030-intro-to-secondary-metabolism-and-biosynthesis.ppt

Introduction to Secondary
Metabolism and the Biosynthesis of
Natural Products
Mohit Kumar
Class Bsc Biotech.
Roll No 226577
Mata Gujri Collage
PRIMARY METABOLITES INTERMEDIATE METABOLITES SECONDARY METABOLITES
CO2
+
H2O Glucose
Polysaccharides
Pentose phosphate
Erythrose-4-phosphate
Phosphoenol pyruvate
Shikimate
Aromatic compounds
(C6
-
C1; C6
-
C2)
Phenylpropanoids (C 6
-
C3)
Lignans
Pyruvate
Citric acid
cycle
Aromatic
amino acids
Aliphatic
amino acids
Aromatic alkaloids
Mixed alkaloids
Aliphatic alkaloids
Acetyl-
CoA Polyketides Polyphenols
Phenylpropanoids
Flavonoids
Fatty acids
Polyacetylenes
Prostaglandins
Mevalonic acid
Terpenes
Steroids
Carotenoids
+
NH3
Iridoids
Aliphatic
amino acids
Alkaloids

2
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) 3
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.
SCoA
O CO2H
CH3
HO
OH
CO2H
OH
OH
HO
NH2
R
CO2H
N
NH2
CO2H
H
H2N CO2H
NH2
H2N
CO2H
NH2
HO
CH3
HO
OP
OH

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.

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.
SCoA
O
CO2H
CH3
HO
OH
CO2H
OH
OH
HO
NH2
R
CO2H
N
NH2
CO2H
H
H2N CO2H
NH2
H2N
CO2H
NH2
Biosynthesis: detailed study of the step-wise formation of
secondary metabolites. At more detailed levels, the specific
enzymes, genes and signals are also identified.
HO
CH3
HO
OP
OH

PRIMARY METABOLITES INTERMEDIATE METABOLITES SECONDARY METABOLITES
CO2
+
H2O Glucose
Polysaccharides
Pentose phosphate
Erythrose-4-phosphate
Phosphoenol pyruvate
Shikimate
Aromatic compounds
(C6
-
C1; C6
-
C2)
Phenylpropanoids (C 6
-
C3)
Lignans
Pyruvate
Citric acid
cycle
Aromatic
amino acids
Aliphatic
amino acids
Aromatic alkaloids
Mixed alkaloids
Aliphatic alkaloids
Acetyl-
CoA Polyketides Polyphenols
Phenylpropanoids
Flavonoids
Fatty acids
Polyacetylenes
Prostaglandins
Mevalonic acid
Terpenes
Steroids
Carotenoids
+
NH3
Iridoids
Aliphatic
amino acids
Alkaloids
Overview of
Secondary
Metabolism
* Metabolites found in
higher organisms only
*
*
*
SCoA
O
CO2H
CH3
HO
OH
CO2H
OH
OH
HO
NH2
R
CO2H
N
NH2
CO2H
H
H2N CO2H
NH2
H2N
CO2H
NH2

7
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 ().
()

Biogenetic classification of natural products.
Biogenesis Intermediate Structural Types
Acetogenins (n x C2) acetyl CoA fats and lipids,
macrolides, phenols
Terpenoids (n x C5) mevalonic acid,
methyl erythritol phosphate
monoterpenes, sesquiterpenes,
diterpenes, triterpenes, steroids
carotenoids
Shikimates shikimic acid, prephenic acid phenylpropanoids, phenols
flavonoids
Aliphatic alkaloids lysine, ornithine aliphatic alkaloids
Aromatic alkaloids phenylalanine, tyrosine,
tryptophan
aromatic alkaloids

The basic biogenetic and structural groups: Acetogenins
a. Acetogenins: Acetyl CoA  fats, polyketides
CH3
C
S
O
CoA = S-CoA
O
S-CoA
O
n x
CO2H
lauric acid
OH
CH3
CO2H
6-methylsalicylic acid

The basic biogenetic and structural groups: Terpenoids
b. Isoprenoids: MVA  terpenes, steroids; MEP  carotenoids
=
CO2H
OH
H3C OH
"isoprene" mevalonic acid
n x
OH
menthol
HO
lanosterol
-carotene

HO
CH3
HO
OP
OH
methyl erthritol phosphate

c. Shikimates: Shikimic acid  phenylpropanoids
CO2H
OH
OH
HO
PO CO2
-
OH
-
O2C CO2
-
O
shikimic acid prephenate
chorismic acid
CO2H
OH
O CO2H
p-hydroxybenzoic acid
CO2H
OH
CO2H
OH
OH
CO2H
NH2
R
caffeic acid R=H, phenylalanine
R=OH, tyrosine
The basic biogenetic and structural groups: Shikimates

d. Aliphatic alkaloids: Lysine  aliphatic alkaloids
H2N CO2H
H2N
ornithine
CH3N
OH
tropine
e. Aromatic alkaloids: Phenylalanine  aromatic alkaloids
phenylalanine
CO2H
NH2
ephedrine
N(H)CH3
HO
CH3
NCH3
CH3
CH3O
HO
pellotine
The basic biogenetic and structural groups: Alkaloids

Exercise
The following cytotoxic anthraquinone
derivative was recently isolated from the stem
bark of Goniothalamus marcanii Craib.
Propose its biogenetic origin. Highlight the
appropriate atoms in the molecule.
N
O
O CH3
OCH3
O
OH
H
marcanin D
NCH3
CH3O
HO
CH3O
CH3O
OH
Propose its biogenetic origin of the following
alkaloid. Highlight the appropriate atoms in the
molecule.

Chemistry of Natural Products (Dayrit) 14
Exercises 2 & Answers
The following cytotoxic anthraquinone
derivative was recently isolated from the
stem bark of Goniothalamus marcanii
Craib. Propose its biogenetic origin.
Highlight the appropriate atoms in the
molecule.
Propose the biogenetic origin of the following
alkaloid. Highlight the appropriate atoms in the
molecule.
From Acyl-CoA From Methyl
methionine
N
O
O CH3
OCH3
O
OH
H
marcanin D
From Methyl
methionine
From Shikimate
7 AcylCoA’s + 2
methyl
methionines
2 Phenylalanines/
Tyrosines + 2
methyl methionines
NCH3
CH3O
HO
CH3O
CH3O
OH

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.

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)

Evolution of secondary metabolism in higher plants)
• 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.

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

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.

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

Some types of biosynthetic pathways:
1. Simple linear process A B C ..... X Y
2. Modified linear process
A B Y Z
C D
M N
3. Convergent process A B C
D E
Y
4. Branching process A B C D .......... Y
E
F
G
5. Metabolic grid A B C
D E F
G H Y

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.
2. Although compounds are usually transformed from simple
structures to more complex ones, this is not always the case.
Y
X
.
.
.
.
.
C
B
A
C
B
A
D
Y Z
N
M
C
B
A
D F
E
H Y
G

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.

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
organism’s 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.

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.

Strategies for studying secondary metabolism:
Enzyme control
Experiment Biosynthetic process Comments
Overall process
A B C D
Ea Eb Ec
Exp. 1 Ea
A B C D
x x x A accumulates when enzyme Ea is
blocked; B, C and D are not formed
Exp. 2 Ea Eb
A B C D
x x B accumulates when enzyme Eb is
blocked; C and D are not formed
Exp. 3 Ea Eb Ec
A B C D
x C accumulates when enzyme Ec is
blocked; D is not formed
Example: the biosynthetic sequence in a linear process using
mutants or enzyme inhibitors

Type Isotope used Method of
Detection
Comments
Radioactive 3
H, 14
C scintillation Advantages: High sensitivity, requires only a
small amount of material
Disadvantage: special procedures required
due to radioactivity
Non-
radioactive
2
H,
13
C,
19
F NMR, MS Advantage: Structural information available
Disadvantages: Relatively lower sensitivity;
expensive instrumentation
Strategies for studying secondary metabolism:
Metabolite control

Examples of isotopically-label compounds used in
biosynthetic studies:
.
.
= 13C or 14C
H3C
S CO2H
NH2
.
methionine
H3C
C
OH
O
D3C
C
OH
O
H3C
C
OH
O
.
.
acetic acid
-O2C OP
CH3
HO
.
mevalonate
2 5
CO2H
NH2
.
phenylalanine
5
2
.
-O2C OP
CH3
HO
D
D
-O2C OP
CH3
HO
D
D
2 5

a. Skimmianine, in Choisya ternata (Grundon, Harrison and Spyropoulos, Chem. Comm., 51, 1974).
N
H
O
T
T
CH3O
N
CH3O
O
T
. .
3H : 14C = 2 : 1
Skimmianine
3H : 14C = 1.1 : 1

b. Ephedrine, in Ephedra distachya (Yamasaki, Sankawa and Shibata, Tetrahed. Lett., 4099, 1969).
CO2
-
NH3
+
T5
.
T5
OH
CH3
N(H)CH3
D,L-phenylalanine (-) ephedrine
[14C = nil]
c. Tyrosine, in Psuedomonas (Bowman, Gretton and Kirby, J. Chem. Soc. Perkin I, 218, 1973).
CO2
-
NH3
+
T
. CO2
-
NH3
+
HO
T
phenylalanine
tyrosine
.

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

Major biosynthetic transformations
Reaction
Classification
General equation Comments
1. Hydrolysis
R1 OR2
O
R1 OH
O
+ R OH
2
Common transformation.
2. Esterification
R1 OH
O
+ R OH
2
R1 OR2
O Common transformation.
3. Oxidation
a. C-H  C-OH
[ OH]
R1 R2
Ha
Hb
.
R1 R2
OH
Hb Generally stereospecific.
b. Epoxidation [O]
O
Generally stereospecific
Reaction
Classification
c. Double bond
oxidation
R1 R3
R4
R2
[2 O] R1
R2
O
R4
R3
O

Reaction
Classification
d. Dehydrogenation H
H
H
H
-2H
H
H
e. Halogenation H Cl
4. Reduction
a. e- transfer + H+ H
H
+2H
H
H
H
H
[H] = e- transfer, then + H+
b. deoxygenation
R1 R2
O
R1 R2
OH
H
R1 R2
H
H

Reaction
Classification
5. C-C bond formation
a. Radical coupling Commonly observed
in aromatic and
conjugated systems
OH
-H.
O. O
.
coupling
HO OH
b. Claisen
condensation
R2
O
R1 R2
O
R1
R3
O
R COX
base
3
+ X
_
Very common reaction,
e.g., in lenghtening of
polyketide chain
c. Aldol
R1
R2
O
+ R3 H
O
base
R1
R2
O
R3 OH
base
R1
R2
O
R3

Reaction
Classification
6. Nucleophilic
substitution, Sn2
CH3
S
CH2
-CH2
-CH(NH2)CO2H
R
+
+
H
O
R1 CH3
O
R1
Conversion of alcohol to
methyl ether. Methyl
methionine is usual
methyl source.
7. Elimination
reaction, E2
R2
R1
OH
H
base
R2
R1
-OH is usually converted to
–OPP which becomes
leaving group
8. Cationic
rearrangement
a. 1,2-methyl
migration
CH3
H
CH3
+
+
b. Wagner-
Meerwein shift
+ +
Common in monoterpenes
and sesquiterpenes.

Reaction
Classification
9. Orbital symmetry-
controlled
a. 3,3,-sigmatropic
shift
O
1
2
3
3
2
1 1
2
3
3
2
1
O
Not commonly observed.
10. Carboxylation
R1
R2
O
base
CO2 R1
R2
O
CO2
-
Commonly observed in
activation of -position
for nucleophilic attack.
11. Decarboxylation
R1
R2
O
CO2
-
2
-CO
R1
R2
O Usually observed together
with carboxylation to
remove carboxylic
activating group.

Most of the biosynthetic reactions are mediated by specific
enzymes. Enzymes have five fundamental properties:
Enzymes in biosynthesis
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.

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).

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
R1R2, reduction of the carbonyl to an alcohol produces a
chiral center at the carbon.
R1 R2
Ha
Hb
pro-S
pro-R
O
R1
R2
re-face
si-face

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.

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.

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.

Some types of
control of
biosynthetic
activity through
the action of
metabolites on
enzymes.
A. Negative feedback by one of the products: A B C D
B. Negative feedback by a combination D
of products: A B C }
E
C. Selective positive / negative feedback by products:
C D
A B
E F
(-)
D+E
(-)
D
D
(-)
(+)
F
D. Allosteric control: E(+)=enzyme in active form;
E(-)=enzyme in inactive form; A=substrate; B= product;
P=positive effector; N=negative effector
E(-)
E(+)
N P
A B

Schematic
representation of
the mechanisms
for inducing or
repressing gene
function.
Chromosome
Operator Gene 1 Gene 2 Gene 3
Enzyme 1 Enzyme 2 Enzyme 3
A B C D
A. General mechanism
B. Control by induction of transcription of enzyme synthesis by I.
Operator
Operator + I Operator
Operator - I
(inactive biosynthesis) (active biosynthesis)
(inactive enzyme
degradation)
(active enzyme
degradation)
Operator - I
Operator - R
+ R
Operator
Operator
C. Control by repression of enzyme degradation by R.

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

The IUB number and classification of enzymes
Main Classes and Subclasses Main Classes and Subclasses
1: Oxidoreductase
1.1: acts on the CH-OH group of donors
1.2: acts on the aldehyde or keto group of donors
1.3: acts on the CH-CH group of donors
1.4: acts on the CH-NH2 group of donors
1.5: acts on the C-NH group of donors
1.6: acts on (reduced) NADH or NADPH as a donor
of H
-
1.7: acts on other nitrogenous compounds as donor
1.8: acts on sulphur groups as donor
1.9: acts on haem groups as donor
1.10: acts on diphenols and related substances as
donor
1.11: acts on H2O2 as electron acceptor
1.12: acts on H2 as donor
1.13: acts on single donors with incorporation of
oxygen (oxygenases)
1.14: acts on paired donors with incorporation of
oxygen into one donor (hydrolase).
2: Transferase
2.1: transfers one-carbon group
2.2: transfers aldehyde or ketone
2.3: acyltranferase
2.4: glycosyltransferase
2.5: transfers other alkyl groups
2.6: transfers nitrogenous groups
2.7: transfers phosphorous-containing groups
2.8: transfers sulphur-containing groups
3: Hydrolase
3.1: hydrolysis of the ester bond
3.2: hydrolysis of the glycosyl bond
3.3: hydrolysis of the ether bond
3.4: hydrolysis of the peptide bond
3.5: hydrolysis of C-N bond other than the peptide
bond
3.6: hydrolysis of the acid-anhydride bond
3.7: hydrolysis of C-C bond
3.8: hydrolysis of the C-halide bond
3.9: hydrolysis of the P-N bond
4: Lyase
4.1: lysis of C-C bond
4.2: lysis of C-O bond
4.3: lysis of C-N bond
4.4: lysis of C-S bond
4.5: lysis of C-halide bond
4.99: others
5: Isomerase
5.1: racemization and epimerization
5.2: cis-trans isomerization
5.3: intramolecular oxidoreduction, e.g. aldehyde-
ketone, keto-enol, double bond migration
5.4: intramolecular group transfers
5.99: other isomerizations
6: Ligase
6.1: formation of C-O bond
6.2: formation of C-S bond
6.3: formation of C-N bond
6.4: formation of C-C bond

The four major types of biological oxidation reactions
catalyzed by oxidoreductases
Type of
Oxidation
Description Schematic Reaction and Examples
Dehydrogenase Removes of two H atoms from the
substrate, and transfers this to
another organic compound. The H-
acceptor, A, is a coenzyme.
SH2 + A  S + AH2
R
CH2
CH2
R
R R
H H
R
CH OH
R
R
C O
R
R R
H H
O
R
CH2
CH2
R
Oxidase Removes two H atoms from the
substrate and utilizes O2 or H2O2 as
the H-acceptor.
SH2 + ½O2  S + H2O
SH2 + H2O2  S + 2H2O
OH
OH
O
O
2
O
1/2

The four major types of biological oxidation reactions
catalyzed by oxidoreductases
Type of
Oxidation
Description Schematic Reaction and Examples
Monooxygenase Adds one O atom to the substrate. A
is a coenzyme.
S + AH2 + O2  SO + A + H2O
R R
H H
R R
H H
O
CH R
CH2
OH
R
R
CH2
CH2
R
R
C
H
O
R
C
OH
O
Dioxygenase Adds two O atoms to the substrate S + O2  SO2
R1 R2
H H
O2
R1
H
O
R2
H
O
+

Elimination and rearrangement reactions following oxidation
R
O
CH3 R
O
CH2
O-H
R OH + HCHO
[O]
A. Demethylation: Methyl ether to alcohol
[O]
+ HCHO
R1
N
R2
CH3
CH2
O-H
N
R1
R2
R1
NH
R2
B. Demethylation: Methyl amine to amine
C. Formation of phenyl methylenedioxy ring
O-CH3
OH
[O]
O-
CH2
OH
OH O
CH2
O
-H2O

D. Aromatic ring opening reaction (mono-oxygenase)
[O]
O O
E. Aromatic ring opening reaction (dioxygenase)
[O ]
OH
OH
2
OH
OH
O
O
+
_
OH
OH
O
O
H
CO2H
CHO
OH
F. Oxidation of aromatic ring: NIH shift (hydride shift); R = alkyl group
O
R
D
H
[O]
R
D
R
O
H
D
R
OH
D
isotope
effect
hydride
shift

R-
O
OH
R-
O
H
O
R-O
[O]
O
R-O H
H
G. Para oxidation of aromatic ring.
_
+
H. Oxidative decarboxylation of aromatic carboxylic acid.
[O]
CO2
_ _
O
O
O
-CO2
OH

Oxidative coupling of phenols
OH
H3C
A. Illustration of phenoxy radical formation, resonance stabilization and coupling: Pummerer's ketone.
base
O
H3C
_
-e
_ O
H3C
H
.
.
O
H3C H
O
H3C
.
.
O
H3C
.
O
H3C
O
H3C H
. +
O
H3C
O
H CH3
OH
H3C
O
CH3
O
H3C
OH
CH3

Oxidative coupling of phenols
B. Some important phenolic structures which can undergo phenolic coupling.
OH OH
*
*
*
* *
*
*
*
HO O
O
OH
OH
HO
*
*
OH
CH3
CHO
HO
HO
H3C CH3
CO2H
OH
HO
HO CH2OH
HO
O-
CH3
*
*
*

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
S
(Adenosyl)
H2N
CO2H
+ -H
+
R OCH3
B. Glycosylation of an alcohol with glycosyl phosphate as alkylating agent.
O
OH
OH
HO
HO
OP
HO R
Gly
O
R

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.
R CH2
O
O
O
_
-CO2
_
R CH2
O
R CH2
O
_
H3C S-CoA
O R CH3
O
O
OPP
D. Sn2 displacement of pyrophosphate.
OPP
H 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.

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.

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

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.

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.

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.

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.

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.

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

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 plant’s genome whose
expression can be controlled at the level of the gene.

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