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3.

Introduction to Secondary
Metabolism and the Biosynthesis of
Natural Products

RA Macahig
PRIMARY METABOLITES INTERMEDIATE METABOLITES SECONDARY METABOLITES

+ Glucose Pentose phosphate


CO 2 H2O Erythrose-4-phosphate
Aromatic compounds
(C6-C1; C6-C2)
Shikimate
-
Phenylpropanoids (C 6 C3)

FM Dayrit
Phosphoenol pyruvate + Lignans
NH 3
Polysaccharides

Aromatic Aromatic alkaloids


amino acids
Citric acid Mixed alkaloids
cycle Pyruvate Aliphatic
amino acids
Aliphatic alkaloids

Acetyl-CoA Polyketides Polyphenols


Flavonoids
Phenylpropanoids

Polyacetylenes
Fatty acids Prostaglandins

Terpenes
Mevalonic acid
Steroids
Carotenoids

Iridoids

Alkaloids
Aliphatic
amino acids
Introduction
Metabolism: (Gr. metabole = change) the totality of the
chemical changes in living cells which involves the buildup and
breakdown of chemical compounds.
Primary metabolism: biosynthesis, utilization and breakdown
of the essential compounds and structural elements of the living
organism, such as: sugars and polysaccharides; amino acids,
peptides and proteins (including enzymes); fatty acids; and
nucleotides. The starting materials are CO2, H2O and NH3. All
organisms possess similar primary metabolic pathways and use
similar primary metabolites.

3. Secondary metabolites and Biosynthesis (Dayrit) 2


Introduction
Secondary metabolism: refers to the biosynthesis, utilization
and breakdown of smaller organic compounds found in the
cell. These compounds, called secondary metabolites, arise
from a set of intermediate building blocks : acetyl coenzyme A
(acetyl-CoA), mevalonic acid (MVA) and methyl erythritol
phosphate (MEP), shikimic acid, and the amino acids
phenylalanine/tyrosine, tryptophan, ornithine and lysine.
HO CH3
CO2H NH2
CO2H H2N
NH2 CO2H
O CO2H R
OH
CO2H NH2
SCoA HO OH
HO CH3
OH NH2 H2N CO2H
OH N
H
HO
OP

3. Secondary metabolites and Biosynthesis (Dayrit) 3


Introduction
Relationship between primary and secondary metabolism:
The processes and products of primary metabolism are
similar in most organisms, while those of secondary
metabolism are more specific.
In plants, primary metabolism is made up of photosynthesis,
respiration, etc., using CO2, H2O, and NH3 as starting
materials, and forming products such as glucose, amino
acids, nucleic acids. These are similar among different
species.
In secondary metabolism, the biosynthetic steps, substrates
and products are characteristic of families and species.
Species which are taxonomically close display greater
similarities (and metabolites); those which are distant have
greater differences.
3. Secondary metabolites and Biosynthesis (Dayrit) 4
Introduction
Biogenesis: overview of the origin of compounds starting from
the set of intermediate building blocks: acetyl-CoA, MVA and
MEP, shikimic acid, and the amino acids phenylalanine and
tyrosine, tryptophan, ornithine and lysine.
HO CH3 CO2H NH2
CO2H H2N
O NH2 CO2H
R
CO2H
OH
SCoA CO2H NH2
HO OH
HO CH3
OH NH2 H2N CO2H
OH N
H
HO
OP

Biosynthesis: detailed study of the step-wise formation of


secondary metabolites. At more detailed levels, the specific
enzymes, genes and signals are also identified.
3. Secondary metabolites and Biosynthesis (Dayrit) 5
PRIMARY METABOLITES INTERMEDIATE METABOLITES SECONDARY METABOLITES
CO2H

+ Glucose Pentose phosphate


CO 2 H2O Erythrose-4-phosphate HO OH
Aromatic compounds
OH
(C 6-C1; C6-C2)
Shikimate
-
Phenylpropanoids (C 6 C3)
Phosphoenol pyruvate + Lignans
NH 3
Polysaccharides CO2H

R
NH2

CO2H
Aromatic
amino acids
Aromatic alkaloids
*
Citric acid N
NH2 Mixed alkaloids
cycle Pyruvate
H
Aliphatic
amino acids
Aliphatic alkaloids
NH2

H2N CO2H

Acetyl-CoA Polyketides Polyphenols


SCoA

Phenylpropanoids
Flavonoids
*
Polyacetylenes
Fatty acids Prostaglandins
HO CH3

Overview of Mevalonic acid


Terpenes
Steroids
CO2H
Secondary OH Carotenoids

Metabolism Iridoids

* Metabolites found in 3. Secondary metabolites and Biosynthesis (Dayrit)


Aliphatic
Alkaloids
6 *
amino acids
higher organisms only
Metabolite linkage map
representing primary and
secondary plant metabolism
in opium poppy. The circles
associated with each
metabolite indicate whether
the metabolite was detected
(), not detected () or
masked ().

7
(Zulak et al. BMC Plant Biology 2008 8:5; www.biomedcentral.com)
Biogenetic classification of natural products.

3. Secondary metabolites and Biosynthesis (Dayrit) 8


The basic biogenetic and structural groups: Acetogenins

3. Secondary metabolites and Biosynthesis (Dayrit) 9


The basic biogenetic and structural groups: Terpenoids

HO CH3
OH

HO
OP
methyl erthritol phosphate

3. Secondary metabolites and Biosynthesis (Dayrit) 10


The basic biogenetic and structural groups: Shikimates
c. Shikimates: Shikimic acid phenylpropanoids

O
- CO2H
CO2H PO CO2 - -
O2C CO2

HO OH O CO2H
OH OH
OH

shikimic acid chorismic acid prephenate

CO2H NH2
CO2H
CO2H

OH OH
OH R
p-hydroxybenzoic acid
caffeic acid R=H, phenylalanine
R=OH, tyrosine

3. Secondary metabolites and Biosynthesis (Dayrit) 11


The basic biogenetic and structural groups: Alkaloids
d. Aliphatic alkaloids: Lysine aliphatic alkaloids

CH3N

H2N H2N CO2H

ornithine OH
tropine

e. Aromatic alkaloids: Phenylalanine aromatic alkaloids

NH2 N(H)CH3
HO
CO 2H CH3
CH3O

NCH3
HO
CH3
phenylalanine ephedrine
pellotine

3. Secondary metabolites and Biosynthesis (Dayrit) 12


Exercise

The following cytotoxic anthraquinone O H


derivative was recently isolated from the stem
N O
bark of Goniothalamus marcanii Craib.
Propose its biogenetic origin. Highlight the
appropriate atoms in the molecule. OCH3
OH O CH3

marcanin D

Propose its biogenetic origin of the following CH3O

alkaloid. Highlight the appropriate atoms in the NCH3


molecule. HO

CH 3O

CH 3O

OH
Exercises 2 & Answers
The following cytotoxic anthraquinone From Acyl-CoA From Methyl
derivative was recently isolated from the methionine
O H
stem bark of Goniothalamus marcanii
Craib. Propose its biogenetic origin. N O

Highlight the appropriate atoms in the


molecule. OCH3
OH O CH3
7 AcylCoAs + 2
marcanin D methyl
methionines

CH3O
Propose the biogenetic origin of the following
alkaloid. Highlight the appropriate atoms in the NCH3
HO
molecule.
From Shikimate CH 3O

CH 3O
From Methyl 2 Phenylalanines/
methionine OH Tyrosines + 2
methyl methionines
Chemistry of Natural Products (Dayrit) 14
Phylogenetics and natural products
Prevalence of secondary metabolites in various organisms:
Bacteria and Fungi: Fats & lipids, Acetogenins, Terpenes
Plants: +Phenylpropanoids, +Alkaloids
Variations of secondary metabolism exist in various organisms.
For example, recently a second pathway in the biosynthesis of
terpenes in plants was discovered. The first pathway is the
better-known mevalonic acid (MVA) pathway; the second
pathway is the methyl erythritol phosphate (MEP) pathway
which operates in the chloroplast.
Many of the early biosynthetic studies were conducted using
bacteria, in particular E. coli. It is possible that processes in
higher organisms differ, and that revisions may appear in the
future.
3. Secondary metabolites and Biosynthesis (Dayrit) 15
Phylogenetics and natural products:
Evolution of terpene biosynthesis in plants
Acetate

Mevalonate

C10 Iridoids Indole alkaloids


(Labiatae) (Apocynaceae)

C15 Sesquiterpenes Sesquiterpene lactones


(Myrtaceae) (Compositae)

C20 Diterpenes Diterpene acids


(Euphorbiaceae) (Leguminosae)

C30 Steroidal alkaloids


(Solanaceae)

3. Secondary metabolites and Biosynthesis (Dayrit) 16


Evolution of secondary metabolism in higher plants
(http://www.uk.plbio.kvl.dk/plbio/students-projects/evolution-sec-metaboites.pdf)

Cytochromes P450 and family 1


glycosyltransferases are key
enzymes in biosynthesis of
secondary metabolites found in
higher plants. Genomic and cDNA
sequencing programs of a number
of model plants have unravelled a
wealth of information on genes
and genomes giving better
understanding of evolution in
terrestrial plants.
Deduced sequences of genes can be used in the analysis of
phylogenetic trees to obtain their evolutionary relationship.

3. Secondary metabolites and Biosynthesis (Dayrit) 17


Introduction to Biosynthesis
This section will focus on the chemical transformations of
biosynthesis. It will also survey the enzymes which are
responsible for these transformations.

Natural products are unparalleled in the diversity and


complexity of chemical structures. Despite the complexity of
natural products, it should be emphasized that biosynthesis
proceeds by discrete chemically reasonable steps. That is, no
matter how complicated a natural product compound is, one
can rationalize its biosynthesis using a series of simple
chemical transformations,.

3. Secondary metabolites and Biosynthesis (Dayrit) 18


Why study the biosynthetic pathway?
The determination of the biosynthetic pathway enables us to
understand the relationships and dynamic flow of the compounds
that are present in a living cell.
The objective of the study of a biochemical sequence is to be
able to identify the intermediates and the product. However,
there are cases when this is not so obvious. During the chemical
extraction process, we obtain many of these compounds and the
problem is to determine the sequence of their formation.
An understanding of a biosynthetic sequence can help us identify
the enzymes and genes, understand the relationships among
different organisms (such as symbiosis, plant-insect interactions,
etc). An understanding of biosynthesis is part of a complete
understanding of plant biology, ecology and biodiversity.
3. Secondary metabolites and Biosynthesis (Dayrit) 19
An understanding of biosynthesis is very useful!
It enables us to classify the diversity and complexity of natural
products structures.
It reveals the functional relationships among natural products in
a dynamic context.
It provides essential information which enables us to control or
manipulate the formation of desired metabolites.
It opens up possible directions in biotechnology and molecular
biology through the study of enzymes (proteomics) and
genomics:
Genomics + Proteomics + Biosynthesis = Metabolonomics

3. Secondary metabolites and Biosynthesis (Dayrit) 20


Some types of biosynthetic pathways:
1. Simple linear process A C ..... X Y
B

2
.Mo
difie
dlin
earp
roc
ess C D

A B Y Z

M N

3
.Co
nve
rg
entp
ro
ces
s A B C

Y
D E

4
.Bra
nch
in
gpro
ces
s A B C D.......... Y

E G

5
.
M e
t
abo
l
i
c g
r
i
d A B C

D E F

3. Secondary metabolites and BiosynthesisG


(Dayrit) H Y 21
Some comments on biosynthetic pathways:
1. A compound is an obligatory intermediate if its formation is
required for the biosynthetic process to continue and there are
no alternative pathways. Such is the case for the compounds
in a linear pathway. In comparison, a metabolic grid provides
many alternative routes to the product.
A B C

C D
D E F
A B C . .. .. X Y A B Y Z

M N
G H Y

2. Although compounds are usually transformed from simple


structures to more complex ones, this is not always the case.

3. Secondary metabolites and Biosynthesis (Dayrit) 22


Some comments on biosynthetic pathways:
3. Different organisms may produce the same types of
compounds through different pathways (e.g., convergent
evolution), even if they are widely separated
phylogenetically.
4. Some compounds may be produced by the same organism
via more than one biosynthetic path. That is, there may be
more than one path available, such as in a modified linear
process or metabolic grid.
5. Even if the same compound is present in two different
organisms, it is possible that they are formed via different
pathways. This, however, is more likely for metabolites with
simple structures.

3. Secondary metabolites and Biosynthesis (Dayrit) 23


Some comments on biosynthetic pathways:
6. The production of secondary metabolites depends on genetic
and environmental factors. That is, secondary metabolites
may be present in the organism in various amounts depending
on the time of day or season, at particular stages of the
organisms life, or in response to certain environmental
stimuli (e.g., production of defense compounds).
7. Because these compounds are produced by specific enzymes
and precursors, it can be assumed that they are produced in
specific parts or organelles of the plant.
8. Secondary metabolites are probably in a state of dynamic
flux, being produced and broken down constantly. Some
compounds, however, may be stored in specific organelles
and have more constant presence.
3. Secondary metabolites and Biosynthesis (Dayrit) 24
General strategies for studying secondary metabolism:

1. Enzyme control. If the enzymes in the biosynthetic


pathway are known or have been isolated, these enzymes
can be blocked either by introducing enzyme inhibitors or
by causing mutations which alter the activities of these
enzymes.
2. Metabolite control. Many secondary metabolites are
controlled by a feedback mechanism. It is reasonable to
assume that there is a steady-state condition operating in the
organism where the concentrations of the metabolites are
maintained at some level. Effect on biosynthesis may be
negative (inhibitory) or positive.

3. Secondary metabolites and Biosynthesis (Dayrit) 25


Strategies for studying secondary metabolism:
Enzyme control
Example: the biosynthetic sequence in a linear process using
mutants or enzyme inhibitors
Experiment Biosynthetic process Comments

Overall process Ea Eb Ec
A B C D

Exp. 1 Ea
x x x A accumulates when enzyme Ea is
A B C D
blocked; B, CandDarenot formed

Exp. 2 Ea Eb
x x B accumulates when enzyme Eb is
A B C D
blocked; CandDarenot formed

Exp. 3 Ea Eb Ec
x C accumulates when enzyme Ec is
A B C D
blocked; Disnot formed

3. Secondary metabolites and Biosynthesis (Dayrit) 26


Strategies for studying secondary metabolism:
Metabolite control
Type Isotope used Method of Comments
Detection

3 14
Radioactive H, C scintillation Advantages: High sensitivity, requires only a
small amount of material
Disadvantage: special procedures required
due to radioactivity
2 13 19
Non- H, C, F NMR, MS Advantage: Structural information available
radioactive
Disadvantages: Relatively lower sensitivity;
expensive instrumentation

3. Secondary metabolites and Biosynthesis (Dayrit) 27


Examples of isotopically-label compounds used in
biosynthetic studies:

3. Secondary metabolites and Biosynthesis (Dayrit) 28


Examples of isotopically-label compounds used in
biosynthetic studies:
a. Skimmianine, in Choisya ternata (Grundon, Harrison and Spyropoulos, Chem. Comm., 51, 1974).

.
CH3O T T CH3O

. T

N O N O
H
Skimmianine

3H : 14C = 2 : 1 3H : 14C = 1.1 : 1

3. Secondary metabolites and Biosynthesis (Dayrit) 29


Examples of isotopically-label compounds used in
biosynthetic studies:
b. Ephedrine, in Ephedra distachya (Yamasaki, Sankawa and Shibata, Tetrahed. Lett., 4099, 1969).

OH

.
NH3+
CO2-
CH3

N(H)CH3

T5 T5

D,L-phenylalanine (-) ephedrine


[14C= nil]

c. Tyrosine, in Psuedomonas (Bowman, Gretton and Kirby, J. Chem. Soc. Perkin I, 218, 1973).

.
CO2-
. CO2-

NH3+ NH3+
T HO
T
phenylalanine
tyrosine

3. Secondary metabolites and Biosynthesis (Dayrit) 30


Major chemical transformations
in Biosynthesis
1. Hydrolysis
2. Esterification
3. Oxidation
4. Reduction
5. C-C Bond formation
6. Nucleophilic substitution
7. Elimination reaction
8. Cationic rearrangement

3. Secondary metabolites and Biosynthesis (Dayrit) 31


Major biosynthetic transformations
Reaction General equation Comments
Classification

b
.Ep
oxid
atio
n [O
] G
ene
rallys
tere
osp
ecific
O

c. Doublebond R1 R3 [2O] R1 R3
oxidation O O
R2 R4 R2 R4

3. Secondary metabolites and Biosynthesis (Dayrit) 32


Major biosynthetic transformations
Reaction General equation Comments
Classification

d
.De
hyd
rog
ena
tio
n H
H
H -2
H
H
H H

H C
l
e
.Ha
log
ena
tio
n

b. deoxygenation O
H OH H H

R1 R2 R1 R2 R1 R2

3. Secondary metabolites and Biosynthesis (Dayrit) 33


Major biosynthetic transformations
Reaction General equation Comments
Classification

5. C-C bond formation


a. Radical coupling OH O. O Commonly observed
-H . coupling in aromatic and
HO OH
conjugated systems
.

b. Claisen O Verycom monreaction,


O R COX
condensation 3 R1
_ e.g., inlenghteningof
R base R +X
1
R
2 polyketidechain
2

R3
O

O
c. Aldol O O
R1
R1
R2
R + R H base
2 3

O R3 OH

R1
R2
base
R3

3. Secondary metabolites and Biosynthesis (Dayrit) 34


Major biosynthetic transformations
Reaction General equation Comments
Classification

b
.Wag
n e
r- C
ommoninm onote
rpen
es
M
eerwe
ins
hift ands
esquite
rpe
n es
.
+ +

3. Secondary metabolites and Biosynthesis (Dayrit) 35


Major biosynthetic transformations
Reaction General equation Comments
Classification

3. Secondary metabolites and Biosynthesis (Dayrit) 36


Enzymes in biosynthesis
Most of the biosynthetic reactions are mediated by specific
enzymes. Enzymes have five fundamental properties:
1. increase in reaction rate - enzymes are catalysts which
increase the forward and reverse rates of a chemical step.
2. kinetic control - Enzymes are subject to various types of
control, such as pH and feedback.
3. chemoselectivity - Enzymes can distinguish functional
groups. For example, in an oxidation reaction: C-H C-
OH, chemoselectivity allows the differentiation between
various types of C-H, such as primary, secondary and
tertiary alkyl, olefinic and aromatic positions.

3. Secondary metabolites and Biosynthesis (Dayrit) 37


Enzymes in biosynthesis
4. regioselectivity - Regioselectivity is the ability of select
only one site of reaction from a number of possibilities of
the same functional group. For example, in a long chain
saturated fatty acid, the initial site of dehydrogenation is
typically 9,10. In a sugar, or a compound with many
-OH groups, the position of methylation is specific.
5. stereoselectivity - This refers to the chiral recognition of
substrates (compare with chemoselectivity).

3. Secondary metabolites and Biosynthesis (Dayrit) 38


Stereoselectivity in biosynthesis
Classification of stereoselectivity:
Enantioselective - The reactants are enantiomeric and the
enzyme reacts with only one enantiomer.
Prochiral - The carbon reaction center, CH2(R1)(R2), is not
chiral, but becomes chiral with substitution of one of the
hydrogens. In the case of a ketone, (R1)(R2)C=O, where
R1R2, reduction of the carbonyl to an alcohol produces a
chiral center at the carbon.
pro-R pro-S re-face
Hb Ha
R2 O
R1 R2 R1

si-face
3. Secondary metabolites and Biosynthesis (Dayrit) 39
Control of enzyme activity
An organism must be able to regulate its enzymes so that it
can coordinate its many biosynthetic activities and respond
to its environment. It is reasonable to assume that the
organism derives an advantage or fulfills a need when it
biosynthesizes secondary metabolites. Therefore, careful
control of their biosynthesis is an important ability.
There are two major types of control of biosynthesis:
inhibition of a specific enzyme by one of the
metabolites (protein inhibition); and
regulation by induction or repression of gene
expression.

3. Secondary metabolites and Biosynthesis (Dayrit) 40


Inhibition of enzyme activity
Feedback inhibition is one common mode of biosynthetic
regulation in which the changing concentration of a product
attenuates (decreases) the activity of an enzyme.
Allosteric control (Greek: allos, other + stereos, space or
solid) occurs when the binding of the substrate is
selectively increased or decreased by the binding of another
species at a different (allosteric) site on the enzyme.

3. Secondary metabolites and Biosynthesis (Dayrit) 41


Types of feedback control of biosynthesis.
1. Simple mass action: In a reversible process, if the ratio of
the concentrations of products over those of reactants, [P]/
[R], is not equal to the equilibrium constant, K, then the
equilibrium will shift accordingly.
2. Reversible competitive inhibition of the enzyme by the
product: In this case, the product slows down its own
formation by inhibition of the enzyme.
3. Product or reactant interacts with the DNA or RNA to
induce or repress the synthesis of the enzymes which are
responsible for the biosynthesis.

3. Secondary metabolites and Biosynthesis (Dayrit) 42


D

(-)
A. Negative feedback by one of the products: A B C D

D+E
(-)
B. Negative feedback by a combination D
Some types of of products: A B C }

control of E

biosynthetic C. Selective positive / negative feedback by products: D

activity through (-)


C D
the action of A B

metabolites on E F
(+)
enzymes. F

D. Allosteric control: E(+)=enzyme in active form; E(-)


E(-)=enzyme in inactive form; A=substrate; B= product;
P=positive effector; N=negative effector N P

E(+)
A B

3. Secondary metabolites and Biosynthesis (Dayrit) 43


A. General mechanism
Chromosome

Operator Gene 1 Gene 2 Gene 3

Schematic
representation of Enzyme 1 Enzyme 2 Enzyme 3

the mechanisms
A B C D
for inducing or
repressing gene B. Control by induction of transcription of enzyme synthesis by I.

function.
Operator + I Operator- I
Operator

(inactive biosynthesis) (active biosynthesis)

C. Control by repression of enzyme degradation by R.

Operator + R Operator
Operator -- R
I

(active enzyme (inactive enzyme


degradation) degradation)

3. Secondary metabolites and Biosynthesis (Dayrit) 44


Enzyme classification (EC) system

Classification (EC) Type of reaction catalyzed


-
1: Oxidoreductase oxidation-reduction: transfer of e from a donor which is
oxidized to an acceptor which is reduced
2: Transferase transfer of functional groups
3: Hydrolase hydrolysis, for example, of ester or amide groups, or
esterification
4: Lyase elimination of a group of adjacent groups of atoms to form a
double bond, or addition of a group of atoms to a double
bond
5: Isomerase conversion of a compound into its isomer
6: Ligase bond formation accompanied by ATP hydrolysis; also known
as synthetase

3. Secondary metabolites and Biosynthesis (Dayrit) 45


The IUB number and classification of enzymes

3. Secondary metabolites and Biosynthesis (Dayrit) 46


The four major types of biological oxidation reactions
catalyzed by oxidoreductases
Type of Description Schematic Reaction and Examples
Oxidation
D ehyd ro gen ase R em o ves o f tw o H atom s fro m the SH2 + A S + AH2
su bstrate, and tra n sfers th is to R R
a nother o rg anic com po un d. T h e H - R C H2
a ccepto r, A , is a co enzym e. C H2 R
H H

R R
CH OH C O
R R
R R
R C H2
C H2 R
H H
O

Oxidase RemovestwoHatomsfromthe SH2+O2 S+H2O


substrateandutilizesO2or H2O2as
SH2+H2O2S+2H2O
theH-acceptor.
OH O
1/2O2

OH O

3. Secondary metabolites and Biosynthesis (Dayrit) 47


The four major types of biological oxidation reactions
catalyzed by oxidoreductases
Type of Description Schematic Reaction and Examples
Oxidation
M o no o xyg ena s e A d d s on e O a to m to the su b stra te. A S + AH2 + O 2 SO + A + H2O
is a co en zym e. R R R R

H H H H
O
R C H2
R CH2 CH R
C H2 R
OH

O O

C C
R H R OH
Dioxygenase AddstwoOatomstothesubstrate S+O2SO2
R1 R2 R1 R2
O2
O + O
H H H H

3. Secondary metabolites and Biosynthesis (Dayrit) 48


Elimination and rearrangement reactions following oxidation
A. Demethylation: Methyl ether to alcohol

R CH3 [O]
R CH2 + HCHO
O R OH
O O-H
B. Demethylation: Methyl amine to amine

R1 [O] R1 CH2 R1
N CH3 N O-H NH + HCHO
R2 R2
R2

C. Formation of phenyl methylenedioxy ring

O-CH3 O-CH2 OH O
[O] -H2O CH2

OH O
OH

3. Secondary metabolites and Biosynthesis (Dayrit) 49


Elimination and rearrangement reactions following oxidation
D. Aromatic ring opening reaction (mono-oxygenase)

[O]
O O

E. Aromatic ring opening reaction (dioxygenase)

O O
O H O
OH OH
[O2] OH CHO
_ CO2H
OH +
OH OH OH
F. Oxidation of aromatic ring: NIH shift (hydride shift); R = alkyl group

D hydride
D O isotope
[O] shift OH
effect
O
R H
R R R D
H D

3. Secondary metabolites and Biosynthesis (Dayrit) 50


Elimination and rearrangement reactions following oxidation
G. Para oxidation of aromatic ring.
H O
[O] OH
O _ H
+
R-O -
RO R-O R-O
H

H. Oxidative decarboxylation of aromatic carboxylic acid.


_ O _
CO2 O OH
[O] -CO2
O

3. Secondary metabolites and Biosynthesis (Dayrit) 51


Oxidative coupling of phenols
A. Illustration of phenoxy radical formation, resonance stabilization and coupling: Pummerer's ketone.

H
_
OH O _ . O O
base -e
.
H3C H3C H
H3C H3C

O. O

.
H3C H3C

O O O
O
+
. .
H3C H H3C
H3C H CH3

OH O
O OH

H3C
H3C CH3
CH3

3. Secondary metabolites and Biosynthesis (Dayrit) 52


Oxidative coupling of phenols
B. Some important phenolic structures which can undergo phenolic coupling.

OH OH HO O OH CHO OH

* * HO
* * *

HO OH HO CH3
* * * * *
O
H3C CH3

HO CO2H CH2OH
*
*
*
HO HO

OH O-CH3

3. Secondary metabolites and Biosynthesis (Dayrit) 53


Carbon-carbon bond formation by Sn2 displacement of a
stable nucleophile on an electrophilic alkylating agent.
A. Methylation of alcohol or amine with S-adenosyl-L-methionine as alkylating agent..

R OH H3C + (Adenosyl) +
-H R OCH3
S

H2N
CO2H

B. Glycosylation of an alcohol with glycosyl phosphate as alkylating agent.

HO R
HO O
OP Gly R
O
HO OH
OH

3. Secondary metabolites and Biosynthesis (Dayrit) 54


Carbon-carbon bond formation by Sn2 displacement of a
stable nucleophile on an electrophilic alkylating agent.
C. Alkylation of a stabilized carbanion with acetyl CoA as alkylating agent.
_ O
O O O _
-CO2 O O
R CH2
_
R CH2 O R CH2
O R CH3

H3C S-CoA

D. Sn2 displacement of pyrophosphate.

OPP + _
-H , -OPP
H H
OPP
OPP

Note: One common series of reactions for Sn2 displacement is:


phosphorylation of R-OH group R-OPP-, followed by
Sn2 displacement of OPP- by nucleophile.
3. Secondary metabolites and Biosynthesis (Dayrit) 55
Control of biosynthesis in plants
Plants exercise control over the biosynthesis in several
ways:
First, the enzymes are coded for separately allowing better
control of each enzyme.
Second, several of the enzymes exist in more than one
form. It is believed that the existence of isozymes allows the
plant better regulation of biosynthesis.
Third, some of the biosynthetic transformations can take
more than one pathway.

3. Secondary metabolites and Biosynthesis (Dayrit) 56


Control of biosynthesis in plants: alternative
pathways to tyrosine (a modified linear process)
CH2CCO2H
O
prehenate 4-hydroxyphenylpyrivate
dehydrogenase, transaminase, pyridoxal-5'-
NAD + phosphate
CH2CHCO2H
OH
NH2
HO2C CH2CCO2H 4-Hydroxy
phenylpyruvic acid
O
4-hydroxyphenylpyrivate
transaminase, pyridoxal-5'- pretyrosine
phosphate dehydrogenase, OH
NAD +
OH Tyrosine
Prephenic acid HO2C CH2CHCO2H
NH2

OH
Pretyrosine

3. Secondary metabolites and Biosynthesis (Dayrit) 57


Localization of enzymes

One of the important phenomena of living organisms is cell


structure and differentiation. This means that many functions
of cells are localized in certain parts of the cell and that
different types of cells within the same organism have
different functions.
Enzymes of different types can be found in all parts of the
cell. While many types of enzymes are assumed to function
in the cytosol, some enzymes are known to be localized in
specific parts of the cell and be active only under certain
conditions.

3. Secondary metabolites and Biosynthesis (Dayrit) 58


Localization of enzymes

One well studied system is fatty acid synthase. Fatty acids


play different roles in the organism. First, fatty acids are a
form of energy storage; second, fatty acids are essential
constituents of the cell membrane; third, fatty acids are
sometimes found to be components of other natural products
(R-OH) being attached as esters.
Consistent with this observation, the synthesis of fatty acids
takes place in three different sites of the cell and is mediated
by three enzymatic systems: the mitochondrial system, the
cytoplasmic system, and the microsomal system.
We will discuss this further when we cover fats.

3. Secondary metabolites and Biosynthesis (Dayrit) 59


Comments regarding biosynthetic mechanisms
There are three approaches to the study of natural products:
Classification of natural products according to activity, such
as pharmacological activity (e.g., antioxidants) or ecological
function.
Classification based on structural types and physico-chemical
properties, for example, phenolics, glycosides, etc.
Classification according to biogenetic origins or biosynthetic
pathways.

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Advantages of the approach of biosynthesis
It follows established principles and mechanisms of organic
chemistry.
This approach readily links with the fields of biochemistry,
genetics, ecological interactions and evolutionary
development.
It also provides insight into the structural relationships
among secondary metabolites.

The biosynthetic mechanism can be used to guide further


research into the search for enzymes and genes.

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Tips on biosynthetic mechanisms
How does one judge a good from a bad biosynthetic
mechanism?
1. A good mechanism is based on precedent: it should
follow patterns of known biosynthetic transformations.
2. If appropriate, the mechanism should start with
intermediate metabolites which are already well known.
3. It should use known enzymatic transformations.
4. There should be economy of reaction.
5. The transformations should not be too cluttered.

3. Secondary metabolites and Biosynthesis (Dayrit) 62


Summary
1. All secondary metabolites, no matter how complex, are
biosynthesized via discrete chemically-reasonable steps. The
biosynthetic transformations are classified as follows:
1. hydrolysis
2. esterification
3. oxidation: hydroxylation, epoxidation or oxygenation of alkene,
dehydrogenation, halogenation
4. reduction: hydrogenation, deoxygenation
5. carbon-carbon bond formation: aromatic radical coupling,
Claisen condensation, aldol condensation
6. Cationic rearrangement: 1,2-migration, Wagner-Meerwein
7. Rearrangement under control of orbital symmetry
8. Sn2 displacement
9. E2 elimination
10. carboxylation / decarboxylation
3. Secondary metabolites and Biosynthesis (Dayrit) 63
Summary
2. Each step is presumed to be mediated by a specific enzyme.
All chemical transformations are accounted for by the
system of six enzyme classes:
1. oxidoreductase
2. transferase
3. hydrolase
4. lyase
5. isomerase
6. ligase
3. The enzymes are located in specific parts of the cell, and in
some cases may be immobilized on a membrane.
4, The enzymes are coded for in the plants genome whose
expression can be controlled at the level of the gene.

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