CH620244A5 - Process for the microbiological preparation of a deacylpepsidin - Google Patents

Description

620244 2

PATENT CLAIMS noyI-alanyl-4-amino-3-hydroxy-6-methyIheptanoic acid of form

1. Process for the microbiological production of the seed (I)

cyl-pepsidins valyl-valyl-4-amino-3-hydroxy-6-methyl-hepta-

H, C CH .. H-.C CH,

\ / 3 3 \ / 3

H, C CH, H, C CH, CH CH

3 \ / 3 3 \ / J

CH CH CH ~ OH CH, CH-, OH (I)

I! I ^ I »J 1 ^ 1

H2 K-CH-CO-NM-CH-CO-MH-CH-Cli-CHg-CO-NH-CH-CO-NH-CH-CH-CH2-COOH

characterized in that the N-acyl pentapeptide N-acyl-valyl-valyl-4-amino-3-hydroxy-6-methyl-heptanoyl-ala-nyl-4-amino-3-hydroxy-6-methylheptanoic acid of the formula (II )

\ / CU

H, C CH,

J \ / 3

H, C CH.,

3 \ / 3

H..C, CH,

V 3

CH

i

CH

i

CHp Oli

CH

i ->

CH!

CH.

OR

(11 /

R-NH-CH-CO-NH-CH-CO-NH-CH-0H-CH2-CO-HH-CH-CO-NH-CH ~ CH-CH ^ -COCH

in which R stands for an acyl group, by means of contacting with bacteria of the Bacillus pumilus type or with raw and enriched acellular culture filtrates and enzymes which are produced by the bacterial species mentioned, converted to the product of the formula (I). a

2. The method according to claim 1, characterized in that the implementation time is between 3 and 60 hours.

3. The method according to claim 1, characterized in that the bacteria are those of the strain Bacillus pumi- 40 lus Kawaguchi EF 49-210 (ATCC No. 31132).

4. The method according to claim 1, characterized in that the bacteria are those of the strain Bacillus pumilus IFO-12092.

5. The method according to claim 1, characterized in that the bacteria are those of the strain Bacillus pumilus IFO-12HO.

6. The method according to claim 1, characterized in that the bacteria of the species Bacillus pumilus in the form of whole or dried cells and the acellular preparations in the form of culture solution, filtrates of the culture solution and / or enzyme preparations are used.

7. The method according to claim 1, characterized in that the N-acylpentapeptide of the formula (II) is present as a starting material in a mixture which contains culture filtrate from the culture filtrate and products extracted from the culture filtrate by means of an organic solvent and dried products of that microorganism, which produces microbiological N-acylpentapeptides.

8. The method according to claim 1, characterized in that Co ++ salts are added to the reaction mixture.

9. The method according to claim 8, characterized in that the Co ++ salt is C0CI2 or C0SO4.

10. The desacyl-pepsidine valyl-valyl-4-amino-3-hydroxy-6-methyl-heptanoyl-alanyl-4-amino-3-hydroxy-6-methyl-heptanoic acid of the formula (I)

H-jC CH3 H3C CH3

\ / V '

\ /

HC CH3 H3C CH CH CH

(I)

CH CH CH. OH CH CH "OH

I I. I 2 I I 3 I 2 I

H N-CH-CO-NH-CH-CO-NH-CH-CH-CHj-CO-NH-CH-CO-NH-CH - CH-CH COOH

produced by the method according to claim 1.

620244

The present invention described relates to a process for the microbiological production of desacyl pepsidine

H ^ C

CH.

Valyl-valyl4-amino-3-hydroxy-6-methyl-heptanoyI-alanyI4-amino-3-hydroxy-6-methylheptanoic acid of the formula (I)

H ^ C

CH.

H.C CH,

3 \ / 3

CH

t

H, C CH,

\ / 3

CR

i

CH}

CH,

OH

i

CH,

t ->

CH!

CH.

OH

i

I)

H2K-CH-CO-K-H-CH-CO-NH-CH-CH-CH2-CO-NH-CH-CO-N1I-CH-CH-CH2-C00H

The starting material for the preparation of the desacyl-pepsidine of the formula (I) is the N-acyl-valyl-valyl4-amino-3-hydroxy-

6-methyl-heptanoylalanyl4-amino-3-hydroxy-6-methylheptanoic acid of the formula (II)

CH ..

V J

CH

i

3C \

»C CH,

V

CH

i

\ /

CH

1

CH.

CH.

OH

t

H.C

CH

CH

i

3rd

\ / CH

t

CH,

3rd

QH

i

(i t;

R-NH-CH-CO-NH-CH-CG-NH-CH - CH-CH? -CO ~ NH-CH-CO-NH-CH- CH-CH2-COCH

where R represents an acyl group.

The described desacyl-pepsidine has strong activities as a pepsin inhibitor and is therefore useful in the medicinal treatment of gastric ulcers.

The process according to the invention for the microbiological production of the desacyl-pepsidine valyl-valyl4-amino-3-hydroxy-6-methyl-heptanoylalanyl4-amino-3-hydroxy-6-methyl-heptanoic acid of the formula (I) is defined in claim 1.

More than 10 compounds of N-acylpentapeptides of the formula (II) are already known. The connection with R = acety-tyl was discovered, for example, by N. Kuwana. The compound is disclosed in U.S. Patent Nos. 3,819,486 and 3,878,185, and in J. Patanm. No. 39446/73 and bears the designation «S-PI» or «Pepsidine C». In the mentioned J. Patanm. those compounds with R = butyryl and propionyl are also described; they are called "Pepsidine A" or. "Pepsidine B".

Furthermore, by Umezawa et al. N-acylpentapeptides with R = isovaleryl or straight-branched aliphatic acyl groups with 5 to 16 carbon atoms in Japanese Patent Publication No. 8996/72, in Japanese Laid-Open Publication No. 29582/72 and in Japanese Publication No. 41590/74. In the documents mentioned, the products are referred to as “pepstatin”.

It is known that all of the N-acylpentapeptides described have strong pepsin-inhibitory activities.

The N-acylpentapeptides are obtained by microbiological cultures from various Actinomycetes. The above-mentioned desacyl-pepsidine is obtained, for example, from the cultures of Streptomyces naniwaensis EF 44-201. The pepstatins mentioned can be obtained, for example, from cultures of Streptomyces testaceus. However, the N-acylpentapeptides obtained from the Actinomycetes cultures mentioned are normally not homogeneous products. Rather, they represent a mixture of different, up to more than 10, N-acylpentapeptides, which have different acyl radicals R and have similar properties. In order to obtain pure, special N-acylpentapeptides from these mixtures, very complicated and complicated separation processes are necessary. However, this also greatly reduces the yield of the end product sought. m Desacyl-pepsidine with the free, N-containing terminal group has the same basic structure as the homologous N-acylpentapeptides mentioned above. The process according to the invention gives the desacyl-pepsidine from the mixture of the N-acylpentapeptides; this can then be converted into a desired N-acylpentapeptide by 3-5 a special acylating agent. This is thus obtained in pure form and with an increased yield.

Likewise, desacyl-pepsidine can of course be used as an intermediate for the synthesis of further, as yet unknown, N-acylpentapeptides.

A bacterial strain that is used in the method according to the invention is Bacillus pumilus EF49-210 (nov. Sp.). This strain was isolated from the ground at Kawaguchiko machi, Yamanashi Prefecture, Japan. The microbiological properties of the strain are as follows:

I. Microscopic properties (broth agar nutrient medium)

1. Morphology: rods with rounded ends, which are available ■ individually, in pairs or (rarely) in a triple formation.

2.Size: 0.4 to 0.7 times 1.0 to 4.0 n.m.

3. Motility: agile

4. Flagella: Peritrichous

5. Spores: trained

:> 6. Coloring, grief: positive

7. Rapid staining, acidity: negative

II. Observation on culture

1. On an inclined broth agar medium (at 30 ° C, for a b'i day): growth by flat expansion, color milky yellow to slightly yellow, slightly shimmering, no discoloration of the nutrient medium.

2. On a flat broth agar culture medium (30 ° C, for 1 to 7 days): grew by flat expansion, dendroitic, milky-yellowish, slightly yellowish, smooth colony, slightly shimmering and translucent to opaque, no soluble pigment .

3. In the culture broth (at 30 ° C, for 2 days): skin formation on the surface, cloudiness is weak and even.

J

620244

4. In gelatin medium (at 20.27 and 30 ° C, for 3 days): weak growth, but gelatin is liquefied, not clear compared to stab culture.

5. In litmus milk (at 30 ° C, for 6 days): Rather acidic, slightly liquefied after a day's culture, no coagulation.

6. On the cut potato surface (at 30 ° C, for 2 days): moist growth and slimy expansion, potato darkened.

III. Physiological properties:

1. Nitrate reduction: negative

2. Nitrogen elimination: negative

3. Methyl red test: positive

4. Voges-Proskauer reaction: positive

5. Indole formation: negative

6. H2S formation: negative

7. Starch hydrolysis: negative

8. Use of citric acid: positive in Koser's medium, negative in Symmon's medium

9. Use of inorganic nitrogen: weakly positive in potassium nitrate, weakly positive in ammonium sulfate

10. Formation of pigments: negative

11. Urease: negative

12. Oxidase: positive

13. Catalase: positive

14. Growth parameters: a) pH (culture broth as nutrient medium at 30 ° C., for 2 days with shaking) growth in the sterilized medium at pH between 5 and 9.8;

b) temperature (culture broth as nutrient medium, with shaking for 2 days) grew at a temperature between 20 ° C and 45 ° C;

c) salt concentration (culture broth as nutrient medium, with shaking for 2 days) grew within a day in NaCl concentrations between 0.5 to 7.0%; Grew in NaCl concentration of 10% within two days.

15. Oxygen requirement: optional anaerobic

16.0-F test according to Hugh-Leifson: formation of acid both aerobic and anaerobic; no gas formation.

17. Fermentation test and acid formation with carbohydrates:

No.

Carbohydrate

Acid formation aerobic anaerobic

1

Arabinose

+

2nd

Xylose

+

±

3rd

glucose

+

+

4th

Mannose

+

+

5

Fructose

+

+

6

Galactose

±

±

7

Maltose

-

+

8th

Sucrose

+

+

9

Lactose

-

+

10th

Trehalose

+

+

11

Sorbitol

-

+

12

Mannitol

-

±

13

Inositol

+

+

14

Glycerol

+

±

15

Strength

-

±

No gas formation was observed in any of the carbohydrates.

Acidic (+), slightly acidic (±), not acidic (-).

By comparing the specifications given above with those in Bergey, Manual of Determinative Bacterio-Iogy, 7th edition, it can be assumed that the bacterial culture described is a variant of Bacillus pumilus.

The strain was therefore called Bacillus pumilus Kawaguchi. The strain EF 49-210 (ATCC No. 31132) was deposited on August 16, 1974 with the Agency of Industriai Science and Technology, Fermentation Research Institute in Japan under the name FERM-P No. 2677. It has also been found that two further strains of this type of bacteria, namely Bacillus pumilus IFO-12092 and Bacillus pumilus IFO-12110, both of which are kept at the Institute of Fermentation in Osaka, Japan, can be used in the method according to the invention.

Various media and conditions can be used for the cultivation of the microorganisms used in the process according to the invention, provided that the microorganism can grow therein and fully develop its desired activities. For example, any organic or inorganic nitrogen sources such as peptones, meat extract, soaked grain liquid, soybean hydrolyzate, soybean extract, starch extract, inorganic ammonium salts and the like may be present in the nutrient medium. Furthermore, the nutrient medium can contain any carbon source such as molasses, dextrose, starch and the corresponding hydrolysates. Inorganic salts can also be present in the media. The cultivation is usually carried out with shaking or aeration at temperatures between 20 and 45 ° C, the duration is 1 to 7 days.

The deacylating effect of Bacillus pumilus has been found to be both intra- and extracellular. It is therefore possible in the process according to the invention to isolate the type of bacteria as culture broth, filtrate of the culture broth,

use whole cells, dry cells and / or enzyme preparations of these components.

The starting material for carrying out the method according to the invention, i. H. the N-acylpentapeptide need not always be in a purified form. For example, mixtures of different N-acyl pentapeptides can be used. These can be separated by different cleaning stages so that filtrates from the culture broth and salted-out products of this filtrate can be used. Products which have been extracted from the culture filtrate by means of organic solvents and from which the solvent has subsequently been separated off can also be used.

When carrying out the process according to the invention, the reaction conditions, such as concentrations, temperatures, pH and the like, are not critical. They can be varied over a wide range, depending on the type of microorganism used and the N-acylpentapeptide used. If the deacylation of the N-acylpentapeptide is carried out, for example, using Bacillus pumilus Kawaguchi EF 49-210, the following reaction conditions are advantageous: pH 4 to 10, temperature 30 to 50 ° C., reaction time: 3 to 60 hours. By adding cobalt ++ salts such as cobalt chloride and cobalt sulfate, etc., the yield of desacyl-pepsidine is increased.

The separation and purification of the desacyl pepsidine from the reaction mixture can be carried out by conventional methods such as solvent extraction, column chromatography, fractional crystallization, recrystallization and the like.

To detect the formation of the desacyl-pepsidine according to the invention, thin-layer chromatography can be used according to the following procedure:

A drop of the reaction solution is placed on a thin layer plate of silica gel G (product and brand of Merck AG). The development is then either with a solvent mixture of N-butyl alcohol, acetic acid and water in a volume ratio of 3: 1: 1, or

4th

5

10th

15

20th

25th

30th

i-5

4 «

4-3

10th

3>

h (>

t 3

with a solvent mixture of N-butyl alcohol, acetic acid, water and butyl acetate in a volume ratio of 4: 1: 1: 4. The desacyl-pepsidine then appears as a spot which also reacts positively in the ninhydrin reaction and in the Rydon-Smith reaction at Rf = 0.48 with the first solvent mixture.

Another way of detecting desacyl pepsidine is by using casein plates:

A test slide, as described above, is developed and dried and then placed on a flat base of agar containing casein. A filter paper moistened with the pepsin solution is then placed on the agar surface. The mixture is then allowed to react at 30 ° C. overnight to determine the undegraded casein.

The desacyl-pepsidine, which has been produced according to the method according to the invention, has the following physico-chemical properties:

a) Appearance: white needles b) Solubility: Easily soluble in acetic acid and methanol, soluble in ethanol, n-butanol and pyridine, slightly soluble in acetone and ethyl ether.

c) Color reaction: positive both in the ninhydrin reaction and in the Rydon-Smith reaction.

d) UV absorption spectrum: A 0.1% solution in methanol only shows the final absorption of the peptide bond; no maximum absorption between 250 µm to 370 µm.

e) IR absorption spectrum: This is shown in the attached FIG. 1.

f) Amounts of amino acids: After 72 hours of hydrolysis with 6N HCl at 110 ° C., the pattern was examined using an amino acid analyzer. The molar ratio of alanine to valine has been found to be 1: 2.

g) Molecular weight and structural formula: A sample was acetylated with an acid chloride and then esterified with diazomethane to give the corresponding methyl ester. The compound obtained was analyzed in the mass spectrometer and a molecular weight of 657 was calculated. The molecular weight of the desacyl pepsidine was calculated to be 601, which corresponds to the theoretical value. The peak of a split-off part of the compound coincided with the calculated value, which was calculated from the structural formula of desacyl-pepsidine according to the formula (I).

The acetylated product of the sample listed above behaved identical to pepsidine C on thin-layer chromatographic plates and the esterified product identical to the methyl ester of pepsidine C.

h) Pepsin-inhibitory activity: The pepsin-inhibitory activity of desacyl-pepsidine was determined according to the method according to S. Murao and S. Satoi in Agr. Biol. Chem. Japan 34 (8) 1265-1267 (1970).

It has been demonstrated that the amount of desacyl-pepsidine which gives 50% inhibition against 100 | xg pepsin is 0.82 | xg. The corresponding amount of pepsidine C is 0.86 ng.

The following examples illustrate some preferred embodiments of the method according to the invention; but in no way serve to limit it.

example 1

In a 0.5 liter Sakaguchi bottle, 0.1 liter of a liquid medium (pH 7) was added, which contained 1% meat extract, 1% peptone and 0.5% NaCl. The medium was then sterilized at 120 ° C for 10 minutes. In this way, 20 such bottles were prepared.

Each bottle was then washed using 1 ml of a slurry of the bacterial strain Bacillus pumilus Kawaguchi EF

620244

49-210 vaccinated. This strain had previously been dormant in a similar nutrient solution for 24 hours at 30 ° C and then cultured for 72 hours at 30 ° C with shaking.

After the cultivation was completed, the cells were separated from the slurry by centrifugation. 4 l of cold acetone were then added dropwise and with vigorous cooling to the centrifugates which had been poured together. The precipitate obtained was suspended in distilled water and 50 ml of suspension was obtained.

In addition, 10 g of a mixture of N-acylpentapeptides (pepsidine C, pepsidine B and pepsidine A in a weight ratio of 94: 4: 2) were dissolved in water and neutralized with 6N NaOH, whereby 500 ml of an aqueous solution was obtained. 10 ml of the suspension of the acetate precipitate obtained above were added to this aqueous solution. The mixture, which had a pH of 8.5, was then stirred at 37 ° for 5 hours.

The resulting reaction solution was extracted three times with 500 ml of N-butyl alcohol. The combined N-butyl alcohol extracts were combined, evaporated to dryness under reduced pressure and 9.8 g of residue were thus obtained.

This residue was then dissolved in 90 ml of a solvent mixture. This mixture contained N-butyl alcohol, acetic acid and n-butyl acetate in a volume ratio of 4: 1: 1: 4. The solution obtained was placed on a column which was filled with 500 g of silica gel. The solution was separated chromatographically by eluting according to the procedure given above.

About 0.75 l of the main fraction were then evaporated to dryness under reduced pressure. 0.7 g of residue were thus obtained. By recrystallizing the residue from ethyl alcohol, 0.3 g of crude, crystalline material was obtained. Further recrystallization from ethyl alcohol gave 0.17 g of desacyl-pepsidine in the form of white needles.

Melting point: The material decomposes at 193 ° to 199 ° C without a clear melting point being observed.

[a] o ° = -57 ° to -60 ° (C = 1, methanol) Elemental analysis for CisHssNsOs • H2O

C%

H%

N%

Calculated

56.19

9.26

11.29

Found

56.39

8.99

11.27

Example 2

15 l of a liquid nutrient medium (pH 7) which contained 1% meat extract, 1% peptone and 0.5% NaCl were placed in a 30 l fermentation vessel. The liquid medium was then sterilized at 120 ° C for 10 minutes.

In addition, a strain of Bacillus pumilus EF 49-210 was cultivated, in which the culture was built up for 24 hours in a similar nutrient medium at 30 ° C. with stirring.

The contents of the fermentation vessel were inoculated using 0.2 l culture broth from strain EF 49-210. The new cultivation was carried out under the following conditions:

Time: 24 h

Temperature: 30 ° C aeration: 151 / min stirring speed: 350 U / min

5

5

'0

i5

20th

25th

30th

Vj

4 (i

45

hl)

620244

After the cultivation was completed, the microorganisms were removed from the culture broth by centrifugation. Amount of ammonium sulfate was added to 12 l of centrifugate until 80% saturation was reached. The mixture was then left in the refrigerator for 3 days. The precipitate formed was suspended in distilled water and 750 ml of suspension were obtained.

A mixture of 100 ml of the suspension mentioned, 400 ml of a neutralized aqueous solution with 8 g of pepsidine C, 292 ml of a 1/15 m phosphate buffer (pH 5.5) and 8 ml of a 5 × 10 −2 m COCh solution was then prepared This mixture was kept in the incubation cabinet for 40 hours at 37 ° C. The reaction mixture was extracted three times with 800 ml of N-butyl alcohol. The combined extracts were evaporated to dryness. The residue was dissolved in a minimal volume of solvent mixture of N-butyl alcohol, Acetic acid, water and N-butyl acetate in a volume ratio of 4: 1: 1: 4. The solution was then separated by means of silica gel column chromatography.

2 l of the main fraction were evaporated to dryness and gave 6 g of white powder. After double recrystallization from ethanol, 2 g of desacyl-pepsidine were obtained, which was in the form of white needles.

Example 3

100 mg of desacyl-pepsidine, as obtained in Example 1, was dissolved in 100 ml of pyridine. 1.2 ml of a 20 times diluted acetyl chloride solution in acetone was then added dropwise to the pyridine solution, the whole being cooled with ice. The mixture was left overnight. An aliquot of the reaction mixture was then subjected to silica gel thin layer chromatography, using mixtures of N-butanol, acetic acid, water and n-butyl acetate in a volume ratio of 4: 1: 1: 4 as the developing solvent. The actual determination was then carried out using the ninhydrin reaction, the Rydon-Smith reaction and the casein plate method. The investigated material showed an Rf = 0.49 and it was identified as authentic pepsidine C.

The reaction solution was evaporated to dryness. The residue was taken up in 100 ml of methanol and then 17 ml of an ethereal solution of diazomethane was added to the solution. The solution was left at room temperature for 30 hours and then evaporated to dryness. The residue was dissolved in methanol. 34 mg of methyl ester of pepsidine C were crystallized out by adding ether.

Example 4

According to the procedure in Example 3 and using isovaleryl chloride instead of acetyl chloride, a product with Rf = 0.64 was produced, which was identified as authentic pepstatin A by thin layer chromatography.

Example 5

1 ml of the culture broth of the strain EF 49-210, as is obtained according to the procedure in Example 1, was mixed with 1 ml of an aqueous solution of 10 mg of pepsidine C in water, which had been neutralized with a 6N NaOH solution. The resulting solution was shaken at 37 ° C for 5 hours.

The reaction solution was examined by means of silica gel thin layer chromatography using a mixture of N-butanol, acetic acid and water in a volume ratio of 3: 1: 1 as the developing solvent. The product obtained shows an Rf = 0.48 and is identified as authentic desacyl-pepsidine.

Example 6

Desacyl-pepsidine was prepared in accordance with the procedure in Example 5, in contrast to the example mentioned starting from pepsidine B or pepsidine A. The product was again analyzed by thin layer chromatography.

Example 7

According to the procedure in Example 5, desacyl-pepsi-din was prepared by using N-isovalerylpene tapeptide (pepstatin A) instead of pepsidine. The product was again analyzed by means of thin layer chromatography.

Example 8

Desacyl-pepsi-din was prepared in accordance with the procedure in Example 5, with N-n-hexanoyl-pentapeptide or N-n-deca-noyl-pentapeptide being replaced by pepsidine C. The product was again analyzed by thin layer chromatography.

Example 9

Desacyl-Pepsi-din was prepared according to the procedure in Example 5, but Bacillus pumilus IFO 12092 or Bacillus pumilus IFO 12110 was used instead of Bacillus pumilus Kawaguchi EF 49-210. The product was again determined by means of thin layer chromatography.

Example 10

Desacyl-pepsidine was prepared according to the procedure in Example 5, but 1 ml of the culture broth of EF 49-210 was replaced by 10 mg of bacterial cells which had been dried from acetone. The product was again determined by means of thin layer chromatography.

6

3rd

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20th

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50

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G

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