Chemical Studies on Polymyxin.¹ I. Isolation and Preliminary Purification

Abstract

Isolation and Preliminary Purification of Polymyxin 3771 corresponding alcohol, tryptophol (IV), by the excellent method of Nystrom and Brown.9 An ether solution of III was treated with lithium aluminum hydride, suspended in ether, to give IV in good yield (65%). Experimental 3-Indoleacetic Acid (III).-Gramine (II) was prepared by the Mannich reaction on indole (I) according to the method of Kuhn and Stein.7 A mixture of 25.0 g. of gramine (II), 35.2 g. of sodium cyanide, 280 ml. of 95% ethyl alcohol and 70 ml. of water was boiled for eighty hours.To the cooled reaction mixture was added 350 ml. of water.The solution was treated with Norite, filtered, concentrated under reduced pressure until all the alcohol had been removed, cooled to 5°and filtered. The solid on the funnel (7.0 g., m. p. 145-150°) was recrystallized from alcohol and ether to give 5.0 g. of 3-indoleacetamide, m. p. 149-151 °. A sample of the amide, prepared for analysis by recrystallization from absolute alcohol and petroleum ether, was found to melt at 153 °. The amide has been previously reported to melt at 150-151 °.10>6

Fermentation

“…Various media containing meat extract, peptone, and corn-steep liquor have been described by Waksman and Schatz as suitable for the production of streptomycin (815), LePage and Campbell (119) reported that yeast extract-salts media are acceptable. Soybean meal has been demonstrated to be a suitable nutrient for streptomycin production (158,178,800).…”

“…Rake, Koerber, and Donovick {154) claim that an aqueous acid treatment will also aid in recovering streptomycin from the solids of the culture. Techniques for inducing mutations in microorganisms, which have worked so successfully in producing high yielding mutants of penicillin-producing microorganisms, have been applied to Streptomyces griseus by Savage (178). Using ultraviolet and x-ray irradiation techniques, Savage obtained mutants which gave streptomycin yields greater than 1000 y per ml.…”

“…Polymyxin. Polymyxin is a general term for a group of antibiotics produced by strains of Bacillus polymyxa (25,178,198) and includes another antibiotic originally called aerosporin (3). The polymyxins described to date, namely, A, B, C, D, and E, have similar antibacterial action but vary considerably in toxicity and are all unique in their specificity for Gram-negative bacteria (29,198).…”

Comparative Biological Studies of Polymyxin and “Aerosporin”

“…The quantitative comparison for each route of administration was based on dosagemortality curves, derived from graded doses of each drug, with groups of ten or twenty mice per dose. Results of these experiments are shown in detail in TABLE 9 and are summarized in TABLE 10.…”

Chemical Studies on Polymyxin B 1

“…It was purified by adsorption onto carbon after clarification of the fermentation liquors, elution with acid-methanol and precipitation with acetone. Further purification by Shepherd et al (6) involved butanol extraction, fractional precipitation of the picrate and conversion to the hydrochloride. Hydrolyzates of polymyxin B (7), another basic polypeptide produced by strain CN 1419 of B. polymyxa (Wellcome Foundation Culture Numbers), show the presence of D-leucine and phenylalanine as well as the three constituents common to all the polymyxins.…”