BIOSYNTHESIS OF OPIUM ALKALOIDS

14C-Tyrosine

fed to opium poppy seedlings

HO

CH2

CH

hydroxylase O2
HO

COOH

NH2

tyrosine

If labeled DOPA is fed only one label is found.

Two labels are incorporated into papaverine and norlaudanosoline, implying that two tyrosines are used.

CH2

CH

PLP

HO

COOH

NH2

DOPA

PLP
HO CH2

HO

CH2

HO

HO

CH2 NH2

Mannich + Arom. Subst.

MeO MeO MeO MeO

dopamine

HO

SAM
FAD

HO HO HO

NH

thebaine codeine

*
norlaudanosoline

papaverine
(in all poppies)

morphine

14CO 2
HO HO HO HO NH

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.
The same result is obtained with carbon-14 labeled tyrosine.

norlaudanosoline

thebaine 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.

HO HO HO HO

NH

*
SAM

MeO MeO MeO MeO

N CH3

MeO MeO

N CH3

*
laudanosine

FAD

MeO MeO

*
papaverine

norlaudanosoline

SAM

Norlaudanosoline is incorporated into papaverine and thebaine at a higher rate than tyrosine - its closer.
MeO HO HO MeO

HO HO HO HO

* N CH * SAM
3

* N CH *
(-)-reticulene

redrawn (folded) on the next slide ..

laudanosoline

In a separate set of experiments, it can be shown that the methyl groups originate from methionine (14C labeled CH3) and hence SAM.

MULTIPLE LABELS
* MeO
HO

.
T OH

* MeO
O

N CH3

.
T

N CH3

MeO

MeO

(-)-reticulene (5 labels)

thebaine (all labels intact !)

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
* MeO
HO

.
T OH

* MeO
[O]
O

N CH3

. .
O

.
T

* MeO
O H

N CH3

.
T O

coupling
MeO

N CH3

MeO

MeO

* * MeO
HO

(-)-reticulene (5 labels)

enolization

.
T O

N CH3

MeO

salutaridine

ORIGINAL HYPOTHESIS (ALA PUMMERERS KETONE)

IT MISLEAD THEM

* MeO
HO

.
T O

* MeO
Michael addition
O

N CH3

.
T O

N CH3

MeO

MeO

(+)-salutaridine

* * MeO

NADPH

THEBAINE

-H2O

O H

.
T OH

N CH3

MeO

H+

REVISION OF ROUTE TO THEBAINE

* MeO
HO

.
T O

* MeO
NADPH
HO

N CH3

.
T OH

N CH3

MeO

MeO

salutaridinol allylic displacement

(+)-salutaridine

Labeled salutaridine is incorporated at a high rate into thebaine, codeine and morphine.

* MeO
THEBAINE
O

Labeled salutaridinol (both -OH epimers) is also incorporated at a high rate.

.
T

N CH3

MeO

THE FINAL INTERCONVERSIONS

MeO
MeO
MeO

O H MeO

N CH3

+ H2O

O O H

N CH3 - H2O

O H O

N CH3

MeO H

thebaine

neopinone isomerization of double bond

hemiketal

HO

MeO

MeO

demethylation
O H HO N CH3

NADPH
O H N CH3

O H O

N CH3

HO

morphine

codeine

codeinone

Labeled neopinone is converted to codeine in the plant.

ANOTHER REVISION

* MeO
HO

.
T OH

* MeO
HO

N CH3 MeO

.
OH

*MeO
HO

CH3 MeO

.
H OH

N CH3

MeO

* *MeO
HO

(+)-reticulene

1,2-dihydroreticulene

(-)-reticulene

TRITIUM IS LOST

.
T OH

N CH3

MeO

TRITIUM SURVIVES

(+)-Reticulene appears to be formed first, and than converted to (-)-reticulene through the 1,2-dihydroreticulene intermediate. (-)-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 doesnt 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.

ASSIGNMENTS Read: Hesse Alkaloids - Natures 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.