BIOSYNTHESIS OF OPIUM ALKALOIDS

* * * * * * * * 14C-Tyrosine fed to opium poppy seedlings Two labels are incorporated into papaverine and norlaudanosoline, implying that two tyrosines are used. 14C-Tyrosine fed to opium poppy seedlings * hydroxylase tyrosine O2 If labeled DOPA is fed only one label is found. * PLP DOPA PLP * * Mannich + Arom. Subst. dopamine * * thebaine SAM * * FAD codeine papaverine norlaudanosoline morphine (in all poppies)

14CO2 FEEDING EXPERIMENT This order was established by growing the poppies in a greenhouse with 14CO2 and watching the order of appearance and buildup of the the radioactivity At first the rate of incorporation is greater for thebaine than for codeine or morphine. After several days, the rate of incorporation into morphine is greatest. This indicates that thebaine is formed first, and that codeine and morphine follow in order. norlaudanosoline thebaine The same result is obtained with carbon-14 labeled tyrosine. codeine morphine Also, labeled thebaine is converted to both codeine and morphine, but not the other way around. Finally, codeine is demethylated to morphine; the reverse is not observed.

* * * * * * * * * * redrawn (folded) on the next slide ….. SAM FAD norlaudanosoline laudanosine papaverine SAM Norlaudanosoline is incorporated into papaverine and thebaine at a higher rate than tyrosine - it’s closer. * * redrawn (folded) on the next slide ….. * * SAM In a separate set of experiments, it can be shown that the methyl groups originate from methionine (14C labeled CH3) and hence SAM. laudanosoline (-)-reticulene

. . MULTIPLE LABELS * * * * not an easy synthesis ! thebaine (all labels intact !) (-)-reticulene (5 labels) not an easy synthesis ! When multiple labels survive intact it is a good indication that the incorporation study is a very valid one!

. . . . OXIDATIVE COUPLING * * * . . * * * * * [O] coupling enolization (-)-reticulene (5 labels) * . * salutaridine

. . . * * * * * * ORIGINAL HYPOTHESIS (ALA PUMMERERS KETONE) THEBAINE IT MISLEAD THEM * * . . Michael addition * * (+)-salutaridine NADPH * . -H2O THEBAINE * H+

. . . * * * * * * REVISION OF ROUTE TO THEBAINE THEBAINE NADPH salutaridinol * * (+)-salutaridine allylic displacement Labeled salutaridine is incorporated at a high rate into thebaine, codeine and morphine. * . Labeled salutaridinol (both -OH epimers) is also incorporated at a high rate. THEBAINE *

THE FINAL INTERCONVERSIONS + H2O - H2O neopinone thebaine hemiketal isomerization of double bond demethylation NADPH morphine codeine codeinone Labeled neopinone is converted to codeine in the plant.

. . . . ANOTHER REVISION * * * * * * * * TRITIUM IS LOST TRITIUM + * * * (+)-reticulene 1,2-dihydroreticulene (-)-reticulene TRITIUM IS LOST * . (+)-Reticulene appears to be formed first, and than converted to (-)-reticulene through the 1,2-dihydroreticulene intermediate. TRITIUM SURVIVES * (-)-Reticulene is incorporated with the tritium label intact. (+)-Reticulene loses the label. (-)-reticulene

WHAT WAS NOT SHOWN Remember, each of the labeled compounds had to be synthesized with high specificity. Dilution compounds had to isolated or synthesized. Each of the destination compounds had to be specifically degraded to find the position of the label. At times the percentage incorporation was important to know - this requires painstaking work. Most times, fortunately, it is sufficient just to know where the label ended up. All of these plants had to be grown and tended to. Manipulation of radioactive waste was a major problem (14C has a half-life of 5730 years) it doesn’t go away if spilled.

Much of this work was done by Sir Alan Rushton Battersby (b. 1925) and his co-workers. Manchester College, England Ph.D. Dundee (1959) Reader at Bristol, Liverpool and Cambridge OPTIONAL READING A.R. Battersby and B.J.T. Harper, “Biogenesis of Morphine”, Chemistry and Industry 1958, 364. A.R. Battersby, R. Binks, D.J.Le Count, “Biosynthesis of Morphine”, Proceedings of the Chemical Society, 1960, 287.

Alkaloids - Nature’s Cure or Curse, Wiley-VCH (2000) ASSIGNMENTS Read: Hesse Alkaloids - Nature’s Cure or Curse, Wiley-VCH (2000) 8.2.4 “Alkaloid Biogenesis in Papaver Somniferum”, pp 272-277 Two of the degradation procedures will be found here in Figures 8.16 (C9) and 8.17 (C16). You will already be familiar with some of this chemistry as we learned it in the discussion of the structure proof of morphine.