CH625803A5 - Process for the preparation of thienamycin and its isomers deacetyl-890 A1 and 890 A3 - Google Patents

Description

The invention has for its object to provide a method for producing new and valuable antibiotics which inhibit the growth of various gram-negative and gram-positive microorganisms in a highly effective manner, which is based on enzymatic deacetylation of the compounds 890 Ai and 890 A3 and is also suitable for deacetylating N-acetylthienamycin.

This object is achieved by the method according to the invention, which is defined in patent claim 1.

Suitable sources of an amidohydrolase capable of hydrolyzing the N-acetyl group are strains of the microorganism Protaminobacter ruber which produce aminohydrolase. The enzyme produced by Protaminobacter ruber is N-acetylthienamycin amidohydrolase, a member of the subgroup of enzymes, which according to the recommended enzyme class of the "International Union of Pure and Applied Chemistry" and the "International Union of Biochemistry" as EC 3.5.1 referred to as.

The new antibiotic substances produced by the process according to the invention, which are referred to here as desacetyl-890 Ai and desacetyl-890 A3, and also the equally available thienamycin can be present in dilute form as a crude concentrate and in pure form.

The microorganism capable of performing the deacetylation process was isolated from a soil sample and identified as belonging to the Protaminobacter ruber species based on classifying studies. In the culture collection of Merck & Co., Inc., Rahway, N.J., it was designated MB-3528. A culture of the microorganism is permanent on November 21, 1975 in the culture collection of the Northern Regional Research Laboratories, Nothern Utilization Research and Development Division, Agricultural Research Service, U.S. Department of Agriculture, Peoria, Illinois, has been assigned the identification number NRRL B-8143.

The morphological and cultural properties of Protaminobacter ruber NRRL B-8143 as well as the

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Details of the use of carbon and nitrogen and the biochemical reactions of the microorganism are given below:

Morphology 5

The cells are rod-shaped with rounded ends, 0.9-1.2 X 2.3-4.6 n in size and occur individually or in pairs. 24 and 48 hour old cells are stained gram negative with a granular appearance. The grains, especially the polar grains, are colored black by Sudanese Black B. The 10 cells are mobile at 28 ° C, but mobility is questionable at 37 ° C.

Cultural characteristics Colonies on nutrient agar are initially thin, punctiform, semi-transparent and colorless; then they become low-convex, 1S opaque, smooth, completely (edge entire), slightly dry in consistency and pink to pink pigmented.

Cultures in broth are evenly cloudy without cuticles.

The pigment generation is independent of light or the 20 temperatures examined (28 ° C and 37 ° C). The pigment is soluble in acetone, but insoluble in water or chloroform.

At 28 ° C the growth on nutrient agar and brain-heart-infusion agar takes place somewhat slowly but well under aerobic conditions; at 37 ° C the growth is moderate to good, but 2S slower; no growth takes place at 50 ° C.

Exploitation of C and N sources When using a basal salt medium with ammonium sulfate as the N source, growth with arabinose is good, 30

moderate with xylose and poor with dextrose, fructose, mannose, rhamnose, lactose, maltose, sucrose, raffinose, cellulose, inositol and mannitol.

N-acetylethanolamine can be used as the only C and N source.

In OF basal medium (Difco Laboratories, Detroit, Michigan), no acid or gas is formed from dextrose or lactose under aerobic or anaerobic conditions.

Biochemical reactions The biochemical reactions are based on standard methods, which are described in the “Manual of Microbiological Methods”, published by the Society of American Bacteriologists, McGraw-Hill Book Co., New York, 1957.

Catalase - positive Oxidase - negative Starch is not hydrolyzed Casein is not hydrolyzed Gelatin is not liquefied

Litmus milk remains unchanged in its consistency, but becomes somewhat alkaline indole - negative H2S - negative after 7 days

Nitrates are not reduced urease-positive

Lysine and ornithine decarboxylase negative.

N-acetylthienamycin, 890Ai and 890A3 are the names for isomers of the antibiotic of the structural formula

CH3-CH (Om s-ch2-ch2

di)

N-acetylthienamycin, its description, and a method for making the same are described in U.S. Patent Application Serial No. 634,301, and the compounds 890Ai and 890A3, their description and methods for making the same are found in U.S. Patent Application Serial No. 634,300.

40 The new antibiotics produced according to the invention, namely desacetyl 890Ai and desacetyl 890A3, are isomers of thienamycin of the structural formula

45

ch3-ch (oh)

-ch2-ch2-nh2

(I)

cooh and are produced by enzymatic hydrolysis of 890Ai or ss 890A3 with the aid of an amidohydrolase, which occurs, for example, in species of the genus Protaminobacter.

An amidohydrolase which is particularly suitable for the N-deacetylation of N-acetylthienamycin, 890Ai and 890A3 is N-acetylthienamycin admidohydrolase. 60

A surprising homology between N-acetylethanolamine and N-acetylthienamycin was found in that extracts of microorganisms with the enzyme N-acetylethanolamine amidohydrolase, which has not yet been described, are in many cases able to hydrolyze N-acetylthienamycin. It has also been found that amidohydrolases capable of hydrolyzing N-acetylthienamycin also have the ability to convert compounds 890Ai and 890A3 to their new deacetyl forms. To obtain such microorganisms, one can select those strains which are able to use N-acetylethanolamine for their growth and then examine these microorganisms for the presence of N-acetylthienamycin amide hydrolase.

The antibiotic thienamycin, which is obtained from N-acetylthienamycin by the N-deacetylation process according to the invention, is a valuable antibiotic. Its description, manufacture, and commercial utility are disclosed in U.S. Patent Application Serial No. 526,992. The antibiotics Desacetyl-890Ai and Desacetyl-890A3 are new and valuable antibiotics.

A feature of the invention is the new deacetylation

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of N-acetylthienamycin. There are two sources for N'Acetyl-thienamycin. N-acetylthienamycin is produced by fermentation of a liquid with the microorganism Streptomyces cattleya NRRL-8057. This microorganism also produces thienamycin, which can then be chemically N-acetylated.

Based on extensive classifying studies, the microorganism Streptomyces cattleya isolated from a soil sample was identified as Actinomycete and designated MA-4297 in the culture collection by Merck & Co., Inc., Rahway, N.J. A culture of this microorganism is permanently in the culture collection of the Northern Regional Research Laboratories, Northern Utilization Research and Development Division, Agricultural Research Service, U.S. Department of Agriculture, Peoria, Illinois, has been assigned the identification number NRRL 8057.

The compounds desacetyl-890Ai and desacetyl-890A3 produced according to the invention are valuable antibiotics which are active against a wide variety of gram-positive and gram-negative bacteria and are therefore used both in human medicine and in veterinary medicine. The compounds can be used as antibacterial drugs to treat infections caused by gram-positive or gram-negative bacteria. e.g. against Staphylococcus aureus, Proteus mirabilis, Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae and Pseudomonas aeruginosa. The antibacterial agents can also be used as an additive to animal feed, for food preservation and as a disinfectant. For example, they can be used in aqueous compositions in concentrations of 0.1 to 100 parts of antibiotic per million parts of solution, or preferably in concentrations of 1 to 10 parts of antibiotic per million parts of solution, to promote the growth of harmful bacteria on medical and dental equipment destroy and inhibit, as well as bactericides for technical applications, e.g. in water-based paints, in the white water of paper mills and to inhibit the growth of harmful bacteria.

The antibiotics described can be used in the various pharmaceutical preparations as sole active ingredients or in combination with one or more other antibiotics or with one or more pharmacologically active substances. As an example of the former case, an aminocyclitol antibiotic, such as gentamicin, can be administered together with the antibiotics described in order to limit the possibility of the occurrence of resistant organisms to a minimum. As an example of the latter case, diphenoxylate and atropine can be combined in dosage forms for the treatment of gastrointestinal diseases. The antibiotics can be used in the form of capsules, tablets, powders, liquid solutions or as a suspension or elixirs. They can be given orally, locally, intravenously or intramuscularly.

Oral delivery tablets and capsules may be in unit dosage form and conventional excipients such as binders e.g. Syrup, acacia, gelatin, sorbitol, tragacanth or polyvinyl pyrrolidone; Fillers such as lactose, sugar, corn starch, calcium phosphate, sorbitol or glycine; Lubricants, e.g. Magnesium stearate, talc, polyethylene glycol, silicon dioxide; decay promoting agents, e.g. Contain potato starch or harmless wetting agents such as sodium lauryl sulfate. The tablets can be coated by methods known per se. Oral liquid preparations can be in the form of aqueous or oily suspensions, solutions, emulsions, syrups, elixirs, etc. or as a dry product for mixing with water or other carriers before use. Such liquid

Preparations can be conventional additives such as suspending agents e.g. Sorbitol syrup, methyl cellulose, glucose / sugar syrup, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminum stearate gel or hydrogenated edible fats, emulsifiers, e.g. Lecithin, sorbitan monooleate or acacia resin; non-aqueous vehicles, such as edible oils, e.g. B. almond oil, fractionated coconut oil, oily esters, propylene glycol or ethyl alcohol; Preservatives, e.g. p-Hydroxyben-zoesäuremethyl- or -propylester or sorbic acid, contain. Suppositories contain e.g. conventional suppository bases, such as cocoa butter or other glycerides.

Means for injection can be in unit dose form in ampoules or in multiple dose form in containers with the addition of preservatives. The agents can take the form of suspensions, solutions, emulsions in oily or aqueous vehicles and contain formulating agents such as suspending, stabilizing and / or dispersing agents. The active ingredient may also be in powder form and, before use, with a carrier, e.g. sterile, pyrogen-free water.

The agents may also be in a form suitable for absorption by the mucous membranes of the nose and throat or the bronchial tissues, e.g. as powder or liquid spray or inhalation agent, pastilles, throat paints, etc. For the treatment of the eyes or ears, the preparations can be used as individual capsules, in liquid or semi-liquid form or as drops, etc. For local application, they can be present in hydrophobic or hydrophilic bases as ointments, creams, lotions, paints, powders, etc.

In addition to a carrier, the agents can contain other constituents, such as stabilizers, binders, oxidation retardants, preservatives, lubricants, suspending agents, viscosity-influencing agents or flavorings and the like.

In veterinary medicine, such as in the treatment of chickens, cows, sheep, pigs and the like, the agents can e.g. are formulated as preparations for injecting into the breast that are long-term effects or release the active ingredient quickly.

The dose to be administered depends largely on the condition of the animal to be treated, the weight of the animal and the type of infection, the type and frequency of administration, with parenteral administration for general infections and oral administration for intestinal infections being preferred.

For the treatment of bacterial infections in humans, the compounds described can be administered orally or parenterally by conventional methods for the treatment with antibiotics in amounts of 2 to 600, preferably 5 to 100 mg / kg / day and preferably divided into individual doses and e.g. be given three to four times a day. Unit dosage forms can be used, e.g. Contain 25, 250, 330,400 or 1000 mg of active ingredient together with physiologically acceptable carriers or extenders. The dose units are expediently in the form of liquid preparations, such as solutions or suspensions, or as solid substances in tablets or capsules. The optimal dose for each individual case depends on the type and severity of the infection, with smaller doses being used for the treatment of children; all such design rules are known to the person skilled in the art.

The antibiotic thienamycin can be administered in the same way as the antibiotics Desacetyl-890Ai and Desacetyl-890A3.

Analytical method for antibiotic N-acetylthienamycin

The percentages given below are by weight unless otherwise stated.

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I. Bioanalysis

Antibacterial activity analyzes are carried out by the following disc diffusion method using Vibrio percolans ATCC 8461 or Staphylococcus aureus ATCC 6538P as the test organism.

Panels with Vibrio percolans ATCC 8461 are manufactured as follows:

A freeze-dried culture of Vibrio percolans ATCC 8461 is suspended in 15 ml of a sterile medium containing 8 g / 1 "dif co" nutrient broth and 2 g / 1 yeast extract in distilled water (hereinafter abbreviated as NBYE). The culture is incubated overnight in a rotating shaker at 28 ° C. This culture is used to inoculate the surface of slant tubes containing 1.5% agar in NBYE and the inoculated slant tubes are incubated overnight at 28 ° C and then stored in the refrigerator.

The cooled oblique tubes made from a single freeze-dried culture are used for a period of up to 4 weeks after their preparation as follows: An eyelet with seed from the oblique tube is dispersed in a 250 ml Erlenmeyer flask in 50 ml NBYE. The culture is incubated overnight in a rotating shaker at 28 ° C and then diluted to a density that results in 50% light transmission at 660 nm. 33.2 ml of this diluted culture are added to a volume of 11 NBYE containing 15 g agar and kept at 46 ° C. The inoculated, agar-containing medium is poured into petri dishes made of plastic of 100 × 15 mm in quantities of 5 ml per dish, cooled and kept at 2 to 4 ° C. up to 5 days before use.

Plates with Staphylococcus aureus ATCC 6538P are made as follows:

A culture of the analytical organism Staphylococcus aureus ATCC 6538P developed overnight in nutrient broth containing 0.2% yeast extract is diluted with nutrient broth containing 0.2% yeast extract to a suspension with an optical transmittance of 55% at a wavelength of 660 nm. This suspension is added to «Difco» nutrient agar, which has been supplemented with 2.0 g / 1 «Difco» yeast extract and is at a temperature of 47 to 48 ° C, giving a mixture which is 33.2 contains ml of the suspension per liter of agar. 5 ml of this suspension are poured into Petri dishes with a diameter of 85 mm and these plates are cooled and kept at 4 ° C. until used (for a maximum of 5 days).

Samples of the antibiotic to be analyzed are diluted with pH 7 phosphate buffer solution. Filter paper discs with a diameter of 6.35 or 12.7 mm are immersed in the test solution and placed on the surface of the analysis plates. The plates are incubated overnight at 37 ° C, after which the diameter of the zone of inhibition is determined in millimeters. The diameter of the inhibition zone in millimeters determines the relative strength.

II. Absorbance extinguished by hydroxylamine

The proportion of the absorbance measured at 301 nm, which can be attributed to the content of impure samples in the antibiotic, is determined by selective extinction of this absorbance (with simultaneous inactivation of the antibiotic activity) by reaction with dilute hydroxylamine.

Samples containing the antibiotic to be tested are prepared in 0.01 molar potassium phosphate buffer solution (pH 7) so that they have an initial A301 between 0.1 and 1.0. Freshly prepared, neutralized hydroxylamine (NH20H HCl + Na0H up to a final pH

Value of 7) is added up to a final concentration of 10 mmolar, after which the reaction is allowed to proceed at room temperature for at least 30 minutes. If the A30i value obtained in this way is subtracted from the original reading (after correction for the dilution by the added reagent), the absorbance extinguished by hydroxylamine is obtained. Solutions of pure N-acetylthienamycin show an absorbance of 96.0% which can be extinguished by hydroxylamine.

Analytical method for thienamycin I. Bioanalysis

Unless otherwise stated, analyzes of the antibacterial activity are carried out according to the following disk diffusion method. The analysis plates are made as described above for the analysis of N-acetylthiena-mycin.

Samples of the antibiotic to be analyzed are diluted with pH 7 phosphate buffer solution. Filter paper disks with a diameter of 12.7 mm are immersed in the test solution and placed on the surface of the analysis plates; two slices of each sample are usually placed on a plate facing each other. The plates are incubated overnight at 37 ° C and the zone of inhibition is measured in millimeters in diameter. The zone of inhibition measured in millimeters determines the relative strengths or, when compared to a purified reference standard such as cephalo-thin, the strength of the antibiotic in units / ml. The activity unit is based on standard cephalothin solutions of 8.4, 2 and 1 y / ml. One unit is defined as the amount which, according to the calculation, produces the same inhibition as 1 y cephalothin per milliliter, this inhibition zone having a diameter between 16 and 21 mm.

II. Absorbance Extinguished by Hydroxyalamine The proportion of the absorbance measured at 297 nm, which can be attributed to the content of impure samples in the antibiotic, is determined by the selective extinction of this absorbance (with simultaneous inactivation of the antibiotic activity) by reaction with dilute hydroxylamine.

Samples are prepared in 0.01 molar potassium phosphate buffer solution (pH 7.0) so that they have an initial A297 between 0.05 and 2.0. Freshly prepared, neutralized hydroxylamine (NH2OH • HCl + NaOH to a final pH of 7) is added to a final concentration of 10 mmolar, after which the reaction is allowed to proceed at room temperature for at least 30 minutes. Subtracting the A297 value obtained in this way from the original reading (after correction for the dilution by the added reagent) gives the absorbance extinguished by hydroxylamine. Solutions of pure thienamycin show an absorbance of 94.5% which can be extinguished by hydroxylamine.

Analysis methods for 890Ai and 890A3 I. Microbiological analysis An agar plate disc diffusion method is used, using either Vibrio percolans ATCC 8461 or Salmonella gallinarum MB-1287 as test organism. A purified sample of the 890Ai antibiotic is used as a standard.

Plates containing Vibrio percolans ATCC 8461 are made as described above under I. Bioanalysis of N-acetylthienamycin.

The plates containing Salmonella gallinarum MB-1287 are produced as follows:

A sealed tube containing frozen and freeze-dried Salmonella gallinarum MB-1287 cells in Lean5

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contains milk and has been closed under vacuum, is opened and inoculated on 15 ml of brain-heart-infusion broth. The cells are allowed to grow overnight without shaking at 37 ° C and the culture is inoculated on slant tubes containing 0.8% BBL broth + 0.2% "Difco" yeast extract + 1.5% agar. After growing overnight at 37 ° C, an eyelet of culture is transferred from the slant tube into a flask containing 50 ml of 0.8% nutrient broth + 0.2% yeast extract. The flask is shaken overnight and 11 of 0.8% nutrient broth + 0.2% yeast extract +1.5% agar, which has been previously sterilized and cooled to 48 ° C, is inoculated with 20 ml of this culture. The inoculated NBYE agar is immediately poured into plastic petri dishes of 100 X 15 mm (5 ml per dish) and the plates are kept at 0 to 3 ° C until use.

Filter paper disks with a diameter of 12.7 mm are dipped into the solution to be analyzed and placed on the agar. According to another method, the slices can be loaded by pipetting 0.1 ml of the solution onto a dry slice and then placing the slice on the agar. After a suitable incubation (for Salmonella gallinarum MB-1287 for 9 to 18 hours at 37 ° C, for Vibrio percolans ATCC 8461 for 12 to 24 hours at 25 ° C) the diameter of the zone of inhibition is measured. If necessary, dilutions of the solutions to be analyzed are prepared in 0.05 molar potassium phosphate buffer (pH 7.4; hereinafter abbreviated as KPB) or in demineralized water.

The strength is calculated as follows: A slope is determined by measuring the zone diameter of a solution of the antibiotic 890Ai or 890A3 and a fourfold dilution of this solution (in KPB). Two slices of each concentration are analyzed on a single plate and the mean zone size is determined at each concentration. The gradient is equal to half the difference between the mean values of the zone sizes. The strength is then calculated using the following equation:

Strength (units / ml) =

([D-Ds] log 2 \ —G f "ll /

In the above equation, D is the average diameter of the zones formed by the unknown sample, Ds is the average diameter of the standard zones and "dilution" is the degree to which the unknown sample has been diluted prior to analysis. If no standard is used, Ds is assumed to be 25 mm and the size (thickness of the standard) to be 1 unit / ml if Vibrio percolans ATCC 8461 is used as the test organism. Pure 890Ai is determined to have a strength of 250 units per unit hydroxylamine-extinct absorbance at 300 nm when used as a standard. Accordingly, the strength of the antibiotic 890A3 is 31 units per unit of the absorbance at 300 nm which can be extinguished by hydroxylamine.

For analyzes on plates with Salmonella gallinarum MB-1287, the gradient is determined in the same way as with Vibrio percolans ATCC 8461. If no standard is used, only relative strengths are calculated. If no slope is measured, a value of 2.3 mm is assumed. The strength calculations are carried out as in the analysis with Vibrio percolans ATCC 8461. If an 890Ai standard is not used, a control of penicillin G at 250 y / ml can be used to demonstrate that the sensitivity of the organisms is in the normal range.

II. Analysis method for determining «890 analysis units»

The conventional disk-plate analysis method is used with disks with a diameter of 12.7 mm. One works with routinely poured 10 ml per plate, inoculates with Vibrio percolans (ATCC 8461) and uses as standard cephaloridine. Four concentrations of cephalori-din, namely 3.12, 6.25, 12.5, and 25 y / ml form the standard, the concentration of 12.5 y / ml being used as the reference solution. The zone diameters on a 5 ml plate for the standard are as follows:

Concentration, y / ml

Zone diameter, mm

3.12

16.8

6.25

22.3

12.5

25.0

25th

29.6

One unit is defined as the amount of antibiotic that creates a zone of inhibition of 25 mm on a 5 ml plate. Therefore, in this analysis, a concentration of cephaloridine of 12.5 y / ml is considered to be a unit of 890A equivalent. Since the slope of the line for cephaloridine is 4.0, the strength calculations of a sample are made using a 4.0 slope.

III. Hydroxylamine reaction The two antibiotics 890Ai and 890A3 react with hydroxylamine to form a substance that greatly reduces the absorbance at 300 nm. This provides a basis for quantitative analysis on the 890Ai and 890A3 antibiotics.

The solution to be analyzed is adjusted to 0.05 molar in a potassium phosphate solution (pH 7.4) by adding V20 space part of a solution of 0.8 molar K2HPO4 and 0.2 molar KH2PO4. Then V100 Raumteü 1 molar hydroxylamine hydrochloride is added and the absorbance at 300 nm is determined at intervals of V2 to 2 minutes. The reaction is carried out at 22 to 28 ° C. A first order kinetic response is assumed and the half-life is determined from the decrease in absorbance during the first 10 minutes. From this half-life, the point in time beyond which no further decrease in the absorbance should be observed is estimated, and the observations are continued beyond this point in time. If no further decrease is observed beyond this point in time, the total decrease in absorbance (corrected for the dilution effect and the absorbance of the hydroxylamine) is set as "hydroxylamine-extinct absorbance at 300 nm" (hereinafter abbreviated as HAEA300). If the absorbance decreases beyond this time, the rate of decrease in background absorbance is calculated and the observed decrease at that time is corrected for the decrease in background absorbance, assuming that the decrease in background absorbance is linear with time. The corrected value is then recorded as HAEA300.

The number of HAEA300 units is equal to the value of HAEA300 multiplied by the volume in ml.

Method for isolating organisms producing N-acetylthienamycin amidohydrolase A suspension of 1 g of fertile lawn soil in 100 ml of sterile phosphate buffer saline of the following composition is prepared:

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Phosphate buffer saline

NaCl

8.8 g

1-molar phosphate buffer, pH 7.5 *

10 ml of distilled water

1000 ml

* 1 molar phosphate buffer, pH 7.5

16 ml of 1 molar KH2P04 solution are mixed with 84 ml of 1 molar KîHPCU solution. The pH of the phosphate buffer is adjusted to 7.5 by adding a little 1-molar KH2PC> 4 solution or 1-molar K2HP04 solution.

Portions of this 1% stock suspension are used to make 10-fold, 100-fold, and 100-fold dilutions.

Portions of the stock suspension of 1 ml each or portions of the 10-fold, 100-fold and 100-fold dilutions of 1 ml are added to 2 ml of sterile, 1.0% agar solution at 48 ° C. The mixtures are quickly poured over the surface of sterile Petri dishes 85 mm in diameter, each containing 20 ml of Medium A of the following composition:

Medium A

KH2PO4 3.0 g

K2HPO4 7.0 g

MgS04 0.1 g distilled water 1000 ml

N-acetylethanolamine solution * 8.5 ml

* N-acetylethanolamine solution N-acetylethanolamine is diluted tenfold with water and membrane sterilized. This solution is added after the autoclave treatment.

20 g agar is added for solid media.

The petri dishes are incubated at 28 ° C for 18 days. Colonies that are well isolated are selected and streaked onto Medium B. Medium B has the following composition:

Medium B

Tomato porridge

40 g

Ground oatmeal

15 g distilled water

1000 ml pH adjusted to 6 with NaOH. For solid media, 20 g agar are added.

Individual clones are selected and grown on medium B slant tubes at 28 ° C for 2 days.

Part of the slant tube culture is used to make a 250 ml Erlenmeyer flask containing 50 ml Medium A, a 250 ml Erlenmeyer flask containing 50 ml supplemented Medium B (supplemented after autoclaving with 0.4 ml of a membrane-sterilized solution of water to the 10-fold diluted N-acetylethanolamine), and a 250 ml Erlenmeyer flask containing 50 ml of medium C. Medium C has the following composition:

Medium C

Dextrose 20 g

Pharmamedia 8 g

Corn steep liquor

(on a wet basis) 5 g distilled water 1000 ml pH adjusted to 7 with NaOH or HCl N-acetylethanolamine solution * 8.5 ml

* N-acetylethanolamine solution

N-acetylethanolamine is diluted tenfold with water and membrane sterilized. This solution is added after the autoclave treatment.

The flasks are shaken for 4 days at 28 ° C at 220 rpm (stroke 5.1 cm). 30 ml of each flask contents are centrifuged at 8000 rpm for 15 minutes. The protruding part is removed so that only enough remains to form a thick suspension of cells and medium solids. Half of the suspension is sonicated in a Branson Model LS-75 sonicator with a 12.7 mm probe to rupture the cells. The output power is set to position No. 4, and one works with four successive sonication cycles of 15 seconds each, the suspension being cooled in ice water during and between the individual sonication processes. To examine the whole cell preparation or the sonication product for N-acetylthienamycin amidohydrolase activity, proportions of both suspensions are analyzed by incubating 5 ml of these suspensions with solutions containing 20 μl of a solution of 1.586 mg N-acetylthienamycin per ml 10 mmolar Potassium phosphate buffer solution (pH 7) and 10 | il 0.2 molar potassium phosphate buffer solution (pH 7.4). Control experiments are carried out on the one hand with antibiotic and buffer alone and on the other hand with cell suspensions that do not contain an antibiotic. After incubating these mixtures overnight at 28 ° C., 2 ^ 1 portions are removed and applied to cellulose-coated thin-layer chromatography plates (TCL plates), which are then mixed with a mixture of 70% by volume of ethyl alcohol and 30% by volume of water be developed. After air drying, the TLC plates are placed on Staphylococcus aureus ATCC 6538P analytical plates for 5 minutes. The TLC plates are removed and the analysis plates are incubated overnight at 37 ° C.

The analysis plates are made as described above.

The activity of N-acetylthienamycin amidohydrolase in the incubated mixtures results from a bioactive range at Rf 0.44-0.47, which is attributable to thienamycin. The unreacted, bioactive N-acetylthienamycin appears at Rf 0.7-0.89. This method enables the isolation of N-acetylethanolamine amidohydrolase-producing microorganisms with N-acetylthienamycin amidohydrolase activity.

example 1

Deacetylation of N-acetylthienamycin A suspension of 1 g of fertile lawn soil is prepared in 100 ml of sterile phosphate buffer saline of the following composition:

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Phosphate buffer saline

NaCl

8, fig

1-molar phosphate buffer, pH 7.5 *

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ml of distilled water

1000

ml

* 1 molar phosphate buffer, pH 7.5

16 ml of 1 molar KHhPCk solution are mixed with 84 ml of 1 molar K2HPC> 4 solution. The pH of the phosphate buffer is adjusted to 7.5 by adding approximately 1 molar KH2PC> 4 solution or 1 molar 1 & HPCU solution.

Portions of this 1% stock suspension are used to make 10-fold, 100-fold and 100-fold dilutions.

Portions of the stock suspension of 1 ml each or portions of the 10-fold, 100-fold and 100-fold dilutions of 1 ml are added to 2 ml of sterile, 1.0% agar solution at 48 ° C. The mixtures are quickly poured over the surface of sterile Petri dishes 85 mm in diameter, each containing 20 ml of Medium A of the following composition.

Medium A

KH2PO4 3.0 g

K2HPO4 7.0 g

MgS04 0.1 g distilled water 1000 ml

N-acetylethanolamine solution * 8.5 ml

* N-acetylethanolamine solution

N-Acetylethanolamine is diluted tenfold with water and membrane sterilized. This solution is added after the autoclave treatment.

20 g agar is added for solid media.

The petri dishes are incubated at 28 ° C for 18 days. A well-isolated colony is selected and spread on a Petri dish with medium B. Medium B has the following composition:

Medium B

Tomato porridge

40 g

Ground oatmeal

15 g distilled water

1000 ml pH adjusted to 6 with NaOH. 20 g agar is added for solid media.

A single clone is selected and grown on a slant tube with medium B at 28 ° C for 2 days. Part of the culture of this slant tube is spread on the surface of six slant tubes made with Medium B. These oblique tubes are incubated at 28 ° C for 2 days. This culture has been identified as Protaminobacter ruber and has been deposited in the culture collection of Merck & Co., Inc. Rahway, New Jersey under the designation MB-3528.

A portion of the Protaminobacter ruber MB-3528 slant tube culture is used to inoculate a 250 ml Erlenmeyer flask containing 50 ml Medium C of the following composition:

Medium C

Dextrose

20th

S

Pharmamedia

8th

S

Corn steep liquor (on a wet basis)

5

g distilled water

1000

ml pH adjusted to 7 with NaOH or HCl

N-acetylethanolamine solution *

8.5 ml

* N-acetylethanolamine solution

N-Acetylethanolamine is diluted tenfold with water and membrane sterilized. This solution is added after the autoclave treatment.

The flask is shaken at 28 ° C at 220 rpm (stroke 5.1 cm) for 4 days. 25 ml of the flask contents are centrifuged at 8000 rpm for 15 minutes. The supernatant is removed and the cells on the surface of the medium solids are scraped into 0.5 ml of 0.05 molar potassium phosphate buffer solution (pH 7.4). The suspension thus obtained is subjected to ultrasonic treatment in a Branson sonication device, model LS-75, with a 12.7 mm probe at setting No. 4 in order to tear the cells. One works with four sonication cycles of 15 seconds each and cools the suspension in ice water during and between the individual treatment periods. 10 (.il of the sonication product are mixed with 25 (il of a solution containing 840 7 / ml N-acetylthienamycin in 10 mmolar potassium phosphate buffer solution (pH 7)) and incubated overnight at 28 ° C. Control is attempted performed on the one hand with antibiotic and buffer alone and on the other hand with sonicated cells and buffer without antibiotic After incubation overnight at 28 ° C., 2 ul each are applied to cellulose-coated TLC plates which are mixed with a mixture of 70% by volume ethyl alcohol and 30% by volume of water are developed and after drying in air, the TLC plates are placed on analysis plates of Staphylococcus aureus ATCC 6538P for 5 minutes.

The analysis plates are prepared as described above under I. Bioanalysis for N-acetylthienamycin.

The TLC plates are removed and the analysis plates are incubated overnight at 37 ° C. In addition to the bioactive spot for unreacted N-acetylthienamycin at Rf 0.7-0.89, a bioactive spot at Rf 0.44-0.47 is observed, which is due to thienamycin. The control incubation mixtures of antibiotic + buffer, sonicated cell suspension + buffer and a sample of antibioticum + buffer, to which the sonicated cell suspension was added just before TLC analysis, show no bioactive material at Rf 0.44-0.47 .

Example 2 Deacetylation of 890Ai A portion of the Protaminobacter ruber MB-3528 slant tube culture is used to inoculate a 250 ml Erlenmeyer flask containing 50 ml Medium C of the following composition:

Medium C

Dextrose 20 g

Pharmamedia 8 g

Corn steep liquor (on a wet basis) 5 g distilled water 1000 ml pH adjusted to 7 with NaOH or HCl

N-acetylethanolamine solution * 8.5 ml

* N-acetylethanolamine solution N-acetylethanolamine is diluted tenfold with water and membrane-sterilized. This solution is added after the autoclave treatment.

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The flask is shaken at 28 ° C at 220 rpm (stroke 5.1 cm) for 4 days. 25 ml of the flask contents are centrifuged at 8000 rpm for 15 minutes. The supernatant is removed and the cells on the surface of the medium solids are scraped into 0.5 ml of 0.05 molar potassium phosphate buffer solution (pH 7.4). The suspension thus obtained is subjected to ultrasonic treatment in a Branson sonication device, model LS-75, with a 12.7 mm probe at setting No. 4 in order to tear the cells. One works with four sonication cycles of 15 seconds each and cools the suspension in ice water during and between the individual treatment periods. 10 | xl of the sonication product are mixed with 25 | xl of an 890Ai solution which contains 4.85 optical density units at 300 nm per ml that can be erased with hydroxylamine. Control experiments are carried out on the one hand with antibiotic and buffer and on the other hand with the sonicated cell suspension with buffer, but without antibiotic. After the overnight incubation at 28 ° C., 5 and 1 portions are applied to a cellulose-coated TLC plate, which is developed with a mixture of 70% by volume of ethanol and 30% by volume of water. After air drying, the TLC plate is placed on a Staphylococcus aureus ATCC 6538P analytical plate for 5 minutes.

The analysis plates are made as described above.

The TLC plate is removed and the analysis plate incubated overnight at 37 ° C. In addition to the spot at Rf 0.7-0.89 for the unchanged bioactive 890Ai, a new bioactive spot at Rf 0.44-0.47 is observed, which can be attributed to deacetyl-890 Ai. Control incubation mixtures from antibiotic + buffer and from sonicated cells + buffer yield no bioactive material at Rf 0.44-0.47.

Example 3 Deacetylation of 890A3 The antibiotic 890A3 is deacetylated according to the procedure described in Example 2 for the deacetylation of 890Ai.

Example 4 Preparation of Desacetyl-890Ai Six 250 ml Erlenmeyer inoculation flasks, each containing 50 ml of medium C, are inoculated with part of a slant tube culture from Protaminobacter ruber MB-3528. Medium C has the following composition:

Medium C

Dextrose 20 g

Pharmamedia 8 g

Corn steep liquor (on a wet basis) 5 g distilled water 1000 ml pH adjusted to 7 with NaOH or HCl

N-acetylethanolamine solution * 8.5 ml

* N-acetylethanolamine solution

N-Acetylethanolamine is diluted tenfold with water and membrane sterilized. This solution is added after the autoclave treatment.

The flasks are shaken at 28 ° C at 220 rpm (stroke 5.1 cm) for one day.

42 2-liter production flasks, each containing 400 ml of medium C, are inoculated with 7 ml of the culture from these inoculation flasks. These production flasks are shaken for 6 days at 28 ° C at 220 rpm (stroke 5.1 cm). The contents of the flask are combined and centrifuged at 8000 rpm for 15 minutes. The cells are scraped from the lump of medium solids and mixed with 0.05 molar potassium phosphate buffer solution (pH 7.4) to a final volume of 1600 ml. This suspension is again centrifuged at 8000 rpm for 15 minutes. The cells are scraped off the lump of medium solids again and mixed with 0.05 molar potassium phosphate buffer solution (pH 7.4) to a final volume of 160 ml. This suspension is cooled to 5 ° C and 15 ml portions are successively subjected to the ultrasonic treatment described above in 15 second cycles until no further decrease in turbidity is observed when the suspension is diluted 500 times in phosphate buffer saline of the following composition will be produced:

Phosphate buffer saline

NaCl

8.8 g

1-molar phosphate buffer, pH 7.5 *

10 ml of distilled water

1000 ml

* 1-molar phosphate buffer, pH 7.5 16 ml 1-molar KH2P04 solution are mixed with 84 ml 1-molar K2HP04 solution. The pH of the phosphate buffer is adjusted to 7.5 by adding a little 1-molar KH2PO solution or 1-molar K2HP04 solution.

250 mg of Antibioticum 890Ai are added to 11 0.005-molar potassium phosphate buffer solution (pH 7.4). This mixture is mixed with 160 ml of the sonication extract from Protaminobacter ruber, which contains N-acetylthienamycin amidohydrolase. The mixture is slowly stirred with a magnetic stirrer at 28 ° C for 20 hours. The mixture is then centrifuged at 10,000 g for 15 minutes and the supernatant removed, cooled to 5 ° C and adjusted to pH 4.5 ± 0.2 with acetic acid. The separation of the desacetyl-890Ai from the non-hydrolyzed antibiotic and from other components of the reaction mixture is carried out as follows, using a disk diffusion bioanalysis against Staphylococcus aureus ATCC 6538P and measurements of the absorbance at 297 nm which can be extinguished with hydroxylamine (as under the heading «Analysis method for thienamycin ») to monitor the performance of cleaning procedures.

The acidified, centrifuged reaction mixture is adsorbed at a rate of 12 ml / min on 120 ml «Dowex» -50X4 in the sodium form (300-840 n). The adsorbate is washed with 120 ml of demineralized water and then eluted at a rate of 6 ml / min with 2% aqueous pyridine solution. As soon as 75 ml of eluate have passed through the column, the following 240 ml are combined, concentrated to 25 ml and the concentrate is adjusted to a pH of 7.

The 25 ml concentrate is adsorbed at a rate of 1 ml / min on 25 ml "Dowex" -l X2 resin in the chloride form (150-300 u). The resin is eluted with demineralized water at the same rate. After passage of 25 ml through the column, the following 50 ml are combined, neutralized to a pH of 7 and concentrated to 10 ml. This concentrate is adjusted to pH 6.3 with acetic acid and placed in a bed of «Dowex» -50X8 resin (37-74 ix) in the 2,6-lutidinium form, which has a diameter of 1 cm and a Has a height of 50 cm and has previously been brought into equilibrium with 0.1 molar 2,6-lutidina acetate buffer (pH 6.3). The buffer is eluted at a rate of s

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1 ml / min. As soon as 25 ml of eluate have run out of the column, the following 35 ml are combined and freeze-dried.

The freeze-dried solids are dissolved in 0.5 ml of 0.1 molar 2,6-lutidine acetate buffer (pH 7.0). The solution is introduced into a column with "Bio-Gel" P-2 (37-74 u), which has a diameter of 1 cm and a height of 50 cm and has previously been brought into equilibrium with this buffer. Then the gel is developed with the same buffer at a rate of 0.5 ml / min. After 25 ml of eluate has passed through the column, the following 10 ml are combined and freeze-dried.

The freeze-dried solids are dissolved in 4 ml of distilled water and placed in a column with a diameter

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water of 1.7 cm entered, which was charged with 90 ml of pre-washed XAD-2 and equilibrated at 5 ° C with distilled water. Before use, the XAD-2 is divided in succession with 1) 5 parts in NaOH and then with demineralized water until the process is neutral, 2) 5 parts in HCl and then with demineralized water until the process is neutral, 3) Washed 5 parts each of methanol, acetone, 0.001 molar tetranetrium EDTA solution and finally distilled water. After the sample has been added, two portions of 2 ml of distilled water are poured in. The column is washed at a rate of 2 ml / min with distilled water. The eluate is collected in 4 ml fractions. Fractions 25 to 58 are combined and freeze-dried; desacetyl-890Ai is obtained.

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Claims (7)

625 803 2 PATENT CLAIMS 1. Process for the preparation of thienamycin of the structural formula CK -CH (OH) '"2 ~" "2 (I) COOH or of its isomers referred to as desacetyl-890 Ai and desacetyl-890 A3, characterized in that N-acetylthienamycin of the structural formula CH, -CH (0H) 5 0 II S-CH2CH2-NK-C-CH (II) COOK or its isomer, referred to as compound 890 Ai or 890 A3, is brought into intimate contact with an amidohydrolase which is capable of hydrolyzing the N-acetyl group. 2. The method according to claim 1, characterized in that an N-acetylthienamycin amidohydrolase is used as the amidohydrolase. 3. The method according to claim 1, characterized in that an N-acetylethanolamine amidohydrolase is used as the amidohydrolase. 4. The method according to claim 1, characterized in that one uses an amidohydrolase, which was obtained by an amidohydrolase-producing strain of the microorganism Protaminobacter ruber. 5. The method according to claim 4 for the preparation of thienamycin, characterized in that bringing N-acetylthienamycin into intimate contact with the enzyme N-acetylthi enamycin amidohydrolase produced by Protaminobacter ruber. 6. The method according to claim 4 for the preparation of Desace-tyl 890 Ai, characterized in that the compound 890 Ai is brought into intimate contact with the enzyme N-acetylthienamycin amidohydrolase produced by Protaminobacter ruber. 7. The method according to claim 4 for the production of deacetyl-890 A3, characterized in that the compound 890 A3 is brought into intimate contact with the enzyme N-acetylthienamycin amidohydrolase produced by Protaminobacter ruber. 30th 35 40 45 SO The discovery of the remarkable antibiotic properties of penicillin has sparked great interest in this area, which has led to the discovery of many other valuable antibiotic substances; such antibiotics are e.g. other penicillins, streptomycins, bacitracin, tetracyclines, chloramphenicol, erythromycins and the like. In general, the antibacterial activity of each of these antibiotics extends to certain clinically important pathogenic bacteria. So some mainly only work against gram-positive types of bacteria. In the course of the widespread use of previously known antibiotics for the treatment of bacterial infections, the acquired resistance has led to the emergence of difficult resistance problems. 65 The shortcomings of the known antibiotics have therefore prompted further research to find other antibiotics which are active against a broad range of pathogens and also against resistant strains of particular microorganisms.