JPH0912477A - Novel substance DQ90A, method for producing the same, and protease inhibitor containing the DQ90A - Google Patents

Abstract

(57) [Summary] (Modified) [Purpose] To provide a novel compound having proteolytic enzyme inhibitory activity. [Structure] A novel substance DQ90A represented by the formula (I), a method for producing the same, and a protease inhibitor containing the substance as an active ingredient.

Description

Detailed Description of the Invention

[0001]

The present invention relates to a novel substance DQ90A,
The present invention relates to a method for producing the same and a protease inhibitor containing DQ90A as an active ingredient.

[0002]

2. Description of the Related Art Many proteolytic enzyme inhibitors are already known as research reagents and anti-inflammatory agents. For example, the leupeptin analog Ac-Leu-L.
eu-Nle-al, Saito et al., Neuroscience Lette
rs, 89, 102 (1988).
In addition, tyrostatin was identified by Oda et al., Agric. Biol.
Chem., 53, 405 (1989)]. However, in order to solve various research problems, the advent of new compounds having proteolytic enzyme inhibitory activity is always desired.

DISCLOSURE OF THE INVENTION An object of the present invention is to provide a novel compound having a protease inhibitory activity.

The present inventors have completed the present invention by isolating a novel substance DQ90A having proteolytic enzyme inhibitory activity from the culture solution of a strain belonging to the genus Streptomyces. The present invention provides a novel substance DQ90A represented by the formula (I).

[0003]

Embedded image

The present invention also provides a protease inhibitor containing the compound DQ90A as an active ingredient. The present invention also provides a method for producing a DQ90A substance, which comprises culturing a DQ90A substance-producing bacterium belonging to the genus Streptomyces and collecting the DQ90A substance from the culture.
As the DQ90A substance-producing bacterium of the present invention, any one can be used as long as it belongs to the genus Streptomyces and has a DQ90A substance-producing ability. Specifically, Streptmyces halstedii 272
3-SV2 strain (hereinafter referred to as "DQ-90 strain") can be advantageously used. DQ-90 strain The DQ-90 strain found by the present inventors as a strain of the genus Streptomyces having a DQ90A substance-producing ability has the following contents.

1) Origin and Deposit No. DQ-90 strain is an actinomycete isolated from a soil sample collected at Hakone Pass in Shizuoka Prefecture. It has been deposited and has obtained the number of "Micromachine Research Deposit No. 5135" (FERM BP-5135). 2) Mycological Properties The mycological properties of the DQ-90 strain are as follows. The characterization of actinomycete DQ-90 strain is "Patent Office Industry-Specific Examination Standards"
Was carried out according to the method described in. The basal hypha of this strain is not broken. The aerial hyphae form the main axis, and 10 to 50 or more straight or curved spore chains are formed at the tips branched irregularly therefrom. This spore chain is a bird's nest (ne
St-like) dense lumps are formed. Spores are non-motile, cylindrical or oblong, width 0.3-0.5 μm, length
From 1.0 to 1.2 μm, the spore surface is smooth. Sclerotia, sporangia and other special forms are not observed. The cell wall chemotype is type I, which contains LL diaminopimelic acid. The culture properties are shown in Table 1. The flora color on the surface of the community belongs to the gray series. The back side color is unclear and does not change with pH. The diffusible pigments were light orange to light brownish gray.

[0006]

[Table 1] Table 1 Culture properties Medium Bacterial surface color of colony surface Backside color of colony Diffusible pigment sucrose, no aerial hyphae, light brown grey, no nitrate agar (3ec) Glucose / As gray series light brown, pale orange paragine agar (2fe) (4le to 4ni) (4ea) ) Glycerin ・ As Gray series Light tea taste Gray Light tea taste Gray Paragin Agar (2fe) (4ge) (4ec) Inorganic salt / starch agar Gray series Light tea taste gray None (5fe) (4ge) Tyrosine agar Gray series Light yellow brown None ( cd ~ (3gc ~ 3le) Nutrient agar No aerial hypha No pale yellow (2db) Yeast / malt agar gray series Reddish brown Light brown (5fe) (6le ~ 6li) (4gc) Oatmeal agar Gray series Light brown to brown Akira Chami gray (5fe) (4lg~4ni) (5ec ) * () in the color harmony Maniyuaru (container copolyarylene configuration of America, 1950) color plate U De. The physiological properties are shown in Table 2. This strain is mesophilic and uses assimilation ability of carbon source glucose, arabinose, xylose, and fructose. Based on the morphological characteristics and cell wall chemotype of this strain, it is located in the genus Streptmyces (hereinafter abbreviated as S.).

[0007]

[Table 2] Table 2 Physiological properties Growth temperature range 20-37 ℃ Optimum temperature 20-30 ℃ Melanin pigment Tyrosine agar-Peptone yeast iron agar ± Tryptone yeast broth-Hydrolysis of starch + Liquefaction of gelatin-Peptone of skim milk powder-Coagulation of skim milk powder + Reduction of nitrate + Assimilation of carbon source D-glucose + L-arabinose + D-xylose + D-fructose + sucrose + L-rhamnose-raffinose- i -inositol- D-mannitol-

[0008] Based on the above-mentioned various properties, "Bacterial name approval list,
1980 ”and subsequent S.R. We searched for species of the genus and selected closely related species. S. When the diagnostic properties of S. nigrifaciens were selected, this strain and S. nigrifaciens were selected. Nigrifaciens ( S.nigrif
The characteristics of aciens ) are slightly different in the assimilation ability of carbon source, but the others are in good agreement (Table 3). However
Bergey's Manual of Systematic Bacteriology Vol.
4, Williams et al. Niglifaciens was introduced into S. Synonym for S.halstedii
And Therefore, this strain 2723-SV2 is a strain of Streptmyces halstedii .
2723-SV2 strain.

[0009]

[Table 3] Table 3 Comparison between this strain and related species This strain Streptomyces Streptomyces 2732-SV2 Niglifasciens Halsteady spore chain morphology Straight / curved ＋ ＋ spore surface smooth ＋ ＋ ＋ bacterial color gray ＋ ＋ ＋ reverse color unclear ＋ ＋ ＋ pH diffusive Pigmentation + +-pH sensitivity --- Melanin pigment production --- Hydrolysis of starch + + + Reduction of nitrate + + + Growth temperature 10 ° C --- -37 ° C + + + 45 ° C ----Assimilation of carbon source arabinose + + + xylose + + + inositol - - - mannitol - + - rhamnose - - - raffinose - - - sucrose + - -

Cultivation method of DQ90A substance-producing bacteria DQ90A substance is DQ9 belonging to the genus Streptomyces.
It can be produced by aerobically culturing the OA substance-producing bacterium in an appropriate medium, and separating and purifying the target product from the culture. The medium is a medium containing nutrients that can be utilized by ordinary microorganisms. As the nutrient source, known nutrients conventionally used for cultivation of actinomycetes can be used. Specifically, as the carbon source, glucose, starch syrup, dextrin,
Starch, molasses, fats and oils can be used. As the nitrogen source, organic substances such as soybean flour, wheat germ, cottonseed meal, corn steep liquor, meat extract, peptone, yeast extract and inorganic substances such as ammonium sulfate and ammonium nitrate can be used. If necessary, sodium, potassium, calcium, magnesium, cobalt, chlorine,
Inorganic salts capable of producing phosphoric acid, sulfuric acid and other ions can be added. In addition, organic and inorganic substances that help the growth of bacteria and promote the production of DQ90A substance can be added appropriately.

As the culturing method, the aerobic liquid culturing method is most suitable as in the generally used antibiotic production method. A suitable temperature for culturing is 20 to 30 ° C., but in most cases, culturing is performed at around 27 ° C. The production of the DQ90A substance varies depending on the medium and the culture temperature, but in liquid culture, the maximum accumulation is usually reached in 1 to 3 days. In this way, the target substance is isolated and purified from the culture in which the DQ90A substance has accumulated. Method for Purifying DQ90A Substance In collecting the DQ90A substance obtained by the present invention from a culture, usual separation means utilizing its properties, for example, filtration, centrifugation, dialysis, concentration, drying, freezing,
Adsorption, desorption, a method utilizing the difference in solubility in various organic solvents, means such as chromatography and the like can be used alone or in combination to extract and purify. For example,
The synthetic adsorbent HP-20 (manufactured by Mitsubishi Chemical Co., Ltd.), ODS column chromatography, MPLC, and HPLC can be appropriately combined and carried out. The DQ90A substance thus obtained had the following physicochemical properties, and as a result of analysis of various spectral data, it was found to have the chemical structure represented by the above formula (I).

Physicochemical properties (1) Appearance: White powder (2) Melting point: 215 to 217 ° C. (decomposition) (3) Molecular formula: C ₂₈ H ₃₉ N ₃ O ₅ (4) High resolution mass spectrum (m / z) ): 498.2942 (M + H) ⁺ measured value: 498.2970 Calculated value (5) Specific rotation: -28.6. (C = 0.028, methanol) (6) Solubility: Soluble in methanol and dimethylsulfoxide, but insoluble in water and chloroform. (7) Ultraviolet absorption spectrum λ max nm (in methanol): 259 (1200), 269 (1700), 279 (1700)

(8) Infrared absorption spectrum (KBr disk method) Shown in FIG. : 3269, 2960 to 2870, 1633, 1540 to 1440, 1387, 1223 (cm ⁻¹ ) (9) ¹ H NMR spectrum (500 MHz, heavy dimethyl sulfoxide). Shown in FIG. (10) ¹³ C NMR spectrum (125 MHz, heavy dimethyl sulfoxide) Shown in FIG.

Biological activity of DQ90A substance The inhibitory action of the DQ90A substance according to the present invention on the proteolytic enzyme papain will be described. Here, a synthetic substrate of papain, BANA (α-N-benzoil-DL-arginine
-Β-naphtylamide hydrochroride) Evaluation was made by inhibition of hydrolysis reaction. [Reagent] a) Buffer solution (prepared at the time of use) 32.4 mg L-cysteine / 100 ml ^* EDTA
Phosphate buffer (pH 6.0) ^* EDTA phosphate buffer _{(pH 6.0) 0.495g Na 2 EDTA} 12.0 g KH 2 PO 4 2.14 g Na 2 HPO 4 · 2H 2 O / dH ₂ O 1
000 ml b) Substrate 40 mg BANA / 1 ml DMSO (dimethyl sulfoxide)

C) Coupling reagents (equal amounts of (I) and (II) are mixed and used within 30 minutes) (I) 10 mM PCMB (mercuric parachlorobenzoate) / 50 mM EDTA 2.40 g NaOH 18.6 g Na ₂ EDTA 3.57 g PCMB 950 ml Adjust the pH to 6.0 with dH ₂ O HCl, and add 1000 ml with dH ₂ O.
And (II) 0.5mg FAST GARNET GBC / 1ml 4% Twee
n 20 d) Enzyme solution Papain (3000 U / mg) was added to 0.01% BSA.
Dilute (bovine serum albumin). (The dilution was performed so that the absorbance of the standard reaction solution at 520 nm was about 1.5.) [Method] The enzyme solution (d), the buffer solution (a) and the sample are mixed and preincubated at 40 ° C. (2 ml in total) To this, substrate (b) was added and mixed, and reacted at 40 ° C. for 10 minutes. After 10 minutes, 2 ml of the coupling reagent (c) was added to stop the reaction, and after standing for 10 minutes, the absorbance at 520 nm was measured with a spectrophotometer. The activity was determined by the inhibition against the standard.

[0016]

[Table 4] [Reaction system] ──────────────────────────── Blank standard test enzyme-0.5 ml 0.5 ml buffer 1.9 ml 1.4 ml 1.4 ml Methanol 0.1 ml 0.1 ml-Sample-0.1 ml Substrate 50 μl 50 μl 50 μl Sample: (Methanol solution of DQ90A substance) As a result, 50% activity of proteolytic enzyme papain of DQ90A substance The inhibitory concentration (IC ₅₀ value) was 0.1 μg /
ml. Thus, since the DQ90A substance of the present invention has an inhibitory action on the proteolytic enzyme papain, it may be used as a research reagent and also as an anti-inflammatory agent.

[0017]

INDUSTRIAL APPLICABILITY According to the present invention, it was possible to provide a novel compound DQ90A having an inhibitory action on the proteolytic enzyme papain and useful as a research reagent or an anti-inflammatory agent.
Next, the present invention will be described by way of examples.

[0018]

EXAMPLES Example 1 1) Preparation of Seed Mother 1 The medium used was prepared by dissolving the components having the following composition in 1 liter of water and adjusting the pH to 72. Soluble starch 10.0 g Molasses 10.0 g Polypeptone 10.0 g Meat extract 10.0 g Dispensed 15 ml of the above medium into a large test tube and sterilized, and then sterilized Streptomyces Halstein 2723-SV2 strain (hereinafter DQ-90 strain) from slant. 1 platinum loop inoculation, 27 ℃
Seed mother 1 was cultured for 1 day with shaking.

2) Preparation of Seed Mother 2 The medium used was prepared by dissolving the components having the following composition in 1 liter of water and adjusting the pH to 7.0. Glycerin 20.0 g Molasses 10.0 g Casein 5.0 g Polypeptone 1.0 g Calcium carbonate 4.0 g 100 ml of the above medium was added to a 500 ml Erlenmeyer flask (7
Bottle), sterilized, and then seed seed 1 (1 ml each)
Was inoculated and cultured with shaking at 27 ° C. for 1 day. 3) Culture The culture medium used is the one shown in 2). This medium 2
7 liters were placed in a 50 liter jar fermenter, sterilized, and then seeded with the seed mother 2 (total of 7), and stirring speed was 200 rpm. The culture was carried out with aeration and stirring at 27 ° C. for 1 day under the conditions of an aeration rate of 30 liters / minute.

4) Purification of DQ90A substance After culturing under the above conditions, the culture supernatant obtained by centrifuging 80 liters of the culture broth was adsorbed on a "Diaion HP-20" (manufactured by Mitsubishi Chemical Corporation) column. This with water, then 30%
After washing with methanol, 70% methanol was passed through, and the obtained 70% methanol solution was concentrated to dryness. The crude active fraction thus obtained was applied to a MPLC LiChroprep C18 (MERCK) column to obtain a fraction containing the DQ90A substance.
After further concentration of this fraction, HPLC TSKgel-80TM ODS (TOS
OH column) provided 100 mg of DQ90A material.

[Brief description of the drawings]

FIG. 1 is an infrared absorption spectrum of DQ90A substance.

FIG. 2 is a 500 MHz ¹ H NMR spectrum of DQ90A substance in a heavy dimethyl sulfoxide solution.

FIG. 3 is a 125 MHz ¹³ C NMR spectrum of DQ90A substance in a heavy dimethyl sulfoxide solution.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Reference number within the agency FI technical display location C12R 1: 465) C07M 7:00 (72) Inventor Nobuaki Tsuge 1-Mirikaeicho, Higashiosaka City, Osaka Prefecture 5-7 House Food Co., Ltd. (72) Inventor Yoji Hirao 1-5-7 Mikitei-cho, Higashiosaka City, Osaka Prefecture House Food Co., Ltd.

Claims (3)

[Claims] 1. A novel substance DQ90 represented by the formula (I).
A. Embedded image 2. A protease inhibitor containing the compound DQ90A according to claim 1 as an active ingredient. 3. A DQ90A belonging to the genus Streptomyces
A method for producing a DQ90A substance, which comprises culturing a substance-producing bacterium and collecting the DQ90A substance from the culture.