Biosynthesis

BY R. B. HERBERT

Terpenoid lndole Alkaloids. — Monoterpenes have been the subject of a comprehensive review which includes these alkaloids. Indole alkaloids have been classified according to their biogenesis and another review discusses the application of tritium labelling in biosynthetic studies on these alkaloids as well as several others.

An interesting new technique has been applied to the study of indole alkaloid biosynthesis: the alkaloids in Vinca rosea seedlings were examined after the administration of DL-[2'-14C]tryptophan (ca. 30% incorporation) and the appearance and disappearance of radioactivity noted as a function of time. The technique is thus similar to the widely used method of 14CO2 feeding. The results were in accord with those obtained earlier by precursor feeding. In particular, geissoschizine (3), preakuammicine (5), and tabersonine (7) were confirmed as important ('dynamic') intermediates in the biosynthesis of other alkaloids, whereas ajmalicine (4), catharanthine (8), coronaridine (9), and vindoline (10) appeared as end-products of biosynthesis. An unknown alkaloid was the first to be labelled in the experiment. It showed rapid turnover and is thus an important precursor for other alkaloids. It appeared that it could be (2), lying between vincoside (1) and geissoschizine (3).

The precursor role of tabersonine (7) for the Aspidosperma and Iboga alkaloids was further strengthened by feeding radioactive tabersonine to V rosea and examining the products as for tryptophan. It was suggested, in view of the large differences of incorporation observed for the two classes of alkaloid, that tabersonine might be formed as the racemate, one enantiomer of which [the ( -)-form] would give vindoline (10) and the other [the unknown (+)-form] catharanthine (8) and coronaridine (9).

Stemmadenine (6) did not show the characteristics, in these experiments, of a dynamic intermediate. This could be explained, however, as the consequence of an equilibration with preakuammicine (5) which is enzyme bound, or if stemmadenine is a stabilized form of preakuammicine (5), and is involved in biosynthesis via (5).

The secondine group of alkaloids are interesting biosynthetically as derivatives of (11) or (12), which are regarded as likely intermediates in the biosynthesis of indole alkaloids with rearranged monoterpenoid units. Accordingly, 16,17-dihydrosecodin-17-ol (13) and secodine (14) have been tested as precursors for uleine (15), apparicine (16), vincamine (17), minovine (18), vindoline (10), and catharanthine (8), as well as ajmalicine (4), which has an unrearranged unit. All incorporations were similarly low except for those for secodine into vindoline (10) in Vinca rosea and apparicine (16) in Aspidosperma pyricollum, which were significantly higher. When [carboxy-14C; ar-3H]secodine was used, both of these alkaloids were formed without change in isotope ratio, indicating intact incorporations of the precursor. Degradation of the vindoline showed that the 14C label was located at C-14 and thus, reasonably, the methoxycarbonyl group of secodine (14) was transferred as such into vindoline (10). [carboxy-14C; 19-3H]Secodine was incorporated without change in isotope ratio. On the other hand, [carboxy-14C;3,14,15, 21-3H4]secodine was converted into vindoline with loss of 60% of the tritium label, consistent with its involvement in vindoline biosynthesis via the more highly oxidized (11) or (12). The same precursor was incorporated into apparicine (16) and although the results are regarded as preliminary, the methoxycarbonyl carbon appears to be retained ; tritium was lost (48%) from the piperidine ring, again consistent with involvement of an intermediate similar to that which yields vindoline.

Two new acidic terpenes have been isolated from V. rosea and rigorously characterized as secologanic acid (19) and secologanoside, which was studied as its methylation-acetylation product (21). [14C]Loganic acid (22) was efficiently incorporated into secologanic acid in V. rosea as well as secologanin (20) and loganin (23). A similar efficient conversion of loganin into secologanic acid was recorded. Taking into account the purification of a methyltransferase from V. rosea capable of methylating loganic and secologanic acids, these results suggest a role for these acids in indole alkaloid biosynthesis, and the incorporation of sweroside (24) into the alkaloids may be via secologanic acid (19). Sweroside also serves as a precursor for reserpinine and quinine.

The terpenoid moieties of indole alkaloids have been proved to arise from mevalonate. Although labelled acetate can be specifically incorporated into many terpenoid compounds via mevalonate1 it is not a specific precursor for the indole alkaloids. This has prompted a search for a source other than acetate. When intermediates of glycolysis and the Krebs cycle, leucine, and glycollic acid were tested as precursors for cephaeline (25) in Cephaelis acuminata, randomization of the labels was observed. 17 [2-14C]Glycine, however, was incorporated with specificity; cephaeline (25) was labelled at C-16 but not C-15. In Rauwolfia serpentina, ajmaline (26) was labelled at C-18 but not C-19 and reserpine predominantly in the reserpic acid moiety. The labelled positions showed 15 — 18% of the total activity compared with 20% calculated for utilization of glycine as a C2 unit via mevalonate (Scheme 1). The results for cephaeline were obtained in the summer with four-five-year-old plants. Winter feeding to two-year-old plants, however, gave non-specific incorporation into the terpenoid portions of cephaeline and ipecoside. Similar non-specific incorporation of glycine has been observed for vindoline, ajmalicine, and the Strychnos alkaloids. On the other hand, specific incorporation of [2-14C]glycine into gentianine (27) has been recorded. L-Leucine has been found to be incorporated into vindoline and catharanthine as inefficiently as acetate. Inhibition of protein synthesis in the plant by puromycin caused a measurable but not dramatic increase in leucine incorporation into the alkaloids.

Ergot. —...