Synthesis of pyrrolidine ring of nicotine from several C14-labeled metabolites by Nicotiana rustica.

The pyrrolidine ring of nicotine has been shown to be formed from ornithine, glutamic acid, and proline (l-5). Although little information is available concerning the interrelationship of these three amino acids in higher plants, Al-pyrrolinedcarboxylate (in equilibrium with glutamic semialdehyde) has been shown to be a key intermediate in their interconversion in animals and microorganisms (6, 7). Al-Pyrroline&carboxylate5-Cr4, which might, therefore, be a precursor of the pyrrolidine ring of nicotine was fed to tobacco plants. Degradation studies on radioactive nicotine isolated from these plants showed that the Cl4 present in the pyrrolidine ring was evenly distributed between carbon atoms 2 and 5 (8). When glutamic acid-2-Cl4 or ornithine-2-04 was administered to tobacco plants, the pyrroliclme ring of nicotine was also labeled nearly equally in the 2 and 5 positions (3, 4). These results suggest that in the tobacco plant glutamic acid and ornithine are converted to Alpyrroline-5-carboxylate, which upon decarboxylation gives rise to a symmetrical compound in which the 5 position of the original Al-pyrroline ring becomes equivalent to the 2 position. This symmetrical intermediate appears then to be incorporated directly into nicotine. Acetate has also been studied as a possible precursor of the pyrrolidine ring of nicotine (9). Partial degradation of nicotine from plants fed either acetate-l-04 or acetate-2-04 indicated that the location of Cl4 in the pyrrolidine ring was compatible with the conversion of acetate through the tricarboxylic acid cycle to a-ketoglutarate, the amination of cY-ketoglutarate to glutamate, and the conversion of glutamate to the pyrrolidine ring with the symmetrical compound mentioned earlier as an intermediate. The degradation procedure, however, was not complete enough to establish such a pathway with certainty. In the present study, a more complete degradation of nicotine from plants fed acetate-l-U4 for 168 hours revealed that the radioactive carbon in the pyrrolidine ring was equally distributed between carbons 2 and 5. The distribution of Cl4 in the pyrrolidine ring after feeding labeled acetate, propionate, glycerol, and aspartate for various periods of time was in agreement with the hypothesis that all of these compounds were converted by way of the tricarboxylic acid cycle to glutamate and thence to the pyrrolidine ring.