Development of New Chromatography Method for Separation of Tetraene Macrolide Antibiotic Tetramycin Components

Valery V. Belakhov, Schulich Faculty of Chemistry, Technion, Haifa, Israel (chvalery@technion.ac.il)
Alexander V. Garabadzhiu, Laboratory Of Molecular Pharmacology, Saint-petersburg State Technological Institute (technical University), Saint-petersburg, Russia

Antifungal antibiotic tetramycin B discovered by German researchers is a member of tetraene macrolide antibiotics family. Tetramycin, as a complex of two components tetramycin A and tetramycin B, was prepared via the microbiological synthesis by Streptomyces noursei as the producer. Chemical modification of polyene macrolide antibiotics is known to afford less toxic derivatives with improved chemotherapeutical properties and extended range of biological activity. In order to separate complex of tetramycin for two components, such as tetramycin A and tetramycin B, followed by using them for chemical modification, we developed a new chromatography method.

The developed chromatography method for the separation of complex of tetramycin consists of two stages. At the first stage, complex of tetramycin (1.5 g) was dissolved in 6 mL methanol, and solution was loaded on the silica gel 60 column (Fluka, 65 x 2.7 cm) after filtration. Approximately, 50 g of silica gel was packed into the column and the total volume was 120 mL. The column was eluted with methanol-chloroform (3:2, v/v) at a flow rate of about 1.5 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 tetramycin. At the second stage, the earlier purified complex of tetramycin 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 30-45 min, 65 % methanol. The following rate was 7 mL/min, and effluent was continuously monitored at 304 nm. The fractions containing components of tetramycin A and tetramycin B were collected, concentrated and analyzed by NMR-, IR-, UV- and mass-spectroscopy.