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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.