Biosynthesis of unusual monocyclic alkenes by the diatom Rhizosolenia setigera (Brightwell) G. Masse´ a,b , S.T. Belt a, *, S.J. Rowland a a Petroleum & Environmental Geochemistry Group, University of Plymouth, Drake Circus, Plymouth PL4 8AA, UK b ISOMer, Faculte ´ des Sciences et des Techniques, Universite ´ de Nantes, 2, Rue de la Houssinie `re, 44027 Nantes, Cedex 03, France Received 14 October 2003; accepted 25 February 2004 Abstract Novel, polyunsaturated monocyclic sester- and triterpenes isolated from the diatom Rhizosolenia setigera (Brightwell), are biosynthesised mainly via the mevalonate pathway. The experiments involved incubation of the alga with [1- 13 C]acetate, isolation of the alkenes by extraction and silver ion HPLC, followed by determination of the labelling pattern of one of the monocyclic triterpenes by 13 C-NMR spectroscopy. In addition, the extent of 13 C incorporation was also measured by mass spectrometry which revealed that the involvement of the mevalonate route in the biosynthesis of these cyclic compounds was less than for the co-occurring acyclic highly branched isoprenoid alkenes. # 2004 Elsevier Ltd. All rights reserved. Keywords: Rhizosolenia setigera; Bacillariophyceae; Diatoms; Isoprenoid alkenes; mevalonate; biosynthesis; monocyclics 1. Introduction The widespread planktonic diatom Rhizosolenia setigera (Brightwell), biosynthesises a number of straight-chain (Sinninghe Damste´ et al., 1999, 2000; Rowland et al., 2001a), highly branched isoprenoid (HBI; Volkman et al., 1994, 1998; Belt et al., 2001; Rowland et al., 2001a) and monocyclic alkenes (Belt et al., 2003). Representative structures of these three groups of compounds are shown in Fig. 1. In culture, factors such as salinity, temperature, algal strain and position in the life cycle all influence the HBI and cyclic alkene distributions, and these have been described in detail elsewhere (Rowland et al., 2001a; Belt et al., 2002). Biosynthesis of the HBI alkenes, which have unknown function in the alga, has been studied by a series of experiments involving selective pathway inhibition, 13 C and 2 H isotopic incorporation from labelled precursors, determination of labelling patterns by NMR spectroscopy, and measurement of the 13 C/ 12 C isotope ratios of HBIs isolated from algae grown under natural conditions (Masse´ et al., 2004). The HBIs and sterols are biosynthesised under these conditions, mainly by the well-known mevalonate (MVA) pathway (e.g. Rohmer, 1999). This is in contrast to plastidic phytol (from chlorophyll a) which is produced by the methylerythritol (MEP) route. This divergence of pathways for presumed cytosolic and known plastidic isoprenoids is similar to that observed in some other diatoms (Cvejic´ and Rohmer, 2000). However, it was noted previously that the (then unknown) cyclic alkenes produced by Rhizosolenia setigera had 13 C/ 12 C ratios which were somewhat isotopically distinct from both the HBI alkenes and from phytol under all culture conditions (Rowland et al., 2001a). For example, compared with phytol, the HBI and cyclic alkenes were consistently 2% and 1% depleted in 13 C respectively. Subsequently the cyclic alkenes were identified as V–VII by isolation and characterisation by NMR spectroscopy (Belt et al., 2003). The structures of the cyclic alkenes are thus unrelated to the HBI alkenes. The former may reasonably be assumed to be biosynthesised from coupling of a geranyl (C 10 ) moiety to either a farnesyl (C 15 ) or geranylgeranyl unit (C 20 ) at C-7 (Fig. 2) to yield C 25 and C 30 compounds respectively. By contrast, the HBI alkenes are likely biosynthesised via coupling of geranyl and farnesyl or two farnesyl units at C-6 (Fig. 2). 0031-9422/$ - see front matter # 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2004.02.019 Phytochemistry 65 (2004) 1101–1106 www.elsevier.com/locate/phytochem * Corresponding author: Tel.: +44-1752-233042; fax: +44-1752- 233035. E-mail address: s.belt@plymouth.ac.uk (S.T. Belt).