Nicotine demethylation in Nicotiana cell suspension cultures: N′-formylnornicotine is not involved

• Published in: Phytochemistry (Oxford) Vol. 66; no. 20; pp. 2432 - 2440
• Main Authors: Bartholomeusz, Trixie Ann, Bhogal, Ramneek K., Molinié, Roland, Felpin, François-Xavier, Mathé-Allainmat, Monique, Meier, Anna-Carolin, Dräger, Birgit, Lebreton, Jacques, Roscher, Albrecht, Robins, Richard J., Mesnard, François
• Format: Journal Article
• Language: English
• Published: OXFORD Elsevier Ltd 01.10.2005; Elsevier
• Subjects: alkaloids; Alkaloids - metabolism; Biochemistry & Molecular Biology; Biological and medical sciences; Cell Culture Techniques - methods; cell suspension culture; Cells, Cultured; Chemical constitution; cotinine; Cotinine - metabolism; cultured cells; Demethylation; Fundamental and applied biological sciences. Psychology; Gas Chromatography-Mass Spectrometry; isotope labeling; Life Sciences & Biomedicine; Methylation; myosmine; N'-carboethyoxynornicotine; N'-formylnornicotine; Nicotiana - cytology; Nicotiana - metabolism; Nicotiana plumbaginifolia; Nicotine; Nicotine - analogs & derivatives; Nicotine - metabolism; Nitrogen Isotopes - metabolism; Nornicotine; plant biochemistry; plant physiology; Plant physiology and development; Plant Sciences; Science & Technology; Solanaceae; Tissue cultures, protoplasts; tobacco; Demethylation; Nicotiana plumbaginifolia; Solanaceae; Nornicotine; N′-Formylnornicotine; Nicotine; demethylation; OXIDATION; TOBACCO ALKALOIDS; nicotine; METABOLISM; DEGRADATION; nornicotine; N '-formylnornicotine; Cell culture; Incorporation; Metabolite; Metabolism; Gas chromatography; Alkaloid; Suspension culture; Dicotyledones; Angiospermae; Spermatophyta; N'-Formylnornicotine
• ISSN: 0031-9422; 1873-3700
• DOI: 10.1016/j.phytochem.2005.07.005

Label from [ 13C, 2H 3-methyl]nicotine fed to Nicotiana plumbaginifolia suspension cell cultures is incorporated into cotinine but to a much lesser extent into N′-formylnornicotine. This labelling pattern directly shows that N′-formylnornicotine is not intermediate in the demethylation mechanism, but is formed by the condensation of nornicotine with formaldehyde. Nicotine or nornicotine enriched with stable isotopes in either the N′-methyl group or the pyrrolidine-N were fed to Nicotiana plumbaginifolia suspension cell cultures that do not form endogenous nicotine. The metabolism of these compounds was investigated by analysing the incorporation of isotope into other alkaloids using gas chromatography–mass spectroscopy (GC–MS). Nicotine metabolism primarily resulted in the accumulation of nornicotine, the N′-demethylation product. In addition, six minor metabolites appeared during the course of nicotine metabolism, four of which were identified as cotinine, myosmine, N′-formylnornicotine and N′-carboethoxynornicotine. While cotinine was formed from [ 13C, 2H 3-methyl]nicotine without dilution of label, N′-formylnornicotine was labelled at only about 6% of the level of nicotine and N′-carboethoxynornicotine was unlabelled. Feeding with [1′- 15N]nornicotine resulted in incorporation without dilution of label into both N′-formylnornicotine and N′-carboethoxynornicotine. This pattern strongly indicates that, while nornicotine and cotinine are derived directly from nicotine, N′-formylnornicotine and N′-carboethoxynornicotine are metabolites of nornicotine. Thus, it is directly demonstrated that N′-formylnornicotine is not an intermediate in nicotine demethylation.