102

4. Biosynthesis of Natural Products Derived from Shikimic Acid

4.1. Phenyl-Propanoid Natural Products (C6-C3)

The biosynthesis of the aromatic amino acids occurs through the shikimic acid pathway, which is found
in plants and microorganisms (but not in animals). We (humans) require these amino acids in our diet,
since we are unable to produce them. For this reason, molecules that can inhibit enzymes on the shikimate
pathway are potentially useful as antibiotics or herbicides, since they should not be toxic for humans.

COO

COO
NH3
R = H Phenylalanine
NH3 R = OH Tyrosine Tryptophan
R N
H

The aromatic amino acids also serve as starting materials for the biosynthesis of many interesting natural
products. Here we will focus on the so-called phenyl-propanoide (C6-C3) natural products, e.g.:

OH OH
OH

O HO O
HO OH HO

Chalcone
OH O a Flavonone OH O a Flavone
OH O
OH
OH
O Ar O
RO
O O
HO
O
OH O
OR OH
Anthocyanine Podophyllotoxin
OH O a Flavonol MeO OMe
OMe
OH COOH

Cinnamyl alcohol HO O O
Cinnamic acid
OH (Zimtsäure) Umbellierfone
OH
a Coumarin)
MeO
OH
O COOH
HO Polymerization
OH
Wood
OH HO OH
O
OH
MeO
OMe Shikimic acid
O
HO

4.2. Shikimic acid biosynthesis

The shikimic acid pathway starts in carbohydrate metabolism. Given the great social and industrial
significance of this pathway, the enzymes have been intensively investigated. Here we will focus on the
mechanisms of action of several key enzymes in the pathway. The following Scheme shows the pathway
to shikimic acid:
 103

COO- COO-
Phosphoenolpyruvate HO COO-
2-O P-O O
3 2-O DHQ-Synthase
3P-O
2-O
3P-O DAHP-Synthase HO OH O OH
O OH
HO OH
OH 3-Deoxy-D-arabinoheptulo- Dehydroquinate
sonate-7-phosphate (DAHP) (DHQ)
D-Erythrose-4-phosphate
COO- COO- COO-
Shikimate Shikimate
Dehydroquinase Dehydrogenase Kinase

O OH OH 2-O P-O OH
HO 3
OH OH
OH
Dehydroshikimate Shikimate Shikimate-3-phosphate
COO-
COO-
2-O COO- COO- OH
3P-O Chorismate
Synthase
O COO-
EPSP-Synthase Isochorismate
2-O O COO- O
3P-O COO-
OH Vitamin K
OH
5-Enolpyruvylshikimate- O
Chorismate
3-phosphate (EPSP) HOOC
Anthranilate Chorismate COOH
Synthase Mutase

COO- COO- ⊕
⊕ OH
NH3 NH3 Aminodeoxychorismate
Tryptophan Synthase Prephenic acid
O COO-
Anthranilic acid
COO- COO-
COO-
COO-
THF
HO NH3 NH3
O COO- ⊕ ⊕
p-Aminobenzoic acid Tyrosine
⊕NH3 ⊕ NH3 Phenylalanine

DAHP-Synthase
At first sight this seems to be a straightforward Aldol-like reaction between phosphoenolpyruvate (PEP)
und erythrose-4-phosphate. However, for unknown reasons, Nature has made this more complicated than
it appears:

-O O COO-
P DAHP-Synthase COO-
-O
O
O
2-O
2-O 3P-O
3P-O
O
HO HO OH
OH OH

Experiments with 18O-labelled PEP have shown that all of the 18O label is lost with phosphate - none is
incorportated into the aldol-product. Other labelling experiments with Z-[3-3H]-PEP have shown that the
reaction proceeds stereospecifically, even with respect to the new prochiral center in the product. The Si-
face of the PEP must add to the Re-face der carbonyl group. A likely mechanism is :
 104

O
-O O COO- O COO-
O
P -O COO-
O P
-O
HB O O
-O HB 2-O
3P-O
HA HA
2-O 2-
O3P-O H
3P-O HO OH
O OH
HO H HO H OH
OH OH

3-Dehydroquinate Synthase
This is a very interesting enzymic reaction. At first sight, it is not clear what the reaction mechanism is.
The enzyme needs NAD+ as coenzyme, but this is not consumed during the reaction (no net redox
change):

1
COO- HO COO-
O 2
DHQ-Synthase
2-O
3P-O
6 4 NAD+
5 O OH
HO OH
OH OH
3-Deoxy-D-arabinoheptulo- Dehydroquinate
sonate-7-phosphate (DAHP) (DHQ)

It was shown that when DAHP is labelled at C5 or C6 with 2H (deuterium), then a significant kinetic
isotope effect on the reaction rate can be observed (i.e. slower with the deuterated substrates). This
implies that both the C(6)-H and the C(5)-H bonds are cleaved during the reaction. The following
mechanism was suggested:

H O
HO HO
H
O
HOOC O HOOC O
O-P O
H
OH OH P O
-
+ NADH O
H
NAD O-
HO O
OH HO
HOOC O
HOOC O
O
H OH

H H
HO HO
H
OH O
-O HOOC DHQ
HOOC
O
O OH

This mechanism has been suggested, on the basis of studies carried out over many years. At first sight the
enzyme appears to catalyze: 1) a redox reaction, 2) an elimination, 3) another redox reaction, 4) an aldol-
like reaction. At least the chemical logic of oxidizing the alcohol group then becomes clear.

How does one active site achieve all this ??

 105

Modifications to the phosphate at C-7 have a dramatic effect on rate, suggesting that it plays an active
role in the elimination step.

It is known that the labelled substrates 7S- und 7R-[7-3H]-DAHP are converted into labelled products
with overall inversion of configuration at C7. So the C-C bond-forming step also proceeds
stereospecifically (Proc. Natl. Acad. Sci.USA 1970, 67, 1669). In a model study, however, it was also
shown that the the aldol-like reaction can proceed rapidly and also stereospecifically without catalysis by
the enzyme (JACS, 1988, 110, 1628):

H H H
HO HO HO
OH H
hν, 0oC OH OH
HOOC O HOOC O HOOC
H H
D O
O OH OH
O D D
NO2

Apparently, the steps that really need the catalytic action of the enzyme, in order to achieve rapid
turnover, are those involving the redox changes (alcohol ketone) with the coenzyme NAD. The
catalytic power of the enzyme appears to be focused on making these steps fast, and perhaps is less
crucial for providing catalysis for the elimination and aldol-like reactions, which proceed fast anyway if
the substrate is bound in an optimal conformation.

EPSP-Synthase
The sixth step in shikimic acid biosynthesis is the EPSP-synthase reaction. This enzyme has been
intensively investigated, not least because it is the target of the commercially important herbicide
Glyphosate, which inhibits the enzyme :

COO-
COO-
COO- 2-O
⊕
2-O
3P-O
+ Pi 3P NH2
2-O
3P-O O COO-
OH COO-
2-O
3P-O OH
5-Enolpyruvylshikimic acid- Glyphosate
OH EPSP-Synthase 3-phosphate (EPSP)

Glyphosate is effective in killing a wide variety of plants, including grasses, broadleaf, and woody plants.
It has a relatively small effect on some clover species. By volume, it is one of the most widely used
herbicides. It is commonly used for agriculture, horticulture, and silviculture, as well as garden
maintenance (including home use). Some crops have been genetically engineered to be resistant to
glyphosate. Glyphosate was first sold by Monsanto under the tradename "Roundup".

Mechanism of the EPSP synthase reaction ?

-- the phosphate group is lost from PEP with cleavage of the C-O bond, not the P-O bond.
-- If the enzymic reaction is carried out in D2O, then deuterium is incorporated into the product, and is
found equally distributed between the E- and Z-positions in the enolpyruvyl group.
-- If [3-2H2]PEP is used as substrate in H2O then 2H is lost in equal amounts from the E- und Z-
positions in the enolpyruvyl group in the product.

These observations have led to the proposal of an addition-elimination sequence, as shown below:
 106

COO
COO COO H
CH2
2-O
3P-O

2-O O
2-O OH 3P-O COO
3P-O EPSP-Synthase OH
OH OPO32

In one key experiment, the existence of the tetrahedral intermediate was proven. The enzyme (800µM)
+S3P (800µM) + 2-[13C]-PEP (1mM) was mixed for 5s, and then quenched with Et3N. Ion exchange
chromatography of the resulting products gave a small amount of the intermediate that could be
characterized.

Glyphosate is a potent inhibitor of EPSP synthase. The inhibition ist competitive with respect to PEP (Ki
= 1µM) but non-competitive with respect to S3P (Eur. J. Biochem. 1984, 143, 351).

E + S ES E + P

E + S ES E + P
EI

EI + S ESI

E + S ES E + P

ESI

Crystallographic studies have revealed how the substrate, intermediate, and glyphosate bind at the active
site of the enzyme. A substrate analogue Z-3-fluoro-PEP acts as a pseudosubstrate and forms a relatively
stable tetrahedral intermediate that could be crystallized on the enzyme (Mol. Microbiol. 2004, 51, 963).

Chorismate Mutase
The chorismate mutase reaction involves formally a Claisen rearrangement. This reaction occurs at a
measurable rate in aqueous solution even in the absence of the enzyme (t1/2 in water at 50oC ≈ 90 min),
but the reaction is accelarated about ≈106 fold by the enzyme :
 107
COOH
COO-
Chorismate HOOC
Mutase O

O COO-
OH
Prephenic acid
Chorismate OH

The enzymic and the spontaneous reactions could proceed through either chair-like or boat-like transition
states. The stereochemical consequences, however, are different:
COO

COO- O
O COO-
COO-
boat-like TS

COO- OH
OH

O
-OOC
HO COO-
O COO-
COO-
chair-like TS O
COO-
OH
OH
The stereochemical course of both enzymic and spontaneous reactions has been studied, and both have
been shown to proceed through chair-like transitions states (JACS, 1984, 106, 2701; JACS 1985, 107,
5306).

Other kinetic and spectroscopic studies have shown that the enzymic reaction most likely is a more-or-
less concerted pericyclic reaction. The slowest step appears to be release of product (prephenate) from the
enzyme (Biochemistry, 1990, 29, 8872).

Prephenate dehydrogenase and prephenate dehydratase

The conversion of prephenate to p-hydroxyphenylpyruvate is catalyzed by the enzyme prephenate
dehydrogenase, which requires NAD+. Kinetic isotope studies have suggested that the reaction proceeds
in a concerted manner, as shown below :

O
OOC
COOH Prephenate dehydrogenase COOH

O
HO
HO H N R NAD + NADH

H 2NOC

Finally a transaminase (PLP-dependent) converts the α-ketoacid into the amino acid tyrosine. For the
production of phenylalanine, the enzyme prephenate dehydratase produces first phenylpyruvate, and then
again by transamination the amino acid Phe :

O
OOC Prephenate
COOH Transaminase COOH
COO dehydratase
O NH2

HO H
 108
Chorismate also plays a key role as precursor to several other very important natural products, including
the amino acids tryptophan, p-aminobenzoic acid, as well as p-hydroxybenzoic acid and salicyclic acid.

4.3. Coumarins, Flavonoids und Lignans

Phenylalanine and tyrosine also act as precursors to a large variety of C6C3-Phenylpropanoide natural
products in plants:
O

COOH
OH OH
NH2
X HO
cinnamic acid p-coumaryl alcohol
MeO
OH
coniferyl alcohol
HO
Two interesting coumarin derivatives are dicumarol und warfarin, which can prevent blood clotting and
are used clinically to treat thrombosis :

HO Ph O
OH OH

O O
O O O O O O
coumarin
Dicumarol Warfarin
Flavonoids and stilbenes are products from a pathway that uses cinnamoyl-CoA as starter unit, and
extends the chain with malonyl-CoA extender units, just like in polyketide biosynthesis. Flavonoids such
as Quercitin (in red wine) and catechins (in tea) can act as anti-oxidants. Flavonoids contribute to plant
flower colours; yellow from chalcones and flavonols; red, blue and violet from anthocyanidins. Many of
these are also found in glycosylated forms in plants. Resveratrol (red wine) has recently been shown to
promote longevity in animals:

OH OH OH
OH
HO O HO O HO O
OH OH
HO

OH OH
OH O OH O OH
OH Resveratrol
(a stilbene) Naringenin Quercetin Cyanidin
(a flavanone) (a flavonol) (an anthocycanidin)
Cinnamic acid is also used for the biosynthesis of lignin. Apart from cellulose, lignin is the main
component of wood. Lignin is a high molecular weight polymeric material, produced by polymerization
of coniferyl alcohol.
MeO
OH O
O OH
OH
O
HO
NH2 MeO O
MeO O MeO OMe
O HO
OMe
O
OH OH HO O
(E)-Coniferyl alcohol MeO OH
Tyrosine
O O
O
HO
OMe OMe
HO
OH O
O
O
OMe MeO OMe
O
representative section OH
OH
MeO
O of a molecule of lignin MeO
O
O
HO
Pinoresinol
OMe HO
O
HO O
HO

MeO
O OMe
OH
 109
Plant cell walls are complex structures composed mostly of lignocellulose — the most abundant organic
material on Earth — a matrix of cross-linked polysaccharide networks, glycosylated proteins, and lignin.
This matrix has three main components: cellulose (38–50%), hemicellulose (17–32%), and lignin (15–
30%).

Cellulose is a polysaccharide consisting of a linear chain of several hundred to more than 10,000 D-
glucose units linked by β-1,4 bonds. This bonding motif differs from the α-1,4 glucose linkage of starch,
such as corn starch that comes from corn kernels.

This structural difference proves to be quite significant. Cellulose chains are linear and somewhat rigid,
but starch takes on a coiled chain structure. That makes the cellulose chains amenable to forming
numerous hydrogen bonds, which, unlike starch, leads the cellulose chains to assemble into cablelike
bundles of crystalline fibrils that have high tensile strength and are resistant to hydrolysis to glucose.

Hemicellulose is also a polysaccharide, but it is typically made up of chains of xylose interspersed with
side chains containing arabinose, galactose, mannose, glucose, acetyl, and other sugar groups, depending
on plant type. Hemicellulose contains 500 to 3,000 sugar units and includes a small amount of pectin,
another polysaccharide, with which it forms a cross-linked network.

Lignin is a cross-linked macromolecule composed of three types of substituted phenols

(phenylpropanoids). It fills the spaces in the cell wall between cellulose, hemicellulose, and pectin and is
covalently linked to hemicellulose. Lignin resembles a kind of phenol-formaldehyde resin that acts like
glue to hold the lignocellulose matrix together. Lignin helps provide additional strength to cell walls and
resistance to insects and diseases (C & E News, 2008, Dec. 8, p.15).
 110
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:
e.g. Pyrrolidine, Pyrrolizidine and Tropane Alkaloids
MeN HO OH
Me O
NH3
O
H 3N O
COO OH
O N
Ornithine N
Hygrine Scopolamine Retronecine
Me
MeN Ph

Tropinone
O

e.g. Piperidine, Pyridine und Quinolizidine Alkaloids

OH
NH3 O
H3N COO N N
Me N N
H
Lysine Coniine N Me
Me Nicotine Lupinine
N-Methylpelletierine
N

N
N O
Sparteine
Lycopodine
z.B. Isoquinoline Alkaloids
MeO
MeO
COO
NMe
N HO
MeO
NH3
R MeO OMe
Papaverine
Phenylalanine
Autumnaline
Tyrosine MeO OMe
HO OH
MeO
NHAc HO
MeO O
NH2
MeO NMe HO
O Dopamine
Colchicine Morphine
OMe HO

z.B.Indole Alkaloids
N
COO
NH2 N
N
NH3
H MeO N OAc
N N
Tryptophan H Me OH
H
Tryptamine COOMe
+ Terpene MeOOC
Vindoline
OH
Geissoschizine
N

N N
H OH N
MeOOC
N
H COOMe
Vinblastine
MeO N OAc Catharanthine
Me OH
MeOOC
 111
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:

Hydroxylase
Decarboxylase HO
(PLP) NH2 HO
HO
NH2
HO NH
COOH HO
H
NH2 O COOH CHO
HO
Tyrosin HO
Transaminase Decarboxylase
(PLP) (TPP) HO Norcoclaurine
HO

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:

HO HO
HO O
NH2 NH
HO NH NH HO
HO HO H
H

CHO
HO
HO HO
Norcoclaurine
HO

Next, the norcoclaurine is converted into (S)-reticuline :

HO MeO
SAM SAM Hydroxylase
NH N-Me
HO HO
H SAM H
HO

HO Me-O
Norcoclaurine Reticuline

Reticuline is used for the biosynthesis of many other benzylisoquinoline alkaloids, amongst others, the
so-called aporphine alkaloids, e.g.:

MeO MeO
MeO
NMe NMe
NMe HO MeO
HO H H
H HO MeO
HO

MeO MeO Glaucine

MeO
 112

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.:

MeO MeO MeO

MeO 2 FeII
-2H+ NMe NMe NMe
NMe O O O
HO H H
H H
2 FeIII

MeO K3Fe(CN)6 MeO MeO

MeO
O O O
OH

MeO MeO MeO

NMe NMe NMe

O HO HO
H H H + H
H HO

MeO MeO
MeO
O OH
ortho-para ortho-ortho

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.:

0 HO HO O
electrons OH O O
OH2 + O2 O OH

FeIII FeIV Fe Fe
S-Cys + H2O
S-Cys S-Cys
S-Cys
H2O
P450 enzyme compound-I
(resting state)

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.

 113

(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:

from [2-14 C]-Tyrosin

MeO oxid.
MeO MeO phenol
coupling
NMe NMe HO ortho-
HO Oxid. HO
H H para
HO Salutaridine
HO
Red. N-Me

MeO MeO MeO

(S)-Reticuline OH
(R)-Reticuline

Salutaridine is found as a minor alkaloid constituent in the opium poppy:

MeO MeO AcOH MeO

CoASH
Reduction Acetyl-CoA
HO HO
O
N-Me N-Me N-Me

MeO MeO MeO

O Salutaridinol OH Thebaine

MeO MeO
MeO

O O
O
N-Me
N-Me N-Me
O
O O
Codeinone
OH Neopinone

MeO HO

O O
N-Me N-Me
HO HO
Codeine Morphine
 114
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.

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:

1) MeI / K2CO3 /
HO MeOH MeO MeO
2) Ag2O, then EtONa /
pyrolysis EtOH, Δ
HO
O O
N-Me NMe2
+
HO Morphine HO 1) Ac2O NMe2
2) CrO3 EtO

MeO MeO MeO

H2SO4 1) H2O2
O Δ O 2) NaOH/H2O AcO O
COOH
O O 3) H3O
O

NaOH/
Me2SO4
MeO MeO
heat/ H+
MeO MeO + CO2

HOOC

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 for
 115
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:

Tyrosine MeO
COOH MeO
NMe NHAc
NH2 HO
HO MeO
H
Colchicum MeO MeO
H 2N COOH
O

MeO OMe
(S)-Autumnaline Colchicine
Phenylalanine OH

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.

Dopamine
HO HO
HO
NH2 N NH
HO HO
HO
H
CHO
Tyrosine cf. above

Phenylalanine
OH OH
OH
HO
HO
N-Me
N-Me MeO
MeO MeO
MeO
MeO OH
Isoandrocymbine MeO O
(S)-Autumnaline

O-Methylandrocymbine has been isolated from Androcymbium melanthioides. The later steps have not
been proven, but may involve the following reactions:

Oxidation

MeO MeO HO MeO

NH-Me NHMe N-Me
MeO MeO MeO
MeO MeO MeO

MeO O MeO O MeO O

O-Methylandrocymbine

O
MeO MeO Me
NH-Me NH
MeO H Demethylation MeO
MeO Colchicine
MeO
O Acetylation O
HCHO
OMe OMe
 116

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:

HO
L-Phe
HO
HO CHO
SAM
HO
H2N
HO NH
L-Tyr
OH
Norbelladine
Thereafter, three different modes of phenol coupling are seen:
HO HO para-ortho-
coupling HO

MeO MeO
MeO
NH NH
HO HO N
HO
4'-O-methylnorbelladine OH
OH Norpluvine
MeO

NH
HO OH
NMe
HO
HO para-para-
coupling
O
MeO O
ortho-para- N
coupling MeO O
O Lycorine
HO NH

NMe O
HO
OH
MeO
MeO
N OMe
HO
Oxocrine OH
O NMe MeO

HO N
MeO
Galanthamine Haemanthamine
 117
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):

COO R
NH2 R = H Tryptamine
NH3 R = OH Serotonin
N
N H
H

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.

N
N
N
N
H N
H
MeO N
H OAc H COOMe
Me OH
MeOOC Catharanthine
MeOOC
Vindoline
Geissoschizine OH
N
N H N
N
H OH
H MeOOC N
N
H N H
H
MeOOC Strychnine
OH O O Vinblastine
H MeO N OAc
Stemmadenine Me OH
MeOOC

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):
 118

HO Me
CHO
H O-Glu
O-Glucose

OH H O
O MeOOC
MeOOC
Loganin Secologanin
Geraniol
Secologanin is a glucoside, which can be cleaved by hydrolysis under acidic conditions:

OH
HO OH
O
CHO
OH H3O
O

O
MeOOC

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):

NH2 NH
N NH N
N
H CHO H
H OGlu
O-Glu
O-Glu
O O
MeOOC MeOOC
O
MeOOC Strictosidine

Strictosidine is then a key intermediate in the formation of over 1000 different indole-terpene alkaloids
(Nat. Prod. Rep. 2012, 29, 1176).
 119
For example, the Corynanthe alkaloids:

Glucose

NH NH N
N N N
H H H
O-Glu CHO
H
O Acetal OH OH
MeOOC MeOOC MeOOC

N
NADPH N
N N
N H
H H

OH MeOOC
H
MeOOC O
MeOOC OH
Geissoschizine

NADPH N N
N N
N
N H H
H H H (Imine
Me
reduction) H
H O 2 NADPH
Ajmalicine O MeOOC MeOOC
MeOOC OH

N
N
H H

Yohimbine H
MeOOC
OH

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,
 120
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:

OHC OHC
see above COOMe
NH COOMe
N N
H N N
OGlu N
H H H H
O
MeOOC Polyneuridine Aldehyde
Dehydrogeissoschizine
Strictosidine
MeOH
CO2
O
AcO Acetyl-CoA
AcO H H

N N OH N
N N N
H H H
Oxidation H
16-epi-vellosimine
Vomilenine Vinorine

NADPH Reduction

HO
AcO AcO
NADPH Hydrolysis
N N OH
N N H
N N OH OH
SAM Me
HH H H H
H Reduction H
Ajmaline H
17-O-Acetylnorajmaline H
Dihydrovomilenine

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 -
 121

Hypothetical

H N
N N N
N N
H
H H H H
H
H H
MeOOC CHO
MeOOC CHO MeOOC CHO
Geissoschizine

Redox N
N changes N

N
N N
H
H CHO
MeOOC CH2OH MeOOC MeOOC CHO
preakuammicine
Hypothetical
NADH
N N
N

N N
N H
H H
MeOOC CH2OH
MeOOC CH2OH MeOOC
stemmadenine dehydrosecodine

N
N Oxidation N

N
N H
HO N H MeOOC
H COOMe COOMe
Tabersonine
16-Hydroxytabersonine
N
+ H 2O

2 x SAM N
H COOMe
N N
Oxidation
Catharanthine
Acetyl-CoA
OH N
MeO N MeO N
Me HO Me HO COOMe
COOMe
Desavetoxyvindoline Deacetylvindoline N OCOCH3
MeO
Me HO COOMe

Vindoline

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
 122
(which is dose-limiting), gastrointestinal toxicity, potent vesicant (blister-forming) activity, and
extravasation injury (forms deep ulcers).
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.

Peroxidase
N HO
H2O2 N N

N N
N
H COOMe H
H COOMe COOMe
Catharanthine

Coupling N

N
MeO N OCOCH3
N Me HO COOMe
H
MeOOC N
Vindoline
Reduction

MeO N OAc
N
Me OH
MeOOC
N
H
MeOOC N
N
[O]

MeO N OAc N
Me OH Reduction H OH
MeOOC MeOOC N

MeO N OAc
R OH
Vinblastine (R = Me) MeOOC
Vincristine (R = CHO)

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:
 123

N N O
N

N N N
H COOMe H COOMe H COOMe
Catharanthine

+
N N

N
H MeO N OCOCH3
MeOOC N
Me HO COOMe

Vindoline
CONH2 MeO N OAc
Me OH
MeOOC
N

COOH N

- 40 oC N
H
MeOOC N

N
MeO N OAc
N Me OH
H OH MeOOC
MeOOC N

1) FeCl3, air
2) NaBH4
MeO N OAc
Me OH
Vinblastine 5 steps. 40% yield overall
MeOOC

(see also: JACS, 2008, 130, 420; JACS 2009, 131, 4904).

Finally, note that strictosidine is also the precursor to the quinoline alkaloids, including the important
anti-malarial drug quinine. But that's another story......

CHO
NH N
N N
H H N
O-Glu N
H
H
O OH
MeOOC MeOOC

HO O
N N

MeO
CHO
NH2
N

Quinine