Antibiotic U-62,162 and process of making

BRIEF SUMMARY OF THE INVENTION
Antibiotic U-62,162 is producible in a fermentation under controlled conditions using a biologically pure culture of the new microorganism Streptomyces verdensis, Dietz and Li sp.n., NRRL 12256.
Antibiotic U-62,162 is active against various Gram-positive bacteria. Further, the base addition salts of antibiotic U-62,162 are also active against these bacteria. Thus, antibiotic U-62,162 and its salts can be used to disinfect washed and stacked food utensils contaminated with S. aureus. They can also be used as disinfectants on various dental and medical equipment contaminated with S. aureus. Still further, antibiotic U-62,162 and its salts can be used as a bacteriostatic rinse for laundered clothes, and for impregnating papers and fabrics; and they are also useful for suppressing the growth of sensitive organisms in plate assays and other microbiological media.
DETAILED DESCRIPTION OF THE INVENTION
Chemical and Physical Properties of Antibiotic U-62,162:
Molecular Weight: 419 (high resolution spectrometry)
Molecular Formula: C.sub.23 H.sub.33 NO.sub.6.
Color and Form of Crystals: Off-white needles.
Ultraviolet Absorption Spectrum:
The UV spectrum of antibiotic U-62,162 is shown in FIG. 2 of the drawings. The solution of antibiotic U-62,162 in methanol displayed absorption as follows:
______________________________________Solvent .lambda. max a Absorptivity(.epsilon.)______________________________________Methanol 235 nm 34.36 14,400 276 sh 19.49 8,1500.01N H.sub.2 SO.sub.4 in MeOH 232 35.17 14,750 276 19.37 8,100______________________________________
Melting Point: 96.degree.-98.degree..
Infrared Absorption Spectrum:
Antibiotic U-62,162 has a characteristic infrared absorption spectrum in a mineral oil mull as shown in FIG. 1 of the drawings. Peaks are observed at the following wave lengths.
______________________________________Band BandFrequency.sup.1 Intensity.sup.2 Frequency Intensity______________________________________3511 61 1191 353420 46 1156 683388 34 1140 713374 32 1115 763025 45, sh. 1096 552955 1 1066 552925 0 1060 552870 5 1046 732855 3 1038 742730 68 1009 642680 69 973 421702 6 933 641685 11 922 641675 5 906 711653 27 882 231638 41 853 661518 5 834 611465 22 801 471457 25 767 821434 35 733 751414 38 720 691378 16 697 621366 19 679 681328 52 638 391300 61 614 371276 441260 481246 481230 471203 45______________________________________ .sup.1 Wavenumbers (cm.sup.-1) .sup.2 Percent transmittance (% T), sh, = shoulder.
Intensity at 3800 cm.sup.-1 is 85%T.
Minimum intensity at 1993 cm.sup.-1 is 91% T.
.sup.13 C-Nuclear Magnetic Resonance (NMR) Spectrum:
The .sup.13 C-NMR spectrum of antibiotic U-62,162 is shown in FIG. 4 of the drawings. The .sup.13 C-NMR spectrum was observed on a Varian CFT-20 Spectrometer on a solution (ca. 0.5 ml., ca. 200 mg./ml.) of the sample of the antibiotic in deutero-acetone (d.sub.6 -acetone). The spectrum was calibrated against internal tetramethylsilane and frequencies were recorded in ppm downfield from tetramethylsilane.
Proton Magnetic Resonance (.sup.1 H-NMR) Spectrum:
The .sup.1 H-NMR spectrum of antibiotic U-62,162 at 100 MHZ is shown in FIG. 3 of the drawings. The .sup.1 H-NMR spectrum was observed on a Varian XL-100-15 Spectrometer on a solution (ca. 0.5 ml., ca. 150 mg./ml.) of the sample of the antibiotic in deutero-acetone (d.sub.6 -acetone). The spectrum was calibrated against internal tetramethylsilane and frequencies were recorded in ppm downfield from tetramethylsilane.
Solubilities:
Antibiotic U-62,162 is soluble in lower alcohols (1-4 carbons), esters, for example, ethylacetate and methylacetate, ethers, for example, diethylether, tetrahydrofuran and dioxane, aromatic hydrocarbons, for example, benzene and toluene, dimethylformamide and dimethylsulfoxide, and lower ketones (3-6 carbons).
Antimicrobial Spectrum of Antibiotic U-62,162:
Antibiotic U-62,162 is active against various Gram-positive bacteria as shown in the following tables.
Assay:
The antibacterial assay is a standard agar dilution assay. The MIC is determined by standard methods using two-fold dilutions of the antibiotic in Brain Heart Infusion Broth (Difco Lab., Detroit, Michigan). The inocula are overnight cultures of the test organisms, diluted so that the final population contains approximately 10.sup.5 cells/ml. The solutions are incubated at 28.degree. to 37.degree. C. for 24 hours. The lowest antibiotic concentration which allows no growth=MIC or minimum inhibitory concentration.
______________________________________ Minimum InhibitoryMicroorganism Concentration (mcg/ml)______________________________________Staphylococcus aureus UC 76 1.0Staphylococcus aureus UC 6685 1.0Staphylococcus aureus UC 6690 1.0Streptococcus pyogenes UC 152 125Streptococcus faecalis UC 694 7.8Enterococcus sp. UC 701 7.8Escherichia coli UC 45 >1000Klebsiella pneumoniae UC 58 >1000Salmonella schottmuelleri UC 126 >1000Pseudomonas aeruginosa UC 95 >1000Streptococcus pneumoniae UC 41 1.0______________________________________
Antibiotic U-62,162 was also tested for activity against a group of ampicillin-resistant and ampicillin-susceptible H. influenzae type b. The following results show that U-62,162 has moderate activity against all of the cultures tested.
A solution of the compound was prepared in dimethylsulfoxide (DMSO), diluted in Brain Heart Infusion Broth (BHIB) and MIC's were determined using the standard agar-dilution method.
______________________________________Organism UC# MIC (.mu.g/ml)______________________________________H. influenzae 6582* 31.25H. influenzae 6583* 31.25H. influenzae 6585* 31.25H. influenzae 6586* 31.25H. influenzae 6587* 31.25H. influenzae 6589* 31.25H. influenzae 6592* 15.6H. influenzae 6595* 31.25H. influenzae 6596* 31.25H. influenzae 6597+ 31.25H. influenzae 6598+ 31.25H. influenzae 6482+ 62.5H. influenzae 6483* 31.25H. influenzae 6484* 31.25H. influenzae 6485+ 31.25H. influenzae 6486+ 31.25______________________________________ *Ampicillin resistant +Ampicillin susceptible
"UC" is a registered trademark of The Upjohn Company Culture Collection. These cultures can be obtained from The Upjohn Company, Kalamazoo, Mich., upon request.
THE MICROORGANISM
The microorganism used for the production of antibiotic U-62,162 is a biologically pure culture of Streptomyces verdensis, Dietz and Li sp.n., NRRL 12256.
A subculture of this microorganism can be obtained from the permanent collection of the Northern Regional Research Laboratory, U.S. Department of Agriculture, Peoria, Ill., U.S.A. Its accession number in this depository is NRRL 12256. It should be understood that the availability of the culture does not constitute a license to practice the subject invention in derogation of patent rights granted with the subject instrument by governmental action.
The microorganism of this invention was studied and characterized by Alma Dietz and Grace P. Li of The Upjohn Research Laboratories.
Streptomyces verdensis, Dietz and Li sp.n., which produces antibiotic U-62,162, is characterized and considered to be a new species of the genus Streptomyces. The culture conforms to the general characteristics of the genus (Pridham, T. G. and H. D. Tresner, 1974, Part 17, Actinomycetes and related organisms. Family VII. Streptomycetaceae Waksman and Henrici 1943. Genus I. Streptomyces. p. 748. In R. E. Buchanan and N. E. Gibbons (ed.), Bergey's Manual of Determinative Bacteriology, 8th ed., the Williams and Wilkins Co., Baltimore) but can be readily differentiated from described species of the genus (Pridham, T. G. and H. D. Tresner, 1974, Part 17, Actinomycetes and related organisms. Family VII. Streptomycetaceae Waksman and Henrici 1943. Genus I. Streptomyces. Table 17.46a-d Green Series. p. 825. In R. E. Buchanan and N. E. Gibbons (ed.), Bergey's Manual of Determinative Bacteriology, 8th ed. The Williams and Wilkins Co., Baltimore), in literature and patent descriptions available to us, and from cultures in the Upjohn Culture Collection (UC.RTM.). It can also be differentiated from cultures cited in Skerman et al. (Skerman, V. B. D., V. McGowan, and P. H. A. Sneath. 1980. Approved lists of bacterial names. Int. J. of Syst. Bacteriol. 30:225-420).
The culture belongs to the distinctive "green group cultures" cited in Argoudelis et al. (Argoudelis, A. D., J. H. Coats, and T. R. Pyke. 1972. Lincomycin production. U.S. Pat. No. 3,697,380) and in Hanka et al. (Hanka, L. J., A. Dietz, S. A. Gerpheide, S. L. Kuentzel, and D. G. Martin. 1978. CC-1065 (NSC-298233), A new antitumor antibiotic. Production, in vitro biological activity, microbiological assays and taxonomy of the producing microorganism. J. Antibiotics. 31:1211-1217). It is most similar to, but can be differentiated from, the subject cultures S. espinosus and S. zelensis of the last two references. This is shown in Table 4. On the basis of its color pattern it falls into a new color group orange yellow, gray, orange yellow in the Key for Identification of Actinomycetes with Emphasis on Streptomyces [Barnes, R. E., A. Dietz, and G. P. Li. 1980. Key for identification of actinomycetes with emphasis on the genus Streptomyces, p. 56-140. In A. Dietz and D. W. Thayer (ed.), Actinomycete Taxonomy (Procedures for Studying Aerobic Actinomycetes with Emphasis on the Streptomycetes). SIM Special Publication Number 6. Soc. for Ind. Microbiol. Arlington, VA.]. The new soil isolate has a distinctive green color on maltose-tryptone agar. Because of this distinctive color we propose that the culture, which is considered a new streptomycete species, be designated Streptomyces verdensis Dietz and Li sp.n. The name is derived from "verde" the Spanish name for "green."
Color Characteristics.
Aerial growth predominantly gray-green to gray. Melanin-negative. The primary reference colors of the culture on Ektachrome [Dietz. A. and D. W. Thayer (ed.). 1980. Actinomycete Taxonomy (Procedures for Studying Aerobic Actinomycetes with Emphasis on the Streptomycetes). SIM Special Publication Number 6. Soc. for Ind. Microbiol. Arlington, VA.] are given in Table 1. Secondary reference colors are given in Table 2. The culture may be placed in the yellow (Y) and green (GN) color series of Tresner and Backus (Tresner, H. D., and E. J. Backus. 1963. System of color wheels for streptomycete taxonomy. Appl. Microbiol. 11:335-338.
Microscopic Characteristics.
Spore chains short, straight to flexuous to open spiral. Spores are appressed and are predominantly spherical. The spore surface is spiny to hairy.
General Cultural Characteristics.
See Table 3.
Carbon Utilization.
Growth on carbon compounds was determined by the procedures of Shirling and Gottlieb (Shirling, E. B., and D. Gottlieb. 1966. Methods for characterization of Streptomyces species. Int. J. Syst. Bacteriol. 16:313-340). The culture grew well on the positive control, D-glucose. It also exhibited strong utilization of D-xylose, inositol, D-mannitol, D-fructose, and rhamnose. Growth was positive on L-arabinose and doubtful on sucrose and raffinose. The culture did not grow on cellulose or the negative control, basal medium without added carbon compound.
Temperature.
Growth was good at 24.degree.-37.degree. C., moderate at 18.degree. and 45.degree. C. and doubtful at 55.degree. C. Media used for temperature studies were Bennett's, Czapek's sucrose, and maltose-tryptone agars.
Whole Cell Analysis.
L-diaminopimelic acid was detected.
Antibiotic-Production.
The culture produces antibiotic U-62,162.
Culture Source.
Soil from Arkansas.
Type Strain.
Streptomyces verdensis NRRL 12256.
The methods used were those cited in Becker et al. (Becker, B., M. P. Lechevalier, and H. A. Lechevalier. 1966. Chemical composition of cell wall preparations from strains of various form-genera of aerobic actinomycetes. Appl. Microbiol. 13:236-243.), Dietz (Dietz, A. 1954. Ektachrome transparencies as aids in actinomycete classification. Ann. N. Y. Acad. Sci. 60:152-154; Dietz, A. 1967. Streptomyces steffisburgensis sp. n. J. Bacteriol. 94:2022-2026), Dietz and Mathews (Dietz, A., and J. Mathews. 1971. Classification of Streptomyces spore surfaces into five groups. Appl. Microbiol. 21:527-533), and in part those cited in Shirling and Gottlieb (Shirling, E. G., and D. Gottlieb. 1966. Methods for characterization of Streptomyces species. Int. J. Syst. Bacteriol. 16:313-340). All of the above have been incorporated in Dietz and Thayer [Dietz, A., and D. W. Thayer (ed.). 1980. Actinomycete Taxonomy (Procedures for Studying Aerobic Actinomycetes with Emphasis on the Streptomycetes). SIM Special Publication Number 6. Soc. for Ind. Microbiol. Arlington, VA].
TABLE 1______________________________________Primary Reference Color Characteristics.sup.a ofStreptomyces verdensis(Appearance on Ektachrome.sup.b) Surface Reverse Chip ChipAgar Medium No. Color Name No. Color Name______________________________________Bennett's 89 pale yellow 83 brilliant yellowCzapek's 264 light gray 264 light graysucroseMaltose- 149 pale green 88 dark yellowtryptonePeptone-iron 68 strong orange 68 strong orange yellow yellow0.1% 93 yellowish gray 73 pale orangeTyrosine yellowCasein Starch 148 very pale green 90 grayish yellow______________________________________ .sup.a Cultures were photographed on Ektrachrome after seven days incubation at 28.degree. C. Color determination was based on comparison with NBS color chips (SP 440. Color: Universal Language and Dictionary of Names. U.S. Government Printing Office, Washington, D.C. 20402; SPM 2106. ISCCNBS Centroid Color Charts. Office of Standard Reference Material, Roo B311, Chem. Building, National Bureau of Standards, Washington, D.C. 20234). .sup.b Dietz. A. and D. W. Thayer (ed.) 1980. Actinomycete Taxonomy (Procedures for Studying Aerobic Actinomycetes with Emphasis on the Streptomycetes). SIM Special Publication Number 6. Soc. for Ind. Microbiol. Arlington, VA.
TABLE 2______________________________________ Characteristics ofSecondary Reference Color.sup.a Streptomyces verdensis Determina-Agar Medium tion Chip No. Color Name______________________________________Bennett's S 112 light olive gray R 70 light orange yellow P -- --Czapek's sucrose S 112 light olive gray R 93 yellowish gray P -- --Maltose-tryptone S 122 grayish yellow green R 70 light orange yellow P -- --Yeast extract- S 109 light grayish olivemalt extract R 70 light orange yellow(ISP-2) P -- --Oatmeal S 109 light grayish olive(ISP-3) R 73 pale orange yellow P -- --Inorganic S 122 grayish yellow greensalts starch R 73 pale olive yellow(ISP-4) P 79 grayish yellow brownGlycerol- S 122 grayish yellow greenasparagine R 90 grayish yellow(ISP-5) P -- --______________________________________ S = Surface; R = Reverse; P = Pigment .sup.a Color determinations made after fourteen days incubation at 28.degree. C. Color determination based on comparison with NBS color chip (SP 440. Color: Universal Language and Dictionary of Names. U.S. Government Printing Office, Washington, D.C. 20402; SPM 2106. ISCCNBS Centroid Color Charts. Office of Standard Reference Material, Room B311, Chem. Building, National Bureau of Standards, Washington, D.C. 20234).
TABLE 3__________________________________________________________________________General Cultural Characteristics of Streptomyces verdensis Surface Reverse OtherMedium Color Color Pigment Characteristics__________________________________________________________________________AgarPeptone-Iron Heavy gray- Olive tan -- Melanin green negativeCalcium Pale gray Cream -- Malatemalate solubilizedGlucose Gray-green Light green -- --asparagine with fea- thery color- less edgeSkim milk Grayish vege- Orange Orange Casein tative solubilizedTyrosine Gray-green Olive yellow- Light tan Tyrosine tan solubilizedXanthine Gray-green Olive yellow Light tan Xanthine not solubilizedNutrient Gray-green Greenish yellow -- Starchstarch solubilizedYeast extract Heavy gray- Olive-yellow- Light olive --malt extract green tanyellow-tanPeptone-yeast Trace pink- Tan -- Melaninextract-iron white on negative(ISP-6) colorless vegetativeTyrosine Gray-green Gray-green- -- Melanin(ISP-7) to gray cream negativeGelatinPlain Pink-cream -- Trace olive Liquifaction- surface ring 3/4Nutrient Pink-cream -- Pale yellow- Liquifaction- surface ring tan 1/2BrothSynthetic -- -- -- Colorless floc-nitrate culent bottom growth Nitrate reduced to nitrite.Nutrient Colorless -- -- Pale yellownitrate pellicle flocculent bottom growth Nitrate reduced to nitrite.Litmus milk Blue surface -- Purple Trace pepton- ring with ization pH 7.39 trace white aerial__________________________________________________________________________
TABLE 4______________________________________Differentiation of S. verdensis, S. zelensis and S. espinosusGrowthCondition S. verdensis S. zelensis S. espinosus______________________________________Calcium Malate solu- Malate not Malate notmalateagar bilized solubilized solubilizedSynthetic Reduction Reduction No reductionnitratebrothGelatin Lique- Partial Complete PartialfactionGrowth on Moderate Moderate HeavyCarbonCompounds inSyntheticMedium:L-arabinoseSurcose Doubtful None NoneRhamnose Heavy None HeavyRaffinose Doubtful None NoneTemperature 18-45C 18C-45C 18C-55CRangeAntibiotics U-62,162 CC-1065 LincomycinProducedSpore Surface Spiny to Spiny or Thorny to hairy thorny spiny to hairySpores Appressed and Appressed Well differen- and tiated also dissemi- natedBottom tube Orange Yellowish Orangecolors. Key yellow, brown, yellow,based on color gray, greenish grey, reddishpattern orange yellow, orange yellow yellow (New color group)______________________________________ .sup.a Barnes, R. E., A. Dietz, and G. P. Li. 1980. Key for identificatio of actinomycetes with emphasis on the genus Streptomyces, p. 56140. In A. Dietz and D. W. Thayer (ed.). Actinomycrete Taxonomy (Procedures for Studing Aerobic Actinomycetes with Emphasis on the Streptomycetes). SIM Special Publication Number 6. Soc. for Ind. Microbiol. Arlington, Va.
The compound of the invention process is produced when the elaborating organism is grown in an aqueous nutrient medium under submerged aerobic conditions. It is to be understood, also, that for the preparation of limited amounts surface cultures and bottles can be employed. The organism is grown in a nutrient medium containing a carbon source, for example, an assimilable carbohydrate, and a nitrogen source, for example, an assimilable nitrogen compound or proteinaceous material. Preferred carbon sources include glucose, brown sugar, sucrose, glycerol, starch, cornstarch, lactose, dextrin, molasses, and the like. Preferred nitrogen sources include cornsteep liquor, yeast, autolyzed brewer's yeast with milk solids, soybean meal, cottonseed meal, cornmeal, milk solids, pancreatic digest of casein, fish meal, distillers' solids, animal peptone liquors, meat and bone scraps, and the like. Combinations of these carbon and nitrogen sources can be used advantageously. Trace metals, for example, zinc, magnesium, manganese, cobalt, iron, and the like, need not be added to the fermentation media since tap water and unpurified ingredients are used as components of the medium prior to sterilization of the medium.
Production of the compound by the invention process can be effected at any temperature conducive to satisfactory growth of the microorganism, for example, between about 18.degree. and 40.degree. C., and preferably between about 20.degree. and 28.degree. C. Ordinarily, optimum production of the compound is obtained in about 1 to 5 days. It is critical that the fermentation pH be controlled below 8.5 because U-62,162 is unstable at this pH and higher.
When growth is carried out in large vessels and tanks, it is preferable to use the vegetative form, rather than the spore form, of the microorganism for inoculation to avoid a pronounced lag in the production of the compound and the attendant inefficient utilization of the equipment. Accordingly, it is desirable to produce a vegetative inoculum in a nutrient broth culture by inoculating this broth culture with an aliquot from a soil, liquid N.sub.2 agar plug, or a slant culture. When a young, active vegetative inoculum has thus been secured, it is transferred aseptically to large vessels or tanks. The medium in which the vegetative inoculum is produced can be the same as, or different from, that utilized for the production of the compound, so long as a good growth of the microorganism is obtained.
A variety of procedures can be employed in the isolation and purification of the compound produced by the subject invention from fermentation beers. Isolation can be accomplished by extraction with solvents such as methylene chloride, butanol, ethyl acetate and the like; and silica gel chromatography can be used to purify crude preparations of the antibiotic.
In a preferred recovery process, the compound produced by the subject process is recovered from the culture medium by separation of the mycelia and undissolved solids by conventional means, such as by filtration or centrifugation and solvent extraction of the filtered broth. The filtrate can be extracted with a solvent for antibiotic U-62,162, for example, methylene chloride, and the extract evaporated under reduced pressure to an aqueous concentrate. This preparation can be purified by chromatography on silica gel. The solvent system used for the chromatography is CHCl.sub.3 :MeOH (94:6) (v/v). The solvent system toluene:absolute ethanol (97:3) can also be used.
Further purification is achieved by use of countercurrent distribution using an appropriate solvent system, for example, ethylacetate:ethanol (95%):cyclohexane:water (2:3:3:2, v/v). Material obtained from this purification can be recrystallized from a suitable solvent, for example, ethylacetate and Skellysolve B mixtures to afford essentially pure antibiotic U-62,162.
Salts of antibiotic U-62,162 can be formed with inorganic or organic bases. Such salts can be prepared, as for example, by suspending antibiotic U-62,162 in water, adding one mole equivalent of a base, and freeze-drying to provide a dried residue consisting of the U-62,162 salt. Antibiotic U-62,162 salts with inorganic cations which can be formed include the sodium, potassium, and calcium salts. Other salts of U-62,162, including those with inorganic bases such as primary, secondary, and tertiary monoamines as well as with polyamines, also can be formed using the above-described or other commonly employed procedures. Other valuable salts are obtained with therapeutically effective bases which impart additional therapeutic effects thereto. Such bases are, for example, the purine bases such as theophyllin, theobromine, caffeine, or derivatives of such purine bases; antihistaminic bases which are capable of forming salts with weak acids, pyridine compounds such as nicotinic acid amide, isonicotinic acid hydrazide and the like; phenylalkylamines such as Adrenaline, ephedrine, and the like; choline, and others. Salts of U-62,162 can be used for the same biological purposes as the parent compound.

The following examples are illustrative of the process and product of the invention, but are not to be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.
EXAMPLE 1
A. Fermentation
A biologically pure culture of Streptomyces verdensis Dietz and Li sp.n., NRRL 12256, is used to inoculate 500-ml. Erlenmeyer flasks containing 100 ml of sterile medium of the following composition:
______________________________________ g/liter______________________________________Glucose monohydrate 25.0Pharmamedis 25.0Tap water, q.s. 1 liter______________________________________
The seed medium post-sterilization pH is .about.6.5. The seed inoculum is grown for three days at 28.degree. C. on a Gump rotary shaker operating at 250 rpm and having a 21/2 inch stroke.
Seed inoculum, prepared as above, is used to inoculate the fermentation at 5% rate. The fermentation is conducted in a Virtis fermenter equipped with a continuous pH control and set to maintain the pH of the fermentation between 6.5 and 7.5. The aeration rate is 6 liters of air/min., using a 15 liter fermenter vessel with 10 liters of the fermentation medium and maintaining the temperature at 28.degree. C. Fermentation medium has the following composition:
______________________________________ g/liter______________________________________Brer Rabbit Molasses 30.0Cerelose 5.0Wilson's Liquid Pepton 10.0Sodium glutamate 2.0Calcium carbonate 5.0Tap water, pH adjusted to 7.0 prior to sterilization.______________________________________
B. Recovery
A 4-liter quantity of whole beer prepared as described above, is filtered at beer pH (pH 7.25 ) without addition of filteraid, affording 3850 ml of filtered beer, 4 BU/ml (S. aureus 6029). The filtrate is then acidified to pH 4.5 using 6 N H.sub.2 SO.sub.4 with efficient agitation. It is extracted twice, first with 1500 ml of methylene chloride, then with 1000 ml of that solvent. The combined extracts are washed with 500 ml of water and evaporated to dryness on a rotary evaporator at a pot temperature of 30.degree.. The residue, 270 mg, assayed 37 BU/mg of antibiotic U-62,162.
C. Purification
(1) Chromatography
Buffered (pH 5.8) silica gel is prepared by treating 1 kg of silica gel 60 with a solution of 54.4 g of KH.sub.2 PO.sub.4 in 800 ml of water. This is dried 16 hours at 110.degree.. A 1 inch (diameter) column containing 60 g of buffered (pH 5.8) silica gel is poured in a slurry with chloroform. To this is added 250 mg of crude U-62,162, prepared as described above, in chloroform. The column is developed first with 120 ml of CHCl.sub.3, then with CHCl.sub.3 :MeOH (94:6 v/v). Fractions of 5 ml each are collected beginning when the mixed solvent is introduced. They are assayed by plating 12 mm discs containing 80 ml aliquots on S. aureus 6029 trays. Fractions 55-65, containing most of the activity, are pooled and evaporated, 90 mg, 182 BU/mg, 17,200 total BU.
(2) Countercurrent Distribution:
A preparation of antibiotic U-62,162, as described above, (3 g) is distributed for 300 transfers in the two-phase solvent system afforded by ethyl acetate:ethanol (95%):cyclohexane:water (2:3:3:2 v/v). Phase volumes are 10 ml each. Fractions 120-150 are combined and the residue from these after evaporation affords essentially pure crystalline antibiotic U-62,162, 1 g, assaying 800-1500 BU/mg against S. aureus 6029. A BU is the amount of material distributed on a 12 mm disc which will give a 20 mm zone of inhibition.
(3) Recrystallization
Crystalline antibiotic U-62,162, obtained as described above, is recrystallized by dissolving 100 mg in 1 ml of ethylacetate and diluting with Skellysolve B (isomeric hexanes) until cloudiness results from crystal formation. The crystals are recovered by standard methods.

Antibiotic U-62,162 and process of making

Information

Patent Number

Date Filed

Date Issued

Inventors

Original Assignees

Examiners

Agents

CPC

US Classifications

Field of Search

US

International Classifications

Abstract

Description

Claims

US Referenced Citations (1)