Mechanism–Based Inhibitors for Copper Amine Oxidases

MECHANISM–BASED INHIBITORS FOR COPPER AMINE OXIDASES:

SYNTHESIS, MECHANISM, AND ENZYMOLOGY

BO ZHONG

Submitted in partial fulfillment of the requirements

For the degree of Doctor of Philosophy

Thesis Adviser: Dr. Lawrence M. Sayre, Dr Irene Lee

Department of Chemistry

CASE WESTERN RESERVE UNIVERSITY

January, 2010 CASE WESTERN RESERVE UNIVERSITY

SCHOOL OF GRADUATE STUDIES

We hereby approve the thesis/dissertation of

______

candidate for the ______degree *.

(signed)______(chair of the committee)

______

(date) ______

*We also certify that written approval has been obtained for any proprietary material contained therein. Table of Contents

Table of Contents ...... I

List of Tables ...... V

List of Figures ...... VI

List of Schemes ...... XI

Acknowledgements ...... XIV

List of Abbreviations ...... XV

Abstract ...... XVII

Chapter 1. Introduction ...... 1

1.1 General introduction to copper amine oxidases (CAOs) ...... 2

1.2 Mechanism for CAOs-catalyzed turnover...... 3

1.3 General introduction to mechanism-based inhibitors...... 8

1.4 Kinetics for mechanism-based inhibition ...... 9

1.5 References ...... 13

Chapter 2. Inactivation of two different copper amine oxidases by allenic amines 15

2.1 Introduction ...... 16

2.2 Results ...... 18

2.2.1 Synthesis...... 18

2.2.2 Inactivation of bovine plasma amine oxidase by allenic amines ...... 20

2.2.3 Inactivation of Arthrobacter globiformis amine oxidase (AGAO) by allenic

amines...... 27

2.2.4 Structure of AGAO-inhibitor rac-2 complex ...... 29

2.3 Discussion and conclusion ...... 30

2.4 Experimental procedures ...... 38

2.5 References ...... 47

Chapter 3. Inactivation of bovine plasma amine oxidase (BPAO) by acetic acid-3- amino-propenyl ester ...... 49

3.1 Introduction ...... 50

3.2 Results and discussion ...... 53

3.2.1 Synthesis...... 53

3.2.2 Enzyme assay of inhibition of BPAO by (E)- and (Z)-acetic acid-3-amino-

propenyl ester (1 and 2) ...... 54

3.2.3 Metabolism of acetic acid (E)-3-amino-propenyl ester (1) by BPAO ...... 56

3.2.4 Enzyme assay of inhibition of BPAO by aldehyde product 3-(acetyloxy)-(E)-2-

propenal and its hydrolytic product MA ...... 59

3.2.5 Evaluation of 3-amino-propionaldehyde and its analogs as inhibitors and

substrates of BPAO ...... 60

3.3 Mechanistic conclusion ...... 72

3.4 Experimental procedures ...... 76

3.5 References ...... 85

Chapter 4. Kinetic analysis of propargylamine derivatives as both substrates and inhibitors for bovine plasma amine oxidase (BPAO) and Arthrobacter globiformis amine oxidase (AGAO) ...... 87

4.1 Introduction ...... 88

4.2 Results and discussion ...... 92

4.2.1 Steady-state kinetic parameters for AGAO/BPAO-catalyzed oxidation of

propargylamine derivatives ...... 92

4.2.2 Kinetic parameters for inactivation of AGAO/BPAO by propargylamine

derivatives ...... 94

4.2.3 Inactivation of substrate-reduced BPAO by α, β-unsaturated aldehydes ...... 99

4.2.4 Anaerobic inactivation of BPAO by propargylamine derivatives ...... 102

4.3 Conclusions ...... 104

4.4 Experimental procedures ...... 105

4.5 References ...... 111

III

Chapter 5. Investigate structural basis of inactivation of bovine plasma amine oxidase (BPAO) by 5-aryl-3-pentynamines ...... 114

5.1 Introduction ...... 115

5.2 Results ...... 116

5.2.1 Synthesis...... 116

5.2.2 Irreversible inactivation of BPAO by 5-aryl-3-pentynamines (1-3) ...... 117

5.2.3 Mechanism of inactivation of BPAO by 5-aryl-3-pentynamines...... 121

5.2.3.1 Covalent adduct between BPAO and 5-aryl-3-pentynamines...... 121

5.2.3.2 Immunochemical analysis of inactivation of enzyme BPAO by inhibitor 3 .. 126

5.2.3.3 Attempt to resolve the structural basis of inactivation of BPAO by inhibitor 2

using enzymatic digestion and LC/MS ...... 127

5.3 Discussion ...... 130

5. 4 Conclusions ...... 134

5. 5 Experimental procedures ...... 135

5.6 References ...... 144

Appendix ...... 146

Bibliography ...... 152

List of Tables

Chapter 2 ...... 15

Table 2.1 The inhibitory potencies and reversibility of inhibition on BPAO by allene compounds ...... 21

Table 2.2 The inhibitory potencies and reversibility of inhibition on AGAO by allene compounds ...... 28

Chapter 4 ...... 87

Table 4.1 Kinetic parameters for metabolism of PBA, MPBA [1, 1-2H] PBA and [1, 1-2H]

MPBA, by BPAO and AGAO ...... 94

Table 4.2 Kinetic parameters for inactivation of AGAO and BPAO by PBA, MPBA [1,

1-2H] PBA and [1, 1-2H] MPBA...... 98

List of Figures

Chapter 2 ...... 15

Figure 2.1 Structures of allene compounds ...... 18

Figure 2.2 (A) Time-dependent inactivation of BPAO by various concentrations of rac-1 and (B) Kitz and Wilson replot of the data...... 22

Figure 2.3 Inactivation of BPAO by various concentrations of (S)-3 ...... 24

Figure 2.4 Inactivation of BPAO by various concentrations of (R)-3 ...... 24

Figure 2.5 Time-dependent inactivation of BPAO by various concentrations of 4...... 25

Figure 2.6 Inactivation of BPAO by various concentrations of 5 ...... 26

Figure 2.7 Inactivation of AGAO by various concentrations of rac-2 ...... 29

Figure 2.8 Electron density map of TPQ-rac-2 adduct with top view (A) and bottom view

(B) ...... 30

Chapter 3 ...... 49

Figure 3.1 Structures of (E)- and (Z)-isomer of acetic acid-3-amino-propenyl ester ...... 53

Figure 3.2 Time-dependent inactivation of BPAO (0.6 µM) by various concentrations of

acetic acid (E)-3-amino-propenyl ester (1) ...... 55

Figure 3.3 Time-dependent inactivation of BPAO (0.6 µM) by various concentrations of

acetic acid (Z)-3-amino-propenyl ester (2) ...... 56

Figure 3.4 Difference spectra (against native BPAO) obtained from the reaction of BPAO

with acetic acid (E)-3-amino-propenyl ester (1) ...... 57

Figure 3.5 Hydrolysis of 3-(acetyloxy)-(E)-2-propenal in pH 7.2 phosphate buffer ...... 58

Figure 3.6 Elution profile of DNPH-derivatized MA ...... 59

Figure 3.7 Time-dependent inactivation of BPAO (0.7 µM) by 3-(acetyloxy)-(E)-2- propenal...... 60

Figure 3.8 Time-dependent inactivation of BPAO (0.66 µM) by 3-amino- propionaldehyde ...... 62

Figure 3.9 Competitive inhibition of BPAO by different concentrations of 3-amino- propionaldehyde in pH 7.2, sodium phosphate buffer. (A) Lineweaver-Burk plots; (B)

The corresponding Dixon plots ...... 64

Figure 3.10 Docking of 3-amino-propionaldehyde into the active site of BPAO ...... 65

Figure 3.11 Docking of 3-amino-1-phenyl-1-propanone into the active site of BPAO. .. 66

VII

Figure 3.12 (A) Difference spectra (against the spectrum at 0 min) obtained from the

reaction of BPAO (0.7 µM) with 4-amino-2-butanone (100 µM) (B) Difference spectra

(against the spectrum at 0 min) obtained from reaction of BPAO (0.7 µM) with 3-amino-

1-phenyl-1-propanone (20 µM) ...... 68

Figure 3.13 Inhibition of BPAO by various concentrations of 3-amino-1-phenyl-1-

propanone (A) (CBPAO = 1.48 µM) and 4-amino-2-butanone (B) (CBPAO = 0.83 µM) ..... 70

Figure 3.14 Correlation between the time-dependent recovery of BPAO activity upon incubation with 3-amino-1-phenyl-1-propanone and metabolism of 3-amino-1-phenyl-1- propanone by BPAO over time ...... 71

Chapter 4 ...... 87

Figure 4.1 (A) Time course of enzymatic oxidation of benzylamine (10 mM) in the presence of MPBA at different concentrations and (B) plot of apparent inactivation constant kobs as a function of inhibitor concentration ...... 97

Figure 4.2 Time-dependent inhibition of benzylamine-reduced BPAO (0.78 µM) by 4-(4-

methylphenoxy)-2-butynal (80 µM) under anaerobic condition and inhibition of native

BPAO by the same amount of this aldehyde under the same condition ...... 101

VIII

Figure 4.3 Time-dependent inhibition of benzylamine-reduced BPAO (0.74 µM) by 4-

phenoxy-2-butynal (80 µM) under anaerobic condition and inhibition of native BPAO by

the same amount of this aldehyde under the same condition ...... 101

Figure 4.4 Time course of inactivation of BPAO (0.75 µM) by MPBA of various

concentrations, (■) 2 µM, under anaerobic condition for 60 min, (○) 20 µM, under anaerobic condition for 3min, then exposure to O2 (arrow), and partition ratio plot (inset).

...... 102

Chapter 5 ...... 114

Figure 5.1 Time course of inactivation of BPAO (0.6 µM) by different concentrations of inhibitor 1 ...... 118

Figure 5.2 Time-dependent inactivation of BPAO (0.6 µM) by different concentrations of inhibitor 2 ...... 119

Figure 5.3 Time-dependent inactivation of BPAO (0.6 µM) by various concentrations of inhibitor 3 (upper), and Kitz and Wilson replot of the data (lower) ...... 120

Figure 5.4 Inhibitor 2-labeled BPAO...... 122

Figure 5.5 Electrospray mass spectra of the native BPAO (lower) and inhibitor 2- inactivated BPAO (upper). The peaks were numbered as 1, 2, 3, 4, 5, 6, 7, 8, 9, from the left to the right for both the native and derivatized BPAO...... 123 IX

Figure 5.6 Spectral change upon addition of aqueous inhibitor 1 (157.5 µM) to the native

BPAO (84.4 µM) ...... 125

Figure 5.7 Spectral change upon addition of aqueous inhibitor 3 (272.5 µM) to the native

BPAO (82.5 µM) ...... 126

Figure 5.8 HPLC elution profiles of the chymotryptic digests of BPAO inactivated by inhibitor 2 ...... 128

Figure 5.9 Tandem mass spectra for the peak with absorption at 380nm detected in the chymotryptic digests (lower), compared to the tandem mass spectra of the authentic sample 5-(4-nitroanilino)-3-pentynal (upper) ...... 129

List of Schemes

Chapter 1 ...... 1

Scheme 1.1 Transamination mechanism ...... 4

Scheme 1.2 Two different mechanisms proposed for the oxidative half reaction ...... 7

Scheme 1.3 Classic mode for mechanism-based inhibition ...... 10

Chapter 2 ...... 15

Scheme 2.1 Cu (I) –mediated SN2’ reaction for synthesis of allene compounds...... 20

Scheme 2.2 Overlap of the structures of (S)-3 and (R)-3 ...... 32

Scheme 2.3 Proposed mechanism for the reaction between AGAO and α-allenic amine

rac-2 (including productive turnover and inhibition) ...... 36

Chapter 3 ...... 49

Scheme 3.1 Proposed addition-elimination mechanism for inactivation of BPAO by 3-

haloallylamines ...... 51

Scheme 3.2 Synthesis of (E)- and (Z)- isomer of acetic acid-3-amino-propenyl ester (1 and 2) ...... 54

Scheme 3.3 TPQ adducts possibly formed from the reaction of BPAO with acetic acid

(E)-3-amino-propenyl ester (1) ...... 61

Scheme 3.4 Proposed mechanism for inactivation of BPAO by acetic acid-(E)-3- aminopropenyl ester (1) ...... 75

Chapter 4 ...... 87

Scheme 4.1 Proposed mechanism for inactivation of AGAO by propargylamine derivatives ...... 89

Scheme 4.2 Structures of MPBA, [1, 1-2H] MPBA, PBA, and [1, 1-2H] PBA ...... 91

Scheme 4.3 Classic minimal kinetic mode for mechanism- based inhibition ...... 95

Scheme 4.4 Proposed mechanism for inactivation of BPAO by propargylamine

derivatives ...... 104

Chapter 5 ...... 114

Scheme 5.1 Synthesis of 5-aryl-3-pentynamines (1-3) ...... 117

Scheme 5.2 Synthesis of t-Boc derivative of 5-chloro-3-pentynamine ...... 117

Scheme 5.3 Synthesis of 1-(4-nitroanilino)-5, 5-diethoxy-2-pentyne ...... 117

XII

Scheme 5.4 Alkylation of the reductively aminated TPQ by allenic aldehyde products

...... 133

Scheme 5.5 Proposed mechanism for inactivation of BPAO by homopropargylamine derivatives ...... 134

XIII

Acknowledgements

First of all, I would like to express my deepest thanks to my thesis advisor, Dr.

Lawrence M. Sayre, for his invaluable guidance and constant support during my graduate study. In his laboratory, I have learned to do the research independently and confidently.

And for the past few years, Dr. Sayre's intelligence and diligence and passion for science

have made a deep impression on me. I am lucky and proud to be a member of his group.

Secondly, I would express my thanks to my second advisor Dr. Irene Lee for her

profound suggestions in my research for the past one year, Dr. Vernon Anderson for his

generous help on TOF-MS experiments. I also thank my committee, Dr. Mary D.

Barkley, Dr. Gregory P. Tochtrop, Dr. John J. Mieyal, and Dr. Mark Chance for spending

their valuable time in reviewing my thesis.

I also want to thank Dr. Jim Faulk for help on HPLC-MS technical support and Dr.

Dale Ray for help on NMR technical support.

My sincere thanks also go to all the colleagues (current and former) in Dr. Sayre's

lab, Dr. Keqing Ling, Dr. Yuming Zhang, Dr. De Lin, Dr. Yanwen Chen, Dr. Xiaochun

Zhu, Xiaoxia Tang, Jianye Zhang, Detao Gao, Xibo Li for helpful suggestion and

discussion during my graduate studies.

Finally, my thanks go to my parents and my husband. Without their constant

encouragement and support, I could not finish my studies.

Thank all the people who have ever helped me during my graduate study.

XIV

List of Abbreviations

AGAO Arthrobacter globiformis amine oxidase

BPAO bovine plasma amine oxidase

BSA bovine serum albumin t-Boc tert-butyloxycarbonyl

CAO copper amine oxidase

DAO diamine oxidase

DCM dichloromethane

DMAP 4-dimethylaminopyridine

DMSO dimethylsulfoxide

DMF dimethylformamide

DNPH 2,4-dinitrophenylhydrazine

ECAO Escherichia coli amine oxidase

EPAO equine plasma amine oxidase

FAB fast atom bombardment

HPAO Hansenula polymorpha amine oxidase

HPLC high-performance liquid chromatography

HRMS high resolution mass spectra

HVAP-1 human vascular adhesion protein-1

KDAO kidney diamine oxidase

MA malondialdehyde

MAO monoamine oxidases

NBT nitroblue tetrazolium

PCC pyridinium chlorochromate

PPLO pichia pastoris lysyl oxidase

PSAO Pisum sativum amine oxidase

PSB product Schiff base

SDS sodium dodecyl sulfate

SSAO semicarbazide-sensitive amine oxidase

SSB substrate Schiff base

THF tetrahydrofuran

TPQ 2,4,5-trihydroxyphenylananine quinone

XVI

Mechanism-based Inhibitors for Copper Amine Oxidases:

Synthesis, Mechanism, and Enzymology

Abstract

BO ZHONG

Development of mechanism-based inhibitors and the related inhibition mechanism study have always been significant studies in the field of copper amine oxidases (CAOs).

In Chapter 2, synthesis of several analogs of 1-amino-2,3-butadiene, which contain mono- or disubstitution at C4, and screening against bovine plasma amine oxidase

(BPAO) and Arthrobacter globiformis amine oxidase (AGAO) are described. In the case

of AGAO, the crystal structure of the enzyme derivitized with 6-phenylhexa-2,3-

dienylamine has been obtained. This structure reveals the covalent attachment of the

aldehyde product generated from amine oxidation to the amino group of the reduced TPQ

cofactor.

In Chapter 3, evaluation as inhibitor of BPAO of a novel activated allylamine

derivative acetic acid-3-amino-propenyl ester is described. Both the E- and Z-isomers

display two modes of inactivation of BPAO, reversible and irreversible. In the reversible

inactivation pathway, (E)-acetic acid-3-amino-propenyl ester (1) acts as a substrate of

BPAO to induce the temporary reversible inactivation of BPAO. A new inactivation

mechanism was proposed for the permanent inactivation of BPAO by (E)-acetic acid-3-

XVII

amino-propenyl ester (1), which involves hydrolysis of product Schiff base at the ester

bond followed by tautomerizaiton of TPQ adduct I to II (hydrolysis-tautomerization

mechanism).

In Chapter 4, kinetic analyse of propargylamine derivatives 4-phenoxy-2-butynamine

(PBA) and 4-(4-methylphenoxy)-2-butynamine (MPBA) as both substrates and inhibitors of BPAO is described. The substrate activity, the inhibitory activity, and the corresponding α-C deuterium isotope effects on both activities were determined for both compounds vs. BPAO. To obtain further insights into the inactivation mechanism, the inactivation of the native BPAO and the substrate-reduced BPAO by the turnover product

α, β-unsaturated aldehydes was evaluated. The results can be reconciled by a mechanism where the turnover product α, β-unsaturated aldehyde forms an adduct with the amino group of the reduced TPQ cofactor.

In Chapter 5, synthesis of several Ar-X-extended derivatives of homopropargylamine

(X=NH, O) and evaluation of them as BPAO inhibitors are described. The structural basis of inactivation was probed by UV-Vis spectrophotometric studies, mass spectrometry, and immunochemical analysis.

XVIII

Chapter 1 Introduction

1.1 General introduction to copper amine oxidases (CAOs)

Copper amine oxidases (CAOs) are a family of redox active enzymes, which catalyze

oxidative deamination of primary amines to aldehydes with the concomitant reduction of

1 O2 to H2O2 (Eq. (1.1)). CAOs are ubiquitous in nature, occurring in plants, yeast,

microorganisms, and mammals.2 The physiological functions of CAOs are dependent on

the source of the enzymes. In microorganisms, the catalytic turnover products from

oxidation of low molecular weight amines by CAOs are alternative sources of carbon and

nitrogen.3 In plants and mammals, CAOs play important roles in the metabolism of biogenic amines (including mono-, di-, and polyamines and neurotransmitters) involved in growth, cell division, differentiation and the stress response, where metabolism in some cases has cytotoxic consequences.3

CAO RCHO + H O + NH (1.1) RCH2NH2 + O2 + H2O 2 2 3

Presently, three general classes of CAOs including tissue-associated amine oxidase4,

diamine oxidase5 and retina-specific amine oxidase6 have been described from

mammalian resources. The tissue-associated amine oxidase is commonly designated as

semicarbazide-sensitive amine oxidase (SSAO) and found to be abundant in endothelium,

adipose, and smooth muscle.7 Plasma amine oxidase is another form of SSAO.

Experiments with genetically modified mice have demonstrated that the tissue-associated

amine oxidase is the only source of circulating plasma amine oxidase.8 Another type of

mammalian CAO is diamine oxidase (DAO), which displays distinct substrate specificity for diamines.5 DAO is also distinct in sequence homology from the plasma and the

tissue-bound SSAO.5

1.2 Mechanism for CAOs-catalyzed turnover

Most enzymes in the family of CAOs contain 2,4,5-trihydroxyphenylalanine quinone

(TPQ) cofactor, which is derived from the post-translational modification of an active site tyrosine residue.9, 10 Due to the presence of an active carbonyl organic cofactor at the

active site, CAOs are highly reactive to carbonyl reagents such as semicarbazide and

phenylhydrazine.11

The TPQ cofactor, copper ion, and the amino acid residues at the enzyme active site

form a specific structure, which endows this type of enzymes with a specific catalytic

function. During the catalytic turnover, the TPQ cofactor catalyzes oxidative deamination

of primary amines to aldehydes through a well-described transamination mechanism as

illustrated in Scheme 1.1.12-14 First, the primary amine condenses with TPQ to generate a

substrate Schiff base (SSB). This Schiff base is then tautomerized to generate a product

Schiff base (PSB). Hydrolysis of PSB affords a reduced aminated TPQ (TPQamr) and an

aldehyde. This is known as the reductive half-reaction. In the oxidative half-reaction,

TPQamr is oxidized by O2 to form quinonimine. The latter then undergoes hydrolysis to release ammonia and regenerate the TPQ cofactor.

The present understanding of the catalytic mechanism in CAOs is based on studies of crystal structures, optical spectroscopy, mechanism-based inhibitors, model compounds, and site-directed mutagenesis. The cofactor TPQ exists as an oxoanion due to the acidity

O O OH 2 B: RCH2NH2 4 O O 5 O BH O NH TPQ NH SSB PSB CHR CH2R NH3 H2O O OH

H2O RCH=O O H O 2 O 2 2 HO NH2 NH2 TPQamr Scheme 1.1 Transamination mechanism

of the 4-hydroxyl group. Nucleophilic addition of an amine occurs exclusively at the C5

position of TPQ (See the TPQ structure in Scheme 1.1). Firstly, electrophilicity at the C2

and C4 positions is reduced by resonance. Additionally, as showed by the crystal structure

of bovine plasma amine oxidase (BPAO), TPQ is oriented to expose C5 to the

15 substrate-binding site, which indicates the substrate accessibility to C5. The X-ray

crystal structure of Escherichia coli amine oxidase (ECAO) derivatized by 2-hydrazino pyridine reveals a substrate Schiff base-like species at the enzyme active site.16

Rapid-scanning stop-flow experiments with BPAO under anaerobic conditions

demonstrated the existence of a transient species with a λmax at 340 nm in the oxidation of

benzylamine.17 This species was proposed to be the substrate Schiff base stabilized by the

strong electrostatic interaction between the protonated imine nitrogen at C5 and C4

oxoanion of TPQ. In contrast to substrate Schiff base, product Schiff base has never been

directly detected under normal catalytic turnover conditions. However, kinetic studies of

BPAO-catalyzed oxidation of substrate benzylamine suggest that the conversion of the

substrate Schiff base to the product Schiff base proceeds through C-H cleavage at the C1

position of amine by a catalytic base. A large deuterium isotopic effect

D ( (kKcat M )= 14.9 ) for the oxidation of benzylamine by BPAO indicates proton

abstraction is the rate-limiting step for the reductive half reaction.11 The slow rate for

proton abstraction leads to the building up of substrate Schiff base, which explains the

observation of this intermediate in the transient state.17 Moreover, X-ray crystallographic studies of enzyme structures reveal a strictly conserved aspartate close to the C5 position

of TPQ in the active site of all CAOs. In Hansenula polymorpha amine oxidase (HPAO),

it corresponds to D319 and mutation of the Asp to Asn completely eliminated the

catalytic activity of HPAO, suggesting that D319 acts as a catalytic base in the reductive

half reaction 18.

The mechanism of the oxidative half reaction is more complex than that of the

reductive half reaction. Whether the reduced cofactor TPQamr is oxidized directly by O2

or oxidized indirectly via the active site copper is very important aspect for the mechanism of the oxidative half reaction.12, 19, 20 In the past, it was generally agreed that

2+ intermolecular electron transfer between TPQamr and Cu forms the semiquinone form of

+ + 2+ TPQ (TPQsem) and Cu , then Cu binds with O2 to form Cu -superoxide. This type of

mechanism is shown in Scheme 1.2A19. Recent experimental results support the second

mechanism as shown in Scheme 1.2B19. In this mechanism, oxygen first binds to a

hydrophobic pocket near the copper ion at the enzyme active site where an electron is

transferred from TPQamr to oxygen. The superoxide generated from the reduction of

oxygen can move to and coordinate with the copper ion, which facilitates transferring a

second electron and two protons from TPQsem. Finally, the bound hydrogen peroxide is released from the copper ion and quinonimine undergoes hydrolysis to afford ammonium and TPQ.

Kinetic studies of HPAO and its cobalt-substituted form (Co-HPAO)20 showed

2+ 2+ replacement of Cu by Co alters the Km for O2 but has a relatively small effect on kcat

at all the pH values studied. Under the saturating O2 concentration, the kcat value is

essentially identical for both HPAO and Co-HPAO at pH 7.1, which indicates that

Co-HPAO has a fully catalytic competence. The large Km for O2 with Co-HPAO is attributed to the fact that Co-HPAO reacts with O2 without the ionization of Co-water

complex at the experimental pH range. Moreover, the redox potential for Co2+ to Co+ is

expected to be ca. 800 mV more negative than that for reduction of Cu2+ to Cu+, which

2+ 12 makes the electron transfer from TPQamr to Co highly unfavorable, so the reduction of

metal ion is not necessarily involved in the oxidative half reaction.

O2 HO HO HO

NH2 NH2 NH2 OH OH OH TPQamr TPQsem O2 2+ + His Cu His Cu His Cu2+ His His His His His His

H2O O O

+ H2O2, NH4 NH2 O O O TPQ

OH O2H2 2 OH OH2 2+ 2 His Cu2+ His Cu His His His His

O 2 HO HO HO

NH NH2 NH2 2 OH OH OH O TPQamr O2 TPQsem 2

2+ 2+ His Cu2+ His Cu His Cu His His His His His His

H2O HO O O

H O , NH + NH2 NH2 2 2 4 O OH O O TPQ O2 O2H2 OH2

2+ OH2 2+ OH2 His Cu2+ His Cu His Cu His His His His His His

Scheme 1.2 Two different mechanisms proposed for the oxidative half reaction

X-ray crystal structures for CAOs reveal an entrance for O2 at the dimer interface

21 with the subunits held together by protruding arms. This site leads to the O2 binding

pocket at the enzyme active site. A binding cavity for O2 was first identified at the active

site of HPAO, which is equatorial to the copper and defined by three hydrophobic amino

22 acids Tyr 407, Leu 425, and Met 634. The effects of those residues on kKcat m (O2)

were investigated by mutation of HPAO. A series of mutants at Met 634 (to Phe, Leu and

Gln) were prepared in HPAO and characterized with regard to kKcat m (O2), indicating

that Met 634 affects the O2 activity intensely.

1.3 General introduction to mechanism-based inhibitors

According to Copeland’s definition, mechanism-based inhibitors generally refer to the compounds that can be converted to reactive species by the enzyme. These reactive species can act as (1) an affinity label, or (2) a transition state analogue, or (3) a very tight binding reversible inhibitor, prior to their release from the enzyme active site, resulting in enzyme inactivation.23 Since mechanism-based inhibitors rely on enzyme catalysis to

generate the inhibitory species, they can be very specific to the target enzyme. For some

mechanism-based inhibitors that are transformed to a species as an affinity label, enzyme

inactivation will only result if there is an active-site nucleophile suitably positioned to trap the reactive species before it dissociates. In some cases, reactive species which

escape from the enzyme into bulk solution may covalently bind to multiple sites on the

surface of the enzyme to inactivate the enzyme. However, this kind of inactivation is not

8 considered to be mechanism-based. Silverman and coworkers call this kind of inactivation “metabolism-dependent inactivation”.24

A set of criteria has been generated for characterization of mechanism-based inhibitors.24 The key criteria that are pertinent to this thesis are:

1. Loss of enzyme activity must be time-dependent.

2. Inactivation kinetics must be saturable. Namely, the rate of inactivation is

proportional to low inhibitor concentrations, but independent of high

concentrations.

3. The substrate can slow down mechanism-based inactivation.

4. Inactivation must be irreversible (e. g. enzyme activity does not return upon

dialysis or gel filtration of the inactivated enzyme).

5. The stoichiometry of inhibitor to enzyme active site must be 1: 1.

6. Mechanism-based inhibition depends on enzyme catalytic ability. Namely, a

catalytic step is involved in the conversion of an inhibitor to a reactive

intermediate.

1.4 Kinetics for mechanism-based inhibition

A general scheme describing a mechanism-based inhibition scenario is shown in

Scheme 1.3, where E, I and Q refer to enzyme, inhibitor and product respectively, and EI

’ stands for the first intermediate, EI the second intermediate, and EIinact the inactivated enzyme.25 It is characteristic of mechanism-based inhibitor that metabolism and enzyme

k 1 k2 ' k4 E I EI EI EIinact k-1 k3

E Q Scheme 1.3 Classic mode for mechanism-based inhibition

inactivation proceed concurrently. There are two processes that can then follow, after an enzyme catalyzes the conversion of a mechanism-based inhibitor to its reactive form (EI’).

The reactive product can either be released (k3) or it may bind covalently to the enzyme

25, 26 (k4).

The kincat and KI values are two important kinetic constants for mechanism-based inhibitors. When the plot of the log percent of the remaining enzyme activity versus time displays the pseudo first-order kinetics, kincat and KI can be determined by constructing

Kitz and Wilson plots and fitting to eq. (1.2), where t1/2 is the half life for

0.69 0.69 KI t1/2 = + (1.2) kinact kI inact [ ] inactivation and [I] is the inhibitor concentration.27 Based on the work of Kitz and Wilson,

Jung and Metcalf derived eq.(1.3), where E is the concentration of active enzyme, [I] is the inhibitor concentration.28Waley applied the steady-state hypothesis to Scheme 1.3 and derived an eq.(1.4), where a is the enzyme concentration and s is the inhibitor concentration at a given time. The expressions for the constants A and B are eq. (1.5) and

(1.6).26

∂ ln E kI[] = inact (1.3) ∂+t KII [] −dln a As = (1.4) dt B+ s kk A = 24 (1.5) kkk234++

kk+ kk+ B = −1234 (1.6) k1 kkk 234++

Another important parameter related to mechanism-based inactivation is the partition ratio. According to Copeland’ definition, the partition ratio is the ratio of the number of inhibitor molecules converted and released as products relative to each turnover leading

26 to enzyme inactivation. According to Scheme 1.3, the partition ratio is defined by k3/k4.

The most efficient mechanism-based inactivator has a partition ratio of 0, which means

each turnover leads to an inactivated enzyme. For a inhibitor with a high partition ratio, a

significant portion of the activated inhibitory species are released from the enzyme active

site and can potentially modify other proteins and lead to toxicity.23 So this value is also

used to evaluate whether an inhibitor is suitable for clinical use.

Generally, by titration of a known amount of enzyme with inhibitor and allowing the

reaction to go to completion, the partition ratio of the inhibitor can be determined. The

remaining enzyme activity is measured for each inhibitor concentration and a plot of the

remaining enzyme activity relative to the noninactivated control versus [I]/[E] is

constructed.26 The intercept on the x-axis defines the stoichiometry of inhibitor required

to inactivate one enzyme molecule. Assuming a stoichiometry of 1:1 for irreversible

inactivation, then the partition ratio can be determined by subtracting 1 from the intercept value. Sometimes the turnover product can inhibit the enzyme reversibly and

competitively. So in the cases of high levels of turnover, when the turnover product builds

up, the enzyme will not be able to be inactivated by further inhibitors.24 Consequently,

the partition ratio plot has significant upward curvature at high value of [I]/[E] and the

abscissa may never be reached even at high concentrations of inhibitor. Therefore, when

partition ratio plots show curvature at high values of [I]/[E], the linear part at low values

of [I]/[E] was chosen for extrapolation to the abscissa to determine the intercept.

The IC50 value for mechanism-based inhibitor refers to the inhibitor concentration

that is needed to induce a 50% loss of enzyme activity under a specific set of reaction

conditions. Although this value is important for the quantitative measurement of inactivation potencies of mechanism-based inhibitors, there are few references about introduction of the methods for determining the IC50 value for mechanism-based

inhibitors. In the Dr Sayre’s lab, the general procedure for determination of the IC50 value involves introducing amounts of inhibitor to a known concentration of enzyme, and the reaction is allowed to reach completion where no further loss of enzyme activity can be detected. A plot of the percent remaining enzyme activity vs. [I] is constructed and the

IC50 value is identified from the point where fractional activity is 50%. Moreover for some mechanism-based inhibitors which display pseudo first-order kinetics, a plot of the

percent remaining enzyme activity at the specific time versus [I] is constructed and the

IC50 value determined from this plot should indicate the specific time (e. g. 5 min IC50 ).

1.5 References 1. Messerschmidt, A.; Huber, R.; Poulos, T.; Wieghardt, K.; Editors, Handbook of Metalloproteins, Volume 2. 2001; p 785 pp. 2. Knowles, P. F.; Dooley, D. M., Amine oxidases. Met. Ions Biol. Syst. 1994, 30, 361-403. 3. Klinman Judith, P., The multi-functional topa-quinone copper amine oxidases. Biochim Biophys Acta 2003, 1647, (1-2), 131-7. 4. Jakobsson, E.; Nilsson, J.; Ogg, D.; Kleywegt Gerard, J., Structure of human semicarbazide-sensitive amine oxidase/vascular adhesion protein-1. Acta Crystallogr D Biol Crystallogr 2005, 61, (Pt 11), 1550-62. 5. Shepard Eric, M.; Smith, J.; Elmore Bradley, O.; Kuchar Jason, A.; Sayre Lawrence, M.; Dooley David, M., Towards the development of selective amine oxidase inhibitors. Mechanism-based inhibition of six copper containing amine oxidases. Eur J Biochem 2002, 269, (15), 3645-58. 6. Kim, J.; Zhang, Y.; Ran, C.; Sayre, L. M., Inactivation of bovine plasma amine oxidase by haloallylamines. Bioorg. Med. Chem. 2006, 14, (5), 1444-1453. 7. O'Rourke Anne, M.; Wang Eric, Y.; Miller, A.; Podar Erika, M.; Scheyhing, K.; Huang, L.; Kessler, C.; Gao, H.; Ton-Nu, H.-T.; Macdonald Mary, T.; Jones David, S.; Linnik Matthew, D., Anti-inflammatory effects of LJP 1586 [Z-3-fluoro-2-(4-methoxybenzyl)allylamine hydrochloride], an amine-based inhibitor of semicarbazide-sensitive amine oxidase activity. J Pharmacol Exp Ther 2008, 324, (2), 867-75. 8. Stolen, C. M.; Yegutkin, G. G.; Kurkijaervi, R.; Bono, P.; Alitalo, K.; Jalkanen, S., Origins of Serum Semicarbazide-Sensitive Amine Oxidase. Circ. Res. 2004, 95, (1), 50-57. 9. Janes, S. M.; Palcic, M. M.; Scaman, C. H.; Smith, A. J.; Brown, D. E.; Dooley, D. M.; Mure, M.; Klinman, J. P., Identification of topaquinone and its consensus sequence in copper amine oxidases. Biochemistry 1992, 31, (48), 12147-54. 10. Janes, S. M.; Mu, D.; Wemmer, D.; Smith, A. J.; Kaur, S.; Maltby, D.; Burlingame, A. L.; Klinman, J. P. A new redox cofactor in eukaryotic enzymes: 6-hydroxydopa at the active site of bovine serum amine oxidase. Science 1990, 248, 981-7. 11. Hartmann, C.; Klinman, J. P., Structure-function studies of substrate oxidation by bovine serum amine oxidase: relationship to cofactor structure and mechanism. Biochemistry 1991, 30, (18), 4605-11. 12. Mure, M.; Mills, S. A.; Klinman, J. P., Catalytic Mechanism of the Topa Quinone Containing Copper Amine Oxidases. Biochemistry 2002, 41, (30), 9269-9278. 13. Lee, Y.; Sayre, L. M., Model studies on the quinone-containing copper amine oxidases. Unambiguous demonstration of a transamination mechanism. J. Am. Chem. Soc. 1995, 117, (48), 11823-8. 14.Mure, M.; Klinman, J. P., Model Studies of Topaquinone-Dependent Amine Oxidases.

2. Characterization of Reaction Intermediates and Mechanism. J. Am. Chem. Soc. 1995, 117, (34), 8707-18. 15. Lunelli, M.; Di Paolo, M. L.; Biadene, M.; Calderone, V.; Battistutta, R.; Scarpa, M.; Rigo, A.; Zanotti, G., Crystal structure of amine oxidase from bovine serum. J. Mol. Biol. 2005, 346, (4), 991-1004. 16. Wilmot, C. M.; Murray, J. M.; Alton, G.; Parsons, M. R.; Convery, M. A.; Blakeley, V.; Corner, A. S.; Palcic, M. M.; Knowles, P. F.; McPherson, M. J.; Phillips, S. E., Catalytic mechanism of the quinoenzyme amine oxidase from Escherichia coli: exploring the reductive half-reaction. Biochemistry 1997, 36, (7), 1608-20. 17. Hartmann, C.; Brzovic, P.; Klinman, J. P., Spectroscopic detection of chemical intermediates in the reaction of para-substituted benzylamines with bovine serum amine oxidase. Biochemistry 1993, 32, (9), 2234-41. 18. Plastino, J.; Green, E. L.; Sanders-Loehr, J.; Klinman, J. P., An unexpected role for the active site base in cofactor orientation and flexibility in the copper amine oxidase from Hansenula polymorpha. Biochemistry 1999, 38, (26), 8204-16. 19. Schwartz, B.; Olgin, A. K.; Klinman, J. P., The role of copper in topa quinone biogenesis and catalysis, as probed by azide inhibition of a copper amine oxidase from yeast. Biochemistry 2001, 40, (9), 2954-63. 20. Mills, S. A.; Goto, Y.; Su, Q.; Plastino, J.; Klinman, J. P., Mechanistic Comparison of the Cobalt-Substituted and Wild-Type Copper Amine Oxidase from Hansenula polymorpha. Biochemistry 2002, 41, (34), 10577-10584. 21. Li, R.; Klinman, J. P.; Mathews, F. S., Copper amine oxidase from Hansenula polymorpha: the crystal structure determined at 2.4 .ANG. resolution reveals the active conformation. Structure (London) 1998, 6, (3), 293-307. 22. Su, Q.; Klinman, J. P., Probing the mechanism of proton coupled electron transfer to dioxygen: the oxidative half-reaction of bovine serum amine oxidase. Biochemistry 1998, 37, (36), 12513-25. 23. Copeland, R. A.; Editor, Evaluation of Enzyme Inhibitors in Drug Discovery: A Guide to Medicinal Chemists and Pharmacologists. 2005; p 296 pp. 24. Silverman, R. B., Mechanism-based enzyme inactivators. Methods Enzymol 1995, 249, 240-83. 25. Waley, S. G., Kinetics of suicide substrates. Practical procedures for determining parameters. Biochem. J. 1985, 227, (3), 843-9. 26. Waley, S. G., Kinetics of suicide substrates. Biochem. J. 1980, 185, (3), 771-3. 27. Kitz, R.; Wilson, I. B., Esters of methanesulfonic acid as irreversible inhibitors of acetylcholinesterase. J. Biol. Chem. 1962, 237, 3245-49. 28. Jung, M. J.; Metcalf, B. W., Catalytic inhibition of gamma-aminobutyric acid - alpha-ketoglutarate transaminase of bacterial origin by 4-aminohex-5-ynoic acid, a substrate analog. Biochem Biophys Res Commun 1975, 67, (1), 301-6.

Chapter 2 Inactivation of two different copper amine oxidases by allenic amines

2.1 Introduction

The topaquinone-dependent copper amine oxidases (CAOs) catalyze oxidative

deamination of primary amines to aldehydes at the expense of reduction of O2 to H2O2

via well-established transamination mechanism as described in Chapter 1. Human

enzymes in this family have attracted much recent attention due to the understanding of

their physiological and pathological significance.1, 2 Four types of human TPQ-dependent

CAOs have been described: semicarbazide-sensitive amine oxidase (SSAO),2 soluble

plasma amine oxidase,3 kidney diamine oxidase (KDAO),4 retina-specific amine

oxidase.5 Although all the CAOs are inhibited by semicarbazide, only mammalian tissue- associated (endothelium, adipose, and smooth muscle) amine oxidases are designated as

SSAO. Recently, Jakobsson and coworkers have resolved the crystal structure of human

vascular adhesion protein-1 (HVAP-1).2 HVAP-1 is known as a SSAO and plays a role in migration of leukocytes from the circulation to inflammation sites and regulation of developing the inflammatory reaction, making this enzyme an important anti- inflammatory target.6 The role of SSAO in the pathophysiology of diabetes has also been

extensively investigated. Elevated SSAO activity is associated with diabetes, particularly

in patients with vascular complications.7, 8 The SSAO-mediated production of aldehyde and hydrogen peroxide has been proposed to contribute to the aforementioned disease.

Overall, SSAO is a tremendously promising target for pharmaceutical intervention

based on the significant pathological roles ascribed to it. The development of small

molecule SSAO inhibitors should aid to drug discovery in this area. So far some

hydrazine derivatives have been found to be effective inhibitors of SSAO/VAP-1,

reducing the clinical symptoms of inflammation in rodents.9 However most of those known hydrazine derivatives have limited potential for human drug use due to the fact that they either lack selectivity with respect to other CAOs and the FAD-dependent monoamine oxidases (MAOs) or contain highly reactive structural elements.10

The Sayre lab proposes that the design of mechanism-based SSAO inhibitors as drugs is a promising field, because specificity can be achieved at two different levels: (1) noncovalent binding and (2) whether metabolism of the molecule leads to covalent modification. In the 1970’s, 1-amino-2,3-butadiene was found to be an inhibitor of

MAO-B.11 Recently, this compound prepared in an effort to understand the inactivation

mechanism of its isomer 3-amino-1-butyne, was accidentally discovered to be another highly potent inhibitor for BPAO.12 It does not have much selectivity in inhibiting BPAO

and MAO-B, likely because it is a molecule of small size without a recognition motif. To

explore the structural dependence of enzyme inactivation by 1-amino-2,3-butadiene, we synthesized and evaluated the C4-monosubstituted and C4-disubstituted analogs and

homoallenylamine shown in Figure 2.1. Our preliminary structure-activity relationship studies using BPAO as a model for human SSAO reveals that C4-monosubstitution (alkyl

or phenyl) is well tolerated; C4-disubstitution and extension of one carbon to yield compound 6 significantly decreased or nearly eradicated inhibitory potency. Significant

selectivity is observed for C4-monosubstituted analogs (versus Arthrobacter globiformis

amine oxidase (AGAO)). Additionally, determination of crystal structures of AGAO

derivitized with rac-2 reveals that enzyme inactivation involves alkylation of the

aminoresorcinol forms of TPQ cofactor with electrophilic aldehyde turnover product.

C Et Ph C NH2 C NH2 NH2 1-amino-2,3-butadiene rac-1 rac-2

Ph Ph C Ph C C NH2 NH2 NH2 rac-3 (R)-3 (S)-3

Et Ph C C C Et NH2 Ph NH2 NH 6 2 4 5

Figure 2.1 Structures of the allene compounds

2.2 Results

2.2.1 Synthesis

For quick access to certain racemic allenyl amines, Cu (I)-mediated one-pot reaction of t-Boc-protected propargylamine with the corresponding aldehyde followed by

deprotection of the amino group was used to generate rac-1 and rac-2.13 This method was

previously described for the preparation of 1-amino-2,3-butadiene using paraformaldehyde with a moderate yield of about 45%. Reaction with propylaldehyde or

3-phenylpropylaldhyde only afforded the corresponding product with a yield of about 10-

20%. No product was detected when benzaldehyde was used. Compound 6 was prepared similarly through the reaction of t-Boc protected homopropargylamine with paraformaldehyde to provide 20% yield.

The Cu (I)–mediated SN2’ reaction as shown in Scheme 2.1 is a more efficient

method for preparing 1-amino-2,3-butadiene derivatives containing mono- or

14 disubstitution at C4. Optically pure 4-phenyl-buta-2,3-dienylamines were prepared from the corresponding optically pure propargyl mesylate HC≡CC(OMs)HPh through an

SN2’ reaction with the lithium anion of Ph2C=NCH3 with high stereoselectivity. The

enantiomeric purity was determined by derivatization of α-allenic amine with (1S)-(+)-

10-camphorsulfonyl chloride and then analysis by 1H-NMR. The chemical shift of the

protons at C10 of N- 4-phenyl-buta-2,3-dienyl-10-camphorsulfonamide can be well resolved for both diastereoisomers.

Ph Ph Ph H N a, b N c 2 R1 N R1 C C Ph R2 R2

rac-3: R1=H, R2=Ph (S)-3: R1=H, R2=Ph (R)-3: R1=Ph, R2=H 4: R1=R2= Et 5: R1=R2= Ph

Reagent: a) t-BuLi, CuCN, LiCl; b) Propargyl mesylate; c) (CO2H)2

Scheme 2.1 Cu (I)–mediated SN2’ reaction for synthesis of allene compounds

2.2.2 Inactivation of BPAO by allenic amines

The inhibitory potency of α-allenic amine derivatives on BPAO and the reversibility of inhibition were evaluated, as summarized in Table 2.1. The reversibility of inhibition of BPAO was checked by gel filtration and 24 h dialysis, since mechanism-based inhibition involves covalent attachment of the inhibitor to the enzyme and therefore removal of excess inhibitor by dialysis or gel filtration should not affect the inactivated enzyme. In contrast to the C4-disubstituted analogs (4 and 5), the C4-monosubstituted analogs (rac-1, rac-2, (R)-3, (S)-3) exhibited remarkably potent and concentration- dependent inhibition on enzyme BPAO. Rac-1 and rac-2 are slightly more potent than the parent compound 1-amino-2,3-butadiene, with 5 min IC50 values that are less than 1µM.

At 1 µM inhibitor, BPAO lost about 75% activity within the 5min of incubation with rac-

1 and 90% with rac-2 at 30 ℃. These results suggest that both inhibitors have a very low

KI and large kinact values. To determine the kinetic parameters for BPAO inactivation, the

inactivation kinetics by rac-1 were monitored at 2 ℃, since the decrease of the

incubation temperature can decrease inactivation rate such that higher concentrations of

inhibitor can be used. As in the case for 1-amino-2,3-butadiene, rac-1 displayed a

pseudo-first-order loss of BPAO activity at 2 ℃ (Figure 2.2). The Kitz and Wilson plot 15

shows the inactivation rate data approximate to the 0-0 origin. The inactivation kinetics

-1 -1 are therefore described with an apparent bimolecular term: kinact/KI = 42 mM min at 2

℃. This value is comparable to the value obtained for 1-amino-2,3-butadiene (kinact/KI =

60 mM-1 min-1).16

Table 2.1 The inhibitory potency and reversibility of inhibition on BPAO by allene

compounds Reversibility of inhibition Reversibility of inhibition Compound IC 50 by Gel filtration by 24h dialysis rac-1 <1.0 µM a Irreversible Irreversible rac-2 <1.0 µM a Irreversible Irreversible (R)-3 1.2 µM b Irreversibled Partly reversibled, e (S)-3 1.3 µM b 4 1 mM c Irreversible Irreversible 5 500 µM b completely reversible n.d. f 6 350 µM a n.d n.d. f

a b c d 5 min IC50; Apparent plateau; 40 min IC50; Reversibility of inhibition on BPAO was

evaluated for rac-3; e Enzyme activity at the end of incubation before 24 h dialysis was

0%; after 24 h dialysis, enzyme activity partly recovered to 12.5%. f Not determined.

A 100

0 µM 1 µM 2 µM 10 4 µM Remaining enzyme activityRemaining enzyme ,% 6 µM

0 2 4 6 8 10 12 14 16 Time, min

B 18

, min 8 1/2 t 6

0 0 0.0 0.2 0.4 0.6 0.8 1.0 1/[I], uM-1

Figure 2.2 (A) Time-dependent inactivation of BPAO by various concentrations of rac-1

at 2℃ and (B) Kitz and Wilson replot of the data.

Rac-3 also displayed potent inhibition of BPAO with an IC50 of approximately 1 µM.

However, rac-3 exhibited an inhibition pattern different from rac-1 and rac-2. At 1 µM of rac-3, loss of enzyme activity occurred within the first 1 min, reaching a plateau at

around 60% remaining enzyme activity. At higher inhibitor concentration, a lower level

of the remaining enzyme activity was reached. Therefore for BPAO, rac-3 has a

partitioning between inactivation and turnover. Since all three compounds (rac-1, rac-2,

and rac-3) used in the preliminary screening experiments are racemic mixtures, it would be of interest to determine the relative inactivation potency of both enantiomers of each compound. Compared with inhibitor rac-3, inhibitor rac-1 and rac-2 are structurally more flexible with an alkyl chain (CH2CH3, CH2CH2Ph) at C4. Both enantiomers of rac-1

and rac-2 may be able to adjust the conformation of the alkyl chains to best fit the active

site of BPAO, thereby weakening the differentiation of both enantiomers by BPAO. Rac-

3 which contains only a phenyl ring at the C4, is structurally more rigid, therefore the R

and S enantiomer could interact differently with the enzyme active site residues, which

would result in different inactivation potency. To test this hypothesis, (R)-3 and (S)-3

were synthesized and evaluated. Unexpectedly, both enantiomers exhibited similar

potency towards the inhibition of BPAO (Figure 2.3 and 2.4). At 1 µM (S)-3, the enzyme

activity decreased to around 40% in 1 min and then slightly recovered to 60% in another

few minutes arriving at a plateau. However, for the same concentration of (R)-3, loss of

enzyme activity occurred at the first 1 min, followed by a plateau at the same level as (S)-

3. For both enantiomers, there was partitioning behavior (turnover and inactivation) and the partition ratio was determined to be approximately 3. Highly potent inactivation of

BPAO by 1-amino-2,3-butadiene and its C4-monosubstituted analogs indicates that the

23 essential structural unit for allenic amine inactivation is C=C=CCH2NH2. Simple extension of 1-amino-2,3-butadiene by replacement of α-CH2 with CH2CH2 yielded compound 6, which provided a 300-fold drop in the measured 5 min IC50.

100 0 µM 1 µM 80 2 µM 4 µM

40 Remaining enzyme activityRemaining enzyme 20

0 5 10 15 20 25 30 Time/min

Figure 2.3 Inactivation of BPAO by various concentrations of (S)-3 at 30℃

100 0 µM 1 µM 80 2 µM 4 µM

40 Remaining enzyme activityRemaining enzyme 20

0 5 10 15 20 25 30 Time/min

Figure 2.4 Inactivation of BPAO by various concentrations of (R)-3 at 30℃

In contrast to the C4-monosubstituted analogs, the C4-disubstituted analogs are weak inhibitors. Compound 4 exhibited significantly weaker time and concentration-dependent

inhibition of BPAO, with a 40min IC50 of 1 mM (Figure 2.5). Accordingly, we wondered

whether the C4-disubstituted analogs are good substrates of BPAO. Since the metabolism

of substrates by BPAO is accompanied by the reduction of oxygen, the oxygen

consumption can be measured to evaluate the substrate activity. No oxygen uptake was

detected when incubated with BPAO, indicating that inhibitor 4 cannot be metabolized

by BPAO. Compound 5 is also a weak inhibitor for BPAO with an IC50 value of more

than 500 µM. Loss of enzyme activity occurred within 1 min, followed by a plateau at a

level of activity that decreased with increasing inhibitor concentration (Figure 2.6).

100

80 Control 0.5mM 60 1mM 2mM

40 Remaning enzyme activity enzyme Remaning 20

0 20 40 60 80 Time/min

Figure 2.5 Time-dependent inactivation of BPAO by various concentrations of 4 at 30℃

110 control µ µ 105 50 M 125 M 250 µM 500 µM 100

75 70

Remaining jenzymeactivity 65

55 0 5 10 15 20 25 30 Time/min

Figure 2.6 Inactivation of BPAO by various concentrations of 5 at 30 ℃

An assay of enzyme activity following the separation of enzyme from inhibitor by

gel filtration or 24 h dialysis confirmed the irreversibility of inactivation of BPAO by

rac-1, rac-2, and 4. Twenty four hours dialysis of the rac-3 inactivated enzyme was

associated with slight recovery of activity by 12.5%. Moreover, inactivation of BPAO by

5 was completely reversible as indicated by the complete recovery of the enzyme activity after gel filtration. Generally, the irreversible inactivation of enzyme by inhibitor implicates a stable covalent adduct formed between enzyme and inhibitor. Whether TPQ forms a stable covalent adduct with an inhibitor can be determined by the performance of the redox cycling assay with nitroblue tetrazolium (NBT) on the denatured inactivated enzyme. This assay determines the competency of the quinone cofactor to mediate the

O2-dependent chemical deamination of glycinate. The TPQ loses this redox competency,

when it forms a stable covalent adduct with an inhibitor. As in the case of 1-amino-2,3-

butadiene, the redox cycling assay with NBT on the denatured enzyme following

inactivation by those potent C4-monosubstituted analogs (rac-1, rac-2, and rac-3) was indistinguishable from the denatured control enzyme, indicating complete quinone cofactor redox competence following denaturation of the inactivated enzyme.

2.2.3 Inactivation of AGAO by allenic amines

AGAO is a bacterial CAO. The inhibitor screening always performs on AGAO as well as BPAO to elucidate the factors that govern the inhibitor selectivity and potency.17,

18 The inhibitory potency of α-allenic amine derivatives on AGAO were evaluated and

their reversibility of inhibition was assayed by 24 h dialysis (compound 6 was not tested)

(Table 2.2). All the α-allenic amines including 1-amino-2,3-butadiene, C4- monosubstituted and disubstituted analogs (rac-1, rac-2, (R)-3, (S)-3, 4, and 5) exhibit modest or weak inhibition towards AGAO. As shown in Figure 2.7 for rac-2 (1-amino-

2,3-butadiene, (R)-3, and (S)-3 behaved similarly), loss of enzyme activity occurs within the first 1 min of incubation, reaching a plateau at a level of activity that decreases with increasing concentration of rac-2. This behavior is expected for the classical partitioning of a mechanism-based inhibitor between inactivation and turnover. Rac-1, 4 and 5 display a slightly different inactivation pattern. At the lower inhibitor concentration, there is an

initial drop of enzyme activity in the first 1min followed by partial recovery until a

plateau is reached. At 200 µM of rac-1, the activity of AGAO decreases to 36% in 1 min

and then increases to a plateau at 66 % activity over 30 min; At 100 µM of 5, the activity

of AGAO decreases to 31% in 1min and then increases to a plateau at 94% over 30 min.

The general explanation for these observations is that those compounds (rac-1, 4 and 5)

act as both inhibitor and substrate of AGAO; the enzyme inactivation observed at the

beginning of incubation period is ascribed to the potent reversible competitive inhibition

superimposed on the turnover-dependent inactivation; with the metabolism of inhibitor

by enzyme, the inhibitor concentration deceases and the competitive inhibition is

eliminated gradually.18, 19

Table 2.2 The inhibitory potency and reversibility of inhibition on AGAO by allene

compounds Reversibility of inhibition by 24 h dialysis

Compound IC50 Enzyme activity at the end of After 24 h preincubation before 24 h dialysis dialysis 1-amino-2,3- 64 µM 2% 21% butadiene rac-1 255 µM 0 4% rac-2 45 µM 1% 4% (R)-3 100 µM 0 44% (S)-3 65 µM 0 56% 4 530 µM 6% 10% 5 440 µM 0 29%

Less than 5% recovery of enzyme activity was detected after 24 h dialysis of the

inactivated AGAO by rac-1, rac-2, and 4 (4% for rac-1 and 4, 3% for rac-2). These results indicate that all three compounds are irreversible inhibitors for AGAO and probably form stable covalent adducts with AGAO. However, when AGAO was incubated with 1-amino-2,3-butadiene, 5, (R)-3 and (S)-3 respectively, partial recovery of enzyme activity was detected after 24 h dialysis of the inactivated AGAO.

100

60 Control 10µM 30µM 40 60µM 100µM

20 The remainingThe activity enzyme

0 10 20 30 40 50 60 Time/min

Figure 2.7 Inactivation of AGAO by various concentrations of rac-2 at 30℃

2.2.4 Structure of AGAO-inhibitor rac-2 complex

Residual Fobs- Fcalc electron-density differences observed during the refinement of the

AGAO complex with rac-2 was interpreted in terms of this inhibitor being bound at the

enzyme active site (Figure 2.8) (In collaboration with Dr Jules Mitchell Guss’s lab,

University of Sydney). The distinctive electron-density of rac-2 indicates that it is linked

covalently to the TPQ cofactor with a new bond formed between the O5 of TPQ (or the N

of TPQamr) and the C3 of rac-2. However, the cumulative double bond (C=C=C) in rac-2

is unreactive. Once rac-2 is metabolized by AGAO to form the corresponding aldehyde,

the central carbon (C3) of the diene becomes remarkably electrophilic. Therefore the

observed electron density of TPQ-rac-2 adduct can be rationalized in terms of nucleophilic attack by the amino group of the reductively aminated TPQ (TPQamr) at the

C3 of the corresponding turnover product of rac-2.

Figure 2.8 Electron density map of TPQ-rac-2 adduct with top view (A) and bottom view (B).

2.3 Discussion and conclusion

In this study, we screened a series of allenic amines against BPAO and AGAO to

investigate the inhibitory potency and selectivity. All the C4-monosubstituted analogs of

1-amino-2,3-butadiene reported to date are very potent and irreversible inhibitors of

BPAO. Comparison of the inhibitory potency of the C4-monosubstituted analogs with

that of 1-amino-2,3-butadiene indicates that mono-substitution at C4 does not impair the inhibitory potency. C4-monosubstituted analogs are well suited for retention of inhibitor

potency. Additionally, 1-amino-2,3-butadiene is a distinct inactivation unit in contrast to propargylamine and (E)-3-chloroallyamine reported previously. Propargylamine and (E)-

3-chloroallyamine are another two potent inactivation constructs for BPAO with IC50

values of 3 µM and 2 µM respectively.20, 21 Incredibly, simple extension of one carbon

(replacement of the H at C3 with CH3) to yield 2-butynamine results in a 200-fold drop in

22 inhibition potency in terms of IC50 value; (Z)-3-Chloro-2-buten-1-amine does not show

any inhibition on BPAO up to 1 mM.

It is intriguing that both (R)-3 and (S)-3 display nearly the same potency for

inactivation of BPAO. Our interpretation of this finding is that both enantiomers must

bind in a similar manner to the active site of BPAO. As shown in Scheme 2.2, the

structures of (R)-3 and (S)-3 can overlap considerably except that the pro-R α-proton of

(R)-3 and the pro-S α-proton of (S)-3 are oriented to the opposite position. Therefore, only subtle movements of enzyme active site residues would be required to accommodate each enantiomer.

(pro-R) Ha NH2 (pro-S) C Hb C NH2 Ha Hb (pro-R) (S)-3 (pro-S) (R)-3

Scheme 2.2 Overlap of the structures of (S)-3 and (R)-3.

Unlike the C4-monosubstituted analogs, the C4-disubstituted analogs 4 and 5 are

branched molecules. The inhibitory data obtained for the C4-disubstituted analogs 4 and 5

demonstrate that lateral branching remarkably reduces inhibitory potency for BPAO.

Additionally, no oxygen uptake was detected when BPAO was incubated with compound

4 and 5 respectively, indicating compound 4 and 5 are not substrates of BPAO. These

results suggest the branched molecules are less favorably accommodated by the enzyme

active site.

AGAO activity was affected by a much higher concentration of 1-amino-2,3-

butadiene (IC50 = 64 µM), so this parent compound is not an effective inactivation unit

for AGAO. All of the C4-monosubstituted and disubstituted analogs developed here

exhibited only modest or weak inhibition on AGAO, with IC50 values in the range of 45-

530 µM. Interestingly, in the cases of 1-amino-2,3-butadiene, rac-1 and (S)-3, all three

inhibitors have similar IC50 values, but the reversibility of their AGAO inhibition is

different .

Relevant to understanding the mechanistic basis of the inactivation potency of α-

allenic amines are phenylhydrazine titration 23and nitroblue tetrazolium (NBT) redox cycling assays 24of 1-amino-2,3-butadiene-inactivated BPAO as reported by Qiao et al.12

Treatment of 1-amino-2,3-butadiene-inactivated BPAO with phenylhydrazine does not

generate the characteristic 450 nm absorbance due to derivatization of TPQ cofactor. This

result indicates either that 1-amino-2,3-butadiene modifies the TPQ cofactor or that this

inhibitor modifies an active-site residue in a manner that sterically blocks access of

phenylhydrazine. The NBT staining for the denatured BPAO inactivated by 1-amino-2,3-

butadiene is indistinguishable from that for the denatured control enzyme, indicating the

complete quinone cofactor redox competence following denaturation of the inactivated

enzyme. Similar NBT staining results were also obtained for the denatured BPAO

inactivated by rac-1, rac-2, rac-3, respectively. Although phenylhydrazine titration was

only conducted on the parent compound 1-amino-2,3-butadiene, we assume a common

mechanism for the potent inhibition of BPAO by α-allenic amines including 1-amino-2,3-

butadiene and its C4-monosubstituted analogs since they contain the same inactivation

unit. Thus the finding of a lack of phenylhydrazine reactivity but retention of cofactor

redox activity for the inactivated BPAO demonstrate that 1-amino-2,3-butadiene and its

C4-mono-substituted analogs inactivate BPAO either by covalent modification of an

active-site residue that blocks substrate access to the quinone cofactor or by modifying the cofactor in a form that can only be reversed upon enzyme denaturation. At this time,

we tentatively interpret the mechanism of BPAO inactivation simply in terms of

transaminative turnover of α-allenic amine (RCH=C=CHCH2NH2) to yield highly

electrophilic aldehyde (RCH=C=CHCHO) (either as free aldehyde or in imine linkage to

the reduced aminoresorcinol form of the cofactor) which then alkylates an active site

residue. Alternatively, free allenic aldehyde product could alkylate the reduced cofactor

prior to release into bulk solvent.

The mechanism of AGAO inactivation by rac-2 was revealed by the crystal structure

of AGAO derivatized by rac-2. This structure revealed an inactivation mechanism involving covalent attachment of the allenic aldehyde turnover product to the amino group of the reduced TPQ cofactor. Rac-2 was a moderately potent AGAO inhibitor which displayed a plateau behavior (see Figure 2.7), suggestive of partitioning between turnover and inactivation. A reaction mechanism which accommodates both processes

(turnover and inactivation) is shown in Scheme 2.3. The productive turnover follows the well-known transamination mechanism (Scheme 2.3, path A). After α-allenic amine condenses with TPQ to give a substrate Schiff base, a conserved Asp acts as a catalytic base to abstract a proton at Cα of allenic amine, inducing tautomerization to generate a product Schiff base. The product Schiff base hydrolyzes to yield α-allenic aldehyde product and reductively aminated cofactor. The latter is then oxidized to regenerate

oxidative TPQ cofactor at the expense of reduction of O2 to H2O2 with the hydrolytic

release of NH3. The inactivation occurs after the hydrolysis of product Schiff base.

Instead of releasing into the bulk solvent, the α-allenic aldehyde product is attacked at the electron-deficient C3 by the amino group of reduced aminated TPQ. This Michael-type

addition reaction results in the formation of reduced TPQ-6-phenyl-hex-2-enal derivative as observed in the crystal structures. A similar inactivation mode was previously observed in inactivation of AGAO by 4-(aryloxy)-2-butynamine.17 Basically this type of

inactivation is affected by two factors: (1) the inherent chemical reactivity of reduced

aminated TPQ and product aldehyde and (2) whether the reduced TPQ-aldehyde adduct

can be accommodated at the active site of AGAO through the localized structural

arrangement. Assuming that all the other α-allenic amines inactivate AGAO via the same

mechanism as rac-2, the weaker potency of C4-disubstituted derivatives with respect to

the parent compound and C4-monosubstituted derivatives suggest that the corresponding

aldehydes of C4-disubstituted derivatives are weaker Michael acceptors due to the steric

hindrance caused by two substituent groups at C4. Weaker inhibition of C4-disubstituted

α-allenic amines can also be attributed to the less favorable accommodation of those terminal branched molecules by AGAO. Compared with the branched molecules, the linearly extended molecules have a much more favorable van der Waals interaction with enzyme AGAO since the terminal extended aryl group can project into the hydrophobic binding pocket, as noticed for 4-(aryloxy)-2-butynamine.17 A noticeable feature about

inactivation of AGAO by 1-amino-2,3-butadiene, (R)-3, (S)-3, and 5, is that 24 h dialysis of the inactivated AGAO was associated with partial recovery of enzyme activity. The enzyme activity recovery may reflect the instability of the TPQamr-aldehyde adduct at the active site of AGAO, since the adduct shown in Scheme 2.3 could eventually be hydrolyzed (e.g., by addition-elimination reaction). However, for rac-2, no recovery of

O R O C NH3

O -H O O 2 R O HN C

H NH3 B H2O

H2O2

O2 OH OH OH A B -O O H2O HO HO HN H2O NH2 O NH2 C C C R R R

OH OH

HO O O NH O NH

R H+ R

Scheme 2.3 Proposed mechanism for the reaction between AGAO and α-allenic amine

rac-2 (including productive turnover and enzyme inhibition)

activity was detected after 24 h dialysis of the inactivated AGAO, indicating that the

reduced TPQ-6-phenyl-2-hexaenal adduct is stable. A possible explanation for the

stability of this adduct is the terminal phenyl group of rac-2 projects into the hydrophobic substrate channel of AGAO in a mode that blocks water access to the enamine structure of the adduct.

As mentioned above, the reduced TPQ modification by 4-(aryloxy)-2-butynal was initially disclosed in the case of inactivation of AGAO by 4-(aryloxy)-2-butynamine.17

Our result represents another case for the reduced TPQ as an effective nucleophile

involved in AGAO inactivation by α-allenic amine. Since this kind of inactivation mode is a consequence of the inherent chemical reactivity of the reduced TPQ and the aldehyde product, similar reactions may be involved in BPAO with the same class of inhibitors.

This hypothesis is also supported by structural analysis of AGAO and BPAO, showing that both enzymes share significant structural similarities in the active site and the substrate entry channel, albeit the relatively low overall sequence identity (about 20%).

Firstly, the active sites for both enzymes contain TPQ and a copper ion, where the copper ion is coordinated by three conserved histidines. A conserved aspartic acid at the active site acts as a general base in proton abstraction during the transaminative turnover process. Moreover, for both BPAO and AGAO, there is a hydrophobic channel leading from the active site to the enzyme surface, which appears to be important for both substrate recognition and inhibitor binding.25, 26 Additional structural analysis of the

active site and the substrate entry channel of BPAO and AGAO implicates that the

structural basis of inactivation by the same class of inhibitors such as α-allenic amine

compounds may be identical for both enzymes.

Regardless of what kind of mechanism (either alkylation of active site residue or

TPQamr) is involved in the inactivation of BPAO by α-allenic amines, the parent compound 1-amino-2,3-butadiene and its mono-substituted analogs are the most potent inhibitors thus far described for BPAO. Synthesis of the second generation of α-allenic amine derivatives and evaluation of their inhibitory potency on human SSAO and

selectivity relative to MAOs are in progress.

2.4 Experimental procedures

General procedures

NMR spectra were run on a Varian 200 MHz (13C-NMR at 50 MHz) or 400 MHz

(13C-NMR at 100 MHz) with chemical shifts referenced to the residue proton peak in the

deuterated solvents. High resolution mass spectra (HRMS) were obtained by electron

impact ionization (20-40 eV) or fast atom bombardment (FAB) on a Kratos MS25RFA

instrument. Benzylamine assays were conducted on a Perkin-Elmer Lambda 25 UV-vis

spectrophotometer using UV Winlab software V 2.85, with constant temperature being

maintained by peltier thermstatting. BPAO was purified by Dr Dooley’s group, Montana

State University.

Synthesis

Hexa-2,3-dienylamine hydrochloride (rac-1)

A dioxane solution of the t-Boc protected propargylamine, propionaldehyde, diisopropylamine and freshly prepared CuBr, was heated for 12 h under argon.

Quenching with 1 N acetic acid, extraction with diethyl ether, drying with anhydrous

Na2SO4, evaporation of diethyl ether, and silica gel flash chromatography of the crude

product afforded the t-Boc derivative of 1-amino-2,3-hexadiene (20%). 1H-NMR

(CDCl3): δ 5.29 (1H, m), 5.19 (1H, m), 4.65 (1H, brs), 3.66 (2H, brm), 1.98 (2H, m), 1.42

13 (9H, s), 0.98 (3H, t, J = 7.4); C-NMR (CDCl3): δ 202.88, 155.74, 95.74, 89.85, 79.26,

39.38 , 28.41, 21.79 , 13.35 . Deprotection of t-Boc derivative of 1-amino-2,3-hexadiene with 3 N HCl at room temperature followed by removal of the solvent and excess HCl in

1 vacuum afford 1-amino-2,3-hexadiene hydrochloride. H-NMR (CD3OD): δ 5.52 (1H, m),

5.34 (1H, m), 3.51 (2H, dd, J = 2.6 and 6.2), 2.10 (2H, m), 1.06 (3H, t, J = 7.4); 13C-NMR

+ (CD3OD): δ 205.66, 97.72, 86.28, 39.68, 22.57, 13.72; HRFABMS MH m/z obsd 98.

09718, C6H12N required 98.09697

6-Phenyl-hexa-2,3-dienylamine hydrochloride (rac-2)

6-Phenyl-hexa-2,3-dienylamine hydrochloride was prepared according to the same

procedure as above using 3-phenylpropionaldehyde instead of propionaldehyde. 1H-NMR

(CD3OD): δ 7.24 (5H, m), 5.46 (1H, m), 5.29 (1H, m), 3.39 (2H, brm), 2.75 (2H, m),

2.38 (2H, m); 13C NMR (CDCl3): 206.25, 142.75, 129.74, 129.49, 127.14, 95.22, 86.06,

+ 39.69, 36.20, 31.24; HRFABMS MH m/z obsd 174.12804, C12H16N required 174.12827

1-Amino-3,4-pentadiene hydrochloride (6)

1-amino-3,4-pentadiene was prepared according to the same method as above from

the reaction of t-Boc protected homopropargylamine with formaldehyde in 20% yield.

1 H-NMR (CD3OD): δ 5.169 (1H, m), 4.817 (2H, m), 3.013 (2H, t, J = 7.2), 2.346 (2H,

13 + m); C NMR (CDCl3): 209.17, 165.39, 85.03, 75.22, 38.78, 26.11; HRFABMS MH m/z

obsd 84.08173, C5H10N required 84.08132,

Racemic 4-Phenyl-buta-2,3-dienylamine hydrochloride (rac-3)

Benzophone (9.1 g, 0.05 mol) was added to methyl amine (15.5 g, 0.5 mol) in 250 mL benzene with stirring under argon. The solution was cooled with ice-water bath. A solution of titanium (IV) chloride (5.7 g, 0.03 mol) in 50 mL benzene was added slowly.

The solution was kept stirring at room temperature for about 3 days until TLC showed benzophone disappeared completely. After reaction, the reaction mixture was filtered and

the solvent benzene was evaporated under reduced pressure. The residue was distilled

under reduced pressure to give the pure final product N-methyl benzophenone imine. 1H-

NMR (CDCl3): δ 7.64-1.16 (10H, m), 3.28 (3H, s).

N-methyl benzophenone imine (0.488 g, 2.5 mmol) in 2.5 mL THF was added to a

solution of t-BuLi (1.5 mL, 1.7 M in pentane) in 15 mL THF. After the mixture was

stirred for 5 min, a solution of dry CuCN (0.27 g) and dry LiCl (0.255 g) in 10 mL THF

was added. The solution was stirred under argon and kept under -78 ℃ before use. 1mL

of 2.5 M n-BuLi in hexane was added to racemic 1-phenyl-propargyl alcohol (2.5 mmol,

0.33 g) in 25 mL THF with vigorous stirring under argon at -78 ℃. After about 3 min, methanesulfonyl chloride was added immediately. The temperature was kept at -78 ℃.

After several minutes, to this methane sulfonate solution was transferred the copperic

reagent solution prepared beforehand. The reaction mixture was stirred under argon at -

78 ℃ for about 2h and then dry-ice acetone bath was removed. 10 mL water was added to quench the reaction. THF was evaporated under reduced pressure and the residue was extracted with ethyl ether (20 mL×3). Ethyl ether layers were combined and ethyl ether was evaporated. To the residue was added a solution of 0.2772 g oxalic acid dihydrate in

7 mL ethanol/ethyl ether (1: 6) and the mixture was stirred for 1 h. The white solid was filtered and washed thoroughly with ethyl ether. The solid was dissolved in 1 N NaOH solution and ethyl ether (20 mL × 3) was added to extract the free base. Ethyl ether layers were combined and dried with anhydrous sodium sulfate. Ethyl ether was evaporated to get ride of the trace of methyl amine. The free primary amine was transformed to hydrogen chloride salt by adding HCl-ethyl ether solution. The solid was filtered and

1 crystallized in methanol: ethyl acetate. H-NMR (CD3OD): δ 7.36 (5H, m), 6.55 (1H, dt,

13 J = 2.8, 6.4), 5.80 (1H, q, J = 6.4), 3.66 (2H, dd, J = 2.8, 6.4); C NMR (CD3OD): δ

207.01, 134.17, 129.83, 128.88, 128.36, 99.04, 89.79, 39.30.

Optical pure 4-Phenyl-buta-2,3-dienylamine hydrochloride ((S)-3 and (R)-3)

R and S enantiomers of 4-Phenyl-buta-2,3-dienylamine hydrochloride were prepared using the corresponding optical pure 1-phenyl-propargyl alcohol as starting material according to the same method for preparation of the racemic compound. 1H-NMR and

13C-NMR of both enantiomers were identical with those of racemic compound above.

+ HRFABMS of S enantiomer MH m/z obsd 146.09726, C10H12N required 146.09697;

+ [a]D+240.7 (0.745, MeOH); HRFABMS of R enantiomer MH m/z obsd 146.09723,

C10H12N required 146.09697, [a]D-248.5 (1.75, MeOH).

4-Ethyl-hexa-2,3-dienylamine hydrogen oxalate and 4,4-diphenyl-buta-2,3-dienylamine hydrochloride (4 and 5)

The free bases of both compounds were prepared according to the same method above. To the free base of 4-ethyl-hexa-2,3-dienylamine in ethyl ether was added a solution of oxalic acid in ethyl ether. The solid was filtered and recrystallized in

1 methanol. H-NMR (CD3OD): δ 5.334 (1H, m), 3.487 (2H, d, J = 6.4), 2.049 (4H, m),

13 1.039 (6H, t, J = 7.6). C-NMR (CD3OD): δ 201.601, 165.428, 112.297, 86.429, 39.08,

25.044, 11.420; HRFABMS MH+ m/z obsd 126.12886, MH+ (+ Glycerol ) m/z obsd

218.17635, C8H16N required 126.12827

To the free base of 4, 4-diphenyl-buta-2,3-dienylamine in ethyl ether was added HCl-

ethyl ether solution. The solid was filtered and recrystallized in methanol. 1H-NMR

13 (CD3OD): δ 7.348 (10H, m), 5.894 (1H, t, J = 6.8), 3.724 (2H, d, J = 6.8). C-NMR

(CD3OD): δ206.14, 135.632, 128.530, 128.469, 127.897, 113.205, 87.726, 38.592;

+ HRFABMS MH m/z obsd, 222.12641, C16H16N required 222.12827

Determination of optical purity of enantiomeric compounds

Enantiomeric purity was assayed by derivatization of enantiomers with

enantiomerically pure agent and then analysis of its 1H-NMR. Here commercial available

(1S)-(+)-10-camphorsulfonyl chloride was used as chiral derivatizing agent. N-4-phenyl- buta-2,3-dienyl-(1S)-10-camphorsulfonamide were prepared as the following procedure.

To a solution of racemic 4-phenyl-buta-2,3-dienylamine hydrochloride (30 mg, 0.165 mmol) and triethylamine (35.5 mg, 0.352 mmol) in 5 mL dry dichloromethane under argon at -20 ℃ was added a solution of (1S)-(+)-10-camphorsulfonyl chloride (44 mg,

0.176 mmol) in 3 mL dichloromethane. The reaction mixture was stirred for 3 h at room temperature until the TLC showed the reaction was complete. The reaction mixture was washed with cold water (5 mL × 3) and the organic layer was evaporated under vacuum.

The residue was subjected to column chromatography to give oil, which contained equal

amount of N-(S)-4-phenyl-buta-2,3-dienyl-(1S)-10-camphorsulfonamide and N-(R)-4- phenyl-buta-2,3-dienyl-(1S)-10-camphorsulfonamide. Both diastereomers can not be separated by column chromatography. H-NMR (CDCl3): δ 7.20-7.35 (5H, m), 6.37-6.30

(1H, m), 5.76-5.66 (1H, m), 5.32 (1H, m), 3.93 (2H, m), 3.51, 3.43 (1H, d, J = 16, R

enantimer derivative), 3.49, 3.41 (1H, d, J = 16, S enantimer derivative), 2.99, 2.91(1H,

d, J = 16, S enantiomer derivative), 2.98, 2,90 (1H, d, J = 16, R enantiomer), 1.85-2.45

(6H, m),1.41 (1H, m), 1.018, 1.011 (s, total 3H, R and S enantiomer derivative

respectively), 0.871, 0.851 (s, total 3H, S and R enantiomer derivative respectively);

+ HRFABMS MH m/z obsd 360.16311, C20H26NO3S required 360.16333

N-(R)-4-phenyl-buta-2,3-dienyl-(1S)-10-camphorsulfonamide was prepared in the

1 same way. H-NMR (CDCl3): δ 7.20-7.35 (5H, m), 6.34 (1H, m), 5.71 (1H, q, J = 4), 5.30

(1H, br), 3.92 (2H, m), 3.51, 3.43 (1H, d, J = 16), 2.98, 2.90 (1 H, d, J = 16), 1.85-2.45

(6H, m),1.41 (1H, m), 1.02 (s, 3H), 0.85 (s, 3H); HRFABMS MH+ m/z obsd 360.16317,

C20H26NO3S required 360.16333

Time-dependent inactivation of BPAO/AGAO by candidate inhibitors

A 0.9 mL aliquot of a solution of candidate inhibitor in 100 mM potassium phosphate

buffer, pH 7.2, was mixed with 0.1 mL enzyme stock solution (6-8 µM BPAO; 20 µM

AGAO) and incubated at 30℃aerobically. Aliquots (0.1mL) were periodically withdrawn

using disposable calibrated Drummond micropipettes and diluted with 1.0 mL of

44 benzylamine (5 mM in 50 mM sodium phosphate buffer, pH 7.2) in a 1 cm quartz cuvette

(1.5 mL volume). The rate of oxidation of benzylamine to benzylaldehyde was measured by recording the increase in absorbance at 250 nm for 40 seconds. All percent activities refer to ratios of measured benzaldehyde formation slopes to those obtained on a control incubation lacking inhibitor.

BPAO concentrations were in terms of active site as determined from the absorbance change at 250 nm for benzaldehyde formation using an activity of 0.48 units/mg of

-1 -1 protein for the pure monomer of molecular weight 85 kDa and Δε250 = 12800 M cm for benzaldehyde. AGAO concentration in terms of active site was determined by titration of TPQ with phenylhydrazine.

Irreversibility of enzyme inhibition

(a) By gel-filtration. The irreversibility of the inhibition was checked by applying enzyme preparations (0.3 mL) which were 90–95% inhibited to a PD-10 column, equilibrated with 100 mM sodium phosphate buffer (pH 7.2), to separate non-covalently bound small molecules. Control runs established that no enzyme elutes in the first 50 drops, and that in the subsequent 10 aliquots of 5 drops each, enzyme eluted in aliquots

3–6. Percent activity following gel-filtration was determined by comparing the activity between two gel-filtrated incubations of enzyme in the absence and presence of inhibitor.

The same initial protocol had to be worked out each time the column was changed.

(b) By dialysis. The irreversibility of the inhibition was also checked by adding 0.5 mL enzyme preparations which were 90–95% inhibited to dialysis tubing (6.4 mm

Spectrum Spectro/Pro membrance, Mw cutoff 12,000–14,000), following by dialysis against 100 mM sodium phosphate buffer, pH 7.2, at room temperature for periods up to

24 h. Percent activity was determined by comparing the enzyme activity between two dialysis experiments, following incubation of enzyme in the absence and presence of inhibitor.

Nitroblue tetrazolium (NBT) redox cycling assay

Purified BPAO (20 µM) was incubated with amounts of candidate inhibitor that achieved 97–99% inactivation. Inhibited and control enzyme samples (40 µl) were mixed with 20 µl of standard denaturing buffer (10% SDS with 0.5 M 2-mercaptoethanol) and the mixture was heated at 100 ℃for 6 min before application in duplicate to two halves of a polyacrylamide slab gel (6% acrylamide with 0.16% bisacrylamide). After running the gel (at 0.02 amp constant current, voltage near 200 V), the gel was cut in half and the two halves were stained with either Coomassie blue (0.25% Coomassie in 50% methanol,

7% acetic acid) or 0.24 mM NBT in 2M potassium glycinate (pH 10) for 120 min in the dark. No alteration of the electrophoretic properties of the enzyme or the intensity of

Coomassie staining was observed.

2.5 References 1. Dunkel, P.; Gelain, A.; Barlocco, D.; Haider, N.; Gyires, K.; Sperlagh, B.; Magyar, K.; Maccioni, E.; Fadda, A.; Matyus, P., Semicarbazide-sensitive amine oxidase/vascular adhesion protein 1: recent developments concerning substrates and inhibitors of a promising therapeutic target. Curr. Med. Chem. 2008, 15, (18), 1827-1839. 2. Jakobsson, E.; Nilsson, J.; Ogg, D.; Kleywegt Gerard, J., Structure of human semicarbazide-sensitive amine oxidase/vascular adhesion protein-1. Acta Crystallogr D Biol Crystallogr 2005, 61, (Pt 11), 1550-62. 3. Boomsma, F.; Bhaggoe, U. M.; van der Houwen, A. M. B.; van den Meiracker, A. H., Plasma semicarbazide-sensitive amine oxidase in human (patho)physiology. Biochim. Biophys. Acta, Proteins Proteomics 2003, 1647, (1-2), 48-54. 4. Elmore Bradley, O.; Bollinger John, A.; Dooley David, M., Human kidney diamine oxidase: heterologous expression, purification, and characterization. J Biol Inorg Chem 2002, 7, (6), 565-79. 5. Zhang, Q.; Mashima, Y.; Noda, S.; Imamura, Y.; Kudoh, J.; Shimizu, N.; Nishiyama, T.; Umeda, S.; Oguchi, Y.; Tanaka, Y.; Iwata, T., Characterization of AOC2 gene encoding a copper-binding amine oxidase expressed specifically in retina. Gene 2003, 318, 45-53. 6. Stolen Craig, M.; Marttila-Ichihara, F.; Koskinen, K.; Yegutkin Gennady, G.; Turja, R.; Bono, P.; Skurnik, M.; Hanninen, A.; Jalkanen, S.; Salmi, M., Absence of the endothelial oxidase AOC3 leads to abnormal leukocyte traffic in vivo. Immunity 2005, 22, (1), 105- 15. 7. O'Sullivan, J.; Unzeta, M.; Healy, J.; O'Sullivan, M. I.; Davey, G.; Tipton, K. F., Semicarbazide-sensitive amine oxidases: enzymes with quite a lot to do. Neurotoxicology 2004, 25, (1-2), 303-315. 8. Gokturk, C.; Nordquist, J.; Sugimoto, H.; Forsberg-Nilsson, K.; Nilsson, J.; Oreland, L., Semicarbazide-sensitive amine oxidase in transgenic mice with diabetes. Biochem. Biophys. Res. Commun. 2004, 325, (3), 1013-1020. 9. Lazar, L.; Szakonyi, Z.; Forro, E.; Palko, M.; Zalan, Z.; Szatmari, I.; Fulop, F., Synthesis of hydrazino alcohols with anti-inflammatory activity. Acta Pharm Hung 2004, 74, (1), 11-8. 10. Matyus, P.; Dajka-Halasz, B.; Foeldi, A.; Haider, N.; Barlocco, D.; Magyar, K., Semicarbazide-sensitive amine oxidase: Current status and perspectives. Curr. Med. Chem. 2004, 11, (10), 1285-1298. 11. Krantz, A.; Kokel, B.; Sachdeva, Y. P.; Salach, J. I.; Detmer, K.; Claesson, A.; Sahlberg, C., Inactivation of mitochondrial monoamine oxidase by beta ,gamma ,delta - allenic amines. Monoamine Oxidase: Struct., Funct., Altered Funct., [Proc. Symp.] 1979, 51-70. 12. Qiao, C.; Jeon, H.-B.; Sayre, L. M., Selective Inhibition of Bovine Plasma Amine Oxidase by Homopropargylamine, a New Inactivator Motif. J. Am. Chem. Soc. 2004, 126, (25), 8038-8045.

13. Casara, P.; Jund, K.; Bey, P., General synthetic access to alpha -allenyl amine and alpha -allenyl-alpha -amino acids as potential enzyme-activated irreversible inhibitors of PLP-dependent enzymes. Tetrahedron Lett. 1984, 25, (18), 1891-4. 14. Elsevier, C. J.; Vermeer, P., Synthesis and stereochemistry of allenes. Part 3. Highly stereoselective synthesis of chiral alkyl allenes by organocopper(I)-induced anti 1,3- substitution of chiral propynyl esters. J. Org. Chem. 1989, 54, (15), 3726-30. 15. Kitz, R.; Wilson, I. B., Esters of methanesulfonic acid as irreversible inhibitors of acetylcholinesterase. J. Biol. Chem. 1962, 237, 3245-49. 16. Qiao, C. New copper amine oxidase probes: Synthesis, mechanism, and enzymology. Ph. D. Thesis, Case Western Reserve University, OH, 2004. 17. O'Connell Kimberly, M.; Langley David, B.; Shepard Eric, M.; Duff Anthony, P.; Jeon, H.-B.; Sun, G.; Freeman Hans, C.; Guss, J. M.; Sayre Lawrence, M.; Dooley David, M., Differential inhibition of six copper amine oxidases by a family of 4-(aryloxy)-2- butynamines: evidence for a new mode of inactivation. Biochemistry 2004, 43, (34), 10965-78. 18. Shepard, E. M.; Smith, J.; Elmore, B. O.; Kuchar, J. A.; Sayre, L. M.; Dooley, D. M., Towards the development of selective amine oxidase inhibitors: mechanism-based inhibition of six copper containing amine oxidases. Eur. J. Biochem. 2002, 269, (15), 3645-3658. 19. Qiao, C.; Ling, K.-Q.; Shepard, E. M.; Dooley, D. M.; Sayre, L. M., Mechanism- Based Cofactor Derivatization of a Copper Amine Oxidase by a Branched Primary Amine Recruits the Oxidase Activity of the Enzyme to Turn Inactivator into Substrate. J. Am. Chem. Soc. 2006, 128, (18), 6206-6219. 20. Hevey, R. C.; Babson, J.; Maycock, A. L.; Abeles, R. H., Highly specific enzyme inhibitors. Inhibition of plasma amine oxidase. J Am Chem Soc 1973, 95, (18), 6125-7. 21. Jeon, H.-B.; Sayre, L. M., Highly potent propargylamine and allylamine inhibitors of bovine plasma amine oxidase. Biochem. Biophys. Res. Commun. 2003, 304, (4), 788-794. 22. Chen, Y. Structural basis of substrate and inhibitory activity of bovine plasma amine oxidase. Ph. D. Thesis, Case Western Reserve University, OH, 2008. 23. Janes, S. M.; Klinman, J. P., An investigation of bovine serum amine oxidase active site stoichiometry: evidence for an aminotransferase mechanism involving two carbonyl cofactors per enzyme dimer. Biochemistry 1991, 30, (18), 4599-605. 24. Paz, M. A.; Fluckiger, R.; Boak, A.; Kagan, H. M.; Gallop, P. M., Specific detection of quinoproteins by redox-cycling staining. J Biol Chem 1991, 266, (2), 689-92. 25. Wilce, M. C.; Dooley, D. M.; Freeman, H. C.; Guss, J. M.; Matsunami, H.; McIntire, W. S.; Ruggiero, C. E.; Tanizawa, K.; Yamaguchi, H., Crystal structures of the copper- containing amine oxidase from Arthrobacter globiformis in the holo and apo forms: implications for the biogenesis of topaquinone. Biochemistry 1997, 36, (51), 16116-33. 26. Lunelli, M.; Di Paolo, M. L.; Biadene, M.; Calderone, V.; Battistutta, R.; Scarpa, M.; Rigo, A.; Zanotti, G., Crystal structure of amine oxidase from bovine serum. J. Mol. Biol. 2005, 346, (4), 991-1004.

Chapter 3 Inactivation of bovine plasma amine oxidase by acetic acid-3-amino-propenyl ester

3.1 Introduction

Early in 1973, Abeles and coworkers reported that amines containing an unsaturated

C-C bond at the β-position, such as propargylamine and 2-chloroallylamine, can inactivate bovine plasma amine oxidase (BPAO).1 Based on this discovery, our lab has

directed significant effort to the design and synthesis of new mechanism-based inhibitors

for copper amine oxidases (CAOs). To this end, the most important findings are the

development of two classes of inhibitors: homopropargylamine and 3-aryl-3-pyrrolines.

Homopropargylamine is a potent and selective mechanism-based inhibitor for BPAO

(versus rat liver mitochondrial monoamine oxidase (MAO)).2 3-Phenyl-3-pyrroline acts

as an inhibitor against BPAO but as a substrate of MAO-B purified from beef liver.3

Another endeavor in developing inhibitors for CAOs is the evaluation of inhibitory

potencies of some of those molecules initially developed as inhibitors of the

flavin-dependent enzyme MAO.4, 5 Evaluation of (E)- and (Z)-3-chloroallylamine as inhibitors of MAO reveals that the Z-isomer but not the E-isomer inhibits MAO.

However (E)-3-chloroallylamine exhibits potent time- and concentration-dependent inhibition of BPAO, with an IC50 value of 2 µM. This result suggests that

(E)-3-chloroallylamine is a highly selective inhibitor of BPAO as opposed to MAO.

Moreover, various 2-phenyl-3-haloallylamine analogs that McDonald and coworkers had originally developed for inhibition of MAO were found to be potent inhibitors of BPAO.6

The mechanism for inactivation of BPAO by 3-chloroallylamine E- and Z-isomers and

various 2-phenyl-3-haloallylamines are generally proposed to be through an

4, 5 addition-elimination process (Scheme 3.1). The C3 of 3-haloallylamines would become very reactive to displacement of halogen by an active-site nucleophile at the product-Schiff base stage of turnover (See path A in Scheme 3.1), or immediately following hydrolytic generation of the corresponding aldehyde (See path B in Scheme

3.1). The proposed addition-elimination inactivation mechanism is consistent with the greatly enhanced inhibitory potencies of 2-phenyl-3-haloallylamines with respect to

2-phenylallylamine, (E)- and (Z)-3-chloroallylamine with respect to allylamine (substrate of BPAO).

H2N OH O

Ph O (H) X HO O N Ph O (H) X

Ph (H) A B X Nu

OH O

Ph (H) Nu HO N

Ph (H) Nu Scheme 3.1 Proposed addition-elimination mechanism for inactivation of BPAO by

3-haloallylamines

(E)-3-chloroallylamine is the most potent and selective (versus MAO) inhibitor

against BPAO among all the 3-haloallylamines we have screened. We planned to generate analogs for this inhibitor by covalently attaching to its C3 appropriate binding

ligands for enzyme recognition, in order to achieve both potency and selectivity.

However replacement of H at C3 with CH3 to yield (Z)-3-chloro-2-butenamine nearly eradicates the inhibitory potency. The weakened inhibition could be attributed to an increase in steric hindrance effect at C3, which makes the corresponding product Schiff base (or the aldehyde product) a weaker Michael acceptor for an active-site nucleophile in an addition–elimination reaction. This preliminary result suggests that

(E)-3-chloroallylamine is not the inactivation unit suitable for our inhibitor-developing strategy.

In this work, we studied two new allylamine derivatives, (E)- and (Z)- acetic acid-3-amino-propenyl ester (1 and 2) (see Figure 3.1). Compared with

3-chloroallylamine, replacement of Cl with CH3CO2 in acetic acid-3-amino-propenyl ester does not substantially increase the steric hindrance effect at C3; In addition, CH3CO2

is a stronger electron withdrawing group than Cl, therefore acetic acid-3-amino-propenyl

ester may display an increased inhibitory potency due to the electronic activation effect.

More importantly, acetic acid-3-amino-propenyl ester allows for structural variation, which may offer opportunities for fine-tuning selectivity of inhibitor design. Replacement of CH3CO2 in acetic acid-3-amino-propenyl ester with RCH2CO2 results in a series of

new derivatives, where the R group can be any binding ligand for enzyme recognition

sites. As a possible promising inactivation unit acetic acid-3-amino-propenyl ester should

provide important insights.

O O NH2 O NH2 O

1 2

Figure 3.1 Structures of (E)-isomer (1) and (Z)-isomer (2) of acetic acid-3-amino-

propenyl ester

3.2 Results and discussion

3.2.1 Synthesis

The (E)- and (Z)- isomers of acetic acid-3-amino-propenyl ester (1 and 2) were

prepared according to the method shown in Scheme 3.2. N-t-Boc-3-aminopropanal was

obtained from oxidation of the corresponding alcohol by pyridinium chlorochromate

(PCC). Acylation of the enol form of N-t-Boc-3-aminopropanal was carried out under

basic condition using 4-dimethylaminopyridine (DMAP) as a catalyst.7 A mixture of

N-t-Boc protected trans and cis isomers were obtained at a ratio of 3: 2, which could not

be separated by column chromatography. Following the deprotection of t-Boc group and fractional crystallization, pure (E)- and (Z)-isomer (1 and 2) were obtained.

BocNH a BocNH b BocNH c OAc OH O

HCl.H2N d HCl.H2N OAc HCl.H2N OAc OAc

Reagents: a) PCC; b) DMAP, triethylamine, acetyl anhydride; c) dry hydrochlordie gas; d) fractional crystallization. Scheme 3.2 Synthesis of (E)- and (Z)- isomer of acetic acid-3-amino-propenyl ester (1 and 2)

3.2.2 Enzyme assay of inhibition of BPAO by (E)- and (Z)-acetic acid-3-amino-propenyl ester (1 and 2)

Inhibition of BPAO by both acetic acid-3-amino-propenyl ester isomers (1 and 2) was followed by the standard benzylamine assay (Figure 3.2 and 3.3). Comparing the 40 min

IC50 values (20 µM for 1 and 220 µM for 2) reveals that the trans isomer (1) is about

10-fold more potent. As shown in Figure 3.2 and 3.3, both isomers displayed a rapid loss

of enzyme activity after the first few minutes, followed by a slow recovery in activity. At

20 µM of the trans isomer (1), the enzyme activity decreased to 10% in 3 min, then recovered back to 60% in 80 min. Generally, it is believed that there is potent reversible competitive inhibition superimposed on turnover-dependent inactivation. However, gel filtration of the 93% inactivated enzyme which was induced by incubation with 20 µM of the trans isomer (1) for 3 min only recovered enzyme activity by 15%. This suggests a very tight covalent or noncovalent binding between the enzyme and the inhibitor. The recovery of enzyme activity over time following incubation with the trans isomer (1) also

54 demonstrated that it was being metabolized by the enzyme. Moreover, following 24 h dialysis, a sample of enzyme inactivated by 20 µM of the trans isomer (1) by 49% exhibited enzyme activity recovery by 26%. The incomplete recovery of enzyme activity indicated that there was permanent inhibition.

100

40 Control 5µM 10µM Remaining enzyme activityRemaining enzyme µ 20 15 M 20µM

0 20 40 60 80 Time/min

Figure 3.2 Time-dependent inactivation of BPAO (0.6 µM) by various concentrations of

(E)- acetic acid-3-amino-propenyl ester (1) at 30℃

100 control 90 100 µM 200 µM 80 300 µM 70

Remaining enzyme activityRemaining enzyme 20

0 0 20 40 60 time/min

Figure 3.3 Time-dependent inactivation of BPAO (0.6 µM) by various concentrations of

(Z)-acetic acid-3-amino-propenyl ester (2) at 30℃

3.2.3 Metabolism of (E)-acetic acid-3-amino-propenyl ester (1) by BPAO

Coincident to the time-dependent recovery of enzyme activity, the UV-vis spectra of the incubation solution of BPAO with (E)-acetic acid-3-amino-propenyl ester (1) changed over time, suggesting that the trans isomer (1) was being metabolized by BPAO.

Metabolism of the trans isomer (1) by BPAO was monitored by UV-vis spectrophotometer. The difference UV-vis spectra (against native BPAO) were obtained as shown in Figure 3.4. Upon mixing the enzyme and the trans isomer (1), a new peak at

235 nm appeared immediately, which does not correspond to the inhibitor. The peak at

235 nm decreased slowly and another new peak developed at 266 nm over time.

266nm

0.40

0.35

0.30

0.25 235nm

A 0.20

0.15

0.10

0.05

0.00

220 240 260 280 300 320 340 nm

Figure 3.4 Difference spectra (against native BPAO) obtained from the reaction of BPAO

(0.7 µM) with (E)-acetic acid-3-amino-propenyl ester (1) (20 µM) in 100 mM phosphate

buffer, pH 7.2, 30℃, monitored by UV-vis spectrophotometer with 2 min interval.

In terms of transaminative turnover, BPAO can catalyze the conversion of (E)-acetic

acid-3-amino-propenyl ester (1) to 3-(acetyloxy)-(E)-2-propenal. The authentic sample of

3-(acetyloxy)-(E)-2-propenal was independently prepared according to the published

synthetic method.8 This aldehyde hydrolyzed rapidly in pH 7.2 phosphate buffer, accompanied with the peak at 235 nm decreasing over time. At the same time, a new peak at 266 nm increased, which is attributed to the hydrolytic product malondialdehyde (MA)

9(Figure 3.5). So the difference spectra (against native BPAO) obtained from the reaction

of BPAO with (E)-acetic acid-3-amino-propenyl ester (1) showed that the aldehyde product 3-(acetyloxy)-(E)-2-propenal was generated, which subsequently hydrolyzed to

generate MA in phosphate buffer.

0.12 266nm

0.10 235nm 0.08

0.06 A

0.04

0.02

0.00

-0.02 220 240 260 280 300 320 340 wavelength/nm

Figure 3.5 Hydrolysis of 3-(acetyloxy)-(E)-2-propenal in pH 7.2 phosphate buffer, at

30℃, monitored by UV-vis spectrophotometer at 2 min time interval.

Formation of MA in the incubation solution of BPAO with (E)-acetic acid-3-amino-propenyl ester (1) was further confirmed by HPLC analysis. MA is eluted shortly within the solvent front, so 2,4-dinitrophenylhydrazine (DNPH) is generally used

as a derivatizing reagent to detect MA in HPLC analysis.10 MA reacts readily with DNPH

in an acidic condition to give 1-(2,4-dinitrophenyl) pyrazole. Elution profile of MA stock

solution treated with DNPH showed two peaks. The peak at 14.25 min corresponds to

DNPH and the other one at 16.01 min is attributed to 1-(2, 4-dinitrophenyl) pyrazole.

DNPH-treatment of the incubation solution of BPAO and (E)-acetic

acid-3-amino-propenyl ester (1) followed by HPLC analysis also showed the peak of 1-(2,

4-dinitrophenyl) pyrazole at 16.01 min (Figure 3.6).

42 36 DNPH-MA 30 24 18 DNPH

B 12 6 0 -6 5 10 15 20 25 DNPH-MA 140 120 100 80 60

A DNPH 40 20 0 -20 5 10 15 20 25 Time/min

Figure 3.6 Elution profile of DNPH-derivatized MA. (A) MA stock solution treated with

DNPH, detected at 310 nm; (B) BPAO incubated with (E)-acetic acid-3-amino-propenyl ester (1) for half an hour, then treated with DNPH, detected at 310 nm.

3.2.4 Enzyme assay of inhibition of BPAO by aldehyde product

3-(acetyloxy)-(E)-2-propenal and its hydrolytic product MA

The identification of metabolites of (E)-acetic acid-3-amino-propenyl ester (1) by

BPAO disclosed the substrate activity of this inhibitor (vs. BPAO). In considering possible mechanisms for the inactivating effect of (E)-acetic acid-3-amino-propenyl ester

(1), it was desirable to evaluate the effect of incubating the enzyme with the aldehyde

product 3-(acetyloxy)-(E)-2-propenal and its hydrolytic product MA. Data shown in

Figure 3.7 demonstrate that 3-(acetyloxy)-(E)-2-propenal exerts time-dependent

reversible inhibition of BPAO at the relatively high concentrations. MA at a

concentration up to 80 µM doses not show any effects on BPAO activity at the measured

time period (0-80 min, data are not shown here). The inactivating effect on BPAO by

(E)-acetic acid-3-amino-propenyl ester (1) may not be attributed to the corresponding

aldehyde product or its hydrolytic product MA.

100

95 control 20uM 90 200uM

Remaining enzyme activityRemaining enzyme 80

75 0 2 4 6 8 10 time/min

Figure 3.7 Time-dependent inactivation of BPAO (0.7 µM) by 3-(acetyloxy)-(E)-

2-propenal at 30℃

3.2.5 Evaluation of 3-amino-propionaldehyde and its analogs as inhibitors and

substrates of BPAO

According to the transamination mechanism, metabolism of (E)-acetic acid-3-amino-propenyl ester (1) by BPAO goes through the stage of formation of product

Schiff base between 1 and cofactor TPQ. As an alternative to hydrolysis of the imine to

afford the product aldehyde, the product Schiff base may hydrolyze at the ester bond to

give TPQ derivatives (I and II) (see Scheme 3.3), which induces the inhibition on enzyme

BPAO. So we evaluated the interaction of BPAO with 3-amino-propionaldehyde, since

this is the amine that would derivatize TPQ to form the same TPQ adducts (see Scheme

3.3).

O OH O O O O HO + H3 O O NH2 N O HO

O NH2

O NH2 O

+ H3 O

O CH3CO2H O OH OH OH

HO HO HO HO N N N HN

O O O HO I II

Scheme 3.3 TPQ adducts (I and II) possibly formed from the reaction of BPAO with

acetic acid (E)-3-amino-propenyl ester (1)

Interestingly, 3-amino-propionaldehyde was previously reported as a potent inhibitor

of pea diamine oxidase.11 Since this report, no further study has been conducted. In this

work, 3-amino-propionaldehyde exerted potent and time-dependent inhibition of BPAO

(Figure 3.8). Loss of enzyme activity occurred in the first 20-30 min for all the inhibitor concentrations, followed by a plateau at the different levels. This behavior does not represent the partitioning between turnover and inactivation, since no turnover product

(MA) or oxygen consumption was detected from the incubation solution of BPAO with

3-amino-propionaldehyde. However, this time-dependent inactivation implicates that

3-amino-propionaldehyde is a slow binding inhibitor of BPAO. The plateaus reached after 20-30 min preincubation of BPAO with 3-amino-propionaldehyde suggest that the equilibrium between the enzyme and the enzyme-inhibitor complex is established slowly.

105 100 95 5 µM µ 90 10 M µ 85 15 M 20 µM 80 25 µM 75 70 65 60 55 50 45 remaining activity enzyme 40 35 30 25 0 10 20 30 40 50 60 time/min

Figure 3.8 Time-dependent inactivation of BPAO (0.66µM) by 3-amino-propionaldehyde

at 30℃

The dissociation constants of this inhibitor-enzyme complex were determined under both conditions (with and without preincubation with enzyme) (see experimental section).12 A Lineweaver-Burk plot was constructed from the initial rates of oxidative deamination of varying concentrations of benzylamine by BPAO in the presence of varying concentrations of 3-amino-propionaldehyde without preincubation with BPAO

(Figure 3.9 (A)). Plots of the reciprocal of initial reaction rates vs. the reciprocal of different benzylamine concentrations at varying concentrations of

3-amino-propionaldehyde pass the same point on the y-axis, suggesting that

3-amino-propionaldehyde is acting on the active site of BPAO. The corresponding Dixon plot was constructed to determine the dissociation constant of the complex of BPAO with

3-amino-propionaldehyde (Ki = 0.26 mM) (Figure 3.9 (B)). Preincubation of BPAO with varying concentrations of 3-amino-propionaldehyde for 30 min followed by adding

* substrate benzylamine yielded an apparent Ki value (Ki = 0.067 mM), which is nearly 4 fold smaller than that observed without preincubation. This result is consistent with slow binding inhibition of BPAO by 3-amino-propionaldehyde.

(A)

3-amino-propionaldehyde 80000 0µM 500µM 400µM 200µM 60000 100µM

1/V 40000

20000

0 0.0 0.2 0.4 0.6 0.8 1.0 1/ [benzylamine], mM-1

(B)

80000

60000 1/V

40000

20000

-0.2 0.0 0.2 0.4 0.6 [3-amino-propionaldehyde], mM

Figure 3.9 Competitive inhibition of BPAO by different concentrations of

3-amino-propionaldehyde in pH 7.2, 100 mM sodium phosphate buffer at 30℃. (A)

Lineweaver-Burk plots; (B) The corresponding Dixon plots.

The interaction between BPAO and inhibitor 3-amino-propionaldehyde was modeled

by a docking simulation (Figure 3.10). In the best-scored productive docking mode of

3-amino-propionaldehyde with BPAO, the substrate amino group is interacting with

Asp385 by electrostatic interaction as most of the simple alkyl monoamine substrates,

while the short alkyl chain is nearly parallel to the phenyl ring of Tyr 383. A hydrogen

bond could be formed between the terminal carbonyl group of 3-amino-propionaldehyde

and hydroxyl group of Tyr 383 as the distance between two oxygen atoms is only 3.13 Å.

The binding mode of 3-amino-propionaldehyde at the active site of BPAO shows that this

compound has less interaction with the hydrophobic binding pocket (defined by Tyr383,

Phe388, Phe393, Met467, and Leu468) due to its short alkyl chain. We then asked

whether 3-amino- propionaldehyde acts as an inhibitor rather than a substrate of BPAO in

light of the poor interaction between the enzyme and this compound. In order to address

Figure 3.10 Docking of 3-amino-propionaldehyde into the active site of BPAO

this speculation, we evaluated two analogs: 4-amino-2-butanone and 3-amino-1- phenyl-1-propanone. Replacement of H at C1 of 3-amino-propionaldehyde with methyl and phenyl group respectively, would improve the binding to the enzyme. Docking simulation showed that, while the ethyleneamine portion of both analogs is interacting with Asp 385 and Tyr 383 in a similar manner as seen in 3-amino-propionaldehyde, the methyl group in 4-amino-2-butanone and the phenyl group in 3-amino-1- phenyl-

1-propanone are interacting with hydrophobic residues Phe 388 and Phe 393 (Figure

3.11).

Figure 3.11 Docking of 3-amino-1-phenyl-1-propanone into the active site of BPAO

Metabolism of both analogs by BPAO was followed by UV-vis Spectrophotometer.

4 -1 The expected turnover product is acetoacetaldehyde ( λmax = 280 nm, ε = 1.77×10 M

-1 2, 13 cm ) for 4-amino-2-butanone and benzoylacetaldehyde (λmax = 318nm, ε = 1.08×

104 M-1 cm-1) for 3-amino-1-phenyl-1-propanone. The spectral changes in Figure 3.12

match those of the expected turnover products, indicating that both analogs can be

metabolized by BPAO to generate the corresponding aldehyde. A nonlinear least-squares fit of the product formation curves yielded first-order rate constants (k) of 0.0011 min-1

-1 (t1/2 630 min) for 4-amino-2-butanone and 0.1066 min (t1/2 6.5 min) for

3-amino-1-phenyl-1-propanone, respectively. The half-life for metabolism of

4-amino-2-butanone is 97-fold longer than for 3-amino-1-phenyl-1-propanone.

A 0.035 280nm 0.030 0.04 0.025

0.020 280 0 -A 280 t

A 0.015

0.010 0.03 0.005 0.000 0 2 4 6 8 10 12 14 16 18 20 Time/min A ∆ 0.02

0.01

0.00 260 280 300 320 nm

0.30 B 318nm 0.25

0.25 0.20

318 0 0.15 -A

0.20 318 t A

0.10 0.15 0.05

0 2 4 6 8 10 12 14 16 18 20 0.10 Time/min

A 0.05 ∆

0.00

-0.05

-0.10

-0.15 246nm -0.20 240 270 300 330 360 390 420 nm

Figure 3.12 (A) Difference spectra (against the spectrum at 0 min) obtained from reaction

of BPAO (0.7 µM) with 4-amino-2-butanone (100 µM) in 100 mM phosphate buffer, pH

7.2, 30℃, monitored by UV-vis spectrophotometer with 2 min interval. Insets:

time-dependent plots of absorbance growth at 280 nm and nonlinear least-squares fits of

the product formation plots according to the equation At −= A00( A∞ − A )(1 − exp( − kt )) .

(B) Difference spectra (against the spectrum at 0 min) obtained from the reaction of

BPAO (0.7 µM) with 3-amino-1-phenyl-1-propanone (20 µM) in 100 mM phosphate buffer, pH 7.2, 30℃, monitored by UV-vis spectrophotometer with 2 min interval. Insets: time-dependent plots of absorbance growth at 318 nm and nonlinear least-squares fits of

the product formation plots according to the equation At −= A00( A∞ − A )(1 − exp( − kt )) .

Note that the negative peak at 246 nm corresponds to 3-amino-1-phenyl-1-propanone.

The inhibition of BPAO by both analogs 4-amino-2-butanone and

3-amino-1-phenyl-1-propanone displayed two distinct kinetic patterns (Figure 3.13).

3-Amino-1-phenyl-1-propanone induced rapid inhibition of BPAO in one minute followed by nearly complete recovery of enzyme activity. In contrast, time course of inactivation of BPAO by 4-amino-2-butanone was biphasic, where enzyme activity decreased in the first 20-40 min and then recovered very slowly at the measured inhibitor concentrations.

100

80 Control 100 µM 60 200 µM µ 400 M

40 Remaining enzyme activityRemaining enzyme

0 20 40 60 80 100 Time/min

110

100 control 90 50 µM 100 µM 80 200 µM 70

Remaining enzyme activityRemaining enzyme 20

0 0 20 40 60 80 100 120 Time/min

Figure 3.13 Inhibition of BPAO by various concentrations of 3-amino-1-phenyl-1-

propanone (A) (CBPAO = 1.48 µM) and 4-amino-2-butanone (B) (CBPAO = 0.83 µM) at

30℃.

The initial rapid loss of activity of BPAO upon interacting with

3-amino-1-phenyl-1-propanone or 4-amino-2-butanone is due to its competition with benzylamine for the enzyme active site, since both analogs can be metabolized by BPAO.

Moreover, metabolism of each analog by the enzyme eliminates its competitive inhibition gradually, which leads to the recovery of enzyme activity over time. In the case of the interaction of BPAO (1.48 µM) with 3-amino-1-phenyl-1-propanone (100 µM), we observed the eventual recovery of enzyme activity (up to 95%) after approximately 30 min accompanied with complete metabolism of 3-amino-1-phenyl-1-propanone (Figure

3.14). The much slower recovery of enzyme activity when BPAO was incubated with

4-amino-2-butanone than with 3-amino-1-phenyl-1-propanone may be consistent with the

slower turnover rate for the former in comparison to the latter.

100 M µ

80 90

40 80

20 Remaining enzyme activityRemaining enzyme

[3-amino-1-phenyl-1-propanone]/ 70 0

0 10 20 30 Time/min

Figure 3.14 Correlation between the time-dependent recovery of BPAO activity upon incubation with 3-amino-1-phenyl-1-propanone (□) and metabolism of

3-amino-1-phenyl-1-propanone by BPAO over time (■).

Evaluation of substrate activities and inhibition potencies of

3-amino-propionaldehyde and its two analogs reveals some interesting behaviors for those compounds. The general order of substrate activity is

3-amino-1-phenyl-1-propanone > 4-amino-2-butanone >> 3-amino-propionaldehyde; however, in terms of inhibition potency, the opposite rank order is observed:

3-amino-propionaldehyde > 4-amino-2-butanone > 3-amino-1-phenyl-1-propanone. The docking simulations of each compound at the active site of BPAO indicate that the ethyleneamine portion of the three compounds is interacting with Asp 385 and Tyr 383 in a similar manner, while the methyl group in 4-amino-2-butanone and especially the phenyl group in 3-amino-1-phenyl-1-propanone are interacting with the hydrophobic residues (Phe 388 and Phe 393). The significantly improved substrate activity of

3-amino-1-phenyl-1-propanone compared to 3-amino-propionaldehyde can be attributed to the improved binding to BPAO as seen in the docking simulation. In other words, transaminative turnover of 3-amino-1-phenyl-1-propanone by BPAO is facilitated by the catalytic assistance from the hydrophobic residues at the active site. In the case of

3-amino-propionaldehyde, lack of interaction with the hydrophobic region of the enzyme may prevent it from successful metabolism by the enzyme. Inhibition kinetic studies demonstrate that 3-amino-propionaldehyde is a slow binding inhibitor of BPAO. The inhibition follows a mechanism in which a weak complex is rapidly formed, followed by slow isomerization to a tight complex.

3.3 Mechanistic conclusion

In this study, acetic acid-(E)-3-aminopropenyl ester (1) as a new allylamine derivative was synthesized and evaluated as a BPAO inhibitor. Time profile of inhibition on BPAO showed two types of inhibition: temporary reversible inhibition and permanent irreversible inhibition. Acetic acid-(E)-3-aminopropenyl ester (1) can be metabolized by

BPAO, which was confirmed by identification of its corresponding aldehyde product

(3-(acetyloxy)-(E)-2-propenal) and secondary product (MA). The substrate activity of acetic acid-(E)-3-aminopropenyl ester (1) versus BPAO explains the temporary reversible

inhibition of BPAO upon incubation with this compound. The similar behavior was

previously observed for (S)-ethyl 4-amino-4,5-dihydrothiophene-2-carboxylate as a

temporary inhibitor and a substrate of BPAO.14

In terms of transaminative oxidation mechanism, the C3 of acetic

acid-(E)-3-aminopropenyl ester (1) would become an electrophilic reactive site at the

product Schiff base stage of turnover, or immediately following hydrolytic generation of

the corresponding aldehyde, 3-(acetyloxy)-(E)-2-propenal (Scheme 3.4). It is tempting to consider the addition-elimination mechanism for the permanent inactivation of BPAO by acetic acid-(E)-3-aminopropenyl ester (1) (See path A and B in the Scheme 3.4), as proposed in the cases of the simple 3-chloroallylamine and 2-phenyl-3-haloallylamines.4,

5 The eventual partial recovery of activity seen upon dialysis seems to be consistent with

such a mechanism, since the adducts shown could eventually hydrolyze (e.g., by a second

addition–elimination reaction).

However, the product Schiff base between TPQ and acetic acid-(E)-3-aminopropenyl

ester (1) would hydrolyze at the position of the ester bond, as hydrolysis of

3-(acetyloxy)-(E)-2-propenal as observed. Therefore we proposed an alternative inactivation mechanism which involves hydrolysis of product Schiff base at the ester bond followed by tautomerizaiton of TPQ adduct I to II (see path C in Scheme 3.4). Such

a mechanism would be also consistent with the eventual partial recovery of enzyme activity seen upon dialysis of acetic acid-(E)-3-aminopropenyl ester (1) inactivated

BPAO, since TPQ adduct I could hydrolyze slowly to release the reductively aminated

TPQ and then the equilibrium would favor the conversion of TPQ adduct II to I. In view of this possible inactivation mechanism, we evaluated the interaction between BPAO and

3-amino-propionaldehyde, since this amine compound could react with TPQ to form the same TPQ adducts (I and II). 3-Amino-propionaldehyde was demonstrated as a slow binding inhibitor of BPAO, which follows a mechanism in which a weak complex is rapidly formed, followed by slow isomerization to a tight complex. The comparative studies of 3-amino-propionaldehyde and its two analogs in terms of substrate activity and inhibition potency (versus BPAO) revealed that BPAO is incapable of efficient metabolism of 3-amino-propionaldehyde due to the fact this simple compound lacks interaction with the hydrophobic binding region of the enzyme.

3-Amino-propionaldehyde could react with TPQ to form some intermediates as a normal substrate. However, the reaction would be very slow without leading to any detectable product at our measured time period. Those intermediates formed between

3-amino-propionaldehyde and TPQ would build up gradually, which could account for the observed slow time-dependent inhibition of BPAO by 3-amino-propionaldehyde.

OH O

NH2

[O] O O O

H2O

O OH OH O OH O O O NH + 2 HO + HO H O H3 O HO O N N N HN O C CH3CO2H

O HO O O O O I II

Nu H2O

OH O A B

HO O N O

Nu Nu

Scheme 3.4 Proposed mechanism for inactivation of BPAO by acetic acid-(E)-3-aminopropenyl ester (1)

Additional work will be needed to clarify which kind of enzyme modification is responsible for inactivation of BPAO by acetic acid-(E)-3-aminopropenyl ester (1).

Determination of X-ray cystal structure of the inhibitor in complex with enzyme BPAO is the optimal tool, which will not only unambiguously reveal the inactivation mode but also provide the details about structural basis of enzyme inactivation.

3.4 Experimental procedures

General procedures

1H-NMR spectra were obtained on a Varian Gemini 200 MHz (13C-NMR at 50 MHz)

instrument, with chemical shifts being referenced to TMS or the solvent peak. UV-vis

spectra were recorded on a Perkin-Elmer Lambda 20 UV-vis spectrophotometer. All

solvents, reagents, and organic fine chemicals were the purest available from commercial

sources. Pure BPAO sample was provided by Dr Dooley’s group. Docking simulation was conducted by the Accelrys Discovery Studio software package (PC version 1.7). MA

in the incubation solution of BPAO with (E)-acetic acid-3-amino-propenyl ester (1) were detected by HPLC analysis (Hewlett-Packard 1050 system) using a 5 µM 4.6×250 mm reversed-phase C18 column (Agilent ZORBAX SB-C18), a flow rate of 1 mL/min, and a gradient mobile phase composed of HPLC-grade solvents A [5% aqueous CH3CN containing 0.02% (v/v) TFA] and B [95% aqueous CH3CN containing 0.02% (v/v) TFA] according to the following program: 0% B to 80% B 0-20 min, 80% B to 80% B 20-25 min, 80% B to 0% B 25-30 min.

Synthesis

(E)-acetic acid-3-amino-propenyl ester (1) and (Z)-acetic acid-3-amino-propenyl ester (2)

To a suspension of pyridinium chlorochromate (15 g, 69.5 mmol) and celite (10 g) in anhydrous dichloromethane (100 mL) was added a solution of N-Boc-3-amino-1- propanol (8.8 g, 50.3 mmol) in anhydrous dichloromethane (20 mL). The mixture was

stirred at room temperature until TLC showed the staring material alcohol disappeared

completely. The mixture was diluted with ethyl ether (150 mL) and filtered. The filter

cake was washed with ethyl ether (100 mL × 2). The filtrate was combined and

evaporated under vacuum. The residue was subjected to the flash column chromatograph

1 to give pure N-Boc-3-aminopropanal. Yield: 80%; H-NMR (CDCl3): δ 9.82 (1H, s), 3.42

(2H, m), 2.72 (2H, t, J = 7.6Hz), 1.44 (9H, s)

N-Boc-3-aminopropanal (6 g, 34.7 mmol) was stirred with DMAP (1.27 g, 10.41 mmol), triethylamine (7 g, 34.7 ×2 mmol) and acetyl anhydride (14 g, 34.7 ×4 mmol)

at about 50℃ for 24 h under argon. After reaction, 150 mL dichloromethane was added

to the reaction mixture. Dichloromethane layer was wished with saturated sodium

bicarbonate aqueous solution and water, dried with anhydrous sodium sulfate, and then

evaporated under vacuum. The residue was subjected to column chromatograph to afford

2.3 g oil, which contained both trans and cis isomer with ratio 3:2. Yield: 31%. 1H-NMR

(CDCl3): Trans isomer δ 7.23 (1H, dt, J = 12.5, 1.4), 5.46 (1H, dt, J = 12.5, 6.96), 4.55

(1H, br), 3.73 (2H, t, J = 6), 2.13(3H, s), 1.45(9H, s); Cis isomer δ 7.10 (1H, dt, J = 6.46,

1.4), 4.96 (1H, q, J = 6.74), 4.55 (1H, br), 3.88 (2H, t, J = 6.2), 2.16 (3H, s), 1.45 (9H, s)

The 40 mL ethyl ether solution of Boc-protected acetic acid-3-amino-propenyl ester

(0.5 g,) was bubbled with dry HCl gas until TLC shows the starting material disappeared.

To the reaction mixture was added more ethyl ether and more solid precipitated. Ethyl ether was evaporated under vacuum to give yellow solid. HCl residue contaminated in the solid was removed by adding and evaporating ethyl ether several times. Step

crystallization of the trans and cis mixture in ethyl alcohol: ethyl acetate afforded pure

1 trans and cis isomer respectively. Trans isomer H-NMR (CD3OD): δ 7.525 (1H, d, J =

13 12.4), 5.53 (1H, dt, J = 7.6, 12.4), 3.56 (2H, d, J = 7.6), 2.14 (3H, s). C-NMR (CD3OD):

δ 169.01 (+), 142.72 (-), 106.80 (-), 38.46 (+), 20.36(-); HRFABMS MH+ m/z obsd

1 116.07229, C5H10NO2 required 116.07115; Cis isomer H-NMR (CD3OD): δ 7.37 (1H, dt,

J = 6.5, 1.4), 5.075 (1H, td, J = 6.5, 7.3), 3.70 (2H, d, J = 7.3), 2.20 (3H, s). 13C-NMR

+ (CD3OD): δ 168.59 (+), 140.06 (-), 105.1 (-), 35.04 (+), 20.43 (-); HRFABMS MH m/z

obsd 116.07218, C5H10NO2 required 116.07115

3-(Acetyloxy)-(E)-2-propenal

20 g AG 50W-X8 (hydrogen form) resin was added to the suspension of

1,1,3,3-tetraethoxypropane (5.5 g, 25 mmol) in 100 mL water. The mixture was stirred at

room temperature for about 1 h and TLC showed the starting material disappeared

completely. The resin was filtered and the pH value of the filtrate was adjusted to 7 by

addition of aqueous NaOH solution. Finally, concentration of the solution to a small

volume and precipitation with acetone afforded crude sodium salts of malondialdehyde 2

1 g, which is used for the next step without purification. H-NMR (D2O): δ 8.48 (d, J = 10),

5.14 (t, J = 10)

Acetyl chloride (1.7 g, 22 mmol) was added dropwise at 0℃to a suspension of

sodium salts of malondialdehyde (1.9 g, 20 mmol) in dichloromethane with stirring. After

addition was complete, the ice bath was removed. The mixture was stirred at room

temperature for 3 h. Dichloromethane was evaporated and residue was subjected to flash

column chromatograph to afford oil product. Part of the product was hydrolyzed on the

1 column. H-NMR (CDCl3): δ 9.58 (1H, d, J = 8.4), 8.21 (1H, d, J = 12.8), 6.0(1H, dd, J

13 = 8.4, 12.8), 2.29 (3H, s). C-NMR (CDCl3): δ 191.608, 166.687, 156.694, 116.813,

20.81

Sodium salt of benzoylacetaldehyde

Sodium (1.38 g, 60 mmol) was added to 10 mL methanol with stirring. After the

reaction was complete, 70 mL ethyl ether and ethyl formate (6.1 g, 82.4 mmol) was added slowly. After addition, acetophenone (8.9 g, 74 mmol) was added drop by drop.

The solution was stirred at room temperature until lots of white solid was formed. The formed sodium salt of benzoylacetaldehyde was filtered, washed with ethyl ether, and dried under vacuum. 1H-NMR (DMSO): δ 9.29 (br) (S trans conformation) , 5.376 (br) (S

trans conformation) , 8.448(d, J = 3.9) (U conformation), 5.454 (d, J = 3.9) (U

conformation) , 7.761, 7.662, 7.316, 7.287. 1H-NMR assignment was based on the reference.15

UV-vis spectra of benzoylacetaldehyde of different concentrations in 100 mM

phosphate buffer, pH 7.2, were recorded and λmax was determined to be 318 nm. The plot

of absorbance at 318 nm vs concentration was constructed and fitted to eq. (3.1) to determine the molar absorption coefficientε .

A= ε lc (3.1)

where l is the optical length, A the absorbance at the specific wavelength, c the

concentration of the measured compound.

3-Amino-propionaldehyde

To 0.1 M HCl solution (5.714 mL) was added 1-amino-3, 3’-ethoxylpropane (8.4 mg,

0.057 mmol), then sealed and heated with boiling water for several minutes. The solution

was diluted to the different concentration and used for enzyme assay directly.

4-Amino-2-butanone

To the 40 mL 6 N HCl solution in a high pressure bottle was added

homopropargylamine hydrochloride (0.25 g, 2.4 mmol) and mercuric acetate (0.76 g, 2.4

mmol). The mixture was heated at 100℃with stirring for overnight. The solvent was

evaporated and 1H-NMR of crude product showed that there were two major product

4-amino-2-butanone hydrochloride and 3-Chloro-but-3-enylamine. To the crude product

was added water and THF, sodium carbonate was added to adjust pH value to 9-10, and

then di-t-butyl dicarbonate (0.6 g, 3 mmol) was added. The mixture was stirred for 1 h.

THF was evaporated under vaccum and ethyl ether was added to extract water layer.

Combined ethyl ether was dried with anhydrous sodium sulfate and ethyl ether was evaporated. The residue was subjected to column chromatography to afford Boc protected 4-amino-2-butanone. To the Boc protected 4-amino-2-butanone was added 3 N

HCl ethyl alcohol solution. The solution was stirred for half an hour and evaporated to

1 afford oil. H-NMR (CD3OD): δ 3.145 (2H, t, J = 6), 2.928 (2H, t, J = 6), 2.201 (3H, s)

3-Amino-1-phenyl-1-propanone

The 3-chloropropiophenone (0.5 g) was dissolved in DCM and shaken with 1N

NaOH aqueous solution. The aqueous layer was washed twice with DCM and the

combined organic extracts were dried with anhydrous magnesium sulfate, filtered and

evaporated. The residue was mixed with 0.6 g potassium phthalimide in anhydrous DMF

(5 mL). The solution was heated at 100 ℃ for 2 h under argon protection. After the

solution was cool, 50 mL water was added. The precipitate was filtered and washed with

water. The solid was crystallized in ethyl alcohol and finally 0.34 g pure colorless product

1 was obtained. H-NMR (CDCl3): δ 7.947 (m), 7.85 (m), 7.720 (m), 7.565 (m), 7.454 (m),

4.15 (dd, J = 7.6, 6.4), 3.43 (dd, J = 7.6, 6.4)

0.34 g β-phthalylpropiophenone was added to the mixture of 8 mL acetic acid and 8

mL concentrated hydrochloride acid. The suspension was heated at 100℃ with stirring

for abut 16 h. Then the mixture was evaporated to a small volume (not to dryness). The

10 mL water was added and phthalic acid and unreacted β-phthalylpropiophenone were filtered. The filtrate was then allowed to evaporate to dryness. The solid was crystallized

1 from ethanol and ethyl acetate. H-NMR (D2O): δ 8.01 (2H, m), 7.71 (2H, m), 7.57 (1H,

13 m), 3.55 (2H, t, J = 6), 3.42 (2H, t, J = 6); C-NMR (CD3OD): δ 197.6, 136.17, 133.78,

128.74, 128.03, 110.00, 35.26, 34.75

Standard benzylamine assay

Time-dependent inactivation of BPAO by candidate inhibitors was followed by the standard benzylamine assay. A 0.9 mL aliquot of a solution of candidate inhibitor in 100 mM potassium phosphate buffer, pH 7.2, was mixed with 0.1 mL enzyme stock solution

(6-8 µM BPAO) and incubated at 30℃ aerobically. Aliquots (0.1 mL) were periodically withdrawn using disposable calibrated Drummond micropipettes and diluted with 1.0 mL of benzylamine (5 mM in 50 mM sodium phosphate buffer, pH 7.2) in a 1 cm quartz cuvette (1.5 mL volume). The rate of oxidation of benzylamine to benzylaldehyde was measured by recording the increase in absorbance at 250 nm for 40 seconds. All percent activities refer to ratios of measured benzaldehyde formation slopes to those obtained on a control incubation lacking inhibitor.

Determination of the dissociation constants of the BPAO complex with 3-amino- propionaldehyde

Initial rates for four concentrations of benzylamine at each of four concentrations of

3-amino-propionaldehyde were measured in order to determine the inhibition mechanism.

3-amino-propionaldehyde was treated as a slow binding inhibitor. Characterization of the inhibition mechanism employed the initial rates measured before the onset of slow binding inhibition. The Lineweaver-Burke plot was constructed from those data and then fitted to eq. (3.2), which describes competitive inhibition.

[I ] 11Km (1+ K ) 1 = + i . (3.2) v Vmax V max [] BA

Here, v is the initial rate, Vmax the maximal rate, Km the Michaelis constant, I the inhibitor,

and Ki the dissociation constant. The dissociation constant was determined by

constructing the corresponding Dixon plot. Data for each substrate concentration fall on

straight lines that intersect at [I] = -Ki and 1/v = 1/Vmax.

* The overall dissociation constant, Ki , for 3-amino-propionaldehyde was measured by preincubation of BPAO in a 0.1 mL solution containing 100 mM sodium phosphate buffer and various concentrations of 3-amino-propionaldehyde for 30 min. This preincubated solution was then mixed with 1 mL of 5 mM benzylamine, and production of benzaldehyde at 250 nm was monitored. The preliminary standard benzylamine assay showed that the 30 min preincubation time was sufficient for full equilibration of inhibitor binding; thus the measured rates reflect the slow binding inhibition of BPAO.

* The value of Ki was determined by fitting initial rates to eq. (3.3).

V[ BA] 1+α []IK = max i v * (3.3) []I 1+α []IKi Km 1++[BA] Ki

* where Ki is the slow binding dissociation constant and α is the ratio of the final reaction

volume to the volume of the preincubation mixture.

Monitoring of metabolism of 3-amino-1-phenyl-1-propanone by BPAO by UV-vis

spectrophotometer

1.48 µM BPAO was incubated with 100 µM 3-amino-1-phenyl-1-propanone in pH

7.2 phosphate buffer at 30℃, absorption at 318 nm was recorded over time until a constant value was reached after around 30 min. The increase of absorbance at 318 nm

4 -1 corresponds to the production of benzoylacetaldehyde (λmax = 318 nm, ε = 1.08×10 M

cm-1). Based on it the remaining amount of 3-amino-1-phenyl-1-propanone in the incubation solution can be determined, since a clean isosbestic conversion of

3-amino-1-phenyl-1-propanone to benzoylacetaldehyde was observed

spectrophotometrically.

Docking simulation

The crystal structure of native BPAO (1TU5) was obtained from PDB bank (www. pdb.org) and loaded on the Discovery Studio software. Bond orders and charges of the

TPQ cofactor were corrected manually, since the program can not add hydrogens and

charges correctly to this post-translationally modified residue. All the water molecules were removed. The active site was then identified, and the corresponding energy grid was set automatically by the program. The program-defined energy grid was too diffuse, so manual adjustment was needed in order to generate the productive docking modes. In our cases, only those binding modes where the ammonium group of the molecule was sufficiently close to the TPQ cofactor and/or Asp385 were considered productive. All the molecules (ligand structures) were drawn by ChemDraw Ultra 10.0 (Cambridge Soft

Corp., US) and then energy-optimized by MM2 and saved in MOL format and then

84 loaded on the Discovery Studio software. The “energy grid forcefield” was set as

“Drieding”. The “conformation search include electrostatic energy” was allowed, the

“conformation search number of Monte Carlo trials” was set as 30000, and the “pose saving perform clustering” was allowed, with “pose saving maximum cluster per molecule” being set as 100. Each output pose represents the energy minimum of a cluster of poses and is thus considered as a docking mode. The “scoring scores” option was set as LigScore2. Other parameters were set as default. After calculation, the output poses were ranked according to the Lig-Score2 scores, visualized and analyzed.

3.5 References 1. Hevey, R. C.; Babson, J.; Maycock, A. L.; Abeles, R. H., Highly specific enzyme inhibitors. Inhibition of plasma amine oxidase. J Am Chem Soc 1973, 95, (18), 6125-7. 2. Qiao, C.; Jeon, H.-B.; Sayre, L. M., Selective Inhibition of Bovine Plasma Amine Oxidase by Homopropargylamine, a New Inactivator Motif. J. Am. Chem. Soc. 2004, 126, (25), 8038-8045. 3. Lee, Y.; Ling, K.-Q.; Lu, X.; Silverman, R. B.; Shepard, E. M.; Dooley, D. M.; Sayre, L. M., 3-Pyrrolines Are Mechanism-Based Inactivators of the Quinone-Dependent Amine Oxidases but Only Substrates of the Flavin-Dependent Amine Oxidases. J. Am. Chem. Soc. 2002, 124, (41), 12135-12143. 4. Kim, J.; Zhang, Y.; Ran, C.; Sayre, L. M., Inactivation of bovine plasma amine oxidase by haloallylamines. Bioorg. Med. Chem. 2006, 14, (5), 1444-1453. 5. Jeon, H.-B.; Sayre, L. M., Highly potent propargylamine and allylamine inhibitors of bovine plasma amine oxidase. Biochem. Biophys. Res. Commun. 2003, 304, (4), 788-794. 6. McDonald, I. A.; Lacoste, J. M.; Bey, P.; Palfreyman, M. G.; Zreika, M., Enzyme-activated irreversible inhibitors of monoamine oxidase: phenylallylamine structure-activity relationships. J. Med. Chem. 1985, 28, (2), 186-93. 7. Cousineau, T. J.; Cook, S. L.; Secrist, J. A., III, A convenient procedure for the formation of enol acetates under basic conditions. Synth. Commun. 1979, 9, (3), 157-63. 8. Protopopova, T. V.; Skoldinov, A. P., beta -Acyloxyacroleins. Zh. Obshch. Khim. FIELD 1958, 28, 240-3. 9. Kwon, T.-W.; Van der Veen, J., Ultraviolet and infrared absorption spectra of

malonaldehyde in organic solvents. J. Agr. Food Chem. 1968, 16, (4), 639-42. 10. Kawai, S.; Kasashima, K.; Tomita, M., High-performance liquid chromatographic determination of malonaldehyde in serum. J. Chromatogr., Biomed. Appl. 1989, 495, 235-8. 11. Awal, H. M. A.; Hirasawa, E., 1,3-Diaminopropane is a suicide substrate for pea diamine oxidase. Phytochemistry 1995, 39, (3), 489-90. 12. Liu, W.; Rogers, C. J.; Fisher, A. J.; Toney, M. D., Aminophosphonate Inhibitors of Dialkylglycine Decarboxylase: Structural Basis for Slow Binding Inhibition. Biochemistry 2002, 41, (41), 12320-12328. 13. Martinez, A. M.; Cushmac, G. E.; Rocek, J., Chromic acid oxidation of cyclopropanols. J. Am. Chem. Soc. 1975, 97, (22), 6502-10. 14. Qiao, C.; Ling, K.-Q.; Shepard, E. M.; Dooley, D. M.; Sayre, L. M., Mechanism-Based Cofactor Derivatization of a Copper Amine Oxidase by a Branched Primary Amine Recruits the Oxidase Activity of the Enzyme to Turn Inactivator into Substrate. J. Am. Chem. Soc. 2006, 128, (18), 6206-6219. 15. Esakov, S. M.; Petrov, A. A.; Ershov, B. A., Mesomeric anions. IV. Stereochemistry of enolate anions of beta -oxoaldehydes studied by a PMR spectroscopic method. Zh. Org. Khim. 1975, 11, (4), 680-91.

Chapter 4 Kinetic analysis of propargylamine derivatives as both substrates and inhibitors for bovine plasma amine oxidase and arthrobacter globiformis amine oxidase

4.1 Introduction Based on Abeles’s pioneering work on propargylamine,1 amines containing an

unsaturated bond at the β, γ-position have been developed and evaluated as mechanism-based inhibitors for various CAOs.2-4 The 4-aryloxy-2-butynamine series exhibit selective inhibition against six different CAOs, which include equine plasma amine oxidase (EPAO), Pisum sativum amine oxidase (PSAO), Pichia pastoris lysyl oxidase (PPLO), bovine plasma amine oxidase (BPAO), human kidney diamine oxidase

(HKDAO), and Arthrobacter globiformis amine oxidase (AGAO)5. The observed

selectivity could be attributed to the differences in the electrostatic properties and dimensions of the enzyme active sites and/ or mechanisms of enzyme inactivation. To obtain insights into the structural basis of enzyme inactivation, the crystal structures of

AGAO incubated with 4-(4-methylphenoxy)-2-butynamine (MPBA) and

4-(2-naphthyloxy)-2-butynamine (NBA) were determined. The crystal structures reveal a covalent adduct formed between the oxidized amine product,

4-(4-methylphenoxy)-2-butynal or 4-(2-naphthyloxy)-2-butynal with TPQamr, implicating

that the reduced TPQ is trapped by the product aldehyde, thereby rendering enzyme

inactivation (Scheme 4.1).5

O O OH B: RCH2NH2

O O O BH NH NH O TPQ SSB PSB CHR CH2R

H2O R: OH OAr OH

HO HO NH2 NH O O

ArO ArO

Scheme 4.1 Proposed mechanism for inactivation of AGAO by propargylamine

derivative

Many catalytic mechanism studies of BPAO and inhibitor developments based on

BPAO have been reported .6-9 BPAO shares an 82% sequence homology with human

vascular adhesion protein-1 (HVAP-1) and is considered a valid model system for human

SSAO/HVAP-1. At present, high resolution structures of native BPAO and BPAO complexed with clonidine have been reported.10, 11 By comparison, AGAO shares 20%

sequence identity with BPAO and complexes of AGAO bound to propargylamine-based

inhibitors have been previously solved.5 Structural analysis of AGAO and BPAO showed

that both enzymes share similarities in the active site and the substrate entry channel.10, 12

For example, in both enzymes, the copper ion is coordinated by three conserved histidines. The cofactor TPQ in BPAO is positioned in a productive conformation where the reactive carbonyl group at C5 in TPQ is exposed to the substrate-binding site and the

active site base. AGAO also has a productive conformation, but a nonproductive

conformation also exists. The two conformations differ by a 180º rotation of the TPQ ring in the enzyme active site.12 Moreover, both BPAO and AGAO contain a hydrophobic

channel leading from the active site to the enzyme surface, which is important for

substrate recognition and inhibitor binding. In the BPAO structure, the channel is defined mainly by the presence of Y383, Y472, L468, M467, F393, F388 and Y175. Residue

D385 and TPQ lie at the bottom of the channel, forming the most negative site of the cavity. The internal surface of the channel in AGAO is also characterized by hydrophobic residues (Y 302, Y 307, F 105, P136, I379, W168 and W359).

As the active site and substrate entry channel of both BPAO and AGAO share structural similarities, it is conceivable that enzyme inhibition by 4-aryloxy-2- butynamines adopt the same general mechanism depicted in Scheme 4.1. Comparing the inhibition profiles of MPBA and 4-phenoxy-2-butynamine (PBA) towards BPAO and

AGAO further revealed that these two inhibitors displayed similar potency towards

AGAO, but a 10-fold difference in potency towards BPAO, suggesting inhibition selectivity is attained in the latter.4, 5 Therefore understanding the molecular basis governing the observed selectivity will provide insights for further design and optimization of selective inhibitors.

According to Scheme 4.1, enzyme inactivation occurs after C-H bond cleavage, which is the same step involved in the regular enzyme turnover. Thereafter, the aldehyde product inactivates the amine oxidase by derivatizing TPQ. Alternatively, the enzyme can be regenerated by a competing pathway, where the reduced TPQ is reoxidized before the product aldehyde can react with the cofactor. To evaluate the extent to which each of the aforementioned steps contributes to inhibition potency and selectivity, we determined the

steady-state kinetic parameters for BPAO- and AGAO-catalyzed oxidation of MPBA, [1,

1-2H] MPBA, PBA, and [1, 1-2H] PBA (Scheme 4.2). Our kinetic data suggest that

inactivation of BPAO and AGAO occurs after the hydrolysis of product Schiff base

(PSB), and the resulting α, β-unsaturated aldehyde product is responsible for enzyme inactivation. Moreover the observation that TPQamr acted as an effective nucleophile in the formation of a covalent adduct in AGAO led us to investigate whether BPAO inactivation by propargylamine derivatives involved alkylation of TPQamr rather than

active site residues by the α, β-unsaturated aldehyde products. We observed that α,

β-unsaturated aldehyde products corresponding to MPBA and PBA induced faster

inactivation of the substrate-reduced BPAO than the native BPAO, suggesting that BPAO

inactivation is mostly associated with the reaction between TPQamr and aldehyde products.

Furthermore, MPBA and PBA induced more potent inactivation of BPAO under anaerobic incubation condition than aerobic condition. The less inactivation potency of both compounds under aerobic condition implicates that TPQamr reoxidaiton is

competitive to the inhibition reaction. Collectively, the mechanism of inactivation of

BPAO by this class of inhibitors can be rationalized in terms of transaminative turnover

of those compounds to the corresponding α, β-unsaturated aldehyde products, the latter

are capable of covalent alkylation of TPQamr before they release from the active site.

(D)H H(D) O (D)H H(D) O

H2N H2N

2 2 MPBA; [1, 1 - H] MPBA PBA; [1, 1- H] PBA Scheme 4.2 Structures of MPBA, [1, 1-2H] MPBA, PBA, and [1, 1-2H] PBA

4.2 Results and discussion

4.2.1 Steady-state kinetic parameters for the AGAO/BPAO-catalyzed oxidation of

PBA, [1, 1-2H] PBA, MPBA, and [1, 1-2H] MPBA

The x-ray crystal structure of enzyme AGAO in complex with the inhibitor MPBA reveals the presence of a covalent adduct formed between TPQamr and the corresponding aldehyde product generated from the oxidation of MPBA.5 As a mechanism-based inhibitor of AGAO, MPBA displays a partitioning between normal turnover and enzyme inactivation. When AGAO catalyzes the conversion of MPBA to the corresponding aldehyde product, there are two processes that can follow. The aldehyde product can be released from the active site (normal substrate metabolism) or it can react with the reduced TPQ to form an adduct (enzyme inactivation). Presumably, this kind of inactivation mode is dependent on the relative rates of the different steps occurring in the enzyme-catalyzed reaction, which includes the release of product aldehyde and enzyme reoxidation. In this study, the deuterium isotope effects on kcat/Km and kcat were measured and compared to the intrinsic isotope effect on the rate constant for C-H bond cleavage to evaluate the relative rates of different reaction steps along the enzymatic reaction pathway.

Steady-state kinetic parameters for the oxidation of PBA and MPBA and the corresponding deuterium-labeled compounds by AGAO are summarized in Table 4.1.

D Inverse deuterium isotope effects kcat of 0.8 were obtained for both compounds. These values may reflect a combination of an insignificant primary isotope effect and an inverse

secondary isotope effect. It is plausible that the inverse secondary effect occurs when the

coordination at the reaction center increases from sp2 to sp3 (NH=CD→NH-CDOH) at the transition state of the product Schiff base hydrolysis step. The negligible kinetic isotope effects on kcat indicate that C-H bond cleavage does not constitute the

rate-limiting step. This observation is consistent with the previous report that the reaction

of AGAO with 2-phenylethylamine and tyramine is not rate-limited by C-H bond cleavage 13. As noted in Northrop’s studies on deuterium isotope effects in minimal

D kinetic mechanism by simulation, the magnitude of Vmax is considerably more sensitive

to changes in the rate of the postcatalytic than the precatalytic steps 14, 15. Therefore the

minor deuterium isotope effects on kcat may suggest the reaction steps occurring after C-H

bond cleavage dominate the overall reaction rate. Moreover, the copper-dependent oxidation of TPQamr may be slow, so TPQamr can build up and react with the aldehdye product generated from the oxidation of MPBA by AGAO.

Similarly, insignificant deuterium isotope effect on kcat/Km and kcat were observed for

the BPAO-catalyzed oxidation of PBA and MPBA (Table 4.1). The deuterium isotope

effects on kcat/Km are 3.9 and 1.9 for MPBA and PBA respectively. These values are much

lower than the one measured for the benzylamine substrate, which is 14.9 7. In BPAO,

C-H bond cleavage is the rate-limiting step for catalytical oxidation of benzylamine.

However, for BPAO-catalyzed oxidation of MPBA and PBA, the later steps (possibly the

hydrolysis of the product Schiff base or enzyme reoxidation) subsequent to C-H bond

cleavage likely contribute to the rate-limiting step.

Table 4.1 Kinetic parameters for the oxidation of PBA, [1, 1-2H] PBA, MPBA, and [1,

1-2H] MPBA, by AGAO and BPAO

kcat (H) Km (H) kcat (D) Km (D) D D Enzyme Compound kcat kcat/Km (min-1) (µM) (min-1) (µM) PBA / 30.5 ± 0.7 0.3 ± 0.1 38.4 ± 1.4 1.2 ± 0.2 0.8 3.2 [1, 1-2H] PBA AGAOa MPBA / 36.3 ± 1.2 0.7 ± 0.2 45.8 ± 1.9 2.0 ± 0.4 0.8 2.3 [1, 1-2H] MPBA PBA / 86.7 ± 3.3 2.6 ± 0.3 82.2 ± 8.2 4.7 ± 1.3 1.1 1.9 [1, 1-2H] PBA BPAOa MPBA / 33.3 ± 2.3 2.5 ± 0.6 15.7 ± 0.3 4.6 ± 0.3 2.1 3.9 [1, 1-2H] MPBA

a 0.2 µM AGAO was used for the kinetic analysis of PBA and [1, 1-2H] PBA , 0.4 µM

AGAO for MPBA and [1, 1-2H] MPBA, 0.1 µM BPAO for PBA and [1, 1-2H] PBA , 0.6

µM BAPO for MPBA and [1, 1-2H] MPBA.

4.2.2 Kinetic parameters for the inactivation of AGAO and BPAO by PBA, [1, 1-2H]

PBA, MPBA, and [1, 1-2H] MPBA

The inactivation parameters and deuterium isotope effects provided key insights into

the mechanism of inactivation of BPAO by PBA and MPBA. The inhibition potencies of

MPBA and PBA on enzyme BPAO and AGAO were previously evaluated by the standard

benzylamine assay, but the inhibition data did not display the pseudo first order kinetic

pattern, which precluded the determination of KI and kinact by the Kitz and Wilson

analysis.16 In this work, the method of progress curve was used to determine the

inactivation kinetic parameters.17 The classic minimal kinetic mode in

k E I 1 k2 ' kinact EI EI EIinact k-1 kq

E Q

Scheme 4.3 Classic minimal kinetic mode for mechanism-based inhibition

Scheme 4.3 was used to describe the reaction between enzyme and propargylamine

derivatives which entails inhibitor oxidation and subsequent enzyme inactivation. EI

represents the enzyme-inhibitor complex formed by rapid inhibitor binding and EI’

represents another important enzyme-inhibitor complex. Enzyme inactivation branches

off immediately after the formation of EI’. There may be multiple steps leading from EI

’ 18 to EI . In our case, k2 represents the net rate constant of all the catalytic steps for the

conversion of inhibitor to reactive species by enzyme. Generally, BPAO inactivation

(kinact) by propargylamine derivatives is proposed to be associated with product Schiff

bases or aldehyde products.4 If enzyme inactivation occurs subsequent to the step of formation of product Schiff base, the catalytic steps (k2) encompass the step of formation

of substrate Schiff base and the C-H bond cleavage. However, if enzyme inactivation occurs subsequent to the step of hydrolysis of product Schiff base, the catalytic steps (k2)

encompass not only the formation of substrate Schiff base and the C-H bond cleavage but

also the product Schiff base hydrolysis. In both cases, k2 is a deuterium isotope sensitive

kinetic parameter. In the latter case, the magnitude of deuterium isotope effect on k2 is

sensitive to the change in the rate of product Schiff base hydrolysis.

Figure 4.1 (A) demonstrated the representative time courses of production of

benzaldehyde by the BPAO-catalyzed benzylamine oxidation in the presence of various

concentrations of MPBA. Those progress curves were fit individually to eq. (4.2) to

obtain the apparent inactivation constant kobs. The apparent inactivation constant kobs

increases nonlinearly with the increase of inhibitor concentration (Figure 4.1 (B)). Fitting

eff -1 eff the plot of kobs versus [I] to eq. (4.3) yielded kinact() H = 0.93 ± 0.04 min and KIH() =

1.06 ± 0.10 µM. A similar analysis was also performed for the inhibition of BPAO by [1,

1-2H] MPBA, PBA, and [1, 1-2H] PBA as well as the inhibition of AGAO by all the

inhibitor compounds. The inactivation kinetic parameters are summarized in Table 4.2.

(A) 0.16 µ 0.14 1 M 2µM 0.12 3µM 5µM 0.10 8µM 10µM 0.08 20µM

0.06

0.04 Absorbance in 250nm

0.02

0.00

0 1 2 3 4 5 Time/min

(B) 0.7

0.6

0.5

0.4 obs

K 0.3

0.2

0.1

0.0 0 5 10 15 20 [I]/µM

Figure 4.1 (A) Nonlinear least-squares fitting of progressive curves from the inhibition of

BPAO-catalyzed metabolism of benzylamine by inhibitor MPBA to eq. (4.2). The initial

substrate benzylamine concentration was 10 mM. The inhibitor concentrations were (■) 1

µM, (●) 2 µM, (▲) 3 µM, (▼) 5 µM, (◄) 8 µM, (►) 10 µM, (◊) 20 µM); (B) Nonlinear

least-squares fitting of the plot of apparent inactivation constant kobs versus inhibitor concentration to eq. (4.3)

Table 4.2 Kinetic parameters for the inactivation of AGAO and BPAO by PBA, [1, 1-2H]

PBA, MPBA, and [1, 1-2H] MPBA

eff eff eff eff kinact() H KIH() kinact() D KID() D eff D eff eff Enzyme Compound ( kinact ) ( kinact / K I ) (Min-1) (µM) (Min-1) (µM) PBA / 0.55 ± 0.24 ± 0.14 ± 0.34 ± 0.09 1.62 0.94 [1, 1-2H] PBA 0.25 0.15 0.07 AGAO MPBA/ 1.02 ± 0.15 ± 0.88 ± 0.11 ± [1, 1-2H] 1.16 0.85 0.24 0.05 0.25 0.05 MPBA PBA / 0.23 ± 12.6 ± 0.19 ± 8.1 ±1.7 1.21 0.78 [1, 1-2H] PBA 0.01 2.5 0.01 BPAO MPBA/ 0.93 ± 1.06 ± 0.51 ± 2.8 ± [1, 1-2H] 1.82 4.81 0.04 0.1 0.02 0.25 MPBA

eff eff According to the definition of kinact and KI (see the experimental section),

eff eff kKinact I equates to kk2inact k 11 k− () kq+ k inact . Since kinact, kq, k-1, and k1 are not isotope

D eff eff D sensitive, (kKinact I ) is approximate to k2 . Deuterium isotope effects on the inactivation of AGAO by PBA and MPBA were determined to be almost unity (0.94) and

D eff eff eff eff D (0.85) on kKinact I . The magnitude of (kKinact I ) (or k2) indicates that C-H bond

cleavage is not rate-limiting step for the conversion of PBA and MPBA to the

D eff eff D corresponding aldehyde products by AGAO. (kKinact I ) (or k2 ) was found to be

0.78 and 4.81 for the inactivation of BPAO by PBA and MPBA respectively. Compared

with the intrinsic deuterium isotope effect ( Dk =13.5)15 for C-H bond cleavage in BPAO,

the magnitude of observed deuterium isotope effect on k2 is small, especially for PBA.

The catalytic steps (k2) may encompass the step of product Schiff base hydrolysis which

dominates k2. Therefore for both inhibitors PBA and MPBA, BPAO inactivation may occur subsequent to the step of product Schiff base hydrolysis and the corresponding turnover product aldehydes may be responsible for BPAO inactivation.

4.2.3 Inactivation of the substrate-reduced BPAO by α, β-unsaturated aldehydes

The study of deuterium isotope effects on the inactivation of enzyme BPAO by both inhibitors MPBA and PBA suggests that enzyme inactivation occurs subsequent to the hydrolysis of product Schiff bases and the resulting aldehyde products should be responsible for enzyme inactivation. Further experiments were conducted to study the inactivation of enzyme BPAO by the independently synthesized aldehydes.

4-Phenoxy-2-butynal has been previously reported to be a less potent inhibitor for BPAO, which accounts for the much slower inactivation of enzyme after complete metabolism of

PBA4. Effects of 4-(4-methylphenoxy)-2-butynal and 4-phenoxy-2-butynal on both the

native BPAO and the benzylamine-reduced BPAO were measured for comparison. The pseudo-first-order rate constant (k) for inactivation of the benzylamine-reduced BPAO by

-1 4-(4-methylphenoxy)-2-butynal was measured to be 0.92 ± 0.23 min (t1/2 0.75 min),

which is ~31-fold faster than that for inactivation of the native BPAO by the same

aldehyde product (Figure 4.2). Similarly, 4-phenoxy-2-butynal is also a more potent

inhibitor of the reduced BPAO (Figure 4.3). The pseudo-first-order rate constant (k) was

-1 determined to be 0.012 ± 0.001 min (t1/2 57.7 min) for inactivation of the

-1 benzylamine-reduced BPAO and 0.003 ± 0.001 min (t1/2 231 min) for inactivation of the

native BPAO respectively. The similar phenomena were previously reported for indoleacetaldehyde and lentil seedling copper amine oxidase (LSAO)19.

Indoleacetaldehyde did not inhibit the native LSAO but the substrate-reduced LSAO.

Medda et al. interpreted this behavior in terms of initial formation of an unstable neutral

product Schiff base between TPQamr and indoleacetaldehyde followed by rearrangement to a stable enamine-derivative. In our case, the TPQamr as a nucleophile may directly add

to the C3 of α, β-unsaturated aldehyde to form a stable adduct, leading to the fast

inactivation of the reduced BPAO. An alternative interpretation is formation of a neutral

product Schiff base between the reduced TPQ and the aldehyde. However, the formation

of the neutral product Schiff base may not account for the irreversible inactivation of

BPAO by MPBA and PBA, since the reduced TPQ could be released by hydrolysis of the

neutral form of product Schiff base upon dialysis and immediately reoxidized by O2 to

regenerate the oxidative TPQ leading to the recovery of enzyme activity. Additionally,

compared to 4-phenoxy-2-butynal, 4-(4-methylphenoxy)-2-butynal is much more potent in inactivation of the reduced BPAO, which may account for the different inhibition

potencies of the corresponding inhibitors.

100

20 The remainingThe activity enzyme

0 50 100 Time/min

Figure 4.2 Time-dependent inhibition of the benzylamine-reduced BPAO (0.78 µM) by

4-(4-methylphenoxy)-2-butynal (80 µM) under anaerobic condition (□) and inhibition of the native BPAO by the same amount of the aldehyde under the same condition (■)

110

100

60 The remainingThe activity enzyme

0 10 20 30 40 50 60 Time/min

Figure 4.3 Time-dependent inhibition of the benzylamine-reduced BPAO (0.74 µΜ) by

4-phenoxy-2-butynal (80 µM) under anaerobic condition (□) and inhibition of the native

BPAO by the same amount of the aldehyde under the same condition (■)

101

4.2.4 Anaerobic inactivation of BPAO by propargylamine derivatives

The product aldehydes can induce more potent inactivation on the substrate-reduced

BPAO than the native BPAO, suggesting that inactivation of BPAO by MPBA and PBA is

due to the reaction between the reduced TPQ and aldehyde products. Under aerobic

incubation condition, the reduced TPQ is either oxidized by O2 to regenerate the

oxidative TPQ or trapped by product aldehydes. If O2 is totally abolished, more potent

inhibition on BPAO would be expected for both inhibitors. Therefore, the evaluation of

BPAO inactivation by MPBA and PBA was conducted under anaerobic conditions to

address this hypothesis

100 100 80

60 80 40

20 60 0 Remaining enzyme activityRemaining enzyme at plateau (%)

0.0 0.5 1.0 1.5 2.0 2.5 40 [I]/[E]

20 The remainingThe activity enzyme

0 O2 0 20 40 60 Time/min

Figure 4.4 Time course of inactivation of BPAO (0.75 µM) by MPBA of various

concentrations, (■) 2 µM, under anaerobic condition for 60 min, (○) 20 µM, under

anaerobic condition for 3 min, then exposure to O2 (arrow), and partition ratio plot

(inset).

102

20 µM MPBA induced more than 90% inactivation of BPAO in 3 min and exposure

of the inactivated enzyme solution to air did not lead to enzyme activity recovery. Even 2

µM MPBA can induce the loss of enzyme activity up to 90% in 5 min (Figure 4.4).

Inactivation of BPAO was tested at the even lower concentrations of MPBA under anaerobic conditions (data not shown). The partition ratio was estimated to be 1~2, which is much smaller than the value (18) obtained under aerobic conditions4. So MPBA is a

much more potent inhibitor under anaerobic conditions than aerobic conditions. Similarly,

more potent inactivation of BPAO by PBA was also observed under anaerobic conditions

(data not shown). O2 may help protect enzyme against inhibition by stimulating turnover

by some mechanism under aerobic conditions. Under anaerobic conditions, the oxidative

half reaction is completely abolished and the reduced TPQ can build up (Scheme 4.4).

The product aldehydes generated at the enzyme active site could reorient and react with

the reduced TPQ to induce enzyme inactivation. Additionally, the complete TPQ redox

competence showed by nitroblue tetrazolium redox cycling assay of MPBA-inactivated

BPAO 4 indicates this kind of modification is reversed upon enzyme denaturation and

then the cofactor regains the redox activity.

103

[O]

O O OH OH OH

H2O NH3 O O O HO HO NH O NH NH2 O NH O

R R

R R R=phenoxy, p-methyphenoxy R

Scheme 4.4 Proposed mechanism for inactivation of BPAO by propargylamine

derivatives

4.3 Conclusions

Propargylamine derivatives MPBA and PBA are both inhibitors and substrates of

BPAO and AGAO. According to the well-known transamination mechanism, C-H bond cleavage is involved in the enzyme-catalyzed metabolism of both compounds. A study of deuterium isotope effects on the rates of normal turnover and enzyme inactivation helps

to establish where enzyme inactivation branches away from the normal metabolism

process. Insignificant deuterium isotope effects on substrate activity indicate that the

oxidation of PBA and MPBA by BPAO and AGAO is not rate-limited by C-H bond

D eff eff cleavage. Magnitude of deuterium isotope effects (kKInact I ) suggests that inactivation

of BPAO by MPBA and PBA occurs after the product Schiff base hydrolysis and the

resulting product aldehydes should be associated with enzyme inactivation. Additionally,

the reduced TPQ has been proved to be a prerequisite to the efficient enzyme inactivation.

104

Based on all of that information, the main inactivation pathway for BPAO by

propargylamine derivatives is proposed to be through the reaction between the reduced

TPQ and the corresponding aldehyde products. This work may provide insights for synthesis of more efficient mechanism-based inhibitors for copper amine oxidases.

4.4 Experimental procedures

General procedures

1H-NMR spectra were obtained on Varian 200 or 400 MHz spectrometers (13C-NMR

at 50 or 100 MHz), with the chemical shifts being referenced to TMS or the solvent peak.

Thin layer chromatograph was run on 0.25 mm silicon gel plate with fluorescent indicator at 254 nm. O2 uptake was monitored using a Yellow Springs Instruments 5300 biological

oxygen meter. Benzylamine assays were conducted on a Perkin-Elmer Lambda 25

UV-Vis spectrophotometer using UV Winlab software V 2.85, with constant temperature

being maintained by peltier thermstatting. Pure enzyme BPAO and AGAO were provided

by Dr David M. Dooley’s group, Montana State University.

Synthesis

MPBA and PBA

Both compounds were prepared according to the reference 4. MPBA · HCl: 1H-NMR

(D2O): δ 7.05 (2H, m), 6.81(2H, m), 4.65 (2H, t, J = 2), 3.68 (2H, t, J=2), 2.11 (3H, s);

1 PBA · HCl: H-NMR (D2O): δ 7.24 (2H, m), 6.91 (3H, m), 4.69 (2H, t, J = 2), 3.69 (2H,

105

t, J = 2)

[1, 1 - 2H] MPBA and [1, 1- 2H] PBA

A solution of p-cresol (30 mmol, 3.24 g), 80% 3-bromopropyne in toluene (7.08 g), sodium hydroxide (45 mol, 1.8 g), tetra-butylamine chloride (2 g) in THF (60 mL) and water (30 mL) was stirred at room temperature. After reaction was complete, THF was evaporated and water layer was extracted with ethyl ether. The combined ether layer was washed with water, dried over anhydrous sodium sulfate and concentrated under vacuum.

The residue was subjected to flash column chromatography to give pure product

1 3-(4-methylphenoxy)-propyne. H-NMR (CDCl3) δ 7.11 (2H, m), 6.89 (2H, m), 4.67 (2H,

d, J = 2.4), 2.51 (1H, t, J=2.4), 2.30 (3H, s); To a solution of 3-(4-methylphenoxy)-

propyne (14.8 mmol, 2.16 g) in THF (100 mL) was added n-butyl lithium (16.3 mmol,

6.5 mL) at -78℃ under Ar with stirring. After 15 minutes, the cooling bath was removed

and then perduterioformaldehyde (17.8 mmol, 0.568 g) was added quickly. The mixture

was kept stirring for several hours until the TLC showed that 3-(4-methylphenoxy)-

propyne disappeared completely. Ice cold water was added to quench the reaction. THF was evaporated and water layer was extracted with ethyl ether. Combined ether layer was dried over anhydrous sodium sulfate and then concentrated. The residue was subjected to

flash column chromatography to give [1, 1 - 2H] 4-(4-methylphenoxy)-2-butyn-ol in 84%

1 yield. H-NMR (CDCl3) δ 7.09 (2H, m), 7.86 (2H, m), 4.68 (2H, s), 2.29 (2H, s); To a solution of [1,1-2H]-4-(4-methylphenoxy)-2-butyn-ol (12.4mmol, 2.2g) and diisopropyl-

106

ethylamine (18.6 mmol, 2.4 g) in dichloromethane (100 mL) at 0℃ under Ar was added

methylsulfonyl chloride (14.9 mmol, 1.7g). The mixture was kept stirring at room

temperature. After reaction was complete, ice cold water was added to the solution.

Organic layer was washed twice with ice cold water and then evaporated to give crude

mesylated [1,1-2H]-4-(4-methylphenoxy)-2-butyn-ol. A solution of this intermediate in

ethanol (40 mL) and ammonium hydroxide (40 mL) was stirred for overnight and then concentrated to give crude product. Crude product was then reacted with di-t-butyl dicarbonate in the mixture of THF and aqueous sodium carbonate. THF was evaporated and aqueous layer was extracted with ethyl ether. Combined ether layer was concentrated and residue was subjected to flash column chromatography to give t-Boc derived [1, 1-

2 1 H] MPBA. H-NMR (CDCl3): δ 7.09 (2H, m), 6.84 (2H, m), 4.65 (2H, s), 2.29 (3H, s),

1.44 (9H, s); A solution of t-Boc derived [1, 1- 2H] MPBA (2 g) dissolved in the mixture

of ethanol (30 mL) and concentrated HCl (10 mL) was stirred for1 h and concentrated to

dryness. The solid was discolored with activated charcoal and recrystallized in ethanol to

give white solid 0.3 g. 1H-NMR (DMSO): δ 8.49 (3H, s), 7.08 (2H, m), 6.85 (2H, m),

13 4.80 (2H, s), 2.21 (3H, s); C-NMR (D2O): δ 154.57, 132.24, 130.28, 115.19, 82.36,

78.47, 56.09, 19.62

[1, 1- 2H] PBA hydrochloride was prepared according to the same method.

1 3-phenoxyl-propyne: H-NMR (CDCl3) :δ 7.32 (2H, m), 7.00 (3H, m), 4.70 (2H, d, J =

2 1 2.4), 2.53 (1H, dt, J = 2.4, 1.2)); [1, 1 - H] 4-phenoxy-2-butyn-ol: H-NMR (CDCl3): δ

7.30 (2H, m), 6.97 (3H, m), 4.718 , 4.714 (dd, 2H, J=1.6); t-Boc derived [1, 1 - 2H] PBA:

107

1 2 H-NMR (CDCl3): δ 7.29 (2H, m), 6.96 (3H, m), 4.68 (2H, s), 1.44 (9H, s); [1, 1 - H]

PBA · HCl: 1H-NMR (DMSO): δ 8.54 (3H, s), 7.30 (2H, m), 6.96 (3H, m), 4.84 (2H, s),

13 C-NMR (CD3OD) 157.84, 129.41, 121.37, 114.7, 82.7, 78.16, 55.27

4-(4-Methylphenoxy)-2-butynal and 4-phenoxy-2-butynal

4-(4-methylphenoxy)-2-butynal and 4-phenoxy-2-butynal were prepared from the

reaction of lithium acetylides and DMF according to Michel Journet’s procedure20.

1 4-phenoxy-2-butynal H-NMR (CDCl3): δ 9.22 (1H, t, J = 0.4), 7.33 (2H, m), 7.04 (1H,

13 m), 6.97 (2H, m), 4.89 (2H, d, J = 0.4); C-NMR (CDCl3): δ 176.28, 157.22, 129.79,

1 122.21, 114.90, 90.68, 85.86, 55.51; 4-(4-methylphenoxy)-2-butynal H-NMR (CDCl3): δ

13 9.22 (1H, s), 7.12 (2H, m), 6.86 (2H, m), 4.85 (2H, s), 2.31 (3H, s); C-NMR (CDCl3): δ

176.37, 155.27, 131.80, 130.35, 114.97, 91.05, 85.95, 55.94, 20.76

Turnover kinetics of PBA, [1, 1- 2H] PBA, MPBA and [1, 1- 2H] MPBA,

Enzymatic metabolism of deuterated and protonated compounds was evaluated by

oxygen uptake measurement. Rates of oxygen uptake for both deuterated and protonated

compounds were determined in 3 mL reaction solutions containing known concentration

of enzyme and 100 mM sodium phosphate buffer, pH 7.2, 30℃. Reaction was initiated

by adding various concentrations of compound. Initial rate was obtained from the slope

of the tangent along the initial part of progressive curve and analyzed by fitting to eq. (4.1)

using software original 8 SR4.

108

υ = []Ekcat []( S K m + []) S (4.1)

Kinetic parameters kcat and Km were obtained. The concentrations of active BPAO were

determined from the rate of benzylamine oxidation as described previously 21. AGAO concentration in terms of active site was determined by titration of TPQ with

22 D D phenylhydrazine. kcat and kcat/Km were determined by kkcat() H cat () D and

(kKcatH() mH ()) ( kK catD () mD ()) respectively.

Inhibition kinetics of benzylamine oxidation

Enzymatic oxidation of 10 mM substrate benzylamine in the presence of different concentrations of inhibitor compound in 100 mM sodium phosphate buffer, pH 7.2, was initiated by adding enzyme solution (the final concentration for AGAO is 0.2 µM and for

BPAO is 0.09 µM). Production of benzaldehyde was monitored at 250 nm over time. The

obtained progress curves were fit individually to eq. (4.2)

[P]t = A0 (1− exp(−kobst)) (4.2)

eff where kobs is the apparent inactivation constant. The kinetic parameters kinact and

eff KI were determined by fitting the plot of kobs versus inhibitor concentration to eq. (4.3)

using software original 8 SR4.

k eff .[I ] = inact kobs eff (4.3) K I .(1+ [S]/ K M ) + [I ]

eff eff where kinact and KI were defined by eq. (4.4) and (4.5) respectively.

109

eff k2 kkinact = ()⋅ inact (4.4) kkq++ inact k2

+ eff kkq inact k−1 KI = ()⋅ (4.5) kkq++ inact k21 k

Inhibition of BPAO by MPBA and PBA under both aerobic and anaerobic condition

BPAO was incubated with various concentration of inhibitor in 1 mL of 100mM air-saturated sodium phosphate buffer, pH 7.2, 30℃. 100 µl aliquots was removed

periodically and diluted to 1 mL of 5 mM benzylamine solution in 50 mM sodium

phosphate buffer, pH 7.2. The rate of production of benzaldehyde was monitored for the

first 40 seconds. Control experiment without inhibitor was set up at the same time.

Anaerobic experiments were setup according to the reference 9. 1.1 mL anaerobic

incubation solution was prepared in 3.5 mL vial containing 0.75 µM BPAO in 100 mM

sodium phosphate buffer, pH 7.2 and sealed with a septum. One inlet and one outlet

needles were attached to the vial, with the inlet one connected to deoxygenated

humidified argon gas line and the outlet one leading to atmosphere. Argon stream was

directed at the surface of enzyme solution for 3 h at room temperature. The outlet needle

was removed and inlet one was still kept to maintain positive argon pressure in the vial.

The enzyme solution was incubated at 30 ℃ for 10 min and then 100 µl aliquot was

withdrawn with air-tight injector for enzyme activity assay. This value was used as

control. 10 µl O2-depleted aqueous inhibitor of different concentration was injected into

the remaining incubation solution and the enzyme activity was assayed over time. To

110

check whether there was O2-dependent recovery of activity for BPAO anaerobically inactivated by MPBA, the same experiment was set up as above. After O2-depleted

inhibitor solution was injected and the enzyme-inhibitor solution was incubated at 30 ℃

for 3 or 30 min, the septum was removed, the incubation solution was blown and bubbled

occasionally with air for 30 seconds, and then enzyme activity was assayed over time.

Anaerobic inhibition of substrate-reduced BPAO by 4-(4-methylphenoxy)-2-buty

-nal and 4-phenoxy-2-butynal

A 1.1 mL anaerobic enzyme solution containing 0.78 µM BPAO was prepared in the

same way as above. After deoxygenating for 3 h, O2-depleted benzylamine solution was

injected to reach the final concentration of 20 µM. The solution was incubated for 40 min at 30 ℃ and then 100 µl aliquot was withdrawn for enzyme activity assay. This value was used as control. Following injection of O2-depleted 4-(4-methylphenoxy)-2-butynal or 4-phenoxy-2-butynal in ethanol with the final concentration of 80 µM, enzyme activity was assayed over time.

4.5 Reference 1. Hevey, R. C.; Babson, J.; Maycock, A. L.; Abeles, R. H., Highly specific enzyme inhibitors. Inhibition of plasma amine oxidase. J Am Chem Soc 1973, 95, (18), 6125-7. 2. Jeon, H.-B.; Lee, Y.; Qiao, C.; Huang, H.; Sayre, L. M., Inhibition of bovine plasma amine oxidase by 1,4-diamino-2-butenes and -2-butynes. Bioorg. Med. Chem. 2003, 11, (21), 4631-4641. 3. Jeon, H.-B.; Sayre, L. M., Highly potent propargylamine and allylamine inhibitors of bovine plasma amine oxidase. Biochem. Biophys. Res. Commun. 2003, 304, (4), 788-794.

111

4. Jeon, H.-B.; Sun, G.; Sayre, L. M., Inactivation of bovine plasma amine oxidase by 4-aryloxy-2-butynamines and related analogs. Biochim. Biophys. Acta, Proteins Proteomics 2003, 1647, (1-2), 343-354. 5. O'Connell Kimberly, M.; Langley David, B.; Shepard Eric, M.; Duff Anthony, P.; Jeon, H.-B.; Sun, G.; Freeman Hans, C.; Guss, J. M.; Sayre Lawrence, M.; Dooley David, M., Differential inhibition of six copper amine oxidases by a family of 4-(aryloxy)-2-butynamines: evidence for a new mode of inactivation. Biochemistry 2004, 43, (34), 10965-78. 6. Hartmann, C.; Brzovic, P.; Klinman, J. P., Spectroscopic detection of chemical intermediates in the reaction of para-substituted benzylamines with bovine serum amine oxidase. Biochemistry 1993, 32, (9), 2234-41. 7. Hartmann, C.; Klinman, J. P., Structure-function studies of substrate oxidation by bovine serum amine oxidase: relationship to cofactor structure and mechanism. Biochemistry 1991, 30, (18), 4605-11. 8. Qiao, C.; Jeon, H.-B.; Sayre, L. M., Selective Inhibition of Bovine Plasma Amine Oxidase by Homopropargylamine, a New Inactivator Motif. J. Am. Chem. Soc. 2004, 126, (25), 8038-8045. 9. Qiao, C.; Ling, K.-Q.; Shepard, E. M.; Dooley, D. M.; Sayre, L. M., Mechanism-Based Cofactor Derivatization of a Copper Amine Oxidase by a Branched Primary Amine Recruits the Oxidase Activity of the Enzyme to Turn Inactivator into Substrate. J. Am. Chem. Soc. 2006, 128, (18), 6206-6219. 10. Lunelli, M.; Di Paolo, M. L.; Biadene, M.; Calderone, V.; Battistutta, R.; Scarpa, M.; Rigo, A.; Zanotti, G., Crystal structure of amine oxidase from bovine serum. J. Mol. Biol. 2005, 346, (4), 991-1004. 11. Holt, A.; Smith, D. J.; Cendron, L.; Zanotti, G.; Rigo, A.; Di Paolo, M. L., Multiple binding sites for substrates and modulators of semicarbazide-sensitive amine oxidases: kinetic consequences. Mol. Pharmacol. 2008, 73, (2), 525-538. 12. Wilce, M. C.; Dooley, D. M.; Freeman, H. C.; Guss, J. M.; Matsunami, H.; McIntire, W. S.; Ruggiero, C. E.; Tanizawa, K.; Yamaguchi, H., Crystal structures of the copper-containing amine oxidase from Arthrobacter globiformis in the holo and apo forms: implications for the biogenesis of topaquinone. Biochemistry 1997, 36, (51), 16116-33. 13. Murakawa, T.; Okajima, T.; Kuroda, S. i.; Nakamoto, T.; Taki, M.; Yamamoto, Y.; Hayashi, H.; Tanizawa, K., Quantum mechanical hydrogen tunneling in bacterial copper amine oxidase reaction. Biochem. Biophys. Res. Commun. 2006, 342, (2), 414-423. 14. Northrop, D. B., Minimal kinetic mechanism and general equation for deuterium isotope effects on enzymic reactions: uncertainty of detecting a rate-limiting step. Biochemistry 1981, 20, (14), 4056-61. 15. Wilmot, C. M.; Murray, J. M.; Alton, G.; Parsons, M. R.; Convery, M. A.; Blakeley, V.; Corner, A. S.; Palcic, M. M.; Knowles, P. F.; McPherson, M. J.; Phillips, S. E., Catalytic mechanism of the quinoenzyme amine oxidase from Escherichia coli: exploring

112 the reductive half-reaction. Biochemistry 1997, 36, (7), 1608-20. 16. Kitz, R.; Wilson, I. B., Esters of methanesulfonic acid as irreversible inhibitors of acetylcholinesterase. J. Biol. Chem. 1962, 237, 3245-49. 17. Wimalasena, K.; Haines, D. C., A general progress curve method for the kinetic analysis of suicide enzyme inhibitors. Anal. Biochem. 1996, 234, (2), 175-82. 18. Waley, S. G., Kinetics of suicide substrates. Biochem. J. 1980, 185, (3), 771-3. 19. Medda, R.; Padiglia, A.; Agro, A. F.; Pedersen, J. Z.; Lorrai, A.; Floris, G., Tryptamine as substrate and inhibitor of lentil seedling copper amine oxidase. Eur. J. Biochem. 1997, 250, (2), 377-382. 20. Journet, M.; Cai, D.; DiMichele, L. M.; Larsen, R. D., Highly efficient synthesis of alpha ,beta -acetylenic aldehydes from terminal alkynes using DMF as the formylating reagent. Tetrahedron Lett. 1998, 39, (36), 6427-6428. 21. Ling, K.-Q.; Sayre, L. M., Discovery of a Sensitive, Selective, and Tightly Binding Fluorogenic Substrate of Bovine Plasma Amine Oxidase. J. Org. Chem. 2009, 74, (1), 339-350. 22. Kishishita, S. i.; Okajima, T.; Kim, M.; Yamaguchi, H.; Hirota, S.; Suzuki, S.; Kuroda, S. i.; Tanizawa, K.; Mure, M., Role of Copper Ion in Bacterial Copper Amine Oxidase: Spectroscopic and Crystallographic Studies of Metal-Substituted Enzymes. J. Am. Chem. Soc. 2003, 125, (4), 1041-1055.

113

Chapter 5 Investigate structural basis of inactivation of bovine plasma amine oxidase by 5-aryl-3-pentynamines

114

5.1 Introduction

Recently our lab reported that homopropargylamine acts as a new inactivating unit to

induce a potent time-dependent irreversible inhibition on BPAO.1 This preliminary study

demonstrated that titration of homopropargylamine-inactivated BPAO with

phenylhydrazine failed to show the characteristic 450 nm absorbance band, indicating

that the inhibitor modifies either the cofactor TPQ or an active site residue in a manner which prevents phenylhydrazine from accessing the TPQ. Additionally, according to this study,1 the nitroblue tetrazolium (NBT) redox cycling assay showed that the denatured

inactivated BPAO retained complete cofactor redox activity compared to the denatured

control BPAO. These results suggest that enzyme inactivation likely results from either

alkylation of an active site residue or modification of TPQ cofactor in a manner that

reverses upon enzyme denaturation. Another conclusion based on this previous study is

that the enzyme inactivation should involve the highly reactive aldehyde

H2C=C=CH-CH=O (tautomer of theoretical turnover product HC≡C-CH2-CH=O), either

as free aldehyde or in the imine linkage to the reductively aminated TPQ.1 So far, the

structural basis of enzyme inactivation is still a mystery.

We prepared three chromophore-labeled homopropargylamine derivatives,

5-(4-nitrophenoxy)-3-pentynamine (1), 5-(4-nitroanilino)-3-pentynamine (2), and 5-(2,

4-dinitrophenoxy)-3-pentynamine (3), and evaluated their inhibitory activities towards

BPAO. Like homopropargylamine, no plateau of enzyme activity loss was observed for

115

the three derivatives, which indicated a very efficient inactivation without competitive

turnover. If the inactivation involves a covalent modification of BPAO by these types of

chromophore-labeled inhibitors, then the inactivated BPAO should demonstrate an

association with the chromophore. In the favorable cases, the appropriate enzymatic

digest of inactivated enzyme followed by HPLC analysis might reveal the

chrommophore-labeled peptides. In this work, we used different methods to characterize

the BPAO as it was inactivated by different inhibitors. An alternative inactivation

mechanism consistent with the present experimental data is proposed here.

5.2 Results

5.2.1 Synthesis of 5-aryl-3-pentynamines (1-3) and 1-(4-nitroanilino)-5,

5-diethoxy-2-pentyne

5-Aryl-3-pentynamines (1-3) were prepared by nucleophilic substitution reaction between the t-Boc derivative of 5-chloro-3-pentynamine and the corresponding

(substituted) aniline/phenol followed by deprotection of the amino group (Scheme 5.1).

The t-Boc derivative of 5-chloro-3-pentynamine was prepared by alkylation of the t-Boc derivative of homopropargylamine with paraformaldehyde, followed by conversion of the

2 primary alcohol to the chloride using PPh3/CCl4 (Scheme 5.2). 1-(4-Nitroanilino)-5,

5-diethoxy-2-pentyne, the acetal precursor of 5-(4-nitroanilino)-3-pentynal, was prepared by a similar method (Scheme 5.3).

116

Ar-X-H HCl Ar X NH Cl NHBoc NaI Ar X NHBoc 2 1-3 O NH O

NO2 Ar-X =

NO2 NO2 NO2 1 2 3

Scheme 5.1 Synthesis of 5-aryl-3-pentynamines (1-3)

H HCHO PPh3 HO NHBoc Cl NHBoc NHBoc n-BuLi CCl4

Scheme 5.2 Synthesis of t-Boc protected 5-chloro-3-pentynamine

H HCHO PPh3 OEt n-BuLi HO OEt CCl4 Cl OEt EtO EtO EtO

4-nitroaniline O2N NH OEt NaI EtO

Scheme 5.3 Synthesis of 1-(4-nitroanilino)-5, 5-diethoxy-2-pentyne

5.2.2 Irreversible inactivation of BPAO by 5-aryl-3-pentynamines (1-3)

Inactivation of BPAO by inhibitor 1 showed time- and concentration-dependent loss of enzyme activity (Figure 5.1). At 1µM of inhibitor 1, the enzyme lost about 90% activity in 5 min. No plateau behavior was observed during the assay period, indicating

117

that there was almost no competing productive turnover for this inhibitor. Compared with

homopropargylamine (30 min IC50 of 2.9 µM), inhibitor 1 is much more potent for

inactivation of BPAO. No enzyme activity was recovered after gel filtration or 24 h

dialysis, which implicated the formation of a stable covalent adduct between BPAO and

inhibitor 1.

100 1µM 2µM 80

20 Remaining enzyme activityRemaining enzyme

0 5 10 15 20 25 30 Time/min

Figure 5.1 Time course of inactivation of BPAO (0.6 µM) by different concentrations of

inhibitor 1 at 30℃

The time course of inactivation of BPAO at various concentrations of inhibitor 2 is

shown in Figure 5.2. The 5 min IC50 value for this inhibitor is less than 1 µM. As with 1,

loss of enzyme activity is very rapid in the first 5 min, which may suggest that both

inhibitors 1 and 2 have a preferred binding at the enzyme active site and a fast

inactivation rate. Inhibitor 2 is also an irreversible inhibitor for BPAO, since no enzyme

118 activity recovery was seen after gel filtration or 24 h dialysis. Moreover, inhibitor 2

4 contains a chromophore with λmax 380 nm (ε = 1.6 × 10 ). If BPAO is inactivated through the formation of a covalent complex with inhibitor 2, the inactivated BPAO should take on color with this chromophore.

100 1µM 2µM 4µM 80

20 Remaining enzyme actvityRemaining enzyme

0 5 10 15 20 time/min

Figure 5.2 Time-dependent inactivation of BPAO (0.6 µM) by different concentrations of inhibitor 2 at 30℃

Compared with inhibitors 1 and 2, inhibitor 3 is less potent in inhibition of BPAO. 15 µM of inhibitor 3 induced a loss of 60% enzyme activity in 3min. The loss of enzyme activity follows pseudo-first order kinetics, which allows the determination of inactivation kinetic parameters by a Kitz and Wilson plot (Figure 5.3).3 Enzyme saturation was observed for

-1 inhibitor 3. The kinact was determined to be 0.11 min and KI was 6.4 µM. As inhibitor 1 and 2, inhibitor 3 inactivated BPAO irreversibly. Since inhibitors 1-3 contain the same

119 inactivation unit (C≡CCH2CH2NH2), they should inactivate enzyme BPAO through a similar mechanism analogous to the parent compound homopropargylamine.

100

1µM 4µM 8µM

Remaining Enzyme activityRemaining Enzyme 10µM 15µM

1 0 10 20 30 40 50 Time/min

25 1/2 t 20

0.0 0.2 0.4 0.6 0.8 1.0 1/[I]

Figure 5.3 Time-dependent inactivation of BPAO (0.6 µM) by various concentrations of inhibitor 3 (upper), and Kitz and Wilson replot of the data (lower).

120

5.2.3 Mechanism of inactivation of BPAO by 5-aryl-3-pentynamines

5.2.3.1 Covalent adducts between enzyme BPAO and 5-aryl-3-pentynamines

Even though no recovery of enzyme activity after gel filtration or 24 h dialysis implicated a stable covalent adduct of BPAO with 5-aryl-3-pentynamines, further experiments were carried out to support this conclusion.

As mentioned earlier, inhibitor 2 contains a chromophore with λmax 380 nm (ε = 1.6×

104). If BPAO is covalently modified by inhibitor 2, that should endow BPAO with this

special chromophore. To demonstrate the covalent labeling of BPAO, the enzyme BPAO

was incubated with inhibitor 2 for 2 h. After denaturation with urea and β-mecaptoethanol,

the reaction mixture was gel-filtered to remove excess inhibitors from the enzyme using a

Pd-10 column, and the absorbances at 280 nm and 380 nm corresponding to BPAO and

inhibitor 2 respectively were determined for all of the collected fractions. The results

showed that enzyme BPAO coeluted with inhibitor 2 (Figure 5.4), which supports the

conclusion that inhibitor 2 forms a covalent bond with BPAO. The same experiment was

repeated for three different enzyme concentrations (6.8, 13.6 and 20.4 mg/mL). Based on

the yield of BPAO and inhibitor 2, the stoichiometry of inhibitor to enzyme was

determined to be around 2:1 (see experimental procedures). Because mechanism-based

inhibition depends on enzyme catalysis, there can not be more than one inhibitor bound

to the enzyme active site. In the case of dimeric enzyme BPAO, the stoichiometry of

inhibitor to enzyme could be more than 1:1.

121

0.35 280nm 380nm 0.30

0.25

0.20 A 0.15

0.10

0.05

0.00 1 2 3 4 5 6 7 8 9 10 Fraction

Figure 5.4 Inhibitor 2-labeled BPAO

Moreover, electrospray ionization spectrometry was used to reconfirm the formation of an adduct between BPAO and inhibitor 2. Gas-phase macromolecular ions are formed directly from solution via protonation and ion evaporation. Multiple charging produces a series of molecular ions with dramatically reduced mass-to-charge ratios, which allows for determining the molecular weight of large proteins. Shown in Figure 5.5 are the electrospray mass spectra of the native BPAO and inhibitor 2-inactivated BPAO. The nine well-resolved peaks were observed in the m/z range 1800 to 2000. The spaces from peak

1 to 3, 4 to 6, 7 to 9, are 36.3, 39.3 and 43.5 respectively for the native BPAO and the corresponding spaces are 35.9, 40.8 and 44.4 for the derivatized BPAO. Unequal distances between peaks indicate they are real protein peaks. Both the native and

122

derivatized BPAO produced charge distribution profiles in the high m/z range

(1800-2000). Charge states ranging from 94+ to 86+ were observed for the native BPAO,

which were assumed to be identical to those of the corresponding peaks in the derivatized

BPAO. Based on the charge states and m/z maxima, average molecule weight was determined to be 170355.1 and 170657.3 for the native BPAO and the modified BPAO respectively. The molecular weight difference between them showed the stoichiometry of inhibitor to enzyme is 1.4, indicating more than one inhibitor was incorporated per BPAO dimer.

3.8 86+ 3.6 3.4 9 8 3.2 7 3.0 6 5 2.8 4 94+ 3 2.6 2 2.4 1

Intensity 2.2 2.0 86+ 1.8 94+ 1.6 4 5 6 7 8 1 2 3 9 1.4 1.2 1.0 1800 1850 1900 1950 2000 m/z

Figure 5.5 Electrospray mass spectra of the native BPAO (lower) and inhibitor

2-inactivated BPAO (upper). The peaks were numbered as 1, 2, 3, 4, 5, 6, 7, 8, 9, from the

left to the right for both the native and derivatized BPAO.

123

The most convincing evidence for the formation of a covalent BPAO-inhibitor adduct

was obtained by UV-vis spectrophotometric analysis of the reaction between BPAO and inhibitor. Adding of inhibitor 1 (157.5 µM) to enzyme BPAO (84.4 µM) in 100 mM

phosphate buffer, pH 7.2, immediately caused bleaching of the TPQ chromophore at 482

nm and the appearance of new peak at around 345 nm (Figure 5.6). Another new peak at

around 406 nm increased slowly, which is concomitant with a slight decrease at 345 nm

absorption. The 345 nm absorption is close to the absorption maximum (340 nm)

reported for the substrate Schiff base between BPAO and benzylamine.4 Moreover, the

405 nm absorption maximum was previously observed for a protonated analog of product

Schiff base. The product Schiff base which was prepared from the reaction of

4-amino-6-t-butylresorcinol and benzaldehyde has λmax at 368 nm and protonation of this

4 product Schiff base causes λmax to shift to 405nm. The reaction of BPAO with inhibitor 3

was also monitored using UV-vis spectra (Figure 5.7). Immediately upon the addition of

inhibitor 3 to enzyme BPAO, a broad absorption band (300-450 nm) increased with loss

of the TPQ cofactor absorption at 480 nm. This broad absorption band is composed of

two unresolved peaks (nearly at 345 and 405nm). Generally, the reaction of BPAO with

inhibitor 1 and 3 is assumed to follow the same pathway. The BPAO displayed different

spectral changes upon mixing with inhibitors 1 and 3, respectively, and this could be

attributed to different reaction rates between BPAO and inhibitors. When BPAO reacts

with 2 equivalents of inhibitor 1, the TPQ absorbance change at 480 nm appears to be

124

rapid in the initial 30 seconds, which accounts for nearly 78% of the total change at this

wavelength. In contrast, for the reaction of BPAO with 3 equivalents of inhibitor 3, the

TPQ absorbance change in the initial 30 seconds is insignificant. However, the absorption

of TPQ cofactor cannot be recovered after 24 h dialysis for both inhibitors. The reaction

of BPAO with inhibitor 2 was not monitored by UV-vis spectra because inhibitor 2 has

strong absorption at 380 nm.

0.3 a: native BPAO b: 0.5min c: 2.5min d: 4.5min 0.2 e: 6.5min h f: 8.5min g g: 10.5min f h: after dialysis A e 0.1 d c b a

0.0

300 350 400 450 500 550 wavelength/nm

Figure 5.6 Spectral change upon addition of aqueous inhibitor 1 (157.5 µM) to the native

BPAO (84.4 µM). The spectra b-g were recorded at 2 min interval. The arrows indicate

the direction of spectral change. The spectrum h was recorded after dialysis of enzyme-

inhibitor mixture against 0.1 M phosphate buffer, pH 7.2, for 24 h.

125

0.4 a: native BPAO b: 0.5min c: 2.5min 0.3 d: 4.5min e: 6.5min e f: 8.5min c d f g: after dialysis 0.2 A

g b 0.1 a

0.0

300 400 500 600 wavelength/nm

Figure 5.7 Spectral change upon addition of aqueous inhibitor 3 (272.5 µM) to the native

BPAO (82.5 µM). The spectra b-f were recorded at 2 min interval. The arrows indicate

the direction of spectral change. The spectrum g was recorded after dialysis of enzyme-

inhibitor mixture against 0.1 M phosphate buffer, pH 7.2, for 24 h.

5.2.3.2 Immunochemical analysis of inactivation of enzyme BPAO by inhibitor 3

The irreversible inactivation of BPAO by inhibitor 3 indicates covalent enzyme

modification. We sought to obtain confirmatory evidence through western blotting

analysis. The enzyme BPAO was incubated with inhibitor 3 until a benzylamine assay showed the enzyme lost all activity. The inactivated BPAO was subjected to gel-electrophoresis, followed by Western blotting with anti-DNP antibody and alkaline phosphatase conjugated-secondary antibody (see experimental section). The native BPAO was used as a control. The chromogenic visualization showed that the inactivated BPAO was indistinguishable from the native BPAO in terms of the intensity of the bands.

126

5.2.3.3 Attempted resolution of the structural basis of inactivation of enzyme BPAO by inhibitor 2 using enzyme digestion and LC/MS

The combination of enzyme digestion and LC/MS method was used to study the nature of the BPAO-inhibitor adduct. Enzyme BPAO was incubated with excess inhibitor

2 for 2 h at 30℃ and a benzylamine assay showed the remaining enzyme activity was no more than 5% as compared with the control enzyme. Several procedures were attempted for proteolytic digestion and peptide isolation (see the experimental procedures). In the initial experiment, inactivated BPAO was digested by chymotrypsin followed by reverse-phase HPLC analysis (Figure 5.8). Monitoring of the chromophore (at 380 nm) revealed only one dominant peak with a retention time of 48 min. This peak can not be attributed to inhibitor 2 because of their different retention times. The same dominant peak was obtained by digestion with trypsin, thermolysin, and proteinase K respectively, implicating that the observed peak with absorption at 380 nm was not an

127

250

200

150 380nm 100

0 200

150

100 280nm 50 absrobance at 0 1500

1200

900

220nm 600

300

0 20 40 60 80 100 120 Time/min

Figure 5.8 HPLC elution profiles of the chymotryptic digest of BPAO inactivated by

inhibitor 2

inhibitor-labeled peptide but a small molecule, because those proteinases have different peptide bond cleavage specificity. This small molecule was confirmed to be

5-(4-nitroanilino)-3-pentynal by comparing it to the authentic sample which was prepared from mild acidic hydrolysis of the acetal precursor

1-(4-nitroanilino)-5,5-diethoxy-2-pentyne. The small molecule detected in the enzyme digest has the same retention time and tandem mass spectra as the authentic compound

5-(4-nitroanilino)-3-pentynal (Figure 5.9). The reason for the failure to observe inhibitor

128

2-labeled peptides was probably that the enzyme-inhibitor adduct dissociated to release the molecule 5-(4-nitroanilino)-3-pentynal throughout the enzymatic digestion. Finally, chemical reagent cyanogen bromide was used for protein cleavage. Still a dominant small

+ MH -H2O 100 201.03 MS2 219.29

80 + MH -NO2 173.00 60

40 204.90 + 218.92 20 MH -NO 189.03 0 120 140 160 180 200 220 240

100 210.02 MS2 219.21

80 173.03 Relative (%) Abundance 60

40 204.88 218.98 20 174.03 189.07 0 120 140 160 180 200 220 240 m/z

Figure 5.9 Tandem mass spectra for the peak with absorption at 380 nm detected in

chymotryptic digest (lower), compared to the tandem mass spectra for the authentic

sample 5-(4-nitroanilino)-3-pentynal (upper)

molecule was observed by HPLC analysis with UV-vis detection at 380 nm. Mass spectra

of this molecule showed two isotope ion peaks at m/z 297.35 and 299.32 with equal

abundance, which indicated it contained a bromine atom. Presumably, it came from the

reaction between 5-(4-nitroanilino)-3-pentynal and cyanogen bromide. No additional

129

experiments were done for further identification.

5.3 Discussion

The structure-inhibitory profiles of three homopropargylamine derivatives 1-3 were reported here. The simple extension of homopropargylamine by incorporating aryl groups to yield inhibitors 1 and 2 increases the inhibition potency remarkably. This increase in inhibition potency may reflect the favorable interactions between the aryl groups of the inhibitors and amino acid residues at the enzyme substrate channel. However, replacement of the 4-nitrophenoxy group of inhibitor 1 with the 2, 4-dinitrophenoxy

group impairs the inhibition potency greatly. The inhibition potencies of this class of

inhibitors are very sensitive to the structure changes at the C terminus of the reaction

center. Despite the differences in inhibition potency, homopropargylamine and the three

derivatives share several characteristics: (1) the same inactivation unit; (2)

time-dependent irreversible inactivation on enzyme BPAO; (3) the partition ratio is nearly

zero. A common inactivation mechanism is implicated for this class of inhibitors.

In this work, UV-vis analysis of the fractions from gel filtration of inhibitor

2-inactivated BPAO showed that a covalent adduct was formed between inhibitor 2 and enzyme, which is capable of surviving the mild denaturing conditions used in this experiment (urea and β-mecaptoethanol). The calculated stoichiometry of inhibitor to

enzyme is close to the theoretical value (2:1) for dimeric BPAO. However lower

130

enzyme-inhibitor stoichiometry (1.4:1) is obtained from the mass spectra of the native

BPAO and inhibitor 2-derivatized BPAO. As we know, it may not be necessary to modify all of the active sites within the BAPO dimer to effect total inactivation of BPAO.5 The

reason for higher inhibitor-enzyme stoichiometry is unknown.

The UV-vis spectrophotometric analysis of the reaction of both inhibitors 1 and 3

with BPAO provides further insights into the nature of inactivation of BPAO by this class

of inhibitors. Both inhibitors behave like normal substrates, form substrate Schiff bases

with TPQ and then tautomerize to product Schiff bases. After 24 h dialysis, the UV-vis

spectra showed that the peaks of the product Schiff bases disappeared. If the product

Schiff bases hydrolyzed to generate aldehydes, the absorption of oxidative TPQ would be

recovered after the rapid oxidation of the reductively aminated TPQ. Here no recovery of

oxidative TPQ or enzyme activity implicated that the product Schiff bases may isomerize

to other TPQ-derivatized species. Further information was gained by the performance of

the redox cycling assay with nitroblue tetrazolium on the denatured inactivated enzyme.

The denatured inhibitor 1-inactivated BPAO showed complete quinone cofactor redox

competence as compared to the denatured control sample. Similar results were obtained

for the BPAO inactivated by inhibitors 2 and 3. Moreover, immunochemical analysis of

the denatured inhibitor 3-inactivated BPAO using 2, 4-DNP antibody did not show any

immunoreactivity. These results together demonstrate that this class of inhibitors may

inactivate BPAO by modifying the cofactor TPQ in a form that is reversed under the

131

denaturing conditions used in SDS-PAGE (see experimental section).

Enzymatic digestion of the inactivated BPAO by inhibitor 2 followed by separation by HPLC failed to reveal any inhibitor 2-labeled peptides. The aldehyde corresponding to inhibitor 2 was detected from the proteolytic digests. Enzymatic digestion over time monitored by HPLC showed that the aldehyde was generated during the digestion process.

Presumably, enzyme-inhibitor adducts collapse and result in the release of the aldehyde.

In previous studies of inactivation of BPAO by homopropargylamine, it was proposed

that the highly reactive allenic product H2C=C=CH-CHO, either as a free aldehyde or in

imine linkage to the reductively aminated TPQ, could be formed by metabolism of the

inhibitor by BPAO. This allenic aldehyde or imine could either alkylate the active site

residue or modify TPQ in a manner that is reversed upon enzyme denaturation.1 Evidence

for the reductively aminated TPQ acting as a nucleophile to form a covalent adduct with

α, β-unsaturated aldehyde turnover product has been presented by O’Connell et al., who determined the crystal structure of the complex of Arthrobacter globiformis amine oxidase (AGAO) and 4-aryoxy-2-butynamine.6 So in the case of BPAO with

homopropargylamine or its derivatives, the allenic aldehyde product might alkylate the

reductively aminated TPQ to induce enzyme inactivation (see Scheme 5.4). However, if

this is true, we should expect to see the other hydrolytic small molecules rather than the

product aldehyde released from the TPQ adduct during the process of enzymatic

digestion.

132

OH OH OH

R C + kinact HO HO O HO HO NH NH2 O NH

R R

R: substituting group Scheme 5.4 Alkylation of the reductively aminated TPQ by allenic aldehyde products

We are proposing a new mechanism for inactivation of BPAO by homopropargylamine derivatives (Scheme 5.5) based on the experimental results in this work. Initially, TPQ reacts with a homopropargylamine derivative to form a substrate

Schiff base 4. The latter is subsequently isomerized to a product Schiff base 5. Instead of hydrolysis to give aldehyde and reductively aminated TPQ, the product Schiff base 5 may tautomerize to its stable enamine form 6. During enzymatic digestion or under the denaturing condition, the product Schiff base 5 is exposed to aqueous solvent and rapidly hydrolyzes, shifting the equilibrium in favor of the conversion of the enamine 6 to the product Schiff base 5. Following the hydrolysis of the product Schiff base 5, the aldehyde is released and the reductively aminated TPQ is oxidized by O2 to regenerate TPQ

cofactor.

133

O O OH OH

H2O O + O O O HO NH O HN HN 2 R NH3 4 R 5 R R

R: substituting group OH

HO HN

6 R Scheme 5.5 Proposed mechanism for inactivation of BPAO by homopropargylamine

derivatives

5. 4 Conclusions

Three homopropargylamine derivatives 1-3 were prepared and evaluated as inhibitors

of BPAO. Like the parent compound homopropargylamine, all of them displayed time-dependent irreversible inactivation on BPAO with a ranked order of potency of 1 >

2 > 3 ≈ homopropargylamine. The structure-inhibitory profiles indicated that the presence of the aryl group can greatly modulate inactivation potency of all these inhibitors. All of the inhibitors exhibited evidence of covalent modification of enzyme BPAO. Specifically,

monitoring the reaction of BPAO with inhibitors by UV-vis spectrophotometer

demonstrated the BPAO inactivated by inhibitors 1 and 3 lost the characteristic

absorption of TPQ cofactor. The denatured BPAO inactivated by inhibitor 3 did not show

134

any immune reactivity to 2, 4-DNP antibody. For all of the homopropargylamine derivatives in this work, the cofactor TPQ regains complete redox reactivity upon

denaturation of the inactivated enzyme, suggesting that this type of inhibitor may modify

the cofactor TPQ in a manner that is reversed upon denaturation of enzyme. We proposed

that the normal product Schiff base may tautomerize to its stable enamine form and the latter is probably favored by the enzyme active site structures. Under denaturing conditions or during enzymatic digestion process, the normal product Schiff base would be exposed to bulk solution and rapidly hydrolyze to afford the corresponding aldehyde.

The enzymatic digestion of inhibitor 2-derivatized BPAO followed by HPLC separation

revealed no inhibitor 2-labled peptides except the corresponding aldehyde, which seems

to be consistent with the inactivation mechanism we propose here. Additional work will be needed to provide the confirmatory evidence for this inactivation mechanism.

5. 5 Experimental procedures

General procedures

All NMR spectra were obtained at ambient temperature, on a Varian Gemini 400

MHz instrument. Chemical shifts were referenced to the residual proton peaks in the deuterated solvents. Benzylamine assays were conducted on a Perkin-Elmer Lambda 25

UV-vis spectrophotometer using UV Winlab software V 2.85, with constant temperature being maintained by peltier thermstatting. High-resolution mass spectra (HRMS) were

135

obtained by electron impact ionization (EI) (20-40 eV) or fast atom bombardment (FAB) on a Kratos MS25RFA instrument. The concentration of protein solutions was by centrifugal evaporation under reduced pressure with a Savant Speed Vac SC110 system

(Forma Scientific, Inc., Marietta, OH). Pure enzyme BPAO was provided by Dr David M.

Dooley’ group, Montana State University. The anti-DNP antibody (developed in goat) and anti-goat lgG (whole molecule) alkaline phosphate conjugate were purchased from

Sigma.

Synthesis t-Boc derivative of 5-chloro-3-pentynamine

To a stirred solution of t-butyl-3-butynylcarbamate (2.5 g, 15 mmol) in THF (100 mL) at -78 ℃ was added n-butyllithium (2.5 M, 14 mL) over a period of 5 minutes. After stirring for another 20 minutes, the solution was warmed to room temperature and paraformaldehyde (1.1 g, 36 mmol) was added in one portion. The mixture was refluxed for 4 h. The reaction was quenched with ice-cold water. The mixture was extracted with ethyl ether. The combined ethyl ether layer was washed with saturated NH4Cl solution,

dried with anhydrous Na2SO4, and then evaporated in vacuum to give oil residue.

Purification by column chromatograph gave pure t-butyl-5-hydroxy-3-pentynylcarbamate

1 (2.6 g). H-NMR (CDCl3): δ 4.912 (1H, br), 4.255 (2H, dt, J = 2.2, 5.6), 3.278 (2H, q, J =

6), 2.412 (2H, tt, J = 5.6, 2.2), 2.152 (1H, br), 1.453 (9H, s)

136

A solution of t-butyl-5-hydroxy-3-pentynylcarbamate (2.6 g, 13 mm) and carbon tetrachloride (2.3 g, 14.6 mm) in dry methylene chloride (100 mL) was cooled to 0 ℃. A solution of triphenylphosphine (3.8 g, 14.6 mm) in 10 mL methylene chloride was added drop by drop. The mixture was warmed to room temperature and stirred for overnight.

The solvent was evaporated under vacuum and ethyl ether was added to the residue. The solid was filtered and the filtrate was evaporated to dryness to give residue, which was purified by column chromatograph to give t-Boc derivative of 5-chloro-3-pentynamine

1 (2.2 g). H-NMR (CDCl3): δ 4.81 (1H, br), 4.143 (2H, t, J = 2.2), 3.281 (2H, q, J = 6),

2.441 (2H, m), 1.455 (9H, s)

5-Aryl-3-pentynamines (1-3)

A mixture of anhydrous potassium carbonate (8.29 g) and the corresponding

(substituted) phenol or aniline (20 mm) in dry DMF (45 mL) was heated to 60 ℃ for half an hour under argon atmosphere. The mixture was then cooled to room temperature and t-Boc derivative of 4-chloro-2-butynamine (4.07 g, 20 mm) and sodium iodide (3 g,

20 mm) was added. The mixture was stirred for 3 hours at about 90 ℃. The mixture was poured into ice-cold water with stirring, extracted with methylene chloride. The combined organic layer was washed with water and concentrated under vacuum. The residue was applied to column chromatography to give t-Boc derivative of the final product. A solution of the t-Boc derivative in the mixture of concentrated hydrochloride

137

(10 mL) and ethanol (20 mL) was stirred at room temperature for 1 h and concentrated to

dryness. The obtained solid was recrystallized in methanol to give pure product.

1 5-(4-nitrophenoxy)-3-pentynamine (1): H-NMR (D2O): δ 8.06 (2H, m), 6.96 (2H, m),

4.73 (2H, t, J = 1.6), 3.01 (2H, t, J = 6.6), 2.53 (2H, m); 13C-NMR (DMSO): δ 163.18,

141.92, 126.49, 116.13, 84.85, 77.51, 57.47, 38.21, 17.63; HRFABMS MH+ m/z obsd

221.09420, C11H13N2O3 required 221.09261. 5-(4-nitroanilino)-3-pentynamine (2):

1 H-NMR (D2O) δ 7.96 (2H, m), 6.62 (2H, m), 3.89 (2H, t, J = 2.2), 2.96 (2H, t, J = 6.6),

2.44 (2H, tt, J = 2.2, 6.6); 13C-NMR (DMSO) δ 154.37, 137.04, 126.7, 112.04, 79.53,

+ 79.51, 38.49, 32.79, 17.62; HRFABMS MH m/z obsd 220.10944, C11H14N3O2 required

1 220.1086. 5-(2, 4-dinitrophenoxy)-3-pentynamine (3): H-NMR (CD3OD): δ 8.74 (1H, d,

J=3.2), 8.51 (1H, dd, J = 2.6, 9.4,), 7.60 (1H, d, J = 9.6), 5.11 (2H, t, J = 2.4), 3.09 (2H, t,

13 J = 6.8), 2.68 (2H, tt, J = 2, 6.8,); C-NMR (DMSO): 155.061, 140.773, 139.522,

129.781, 121.847, 117.042, 86.147, 76.573, 59.234, 38.103, 17.674; HRFABMS MH+

m/z obsd 266.07794, C11H12N3O5 required 266.0777

1-(4-Nitroanilino)-5, 5-diethoxy-2-pentyne

The starting material 4, 4-diethoxy-1-butyne was prepared by the reaction of propargyl bromide with triethyl orthoformate in the presence of aluminum amalgam as a catalyst.7 The remaining procedures were referred to the preparation of

5-aryl-3-pentynamines.

138

1 5, 5-diethoxy-2-pentyn-1-ol: H-NMR (CDCl3): δ 4.651 (1H, t, J = 5.6), 4.256 (2H,

m), 3.684 (2H, m), 3.564 (2H, m), 2.574 (2H, dt, J = 5.6, 2,), 1.229 (6H, t, J = 6.8,).

1 1-chloro-5, 5-diethoxy-2-pentyne: H-NMR (CDCl3): δ 4.649 (1H, t, J = 6,), 4.148 (2H, t,

J = 2.4,), 3.666 (2H, m), 3.581 (2H, m), 2.588 (2H, dt, J = 5.6, 2.4,), 1.221 (6H, t, J = 7).

1 1-(4-nitroanilino)-5, 5-diethoxy-2-pentyne: H-NMR (CDCl3): δ 8.113 (2H, m), 6.615

(2H, m), 4.728 (1H, br), 4.605 (1H, t, J = 5.6), 4.005 (2H, m), 3.636 (2H, m), 3.536 (2H,

13 m), 2.528 (2H, dt, J = 2, 5.6,), 1.192 (6H, t, J = 7,); C-NMR (CDCl3): δ 152.49, 139.02,

126.41, 111.96, 100.80, 80.14, 62.08, 33.78, 25.14, 15.42; HRFABMS MH+ m/z obsd

292.14254, C15H20N2O4 required 292.142307.

Enzymology

Time-dependent inactivation of BPAO by inhibitors was determined by taking

aliquots over time of primary incubations of BPAO with inhibitors and assaying for

conversion of benzylamine to benzaldehyde at 250 nm. Irreversibility of BPAO inhibition

was checked by gel filtration or after 24 h dialysis, with the before and after activity

assays varying no more than 5% in the cases of irreversible inhibitors.

The stoichiometry of BPAO-inhibitor complex was determined using inhibitor 2. 50

µl pure enzyme BPAO (13.6 mg/mL) in 100 mM sodium phosphate buffer, pH 7.2, was incubated with 3 µl aqueous 5-(4-nitroanilino)-3-pentynamine (10 mM) at 30 ℃. After 1 h, the enzyme-inhibitor mixture was denatured by adding 28 mg urea and 2 µl

139

β-mercaptoethanol. The mixture was gel-filtered using a Pd-10 column and the filtrate

was collected in 0.5 mL fractions. Absorbance at 280 nm and 380 nm was determined for

each fraction. A control experiment was set up similarly with the exception of

denaturation of enzyme BPAO with urea and β-mercaptoethanol before incubation with

5-(4-nitroanilino)-3-pentynamine. The stoichiometry of inhibitor-enzyme complex can be obtained by eq. (5.1)

AAA380+ 380 + 380 +⋅⋅⋅ C ε ( i ii++12) I = 280 (5.1) ε 280 280 280 CE 380 ( AAA+ii++12 + +⋅⋅⋅) i

511−− where ε 280 =2.96 × 10 M cm (converted from the percent solution extinction

1% 411−− coefficient E1cm =17.4 for BPAO) and ε380nm =1.6 × 10 M cm .

Quadrupole-time of flight (TOF) MS analysis of the inhibitor 2-derivatized BPAO

Derivatized BPAO sample was prepared by incubation of 26 µl pure BPAO (4 mg/mL) with 2 µl aqueous solution of inhibitor 2 (10 mM) for 2 h, followed by gel filtration to remove excessive inhibitors using water as eluent. Derivatized BPAO was dried by speed vacuum and reconstructed by adding 50% (v/v) acetonitrile in water containing 0.1% formic acid. The native BPAO sample in the absence of inhibitor 2 was prepared in the same way. Mass spectra of the native BPAO and derivatized BPAO were acquired on a

Q-star XL quadrupole/time of flight mass spectrometer (Applied Biosystems-MDS sciex,

Foster city, CA, USA) equipped with a nanoelectrospray ion source. The enzyme sample was introduced to the mass spectrometer by direct infusion through a capillary with the

140 flow rate at 1 µl/min. The ion spray voltage was set at 2.2 kV and interface heater temperature was 180 ℃.

Nitroblue tetrazolium (NBT) redox cycling assay and immunochemical analysis

A solution of purified BPAO (final concentration 4 mg/mL) and 1 mM 3 in 0.1 M phosphate buffer, pH 7.2, was incubated at 30 °C for 2 h, resulting in enzyme preparations that had <5% activity in comparison to a control sample incubated in the absence of inhibitor. Aliquots (20 µl) of control and inhibitor-treated solutions of BPAO were diluted with 10 µl denaturing solution (2% mercaptoethanol, 10% glycerol, 4% SDS,

0.03% bromphenol blue) and then heated at 100 °C for 6 min. The molecular weight marker and denatured solutions were applied to a polyacryamide slab gel (6% acrylamide with 0.16% bis-acrylamide) in duplicate. The two gels were run at a constant current of

0.04 amp (voltage near 200 V) and was stopped when the bromphenol blue dye reached the bottom. One gel was stained with 0.24 mM nitroblue tetrazolium in 2 M potassium glycinate, pH 10.0, for 2 h in the dark, while the other one was electroblotted to a nitrocellulose membrane using TE 22 Mighty Small Transphor Unit.

The nitrocellulose membrane was removed from the electroblotting apparatus and incubated for 1 h with 0.1 M tris-buffered saline containing 0.1% (v/v) Tween-20

(TBS-Tween) and 1% (w/v) bovine serum albumin (BSA). The blocking buffer was discarded and the membrane was incubated for 1 h at room temperature with 0.01 mg/mL

141

anti-DNP primary antibody solution (developed in goat) in TBS-Tween containing 1%

(w/v) BSA. The membrane was then washed three times (10 min each) with TBS-Tween

and incubated for another 1 h at room temperature with a 1:5000 dilution of anti-goat lgG

secondary antibody (alkaline phosphate conjugated) in TBS-Tween containing 1% (w/v)

BSA. The membrane was washed again three times (10 min each) with TBS-Tween and

then visualized using BCIP/NBT visualization stock solution. BCIP/NBT stock solution

was prepared according to the procedures described by E. Harlow and D. Lane.8

HPLC and LC-MS/MS analysis of the digested inhibitor 2-derivatized BPAO

50 µL of 5.73 mg/mL purified BPAO in 0.1 M potassium phosphate buffer, pH 7.2,

was incubated with 17 µL of 10 mM inhibitor 2 at 30 ℃ until enzyme assay showed no

more than 5% enzyme activity compared to a control sample incubated in the absence of inhibitor. The enzyme-inhibitor incubation solution was applied to a PD-10 column to

remove the excess inhibitor and the buffer salts. The eluted enzyme BPAO was

concentrated to dryness and redissolved in 50 µL of 50 mM tris buffer (pH 8) containing

6 M urea. The mixture was incubated at 37 ℃ for 1 h and then 250 µL of 50 mM

NH4HCO3 was added to dilute urea to 1 M. Proteinase was added at a ratio of BPAO:

Proteinase of 50:1 (w/w) to digest BPAO at 37 ℃. After 24 h, another equal amount of proteinase was added with additional 24 h incubation at 37℃. Four different proteinases

(trypsin, chymotrypsin, proteinase K and thermolysin) were tried in order to achieve the

142

best yield of inhibitor-modified peptides. After digestion was completed, the digested

BPAO sample was concentrated to around 50 µL and then stored at -20℃ for HPLC or

HPLC-MS/MS analysis.

Nonenzymatic digestion of enzyme BPAO was tried by using cyanogen bromide. To

50 µL of 6.8 mg/mL pure BPAO was added 3 µL of 10 mM inhibitor 2 and then incubated at 30℃. Following the complete inactivation of BPAO by inhibitor 2, the enzyme BPAO was precipitated by adding 100 µL of 15% (w/v) trichloroacetic acid and washed with 200 µL acetone twice and then dried. The enzyme BPAO was redissolved in

100 µL of 70% trifluoroacetic acid containing about 5 M cyanogen bromide. The digestion was done in a sealed tube at the room temperature. After 3 days, the digested

BPAO sample was dried by Speed Vac to remove the toxic gas. The sample was

redissolved in 70% trifluoroacetic acid before analysis.

Inhibitor 2-derivatized BPAO digests were subjected to analysis by HPLC (Shimadzu

Scientific Instruments) using a 5 µM 2.1×250 mm Reversed-Phase C18 column (Grace

Vydac), a flow rate of 200 µL/min, and a gradient mobile phase composed of

HPLC-grade solvents A [5% aqueous CH3CN containing 0.02% (v/v) TFA] and B [95% aqueous CH3CN containing 0.02% (v/v) TFA] according to the following program: 0%

B to 20% B 0-70 min, 20% B to 35% B 70-120 min, 35% B to 80% B 120-130 min, 80%

B to 100% B 130-140 min, 100% B to 0% B 140-145 min. The absorbance at three different wavelengths (220 nm and 280 nm and 380 nm) was monitored continuously.

143

LC-MS/MS analysis of inhibitor 2-derivatized BPAO digests was performed on a

HPLC (Thermo Electron Corporation) interfaced with Finnigan LCQ Deca XP Max Mass

Spectrometer (Thermo Electron Corporation). Firstly, proteolytic peptides were separated

by HPLC using a 5 µM 2.1×250 mm Reversed-Phase C18 column (Grace Vydac), a

flow rate of 200 µL/min, and a gradient mobile phase composed of HPLC-grade solvents

A [5% aqueous CH3OH containing 0.1% (v/v) FA] and B [95% aqueous CH3OH

containing 0.1% (v/v) FA] according to the following program: 0% B to 15% B 0-60 min,

15% B to 60% B 60-120 min, 60% B to 90% B 120-140 min, 90% B to 100% B 140-150

min, 100% B to 100% B 150-160 min, 100% B-0% B 160-175 min. The eluate from the

column was introduced into the mass spectrometry which was set in the positive mode.

Nitrogen was used as the sheath and auxiliary gas. The heated capillary temperature was

300 ℃, the source voltage was 4.50 kV, and the capillary voltage was 23.00 V. Two scan

events were used: (1) m/z 100-2000 full scan MS and (2) data-dependent full scan

MS/MS on the most intense ion from the MS full spectrum or from the parent mass list.

The spectra were recorded using dynamic exclusion of previously analyzed ions for 0.5 min with five repeats and a repeat duration of 0.5 min. The MS/MS collision energy was set to 35%. All data were processed with software Xcalibur 2.0.

5.6 References 1. Qiao, C.; Jeon, H.-B.; Sayre, L. M., Selective Inhibition of Bovine Plasma Amine Oxidase by Homopropargylamine, a New Inactivator Motif. J. Am. Chem. Soc. 2004, 126, (25), 8038-8045.

144

2. Zhang, N.; Casida, J. E., Novel Irreversible Butyrylcholinesterase Inhibitors: 2-Chloro-1-(substituted-phenyl)ethylphosphonic Acids. Bioorg. Med. Chem. 2002, 10, (5), 1281-1290. 3. Kitz, R.; Wilson, I. B., Esters of methanesulfonic acid as irreversible inhibitors of acetylcholinesterase. J. Biol. Chem. 1962, 237, 3245-49. 4. Kishishita, S. i.; Okajima, T.; Kim, M.; Yamaguchi, H.; Hirota, S.; Suzuki, S.; Kuroda, S. i.; Tanizawa, K.; Mure, M., Role of Copper Ion in Bacterial Copper Amine Oxidase: Spectroscopic and Crystallographic Studies of Metal-Substituted Enzymes. J. Am. Chem. Soc. 2003, 125, (4), 1041-1055. 5. Copeland, R. A.; Editor, Evaluation of Enzyme Inhibitors in Drug Discovery: A Guide to Medicinal Chemists and Pharmacologists. 2005; p 296 pp. 6. O'Connell Kimberly, M.; Langley David, B.; Shepard Eric, M.; Duff Anthony, P.; Jeon, H.-B.; Sun, G.; Freeman Hans, C.; Guss, J. M.; Sayre Lawrence, M.; Dooley David, M., Differential inhibition of six copper amine oxidases by a family of 4-(aryloxy)-2-butynamines: evidence for a new mode of inactivation. Biochemistry 2004, 43, (34), 10965-78. 7. Stoller, A.; Mioskowski, C.; Sepulchre, C.; Bellamy, F., Synthesis of new 5- and 6-exomethylenic arachidonic acid analogs. Tetrahedron Lett. 1990, 31, (3), 361-4. 8. Harlow, E.; Lane, D., Using Antibodies: A Laboratory Manual. 1999; p 495 pp.

145

Appendix

1. Enantiomeric purity of 4-phenyl-buta-2,3-dienylamine was determined by using chiral derivatizing agent (1S)-(+)-10-camphorsulfonyl chloride and 1H-NMR analysis

(Chapter 2). The chemical shifts of the protons at C10 of N-4-Phenyl-buta-2,3-dienyl-10-

1 camphorsulfonamide were well resolved for both diastereoisomers. H-NMR spectra of

N-(S)-4-phenylbuta-2,3-dienyl-(1S)-10-camphorsulfonamide (Figure A) and a 1:1

mixture of N-(S) and N-(R)-4-phenylbuta-2,3-dienyl-(1S)-10-camphorsulfonamide

(Figure B).

Me 2 Me Me1 2 Me1

R1 Ha + H2N C Ha O R2 O R Hb S 1 Cl Hb S O O NH C O O R2

Figure A

146

Figure B

2. Time-dependent inactivation of AGAO by various concentrations of allene compounds (Chapter 2).

20 µM 50 µM 100 C 80 µM NH2 100 µM

40 The remainingThe activity enzyme 20

0 10 20 30 40 50 60 Time/min

147

µ Et 100 M 100 C 200µM NH2 400µM 500µM 80

20 The remainingThe activity enzyme

0 10 20 30 40 50 60 Time/min

10 µM 110 Ph 100 µM C 500 µM 100 NH2 1 mM 90 80 70 60 50 40 30 20

The remainingThe activity enzyme 10 0 -10 0 10 20 30 40 50 60 Time/min

148

10µM 110 Ph µ C 50 M µ 100 NH 80 M 2 100µM 90

30 The remainingThe activity enzyme 20

10 0 10 20 30 40 50 60 Time/min

Et C Et NH2 100

80 100µM 300µM 60 600µM 1mM

40 The remainingThe activity enzyme 20

0 10 20 30 40 50 60 Time/min

149

Ph 110 C Ph NH 100 2

80 60µM 70 100µM 60 200µM 500µM 50

20 The remainingThe activity enzyme 10

0 0 10 20 30 40 50 60 Time/min

3. Time-dependent inactivation of BPAO by various concentrations of MPBA under aerobic condition (Chapter 4)

NH2 100 O

50 Control 5µM 40 10µM 30 20µM 80µM 20 The remainingThe activity enzyme 10

0 0 10 20 30 40 50 Time/min

150

4. Time-dependent inactivation of BPAO by various concentrations of PBA under aerobic condition (Chapter 4)

110 Control 50 µM 100 O NH2 100 µM 90 150 µM µ 80 200 M

20 The remainingThe activity enzyme 10

0 0 10 20 30 40 50 60 Time/min

5. Time-dependent inactivation of BPAO by various concentrations of PBA under anaerobic condition (Chapter 4)

100 O NH2 5µM 25µM 50µM 80

20 The remainingThe activity enzyme

0 0 5 10 15 20 Time/min

151

Bibliography 1. Awal, H. M. A.; Hirasawa, E., 1,3-Diaminopropane is a suicide substrate for pea diamine oxidase. Phytochemistry 1995, 39, (3), 489-90.

2. Boomsma, F.; Bhaggoe, U. M.; van der Houwen, A. M. B.; van den Meiracker, A. H., Plasma semicarbazide-sensitive amine oxidase in human (patho)physiology. Biochim. Biophys. Acta, Proteins Proteomics 2003, 1647, (1-2), 48-54

3. Boomsma, F.; Hut, H.; Bhaggoe, U.; van der Houwen, A.; van den Meiracker, A., Semicarbazide-sensitive amine oxidase (SSAO): from cell to circulation. Med. Sci. Monit. 2005, 11, (4), RA122-RA126.

4. Casara, P.; Jund, K.; Bey, P., General synthetic access to alpha -allenyl amine and alpha -allenyl-alpha -amino acids as potential enzyme-activated irreversible inhibitors of PLP-dependent enzymes. Tetrahedron Lett. 1984, 25, (18), 1891-4.

5. Chen, Y.; Structural basis of substrate and inhibitory activity of bovine plasma amine oxidase. Ph. D. Thesis, Case Western Reserve University, OH, 2008.

6. Cousineau, T. J.; Cook, S. L.; Secrist, J. A., III, A convenient procedure for the formation of enol acetates under basic conditions. Synth. Commun. 1979, 9, (3), 157-63.

7. Copeland, R. A.; Editor, Evaluation of Enzyme Inhibitors in Drug Discovery: A Guide to Medicinal Chemists and Pharmacologists. 2005; p 296 pp.

8. Di Paolo, M. L.; Scarpa, M.; Corazza, A.; Stevanato, R.; Rigo, A., Binding of cations of group IA and IIA to bovine serum amine oxidase: effect on the activity. Biophys. J. 2002, 83, (4), 2231-2239.

9. Di Paolo, M. L.; Stevanato, R.; Corazza, A.; Vianello, F.; Lunelli, L.; Scarpa, M.; Rigo, A., Electrostatic compared with hydrophobic interactions between bovine serum amine oxidase and its substrates. Biochem. J. 2003, 371, (2), 549-556.

10. Dura, E.; Meszaros, Z.; Salacz, G.; Magyar, K.; Romics, L.; Karadi, I., Serum semicarbazide-sensitive amine oxidase activity in patients with retinopathy in type 2 diabetes mellitus. Diabetes Res. 2001, 35, (4), 127-141.

11. Ekblom, J., Potential therapeutic value of drugs inhibiting semicarbazide-sensitive amine oxidase: vascular cytoprotection in diabetes mellitus. Pharmacol. Res. 1998, 37, (2), 87-92.

152

12. Elmore Bradley, O.; Bollinger John, A.; Dooley David, M., Human kidney diamine oxidase: heterologous expression, purification, and characterization. J Biol Inorg Chem 2002, 7, (6), 565-79.

13. Elsevier, C. J.; Vermeer, P., Synthesis and stereochemistry of allenes. Part 3. Highly stereoselective synthesis of chiral alkyl allenes by organocopper (I)-induced anti 1,3- substitution of chiral propynyl esters. J. Org. Chem. 1989, 54, (15), 3726-30.

14. Esakov, S. M.; Petrov, A. A.; Ershov, B. A., Mesomeric anions. IV. Stereochemistry of enolate anions of beta -oxoaldehydes studied by a PMR spectroscopic method. Zh. Org. Khim. 1975, 11, (4), 680-91.

15. Gokturk, C.; Nordquist, J.; Sugimoto, H.; Forsberg-Nilsson, K.; Nilsson, J.; Oreland, L., Semicarbazide-sensitive amine oxidase in transgenic mice with diabetes. Biochem. Biophys. Res. Commun. 2004, 325, (3), 1013-1020.

16. Harlow, E.; Lane, D., Using Antibodies: A Laboratory Manual. 1999; p 495 pp.

17. Hartmann, C.; Brzovic, P.; Klinman, J. P., Spectroscopic detection of chemical intermediates in the reaction of para-substituted benzylamines with bovine serum amine oxidase. Biochemistry 1993, 32, (9), 2234-41.

18. Hartmann, C.; Klinman, J. P., Structure-function studies of substrate oxidation by bovine serum amine oxidase: relationship to cofactor structure and mechanism. Biochemistry 1991, 30, (18), 4605-11.

19. Hevey, R. C.; Babson, J.; Maycock, A. L.; Abeles, R. H., Highly specific enzyme inhibitors. Inhibition of plasma amine oxidase. J Am Chem Soc 1973, 95, (18), 6125-7.

20. Holt, A.; Smith, D. J.; Cendron, L.; Zanotti, G.; Rigo, A.; Di Paolo, M. L., Multiple binding sites for substrates and modulators of semicarbazide-sensitive amine oxidases: kinetic consequences. Mol. Pharmacol. 2008, 73, (2), 525-538.

21. Jakobsson, E.; Nilsson, J.; Ogg, D.; Kleywegt Gerard, J., Structure of human semicarbazide-sensitive amine oxidase/vascular adhesion protein-1. Acta Crystallogr D Biol Crystallogr 2005, 61, (Pt 11), 1550-62.

153

22. Janes, S. M.; Palcic, M. M.; Scaman, C. H.; Smith, A. J.; Brown, D. E.; Dooley, D. M.; Mure, M.; Klinman, J. P., Identification of topaquinone and its consensus sequence in copper amine oxidases. Biochemistry 1992, 31, (48), 12147-54.

23. Janes, S. M.; Mu, D.; Wemmer, D.; Smith, A. J.; Kaur, S.; Maltby, D.; Burlingame, A. L.; Klinman, J. P. A new redox cofactor in eukaryotic enzymes: 6-hydroxydopa at the active site of bovine serum amine oxidase. Science 1990, 248, (4958), 981-7.

24. Janes, S. M.; Klinman, J. P., An investigation of bovine serum amine oxidase active site stoichiometry: evidence for an aminotransferase mechanism involving two carbonyl cofactors per enzyme dimer. Biochemistry 1991, 30, (18), 4599-605.

25. Jeon, H.-B.; Sayre, L. M., Highly potent propargylamine and allylamine inhibitors of bovine plasma amine oxidase. Biochem. Biophys. Res. Commun. 2003, 304, (4), 788-794.

26. Jeon, H.-B.; Lee, Y.; Qiao, C.; Huang, H.; Sayre, L. M., Inhibition of bovine plasma amine oxidase by 1,4-diamino-2-butenes and -2-butynes. Bioorg. Med. Chem. 2003, 11, (21), 4631-4641.

27. Jeon, H.-B.; Sun, G.; Sayre, L. M., Inactivation of bovine plasma amine oxidase by 4- aryloxy-2-butynamines and related analogs. Biochim. Biophys. Acta, Proteins Proteomics 2003, 1647, (1-2), 343-354.

28. Journet, M.; Cai, D.; DiMichele, L. M.; Larsen, R. D., Highly efficient synthesis of alpha ,beta -acetylenic aldehydes from terminal alkynes using DMF as the formylating reagent. Tetrahedron Lett. 1998, 39, (36), 6427-6428.

29. Jung, M. J.; Metcalf, B. W., Catalytic inhibition of gamma-aminobutyric acid - alpha- ketoglutarate transaminase of bacterial origin by 4-aminohex-5-ynoic acid, a substrate analog. Biochem Biophys Res Commun 1975, 67, (1), 301-6.

30. Karadi, I.; Meszaros, Z.; Csanyi, A.; Szombathy, T.; Hosszufalusi, N.; Romics, L.; Magyar, K., Serum semicarbazide-sensitive amine oxidase (SSAO) activity is an independent marker of carotid atherosclerosis. Clin. Chim. Acta 2002, 323, (1-2), 139- 146.

31. Kawai, S.; Kasashima, K.; Tomita, M., High-performance liquid chromatographic determination of malonaldehyde in serum. J. Chromatogr., Biomed. Appl. 1989, 495, 235-8.

154

32. Kim, J.; Zhang, Y.; Ran, C.; Sayre, L. M., Inactivation of bovine plasma amine oxidase by haloallylamines. Bioorg. Med. Chem. 2006, 14, (5), 1444-1453.

33. Kishishita, S. i.; Okajima, T.; Kim, M.; Yamaguchi, H.; Hirota, S.; Suzuki, S.; Kuroda, S. i.; Tanizawa, K.; Mure, M., Role of Copper Ion in Bacterial Copper Amine Oxidase: Spectroscopic and Crystallographic Studies of Metal-Substituted Enzymes. J. Am. Chem. Soc. 2003, 125, (4), 1041-1055.

34. Kitz, R.; Wilson, I. B., Esters of methanesulfonic acid as irreversible inhibitors of acetylcholinesterase. J. Biol. Chem. 1962, 237, 3245-49.

35. Krantz, A.; Kokel, B.; Sachdeva, Y. P.; Salach, J. I.; Detmer, K.; Claesson, A.; Sahlberg, C., Inactivation of mitochondrial monoamine oxidase by beta ,gamma ,delta - allenic amines. Monoamine Oxidase: Struct., Funct., Altered Funct., [Proc. Symp.] 1979, 51-70.

36. Kwon, T.-W.; Van der Veen, J., Ultraviolet and infrared absorption spectra of malonaldehyde in organic solvents. J. Agr. Food Chem. 1968, 16, (4), 639-42.

37. Lazar, L.; Szakonyi, Z.; Forro, E.; Palko, M.; Zalan, Z.; Szatmari, I.; Fulop, F., Synthesis of hydrazino alcohols with anti-inflammatory activity. Acta Pharm Hung 2004, 74, (1), 11-8.

38. Lee, Y.; Sayre, L. M., Model studies on the quinone-containing copper amine oxidases. Unambiguous demonstration of a transamination mechanism. J. Am. Chem. Soc. 1995, 117, (48), 11823-8.

39. Lee, Y.; Ling, K.-Q.; Lu, X.; Silverman, R. B.; Shepard, E. M.; Dooley, D. M.; Sayre, L. M., 3-Pyrrolines Are Mechanism-Based Inactivators of the Quinone-Dependent Amine Oxidases but Only Substrates of the Flavin-Dependent Amine Oxidases. J. Am. Chem. Soc. 2002, 124, (41), 12135-12143.

40. Li, R.; Klinman, J. P.; Mathews, F. S., Copper amine oxidase from Hansenula polymorpha: the crystal structure determined at 2.4 .ANG. resolution reveals the active conformation. Structure (London) 1998, 6, (3), 293-307.

41. Ling, K.-Q.; Sayre, L. M., Discovery of a Sensitive, Selective, and Tightly Binding Fluorogenic Substrate of Bovine Plasma Amine Oxidase. J. Org. Chem. 2009, 74, (1), 339-350.

155

42. Liu, W.; Rogers, C. J.; Fisher, A. J.; Toney, M. D., Aminophosphonate Inhibitors of Dialkylglycine Decarboxylase: Structural Basis for Slow Binding Inhibition. Biochemistry 2002, 41, (41), 12320-12328.

43. Lunelli, M.; Di Paolo, M. L.; Biadene, M.; Calderone, V.; Battistutta, R.; Scarpa, M.; Rigo, A.; Zanotti, G., Crystal structure of amine oxidase from bovine serum. J. Mol. Biol. 2005, 346, (4), 991-1004.

44. Lyles, G. A.; Marshall, C. M.; McDonald, I. A.; Bey, P.; Palfreyman, M. G., Inhibition of rat aorta semicarbazide-sensitive amine oxidase by 2-phenyl-3- haloallylamines and related compounds. Biochem Pharmacol 1987, 36, (17), 2847-53.

45. Martinez, A. M.; Cushmac, G. E.; Rocek, J., Chromic acid oxidation of cyclopropanols. J. Am. Chem. Soc. 1975, 97, (22), 6502-10.

46. Mathys, K. C.; Ponnampalam, S. N.; Padival, S.; Nagaraj, R. H., Semicarbazide- sensitive amine oxidase in aortic smooth muscle cells mediates synthesis of a methylglyoxal-AGE: implications for vascular complications in diabetes. Biochem. Biophys. Res. Commun. 2002, 297, (4), 863-869.

47. Matyus, P.; Dajka-Halasz, B.; Foeldi, A.; Haider, N.; Barlocco, D.; Magyar, K., Semicarbazide-sensitive amine oxidase: Current status and perspectives. Curr. Med. Chem. 2004, 11, (10), 1285-1298.

48. McDonald, I. A.; Foot, J.; Yin, P.; Flening, E.; van Dam, E. M., Semicarbazide sensitive amine oxidase and vascular adhesion protein-1: one protein being validated as a therapeutic target for inflammatory diseases. Annu. Rep. Med. Chem. 2007, 42, 229-243.

49. Messerschmidt, A.; Huber, R.; Poulos, T.; Wieghardt, K.; Editors, Handbook of Metalloproteins, Volume 2. 2001; p 785 pp.

50. Mills, S. A.; Goto, Y.; Su, Q.; Plastino, J.; Klinman, J. P., Mechanistic Comparison of the Cobalt-Substituted and Wild-Type Copper Amine Oxidase from Hansenula polymorpha. Biochemistry 2002, 41, (34), 10577-10584.

156

51. Mura, A.; Pintus, F.; Fais, A.; Porcu, S.; Corda, M.; Spano, D.; Medda, R.; Floris, G., Tyramine oxidation by copper/TPQ amine oxidase and peroxidase from Euphorbia characias latex. Arch. Biochem. Biophys. 2008, 475, (1), 18-24.

52. Murakawa, T.; Okajima, T.; Kuroda, S. i.; Nakamoto, T.; Taki, M.; Yamamoto, Y.; Hayashi, H.; Tanizawa, K., Quantum mechanical hydrogen tunneling in bacterial copper amine oxidase reaction. Biochem. Biophys. Res. Commun. 2006, 342, (2), 414-423.

53. Mure, M.; Mills, S. A.; Klinman, J. P., Catalytic Mechanism of the Topa Quinone Containing Copper Amine Oxidases. Biochemistry 2002, 41, (30), 9269-9278.

54. Mure, M.; Klinman, J. P., Model Studies of Topaquinone-Dependent Amine Oxidases. 2. Characterization of Reaction Intermediates and Mechanism. J. Am. Chem. Soc. 1995, 117, (34), 8707-18.

55. Northrop, D. B., Minimal kinetic mechanism and general equation for deuterium isotope effects on enzymic reactions: uncertainty of detecting a rate-limiting step. Biochemistry 1981, 20, (14), 4056-61.

56. O'Connell Kimberly, M.; Langley David, B.; Shepard Eric, M.; Duff Anthony, P.; Jeon, H.-B.; Sun, G.; Freeman Hans, C.; Guss, J. M.; Sayre Lawrence, M.; Dooley David, M., Differential inhibition of six copper amine oxidases by a family of 4-(aryloxy)-2- butynamines: evidence for a new mode of inactivation. Biochemistry 2004, 43, (34), 10965-78.

57. O'Rourke Anne, M.; Wang Eric, Y.; Miller, A.; Podar Erika, M.; Scheyhing, K.; Huang, L.; Kessler, C.; Gao, H.; Ton-Nu, H.-T.; Macdonald Mary, T.; Jones David, S.; Linnik Matthew, D., Anti-inflammatory effects of LJP 1586 [Z-3-fluoro-2-(4- methoxybenzyl)allylamine hydrochloride], an amine-based inhibitor of semicarbazide- sensitive amine oxidase activity. J Pharmacol Exp Ther 2008, 324, (2), 867-75.

58. O'Sullivan, J.; Unzeta, M.; Healy, J.; O'Sullivan, M. I.; Davey, G.; Tipton, K. F., Semicarbazide-sensitive amine oxidases: enzymes with quite a lot to do. Neurotoxicology 2004, 25, (1-2), 303-315.

157

59. Palcic, M. M.; Klinman, J. P., Isotopic probes yield microscopic constants: separation of binding energy from catalytic efficiency in the bovine plasma amine oxidase reaction. Biochemistry 1983, 22, (25), 5957-66.

60. Paz, M. A.; Fluckiger, R.; Boak, A.; Kagan, H. M.; Gallop, P. M., Specific detection of quinoproteins by redox-cycling staining. J Biol Chem 1991, 266, (2), 689-92.

61. Plastino, J.; Green, E. L.; Sanders-Loehr, J.; Klinman, J. P., An unexpected role for the active site base in cofactor orientation and flexibility in the copper amine oxidase from Hansenula polymorpha. Biochemistry 1999, 38, (26), 8204-16.

62. Protopopova, T. V.; Skoldinov, A. P., beta -Acyloxyacroleins. Zh. Obshch. Khim. 1958, 28, 240-3.

63. Qiao, C. New copper amine oxidase probes: Synthesis, mechanism, and enzymology. Ph. D. Thesis, Case Western Reserve University, OH, 2004.

64. Qiao, C.; Ling, K.-Q.; Shepard, E. M.; Dooley, D. M.; Sayre, L. M., Mechanism- Based Cofactor Derivatization of a Copper Amine Oxidase by a Branched Primary Amine Recruits the Oxidase Activity of the Enzyme to Turn Inactivator into Substrate. J. Am. Chem. Soc. 2006, 128, (18), 6206-6219.

65. Qiao, C.; Jeon, H.-B.; Sayre, L. M., Selective Inhibition of Bovine Plasma Amine Oxidase by Homopropargylamine, a New Inactivator Motif. J. Am. Chem. Soc. 2004, 126, (25), 8038-8045.

66. Salmi, M.; Jalkanen, S., VAP-1: an adhesin and an enzyme. Trends Immunol 2001, 22, (4), 211-6.

67. Schwartz, B.; Olgin, A. K.; Klinman, J. P., The role of copper in topa quinone biogenesis and catalysis, as probed by azide inhibition of a copper amine oxidase from yeast. Biochemistry 2001, 40, (9), 2954-63.

68. Silverman, R. B., Mechanism-based enzyme inactivators. Methods Enzymol 1995, 249, 240-83.

158

69. Silverman. R. B.; Mechanism-based enzyme inactivation: Chemistry and Enzymology, CRC Press, Inc.: Boca Raton, FL, 1988

70. Stolen Craig, M.; Marttila-Ichihara, F.; Koskinen, K.; Yegutkin Gennady, G.; Turja, R.; Bono, P.; Skurnik, M.; Hanninen, A.; Jalkanen, S.; Salmi, M., Absence of the endothelial oxidase AOC3 leads to abnormal leukocyte traffic in vivo. Immunity 2005, 22, (1), 105-15.

71. Stolen, C. M.; Yegutkin, G. G.; Kurkijaervi, R.; Bono, P.; Alitalo, K.; Jalkanen, S., Origins of Serum Semicarbazide-Sensitive Amine Oxidase. Circ. Res. 2004, 95, (1), 50- 57.

72. Stoller, A.; Mioskowski, C.; Sepulchre, C.; Bellamy, F., Synthesis of new 5- and 6- exomethylenic arachidonic acid analogs. Tetrahedron Lett. 1990, 31, (3), 361-4.

73. Su, Q.; Klinman, J. P., Probing the mechanism of proton coupled electron transfer to dioxygen: the oxidative half-reaction of bovine serum amine oxidase. Biochemistry 1998, 37, (36), 12513-25.

74. Waley, S. G., Kinetics of suicide substrates. Practical procedures for determining parameters. Biochem. J. 1985, 227, (3), 843-9.

75. Waley, S. G., Kinetics of suicide substrates. Biochem. J. 1980, 185, (3), 771-3.

76. Wilce, M. C.; Dooley, D. M.; Freeman, H. C.; Guss, J. M.; Matsunami, H.; McIntire, W. S.; Ruggiero, C. E.; Tanizawa, K.; Yamaguchi, H., Crystal structures of the copper- containing amine oxidase from Arthrobacter globiformis in the holo and apo forms: implications for the biogenesis of topaquinone. Biochemistry 1997, 36, (51), 16116-33.

77. Wilmot, C. M.; Murray, J. M.; Alton, G.; Parsons, M. R.; Convery, M. A.; Blakeley, V.; Corner, A. S.; Palcic, M. M.; Knowles, P. F.; McPherson, M. J.; Phillips, S. E., Catalytic mechanism of the quinoenzyme amine oxidase from Escherichia coli: exploring the reductive half-reaction. Biochemistry 1997, 36, (7), 1608-20.

78. Wimalasena, K.; Haines, D. C., A general progress curve method for the kinetic analysis of suicide enzyme inhibitors. Anal. Biochem. 1996, 234, (2), 175-82.

159

79. Zaimis, E. J., The synthesis of methonium compounds, their isolation from urine, and their photometric determination. Br J Pharmacol Chemother 1950, 5, (3), 424-30.

80. Zhang, Q.; Mashima, Y.; Noda, S.; Imamura, Y.; Kudoh, J.; Shimizu, N.; Nishiyama, T.; Umeda, S.; Oguchi, Y.; Tanaka, Y.; Iwata, T., Characterization of AOC2 gene encoding a copper-binding amine oxidase expressed specifically in retina. Gene 2003, 318, 45-53.

160