1
5. Biosynthesis of Alkaloid Natural Products 5.1. Alkaloids are derived from amino acids Nitrogen-containing compounds, with a slightly basic character, have been isolated from many different organisms, mostly plants and microorganisms, and are biosynthesized from amino acids - these are called alkaloids. There are probably over 10'000 known alkaloids, having very diverse structures. They can nevertheless be classified into families, on the basis of structural similarities and the amino acids that are used for their biosynthesis Some alkaloids are also produced using building blocks derived from other secondary metabolic pathways, such as terpenoids, polyketides and peptides. Some of the important classes of alkaloid are shown below:
N
Me
O
N ON
N
MeN
O
O
O
Ph
OHN
HO
NH
NH2
N
H
N
COOMe
N
H N
Me
N
MeOOC
OH
N
NH
OAcOH
N
OH
HO
HONH2
N
MeO
MeO
MeO
MeO
NMe
HO
O
HO
NHAc
OMe
OMeO
MeO
MeO
COOMe
MeOOC
NMe
N
OAcOH
MeOOC
OH
MeO
N-Methylpelletierine
Lycopodine
Scopolamine Retronecine
Dopamine
Papaverine
Colchicine
Catharanthine
Vindoline
Geissoschizine
Vinblastine
H3N
NH3
COO
Lysine
H3N
NH3
COO
Ornithine
NH3
COO
R
PhenylalanineTyrosine
NH
NH3
COO
Tryptophan
N
Me O
MeHygrine
e.g. Pyrrolidine, Pyrrolizidine and Tropane Alkaloids
MeN
O
Tropinone
Sparteine
e.g. Piperidine, Pyridine und Quinolizidine Alkaloids
N
OH
Lupinine
NH
MeConiineN
N
Me
Nicotine
z.B. Isoquinoline Alkaloids
NMe
MeO
HO
OMe
OMe
OH
Autumnaline
Morphine
z.B.Indole Alkaloids
Tryptamine+ Terpene
MeO
2
5.2. Benzylisoquinoline Alkaloids Of special interest within the family of isoquinoline alkaloids are those containing the 1-benzyl(tetrahydro)isoquinoline skeleton, which are found in many different plants. Studies on the biosynthesis of these compounds made progress as soon as radioactively labelled compounds (14C and 3H) became available. Potential precursors could be fed to intact plants, and later the natural prodicts could be isolated from the plants, and then analyzed chemically to detemine whether, and if so, where the radioactive labels had been incorporated. In this way, it was shown that the benzylisoquioline alkaloids are constructed from two molecules of tyrosine:
HONH2
HO
NH
HO
HO
HO
H
HONH2
COOH
Tyrosin
HO
HONH2
HO
CHOO COOH
Norcoclaurine
Decarboxylase(PLP)
Transaminase(PLP)
Hydroxylase
Decarboxylase(TPP)
The formation of norcoclaurine is catalyzed by an enzyme, which in effect catalyzes a Pictet-Spengler-Reaction. The reaction shown actually occurs spontaneously in aqueous solution, but then slowly gives racemic product, whereas the enzymic reaction runs much faster and gives optically pure product: HO
HONH2
HO
CHO
NH
HO
HO
HO
H
Norcoclaurine
NH
HO
HO
HO
NH
O
HO
HO
H
Next, the norcoclaurine is converted into (S)-reticuline :
NH
HO
HO
HO
H
Norcoclaurine
HydroxylaseN-Me
MeO
HO
Me-O
HHO
Reticuline
SAM SAM
SAM
Reticuline is used for the biosynthesis of many other benzylisoquinoline alkaloids, amongst others, the so-called aporphine alkaloids, e.g.:
NMe
MeO
HO
HO
MeO
H
NMe
MeO
HO
HO
MeO
H
NMe
MeO
MeO
MeO
MeO
H
Glaucine
3
An important step here is the formation of a direct aryl-aryl bond. This occurs in an oxidative phenol coupling reaction. Nature has evolved a series of hemoproteins of the cytochrome P450 family that catalyze specific oxidative phenol coupling reactions (not hydroxylations, compare earlier). Such coupling reactions are well known in synthetic chemistry, where they can be carried out with phenolic compounds, under basic conditions, using K3Fe(CN)6 as oxidizing agent, e.g.:
NMe
MeO
HO
MeO
H
OH
NMe
MeO
O
MeO
H
O
NMe
MeO
O
MeO
H
O
NMe
MeO
O
MeO
H
O
NMe
MeO
HO
MeO
H
OH
2 FeIII
-2H+
2 FeII
NMe
MeO
HO
HO
MeO
H+
ortho-para ortho-ortho
K3Fe(CN)6
NMe
MeO
O
MeO
H
O
HH
Such reactions tend to produce mixtures of products, because the free radical intermediates can often couple in more than one way. The enzymes, however, catalyze only one pathway specifically. The mechanisms of the enzymic reactions are not well understood, but require molecular oxygen as well as the hemoprotein (P450). The oxidizing power of compound-I is used to drive the coupling reaction, e.g.:
OH2
S-Cys
FeIII
0
P450 enzyme(resting state)
electrons+ O2 O
FeIV
S-Cys
OH HO
OH
Fe
S-Cys
O HO
Fe
S-Cys
O O
+ H2O
H2Ocompound-I
Oxidative phenol coupling reactions are often found in alkaloid biosynthesis. Perhaps the best-known example occurs during the biosynthesis of morphine. Morphine is a highly-potent opiate analgesic drug and is the principal active agent in opium and the prototypical opioid. It is also a natural endocrine product in humans and other animals. Like other opiates, e.g., diacetylmorphine (heroin), morphine acts directly on the central nervous system (CNS) to relieve pain, and at synapses of the nucleus accumbens in particular. Studies done on the efficacy of various opioids have indicated that, in the management of severe pain, no other narcotic analgesic is more effective or superior to morphine. Morphine is highly addictive when compared to other substances; tolerance, physical and psychological dependences develop very rapidly. The word "morphine" is derived from Morpheus, one of the Greek gods of dreams. The opium poppy is Papaver somniferum.
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(R)-Reticuline is an important intermediate in the biosynthesis of morphine, and is produced by racemization of (S)-reticuline in a redox process, as shown below:
NMe
MeO
HO
HO
MeO
H
MeO
HO
MeO
OH
N-Me
MeO
HO
MeO
O
N-Me
(S)-Reticuline(R)-Reticuline
Salutaridine
oxid.Phenol-Coupling
Oxid.
Red.
Salutaridine is found as a minor alkaloid constituent in the opium poppy:
MeO
O
MeO
N-Me
HO
O
HO
N-Me
MeO
O
HO
N-Me
MeO
HO
MeO
N-Me
MeO
HO
MeO
N-Me
O OH
MeO
O
O
N-Me
Codeine Morphine
Thebaine
Reduction
Salutaridinol
Acetyl-CoA
AcOHCoASH
Neopinone
MeO
O
O
N-Me
OH
MeO
O
O
N-Me
Codeinone
5
The biosynthesis of morphine in the opium poppy was one of the first alkaloid pathways to be elucidated with the aid of 14C-labelled precursors. It was shown that [2-14C]-tyrosine is incorporated into morphine, with the 14C label appearing at the positions indicated above. This was proven, by degrading the 14C-labelled morphine in the following way:
HO
O
HO
N-Me
Morphine
1) MeI / K2CO3 / MeOH2) Ag2O, then pyrolysis
MeO
O
HO
NMe2
EtONa / EtOH, !
MeO
HO
EtONMe2
+
1) Ac2O2) CrO3
MeO
AcOO
O
1) H2O2
2) NaOH/H2O
3) H3O
COOH
MeO
O
O
MeO
O
O
H2SO4
!
NaOH/Me2SO4
MeO
MeO
HOOC
heat/ H+
MeO
MeO + CO2
Another interesting benzylisoquinoline alkaloid is colchicine. Colchicine was originally extracted from plants of the genus Colchicum (Autumn crocus, Colchicum autumnale, also known as the "Meadow saffron"). Originally used to treat rheumatic complaints and especially gout, it was also prescribed for its cathartic and emetic effects. Its present medicinal use is mainly in the treatment of gout; it is also being investigated for its potential use as an anti-cancer drug. Colchicine inhibits microtubule polymerization by binding to tubulin, one of the main constituents of microtubules. Tubulin is essential for mitosis, and therefore colchicine effectively functions as a "mitotic poison" or spindle poison. Since one of the defining characteristics of cancer cells is a significantly increased rate of mitosis, this means that cancer cells are significantly more vulnerable to colchicine poisoning than are normal cells. However, the therapeutic value of colchicine against cancer is (as is typical with chemotherapy agents) limited by its toxicity against normal cells. In 2008, the Botanic Gardens Conservation International (representing botanic gardens in 120 countries)
stated that "400 medicinal plants are at risk of extinction, from over-collection and deforestation, threatening the discovery of future cures for disease." These included Yew trees (the bark is used for the cancer drug taxol (paclitaxel)); Hoodia (from Namibia, source of weight loss drugs); half of Magnolias (used as Chinese medicine for 5,000 years to fight cancer, dementia and heart disease); and Autumn crocus (for gout). The group also found that 5 billion people benefit from traditional plant-based medicine for health care.
Early labelling experiments showed that tyrosine and phenylalanine are required for colchicine biosynthesis, and that autumnaline is a key intermediate. However, the Phe provides a C6C3 unit rather than a C6C2 fragment:
6
NHAc
OMe
O
MeO
MeO
MeO
Colchicum
Colchicine
Tyrosine
Phenylalanine
NMe
MeO
HO
MeO
MeO
OH
H
(S)-Autumnaline
NH2HO
COOH
COOHH2N
The seven membered tropolone ring was shown by labelling experiments to originate by ring expansion of the tyrosine-derived aromatic ring, including the adjacent benzylic carbon atom.
MeO
N-Me
MeO OH
MeO
HO
NH2
CHO
HO
HO
OH
NH
HO
HO
OH
H
(S)-Autumnaline
Phenylalanine
Tyrosine
Dopamine
cf. above
N
HO
HO
OH
MeO
N-Me
MeO O
MeO
HO
Isoandrocymbine
Androcymbine has been isolated from Androcymbium melanthioides. The later steps have not been proven, but may involve the following reactions:
NH
OMe
O
MeO
MeO
MeO
HO
NH-Me
MeO O
MeO
MeO
MeO
NHMe
MeOO
MeO
MeO
NH-Me
OMe
O
MeO
MeO
MeO
H
MeO
N-Me
MeO O
MeO
MeO
Androcymbine
Colchicine
Oxidation
HO
OMe
HCHO
Demethylation
Acetylation
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Various types of alkaloids are encountered in the daffodil family, called the Amaryllidaceae alkaloids (Amaryllidaceae is the botanical name of a family of flowering plants. The plants are herbaceous perennials that grow from bulbs, often with showy flowers). The Amaryllidaceae family includes Amarylis, Narcissus
and Galanthus, and the alkaloid content of bulbs from most members makes them toxic. However, galanthamine from daffoldils and snowdrops is currently an important drug for the treatment of the symptoms of Alzheimer's disease. The natural sources of galanthamine are certain species of daffodil and because these species are scarce and because the isolation of galanthamine from daffodil is expensive (a 1996 figure
specifies 50,000 US $ per kilogram; the yield from daffodil is 0.1-0.2% dry weight) alternative synthetic sources have been developed. Galanthamine acts as a competitive inhibitor of acetylcholinesterase, and enhances cognitive functions by raising acetylcholine levels in brain areas lacking cholinergic neurons. It does not cure the condition, but merely slows the rate of cognitive decline. Phe and Tyr are again the starting materials used for the biosynthesis of the Amaryllidaceae alkaloids:
L-Phe
HO
HO CHO
H2N
OH
L-TyrNH
HO
HO
HO
Norbelladine
SAM
Thereafter, three different modes of phenol coupling are seen:
NH
MeO
HO
HO
4'-O-methylnorbelladine
NH
MeO
HO
HO
N
MeO
HO
HO
N
HO
OH
O
O
Norpluvine
Lycorine
NH
MeO
HO
OH
para-ortho-coupling
NH
MeO
HO
O
N
MeO
HO
O
N
MeO
HO
OMe
OHOxocrine
Haemanthamine
para-para-coupling
OH
NMe
HO
MeO
O
NMe
HO
MeO
ortho-para-coupling
NMeO
MeO
OH
Galanthamine
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5.3. Indole Alkaloids (see Nat. Prod. Rep. 2006, 23, 532) The simplest representative of the indole alkaloids are the natural amines tryptamine und serotonin, which are biosynthesized from the amino acid tryptophan (Trp):
N
NH3
COO
H
N
NH2
H
RR = H TryptamineR = OH Serotonin
Serotonin is a monoamine neurotransmitter synthesized in serotonergic neurons in the central nervous system (CNS), and enterochromaffin cells in the gastrointestinal tract of animals including humans. Serotonin is also found in many mushrooms and plants, including fruits and vegetables. Serotonin is believed to play an important role as a neurotransmitter, in the modulation of anger, aggression, body temperature, mood, sleep, sexuality, and appetite as well as stimulating vomiting. The vinca alkaloids are a very interesting class of indole alkaloids, and include vinblastine, vincristine, vindesine and vinorelbine. These alkaloids are produced by plants of the genus Catharanthus. Catharanthus (Madagascar Periwinkle) is a genus of eight species of herbaceous perennial plants, seven endemic to the island of Madagascar, the eighth native to the Indian subcontinent in southern Asia. One species, C. roseus,
has been widely cultivated, and after introduction has become an invasive species in some areas. C. roseus has also gained interest from the pharmaceutical industry; the alkaloids vincristine and vinblastine from its sap have been shown to be an effective treatment for leukaemia. Although the sap is poisonous if ingested, some 70 useful alkaloids have been identified from it. In Madagascar, extracts have been used for hundreds of years in herbal medicine for the treatment of diabetes, as hemostatics and tranquilizers, to lower blood pressure, and as disinfectants. The extracts are not without their side effects, however, which include hair loss.
N
Me
N
Vindoline
H
H
N
H
N
MeOOCOH
Stemmadenine
MeOOC
OH
N
H
N
COOMe
N
H
N
MeOOC
OH
N
H
N
MeOOC
N
Me
N
OAc
OH
MeOOC
OH
MeO
Catharanthine
Geissoschizine
Vinblastine
N
O
N
O
H
H
H
H
StrychnineH
OAcMeO
The structures of these alkaloids reveal that not only Trp is required for the biosynthesis. A C10 fragment is also needed, and is provided from terpene metabolism. Strychnine biosynthesis also incorporates one acetate unit (in red above). The important C10 fragment is produced from geraniol, and is called secologanin:
9
Geraniol
OH O
MeHO
MeOOC
O-Glucose
H
H
Loganin
OMeOOC
CHO
O-Glu
Secologanin
Secologanin is a glucoside, which can be cleaved by hydrolysis under acidic conditions:
O
MeOOC
CHO
O
O
HOOH
OH
OH H3O
The formation of the indole alkaloids begins with the condensation of tryptamine and secologanin, catalyzed by strictosidine synthase (STR, see below):
O
MeOOC
CHO
O-Glu
N
NH2
HN
NH
H
OMeOOC
O-Glu
NNH
H
OMeOOC
OGlu
Strictosidine Strictosidine is then a key intermediate in the formation of over 1000 different indole-terpene alkaloids.
10
For example, the Corynanthe alkaloids:
NNH
H
OMeOOC
O-Glu
Glucose
NNH
H
OH
CHO
MeOOC
NN
H
OHMeOOC
N
MeOOC
O
NN
H
MeOOC
OH
H
Yohimbine
NN
H
OHMeOOC
NN
H
OHMeOOC
H
H
Geissoschizine
NADPH
NN
H
OMeOOC
NADPH
NN
H
OMeOOC
Me
Ajmalicine
H
H
H
Acetal
H
NN
H
MeOOC
OH
H
H
2 NADPH
(Imine reduction)
Yohimbine is the principal alkaloid of the bark of the West-African evergreen Pausinystalia yohimbe Pierre (formerly Corynanthe yohimbe), family Rubiaceae (Madder family). There are 31 other yohimbane alkaloids found in Yohimbe. In Africa, yohimbine has traditionally been used as an aphrodisiac. Yohimbine hydrochloride is a standardized form of yohimbine that is available as a prescription drug in the United States, and has been shown to be effective in the treatment of male impotence. Yohimbine hydrochloride has also been used for the treatment of sexual side effects caused by some antidepressants, female hyposexual disorder, as a blood pressure boosting agent in autonomic failure, xerostomia, and as a probe for noradrenergic activity. Ajmaline was first isolated from the roots of Rauwolfia serpentina, a species of flowering plant in the
family Apocynaceae. It is one of the 50 fundamental herbs used in traditional Chinese medicine, where it has the name shégēn mù (蛇根木) or yìndù shémù (印度蛇木). The extract of the plant has also been used for millenia in India — it was reported that Mahatma Gandhi took it as a tranquilizer during his lifetime. Ajmaline is a class Ia antiarrhythmic agent, a group of pharmaceuticals that are used to suppress fast rhythms of the heart (cardiac arrhythmias), such as atrial fibrillation, atrial flutter, ventricular tachycardia,
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and ventricular fibrillation. Ajmaline functions by blocking Na-channels in cell membranes. Rauwolfia caffra is the South African quinine tree. Rauwolfia serpentina, or Indian Snakeroot or Sarpagandha, contains a number of bioactive chemicals, including ajmaline, deserpidine, rescinnamine, serpentinine, and yohimbine. Reserpine is an alkaloid first isolated from R. serpentina, and was widely used as an antihypertensive drug. It had drastic psychological side effects and has been now replaced by blood-pressure-lowering drugs that lack such adverse effects. But in herbal use it is a safe and effective resource for hypertensive patients. The pharmaceutical companies have stopped producing this drug as reserpine or deserpedine. It is only available currently in the U.S. as a herbal medicine over the Internet. The pathway to ajmaline has been well documented, although few mechanistic studies have been reported so far on the biosynthetic enzymes:
NNH
H
OMeOOC
OGlu
Strictosidine
NN
H H
Dehydrogeissoschizine
OHC
COOMe
NN
H H
OHCCOOMe
MeOHCO2
NN
H H
H
N N
H
AcO
N N
H
AcO
OH
N N
HH
AcO
OHH
H
NADPH
N N
MeH
HO
OHH
H
SAM
Polyneuridine Aldehyde
16-epi-vellosimineVomilenine
Dihydrovomilenine
Ajmaline
N N
H
AcO
OHHH
17-O-Acetylnorajmaline
Vinorine
Acetyl-CoA
NADPH
see above
O
H
Oxidation
Reduction
Reduction
Hydrolysis
Catharanthine is a member of the so-called iboga family of indole alkaloids. It is one of the many alkaloids
present in Catharanthus roseus. It is produced along with many other Catharanthus alkaloids by factory farming in China. It can be used as a starting material for the synthesis of the anti-tumor drugs, vinblastine and vincristine. Vindoline (an Aspidosperma alkaloid) is another important component of the bis-indole alkaloids, typified by vinblastine and vincristine, also produced by C. roseus. Some of the biosynthetic steps have been documented, but the enzymes have not yet been studied in detail. A fascinating proposal was made to explain how catharanthine and vindoline might be produced from geissoschizine. Tabersonine is a
known intermediate, and the steps from tabersonine have been established; the rest is hypothetical -
12
NN
H
CHOMeOOC
H
H
Geissoschizine
NN
H
CHOMeOOC
H
N
N
CHOMeOOC
HN
N
MeOOCCHOH
N
N
MeOOC CH2OH
N
N
MeOOC CH2OH
H
N
N
COOMe
Tabersonine
N
N
COOMe
HO
N
N
COOMe
MeO
HH
Me HO
16-Hydroxytabersonine
Desavetoxyvindoline
N
N
COOMe
MeOMe HO
OH
Deacetylvindoline N
N
COOMe
MeOMeHO
OCOCH3
Vindoline
+ H2O
2 x SAM
Hypothetical
NN
H
CHOMeOOC
H
H
H
Redoxchanges
N
H
N
COOMe
Catharanthine
N
N
MeOOC
H
N
N
MeOOCH
Oxidation
Acetyl-CoA
Oxidation
preakuammicine
stemmadenine
N
N
MeOOC CH2OH
H
dehydrosecodine
NADHHypothetical
Vinblastine and vincristine are anti-mitotic drugs used to treat certain kinds of cancer, including Hodgkin's lymphoma, non-small cell lung cancer, breast cancer and testicular cancer. They bind to tubulin, thereby inhibiting the assembly of microtubules. They are M phase cell cycle specific, since microtubules are a component of the mitotic spindle and the kinetochore, which are necessary for the separation of chromosomes during anaphase of mitosis. Toxicities include bone marrow suppression (which is dose-limiting), gastrointestinal toxicity, potent vesicant (blister-forming) activity, and extravasation injury (forms deep ulcers).
13
The coupling of catharanthine and vindoline can be catalyzed by a relatively non-specific peroxidase (a hemoprotein). It is possible that a similar enzyme specifically catalyzes this coupling in C. roseus.
N
H
N
MeOOC
N
Me
N
OAc
OH
MeOOC
MeO
Vinblastine (R = Me)Vincristine (R = CHO)
N
H
N
COOMe
Catharanthine
N
H
N
COOMe
Peroxidase
H2O2HO
N
H
N
COOMe
N
N
COOMe
MeOMeHO
OCOCH3
Vindoline
N
H
N
MeOOC
N
Me
N
OAc
OH
MeOOC
MeO N
H
N
MeOOC
N
R
N
OAc
OH
MeOOC
OH
MeO
Reduction
[O]
Reduction
Coupling
Vinblastine is only present at low levels in C. roseus (0.0002% of dry leaf wt). Over 500 kg of catharanthus is needed to produce 1g of pure vincristine. Much effort has been put into the synthesis of the dimeric alkaloids, starting from the monomers, which can be isolated from the plant in much higher yields. One example is shown below:
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N
H
N
COOMe
Catharanthine
N
H
N
COOMe
O
N
H
N
MeOOC
N
Me
N
OAc
OH
MeOOC
MeO
Vinblastine
N
H
N
COOMe
N
N
COOMe
MeOMeHO
OCOCH3
Vindoline
N
H
N
MeOOC
N
Me
N
OAc
OH
MeOOC
MeO
N
H
N
MeOOC
N
Me
N
OAc
OH
MeOOC
OH
MeO
N
CONH2
COOH
- 40 oC
1) FeCl3, air2) NaBH4
+
5 steps. 40% yield overall (see also: J.Am.Chem.Soc., 2008, 130, 420). Finally, note that strictosidine is also the precursor to the quinoline alkaloids, including the important anti-malarial drug quinine.
NNH
H
OMeOOC
O-Glu
NN
H
OHMeOOC
H
NH
CHO
N
NH2
CHO
ON
N
NHO
MeO
Quinine