EP0791069A1

EP0791069A1 - Microbial transformation process for obtaining antibiotic ge 2270 factor d 2?

Info

Publication number: EP0791069A1
Application number: EP95935929A
Authority: EP
Inventors: Ameriga Lazzarini; Linda Cavaletti
Original assignee: Biosearch Italia SpA
Current assignee: Biosearch Italia SpA
Priority date: 1994-11-08
Filing date: 1995-10-11
Publication date: 1997-08-27
Also published as: WO1996014427A1; AU3805295A; JPH10508476A

Abstract

The present invention refers to a microbial transformation process for obtaining antibiotic GE 2270 factor D2, which comprises contacting antibiotic GE 2270 factor A with a culture of a Streptosporangium vulgare strain.

Description

MICROBIAL TRANSFORMATION PROCESS FOR OBTAINING ANTIBIOTIC GE 2270 FACTOR D2

The present invention refers to a microbial transformation for obtaining a compound of formula I

The compound represented by the above formula I is a known compound and more precisely it is one of the single factors of the antibiotic complex GE 2270. namely factor D2.

Such an antibiotic complex has been first disclosed in US patent 5202241. As described in the above cited patent, the antibiotic GE 2270 complex is obtained by cultivating the strain Planobispora rosea ATCC 53773 (or suitable mutant or variant thereof) in an aqueous medium containing assimilable sources of carbon, nitrogen and inorganic salts. By purification of the fermentation products, the main factor of the complex is isolated, which has been named GE 2270 factor A; the chemical structure of this compound may be represented by the above formula I, with the only difference that a -CH2-O-CH3 group is linked to the thiazolic ring E, instead of the -CH2-OH group.

A method for both selectively increasing the yield of the factor A and the overall yield of the antibiotic complex is disclosed in the International Patent

Application PCT/EP93/1907 (designating also US), Publ. No. WO 94/05798. Such a result is achieved by adding vitamin B12 (cyanocobalamin), or its analogs having vitamin Bi2~like activity wherein the cyano group is replaced by another ligand, to the fermentation medium employed for production of the GE 2270 antibiotic complex; vitamin B12 or its analogs may be added as such or as a component of a more complex material (e.g. fish meal) and the amount added to the fermentation broth ranges from 0.005 to 5 ppm.

The isolation and purification of the minor factors (i.e. Bi, B2, Ci, C2, Di, D2, E and T) of the GE 2270 complex, as well as their antimicrobial activity, are described in EP Patent 451486 (corresponding to US Serial No. 08/460177).

These minor factors, as well as the main factor A, are antimicrobial agent mainly active against gram positive bacteria and gram positive as well as gram negative anaerobes. They appear to be very active also in Staphylococcal endocarditis without any cross- resistance with methicillin, aminogl cosides or glycopeptide antibiotics.

The above cited patent application first disclosed the chemical structure of factor D2 (and of the other minor factors), which structure was assigned on the basis of the physico-chemical data reported in said Patent Application. However, further studies on the degradation products of the GE2270 factors have suggested that the surmised a inoacid sequence was not correct, as the two a inoacids marked with the letters D and E are actually in an opposite sequence in comparison with the formula reported in EP 451486; therefore the present formula I has been proposed for correctly representing the structure of the antibiotic GE 2270, in the specific of the D2 factor.

At present, no simple way for producing considerable amounts of the antibiotic GE 2270 factor D2 is available.

Object of the present invention is therefore to provide a new process which allows to obtain a substantial amount of the GE 2270 factor D2.

This purpose is achieved, according to the present invention, by microbiologically transforming the main component of the antibiotic compound GE 2270, i.e. the A factor, either as a single component or as a complex; the result is a O-demethylation of the -CH2-O-CH3 moiety linked to the thiazolic ring E of the factor A, thus obtaining the desired compound of formula I, wherein said moiety is -CH2-OH, i.e. the factor D2. Biotransformations of known molecules with enzymatic reactions by using suitable tranforming microorganisms, although being a known technique since long (see Sariaslani F.S. and Rosazza J.P.N., Enzyme Microb. Technol., 1984, vol 6, 242-253), generally present the limitation that the transforming microorganism performs a specific reaction on a specific compound. Only sometimes the microorganism is able to perform the same transformation on more than one substrate of a specific class and more seldom on compounds of different classes; very rarely the same microorganism performs different biotransformations on different substrates.

Thus, when a certain microorganism is known for performing a particular transformation on a specific substrate, there is no certainty that the same microorganism would perform the same or a different reaction on a different substrate.

In the specific case, a number of microorganisms are known which are useful for performing O-demethylations on a variety of substrates.

Among these, the species Streptomvces griseus shows a considerable O-dealkylating activity with respect to several alkaloids, terpenes and other substrates (see Sariaslani and Rosazza, cited above), while the species Bacillus megaterium has been found able to O-demethylate the ansatomicin substrates, as described by Izawa M. et al. in J. of Antibiot., 34, (1981b), p. 1587 and in US patent 4361650. Also fungi may be conveniently employed for O-demethylating specific substrates; for instance, Microsporum canis may be employed for the 0- demethylation of Griseofulvin, according to Boothroyd B. et al., Biochem. J., 80 (1960), p. 34. However, no one of the above species, as well as a number of other microorganisms known for their O-demethylating properties, are able to perform the O-demethylating reaction on the antibiotic GE 2270 factor A.

According to the process of the present invention, the biotransformation of GE 2270 factor A into factor D is achieved with the species Streptosporangium vulgare; at present, no strain of this species are known as having O-demethylating activity on some substrate.

Example of Streptosporangium vulgare species which may conveniently be used for the process of the present invention are any Streptosporangium vulgare strain which is able to perform the biotransformation of factor A into factor D2, such as Streptosporangium vulgare ATCC 33329 or Streptosporangium vulgare ATCC 21906.

Preferably, strain Streptosporangium vulgare ATCC 33329, or a variant or mutant thereof which is able to transform GE 2270 factor A into factor D2, is employed for the biotransformation process of the present invention. The acronym "ATCC" refers to the "American Type Culture Collection", Rockville, Maryland, U.S.A., where the strain has been deposited with the above collection number; this strain has also been deposited in other culture collection, such as the "Centraalbureau voor Schimmercultures", Baarn, Netherlands (collection number: CBS 433.61), the "Northern Utilization Research and Development Division", U.S. Dept. of Agriculture, Peoria, Illinois, U.S.A. (collection number: NRRL B- 2633) and the "USSR Research Institute for Antibiotics", Moscow, USSR (collection number: RIA 765). The above strain was first described by Nonomura H. and Ohara Y. on the Journal of Fermentation Technology 38. 405, 1960. The cultural characteristics of this strain are reported in the following table 1.

TABLE I

Cultural characteristics of Strept. vulg. ATCC 33329

Cultural characteristics

MEDIUM SM = Substrate micelyum AM = Aerial mycelium

Medium No. 2t (Yeast SM: abundant, wrinkled, amber extract-malt agar) to vinaceous AM: abundant, powdery whitish

Medium No. 3t SM: moderate, yellowish (Oatmeal agar) AM: good, whitish

Medium No. 4t SM: poor, smooth and thin, (Inorganic salts- yellow starch agar) AM: none

Medium No.5t SM: poor, smooth surface (Glycerol asparagine AM: none agar)

Medium No. 6t SM: poor, slightly wrinkled (Peptone-yeast amber color extract iron agar) AM: none

Medium No.7t SM: poor, slightly wrinkled (Tyrosine agar) light brown AM: none

Oatmeal agar SM: good, wrinkled, pale coral AM: good, powdery, whitish

Hickey and Tresner's SM: abundant, wrinkled, dark agar brown-reddish AM: good, whitish

Bennett's agar SM: abundant, wrinkled, light amber-brown AM: good, whitish

Czapek glucose agar SM: poor, smooth and thin, amber colored AM: none

Glucose asparagine SM: poor,smooth, yellowish agar AM: none TABLE I Continued...

Cultural characteristics

MEDIUM SM - = Substrate micelyuπt

AM = - Aerial mycelium

Nutrient agar SM: good, wrinkled surface.amber to vinaceous AM: poor, pinkish

Potato agar SM: good, amber colored AM: poor, whitish

Calcium malate agar SM: poor-, smooth and flat hyaline AM: none

Skim milk agar SM: good, wrinkled surface deep orange AM: none

Egg agar SM: very scant, hyaline AM: none

Peptone glucose agar SM: poor, wrinkled, cream AM: none

Agar SM: very scant, hyaline AM: traces, whitish

Loeffler serum SM: poor, hyaline to cream

AM: none

Potato SM: scant, cream

AM: none

Gelatin SM: scant, cream AM: none

Cellulose agar SM: very scant, hyaline AM: none

Czapek agar SM: scant, thin hyaline AM: poor, whitish t = the number of the culture media refers to those given by Shirling and Gottlieb, Methods for characterization of Streptomyces species, Int. J. Syst, Bact., 16:313-338, 1966

As disclosed in US patent 3827941, the Streptosporangium vulgare ATCC 33329 strain has been utilized for the 11-deacetylation of Erythromycin B. The GE 2270 factor D2 producing variant or mutant of the preferred strain may be obtained according to the common technical knowledge. For instance, suitable variants of the

Streptosporangium vulgare ATCC 33329 strain may be obtained by seeding the original strain on a culture medium (such as one of those listed hereinafter) which contains an antimicrobial effective amount (up to 50 μg/ l) of antibiotic GE 2270 factor A; when colonies are observed which show a higher growth with respect to the others in the presence of the antibiotic, these are selected and transferred on a new culture medium, fermented and employed for the biotransformation process. This methodology may involve either a single step or serial repeated steps; as known in the art, the more the steps performed, the higher the homogeneity of the strain obtained.

Also GE 2270 factor D2 producing mutants of Streptosporangium vulgare ATCC 33329 strain may be derived therefrom, according to the usual mutagenic techniques. For example by mutagenic irradiation, e.g. with high frequency waves, ultraviolet, radioactive or X-rays, or by means of chemical mutants such as nitrous acid, nitrosoguanidine, N-methyl-N'-nitro-N- nitrosoguanidine, ethylenimine and the like.

As stated above, the biotransformation process of the present invention comprises contacting an amount of antibiotic GE 2270 factor A, either in the purified form or in admixture with the other minor factors, with a culture of a Streptosporangium vulgare strain, preferably the Streptosporangium vulgare ATCC 33329 strain, or a variant or mutant thereof which is able to transform GE 2270 factor A into factor D2. According to a preferred embodiment of the present invention (procedure A), pure GE 2270 factor A is added to a culture of a suitable Streptosporangium vulgare strain.

Alternatively, the antibiotic GE2270 complex (containing the A factor as the main component) may be added to a culture of the biotransfor ing Streptosporangium vulgare strain.

According to another preferred embodiment (procedure B), the mycelium containing the crude GE 2270 factor A which results from the fermentation of a Planobispora rosea antibiotic producing strain is filtered and washed, afterwards it is directly added to a culture of Streptosporangium vulgare. According to this second embodiment, any mycelium obtained from the fermentation of a Planobispora rosea antibiotic GE 2270 producing strain may be employed; preferably, the mycelium obtained from the fermentation of Planobispora rosea ATCC 53773 is used; particularly preferred is the one obtained from the fermentation according to the above cited PCT/EP93/01907.

The medium used for cultivating the microorganisms suitable for the mycelial growth of both the Planobispora rosea and Streptosporangium vulgare strains as well as for the biotransformation process of the present invention may be any fluid or solid medium containing the nutrients which the particular microorganisms are able to utilize, although a fluid medium is preferable for commercial scale operations.

The culture medium is prepared using the carbon sources which will be assimilated by the microorganism and the nitrogen sources, inorganic materials, trace nutrients, etc., which will be digested by the microorganism. Thus, as such carbon sources, there may be employed glucose, lactose, sucrose, maltose, dextrin, starch, glycerin, mannitol, sorbitol, fats and oils (e.g. soyben, oil, lard oil, chicken oil etc.,) and the like, while the nitrogen sources may be meat extract, yeast extract, dried yeast, soyben meal, corn steep liquor, peptone, tryptone, cottonseed meal, spent molasses, urea, ammonium salts (e.g. ammonium sulfate, ammonium chloride, ammonium nitrate, ammonium acetate, etc.) and and the like. The medium may further contain appropriate amounts of inorganic or organic salts such as salts of phosphoric acid, boric acid, hydrochloric acid, nitric acid, sulfuric acid, carbonic acid, acetic acid and propionic acid with sodium, potassium, calcium, magnesium, iron, manganese, zinc, cobalt, nickel and the like. The medium may also contain other components such as amino acids (e.g. glutamic acid, aspartic acid, alanine, glycine, lysine, methionine, proline, etc.), peptides (e.g. dipeptides, tripeptides, etc.), vitamins (e.g. Bi, B2 nicotinic acid, B12, C, E, etc.), nucleic acids (e.g. purine, pyrimidine and their derivatives, etc.) and the like. It is, of course, possible to add inorganic or organic acids, alkalies, buffer, etc. for the purpose of adjusting the pH of the medium, or to add suitable amounts of oils, surfactants, etc. for defoaming purposes.

Examples of fermentation media suitable for both the mycelial growth and the biotransformation of the process of the present invention are illustrated in the following table II: TABLE II

Medium Composition

Ml: 30 g/1 oat meal; 20 g/1 agar; pH 6.5

M2: 25 g/1 glucose, 1 g/1 yeast extract,

10 g/1 soybean meal, 4 g/1 peptone, 4 g/1 meat extract, 5 g/1 CaCθ3; pH 6.6

M3: 20 g/1 glucose, 2 g/1 yeast extract, 8 g/1 soybean meal, 1 g/1 NaCl, 4 g/1 CaCOβ; pH 6.8

M4: 10 g/1 glucose, 4 g/1 peptone, 4 g/1 yeast extract, 0.5 g/1 MgSθ4'7H2θ, 2 g/1 KH2PO4, 4 g/1 K2HPO4; pH 7

M5: 20 g/1 dextrin, 5 g/1 peptone, 5 g/1 yeast extract, 5 g/1 meat extract; pH 7.5

M6: 10 g/1 peptone, 24 g/1 malt extract, 40 g/1 maltose.

M7: 35 g/1 soluble starch, 5 g/1 hydrolysed casein, 8 g/1 yeast extract, 3.5 g/1 meat extract, 3.5 g/1 soybean meal, 10 g/1 glucose,

2 g/1 CaC0₃, 0.1 g Vitamin B12, 0.05% Hodag® AFM-5, pH 7.2.

M8: 20 g/1 soluble starch; 5 g/1 polypeptone;

3 g/1 yeast extract; 2 g/1 meat extract; 2 g/1 soybean meal; 1 g/1 CaCOβ, 0.05% Hodag® AFM-5; pH 7.0.

In general, the microbial strains are pre- cultured in flasks, thus obtaining the so-called "seed medium", which is in turn used to seed the fermentation flasks or jars. Any of the above listed culture media may be employed for the pre-culture of Streptosporangium vulgare. although the M4 medium is the preferred one.

The pre-culture is performed at a temperature of from 20^βC to 40^βC, preferably from 24°C to 32^βC, for a period of from 2 to 4 days.

The fermentation may be carried out by any procedure such as stationary, shake or aerobic stirred culture; preferably shaking or surface culture are employed, particulary preferred being the fermentation on a rotary shaker.

According to the above outlined procedure A, the "seed medium" is then inoculated into a flask or jar containing a culture medium which may be either the same as the one used for the pre-culture or a different one. Also in this case, any of the above listed fermentation media may be conveniently employed; among these M2 and M3 are preferred, particularly preferred being M2.

In the flask or jar fermenters, it is desirable to conduct both the fermentation and the biotransformation reaction under submerged aerobic culture conditions, at a temperature of from 20°C to 40^βC, preferably from 24^βC to 32^βC.

Agitation and aeration of the culture mixture may be accomplished in a variety of ways. Agitation may be provided by a propeller or similar mechanical agitation equipment, by revolving or by shaking the fermentor, by various pumping equipment or by passage of sterile air through the medium. Aeration may be effected by passing sterile air through the fermentation mixture. After from 3 to 8 days from the inoculation of the pre-culture into the flask or jar, preferably after 5 or 6 days, the GE 2270 (single factor A or complex) is added to the culture.

The concentration of the single factor A in the culture mixture may range from 10 μg/ml to 400 mg/ml, preferably from 50 μg/ml to 200 μg/ml.

The antibiotic compound is preferably added as a solution with a water-miscible organic solvent.

The term "water-miscible organic solvent" as used in this application, is intended to have the meaning currently given in the art to this term and refers to solvents that, at the conditions of use, are miscible with water in a reasonably wide concentration range. Suitable water-miscible organic solvent are lower alkanols, e.g. (Cι-C3)alkanols such as methanol, ethanol or propanol; phenyl(Cι-C3)- alkanols such as benzyl alcohol; lower ketones, e.g. (C3~C4)ketones such as acetone or ethylmethylketone; cyclic ethers such as dioxane or tetrahydrofurane; glycols and their products of partial etherification, such as ethylene glycol, propylene glycol or ethylene glycol onomethyl ether; lower amides such as dimethylformamide or diethylformamide; dialkylsulfoxides such as dimethylsulfoxide, and mixtures thereof. Preferably acetone is employed.

As known in the art, small amounts of the above listed water-miscible solvents may be added to the culture mixture for increasing the yield of the microbial process. Among these solvents, preferred are acetone, DMSO, acetonitrile, DMF, ethanol and n-butanol, particularly preferred being acetone, acetonitrile and ethanol.

Preferably, an amount of from 1% to 10% (v/v) of solvent is added to the fermentation mixture.

Addition of defoaming agents to antibiotic producing microbial cultures is also known to enhance the yield of the microbial process and, therefore, such compounds may also be employed in the process of the present invention; preferably, non-ionic surfactants may be employed, particularly preferred being polyox ethylene derivatives or mixtures of polyoxyethylene and polyoxyethylene derivatives, such as Tween 80® (Sorbitan mono-oleate polyoxyethylene), Triton X-100® (Polyethylene glycol p-isooctylphenyl ether) or Hodag AFM-5® (mixture of unesterified polyethylene glycols and mono- and di-stearates of polyethylene glycols).

Preferably, an amount of from 0.1% to 5% of defoaming agent is added to the fermentation mixture.

During fermentation, the biotransformation of the antibiotic can be monitored by testing broth or mycelial extract samples for antibiotic activity, for instance by means of TLC/HPLC procedures.

The monitoring data suggest that the reaction may be considered completed after from 1 to 3 days from the addition of GE 2270 factor A; in general, negligible increases in product yield are detected after 48 hours.

According to procedure B of the present invention, the seed medium containing a Streptosporangium strain (preferably the strain Streptosporangium vulgare ATCC 33329), prepared as above described, is inoculated into a flask or jar.

After from 3 to 8 days from the inoculation of the pre-culture into the flask or jar, preferably after 5 or 6 days, the filtered and washed mycelium containing antibiotic GE 2270 factor A, which results from a grown culture of Planobispora rosea is directly added into the flask or jar.

As for procedure A, organic solvents and/or surfactants like the above cited may conveniently be added to the culture mixture.

The reaction may be monitored according to the above cited analytical techniques; after from 1 to 3 days from the addition of GE 2270 factor A the reaction may be considered as completed; in general, negligible increases in product yield are detected after 48 hours.

When the biotransformation reaction is completed, according to either procedure A or B, the GE 2270 factor D2 may be recovered (together with untransformed factor A and, if present, the other minor factors) from the mycelium or the fermentation broth of the producing microorganism according to the known per se techniques such as extraction with solvents, precipitation by adding non-solvents or by changing the pH of the solution, partition chromatography, adsorption chromatography, reverse- phase partition chromatography, molecular exclusion chromatography and the like.

Suitable recovering techniques, either from the mycelium or from the fermentation broth, are disclosed in the above cited EP patent 451486 (corresponding to US Pat. Appl. Ser. No. 08/460177, which is herein incorporated by reference).

A preferred procedure for recovering the antibiotic substances of the invention from the mycelium includes extracting the filtered or centrifuged mycelium with a water-miscible organic solvent, concentrating the extracts and recovering the crude antibiotic substance by precipitation, optionally with the addition of a precipitating agent, by extraction of the aqueous residue with a water-immiscible organic solvent or by adsorption chromatography followed by elution of the desired product from the absorption matrix.

A preferred procedure for recovering the antibiotic substances of the invention from the fermentation broth includes extraction with a water- immiscible organic solvent, followed by precipitation from the concentrated extracts, optionally by adding a precipitating agent, or by further extraction of an aqueous residue thereof with a water-immiscible solvent. Alternatively, the fermentation broth can be contacted with an adsorption matrix followed by elution with a polar elution mixture. This chromatographic procedure can also be applied to a concentrated extract obtained from the fermentation broth instead of on the broth itself.

Meaning and examples of water-miscible organic solvents which may be employed in the extraction of the antibiotic substances of the invention from the mycelial mass are as same as those previously disclosed along the present specification; in this case, dimethylsulfoxide (DMSO) is preferably employed.

The term "water-immiscible solvent" as used in this application, is intended to have the meaning currently given in the art to this term and refers to solvents that at the conditions of use are slightly miscible or practically immiscible with water in a reasonably wide concentration range, suitable for the intended use.

Examples of water-immiscible organic solvents that can be used in the extraction of the antibiotic substance of the invention from the fermentation broth or from the aqueous residue of the mycelium extraction are: the usual hydrocarbon solvents which may be linear, branched or cyclic such as hexane or cyclohexane; halogenated hydrocarbons such as chloroform, carbon tetrachloride, dichloroethane, fluorobro oethane, dibro oethane, trichloropropane, chlorotriflυorooctane and the like; aromatic hydrocarbons such as benzene, toluene, xylene and the like; esters of at least four carbon atoms, such as ethyl acetate, propyl acetate, ethyl butyrrate, and the like; alkanols of at least four carbon atoms which may be linear, branched or cyclic such as butanol, 1-ρentanol, 2-ρentanol, 3-ρentanol, 1-hexanol, 2-hexanol, 3-hexanol, 3,3-dimethyl-l- butanol, 4-methyl-l-pentanol; 3-methyl-l-pentanol, 2,2-dimethyl-3-ρentanol, 2,4-dimethyl-3-pentanol, 4,4-dimethyl-2-pentanol, 5-methyl-2-hexanol, 1- heptanol, 2-heρtanol, 5-methyl-l-hexanol, 2-ethyl-l- hexanol, 2-methyl-3-hexanol, 1-octanol, 2-octanol, cyclopentanol, 2-cyclopentylethanol, 3-cyclopentyl-l- propanol, cyclohexanol, cycloheptanol, cyclooctanol, 2,3-dimethylcyclohexanol, 4-ethy1cyclohexanol, cyclooctylmethanol, 6-methyl-5-hepten-2-ol, 1- nonanol, 2-nonanol, 1-decanol, 2-decanol and 3- decanol; straight or branched alkyl ethers and mixture thereof such as ethyl ether, propyl ether, 5 butyl ether, etc; and mixtures or functional derivatives thereof. Preferably, n-butanol is employed.

Examples of precipitating agents are petroleum ether, lower alkyl ethers, such as ethyl ether, propyl ether and butyl ether, and lower alkyl ketones such as acetone. Preferably, petroleum ether is employed.

As known in the art, product extraction may be •_jc improved by salting or by adding a proper organic salt forming a ion pair with the antibiotic which is soluble in the extraction solvent.

An alternative procedure for recovering the

20 antibiotic substances of the invention from the aqueous solution of the extracted mycelium, when said solution contains a substantial amount of an organic solvent, is to azeotropically distill water from it.

Generally, this requires adding a solvent 25 capable of forming minimum azeotropic mixtures with water, followed by the addition of a precipitating agent to precipitate the desired product, if necessary. Representative examples of organic solvents capable of forming minimum azeotropic

30 mixtures with water are n-butanol, benzene, toluene, butyl ether, carbon tetrachloride, chloroform, cyclohexane, 2,5-dimethylfurane, hexane and m-xylene; the preferred solvent being n-butanol.

35 Preferably, the recovery of the antibiotic substance of the invention is made from the mycelium, which is filtered, washed and extracted with a water- miscible organic solvent, such as acetone. After concentration, the mixture is extracted with a water immiscible solvent, such as n-butanol, concentrated and the crude product is precipitated by adding a precipitating agent, such as petroleum ether.

After recovery of the crude mixture as described above, it might be necessary to submit it to a further purification/concentration step before separating the single antibiotic substance of the invention. A chro atographic procedure is the first choice, in this case.

The obtained crude mixture contains the desired factor D2 together with untransformed factor A and, if the starting material was crude GE 2270, small amounts of the other minor factors.

The separation of factor D2 from the other antibiotic factors is generally performed by means of per se known chromatographic techniques, which comprise partition chromatography, reverse-phase partition chromatography, flash chromatography, affinity chromatography, HPLC techniques and the like.

Examples of stationary phases, which are chosen on the basis of the specific chromatographic technique to be applied and that can be conveniently used in the above mentioned step, are silica gel (e.g. ICN Biomedicals silica 32-62, 60A), silanized silica gel (e.g. Hibar Lichrosorb RP 18; Beckman Ultrasphere ODS), allumina, diatomaceous earth, carbon, polystyrene resins (e.g. A berlite XAD2 or XAD4, Rohm and Haas; Dowex M112, Dow Chemical Co.; and Diaion HP 20, Mitsubishi), acrylic resins (e.g. XAD7 or XAD8, Rohm and Haas), polyamide resins such as polycaprolactames, nylons and cross-linked polyvinylpyrrolidones (e.g. Polyamide-CC 6, Polyamide-SC 6, Polyamide-CC 6.6, Polyamide-CC 6AC and Polyamide-SC 6AC, Macherey-Nagel & Co., West Germany; PA 400, M. Woelm AG, West Germany; and the polyvinylpyrrolidone resin PVP-CL, Aldrich Chemie GmbH & Co., KG, West Germany) and controlled pore cross-linked dextrans (e.g. Sephadex LH-20, Pharmacia Fine Chemicals, Ab).

The preferred eluting phase depends on the specific stationary phase.

For instance, when silica gel or alumina is employed, preferred solvents are halogenated hydrocarbons, lower alkanols, ethers, higher ketones and mixtures thereof; lower ketone such as acetone or a lower alcohol such as ethanol may be used with carbon as stationary phase; polar solvent mixtures of water-miscible solvents are preferred eluents for polystyrene or acrylic resins, while aqueous mixtures of water-miscible solvents are preferred for polyamide resins.

According to a preferred embodiment of the invention, the chromatographic separation of GE 2270 factor D2 from the other factors is made by means of flash column chromatography on silica gel with a mixture methanol÷dichloroethane as mobile phase.

Alternatively, HPLC separation systems may be employed, using silanized silica gel as stationary phase and mixtures of ammonium formiate and acetonitrile as mobile phase. The following examples are given for further illustrate the invention in more detail.

The following experimental conditions are applied:

FERMENTATION MEDIA

M2: 25 g/1 glucose, 1 g/1 yeast extract, 10 g/1 soybean meal, 4 g/1 peptone, 4 g/1 meat extract, 5 g/1 CaCθ3; pH 6.6

M3: 20 g/1 glucose, 2 g/1 yeast extract, 8 g/1 soybean meal, 1 g/1 NaCl, 4 g/1 CaCθ3; pH 6.8

M7: 35 g/1 soluble starch; 5 g/1 hydrolysed casein; 8 g/1 yeast extract; 3.5 g/1 meat extract; 3.5 g/1 soybean meal; 10 g/1 glucose; 2 g/1 CaC03; 0.1 g Vitamin B12; 0.05% Hodag® AFM-5; pH 7.2.

M8: 20 g/1 soluble starch; 5 g/1 polypeptone; 3 g/1 yeast extract; 2 g/1 meat extract; 2 g/1 soybean meal; 1 g/1 CaCθ3, 0.05% Hodag® AFM-5; pH 7.0.

CHROMATOGRAPHIC CONDITIONS

HPLC

- Instrument: 9091 Varian equipped with 9092 UV detector set at 254 run and a 9095 autosampler;

- Mobile phases: A)0.2% aqueous NH4ΦCOOΘ/CH3CN (9:1);

B)0.2% aqueous NH4©C00θ/CH₃CN (3:7); Analytical HPLC

- Column: Beckman Ultrasphere ODS (reverse phase,

Ci8~alkyl silanized silica gel), 25 cm, i.d. 4.6 mm,

5 μm particles;

- Linear gradient from 40% to 75% of B in 25 min, with a flow rate of 1.5 ml/min

Semi-preparative HPLC

- Column: Hibar Lichrosorb RP 18 (reverse phase, Ciβ-alkyl silanized silica gel), 25 cm, i.d. 10 mm, 7 μm particles;

- Step gradient (minutes/%A): 0/60; 35/40; 10/50; flow rate: 5 ml/min

Flash Chromatography:

Silica gel: ICN Biomedicals silica 32-62, 6θl Mobile phase: 8% CH3OH, 92% CH₂C1₂ Air pressure: 2 ATM Column: cm 15 x 4.5 id

SPECTROSCOPY FAB-MS recorded on a Finnigan TSQ 700 triple quadrupole mass spectrometer operating in FAB positive ion mode, equipped with a Xe saddle field atom gun at 8 kV. The sample is dissolved in a 1:1 mixture of DMSO and m-NBA immediately before the analysis.

IH-NMR spectrum recorded in DMS0-d6 solution with TMS as internal standard (0.00 pp ), at 500 MHz with a Bruker AM-500 spectrometer equipped with an Aspect 3000 computer. Fermentation of Planobispora rosea ATCC 53773

EXAMPLE 1 Planobispora rosea ATCC 53773 from a liquid frozen stock culture is used to seed M8 medium containing flasks at 3%.

After 48 hours of incubation on a rotary shaker at 28°C and 200 rpm, a 500 ml flask containing 100 ml of culture medium M7 is seeded with 3 ml of grown culture.

The flask is incubated for 7 days at 28°C on a rotary shaker at 28°C and 200 rpm.

The mycelium is then centrifυged, washed with saline solution and the supernatants discarged. A small amount of saline solution (about 20% with respect to the mycelial cake) is then added to resuspend the mycelium.

HPLC analysis shows a content of GE2270 factor A of about 120 μg/ml of mycelial suspension.

Biotransformation of antibiotic GE2270 factor A

EXAMPLE 2 Streptosporangium vulgare ATCC 33329 from a liquid frozen stock culture is used to seed M4 medium containing flasks at 3%.

After 72 hours of incubation on a rotary shaker at 28°C and 200 rpm, 4 litres of M2 medium contained in a five-litre jar are seeded with 200 ml (5%) of grown culture. The jar is aerated by bubbling air at 2 1/min, agitated and maintained at 28°C.

120 hours after inoculation, 800 mg of GE2270 factor A dissolved in 40 ml DMSO are added to the fermentation broth (200 μg/ml) and the fermentation is continued for further 48 hours.

HPLC analysis shows a total amount of 76 μg/ml of GE2270 factors A and D2 in the fermentation broth, the 19% of which is the factor D2.

EXAMPLE 3 The same procedure of Example 2 is followed, but adding 250 g of GE2270 factor A in 40 ml DMSO to the fermentation broth (62.5 μg/ml).

HPLC analysis shows a total amount of 51 μg/ml of GE2270 factors A and D2 in the fermentation broth, the 55% of which is the factor D2.

EXAMPLE 4

Streptosporangium vulgare ATCC 33329 is incubated according to Example 2, and fermented accordingly but using M3 as fermentation medium instead of M2.

120 hours after inoculation, 450 mg of GE2270 factor A dissolved in 45 ml DMSO are added to the fermentation broth (112.5 μg/ml) and the fermentation is continued for further 48 hours.

HPLC analysis shows a total amount of 100 μg/ml of GE2270 factors A and D2 in the fermentation broth, the 28% of which is the factor D2.

EXAMPLE 5 Example 2 is repeated, but a 500 ml flask containing 100 ml of M2 medium, instead of the 5 litre jar, is seeded with 5 ml of S. vulgare grown culture. 120 hours after inoculation, 10 mg of GE 2270 factor A in 1 ml of DMSO are added to the fermentation broth (100 μg/ml). HPLC analysis after 48 hours shows a total amount of 46 μg/ml of GE2270 factors A and 2, the 26% of which is the factor D2.

EXAMPLE 6

Example 5 is repeated in four different flasks, but adding to each flask (1 to 4) 20 μg/ml, 40 μg/ml, 80 μg/ml and 150 μg/ml of GE 2270 factor A, respectively; the amounts of factor D2 with respect to the total amount of GE2270 factors A and D2 in the fermentation broth, determined by HPLC analysis, are approximately the following: 1) 50%; 2) 36%; 3) 26% and 4) 20%.

EXAMPLE 7 By following the procedure of example 6, but using M3 as fermentation medium, the following amounts of factor D2 with respect to the total amount of GE2270 factors A and D2 in the fermentation broth are determined by HPLC analysis in the four flasks: 1) 37%; 2) 43%; 3) 37% and 4) 18%.

EXAMPLE 8 Example 5 is repeated in 5 different flasks, but to each flask (1 to 5) is respectively added: 1) 0.2 ml Hodag® AFM-5; 2) 0.4 ml Hodag® AFM-5; 3) 0.2 ml of Tween 80®; 4) 4 ml acetone; 5) 2 ml DMF.

The amounts of factor 2 with respect to the total amount of GE2270 factors A and D2 in the fermentation broth of the five flasks, determined by HPLC analysis, are approximately the following: 1) 33%; 2) 30%; 3) 38%; 4) 33% and 5) 21%. EXAMPLE 9

S. vulgare ATCC 33329 is fermented as described in Example 5; after 7 days from the inoculation, 4.5 of the washed mycelium of P. rosea obtained according to

Example 1 are added to 10 ml of the S. vulgare culture and the iture is transferred to a 50 ml fermentation flask. This reference flask is marked with No. 1.

Four further 50 ml flasks (2 to 5) are prepared as above described, each flask further containing: 2) 0.3 ml of Hodag AFM-5®, 3) 0.6 ml of acetone; 4) 0.6 ml of aceto-nitrile;; 5) 0.6 ml of ethanol.

After 48 hours fermentation, the amounts of factor D₂ with respect to the total amount of GE2270 factors A and D2 in the fermentation broth of the five flasks, determined by HPLC analysis, are approximately the following: 1) 19%; 2) 31%; 3) 54%; 4) 24%; 5) 53%.

Recovery of crude antibiotic GE2270 factor D? from the fermentation broth

EXAMPLE 10 After the fermentation according to Example 2 is completed, the mycelium is collected using a Buchner filter, washed with 1 volume of water and extracted with 2 volumes of acetone.

The acetone/water solution is concentrated on a rotary evaporator and the obtained solution is extracted with 3 volumes of n-butanol, concentrated to small volume, precipitated with 10 volumes of petroleum ether and filtered.

The residue is washed with petroleum ether, dried, dissolved in DMSO and freeze dried, obtaining 500 mg of crude powder containing both GE2270 factor A and factor D2, together with the other minor factors.

EXAMPLE 11 After the fermentation according to Example 4 is completed, the same procedure as in example 6 is followed, obtaining 300 mg of crude powder containing both GE2270 factor A and factor D2, together with the other minor factors.

Separation of GE2270 factor D?

EXAMPLE 12

500 mg of the crude powder obtained according to Example 10 are loaded on the top of a flash chromatography column. After elution, fractions from 36 to 70 are collected, pooled and evaporated on a rotary evaporator.

A solid is obtained which is then dissolved in DMSO and freeze-dried, yielding 51 mg of the substantially pure title compound as a white powder.

EXAMPLE 13 10 mg of the crude powder obtained according to Example 11 are purified by semi-preparative HPLC; the fractions containing the factor D2 are collected, pooled and evaporated, yielding 1 mg of substantially pure title compound as a white powder. The physico-chemical characteristics of the obtained GE2270 factor D2 are the following:

Molecular weight (FAB-MS spectrum): 1275 amu

IH-NMR spectrum: 9.00, d, (NH); 8.69, br s (2NH's); 8.59, s, 8.53, s, 8.29, s and 7.35, s, (thiazole CH's); 8.38, m, (glycine NH); 8.40 and 8.26 (m), (Py.CH's); 7.37-7.18, m, (aromatic CH's, primary amide NH); 6.97, s, (primary amide NH); 6.03, d and t, (2 OH's); 5.28-5.16, m (αCH's); 5.03, m, (βCH); 4.97, m, (CH₂(OH)]; 4.79 and 4.55, (CH₂ of oxazoline); 3.97-3.76, m, (CH2 of glycine and CH's of prolineamide); 2.71, m and 1.28, m, (CH2 of N-methylasparagine); 2.18-1.89, m, (isopropyl CH and prolineamide C& s); 0.88, d and 0.84 d (valine CH3's).

Retention time relative to factor A, according to the above HPLC analytical system: 0,82

Claims

1. Process for preparing a compound of formula I

which comprises contacting antibiotic GE 2270 factor A with a culture of a Streptosporangium vulgare strain.

2. Process according to claim 1 wherein the Streptosporangium vulgare strain is the Streptosporangium vulgare ATCC 33329 strain, or a variant or mutant thereof which is able to transform GE 2270 factor A into factor D2.

3. Process according to claims 1 or 2 wherein substantially pure antibiotic GE 2270 factor A is added as a solution with a water-miscible organic solvent to the culture of the Streptosporangium vulgare strain.

4. Process according to claims 1 or 2 wherein antibiotic GE 2270 complex is added as a solution with a water-miscible organic solvent to the culture of the Streptosporangium vulgare strain.

5. Process according to claims 3 or 4 wherein the water-miscible organic solvent is selected from (Cι~ C3)-alkanols, ρhenyl(Cι-C3)alkanols, lower ketones, cyclic ethers, glycols and their products of partial etherification, lower amides, dimethylsulfoxide and mixtures thereof.

6. Process according to claims 1 or 2 wherein a filtered and washed mycelium containing antibiotic GE 2270 factor A, which results from the fermentation of a Planobispora rosea antibiotic GE 2270 producing strain is added to the culture of the Streptosporangium vulgare strain.

7. Process according to claims 1 to 6 wherein a water-miscible organic solvent is added to the culture mixture.

8. Process according to claim 7 wherein the water- miscible organic solvent is selected from acetone,

DMSO, acetonitrile, DMF, ethanol and n-butanol.

9. Process according to claims 7 or 8 wherein the water-miscible organic solvent is added in an amount of from 1% to 10% (v/v) to the fermentation mixture.

10. Process according to claims 1 to 9 wherein a defoaming agent is added to the culture mixture.

11. Process according to claim 10 wherein the defoaming agent is a non-ionic surfactant.

12. Process according to claim 11 wherein the non- ionic surfactant is a polyoxyethylene derivative or a mixture of polyoxyethylene and polyoxyethylene derivatives.

13. Process according to claims 10 to 12 wherein the defoaming agent is added in an amount of from 0.1% to 5% (v/v) to the fermentation mixture.

14. Process according to any one of claims 1 to 13 wherein the crude GE 2270 factor D2 is recovered from the mycelium or the fermentation broth of the producing microorganism by means of extraction with solvents, precipitation by adding non-solvents or by changing the pH of the solution, partition chromatography, adsorption chromatography, reverse-phase partition chromatography or molecular exclusion chromatography.

15. Process according to claim 14, wherein the crude GE 2270 factor D2 is recovered from the mycelium, which includes: extracting the filtered and washed mycelium with a water-miscible organic solvent, concentrating the extracts, extracting the obtained mixture with a water immiscible organic solvent and precipitating the crude GE2270 factor D2 by adding a precipitating agent.

16. Process according to claim 15 wherein: a) the water-miscible organic solvent is selected from (Cι~C3)-alkanols, phenyl(Cι-C3)alkanols, lower ketones, cyclic ethers, glycols and their products of partial etherification, lower amides, dialkylsulfoxides and mixtures thereof; b) the water immiscible organic solvent is selected from linear, branched or cyclic hydrocarbon solvents; halogenated hydrocarbons; aromatic hydrocarbons; esters of at least four carbon atoms; linear, branched or cyclic alkanols of at least four carbon atoms; straight or branched alkyl ethers and mixtures or functional derivatives thereof; c) the precipitating agent is selected from petroleum ether, lower alkyl ethers and lower alkyl ketones.

17. Process according to claim 15 wherein: a) the water-miscible organic solvent is selected from methanol, ethanol, propanol, benzyl alcohol, acetone, ethylmethylke one, dioxane, tetrahydrofurane, ethylene glycol, propylene glycol, ethylene glycol monomethyl ether, dimethylformamide, diethylformamide dimethylsulfoxide and mixtures thereof; b) the water immiscible organic solvent is selected from hexane, cyclohexane, chloroform, carbon tetrachloride, dichloroethane, fluorobromoethane, dibro oethane, trichloropropane, chlorotrifluorooctane, benzene, toluene, xylene, ethyl acetate, propyl acetate, ethyl butyrrate, butanol, 1-pentanol, 2- pentanol, 3-pentanol, 1-hexanol, 2-hexanol, 3-hexanol, 3,3-dimethyl-l-butanol, 4-methyl-l-pentanol; 3-methyl- 1-ρentanol, 2,2-dimethyl-3-pentanol, 2,4-dimethyl-3- pentanol, 4,4-dimethyl-2-pentanol, 5-methyl-2-hexanol, 1-heptanol, 2-heptanol, 5-methyl-l-hexanol, 2-ethyl-l- hexanol, 2-methyl-3-hexanol, 1-octanol, 2-octanol, cyclopentanol, 2-cyclopentylethanol, 3-cyclopentyl-l- propanol, cyclohexanol, cycloheptanol, cyclooctanol, 2,3-dimethylcyclohexanol, 4-eth 1cyclohexanol, cyclooctylmethanol, 6-methyl-5-hepten-2-ol, 1-nonanol, 2-nonanol, 1-decanol, 2-decanol, 3-decanol, ethyl ether, propyl ether, butyl ether and mixtures thereof; c) the precipitating agent is selected from petroleum ether, butyl ether and acetone.

18. Process according to claim 15 wherein the water- miscible organic solvent is acetone, the water immiscible organic solvent is n-butanol and the precipitating agent is petroleum ether.

19. Process according to anyone of claims 14 to 18, wherein the crude GE 2270 factor D2 is purified by means of a chromatographic technique selected from partition chromatography, reverse-phase partition chromatography, flash chromatography, affinity chromatography and HPLC.

20. Process according to claim 19 wherein the crude GE 2270 factor D2 is purified by means of flash column chromatography on silica gel with a mixture methanol and dichloroethane as mobile phase.

21. Process according to claim 19 wherein the crude GE 2270 factor D2 is purified by means of HPLC with silica gel as stationary phase and a mixture of ammonium formiate and acetonitrile as mobile phase.

22. Use of a microbial strain of the genus Streptosporangium vulgare for the O-demethylating biotransformation of an organic substrate.

23. Use of a microbial strain according to claim 22 wherein said microbial strain is Streptosporangium vulgare ATCC 33329.

24. Use of a microbial strain according to claims 22 or 23 wherein the organic substrate to be 0- demethylated is antibiotic GE 2270 factor A.