange resins for chromatography, such as Amberlite CG-50 (
Manufactured by Rohm and Haas, USA, product name) Hydrogen type, resin for gel filtration chromatography, such as Toyopearl H
It can also be purified using W-40 (manufactured by Toyo Soda Kogyo Co., Ltd., trade name). Also, hydrous alcohol and alcohol-
High purity antibiotic 446-33-1 can also be obtained by repeating recrystallization using a mixed solvent such as ethyl acetate.

Next, examples of the present invention will be shown, but these are merely examples, and the present invention is not limited thereto.

Example 1 Streptomyces sp. 446-33 strain (St
reρtomyces sp, 446-53) (Feikoken Bacteria No. 7416) was used as the seed, and the medium was glycerol 1.8%, polypeptone 0.6%, beef extract 0.5%, sodium chloride 0.3 % composition was adjusted to pH 6.9 and then sterilized at 121'C for 15 minutes. Inoculum - 100 m of the above medium containing a loopful of platinum in a 500 mff Erlenmeyer flask.
j! and cultured with shaking at 28°C for 2 days. This seed culture solution is 100m1A! Twenty-five 500 mA Erlenmeyer flasks containing the above-mentioned medium were inoculated at a rate of 3% each, and cultured at 28° C. for 6 days with rotary shaking culture a (180 revolutions per minute).

500m7! Culture 2.51 obtained by combining cultures from 25 Lurenmeyer flasks was diluted with 2N-hydrochloric acid.
After adjusting the temperature to 17, centrifugation was performed. The obtained supernatant liquid was passed through a Diaion HP20 resin column (200 mj2) to adsorb the target substance, and after washing with water, LOOOmj2 of 50
Elution was performed with % aqueous acetone. ? 200 mβ of the active fraction obtained by discharging was concentrated under reduced pressure and further freeze-dried to obtain 1.80 g of brown powder. This brown powder was placed on a Toyopearl HMI-40F gel column (manufactured by Toyo Soda Kogyo Co., Ltd., 450 mJ) packed with °ζ using methanol, developed and eluted with methanol, and active fractions (fraction numbers 12 to 14,
20mj! / fraction) was obtained.

This active fraction 5Q m 11 was evaporated to dryness under reduced pressure,
0.50 g of brown solid was obtained. The brown solid obtained was 0.
50g was dissolved in a small amount of methanol, placed on a silica gel column (Wako Gel C-200, 400m# manufactured by Wako Pure Chemical Industries, Ltd.) packed with chloroform, developed with chloroform, and then mixed with chloroform:methanol ('3:1). Develop and elute with a mixed solvent to obtain active fractions (fraction numbers 25 to 3).
No. 6, 10m117 fraction) was obtained. This active fraction 120
m1 was evaporated to dryness under reduced pressure to obtain 200 ml of pale yellow powder. This was further placed on a silica gel column (150m#) filled with chloroform, developed with chloroform, and then developed and eluted in sequence using a mixed solvent of chloroform:methanol (5:1) and chloroform:methanol (3:1). did. 30 m 12 of this active fraction was concentrated under reduced pressure and the precipitate obtained by adding ethyl acetate was filtered off and dried to obtain 1.80 ml of white powder of antibiotic 446-33-1. The physical and chemical properties of this thing are
This was consistent with the above-mentioned physical and chemical properties.

Example 2 As a seed culture, 2 m of the seed culture solution cultured in the medium of Example 1
1 was added to a test tube with a screw plug containing 2 m6 of a previously sterilized 40% sucrose aqueous solution and mixed.
Those stored frozen at 0°C were used. This seed culture solution was returned to room temperature, and 500 mj+ of the medium of Example 1 was added.
m/vol. Erlenmeyer flasks and at 28°C.
The cells were cultured with shaking for 1 day. This seed culture solution was inoculated at a rate of 3% into 50 Erlenmeyer flasks each containing 100 mN of medium and having a capacity of 500 m It, and cultured with shaking at 28°C for 6 days. In this case, the medium contains 2.0% glycerol, 0.5% glucose, and 0.6% polypeptone.
, beef extract 0.5%, and sodium chloride 0.3% were adjusted to pH 6.9 and then sterilized at 121° C. for 15 minutes before use.

Obtained culture5. ONs were centrifuged to obtain a culture supernatant. This was applied to a column of Diaion HP20 resin (500 m
#) to adsorb the target substance.

After washing with water, 1.57! The active fraction (550 mf) obtained by elution with 50% aqueous acetone was distilled under reduced pressure to remove acetone, and then freeze-dried to obtain 3.5 g of brown powder. This was dissolved in methanol and placed on a silica gel column (700 m4) filled with chloroform, developed with chloroform, and then chloroform:methanol (5:
1) Develop and elute in order using a mixed solvent of chloroform:methanol (3:1) to obtain the active fraction (fraction number 210).
No. to No. 240, 20 m#/fraction) were obtained. This active fraction was concentrated under reduced pressure, and the white precipitate obtained by adding ethyl acetate was filtered off and dried under reduced pressure to obtain 0.90 g of pale yellow powder.

After dissolving this in water and adjusting the pH to 7.5, weakly acidic cation exchange resin Amberlite CG-50 (hydrogen type 2
50 m/) to adsorb the target substance.

Next, wash with water, further elute with 0.05N hydrochloric acid,
Active category 330■11! I got it. The obtained active section was heated to 20 mA under reduced pressure! This was placed on a Toyopearl HW-40F column (340 ml) filled with water, and developed and eluted with water to obtain 50 ml of active fraction. This was lyophilized and 390mg of white powder of antibiotic 446-53-1 was obtained.
I got it. The physicochemical properties of this substance were consistent with the above-mentioned physicochemical properties.

[Brief explanation of drawings]

FIG. 1 shows the ultraviolet absorption spectrum (measured in methanol) of antibiotic 446-33-1, and FIG. 2 shows the infrared absorption spectrum (KBr method). Figure 1 Water absorption (nm) Procedure? 1lii'E" 59.10.23 Showa Year Month Day 1, Incident Indication 1982 Patent Office No. 116315 2
, Title of the invention Slanted pile organism'i! f446-33-1 and its manufacturing method 3゜Relationship with the case of the person making the amendment Applicant name: Kanto Ishii Pharmaceutical Co., Ltd. 4, Agent 5, Date of amendment order Voluntary 7, Contents of amendment 1 (1) Details The claims of the book are amended as shown in the attached sheet. (2) Delete “Also, it is only for the purpose of indicating...” on page 7, lines 6 to 10 of the same book. (3) Change “basic” to “weakly acidic” on page 8, line 9 of the same book. correct. (4) “For example” in the same book, page 28, line 14
I am corrected. (5) In the same book, page 31, line 12, "alcohol-ethyl acetate, etc." is corrected to "alcohol and ethyl acetate, etc.". Claims 1) New antibiotics having the following physical and chemical properties 446-53
--1゜111 Elemental analysis values: C48, 50%, H5, 49%, O33.71%, N12.30% (2) Molecular weight: Mass number 570 in secondary ion mass spectrometry (SI-MS) analysis (M+H)”, 592 (M+N
a) ", 60 From the observation of (M+K)" in B, the molecular weight is concluded to be 569. (3) Melting point: 173-176°C (4) Ultraviolet absorption spectrum: Maximum absorption in methanol (IMeOHnm(t)) is 214 (16200")
), a x 257 (shoulder 9800) is shown. As shown in Figure 1. (5) Infrared absorption spectrum: The infrared absorption spectrum measured with KBr is as shown in FIG. The wavelengths showing maximum absorption are as follows. The unit is (2) 1°3400.2930.1680.15
15.1270.1100° (6) Solubility in solvents: Very soluble in water and methanol, ethanol,
It is soluble in acetone, slightly soluble in chloroform, and insoluble in ethyl acetate, benzene, and n-hexane. (7) Color reactions: (Positive) Potassium permanganate reaction, Molisch reaction (Negative) Anthrone reaction, Aniline hydrogen phthalate reaction, Ferric chloride reaction, Ninhydrin reaction (8) Basic, neutral, acidic Classification: Weakly acidic (9) Substance color: White QIRf value Rainbow silica gel thin layer chromatography (TLC manufactured by Merck & Co.)
The Rf value when the plate (silica gel 60F 1 thickness 0.25 m) was measured in the usual manner is as follows. (a) Chloroform: methanol (2: 1) Rf value 0.43 (b) Benzene: methanol (2: 1) Rf value 0.
21? Q n-Pucnol: Acetic acid: Water (3: 1 j 1)
Rf value 0.43 01) Uracil and lysine are present in the acid hydrolyzate. (1) 'HNMR (400MHz, D20) δ(p
pm): 1.37 (IH), 1.64 (ill), 1.
78 (IH), 1.84 (IH), 1.93 (1) 1
), 2.00 (IH), 3.3 (1)1), 3.32
(3B,,s), 3.77 ←■, t), 4.2
3 (1)1), 4.4 (IHlt), 4.50
←■, t), 4, 53 (ill), 4.65 (1
8), 4.80 (IH), 5.4 (IHSd)
, 5.78 (IHld) , 5.88 (IH,d)
, 6.04 (IH) , 7.78 ←■, d), Q
"CNMR (L, OOMHz, D20) δ (pp
m): 2 B, 381 tl, 28.6 1 (t)
, 31.33 (tl, 42.33 (tl, 53, 1
01dl, 5 B, T Otq>, 62, 7 2
+dl, 65.7 2 (dl, 72, 80 +d
), 76.42 +d), 78, 97 (dl, 8
2.39 (dl, 90, 7 1 +d+, 100.
OO(dl, 102, 48 (d), 109.92
(dl, 141, 45 +d+, 142.06 +
31.151, 62 (S), 161.74 (S)
, 166, 3013), 173.20 +31.17
6.47 (31, 2) Antibiotics belonging to the genus Streptomyces 446-33
-1 production bacteria were cultured, and antibiotic 446 was extracted from the culture.
New antibiotic 44 characterized by collecting -33-1
6-33-1 manufacturing method. 3) The producing bacterium is Streptomyces griseus 446-
S 3- (Streptomyces grise
446-53).

Claims (1)

[Claims] 1) New antibiotic 446-33 having the following physical and chemical properties
-1゜ (1) Elemental analysis values: C48, 50%, H5, 49%, 033.71%, N12.30% (2) Molecular weight: In secondary ion mass spectrometry (SI-MS) analysis, mass number 570 (M+H)”, 592 (M+N
From the fact that (M0K)'' is observed at a)'', 608, it is concluded that the molecular weight is 569. (3) Melting point: 173-176°C (4) Ultraviolet absorption spectrum: Absorption maximum (λMeOHnmax (ε)) in methanol is 214 (16200), 257 (shoulder 98
00). As shown in Figure 1. Also, aqueous solution,
The ultraviolet absorption spectrum in 0.1N hydrochloric acid and 0.1N sodium hydroxide aqueous solution does not show a characteristic absorption maximum, but only shows terminal absorption. (5) Infrared absorption spectrum: The infrared absorption spectrum measured with KBr is as shown in FIG. The wavelengths showing maximum absorption are as follows. The unit is am-'. 3400.2930.1680.1
515.1270.1100° (6) Solubility in solvents: Very soluble in water and methanol, ethanol,
Soluble in acetone, slightly soluble in chloroform, and insoluble in ethyl acetate, benzene, and n-hexane. (7) Color reaction: (Positive) Potassium permanganate reaction, Molisch reaction (Negative) Anthrone reaction, Aniline hydrogen phthalate reaction, Ferric chloride reaction, Ninhydrin reaction (8) Basic, neutral, acidic Classification: Color of basic (9) substance: White Color Q@Rf value Rainbow silica gel thin layer chromatography (Merck TLC)
Plate, silica gel 60F2S4, thickness 0.25m)
The Rf value when this is carried out in the usual manner is as follows. (a) Chloroform:methanol (2:1) Rf value 0
．． 43 (b) Benzene:methanol (2:1) Rf value 0.
21 (1) n-butanol:acetic acid:water (3:1:1)
Rf value 0.43 (11) Uracil and lysine are present in the acid hydrolyzate (■). (12) 'HNMR(,400MH2,020)δ(
ppm): 1, 37 (LH), 1. 64 (
IH), 1,. 78 (IH), 1. 84 (I
H), 1, 93 (IH), 2. 00 (LH
) , 3, 3 (11-1> , 3. 32 (3H
, s) , 3, 77 (IH, t) , 4. 23
(IH) , 4, 4 (IH, t) , 4. 50(
IHS t), 4, 53 (IH), 4. 6
5 (LH), 4, 80 (IH), 5. 4
(IH, d) , 5, 78 (IH, d) , 5. 8
8 (IH, d), 6, 04 (LH), 7. 7
8 (IH, d) (13) ”'(: NMR (100
MH2, DgO) δ<ppm>: 28, 38 (t
), 28. 61 (t), 31, 33 (t)
, 42. 33 (t), 53, 10 (d)
, 58. 70 (q), 62, 72 (d),
65. 72 (d), 72, 80 (d), 7
6, 42 (d), 78, 97 (d), 82
．． 39 (d), 90, 71 (d), 100.
00 (d), 102, 48 (d), 109
．． 92(d), 141, 45(d), 14
2.06 (s), 151, 62 (,s), 1
61. 74 (s), 166, 30 (s),
173. 20 (s), 176, 47 (s) 2) Antibiotic 446-3 belonging to the genus Streptomyces
Cultivate the producing bacteria of 3-1 and extract antibiotic 44 from the culture.
New antibiotic 4 characterized by collecting 6-33-1
46-33-1 manufacturing method. 3) The producing bacterium is Streptomyces griseus 446-
33 (Streptomyces griseus
446-33).