DEVELOPMENT OF NEW CHROMATOGRAPHY METHOD FOR SEPARATION OF HEPTAENE MACROLIDE ANTIBIOTIC HAMYCIN COMPONENTS

Valery Belakhov, Schulich Faculty of Chemistry, Technion - Israel Institute of Technology, Haifa, Israel (chvalery@technion.ac.il)

Enter the body of your abstract hereHamycin is a heptaene macrolide antibiotic discovered and developed at the Research Centre of Hindustan Antibiotics Ltd (Pimpri, Pune, India).  It has been introduced into the Indian market exclusively for agricultural use as a potent fungicide. Hamycin is produced by Streptomyces pimprina Thirum., and available only as a complex mixture of four components. In order to separate two major components hamycin A and hamycin B for study of their biological activity against various phytopathogenic fungi and harmful arthropods, we developed a new chromatography method.

The developed chromatography method for the separation of complex of hamycin consists of two stages. At the first stage, complex of hamycin (1.0 g) was dissolved in 5 mL methanol, and solution was loaded on the silica gel 60 column (Fluka, 50 x 2.5 cm) after filtration. Approximately, 50 g of silica gel was packed into the column and the total volume was 130 mL. The column was eluted with methanol-chloroform (5:3, v/v) at a flow rate of about 1.2 mL/min, and the effluent was then collected in each 10 mL fraction. All fractions with complex of this antifungal antibiotic were combined and concentrated under reduced pressure at 40^oC. Thus, all admixtures were removed from complex of hamycin. At the second stage, the earlier purified complex of hamycin was additionally purified and separated by the preparative HPLC using various Venusil XBP (L) columns at 30^oC. The mobile phase was methanol and water in the gradient mode as follows: 0-30 min, 60 % methanol and 40-55 min, 65 % methanol. The following rate was 5 mL/min, and effluent was continuously monitored at 384 nm. The fractions containing components of hamycin A and hamycin B were collected, concentrated and analyzed by NMR-, IR-, UV- and mass-spectroscopy.