Download 1. Introduction to Natural Products Chemistry

3. Introduction to Secondary
Metabolism and the Biosynthesis of
Natural Products
RA Macahig
FM Dayrit
PRIMARY METABOLITES
CO 2
+
H2O
Glucose
INTERMEDIATE METABOLITES
SECONDARY METABOLITES
Pentose phosphate
Erythrose-4-phosphate
Shikimate
Phosphoenol pyruvate
+
NH 3
Polysaccharides
Aromatic
amino acids
Citric acid
cycle
Aromatic compounds
(C 6-C1; C6-C2)
Phenylpropanoids (C
Lignans
Aromatic alkaloids
Mixed alkaloids
Pyruvate
Aliphatic
amino acids
Aliphatic alkaloids
Acetyl-CoA
Polyketides
Polyphenols
Flavonoids
Phenylpropanoids
Fatty acids
Mevalonic acid
Polyacetylenes
Prostaglandins
Terpenes
Steroids
Carotenoids
Iridoids
Aliphatic
amino acids
Alkaloids
6
C3)
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
NH2
CO2H
CO2H
CO2H
O
SCoA
R
OH
HO
HO CH3
OH
HO
H2N
NH2
CO2H
OH
OH
N
H
NH2
CO2H
NH2
H2N
CO2H
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
NH2
CO2H
CO2H
O
R
CO2H
CO2H
OH
SCoA
HO
HO CH3
OH
HO
H2N
NH2
CO2H
OH
OH
N
H
NH2
NH2
H2N
CO2H
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
CO 2
+
H2O
Pentose phosphate
Erythrose-4-phosphate
Glucose
HO
OH
OH
Shikimate
Phosphoenol pyruvate
+
Polysaccharides
NH 3
CO2H
NH2
R
CO2H
Citric acid
cycle
Pyruvate
N
H
Aromatic compounds
(C 6-C1; C6-C2)
Phenylpropanoids (C
Lignans
6
C3)
*
Aromatic alkaloids
Aromatic
amino acids
Mixed alkaloids
NH2
Aliphatic
amino acids
Aliphatic alkaloids
H2N
CO2H
NH2
NH2
H2N
CO2H
O
SCoA
Acetyl-CoA
Polyketides
Polyphenols
Flavonoids
Phenylpropanoids
Fatty acids
HO
Overview of
Secondary
Metabolism
* Metabolites found in
higher organisms only
Polyacetylenes
Prostaglandins
CH3
Mevalonic acid
CO2H
*
OH
Terpenes
Steroids
Carotenoids
Iridoids
3. Secondary
metabolites and Biosynthesis (Dayrit)
Aliphatic
amino acids
Alkaloids
6
*
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.
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
3. Secondary metabolites and Biosynthesis (Dayrit)
8
The basic biogenetic and structural groups: Acetogenins
a. Acetogenins: Acetyl CoA  fats, polyketides
O
O
C
CH3
CoA
S
=
S-CoA
CO2H
O
nx
lauric acid
S-CoA
CO2H
CH3
OH
6-methylsalicylic acid
3. Secondary metabolites and Biosynthesis (Dayrit)
9
The basic biogenetic and structural groups: Terpenoids
b. Isoprenoids: MVA  terpenes, steroids; MEP  carotenoids
H3C
HO CH3
OH
OH
OH
=
HO
CO2H
"isoprene"
mevalonic acid
OP
methyl erthritol phosphate
nx
OH
HO
lanosterol
menthol
 -carotene
3. Secondary metabolites and Biosynthesis (Dayrit)
10
The basic biogenetic and structural groups: Shikimates
c. Shikimates: Shikimic acid  phenylpropanoids
O
CO2H
HO
PO
-
CO2
CO2H
-
O
OH
-
O2C
OH
OH
shikimic acid
chorismic acid
CO2
CO2H
CO2H
OH
prephenate
NH2
CO2H
CO2H
OH
p-hydroxybenzoic acid
OH
OH
caffeic acid
3. Secondary metabolites and Biosynthesis (Dayrit)
R
R=H, phenylalanine
R=OH, tyrosine
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
CO2H
HO
CH3
CH3O
NCH3
HO
phenylalanine
ephedrine
3. Secondary metabolites and Biosynthesis (Dayrit)
CH3
pellotine
12
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.
H
O
N
O
OCH3
OH
O
CH3
marcanin D
Propose its biogenetic origin of the following
alkaloid. Highlight the appropriate atoms in the
molecule.
CH3O
NCH3
HO
CH3O
CH3O
OH
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.
From Methyl
methionine
From Acyl-CoA
O
H
N
O
OCH3
OH
O
CH3
marcanin D
Propose the biogenetic origin of the following
alkaloid. Highlight the appropriate atoms in the
molecule.
From Shikimate
From Methyl
methionine
Chemistry of Natural Products (Dayrit)
7 AcylCoA’s + 2
methyl
methionines
CH3O
NCH3
HO
CH3O
CH3O
OH
2 Phenylalanines/
Tyrosines + 2
methyl methionines
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
(Labiatae)
Indole alkaloids
(Apocynaceae)
C15
Sesquiterpenes
(Myrtaceae)
Sesquiterpene lactones
(Compositae)
C20
Diterpenes
(Euphorbiaceae)
Diterpene acids
(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
B
2. Modified linear process
C
A
D
B
Y
M
3. Convergent process
A
Y
Z
N
B
C
Y
D
4. Branching process
A
B
E
C
D
E
G
.......... Y
F
5. Metabolic grid
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.
C
A
B
C . .. .. X
Y
A
B
C
D
E
F
G
H
Y
D
B
Y
M
A
N
Z
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
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.
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
Overall process
Exp. 1
Exp. 2
Exp. 3
Biosynthetic process
A
A
A
A
Ea
Ea
Ea
Ea
B
Eb
x
B
B
B
Eb
Eb
C
Ec
Comments
D
x
C
x
D
A accumulates when enzyme Ea is
blocked; B, C and D are not formed
x
C
x
D
B accumulates when enzyme Eb is
blocked; C and D are not formed
x
D
C accumulates when enzyme Ec is
blocked; D is not formed
C
Ec
3. Secondary metabolites and Biosynthesis (Dayrit)
26
Strategies for studying secondary metabolism:
Metabolite control
Type
Isotope used
3
Radioactive
H,
14
C
Method of
Detection
Comments
scintillation
Advantages: High sensitivity, requires only a
small amount of material
Disadvantage: special procedures required
due to radioactivity
Nonradioactive
2
H, 13C, 19F
NMR, MS
Advantage: Structural information available
Disadvantages: Relatively lower sensitivity;
expensive instrumentation
3. Secondary metabolites and Biosynthesis (Dayrit)
27
Examples of isotopically-label compounds used in
biosynthetic studies:
.
= 13C or 14C
.
S
.
CO2H
H3C
O
O
C
C
NH2
CH3
-O C
2
HO
OP
2
D
5
-O C
2
H3C
OH
acetic acid
methionine
HO
C
OH
D3C
OH
H3C
..
O
.
2
.
CH3
HO
OP
-O C
2
D
D
CO2H
CH3
OP
2
5
.
5
NH2
D
mevalonate
3. Secondary metabolites and Biosynthesis (Dayrit)
phenylalanine
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
N
H
3H : 14C = 2 : 1
.
O
T
CH3O
N
.
T
O
Skimmianine
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
.
CH3
CO2-
NH3+
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+
T
NH3+
HO
T
phenylalanine
tyrosine
3. Secondary metabolites and Biosynthesis (Dayrit)
30
Major chemical transformations
in Biosynthesis
1. Hydrolysis
2. Esterification
3.
4.
5.
6.
7.
8.
Oxidation
Reduction
C-C Bond formation
Nucleophilic substitution
Elimination reaction
Cationic rearrangement
3. Secondary metabolites and Biosynthesis (Dayrit)
31
Major biosynthetic transformations
Reaction
Classification
General equation
O
O
1. Hydrolysis
R1
2. Esterification
Comments
+ R2 OH
R1
OR2
OH
O
O
+ R 2 OH
R1
Common transformation.
R1
OH
Common transformation.
OR2
3. Oxidation
a. C-H  C-OH
Hb Ha
R1
R2
b. Epoxidation
c. Double bond
oxidation
R1
R2
Generally stereospecific
[O]
R1
R3
[2 O]
O
R1
R3
O
R2
R4
Generally stereospecific.
Hb OH
[.OH]
R2
O
R4
3. Secondary metabolites and Biosynthesis (Dayrit)
32
Major biosynthetic transformations
Reaction
Classification
General equation
H
H
d. Dehydrogenation
Comments
H
-2H
H
H
H
Cl
H
e. Halogenation
4. Reduction
a. e- transfer + H+
+2H
H
H
H
O
b. deoxygenation
R1
H
R2
[H] = e- transfer, then + H+
H
H
H
R1
OH
R2
H
R1
H
R2
3. Secondary metabolites and Biosynthesis (Dayrit)
33
Major biosynthetic transformations
Reaction
Classification
General equation
Comments
5. C-C bond formation
a. Radical coupling
O.
OH
-H
Commonly observed
in aromatic and
OH
conjugated systems
O
.
coupling
HO
.
b. Claisen
condensation
O
O
R1
R3COX
base
R2
_
R1
R2
+ X
Very common reaction,
e.g., in lenghtening of
polyketide chain
O
R3
O
c. Aldol
O
R1
O
R1
R2
+
R3
H
O
R1
R2
base
R3
OH
R2
base
R3
3. Secondary metabolites and Biosynthesis (Dayrit)
34
Major biosynthetic transformations
Reaction
Classification
General equation
R
6. Nucleophilic
substitution, Sn2
S
CH3
7. Elimination
reaction, E2
Comments
+
+
CH2-CH2-CH(NH2)CO2H
O
H
O
R1
CH3
R1
OH
R2
R1
R2
R1
base
Conversion of alcohol to
methyl ether. Methyl
methionine is usual
methyl source.
-OH is usually converted to
–OPP which becomes
leaving group
H
8. Cationic
rearrangement
CH3
a. 1,2-methyl
migration
+
H
+
CH3
b. WagnerMeerwein shift
Common in monoterpenes
and sesquiterpenes.
+
+
3. Secondary metabolites and Biosynthesis (Dayrit)
35
Major biosynthetic transformations
Reaction
Classification
General equation
Comments
9. Orbital symmetrycontrolled
O
2
2
O
2
3
1
10. Carboxylation
3
1
3
1
a. 3,3,-sigmatropic
shift
R1
2
3
1
O
O
R2
CO 2
base
Not commonly observed.
R1
R2
Commonly observed in
activation of -position
for nucleophilic attack.
CO2-
O
11. Decarboxylation
R1
O
R2
-CO2
R1
R2
CO2-
3. Secondary metabolites and Biosynthesis (Dayrit)
Usually observed together
with carboxylation to
remove carboxylic
activating group.
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  COH, 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
R1R2, reduction of the carbonyl to an alcohol produces a
chiral center at the carbon.
pro-S
pro-R
Hb Ha
R1
R2
re-face
R2
R1
O
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
(-)
Some types of
control of
biosynthetic
activity through
the action of
metabolites on
enzymes.
B. Negative feedback by a combination
of products:
D
A
B
C
}
E
C. Selective positive / negative feedback by products:
D
(-)
A
C
D
E
F
B
(+)
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(-)
N
P
E(+)
A
3. Secondary metabolites and Biosynthesis (Dayrit)
B
43
A. General mechanism
Chromosome
Operator
Schematic
representation of
the mechanisms
for inducing or
repressing gene
function.
Gene 1
Enzyme 1
A
Gene 3
Gene 2
Enzyme 2
B
Enzyme 3
C
D
B. Control by induction of transcription of enzyme synthesis by I.
Operator
+
Operator- I
Operator
I
(active biosynthesis)
(inactive biosynthesis)
C. Control by repression of enzyme degradation by R.
Operator
+
R
(active enzyme
degradation)
3. Secondary metabolites and Biosynthesis (Dayrit)
Operator
Operator -- R
I
(inactive enzyme
degradation)
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
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).
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
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
5: Isomerase
5.1: racemization and epimerization
5.2: cis-trans isomerization
5.3: intramolecular oxidoreduction, e.g. aldehydeketone, keto-enol, double bond migration
5.4: intramolecular group transfers
5.99: other isomerizations
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
6: Ligase
6.1: formation of C-O bond
6.2: formation of C-S bond
6.3: formation of C-N bond
3. Secondary metabolites and Biosynthesis
(Dayrit)
6.4: formation
of C-C bond
46
The four major types of biological oxidation reactions
catalyzed by oxidoreductases
Type of
Oxidation
Dehydrogenase
Description
Removes of two H atoms from the
substrate, and transfers this to
another organic compound. The Hacceptor, A, is a coenzyme.
Schematic Reaction and Examples
SH2 + A
 S + AH2
R
R
R
CH2
CH2
R
H
R
H
R
CH OH
C O
R
R
R
R
R
CH2
Oxidase
Removes two H atoms from the
substrate and utilizes O 2 or H2O2 as
the H-acceptor.
CH2
R
H
O
H
SH2 + ½O2  S + H2O
SH2 + H2O2  S + 2H2O
O
OH
1/2 O 2
OH
3. Secondary metabolites and Biosynthesis (Dayrit)
O
47
The four major types of biological oxidation reactions
catalyzed by oxidoreductases
Type of
Oxidation
Monooxygenase
Description
Schematic Reaction and Examples
S + AH2 + O2  SO + A + H2O
Adds one O atom to the substrate. A
is a coenzyme.
R
H
R
R
H
H
R
O
R
R
CH2
CH2
CH
OH
O
C
Dioxygenase
R
R
O
R
CH2
H
C
H
R
OH
S + O2  SO2
Adds two O atoms to the substrate
R1
R2
O2
R2
R1
O + O
H
H
3. Secondary metabolites and Biosynthesis (Dayrit)
H
H
48
Elimination and rearrangement reactions following oxidation
A. Demethylation: Methyl ether to alcohol
R
O
[O]
CH3
R
O
CH2
R OH
O-H
+ HCHO
B. Demethylation: Methyl amine to amine
R1
R2
N
CH3
[O]
R1
N
CH2
R1
O-H
NH + HCHO
R2
R2
C. Formation of phenyl methylenedioxy ring
O-CH3
OH
O
O-CH2 OH
[O]
-H2O
OH
3. Secondary metabolites and Biosynthesis (Dayrit)
CH2
O
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
OH
H
OH
[O2]
O
CHO
CO2H
OH
_
+
OH
OH
O
OH
OH
F. Oxidation of aromatic ring: NIH shift (hydride shift); R = alkyl group
D
D
[O]
hydride
shift
O
O
R
R
H
OH
H
R
3. Secondary metabolites and Biosynthesis (Dayrit)
isotope
effect
D
R
D
50
Elimination and rearrangement reactions following oxidation
G. Para oxidation of aromatic ring.
H
O
OH
[O]
O
R-O
R-O
H
_
H
+
R-O
H. Oxidative decarboxylation of aromatic carboxylic acid.
_
O
_
CO2
O
-CO 2
[O]
O
3. Secondary metabolites and Biosynthesis (Dayrit)
R-O
OH
51
Oxidative coupling of phenols
A. Illustration of phenoxy radical formation, resonance stabilization and coupling: Pummerer's ketone.
H
_
OH
O
base
H3C
-e
.
_
O
O
.
H
H3C
H3C
H3C
O.
O
.
H3C
H3C
O
+
.
H
H3C
.
H3C
H3C
O
H3C
O
O
H
O
CH3
OH
OH
H3C
CH3
3. Secondary metabolites and Biosynthesis (Dayrit)
O
CH3
52
Oxidative coupling of phenols
B. Some important phenolic structures which can undergo phenolic coupling.
OH
OH
HO
*
*
*
OH
*
HO
*
O
CHO
HO
*
*
O
*
OH
CO2H
*
HO
OH
*
HO
H3C
HO
OH
*
CH3
CH3
CH2OH
*
*
HO
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..
+
-H
H3C + (Adenosyl)
S
R OH
R OCH3
H2N
CO2H
B. Glycosylation of an alcohol with glycosyl phosphate as alkylating agent.
HO R
HO
OP
O
HO
Gly
O
R
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
-CO2
_
R
CH2
R
R
O
O
O
_
CH2
CH2
O
O
H3C
O
R
CH3
S-CoA
D. Sn2 displacement of pyrophosphate.
OPP
H
H
_
+
-H , -OPP
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
dehydrogenase,
NAD +
4-hydroxyphenylpyrivate
transaminase, pyridoxal-5'phosphate
CH2CHCO2H
OH
HO2C
CH2CCO2H
O
4-hydroxyphenylpyrivate
transaminase, pyridoxal-5'phosphate
pretyrosine
dehydrogenase,
NAD +
OH
Prephenic acid
NH2
4-Hydroxy
phenylpyruvic acid
HO2C
OH
Tyrosine
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 physicochemical properties, for example, phenolics, glycosides, etc.
• Classification according to biogenetic origins or biosynthetic
pathways.
3. Secondary metabolites and Biosynthesis (Dayrit)
60
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.
3. Secondary metabolites and Biosynthesis (Dayrit)
61
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.
2.
3.
4.
5.
6.
7.
8.
9.
10.
hydrolysis
esterification
oxidation: hydroxylation, epoxidation or oxygenation of alkene,
dehydrogenation, halogenation
reduction: hydrogenation, deoxygenation
carbon-carbon bond formation: aromatic radical coupling,
Claisen condensation, aldol condensation
Cationic rearrangement: 1,2-migration, Wagner-Meerwein
Rearrangement under control of orbital symmetry
Sn2 displacement
E2 elimination
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 plant’s genome whose
expression can be controlled at the level of the gene.
3. Secondary metabolites and Biosynthesis (Dayrit)
64