Separation of WAP-8294A components, a novel anti-methicillin-resistant staphylococcus aureus antibiotic, using high-speed counter-current chromatography.

The WAP-8294A complex was isolated from the fermentation broth of Lysobacter sp. WAP-8294, whose major component, WAP-8294A2, showed a strong activity against Gram-positive bacteria including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci in vitro, and also exhibited a potent activity against MRSA in vivo. The previous separation procedure using the conventional chromatographic methods was laborious and time-consuming, and the recovery of the desired compound was often unsatisfactory. In the present study, high-speed counter-current chromatography (HSCCC) was applied to the separation of the main components of the WAP-8294A complex. Due to the high polarity of the target compounds, we selected a hydrophilic two-phase solvent system composed of n-butanol-ethyl acetate-aqueous 0.005 M trifluoroacetic acid (1.25:3.75:5, v/v/v) which provided a suitable range of partition coefficient values for these compounds. Although the settling time of this solvent system was much longer than the optimum range, suggesting a low retention of the stationary phase under the standard experimental conditions, the separation was successfully performed at the low flow-rate of 0.5 ml/min. A sample size of 25 mg yielded pure fractions of three components (1-6 mg). The identification of each component was carried out by HPLC and fast atom bombardment mass spectrometry. The method will contribute to the clinical development of WAP-8294A2 as an anti-MRSA agent.