Lienhwalides: Unique Tropolone–Maleidride Hybrids from <i>Hypoxylon lienhwacheense</i>

Hypoxylon lienhwacheense, a fungal species with an unclear taxonomic placement within the Hypoxylaceae, presents a highly rare stromatal secondary metabolite profile. Isolation of its major stromatal constituents leads to the discovery of a novel tropolone-maleidride hybrid molecule, lienhwalide A 5, in addition to the known cordyanhydride B 6, its new derivative 7, and binaphthalenetetraol 8. Unexpectedly, Hypoxylon lienhwacheense produces in liquid cultures various lienhwalide A congeners 9-11. Their structures and relative configurations are elucidated using high-resolution mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy, with their absolute configurations determined using X-ray analysis of a semisynthetic brominated derivative of 9 and synthesizing α-methoxy-α-trifluoromethylphenylacetyl esters of 11. Feeding experiments with ¹³C-labeled precursors (¹³C-methionine; 1-¹³C- and U-¹³C₆-glucose) reveal insights into the biogenesis of tropolone and maleidride moieties, according to ¹³C couplings and incredible natural abundance double quantum transfer NMR data. Genome analysis identifies two separate biosynthetic gene clusters responsible for these moieties, and heterologous expression experiments provide further insights into the interplay of both clusters during the biosynthesis of these hybrid natural products. Remarkably, lienhwalides exhibit reduced toxicity and enhance antibacterial selectivity compared to related fungal tropolones.