The biosynthetic origin of oxygen functions in phenylphenalenones of Anigozanthos preissii inferred from NMR- and HRMS-based isotopologue analysis

The biosynthetic origin of 9-phenylphenalenones and the sequence according to which their oxygen functionalities are introduced were studied using nuclear magnetic resonance (NMR) spectroscopy and high-resolution electrospray ionization mass spectrometry (HRESIMS). (13)C-labelled precursors were administered to root cultures of Anigozanthos preissii, which were simultaneously incubated in an atmosphere of (18)O(2). Two major phenylphenalenones, anigorufone and hydroxyanigorufone, were isolated and analyzed by spectroscopic methods. Incorporation of (13)C-labelled precursors from the culture medium and (18)O from the atmosphere was detected. O-Methylation with (13)C-diazomethane was used to attach (13)C-labels to each hydroxyl and thereby dramatically enhance the sensitivity with which NMR spectroscopy can detect (18)O by means of isotope-induced shifts of (13)C signals. The isotopologue patterns inferred from NMR and HRESIMS analyses indicated that the hydroxyl group at C-2 of 9-phenylphenalenones had been introduced on the stage of a linear diarylheptanoid. The oxygen atoms of the carbonyl and lateral aryl ring originated from the hydroxyl group of the 4-coumaroyl moiety, which was incorporated as a unit.