Process for producing 4-thiouracil

BACKGROUND OF THE INVENTION
The compound, 4-thiouracil, produced by the novel process of the subject invention has been known since about 1945. It has been described in the literature as an antibacterial, antitumor agent, useful in photographic processes and as a plant growth stimulator. The compound has been prepared in the prior art by chemical means. There is no known prior art which describes the production of 4-thiouracil by a microbiological process.
BRIEF SUMMARY OF THE INVENTION
The novel process of the subject invention produces a high yield of the compound 4-thiouracil. This process is conducted by culturing the new microorganism Streptomyces libani subsp. soldani, NRRL 8173, and its strains NRRL 8174 and NRRL 8175, in an aqueous nutrient medium under aerobic conditions. 4-Thiouracil is isolated from the fermentation beer by procedures generally known in the antibiotic art, for example, filtration, solvent extraction, partition chromatography, adsorption onto charcoal and the like.
DETAILED DESCRIPTION OF THE INVENTION
The compound produced by the novel process of the subject invention, 4-thiouracil, can be shown by the following structural formula: ##STR1##
This compound is active against a variety of bacteria, for example, Staphylococcus aureus, Bacillus subtilis, Escherichia coli, and Proteus vulgaris. Accordingly, 4-thiouracil can be used in various environments to eradicate or control these microorganisms. For example, it can be used for treating breeding places of silkworms and to prevent or minimize infections caused by Bacillus subtilis. Since 4-thiourcil is active against E. coli, it can be used to reduce, arrest, and eradicate slime production in papermill systems caused by this microorganism.
THE MICROORGANISM
The microorganism used for the production of 4-thiouracil is Streptomyces libani subsp. soldani. 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 8173. 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 of the Upjohn Research Laboratories.
A new soil isolate NRRL 8173 has been compared with two strains and with the type culture Streptomyces libani. The new culture was considered to be most similar to S. libani in its color pattern on Ektachrome and in its microscopic characteristics. Detailed comparisons are given in Tables 1-5. A number of isolates of S. libani are characterized by Baldacci and Grein [Baldacci, E. and A. Grein. 1966. Streptomyces avellaneus and Streptomyces libani; two new species characterized by a hazel-nut brown (avellaneus) aerial mycelium. Giornale di Microbiologia 14: 185-198]. The characterizations reveal the natural variability of the culture. The same variability is noted between NRRL 8173 and the strains NRRL 8174 and NRRL 8175. S. libani var. soldani may be placed in the Gray series of Table 17.42f (Part 17. Actinomycetes and Related Organisms) of the Eighth Edition of Bergey's Manual of Determinative Bacteriology [Buchanan, R. E., and N. E. Gibbons. 1974. Bergey's Manual of Determinative Bacteriology, Eight Edition, The Williams and Wilkins Co., Baltimore]. The type culture, S. libani, is listed in Table 17.42f. An additional characterization of S. libani is found in Shirling and Gottlieb [Shirling, E. B. and D. Gottlieb. 1972. Cooperative description of type strains of Streptomyces. V. Additional descriptions. Int. J. Syst. Bacteriol. 22: 265-394]. Strong similarities are noted between the new soil isolate and S. libani in Tables 1-5 and in the references cited. The natural variability of the cultures and, in particular, the difference in antibiotic production require the new isolate to be considered a new subspecies. Therefore, in accordance with the rules set forth in the International Code of Nomenclature of Bacteria [International Code of Nomenclature of Bacteria, 1976 Revision. 1975. Edited by S. P. LaPage, p. H. A. Sneath, E. F. Lessel, V. B. D. Skerman, H. P. R. Seeliger and W. A. Clark. Published for the International Association of Microbiological Societies by the American Society for Microbiology, Washington, D.C.]the name Streptomyces libani subsp. soldani Dietz subsp. nova is proposed.
Color Characteristics
The cultures are melanin-negative and have white and gray aerial growth. Color characteristics of the cultures on Ektachrome are given in Table 1. Reference color characteristics are given in Table 2. The cultures may be placed in the White (W) or Gray (GY) 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].
Microscopic Characteristics
Spores with a smooth surface are found in spiral spore chains. More than 10 spores occur per chain. The spore shape may be oval, reniform or spherical. Microscopic determinations were made following the procedures 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] and Dietz and Mathews [Dietz, A. and J. Mathews. 1971. Classification of Streptomyces spore surfaces into five groups. Appl. Microbiol. 21:527-533].
Cultural and Biochemical Characteristics
Cultural and biochemical characteristics are cited in Table 3.
Carbon Utilization
Carbon utilization is cited in Tables 4 and 5.
Temperature
The cultures grew well in the temperature range of 18.degree. -37.degree. C. Growth did not occur at 45.degree. or 55.degree. C. Media used for temperature studies were Bennett's, Czapek's sucrose, maltose-tryptone and Hickey-Tresner agars.
Antibiotic-Producing Properties
S. libani subsp. soldani NRRL 8173produces 4-thiouracil. S. libani UC-5629 produces the libanomycin complex.
Source: Soil.
Type Culture: Streptomyces libani Baldacci and Grein, UC-5629, IMRU 3915.
Variety: Streptomyces libani subsp. soldani Dietz subsp. nova.
Strains: Streptomyces libani subsp. soldani strain W Streptomyces libani subsp. soldani strain P
Table 1__________________________________________________________________________Appearance of Streptomyces libani subsp. soldani NRRL 8173 and theStrains NRRL 8174 and NRRL 8175 vs. S. libani UC-5629 on Ektachrome* Determi-Agar Medium nation NRRL 8173 NRRL 8174 NRRL 8175 UC-5629__________________________________________________________________________Bennett's S White Lavender-gray Trace gray Lavender-gray R Pale yellow cream Yellow Pale tan Pale yellow-tanCzapek's S White White Fair white Whitesucrose R Cream Pale yellow Pale tan Pale yellowMaltose S White Gray white Fair white Lavender-graytryptone R Pale yellow Yellow-tan Pale yellow- Pale yellow-tan tanPeptone-iron S Trace white Trace white -- Trace lavender- gray R Yellow-tan Yellow Yellow-tan Yellow0.1% Tyrosine S Trace white Trace white -- Trace lavender- gray R Red-pink Red-pink Deep red Pale yellowCasein strarch S White Pale gray Pale cream Trace lavender- gray R Very pale cream Pale yellow Lavender gray Pale yellow__________________________________________________________________________ *Dietz, A. 1954. Ektachrome transparencies as aids in actinomycete classification. Ann. N. Y. Acad. Sci. 60:152-154. S = Surface R = NOTE: "UC" is a registered trademark and refers to The Upjohn Company Culture Collection.
Table 2__________________________________________________________________________Reference Color Characteristics of S. libani subsp. soldani vs. S.libani De- ter- mi- ISCC-NBS Color-Name Charts Illustrated with Centroid Colors* na- S. libani var. soldaniAgar Medium tion NRRL 8173 NRRL 8174__________________________________________________________________________Bennett's S 86 l.Y Light Yellow 93 y Gray Yellowish gray under 214 p.V Pale violet R 84 s.Y Strong yellow 89 p.Y Pale yellow P 84 s.Y Strong yellow 90 gy. Y Grayish yellowCzapek's S 9 pk White Pinkish white 263 White Whitesucrose R 52 l.0 Light orange 73 p.OY Pale orange yellow P 28 l.y Pink Light yellowish pink 70 l.OY Light orange yellowMaltose- S 93 y Gray Yellowish gray 263 White Whitetryptone R 83 brill.Y Brilliant yellow 89 p.Y Pale yellow P -- 71 m OY Moderate orange yellowHickey- S 92 y White Yellowish white 9 pk White Pink whiteTresner R 87 m.Y Moderate yellow 70 l.OY Light orange yellow P -- 71 m OY Moderate orange yellowYeast extract- S 86 l.Y Light yellow 263 White Whitemalt extract under(ISP-2) 294 p.V Pale violet R 91 d.gy.Y Dark grayish yellow 70 l.OY Light orange yellow P -- --Oatmeal S 261 l.gy.p R Light grayish purplish red 93 y Gray Yellowish gray(ISP-3) R 89 p.Y Pale yellow 89 p.Y Pale yellow P -- 90 gy.Y Grayish yellowInorganic S 86 l.Y Light yellow 93 y Gray Yellowish graysalts-starch over(ISP-4) 244 p.r P Pale reddish purple R 83 brill. Y Brilliant yellow 89 p.Y Pale yellow P 87 m.Y Moderate yellow 90 gy.Y Grayish yellowGlycerol- S 89 p.Y Pale yellow 265 med. Gy Medium grayasparagine over(ISP-5) 261 l.gy.p R Light grayish purplish red R 87 m.Y Moderate yellow 89 p.Y Pale yellow P 87 m.Y Moderate yellow 90 gy.Y Grayish yellow De- ter- mi- ISCC-NBS Color-Name Charts Illustrated with Centroid Colors* na- S. libani var. soldani S. libaniAgar Medium tion NRRL 8175 UC-5629__________________________________________________________________________Bennett's S 93 y Gray Yellowish gray 93 y Gray Yellowish gray R 70 l.OY Light orange yellow 67 brill.OY Brilliant orange yellow P 67 brill.OY Brilliant orange-yellow 68 s.OY Strong orange yellowCzapek's S 92 y White Yellowish white 253 White Whitesucrose R 76 l.y Br Light yellowish brown 73 p.OY Pale orange yellow P 77 l.y Br Light yellowish brown 29 m.y Pink Moderate yellowish pinkMaltose- S 92 y White Yellowish white 93 y Gray Yellowish graytryptone R 89 p.Y Pale yellow 83 brill. Y Brilliant yellow P 76 l.y Br Light yellowish brown 84 s.Y Strong yellowHickey- S 92 y White Yellowish white 9 pk White Pinkish whiteTresner R 73 p.OY Pale orange yellow 73 p.O Pale orange yellow P 76 l.y Br Light yellowish brown 67 brill.OY Brilliant orange yellowYeast extract- S 9 pk White Pinkish white 263 White Whitemalt extract(ISP-2) R 57 l.Br Light brown 70 l.OY Light orange yellow P 42 l.r Br Light reddish brown 71 m.OY Moderate orange yellowOatmeal S 93 y Gray Yellowish gray 93 y Gray Yellowish gray(ISP-3) R 70 l.OY Light orange yellow 89 p.Y Pale yellow P 71 m.OY Moderate orange yellow 101 1.Gray light grayInorganic S 92 y White Yellowish white 81 d.gy.yBr Dark grayish yellowishsalts-starch brown(ISP-4) R 70 l.OY Light orange yellow 73 p.OY Pale orange yellow P 71 m.OY Moderate orange yellow 73 d.OY Dark orange yellowGlycerol- S 93 y Gray Yellowish gray 9 pk White Pinkish whiteasparagine(ISP-5) R 70 l.OY Light orange yellow 73 p.OY Pale orange yellow P 71 m.OY Moderate orange yellow 71 m.OY Moderate orange__________________________________________________________________________ yellow *Kelly, K. L. and D. B. Judd. 1955. The ISCC-NBS method of designating colors and a dictionary of color names. U.S. Dept. Comm. Circ. 553. S = Surface R = Reverse P = Pigment
Table 3__________________________________________________________________________Cultural and Biochemical Characteristics Determi- S. libani subsp. soldani S. libaniMedium nation NRRL 8173 NRRL 8174 NRRL 8175 UC-5629__________________________________________________________________________AgarPeptone-iron S -- White -- Trace pale gray R Colorless Yellow Yellow-tan Pale cream-tan P -- -- -- -- O Melanin negative Melanin negative Melanin negative Melanin negativeCalcium malate S Fair white Trace white -- Trace pale gray R White or colorless Colorless Colorless Colorless or pale gray P -- -- -- -- O Malate solubilized Malate not solu- Malate not solu- Malate not solu- bilized bilized bilizedGlucose S Cream White with trace Trace white Pale gray-whiteasparagine gray R Pink-tan Cream Pale tan Pale cream-pink P Pink -- Trace pink Pale pinkSkim milk S Trace white on edge White on edge -- Pale gray-white R Pink-orange Colorless Colorless and Orange-tan trace orange P Pink-orange -- -- Orange-tan O Casein hydrolyzed Casein not Casein not Casein not hydrolyzed hydrolyzed to hydrolyzed trace hydrolyzed around growthTyrosine S Gray Pale cream-pink -- Pale cream-white R Red-tan Light red-tan Red-tan Yellow P Red-tan Light red-tan Red-tan Pale yellow O Tyrosine solu- Tyrosine solu- Tyrosine solu- Tyrosine solu- bilized bilized bilized bilizedXanthine S Cream white Pale cream white Fair pale gray Cream white R Cream Cream Cream Pale yellow P Pale cream Pale cream Cream Pale yellow O Xanthine not Xanthine not Xanthine solu- Xanthine solu- solubilized solubilized bilized bilizedNutrient starch S Cream white Cream white -- Cream white R Cream Cream Cream Pale yellow P -- Pale Cream Pale cream -- O Starch solubilized Starch solubilized Starch solubilized Starch solubilizedYeast extract- S Cream Pale gray-cream -- Pale gray-creammalt extract R Yellow-tan Pale cream-brown Pale yellow-tan Yellow-tan P -- Pale olive-gray Pale yellow-tan Yellow-tanPeptone-yeast S White Cream white -- Very pale gray-whiteextract-iron R Yellow Pale tan Yellow-tan Yellow-tan(ISP-6) P -- -- Pale yellow-tan Yellow-tanL-Tyrosine S Lavender-gray- Lavender-gray- Pale lavender- Cottony pale-gray(ISP-7) white cream with gray-cream with white flecks black exudate R Pale tan Pale olive Olive-tan Cream-tan (reddish at base P Pale tan Pale olive Pale olive-tan --GelatinPlain S Colorless surface White surface Colorless surface Colorless surface ring pellicle ring ring P -- Olive -- Olive brown to olive O Liquefaction No liquefaction No liquefaction Liquefied in olive- complete brown area = trace liquefactionNutrient S Colorless surface White surface Colorless surface Colorless surface ring pellicle ring ring P -- Pale tan -- -- O Liquefaction No liquefaction No liquefaction No liquefaction completeNitrate BrothSynthetic S White aerial on -- -- -- surface ring P -- -- -- -- O Trace flocculent Trace flocculent Flocculent bottom Compact bottom bottom growth bottom growth growth growthSynthetic O Nitrate not Nitrate not Nitrate not Nitrate not reduced reduced reduced reduced Red with Zn dust Red with zn dust Red with zn dust Red with zn dustNutrient S Trace white aerial Pale gray-white -- -- growth on aerial growth surface ring on surface ring P -- -- -- -- O Flocculent bottom Flocculent bottom Pelliate like Flocculent bottom growth growth bottom growth growth Nitrate not Nitrate not Nitrate not Nitrate not reduced reduced reduced reduced Orange with Zn Orange with zn Orange with zn Orange with zn dust dust dust dustLitmus milk S Pale gray-pink Pale lavender-gray -- Pale gray-pink aerial growth on aerial growth on aerial growth pink-maroon pale salmon reddish-purple surface ring surface ring surface ring P -- -- -- -- O Peptonization 2/3 No peptonization Peptonization 2/3 Peptonization 2/3 Litmus reduced Litmus changed to Partial decoloriza- and coagulation lavender gray tion 1/3 pH 6.5 pH 7.1 pH 6.5 pH 6.7__________________________________________________________________________ S = Surface R = Reverse P = Pigment O = Other characteristics
Table 4__________________________________________________________________________Growth on Carbon Compounds in the Synthetic Medium ofPridham and Gottlieb* S. libani subsp. soldani S. libani__________________________________________________________________________ NRRL 8173 NRRL 8174 NRRL 8175 UC-5629CONTROL + (-) (-) (+)__________________________________________________________________________1. D-Xylose + + + +2. L-Arabinose + (+) (+) (+)3. Rhamnose + (-) (-) (+)4. D-Fructose + + + +5. D-Galactose + + + +6. D-Glucose + + + +7. D-Mannose + + + +8. Maltose + + + +9. Sucrose + + + +10. Lactose + (-) (-) +11. Cellobiose + (-) (-) (+)12. Raffinose + + + +13. Dextrin + + + +14. Inulin + (-) (-) (+)15. Soluble Starch + + + +16. Glycerol + + + +17. Dulcitol (+) (-) (-) (+)18. D-Mannitol + + + +19. D-Sorbitol (+) + + +20. Inositol + + + +21. Salicin (-) - - -22. Phenol - - - -23. Cresol - - - -24. Na Formate - - - -25. Na Oxalate (+) (-) (-) (-)26. Na Tartrate (+) (-) (-) (-)27. Na Salicylate - - - -28. Na Acetate (+) (+) (+) (+)29. Na Citrate + (+) (+) (+)30. Na Succinate + (+) (+) (+)__________________________________________________________________________ *Pridham, T. G. and D. Gottlieb. 1948. The utilization of carbon compound by some Actinomycetales as an aid for species determination. J. Bacteriol 56: 107-114. + = Good growth (+) = Moderate growth (-) = Poor growth - = No growth
Table 5__________________________________________________________________________Growth on Carbon Compounds in the Synthetic Medium ofShirling and Gottlieb *__________________________________________________________________________ S. libani subsp. soldani S. libaniCONTROL NRRL 8173 NRRL 8174 NRRL 8175 UC-5629__________________________________________________________________________Negative + - - +(syntheticmedium only)Positive + + + +(syntheticmedium withD-glucose)1. Arabinose + + + ++2. Sucrose ++ ++ ++ ++3. D-Xylose ++ + ++ +4. Inositol ++ ++ ++ ++5. D-Mannitol + ++ ++ ++6. D-Fructose + + ++ ++7. Rhamnose + .+-. - -8. Raffinose ++ .+-. .+-. +9. Cellulose - - - -__________________________________________________________________________ * Shirling, E. B. and D. Gottlieb. 1966. Methods for characterization of Streptomyces species. Int. J. Syst. Bacteriol.
4-Thiouracil 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' solubles 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 4-thiouracil can be effected at any temperature conducive to satisfactory growth of the micro-organism, 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 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 4-thiouracil 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, agar plug, stored frozen above liquid N.sub.2, 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 4-thiouracil, so long as a good growth of the microorganism has been obtained.
A variety of procedures can be employed in the isolation and purification of 4-thiouracil from fermentation beers, for example, solvent extraction, partition chromatography, silica gel chromatography, liquid-liquid distribution in a Craig apparatus, adsorption on resins, and crystallization from solvents.
In a preferred recovery process 4-thiouracil is recovered from its culture medium by separation of the mycelia and undissolved solids by conventional means, such as by filtration or centrifugation. The antibiotic is then recovered from the filtered or centrifuged broth by extraction. For the extraction of 4-thiouracil from the filtered broth, water-immiscible organic solvents in which it is soluble, for example, chloroform, ethylene dichloride, ethyl acetate (preferred), and methylene chloride can be used. Advantageously, the extraction is carried on after the filtered beer is adjusted to an approximate pH 7.0 with a mineral acid. The solvent extracts are combined and evaporated to dryness under vacuum. Alternately, 4-thiouracil can be recovered from the filtered fermentation beer by use of a resin which comprises a non-ionic macroporous copolymer of styrene crosslinked with divinylbenzene. Resins of this type are disclosed in U.S. Pat. No. 3,515,717. Examples of this type of resin are Amberlite XAD-2, XAD-4, XAD-12. These resins can be obtained from the Rohm and Haas Chemical Company. Further, anion exchange resins can be used to recover 4-thiouracil from the filtered fermentation broth. Examples of such resins are Dowex-1 and Dowex-2 in the hydroxide form. These resins can be obtained from the Dow Chemical Company. When an anion exchange resin is used in the recovery process, the 4-thiouracil can be eluted from the resin by adding a polar, water-miscible solvent to the aqueous stream. Examples of such solvents are acetone, methanol, i-propanol, ethanol, dioxane or tetrahydrofuran. The organic solvent can then be removed in vacuo and the 4-thiouracil extracted from the aqueous using one or more of the above solvents. If a solid forms on removal of the organic solvent, then the product can be recovered by filtration or lyophilization. A further alternate recovery process is to pass the filtered beer over activated charcoal and eluting the 4-thiouracil, as described above.
Purification of the 4-thiouracil preparation, obtained as described above, can be achieved by the use of silica gel or alumina chromatography using a pair of compatible lipophilic and hydrophilic solvents such as CHCl.sub.3 /MeOH, CHCl.sub.3 /EtOH, EtOAc/acetone, BuOAc/MeOH, EtOAc/MeOH (preferred), and the like.

Hereinafter are described non-limiting examples of the process of the subject invention. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.
EXAMPLE 1
Part A. Fermentation
One agar plug of Streptomyces libani subsp. soldani, NRRL 8173, stored above liquid nitrogen, is used to inoculate each of a series of 500-ml Erlenmyer flasks each containing 100 ml of sterile seed medium consisting of the following ingredients:
______________________________________Glucose monohydrate 25 g/literPharmamedia * 25 g/literTap water q.s. 1 liter______________________________________ * Pharmamedia is an industrial grade of cottonseed flour produced by Traders Oil Mill Company, Fort Worth, Texas.
The seed medium presterilization pH is 7.2. The seed inoculum is grown for 3 days at 28.degree. C. on a Gump rotary shaker operating at 250 r.p.m. and having a 21/2 inch stroke.
Seed inoculum (5%), prepared as described above, is used to inoculate a series of 500 ml fermentation flasks, each containing 100 ml of sterile fermentation medium consisting of the following ingredients:
______________________________________Glycerol 10 g/literYeast Extract 2.5 g/literBrer Rabbit Molasses 10 g/literTryptone * 5 g/literTap water q.s. 1 liter______________________________________ * Difco Laboratories, Detroit, Michigan.
The inoculated fermentation flasks are incubated at a temperature of 28.degree. C. for 5 days while being shaken on a Gump rotary shaker operating at 250 r.p.m. and having a 21/2 inch stroke. Foaming in the fermentation flasks is controlled by the antifoam agent UCON (a synthetic defoamer supplied by Union Carbide, N.Y., N.Y.). A representative 5-day fermentation has the following titers of antibiotic in the fermentation broth:
______________________________________Day Assay, BU/ml______________________________________2 6.73 15.04 10.05 16.0______________________________________
The assay is a disc plate biounit assay using the bacterium Bacillus subtilis grown on a synthetic medium. The synthetic medium has the following composition:
______________________________________Na.sub.2 HPO.sub.4 .sup.. 7H.sub.2 O 1.7 gKH.sub.2 PO.sub.4 2.0 g(NH.sub.4).sub.2 SO.sub.4 1.0 gMgSO.sub.4 0.1 gGlucose 2.0 gBacto Agar * 15.0 gDistilled water 1 literMetallic ion stock solution ** 1 ml______________________________________ * Bacto Agar obtained from Difco Laboratories, Detroit, Michigan. ** Metallic ion stock solution consists of the following:
NaMoO.sub.4 .sup.. 2H.sub.2 O 200 .mu.g/mlCoCl.sub.2 100 .mu.g/mlCuSO.sub.4 100 .mu.g/mlMnSO.sub.4 2 mg/mlCaCl.sub.2 25 mg/mlFeCl.sub.2 .sup.. 4H.sub.2 O 5 mg/mlZnCl.sub.2 * 5 mg/ml______________________________________ * ZnCl.sub.2 has to be dissolved separately using a drop of 0.1N HCl for 10 ml of water. The stock solution is heated to bring all the compounds i solution, kept standing for 24 hours, and sterile filtered.
This medium is inoculated with a spore suspension of B. subtilis (1.5 .times. 10.sup.10 cells/ml) at a rate of 0.5 ml/liter. The beer samples are applied to 12.5 mm diameter adsorbent paper discs (0.08 ml/disc), which are then applied to the surface of the assay plate. The assay system is incubated overnight at 37.degree. C., and the zones of inhibition are measured. The potency of the sample is related to the diameter of the inhibition zone by means of the usual standard curve.
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.
Part B. Recovery
Whole fermentation beer (800 ml) at pH 8.0 is filtered with the aid of diatomaceous earth as a filter aid after the adjustment to pH 7.0 with 3N HCl. The resulting 750 ml of filtrate is extracted twice with 2-750 ml portions of ethyl acetate. The organic phases are combined, washed with a saturated sodium chloride solution, dried with magnesium sulfate, filtered and concentrated on a rotary evaporator; yield 166.0 mg of a preparation of 4-thiouracil.
Part C. Purification
A preparation of 4-thiouracil (116 mg), prepared as described above, is dissolved in about 5 ml of ethyl acetate and the solution is injected via a syringe onto a size C Merck pre-packed column of silica gel. The column is developed isocratically at 5 ml/min. to tube -35 whereupon the solution is switched to 10:1 EtOAc:MeOH (v/v). The 25 ml fractions are assayed by dipping 12.7 mm pads and placing them onto agar trays seeded with Salmonella schottmuelleri. The trays are incubated for 18 hours at 32.degree. C. and the zones are read. Tubes 23-28 which give zones ranging from 24-38 mm are combined. Concentration of this pool gives 112.2 mg. of essentially pure 4-thiouracil.
EXAMPLE 2
By substituting Streptomyces libani subsp. soldani, strain W, NRRL 8174for S. libani subsp. soldani, NRRL 8173 in Example 1, there is obtained 4-thiouracil which can be recovered in the essentially pure form by the procedures disclosed in Example 1.
EXAMPLE 3
By substituting Streptomyces libani subsp. soldani, strain P, NRRL 8175 for S. libani subsp. soldani, NRRL 8173 in Example 1, there is obtained 4-thiouracil which can be recovered in the essentially pure form by the procedures disclosed in Example 1.

Process for producing 4-thiouracil

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