sch 511 lecture introduction primary and secondary metabolites coenzyme 2010
TRANSCRIPT
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SCH 511
CHEMISTRY OF
PRIMARY
AND
SECONDARY
METABOLITES
SCH 511
Dr. Solomon Derese 1
SCH 511
SCH 511 – Chemistry of Primary and Secondary Metabolites
Teaching and Examination Schedule (2008/09) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Date/
Month
7/3 14/3 21/3 28/3 4/4 11/4 18/4 25/4 2/5 9/5 16/5 23/5 30/5 6/6 13/6
Teaching
CAT-I
CAT-II
Revision
Exam
Time table Thursday 5:30 – 8:30 p.m.
Examination policy 2 CATs & Assignments (30%) Final Exam (70%)
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Introduction (Primary and Secondary metabolites. Enzymes and Cofactors).
Secondary metabolites derived from Acetate (Fatty Acids and Polyketides).
Secondary metabolites derived from Mevalonate (The Terpenoids).
Course Outline
Primary metabolites (Carbohydrates and amino acids)
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Secondary metabolites derived from mixed biosynthetic origin (The flavonoids).
Secondary metabolites derived from amino acids (The alkaloids).
Secondary metabolites derived from shikimic acid (Phenyl propanoids and Lignans).
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Photosynthesis
OUTLINE OF THE COURSE
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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.
General course objectives
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ENZYMES AND COENZYMES
INTRODUCTION
PRIMARY AND SECONDARY METABOLITES
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.
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For instance, plants are very efficient at synthesizing organic compounds via photosynthesis from inorganic materials found in the environment.
Organisms vary widely in their capacity to synthesize and transform chemicals.
While other organisms such as animals and microorganisms rely on obtaining their raw materials in their diet, e.g. by consuming plants.
INTRODUCTION
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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:
2. Secondary metabolites
1. Primary metabolites and
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Primary metabolites
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.
Definition
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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.
Examples of primary metabolites are sugars (carbohydrate), amino acids, nucleotides, common “fats” and polymers such as proteins, DNA, RNA, lipids and Polysaccharides.
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Primary metabolism comprises the chemical processes that every plant must carry out every day in order to survive and reproduce its line.
Photosynthesis Glycolysis
Citric Acid Cycle Synthesis of amino acids Transamination
Synthesis of enzymes
Synthesis of coenzymes
Synthesis of structural materials Duplication of genetic material
Reproduction of cells (growth)
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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.
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CO2 + H2O
O
OHOH
HOHO
HO
Photosynthesis
Glucose Glycolysis
Phosphoenol pyruvate
Pyruvate
Erythrose-4-phosphate
Acetyl coenzyme A
Acetoacetyl coenzyme A
Malonyl coenzyme A
Shikimate
Mevalonate
CO2
PO
CO2
O
SCoA
O
SCoA
O
O
+ PO
HO
OH
O
CO2
HO
OH
OH
SCoA
OCO2
HOCO2
HO
Krebs cycle
Amino acids } Peptides Proteins
Porphyrins
Fatty acids
Primary metabolism
Polysaccharides Nucleic acids
Primary metabolites
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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.
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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.
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EXAMPLES OF SECONDARY METABOLITES
Poppy (Papaver somniferum and P. sestigerum)
Morphine is found only in Poppy (Papaver somniferum and P. sestigerum).
HO
O
HOH N
MORPHINE
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Pyrethrin I, R = CH3 Pyrethrin II, R = CO2CH3
Are exclusively synthesized by the genera Chrysanthemum and Pyrethrum of the Compositae family.
Chrysanthemum cinerariaefolium
R
O
O
O
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O O
CH3
CH3
O
H3CONHO
OH
O
H3C CH3OH
OOOH
O
CH3
O
O
H
Taxus brevifolia Taxol
-anticancer
Taxol Dr. Solomon Derese
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Taxus brevifolia Taxol
-anticancer
Papaver somniferum
Morpine, Codeine
- eases pain; suppresses coughing
Ephedra sinica Pseudoephedrine - reduces nasal
congestion
Catharanthus roseus Vinblastine -anti-cancer
Datura stramonium Scopolamine -eases motion
sickness
Filipendula ulmaria Aspirin
- reduces pain & imflammation
Cinchona pubescens
Quinine -anti-malaria
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WHY DO PLANTS PRODUCE SECONDARY METABOLITES?
INTERACTION Survival in the environment
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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 the environment are mediated by secondary metabolites.
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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. Dr. Solomon Derese
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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.
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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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Some examples are rishitin, which is produced by the potato, Solanum tuberosum, and phaseolin, which is produced by the bean, Phaseolus vulgaris.
A phytoalexin is a natural product produced by plants in response to stress.
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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 interspecies competition
For example Polygonum senegalense has been shown to significantly inhibit lettuce seedling growth.
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To protect themselves from excessive UV-B radiation.
Exposure to UV radiation induces the biosynthesis of UV-absorbing compounds.
To facilitate the reproductive processes
Coloring agents
Attractive smells ( e.g. terpenoids)
They produce colourful pigments to attract insects for pollination.
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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.
Plant genomes are variously estimated to contain 20,000–60,000 genes, and perhaps 15–20% 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.
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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
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- Pharmaceuticals - Stimulants (psychoactive) - Flavors, spices, perfumes - Dyes - Natural insecticides - Functional foods
Human Uses
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Medicines
N
OH
H
N O
OMe
CN
(used to treat respiratory aliments such as asthma)
Ephedrine
(from Castor oil, Purgative)
Ricinine
Ephedra (Ephedraceae)
Ricinus communis (Euphorbiaceae)
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OH
Geraniol
OH
Linalolfrom Lavandula angustifolia
Perfumes
(Geranium oil) (Oil of lavender/Coriander oil)
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Spices
MeO
HO
Eugenol (cloves)
H
O
Cinnamaldehyde(from Cinnamonum species)
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Narcotoics and Hallucinogens
O
HO
HO
NH H
Morphine (opium)From Papaver somniferum
O
OH
(CH2)4CH3
Tetrahydrocannabinol (hashish, marijuana)From Cannabis sativa
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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.
TAXONOMICAL USE
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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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MAIZE
Striga (Witch weed)
STEMBORERS
Striga (witchweeds) infest ~40% of arable land in the savanna region causing 10-100% losses (7-13b US$) in crop yield.
A 10% reduction in stemborers in eastern and southern Africa means a savings of US$ 25 million per annum, or enough food to feed 27 million people.
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“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
Napier grass Maize Napier grass Maize Maize Desmodiun Desmodiun Desmodium Desmodium
Solution
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Mrs Ouso,
Lambwe Valley, Kenya
BEFORE
AFTER
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Push - Pull
Napier grass
Desmodium Maize
Maisha bora
Unique technique for (i) control stemborers, (ii) the parasitic weed Striga and (iii) to improve soil fertility & increase maize yields
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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).
Distinction between Primary and Secondary metabolites
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OO
H
Kaurenoic acid Abietic acid
OO
H
Primary metabolite Secondary metabolite
Is an essential intermediate in the synthesis of gibberellins, i.e., growth hormones found in all plants.
Is a resin component largely restricted to members of the Fabaceae and Pinaceae family.
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NH O
O
HNH O
OH
Primary metabolite Secondary metabolite
Proline Pipecolic acid Essential amino acid Alkaloid
In the absence of a valid distinction based on either structure or biochemistry, we return to a functional definition.
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Primary products participating in nutrition and essential metabolic processes inside the plant.
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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Despite the diversity of secondary metabolites, a few key intermediates in primary metabolism supply the precursors for most secondary products.
How do plants synthesize these compounds?
This is what this unit is all about.
Secondary metabolites are produced by pathways derived from primary metabolic routes.
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CO2 H2O Pi N2 hν
HO
N
H
O
N
QUININE
O
O
O O
H H
H
ARTEMISININ
CAMPHOR
OMe
MeMe
N
N
NICOTINEO
N
N
NN
O
CAFFEINEOO
O
OO
MeO
OH
H
(+)-Usararotenoid-CDr. Solomon Derese
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CO2 + H2O
O
OHOH
HOHO
HO
Photosynthesis
Glucose Glycolysis
Phosphoenol pyruvate
Pyruvate
Erythrose-4-phosphate
Acetyl coenzyme A
Acetoacetyl coenzyme A
Malonyl coenzyme A
Shikimate
Mevalonate
CO2
PO
CO2
O
SCoA
O
SCoA
O
O
+ PO
HO
OH
O
CO2
HO
OH
OH
SCoA
OCO2
HOCO2
HO
Krebs cycle
Amino acids
Primary metabolism Secondary metabolites
Alkaloids
Fatty acids & Polyketides
Terpenoids
Shikimate Metabolites (Cinnamic acid derivatives Aromatic compounds Lignans, flavonoids)
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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.
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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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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.
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The most restrictive constraints is a consequence of two unavoidable facts about living cells:
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.
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The Role of Enzymes in Natural Product Chemistry
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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Ene
rgy
stat
e
Reaction
Without enzyme
With enzyme
DG=-RTlnKeq
Enzymes lower the activation energy of reactions
AEw/o
AEw
However, enzymes enhance rates of these reactions by as much as 1012.
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The hallmarks of enzyme catalysis are: speed, selectivity and specifcity.
A property of the reaction catalysed by the enzyme, being the production of a single regio- and stereo-isomer of the product.
Selectivity
Specificity
The ability of the enzyme to select a certain substrate or functional group out of many.
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The catalytic activity of many enzymes depends on the presence of small non protein molecules termed as cofactors.
Coenzymes are organic molecules that are required by certain enzymes to carry out catalysis.
Cofactors may be are often classified as inorganic substances such as Mg2+, Zn2+, Fe2+, Fe3+, etc. or small organic molecules known as coenzymes.
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ATP
CoASH SAM
DMAPP
BIOTIN
NAD(P)+
NAD(P)H
PLP
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I. ATP (Adenosine TriPhosphate) ATP activates a chemical reaction that is thermodynamically unfavorable.
R-OH :Nuc
R-Nuc -:OH Thermodynamically unfavourable highly Endothermic reaction.
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Consider a chemical reaction that is thermodynamically unfavorable without an input of energy, a situation common to many biosynthetic reactions.
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The carbinol carbon of an alcohol is electrophilic; however, the -OH ion is a poor leaving group.
The hydroxyl group can be converted to the tosylate ester, which acts as a very good leaving group.
SO
OO
RCH3
CNuc - + Nuc + OH-O H Cd+ d-
IN VITRO (IN A TEST TUBE)
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R OH
Cl SO
OCH3:
..O
+SO
OCH3
R
H
N..
O SO
OCH3R
Formation of Tosylate Ester
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d+ d- :Nuc O SO
OCH3R
O SO
OCH3
R Nuc
+
A resonance stabilized leaving group
An energetically unfavorable reaction is biosynthetically driven by linking it to an energetically favorable reaction, such as the hydrolysis of ATP.
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ON
N
N
N
NH2
OPO
OPO
OOP
O
OHO
HOOH
O
Adenosine AMP
ADP
Phophoester bond Phosphoanhydride
bonds
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ON
N
N
N
NH2
OPO
OPO
OOP
O
OHO
HOOH
OSoft Nu:Hard Nu:
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.
The phosphoanhydride bonds are effective stores of chemical potential energy.
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In many structures, the abbreviation P is used to represent the phosphate (orthophosphate) group and PP the diphosphate (or pyrophosphate) group:
OPO
OOP
O
OOP
O
O
P (orthophosphate) PP (Diphosphate)
When a new reaction is initiated in nature, very often the first step is a reaction with ATP to make the compound more reactive.
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ON
N
N
N
NH2
OPO
OPO
OOP
O
OOH
OHOH
OR OH..
..+
ON
N
N
N
NH2
OPO
OPO
OO
OHOH
O
PO
OHO
O+
R
H
:....
+
ON
N
N
N
NH2
OPO
OPO
OHO
OHOH
OPO
OHO
OR
+
Mg2+/Mn2+ Enzyme ATP
ADP
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The phosphorylated alcohol is then activated towards nucleophilic displacement:
H Y+
R
PO
OHO
O:....
+
PO
OHO
HOR Y +
PO
OHO
ORH Y
R OH R Y:Y-H
+ -:OHATP ADP
In summary
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So, overall the endothermic process
R OH R Y:Y-H
+ -: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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More generally the hydrolysis of n ATP molecules change the equilibrium ratio of a coupled reaction by a factor of 108n.
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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THERMODYNAMICALLY UNFAVORABLE
IN SUMMARY
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II. Coenzyme A
Coenzyme A is one of the most important acyl-transfer and a-carbon activating reagents in living organisms.
Acylation and formation of C-C bond formation a to C=O.
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IN VITRO (IN A TEST TUBE)
O
O
RY
OR OH..
:+ :YH +
O+
O
R
STRONG ACID OR BASE
Ester
Acyl substitution reaction SCH 302
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a-substitution reaction
O
O
RH H
O
O
R
..:
+ R1-X
R1
:BASE
a-Hydrogens
The a-Hydrogens are acidic because they can easily be picked by a base forming a resonance stabilized enolates.
O
O
RH H
..:
O
O
R.. O
O
R
:BASE
ENOLATE
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Coenzyme A (CoASH)
ON
N
N
N
NH2
OPO-
O
OP
O-
O
O
OOH
P O-O-O
N
HO
O
H
NHS
O
H
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Acyl substitution reaction
a-substitution reaction
This reactions can go readily in a biological system (in vivo) with out any acid or a base.
SCoAR
O+ YH
YR
O+ CoASH
SCoAR
O+ R1X
SCoAR
O
R1
+ HX
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CoASH
Y
SCoAROXR
O
R'-LG
O
SCoAO
YRO
SCoARO
R'
SCoARO
OH
SCoARO
O
ACYL TRANSFER
α-CARBON ALKYLATION
ALDOL REACTIONS
CLAISEN-type C-ACYLATION
Can act as a nucleophile or electrophile
Enzyme
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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.
OR
OR'
H
H .... O
RO-
R'
H
H
+
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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.
SRO
CoA
H
H .... SR
O-
CoA
H
H
+
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Z
OR
H HY
OR
H H
Y H
R' X
Z
OR
H R'
ACYL SUBSTITUTION
- SUBSTITUTION
REQUIRE THE USE OF STRONG ACIDS AND BASES
IN SUMMARY SCH 511
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SCoA
OR
H H
CoASH
SCH 511
III.Methylation reaction in biological systems
IN VITRO Williamson Ether Synthesis
R O H
R O CH3
..
..:OH
_
R O:....
_
CH3 X
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OPO
OPO
OOP
O
OOH
OO N
NN
NNH2
OH OH
S+
NH2
O OH
CH3
+
CH3S
NH2
OOH
ON
N
N
N
NH2
OPO
OPO
OOP
O
OOH
OHOH
O
L-methionine
ATP
S-AdenosylMethionie (SAM) SAM acts as a versatile O-, C-, N- & S- Methylating agent in vivo
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O N
NN
NNH2
OH OH
S+
NH2
O OH
CH3OHNH2 ..
.. ..OR
OMe
NMe
H
O N
NN
NNH2
OH OH
SNH2
O OH
OR +
O and N alkylation using SAM
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..
.. ..OH OH OH
.._
+
_
+
O N
NN
NNH2
OH OH
S+
NH2
O OH
CH3
OHCH3
+ OH
CH3
+
OHCH3
Aromatization
OH
CH3Aromatization
C alkylation using SAM
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S+
NH2
O OH
CH3
Ad
S-AdenosylMethionie (SAM)
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IV. Dimethylallylation The dimethylallyl group is a very common substituent in secondary metabolites.
OPPDimethylallyl pyrophosphate (DMAPP)
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OPP
a b
-:Nu
Reverse prenylation Prenylation Nu
Nu
Enzyme (Mg2+ or Mn2+)
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O
HO O-
ATP
Carbonic acid
O
ONu
V. Carboxylation
Biotin in the presence of bicarbonate, ATP and Mg2+ enables nucleophile carboxylation in vivo:
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N'-Carboxybiotin
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VI. OXIDATION AND REDUCTION REACTIONS IN BIOLOGICAL SYTEMS
OH
OH
OH
OH
[H][O]
[O]
[H]
IN VITRO
PCC KMnO4, -:OH, Heat ii. H3O+
i. LiAlH4 ii. H3O+
i. LiAlH4 ii. H3O+
i. NaBH4 ii. H3O+
OR
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NAD+
NADP+
R = -H R = -PO3H
Nature’s Hydride Reducing / Oxidizing Coenzyme (reagent) membranes
cytosol
N
NN
NN
CNH2
O
O
CH2 O P OO
OPO
OO CH2 O
OH OH
OH O
NH2
R - -
+
The two forms differ by a phosphate group which also controls the location in the cell.
NICOTINE ADENINE DINUCLEOTIDE
N
CNH2
O
R+
96 Dr. Solomon Derese
17
SCH 511
NAD+ and NADP+ ARE HYDRIDE ACCEPTORS
OXIDATION
N
CNH2
O
R+
CH3 H
O
H
H
RS
C
O
H
N
CNH2
O
R
HH
..
RS
Hydride transfers
REDUCTION
:B-Enz H-B-Enz
NAD+
NADP+
NADH
NADPH
NADH and NADPH ARE HYDRIDE DONORS
Ethanol
Acetaldehyde
Unlike ordinary chemical reagents, these coenzymes function reversibly.
OXIDATION
REDUCTION
Dehydrogenase
97 Dr. Solomon Derese
SCH 511
98
Since NADP(H) has a prochiral center, and many enzymes can differentiate between the hydrogens HR and HS, the process is usually stereospecific.
SCH 511
99
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].
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.
Dr. Solomon Derese
SCH 511
100
N
H H
OH
HO OH
R
Me O
OO
O
H2NNH
Enzyme
Mg2
The enzyme binds both the 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.
Dr. Solomon Derese
SCH 511
101
Oxi
datio
n R
eact
ions
M
edia
ted
by N
AD
(P)+
Dr. Solomon Derese
SCH 511
102
VII.Hydroxylation and epoxidation reactions in biological systems
EPOXIDATION OF ALKENES
R
RR
R R
RR
R OCHCl3 or CCl4
Epoxide (oxirane)
Peracid (peroxyacid)
Commonly used per-acid
Cl
O
OO
H
mCPBADr. Solomon Derese
18
SCH 511
103
R
R
R
R
R
R
R
RO
O2, H+, NADPH
monooxygenase
The enzyme monooxygenase catalyzes the insertion of an oxygen atom across a carbon-carbon double bond to form an epoxide.
Dr. Solomon Derese
SCH 511
104
Synthesis of Phenols
Dr. Solomon Derese
SCH 511
105
R-H + O2 + NADPH + H+
R-OH + H2O + NADP+
Mono-oxygenase
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.
Dr. Solomon Derese
SCH 511
106
With mono-oxygenases, the second oxygen atom from O2 is reduced to water by an appropriate hydrogen donor, NAD(P)H.
O2H OH
NADPH-active form of oxygen (O2- superoxide) is used. -transfer of one atom from molecular oxygen -radical mechanism
Dr. Solomon Derese
SCH 511
107
H OHO2 , NADPH
enzyme
Mechanism
HH
R
HH
R
O
O2
H
H
R
O:
+
R
O
H
H
R
OH
H
Enolization
NIH SHIFT
Dr. Solomon Derese
SCH 511
108
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 OH
D
Dr. Solomon Derese
19
SCH 511
109
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.
This reaction, of course, produces racemic amines.
Dr. Solomon Derese
NaCNBH3 or NaBH4 [H] ≡
SCH 511
110
For this transformation nature uses a substituted pyridine called PyridoxaL Phosphate (PLP) which in a reversible reaction yield a stereospecific product.
PyridoxaL Phosphate (PLP)
Dr. Solomon Derese
SCH 511
111
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.
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.
Dr. Solomon Derese
SCH 511
112
Imine between enzyme and pyridoxal
N
OH
Me
O
OH
H
PO
O OH
Pyridoxal phosphate
Lysine residue
Dr. Solomon Derese
SCH 511
113
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 a-keto-acids.
a-Keto acid a-Amino acid
R
OO
OH
R
NH2
O
OH
Aminotransferase
PLP Dr. Solomon Derese
SCH 511
114
Imine between enzyme and pyridoxal
Imine between amino acid and pyridoxal
N
OH
Me
O
N
H
PO
O OH
HN
O
NH
OAminotransferase
R
H2N
HO
O
H
N
OH
Me
O
N
H
PO
O OH
RH
O
OH
Dr. Solomon Derese
20
SCH 511
115
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.
N
OH
Me
O
N
H
PO
O OH
RO
HH
N
OH
Me
O
N
H
PO
O OH
RO
H
O O
new imine old imine
Dr. Solomon Derese
SCH 511
116
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.
Dr. Solomon Derese
SCH 511
117
N
OH
Me
O
N
H
PO
O OH
RO
H
O
Reversing this reaction makes an amino acid stereospecifically out of an a-keto-acid.
a-keto-acid Pyridoxamine phosphate
Dr. Solomon Derese
SCH 511
118
R
OO
OH
R
NH2
O
OH
Pyridoxalphosphate
Pyridoxalphosphate (PLP)
Aminotransferase
a-Amino acid a-Keto acid
TRANSAMINATION
Dr. Solomon Derese
SCH 511
119
Several different kinds of amino acid transformations are catalyzed by PLP-requiring enzymes.
The most common transformations are decarboxylation, transamination, racemization (the interconversion of L- and D-amino acids), Ca -- Cb bond cleavage, and a,b-elimination.
NH3
H
O
O
Ra
b
Dr. Solomon Derese
SCH 511
120
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.
N
OH
Me
O
N
H
PO
O OH
HN
O
NH
OAminotransferase
R
H2N
HO
O
H
N
OH
Me
O
N
H
PO
O OH
RH
O
OH
Dr. Solomon Derese
21
SCH 511
121
Decarboxylation
Dr. Solomon Derese
SCH 511
122
MECHANISM
Dr. Solomon Derese
SCH 511
123
Racemization
Dr. Solomon Derese
SCH 511
124
Ca -- Cb bond cleavage
Dr. Solomon Derese
SCH 511
125
R
NH2
H O
OH
Bond broken in transamination,
racemization and a,b - elimination
Bond broken in Ca-Cb cleavage
Bond broken in decarboxylation
Dr. Solomon Derese
SCH 511
126
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,b-elimination 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.
Dr. Solomon Derese
22
SCH 511
127
Control over the choice of reaction arises because the different enzymes bind the substrate–pyridoxal imine in different ways. Decarboxylases bind so that the C–C 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.
Dr. Solomon Derese
SCH 511
128
Racemases and transaminases bind the substrate–pyridoxal imine so that the C–H bond is parallel to the p orbitals in the ring so that proton removal can occur. Enzymes do not speed reactions up indiscriminately—they can selectively accelerate some reactions at the expense of others, even those involving the same reagents.
Dr. Solomon Derese
SCH 511
129
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.
Dr. Solomon Derese