Objective  To study the tautomerism of ascomycin in polar solvents, and isolate, purify and identify the structure of tautomeric compound of ascomycin.

Methods  The equilibrium of ascomycin and its tautomers in acetonitrile and acetonitrile-water solutions was analyzed by reversed-phase HPLC. The separation and purification of isomer was achieved through preparative HPLC to obtain high-purity samples. The structure was confirmed by high-resolution mass spectrometry and 2D nuclear magnetic resonance.

Results  Two tautomeric compounds were detected in acetonitrile and acetonitrile-water solutions, and the tautomeric equilibrium of ascomycin shifts faster remarkably in acetonitrile-water solution than acetonitrile. The structure of tautomeric compound of ascomycin was (3S,4R,5S,8R,9E, 12S,14S,15R,16S,18R,19S,26aS)-8-Ethyl-5,6,8,11,12,13,14,15,16,17,18,19, 24,25,26,26a-hexadecahydro-5,19-dihydroxy-3-{(E)-2-[(1R,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylvinyl}-14,16-dimethoxy-4,10,12,18-tetramethyl-15,19-epoxy-3H-pyrido[2,1-c][1,4]oxaazacyclotricosine-1,7,20,21-(4H,23H)-tetrone.

Conclusion  The tautomer of ascomycin is primarily the C19 epimer in polar solvents. The method about preparation of ascomycin epimer compound by reverse phase HPLC is effective and efficient. This study provides a basis for establishing the quality standard of ascomycin.

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