Structure and Biosynthesis of Perochalasins A–C, Open-Chain Merocytochalasans Produced by the Marine-Derived Fungus <i>Peroneutypa</i> sp. M16

Novel open-chain merocytochalasans, perochalasins A-C (<b>1</b>-<b>3</b>), containing an unusual N-O six-membered heterocyclic moiety, were isolated from cultures of the marine-derived <i>Peroneutypa</i> sp. M16 fungus, along with cytochalasin Z₂₇ (<b>4</b>), cytochalasin Z₂₈ (<b>5</b>), [12]-cytochalasin (<b>6</b>), and phenochalasin B (<b>7</b>). The structures of compounds <b>1</b>-<b>3</b> were established by analysis of the spectroscopic data. Full genome sequencing of <i>Peroneutypa</i> sp. M16 enabled the identification of a cytochalasan biosynthetic gene cluster and a proposal for the biosynthetic assembly of perochalasins. The proposal is supported by the nonenzymatic conversion of phenochalasin B (<b>7</b>) into <b>1</b>-<b>3</b>, based on isotope-labeled hydroxylamine (¹⁵NH₂OH and ND₂OD) feeding studies <i>in vivo</i> and <i>in vitro</i>. In contrast to other merocytochalasans, these are the first cytochalasans confirmed to arise via nucleophilic addition and at a distinct location from the reactive macrocycle olefin, potentially expanding further the range of merocytochalasans to be discovered or engineered. Cytochalasin Z₂₇ (<b>4</b>) exhibited antiplasmodial activities in the low micromolar range against the chloroquine-sensitive <i>Plasmodium falciparum</i> 3D7 strain as well as against resistant strains of the parasite (Dd2, TM90C6B, and 3D7r_MMV848).