the organic chemistry of enzyme-catalyzed reactions chapter 4 monooxygenation
TRANSCRIPT
The Organic Chemistry of Enzyme-Catalyzed Reactions
Chapter 4
Monooxygenation
C H
R
1
R
3
R
2
C OH
R
1
R
3
R
2
C C
R
3
R
1
R
2
R
4
C C
R
3R
1
R
2
R
4
O
Ar H Ar OH
N H
R
1
R
2
N OH
R
1
R
2
O
n
O
O
n+1
S
R
1
R
2
S O
R
1
R
2
CH R
2
NH2
R
1
C R
2
NH2
R
1
OH
B
+
H
C R
2
R
1
O
+ NH4
+
:
Table 4.1. Typical reactions catalyzed by monooxygenases
Monooxygenation
Internal Monooxygenase
Scheme 4.1
Reaction catalyzed by lactate oxidase from Mycobacteria
Flavin-dependent Hydroxylases
No external reducing agent required
+ O2E•FMN
+ CO2 + H2O
4.1
HO
C COOH
H
CH3 CH3COOH
One Turnover Experiment (enzyme concentration in excess
over substrate)
Scheme 4.2
Acting like an oxidase
The lactate oxidase reaction under anaerobic conditions
HO
C COOH
H
CH3
4.2
CH3C COOH
OFMN FMNH2
Scheme 4.3
If O2 is added first, then [14C]pyruvate, pyruvate is unchanged and H2O2 is formed. Therefore, pyruvate is an intermediate.
Model study:
Reaction of Reduced Lactate Oxidase with Pyruvate and Oxygen
H3C C
O
COO-E•FMNH2 +14
E•FMN + CH314COO- + H2O + CO2+ O2
CH3CCOOH + H2O2
O
CH3COOH + CO2 + H2O
Scheme 4.4
like DAAO
flavin hydroperoxide acts as a nucleophile
Possible Mechanisms for Lactate Oxidase
CH3 C
H
OH
COOH
B:
CH3 C COOH
OH
CH3 C
O
COO-
NH
RN
NH
HN O
O
18O2
N
RN
NH
N O
O18O
18O
O O HC
O
COO-
O-
18O
CH3C
B: H
NH
RN
NH
N O
O18O
18O
C CCH3
O-
O
O
B+H- CH3C
18O
O-
N
RN
NH
N O
O18OH
B+H
b
4.3
b
4.4
b
b aFMN
- H218O
- CO2
18 18
Enzyme bound
4.6
via a mechanism such asshown in Scheme 3.33
H
:B
CH3 C
O
COO-
4.5
H218O + CO2 +
-FMN
+
CH3C
O
C
18O
18OH
via a mechanism such as shown in Schemes 3.43 or 3.44
O
O
FMN
a
HB:
H3C
B+H
a
FMNH-
electrophilicsubstrate
Scheme 4.5
External Monooxygenases
NAD(P)H reduction of flavin
N
RN
NH
N O
O
NH
RN
NH
HN O
O
NAD(P)H NAD(P)+
O2
H2O2
O2 activation
Activated O2 is probably in the form of flavin hydroperoxide
Nucleophilic Substrates
stopped-flow spectroscopic evidence for boxed intermediates
flavin hydroperoxide acts as electrophile
electrophilic aromatic substitution
Mechanism proposed for flavin-dependent hydroxylases
Scheme 4.6
Substrate NADH Substrate
NADH NAD+
NAD+
H+
NH
RN
NH
N O
OO
O
O2
NH
RN
NH
N O
O
-OOC
OH
_
-OOC
OH
E
Substrate
E
NH
RN
NH
N O
OOB+ H
OH
O
N
RN
NH
N O
OOH
B:
H
O
:BHO H OH
OH
see Scheme 3.33
- H2O
H
:B
BH
+
-OOC
-OOC -OOC
E FAD FADH-FAD
FAD
log Vmax for hydroxylation vs pKa linear free energy relationship = -0.5
4.8
N
RN
NH
N O
O
X
Hammett Study
p-hydroxybenzoate hydroxylase
Consistent with electrophilic aromatic substitution
(Electron deficient mechanism)
Electrophilic Substrates
Scheme 4.7long-chain aldehydes (electrophilic substrates)
Reaction catalyzed by bacterial luciferase
FMN, O2+ hνRCOOH
NADHRCHO
Scheme 4.8
on warming
isolated by cryoenzymology (-30 C in mixed aqueous-organic media)
detected spectro-photometrically
However, with 8-substituted FMN analogues rate increases with decreasing one electron oxidation potentials of analogues
Nucleophilic Mechanism for Bacterial Luciferase
- RCOOH
NH
RN
NH
N O
O
O
O
NH
RN
NH
N O
O
O
O
OH
H
R
:BNH
RN
NH
N O
OOH
N
RN
NH
N O
OOHB:
H
H
R
HO
hν
*
:B
H B
FMN
BH
-H2O
electrophilicsubstrates
Scheme 4.9
SET
Chemically Initiated Electron Exchange Luminescence (CIEEL) Mechanism for
Bacterial Luciferase
NH
RN
NH
N O
O
OO OH
R
O
OHR
R CO
OH
NH
RN
NH
N O
OOH
NH
RN
NH
N O
OOH
R OH
O
H
B
R CO
OH
FMN
H
BH
-H2O-H+
Scheme 4.10
Alternative One-electron Mechanism via a Dioxirane
SET
kx/kh vs. p
for 8-substituted flavins
Dioxirane mechanism for bacterial luciferase
= -4
NH
RN
NH
N O
R
O
O
O
HO
NH
RN
NH
N O
R
O
H
OH
OO
R H
OOB H
NH
RN
NH
N O
O
O
O
H
R
HO
:B
NH
RN
NH
N O
OOH
R O
O
NH
RN
NH
N O
OOH
:B
N
RN
NH
N O
OOHB:
H
determining step
*
hν
FMN-H2O
rate
(facilitated by e- donation)
Inconsistent with Baeyer-Villiger mechanism ( values +0.2 to 0.6)
Scheme 4.11
Migratory aptitude - more e- donating group migrates (in the case above, R)
Baeyer-Villiger Oxidation of Ketones
R R'
O
Ar C
O
O O
O
C
O
R'R
O Ar
O
RO R'
O
+ ArCOO-+
Scheme 4.12
Ketone Monooxygenases - an Example of a Baeyer-Villiger Oxidation
C4a-FAD hydroperoxide intermediate detected
Reaction catalyzed by cyclohexane oxygenase
O O
O
+ NADPH + 18O2
enzyme+ H2
18O
18
FAD
Scheme 4.13
same migratory aptitudes as nonenzymatic reaction
Other Reactions Catalyzed by Cyclohexanone Oxygenase
PhMe
Ph O Me
O
O
R = alkyl
RCHO RCOOH
Scheme 4.14
Cyclohexane Oxygenase Proceeds with Retention of Configuration (like nonenzymatic)
O O
OH
D H
D
Scheme 4.15
Same migratory aptitude as nonenzymatic (3° > 2° > 1° > Me)
Migratory Aptitude of Cyclohexanone Oxygenase-catalyzed Reaction
O
O O
O
O
no loss of D (like nonenzymatic reaction)
4.9
ODD
DD
Scheme 4.16
Baeyer-Villiger-type Mechanism Proposed for Cyclohexanone Oxygenase
O O
O
NH
RN
NH
N O
O
O
O
H:B
NH
RN
NH
N O
O
O
O
O
N
RN
NH
N O
OOHB:
H
H B
-H2OFAD
electrophilic substrate
Scheme 4.17
same as nonenzymatic reaction
Reaction of Cyclohexanone Oxygenase with Boranes
B(OMe)2 B
O O FAD
OMeOMe
BOMe
OMe OHOhydrolysis
Scheme 4.18
when R1 = R2 = Me 1 : 20 R1 = H; R2 = Me 1 : 1
(same as nonenzymatic reaction)
Reactions Catalyzed by Ketone Monooxygenase
O R1
R2O
O
R1
R2
O R2
R1
O
+
4.10 4.11 4.12
Scheme 4.19
>95% ee
Reactions Catalyzed by the Ketone Monooxygenase from A. calcoaceticus
O R1
R2O
O
R1
R2
O R2
R1
O
+
4.10 4.13 4.14
A. calcoaceticusHH HH
R1 = R2 = H (1S,5R) (1R,5S)
R1 = H, R2 = CH3 (1S,5S) (1R,5S)
1S 1R 5S5R
>95% ee
racemate
Scheme 4.20
Reactions Catalyzed by the Ketone Monooxygenase from P. putida
>95% ee
1S1R 5S 5R
>95% ee
50% ee
O R1
R2O
O
R2
R1
O R1
R2
O
+
4.10 4.15 4.16
P. putidaHH HH
R1 = R2 = H (1S,5R)(1R,5S)
R1 = H, R2 = CH3 (1R,5R) (1S,5R)
racemate
pteridine ring
N
NN
N
4.17
Pterin-dependent Monooxygenases
aromatic hydroxylation
• Fe2+ also required for activity
• Only a few enzymes require tetrahydrobiopterin• Important in biosynthesis of dopa, norepinephrine, epinephrine, and serotonin• Reactions similar to flavoenzymes
NH
NHN
NH
O
NH2
4.18
OH
H3C
HO H
H
Tetrahydrobiopterin
Scheme 4.21
Comparison of the Dihydrobiopterin and Tetrahydrobiopterin with Oxidized Flavin
and Reduced Flavin
NH
NN
N
OH
HO
O
NH2
NH
NHN
NR
NH
O
NH2
NRN
N
O
O
NH
HN
HN
NH
R
NH
O
NH2
NHN
NH
R
NH
O
NH2
HN
RN
NH
O
O
pteridine
reductase(NADPH)
reductase(NADPH)
+
dihydropteridine
4.19 4.20 4.21a
4.21b
oxidized flavin reduced flavin
Scheme 4.22
NIH shift
[1,2] migration
Similar to flavin hydroxylases except 2H washed out with flavoenzymes
Reaction Catalyzed by Phenylalanine Hydroxylase
X COO-
NH3+
H18O COO-
NH3+
+ 18O2 + H4-pterin
X = 2H, 3H, Cl, Br, alkyl
Phe
hydroxylase
+ H218O + H2-pterin
X
4.22
NH
HN
NH
N
OO
NH2
R
O
H
+
Possible Intermediate
Scheme 4.23discussed with heme-dependent enzymes
Mechanism of the Reaction Catalyzed by Tetrahydrobiopterin-dependent Monooxygenases
NH
HN
NH
N
O18O
NH2
R
18OB+ H
X R
R
H18O
X X
18O
R
H
RH18O
X
RH18O
H X
B:
RH18O
X
see Scheme 3.33
NIH shift
+
4.23
H2-pterin + H218O
+
+
NH
HN
NH
HN
O
NH2
R
H
18O2
nucleophilic substrate
Scheme 4.24
Evidence for Arene Oxide Intermediate
Reaction of dihydrophenylalanine with phenylalanine hydroxylase
COO-
NH3+
COO-
NH3+
Ohydroxylase
Phe
4.24 4.25
Scheme 4.25
Incubation with [4-2H]Phe should favor formation of
Arene Oxide Mechanism Proposed for Tetrahydrobiopterin-dependent Monooxygenases
m-Tyr
Tyr
Therefore, not an arene oxide intermediate
m-Tyr (isotope effect), and [3,5-2H2]Phe should favor Tyr, but they do not.
COO-
NH3+
COO-
NH3+
O
COO-
NH3+
O
COO-
NH3+
O
H2H
H
2H
COO-
NH3+
HO
COO-
NH3+
HO
2H
hydroxylase
Phe
H 2H
2H
2H
Scheme 4.26
Fe
as X is larger
The larger the size of X, the more m-Tyr product
Cationic Mechanism Proposed for Tetrahydrobiopterin-dependent Monooxygenases
m-Tyr
= -5(cation-like TS‡)
NH2
COOHR
COO-
NH3+
Xhydroxylase
PheCOO-
NH3+
X
O
YCOO-
NH3+
HO
COO-
NH3+
HO
X
COO-
NH3+
X
OH
Y
COO-
NH3+
X
HO
These species could account for alkyl hydroxylation products (heme chemistry), e.g. with
hydroxylation here
NH
HN
NH
N
RO
O
Fe2+
O
NH2+
4.25a
NH
HN
NH
N
R
OHO
Fe4+
O
NH2+
4.25b
NH2
COOHH3C
Alternative Species
Heme
Cytochrome P450s (>500 different isozymes) require NAD(P)H and O2
4.26
N N
NN
COOHCOOH
FeIII
Protection from xenobiotics
Heme-Dependent Monooxygenases
Reactions Catalyzed by Heme-dependent Monooxygenases
Substrate ProductAlkane Alcohol
Alkene Epoxide
Arene Arenol or arene oxide
R2NH, R2O, R2S RNH2, ROH, RSH + RCHO
R3N, R2S R3N-O, R2S-O
RCH2X RCHO + HX
RCH2OH RCHO
RCHO RCOOH
+ +- -
S
FeIII
O
N
N N
N
HH
S
FeIII
N N
N N
S
FeII
N N
N N
S
FeIII
O
N
N N
N
O
S
FeIII
O
N
N N
N
O
S
FeIII
O
N
N N
N
OH
S
FeIII
O
N
N N
N
S
FeVN
N N
N
4.27
4.294.30 4.31
4.32
S
FeIVN
N N
N
4.33d
NAD(P)H
FMN
O
FMN
:O
4.28
4.33a4.33c
S
FeIV
O
N
N N
N
NAD(P)+
:: : :
4.33b
:
B H
: :
H B
R-H
FMN
FMN
FAD
R-H
FMNH
-H2O
FADH
O2
R-O-H
R-H R-H R-H R-H
R-H
Scheme 4.27
low-spin state high-spin state
In P450cam Thr-252
means isolated and characterized
FeIII more readily accepts e-
cytochrome P450 reductase
calculations favor this structure
Molecular Oxygen Activation by Heme-dependent Monooxygenases
(requires NADPH)
Scheme 4.28
Alkane Hydroxylation
3° > 2° > 1°
Intermolecular isotope effect < 2 (suggests C-H cleavage is not the rate-determining step)
retention of configuration
Two-step radical mechanism with oxygen rebound for alkane oxygenation by heme-
dependent monooxygenases
H C R'
R
R''
HO C R'
R
R''S
FeIV
O
N
N N
N
C R'
R
R''
rebound
: :
oxygen
4.284.33b 4.34
S
FeIV
OH
N
N N
N
S
FeIII
N N
N N
C-H cleavage during catalysisIntramolecular isotope effect > 11
Scrambling of stereochemistry supports 2-step radical mechanism
Scheme 4.29
Products from the Reaction of all Exo-2,3,5,6-tetradeuterionorbornane with the CYP2B4 Isozyme of Cytochrome P450
D
DD
D
D
DOH
H
D
D
DOH
D
D
D
DH
OH
D
D
DD4.35
4.37
4.39
4.36
OH
D
4.38
CYP2B4
Scheme 4.30
Radical Clocks - detection of radical intermediates
known
The rate of hydroxylation can be calculated (lifetime of radical intermediate)
Radical clock approach for determination of reaction rates in radical rearrangement reactions
substrate H substrate OHsubstratekOH
kr
rearrangedsubstrate OH
rearrangedsubstrate
kOH = kr (substrate-OH / rearranged substrate-OH)
Scheme 4.31
Example of Radical Clock
a
From kr = 2 109 s-1 and the ratio of a/b, can calculate kOH = 2.4 1011 s-1
b
Cytochrome P450-catalyzed monooxygenation of a cyclopropane analogue
H
S
FeIV
ON
N N
NHO
OH
kOH
kr
::
S
FeIV
OHN
N N
N
Scheme 4.32Perdeuteration (CD3) gives
Cytochrome P450-catalyzed Oxidation of Trans-1-methyl-2-phenylcyclopropane
FeIV O H
FeIV OH
HO
4.42
OH4.43
FeIV OH
b
3 x 1011 s-1
4.44
4.40
a
kOH
4.41
kr
OH
a
increased pathway b called metabolic switching
HH
FeIV O
H
OH
OH
4.45 kr
kOH
a
b
b
Scheme 4.33
Evidence against a True Radical Intermediate
3 1011 s-1
very little
kOH has to be faster than the decomposition of a TS‡ (6 1012 s-1); therefore propose carbocation after oxidation step
Another ultrafast radical clock reaction catalyzed by cytochrome P450
Scheme 4.34
based on nonenzymatic reactions
With CYP2B1 - mostly unrearranged, but small amount of both 4.48 and 4.51; therefore radical lifetime is 70 fs
A Hypersensitive Radical Probe Substrate to Differentiate a Radical from a Cation
Intermediate Generated by Cytochrome P450
OPh
CH3
OPh
CH2
O
Ph
O
Ph
OPh
CH2 Ph
O
Ph
O OH
Ph
H O
4.47
a
4.48
b
+
4.49
HO
Ph
H
4.50
O
4.46
4.51
-tert-butanol
HO
HO-
Scheme 4.35
General conclusion:More than one oxidizing species involving more than one pathway with multiple high-energy heme complexes (radical and cation)
A Concerted, but Nonsynchronous, Mechanism Proposed for Cytochrome P450
H R
O
Fe(IV)
H R
O
Fe
H R
O
Fe
H R
O
Fe
H R
O
Fe(III)
70 fs
Scheme 4.36
Alkene Epoxidation
lifetime?
Two-step radical mechanism with oxygen rebound for alkene oxygenation by heme-
dependent monooxygenases
:
S
FeIV
O
N
N N
N
4.52
rebound"
:
S
FeIV
O
N
N N
N
S
FeIII
N N
N N
R'R
"oxygen
R R'
R'R
O
Evidence for Short-lived Radical
only
cyclopropyl/carbinyl radical rearrangement not detected
Scheme 4.37
Cytochrome P450-catalyzed epoxidation of trans-1-phenyl-2-vinylcyclopropane
Ph Ph4.55
O
4.56
S
FeIV
O
N
N N
NPh
P450
Arene Hydroxylation
Isolation of first arene oxide
Scheme 4.38 Is it an intermediate or side product?
Cytochrome P450-catalyzed formation of an arene oxide
O
4.57
P450
Scheme 4.39
Evidence for a Cyclohexadienone Intermediate
eithersame product and 2H incorporation from both isomers
A common intermediate in the oxygenation of naphthalene
O2H
H
OH
H2
O
H
2HOH
4.58
2H (H)
Should have observed 1- and 2-hydroxynaphthalene because ofan isotope effect
OH
H
O
H
H
O
H
H
O
b
H
H
a
Scheme 4.40
concerted
stepwise
Evidence against concerted: 1) no deuterium isotope effect
2) Hammett plot shows large -
Concerted (pathway a) and Stepwise (pathway b) Mechanisms for the Potential Conversion of an
Arene Oxide to a Cyclohexadienone
(carbocation intermediate)
RD
R
OD
R
H
HO R
D
O
R
S
FeIV
O
N
N N
N
S
FeIV
O
N
N N
N
D
D
:
S
FeIII
N N
:
N N
: :
D
R
a
a
b
b
a
4.59a
S
FeIII
O
N
N N
N
reboundoxygen
: :
electron
4.59b
c
transfer
Scheme 4.41
Isotope Effect and Hammett Studies Indicate either Radical or Cation (or both) Intermediates, but not Arene Oxide
reasonable
unfavorable
favorableNIH shift
Electrophilic additionwhen R is o/p directing, get mostly p productwhen R is m-directing, get m and p products
Mechanism proposed for heme-dependent oxygenation of aromatic compounds
Sulfur Oxygenation
Scheme 4.42
Linear free energy relationship: log kcat vs. one-electron oxidation potential as well as +
Electron transfer mechanism proposed for heme-dependent oxygenation of sulfides
:
RS
R'
S
FeIV
O
N
N N
N
RS
R'
S
FeIV
O
N
N N
N
RS
R'
..
rebound
:
S
FeIII
N N
:..
N Noxygen
:O::
CH3
S
X
Scheme 4.43
N-DealkylationElectron transfer mechanism proposed for
heme-dependent oxygenation of tertiary amines
R
N
R'
S
FeIV
O
N
N N
N
S
FeIV
O
N
N N
N
R''
R
N
R'
R''
HH
S
FeIV
OH
N
N N
N
R
N
R'
R''
H
R
N
R'
R''
H
R
N
R'
: ::
R''
:
H
..
HO:
rebound
RN
oxygen
H
R'
:..
+R"CHO
4.604.61
4.62
:
H+
With primary and secondary amines hydrogen atom abstraction mechanism favored (see next slide)
Scheme 4.44
O-Dealkylation
Not electron transfer mechanism--
Hydrogen atom abstraction mechanism proposed for heme-dependent oxygenation of ethersR
O
S
FeIV
O
N
N N
N
S
FeIII
N N
N N
R'
S
FeIV
OH
N
N N
N
: :
rebound
oxygen
H H
:
R
O R'
H
+R'CHO
.. R
O R'
HHO::
ROH
H+
oxidation potential for oxygen is too high
C-C Bond Cleavage
Scheme 4.45
androstenedione estrone
Reaction catalyzed by aromatase
CH3
O
O
HO
O
+ HCO2H3 NADPH
3 O2
4.63 4.64
19
Scheme 4.46
also a substrate
also a substrate
Fate of the Atoms during Aromatase-catalyzed Conversion of Androstenedione to Estrone
4.66
CH3
O
O
O
O
O
O
O
O
4.63
NADPH
H
NADPH
4.67
HR HS H
2
4.65
2
H
H
NADPH
HO
O
++
4.64
H
H
-H2
A B
C D
+H+
-H2
+H+
H
H
+H+O2
HC OHH2O
H
First two oxygenation steps proceed by normal heme hydroxylation mechanism
Scheme 4.47
heme peroxide
like Fl-OO- addition to aldehydes
Three Possible Mechanisms for the Last Step in the Aromatase-catalyzed Oxygenation of
Androstenedione
O
O OHOOFe+3
O HO
HFe+3 O
Fe+3 OH
Fe+4 O
HO
HO
HO
O
Fe+4 OH
O
C H
O
O
OOFe+4
O
O OFe+4
O
O
CH
O
Fe+4
HO
H
OHFe+4
CH
O
4.64
O O Fe+3OH
O
O
4.64 +(2)
OFe+3
4.64 +(3)
Fe+4 OH
Fe+4 O
Fe3+ + HCOOH
(1)- HCOOH
H
Fe3+ + HCOOH
HO
HO
OH3C
HHD
HO
HO
H
HD
OH
1716
21
1716
+
4.68 4.69
NADPHH3CCOOH
O2
Evidence for Heme Peroxide Mechanism
retained
FeIV-O• would have abstracted a C21 CH3 hydrogen or a C16 or C17 H
Scheme 4.48
Oxidation of pregnenolone, catalyzed by an isozyme of cytochrome P450 (P45017)
HO
HO
OH3C
H
HDFeIV O
HO
HO
OH2C
H
HD
4.70
4.71
+ CH2=C=O
HO
HO
H
HD
FeIV OH
FeIV OH
FeIII
17
HO
HO
H
HD
1617
1617
16
17
OH
16
2H2O
H2C2HCOO2H
Scheme 4.49
In 2H2O, ketene would give H2C2HCOO2H; no 2H found in CH3 group of acetate, therefore not FeIV-O•
retained
Hydrogen Atom Abstraction Mechanism, Using a Heme Iron Oxo Species, for the P45017-catalyzed
Oxygenation of Pregnenolone
Scheme 4.50
Mutation of Thr-302 (T302A) in P450 2B4 (needed for formation of iron oxo species) decreased hydroxylation activity, but increased deacylation (nucleophilic) activity
Evidence for a Nucleophilic Mechanism, Using Heme Peroxy Anion Followed by a Radical Decomposition of the Heme
Peroxide, for the P45017-catalyzed Oxygenation of Pregnenolone
H3C O H3C
O
OH
O FeIII
H3CO
OH
O
H
CH3COOH O FeIII
17
FeIIIOH
O O FeIII
H B
O-FeIII
+H+
FeIII O OHO
302T
FeIII O OH2 FeIV OH
Scheme 4.51
Further Evidence for Heme Peroxide
isolated, also a substrate
Nucleophilic mechanism, using heme peroxy anion followed by a Baeyer-Villiger rearrangement, for the lanosterol 14-methyl demethylase-catalyzed oxygenation of lanosterol
HO
CH3 HO
C8H15
C O
HHO
C8H15
C
OH
OH
O
FeIII
–OFeIII
2 NADPH
2 O2
FeIII OO
HO
C8H15
O- HCO2H
H
O- H2O
no O2 /NADPH
4.73
4.72
H
14
Baeyer-Villigerrearrangement
HO
C8H15
4.74
H+
Scheme 4.52
Nucleophilic Mechanism, Using Heme Peroxy Anion, Followed by a Radical Decomposition of the Heme Peroxide, for the
Lanosterol 14-Methyl Demethylase-catalyzed Oxygenation of Lanosterol Would Not Give the Baeyer-Villiger Product
HO
CH3 HO
C8H15
C O
H HO
C8H15
C
OH
OH
O
FeIIIFeIII OO
2 O2
HO
C8H15
C
OH
OH
O
FeIII
HO
C8H15
H
O
FeIII
2 NADPH
-HCOOH
HO
C8H15
4.72
14
4.74
H+
-FeIII-OH
No formate ester formed
Synthesized to test Baeyer-Villiger mechanism with aromatase - no estrone
Maybe aromatase and P45017 have different mechanisms from that of lanosterol 14-methyl demethylase
4.75
O
O
O
H
O
Scheme 4.53
gives aromatase product
Model Studies on the Mechanism of Aromatase
OTHP
TBDMSO
OOTHP
TBDMSO
OTHP
O
O
4.76
OTHP
O
4.77
no estrone
O
+ HCO2H
O
H2O2
H2O2
Scheme 4.54
Revised Aromatase Mechanism
Mechanism proposed for aromatase initiated by dienol formation
O
O
O
HB
O
HO
O O
HO
OHOFeIIIO
O
HO
OHOFeIIIO
O
HO
FeIIIOHaHb
O
HO
Ha
4.78
4.79
-FeIIIOHb
-HCOOHB H
FeIIIOO
Nonheme Iron Oxygenation
Methane monooxygenases
binuclear iron cluster
CH4 CH3OH
4.80
FeIII
OFeIII
HN
O O N
N N
His246His147
Glu144
COO OOC Glu209Glu114
H2O O
O Glu243
Scheme 4.55
*
soluble methane monooxygenase
XAS and Mössbauer spectroscopy support 4.83a, not 4.83b
Studies with the hypersensitive cyclopropane probe (4.46,
Scheme 4.34) and methylcubane indicate a cation, not radical,
intermediate
Therefore mechanism like P450
Binuclear Ferric Cluster of Methane Monooxygenase
+2H+
FeIIIO
FeIII
H
FeIIO
FeII
H
FeIIIO
FeIII
H
FeIVO
FeIV
H
FeIIIO
FeIII
2 NADPH 2 NADP+
4.824.81
H
2
FeIVO
FeIV
4.83b 4.84b
H
4.83a 4.84a
-H2
Scheme 4.56
Copper-dependent Oxygenation
from ascorbic acid
Optimal activity with 2 CuII per subunit
one CuII catalyzes e- transfer from ascorbate
one CuII catalyzes oxygen insertion into substrate
Reaction catalyzed by dopamine -monooxygenase
+ O2HONH2
2e-
2H+
HO
HONH2
HO
OHH
+ H2O
4.85 4.86
Scheme 4.57Hammett plot = -1.5 fits better to than
+, suggesting a radical with a polar TS‡
+H+
Mechanism Proposed for Dopamine -Monooxygenase
CuII
O
OH
ArNH3
+
H
OH
CuII
O
ArNH3
+
H
O
CuII
O
ArNH3
+
OH
CuII
O
ArNH3
+
OH
CuII
O
ArNH3
+
HO
CuII
OH
ArNH3
+
HO
4.87 4.88
H2O