LABELING EXPERIMENTSLABELING EXPERIMENTS

BIOSYNTHESIS

RADIOACTIVE LABELLING STUDIESRADIOACTIVE LABELLING STUDIES

The idea here is to “feed” the plant a radioactivelylabeled precursor and then monitor what happensto the radioactive atom(s) when they are taken in(“incorporated”) by the plant.

Where do the labels go - to which specific atoms?

What is the time sequence of incorporation? Whichatoms acquire the label first?

What is the percentage incorporation? How much of the labeled material appears in the plant compound?

Do precursors which are closer to the final producthave higher percentages of incorporation than thosesuspected to be farther away (earlier in the sequence)?

How do you feed the plant the labeled compound?

SOME FEEDING METHODSSOME FEEDING METHODS

FEEDING METHODS FOR HIGHER PLANTSFEEDING METHODS FOR HIGHER PLANTS

NUTRIENTS

(1) HYDROPONIC

stem

bud

bulb

(4) DIRECT INJECTION

(2) PAINT LEAVES

(5) WICK METHOD

(3) SHALLOW TRAY

excised buds, leaves, roots intray of nutrients

compound entersroot hairs

compoundenters leafstomata

compoundenters plantthrough wicknutrients

*CO2

(6) SEALED ATMOSPHERE

greenhouseor bag

(7) TISSUE OR CELL CULTURE

Shaker - Heater

NUTRIENT BROTH

supplies

products

(8) CELL-FREE CULTURE

like brewing orfermentationof beer

heater

cell contents,enzymes andcoenzymes andnutrients

thin slices

Cells are lysedto spill contents.

So far, methods 7&8 work best for the lower plants, like fungi and yeasts.

SOME PROBLEMS ENCOUNTEREDSOME PROBLEMS ENCOUNTEREDIN RADIOACTIVE LABELING STUDIESIN RADIOACTIVE LABELING STUDIES

SOME PROBLEMS WITH LABELING STUDIESSOME PROBLEMS WITH LABELING STUDIES

Not all substances can cross the cell wall. Plant cells have adouble wall. The wall has two cellular membranes, both of which have to be crossed in order to incorporate the labeled compound.

*C A

B C Z

Even though compound C is apart of the biosynthetic sequencethat leads to natural product Zit will not be incorporated if itcannot cross the cell wall.compound C with a

radioactive atom,perhaps 14C insteadof 12C.

cell nucleuscytosol

Most synthesis takes place in the cytosol (fluid content of thecell, or cytoplasm), but sometimes synthesis takes place usingenzymes that are bound to a membrane or are located in thecell’s nucleus. This poses additional problems for incorporation.

other “organelles”(cellular structures that have membranes)

cell wall

CROSSING CELL WALLS

FALSE INCORPORATION OR NON-INCORPORATION

A B C D E Z

M M N O

Sometimes a substance will be incorporated, but is not actuallyin the true biosynthetic pathway (ABCDE Z). It can be convertedin the cell to a compound that is subsequently merged into thebiosynthetic pathway.

A B CCOOH

COOH

COOH

COOH

COOH

COOH

Z

COOEt

COOEt

COOEt

COOEt

Succinic acid is in the pathway to Z but cannotcross the cell wall barrier -incorporation studies fail.

Diethyl succinate is notin the pathway to Z butcan cross the cell wallbarrier. After hydrolysis,it is incorporated. One could incorrectly conclude diethyl succinate is a precursor to Z.

EXAMPLE

GENERAL INCORPORATION - TOTAL DISPERSALlow percentage incorporation

Certain substances, like glucose or acety-CoA, can be considered as potential precursors to everything in the plant. If labeled glucose is fed to the plant everything becomes radioactive, but with low incorporation rates (percentages).

A

B C D E Z

W X YQ R SL M N

A

Obviously these studies must be performed by persons with a greatdeal of experience and knowledge.

QUININEQUININE

Cinchona spp

Stripping Bark

Gin and Tonic “gin and it”

Bark of the Cinchona tree is stripped andpowdered as a source of Quinine.

Quinine is a febrifuge ( lowers bodytemperature, literally “chases fever away”)and has been used for centuries to controlthe high-fever symptoms of malaria.

Chinchona trees are found on the slopes ofthe Andes in Peru (3000-9000 feet). Some years after the Spanish Conquest (about 1630),Jesuit missionaries learned about the tree it from the indian natives.

Samples sent to Spain were reputed to have cured the spanish Countess of Chinchon, hence the name Cinchona. The indian name for the tree was quina, hence quinine.

For many decades it was referred toas “Jesuit powder” or “Peruvian bark”.

Anopheles

PlasmodiumProtozoan responsible for malaria

Mosquitos carry the plasmodiummicroorganism as a parasite.

This microorganism has anunbelievably complex life cycleincluding asexual stages.

The high fever due to infectioncomes from one stage of the reproductive cycle while theparasite is in a human host.These fevers can be high enough to kill.

Dutch planted whole plantationsof these trees in the Dutch EastIndies.

QUININE - A CASE STUDYQUININE - A CASE STUDY

FIRST - A COUPLE OF REACTIONSFIRST - A COUPLE OF REACTIONS WE WILL USEWE WILL USE

HOFMANN REARRANGEMENTHOFMANN REARRANGEMENTDIFFERENT FROM THE HOFMANN ELIMINATION AND THE HOFMANN DEGRADATION

R C NH2

ONaOH Br2 R NH2

amide amine

* *

retains stereochemistry

Recall how to make an amide:

R C Cl

O

R C OH

O

R C NH2

OSOCl2 NH3

conc aq.

The reagent is sometimes designated as sodium hypobromite (NaOBr)

2 NaOH + Br2 HOBr + NaBr + NaOH NaOBr + NaBr + H2O

Br-BrH-O:..

.. H-O:..

.. H-OBr

HOFMANN REARRANGEMENTHOFMANN REARRANGEMENTTHE MECHANISM

R C N

O

H

HR C N

O

HR C N

O

Br

H

Br-Br

:OH-..

..:OH-..

..

R C N

O

BrR C N

O

WOLFF REARRANGEMENT(nitrene)

R N C O

:OH-..

..

R NC

O

OH

H

HO-H

..

-CO2

:OH-..

..R N

H

H

..

....

-

..

....

..

..

..

..

REMEMBER THECURTIUS REACTIONALSO HAD A WOLFFREARRANGEMENT

-

CHICHIBABIN REACTIONCHICHIBABIN REACTION

N NH2N H

NH2

H NH2

..

..

..

.. ....

N H

NH2

..

..

..- -

NaNH2

NH3 (liq)

nucleophilic substitution in a pyridine ring

+ H2 + NH2

.. -

..

hydridetransfer

also works with other strong bases like alkyllithiums

N.. N Ph..

PhLi

toluene

:Ph-

THE PLANTHE PLAN

Wick feeding of Cinchona succuriba

N

NH

OH

MeO

IT WAS SUSPECTED THAT THE PRECURSORS WEREIT WAS SUSPECTED THAT THE PRECURSORS WERE TRYPTOPHAN AND GERANIOLTRYPTOPHAN AND GERANIOL

N

NH2

H

COOH

OH

tryptophan

geraniol

quinine

AMINO ACIDAMINO ACID

TERPENOIDTERPENOID

NH

N

OHA big hint that tryptophan,an indole, was a precursorto the quinoline ring was found in cinchonamine which is also found in the cinchona plant, along withthe quinine.cinchonamine

Note that this is a “hybrid”alkaloid coming from bothterpenoid and amino acidsources.

quinolinering

quinuclidinering

Neither labeled tryptophan nor geraniol were commercially available and had to be synthesized.

STRATEGYSTRATEGY

Generally large compounds with labels are not commercially available.Only small labeled compounds can be purchased

14CO2 Na14CN 15NH3 H2C=O14CH3COOH D2O H2O18

NaC15N

H14COONa

Any synthesis has to incorporate labels from these simple compounds.

In the case of quinine, they wished to label three different positions intryptophan, two different carbons and and the ring nitrogen. In additionthey wished to be able to label each position individually, as well as allthree together. This would be most efficient if a single synthesis couldbe used for whichever label(s) were desired. Two different positions were of interest in geraniol.

The label positions are shown on the next slide.

E. Leete, Accounts of Chemical Research , Feruary 1969

N

NH2

H

COOH OH

SYNTHETIC TARGETS FOR FEEDING EXPERIMENTSSYNTHETIC TARGETS FOR FEEDING EXPERIMENTS

carbons = 14C

nitrogens = 15N

Because labeled compounds are quite expensive to purchase, and becauseradioactive waste is difficult to deal with, the synthetic method had to be workedout and optimized with non-radioactive atoms prior to making the compounds for the feeding experiment.

Members of the Leete group had to come up with an efficient synthesis whichproceeded in high yield at each step.

The partial synthesis of tryptophan on the next slide gives you an idea of what was involved. The synthesis of labeled geraniol is not given here.

THE SYNTHESIS OF LABELED TRYPTOPHANTHE SYNTHESIS OF LABELED TRYPTOPHAN

CH3

Cl

O

CH3

OH

O

CH3

NH2

O

15NH3

CH3

NH2

HOFMANNREARRANGEMENT

CH3

NHC

O

CH3

H14COOH

N

H

SOCl2

KOtBu

tBuOH

N

H

NCH3

CH3

HC

H

O(CH3)2NH

H+

MANNICH

CH NH

COOEt

CN

C

O

CH3

N

H

C NH

CCH3

OCOOEt

CN

N

H

CH2 C NH2

H

HOOC

NaOHH2O

synthesis omitted

MAJOR STEPS ONLY

RESULTS OF THE FEEDING EXPERIMENTRESULTS OF THE FEEDING EXPERIMENT

N

NH2

H

COOH OH

N

NH

OH

MeO The positions of the labels weredetermined by degration ofthe quinine as shown on thenext slide.

This procedure was also workedout before the feeding experiment.

DEGRADATION OF QUININEDEGRADATION OF QUININE

N

NH

OH

MeO

N

MeO

COOH

N

MeO

CO2

N2

N

MeO

Ph

Ph-COOH

KMnO4CuCrO2

sealed tubeCaO, CuO

ChichibabinPhLi toluene

Degradation of theQuinoline Ring

FINDING THE LABELS FROMTRYPTOPHAN

N

NH2

H

COOH

1) CH3I2) KMnO4 /

DEGRADATION OF QUININEDEGRADATION OF QUININE

N

NH

OH

MeO

Degradation of theQuinuclidine Ring

N

NH

OH

MeOH2 / Pt

Kuhn-Roth OxidationCrO3 / H2SO4

HOOC-CH3 HOOC-CH2-CH3+

1) SOCl22) NH3

3) NaOH, Br2

1) SOCl22) NH3

3) NaOH, Br2

CO2 + CH3NH2 CO2 + CH3CH2NH2

FINDING THE LABELS FROMGERANIOL

HOFMANNREARR.

OH

INVERSE ISOTOPIC DILUTION METHODSINVERSE ISOTOPIC DILUTION METHODSHow in the devil do you get enough material to do these degradations?

You would have to do the feeding experiment on hundreds of seedlingsto get enough labeled quinine to do this study.

Fortunately, you don’t need large quantities of the labeled quinine.

Since very small amounts of radioactivity can be measured, you can “dilute”the radioactive quinine with a large amount of non-radioactive quinine fromanother source and work on the mixture.

In fact, you don’t even have to purify the radioactive quinine. You justgrind the seedlings to a fine powder, extract the alkaloids (mg), and then add a large amount (g) of non-radioactive quinine. Continue heating.

When you are finished, crystallize the quinine. The radioactive material (mg)will be swept into the large amount of non-radioactive quinine (g) and willco-crystallize with it (g) + (mg).

Multiple crystallizations are performed until the mixture gives a constantcount in the scintillation counter. This gets rid of any non-quinine radio-active compounds. Degradation can be done on this “diluted” mixture.

A CLOSE LOOK AT THE RESULTSA CLOSE LOOK AT THE RESULTS

N

NH

OH

MeO

N

NH2

H

COOH

adjacent,

adjacent,

Rearrangementof , ,

OH

SOME SURPRISES !

Any proposal for the biosynthetic pathway must explain the rearrangements of the labels.

still adjacentbut order reversed

These transformations of geraniol were known from previous work withother terpene biosynthetic pathways. The trialdehyde was a well-docu-mented intermediate.

OH

O

OH

CH3

Oglucose

MeOOC

O

CH2

Oglucose

MeOOC

CHO

loganin secologaningeraniol

A PREVIOUSLY KNOWN TERPENE PATHWAYA PREVIOUSLY KNOWN TERPENE PATHWAY

CHO

CHO

CHO

trialdehyde

If you are curious, you can findthe details of this pathway inany text on biosynthesis - lookup loganin, secologanin or “iridoids” or the reading assignment on the last slide.

o

-Coxidations andbond formation

THE PROPOSED BIOSYNTHETIC PATHWAYTHE PROPOSED BIOSYNTHETIC PATHWAY

CHO

CHO

CHO

NH

N

COOH

CHO

..

NH

N

COOH

cinchonamine

NN

COOHOH

(next slide)

N

NH2

H

COOH

this breaks the and labels

C N

OH

NH2 O

CHO

..N

NO

OH

- H2O

NADPH

1) O2 (hydroxylase)2) SAM

N

NH

OH

MeOquinine

this joins the and labels

ASSIGNMENTS

Read:

HesseAlkaloids - Nature’s Cure or Curse, Wiley-VCH (2000)Chapter 7 “Alkaloids and Chemotaxonomy”, pp 237-256,especially Section 7.2 which discusses the formation ofsecologanin and other iridoids from geraniol.

TERPENOID INDOLE ALKALOIDSTERPENOID INDOLE ALKALOIDS

IRIDOIDS

There are a variety of alkaloids that have tryptophanand geraniol as their precursors.

The geraniol precursor can yield a variety of skeletalpieces as it rearranges readily via a pathway containingtwo intermediates called loganin and secologanin.

OH

O

OGlu

OH

MeOOC

C

O

OGlu

O

MeOOC

H

geraniol loganin secologanin

C

O

OGlu

O

MeOOC

H

secologanin

Corynanthetype

Aspidospermatype

Ibogatype

SECOLOGANINSECOLOGANINSecologanin has so much functionality that it can open bonds, rotate and make new bonds, easily rearranging the carbon skeleton. Three principalskeleton types are shown below. These combine with tryptamine to formalkaloids (some examples are given).

N

N

H

COOMe

N

N

HCOOMeN

N

H

COOMe

tabersonine

akuammicine

catharanthine

IRIDOIDSIRIDANESKELTON

( )sometimes acarbon is lost C10 => C9

N

NH

OH

MeO Quinine

NN

O

HH

H

CH3

H

H

MeOOC

NHN

O

H

OHMeOOC

NN

HH

H

H

OH

H

Ajmalicine