Biosynthesis of antimalarial lignans from Holostylis reniformis Gisele B. Messiano, Tito da Silva, Isabele R. Nascimento, Lucia M.X. Lopes * Instituto de Química, São Paulo State University, UNESP, C.P. 355, 14801-970 Araraquara, SP, Brazil article info Article history: Received 16 October 2007 Received in revised form 27 June 2008 Available online 21 March 2009 Keywords: Holostylis reniformis Aristolochiaceae Lignans Neolignans Aryltetralone lignans Epoxylignans Biosynthesis Propenylphenols abstract Holostylis reniformis biosynthesizes 8-8 0 linked lignans without 9,9 0 -oxygenation. To elucidate the biosyn- thetic pathways to these lignans, the reputed precursors [U- 14 C]phenylalanine, [9- 3 H 1 ]coniferyl alcohol, and [9- 3 H 1 ]isoeugenol were administered to roots of the plant, which led to the incorporation of 3 H and 14 C into ten 2,7 0 linked-lignans (aryltetralone lignans) and two 7,7 0 -epoxylignans (furan lignans). These administration experiments demonstrated that the lignans were propenylphenol-derived and that H. reniformis can exhibit regioselective control over radical–radical coupling (via isoeugenol radicals). Reg- iospecific control over propenylphenol-derived lignan biosynthesis was observed, together with diaste- reoselective control of C2–C7 0 bond formation for the aryltetralone lignans (7 0 R). These experiments provide evidence that isoeugenol is a biosynthetic intermediate to the aryltetralone and furan lignans. Ó 2009 Elsevier Ltd. All rights reserved. 1. Introduction Lignans are a very structurally diverse class of vascular plant natural products. They are typically dimers and/or higher oligo- mers (Moss, 2000). Lignans vary substantially in oxidation level, substitution pattern, and the chemical structure of their basic car- bon framework. In addition to structural diversity, lignans show considerable diversity in terms of enantiomeric composition, bio- synthesis, and phylogenetic distribution (Umezawa, 2003). Based on structural (chemotaxonomical) differences, lignans can be read- ily separated into distinct coupling-type subclasses (Gottlieb, 1978; Umezawa, 2003; Davin and Lewis, 2005). An examination of their corresponding structures and distribution in nature illus- trates the existence of a large number of distinct phenoxyl radi- cal–radical coupling modes, including those that are either stereoselective and/or regiospecific in coupling origin (Davin and Lewis, 2005). The first demonstration of phenoxyl radical–radical coupling control was reported during the investigation of (+)- pinoresinol formation from coniferyl alcohol in Forsythia species (Paré et al., 1994; Davin et al., 1997). The dirigent protein was pro- posed to bind and orient coniferyl alcohol-derived radicals in such a way as to enable 8,8 0 coupling at the si–si face with subsequent intramolecular cyclization to afford (+)-pinoresinol (Davin and Le- wis, 2005). The biosyntheses of chavicol and eugenol have been shown to occur via the phenylpropanoid pathway to p-coumaryl alcohol. Activation of the side-chain alcohol of p-coumaryl and coniferyl alcohols, e.g. via esterification, to form a more facile leaving group via reductive elimination has been demonstrated (Koeduka et al., 2006; Vassão et al., 2006). This was shown to be the case using p-coumaryl/coniferyl esters as potential substrates for a NAD(P)H-dependent reductase to afford chavicol and eugenol. Lignans present in Larrea tridentata demonstrate an additional means of control of radical–radical coupling. Instead of being of monolignol origin, these lignans are derived from propenyl/ allylphenols (presumably from anol) (Davin and Lewis, 2003, 2005). Pathways for the formation of aryltetralin lignans with oxygenation at C-9,9 0 have been proposed. The overall pathway for the formation of podophyllotoxin, for example, has been shown to involve coniferyl alcohol, (+)-pinoresinol, (+)-mataires- inol, yatein, and deoxypodophyllotoxin (Seidel et al., 2002; Fuss, 2003). Lignans (1–19) and neolignans (20 and 21) have been iso- lated from the roots of H. reniformis (da Silva and Lopes, 2004, 2006; Andrade-Neto et al., 2007)(Fig. 1). Of these, lignans 6, 12, 13, and 15 have attracted much interest due to their antiplasmodial activity (da Silva et al., 2004; Andrade-Neto et al., 2007). All of these lignans and neolignans are apparently derived from propenylphenols and lack the oxygenated func- tionalities at C-9,C-9 0 which are characteristic of many other phenylpropanoids. The aim of this work was to contribute to the elucidation of the biosynthetic pathways of lignans without oxygenation at C-9,C-9 0 especially those from H. reniformis, by in vivo conversion of phenyl- propanoid intermediates into aryltetralone lignans. 0031-9422/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2009.02.008 * Corresponding author. Tel.: +55 16 3301 6663; fax: +55 16 3301 6692. E-mail address: lopesxl@iq.unesp.br (L.M.X. Lopes). Phytochemistry 70 (2009) 590–596 Contents lists available at ScienceDirect Phytochemistry journal homepage: www.elsevier.com/locate/phytochem