KR950014463B1 - Preparation method of antibiotic chloropolysporin c - Google Patents

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Description

Method for preparing antibiotic chloropolisporin C

1, 2 and 3 show the ultraviolet absorption spectrum, the infrared absorption spectrum and the nuclear magnetic resonance spectrum of chloropolisporin B, respectively,

4, 5 and 6 show the ultraviolet absorption spectrum, infrared absorption spectrum and nuclear magnetic resonance spectrum of chloropolisporin C, respectively.

The present invention relates to a process for the preparation of the antibiotic chloropolisporin C from the related compound chloropolisporin B. Chloropolisporins B and C exhibit antimicrobial activity and are glycopeptide antibiotics useful as a preventive and therapeutic agent for infections and as growth promoters in animals.

As resistance to known antibiotics is increasing in common pathogens, there is an urgent need for a broader class of antibiotics available for these resistant bacteria. Furthermore, various antibiotics, such as chloramphenicol, aureomycin, vancomycin and aboparcin, have been administered or suggested to farm animals, including poultry and ruminants and pigs, for the prevention of growth, to promote growth or to produce milk. It is becoming. However, the basic disadvantage of using antibiotics in this field is that traces of antibiotics or their metabolites can be found in animal products that humans consume (eg eggs, milk or meat), and the risk of allergy of these residues in some parts of society. Is increasing. Therefore, farmers have a significant need for antibiotics that produce the desired growth promoting effect and have little or no residual in animal products.

In U.S. Patent Application No. 84304765.5 (published as EP-A-l32349) assigned to the assignee, Micropolyspora sp. An antibiotic called "chloropolyporin" has been described that has been isolated from the culture medium of the microorganism identified with SANK 60983. It was then discovered that the same microorganism and other microorganisms in the micropolispora produced two new antibiotics that had a high effect on Gram-positive bacteria and significantly improved their use as animal growth promoters. These two antibiotics, called chloropolisporin B and chloropolisporin C, methods of making by culturing microorganisms of the genus Micropolispor and their therapeutic and livestock therapeutic uses are disclosed in European Patent Application No. 86300176.4 (EP-A-187722). ) Is the subject of.

When chloropolyporin B and C are prepared by microbial culture, the yield of chloropolyporin C in the resulting culture broth is generally lower than the yield of chloropolyporin B. On the other hand, it was found that chloropolisporin C exhibits high antibacterial activity against more bacteria than chloropolisporin B. Therefore, there is a need for a method in which chloropolyporin C is a more potent ingredient, for example as a food additive, and can be selectively used to produce chloropolyporin C.

According to the present invention, it has now been found that chloropolyporin C can be prepared by selective hydrolysis of chloropolyporin B with rhamnosidase.

It is known that chloropolyporins B and C can be represented by the following planar structural formula (I).

here,

In the case of chloropolisporin B

R ¹ represents a 1-L-ristosamine residue;

R ² represents an lD-mannose residue;

R ³ represents a 1-D-glucose residue;

R ⁴ represents a 1-L-rhamnose residue;

In the case of chloropolisporin C

R ¹ represents a 1-L-ristosamine residue;

R ² represents a 1-D-mannose residue;

R ³ represents a 1-D-glucose residue;

R ⁴ represents a hydrogen atom

(Ristosamine is 3-amino-2,3,6-trideoxy-L-ribohexopyranose).

Accordingly, chloro Indianapolis Lin B, and C are only different substituents represented by the general formula -OR ^4. Various asymmetric carbon atoms represented by the general formula (I) in the colopolisporin B and C may have a specific configuration, but will not be described herein.

The process of the invention is an enzymatic hydrolysis reaction and can be carried out under conditions such as reaction temperature, reaction time and pH which are generally well known in this type of reaction. For example, it may be carried out at 20-50 ° C., preferably 30-40 ° C., most preferably about 37 ° C., with or without stirring in solution. For this reaction, known aqueous solvents such as, for example, 0.01-0.1 N phosphate buffer or Tris-HCl buffer solution can be used. The pH of the reaction solution is in the range of Boton 4-8, preferably 5-6. The reaction time depends on the reaction conditions such as temperature, pH and concentration of enzyme and chloropolyporin B substrate, and generally ranges from 5 hours to 3 days, preferably from 15 to 24 hours.

Enzyme preparations used in the methods of the invention usually have a rhamnosidase activity of 100 to 1000 units / mg, preferably 200 to 500 units / mg. The enzyme may generally be used in an amount corresponding to the weight ratio of rhamnosidase to chloropolyporin B in the range of 1: 0.01 to 1:50, more preferably l: 0.05 to 1:20.

The kind of rhamnosidase formulation used in the method of the present invention is not particularly limited as long as it has appropriate rhamnosidase activity. In practice, α-L-lamnosidase (enzyme classification number 3.2.1.40) is generally used. The enzyme may be used in pure form or in the form of a crude rhamnosidase which is commercially prepared from various microorganisms and further contains other enzymes and / or other substances besides the rhamnosidase itself. However, these other substances should not have an unacceptable adverse effect on the reaction process.

Examples of ramnosidase formulations that can be used in the method of the present invention include "Naringinase" and "Sclelase", which are manufactured by Japan's Sangha Kabushiki Kaisha, and "Gumidanase", which is manufactured by Tanabe Seiyaku Kabushi Kaisha. Naringinase is an enzyme preparation whose main enzyme component is α-lamnosidase and contains a small amount of β-glucosidase, and is produced by culturing Aspergillus usamii microorganisms or microorganisms of penicillium species. do. Sclease is a rich product of tinatinas containing naringinase , and is prepared by culturing Coniothrium diplodiella ( Aspergillus usamii ) or Aspergillus niger ( Spergillus niger ). Gumidanase is an α-lamnosidase derived from Aspergillus niger.

Instead of using the soluble form of the rhamnosidase formulation, the enzyme itself is immobilized on a suitable carrier, or a rhamnosidase-producing microorganism, such as a naringinase-producing microorganism such as Aspergillus Usami or Immobilized enzyme preparations can be used, such as preparations containing immobilized cells of sclease-producing microorganisms such as Pergillus niger. If such immobilized forms are used, the inhibition, materials and techniques used for immobilization of enzymes or enzyme-producing microorganisms can be selected from those commonly used for this purpose in the enzyme art.

The process of the present invention can be carried out by using a solution of chloropolisporin B previously separated in crude form or in pure form as a starting material, and the concentration of chloropolyporin B in the reaction solution is not particularly limited. However, according to a preferred embodiment of the invention the starting material contains chloropolisporin B, or a mixture of chloropolisporins B and C, obtained by culturing the chloropolisporin-producing micropolispor strain by a method as already mentioned. It may be a trillion cultures. In this process, it is not necessary to isolate or purify the chloropolyporin B starting material prior to entering the manufacturing process of the invention, which method is particularly advantageous for large scale commercial production. For example, the concentration of chloropolysporin B in the crude culture broth may be 1,000-10,000 γ / ml. More generally, the concentration of chloropolyporin B in the reaction solution is usually at least 1-5 γ / m 1.

At the end of the reaction, clopolisporin C formed by enzymatic hydrolysis can be recovered from the reaction mixture and purified by various techniques known in the art of producing biochemicals. For example, "Dion" (registered trademark) HP 20) such as Mitsubishi Chemical Co., Ltd. or "Amberlite" (registered trademark) XAD-2 or XAD-4 (Romend Haas Company) It may be adsorbed on an adsorbent resin and eluted from the resin with a suitable eluent such as aqueous acetone or aqueous methanol. Another purification method that can be used is reverse phase chromatography with silanized silica having a uniform particle size, preferably in the range of 0.06 to 0.2 mm. The eluent used in this technique may suitably be a mixture of aqueous ammonium formate and acetonitrile, a mixture of trifluoroacetic acid and acetonitrile, or a mixture of phosphate buffer and acetonitrile, with the most preferred eluent being about 85 parts by volume Mixture containing 0.2% trifluoroacetic acid and about 15 parts by volume of acetonitrile.

Thin film chromatography and high performance liquid chromatography can be used to control the concentrations of starting materials and reaction products through reactions including purification steps.

Since the structures of chloropolyporins B and C have not been fully revealed, the characteristics described below together with the accompanying drawings will be described.

Chloropolyporin B as sulfate thereof has the following properties:

(a) amorphous white powder, in water-soluble form;

(b) Specific light intensity: [α] ²⁵ -64.5 ° (c-1.04, 0.1N hydrochloric acid, sodium D-ray);

(c) Elemental Analysis: C; 48.33%, H; 5.05%, N; 5.48%, C1; 5.11%, S; 1.00%;

(d) substances obtained by acid hydrolysis;

Neutral saccharides: glucose, mannose and rhamnose; Amino acid: 3-chloro-4-hydroxyphenyl glycine and N -methyl- R -hydroxyphenylglycine;

(e) UV absorption spectrum:

As shown in FIG. 1 of the accompanying drawings, it has a maximum absorbance lambda max at 280 nm (E _lcm -51) in _0.1N hydrochloric acid solution. Absorbance E is measured at a concentration of 1% w / v;

(f) UV absorption spectrum:

The infrared absorption spectrum (γ cm ^-1 ) measured on the KBr disk is shown in FIG.

(g) nuclear magnetic resonance spectrum:

Nuclear magnetic resonance spectra (δ ppm) measured at 270 MHz in deuterated dimethyl sulfoxide using tetramethylsilane as internal standard are shown in FIG.

(h) Solubility:

Soluble in water, poorly soluble in methanol and acetone, and insoluble in ethyl acetate, couloform and benzene;

(i) color reaction:

Positive for ninhydrin and lidon-smith reaction;

(j) thin layer chromatography

Rf value-0.65, using cellulose sheet (Eastman) as adsorbent and 15: 10: 3: 12 volume mixture of butanol, pyridine, acetic acid and water as developing solvent;

(k) high pressure paper electrophoresis:

Use DO filter paper 51A (3300 volts / 60 cm, 1 hour) in 0.1 ml Tris-HCl buffer at pH 7.5, and travel distance from the original to the cathode (Basilus Subtilis PCI 219 by bioautomation). Detection) is 4 cm;

(1) Molecular formula:

C ₈₃ H ₈₉ O ₃₄ N ₈ C1 ₃ .0.5H ₂ SO ₄ .10H ₂ O;

(m) Molecular Weight:

The molecular weight measured by FAB-MS is 1846 (MH <+>, 1847). "FAB-MS" is a fast atom bombardment mass spectroscope.

Chloropolyporin C as sulfate thereof has the following properties:

(a) amorphous white powder, in water-soluble form;

(b) Specific light intensity: [α] ²⁵ -64.4 ° (c-1.08, 0.1N hydrochloric acid, sodium D-ray);

(c) Elemental analysis: C; 50.53%, H; 4.69%, N; 6.14%, C1; 5.82%, S; 1.12%:

(d) Substances obtained by acid hydrolysis:

Neutral saccharides: glucose, mannose, amino acids: 3-chloro-4-hydroxyphenylglycine and N -methyl- P -hydroxyphenylglycine;

(e) UV absorption spectrum:

As shown in FIG. 1 of the accompanying drawings, it has a maximum absorbance lambda max at 280 nm (E _lcm〓57 ) in _0.1N hydrochloric acid solution. Absorbance E is measured at a concentration of 1% w / v;

(f) UV absorption spectrum:

The infrared absorption spectrum (γ cm ^-1 ) measured on the KBr disk is shown in FIG.

(g) nuclear magnetic resonance spectrum:

The nuclear magnetic resonance spectrum (δ ppm) measured at 400 MHz in deuterated dimethyl sulfoxide using tetramethylsilane as an internal standard is shown in FIG.

(h) Solubility:

Soluble in water, poorly soluble in methanol and acetone, and insoluble in ethyl acetate, chloroform and benzene;

(i) color reaction:

Positive for ninhydrin and lidon-smith reaction;

(j) thin layer chromatography

Rf value-0.65, using cellulose sheet (Eastman) as adsorbent and 15: 10: 3: 12 volume mixture of butanol, pyridine, acetic acid and water as developing solvent;

(k) Molecular formula:

C ₇₇ H ₇₉ O ₃₀ N ₈ Cl ₃ .0.5H ₂ SO 4 .5H ₂ O;

(1) Molecular Weight:

The molecular weight measured by FAB-MS is 1700 (MH <+>, 1701).

The acute toxicity (LD ₅₀ ) value measured by intravenous administration to mice (ICR, male, 5 weeks old) is 215 mg / kg for chloropolyporin B and 250 mg / kg for chloropolyporin C.

The minimum inhibitory concentrations (MIC) of chloropolisporin B and C for various Gram-positive and Gram-negative bacteria are determined by a 2-fold agar dilution method using Mueller-Hinton Agar Medium (Difco) MIC for was determined using GAM agar medium (Nisyi Products). The results are shown in Tables 1 and 2.

TABLE 1

TABLE 2

From the results listed in the table, chloropolisporin B and C are aerobic grams such as Staphylococcus aureus, Staphylococcus epidermidis Enterococcus paecalis, Bacillus subtilis and Mycobacterium smeg Maris. Positive bacteria and anaerobic grams such as Eubacterium cylindroides, Peptococcus asacaroliticus, Propionibacterium acnes, Chlosterridium symbiosum, Clostridium perfringens and Clostridium difficile It is effective against modal bacteria.

Comparing the above-described chemical, physical and biological properties of chloropolyporin B and C with those of known antibiotics, they are like vancomycin, aboparcin α and β, actinoidins A and B or A-35512 B. It can be seen that it belongs to the chlorine-containing glycopeptide antibiotics. However, chloropolyporins B and C can be clearly distinguished from these known antibiotics based on the differences shown in Table 3 below. In particular, they have different neutral saccharide components and different amino acids are produced upon acid hydrolysis. Moreover, different distances are traveled in high pressure paper electrophoresis (HVPE 3300 volts / 60 cm, 1 hour, pH7.5, 0.1M tris-hydrochloric acid buffer solution) and have different chlorine content.

TABLE 3

The value reported as “distance” is the travel distance in high pressure paper electrophoresis measured in bioautomation using Bacillus subtilis PCI 219 as the test microorganism.

From the above findings, it can be seen that chloropolisporin B and C can be used as antibiotics for various diseases caused by bacterial infection. The route of administration can vary widely and can be parenteral (eg, subcutaneous, intravenous or intramuscular injection, or suppository) or oral (tablet, capsule, powder or granule form). The dosage will of course vary depending on the type of name to be treated, the age, condition and weight of the patient, the route and time of administration, but in adult patients 0.1 to 1.0 g per day is preferred, and may be administered once or in divided doses.

Moreover, due to the strong activity of chloropolisporin B and C against infectious bacteria in Clostridium, its utility as a growth promoter for livestock can be expected. Bacteria in chlorostridium, in particular Clostridium perfringens and Clostridium difficile, are often present in the intestines of farm animals and cause diarrhea. Since chloropolyporin B and C have a strong activity against these microorganisms, it inhibits the growth of microorganisms in the intestine to balance the microorganisms in the intestine. This phenomenon improves feed efficiency and contributes to weight gain and milk yield of various farm animals including ruminants, pigs and poultry. Furthermore, chloropolisporin B and C, like other glycopeptide antibiotics, have a low rate of absorption through the digestive tract, so when chloropolisporin B or C is administered to a feed, it can be found in animals and in animals, such as meat, milk or eggs. Rarely remains in animal products. When chloropolyporin B or C is used as an animal growth promoter, oral administration is preferred.

It may be formulated in an edible composition with a suitable carrier or diluent, but in particular it is convenient for the administrator to combine it with a feed or a drink. When chloropolyporin B or C is used as a feed additive, it may be mixed with the feed alone or in combination with other non-toxic edible excipients, nutrients (eg vitamins, minerals or amino acids), other antibiotics, repellents or enzymes. Can be. For administration to animals as growth promoters, chloropolyporin B or C need not be in fully purified form, but may be used in the crude form or in partially purified form obtained at the necessary stages during the extraction and / or purification described above. For use as a growth promoter, chloropolisporin B or C is preferably used in an amount of 1 to 200, more preferably 5 to 60 ppm by weight, based on the feed, beverage or other carrier to which the chloropolyporin B is applied. Or the impurity form of C is used at a concentration having the same activity.

Animals capable of administering chloropolisporin B or C include farm animals (e.g. cattle, horses, pigs, sheep and goats), poultry (e.g. chickens, turkeys and ducks) and pets (e.g. dogs, cats and birds). There is.

Most importantly, when orally administered chloropolisporin B or C to an animal, its growth is effectively promoted, but it is hardly absorbed in the stomach and hardly remains in animal tissue. Therefore, it is the most advantageous in terms of food hygiene that there is little residue of chloropolisporin B or C in the product of the animal to which chloropolisporin B or C is administered (eg milk, meat or egg).

Chloropolisporins B and C were isolated from the soil samples collected from Tochigi, Japan. Prepared by culturing the micropolisphor strain identified as SANK 60983.

Microbial micropolispor sp. SANK 60983 has the properties described hereinafter and is described in the above-mentioned European patent application no. 84304765.5 (Publication EP-A-132349). These characteristics are determined by incubation (in all cases a temperature of 28 ° C.) in various media as defined by the ISP (International Streptomyces Project) or in media recommended by S.A wax only in Volume 2 of “The Actinomycetes”.

1. Morphological characteristics

Strain SANK 60983 grows relatively well in various media. Airborne hyphae are not observed in almost all media, but can sometimes be observed in glycerol-asparagine agar or potato extract-carrot extract agar. At the top and center of the aerial and proliferative mycelium, they usually have short chains of 1 to 20 spores, sometimes more than 20 spores. Pieces of mycelium are not pronounced in the strain and can be observed at later stages of culture.

2. Culture Characteristics

Strain SANK 60983 may exhibit light yellow, tan or yellow gray. No airborne hyphae is observed in most media, and white airborne hyphae are produced in some mediums. No soluble pigment is produced. Table 4 shows the results obtained after 14 days of incubation at 28 ° C. in various standard culture media. The color name and number used are in accordance with the manual "Guide to Color Standard" issued by Nippon Kisai Kisho, Japan.

TABLE 4

3. Physiological Characteristics

Physiological properties of strain SANK 60983 are shown in Table 5.

TABLE 5

In the above table, "+" means positive, "_" means negative, and "±" means about positive.

Coagulation and peptonation of milk have been reported as negative, but can sometimes be positive after long term culture.

4. Whole cell composition

Acid hydrolysates of bacterial cells. blood. It is analyzed by paper chromatography by the method of Lachevalia et al. ("The Actinomycetes Taxonomy", pp225 (1980)). Miso-diaminopimelic acid, arabinose and galactose are Type IV because they are found to be present in the cell wall and the total per cell pattern is Type A. In addition, the characteristic acyl group of the cell wall is described in [J. Gen Appl. Microbia1. 23 . 249) 1977) was found to be an acetyl group.

It is not reported that actinomycetes of known genus can form spores in the middle of mycelia. However, in comparison to other features, this new strain is clearly related to the genus Actinopolisa, Saccharopolisa, Pseudocardia and Micropolisa. However, both actinopolispora and saccharopolispor spores grow at the end of aerial mycelium, the former is a highly basophilic genus, and the characteristic acyl groups of the posterior, cell wall are glycolyl groups. For this reason, the new strain SANK 60983 cannot belong to any of these genera. Strains of the genus Pseudonocardia can grow spores in aerial mycelia and celiac mycelium, as in strain SANK 60983, but their growth site is the tip of the hyphae, moreover, their hyphae grow characteristically by budding. Therefore, strain SANK 60983 may also belong to the genus Pseudocardia.

The difference between the genus Micropolispora and strain SANK 60983 is that the spore growth site of the micropolispor is restricted to the end of the hyphae, while the growth of SANK 60983 occurs at the end and center of the hyphae.

At present, there has been no practical discussion on the classification of these differences, but it seems inappropriate to classify the genus based solely on the difference in its spore growth area. Therefore, it would be most satisfactory to consider strain SANK 60983 as a new species in the micropolispor genus, and therefore micropolispor sp. It was named SANK 60983. However, the classification of microbial strains into certain species, genera, or families should be consistent with those experienced in the study of specific classifications of microorganisms, and the original classification of microorganisms is less common but can be changed after further discussion.

Strain SANK 60983 was deposited on March 10, 1983, with the accession number of FERM P-6985 to the Institute of Microbiological and Industrial Technology, Ministry of Commerce, Industry and Energy, Japan, March 10, 1983. Re-deposited under accession number BP-538. This strain was also deposited with the accession number of KCTC 8283P to the Korea Advanced Institute of Science and Technology on August 14, 1987.

Strain SANK 60983 can produce chloropolisporin B and C. However, as is known, the properties of microorganisms belonging to the common class of actinomycetes can vary considerably, and such microorganisms can cause mutations by natural causes and by artificial methods. Thus, microorganisms classified into the genus Micropolispora and having the characteristic ability of strain SANK 60983 to produce chloropolisporin B and C can also be used to prepare starting materials of the process of the invention.

Cultivation of microorganisms of the genus Micropolispora that produces chloropolisporin B and C can be carried out under conditions conventionally used for culturing actinomycete species, preferably liquid culture under shaking or stirring and aeration. The nutrient medium used for the culturing is completely known, and contains components commonly used for culturing actinomycetes. In particular, the medium may be an assimilable carbon source (e.g., glucose, maltose, sucrose, mannitol, molasses, glycerol, dextrin, starch, soybean oil and cottonseed oil), an assimilable nitrogen source (e.g., soybean wheat, peanuts). Wheat, cottonseed wheat, wheat, corn steep liquor, peptone, broth extract, compressed yeast, yeast extract, sodium nitrate, ammonium nitrate or ammonium sulfate and at least one inorganic salt (e.g. sodium chloride, phosphate, calcium carbonate and trace metal salts) It should contain When the cultivation is carried out in a liquid medium, it is generally preferred to incorporate an antifoaming agent (eg silicone oil, vegetable oil or a suitable surfactant) in the medium.

The cultivation is suitably carried out at a substantially neutral pH and a temperature of 24 to 30 ° C, preferably about 28 ° C.

The production of chloropolyporin B and C in the course of the culturing can be controlled by various conventional microbiological assays (if produced by microbial culture) to control the production of antibiotics, which are rarely needed here. Suitable techniques are, for example, paper disk-agar diffusion assays using Bacillus subtilis PCI 219 or Staphylococcus aureus FDA 209P JC-1 as test microorganisms (e.g., about 8 mm manufactured by TOYODA Corporation) Paper discs in diameter).

The amount of chloropolyporin B and C produced reaches a maximum after 55 to 70 hours of incubation, and it is preferable to separate chloropolyporin from the culture medium before reaching the maximum. However, this period may vary depending on culture conditions and techniques, and shorter or longer periods may be appropriate, depending on the situation. The exact incubation time is assessed on a case-by-case basis by routine experiments using appropriate control techniques as described above.

Chloropolyporins B and C are mainly released in the liquid portion of the culture broth and can therefore be recovered by removing solids, including mycelia, by filtration (preferably with a filter such as diatomaceous earth) or by centrifugation. have. And it can be recovered from the separated liquid portion by known techniques and purified if necessary.

Chloropolyporins B and C are preferably separated from other products of the liquid portion by adsorbents by adsorbing impurities, adsorbing chloropolyporins, adsorbing both separately or together, and eluting chloropolyporin. Various types of adsorbents can be used, for example activated carbon, and Amberlite® XAD-2, XAD-4 or XAD-7 (Rom End Haas), Dion® HP 10, HP 20 Adsorption values such as, CHP20P or HP 50 (manufactured by Mitsubishi Chemical Co., Ltd.) and polyamide gel (manufactured by West Germany Welm Pharma) are found to be particularly satisfactory. Impurities present in the liquid portion pass a solution containing chloropolyporin through the above-described layers or columns of one or more adsorbents, or adsorb chloropolyporin to one or more adsorbents, and separately or together the chloropolyporin into a suitable eluent. Can be removed by eluting. Suitable eluents are mixtures of methanol, acetone or butanol with water.

The chloropolyporins B and C thus obtained can be further purified by various methods. Suitable methods include fractional column chromatography using a cellulose product such as Avicel (registered trademark of Asahi Kasei Kogyo Co., Ltd.) or Sephadex LH-20 (registered trademark of Swedish Pharmacia); Reverse phase column chromatography using reverse phase carrier; Extraction using difference in distribution of chloropolyporin B and C and their contaminated impurities in the solvent; Or countercurrent distribution.

If necessary, the chloropolyporins B and C can be separated from one another by chromatography. A preferred system for this purpose is System 500 (prepared from Waters) using prepack C ₁₈ cartridges. Suitable eluents are buffer mixtures containing acetonitrile adjusted to slightly acidic pH.

Depending on the culture conditions, chloropolyporin B and C may be present in the mycelia from the culture broth and extracted therefrom by known techniques. For example, they are extracted with a hydrophilic organic solvent (e.g. alcohol or acetone) and the solvent is removed from the extract to give a residue which is then dissolved in an aqueous medium. Chloropolyporin can be extracted from the resulting solution and purified as described above.

The chloropolyporins B and C thus obtained have the physical and chemical properties described above as their sulfates. They are usually and preferably separated from the culture broth in water-soluble salt form, and most conveniently in salt form, ie sulfate in form, since chloropolisporin B and C itself (ie free base) are insoluble in water. .

When chloropolyporin B or C is separated in the form of a salt, it can be converted to the free base by a known method using an adsorbent for reverse phase chromatography or an ion exchange resin. Aqueous solutions of salts usually have an acidic pH value, and when the pH value is adjusted to almost neutral, the free base is predominantly precipitated and can be collected by suitable methods such as filtration or centrifugation. However, this product may be contaminated by impurities including traces of salts and therefore needs to be further purified. Thus, a more preferred method is to use, for example, an adsorbent for reverse phase chromatography or a suitable ion exchange resin. However, these compounds are known glycopeptide antibiotics, such as avoparsin, which have properties that are very difficult to separate in the free base form [eg, WJ McGahren ete et , al ., Journal of Antibiotics, XXXVI, 12 , 1671 (1983). ], It is preferably used separately in salt form.

The present invention is described in more detail by the following non-limiting examples illustrating the preparation of chloropolisporin C by enzymatic hydrolysis of chloropolisporin B and the following preparations explaining the preparation of starting materials by microbial culture. do.

Example 1

3.8 g of enzyme Naringinase (manufactured by Sangup Kasei Co., Ltd.) was added to 10.5 g of chloropolyporin B solution dissolved in 2 L of 0.06 M phosphate buffer (pH 5,8), and the mixture was slowly stirred, starting with chloropolyporin B. The reaction is carried out at 37 ° C. for 21 hours until the material can no longer be detected by HPLC.

After completion of the reaction, the reaction mixture was passed through a column containing 370m1 of DIION HP 20 adsorption resin (manufactured by Mitsubishi Chemical Corporation, Kogyo Co., Ltd.), and the column was washed with water, followed by 700m1 of 50% v / v aqueous acetone. Elute. The solvent is evaporated off from the eluent under reduced pressure and the residue is lyophilized to yield 7.8 g of crude product which is then purified by the following method.

The entire crude product is dissolved in 30 ml of 50% v / v aqueous methanol, and the solution is adsorbed onto a 3x50 cm column of Toyopearl HW-40F resin (manufactured by Torea Chemical Co., Ltd.). The column is eluted with 50% v / v aqueous methanol. The eluate is collected in each 20 ml fraction and the fraction in which chloropolisporin C is detected by HPLC is selected. These fractions are combined and the solvent is distilled off under reduced pressure. The residue is acidified to pH 4.0 with hydrochloric acid and lyophilized to yield 5.6 g of hydrochloride of chloropolisporin C.

Example 2

200 mg of enzyme sclease (manufactured by Sangup Kasei Co., Ltd.) was added to a solution of 10 mg of chloropolyporin B dissolved in 5 ml of 0.1 M phosphate buffer, and the mixture was allowed to react for about 24 hours at room temperature with gentle stirring. After completion of the reaction, the mixture is adsorbed onto a column containing 5 ml of Diaion HP 20 resin (manufactured by Mitsubishi Chemical Corporation, Kyokyo Co., Ltd.), the column is washed with water and then eluted with 50% v / v aqueous acetone. Fractions of the eluate containing chloropolyporin C are concentrated under reduced pressure and the residue is lyophilized to give 5 mg of crude product. The crude product can be purified by the following method.

8 g of the crude product containing chloropolyporin C are dissolved in 40 ml of water and the solution is adjusted to pH 3.0 with dilute hydrochloric acid. The solution is adsorbed onto a 4 × 40 cm column of polyamide gel (manufactured by Welch), developed with water, and each 20 ml of fractions are collected. Fractions containing chloropolyporin C (detected by HPLC) are combined and concentrated under reduced pressure. The residue is acidified to pH 4.0 with hydrochloric acid and lyophilized to yield 4.6 g of chloropolisporin C hydrochloride.

(quite)

In a 500 ml Erlenmeyer flask containing Medium A 80 ml with the following components (percent by weight): 1 loop growth of micropolispor sp. Inoculate SANK 60983.

The microorganisms are incubated at 28 ° C. for 84 hours using a 220 rpm rotating shaker.

25 ml of the resulting species culture are inoculated into four Erlenmeyer flasks containing 500 ml of medium B having the following composition (percent by weight).

The microorganisms are incubated at 28 ° C. for 24 hours using a rotary shaker at 220 rpm.

Combine the resulting culture broth. 750 ml of this broth are inoculated into two 30 L bottle fermenters each containing 15 L of medium B, and the microorganisms are incubated at 28 ° C. for 69 hours with aeration and stirring at a rate of 15 L per minute.

After the completion of the reaction, a batch of culture broth separately cultured as described above is combined to obtain a total of 30 L culture broth. A Celite 545 (registered trademark of Jones-Manville Product Corporation, NJ, USA) filter is added to the culture broth and the mixture is filtered to yield 30 L of filtrate. The filtrate is adsorbed onto 3 L of DIION HP 20 (manufactured by Mitsubishi Chemical Corporation, Kyokyo Kogyo Co., Ltd.), and the adsorbent is washed with water and then eluted with 50% v / v aqueous acetone. Acetone is removed from the active fractions by evaporation under reduced pressure and the concentrate obtained is lyophilized to give 44 g of crude powder.

41 g of this powder is dissolved in water, adsorbed onto 1.8 L of Dion HP 20, washed with 5 L of water and 2 L of 10% v / v aqueous acetone, and eluted with 4 L of 50% v / v aqueous acetone. . The active fractions from the elution are collected and concentrated to 1 L by evaporation under reduced pressure. The concentrate is centrifuged at 5000 rpm and the resulting precipitate is dried to give 9.6 g of crude powder containing chloropolyporin B and C.

This crude powder is dissolved in 1 L of 50% v / v aqueous methanol and equilibrated to 200 ml of acidic alumina (prepared by West German Wel Pharmacia) previously formed of 50% v / v aqueous methanol. The adsorbed product is eluted with the same solvent and a total of 1.1 L of active fractions are collected. The combined active fractions are passed through 60 ml of Doch's 21K (OH-) and eluted with water. 1.2 liter of the active fraction from this elution is collected and concentrated to a volume of 30 ml by evaporation under reduced pressure. The concentrate is lyophilized to yield 1.23 g of powder. The powder is dissolved in aqueous hydrochloric acid at pH 4.0 and adsorbed onto 56 g of polyamide (West Germany Welm Pharmacia) filled with water. It is eluted from 20 ml fractions to fraction 80 with 400 ml of water and 1.2 L of methanol. Collect fractions 30-60 and combine. Methanol was distilled off under reduced pressure, and the resulting concentrate was freeze-dried to give 738 mg of white powder containing chloropolyporin B and C.

4.4 g of white crude powder containing chloropolyporin B and C were mixed with 15 parts of acetonitrile and 85 parts of buffer solution (0.2% w / v sodium heptane sulfonate, 2.5% w / v acetic acid and 0.5% w / v concentrated aqueous ammonia). In a mixed solvent of 80 ml, and the solution is adsorbed onto a system 500 chromatograph (manufactured by Waters) using a Prepack C ₁₈ cartridge. It is developed and eluted with the mixed solvent as described above at a flow rate of 100 to 150 m1 per minute. Chloropolyporin B elutes from the solvent after 800 to 1700 ml of eluent passes through the cartridge, and chloropolyporin C elutes after passing of 1700 to 4700 ml of eluent.

The active fractions containing chloropolyporin B are collected and adjusted to pH 7.0. These are evaporated under reduced pressure to distill off acetonitrile. The resulting concentrated solution is adsorbed onto 20 columns of Diaion HP (100 ml), washed with water and eluted with 500 ml of 70% v / v aqueous acetone. The eluate is concentrated by evaporation under reduced pressure and the residue is lyophilized to give a powdered chloropolyporin B heptane sulfonate.

200 mg of this powder is dissolved in 5 ml of water, and 1 ml of a 10% w / v aqueous solution of sodium dodecyl sulfonate is added dropwise to the resulting solution. The precipitate formed is collected by centrifugation at 3000 rpm for 10 minutes. This precipitate is suspended in water and the suspension is again centrifuged at 3000 rpm for 10 minutes to wash the precipitate. Repeat this process three more times to wash the precipitate. The precipitate is dissolved in 3 ml of methanol and the insoluble residue is filtered off. 2 ml of a 0.5 M methanolic solution of triethylamine sulfate is added dropwise and the resulting precipitate is collected by centrifugation at 3000 rpm for 10 minutes. This precipitate is suspended in a small amount of methanol and centrifuged at 3000 rpm for 10 minutes. Repeat this process three more times to wash the precipitate. The precipitate is dissolved in 1.5 ml of water and the insoluble residue is filtered off. The filtrate was lyophilized to yield 65 mg of chloropolisporin B sulfate.

Claims (8)

With his sulfate (a) amorphous white powder, in water-soluble form; (b) Specific light intensity: [α] ²⁵ -64.5 ° (c〓1.04, 0.1N aqueous hydrochloric acid, sodium D-ray); (c) Elemental analysis: C; 48.33%, H; 505%, N; 548%, Cl; 511%, S; 100%, (d) Substances obtained by acid hydrolysis: Neutral saccharides: glucose, mannose and rhamnose; Amino acid 3-cholo-4-hydroxyphenyl glycine and N -methyl- P -hydroxyphenylglycine; (e) UV absorption spectrum: As shown in FIG. 1 of the accompanying drawings, it has a maximum absorbance lambda max at 280 nm (E _lcm -51) in _0.1N hydrochloric acid solution. Absorbance E is measured at a concentration of 1% w / v; (f) infrared absorption spectrum: The infrared absorption spectrum (γ cm ^-1 ) measured on the KBr disk is shown in FIG. (g) nuclear magnetic resonance spectrum: Nuclear magnetic resonance spectra (δ ppm) measured at 270 MHz in deuterated dimethyl sulfoxide using tetramethylsilane as internal standard are shown in FIG. (h) Solubility: Soluble in fire, poor in methanol and acetone, and insoluble in ethyl acetate, chloroform and benzene; (i) color reaction: Positive for ninhydrin and lidon-smith reaction; (j) thin layer chromatography Rf value = 0.65, use of cellulose sheet (Eastman) as adsorbent and 15: 10: 3: 12 volume mixture of butanol, pyridine, acetic acid and water as developing solvent; (k) high pressure paper electrophoresis: Toyo filter paper 51A (3300 volts / 60 cm, 1 hour) in 0.1M tris-hydrochloric acid buffer solution at pH 7.5; Detection) is 4 cm; (1) Molecular formula: C ₈₃ H ₈₉ O ₃₄ N ₈ Cl ₃ .0.5H ₂ SO ₄ .10H ₂ O; (m) Molecular Weight: The molecular weight measured by FAB-MS is the following planar structural formula (I) having the characteristics of ^l 846 (MH +, 1847), wherein R ¹ represents a 1-L-listamine residue and R ² represents 1-D-mannose. Residues, R ³ represents lD-glucose residues, R ⁴ represents 1-L-rhamnose residues), and the enzymatic hydrolysis of the antibiotic chloropolisporin B using rhamnosadases Made with his sulfate (a) amorphous white powder, in water-soluble form; (b) specific light intensity [α] ²⁵ -64.4 ° (cl. 08, 0.1 N aqueous hydrochloric acid, sodium D-ray); (c) Elemental analysis: C; 50.53%, H; 4.69%, N; 6.14%, C1; 5.82%, S; 1.1%; (d) Substances obtained by acid hydrolysis: Neutral saccharides: glucose, mannose; Amino Acids: 3-Chloro-4-hydroxyphenylglycine and N -methyl- P- Hydroxyphenylglycine; (e) UV absorption spectrum: As shown in FIG. 1 of the accompanying drawings, it has a maximum absorbance lambda max at 280 nm (E _lcm -57) in _0.1N hydrochloric acid solution. Absorbance E is measured at a concentration of 1% w / v; (f) infrared absorption spectrum: The infrared absorption spectrum (γ cm ^-1 ) measured on the KBr disk is shown in FIG. (g) nuclear magnetic resonance spectrum: Nuclear magnetic resonance spectra measured at 400 MHz in deuterated dimethyl sulfoxide using tetramethylsilane as internal standard (

ppm) is shown in FIG. (h) Solubility: Soluble in fire, poorly soluble in methanol and acetone, and insoluble in ethyl acetate, chloroform and benzene; (i) color reaction: Positive for ninhydrin and lidon-smith reaction; (j) thin layer chromatography Rf value-0.65, using cellulose sheet (eastman) as adsorbent and 15: 10: 3: 12 volume mixture of butanol, pyridine, acetic acid and water as developing solvent; (k) Molecular formula: C ₇₇ H ₇₉ O ₃₀ N ₈ C1 ₃ .0.5H ₂ SO ₄ .5H ₂ O; (1) Molecular Weight: The molecular weight measured by FAB-MS is the following planar structural formula (1) (wherein, R ¹ represents 1-L-ristosamine residue and R ² represents 1-D-manno) having the characteristics of 1700 (MH +, 1701). An ozone residue, R ³ represents a 1-D-glucose residue, and R ⁴ represents a hydrogen atom).

The method according to claim 1, wherein the hydrolysis is carried out in the range of pH 5-6. The process according to claim 1, wherein the hydrolysis is carried out at a temperature of 30 to 40 ° C. The method of claim 1, wherein the hydrolysis is carried out using an enzyme preparation having an α-lamnosidase activity in the range of 200 units / mg to 500 units / mg. The process according to claim 1, wherein the hydrolysis is carried out at an enzyme concentration whose weight ratio of rhamnosidase to chloropolyporin B is in the range of 1: 0.05 to 1:20. The process according to claim 1, wherein the hydrolysis is carried out in a solution containing 1,000-10,000 γ / m chloropolyporin B. The method of claim 1, wherein the hydrolysis is performed in a culture broth containing chloropolisporin B obtained by culturing the chloropolisporin-producing micropolispor strain. The method of claim 1 wherein the hydrolysis is carried out for a time in the range of 15 to 24 hours.

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