Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
  • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
  • C07K5/06—Dipeptides
  • C07K5/06139—Dipeptides with the first amino acid being heterocyclic

Definitions

• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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...
• Nutrient agar SM good, wrinkled surface.amber to vinaceous AM: poor, pinkish
• Potato agar SM good, amber colored AM: poor, whitish
• Skim milk agar SM good, wrinkled surface deep orange AM: none
• Peptone glucose agar SM poor, wrinkled, cream AM: none
• Agar SM very scant
• hyaline AM traces
• Loeffler serum SM poor, hyaline to cream
• Potato SM scant, cream
• Gelatin SM scant
• cream 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
• 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 ⁇ g/ 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.
• 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.
• 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.
• 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.
• pure GE 2270 factor A is added to a culture of a suitable Streptosporangium vulgare strain.
• 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.
• 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.
• 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.
• 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
• 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.
• 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.
• vitamins e.g. Bi, B2 nicotinic acid, B12, C, E, etc.
• nucleic acids e.g. purine, pyrimidine and their derivatives, etc.
• M2 25 g/1 glucose, 1 g/1 yeast extract
• M3 20 g/1 glucose, 2 g/1 yeast extract, 8 g/1 soybean meal, 1 g/1 NaCl, 4 g/1 CaCO ⁇ ; pH 6.8
• M4 10 g/1 glucose, 4 g/1 peptone, 4 g/1 yeast extract, 0.5 g/1 MgS ⁇ 4'7H2 ⁇ , 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,
• M8 20 g/1 soluble starch; 5 g/1 polypeptone;
• 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.
• seed medium 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 ⁇ C to 40 ⁇ C, preferably from 24°C to 32 ⁇ C, 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.
• 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.
• 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 GE 2270 single factor A or complex
• the concentration of the single factor A in the culture mixture may range from 10 ⁇ g/ml to 400 mg/ml, preferably from 50 ⁇ g/ml to 200 ⁇ g/ml.
• the antibiotic compound is preferably added as a solution with a water-miscible organic solvent.
• 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. (C ⁇ -C3)alkanols such as methanol, ethanol or propanol; phenyl(C ⁇ -C3)- alkanols such as benzyl alcohol; lower ketones, e.g.
• 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.
• acetone is employed.
• water-miscible solvents may be added to the culture mixture for increasing the yield of the microbial process.
• solvents preferred are acetone, DMSO, acetonitrile, DMF, ethanol and n-butanol, particularly preferred being acetone, acetonitrile and ethanol.
• an amount of from 1% to 10% (v/v) of solvent is added to the fermentation mixture.
• 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).
• Tween 80® Bitan mono-oleate polyoxyethylene
• Triton X-100® Polyethylene glycol p-isooctylphenyl ether
• Hodag AFM-5® mixture of unesterified polyethylene glycols and mono- and di-stearates of polyethylene glycols.
• an amount of from 0.1% to 5% of defoaming agent is added to the fermentation mixture.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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, chlorotrifl ⁇ orooctane 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- ⁇
• precipitating agents are petroleum ether, lower alkyl ethers, such as ethyl ether, propyl ether and butyl ether, and lower alkyl ketones such as acetone.
• petroleum ether is employed.
• product extraction may be • j c improved by salting or by adding a proper organic salt forming a ion pair with the antibiotic which is soluble in the extraction solvent.
• 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.
• a water- miscible organic solvent such as acetone.
• a water immiscible solvent such as n-butanol
• 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.
• factor D2 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.
• 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.
• polyamide resins such as polycaprolactames, nylons and cross-linked polyvinylpyrrolidones
• polyamide resins such as polycaprolactames, nylons and cross-linked polyvinylpyrrolidones
• PA 400 M. Woelm AG, West Germany
• the polyvinylpyrrolidone resin PVP-CL Aldrich Chemie GmbH & Co., KG, West Germany
• controlled pore cross-linked dextrans e.g. Sephadex LH-20, Pharmacia Fine Chemicals, Ab.
• the preferred eluting phase depends on the specific stationary phase.
• 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.
• 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.
• HPLC separation systems may be employed, using silanized silica gel as stationary phase and mixtures of ammonium formiate and acetonitrile as mobile phase.
• silanized silica gel as stationary phase
• ammonium formiate and acetonitrile as mobile phase
• 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 CaC ⁇ 3; 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 CaC ⁇ 3; pH 6.8
• M4 10 g/1 glucose, 4 g/1 peptone, 4 g/1 yeast extract, 0.5 g/1 MgS ⁇ 4'7H2 ⁇ , 2 g/1 KH2PO4, 4 g/1 K2HPO4; pH 7
• 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 CaC ⁇ 3, 0.05% Hodag® AFM-5; pH 7.0.
• Ci8 ⁇ alkyl silanized silica gel 25 cm, i.d. 4.6 mm,
• Hibar Lichrosorb RP 18 reverse phase, Ci ⁇ -alkyl silanized silica gel), 25 cm, i.d. 10 mm, 7 ⁇ m particles;
• 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.
• EXAMPLE 1 Planobispora rosea ATCC 53773 from a liquid frozen stock culture is used to seed M8 medium containing flasks at 3%.
• the flask is incubated for 7 days at 28°C on a rotary shaker at 28°C and 200 rpm.
• the mycelium is then centrif ⁇ ged, 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 ⁇ g/ml of mycelial suspension.
• EXAMPLE 2 Streptosporangium vulgare ATCC 33329 from a liquid frozen stock culture is used to seed M4 medium containing flasks at 3%.
• HPLC analysis shows a total amount of 76 ⁇ g/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 ⁇ g/ml).
• HPLC analysis shows a total amount of 51 ⁇ g/ml of GE2270 factors A and D2 in the fermentation broth, the 55% of which is the factor D2.
• Streptosporangium vulgare ATCC 33329 is incubated according to Example 2, and fermented accordingly but using M3 as fermentation medium instead of M2.
• HPLC analysis shows a total amount of 100 ⁇ g/ml of GE2270 factors A and D2 in the fermentation broth, the 28% of which is the factor D2.
• 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 ⁇ g/ml). HPLC analysis after 48 hours shows a total amount of 46 ⁇ g/ml of GE2270 factors A and 2, the 26% of which is the factor D2.
• Example 5 is repeated in four different flasks, but adding to each flask (1 to 4) 20 ⁇ g/ml, 40 ⁇ g/ml, 80 ⁇ g/ml and 150 ⁇ g/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 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.
• 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.
• 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.
• the amounts of factor D 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) 19%; 2) 31%; 3) 54%; 4) 24%; 5) 53%.
• 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.
• 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:

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• 1995
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