Studies on the biosynthesis and metabolites of pyridine alkaloids in Nicotiana species

A GC/MS method has been developed for the routine quantification of nicotine oxidation products, 4-methylamino-1-(3-pyridyl)-1-butanone [pseudo-oxynicotine (PON)], 4-methylamino-4-(3-pyridyl)butanal (MPB), and cotinine, in tobacco using isotopic dilution assay (IDA). PON and MPB were transformed to their cyano derivatives. By using GC/MS selected ion monitoring method, the two nicotine cyano derivatives, along with cotinine, were quantified with the use of 4-trideuteromethylamino-1-(3-pyridyl)-1-butanone (2H 3-PON) was as the internal standard. The levels of PON in lamina are over 100 ppm. This indicates PON is present in sufficient quantity to be considered as the immediate precursor for the formation of NNK in tobacco.;Previous studies have shown that the pyridine ting of nicotine molecule is from nicotinic acid. To better understand the steric and electronic effects of the substitutents on the pyridine ring on the aberrant biosynthesis, several mono or disubstituted nicotinic acid derivatives were fed to hydroponically grown tobacco plants. A GC/MS method was developed for the isolation and identification of the substituted nicotines and/or other pyridine alkaloids. It was observed that 4-methyl-, 5-acetamido-, 5-bromo-, 5-chloro-, 5-iodo-, 5-methylnicotinic acids were incorporated into the corresponding substituted nicotines. Because of steric hindrance, no nicotinic acid derivatives with substituent(s) at 2- or 6-position(s) were utilized by the plants. 5-Amino- and 5-N, N-dimethylaminonicotinic acids could not be incorporated either because of electronic effects. Three new anatabine derivatives were detected in N. tabacum plant parts after feeding of 5-methylnicotinic acid. They were 5-methyl-, 5 '-methyl-, and 5, 5'-dimethylanatabine.;During the study on the feeding of 3, 5-dinicotinic acid to N. tabacum plants, a new pyridine alkaloid was isolated from both the control and treated plants. It was finally identified as 3, 5-bis(1-methyl-2-pyrrolidinyl)pyridine, or 5-(1-methyl-2-pyrrolidinyl)nicotine, or simply as 3, 5-dinicotine. A GC/MS quantitation method was developed with 5-bromonicotine as the internal standard. The highest level was found in the field grown burley roots, around 100 mug/g. Its level in the cigarette smoke was approximately 5 mug/cigarette.