Antibiotic 354 and process for producing same

BACKGROUND OF THE INVENTION
The disclosed fermentation which produces antibiotic 354 also produces the known antibiotic gougerotin. This known antibiotic is described in the publication, JACS 94: 3272 (1972). Gougerotin was also known as aspiculamycin and is disclosed and claimed in U.S. Pat. No. 3,849,398.
BRIEF SUMMARY OF THE INVENTION
Antibiotic 354 is producible in a fermentation under controlled conditions using a biologically pure culture of the new microorganism Streptomyces puniceus subsp. doliceus, NRRL 11160. Concomitantly produced with antibiotic 354 is the known antibiotic gougerotin. Antibiotic 354 is readily separated from gougerotin during the recovery procedure by subjecting a preparation containing the two antiobiotics to absorption on a cellulose column followed by elution with methanol followed by water. Antibiotic 354 elutes with the methanol and gougerotin elutes with the water.
Antibiotic 354 is active against Gram-negative bacteria, and is especially very active against species of Pseudomonas and Proteus. For example, antibiotic 354 is active against Pseudomonas aeruginosa GN-315 (UC 6149) which is resistant to gentamycin, kanamycin and nalidixic acid. Thus, antibiotic 354 can be used to treat topical Pseudomonas infections resistant to gentamycin, kanamycin or nalidixic acid. It can also be used an an oil preservative, for example, as a bacteriostatic agent for inhibiting the growth of Proteus vulgaris which is known to cause spoilage in cutting oils. Also, it is useful in wash solutions for sanitation purposes, as in the washing of hands and the cleaning of equipment, floors, or furnishings of contaminated rooms or laboratories; it is also useful as an industrial preservative, for example, as a bacteriostatic rinse for laundered clothes and for impregnating paper and fabrics; and it is useful for suppressing the growth of sensitive organisms in plate assays and other microbiological media. It can also be used as a feed supplement to promote the growth of animals.

DETAILED DESCRIPTION OF THE INVENTION
Chemical and Physical Properties of Antibiotic 354:
Molecular Weight: 172 (field desorption mass. spec.)
Elemental Analysis: (C.sub.7 H.sub.9 ClN.sub.2 O).sub.2.H.sub.2 SO.sub.4 (M.W. 474)
Found: C, 37.08; H, 4.79; N, 12.38; Cl, 15.52; S, 7.48; O, 22.75.
Ultraviolet Absorption Spectrum:
The ultraviolet absorption maxima of antibiotic 354, as reproduced in FIG. 2 of the drawings, are:
In 0.01 N HCl, .lambda., a, (.epsilon.): 213 nm, 38.54, (6,650) and 251 nm, 9.02, (1,550).
Infrared Absorption Spectrum:
Antibiotic 354, as the sulfate salt, 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 expressed in reciprocal centimeters.
Key: S = Strong M = Medium W = Weak sh = shoulder
______________________________________Band Frequency(Wave Numbers) Intensity______________________________________3170 S (sh)3070 S2950 S (oil)2920 S (oil)2850 S (oil)2750 S (sh)1687 S1572 M1462 S (oil)1377 M (oil)1342 M1300 W1285 W1252 W1217 M1187 M1100 S1062 S (sh) 992 M 975 M 940 M 925 M 890 W 862 M 800 M 730 M 705 M 660 M 608 S______________________________________
Solubilities:
Antibiotic 354 is soluble in water, and poorly soluble in methanol, dimethylsulfoxide and dimethylformamide.
Nuclear Magnetic Resonance (NMR) Spectrum:
The 'H-NMR spectrum of antibiotic 354 (as the sulfate) at 60 megacycles is shown in FIG. 3 of the drawings. The NMR spectrum was observed on a Varian XL-100 Spectrometer on a solution (ca. 0.5 ml., ca. 15% concentration) of the sample of antibiotic 354 in deuterium oxide (D.sub.2 O). The spectrum was calibrated against external tetramethylsilane and the precision of .DELTA..gamma. was >.+-.1 c.p.s. Frequencies were recorded in c.p.s. downfield from tetramethylsilane.
Antibacterial Spectrum of Antibiotic 354:
Antibiotic 354 shows the following zones of inhibition in millimeters (mm) on a standard disc plate assay (12.7 mm assay discs) at a concentration of 1 mg/ml.
______________________________________ Zone OfMicroorganism Inhibition______________________________________Bacillus subtilis 25 mmPseudomonas mildenbergii 30 mm______________________________________
On testing antibiotic 354 by a microplate broth dilution assay using nutrient broth the following spectrum was observed.
______________________________________ Minimum InhibitoryMicroorganism Concentration (mcg/ml)______________________________________Staphylococcus aureus UC 76 31.2Streptococcus fecalis UC 694 125Escherichia coli UC 45 15.6Klebsiella pneumoniae UC 57 31.2Klebsiella pneumoniae UC 58 7.8Salmonella schottmuelleri UC 126 7.8Proteus vulgaris UC 93 15.6Proteus mirabilis 31.2Pseudomonas aeruginosa UC 95 15.6Serratia marcescens UC 131 3.9Shigella flexneri UC 143 15.6Salmonella typhi 15.6______________________________________
"UC.RTM." is a registered trademark of The Upjohn Company Culture Collection. These cultures can be obtained from The Upjohn Company in Kalamazoo, Michigan, upon request.
Antibiotic 354 has been shown to be active against Pseudomonas aeruginosa strains obtained from Bronson Hospital, Kalamazoo, Michigan. These strains were relatively resistant to the well-known antibiotics kanamycin, gentamycin, nalidixic acid, and Polymyxin B. The results of this comparative test, which was performed on a standard agar disc plate assay using 6.35 mm paper discs with 0.03 ml of antibiotic (1 mg/ml) per disc, follows.
______________________________________ Zones of Inhibition (mm) Anti-P. aeruginosa Kana- Genta- Nalidixic Polymyxin bioticStrain No. mycin mycin Acid B 354______________________________________6429 11 14 13 trace 226430 11 14 13 trace 336431 0 trace 12 trace 226433 trace 11 13 trace 216434 13 22 13 trace 206435 0 13 17 trace 246436 0 9 13 9 22______________________________________
Antibiotic 354 was also tested on a nutrient broth dilution test against the same Pseudomonas strains listed above. The test tubes were incubated at 32.degree. C. for 18 hours. The results are as follows:
______________________________________P. aeruginosa Minimum InhibitoryStrain No. Concentration (mcq/ml)______________________________________6429 256430 256431 256433 256434 12.56435 12.56436 25______________________________________
THE MICROORGANISM
The microorganism used for the production of antibiotic 354 and gougerotin is Streptomyces puniceus subsp. doliceus, NRRL 11160.
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 11160. 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.
An actinomycete, isolated from the Upjohn soils screen, was found to be similar in cultural characteristics to the cultures Streptomyces griseus var. purpureus, S. californicus and S. vinaceus. In 1955, Burkholder et al. [Burkholder, P. R. and S. H. Sun, L. E. Anderson, and J. Ehrlich. 1955. The identity of viomycin-producing cultures of Streptomyces Bull. Torrey bot. Cl. 82:108-117], proposed that the viomycin-producing cultures be brought to synonymy in a new variety of S. griseus designated S. griseus var. purpureus. The viomycin-producers are distinguished from S. griseus by their distinctive red-purple reverse and pigment on many media [Buchanan, R. E., and N. E. Gibbons. 1974. Bergey's Manual of Determinative Bacteriology, 8th ed. The Williams and Wilkins Co., Baltimore] [Burkholder, P. R., supra] [Shirling, E. B., and D. Gottlieb. 1968. Cooperative description of type cultures of Streptomyces. II. Species descriptions from first study. Int. J. Syst. Bacteriol. 18:69-189][Shirling, E. B., and D. Gottlieb. 1969. Cooperative description of type cultures of Streptomyces. IV. Species descriptions from the second, third and fourth studies. Int. J. Syst. Bacteriol. 19:391-512]. They do not differ from S. griseus [Shirling, E. B., and D. Gottlieb. 1968. Cooperative description of type cultures of Streptomyces. III. Additional species descriptions from first and second studies. Int. J. Syst. Bacteriol 18:280-399] in their temperature, carbon utilization, and general growth requirements, or in their spore chain or spore surface pattern.
In 1966, Buchanan et al. [Buchanan, R. E., J. G. Holt, and E. F. Lessel, Jr. 1966. Index Bergeyana. The Williams and Wilkins Co., Baltimore] declared S. griseus var. purpureus Burkholder et al. an illegitimate name. S. vinaceus (Mayer et al.) Waks. and Henrici was also declared illegitimate. S. californicus, S. floridae and S. puniceus were considered legitimate names. In Bergey's Manual, 8th ed. [Buchanan, R. E., and N. E. Gibbons, supra], the last three named cultures are cited as type cultures. In Shirling and Gottlieb [Shirling and Gottlieb, supra at 18:69-189 and at 19:391-512], S. californicus, S. puniceus and S. vinaceus are cited as type cultures. The cultural characteristics cited in Burkholder, supra, in Shirling and Gottlieb, supra at 18:69-189, and in Bergey's Manual, 8th ed. [Buchanan, R. E., and N. E. Gibbons, supra] for S. puniceus are in agreement with the characteristics noted for the cultures compared with the new soil isolate. Of these cultures, S. puniceus Patelski (1950) is the earliest described [Burkholder, P. R., supra]. The new isolate exhibits minor differences in color of growth and antibiotic production from the cultures cited. On the basis of these differences, we propose the designation Streptomyces puniceus subsp. doliceus subsp. nov. for this new culture.
The methods used were those cited by 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 Shirling and Gottlieb [Shirling, E. B., and D. Gottlieb. 1966. Methods for characterization of Streptomyces species. Int. J. Syst. Bacteriol. 16:313-340]. S. puniceus subsp. doliceus was compared with the following viomycin-producing cultures to which it appeared most similar on Ektachrome (Table 1): S. griseus var. purpureus NRRL 2423 (UC 2414), S. griseus var. purpureus CBS (UC 2468), S. vinaceus NRRL 2285 (UC 2920), and S. californicus ATCC 3312 (UC 5270).
Taxonomy. Streptomyces puniceus Finlay & Sobin subsp. doliceus Dietz and Li subsp. nov.
Color characteristics. Aerial growth cream to cream pink to lavender-pink. Melanin negative. Appearance on Ektachrome is given in Table 1. Reference color characteristics are given in Table 2. The new culture and S. californicus UC 5270 may be placed in the Gray and Violet color groups of Tresner and Backus [Tresner, H. D., and E. J. Backus. 1963. System of color wheels for streptomycete taxonomy. Appl. Microbiol. 11:335-338]; S. griseus v. purpureus UC 2414 in the Red and Violet color groups; and S. griseus v. purpureus UC 2468 and S. vinaceus UC 2920 in the Gray color group.
Microscopic characteristics. Spore chains long, flexuous (RF) in the sense of Pridham et al. [Pridham, T. G., C. W. Hesseltine, and R. G. Benedict. 1958. A guide for the classification of streptomycetes according to selected groups. Placement of strains in morphological sections. Appl. Microbiol. 6:52-79]. Spore chains may be in tufts. Spores, examined with the scanning electron microscope, are rectangular, appressed, and have a smooth surface which depresses to give a ridged effect.
Carbon utilization. See Tables 3 and 4.
Cultural and biochemical characteristics. See Table 5.
Temperature. All the cultures grew poorly at 18.degree. C., well at 24.degree. C. and very well at 28.degree.-37.degree. C. in 48 hours. There was no growth at 4.degree. C., 45.degree. C. or 55.degree. C. Plates were removed from incubation after 14 days. Plates showing no growth were then incubated at 24.degree. C. All plates from 4.degree. C. showed growth in 24 hours at 24.degree. C.; plates from 45.degree. C. and 55.degree. C. showed no growth with the exception of the plates containing the new culture. This culture grew out from the plates previously incubated at 45.degree. C.
Antibiotic-producing properties. The reference cultures produce the antibiotic viomycin [Burkholder, P. R., supra]. UC 2414 produces the Bacillus subtilis and Klebsiella pneumoniae activities of the new culture. The new culture produces antibiotic gougerotin and antibiotic 354.
Table 1__________________________________________________________________________Appearance of cultures of Ektachrome* De-ter- S. puniceus S. griseus S. griseus mi- subsp. var. var.Agar na- doliceus purpureus purpureus S. vinaceus S. californicusMedium tion NRRL 11160 NRRL 2423 UC 2468 NRRL 2285 ATCC 3312__________________________________________________________________________Bennett's S Lavender-pink Lavender-pink Lavender-pink Lavender-pink Trace lavender-pink R Red-tan Red-tan Red-tan Red-tan Red-tanCzapek's S Pale pink Pink Pink Pale Pink Very slight trace pinksucrose R Pale pink Pink Pale pink Pale pink Pale pinkMaltose S Lavender-pink Lavender-pink Lavender-pink Lavender-pink Very pale pinktryptone R Red-tan Red-tan Red-tan Red-tan Yellow-tanPeptone-iron S -- Trace Trace Trace -- lavender-pink lavender-pink lavender-pink R Yellow-tan Yellow-tan Yellow-tan Yellow-tan Yellow-tan0.1% Tyrosine S Pale pink Pale pink Pale pink Pale pink Trace pale pink R Yellow-tan Red-tan Pale pink Pale pink Red-tanCasein starch S Lavender-pink Lavender-pink Lavender-pink Lavender-pink Very slight trace pink R Gray-tan Gray-tan Gray-tan Gray-tan Gray-tan__________________________________________________________________________ S = Surface R = Reverse *Dietz, A. 1954. Ektachrome transparencies as aids in actinomycete classification. Ann. N.Y. Acad. Sci. 60:152-154.
Table 2.__________________________________________________________________________ Reference Color Characteristics from ISCC-NBS ColorName Charts Illustrated with Centroid Colors* S. puniceas S.griseus S. griseusDe- subsp. var. var.ter- doliceus purpureus purpureus S. vinaceus S. californicusmi- NRRL 11160 NRRL 2423 VC 2468 NRRL 2285 ATCC 3312Agar na- Chip Chip Chip Chip ChipMedium tion No. Color No. Color No. Color No. Color No. Color__________________________________________________________________________Bennett's S 240 Light 32 Grayish 63 Light 63 Light 229 Dark reddish yellowish brownish brownish grayish purple to pink gray gray purple 93 Yellowish gray R 17 Very dark 21 Blackish 19 Grayish 20 Dark 260 Very dark red red red grayish purplish red red P 57 Light 45 Light 60 Light 60 Light 60 Light brown grayish grayish grayish grayish reddish brown brown brown brownCzapek's S 253 Grayish 253 Grayish 93 Yellowish 93 Yellowish 223 Moderatesucrose purplish purplish gray gray purple pink pink R 253 Grayish 237 Strong 93 Yellowish 244 Pale 223 Moderate purplish reddish gray purple pink purple purple P -- -- -- -- -- -- -- -- -- --Maltose S 226 Very pale 32 Grayish 93 Yellowish 93 Yellowish 228 Grayishtryptone purple yellowish gray gray purple pink R 21 Blackish 17 Very dark 62 Dark 81 Dark 257 Very deep red red grayish grayish purplish brown yellowish red brown P 57 Light 45 Light 60 Light 60 Light -- -- brown grayish grayish grayish reddish brown brown brownHickey- S 63 Light 32 Grayish 93 Yellowish 93 Yellowish 227 Pale purpleTresner brownish yellowish gray gray gray pink R 17 Very 260 Very 259 Dark 20 Dark 260 Very dark dark red dark purplish grayish purplish purplish red red red red P 57 Light 45 Light 60 Light 60 Light 60 Light brown grayish grayish grayish grayish reddish brown brown brown brownYeast extract- S 63 Light 32 Grayish 93 Yellowish 93 Yellowish 234 Darkmalt extract brownish yellowish gray gray purplish(ISP-2) gray pink gray R 73 Pale 73 Pale 73 Pale 73 Pale 17 Very dark orange orange orange orange red yellow to yellow to yellow to yellow to 21 Blackish 46 Grayish 46 Grayish 20 Dark -- -- red reddish reddish grayish brown brown red P 57 Light 45 Light 60 Light 60 Light -- -- brown grayish grayish grayish reddish brown brown brownOatmeal S 240 Light 226 Very pale 63 Light 93 Yellowish 257 Very deep(ISP-3) reddish purple brownish gray purplish purple to gray red 93 Yellowish gray R 242 Dark 259 Dark 259 Dark 262 Grayish 17 Very dark reddish purplish purplish purplish red purple red red red P 57 Light 45 Light 60 Light 60 Light 60 Light brown grayish grayish grayish grayish reddish brown brown brown brownInorganic- S 63 Light 32 Grayish 93 Yellowish 93 Yellowish 227 Palesalts brownish yellowish gray gray purplestarch gray pink(ISP-4) R 242 Dark 257 Very 257 Very 259 Dark 21 Blackish reddish deep deep purplish red purple purplish purplish red red red P 57 Light 45 Light 60 Light 60 Light 60 Light brown grayish grayish grayish grayish reddish brown brown brown brownGlycerol- S 63 Light 32 Grayish 93 Yellowish 93 Yellowish 227 Pale purpleasparagine brownish yellowish gray gray(ISP-5) gray pink R 21 Blackish 257 Very 257 Very 259 Dark 21 Blackish red deep deep purplish red purplish purplish red red red P 57 Light 45 Light 60 Light 60 Light 60 Light brown grayish grayish grayish grayish reddish brown brown brown brown__________________________________________________________________________ S = Surface R = Reverse P = Pigment *Kelly, K. L., and D. B. Judd. 1955. The ISCC-NBS method of designating colors and a dictionary of color names. U.S. Dept. of Comm. Circ. 553, Washington, D.C.
Table 3__________________________________________________________________________ Growth on Carbon Compounds in the SyntheticMedium of Pridham and Gottlieb* S. puniceus S. griseus S. griseus subsp. var. var. doliceus purpureus purpureus S. vinaceus S. californicus NRRL 11160 NRRL 2423 UC 2468 NRRL 2285 ATCC 3312__________________________________________________________________________ CONTROL (+) (+) (-) (+) (-) D-Xylose + + + + + L-Arabinose (+) (+) (+) (+) (+) Rhamnose (+) (+) - (+) - D-Fructose + + + + + D-Galactose + + + + + D-Glucose + + + + + D-Mannose + + + + + Maltose + + + + (+) Sucrose (+) (+) (+) (+) (+)10. Lactose (+) (+) (-) (+) + Cellobiose + + + + + Raffinose (+) (+) (+) (+) (-) Dextrin + + + + + Inulin (+) (+) (+) (+) (-) Soluble Starch + + + + + Glycerol + + + + + Dulcitol (+) (+) (-) (+) (-) D-Mannitol + + + + + D-Sorbitol (+) (+) (+) (+) (-)20. Inositol (+) (+) (+) (+) (-) Salicin (+) (+) (+) (+) (+) Phenol - - - - - Cresol - - - - - Na Formate (-) (-) - (-) - Na Oxalate (-) (+) (+) (+) - Na Tartrate (+) (+) (-) (+) (-) Na Salicylate - - - - - Na Acetate + + + + (+).fwdarw.+ Na Citrate + + + + (-)30. Na Succinate + + + + (+)__________________________________________________________________________ += Good growth (+) = Fair growth (-) = Trace growth -= No growth *Pridham, T. G., and D. Gottlieb. 1948. The utilization of carbon compounds by some Actinomycetales as an aid for species determination. J. Bacteriol. 56:107-114.
Table 4__________________________________________________________________________ Growth of Carbon Compounds in the SyntheticMedium of Shirling and Gottlieb* S. puniceus S. griseus S. griseus subsp. var. var. doliceus purpureus purpureus S. vinaceus S. californicus NRRL 11160 NRRL 2423 UC 2468 NRRL 2285 ATCC 3312__________________________________________________________________________Negative Control(Synthetic Medium-ISP-9) .+-. .+-. .+-. .+-. -Positive Control(Synthetic Medium+D-Glucose ++ ++ ++ ++ ++Carbon Compounds:L-Arabinose .+-. + + .+-. +Sucrose - - .+-. - -D-Xylose ++ ++ + + ++Inositol - - - - -D-Mannitol ++ ++ ++ ++ ++D-Fructose + ++ + ++ ++Rhamnose .+-. .+-. .+-. .+-. .+-.Raffinose - - - - -Cellulose - - - - -__________________________________________________________________________ ++ Strong utilization + Positive utilization .+-. Doubtful utilization - Negative utilization *Shirling, E. B., and D. Gottlieb. 1966. Methods for characterization of Streptomyces species. Int. J. Syst. Bacteriol. 16:313-340.
Table 5__________________________________________________________________________Cultural and Biochemical Characteristics De- ter- S. puniceus S. griseus S. griseus mi- subsp. var. var. na- doliceus purpureus purpureus S. vinaceus S. californicusMedium tion NRRL 11160 NRRL 2423 UC 2468 NRRL 2285 ATCC 3312__________________________________________________________________________AgarPeptone-iron S Cream-pink Pale cream- Cream Pale cream- Trace cream pink pink R Yellow-tan Yellow-tan Yellow-tan Yellow-tan Yellow-tan P -- -- -- -- -- O Melanin Melanin Melanin Melanin Melanin negative negative negative negative negativeCalcium S Very slight Cream Trace cream Cream Trace creammalate trace cream- aerial pink R Colorless Colorless Colorless Colorless Colorless P -- -- -- -- -- O Malate not Malate not Malate not Malate not Malate solubilized solubilized solubilized solubilized solubilizedGlucose S Cream-pink Cream Cream Cream Cream-pinkasparagine R Maroon Maroon Pale pink-red Pale pink-red Maroon center, tan center, tan center, cream center, cream center, tan edge edge edge edge edge P Pale pink Pale pink Pale pink Pale pink Pale pinkSkim milk S Very slight Cream Cream Cream-pink -- trace cream- pink R Yellow-tan- Yellow-tan- Yellow-tan- Yellow-tan- Yellow-tan- orange orange orange orange orange P Yellow-tan- Yellow-tan- Yellow-tan- Yellow-tan- Yellow-tan- orange orange orange orange orange O Casein Casein Casein Casein Casein solubilized solubilized solubilized solubilized solubilizedTyrosine S Cream Cream Cream Cream Trace cream R Light tan Tan Tan Tan Pale yellow P Tan Tan Tan Tan Pale yellow O Tyrosine Tyrosine Tyrosine Tyrosine Tyrosine solubilized solubilized solubilized solubilized solubilizedXanthine S Cream Cream Cream Cream Trace cream R Pale cream- Cream-yellow Cream-yellow Cream-yellow Pale yellow tan P Cream-tan to Cream-yellow Cream-yellow Cream-yellow Very pale very pale to pale tan to pale tan to pale tan yellow tan O Xanthine not Xanthine not Xanthine Xanthine not Xanthine solubilzed solubilized slightly solubilized solubilized solubilizedNutrient S Cream Cream Cream Cream Trace creamstarch R Pale cream- Cream-yellow Pale cream- Pale cream- Very pale yellow tan pink-tan pink-tan P None to very Cream-yellow Very pale Very pale -- pale tan to very pale pink-tan pink-tan tan O Starch Starch Starch Starch Starch solubilized solubilized solubilized solubilizedYeast extract- S Pale Pale Cream with Cream with Trace creammalt extract lavender lavender fish-net fish-net with cream with cream edge edge edge edge R Maroon with Maroon with Maroon with Maroon with Pale reddish- tan edge tan edge tan edge tan edge tan P Pale red-tan Pale red-tan Very pale tan Very pale tan Very pale red-tanPeptone-yeast S Colorless Trace cream White White Colorlessextract-iron vegtative aerial on vegtative(ISP-6) colorless vegetative R Colorless Colorless Yellow-tan Yellow-tan Yellow-tan P Trace tan Trace tan Yellow-tan Yellow-tan Yellow-tan O Melanin Melanin Melanin Melanin Melanin negative negative negative negative negativeTyrosine S Mottled Cream Gray-cream Gray-cream Lavender(ISP-7) cream- lavender R Maroon Maroon Maroon Maroon Maroon P Trace pale Trace pale Gray-pink Gray-pink Pale red-tan red-tan red-tan O Melanin Melanin Melanin Melanin Melanin negative negative negative negative negativeGelatinPlain S Trace white Trace white Trace Trace white -- aerial aerial colorless aerial vegetative P Pale yellow Pale yellow Pale yellow Pale yellow Pale yellow O Liquefaction Liquefaction Liquefactin Liquefaction No liquefaction 1/3 1/3 1/3 1/3Nutrient S Trace white Trace white Trace white Trace white -- aerial aerial aerial aerial P Yellow Yellow Yellow Yellow YellowO Liquefaction Liquefaction Liquefaction Liquefaction No liquefaction 1/3 1/3 1/3 1/3Nitrate BrothSynthetic S -- -- -- Cream-pink -- aerial on lavender-pink vegetative pellicle P -- -- -- -- -- O Compact Trace bottom Compact Trace bottom Compact bottom growth growth bottom growth growth bottom growth No reduction No reduction No reduction Reduction No reduction Red with Red with Red with Red with Zn dust Zn dust Zn dust Zn dustNutrient S Cream aerial Lavender Lavender Gray-cream Gray-cream on maroon aerial on aerial on aerial on aerial on ring surface surface maroon ring maroon ring pellicle pellicle P -- -- -- -- -- O Trace bottom Trace bottom Trace bottom Trace bottom Trace bottom growth growth growth growth growth No reduction Reduction Reduction Reduction Reduction Red with Zn dustLitmus Milk S Cream aerial Gray aerial Gray aerial Gray aerial Trace gray on blue on blue on blue-gray on blue-gray aerial on vegetative vegetative ring ring blue-gray ring ring red ring P Slight purple Slight purple -- -- -- O Trace Trace Peptonization Peptonization Peptonization peptonization peptonization good Litmus reduced in one pH 7.07 pH 7.12 pH 7.3 pH 7.07 pH 7.07__________________________________________________________________________ S = Surface R = Reverse P = Pigment O = Other characteristics
The compounds of the invention process are 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 compounds 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 compounds is obtained in about 3 to 15 days. The medium normally remains acidic during the fermentation. The final pH is dependent, in part, on the buffers present, if any, and in part on the initial pH of the culture medium.
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 compounds 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 compounds, 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 compounds produced by the subject invention from fermentation beers, for example, charcoal absorption, 1-butanol extraction, and adsorption on cellulose and cation exchange resins.
In a preferred recovery process the compounds produced by the subject process invention are recovered from the culture medium by separation of the mycelia and undissolved solids by conventional means, such as by filtration or centrifugation.
The antibiotics are then recovered from the filtered or centrifuged broth by absorption on a charcoal column. The charcoal can be eluted by passing 10 to 50% acetone in water (v/v) to remove the antibiotics.
The eluates are pooled and concentrated to an aqueous solution. This solution then is passed over a weak cation exchange resin in the ammonium form, for example, IRC-50 which is supplied by Rohm & Haas, Philadelphia, Pa. The resin can be eluted with an inorganic salt, for example, ammonium chloride, ammonium sulfate (preferred), calcium perchlorate, and the like. Collected fractions are assayed for antibacterial activity as herein described. Fractions showing antibacterial activity can be extracted with 1-butanol to remove impurities. The antibiotics remain in the aqueous phase.
The aqueous phase can be passed over a charcoal column which is then eluted with 25% acetone in water (v/v) and fractions are collected. These fractions are concentrated to an aqueous which is then lyophilized. The next step of the purification process will separate antibiotic 354 from gougerotin.
The lyophilized solid containing antibiotic 354 and gougerotin, described above, is dissolved in a minimum amount of water. This material is injected or layered onto a cellulose column. The column is eluted with methanol and fractions are collected. These fractions contain antibiotic 354. Gougerotin is removed from the cellulose column by eluting the column with water.
Antibiotic 354 is obtained in essentially pure form from the above-described methanol eluates by first concentrating the pooled eluates to a solid and then passing the solid, which has been dissolved in a minimum amount of water, over a strong cation exchange resin, for example, Dowex 50 (supplied by Dow Chemical Co., Midland, Mich.). The column is eluted with a solution of an inorganic salt (ammonium sulfate preferred) and fractions containing essentially pure antibiotic 354 are collected.
Essentially pure gougerotin can be obtained by taking the fractions containing gougerotin from the cellulose column, described above, and passing them over a strong cation exchange resin, as described above for antibiotic 354.
Since antibiotic 354 is a strongly basic compound, procedures involving adsorption on cationic ion exchange resins and elution by organic bases or ammonia can be used to purify crude preparations of antibiotic 354. Also, crude preparations of antibiotic 354 can be purified by transformation to a salt form by treatment with inorganic or organic acids. The base form of the antibiotic can be recovered by neutralization of the acid anion with ammonia or other inorganic or organic bases.
In order to make salts of antibiotic 354 with both inorganic or organic acids, as hereinafter disclosed in exemplary form, it is necessary that the acid be carefully added to an aqueous solution of antibiotic 354 in view of the instability of this antibiotic at acid pH's. Examples of inorganic and organic acids which can be used, but which examples should not be considered limiting, are hydrochloric, sulfuric, phosphoric, acetic, succinic, citric, lactic, maleic, fumaric, pamoic, cholic, palmitic, mucic, camphoric, glutaric, glycolic, phthalic, tartaric, lauric, stearic, salicylic, 3-phenylsalicylic, 5-phenylsalicyclic, 3-methylglutaric, orthiosulfobenzoic, cyclohexanesulfamic, cyclopentanepropionic, 1,2-cyclohexanedicarboxylic, 4-cyclohexenecarboxylic, octadecenylsuccinic, octenylsuccinic, methanesulfonic, benzenesulfonic, helianthic, Reinecke's, dimethyldithiocarbamic, sorbic, monochloroacetic, undecylenic, 4'-hydroxyazobenzene-4-sulfonic, octadecylsulfuric, picric, benzoic, cinnamic, and like acids.
Other procedures for making certain salts are as follows. The sulfate salts can be made by using ammonium sulfate elution from a cation exchange resin. Also, the acetate salts can be made by using pyridinium acetate to elute the antibiotic from cation exchange resins. Further, the chloride salts of antibiotic 354 can be made by using ammonium chloride to elute the antibiotic from a cation exchange resin. The sulfate salts can be converted to the chloride by passing them over an anion exchange resin, for example, Dowex 1 (Cl.sup.-) and Dowex 2 (Cl.sup.-). If the resin is used in the OH.sup.- form, the free base of 354 is isolated.
The salts of antibiotic 354 can be used for the same biological purposes as the parent antibiotic.
Acylates of antibiotic 354 can be made as follows: A sample of antibiotic 354 is dissolved in an excess of a silylating reagent such as TMS-imidazole or bis-TMS-trifluoroacetamide. A catalyst such as trimethylchlorosilane and/or a base such as pyridine may be used but neither is necessary. An acylating reagent such as trifluoroacetyl-imidazole or acetic anhydride is then added. Acylation is rapid and quantitative as judged by combined gas chromatography-mass spectroscopy. The peaks corresponding to silylated antibiotic 354 (both mono and di-silylated derivatives can be present) disappear and a new one appears with longer retention time and with a mass spectrum indicative of an acylated and monosilylated antibiotic 354. This derivative can then be selectively hydrolyzed with methanol or water to give an acylated derivative of antibiotic 354.
Suitable acid-binding agents include amines such as pyridine, quinoline, and isoquinoline, and buffer salts such as sodium acetate. The preferred base is pyridine. Carboxylic acids suitable for acylation include (a) saturated or unsaturated, straight or branched chain aliphatic carboxylic acids, for example, acetic, propionic, butyric, isobutyric, tertbutylacetic, valeric, isovaleric, caproic, caprylic, decanoic, dodecanoic, lauric, tridecanoic, myristic, pentadecanoic, palmitic, margaric, stearic, acrylic, crotonic, undecylenic, oleic, hexynoic, heptynoic, octynoic acids, and the like; (b) saturated or unsaturated, alicyclic carboxylic acids, for example, cyclobutanecarboxylic acid, cyclopentanecarboxylic acid, cyclopentenecarboxylic acid, methylcyclopentenecarboxylic acid, cyclohexanecarboxylic acid, dimethylcyclohexanecarboxylic acid, dipropylcyclohexanecarboxylic acid, and the like; (c) saturated or unsaturated, alicyclic aliphatic carboxylic acids, for example, cyclopentaneacetic acid, cyclopentanepropionic acid, cyclohexaneacetic acid, cyclohexanebutyric acid, methylcyclohexaneacetic acid, and the like; (d) aromatic carboxylic acids, for example, benzoic acid, toluic acid, naphthoic acid, ethylbenzoic acid, isobutylbenzoic acid, methylbutylbenzoic acid, and the like; and (e) aromatic aliphatic carboxylic acids, for example, phenylacetic acid, phenylpropionic acid, phenylvaleric acid, cinnamic acid, phenylpropiolic acid, and naphthylacetic acid, and the like. Also, suitable halo-, nitro-, hydroxy-, amino-, cyano-, thiocyano-, and loweralkoxyhydrocarboncarboxylic acids include hydrocarboncarboxylic acids as given above which are substituted by one or more of halogen, nitro, hydroxy, amino, cyano, or thiocyano, or loweralkoxy, advantageously loweralkoxy of not more than six carbon atoms, for example, methoxy, ethoxy, propoxy, butoxy, amyloxy, hexyloxy, and isomeric forms thereof. Examples of such substituted hydrocarboncarboxylic acids are:
mono-, di- and trichloroacetic acid;
.alpha.- and .beta.-chloropropionic acid;
.alpha.- and .gamma.-bromobutyric acid;
.alpha.- and .delta.-iodovaleric acid;
mevalonic acid;
2- and 4-chlorocyclohexanecarboxylic acid;
shikimic acid;
2-nitro-1-methylcyclobutanecarboxylic acid;
1,2,3,4,5,6-hexachlorocyclohexanecarboxylic acid;
3-bromo-2-methylcyclohexanecarboxylic acid;
4- and 5-bromo-2-methylcyclohexanecarboxylic acid;
5- and 6-bromo-2-methylcyclohexanecarboxylic acid;
2,3-dibromo-2-methylcyclohexanecarboxylic acid;
2,5-dibromo-2-methylcyclohexanecarboxylic acid;
4,5-dibromo-2-methylcyclohexanecarboxylic acid;
5,6-dibromo-2-methylcyclohexanecarboxylic acid;
3-bromo-3-methylcyclohexanecarboxylic acid;
6-bromo-3-methylcyclohexanecarboxylic acid;
1,6-dibromo-3-methylcyclohexanecarboxylic acid;
2-bromo-4-methylcyclohexanecarboxylic acid;
1,2-dibromo-4-methylcyclohexanecarboxylic acid;
3-bromo-2,2,3-trimethylcyclopenanecarboxylic acid;
1-bromo-3,5-dimethylcyclohexanecarboxylic acid;
homogentisic acid, o-, m-, and p-chlorobenzoic acid;
anisic acid;
salicyclic acid;
p-hydroxybenzoic acid;
.beta.-resorcylic acid;
gallic acid;
veratric acid;
trimethoxybenzoic acid;
trimethoxycinnamic acid;
4,4'-dichlorobenzilic acid;
o-, m-, and p-nitrobenzoic acid;
cyanoacetic acid;
3,4- and 3,5-dinitrobenzoic acid;
2,4,6-trinitrobenzoic acid;
thiocyanoacetic acid;
cyanopropionic acid;
lactic acid;
ethoxyformic acid (ethyl hydrogen carbonate); and the like.
The above acylates of antibiotic 354 are useful to upgrade the parent compound, i.e. by acylating the parent compound, then removing the acyl group, the parent compound is isolated in a purer form.
Trimethylsilylation of antibiotic 354 gives a volatile di-TMS derivative (along with a little mono-TMS derivative) which is useful in vapor phase chromatography and mass spectroscopy work. This derivative can be prepared by heating a sample of antibiotic 354 at about 60.degree. C. for about 30 minutes in tetrahydrofuran with an excess of bistrimethylsilylacetamide.
Also, the mono-TMS derivative can be prepared using either trimethylsilylimidazole or bistrimethylsilyltrifluoroacetamide. The monosilylated antibiotic 354 can be acylated in situ using trifluoroacetylimidazole, trifluoroacetic anhydride or acetic anhydride. These are also useful in vpc-mass spectroscopic work and represent a practical route to selective (O vs. N) protection of antibiotic 354.
The following examples are illustrative of the process and products 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 puniceus subsp. doliceus, NRRL 11160, is used to inoculate 500-ml Erlenmeyer seed flasks containing 100 ml of sterile medium consisting of the following ingredients:
______________________________________Glucose 10 g/lYeast Extract 2.5 g/lPeptone 10 g/lDeionized Water q.s. 1 liter______________________________________
The seed medium presterilization pH is 6.5. The seed inoculum is grown for three days at 28.degree. C. on a Gump rotary shaker operating at 250 r.p.m. Seed inoculum, prepared as described above, is used to inoculate 500-ml Erlenmeyer fermentation flasks containing 100 ml of sterile fermentation medium consisting of the following ingredients:
______________________________________Brer Rabbit/Sucrest* 20 ml/1Yeast Extract/Brewer's Yeast** 2 g/lDextrin*** 10 g/lCerelose*** 15 g/lProteose-Peptone #3** 10 g/lPeanut Meal 5 g/lWater q.s. 1 liter______________________________________ *RJR Foods, Inc., New York, NY **Difco Laboratories, Detroit, MI ***CPC International, Inc., Englewood Cliffs, NJ
The presterilization pH is 7.0. The fermentation flasks are inoculated at the rate of 5 ml of seed inoculum per 100 ml of fermentation medium. The fermentation flasks are grown for 3 days at a temperature of 25.degree.-28.degree. C. on a Gump rotary shaker operating at 250 r.p.m.
A representative shake flask fermentation harvested after 3 days shows the following assay pattern against Pseudomonas mildenbergii (UC 3029).
______________________________________Day Assay Results (BU/ml)______________________________________1 02 143 14______________________________________
The assay is an agar disc plate assay using the microorganism P. mildenbergii. The agar medium is buffered with 0.1 M phosphate buffer at a pH of 7.4. A unit volume (0.08 ml) of solution containing the substance to be assayed is placed on a 12.7 mm paper disc which is then placed on an agar plate seeded with the assay organism. The agar plate is then incubated for 16-18 hours at 37.degree. C. A biounit (BU) is defined as the concentration of the antibiotic which gives a 20 mm zone of inhibition under the above assay conditions. Thus, if for example a fermentation beer, or other solution containing the antibiotic, needs to be diluted 1/100 to give a 20 mm zone of inhibition, the potency of such beer or solution is 100 BU per ml.
B. Recovery
(1) Carbon Sorption
Ten liters of shake flask-grown beer, as described above, is stirred with 4 l washed, granular charcoal for 15 minutes. The charcoal is allowed to settle for 10 minutes and the beer is decanted. The charcoal is washed with deionized water until the water remains clear. An overhead paddle stirrer is used in the first step and a 10 l pail in the second step. The charcoal is slurried into a chromatography tube with deionized water. The activities are eluted with 25% acetone in water using gravity flow at the highest possible flow rate. One liter fractions are collected until a yellow color emerges. When the yellow color is almost undectable small fractions are again collected. The yellow fractions (11 l) are concentrated to an aqueous at 35.degree.-40.degree. C./l mm and assayed. The data are presented in Table I below. It is seen that 80% of the UC 3029 biounits are recovered from the column.
Table I__________________________________________________________________________ Pseudomonas Mildenbergii BacillusSample Volume Dilution (UC 3029) subtilis Conc. BU/mg__________________________________________________________________________Whole Beer 10 1 FS* 28 mm 24 mm -- -- 1:2 23 21 -- -- 1:4 trace 20 -- -- 1:8 NZ** 19 -- --Spent Beer 11 1 FS NZ 20 -- --1st Eluate 3 1 FS NZ -- -- --2nd Eluate 9 1 FS 25 -- -- --3rd Eluate 2 1 FS NZ -- -- --Aqueous Pool 8 1 FS 29 23 16.9 0.18 mg/ml 1:2 23 19 (3 BU/ml) 1:4 16 trace__________________________________________________________________________ FS* = Full Strength NZ** = No Zone
(2) IRC-50 Sorption
Carbon eluates from four runs (about 48 l of beer in all), as described above, are pooled to give 33 l of aqueous which assay for a total of 91,000 UC 3029 biounits.
This is passed over 2 pounds of IRC-50 (NH.sub.4.sup.+) in a chromatography tube at 5-6 l/hour. The column is then washed with 4 l of deionized water and eluted with 1 M (NH.sub.4).sub.2 SO.sub.4 solution. The fractions are assayed by UV after diluting them 1:10 with water. On the basis of the UV data, the first two eluates are pooled. The third eluate is saved. The data are presented in Table II below.
Table II______________________________________ Pseudomonas mildenbergii BacillusSample Volume (UC 3029) subtilis A .lambda.______________________________________Spent 33.4 1 NZ NZ 1.4 265 nmWash 3.5 1 NZ NZ 0.24 2651st Eluate 320 ml 21 mm 20 mm 0.62 2652nd Eluate 2000 ml 31 30 5.80 2603rd Eluate 2000 ml 23 20 2.18 2554th Eluate 1000 ml 18 NZ 1.29 2555th Eluate 1000 ml NZ NZ 1.13 255______________________________________
(3) Desalting
Eluate #3 above (2000 ml) is passed over 200 ml granular charcoal in a chromatography tube. The column is washed with 500 ml deionized water. Neither the spent nor the wash has any UV absorbance. The activities are eluted with 700 ml 25% acetone in water. The eluate is concentrated to an aqueous amounting to 500 ml. A 1:10 dilution of this absorbs strongly at 255 nm. Bioautography on cellulose with methanol shows that gougerotin and antibiotic 354 are present.
(4) Ultrafiltration
The desalted aqueous from above is passed over an Amicon UM 2 ultrafilter (Amicon Corp., 21 Hartwell Avenue, Lexington, MA 02173). The retentate shows no activity and is discarded after a wash of one volume. The first filtrate and wash are pooled and lyophilized. The residue weights 4.5 gm. This shows a UV max at 252 nm with an inflection at 212 nm on a strong end absorption.
(5) Separation of Gougerotin From Antibiotic 354
A 68 gm preparation treated essentially as described above (except for the ultrafiltration), assays at 0.68 BU/mg vs. B. subtilis; it is labeled Prep. 216-4. Bioautography shows that it contains gougerotin and antibiotic 354.
A cellulose 300 column measuring 5.0 .times. 150 cm is flushed with methanol at 20 ml/minute (10 psi). The bed volume is 2.9 l.
Thirty grams of the above Prep. 216-4 is dissolved in 65 ml water and the solution is injected onto the column. Some of the solid precipitates when it contacts the methanol but this does not plug the column or otherwise interfere with the procedure. The column is eluted with methanol at 20 ml/minute. The eluate fractions are assayed by UV using 1:10 dilutions. The data are presented in Table III. Fractions 3-8 are pooled and concentrated to give 10.2 gm tan solid. Bioautography shows this to contain antibiotic 354 and no gougerotin. A second injection using the rest of the sample and similarly analyzed yields 12.0 gm of a like preparation.
Table III______________________________________ Pseudomonas mildenbergilFraction Volume Color A.sub.255-265 (UC 3029)______________________________________1 1300 ml Colorless 0.00 --2 1200 ml Colorless 0.21 --3 400 ml Colorless 2.13 30 mm4 400 ml Light Yellow 9.30 405 425 ml Light Yellow 12.8 396 500 ml Light Yellow 12.4 367 500 ml Trace Yellow 8.4 278 500 ml Colorless 1.12 239 500 ml Colorless -- 2010 500 ml Colorless -- 1911 500 ml Colorless -- trace______________________________________
After fraction #11 the solvent is switched to water and the elution is continued at 20 ml/minute. The data are presented in Table IV below. Fractions 2 and 3 are pooled and lyophilized to give 17.9 gm tan solid. A second injection yields 22.1 gm. Bioautography shows this to contain only gougerotin.
Table IV______________________________________Fraction Volume Color A.sub.269______________________________________1 1000 ml Colorless 0.302 1000 ml Yellow 11.03 900 ml Light Yellow 2.644 1400 ml Colorless 0.08______________________________________
The 68 gm sample (216-4) gives 22.2 gm (32.6%) antibiotic 354-containing solids and 40 gm (58.8%) gougerotin-containing solids.
The 22 gm of antibiotic 354 material assays at 0.35 BU/mg vs. B. subtilis and 0.83 BU/mg vs. UC 3029.
The 22 gm of gougerotin material obtained from the second run assays at 0.5 BU/mg vs. B. subtilis and 1.5 BU/mg vs. UC 3029.
Both preparations are grossly impure.
(6) Pure Gougerotin
A pool from two cellulose runs similar to that described above (but ultrafiltered) amounts to 5.8 gm. This is dissolved in 15 ml water and injected onto a 200-400 mesh Amberlite CG-120 (NH.sub.4.sup.+) column (Rohm & Haas, Philadelphia, Pa.) measuring 2.5 .times. 100 cm. This is eluted with a water to 1 M (NH.sub.4).sub.2 SO.sub.4 gradient at 12 ml/minute.
Fractions amounting to 25 ml are collected. Every fifth fraction is assayed (100 .lambda./12.7 mm pad) vs. UC 3029. There is no activity through tube #250. The UV assay is done with 1:10 dilutions of every tenth tube. This shows no 265 nm band through tube #250. The UV data for subsequent tubes is presented in Table V. The B. subtilis zones obtained for tubes 260-370 are very small. Tubes 280-340 are pooled to give 1.4 l solution. This is desalted over 200 ml charcoal in a chromatography tube. The charcoal is washed with water and eluted with 25% acetone in water. The fractions are monitored by UV absorbance at 268 nm. The aqueous eluate is lyophilized to give 1.0 gm of essentially pure gougerotin as a white solid.
Table V______________________________________Fraction A.sub.268 Fraction A.sub.268______________________________________260 0.26 320 1.87270 0.27 330 --280 0.45 340 0.68290 1.30 350 0.32300 2.46 360 0.20310 2.55 370 0.15______________________________________
(7) Pure Antiobiotic 354 As The Sulfate
A pool is made of various fractions which had been treated as described above through the cellulose column step. It amounts to 3.78 gm and is dissolved in 10 ml water. This is injected onto the CG-120 (NH.sub.4.sup.+) column described above for gougerotin and eluted with the same gradient at the same rate. An aliquot of every tenth tube is diluted 1:10 with water and examined by UV and dioassayed. Nothing elutes up to tube #220. The data for subsequent tubes are presented in Table VI. Fractions 248-340 are pooled to give 2.5 l solution with A.sub.252 = 1.27. This is desalted as described for gougerotin using a bed of charcoal measuring 3.5 .times. 28 cm (270 ml) and monitoring at the appropriate wavelengths. The desalted eluate is lyophilized to give 2.36 gm essentially pure antibiotic 354 as a tan solid. It assays at 8 BU/mg vs. UC 3029.
Table Vi______________________________________ Pseudomonas mildenbergil -Fraction A.sub.255 A.sub.212 ( UC 3029)______________________________________230 0.23 1.08 --240 1.10 5.39 --250 1.60 8.20 --260 1.87 8.60 35 mm270 2.75 10.1 40280 2.05 9.00 39290 1.49 6.95 37300 1.03 4.60 33310 0.69 2.41 26320 0.46 1.88 NZ330 0.31 1.25 NZ340 0.20 0.82 NZ350 0.15 0.51 NZ______________________________________
EXAMPLE 2
Acetylation Of Antibiotic 354
A sample of antibiotic 354 is stirred in tetrahydrofuran with pyridine and acetic anhydride. The solvent is removed in vacuo and the residue is partitioned between ethyl acetate and 0.01 N HCl. The aqueous is lyophilized. When the solids are redissolved in water, crystals form and are collected. These crystals are shown by mass spectroscopy to be the mono-N-acetyldehydrochlorinated derivative of antibiotic 354.
EXAMPLE 3
Preparation Of Antibiotic 354.HCl From Antibiotic 354.H.sub.2 SO.sub.4
A 14 mg sample of antibiotic 354.H.sub.2 SO.sub.4 is dissolved in 0.3 ml water and passed over a 0.4 .times. 8 cm column of 100-200 mesh Dowex 2 .times. 8 (Cl.sup.-). Eluted with distilled water and assayed each 3.0 ml fraction with ninhydrin spray after spotting onto a cellulose tlc (thin layer chromatography) plate and by UV absorption (bands at 251 and 211 in a 1:4 ratio). The appropriate pool is lyophilized. The solid residue is judged to be the hydrochloride by mass spectroscopy.
EXAMPLE 4
Preparation Of Antibiotic 354.HOAc From A Mix Of Gougerotin And Antibiotic 354
An aqueous carbon eluate (4 l) containing gougerotin and antibiotic 354 is passed over a column of 200 g Dowex 50 W .times. 8 (H.sup.+). The column is washed with deionized water and eluted with 2.0 M pyridinium acetate buffer at pH 5. Fractions 13-17 (45 ml each) are pooled on the basis of bioactivity (12.7 mm pads, agar tray) vs. UC 3029 an lyophilized. The solids give bioautographic patterns indicating that the main activity is antibiotic 354 which must be in the acetate form. The mixture of acetates so obtained is separated into gougerotin acetate and antibiotic 354 acetate by cellulose chromatography as described above.

Antibiotic 354 and process for producing same

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