Alkaloids

MeHC

Alkaloids

O

N

O

O

O

OH

Heroin(diacetyl morphine or diamorphine)a semisynthetic derivative of morphine

O

N

HO

HO

H

MorphinePapaver somniferum(Plant)

O

N

MeO

HO

H

Codeine(methylmorphine)

NH

N MeO

HO H

lysergic acidergot fungi

NH

ConiineNeurotoxin from Conium maculatum(Plant)

HO

MeOOMe

OBz

OHOAcOMe

OH

MeO

N

AconitineToxin from aconite species(plant)

N O

O

H

N

H

H

StrychnineToxin from Strychnos nux-vomica(tree)

N

NH

NicotineStimulant from the Nicotiana (Nightshade) genus (Plants)

NCl HN

EpibatidinePain killer (200 x more potent than morphine)Epipedobates tricolor(Frog)

N

NN

NO

O

Me

Me

Me

CaffeineIsolated from plants - first 1819StimulantAlkaloid

NMe O

O

OMeO

CocaineErythroxylum coca (Plant)Alkaloid

Cocaine

The Coca plant (Erythroxylum coca) grows in NE parts of South America. Used for at least 3000 years by natives. Introduced into Europe during 16th century. Popular in drinks, including coca-cola.

Albert Niemann (Gottingen) isolated pure cocaine in 1859.

First synthesised and its structure elucidated in 1898 (Richard Willstater, Munich, Nobel prize 1915).

Pure cocaine used as an anaesthetic - e.g. Koller 1884 experimented on his own eye. Freud wrote about the euphoric effects of ingestion of pure cocaine at the same time.

Erythroxylum coca

NMe

O

O

OMeO

Cocaine

O

Cocaineisatropanealkaloid-namedafterthe7Ntropinone nucleus. Other highly bioactivecompounds in this class include scopolamine andhyoscyamine species e.g. thenightshade family of plants). Atropine is animportant pharmaceutical - it is a racemic mixture of hyoscyamine enantiomers.

NMe

NMe

Hyoscyamine

Scopolamine

O

O

OH

O

O

OH

H H

NMe

O

O

OH

Atropine

NMe

O

Tropinone

Efforts to Understand CocaineBiosynthesis led to important developments in methodology for thewider investigation of secondary metabolismin plants and microorganisms

The concepts of Labelling and Dilution

LabelledPrecursors

CELL

much does notpenetrate the

cell

much is diverted

much is diverted

endogenous unlabelledprecursor in excess

endogenous unlabelledprecursor

Biosynthesis

Isolated material ready for analysis - - here 1/12 = 8.3% incorporation

endogenous labelled

precursor

Isotope natural abundances

2H - 0.015%

3H - trace *

13C - 1.1%

14C - 0.0000000001% *

15N - 0.37%

18O - 0.2%

33S - 0.75%

34S - 4.21%

35S - trace *

* radioisotope

Notes

Label is inevitably diluted - it is usual for only a small proportion to be incorporated.

A very good level of labelling would be 10% - i.e. 10% of the isolated compound contains a labelled fragment.This could easily be detected by 13C NMR (where 1.1% of endogenous molecules are labelled.

1% incorporation could be detected as an approx doubling of a 13C peak.

0.1% incorporation would be hard to observe by 13C NMR - but easy by 2H NMR (2H natural abundance is 0.015%)

Experiment by Edward Leete (Chem Commun, 1980, p1170)

O

OHH2NNH2

= 14C Erythroxylum coca

28 days

extract leaves& purify

NMe

O

O

OMeO

6.4 x 108 dpm

283mg, 2.4 x 105 dpm / mmollabelled ornithine

painted onleaves

NMe

HO

O

OHO

aq HCl, Δ

ecgonidine2.4 x 105 dpm / mmol

benzoatecold

N OO

Me2.5 x 105 dpm / mmol

CrO3

H2SO4NMe

PhPh

2.4 x 105 dpm / mmol

Ph

O

OH

KMnO4

1.2 x 105 dpm / mmol

15

15

This experiment proves that either or both of the bridgehead carbons 1 and 5 derive from C-5 of ornithine.

Experiment required extensive small scale degradation to determine the location of the label. Hard work!

1/51/51/51/5

1/5

PhMgBr

Radio-labelling

Next - Formulate a Biosynthetic Hypothesis:

O

OHH2NNH2

H2NNH2

HNOMe

symmetrical intermediate

NMe

+

X

O O

N

O

X

O

N

O

X

O MeMe+N

MeX

O

O

NMe

XO

OH

Cocaine

020406080100120140160180PPM

NH

= 13CErythroxylum coca

28 days

extract leaves& purify

painted onleaves

020406080100120140160180PPM

NO

O

OO1 2

34

5

6 7

89

10

1112

1314

15

1 23 45 6 789 101112

13 14

15

OEt

EtO

13C spectrum of unlabelled cocaine 13C spectrum of cocaine from experiment

Me

Stable Isotope labelling

Much better experiment - less work and 13C NMR precisely locates position of label.

Enhancement of C-5 signal supports intermediacy of the N-methyl pyrrolinium species.

Natural abundance of 13C is 1.1% - detected a 0.25% incorporation

1 23 4

5

6 789 101112

13 14

15

= 13C Erythroxylum coca

28 days

extract leaves& purify

painted onleaves

NO

O

OO1 2

34

5

6 7

89

10

1112

1314

15N

Me

O O

OMe

J = 60 Hz

A

B

C

DE

Expansion of 13C NMR spectrum for C-2

A = doublet due to coupling to C-9 (incorporated 13C)B = C-2 of cocaine (natural abundance)C = C-2 of trans-cinnamoylcocaine (minor impurity)D = C-2 of cis-cinnamoylcocaine (minor impurity)E = doublet due to coupling to C-9 (incorporated 13C)

Dual label experiment

Dual 13C labelling enhances sensitivity - no enhancement could be observed at C-2 due to ca 0.08% incorporation compared to 1.1% natural abundance.

Coupling to C-9 proves intact unit has been incorporated. Coupling is only observed because the small proportion of molecules with 13C at C-2 also have 13C at C-9.

In an unlabelled 13C spectrum no 13C-13C coupling is observed because the chance of observing two contiguous 13C labels is 0.011 x 0.011 = 0.000121

Datura innoxia

roots homogenized

and centrifugedto make a cell-free extract(CFE)

N

O

Tropinone

N

OH

H

Tropine

N

H

OH

ψ-Tropine

+2h, 20 ˚CNAD(P)H

Two enzymes were eventually purified from the CFE - one which synthesises Topine and a second which synthesises ψ-tropine. The ψ-tropine reductase has been crystallised and studied. SeeStructure of tropinone reductase-II complexed with NADP+ and pseudotropine at 1.9 A resolution: implication for stereospecific substrate binding and catalysis.Yamashita, A., Kato, H., Wakatsuki, S., Tomizaki, T., Nakatsu, T., Nakajima, K., Hashimoto, T., Yamada, Y., Oda, J., Biochemistry 1999, 38, 7630-7637.

Cell Free extract and purified enzymes....

Crystal structure of tropinone reductase II bound to NADPH and ψ-tropine.

Experiment by Edward Leete and Leo Vining (Tet. Lett, 1975, 47, p4103)

More isotopic labelling experiments - microbial cultures

Beauveria bassianaAn isect pathogenic fungus....

Can be grown in the lab on agar plates

and in liquid media as'shake cultures' which are characteristically orange

HO

N

OOH

OOH

Tenellin - yellow

O

O

O

OOH

HO

OH

HO

Oosporein - red

NH2

O

OH

= 13C

feed to cultures

7 days4

5

Phenylalanine

ca 4% incorporation of labelled phenylalanine - note enhanced doublets from now adjacent labels

+

AlkaloidsThe opium poppy, exuding latex which contains the opiatesPapaver somniferum is used as a commercial crop in numerous countries including the UKit yields around 1.5Kg pure morphine per hectare.

O

N

HO

HO

H

MorphinePapaver somniferum(Plant)

The latex is rich in opiates, but all parts of the plant contain valuable material.

The Biosynthesis of morphine is well understood chemically and genetically.

Morphine is ultimately derived from the amino acid tyrosine.

Alkaloids

The mechanism of a Pictet-Spenglerase - Strictosidine Synthase

NH

NH2

Tryptamine

O

OGlc

OH

O

MeO

Secologanin(a monoterpene)

NH

NH

O

OGlc

MeOOStrictosidine

Strictosidine

Synthase

Vinblastine (Anti-cancer)Yohimbine (Sexual disorders)Ajmaline (Anti-arrhythmic)Camptothecin (Anti-cancer)Strychnine (Toxin)

+

Tyr 151

Glu 309

His 307

Secologanin

See O'Connor et al, J. Am. Chem. Soc., 2008, 130, 710-723.

Alkaloids

Cocaine Biosynthesis

O

OHH2NNH2

Ornithine(5 Carbons)

NH2 NH2

putrescine

NH NH2Me

SAMdecarboxylase

(PLP)

Aminotransferase

(PLP) NH OMe

NMe

Similarly

O

OHNH2

H2N

Lysine(6 carbons)

NMe

Piperidine Alkaoidsvia cadaverine

Pyrrolidine alkaloids

NMe

O SCoA

SCoA

O

NMe

O

SCoA

O

O SCoA

P450NMe

O

O SCoA

HO

H+-H2O

NMe

O

O SCoA

ONMe

O

SCoAOHNMe

O

OMeONMe

O

OMe

O

Cocaine

NH

e.g. Coniine

Toxic principal of Hemlock (plant).Used by Socrates to commit suicide.First alkaloid to be synthesisedby Ladenburg in 1886.