FULL PAPER DOI: 10.1002/ejoc.200700455 Hemivannusal and Prevannusadials – New Sesquiterpenoids from the Marine Ciliate Protist Euplotes vannus: The Putative Biogenetic Precursors of Dimeric Terpenoid Vannusals Graziano Guella,* [a] Emanuela Callone, [a] Graziano Di Giuseppe, [b] Rita Frassanito, [a] Francesco P. Frontini, [b] Ines Mancini, [a] and Fernando Dini [b] Keywords: Terpenoids / Marine ciliates / Structure elucidation / Ab initio calculations / Endosymbionts Hemivannusal (6), a bicyclic sesquiterpenoid with an unprec- edented skeleton, has been isolated from the marine ciliate Euplotes vannus (strain TB6). The relative configuration of 6 has been established through an extended conformational search performed by molecular mechanics and refined through ab initio quantum chemical calculations. Another strain (CM1) of the same morphospecies has been found to produce the linear prevannusadial A (7) and the monocyclic prevannusadial B (8), which could represent key intermedi- ates for the biosynthesis both of hemivannusal (6) and of the C30 terpenoids vannusals A and B (5a and 5b), the latter pair being metabolites isolated from conspecific strains (Sil21 and Introduction Massive cell cultures of unicellular marine ciliates of the genus Euplotes (Ciliophora) have been demonstrated to be a rich source of secondary metabolites (Scheme 1). In par- ticular, the cosmopolitan morphospecies Euplotes crassus has yielded highly strained acetylated sesquiterpene hemia- cetals (euplotane skeleton). Among them, euplotin C (1), [1] the structure of which has been characterized up to the ab- solute configuration level, [1c] has been demonstrated to be strongly bioactive, as it: 1) is able to kill, at the μ level, strains of euplotin nonproducing Euplotes , [1a] 2) has cyto- toxic effects on free-living and parasitic protozoa, yeasts, and bacterial strains, [2] and finally 3) shows remarkable cytotoxic and apoptotic inducing action towards mouse (AtT-20) and rat (PC12) tumor cell lines. [3] Other terpenoids with unprecedented new skeletons have also been isolated from representatives of the genus Eu- plotes . Among sesquiterpenoids, we cite here raikovenal (2a), [4a] isolated from Euplotes raikovi and built around a [a] Laboratorio di Chimica Bioorganica, Dipartimento di Fisica, Università di Trento, 38050 Povo-Trento, Italy Fax: +39-461-882009 E-mail: guella@science.unitn.it [b] Dipartimento di Biologia, Università di Pisa, Pisa, Italy Supporting information for this article is available on the WWW under http://www.eurjoc.org or from the author. © 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Eur. J. Org. Chem. 2007, 5226–5234 5226 BUN3) of the same E. vannus morphospecies. The occurrence of hemivannusal (6) in mass cell cultures both of an E. vannus endosymbiont-free strain and of a penicillin-treated E. vannus bacteria-infected strain strongly suggests that its biosynthesis is coded by the ciliates and not by prokaryotic endosymbiont genes. The pattern of the secondary metabo- lites isolated so far from different strains of E. vannus allows us not only to assign them a chemotaxonomic significance, but also to propose a unifying picture of their biogenesis. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2007) Scheme 1. Sesquiterpenoids isolated from marine ciliates of the ge- nus Euplotes . bicyclo[3.2.0]heptane ring system, and rarisetenolide (3a), [5] isolated from Euplotes rariseta and containing an octahyd- roazulene moiety. Among diterpenoids, focardin (4), [6] first obtained from an Antarctic strain of Euplotes focardii, de- serves special mention since it represents a reliable taxo- nomic marker of the morphospecies, characterizing all pop-