372 



DESCRIPTIONS OF ANTIBIOTICS 



CHs = 9.5% (2;. Forms a biologically inactive, 

 yellow addition j^roduct with bromine in CCU : 

 C33H4709N.2Br3 (2) or C32H4609N2Br3 (1). Addition 

 product has no definite melting point, chars at 

 >100°C, gives positive FeCl3 test, and has two 

 weak maxima in ultraviolet light at 255 and 290 

 to 295 m^u (1, 2). Calcium salt: Soluble in alcohol 

 and methylene chloride. Insoluble in water and 

 hydrocarbons. Na salt: Buff-colored powder. Sol- 

 uble in water, ethanol, and chloroform, but not in 

 hydrocarbons. K salt: Same solubility as Na salt 

 (1, 2). Infrared spectra of these salts given in 

 reference 2. 



Biological activity: Active on gram-positive bac- 

 teria, including B. cereus, Corynebacterium diph- 

 theroides, D. pneumoniae, Erysipelothrix rhusi- 

 ophathiae, Listerella vionocytogenes, Clostridia, 

 mycobacteria, streptococci, and Nocardia aster- 

 oides. Less active on micrococci. Not active on 

 gram-negative bacteria, except PasteureUa multo- 

 cida. Not active on fungi or viruses. Optimally ac- 

 tive at pH 7.0. Partially bound by serum; not af- 

 fected by cj'steine, methionine, or penicillinase. 

 Effective against Str. hemolyticus, D. pneumoniae, 

 and P. multocida infections in mice. Not effective 

 in K. pneumoniae, Sal. typhi, or Sal. paratyphi B 

 infections (1). 



Toxicity: LDso (mice) 533 mg per kg intraperi- 

 toneally (in 1 per cent sodium carbo.xymethylcel- 

 lulose) (1). 



References: 



1. DeBoer,C.e/a/. Antibiotics Ann. 1955-1956, 



pp. 880-902. 



2. British Patent 779, 570, July 2-1, 1957. 



Streptomycin 



Produced by: Streptomyces griseus (135), S. 

 bikiniensis (13), S. olivaceus (36), S. mashuensis 

 (82), Streptomyces sp. resembling S. roseochromo- 

 genes (65), and Streptomyces sp. reseml)ling S. 

 olivaceus (52). 



Synonym: Streptomycin A. 



Method of extraction: I. Adsorbed from broth 

 on IRC-50 or other weak carboxylic acid cation 

 exchange resin at pH 7.0. Resin washed with water, 

 then 1 per cent citric acid, and adjusted to pH 

 6.0 with triethylamine, monoethanolamine, or 

 similar amines, and eluted with hydrochloric or 

 sulfuric acid. The amine displaces contaminating 

 metal ions and is eluted as the sulfate wath strep- 

 tomycin, but does not precipitate from the eluate 

 with streptomycin on addition of methanol (118). 

 Metallic contaminants can also be removed from 

 the eluate without the use of amines by a seques- 

 tering agent such as ethylenediaminetetraacetic 



acid. The antibiotic is re-adsorbed on IRC-50 aV 

 pH 6.8 and eluted with sulfuric acid at pH 2.0. 

 Eluate neutralized with IR4B resin, deionized on a 

 "monobed" of IR-120-IR4B, and freeze dried 

 (113). Another process uses resins IRC-50, XE 89, 

 and Dowex 50X16 in succession; the first two 

 to al)sorb the antibiotic, the last to remove im- 

 purities (120). II. Whole broth acidified with 

 HCl or H2SO4 to pH 2 to 4, stirred with charcoal, 

 and filtered. Filtrate neutralized (pH 6 to 8) and 

 adsorbed on charcoal. Charcoal washed with 

 water, aqueous methanol or ethanol, and eluted 

 with 0.8 N formic acid in methanol-water (1:1), 

 0.1 N methanolic HCl, dilute acids, 60 per cent 

 aqueous acetone containing HCl, or 5 to 10 per 

 cent acetone-water containing H2SO4 (pH 2.5). 

 Eluates give a precipitate of streptomycin on addi- 

 tion of acetone to 75 per cent, or can be neutral- 

 ized, concentrated under reduced pressure, and 

 taken up in methanol, followed by acetone pre- 

 cipitation. Purified by chromatography on alu- 

 mina (sulfuric acid-washed) at pH 5 to 6, or on 

 Darco G-60. Methanol or aqueous methanol is used 

 as solvent and developer. Eluates concentrated to 

 remove the methanol, then lyophilized. Further 

 purification by salt conversion (picrate — > phos- 

 photungstate —> sulfate/helianthate — > trihydro- 

 chloride; or trihydrochloride — > CaCL complex) 

 (4, 7, 9, 15, 25). IIIA. Streptomycin is quantita- 

 tively precipitated from broths with orange II 

 (sodium p-(2-hydroxy-l-naphthylazo) benzene sul- 

 fonate) at pH 5.5, or with Najjhthol blue-black 

 (disodium salt of 8-amino-7-/>-nitrophenylazo-2- 

 phen3-lazo-l-naphthol-3,6-disulfonic acid). Such 

 precipitates canbe converted (40,41) to useful salts, 

 such as the sulfate, by suspending them in alcohols 

 or Cellosolves, or aqueous mixtures of these, treat- 

 ing the suspension with an amine salt (e.g., tri- 

 ethylamine sulfate) to give a precipitate of strep- 

 tomycin sulfate from the anhydrous solvents. 

 (Acetone must be added to aeiueous mixtures to 

 give a precipitate.) IIIB. Wetting agents, such as 

 the "Tergitols" (sodium salts of synthetic higher 

 aliphatic alcohols), "Duponols" (partial sulfuric 

 acid esters of higher aliphatic alcohols), aromatic 

 sulfonic acids, sulfonated oils, and "Ultrawet," 

 precipitate streptomycin from broths or partiallj^ 

 purified solutions. For example: Tergitol 7 (so- 

 dium sulfate derivative of 3,9-diethyltridecanol-6) 

 in amyl or butyl alcohols is added to streptomycin- 

 containing solutions at pH 3.3 to 7.5 to give pre- 

 cipitated streptomycin "tergitate." Crystallized 

 from methanol-water. Aging for >8 days increases 

 the yield of streptomycin inorganic salts because 

 of tautomerism (see "Chemical and Physical 



