KINETICS AND MECHANISTIC STUDY OF ADENOSINE MONOPHOSPHATE

DISODIUM SALT (AMPNA2) IN ACIDIC AND ALKALINE MEDIA

By

BELJIT KAUR

A dissertation submitted to the Department of Chemical Science,

Faculty of Science,

Universiti Tunku Abdul Rahman,

in partial fulfillment of the requirements for the degree of

Master of Science

March 2017

ii

ABSTRACT

KINETICS AND MECHANISTICTIC STUDY OF ADENOSINE

MONOPHOSPHATE DISODIUM SALT (AMPNA2) IN ACIDIC AND

ALKALINE MEDIA

Beljit Kaur

Phosphate ester hydrolysis essential in intracellular signaling, energy storage

and production, information storage and DNA repair. However, the mechanism

of this process remains poorly understood. In this investigation, adenosine

monophosphate disodium salt (AMPNa2) was used as the model substrate to

mimic phosphate ester linkages that are present in a natural nucleotide,

adenosine monophosphate (AMP) and understand the mechanism of phosphate

ester hydrolysis in natural AMP. Hydrolysis of AMPNa2 was carried out in

alkaline, acidic and neutral conditions ranging from pH 0.30-12.71 at 60 °C and

the reaction was monitored spectrophotometrically by using a UV-Vis

Spectrophotometer. The rate ranged between (1.20 ± 0.10) × 10-7 s-1 to (4.44 ±

0.05) × 10-6 s-1 at [NaOH] from 0.0008 M to 1.0000 M. The second-order base-

catalyzed rate constant, kOH obtained was 4.32 × 10-6 M-1 s-1 and uncatalysed

rate constant, ko obtained was 6.30 × 10-8 s-1. In acidic conditions, the rate

ranged between (1.32 ± 0.06) × 10-7 s-1 to (1.67 ± 0.10) × 10-6 s-1 at [HCl] from

0.01 M to 1.00 M. Second-order acid-catalyzed rate constant, kH obtained was

1.62 × 10-6 M-1 s-1 and uncatalysed rate constant, ko obtained was 1.03 × 10-8 s-

1. Rate of reaction for the neutral region was studied by extrapolating the rates

iii

of the acid and base catalyzed reactions to neutral pH. The hydrolytic product

characterization was confirmed with Fourier Transform Infrared Spectroscopy

and Liquid Chromatography Mass Spectrometry (LC-MS). Mechanisms were

proposed to explain the hydrolysis of natural AMP in accordance with the

characterization results for both acidic and basic conditions. In conclusion, the

cleavage of phosphate ester bond in adenosine monophosphate disodium salt

(AMPNa2) readily occurred in basic and acidic conditions. N-glycosidic

cleavage also occurred in acidic medium. This investigation has provided us

more information on the kinetics and mechanism of cleavage of natural AMP

and also the enzymes that facilitates its cleavage.

iv

ACKNOWLEDGEMENTS

I would like to thank Associate Professor Dr. Sim Yoke Leng and

Assistant Professor Dr. Yip Foo Win for their patient supervision, guidance,

supports and encouragements throughout the period of this research work.

I would like to thank University Tunku Abdul Rahman for funding my

research. I am also grateful to the lab officers of the Faculty of Science of

University Tunku Abdul Rahman for their help and assistance throughout the

period of this study.

Last but not least, I am grateful to God, for giving me the courage and

strength. Special thanks to my mother and friends who always encouraged me.

v

APPROVAL SHEET

This dissertation/thesis entitled “KINETICS AND MECHANISTIC STUDY

OF ADENOSINE MONOPHOSPHATE DISODIUM SALT (AMPNA2) IN

ACIDIC AND ALKALINE MEDIA” was prepared by BELJIT KAUR and

submitted as partial fulfillment of the requirements for the degree of Master of

Science at Universiti Tunku Abdul Rahman.

Approved by:

___________________________

(Dr. SIM YOKE LENG)

Date:

Supervisor

Department of Chemical Science

Faculty of Science

Universiti Tunku Abdul Rahman

___________________________

(Dr. YIP FOO WIN)

Date:

Co-supervisor

Department of Chemical Science

Faculty of Science

Universiti Tunku Abdul Rahman

vi

FACULTY OF SCIENCE

UNIVERSITI TUNKU ABDUL RAHMAN

Date:_________________

SUBMISSION OF THESIS

It is hereby certified that Beljit Kaur_ (ID No: 1308002) has completed

this dissertation entitled “Kinetics and mechanistic study of adenosine

monophosphate disodium salt (AMPNa2) in acidic and alkaline media”

under the supervision of Dr. Sim Yoke Leng (Supervisor) from the

Department of Chemical Science, Faculty of Science, and Dr. Yip Foo Win

(Co-Supervisor)* from the Department of Chemical Science, Faculty of

Science.

I understand that University will upload softcopy of my dissertation in pdf

format into UTAR Institutional Repository, which may be made accessible

to UTAR community and public.

Yours truly,

____________________

(Beljit Kaur)

vii

DECLARATION

I Beljit Kaur hereby declare that the dissertation is based on my original work

except for quotations and citations which have been duly acknowledged. I also

declare that it has not been previously or concurrently submitted for any other

degree at UTAR or other institutions.

Name ________________

(BELJIT KAUR)

Date ____________________

viii

TABLE OF CONTENTS

Page

ABSTRACT ii

ACKNOWLEDGEMENTS iv

APPROVAL SHEET v

SUBMISSION SHEET vi

DECLARATION vii

LIST OF TABLES xi

LIST OF FIGURES xii

LIST OF ABBREVIATIONS xv

CHAPTER

1.0 INTRODUCTION 1 1.1 Phosphate esters 1

1.2 Type of Phosphate Esters 2

1.3 Phosphate Ester Cleavage Studies 5

1.3.1 Phosphate ester cleavage studies with natural

substrates 5

1.3.2 Phosphate ester cleavage studies with

non-natural substrates 6

1.3.3 Nucleotides/ nucleosides as phosphate ester

Models 6

1.4 Adenosine Monophosphate, Adenosine and Adenine 8

1.5 Mechanism of Cleavage by Enzymes 11

1.6 Importance of Studying Cleavage of Phosphate Esters,

AMP and Adenosine 13

1.7 Problem Statement 15

1.8 Objectives 16

1.9 Scope of Work 17

2.0 LITERATURE REVIEW 19

2.1 Acid Base Catalysis Studies of Phosphate Monoesters 19

2.1.1 Specific acid and base catalysis of phosphate

monoesters 19

2.1.2 General acid and base catalysis of phosphate

monoesters 23

2.2 Possible Pathways of Phosphate Monoesters Hydrolysis 26

2.3 Choice of Substrate 27

2.4 Mechanisms Competing with P-O Bond Cleavage 31

2.4.1 C-O Bond Cleavage 31

2.4.2 N-glycosidic bond cleavage 32

2.5 Ionic Strength 34

2.6 Characterization Methods 35

2.6.1 NMR Spectroscopy 35

ix

2.6.2 Mass Spectrometry 36

2.6.3 FTIR Spectroscopy 37

2.6.4 UV/Vis Spectroscopy 38

3.0 MATERIALS AND METHODS 42

3.1 Materials Used 42

3.2 Apparatus 43

3.3 Procedure 43

3.3.1 Kinetic Study of the Nucleotide Analogue, 43

AMPNa2

3.3.2 Characterization of the Products 47

3.3.2.1 UV-Vis Spectrometry 47

3.3.2.2 Fourier Transform Infrared Spectroscopy 48

(Perkin Elmer Spectrum ex1)

3.3.2.3 LC-MS Spectrometry 48

3.4 Derivation of the Equation used for all Kinetic 49

Measurements

4.0 RESULTS AND DISCUSSION 53

4.1 Hydrolysis of AMPNa2 in Alkaline, Acidic and Neutral 53

Media

4.2 Specific Base Hydrolysis of AMPNa2 54

4.2.1 UV-Vis Spectrum of Hydrolysis of AMPNa2 in 54

Alkaline Medium

4.2.2 Kinetic Study of Hydrolysis of AMPNa2 55

in Alkaline Medium

4.2.3 Mechanism of Hydrolysis of AMPNa2 in 62

Alkaline Conditions

4.3 Specific Acid Hydrolysis of AMPNa2 75

4.3.1 UV-Vis Spectrum of Hydrolysis of AMPNa2 in 75

Acidic Medium

4.3.2 Kinetic Study of Hydrolysis of AMPNa2 in 77

Acidic Medium

4.3.3 Mechanism of Hydrolysis of AMPNa2 in 83

Acidic Medium

4.4 General Acid and Base Hydrolysis of AMPNa2 91

4.4.1 Spectra of General Acid and Base Hydrolysis 91

of AMPNa2

4.4.2 Kinetic Study of General Acid and Base 93

Hydrolysis of AMPNa2

4.5 pH Rate Profile 100

4.6 NMR Spectroscopy as Characterization Method

for the hydrolysis of AMPNa2 107

4.7 Effect of Ionic Strength on the Rate of Hydrolysis of

AMPNa2 in Alkaline Medium 108

4.8 Comparison with Enzymatic Cleavage Rate of AMP 109

4.8.1 Alkaline Hydrolysis of AMPNa2 109

4.8.2 Acidic Hydrolysis of AMPNa2 110

4.9 Further Studies 113

x

5.0 CONCLUSION 115

xi

LIST OF TABLES

Table

2.1

The values for absorption maxima and extinction

coefficients of several purine and pyrimidine bases

and their derivatives

Page

40

3.1 Materials used in the preparations of sample

solutions

42

3.2 Chemicals used for each pH range for sample

solutions

44

4.1 Values of concentration, pH before, pH after, kobs,

kcalc, Eapp and A∞ for alkaline-hydrolysis of 0.0001

M AMPNa2 at 60 °C

58

4.2 Second-order base-catalysed rate constant values of

phosphate ester hydrolysis in previous study and

present study

62

4.3 Peak assignments of IR absorbance spectra for the

substrate, residue and the filtrate obtained in

alkaline hydrolysis of AMPNa2 at 60 °C

65

4.4 Comparison of FTIR peak assignments for filtrate

and literature spectrum of adenosine

67

4.5 Values of concentration, pH before, pH after, kobs,

kcalc, Eapp and A0 for acidic-hydrolysis of 0.0001 M

AMPNa2 at 60 °C

80

4.6 Peak assignments of IR absorbance spectra for the

substrate, product obtained in acidic hydrolysis of

AMPNa2 at 60 °C

86

4.7 Formula, molecular weight, M+1, and

corresponding fragments for acidic hydrolysis of

AMPNa2 at 60 °C

89

4.8 Values of composition, pH before, pH after, kobs,

Eapp and A∞ for general acid hydrolysis of 0.0001 M

AMPNa2 at 60 °C

96

4.9 Values of composition, pH before, pH after, kobs,

Eapp and A0 for general base of 0.0001 M AMPNa2

at 60 °C

97

4.10 Rate of hydrolysis of acidic hydrolysis of acid

nucleosides at pH 1 and 37 °C

112

xii

LIST OF FIGURES

Figures

1.1

Mechanism for hydrolysis of phosphate monoesters

Page

3

1.2 Possible mechanisms for hydrolysis of trimethyl

phosphate

4

1.3 Structure of natural AMP 9

1.4 Structure of adenosine 9

1.5 Structure of adenine 11

2.1 The possible pathways of phosphate monoester in

specific acidic medium

21

2.2 Possible mechanisms of phosphate monoester

hydrolysis in basic conditions

22

2.3 Mechanism of general acid catalysis in phosphate

monoester

26

2.4 Structure of adenosine monophosphate disodium

salt

28

2.5 Reaction pathway for P-O bond cleavage of AMP

using UV-photodissociation

30

2.6 Mechanism of C-O bond cleavage in protonated

adenosine monophosphate

31

2.7 Mechanism for glycosidic bond cleavage by

intramolecular E2 reaction

33

2.8 Mechanism for glycosidic bond cleavage by

heterolytic cleavage

34

4.1 UV-Vis absorption spectrum of alkaline hydrolysis

of AMPNa2 at [NaOH] 1.0 M at 60 °C

54

4.2 Alkaline hydrolysis of AMPNa2 in the presence of

[NaOH] 1.0 M at 60 °C. A decrease in the

absorbance with time was observed and the solid

line was drawn through the calculated absorbance

values with kobs = 4.44 × 10-6 s-1, Eapp = 8466 ± 44

M-1 cm-1, and A∞ = 0.294 ± 0.003 using Equation 3

56

4.3 Pseudo-first-order rate constant, kobs versus [NaOH]

for alkaline hydrolysis of 0.0001 M AMPNa2 at

60 °C calculated using Equation 3

59

4.4 Proposed mechanism of alkaline-hydrolysis of

AMPNa2 under basic condition with OH- acting as

a nucleophile

63

4.5 Comparison of FTIR spectra of the substrate,

residue and the filtrate for alkaline hydrolysis of

0.0001 M AMPNa2 in the presence of [NaOH] 1.0

M at 60 °C

64

4.6 FTIR spectrum of adenosine obtained from Spectral

Database for Compounds SDBS

67

4.7 Proposed mechanism for the formation of the final

product

68

xiii

4.8 Positive-ion LC-MS spectrum of the final product

of alkaline hydrolysis of 0.0001 M AMPNa2 in

[NaOH] 1.0 M at 60 °C

69

4.9 Positive-ion LC-MS spectrum of freshly prepared

0.0001 M AMPNa2 in distilled water

70

4.10 Structure of protonated AMPNa2 corresponding to

m/z = 393.2102

71

4.11 Structure of protonated AMPNa2 corresponding to

m/z =349.1837

71

4.12 Positive-ion LC-MS spectrum of AMPNa2 in

distilled water undergoing self-hydrolysis

72

4.13 Mechanism of AMPNa2 undergoing self-hydrolysis

into adenosine

73

4.14 Possible structure for residue (adenine phosphate) 74

4.15 UV-Vis absorption spectrum of acidic hydrolysis of

AMPNa2 in [HCl] 1.0 M at 60 °C

76

4.16 Acidic hydrolysis of AMPNa2 in the presence of

[HCl] 1.0 M at 60 °C. An increase in the absorbance

with time was observed and the solid line was

drawn through the calculated data points with kobs =

1.67 × 10-6 s-1, Eapp = 3688 ± 91 M-1 cm-1, and A∞ =

0.757 ± 0.009

78

4.17 Pseudo-first-order rate constant, kobs versus [HCl]

for acidic hydrolysis of 0.0001 M AMPNa2 at 60 °C

calculated using Equation 4

81

4.18 Proposed mechanism of acidic hydrolysis of

AMPNa2 under acidic condition with H+ acting as a

protonating agent

84

4.19 Comparison of IR spectra of AMPNa2 and product

of acidic hydrolysis of 0.0001 M AMPNa2 in the

presence of [HCl] 1.0 M at 60 °C

85

4.20 The mechanism of N-glycosidic bond cleavage of

AMPNa2 in acidic conditions at 60 °C

87

4.21 Positive-ion LC-MS spectrum of the product of

acidic hydrolysis of 0.0001 M AMPNa2 in [HCl] 1.0

M at 60 °C

88

4.22 UV-Vis absorption spectrum of general acid

hydrolysis of AMPNa2 in glycine-HCl at pH 1.82 at

60 °C

92

4.23 UV-Vis absorption spectrum of general base

hydrolysis of AMPNa2 in TRIS-HCl at pH 8.03 at

60 °C

93

4.24 General acid hydrolysis of AMPNa2 at pH 1.82 in

20:80% glycine-HCl at 60 °C. A decrease in the

absorbance with time was observed and the solid

line was drawn through the calculated absorbance

values with kobs = 4.21 × 10-7 s-1, Eapp = 1533 ±

474 M-1 cm-1, and A∞ = 0.850 ± 0.051 using

Equation 3

94

xiv

4.25 General base hydrolysis of AMPNa2 at pH 8.03 in

80%:20% TRIS-HCl at 60 °C. An increase in the

absorbance with time was observed and the solid

line was drawn through the calculated data points

with kobs = 9.00 × 10-8 s-1, Eapp = 2335 ± 743 M-1

cm-1, and A∞ = 1.087 ± 0.004 using Equation 4

95

4.26 UV-Vis absorption spectrum of general acid and

base hydrolysis of AMPNa2 in HEPES buffer at pH

7.06 at 60 °C

98

4.27 Absorbance versus time for hydrolysis of AMPNa2

at pH 7.03 in 50:50% HEPES: NaOH at 60 °C. No

consistent changes observed on the absorbance

values

99

4.28 A plot of log kobs against pH of 18 samples for the

hydrolysis of AMPNa2 at 60 °C in reaction media

with various concentration

100

4.29 Structure of TRIS buffer 102

4.30 Mechanism of hydrolysis of AMPNa2 in TRIS-HCl

medium

103

4.31 Structure of glycine acidified with hydrochloric

acid

104

4.32 Mechanism of hydrolysis of AMPNa2 in glycine

buffer

105

4.33 The possible mechanism of anomerization of

adenosine at pH 7

106

xv

LIST OF ABBREVIATION

AMP

Adenosine Monophosphate

AMPNa2

Adenosine Monophosphate Disodium Salt

kcalc

Calculated rate constant

kobs

Observed rate constant

PO

Phosphodiester

AP

Alkaline Phosphatase

UV-Vis

Ultraviolet Visible

FTIR

Fourier Transform Infrared

LCMS

Liquid Chromatography Mass Spectrometry

MES

2-(N-morpholino)ethanesulfonic acid

TRIS

tris(hydroxymethyl)aminomethane

HEPES

4-(2-hydroxyethyl)-1-piperazineethanesulfonic

acid

cAMP

Cyclic Adenosine Monophosphate

cGMP

Cyclic Guanosine Monophosphate

RNA

Ribonucleic acid

AMPase

Adenosine monophosphate nucleotidases

DNA

Deoxyribonucleic acid

HPLC

High Performance Liquid Chromatography

FDA

Food and Drug Administration

xvi

CHAPTER 1

INTRODUCTION

1.1 Phosphate Esters

Phosphate esters are the most common chemical functional group in our

body as it involves many processes in the human body. Among some of the

important processes are the production of cellular energy which involves ATP and

phosphoenolpyruvate, essential part of nucleic acids, as an important component of

cell membrane, and most importantly storage of genetic information.

Phosphodiester linkages that are found in RNA and DNA are suitable for the storage

of genetic information as these linkages are very stable (Banaszczyk, 1989).

It is often very hard for chemists to study the mechanism of phosphate esters

as the cleavage rates are extremely slow in neutral conditions and also due to its

complicated mechanism (Banaszczyk, 1989). These phosphate esters are highly

stable as they have an estimated half-life of 3 × 109 years at pH 6.8 and 25 °C and

only selected nucleases and phosphatases can accelerate the cleavage rate by factors

up to 1016 and 1021 (Desbouis et al., 2012).

2

1.2 Types of Phosphate Esters

Phosphate esters can be divided into three main categories, phosphate

monoester, phosphate diester and phosphate triester. Among all the three phosphate

esters, phosphate monoesters have the most complex mechanism as there are three

possible mechanisms for the reaction to proceed depending on the pH of the

reaction. The reaction could proceed through P-O bond cleavage, C-O bond

cleavage and alcohol elimination through a metaphosphate intermediate. Generally,

the hydrolysis proceeds through P-O bond cleavage through a metaphosphate

intermediate at ambient temperatures at pH >1. An example of a phosphate

monoester that goes thru hydrolysis following this mechanism is methylphosphate.

Evidence has been found that hydrolysis of phosphate monoester could proceed

through a dissociative pathway rather than an associative pathway. Dissociative

pathway proceeds via the hydrated PO-3 and involves the formation of a

metaphosphate (Banaszczyk, 1989; Florián et al., 1998). At highly acidic

conditions such as pH < 0, the hydrolysis of phosphate esters may proceed via a C-

O bond cleavage. In the hydrolysis of methyl phosphate, water attacks the

phosphorus centre leading to P-O cleavage only occur from pH 0-1 (Banaszczyk,

1989). Figure 1.1 shows the reaction pathway of hydrolysis of phosphate

monoester.

3

Figure 1.1: Mechanism for hydrolysis of phosphate monoesters (Banaszczyk,

1989).

Phosphate diesters have the slowest cleavage reaction rates among all the

types of phosphate esters as phosphate diesters are extremely stable. There have

been few studies on the mechanism of phosphate diester cleavage due to the fact

that it is stable. In the hydrolysis of dimethylphosphate (DMP) and

dibenzylphosphate, C-O bond cleavage was observed and the hydrolysis of the

anion of each diester proceeded very slowly (Banaszczyk, 1989). Accurate

measurements of the displacement process were not done due to the slowness of

the hydrolysis. However, the rates of hydrolysis of five membered ring cyclic esters

were far greater than the rates of hydrolysis of simple dialkyl phosphate esters.

Phosphate diesters with hydroxyl and carboxyl neighbouring groups were

hydrolysed at higher rates. The hydrolysis proceeded with ease in phosphate esters

with good leaving groups such as bis-(p-nitrophenyl)phosphate (BNPP) and bis-

(2,4-dinitrophenyl)phosphate (BDNPP) through bimolecular nucleophilic attack at

the phosphorus atom (Banaszczyk, 1989). Density functional theory experiments

often included dimethylphosphate to figure out the potential energy surface for

phosphodiester hydrolysis. It was found that water and hydroxide ion both acted as

nucleophiles although they may differ in their involvement in the mechanism. With

4

hydroxide, the cleavage was a one-step mechanism while with water acting as the

nucleophile, it was much more complex as it involved a total of three steps and two

intermediates (Ribeiro et al., 2010).

Among the three phosphate esters, phosphate triesters are the most easily

hydrolysed. This is due to the lack of repulsive interaction from the negatively

charged oxygen at the phosphorane group towards the incoming nucleophile.

However, the subsequent steps of the hydrolysis are quite slow. At pH > 10 the

hydrolysis proceeds through P-O bond cleavage while at pH < 10, the hydrolysis

proceeds through C-O bond cleavage as depicted in Figure 1.2 (Banaszczyk, 1989).

Figure 1.2: Possible mechanisms for hydrolysis of trimethyl phosphate

(Banaszczyk, 1989)

Cyclic phosphotriesters such as methyl ethylene phosphate (MEP) has a

hydrolysis rate six orders of magnitude faster than trimethyl phosphate. This is due

P-O bond cleavage C-O bond cleavage

5

to the presence of stereoelectronic effect and release of ring strain during transition

state of the hydrolysis reaction (Banaszczyk, 1989).

1.3 Phosphate Ester Cleavage Studies

1.3.1 Phosphate ester cleavage studies with natural substrates

Previously, naturally occurring DNA or RNA sequences such as the 20-mer

sequence (which has 20 nucleotides) and the 31-mer sequence (which has 31

nucleotides) have been utilized in order to understand their cleavage activity

(Desbouis et al., 2012). However, these sequences are troublesome because they

involve separation, detection and identification of cleavage products (Desbouis et

al., 2012). Among the processes involved are radio-labelling, gel purification and

identifying the cleavage pattern after subjecting to cleavage. The cleavage patterns

are detected by running the reactions on denaturing gel and radioactivity detection

by phosphorimaging (Forconi and Herschlag, 2009). As an alternative to the

problems encountered from using naturally sequences, oligomers were used as they

can be separated using ion-exchange HPLC. Besides that, deribonucleotides and

chimeric DNA/RNA molecules have also been employed (Desbouis et al., 2012).

6

1.3.2 Phosphate ester cleavage studies with non-natural substrates

Recently, cyclic monophosphate nucleotides, such as 2’3’-cyclic guanosine

monophosphate (cGMP) are often used to mimic phosphate ester bonds. Since then,

many non-natural substrates have been employed to mimic phosphate ester

linkages. Among other non-natural mimics of phosphate ester linkages include bis

(4-nitrophenyl) phosphate (BNPP), 2-hydroxypropyl-4-nitrophenyl phosphate and

so on (Desbouis et al., 2012). Most studies involving the enzyme Alkaline

Phosphatase have employed p-nitrophenyl-phosphate as a substrate, as it offers a

colorimetric assay (O’Brien and Herschlag, 2002). Besides efforts to mimic the

phosphate ester linkage, various artificial mimics of enzymes have been developed

over the past few years as these synthetic products serve as useful models to provide

detailed information on the mechanism of phosphate ester cleavage (Desbouis et

al., 2012; Korhonen, 2011; Zagórowska et al., 1998).

1.3.3 Nucleotides/ nucleosides as phosphate ester models

Nucleotides consist of three components which are nitrogen base, pentose

sugar and a phosphate residue. Nucleosides consist of purine and pyrimidine bases

joined to a pentose sugar. Nucleotides are phosphate esters of nucleosides

(Blackburn et al., 2006). Over the past few years numerous mechanistic studies

7

have been carried out on nucleotide model compounds to gain insight on the

mechanism of RNA cleavage (Zagórowska et al 1998).

This increased interest of chemistry in nucleotides boils down to the

antibacterial, antiviral and anticancer properties of nucleotides (Das et al., 2005).

Nucleotide analogues are often employed to target viral RNA and DNA

polymerases (Kuchta, 2011). Apart from being essential component of DNA and

RNA, nucleotides are also part of regulatory factors in various metabolic pathways.

When inhibition of enzyme occurs in these metabolic pathways, RNA and DNA

synthesis will be inhibited as well, and results in cell death. Nucleoside analogs can

be transported into cells and metabolized by cells and these analogs can then

interfere with natural nucleotides’ metabolism. Nucleotide analogs are also capable

of interfering with DNA and RNA synthesis. Sulfamethoxazole is an antibiotic that

performs its function this way. It inhibits biosynthesis of folic acid, by targeting

dihydropteroate synthetase and results in incomplete biosynthesis of purine and

pyrimidine nucleotides (Sun and Wang, 2013).

Like nucleotide, nucleosides show good important anticancer and antiviral

properties (Hunsucker et al., 2005; Kuchta, 2011). They also act as antimetabolites

which means that they can act as drugs to inhibit DNA synthesis. Nucleoside

analogs can mimic naturally occurring nucleosides. Nucleoside analogs act as

anticancer agents by inhibiting nuclear DNA polymerases and incorporation into

8

nuclear DNA which leads to chain termination. Nucleoside analogs can perform as

antivirals by reversing the transcriptase of retroviruses or DNA polymerases of

DNA viruses. However, mechanisms of these analogs are only partially understood

(Hunsucker et al., 2005). Highly modified nucleosides, for instance carbocyclic,

heterosubstituted, aromatic, fluorinated, acyclic analogues have been prepared as

an effort to search for new antiviral agents (Wójtowicz-Rajchel, 2012).

At least 50% of the drugs approved by United States Food and Drug

Administration (FDA) comprises of anticancer and antiviral drugs that were

produced from nucleoside and nucleobase analogs. Herpes viral infections and HIV

infections have been treated with acyclic guanosine analogs and nucleoside analogs

respectively. Except for acyclic guanosine analogs, narrow therapeutic index and

harmful side effects were noticed in the nucleoside and nucleobase analogs that

were employed as medication to combat cancer and viral infections. Nucleoside

analog based antibiotics have also been developed (Sun and Wang, 2013).

1.4 Adenosine Monophosphate, Adenosine and Adenine

Adenosine Monophosphate (AMP) is also known as 5’-adenylic acid. AMP

is a nucleotide and also performs the role of a monomer in RNA. It consists of a

phosphate group, a ribose sugar and a nucleobase (adenine). The structure of AMP

and adenosine are shown in Figure 1.3 and Figure 1.4 respectively.

9

Figure 1.3: Structure of natural AMP

(National Centre for Biotechnology Information)

Figure 1.4: Structure of adenosine

(National Centre for Biotechnology Information)

Adenosine 5’-monophosphate is catabolised into adenosine by ecto-5’-

AMPases such as CD73 and endo-5’-AMPases such as cytosolic 5’-nucleotidase.

These enzymes are responsible to catalyse dephosphorylation of nucleoside

monophosphates to their corresponding nucleosides. These enzymes metabolize 5’-

AMP to adenosine (Jackson, 2011; Hunsucker et al., 2005). These enzymes play an

10

important role in energy production as they balance the levels of purine and

pyrimidine nucleoside triphosphates (Hunsucker et al., 2005; Borowiec et al.,

2006).

While CD73 is only specific to nucleoside monophosphates, whereby

(AMP adenosine), there is another class of enzyme called alkaline

phosphatases (AP) that metabolize more substrates. These substrates include

pyrophosphate, p-nitrophenylphosphate, and 5’-nucleotides. APs metabolize ATP

ADP AMP adenosine (Bontemps et al., 1983; Picher et al., 2003).

Phosphatases can accelerate the rate of reaction by 1021 fold (Desbouis et

al., 2012). Alkaline phosphatases are highly specific and have higher Km values,

and they have a more alkaline pH optimum (Millán, 2006). Its catalytic site contains

two Zn2+ and one Mg2+ ions. Alkaline phosphatases are highly catalytic and have

high affinity for their substrates (Desbouis et al., 2012).

Adenosine in turn can form adenine in the presence of nucleoside N-

ribohydrolases. This enzyme cleaves the N-glycosidic bond in adenosine. Acid-

catalysed and enzymatic hydrolysis both indicate similar mechanisms whereby the

adenosine gets protonated at N7 position and is followed by a nucleophilic attack

at C’1. Protonation at N7 facilitates the cleavage reaction by lowering electron

density of the leaving group (Versées et al., 2002). The most proficient enzymes

11

ever described are ricin A-chain and Trypanosome brucei nucleoside ribohydrolase

and the reported rate constants are 30 s-1 and 18 s-1 respectively (Stockbridge et al.,

2010). The equation of hydrolysis of adenosine to adenine is shown in Equation 1.

Adenosine + H2O adenine + ribose (Equation 1)

(Versées et al., 2002)

The structure of adenine is as shown in Figure 1.5.

Figure 1.5: Structure of adenine

(National Centre for Biotechnology Information)

1.5 Mechanism of Cleavage by Enzymes

Cleavage of phosphate esters are done with ease by enzymes. Among the

steps that are involved in enzymatic cleavage of phosphate esters are that the

substrate is positioned and activated towards nucleophilic attack. This activation is

assisted by Lewis acid metal ion coordination and also hydrogen binding

Nucleoside ribohydrolase

12

interactions with protonated side chains or closely positioned active side residues.

The next step involves the formation of a pentavalent phosphorus transition state.

This transition state formation is due to extensive supply of positive charge

originating from the metal ions and the active side residues. The transition state

then is attacked by a nucleophile. Lastly, metal ions or active site residues stabilize

leaving groups which results in departure of leaving groups (Desbouis et al., 2012)..

All these steps of enzymatic cleavage employ general-acid base catalysis or

metal-ion assisted catalysis where protons are accepted and donated by enzyme

functional groups. Amino acid side chains are responsible for proton donations and

acceptance (Bevilacqua, 2003; Desbouis et al., 2012; Ferŕe-D’Amaŕe et al., 2010).

General acid base catalysis stabilizes unfavorable changes that develop in the

structure during the transition state, activates weak nucleophiles and stabilizes poor

leaving groups (Bevilacqua, 2003). Enzymes are perfect for proton transfer as their

functional groups are positioned close to the nucleophile and the leaving group.

Besides that, these functional groups have pKa values near neutrality (Bevilacqua,

2003).

Enzymes such as selected nucleases can accelerate hydrolysis of the P-O

bonds by 1016 and phosphatases can accelerate the rate of hydrolysis of the P-O

bonds by 1021 (Desbouis et al., 2012). Alkaline phosphatase (AP) which catalyses

the hydrolysis of phosphate monoesters contains at least two to four Zn2+ and Mg2+

13

per dimer which might play a role enhancing the enzyme activity by stabilizing its

structure and increasing nucleophilicity of the phosphorus centre and activating the

nucleophile (Eguzozie, 2008). For the enzyme AP, arginine residue (Arg 166) is

responsible in substrate binding and stabilizing transition state. The Zn(II) is

responsible for stabilizing the leaving group. The alcohol group present in serine

side-chain of AP acts as a nucleophile here (Desbouis et al., 2012).

In the enzymatic cleavage of N-glycosidic bond cleavage of adenosine to

produce adenine, acid base catalysis is also employed by nucleoside N-

ribohydrolases. X-ray crystal structures of adenosine hydrolysis in the presence of

nucleoside N-ribohydrolase have indicated that histidine in the enzyme active site

plays the role of the general acid. This general acid protonates the leaving group to

facilitate the N-glycosidic bond cleavage. Meanwhile, aspartate present in the

active site facilitates the N-glycosidic bond cleavage by acting as a general base. It

abstracts a proton from the water molecule which in turn acts as a nucleophile

(Versées et al., 2002).

1.6 Importance of Studying Cleavage of Phosphate Esters, AMP and

Adenosine

Adenosine plays important roles such as energy transfer and as an inhibitory

neurotransmitter whereby it promotes sleep and suppresses arousal. It also

increases blood flow and reduces rate and force of contraction in the heart (Clark

14

et al., 2012; Sala-Newby et al., 1999). Adenosine plays a role in regulating

epithelial functions in human airways which is an important defence against

bacteria. Therefore, it is important to figure out the mechanism of adenosine

production. Information on the adenosine production will benefit us to create

artificial enzymes that are capable to catabolize natural AMP to adenosine (Picher

et al., 2003). Adenine performs functions in signal transferring and is a major

component in the genetic code in the DNA and RNA whereby adenine is one of

purine bases in nucleic acids (Mehta et al., 2015; Olkowski, 2012).

Previously, many nucleoside analogs have been developed due to extensive

studies on human purine metabolism. These analogues are currently used in the

treatment of cancer, parasitic and viral infections (Buckoreelall et al., 2011; Naito

et al., 1985). Nucleotidases also dephosphorylate nucleoside analogues that are

employed in the treatment of cancer and virus infections and could also resist the

action of analogues. For instance, metabolic pathway of mycobacteria (agent for

tuberculosis) have been studied to understand the enzymes involved in the

metabolic pathway. This would lead to drug discovery (Buckoreelall et al., 2011).

These enzymes play an important role in energy production as they balance the

levels of purine and pyrimidine nucleoside triphosphates (Hunsucker et al., 2005;

Borowiec et al., 2006).

15

Many life processes involve phosphate esters transformations and these

transformations are facilitated by highly specific enzymes (Banaszczyk, 1989). It

is important to gain understanding on how these enzymes can facilitate phosphate

monoester hydrolysis as this information will benefit us in future research.

1.7 Problem Statement

Despite extensive research on phosphate esters, adenosine nucleotides and

nucleosides, the exact mechanisms are poorly understood. Therefore, more research

has to be carried out to understand phosphate ester hydrolysis as this will allow us

to understand how exactly the reaction proceeds. Previous studies focused on the

cleavage products and kinetics but not on the transition states of the cleavage

reaction. Therefore, it is necessary to carry out characterization of the transition

state to understand the cleavage mechanism. Uncatalysed hydrolysis of phosphate

esters are extremely slow (Desbouis et al., 2012). Among the parameters that can

significantly affect the cleavage reaction are pH, temperature, buffer concentration

and reaction time. Initial set of experiments are necessary to optimize these

conditions (Forconi and Herschlag, 2009). Therefore, different reaction media have

to be created to incorporate all these parameters.

Previously, research on adenosine nucleosides cleavage has been carried out

using natural nucleosides and natural nucleobases and techniques such as gel

16

electrophoresis and gel filtration chromatography. Temperature was maintained at

37 °C and pH was maintained at 7. Enzymes were obtained from bacteria and

biologically obtained such as rat hearts to cleave adenosine to adenine

(Buckoreelall et al., 2011; Naito et al., 1985).

In most conditions, the substrates are cleaved too fast, resulting in unclear

information on the exact mechanism. Enzymes are also denatured at high

temperatures and extreme pH. Enzymes also have to be purified prior to usage

(O’Brien and Herschlag, 2002). To overcome this problem, non-enzymatic system

has to be created mimic the action of these enzymes to cleave the phosphate ester

bond (Komiyama et al., 1999).

1.8 Objectives

The aims of this study are:-

1. To utilize adenosine monophosphate disodium salt, AMPNa2 (phosphate

monoester) as a substrate model for the phosphate ester bond cleavage studies in

natural adenosine monophosphate (AMP)

2. To understand the kinetics and mechanism of cleavage that occurs in natural

AMP which provides further information on how enzymes performs their

functions in cleaving this phosphate monoester.

17

3. To study the effects of acids and bases for the bond cleavage reaction of

adenosine monophosphate disodium salt

1.9 Scope of Work

This study conducted will focus on the application of adenosine

monophosphate disodium salt, AMPNa2 as a model of natural AMP. The hydrolysis

of this phosphate ester will be carried out in various pH covering acidic and basic

range to study the effects of general and specific acid and base catalysis in the

cleavage of natural AMP.

Kinetics of the hydrolysis will be calculated at certain pH value and this will

provide information on the effects of the acid and base catalysis in the cleavage.

The kinetics data will be able to provide evidence if acid and base catalysis is a

good catalyst in natural AMP cleavage. These reaction rates will be compared to

reaction rates in other systems and reaction rates with different types of substrate

model. These reaction rates will also be compared to enzymatic rates and

background reaction for the hydrolysis of AMPNa2 to investigate if acidic and

alkaline media have provided rate enhancements.

18

Since it is well known that phosphate esters are very stable and react very

slowly, this investigation involves thermostating the reaction mixture at 60 °C to

speed up the reaction. Characterization of the product will be carried out using

Liquid Chromatography Mass Spectrometry and Infrared Spectroscopy. The

kinetic data paired with characterization results will be able to help us to deduce

the mechanisms of the cleavage of the phosphate ester bond or other possible

cleavage in adenosine monophosphate disodium salt, AMPNa2. By understanding

the mechanisms of cleavage in simple phosphate esters, we will be able to deduce

the mechanism of cleavage in natural AMP. This in turn will provide information

on how enzymes such as Alkaline Phosphatase, ecto-5’-AMPases and endo-5’-

AMPases, and nucleoside N-ribohydrolases perform their functions. The effect of

ionic strength on the rate of hydrolysis of AMPNa2 will be investigated by varying

the ionic strength throughout the investigation.

19

CHAPTER 2

LITERATURE REVIEW

2.1 Acid Base Catalysis Studies of Phosphate Monoesters

The concept behind most acid base catalysis studies originates from the role

of metal ions in hydrolysis of phosphate monoesters. Metal ions serves as a general

acid catalyst where it neutralizes negative charges on the phosphate so that a

nucleophile could attack the phosphorus centre (Eguzozie, 2008). Acid base

catalysis in monophosphate nucleotides can be divided into specific acid base

catalysis and general acid base catalysis (Widlanski and Taylor, 1999).

2.1.1 Specific acid and base catalysis of phosphate monoesters

Specific acid and specific base catalysis is provided by hydronium ion, H+

and the hydroxide ion, OH- respectively. H2O has the ability to dissociate into H+

and OH-. Base and acid catalysis can significantly accelerate the rate of hydrolysis

because the hydroxide and hydronium ions act as catalysts which provide an

alternative pathway for the reaction to proceed. This alternative pathway is more

favourable energetically.

20

In specific acid catalysis, this is done by the hydronium ion when it

withdraws electron density from the atom bearing the leaving group. This in turn

makes the atom more susceptible to nucleophilic attack (Larson and Weber, 1994).

The hydrolysis of monomethyl phosphate is catalysed by strong acid whereby

proton is transferred to the leaving group. This results in the formation of an

unstable metaphosphate ion intermediate. This indicates that the leaving group has

to be protonated in advance. This intermediate reacts with water rapidly to produce

inorganic phosphate. The proton transfer to the phosphate also facilitates cleavage

by increasing negative charges in the phosphate group. This results in repulsive

force on the leaving group. At pH less than 1, C-O and P-O bond cleavage

competes. Figure 2.1 below depicts the possible ways how proton can be transferred

to the leaving group resulting in the cleavage. In (I), I zwitterion ion is formed by

a pre-equilibrium proton transfer. In (II), a four membered ring results in a

concerted proton transfer which results in P-O cleavage. In (III), a water molecule

participates and proton transfer is achieved through a six membered ring (Jubian,

1991).

21

Figure 2.1: The possible pathways of phosphate monoester in specific acidic

medium (Jubian, 1991).

Theoretical study on monoester phosphates, namely methyl phosphate and

p-nitrophenyl phosphate included explicit hydrogen bonding interactions. It was

noticed that these bonding posed a significant effect whereby the hydrogen bonding

protonates P-O in the transition state (Duarte et al., 2015). pH rate profiles of most

monoalkyl phosphates are maximum between pH 3 to 5. pKa values for the first

step of dissociation of methyl phosphate is 1.54 and the pKa values for the first step

of dissociation of methyl phosphate is 6.31. The highest rate of hydrolysis of methyl

22

phosphate is at pH 4 is due to the highest monoanion concentration in the solution

(Shabarova and Bogdanov, 1994).

Specific base catalysis can accelerate rate of hydrolysis due to the fact that

OH- is a better nucleophile than H2O by 108 towards the phosphorus atom.

Therefore, a reaction whereby OH- acts as a nucleophile proceeds faster than a

reaction where H2O acts as a nucleophile (Larson and Weber, 1994). Base-

catalysed hydrolysis of phosphate ester proceeds through a BP2 reaction, stands for

base-catalysed, phosphoryl-oxygen fission, bimolecular reaction analogous to an

SN2 reaction. Here, hydroxide ion attacks the phosphorus atom and this is known

as the rate limiting step of this reaction. The cleavage can also proceed through

other mechanisms but BP2 often dominates (Hilal, 2006). Figure 2.2 depicts the

possible pathways of phosphate monoester hydrolysis. Mechanism A stands for

dissociative pathway, B stands for associative pathway and C stands for concerted

pathway.

Figure 2.2: Possible mechanisms of phosphate monoester hydrolysis in basic

conditions (Duarte et al., 2013)

23

Theoretical study on base-catalysed phosphates monoester in have been

carried out previously. When water is employed as a nucleophile, a substrate-

assisted mechanism takes place whereby proton is transferred to the phosphate (a

result of high pKa difference). The hydroxide ion then acts as a nucleophile and

subsequently attacks the phosphate monoanion. This mechanism was noticed in

computational study of methyl phosphate and p-nitrophenyl phosphate (Duarte et

al., 2015; Spillane, 2004). It was also calculated that the rate of the hydroxide ion

attack on the neutral phosphate monoester is very fast (Florián and Warshel, 1997).

In metal complex study of hydrolysis of p-nitrophenyl phosphate, a metal

hydroxide was employed to attack the coordinated phosphate to produce 4-

nitrophenol and phosphate derivative indicating the role of hydroxide ion as a

nucleophile (Eguzozie, 2008). In an investigation involving acyl phosphate

monoesters, there was a relationship noticed between the concentration of the

hydroxide ion and the metal ion catalysed process indicating that coordinated

hydroxide acts as a nucleophile. Hydrolysis without metal ions was also carried out

and rate of hydrolysis was promoted by the presence of hydroxide ions (Kluger and

Cameron, 2002).

2.1.2 General acid and base catalysis of phosphate monoesters

General acid catalysis plays a role in many biological processes and can

occur in the absence of a catalytic metal, whereby the P-O bond is cleaved. In

24

simple models, acid-base catalysis has been noticed whereby nucleophiles are

phosphorylated. In the hydrolysis of 2-(2’-imidazolium) phenyl hydrogen

phosphate (IMPP) described the hydrogen bonding between the aryl oxygen

leaving group by a nearby imidazolium NH. Protonation of this aryl oxygen leaving

group strongly favours P-O bond cleavage. The P-O bond cleavage occurs as a

result of water molecule attack on the phosphorus centre (Brandão et al., 2007).

General acid or general base catalysis also known as buffer catalysis is the

catalysis performed by all Brønsted acids and/or bases. In laboratories, buffer salts

are commonly used to control the pH. A mathematical model developed by Perdue

and Wolfe states that buffer catalysis may be significant if the buffer concentrations

are greater than 0.001 M (Larson, 1994). This catalysis is common in enzyme

catalysed systems involving proton transfer. General acid-base catalysis provides

acceleration by 10- to 100-fold. Among the buffers that have been employed in

general acid and general base catalysis of phosphate monoesters are NaAcetate (pH

4.4-6.0), NaMES (pH 4.8-6.9), NaMOPS (pH 5.9-7.9), NaCHES (pH 8.0-9.8) and

NaCAPS (pH 9.4-11.4) (O’Brien and Herschlag, 2002). The effects of buffers such

as Tris, Glycine, and Tricine on the activity of alkaline phosphatase has been

studied by employing p-nitrophenyl phosphate as a substrate. It was found that the

activity alkaline phosphatase was higher in Tris buffer than Glycine and Tricine

buffer (Hethey et al., 2002). However, in some cases buffers can inhibit the

hydrolysis. For instance, in the metal ion promoted hydrolysis of benzoyl methyl

25

phosphate, increasing the concentration of EPPS buffer showed inhibition of the

rate of hydrolysis (Kluger and Cameron, 2002).

In the mechanisms proposed by Kirby and his co-workers on the

mechanisms of phosphate monoester of 8-(dimethylamino)-1-naphthol, water

could act as a nucleophile. However, if water is replaced with a better nucleophile

the reaction would proceed faster (Kirby et al., 2004). In this investigation, a

nucleophilic attack on PO32- was evident and general acid catalysis was exhibited

by neighbouring dimethylammonium group. Dimethylammonium group together

with positive charge that was attached to the electrophilic phosphorus centre were

responsible in neutralizing the repulsive electrostatic effects. This justifies the role

of amino side chains such as lysines, histidines and arginines that are present in

active sites of most enzymes that catalyse reactions of phosphate monoesters (Kirby

et al., 2004). Another phosphate monoester that employed general acid catalysis is

salicyl phosphate. Here also, intramolecular general acid catalysis resulted in the

attack of water and amine nucleophiles on the phosphorus resulting on P-O bond

cleavage (Kirby et al., 2005). Mechanism of protonation of leaving group in

salicylic acid is as shown in Figure 2.3.

26

Figure 2.3: Mechanism of general acid catalysis in phosphate monoester

(Kirby et al., 2005)

2.2 Possible Pathways of Phosphate Monoesters Hydrolysis

Besides P-O bond cleavage, C-O bond cleavage is also possible along with

alcohol elimination through a metaphosphate intermediate (Banaszczyk, 1989).

Generally, hydrolysis of phosphate esters involves mechanism that is analogous to

SN2 mechanism and can undergo acid-catalysed hydrolysis, base-catalysed

hydrolysis and general base-catalysed (neutral) hydrolysis. Alkaline or base-

catalysed hydrolysis may result in different products than neutral catalysed

hydrolysis as hydroxide ion is 108 times a better nucleophile than water towards the

phosphorus atom (Hilal, 2006).

27

2.3 Choice of Substrate

Phosphate monoesters are of interest as these compounds have low

reactivity. Therefore, only phosphate monoesters with good leaving groups are

preferable as they are able to react quickly (Duarte et al., 2015). Previously, simple

phosphate ester models such as p-nitrophenyl phosphate and phenyl phosphate have

been used as substrate models of phosphate ester linkage (O’Brien et al., 2002;

Hethey et al., 2002). In choosing the suitable substrate, aryl phosphates are

preferable as they are easy to analyse due to the cleavage rate of aryl phosphates

are much faster than alkyl phosphates. Aryl substances also release aryloxide

leaving groups which are easier to study by UV/Vis spectroscopy (Jenkins et al.,

1999). Previous pH dependency investigations involving Alkaline Phosphatase also

employed aryl phosphates as substrates (O’Brien and Herschlag, 2002).

There has been extensive research in the recent years on adenosine and its

corresponding nucleotides as they are biomolecules that are involved in energy

production and substrates for various cellular biochemical processes (Qian et al.,

2004). Previously, 3’,5’-cAMP-adenosine and 2’,5’-cAMP has also been used in

various investigations (Jackson, 2011, Jenkins et al., 1999). There is evidence of

intracellular metabolism to 5’-AMP by endo-3’,5’-cAMP-3’-phosphodiesterases.

This 5’-AMP then is metabolized to adenosine by ecto-5’-AMPases (Jackson,

2011).

28

In this investigation, adenosine monophosphate disodium salt, AMPNa2

will be used as a model substrate to mimic the phosphate ester bond in phosphate

monoesters. Adenosine monophosphate disodium salt, or AMPNa2 is a nucleotide

analogue that is known for its antiviral, anticancer and antibacterial properties.

Understanding the kinetics and mechanism of this analogue will give us insights on

prospects to develop drugs using this nucleotide. At the same time, understanding

how certain enzymes function to hydrolyse this substrate could lead to drug

discovery. For instance, nucleoside hydrolase is not present in mammals and

therefor they appear as an attractive target for drug design against pathogens

(Versées et al., 2002). Figure 2.4 shows the structure of adenosine monophosphate

disodium salt.

Figure 2.4: Structure of adenosine monophosphate disodium salt

Besides that, adenosine monophosphate is present in the human body and it

is broken down to adenosine by Alkaline Phosphatase and ecto-5’-AMPases and

endo-5’-AMPases (Skoog, 1986). Natural adenosine monophosphate can also be

hydrolysed into adenine and ribose 5-phosphate by adenosine monophosphate

nucleosidase (Skoog, 1986). This reaction is known as depurination of nucleotides

or N-glycosidic bond cleavage. This cleavage is acid-catalysed (Nelson and Cox,

29

2013). Carrying out study on adenosine monophosphate disodium salt provides

useful knowledge on how this enzymes carries out its function in terms of

mechanism. Kinetics study on adenosine monophosphate disodium salt using acid

base catalysis also allows the confirmation of the principles used by these enzymes

in breaking down adenosine monophosphate and therefore allowing the

development of synthetic enzymes in the future to mimic the actions of these

enzymes.

AMP yields the highest Vmax/Km value whereby the Vmax for AMP hydrolysis

is 34 IU/mg of protein and Km is 0.12 mM. (Naito et al., 1985; Skoog, 1986). AMP

is also an excellent substrate model because it allows us to measure the absorbance

of the reaction at 259.5 nm using UV-Vis spectroscopy without any interference

due to the presence of an aryloxide group (Tuan, 2014; Jenkins et al., 1999).

Besides that, AMP has been employed in non-enzymatic hydrolysis too and

resulted in cleavage of the phosphate ester bond and adenosine. In a study of metal

complex promoted bond cleavage of phosphate esters, a Co(III) complex was

employed to cleave adenosine monophosphate (AMP) by stirring two equivalents

of [(trpn)Co(OH2)2]3+ with adenosine monophosphate disodium salt in water for 6

hours in 25 degrees. This resulted in adenosine and [((trpn)Co)2PO4]3+ in

quantitative yield. The [(trpn)Co(OH2)2]3+ was added into AMP, and a cobalt

complex adduct of the phosphate monoester was formed. This complex was stable

in water, however, with addition of [(trpn)Co(OH2)2]2+, it was hydrolysed into

30

[((trpn)Co)2PO4]3+. The formation of [((trpn)Co)2PO4]

3+ showed a linear

relationship to the addition of [(trpn)CO(OH2)2]2+. The kinetics study was carried

out by monitoring the increase/decrease in 31P NMR and also 1H NMR. The second-

order rate constant for the formation of [((trpn)Co)2PO4]3+ from the phosphate

cobalt complex and AMP is 3.6 ± 0.5 × 10-3 M-1 s-1 (Chin and Banaszxzyk, 1989).

In a UV-photodissociation of non-cyclic and cyclic mononucleotides

experiment, UV radiation was carried out on deprotonated AMP, and fragmentation

of AMP occurred into general classes of fragments. Phosphate based products

fragments were observed, indicating that phosphate sugar backbones were cleaved

during the photodissociation. Fragment with the highest ratio was PO-3, followed

by H2PO4- (Marcum et al., 2011). Figure 2.5 suggests the mechanism that could

have been employed by deprotonated AMP under UV photodissociation to carry

out P-O bond cleavage (Marcum et al., 2011).

Figure 2.5: Reaction pathway for P-O bond cleavage of AMP using UV-

photodissociation (Marcum et al., 2011)

31

2.4 Mechanisms Competing with P-O Bond Cleavage

2.4.1 C-O bond cleavage

It is possible for 5’-monophosphates to undergo C-O bond cleavage

whereby the phosphate group abstracts a proton from the sugar leading to an E2

type elimination forming H2PO4- (Marcum et al., 2011). In another study of

hydrolysis of 3,5’-cyclic monophosphates, there was competition between C-O and

P-O bond cleavage (Varila et al., 1997). C-O bond cleavage is possible in all three

types of phosphate esters, be it, phosphate monoesters, diesters or triesters

(Banaszczyk, 1989). Figure 2.6 depicts C-O bond cleavage in protonated adenosine

monophosphate.

Figure 2.6: Mechanism of C-O bond cleavage in protonated adenosine

monophosphate (Marcum et al., 2011).

32

2.4.2 N-glycosidic bond cleavage

Natural adenosine monophosphate can also be hydrolysed into adenine and

ribose 5-phosphate by adenosine monophosphate nucleosidase (Skoog, 1986). This

reaction is known as depurination of nucleotides or N-glycosidic bond cleavage.

This cleavage is acid-catalysed (Nelson and Cox, 2013). The equation of how this

enzyme performs its function is shown in Equation 2.

AMP adenine + ribose 5-phosphate (Equation 2)

(Schramm, 1974)

Efficiency of AMP nucleosidase is due to the fact that they have

interdependent modifier sites that can be occupied by MgATP2-, ATP4- or MgPPi to

produce enzyme-activator complexes. MgATP2- is the most effective activator

compared to the rest in the reports. With the saturated presence of this activator, the

rate was increased by 100- to 400- fold over compared with the absence of these

activator.

Non-enzymatic hydrolysis of adenosine monophosphate also produced

adenine indicating that it is possible for adenosine monophosphate cleavage to

proceed through N-glycosidic cleavage. In a UV- photodissociation study of

33

mononucleotides, fragments of protonated bases, B- were observed. The scheme of

how these fragments are formed is shown in Figure 2.7.

Figure 2.7: Mechanism for glycosidic bond cleavage by intramolecular E2

reaction producing adenine (Marcum et al., 2011)

This mechanism, involves proton transfer from the sugar to the phosphate

group, consequently leading to loss of nucleobase in E2-type elimination process.

The B- ion that is formed remains attached to the remainder molecule and slowly

dissociates, resulting as a B- fragment as shown in Figure 2.7 (Marcum et al., 2011).

Another mechanism that involves the formation of B- products is shown in Figure

2.8. This mechanism involves a direct heterolytic bond cleavage of the N-glycosidic

bond. This leads to the formation of a B- and zwitterionic fragment. This

zwitterionic fragment is resonance stabilized by the sugar oxygen (Marcum et al.,

2011).

34

Figure 2.8: Mechanism for glycosidic bond cleavage by heterolytic cleavage

(Marcum et al., 2011)

2.5 Ionic strength

During biochemical processes, ionic strength of a solution is often

neglected. However, ionic strength can affect acid dissociation, and subsequently

change kinetic parameters. Ionic strength is also known as I (Kennedy, 1990).

When salt is added to a solution with constant buffer concentration, the activity

coefficient of species (i) is affected. The Debye-Hückel theory is accurate to predict

how activity coefficients change with respect to ionic strength. This theory can be

applied for solutions with low ionic strengths which are less than 0.1 M.

Unfortunately, this theory cannot be employed to predict activity of the ions at high

ionic strengths (Smith and Collins, 2011).

Alkaline phosphatase activity has depicted a dependency on ionic

environment (Hethey et al., 2002). NaCl and KCl has been used hydrolysis of

phosphate monoesters to maintain ionic strength of a solution (Awadhiya et al.,

35

2011; Kluger et al., 2002; O’Brien et al., 2002; Tiwari et al., 2005). For example,

the rate of hydrolysis of mono-2-methyl-5-nitroaniline in conjugate acid and neutral

species included NaCl to maintain ionic strength. The effect of ionic strength on

the rate of hydrolysis was carried out by varying the ionic strength. It was found

that the rate of hydrolysis of mono-2-methyl-5-nitroaniline increased with the

increase of ionic strength thus concluding that this acid-catalysed hydrolysis is

subjected to positive ion effect (Awadhiya and Bhoite, 2011). In another

experiment of adenosine decomposition in anionic buffers, rate of glycosidic

cleavage increased by 2-fold when the concentration of KCl was increased from

0.0-0.1 M. This implies the effect of ionic strength on the rate of cleavage of

glycosidic bond (Stockbridge et al., 2010).

2.6 Characterization Methods

2.6.1 NMR Spectroscopy

Often with investigation involving metal complex, NMR spectroscopy is

used. Chin and his co-workers (1989) employed 31P and 1H NMR when studying

the hydrolysis of adenosine monophosphate with a binuclear Co (III) complex. 31P

NMR has been widely used to monitor the hydrolysis of ATP, diphosphate, and

simple phosphate monoesters where Co(III) complexes are employed to promote

hydrolysis. As the number of Co(III)-phosphate oxygen bonds increased, the signal

of the phosphate was in the downfield region in the 31P NMR spectrum (Chin and

36

Banaszczyk, 1989). In another study by Stockbridge to cleave the N-glycosidic

bond, the rate of the cleavage was determined by observing the appearance of

adenine using 1H NMR (Stockbridge et al., 2010).

2.6.2 Mass Spectrometry

There has been extensive growth in mass spectrometry field (MS), and

coupling of mass spectrometry with separation techniques. Many HPLC-MS

systems have been used worldwide. This is due to the ability of MS in the

identification of chromatographic peaks. Shortly after, invention of electrospray

ionization (ESI) took place. ESI could be used to identify various classes of bio

(macro) molecules such as proteins, nucleotides, nucleosides, polysaccharides and

phospholipids. Interpretation of mass spectra involves molecular weight

determination. Electrospray ionization coupled with mass spectrometry is an

excellent tool for identifying the composition of the products in this investigation

due to the nature of the substrate which is a nucleotide (Holčapek et al., 2010). ESI-

MS have been previously employed to identify the presence of adenine and

adenosine and therefore ESI-MS is a great tool that can be used to identify the

products of this investigation (Zhao et al., 2013). Electrospray ionization coupled

with mass spectrometry is helpful in determining the number of phosphate groups

present and identify ribose or phosphate substitutions. Positive-ion mode spectra of

nucleotides contain information of nucleobase-derived structure such as adenine

37

(m/z = 134), guanine (m/z = 150), cytosine (m/z = 111) and uridine (m/z = 112)

(Strzelecka et al., 2017).

In the ESI-MS spectrum of adenosine monophosphate, the peaks that can

be expected are at m/z 348, 268, 136 and 597. Fragment at m/z = 348 represents

[AMP + H]+, while the fragment at m/z = 695 represents [2AMP + H]+. Fragment

at m/z = 268 represents adenosine, which is formed if the P-O bond is cleaved while

fragment at m/z = 136 represents adenine, which indicates that N-glycosidic bond

has been cleaved. The appearance of these fragments will be very helpful in this

investigation as they may provide information on the mechanism employed by

adenosine monophosphate disodium salt in acidic and alkaline media (Liu et al.,

2006; Skoog, 1986; Chin et al., 1989).

2.6.3 FTIR Spectroscopy

Through infrared spectroscopy we are able to detect bands for adenine,

guanine, thymine, and cytosine. The appearance and disappearance of these bands

throughout the investigation can provide us with information on the breaking and

closing of glycosidic bond in adenosine monophosphate disodium salt (Mello and

Vidal, 2012). C-N glycosidic bond corresponds to absorption band around 1458

cm-1 (Agarwal et al., 2014; Stuart, 2004). The ribose phosphate skeletal motions

38

corresponds to the band around 970-916 cm-1. The disappearance of these band in

the product could indicate a P-O bond cleavage (Stuart, 2004).

In previous investigations, FTIR spectrum of adenosine monophosphate salt

have been obtained. The bands that gained attention would be at 1091 cm-1 and 976

cm-1, which represent the presence of phosphate group. This FTIR spectrum serves

as a reference for us in this investigation (Theophanides and Sandorfy, 2012).

2.6.4 UV/Vis Spectroscopy

In previous studies of phosphate monoesters, the release of phenols were

measured using a UV-spectrophotometer in order to study the kinetics of hydrolysis

of p-nitrophenyl phosphate (Kirby and Varvoglis, 1996). UV-Vis spectrometry is a

great tool in studying kinetics as a huge number of wavelengths can be employed

for the evaluation of the similar investigation, resulting in improved results. The

most suitable wavelength can be selected for evaluation which is beneficial if the

reactant or the products absorbs at a particular wavelength (Perkampus, 1992)

When studying aryl phosphate esters, UV/Vis spectroscopy is the most

relevant method for compound identification as aryloxide groups are easily

observed in UV/Vis spectrum (Jenkins et al., 1999). As shown in the Table 2.1, UV

39

spectroscopy not only allows detection of purine and pyrimidine bases, but also

their derivatives. This allows us to identify nucleotides bases with ease as it is, or

even when the bases have broken down.

This is also a great tool to compare initial and final substance or as the slight

change in the absorption maximum could indicate breakage or formation of new

bonds. For example, the maximum absorption for adenine is 260.5 nm and the

maximum absorption for adenosine is 259.5 nm. This slight difference could

indicate if adenosine breaks down into adenine. Table 2.1 lists the values for

absorption maxima and extinction coefficients of several purine and pyrimidine

bases and their derivatives.

40

Table 2.1: The values for absorption maxima and extinction coefficients of

several purine and pyrimidine bases and their derivatives (Tuan, 2014)

Absorption Maximum (λmax)

nm

Extinction Coefficient (ԑ)

(cm2.mol-1)

Adenine 260.5 13.4 × 103

Adenosine

259.5

14.9 × 103

Guanine

275.0

8.1 × 103

Guanosine

276.0

9.0 × 103

Cytidine

271.0

9.1 × 103

Cytosine

267.0

6.1 × 103

Uracil

259.5

8.2 × 103

Uridine

261.1

10.1 × 103

Thymine

264.5

7.9 × 103

Thymidine

267.0

9.7 × 103

In an investigation carried out by Stockbridge and his colleagues, adenosine

decomposed into adenine at pH 7. This was justified by the decreased amount of

adenosine as the amount of adenine increased. UV Spectroscopy was also

employed here to justify the disappearance of adenosine and appearance of adenine.

There was a slight shift in wavelength to the right over the course of time, from

260- 261 nm. Adenosine is responsible for the absorption at 260-261 nm

(Stockbridge et al., 2010). In a study where 4-nitrophenylphosphate was also

employed as a substrate model, the UV-Vis spectroscopy was employed. When 4-

nitrophenyl phosphate was hydrolysed into 4-nitrophenyl, it was easy to determine

41

the reduction of 4-nitrophenylphosphate amount and increase of 4-nitrophenyl due

to the fact that both 4-nitrophenylphosphate and 4-nitrophenyl have distinct

absorption maximum at 310 nm and 400 nm respectively (Eguzozie, 2008). The

rate of methanolysis of benzoyl methyl phosphate also employed UV Spectroscopy

by inspecting the decrease of absorbance at 240nm. UV scans of the product

depicted a shift of absorption maximum (λmax) to 227nm, which is the absorption

maximum (λmax) for methyl benzoate indicating the presence of methanolysis

reaction (Kluger and Cameron, 2002).

42

CHAPTER 3

MATERIALS AND METHODS

3.1 Materials Used

All the chemicals, solvents and reagents were obtained and used without

further purification. All the chemicals or reagents used are listed in Table 3.1.

Table 3.1: Materials used in the preparations of sample solutions

Materials

Manufacturer

Purity

Adenosine 5’-monophosphate disodium salt, (AMPNa2)

Sigma-Aldrich

≥ 99%

Hydrochloric acid

Fisher Scientific

≥ 37%

Sodium hydroxide

QRëc

≥ 99%

Glycine

R&M Chemicals

≥ 99%

Tris(hydroxymethyl)aminomethane, (TRIS)

Fisher Scientific

≥ 99.8%

Sodium chloride

QRëc

≥ 99%

Citric acid

R&M Chemicals

≥ 99.5%

Sodium citrate

Fisher Scientific

≥ 99%

2-(N-morpholino)ethanesulfonic acid, (MES)

Fisher Scientific ≥ 98%

4-(2-hydroxyethyl)-1-piperazineethanesulfonic

acid, (HEPES)

Fisher Scientific ≥ 99%

43

3.2 Apparatus

1.0, 10.0, 25.0 mL pipettes were used to measure and transfer volumes of

liquids that were used in this experiment. Sodium hydroxide, sodium chloride,

hydrochloric acid, glycine-HCl, TRIS-HCl, citrate buffer, MES-NaOH, HEPES-

NaOH and adenosine monophosphate disodium salt solutions were prepared at

various concentrations in volumetric flasks. Sample solutions were prepared in 20

mL sample vials and stored in a water bath set at 60 °C. Microsyringe was used to

measure a small amount of AMPNa2 (0.02 mL) accurately before it is injected into

the sample solutions.

3.3 Procedure

3.3.1 Kinetic Study of the Nucleotide Analogue, AMPNa2

A 0.01 M solution of AMPNa2 was prepared in a 5 mL volumetric flask by

diluting 0.197 g of AMPNa2 in distilled water. Meanwhile, sample solutions of 20

mL each covering pH range from pH 0.30-12.71 were prepared at [AMPNa2] =

0.0001 M, [HCl] = 0.01-1.00 M, [NaOH] = 0.0008-1.0000 M, [glycine] = 20-80%,

[citrate] = 20-40%, [MES] = 40-60%, [HEPES] = 10-90%, [TRIS] = 80-90%,

[NaCl] = 0.2-1.0 M according to Table 3.2. The more detailed composition of these

chemicals for each sample produced are shown in Appendix A-G.

44

Table 3.2: Chemicals used for each pH range for sample solutions

Chemical

pH Range

Hydrochloric acid

0.30-1.83

Glycine buffer

1.82-2.70

Citrate buffer

3.91-4.21

MES buffer

5.76-6.15

HEPES buffer

6.04-7.13

TRIS buffer

8.03-8.42

Sodium hydroxide

9.95-12.71

NaCl served to control the ionic strength of the solutions. Samples with pH

12.08 and pH 12.21 have the same concentration of sodium hydroxide, but different

composition of NaCl. This is to study the effect of ionic strength on the rate of the

reaction. For [NaOH] less than 0.2 M, ionic strength was maintained at 0.2 M, while

for [NaOH] more than 0.2 M, ionic strength was increased to 1.0 M.

All these sample solutions were stored in a water bath at 60 °C at all times.

The pH values of each sample were taken before and after the reaction has gone

into completion. The substrate, which is a small amount of 0.02 mL was added into

the solution and made up to 20 mL. The 0.02 mL AMPNa2, was measured using a

microsyringe to increase accuracy. As soon as the substrate was added into the

sample solution, it was quickly shaken to ensure the substrate was mixed with the

45

rest of the solution and quickly then added into a cuvette and placed in the UV-Vis

spectrophotometer. The absorbance was measured until the reaction is completed,

around 8 half-lives. Absorbance values were taken at 260-270 nm.

The rate of reaction of each sample with decreasing absorbance value was

calculated with the following equation:-

Aobs = Eapp [X0] exp (– kobs t) + A∞ (Equation 3)

where, Eapp is apparent molar extinction coefficient of the reaction mixture,

A∞ is absorbance at reaction time, t = ∞

kobs is pseudo-first-order rate constant

[X0] represents the initial concentration of substrate, AMPNa2

The rate of reaction of each sample with increasing absorbance value was

calculated with the following equation:-

46

Aobs = Eapp [X0] {1-exp (– kobs t)} + A0 (Equation 4)

where, Eapp is apparent molar extinction coefficient of the reaction mixture,

A0 is absorbance at reaction time, t = 0

kobs is pseudo-first-order rate constant

[X0] represents the initial concentration of substrate, AMPNa2

Observed rate constants, kobs are plotted against concentrations. Theoretical

rate constants, kcalc were also calculated and also plotted on the same graph as a

solid line. Equation 5 was employed for alkaline media while Equation 6 was

employed for acidic media.

kobs = kb[OH-] + k0 (Equation 5)

kobs = ka[H+] + k0 (Equation 6)

where, kobs represents pseudo-first-order rate constant of the reaction, kb represents

second-order base-catalysed rate constant, ka represents second-order acid-

catalysed rate constant and k0 represents uncatalysed rate constant for the cleavage

of P-O bond in AMPNa2.

47

3.3.2 Characterization of the Products

Apart from kinetics measurements, characterization of the products was

carried out in order to be able to deduce the mechanism of the reaction. For some

samples, ranging from pH 11.81-12.71, white precipitate was noticed at the bottom

of the solutions. To figure out the composition of this white precipitate, it was

scanned with the following characterization methods. The solutions were first

filtered using a synthered funnel. The white precipitate was left as a residue. This

residue was then dried in an oven set at 50 °C for two days prior to analysis.

3.3.2.1 UV-Vis Spectrometry

This instrument was employed to carry out spectral measurements until

reaction goes into completion. Reaction is said goes into completion when the

absorbance values for three consecutive spectral measurements are giving the same

value. In this study, a reaction goes into completion in around eight half-lives. Apart

from providing kinetic data, UV-Vis analysis was able to provide information on

absorption maximum which allows us to deduce the composition of the compound

being scanned. The UV-Vis analysis was performed with Perkin Elmer Lambda

35 double beam spectrometer. The spectra were obtained within a range of 190-450

nm. Deuterium and tungsten lamps were used to provide illumination to pass

through a cuvette with a path length of 1 cm.

48

3.3.2.2 Fourier Transform Infrared Spectroscopy (Perkin Elmer Spectrum

ex1)

The spectrum of the substrate, AMPNa2 was scanned before the

investigation to ensure the purity of the substrate. The spectrum of the substrate

was then compared with literature spectrum. The spectra of the residue and the

filtrate from the filtration of sample solution were also obtained. This method of

characterization was employed to determine the functional groups of the products.

The infrared spectra of liquid and solid samples were obtained in a range of 4000-

650 cm-1 and 4000-400 cm-1 respectively, with a total of eight scans. Solid samples

were prepared by incorporating the samples in a potassium bromide (KBr) disk.

Liquid samples were prepared by dissolving the sample in water and then poured

into zinc selenide crystal of Perkin Elmer Horizontal Attenuated Total Reflective

Accessory.

3.3.2.3 LC-MS Spectrometry

The acidic medium and basic medium products were analysed with 6520

Accurate-Mass Q-TOF LC/MS by Agilent Technology mass spectrometer with an

electrospray ionization source operated in positive ion mode. This was controlled

by the acquisition and qualitative analysis software. The mobile phase consists of

H2O and methanol with a ratio of 70:30%. Isocratic elution was performed. LC-MS

49

spectra were recorded from m/z 100 to 1000 at a flow rate of 1mL/min at room

temperature.

3.4 Derivation of the Equation used for all Kinetic Measurements

All kinetics were carried out in pseudo-first order reaction conditions. For

example, the base-catalysed hydrolysis with various [NaOH] concentrations is

illustrated as follows whereby A represents the reactant, while P represents the

product.

A P (Equation 3.1)

The rate law of the reaction in Equation 3.2 can be expressed as:

𝑅𝑎𝑡𝑒 = −𝑑[A]

𝑑𝑡= −

𝑑[OH−]

𝑑𝑡= +

𝑑[P]

𝑑𝑡= kOH[OH−][A] (Equation 3.2)

whereby [A] and [OH-] represents the concentrations of A and basic ion at reaction

time, t, respectively and kOH is the second-order rate constant for the base-catalysed

hydrolysis of A. In order to maintain pseudo-first order conditions, [OH-] must be

present in excess for the base-catalysed hydrolysis of A to follow the Equation 3.3.

kOH[OH-]

50

𝑅𝑎𝑡𝑒 = kobs[A] (Equation 3.3)

where kobs represents the observed pseudo-first order rate constant and kobs =

kOH[OH−]

From Equation 3.2 and 3.3:

𝑅𝑎𝑡𝑒 = −𝑑[A]

𝑑𝑡= kobs[A] (Equation 3.4)

Rearranging Equation 3.4 yields

1

[A] 𝑑[A] = −kobs𝑑𝑡 (Equation 3.5)

Integration of Equation 3.5 gives Equation 3.6 which is rearranged to yield

Equation 3.7:

ln[A]

[A]0= −kobs 𝑡 (Equation 3.6)

[𝐴] = [A]0 exp(−kobs 𝑡) (Equation 3.7)

51

where [A]0 represents the initial concentration of A and [A] is the concentration of

A at any reaction time, t. By employing Beer-Lambert’s law: E[A]l where l is unity,

the product of E and [A] would yield absorbance, A. Thus, the observed absorbance

of the reaction mixture, Aobs which constitutes both the reactant, A and product, P

is determined to be:

Aobs = E[A][A] + E[P][P] (Equation 3.8)

where E represents molar extinction coefficient of a particular species. It is

determined from Equation 3.1 that

[A]0 = [A] + [P] (Equation 3.9)

therefore,

[P] = [A]0 − [A] (Equation 3.10)

By substituting Equation 3.10 into Equation 3.8 yields:

52

Aobs = E[A][A] + E[P][P]

= (EA − EP)[A] + E[P][𝐴]0 (Equation 3.11)

Assuming EA − EP= 𝐸𝑎𝑝𝑝 and EP[A]0 = A∞, Equation 3.11 is simplified into:

Aobs = Eapp[A] + A∞ (Equation 3.12)

Upon substitution of Equation 3.7 into Equation 3.12 and taking [A]0 = [X]0 gives

Equation 3.

Aobs = Eapp [X0] exp (– kobs t) + A∞ (Equation 3)

Equation 3 is employed for any reaction that is monitored as the disappearance

of reactants. In present study, reactions were also monitored by appearance of

product, therefore rearrangement of Equation 3 will generate Equation 4.

Aobs = Eapp [X0] {1-exp (– kobs t)} + A0 (Equation 4)

53

CHAPTER 4

RESULTS AND DISCUSSION

4.1 Hydrolysis of AMPNa2 in Alkaline, Acidic and Neutral media

In this experiment, a substrate was added into sample solution to make up

20 mL and then stored in a water bath at 60 °C covering the pH range from pH

0.30-12.71. The samples were stored in a water bath at 60 °C to speed up the

reaction. The reaction was monitored spectrophotometrically using a UV-Vis

spectrophotometer. The substrate used in this experiment was adenosine

monophosphate disodium salt, AMPNa2. In this investigation, AMPNa2 mimics the

phosphate ester bond in cellular AMP. Hydrolysis of AMPNa2 was carried out in

acidic, alkaline and neutral media.

54

4.2 Specific Base Hydrolysis of AMPNa2

4.2.1. UV-Vis Spectrum of Hydrolysis of AMPNa2 in Alkaline Medium

For specific base hydrolysis, the pH ranged from pH 9.95-12.71 with

[NaOH] = 0.0008-1.0000 M. Figure 4.1 shows the UV-Vis absorption spectrum of

alkaline hydrolysis of AMPNa2 at [NaOH] 1.0 M at 60 °C. Absorption spectrum I

refers to the first UV-Vis absorption spectrum which was taken at t = 15 s. Spectral

measurements were carried out until t = 1, 483, 260 s which is spectrum labelled

by VI.

Figure 4.1: UV-Vis absorption spectrum of alkaline hydrolysis of AMPNa2 at

[NaOH] 1.0 M at 60 °C

220.0 225 230 235 240 245 250 255 260 265 270 275 280 285 290 295 300 305 310.0

0.00

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2

1.3

1.4

1.5

1.6

1.7

1.8

1.9

2.0

2.1

2.2

2.3

2.4

2.50

nm

A (I) t = 15 s

(II) t = 77,280 s

(III) t = 198,240 s

(IV) t = 456,540 s

(V) t = 894,120 s

(VI) t = 1, 483, 260 s

55

It can be seen in Figure 4.1 that the absorption maximum (λmax) is around

260.0 nm. This correlates with the absorption maximum (λmax) of adenosine which

is 259.5 nm (Tuan, 2014). The reaction began from t = 15 s until it reached reaction

completion at t = 1, 483, 260 s. As time of the reaction progressed from t = 15 s to

t = 1, 483, 260 s, it could be seen that the absorbance value decreased. This shows

that the concentration of the reactants has decreased as the reaction was

progressing. At the beginning of the reaction, the hydrolysis proceeded at a very

fast rate and then slowly decreased and the last few absorbance data recorded

similar values which indicated that the reaction has gone into completion. The

hydrolysis of AMPNa2 in alkaline condition was a one step process and it did not

involve the presence of a transition state.

4.2.2 Kinetic Study of Hydrolysis of AMPNa2 in Alkaline Medium

Figure 4.2 shows the graph of absorbance against time for AMPNa2 in the

presence of [NaOH] 1.0 M at 60 °C. The absorbance values were taken at 260.0

nm.

56

Figure 4.2: Alkaline hydrolysis of AMPNa2 in the presence of [NaOH] 1.0 M

at 60 °C. A decrease in the absorbance with time was observed and the solid

line was drawn through the calculated absorbance values with kobs = 4.44 × 10-

6 s-1, Eapp = 8466 ± 44 M-1 cm-1, and A∞ = 0.294 ± 0.003 using Equation 3

It can be seen that the absorbance decreased with time following a first order

reaction. The slope of the graph provided us with information of the rate of reaction

whereby in the beginning of the hydrolysis, the reaction proceeded very quickly

until it reached a stage around 6.0 × 105 s where the slope of the graph was not so

steep. This slope indicated that the rate of reaction is slower until the reaction

completed. The reaction is said to have gone into completion when there is no

significant increase or decrease in the absorbance as shown in the figure above from

t = 10.0 × 105 s and t = 13.0 × 105 s. The rate or reaction can then be calculated by

observing the amount of reactant that reduced with reference to time. The rate of

reaction of this particular sample was calculated by Equation 3.

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0

Ab

sorb

ance

Time (s) × 105

57

Aobs = Eapp [X0] exp (– kobs t) + A∞ (Equation 3)

where, Eapp is apparent molar extinction coefficient of the reaction mixture, A∞ is

absorbance at reaction time, t = ∞, kobs is pseudo-first-order rate constant, and [X0]

represents the initial concentration of substrate, AMPNa2

A total of twelve reactions were carried out with different sodium hydroxide

and sodium chloride concentrations and the rate of reactions were calculated. Table

4.1 shows the concentration of NaOH, pH of sample before and after the reaction

at 60 °C, observed rate of reaction (kobs), calculated rate of reaction (kcalc), Eapp, A∞,

and ∑di2 for all the reaction mixtures in this investigation. For [NaOH] less than

0.2 M, ionic strength was maintained at 0.2 M, while for [NaOH] more than 0.2 M,

ionic strength was increased to 1.0 M. There were two samples of 0.2 M [NaOH],

one with [NaCl] of 0.2 M and the other with [NaCl] of 1.0 M. The rate of reaction

of these two samples were compared to study the effect of ionic strength on the rate

of hydrolysis of AMPNa2 in alkaline condition.

58

Table 4.1: Values of concentration, pH before, pH after, kobs, kcalc, Eapp and A∞

for alkaline-hydrolysis of 0.0001 M AMPNa2 at 60 °Ca

[NaOH]

pH

beforeb

pH

afterc

107

kobs/s-1

107

kcalc/s-1

Eapp/M-

1 cm-1

A∞

∑di2d

0.0008

9.95

9.76

(1.20 ±

0.10 d)

0.65

2718 ±

78 d

0.857 ±

0.007 e

1.67 ×

10-3

0.0020 10.28 10.35 (1.66 ±

0.17)

0.72 1482 ±

55

1.095 ±

0.004

1.89 ×

10-3

0.0100 10.98 10.78 (1.70 ±

0.35)

1.06 1691 ±

154

0.915 ±

0.017

2.21 ×

10-3

0.0200 11.18 11.07 (1.98 ±

0.14)

1.50 4177 ±

126

0.659 ±

0.013

2.55 ×

10-3

0.0400 11.46 11.94 (2.17 ±

0.10)

2.36 3519 ±

73

0.743 ±

0.008

1.10 ×

10-3

0.0500 11.62 11.64 (1.47 ±

0.27)

2.79 3216 ±

361

0.764 ±

0.038

2.68 ×

10-3

0.1000 11.91 11.87 (3.95 ±

0.58)

4.95 3422 ±

196

0.791 ±

0.022

4.38 ×

10-3

0.2000 (ionic

strength 0.2 M)

12.08 12.41 (8.69 ±

0.41)

9.27 4590 ±

75

0.653 ±

0.008

1.30 ×

10-3

0.2000 (ionic

strength 1.0 M)

12.21 12.56 (10.20

± 0.21)

9.27 6245 ±

43

0.578 ±

0.004

9.26 ×

10-4

0.4000 12.43 12.60 (17.70

± 0.68)

17.90 7221 ±

104

0.425 ±

0.011

1.72 ×

10-3

0.5000 12.51 12.44 (21.40

± 0.82)

22.20 8353 ±

124

0.382 ±

0.013

2.47 ×

10-3

1.0000 12.71 12.86 (44.40

± 0.50)

43.80 8466 ±

44

0.294 ±

0.003

6.80 ×

10-4

a Reaction conditions for alkaline hydrolysis of AMPNa2 as shown in Appendix B

b pH was taken after all the ingredients were added except substrate at temperature 60°C c pH was taken after reaction is completed at temperature 60°C d Residual error of calculated data points to the observed data points e Error limits are standard deviations

59

The observed rate constants, kobs were plotted against [NaOH]

concentrations to clearly observe the effect of [NaOH] concentrations on the rate

of reaction as shown in Figure 4.3.

Figure 4.3: Pseudo-first-order rate constant, kobs versus [NaOH] for alkaline

hydrolysis of 0.0001 M AMPNa2 at 60 °C calculated using Equation 3

Theoretical rates of reaction, kcalc for each [NaOH] were calculated from

Equation 3 and also plotted on the graph as the solid line. As seen in the graph, the

calculated rates of reaction, kcalc does not deviate far from the observed rates of

reaction, kobs. The graph also depicts that as the concentration of sodium hydroxide

increases, the observed rate of reaction, kobs increases as well.

y = 4.32 × 10-6x + 6.30 × 10-8

R² = 1

0

5

10

15

20

25

30

35

40

45

50

0.0 0.2 0.4 0.6 0.8 1.0

Pse

udo

-Fir

st-O

rder

Rat

e C

on

stan

t, 1

07

kob

s

[NaOH] (M)

Kobs kcalc

60

kobs = kb [OH-] + k0 (Equation 5)

where, kobs represents pseudo-first-order rate constant of the reaction, kb represents

second-order base-catalysed rate constant and k0 represents uncatalysed rate

constant for the cleavage of P-O bond in AMPNa2. From Equation 5, the kb and k0

obtained were 4.32 × 10-6 M-1 s -1 and 6.30 × 10-8 s-1 respectively.

Equation 5 allows estimation of the contribution of specific base catalysis

on alkaline hydrolysis of AMPNa2 at any desired [OH-]. The linear relationship

between kobs and [OH-] will also allow us to determine the rate constants of

AMPNa2 hydrolysis at lower pH, as the rates of reaction at these pH values are

extremely slow. It was observed that the rate constant, kobs increased with the

increase of pH under basic conditions. The highest pH value in our investigation is

12.71.

As the pH increases, the concentration of hydroxide ion increases as well.

The hydrolysis of AMPNa2 was catalysed by hydroxide ions, whereby hydroxide

ion acts as nucleophile to attack the phosphorus centre. Base-catalysed hydrolysis

of phosphate ester proceeds through a BP2 reaction, stands for base-catalysed,

phosphoryl-oxygen fission, bimolecular reaction analogous to an SN2 reaction. The

attack of hydroxide ion on the phosphorus atom is known as the rate limiting step

of this hydrolysis (Hilal, 2006). As most SN2 reaction, increasing concentration of

61

the nucleophile increases the rate of the reaction (Singh, 2004). This explains the

pH dependency in the hydrolysis of AMPNa2. It was previously calculated that the

rate of the rate of the hydroxide ion attack on the neutral phosphate monoester is

very fast (Florián and Warshel, 1997). This trend was also noticed in the hydrolysis

of benzoyl methyl phosphate. Hydrolysis of benzoyl methyl phosphate was carried

out in the absence of metal ions and it was noticed that the hydrolysis of benzoyl

methyl phosphate was promoted by hydroxide ions, providing a second-order base-

catalysed rate of 3.4 × 10-1 M-1 s-1. A pH dependency was noticed in the hydrolysis

of benzoyl methyl phosphate, similar to the base-catalysed hydrolysis of AMPNa2

(Kluger and Cameron, 2002)

Table 4.2 summarizes the second-order base-catalysed rate constant for

diesters and triesters at 25 °C for phosphate ester bond cleavage that have been

previously reported and the second-order base-catalysed rate constant for AMPNa2

at 60 °C for phosphate ester bond cleavage.

62

Table 4.2: Second-order base-catalysed rate constant values of phosphate ester

hydrolysis in previous study and present study (Schroeder et al., 2006)

Ester kb at 25 °C, M-1 s-1

bis-3-(4-carboxyphenyl) neopentyl phosphate 1.00 × 10-6

Trimethylphosphate 1.60 × 10-4

Triethylphosphate 8.20 × 10-6

Triphenylphosphate

Benzoyl Methyl Phosphate

0.25

3.4 × 10-1

AMPNa2 (this study) (60°C) 4.32 × 10-6

Table 4.2 allows us to compare the rate of reaction for base-catalysed P-O

hydrolysis for diesters, triesters and monoesters. As can be seen in the table above,

the rate is the slowest for diesters and fastest for triesters.

4.2.3 Mechanism of Hydrolysis of AMPNa2 in Alkaline Conditions

The mechanism of hydrolysis of AMPNa2 in basic conditions are shown in

Figure 4.4.

63

Figure 4.4: Proposed mechanism of alkaline-hydrolysis of AMPNa2 under

basic condition with OH- acting as a nucleophile (Marcum et al., 2011; Chin et

al., 1989; Duarte et al., 2013)

The ions present in the basic medium are OH-, Na+, and Cl-. The hydroxide

ion serves as a nucleophile to attack the phosphorus centre. This provides an insight

into the role of hydroxide ion in catalysing the reaction and phosphate ester

cleavage proceed through specific base catalysis. The role of hydroxide ion as a

nucleophile was proposed by previous studies involving p-nitrophenyl phosphate

and benzoyl methyl phosphate (Duarte et al., 2015; Spillane, 2004; Kluger et al.,

2002). The mechanism of AMPNa2 cleavage in the presence of specific base

catalyst is proposed to be initiated with a nucleophilic attack on the phosphorus

centre as depicted in Figure 4.4. This in turn results in the cleavage of P-O bond

followed by the production of Na2PO4H and deprotonated adenosine.

The hydrolysis of AMPNa2 in 1.0 M [NaOH] produced an insoluble white

precipitate. This precipitate was then filtered and dried. To further verify this

mechanism, the cleavage products were isolated and FTIR spectra of AMPNa2, the

filtrate and the residue (powder) were obtained and compared as shown in Figure

64

4.5. Individual FTIR spectra of AMPNa2, the filtrate and the residue of the alkaline

hydrolysis are attached in Appendix H-J respectively.

Figure 4.5: Comparison of FTIR spectra of the substrate, residue and the

filtrate for alkaline hydrolysis of 0.0001 M AMPNa2 in the presence of [NaOH]

1.0 M at 60 °C

Table 4.3 consists of the peak assignments of IR absorbance spectra given

to the substrate which is AMPNa2, the residue and the filtrate obtained in alkaline

hydrolysis of AMPNa2 at 60 °C. As seen in Table 4.3, the presence of the hydroxy,

adenine, phosphate and phosphate ester groups in AMPNa2 can be confirmed.

65

Table 4.3: Peak assignments of IR absorbance spectra for the substrate,

residue and the filtrate obtained in alkaline hydrolysis of AMPNa2 at 60 °C

(Stuart, 2004; Mello et al; 2012; Theophanides et al., 2012; Tiwari et al., 2005))

Absorption (cm-1)

Assignment

Filtrate

Residue

Adenosine 5’-Monophosphate

disodium salt, AMPNa2

3238

3456

3424

Hydroxy group

1635

1654

1649

Adenine

1016

1093

Phosphate

978

Phosphate ester,

ribose phosphate

skeletal motions

The FTIR spectrum of AMPNa2 was obtained to ensure the purity of the

AMPNa2 and to confirm the structure of AMPNa2 used in this investigation (Tiwari

et al., 2005). In previous studies, where the FTIR spectrum of adenosine

monophosphate disodium salt were obtained, the presence of phosphate group were

confirmed by the presence of absorption band at 1091 cm-1 (Theophanides and

Sandorfy, 2012). Absorption band at 976 cm-1 corresponds to phosphate ester or

also known as ribose phosphate skeletal motions. (Theophanides et al., 2012;

Stuart, 2004). These absorption bands were similar to the ones obtained for

AMPNa2 in this investigation.

66

In the FTIR spectrum of AMPNa2 obtained in this investigation, there were

two absorption bands at 978 cm-1 and 1093 cm-1 representing phosphate ester band

and phosphate group band respectively. In the filtrate and the residue, there was no

absorption observed at around 970 cm-1, indicating disappearance of phosphate

ester bond. This confirms that phosphate ester bond was cleaved producing a

phosphate salt (residue) and an adenosine salt (filtrate). The filtrate had two

absorption bands at 3238 cm-1 and 1635 cm-1 representing a hydroxy group and an

adenine group respectively indicated the filtrate is adenosine (Stuart, 2004; Mello

et al., 2012). The spectrum of the filtrate was also compared with literature

spectrum of adenosine and similarity was noticed (Spectral Database for Organic

Compounds SDBS). Absorption band at 1396 cm-1 was also present which

indicated that N-glycosidic bond was still present in the product (Agarwal et al.,

2014). This indicates that N-glycosidic bond cleavage did not take place in the

alkaline hydrolysis of adenosine monophosphate disodium salt.

This method of FTIR spectra comparison have been carried out before in

the hydrolysis of mono-2-methyl-5-nitroaniline phosphate (Awadhiya and Bhoite,

2011). Literature infrared spectrum of adenosine obtained is shown in Figure 4.6.

Table 4.4 shows the comparison of FTIR peak assignments for filtrate obtained in

this investigation and literature spectrum of adenosine obtained from Spectral

Database for Organic Compounds SDBS.

67

Figure 4.6: FTIR spectrum of adenosine obtained from Spectral Database for

Compounds SDBS

Table 4.4: Comparison of FTIR peak assignments for filtrate and literature

spectrum of adenosine (Stuart, 2004; Mello et al., 2012)

Absorption (cm-1)

Assignment

Filtrate

Literature spectrum of adenosine

3238

3166

Hydroxy group

1635

1667

Adenine

UV-Vis spectra of alkaline hydrolysis (Figure 4.1) also depicted that

absorption maximums (λmax) of the spectra did not deviate from 260 nm throughout

the hydrolysis which indicates that adenosine was not broken down into adenine.

68

The absorption maximum (λmax) of adenosine which is 259.5 nm while the

absorption maximum (λmax) of adenine is 260.5. (Tuan, 2014). If adenosine was

broken down into adenine, slight deviation would have been seen in the spectra.

Therefore, it is evident that adenosine was produced in the alkaline hydrolysis of

AMPNa2. Adenosine that was produced in alkaline medium goes through further

reaction as shown in Figure 4.7.

Figure 4.7: Proposed mechanism for the formation of the final product

Adenosine gets further deprotonated as shown in Figure 4.7. This

deprotonation occurred due to the presence of OH- and Cl- which act as

deprotonating agents (Pavia, et al., 2005; Hon, 1996). This loss of hydrogen atoms

in fragments have been noticed in previous fragmentation of adenine molecules

(Minaev et al., 2014). In solutions involving negative ions, it is possible to observe

abstraction of hydrogen atoms by hydroxide ions and this explains why the formula

weight of adenosine reduced to 261.1937 (Nibbering, 1985). Abstraction of

hydrogen atom from adenosine is favourable as the lone pair electrons of oxygen

in the aromatic ring can be delocalised. Hydrogen atoms can also be abstracted from

69

C-H bonds. Theoretically, adenosine has a peak at m/z =268.2. Since abstraction of

hydrogen atoms were highly possible, the expected peak corresponding to

adenosine should be lesser than m/z =268.2 but not deviate to far from this value

(Zhao et al., 2013). Therefore it is safe to say that the peak with m/z =262

corresponds to adenosine that has gone through hydrogen abstraction in the

presence of hydroxide ions (Zhong et al., 2017). To verify this mechanism,

positive-ion LC-MS spectrum of the product (filtrate) was obtained as shown in

Figure 4.8.

Figure 4.8: Positive-ion LC-MS spectrum of the final product of alkaline

hydrolysis of 0.0001 M AMPNa2 in [NaOH] 1.0 M at 60 °C

70

From the mechanism in Figure 4.4 and Figure 4.7, the peaks that can be

expected from the spectrum are m/z =141.9588 + 1 and m/z =261.1937 + 1. The

peaks in the positive-ion LC-MS spectrum that corresponds to these values are m/z

=142.9658 and m/z =262.9397.

LC-MS spectrum of AMPNa2 dissolved in only distilled water was also

obtained at two different times to study if self-decomposition of AMPNa2 takes

place over time. Figure 4.9 shows the positive-ion LC-MS spectrum of freshly

prepared AMPNa2 in distilled water.

Figure 4.9: Positive-ion LC-MS spectrum of freshly prepared 0.0001 M

AMPNa2 in distilled water

71

As can be seen from the positive-ion LC-MS spectrum, two major peaks

obtained were at m/z =393.2102 and m/z =349.1837. The m/z =393.2102 peak

corresponds to the protonated AMPNa2, m/z =392.1923+1 as shown in Figure 4.10.

Figure 4.10: Structure of protonated AMPNa2 corresponding to m/z

=393.2102

The peak with m/z =349.1837 corresponds to the compound that is formed

when the Na+ groups were replaced by H+ whereby m/z =348.2286+1. The structure

of this fragment is shown in Figure 4.11.

Figure 4.11: Structure of protonated AMPNa2 corresponding to m/z =349.1837

72

These assignments of the fragments were also well documented by other

researchers (Lorenzetti et al., 2007; Qian et al., 2004). Positive-ion mass spectrum

of AMPNa2 that was dissolved in distilled water and left aside for a period of 3

months was also obtained and the positive-ion mass spectrum is as shown in Figure

4.12.

Figure 4.12: Positive-ion LC-MS spectrum of AMPNa2 in distilled water

undergoing self-hydrolysis

It can be seen in the spectrum that there were no fragments at m/z =393 or

m/z =349, and the majority abundance was at peak with m/z =268.1050. This peak

corresponds to (adenosine + 1) (Van Dycke et al., 2010). This indicates that

AMPNa2 breaks down into adenosine without the presence of OH-, but at a slower

rate due to the fact that water is weaker nucleophile (Hilal, 2006). Water here acts

as a nucleophile and the mechanism is depicted in Figure 4.13.

73

Figure 4.13: Mechanism of AMPNa2 undergoing self-hydrolysis into

adenosine (Hilal, 2006; Marcum et al., 2011; Larson et al., 1994, Jubian,

1991; Duarte et al., 2015; Spillane, 2004)

Water acts as nucleophile by first protonating the leaving group. Then the

hydroxide ion rapidly attacks the phosphorus centre. This mechanism is known as

substrate-assisted cleavage of P-O bond in phosphate esters. The proton transfer to

the phosphate also facilitates cleavage by increasing negative charges in the

phosphate group. This results in repulsive force on the leaving group (Jubian,

1991). This was also proven by theoretical study on the hydrolysis of monoester

phosphates, such as methyl phosphate and p-nitrophenyl phosphate (Duarte et al.,

2015; Spillane, 2004).

This has indicated there was a presence of uncatalysed hydrolysis of

AMPNa2 at pH 7 and the predominant mechanism is P-O bond cleavage. It is

important to note that the products formed from the hydrolysis in basic conditions

and the products formed from neutral hydrolysis (where water acts as a nucleophile)

were slightly different. This was due to the presence of OH- and Cl- in the alkaline

medium acting as a deprotonating agent (Pavia et al., 2005; Hon, 1996). The

mechanisms employed in these two different media was also different.

74

It was concluded that the residue was a phosphate salt due to the fact that it

has an absorption band at 1016 cm-1 which represents a phosphate absorption band.

This residue also had a slight absorption band for adenine. It could be possible that

adenosine that was formed further broke down into adenine and this free adenine

might have reacted with the phosphate group to form adenine phosphate salt. This

adenine phosphate is a known compound with a molecular formula of C5H8N5O4P

(Kim, 2016). The possible structure of this compound is shown in Figure 4.14.

Figure 4.14: Possible structure for residue (adenine phosphate) (Kim, 2016).

The FTIR spectra of the substrate, filtrate and residue further confirmed the

proposed mechanism which produced phosphate salt and adenosine as shown in

Figure 4.4 and Figure 4.7. LC-MS data also confirmed the presence of adenosine

in the product. This method of characterization which involves a combination of

LC-MS and infrared spectra have been employed in previous hydrolysis studies of

alkyl hydrogen methylphosphonates (Keay, 1965).

In previous investigation of UV-photodissociation of AMP, similar

products were obtained whereby phosphate based fragments were found in high

abundance especially PO-3. Marcum and his co-workers (2011) proposed a

75

mechanism whereby the phosphate ester bond was broken and produced PO-3 and

adenosine. A metal complex study aiming to cleavage cleave adenosine

monophosphate (AMP) also resulted in the formation of adenosine (Chin and

Banaszxzyk, 1989). Enzymatic hydrolysis of AMP by enzymes such as Alkaline

Phosphatase (AP) also produced the same products as proposed in the above

mechanism (Millán, 2006).

4.3 Specific Acid Hydrolysis of AMPNa2

4.3.1 UV-Vis Spectrum of Hydrolysis of AMPNa2 in Acidic Medium

Acidic hydrolysis of AMPNa2 was also carried out and reaction was

monitored spectrophotometrically using a UV-Vis spectrophotometer. For specific

acid hydrolysis, reactions were carried out at pH ranged from pH 0.30-1.83 with

[HCl] = 0.01-1.00 M. Figure 4.15 shows the UV-Vis absorption spectrum of acidic

hydrolysis of AMPNa2 in [HCl] 1.0 M at 60 °C. Absorbance spectrum I refers to

the first UV-Vis absorption spectrum which was taken at t = 15 s. Spectral

measurements were carried out until t = 4, 048, 800 s which is labelled by spectrum

III.

76

Figure 4.15: UV-Vis absorption spectrum of acidic hydrolysis of AMPNa2 in

[HCl] 1.0 M at 60 °C.

The hydrolysis of AMPNa2 in acidic condition was slightly different than

the hydrolysis of AMPNa2 in alkaline condition. Unlike hydrolysis of AMPNa2 in

alkaline conditions which did not have a transition state, hydrolysis of AMPNa2 in

acidic conditions involved the formation of a transition state. The hydrolysis began

at t = 0 s and the absorbance decreased progressively until t = 72, 480 s. At t = 72,

480 s, the absorbance did not increase or decrease but remained until t = 421, 440

s. This constant absorbance signified the transition state of the hydrolysis reaction.

After t = 421, 440 s, the absorbance value increased until t = 4, 048, 800 s whereby

at this time the reaction has completed. This indicated that the hydrolysis of

AMPNa2 in acidic condition was a two-step process.

210.0 215 220 225 230 235 240 245 250 255 260 265 270 275 280 285 290 295 300 305 310 315 320.0

0.00

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2

1.3

1.4

1.5

1.6

1.7

1.8

1.9

2.0

2.1

2.2

2.3

2.4

2.50

nm

A

(I) t = 15 s

(III) t = 4, 048, 800 s (End of reaction)

(II) t = 72, 480 s to

421, 440 s

77

Another difference noticed in the hydrolysis of AMPNa2 in acidic condition

with hydrolysis of AMPNa2 in alkaline condition was that in acidic condition, as

the reaction progresses, there was a shift of absorption maximum values. At the

beginning of the hydrolysis, the absorption maximum value was around 257.0 nm

and the reaction ends with absorption value at 265.0 nm. This shift can be explained

as AMPNa2 generally has an absorption maximum of 259.5 nm, where adenosine

group is responsible for this absorption. The end product of acidic hydrolysis was

adenine, which has an absorption maximum (λmax) of 260.5 nm (Tuan, 2014).

Therefore, as the amount of adenosine decreased and the amount of adenine

increased, the absorption maximum (λmax) shifted to the right. This shift to the right

was also demonstrated by Stockbridge and co-workers (2010). In Stockbridge’s

investigation, adenosine was decomposed at pH 7 over 24 hours at 150 °C followed

by break down of adenosine into adenine. In their investigation, the UV-Vis

spectrum also demonstrated a shift to the right as adenosine decreased and adenine

increased. The hydrolysis of AMPNa2 in acidic condition took a much longer time

to reach completion when compared with hydrolysis of AMPNa2 in alkaline

condition which indicated a slower rate compared to alkaline hydrolysis.

4.3.2 Kinetic Study of Hydrolysis of AMPNa2 in Acidic Medium

Figure 4.16 shows the graph of absorbance against time for AMPNa2 in the

presence of [HCl] 1.0 M at 60 °C.

78

Figure 4.16: Acidic hydrolysis of AMPNa2 in the presence of [HCl] 1.0 M at 60

°C. An increase in the absorbance with time was observed and the solid line

was drawn through the calculated absorbance values with kobs = 1.67 × 10-6 s-1,

Eapp = 3688 ± 91 M-1 cm-1, and A0 = 0.757 ± 0.009 using Equation 4

It is important to note that in acidic conditions the absorbance values were

increasing over time. For simplicity, the absorbance values in this acidic conditions

were taken at 270.0 nm due to the fact that in acidic spectrum there was a shift of

absorption maximum to the right. The reason why the absorption values were

increasing was that in this acidic mechanism, quantity of adenosine was decreasing

while more adenine was being produced. This absorbance values here represent the

amount of adenine that was produced over time.

0.70

0.75

0.80

0.85

0.90

0.95

1.00

1.05

1.10

1.15

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0

Ab

sorb

ance

Time (s) × 105

79

The slope of the graph can provide us with information of the rate of

reaction whereby in the beginning of the hydrolysis, the reaction proceeds very

quickly until it reaches a stage around 15.0 × 105 s where the slope of the graph less

steep. This slope indicates that the rate of reaction was slower until the reaction

goes into completion. The reaction is said to have gone into completion when there

is no significant increase or decrease in the absorbance as shown in the Figure 4.16

from t = 30.0 × 105 and 45.0 × 105 s. The rate of reaction of this particular sample

was calculated with the following formula.

The rate of reaction was calculated using the following equation:-

Aobs = Eapp [X0] {1-exp (– kobs t)} + A0 (Equation 4)

where, Eapp is apparent molar extinction coefficient of the reaction mixture, A0 is

absorbance at reaction time, t = 0, kobs is pseudo-first-order rate constant, [X0]

represents the initial concentration of substrate, AMPNa2.

A total of four reactions were carried out under different hydrochloric acid

concentrations and the rate of reactions were calculated. Table 4.5 shows the

concentration of HCl, pH of sample before and after the reaction at 60 °C, observed

80

rate of reaction (kobs), calculated rate of reaction (kcalc), Eapp, A0, and ∑di2 for all the

reaction mixtures in this investigation.

Table 4.5: Values of concentration, pH before, pH after, kobs, kcalc, Eapp and A0

for acidic-hydrolysis of 0.0001 M AMPNa2 at 60 °Ca

[HCl]

pH

beforeb

pH

afterc

107 kobs/s-

1

107

kcalc/s-1

Eapp/M-1

cm-1

A0

∑di2d

0.01

1.83

1.95

(1.32 ±

0.06d)

0.27

4500 ±

77 e

0.744 ±

0.005 e

2.27 ×

10-3

0.04 1.38 1.25 (0.32 ±

0.07)

0.75 10004 ±

141

0.468 ±

0.016

7.48 ×

10-4

0.40 0.51 0.45 (5.52 ±

0.58)

6.57 3687 ±

164

0.764 ±

0.010

3.08 ×

10-3

1.00 0.30 0.09 (16.70 ±

1.00)

16.3 3688 ±

91

0.757 ±

0.009

3.36 ×

10-3

a Reaction conditions for acidic hydrolysis of AMPNa2 as shown in Appendix A

b pH was taken after all ingredients were added except substrate at temperature 60 °C c pH was taken after reaction is completed at temperature 60°C d Residual error of calculated data points to the observed data points e Error limits are standard deviations

The observed rate constants, kobs were plotted against [HCl] concentrations

to clearly observe the effect of [HCl] concentrations on the rate of reaction as

depicted in Figure 4.17.

81

Figure 4.17: Pseudo-first-order rate constant, kobs versus [HCl] for acidic

hydrolysis of 0.0001 M AMPNa2 at 60 °C calculated using Equation 4

Theoretical rate of reaction, kcalc for each [HCl] were calculated and also

plotted on the graph as the solid line. As seen in the graph, the calculated rate of

reactions, kcalc does not deviate far from the observed rate of reactions, kobs. The

graph also depicted that as the concentration of hydrochloric acid increased, the

observed rate of reaction, kobs increased.

The solid line is drawn through the calculated data points in Figure 4.17

follows Equation 6,

y = 1.62 × 10-6x + 1.03 × 10-8

R² = 1

0

2

4

6

8

10

12

14

16

18

0 0.2 0.4 0.6 0.8 1 1.2

Pse

udo

-Fir

st-O

rder

Rat

e C

onst

ant,

10

7k

ob

s

[HCl] (M)

kobs kcalc

82

kobs = ka [H+] + k0 (Equation 6)

where, kobs represents pseudo-first-order rate constant of the reaction, ka represents

second-order acid-catalysed rate constant and k0 represents uncatalysed rate

constant for the cleavage of P-O bond in AMPNa2. From Equation 6, the ka and k0

obtained were 1.62 × 10-6 M-1 s -1 and 1.03 × 10-8 s-1 respectively.

Equation 6 allows estimation of the contribution of specific acid catalysis

on the rate constant of AMPNa2 hydrolysis at any desired [H+]. The linear

relationship between kobs and [H+] will also allow us to determine the rate constants

of AMPNa2 hydrolysis at higher pH, as the rates of reaction at these pH values are

extremely slow. It was observed that the rate constant, kobs increased with the

decrease of pH under acidic conditions.

At low pH conditions, or as the concentration of HCl increased, the quantity

of H+ or hydronium ion in the solution increased. In specific acid catalysis, the H+

ion enhanced the rate of the reaction by providing an alternative mechanism which

was more favourable energetically. Hydronium ion did this by withdrawing the

electron density from the phosphorus atom that held the adenosine leaving group

making the phosphorus atom more susceptible to nucleophilic attack by the chloride

ion during the phosphate ester cleavage (Larson and Weber, 1994). Alternative

mechanism was also provided by the hydronium ion during acid-catalysed

83

depurination as hydronium ion protonates N7 of the adenine ring, the leaving group

and lowered the energetics of the transition state (An et al., 2014).

The contribution of H+ ion on the rate of reaction of neutral phosphate

monoesters was also explained in previous investigations. In the hydrolysis of

mono-4-bromo, 2,6-dimethylphenyl phosphate, the rate of hydrolysis increased as

the concentration of HCl increased (Tiwari et al., 2005). Hydrolysis of benzoyl

methyl phosphate in acidic media at constant ionic strength depicted a linear

relationship between that rate of hydrolysis and the concentration of hydronium

ion. This indicated that the hydrolysis of benzoyl methyl phosphate was subject to

hydronium ion catalysis. The second-order acid-catalysed rate constant of benzoyl

methyl phosphate was 3.1 × 10-6 M-1 s-1 (Kluger and Cameron, 2002). pH rate

profiles of most monoalkyl phosphates were maximum between pH 3 to 5

indicating that the presence of hydronium ion accelerates the phosphate ester

cleavage.

4.3.3 Mechanism of Hydrolysis of AMPNa2 in Acidic Medium

The ions present in the acidic medium were H+, Na+, and Cl-. The hydrolysis

of AMPNa2 in acidic medium differed from the hydrolysis of AMPNa2 in basic

medium. AMPNa2 undergoes two different cleavage reactions in acidic medium.

84

The first reaction of the bond cleavage in AMPNa2 involved phosphate ester

cleavage as shown in Figure 4.18.

Figure 4.18: Proposed mechanism of acidic hydrolysis of AMPNa2 under

acidic condition with H+ ion acting as a protonating agent (Kwan, 2005; Hilal

2006; Tiwari et al., 2005))

As depicted in Figure 4.18, the oxygen from the leaving group gets

protonated due to the high concentration of H+ ions in acidic medium. Cl- also acts

as a nucleophile and attacks the phosphorus centre. This resulted in the cleavage of

the phosphate ester bond (Allen et al., 1994; Fish et al., 2006; Larson et al., 1994).

This mechanism is in accordance with acid-catalysed hydrolysis of previous studies

including monomethyl phosphate and mono-4-bromo, 2,6-dimethylphenyl

phosphate whereby the leaving group was protonated before nucleophilic attack

(Jubian, 1991; Tiwari et al., 2005). The role of hydrogen bonding in acid-catalysed

phosphate ester cleavage was also depicted in theoretical studies of monoester

phosphates (Duarte et al., 2015).

Infrared spectrum of the product of acidic hydrolysis of AMPNa2 was

obtained to verify the mechanism in Figure 4.18. There was no insoluble powder

85

formed in acidic hydrolysis therefore only one infrared spectrum was obtained.

Comparison of the infrared spectrum of AMPNa2 and the final product of the acidic

cleavage indicated the disappearance of the phosphate ester, ribose phosphate

skeletal motions around 970 cm-1. This indicated the phosphate ester bond cleavage.

The comparison of the infrared spectra of AMPNa2 and product of acidic hydrolysis

is shown in Figure 4.19. Individual infrared spectra of AMPNa2 and the product of

acidic hydrolysis are attached in Appendix H and Appendix K respectively.

Figure 4.19: Comparison of FTIR spectra of AMPNa2 and product of acidic

hydrolysis of 0.0001 M AMPNa2 in the presence of [HCl] 1.0 M at 60 °C

Specific peak assignments on the functional groups of AMPNa2 and the

product of acidic hydrolysis are presented in Table 4.6.

2923 1649 1093

978

AMPNa2

Acidic product

3424

3338

1636

1459

86

Table 4.6: Peak assignments of IR absorbance spectra for the substrate, and

the hydrolytic product obtained in acidic hydrolysis of AMPNa2 at 60 °C

(Stuart, 2004; Mello et al; 2012; Theophanides et al., 2012; Agarwal et al.,

2014)

Absorption (cm-1)

Assignment

Acidic

Product

Adenosine 5’-Monophosphate

disodium salt, AMPNa2

3338

3424

Hydroxy group

1636

1649

Adenine

1459

C-N glycosidic bond

1093

Phosphate

978

Phosphate ester, ribose

phosphate skeletal motions

The infrared spectrum in Figure 4.19 confirms the mechanism proposed in

Figure 4.18. Besides phosphate ester and phosphate band disappearance, the

absorption responsible for N-glycosidic bond also disappeared in the product. As

depicted on spectrum on Figure 4.19 and Table 4.6, absorption band around 1459

cm-1 that was present in AMPNa2 was not present in the acidic product (Agarwal et

al., 2014). This indicated that N-glycosidic bond cleavage occurred. After the

phosphate ester bond cleavage, adenosine undergoes N-glycosidic bond cleavage

or acid-catalysed depurination producing adenine and ribose sugar. The mechanism

of acid-catalysed depurination is shown in Figure 4.20.

87

Figure 4.20: The mechanism of N-glycosidic bond cleavage of AMPNa2 in

acidic conditions at 60 °C (An et al., 2014; Jobst et al., 2016)

Acid-catalysed depurination of adenosine began with attack of H+ on N7 of

adenine and this lead to the formation of a monoprotonated intermediate due to high

concentration of H+ under highly acidic conditions. This caused a series of charge

redistribution. This in turn caused N-glycosidic bond cleavage of the C’1 from the

ribose ring and N7 of the adenine ring. This provided an insight into the role of

hydrogen ion in catalysing the depurination of adenosine as H+ protonated the

leaving group as depicted in Figure 4.20. This results in the formation of protonated

ribose compound and double protonated adenine (C5H7N5) (An et al., 2014; Jobst

et al., 2016). At highly strong acidic conditions, it is possible that double

protonation of the adenine has accelerated the hydrolysis. This double protonation

occurs at N3, where hydrogen ions were abstracted towards nitrogen and resulted

in the formation of a double protonated adenine as shown in Figure 4.20 (An et al.,

2014).

88

To verify the mechanisms proposed in Figure 4.20, positive-ion LC-MS

spectrum of the product was obtained as shown in Figure 4.21.

Figure 4.21: Positive-ion LC-MS spectrum of the product of acidic hydrolysis

of 0.0001 M AMPNa2 in [HCl] 1.0 M at 60 °C

Table 4.7 lists of the formula of products, molecular weight (M), ionized

(M+1) and values of fragments of the LC-MS spectrum that corresponds to the

formula of products based on Figure 4.21.

89

Table 4.7: Formula, molecular weight, M+1, and corresponding fragments for

acidic hydrolysis of AMPNa2 at 60 °C

Formula

Molecular

weight, M

M+1

m/z

C5N7H5 (protonated

adenine)

137.1426

138.1426

138.9065

C5H11O4 (protonated

ribose)

135.1379

136.1379

136.0622

Theoretically, adenine corresponds to m/z =136.1 and according to previous

study, protonated adenine is corresponds to peak with m/z =137 (Zhao et al., 2013).

Since, in the present study, due to high concentration of H+ ions, adenine gets

double protonated, therefore peak that can be expected is around m/z =138.1426

(Dwivedi et al., 2010). As time progresses, according to the mechanisms proposed

for acidic hydrolysis of AMPNa2 as shown in Figure 4.20, the quantity of adenosine

decreased while the quantity of adenine increased. This proposal is in agreement

with the UV-Vis absorption spectrum of hydrolysis of AMPNa2 throughout the

reaction, whereby from the initial stage of reaction and towards the end of the

reaction, there was a shift of absorption towards the right as shown in Figure 4.15.

This is also in accordance with absorption maximum values of adenosine and

adenine which is at 259.5 nm and 260.5 nm respectively (Tuan, 2014). As the

reaction progressed, more adenosine was broken down to produce more adenine.

Therefore, we can expect the absorption maximum values to be shifted. After

absorption maximum was shifted, the absorption values increased back again

indicating more adenine was being produced. The combination of UV-Vis

90

absorption spectrum, FTIR spectra and LC-MS spectrum of the product have

indicated that P-O bond and N-glycosidic bond have been cleaved. It was also

confirmed that adenine has been produced. This method of characterization which

involves a combination of LC-MS and infrared spectra have been employed in

previous hydrolysis studies of alkyl hydrogen methylphosphonates (Keay, 1965).

In previous investigation of UV-photodissociation of AMP, similar

products were obtained whereby adenine fragments were found (Marcum et al.,

2011). The mechanism proposed was also documented by other researchers (An et

al., 2014; Stockbridge et al., 2010). These products were also produced during the

enzymatic hydrolysis of AMP. Alkaline phosphatase first cleaves the P-O bond in

AMP, producing adenosine and nucleoside N-ribohydrolase cleaves the N-

glycosidic bond (Bontemps et al., 1983; Picher et al., 2003). Adenosine

monophosphate nucleosidase is another enzyme that directly cleaves the cleavages

the N-glycosidic bond in AMP and produces adenine and ribose 5-phosphate

(Skoog, 1986). N-glycosidic bond cleavage is acid-catalysed which explains the

occurrence of this cleavage only in acidic cleavage of AMPNa2 (Nelson and Cox,

2013).

As proposed by the kinetic data, the hydrolysis of AMPNa2 under acidic

conditions exhibited a linear relationship between rate constant and [H+]. This is

due to the fact that protonation of N7 has lowered the energetics of the transition

91

state by ~10kcal/mol. With the decrease of pH values, amount of protonated

adenine increased and therefore increasing the rate of depurination (An et al., 2014).

Electron density from the N-glycosidic bond was withdrawn due to positive charges

on the base causing it to be weakened (Jobst et al., 2016). Non-enzymatic cleavage

of adenosine also resulted in the formation of adenine (Stockbridge et al., 2010).

4.4 General Acid and Base Hydrolysis of AMPNa2

4.4.1 Spectra of General Acid and Base hydrolysis of AMPNa2

Specific base catalysis was carried out in pH ranging from pH 9.95-12.71

while specific acid catalysis was carried out for pH ranging from pH 0.30-1.83. In

order to study the effect of general acid and base catalysis on the rate of hydrolysis

of adenosine monophosphate disodium salt, various buffers were prepared at pH

from pH 2.61-9.95 by using buffer solutions such as glycine, citrate, MES, HEPES

and TRIS buffers.

However, only catalysis using glycine and TRIS buffers successfully

provided kinetic data. We were not able to obtain kinetic data for AMPNa2

hydrolysis in media involving citrate, MES and HEPES buffers as the absorbance

changes were inconsistent for a long period of time. The explanation for this

behaviour will be discussed later on. The spectrum of hydrolysis of AMPNa2 in

92

glycine showed the same pattern as acidic hydrolysis whereby there was a shift of

the maximum absorption. This was due to the breakdown of adenosine which has

an absorption maximum of 259.5 nm into adenine which has an absorption

maximum of 260.5 nm. Figure 4.22 is the UV-Vis spectrum for hydrolysis of

AMPNa2 in a medium prepared with a ratio of 20:80% glycine and hydrochloric

acid with a pH of 1.82. Absorbance spectrum I represents the UV-Vis spectrum for

hydrolysis of AMPNa2 at t = 15 s, while absorbance spectrum II represents the UV-

Vis spectrum for hydrolysis of AMPNa2 at t = 2, 598, 660 s (at the end of the

investigation).

Figure 4.22: UV-Vis absorption spectrum of general acid hydrolysis of

AMPNa2 in glycine-HCl at pH 1.82 at 60 °C

Figure 4.23 is the UV-Vis spectrum for hydrolysis of AMPNa2 in a medium

prepared with a ratio of 80:20% TRIS and hydrochloric acid with a pH of 8.03.

230.0 235 240 245 250 255 260 265 270 275 280 285 290 295 300 305 310.0

0.00

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2

1.3

1.4

1.5

1.6

1.7

1.8

1.9

2.0

2.1

2.2

2.3

2.4

2.5

2.6

2.7

2.8

2.91

nm

A

(I) t = 15 s

(II) t = 2, 598, 660 s

(end of reaction)

93

Absorbance spectrum labelled I represents the UV-Vis spectrum for hydrolysis of

AMPNa2 at t = 15 s, while spectrum labelled II represents the UV-Vis spectrum for

hydrolysis of AMPNa2 at t = 9, 076, 020 s (at the end of the investigation).

Figure 4.23: UV-Vis absorption spectrum of general base hydrolysis of

AMPNa2 in TRIS-HCl at pH 8.03 at 60 °C

4.4.2 Kinetic Study of General Acid and Base Hydrolysis of AMPNa2

Figure 4.24 and Figure 4.25 depict the graph of absorbance against time for

AMPNa2 in the presence of glycine (pH 1.82) and TRIS base (pH 8.03) at 60 °C

respectively. The absorbance values were taken at 260 nm.

230.0 235 240 245 250 255 260 265 270 275 280 285 290 295 300 305 310.0

0.00

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2

1.3

1.4

1.5

1.6

1.7

1.8

1.9

2.0

2.1

2.2

2.3

2.4

2.5

2.6

2.7

2.8

2.91

nm

A

(I) t = 15 s

(II) t = 9, 076, 020 s (end of reaction)

94

Figure 4.24: General acid hydrolysis of AMPNa2 at pH 1.82 in 20:80% glycine-

HCl at 60 °C. A decrease in the absorbance with time was observed and the

solid line was drawn through the calculated absorbance values with kobs = 4.21

× 10-7 s-1, Eapp = 1533 ± 474 M-1 cm-1, and A∞ = 0.850 ± 0.051 using Equation 3

0.94

0.95

0.96

0.97

0.98

0.99

1.00

1.01

1.02

1.03

1.04

1.05

0 5 10 15 20 25 30

Ab

sorb

ance

Time (s) × 105

95

Figure 4.25: General base hydrolysis of AMPNa2 at pH 8.03 in 80:20% TRIS-

HCl at 60 °C. An increase in the absorbance with time was observed and the

solid line was drawn through the calculated absorbance values with kobs = 9.00

× 10-8 s-1, Eapp = 2335 ± 743 M-1 cm-1, and A0 = 1.087 ± 0.004 using Equation 4

A total of two reactions were also carried out under different glycine buffer

compositions and the rate of reactions were calculated. Table 4.8 shows the

percentage of glycine buffer in the form of free acid, pH of sample before and after

the reaction at 60°C, observed rate of reaction (kobs), Eapp, A0, and ∑di2 for all the

reaction mixtures in this investigation.

1.08

1.10

1.12

1.14

1.16

1.18

1.20

1.22

1.24

0 10 20 30 40 50 60 70 80 90 100

Ab

sorb

ance

Time (s) × 105

96

Table 4.8: Values of composition, pH before, pH after, kobs, Eapp and A∞ for

general acid hydrolysis of 0.0001 M AMPNa2 at 60 °Ca

Composition

of glycine in

form of free

acid

pH

beforeb

pH

afterc

108

kobs/s-1

Eapp/M-1

cm-1

A∞

∑di2d

20%

1.82

1.75

(42.1 ±

22.9 d)

1533 ±

474 d

0.850 ±

0.051 e

2.89 × 10-4

40%

2.00

1.95

(72.5 ±

22.5)

928 ±

123

0.936 ±

0.014

2.56 × 10-4

a Reaction conditions for general acid hydrolysis of AMPNa2

as shown in Appendix C

b pH was taken after all ingredients were added except substrate at temperature 60°C c pH was taken after reaction is completed at temperature 60°C d Residual error of calculated data points to the observed data points e Error limits are standard deviations

A total of two reactions were also carried out under different TRIS buffer

compositions and the rate of reactions were calculated. Table 4.9 shows the

percentage of TRIS buffer in the form of free base, pH of sample before and after

the reaction at 60°C, observed rate of reaction (kobs), Eapp, A∞, and ∑di2 for all the

reaction mixtures in this investigation.

97

Table 4.9: Values of composition, pH before, pH after, kobs, Eapp and A0 for

general base of 0.0001 M AMPNa2 at 60 °Ca

Composition of

TRIS in form

of free base

pH

beforeb

pH

afterc

108

kobs/s-1

Eapp/M-

1 cm-1

A0

∑di2d

80%

8.03

8.25

(9.00 ±

4.16 d)

2335 ±

743 d

1.087 ±

0.004 e

3.15 ×

10-4

90%

8.42

8.35

(4.94 ±

1.08)

2228 ±

194

1.093 ±

0.008

2.47 ×

10-3

a Reaction conditions for general base hydrolysis of AMPNa2

as shown in Appendix D

b pH was taken after all ingredients were added except substrate at temperature 60°C c pH was taken after reaction is completed at temperature 60°C d Residual error of calculated data points to the observed data points e Error limits are standard deviations

Figure 4.26 is the absorption spectrum for hydrolysis of AMPNa2 in 50:50%

HEPES-NaOH buffer at pH 7.06. Spectrum labelled I refers to the first UV-Vis

absorption spectrum which was taken at t = 15 s. Spectral measurements were

carried out until t = 34, 627, 620 s which is labelled by VI.

98

Figure 4.26: UV-Vis absorption spectrum of general acid and base hydrolysis

of AMPNa2 in HEPES buffer at pH 7.06 at 60 °C

Figure 4.27 shows the absorbance versus time plot for the hydrolysis of

AMPNa2 at pH 7.06 in the presence of 50:50% HEPES-NaOH buffer. As it can be

seen, the absorbance value decreased and increased over time. At first, the

absorbance value increased up to 1.1, and then reduced sharply, increased back

again and started decreasing again. This pattern did not allow a proper rate of

reaction to be calculated. This was also observed in the hydrolysis of AMPNa2 in

MES and citrate buffer. This is due to the fact that HEPES, MES and citrate

inhibited the hydrolysis of AMPNa2. This slight inhibition caused by HEPES was

220.0 225 230 235 240 245 250 255 260 265 270 275 280 285 290 295 300 305 310 315 320 325 330.0

-0.01

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2

1.3

1.4

1.5

1.6

1.7

1.8

1.9

2.0

2.1

2.2

2.3

2.4

2.50

nm

A

(III) t = 10, 600, 380 s

(IV) t = 34, 627, 620 s

(I) t = 15 s

(II) t = 5, 146, 980 s

99

also noticed in the hydrolysis of cAMP by Cu(II) Terpyridine. Besides that,

background (buffer) hydrolysis was also high in HEPES (Jenkins et al., 1999). It

was also mentioned in previous general acid and base studies that using buffers as

medium can be quite troublesome as buffers can be inefficient as it causes many

experimental problems (Oivanen et al., 1998). MES buffer has also inhibited the

hydrolysis of nitrophenyl phosphate whereby spectroscopic changes were observed

in the UV-Vis spectra of nitrophenyl phosphate in MES (Chernobryva, 2012). In

another study of metal promoted sugar phosphate hydrolysis carried out in citrate

buffer, no significant increase was noticed in the rate of reaction. Buffers are known

to decrease the promotion of metal ion in the hydrolysis of nucleoside phosphate

(Huang and Zhang, 2011).

Figure 4.27: Absorbance versus time for hydrolysis of AMPNa2 at pH 7.03 in

50:50% HEPES: NaOH at 60 °C. No consistent change observed on the

absorbance values.

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

0 5 10 15 20 25 30 35 40

Ab

sorb

ance

Time (s) × 106

100

4.5 pH Rate Profile

Log kobs versus pH plots are helpful in determining the contribution of acid,

neutral and base in the hydrolysis reaction (Larson and Weber, 1994). Rate

constants of the hydrolysis of AMPNa2 were determined at various pH values

ranging from pH 0.30-12.71. Figure 4.28 depicts the log kobs plot against pH for all

the samples prepared in this investigation.

Figure 4.28: A plot of log kobs against pH of 18 samples for the hydrolysis of

AMPNa2 at 60 °C in reaction media with various concentration.

-13.00

-12.00

-11.00

-10.00

-9.00

-8.00

-7.00

-6.00

-5.00

0 2 4 6 8 10 12 14

log k

ob

s (s

-1)

pH

Theoretical value

Experimentally determined Background rate

Experimentally

determined

101

As it can be observed from Figure 4.28, a pH dependency of the rate of

hydrolysis of AMPNa2 was observed in highly alkaline and highly acidic regions.

A linear relationship was observed from pH 11.81-12.71 and pH 0.30-1.83

respectively. This correlation was due to the increasing concentration of hydroxide

and hydronium ions at these pH values. Unfortunately, experimental determination

of kobs from pH 2-10 does not fit to the linear lines. The rates of reaction from pH

in this region were pH independent. The rate constants between these pH are

scattered along the dotted line which have a rate constant around 10-7 s-1.

As can be seen from Figure 4.28, theoretical value for the rate constant for

close to neutral pH which is around pH 6 was determined by extrapolating the lines

from the acidic and the basic regions (Stockbridge et al., 2010). These two lines

intersect each other which indicates a theoretical rate constant at about 10-11 s-1.

Comparing to the rate constant estimated by dotted line, there was a rate

enhancement of 104 s-1 from the theoretical rate constant and the experimentally

determined rate constants.

Background rate for the hydrolysis of AMPNa2 was also determined, which

had a value of 4.64 × 10-8 s-1. As we compare the background rate and the rate of

hydrolysis that was provided by buffers, hydrochloric acid and sodium hydroxide,

it can be noticed that there was a rate enhancement. In the highly acidic and alkaline

region, the rate enhancements was provided by hydronium and hydroxide ion

102

respectively. It is also important to note that background rate was far higher than

the theoretical rate at about pH 7. This is due to the fact that at pH, water acted as

a nucleophile to attack the phosphorus centre (Banaszczyk, 1989; Ribeiro et al.,

2010; Duarte et al., 2015; Spillane, 2004; Brandão et al., 2007).

In pH values closer to neutral, the rate enhancement might be due to the

buffer catalysis provided by glycine and TRIS buffer. TRIS buffer here acts as a

nucleophile which attacks the phosphorus centre while glycine acts as a protonating

agent for the leaving group. These rate enhancements provided by TRIS and glycine

have been noticed in previous cleavage of P-O bond in phosphate monoesters in

the presence of Alkaline Phosphatase (Hethey et al., 2002).

A mixture of hydrochloric acid and TRIS was employed to obtain pH at pH

lower than 8.42. The rate enhancement obtained was due to the fact that TRIS is a

nucleophile as it has lone pair electrons on its nitrogen atom. The nitrogen atom is

only attached to one carbon atom and two hydrogen atoms as shown in Figure 4.29.

Figure 4.29: Structure of TRIS buffer (Hethey et al., 2002)

103

TRIS may act as a great nucleophile by attacking the phosphorus centre and

causing the bond cleavage. The oxygen then attracted H+ that is readily available

in the medium due to HCl present. This reaction produced adenosine and a

phosphate combined with TRIS compound (Hethey et al., 2002). The proposed

mechanism is depicted in Figure 4.30.

Figure 4.30: Mechanism of hydrolysis of AMPNa2 in TRIS-HCl medium

(Hethey et al., 2002; Marcum et al., 2011)

In the cleavage of p-nitrophenyl phosphate in TRIS buffer in previous study,

TRIS acts a nucleophile and attacks the enzyme-bound phosphate (Hethey et al.,

2002). The rate constants obtained by using TRIS as a nucleophile were much lower

than the rate constants obtained by using hydroxide ions due to the fact that OH- is

a stronger nucleophile as it is negatively charged compared to the neutral TRIS

molecule (Sloop, 2010).

Glycine is the simplest amino acid found in protein with a formula of

H2CH2CO2H. Glycine has both acidic and basic character. The O-H hydrogen

104

atoms display acidic behaviour while the nitrogen atoms display basic properties

(Olmsted and Williams, 1997). The glycine buffer used in this investigation was

prepared with hydrochloric acid and the hydrogen ion causes the carboxylate group

to be converted into carboxylic acid (Oswald, 2016). The structure of glycine is as

shown in Figure 4.31.

Figure 4.31: Structure of glycine acidified with hydrochloric acid (Oswald,

2016)

General acid catalysis in the presence of glycine works whereby it begins

with protonation of the leaving group by the glycine followed by a water molecule

attacking the phosphorus centre and causing the phosphate ester cleavage. The role

of glycine in protonating the leaving group is in accordance with previous general

acid catalysis of phosphate monoesters. This protonation facilitated cleavage by

neutralizing the repulsive electrostatic effects at the phosphorus centre (Kirby et al.,

2004; Kirby et al., 2005). This results in the formation of adenosine and a

deprotonated glycine molecule (Edwards, 1950). The mechanism is shown in

Figure 4.32.

105

Figure 4.32: Mechanism of hydrolysis of AMPNa2 in glycine buffer (Edwards,

1950; Marcum et al., 2011; Chin et al., 1989; Kirby et al., 2004; Kirby et al.,

2005).

The rates of hydrolysis of AMPNa2 in glycine-HCl were much lower than

the rate of hydrolysis of AMPNa2 in just hydrochloric acid. This is due to the fact

that in strongly acidic conditions, the amount of H+ ions in the medium was quite

high and H+ ions were readily available. This is because HCl is a strong acid which

dissociates completely in water (Klein, 2013). When glycine was used as a medium,

the H+ ions were not free form and had to be abstracted from the glycine.

The rates of reaction at pH close to physiological rates were not able to be

determined from pH 3 and pH 8. At these pH values, the ribose ring of the adenosine

in AMPNa2 opened and re-closed to yield other adenosine anomers such as

furanoside and pyranosides namely adenine α-ribofuranoside, adenine β-

ribopyranoside and adenine α- ribopyranoside. This ribose ring opening was not

observed in acidic or basic hydrolysis. The opening and closing of the ribose ring

formed anomers and also could form adenine making it difficult to determine the

106

rates of the reaction. The concentration of products increased and decreased over

time. This anomerization was also observed in previous studies (Stockbridge et al.,

2011). Figure 4.33 shows the mechanism for the possible mechanism of adenosine

anomerization at pH 7.

Figure 4.33: The possible mechanism of anomerization of adenosine at pH 7

(Stockbridge et al., 2011)

107

Besides that, for sample reactions from pH 6-7, HEPES: NaOH mixture was

used as a buffer. It could be possible that the hydrolysis of AMPNa2 at neutral pH

was not successful due to the fact that HEPES inhibited the hydrolysis. This slight

inhibition was also noticed in the hydrolysis of cAMP by Cu(II) Terpyridine.

Besides that, background (buffer) hydrolysis was also high in HEPES (Jenkins et

al., 1999). It was previously mentioned that buffers could inhibit the hydrolysis

reaction. This inhibition was noticed in metal ion promoted hydrolysis of benzoyl

methyl phosphate (Kluger and Cameron, 2002).

4.6 NMR Spectroscopy as Characterization Method for Hydrolysis of

AMPNa2

NMR Spectroscopy is widely used to monitor the hydrolysis of ATP,

diphosphate, and simple phosphate monoesters. However, in this investigation it

was not possible to carry out NMR Spectroscopy for the characterization of the

product of the hydrolysis of Adenosine Monophosphate disodium salt. The solid

powder that was produced in this hydrolysis was not readily soluble in solvents

such as acetonitrile, cyclohexane, ethanol, toluene and many more solvents. The

solid sample was only soluble in acetic acid. However, there were constraints in

obtaining deuterated acetic acid and other deuterated solvents. Therefore, NMR

spectroscopy of the solid product was not able to be carried out. However, LC-MS

spectrometry combined with FTIR Spectroscopy were sufficient to provide

108

adequate data on the mechanisms of the hydrolysis and products of hydrolysis of

AMPNa2. This combination of LC-MS spectrometry and FTIR Spectroscopy for

characterization of hydrolysis products were carried out in previous hydrolysis

studies of alkyl hydrogen methylphosphonates as well (Keay, 1965).

4.7 Effect of Ionic Strength on the Rate of Hydrolysis of AMPNa2 in Alkaline

Medium

In this investigation, the ionic strength was varied between 0.2 and 1.0 M

to study the effect of ionic strength on the rate of hydrolysis of AMPNa2. In a typical

reaction with 0.2 M [NaOH], the ionic strength was varied from 0.2 M and 1.0 M.

The rates of reaction were recorded in Table 4.1. For reaction sample of 0.2 M

[NaOH] with an ionic strength of 0.2 M, the rate of reaction obtained was (8.69 ±

0.41) × 10-7 s-1 while for reaction sample of 0.2 M [NaOH] with an ionic strength

of 1.0 M, the rate of reaction obtained was (10.2 ± 0.21) × 10-7 s-1. It was noticed

that as the ionic strength increases 5-fold from 0.2 M to 1.0 M, the rate of reaction

increases by only 17.38% indicating that the specific base catalysis was subjected

to mild positive salt effect. This positive-ion effect was also observed in the

hydrolysis of mono-2-methyl-5-nitroaniline phosphate where by the ionic strengths

were varied with NaCl. As the ionic strength increased, the rate of reactions

increased as well (Awadhiya and Bhoite, 2011). These rates have been related with

the ionic strength by the Debye-Hückel equation (Smith, and Collins, 2011).

109

4.8 Comparison with Enzymatic Cleavage Rate of AMP

4.8.1 Alkaline Hydrolysis of AMPNa2

The role of basic hydrolysis in this investigation mimics the function of

ecto-5’-AMPases such as CD73 and endo-5’-AMPases such as cytosolic 5’-

nucleotidase. These enzymes metabolize 5’-AMP to adenosine (Jackson, 2011).

While CD73 is only specific to nucleoside monophosphates (AMP adenosine),

there is another class of enzyme called alkaline phosphatases (AP) that metabolize

more substrates. These substrates include pyrophosphate, p-nitrophenylphosphate,

and 5’-nucleotides. APs metabolize ATP ADP AMP adenosine

(Bontemps et al., 1983; Picher et al., 2003). This investigation also gave insights

on how these enzymes could function in our body and the products could be formed.

The products of this investigation were similar to the products formed by enzymatic

cleavage of adenosine monophosphate (Jackson, 2011; Bontemps et al., 1983;

Picher et al., 2003).

The rate of base-catalysed hydrolysis is 4.32 × 10-6 at 60 °C. This rate was

compared with the rate of enzymatic cleavage of phosphate esters. Alkaline

Phosphatase can provide rate acceleration by 1017 and with nitrophenyl phosphate

as a substrate, Alkaline Phosphatase has an efficiency of 4.5 × 106 M-1 s-1 (Desbouis

et al., 2012; Petsko et al., 2004). It is obvious that the rate of phosphate ester

cleavage provided by enzymes is far faster than the rate of non-enzymatic cleavage

110

provided by base-catalysed cleavage in this investigation. This is due to the fact

that enzymes such as Alkaline Phosphatases are highly specific and have higher Km

values, and they have a more alkaline pH optimum (Millán, 2006). Its catalytic site

contains two Zn2+ and one Mg2+ ions. Alkaline phosphatases are highly catalytic

and have high affinity for their substrates (Desbouis et al., 2012).

The hydroxide ion which functions as the nucleophile in this investigation

is analogous to the serine alkoxide that is present in Alkaline Phosphatase active

site. In Alkaline Phosphatase, the alkoxide is activated by Zn2+ which facilitates the

formation of a reactive alkoxide. The Zn(II) is also responsible for stabilizing the

leaving group. Serine alkoxide acts as a nucleophile and attacks the phosphorus

centre causing the P-O bond cleavage in phosphate monoesters. Similar to the

mechanism of Alkaline Phosphatase, in the present investigation the hydroxide ion

acts as nucleophile and attacks the phosphorus centre causing the P-O bond

cleavage (O’Brien et al., 2002; Desbouis et al., 2012).

4.8.2 Acidic Hydrolysis of AMPNa2

The rate of acid-catalysed hydrolysis of AMPNa2 is 1.62 × 10-6 M-1 s -1 at 60

°C. It wouldn’t be right to directly compare this rate of acid-catalysed hydrolysis

of AMPNa2 to the rate adenosine monophosphate nucleosidase. This is because

adenosine monophosphate nucleosidase directly cleaves the N-glycosidic bond

111

cleavage and produces adenine and ribose 5-phosphate. In non-enzymatic acid-

catalysed hydrolysis of AMPNa2, phosphate ester bond was broken prior to N-

glycosidic bond cleavage. Therefore, the products formed were also slightly

different. The common product was adenine in both enzymatic and non-enzymatic

cleavage. It is best to conclude that the hydrolysis provided by this investigation is

similar to combined reaction of Alkaline Phosphatase (AMPNa2 adenosine)

and nucleoside N-ribohydrolase (adenosine adenine) (Bontemps et al.,

1983; Picher et al., 2003).

There has been no evidence about Alkaline Phosphatase having an acidic

residue that facilitates the cleavage reaction by protonating the leaving group in P-

O bond cleavage. However, this protonation is facilitated by the Zn+ as they

stabilize the leaving group. The role performed by hydronium ions, H+ in present

study, is analogous to the role performed by Zn+ in Alkaline Phosphatase (O’Brien

and Herschlag, 2002). In the hydrolysis of adenosine to adenine in present study,

H+ ion facilitates the hydrolysis by protonating the N7 of the adenine ring. This role

performed by H+ ion is analogous to the role performed by Histidine in nucleoside

N-ribohydrolase.

112

Table 4.10 shows rate of hydrolysis of acidic hydrolysis of nucleosides at

pH 1 and 37 °C.

Table 4.10: Rate of hydrolysis of acidic hydrolysis of nucleosides at pH 1 and

37 °C (Jobst et al., 2016)

Nucleoside

k/s-1

Depyrimidination

Depurination

2’-deoxyadenosine

4.30 × 10-4

2’-deoxyguanosine

8.30 × 10-4

2’-deoxycytidine

1.10 × 10-7

2’-deoxyuridine

< 1.00 × 10-7

2’-deoxythymidine

2.00 × 10-8

Adenosine

3.60 × 10-7

Guanosine

9.36 × 10-7

Cytidine

< 1.00 × 10-9

Uridine

< 1.00 × 10-9

As can be seen in the table above, the rate of depurination is faster than the

rate of depyrimidination due to the fact that purines have a tendency to become

deprotonated. This in turn destabilizes N-glycosidic bond. Besides that, purines

have dual rings whereby positive charges are delocalized more effectively (Jobst et

al., 2016).

113

This investigation also gave insights on how these enzymes could function

to catalyse N-glycosidic bond cleavage. This understanding is great in order to be

able to develop synthetic enzymes to mimic the function of adenosine 5’-

monophosphate nucleosidase and nucleoside N-ribohydrolases.

4.9 Further Studies

Different nucleotide analogue studies has gained attention mainly due to

their importance in clinical analysis and food analysis (Landers, 2007; Wiens et al.,

2013). The AMPNa2 used as subject in this research could be further studied for its

uses as cancer markers, diagnostic marker for human immunodeficiency virus

(HIV), and therapeutic agents due to the antiviral, antitumoral, and

antiimmunostimulatory properties of common diseases (Hunsucker et al., 2005;

Paleček et al., 2012). AMPNa2 could be used in interference studies whereby

analogues are incorporated at random positions trough in vitro transcription. This

can be done on RNA molecules at low frequency. By doing this, we will be able to

identify the specific positions which are crucial for folding and catalysis. (Jaikaran

et al., 2008).

In order to employ these analogues in clinical analysis, the kinetic and

mechanism of the hydrolysis of these analogues have to be extensively studied to

further understand how these analogues work in our body. This model should be

114

employed for future kinetics and mechanistic studies as a phosphate ester model

and this model should be employed in enzymatic and non-enzymatic studies. Effect

of different parameters on the rate of hydrolysis could be investigated such as ionic

strength and energy profile of the anomerization at physiological pH (Stockbridge

et al., 2010).

Besides that, synthetic enzymes to mimic the function of adenosine 5’-

monophosphate nucleosidase, ecto-5’-AMPases, endo-5’-AMPases, nucleoside N-

ribohydrolases, and Alkaline Phosphatases can be developed as we have already

understood the mechanism of these enzymes through this investigation. These

enzymes cleave the phosphate ester bond and N-glycosidic bond and they employ

the same general acid and base mechanism as studied in this investigation (Oivanen

et al., 1998).

115

CHAPTER 5

CONCLUSION

Phosphate esters are essential in many processes of the human body. For

example, phosphate esters are essential in the production of cellular energy,

essential part of nucleic acids, as an important component of cell membrane, and

most importantly storage of genetic information. Phosphate esters are extremely

stable and it is often very hard for chemists to study the mechanism of phosphate

esters as the cleavage rates are extremely slow in neutral conditions and also due to

its complicated mechanism. There has been extensive study on phosphate diesters

and triesters. However, there has been very little study on phosphate monoesters.

In this study, AMPNa2 was employed as a phosphate monoester substrate

model to further understand various bond cleavage of phosphate monoesters. This

hydrolysis was carried out in acidic and basic media. The phosphate bond in

AMPNa2 mimics the phosphate ester bond found in phosphate monoesters whereas

the acidic and basic media mimics the action of enzymes such as ecto-5’-AMPases

and endo-5’-AMPases, Alkaline Phosphatases, adenosine monophosphate

nucleosidase and nucleoside N-ribohydrolases.

116

In this study, hydrolysis of adenosine monophosphate disodium salt,

AMPNa2 was carried out in pH values covering pH 0.30-12.71 to study acid and

base hydrolysis of AMPNa2 at 60 °C. Specific base hydrolysis was carried out by

using sodium hydroxide with various concentrations to vary the pH values, while

specific acid hydrolysis was carried out by using hydrochloric acid with various

concentrations to vary pH values as well. For pH values closer to physiological pH,

buffers were used to vary the pH to create a general acid and general base hydrolysis

environment. Sample reactions covering the pH range from pH 0.30-12.71 and a

small amount of AMPNa2 was added. Spectral measurements using a UV-Vis

spectrophotometer were carried out until the reaction goes into completion of about

8 half-lives. Rate of reactions were calculated to find out the optimum pH for the

hydrolysis reaction. Characterization of products was also carried out by employing

FTIR spectroscopy and LC-MS spectrometry.

It was found that basic hydrolysis of AMPNa2 proceeds in a one-step

mechanism. It was also found that the rate of this phosphodiester cleavage increases

with the increase of pH conditions in alkaline region indicating that base hydrolysis

can accelerate AMPNa2 hydrolysis. This is due to the fact that at high pH values,

the concentration of OH- ions increased as well. The OH- ions here act as

nucleophiles and they attacked the phosphorus centre causing the cleavage. Pseudo-

first-order rate constants, kobs of each sample solution were calculated and plotted

against [NaOH] concentrations to clearly observe the effect of [NaOH]

concentrations on the rate of reaction. Theoretical rates of reaction, kcalc for each

117

[NaOH] were calculated and it was found that the theoretical rate of reactions, kcalc

did not deviate far from the observed rate of reactions, kobs. Rate constants of

hydrolysis of AMPNa2 in 60 °C were determined where the rate ranged from (1.20

± 0.10) 10-7 s-1 to (4.44 ± 0.05) × 10-6 s-1 at [NaOH] from 0.0008 M to 1.0000 M.

Pseudo-first-order rate constant against [NaOH] showed a linear relationship.

Second-order base-catalysed rate constant, kOH obtained was 4.32 × 10-6 M-1 s-1 and

uncatalysed rate constant, k0 obtained was 6.30 × 10-8 s-1 at 60 °C.

The function performed by OH- ions here is in coherence with the function

of enzymes in the phosphate ester cleavage in phosphate monoesters, such as

natural adenosine monophosphate. The rate achieved in this investigation was far

from the one that is associated with phosphatases due to many reasons. Alkaline

phosphatases are highly specific, they have a more alkaline pH optimum, are highly

catalytic and have high affinity for their substrates. Although the rate obtained in

this investigation does not match up to the rate provided by enzymes, this

investigation has definitely provided insights on how these enzymes could function

in our body and the products that were formed. The products that were expected

after the cleavage were adenosine and phosphate salt. LC-MS spectra and infrared

spectra indicated the presence of these products. The infrared band responsible for

the phosphate bond at around 978 cm-1 was present in the infrared spectrum of

AMPNa2 but absent in the infrared spectra of the products.

118

In acidic hydrolysis of AMPNa2, the hydrolysis involved a two-step

mechanism. In acidic hydrolysis, two types of cleavage were noticed, phosphate

ester hydrolysis and N-glycosidic bond cleavage occurred producing adenine. This

indicated that specific acid hydrolysis can accelerate the rate of phosphate ester

cleavage and glycosidic cleavage of AMPNa2. The result also indicated that in

acidic medium, there was a competition between phosphate ester bond cleavage

and N-glycosidic bond cleavage.

It was also found that the rate of this phosphodiester cleavage and acid-

catalysed depurination increases with the decrease of pH in the acidic region. This

was due to the fact that at low pH values, the concentration of H+ ions or hydronium

ions increased as well. In specific acid catalysis of AMPNa2, the H+ ion protonates

the adenine leaving group leading to cleavage. Pseudo-first-order rate constants,

kobs of each sample solution were calculated and plotted against [HCl]

concentrations to clearly observe the effect of [HCl] concentrations on the rate of

reaction. Theoretical rate of reaction, kcalc for each [HCl] were calculated and it was

found that the theoretical rate of reactions, kcalc did not deviate far from the observed

rate of reactions, kobs. Rate constants of hydrolysis of AMPNa2 in acidic medium at

60 °C were determined where the rate ranged from (1.32 ± 0.06) × 10-7 s-1 to (16.7

± 1.0) × 10-7 s-1 at [HCl] from 0.01 M to 1.00 M. Pseudo-first-order rate constant

against [HCl] showed a linear relationship. Second-order acid-catalysed rate

constant, kH obtained was 1.62 × 10-6 M-1 s-1 and uncatalysed rate constant, k0

obtained was 1.03 × 10-8 s-1 at 60 °C.

119

The function performed by H+ ions here are in tally with the functions of

enzymes. The rates obtained in this investigation were compared with the rate of

depurination that is carried out by enzymes. Rate of enzymatic hydrolysis of AMP

to adenosine and subsequently adenine are far from the rate obtained in this

investigation mainly due to the fact that these enzymes have activators and are

highly specific for their substrate. Although the rate obtained in this investigation

does not match up to the rate provided by enzymes, this investigation has definitely

also provided insights on how these enzymes could function in our body and the

products that were formed. Initially, the P-O bond in AMP was broken, producing

adenosine. Enzymatic depurination of adenosine results in adenine, therefore

adenine can be expected in the products. LC-MS spectra and infrared spectra

indicated the presence of adenine and disappearance of the phosphate ester bond

were noticed. The disappearance of N-glycosidic bond was noticed.

The insights on the mechanisms provided in this investigation justifies the

presence of acid and base hydrolysis in the phosphate ester cleavage of phosphate

monoesters by enzymes. This indicated that H+ and OH- ions cam act as catalysts

for the hydrolysis of phosphate esters. The role of H+ as proposed in this

investigation is to protonate the leaving group and this proposal is in coherence

with the postulation made by previous investigators. Similarly, OH- facilitates

catalysis by acting as a nucleophile and attacking the phosphorus centre and this

mechanism has been confirmed by previous research. In buffer hydrolysis, only

hydrolysis of AMPNa2 in glycine and TRIS buffer were successful. Hydrolysis of

120

AMPNa2 in glycine and TRIS showed linear dependency of pseudo-first-order rate

constants with different pH controlled by buffers. As the concentration of glycine

and TRIS buffer were increased respectively, the rate of hydrolysis of AMPNa2

increased as well.

As previously mentioned, besides phosphate ester cleavage, there are many

competing mechanisms in phosphate monoester hydrolysis such as C-O bond

cleavage, depurination or depyrimidination and ribose ring opening. In this

investigation, depurination or N-glycosidic bond cleavage was noticed in acidic

media. At pH 7, ribose ring opening was also observed whereby ribose ring of

adenosine could open and re-close to yield anomers. This phenomenon was not

noticed in acidic or basic hydrolysis.

Rate constants for the hydrolysis of AMPNa2 were determined at various

pH values ranging from pH 0.30-12.71 and log kobs against pH were plotted for all

the samples prepared in this investigation. A pH dependency of the rate of

hydrolysis of AMPNa2 was observed in highly alkaline and highly acidic regions.

This correlation is due to the increasing concentration of hydroxide and hydronium

ions at these pH values. Due to the fact that experimental determination of rate

constant at close to neutral pH was not able to be determined (due to ring opening)

and some points were pH independent, theoretical value for the rate constant for

close to neutral pH which is around pH 6 was determined by extrapolating the lines

121

from the acidic and the basic region. These rates were compared with the rates of

samples that were determined experimentally. There was a rate enhancement from

the theoretical rate constant and the experimentally determined rate constants by

104 fold. Background rate for the hydrolysis of AMPNa2 was also determined,

which had a value of 4.64 × 10-8 s-1. As we compare the background rate and the

rate of hydrolysis that was provided by buffers, hydrochloric acid and sodium

hydroxide, it can be noticed that there was a rate enhancement. This indicated that

acid and base can act as excellent catalysts in the hydrolysis of AMPNa2.

In pH values closer to neutral, the rate enhancement might be due to the

buffer catalysis provided by glycine and TRIS buffer. This rate enhancement was

due to the buffer catalysis that was provided by glycine and TRIS buffer. TRIS

buffer here acts as a nucleophile which attacks the phosphorus centre while glycine

acts as a protonating agent for the leaving group. These rate enhancements provided

by TRIS and glycine have been noticed in previous cleavage of P-O bond in

phosphate monoesters in the presence of Alkaline Phosphatase.

Ionic strength was varied to show the effect of ionic strength on the

hydrolysis of AMPNa2 in the same hydroxide concentrations by using sodium

chloride. It was noticed that as the ionic strength increases 5-fold from 0.2-1.0 M,

the rate of reaction increases by only 17.38% indicating that the specific base

catalysis was subjected to positive salt effect. This increase isn’t enough to justify

122

the effect of ionic strength on the rate of hydrolysis therefore more studies should

be carried out with ionic strength as a parameter.

The analogue used in this investigation should be used further in clinical

industry and subsequently lead to drug discovery. Due to its antiviral, antitumoral,

and antiimmunostimulatory properties, AMPNa2 is a great model to mimic the

phosphate ester bond. This model should be employed for future kinetics and

mechanistic studies as a phosphate monoester substrate model. Effect of different

parameters on the rate of hydrolysis could be investigated such as ionic strength

and temperature. AMPNa2 could be used in future studies on phosphate monoesters.

123

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132

Appendix A

Hydrochloric acid

pH

AMP

NaCl

HCl

H2O

Total

Volume

0.30

0.20 mL of 0.01

M

-

10 mL of

2.000 M

9.80 mL

20 mL

0.51 0.20 mL of 0.01

M

6.00 mL of

2.0 M

8 mL of

1.000 M

5.80 mL 20 mL

1.38 0.20 mL of 0.01

M

6.40 mL of

0.5 M 8 mL of

0.100 M

5.40 mL 20 mL

2.61 0.20 mL of 0.01

M

7.92 mL of

0.5 M 8 mL of

0.005 M

3.88 mL 20 mL

133

Appendix B

Sodium hydroxide

pH

AMP

NaCl

NaOH

H2O

Total

Volume

9.95

0.20 mL of

0.01 M

7.97 mL of

0.5 M

8 mL of

0.002 M

3.83

mL

20 mL

10.28 0.20 mL of

0.01 M

7.92 mL of

0.5 M

8 mL of

0.005 M

3.88

mL

20 mL

10.98 0.20 mL of

0.01 M

7.60 mL of

0.5 M

10 mL of

0.020 M

2.20

mL

20 mL

11.18 0.20 mL of

0.01 M

7.20 mL of

0.5 M

8 mL of

0.050 M

4.60m

L

20 mL

11.46 0.20 mL of

0.01 M

6.40 mL of

0.5 M

8 mL of

0.100 M

5.40

mL

20 mL

11.62 0.20 mL of

0.01 M

6.00 mL of

0.5 M

10 mL of

0.100 M

3.80

mL

20 mL

11.91 0.20 mL of

0.01 M

4.00 mL of

0.5 M

10 mL of

0.200 M

5.80

mL

20 mL

12.08 (ionic

strength 0.2 M)

0.20 mL of

0.01 M

- 8 mL of

0.500 M

11.80

mL

20 mL

12.21 (ionic

strength 1.0 M)

0.20 mL of

0.01 M

8.00 mL of

2.0 M

8 mL of

0.500 M

3.80

mL

20 mL

12.43 0.20 mL of

0.01 M

6.00 mL of

2.0 M

8 mL of

1.000 M

5.80

mL

20 mL

12.51 0.20 mL of

0.01 M

5.00 mL of

2.0 M

5 mL of

2.000 M

9.80

mL

20 mL

12.71 0.20 mL of

0.01 M

- 10 mL of

2.000 M

9.80

mL

20 mL

134

Appendix C

Glycine-HCl

pH

AMP

Glycine

HCl

NaCl

H2O

Total

Volume

1.80

0.20 mL

of 0.01 M

1 mL of 1

M

1.6 mL of

0.5 M

3.2 mL of

1 M

14.0 mL

20 mL

2.00 0.20 mL

of 0.01 M

1 mL of 1

M 1.2 mL of

0.5 M 3.4 mL of

1 M 14.2 mL 20 mL

2.70 0.20 mL

of 0.01 M

1 mL of 1

M 1.0 mL of

0.2 M 3.8 mL of

1 M 14.0 mL 20 mL

135

Appendix D

TRIS-HCl

pH

AMP

TRIS

HCl

NaCl

H2O

Total

Volume

8.03

0.20 mL of

0.01 M

1 mL of 1

M

1 mL of

0.20 M

3.8 mL of

1 M

14.0 mL

20 mL

8.42 0.20 mL of

0.01 M

1 mL of 1

M 10 mL of

0.01 M

3.9 mL of

1 M 4.9 mL 20 mL

136

Appendix E

Citrate buffer

pH

AMP

Sodium

citrate

Citric

acid

NaCl

H2O

Total

Volume

3.91

0.20 mL of

0.01 M

6 mL of 0.1

M

1 mL of

1 M

3.4 mL of

1 M

9.4 mL

20 mL

4.21 0.20 mL of

0.01 M

8 mL of 0.1

M 1 mL of

1 M

3.2 mL of

1 M 7.6 mL 20 mL

137

Appendix F

MES buffer

pH

AMP

MES

NaOH

NaCl

H2O

Total

Volume

5.76

0.20 mL

of 0.01 M

4 mL of 0.1

M

1 mL of 1

M

3.6 mL

of 1 M

11.2 mL

20 mL

6.15 0.20 mL

of 0.01 M

6 mL of 0.1

M 1 mL of 1

M

3.4 mL

of 1 M 9.4 mL 20 mL

138

Appendix G

HEPES buffer

pH

AMP

HEPES

NaOH

NaCl

H2O

Total

Volume

6.04

0.20 mL

of 0.01 M

1 mL of 1 M

2 mL of

0.05 M

3.9 mL

of 1 M

12.9 mL

20 mL

6.63

0.20 mL

of 0.01 M

1 mL of 1 M 3 mL of

0.05 M 3.7 mL

of 1 M 12.1 mL 20 mL

7.06

0.20 mL

of 0.01 M

1 mL of 1 M 5 mL of

0.10 M 3.5 mL

of 1 M 10.3 mL 20 mL

7.21

0.20 mL

of 0.01 M

1 mL of 1 M 6 mL of

0.10 M 3.4 mL

of 1 M 9.4 mL 20 mL

7.65

0.20 mL

of 0.01 M

1 mL of 1 M 8 mL of

0.10 M 3.2 mL

of 1 M 7.6 mL 20 mL

8.13 0.20 mL

of 0.01 M

1 mL of 1 M 9 mL of

0.10 M 3.1 mL

of 1 M 6.7 mL 20 mL

139

APPENDIX H

FTIR Spectrum of AMPNa2

140

APPENDIX I

FTIR Spectrum of Filtrate of Alkaline Hydrolysis of AMPNa2

141

APPENDIX J

FTIR Spectrum of Residue of Alkaline Hydrolysis of AMPNa2

142

APPENDIX K

FTIR Spectrum of Acidic product of hydrolysis of AMPNa2