3. Introduction to Secondary Metabolism and the Biosynthesis of Natural Products

3. Introduction to Secondary 3. Introduction to Secondary Metabolism and the Biosynthesis of Metabolism and the Biosynthesis of

Natural ProductsNatural Products

RA Macahig

FM Dayrit

PRIMARY METABOLITES INTERMEDIATE METABOLITES SECONDARY METABOLITES

CO2 + H2O Glucose

Polysaccharides

Pentose phosphateErythrose-4-phosphate

Phosphoenol pyruvate

Shikimate

Aromatic compounds(C6

-C1; C6-C2)

Phenylpropanoids (C 6-C3)

Lignans

PyruvateCitric acidcycle

Aromaticamino acids

Aliphaticamino acids

Aromatic alkaloids

Mixed alkaloids

Aliphatic alkaloids

Acetyl-CoA Polyketides Polyphenols

Phenylpropanoids

Flavonoids

Fatty acidsPolyacetylenesProstaglandins

Mevalonic acidTerpenesSteroidsCarotenoids

+NH3

Iridoids


Alkaloids

3. Secondary metabolites and Biosynthesis (Dayrit) 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.


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

CH3HO

OH

CO2H

OH

OH

HO

NH2R

CO2H

NNH2

CO2H

H

H2N CO2H

NH2

H2NCO2H

NH2

HO

CH3HO

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

OCO2H

CH3HO

OH

CO2H

OH

OH

HO

NH2R

CO2H

NNH2

CO2H

H

H2N CO2H

NH2

H2NCO2H

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

CH3HO

OP

OH


PRIMARY METABOLITES INTERMEDIATE METABOLITES SECONDARY METABOLITES

CO2 + H2O Glucose

Polysaccharides

Pentose phosphateErythrose-4-phosphate

Phosphoenol pyruvate

Shikimate

Aromatic compounds(C6

-C1; C6-C2)

Phenylpropanoids (C 6-C3)

Lignans

PyruvateCitric acidcycle

Aromaticamino acids


Aromatic alkaloids

Mixed alkaloids

Aliphatic alkaloids

Acetyl-CoA Polyketides Polyphenols

Phenylpropanoids

Flavonoids

Fatty acidsPolyacetylenesProstaglandins

Mevalonic acidTerpenesSteroidsCarotenoids

+NH3

Iridoids


Alkaloids

Overview of Secondary

Metabolism

* Metabolites found in

higher organisms only

*

*

*SCoA

O

CO2H

CH3HO

OH

CO2H

OH

OH

HO

NH2R

CO2H

NNH2

CO2H

H

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 ().

(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


The basic biogenetic and structural groups: Acetogenins

a. Acetogenins: Acetyl CoA fats, polyketides

CH3

CS

O

CoA = S-CoA

O

S-CoA

O

n x

CO2H

lauric acid

OHCH3

CO2H

6-methylsalicylic acid


The basic biogenetic and structural groups: Terpenoidsb. Isoprenoids: MVA terpenes, steroids; MEP carotenoids

=

CO2H

OH

H3C OH

"isoprene" mevalonic acid

n x

OH

menthol

HO

lanosterol

-carotene

HO

CH3HO

OP

OH

methyl erthritol phosphate


c. Shikimates: Shikimic acid phenylpropanoids

CO2H

OH

OH

HO

PO CO2-

OH

-O2C CO2

-

O

shikimic acid prephenatechorismic 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 CO2HH2N

ornithine

CH3N

OHtropine

e. Aromatic alkaloids: Phenylalanine aromatic alkaloids

phenylalanine

CO2H

NH2

ephedrine

N(H)CH3

HOCH3

NCH3

CH3

CH3O

HO

pellotine

The basic biogenetic and structural groups: Alkaloids

ExerciseExercise

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 & AnswersExercises 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 plantsAcetate

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 (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.


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

21

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 Y3. Secondary metabolites and Biosynthesis (Dayrit)


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.

YX.....CBA

C

BA

D

Y Z

NM

CBA

D FE

H YG



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 DEa Eb Ec

Exp. 1 EaA B C Dx x x A accumulates when enzyme Ea is

blocked; B, C and D are not formed

Exp. 2 Ea Eb A B C Dx x B accumulates when enzyme Eb is

blocked; C and D are not formed

Exp. 3 Ea Eb EcA B C Dx 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 ofDetection

Comments

Radioactive 3H, 14C scintillation Advantages: High sensitivity, requires only asmall amount of material

Disadvantage: special procedures requireddue to radioactivity

Non-radioactive

2H, 13C, 19F 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

H3CS CO2H

NH2

.methionine

H3CC

OH

O

D3CC

OH

O

H3CC

OH

O..acetic acid

-O2C OP

CH3HO

.mevalonate

2 5

CO2H

NH2

.

phenylalanine

52

.-O2C OP

CH3HO

DD

-O2C OP

CH3HO

DD

2 5



a. Skimmianine, in Choisya ternata (Grundon, Harrison and Spyropoulos, Chem. Comm., 51, 1974).

N

H

O

TTCH3O

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. Hydrolysis2. 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 OH2

Common transformation.

2. Esterification

R1 OH

O+ R OH2

R1 OR2

O

Common transformation.

3. Oxidation

a. C-H C-OH [ OH]

R1 R2

HaHb.

R1 R2

OHHb

Generally stereospecific.

b. Epoxidation [O]

OGenerally stereospecific

ReactionClassification


c. Double bondoxidation

R1 R3

R4R2

[2 O] R1

R2

O

R4

R3

O


Major biosynthetic transformationsReaction

ClassificationGeneral equation Comments

d. Dehydrogenation H

H

H

H

-2HH

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

OHH

R1 R2

HH





5. C-C bond formation

a. Radical coupling Commonly observedin aromatic andconjugated systems

OH

-H.

O . O

.

couplingHO OH

b. Claisencondensation

R2

O

R1 R2

O

R1

R3O

R COX

base3

+ X_

Very common reaction,e.g., in lenghtening ofpolyketide chain

c. Aldol

R1R2

O

+ R3 H

O

base

R1R2

O

R3 OH

base

R1R2

O

R3





6. Nucleophilic substitution, Sn2

CH3

SCH2

-CH2-CH(NH2)CO2H

R

+

+H

OR1 CH3

OR1

Conversion of alcohol to methyl ether. Methyl methionine is usual methyl source.

7. Elimination reaction, E2 R2

R1

OH

H

baseR2

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 monoterpenesand sesquiterpenes.





9. Orbital symmetry-controlled

a. 3,3,-sigmatropic shift

O1

23

32

1 12

3

32

1O

Not commonly observed.

10. Carboxylation

R1R2

O

baseCO 2 R1

R2

O

CO2-

Commonly observed in activation of -position for nucleophilic attack.

11. Decarboxylation R1

R2

O

CO2-

2-COR1

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 R1R2, reduction of the carbonyl to an alcohol produces a chiral center at the carbon.

R1 R2

HaHbpro-Spro-R

OR1

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.

OperatorOperator + I OperatorOperator - I

(inactive biosynthesis) (active biosynthesis)

(inactive enzyme

degradation)

(active enzyme

degradation)

Operator - IOperator - R+ ROperatorOperator

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, oresterification

4: Lyase elimination of a group of adjacent groups of atoms to form adouble bond, or addition of a group of atoms to a doublebond

5: Isomerase conversion of a compound into its isomer

6: Ligase bond formation accompanied by ATP hydrolysis; also knownas 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 ofOxidation

Description Schematic Reaction and Examples

D ehydrogenase R em oves o f tw o H a tom s from thesubstrate, and transfers th is toanother organic com pound. The H -acceptor, A , is a coenzym e.

SH 2 + A S + A H2

RC H 2

C H2R

R R

H H

R

C H O H

R

R

C O

R

R R

H HO

RC H 2

C H 2R

Oxidase Removes two H atoms from thesubstrate and utilizes O2 or H2O2 asthe H-acceptor.

SH2 + ½O2 S + H2O

SH2 + H2O2 S + 2H2O

OH

OH

O

O

2O1/2


The four major types of biological oxidation reactions catalyzed by oxidoreductases

Type ofOxidation

Description Schematic Reaction and Examples

M onooxygenase A dds one O a tom to the substra te. Ais a coenzym e.

S + A H 2 + O 2 S O + A + H 2OR R

H H

R R

H HO

C H RC H2

O H

RR

C H2

C H2R

RC

H

O

RC

O H

O

Dioxygenase Adds two O atoms to the substrate S + O2 SO2

R1 R2

H H

O2R1

H

O

R2

H

O+


Elimination and rearrangement reactions following oxidation

RO

CH3 RO

CH2O-H R OH + HCHO

[O]

A. Demethylation: Methyl ether to alcohol

[O] + HCHOR1

NR2

CH3CH2

O-HNR1

R2

R1NH

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

2OH

OH

OO

+

_ OH

OH

OO

H

CO2HCHO

OH

F. Oxidation of aromatic ring: NIH shift (hydride shift); R = alkyl group

O

R

D

H

[O]

R

D

R

O

H

DR

OH

D

isotopeeffect

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

OO

-CO2OH


Oxidative coupling of phenols

OH

H3C

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

baseO

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

OHHO*

*

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.

OOH

OHHO

HOOP

HO R

GlyO

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

OO

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-Hydroxyphenylpyruvic acid

Tyrosine

Pretyrosine

prehenatedehydrogenase,NAD+

4-hydroxyphenylpyrivatetransaminase, pyridoxal-5'-phosphate

4-hydroxyphenylpyrivatetransaminase, pyridoxal-5'-phosphate

pretyrosinedehydrogenase,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. esterification3. oxidation: hydroxylation, epoxidation or oxygenation of alkene,

dehydrogenation, halogenation 4. reduction: hydrogenation, deoxygenation5. carbon-carbon bond formation: aromatic radical coupling,

Claisen condensation, aldol condensation 6. Cationic rearrangement: 1,2-migration, Wagner-Meerwein7. 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. oxidoreductase2. transferase 3. hydrolase 4. lyase 5. isomerase6. 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.

Documents

3. Introduction to Secondary Metabolism and the Biosynthesis of Natural Products