JPS5932120B2 - Method for producing 9-β-D arabinofuranosyl adenine - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing the compound 9-β-D arabinofuranosyladenine (hereinafter abbreviated as CI).

The compounds of formula CI,l are known substances, for example J
our own of the american
Chemical Society Vol. 82, 264
8-2649, 1960.

The present inventors have conducted various studies on the mycological properties of the actinomycete strain AM-3279, which was newly isolated from the soil of Kitakoma District, Yamanashi Prefecture, and the fermentation products produced by this bacterium. The microorganism is a new strain (Streptomyces herbaceus) that belongs to the genus Streptomyces but does not belong to any known species.
), and that when the above-mentioned microorganism is cultured in a medium, a compound [■] having antiviral and herbicidal effects is produced and accumulated in the culture, and that the compound [■] has been accumulated in this way. ] was found to be easily separated and collected from the culture medium, leading to the completion of the present invention.

The method for producing CI, 1 will be described in detail below. A, AM-3
Identification of Strain 279 The mycological properties of the AM-3279 strain producing CI) are as follows.

(1) Morphological characteristics This strain does not form prespores, and the spore-bearing hyphae have the morphology of the genus Streptomyces, which is simply branched. live.

The tip of the aerial hyphae has a spiral shape, and a chain of 10 or more spores is observed.

The size of the spore is 0.6 μ x 1.0 to 1.1 μ, circular, and its surface has a smooth structure.

(2) Properties on the medium The properties when cultured on various media at 27°C for 2 weeks are as shown in Table 1.

(3) Physiological properties The physiological properties of AM-3279 strain are shown in Table 2.

(4) Carbon source assimilation The carbon source assimilation ability of AM-3279 strain on Prudham-Gottlieb agar medium is shown in Table 3.

As mentioned above, AM-3279 strain (1) The tips of aerial hyphae exhibit a linear shape.

(2) Spores are smooth.

(3) Both synthetic media and protein-containing media show pale yellow, yellow, or yellow-brown growth.

(4) The color tone of aerial mycelium varies slightly depending on the culture medium, but it is green or pale green, with green as the base tone.

(5) Almost no soluble pigments are produced, with the exception of 2 to 30.

It showed this characteristic.

Based on these characteristics, Waxman [The Actinomycetes Volume 2] [International Journal of Systematic Bacteriology Volume 1]
Vol. 8, pp. 69-189 and pp. 279-392 (1968)
, Vol. 19, pp. 391-512 (1969), and Vol.
2, pp. 265-392 (1972),” “Purge Aids.
Manual of Determinative Bacteriology, 8th Edition (1974)
us), Streptomyces-bilitosporus (S tr
eptomyces viridosporus) is mentioned as a similar bacterial species.

In addition, the producing bacterium [■] is Streptomyces antibiotics.
Ibiotics) (Ruskin et al. [Handbook of Microbiology Volume 2]) is known.

Therefore, a comparison with these three strains was made according to the above-mentioned "Purge Aids Manual of Determinative Bacteriology, 8th Edition". However, the spore surface of AM-3279 strain is smooth and does not assimilate sucrose, and the surface of Streptomyces viritosporus spores is also spiny, and Streptomyces antibiotics is generally It forms gray spores on agar medium, settles in a flexible manner, and is chromogenic, but the spores of AM-3279 strain are light green or green, settle in a spiral shape, and are non-chromogenic. They are clearly distinguished in one respect.

The above differences are that Streptomyces hirsutus and Streptomyces viritosporus are [Purges Manual of Determinative Bacteriology, 8th Edition (1974, according to the classification method of fragments, 17.46b (G
N; S; C-; 5PY), Streptomyces antibiotics is 17.42a (GY; RF; C-; SM), and strain AM-3279 is (GN; S
;C-;SM).

In addition, strains belonging to (GN; S; C-; SM), such as this strain, are published in "Manual of Systematic Bacteriology Volume 8" and "International Journal of Systematic Bacteriology". stomach"
There is no mention at all.

Therefore, the actinomycetes that produce CI) of the present invention are recognized as a new bacterial species, and because they grow green aerial hyphae, they are known as Streptomyces herbaceus.
erbaseus).

This strain has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as Application No. 4334 for consignment of microorganism storage.

B. Culture of AM-3279 strain In culturing the present bacterium, a normal actinomycete culture method is generally used.

Various nutritional sources are used for culture, and carbon sources include glucose, starch, mannitol,
Fructose, galactose, rhamnose, etc. are used alone or in combination.

Inorganic and organic nitrogen sources are used, including ammonium chloride, ammonium sulfate, urea, ammonium nitrate, sodium nitrate, and natural nitrogen sources such as peptone, meat extract, yeast extract, dried yeast, corn steep liquor, Soybean flour, soybean flour, casamino acids, soluble vegetable proteins, etc. can be used alone or in combination.

In addition to adding inorganic salts such as table salt, potassium chloride, calcium carbonate, and phosphates, the growth of this bacterium and the compound CI)
without interfering with the addition of organic or inorganic substances that promote the production of

The culture method is liquid culture method, especially deep stirring.

Culture method is most suitable.

It is desirable to culture at a culture temperature of 27 to 35°C and a pH around neutral.

When culture is carried out in a liquid culture for usually 2 to 5 days, the substance of the present invention (CI) is produced and accumulated in the culture solution.

Changes over time in the titer of [I] produced in the culture solution as the culture progresses can be determined by comparing the culture filtrate with filter paper (Toyo Roshi A6.5).
0), developed with a saturated solution of developing solvent n-butanol and water, cut out the section of CI) of RfO828, eluted with methanol, and measured by a quantitative method based on its absorbance at 260 mμ.

Stop the culture when the production amount in the culture medium reaches the maximum,
The bacterial cells are separated by filtration, and the target product is purified and isolated from the resulting culture filtrate.

Purification and isolation of C1 Compound CI) For purification and isolation of the culture filtrate, the substance of the present application can be purified by separation and purification methods generally used to isolate metabolic products of microorganisms from their culture fluids.

In the case of deep culture method, it is preferable to carry out as follows.

That is, the mycelium is filtered or centrifuged, the filter cake is thoroughly washed with water, the washings and filtrate are combined and concentrated under reduced pressure to about one-twelfth of its original volume.

The concentrated solution is cooled for a long period of time (from several hours to several days depending on its volume) at about 5° C., and the precipitated solid is filtered off using diatomaceous earth.

The filter cake is thoroughly extracted with boiling water, and the extracts are combined, concentrated, and then cooled to about 5° C. until complete precipitation occurs.

The precipitated crystalline [■] is filtered out and further purified by repeated recrystallization from boiling water.

According to another method, the substance of the present application can also be obtained from the deep culture solution by employing the following adsorption technique.

That is, the crude product is adsorbed by treating the culture filtrate with activated carbon or other adsorbent, strongly basic ion exchange resin.

This adsorption can be done in batch mode and
It can also be carried out by continuous flow through the adsorption tower.

In the batch method, the filtrate is added with activated carbon adsorbent at 0.1
~0.6% w/v, preferably 0.35-0.40% w/v is added and the mixture thus obtained is stirred for 1-3 hours.

The crude product is isolated by eluting the activated carbon adsorbent with aqueous acetone or aqueous lower alkanol and evaporating the eluent under reduced pressure.

Alternatively, the culture filtrate is made alkaline (pH 10,0) and the substance of the present invention is extracted with an organic solvent such as ethyl acetate.

The crude product is isolated from this extract by removing the solvent by concentration under reduced pressure.

The solid residue thus obtained is repeatedly crystallized from water or lower alkanols or purified by the following method.

That is, the solid residue is extracted with a water-immiscible liquid alkanol such as n-butanol, and the extract is poured into a silica gel column (PH 5-6).

This silica gel column was mixed with chloroform:methanol (1:1).
), evaporation of the eluent, and crystallization of the solid obtained from water or a lower alkanol to obtain the substance CI).

The results of infrared spectroscopic analysis of this substance confirmed that its absorption characteristics were the same as those of 9-β-D arabinofuranothreanine (Fig. 1).

The product CI) produced by the method of the present invention is useful as an anticancer agent against leukemia or as a herbicide, and was approved by the US FDA in 1976 as an anti-herpetic agent.

Hereinafter, it will be explained in detail using examples.

Example 1 Streptomyces herbaceus
No. 4), glucose 4.0%, soybean flour 1.0%, potassium chloride 0.4%, yeast extract 0.25%, meat extract 0
61%, sulfuric acid 77-E = medium containing 0.5% rum, 0.02% dipotassium phosphate, and 0.3% calcium carbonate (pH 7.2 before sterilization) was inoculated into 10 ml (24% diameter test tube). A seed culture solution was obtained by culturing with shaking at 30° C. for 3 days.

Add 2 ml of this seed culture to 100 ml of a medium with the same composition as above.
Inoculate a 500 m Erlenmeyer flask containing
Culture was performed at 30°C in an Or, p, m shaking culture machine.

144 μft /rn/ after 96 hours of culture! production volume has been reached.

At this point, the culture was stopped and purification was performed.

This culture solution 11 was centrifuged at 7000 r, p, m, and the supernatant was adjusted to pH 3.0 with hydrochloric acid.
Impurities were extracted twice with 500ml and 300ml of ethyl acetate.

This extraction residue was adjusted to pH 10.0 with 40% caustic soda and extracted twice with 500 ml and 300 ml of ethyl acetate.

This ethyl acetate layer was concentrated to obtain 150 ml of concentrate.

Add this concentrate to chromatographic silica gel 2? The mixture was subjected to column chromatography.

CI) was eluted in chloroform:methanol (1:1).

Crude crystals were obtained by concentrating this eluate under reduced pressure.

Reconcile with boiling methanol. Purified by crystallization.

The melting point was 259-260°C.

The yield of purified CI, 1 was 30 μl.

Example 2 A liquid medium 451 having the same composition as shown in Example 1 was added to 3
Dispense 151 of each into 3 OZ capacity jar fermenters, inoculate each 300 ml of seed culture that had been sterilized and cultured for 72 hours in a medium with the same composition.
Culture was carried out at 0°C for 67 hours (aeration rate 151/min, rotation speed 300 r, p, m, internal pressure 0.5 kg/crA
).

This culture filtrate was transferred to a Sharpless centrifuge 10000r, p.
The bacterial cells were removed with m, and the resulting supernatant was concentrated under reduced pressure to a concentration of 51%, and then this concentrate was treated with activated carbon (e.g., purified Shirasagi) iooy, stirred at room temperature for 1 hour, and then filtered. did.

After washing the activated carbon cake with 31 parts of water, it is extracted three times with 51 parts of 50% aqueous acetone.

These three aqueous acetone extractions were combined and concentrated under reduced pressure to about 500 ml, then cooled to 5° C. for 48 hours.

The thus precipitated CI) was isolated and purified by successive crystallization from boiling methanol.

The melting point was 259-260°C and the yield was 910m9.

Example 3 Liquid medium 2001 having the same composition as shown in Example 1 was
Prepared in two fermentation tanks of 0.001 volume, after sterilization, seed culture solution that had been cultured for 72 hours in a medium of the same composition was inoculated into each tank at a concentration of 2.41, and cultured at 30°C for 77 hours. (Air flow rate 10B'/min, rotation speed 30 Or, p
, m,, internal pressure 0.5 kg/aA).

After the cultivation was completed, the bacterial cells were removed using a filter press filtration machine to obtain filtrate 2001 (containing some tank washing liquid).

40 (l) of activated carbon (special Shirasagi) was added to this liquid, and the mixture was stirred for 2 hours to adsorb the crude product, which was then filtered.

Next, pour this activated carbon cake into boiling ion exchange water 301 and 20
1 was eluted twice.

This extract was concentrated to about 1/20 and cooled to 5°C.
I left it there day and night.

The obtained precipitate was filtered, dissolved in boiling methanol, and heated at 5°C.
It was crystallized by cooling to .

This crude crystal was repeatedly recrystallized to obtain 5.11.

The melting point was 258-260°C.

[Brief explanation of drawings]

Figure 1 shows that the substance produced by the method of the present invention is 9-β.
It is a characteristic absorption diagram of an infrared absorption spectrum showing that it is -D arabinofuranosyl adenine.

Claims (1)

[Scope of Claims] 1 Actinomycete Streptomyces strain Streptomyces herbaceus
9-β-D arabinofuranosyl adenine expressed by the formula was cultured aerobically in a medium containing a carbon source, a nitrogen source, inorganic salts, and vitamins. A method for producing the compound, which comprises producing and accumulating the compound and collecting it from a medium.