ABBOTT 29119, from Streptomyces erythreus

HUNG, P. P. (Abbott Laboratories, North Chicago, Ill.), C. L. MARKS, AND P. L. TARDREW. Isolation and characterization of a new antibiotic, ABBOTT 29119, from Streptomyces erythreus. Appl. Microbiol. 13:216-317. 1965.-A new antibiotic was isolated from fermentations of Streptomyces erythreus designed for erythromycin A production. Isolation of this compound was accomplished by use of ion-exchange chromatography and countercurrent distribution. It crystallized from methyl isobutyl ketone in colorless acicular plates which melted at 127 to 130 C. The empirical formula was C42H75NO14 with a molecular weight of 820. Electrometric titration in 66% dimethylformamide showed an apparent pKa of 8.4. Nuclear magnetic resonance, infrared, and ultraviolet absorption indicated a close resemblance of the compound to known erythromycins, although its chromatographic behavior and solubility differed from those of the known erythromycins. Its antimicrobial spectrum was similar to erythromycin A, but the minimal inhibitory concentrations were much higher. Fermentations of Streptomyces erythreus ER598 (a high erythromycin-yielding mutant isolated at Abbott Laboratories) produce erythromycin A as well as other related compounds. This paper concerns the isolation and characterization of a new antibiotic, ABBOTT 29119, from the fermentation. Chemical, physical, and biological properties of the crystalline compound are described. MATERIALS AND METHODS Production and isolation. ABBOTT 29119 was a by-product of regular fermentations designed to produce erythromycin A. Because of its greater solubility in methylene chloride, the antibiotic remained in the mother liquor when the major product, erythromycin A, was crystallized. The mother liquor was then concentrated to a syrup which solidified upon cooling. This residue contained a few per cent of the antibiotic to be isolated by the following procedures. After 1 kg of the residue was dissolved in 2 liters of methyl isobutyl ketone, the solution was extracted with an equal volume of phosphate buffer (0.1 M, pH 6.5) to remove preferentially erythromycins A and B. The extraction was repeated nine more times. The methyl isobutyl ketone solution was then chilled to -40 C, and silicon compounds in the residue were removed by filtration. The filtrate was passed through a column containing Dowex 50WX2 which was buffered at pH 6.3. The elution of the absorbed antibiotics was carried out with an ammonium chloride solution (5%, pH 8.3). Compounds in the eluates were extracted into chloroform, and the extracts were evaporated to dryness. About 10 g of crude ABBOTT 29119 were obtained in a typical run. The crude antibiotic was further purified by countercurrent distribution by use of the two-phase system of Pettinga, Stark, and Van Abeele (1954); it was separated from residual erythromycins A and B and was contained in tubes 95 to 120 in a 120transfer run. Upon concentration of the combined organic phase, the new antibiotic crystallized as colorless acicular plates. Recrystallization was accomplished by dissolving the antibiotic in warm ethyl alcohol to which cold water was added slowly until the haze point. Crystals formed on standing at room temperature. RESULTS AND DISCUSSION Crystalline ABBOTT 29119 melts between 127 and 130 C and is virtually insoluble in water and soluble in common organic solvents. The recrystallized sample was ash-free and gave the following elemental analysis for C42H75NO14. Calculated: C, 61.70; H, 9.18; 0, 27.40; N, 1.72. Found: C, 61.85; H, 9.27; 0, 26.30, 27.11; N, 1.78. Titration of the crystalline antibiotic in 66% dimethylformamide showed an apparent pKa of 8.4 and equivalent weight of 820. X-ray diffraction on a single crystal provided the following information: space group, C2H-P21/m; lattice dimension, a = 18.74A, b = 19.69A, c = 13.76A, ,B = 107.09'; density, 1.126 g/cm3; and molecular weight, 822.5. 216 on O cber 6, 2017 by gest ht://aem .sm .rg/ D ow nladed fom NEW ANTIBIOTIC FROM STREPTOMYCES ERYTHREUS