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The inherent structural instability of some physalins has hampered the isolation and identification of these compounds for approximately 50 years, and an effective method to overcome these challenges remains unavailable. In the present study, the unprecedented tautomerization mechanism of unstable physalins was elucidated by performing isotopic labeling experiments and DFT calculations, which led to the successful separation of tautomers and isolation of highly pure products for the first time. As a result, 15 new physalins, physaminins A-O (