Mechanism of nicotine N-demethylation in tobacco cell suspension cultures

Radioactive feeding experiments, in which DL-[pyrrolidine-2'-C-14]-nicotine was added to 10-day-old cultures, confirmed that the kinetic pattern of this N-demethylation was similar to that of non-radioactive nicotine, and that nornicotine, the major product, was produced intracellularly with a maximum percentage conversion of about 70%. The appearance of nornicotine paralleled the disappearance of the added nicotine, although small amounts of radioactive metabolites other than nornicotine were also observed. One of these metabolites was tentatively identified by GC-MS as N-formyl-3'-nornicotine which is probably a side-product rather than an intermediate in nicotine N-demethylation. Nornicotine produced from added (-)-nicotine by the cultures was determined using both polarimetry and chiral GC, and shown to be exclusively one enantiomer. This provided strong evidence that the mechanism of this reaction in cultured cells was unlikely to involve opening of the pyrrolidine ring and, therefore, differed from that previously hypothesized for the tobacco plant. The possible mechanism for this reaction is also discussed.