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The Role of Enzymes in Natural Product Chemistry
Biosynthetic reactions are reversible and arecatalyzed by enzymes (enzymes are proteinswhich catalyze biological reactions).
Enzymes catalyse the same types ofreactions that are utilized in any organicchemistry laboratory: oxidation, reduction,alkylation, hydrolysis, hydroxylation,elimination etc.
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Ener
gy st
ate
Reaction
Without enzyme
With enzyme
DG=-RTlnKeq
Enzymes lower the activation energy of reactions
AEw/o
AEw
However, enzymes enhance rates of thesereactions by as much as 1012.
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The hallmarks of enzyme catalysis are:speed, selectivity and specificity.
A property of the reaction catalyzed by theenzyme, being the production of a singleregio- and stereo-isomer of the product.
Selectivity
Specificity
The ability of the enzyme to select a certainsubstrate or functional group out of many.
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The catalytic activity of many enzymesdepends on the presence of small nonprotein molecules termed as cofactors.
Coenzymes are organic molecules that arerequired by certain enzymes to carry outcatalysis.
Cofactors are classified as inorganicsubstances such as Mg2+, Zn2+, Fe2+, Fe3+, etc.or small organic molecules known ascoenzymes.
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Dr. Solomon Derese 67
Cofactors are complex molecules that function incombination with enzymes. Enzymes act ascatalysts to control the rate of biologicaltransformations and, as such, are themselvesunchanged as a result of a reaction. Cofactors, onthe other hand, act as reagents to accomplish netchemical conversions and are transformed.
Cofactors are the biochemical equivalents oflaboratory reagents, except that cofactors arealways recycled.
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ATP
CoASHSAM
DMAPP
BIOTINNAD(P)+
NAD(P)H
PLP
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I. ATP (Adenosine TriPhosphate)ATP activates a chemical reaction that isthermodynamically unfavorable.
R-OH:Nuc
R-Nuc -:OHA thermodynamically unfavorable highlyendothermic reaction, because the hydroxyl is abad leaving group.
Dr. Solomon Derese
Consider a chemical reaction that isthermodynamically unfavorable without an inputof energy, a situation common to manybiosynthetic 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 thetosylate ester, which acts as a very good leavinggroup.
d+ 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
Good leaving groupDr. Solomon Derese
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d+ d-:Nuc O SO
OCH3R
O SO
OCH3
R Nuc
+
A resonance stabilized leaving group
An energetically unfavorable reaction isbiosynthetically driven by linking it to anenergetically favorable reaction, such as thehydrolysis of ATP.
Dr. Solomon Derese
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73ATP
ON
N
N
N
NH2
OPO
OPO
OOP
O
OHO
HOOH
O
Adenosine
AMPADP
Phophoester bond
Phosphoanhydridebonds
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ATP can be attacked by hard nucleophiles at aphosphate group (usually the end one) or by softnucleophiles at the CH2 group on the sugar.
The phosphoanhydride bonds are effective storesof chemical potential energy.
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In many structures, the abbreviation P is used torepresent the phosphate (orthophosphate) groupand PP the diphosphate (or pyrophosphate)group:
OPO
OOP
O
OOP
O
O
P (orthophosphate) PP (Diphosphate)
When a new reaction is initiated in nature, veryoften the first step is a reaction with ATP to makethe 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 activatedtowards nucleophilic displacement:
H Y+
R
PO
OHO
O:....
+
PO
OHO
HOR Y +
PO
OHO
ORH Y
R OH R Y:Y-H
+ -:OH
ATP ADP
In summary
Dr. Solomon Derese
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So, overall the endothermic process
R OH R Y:Y-H
+ -:OH
has been achieved by ‘coupling’ the process tothe ‘hydrolysis of ATP’.
In general, the exothermicity associated withphosphorylation shifts the equilibria of ‘coupled’process by a factor of ≈108 .
In other words, coupling the hydrolysis of ATPwith the conversion of ROH to RY can change theequilibrium ratio of ROH to RY by 108.
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More generally the hydrolysis of n ATPmolecules change the equilibrium ratio of acoupled reaction by a factor of 108n.
Thus, a thermodynamically unfavorablereaction sequence can be convertedinto a favorable one by coupling it withthe hydrolysis of a sufficient number ofATP 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 importantacyl-transfer and a-carbon activatingreagents in living organisms.
Acylation and formation of C-C bondformation 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
Dr. Solomon Derese
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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 caneasily be picked by a base forming a resonancestabilized 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 anucleophile orelectrophile
Enzyme
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Important contributor
The P orbitals of C and O are in the samegroup such that they can effectively overlapand 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 groupssuch that they cannot effectively overlap and forma p-bond.
The C-S bond is longer and weaker than the C-Obond.
SRO
CoA
H
H .... SR
O-
CoA
H
H
+
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Require the use OfStrong Acids and Bases
IN SUMMARY
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SCH 511
III.Methylation reaction inbiological systems
IN VITRO Williamson Ether Synthesis
R O H
R O CH3
..
..:OH
_
R O:....
_
CH3 X
91Dr. Solomon Derese
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92
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. DimethylallylationThe dimethylallyl group is a very commonsubstituent in secondary metabolites.
OPPDimethylallyl pyrophosphate (DMAPP)
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OPP
ab
-:Nu
Reverse prenylation PrenylationNu
Nu
Enzyme (Mg2+ or Mn2+)
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V. Carboxylation of Nucleophiles
Dr. Solomon Derese
Carbonic acid
Biotin in the presenceof bicarbonate, ATP andMg2+ enablesnucleophilecarboxylation in vivo:
O
O OP
O–
O
O–
H
N'-Carboxybiotin
ATP
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VI. OXIDATION AND REDUCTIONREACTIONS IN BIOLOGICAL SYTEMS
OH
OH
OH
OH
[H][O]
[O]
[H]
IN VITRO
PCCKMnO4, -:OH, Heat ii. H3O+
i. LiAlH4 ii. H3O+
i. LiAlH4 ii. H3O+
i. NaBH4 ii. H3O+
OR
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SCH 511
NAD+
NADP+
R = -HR = -PO3H
Nature’s HydrideReducing / OxidizingCoenzyme (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 groupwhich also controls the location in the cell.
NICOTINE ADENINE DINUCLEOTIDE
100Dr. Solomon Derese
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NAD+ and NADP+ ARE HYDRIDE ACCEPTORS
OXIDATION
CH3 H
O
H
H
RS
C
O
H
N
CNH2
O
R
HH
..
RS
Hydride transfers
REDUCTION
:B-EnzH-B-Enz
NAD+
NADP+
NADH
NADPH
NADH and NADPH ARE HYDRIDE DONORS
Ethanol
Acetaldehyde
Unlike ordinary chemical reagents, these coenzymesfunction reversibly.
OXIDATION
REDUCTION
Dehydrogenase
101Dr. Solomon Derese
+
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Since NADP(H) has a prochiral center, andmany enzymes can differentiate between thehydrogens HR and HS, the process is usuallystereospecific.
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103
Other enzymes are specific for the HShydrogen. In each instance, one :H- (of thecofactor) and H+ are utilized. This process isdepicted as 2[H].
In the example given above, the hydridefrom ethanol enters from above the plane ofthe ring, and it is this same hydrogen, HR,which is transferred to acetaldehyde in thereverse process.
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N
H H
OH
HO OH
R
Me O
OO
O
H2NNH
Enzyme
Mg2
The enzyme binds both thesubstrate (pyruvic acid) andthe reagent (NADH) in aspecific way so that thehydride is delivered to oneenantiotopic face of theketone. A magnesium(II)cation, also held by theenzyme, binds the carbonylgroup of the amide of NADHand the ketone in pyruvate.only the top H atom (asdrawn) of the diastereotopicCH2 group in NADH shouldbe transferred to pyruvate.
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Oxi
dation
Rea
ctio
nsM
edia
ted
byN
AD
(P)+
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VII.Hydroxylation and epoxidationreactions 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
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R
R
R
R
R
R
R
RO
O2, H+, NADPH
monooxygenase
The enzyme monooxygenase catalyzesthe insertion of an oxygen atom acrossa carbon-carbon double bond to forman epoxide.
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Synthesis of Phenols
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R-H + O2 + NADPH + H+
R-OH + H2O + NADP+
Mono-oxygenase
Oxygenases catalyze the direct addition ofmolecular oxygen to the substrate. They aresubdivided into mono- and di-oxygenasesaccording to whether just one or both of theoxygens are introduced into the substrate.
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With mono-oxygenases, the second oxygen atomfrom O2 is reduced to water by an appropriatehydrogen donor, NAD(P)H.
O2H OH
NADPH-active form of oxygen (O2- superoxide) is used.-transfer of one atom from molecular oxygen-radical mechanism
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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
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An NIH shift is a chemical rearrangement wherea hydrogen atom on an aromatic ring undergoesan intramolecular migration primarily during ahydroxylation reaction. This process is alsoknown as a 1,2-hydride shift.
D OH
D
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VIII. Reductive Amination in Nature
One of the best methods of amine synthesisin the laboratory is reductive amination, inwhich an imine (formed from a carbonylcompound and an amine) is reduced to asaturated amine.
This reaction, of course, produces racemicamines.
Dr. Solomon Derese
NaCNBH3 or NaBH4[H] ≡
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For this transformation nature uses asubstituted pyridine called PyridoxaLPhosphate (PLP) which in a reversiblereaction yield a stereospecific product.
PyridoxaL Phosphate (PLP)Dr. Solomon Derese
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PLP is a coenzyme and it is carried around onthe side chain of a lysine residue of theenzyme. Lysine has a long flexible side chainof four CH2 groups ending with a primaryamine (NH2). This group forms an imine withPLP.
It uses an amine transfer rather than a simplereductive amination, and the family ofenzymes that catalyse the process is thefamily of aminotransferases.
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Imine between enzymeand pyridoxal
N
OH
Me
O
OH
H
PO
O OH
Pyridoxal phosphate
AminotransferaseHN
O
NH
O
NH2
N
OH
Me
O
N
H
PO
O OH
HN
O
NH
OAminotransferase
Lysineresidue
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When reductive amination or its reverse isrequired, the pyridoxal is transferred fromthe lysine imine to the carbonyl group of thesubstrate to form a new imine of the samesort. The most important substrates for PLPare the amino acids and their equivalent a-keto-acids.
a-Keto acid a-Amino acid
R
OO
OH
R
NH2
O
OH
Aminotransferase
PLPDr. Solomon Derese
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Imine between enzymeand pyridoxal
Imine between aminoacid and pyridoxal
N
OH
Me
O
N
H
PO
O OH
HN
O
NH
OAminotransferase
R
H2N
HO
O
H
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By using the protonated nitrogen atom ofthe pyridine as an electron sink, the aproton of the amino acid can be removed toform a new imine at the top of the moleculeand 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 imineold imine
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Now the electrons can return through thepyridine ring and pick up a proton at thetop of the molecule. The proton can bepicked up where it came from, but morefruitfully it can be picked up at the carbonatom on the other side of the nitrogen.
Hydrolysis of this imine releasespyridoxamine and the keto-acid. All thenatural amino acids are in equilibrium withtheir equivalent a-keto-acids by thismechanism, catalysed by anaminotransferase.
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H2O
N
OH
Me
O
NH2
H
PO
O OH
RO
H
H
O
O
Reversing this reaction makes an amino acidstereospecifically out of an a-keto-acid.
a-keto-acidPyridoxamine phosphate
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R
OO
OH
R
NH2
O
OH
a-Amino acid a-Keto acid
TRANSAMINATION
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Several different kinds of amino acidtransformations are catalyzed by PLP-requiring enzymes.
The most common transformations aredecarboxylation, transamination,racemization (the interconversion of L- andD-amino acids), Ca -- Cb bond cleavage, anda,b-elimination.
NH3
H
O
O
Ra
b
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The first step in all PLP-requiring enzymes isa reaction between the amino group of theamino acid and the Imine between enzymeand pyridoxal PLP forming an imine.
N
OH
Me
O
N
H
PO
O OH
HN
O
NH
OAminotransferase
R
H2N
HO
O
H
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Decarboxylation
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MECHANISM
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Racemization
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Ca -- Cb bond cleavage
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Bond broken in transamination,
racemization and a,b - elimination
Bond broken in Ca-Cbcleavage
Bond broken in decarboxylation
Dr. Solomon Derese
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