JPS5825658B2 - Desacyl-pepcidin - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel desacylupepcidin (hereinafter referred to as DA-pepcidin). DA-pepcidin of the present invention is a pentapeptide having the following structural formula (I), namely valyl-valyl-4-
Amino-3-hydroxy-6-methyl-heptanoyl-
Alanyl-4-amino-3-hydroxy-6-methyl-
It is heptanoic acid. The DA-pepcidin of the present invention is derived from N-7 silupentapeptidoni, Bacillus spp., as defined hereinafter.
It can be produced by reacting with a culture solution, cells, or a treated product of a bacterium that belongs to N.s sp, ) and has the ability to deacylate the N-acyl-pen tapeptide. The N-acyl pentapeptide used as a starting material in the production of DA-pepcidin has the following general formula (T
I) Pentapeptides having the formula C, in which R is an acyl group, i.e. N-acyl-valyl-valyl-4-amino-3
-Hydroxy-6-methyl-hebutanoyl-alanyl-
4-amino-3-hydroxy-6-methyl-heptanoic acid. As N-acyl pentapeptide (II), several types of compounds with different acyl moieties are already known. For example, a compound in which R is acetyl was discovered by Kuwana et al., one of the inventors of the present invention, and is called "5-PI'".
Or as 1 Pepcidin C91, patent application 1984-3590.
No. 0 and Japanese Patent Application No. 48-39446. In Japanese Patent Application No. 48-39446, compounds in which R is butyryl and propionyl are shown as "pepcidin AII" and "pepcidin B 9+, respectively." Kaisho 47
-29582 and JP-A No. 49-41590,
N-acyl pentapeptides in which R is a linear or branched aliphatic acyl group having 5 to 16 carbon atoms, such as invaleryl, are disclosed as 1 pepstatins. Lupenta pegtides can be obtained by culturing various actinomycetes. For example, the above-mentioned pepcidins can be obtained by culturing Streptomyces naniwaensis.
waensis) EF 44-201 strain [Kohakken Bacteria No. 278]. In addition, pepstatins are, for example, Streptomyces testaceus (Streptomyces testaceus).
) is obtained by culturing. The bacteria used in the production of DA-pepcidin of the present invention is a new strain of Bacillus EF49-21 isolated by the present inventors from the soil of Kawaguchiko Town, Yamanashi Prefecture.
Particularly preferred is 0 stocks. Its mycological properties are shown below: [0Microscopic observation (broth agar medium) 1. Morphology: rods with rounded ends, singly or in pairs,
Rarely three in a row. 2. Size; 0.4-0.7X1. O~4.0μ3, motility; present. 4. Flagella; present (periflalagella). 5. Spore; formed. 6. Durham staining; positive. 7. Anti-acidity; negative. Culture findings 1, broth agar slant medium (30°C, 1 day) Growth spreads flat, milky yellow to slightly yellow, with a weak luster. No change in medium color. 2. Broth agar plate medium (30℃, 1~
7 days) Growth spreads out flat like a tree branch, milky yellow to slightly yellow in color, and the colony is smooth, slightly glossy, and translucent to opaque. No diffusible dye. 3. Meat juice liquid medium (30°C, 2 days) Forms a bacterial film on the surface. The turbidity in the liquid is weak (uniform. 4 Gelatin medium (20℃, 2
(7℃, 30℃, 3 days) Growth is weak, but it liquefies gelatin. It is not clear with puncture. 5. Litmus milling (30°C, 6 days) After 6 days of culture, it becomes slightly acidic and weak liquefaction occurs. No clotting. 6. Potato (30℃, 2 days) It becomes moist and spreads like mucus, and the potato turns brown. Sub-physiological properties■, nitrate reducing property; negative. 2. Denitrification reaction; negative. 3 Methyl red test; positive. 4. VP reaction; positive. 5. Indole production; negative. 6. Hydrogen sulfide production; negative. 7. Starch hydrolysis; negative. 8. Utilization of citric acid; Coser medium positive. Simmons medium negative. 9. Use of inorganic nitrogen sources; potassium nitrate is slightly positive, ammonium sulfate is slightly positive. 10. Pigment production; negative. 11. Urease; negative. I2. Oxidase; positive. I3. Catalase; positive. 14. Growth range; (a) pH (broth liquid medium 30°C shaking for 2 days) sterile medium p
Grows from H5 to 9.8. (b) Temperature (2 days of shaking in broth liquid medium) Grows at 20°C to 45°C. (C) Salt concentration (broth liquid medium shaking at 30°C) NaC1O,
It grows in one day at 5-7.0%. Grows in 2 days on 110% NaC. 15. Oxygen demanding facultative anaerobic. I6. Rifunn method sugar metabolism: Acid is produced in both aerobic and anaerobic conditions, and no gas generation is observed. 17. The fermentability of sugar and the production of acid are as follows; (sugar)
Acid production (aerobic) (anaerobic) ■ Arabinose ±■ Xylose ■ Glucose
+ Ten (sugar) Acid production (aerobic)
(anaerobic) ■ Mannose + 10 ■ Fructose - 10 ■ Galactose 2 ■ Maltose
■ Sucrose 10
＋■Lactose [phase] Trehalose
10 @ Inojit
100 Glycerin
±0 Starch No gas generation was observed. The above properties are summarized in the Purging Aids Manual of Determinative Bacteriology (Bergey
s Manual of Determinati
When compared with the description in the 7th edition of ve13acteriology, this bacterium is B.
The present inventors identified this bacterium as belonging to Bacillus pumilus (B. illus pumilus).
acillus pun illuskawaguchi
) was named. The strain EF49-210 of this bacterium has been deposited with the National Institute of Microbial Technology, Agency of Industrial Science and Technology as Microbiology Research Institute No. 2677. Known bacteria Bacillus maintained at the Fermentation Research Institute
Bacillus pumilus
It has been found that IFO-12092 and IFO-12110 can also be suitably used for the production of DA-pepcidin. When culturing the microorganisms used in the production of DA-pepcidin, any medium composition and culture conditions may be used as long as the microorganisms can grow and sufficiently express the desired activity. Organic or inorganic nitrogen sources such as peptone, meat extract, corn steep liquor, soy protein digest, soy infusion, yeast extract, inorganic ammonium salts, etc.;
A medium containing an appropriate carbon source such as glucose, starch and its hydrolyzate; and inorganic salts can be selected. Cultivation is usually carried out at 20 to 45°C for 1 to 7 days with shaking or aeration. The resulting culture solution, culture sterilization solution, live bacterial cells, dried bacterial cells, salting out products from the culture sterilization solution, precipitates with organic solvents, or other processed products at various stages are subjected to DA.
- Can be used in the production of pepcidin. The raw material N-acyl pentapeptide does not need to be a specific single isolated and purified N-acyl pentapeptide. When culturing the actinomycetes, usually several to several types of N-acyl pentapegtides with different acyl moieties are produced at the same time, but such a mixture can also be used as a raw material, and therefore, in practice, For example, N-acyl pentapeptide-containing materials at each purification step leading to crystallization are preferably used, such as a culture liquid of N-acyl pentapeptide-producing bacteria, a salted-out product thereof, or a dried product extracted with an organic solvent. . When producing DA-pepcidin, reaction conditions such as concentration, temperature, pH, etc. can be appropriately varied within a wide range depending on the type of bacterial strain used, the composition of the raw material N-acyl pentapeptide, etc. For example, when performing a deacylation reaction of a Bacillus pumilus-kawaktisyl pentapeptide mixture, p
It is preferred to react at a pH of about 4-10, especially pH 6-9, and a temperature of about 30-50°C for about 3 to 60 hours. The production rate of DA-pepcidin tends to increase with the addition of co+hydrogen compounds such as CoC12 and COS04. Separation and purification of the target DA-pepcidin from the reaction mixture is as follows:
This can be done by a conventional method. For example, the reaction mixture may be filtered in advance to remove solid matter, if necessary, and then extracted with an organic solvent such as n-butanol, and then the target product can be separated by removing the solvent. Purification of crude DA-pepcidin can be carried out by conventional methods such as column chromatography using a cation exchange resin or silica gel, fractional crystallization using an organic solvent such as methanol, ethanol, etc., or recrystallization. To confirm the production of DA-pepcidin of the present invention, the following method can be used: attaching a sample solution to a thin plate of silica gel G (manufactured by Merck, trade name);
n-Butyl alcohol:acetic acid:water (volume ratio 3:I:I)
Thin layer chromatography is performed using a mixed solvent (I) of n-butyl alcohol:acetic acid:water:butyl acetate (volume ratio 4:I:I:4). DA-pepcidin has Rf=0.48 [mixed solvent (I)
] are detected as ninhydrin reaction-positive and Lydon-Smith reaction-positive spots. Furthermore, after transferring the thin layer plate spread and dried in this manner onto an agar plate containing casein, a sheet of paper containing a pepsin solution was pasted on the plate and the plate was reacted overnight at 30°C to remove casein. It can also be detected as a decomposition spot (this method is hereinafter referred to as the casein plate method). The DA-pepsin of the present invention has the following physicochemical properties: (+) Appearance: White needle-like crystals (n) Solubility: Acetic acid, easily soluble in methanol, soluble in ethanol, n-butanol, pyridine, acetone, ethyl ether Hardly soluble in. (m) Positive for color reaction ninhydrin reaction and Lydon-Smith reaction. 6V) Ultraviolet absorption spectrum 0.1% methanol solution shows only terminal absorption based on peptide bonds and does not show absorption maximum between 250 and 370 mμ. (v) Infrared absorption spectrum shown in FIG. 1) Amino acid composition After hydrolyzing with 6N-HCI for 72 hours, the ratio of alanine to valine in the sample was measured with an amino acid analyzer, and the ratio of alanine to valine in the sample was I:2. Q: Molecular weight and structural formula: Mass spectrum analysis results for a compound obtained by acetylating a sample using the acid chloride method and then methyl esterifying using the diazomethane method. The value was 601. Furthermore, the fragment peak also coincided with the calculated value from the structural formula (I) of DA-pepcidin. The acetylated product of the above sample was found to be pepcidin C in thin layer chromatography, and the esterified product thereof was similarly found to be pepcidin C methyl ester. Vil pepsin inhibitory activity Murao and Satoi Agr, Biol, Chem. Japan 34(8) 1265-(1970)
As a result of measurement according to the method reported in
It was found that it inhibits DA-pepcidin of the present invention has a strong pepsin inhibitory activity as described above, and is therefore useful for the prevention and treatment of various diseases, such as gastrointestinal ulcers, based on the secretion source of pepsin. DA-pepcidin can also be easily converted into N-acyl pentapeptide derivatives having various acyl groups by applying conventional acylation methods,
These derivatives are expected to have useful properties such as pepsin inhibitory activity, and are therefore useful as intermediates thereof. Embodiments of the present invention will be illustrated below with reference to Examples, but these are merely illustrative and do not limit the present invention. Example I 0.1 J of a pH 7 liquid medium containing 1% meat extract, 1% peptone, and 5% NaC1O was charged into a 0.5 J Sakalo flask and sterilized by heating at 120° C. for 10 minutes.゛Inoculate 1 ml of the culture solution (Bacillus pumilus Kawaguchi strain EF 49-210, which was previously cultured in a medium of the same composition at 30°C for 24 hours into 20 flasks prepared above) and inoculate at 30°C for 72 hours. The culture was cultured with shaking. After the culture was completed, the culture sterilization solution obtained by removing the bacterial cells by centrifugation was combined, and cold acetone 41 was added dropwise under cooling. The generated precipitate was separated and collected, and suspended in distilled water. 50rn
1 of suspension. On the other hand, as a raw material, 500 ml of an aqueous solution was prepared by neutralizing and dissolving an N-acyl pentapeptide mixture IOy containing pepcidin C, pepcidin B, and pepcidin A in a weight ratio of 94:4:2 with 6N-NaOH, and to this was added the acetone 10 d of a suspension of the precipitate was added, and the mixture was stirred at 37° C. and pH 8.5 for 5 hours. The resulting reaction solution was mixed with 500 rIL of n-butyl alcohol.
The n-butyl alcohol layers were combined and concentrated to dryness under reduced pressure to obtain a residue 9.81. This residue was then dissolved in a mixed solvent 9 consisting of n-butyl alcohol:acetic acid:water:ethyl acetate (4:I:I:4 volume ratio).
After dissolving it in 0rnl and loading it onto a column containing 500 g of silica gel, column chromatography was carried out by developing and eluting with the same mixed solvent as above. About 0.75 J of the obtained chromatographic main fraction was concentrated to dryness under reduced pressure to obtain a residue of 0.72 J. This residue was crystallized with ethyl alcohol to give a crude crystal of 0.00%. :1 was obtained, which was further recrystallized from ethyl alcohol to obtain 0.171 white needle-like crystals of DA-pepcidin.
I got it. Melting point: Decomposes at 193-199°C and does not show a clear melting point. [α] -57 to -60° (C=1.methanol) Elemental analysis value: 0% as C29H55N508/H2O
N% N% Calculated value 56.19 9.26 11.29 Measured value 5
6.39 8.99 11.27 Example 2 Meat extract 1%, peptone 1% and sodium chloride 0.
A 301 volume jar fermenter was filled with liquid medium (pH 7) I51 containing 5% and then the medium was sterilized at 120° C. for 10 minutes. On the other hand, 0.2 l of a culture solution of the EF49-210 strain, which had been cultured with shaking at 30° C. for 24 hours in a medium having the same composition as above, was inoculated into the jar fermenter, and cultured under the following conditions. Culture time: 24 hours Culture temperature: 30°C Aeration rate: 15 J/min Stirring rotation speed: 350 revolutions/min After culturing, remove the bacterial cells by centrifugation. Add ammonium sulfate to 12 J of culture sterilization solution to a saturation level of 80%. In addition to 3
Refrigerated for a day. The generated precipitate was separated and collected, suspended in distilled water, and 750I
A suspension of 711 was obtained. 100 ml of this suspension. 400rrLl of neutralized aqueous solution containing pepcidin C3fI, 1715M phosphate buffer (pH 5,5) 2c+2d and COCl
A mixture of 2 solutions (5XIO"M) and 8rui
Incubated at ℃ for 40 hours. The reaction mixture was diluted with n-butyl alcohol (800wLlX3
), and the extracts were combined and evaporated to dryness. The residue was dissolved in the smallest possible amount of n-butanol:acetic acid:water:butyl acetate (4:I:I:4 volume ratio) mixed solvent, and subjected to silica gel column chromatography.
Eluted with the same solvent. Main fraction 21 was collected and evaporated to dryness to obtain white powder 62. This powder was recrystallized twice from ethanol to obtain DA-pepcidin 21 as a white needle. Really ＾

[82li 3] 100 ml of DA-pepcidin obtained by the method of Example I was dissolved in pyridine]orILl, 1.2 rnl of a 20-fold dilution of acetyl chloride in acetone was added dropwise to this solution under water cooling, and the solution was left overnight at room temperature. I left it there. A portion of this reaction solution was subjected to silica gel thin layer chromatography developed with n-butanol:acetic acid:water:butyl acetate (volume ratio 4:I:I:4), and a ninhydrin reaction was performed.
As a result of detection by Lydon-Smith reaction and casein plate method, the product showed Rf=0.49, which completely matched the standard pepcidin C. The reaction solution was concentrated to dryness, and the residue was dissolved in 10 ml of methanol. Add 17 ru of ether solution of diazomethane to the generated solution.
was added and left at room temperature for 3 hours. The reaction solution was then concentrated to dryness, the residue was dissolved in methanol, and ether was added to obtain 34 parts ml of crystals of pepcidin C1 methyl ester. Example 4 Culture solution 1 rIll of EF49-210 strain cultured in the same manner as in Example I and pepcidin Cl0m9 were mixed in 6N-N
Mix with 1 ml of an aqueous solution neutralized with aOH,
It was shaken at ℃ for 5 hours. This reaction solution was subjected to silica gel thin layer chromatography developed with n-butanol:acetic acid:water (volume ratio 3:]:I), and as a result, DA-pepcidin with Rf = 0.48 was found (a matching product spot was found). Example 5 According to Example 4, a reaction was carried out using pepcidin B or pepcidin A instead of pepcidin C, and both
The production of A-pepcidin was confirmed by thin layer chromatography. Example 6 According to Example 4, a reaction was carried out using N-invaleryl pentapeptide (pepstatin A) instead of pepcidin C, and the same reaction was carried out by thin layer chromatography.
Production of DA-pepcidin was confirmed. Example 7 According to Example 4, the reaction was carried out using N-n-hexanoyl pentapeptide or N-n-decanoyl pentapeptide in place of pepcidin C, and the same (DA-pepcidin by thin layer chromatography) The generation of was confirmed.

[Brief explanation of the drawing]

FIG. 1 shows the infrared absorption spectrum of DA-pepcidin.

Claims (1)

[Claims] 1st order structural formula Desacyl-pepcidin, which has