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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
REFERENCES
Agarwal, S., Jangir, D.K., Mehrotra, R., Lohani, N. and Rajeswari, M.R., 2014. A
Structural Insight into Major Groove Directed Binding of Nitrosourea Derivative
Nimustine with DNA: A Spectroscopic Study. PLoS ONE, 9(8): e104115. doi:
10.1371/journal.pone.0104115
Allen, D.W. and Walker, B.J., 1994. Organophosphorus Chemistry, Volume
25.Cambridge: The Royal Society of Chemistry, pp. 93.
An, R., Jia, Y., Zhang, Y., Dong, P., Li, J. and Liang, X., 2014. Non-enzymatic
depurination of nucleic acids: Factors and mechanisms. PLoS ONE, 9(12):
e115950. doi: 10.1371/journal.pone.0115950
Awadhiya, P. and Bhoite, S.A., 2011. Kinetic study of hydrolysis of mono-2-
methyl-5- nitroaniline phosphate. International Journal of ChemTech Research,
3(4), pp. 1751-1757.
Banaszczyk, M.G., 1989. Artificial Hydrolytic Enzymes: Part 1 Hydrolysis of
phosphate esters catalysed by cobalt(III) complexes. PhD thesis, McGill
University, Canada.
Bevilacqua, P.C and Yajima, R., 2006. Nucleobase catalysis in ribozyme
mechanism. Current Opinion in Chemical Biology, 10, pp. 455-464.
Blackburn, G.M., Gait, M.J., Loakes, D., Williams, D.M., (eds.), 2006. Nucleic
acids in chemistry and biology, 3rd ed. Cambridge: Royal Chemistry Society, pp.
14-15.
Bontemps, F., Van Den Berghe, G. and Hers, H., 1983. Evidence for a substrate
cycle between AMP and adenosine in isolated hepatocytes. Proceedings of the
National Academy of Sciences of the United States of America, 80, pp. 2829-2833.
Borowiec, A., Lechward, K., Tkacz-Stachowska, K. and Skladanowski, C., 2006.
Adenosine as a metabolic regulator of tissue function: production of adenosine by
cytoplasmic 5’-nucleotidases. Acta Biochimica Polonica. 53(2), pp. 269-278
Brandão, T.A.S, Orth, E.S., Rocha, W.R., Bortoluzzi, A.J., Bunton, C.A. and
Nome, F. 2007. Intramolecular General Acid Catalysis of the Hydrolysis of 2-(2’-
Imidazolium)phenyl Phosphate, and Bond Length−Reactivity Correlations for
Reactions of Phosphate Monoester Monoanions. Journal of the American Chemical
Society, 72(10), pp. 3800-3807
124
Buckoreelall, K., Wilson, L. and Parker, W.B. 2011. Identification and
characterization of two adenosine phosphorylase activities in mycobacterium
smegmatis. Journal of Bacteriology. 193(20), pp. 5668-5674.
Chernobryva. M., 2012. Development of Metal-based Catalysts for Phosphate Ester
Hydrolysis. PhD thesis, University of London, England.
Chin, J. and Banaszxzyk, M., 1989. Highly efficient hydrolytic cleavage of
adenosine monophosphate resulting in a binuclear Co(III) complex with a novel
doubly bidendate µ4-phosphato bridge. Journal of the American Chemical Society,
111(11), pp. 4103-4105.
Clark, M.A., Finkel, R., Rey, J.A. and Whalen, K., 2012. Lippincott’s Illustrated
Reviews. Pharmacology. 4th ed. Baltimore: Wolters Kluwer Health/Lippincott
Williams & Wilkins
Das, S.R., Fong, R. and Piccirilli, J.A., 2005. Nucleotide analogues to investigate
RNA structure and function. Current Opinion in Chemical Biology, 9(6), pp. 585-
593.
Desbouis, D., Troitsky, I.P., Belousoff, M.J., Spiccia, L. and Graham, B., 2012.
Copper(II), zinc(II) and nickel(II) complexes as nuclease mimetics. Coordination
Chemistry Reviews, 256, pp. 897-937.
Duarte, F., Amrein, B.A. Amrein, Kamerlin, S.C.L., 2013. Modeling catalytic
promiscuity in the alkaline phosphatase superfamily. Physical Chemistry Chemical
Physics, 15, pp. 11160-11177.
Duarte, F., Åqvist, J., Williams, and Kamerlin, S.C.L., 2015. Resolving Apparent
Conflicts between Theoretical and Experimental Models of Phosphate Monoester
Hydrolysis. Journal of American Chemical Society, 137, pp. 1801-1093
Dwivedi, P., Schultz, A. J., and Hill, Jr., H.H., 2010. Metabolic Profiling of Human
Blood by High Resolution Ion Mobility Mass Spectrometry (IM-MS). International
Journal of Mass Spectrometry, 298(1-3), 78-90.
Edwards, L.J., 1950. The hydrolysis of aspirin: A determination of the
thermodynamic dissociation constant and a study of the reaction kinetics by ultra-
violet spectrophotometry. Transactions of the Faraday Society, 46, pp. 723-735.
Eguzozie, K.U., 2008. Metal ion mediated hydrolysis of 4-nitrophenylphosphate in
microemulsion media: Catalytic versus stoichiometric effects. MSc thesis,
University of South Africa, South Africa.
Fish, C., Green, M., Kilby, R.J., Lynam, J.M., McGrady, J.E., Pantazis, D.A.,
Russell, C.A., Whitwood, A.C. and Willans, C.E., 2006. A New Reaction Pathway
125
in Organophosphorus Chemistry: Competing SN2 and AE’ Pathways for
Nucleophilic Attack at a Phosphorus–Carbon Cage Compound. Angewandte
Chemie International Edition, 45, pp. 3628-3631.
Florián, J. and Warshel, A., 1997. A fundamental assumption about OH- attack in
phosphate ester hydrolysis is not fully justified. Journal of American Chemical
Society, 119(23), pp. 5473-5474.
Florián, J. and Warshel, A., 1998. Phosphate ester hydrolysis in aqueous solution:
Associative versus dissociative mechanisms. Journal of Physical Chemistry B,
102(4), pp. 719-734.
Forconi, M. and Herschlag, D., 2009. Metal ion-based RNA cleavage as a
structural probe. Methods in Enzymology, 468, pp. 91-106.
Hethey, J., Lai, J., Loutet, S., Martin, M. and Tang, V., 2002. Effects of Tricine,
Glycine and TRIS buffers on Alkaline Phosphatase activity. Journal of
Experimental Microbiology and Immunology, 2, pp. 33-38.
Hilal, S. H. 2006. Estimation of hydrolysis rate constants of carboxylic acid ester
and phosphate ester compounds in aqueous systems from molecular structure by
SPARC. Ecosystems Research Division National Exposure Research Laboratory,
United States Environmental Protection Agency, Athens, Georgia.
Holčapek, M., Jirásko, R. and Lísa, M., 2010. Basic rules for the interpretation of
atmospheric pressure ionization mass spectra of small molecules. Journal of
Chromatography A, 1217(25), pp. 3908-3921.
Hon, D.N.S. (eds.), 1996. Chemical Modification of Lignocellulosic Materials.
New York: Marcel Dekker, pp. 142
Huang, X. and Zhang, J., 2011. Hydrolysis of glucose-6-phosphate in aged, acid-
forced hydrolysed nanomolar inorganic iron solutions—an inorganic biocatalyst?
RSC Advances. 2, pp. 199-208
Hunsucker, S.A., Mitchell, B.S. and Spychala, J., 2005. The 5’-nucleotidases as
regulators of nucleotide and drug metabolism. Pharmacology & Therapeutics,
107, pp. 1-30.
Jaikaran, D., Smith, M.D., Mehdizadeh, R., Olive, J. and Collins, R.A., 2008. An
important role of G638 in the cis-cleavage reaction of the Neurospora VS
ribozyme revealed by a novel nucleotide analog incorporation method. RNA, 14,
pp. 938-949.
Jackson, E.K., 2011. The 2’,3’-cAMP-adenosine pathway. American Journal of
Physiology-Renal Physiology, 301, pp. 1160-1167
126
Jenkins, L.A., Bashkin, J.K., Pennock, J.D., Florián, J. and Warshel, A., 1999.
Catalytic hydrolysis of adenosine 2’,3’-cyclic monophosphate by Cu-II terpyridine.
Inorganic Chemistry, 38(13), pp. 3215-3222.
Jobst, K.A., Klenov, A., Neller, K.C.M. and Hudak, K.A., 2016. Effect of
depurination on cellular and viral RNA. In: Jurga, S., Erdmann, V.A. and
Barciszewski, J. (eds.). Modified Nucleic Acids in Biology and Medicine,
Switzerland: Springer International Publishing. pp. 273-292.
Jubian, V., 1991. Relationship between ligand structure and reactivity for copper
(II) complex mediated hydrolysis of phosphate diesters, carboxylic esters and
amides. PhD thesis, McGill University, Canada.
Keay, L. The preparation of and hydrolysis of alkyl hydrogen methylphosphonates.
Canadian Journal of Chemistry. 43. pp. 2367-2639
Kennedy, C.D., 1990. Ionic strength and the dissociation of acids. Biochemical
Education. 18(1), pp. 35-40
Kim, S. et al., 2016. PubChem Substance and Compound databases. Nucleic Acids
Resolution. 44(D1), pp. D1202-D1213.
Kirby, A.J., Dutta-Roy, N., da Silva, D., Goodman, J.M., Lima, M.F., Roussev, C.D.
and Faruk., N., 2005.
Intramolecular General Acid Catalysis of Phosphate Transfer. Nucleophilic Attack
by Oxyanions on the PO32- Group. Journal of the American Chemical Society,
127(19), pp. 7033-7040.
Kirby, A.J., Lima, M.F., Silva, D. and Nome, F., 2004. Nucleophilic attack by
oxyanions on a phosphate monoester dianion: The positive effect of a cationic
general acid. Journal of the American Chemical Society, 126(5), pp. 1350-1351.
Kirby, A. J. and Varvoglis, A.G., 1996. The reactivity of phosphate esters.
Monoester hydrolysis. Journal of the American Chemical Society, 89(2), pp. 415-
423.
Klein, B.G., 2013. Homeostatis. Cunningham's Textbook of Veterinary Physiology.
5th ed. St. Louis: Elsevier. pp. 550.
Kluger, R. and Cameron, L.L, 2002. Activation of acyl phosphate monoesters by
lanthanide ions: enhanced reactivity of benzoyl methyl phosphate. Journal of the
American Chemical Society. 124 (13), pp. 3303-3308
Komiyama, M., Takeda, N. and Shigekawa, H., 1999. Hydrolysis of DNA and RNA
by lanthanide ions: mechanistic studies leading to new applications. Chemical
Communications, 16, pp. 1443-1451
127
Korhonen, H., 2011. The significance of hydrogen bonding interactions in the
cleavage of RNA. PhD thesis, University of Turku, Finland.
Kwan, E.E., 2005. Factors affecting the relative efficiency of general acid catalysis.
Journal of Chemical Education, 82(7), pp. 1026-1030.
Kuchta, R.D., 2011. Nucleotide analogues as probes for DNA and RNA
polymerases. Current Protocols in Chemical Biology, 2(2), pp. 111-124
Landers, J.P. (ed.), 2007. Handbook of capillary and microchip electrophoresis and
associated microtechniques, 3rd ed. Boca Rotan: CRC Press.
Larson, R.A. and Weber, E.J., 1994. Reaction mechanism in environmental organic
chemistry. Boca Raton: Lewis Publishers, CRC Press. pp. 103-147
Liu, H., Zhao, C., Lu, J., Liu, M., Zhang, S., Jiang, Y., and Zhao, Y., 2006.
Simultaneous measurement of trace monoadenosine and diadenosine
monophosphate in biomimicking prebiotic synthesis using high-performance liquid
chromatography with ultraviolet detection and electrospray ionization mass
spectrometry characterization. Analytic Chimica Acta. 566(1), pp. 99-108
Lorenzetti, R., Lilla, S., Donato, J.L. and de Nucci, G., 2007. Simultaneous
quantification of GMP, AMP, cyclic GMP and cyclic AMP by liquid
chromatography coupled to tandem mass spectrometry. Journal of
Chromatography B., 859, pp. 37-41.
Marcum, J.C., Kaufman, S.H., and Weber, J.M., 2011. UV-photodissociation of
non-cyclic and cyclic mononucleotides. International Journal of Mass
Spectrometry, 303, 129-136.
Mehta, B. and Mehta, M., 2015. Mini Essay II. Organic Chemistry. 2nd ed. Prentice-
Hall of India Pvt. Ltd.
Mello, M.L.S. and Vidal, B.C., 2012. Changes in the infrared microspectroscopic
characteristics of DNA caused by cationic elements, different base richness and
single-stranded form. PLoS ONE, 7(8): e43169. doi:10.1371/journal.pone.0043169
Millán, J.L., 2006. Alkaline Phosphatases: Structure, substrate specificity and
functional relatedness to other members of a large superfamily of enzymes.
Purinergic Signalling, 2(2), pp. 335-341
Minaev, B.F., Shafranyosh, M.I., Svida, Y.Y., Sukhoviya, M.I., Shafranyosh, I.I.,
Baryshnikov, G.V., and Minaeva, V.A., 2014. Fragmentation of the adenine and
guanine molecules induced by electron collisions. The journal of Chemical Physics,
140, pp.175101-175115.
128
Naito, Y. and Lowenstein, J.M., 1985. 5’-Nucleotidase from rat heart membranes.
Biochemical Journal, 226(3), pp. 645-651.
Nelson, D.L. and Cox, M.M., 2013. Nucleotides and Nucleic Acids. In: Lehninger
principles of biochemistry, 6th ed. New York: Worth Publishers.
Nibbering, N.M.M., 1985. Fourier-transform ion cyclotron resonance. In: Rose,
M.E. (ed.). Specialist Periodical Reports: Mass Spectrometry. 8, London: Royal
Society of Chemistry.
O’Brien, P.J. and Herschlag, D., 2002. Alkaline Phosphatase revisited: Hydrolysis
of alkyl phosphates. Biochemistry, 41(9), pp. 3207-3225.
Oivanen, M., Kuusela, S. and Lönnberg, H., 1998. Kinetics and mechanisms for the
cleavage and isomerization of the phosphodiester bonds of RNA by Brønsted acids
and bases. Chemical Reviews, 98(3), pp. 961-990.
Olkowski, E.D. 2012. Postmodern Philosophy and the Scientific Turn. Indiana
University Press. pp. 176
Olmsted, J. and Williams, G.M., 1997. Chemistry: The Molecular Science. 2nd ed.
Iowa: Wm. C. Brown Publishers
Oswald, N., 2016. How SDS-PAGE works. [Online]. Available at:
http://bitesizebio.com/580/how-sds-page-works/ [Accessed: 30 November 2016]
Paleček, E., & Bartošik, M., 2012. Electrochemistry of nucleic acids, Chemical
Reviews, 112, pp. 3427-3481.
Pavia, D.L., Lampman, G.M., Kriz, G.M. and Engel, R.G., 2005. Introduction to
organic laboratory techniques: A small scale approach, 2nd ed. California:
Thomson Learning, pp. 718.
Picher, M., Burch, L.H., Hirsh, A.J., Spychala, J. and Boucher, R.C., 2003. Ecto 5_-
nucleotidase and nonspecific alkaline phosphatase: Two AMP-hydrolyzing
ectoenzymes with distinct role in human airways. The Journal of Biological
Chemistry, 278(15), pp. 13468-13479
Petsko, G.A. and Ringe, D., 2004. From Structure to function. In: Protein Structure
and Function. London: New Science Press Ltd. pp. 63
Perkampus, H.H., 1992. UV-Vis Spectroscopy and its applications. 1st ed. Springer-
Verlag Berlin Heidelberg. pp.179
129
National Centre for Biotechnology Information. PubChem Compound Database,
n.d., Adenine. [Online]. Available at:
https://pubchem.ncbi.nlm.nih.gov/compound/adenine#section=2D-Structure
[Accessed: 9th December 2017].
National Centre for Biotechnology Information. PubChem Compound Database,
n.d., Adenosine. [Online]. Available at:
https://pubchem.ncbi.nlm.nih.gov/compound/adenosine#section=2D-Structure
[Accessed: 9th December 2017].
National Centre for Biotechnology Information. PubChem Compound Database,
n.d., Adenosine 5’-monophosphate. [Online]. Available at:
https://pubchem.ncbi.nlm.nih.gov/compound/5_-adenylic_acid#section=2D-
Structure [Accessed: 9th December 2017].
Qian, T., Cai, Z. and Yang, M.S., 2004. Determination of adenosine nucleotides in
cultured cells by ion-pairing liquid chromatography-electrospray ionization mass
spectrometry. Analytical Biochemistry, 325(1), pp.77-84
Ribeiro, A.J.M., Ramos, M.J. and Fernandes, P.A., 2010. Benchmarking of DFT
functionals for the hydrolysis of phosphodiester bonds. Journal of Chemical theory
and Computation, 6(8), pp. 2281-2292.
Sala-Newby, G.B., Skladanowski, A.C. and Newby., A.C., 1999. The mechanism
of adenosine formation in cells. The Journal of Biological Chemistry, 274(25), pp.
17789-17793.
Schramm, V.L., 1974. Kinetic properties of allosteric adenosine monophosphate
nucleosidase from Azotobacter vinelandii. The Journal of Biological Chemistry,
249(6), pp. 1729-1736.
Schroeder, G.K., Lad, C., Wyman, P., Williams, N.H. and Wolfenden, R., 2006. The
time required for water attack at the phosphorus atom of simple phosphodiesters
and of DNA, Proceedings of the National Academy of Sciences of the United States
of America.,103(11), pp. 4052-4055.
Shabarova, Z.A. and Bogdanov, A.A, 1994. Properties of Nucleosides. In:
Advanced Organic Chemistry of Nucleic Acids. Weinheim: VCH
Verlagsgesellschaft mbH
Singh, M. S., 2004. Advanced Organic Chemistry: Reactions and Mechanisms.
India: Dorling Kindersley, pp. 106.
130
Skoog, M.T., 1986. Mechanism and activation for allosteric adenosine 5’-
monophosphate nucleotidase. The Journal of Biological Chemistry, 261(10), pp.
4451-4459
Spectral Database for Compounds SDBS, n.d. Infrared spectra of adenosine
[Online].Available at:
http://sdbs.db.aist.go.jp/sdbs/cgibin/direct_frame_disp.cgi?sdbsno=828
[Accessed: 28 November 2016].
Sloop, J.C., 2010. Succeeding in organic chemistry: Systematic problem-solving
approach to mastering structure, function and mechanism. Indiana: Authorhouse.
Smith, M.D. and Collins, R.A., 2011. Use of ribozyme cleavage kinetics to measure
salt-induced changes in solution pH. Analytical Biochemistry, 415(1), pp. 12-20.
Spillane, W.J. 2004. Reactions of carboxylic, phosphoric and sulfonic acids and
their derivatives. In: Knipe, A.C, (eds.). Organic Reaction Mechanisms 2000: An
annual survey covering the literature dated December 1999 to December 2000.
John Wiley & Sons.
Strzelecka, D. Chmielinski, S. Bednarek, S., Jemielity, J. and Kowalska. 2017.
Analysis of mononucleotides by tandem mass spectrometry: investigation of
fragmentation pathways for phosphate- and ribose-modified nucleotide analogues.
Scientific Reports. 7. doi: 10.1038/s41598-017-09416-6
Stuart, B. H., 2004. Infrared spectroscopy: Fundamental and applications. Sydney:
John Wiley and Sons ltd, pp. 72-152.
Stockbridge, R.B., Schroeder, G. and Wolfenden, R., 2010. The rate of spontaneous
cleavage of the glycosidic bond of adenosine. Bioorganic Chemistry, 38(5), pp.
224-228.
Sun, R. and Wang, L., 2013. Inhibition of Mycoplasma pneumonia growth by FDA-
approved anticancer and antiviral nucleoside and nucleobase analogs. BMC
Microbiology, 13(184), pp. 1-12
Theophanides, T. and Sandorfy, C. (eds.), 2012. Spectroscopy of biological
molecules: Theory and applications-Chemistry, Physics, Biology and Medicine.
Quebec: D. Reidel Publishing Company. pp. 147
Tiwari, B.K., Kadam, R., Dixit, V.K., Agarwal, A. and Parihar, P.S., 2005.
Kinetics and mechanism of the hydrolysis of 4-bromo-2,6-dimethylphenyl
phosphate monoester. Asian Journal of Chemistry, 17(1), pp. 109-116.
Tuan, V. (ed.), 2014. Biomedical photonics handbook: Fundamentals, devices and
techniques, 2nd ed. Tennessee: CRC Press
131
Van Dycke, A. et al., 2010. Quantitative analysis of adenosine using Liquid
Chromatography/Atmospheric Pressure Chemical Ionization-tandem Mass
Spectrometry (LC/APCI-MS/MS). Journal of Chromatography B: Analytical
Technologies in the Biomedical and Life Sciences, 878(19), pp. 1493-1498.
Varila, J., Hankamäki, T., Oivanen, M., Koole, L.H., and Lönnberg, H. 1997.
Hydrolysis of the cis-phenyl ester of thymidine 3’,5’-Cyclic Monophosphate: pH-
dependent competition between depyrimidination and phosphotriester hydrolysis
via CO and PO bond ruptures. Journal of Organic Chemistry, 62(4), pp. 893-898.
Versées, W., Decanniere, K., Holsbeke, E.V., Devroede, N. and Steyaert, J., 2002.
Enzyme-substrate interactions in the purine specific nucleoside hydrolase from
Trypanosoma vivax, Journal of Biological Chemistry, 277(18), pp. 15938-15946
Wiens, A. et al., 2013. Comparative efficacy of oral nucleoside or nucleotide
analog monotherapy used in chronic hepatitis B: a mixed-treatment comparison
meta-analysis, Pharmacotherapy, 33(2), pp. 144-151.
Widlanski, T.S. and Taylor, W., 1999. Chemistry and Enzymology of Phosphatases
In: Barton, D. and Meth-Cohn, O. (eds.). Comprehensive Natural Products
Chemistry. Oxford: Pergamon, pp. 139-162
Wójtowicz-Rajchel, H., 2012. Synthesis and applications of fluorinated nucleoside
analogues. Journal of Fluorine Chemistry, 143, pp. 11-48
Zagórowska, I., Kuusela, S. and Lönnberg, H., 1998. Metal ion-dependent
hydrolysis of RNA phosphodiester bonds within hairpin loops. A comparative
kinetic study on chimeric ribo/2’-O-methylribo oligonucleotides. Nucleic Acids
Research, 26(14), pp. 3392-3396.
Zhao, H., Wang, X., Li, H., Yang, B., Yang, H. and Huang, L., 2013.
Characterization of Nucleosides and Nucleobases in Natural Cordyceps by HILIC–
ESI/TOF/MS and HILIC–ESI/MS. Molecules. 18. pp. 9755-9769
Zhong, H., Zhang, J., Tang, X., Zhang, W., Jiang, R., Chen, D., Wang, P. and Yuan,
Z. Mass spectrometric monitoring of interfacial photoelectron transfer and imaging
of active crystalline facets of semiconductors. Nature Communications, 8
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
Recommended