112 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:
NMe
O
N ON
N
MeN
O
OO
Ph
OHN
HO
NH
NH2
NH
N
COOMe
NH N
Me
N
MeOOCOH
N
NH
OAcOH
N
OH
HO
HONH2
N
MeO
MeOMeO
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
H3NNH3
COOOrnithine
NH3
COO
RPhenylalanineTyrosine
NH
NH3
COO
Tryptophan
N
Me O
MeHygrine
e.g. Pyrrolidine, Pyrrolizidine and Tropane Alkaloids
MeN
OTropinone
Sparteine
e.g. Piperidine, Pyridine und Quinolizidine Alkaloids
N
OH
Lupinine
NH
MeConiine N
NMe
Nicotine
z.B. Isoquinoline Alkaloids
NMe
MeO
HO
OMe
OMe
OH
Autumnaline
Morphine
z.B.Indole Alkaloids
Tryptamine+ Terpene
MeO
113 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:
The formation of norcoclaurine is catalyzed by an enzyme, which in effect catalyzes a Pictet-Spengler-Reaction (see Angew.Chem.Int.Ed 2011,50,8538). 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:
Next, the norcoclaurine is converted into (S)-reticuline :
Reticuline is used for the biosynthesis of many other benzylisoquinoline alkaloids, amongst others, the so-called aporphine alkaloids, e.g.:
HO NH2
HO
NH
HO
HO
HO
HHO NH2
COOH
Tyrosin
HO
HO NH2
HO
CHOO COOH
Norcoclaurine
Decarboxylase(PLP)
Transaminase(PLP)
Hydroxylase
Decarboxylase(TPP)
HO
HO NH2
HO
CHO
NH
HO
HO
HO
H
Norcoclaurine
NH
HO
HO
HO
NH
O
HO
HO
H
NH
HO
HO
HO
H
Norcoclaurine
HydroxylaseN-Me
MeO
HO
Me-O
HHO
Reticuline
SAM SAM
SAM
NMe
MeO
HO
HO
MeO
H
NMe
MeO
HO
HO
MeO
H
NMe
MeO
MeO
MeO
MeO
H
Glaucine
114 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.:
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.:
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.
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
OH2
S-Cys
FeIII
0
P450 enzyme(resting state)
electrons+ O2 O
FeIV
S-Cys
OH HOOH
FeS-Cys
O HO
FeS-Cys
O O
+ H2O
H2Ocompound-I
115
(R)-Reticuline is an important intermediate in the biosynthesis of morphine, and is produced by racemization of (S)-reticuline in a redox process (Science, 2015, 349, 309) as shown below:
Salutaridine is found as a minor alkaloid constituent in the opium poppy:
NMe
MeO
HO
HO
MeO
H
MeO
HO
MeOOH
N-Me
(S)-Reticuline (R)-Reticuline
Salutaridine
oxid.phenolcoupling
Oxid.
Red.
from [2-14C]-Tyrosin
ortho-para
NMe
MeO
HO
HO
MeO
H
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
116 The biosynthesis of morphine in animals, including humans, occurs in a very similar way, with many common intermediates and enzymic reactions (see JBC, 2015, 290, 20200). In recent years major advances have been made in engineering yeast strains to produce opioids, e.g. thebaine, hydrocodone (Science 2015, 349, 1095). A microbial-based manufacturing process may overcome many of the problems associated with poppy-based agricutural methods (ACIE, 2016, 55, 1248-50). 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:
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 botanical 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.
HO
O
HO
N-Me
Morphine
1) MeI / K2CO3 / MeOH2) Ag2O, then pyrolysis
MeO
O
HO
NMe2
EtONa / EtOH, Δ
MeO
HO
EtONMe2
+
1) Ac2O2) CrO3
MeO
AcO O
O
1) H2O22) NaOH/H2O
3) H3O
COOH
MeO
O
O
MeO
O
O
H2SO4Δ
NaOH/Me2SO4 MeO
MeO
HOOC
heat/ H+MeO
MeO + CO2
117 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:
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.
O-Methylandrocymbine has been isolated from Androcymbium melanthioides. The later steps have not been proven, but may involve the following reactions:
NHAc
OMe
OMeO
MeO
MeO
Colchicum
Colchicine
Tyrosine
Phenylalanine
NMe
MeO
HO
MeO
MeOOH
H
(S)-Autumnaline
NH2HO
COOH
COOHH2N
MeON-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
MeON-Me
MeO O
MeO
HO
Isoandrocymbine
NH
OMe
OMeO
MeO
MeO
MeONH-Me
MeO O
MeO
MeO
MeONHMe
MeO O
MeO
MeO
NH-Me
OMe
OMeO
MeO
MeO
H
MeON-Me
MeO O
MeO
MeO
O-Methylandrocymbine
Colchicine
Oxidation
HO
OMe
HCHO
Demethylation
Acetylation
*
*
118 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:
Thereafter, three different modes of phenol coupling are seen:
L-Phe
HO
HO CHO
H2N
OH
L-Tyr NH
HO
HO
HO
Norbelladine
SAM
NH
MeO
HO
HO
4'-O-methylnorbelladine
NH
MeO
HO
HO
N
MeO
HO
HO
N
HOOH
O
O
Norpluvine
Lycorine
NH
MeO
HO
OH
para-ortho-coupling
NH
MeO
HO
O
N
MeO
HO
O
N
MeO
HO
OMeOHOxocrine
Haemanthamine
para-para-coupling
OH
NMe
HO
MeO
O
NMeHO
MeO
ortho-para-coupling
NMeO
MeO
OH
Galanthamine
119 5.3. Indole Alkaloids The simplest representative of the indole alkaloids are the natural amines tryptamine und serotonin, which are biosynthesized from the amino acid tryptophan (Trp):
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 loss of hair.
The structures of these alkaloids reveal that not only Trp is required for the biosynthesis (see Nat. Prod. Rep. 2006, 23, 532). 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 (Nat. Comm. 2014, 5, ncomms4606):
N
NH3
COO
HN
NH2
H
RR = H TryptamineR = OH Serotonin
NMe
N
Vindoline
H
H
NH
N
MeOOC OHStemmadenine
MeOOCOH
NH
N
COOMe
NH
N
MeOOCOH
NH
N
MeOOC
NMe
N
OAcOH
MeOOC
OH
MeO
Catharanthine
Geissoschizine
Vinblastine
N
O
N
O
H
HH
H
Strychnine H
OAcMeO
120
Secologanin is a glucoside, which can be cleaved by hydrolysis under acidic conditions:
The formation of the indole alkaloids begins with the condensation of tryptamine and secologanin, catalyzed by strictosidine synthase (STR, see below) (compare with Pictet-Spengler reaction):
Strictosidine is then a key intermediate in the formation of over 1000 different indole-terpene alkaloids (Nat. Prod. Rep. 2012, 29, 1176).
Geraniol
OH O
MeHO
MeOOC
O-Glucose
H
H
Loganin
OMeOOC
CHOO-Glu
Secologanin
OMeOOC
CHOO
OHO
OHOH
OH H3O
OMeOOC
CHOO-Glu
NNH2
HN
NH
H
OMeOOC
O-Glu
NNH
H
OMeOOC
OGlu
Strictosidine
121 For example, the Corynanthe alkaloids:
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,
NNH
H
OMeOOC
O-Glu
Glucose
NNH
H
OHCHO
MeOOC
NN
H
OHMeOOC
N
MeOOCO
NN
H
MeOOCOH
H
Yohimbine
NN
H
OHMeOOC
NN
H
OHMeOOC
H
H
Geissoschizine
NADPH
NN
H
OMeOOC
NADPH
NN
H
OMeOOC
Me
Ajmalicine
H
H
H
AcetalH
NN
H
MeOOCOH
H
H
2 NADPH
(Imine reduction)
122 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:
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 (Review: Nat. Prod. Rep. 2006, 23, 532-47) -
NNH
H
OMeOOC
OGlu
Strictosidine
NN
H H
Dehydrogeissoschizine
OHCCOOMe
NN
H H
OHCCOOMe
MeOHCO2
NN
H H
H
N N
H
AcO
N N
H
AcO
OH
N NH
H
AcO
OHH
H
NADPH
N NMe H
HO
OHH
H
SAM
Polyneuridine Aldehyde
16-epi-vellosimineVomilenine
DihydrovomilenineAjmaline
N N
H
AcO
OHHH
17-O-Acetylnorajmaline
Vinorine
Acetyl-CoA
NADPH
see above
O
H
Oxidation
Reduction
Reduction
Hydrolysis
123
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).
NN
H
CHOMeOOC
H
H
Geissoschizine
NN
H
CHOMeOOCH
N
N
CHOMeOOC
HN
N
MeOOCCHOH
N
N
MeOOC CH2OH
N
N
MeOOC CH2OHH
N
N
COOMeTabersonine
N
N
COOMeHO
N
N
COOMeMeO
HH
Me HO
16-Hydroxytabersonine
Desavetoxyvindoline
N
N
COOMeMeO
MeHO
OH
Deacetylvindoline N
N
COOMeMeO
MeHO
OCOCH3
Vindoline
+ H2O
2 x SAM
Hypothetical
NN
H
CHOMeOOC
H
H
H
Redoxchanges
NH
N
COOMeCatharanthine
N
N
MeOOC
H
N
N
MeOOCH
Oxidation
Acetyl-CoA
Oxidation
preakuammicine
stemmadenine
N
N
MeOOC CH2OHH
dehydrosecodine
NADH Hypothetical
124 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.
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 early example is shown below (J. Chem. Soc. Chem Comm. 1975, 670; JACS 1976, 98, 7017; Helv. Chim. Acta, 1976, 59, 2858):
NH
N
MeOOC
NMe
N
OAcOH
MeOOC
MeO
Vinblastine (R = Me)Vincristine (R = CHO)
NH
N
COOMeCatharanthine
NH
N
COOMe
PeroxidaseH2O2
HO
NH
N
COOMe
N
N
COOMeMeO
MeHO
OCOCH3
Vindoline
NH
N
MeOOC
NMe
N
OAcOH
MeOOC
MeO NH
N
MeOOC
NR
N
OAcOH
MeOOC
OH
MeO
Reduction
[O]
Reduction
Coupling
125
(see also: Accts Chem. Res. 2015, 48, 653-62; JACS, 2008, 130, 420; JACS 2009, 131, 4904; JACS 2012, 134, 13240; JACS 2016, 138, 8376-9; Org. Lett. 2012, 14, 1428). Finally, note that strictosidine is also the precursor to the quinoline alkaloids, including the important anti-malarial drug quinine. But that's another story......
NH
N
COOMeCatharanthine
NH
N
COOMe
O
NH
N
MeOOC
NMe
N
OAcOH
MeOOC
MeO
Vinblastine
NH
N
COOMe
N
N
COOMeMeO
MeHO
OCOCH3
Vindoline
NH
N
MeOOC
NMe
N
OAcOH
MeOOC
MeONH
N
MeOOC
NMe
N
OAcOH
MeOOC
OH
MeO
N
CONH2
COOH
- 40 oC
1) FeCl3, air2) NaBH4
+
5 steps. 40% yield overall
NNH
H
OMeOOC
O-Glu
NN
H
OHMeOOC
H
NH
CHO
N
NH2CHO
O N
N
NHO
MeO
Quinine