JP2966859B2 - Novel bioactive substance dioctatin and method for producing the same - Google Patents

Description

The present invention relates to a novel bioactive substance, dioctatin.
n) and a method for producing the same.

[Conventional technology]

The enzymatic activity of dipeptidyl aminopeptidase II is significantly increased in collagen diseases such as systemic lupus erythematosus and rheumatoid arthritis, and is suggested to be closely related to the pathogenesis [M. Hagihara et al., Clinical・ Chemistry (Clinical Chemistry), Vol. 33, No.
1463-1465, 1987]. As a dipeptidyl aminopeptidase II inhibitor produced by microorganisms, Puromycin has been reported by JK McDonald et al. [AJ Barret, Ed., Proteases in. Manmarian Cells and Tissues (Proteases in
mammalian cells and tissues), pp. 311-391, North-Holland Publishing Company, Amsterda
m), 1977].

[Problems to be Solved by the Invention] Puromyin is dipeptidyl aminopeptidase
It is known to inhibit II and also inhibit other aminopeptidases. Therefore, specific inhibitors of aminopeptidase II are desired.

An object of the present invention is to provide such a highly specific physiologically active substance and a method for producing the same.

[Means for solving the problem]

In summary, the first invention of the present invention relates to a novel bioactive substance dioctatin, and has the following general formula I: [Wherein, R represents hydrogen (B) or a methyl group (A)], which is a physiologically active substance dioctatin or a salt thereof.

The second invention of the present invention relates to a method for producing a novel bioactive substance, dioctatin, which comprises culturing a dioctatin-producing bacterium belonging to the genus Streptomyces and, from the culture, the physiological activity represented by the above general formula I. It is characterized in that at least one of the substances dioctatin A and B is separated and collected.

An example of a dioctatin-producing bacterium belonging to the genus Streptomyces is Streptomyces sp. SA-2581 (Streptomyces sp. SA-258) isolated by the present inventors.
1) There is. The bacteriological properties of this strain are as follows.

This strain is an actinomycete with a hyphal width of 1 μm or less, and the spore chain on the aerial mycelium of the actinomycete is open spiral under an optical microscope. Under an electron microscope, rod-shaped spores are observed, and the spore surface is smooth. As a component of the cell wall, L, L-diaminopimelic acid is detected,
Electron microscopy shows no spore capsule or other characteristic structures. Thus, this bacterium belongs to the genus Streptomyces. Tables 1 to 3 show the culture properties, physiological properties, and the presence or absence of the ability to assimilate sugars of this bacterium.

Regarding the description of the colors in the various media, the color harmony manual of Container Corporation of America (Color harmony manual) was used as the standard shown in parentheses. All property tests were determined after 2 weeks culture at 27 ° C.

Table 2 Physiological properties Liquefaction of physiological gelatin Positive starch hydrolysis Positive casein hydrolysis Positive urea hydrolysis Positive esculin hydrolysis Negative Catalase test Positive Cellulase activity Negative Tyrosinase activity Positive Nitrate reduction Positive Peptone conversion of skim milk Positive skim milk coagulation Negative production of hydrogen sulfide Negative Salt tolerance concentration 6% Growth temperature 15-34 ° C Table 3 Utilization of sugar Utilization D-glucose positive D-fructose positive L-arabinose negative N-D-xylose negative L-rhamnose positive sucrose False positive Raffinose positive D-mannitol positive inositol False negative or higher indicates that the bacterium belongs to the genus Streptomyces.

In addition, SA-2581 strain was applied for deposit at the Institute of Microbial Industry and Technology of the National Institute of Advanced Industrial Science and Technology, and on June 7, 1989,
No. (FERM P-10778). The strains that can be used in the present invention include all the dioctatin-producing bacteria belonging to the genus Streptomyces, including the above-mentioned strains and mutants thereof.

Follow-up during the culture and purification steps of dioctatin was carried out using the following method: anti-dipeptidyl aminopeptidase
It was performed based on the measurement of II activity. For the measurement, the measurement method of JK McDonald et al. [The Journal of Biological Chemistry (The Journa
l of Biological Chemistry), Volume 243, Volumes 4143-4150
P. 1968]. That is, 0.025 ml of an aqueous solution of 3.2 mM lysyl-alanine-β-naphthylamide.
To a mixture of 0.1 M 3,3-dimethylglutarate buffer 0.1 ml and a solution containing the sample 0.05 ml was added 0.025 ml of dipeptidyl aminopeptidase II solution partially purified from rat spleen homogenate by ammonium sulfate fractionation. At 37 ° C. for 1 hour. After the reaction, 0.5% containing 10% of polyoxyethylene (20) sorbitan monolaurate (manufactured by Wako Pure Chemical Industries, Ltd.) and 0.2% of Fast Garnet GBC salt (Sigma Chemical Company, Sigma, USA)
The reaction was stopped by adding 0.1 ml of a sodium citrate buffer (pH 3.78), and the absorbance (a) at 525 nm was measured. At the same time, the absorbance (b) of the control when the same reaction was carried out except for only the sample was measured. At this time, the absorbances (a ') and (b') of each of the blind tests were also measured. In the blind test, a buffer solution (pH 3.78) was added first, then an enzyme was added, and color development was performed without performing an enzyme reaction. The dipeptidyl aminopeptidase II inhibitory activity (%) was determined by the formula [1-
(A-a ') / (bb') '× 100.
The amount of the inhibitor that shows 50% inhibition by this assay (0.2 ml system) is defined as one unit. In this assay, pure dioctatin A and dioctatin B were 0.19 μg / ml and 0.89 μg, respectively.
50% dipeptidyl aminopeptidase II at a concentration of g / ml
Inhibited.

A culture containing dioctatin can be obtained by inoculating a dioctatin-producing bacterium belonging to the genus Streptomyces into a nutrient-containing medium and growing aerobically. As a nutrient source, one that can be used as a nutrient source for actinomycetes is used.
For example, commercially available peptone, meat extract, corn steep liquor, cottonseed flour, peanut flour, soy flour, yeast extract, NZ-amine, hydrolyzate of casein, sodium nitrate, ammonium nitrate, nitrogen sources such as ammonium sulfate, and Commercially available glycerin, sucrose, starch, glucose, galactose, mannose, carbohydrates such as molasses, or a carbon source such as fat, and salt,
Inorganic salts such as phosphate, calcium carbonate and magnesium sulfate can be used. Other trace metal salts as needed,
Fluids, plants, mineral oils and the like as antifoaming agents can also be added. Any of these can be used as long as they are used by the producing bacteria and are useful for producing dioctatin, and all known actinomycete culture materials can be used. Liquid culture is preferable for mass production of dioctatin, and the culture temperature can be applied within a range in which the producing bacterium grows and produces dioctatin.
34 ° C, preferably 25-30 ° C. The cultivation can be performed by appropriately selecting the above-described conditions according to the properties of the dioctatin-producing bacterium to be used. Dioctatin is present in both the culture filtrate and the cells. From the culture filtrate, it can be extracted with a water-immiscible organic solvent such as butanol at a pH of 2 or less, and after adsorption on an organic adsorbent such as Diaion HP-20, it can be extracted with acidic aqueous methanol, acidic aqueous acetone, etc. Can be eluted. After extracting the cells with an organic solvent such as acidic aqueous methanol or acidic aqueous acetone, the extract is concentrated under reduced pressure, and the solvent can be further extracted in the same manner as the culture filtrate. In addition to the above-described methods, known methods used for collecting fat-soluble substances, for example, adsorption chromatography, gel filtration chromatography, scraping from thin-layer chromatography, appropriately combining high-performance liquid chromatography, and the like,
Alternatively, it can be collected purely by repeating.

Table 4 shows the shapes, mass spectrometry, molecular formula, specific rotation, ultraviolet absorption maximum, infrared absorption maximum, solubility, and Rf value of silica gel thin-layer chromatography of dioctatin A and B. 1 and 2 show infrared absorption spectra of dioctatin A and dioctatin B by potassium bromide tablets, respectively. In each figure, the horizontal axis is the wave number (cm ^-1 )
And the vertical axis indicates transmittance (%). FIG. 3 shows a 400 MHz proton nuclear magnetic resonance spectrum of dioctatin A hydrochloride, and FIG. 4 shows a 400 MHz proton nuclear magnetic resonance spectrum of dioctatin B hydrochloride in deuterated dimethyl sulfoxide. In each figure, the horizontal axis represents chemical shift (ppm). The internal standard is tetramethylsilane. Furthermore, dioctatin A hydrochloride,
¹³ C nuclear magnetic resonance spectrum data of deoctatin and dioctatin B hydrochloride in deuterated dimethyl sulfoxide
It is shown in the table.

According to the above physicochemical properties, dioctatins A and B have structures of the following formulas II and III, respectively.

Examples of dioctatin salts include salts with pharmaceutically acceptable cations at the carboxyl group of dioctatin, for example, cations of alkali metals such as sodium and potassium, and alkaline earth metals such as calcium and magnesium.

Further, there are pharmaceutically acceptable anions at the amino group of dioctatin, for example, salts with mineral acids such as hydrochloric acid, sulfuric acid and phosphoric acid.

Dioctatin is a substance with low toxicity. In an acute toxicity test for mice, dioctatin A
No deaths were observed when 250 mg / kg or 250 mg / kg of dioctatin B was administered.

〔Example〕

Next, the present invention will be described with reference to examples, but the present invention is not limited to these examples. In addition,% in each case is% by weight unless otherwise specified.

Example 1 Actinomyces Streptomyces s Cultured on Agar Slope Medium
p.SA-2581 (Streptomyces sp. SA-2581) strain [
No. 10778 (FERM P-10778)] to glycerol I5
%, Fumarma Media (Traders Oil Mill
100% liquid medium (pH 7.4) consisting of 1.5%
Inoculate one platinum loop into each dispensed Erlenmeyer flask with waffle
After shaking at 27 ° C for 3 days. This is called seed culture
Jar incubator 4 containing the sterilized medium 1.5
Inoculate 400ml into the base, 27 ° C, aeration 10 per minute, or
6 days at 100 rpm
Pronal 501 (Shin-Etsu Chemical Co., Ltd.)
]. Celite is used as a filter aid for the culture solution thus obtained.
After filtration, the filtrate and the cells were separated. Filtrate 41 (3.2 × 10⁷
(Including inhibitor of unit)
20 [Mitsubishi Kasei Co., Ltd.] column and water 16
Acetone solution 16 containing 50% (by volume) 0.1N hydrochloric acid
Eluted. The obtained eluate 16.8 (2.15 × 10⁷Uni
) Was concentrated under reduced pressure to distill off acetone, and water was added to add
And This is extracted with butanol 3 and washed with water.
Tanol layer 3 (1.87 × 10⁷1 unit ammo per unit)
Near water was added for neutralization, and the mixture was concentrated under reduced pressure. Solids such as bacteria
The solution is a methanol solution containing 20% (by volume) 0.25N hydrochloric acid.
Extracted with liquid 10. This extract 10 (3.28 × 10⁷Unity
G) was concentrated under reduced pressure to remove methanol, and the filtrate was concentrated.
Extract with butanol in the same way as
Add ammonia water to neutralize and concentrate under reduced pressure to give a syrup
A concentrated solution was obtained. This is a syrupy concentrate derived from the culture filtrate.
Combine with the contracted solution and dissolve in hydrochloric acid acidic methanol to make 600 ml,
This is neutralized with 1N aqueous ammonia to precipitate a precipitate.
Let out. The precipitate is collected by filtration, washed with water, and dried under reduced pressure.
After drying, 1.36 g of a coarse powder was obtained. Add 100 ml of methanol to this.
Dissolve 20% hydrochloric acid dropwise and add 13.6 g of silica gel
And concentrated to dryness under reduced pressure. Next, 122 g of this silica gel
Apply the mixed solvent (Chromate) to a packed 3.2 cm ID column.
Elution was performed with chloroform: methanol: acetic acid = 100: 30: 1).
An active fraction of two components was obtained. Active substance eluted later
Dioctatin A and dioctane as active substance
It was named tatin B. Collect each active fraction and decompress
It was concentrated to dryness below. Dissolve each in hydrochloric acid acidic methanol
Dissolve 1N ammonia water and add active substance
Was precipitated. This is collected by filtration, washed with water, and dried under reduced pressure.
And 662 mg of white powder of dioctatin A and dioctatin
180 mg of white powder of B were obtained. Dioctatin A and dioc
Statins B were added at concentrations of 0.19 μg / ml and 0.89 μg / ml, respectively.
50 dipeptidyl aminopeptidase II from rat spleen
% Inhibition.

〔The invention's effect〕

As described in detail above, dipeptidyl aminopeptidase closely related to autoimmune diseases according to the present invention
A novel bioactive substance dioctatin that specifically inhibits II has been provided. Dioctatin is also useful as an immunosuppressant because it significantly suppresses a wide range of concentrations in a mixed lymphocyte reaction using spleen vesicles from two different strains of rats. On the other hand, dioctatin significantly promotes the uptake of tritiated thymidine by proliferating cells in the culture of rat splenocytes (cells passing through nylon wool) pretreated with concanavalin A, and is expected to act as an immunomodulator.

[Brief description of the drawings]

1 and 2 are infrared absorption spectra of dioctatin A and B by potassium bromide tablets, respectively, and FIGS. 3 and 4 are 400 MHz proton nuclear magnetism in deuterated dimethyl sulfoxide of dioctatin A and B, respectively. It is a resonance spectrum figure.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI (C12N 1/20 C12R 1: 465) (72) Inventor Keiji Ogawa 2-20-9 Kinugaoka, Hachioji-shi, Tokyo (58) Survey ⁶ (Int. Cl. ⁶ , DB name) C07K 5/023 C12P 21/00-21/06 CA (STN) REGISTRY (STN) BIOSIS (DIALOG) WPI (DIALOG)

Claims (2)

(57) [Claims] (1) The following general formula (I): (Wherein R represents hydrogen or a methyl group). Dioctatin, a physiologically active substance, or a salt thereof. 2. A method for producing dioctatin, a physiologically active substance, comprising culturing a dioctatin-producing bacterium belonging to the genus Streptomyces, and isolating and collecting at least one of the physiologically active substances, dioctatin, according to claim 1. .