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