Product Chemistry · protein molecules termed as cofactors. Coenzymes are organic molecules that...
67
SCH 511 63 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. Dr. Solomon Derese
Transcript of Product Chemistry · protein molecules termed as cofactors. Coenzymes are organic molecules that...
SCH 511
63
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.
Dr. Solomon Derese
SCH 511
64
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.
Dr. Solomon Derese
SCH 511
65
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.
Dr. Solomon Derese
SCH 511
66
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.
Dr. Solomon Derese
SCH 511
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.
SCH 511
68
ATP
CoASHSAM
DMAPP
BIOTINNAD(P)+
NAD(P)H
PLP
Dr. Solomon Derese
SCH 511
69
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.
+
SCH 511
70
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)
Dr. Solomon Derese
SCH 511
71
R OH
Cl SO
OCH3:
..O
+SO
OCH3
R
H
N..
O SO
OCH3R
Formation of Tosylate Ester
Good leaving groupDr. Solomon Derese
SCH 511
72
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
SCH 511
73ATP
ON
N
N
N
NH2
OPO
OPO
OOP
O
OHO
HOOH
O
Adenosine
AMPADP
Phophoester bond
Phosphoanhydridebonds
Dr. Solomon Derese
SCH 511
74
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.
Dr. Solomon Derese
SCH 511
75
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.
Dr. Solomon Derese
SCH 511
76
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
Dr. Solomon Derese
SCH 511
77
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
SCH 511
78
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.
Dr. Solomon Derese
SCH 511
79
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.
Dr. Solomon Derese
SCH 511
Dr. Solomon Derese 80
Thermodynamically Unfavorable
IN SUMMARY
SCH 511
81
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.
Dr. Solomon Derese
SCH 511
82
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
SCH 511
83
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
Dr. Solomon Derese
SCH 511
84
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
Dr. Solomon Derese
SCH 511
85
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
Dr. Solomon Derese
SCH 511
86
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
Dr. Solomon Derese
SCH 511
87
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
+
Dr. Solomon Derese
SCH 511
88
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
+
Dr. Solomon Derese
SCH 511
Dr. Solomon Derese 89
Require the use OfStrong Acids and Bases
IN SUMMARY
SCH 511
Dr. Solomon Derese 90
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
SCH 511
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
Dr. Solomon Derese
SCH 511
93
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
Dr. Solomon Derese
SCH 511
94
..
.. ..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
Dr. Solomon Derese
SCH 511
95
S+
NH2
O OH
CH3
Ad
S-AdenosylMethionie (SAM)
Dr. Solomon Derese
SCH 511
96
IV. DimethylallylationThe dimethylallyl group is a very commonsubstituent in secondary metabolites.
OPPDimethylallyl pyrophosphate (DMAPP)
Dr. Solomon Derese
SCH 511
97
OPP
ab
-:Nu
Reverse prenylation PrenylationNu
Nu
Enzyme (Mg2+ or Mn2+)
Dr. Solomon Derese
SCH 511
98
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
SCH 511
99
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
Dr. Solomon Derese
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
SCH 511
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
+
SCH 511
102
Since NADP(H) has a prochiral center, andmany enzymes can differentiate between thehydrogens HR and HS, the process is usuallystereospecific.
SCH 511
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.
Dr. Solomon Derese
SCH 511
104
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.
Dr. Solomon Derese
SCH 511
105
Oxi
dation
Rea
ctio
nsM
edia
ted
byN
AD
(P)+
Dr. Solomon Derese
SCH 511
106
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
SCH 511
107
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.
Dr. Solomon Derese
SCH 511
108
Synthesis of Phenols
Dr. Solomon Derese
SCH 511
109
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.
Dr. Solomon Derese
SCH 511
110
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
Dr. Solomon Derese
SCH 511
111
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
112
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
Dr. Solomon Derese
SCH 511
113
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] ≡
SCH 511
114
For this transformation nature uses asubstituted pyridine called PyridoxaLPhosphate (PLP) which in a reversiblereaction yield a stereospecific product.
PyridoxaL Phosphate (PLP)Dr. Solomon Derese
SCH 511
115
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.
Dr. Solomon Derese
SCH 511
116
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
Dr. Solomon Derese
SCH 511
117
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
SCH 511
118
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
Dr. Solomon Derese
SCH 511
119
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
Dr. Solomon Derese
SCH 511
120
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.
Dr. Solomon Derese
SCH 511
121
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
Dr. Solomon Derese
SCH 511
122
R
OO
OH
R
NH2
O
OH
a-Amino acid a-Keto acid
TRANSAMINATION
Dr. Solomon Derese
SCH 511
123
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
Dr. Solomon Derese
SCH 511
124
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
Dr. Solomon Derese
SCH 511
125
Decarboxylation
Dr. Solomon Derese
SCH 511
126
MECHANISM
Dr. Solomon Derese
SCH 511
127
Racemization
Dr. Solomon Derese
SCH 511
128
Ca -- Cb bond cleavage
Dr. Solomon Derese
SCH 511
129
Bond broken in transamination,
racemization and a,b - elimination
Bond broken in Ca-Cbcleavage
Bond broken in decarboxylation
Dr. Solomon Derese
