The Organic Chemistry of Enzyme-Catalyzed Reactions Chapter 13 Rearrangements

1
The Organic Chemistry of Enzyme-Catalyzed
Reactions Chapter 13Rearrangements
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Rearrangements
Pericyclic Reactions - concerted reactions in
which bonding changes occur via reorganization of
electrons within a loop of interacting orbitals
3
Sigmatropic Rearrangements
3,3 sigmatropic rearrangement
General form of the Claisen rearrangement
Scheme 13.1
4
Chorismate Mutase-catalyzed Conversion of
Chorismate to Prephenate
Scheme 13.2
chorismate
prephenate
A step in the biosynthesis of Tyr and Phe in
bacteria, fungi, plants
5
Conformation of Chorismate in Solution
Required conformer for Claisen rearrangement
(10-40 observed in solution from NMR spectrum)
chair-like TS
6
Stereochemical outcome if chorismate mutase
proceeds via chair and boat transition states,
respectively, during reaction with
(Z)-9-3Hchorismate
Evidence for Chairlike Transition State
Scheme 13.3
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Chemoenzymatic degradation of the prephenate
formed from the chorismate mutase-catalyzed
conversion of (Z)-9-3Hchorismate to determine
the position of the tritium
To Determine the Position of the 3H
Scheme 13.4
Z-9- 3Hchorismate 20 3H release E-9-
3Hchorismate 67 3H release
Therefore, chair TS
8
Five Hypothetical Stepwise Mechanisms for the
Reaction Catalyzed by Chorismate Mutase
4
Figure 13.1
2 inverse isotope effect on C-4 (sp2 ? sp3)
therefore not 1-3 (sp3 ? sp2)
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Both are substrates
mechanism 5 excluded
mechanisms 1, 2, 5 excluded
16 mutants made to show neither general acid-base
catalysis (mechanisms 1-3, 5) nor nucleophilic
catalysis (mechanism 4) is important
Conclusion pericyclic
Function of the enzyme is to stabilize the chair
transition state geometry
10
General form of Cope (A) and oxy-Cope (B)
reactions
Oxy-Cope Rearrangement
Cope
oxy-Cope
Scheme 13.5
Neither observed yet by an enzyme, but a
catalytic antibody has been raised
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Oxy-Cope Rearrangement Catalyzed by an Antibody
Scheme 13.6
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hapten to raise the antibody
13
2,3 Sigmatropic Rearrangement Catalyzed by
Cyclohexanone Oxygenase
Scheme 13.7
14
42 Cycloaddition (Diels-Alder) Reaction
boat like TS
Scheme 13.9
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An Intramolecular Diels-Alder Reaction Catalyzed
by Alternaria solani
Scheme 13.10
solanopyrones
in aqueous solution exo endo is 3 97
(nonenzymatic)
enzymatic exo endo is 53 47
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An Antibody-Catalyzed Diels-Alder Reaction
Scheme 13.11
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Hapten used
18
This hapten gives an antibody that makes only
endo product
This hapten gives an antibody that makes only exo
product
19
An acid-catalyzed acyloin-type rearrangement
Rearrangements via a Carbenium Ion
acid-catalyzed
1,2 alkyl migration
acyloins
Scheme 13.14
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Reactions Catalyzed by Acetohydroxy Acid
Isomeroreductase
Scheme 13.15
21
substrate
Kinetically-competent intermediate
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Proposed Acyloin-type Mechanism for Acetohydroxy
Acid Isomeroreductase
Scheme 13.16
intermediate
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Conversion of squalene to lanosterol
Cyclizations Sterol biosynthesis
cholesterol
lanosterol
squalene
Scheme 13.17
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Initial Mechanism Proposed for 2,3-Oxidosqualene-
lanosterol Cyclase
2,3-oxidosqualene-lanosterol cyclase
not isolated
squalene 2,3-epoxidase
17?
anti-Markovnikov (to get 6-membered ring)
protosterol
squalene
Scheme 13.18
lanosterol
7 stereogenic centers
(128 possible isomers)
only isomer formed
Isotope labeling shows the 4 migrations are
intramolecular Covalent catalysis proposed to
control stereochemistry
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Use of 20-oxa-2,3-oxidosqualene to determine the
stereochemistry at C-17 of lanosterol from the
reaction catalyzed by 2,3-oxidosqualene-lanosterol
cyclase
Evidence for 17? Configuration
O instead of CH2
17?
Scheme 13.19
no covalent catalysis needed
17?
isolated
26
Use of (20E)-20,21-dehydro-2,3-oxidosqualene to
determine the stereochemistry at C-17 of
lanosterol from the reaction catalyzed by
2,3-oxidosqualene-lanosterol cyclase
Further Support for Structure of Protosterol
17?
Scheme 13.20
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Chemical model for the conversion of protosterol
to lanosterol
Model Study for Stereospecificity and Importance
of 17? Configuration
Scheme 13.21
17?
17?
90
With the 17? isomer a mixture of C-20 epimers is
formed
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Mechanism proposed for the formation of the minor
product isolated in the 2,3-oxidosqualene
cyclase-catalyzed reaction with
20-oxa-2,3-oxidosqualene
Evidence that the Cyclization Is Not Concerted
does not come from a concerted reaction
not when XCH2
Markovnikov addition
ring expansion
Scheme 13.22
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Evidence for Carbocation Intermediate
no reaction without methyls - suggests initial
epoxide opening
Vmax/Km for R CH3, H, Cl 138, 9.4,
21.9 pmol ?g-1h-1?M-1
correlates with carbocation stabilization (CH3 gt
Cl gtH)
30
Squalene synthase-catalyzed conversion of
farnesyl diphosphate to squalene via presqualene
diphosphate
Squalene Biosynthesis
farnesyl diphosphate
squalene
presqualene diphosphate
Scheme 13.23
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Mechanism proposed for the conversion of
presqualene to squalene by squalene synthase
Rearrangement of Presqualene Diphosphate to
Squalene
squalene
Scheme 13.24
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Mechanisms proposed for the squalene
synthase-catalyzed hydrolysis of presqualene
diphosphate to several different products in the
absence of NADPH
In the Absence of NADPH there is a Slow
Hydrolysis Evidence for 13.56 and 13.57
Scheme 13.25
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Use of dihydro-NADPH to provide evidence for the
formation of intermediate 13.57 in the reaction
catalyzed by squalene synthase
Support for Intermediate 13.57
dihydro-NADPH
unreactive NADPH to mimic bound NADPH
Scheme 13.26
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Rearrangements Via Radical Intermediates
DNA Photolyase UV light causes DNA damage
Reactions catalyzed by DNA photolyase and (6-4)
photolyase
visible h? used as a substrate for
photoreactivation
cyclobutane pyrimidine dimer
both types carcinogenic, mutagenic
Scheme 13.27
(6-4) photoproduct
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Other Cofactors Used by Photolyases
reduced FADH-
N5,N10-methenyl H4PteGlun
These act as photoantennae to absorb blue light
and transmit to the FADH-
8-OH-7,8-didemethyl-5-deazariboflavin
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Mechanism Proposed for DNA Photolyase
Scheme 13.28
EPR evidence
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Proposed Mechanism for the Formation of the (6-4)
Photoproduct
Scheme 13.29
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Mechanism Proposed for (6-4) Photolyase
Scheme 13.30
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Coenzyme B12 Rearrangements
adenosylcobalamin
(coenzyme B12)
(vitamin B12)
40
5?-deoxyadenosyl
abbreviation for coenzyme B12
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Bioynthesis of coenzyme B12
Conversion of Vitamin B12 to Coenzyme B12
B12r
2nd known reaction at C-5? of ATP
B12s
Scheme 13.31
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Light Sensitivity of the Co-C Bond of Coenzyme B12
Scheme 13.32
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(No Transcript)
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General Form of Coenzyme B12-Dependent
Rearrangements
X is alkyl, acyl, or electronegative group
Scheme 13.33
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Three Examples of Coenzyme B12 Rearrangements
Figure 13.2
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Mechanism for Diol Dehydratase and Ethanolamine
Ammonia-Lyase
Stereospecific conversion of (1R,2R)-1-2H-1-14C
propanediol to (2S)-2-2H-1-14Cpropionaldehyde
catalyzed by diol dehydratase
R
R
Scheme 13.34
(2S)
(1R, 2R)
No incorporation of solvent protons therefore no
elimination of water (enol would form)
Stereospecific 1,2 migration of the pro-R H
with inversion
kH/kD 10-12
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Stereospecific Conversion of (1R,2S)-1-2H-1-14C
propanediol to 1-2H-1-14Cpropionaldehyde
Catalyzed by Diol Dehydratase
S
R
(1R, 2S)
Scheme 13.35
With the (1R, 2S) epimer, the pro-S H migrates
therefore stereochemistry at C-2 determines which
C-1 H migrates
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Stereospecificity of Elimination of Water
Diol dehydratase-catalyzed conversion of
(2S)-1-18Opropanediol to 18Opropionaldehyde
(A) and of (2R)-1-18Opropanediol to
propionaldehyde (B)
(2S)-1-18O
Scheme 13.36
(2R)-1-18O
The same OH is eliminated (pro-R) regardless of
which C-1 H migrates
Therefore the C-1 H and the C-2 OH migrate from
opposite sides giving inversion at both C-1 and
C-2
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Crossover Experiment to Show that Diol
Dehydratase Catalyzes an Intermolecular Transfer
of a Hydrogen from C-1 to C-2
Scheme 13.37
Therefore, hydrogen transfer is intermolecular
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Time Course for Incorporation of Tritium from
1-3Hpropanediol into the Cobalamin of Diol
Dehydratase
Figure 13.3
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Determination of the Site of Incorporation of 3H
into Coenzyme B12
Aerobic and anaerobic photolytic degradation of
coenzyme B12 to locate the position of the
tritium incorporated from 1-3Hpropanediol in a
reaction catalyzed by diol dehydratase
1/2 3H lost
no 3H here
3H here
all 3H retained
Scheme 13.38
no 3H here
3H here
Reconstitution of the isolated 3H coenzyme B12
into apoenzyme with propanediol gives
2-3Hpropionaldehyde. All 3H transferred from
3H coenzyme B12
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Synthesized (R,S)-5?-3H Coenzyme B12 Transfers
All 3H to the Product Randomly
possible intermediate to equilibrate the C-5?
protons
13.88 isolated with substrates that cannot
rearrange
Coenzyme B12 is the hydrogen transfer agent.
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Formation of 5?-deoxyadenosine, cob(II)alamin,
and substrate radicals during coenzyme
B12-dependent reactions
Proposed Rationalization for EPR Spectrum of
Co(II) Carbon Radicals
Scheme 13.39
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Mechanism(s) Proposed for Diol Dehydratase
The part shown in the dashed box is even more
speculative than the rest of the mechanism
Not clear if important
Scheme 13.40
Radicals observed in EPR spectrum
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Chemical Model Study for a Proposed Diol
Dehydratase-catalyzed Rearrangement Involving a
Co(III)-olefin ?-Complex
The trapezoid represents the cobaloxime ligand
Scheme 13.41
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The Fenton reaction as a model for a proposed
diol dehydratase-catalyzed free radical
rearrangement
A Cobalt Complex Is Not Necessary
Scheme 13.42
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Another Chemical Model Study for a Proposed Diol
Dehydratase-catalyzed Free Radical Rearrangement
(the cobalt complex is just to initiate the
reaction by radical generation)
Scheme 13.43
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Carbon Skeletal Rearrangements
Stepwise (a) versus concerted (b) mechanisms for
the methylmalonyl-CoA mutase-catalyzed generation
of 5?-deoxyadenosine, cob(II)alamin, and
substrate radical
Scheme 13.44

EPR confirms Co(II) organic radical Crystal
structures with and without substrates bound show
the active site closes upon substrate binding -
shields radical intermediates
Co-C cleavage is 21 times faster with (CH3)MM-CoA
than with (CD3)MM-CoA.
Therefore, Co-C and C-H cleavage are concerted.
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Six Possible Pathways for the Conversion of
Methylmalonyl-CoA Radical to Succinyl-CoA Radical
Catalyzed by Methylmalonyl-CoA Mutase
Ab initio calculations disfavor pathway e No
concensus about the others
Figure 13.4
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Ribonucleotide Reductase
Converts ribonucleotides to deoxyribonucleotides
Results are different from other coenzyme B12
enzymes

• 0.01-0.1 of 3H from 3-3HUTP is released

• no 3H from 3-3HUTP found in adenosylcobalamin

• no crossover between 3-3HUTP ATP

• 3-3HUTP gives 3-3HdUTP

• 3H in 5?-3Hadenosylcobalamin is washed out in
  the
• absence of substrate

• adenosylcobalamin ? 5?-deoxyadenosine Co(II)

By EPR formation of Co(II) corresponds to
formation of 5?-deoxyadenosine and the
generation of a thiiyl radical (Cys-408)
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Mechanism Proposed for Coenzyme B12-dependent
Ribonucleotide Reductase
rates of formation are identical therefore,
concerted reaction
Scheme 13.45
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Mechanism Proposed for Reducing and
Reestablishing the Active Site of Coenzyme
B12-dependent Ribonucleotide Reductase
Scheme 13.46
regenerates active site for next cycle reduced
by thioredoxin
electrons are transferred to active-site disulfide
The function of the cobalamin in this enzyme is
to initiate the radical reaction by abstraction
of H from Cys-408
63
Other Ribonucleotide Reductases Use Other
Radicals to Abstract a H from an Active Site Cys
Cofactors for class I (13.118), class III
(13.119), and class IV (13.120) ribonucleotide
reductases
Figure 13.5
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Reaction Catalyzed by Lysine 2,3-Aminomutase
pro-R
pro-R
L-?-Lys
L-?-Lys
Scheme 13.47
Requires PLP, SAM, 4Fe-4S, and a reducing agent
Transfers 3-pro-R H of L-?-Lys to 2-pro-R of
L-?-Lys with migration of 2-amino of L-?-Lys to
C-3 of L-?-Lys
No exchange with solvent
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With (S)-5?-3Hadenosylmethionine, 3H ends up in
both L-?-Lys and L-?-Lys
One equivalent of Met and 5?-deoxyadenosine are
formed with L-?-3-3HLys.
1-6 of 3H ends up in SAM
C-S bond is stable, unlike C-Co bond
In the presence of a reducing agent, 4Fe-4S is
observed in the EPR, which reduces SAM to Met
and 5?-deoxyadenosyl radical
It appears that SAM is functioning like coenzyme
B12
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Mechanism Proposed for Lysine 2,3-Aminomutase
not observed in EPR
unique function for PLP
EPR detects organic radicals 13C label shows
product radical 13.126 in EPR spectrum
Scheme 13.48
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Model Study for New Function of PLP
Chemical model study to test the proposed
rearrangement mechanism for lysine 2,3-aminomutase
Scheme 13.49
68
To Get Evidence for Substrate Radical (13.124)
stabilize ?-radical
69
Lysine 2,3-aminomutase-catalyzed rearrangement of
4-thialysine to generate a more stable substrate
radical
Evidence for Substrate Radical Formation
EPR detected
isolated
Scheme 13.50