Abstract:
Microbiological process for preparing the known antibiotics, antibiotic A-396-1 also known as U-50,147 and L-dihydrophenylalanine also known as U-51,748, by use of the new microorganism Streptomyces lemensis, NRRL 8170 using controlled fermentation conditions. These antibiotics are active against Gram-positive and Gram-negative bacteria. Accordingly, they can be used in various environments to eradicate or control such bacteria.

Description:
BACKGROUND OF THE INVENTION 
     Antibiotic A-396-1 is a known antibiotic described in The Journal of Antibiotics, Vol. 23, No. 6, pages 291-294 (1970) and The Journal of Antibiotics, Vol. 23, No. 11, pages 569-571 (1970). Antibiotic L-dihydrophenylalanine is a known antibiotic described in J. Org. Chem., 33, 1777 (1968). 
     BRIEF SUMMARY OF THE INVENTION 
     The novel process of the invention consists of using the new microorganism Streptomyces lemensis, NRRL 8170, and employing controlled fermentation conditions, to produce the known antibiotic A-396-1, hereinafter referred to as U-50,147, and L-dihydrophenylalanine, hereinafter referred to as U-51,738. 
     The antibiotics produced by the subject process have the property of adversely affecting the growth of Grampositive bacteria, for example, Staphylococcus aureus and Bacillus subtilis, and Gram-negative bacteria, for example, Escherichia coli and Klebsiella penumoniae. Accordingly, these antibiotics can be used to disinfect washed and stacked food utensils contaminated with S. aureus; they also can be used as disinfectants on various dental and medical equipment contaminated with S. aureus. Further, these antibiotics can be used for suppressing the growth of sensitive organisms in plate assays and other biological media. 
     DETAILED DESCRIPTION OF THE INVENTION 
     The microorganism used for the production of antibiotics U-50,147 and U-51,738 is Streptomyces lemensis, NRRL 8170. 
     A subculture of this microorganism can be obtained from the permanent collection of the Northern Regional Research Laboratory, U.S. Department of Agriculture, Peoria, Illinois, U.S.A. Its accession number in this depository is NRRL 8170. 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 streptomycete isolated from a soil sample has been studied and found to differ in macroscopic, microscopic and biochemical properties from members of the Genus Streptomyces characterized in Bergey&#39;s Manual of Determinative Bacteriology [Buchanan, R.E., and N.E. Gibbons. 1974. Bergey&#39;s Manual of Determinative Bacteriology, Eight Edition, The Williams and Wilkins Co., Baltimore], from cultures characterized in Shirling and Gottlieb [Shirling, E.G., and D. Gottlieb. 1968. Cooperative description of type cultures of Streptomyces. II. Species descriptions from first study. 18:69-189] [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] [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] [Shirling, E.B., and D. Gottlieb. 1972. Cooperative description of type strains of Streptomyces. V. Additional descriptions. Int. J. Syst. Bacteriol. 22:285-394], and from &#34;type&#34; cultures in the Upjohn Culture Collection. 
     S. lemensis could be placed in Gray or Red series of melanin-negative cultures with smooth spores in spiral spore chains. The new culture is readily distinguished from the cultures in the references cited by its color pattern, its utilization of carbon compounds in synthetic media [Pridham, T.G., and D. Gottlieb. 1948. The utilization of carbon compounds by some Actinomycetales as an aid for species determination. J. Bacteriol. 65:107-114] [Shirling, E.B., and D. Gottlieb. 1966. Methods for characterization of Streptomyces species. Int. J. Syst. Bacteriol. 16:313-340], and its production of the antibiotics N-demethylhygromycin B and dihydrophenylalanine. 
     N-demethylhygromycin B (A396-1) is produced by Streptoverticillium eurocidicus, Junichi Shoji, et al. 1970. Isolation of a new water-soluble basic antibiotic A-396-I, J. Antibiotics 23, 291-294; and Tamura, Atsushi, Ryuje Furuta and Hirotada Kotami. 1975. Antibiotic A-16316-C, a new water-soluble basic antibiotic. The Journal of Antibiotics XXVIII: 260-265. The verticillate spore chains of S. eurocidicus make additional comparisons with S. lemensis unnecessary. 
     Production of dihydrophenylalanine by an actinomycete has not been reported in the literature. 
     The distinctive properties cited for the culture characterized require it to be considered a new species of Streptomyces designated Streptomyces lemensis sp.n. This is to be considered the type species. Should variants be designated, the type species will become the type subspecies (Streptomyces lemensis sub.sp. lemensis). The culture is designated a new species in accordance with the rules of nomenclature set forth in the International Code of Nomenclature of Bacteria [Lapage, S. P., P.H.A. Sneath, E.F. Lessel, V.B.D. Sherman, H.P.R. Seeliger and W.A.Clark. 1975. International Code of Nomenclature of Bacteria. Amer. Soc. for Microbiology, Washington, D. C.]. 
     Streptomyces lemensis Dietz sp.n. 
     Color characteristics. Aerial growth lavender-gray-tan or grayish reddish brown. Melanin- negative. Appearance on Ektachrome is given in Table 1. Reference color characteristics are given in Table 2. The culture may be placed in the Red (R) 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. Smooth spores with convolutions are found in spiral spore chains. The chains contain more then ten appressed spores. Microscopic determinations followed the procedure 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 was determined following the procedures of Pridham and Gottlieb, supra, J. Bacteriol. 56:107-114, and Shirling and Gottlieb, supra, Int. J. Syst. Bacteriol. 16:313-340. In the former the culture grew well on the basal medium (no carbon compound added) and on D-xylose, L-arabinose, D-fructose, D-galactose, D-glucose, D-mannose, maltose, sucrose, lactose, cellobiose, raffinose, dextrin, soluble starch, glycerol, D-mannitol, sodium acetate, sodium citrate, and sodium succinate; moderately on rhamnose, inulin, dulcitol, D-sorbitol, inositol, and sodium tartrate; and poorly on sodium oxalate. There was no growth on salicin, phenol, cresol, sodium formate, or sodium salicylate. In the latter growth was strong on the positive control (basal medium plus D-glucose), D-xylose, D-mannitol, and D-fructose; positive on the negative control (basal medium without carbon compound), L-arabinose, sucrose, rhamnose, and raffinose; and negative on inositol and cellulose. 
     Temperature. The culture had good vegetative and aerial growth at 18° to 37° C. There was smooth vegetative growth after 24 hours at 45° C. The culture did not grow at 55° C. The media used for temperature studies were Bennett&#39;s, Czapek&#39;s sucrose, maltose-tryptone, and Hickey-Tresner agars. 
     
                       TABLE 1______________________________________Appearance of Streptomyces lemensis on Ektachrome*______________________________________Agar Medium Surface        Reverse______________________________________Bennett&#39;s   Lavender-gray-red                      Gray-red-tanCzapek&#39;s sucrose       Trace lavender-gray                      Trace gray-yellowMaltose-tryptone       Lavender-gray-red                      Red-tanPeptone-iron       --             Yellow0.1% Tyrosine       Trace lavender-gray                      Pale YellowCase in-starch       Lavender-gray  Pale lavender-gray______________________________________ *Dietz, A. 1954. Ektachrome transparencies as aids in actinomycete classification. Ann. N.Y. Acad. Sci. 60:152-154. 
    
     
                                           Table 2__________________________________________________________________________Reference Color Characteristics of Streptomyces lemensis__________________________________________________________________________   De-   ter-   mi-      ISCC-NBS Color-Name Charts   na-      illustrated with Centroid Colors *Agar Medium   tion      Chip No.            Color__________________________________________________________________________Bennett&#39;s   S  45    l.gy. r Br                   Light Grayish Reddish Brown   R  75    deep y Br                   Deep Yellowish Brown      78    d. y. Br                   Dark Yellowish Brown   P  79    l.gy. yBr                   Light Grayish Yellowish BrownCzapek&#39;s   S  73    p. OY  Pale Orange Yellow sucrose   R  73    p. OY  Pale Orange Yellow   P  --    --     --Maltose-   S  45    l.gy. r Br                   Light Grayish Reddish Brown tryptone   R  76    1y.y Br                   Light Yellowish Brown   P  79    l. gy. y Br                   Light Grayish Yellowish BrownHickey- S  45    l.gy. r Br                   Light Grayish Reddish Brown Tresner   R  68    s. OY  Strong Orange Yellow   P  79    l.gy. y Br                   Light Grayish Yellowish BrownYeast extract-   S  45    l.gy. r Br                   Light Grayish Reddish Brown malt extract   R  17    m. OY  Moderate Orange Yellow (ISP-2)   P  79    l.gy. y Br                   Light Grayish Yellowish BrownOatmeal S  45    l.gy. r BR                   Light Grayish Reddish Brown (ISP-3)   R  67    brill. OY                   Brilliant Orange-Yellow   P  --    --     --Inorganic-   S  45    l.gy. r Br                   Light Grayish Reddish Brown salts-starch   R  74    s. y Br                   Strong Yellowish Brown (ISP-4)   P  72    d. OY  Dark 0range YellowGlycerol-   S  60    l.gy. Br                   Light Grayish Reddish Brown asparagine   R  71    ml OY  Moderate Orange Yellow (ISP-5)   67    brill. OY                   Brilliant Orange Yellow   P  79    l.gy. y Br                   Light Grayish Yellowish BrownS = 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__________________________________________________________________________Cultural and Biochemical Characteristics of Streptomyces__________________________________________________________________________lemensisMedium    Surface    Reverse  Other Characteristics__________________________________________________________________________ AgarPeptone-iron     --         Pale colorless                         No pigment                or cream Melanin negativeCalcium malate     --         Colorless                         No pigment                         Malate not solubilizedGlucose asparagine     lavender-gray-cream                Orange-tan                         Pale orange-tan pigmentSkim milk Trace gray-white                Pale orange-tan                         Pale orange-tan pigment                         Casein solubilized under                         growth to around growthTyrosine  pale gray-pink                Pale cream                         Pale cream pigment                         Tyrosine not solubilizedXanthine  Trace gray-pink                Very pale cream                         Pale cream pigment                         Xanthine solubilizedNutrient starch     Pale gray-pink                Pale cream                         No pigment                         Starch partially solubilizedYeast extract-     Good lavender-gray                Red-tan with                         Pink-tan pigment malt extract        pale tan edgePeptone-yeast     --         Pale yellow-tan                         Pale yellow-tan pigment extract-iron (ISP-6)Tyrosine (ISP-7)     Good lavender-gray                Pale red-tan                         Pale red-tan pigment GelatinPlain     --         --       Liquefaction - 1/4Nutrient  --         --       Liquefaction - 1/4 to                         complete BrothSynthetic nitrate     Colorless flaky                --       No pigment      surface pellicle   Trace growth throughout                         Flocculent bottom growth                         Nitrate reduced to nitriteNutrient nitrate     Colorless surface                --       Flocculent bottom growth      pellicle           Nitrites not foundLitmus milk     Gray aerial growth                Blue-gray                         Peptonization - 2/3      or surface ring    Coagulation - 1/3                         Decolorization - 1/3                         pH 6.73-7.55__________________________________________________________________________ 
    
     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&#39;s yeast with milk solids, soybean meal, cottonseed meal, cornmeal, milk solids, pancreatic digest of casein, fish meal, distillers&#39; 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° and 40° C., and preferably between about 20° and 28° 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 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 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, 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 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 adsorption on a resin column. Non-ionic (preferred) as well as cationic exchange resins can be used. Both the carboxylic acid and sulfonic acid types can be used. Suitable non-ionic resins include resins comprising a non-ionic macro porous copolymer of styrene crosslinked with divinylbenzene. Non-ionic resins of this type are marketed under the trade names Amberlite XAD-2 and XAD-4, disclosed in U.S. Pat. No. 3,515,717. Suitable carboxylic acid resins include the polyacrylic acid resins obtained by the copolymerization of acrylic acid and divinylbenzene by the procedure given on page 87 of Kunin, Ion Exchange Resins, 2nd Ed. (1958). John Wiley and Sons, Inc. Carboxylic acid cation exchange resins of this type are marketed under the trade names Amberlite IRC-50 and Zeokarb 226. Suitable sulfonic acid resins include sulfonated polystyrene resins crosslinked with divinylbenzene obtained by the procedure given on page 84 of Kunin, supra. Sulfonated cation exchange resins of this type are marketed under the trade names Dowex-50, Amberlite IR-120, Nalcite HCR, Chempro C-20, Permutit Q, and Zeokarb 225. 
     In the preferred process, using the non-ionic resin, the filtrate from the filtered fermentation beer is passed over the resin. The spent is collected. The column is then washed with water and the aqueous wash is collected. This spent and aqueous wash contain Antibiotic U-50,147. This antibiotic is recovered from these materials by chromatography over a carboxylic acid cation exchange resin or a sulfonic acid type resin as described above. 
     The material is passed over the cation exchange resin. The column is eluted with a basic solution, for example 0.25 N aqueous ammonium hydroxide (preferred) , and other bases like 0.25 N aqueous sodium hydroxide, potassium hydroxide, and pyridinium acetate buffers prepared by mixing pyridine and acetic acid. Antibacterially-active fractions are collected. The active fractions are purified by use of a strongly basic anion exchange resin to remove impurities. Suitable anion exchange resins for this purpose are obtained by chlormethylating by the procedure given on pages 88 and 97 of Kunin, supra, polystyrene crosslinked, if desired, with divinylbenzene prepared by the procedure given on page 84 of Kunin, supra, and quaternizing with trimethylamine, or dimethylethanolamine by the procedure given on page 97 of Kunin, supra. Anion exchange resins of this type are marketed under the trade names Dowex-1, Dowex-2, Dowex-3, Dowex-2K, Amberlite IRA-400, Amberlite JB-45, Duolite A-102, and Ionac A-300. Dowex-1(OH) is the preferred resin. Antibacterially-active fractions from the anion exchange resin are concentrated to drynes to give an essentially pure preparation of Antibiotic U-50,147. 
     Antibiotic U-51,738 is recovered from the fermentation beers of the subject invention process by first filtering the beer and then passing the filtrate over a non-ionic resin as described above. Antibiotic U-50,147 is not adsorbed onto the non-ionic resin, but, rather, passes through or is washed off the resin by water. On the other hand, antibiotic U-51,738 is adsorbed on the non-ionic resin and is removed by elution with a lower-alkanol-water solution. Preferably, methanol-water (90:10) is used to elute antibiotic U-51,738 from the non-ionic resin. Fractions containing antibiotic U-51,738, as determined by antibacterial testing hereinafter described, are combined and concentrated in vacuo. Insoluble crystalline material is separated during the concentration to give an essentially pure crystalline preparation of antibiotic U-51,738. This material is then recrystallized from a lower alcohol (ethanol is preferred) and water, to give an essentially pure crystalline preparation of antibiotic U-51,738. 
     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 
     A soil stock of Streptomyces lemensis, NRRL 8170, is used to inoculate a series of 500-ml Erlenmeyer flasks, each containing 100 ml of sterile seed medium consisting of the following ingredients: 
     
         ______________________________________Glucose monohydrate     10 g/literBacto peptone (Difco)   10 g/literBacto Yeast Extract (Difco)                  2.5 g/literDeionized Water         Balance______________________________________ 
    
     The seed inoculum is grown for 96 hours at 28° C. on a Gump rotary shaker operating at 250 rpm and having a 2 1/2 inch stroke. 
     Seed inoculum (5%) prepared as described above, is used to inoculate a series of 500 ml Erlenmeyer fermentation flasks containing 100 ml of sterile fermentation medium consisting of the following ingredients: 
     
         ______________________________________Cerelose           14 g/literStarch (Buffalo)*  12.5 g/literCottonseed meal**  40 g/literTap water q.s.     1 liter______________________________________  *CPC International, Englewood Cliffs, N.J. **Southern Cotton Oil Division, Hunt Foods and Industries, Newport, Arkansas. 
    
     The pH of the fermentation medium is adjusted to 7.2 with an aqueous solution of sodium hydroxide before sterilization. The inoculated fermentation flasks are incubated at 28° C. on a rotary shaker operating at 250 rpm. The fermentation is monitored by assaying samples with the microorganisms Bacillus subtilis and Penicillium oxalicum to give the total assay for antibiotics U-50,147 and U-51,738. A representation 5-day fermentation has the following titers of antibiotics in the fermentation broth: 
     
         ______________________________________    Assay In BU/mlDay        B. subtilis    P. oxalicum______________________________________2           2.0           1.63           7.0           2.54          10.0           4.05          10.0           3.2______________________________________ 
    
     The assay is a disc-plate biounit assay on standard culture medium. The B. subtilis plates are prepared with Streptomycin Assay Agar with Yeast Extract (Antibiotic Medium No. 5, BBL, Cockeysville, Md.), and the P. oxalicum plates are prepared with Sabouraud Dextrose Agar (Difco Laboratories, Detroit, Michigan). The B. subtilis assay plates are incubated at 32° C. for 18 hours and the P. oxalicum assay plates are incubated at 28° C. for 18 hours. 
     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 
     (1) Filtration - Fermentation broth, as described above, ca. 10 liters, is filtered with the aid of diatomaceous earth. The filtrate is then subjected to chromatography over Amberlite XAD-4 as disclosed below. 
     (2) Chromatography Over Amberlite XAD-4 - The column is prepared from 750 ml of Amberlite XAD-4 resin packed in water. Filtrate, obtained as described above, ca. 7.5 liters, is passed over the column at a rate of 30 ml/min. The spent filtrate is collected as one fraction (SPENT). The column is then washed with 2 liters of water at a rate of 30 ml/min. The aqueous wash is also collected as one fraction (AQUEOUS). The column is then eluted with 90% aqueous methanol. Fractions of 20 ml are collected. Results follow: 
     
         __________________________________________________________________________          Zone Size (mm)           B. subtilis                 P. vulgaris                        P. oxalicum__________________________________________________________________________STARTING MATERIAL          31     17     20 (Filtrate)SPENT          27     0      tracesAQUEOUS        25     0      090% METHANOLIC ELUATESFraction No. 5             22     0      010             20     0      015             20.5   0      020             22     0      025             24     0      030             35     26     4235             33     25     4140             32     23     4045             31     21     3450             29     17     2155             27.5   traces 1660             26     0      traces65             25     0      traces70             24     0      075             23.5   0      080             24     0      085             23     0      090             23     0      095             22.5   0      0100            22     0      0__________________________________________________________________________ 
    
     Thin layer chromatography (tlc) using Eastman Cellulose Chromagram (13255) as support and a solvent consisting of 96% H 2  O and 4% butanol is used to analyze or monitor the recovery operation. The presence of the desired antibiotics are shown by bioautography using the microorganisms P. oxalicum, K. pneumoniae, and S. lutea. This tlc shows that the spent filtrate and the aqueous wash contains antibiotic U-50,147. These two fractions are combined and labeled ADA-71A. Methanolic fractions 30-60 from the Amberlite XAD-4 column are shown by tlc to contain antibiotic U-51,738. 
     Preparations similar to ADA-71A are prepared from additional fermentation broth to give ca. 36 liters of material containing antibiotic U-50,147. The isolation of U-50,147 from this material is accomplished by chromatography over Amberlite IRC 50 (H + ), described below. 
     (3) Chromatography Over Amberlite IRC-50 (H + ) - The column is prepared from 1.5 liter of Amberlite IRC-50 in the hydrogen form. The starting material, 36 liters of the combined filtrates-wash, is adjusted to pH 3.0 with aqueous sulfuric acid. Insoluble material is separated by filtration. The clear filtrate is adjusted to pH 8.5 with aqueous sodium hydroxide and is passed over the column. The spent is collected as one fraction (SPENT). The column is washed with 12 liters of water. The aqueous wash is collected as one fraction (AQUEOUS). The column is washed with 6 liters of 0.25 N aqueous ammonium hydroxide collected as one fraction (0.25 N AMMONIA). The column is eluted with 1 N aqueous ammonium hydroxide. Fractions of 20 ml are collected. Results follow: 
     
         __________________________________________________________________________       Zone Size (mm)        B. subtilis             K. pneumoniae                    P. vulgaris                          P. oxalicum__________________________________________________________________________SPENT       0     0      --    --AQUEOUS     0     0      --    --0.25 N AMMONIA       0     0      --    --1 N AMMONIAFraction No. 10         0     0      --    -- 20         0     0      --    -- .          .     .      .     . .          .     .      .     .230         0     0      --    --240         21    traces --    --250         31    24     --    --260         32    26     --    --270         36    28.5   --    --280         37    31     --    --290         39    33     --    --300         40    33     --    --310         43.5  39     26    32320         43    37.5   25.5  31330         42    38     26    31340         42.5  37.5   25    30350         37    23     20    23360         19    17.5   0     0370         traces             0      0     0390         traces             0      0     0400         0     0      0     0 .          .     .      .     .570         0     0      0     0__________________________________________________________________________ 
    
     Fractions 250-279 are combined and concentrated to dryness in vacuo to give Preparation ADA-116.1, 890 mg. 
     Fractions 280-350 are treated similarly to give Preparation ADA-116.2, 6.28 g. 
     Preparations ADA-116.1 and ADA-116.2 contain antibiotic U-50,147 only (by tlc). Testing of preparation ADA-116.1 and ADA-116.2 shows the following: 
     
         ______________________________________      Zone Size (mm)       ADA-116.1* ADA-116.2*______________________________________P. oxalicum  30            32P. vulgaris  24            26K. Pneumoniae        36            39B. subtilis  39            41______________________________________ *Solutions of 10 mg/ml in water are tested. 
    
     Preparations ADA-116.1 and ADA-116.2 are combined and purified by Dowex-1 (OH - ) chromatography as described below. 
     (4) Chromatography Over Dowex-1 (OH - ) - The column is prepared from 400 ml of Dowex-1 (Cl - ) (Dow Chemical Co., Midland, Michigan). 
     Six liters of 2 N aqueous sodium hydroxide is passed over the column at a rate of 10 ml/min. The column is then washed with water until the pH of the effluent is ca. 8.0. 
     Preparations ADA-116.1 and ADA-116.2 are combined (ca. 7.1 g) dissolved in 25 ml of water and passed over the column at a rate of 4 ml/min. The column is eluted with water. Fractions of 20 ml are collected. Testing shows the following: 
     
         ______________________________________     Zone Size (mm)Fraction No.       B. subtilis    K. pneumoniae______________________________________3           0              06           0              09           27             2012          25             1815          18.5           traces18          16             021          traces         024          17             027          18             traces30          21             traces33          22             1536          22             1539          23             1642          24             1645          24             1748          24             1751          25             1854          26             1957          27             2060          30             2362          30             2266          30             2269          30             22--          --             --60          24             20.565          25.5           2270          25.5           21.575          26             22.580          27             2485          28             24.590          28.5           25.595          30             27100         31             28.5110         32             27.5120         32             29130         33             30140         33.5           31150         34             31.5160         34.5           31170         35             32180         35             32190         35             32200         35             32210         35             30220         34             30230         33             30240         32             29250         31.5           29.5260         31             29.5270         31             29280         30             28.5290         30             27.5300         29             27.5310         28.5           26320         28             26330         27             25340         26             24350         26.5           23360         26             22370         26             22380         26             22390         25.5           22400         26             22.5410         24             21420         24             21430         23             20.5440         23.5           20450         23             20460         23             20470         23             19480         23             19490         23             20500         22.5           20510         22.5           20520         22             19530         22             19540         21.5           18.5550         21             18______________________________________ 
    
     The following pools are made. Each pool is concentrated to dryness to give the following preparations. 
     
         ______________________________________Pool I  Fractions 8-18  ADA-123.1,  360 mgPool II Fractions 30-50 ADA-123.2,  570 mgPool III   Fractions 51-90 ADA-123.3,  333 mgPool IV Fractions 91-200 Fractions 201-300                   ADA-123.4,  ADA-123.5,                               See  BelowPool VI Fractions 301-500                   ADA-123.6,  330 mg______________________________________ 
    
     Preparations ADA-123.4 and ADA-123.5 are combined and kept as ADA- 123,.4A, 1.64 g. 
     Preparation ADA-123.4A contains essentially pure (tlc and paper chromatography) antibiotic U-50,147. 
     Antibiotic U-50,147 has the following characteristics: 
     a. Infrared 
     The IR bands in Nujol and KBr are presented in Tables A (Nujol) and B (KBr) as follows: 
     TABLE A 
     Band Tabulation of the Infrared Spectrum of Antibiotic U-50,147 (Nujol Mull) 
     
         ______________________________________Band Frequency(Wave Numbers)   Intensity______________________________________3350             S3290             S2960             N, S2920             N, S2850             N, S2730             M1640             M, sh1588             S1462             N, S1378             N, S1368             S, sh1342             M1245             M1152             S1078             S1040             S1008             S940              M892              M860              M808              M790              M722              N, M670              M______________________________________ Key: S = Strong, M = Medium, W = Weak, sh = Shoulder, N = Nujol   28 
    
     TABLE B 
     Band Tabulation of the Infrared Spectrum of Antibiotic U-50,147 (KBr Pellet) 
     
         ______________________________________Band Frequency(Wave Numbers)   Intensity______________________________________3410             S3370             S3290             S, sh2920             M2880             M2700             M, sh1630             M, sh1590             M1458             M1385             M1339             M1242             M1152             S1075             S1038             S1005             S938              M890              M805              M790              M730              M700              M668              M______________________________________ Key:  S = Strong, M = Medium, W = Weak, sh = Shoulder 
    
     b. UV Absorption Spectrum 
     Antibiotic U-50,147 does not show any UV maxima between 220-400 mn. 
     c. Titration Data 
     Potentiometric titration in water using aqueous hydrochloric acid shows an equivalent weight of 192. 
     d. Elemental Analyses 
     Calculated for C 19  H 35  N 3  O 13  : C, 44.44; H, 6.82; N, 8.18; O, 40.55. Found: C, 41.78; H, 6.92; N, 8.64. 
     e. Molecular Weight 
     The molecular weight determined by field desorption mass spectrometry is found to be 513. Field desorption mass spectra are obtained on the CH5DF mass spectrometer. 
     f. Optical Rotation 
     [α] D   25  +13° (C, 0.95, water) 
     g. Solubilities 
     Antibiotic U-50,147 (both the free base and the salts) is soluble in water and lower alcohols (methanol, ethanol). The antibiotic is insoluble in higher alcohols, acetone, ethyl acetate, chlorinated hydrocarbon and saturated hydrocarbon solvents. 
     EXAMPLE 2 
     Isolation of Antibiotic U-51,738 
     The methanolic fractions obtained in Example 1, Part B (2) from the Amberlite XAD-4 resin are combined and the solution is concentrated in vacuo. Insoluble crystalline material of antibiotic U-51,738 is separated during the concentration; yield 1.8 grams. 700 mg of this material is recrystallized from 60 ml of ethanol and 10 ml of water to give an essentially pure crystalline preparation of antibiotic U-51,738. The crystalline material is in the form of colorless needles. Antibiotic U-51,738 is a known antibiotic, L-dihydrophenylalanine, having the following structural formula: ##STR1## 
     This compound has the following reported characteristics: (see J. Org. Chem. 33, 1779 [1968]). 
     Anal. Calcd. for C 9  H 13  NO 2  : C, 64.7; H, 7.84; N, 8.38. Found: C, 65.0; H, 7.86; N, 8.53. IR bands: KBr μ max  3030-2850, 2620, 1590-1560, 1480, 1390, 1220, 1135, 965, 895, 855 cm -   1 .