The Organic Chemistry of Enzyme-Catalyzed Reactions Chapter 4 Monooxygenation

Monooxygenation Table 4.1. Typical reactions catalyzed by monooxygenases

Reaction catalyzed by lactate oxidase from Mycobacteria Internal Monooxygenase Flavin-dependent Hydroxylases Reaction catalyzed by lactate oxidase from Mycobacteria Scheme 4.1 No external reducing agent required

The lactate oxidase reaction under anaerobic conditions One Turnover Experiment (enzyme concentration in excess over substrate) The lactate oxidase reaction under anaerobic conditions Acting like an oxidase Scheme 4.2

Reaction of Reduced Lactate Oxidase with Pyruvate and Oxygen 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:

Possible Mechanisms for Lactate Oxidase like DAAO flavin hydroperoxide acts as a nucleophile electrophilic substrate Scheme 4.4

NAD(P)H reduction of flavin External Monooxygenases NAD(P)H reduction of flavin O2 activation Scheme 4.5 Activated O2 is probably in the form of flavin hydroperoxide

Mechanism proposed for flavin-dependent hydroxylases Nucleophilic Substrates Mechanism proposed for flavin-dependent hydroxylases stopped-flow spectroscopic evidence for boxed intermediates flavin hydroperoxide acts as electrophile electrophilic aromatic substitution Scheme 4.6

Hammett Study p-hydroxybenzoate hydroxylase log Vmax for hydroxylation vs pKa linear free energy relationship  = -0.5 (Electron deficient mechanism) Consistent with electrophilic aromatic substitution

Reaction catalyzed by bacterial luciferase Electrophilic Substrates Reaction catalyzed by bacterial luciferase long-chain aldehydes (electrophilic substrates) Scheme 4.7

Nucleophilic Mechanism for Bacterial Luciferase on warming isolated by cryoenzymology (-30 C in mixed aqueous-organic media) electrophilic substrates Scheme 4.8 detected spectro-photometrically However, with 8-substituted FMN analogues rate increases with decreasing one electron oxidation potentials of analogues

Chemically Initiated Electron Exchange Luminescence (CIEEL) Mechanism for Bacterial Luciferase SET Scheme 4.9

Dioxirane mechanism for bacterial luciferase Alternative One-electron Mechanism via a Dioxirane Dioxirane mechanism for bacterial luciferase SET kx/kh vs. p for 8-substituted flavins  = -4 (facilitated by e- donation) Scheme 4.10 Inconsistent with Baeyer-Villiger mechanism ( values +0.2 to 0.6)

Baeyer-Villiger Oxidation of Ketones Scheme 4.11 Migratory aptitude - more e- donating group migrates (in the case above, R)

Reaction catalyzed by cyclohexane oxygenase Ketone Monooxygenases - an Example of a Baeyer-Villiger Oxidation Reaction catalyzed by cyclohexane oxygenase Scheme 4.12 C4a-FAD hydroperoxide intermediate detected

Other Reactions Catalyzed by Cyclohexanone Oxygenase same migratory aptitudes as nonenzymatic reaction Scheme 4.13

Cyclohexane Oxygenase Proceeds with Retention of Configuration (like nonenzymatic) Scheme 4.14

Migratory Aptitude of Cyclohexanone Oxygenase-catalyzed Reaction Same migratory aptitude as nonenzymatic (3° > 2° > 1° > Me) Scheme 4.15

no loss of D (like nonenzymatic reaction)

Baeyer-Villiger-type Mechanism Proposed for Cyclohexanone Oxygenase electrophilic substrate Scheme 4.16

Reaction of Cyclohexanone Oxygenase with Boranes same as nonenzymatic reaction Scheme 4.17

Reactions Catalyzed by Ketone Monooxygenase when R1 = R2 = Me 1 : 20 R1 = H; R2 = Me 1 : 1 (same as nonenzymatic reaction) Scheme 4.18

Reactions Catalyzed by the Ketone Monooxygenase from A. calcoaceticus >95% ee >95% ee racemate Scheme 4.19

Reactions Catalyzed by the Ketone Monooxygenase from P. putida 50% ee >95% ee >95% ee racemate Scheme 4.20

Pterin-dependent Monooxygenases aromatic hydroxylation pteridine ring

Tetrahydrobiopterin Fe2+ also required for activity Only a few enzymes require tetrahydrobiopterin Important in biosynthesis of dopa, norepinephrine, epinephrine, and serotonin Reactions similar to flavoenzymes

Comparison of the Dihydrobiopterin and Tetrahydrobiopterin with Oxidized Flavin and Reduced Flavin Scheme 4.21

Reaction Catalyzed by Phenylalanine Hydroxylase NIH shift [1,2] migration Scheme 4.22 Similar to flavin hydroxylases except 2H washed out with flavoenzymes

Possible Intermediate

Mechanism of the Reaction Catalyzed by Tetrahydrobiopterin-dependent Monooxygenases nucleophilic substrate discussed with heme-dependent enzymes Scheme 4.23

Reaction of dihydrophenylalanine with phenylalanine hydroxylase Evidence for Arene Oxide Intermediate Reaction of dihydrophenylalanine with phenylalanine hydroxylase Scheme 4.24

Arene Oxide Mechanism Proposed for Tetrahydrobiopterin-dependent Monooxygenases Tyr Scheme 4.25 m-Tyr Incubation with [4-2H]Phe should favor formation of m-Tyr (isotope effect), and [3,5-2H2]Phe should favor Tyr, but they do not. Therefore, not an arene oxide intermediate

The larger the size of X, the more m-Tyr product Cationic Mechanism Proposed for Tetrahydrobiopterin-dependent Monooxygenases Fe as X is larger Scheme 4.26 m-Tyr The larger the size of X, the more m-Tyr product = -5 (cation-like TS‡)

Alternative Species These species could account for alkyl hydroxylation products (heme chemistry), e.g. with hydroxylation here

Heme-Dependent Monooxygenases Cytochrome P450s (>500 different isozymes) require NAD(P)H and O2 Protection from xenobiotics

Reactions Catalyzed by Heme-dependent Monooxygenases + - + -

Molecular Oxygen Activation by Heme-dependent Monooxygenases (requires NADPH) In P450cam Thr-252 low-spin state high-spin state FeIII more readily accepts e- cytochrome P450 reductase calculations favor this structure Scheme 4.27 means isolated and characterized

Alkane Hydroxylation Two-step radical mechanism with oxygen rebound for alkane oxygenation by heme-dependent monooxygenases 3° > 2° > 1° retention of configuration Scheme 4.28 (suggests C-H cleavage is not the rate-determining step) Intermolecular isotope effect < 2 Intramolecular isotope effect > 11 C-H cleavage during catalysis

Products from the Reaction of all Exo-2,3,5,6-tetradeuterionorbornane with the CYP2B4 Isozyme of Cytochrome P450 Scheme 4.29 Scrambling of stereochemistry supports 2-step radical mechanism

Radical Clocks - detection of radical intermediates Radical clock approach for determination of reaction rates in radical rearrangement reactions known Scheme 4.30 The rate of hydroxylation can be calculated (lifetime of radical intermediate)

Cytochrome P450-catalyzed monooxygenation of a cyclopropane analogue Example of Radical Clock Cytochrome P450-catalyzed monooxygenation of a cyclopropane analogue a Scheme 4.31 b From kr = 2  109 s-1 and the ratio of a/b, can calculate kOH = 2.4  1011 s-1

Cytochrome P450-catalyzed Oxidation of Trans-1-methyl-2-phenylcyclopropane Scheme 4.32 Perdeuteration (CD3) gives increased pathway b called metabolic switching

Another ultrafast radical clock reaction catalyzed by cytochrome P450 Evidence against a True Radical Intermediate Another ultrafast radical clock reaction catalyzed by cytochrome P450 3  1011 s-1 Scheme 4.33 very little kOH has to be faster than the decomposition of a TS‡ (6  1012 s-1); therefore propose carbocation after oxidation step

A Hypersensitive Radical Probe Substrate to Differentiate a Radical from a Cation Intermediate Generated 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 Concerted, but Nonsynchronous, Mechanism Proposed for Cytochrome P450 Scheme 4.35 General conclusion: More than one oxidizing species involving more than one pathway with multiple high-energy heme complexes (radical and cation)

Alkene Epoxidation Two-step radical mechanism with oxygen rebound for alkene oxygenation by heme-dependent monooxygenases lifetime? Scheme 4.36

Evidence for Short-lived Radical Cytochrome P450-catalyzed epoxidation of trans-1-phenyl-2-vinylcyclopropane only cyclopropyl/carbinyl radical rearrangement not detected Scheme 4.37

Cytochrome P450-catalyzed formation of an arene oxide Arene Hydroxylation Isolation of first arene oxide Cytochrome P450-catalyzed formation of an arene oxide Scheme 4.38 Is it an intermediate or side product?

A common intermediate in the oxygenation of naphthalene Evidence for a Cyclohexadienone Intermediate A common intermediate in the oxygenation of naphthalene either same product and 2H incorporation from both isomers Should have observed 1- and 2- hydroxynaphthalene because of an isotope effect Scheme 4.39

Concerted (pathway a) and Stepwise (pathway b) Mechanisms for the Potential Conversion of an Arene Oxide to a Cyclohexadienone concerted stepwise Scheme 4.40 Evidence against concerted: 1) no deuterium isotope effect 2) Hammett plot shows large - (carbocation intermediate)

Electrophilic addition Isotope Effect and Hammett Studies Indicate either Radical or Cation (or both) Intermediates, but not Arene Oxide Mechanism proposed for heme-dependent oxygenation of aromatic compounds reasonable unfavorable NIH shift favorable Electrophilic addition when R is o/p directing, get mostly p product when R is m-directing, get m and p products Scheme 4.41

Sulfur Oxygenation Electron transfer mechanism proposed for heme-dependent oxygenation of sulfides Scheme 4.42 Linear free energy relationship: log kcat vs. one-electron oxidation potential as well as +

N-Dealkylation Electron transfer mechanism proposed for heme-dependent oxygenation of tertiary amines Scheme 4.43 With primary and secondary amines hydrogen atom abstraction mechanism favored (see next slide)

O-Dealkylation Hydrogen atom abstraction mechanism proposed for heme-dependent oxygenation of ethers Scheme 4.44 Not electron transfer mechanism-- oxidation potential for oxygen is too high

Reaction catalyzed by aromatase C-C Bond Cleavage Reaction catalyzed by aromatase androstenedione estrone Scheme 4.45

Fate of the Atoms during Aromatase-catalyzed Conversion of Androstenedione to Estrone also a substrate also a substrate Scheme 4.46 First two oxygenation steps proceed by normal heme hydroxylation mechanism

Three Possible Mechanisms for the Last Step in the Aromatase-catalyzed Oxygenation of Androstenedione heme peroxide like Fl-OO- addition to aldehydes Scheme 4.47

Evidence for Heme Peroxide Mechanism Oxidation of pregnenolone, catalyzed by an isozyme of cytochrome P450 (P45017) retained Scheme 4.48 FeIV-O• would have abstracted a C21 CH3 hydrogen or a C16 or C17 H

Hydrogen Atom Abstraction Mechanism, Using a Heme Iron Oxo Species, for the P45017-catalyzed Oxygenation of Pregnenolone Scheme 4.49 retained In 2H2O, ketene would give H2C2HCOO2H; no 2H found in CH3 group of acetate, therefore not FeIV-O•

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 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

Further Evidence for Heme Peroxide Nucleophilic mechanism, using heme peroxy anion followed by a Baeyer-Villiger rearrangement, for the lanosterol 14-methyl demethylase-catalyzed oxygenation of lanosterol Scheme 4.51 isolated, also a substrate

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 No formate ester formed Scheme 4.52

Synthesized to test Baeyer-Villiger mechanism with aromatase - no estrone Maybe aromatase and P45017 have different mechanisms from that of lanosterol 14-methyl demethylase

Model Studies on the Mechanism of Aromatase gives aromatase product Scheme 4.53

Mechanism proposed for aromatase initiated by dienol formation Revised Aromatase Mechanism Mechanism proposed for aromatase initiated by dienol formation Scheme 4.54

Nonheme Iron Oxygenation Methane monooxygenases binuclear iron cluster CH4  CH3OH

Binuclear Ferric Cluster of Methane Monooxygenase * 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

Reaction catalyzed by dopamine -monooxygenase Copper-dependent Oxygenation Reaction catalyzed by dopamine -monooxygenase from ascorbic acid Scheme 4.56 Optimal activity with 2 CuII per subunit one CuII catalyzes e- transfer from ascorbate one CuII catalyzes oxygen insertion into substrate

Mechanism Proposed for Dopamine -Monooxygenase Scheme 4.57 Hammett plot  = -1.5 fits better to  than +, suggesting a radical with a polar TS‡