Biosynthesis of tetraoxygenated phenylphenalenones in Wachendorfia thyrsiflora.

The biosynthetic origin of 1,2,5,6-tetraoxygenated phenylphenalenones and the sequence according to which their oxygen functionalities are introduced during the biosynthesis in Wachendorfia thyrsiflora were studied using two approaches. (1) Oxygenated phenylpropanoids were probed as substrates of recombinant W. thyrsiflora polyketide synthase 1 (WtPKS1), which is involved in the diarylheptanoid and phenylphenalenone biosynthetic pathways, (2) Root cultures of W. thyrsiflora were incubated with (13)C-labelled precursors in an (18)O2 atmosphere to observe incorporation of the two isotopes at defined biosynthetic steps. NMR- and HRESIMS-based analyses were used to unravel the isotopologue composition of the biosynthetic products, lachnanthoside aglycone and its allophanyl glucoside. Current results suggest that the oxygen atoms decorating the phenalenone tricycle are introduced at different biosynthetic stages in the sequence O-1→O-2→O-5. In addition, the incubation of W. thyrsiflora root cultures with (13)C-labelled lachnanthocarpone established a direct biosynthetic precursor-product relationship with 1,2,5,6-tetraoxygenated phenylphenalenones.

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.

O-Methylation of phenylphenalenone phytoalexins in Musaacuminata and Wachendorfia thyrsiflora.

Biosynthetic O-methylation at various sites along the backbone of inducible phenylphenalenones in Musaacuminata var. "Williams" (Musaceae) and Wachendorfiathyrsiflora (Haemodoraceae) was investigated using 13C-labelled precursors. The inducibility of O-methylated metabolites was demonstrated in both species and the origin of methoxyl group from [methyl-13C]L-methionine was confirmed. In addition to known phenylphenalenones, a methoxylated metabolite, 4-(4-hydroxy-3-methoxy-phenyl)-benzo[de]isochromene-1,3-dione, was detected and its structure elucidated mainly by NMR spectroscopic techniques. The experiments were used to discriminate methionine-derived and artificial methoxy groups formed during methanolic extraction. Finally, demethylation of 4'-methoxycinnamic acid and subsequent conversion to 3',4'-methylenedioxycinnamic acid was demonstrated in M.acuminata.