Stereoselectivity of the demethylation of nicotine piperidine homologues by Nicotiana plumbaginifolia cell suspension cultures Trixie Ann Bartholomeusz a,b,1 , Roland Molinie ´ c,1 , Albrecht Roscher b , Franc ¸ois-Xavier Felpin d , Franc ¸oise Gillet a , Jacques Lebreton d , Franc ¸ois Mesnard a, * , Richard J. Robins c, * a Laboratoire de Phytotechnologie, EA 3900, Universite ´ de Picardie Jules Verne, Faculte ´ de Pharmacie, 1 rue des Louvels, 80037 Amiens, France b Ge ´nie Enzymatique et Cellulaire, CNRS UMR6022, Universite ´ de Picardie Jules Verne, 33 rue St. Leu, 80039 Amiens, France c Laboratoire dÕAnalyse Isotopique et Electrochimique de Me ´tabolisme, CNRS UMR6006, Universite ´ de Nantes, Faculte ´ des Sciences et Techniques, 2 rue de la Houssinie `re, 44322 Nantes, France d Laboratoire de Synthe `se Organique, CNRS UMR6513, Universite ´ de Nantes, Faculte ´ des Sciences et Techniques, 2, rue de la Houssinie `re, 44322 Nantes, France Received 20 May 2005; received in revised form 11 July 2005; accepted 11 July 2005 Abstract The metabolism of (R,S)-N-methylanabasine and (R,S)-N-methylanatabine has been studied in a cell suspension culture of Nicotiana plumbaginifolia. Both substrates are effectively demethylated, anabasine or anatabine, respectively, accumulating in the medium. Similarly, there is strong stereoselectivity for the (R)-isomers of both substrates. The kinetics of metabolism of (R,S)-N- methylanabasine differ significantly from those of nicotine in that no further degradation of the initial demethylation product occurs. (R,S)-N-Methylanatabine, however, shows kinetics closer to those of nicotine, with loss of alkaloid from the system. Fur- thermore, (R,S)-N-methylanabasine does not diminish (S)-nicotine demethylation, indicating a lack of competition. However, the metabolism of (S)-nicotine is affected by the presence of (R,S)-N-methylanabasine. Hence, the demethylation of the piperidine homologues of nicotine is seen to be similar but not identical to that of the pyridine analogues. The implications of these different metabolic profiles in relation to the demethylation activity are discussed. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Nicotiana plumbaginifolia;(R,S)-N-Methylanabasine; (R,S)-N-Methylanatabine; (S)-Nicotine; Demethylation; Cell suspension culture; Chiral selectivity 1. Introduction The principal alkaloid found in most Nicotiana spe- cies is (S)-nicotine (Fig. 1), the chiral centre at C2 0 being established during the condensation of N-methylpyrro- linium ion with ÔactivatedÕ nicotinic acid. It is usually accompanied by smaller amounts of other alkaloids (Leete, 1983; Saitoh et al., 1985). While some of these – such as nornicotine, cotinine and myosmine – are bio- synthetically derived from nicotine, the extent to which the configuration at the C2 0 is conserved is variable (Armstrong et al., 1999; Leete, 1983; Mesnard et al., 2001). In a few species, such as Nicotiana alata and N. otophora, nornicotine can accumulate in roots to levels as high as, or even higher than, nicotine. 0031-9422/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2005.07.012 * Corresponding authors. Tel.: +33 3 2282 7494; fax: +33 3 2282 7469 (F. Mesnard); Tel.: +33 2 5112 5701; fax: +33 2 5112 5712 (R. Robins). E-mail addresses: francois.mesnard@u-picardie.fr (F. Mesnard), richard.robins@univ-nantes.fr (R.J. Robins). 1 These two authors participated equally in the experimental work reported in this presentation. www.elsevier.com/locate/phytochem Phytochemistry 66 (2005) 1890–1897 PHYTOCHEMISTRY