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SCH 511

SCH 511

SCH 511
CHEMISTRY OF PRIMARY AND SECONDARY METABOLITES
Dr. Solomon Derese 1
Date/ Month Teaching CAT-I CAT-II Revision Exam

SCH 511 Chemistry of Primary and Secondary Metabolites Teaching and Examination Schedule (2008/09)
1 7/3 2 14/3 3 21/3 4 28/3 5 4/4 6 11/4 7 18/4 8 25/4 9 2/5 10 9/5 11 16/5 12 23/5 13 30/5 14 6/6 15 13/6

Time table Thursday 5:30 8:30 p.m.

Examination policy 2 CATs & Assignments (30%) Final Exam (70%)


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Course Outline
Introduction (Primary and Secondary metabolites. Enzymes and Cofactors). Primary metabolites (Carbohydrates and amino acids) Secondary metabolites derived from Acetate (Fatty Acids and Polyketides). Secondary metabolites derived Mevalonate (The Terpenoids).
Dr. Solomon Derese

Secondary metabolites derived from shikimic acid (Phenyl propanoids and Lignans). Secondary metabolites derived from mixed biosynthetic origin (The flavonoids). Secondary metabolites derived amino acids (The alkaloids). from

from
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OUTLINE OF THE COURSE


Photosynthesis

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General course objectives To give the learner an overview of the different types of primary and secondary metabolites that plants and microorganisms can biosynthesize and understand the mechanism of formation of the major classes of secondary metabolites.
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INTRODUCTION PRIMARY AND SECONDARY METABOLITES ENZYMES AND COENZYMES

LEARNING OBJECTIVES
To understand the general differences between primary and secondary metabolism. To appreciate the origins of secondary metabolites; Recognize the major building blocks that are used by nature to synthesize secondary metabolites To understand the ecological roles played by secondary metabolites
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To understand the molecular mechanisms of the most important enzyme catalyzed reactions in primary and secondary metabolism, and associated coenzyme. To appreciate the similarities and differences between chemical reaction in the cell and the test tube.

INTRODUCTION
Organisms vary widely in their capacity to synthesize and transform chemicals. For instance, plants are very efficient at synthesizing organic compounds via photosynthesis from inorganic materials found in the environment. While other organisms such as animals and microorganisms rely on obtaining their raw materials in their diet, e.g. by consuming plants.

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Plants produce chemical compounds as part of their normal metabolic functions. The compounds synthesized by plants may be divided into two major groups: 1. 2. Primary metabolites and Secondary metabolites

Primary metabolites Definition


Primary Metabolites are compound synthesized by plants which are needed for growth and development. They are universal and essential components needed for the survival of living organisms needed to create and maintain life.

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Examples of primary metabolites are sugars (carbohydrate), amino acids, nucleotides, common fats and polymers such as proteins, DNA, RNA, lipids and Polysaccharides.

Primary metabolism comprises the chemical processes that every plant must carry out every day in order to survive and reproduce its line. Photosynthesis Glycolysis Synthesis of enzymes Synthesis of amino acids Citric Acid Cycle Transamination Synthesis of coenzymes Reproduction of cells (growth) Synthesis of structural materials Duplication of genetic material Dr. Solomon Derese

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The compounds are involved in the energy regulation of organisms and with growth and development of tissues; in short, they are the building blocks of organisms.
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Primary metabolism CO2 + H2O


Photosynthesis
HO O

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Despite the extremely varied characteristics of living organisms, the pathways for generally modifying and synthesizing carbohydrates, fats, proteins and nucleic acids are found to be essentially the same in all organisms.

Primary metabolites
Polysaccharides Nucleic acids

Glycolysis

HO HO OH OH

Glucose

CO2

PO

CO 2

HO OH

HO OH

OH

PO

Phosphoenol pyruvate

Erythrose-4-phosphate
CO2

Shikimate

Pyruvate
O

Amino acids

Peptides Proteins Porphyrins

SCoA

Krebs cycle
O

SCoA CO2

Fatty acids

Acetyl coenzyme A
SCoA O
HO

Malonyl coenzyme A
HO CO2

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Acetoacetyl Derese Dr. Solomoncoenzyme A

Mevalonate

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The scheme outlines how metabolites from the fundamental processes of photosynthesis, glycolysis, and the Krebs cycle are tapped off from energy-generating processes to provide biosynthetic intermediates. Animals, including humans, and most microorganisms depend directly or indirectly on plants as a source of food. Plant cells contain far more compounds than are produced by the basic primary metabolism.
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In contrast to these primary metabolic pathways, which synthesize, degrade, and generally interconvert compounds commonly encountered in all organisms, there also exists an area of metabolism concerned with compounds which have a much more limited distribution in nature. In addition to primary metabolites, plants also produce a vast and diverse assortment of organic compounds, the great majority of which do not appear to participate directly in growth and development.
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These substances, traditionally referred to as secondary metabolites, often are differentially distributed among limited taxonomic groups within the plant kingdom. Secondary metabolism comprises the chemical processes that are unique to a given plant, and are not universal.

Secondary metabolites, are found in only specific organisms, or groups of organisms, and are an expression of the individuality of species. Secondary metabolites are those metabolites which are often produced in a phase of subsequent to growth, have no function in growth (although they may have survival function), are produced by certain restricted taxonomic groups of plants.
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Examples of secondary metabolites include fatty acids, terpenoids, anthraquinones, flavonoids, phenylpropanoids, alkaloids etc. some of which have good pharmacological properties.

EXAMPLES OF SECONDARY METABOLITES


HO

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O HN HO MORPHINE
Poppy (Papaver somniferum and P. sestigerum)

Morphine is found only in Poppy (Papaver somniferum and P. sestigerum).


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O O R

Pyrethrin I, R = CH3 Pyrethrin II, R = CO2CH3 Chrysanthemum cinerariaefolium

Are exclusively synthesized by the genera Chrysanthemum and Pyrethrum of the Compositae family. 23
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Taxus brevifolia Taxol -anticancer

Datura stramonium Scopolamine -eases motion sickness

Taxus brevifolia Taxol -anticancer

O O H3C O NH H3C O O OH OH CH3 CH3 O H O O CH3 O O


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O CH3

OH

Filipendula ulmaria Aspirin - reduces pain & imflammation

Papaver somniferum Morpine, Codeine - eases pain; suppresses coughing

Ephedra sinica Pseudoephedrine - reduces nasal congestion Cinchona pubescens Quinine -anti-malaria

Catharanthus roseus Vinblastine -anti-cancer

Taxol
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WHY DO PLANTS PRODUCE SECONDARY METABOLITES?

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Irrespective of taxon, the chemicals that play a prominent role in interspecific interactions are rarely the same substances used by an organism to meet the daily challenges of living. Their interactions with environment are mediated secondary metabolites. the by

INTERACTION

Survival in the environment


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The function or importance of secondary metabolites in plants is ecological. It is for interactions between the plants and their environment. Plants can't run away. Plants can't run away and so they developed weapons to protect themselves. These weapons may be as simple as spines but are often complex chemical compounds.
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Plants synthesize secondary metabolites: To defend themselves against herbivores, microbes, viruses. Plants produce repellants to protect themselves from predators and antimicrobial to fight off parasites. Fungi attack plants by producing phytotoxins together with enzyme that digest plant tissues. In response to this attack the plant produces phytoalexins, which act as natural antifungal agents.
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A phytoalexin is a natural product produced by plants in response to stress. Some examples are rishitin, which is produced by the potato, Solanum tuberosum, and phaseolin, which is produced by the bean, Phaseolus vulgaris.

For interspecies competition


Chemical interactions among plants have long been recognized. Certain plants produce secondary metabolites which have the ability to inhibit growth and germination of other plants. For example Polygonum senegalense has been shown to significantly inhibit lettuce seedling growth.

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To facilitate the reproductive processes Coloring agents They produce colourful pigments to attract insects for pollination. Attractive smells ( e.g. terpenoids) To protect themselves from excessive UV-B radiation. Exposure to UV radiation induces the biosynthesis of UV-absorbing compounds.
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This requirement for secondary metabolites to have highly diverse biological activities has led plants to accumulate a vast number of compounds.

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Plant genomes are variously estimated to contain 20,00060,000 genes, and perhaps 1520% of these encode enzymes for secondary metabolism, while the genetic complement of the fruit fly (Drosophila melanogaster) is substantially lower (13,601 predicted genes).
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One explanation for this discrepancy in the relationship between biological and genetic complexity may lie in the differences between the ways that plants and animals protect themselves against predators, pests, diseases, and abiotic stress. Animals have developed nervous and immune systems that enable them to detect and respond to danger, and they are capable of avoiding perilous situations.

By contrast, plants cannot escape from their biotic and abiotic stressors, being linked to the ground by means of their root system, and therefore they must stay and protect themselves. Plants, as sessile (permanently attached) organisms, evolve and exploit metabolic systems to create a rich repertoire of complex natural products that hold adaptive significance for their survival in challenging ecological niches on earth.
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The pattern of secondary metabolites in a given plant is complex; it changes in a tissue- and organ-specific way; regularly, differences can be seen between different developmental stages (e.g., organs important for survival and reproduction have the highest and most potent secondary metabolites), between individuals, and between populations

Human Uses
- Pharmaceuticals - Stimulants (psychoactive) - Flavors, spices, perfumes - Dyes - Natural insecticides - Functional foods

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Medicines
OH N H

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Perfumes
OH
OH

Ephedra (Ephedraceae) (used to treat respiratory aliments such as asthma)

Ephedrine
OMe CN

Ricinus communis (Euphorbiaceae)


N O

Geraniol

Linalol from Lavandula angustifolia

(from Castor oil, Purgative)

(Geranium oil)

(Oil of lavender/Coriander oil)

Ricinine
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Spices
MeO HO Eugenol (cloves)
Cinnamaldehyde (from Cinnamonum species) O

Narcotoics and Hallucinogens


HO

H
O N H HO Morphine (opium) From Papaver somniferum H

OH

(CH2)4CH3

Tetrahydrocannabinol (hashish, marijuana) From Cannabis sativa

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TAXONOMICAL USE Since these compounds are usually restricted to a much more limited group of organisms, they have long been of prime importance in taxonomic research-Chemotaxonomy.

Individual secondary metabolites may be common to a number of species or may be produced by only one organism. Related species often have related patterns of secondary metabolite production and so a species can be classified according to the secondary metabolites they produce. Such a classification is known as chemical taxonomy or chemotaxonomy.

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SCH 511

MAIZE

STEMBORERS

A 10% reduction in stemborers in eastern and southern Africa means a savings of US$ 25 million per annum, or enough food to feed 27 million people.

Solution Push Pull for striga and stemborer control


Pull Volatile chemicals from Napier border attract moths to lay eggs Push Volatile chemicals from Desmodium intercrop repel moths

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Striga (Witch weed)

Striga (witchweeds) infest ~40% of arable land in the savanna region causing 10100% losses (7-13b US$) in crop yield.
Napier grass Maize Desmodium Desmodiun Maize Desmodium Desmodiun Maize Napier grass

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BEFORE
Mrs Ouso, Lambwe Valley, Kenya

Push - Pull
Unique technique for (i) control stemborers, (ii) the parasitic weed Striga and (iii) to improve soil fertility & increase maize yields Napier grass

Maisha bora

AFTER Maize
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Desmodium
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Distinction between Primary and Secondary metabolites Primary and secondary metabolites cannot readily be distinguished on the basis of precursor molecules, chemical structures, or biosynthetic origins. For example, both primary and secondary metabolites are found among the diterpenes (C20) and triterpenes (C30).
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O O H
O O H

Kaurenoic acid Is an essential intermediate in the synthesis of gibberellins, i.e., growth hormones found in all plants. Primary metabolite
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Abietic acid Is a resin component largely restricted to members of the Fabaceae and Pinaceae family. Secondary metabolite
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O N H O H

N H O

Primary products participating in nutrition and essential metabolic processes inside the plant.
H

Proline Essential amino acid Primary metabolite

Pipecolic acid Alkaloid Secondary metabolite

Natural (secondary) products influencing ecological interactions between the plant and its environment. Secondary metabolites are organic compounds that are not directly involved in the normal growth, development or reproduction of organisms.
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In the absence of a valid distinction based on either structure or biochemistry, we return to a functional definition.
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How do plants compounds?

synthesize

these
HO O

N H
Me

Me

SCH 511
Me O

H
OO

CAMPHOR

This is what this unit is all about.


N QUININE

O O

ARTEMISININ

Despite the diversity of secondary metabolites, a few key intermediates in primary metabolism supply the precursors for most secondary products. Secondary metabolites are produced by pathways derived from primary metabolic routes.
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CO2 H2O Pi N2 h
O N N N
MeO O H O OH O O

N N NICOTINE

O CAFFEINE

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(+)-Usararotenoid-C

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Primary metabolism CO2 + H2O


Photosynthesis
HO O

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Secondary metabolites

Glycolysis

HO HO OH OH

Glucose

CO2

PO

CO 2

HO OH

HO OH

OH

PO

Shikimate Metabolites (Cinnamic acid derivatives Aromatic compounds Lignans, flavonoids)

Phosphoenol pyruvate

Erythrose-4-phosphate
CO2

Shikimate

The most important building blocks employed in the biosynthesis of secondary metabolites are derived from the intermediates acetyl coenzyme A (acetyl-CoA), shikimic acid, mevalonic acid.

Pyruvate
O

Amino acids

Alkaloids

SCoA

Krebs cycle
O

SCoA CO2

Fatty acids & Polyketides

Acetyl coenzyme A
SCoA O
HO

Malonyl coenzyme A
HO CO2

Terpenoids
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Acetoacetyl coenzyme A Dr. Solomon Derese

Mevalonate

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Synthesis in The Cell vs. The Flask


Just like the synthesis of organic compounds in the laboratory, the synthesis of organic compounds in the cell requires reactions that can be used to form the carbon-carbon and carbon-heteroatom bonds of the target compounds. However, because the biosynthesis of secondary metabolites occur in a living cell, there are quite several quite restrictive constraints on the reaction which may be used to convert one metabolite into another.
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The most restrictive constraints is a consequence of two unavoidable facts about living cells: A living cell is mainly water, so many of the organic reactions must be carried out in aqueous solution; and If the pH of the cell deviates even by a half pH unit (in cells the pH is 7.5), the cell dies. Consequently, whenever a cell synthesizes an organic compound it must do so in aqueous environment using reactions that do not require strong acids or bases.
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One way in which a cell avoids some of the difficulties imposed by these constraints is to use enzymes as catalyst for many reactions: the enzyme function by bringing the reactants together in a suitable orientation, and they also function by producing a microenvironment conducive to the reaction.

The Role of Enzymes in Natural Product Chemistry

SCH 511

Biosynthetic reactions are reversible and are catalyzed by enzymes (enzymes are proteins which catalyze biological reactions). Enzymes catalyse the same types of reactions that are utilized in any organic chemistry laboratory: oxidation, reduction, alkylation, hydrolysis, hydroxylation, elimination etc.
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10

However, enzymes enhance rates of these reactions by as much as 1012.


Without enzyme
AEw/o AEw

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The hallmarks of enzyme catalysis are: speed, selectivity and specifcity. Selectivity The ability of the enzyme to select a certain substrate or functional group out of many. Specificity

Energy state

With enzyme
DG=-RTlnKeq

Reaction

Enzymes lower the activation energy of reactions 61


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A property of the reaction catalysed by the enzyme, being the production of a single regio- and stereo-isomer of the product.
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The catalytic activity of many enzymes depends on the presence of small non protein molecules termed as cofactors. Cofactors may be are often classified as inorganic substances such as Mg2+, Zn2+, Fe2+, Fe3+, etc. or small organic molecules known as coenzymes. Coenzymes are organic molecules that are required by certain enzymes to carry out catalysis.
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ATP CoASH BIOTIN DMAPP


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NAD(P)H NAD(P)+ SAM PLP


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I.

ATP (Adenosine TriPhosphate)

ATP activates a chemical reaction that is thermodynamically unfavorable. Consider a chemical reaction that is thermodynamically unfavorable without an input of energy, a situation common to many biosynthetic reactions.

IN VITRO (IN A TEST TUBE) The carbinol carbon of an alcohol is electrophilic; however, the -OH ion is a poor leaving group.
Nuc
-

d+

C O H

d-

Nuc C

+ OH

:Nuc R-OH R-Nuc


-:OH

The hydroxyl group can be converted to the tosylate ester, which acts as a very good leaving group.
O CH3 S O R O
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Thermodynamically unfavourable Endothermic reaction.


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highly
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Formation of Tosylate Ester


O .. R O: H Cl S O CH3

R
+

O O S CH3

O
:Nuc

R Nuc
CH3

R O S

H
.. N

d+ d-

+
O CH3

O S O

O R O S O CH3

A resonance stabilized leaving group An energetically unfavorable reaction is biosynthetically driven by linking it to an energetically favorable reaction, such as the 68 hydrolysis of ATP.
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Good leaving group


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Phophoester bond Phosphoanhydride bonds

NH2 N N N

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The phosphoanhydride bonds are effective stores of chemical potential energy.


NH2 Hard Nu: O O O O Soft Nu: O O OH HO O N N N N

O O

O O

O O O

HO P O P O P O

HO P O P O P O

OH HO
Adenosine AMP ADP
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ATP

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ATP can be attacked by hard nucleophiles at a phosphate group (usually the end one) or by soft nucleophiles at the CH2 group on the sugar. 70
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NH2 N .. R OH .. O + O O O O O OH HO O N N N

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When a new reaction is initiated in nature, very often the first step is a reaction with ATP to make the compound more reactive. In many structures, the abbreviation P is used to represent the phosphate (orthophosphate) group and PP the diphosphate (or pyrophosphate) group:
O P O O O O O O P O P O

HO P O P O P O

ATP
Mg2+/Mn2+
R
+

Enzyme
N

NH2 N N

O O P OH + O

O O .. :O P O P O .. O O

N O OH HO

NH2 N R O O P OH O + O O O O OH HO O N N N

HO P O P O

ADP
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P (orthophosphate)
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PP (Diphosphate)
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The phosphorylated alcohol is then activated towards nucleophilic displacement:


H Y R O O P OH O

So, overall the endothermic process


:Y-H R OH R Y

H Y R + O .. :O P OH .. O

-:OH

has been achieved by coupling the process to the hydrolysis of ATP. In general, the exothermicity associated with phosphorylation shifts the equilibria of coupled process by a factor of ~108 . In other words, coupling the hydrolysis of ATP with the conversion of ROH to RY can change the equilibrium ratio of ROH to RY by 108.
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R Y +

O HO P OH O

In summary
R OH :Y-H R Y
ATP ADP

-:OH

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More generally the hydrolysis of n ATP molecules change the equilibrium ratio of a coupled reaction by a factor of 108n.

IN SUMMARY
THERMODYNAMICALLY UNFAVORABLE

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Thus, a thermodynamically unfavorable reaction sequence can be converted into a favorable one by coupling it with the hydrolysis of a sufficient number of ATP molecules in a new reaction.
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II. Coenzyme A
Acylation and formation of C-C bond formation a to C=O. Coenzyme A is one of the most important acyl-transfer and a-carbon activating reagents in living organisms.

IN VITRO (IN A TEST TUBE) Acyl substitution reaction


O .. O: R + :YH Y

SCH 302
O

STRONG ACID OR BASE

R OH

Ester

O O R
78
+

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a-substitution reaction
O H .. O: H R + R1-X R1 :BASE O

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NH2 O N O N H HO O
-O

N N

O R

HS N H

OO P O O

OP O O O

a-Hydrogens The a-Hydrogens are acidic because they can easily be picked by a base forming a resonance stabilized enolates.
O H .. O: H R :BASE .. O R ENOLATE
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OH P
-O

Coenzyme A (CoASH)

O O R

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This reactions can go readily in a biological system (in vivo) with out any acid or a base. Acyl substitution reaction
O R SCoA + YH R

Y
R

O Y

ACYL TRANSFER Enzyme R'-LG


O R SCoA

O R R' SCoA

O + CoASH Y

O R

CoASH

-CARBON ALKYLATION
O

O R SCoA OH

a-substitution reaction
O R SCoA + R1X R R1
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O + HX SCoA
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Can act nucleophile electrophile

as

a or

ALDOL REACTIONS
O SCoA
O R O SCoA

CLAISEN-type C-ACYLATION
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H H

O .. R' O .. R

H H

OO
+

H H

O .. CoA S .. R

H H

OS
+

CoA

R'

Very minor contributor The P orbitals of C and S are in different groups such that they cannot effectively overlap and form a p-bond. The C-S bond is longer and weaker than the C-O bond.
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Important contributor

The P orbitals of C and O are in the same group such that they can effectively overlap and form a p-bond.
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O R H H R' O

IN SUMMARY
Y H Z R H X

O Y H
R H H

CoASH
O SCoA

ACYL SUBSTITUTION

Z H R' - SUBSTITUTION
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REQUIRE THE USE OF STRONG ACIDS AND BASES

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III.Methylation reaction biological systems


_ .. :OH ..

in

H3C
S

SCH 511

L-methionine
H2N OH O

IN VITRO Williamson Ether Synthesis


O O O O O O

NH2 N N N N

R O H

_ .. R O: ..
H3C S
H2N
+

HO P O P O P O O

ATP
OH HO

N O N N OH

NH2 N O O O O O

H3C X
R O CH3
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HO P O P O P O O

OH

HO

S-AdenosylMethionie (SAM) SAM acts as a versatile O-, C-, N- & S- Methylating 88 agent in Derese vivo Dr. Solomon

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O and N alkylation using SAM


.. NH2 OR .. OH ..
H2N O OH

C alkylation using SAM


NH2 N

H3C S
+

N O N N OH

_ ..
..

OH ..

_ ..

OH

OH
+

HO

H3C S
H2N
+

N O N N OH

NH2 N

O Me OR H N Me S

N O N N OH

NH2 N

OH

HO

Aromatization

H3C
+

Aromatization

H3C OH
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H2N O OH

OH CH3

OH

HO

OH
+

CH3

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IV. Dimethylallylation
H3C S Ad
H2N O OH
+

The dimethylallyl group is a very common substituent in secondary metabolites.

OPP
Dimethylallyl pyrophosphate (DMAPP)

S-AdenosylMethionie (SAM)

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-:Nu

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V. Carboxylation
O ATP HO OCarbonic acid

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b Enzyme (Mg2+ or Mn2+)

OPP

Nu
Nu
Prenylation
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Biotin in the presence of bicarbonate, ATP and Mg2+ enables nucleophile carboxylation in vivo:
O

N'-Carboxybiotin
O
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Reverse prenylation
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Nu

VI. OXIDATION AND REDUCTION REACTIONS IN BIOLOGICAL SYTEMS


IN VITRO
OH

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NICOTINE ADENINE DINUCLEOTIDE


O C NH2 NH2 O O O O O CH2 O P O P O CH2 OH O R O N N N

O
N

+
N

C N R

NH2

[O]

[H]

PCC
H O

i. NaBH4 ii. H3O+ OR i. LiAlH4 ii. H3O+


KMnO4, -:OH, Heat ii. H3O+

HO OH

[O]

H O HO
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Natures Hydride Reducing / Oxidizing NAD+ R = -H membranes Coenzyme (reagent) NADP+ R = -PO3H cytosol

i. LiAlH4 ii. H3O+


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[H]

The two forms differ by a phosphate group which also controls the location in the cell.
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NAD+ and NADP+ ARE HYDRIDE ACCEPTORS


O
H3 C

:B-Enz

Dehydrogenase
O C H
S

H-B-Enz
Acetaldehyde

OXIDATION

H HR S

Since NADP(H) has a prochiral center, and many enzymes can differentiate between the hydrogens HR and HS, the process is usually stereospecific.

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REDUCTION

Ethanol

O C NH2
REDUCTION OXIDATION

H .. N R

HR

O C NH2

NAD+ NADP+

N R

NADH NADPH

Hydride transfers
NADH and NADPH ARE HYDRIDE DONORS

Unlike ordinary chemical reagents, coenzymes function reversibly.


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these
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In the example given above, the hydride from ethanol enters from above the plane of the ring, and it is this same hydrogen, HR, which is transferred to acetaldehyde in the reverse process. Other enzymes are specific for the HS hydrogen. Other enzymes are specific for the HS hydrogen. In each instance, one :H- (of the cofactor) and H+ are utilized. This process is depicted as 2[H].
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O H Me H

O H2N O O

Enzyme The enzyme binds both the NH

Mg2

N H O R

HO

OH

substrate (pyruvic acid) and the reagent (NADH) in a specific way so that the hydride is delivered to one enantiotopic face of the ketone. A magnesium(II) cation, also held by the enzyme, binds the carbonyl group of the amide of NADH and the ketone in pyruvate. only the top H atom (as drawn) of the diastereotopic CH2 group in NADH should be transferred to pyruvate.
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Oxidation Reactions Mediated by NAD(P)+

VII.Hydroxylation and epoxidation reactions in biological systems


EPOXIDATION OF ALKENES
R R R R Peracid (peroxyacid) CHCl3 or CCl4 R R

R O R Epoxide (oxirane)

Commonly used per-acid


O O O H

Cl
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mCPBA

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SCH 511

SCH 511

The enzyme monooxygenase catalyzes the insertion of an oxygen atom across a carbon-carbon double bond to form an epoxide.
R R R R O2, H+, NADPH monooxygenase R R O R R

Synthesis of Phenols

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SCH 511

SCH 511

R-H + O2 + NADPH + H+
Mono-oxygenase

With mono-oxygenases, the second oxygen atom from O2 is reduced to water by an appropriate hydrogen donor, NAD(P)H.

R-OH + H2O + NADP+


Oxygenases catalyze the direct addition of molecular oxygen to the substrate. They are subdivided into mono- and di-oxygenases according to whether just one or both of the oxygens are introduced into the substrate.
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O2

OH

NADPH
-active form of oxygen (O2- superoxide) is used. -transfer of one atom from molecular oxygen -radical mechanism

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SCH 511

SCH 511

O2 , NADPH
H OH

enzyme

Mechanism
R
O2
+

An NIH shift is a chemical rearrangement where a hydrogen atom on an aromatic ring undergoes an intramolecular migration primarily during a hydroxylation reaction. This process is also known as a 1,2-hydride shift.

D D OH

H H H

H
H

:O

NIH SHIFT
R

Enolization
H OH

H H O

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SCH 511

VIII. Reductive Amination in Nature One of the best methods of amine synthesis in the laboratory is reductive amination, in which an imine (formed from a carbonyl compound and an amine) is reduced to a saturated amine.

For this transformation nature uses a substituted pyridine called PyridoxaL Phosphate (PLP) which in a reversible reaction yield a stereospecific product.

SCH 511

[H] NaCNBH3 or NaBH4 This reaction, of course, produces racemic amines.


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PyridoxaL Phosphate (PLP)


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SCH 511

SCH 511

It uses an amine transfer rather than a simple reductive amination, and the family of enzymes that catalyse the process is the family of aminotransferases. PLP is a coenzyme and it is carried around on the side chain of a lysine residue of the enzyme. Lysine has a long flexible side chain of four CH2 groups ending with a primary amine (NH2). This group forms an imine with PLP.
111

Lysine residue
H O P O O N H Me OH OH O

Pyridoxal phosphate
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Imine between enzyme and pyridoxal


112

When reductive amination or its reverse is required, the pyridoxal is transferred from the lysine imine to the carbonyl group of the substrate to form a new imine of the same sort. The most important substrates for PLP are the amino acids and their equivalent aketo-acids.
NH2 R O O H
Aminotransferase

SCH 511

SCH 511

Aminotransferase
O H N N H O
O

R H2N

H O H O
O OH P O O

H R O N OH H

N O P O O N H Me OH OH

O R O O H
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N H

Me

Imine between enzyme and pyridoxal

Imine between amino acid and pyridoxal

a-Amino acid
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PLP

a-Keto acid
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SCH 511

SCH 511

By using the protonated nitrogen atom of the pyridine as an electron sink, the a proton of the amino acid can be removed to form a new imine at the top of the molecule and an enamine in the pyridine ring.
O H R O H N O OH O N H Me O P O N H Me R O H O N OH

Now the electrons can return through the pyridine ring and pick up a proton at the top of the molecule. The proton can be picked up where it came from, but more fruitfully it can be picked up at the carbon atom on the other side of the nitrogen. Hydrolysis of this imine releases pyridoxamine and the keto-acid. All the natural amino acids are in equilibrium with their equivalent a-keto-acids by this mechanism, catalysed by an aminotransferase.

old imine
O P O OH

new imine
OH

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O R O N O P O O N H Me OH OH H

SCH 511

SCH 511

TRANSAMINATION
NH2 R O O H

Aminotransferase
R O

O H

a-Amino acid Pyridoxamine phosphate a-keto-acid

a-Keto acid

Pyridoxal phosphate (PLP)

Reversing this reaction makes an amino acid stereospecifically out of an a-keto-acid.


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Pyridoxal phosphate
118

Several different kinds of amino acid transformations are catalyzed by PLPrequiring enzymes.
NH3
R

SCH 511

SCH 511

The first step in all PLP-requiring enzymes is a reaction between the amino group of the amino acid and the Imine between enzyme and pyridoxal PLP forming an imine.
Aminotransferase
O H N N H
O

a b
H O

O
O

R H2N

H O H O
O OH P O O

H R O N OH H

The most common transformations are decarboxylation, transamination, racemization (the interconversion of L- and D-amino acids), Ca -- Cb bond cleavage, and a,b-elimination. 119
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N O P O O N
Dr. Solomon DereseH

OH OH

N H

Me

Me
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SCH 511

SCH 511

Decarboxylation

MECHANISM

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SCH 511

Racemization

Ca -- Cb bond cleavage

SCH 511

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SCH 511

SCH 511

Bond broken in transamination, racemization and a,b - elimination

H R
Bond broken in Ca-Cb cleavage

O OH
Bond broken in decarboxylation

NH2

In each of these transformations, one of the bonds to the of the amino acid substrate is broken in the first step of the reaction. Decarboxylation breaks the bond joining the carboxyl group to the a-carbon; transamination, racemization, and a,belimination break the bond joining the hydrogen to the a-carbon; and Ca -- Cb bond cleavage breaks the bond joining the R group to the a-carbon.

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SCH 511

SCH 511

Control over the choice of reaction arises because the different enzymes bind the substratepyridoxal imine in different ways. Decarboxylases bind so that the CC bond to be broken is held orthogonal to the pyridine ring and parallel to the p orbitals in the ring. Then the bond can be broken and CO2 can be lost.

Racemases and transaminases bind the substratepyridoxal imine so that the CH bond is parallel to the p orbitals in the ring so that proton removal can occur. Enzymes do not speed reactions up indiscriminatelythey can selectively accelerate some reactions at the expense of others, even those involving the same reagents.

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SCH 511

A catalyst influences the magnitude of the activation energies for the forward and reverse reactions but does not affect the potential energies of the reactants or products . A catalyst, therefore, influences the rate at which an equilibrium is established but not its position. In lowering the activation energy barrier for the forward reaction, a catalyst accelerates the rate of formation of product.
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