JPS62114947A - D-beta-lysylmethanediamine and production thereof - Google Patents

Abstract

NEW MATERIAL:D-beta-Lysylmethanediamine shown by the formula and its acid addition salt. USE:An antibacterial agent, an antibiotic effective against Gram-positive bacteria, having immunologically enhancing action. Useful as a raw material for antibiotic deoxynegamycin effective against Gram-positive bacteria. PREPARATION:A bacterium [e.g., MD743-GF4 strain] belonging to the genus Streptomyces, capable of producing D-beta-lysylmethanediamine is inoculated into a nutritive source-containing medium, cultivated under an aerobic condition, adsorbed on active carbon, etc., and eluted with an aqueous solution of methanol to give the aimed substance. Properties: D-'-lysylmethanediamine which is a strongly basic substance and isolated as 1/2 carbonate is hygroscopic colorless powder; specific rotatory power of 1/2 carbonate [alpha]D<26>=-7.4 deg. (C 0.5, water) and [alpha]D<26>=-21.3 deg. (C 0.46, 0.08N hydrochloric acid); elemental analysis value (%) C 44.75, H 8.63, N 26.50, O 20.62; molecular formula C7H18N4O.1/2H2CO3, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel antibiotic, D-betalysylmethanediamine, and to a method for producing this compound by culturing a microorganism belonging to the genus Streptomyces.

The present inventors isolated actinomycetes M D743-GF from a soil sample within the premises of the Microbial Chemistry Research Institute in Kamiosaki, Tokyo's Parts Industry Ward.
It was discovered that strain A could be cultured to produce and accumulate D-peterinolmethanezoamine, and a new compound, D-peterinolmethanediamine, could be collected from the culture.

D-peter lysolmethanoamine is an antibiotic effective against Durham-positive bacteria, and also has an immune-enhancing effect. In addition, D-peterinone obtained by hydrolysis of D-betalysylmethanoamine is useful as a starting material for the synthesis of deoxynegamycin, an antibiotic effective against Durham-negative bacteria invented by the present inventors. Japanese Patent Application Publication No. 5
2-5'? No.l12, published on May 16, 1978).

The first gist of the present invention is D-betalysylmethanecyamine represented by the following formula (1) N and its acid addition salt as a new compound.

The physicochemical and biological properties of this new compound are as follows. Amines are hygroscopic, colorless powders with no definite melting point. The specific optical rotation of this 1/2 carbonate is [α]D
= −7,4°(co, s, water) and [α), =−
21.3° (co, 46,0.08N hydrochloric acid), and the elemental analysis values are C44.75 and H8.63.

N 26.50%, 020.62%, C7
Molecular formula of H18N40-I/2H2C03 (theoretical value:
C43,8'? %, H'? , 33 s, N27.
30%, 019.49chi) was estimated, and mass spectrometry (
SIMS) by m/z l 75 (MH)
showed that. The Kutle curve of the ultraviolet absorption of D-betalysylmethanediamine in a water bath solution does not show any specific absorption. The infrared absorption spectrum curve measured with potassium bromide tablets is as shown in FIG. 1 of the attached drawings. In heavy water (pD
Chemical shift of the nuclear magnetic resonance spectrum measured in 6) (
δ, ppm) are as shown in Table 1.

1 4.56 s 2H46,6+3
174.31!4 2.76 dd
IH37,4+2.90 dd 1) 1 5 3.77 br IH49,3d7
24.0 +83.09 br 2H4
0,2t D-betalysylmethanecyamine is well soluble in water, but poorly soluble or insoluble in common organic solvents.

ninhydrin reaction and Lydon-Smith reaction. Thin layer chromatography of cellulose (Avicel SF, Funakoshi Pharmaceutical Co., Ltd.) (developing system Niglo-inol-pyricine-acetic acid-water, +5:10:3:12 volume) revealed Rf.
Migration of alanine was performed by high-pressure paper electrophoresis (3.300 V, 15 min) using formic acid, acetic acid, and water (1:3:36 volume). When the mobility is 1.0, the mobility of D-betalysylmethanecyamine is 2.50.

Picrate of D-betalysylmethanecyamine is a yellow crystal with a decomposition point of 18+-1g3°C, and elemental analysis values: C34.74*, H3, 21%, N 21.4.
0chi, 040.95%, C7H18N40・3C6
H3N50. Theoretical value of (C34,gs%,
H3, 16 da 6. N21. +3 la 6.

D-betalysylmethanecyamine obtained in the present invention (
1/2 carbonate), Staphylococcus aureus (Smith strain), Bacillus subtilis (B-558) measured on 0.5% pegtone agar flat root.
strain and PCI219 strain) were each 50 mcg/-. Furthermore, hysozo red blood cells (SRBC) were administered as an antigen to mice that had been intravenously injected with D-betalysylmethanecyamine every day, and after a few days, the spleen was removed and the number of antibody-producing cells was determined for the spleen cells. As a result of measurement, it was found that the number of antibody-producing cells was increased compared to the control test, and D-betalysylmethanecyamine was recognized to have a humoral immunity-enhancing effect. In addition, 5RBC was administered as an antigen to mice that had been intraperitoneally administered with D-beta-lysylmethanecyamine every day, and after the establishment of immunity to 5RBC, 5RBC was administered again, and then the mouse paws were injected with D-betalysylmethanecyamine intraperitoneally. Delayed Type Hypersensitivity (Delayed Type Hypersensitivity) is a method that calculates the increase in thickness, that is, the rate of increase in edema.
ivity), it was found that the cell-mediated immunity-enhancing effect was observed.

In an acute toxicity test of D-betalysylmethanecyamine administered intravenously to mice, there were no deaths at a dose of 250 μ/kg.

The D-betalysylmethanecyamine according to the first invention can be obtained as a free base or its hydrate or charcoal salt, but it can also be prepared by adding a pharmaceutically acceptable acid in a conventional manner. It can be any non-toxic acid addition salt. Examples of the acid to be added include inorganic salts such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, acetic acid, malic acid, citric acid, ascorbic acid, methane, etc. Organic acids such as sulfonic acid and picric acid are used.

The second gist of the present invention is to cultivate D-betalysylmethanediamine-producing bacteria belonging to the genus Streptomyces, and to collect D-betalysylmethanediamine from the culture. It is in the manufacturing method of Noamine.

According to the method of the invention, D-betalysylmethanediamine is obtained in pure or crude form,
It can also be collected in solution or solid form.

An example of a D-betalysylmethanediamine-producing bacterium is an actinomycete that was isolated from the soil on the premises of the Institute of Microbial Chemistry in February 1970, and was assigned the strain number MD743-GF4.

The mycological properties of the MD743-(, FA strain are as shown below. 01, Morphology The MD743-GF'4 strain forms 9 spiral aerial hyphae with branched basal hyphae, and rings. No live branches were observed.Mature spore chains were found to be 20 (!r, J or more), and the size of the spores was 0.6 to 0.7
The surface of the spore is smooth, measuring 1.4 to 1.7 microns.

2. Regarding the description of the growth state color for each medium, the standards shown in [ ] are for containers and
Container Corporation of America Color Bar Sony 6 Manual
ion of America's Co1or harm
onemanual) was used.

(Sucrose/nitrate agar medium (27″c culture) Bright straw ash [4ge, Rose
Beige] aerial mycelium grows loosely and dissolves +'
QHFJ element is not recognized.

(2) Glycerin/nitrate agar medium (27°C culture)
Light yellow C2gc, Bamboo] ~ Nibu yellow [3
pC1Amber], white aerial mycelium grows loosely on the growth, and the soluble pigment has a yellowish tinge.

(3) Glucose-asparagine agar medium (27
C4ge-Rose Beige on the growth of pale yellow C2gc, Bamboo)
] Aerial mycelium grows on it, and no soluble pigment is observed.

(4) Yeast/malt agar medium (l5p-medium 2
．． 27℃ culture II) Light yellow tea C3ng, YellowM
A bright straw ash C3dc, Na
tral ~5 fe, Ashes], and no soluble pigments are observed.

+51 # - Tomil agar jib (ISP-medium 3.27c'c culture) light yellow [3ie, Gamel
] On the growth of bright straw ash C5fe, Ashes
], and no soluble pigments are observed.

(6) Starch/inorganic salt agar medium (l5P-medium 4
, 27°C culture) Colorless to light brown C31g, Lt Br
oi) on the development of bright straw ash [5fe, As
hes], and no soluble pigments are observed.

(7) Glycerin-asparagine agar medium (l5P
-Medium 5°27'C culture) Light brown C31)g, Golden BroWn]
On the growth of white to light straw ash C5fe, Ashes
], and the soluble pigment is only yellowish.

17 ng Tyrosine agar medium (l5P-medium 7.27°C culture) Light tea C, 3 ng, Yellow Maple
] On the growth of bright gray C3dC, Natural
] ~Bright straw ash [: 5 fe.

Aerial mycelia of Ashes] are attached, and almost no soluble pigment is observed.

(8) Growth on malic acid lime agar medium (2n culture): pale yellow C2ea, Lt Wheat], aerial mycelium exhibits a pale white color, and no soluble pigment is observed.

(9) Nutrient agar medium (27℃ culture) growth is light tea (
2pi, M, 1sLard Brown), no aerial mycelia were attached, and no soluble pigment was observed.

αG starch agar medium (27°C culture) light yellow (2g
c, Bamboo] on the development of Akuru (, -.

Straw ash [: 3 dC, Natural ~5 fe,
Aerial mycelium of Ashes] is attached, and no soluble pigment is observed.

αυ Skimmed milk (cultured at 37°C) Pale yellow C2ic, I (oney gold) Aerial mycelia of brown color are grown on the growth, and the soluble pigment is only slightly brownish.

+13 Gelatin puncture culture In simple gelatin medium (20°C culture), light yellow tea C3
ne, Topaz], a thin white to gray aerial mycelium grows on it, and the soluble pigment is brownish. In glucose-Happton-gelatin medium (cultured at 27°C), the growth was light yellow brown (2ic.

Honey Gold), aerial mycelium did not grow,
Soluble pigments exhibit a dark brown color.

a3 Cellulose (cultured at 27°C) Brown-colored aerial mycelium grows on the colorless growth, and no soluble pigments are observed.

3. Physiological properties fil Growth temperature range Yeast starch agar (yeast extract (Inugo Nutrient Chemicals N) 0.2%, soluble starch (Kokan Kagaku) 1.0
%, string agar 3. Os, pf(7,0) woyoi, 2
0°C924°C927°C, 30'C, 37°C, 5
As a result of testing at each temperature of 00C, growth occurred at all temperatures except 50'C, but the optimum growth temperature was 24°C.
It is thought to be around 27°C.

(21 Liquefaction of gelatin (15% simple gelatin, 20'C
Culture: Glucose-Begton-Gelatin, 270C culture) Liquefaction begins around the 14th day after cultivation in simple gelatin medium, and around 10th day after cultivation in the case of glucose-begton-gelatin medium.

Both effects are moderate.

(3) Hydrolysis of starch (starch/inorganic salt agar medium and starch agar medium, both cultured at 27°C)
In starch inorganic salt agar media, ice-melting properties have been found to occur from around the third day after cultivation, and in starch agar media, from around seven days after cultivation, and the effect is stronger.

◆ (4) Coagulation and vegtonization of skim milk (10% skim milk (Difco), cultured at 37°C) Weak vegtonization begins around the 9th day after culture and is completed around the 13th day. Its effect is strong.

No coagulation is observed.

(5) Production of melanin-like pigments (Tryptone Yeast Zoros, l5P-Medium 1: Beptone Yeast Zoros,
Iron agar.

15P-medium 6; tyrosine agar, 15P-medium 7°, both cultured at 27°C) Production of melanin-like pigments was observed in both media.

The effect is moderate for tryptone yeast gloss, strong for begtone yeast and iron agar, and weak for tyrosine agar.

(6) Utilization of carbon source (Gritnomuco9Treep agar medium.

rsp-medium 9.27°C culture) Using glucose and raffinose to produce WL, L-arabinose, D-xylose, D-fructose,
Sucrose, inositol.

L-ymnose and D-mannitol are used.

(7) Dissolution of malate lime (malate lime agar, 27°
C culture) Dissolution of lime malate was observed, and the effect was moderate to strong. (8) Nitrate reduction reaction (peptone water containing 0.1% potassium nitrate.

l5P-medium g, 27″c culture) It is positive.

To summarize the above characteristics, strain MD743-GFA belongs to the genus Streptomycetes, and the 2,6-diaminopimelic acid contained in the cell wall is of the LL-type. The hyphae have a spiral formation and no whorled branches are observed. Also, the surface of the spore is smooth. In various media, colorless to light yellow brown, or white to light brown aerial mycelia grow on the growing light brown, with almost no soluble pigments or a slight yellowish tinge. The melanin-like pigment is positive, the proteolytic ability is moderate, and the starch ice-melting ability is strong.

Based on these properties, a search for known bacterial strains closely related to the MD743-GFA strain revealed the following species.

i.e., Streptomyces natushpilev si x
(Streptomyces nashvillen)
sis, Literature International Journal
al of SystematicBa6teriol
ogy, vol. 19, p. 455, +969: Antib
iotics and chomotherapy,
Volume II, 312 Sada.

+961), Streptomyces misakiensis (
Streptomyces m1sakienais.

LiteratureInternational Journal o
f Systematic Bacteriology,
1g, p. 348, +968), and Streptomyces xanthocidi
cus, Literature International Journal
al of Systematic Bacteriol
ogy, Vol. 22, p. 372, +972). These three ISP strains were obtained and compared with the MD743-024 strain. As a result, Streptomyces misakiensis has extremely weak proteolytic ability and uses sugars to produce D-xylose and D-7 lactose.

It is distinguished from the MD743-GFA strain in that it uses sucrose. On the other hand, the Streptomyces ,M
It showed major differences from the D743-024 strain. 7. Remaining Streptomyces natuschvirensis and u, iJ, H. The M results are shown in Table 2 below.

As shown in Table 2, there is good agreement with MD743-GFA strain Tostrezotomyces natushpirensis except for the availability of L-arabinose and D-xylose.

Regarding the utilization of L-arabinose by the MD743-CF4 strain, fluctuations were observed in repeated experiments, and there were cases where a slight amount was utilized. Considering this point, it is thought that the only definite difference between the two is the use of D-xylose.

From these facts, MD743-CF4 strain is Streptomyces nashvi/I/x
It was identified as Streptomyces naahvillensis MD743-cr4.

The MD743-CF'4 strain was submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology for storage entrustment on September 7, 1985, and the accession number is Fiber Science and Technology Research Institute No. 8442.

Streptomyces tend to undergo mutations both artificially and naturally, and the term Streptomyces nasgiluensis referred to in the present invention includes all such mutants. D- in the present invention
The beta-lysylmethanediamine-producing bacteria produce D-beta-linolmethanediamine and produce the above MD743-C.
The method of the present invention includes all strains belonging to the genus Streptomyces that produce D-betalysylmethanediamine, including strains that cannot be clearly distinguished from the F4 strain and its mutants. Inoculating a diamine-producing bacterium, particularly the above-mentioned Ml)743-CF4 strain into a nutrient-containing medium that can be used by ordinary microorganisms, and culturing it.
Preferably, by growing under aerobic conditions, D-betalysylmethanoamine is mainly produced and accumulated in the culture solution, and the target product is further separated from the culture, particularly from the culture solution.

As the nutrient source in the medium used, any known nutrient source used as a nutrient source for actinomycetes can be used. For example, commercially available soybean flour, peanut flour, cottonseed flour, dry yeast,
Nitrogen sources such as meat extract, casein, corn steep liquor, sodium nitrate, sulfuric acid, and commercially available glucose, 7fj cutol, 61, glycerin, maltose, dextrin, sucrose, Carbohydrates such as lactose, or carbon sources such as soybean oil and fat, as well as salt, calcium carbonate, magnesium sulfate,
Inorganic salts such as manganese chloride, ammonium sulfate, phosphates, and various amino acids can be used.

Any of these materials may be used as long as they are utilized by D-betalysylmethanediamine-producing bacteria and useful for producing D-betalysylmethanediamine, and all known culture materials for actinomycetes can be used. For large-scale production, liquid aeration stirring culture is preferable, and it can be applied within a culture temperature range that allows D-betalysylmethanediamine-producing bacteria to grow and produce D-betalysylmethanediamine, but particularly preferred is 25% The incubation of OD-beta-lysylmethanediamine is continued until sufficient accumulation of eA,D-peter-linol-methanezoamine is obtained, and the culture is usually carried out for 2 to 7 days. Using Pacilus subtilis strain PCI219 as a test bacterium, the purified D-beta-lysylmethanecyamine obtained in the present invention was purified by using the ordinary cylindrical-plane root method used for the determination of antibiotics.
It was used as a standard material as 00O (unit)/q.

D-betalysylmethanediamine and its salts are highly soluble in water and are present primarily in the liquid portion of the culture of D-betalysylmethanediamine producing bacteria. Since D-betalysylmethanediamine in liquids is not substantially extracted by organic solvents such as getanol, butyl acetate, and chloroform, treatment with these solvents can be used if necessary for the removal of contaminants@culture medium or D-betalysylmethanediamine in an aqueous solution can be collected by adsorption using various adsorbents. When activated carbon is used as an adsorbent, the adsorbed antibiotics are weakly acidic water, weakly acidic methanol water, and Glo! It is eluted with ethanol water, acetone water, etc.

Also, D-peterinolmethanezoamide/ is adsorbed to a cation exchange resin in good yield and eluted with a suitable eluent due to its basic nature.

This adsorption/elution method is the most suitable method for collecting from a large amount of culture fluid. As a cation exchanger, Annoglite IRC-50, CG, which has carboxylic acid as an active group,
-50 (e+-mu-7-7-/%-S), Rewacit GNP (Bayer), CM-Sephadex (Pharmacia) Natono H type, Na type, NH4 type, etc., and mixed types thereof is used, and the adsorbed antibiotic is eluted with good yield by acidic water, dilute ammonia water, an aqueous solution of inorganic salt, etc., and 0.2-IN hydrochloric acid and 0.2N-IN ammonia water are usually used. Since D-betalysylmethanecyamine is not substantially adsorbed on anion exchange resins, it is a preferred material for neutralizing acidic solutions and removing acidic contaminants.

D-betalysylmethanecyamine can be purified in a pure manner by appropriately combining or repeating the above-mentioned extraction methods and separation methods.

The present invention will be explained below with reference to Examples. Since the properties of D-betalysylmethanecyamine have been clarified by the present invention, the MA of D-betalysylmethanecyamine is Various m methods can be devised. Therefore, the present invention is not limited to the examples, and the modification means of the examples as well as the properties of D-peterizolmethanoamine clarified by the present invention may be applied to known methods. It includes all methods for producing, concentrating, extracting, and purifying D-betalysylmethanecyamine.

Example 1 Streptomyces natushvirensis MD743-GFA strain cultured on agar slant medium
No. 2), galactose 2.0%, dextrin 2.0
%, Begton (Bactosoiton, manufactured by Difco) 1.01
0.5 inch corn steep liquor (manufactured by Ajinomoto),
A liquid medium containing 0.2% ammonium sulfate and 0.2% calcium carbonate (110ml in a 5000 Erlenmeyer flask) was inoculated and cultured with rotary shaking (180 revolutions per minute) at 28°C for 2 days to obtain a seed culture. Ta. This seed culture was inoculated into the same medium (IIO+t/) as above and grown under the same conditions for 9 hours.
0 Erlenmeyer flasks were cultured for 3 days, 90 this culture solution was collected and passed through a 9000-pI solution (pI-1
6,0,80mcg/d) was obtained. Remove this R liquid/
G-Light I RC-50 (NH4 type: H type, 7:3)
Passed through a column packed with 550- to adsorb D-betalysylmethanecyamine, and after washing with water (1・+00-), 1
．． Elution was performed with 2N aqueous ammonia. Active eluent (1370
m/) was collected and concentrated and dried under reduced pressure to obtain a crude powder of 636+n9(
644mcg/+W) was obtained.

This coarse powder was dissolved in IQ++y water, adsorbed in a column filled with 60- of Anno-Nite CG-50 (NH4 type), water + zo- (fraction 1-18), 300 m of 0.6N ammonia water (min. After washing with
1.2N ammonia water 30 o-(fraction 6l-1ot)
It was eluted. Fractions 77-95 were combined and concentrated to dryness under reduced pressure.
Upon further drying, the heptacarbonate of D-betalysylmethanecyamine was obtained as a colorless powdery substance (hygroscopic). The yield is 370 Trq (1,000 mcg/m
y), the yield of the culture solution was 50.8 h. Specific rotation of this substance [α] 0-7.4° (C0,5, water)
showed that. When this substance absorbed moisture, it became colorless and eye-shaped.

[Brief explanation of drawings]

FIG. 1 shows an infrared absorption spectrum curve of D-betalysylmethanecyamine carbonate according to the invention measured in a potassium bromide tablet.

Claims (1)

[Claims] 1. D-betalysylmethanediamine and its acid addition salt represented by the following formula (I) ▲Mathical formula, chemical formula, table, etc.▼(I). 2. A method for producing D-betalysylmethanediamine, which comprises culturing a D-betalysylmethanediamine-producing bacterium belonging to the genus Streptomyces and collecting D-betalysylmethanediamine from the culture.