02 Whole

An investigation of transition metal complex chemistry:
enzyme mimicry and Zn(II) detection
Hilary Ceywood Coleman
A thesis submitted for the degree of

Doctor of Philosophy
in
The University of Adelaide
Department of Chemistry
June 2015
CONTENTS
ABSTRACT ……..v
DECLARATION vii
ACKNOWLEDGEMENTS viii
ABBREVIATIONS x
CHAPTER 1 1
1.1 Metalloenzymes 2
1.1.1 Introduction 2
1.1.2 Features of metalloenzymes – Carbonic Anhydrase 2
1.1.3 Ester hydrolysis – the Hydrolases 6
1.2 Hydrolase Mimics 9
1.2.2 Previous metallo-amine and -cyclodextrin hydrolase mimic systems 11
1.3 The use of cyclodextrins in ligand design 15
1.3.1 Cyclodextrin structure 15
1.3.2 Cyclodextrin host-guest complexes 16
1.4 Possible mechanisms of enzyme mimic activity 17
1.5 Enzyme kinetics investigation 19
1.6 Aims of this research 22
1.6.1 Ligand suite 23
1.6.2 Potentiometric titrimetry 26
1.6.3 UV-Vis absorbance spectrophotometry 28
1.7 References 30
CHAPTER 2 38
2.1 Introduction 39
2.1.1 The properties of Tren and Tacn 39
2.1.2 Previous studies 41
2.2 The pKas of protonated tren, Me6tren, tacn and Me3tacn, and stability constants
of their metal complexes 42
i
2.2.1 Determination of protonated tren, Me6tren, tacn and Me3tacn pKas 42
2.2.2 Tren and Me6tren metal complex characterisation 50

2.2.3 Tacn and Me3tacn metal complex characterisation 62
2.3 Conclusions 70
2.4 References 72
CHAPTER 3 77
3.1 Introduction 78
3.1.1 Metallo-cyclodextrins 78
3.1.2 New ligands βCDMe5tren and βCDMe2tacn 80
3.2 The pKas of the protonated βCDtren, βCDMe5tren, βCDtacn and
βCDMe2tacn and stability constants of their metal complexes 80
3.2.1 Determination of protonated βCDtren and βCDMe5tren pKas 80

3.2.2 Determination of protonated βCDtacn and βCDMe2tacn pKas 84
3.2.3 βCDtren and βCDMe5tren metal complex characterisation 88
3.2.4 βCDtacn and βCDMe2tacn metal complex characterisation 98
3.3 Conclusions 106
3.4 References 109
CHAPTER 4 112
4.1 Introduction 113
4.1.1 Functionalised β-cyclodextrins in enzyme mimicry 113
4.1.2 4-Nitrophenyl acetate hydrolysis as a model reaction 114
4.1.3 The Michaelis-Menten enzyme kinetics model 117
4.1.4 Adaptation of the Michaelis-Menten model to catalysis by the enzyme
mimics [M(βCDtren)(OH)(H2O)]+ 122
4.1.5 Aims and methods of this study 127

4.2 Kinetics analysis of the βCDtren/Zn2+ and βCDtren/Cu2+ systems 136
4.2.1 Experimental preparations 136
4.2.2 The Zn2+/βCDtren system 138
4.2.3 The Cu2+/βCDtren system 151
4.2.4 Discussion of the Zn2+/βCDtren and Cu2+/βCDtren systems – are they
successful enzyme mimics? 159
ii
4.3 Analysis of other possible catalytic species – free metal, free ligand, free βCD 162
4.4 Mechanistic information gained from qualitative UV-Vis absorbance experiments 172
4.5 Summary of findings 188
4.6 References 190
CHAPTER 5 196
5.1 Introduction 197
5.2 Chapter 2 – Potentiometric titration characterisation of the Foundation Polyamines 197
5.3 Chapter 3 – Potentiometric titration characterisation of the β-cyclodextrin-
Functionalised Polyamines 199
5.4 Chapter 4 – Kinetic characterisation of the enzyme mimic systems 201

5.4.1 Summary of findings 201
5.4.2 Short term Future Work 202
5.4.3 Expansion of the original ligand suite 203
5.5 References 205
CHAPTER 6 206
6.1 Physiological Zn(II) 207
6.1.2 Developments in detection methods 209
6.1.3 The mechanism of the quinoline fluorophores: Chelation Enhanced
Fluorescence 210
6.1.4 The chemistry of Zinquin 211

6.2 Aims of this research 214
6.2.1 Potentiometric titrations 215
6.2.2 Electronic spectroscopy 216
6.3 References 219
CHAPTER 7 224
7.1 Potentiometric titration of 2-((E)-2-Phenyl)ethenyl-8-(N-4-methylbenzenesulfonyl)
aminoquinol-6-yloxyacetic Acid (stZQA) for ligand pKa determination 225
7.2 UV-Vis characterisation of 2-((E)-2-Phenyl)ethenyl-8-(N-4-

methylbenzenesulfonyl)aminoquinol-6-yloxyacetic Acid 229
iii
7.2.1 UV-Vis titration: determination of stability constants for Zn(II) complexes
of stZQA 229
7.2.2 Photoisomerism of the stZQA ligand 234

7.2.3 Binding of metals other than Zn(II) 236
7.3 Fluorescence characterisation of 2-((E)-2-Phenyl)ethenyl-8-(N-4
methylbenzenesulfonyl)aminoquinol-6-yloxyacetic Acid (stZQA) 239
7.3.1 Determination of Adventitious Zn(II) 239

7.3.2 Fluorescence signal with other metals: stZQA, a selective probe? 241
7.3.3 Fluorescence titration: determination of stability constants for Zn(II)
complexes of stZQA 243
7.4 Conclusions and Future Work 249

7.5 References 252
CHAPTER 8 254
8.1 General 255
8.1.1 Measurements 255
8.1.2 Materials 256
8.1.3 Data analysis tools 257
8.2 Experimental for Part I, Chapters 2 and 3 260
8.2.1 Preparation of compounds 260
8.2.2 Procedures for potentiometric titrations (Part I) 262
8.3 Experimental procedures for Part I, Chapter 4 266
8.3.1 Preparation for UV-Vis kinetics studies (Part I) 266
8.4 Experimental for Par II, Chapter 7 – stZQA 270
8.4.1 Procedures for potentiometric titrations (Part II) 270
8.4.2 UV-Visible studies (Part II) 271
8.4.3 Fluorescence studies (Part II) 272
8.5 References 274
APPENDIX A 276
APPENDIX B 288
APPENDIX C 309
iv
ABSTRACT
This thesis presents research in the field of transition metal complex chemistry.
The major project, reported in Part I, is focussed on the mimicking of enzymes in order to
produce simple catalyst molecules that enhance the rate of ester hydrolysis. This work involved a
systematic characterisation of a suite of potential enzyme mimics which were based on the simple
template molecules tris(2-aminoethyl)amine (tren) and 1,4,7-triazacyclononane (tacn). The
effects of substituting tren and tacn with methyl groups and β-cyclodextrin and then complexing
these with Zn2+, Cu2+, Cd2+ and Ni2+ were examined. The aim was to produce stable complexes
with lowered pKas for their associated aqua ligands (‘pKaH2O’), such that the catalytically active
hydroxo ligands (responsible for the ester hydrolysis) were readily available over a broad pH
range.
The thirty two resulting transition metal complex systems were characterised by
potentiometric titration under identical experimental conditions (aqueous NaClO4 solution, I =
0.10 mol dm-3, 298.2 K). For selected systems which were characterised by large stability
constants and relatively low aqua ligand pKaH2Os, their ability to enhance the rate of hydrolysis of
4-nitrophenyl acetate was measured using UV-Visible spectroscopy. A modified Michaelis-
Menten enzyme kinetics analysis was developed specifically for this study. Information about the
possible mechanism of catalysis was also obtained by a qualitative investigation of the UV-Vis
spectra of several systems over the course of the reaction.
The chapters comprising Part I therefore describe the synthesis of the ligands (including two
new compounds; 6A-{2-[bis(2-dimethylamino)ethyl]amino}-6A-deoxy-β-cyclodextrin
(βCDMe5tren) and 6A-(1,4,7-trimethyl-1,4,7-triazacyclononan-1-yl)-6A-deoxy-β-cyclodextrin
(βCDMe2tacn)), potentiometric titrations to establish speciation of the systems and the
subsequent monitoring of the hydrolysis of 4-nitrophenyl acetate using UV-Vis spectroscopy.
The secondary project, reported in Part II, is focussed on the detection of physiological Zn2+
using a newly characterised ligand; 2-((E)-2-phenyl)ethenyl-8-(N-4-
methylbenzenesulfonyl)aminoquinol-6-yloxyacetic acid. A styryl functional group was added to
the commercially available Zn2+-selective fluorophore “Zinquin” which detects physiological
Zn2+ by complexing it and producing a fluorescent signal exclusively on Zn2+ binding. The
v
intention of the styryl addition was to enhance the bulk of the Zinquin analogue and thereby hope
to improve the selectivity of this fluorophore for free, intracellular Zn2+ over the Zn2+ found in
the structures and catalytic centres of proteins and enzymes. This styryl Zinquin derivative was
characterised using potentiometric titrimetry, UV-Vis spectroscopy and fluorimetry. This
includes an analysis of the photoisomerism introduced by the styryl functionality. The
absorbance and fluorescence characteristics of the free and Zn2+-complexed ligand were
measured and stability constants for the Zn2+ complexes were determined. The results were
compared to Zinquin under the same experimental conditions. Like Zinquin, the new styryl
analogue was found to be Zn2+ selective.
vi
ACKNOWLEDGEMENTS
There are so many people who deserve my heartfelt thanks, that their names could fill another
thesis.
Thank you to Prof Stephen Lincoln for your supervision over many years, and particularly
your encouragement in gaining Chemistry communication and education skills.
Thank you very much to Associate Professor Chris Sumby for your support and editing
efforts.
Thank you to past members of Lab 12 who became my second family (Corri, Renée, Jacquie,
Lara, Charlotte, Jianjia and Sean).
Thank you to the Lab 8 members for your many years of support and friendship (Jo, Trang,
Hamish, Noby and Tien) and, in particular, thank you to Truc for your invaluable help at the end
of this work and for providing me with βCDMe2tacn.
Thanks also to Dr. Bruce May for synthesising the stZQA compound used in this study.
Many thanks to all the other members of the Chem Department who filled my time with such
wonderful memories and who made me a better scientist and teacher – in particular Nat, Jason,
Erin, Sean, Anton, Claire, Jason, Alex, Danielle, Jon, David and Natalie.
Thank you to Pamela for all your support, above and beyond the norm.
Gerald, thank you for turning me into the scientist I am. You are greatly missed.
Ray, thank you for the wonderful opportunity of working with you and producing something
to be proud of during my studies.
Ann, thank you for your passion for chemistry and teaching that sparked mine.
Thank you to Jo, thank you to Ricky, Steve and Karl, and thank you to Andrew, Jamie,
Micah, Laura and Eric, for the literally hundreds of hours of audio company during all the long,
lonely and late hours in the lab.
Thank you to so many of my friends who put up with, and supported me over so many lab-
and desk-bound years: Sally, Kimberley, Abby and Bec (the Hackies), Katie (the Top Half of
viii
Miss Christine Felton), Fi (oh my Darwin), Joanne and Richard (my favourite Meyers), Christine
and Kathryn (you rock), Shusuke (thanks for all the coffee), Tony (thanks for all the words).
Thank you to grandparents, aunts, uncles and cousins for all your support and encouragement.
Many thanks to Patrick (Perchik) – you are the best brother a person could hope for. (FAD.)
Finally, there are several people without whom this study would simply not have been
accomplished –
Corri, completing this is as much for you as it is for me. You are a supervisor, postdoc, friend,
teacher and sister all in one crucible.
Mum and Dad, mcsquared, I would be absolutely nowhere without you. Thank you for your
never ending love and support.
“It was, he thought, the difference between being dragged into the arena to face a battle to the
death and walking into the arena with your head held high. Some people, perhaps, would say that
there was little to choose between the two ways, but Dumbledore knew - and so do I, thought
Harry, with a rush of fierce pride, and so did my parents - that there was all the difference in the
world.”
Harry Potter and the Half-Blood Prince
ix
ABBREVIATIONS
1. General
δ chemical shift (parts per million)
υ frequency (s-1)
Φ quantum yield
λ wavelength (nm)
Ainfinity UV-Vis absorbance at an infinite reaction time
c speed of light, 3 × 108 m s-1
CHEF chelation enhanced fluorescence
dm decimeter
E enzyme (Pat I)
E observed electrode potential (millivolts) (Part II)
E0 standard electrode potential (millivolts)
EPR electron paramagnetic resonance
E•S enzyme-substrate Michaelis complex
et al. et alia
F Faraday constant, 9.6487 × 104 C mol-1
h Planck’s constant, 6.63 × 10-34 J s
[H+] proton concentration (mol dm-3)
I constant ionic strength (mol dm-3)
J coupling constant (context; NMR) or joules
K stability constant
K1 stability constant for the 1:1 (M2+:ligand) transition metal complex

x
K2 stability constant for the 2:1 (M2+:ligand) transition metal complex
Ka acid dissociation constant
KaH2O#1 acid dissociation constant for the first aqua ligand deprotonation
KaH2O#2 acid dissociation constant for the second aqua ligand deprotonation
KaH2O#3 acid dissociation constant for the third aqua ligand deprotonation
kcat rate constant (s-1) for the catalysed rate of hydrolysis of 4-nitrophenyl
acetate
kobs rate constant for the overall observed rate of hydrolysis of 4-nitrophenyl
acetate
kun rate constant for the uncatalysed rate of hydrolysis of 4-nitrophenyl acetate
KD dissociation constant for the reformation of the free enzyme and substrate
from the Michaelis complex (E•S)
KM Michaelis constant
L free, deprotonated ligand
LH+ free, monoprotonated ligand
LH22+ free, diprotonated ligand
LH33+ free, triprotonated ligand
LH44+ free, tetraprotonated ligand
MHz Megahertz
[MLH]3+ 1:1 M2+:monoprotonated ligand complex (Part I)
[ML]2+ 1:1 M2+:deprotonated ligand (Part I)
1:1 stZQA:M2+ (Part 2)
[MLOH]+ 1:1:1 M2+:ligand:hydroxo
[ML(OH)2] 1:1:2 M2+:ligand:2×hydroxo

xi
[M(L)2]2+ 1:2 M2+:2×ligand
Note: the five complexes above may or may not contain water ligands as is
noted for each specific example in the text.
mV millivolts
nm nanometer
NMR nuclear magnetic resonance
pH -log[H+]
pKa -log[Ka]
pKaH2O#1 -log[KaH2O#1]
ppm parts per million
R Ideal gas constant, 8.314 J K-1 mol-1
S substrate (in this study; ester 4-nitrophenyl acetate – Part I)
S0 ground electronic state (Part II)
S1 first excited electronic state (Part II)
T Temperature (˚C or Kelvin)
tinfinity An infinite time into a reaction
UV-Vis UV-Visible
v0 ground vibrational state
v1 first excited vibrational state
Zn2+Ad adventitious Zn2+
xii
2. Chemicals
ACES 2-(carbamoylmethylamino)ethanesulfonic acid (buffer)
βCD β-cyclodextrin
βCDMe2tacn 6A-(1,4,7-trimethyl-1,4,7-triazacyclononan-1-yl)-6A-deoxy-β-cyclodextrin
βCDMe5tren 6A-{2-[bis(2-dimethylamino)ethyl]amino}-6A-deoxy--cyclodextrin
βCDtacn 6A-(1,4,7-triazacyclononan-1-yl)-6A-deoxy--cyclodextrin
βCDtacdo 6A-(1,5,9-triazacyclododecan-1-yl)-6A-deoxy--cyclodextrin
βCDtren 6A-{2-[bis(2-aminoethyl)amino]ethylamino}-6A-deoxy--cyclodextrin
Bicine 2-(bis(2-hydroxyethyl)amino)acetic acid (buffer)
DEPP 1,4-diethylpiperazine (buffer)
HEPES 2-[4-(2-hydroxyethyl)piperazin-a-yl]ethanesulfonic acid (buffer)
Me3tacn 1,4,7-trimethyl-1,4,7-triazacyclononane
Me6tren tris(2-dimethylaminoethyl)amine
MES 2-(N-morpholino)sulfonic acid (buffer)
MOPS 3-morpholinopropane-1-sulfonic acid (buffer)
PIPES 1,4-piperazinediethanesulfonic acid (buffer)
Tacn 1,4,7-triazacyclononane
TAPSO 3-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]-2-
hydroxypropane-1-sulfonic acid (buffer)
TEEN N-N-N'-N'-tetraethylethylenediamine (buffer)
TES 2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic
acid (buffer)
Tren tris(2-aminoethyl)amine
xiii
TRIS 2-amino-2-hydroxymethyl-propane-1,3-diol (buffer)
stZQA 2-((E)-2-Phenyl)ethenyl-8-(N-4-methylbenzenesulfonyl)aminoquinol-6-
yloxyacetic Acid (styryl Zinquin A)
ZQA 2-methyl-8-p-toluenesulfonamido-6-quinolyloxyacetic acid (Zinquin A)
xiv
Hilary Coleman Chapter 1. Introduction Part I: Metallo-amine complexes as enzyme mimics
CHAPTER 1
INTRODUCTION PART I: METALLO-AMINE

COMPLEXES AS ENZYME MIMICS
1
1.1 Metalloenzymes
1.1.1 Introduction
Metalloenzymes are essential for life and are indispensable elements in many biological
processes including respiration and photosynthesis.1-3 Without them, organisms would be unable
to carry out reactions on the timescales that are required for existence. As just one of many
examples, the half-life for hydrolysis of the phosphate ester backbone of DNA is assessed to be
more than 130 000 years in the absence of a metalloenzyme catalyst, in water at pH 7 and 25°C.4-
7
The process of phosphate ester hydrolysis is indispensably required for both DNA replication
and repair, and is therefore vital to life.8 Some phosphodiesterase enzymes reportedly increase the
ester cleavage rate by up to a factor of 1015-16 which is a remarkable kinetic improvement.9
Without the presence of these biological catalysts the reactions would take place on a timescale
that is incompatible with life.
Nature has developed a spectacular catalogue of metalloenzymes to enhance the rates of

many life-sustaining reactions under the relatively mild conditions of human physiology; 37°C
and essentially in aqueous solution. For the transition metal zinc alone, there are over 1000
documented metalloenzymes. They fall into all six of the International Union of Biochemistry
and Molecular Biology’s established enzyme categories; oxidoreductases, transferases,
hydrolases, lyases, isomerases and ligases.10-13 Whilst metalloenzymes are intricate in structure
and function, this complexity arises from some three billion years of evolution and makes the
enzymes highly efficient and selective. Quite apart from the challenge of seeking to mimic
enzyme activity, the ability to gain this capability for practical application in a laboratory setting
is highly desirable. Consequently, the investigation of metalloenzyme structure, function and
mechanism is of great intrinsic interest as well as being of considerable significance from both
industrial and environmental standpoints.
1.1.2 Features of metalloenzymes – Carbonic anhydrase

More than one third of all naturally-occurring enzymes contain metals that are essential for
their biological function.14, 15 These metalloenzymes consist of a metal ion attached to the overall
protein structure by amino acid residues. Whilst a single metal ion is the active site of many
2
metalloenzymes, some incorporate multiple metal ions in their skeletons which contribute to the
highly specific structure that is vital to the enzyme activity.
Metalloenzymes work as catalysts by providing an alternative reaction pathway of lower

activation energy than the uncatalysed pathway of a given reaction. Around the metal centre there
is usually an environment which is selectively tailored to capture the reactant (substrate) and
activate it towards reaction. One such example is the carbonic anhydrase enzyme family, a group
of metalloenzymes that catalyse the reversible hydration of carbon dioxide – an essential reaction
in all life domains (Bacteria, Archaea and Eukarya).16-18 Carbonic anhydrase is associated with
many crucial pathological and physiological processes involved with maintaining the acid-base
balance in blood and tissues, solubilising CO2 for transport, homeostasis and respiration.19
At the active site of carbonic anhydrase is a zinc(II) ion situated in a protein cavity. The Zn2+
is coordinated in a tetrahedral arrangement by three histidine side chains (His-94, His-96 and His-
119) and a water (aqua) ligand.20 This change in environment around the Zn2+ due to the histidine
and other nearby amino acid moieties, changes its reactivity and particularly the reactivity of the
associated aqua ligand.21-24 This causes the aqua ligand to be more acidic than it would otherwise
be as a bulk water molecule and it can therefore exist in the deprotonated hydroxide form
(hydroxo ligand, OH ̅) at physiological pH. Figure 1.1 displays a ribbon diagram of human
carbonic anhydrase II and a close-up of the histidine residues and hydroxo ligand coordinated to
the Zn2+ centre. It can also be seen that the cleft holds the carbon dioxide substrate in close
proximity to the hydroxo ligand, thereby increasing the effective concentration of the reacting
species – a common feature of enzymes.
The reaction shown in Equation 1.1 occurs relatively slowly in the absence of a catalyst. At
25°C this reaction of carbon dioxide and water to form carbonic acid has a half-life of the order
of 180 s which is tardy on the timescale required for cellular activity.25
CO2 + H2O ⇌ H2CO3 ⇌ HCO3 ̅ + H+ 1.1
However, carbonic anhydrase produces some of the greatest rate enhancements observed for
any enzyme.26 A catalytic rate constant, kcat, of approximately 106 s-1 is observed for the
hydration of CO2 (Equation 1.1) in the presence of human carbonic anhydrase II – the enzyme
accelerates the hydration of CO2 molecules to a million reactions per second.22, 27
3
Figure 1.1. The human carbonic anhydrase II metalloenzyme (a) and a close-up of its active site
(b). The protein chain is depicted as yellow and pink ribbons, the Zn2+ ion is shown as the grey
ball, the carbon dioxide molecule is highlighted here in green and the red spheres in (a) are the
oxygen atoms of the water molecules surrounding the enzyme. In the close-up, the histidine
residues are displayed by rod diagrams (blue = nitrogen, grey = carbon, red = oxygen) as is the
hydroxo ligand associated with the Zn2+ (red = oxygen).28, 29
4
In an aqueous solution, autoprotolysis occurs with a pKa of 14. By comparison, the first
protolysis of [Zn(H2O)6]2+ is characterised by a pKa ~ 9 whilst the Zn2+-coordinated aqua ligand
in carbonic anhydrase has a pKa of 6.8-7.9; up to two pH units lower.16,20,30,32 Consequently,
carbonic anhydrase supplies a nucleophilic hydroxo ligand at the physiological pH.
The influence of local hydrophobicity on the pKa of an aqua ligand was first demonstrated in
1974.31 Complexes of Co2+, Ni2+, Cu2+and Zn2+ with tris(2-aminoethyl)amine (tren) and tris(2-
dimethylaminoethyl)amine (Me6tren, Figure 1.2) show a marked decrease in the pKa of the
associated aqua ligand when the hydrophobicity of the complex is increased on substitution of
tren for Me6tren in each of the metal complexes.32 This decrease in aqua ligand pKa resembles
the effect on the aqua ligand of the presence of the histidine residues (and other nearby amino
acids) in the highly intricate structure of carbonic anhydrase which is proposed to contribute to
the enzyme function.20 Accordingly, it is of considerable interest to investigate in potential
enzyme mimics the effect of this decreased aqua ligand pKa that arises from this hydrophobic
effect.
Figure 1.2. The ligands tren (a) and Me6tren (b).
As a consequence of the presence of the hydroxo ligand, the hydration of carbon dioxide by
carbonic anhydrase is proposed to follow the mechanism below, where E represents the bulk of
the carbonic anhydrase enzyme:
EZnOH ̅ + CO2 ⇌ EZn(OH¯)CO2 ⇌ EZnHCO3¯ ⇌ EZnH2O + HCO3¯ 1.2
EZnH2O ⇌ EZnOH¯ + H+ 1.3
5
Equation 1.2 characterises the simplified process whereby the hydroxo ligand makes a
nucleophilic attack on the carbon dioxide in the active site cavity of the enzyme. This facilitates
the formation of bicarbonate which then leaves the active site to be replaced by an aqua ligand.
Equation 1.3 shows the loss of a proton from this aqua ligand to reform the hydroxo ligand. It is
this second process that occurs at a reduced pH in the presence of carbonic anhydrase.
As depicted in Figure 1.1, the Zn2+ is situated at the centre of the enzyme at the bottom of a
15 Å deep cavity which houses the CO2 substrate for reaction.20 This feature coupled with the
production of the hydroxo ligand at physiological pH makes carbonic anhydrase an extremely
efficient metalloenzyme.
1.1.3 Ester hydrolysis – the Hydrolases

The ester hydrolysis reaction is ubiquitous in both nature and industry. From physiological
reactions (such as ATP hydrolysis32) to the production of soap and detergents (saponification33)
to manufacturing biofuels34 to organic synthesis35, humans would have neither quality of life, nor
life at all without ester hydrolysis. Consequently, the pursuit of efficient catalysts for this reaction
has potentially both industrial and environmental importance. Since enzymes are biological
catalysts whose features have been honed and perfected over some three billion years of
evolution, their structures and mechanisms are extremely valuable in guiding research in this
area.
The third class of enzymes set out by the International Union of Biochemistry and Molecular
Biology contains the “hydrolases” – enzymes that catalyse the hydrolysis of a chemical bond. Of
particular interest to this study is the subclass EC 3.1 – hydrolases acting on ester bonds. Of these
hydrolases, there are 21 reported types of esters that they hydrolyse, and there are 90 specifically
listed carboxylic ester hydrolases alone.36 The term “esterase” was coined to describe enzymes
that catalyse the hydrolysis of carboxylic esters.
Figure 1.3 shows four esters that undergo hydrolysis and play a significant role in human
physiology. Structure a) depicts a triglyceride (TG), an ester formed from glycerol and three fatty
acids. Triglycerides are the main constituents of animal fats and vegetable oils and, when
hydrolysed, they form soaps and detergents.33 Structure b) is that of adenosine triphosphate
(ATP) which is present in every cell and transports and provides the energy required for every
6
physiological activity.37 The human body continuously recycles ATP and produces and consumes
its own weight in ATP every day.38 Structure c) is that of paraoxon, a phosphate ester that is a
powerful insecticide. Phosphate esters are used in a wide variety of pesticides which are
experiencing a large global growth in manufacture for their employment in agricultural food
production. Phosphate esters also function as nerve agents such as sarin gas which was used in
1995 in an act of domestic terrorism in Tokyo.39 The toxicity of pesticides like paraoxon to
species other than insects is well documented.40, 41 The final structure, d), is that of acetylcholine
(ACh). Acetylcholine is one of the main neurotransmitters in both the peripheral and central
nervous systems and is responsible for much of the stimulation of muscle tissue.42
Figure 1.3. Four ubiquitous esters. An example of a triglyceride a, adenosine triphosphate (ATP)
b, insecticide paraoxon c, and acetylcholine (ACh) d.
There are some similarities in the structures of paraoxon and acetylcholine and this is not
coincidental. The action of paraoxon as a pesticide stems from its ability to inhibit the enzyme
acetylcholine esterase by being a close match in the active site for the normal substrate molecule,
ACh.43 Once the substrate ACh has performed its nerve transmission function it must be
metabolised so that more neurotransmissions may occur. The enzyme acetylcholine esterase
7
breaks down ACh very quickly but this can be irreversibly inhibited by the presence of paraoxon
in its active site. If the ACh is not hydrolysed after use, subsequent neurotransmissions (required
for respiration, for example) cannot occur and this leads to subject (insect) death.44
Figure 1.4 below shows the active site of the acetylcholine esterase enzyme after an
interaction with the toxic nerve agent sarin (a phosphate ester similar to paraoxon). The pocket in
the centre of the enzyme which usually receives the ACh substrate contains three amino acid
residues (histidine, serine and glutamate) which interact with the neurotransmitter. On exposure
to sarin however, the histidine residue becomes phosphorylated and can no longer participate in
ACh hydrolysis.44 This leads to subject (human) paralysis and finally death. Consequently, an
ability to hydrolyse these toxic esters is a desirable one in carrying out soil and water remediation
and chemical warfare stockpile destruction.45,46
Figure 1.4. The active site of acetylcholine esterase that has been attacked by the nerve agent
sarin. The active site residues serine (Ser-200), histidine (His-440) and glutamate (Glu-327) are
represented by the grey (carbon), blue (nitrogen) and red (oxygen) rods and the serine can be
seen bonded to a methylphosphonate group (orange) which inhibits the esterase activity.29, 44, 47
Figure 1.3 shows just four of many physiologically and environmentally significant esters that
undergo hydrolysis reactions. With the ubiquity of the hydrolysis reaction in nature and industry,
mimicking enzymes of the hydrolase family could have wide-reaching applications.
8
1.2 Hydrolase Mimics
1.2.1 Introduction
Physiological reactions occur under very mild conditions (37°C and in essentially aqueous
solution), but only with the aid of the biological catalysts; enzymes. Despite our bodies handling
these reactions with consummate ease, we cannot perform many of the same reactions on the
bench top with the same rate and yield. Simple synthetic catalysts often fail to approach the high
selectivities and bifunctional or even polyfunctional catalytic actions of natural enzymes.48
Enzyme-catalysed reactions include oxidation and reduction, transferal of a functional group

between species, hydrolyses, bond breaking and formation, structural rearrangement between
isomers of an individual molecule and the joining of two molecules.36 Control over such a broad
spectrum of chemical processes is essential in the body and highly sought after in the laboratory.
However, whilst enzymes are naturally occurring, the complexity of structure that gives them
their activity also makes them virtually impossible to artificially synthesise. Therefore, simplified
enzyme mimics have been sought in all six enzyme classes.49-54 These more basic analogues must
ideally still contain the key features of enzymes but without the synthetic obstacles; a selective
pocket to attract and capture the substrate and an active site which promotes or enhances the
desired reaction.
In the pursuit of metalloenzyme mimics, metal complexes have been extensively studied for
potential catalytic activity towards ester hydrolysis.55-62 The broadly accepted general mechanism
for ester hydrolysis in this format involves the acid dissociation of a metal-bound aqua ligand (to
form the nucleophilic hydroxo ligand), the coordination of the ester substrate to the metal ion
followed by the attack of the metal-bound hydroxo ligand on the ester.62, 63
(This is the same
mechanism experienced by carbon dioxide interacting with carbonic anhydrase.) The enzyme-
mimicking features employed by this approach include production of the nucleophilic hydroxo
ligand, activation of the ester, and increasing the proximity of the reactants (the template
effect64). Therefore, the identities of the ligand(s) and metal ion in the metal complex will affect
the overall catalytic efficiency and their variation can lead to the optimisation of the end product
enzyme mimic.
9
There have been a variety of proposals for the design and function of enzyme mimics. Figure
1.5 illustrates a proposed generic ester hydrolysis mechanism for a phosphate ester, aided by a
metal complex that is functioning as an enzyme mimic.62 In this example only the metal centre,
M2+, the aqua and hydroxo ligands, and the interaction between M2+ and the phosphate ester are
shown.
Figure 1.5. A proposed ester hydrolysis mechanism for a phosphate diester by a metal complex
enzyme mimic.62
The aim of the work presented in this thesis was to investigate a family of ligands combined
with four metal ions to produce potential enzyme mimics that would reveal information about the
relative importance of each ligand and metal ion and potentially lead to predictive powers in
proposing metal complexes as enzyme mimics. The complexes explored in this study are tren,
Me6tren, tacn (1,4,7-triazacyclononane) and Me3tacn (1,4,7-trimethyl-1,4,7-triazacyclononane)
with the metal ions Zn2+, Cu2+, Ni2+ and Cd2+ (see Figure 1.9 in Section 1.5.1). However, whilst
the metal complexes formed by these ligands and metal ions will provide the hydroxo ligand
required for ester hydrolysis (through aqua ligand deprotonation), they are missing the selective
10
cavity to capture the substrate that is expected of an efficient enzyme mimic. Without this
substrate capture, the rate of reaction relies on the random encounters of the reacting species.65
One solution is to employ the use of cyclodextrins.39, 46, 66-69

They contain a hydrophobic
annulus that can include esters, acids, alcohols and amines,70 and can be readily modified to be
substituted with the four metal ion-complexing ligands reported above.71 This allows for the
relatively simple synthesis of species which contain a hydrophobic pocket to capture the ester
substrate (the cyclodextrin) in close proximity to the activated reactant (the aqua ligand that
undergoes acid dissociation to form the nucleophilic hydroxo ligand). Therefore in this study β-
cyclodextrin (βCD) was substituted with tren, Me6tren, tacn and Me3tacn and these new ligands
were also combined with the metal ions Zn2+, Cu2+, Ni2+ and Cd2+ (see Figure 1.9 in Section
1.5.1) to explore the resulting complexes’ potential enzyme mimic abilities.
1.2.2 Previous metallo-amine and -cyclodextrin hydrolase mimic systems

In 1990, (1,5,9-triazacyclododecane)zinc(II) ([Zn(tacdo)]2+, see Figure 1.6a) became the first
model complex to successfully mimic the Zn2+ centre in carbonic anhydrase.72 With an aqua
ligand pKa of 7.3, this complex remains a competitive carbonic anhydrase mimic.73, 74
An
advantage in the employment of macrocyclic polyamines to build environments for Zn2+
coordination in modelling zinc enzymes is the large thermodynamic stability of these complexes
which permits otherwise unstable Zn2+ polyamine derivatives to be formed at pH 7 – 8 in
aqueous solution.75 However, this [Zn(tacdo)]2+ complex (along with other metallo-amine
hydrolase mimics) does not contain a hydrophobic pocket for substrate capture.
Unmodified βCD has been shown to enhance the rate of hydrolysis of the ester 4-nitrophenyl
acetate above the uncatalysed rate, involving a mechanism in which the ester includes in the βCD
annulus (Figure 1.6b). However, the experimental conditions for these experiments involved a
pH ≥ 10 at which there would be some deprotonation of the 1˚ and 2˚ cyclodextrin rim hydroxyl
groups which then react with the ester.76 Combining both the metallo-amine complex with a
cyclodextrin, a metal complex-functionalised cyclodextrin was originally synthesised more than
40 years ago77 and its ability to enhance the rate of the hydrolysis of ester 4-nitrophenyl acetate
was tested. This metallo-cyclodextrin is shown in Figure 1.6c along with other examples of
metallo-amine and -cyclodextrin enzyme mimics. Since then this has become a fruitful area of
research.78, 79
The hydroxy(6A-(1,5,9-triazacyclododecanyl)-6A-deoxy-β-cyclodextrin)zinc(II)
11
complex, [Zn(βCDtacdo)]2+, is very similar to some of the metallo-cyclodextrin systems

investigated in this study and has already been reported in the literature (Figure 1.6d).66 The
mechanism through which it catalyses ester hydrolysis is also included in Figure 1.7. Whilst the
[Zn(βCDtacdo)]2+ complex is not nearly as intricate nor specialised as an enzyme, the rate of
acceleration observed for the ester hydrolysis over the uncatalysed rate was 291-fold.66 The
above system, and similar complexes, brings the reacting species (the ester and hydroxo ligand)
together in close proximity in an assembly similar to a physiological Michaelis complex; an
established strategy in catalyst design using supramolecular chemistry.78 However, an enzyme
mimic must undergo many catalytic cycles without deactivating if it is to be considered a true
catalyst. Therefore, the 4-nitrophenolate product in the cyclodextrin annulus and the acetate
group bound to the Zn2+ (Figure 1.7) must both be displaced if catalyst poisoning is to be
avoided. A labile aqua ligand at the Zn2+ centre must then undergo acid dissociation again to
form the incipient hydroxo ligand and allow the next catalytic cycle to occur.
Finally, most recently in 2015 a dinuclear, divalent Co2+ system has been found to enhance
the hydrolysis rate of both 4-nitrophenyl phosphate and bis(4-nitrophenyl) phosphate esters, the
efficiencies of which have rarely been reported for synthetic Co2+ complexes before (Figure
1.6e).80 All of these systems shown in Figure 1.6 reportedly enhance the rate of ester hydrolysis
to varying extents. However, the results are not quantitatively compared here because the studies
involve difference esters and different experimental conditions. The advantage of the systematic
investigation completed in this thesis is the ability to directly compare each system in order to
determine which features are responsible for producing successful enzyme mimics. In this study,
the ligands tren, Me6tren, tacn and Me3tacn were attached to β-cyclodextrin to investigate the
possible contribution of the hydrophobic cavity, represented by its hydrophobic annulus, towards
enzyme mimicry. All the ligands displayed in Figure 1.9 (Section 1.5.1) were complexed by Zn2+,
Cu2+, Ni2+ and Cd2+ and the catalytic activity of some of the most promising of these were tested
with the ester 4-nitrophenyl acetate.
12
Figure 1.6. Examples of metallo-amine and –cyclodextrin hydrolase mimic systems: a72, b76, c77,
d66 and e80.
13
Figure 1.7. Hydrolysis of 4-nitrophenyl acetate catalysed by hydroxy(6A-(1,5,9-triazacyclododecanyl)-6A-deoxy-β-cyclodextrin)zinc(II). The

“Michaelis complex” is the complex formed by the enzyme and the substrate (see Section 1.4).
14
1.3 The use of cyclodextrins in ligand design
1.3.1 Cyclodextrin structure

Cyclodextrins (CDs) are cyclic oligosaccharides consisting of 1,4-linked D-glucopyranose
units as depicted in Figure 1.8. They are formed from the digestion of starch by the CD
transglycosylase enzyme that is derived from the bacterium Bacillus Macerans.81, 82 This enzyme
can vary the number of glucose units per CD ring from 6-13.83 The three most common
cyclodextrins are αCD, βCD and γCD, comprised of six, seven and eight glucopyranose units,
respectively. The 1,4-linked D-glucopyranose assembly gives αCD, βCD and γCD their
characteristic truncated cone form.
Figure 1.8. General structural representation of a cyclodextrin showing the individual

glucopyranose units. For α-, β- and γ-cyclodextrin n = 1, 2 and 3, respectively. The carbon atoms
are numbered on ring A and the hydroxyl groups and hydrogens are discussed by referring to
which of these carbon atoms they are bonded to.
The attraction of cyclodextrins as a productive field of research stems from their distinctive
structure; an exterior with many hydrophilic hydroxyl groups and an annulus with a hydrophobic
interior. The location of the hydroxyl groups pointing outwards is responsible for the water-
solubility of cyclodextrins.84 Whilst the inner rim of the CD annulus is lined with hydrogen atoms
(hydrogens H3 and H5 point inwards to the cavity) and glycosidic oxygen bridges (Figure 1.8).
The non-bonding electron pairs of these glycosidic bridges are directed towards the inside of the
15
annulus, contributing to its relatively hydrophobic nature. The interior annular radii increase from
5.70 Å to 7.80 Å to 9.50 Å for αCD, βCD and γCD, respectively.85 Consequently CDs have the
ability to encapsulate a broad range of molecules, or “guest” species, to form host-guest, or
“inclusion”, complexes through non-covalent interactions.86 This is similar to the manner in
which substrates are bound by enzymes.
Cyclodextrin complexing abilities may be modified by substitution at one or more of the C2,
C3 or C6 hydroxyl sites.71, 87-90 There are many possible modifications which include, but are by
no means limited to, polymers,91 amines,71 alkyl groups,92, 93
, tosyl and pyrene moieties,94
sulfates,95 charged species96 and chromophores.97 Since cyclodextrins are naturally occurring
compounds they are suitable for many applications in which toxicity and scale of production are
factors. Thousands of tonnes of CDs are produced globally each year.82 These cyclodextrins and
their derivatives are utilised in a broad spectrum of activities such as drug solublising and
delivery,98 polymer manufacture,99 synthetic chemistry (e.g. olefin reduction, aldehyde alkylation
and hydrogenation and the C-C coupling reaction),100 the food industry101 and in biomimicry as
artificial enzymes.78, 90
1.3.2 Cyclodextrin host-guest complexes

It is the properties of the CD annulus that make cyclodextrins able to differentiate between
molecular guests according to their characteristics such as functional groups, length, diameter and
chirality, and to selectively complex these species in aqueous solution.87, 89, 102 Although they do
not approach the affinities achieved by enzymes, this ability of CDs and their modified
derivatives to discriminate between, and complex, guests is analogous to enzymes binding
specific substrates at their own active sites.103 This property makes CDs very appropriate for
investigation in the design and production of enzymes mimics.78
The CD annular radius has a large influence on guest selection in host-guest complexation,
and consequently is an important parameter in enzyme mimic design. Furthermore, guest
complexation in the CD annulus may advantageously alter the properties of the guest molecule
including increasing its water solubility, its stability in the presence of air and light and its
reactivity.70, 104
16
Host-guest complex formation is strongly influenced by the closeness of fit between the guest
molecule and the host annulus. No covalent bonds are formed or broken during host-guest
complex formation. The complexes produced are very dynamic in nature, and their stability
depends on the local interactions occurring between the guest and the microenvironment of the
cyclodextrin cavity. A substantial contribution to the driving force behind host-guest complex
formation is the release of water molecules from within the annulus into the bulk solvent.84 Prior
to complexation, the cavity is occupied by water molecules that interact weakly with the annular
interior, by comparison with the interactions between water molecules in the bulk solvent. Their
displacement by a hydrophobic guest increases the hydrogen bonding between the released water
molecules and those in the solvent, reduces the repulsive forces between the hydrophobic guest
and the aqueous environment and allows for more favourable interactions as the hydrophobic
guest inserts into the apolar cyclodextrin cavity.104 Thus, the host-guest complex represents an
energetically more favourable state than does the separate host and guest.
In this study, βCD and its amine-substituted derivatives complex the ester 4-nitrophenyl
acetate in a similar manner to that in the mechanism displayed in Figure 1.7. The formation of the
host-guest complex formed between unmodified βCD and 4-nitrophenyl acetate is characterised
by logK ≈ 2 at pH 10.0 and 298 K, where K is the complexation constant which is equivalent to
the reciprocal of the Michaelis constant KM.76
1.4 Possible mechanisms of enzyme mimic activity

The metallo-βCDs investigated in this study (and, indeed, any enzymes or their proposed
mimics) may enhance the rate of ester hydrolysis via either catalysis and/or promotion of the
reaction. A catalyst will enhance the rate by providing an alternative reaction pathway of lower
activation energy and will not be consumed in the reaction. A promoter may enhance the rate
when combined with other catalytic species (but without being catalytic on its own) or may
enhance the rate but be consumed as a stoichiometric reagent in the process.
As noted in Section 1.2.1, the broadly accepted mechanism of activity for metallo –amine and
–βCD species in the literature involves the acid dissociation of a metal-bound aqua ligand (to
form the nucleophilic hydroxo ligand), the coordination of the ester substrate to the metal ion
followed by the attack of the metal-bound hydroxo ligand on the ester. The acetate product is
17
then displaced by labile aqua ligands and the hydroxo group is re-formed. This is a reasonable
postulate as the individual components that comprise the amine-modified metallo-βCD
compounds synthesised in this study (M2+, amine, βCD) have been shown to exhibit aspects of
this mechanism on their own. Metal cations on their own have been shown to enhance the rate of
ester hydrolysis by a mechanism which involves the coordination of the ester substrate to the
metal centre.56, 105-107
Similarly, metallo -amine and -βCD complexes are described in the
literature as catalysing ester hydrolysis reactions by promoting the formation of hydroxo ligands
at a depressed pH and then coordinating the ester to the metal to increase the effective
concentration of ester and hydroxide reactants.68, 72, 108-111
It has also been shown that 4-
nitrophenyl acetate includes in the annulus of βCD in an orientation that is favourable for
interaction with a metal-bound hydroxo ligand.76
This broadly accepted general metal-hydroxo mechanism follows the definition of catalysis
(rather than promotion) in that the metallo-βCD complex is regenerated at the end of the cycle
and is free to catalyse further hydrolysis reactions. Preliminary spectrophotometric data for the
hydrolysis of 4-nitrophenyl acetate in the presence of the metallo-βCD complexes synthesised in
this study indicates the mechanism likely involves initial binding of the ester to Cu2+ but not
necessarily for Zn2+(see Section 4.4). Interestingly, experiments elucidating the hydrolysis of 4-
nitrophenyl acetate by another amine-functionalised βCD Zn2+ complex ([Zn(βCDtacdoOH)]2+),
also suggests this mechanism does not involve coordination of the ester to Zn2+.66 Because
understanding the mechanism is critical to designing future potential enzyme mimics, the future
work described in Chapter 5 involves elucidating the mechanisms fully.
However, several other hydrolysis mechanisms (fitting the definitions of both catalysis and
promotion) are feasible for the complexes synthesised in this study. For example, the metal-
carbonyl mechanism (sometimes known as the Lewis acid activation mechanism) involves the
inner sphere binding of the carbonyl of the ester substrate to the complex metal cation which
activates the carbonyl towards attack by a solvent water molecule, for example. Although a
possible mechanism for the reactions studied herein, the kinetics analysis presented in Chapter 4
indicates that a metal-bound hydroxo ligand is responsible for the rate enhancement, providing
evidence for the broadly accepted metal-hydroxo mechanism discussed above (see Sections 4.2.2
and 4.2.3).
18
Direct nucleophilic acyl substitution by free (unbound) a primary or secondary amine-

substituted βCD, and general base-catalysed hydrolysis in the presence of a tertiary amine-
substituted βCD are also proposed in Chapter 4 as potential mechanisms of activity (Figure 4.21).
The nucleophilic acyl substitution mechanism involves the amine-substituted βCD directly
attacking the carbonyl as a reactant to form the phenolate and amide products. The general base
mechanism involves a tertiary amine-substituted βCD promoting hydroxide formation in solvent
water, which then attacks the carbonyl.
Kinetics experiments and preliminary spectrophotometric data for the hydrolysis of the model
ester 4-nitrophenyl acetate in the presence of some the free and complexed ligands presented in
Section 1.6.1 do yield results that help narrow the likely mechanism of enzyme mimic activity.
These results and discussion are presented in Chapters 4 and 5.
1.5 Enzyme kinetics investigation

Working on the “lock and key” concept for enzyme activity proposed by Emil Fischer in the
1890s,112 French chemist Victor Henri reported in 1903 that enzyme-mediated reactions were
initiated when the enzyme formed a bond with the substrate.113 Building on this concept, ten
years later Leonor Michaelis and Maud Menten published their now classic work that
transformed the study of enzyme kinetics.114, 115
In this paper, they reported a study of the
kinetics of the enzyme saccharase, which catalyses the hydrolysis of sucrose into fructose and
glucose. They showed that the rate of an enzyme-catalysed reaction has a hyperbolic dependency
on the concentration of the substrate, providing direct evidence to support the hypothesis that
enzyme activity proceeds through the formation of an enzyme-substrate complex; now often
referred to as the Michaelis complex. They included this relationship in their now renowned
Michaelis-Menten equation, which for the first time provided an indication of an enzyme’s
affinity for its substrate.116
To investigate any single-substrate bimolecular enzyme-catalysed reaction, the equilibria

shown in Equation 1.4 are considered:117
k1 k2
E + S ⇌ E•S ⇌ E + P 1.4
k-1 k-2
19
where E, S and P represent the free enzyme, free substrate and product, respectively. The
enzyme-substrate complex, or Michaelis complex, is shown as E•S, and rate constants k1 and k-1
represent fast processes controlling the formation of E•S such that k-1/k1 = KM, the Michaelis
constant. The rate constant k2 characterises the formation of the product by E•S and k-2
characterises the reversal of this reaction, although this seldom occurs.
A common method used in enzyme kinetic experiments, as in the experiments of Michaelis

and Menten, is to follow the reaction only in its early stages to avoid a buildup of product that
could potentially inhibit the enzyme and complicate the interpretation. Under this regime only
approximately the first 10% of the reaction is followed and Equation 1.4 is rewritten as 1.5 where
kcat is now the rate constant characterising the reaction of E•S to give the product, P, and no
reverse reaction is considered.
k1 kcat
E + S ⇌ E•S → E + P 1.5
k-1
Subsequently, P is released from the successor of E•S, (E•P), to regenerate E to repeat the
catalytic cycle. The rate constant, kcat, is also known as the “turnover number” and represents the
maximum number of substrate molecules one enzyme can convert into product per unit time.118
Thus, carbonic anhydrase (Section 1.1.2) has one of the largest turnover numbers known, kcat = 1
× 10-6 s-1 at 25°C. This translates into a 1 × 10-6 mol dm-3 solution of the enzyme generating 1
mol dm-3 of carbonic acid, H2CO3, every second from CO2 and H2O (Equation 1.1).27
The maximum achievable reaction rate, Vmax, is reached when as much substrate as possible is
complexed by the enzyme, which itself exists completely in the form of the Michaelis complex,
and the maximum [E•S] concentration is therefore attained for that system. Under these
conditions, the enzyme is saturated and further substrate addition will not increase the rate of
reaction beyond the maximum, Vmax. This is the saturation kinetics condition predicted by the
Michaelis-Menten model and which is revealed in a graph of initial reaction rate, V0, versus
substrate concentration which plateaus at a limiting V0 = Vmax beyond a particular substrate
concentration for a given enzyme mimic concentration.
The equilibrium characteristics of the enzyme mechanism may also be analysed according to
Equation 1.6 in which KA is the association equilibrium constant for the formation of the E•S
complex and KD is the corresponding dissociation constant. The size of KA directly indicates the
20
strength of the enzyme affinity for the substrate, but frequently 1/KA = KD, the dissociation
constant is quoted as KM, the Michaelis constant which is equal to the substrate concentration at
which the reaction rate is half of Vmax.
KA kcat
E + S ⇌ E•S → E + P 1.6
KD (= KM)
Accordingly, to characterise new enzyme mimics it is essential to determine kcat and KM to

determine the mimics’ overall catalytic efficiencies and to identify the mimic features which
affect the magnitudes of kcat and KM. Both kcat and KM may be determined through the seminal
equation (1.7) described in the original work of Michaelis and Menten (discussed further in
Section 4.1.3).114 Thus, a plot of V0 against substrate concentrations, [S], which are greatly in
excess of a constant enzyme mimic concentration, will yield Vmax (= kcat[E]) and KM provided that
KM is sufficiently small for V0 to approach Vmax in magnitude.
V0 = Vmax[S]/(KM + [S]) 1.7
Under biological conditions the substrate is in large excess over the enzyme. Because [S] >>
[E] this causes the described reaction to follow pseudo-first order kinetics and therefore pseudo-
first order rate constants, kobs, may be measured for the evolution of the product. However, there
is a potential for the product to be complexed by the enzyme mimic in competition with the
remaining unreacted substrate. If this interaction is significant, some of the enzyme mimic will no
longer be available to catalyse the reaction of the substrate. This process is referred to as “catalyst
poisoning” and must be allowed for in enzyme mimic kinetics studies. As discussed earlier,
cyclodextrins have the ability to form host-guest complexes with a wide range of guest species in
solution. Consequently, cyclodextrin-based enzyme mimics are prime candidates for catalyst
poisoning as the catalysed reaction progresses and the product concentration increases. For
example, it is known that the logarithm of the stability constant for the host-guest complex
formed between βCD and the hydrolysis product 4-nitrophenolate, logK = 2.97,70 is larger than
that for corresponding ester substrate 4-nitrophenyl acetate, logK ~2.076, at pH 10 and 298.2 K in
aqueous solution.
The answer – employed in many studies in which catalyst poisoning is a potential problem –
is to invert the ratio of substrate and enzyme such that the enzyme mimic is in excess
concentration.66, 76, 111, 119, 120 If the substrate is kept at a maximum of one tenth of the enzyme
21
mimic concentration then its hydrolysis product can complex a maximum of 10% of the enzyme
mimic and will therefore have minimal influence on the substrate hydrolysis. Thus, the kinetics
of substrate hydrolysis will remain pseudo-first order as the enzyme mimic concentration is
always in great excess over the substrate
The saturation kinetics predicted by the Michaelis-Menten model still occur when [E] >> [S]
as shown in Sections 4.1.3, 4.2.2 and 4.2.3. According to this model, as the enzyme mimic
concentration increases for a constant substrate concentration, the observed rate constant for the
hydrolysis, kobs, will increase until all of the substrate is bound to the enzyme mimic and the
maximum hydrolysis rate is reached. A modified version of the Michaelis-Menten equation, 1.8,
is used in this thesis to determine kcat and KM and its new derivation is discussed in Section 4.1.3:
kobs = (KMkun + kcat[E])/(KM + [E]) 1.8
In Equation 1.8, kobs is the observed rate constant, replacing initial rate, V0, in the
conventional Michaelis-Menten equation, KM is the conventional Michaelis constant, kun is the
uncatalysed rate constant for the hydrolysis (the rate constant for the hydrolysis that will occur in
the absence of an enzyme mimic, solely due to the hydroxide concentration dictated by the pH of
the reaction solution), kcat is the conventional catalysed rate constant (for the hydrolysis in the
presence of an enzyme mimic) and [E] is the enzyme mimic concentration. Thus, under the
limiting conditions when [E] = 0, kobs = kun, and when KM << [E], kobs = KMkun + kcat.
1.6 Aims of this research

Quite apart from the intrinsic interest in their chemistry, enzyme mimics are one facet of the
hunt for efficient, inexpensive and easily-producible catalysts being sought for industrial,
economic and environmental reasons. Therefore the overall objective of the research presented
here is to examine the features of naturally occurring catalysts, or enzymes, which may be
mimicked with simpler synthetic and more robust analogues. More specifically, the premise is to
use carbonic anhydrase and the family of esterases as the basis for the design and preparation of a
suite of metal complexes which mimic enzyme activity. The aim is to prepare stable metal
complexes that selectively capture the ester substrates and to generate in close proximity a
hydroxo ligand which makes a nucleophilic attack on the ester carbonyl carbon.
22
The pursuit of such enzyme biomimicry in this study has two parts. First, appropriate ligands
and their divalent metal complexes which bear hydroxo ligands at close to the physiological pH
are synthesised and then characterised by potentiometric titration. In this way the nature of the
metal complexes and their suitability for testing as potential enzyme mimics are established. A
critical part of this phase is the determination of the pKa of the aqua ligand which deprotonates to
give the hydroxo ligand. This work is presented in Chapters 2 and 3. In the second phase, the
kinetic and mechanistic aspects of hydrolysis of the carboxylate ester 4-nitrophenyl acetate by the
selected metal complexes are determined using UV-Vis spectrophotometric methods. This work
is presented in Chapter 4.
1.6.1 Ligand suite

The ligands tris(2-aminoethyl)amine, 1 tren, and 1,4,7-triazacyclononane, 2 tacn, were
selected as the core compounds for this study to be centred around (see Figure 1.9). They were
chosen for their similarity to the active site of carbonic anhydrase where the imidazole nitrogens
of three histidine residues coordinate Zn2+ along with an aqua ligand.17, 22 Furthermore, nitrogen
donor atoms feature strongly in biological systems and the amine groups of tren and tacn
coordinate strongly to the four metal ions selected, Zn2+, Cu2+, Ni2+ and Cd2+. An investigation of
these systems and their derivatives also lends itself to a comparison of the effects of the acyclic
structure of tren contrasted with the macrocyclic nature of tacn. The pKas of protonated tren and
tacn, the stability constants of the complexes formed by tren and tacn with Ni2+, Cu2+, Zn2+ and
Cd2+, and the pKas of the aqua ligands coordinated in these metal complexes were determined by
potentiometric titrimetry in aqueous solution.
Methylation of tren and tacn to form tris(2-dimethylaminoethyl)amine, 3 Me6tren, and 1,4,7-

trimethyl-1,4,7-triazacyclononane, 4 Me3tacn, was carried out in order to increase the
hydrophobicity of the ligands. Potentiometric titrations identical to those described above were
carried out to determine the effect of methylation in Me6tren and Me3tacn and their Zn2+, Cu2+,
Ni2+ and Cd2+ complexes. Of particular interest was the effect of methylation on the pKas of the
aqua ligands of the metal complexes. The tactic of creating a hydrophobic environment by
methylation in order to lower an aqua ligand pKa has already been demonstrated and is of
particular focus in this study.31
23
The other feature of carbonic anhydrase that was employed in this study is its ability to bind
the substrate (CO2 or H2CO3) in a hydrophobic cavity in close proximity to the nucleophilic
hydroxo ligand. Substituting β-cyclodextrin with tren, tacn, methylated tren and methylated tacn
incorporates a hydrophobic pocket which complexes the ester to be hydrolysed (4-nitrophenyl
acetate in this study) adjacent to the metal complexes. The methylated ligands 6A-{2-[Bis(2-
dimethylamino)ethyl]amino}-6A-deoxy--cyclodextrin, 5 βCDMe5tren, and 6A-(1,4,7-trimethyl-
1,4,7-triazacyclononan-1-yl)-6A-deoxy-β-cyclodextrin, 6 βCDMe2tacn, are new ligands and their
syntheses are described in Section 8.2.1. Figure 1.9 on the following page displays the whole
ligand suite.
24
Figure 1.9. The suite of ligands used for the synthesis of enzyme mimic metal complexes in this
study
25
Using Zn2+, Cu2+, Ni2+, and Cd2+, 32 metal complexes were formed with the eight ligands in
Figure 1.9. These were all characterised by potentiometric titration, and some tested for catalytic
activity. In the work presented in this study only Zn2+ and Cu2+ complexes were subjected to
kinetic analyses as these metal ions often form the metal centre in enzymes encountered in
biology. These complexes also appeared to be the most promising from their potentiometric
titration data. However, all of the systems characterised could be tested as part of Future Work.
Conclusions were then drawn as to the success of the investigated features (metal ion,
hydrophobicity due to methylation, presence of βCD annulus) in increasing the rate of ester
hydrolysis and thereby mimicking an enzyme. Some aspects of these systems have previously
been examined and these are referred to in the relevant chapters. The significance of the research
reported here is that it constitutes a systematic investigation of the complexation and kinetic
characteristics of every species under standardised conditions. A large catalogue of standardised
information such as this is important in understanding catalyst features and therefore enzyme
mimic design.
1.6.2 Potentiometric titrimetry

The pKa values for the free protonated forms of all eight ligands were determined using
potentiometric titrimetry together with the stability constants of the corresponding Zn2+, Cu2+,
Ni2+, and Cd2+ complexes and the pKa values of the associated aqua ligand(s) in their metal
complexes. Over the course of each potentiometric titration the pH increases from ~2 to ~ 11 as
sodium hydroxide titrant is added. Equilibria a) – f) in Figure 1.10 illustrate the equilibria
involved in the titrations as typified by the Zn2+/tren system. Characteristics specific to each
M2+/ligand combination dictate which species form during the titration as is discussed in
Chapters 2 and 3.
For tren shown in Figure 1.9, and the other seven ligands studied, deprotonation of the amine
group must occur prior to M2+ coordination (although there are some examples for which full
deprotonation is not required – see Chapters 2 and 3). Accordingly, the potentiometric titrations
carried out on solutions of the ligand and a particular M2+ are a measure of the competition
between the M2+ and H+ for the lone pairs of electrons on the ligand’s the amine nitrogen donor
atoms. When the ligands deprotonate or are involved in metal complexation, a change in
26
pH (= –log[H+]) occurs from which the corresponding pKas and metal complex stability constants
may be determined through potentiometric titration.121
Ka4
a) trenH44+ ⇌ trenH33+ + H+
Ka3
b) trenH33+ ⇌ trenH22+ + H+
Ka2
c) trenH22+ ⇌ trenH+ + H+
Ka1
d) trenH+ ⇌ tren + H+
K1
e) tren + Zn2+ ⇌ [Zn(tren)]2+
KaH2O
f) [Zn(tren)(H2O)]2+ ⇌ [Zn(tren)(OH)]+ + H+
Figure 1.10. Equilibria characterised by potentiometric titration, typified by the Zn2+/tren

system. a), b), c) and d) represent tren free ligand deprotonation constants, e) represents Zn2+
complex formation (aqua ligands not shown) and f) represents an aqua ligand deprotonation.
In each titration the variation of the observed potential (millivolts), E, is described by the
modified Nernst Equation 1.9:122
E = E0 + (RT/F)ln[H+] 1.9
where E0 is the standard potential of the electrode (millivolts), R is the ideal gas constant (8.314 J
K-1 mol-1), T is the temperature in Kelvin, F is the Faraday constant (9.6487 × 104 C mol-1) and
[H+] is the proton concentration (mol dm-3).
27
At 298.2 K, the pH of a solution is therefore given by Equation 1.10:
pH = (E0 – E)/59.15 1.10
When all of the equilibria occurring during a titration are identified, the concentrations of
their constituent species may be calculated along with the equilibrium constants. The fitting
program Hyperquad 2008 was used to fit the appropriate pH dependent algorithms to the
potentiometric data to simultaneously derive the values of all acid dissociation constants and
metal complex stability constants.123 A non-linear least-squares method is employed to minimise
the sum of the squares of the residuals in order to determine the best fit of the algorithm to the
experimental data. The speciation plots associated with the titration experiments are presented in
the Appendices and the corresponding pKas and stability constants are reported in Chapters 2 and
3. The kinetics experiments were each run at a designated pH such that the concentrations of the
enzyme mimics in their aqua and hydroxo ligand forms, and any free ligand and M 2+
concentrations were precisely known.
1.6.3 UV-Vis absorbance spectrophotometry

The kinetics of the hydrolysis of 4-nitrophenyl acetate were followed using UV-Vis
spectrophotometry. This is a standard method of measurement for this reaction.58, 66, 68, 72, 124-126 In
each experiment the evolution of the 4-nitrophenolate product was followed at 400 nm for a
minimum of 2.5 half-lives, and the absorbance at infinite time (i.e. when then the 4-nitrophenyl
acetate hydrolysis was complete) was recorded (Section 4.1.2). For each enzyme mimic (metal
complex) two different experiments were performed:
1) Buffered at the pH of the pKa of the aqua ligand, kinetic runs at a constant initial
4-nitrophenyl acetate concentration and over a range of excess enzyme mimic
concentrations were monitored and the observed rate constants, kobs, and hydrolysis half-
lives were determined.
2) Kinetic pH profiles were developed by performing kinetic runs buffered at several pHs
within an approximate pH range of 1.5 units on either side of the pKa of the aqua ligand of
the enzyme mimic. Analysis of this data showed the hydroxo ligand to be the catalytic
species.
28
The potential enzyme mimics were selected on the basis that their stability constants
determined by potentiometric titration were of sufficient magnitude to ensure that only very small
amounts of free ligand and M2+ were present at the pH of kinetic study so these species would not
contribute to the hydrolysis. Also, the pKa of the aqua ligand of the potential enzyme mimics
must not require a pH for its formation at which M2+ hydroxide species might precipitate.
UV-Vis absorption spectra were recorded over the ranges 395-405 nm and 225-500 nm,
depending on the reaction rate. Rapid reactions could only be followed over the smaller
wavelength range to ensure that enough data points were collected. The 225-500 nm range
encompasses both the 4-nitrophenolate product absorbance peak (λmax at 400 nm) as well as the
4-nitrophenyl acetate reactant absorbance peak (λmax at 271 nm).127 An investigation of the
changes in the absorption amplitudes of these peaks also gave some indication of reaction
mechanism. The variation of the derived kobs with the ratio of 4-nitrophenol acetate substrate to
enzyme mimic facilitated the determination of kcat and KM through the modified Michaelis-
Menten equation, Equation 1.8, for each of the enzyme mimics.
29
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37
Hilary Coleman Chapter 2. Potentiometric Titrations of the Foundation Polyamines
CHAPTER 2
POTENTIOMETRIC TITRATIONS OF THE

FOUNDATION POLYAMINES
38
2.1 Introduction
2.1.1 The properties of Tren and Tacn

Tren (tris(2-aminoethyl)amine 1) and tacn (1,4,7-triazacyclononane 2) were chosen as the
foundation ligands on which to design this study. The whole suite of ligands is shown in Chapter
1 (Figure 1.9) and it can be seen that tren and tacn are the basic building blocks upon which the
other ligands are built. This suite of ligands for potentiometric and kinetics investigation was
planned with the aim of testing the enzyme biomimicry activity of some of their metal
complexes. Therefore, to gain maximum insight the ligands were selected to share similarities
with the binding sites of enzymes, as well as to allow for other comparisons such as denticity,
acyclic versus macrocyclic character, the effects of alkylation and the nature of their
supramolecular chemistry. Tren and tacn continue to be used to this day, as the foundation on
which to build new derivative ligands for transition metal complex formation.1, 2
The
potentiometric characterisation of tren, Me6tren (tris(2-dimethylaminoethyl)amine), tacn and
Me3tacn (1,4,7-trimethyl-1,4,7-triazacyclononane) is reported in this chapter.
Tren was selected because it is a classic ligand in metal coordination chemistry,3-5 first
introduced in 1925.6 It is tetradentate and often favours the formation of high-spin,7 five-
coordinate complexes with transition metals.4 Tren’s tripodal structure allows it to wrap its
unrestricted pendant arms around a metal ion in a cage-like fashion. In a trigonal bipyramidal or
octahedral geometry the four nitrogens bind to the metal ion leaving one or two further co-
ordination sites, respectively, for a solvent molecule or other ligand.
Figure 2.1 shows the crystal structure of [Cu(tren)(H2O)]2+.8 In this particular structure the
[Cu(tren)(H2O)]2+ complex is part of a much larger supramolecular network in which it resides in
the interstices between layers of [NaCr(ox)3]2-. Interactions with this network are likely to affect
its structure, however it remains a good example of the archetypal coordination chemistry of the
tren ligand. Due to the compressed geometry of the complex, the axial bonds are shorter than the
equatorial bonds. This results in an increased coordinative interaction for the aqua ligand which is
responsible for its augmented reactivity.9 Consequently there has been interest in using tren
systems as potential catalysts (enzyme mimics) for ester hydrolysis.10-12
39
Tacn was also chosen for its longstanding presence in the literature and because it has been
the subject of extensive investigation.13, 14 This ligand is tridentate and its cyclic structure restricts
its flexibility and governs its chemistry, in contrast to the acyclic structure of tren which
possesses flexible pendant arms.
Figure 2.1. Ball and stick crystal structure of [Cu(tren)(H2O)] 2+ as part of a hybrid solid
consisting of 2D supramolecular networks.8, 15
Copper is shown in green, nitrogen is blue,
carbon is grey, hydrogen is white and oxygen, red.
Due to its constrained structure, tacn must coordinate in a trigonal manner to one face of a
metal ion in a complex.16, 17
This affects the size of the stability constants for the complexes
formed with some metals. However, despite its small size, tacn shows a strong macrocyclic
effect18, 19 which is manifested in its increased thermodynamic stability with respect to chelated
acyclic triamines.14, 16 In every transition metal case but copper(II), the tacn complexes are found
to be more stable than those formed with the analogous linear ligand, diethylenetriamine.13 This
break in trend for copper(II) is likely due to the influence of its d9 electronic configuration.
Coincidentally, the stereochemical arrangement of the three amine nitrogens on tacn bears
some resemblance to the arrangement of the nitrogens of the three protein histidine residues that
bind to zinc(II) in carbonic anhydrase. In this metalloenzyme, zinc(II) is tetrahedrally coordinated
to three nitrogens of the histidine residues and the reactive aqua molecule.20 Tacn may therefore
provide a mimic of this environment as a tridentate ligand, at least in terms of geometry.
As is the case with the tren metal(II) complexes, aqua ligands present in the tacn metal(II)
complexes likewise display altered reactivity.21-23 Consequently some tacn systems have also
been investigated for their potential enzyme mimicry in ester hydrolysis.12, 21, 22
40
As is illustrated by the mechanism shown in Chapter 1 (Figure 1.5), it is the production of the
nucleophilic hydroxo ligand that is key to the ester hydrolysis reaction. The methylated versions
of tren and tacn were therefore also chosen for study as ligand methylation has been shown to
decrease the pKas of aqua ligands in the corresponding metal complexes.24 Methylation of N-
donor sites has been a topic of considerable interest due to the resulting influence on the stability
and structure of the complexes formed by these ligands.7, 25-29
2.1.2 Previous studies

Some of the pKas and stability constants for the ligands and metal complexes in this study
have been previously determined. However, in order to make reliable comparisons across all
titration and kinetic work (including results reported in Chapters 3 and 4), all experiments were
carried out under identical experimental conditions to ensure uniformity across the data.
Some of the literature referenced in this chapter includes studies in different solvent systems,
at different temperatures, using different ionic strength salts and concentrations, and using
different determination techniques (exemplified by potentiometric titration, 1H NMR and UV-Vis
spectroscopy). Some observations on the variability of the data in the literature are outlined
below.
A 33% aqueous methanol (v/v) solvent system reportedly yields ligand pKas for tren that are
0.2 – 0.3 log units larger than the averages for these values recorded in aqueous solution.11 The
size of this difference is outside of the error range reported for the individual pKas. The use of
DMSO for the titration solvent appears to promote the formation of a stable 2:1 L:M complex
between tren and cadmium(II) that is not observed in any of the aqueous studies.29 Furthermore,
within a set of aqueous solution experiments, a 10 K rise in temperature lowers the ligand pKas of
tren and Me6tren by approximately 0.1 log units and a 20 K rise in temperature can alter the pKa
of an aqua ligand in the tren complex formed with Zn(II) by > 0.4.30
The identity of the salt used to maintain the constant ionic strength may also cause a large
variation in the results obtained.31 Furthermore, differences in ionic strength for the same salt can
also have a noticeable effect. At 298.2 K in 0.1 mol dm-3 NaClO4, the ligand pKa values for
triprotonated tren, trenH33+, are reported as pKa1 = 10.12, pKa2 = 9.41 and pKa3 = 8.42,31 but the
analogous values in 1.0 mol dm-3 are 10.42, 9.88 and 8.92.27 It is self-evident from these
41
observations that it was important to determine all characteristics of the systems studied here
(even data previously reported) to ensure consistency in the data obtained and its interpretation.
2.2 The pKas of protonated tren, Me6tren, tacn and Me3tacn, and
stability constants of their metal complexes
Samples of tren and tacn were purchased from Sigma Aldrich and Me6tren, tacn and Me3tacn
were synthesised according to the literature. All titrations were performed in duplicate with
reproducible results. Even for systems that have already been characterised in the literature, the
fitting in this study involved attempts to fit all possible species and all possible combinations of
these species until the best fit was achieved. Given potentiometric titration data fitting cannot be
used to definitively determine complex geometry, the fitting in this study was also informed by
data obtained by other techniques and reported in the literature (see Sections 2.2.2 and 2.2.3). The
data fits obtained in this study are good ( values routinely 2.0 or less). Consistent with the
literature, the errors reported for the titration data are derived from the Hyperquad fitting program
and reflect the congruity between the fit and experimental data. Estimated experimental errors are
typically a maximum of ± 3%. See Sections 8.1.3 and 8.2.1.
2.2.1 Determination of protonated tren, Me6tren, tacn and Me3tacn pKas

Acid dissociation constants, pKas, were determined by potentiometric titrimetry for the fully
protonated forms of ligands 1, 2, 3 and 4 (see Figure 1.9) at 298.2 K and in aqueous NaClO4 (I =
0.10 mol dm-3) as described in Section 8.2.2. Tren (1) and Me6tren (3) have four protonation sites
and are characterised by four acid dissociation constants, Ka1, Ka2, Ka3 and Ka4. Tacn (2) and
Me3tacn (4) have three protonation sites and are characterised three acid dissociation constants,
Ka1, Ka2 and Ka3. These are defined by Equations 2.1 to 2.4. The pKas that were determined in this
study are displayed in Table 2.1 for tren and Me6tren and in Table 2.2 for tacn and Me3tacn. They
may be compared with the literature values for these ligands (also recorded in Tables 2.1 and 2.2)
under several sets of experimental conditions.
As expected for all four ligands, the pKas decrease from pKa1 to pKa3 (or pKa4 in the instance
of tren and Me6tren). This is due to a combination of charge and statistical effects. As each
subsequent amine nitrogen becomes protonated this results in fewer remaining sites for an H+
42
from solution to attach at, thereby lowering the probability of protonation (and pKa) with each
successive H+ addition. The more significant contributing factor, however, is the increasing
overall positive charge on each ligand as the amine nitrogens are sequentially protonated. This
causes repulsion between the added protons, resulting in successively lower pKas for each added
H+.
Ka1
LH+ ⇌ L + H+ where Ka1 = ([L][H+])/[LH]+ 2.1
Ka2
LH22+ ⇌ LH+ + H+ where Ka2 = ([LH+][H+])/[LH22+] 2.2
Ka3
LH33+ ⇌ LH22+ + H+ where Ka3 = ([LH22+][H+])/[LH33+] 2.3
Ka4
(2.4: tren systems only)
The effect of adding successive protons to an acyclic ligand as compared to a macrocyclic

ligand may be seen by comparing tren and tacn. As is seen from Tables 2.1 and 2.2, the pKas of
protonated tren decrease from pKa1 to pKa3 to a lesser extent than do those of the macrocycle tacn.
This is because the amine nitrogens in tren are more spatially separated on individual pendant
arms and are therefore not confined in close proximity to each other. Unlike in macrocyclic
polyamines such as tacn, whose nitrogens are constrained near each other, there is less
electrostatic repulsion between the added protons, resulting in a smaller decrease in subsequent
pKas by comparison with those of tacn.
Of the ligands investigated here, tren and Me6tren have the most extensive data reported in
the literature. The results obtained from this study agree well with the literature values as
reported in Table 2.1. The closest agreement between the data from this study and from the
literature is with studies a, b and g in which the ionic strengths are most closely matched to the
conditions in this study.
43
Ligand LH+ LH2+ LH3+ LH4+

(L) pKa1 pKa2 pKa3 pKa4
tren This study 10.21 ± 0.004 9.45 ± 0.005 8.31 ± 0.006 1.74 ± 0.03
a 10.14 9.43 8.41 2.60
b 10.12 9.41 8.42
c 10.42 9.88 8.92
d 10.43 9.87 9.01
Me6tren This study 9.40 ± 0.02 8.79 ± 0.02 7.37 ± 0.02 1.52 ± 0.24
e 10.13 9.32 8.17
f 9.98 9.31 8.11
g 9.50 8.68 7.44 <1.5
a: Ref32 I = 0.10 mol dm-3 (KCl), 298.2 K, b: Ref31 I = 0.10 mol dm-3 (NaClO4), 298.2 K, c & e: Ref27 I = 1.0
mol dm-3 (NaClO4), 298.2 K, d & f: Ref30 I = 1.0 mol dm-3 (NaClO4), 298.2 K, g: Ref33 I = 0.15 mol dm-3
(NaCl), 298.2 K.
Table 2.1. pKas for the ligands tren and Me6tren determined by potentiometric titration at 298.2
K in aqueous NaClO4 (I = 0.1 mol dm-3). Analogous literature values determined by
potentiometric titration in aqueous solution are also shown (conditions indicated).
The larger errors associated with pKa4 for both tren and Me6tren in this study are due to the
small size of these constants. The potentiometric titration technique loses accuracy as the pKas
approach 2 as a lower pH limit and 11 as an upper limit. Consequently, when the pKas are outside
this pH range, larger errors are sometimes observed. This may be responsible for the difference
between the reported pKa4 value for tren32 and the one measured in this study. It should be noted
that none of the more recent studies appear to fit for, or report, a value for pKa4 for tren because
its magnitude is small. However, the pKa1 - pKa3 values are in good agreement with the literature
values and the inclusion of pKa4 in the pH variation algorithm improves the fitting for the
titrations performed in this study, particularly in which the metal complex stability is low. A
typical titration curve (raw data) and fit for Me6tren are shown in Figure 2.2. The derived pKa
values are averaged from two identical titrations. A speciation plot based on the pKa values for
Me6tren recorded in Table 2.1 is also shown in Figure 2.3. (Figures for the remaining ligands can
be found in Appendix A.
44
Ligand LH+ LH2+ LH3+
(L) pKa1 pKa2 pKa3
tacn This study 10.47 ± 0.03 6.96 ± 0.02 2.39 ± 0.01

a 10.68 6.86 2.1
b 10.42 6.82 strong
c 10.47 6.8 < 2.5
d 10.4 6.9 0.7
Me3tacn This study 11.96 ± 0.05 5.17 ± 0.03 1.88 ± 0.06
e 11.7 5.2 0.6
a: Ref13 I = 0.1 mol dm-3 (KNO3), 298.2 K, b: Ref16 I = 0.1 mol dm-3 (KNO3), 298.2 K, I =
0.1 mol dm-3 (KNO3), 298.2 K, c: Ref34 I = 0.1 mol dm-3, 298.2 K, d & e: Ref35, 298.2 K
(determined by 1H NMR titration).
Table 2.2. pKas for the ligands tacn and Me3tacn determined by potentiometric titration at 298.2
K in aqueous NaClO4 (I = 0.1 mol dm-3). Analogous literature values determined by
potentiometric titration in aqueous solution are also shown (conditions indicated).
A comparison of the pKas characterising tren and Me6tren (Table 2.1) shows those of Me6tren
to be less than those for tren. This reflects the stronger basicity of the amine nitrogens of the non-
methylated ligand tren over those of the hexa-methylated ligand Me6tren. Inductive effects dictate
that the basic nature of amines should follow this trend of decreasing basicity: tertiary amine
Me3N > secondary amine Me2NH > primary amine MeNH2 as has been observed in the gas
phase.36 However, solvent and steric effects may alter this trend in aqueous phase experiments.36,
37
Interaction of the Lewis base nitrogen lone pair with the Lewis acid proton is hindered by the
presence of bulky substituents, as is the ability of solvent water to hydrogen bond to the
positively charged conjugate acid, N-H+. Consequently N-methylation of the tren amine
substituents to give Me6tren is predicted to decrease the basicity of the amine groups and
decrease their pKas and this was observed for protonated Me6tren in this study.
45
11.5
10.5
9.5
8.5
7.5 Observed
pH
6.5 Fit
5.5
4.5
3.5
2.5
0.67 0.77 0.87 0.97 1.07 1.17 1.27
Titre (mL)
Figure 2.2. Potentiometric titration curve for the determination of Me6tren ligand pKas –
observed points (◊) and fit (-), resulting in the values reported in Table 2.1 (‘goodness of fit’, σ =
1.1). Aqueous NaClO4 (I = 0.10 mol dm-3), 298.2 K. The initial [Me6tren] = 1.0 × 10-3 mol dm-3,
the added [HClO4] = 1.0 × 10-2 mol dm-3, and the titrant [NaOH] = 0.1 mol dm-3.
100
90
% speciation relative to [L]total
80 free L
70
LH⁺
60
50 LH₂²⁺
40
30 LH₃³⁺
20
LH₄⁴⁺
10
0
3 5 7 9 11
pH
Figure 2.3. Speciation plot showing the variation of the protonation of Me6tren with pH, relative
to the total concentration of all Me6tren and its protonated species being equivalent to 100%.
Me6trenH44+ (LH4 4+
), Me6trenH33+ (LH3 3+
), Me6trenH22+ (LH2 2+
), Me6trenH+ (LH+) and
Me6tren (free L).
46
The pKas of tacn and Me3tacn determined in this study are also in good agreement with those
reported in the literature (Table 2.2). The larger pKa1 values of tacn (10.47) and Me3tacn (11.96),
as compared with those of tren (10.21) and Me6tren (9.40), typify the acid-base chemistry of the
polyazamacrocycles. Generally, due to the restricted polyazamacrocyclic conformation, nitrogen
lone pairs may overlap to produce a region of unusually high electron density in the macrocyclic
cavity and thereby increase the electrostatic attraction for the first proton.38 This is illustrated by
comparison of tacn with the analogous tridentate linear ligand, diethylenetriamine. The pKa1 for
monoprotonated diethylenetriamine is 9.70 while that for monoprotonated tacn is 10.47,
consistent with basicity increasing on cyclisation.35
Unlike for the comparison between tren and Me6tren, the predicted trend of basicity (and
therefore pKa) decrease on methylation of tacn to give Me3tren is not observed. This is due to the
lowest energy conformations of tacn and Me3tacn that are imposed by the cyclic ligand structure,
pre- and post-protonation.39 Figure 2.4 displays the symmetrical conformation of tacn (a), the
lowest energy tacn conformation (b), the mono-protonated tacnH+ conformation (c), the lowest
energy Me3tacn conformation (d), and finally the mono-protonated Me3tacnH+ conformation as
deduced from gas phase molecular mechanics (MM) calculations.40 The lowest energy
conformation of tacn (b) involves one of the amine hydrogens (prior to protonation) pointing into
the cavity of the macrocycle. In this arrangement it can hydrogen bond to the two remaining ring
nitrogens. Thus, when the first protonation takes place the proton must be added to the outside of
the ring (c) and no new amine hydrogen bonds result.
Due to the steric bulk of the methyl groups, the lowest energy conformation of Me3tacn (d)
does not contain a cavity-directed hydrogen. Consequently the first protonation most likely
involves a proton addition into the ring cavity whereby hydrogen bonding with the remaining
ring nitrogens occurs. This model is supported by the crystal structure of the mono-protonated
Me3tacnH+ perchlorate salt, which contains a proton directed into the macrocycle cavity.41 The
drop in strain energy that accompanies the protonation step for Me3tacn is responsible for its
otherwise anomalously high pKa1. This protonation trend may be extrapolated to all N-
functionalised tacn derivatives and indeed the characteristic of high first protonation constant
relative to tacn is a common feature of these ligands.32, 40
47
Figure 2.4. The molecular mechanics (MM) generated structures of the free ligands tacn (a, b
and c) and Me3tacn (d and e) taken directly from reference 40. Structures a and b depict tacn
with a hydrogen pointing both outside (symmetrical, higher energy conformation a) and inside
(asymmetrical, lower energy conformation b) the ring cavity. Structure c is protonated tacn,
tacnH+. Structures d and e show Me3tacn and protonated Me3tacn, Me3tacnH+. The
conformational energies shown have been converted from kcal mol-1 in reference 40
to kJ mol-1
here.
The pKa3 values determined for tacnH33+ and Me3tacnH+ in this study by potentiometric
titration are larger than (by more than triple) two of the literature pKa3 values reported in Table
2.2 (literature d and e). However, the latter two pKa3 values were determined using 1H NMR
spectroscopy and variations of ionic strength in these studies may have affected the pKa3 values
obtained.35
A typical titration curve is shown for tacn in Figure 2.5 and the characterisation of the tacn
system resulting from the fit for this pH variation data was used to produce the speciation plot in
Figure 2.6.
48
11.5
10.5
9.5
8.5
7.5 Observed
pH
6.5 Fit
5.5
4.5
3.5
2.5
0.62 0.72 0.82 0.92 1.02 1.12
Titre (mL)
Figure 2.5. Potentiometric titration curve for the determination of tacn ligand pKas in aqueous
NaClO4 (I = 0.10 mol dm-3) at 298.2 K – observed points (◊) and fit (-), resulting in the values
reported in Table 2.2. The initial [tacn] = 1.0 × 10-3 mol dm-3, the added [HClO4] = 1.0 × 10-2
mol dm-3, and the titrant [NaOH] = 0.1 mol dm-3.
This plot shows that the mono- and di-protonated tacn ligands (LH+ and LH22+) are the
predominant species in solution over the majority of the titration range. Although the “observed”
data points and “fit” agree well in Figure 2.5, it can be seen that the pH regions where the points
do not completely overlay are approximately 4.5 and 8. With reference to Figure 2.6, it is
apparent that these two regions are those in which tacnH22+ and tacnH+ are in great excess.
Conventionally (and if required to achieve the fit) titration data points may be ignored in the fit if
any species is present at 95% formation or above at those points.42 This is because small changes
in species concentrations cannot be detected when one species is in great excess and
consequently the data may be less reliable over these ranges of the curve.
49
100
90

80 free L
70
60
LH⁺
50
40
LH₂²⁺
30
20
LH₃³⁺
10
0
3 5 7 9 11
pH
Figure 2.6. Speciation plot showing the variation of the protonation of Me3tacn with pH, relative
to the total concentration of all Me3tacn and its protonated species being equivalent to 100%
speciation. Me3tacnH33+ (LH3 3+), Me3tacnH22+ (LH2 2+), Me3tacnH+ (LH+) and Me3tacn (free L).
Overall the pKa values determined in this study for all four ligands agree well with those
reported in the literature. Consequently, they were incorporated into the data fitting for the
stability constants of their Zn2+, Cu2+, Ni2+ and Cd2+ complexes.
2.2.2 Tren and Me6tren metal complex characterisation

Complex formation of the ligands 1 and 3 with the Zn2+, Cu2+, Ni2+ and Cd2+ was
characterised by potentiometric titrations of solutions containing [L]total to [M2+]total ratios of both
1:1 and 1:1.5. The magnitudes of the stability constants and the corresponding speciations
derived from both sets of data were compared in order to ensure that all states of protonation of
both the free ligands and the metal complexes had been accounted for in the data analyses. The
experiments were carried out in duplicate at 298.2 K in aqueous NaClO4 (I = 0.10 mol dm-3) as
described in Section 8.2.2 and the data reported represents the average of two titrations.
In principle there are several permutations of [M(LHn)](2+n)+ complexes that may form during
a titration and not all are necessarily present for each concentration ratio of LHnn+ to M2+ or each
combination of specific L and M2+.42, 43 However, the species incorporated into the equilibria in
50
the algorithms used to best fit the experimental pH variation of the titration are greatly influenced
by the stereochemistry, denticity and pKas of LHnn+, and the coordination number range,
preferred stereochemistry and ionic radius of M2+.
Tren and Me6tren are tripodal, tetradentate ligands. Therefore, the complexes they form may
have geometries resulting from a coordination number of four or higher, depending on the
preferred coordination numbers of M2+ and the identities of other ligands present. Both ligands
form 1:1 [M(L)]2+ complexes with the first row transition metal ions Zn2+, Cu2+ and Ni2+ as well
as the second row metal ion Cd2+. Studies of these [M(L)]2+ complexes reveal them to be five-
coordinate with a distorted trigonal bipyramidal geometry (as shown in Figure 2.1).3, 4, 9, 10, 44 The
logK1 stability constant for the reaction and equilibrium constant expression are described by
Equation 2.5, and the results from this study are shown in Table 2.3 along with analogous
literature values.
K1
L + M2+ ⇌ [M(L)]2+ where K1 = [M(L)2+]/([L][M2+]) 2.5
The measured logK1 values for tren in this study match closely with those in the literature,
particularly for the metals Zn2+, Cu2+ and Ni2+. However, logK1 for [Cd(tren)]2+ is slightly lower
than the reported values. In the study d27 (Table 2.3) the logK1 values are consistently higher than
the other literature values and those determined in this study. This is most likely due to the ten-
fold increase in the ionic strength, I = 1.0 mol dm-3 (NaClO4) in that citation. It was also noted on
analysis of the tren/Cd2+ titrations in this study that there is evidence of slow equilibria as
indicated by the electrode not stabilising on addition of NaOH titrant, even after the maximum
wait time of 5 minutes had elapsed. Although it is not evident from the shape of the
potentiometric titration curves, this occurred over two portions of the titrations in the pH range
3.5 – 9.5. Reference d reports the collection of 80 – 100 data points for each titration which may
compromise the results if some of these are unreliable due to the slow equilibrium processes. The
titrations performed to produce the results reported in this thesis collected between 400 and 500
data points per titration.
51
Complex Ligand Zn2+ Cu2+ Ni2+ Cd2+
(L) logK1 logK1 logK1 logK1

This
[M(L)]2+ tren 14.50 ± 0.007 18.92 ± 0.008 14.32 ± 0.02 11.83 ± 0.01
study
a 14.6 18.8 14.8 12.3
b 14.5 18.5 14.6
c 14.65 18.8 14.8
d 15 19.58 14.95 12.35
This
Me6tren 8.75 ± 0.01 14.75 ± 0.008 5.95 ± 0.03 6.08 ± 0.02
study
e 9.75 15.65 7.14 7.32
f 9.68
g 15.43
a: Ref46, I = 0.1 mol dm-3 (NaCl), 293.2 K, b: Ref31, I = 0.1 mol dm-3 (NaClO4), 298.2 K, c: Ref47, I = 0.5 mol
dm-3
(KCl), 299.0 K, d & e: Ref27, I = 1.0 mol dm-3 (NaClO4), 298.2 K, f: Ref30, I = 1.0 mol dm-3 (NaClO4), 298.2 K,
g: Ref33, I = 0.15 mol dm-3 (NaCl), 298.2 K.
Table 2.3. Potentiometrically determined logK1 values for the [M(L)] 2+ complexes of tren and
Me6tren formed with Zn2+, Cu2+, Ni2+ and Cd2+ at 298.2 K in aqueous NaClO4 (I = 0.10 mol dm-
3
) in this study. Literature values determined by potentiometric titrations in aqueous solution
under the conditions indicated are also shown.
The size of logK1 for the [M(L)]2+ complexes when L = tren varies with the M2+ in the
sequence: Cu2+ > Zn2+ > Ni2+ > Cd2+. The lowest logK1 value for complexation with Cd2+ is
likely to be due to the increased ionic radius of this second row transition metal ion by
comparison with its first row counterparts. The coordination of the pendant arms of tren and
Me6tren within the equatorial plane of a trigonal bipyramidal or octahedral geometry is probably
more difficult with a larger ionic radius. The remaining trend for Cu2+, Zn2+ and Ni2+ follows the
Irving-Williams Series.45
The logK1 values for the [M(L)]2+ complexes of Me6tren follow a similar trend. However two
differences are immediately noticeable. Firstly, the stability constants when L = Me6tren are all
lower by several orders of magnitude than those when L = tren. This is attributable to steric
52
effects whereby the methyl groups hinder the coordination of the nitrogen lone pairs to the metal
centre.48, 49
This has been elegantly illustrated by the measurement of the logK1 of [Cd(L)]2+
where L = tren, Me3tren (each pendent nitrogen is mono-methylated) and Me6tren in DMSO.29
The [M(L)]2+ logK1 values decrease sequentially as the methylation of the tren ligand increases:
12.32, 9.87 and finally 4.18 (note the largest decrease occurs upon complete methylation).
Furthermore, the higher basicity of tren (29.71) relative to that of Me6tren (27.08) as measured by
the sum of the logarithms of their stepwise protonation constants (shown in parentheses) suggests
the formation of more stable [M(L)]2+ when L = tren.
Secondly, [M(L)]2+ when L = Me6tren and M2+ = Ni2+, has a particularly low logK1 (smaller
than that of when M2+ = Cd2+). It is hypothesised that the overall logK1 trend for [M(L)]2+ when L
is either tren or Me6tren is significantly influenced by the ability of M2+ to adopt a coordination
number of five.27 The decrease of logK1 on going from tren to Me6tren for the [M(L)]2+
complexes is: Cu2+ 4.17 < Zn2+ 5.75 = Cd2+ 5.75 < Ni2+ 8.41. As reported, the decrease in
stability between tren and Me6tren complexes, [M(L)]2+, is twice as large when M2+ = Ni2+ than
when M2+ = Cu2+. This may suggest that whilst Cu2+ readily forms five-coordinate, trigonal
bipyramidal complexes even with a bulkier ligand, Zn2+ and Cd2+ are less capable of this and Ni2+
is the least able of all. Whilst flexibility of coordination number is well-documented for Cu2+,
Zn2+ and Cd2+,50-52 octahedral coordination is more prevalent for Ni2+.53 This appears to be
reflected in the relatively low logK1 of [Ni(L)]2+ by comparison with logK1 for its Cu2+, Zn2+ and
Cd2+ analogues when L = Me6tren, and this probably signals greater steric strain in five-
coordinate [Ni(Me6tren)H2O]2+.
All of the logK1 values for the Me6tren complexes determined in this study are lower than the
values reported in the literature. However, none of the literature values were obtained under the
same experimental conditions (ionic strength and supporting electrolyte) as those of this study.
As discussed in Section 2.1.2, such differences can significantly affect the stability of [M(L)]2+
complexes. The protonated ligand pKa values used in the fitting agree with literature values
obtained under similar conditions and the trend in [M(L)]2+ stabilities obtained in this study is in
agreement with the literature.
Early studies of the tren and Me6tren complexes showed them to be of the form [M(tren)X]m+
and [M(Me6tren)X]m+, respectively, where M2+ is a transition metal ion, X is a monodentate
ligand and m+ is the overall complex charge depending upon the charge of X. With this
53
stoichiometry it is possible that these complexes may be four-coordinate or five-coordinate

depending on the denticity of tren and Me6tren. It is known that tren may act as both a tridentate
or tetradentate ligand.29, 54, 55 (When a multidentate ligand binds to a metal ion with fewer than its
maximum number of donor atoms this is sometimes referred to as hypodenticity.56) X-ray
crystallography and solution experiments of their complexes suggest that tren and Me6tren act as
tetradentate ligands with the monodentate X occupying the fifth coordination site in a five-
coordinate complex.3, 11, 57, 58
However, over the broad pH range investigated during a
potentiometric titration it may be possible to observe other species. Crystals for X-ray study are
grown under a specific set of conditions (at a given pH) and UV-Vis solution studies are often
carried out at a pH where protonation of a coordinated tren or Me6tren is unlikely. However,
when complex formation is studied potentiometrically over a pH range of 2 – 11, there is
evidence that both protonated tren and Me6tren act as hypodentate ligands.27, 55 Evidence for such
species is adduced from the systematic pH variation observed in the potentiometric titration data,
and their occurrence is intuitively reasonable. When all four amine groups of tren and Me6tren
are deprotonated, these ligands are likely to exhibit tetradenticity, but it is conceivable that
complex formation may occur before the all of the amine donor atoms are deprotonated. An
example of such a complex is displayed in Figure 2.7 for Zn2+, where it can be seen that the
uncoordinated and protonated arm is not in close proximity to Zn2+ in the postulated five-
coordinated [Zn(trenH)(H2O)2]3+ complex. Without the fourth nitrogen attached, it may be
hypothesised that a second aqua ligand coordinates to preserve trigonal bipyramidal geometry in
[Zn(trenH)(H2O)2]3+. (There is some spectrophotometric evidence that the trenH+ complex with
Cu2+ is square pyramidal, however there are large errors associated with this analysis because this
complex was only a minor species in solution.55 Further investigation would be required as part
of Future Work for the precise geometries of these complexes to be confirmed.) The
corresponding equilibrium for the formation of the general [M(LH)]3+, (where only the LH+
ligand is shown irrespective of the occupancy of the other coordination sites on M2+), along with
the stability constant expression are shown in Equation 2.6. The [M(LH)]3+ complexes of the
mono-protonated ligands are assigned the stability constant K1′, and the corresponding logK1′.
54
Figure 2.7. Mono-protonated trenH+ acting as a hypodentate ligand in a trigonal bipyramidal

complex with Zn2+: [Zn(trenH)(H2O)2] 3+.
The logK1′ values for the [M(LH)]3+ complexes determined in this study as the average of two
sets of duplicated potentiometric titrations of solutions in which the ratio of [L]total to [M2+]total
was 1:1 appear in Table 2.4 along with the corresponding literature values.
K1′
LH+ + M2+ ⇌ [M(LH)]3+ where K1’ = [M(LH)3+]/([LH+][M2+]) 2.6
A speciation plot for the tren/Cu2+ system that is representative of the [M(LH)]3+ species
formation is shown in Figure 2.8 based on the constants derived from the fit of the titration data
seen in Figure 2.9. It is seen that [M(LH)]3+ is present over the pH range ~3 to 6. As the pH
increases and the trenH+ is deprotonated, the [M(LH)]3+ complex is replaced by the far more
stable [ML]2+ complex. As the [ML]2+ complex reaches close to 100% formation, the [MLOH]+
hydroxide species begins to form (approximately pH 6.5 and titre = 0.9705 mL in Figures 2.8 and
2.9) and the [MLH]3+ species is no longer present. It is seen from Table 2.4 that the logK1′ values
determined in this study are similar to those reported in the literature despite the differences in
ionic strength concentration. However, no logK1’ values appear in the literature for [Zn(trenH)]3+
and [Ni(trenH)]3+.27 Their inclusion in the models for titration data in this study consistently
results in a better fit and therefore these species cannot be ignored.
55
(L) logK1' logK1' logK1' logK1'
[M(LH)]3+ tren This study 9.13 ± 0.07 12.74 ± 0.02 9.68 ± 0.02 7.56 ± 0.07
a 13.22 7.56
b 12.74
Me6tren This study 5.43 ± 0.07 9.70 ± 0.03 3.82 ± 0.06
c 5.31 9.53 3.77
a & c: Ref27, I = 1.0 mol dm-3 (NaClO4), 298.2 K b: Ref55, I = 0.15 mol dm-3 (NaClO4), 298.2 K.
Table 2.4. Potentiometrically determined logK1′ for the mono-protonated ligand complexes
[M(trenH)] 3+ and [M(Me6trenH)] 3+ formed with Zn2+, Cu2+, Ni2+ and Cd2+ at 298.2 K in
aqueous NaClO4 (I = 0.10 mol dm-3). Literature values determined by potentiometric titrations in
aqueous solution under the conditions indicated are also shown.
100
90
free L
80
LH⁺
70
LH₂²⁺
60
LH₃³⁺
50
LH₄⁴⁺
40
[MLH]³⁺
30
[ML]²⁺
20
[M(L)(OH)]⁺
10
[M(L)(OH)₂]
0
3 4 5 6 7 8 9 10 11
pH
Figure 2.8. Speciation plot showing the variation of the protonation of tren and the formation of
its Cu2+ complexes as pH is varied relative to the total concentration of all tren and its
protonated and complexed forms being equivalent to 100% speciation: trenH44+ (LH44+),
trenH33+ (LH33+), trenH22+ (LH22+), trenH+ (LH+), tren (free L), complex [Cu(trenH)] 3+
([MLH] +), complex [Cu(tren)] 2+ ([ML] 2+), complex [Cu(tren)(OH)] + ([ML(OH)] +) and complex
[Cu(tren)(OH)2] ([ML(OH)2]).
56
In every case, the logK1′ values for [M(LH)]3+ are smaller than those of the corresponding
logK1 values for the analogous [M(L)]2+ complexes. This is likely due to the electrostatic
repulsion between LH+ and M2+ lessening the overall ligand to metal ion binding attraction in
[M(LH)]3+ by comparison with that in [M(L)]2+.
11.5
10.5
9.5
8.5
7.5 Observed
pH
6.5 Fit
5.5
4.5
3.5
2.5
0.57 0.67 0.77 0.87 0.97 1.07 1.17 1.27
Titre (mL)
Figure 2.9. Potentiometric titration curve for the characterisation of the species present in the
tren/Cu2+ system in aqueous NaClO4 (I = 0.10 mol dm-3) at 298.2 K – titration points (◊) and fit
(-), resulting in the values reported in Tables 2.3, 2.4 and 2.5. The initial [tren] = 1.0 × 10-3 mol
dm-3 and [Cu2+] = 1.0 × 10-3 mol dm-3, the added [HClO4] = 1.0 × 10-2 mol dm-3, and the titrant
[NaOH] = 0.10 mol dm-3.
The [M(trenH)]3+ logK1′ and the [M(tren)]2+ logK1 values increase in the order: M2+ = Cu2+ >
Ni2+ ↔ Zn2+ > Cd2+ with those for Ni2+ and Zn2+ being quite similar for both complexes, and the
overall trend in complex stability is broadly in accord with the Irving-Williams Series (Ni2+ <
Cu2+ > Zn2+). It is unclear why the [Ni(trenH)] 3+ and [Zn(trenH)]3+ complexes have not been
reported in the literature. The [M(Me6trenH)]3+ logK1′ and the [M(Me6tren)]2+ logK1 increase in
the sequence: M2+ = Cu2+ > Zn2+ > Ni2+ both consistent with the literature as is the absence of
[Cd(Me6trenH)]3+. The absence of [Cd(Me6trenH)]3+ at detectable levels may be attributable to
the large size of Cd2+ relative to Zn2+ (both are d10 metal ions) coupled with the steric bulk of the
57
methyl groups bringing the protonated amine in [Cd(Me6trenH)]3+ into close proximity to the
Cd2+ surface and thereby increasing electrostatic repulsion.
The final complexes to be considered in the characterisation of these metal complexes are
[M(L)(OH)]+ in which an aqua ligand is deprotonated to give a nucleophilic hydroxo ligand, and
which are central to this study. This deprotonation is characterised by KaH2O in Equation 2.7.
Thus, the [M(L)(H2O)]2+ complex is a five-coordinate, trigonal bipyramidal complex with either
one tetradentate tren or Me6tren ligand and one aqua ligand present which at the appropriate pH
deprotonates to give [M(L)(OH)]+.
KaH2O
[M(L)(H2O)]2+ ⇌ [M(L)(OH)]+ + H+ where KaH2O = [M(L)(OH)+][H+]/([M(L)(H2O)2+]) 2.7
Interestingly, although all of the metal ions considered in this study can in principle form
octahedral complexes and bind two aqua ligands, the UV-Vis absorbance spectra of the tren and
Me6tren complexes in solution are usually consistent with a trigonal bipyramidal structure.50, 57-59
This trigonal bipyramidal geometry is also found in the solid-state as exemplified by a the X-ray
crystallographic studies of [Cu(tren)(H2O)]2+ and [Zn(tren)X]2+ where X = H2O, Cl-, NCS-).8,11,51
The axial M2+-OH2 bond in [M(L)(H2O)]2+ is shorter than the axial M2+-N(tertiary) bond in
[M(tren)(H2O)]2+ and [M(Me6tren)(H2O)]2+. This results in substantial polarization of the M2+-
OH2 bond and a consequently increased acidity of the aqua ligand.
The pKaH2O values of the aqua ligands of [M(tren)(H2O)]2+ and [M(Me6tren)(H2O)]2+

determined in this study together with those from the literature are shown in Table 2.5 along with
the corresponding first pKaH2O of the corresponding [M(H2O)6]2+ complexes. (The aqua ligand
deprotonations for hexa-aqua metal complexes, [M(H2O)6]2+, may be determined by
potentiometric titration in exactly the same way as the analogous values for [M(L)(H2O)]2+
complexes. They are also required information for the fitting of data when analysing the titrations
of L/M2+ systems because they explain the acid-base chemistry of free, uncomplexed metal that
may be present if the stability of the [ML]2+ species is not strong.)
The pKaH2O values of the [M(tren)(H2O)]2+ and [M(Me6tren)(H2O)]2+ complexes follow the
trends of those reported in the literature although they are slightly lower than the literature values
in some cases. However, only one of the literature studies31 was carried out under the same
58
conditions as in this study and congruently the reported pKaH2O = 9.17 for [Cu(tren)(H2O)]2+ is
the closest match to any of the values determined in this study: 9.11. Contrarily, this source also
quotes a pKaH2O = 9.80 for [Ni(tren)(H2O)]2+ whilst two other studies24, 27 and this study show this
pKaH2O is >11 – thus the value of 9.80 appears to be anomalous.
(L) pKaH2O pKaH2O pKaH2O pKaH2O

This
[M(L)(OH)]+ tren 9.97 ± 0.02 9.11 ± 0.02 11.37 ± 0.02 10.21 ± 0.03
study
a 10.4 9.4 >11 10.9
b 10.26 9.73 >11
c 9.17 9.80
d 10.68
This
Me6tren 8.61 ± 0.02 8.40 ± 0.03 8.81 ± 0.03 8.45 ± 0.03
study
e 8.95 8.1 9.6 8.6
f 9.00 8.52 9.53
g 8.86
This
[M(H2O)5(OH)]+ H2 O
study 8.97 8.16
h 9.86 10.08
a & e: Ref27, I = 1.0 mol dm-3 (NaClO4, 298.2 K, b & f: Ref24, I = 0.1 mol dm-3 (NaClO4), 298.2 K, c: Ref31, I =
0.1 mol dm-3 (NaClO4), 298.2 K, d & g: Ref30, h: Ref60, I = 0.1 mol dm-3, 298.2K.
Table 2.5. Potentiometrically determined aqua ligand pKaH2O values for [M(tren)(H2O)] 2+,
[M(Me6tren)(H2O)] 2+ and [M(H2O)6] 2+ in aqueous NaClO4 (I = 0.10 mol dm-3) at 298.2 K.
Literature values determined by potentiometric titrations in aqueous solution under the
conditions indicated are also shown.
The [Cu(tren)(H2O)]2+ and [Cu(Me6tren)(H2O)]2+ complexes are characterised by the lowest

pKaH2O in each series which may be a consequence of the Cu2+ d9 electronic configuration which
causes tetragonal Jahn-Teller distortions in six-coordinate Cu2+ complexes.61 This may indicate
59
that the Cu2+ d9 electronic configuration may cause a shortening of the Cu2+-OH2 bond and
therefore greater polarisation of the aqua ligand in five-coordinate [Cu(tren)(H2O)]2+ and
[Cu(Me6tren)(H2O)]2+ with a corresponding decrease in pKaH2O. Interestingly, a previous study of
[M(tren)(H2O)]2+ and [M(Me6tren)(H2O)]2+ where M2+ = Co2+, Ni2+, Cu2+ and Zn2+ suggests that
the M2+–OH2 bond distance is the key factor determining the trend in pKaH2O and that this is
dependent on the identity of the M2+.24 For the similar complex [M(Me6tren)Br]+ where M =
Ni2+, Cu2+ and Zn2+ the trend in M–Brˉ bond distance is Cu2+ < Zn2+ < Ni2+ which may be
explained in terms of the differing d-orbital occupancy of the M2+.59 Characterisation of similar
variations in the M2+-OH2 distances in [M(tren)(H2O)]2+ and [M(Me6tren)(H2O)]2+ would
determine if there was a similar trend in these distances, the relative extent of aqua ligand
polarization and the corresponding values of the associated pKaH2Os.
The aqua ligand pKaH2O values of the [M(Me6tren)(H2O)]2+ complexes listed in Table 2.5 are
lower than those of the corresponding [M(tren)(H2O)]2+ complexes. This is in accord with the
theory that the hydrophobic pocket in carbonic anhydrase contributes towards lowering the
pKaH2O of the aqua ligand of Zn2+ at the active site; and that this effect is reproducible by simple
mimicry as was first published in Nature in 1974.24 In each case, methylation of the tren amine
groups of [M(tren)(H2O)]2+ increases the hydrophobicity of the aqua ligand environment in
[M(Me6tren)(H2O)]2+ and results in substantial decreases in log KaH2O corresponding to a decrease
in KaH2O five to > 500 fold. Whilst the aqua ligand pKaH2O values for [M(tren)(H2O)]2+ are higher
than those of the first pKaH2O values of the corresponding [M(H2O)6]2+ (Table 2.5), the pKaH2O
values of [M(Me6tren)(H2O)]2+ where M2+ = Zn2+, Ni2+ and Cd2+ are smaller than the pKaH2O
values of the corresponding [M(H2O)6]2+. The Cu2+ complexes are the exception to this
observation.
There is also some literature evidence for the tren/Cu2+ system that these hydroxide species
may dimerise in solution.22, 62
Once the water ligand has been deprotonated, a pair of these
complexes bonds together with a central square arrangement of the two copper cations bridged by
two hydroxo ligands. This dimer species was added to the fitting models for the tren complexes
but it resulted in either excessive errors or an overall failure to fit.
Although the literature most often references the five coordinate, trigonal bipyramidal
complexes with only one water ligand present, all of the metals in this study are capable of
forming stable octahedral complexes. Therefore, to be comprehensive, a second aqua ligand
60
pKaH2O was added to the fits for all titrations (pKaH2O#2). As expected (from the lack of presence in
the literature and as predicted due to the bulk of Me6tren), this species could not be made to fit to
any of the Me6tren titration data. Space filling models indeed show the metal centre to be very
crowded when coordinated to hexa-methyl tren.24 Similarly, the titrations of tren with Zn2+, Ni2+
and Cd2+ also did not support the occurrence of a second aqua deprotonation.
However, for the tren/Cu2+ system, the presence of a second aqua ligand that undergoes
deprotonation did improve the fit for both duplicate runs of the 1:1 and 1:1.5 L:M2+ solutions (ie
in all four titrations for this system). The complex formed is [Cu(tren)(H2O)2] and the determined
pKaH2O#2 for the second deprotonation was 11.61 ± 0.03. This di-aquo, octahedral geometry for
tren/Cu2+ is supported when investigating the enthalpy and entropy changes associated with the
formation of this complex. There is a lower than average enthalpy change associated with the
formation of [Cu(tren)]2+ and this value and the corresponding entropy change are much closer to
those of similar octahedral complexes.63 The tren/Ni2+ system is also proposed to favour six-
coordinate stereochemistry with two aqua ligands,24, 63
but with a first aqua deprotonation
measured at >11, the second is definitely outside of the range possible for potentiometric
titration.
There is also some evidence of these octahedral di-aquo species in the βCD-containing
complex analogues which is discussed in Chapters 3 and 4. The question of complex geometry
(and therefore number of potentially acidic aqua ligands present) is a relevant one when
considering both data fitting and future kinetics work. This factor may have an impact on the
catalyst efficiency and hydrolysis mechanism10, 11, 22
and therefore must be investigated
thoroughly.
Finally, it should also be noted that there is literature evidence for the formation of two 2:1
complexes ([M(L)2]2+) – in the tren/Ni2+ system54 and the tren/Cd2+ system29 (Me6tren is too
bulky a ligand to allow these species to form). Both of these complexes would almost certainly
require hypodenticity of both tren ligands in order to keep a stable geometry. However, neither of
these species could be fit with the titration data obtained in this study. This may be explained by
the different conditions used to form these complexes. The [Ni(tren)2]2+ complex in the literature
was prepared as a solid with Cl¯ counter ions from ethanol for crystallographic analysis (ie not
characterised by potentiometric titration). Both the solvent used and the counter ion (which differ
between this literature source and the work conducted in this study) could affect the formation of
61
this species. The [Cd(tren)2]2+ species is characterised by potentiometric titration and has a
reported logK2 stability constant of 5.07. However the solvent used is DMSO and there are no
published values for the equivalent species in water.
2.2.3 Tacn and Me3tacn metal complex characterisation

Complex formation of the ligands 2 and 4 (Figure 1.9) with the Zn2+, Cu2+, Ni2+ and Cd2+ was
characterised by potentiometric titrations of solutions containing [L]total to [M2+]total ratios of
either 1:1 or 1:1.5. The magnitudes of the stability constants and the corresponding speciations
derived from both sets of data were compared in order to ensure that all states of protonation of
both the free ligands and the metal complexes had been accounted for in the data analyses. Each
experiment was carried out in duplicate at 298.2 K in aqueous NaClO4 (I = 0.10 mol dm-3) as
described in Section 8.2.2 and the data reported represents the average of two titrations.
Tacn and Me3tacn are tridentate macrocyclic ligands that coordinate facially to the metal
centre.16, 17 They may potentially form both 1:1 ([M(L)]2+) and 2:1 (bis – [M(L)2]2+) complexes
containing a probable maximum octahedral geometry. However, the literature suggests that
whilst tacn is capable of forming both these complex species, its methylated derivate Me3tacn is
too bulky for the bis-ligand geometry.28, 64 The equation that describes the formation of the 1:1
species is shown in Equation 2.5 and the results for tacn and Me3tacn are shown in Table 2.6
along with the analogous literature values.
The values obtained in this study correspond well to those reported in the literature. There are
several discrepancies about the existence of species, however, found between this study and the
literature and also within the literature itself. These are addressed below, but when this study and
the literature are in agreement about the presence of a species, the size of the corresponding
stability constant matches well.
For each of the four metals there is debate in the literature about the stability constants for the
[M(L)2]2+ bis complexes. Indeed, the Cu2+ and Ni2+ complexes have been synthesised,16, 64 but not
all potentiometric experiments have been able to yield the logK2 stability constant values for
these bis-ligand species. (This is not surprising as the specific temperature, solvent, pH, reaction
time and concentration conditions required for complex preparation are not necessarily met
during the course of a titration.) In fact, the majority of potentiometric experiments report only
62
the formation constant for the [ML]2+ species and not the second [M(L)2]2+ bis-ligand species.39,
64
Interestingly, when these logK2 formation constants are reported they tend to be only 1-2 log
units smaller than the logK1 values, indicating quite stable complexes. However, the same studies
that report the bis-ligand complex stability constants also omit the formation of the corresponding
complex hydroxides (see Figures 2.8 and 2.9 for the speciation plot and titration curve involving
these complex hydroxide species for the tren/Cu2+ system). This is problematic because the
hydroxide species (eg. [ML(OH)]+) definitely form during the course of a titration and tend to
dominate the second half of the titration curves. Their absence from these studies raises questions
about the fitting models used.

This
[M(L)]2+ tacn 11.58 ± 0.02 15.00 ± 0.03 11.06 ± 0.01 9.53 ± 0.05
study
a 11.7 15.1 13.6 9.5
b 11.3 15.4 12.4 9.5
c 11.62 15.52 16.24*
This
Me3tacn 9.74 ± 0.03 14.01 ± 0.01 6.46 ± 0.06 9.07 ± 0.04
study
d 12.3*
e 7.51
a: Ref13, I = 0.1 mol dm-3 (KNO3), 298.2 K, b: Ref34 (conditions not listed), c: Ref16, I = 0.1 mol dm-3 (KNO3),
298.2 K, d: Ref65, I = 0.1 mol dm-3 (NaClO4), 298.2 K, e: Ref28, I = 0.1 mol dm-3 (NEt4ClO4), DMSO solvent
system. *“Out of cell” method used for determination due to slow equilibria.
Table 2.6. Potentiometrically determined logK1 values for the [M(L)] 2+ complexes of tacn and
Me3tacn formed with Zn2+, Cu2+, Ni2+ and Cd2+ at 298.2 K in aqueous NaClO4 (I = 0.1 mol dm-3)
in this study. Literature values determined by potentiometric titrations in aqueous solution under
the conditions indicated are also shown.
An excellent illustration of this is the 1976 study by Yang and Zompa.16 Bis tacn complexes
of both Cu2+ and Ni2+ ([Cu(tacn)2]2+ and [Ni(tacn)2]2+) were synthesised, confirmed and analysed,
63
but only the [ML]2+ complexes along with the first aqua ligand deprotonation (e.g. [Cu(tacn)]2+
and [Cu(tacn)(OH)]+) could be observed as dominant species over the titration pH range. The
inconsistencies in the literature are noted.64 Consequently, species are only reported in this study
if they could be fit in every titration (duplicate runs for both 1:1 and 1:1.5 L:M 2+ concentration
ratios) and without an excessive error present in the Hyperquad fitting software.66
The measured logK1 stability constants for both tacn and Me3tacn again follow the same trend
predicted by the Irving-Williams series as for tren and Me6tren: Cu2+ > Zn2+ > Ni2+. Identical to
tren and Me6tren, the least stable complex is formed with cadmium (due to this ion’s larger
radius) until ligand methylation occurs and causes the [Ni(Me3tacn)]2+ complex to become the
least stable of that series.
Whilst the tacn and Me3tacn complexes with Cu2+ are still the most stable, they are less stable
than might be expected for a macrocycle when comparing them to other acyclic tridentate
ligands.16 The macrocylic effect18 does not appear to be as strong for the Cu2+ complexes as it
does for those of Zn2+, Ni2+ and Cd2+. This may be a result of the Cu2+ preference for meridional
coordination of a tridentate ligand coupled with the necessary facial coordination of these ligands
due to their small cyclic structure.41
Predictably, the logK1 stability constants for complexes with a specific M2+ decrease on going
from the tacn to the Me3tacn coordinating ligand. Following the same chemistry as tren and
Me6tren, the methyl groups destabilise the complexes by hindering the coordination of the
nitrogen lone pairs to the metal centre. Also, the basicity of tacn (19.82) relative to that of
Me3tacn (19.01) as measured by the sum of the logarithms of their stepwise protonation constants
(provided in parentheses) would suggest the formation of more stable complexes for this ligand.
For any given complex, the most discrepancies found in the literature are for [Ni(tacn)]2+ and
this is caused by the slow equilibria that occur during its titration. For the experiments in this
study the titration method for stability constant determination involved implementing a maximum
wait time between subsequent titrant additions by the automated burette (see Section 8.2.2). The
electrode-measured mV reading is recorded when it is stable (consistent within a designated mV
range drift – in this case 0.3 mV). The maximum wait time is the time at which the mV reading
will be recorded if stabilisation during that period has not been achieved. Overwhelmingly across
all titrations, this stability was achieved swiftly and most readings were recorded in less than 30s
after titrant addition, indicating that the equilibria were established quickly on addition of each
64
new aliquot of NaOH(aq). An initial maximum wait time of 300s was implemented – well above
the time required for most readings to stabilise.
However, on analysis of the tacn/Ni2+ titration data, between 11 and 17% of all data points
taken (>500) were recorded right on the maximum wait time. This means that these mV readings
were taken before equilibrium had been reached and are not a reflection of the final stable
speciation at each data point. Furthermore, these values occur in the pH range from ~4.2 – 9.4,
the central part of the curve. When the Me3tacn/Ni2+ system was investigated, the maximum wait
time was extended to 1200s, however the curves were still difficult to fit. The values reported are
the best fits achieved for these titrations.
Even with the smaller wait time of 300s these titrations took approximately 12 hours to
complete. Exceedingly long titrations are not ideal as the electrode may be exposed to precipitate
for long periods of time and its E0 may drift during this time. One study overcame the slow
equilibrium problem by performing an unconventional titration using an “out of cell” method.67
Yang and Zompa16 equilibrated separate solutions at the desired pH for each titration point for
100 hours before individual measurement. (Similar solutions were subsequently found to require
3 to 4 months for proper equilibration.68) This results in a substantially larger logK1 stability
constant (16.24) than those obtained in other studies (13.613 and 12.434) and in this thesis (11.06).
Whilst the out of cell method avoids inaccuracies arising from a long titration time for the
electrode and taking measurements before equilibrium has been reached, it introduces them in
having fewer data points and in producing and maintaining consistent titration solutions over
very long periods. Potentiometric titrations collect well over 500 data points for a single solution
and, though the literature reports do not specify, it is assumed that 500 individual solutions were
not prepared and tested. Consequently neither method is ideal but the slow equilibrium is a
barrier intrinsic to the experiment and cannot be avoided. However, if enough solutions are
prepared and maintained precisely, the out of cell method is likely to be more reliable.
Unlike for tren and Me6tren, no evidence of hypodenticity in the form of a mono-protonated
ligand binding to M2+ ([M(LH)]3+) was found for tacn and Me3tacn. The species [M(LH)]3+ could
not be made to fit the data consistently for any L-M2+ combination. This is anticipated because
unlike with the free, pendant arms of tren and its derivatives, the nitrogens of tacn and Me3tacn
are all held in close proximity to the coordinating metal cation. The repulsion that would occur
between the constrained protonated nitrogen and M2+ would stop any approach of the ligand and
65
metal cation and destabilise any complex that could form. This finding is supported by the
literature.14, 39
Aqua ligands present on these tacn and Me3tacn metal complexes also undergo an increase in
reactivity in the same way as those present in the tren and Me6tren metal complexes. Since tacn
and Me3tacn are tridentate ligands and do not exhibit hypodenticity, it is possible to have one,
two or three potentially acidic aqua ligands also coordinated. The resulting complexes would be
either four-coordinate, five-coordinate or octahedral in geometry. A consideration of the
preferred geometry of each metal was helpful in deciding how many of these hydrolysed species
might be suitable in the data fitting models. Furthermore, whilst it is possible to coordinate three
aqua ligands and produce a stable octahedral complex, the third water deprotonation would likely
be unfavourable as it would produce a third negatively charged hydroxo ligand and a negatively
charged complex. A third aqua deprotonation would also have to occur at very high pH for these
complexes, likely outside of the range the electrode could measure. Therefore testing of the fits
was limited to the [ML(OH)]+ and [ML(OH)2] hydroxide species. The formation of the first
hydroxo species is described by Equation 2.7 and the second is described by Equation 2.8 below.
The aqua deprotonation results from this study are reported in Table 2.7 along with the
corresponding literature values.
KaH2O#2
[M(L)(H2O)(OH)]+ ⇌ [M(L)(OH)2] + H+ where KaH2O#2 = [M(L)(OH)2][H+]/([M(L)(H2O)(OH)+])
2.8
It should be noted that the absence of a hydroxo species in the fit does not necessarily mean
the parent aqua ligand is not present in the geometry, only that it does not undergo a
deprotonation that is measureable during the range of the titration. An example of this is the
Me3tacn/Zn2+ system. The crystal structure of [Zn(Me3tacn)(H2O)3](NO3)2 reveals an octahedral
geometry for the [Zn(Me3tacn)(H2O)3]2+ with the tridentate Me3tacn and three monodentate aqua
ligands.23 However, titration experiments performed as part of the same study yield only two
aqua ligand pKaH2Os. With a second deprotonation reported at 12.3, the third would be measured
at a pH outside of the range of the titration if it did occur. The identification of hydroxo species
can therefore not be used to automatically assign the complex geometry.
66
(L) pKaH2O (1&2) pKaH2O (1&2) pKaH2O (1&2) pKaH2O (1&2)

[M(L)(OH)] This
+ tacn 8.90 ± 0.11 7.99 ± 0.04 11.66 ± 0.06 9.03 ± 0.03
study
a 7.3
b 7.9
Me3tac This
10.20 ± 0.09 7.85 ± 0.03 8.15 ± 0.20* 9.46 ± 0.05
n study
c 7.85
d 10.9
This
[M(L)(OH)2] tacn 11.35 ± 0.05
study
Me3tac This
11.30 ± 0.09 9.64 ± 0.03 12.03 ± 0.20* 10.92 ± 0.05
n study
e 12.3
a: Ref21, I = 0.1 mol dm-3 (NaClO4), 298.2 K, b: Ref13 I = 0.1 mol dm-3
(KNO3), 298.2 K, c: Ref22, I = 0.38 mol dm-3 (NaClO4), 323.2 K determined spectrophotometically,
d & e: Ref23, I = 0.1 mol dm-3 (NaNO3), 298.2 K. *Values reported despite fit resulting in
excessive error due to unavoidable slow equilibrium processes.
Table 2.7. Potentiometrically determined aqua ligand pKaH2O values for [M(tacn)(H2O)] 2+,
[M(Me3tacn)(H2O)] 2+, [M(tacn)(H2O)(OH)] + and [M(Me3tacn)(H2O)(OH)] + in aqueous NaClO4
(I = 0.10 mol dm-3) at 298.2 K. Literature values determined by potentiometric titrations in
aqueous solution under the conditions indicated are also shown.
There is no common trend of pKaH2O across the metals for both tacn and Me3tacn series.
Furthermore the results for both ligands complexed with Ni2+ should be treated with caution due
to the slow equilibrium issues already discussed. However it is noteworthy that, like with tren
and Me6tren, the L-M2+ pairing with largest logK1 stability constant also produced the lowest
aqua ligand pKaH2O#1: tacn and Me3tacn with Cu2+. Whilst these complexes are depicted in the
literature as being five-coordinate with two water ligands present, only one aqua pKaH2O is
reported in these sources.21, 22 However, the first aqua deprotonations for [Cu(tacn)(H2O)2]2+ and
[Cu(Me3tacn)(H2O)2]2+ are at a low pH, suggesting that if a second aqua ligand is indeed present,
67
its deprotonation is likely to occur at a measureable pH. The second acidic aqua ligand species
was therefore included in all the copper fitting and found to fit well.
Given the large jump in pKaH2O on going from the first aqua ligand deprotonation to the
second for the tacn/Cu2+ system (+3.36 log units), it may be the case that the first deprotonation
constants (pKaH2O#1) for tacn with the other metals are too high to allow the second to be
measured. These complexes are likely to be five- or six-coordinate with more aqua ligands
present, but the inclusion of the second hydroxo species [ML(OH)2] in the models for tacn with
the other metals resulted in excessive errors or an overall failure to fit.
In contrast to tren and Me6tren, methylation of tacn does not appear to consistently lower the
associated aqua ligand pKaH2Os. In fact, excluding Ni2+, it is only in the complexes with Cu2+ for
which this occurs. The theory behind the methylation is that it increases the hydrophobicity of the
aqua ligand’s environment, thereby lowering its pKaH2O. The mechanism involves contributions
from both altered aqueous solvent ordering conditions and altered electron donation from the
ligand to the metal upon methylation.24 Therefore if the predicted decrease in pKaH2O is not
observed, the assumption is that the aqua ligands are not experiencing the anticipated increase in
hydrophobicity. This may be explained by the way in which Me3tacn must facially coordinate to
the metal centre due to its constrained, cyclic, tridentate structure. Unlike the floppy, tetradentate
Me6tren ligand which can effectively wrap around a metal centre, Me3tacn can only cap a metal
by attaching to the base or top. The methyl groups are therefore constrained to one side of the
complex and the aqua ligands must coordinate to the other, whilst for Me6tren the methyl groups
are very close to the water ligand.24 This theory is supported by the crystal structure of the free
Me3tacn ligand where the methyl groups point away from the coordinating cavity41 and by the
octahedral crystal structure of [Zn(Me3tacn)(H2O)3]2+ where there are large bond angles
separating the methyl carbons from the water oxygens (see Figure 2.10).23
In Figure 2.10 the crystal structure determined in reference 23 sits alongside the molecular
diagram for clarity.
68
Figure 2.10. The crystal structure of [Zn(Me3tac)(H2O)3] 2+ from reference 23 alongside its
molecular diagram. Noticeably, neither the ethyl chains nor methyl groups are symmetrical
around the zinc cation and the aqua ligands are pointed away from the methyl groups.
The lack of observed water pKaH2O#1 decrease on methylation may also be affected by the
geometry of the complexes. It has been reported for Zn2+ that this pKaH2O value decreases with
decreasing coordination number of the complex.23 Both tacn and Me3tacn Cu2+ complexes have
been prepared and determined to be five-coordinate with two aqua ligands21, 22
and on
methylation of the tacn ligand to Me3tacn the pKaH2O does decrease very slightly. For an identical
complex stereochemistry, this therefore shows a comparison purely based on methylation effects.
(And, justified by the reasoning above, there is not the expected large decrease in pKaH2O.) The
crystal structure of [Zn(Me3tacn)]2+ shows this complex is octahedral and there are two
measureable aqua deprotonations. (This is also supported by the fact that this complex yields two
measurable aqua ligand deprotonations in this study.) However, the corresponding [Zn(tacn)]2+
complex has only one measureable aqua ligand pKaH2O which may indicate it is less than six
coordinate. If the Zn2+ and Cd2+ complexes move from five coordinate in their complexes with
tacn to six-coordinate in their complexes Me3tacn, this may also explain the increase in aqua
ligand pKaH2O for these species upon ligand methylation. Five-coordinate tacn metal complexes
will yield lower water ligand pKaH2Os than six-coordinate Me3tacn metal complexes. Crystal
structures of all of these complexes obtained as part of future work would test this theory. M2+-
oxygen bond lengths for each complex would also be useful in determining if there is a
correlation between this and aqua ligand pKaH2O as there appears to be for the tren and Me6tren
examples.
69
Interestingly, one affect the methylation does appear to have is the depression of the second
aqua ligand pKa – pKaH2O#2. Whilst potentially a change in complex geometry and the structural
constraints on the methylated ligand do not produce the usual decrease in pKaH2O#1, the Me3tacn
metal complexes all have second aqua ligand deprotonations that are low enough to be measured.
This is unlike their tacn counterparts. It is possible that once one hydroxo has been formed, the
methylated environment promotes the second deprotonation where the non-methylated version
does not. However this observation requires further investigation as part of future work.
When designing a catalyst for which a lowered water ligand pKaH2O is required, understanding
how geometry, structure and environment influence this value is very important.
2.3 Conclusions
The characterisation of the 16 transition metal complexes presented in this chapter (ligand
pKas, complex stability constants and water ligand pKaH2Os) agree well with the literature where
similar studies could be found.
From an enzyme-mimic catalyst design perspective, several points from the results are
significant. Firstly the slow equilibrium obstacles involved with the Ni2+ systems makes the
complexes formed with this metal non-ideal for catalyst development. This metal was continued
for characterisation with the remaining ligands described in Chapter 1, Figure 1.9 (see Chapter 3)
to complete this comprehensive study and also to investigate the slow equilibria further. However
these complexes were not chosen for testing of their ester hydrolysis catalyst activity (Chapter 4).
Secondly, for all four ligands the metal that resulted in both the most stable complex
formation and also the lowest aqua ligand pKaH2O (ie the most reactive aqua ligand) was Cu2+.
Stable complexes (evidenced by large stability constants, logK1) are advantageous because they
ensure that the desired complex will be present in high concentrations and that free ligand and
free metal will not exist to any great extent. Free ligand and metal are problematic when testing
ester hydrolysis activity because they can also catalyse the reaction along with the complex
hydroxo species of interest. In Figure 2.8 at the pH equal to the pKaH2O#1 for [Cu(tren)(H2O)2]2+
(9.11) there is < 5% free ligand and the majority of ligand is found as [Cu(tren)] 2+ or
[Cu(tren)(OH)]+. This is ideal.
70
Furthermore, the lower the aqua ligand pKaH2O, the larger the pH range over which this
complex could act as a catalyst. A low aqua ligand pKaH2O for a complex means that the
hydrolysis-performing hydroxo species is present over a pH range at which it would not usually
be able to form naturally in the absence of the complex species.
Thirdly, whilst tren forms more stable complexes with the transition metals in this study, tacn
results in the lower aqua ligand pKaH2Os. Although an exact acyclic versus macrocyclic
comparison cannot be made because the ligands do not have the same denticity, it does appear
that whilst the constrained cyclic nature of tacn diminishes its ability to bind to M2+ as compared
with tren, it does enhance the coordinative forces felt by the aqua ligands also present at the metal
centre.
Finally it is noted that ligand methylation in an effort to create a hydrophobic complex

environment does not always succeed in lowering the associated aqua ligand pKaH2O. In the case
of tren this can be achieved, but for tacn there are geometric and structural influences that likely
override this action. To extrapolate to other ligands for potential complex catalyst application, it
is possible that methylation (or other alkylation) for the purposes of pKaH2O depression will only
be successful for ligands which can impose their hydrophobic moieties upon the remaining
coordination sites (greater steric bulk, more donors etc) and therefore have a closer interaction
with the aqua ligand(s). This can easily be investigated as part of future work by producing
space-filling models of the complexes prior to their experimental examination.
71
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76
Hilary Coleman Chapter 3. Potentiometric Titrations of the β-Cyclodextrin-Functionalised Polyamines
CHAPTER 3
POTENTIOMETRIC TITRATIONS OF THE

β-CYCLODEXTRIN FUNCTIONALISED
POLYAMINES
77
3.1 Introduction
3.1.1 Metallo-cyclodextrins
Many amine-substituted β-cyclodextrins have been prepared and characterised.1-8 Metal
complexes of these β-cyclodextrin derivatives, hereafter referred to as metallo-βCDs, have been
studied extensively as enzyme mimics,9-12 as chiral discriminators between enantiomeric guests13-
16
and for their potential synthetic and industrial uses as molecular reactors,17 as chemical
sensors18 and in the improvement of MRI contrast agents.19-21 These are just some of the
applications for which this class of compounds has been investigated.
The employment of metallo-βCDs in such diverse areas of chemical research highlights their
versatility. This stems from their multifaceted structures (Figure 3.1). The metal-complexing
ligand substituent displaces a primary hydroxyl group on the cyclodextrin rim in the examples
shown in Figure 3.1 and is the basis of the classical metal complex chemistry that the metallo-
βCDs exhibit. Variation of the Mm+ (usually a transition metal or lanthanide cation) or the ligand
substituent has a large effect in determining the character and activity of the metallo-βCD.
Furthermore, covalent attachment of this metal complex to the cyclodextrin essentially pairs the
metal complex chemistry with the host-guest inclusion complex chemistry that has been well
elucidated for native cyclodextrins.22 This allows for interaction between the complexed guest
molecule, free Mm+ and Mm+-coordinated ligands. If the guest molecule reacts with a ligand in the
metal complex (for example if the guest is an ester and the metallo-βCD has a coordinated
hydroxo ligand11) then the metallo-βCD can dramatically increase the effective concentration of
the reacting species. Finally, cyclodextrins are naturally occurring, readily available and non-
toxic, making them extremely attractive for biological and pharmaceutical applications.
Figure 3.1 displays the structures of βCDtren and βCDtacn along with two other substituted
βCD species used for comparison in this study – βCDtrien and βCDtacdo.2 In βCDtren and
βCDtacn the 1°, 2° and 3° amine donor atoms are indicated to highlight the difference in
coordination chemistry between the tren and tacn families investigated in this study.
78
Figure 3.1. The structures of the ligands βCDtren (and βCDMe5tren) and βCDtacn (and
βCDMe2tacn) characterised in this study are shown along with analogues for comparison,
βCDtrien and βCD tacdo. The 1o, 2o and 3o nature of the amine groups is indicated for βCDtren
and βCDtacn.
79
3.1.2 New ligands βCDMe5tren and βCDMe2tacn

The ligands βCDMe5tren and βCDMe2tacn (compounds 7 and 8 in Figure 1.9) whose
synthesis is described in Section 8.2.1, have not been previously reported. The protonated ligand
pKa values, logK1 values for their M2+ complex formation and aqua ligand pKaH2O data and
analysis of trends in these data for the ligands βCDtren, βCDMe5tren, βCDtacn and βCDMe2tacn
complexed with Zn2+, Cu2+, Ni2+ and Cd2+ are reported in this Chapter. A comparison with
relevant data for the ligands tren, Me6tren, tacn and Me3tacn is also presented. Thus, where the
chemical reasoning behind the results reported in this Chapter is identical to that in Chapter 2 the
references therein also apply. The speciation plots that result from the pKa, logK, logK’ and
pKaH2O values that are presented for each complex in this Chapter are contained in Appendix B.
The titration curves for the new ligands (βCDMe5tren and βCDMe2tacn pKas and complexed with
Zn2+, Cu2+, Ni2+ and Cd2+) that are not presented within this Chapter are also found in Appendix
B.
3.2 The pKas of the protonated βCDtren, βCDMe5tren, βCDtacn and

βCDMe2tacn and stability constants of their metal complexes
All titrations were performed in duplicate with reproducible results. Even for systems that
have already been characterised in the literature, the fitting in this study involved attempts to fit
all possible species and all possible combinations of these species until the best fit was achieved.
Where other literature data was available, the data fitting in this study was informed by and
compared to this literature. The data fits obtained in this study are good ( values routinely 2.0 or
less). Consistent with the literature, the errors reported for the titration data are derived from the
Hyperquad fitting program and reflect the congruity between the fit and experimental data.
Estimated experimental errors are typically a maximum of ± 3%. See Sections 8.1.3 and 8.2.1.
3.2.1 Determination of protonated βCDtren and βCDMe5tren pKas

The pKas of the fully protonated forms of the ligands βCDtren, 5, βCDtacn, 6, βCDMe5tren, 7
and βCDMe2tacn, 8, shown in Figure 1.9, were determined by potentiometric titration at 298.2 K
in aqueous NaClO4 (I = 0.10 mol dm-3) as described in Section 8.2.2. Both βCDtren and
βCDMe5tren have four protonation sites and are characterised by pKa1, pKa2, pKa3 and pKa4 as
80
defined by Equations 3.1 to 3.4. A potentiometric titration curve for protonated βCDtren and the
corresponding speciation plot are shown in Figures 3.2 and 3.3, respectively. The corresponding
pKa values for βCDtren and βCDMe5tren are shown in Table 3.1 along with relevant literature
values22 which are in reasonable agreement with the data obtained in this study. They may also be
compared with the corresponding pKa values for tren and Me6tren, also shown in Table 3.1.
Ka1
LH+ ⇌ L + H+ where Ka1 = ([L][H+])/[LH+] 3.1
Ka2
LH22+ ⇌ LH+ + H+ where Ka2 = ([LH+][H+])/[LH22+] 3.2
Ka3
Ka4
As observed for protonated tren and Me6tren, there is a greater overall basicity for the amine
nitrogens of protonated βCDtren as indicated by the summation of pKa1 - pKa4 (28.36) over those
of protonated βCDMe5tren (25.91). Similarly, as for protonated tren and Me6tren, their
protonated βCDtren and βCDMe5tren analogues show a decrease in pKa size from pKa1 to pKa4
consistent with each protonation rendering fewer protonation sites available and increasing the
overall charge thereby increasing electrostatic repulsion as protonation proceeds. All four
systems show a decrease from pKa1 to pKa3 between non-methylated and methylated ligand
versions as protonation proceeds. However, there is a small decrease in pKa4 on going from
protonated tren to protonated Me6tren that contrasts with a small increase in pKa4 which occurs
on going from protonated βCDtren to protonated βCDMe5tren. This is consistent with N-
methylation both hindering amine nitrogen protonation for pKas 1 to 3 and decreasing the ability
of bulk water to hydrogen bond with the positively charged Me2N-H+ groups as the methyl
groups render the local environment partly hydrophobic. The corresponding changes in pKa4 are
smaller, consistent with the central tertiary amines being more distant from the methylation sites.
81
11.5
10.5
9.5
8.5
7.5 Observed
pH Fit
6.5
5.5
4.5
3.5
2.5
0.615 0.715 0.815 0.915 1.015 1.115
Titre (mL)
Figure 3.2. Potentiometric titration curve for the determination of the protonated βCDtren
ligand pKa values – observed points (◊) and best-fit of the pH variation algorithm based on
Equations 3.1 – 3.4 (-), resulting in the pKa values reported in Table 3.1. Aqueous NaClO4 (I =
0.10 mol dm-3), 298.2 K. The initial [βCDtren] = 1.0 × 10-3 mol dm-3, the added [HClO4] = 1.0 ×
10-2 mol dm-3, and the titrant [NaOH] = 0.10 mol dm-3.
Comparison of the pKa1 - pKa4 values of protonated tren with those of protonated βCDtren,
and of the pKa values of protonated Me6tren with those of protonated βCDMe5tren indicates that
attachment of the amine at the C6A position of the βCD moiety causes decreases in pKa1 - pKa3
but an increase in pKa4. The pKa1 - pKa3 values decrease by between 0.25 and 1.3 units on going
from tren to βCDtren, but pKa4 increases by 0.77. Similarly, on going from Me6tren to
βCDMe5tren, pKa1 - pKa3 decrease by between 0.59 and 0.97 units but pKa4 increases by 1.21.
Thus, the second, third and fourth nitrogens to be deprotonated become less basic when attached
to βCD, while the first nitrogen (characterised by pKa4) becomes more basic. (Decreases in the
basicity for polyamines upon attachment to βCD appears to be a general phenomenon as
illustrated by another tetraamine, trien, NH2(CH2)2NH(CH2)2NH(CH2)2NH2. For trienH44+, pKa1
= 9.83, pKa2 = 8.93, pKa3 = 5.40 and pKa4 = 3.0 (overall basicity = 27.16), and for βCDtrienH44+
(in Figure 3.1) the pKa1 = 9.33, pKa2 = 8.22, pKa3 = 5.61 and pKa4 = 3.13 (overall basicity =
26.29). The pattern of pKa variation depends greatly on the structure of the polyamine.2)
82
100
90

80 free L
70
LH⁺
60
50 LH₂²⁺
40
LH₃³⁺
30
20 LH₄⁴⁺
10
0
3 5 7 9 11
pH
Figure 3.3. Speciation plot showing the variation of the protonation of βCDtren with pH, relative
to the total concentration of all βCDtren and its protonated species being equivalent to 100%.
βCDtrenH44+ (LH44+), βCDtrenH33+ (LH33+), βCDtrenH22+ (LH22+), βCDtrenH+ (LH+) and
βCDtren (L).
Ligand LH+ LH22 + LH33+ LH44+

L pKa1 pKa2 pKa3 pKa4
tren This study 10.21 ± 0.01 9.45 ± 0.01 8.31 ± 0.01 1.74 ± 0.03
CDtren This study 9.64 ± 0.02 9.20 ± 0.02 7.01 ± 0.03 2.51 ± 0.04
a 9.85 8.99 6.86 2.6
CDMe5tren This study 8.81 ± 0.07 7.97 ± 0.07 6.40 ± 0.07 2.73 ± 0.09
a: Ref. 23, I = 0.10 mol dm-3 (NaClO4), 298.2 K.
Table 3.1. The pKa values for the tren-based ligands: tren, Me6tren, βCDtren and βCDMe5tren
determined by potentiometric titration at 298.2 K in aqueous NaClO4 (I = 0.10 mol dm-3). The
errors shown are derived from fitting the pH variation algorithm based on Equations 3.1 – 3.4 to
the titration data. Analogous literature values for βCDtren determined by potentiometric titration
in aqueous solution are also shown.
83
The implication of these trends is that by substituting βCD onto either tren or Me6tren to give
βCDtren and βCDMe5tren, respectively, and by methylating βCDtren to give βCDMe5tren, the
amine nitrogen involved in the pKa4 equilibrium (Equation 3.4) becomes more basic and/or the
resulting N-H+ groups more readily hydrogen bond with solvent water molecules. Both of these
features are attributable to the changed environment of the central amine nitrogens of βCDtren
and βCDMe5tren as a result of βCD-substitution. By comparison with the fully hydrated
environment of free tren and Me6tren in aqueous solution, the tren and Me5tren substituents of
βCDtren and βCDMe5tren experience an environment in which they are constrained to be in close
proximity to the six primary hydroxyl groups (none of which dissociate below pH 1224) defining
the narrow end of the βCD annulus (Figure 3.1).
3.2.2 Determination of protonated βCDtacn and βCDMe2tacn pKas
Both βCDtacn and βCDMe2tacn have three protonation sites and are characterised by pKa1,
pKa2 and pKa3 as defined by Equations 3.1 to 3.3. A potentiometric titration curve for protonated
βCDtacn and the corresponding speciation plot is shown in Figures 3.4 and 3.5, respectively. The
corresponding pKa values for βCDtacn and βCDMe5tacn are shown in Table 3.2 along with some
literature values2 which are in reasonable agreement with the data obtained in this study. These
pKa values for βCDtacn and βCDMe5tacn may be compared with the corresponding pKa values
for tren and Me6tren also shown in Table 3.2.
As a consequence of their macrocyclic structures, which constrain their protonated amine

groups in close proximity, the pKa1 - pKa3 values characterising protonated tacn, Me3tacn,
βCDtacn and βCDMe2tacn decrease in larger increments than do those of acyclic protonated tren,
Me6tren, βCDtren and βCDMe5tren whose flexibility allows the protonated amine groups to
maximise distance one from another and thereby minimise electrostatic repulsion. As discussed
in Chapter 2, the variation of pKa1 - pKa3 for tacn and Me3tacn may be rationalised in terms of the
effects of the conformations of their macrocyclic rings on their protonations. Thus, the first
protonation of tacn occurs on the outside of the macrocyclic ring and of Me3tacn occurs on the
inside. The added proton hydrogen bonds to the other two amine groups in the case of Me3tacn
and a lowering of pKa2 and pKa3 on methylation results. However, this effect is not observed for
βCDtacn and βCDMe2tacn. This suggests that addition of βCD to the macrocyclic triamine ligand
influences its conformation such that the first protonation occurs outside the macrocylic ring, and
84
stabilisation of the monoprotonated state through hydrogen bonding between the added proton
and the other two amine groups does not occur for βCDMe2tacnH+ as it does for Me3tacnH+. If
these deductions that the protons of βCDtacnH+ and βCDMe2tacnH+ are added outside the triaza
macrocyclic ring are correct, this indicates that a lone electron pair of one of the amine nitrogens
points away from the triaza macrocyclic ring of βCDtacn and βCDMe2tacn. This is expected to
affect metal complex stabilities and is discussed below.
11.5
10.5
9.5
8.5
7.5 Observed
pH
Fit
6.5
5.5
4.5
3.5
2.5
0.13 0.15 0.17 0.19 0.21 0.23
Titre (mL)
Figure 3.4. Potentiometric titration curve for the determination of the protonated βCDtacn
ligand pKa values: observed points (◊) and best-fit of the pH variation algorithm based on
Equations 3.1 – 3.4 (-), resulting in the pKa values reported in Table 3.1. Aqueous NaClO4 (I =
0.10 mol dm-3, 298.2 K. The initial [βCDtacn] = 1.0 x 10-3 mol dm-3, the added [HClO4] = 1.0 ×
10-2 mol dm-3, and the titrant [NaOH] = 0.10 mol dm-3.
85
100
90

80 free L
70
60 LH⁺
50
40 LH₂²⁺
30
20 LH₃³⁺
10
0
3 5 7 9 11
pH
Figure 3.5. Speciation plot showing the variation of the protonation of βCDtacn with pH,
relative to the total concentration of all βCDtacn and its protonated species being equivalent to
100%. βCDtacnH33+ (LH3+), βCDtacnH22+ (LH22+), βCDtacnH+ (LH+) and βCDtacn (L).

L pKa1 pKa2 pKa3
tacn This study 10.47 ± 0.03 6.97 ± 0.02 2.39 ± 0.01
Me3tacn This study 11.96 ± 0.05 5.17 ± 0.03 1.88 ± 0.06
βCDtacn This study 9.98 ± 0.02 6.00 ± 0.03 2.34 ± 0.04
a 10.0 5.89 2.4
βCDMe2tacn This study 9.80 ± 0.02 4.47 ± 0.03 2.97 ± 0.02
a: Ref. 2, I = 0.10 mol dm-3 (NaClO4), 298.2 K
Table 3.2. The pKa values for the tacn-based ligands: tacn, Me3tacn, βCDtacn and βCDMe2tacn
determined by potentiometric titration at 298.2 K in aqueous NaClO4 (I = 0.10 mol dm-3).
Analogous literature values for βCDtacn determined by potentiometric titration in aqueous
solution are also shown.
86
Addition of the βCD moiety to tacn (to form βCDtacn) and to methylated tacn (to form
βCDMe2tacn) also results in a similar trend to that observed for βCD-substitution on the tren-
based ligands. Thus, pKa1 and pKa2 decrease by 0.49 and 0.96, respectively, on going from
tacnH33+ to βCDtacnH33+, and pKa3 is very similar for both. The trend is more pronounced for the
methylated ligands: on going from Me3tacnH33+ to βCDMe2tacnH33+, pKa1 and pKa2 decrease by
2.16 and 0.70, respectively, but pKa3 increases by 1.09. Again, this shows a decrease in basicity
for the second and third amine nitrogens to be deprotonated during a titration but an increase for
the first once the polyaza ligand has undergone βCD-substitution.
To determine if these observations are isolated to the tacn-based ligands it is of interest to

make comparisons with the triaza-macrocyclic ligands tacdo, Me3tacdo and βCDtacdo (Figure
3.1) whose pKa1 - pKa3 values appear in Table 3.3.25, 26
These ligands differ from the nine-
membered triazamacrocycle tacn-based ligands as they are twelve membered triazamacrocycles.
It is seen that the pKa1 - pKa3 trends individually for tacdo, Me3tacdo and βCDtacdo also decrease
in large steps consistent with the macrocycle constraining the protonated amine groups to be in
close proximity. The value of pKa1 increases on going from tacdo to Me3tacdo, likely as a
consequence of the effect of macrocyclic ring conformation being similar to those operating in
tacn and Me3tacn. There is no report of βCDMe2tacdo in the literature and therefore further
comparisons cannot be made.
Ligand LH+ LH + LH3+
L pKa1 pKa2 pKa3
tacdo a 12.60 7.57 2.41

b 12.3 7.3 2.4
Me3tacdo b 12.8 5.7 2.9
βCDtacdo c 11.24 5.85 2.8
a:Ref25, I = 0.10 mol.dm-3 (KNO3), 298.2 K, b:Ref26 298.2 K nmr titration (D2O). c: Ref2, I = 0.10
mol.dm-3 (NaClO4), 298.2 K.
Table 3.3. Literature data for the pKa values of the triaza macrocyclic ligands tacdo, Me3tacdo
and βCDtacdo.
87
It should be noted that some of the results quoted in this section were produced from titrations
that contained slow equilibrium processes over small sections of the titration curves. This concept
was raised in Chapter 2 and is discussed in further detail in Section 3.2.3. Regardless, all of the
titrations could be fit satisfactorily and in many cases the slow equilibria were only detectable by
analysis of the titrimeter data and were not apparent from observation of the curve or the fit.
3.2.3 βCDtren and βCDMe5tren metal complex characterisation

Formation of the Zn2+, Cu2+, Ni2+ and Cd2+ complexes of βCDtren and βCDMe5tren was
characterised by potentiometric titrations of solutions containing either [CDL]total to [M2+]total
ratios of both 1:1 and 1:1.5. A typical titration curve for the equilibria associated with the
[ZnCDMe5tren(H2O)2]2+ complex and the corresponding speciation plot are shown in Figures
3.6 and 3.7, respectively. (In all cases, titrations were carried out in duplicate at 298.2 K in
aqueous NaClO4 (I = 0.10 mol dm-3) as described in Section 8.2.2, and the reported data
represents the average of two titrations.) The magnitudes of the derived K1, K1′, KaH2O#1, and
KaH2O#2 (Tables 3.4 – 3.6) are consistent with species they characterise (Equations 3.5 – 3.8)
being dominant in solution.
K1
L + M 2+ ⇌ [M(L)]2+ where K1 = [M(L)2+]/[L][M2+] 3.5
K1′
LH+ + M2+ ⇌ [M(LH)]3+ where K1′ = [M(LH)3+]/[LH+][M2+] 3.6
KaH2O#1
[M(L)(H2O)2]2+ ⇌ [M(L)(H2O)(OH)]+ + H+
where KaH2O#1 = [M(L)(H2O)(OH)+][H+]/[M(L)(H2O)22+] 3.7
KaH2O#2
[M(L)(H2O)(OH)]+ ⇌ [M(L)(OH)2] + H+
where KaH2O#2 = [M(L)(OH)2][H+]/[M(L)(H2O)(OH)+] 3.8
88
11.5
10.5
9.5
8.5
Observed
7.5
pH Fit
6.5
5.5
4.5
3.5
2.5
0.129 0.179 0.229 0.279
Titre (mL)
Figure 3.6. Potentiometric titration curve for the determination of the Zn2+/βCDMe5tren system
characteristics described by logK1′, logK1, pKaH2O#1 and pKaH2O#2 – observed points (◊) and best-
fit of the pH variation algorithm based on Equations 3.1 – 3.8 (-), resulting in the values reported
in Tables 3.4 – 3.6. Aqueous NaClO4 (I = 0.10 mol dm-3, 298.2 K. The initial [βCDMe5tren] =
1.0 × 10-3 mol dm-3, the initial [Zn2+] = 1.0 × 10-3 mol dm-3, the added [HClO4] = 1.0 × 10-2 mol
dm-3, and the titrant [NaOH] = 0.10 mol dm-3.
These equations represent the 1:1 complex formation between the completely deprotonated
CDtren and CDMe5tren (L) ligands and M2+ = Zn2+, Cu2+, Ni2+ and Cd2+ (Equation 3.5), the
1:1 complex formation between the mono-protonated ligands CDtrenH+ and CDMe5trenH+
(CDLH+) ligands and M2+ (Equation 3.6), the deprotonation of the first aqua ligand present in
the [M(CDtren(H2O)2]2+ and [M(CDMe5tren(H2O)2]2+ complexes ([M(CDL)(H2O)2]2+)
(Equation 3.7) and the deprotonation of the second aqua ligand from the
[M(CDtren(OH)(H2O)]+ and [M(CDMe5tren(OH)(H2O)]+ complexes
([M(CDL)(OH)(H2O)]+) (Equation 3.8). Best-fitting of a pH-dependent algorithm to the
potentiometric titration data is consistent with all eight M2+ complexes formed with βCDtren and
βCDMe5tren being six-coordinate with two aqua ligands present. The logK1 and logK1′ for the
Zn2+, Cu2+, Ni2+ and Cd2+ of βCDtren and βCDMe5tren are shown in Tables 3.4 and 3.5,
respectively, together with those for the analogous tren and Me6tren complexes in Table 3.5. A
89
potentiometric titration curve for the Zn2+/βCDMe5tren) system and the corresponding speciation
plot are shown in Figures 3.6 and 3.7, respectively.
100
free L
90
LH⁺
80
70 LH₂²⁺
60 LH₃³⁺
50
LH₄⁴⁺
40
MLH⁺
30
20 [ML]²⁺
10 [M(L)(OH)]⁺
0
3 4 5 6 7 8 9 10 11 [M(L)(OH)₂]
pH
Figure 3.7. Speciation plot showing the variation of the protonation of βCDMe5tren and the
formation of its Zn2+ complexes as pH is varied relative to the total concentration of βCDMe5tren
and its protonated and complexed forms being equivalent to 100% speciation: βCDMe5trenH44+
(LH44+), βCDMe5trenH33+ (LH33+), βCDMe5trenH22+ (LH22+), βCDMe5trenH+ (LH+),
βCDMe5tren (free L), [Zn(βCDMe5trenH)(H2O)2] 3+ ([M(LH)] 3+), [Zn(βCDMe5tren)(H2O)2] 2+
([M(L)] 2+), [Zn(βCDMe5tren)(H2O)(OH)] + ([M(L)(OH)] +) and [Zn(βCDMe5tren)(OH)2]
([M(L)(OH)2]). Aqua ligands are not shown.
The order of complex stability for [M(βCDtren)(H2O)2]2+ is Cu2+ > Zn2+ > Ni2+ ≥ Cd2+. The
relatively low value of logK1 for Cd2+ probably reflects the greater ionic radius of Cd2+ which, in
combination with a lower surface charge density, renders meridional coordination of the three
pendant amine groups of βCDtren and βCDMe5tren less favourable than for the smaller Cu2+,
Zn2+ and Ni2+ . This trend in logK1: Cu2+ > Zn2+ > Ni2+ approximately follows the Irving-
Williams Series: Ni2+ < Cu2+ > Zn2+.27
90

logK1 logK1 logK1 logK1
[M(L)]2+ tren This study 14.50 ± 0.01 18.92 ± 0.01 14.32 ± 0.02 11.83 ± 0.01
βCDtren This study 11.99 ± 0.01 17.22 ± 0.02 12.01 ± 0.02 10.47 ± 0.01
a 12.25 17.29 11.65
βCDMe5tren This study 5.61 ± 0.02 11.56 ± 0.06 3.70 ± 0.14 4.29 ± 0.02
a: Ref. 23, I = 0.10 mol dm-3 NaClO4, 298.2 K.
Table 3.4. Potentiometrically determined logK1 values for the [M(L)] 2+ complexes of βCDtren
and βCDMe5tren formed with Zn2+, Cu2+, Ni2+ and Cd2+ at 298.2 K in aqueous NaClO4 (I = 0.10
mol dm-3) in this study. Literature values determined by potentiometric titrations in aqueous
solution under the conditions indicated are also shown.
The three logK1 from the literature for the complexation of βCDtren with Zn2+, Cu2+ and Ni2+
are similar to those determined in this study.23 The largest discrepancy is for Ni2+ for which slow
equilibrium processes were observed during the titrations, similar to those for Ni2+ with tacn and
Me3tacn. For all of the βCD-substituted ligands it was detected that some sections of the titration
curves required longer wait times than for the corresponding free amines. Consequently these
titrations were carried out with a maximum wait time between data points of 1200 s. For
complexes of βCDtren with Cu2+ and Zn2+, most data points were recorded after approximately
20 s and only one or two points required wait times of between 400 and 500 s (out of > 350 data
points). However, for βCDtren with Ni2+ numerous data points required wait times of between
600 and 800 s, with the longest wait time being 900 s. Depending on the titration set-up in the
literature study it is therefore not unexpected that there may be some discrepancy between
determinations of this logK1.
The logK1 values for βCDMe5tren also follow the trend predicted by the Irving-Williams
Series.27 As shown in Table 3.4, methylation of βCDtren decreases logK1 substantially for each
M2+ complex as is also observed for methylation of the free ligand tren to Me6tren (Chapter 2).
Again this can be attributed to steric effects whereby the methyl groups sterically hinder ligand
coordination.28, 29 The logK1 for [Ni(βCDMe5tren)(H2O)2]2+ is the smallest in Table 3.4 and that
for [Ni(Me6tren)(H2O)2]2+ is the second smallest. It has been hypothesised that five-coordinate
91
[Ni(tren)(H2O)]2+ and [Ni(Me6tren)(H2O)]2+ are the dominant species in aqueous solution,30, 31
and this is consistent with only one aqua ligand deprotonation being detected in this work. Only
one aqua ligand deprotonation was detected for [Ni(βCDtren)(H2O)2]2+ consistent either with
five-coordination and only one aqua ligand being bound, or a very high pKaH2O#2 for the second
aqua ligand. As the pKaH2O#1 is 11.37 for deprotonation of the first aqua ligand of
[Ni(βCDtren)(H2O)2]2+ the pKaH2O#2 for the second aqua ligand (if present) is likely to be too
large for determination by potentiometric titration. In contrast two aqua ligand deprotonations
were detected for [Ni(βCDMe5tren)(H2O)2]2+ consistent with a six-coordinate geometry. (The
pKaH2O#1 and pKaH2O#2 data are discussed in more detail below.)

L logK1' logK1' logK1' logK1'
[M(LH)]3+ tren This study 9.13 ± 0.07 12.74 ± 0.02 9.68 ± 0.02 7.56 ± 0.07
Me6tren This study 5.43 ± 0.07 9.70 ± 0.03 3.82 ± 0.06
βCDtren This study 7.96 ± 0.02 11.92 ± 0.03 8.27 ± 0.02 6.92 ± 0.03
a 7.92 11.56 8.46

βCDMe5tren This study 4.03 ± 0.03 8.27 ± 0.05 3.58 ± 0.05
a: Ref. 23, I = 0.10 mol dm-3 NaClO4, 298.2 K.
[M(βCDtrenH)] 3+ and [M(βCDMe5trenH)] 3+ formed with Zn2+, Cu2+, Ni2+ and Cd2+ at 298.2 K
in aqueous NaClO4 (I = 0.10 mol dm-3). Literature values determined by potentiometric titrations
in aqueous solution under the conditions indicated are also shown.
A comparison of the logK1 for the Zn2+, Cu2+, Ni2+ and Cd2+ complexes of tren with those of
CDtren shows that in all cases logK1 for the latter are substantially decreased. A similar
situation applies for the analogous complexes of Me6tren and of CDMe5tren. For the tren and
βCDtren complexes this likely reflects a decrease in the electron-pair donating power of the
secondary amine in βCDtren (attached to βCD at C6A, Figure 3.1) by comparison with that of the
corresponding primary amine of tren. In addition, the other three amine groups of tren when
attached to βCD may experience a change in hydration as a consequence of hydrogen bonding
with the six primary hydroxyl groups of βCD which could also affect the magnitude of logK1.
92
The large βCD moiety may also present some steric hindrance to M2+ complexation. However,
the available data does not allow an assessment of the relative importance of these three factors.
As all of the amine groups of Me6tren and βCDMe5tren are tertiary in nature, the presence of
the βCD substituent in βCDMe5tren is unlikely to affect the relative logK1 values of the M2+
complexes of these two ligands through a change in amine group basicity. Consequently the
decreased logK1 of the M2+ complexes of βCDMe5tren relative to those of Me6tren is likely to
arise dominantly from steric hindrance caused by βCD and changes in ligand amine group
hydration.
A comparison of the logK1 for the Zn2+, Cu2+, Ni2+ and Cd2+ complexes of tren with those of
Me6tren and of those of βCDtren with those of βCDMe5tren shows methylation substantially
decreases logK1 in all cases. This is consistent with the dominant factor causing the decrease in
logK1 being the steric hindrance caused by the methyl groups. The smallest logK1 are those
characterising the M2+ complexes of the βCDMe5tren ligand in which the sterically hindered
tertiary amines and the βCD entity maximise the extent of steric hindrance to M2+ complexation.
The most stable complexes are formed by Cu2+ within the tren, Me6tren, βCDtren and
βCDMe5tren series and these complexes show the smallest decreases in logK1 upon methylation
or addition of βCD. The ionic radius of Cu2+ is the second smallest of the M2+ examined in this
study and its relatively high surface charge density together with the effects of a d9 electronic
configuration are likely to contribute significantly to the large logK1 measured.32
An analysis of the size of the changes in logK1 for each complex upon tren methylation and
substitution by βCD yields interesting insights. The larger the decrease in logK1 from one ligand
to the next for a given M2+, the more of a destabilising effect the particular ligand modification is
responsible for (i.e. methylation or βCD substitution). For Zn2+, Cu2+ and Cd2+ the trends in the
size of logK1 decrease are identical: βCDtren → βCDMe5tren > tren → Me6tren > Me6tren →
βCDMe5tren > tren → βCDtren. Thus, stability of these complexes decreases the most when
βCDtren is methylated, the next most when tren is methylated, an intermediate amount when
Me6tren is substituted with βCD and only a small amount when tren is substituted with βCD.
Typically the decreases in complex stability on addition of the βCD moiety are only 100 – 1000
fold, whereas the decreases on methylation are 10 000 – 1 000 000 fold (based on the logK1
values). This suggests that for these ligands it is the presence of primary amine nitrogens as
93
donor atoms that is the key to producing a highly stable complex and therefore methylation has a
greater destabilising effect than substitution by βCD.
The same analysis for these ligands with Ni2+ reveals the largest decrease in logK1 for any of
the complexes. There are almost identical decreases in logK1 for when the βCDtren ligand is
replaced by βCDMe5tren as compared with tren being replaced by Me6tren for Ni2+ (Table 3.4).
The Ni2+ complexes of the non-methylated ligands in both cases have K1 values approximately
200 000 000 times larger than those comprised of the methylated ligands. The decrease in logK1
when the ligand is substituted by βCD is also very similar for both tren and Me6tren. These
different trends in logK1 for Ni2+ (which is responsible for producing the lowest stability
constants across the four M2+ for the methylated ligands in the tren family) compared to those for
Zn2+, Cu2+ and Cd2+ indicates that differing contributions arising from the factors affecting
complex stability discussed above for Zn2+, Cu2+ and Cd2+ may affect the corresponding Ni2+
complex stabilities.
As highlighted, whilst these trends have been recorded and quantified as part of this study,
their origins have not been examined. The chemistry behind the causation of these trends is not
necessary for the selection and testing of the complexes that would best serve as enzyme mimics
(the aim of this work). Examining the results contained in Table 3.4, the tren-based ligand
complexes with Zn2+ and Cu2+ would likely be more suitable than the complexes with Ni2+ and
Cd2+ because larger stability constants ensure low concentrations of free ligand and free M2+ in
the kinetics experiments as these species might also contribute towards ester hydrolysis.
However, with further work on the methylation of βCD-substituted amines, the trends observed
in this study could be examined more thoroughly as part of Future Work.
As for tren and Me6tren, the employment of Equation 3.6 in the data fitting yielded logK1'
values for the mono-protonated ligands complexing Zn2+, Cu2+, Cd2+ and Ni2+. These results are
reported in Table 3.5 together with the corresponding literature values. The logK1' values agree
well with the literature values23 where the corresponding data could be found (Table 3.5).
(However, Me6trenH+ does not form a reliably detectable complex with Cd2+ and βCDMe5trenH+
does not form a reliably detectable complex with Ni2+.) The magnitudes of logK1′ for the Cu2+,
Zn2+, Ni2+ and Cd2+ complexes of mono-protonated trenH+, Me6trenH+, βCDtrenH+ and
βCDtrenMe5trenH+ are lower than the logK1 values determined for the complexes of the
analogous unprotonated ligands (Table 3.4) as anticipated for the increased repulsion between a
94
positively charged ligand and a positively charged metal centre. In addition, the protonation of
one nitrogen donor atom decreases the ligand denticity from tetradentate to tridentate and as such
the resulting complexes provide further examples of hypodenticity as described in Section 2.2.2.
The trend in logK1' qualitatively follows the Irving-Williams series27 for the Cu2+, Zn2+ and Ni2+
complexes of this ligand series, except in the case of βCDtren where the relative magnitudes of
logK1' for the Zn2+ and Ni2+ complexes are reversed. The lowest logK1' values are exhibited by
the Cd2+ complexes despite Cd2+ possessing the largest ionic radius32 of all four M2+ which might
be expected to decrease the effect of charge repulsion by the positively charged protonated
ligands. Evidently, the larger size of Cd2+, which renders meridional coordination of the three
pendant amine groups of the uncharged ligands less favorable than for the smaller Cu2+, Zn2+ and
Ni2+, also applies in the case of the protonated ligands in which only three nitrogens are available
for coordination.
Aqua ligand acid dissociations characterised by pKaH2O#1 and pKaH2O#2 defined by Equations
3.7 and 3.8 were obtained from the best-fit of the pH variation algorithm to the titration data and
appear in Table 3.7 together with the analogous data for the tren and Me6tren complexes from
Chapter 2. A maximum of two aqua ligands and two acid dissociations are anticipated for
octahedral complexes with tetradentate tren ligands. However, the lack of detection of a second
aqua ligand deprotonation is not a confirmation that the complex is five- rather than six
coordinate, but only indicates only that a second deprotonation was not detected under the
experimental conditions. (For the three literature23 comparisons that can be made for
[Zn(βCDtren)(OH)]+, [Cu(βCDtren)(OH)]+ and [Ni(βCDtren)(OH)]+ the pKaH2O#1 values are very
similar when experimental errors are taken into account. However, no second aqua ligand
deprotonations are reported in the literature so it is unclear whether these species were included
in the data analysis.)
95

(L) pKaH2O#1&2 pKaH2O#1&2 pKaH2O#1&2 pKaH2O#1&2
This
[M(L)(OH)]+ tren 9.97 ± 0.02 9.11 ± 0.02 11.37 ± 0.02 10.21 ± 0.03
study
This
Me6tren 8.61 ± 0.02 8.40 ± 0.03 8.81 ± 0.03 8.45 ± 0.03
study
This
βCDtren 9.14 ± 0.02 8.68 ± 0.05 9.42 ± 0.04 8.78 ± 0.02
study
a 8.9 8.48 9.68
This
βCDMe5tren 8.15 ± 0.02 8.35 ± 0.10 8.26 ± 0.07 8.77 ± 0.07
study
This
[M(L)(OH)2] tren 11.61 ± 0.03
study
This
Me6tren
study
This
βCDtren 10.00 ± 0.02 9.77 ± 0.05 10.35 ± 0.03
study
This
βCDMe5tren 9.98 ± 0.04 10.04 ± 0.10 10.04 ± 0.09 10.16 ± 0.02
study
a: Ref. 23, I = 0.10 mol dm-3 (NaClO4), 298.2 K.
Table 3.6. Potentiometrically determined aqua ligand pKaH2O#1 and pKaH2O#2 values for [M(βCDtren)(H2O)2] 2+ and [M(βCDMe5tren)(H2O)2] 2+ in
aqueous NaClO4 (I = 0.10 mol dm-3) at 298.2 K. Literature values determined by potentiometric titrations in aqueous solution under the
conditions indicated are also shown.
96
A generalisation may be applied to the variation of the pKaH2O#1 values in Table 3.6 and this is
that methylation of a particular ligand decreases the pKaH2O#1 of its M2+ complexes. Thus, the
pKaH2O#1 of the metal complexes of tren are greater than those of the Me6tren analogues and the
pKaH2O#1 of the metal complexes of CDtren are greater than those of the CDMe6tren. This
decrease of pKaH2O#1 upon ligand methylation is consistent with the hypothesis that an increase in
the hydrophobicity of the aqua ligand environment increases the aqua ligand’s acidity.33 It is
anticipated that CD substitution should also affect pKaH2O#1 as the aqua ligand appears likely to
experience a significantly different and probably more hydrophobic environment in the βCDtren
complexes by comparison with that in the analogous tren complexes. Correspondingly, it is
observed (Table 3.6) that for the CDtren complexes of all four M2+ there is a substantial
decrease in the pKaH2O#1 values by comparison with those of the analogous tren complexes. For
the Zn2+, Cu2+ and Ni2+ βCDMe5tren complexes there is much smaller decrease in pKaH2O#1 by
comparison with the analogous Me6tren complexes and for the Cd2+ complexes this trend
reverses. This may be a consequence of the hydrophobicity decrease engendered by the methyl
substituents in each case decreasing the effect of the βCD substitution in the βCDMe5tren
complexes.
The size of the variation of the magnitude of pKaH2O#1 with M2+ for the tren complexes is 2.26
units, which deceases to 0.41 units for the Me6tren complexes. This change is attributable to the
effect of the increased hydrophobicity of the latter ligand in increasing aqua ligand acidity.
Substitution of CD onto tren also decreases the variation of the magnitude of pKaH2O#1 with M2+
for the CDtren complexes, in this case to 0.74 units which is attributable to the effect of the
increased hydrophobicity engendered by the CD substituent. In the case of the βCDMe5tren
complexes the size of the variation of the magnitude of pKaH2O#1 is 0.62 units. Thus, for the last
three systems the effects of hydrophobicity appear substantial in determining the magnitude of
pKaH2O#1.
Beyond the above effects, it appears that a range of factors probably affect the magnitude
variation of the pKaH2O#1 such that they do not vary in a similar sequence across the ligand series
as the identity of M2+ changes. Thus, the pKaH2O#1 for the complexes of βCDtren increase in the
sequence: Cu2+ < Zn2+ < Cd2+ < Ni2+ while the sequence for the complexes of βCDMe5tren is:
Zn2+ < Ni2+ < Cu2+ < Cd2+. In contrast, the pKaH2O#1 for the complexes of tren increase in the
sequence: Cu2+ < Zn2+ < Cd2+ < Ni2+ while the sequence for the complexes of βCDMe5tren is:
Cu2+ < Zn2+ < Cd2+ < Ni2+. The data available in this study do not permit a detailed explanation
97
of these variations, but it is probable that differences in the M2+-OH2 bond distance are an
underlying cause, as was hypothesised in Chapter 2.33 There are insufficient pKaH2O#2 data
available to facilitate an analysis similarly to that presented for pKaH2O#1, other than to note its
variation for the four M2+ complexes of βCDMe5tren is constrained to 0.18 units consistent with
the hydrophobic argument presented above.
3.2.4 βCDtacn and βCDMe2tacn metal complex characterisation

Complex formation of the ligands CDtacn and CDMe2tacn (Figure 3.1) with Zn2+, Cu2+,
Ni2+ and Cd2+ was characterised by potentiometric titrations of solutions containing [L]total to
[M2+]total ratios of both 1:1 and 1:1.5. The magnitudes of the constants and the corresponding
speciations derived from both sets of data were compared in order to ensure that all states of
protonation of both the free ligands and the metal complexes had been accounted for in the data
analyses. Each experiment was carried out in duplicate at 298.2 K in aqueous NaClO4 (I = 0.10
mol dm-3) as described in Section 8.2.2 and the data reported represents the average of two
titrations.
The titration curve and speciation plot for βCDMe2tacn with Cd2+ are shown in Figures 3.8
and 3.9, both of which typify the titration curves and speciation plots for all of the βCDtacn and
βCDMe2tacn complexes investigated in this study. (The speciation plots for all of the complexes
and the titration curves for the new complexes - βCDMe2tacn with Zn2+, Cu2+, Ni2+ and Cd2+ -
can be found in Appendix B.) In the range between the titre values 0.18 mL and 0.21 mL
equilibration was slow such that every point was recorded on the maximum instrumental wait
time of 300 s which introduces the possibility of inaccuracies because the mV readings reported
are may not reflect the final equilibrated composition of the solution at that pH. Therefore,,
although, the fit obtained is reasonable and the errors in the derived logK1 and related parameters
are small (Table 3.7), these results for the βCDMe2tacn/Cd2+ system must be treated with caution.
Similarly slow equilibrium processes were observed in segments of all of the titrations involving
M2+ complexation by βCDtacn and βCDMe2tacn and also in the titrations of the free ligands in
which the pKas of their protonated forms were determined (as previously discussed). This
contrasts with the titrations involving βCDtren and βCDMe5tren for which slow equilibria were
only observed in the presence of Ni2+ in some segments of the titrations and in the determination
of the pKas of the free βCDMe5trenH44+. These differences are consistent with slow
98
conformational change in the nine-membered rings of βCDtacn and βCDMe2tacn affecting the
rate at which equilibrium is achieved during the titrations.
11.5
10.5
9.5
8.5
7.5 Observed
pH
6.5 Fit
5.5
4.5
3.5
2.5
0.125 0.175 0.225 0.275
Titre (mL)
Figure 3.8. Potentiometric titration curve for the determination of the βCDMe2tacn/Cd2+ system
characteristics described by logK1′, logK1, pKaH2O#1, pKaH2O#2 and pKaH2O#3 – observed points (◊)
and best-fit of the pH variation algorithm based on Equations 3.1 – 3.3 and 3.5 – 3.9 (-),
resulting in the values reported in Tables 3.12, 3.13 and 3.14. Aqueous NaClO4 (I = 0.10 mol
dm-3), 298.2 K. The initial [βCDMe2tacn] = 1.0 × 10-3 mol dm-3, the initial [Cd2+] = 1.0 × 10-3
mol dm-3, the added [HClO4] = 1.0 × 10-2 mol dm-3, and the titrant [NaOH] = 0.10 mol dm-3.
There is some evidence that ligands based on the 12-membered macrocycle 1,4,7,10-
tetraazacyclododecane (cyclen), for which a Ni2+ complex was characterised by logK1 = 16.4,34, 35
also produce slow equilibria. When substituted by βCD to form βCDcyclen, slow equilibria were
observed in titrations to determine logK1 for the formation the Zn2+ complex. Similar effects were
observed in titrations of the βCDcyclen/Ni2+ system to the extent that logK1 could not be
determined at all for this complex.36 These observations are also consistent with a combination
of slow conformational change and steric crowding by the CD substituent slowing equilibration.
99
100
90
free L

80
LH⁺
70
LH₂²⁺
60
LH₃³⁺
50
[MLH]³⁺
40
[ML]²⁺
30 [M(L)(OH)]⁺
20 [M(L)(OH)₂]
10 [M(L)(OH)₃]⁻
0
3 5 7 9 11
pH
Figure 3.9. Speciation plot showing the variation of species in the βCDMe2tacn/Cd2+ system as
pH is varied relative to the total concentration of all βCDMe2tacn and its protonated and
complexed forms being equivalent to 100% speciation: βCDMe2tacnH33+ (LH33+),
βCDMe2tacnH22+ (LH22+), βCDMe2tacnH+ (LH+), βCDMe2tacn (free L), complex
[Cd(βCDMe2tacnH)] 3+ ([MLH] +), complex [Cd(βCDMe2tacn)] 2+ ([ML] 2+), complex
[Cd(βCDMe2tacn)(OH)] + ([ML(OH)] +), complex [Cd(βCDMe2tacn)(OH)2] ([ML(OH)2]) and
complex [Cd(βCDMe2tacn)(OH)3] - ([ML(OH)3] -. Aqua ligands are not shown.
Thus, best-fit of a pH variation algorithm based on Equations 3.1 – 3.3 and 3.5 – 3.9
(Equation 3.9 appears below) to the titration data for βCDtacn and βCDMe2tacn in the presence
of either Zn2+, Cu2+, Ni2+ or Cd2+ yielded the logK1 values in Table 3.7 except for the that of the
βCDMe2tacn/Ni2+ as is discussed below. The analogous values for the tacn and Me3tacn systems
from Chapter 2 are also presented for comparison. In principle, the tridenticity and facial
coordination of these ligands allow for both 1:1 [M(L)]2+ and [M(L)2]2+ octahedral complexes to
be formed, but inclusion of this species in the fitting algorithm for the four M2+ studied resulted
in a failure to fit. Thus, it appears that at best [M(L)2]2+ is minor species under the conditions
used, if it forms at all. Nevertheless, a [M(L)2]2+ complex has been reported where L is tacn, but
Me3tacn is considered too bulky for [M(L)2]2+ complex formation.37-40 Consequently, the added
bulk of the βCD moiety to these amines is expected to preclude the possibility of [M(L)2]2+
complex formation. In the [M(L)]2+ complexes of βCDtacn and βCDMe2tacn it is expected that
100
βCD substituent is likely to cause some steric strain and therefore some distortion of the ligand
nine-membered ring which may affect coordination of M2+ in turn.

[M(L)]2+ tacn 11.58 ± 0.02 15.00 ± 0.03 11.06 ± 0.01 9.53 ± 0.05
Me3tacn 9.74 ± 0.03 14.01 ± 0.009 6.46 ± 0.06 9.07 ± 0.04
βCDtacn 8.92 ± 0.01 13.27 ± 0.02 5.80 ± 0.03 7.22 ± 0.04
βCDMe2tacn 8.24 ± 0.03 11.78 ± 0.04 6.37 ± 0.05
Table 3.7. Potentiometrically determined logK1 values for the [M(L)] 2+ complexes of tacn,
Me3tacn, βCDtacn and βCDMe2acn formed with Zn2+, Cu2+, Ni2+ and Cd2+ at 298.2 K in
aqueous NaClO4 (I = 0.10 mol dm-3) in this study.
Similar to tacn and Me3tacn, the βCDtacn and βCDMe2tacn M2+ complexes exhibit a decrease
in logK1 upon methylation which is attributed to the methyl substituents of βCDMe2tacn
hindering coordination of the electron lone pairs of the nitrogen donor atoms to the M2+.
Similarly, the logK1 of the βCDtacn and βCDMe2tacn M2+ complexes are lower than those of
their tacn and Me3tacn analogues, respectively, as a consequence of the CD causing additional
steric hindrance.
Interestingly, a comparison of logK1 changes for tacn imposed either by methylation (to give
Me3tacn) or by βCD substitution (to give βCDtacn) reveals that the substitution of βCD results in
a larger decrease in logK1 than does methylation, such that logK1 for the βCDtacn M2+ complexes
are lower with every metal than they are for the corresponding Me3tacn M2+ complexes, relative
to the analogous tacn M2+ complexes. This is the opposite of the trend that is seen for the tren
M2+ complexes (Table 3.4) where methylation to produce the Me6tren M2+ complexes results in a
much larger decrease in logK1 than does βCD substitution for the βCDtren M2+ complexes. This
appears to be a result of the differing degrees of methylation that the tren and tacn ligands
undergo. It is notable that the size of the logK1 decrease on going from the tren M2+ complexes to
the βCDtren M2+ complexes is very similar for the logK1 decrease on going from the tacn M2+
complexes to the βCDtacn M2+ complexes. (The only exception is for Ni2+ which is another
101
demonstration of the different chemistry for the complexes with this metal ion.) This suggests
that βCD substitution affects the complexing abilities of tren and tacn to a similar extent and that
it is simply the degree of methylation that decides whether methyl group or βCD substitution
dominates the trend in logK1 magnitude. As discussed in Section 2.2.3, methylation has a far
more significant relative effect on tren because it results in twice as many methyl groups being
added by comparison with methylation of tacn and removes all 1° amine donor atoms which have
already been identified as a large contributor to complex stability.
All four tacn-based ligands produce the most stable complexes with the same two metals:
logK1 Cu2+ > Zn2+. Also, for Cu2+, Zn2+ and Ni2+ the stability of the complexes formed follow the
trend predicted by the Irving-Williams Series27 as is a recurring feature for all of the ligands
studied herein. However, when the physical bulk of the ligands is increased, changes in the
relative and absolute magnitudes of logK1 are observed for the complexes of Ni2+ and Cd2+. Thus,
the least sterically hindered ligand tacn forms a complex with Ni2+ (logK1 = 11.06) which is
almost as stable as the complex it forms with Zn2+ (logK1 = 11.58), whereas its complex with
Cd2+ (logK1 = 9.53) is much least stable. However, the Me3tacn (logK1 = 6.46) and CDtacn
Ni2+complexes (logK1 = 5.80) are far less stable, even by comparison with the analogous Cd2+
complexes for which logK1 = 9.07 and 7.22, respectively. This is a little surprising as it might be
expected that the larger size Cd2+ would accentuate the effect of increased steric crowding in the
ligands. Nevertheless, Ni2+ remains less able to coordinate Me3tacn and CDtacn than does Cd2+.
As the extent to which methylation and CD substitution of tacn destabilises the Ni2+ complexes
in the sequence tacn (logK1 = 11.06), Me6tacn (logK1 = 6.46) and CDtacn (logK1 = 5.80) is
greater than that for Zn2+, Cu2+ and Cd2+, it is plausible to argue that the combination of both
methylation and CD substitution in CDMe2tacn renders the logK1 for its Ni2+ complex too
small for detection by the methods used in this study.
The speciation plot for βCDMe2tacn and Cd2+ (Figure 3.9) shows that the hypodentate
[Cd(βCDMe2tacnH)]3+ complex (characterised by logK1′ in Table 3.8) exists over a very similar
pH range to the free βCDMe2tacnH+ ligand. The protonated βCDMe2tacnH+ ligand is potentially
bidentate and positively charged and as a consequence the magnitude of logK1′ (3.49) is
substantially less than that of the logK1 (6.37) characterising the Cd2+ complex formed with
tridentate and neutral βCDMe2tacn. Similar magnitude relationships apply for those systems in
Table 3.7 in which monoprotonated ligands complex M2+ (compare to Table 3.8). No hypodentate
102
complexes were detected for tacn and Me2tacn which is consistent with the CD substituents of
βCDtacn and βCDMe2tacn stabilising hypodenticity.

(L) logK1' logK1' logK1' logK1'
[M(LH)]3+ βCDtacn 6.40 ± 0.04 3.43 ± 0.06
βCDMe2tacn 4.62 ± 0.02 5.83 ± 0.08 3.49 ± 0.05
[M(βCDtacnH)] 3+ and [M(βCDMe2tacnH)] 3+ formed with Zn2+, Cu2+, Ni2+ and Cd2+ at 298.2 K
in aqueous NaClO4 (I = 0.10 mol dm-3).
Also shown in the speciation plot for βCDMe2tacn and Cd2+ (Figure 3.9) are the deprotonated
aqua ligand species [Cd(Me2tacn)(H2O)2(OH)]+ and [Cd(Me2tacn)(H2O)(OH)2]0 characterised by
logKaH2O#1 and logKaH2O#2 in Equations 3.7 and 3.8, respectively, and [Cd(Me2tacn)(OH)3]-
characterised by logKaH2O#3 in Equation 3.9. These data are presented for all four ligands tacn,
Me2tacn, CDtacn and CDMe2tacn complexed with Zn2+, Cu2+, Ni2+ and Cd2+ in Table 3.9.
KaH2O#3
[M(L)(OH)2(H2O)] ⇌ [M(L)(OH)3] ¯ + H+
where KaH2O#3 = [M(L)(OH)3¯][H+]/[M(L)(OH)2(H2O)] 3.9
The most obvious trend in the data in Table 3.9 is that the Cu2+ complexes exhibit the largest
number of acid dissociations of their aqua ligands and that their pKaH2O#1, pKaH2O#2 and pKaH2O#3
magnitudes are less than those of the corresponding Zn2+, Ni2+ and Cd2+ complexes where data is
available. This trend is also followed by the higher pKaH2O#1 and pKaH2O#2 magnitudes for the tren,
Me6tren, CDtren and CDMe5tren complexes of Cu2+ in comparison with the analogous
magnitudes for the corresponding Zn2+, Ni2+ and Cd2+ complexes (Table 3.6).
103
(L) pKaH2O#1,2&3 pKaH2O#1,2&3 pKaH2O#1,2&3 pKaH2O#1,2&3
[M(L)(OH)]+ Tacn 8.90 ± 0.11 7.99 ± 0.04 11.66 ± 0.06 9.03 ± 0.03
Me3tacn 10.20 ± 0.09 7.85 ± 0.03 8.15 ± 0.20 9.46 ± 0.05
βCDtacn 9.68 ± 0.02 7.80 ± 0.02 8.84 ± 0.03 9.47 ± 0.05
βCDMe2tacn 9.27 ± 0.02 5.05 ± 0.093 9.04 ± 0.05
[M(L)(OH)2] Tacn 11.35 ± 0.05
Me3tacn 11.30 ± 0.09 9.64 ± 0.03 12.03 ± 0.20 10.92 ± 0.05
βCDtacn 11.01 ± 0.03 10.03 ± 0.03 11.06 ± 0.05 10.57 ± 0.05
βCDMe2tacn 8.32 ± 0.12 10.18 ± 0.06
[M(L)(OH)3]¯ Tacn
Me3tacn
βCDtacn 11.21 ± 0.03
βCDMe2tacn 9.86 ± 0.12 11.63 ± 0.06
Table 3.9. The pKaH2O values for the aqua ligands associated with complexes of tacn, Me3tacn, βCDtacn and βCDMe2tacn with Zn2+, Cu2+, Ni2+
and Cd2+ determined by potentiometric titration at 298.2 K in aqueous NaClO4 (I = 0.10 mol dm-3).
104
This is coincident with the Cu2+ d9 electronic configuration which engenders Jahn-Teller
distortions. On the basis that shortening of a M2+-OH2 bond is likely to increases aqua ligand
acidity,32 it appears that this may be particularly prevalent in the Cu2+ complexes of the tacn-
based ligand complexes and less so for the tren-based ligand complexes. The most acidic aqua
ligands are present in [Cu(βCDMe2tacn)(H2O)3]2+ for which pKaH2O#1 = 5.05 is the lowest
determined for all of the complexes studied, as are pKaH2O#2 = 8.32 and pKaH2O#3 = 9.96. Thus,
pKaH2O#1 = 5.05 for [Cu(βCDMe2tacn)(H2O)3]2+ is the only pKaH2O#1 of all of the M2+ complexes
studied (Tables 3.6 and 3.9) that is lower (by 1.75 units) than pKaH2O#1 = 6.8 for the single aqua
ligand bound to Zn2+ in carbonic anhydrase.41, 42 All of the other pKaH2O#1 listed in Tables 3.6 and
3.9 are more than 1 unit greater than 6.8. This is significant because increasing the pH range over
which a hydroxo ligand (available to perform ester hydrolysis) is present would be a key feature
of newly designed enzyme mimics. Apart from these observations, there is no consistent overall
trend which differentiates between the acidity of the aqua ligands in the tacn-based ligand
complexes from the tren-based ligand complexes.
While this Chapter and the preceding Chapters have been predominantly concerned with the
characterisation of functionalised β-cyclodextrins and their binary, or 1:1 complexes, ultimately
their ternary or 1:2 complexes become important where the third component (or second ligand
partially included in the cyclodextrin annulus) is present. The ester 4-nitrophenyl acetate is of
major importance in the metalloenzyme mimic studies discussed next in Chapter 4. While 4-
nitrophenyl acetate is hydrolysed in the studies described in Chapter 4, ternary complexes have
been well-characterised where the second ligand does not undergo catalysed hydrolysis.23 One
such example is the βCDtren/[Cu(βCDtren)]2+/[Cu(βCDtren)(S-Trp)]+ system (where S-Trp- is S-
tryptophanate) in which the step-wise stability constants have been separately determined for the
formation of the [Cu(βCDtren)]2+ and [Cu(βCDtren)(S-Trp)]+ as shown in Figure 3.10. It is seen
that while S-tryptophanate is included in the βCDtren, its primary amine group and a carboxylate
oxygen displace two aqua ligands to form two bonds to the copper centre. While there is some
uncertainty as to whether the copper centre in this complex is six-coordinate, as shown in Figure
3.10, or is five-coordinate, S-tryptophanate does likely bind directly to the copper centre because
the stability constant for this ternary complex is greater than that of the analogous βCDtren•S-trp+
inclusion complex.
105
Figure 3.10. Step-wise formation of the binary [Cu(βCDtren)] 2+ and ternary [Cu(βCDtren)(S-
Trp)] + complexes and the step-wise logK characterising them at 298.2 K in aqueous NaClO4 (I =
0.10 mol dm-3).23
3.3 Conclusions
The complex stability constants and the pKa values for sixteen transition metal complexes and
four ligands have been determined through the research described in this Chapter. In some cases,
similar studies have been reported in the literature and it is found that the reported data is in good
agreement with the data obtained in this study.
As a major theme of this study is the use of these metal complexes as enzyme mimics it is
appropriate to assess their suitability for such use. The slow attainment of equilibrium
encountered exemplified by the Ni2+ complexes of tacn, Me3tacn, βCDtacn and βCDMe2tacn
renders them unsuitable for use as enzyme mimics. Consequently, these complexes and others
exhibiting slow attainment of equilibrium were not further considered as enzyme mimics.
On comparison of all 32 complexes now characterised in Chapters 2 and 3, a clear trend

emerged - for all eight ligands the metal that resulted in both the most stable complex formation
and also the lowest water ligand pKaH2O#1 (in all but one case - βCDMe5tren) was Cu2+. Stable
complexes (evidenced by large stability constants, logK1) are advantageous because they ensure
that the desired complex will be present in high concentrations and that free ligand and free metal
will not exist to any great extent. Free ligand and metal are problematic when testing ester
hydrolysis activity because they can also catalyse the reaction along with the complex hydroxide
species of interest. Thus, [Cu(βCDMe2tacn)(H2O)3]2+ is one of the more promising systems for
enzyme mimicry study as at the pH equal to its pKaH2O#1 (5.05) there is < 5% formation of free
metal and ligand combined, and the vast majority of ligand is found as [Cu(βCDMe2tacn)]2+ or
[Cu(βCDMe2tacn)(OH)]+. In the search for more similar amine ligands for enzyme mimicry, it is
106
possible that their Cu2+ complexes will be most successful in terms of complex stability and
promotion of the catalytic hydroxide reactant.
The 32 complexes in this study were investigated in order to produce a well-characterised

catalogue of these species. Comparisons of macrocyclic versus acyclic, tridenticity versus
tetradenticity, 1° versus 2° versus 3° amines, unmethylated versus methylated, free amine versus
βCD-substituted and all across four transition metal cations resulted in a large amount of
information. This has provided an understanding into how each of these features affects ligand
basicity, complex stability and aqua ligand pKaH2O, and allows insight into how intricate enzyme
characteristics may be mimicked in a simplified fashion on target analogues.
This study was successful in using these simplified methods and producing an enzyme mimic
with an aqua pKaH2O even lower than that of carbonic anhydrase. However, formation of the
catalytic hydroxide species is only one factor which influences catalytic efficiency. The aim of
characterising all the complexes in this study by potentiometric titration was also therefore to
select the most promising species for kinetic testing. Undoubtedly the βCD versions of the
ligands are of the most interest as they contain the enzyme-mimicking active site feature of the
hydrophobic annulus to trap the reactant ester. Furthermore these ligand versions also produce
some of the lowest aqua ligand pKaH2Os making them most attractive for catalytic success.
Without the constraints of time and resources, all of the complexes with a large enough logK1
to avoid appreciable formation of free ligand and metal should be tested for their catalytic ability
(kinetic studies). However, synthesis of the ligands in the quantities required for this is time-
consuming and the preparation for, and carrying out of, the kinetics experiments themselves is
lengthy. Therefore two ligands were chosen for kinetic analysis as part of this study – βCDtren
and βCDMe5tren. (It should be noted that whilst the βCDtacn and βCDMe2tacn complexes
produce the lowest pKaH2O#1 values, these were the final systems to be synthesised and were
undergoing potentiometric titration characterisation whilst the kinetics experiments were being
performed. The βCDtren and βCDMe5tren ligands were therefore selected as the most promising
species at the time of analysis. Similar kinetics testing for the βCDtacn versions could be carried
out as part of future work.)
As a broad trend, the complexes of Cu2+ and Zn2+ appear to have more predictable chemistry
than for the other two metals and also tend to produce stable complexes and low aqua pKaH2Os in
general. Furthermore these are the most biologically important of the four metals investigated in
107
this study – Zn2+ and Cu2+ are two of the metals most commonly found at the active sites of
metalloenzymes.43 Consequently it was decided to test the catalytic activity of the βCDtren and
βCDMe5tren complexes with Cu2+ and Zn2+. It was noted, however, that whilst
[Zn(βCDMe5tren)]2+ has the second largest stability constant for this ligand with all four metals,
it is still very low at logK1 = 5.61. Analysis of the titration data revealed that at a pH equal to the
pKaH2O#1 of the corresponding aqua ligand on the complex (the pH at which kinetics experiments
are run), there is 2% formation of free ligand, 4% formation of free Zn2+ and 15% formation of
the insoluble metal hydroxide species Zn(OH)2. It was deemed that this complex was not a
suitable target for kinetics work and therefore the three systems [Zn(βCDtren)]2+,
[Cu(βCDtren)]2+ and [Cu(βCDMe5tren)]2+ were prepared for catalytic analysis. Catalytic analysis
involved several sets of experiments for each complex and these are discussed at length in
Chapter 4.
108
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111
Hilary Coleman Chapter 4. Catalysis testing of the enzyme mimics: the kinetics of 4-nitrophenyl acetate hydrolysis
CHAPTER 4
CATALYSIS TESTING OF THE ENZYME MIMICS:

THE KINETICS OF 4-NITROPHENYL ACETATE
HYDROLYSIS
112
4.1 Introduction
4.1.1 Functionalised β-cyclodextrins in enzyme mimicry

Functionalised β-cyclodextrins (βCDs) have been studied as effective biomimetic compounds
for many years and there are extensive reviews describing this work.1-4 The success of the
application of functionalised β-cyclodextrins in this field is due to the combination of their
availability (as naturally occurring molecules themselves), their ability to bind (and thereby
solubilise) hydrophobic guests in aqueous solution, and the hydroxyl groups present on their rims
allowing βCDs to either directly react with substrates or provide sites for functional group
addition.2 A diagram displaying the βCD annular structure and the six-membered 1,4-
glucopyranose rings that comprise it are shown in Figure 4.1. As detailed in Section 1.3, these
features equip cyclodextrins for the applications in enzyme mimicry and have resulted in their
widespread employment in this field of research.
Figure 4.1. Diagram of β-cyclodextrin (n = 7, βCD) depicting the annular structure and the α-
1,4-linked glucopyranose units in the rigid 4C1 chair configuration which define the annulus. For
αCD and γCD n = 6 and 8 respectively. The 2 and 3 carbons define the wide rim of the annulus,
and the 6 carbons define the narrow rim. These three labelled carbons are possible sites for
functionalisation.
The proposal that cyclodextrins could be used as components of enzyme mimic models was
first raised in the 1950s when initial studies showed that the oxyanions of cyclodextrins could
react with bound pyrophosphates.5, 6 Since then, studies of functionalised cyclodextrins acting as
artificial glycosidase7, esterase8, aminotransferase9 oxidase10, thioester hydrolase11 and
113
epoxidase12 mimics and also as catalysts for cycloadditions13 and for aqueous biphasic
processes14 have been reported. In fact, these are just a small sample of examples that highlights
their prevalent use in the field of biomimicry.2, 4
Central to the present study is the investigation of functionalised cyclodextrins as potential

hydrolase mimics – mimics of enzymes that hydrolyse bonds. More specifically it is aimed at
examining the enzymes that fall into the International Union of Biochemistry and Molecular
Biology subclass 3.1.1 (see Chapter 1), the esterases – enzymes acting on carboxylic ester
bonds.15 Examples in the literature of cyclodextrins employed in the area of ester hydrolysis
include βCD functionalised with organic moieties16-18 (including polymers19, 20
), βCD
functionalised with ligands in metal complexes21-25 and βCD dimers.26-30 For the research
conducted in this chapter the metal complexes of the βCD functionalised ligands discussed in the
preceding chapters are of most interest. However kinetic characterisation of the free βCD amine
ligands synthesised in this study, native βCD and the free metal ions is also undertaken in order
to gain maximum insight into the features of these enzyme mimics which make them most
successful.
4.1.2 4-Nitrophenyl acetate hydrolysis as a model reaction

The ester 4-nitrophenyl acetate has been used prolifically as a substrate in investigations of
the hydrolysis reaction31, 32
to the extent that it is sold by Sigma Aldrich as “4-Nitrophenyl
acetate esterase substrate”.33 Its ubiquitous employment in experiments of this type is the result
of several properties. Firstly it is an activated ester which is susceptible to hydrolysis and it
therefore facilitates a broad range of enzyme mimic investigations. Secondly, when human
carbonic anhydrase II (hCA II) was tested for its ester hydrolysis catalytic activity, the most
active ester substrate discovered was 4-nitrophenyl acetate.34 Since the design of the enzyme
mimics in this study were inspired and informed by the structure of carbonic anhydrase, this ester
was considered to be an ideal substrate for the research described in this Chapter.
4-Nitrophenyl acetate is soluble in water with 5% acetonitrile, as are the hydrolysis products:
4-nitrophenolate and 4-nitrophenol. All three compounds are displayed in Table 4.1 along with
their λmax which differ substantially.35 The rate of the hydrolysis of the 4-nitrophenyl acetate
substrate may therefore be measured by either monitoring the absorbance decrease at 271 nm as
the ester is consumed or the absorbance increase at 320 nm or 400 nm as the 4-nitrophenol or 4-
114
nitrophenolate are produced.36 4-Nitrophenol has a pKa of 7.1537 and therefore the hydrolysis
product formed will be dictated by the pH of the solution. Although the 4-nitrophenol absorption
spectrum partially overlaps with those of 4-nitrophenyl acetate and 4-nitrophenolate, the ester
reactant has virtually no absorbance at 400 nm where 4-nitrophenolate has a large molar
extinction coefficient (approximately 18 500 dm-3 mol-1 cm-1 38, 39) and therefore analysis at this
wavelength is optimal.
Substrate Substrate Substrate
Table 4.1. 4-Nitrophenyl acetate, 4-nitrophenolate and 4-nitrophenol absorbance maxima,

λmax.35
It is the combination of these characteristics (reactivity, max) that makes 4-nitrophenyl

acetate an ideal model substrate in studies which investigate the catalytic activity of βCDs
towards ester hydrolysis.16-18, 21, 23, 25, 27, 28, 40 It is therefore employed in this study as it is a model
substrate and the results can be compared with those of other studies by keeping the identity of
the ester consistent.
The key steps in the mechanism proposed for the catalysis of the hydrolysis of 4-nitrophenyl
acetate by the potential enzyme mimics in this study, [M(βCDtren)(H2O)2]2+ (where M is Cu2+ or
Zn2+), are depicted in Figure 4.2. They are based on the Michaelis-Menten mechanism proposed
for enzymic processes (discussed in detail in Section 4.1.3) and the terms derived from this
mechanism are used throughout the remainder of the chapter.
115
Figure 4.2. The mechanism proposed for the hydrolysis of 4-nitrophenyl acetate by the divalent M(II) enzyme mimic [M(βCDtren)(OH)](OH2)] + which is
thought to proceed through either species A, B, C, C′ to E. or A, B, C, C′′, D to E.(It is assumed that M retains a six-coordinate state as aqua ligand
deprotonation occurs.)
116
In Figure 4.2, five steps are shown in which an aqua ligand of A, [M(βCDtren)(H2O)2]2+,
deprotonates to form the hydroxo ligand nucleophile in B, [M(βCDtren)(OH)(H2O)]+. This is
followed by the complexation of 4-nitrophenyl acetate in the CD annulus to form C (which is
likened to the Michaelis complex, E•S, and characterised by the Michaelis constant, KM, as
discussed in Section 4.1.3). Subsequently, the remaining aqua ligand of C is substituted by an
acetate oxygen to give D wherein a nucleophilic attack by the hydroxo ligand on the carbonyl
carbon of 4-nitrophenyl acetate results in hydrolysis to give E which is followed by the release of
acetate and 4-nitrophenolate from E to regenerate A. (The formation of D is not proven in this
study, but as similar species have been shown (or proposed) to occur in related studies it is
incorporated into Figure 4.2 as possible intermediate.41,26, 42-45
Assuming that it occurs, the
displacement of the aqua ligand in C by 4-nitrophenyl acetate is likely to be rapid as both Zn2+
and Cu2+ are very labile metal ions.46,47 Furthermore, in this study there is some evidence that the
ester does interact with the metal centre in the case of the Cu2+ system, although more
investigation should be conducted to determine the definitive mechanism as part of Future Work.
As such, the generation of D could be omitted such that the nucleophilic attack by the hydroxo
ligand could occur still in C’ without the ester initially binding to the metal centre.)
The dihydroxo species, [M(βCDtren)(OH)2], may also act as an enzyme mimic, as has been
found for other enzyme mimic dihydroxo metal complexes,29,46 but mechanistic factors render
this species unavailable as an enzyme mimic (discussed in Section 4.1.5).
4.1.3 The Michaelis-Menten enzyme kinetics model

Enzyme and enzyme mimic kinetics have been analysed using the Michaelis-Menten model
for a century.48 The analysis is based on the following scheme which represents a single-
substrate, bimolecular, enzyme-catalysed reaction:49
k1 k2
E + S ⇌ E•S ⇌ E + P 4.1
k-1 k-2
where E, S and P represent the free enzyme (or enzyme mimic), substrate and product,
respectively and E•S represents the enzyme-substrate complex (or Michaelis complex). The
equivalent representation for the proposed enzyme mimics, [M(βCDtren)(OH)(H2O)]+, appears in
Figure 4.2. The rate constant k1 characterises the association of E and S to form the complex E•S,
117
and k-1 describes the dissociation of E•S. The ratio k-1/k1 = KD the dissociation constant for E•S.48,
50
The rate constants k2 and k-2 characterise the product, P, generation and the regeneration of free
enzyme, E, and the reverse process, respectively. (Usually the reaction is irreversible such that k-2
= 0.)
The kinetics of product formation through the scheme in Equation 4.1 (where k-2 = 0) may be
described by the Michaelis-Menten expression below (Equation 4.2):
V0 = (Vmax[S])/(KM + [S]) 4.2
where V0 is the observed initial rate of reaction (measured by the initial rates method51), Vmax is
the maximum possible initial rate achieved for the reaction in the presence of the enzyme E, [S]
is the concentration of substrate and KM is the substrate concentration at which the reaction rate is
half the maximum value. The derivation of equation 4.2 assumes that the association of S with E
to form E•S, characterised by k1, and the dissociation process characterised by k-1 occur much
more rapidly than the irreversible catalytic process described by k2. Thus, equilibrium ratios of
[E], [S] and [E•S] are very rapidly established and the catalytic step characterised by k2 is the
rate-determining step for the conversion of S to the product, P. (The rate constant k2 is often
rewritten as kcat, as is the case in sections which follow.)
The KM equilibrium constant is therefore the dissociation constant for the E•S complex and
reflects the affinity of the enzyme for its substrate. It is referred to as the Michaelis constant
because Michaelis and Menten first hypothesised that the formation of this complex was a
significant factor in the rate and mechanism of an enzyme’s function.48, 50
In deriving the classical Michaelis-Menten equation (Equation 4.2) from the reaction scheme
in Equation 4.1, there are several assumptions that must be made. As mentioned above, the
binding processes described by k1 and k-1 are generally much faster than the catalytic processes
described by k2 and k-2 such that the reaction immediately reaches steady equilibrium ratios of
[E], [S] and [E•S] and the catalytic process is therefore the rate-determining step.
Secondly, in the initial rates method of study, the catalysed reactions are usually only
followed for the first ≤ 10% of the reaction and only the initial rate, V0, is determined from the
tangent to the curvature of the plot of either reactant or product concentration change with time.
Under these circumstances, the proportion of E which remains associated with P in E•P (the
118
immediate successor complex of E•S) is sufficiently small such that Equation 4.1 becomes
Equation 4.3 in which kcat characterises the rate-determining step:
k1 kcat(RDS)
E + S ⇌ E•S → E + P 4.3
k-1
and the reaction rate is given by the first order rate law:
rate = V0 = kcat[E•S] 4.4
The variation of the concentration of the E•S complex during the reaction is difficult to
measure. However, when the total enzyme concentration is much less than that of the substrate,
[E]total << [S]total, it is assumed that [E•S] is in a steady state such that its concentration is
invariant during the portion of the reaction studied.52 Therefore the rate of E•S formation must be
equal to the rate of E•S dissociation such that:
k1[E][S] = k-1[E•S] + kcat[E•S] 4.5
which may be rearranged to:
[E][S] = [E•S]{(k-1 + kcat)/k1} 4.6
Equation 4.6 is simplified by combining the three rate constants into one overall constant, KM,
the Michaelis constant (Equation 4.7 below).
[E][S] = [E•S]KM 4.7
where KM = (k-1 + kcat)/k1
This is far more streamlined, however Equation 4.7 still contains the component [E•S] which
is difficult to measure (irrespective of whether it is varying or constant). This concentration needs
to be expressed in terms of quantities that are calculable. Because the enzyme concentration at
any point during a reaction is small (much smaller than the substrate concentration under
physiological conditions and therefore under Michaelis-Menten model conditions) and an
unknown proportion converts to [E•S], [E] is equally hard to quantify. However, the total enzyme
concentration, [Etotal], must be known by the experimenter:
[Etotal] = [E] + [E•S] 4.8
119
At any one time, the combined total of enzyme that is free ([E]) and enzyme that is involved
in the enzyme-substrate complex ([E•S]) must be equal to the total amount of enzyme ([Etotal]).
Rearranging Equation 4.8 to solve for [E] and then substituting into 4.7 gives:
([Etotal] – [E•S])[S] = [E•S]KM 4.9
Rearranging to solve for [E•S]:
[E•S] = [Etotal][S]/(KM + [S]) 4.10
Equation 4.10 now expresses [E•S] as a function of known factors, Etotal and S. It may now be
substituted into the original rate law, Equation 4.4, to give:
Rate = V0 = kcat[E•S] = (kcat[Etotal][S])/(KM + [S]) 4.11
In the special case of Equation 4.4 where the rate has reached the maximum, this will occur
under the conditions of [S] >> [E] when 100% of the free enzyme initially present becomes
bound in the E•S complex, i.e. when [E•S] approaches [Etotal], V0 approaches Vmax. This special
case is shown below:
Vmax = kcat[Etotal] 4.12
Equation 4.12 can therefore be substituted into Equation 4.11 to give the final expression
represented in Equation 4.13:
V0 = (Vmax[S])/(KM + [S]) 4.13
The assumptions required in this derivation are collectively known as the Quasi Steady State
Assumption and this has been shown to be valid for all cases of enzyme kinetics described by the
Michaelis-Menten model.52 Under these conditions, the equilibrium constant for the dissociation
of the E•S complex back to E and S (shown in Equation 4.14 below), KD, is equal to the
Michaelis constant, KM.
k1 kcat(RDS)
E + S ⇌ E•S → E + P 4.14
k-1
KD (= KM)
120
where KD conventionally = [product]/[reactant] = [E][S]/[ E•S] and a rearrangement of Equation

4.7 also shows KM = [E][S]/[ E•S]. The Michaelis constant therefore reflects the affinity of the
enzyme for its substrate and its measurement is necessary in characterising any enzyme mimic.
Using Equation 4.13, Vmax and KM may be determined using the initial rates method with
experimental conditions such that the [E] << [S] and between each experiment the [E] is held
small and constant whilst the excess [S] is varied. Under these conditions it follows that the
maximum rate that can be obtained (Vmax) would occur once there is enough substrate present
such that all of the enzyme has become bound in the E•S complex, i.e. when [E0] = [E•S], V0 =
Vmax. (When all of the least-available species is bound in the E•S complex, the rate will have
reached its maximum.) This process is known as saturation kinetics and is predicted by the
Michaelis-Menten model. Furthermore, when there is no substrate present, there will be no
reaction, as is demonstrated by substituting [S] = 0 into Equation 4.13: V0 = (Vmax × 0)/(KM + 0) =
0. A graph of V0 on the y-axis measured experimentally for each substrate concentration on the x-
axis ([S]), should yield a curve as illustrated in Figure 4.3.
Figure 4.3. Generic Michaelis-Menten variation of V0 according to Equation 4.13 under

conditions where the enzyme concentration is held constant and in deficiency, and the substrate
concentration is in excess and varied.
121
The levelling of the curve as the rate reaches a maximum displays the saturation kinetics as
described by the Michaelis and Menten model. Thus, the magnitude of V0 increases with
increasing [S] until all of the enzyme has become bound in the E•S complex, V0 = Vmax and
further increase in [S] produces no further increase in V0. At the other extreme, when [S] = 0, V0
= 0. This predicted variation of V0 with excess [S] is shown in Figure 4.3 from which it is seen
that when V0 = Vmax/2 the [S] at which this relationship holds is equal to KM.
4.1.4 Adaptation of the Michaelis-Menten model to catalysis by the enzyme

mimics [M(βCDtren)(OH)(H2O)]+
Naturally occurring enzymes are very sophisticated and usually accelerate the rate of the
reactions for which they are specific by many orders of magnitude and their regeneration after the
catalytic step is extremely efficient. However, in this study (and others17, 23, 45, 53), the substrate
concentration cannot ideally be in excess over the enzyme mimic under experimental conditions
because this leads to the potential for “catalyst poisoning”. For the reaction specific to this study,
the ester substrate 4-nitrophenyl acetate includes in the βCD annulus as part of the catalytic
mechanism (see Figure 4.2 and Section 4.1.2). However, the phenolate product has many of the
same features as the ester reactant and therefore may also include. (4-nitrophenolate has been
shown to form more stable inclusion complexes with amine-functionalised βCDs in the presence
of M2+ than in the absence of M2+ - due to the attraction of opposite charges - and both of these
species form more stable inclusion complexes in solution than 4-nitrophenolate with native
βCD54.) Contingent on the pH, the corresponding phenol may be present in some proportion as
well and also possibly form a inclusion complex with βCD. Depending on the relative stability
constants of all three inclusion complexes, there is the possibility that the βCD enzyme mimics
may be poisoned if the phenolate and phenol are in competition for βCD complexation with the
ester substrate. The corresponding values have not been determined for the specific enzyme
mimics in this study, however, the log values of the stability constants for the inclusion
complexes formed between free βCD and 4-nitrophenyl acetate, 4-nitrophenolate and 4-
nitrophenol (Kcomplex) at 298 K are displayed in Table 4.2 and they provide a qualitative indication
of the comparative stabilities of the inclusion complexes that may be formed in solution.
122
Substrate Substrate Substrate
a: Ref55 b: Ref56
Table 4.2. Complexation constants, logKcomplex, for the complexation of 4-nitrophenyl acetate, 4-
nitrophenol and 4-nitrophenolate by -cyclodextrin in aqueous solution at 298.2 K.
It can be seen that all three species – the desired ester substrate and its two possible product
structures – have a similar binding strength with βCD. Considering the catalytic mechanism
involves the production on 4-nitrophenol/4-nitrophenolate within the βCD annulus (Figure 4.2),
some form of interaction between the products and the enzyme mimics is therefore highly likely
and this is referred to as “catalyst poisoning”. Indeed, a very similar study, investigating the 4-
nitrophenyl acetate hydrolysis rate-enhancing capability of the Zn2+ complex of another amine-
functionalised βCD moiety (βCDtacdo), found that the persistence of the 4-nitrophenolate
product in the βCD annulus inhibited the catalytic activity.23 The solution employed in this study
is to perform the experiments under experimental conditions of [E] >> [S].
The conventional Michaelis-Menten treatment can therefore not be applied in this study
because the assumptions based on [E]/[S] ratios described in Section 4.1.3 will not hold. In other
sources which involve excess enzyme there are several methods for dealing with this reversal:
some studies simply do not analyse the data in terms of Michaelis-Menten enzyme kinetics and
therefore do not determine Vmax or KM,45 some studies do the experiments under both excess
enzyme and excess substrate conditions and use the latter for Michaelis-Menten enzyme kinetics
analysis57, 58 and finally others develop far more complex mathematical analyses that are similar
to the Michaelis-Menten equation to greater or lesser extents.53, 59, 60
In this study, a simple analysis in excellent agreement with the traditional Michaelis-Menten
model (and therefore also highlighting the saturation kinetics described in Michaelis and
Menten’s theory) has been developed. For this study the ester concentration was held constant
123
and in deficiency in each experiment whilst the enzyme mimic concentration was varied
increasingly from a minimum concentration of ten times the concentration of the ester. The
overall ester hydrolysis reaction is the result of 4-nitrophenyl acetate reacting with hydroxide,
and in the experiments in which the enzyme mimic is present this hydroxide is primarily supplied
by these metal complexes in the form of hydroxo ligands (Figure 4.2). However, in the absence
of the enzyme mimic ([E] = 0) there is still hydroxide present in the bulk solution whose
concentration is pH-dependent: pOH = 14 – pH and pOH = –log[OHˉ]. Therefore unlike the
conventional Michaelis-Menten treatment where zero substrate yields zero reaction (and the
Michaelis-Menten plot has a graph origin of 0, 0), when there is zero enzyme mimic there will
still be a background, uncatalysed ester hydrolysis reaction occurring.
As a consequence of these considerations, the arithmetic of the Michaelis-Menten scheme is

modified as shown in Equation 4.15 which recognises the two possible pathways for the ester to
be converted to the phenolate product: via catalysis with the enzyme mimic or spontaneously
(uncatalysed) by reaction with hydroxide in the bulk solution. Thus, kcat characterises the catalytic
rate determining step and kuncat characterises the irreversible autoprotolysis-generated hydroxide
hydrolysis of 4-nitrophenyl acetate. (For continuity the same symbolism as that used in the earlier
discussion of the Michaelis-Menten scheme is employed here.)
k1 kcat(RDS)
S + E ⇌ E•S → E + P 4.15
k-1
kuncat
The total overall observed rate is therefore contributed to by both the catalysed and
uncatalysed processes and when there is no enzyme present the observed rate must entirely be
due to the uncatalysed reaction. On this basis the rate of hydrolysis is described by the rate law
shown in Equation 4.16 where kobs is the experimentally determined pseudo first order rate
constant for a given excess enzyme mimic, E, concentration.
rate = kobs[S]total = kobs([S] + [E•S]) = kuncat[S] + kcat[E•S] 4.16
where kobs is the experimentally determined, observed overall pseudo first order rate constant for
a given excess enzyme concentration and:
124
i) When no E is present [E•S] = 0 and kobs = kuncat
ii) When all S is complexed in E•S, [S] = 0 and kobs = kcat
Just like the assumptions made for the conventional Michaelis-Menten derivation, it is also
assumed for the scheme above that the processes described by k1 and k-1 occur instantaneously
and therefore equilibrium concentrations of S, E and [E•S] are immediately attained.
Furthermore, the back reaction of the product interacting with the enzyme and reforming the
substrate is ignored. In the Michaelis-Menten model this is ignored because only the very initial
portion of the reactions are measured (V0). In this study, however, the rate constant (kobs) is more
accurately determined from monitoring the hydrolysis over the whole reaction duration. The back
reaction can still be ignored, however, because the equilibrium for the hydrolysis of 4-nitrophenyl
acetate lies very much on the product side.61
As in Section 4.1.3, [E•S] in Equation 4.16 is difficult to experimentally determine and may
be conveniently replaced by more readily measurable quantities. Therefore, just as the Michaelis
constant (the dissociation constant for the enzyme-substrate complex) can be calculated by the
ratio of rate constants in the conventional Michaelis-Menten derivation above (Equation 4.14), it
can also be calculated in terms of reactant and product equilibrium concentrations:
KM = [E][S]/[E•S] 4.17
Rearranging gives the relationship identical to Equation 4.7 which describes [E•S] in terms of
equilibrium enzyme and substrate concentrations:
[E•S] = [E][S]/KM 4.7
Substituting Equation 4.7 into Equation 4.16 yields:
kobs{[S] + ([E][S]/KM)} = kun[S] + kcat([E][S]/KM) 4.18
Which is rearranged to produce the final Equation 4.19 (see Appendix C for full derivation):
This new equation is very similar in format to the traditional Michaelis-Menten equation and
is used for analysis of the kinetics data in this study. It still describes the saturation kinetics for
enzymes as reflected by the curves in Sections 4.2.2 and 4.2.3. This model has also been used
125
successfully in the past for analysis of the rate enhancement of 4-nitrophenyl acetate hydrolysis
by a βCD catalyst.62 However, as modified from Figure 4.3, experimentally determined kobs may
be plotted on the y-axis (rather than initial rate, V0) and [enzyme] may be plotted on the x-axis
(rather than [substrate]). (See Section 4.1.5.)
In Equations 4.15 – 4.19 [E] represents the total enzyme mimic concentration. However, in
[M(βCDtren)(H2O)2]2+ the aqua ligands are poor nucleophiles and it is the hydroxo ligand in
[M(βCDtren)(OH)(H2O)]+ which is the effective nucleophile. Thus, at a given pH the proportion
of the effective enzyme mimic will depend on the pKa of the aqua ligand, as determined by
potentiometric titration. Thus, the amount of [E] in the active form in Equations 4.15 – 4.19 is pH
dependent. Hence, in investigating the variation of the first order rate constant, kobs, with pH there
are two considerations to be made. The first is that in the absence of the enzyme mimic ([E] = 0),
kobs = kuncat = kOH-[OH-] such that under these conditions kobs increases with pH. The second is
that at a particular pH, kobs - kuncat = kobs′ which characterises the contribution of the enzyme
mimic to the hydrolysis of S at that specific pH. The variation of kobs′ as a function of pH for a
given total enzyme mimic concentration is reflected in Equation 4.20:
kobs′ = kcat[M(βCDtren)(OH)(H2O)+]/([M(βCDtren)(OH)(H2O)+] + [M(βCDtren)(H2O)22+])

4.20
which may be simplified to:
kobs′ = kcatKa/(1 + [H+]) 4.21
where Ka is the acid dissociation constant for an aqua ligand of [M(βCDtren)(H2O)2]2+. Thus, the
pH dependence of kobs may be fitted to the Boltzmann expression:
kobs′ = kobs(max)′ + (kobs(min)′ - kobs(max)′)/(1 + (pH/pKa)slope) 4.22
where kobs(max)′ and kobs(min)′ are the maximum and minimum values encompassing the pH range
over which the aqua ligand undergoes complete deprotonation and slope is the slope of the curve
at the inflexion point where pH = pKa.
126
4.1.5 Aims and methods of this study

The aim of this study is to investigate the enzyme mimic catalytic activity of
[Zn(βCDtren)(H2O)2]2+ and [Cu(βCDtren)(H2O)2]2+ and their deprotonated forms as
characterised by potentiometric titration in Chapter 3. This is achieved by determining their
catalytic rate constants for the hydrolysis of the ester 4-nitrophenyl acetate and the affinity of
each complex for this substrate and the pH dependence of both. This involves the collection of
kinetic and related data and analysis thereof. As Future Work more systems should be studied.
However the systems reported here represent optimisation of the experimental procedures and
data analysis that may be used as a model for future investigations.
The [Zn(βCDtren)(H2O)2]2+ and [Cu(βCDtren)(H2O)2]2+ complexes were selected for study as

potential enzyme mimics on the basis of their high stabilities and well-defined pKas. The
deprotonation of their aqua ligands produce hydroxo ligands which are likely to be the
nucleophiles in the hydrolysis of complexed 4-nitrophenyl acetate (Figure 4.2). Accordingly, it
was decided carry out the kinetic catalysis experiments at the first aqua ligand deprotonation
characterised by pKaH2O#1 values of 9.14 and 8.68, respectively (see Table 3.6 in Chapter 3) such
that the active species [Zn(βCDtren)(OH)(H2O)]+ and [Cu(βCDtren)(OH)(H2O)]+ were present in
solution. The deprotonation of the second aqua ligands to give [Zn(βCDtren)(OH)2] and
[Cu(βCDtren)(OH)2] are characterised by pKaH2O#2 values of 10.00 and 9.77, respectively, and as
a consequence these species coexist with [Zn(βCDtren)(H2O)(OH)]+ and
[Cu(βCDtren)(H2O)(OH)]+, respectively, over the appropriate pH range. The consequences of
this coexistence are discussed further later.
In order to conduct the experiments at these constant pHs, the buffer compounds selected
needed to be soluble in water at 298.2 K, not competitively complex the metals under
investigation (Zn2+ and Cu2+) not include in the βCD annulus and not form insoluble precipitate
with any of the other species in solution under these conditions. (Despite the concentration of the
free metal ions in solution being negligible, ensuring the buffer species do not competitively bind
avoids the possibility that such coordination could change the equilibria between the enzyme
mimic metal complexes and the free metal ions.)
Unfortunately these parameters ruled out several common buffers over the pH range required.
Phosphate buffer cannot be used because zinc and copper phosphate are insoluble in water.63
Good's buffers are frequently used in biological and physiological studies64-66 and include ACES
127
(2-(carbamoylmethylamino)ethanesulfonic acid), TES (2-[[1,3-dihydroxy-2-

(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid), HEPES (2-[4-(2-
hydroxyethyl)piperazin-a-yl]ethanesulfonic acid), TAPSO (3-[[1,3-dihydroxy-2-
(hydroxymethyl)propan-2-yl]amino]-2-hydroxypropane-1-sulfonic acid) and Bicine (2-(Bis(2-
hydroxyethyl)amino)acetic acid). However, there are reports of the use of Good’s buffers
interfering with experimental conditions and this has been ascribed to metal complex formation.67
Indeed, all of the aforementioned buffers participate to some degree in metal complexation. This
is not surprising because they all contain combinations of primary and/or secondary alcohol
and/or amine groups. The components of the ligands prepared in this study that make them good
Zn2+ and Cu2+ chelators are their amine functional groups. Buffers containing the same groups
are therefore equally likely to coordinate to the metals of interest, therefore competing with the
ligands that are central to the experiments.
Figure 4.4 displays the structure of the commonly used buffer 2-amino-2-hydroxymethyl-
propane-1,3-diol or tris(hydroxymethyl) aminomethane, referered to as “tris” (though not one of
Good’s) alongside one of the foundation ligands used in this study, tren. Their structures are
similar and the corresponding logK1 stability constants for tris with Zn2+, Cu2+, Ni2+ and Cd2+ are
1.94, 4.05, 2.74 and 1.94 respectively (determined by potentiometric titrimetry in aqueous KNO3
(I = 0.1 mol dm-3) at 298.2 K).68 These are much smaller than the same stability constants for tren
as measured in this study and recorded in Table 2.3. However they do indicate that this buffer
will interact with the species of interest. Furthermore they are more similar in size to some of the
stability constants for the other tren-based ligands such as Me6tren and βCDMe5tren in this study
and the intent is to extend these preliminary kinetics experiments to the other complex systems as
part of Future Work.
Figure 4.4. Tris buffer and tren, both of which complex Zn2+, Cu2+, Ni2+ and Cd2+.
128
It is hypothesised that steric hindrance around the buffer’s donor atoms (such as nitrogens
present as tertiary amines rather than primary amines) could prevent the buffer compounds from
binding to metal cations and that this is why the Good’s buffers MES (2-(N-morpholino)sulfonic
acid), MOPS (3-morpholinopropane-1-sulfonic acid) and PIPES (1,4-piperazinediethanesulfonic
acid) do not cause the same interference as their counterparts.69 MES, MOPS and PIPES contain
only tertiary, ring nitrogens. This chemistry is exactly what is displayed in Chapters 2 and 3 of
this study, where methylation of the tren- and tacn-based ligands causes a decrease in the stability
constants of the complexes they form with the four metals. Therefore several new buffer
structures have been proposed to cover the same pH range as the Good’s buffers but without the
interferences arising from freely-binding amine and alcohol components.69 The buffer DEPP
(1,4-diethylpiperazine, Figure 4.5) was selected for this work. DEPP has a pKa of 8.83 (aqueous
NaNO3 (I = 0.1 mol dm-3), 298.2 K) which is ideal for the complexes in this study.
Figure 4.5. The buffer 1,4-diethylpiperazine, DEPP, in its diprotonated form.
It is now appropriate to consider the potential influence of the deprotonations of the aqua
ligands of [Zn(βCDtren)(H2O)2]2+ and [Cu(βCDtren)(H2O)2]2+ on the mechanism of their
catalysis of the hydrolysis of 4-nitrophenyl acetate using [M(βCDtren)(H2O)2]2+ as a general
example. This discussion is aided by Figure 4.6 found on the following page.
129
Figure 4.6. Protonation/deprotonation equilibria of aqua ligands in species derived from [M(βCDtren)(H2O)2] 2+ in the alternative mechanisms
possible for the enzyme mimic catalysed hydrolysis of 4-nitrophenyl acetate.
130
In Figure 4.6, the species A – D are identical to the species shown in the proposed possible
mechanisms for the hydrolysis of 4-nitrophenyl acetate in Figure 4.2 where C is the Michaelis
complex. The additional species F, G and H exist in protonic equilibrium with them. The di-aqua
species [M(βCDtren)(H2O)2]2+, A, is likely to complex 4-nitrophenyl acetate as shown in species
F (which is in protonic equilibrium with C) and may then undergo substitution by 4-nitrophenyl
acetate to form G which is in protonic equilibrium with D. While the pKaH2O#1 of the aqua ligand
of A is directly determined by potentiometric titration, complexation of 4-nitrophenyl acetate in
the annulus of F may modify it to pKaH2O#1′ and with coordination of 4-nitrophenyl acetate in G
further modification to pKaH2O#1′′ will occur. Neither pKaH2O#1′ nor pKaH2O#1′′ can be titrimetrically
determined as both species F and G, have conjugate bases C and D, respectively, which
depending upon the catalytic enzyme mimic mechanism operating are likely to be catalytically
active. Nevertheless the deprotonations of the aqua ligands in species F and G are important
considerations for interpretation of the pH profile of 4-nitrophenyl acetate hydrolysis as is
discussed in section 4.2. Similar considerations apply to pKaH2O#2′ and pKaH2O#2′′ of B and C,
respectively.
As neither F nor G contain hydroxo ligands they are unlikely make significant direct
contributions to the catalysis process. However, the [M(βCDtren)(OH)2]2+ species H may
complex 4-nitrophenyl acetate which subsequently substitutes on the M centre to give D. Thus,
if, as is assumed here for the experimental pH range of this study (section 4.2), the dominant
pathway for the hydrolysis of 4-nitrophenyl acetate is through either species C or D, species H
and I are unlikely to have significant direct effects on the catalysis process either. However, they
are interrelated through protonic equilibria and this impinges on the extent to which species C
and D form and the rate of catalysed hydrolysis. The interpretation of the 4-nitrophenyl acetate
kinetic data gathered in this study follows the Michaelis-Menten model enzyme kinetics with
some important variations arising from the nature of the two potentially enzyme mimicking
[M(βCDtren)(H2O)2]2+ systems and their deprotonated analogues considered here.
Considering these pH-dependent protonic equilibria, deciding on and maintaining the pH of

these solutions was essential because the pH determines both the concentration of background
hydroxide species present as a consequence of autoprotolysis (pOH = 14 – pH and pOH = -
log[OH¯]) and the speciation of the potential enzyme mimics. Since the hydroxide reactant, OH¯,
is the nucleophile responsible for performing the hydrolysis (Figure 4.2.), its concentration has a
significant effect on the reaction rate. Consequently, keeping the pH constant across all
131
experiments for all complexes was considered, because this would ensure that the hydroxide
present in the bulk solution was at a constant concentration. However, the species of interest – the
hydroxo ligands formed on the complexes – are produced by the deprotonation of aqua ligands at
different pHs, which are unique to each complex. At a constant pH across complexes, the bulk
solution would contain the same concentration of hydroxide, but the complexes themselves
would produce varying amounts of hydroxo ligand, depending on the particular pKaH2O#1 of the
aqua ligand precursor. The decision was therefore made to perform experiments for each
complex buffered at the pH of aqua ligand pKaH2O#1, specific to that complex. Therefore any
differences in hydrolysis rate observed could be attributed directly to the complex and could not
be the result of the pH environment favouring the formation of active hydroxo ligand for one
complex over another. At the pH of the pKaH2O#1 of the aqua ligand for each complex there will
be approximately 50% formation of the hydroxo ligand for a given complex concentration.
Despite the fact that this pH is different for each complex, performing the experiments at these
separate pHs ensures a consistent speciation across every system studied. (Note that because the
complexes in question also undergo a second aqua deprotonation at a pH approximately 1 log
unit from the first, at the pH equal to pKaH2O#1 there will be slightly less than 50% mono-hydroxo
ligand complex formation because a small fraction will have been further converted into the di-
hydroxo ligand complex form. It is hypothesised, however, that both the mono- and di-hydroxo
complexes exist and act in a similar catalytic form once the ester has bound (Figure 4.4, species C
(mono-hydroxo) and I (di-hydroxo) both convert to active species D). Coupled with the fact that
the di-hydroxo ligand complex forms to a small extent, this further conversion is therefore
ignored.)
At pH 8.68 (the pKaH2O#1 of [Cu(βCDtren)(H2O)2]2+) there is approximately five times less

hydroxide in the solution (as a result of autoprotolysis of the solvent water molecules) than at pH
9.14 (the pKaH2O#1 of [Zn(βCDtren)(H2O)2]2+). However the actual difference in bulk solution
hydroxide concentration represented by this increase from pH 8.68 to 9.14 (ignoring the presence
of the complex species) is only 9 × 10-6 mol dm-3. This is two orders of magnitude smaller than
the lowest enzyme mimic complex concentration used in these experiments. The hydroxo
ligand(s) present on the complexes are therefore far more concentrated than the background
hydroxide present in the buffered solutions. Consequently varying the buffered pH to be specific
to the pKaH2O#1 for each individual complex keeps the overall [OH¯] effectively constant, despite
the fact that this means running these experiments at different pHs.
132
Comparisons between complexes at a specific pH can also be drawn by making evaluations

based on their aqua pKaH2Os. For example, at pH 5.05 the only complex producing appreciable
amounts of hydroxo ligand is [Cu(βCDMe2tacn)(H2O)3]2+ (pKaH2O#1 = 5.05, see Section 3.2.4). It
may therefore be presumed to be the most efficient catalyst complex at this pH because it is the
only one that is able to produce the required hydroxo reactant under these conditions. However, if
experiments are performed at this pH (= the pKaH2O#1 of this complex) and the rate is slower than
that of other complexes at their own pHs, then there is clearly a trade-off to be made between the
[OH¯] produced and the intrinsic features of each enzyme mimic that govern the reaction rate.
That a complex can produce a large hydroxo concentration as a result of lowering the aqua
pKaH2O is not necessarily a guarantee of a fast rate (as discussed in the sections below). Therefore,
experiments performed in this manner allow for both comparisons to be made.
Considering the pHs at which these experiments would be performed, a suitable initial ester
concentration had to be determined. The molar absorptivity at 400 nm, ε400, of the absorbing
phenolate product is 18 500 dm3 mol-1 cm-1.38 The pKa of its conjugate acid 4-nitrophenol is
7.1537 and this species does not absorb strongly at this wavelength. Consequently, a consideration
of the percent of the product in the absorbing phenolate form at the relevant pHs coupled with the
phenolate’s molar absorptivity yielded an ideal ester reactant concentration of 4 × 10-5 mol dm-3
in a 1cm path length cuvette. (Note, for the complexes studied in this chapter -
[Zn(βCDtren)(H2O)2]2+ and [Cu(βCDtren)(H2O)2]2+) - the pHs are well above the conjugate acid
4-nitrophenol pKa and therefore the kinetics experiments will produce a reasonable absorbance
signal at 400 nm as most of the product - >97% - will be in the phenolate form. However other
complexes have pKaH2O#1 values (and therefore pHs for kinetics testing) much closer to
([Cu(tacn)]2+, [Cu(Me3tacn)]2+ and [Cu(βCDtacn)]2+) and even lower than ([Cu(βCDMe2tacn)]2+)
the phenol pKa. When the hydrolysis product is formed under these conditions, much of it will
exist in the phenol form. Consequently the wavelength of monitoring may need to be adjusted to
the λmax of the phenol (320 nm) in order to probe these complexes and this is noted for future
work.)
The following three sets of experiments and analyses were performed on the βCDtren/Zn2+
system and the βCDtren/Cu2+ system:
1) Once the pH and ester concentration had been selected, the first set of experiments
involved monitoring the absorbance produced by the phenolate over time for a constant initial
133
ester concentration (of 4 × 10-5 mol dm-3) reacted with varying enzyme mimic complex
concentrations. The complex concentrations started at a minimum of 4 × 10-4 mol dm-3 so that the
complex was always in at least a ten times excess over the substrate. This both allows for pseudo-
first order kinetics analysis (where one reactant is in such excess over the other that its
concentration essentially remains constant over the reaction70) and ensures that even if catalyst
poisoning occurs, an upper limit of 10% of the catalyst can be affected. The complex
concentrations were then increased between 4 × 10-4 and ~ 1.5 × 10-2 mol dm-3 before
precipitation becomes an issue. (These specifications are discussed further in Section 4.1.3.)
For each experiment (with [ester] = 4 × 10-5 mol dm-3 and a [complex] between 4 × 10-4 mol
dm-3 – 1.5 × 10-2 mol dm-3) the absorbance at 400 nm was monitored over time. For the faster
reactions (those containing more concentrated complex) the entire reaction was followed until the
absorbance remained at a constant value indicating that hydrolysis had reached completion. For
the slower reactions (those containing more dilute complex) the reaction was followed for at least
2.5 half-lives and then the cuvettes were thermostatted at 298.2 K and a final absorbance at 400
nm was recorded once the evolution of 4-nitrophenolate had ceased.
These curves of absorbance at 400 nm followed over time for each respective
[ester]:[complex] concentration ratio could bit fit using a nonlinear regression of the form:
y = (y0 – plateau).e(-kobs × time) + plateau 4.23
For absorbance at 400 nm (a.u.) on the y-axis and time (s) on the x-axis, the y0 is therefore the
absorbance at time zero, the plateau is the maximum absorbance reached upon hydrolysis
completion and kobs is the observed rate constant for this specific [ester]:[complex] concentration
ratio, at this pH. Analysis in this manner yielded the observed rate constant, kobs, and half-life of
the reaction for each [complex] (half-life, τ1/2 = ln2/kobs). This fitting equation is known as One
Phase Decay71 and models a first order process. For the two reacting species (ester and
hydroxide) the rate law is shown below:
Rate = k[ester][OH¯] 4.24
However, the hydroxide is in excess over the ester because it is produced and regenerated by
the excess complex catalyst. Consequently the hydroxide concentration changes very little over
the duration of the reaction and can be approximated as constant. The rate law can therefore be
modified to:
134
Rate = k’[ester], where k’ = k[OH¯] 4.25
This is what is referred to as “pseudo first order” kinetics and allows the first order one phase
decay fit to be applied successfully. As shown in Section 4.2 this model is an excellent fit for the
experimental results.
2) Once the observed rate constants were determined for each complex concentration,
Equation 4.15 was used to determine the catalytic rate constant (kcat), and dissociation constant
(KM) of the enzyme-substrate complex ([E•S]) by plotting kobs (s-1) versus [complex] (mol dm-3).
As set out in Section 4.1.3, kcat is the rate constant for the hydrolysis that proceeds via the
catalytic pathway. It provides an indication of how much the catalyst enhances the rate over the
uncatalysed reaction. KM is conventionally referred to as the Michaelis constant and simply
reflects the stability of the enzyme-substrate complex (as depicted in Figure 4.2). Measurement of
these values allows for enzyme mimic characterisation. In order to achieve this, Equation 4.15 is
a modification the traditional Michaelis-Menten equation because both catalysed and uncatalysed
hydrolysis pathways must be considered in this study (Section 4.1.3), unlike for the experimental
conditions used for the conventional Michaelis-Menten model. The algebraic derivation describes
the same overall process however, but the mathematical model must be amended to allow for a
kobs to be measured when there is no complex present. This new equation was therefore written
and applied using GraphPad Prism software.71
3) Finally, a pH-rate profile was built for each complex. At the lowest complex concentration,
experiments were also conducted at pHs in the range ±1.5 units from the pKaH2O#1 (hydroxide
precipitate permitting). For example, for the complex [Zn(βCDtren)(H2O)2]2+ with the pKaH2O#1 of
9.14, kinetics experiments were also run in the range 7.64 – 10.64. At the higher pHs zinc
hydroxide formation and precipitation becomes problematic and renders the results unreliable,
however the kobs values able to be measured will confirm that the hydroxo species formed as a
result of aqua ligand deprotonation is the catalytic species. The kobs values varying with pH can
be fit with a sigmoidal Boltzmann curve25, 28
and if the complexes have a second aqua ligand
deprotonation (pKaH2O#2) then this should be evident on these curves as well. pH-rate profiles that
fit to the sigmoidal model indicate that the catalytic activity is controlled by an acid-base
equilibrium that is described by a pKa at the inflection point of the curve.53, 72-75 The sigmoidal
curve equation has the form:
kobs = (bottom – top)/(1 + ((pH/pKa)slope) + top) 4.26

135
Where “bottom” represents the plateau of minimum kobs at low pH, “top” represents the
plateau of maximum kobs at high pH, pKa represents the central inflection point (and pKa of the
aqua ligand) and “slope” represents the slope of the sigmoidal curve. This produces a graphical
representation of the relationship described in Equation 4.22.
These three sets of analyses thoroughly characterise the catalytic activity of each complex.
On completion, this investigation determines the catalytic rate constant for the complexes, kcat,
the affinity of the complexes for the ester substrate (represented by the dissociation constant, KM)
and confirms the fact that the hydroxo ligand is the catalytic species.
4.2 Kinetics analysis of the βCDtren/Zn2+ and βCDtren/Cu2+ systems
4.2.1 Experimental preparations

Once suitable buffers were selected, preparation for the kinetic studies could proceed. A
detailed description of the kinetics experimental method is provided in the Experimental Chapter
8 (Section 8.3). However, in summary, a stock solution of aqueous 4-nitrophenyl acetate (5%
acetonitrile) was prepared and mixed with a stock solution of enzyme mimic complex (aqueous
NaClO4 (I = 0.1 mol dm-3) and buffer ( = 1.0 × 10-2 – 5.0 × 10-2 mol dm-3). Collection of the
absorbance spectra was initiated at the same time as a comparatively large volume of the complex
stock solution was pipetted into each cuvette on top of a comparatively small volume of the ester
stock solution, ensuring thorough mixing on addition. The cuvette was then placed in the
spectrophotometer and the data points collected in the intervening time (prior to cuvette insertion)
were removed upon analysis. Although this means some points were collected whilst the cuvette
was not in the spectrophotometer, and absorbance reading at time zero is not essential as it can be
extrapolated. However, accurately marking time zero as the instant when the reactant stock
solutions are combined is essential. This method allows for this determination very accurately.
Stock solutions were therefore prepared such that, after combination, the final concentration of
ester in the cuvette was 4 × 10-5 mol dm-3 and the final concentration of complex in the cuvette
varied between 4 × 10-4 and 1.5 × 10-2 mol dm-3.
Unfortunately these stock concentration calculations could not be held constant between the
different complex systems studied. Cuvettes of path length 1.0 cm, 0.5 cm and 0.2 cm all had to
136
be used (depending on the complex concentration) and they each hold different final volumes.
The hexa aqua Zn2+ complex, [Zn(H2O)6]2+, does not have an absorbance spectrum due to its d10
electronic configuration. The hexa aqua Cu2+ complex, [Cu(H2O)6]2+, does absorb although not
over the wavelength range of interest in this study. The complexes of the ligands do produce
absorbance spectra though they tend to dominate the lower energy of the visible region which is
not investigated in this study (500 – 800 nm).76, 77 Indeed, tests of the free ligands showed little
absorbance over the range 225 – 500nm (Section 4.4).
However, on addition of the M2+ and reaction with the ester, the absorbance recorded
increased dramatically and over certain parts of the spectrum. This trend was observed to increase
in intensity as the complex concentration was increased and caused the absorbances recorded to
be well outside of the reliable range of ~ 0 – 1.0 a.u.78 The complex alone was determined to
have some contribution to this increased intensity, but the largest difference was observed on
combination of the complex with the ester. This will be later discussed in more detail, suffice to
say that the species responsible for the large absorbance could not be added to the reference
cuvette because these were the reacting species of interest. Consequently cells of 1.0 cm, 0.5 cm
and 0.2 cm path length were used – decreasing the path length as the complex concentration
increased. This allowed for the desired concentrations to still be investigated whilst producing
absorbance values that were not unreasonably high.
Interestingly, these observations were not constant between complexes. For example, whilst
the [Zn(βCDtren)]2+ complex required a cuvette of 0.5 cm path length at 4 × 10-3 mol dm-3 to
keep the absorbance within a satisfactory range, the corresponding [Cu(βCDtren)]2+ at the same
concentration required a cuvette of 0.2 cm path length. This indicates that the presence of the
Cu2+ metal cation enhances the overall absorbance to a greater extent than the Zn2+. Investigating
which regions of the spectrum are most affected also yielded information relevant to the
mechanism of catalysis, as discussed in Section 4.4.
For all of the data analysis undertaken in this chapter the raw absorbance data was converted
into standard absorbance – that is, the absorbances were re-calculated for a standard 1.0 cm
cuvette. Consequently some very large absorbance values are quoted in this chapter, however
they were not physically measured in this range.
137
4.2.2 The Zn2+/βCDtren system

The characteristics of the [Zn(βCDtren)(H2O)2]2+ complex as determined by potentiometric
titration in Chapter 3 are summarised in Table 4.3. The pKaH2O#1 of the first aqua ligand is 9.14
and therefore the first set of kinetics experiments for this complex was conducted at this pH in
accordance with the reasoning outlined in Section 4.1.3.
Species Parameter and value
CDtrenH44+ pKa4 = 2.51, pKa3 = 7.01, pKa2 = 9.20, pKa1 = 9.64
[Zn(CDtrenH)(H2O)3]3+ logK1′ = 7.96
[Zn(CDtren)(H2O)2]2+ logK1 = 11.99, pKaH2O#1 = 9.14, pKaH2O#2 = 10.00
Table 4.3. Potentiometric titration data characterising the βCDtren/Zn2+ system. Results
determined in aqueous NaClO4 (I = 0.10 mol dm-3) at 298.2 K.
The logK1 characterising [ZnβCDtren(H2O)2]2+ (11.99) is large enough such that at pH 9.14
(at which the effect of this potential enzyme mimic on 4-nitropenyl acetate hydrolysis rate was
studied) there is neither free βCDtren nor its protonated forms, nor [Zn(H2O)6]2+ nor its
deprontonated hydroxide forms present in solution. This is important because free CDtren,
[Zn(H2O)6]2+ and [Zn(H2O)5(OH)]+ accelerate 4-nitropehenyl acetate hydrolysis as is discussed in
Section 4.3 and the [Zn(H2O)4(OH)2] species precipitates, so the absence of appreciable
concentrations of these species is necessary. Figure 4.7 shows the speciation plot for the
βCDtren/Zn2+ system derived from the data found in Table 4.3. The pH at which the kinetics
experiments for this system were run (9.14) is highlighted by a vertical black line and all species
(not solely the ligand-containing species), are displayed. The titration and kinetics experimental
conditions involved a 1:1 ligand:metal concentration ratio (i.e. [ligand] = [M2+]) and therefore the
percentage formations of the ligand and metal species are directly compared on this plot and are
relative to the same overall concentration. This is necessary in order to determine if the presence
of other species (free ligand and metal species as described above) which may interfere with the
hydrolysis investigation. At pH 9.14 it can be seen that the only significant species in solution are
138
equimolar amounts of [Zn(βCDtren)(H2O)2]2+ and [Zn(βCDtren)(H2O)(OH)]+ and a small

amount (~7% formation) of [Zn(βCDtren)(OH)2]. The combination of free βCDtren,
[Zn(H2O)6]2+, [Zn(H2O)5(OH)]+ and [Zn(H2O)4(OH)2] make up less than 0.13% of the overall
solution composition and no precipitation was detected at this pH.
100 pH 9.14 βCDtren

90
βCDtrenH⁺
80 βCDtrenH₂²⁺
70 βCDtrenH₃³⁺
60 βCDtrenH₄⁴⁺
50 [Zn(βCDtrenH)(H₂O)₃]³⁺
40 [Zn(βCDtren)(H₂O)₂]²⁺
30 [Zn(βCDtren)(H₂O)(OH)]⁺
20 [Zn(βCDtren)(OH)₂]
10 [Zn(H₂O)₆]²⁺
0 [Zn(H₂O)₅(OH)]⁺
3 5 7 9 11 [Zn(H₂O)₄(OH)₂]
pH
Figure 4.7. Speciation plot for the system βCDtren/Zn2+ where [βCDtren]total = [Zn2+] total = 1.0
× 10-3 mol dm-3 in aqueous NaClO4 (I = 0.10 mol dm-3) at 298.2K. The speciations are plotted as
percentages of [βCDtren] total = [Zn2+] total as pH is varied. The vertical line represents pH =
pKaH2O#1 = 9.14 for the first aqua ligand of [Zn(CDtren)(H2O)2] 2+.
This speciation plot shows that the βCDtren/Zn2+ system is well suited for studies of its
catalytic effects on the hydrolysis of 4-nitrophenyl acetate. Consequently, experiments to monitor
the absorbance at 400 nm were conducted at pH 9.14, with a constant initial ester concentration
of 4 × 10-5 mol dm-3 and increasing initial [Zn(βCDtren)(H2O)2]2+, [Zn(βCDtren)(H2O)(OH)]+
and [Zn(βCDtren)(OH)2] combined total concentrations of approximately 0, 4.0 × 10-4, 8.0 × 10-4,
1.0 × 10-3, 3.0 × 10-3, 4.0 × 10-3, 6.0 × 10-3, 1.0 × 10-2, 1.2 × 10-2 and 1.5 × 10-2 mol dm-3. This
experimental procedure is similar to those reported in many other studies.17, 18, 23, 25, 53, 73, 79-81
The time dependent change of the UV-visible absorbance spectrum (225 – 500 nm)
accompanying the hydrolysis of 4-nitrophenyl acetate at an initial concentration of 4 × 10-5 mol
dm-3 in the presence of an equilibrium mixture of [Zn(CDtren)(H2O)2]2+,
139
[Zn(CDtren)(H2O)(OH)]+ and [Zn(CDtren)(OH)2] at a combined total concentration = 4.0 ×

10-4 mol dm-3 is shown in Figure 4.8.
1 .5
S ta n d a r d i s e d a b s o r b a n c e
1 .0
0 .5
0 .0
250 300 350 400 450 500
W a v e le n g th ( n m )
Figure 4.8. UV-Vis absorbance change over ~ 8.5 h for a solution containing an initial 4-
nitrophenyl acetate concentration = 4 × 10-5 mol dm-3 in the presence of the equilibrium mixture
of [Zn(CDtren)(H2O)2]2+, [Zn(CDtren)(H2O)(OH)] + and [Zn(CDtren)(OH)2] at a total
combined concentration = 4.0 × 10-4 mol dm-3 in aqueous NaClO4 (I = 0.10 mol dm-3) at pH 9.14
(DEPP buffer concentration = 1 × 10-2 mol dm-3). The arrows indicate the directions of change
of the absorbances of the 4-nitrophenol acetate and 4-nitrophenolate at 271 nm and 400 nm,
respectively, and the isosbestic points indicate that these are the dominant absorbing species
whose concentrations are undergoing change.
The absorbance peaks for the 4-nitrophenol acetate reactant and the 4-nitrophenolate
hydrolysis product can be seen at 271 nm and 400 nm, respectively. 35 For solutions with
equilibrium mixture [Zn(CDtren)(H2O)2]2+, [Zn(CDtren)(H2O)(OH)]+ and
[Zn(CDtren)(OH)2] combined total concentrations in the lower range of those studied, it was
possible to record the absorbance variation over the entire range of 271 nm - 400 nm. However,
as the enzyme mimic concentration was increased and the rate of hydrolysis increased, this range
had to be narrowed to include only the 4-nitrophenolate peak (395 nm – 405 nm) such that
enough measurements could be taken over the short duration of the reaction. (All spectra were
140
run against reference solutions containing 0.10 mol dm-3 NaClO4 and either 1.0 × 10-2 or 5.0 ×
10-2 mol dm-3 DEPP.) As discussed in Section 4.1.3, an algorithm for a first order reaction was
best-fit to the experimental data at 400 nm which represents the increase in 4-nitrophenolate
product concentration with time.
Two typical examples of the increase of the absorbance of 4-nitrophenolate at 400 nm with
time are shown in Figures 4.9 and 4.10 for the lowest and highest enzyme mimic concentrations
([Zn(CDtren)(H2O)2]2+, [Zn(CDtren)(H2O)(OH)]+ and [Zn(CDtren)(OH)2] combined total
concentrations) of 4.0 × 10-4 and 1.5 × 10-2 mol dm-3, respectively.
1 .0
S ta n d a rd is e d a b s o rb a n c e a t 4 0 0 n m
0 .8
0 .6
0 .4
0 .2
0 .0
0 10000 20000 30000 40000
T im e (s )
Figure 4.9. The appearance of 4-nitrophenolate over time in the presence of the equilibrium
mixture of [Zn(CDtren)(H2O)2] 2+, [Zn(CDtren)(H2O)(OH)] + and [Zn(CDtren)(OH)2] at a
total concentration = 4.0 × 10-4 mol dm-3 as indicated by an increase in absorbance at 400 nm.
The initial [4-nitrophenyl acetate] = 4 × 10-5 mol dm-3 and the hydrolysis was carried out in
aqueous NaClO4 (I = 0.10 mol dm-3) at pH 9.14 ([DEPP buffer] = 1.0 × 10-2 mol dm-3). The
solid curve represents the best fit of an algorithm for a first order reaction to the experimental
data at 400 nm. The derived kobs = 1.9 × 10-4 ± ≤ 5% s-1.
141
As discussed, the 4-nitrophenyl acetate concentration is kept constant throughout these

experiments so Figure 4.9 represents the kinetics for which the enzyme mimic is at the minimum
of a 10-fold excess and Figure 4.10 represents the kinetics for with the enzyme mimic is in 375-
fold excess. (It should be noted that in Figure 4.9 the standardised absorbance is the observed
absorbance from a 1.0 cm path length cell, whereas that in Figure 4.10 is the observed absorbance
from a 0.2 cm path length cell multiplied by five in order to measure the absorbances in an
acceptable range but still be able to compare them. This demonstrates the much greater
absorbance observed in the more concentrated solution consistent with the binding of 4-
nitrophenyl acetate to the Zn2+ complexes, as is discussed below.)
2 .6
2 .4
2 .2
2 .0
1 .8
1 .6
1 .4
0 250 500 750 1000 1250 1500
T im e (s )
Figure 4.10. The appearance of 4-nitrophenolate over time in the presence of the equilibrium
mixture of [Zn(CDtren)(H2O)2] 2+, [Zn(CDtren)(H2O)(OH)] + and [Zn(CDtren)(OH)2] at a
total combined concentration = 1.5 × 10-2 mol dm-3 as indicated by an increase in absorbance at
400 nm. The initial [4-nitrophenyl acetate] = 4 × 10-5 mol dm-3 and the hydrolysis was carried
out in aqueous NaClO4 (I = 0.10 mol dm-3) at pH 9.14 ([DEPP buffer] = 5.0 × 10-2 mol dm-3.
The solid curve represents the best fit of an algorithm for a first order reaction to the
experimental data at 400 nm. The derived kobs = 6.7 × 10-3 ± ≤ 5% s-1.
142
The measured pseudo-first order rate constants, kobs, obtained from the fits displayed in
Figures 4.9 and 4.10 and the remaining experiments are reported in Table 4.4 (obtained as
described in Section 4.1.3).
Combined
[Zn(βCDtren)] kobs (s-1) half-life half-life
(mol dm-3) (s) (min)
0 3.7 × 10-5 ± 7 × 10-8 18 969 316.2
-4 -4 -7
4 × 10 1.9 × 10 ± 9 × 10 3697 61.6
-4 -4 -6
8 × 10 3.7 × 10 ± 4 × 10 1878 31.3
-3 -4 -6
1 × 10 6.0 × 10 ± 5.0 × 10 1151 19.2
-3 -3 -5
3 × 10 1.7 × 10 ± 2 × 10 402 6.7
-3 -3 -5
4 × 10 2.6 × 10 ± 2 × 10 271 4.5
-3 -3 -5
6 × 10 3.3 × 10 ± 2 × 10 209 3.5
-2 -3 -5
1.0 × 10 4.8 × 10 ± 7 × 10 145 2.4
1.2 × 10-2 5.5 × 10-3 ± 3 × 10-5 127 2.1
-2 -3 -4
1.5 × 10 6.7 × 10 ± 1 × 10 104 1.7
Table 4.4. Observed first order rate constants, kobs, determined using UV-Vis spectrophotometry
for the hydrolysis of 4-nitrophenyl acetate at a constant initial concentration of 4 × 10-5 mol dm-3
in the presence of varying combined total concentrations of [Zn(CDtren)(H2O)2] 2+,
[Zn(CDtren)(H2O)(OH)] + and [Zn(CDtren)(OH)2] in aqueous NaClO4 (I = 0.10 mol dm-3), at
pH 9.14, 298.2 K. The DEPP buffer concentration = 1.0 × 10-2 mol dm-3for the first seven
solutions studied, and 5.0 × 10-2 mol dm-3for the last three. The errors shown are those obtained
by best-fitting the algorithm for a first order reaction to the absorbance data at 400nm. The
estimated experimental error is ≤ ± 5%.
The remainder of the kinetics curves used to calculate these kobs values are contained in
Appendix C. It should also be noted that the buffer concentration is not constant across all
experiments. The initial buffer concentration of 1.0 × 10-2 mol dm-3 was deemed appropriate and
was consistent with the concentration used for the studies in Chapter 7. However, as the
hydrolysis rates were measured, it was revealed that the resulting kobs would not plateau (as
predicted by the saturation kinetics of enzyme systems) over an enzyme mimic concentration
range less than the initial buffer concentration, as is required for the determination of kcat and KM.
143
For the experiments containing the larger mimic concentrations (1.0 × 10-2 mol dm-3 and above) a
more concentrated buffer was used (5.0 × 10-2 mol dm-3) to retain a sufficient buffer capacity for
these experiments. This reaches the upper limit of DEPP solubility under these conditions.
Ideally, each experiment should be conducted under identical conditions. However, the
concentration range required for Michaelis-Menten analysis is unknown for each particular
system until the experiments are performed. It is unlikely that the change in buffer concentration
adversely affected the results. However, when other systems are investigated as part of Future
Work (for example, the Zn2+/CDMe5tren system), a larger buffer concentration should be used
from the outset.
It is clear from the data in Table 4.4 that kobs (= kuncat) = 3.7 × 10-5 s-1 observed at pH 9.14 in
the presence of DEPP buffer alone increases substantially with increasing concentration of the
[Zn(CDtren)(H2O)2]2+, [Zn(CDtren)(H2O)(OH)]+ and [Zn(CDtren)(OH)2] equilibrium
mixture, consistent with the Zn2+ complexes being effective enzyme mimics. (Recalling that at
pH 9.14, the most prevalent species in solution are [Zn(CDtren)(H2O)2]2+ and
[Zn(CDtren)(H2O)(OH)]+ , Figure 4.7.) Without any enzyme mimic present, the hydroxide in
the bulk solution ([OHˉ] = 1.38 × 10-5 mol dm-3 at pH 9.14) causes 4-nitrophenyl acetate to
hydrolyse with a half-life of more than five hours. The addition of the minimum 4 × 10-4 mol dm-
3
of enzyme mimic causes that half-life to reduce to only one hour. When the enzyme mimic is in
300-fold excess over the ester substrate the hydrolysis half-life decreases to just 2 minutes.
As is reported in the literature, the 4-nitrophenyl acetate ester has its own absorbance
spectrum with a λmax in the UV region at 271 nm.35 Due to the fast speed of many of these
experiments (those with high concentrations of enzyme mimic), or the necessity to perform
multiple experiments at a time, in most cases only a small wavelength range could be probed for
each experiment. This range contained the wavelength of interest 400 nm – eg. 395 – 405 nm.
However, for the slower reactions (those with low concentrations of enzyme mimic) a broader
wavelength range could be investigated – for example 225 – 500 nm. The spectral data obtained
in these experiments made it possible to analyse the decrease in absorbance of the reactant ester
peak as well as the increase in absorbance of the product phenolate peak. Figure 4.8 shows the
full spectra collected over approximately 8.5 hours as 4-nitrophenyl acetate (4 × 10-5 mol dm-3)
was hydrolysed in the presence of the enzyme mimic (combined concentration = 4 × 10-4 mol dm-
3
) at pH 9.14. The arrows indicate the absorbance changes with time.
144
Fitting the variation of absorbance at 400 nm with the One Phase Decay equation described in
Section 4.1.3 produces the curve in Figure 4.9. Fitting the variation of absorbance at 279 nm with
the same equation produces the curve in Figure 4.11. (Note, though the λmax of the 4-nitrophenyl
acetate is 271 nm, the wavelength for ester analysis was chosen to be 279 nm. It can be seen from
Figure 4.8 that there is a larger change in absorbance at this wavelength. There is also a small
peak close to 260 nm assigned to the small amount of 4-nitrophenol product formed at pH 9.14
which it is preferable to avoid. Therefore 279 nm was considered to be a more appropriate
wavelength for analysis.) Analysis at 279 nm reflects the disappearance of the reactant 4-
nitrophenyl acetate over time and analysis at 400 nm reflects the evolution of the 4-
nitrophenolate product over time. Because the mole ratio of ester to phenolate is 1:1, the same
rate constant, kobs, and half-life should therefore be calculated by both analyses.
0 .7
0 .6
0 .5
0 .4
0 .3
0 10000 20000 30000 40000
T im e (s )
Figure 4.11. The disappearance of 4-nitrophenol acetate over time in the presence of the
equilibrium mixture of [Zn(CDtren)(H2O)2] 2+, [Zn(CDtren)(H2O)(OH)] + and
[Zn(CDtren)(OH)2] at a total concentration = 4.0 × 10-4 mol dm-3 as indicated by an decrease
in absorbance at 279 nm. The initial [4-nitrophenyl acetate] = 4 × 10-5 mol dm-3 and the
hydrolysis was carried out in aqueous NaClO4 (I = 0.10 mol dm-3) at pH 9.14 ([DEPP buffer] =
1.0 × 10-2 mol dm-3. The solid curve represents the best fit of an algorithm for a first order
reaction to the experimental data at 279 nm. The derived kobs = 2.0 × 10-4 ± ≤ 5% s-1.
145
Interestingly, this analysis at 279 nm for the experiment with 4 × 10-4 mol dm-3 enzyme
mimic present yielded a kobs of 2.0 × 10-4 s-1 and a half-life of 3499 s (58.3 minutes). This is in
good agreement with the values reported in Table 4.4 for analysis of phenolate production
measured at 400 nm. Unfortunately, most of the experiments in this study could not be
investigated over this full spectral range due to time constraints (both intrinsically due to fast
reaction rates and feasibly due to utilising the spectrophotometer efficiently). However, several
specific cases were investigated over 225 nm – 500 nm because it was hypothesised that
mechanistic information could be gleaned this way. These experiments and their results are
discussed further in Section 4.4.
From the analyses performed at 400 nm reported in Table 4.4, it can also qualitatively be
observed that – over the enzyme concentration range measured – this system experiences the
saturation kinetics which are described by the Michaelis-Menten enzyme kinetics model. Over
the more dilute enzyme mimic concentration range, the increase in kobs (or decrease in half-life)
varies relatively linearly with increasing enzyme mimic concentration. For example, the
hydrolysis half-life for an enzyme mimic concentration of 8 × 10-4 mol dm-3 (31.3 minutes) is
approximately half the hydrolysis half-life for an enzyme mimic concentration of 4 × 10-4 mol
dm-3 (61.6 minutes). However, as the enzyme mimic concentration is increased, the
corresponding change in kobs (and half-life) becomes incrementally smaller and smaller. This
reflects the process whereby over this enzyme mimic concentration range, the ester substrate is
increasingly bound by the [Zn(βCDtren)] complexes until all of it is present as part of the E•S
Michaelis complex, resulting in the maximum hydrolysis reaction rate.
To illustrate this fully, best-fitting the algorithm for Equation 4.27 (which is the equivalent of
Equation 4.19) to the kobs data from Table 4.4 with kuncat = 3.7 × 10-5 s-1 (the kobs determined in the
absence of Zn(II) enzyme mimic as a fixed quantity), yields KM = 3.35 × 10-2 mol dm-3 and kcat =
2.1 × 10-2 s-1 at 298.2 at pH 9.14 (R2 = 0.9977). This represents a 571-fold, kcat/kuncat, increase in
the hydrolysis rate of 4-nitrophenyl acetate. This best-fit and the experimental kobs are shown in
Figure 4.12 and the results are summarised in Table 4.5 below.
kobs = (KMkuncat + kcat[Zn(II) enzyme mimic])/(KM + [Zn(II) enzyme mimic]) 4.27
146
Zn2+/βCDtren
system Parameter Error
-3
pH 9.14 KM (mol dm ) 0.033 ± 0.006
-1
kcat (s ) 0.021 ± 0.003
Table 4.5. Calculated Michaelis constant, KM, and catalysed rate constant, kcat, obtained from
the fit in Figure 4.12 for the hydrolysis of 4-nitrophenyl acetate in the presence of enzyme mimic
system Zn2+/βCDtren. Aqueous NaClO4 (I = 0.10 mol dm-3), pH 9.14 ([buffer] = 1.0 × 10-2 – 5.0
× 10-2 mol dm-3), 298.2 K.
0 .0 0 8
0 .0 0 6
k obs ( s )
-1
0 .0 0 4
0 .0 0 2
0 .0 0 0
0 .0 0 0 0 .0 0 5 0 .0 1 0 0 .0 1 5 0 .0 2 0
-3
[ Z n ( I I ) e n z y m e m im i c ] ( m o l d m )
Figure 4.12. Plot of the experimentally determined kobs against Zn(II) enzyme mimic
concentration (total combined concentration of [Zn(CDtren)(H2O)2] 2+,
[Zn(CDtren)(H2O)(OH)] + and [Zn(CDtren)(OH)2]) at pH 9.14. The solid curve represents the
best fit of the algorithm for Equation 4.19 to the kobs data.
Essentially, as described by Equation 4.19, this graph shows the variation of the kobs with the
enzyme mimic concentration.62 As shown by the scheme in Equation 4.15, kuncat and kcat are the
rate constants for the uncatalysed and catalysed hydrolysis reaction pathways respectively. Under
the experimental conditions these remain constant and the overall observed rate constant (for the
147
combination of the uncatalysed and catalysed hydrolysis pathways) varies as a function of

enzyme mimic concentration (Equation 4.27). The parameters determined from this fit – kcat and
KM – are displayed in Table 4.5.
The data agrees well with the fitting algorithm (evidenced by R2 = 0.9977). The relatively
large errors result because the enzyme concentration range that is workable (before precipitation
occurs) is below the maximum required for the maximum kobs to be reached.
Finally, the pH variation of kobs at 298.2 K with [4-nitrophenyl acetate] = 4 × 10-5 mol dm-3
and [Zn(II) catalyst] = 4 × 10-4 mol dm-3 was determined at pH 8.14 (5.4 × 10-5), 8.64 (9.4 × 10-
5
), 9.14 (1.9 × 10-4), 9.39 (2.9 × 10-4) and 9.64 (3.7 × 10-4) (where the values in brackets are the
determined kobs in s-1) was determined from the time dependence of absorbance at 400 nm as
described above. The curve produced by plotting kobs (s-1) versus pH was then fit using the
Boltzmann sigmoidal model in Section 4.1.3. This graph is shown on the following page in
Figure 4.13 and yielded values for bottom (plateau of minimum kobs), top (plateau of maximum
kobs), inflection point (aqua pKa) and slope (shown in Table 4.6). (It should be noted for this fit
that some of the relative errors are again large because the pH range that could reasonably be
tested before precipitation occurred is far from the pHs required to attain the maximum kobs
plateau. Furthermore only five points are recorded. However, the fit of the data that could be
obtained is satisfactory with an R2 value of 0.9984. Experiments at more pH values should be
undertaken as part of Future Work.)
148
0 .0 0 0 5
0 .0 0 0 4
0 .0 0 0 3
k obs ( s )
-1 0 .0 0 0 2
0 .0 0 0 1
0 .0 0 0 0
8 .0 8 .5 9 .0 9 .5 1 0 .0
pH
Figure 4.13. The variation of kobs with pH for the hydrolysis of 4-nitrophenyl acetate (4 × 10-5
mol dm-3) with [Zn(II) enzyme mimic] (total combined concentration of varying compositions of
[Zn(CDtren)(H2O)2] 2+, [Zn(CDtren)(H2O)(OH)] + and [Zn(CDtren)(OH)2] = 4 × 10-4
moldm-3) in 0.10 mol dm-3 NaClO4 and buffer concentration = 1.0 ×10-2 mol dm-3 at 298.2 K.
The best fit of an algorithm for Equation 4.26 to the kobs data is represented by the solid curve.
Parameter Error
Zn2+/βCDtren
minimum kobs (s-1) 4.8 × 10-5 ± 1.7 × 10-5
system
maximum kobs (s-1) 5.6 × 10-4 ± 1.7 × 10-4
inflection point (aqua pKa) 9.45 ± 0.23
Table 4.6. Summary of minimum and maximum kobs and the aqua ligand pKa from the fit of the
pH-rate profile for the Zn2+/βCDtren system.
Figure 4.14 shows the simulated pH-rate profile curve using the bottom, top, slope and
inflection values determined from the fit of Figure 4.13 (in Table 4.6), extrapolated over the pH
range 5 to 14. These results support the hypothesis that the kinetics characteristics of this system
are controlled by the acid-base equilibrium of the catalytically active hydroxo ligand.
149
0 .0 0 0 6
0 .0 0 0 5
0 .0 0 0 4
k obs ( s )
-1
0 .0 0 0 3
0 .0 0 0 2
0 .0 0 0 1
0 .0 0 0 0
5 6 7 8 9 10 11 12 13 14
pH
Figure 4.14. pH-Dependent rate profile for the hydrolysis of 4-nitrophenyl acetate in the
presence of [Zn(βCDtren)(H2O)2] 2+ and its deprotonated analogues simulated from the
experimentally determined values reported in Table 4.6.
Interestingly, the inflection point of this curve (9.45) is higher than the experimentally
determined pKa of the first aqua ligand deprotonation for this complex (9.14). This is expected
because the first aqua ligand deprotonating to form a single active hydroxo is not the only
catalytic species acting. As the pH is increased the second aqua ligand making up the octahedral
geometry also deprotonates and forms a second active hydroxo. This second aqua deprotonation
occurs with a pKaH2O#2 of 10.0 (Section 3.2.2) which is relatively close to the first aqua pKa and
the species will therefore overlap. The speciation plot in Figure 4.7 confirms a 7% formation of
the di-hydroxo species at the initial pH of investigation. The pH-rate profile for the Zn2+/βCDtren
system is therefore contributed to by both [Zn(βCDtren)(H2O)(OH)]+ and [Zn(βCDtren)(OH)2]
and the overall combination may therefore be expected to have an inflection point between the
two aqua ligand pKas.
It should also be noted that investigations into the mechanism of the catalysis (Section 4.4)
suggest the complexes [Zn(βCDtren)(H2O)(OH)]+ and [Zn(βCDtren)(OH)2] will likely not act as
two different, catalytically distinct species but as identical catalysts. These investigations suggest
that the mechanism involves the ester binding to the M2+ prior to the hydroxo attack on the
carbonyl (see Figure 4.6). For the experimentally supported octahedral geometry of the
150
complexes in question, the ester must replace the H2O on [Zn(βCDtren)(H2O)(OH)]+ or the
second OHˉ on [Zn(βCDtren)(OH)2] in order to bind and then react. By the time the hydrolysis
reaction is taking place, both complexes (regardless of their initial composition) have the form
[Zn(βCDtren)(ester)(OH)] with a single hydroxo present. Therefore, whilst both complexes
[Zn(βCDtren)(H2O)(OH)]+ and [Zn(βCDtren)(OH)2] (with aqua pKaH2Os of 9.14 and 10.0
respectively) contribute to the pH-rate profile, the overall curve is a reflection of how the
proportion of catalyst [Zn(βCDtren)(OH)X] increases with increasing pH, producing an apparent
inflection/pKaH2O#1 of 9.45. As displayed in Figure 4.7, the speciation plot shows that at pH 9.14
there are approximate formations of [Zn(βCDtren)(H2O)(OH)]+ and [Zn(βCDtren)(OH)2] of 47%
and 7%, respectively. However, since both of these species have the same composition once they
are involved in the catalytic mechanism, this equates to a 54% formation of the active species at
this pH. This is likely to be the same case for many similar studies although a discussion of the
consequences of this mechanism is rarely raised.
4.2.3 The Cu2+/βCDtren system

The characteristics of the [Cu(βCDtren)(H2O)2]2+ complex as determined by potentiometric
titration in Chapter 3 are summarised in Table 4.7. The pKaH2O#1 of the first aqua ligand is 8.68
and therefore the first set of kinetics experiments for this complex was conducted at this pH.
Species Parameter and value
CDtrenH44+ pKa4 = 2.51, pKa3 = 7.01, pKa2 = 9.20, pKa1 = 9.64
[Cu(CDtrenH)(H2O)3]3+ logK1′ = 11.92
[Cu(CDtren)(H2O)2]2+ logK1 = 17.22, pKaH2O#1 = 8.68, pKaH2O#2 = 9.77
Table 4.7. Potentiometric titration data characterising the βCDtren/Cu2+ system. Results
determined in aqueous NaClO4 (I = 0.10 mol dm-3) at 298.2 K.
151
In this case the [Cu(βCDtren)(H2O)2]2+ complex has an even higher stability constant (logK1
= 17.22) than the [Zn(βCDtren)(H2O)2]2+ complex. Indeed, at the pH of the pKa of the first aqua
ligand (pKaH2O#1 = 8.68) the speciation data (Figure 4.15 – determined using the results displayed
in Table 4.7) reveals that there is less than 0.1% formation of any free ligand or metal. (This is
important as free CDtren, [Cu(H2O)6]2+ and [Cu(H2O)5(OH)]+ accelerate 4-nitropehenyl acetate
hydrolysis as is discussed in Section 4.3 and the [Cu(H2O)4(OH)2] species precipitates.)
Consequently, as can be observed in Figure 4.15, at the pKaH2O#1 (8.68) the solution is comprised
of approximately 48% [Cu(βCDtren)(H2O)2]2+, 48% [Cu(βCDtren)(H2O)(OH)]+ and 4%
[Cu(βCDtren)(OH)2]. Complexes [Cu(H2O)6]2+, [Cu(H2O)5(OH)]+ and [Cu(H2O)4(OH)2]
constitute < 0.05%. Once again these characteristics suggest that the [Cu(βCDtren)(H2O)2]2+
complex is well-suited for studies of its ability to catalyse the hydrolysis of 4-nitrophenyl acetate.
Consequently, experiments to monitor the increase of the 4-nitrophenolate absorbance at 400 nm
were conducted at pH 8.68, with a constant initial 4-nitrophenyl acetate concentration of 4.0 × 10-
5
mol dm-3 and increasing initial [Cu(CDtren)(H2O)2]2+, [Cu(CDtren)(H2O)(OH)]+ and
[Cu(CDtren)(OH)2] combined total concentrations of 0, 4.0 × 10-4, 8.0 × 10-4, 1.0 × 10-3, 4.0 ×
10-3, 6.0 × 10-3, 1.0 × 10-2 and 1.5 × 10-2 mol dm-3.
100 βCDtren
pH 8.68
90 βCDtrenH⁺
80 βCDtrenH₂²⁺
70 βCDtrenH₃³⁺
60 βCDtrenH₄⁴⁺
50 [Cu(βCDtrenH)(H₂O)₃]³⁺
40 [Cu(βCDtren)(OH)₂]
30 [Cu(βCDtren)(H₂O)(OH)]⁺
20 [Cu(βCDtren)(OH)₂]
[Cu(H₂O)₆]²⁺
10
[Cu(H₂O)₅(OH)]⁺
0
3 5 7 9 11 [Cu(H₂O)₄(OH)₂]
pH
Figure 4.15. Speciation plot for the system βCDtren/Cu2+ where [βCDtren]total = [Cu2+] total = 1.0
× 10-3 mol dm-3 in aqueous NaClO4 (I = 0.10 mol dm-3) at 298.2K. The speciations are plotted as
percentages of [βCDtren] total = [Cu2+] total as pH is varied. The vertical line represents pH =
pKaH2O#1 = 8.68 for the first aqua ligand of [Cu(CDtren)(H2O)2] 2+.
152
Consequently, experiments to monitor the absorbance at 400 nm were conducted at pH 8.68,

with a constant initial ester concentration of 4 × 10-5 mol dm-3 and increasing [Cu(βCDtren)]2+
concentrations of approximately 0, 4.0 × 10-4 mol dm-3, 8.0 × 10-4 mol dm-3, 1.0 × 10-3 mol dm-3,
4.0 × 10-3 mol dm-3, 6.0 × 10-3 mol dm-3, 1.0 × 10-2 mol dm-3 and 1.5 × 10-2 mol dm-3. The
measured pseudo-first order rate constants are reported in Table 4.8 (obtained as described in
Section 4.1.3).
Combined
[Cu(βCDtren)] kobs (s-1) half-life half-life
(mol dm-3) (s) (min)
0 1.7 × 10-5 ± 1.7 × 10-7 41258 687.6
-4 -4 -7
4 × 10 1.0 × 10 ± 6.0 × 10 6649 110.8
-4 -4 -7
8 × 10 2.1 × 10 ± 6.3 × 10 3367 56.1
-3 -4 -7
1 × 10 2.9 × 10 ± 8.7 × 10 2428 40.5
-3 -3 -5
4 × 10 1.5 × 10 ± 2.7 × 10 470 7.8
-3 -3 -6
6 × 10 1.7 × 10 ± 8.6 × 10 409 6.8
-2 -3 -5
1.0 × 10 2.4 × 10 ± 9.2 × 10 287 4.8
-2 -3 -5
1.5 × 10 2.8 × 10 ± 5.3 × 10 244 4.1
Table 4.8. Observed first order rate constants, kobs, determined using UV-Vis spectrophotometry
for the hydrolysis of 4-nitrophenyl acetate at a constant initial concentration of 4 × 10-5 mol dm-3
in the presence of varying combined total concentrations of [Cu(CDtren)(H2O)2] 2+,
[Cu(CDtren)(H2O)(OH)] + and [Cu(CDtren)(OH)2] in aqueous NaClO4 (I = 0.10 mol dm-3), at
pH 8.68, 298.2 K. The DEPP buffer concentration = 1.0 × 10-2 mol dm-3for the first six solutions
studied, and 5.0 × 10-2 mol dm-3for the last two. The errors shown are those obtained by best-
fitting the algorithm for a first order reaction to the absorbance data at 400nm. The estimated
experimental error is ≤ ± 5%.
Firstly, a comparison is made between the changing spectra for the hydrolysis of [4-
nitrophenyl acetate] = 4 × 10-5 mol dm-3 and [enzyme mimic] = minimum concentration = 4 × 10-
4
mol dm-3 between the complex species Zn2+/βCDtren system and Cu2+/βCDtren system. Figure
4.16 displays the change in absorbance spectrum measured over approximately 10 hours as 4-
nitrophenyl acetate (4 × 10-5 mol dm-3) was hydrolysed in the presence of the combined total
153
[Cu(βCDtren)(H2O)2]2+, [Cu(βCDtren)(H2O)(OH)]+ and [Cu(βCDtren)(OH)2] catalyst

concentration (4 × 10-4 mol dm-3) at pH 8.68. The arrows indicate the absorbance changes with
time. This can be directly compared with Figure 4.8 which shows the identical reaction for the
Zn2+/βCDtren system. As noted in this figure, the 4-nitrophenyl acetate reactant has an
absorbance peak at 271 nm and the 4-nitrophenolate product has an absorbance peak at 400 nm.
2 .0
S ta n d a r d i s e d a b s o r b n a c e
1 .5
1 .0
0 .5
0 .0
250 300 350 400 450 500
Figure 4.16. UV-Vis absorbance change with time of a solution containing an intial 4-
nitrophenyl acetate concentration = 4 × 10-5 mol dm-3 and [Cu(CDtren)(H2O)2] 2+,
[Cu(CDtren)(H2O)(OH)] + and [Cu(CDtren)(OH)2] combined total concentration = 4 ×10-4
mol dm-3 at pH 8.68 in aqueous NaClO4 (I = 0.10 mol dm-3) and buffer (1 × 10-2 mol dm-3). The
arrows indicate the directions of change of the absorbances of the 4-nitrophenol acetate and 4-
nitrophenolate at 271 nm and 400 nm, respectively, and the isosbestic points indicate that these
are the dominant absorbing species whose concentrations are undergoing change.
Firstly it should be noted again that the spectra both Figures 4.8 and 4.16 are displayed in
standardised absorbance – that is, the Y-axes are made consistent as though both experiments
were conducted using a 1.0 cm path length cuvette. Some of the absorbance values in Figure 4.16
154
are outside the reliable range. However this experiment was conducted in a 0.5 cm path length
cuvette to obtain accurate results and the absorbance values measured were then converted to
standardised absorbance for comparison. Significantly, the same experiment (with the same
ester:enzyme mimic concentration ratio) for the Zn2+/βCDtren system was performed in a 1.0 cm
cuvette without the need to reduce the path length in order to obtain the absorbance in a
satisfactory range. Whilst the peaks at 400 nm in Figures 4.8 and 4.16 are almost identical, the
peak approximately 270 nm is markedly different. This observation will be discussed further in
Section 4.4.
A comparison of Tables 4.4 and 4.8 also reveals a difference in the experimentally
determined kobs between the Zn2+ and Cu2+ complexes of βCDtren. The rates of reaction with the
Cu2+/βCDtren system are slower than those with the Zn2+/βCDtren system for the same enzyme
mimic concentration. This must, in some part, be contributed to by the slower uncatalysed rate
that occurs at pH 8.68 in comparison with pH 9.14. Both the catalysed and uncatalysed rates
contribute towards the overall observed rate constant, kobs, in accordance with Equation 4.15 and
kuncat is dependent on pH.27 There is almost three times as much hydroxide present in the bulk
solution at pH 9.14 as there is at pH 8.68 and this will have an effect on the measured kobs.
However, even when the contribution from hydroxide in the bulk solution is made equal in
Equation 4.15 for both complexes, the Cu2+/βCDtren system still produces slower rates
suggesting that the intrinsic properties of the complex which governs the rate enhancement are
more significant than the difference in kuncat. (See Section 4.2.4.)
The trend in varying kobs with [enzyme mimic] concentration is very similar between systems.
In the initial stages kobs varies approximately linearly with [enzyme mimic] – when
[Cu(βCDtren)2+] is doubled from 4 × 10-4 to 8 × 10-4 mol dm-3 the kobs halves from 6649 s-1 to
3367 s-1. However, as the complex concentration gets increasingly larger, the reduction in kobs
and the half-life decrease by incrementally smaller amounts.
Just like for the combined enzyme mimics [Zn(βCDtren)(H2O)2]2+,

[Zn(βCDtren)(H2O)(OH)]+ and [Zn(βCDtren)(OH)2], this reflects the process whereby over this
complex concentration range, the ester substrate is increasingly bound by the combined
[Cu(βCDtren)(H2O)2]2+, [Cu(βCDtren)(H2O)(OH)]+ and [Cu(βCDtren)(OH)2] enzyme mimics
until all of it is present as part of the E•S Michaelis complex, resulting in the maximum
hydrolysis reaction rate. To illustrate this fully, the experimentally determined kobs values were
155
plotted against the enzyme mimic (Cu2+ complex) concentration for each experiment. This curve
was then fit with Equation 4.27 (identical to the newly defined Equation 4.19) to determine the
catalytic rate constant, kcat, and the Michaelis constant, KM. Figure 4.17 displays this graph and
Table 4.9 summarises the results.
0 .0 0 4
0 .0 0 3
k obs ( s )
-1
0 .0 0 2
0 .0 0 1
0 .0 0 0
0 .0 0 0 0 .0 0 5 0 .0 1 0 0 .0 1 5 0 .0 2 0
-3
[ C u ( I I ) e n z y m e m im ic ] m o l d m
Figure 4.17. Plot of the experimentally determined kobs against Cu(II) enzyme mimic
concentration (total combined concentration of [Cu(CDtren)(H2O)2] 2+,
[Cu(CDtren)(H2O)(OH)] + and [Cu(CDtren)(OH)2]) at pH 8.68. The solid curve represents the
best fit of the alogorithm for Equation 4.27 to the kobs data.
Essentially, as described by Equation 4.19, this graph shows the variation of the kobs with the
enzyme mimic concentration.62 As shown in the scheme in Equation 4.15, kuncat and kcat are the
rate constants for the uncatalysed and catalysed reaction pathways respectively. Under the
experimental conditions these remain constant, and the overall observed rate constant, kobs, (for
the combination of the uncatalysed and catalysed hydrolysis pathways) varies as a function of
enzyme mimic concentration.
156
Cu2+/βCDtren
system Parameter Error
-3
pH 8.68 KM (mol dm ) 0.0116 ± 2.6 × 10-3
kcat (s-1) 0.00510 ± 6.3 × 10-4
Table 4.9. Calculated Michaelis constant, KM, and catalysed rate constant, kcat, obtained from
the fit in Figure 4.17 for the hydrolysis of 4-nitrophenyl acetate in the presence of enzyme mimic
system Cu2+/βCDtren. Aqueous NaClO4 (I = 0.10 mol dm-3), pH 8.68 ([buffer] = 1.0 × 10-2 – 5.0
× 10-2 mol dm-3), 298.2 K.
The data agrees with the fitting algorithm (evidenced by R2 = 0.9916). Like with the
Zn2+/βCDtren system the relatively large relative errors result because the enzyme mimic
concentration range that is workable (before precipitation occurs) is far from the maximum
required to attain maximum kobs. To further this preliminary work, ideally more experiments
within the concentration range measured above should also be undertaken as part of Future Work
to confirm the shape of this curve. According to this characterisation, the Cu2+/βCDtren system
therefore enhances the rate 303-fold (kcat/kuncat).
Finally, the pH variation of kobs at 298.2 K with [4-nitrophenyl acetate] = 4 × 10-5 mol dm-3
and [Cu(II) catalyst] = 4 × 10-4 mol dm-3 was determined at pH 7.93 (7.9 × 10-5), 8.43 (8.1 × 10-
5
), 8.68 (1.0 × 10-4), 8.98 (1.5 × 10-4) and 9.18 (2.1 × 10-4) (were the values in brackets are the
determined kobs in s-1) was determined from the time dependence of absorbance at 400 nm as
described in Section 4.1.3. The curve produced by plotting kobs (s-1) versus pH was then fit using
the Boltzmann sigmoidal model. This graph is shown in Figure 4.18 and yielded values for
bottom (plateau of minimum kobs), top (plateau of maximum kobs), inflection point (aqua pKa) and
slope (shown in Table 4.10). (It should be noted for this fit that some of the relative errors are
again large because the pH range that could reasonably be tested before precipitation occurred is
far from the pHs required to attain the maximum kobs plateau. Furthermore only five points are
recorded. However, the fit of the data that could be obtained is satisfactory with an R 2 value of
0.9984. Experiments at more pHs should be undertaken as part of Future Work.)
157
Parameter Error
Cu2+/βCDtren
minimum kobs (s-1) 7.6 × 10-5 ± 4.5 × 10-6
system
maximum kobs (s-1) 3.6 × 10-4 ± 1.5 × 10-4
inflection point (aqua pKa) 9.21 ± 0.23
Table 4.10. Summary of minimum and maximum kobs and the aqua ligand pKa from the fit of the
pH-rate profile for the Cu2+/βCDtren system.
0 .0 0 0 2 5
0 .0 0 0 2 0
0 .0 0 0 1 5
k obs ( s )
-1
0 .0 0 0 1 0
0 .0 0 0 0 5
0 .0 0 0 0 0
7 .5 8 .0 8 .5 9 .0 9 .5
pH
Figure 4.18. pH-rate profile experimental curve for the hydrolysis of 4-nitrophenyl acetate (4 ×
10-5 mol dm-3) with enzyme mimic [Cu(βCDtren)] 2+ (4 × 10-4 mol dm-3) at varying pHs. Fit with a
sigmoidal Boltzmann curve.
Figure 4.19 displays the simulated pH-rate profile curve using the bottom, top, slope and
inflection values determined from the fit of Figure 4.19, extrapolated over the pH range 5 to 14.
Again, these results support the concept that the kinetics characteristics of this system are
controlled by the acid-base equilibria of the catalytically active hydroxo ligand forming.
In agreement with the findings for the Zn2+/βCDtren system, the inflection point of this plot
falls between the two experimentally determined aqua ligand pKaH2Os for this complex. As shown
in Table 4.7, pKaH2Os 1 and 2 are equal to 8.68 and 9.77 respectively and the sigmoidal inflection
is 9.21. This is again what might be expected when the two hydroxo-containing complexes –
158
whilst being formed at different pHs – are actually identical in form once the ester has
coordinated.
0 .0 0 0 4
0 .0 0 0 3
k obs ( s )
-1
0 .0 0 0 2
0 .0 0 0 1
0 .0 0 0 0
5 6 7 8 9 10 11 12 13 14
pH
Figure 4.19. pH-Dependent rate profile for the hydrolysis of 4-nitrophenyl acetate in the
presence of [Cu(βCDtren)(H2O)2] 2+ and its deprotonated analogues simulated from the
experimentally determined values reported in Table 4.10.
4.2.4 Discussion of the Zn2+/βCDtren and Cu2+/βCDtren systems – are they

successful enzyme mimics?
There are many different types of approach that have been trialed in order to produce efficient
enzyme mimics,3, 4 including the application of supramolecular chemistry.82, 83 The Zn2+/βCDtren
and Cu2+/βCDtren systems both enhance the rate of 4-nitrophenyl acetate hydrolysis above the
uncatalysed rate by 571- and 303-fold, respectively (kcat/kuncat). They can therefore be deemed
successful enzyme mimics. As discussed in more detail throughout this section, these rate
enhancements cannot compete with carbonic anhydrase which produces a kcat for 4-nitrophenyl
acetate hydrolysis of 2.3 s-1. However, they are comparable with similar mimics. For example,
[Zn(βCDtacdo)OH]+ achieves a rate enhancement for the hydrolysis of 4-nitrophenyl acetate of
291 times over the uncatalysed rate and the Cu2+ complex of dipropylene triamine produces a kcat
of only 7.0 × 10-7 s-1 for the hydrolysis of a similar phosphate ester. Although the work presented
159
in this Chapter is only a preliminary investigation into this type of enzyme mimic, these results
are promising and suggest Future Work in this area may yield further advances.
With the lower aqua pKaH2O#1, [Cu(βCDtren)(H2O)2]2+ is active over a broader pH range than
[Zn(βCDtren)(H2O)2]2+ because its active hydroxo ligand begins to be formed at a lower pH (pH
8.68 versus 9.14). However, for the same extent of mono-hydroxo complex formation (i.e. when
experiments are conducted at the pH = pKaH2O#1) the Zn2+/βCDtren system yields the larger kcat
value than the Cu2+/βCDtren system; 2.1 × 10-2 s-1 versus 5.1 × 10-3 s-1. As initially postulated,
the lowering of the pKaH2O#1 is therefore not the only parameter that predicts enzyme mimic
success.
As described by Equation 4.19, the measured kobs is affected by both kcat and kuncat. The kuncat
contribution in the experiments for the Cu2+/βCDtren system is less than that for the
Zn2+/βCDtren system due to the lower pH used for the Cu2+-containing experiments. Therefore a
direct assessment can only be made by substituting the same kuncat contribution into Equation 4.19
for both species and comparing the resulting calculated kobs. When the kcat and KM values
recorded in Table 4.9 for the Cu2+/βCDtren system are used in Equation 4.19 with the kuncat value
for the Zn2+/βCDtren system (3.7 × 10-5 s-1), the calculated kobs (for every point but that at
complex concentration 4 × 10-4 mol dm-3) values are still smaller than the experimentally
determined values for the Zn2+/βCDtren system. This comparison is not experimentally accurate
but it simply highlights that difference between and effect of kcat between these two complexes is
greater than the effect of kuncat.
It should be noted that the kcat values for both complexes are in reality a reflection of the
proportion of each complex that is in the catalytic form at the investigated pHs. For example, as
discussed for the Zn2+/βCDtren system, at pH 9.14 there is a 47% formation of the catalytically
active [Zn(βCDtren)(H2O)(OH)]+ and a 7% formation of the catalytically active
[Zn(βCDtren)(OH)2]. In other words, at this pH only 54% of the total potential catalyst is in a
catalytically active form that is responsible for the measured kcat and 46% is present as
[Zn(βCDtren)(H2O)2]2+ which is not facilitating any hydrolysis. Therefore if 100% of the
Zn2+/βCDtren complexes were in a catalytically active form, the true kcat would be even larger.
Neither complex reaches the rate enhancements for the hydrolysis of 4-nitrophenyl acetate
that is achieved by carbonic anhydrase. Human carbonic anhydrase I produces a kcat that is two
orders of magnitudes larger than the catalytic rate constants for the complexes investigated in this
160
study.84 However, the complexes in this study do result in better kcat values for 4-nitrophenyl
acetate hydrolysis than similar amine-comprising Zn2+ 23 and Cu2+ 57, 85 complexes when reacted
with similar esters. The measured rate enhancements over the uncatalysed reaction of 571- and
303-fold for the Zn2+/βCDtren and Cu2+/βCDtren systems respectively are also much larger than
the 4-nitrophenyl acetate hydrolysis rate enhancement for the originally published, simplest
deprotonated βCD catalyst under alkaline conditions which achieved only an 8.1-fold increase.62
However, although the results of this study suggest that the addition of the βCD moiety does
function as an effective substrate trap (see Section 4.3), the complexes in this study produce
larger KM values with 4-nitrophenyl acetate than are reported in other investigations. In the
reaction involving the tetradentate, cyclic amine-functionalised βCD ligands βCDtacdo,
βCDcyclen and βCDcyclam, all three complexes with Zn2+ yield smaller Michaelis constants
than [Zn(βCDtren)(H2O)2]2+ 23 and the Cu2+ complex of the linear, bidentate amine-functionalised
βCD ligand, βCDen, yields a smaller Michaelis constant than [Cu(βCDtren)(H2O)2]2+.22 It is
possible therefore that the tacn family of ligands that should be studied as part of the Future
Work may produce smaller KM constants indicative of more stable enzyme-substrate, host-guest
complex formation.
Interestingly, on comparison of the KM values, the Zn2+/βCDtren system has a larger

Michaelis complex dissociation constant than the Cu2+/βCDtren system. Because this equilibrium
constant represents the dissociation of the enzyme-substrate complex ([E•S]), a larger value
indicates a less stable complex. Furthermore this translates that a larger enzyme concentration is
required in order to achieve the maximum [E•S] concentration and therefore the maximum
possible observed rate. The Michaelis constant for the Zn2+/βCDtren system is more than twice
the size of that for the Cu2+/βCDtren system; 0.033 mol dm-3 versus 0.0116 mol dm-3
respectively. Consequently, whilst the [Cu(βCDtren)(OH)]+ complex forms a more stable
interaction with the ester and achieves a maximum rate at a lower enzyme mimic/substrate ratio
than for the [Zn(βCDtren)(OH)]+ complex, it does so with a smaller overall rate enhancement
than is achieved by the [Zn(βCDtren)(OH)]+ complex. (Note here that the [Cu(βCDtren)(OH)]+
and [Zn(βCDtren)(OH)]+ naming protocols have been used here because, as discussed in Section
4.3.2, regardless of the initial composition of the enzyme mimic – [M(βCDtren)(H2O)(OH)]+ or
[M(βCDtren)(OH)2] – the ester must either displace an aqua or hydroxo ligand in order to bind
and react and therefore the overall E•S has the form [M(βCDtren)(OH)(ester)]+.)
161
The fastest ester hydrolysis rate enhancements recorded for βCD-based systems are for βCD
dimer complexes of both Cu2+ and Zn2+.1, 26-28
In these enzyme mimics two cyclodextrins are
tethered by a linker which includes a functional group that will bind to an introduced M2+. The
ester then includes in both annuli simultaneously, causing the carbonyl group to be in the vicinity
of the M2+ and accompanying hydroxo ligand. These achieved rate enhancements can be as high
as 220 000-fold which is far beyond the scope of the complexes investigated in this study.26
However, these dimer moieties require specialised esters whose characteristics (length, bulk and
other functional groups) allow them to bind selectively into both βCD annuli such that the ester
functional group is positioned for direct interaction with the metal complex in the βCD linker.
Indeed, an attempt to use one of these dimers for the hydrolysis of 4-nitrophenyl acetate resulted
in a rate enhancement of only 2-fold over the uncatalysed reaction.28 Therefore, whilst these
dimers represent the current standard and goal in terms of βCD catalytic hydrolysis rate
enhancement, they are more specialised and can only successfully function with specific esters.
In the search for efficient catalysts for general ester hydrolyses for industrial and environmental
purposes, the very fast rate enhancements of the dimer species may be at the cost of their
generality.
4.3 Analysis of other possible catalytic species – free metal, free

ligand, free βCD
In order to fully investigate the properties of these complexes that contribute towards their
catalytic activity, several other kinetics experiments were performed. Each component of the
complexes was tested on its own in order to determine the necessity of these components to the
overall catalyst success. As discussed in Section 1.2, the features of the complexes in this study
were chosen as simplified imitators of the features of enzymes. The hydrophobic annulus of βCD
was selected to mimic the hydrophobic binding pocket of many enzymes, the metals Zn2+ and
Cu2+ were selected as they are prevalent in metalloenzyme active sites, and the amine ligands
were chosen for their similarity to the metal-binding residues of those enzymes. Testing each of
these three components separately allowed for an understanding of their individual significance
in the overall complex catalytic activity.
Therefore, native (free) βCD, free tren, free Me6tren, Zn2+(aq), Cu2+(aq), free βCDtren and free
βCDMe5tren were all tested for their potential to enhance the hydrolysis rate of 4-nitrophenyl
162
acetate. These experiments were performed in an identical fashion to those previously reported –
the formation of the 4-nitrophenolate product over time was followed at its λmax (400 nm) by UV-
Vis spectrophotometery. Plots of this absorbance with time were fit with the equation specified in
Section 4.1.3 to yield pseudo-first order rate constants, kobs. In every experiment the ester
substrate concentration was 4 × 10-5 mol dm-3 (for comparison with the central complexes
investigated) and the potential catalyst concentration was 4 × 10-4 mol dm-3 (the minimum
concentration – a ten-fold excess over the ester). Table 4.11 summarises the experimental
conditions and the kobs that were determined.
Immediately noticeable is the very small kobs measured for free βCD compared with those of
the other species. It is even smaller than the uncatalysed rate constant for the same experimental
conditions (kobs at pH 8.68 in the absence of any potential catalyst species = kuncat = 1.7 × 10-5 s-1
± 1.0% which translates to a half-life of 11.5 hours). The hydrolysis in the presence of free βCD
is more than twice as slow as the uncatalysed hydrolysis at the same pH. Studies have shown that
native βCD does increase the rate of reaction beyond that of the uncatalysed rate. 17, 22, 40, 62
However the experimental conditions in these published studies involve a pH ≥ 10 at which there
will be some deprotonation of cyclodextrin rim hydroxyl groups – the primary (narrow rim) and
secondary (wide rim) hydroxyls of βCD have pKas of 15 – 1686 and 12.287 respectively. The
anionic cyclodextrin is acylated during reaction with the ester and then hydrolysed under the
alkaline conditions in order to regenerate the deprotonated catalytic form.17 However, in the pHs
employed in this work none of the βCD hydroxyl groups will be deprotonated and this
mechanism of ester hydrolysis is therefore prohibited. It is likely, therefore, that this decrease in
hydrolysis rate below the uncatalysed rate indicates that the 4-nitrophenyl acetate is including in
the βCD annulus which is shielding it from reaction. In the uncatalysed experiment the ester
reacts with hydroxide in the bulk solution according to Collision Theory88 but on introduction of
native βCD, the probability of the ester interacting successfully with hydroxide is apparently
decreased. The same finding is reported for the similar analogue dimethyl-βCD.89
163
Potential half-life half-life half-life

kobs (s-1)
enzyme mimic pH (s) (min) (hours)
βCD 8.68 8.3 × 10-6 ± 4.7% 83976 1399.6 23.3
-4
tren 8.68 4.5 × 10 ± 0.4% 1552 25.9 0.4
[Cu(tren)]2+ 9.11 8.2 × 10-6 ± 2.6% 84757 1412.6 23.5
Me6tren 8.68 6.9 × 10-5 ± 1.4% 10080 168.0 2.8
-4
βCDtren 8.68 3.1 × 10 ± 0.4% 2224 37.1 0.6
-5
βCDMe5tren 8.68 9.5 × 10 ± 0.3% 7328 122.1 2.0
-5
Zn2+(aq) 9.14 3.0 × 10 ± 1.9% 23465 391.1 6.5
-5
Cu2+(aq) 8.68 1.3 × 10 ± 2.1% 52891 881.5 14.7
For comparison:
Zn2+/βCDtren 9.14 1.9 × 10-4 ± 0.5% 3697 61.6 1.0
-4
Cu2+/βCDtren 8.68 1.0 × 10 ± 0.2% 6777 113.0 1.9
-5
uncatalysed 9.14 3.7 × 10 ± 0.2% 18969 316.2 5.3
-5
uncatalysed 8.68 1.7 × 10 ± 1.0% 41258 687.3 11.5
Note: the experiments for βCD and [Cu(tren)]2+ where only followed continuously (before
thermostatting to measure the final absorbance at t∞) for approximately the first half-life
(rather than a mimimum of two half-lives) due to their comparatively slow rates and instrumental
time constraints. Satisfactory R2 values (> 0.99) and relative errors were maintained.
Table 4.11. Summarised data - observed rate constants, kobs, and half-lives for the hydrolysis of
4-nitrophenyl acetate ([ester] = constant = 4 × 10-5 mol dm-3) in the presence of different
potential catalysts ([catalyst] = constant = 4 × 10-4 mol dm-3) at the designated pH. Determined
using UV-Vis spectrophotometry in aqueous NaClO4 (I = 0.10 mol dm-3), DEPP buffer (1.0 × 10-
2
mol dm-3), 298.2 K.
One of the modes of orientation for the inclusion of 4-nitrophenyl acetate into the βCD
annulus has been determined and is demonstrated in Figure 4.20.17 The ester functional group is
located at the narrow rim of the cyclodextrin. It is also reported that the addition of an amine
functional group to the C6 position on narrow rim of the βCD annulus has little to no effect on
ester binding geometry and so therefore this is likely to be the orientation that occurs for the
amine-functionalised βCD moieties in this study.17
164
Figure 4.20. An experimentally determined17 orientation of 4-nitrophenyl acetate including into

the annulus of βCD.
Experiments introducing adamantanecarboxylic acid,17 adamantane-1-carboxylate18 and

cyclohexanol21 that competitively complex with βCD and cause a reduction in the ester
hydrolysis rate, demonstrate definitively that 4-nitrophenyl acetate is including in the βCD
annulus as part of the hydrolysis mechanism. (Intuitively there is a possibility for the ester to
react with the deprotonated hydroxyl or a group substituted on the rim without entering the
annulus and therefore these experiments are required.) Similar experiments should be done as
part of this study in Future Work, however this finding for free βCD rate suppression supports the
notion that the βCD is acting like a hydrophobic enzyme pocket and binding the substrate prior to
hydrolysis.
Secondly, in Table 4.11 there is a very large difference recorded between the ester hydrolysis
rate in the presence of free Zn2+(aq) (at pH 9.14) and free Cu2+(aq) (at pH 8.68). Whilst Zn2+(aq)
appears to enhance the rate over the uncatalysed reaction, Cu2+(aq) depresses it, much like free
βCD. At these pHs the predominant species in both solutions is M(OH)2 and both Zn(OH)2 and
Cu(OH)2 are only sparingly soluble in water at 25˚C. Indeed, a small amount of very fine
precipitate was observed in these solutions prior to reaction and consequently these results must
be treated with caution. Despite the presence of precipitate, information was obtained from the
UV-Vis spectra recorded during the course of the reactions. As described in Section 4.4, there is
an enhanced absorbance peak at ~265 nm for the Cu2+ systems over the Zn2+ systems which
suggests that the 4-nitrophenyl acetate binds to the Cu2+ in solution when these reacting species
are combined. (Note, the lack of increased absorbance at this wavelength for Zn2+ does not
discount the ester binding to this metal, it simply means this peak enhancement was not observed
under the same conditions as it was for Cu2+.) This ester –M2+(aq) binding would be instigated on
a collisional time scale (less enduring than the interaction between the ester and the βCD annulus,
for example). However, the measured rate suggests that the probability of 4-nitrophenyl acetate
165
interacting successfully with hydroxide is decreased in the presence of Cu2+(aq). One hypothesis to
explain these observations is that the stereochemistry of the Cu2+ in solution is such that, when
the ester binds, it does so in such a way that is it trans to any hydroxo ligands also bound to Cu2+.
When bound to the Cu2+ (as evidenced by the UV-Vis spectrum) the ester may be prevented from
colliding and reacting with other hydroxide in the bulk solution (particularly if it is insoluble and
precipitates) but may also be bound in an unfavourable orientation to react with the hydroxo
ligands. Conversely, the increased hydrolysis rate in the presence of Zn2+(aq) indicates that the
ester more readily collides and reacts successfully with OHˉ under these conditions. The lack of
enhanced ester peak in the free Zn2+(aq) spectrum could suggest that the ester does not bind to this
metal. Or if binding does occur, the increased rate suggests that the conformation is not such that
the ester reaction with other bound hydroxo ligands or bulk hydroxide is inhibited. Further
investigation into the exact hydrolysis mechanism and the effect of metal ion identity on
hydrolysis rate should be carried out as part of Future Work.
Interestingly, the same result is observed for [Cu(tren)]2+ (and preliminary tests for
[Cu(Me6tren)]2+ and [Zn(Me6tren)]2+). The ester in the presence of these complexes also
undergoes hydrolysis at a rate slower than the uncatalysed rate at a similar pH. This experiment
for [Cu(tren)]2+ was performed at a pH of 9.11 – the pKaH2O#1 of the corresponding aqua
deprotonation for this complex – consistent with the other metal complex investigations. The
mechanism of hydrolysis is assumed to be the same as that shown in Figure 4.2 (without the βCD
pocket), however it occurs very slowly for this complex compared to the other species in Table
4.11. Consistent with native βCD and free Cu2+(aq) the depressed rate below that of the
uncatalysed reaction shows that these reacting species decrease the likelihood of 4-nitrophenyl
acetate reacting successfully with hydroxide (in the bulk solution or a hydroxo ligand bound to
M2+). Unfortunately the same experiment for [Zn(tren)]2+ could not be performed for comparison
because the pH of its pKaH2O#1 is 9.97 and under these conditions precipitation becomes
problematic. However, this result for [Cu(tren)]2+ may provide evidence for a Jahn-Teller
distortion for this complex as postulated in Chapter 2. Again, an enhanced peak in the UV region
of the spectrum in the initial stages of reaction suggests that the ester is binding to the Cu2+. In
the potentiometric titration chapters the Jahn-Teller distortion is hypothesised to shorten the Cu2+
– OH2 bond, thereby further polarising the aqua ligand towards deprotonation and lowering its
pKaH2O (correspondingly, the Cu2+ complexes investigated in this work produce low aqua pKaH2O
values relative to the other metals studied). This shortened bond between Cu2+ and the hydroxo
166
ligand formed may hinder the reaction between this hydroxo and the bound ester. Alternatively
the stereochemistry and therefore ligand orientation on a distorted complex may generally cause
this reaction to be less favourable. Interestingly this result is not unique to this complex and a
similar finding is reported for the complex of Zn2+ with the tetradentate, macrocyclic ligand
cyclam.72 The reaction of 4-nitrophenyl acetate with [Zn(cyclam)]2+ is reportedly too slow to
measure, even under alkaline conditions.
It has also been hypothesised that the ester hydrolysis rate enhancement (kcat/kuncat) produced
by a given potential catalyst is independent of its ground state binding to the ester but instead, in
fact, a function of the transition state binding interaction and the potential catalyst concentration.
32
It is possible, therefore, that the particularly slow rates for Cu(aq)2+ and [Cu(tren)]2+ (and
therefore, by extension, [Zn(cyclam)]2+) are the result of an unfavourable interaction between the
ester hydrolysis transition state and each of these species. However, a much more detailed
investigation would need to be undertaken as part of Future Work in order to determine the
source of the slow rates for these species.
It is most relevant to note that the same slow rate is not observed for the complex involving
the βCD-containing ligand – [Cu(βCDtren)]2+ which is likely a direct result of the incorporation
of βCD which includes the ester and increases the effective concentrations (and possibly even the
favourable orientations) of the reacting species.
The next point to note from Table 4.11 is that the free amines appear to be better at enhancing
the reaction rate than their metal complexes. Firstly, unbound βCDtren catalyses the 4-
nitrophenyl acetate hydrolysis with a half-life of approximately 37 minutes (pH 8.68) whilst the
Zn2+ and Cu2+ complexes with this ligand produce half-lives of 62 minutes (pH 9.14) and 113
minutes (pH 8.68) respectively. There are three possible mechanisms for ester hydrolysis
mediated by these moieties and these are shown in Figure 4.21. Path A represents the familiar
ester binding and hydroxo ligand attack that is proposed for metal complexes of this type.43, 57, 75
However, without a metal cation present this hydrolysis pathway is prohibited. Path B displays
the nucleophilic acyl substitution reaction that occurs between the ester substrate and the free
ligands. This mechanism is only feasible for ligands containing primary and secondary amines
and results in stable acylated products under these conditions17, 18 (as well as the monitored 4-
nitrophenolate). Path C displays the general base catalysed hydrolysis that occurs between the
ester substrate and the free ligands that contain only tertiary amines.
167
Figure 4.21. Possible hydrolysis mechanisms for the reaction of 4-nitrophenyl acetate with the
free amine ligands and metal complexes investigated in this study. Path A displays nucleophilic
attack of the bound hydroxo in the presence of M2+. Path B displays nucleophilic acyl
substitution by 1˚ and 2˚ amines. Path C represents general base catalysed hydrolysis by 3˚
amines. (The amine diagram in Path A is representative of a generic tetradentate ligand. For
tren specifically, three of the nitrogen donor atoms displayed are appended to the fourth.)
168
In the case of all the free primary and secondary amines characterised in this study (tren,
βCDtren, tacn and βCDtacn) the mechanism to produce the 4-nitrophenolate product will not be
the same as that for the reaction with the corresponding metal complexes. For these free, unbound
amines the reaction with the ester substrate will be a nucleophilic acyl substitution instead of
hydrolysis (though both pathways produce the same 4-nitrophenolate product). This general
mechanism for the reaction of the unbound amines with the ester is displayed in Path B of Figure
4.21.
To illustrate, a 1:1 mole ratio reaction of the cyclodextrin 6A-amino-6A-deoxy-β-cyclodextrin

(βCDNH2) with the ester 4-nitrophenyl acetate (pH 10.0, 298.2 K) produces a 71% yield of the
acetamido-substituted βCD (βCDNHCOCH3) that is stable under the experimental conditions.17
Although the same 4-nitrophenolate signal will be produced in these experiments without the M2+
present, these amine species are not catalysts because they are acylated in the process. As can
also be seen in Figure 4.21, that this mechanism requires the lone electron pair on the nitrogen
atom to be free to attack the electrophilic carbonyl carbon. It follows, therefore, that on binding
these amine ligands to M2+ this mode of reaction will be suppressed. This is indeed reported for
the amine, Prop-en (H3CCH2CH2-NH-CH2CH2-NH2), and its copper complex for which it is
explained that the addition of the Cu2+ blocks the nitrogen lone pairs and consequently retards the
nucleophilic acyl substitution rate.22 The same result is also noted for the amine βCDen (6A-(2-
aminoethyl)-6A-deoxy-β-cyclodextrin) and its copper complex, [Cu(βCDen)]2+.22
The amino substituent of βCDen contains one primary and one secondary amine and the
amino substituent of βCDtren in this study contains one tertiary, one secondary and two primary
amines. The nitrogen atoms in these primary and secondary amine functional groups are situated
on flexible pendant arms (increasing the likelihood of finding a favourable geometry of the lone
pair attack on the ester carbonyl carbon) and are all capable of undergoing nucleophilic acyl
substitution reactions with the ester substrate. However, with the addition of M2+ the ester binds
to the metal centre and the reaction mode changes from nucleophilic acyl substitution with the
amine to hydrolysis with a hydroxo ligand also present on M2+.43 The metal complex is likely to
be more rigid than the unbound amine and furthermore the potentiometric titration
characterisation reveals that when the ester is bound to the M2+ there will only be one hydroxo
ligand available for hydrolysis, unlike the multiple possible nitrogen atoms in the unbound
amines. It is therefore not surprising that when the M2+ is added and the reaction mode switches
from stoichiometric acyl substitution to catalytic hydroxo hydrolysis that the rate decreases.
169
However, only the metal complex is catalytic which is an advantage over the free primary and
secondary amines.
When the mechanism is nucleophilic acyl substitution it also therefore plausible that free tren
should be a better at enhancing the reaction rate than βCD-substituted tren, because the free
ligand has more unrestricted amine nitrogens to facilitate attack on the ester (the βCD forms a
bond at one of these nitrogens in order to form the substituted moiety). Indeed, it has been
reported that the efficiency of βCD-substituted amines is less than that of the free amine but does
increase with the increasing number of amine functional groups attached to the βCD.20 (It is also
therefore hypothesised that the free tren and tren-substituted βCD species might be more efficient
catalysts than the free tacn and tacn-substituted βCD species because they contain four amines
rather than three, though this may be investigated as part of the Future Work.)
A comparison of the unbound ligands containing primary and secondary amines is, however,
made more nuanced by the ligand pKas. An evaluation of tren and βCDtren or Me6tren and
βCDMe5tren is not strictly a comparison of free ligand versus βCD-functionalised derivative. Just
as the binding of M2+ suppresses the nucleophilic acyl substitution mechanism, so does
protonation of the N-donor atoms (the lone pair required becomes occupied). At the pH of
testing, each of the amines has a different proportion of protonated species in solution. Table 4.12
shows the percentages of protonated species for each ligand (obtained from the potentiometric
characterisation) in order of decreasing kobs for the pH of the experimental conditions, 8.68.
Species %L % LH+ % LH22+ % LH33+ % LH44+ kobs (s-1)
tren 0.3 10.6 62.4 26.6 < 0.001 4.5 × 10-4

βCDtren 2.4 22.3 73.7 1.6 < 0.001 3.1 × 10-4
βCDMe5tren < 0.001 < 0.001 99.5 0.5 < 0.001 9.5 × 10-5
Me6tren 7.5 39.3 50.7 2.5 < 0.001 6.9 × 10-5
Table 4.12. The percentage speciation of ligands tren, Me6tren, βCDtren and βCDMe5tren at pH
8.68 and the corresponding rate constant for the hydrolysis of 4-nitrophenyl acetate at this pH.
(Percentages rounded to the first decimal place.)
170
The one amine functional group that all of these ligands have in common is the central
tertiary amine connected to the three pendent arms of tren and functionalised tren. This is the
least basic nitrogen (has the lowest pKa) and is represented by the deprotonation of LH44+ to
LH33+.90 In all four cases there is almost none of the tetra-protonated ligand in solution at pH 8.68
which means that Path C in Figure 4.21 is theoretically free to occur for all of these ligands
(though in reality this mechanism may be affected to greater or lesser extents by steric crowding
around the tertiary nitrogen).
Interestingly, the fully deprotonated ligand, L – which has the maximum number of nitrogens
free to react by Paths B and C – evidently does not dictate the hydrolysis rate because the ligand
with the largest proportion of this form in solution (Me6tren) also has the slowest rate.
Furthermore, all of the ligands have a greater than 99% formation of LH33+ + LH22+ + LH+ + L –
i.e. 99% of the combined species in solution have one or more nitrogens free for reaction.
However, when comparing the combinations of the more reactive species (based on their
numbers of deprotonated nitrogens free for reaction), tren has the least of the species LH22+ +
LH+ + L or LH+ + L and Me6tren has the most (Table 4.12). This is the exact opposite of the
trend that might be predicted based on the measured rates.
It might be hypothesised, therefore, that the rate of reaction for Paths B and C in Figure 4.21
is largely contributed to by the proportions of primary, secondary and tertiary amines in the
ligands, and the steric hindrance around the nitrogens in these amine functional groups. All four
ligands contain the tertiary amine at the apex of the tren component tripod, but outside of this,
tren has three primary amines, βCDtren has two primary and one other tertiary and Me6tren and
βCDMe5tren both contain three other tertiary amines. In a comparison between the only two
ligands with the same amine composition – Me6tren and βCDMe5tren – the βCD-functionalised
version produces the faster hydrolysis rate which may be indicative of the βCD annulus
performing its intended role as a substrate trap. Following this hypothesis however, the same
trend would not be the expected result for tren and βCDtren because tren has the larger
proportion of primary amines.
Perhaps the most relevant comparison in the context of this study is one between the
hydrolysis rates affected by a metal complex catalyst without βCD and a metal complex catalyst
containing βCD through Path A, Figure 4.21. This assessment should provide a direct reflection
of the significance of the βCD component in creating an effective enzyme mimic. Comparing the
171
rate constants, kobs, for both [Cu(tren)]2+ and [Cu(βCDtren)]2+ in Table 4.11 it can be seen that the
βCD version facilitates a hydrolysis rate approximately 12 times faster than that of the complex
without the βCD moiety present. The experiment for [Cu(βCDtren)]2+ was even conducted at a
lower pH (8.68) than that of [Cu(tren)]2+ (9.11) so that the uncatalysed rate contribution towards
the overall kobs is less for [Cu(βCDtren)]2+ than for [Cu(tren)]2+ and the βCD version still causes a
larger value. Considering both experiments were conducted at the pH of the first aqua ligand
pKaH2O for both species, the enhanced rate for [Cu(βCDtren)]2+ over [Cu(tren)]2+ is direct
evidence that the βCD moiety increases the rate of reaction and its employment as an enzyme
hydrophobic pocket mimic is constructive. Preliminary data for the complex [Cu(βCDMe5tren)]2+
under the same experimental conditions (pH 8.35, half-life 6.7 hours) also affirms this result.
This is a useful finding as it suggests that the βCD moiety can be an effective addition in enzyme
mimic design.
4.4 Mechanistic information gained from qualitative UV-Vis

absorbance experiments
Studies that use the model ester 4-nitrophenyl acetate in their kinetics experiments report the
absorbance changes at 400 nm because this has been shown to be an accurate way to monitor the
hydrolysis as discussed in Section 4.1.2. However, investigations beyond this wavelength may
yield useful information about the mechanism of reaction but are rarely reported. For a study in
which enzyme mimicry is under scrutiny, understanding how the mimic species performs the
catalysis would be extremely valuable. UV-Vis absorbance spectra over the range 225 – 500 nm
were therefore measured for several systems of interest, both in the presence and absence of the
ester reactant and phenolate product. The ester concentration was kept constant at 4 × 10-5 mol
dm-3 and each potential catalyst investigated was used at a concentration of 4 × 10-4 mol dm-3 (the
minimum excess concentration). It should be noted again that these experiments are preliminary
in nature and provide only a cursory picture of the possible mechanisms for substrate binding and
hydrolysis.
The UV-Vis absorbance spectra of 4-nitrophenyl acetate, 4-nitrophenolate and 4-nitrophenol

have been previously recorded over the wavelength range of interest35, 36 and their wavelengths of
maximum absorbance are recorded in Table 4.13 for comparison.
172
λmax
Species (nm)
4-nitrophenyl acetate 271
4-nitrophenolate 400
4-nitrophenol 320
Table 4.13. Summary of λmax (between 225 – 500 nm) of the potential forms of the substrate and
product in solution during hydrolysis: 4-nitrophenyl acetate, 4-nitrophenolate and 4-nitrophenol.
As is reported in the literature, the 4-nitrophenolate product peak at 400 nm is sufficiently far
from the 4-nitrophenyl acetate reactant peak at 271 nm such that either wavelength can be used to
accurately monitor the hydrolysis and quantify an observed rate constant. There is some overlap
of the phenol peak with both the reactant ester and product phenolate absorbances, however with
a pKa of 7.1537 there will be less than 3% phenol formation at pH 8.68 (the pH at which the
Cu2+/βCDtren system analysis is performed) and 1% phenol formation at pH 9.14 (the pH at
which the Zn2+/βCDtren system analysis is performed). Contributions from this species at 400 nm
can therefore be ignored. The peak at this wavelength for the 4-nitrophenolate product is assigned
to a π to π* intramolecular charge transfer transition whereby an electron is transferred from the
negatively charged phenolate Oˉ moiety to the nitro group.91 The molar absorptivity is almost
twice as large at this wavelength as at the 4-nitrophenyl acetate or 4-nitrophenol peaks, producing
a strong signal at 400 nm.
Firstly, the ester in buffer (in the absence of any potential catalyst) was examined. Please note
that for all of the following spectra these experiments are analysed qualitatively only. Because of
the nature of the kinetics experimental method (see Section 4.1.3) the absorbance at time zero
cannot be measured. Therefore tinitial in these spectra represents an initial measurement at some
point precisely recorded after t0, which will be different for each system depending on each
specific experimental setup and rate of reaction. These spectra can therefore be used to provide a
relative comparison but not a quantitative one. Figure 4.22 displays an initial scan of 4-
nitrophenyl acetate in buffer at pH 8.68 ([ester] = 4 × 10-5 mol dm-3) as well as the spectrum once
hydrolysis completion had been reached and the absorbance measurement at 400 nm had become
constant with time. In the following figures, the reacting species present in solution are shown on
each plot.
173
1 .0
t in itia l
t f in a l
0 .8
0 .6
0 .4
0 .2
0 .0
250 300 350 400 450 500
Figure 4.22. Initial and final spectra of the reaction solution of 4-nitrophenyl acetate (4 × 10-
5
mol dm-3, λmax 272 nm) hydrolysis over time to produce 4-nitrophenolate (λmax 400 nm). 298.2 K,
aqueous NaClO4 (I = 0.10 mol dm-3), buffered at pH 8.68 ([DEPP buffer] = 1.0 × 10-2 mol dm-3).
Hydrolysis half-life = 11.5 hours.
The spectra displayed in Figure 4.22 exemplify the ester-phenolate system. Under the
conditions of measurement (298.2 K, aqueous NaClO4 (I= 0.10 mol dm-3) and buffered at pH
8.68 ([DEPP buffer] = 1.0 × 10-2)) the λmax observed for the initial spectrum (when the
predominant species in solution was the 4-nitrophenyl acetate ester) is 272 nm and the λmax
observed for the final spectrum (when the predominant species in solution was the 4-
nitrophenolate) is 400 nm. Furthermore, the small shoulder in the tfinal spectrum ~265 nm is also
found in the reported spectrum of 4-nitrophenolate.35 These experimentally obtained spectra are
therefore in excellent agreement with the literature and provide a template for evaluation with
when the potential catalysts are introduced.
For comparison with the uncatalysed ester hydrolysis spectra, the same measurements were
recorded for 4-nitrophenyl acetate in the presence of other free potential catalysts. (“Free” in this
context refers to unbound, individual components of the overall metal complex enzyme mimics.)
The initial and final spectra for 4-nitrophenyl acetate (4 × 10-5 mol dm-3) in the presence of free
βCD (4 × 10-4 mol dm-3) are displayed in Figure 4.23. (Again, note, “initial” does not equate to
time “zero” and therefore cannot be quantitatively compared.)
174
1 .0
t in itia l
0 .8 t f in a l
0 .6
0 .4
0 .2
0 .0
250 300 350 400 450 500
5
mol dm-3, λmax 273 nm) hydrolysis over time in the presence of βCD (4 × 10-4 mol dm-3) to
produce 4-nitrophenolate (λmax 400 nm). 298.2 K, aqueous NaClO4 (I = 0.10 mol dm-3), buffered
at pH 8.68 ([DEPP buffer] = 1.0 × 10-2 mol dm-3). Hydrolysis half-life = 23.3 hours.
The spectra in Figures 4.22 and 4.23 are almost identical in the regions of interest in terms of
the wavelengths of maximum absorbance, the relative absorbance values and the shapes of the
curves (including the shoulder at ~265 nm for the 4-nitrophenolate product). This suggests that
the presence of the free βCD has little effect on the electronic properties of both the reactant ester
and product phenolate in solution. However, the kinetics testing of this species shows that the
free βCD impedes the hydrolysis rate, slowing it even further than that of the uncatalysed
reaction at the same pH (Table 4.11). Studies under similar conditions show that the 4-
nitrophenyl acetate does include in functionalised βCD annuli,17, 21, 22 which may shield it from
reaction with hydroxide in the bulk solution.
The next four species investigated were free tren, Me6tren, βCDtren and βCDMe5tren – i.e.
the tren-based ligand family unbound in the absence of M2+. These initial and final spectra are
displayed in Figures 4.24, 4.25, 4.26 and 4.27. Again it is observed that these spectra are very
similar to those for the uncatalysed 4-nitrophenyl acetate experiment and the same hydrolysis in
the presence of free βCD. In the cases of free tren and Me6tren the spectra are virtually identical;
the peaks are neither shifted nor altered in shape (relative height or appearance). This again
175
suggests that whilst the addition of these species certainly enhances the rate (see Table 4.11), the
mode of action does not dramatically alter the electronic (and therefore absorbance) properties of
the 4-nitrophenyl acetate reactant or 4-nitrophenolate product on the UV-Vis radiation
absorbance time scale. The mechanism of reaction for these species likely plays a role in their
lack of impact on the reactant and product absorbance spectra. Whilst free tren and Me6tren do
both participate in the hydrolysis potentially via two separate mechanisms (Figure 4.21), this will
occur on a collisional time scale which is on the order of 10-13 s in solution.92 Therefore it is not
unexpected that their presence has little effect on the reactant and product UV-Vis spectra.
1 .0
t in itia l
0 .8 t f in a l
0 .6
0 .4
0 .2
0 .0
250 300 350 400 450 500
5
mol dm-3, λmax 273 nm) hydrolysis over time in the presence of tren (4 × 10-4 mol dm-3) to
176
1 .0
t in itia l
0 .8 t f in a l
0 .6
0 .4
0 .2
0 .0
250 300 350 400 450 500
5
mol dm-3, λmax 273 nm) hydrolysis over time in the presence of Me6tren (4 × 10-4 mol dm-3) to
However, the βCD-functionalised amines do produce slight differences in the UV region as

seen in Figures 4.26 and 4.27. The 4-nitrophenolate peaks at 400 nm appear unaffected with the
introduction of these species (and still have the same λmax), but the spectra in the region of 250 –
300 nm are altered. The initial 4-nitrophenyl acetate peak is of an enhanced absorbance at tinitial,
as is the final shoulder area of 4-nitrophenolate at tfinal, although the shoulder structure itself is
now indefinable. These spectra also appear to be trailing to a much larger peak in the UV just
outside of the range of the spectrum below 225 nm. It may also be relevant to note that the λmax in
the UV for the experiment containing βCDMe5tren is noticeably hypsochromically shifted
compared to the other spectra evaluated so far which may be the consequence of the Path B
(Figure 4.21) mechanism coupled with the βCD substrate “trap”.
The general mechanisms of reaction for βCDtren and βCDMe5tren are predicted to be the
same as for their non-βCD-substituted versions tren and Me6tren, respectively. However, the
addition of the βCD moiety is likely to alter the interaction between the ester and the amine.
Unfortunately a simple comparison between βCD-substituted and free amine ligands cannot be
made because of the different possible modes of reaction that are affected by the number and type
of substituted amines as discussed in section 4.3. However, between Me6tren and βCDMe5tren in
177
which there is only one possible mode of reaction (Path C, Figure 4.21), addition of the βCD
moiety is observed to increase the rate of reaction. This may be attributable to inclusion complex
formation. Whilst the formation of inclusion complexes is a dynamic process whereby the guest
molecule continuously associates and dissociates, the lifetime of these complexes is measured to
be between 10-5 and 10-8 s, depending on substrate structure and charge.93 This is orders of
magnitude more persistent than the collisional life time between two randomly colliding, free
reactant molecules. Furthermore, organic substrates may preferentially bind in the cyclodextrin
annulus in aqueous solution as characterised by the Kinc inclusion complex equilibrium constant.
Complex formation therefore causes substrates to be bound in close proximity to the amine
reactants and for a longer period than a regular, random collision would last. This may result in
both an enhanced hydrolysis rate as well as an impact on the absorbance spectra as is observed in
this study. To investigate this further, a comparison between the spectra of free tacn and Me3tacn
versus βCDtacn and βCDMe2tacn should be made in Future Work.
1 .0
t in itia l
0 .8 t f in a l
0 .6
0 .4
0 .2
0 .0
250 300 350 400 450 500
5
mol dm-3, λmax 271 nm) hydrolysis over time in the presence of βCDtren (4 × 10-4 mol dm-3) to
178
1 .0
t in itia l
0 .8 t f in a l
0 .6
0 .4
0 .2
0 .0
250 300 350 400 450 500
5
mol dm-3, λmax 262 nm) hydrolysis over time in the presence of βCDMe5tren (4 × 10-4 mol dm-3)
to produce 4-nitrophenolate (λmax 401 nm). 298.2 K, aqueous NaClO4 (I = 0.10 mol dm-3),
buffered at pH 8.68 ([DEPP buffer] = 1.0 × 10-2 mol dm-3). Hydrolysis half-life = 2.0 hours.
The final spectra to analyse are for the potential catalysts involving M2+ (the free metals) and
the complexes [M(βCDtren)]2+ and [M(βCDMe5tren)]2+ (M2+ = Cu2+ and Zn2+). Figure 4.28
displays the spectra in the initial stages of reaction for the hydrolysis in the presence of free Zn2+
and free Cu2+ (4 × 10-4 mol dm-3). (It is noted again that at the respective pHs of these
experiments as quoted in Table 4.11, the predominant species is M(OH)2 which is only sparingly
soluble in water. The measured kobs (that are different from the corresponding kuncat) and the
spectral changes indicate that some interaction between the metal and ester occurred. However
these analyses and the hypotheses drawn from them must be treated with caution.) The 4-
nitrophenyl acetate peak in the UV is noticeably enhanced for the reaction with Cu2+ and this is
interesting to analyse with respect to the measured kobs values for these experiments. Conversely,
the initial spectrum for Zn2+ is extremely similar to that of the uncatalysed experiment (Figure
4.22). It is possible that this observation may indicate that there is no long term interaction
between the ester and Zn2+ (eg, binding) beyond the collisional 10-13 s time scale. (Although the
fact that the presence of this species does increase the hydrolysis rate suggests that some form of
interaction takes place.) The increased (almost doubled) absorbance for the 4-nitrophenyl acetate
peak in the presence of Cu2+, coupled with the depressed hydrolysis rate below the uncatalysed
rate under the same conditions, suggests that the 4-nitrophenyl acetate is interacting with the
179
Cu2+. It should also be noted that similar tests for the buffer compound on its own – DEPP –
indicate that it does not interact with Cu2+ to produce a UV-Vis absorbance spectrum, unlike
some of the conventional Good’s buffers.69 Therefore the most likely scenario is that this
enhanced peak ~ 271 nm in Figure 4.28 is produced by ester-Cu2+ interaction.
1 .0
2+
t in itia l ( Z n )
2+
0 .8 t in itia l ( C u )
0 .6
0 .4
0 .2
0 .0
250 300 350 400 450 500
Figure 4.28. Initial spectra of the reaction solutions of 4-nitrophenyl acetate (4 × 10-5mol dm-3)
in the presence of Zn2+ (4 × 10-4 mol dm-3 at pH 9.14) and Cu2+ (4 × 10-4 mol dm-3 at pH 8.68).
298.2 K, aqueous NaClO4 (I = 0.10 mol dm-3), ([DEPP buffer] = 1.0 × 10-2 mol dm-3).
Hydrolysis half-life = 6.5 hours for the Zn2+ system and 14.7 for the Cu2+ system.
Figures 4.29 and 4.30 display the spectra in the initial and final stages of reaction for 4-
nitrophenyl acetate in the presence of 4 × 10-4 mol dm-3 [Zn(βCDtren)]2+ and [Cu(βCDtren)]2+
respectively. There are some marked differences between the two sets of measurements.
180
1 .5 0
t in itia l
1 .2 5
t f in a l
1 .0 0
0 .7 5
0 .5 0
0 .2 5
0 .0 0
250 300 350 400 450 500
5
mol dm-3, λmax 271 nm) hydrolysis over time in the presence of [Zn(βCDtren)] 2+ (4 × 10-4 mol
dm-3) to produce 4-nitrophenolate (λmax 400 nm). 298.2 K, aqueous NaClO4 (I = 0.10 mol dm-3),
2 .0
t in itia l
t f in a l
1 .5
1 .0
0 .5
0 .0
250 300 350 400 450 500
5
mol dm-3, λmax 271 nm) hydrolysis over time in the presence of [Cu(βCDtren)] 2+ (4 × 10-4 mol
dm-3) to produce 4-nitrophenolate (λmax 400 nm). 298.2 K, aqueous NaClO4 (I = 0.10 mol dm-3),
181
Firstly, the spectra in Figure 4.29 (for the Zn2+/βCDtren system) are very similar in shape and
absorbance to those for the uncatalysed ester hydrolysis shown in Figure 4.22. Most significantly,
the 4-nitrophenyl acetate peak present in the initial stages ~ 271 nm appears to diminish over the
course of the reaction until the ester has been completely hydrolysed in the final stages and there
is no discernible peak. This is not the same for the copper complex in Figure 4.30. Not only is the
ester peak even more markedly enhanced in the initial stages for this reaction, but once the
hydrolysis is complete (as denoted by the characteristic constant absorbance for 4-nitrophenolate
peak at 400 nm) there is still a large absorbance in the UV region. To more thoroughly evaluate
the spectra in Figures 4.29 and 4.30 they must be considered in conjunction with Figure 4.31.
This displays the measured absorbance over the same wavelength range of the same
concentrations of βCDtren and [Cu(βCDtren)]2+ prior to addition of the 4-nitrophenyl acetate
reactant.
1 .0
 C D tre n (n o e s te r)
2+
[C u ( C D tre n )]
0 .8 (n o e s te r )
0 .6
0 .4
0 .2
0 .0
250 300 350 400 450 500
Figure 4.31. UV-Vis absorbance spectra of 4 × 10-4 mol dm-3 free βCDtren and the complex
[Cu(βCDtren)] 2+ both in unbuffered MQ water in the absence of 4-nitrophenyl acetate at
approximately pH 6, 298.2 K, aqueous NaClO4 (I = 0.10 mol dm-3).
Whilst the free βCDtren molecule produces some absorbance over the UV range, the copper
complex [Cu(βCDtren)]2+ has an obvious peak at a very close λmax (262 nm) to that of 4-
nitrophenyl acetate (271 nm). When the concentration of [Cu(βCDtren)]2+ in the same 1.0 cm
cuvette was increased to 1.0 × 10-2 mol dm-3, the absorbance at this wavelength increased to
182
outside of the range measureable by the spectrophotometer and could not be accurately recorded.
This peak is likely attributable to a ligand-to-metal charge transfer transition (LMCT) occurring
within the complex. When the complex [Cu(βCDtren)]2+ interacts with 4-nitrophenyl acetate, the
peak in the UV region in the final spectrum is possibly representative of this LMCT between the
βCDtren ligand and Cu2+, irrespective of the fact that all of the ester has been hydrolysed. This
also explains why the λmax of the UV peak is blue-shifted over the course of the reaction – the
isolated 4-nitrophenyl acetate λmax in the initial stages is 271 nm and the isolated
[Cu(βCDtren)]2+ complex LMCT λmax may be 262 nm.
The same does not appear to occur for the zinc system, [Zn(βCDtren)]2+ in Figure 4.32. There
appears to be some slight absorbance increase in the UV region in the final spectrum, however it
does not take the form of a defined peak and nor is it a large increase. Comparing it with the free
βCDtren spectrum in Figure 4.26 may provide evidence to suggest that this is simply ligand
absorbance of the same concentration.
This same comparison for [Zn(βCDtren)]2+ and [Cu(βCDtren)]2+ can also be made at the
increased complex concentration of 1.0 × 10-3 mol dm-3 (at 100 times excess over the ester).
(Beyond this concentration the reactions become too fast to monitor over the whole wavelength
range 225 – 500 nm.) Figures 4.32 and 4.33 display the absorbance spectra in the initial and final
stages (noting that they display “standardised absorbance” calculated for a 1 cm cuvette but were
measured in shorter path length cuvettes in order to ensure maintaining of the investigated
absorbance at 400 nm inside a reliable range).
Again, the zinc system shows some increase in the UV region, however it is more similar to
the free βCDtren absorbance for this concentration rather than another well-defined peak.
Conversely, the copper system displays a very large increase in the initial reaction stages which
remains as a well-defined, blue-shifted peak post hydrolysis. Again the phenolate peak at 400nm
appears to remain unaffected in either case.
183
2 .0
t in itia l
t f in a l
1 .5
1 .0
0 .5
0 .0
250 300 350 400 450 500
5
mol dm-3) hydrolysis over time in the presence of [Zn(βCDtren)] 2+ (4 × 10-3 mol dm-3) to
produce 4-nitrophenolate. 298.2 K, aqueous NaClO4 (I = 0.10 mol dm-3), buffered at pH 9.14
([DEPP buffer] = 1.0 × 10-2 mol dm-3). Hydrolysis half-life = 19.2 minutes.
4 .0
t in itia l
3 .5
t f in a l
3 .0
2 .5
2 .0
1 .5
1 .0
0 .5
0 .0
250 300 350 400 450 500
5
mol dm-3) hydrolysis over time in the presence of [Cu(βCDtren)] 2+ (4 × 10-3 mol dm-3) to
produce 4-nitrophenolate. 298.2 K, aqueous NaClO4 (I = 0.10 mol dm-3), buffered at pH 8.68
([DEPP buffer] = 1.0 × 10-2 mol dm-3). Hydrolysis half-life = 40.5 minutes.
184
Interestingly the [Cu(tren)]2+ system produces the same findings as [Cu(βCDtren)]2+ as can be
seen in Figure 4.34. Again, the final 4-nitrophenolate peak at 400 nm appears unaffected, but the
initial peak in the UV region is enhanced and remains (blue-shifted) even after the ester is
hydrolysed (as compared to the hydrolysis with free tren in Figure 4.24). It is likely, therefore,
that there is a LMCT transition in the [Cu(tren)]2+ complex similar to that in [Cu(βCDtren)]2+.
(The [Zn(tren)]2+ system could not be analysed because the pH of the pKaH2O#1 is likely too high
to avoid precipitate formation.)
2 .5
t in itia l
2 .0 t f in a l
1 .5
1 .0
0 .5
0 .0
250 300 350 400 450 500
5
mol dm-3, λmax 262 nm) hydrolysis over time in the presence of [Cu(tren)]2+ (4 × 10-4 mol dm-3)
to produce 4-nitrophenolate (λmax 401 nm). 298.2 K, aqueous NaClO4 (I = 0.10 mol dm-3),
However, it was also recognised that the 4-nitrophenyl acetate, 4-nitrophenolate and acetate
ion could be interacting with the Cu2+ in the complex during the course of the hydrolysis and
contributing towards the increased UV absorbance as well as the amine ligands. Considering the
increased affinity of the enzyme mimic for the phenolate product on addition of Cu2+,54 it was
conceded that the presence of the metal may influence the extent of catalyst poisoning, and
therefore the relative extents of interaction between the metal and the ester and phenolate species.
Although not definitive, another experiment was performed to investigate these concepts. The
ester 4-nitrophenyl acetate (4 × 10-5 mol dm-3) was reacted with the unbound amine βCDtren (4 ×
185
10-4 mol dm-3) at pH 8.68 and was monitored until 4-nitrophenolate production had ceased. To
this “t final” solution, Cu2+ was added (to produce a solution equimolar in βCDtren and Cu2+).
The results are displayed in Figure 4.35.
t in itia l
2 .5
t f in a l
2+
t f in a l + C u
2 .0
1 .5
1 .0
0 .5
0 .0
250 300 350 400 450 500
5
mol dm-3, λmax 271 nm) hydrolysis over time in the presence of free βCDtren (4 × 10-4 mol dm-3)
to produce 4-nitrophenolate (λmax 400 nm). Plus the addition of Cu2+(4 × 10-4 mol dm-3) after the
recording of t final. 298.2 K, aqueous NaClO4 (I = 0.10 mol dm-3), buffered at pH 9.11 ([DEPP
buffer] = 1.0 × 10-2 mol dm-3).
Immediately conspicuous is that the 4-nitrophenolate peak at 400 nm does not change at all
on addition of the Cu2+ (λmax nor absorbance). Although it is not definitive, this observation
strongly suggests that the 4-nitrophenolate product producing that signal does not interact with
the Cu2+ in solution. And although not a likely possibility, it also confirms that no back reaction
occurs with the addition of Cu2+ because evidently the amount of phenolate stays constant.
Furthermore, in the absence of Cu2+ the ester peak at 271 nm completely disappears as the
hydrolysis progresses which is as expected. However, once all of the 4-nitrophenyl acetate has
been converted into 4-nitrophenolate, addition of the equimolar amount of Cu2+ causes a large
peak in the UV to appear. This has already been dismissed as unlikely to be due to a Cu2+-
phenolate interaction (because the phenolate peak at 400 nm is unaffected) and cannot be due to a
186
Cu2+-ester interaction as all the ester has been reacted and no back reaction occurs to produce
more. Copper(II) acetate does form a stable, mononuclear complex in aqueous solution.94
However, its absorbance spectrum is akin to that of [Cu(H2O)6]2+ which does have increasing
absorbance below 300 nm but does not exhibit the distinct peak as is seen in this study.
Consequently this peak must be due to a Cu2+- βCDtren interaction and it has the same λmax as for
the complex [Cu(βCDtren)]2+ tested on its own in the absence of the ester or phenolate – 262 nm
(see Figure 4.31). This is particularly interesting because the free βCDtren is predicted to
facilitate the ester hydrolysis in a different mechanism to that of the [Cu(βCDtren)]2+ (Figure
4.21). If βCDtren followed pathway B in Figure 4.21, each hydrolysis would produce a new
acylated product. If this acylated product then bound to Cu2+ post reaction, it might be expected
that the absorbance caused by this ligand-metal interaction could have a shifted λmax in
comparison with the original βCDtren. Given this is not the case, this observation may indicate
that pathway C is a more likely reaction pathway for the hydrolysis of 4-nitrophenyl acetate by
βCDtren.
Although these experiments are not definitive, they do suggest that the ester both includes in
βCD and binds to M2+ as part of the catalytic mechanism. The hydrolysis rate depression in the
presence of native βCD and the rate increase from [Cu(tren)]2+ to [Cu(βCDtren)]2+ indicate that
the βCD hydrophobic annulus is likely forming an inclusion complex with the 4-nitrophenyl
acetate as part of the reaction process. The function of the βCD as part of the ester hydrolysis
mechanism is supported in the literature.17, 21, 22, 62
Despite a likely LMCT band in the UV obscuring the region where evidence of Cu 2+-ester
interaction may be observed in the presence of [Cu(βCDtren)]2+, an enhanced peak in this region
in the presence of free Cu2+ (Figure 4.28) suggests that the ester does bind to the metal. As
discussed, the nonappearance of this peak enhancement in the presence of free Zn2+ does not
disqualify ester binding with this metal, it simply means this evidence was not observed under
these conditions. Indeed, studies reported in the literature suggest that the ester (phosphate or
acetate) does bind to the central metal cation, displacing a solvent or hydroxo ligand as part of the
mechanism. These studies include the metals Co3+,95 Ir3+,41 Cu2+ 57, 75, 96 and Zn2+.42 Furthermore,
when a complex of Zn2+ is found to facilitate ester hydrolysis through a less efficient,
bimolecular mechanism that does not involve the ester binding to the metal, this rate is observed
to be very slow.72 Consequently, because the zinc complex in this study ([Zn(βCDtren)]2+) yields
faster ester hydrolysis than the copper complex ([Cu(βCDtren)]2+) and the copper complex
187
mechanism is likely to involve the ester binding to the Cu2+, then this mechanism is also likely to
follow for the zinc complex.
4.5 Summary of findings

The metallo-βCDs [Zn(βCDtren)]2+ and [Cu(βCDtren)]2+ have been tested for their potential
enzyme mimicking abilities. First, potentiometric titrations were required in order to determine
the speciation of these systems, such that the pH at which their catalytically active hydroxo
ligands were formed, was established. Due to the potential for catalyst poisoning, a new and
modified Michaelis-Menten treatment was then derived in order to analyse the data for systems in
which there is a requirement for an excess of enzyme mimic over ester substrate.
Applying this analysis, both Zn2+/βCDtren and Cu2+/βCDtren systems were shown to exhibit
the saturation kinetics predicted for enzymes. Analysis of the dependence of the rate of
hydrolysis reaction on pH also supported the hypothesis that the kinetics characteristics of these
systems are controlled by the acid-base equilibrium of the catalytically active hydroxo ligand.
Both Zn2+/βCDtren and Cu2+/βCDtren systems produced rate enhancements for the hydrolysis
of 4-nitrophenyl acetate that are commensurate with similar metal complex enzyme mimics. A
comparison between these two systems also showed that the aqua ligand pKas (and therefore
proportion of active hydroxo present in solution) did not dictate the success of the enzyme mimic
because the Cu2+/βCDtren system had the greater concentration of active hydroxo at the pHs
tested but the Zn2+/βCDtren system produced the larger kcat. Further investigation into the factors
that influence success in this type of enzyme mimic should therefore be conducted as part of
Future Work.
A qualitative analysis of the kcat values and UV-Vis spectra of these systems and their free
components also shed some light on the mechanism of reaction. The free (not complexed) tren
and βCDtren species produced faster rates of 4-nitrophenylacetate hydrolysis than their
[M(tren)]2+ and [M(βCDtren)]2+ counterparts. However, the hydrolysis by the free species
possibly follows a different mechanism whereby the amine becomes acylated and therefore is not
a true catalyst. Several of the spectra involving Cu2+-containing species suggest that the ester
interacts with the Cu2+ ion, which likely affects the resulting kcat values. Again, further
188
investigation into these initial observations would help establish which of the enzyme mimic
features contribute towards a successful design.
There are many avenues for Future Work which have been highlighted throughout this
chapter. However, these preliminary results suggest further investigation and optimisation of this
type of enzyme mimic is warranted.
189
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Hilary Coleman Chapter 5. Part I Summary and Future Work
CHAPTER 5
PART I SUMMARY AND FUTURE WORK
196
5.1 Introduction
Part I reports two new compounds, the potentiometric titration characterisation of eight free
ligands and 32 metal complexes, and the full kinetics characterisation of two identified potential
enzyme mimics; the Zn2+/βCDtren and Cu2+/βCDtren systems. These systems displayed the
characteristics that are predicted for Michaelis-Menten enzyme kinetics. This chapter summarises
these findings and outlines the directions Future Work may take.
5.2 Chapter 2 – Potentiometric titration characterisation of the

Foundation Polyamines
In Chapter 2 the foundation ligands (tren Me6tren, tacn and Me3tacn) and their complexes
(with Zn2+, Cu2+, Ni2+ and Cd2+) were characterised by potentiometric titration. The results
agreed well with the literature values where similar studies could be found. Most notably, it was
discovered that Cu2+ produced the most stable complexes for all four ligands and the Cu2+/tren
system was the most stable of all. However, the Cu2+/Me3tacn system produced the lowest
pKaH2O. Both the complex stability and depressed pKaH2O are important factors in the design of
potential enzyme mimics.
Although ligand pKas, complex stability constants (logK) and aqua ligand pKaH2Os were
determined for the four ligands and 16 complexes in Chapter 2, there are several obvious avenues
for Future Work. Firstly, other simple ligands should be similarly characterised to expand the
Foundation ligand set and thereby identify possible trends that could be used predictively when
designing future complexes to be potential enzyme mimics. For example, whilst this study did
investigate both macrocylic ligands (tacn and Me3tacn) and acyclic ligands (tren and Me6tren), a
direct comparison of their results cannot be made because they have different denticities – the
tacn-based ligands are tridentate whilst the tren-based ligands are tetradentate. Expanding the
Foundation ligand set to include cyclen (1,4,7,10-tetraazacyclododecane) and bis-(2-
aminoethyl)amine would allow for comparison with a tetradentate macrocycle and a tridentate
acyclic ligand, respectively. Even more variations on these ligands could be investigated in order
to create a very large catalogue, see Figure 5.1. Furthermore, forms of alkylation other than
methylation could also be undertaken to broaden the foundation ligand set even further (e.g.
ethylation). Different extents of alkylation may also lend further evidence to the fact that
197
increasing the hydrophobicity of the environment around the aqua ligand results in a lowering of
its pKa.1
Figure 5.1. The ligands required for direct comparison with the tren and tacn (a) and examples
of other ligands that could expand the ligand suite (b, c).
198
Because elucidating the catalytic hydrolysis mechanism is critical for planning future enzyme
mimic design, it is necessary to determine each complex geometry and structure in detail. For
example, if efficient 4-nitrophenyl acetate hydrolysis rate enhancements are achieved by a
mechanism in which the ester binds to the metal centre to be attacked by an adjacent hydroxo
ligand, then the stereochemistry of each potential enzyme mimic is important. If the tren-based
ligand systems tend to form five co-ordinate, trigonal bipyramidal complexes in solution,2, 3, 4, 5, 6
then there are not enough coordinative sites on M2+ to host both the hydroxo ligand and the ester
which renders this mechanism impossible for these complexes. Similarly, if the M2+ –OH2 bond
length is a key factor in determining the pKaH2O for the aqua ligand(s) present on each complex1
then this is similarly a characteristic that should be measured. Obtaining the crystal structures of
these complexes would shed light on their preferred geometries and the M2+ –OH2 distances and
possibly lead to an understanding of how these characteristics affect catalytic activity.
If expansion of the foundation ligand set was able to identify trends in the effect of denticity,
macrocylic vs acyclic ligand, geometry and M2+ –OH2 distance on complex stability and aqua
ligand formation, it is also recommended that Future Work involve the production of highly
accurate space-filling models of other potential enzyme mimics. These proposed systems could
then be probed theoretically before the lengthy synthesis process in order to determine their
likelihood of success based on the characteristics elucidated above.
5.3 Chapter 3 – Potentiometric titration characterisation of the β-

Cyclodextrin-Functionalised Polyamines
In Chapter 3 the β-cyclodextrin functionalised ligands (βCDtren, βCDMe5tren, βCDtacn and
βCDMe2tacn) and their complexes (with Zn2+, Cu2+, Ni2+ and Cd2+) were characterised by
potentiometric titration. Again, the results agreed well with the literature values where similar
studies could be found.
Several of the ligands when complexed with Ni2+ were found to exhibit obviously slow
equilibria during their titrations, making them unsuitable for further progression to kinetics
testing. Conversely, for all but one system (involving βMe5tren), Cu2+ was found to produce the
most stable complexes and the lowest aqua pKaH2Os when complexed with the ligands in this
study. Most notably, [Cu(βCDMe2tacn)(H2O)3]2+ is a stable complex (logK1 = 11.78) with a
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pKaH2O#1 lower even that that of carbonic anhydrase.7, 8

Consequently, the first task of Future
Work should be to synthesise enough of the new βCDMe2tacn ligand to undertake the kinetics
testing on its copper complex. Indeed, all of the complexes with large enough stability constants
to limit the formation of free ligand and metal, and low enough pKaH2O values to conduct the
experiments at a pH in which metal hydroxide precipitation does not occur, should have their
kinetics studied. Initially, testing of the Cu2+ and Zn2+ βCDtacn and βCDMe2tacn systems will
afford a good comparison with the conducted in this study.
Secondly, the ligands shown in Figure 5.1 could all be substituted with βCD and similarly
characterised by potentiometric titration, in order to expand the ligand catalogue and identify
possible trends. For example, denticity, geometry, M2+ –OH2 distance and macrocyclic versus
acyclic features may all influence the stability of complexes formed and the extent of depression
of the associated pKaH2O. Consequently, having data for enough systems in order to analyse these
influences may allow for predictive powers when proposing new systems in the future. For
example, of all the metals studied, if Cu2+continues to produce the most stable complexes with
the lowest pKaH2Os then it is likely a good choice for the M2+ of future systems.
As with Section 5.2 (work presented in Chapter 2) crystal structures for the systems
characterised in Chapter 3 could be obtained. Again, this would shed light on the geometries of
the complexes and on the M2+ –OH2 bond distances, both of which likely impact on the suitability
of these complexes as enzyme mimics. Also as with Section 5.2, if expansion of the ligand
catalogue was able to identify trends in the effect of denticity, macrocylic vs acyclic ligand,
geometry and M2+ –OH2 distance on complex stability and aqua ligand formation, it is also
recommended that Future Work involve the production of highly accurate space-filling models of
other βCD substituted potential enzyme mimics. Again, these proposed systems could then be
probed theoretically before the lengthy synthesis process in order to determine their likelihood of
success.
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5.4 Chapter 4 – Kinetic characterisation of the enzyme mimic

systems
5.4.1 Summary of findings

In this study, preliminary results for the enzyme mimicry characterisation of the
Zn2+/βCDtren and Cu2+/βCDtren systems were determined, including calculation of kcat and KM
and a demonstration that the hydroxo ligand is likely to be the catalytically active species.
Furthermore, a new, modified Michaelis-Menten equation was derived, which enabled analysis of
these systems in which the enzyme concentration is in great excess over the substrate.
Both of the systems investigated in this system showed the conventional saturation kinetics
that is predicted for enzymes by Michaelis-Menten kinetics. The kcat values of 0.021 s-1 and 0.005
s-1 for the Zn2+/βCDtren and Cu2+/βCDtren systems respectively, are consistent with studies of
similar metallo-βCD systems with similar esters.9, 57, 85
However, interestingly, although the
Cu2+/βCDtren system has a larger stability constant (logK1) and lower pKaH2O than does the
Zn2+/βCDtren system, the Zn2+/βCDtren system produced a greater hydrolysis rate enhancement
in this study. This indicates that these two factors are not the only (or primary) dictators of the
success of this type of enzyme mimic.
For comparison, the rate constants (kobs) for the hydrolysis of 4-nitrophenyl acetate in the
presence of a ten-fold excess of all of the following species were also determined: free βCD, free
Zn2+, free Cu2+, free tren, free Me6tren, free βCDtren, free βCDMe5tren and the Cu2+/tren system.
Although just preliminary results, this data indicated free βCD decreased the hydrolysis rate even
below that of the uncatalysed rate and that the free amines increased it, even faster than the
corresponding metal complexes of the same amines that were tested. The fact that the hydrolysis
occurred under all of these conditions is evidence for the three proposed potential reaction
mechanisms highlighted in Figure 4.21; metal complex-facilitated nucleophilic attack by a
hydroxo ligand (the main mechanism considered in this study), nucleophilic acyl substitution
(non-catalytic) by non metal-bound 1˚ or 2˚ amines, or general base-catalysed hydrolysis
involving 3˚ amines. With respect to the metal complex-facilitated hydrolysis, the finding in this
thesis that the metal-bound hydroxo ligand is the catalytic species, suggests that the potential
metal-carbonyl mechanism introduced as a possibility in Section 1.4 is not the mechanism of
activity for these enzyme mimic species. (The metal-carbonyl method involves the inner sphere
201
binding of the carbonyl of the ester substrate to the complex metal cation which activates the
carbonyl towards attack by a solvent water molecule, and does not involve a metal-bound
hydroxo ligand.) As noted, however, these are preliminary results and future work is required (as
discussed in Sections 5.4.2 and 5.4.3) to further understand these enzyme mimics’ mechanism of
activity.
Finally, analysis of the UV-Vis spectra between 200 – 500 nm over the course of these
hydrolysis reactions provided a qualitative understanding of the potential mechanism of
hydrolysis. In particular, investigation of the systems involving Cu2+ suggested that the ester
binds to the metal centre during the hydrolysis but the same finding was not necessarily true for
the equivalent Zn2+ systems. Again, it is noted that this work goes beyond the scope of that
presented in the literature and should be considered preliminary in nature and therefore highly
speculative. Further spectrophotometric analysis is in need of attention as part of the Future
Work.
5.4.2 Short term Future Work

Given the results of Chapter 4 are preliminary in nature, Future Work should initially involve
supplementation of the Zn2+/βCDtren and Cu2+/βCDtren system investigations. For example,
more experiments at different [Zn(βCDtren)]2+ and [Cu(βCDtren)]2+ concentrations could
enhance (and reduce the errors for) the graphs used to calculate the kcat and KM values for these
systems. More experiments at different pHs for each of the Boltzmann plots for both systems
could similarly enhance this data (and reduce the errors).
Secondly, because understanding the catalytic hydrolysis mechanism is key to being able to
determine which features of potential future enzyme mimics will likely make them successful,
attempts should be made as part of Future Work to definitively establish a mechanism for this
type of enzyme mimic. In particular, an investigation should be conducted into whether 4-
nitrophenyl acetate binds to M2+ and, if so, what effect this has on the hydrolysis rate
enhancement. One method of investigating this further would be to examine the full UV-Vis
spectra of the kinetics solutions of 4-ntriphenyl acetate with Zn2+/βCDtacn and Cu2+/βCDtacn
systems before and after hydrolysis. In particular, the spectra involving Cu2+ may provide further
evidence that the ester binds to M2+ as part of the mechanism. Unfortunately, static studies of the
structure of the initial metallo-βCD/ester inclusion complex under the conditions used for the
202
experiments in this study would be problematic because as soon as the ester reactant is introduced
it begins to be hydrolysed. However, it may be possible to obtain crystal structures of salts of the
initial metallo-βCD/ester inclusion complex at a pH at which the hydroxo ligand has not formed
(and therefore no mimic-induced hydrolysis will take place). For example, at approximately pH
7.5 in the Zn2+/βCDtren system (Figure 4.7) the [Zn(βCDtren)(H2O)2]2+ complex is at 95%
formation. Hydrolysis will still occur under these conditions but this initial metallo-βCD ester
inclusion complex may be stable enough to obtain crystals for a crystal structure determination. If
so, whether the ester binds to the Zn2+ at this pH would be anticipated to be predictive of whether
it does when the hydroxo ligand is also present. A similar investigation at a pH of approximately
6.4 might achieve the same of the Cu2+/βCDtren system (see Figure 4.15). Alternatively static
studies could be conducted using an inactive substrate – an ester that, unlike 4-nitrophenyl
acetate, is not activated towards hydrolysis. Employment of such an ester may allow for an
investigation of the initial interaction between the enzyme mimic and substrate.
Thirdly, in order to prove that the hydrolysis mechanism involves inclusion of 4-nitrophenyl
acetate in the βCD annulus, the same experiment to determine the rate constant (kobs) for the
reaction of [Zn(βCDtren)(H2O)(OH)]+ with 4-nitrophenyl acetate should be re-run in the presence
of adamantanecarboxylic acid,10 adamantane-1-carboxylate11 and cyclohexanol.12 These species
have been shown to competitively include in βCD and their presence would be expected to
reduce the rate of hydrolysis if, in fact, 4-nitrophenyl acetate does include in βCD as part of the
mechanism proposed in this study (Figure 4.2).
5.4.3 Expansion of the original ligand suite

Next, the full kinetics analysis (as performed for the Zn2+/βCDtren and Cu2+/βCDtren systems
in this study) should be applied to the other potentially promising systems identified in Chapters
2 and 3; tren, Me6tren, tacn, Me3tacn, βCDtacn, βCDMe2tacn and βCDMe5tren complexes with
M2+. Furthermore, the same metal complexes formed with the free and βCD-substituted new
foundation ligands in Figure 5.1 (and others) should also be characterised by potentiometric
titration, kinetics analysis, space filler modelling and crystal structures. Investigation of the full
UV-Vis spectra of these systems before and after catalysing the hydrolysis of 4-nitrophenyl
acetate should also be undertaken. Furthermore, UV-Vis spectra of these systems in the presence
of an inactive substrate should also be obtained in order to better understand the initial enzyme
203
mimic/ester interaction. If possible, crystal structures of all of the metallo-βCD/ester inclusion

complexes should be obtained at a pH at which the metallo-βCD contains only aqua ligands. The
findings of all of these experiments should then be examined relative to the hydrolysis rate
enhancement produced by each system such that overarching conclusions might be drawn about
which features make these systems successful enzyme mimics.
As was the intention of this study, the analysis of a large suite of potential enzyme mimics
may lead to identification of the features of these systems that make them successful or not. This
will hopefully result in predictive power when designing new enzyme mimics into the future.
204
5.5 References
2. M. Ciampolini, N. Nardi and G. P. Speroni, Coord. Chem. Rev., 1966, 1, 222-233.
3. L. Sacconi, Pure Appl. Chem., 1968, 17, 95-127.
4. L. Fabbrizzi and Editor, Beauty in Chemistry: Artistry in the Creation of New Molecules.
[In: Top. Curr. Chem., 2012; 323], Springer GmbH, 2012.
5. X. Xu, A. R. Lajmi and J. W. Canary, Chem. Commun., 1998, 2701-2702.
6. B. F. G. Johnson, F. L. Bowden, C. D. Garner, R. Davis, C. A. McAuliffe, W. Levason,

L. A. P. Kane-Maguire, D. W. Clack, J. Howell, M. Hughes and J. A. McCleverty,
Specialist Periodical Reports: Inorganic Chemistry of the Transition Elements, Vol. 4,
Chem. Soc., 1976.
11. K. Redman, B. L. May, S. D. Kean, P. Clements, C. J. Easton and S. F. Lincoln, J. Chem.

Soc., Perkin Trans. 2, 1999, 1711-1717.
205
Hilary Coleman Chapter 6. Introduction Part II: Physiological Zn(II) Detection
CHAPTER 6
INTRODUCTION PART II: PHYSIOLOGICAL Zn(II)

DETECTION
206
6.1 Physiological Zn(II)
6.1.1 Introduction
Zn2+ is essential for human life.1 It is the second most abundant transition metal ion in human
physiology after Fe2+/Fe3+ 2-4 and this translates to a mass of approximately two to three grams in
an average, adult human.5 This quantity is spread throughout the whole body and Zn2+ is found at
some concentration in all organs, fluids and tissues. The majority of this Zn2+ is intracellular,
though its distribution within this medium is not uniform. Concentrations can range from just 10-9
mol dm-3 in cytoplasm to the millimolar level in vesicles.2, 6 Of the more than 100 000 protein
structures in the Protein Data Bank (PDB) at the time of writing, approximately 10% contain
zinc.7
Physiological Zn2+ is found in three different forms; at the catalytic centre of enzymes, in the
structure of proteins and as pools of ‘free’ (fully hydrated) Zn2+.8 The catalytic and structural
Zn2+ found in enzymes and proteins is known to constitute the majority of intracellular Zn2+.9-14
The final fraction is the free Zn2+, 15 the possible functions of which attract much speculation.
The ubiquity of Zn2+ in living cells is linked to the fact it has been found at the active site of
over 1000 enzymes.16 It is the only metal to be required by metalloenzymes in all six of the
International Union of Biochemistry’s established enzyme categories and is a unique trace
element in this respect.17 Due to its flexible coordination geometry, Zn2+ has diverse roles in
many biological processes.
Zn2+ is inextricably involved in RNA and DNA processes. It is associated with RNA and
DNA synthesis proteins and DNA synthesis has been found to be suppressed as a consequence of
Zn2+ deficiency.8 RNA and DNA metabolism is also regulated by Zn2+ 18-20
and most
polymerases (whose fundamental biological occupation is to synthesise polymers from nucleic
acids) contain Zn2+ cations.21
Unlike the more well-documented processes of the catalytic and structural Zn2+ centres, it is
the function of the free species that is least well understood, although some progress is being
made.22 Large amounts of free Zn2+ are known to be localised in nerve tissues and these are
thought to be active in cell communication.23 24
Human brain tissue contains between 1 × 10-4
and 5 × 10-4 mol dm-3 of this species25 and dysfunction associated with this Zn2+ is related to
207
many severe neurological diseases. Parkinson’s and Alzheimer’s diseases as well as epilepsy and
aggressive forms of dementia are all correlated with zinc metabolism problems. 26-28
Because Zn2+ is an essential requirement in so many physiological functions, a deficiency in

Zn2+ predictably leads to many serious clinical concerns. There are a plethora of conditions (both
common and more infrequent) that are accompanied by low physiological Zn2+ concentration.
However, whether this phenomenon is a result of, or a precursor to, these conditions is not known
for every case. Examples include diabetes,29 asthma,30 sickle cell disease31 and acute
lymphoblastic leukemia.32
Zinc deficiency may also simply be the result of a diet that is poor in this species. Though this
is less common in developed countries, mild forms may become more prevalent as the
consumption of processed foods becomes more commonplace. However, more severe
occurrences are becoming increasingly widespread in developing areas where it is estimated that
nutritional zinc deficiency may affect nearly two billion people.33 In places of poverty and
malnutrition, the diet is deficient in many vital nutrients. However, in many areas not associated
with these difficulties, zinc deficiency still presents an issue when the diet of the population is
predominantly grains-based. In the early stages of the Green Revolution (1960s) cereal
production more than doubled worldwide.34, 35
Through the mass production of cereal grains,
diets moved away from containing as much fruit and green vegetables. Fruit and green vegetables
are more vitamin and mineral rich than the new, more easily up-scaled grain crops.36 As a result,
conditions that may arise from zinc deficiency include anorexia,37 growth retardation, altered
mental state,2 immunodeficiency38 and learning and memory difficulties.39 Ongoing research is
being conducted into methods for improving the zinc nutritional value of crops.40, 41
The role of Zn2+ in all of these physiological processes makes its investigation a significant
one. A greater understanding of its functions would hopefully lead to improved treatments for the
diseases and conditions mentioned and possibly even their prevention. Furthermore, a better
appreciation of the general chemistry of Zn2+ in the body could only be advantageous, given the
ubiquity and significance of this metal cation. Consequently the development and expansion of
Zn2+ detection methods that reliably reveal the concentration and localisation of physiological
Zn2+ could prove extremely beneficial.
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6.1.2 Developments in detection methods

Various methods have been used for Zn2+ detection, some more effectively than others.
However, even the most commonly-engaged detection techniques have disadvantages. Atomic
absorption spectrophotometry is employed in cellular Zn2+ detection (for example42), but only
when the total concentration is required because the procedure involves the homogenisation of
the cells or tissue for analysis. The results therefore only reflect an average sample Zn 2+
concentration and shed no light on the localised concentration, distribution or form of the Zn2+
present (structural, catalytic or free). Accurate methods of detecting subcellular Zn2+ in vivo are
therefore highly sought after. Ideally the technique should be innocuous, sensitive to even very
low concentrations, and afford real-time imaging.
Whereas other biological transition metals can give rise to spectroscopic and magnetic
signals,43 Zn2+ cannot as a result of its electronic configuration: 3d104s0. The lack of possibility
for any d-d orbital electron transitions means that conventional analytical tools cannot be used to
detect Zn2+. As such, UV-visible and electron paramagnetic resonance (EPR) spectroscopy are
67
rendered ineffective. The naturally abundant Zn isotope is NMR-active, although several
practical difficulties, including this isotope’s intrinsic low sensitivity, make 67
Zn NMR an
impractical zinc detection method.44 However, along with several other proposed methods not
investigated here, fluorescence detection has been found to present viable alternative.45, 46 This
has led to the design and exploration of a broad range of ligands which produce a fluorescent
signal when bound to Zn2+.43, 47, 48
Using fluorescence for the detection of physiological Zn2+ involves the employment of a
ligand that selectively recognises Zn2+ and produces a characteristic emission on binding. The
essence of making this approach successful is of course the optimisation of the fluorescent
ligand. As a consequence of this, many types of fluorescence-based Zn2+ probes have been
developed.23, 28, 43, 49-53 Currently, quinoline derivatives have widespread commercial use. These
ligands are fluorophores involving the common quinoline ring component and they are highly
fluorescent on complexation with Zn2+.54 In the past 40 years the skeleton quinoline structure has
been used as a template and built on. Figure 6.1 displays an early prototype along with more
recent compounds. The simplest of these ligands, reported in the 1970s, is 8-aminoquinoline (8-
AQ, 1), comprised solely of the quinoline ring and an amine substituent.54 Enhancements of this
molecule were made, and with the addition of the sulfonamide moiety, 6-methoxy-8-p-
toluenesulfonamido-quinoline (TSQ, 2) became the first sulfonamido compound to be used for
209
the in vitro imaging of Zn2+.55 Twenty five years later, 2-methyl-8-p-toluenesulfonamido-6-

quinolyloxyacetic acid (Zinquin A, ZQA, 3)49 is now a widely used Zn2+ detecting fluorophore.56,
57
Figure 6.1. Major steps in the development of the quinoline Zn2+ sensors: 8-AQ 1 (1970s), TSQ 2
(1987), Zinquin A, ZQA, 3 (1993).
The quest to advance this type of fluorophore is ongoing and designs for quinoline-based
Zn2+ sensors continue to be investigated.58-60 One such development has involved the attachment
of quinoline to derivatives of tacn (1,4,7-triazacyclononane), which was used as a foundation
molecule in Part I of this study due to its zinc-binding properties.61 The development of strategies
to improve solubility in cellular fluid, fluorescence quantum yield and Zn2+ selectivity is in
progress.
6.1.3 The mechanism of the quinoline fluorophores: Chelation Enhanced

Fluorescence
The quinoline derived fluorophores are non-fluorescent (or display negligible fluorescence)
until they encounter Zn2+ and become complexed. This phenomenon is known as chelation
enhanced fluorescence (CHEF) and is utilised in many sensor ligand designs.62
When Zinquin is not bound to Zn2+ its structure is flexible. Unbound, it is free to undergo
uninhibited vibrational and rotational motion. This motion may dissipate any excess energy the
210
ligand might gain by irradiation, for example. This form of dissipation is referred to as non-
radiative decay because it does not involve the emission of visible light. On complexation of
Zn2+, however, the ligand becomes much more rigid. This limits the range of motion that the
ligand may experience which consequently prevents it losing excess energy through its
vibrational modes. Instead the energy is lost as an emission of photons. It is this emission that
constitutes the fluorescence spectrum of the complexed ligand.63 This process is discussed more
thoroughly in Section 6.2.2 where the excitation (absorbance) and subsequent fluorescence
processes are displayed in Figure 6.4.
The attributes of the fluorescence – including the spectral shape, quantum yield (Φ) and
lifetime (τ) – provide information about the fluorescing species. In the case of a fluorescent
sensor, the key to success is producing no fluorescence signal in the absence of the target and
then “switching on” to produce a signal when the target is encountered. For Zinquin this involves
the free, unbound ligand dissipating excess energy via non-radiative decay, the mechanism of
which is then inhibited once it is bound to intracellular Zn2+ such that a fluorescent signal is then
produced. This essential property of chelation enhanced fluorescence may therefore be tested for
all new quinoline-based derivatives by analysing the fluorescence spectrum of the new ligands in
the absence and presence of Zn2+.
6.1.4 The chemistry of Zinquin

There are several criteria a molecule must meet in order to be appropriate for fluorescent Zn2+
sensing. First and foremost it must be Zn2+-selective – if a ligand produces a fluorescent signal
when it is bound to a metal other than Zn2+ then it is not an effective Zn2+ sensor. Zinquin A and
E and other quinoline derivatives will bind to other metals (for example Co2+, Fe2+/3+, Mn2+ and
Cu2+ 64
) but the fluorescence of these complexed ligands is quenched via paramagnetic
quenching. Unpaired electrons of the metal ion may interact with the π electrons of the organic
ligand, thereby providing a pathway for intersystem crossing from an excited singlet state to a
triplet state, from which emission (fluorescence) cannot readily occur.64, 65
Therefore these
complexes do not affect the Zn2+ selectivity.62
Furthermore, for a fluorophore to be used in physiological studies it must be able to pass

through the lipophilic cell membrane whilst also retaining solubility in extracellular and
211
intracellular fluids. The right combination of both polar and non-polar functional groups on the
fluorophore is therefore necessary.
Rapid target delivery, chemical- and photo-stability, pH independent fluorescence and high
sensitivity to even small concentrations are also attributes a sensor should possess.45 And finally,
the ability to differentiate between the different types of Zn2+ is highly desirable because these
different Zn2+ forms are associated with vastly different processes and conditions.
Much work has been done on examining the suitability of Zinquin A and E (see Figure 6.2.)
against all of these criteria.57, 64, 66, 67 The synthesis and characterisation of this fluorophore was
first reported in 1993.49
Zinquin E (4) contains portions that are capable of hydrogen bonding (the amine and ester
functionalities) as well as a hydrophobic aromatic skeleton. This combination of functional
groups makes it able to be transported by the bloodstream as well as to traverse the lipophilic cell
membrane. Once inside the cell it is hydrolysed by cellular esterases such that it becomes Zinquin
A (3). However, at physiological pH the acid will be deprotonated leaving the fluorophore
negatively charged. This charge prevents it from re-crossing the hydrophobic cell membrane and
it is therefore specifically contained in the area of interest.
Stability constants have been determined in organic-aqueous solvent mixtures along with the
corresponding UV-Vis and fluorescence characteristics of the complexes, and in most studies it is
concluded that Zinquin is sensitive to nanomolar Zn2+.6 However, it has been shown that Zinquin
does not differentiate between free and protein-bound Zn2+ and therefore this is an avenue for
improvement.57
212
Figure 6.2. Zinquin E (4) is hydrolysed inside the cell by an esterase to Zinquin A (3) which is deprotonated at cellular pH. The negative charge
stops the fluorophore from leaking back out of the cell through the lipophilic membrane. It may then bind to proteomic, enzymatic or free Zn2+ to
produce a characteristic fluorescent signal on irradiation.
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6.2 Aims of this research

The aim of this research is to synthesise and characterise a Zn2+-selective fluorophore with
enhanced properties over the well-established Zinquin sensor ligand. Potentially the most useful
attribute of a new fluorophore in this area would be the ability to differentiate between the
different forms of physiological Zn2+ - the cations bound in enzymes and proteins versus the free
intracellular Zn2+. As discussed in Section 6.1.1, pools of the free (hexa-aqua complexed) Zn2+
are involved in many serious medical conditions including neurodegenerative diseases. As
depicted in Figure 6.2, Zinquin is compact enough to allow for binding to Zn2+ that is
simultaneously coordinated in the structure of a protein.56 Therefore it is hoped and hypothesised
that adding a bulky substituent to a new Zinquin analogue may result in a preference for binding
to the non-crowded free Zn2+ over the sterically hindered cations in the elaborate structures of
proteins and enzymes. Furthermore it is hypothesised that including the potential for an increased
extent of conjugation on the new ligand may also improve upon Zinquin’s qualities as it should
result in a bathochromic shift of the complex absorbance and emission spectra and potentially a
larger quantum yield.
Therefore the styryl group was selected to be added to Zinquin. Figure 6.3 displays the acid
forms of Zinquin (ZQA) and its styryl-functionalised derivative (stZQA). (These abbreviations
will be used in the remainder of the text.) It can be seen in this figure that the styryl group
contributes both bulk and increased conjugation to the ligand.
Characterisation of the styryl-containing ligand involves potentiometric determination of its

pKas, examination of the absorbance spectra of the free and complexed ligand, and calculation of
stability constants formed with Zn2+ as determined by fluorimetry. These methods are elucidated
below. Because this system has an intended future application in physiological studies, the
experimental conditions chosen must mimic physiological conditions. An aqueous ethanol
solvent system was selected for its resemblance to intracellular fluid. Many studies adopt this
solvent system for cellular work.68-72 Most significantly, previous studies on Zinquin have
utilised this solvent system64, 66 and preliminary investigations of stZQA were undertaken in 25%
v/v aqueous ethanol so as to be able to compare these results with those of past studies.67
The solvent system composition chosen for this study was therefore 25% v/v aqueous ethanol
buffered at pH 6.6 (consistent with previous Zinquin studies) using a buffer compound reported
to not complex Zn2+: NaPIPES.73
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Figure 6.3. Conventional Zn2+-detecting fluorophore Zinquin A (ZQA) and the proposed
derivative with additional styryl functionality, styryl-Zinquin A (stZQA).
6.2.1 Potentiometric titrations

The pKas for the ligand stZQA were determined by potentiometric titrimetry as part of this
study and compared to the corresponding values for Zinquin A. In an identical manner to studies
reported in Chapters 2 and 3, this involved acidification of a sample of the ligand and then
titration against a NaOH solution (0.1 mol dm-3) using a 800 Dosino Metrohm instrument
equipped with a 5 cm3 burette and a 2 cm3 Orion Ross semi-micro combination pH electrode.
Based on the structure of stZQA there were three measurable deprotonation constants; for the
protonated quinolinium nitrogen, the carboxylic acid and the sulfonamide. These are identical to
the deprotonations that occur for ZQA. When the ligand is deprotonated a change in pH (= –
log[H+]) occurs from which the corresponding pKas may be determined through potentiometric
titration.74 In each titration, the variation of the observed potential (millivolts), E, (that is
measured by the electrode as a result of the deprotonation reactions) is described by the modified
Nernst Equation 6.1:75
E = E0 + (RT/F)ln[H+] 6.1
where E0 is the standard potential of the electrode (millivolts), R is the ideal gas constant (8.314 J
K-1 mol-1), T is the temperature in Kelvin, F is the Faraday constant (9.6487 × 104 C mol-1) and
215
[H+] is the proton concentration (mol dm-3). At 298.2 K, the pH of a solution is therefore given by
Equation 6.2:
pH = (E0 – E)/59.15 6.2
When all of the equilibria occurring during a titration are identified, the concentrations of
their constituent species may be calculated along with the equilibrium constants. The fitting
program Hyperquad 2008 was used to fit the appropriate pH dependent algorithms to the
potentiometric data to simultaneously derive the values of the acid dissociation constants.76 A
non-linear least-squares method is employed to minimise the sum of the squares of the residuals
in order to determine the best fit of the algorithm to the experimental data.
Characterisation of the pH-dependent nature of stZQA was required for the subsequent UV-
Vis and fluorescence investigations of its binding and absorption/emission properties. As part of
Future Work, an extension of these potentiometric titrations could be used to determine the
ground state stability constants of this ligand with Zn2+ and other biologically significant metal
cations, as was undertaken in Chapters 2 and 3.
6.2.2 Electronic spectroscopy

The majority of the analysis of fluorophores must necessarily be based on electronic
spectroscopy as both the absorbance and fluorescence properties of the target ligand must be
characterised. The basis of all spectroscopy is the interaction of electromagnetic radiation with
matter. The electromagnetic spectrum encompasses an extremely broad range of energies from
approximately 10-3 to 109 J mol-1 and in this study the ultraviolet and visible region (200 – 800
nm) was used to probe the electronic characteristics of the stZQA ligand.65
Every molecule is comprised of many electronic energy levels, and is said to be in the ground
electronic state (S0) when its electrons occupy the lowest of these levels. A molecule can be
excited from its ground electronic state to an excited electronic state (S1 for example) by the
promotion of an electron into a higher energy level. This may occur when the molecule absorbs
energy in the form of a photon. Because the electrons exist in discrete energy levels, the energy
of the photons absorbed that cause these transitions must be exactly equal to the difference in
energy between the levels traversed. This gives rise to characteristic absorption and emission
216
spectra when the excited species transits back to the original S0 level and emits the excess energy
in the form of light. The electronic transition energy and the wavelength of irradiation involved
are related by the following Equation 6.3.
E = (hc)/λ = hυ 6.3
where E is energy in Joules (J), h is Planck’s constant (= 6.63 × 10-34 J s), c is the speed of light
in a vacuum (= 3 × 108 m s-1), λ is the wavelength in m (which is converted to nm when using
the UV-Vis region for convenience) and υ is the frequency in s-1.
The possible transitions that may occur are displayed for a model species in Figure 6.4. As is
displayed in this figure, each electronic level (Sn) has its own vibrational energy level fine
structure (v0, v1 etc). Upon electronic excitation a molecule is also usually vibrationally excited
and the upper vibrational energy level reached depends upon the energy of the absorbed photon.77
After absorbing the electromagnetic radiation (E = hυ) the molecule is in an excited state that is
less stable than the ground state and it will therefore tend to return to the electronic ground state
to reach the minimum energy (S0). This can occur via several pathways.
The mechanism of interest in this study is fluorescence. This may occur when a molecule (for
example the stZQA ligand complexed to Zn2+) absorbs light to transit to a υ > 0 vibrational level
in an S > 0 electronic level. This is illustrated in Figure 6.4 where the absorbance of incident light
promotes an electron from the ground vibrational level within the electronic ground state (υ0, S0)
to the first vibrational level within the first excited electronic state (υ1, S1). The dashed arrow
then shows the process whereby the molecule shuttles down through the vibrational levels by
non-radiative decay (energy loss through collision) followed by transition to the ground state by
the emission of light at a lower energy (longer λ) because this final conversion is between energy
levels of a smaller energy gap.77
217
Figure 6.4. A schematic energy level diagram displaying the electronic transitions that may
occur for some molecules.
For the organic stZQA ligand in this study these electronic transitions occur on the energy
scale contained within the ultraviolet and visible region of the electromagnetic spectrum.
Experiments with a UV-Vis spectrophotometer (measuring the absorbance over a given
wavelength range) can therefore reveal the absorbance properties of this molecule and
experiments with a fluorimeter (measuring the emission spectra with varying excitation
wavelength) can therefore reveal the fluorescence properties. Both of these are performed as part
of this study to characterise the effectiveness of stZQA as a Zn2+ sensor.
218
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223
Hilary Coleman Chapter 7. Absorbance and Fluorescence Characterisation of Styryl-Zinquin A
CHAPTER 7
ABSORBANCE AND FLUORESCENCE

CHARACTERISATION OF STYRYL-ZINQUIN A
The work presented in this chapter was described in the publication:
“Complexation of Zn2+ by the Fluorophore 2-((E)-2-Phenyl)ethenyl-8-(N-4-

methylbenzenesulfonyl)aminoquinol-6-yloxyacetic Acid: A Preparative, Potentiometric, UV-
visible, and Fluorescence Study” Aust. J. Chem. 2010, 63, 1448-1452.1
224
7.1 Potentiometric titration of 2-((E)-2-Phenyl)ethenyl-8-(N-4-

methylbenzenesulfonyl)aminoquinol-6-yloxyacetic Acid (stZQA) for
ligand pKa determination
Acid dissociation constants, pKas, were determined by potentiometric titrimetry for the fully
protonated form of stZQA at 298.2 K and in 25% aqueous ethanol NaClO4 (I = 0.10 mol dm-3) as
described in Section 7.4. Under acidic conditions this ligand has three sites for deprotonation as
depicted in Figure 7.1 – the sulfonamide, carboxylic and quinolinium protons. These are
characterised by three acid dissociation constants, Ka1, Ka2 and Ka3 which are defined by
Equations 7.1 to 7.3 where L represents the stZQA ligand. The pKas that were determined in this
study are displayed in Table 7.1 along with the corresponding values for the analogue ZQA.
Ka1
(LH)ˉ ⇌ L2ˉ + H+ where Ka1 = ([L2ˉ][H+])/[LHˉ] 7.1
Ka2
(LH2) ⇌ (LH)ˉ + H+ where Ka2 = ([LHˉ][H+])/[LH2] 7.2
Ka3
(LH3)+ ⇌ (LH2) + H+ where Ka3 = ([LH2][H+])/[LH3+] 7.3

(L) pKa1 pKa2 pKa3
stZQA This study 10.46 ± 0.03 4.92 ± 0.03 2.71 ± 0.03
ZQA a 10.13 ± 0.08 4.40 ± 0.02 3.08 ± 0.06
a: Ref2, I = 0.1 mol dm-3 (NaClO4), 25% aqueous ethanol 298.2 K
Table 7.1. pKas for the ligands stZQA and ZQA determined by potentiometric titration at 298.2 K
in 25% aqueous ethanol NaClO4 (I = 0.10 mol dm-3).
225
Figure 7.1. Protonation states of the stZQA

ligand described by the formulae: stZQAH3+,
stZQAH2, stZQAHˉ and stZQA2ˉ.
226
The three acid dissocitation constants measured in this study, pKa1, pKa2 and pKa3, are
assigned to the sulfonamide, carboxylic acid and quinolinium protons, respectively.2 Note that
unlike the amine bases studied in Chapters 2 and 3, only one of these acid dissociations (pKa3)
represents the loss of a proton present under acidic conditions and the other two (pKas 1 and 2)
represent the loss of protons from the neutral molecule causing it to become an anionic species.
This is reflected in the deprotonation scheme in Figure 7.1.
As seen in Table 7.1 the quinolinium proton (pKa3) of stZQA is more acidic than the same
proton on the ZQA analogue. (Alternatively stated, the quinolinium nitrogen of stZQA is less
basic than that of ZQA.) Conversely, the carboxylic acid and sulfonamide protons are less acidic
on stZQA than on ZQA. This is consistent with the hypothesised effects of adding the styryl
functionality to ZQA. The increased conjugation on stZQA relative to ZQA is predicted to
destabilise stZQAH3+ with respect to stZQAH2 to a greater extent than the destabilisation of
ZQAH3+ with respect to ZQAH2. The higher pKa1 and pKa2 of stZQAHˉ and stZQAH2
respectively (relative to ZQAHˉ and ZQAH2) probably reflect the greater delocalisation of the di-
and mono-negative charges of their conjugate bases (stZQA2ˉ and stZQAHˉ respectively) and
decreased electrostatic resistance to deprotonation by comparison with ZQAHˉ and ZQAH2. The
effects of this increased conjugation are also observed in the UV-Vis and fluorescence
spectroscopic investigations of this ligand.
It should also be noted that these pKa values are partially a function of the solvent system
used. The system of 25% aqueous ethanol was selected for this study to emulate the physiological
conditions that this fluorophore might be used in (as discussed in Section 5.2). However, 50%
aqueous ethanol is also a satisfactory cellular fluid mimic and a comparison of the pKas of ZQA
under both sets of conditions reveals that the values may vary by more than one log unit between
solvent systems.2, 3 As anticipated, all of the pKas of ZQA are lower in 50% aqueous ethanol than
in 25% aqueous ethanol, indicating that this species is more readily deprotonated when there is a
larger proportion of H2O (less ethanol) comprising the solvent. This is a consequence of the
greater amount of H2O being available to more effectively solvate the deprotonation products.
Changes in the solvent system may therefore have an impact on the pKa values of stZQA which
should be noted if it is used in a biological system.
A titration curve and fit typical from this study is displayed in Figure 7.2.
227
12.5
11.5
10.5
9.5
8.5 Observed
pH 7.5 Fit
6.5
5.5
4.5
3.5
2.5
0.07 0.095 0.12 0.145 0.17 0.195 0.22
Titre (mL)
Figure 7.2. Potentiometric titration curve for the determination of stZQA ligand pKas – observed
points (◊) and fit (-), resulting in the values reported in Table 7.1. 25% aqueous ethanol NaClO4
(I = 0.10 mol dm-3), 298.2 K. The initial [stZQA] = 1.0 ×10-3 mol dm-3, the added [HClO4] = 9.2
× 10-3 mol dm-3, and the titrant [NaOH] = 0.10 mol dm-3.
The speciation of the stZQA system with varying pH can be determined from these measured
pKas and this is necessary in order to conduct UV-Vis and fluorescence investigations. These
experiments must be conducted at a buffered pH such that the proportions of each form of the
ligand (stZQAH3+, stZQAH2, ZQAHˉ or stZQA2ˉ) are known and kept constant. The form of the
ligand will impact the extent of metal complex formation, the structure of the ligand in solution
and the way it is able to dissipate excess energy upon irradiation (i.e. its absorbance and
fluorescence characteristics). The speciation of the system is therefore key in analysing these
experiments.
A pH of 7.6 was selected for the UV-Vis and fluorimetry investigations because it is
consistent with past Zinquin studies.2 This enables a direct comparison with the corresponding
characterisation of Zinquin A and good predictive power for the behavior of stZQA in a
biological medium. The speciation for stZQA based on the pKas determined from the fit in Figure
7.2 is shown in Figure 7.3 with pH 6.6 highlighted. It can be seen in this plot that at pH 6.6 the
dominant species (over 95% formation) is ZQAHˉ.
228
100
90

80 free L²⁻
70
60 LH⁻
50
40 LH₂
30
20 LH₃⁺
10
0
3 5 7 9 11
pH
Figure 7.3. Speciation of stZQA derived from the titration and fit in Figure 7.2. The pH for the
spectroscopic analyses (6.6) is indicated and the dominant species at this pH is stZQAHˉ.
7.2 UV-Vis characterisation of 2-((E)-2-Phenyl)ethenyl-8-(N-4-

methylbenzenesulfonyl)aminoquinol-6-yloxyacetic Acid
7.2.1 UV-Vis titration: determination of stability constants for Zn(II)

complexes of stZQA
Measurement of the absorbance variation of the ligand stZQA in the absence and presence of
increasing concentrations of Zn2+ was used to characterise the complexes formed in the
stZQA/Zn2+ system. The experiments were performed at pH 6.6 (1.0 × 10-3 mol dm-3 NaPIPES
buffer) in 25% aqueous ethanol NaClO4 (0.10 mol dm-3) at 298.2 K using a UV-VIS-NIR Varian
Cary 5000 spectrophotometer with 1.0 cm matched cuvettes.
Figure 7.4 displays this spectral variation of the ligand at 1.82 × 10-5 mol dm-3 with increasing
[Zn2+](total) over the range 0 – 1.72 × 10-4 mol dm-3. The initial spectrum ([Zn2+]total = 0) shows the
free ligand in the form stZQAHˉ (the dominant species at pH 6.6). As discussed in more detail in
Section 7.3.1 there is a small amount of Zn2+ impurity - known as adventitious Zn2+ - present in
all of the samples due to the ubiquity of this metal. This impurity was quantitatively measured
229
and included in all the following analyses because the sensitivity of stZQA to Zn2+ is likely to
result in the adventitious Zn2+ impacting the results. Therefore, in order to obtain a true
absorbance spectrum of the free ligand (in the absence of any Zn2+ - whether added purposefully
or present as an impurity) the Zn2+ chelator EDTA was added to this sample in order to
preferentially bind the adventitious Zn2+ and produce a spectrum that accurately represented
[Zn2+]total = 0.
As Zn2+ is then added, the remaining proton is lost from stZQAHˉ and complex formation
occurs. The subsequent spectra reflect a combination of free and bound ligand absorbances
(stZQAHˉ, [Zn(stZQA)] and [Zn(stZQA)2]2ˉ), as the speciation changes with varying [Zn2+]total.
1 .0
0 .8
A b so rb a n c e
0 .6
0 .4
0 .2
0 .0
250 300 350 400 450 500
Figure 7.4. Variation of the UV-Vis spectrum of 1.82 × 10-5 mol dm-3 stZQAHˉ in 25% v/v
aqueous ethanol NaClO4 (I = 0.10 mol dm-3) buffered at pH 6.6 (1.0 × 10-3 mol dm-3 NaPIPES
buffer) at 298.2 K with [Zn2+] (total) varying from 0 – 1.72 × 10-4 mol dm-3. Arrows indicate
direction of absorbance change with increasing [Zn2+] (total).
Isosbestic points occur at 336 nm and 375 nm indicating that the free and Zn 2+-complexed
stZQA ligands are the only absorbing species in solution. Due to the filled d10 electronic
configuration (3d10, 4s0) of the zinc cation, there can be no ligand-to-metal-charge transfer
230
(LMCT) bands nor any d-d transitions contributing to these spectra. The results obtained are
therefore purely due to electronic transitions between ligand orbitals. For an organic molecule,
the possible electronic transitions include non-bonding orbital (lone electron pair) to antibonding
orbital (eg. n to π*), and bonding orbital to unfilled antibonding orbital (eg. π to π*).4 The π to π*
transitions tend to be characterised by large molar absorptivities (ε ≥ 1 × 104 dm3 mol-1 cm-1) and
their wavelength of maximum absorbance (λmax) may be shifted depending on the conjugation of
the molecule under investigation. It is likely, therefore, that the peak observed between 300 – 330
nm is the result of one such transition for stZQA and those at longer wavelengths are due to
weaker n to π* transitions. These spectral characteristics are very similar to those displayed by
the ZQA analogue.2
The equilibria described in Equations 7.4 and 7.5 describe the complex formation between
stZQA and Zn2+ under these experimental conditions. The best fit of an algorithm for these
equilibria to the absorbance variation at 1 nm intervals over the range 270 – 450 nm in Figure 7.4
reveals that both 1:1 and 2:1 (L:M) complexes are stable. The determined stability constants for
these complexes from this fit are logK1 = 11.5 ± 0.76 and logK2 = 9.7 ± 0.84.
K1
stZQAHˉ + Zn2+ ⇌ [Zn(stZQA)] + H+ 7.4
where K1 = [Zn(stZQA)][H+]/[stZQAHˉ][Zn2+]
K2
stZQAHˉ + [Zn(stZQA)] ⇌ [Zn(stZQA)2]2ˉ + H+ 7.5
where K2 = [Zn(stZQA)22ˉ][H+]/[stZQAHˉ][Zn(stZQA)]
The contributions made by the individual absorbing species (stZQA present as stZQAHˉ,
[Zn(stZQA)] and[Zn(stZQA)2]2ˉ) are measured by this fit and therefore their individual UV-Vis
spectra are derived. These are displayed in Figure 7.5. It is immediately noticeable that the two
spectra of the ligand involved in the complexes [Zn(stZQA)] and [Zn(stZQA)2]2ˉ contain peaks
that are shifted to longer wavelengths by comparison to the spectrum of the unbound ligand,
stZQAHˉ. It is likely that this bathochromic shift is due to the increased conjugation that occurs
throughout stZQA on metal-binding. Before complexation, the ligand is not held rigidly and is
free to move and therefore the molecular orbitals that extend over the ligand are not restricted in a
231
planar arrangement. Whilst it is possible for conjugation to extend over the entire molecule,
realistically in an unbound form this does not occur because all of the π orbitals are not held in a
fixed, overlapping arrangement. Furthermore, the unbound ligand may also experience more
molecular motion in the form of vibration on the quinoline ring and rotation around the
sulfonamide nitrogen to quinoline single bond. Conversely, when the ligand is bound to Zn2+ it is
likely held in a more rigid, planar arrangement that would enhance the extent of π orbital overlap,
resulting in increased conjugation, a smaller energy gap between orbitals and therefore a larger
wavelength of maximum absorbance.
60
-4
M o la r a b s o r p tiv it y , e , p e r lig a n d × 1 0
(c)
)
40
-1
cm
(b)
-1
m ol
3
(d m
20
(a)
0
250 300 350 400 450 500
Figure 7.5. The UV-Vis spectra of (a) stZQAHˉ (max = 303 nm and  = 3.8 × 104 dm3 mol-1 cm-1
and max = 366 nm and  = 1.9 × 104 dm3 mol-1 cm-1); (b) [Zn(stZQA)] (max = 313 nm and  =
4.8 × 104 dm3 mol-1 cm-1 and max = 378 nm and  = 1.5 × 104 dm3 mol-1 cm-1) and (c)
[Zn(stZQA)2] 2ˉ (max = 312 nm and  = 5.2 × 104 dm3 mol-1 cm-1 and max = 376 nm and  = 1.8
× 104 dm3 mol-1 cm-1) derived from the fitting of an algorithm for the molar absorbance variation
with increasing [Zn2+] total and Equations 7.4 and 7.5 as described in the text.
Also of note in Figure 7.5 is that the y-axis is molar absorptivity per ligand. For the 2:1
complex [Zn(stZQA)2]2ˉ this means the calculated ε was then halved so that a comparison could
be made per ligand rather than per complex. Interestingly when this molar absorptivity per
complex is halved, the spectral shape and intensity are very similar for the ligands present on
232
both the 1:1 and 2:1 complexes. This suggests that the electronic properties of a stZQA ligand
present in a 1:1 complex are very similar to the electronic properties of a stZQA ligand present in
a 2:1 complex, despite the presence of the second ligand. The same result was found when UV-
Vis spectra were experimentally obtained under 1:1 L:M sample concentration conditions and 2:1
L:M sample concentration conditions. This indicates that the electronic properties of the first
ligand are largely unaffected by the addition of a second ligand to the complex. It is therefore
likely that there is little interaction between the two ligands on the 2:1 [Zn(stZQA)2]2ˉ complex
otherwise a larger difference between the 1:1 and 2:1 spectra per ligand may be anticipated.
Table 7.2 shows a comparison of the λmax and corresponding ε in free and Zn2+-bound forms
for the new stZQA ligand and its ZQA precursor. (Note that the table displays the molar
absorptivity of the complex and that the 2:1 complex values have not been adjusted to reflect the
molar absorptivity per ligand in either case of stZQA or ZQA.)
A comparison of each ligand form between stZQA and ZQA shows longer wavelengths of
maximum absorbance for stZQA in every case. This is consistent with the increased conjugation
of stZQA relative to the ZQA analogue caused by the added styryl group; increased conjugation
reduces the energy gap between orbitals and therefore lower energy, longer wavelength light is
absorbed when these transitions occur.
Interestingly there is not a constant trend for molar absorptivity between the derivatives. This
suggests that the probability of electronic transition is contributed to considerably by factors
other than purely the extent of conjugation. For some transitions the stZQA ligand yields a higher
ε but for others the ZQA ligand does.
These results will be discussed further in Section 7.3.3. However, most importantly, an
excitation wavelength for fluorescence studies can be chosen after analysis of these UV-Vis
spectra. It was decided to select an isosbestic point from the experiments displayed in Figure 7.4
so that any observed changes in fluorescence on irradiation at this wavelength could not be due to
differences in the amount of energy absorbed by the different species in solution. The wavelength
336 nm was therefore chosen.
233
Species Wavelength of maximum absorbance (nm)

(Molar Absorptivity (dm3 mol-1 cm-1))
stZQAHˉ This study 303 366

(3.8 × 104) (1.9 × 104)
[Zn(stZQA)] This study 313 378

(4.8 × 104) (1.5 × 104)
[Zn(stZQA)]2ˉ This study 312 376

(1.04 × 105) (3.5 × 104)
ZQAHˉ a 245 336

(4.1 × 104) (4.2 × 103)
[Zn(ZQA)] a 264 361

(3.6 × 104) (3.9 × 103)
[Zn(ZQA)]2ˉ a 264 361

(9.3 × 104) (7.8 × 104)
a: Ref2, I = 0.10 mol dm-3 (NaClO4), 25% aqueous ethanol, 298.2 K, pH 6.6
Table 7.2. Wavelengths of maximum absorbance and corresponding molar absorptivities (per
complex) for the UV-Vis spectra of unbound and Zn2+-complexed stZQA and ZQA in 25% v/v
aqueous ethanol NaClO4 (I = 0.1 mol dm-3), pH 6.6 ([NaPIPES buffer] = 1.0 × 10-3 mol dm-3,
298.2 K.
7.2.2 Photoisomerism of the stZQA ligand

Figures of stZQA have thus far only depicted one possible isomer of this ligand. However,
the added styryl group containing the ethenyl linker and benzene ring allows for the possibility of
both E and Z configurations. (Without this functionality the ZQA analogue does not possess this
same occurrence of structural isomers.) Figure 7.6 displays the conversion of stZQA between the
E and Z isomers which experimentally was discovered to transpire during exposure to laboratory
(i.e. ambient) light. Meticulous conductance of solution preparation and experimentation under
reduced light, and equilibration in complete darkness prior to analysis meant that the ligand was
in 100% E-isomer form when all results were recorded. This was confirmed by TLC and 1H
NMR in which a cis J coupling constant was obtained for the ethenyl protons.2
234
Figure 7.6. E and Z isomers of stZQA converted by exposure to laboratory light over a 15 minute
time scale.
The extent of conjugation on complexation is likely to be significantly different between the

two isomers. In the Z conformation there is likely to be steric hindrance between the benzene ring
and the quinoline body which would cause the ring to bend out of the plane of the rest of the
molecule. Preliminary ab initio molecular modeling calculations suggest this to be the case.5
Therefore the complex forming characteristics and the absorbance spectra of the free and bound
ligands are likely to be altered upon photoisomerisation about the ethenyl bond. This
photoisomerisation was therefore analysed by monitoring the UV-Vis spectrum of the free ligand
over time with continuous exposure to laboratory light. The results are displayed in Figure 7.7.
After approximately 15 minutes a photostationary state is reached in which the equilibrium
proportions of the E-stZQAˉ and Z-stZQAˉ isomers are unknown (differentiation of the 1H NMR
signals is not feasible). The isomerically pure E-stZQAˉ may be regained by refluxing the sample
in darkness with a catalytic amount of p-toluenesulfonic acid.
To avoid any contribution by the Z isomer to the UV-Vis and fluorimetric equilibrium studies
in this chapter, all solution management was carried out under reduced light, and all solutions
were contained in foil-wrapped vessels and allowed to equilibrate for at least 30 min at 298.2 K
in darkness prior to spectroscopic measurement. It should be noted, however, that the presence of
this photoisomerisation makes this new ligand non-ideal as a Zn2+ sensor. It introduces potential
limitations to the use of this ligand in healthcare and health research settings. Depending on how
the fluorophore was stored, practitioners would have to equilibrate the sample prior to use.
235
Furthermore, further investigation into the stability of complexes formed by the Z isomer and
which forms of physiological Zn2+ (catalytic, structural and free) it’s capable of binding to would
be essential. This would require full characterisation of the individual Z isomer and equilibrated
systems. It would be more efficient (and involve fewer possible variables) to use a new
functionality other than the syryl group. Therefore if further improvements to the original
Zinquin (ZQA) ligand were sought as Future Work, a different functionality should be
investigated to increase the conjugation and bulkiness of the ligand without introducing
photoisomerisable groups.
0 .8
0 minute light exposure

0 .6

A b so rb a n c e
0 .4
0 .2
0 .0
250 300 350 400 450
Figure 7.7. The absorbance variation time dependence for E-stZQAˉ on exposure to daylight in
25% v/v aqueous ethanol NaClO4 (I = 0.10 mol dm-3) buffered at pH 6.6 (1.0 × 10-3 mol dm-3
NaPIPES) at 298.2 K.
7.2.3 Binding of metals other than Zn(II)

In order to investigate the suitability of the new ligand stZQA for physiological Zn2+
detection, its binding characteristics in the presence of other metals must be elucidated. Because
Zn2+ is not the only metal cation commonly found in physiological samples,6 stZQA will only be
a successful Zn2+ sensor if it uniquely produces a fluorescence signal when bound to Zn2+. This
236
may be achieved either if the ligand selectively binds to Zn2+ only, or if it binds to other metal
cations but only the zinc complex fluoresces. The UV-Vis spectra of stZQA in the presence of
Ca2+, Cd2+, Cu2+, Co2+ and Ni2+ were therefore also tested to see if complexation occurred.
Samples of stZQA (1.76 × 10-5 mol dm-3) and M2+ (3.63 × 10-4 mol dm-3) were prepared and
examined over the wavelength range 250 – 500 nm. (Without prior knowledge of whether
binding would occur or not, an excess of M2+ was used in order to drive the complexation
equilibrium towards complex formation if stable complexes could, in fact, form.) The resulting
spectra are displayed in Figure 7.8.
0 .8
-
F r e e s tZ Q A H
2+
w ith C a
0 .6 2+
w i th C d
A b so rb a n c e
2+
w i th C u
0 .4 2+
w i th C o
2+
w ith N i
0 .2
0 .0
250 300 350 400 450 500
Figure 7.8. UV-Vis spectra of stZQA (1.76 × 10-5 mol dm-3) unbound and with M2+ (3.63 × 10-4
mol dm-3) in 25% v/v aqueous ethanol NaClO4 (I = 0.10 mol dm-3) and buffered at pH 6.6
(NaPIPES 1.0 × 10-3 mol dm-3), 298.2 K. M2+ = Ca2+, Cd2+, Cu2+, Co2+ and Ni2+.
As can be observed in this figure, the spectra of the unbound ligand and stZQA in the
presence of Ca2+ exactly overlay. This is suggestive of no complex formation with this ligand and
metal cation combination because a change in the ligand absorbance spectrum would be expected
upon metal binding. This result is not unexpected because the calcium cation is classed as a hard
Lewis acid whilst ligands containing nitrogen donor groups are usually borderline or soft Lewis
bases and so complexation is unlikely to occur between the two.7 The remaining metal cations
tested all fall into the borderline or soft Lewis acid categories and consequently are more likely to
237
form complexes with stZQA. Indeed all of the spectra for stZQA in the presence of these metal
cations are shifted to longer wavelengths and are different in shape when compared to the
spectrum of the unbound ligand. This suggests that stZQA forms complexes to some degree of
stability with each of these species. This finding is in agreement with the results of the analogous
ZQA ligand which has measureable stability constants (K1 and K2) with Co2+, Ni2+, Cu2+ and
Cd2+.3 The bathochromic shift in every case again supports the hypothesis that metal-binding
increases the extent of conjugation over the ligand as discussed in Section 7.2.1 for complexation
with Zn2+. Early studies of the most basic Zn2+ sensor analogue, 8-aminoquinoline, (see Section
5.1.2) show that it forms stable complexes with Cu2+, Ni2+, Co2+, Fe2+ and Cd2+ as well as Zn2+.8
It is therefore to be expected that other ligands in the class of quinoline-derived sensors also form
these complexes with varying stabilities dependent on the ligand in question. However, the
formation of stable complexes with metals other than Zn2+ is not problematic as long as these
complexes do not fluoresce, as is the finding for stZQA in Section 7.3.2. Indeed, even a very
recent investigation of a new quinoline-derived Zn2+ sensor reports reduced fluorescence when
the sensor is in the presence of a combination of Zn2+ with Co2+ and Ni2+, indicating that the
sensor forms a stable complexes with these metals but the fluorescence is quenched in the non-
Zn2+ complexes.9 Selective Zn2+ probes are not required to selectively bind to Zn2+, only to
selectively produce a fluorescent signal with Zn2+.
If no complex is formed between stZQA and Ca2+ then this system cannot produce a
fluorescent signal. However there is a possibility for the complexes formed with the other metal
cations to fluoresce, as mentioned above. It is hypothesised that d-d metal electronic transitions
may quench this fluorescence for Co2+, Ni2+ and Cu2+ but this must still be examined in order to
determine the suitability of stZQA as a selective Zn2+ detector. These experiments are presented
and discussed in section 7.3.2; Fluorescence characterisation of stZQA.
238
7.3 Fluorescence characterisation of 2-((E)-2-Phenyl)ethenyl-8-(N-4-

methylbenzenesulfonyl)aminoquinol-6-yloxyacetic Acid (stZQA)
7.3.1 Determination of adventitious Zn(II)

The aim of this study was to characterise the new ligand stZQA in order to assess its potential
application in physiological Zn2+ detection. This involved probing the electronic properties of the
free and bound ligand as well as determining the stability constants of the complexes formed with
Zn2+.
However, Zn2+ is an environmentally ubiquitous metal10 which introduces the problem of

Zn2+ impurity into the experimental work of this chapter. Adventitious Zn2+ from various sources
was identified as an obstacle to Zn2+ sensing in biological samples as early as 1959.11 Due to the
significant sensitivity of the ZQA and its analogues to even small concentrations of Zn2+, the
trace amounts of Zn2+ contamination present in the experimental solutions have to be accounted
for before any analyses can be undertaken. Indeed, initial investigation of the fluorescence of
ZQA in the presence of Zn2+ showed a substantial signal even for samples containing no
experimentally added Zn2+ and the subsequent atomic absorption measurements confirmed a low-
level Zn2+ impurity even in the analytical grade chemicals used in the solution preparation.3
Methods to remove the Zn2+ impurity include adding a zinc chelating agent to detergent and
pre-washing all glassware and utensils in addition to purifying the buffer and solvents used.
However due to the prevalence of Zn2+ and its ability to bind to surfaces such as glass and plastic,
these methods are largely unsuccessful at completely removing the Zn2+ impurity.2 The chosen
remedy in this study was therefore to quantify the adventitious Zn2+ concentration – [Zn2+]Ad – in
the solvent systems used for both UV-Vis and fluorescence experiments prior to their use and
then to incorporate this value into the data analysis. This involved an EDTA fluorescence
titration. The ligand EDTA (ethylenediaminetetraacetic acid) is hexadentate with a very large
binding constant with Zn2+ - K is > 1016.12 It is therefore ideal for this function.
This titration involved measuring the fluorescence (at the λmax) of samples containing 5 × 10-6
mol dm-3 stZQA and increasing concentrations of EDTA; i.e. when no Zn2+ is added by the
experimenter, the fluorescence observed with zero EDTA must be due to the complex formed by
the ligand binding to adventitious Zn2+. This fluorescence should decrease as EDTA is added and
239
competes with stZQA for Zn2+-binding. Because the complex formed between EDTA and Zn2+ is
1:1, a plot of relative fluorescence against [EDTA] (mol dm-3) should yield a straight line with an
x-axis intercept equal to the adventitious Zn2+ concentration. The [Zn2+]Ad determined this way
was 3.9 × 10-7 mol dm-3 for the large scale solvent system prepared for use in both UV-Vis
(Section 7.2.1) and fluorescence titration experiments. This value was therefore incorporated into
the data fitting in both cases; when [Zn2+]added = 0, [Zn2+]total = 3.9 × 10-7 mol dm-3.
Figure 7.9 displays the fluorescence of a) a sample of stZQA with no added Zn2+ or EDTA
(i.e. the ligand in the presence of [Zn2+]Ad only) and b) a sample of stZQA with EDTA.
100
(a)
80
R e la t iv e f l u o r e s c e n c e
60
40
20
(b)
0
450 500 550 600 650
Figure 7.9. Fluorescence spectra of the ligand stZQA (5.56 × 10-6 mol dm-3) in a) the presence of
Zn2+(Ad) and b) in the presence of EDTA (1.0 × 10-5 mol dm-3) in 25% v/v aqueous ethanol
NaClO4 (I = 0.10 mol dm-3) at pH 6.6 (NaPIPES buffer = 1.0 × 10-3) at 298.2 K.
It is clear from this figure that the fluorescence signal produced by stZQA is sensitive to even
small concentrations of Zn2+ and that therefore it is crucial to measure the Zn2+ impurity prior to
experimentation. Without first calculating the [Zn2+]Ad a true characterisation of the fluorophore
cannot be achieved.
240
7.3.2 Fluorescence signal with other metals: stZQA, a selective probe?

Before full characterisation of the stZQA/Zn2+ system was carried out, complexes of this
ligand with the other metal cations - Ca2+, Cd2+, Cu2+, Co2+ and Ni2+ - were investigated for their
potential fluorescence properties, as discussed in Section 7.2.3. The sample solutions used to
obtain the UV-Vis spectra in Figure 7.8 were directly diluted by a factor of ten and irradiated
with the same excitation wavelength as was selected for use with the stZQA/Zn 2+ system: 336
nm. Therefore, if no large fluorescence resulted under these conditions, this method for Zn2+
detection would likely be unaffected by complex formation between stZQA and the other metal
cations present in biological samples. Conversely if a large fluorescence signal for any of these
complexes with cations other than Zn2+ was observed, then stZQA would not be a reliable Zn2+
detector and characterisation of that system would be redundant.
Figure 7.10 displays the fluorescence resulting from irradiation at an excitation wavelength
(λex) of 336 nm of the diluted samples of stZQA with Ca2+, Cd2+, Cu2+, Co2+ and Ni2+ used for
UV-Vis analysis (final [stZQA] = 1.76 × 10-6 mol dm-3 and [M2+] = 3.63 × 10-5 mol dm-3). It
should be noted that no EDTA was added to these samples so the adventitious Zn 2+ impurity was
also present.
25
- 2+
s tZ Q A H + Ca
- 2+
20 s tZ Q A H + Cd
- 2+
s tZ Q A H + Cu
15 - 2+
s tZ Q A H + Co
- 2+
s tZ Q A H + Ni
10
0
450 500 550 600 650
Figure 7.10. Fluorescence spectra of samples containing stZQA (1.76 × 10-6 mol dm-3) in the
presence of Ca2+, Cd2+, Cu2+, Co2+ and Ni2+ (3.63 ×10-5 mol.dm-3), λex = 336 nm, in 25% v/v
aqueous ethanol NaClO4 (I = 0.10 mol dm-3) buffered at pH 6.6 (NaPIPES = 1.0 × 10-3 mol dm-
3
), 298.2 K. (No EDTA present.)
241
As these were qualitative tests, the amount of adventitious Zn2+ present in this solvent system
was not experimentally quantified as described in Section 7.3.1. However, since the UV-Vis
investigations concluded that stZQA appears not to bind to Ca2+ (Figure 7.8) it is likely that the
spectrum recorded for this sample is in fact due to the ligand having complexed with this
adventitious Zn2+ to produce the predicted fluorescence signal. This signal intensity is very small
and is of the size expected for the signal caused by complexation with the Zn2+ impurity. It is also
the largest for all of the M2+ species which may be expected if stZQA does not bind to Ca2+ at all,
leaving the largest concentration of stZQA free for any system, to bind with Zn2+Ad.
Interestingly, the order of the decrease in fluorescence signal at the λmax for each system is in
exact agreement with the order of increasing stability constant for the metal cations and their
complexes formed with the ZQA analogue. The increasing order of logK1 for the complexes
formed with ZQA and M2+ follows the trend Ni2+ < Co2+ < Cd2+ < Zn2+ < Cu2+.3 Although the
complex stability constants between stZQA and these metals have not all been determined (but
should be as part of Future Work), the results displayed in Figure 7.10 suggest that the size of
these constants likely follows the same trend as is found for ZQA.
The Ca2+ does not bind to stZQA and cannot compete with the adventitious Zn2+ in solution
and therefore it is hypothesised that the spectrum observed for this sample is entirely due to the
complex formed between the stZQA and the [Zn2+]Ad. The Ni2+ cation has the next smallest
stability constant with the analogous ZQA ligand and indeed the next highest fluorescence
produced is for the stZQA/Ni2+ sample. This indicates that the Ni2+ competes to some extent with
the [Zn2+]Ad but the [Zn(stZQA)] complex is likely more stable than the [Ni(stZQA)] complex
and so the majority of the complexes in solution will contain Zn2+ as their metal centre and
therefore fluoresce. The small proportion that does contain Ni2+, however, no longer fluoresces
due to electronic transitions in the d8 manifold quenching this signal. Therefore the fluorescence
intensity for this sample is reduced when compared with that of the Ca2+ sample. Subsequently
the Co2+ cation has a slightly larger stability constant with ZQA and this sample with stZQA
yields the next largest fluorescence signal. For the same reasoning above, the Co 2+ should
compete more successfully than Ni2+ for complexation with stZQA but not as successfully as
Zn2+. Therefore the fluorescence intensity is further reduced from that of the stZQA/Ni2+ sample
as a larger proportion of the complexes have their fluorescence quenched by d-d transitions on
Co2+, but the majority of complexes still contain Zn2+ and therefore the signal is still observed.
The Cd2+ cation produces an even lower fluorescence intensity for the same reasoning until the
242
Cu2+ system virtually entirely removes the fluorescence altogether (with the same effect as
adding EDTA). If the stZQA ligand does follow the same stability constant trend as for ZQA, this
is therefore because its logK1 with Cu2+ is higher than that with Zn2+ and the stZQA forms more
stable complexes with this cation and so all of the complexes present in this system contain Cu2+
as the central cation and all of the fluorescence is therefore quenched by its d9 manifold. (It
should be noted that with the same d10 electronic configuration as Zn2+, it is possible that the Cd2+
complexes may produce some fluorescence. There is no evidence of a large signal being
produced in these experiments however and the concentration of Cd2+ in healthy cells is very low
by comparison with Zn2+.13, 14 Therefore significant contributions from Cd2+ can be ignored in
cellular and tissue studies.)
Stability constants for stZQA with all of these metal cations should be determined by
potentiometric titrimetry as part of Future Work. However these qualitative comparisons provide
excellent evidence for the same trend in logK1 as has been measured for the analogous ZQA
ligand. Furthermore, the absence of fluorescence beyond what is expected for the complex
between stZQA and the adventitious Zn2+ present for this experiment suggests that this ligand
does successfully selectively produce a signal for Zn2+. Characterisation of the stZQA/Zn2+
system is therefore warranted.
7.3.3 Fluorescence titration: determination of stability constants for Zn(II)

complexes of stZQA
In an identical manner to the UV-Vis characterisation, measurement of the fluorescence
variation of the ligand stZQA in the absence and presence of increasing concentrations of Zn 2+
was used to characterise the complexes formed in the stZQA/Zn2+ system. The experiments were
performed at pH 6.6 (1.0 × 10-3 mol dm-3 NaPIPES buffer) in 25% aqueous ethanol NaClO4 (0.10
mol dm-3) at 298.2 K using a Varian Cary Eclipse fluorimeter with a 1.0 cm cuvette.
Figure 7.11 displays this variation of the stZQA ligand at a constant concentration of 5.56 ×
10-6 mol dm-3 with increasing [Zn2+]total over the range 3.9 × 10-7 (= [Zn2+]Ad) – 5.0 × 10-5 mol
dm-3. The initial spectrum ([Zn2+]total = 3.9 × 10-7 mol dm-3) reflects the fluorescence produced
when the ligand is complexed to the adventitious Zn2+ in solution. The subsequent spectra reflect
a combination of ligand fluorescence as it is found in varying proportions of [Zn(stZQA)] and
[Zn(stZQA)2]2ˉ as the speciation changes with varying [Zn2+]total.
243
Firstly, a comparison of Figures 7.5, 7.9 and 7.11 provides information on the fluorescence
mechanism of this Zn2+ detector. The absorbance spectrum of stZQAHˉ in the absence of
adventitious Zn2+ is shown in Figure 7.5. Under the same conditions the ligand shows no
fluorescence (Figure 7.9).The fluorescence of stZQA that is induced through coordination in
[Zn(stZQA)] and [Zn(stZQA)2]2ˉ occurs through a chelation enhanced fluorescence (CHEF)
mechanism,15 whereby the coordination of the amide and quinoline nitrogens to Zn2+ forms a
structurally stiffening five-membered chelate ring, which diminishes the fluorescence quenching
which would otherwise occur by energy dissipation through vibrational and rotational modes.
The nitrogen coordination also decreases any residual photoinduced electron transfer effects.16, 17
The stZQA ligand therefore only produces a fluorescence signal when bound to Zn2+ because in
the free form it may dissipate the energy from irradiation through vibrational and rotational
modes that are prohibited in the complexed form. This is the desired situation for a functional
Zn2+ detector.
800
600
400
200
0
450 500 550 600 650
Figure 7.11. Variation of the fluorescence spectrum of 5.56 × 10-6 mol dm-3 stZQA in 25% v/v
aqueous ethanol NaClO4 (I = 0.10 mol dm-3) buffered at pH 6.6 (1 × 10-3 mol dm-3 NaPIPES
buffer) at 298.2 K with [Zn2+] total from 3.9 × 10-7 – 5.0 × 10-5 mol dm-3. The arrow indicates
direction of fluorescence intensity change with increasing [Zn2+] total.
244
The best fit of an algorithm for the sequential equilibria represented by Equations 7.4 and 7.5
to the fluorescence variation at 0.5 nm intervals over the range 430–650 nm also yields both 1:1
L:M and 2:1 L:M metal complex stability constants in agreement with the UV-Vis analysis.
However the size of the stability constants varies between the types of spectroscopy probe used.
The logs of the stability constants obtained through the fluorescence fitting are: logK1 = 10.5 ±
0.20 and logK2 = 11.1 ± 0.1. This fit is displayed in Figure 7.12.
800
R e l a t iv e f lu o r e s c e n c e a t 5 3 4 n m
600
400
O b serv ed
F it
200
0
0 10 20 30 40 50
2+ -6 -3
[Z n ] to t a l  1 0 (m o l d m )
Figure 7.12. Fluorescence of stZQA (5.56 × 10-6 mol dm-3) in the presence of increasing
concentrations of [Zn2+] total (0 – 5.0 × 10-5 mol dm-3) at 534 nm, fitted by Specfit software18, 19 in
the fluorescence spectrum region 430 – 650 nm. Observed points (◊) and fit (-).
In the case of this fluorescence fitting, the logK of the 2:1 complex is larger than that of the
1:1 complex which is not the same as the result obtained via UV-Vis fitting. However, because of
the greater changes in the fluorescence spectra as [Zn2+]total increases by comparison with the
changes and in the UV-Vis spectra, and because of the greater wavelength range of 220 nm
suitable for K1 and K2 derivation from the fluorescence data as compared with the 180 nm
wavelength range for the UV-Vis data, the stability constants obtained by fluorimetry are
considered the more accurate. Furthermore 12 individual spectra were used in the UV-Vis
titration fitting versus 19 spectra for fluorescence. The similar K1 and K2 magnitudes are
245
consistent with the coordination number of [Zn(stZQA)] changing from six (where four aqua
ligands also share the first coordination sphere and stZQA2ˉ is coordinated as a bidentate ligand
through the sulfonamide and quinol nitrogens) to four in tetrahedral [Zn(stZQA)2]2ˉ. This is
likely a consequence of steric interactions between the two bulky stZQA2ˉ ligands and the
coordinative and stereochemical flexibility of d10 Zn2+.
It is also worth noting that whilst the UV-Vis data fitting did not yield the same trend, the
individual UV-Vis absorbance spectra of stZQA2ˉ per ligand in the complex forms [Zn(stZQA)]
and [Zn(stZQA)2]2ˉ do support this finding from the fluorescence fitting. As discussed in Section
7.2.1, the absorbance spectra of the 1:1 and 2:1 complexes are very similar to each other (both the
experimentally- and fit-derived spectra) suggesting that there is little interaction between the two
ligands on the 2:1 complex such that their electronic properties are virtually unaltered. On an
octahedral complex it is likely that steric crowding would result on addition of a second stZQA
ligand and consequently, interaction between these two ligands would be probable. This might be
expected to manifest itself in a more obvious difference between the absorbance spectra of
[Zn(stZQA)] and [Zn(stZQA)2]2ˉ which is not what is experimentally observed. Conversely if the
addition of a second stZQA does result in a change in geometry from octahedral to tetrahedral, it
may be more plausible (with the reduction in crowding) for the two ligands to have less
interaction. For the ZQA analogues [Zn(ZQA)] and [Zn(ZQA)2]2ˉ the stability constants under
the same conditions are logK1 = 10.5 and logK2 = 8.8.2 This is consistent with Zn2+ retaining a
six-coordination in both complexes which may be expected for the smaller, less bulky ZQA
molecule in comparison with stZQA.
Given this hypothesis, it may be plausible that the bulkier stZQA is capable of selectively
identifying the free, intracellular Zn2+ (in the hexa-aqua complex form [Zn(H2O)6]2+) over that
which is bound in proteins and enzymes as compared to ZQA. It has been shown that the less
bulky ZQA derivative readily coordinates to protein-bound Zn2+which means this ligand does not
differentiate between the three different forms of physiological Zn2+ discussed in Chapter 5.20, 21
If the added styryl bulk does induce a geometry change to minimise steric crowding, it is possible
that this styryl version of Zinquin A may be less able to coordinate to Zn2+ that is also
coordinated to other large ligands in the elaborate structures of enzymes and proteins. However,
the trend in 1:1 and 2:1 stability constants would first need to be confirmed by potentiometric
titration in Future Work and this hypothesis should then be investigated in physiological samples.
The ability to detect the location and concentration of free, intracellular Zn2+(aq) would be highly
246
desirable because it is this form of Zn2+ in the brain that is connected with many
neurodegenerative diseases. Determining the location and concentration of this form of Zn2+
(selectively over that found in proteins and enzymes) would therefore by very beneficial.22-24
The fluorescence intensity of the individual spectrum of [Zn(stZQA)] derived from the fitting
procedure applied to the data in Figure 7.11 is slightly greater than that of [Zn(stZQA)2]2ˉ as
displayed in Figure 7.13. This is expected and is observed for many similar complexes because
the introduction of a second ligand results in increased strain in the ligands and therefore greater
fluorescence quenching.2 This corresponds to a similar observation for the complexes of the ZQA
analogue [Zn(ZQA)] and [Zn(ZQA)2]2ˉ and the broader finding that the fluorescence of ZQA2ˉ in
complexes of the general formula [Zn(ZQA)(L)] varies significantly with the nature of L.25
15
(a)
-7
M o la r f lu o r e s c e n c e  1 0
(b)
10
0
450 500 550 600 650
Figure 7.13. The individual fluorescence spectra of (a) [Zn(stZQA)] (λmax = 534 nm and molar
fluorescence = 1.26 × 108 dm3 mol-1) and (b) [Zn(stZQA)2] 2ˉ per stZQA2ˉ ligand (λmax = 537 nm
and molar fluorescence = 1.18 × 108 dm3 mol-1) derived for the fitting of an algorithm for the
fluorescence variation with [Zn2+] total with the equilibria in Equations 7.4 and 7.5 as described in
the text.
The corresponding fluorescence spectra of complexes of the ZQA analogue [Zn(ZQA)] and
[Zn(ZQA)2]2ˉ under the same conditions show λmax = 483 nm and molar fluorescence = 5.56 ×
247
107 dm3 mol-1 for [Zn(ZQA)] and λmax = 483 nm and molar fluorescence = 6.17 × 107 dm3 mol-1.
The shorter wavelengths of maximum fluorescence as compared to the stZQA complexes reflect
the lesser extent of conjugation of the ZQA ligand in contrast with stZQA. (Interestingly whilst
the 2:1 complex involving ZQA produces a larger molar fluorescence than does the 1:1 complex
in 25% aqueous ethanol, this trend is reversed in 50% aqueous ethanol to be in line with the
reasoning above.2 Therefore it is concluded that the solvent system can have an effect on the
fluorescence properties of the ligand species, which should be considered if this ligand is tested
in a cellular fluid medium or other ligands are designed and tested as part of Future Work.)
The excitation wavelength for all of the fluorescence experiments reported in Section 7.3 is
336 nm – as was deemed appropriate after the UV-Vis investigations described in Section 7.2.1.
However, the fluorescence spectrum of the complexed stZQA ligand is dependent on the energy
of irradiation, as discussed in Section 6.2.2. To investigate how the fluorescence changed with
irradiation energy, the excitation wavelength was varied at 1 nm intervals and the resulting
emission spectra over the 430 – 650 nm range were recorded. The resulting three dimensional
plot can be seen in Figure 7.14.
It is observed that an even greater fluorescence signal is produced in the wavelength range of
investigation by irradiating at a lower wavelength than that used in this study – 326 nm, for
example. However, this does not correspond to an isosbestic point in the absorbance spectra
(Figure 7.4) and therefore the fluorescence signal produced is contributed to by the stZQA2ˉ
ligands absorbing to different extents depending on their incorporation in [Zn(stZQA)] or
[Zn(stZQA)2]2ˉ. The fluorescence spectrum produced using the λex of 336 nm does, however,
produce a functional signal over this Zn2+ concentration range.
It should also finally be noted that the constants determined using UV-Vis and fluorescence
titration methods represent excited state, rather than ground state, stability constants. They are
calculated by measuring the spectra produced by the ligand when it has absorbed light – i.e. it is
in an excited state. The ground state stability constants for stZQA with Zn2+ and the other metal
cations investigated in this study may be obtained using potentiometric titration as part of Future
Work. This would also definitively resolve the stability constants for stZQA with Zn2+.
Unfortunately access to the potentiometric titrimeter was limited at the time this particular work
was conducted.
248
Figure 7.14. Three dimensional plot of the fluorescence of stZQA (5.0 × 10-6 mol dm-3) in the
presence of Zn2+ (8.0 ×10-5 mol dm-3) in 25% v/v aqueous ethanol NaClO4 (I = 0.10 mol dm-3)
buffered at pH 6.6 (NaPIPES = 1.0 × 10-3 mol dm-3) at 298.2 K as a function of excitation and
emission wavelength.
7.4 Conclusions and Future Work

UV-Vis and fluorescence characterisation of the complexes formed by the new ligand stZQA
and several biologically significant cations suggest that it is successful Zn2+ sensor. Although
stZQA does bind to metals other than Zn2+ (for example Cd2+, Cu2+, Co2+ and Ni2+) the resulting
complexes do not produce large fluorescence signals when irradiated at 336 nm. Therefore, of the
249
other metals studied, this ligand appears to produce a signal only in the presence of Zn2+ as is
desired for a selective sensor.
The stZQA/Zn2+ system is characterised by stable 1:1 and 2:1 (L:M) complexes with logK1
and logK2 values of 10.5 and logK2 = 11.1 respectively (as determined by fluorescence titration
fitting). The relative size of these stability constants suggests that on addition of the second
stZQA ligand, the complex converts from six-coordinate to four-coordinate, possibly to reduce
the strain between the two bulky ligands. This is not disputed by the experimentally obtained
UV-Vis spectra which show that the stZQA spectrum does not change dramatically on going
from [Zn(stZQA)] to [Zn(stZQA)2]2ˉ as might be expected if the two stZQA ligands on
[Zn(stZQA)2]2ˉ were forced by octahedral complex geometry to interact with each other.
However, there is a discrepancy between the stability constants obtained from fluorescence
titration fitting versus UV-Vis titration fitting. Although the fluorimetric method was deemed
more reliable for several reasons, these constants should be confirmed by potentiometric titration
as part of Future Work.
Crystal structures of both the 1:1 and 2:1 complexes of stZQA and Zn2+ would also be helpful
in characterising this ligand. If the bulk of the added styryl functionality does indeed induce a
stereochemistry change from octahedral to tetrahedral on addition of the second ligand, this may
manifest itself in the inability of this ligand to bind to Zn2+ in physiological samples that is held
in the structures of enzymes and proteins. If this is the case, then it may be hypothesised that the
added bulk could cause the sensor to become specific for intracellular free Zn2+. This would be a
particularly valuable tool and should be examined further as part of Future Work.
Although in theory the stZQA has the characteristics to make it a successful Zn2+ sensor, its
tendency to undergo isomerism in ambient light causes it to be impractical for the intended use in
a healthcare or health research setting. The very fast equilibration to a photostationary state of
unknown proportions of E and Z isomers (which would each be characterised by different
absorbance and fluorescence spectra and different stability constants) presents a barrier towards
simple large scale use of this ligand as compared with its analogue ZQA. However, the results
obtained by adding the styryl functionality are useful and could be applied to a new derivative
with another conjugation- and bulk-increasing functional group that does not also introduce
isomers. The stZQA sensor should also undergo intracellular testing as part of Future Work to
determine whether the styryl functionality has the desired effect characteristic of binding
250
primarily to ‘free; Zn2+. Quinoline-based, fluorescent Zn2+-sensors are still being designed and
tested in the present and so the information gleaned from this study will be helpful in future
investigations.26
251
7.5 References
1. H. C. Coleman, B. L. May and S. F. Lincoln, Aust. J. Chem., 2010, 63, 1448-1452.

Zinc(II) Specific Fluorophore, University of Adelaide, Department of Chemistry, 1999.
3. K. M. Hendrickson, T. Rodopoulos, P.-A. Pittet, I. Mahadevan, S. F. Lincoln, A. D.

Ward, T. Kurucsev, P. A. Duckworth, I. J. Forbes, P. D. Zalewski and W. H. Betts, J.
Chem. Soc., Dalton Trans., 1997, 3879-3882.
4. D. N. Sathyanarayana, Electronic Absorption Spectroscopy and Related Techniques,

Universities Press Limited, Hyderabad, India, 2001.
5. H. Coleman, Honours thesis, A Zn2+ selective probe: synthesis, characterisation and

testing, University of Adelaide, Department of Chemistry, 2006.
6. R. J. P. Williams, Biological Reviews, 1953, 28, 381-412.
7. R. G. Pearson, J. Am. Chem. Soc., 1963, 85, 3533-3539.
8. J. C. Fanning and L. T. Taylor, J. Inorg. Nucl. Chem., 1965, 27, 2217-2223.
9. N. Roy, H. A. R. Pramanik, P. C. Paul and T. S. Singh, Spectrochim. Acta, Part A, 2015,

140, 150-155.
11. B. L. Vallee, Physiol. Rev., 1959, 39, 443-490.
12. A. E. Martell and R. M. Smith, Critical Stability Constants, Plenum Press, USA, 1975.
13. M. Schmid, S. Zimmermann, H. F. Krug and B. Sures, Environ. Int., 2007, 33, 385-390.
14. J. Zhao, B. A. Bertoglio, J. M. J. Devinney, K. E. Dineley and A. R. Kay, Anal. Biochem.,

2009, 384, 34-41.
15. A. W. Czarnik, Acc. Chem. Res., 1994, 27, 302-308.
16. D. G. Whitten, Acc. Chem. Res., 1980, 13, 83-90.
17. M. R. Wasielewski, Chem. Rev., 1992, 92, 435-461.

252
18. Specfit 32 for Windows, Spectrum Software Association, USA
19. H. Gampp, M. Maeder, C. J. Meyer and A. D. Zuberbuehler, Talanta, 1986, 33, 943-951.
20. A. B. Nowakowski and D. H. Petering, Inorg. Chem., 2011, 50, 10124-10133.
21. A. B. Nowakowski, PhD Thesis, Zinc chemical biology: The pursuit of the intracellular
targets of zinquin, University of Wisconsin-Milwaukee, Department of Chemistry, 2013.
22. M. P. Cuajungco and G. J. Lees, Neurobiol. Dis., 1997, 4, 137-169.
23. J.-Y. Koh, Mol. Neurobiol., 2001, 24, 99-106.
24. K. Konoha, Y. Sadakane and M. Kawahara, J. Health Sci., 2006, 52, 1-8.
25. K. M. Hendrickson, J. P. Geue, O. Wyness, S. F. Lincoln and A. D. Ward, J. Am. Chem.

Soc., 2003, 125, 3889-3895.
26. C. Gao, X. Jin, X. Yan, P. An, Y. Zhang, L. Liu, H. Tian, W. Liu, X. Yao and Y. Tang,
Sens. Actuators, B, 2013, 176, 775-781.
253
Hilary Coleman Chapter 8. Experimental
CHAPTER 8
EXPERIMENTAL
254
8.1 General
8.1.1 Measurements
1 13
H and C NMR spectra were recorded on a Varian Gemini 200 spectrometer operating at
199.953 MHz (1H) and 50.4 MHz (13C) and on a Varian Gemini ACP-300 spectrometer operating
at 300.145MHz (1H) and 75.4 MHz (13C). Compounds were dissolved in CDCl3, d6-DMSO or
D2O and peaks were referenced against tetramethylsilane in CDCl3, the residual solvent
resonance (δH = 2.49, δC = 39.5) in d6-DMSO or an external standard
(trimethylsilylpropiosulfonic acid) in D2O. All chemical shifts and coupling constants are quoted
as δ in parts per million (ppm) and hertz (Hz), respectively. The abbreviations singlet (s), doublet
(d), triplet (t), quartet (q) and multiplet (m) refer to the multiplicity of the NMR resonances.
For previously un-characterised, newly synthesised compounds, spectra were also recorded
on a Varian Inova 600 Spectrometer operating at 599.957 MHz. The βCD NMR characterisations
in this study followed the conventions and data recorded in the literature.1, 2
LC-Q mass spectrometry was performed on a Finnigan LCQ instrument. Samples were
dissolved at a concentration of 0.5 mg cm-3 in Milli-Q water, HPLC grade methanol or a mixture
of the two. Elemental analyses were carried out by the Microanalytical Service of the Department
of Chemistry, University of Otago, Dunedin, New Zealand. Samples were dried until constant
mass was attained prior to analysis. Because cyclodextrins and their derivatives have water
molecules associated with their structures, moles of H2O were added to the molecular formula to
give the best fit to the microanalytical data.
Melting points were determined using a Kofler hot-stage apparatus viewed with a Richert
microscope and are uncorrected. Cyclodextrin compounds decompose without melting above
~180°C and these points are annotated with (dec).
UV-Visible absorbance measurements were recorded on a UV-VIS-NIR Varian Cary 5000

spectrophotometer using 1.0 cm, 0.5 cm and 0.2 cm path length matched cuvettes. Fluorescence
spectra were obtained using a Varian Cary Eclipse fluorimeter using a 1.0 cm path length quartz
cuvette. In all cases the cuvettes were thermostatted prior to running at 298.2 K using the Varian
255
Cary temperature controller attached to both instruments. All sample solutions were diluted by
weight from stock solutions using Gilson biological pipettes P20, P100, P200, P1000 and P5000.
Potentiometric titrations were performed using an 800 Dosino Methrohm instrument

equipped with a 5 cm3 burette and either a 2 cm3 or 10 cm3 Orion Ross semi-micro combination
pH electrode. Data collection was performed by the program Tiamo running off a personal
computer and interfaced to the electrode through an 809 Titrando potentiometer with a precision
of ± 0.002 pH units. All titrations were thermostatted at 298.2 K in a water-jacketed titration
vessel (2 cm3 or 10 cm3) that was closed to the atmosphere. Nitrogen was bubbled through
NaClO4 solution (I = 0.10 mol dm-3) to saturate it with solvent (25% aqueous ethanol or Milli-Q
water depending on the experiment type) before it was passed through the titration solution prior
to and during the titration. The reference compartment of the combination electrode was also
filled with NaClO4 solution (I = 0.10 mol dm-3) and allowed to equilibrate over 2 days prior to
use. Each titration solution was magnetically stirred throughout the experiment. The pipettes (2
cm3 and 10 cm3) used to deliver the solutions to the titration vessels were calibrated prior to each
set of experiments by weighing water into a titration vessel to calculate a consistent average
volume delivered and the associated standard error.
8.1.2 Materials
All reagents and starting materials were obtained from Sigma Aldrich, Chem-Supply, Strem
Chemicals, Fluka or Ajax, and were used as received or purified as required using literature
procedures.3 Beta cyclodextrin (βCD) was donated by Nihon Shokuhin Kako Co. The deuterated
solvents for NMR, D2O, d6-DMSO and CDCl3, were sourced from Cambridge Isotope
Laboratories.
Bio-rex 70 resin – for cyclodextrin derivative purification and metal solution standardisation
– was purchased from BioRad Laboratories, Inc., CA and converted to the acid form using 3 mol
dm-3 hydrochloric acid. Metal perchlorate salts – Zn(ClO4)2.6H2O, Cu(ClO4)2.6H2O,
Cd(ClO4)2.6H2O, Ni(ClO4)2.6H2O and Co(ClO4)2.6H2O – were purchased from Aldrich and then
recrystallised and dried under reduced pressure then stored over P4O10 before use. (Caution:
anhydrous perchlorates pose a potential explosion hazard. Care should be taken with their
handling.)
256
Solvent systems for all UV-Vis, fluorescence and titration solutions were either Milli-Q water
(Part I) or 25% aqueous ethanol (Part II). Deionised water was ultrapurified with a Milli-Q
Reagent system to produce water with a specific resistance of > 15 MΩ cm and then boiled and
stored with a CaCl2 drying tube to remove carbon dioxide. Analytical grade ethanol was purified
by fractional distillation under nitrogen.
8.1.3 Data analysis tools

Potentiometric titrations: Hyperquad 2008
The program Hyperquad 20084 (Protonic Software5) was used to determine all pKas and
complex stability constants from the potentiometric titration data. The program iteratively fits
using the least-squares approach by means of the Gauss-Newton-Marquardt algorithm, in which
the sum of squared residuals is minimised. The weighting scheme is based on the theory of
“rigorous” least squares and results in less weighting being afforded to the data points around
where the titration slopes steeply (ie the end points). It can be used reliably to fit data over the pH
ranges in which no species in solution exceeds 95% formation. The data fits obtained in this
study are good – fits routinely produced σ values of 2 or less.6 (Sigma (σ) is a measure of overall
‘goodness of fit’ derived from the sum of squared residuals and has an expected value of 1.) The
values reported represent the average from two titrations and the inclusion of species (identical
for both of the duplicate titrations) which resulted in the best fit (after all possible combinations
of species were tested). Even for systems that had already been characterised in the literature, the
fitting in this study involved attempts to fit all possible species and all possible combinations of
these species until the best fit was achieved. For example, tacn is reported in the literature to have
three measureable pKa values and form a stable [Zn(tacn)]2+ complex. However the equally
feasible species [Zn(tacnH)]3+ and [Zn(tacn)2]2+ were also included in this study’s fitting attempts
but were eventually discarded because their presence did not yield the best fit.
Where fitted results have reliably been obtained, they are reported in this thesis along with
their associated Hyperquad-derived error that reflects the congruity between the fit and the
experimental data (as described above). The overall experimental errors for the derived pKas,
pKaH2Os and stability constants are generally greater than those quoted on the fits due to
experimental errors. These primarily include those involved in solution preparation, pipetting,
257
temperature and electrode drift but also many more that have been identified in the literature.7
These inherent sources of error were minimised by the methods outlined in Section 8.2.2 to
produce experimental error estimates typically of a maximum of ± 3%. Furthermore, to ensure
experimental errors were not excessive, experiments were performed in duplicate from freshly
prepared solutions and the reported results represent and average of the two titrations.
Because elucidating the catalytic hydrolysis mechanism is critical for planning future enzyme
mimic design, it is necessary to determine each complex geometry and structure. It is therefore
noted in the titration chapters that the lack of a second aqua ligand pKa does not discount the
presence of a second aqua ligand, only that, if present, it does not undergo a deprotonation that is
measureable over the range of the titration. (The presence of a second aqua pKa in this case has
implications for whether the complex is 5- or 6-co-ordinate.) Given it is acknowledged that
potentiometric data cannot definitively determine a complex’s geometry, all results were
compared to other titration data in the literature (where available) and other sources of structural
data (e.g. spectrophotometric and crystal structures, where available).
UV-Vis monitoring of kinetics: GraphPad Prism 6
GraphPad Prism 68 was used to determine kobs values, half-life lengths, Michaelis-Menten
constants and kcat from the UV-Vis kinetics data. Because the potential catalyst complexes were
always at a minimum of 10 times in excess of the substrate ester, the reactions followed pseudo-
first order kinetics. The non-linear regression “One Phase Exponential Decay” algorithm was
used to fit for kobs and the half-life by plotting the 4-nitrophenolate absorbance against time. The
model is:
Y = (Y0 – Plateau)(-k ×X) + Plateau 8.1
where Y is the 4-nitrophenolate absorbance (a.u.) at 400 nm (unless otherwise stated), Y0 is the
initial 4-nitrophenolate absorbance at time zero (t0), k is the observed rate constant kobs (s-1), X is
time (s) and Plateau is the maximum 4-nitrophenolate absorbance (ie the absorbance at time
infinity: Absinf).
Once kobs was determined using the procedure above, the modified Michaelis-Menten non-
linear regression algorithm is used to fit for the Michaelis-Menten Constant, KM, and the
258
maximum observed rate constant, kmax. This is achieved by plotting kobs (calculated using
Equation 8.1) against the enzyme mimic concentration and the model is:
Y = (KM×kun + kcat×X)/(KM + X) 8.2
where Y is the kobs (s-1), KM is the Michaelis-Menten constant (the dissociation constant between
the 4-nitrophenyl acetate substrate and enzyme mimic), kun (s-1) is the observed rate constant for
the uncatalysed ester hydrolysis at a given pH, kcat (s-1) is the catalytic rate constant at a given pH
and X is the potential catalyst concentration (mol dm-3).
Complex stability constant determination from UV-Vis and Fluorescence titrations: Specfit
The program Specfit9, 10

was used to calculate the complex stability constants for styryl-
functionalised Zinquin A with Zn2+ from data obtained by UV-Vis and fluorescence
spectroscopy. If all the absorbing or fluorescing species in solution are known for different
sample compositions, then the individual component concentrations may be calculated and, from
this, the complex stability constants. Equation 8.3 describes the observed absorbance when a 1:1
L:M2+ equilibrium exists in solution, where A, εL and εLM represent the total absorbance and
molar absorptivities of the ligand and 1:1 ligand-metal complex ([M(L)]), respectively. Equation
8.4 describes the observed absorbance when a second 2:1 L:M2+ equilibrium exists in solution,
where εL2M represents the molar absorptivity of the 2:1 ligand-metal complex ([M(L)2]). Note in
both systems that the metal, Zn2+, does not absorb and therefore does not contribute an individual
absorbance term to the equations.
A = εL[ligand] + εLM[M(L)] 8.3
A = εL[ligand] + εLM[M(L)] + εL2M[M(L)2] 8.4
For the UV monitored titration, K1 (the stability constant for the 1:1 L:M complex) and K2
(the stability constant for the 2:1 L:M complex) were determined by a fit to either Equation 8.3 or
8.4 over a broad wavelength range at 0.5 nm intervals. Analogous equations apply for the
fluorescence titration and data fitting.
Shortly after completion of the work conducted in this study, the Specfit software product
was discontinued by its vendor and is therefore no longer available.
259
8.2 Experimental for Part I, Chapters 2 and 3
8.2.1 Preparation of compounds

Tris(2-aminoethyl)amine (tren) and 1,4,7-triazacyclononane (tacn) were purchased from
Sigma Aldrich and used as received. Samples of tris(2-dimethylaminoethyl)amine (Me6tren)11,
1,4,7-triazacyclononane (tacn)12, 13
, 1,4,7-trimethyl-1,4,7-triazacyclononane (Me3tacn)11, 12
, 6A-
{2-[bis(2-aminoethyl)amino]ethylamino}-6A-deoxy-β-cyclodextrin (βCDtren) and 6A-(1,4,7-
Triazacyclononan-1-yl)-6A-deoxy-β-cyclodextrin (βCDtacn) were prepared and characterised
according to the literature1 along with their precursors (1,2-di(ρ-toluenesulfonyloxy)ethane,
N,N’,N”-tri(ρ-toluenesulfonyl)diethylenetriamine, 1,4,7-tris(ρ-toluenesolfonyl)diethylene-
triamine-1,7-disodium salt, 1,4,7-tris(ρ-toluenesolfonyl)-1,4,7-triazacyclononane and 6A-O-(4-
methylbenzenesulfonyl)-β-cyclodextrin).13
The functionalised cyclodextrins were freeze dried after purification and then stored in the
dark under refrigeration.
(Please see next page.)
260
6A-{2-[Bis(2-dimethylamino)ethyl]amino}-6A-deoxy--cyclodextrin: βCDMe5tren
This compound was prepared by modification and combination of both the βCD-substitution1
and N-methylating11 literature procedures. A 40% formaldehyde solution (0.90 cm3) was added
to βCDtren (0.99 g, 0.78 mmol) dissolved in H2O (7 cm3). As this solution was stirred, 90%
formic acid (0.35 cm3) was added dropwise. The reaction mixture was then stirred at 60˚C for 7
days. The resulting brown oil was concentrated under reduced pressure and then washed with
chloroform (3 x 10 cm3). The residue was dissolved in a minimum amount of H2O and added to
vigorously stirred acetone. The fine precipitate formed was collected by centrifuge and an
aqueous solution was loaded onto a Biorex cation exchange column (H+ form – 4.5 × 4.5 cm).
The column was washed with H2O (~500 cm3) and then the product was eluted with 0.5 mol dm-3
aqueous ammonia solution (~300 cm3). Water was removed from the basic fractions under
reduced pressure and the product was then re-dissolved in H2O (10 cm3) and dried under reduced
pressure (four times) to remove excess ammonia. The compound was freeze-dried to give the title
compound as a pale yellow product.
Yield: 66%
LCQ-MS: m/z 1333 (M+)
Elemental analysis: Found: C, 47.74; H, 7.40; N, 4.30. Calculation for βCDMe5tren.0.5H2O:
C, 47.42; H, 7.28; N, 4.17.
1
H NMR: δH (D2O) 5.18 (broad singlet, 7H, H1), 3.90 – 4.10 (multiplet, 26H, H3, H5, H6),
3.60 – 3.76 (multiplet, 13H, H2, H4), 3.46 (triplet, 1H, H4A), 3.10 (doublet, 2H, H6A), 2.56 –
2.74 (multiplet, 13H, H6A’, trenH), 2.40 – 2.50 (multiplet, 15H, trenMe).
Melting point: 215˚C (dec)
261
6A-(1,4,7-trimethyl-1,4,7-triazacyclononan-1-yl)-6A-deoxy-β-cyclodextrin: (βCDMe2tacn)
This compound was prepared by Dr. Duc-Truc Pham using the general procedure above for
βCDtacn.
Yield: 57%
LCQ-MS: m/z 1274 (M+)
Elemental analysis: Found: C, 42.89; H, 7.27; N, 3.00. Calculation for βCDMe2tacn.7H2O: C,
42.77; H, 7.05; N, 2.89.
1
H NMR: δH (D2O) 5.15 (broad singlet, 7H, H1) 4.8 (singlet, 7H, H1), 3.98 (triplet, 1H, H5A),
3.80 – 3.94 (multiplet, 25H, H3, H5, H6), 3.60 – 3.74 (multiplet, 13H, H2, H4), 3.46 (triplet, 1H,
H4A), 3.20 (doublet, 1H, H6A), 2.98 – 3.90 (multiplet, 8H, H6A’, tacnH1, tacnH2), 2.86 – 2.96
(tacnH3, multiplet, 4H), tacnMe (2.67, singlet, 6H). (TacnH1 refers to the Hs on the two CH2
groups adjacent to the N linked to βCD, tacnH2 refers to the Hs on the two CH2 groups next to
tacnH1 and tacnH3 refers to the Hs on the two CH2 groups opposite the linked N across the ring.)
Melting point: 202˚C (dec)
8.2.2 Procedures for potentiometric titrations (Part I)

Solution preparation and titration
A summary of the general solution preparation is presented in Table 8.1. All sample solutions
were prepared in Milli-Q water that was boiled for one hour prior to use to remove carbon
dioxide. As the boiled Milli-Q water cooled to room temperature before any solution preparation
(NaOH, KHphthalate or metal perchlorate – see Table 8.1) it was stoppered with a CaCl2(s)
262
packed drying tube to further minimise the extent of carbon dioxide dissolution. Sodium
perchlorate, NaClO4, was employed as the constant ionic strength salt (I = 0.10 mol dm-3) and
this solid was dried under reduced pressure to remove excess H2O before solution preparation.
The sodium hydroxide, NaOH, stock base solution (0.10 mol dm-3) which all samples were
titrated against, was prepared in boiled Milli-Q water and regularly standardised against
potassium hydrogen phthalate (1.0 × 10-2 mol dm-3).
The electrode was calibrated every 2-3 days during experiments by titrating the standardised
stock base solution against a perchloric acid solution (HClO4, 1.0 × 10-2 mol dm-3). The electrode
E0 and solution pKw were determined from the results of these titrations using the modified
Nernst equation:14
E = E0 + (RT/F)×ln[H+] 8.5
where E is the observed potential (volts), E0 is the standard potential of the electrode (volts), R is
the ideal gas constant (8.314 J K-1 mol-1), T is the temperature in Kelvin, F is the Faraday
constant (9.6 487 × 104 C mol-1) and [H+] is the proton concentration (mol dm-3). The standard
potential, E0, of any electrode is not constant over time and therefore needs to be re-determined
on a regular basis. At 298.2 K the pH of a solution is given by:
pH = (E0 – E)/59.15 8.6
Calibration data were analysed by standard computational treatment provided within the
program MacCalib15 to ascertain the values of E0 and pKw. These are essential for all further data
fitting because the standard potential of the electrode will drift and must be determined regularly
in order to minimise potential fitting errors. The diffusion correction terms used were E0 = 2.53
and pKw = 1.116. The initial electrode calibration titrations of the standardised stock base
solution against perchloric acid solution also allowed for exact calculation of the HClO4
concentration. This is also required for all further data fitting because this HClO4 solution was
used to prepare the ligand and ligand/metal solutions for titration, as shown in Table 8.1.
The pKas of the amines were determined by duplicate titration of 10 cm3 or 2 cm3 volumes of
the ligand in HClO4 solution against the 0.10 mol dm-3 standardised NaOH stock solution (under
N2 atmosphere and magnetically stirred at 298.2 K). Constant temperature was maintained by
constantly circulating thermostatted water around the titration vessel for the duration of the
263
experiment. In all of these titrations the concentrations of the ligand and HClO4 were
approximately [ligand]total = 1.0 × 10-3 mol dm-3 and [HClO4]total = 1.0 × 10-2 mol dm-3. Data
fitting for these experiments requires precisely known values for the total number of moles of
ligand and acid as well as the base concentration. Therefore, for the purposes of data analysis, the
exact concentrations in each ligand/acid (L/H+) solution were determined precisely (by weighing
out of the component solutions used) and the stock base was regularly standardised with
potassium hydrogen phthalate. During these titrations the burette was automated to wait a
maximum of 300 s before the next titrant addition if the electrode did not stabilise within 0.3 mV
within this time. (This would only occur if equilibrium was not re-established after the previous
titrant addition.)
Metal perchlorate aqueous stock solutions – Zn(ClO4)2, Cu(ClO4)2, Ni(ClO4)2 and Cd(ClO4)2
– were prepared by first drying the solid salts and then making up standard solutions in boiled
Milli-Q water: 25 cm3, 0.10 mol dm-3. Each solution was then standardised in duplicate using
cation exchange chromatography. A Dowex AG 50W-X2 column was prepared by washing with
three column bed heights of HCl (0.1 mol dm-3) to achieve full protonation, and then rinsing with
Milli-Q water until the solvent running through was no longer acidic. The column was then
loaded with 1.0 cm3 of an approximately 0.1 mol dm-3 solution of the metal perchlorate to be
standardised and eluted with water until the eluent was neutral. The collected acidic eluent
solution was finally titrated against NaOH (0.10 mol dm-3) which had already been standardised
using potassium hydrogen phthalate.
The stability constants for the complexation of each metal ion with each ligand were
determined by duplicate titration of the stock standardised base NaOH (0.10 mol dm-3) with a
solution of ligand/acid/metal (L/H+/M2+) at ratios of L/M2+ of 1:1 and 1:1.5 (under N2 atmosphere
and magnetically stirred at 298.2 K). These solutions were made up in acid, [HClO4]total = 1.0 ×
10-2 and for the 1:1 L/M2+ ratio the concentrations of L and M2+ were approximately [ligand]total =
1.0 × 10-3 mol dm-3 and [M2+]total = 1.0 × 10-3 mol dm-3. For the 1:1.5 L/M2+ ratio the
concentrations of L and M2+ were approximately [ligand]total = 1.0 × 10-3 mol dm-3 and [M2+]total =
1.5 × 10-3 mol dm-3. For the purposes of data analysis, the exact concentrations in each L/H+/M
solution were determined precisely (by weighing out of the stock component solutions used) and
the stock base was regularly standardised with potassium hydrogen phthalate. Similarly, during
these titrations the burette was automated to wait a maximum of 300s before the next titrant
addition if the electrode did not stabilise within 0.3 mV within this time.
264
Ligand solution Metal complex solution
[L] ≈ 1.0 × 10-3 mol dm-3 [M2+] ≈ 1.0 × 10-3 mol dm-
Constant ionic strength Acid solution 3
(1:1 L/M2+ experiments)
I = 0.1 mol dm-3 [H+] ≈ 1.0 × 10-2 mol dm-3 Investigated ligands or
≈ 1.5 × 10-3 mol dm-3
NaClO4 HClO4 0.25 dm3 (10 cm3 (1:1.5 L/M2+ experiments)
electrode)
1.0 dm3 1.0 dm3 or 0.025 dm3 (for 10 cm3
0.025dm3 (2 cm3 electrode)
Made in boiled Milli-Q *Calibrated every 2-3 days electrode) or
water. against stock NaOH base 0.005 dm3 (for 2 cm3
solution. electrode)
[NaOH] ≈ 0.10 mol dm-3 [KHphthalate] = 1 × 10-2 mol dm-3 [Metal perchlorate] ≈ 0.1 mol dm-3
Standardised stock base 0.1 dm3 Zn(ClO4)2, Cu(ClO4)2, Ni(ClO4)2 and

Cu(ClO4)2.
1 dm3 Made in boiled Milli-Q water.
0.025 dm3
Made in boiled Milli-Q water. *Used to standardise the stock base
regularly. Made in boiled Milli-Q water.
*Standardised regularly against *Standardised using cation exchange
potassium hydrogen phthalate. chromatography.
Table 8.1. Summary of solution preparation for the potentiometric titrations described in
Chapters 2 and 3.
265
Table 8.1 shows a summary of the solutions required for potentiometric titrations, their
concentrations and how they were prepared. During all titrations, a maximum time of 300 s wait
was permitted between each titrant addition to allow equilibrium to be established. As previously
stated, ligand pKas, metal complex stability constants (logK) and metal complex associated water
ligand pKas (pKaH2O) were determined by fitting the titration data using the program Hyperquad
20084 (Protonic Software5). Speciation plots were produced using associated software HySS
200916 (Protonic Software5).
Prior to each set of titrations for each ligand, the required pipette (2 cm3 or 10 cm3) was
calibrated by weighing aliquots of Milli-Q water into a clean, dry titration vessel. This weighing
was conducted a minimum of 20 times with consistent technique such that the exact aliquot
volume and associated error were known. (This is required for accurate data fitting.) The pipettes
and volumetric glassware used were all Class A and were washed in a de-metalating detergent
followed by rinsing with Milli-Q water before air drying upside down prior to re-use. The
titration vessels were washed in the same detergent and rinsed with Milli-Q water before oven
drying.
8.3 Experimental procedures for Part I, Chapter 4
8.3.1 Preparation for UV-Vis kinetics studies (Part I)

Each kinetics experiment involved the reaction of the ester, 4-nitrophenyl acetate, with a
potential catalyst in aqueous solution and buffered at the pH equal to the pKaH2O of that potential
catalyst. The potential catalyst species included the βCD, Cu2+(aq), Zn2+(aq), tren, Me6tren,
βCDtren and βCDMe5tren as well as the complexes [Cu(βCDtren)]2+ and [Zn(βCDtren)]2+. As
the ester was hydrolysed, evolution of the product, 4-nitrophenolate, was monitored over time at
400nm. Neither [Zn(H2O)6]2+ nor [Cu(H2O)6]2+ nor βCD absorb over the wavelength range
examined. However, as discussed in Section 4.3 the presence of the βCD-functionalised ligand
and a metal cation does produce a marked absorbance that is even further enhanced in the
presence of the ester. Therefore, to maintain the absorbance readings in a reliable range, cells of
three different path lengths were used: 1.0 cm, 0.5 cm and 0.2 cm. In order for comparison, all
absorbance values are quoted as standardised absorbances, i.e. for path length = 1.0 cm.
266
Buffer solutions
The data analysis from potentiometric titrations in Chapters 2 and 3 yielded pKas for the
water ligands bound to the complexes of interest. From the titration experiments, complexes of
the amines and amine-substituted β-cyclodextrins with Zn2+ and Cu2+ were selected for the
kinetics investigation. The original aim was to design and test simple ligands to mimic enzymes
in the body, and zinc and copper are the most physiologically significant of the four transition
metals used in the titrations. Consequently, each UV-Vis experiment was run at the buffered pH
of the pKa of the associated water ligand for the potential catalyst complex in question. Rather
than conducting all experiments at a uniform pH, this ensured that across all experiments, the
concentration of the hydroxo ligand (shown to be responsible for ester hydrolysis) remained
constant. A comparison of the rates of hydrolysis therefore became a true appraisal of the
catalytic activity of each species, rather than being influenced by the varying percentages of the
aqua ligands that were actually present in the catalytic hydroxo form.
Each buffer was made up as an aqueous solution (1.0 × 10-2 mol dm-3) in boiled Milli-Q water
at constant ionic strength, I = 0.10 mol dm-3 (NaClO4). Several buffers were required in order to
cover the broad range of pKaH2Os measured, and furthermore, the buffer species selected had to
meet several critical criteria. They could not bind the metal cations present, Na+, Zn2+ or Cu2+,
they could not include in the annulus of βCD, nor could they precipitate in aqueous solution at a
concentration of 1.0 × 10-2 mol dm-3 (whether from insolubility or precipitation when exposed to
the metals present). The combination of these criteria rendered many common buffers unusable.
Finally, buffers MES, PIPES, MOPS, DEPP and TEEN17 were selected to cover the pH range
from ~5.5 – 10.0 (above which, precipitation of metal hydroxides becomes an issue). MES,
PIPES, MOPS and TEEN were purchased and DEPP was synthesised according to the
literature.17
Each buffer solution was allowed to equilibrate overnight and the pH re-tested prior to use in
further solution preparation.
267
Reactant solutions – Potential catalysts and 4-nitrophenyl acetate ester
The rate of hydrolysis of 4-nitrophenyl acetate was measured for increasing concentrations of
each potential catalyst. This was achieved by monitoring the production of the 4-nitrophenolate
product which produces a characteristic absorbance peak at 405 nm.
For each new day of experiments, a new 4-nitrophenyl acetate ester stock solution was
prepared. These stocks were prepared just prior to use and refrigerated in between runs when
multiple experiments were begun over a 12 hour period. This ensured no observable hydrolysis
occurred in the stock before it was used in the kinetics runs. Each ester stock solution was of 10
cm3 volume with a 4-nitrophenyl acetate concentration of 6.07 × 10-4 mol dm3, made to the mark
with aqueous NaClO4 (I = 0.10 mol dm3) in boiled Milli-Q water and 2.5% acetonitrile for
solubility. (Note – not buffered.) Ester stocks were prepared in this manner so that the final in-
cell concentration of the ester in each experiment (once it had been combined with the potential
catalyst in the UV-Vis cell) was a constant of 4.0 × 10-5 mol dm-3. This in-cell concentration was
selected because it yielded a convenient absorbance for the 4-nitrophenolate that is produced. All
of the subsequent potential catalyst concentrations were then contingent on this ester
concentration, as discussed in Chapter 4.
Reactant solutions of the potential catalyst species were prepared in 1 or 2 cm3 volumetric
flasks and made up to the mark with the pre-prepared buffer solution for the pH specific to each
potential catalyst. Whilst the buffer stock solutions were allowed to equilibrate overnight, the
potential catalyst solutions for reaction were made on the same day as the UV-Vis experiments
were carried out. The concentrations of these reactant complex solutions were prepared such that
the final in-cell concentrations of the species (once they had been combined with the ester stock
in the UV-Vis cell) were 4 × 10-4 mol dm-3, 1 × 10-3 mol dm-3, 3 × 10-3 mol dm3, 4 × 10-3 mol dm-
3
, 6 × 10-3 mol dm-3, 8 × 10-3 mol dm-3, 1 × 10-2 mol dm-3, 1.2 × 10-2 mol dm-3 and 1.5 × 10-2 mol
dm-3 unless otherwise stated.
268
Vstock
added [Final] in Combined
Cell [Stock] to cell cell (mol Vfinal in
type Species (mol dm-3) (mL) dm-3) cell (mL)
potential
0.2 cm 1.07 × 10-3 0.56 1.0 × 10-2 0.60
catalyst
ester 6.07 × 10-4 0.04 4.0 × 10-5
potential
0.5 cm 1.07 × 10-3 1.26 1.0 × 10-3 1.35
catalyst
ester 6.07 × 10-4 0.09 4.0 × 10-5
potential
1.0 cm 4.3 × 10-4 1.40 4.0 × 10-4 1.50
catalyst
ester 6.07 × 10-4 0.10 4.0 × 10-5
Table 8.2. Summary of stock solution concentrations, initial volumes and final concentrations of
the reactant species present in the 0.2, 0.5 and 1.0 cm cells for final potential catalyst
concentrations of 4 × 10-4 mol dm-3, 1 × 10-3 mol dm-3 and 1 × 10-2 mol dm-3.
These reactant solution specifications meant that the potential catalyst was in between 10-
times and 375-times excess of the ester in every experiment. At a minimum, the potential catalyst
was in 10-times excess of the ester in order to ensure that catalyst poisoning did not occur. For
example, Table 8.2 summarises the stock solution concentrations, initial volumes and final
concentrations of the reactant species present in the 0.2, 0.5 and 1.0 cm UV-Vis cells for final
potential catalyst concentrations of 4 × 10-4 mol dm-3, 1 × 10-3 mol dm-3 and 1 × 10-2 mol dm-3.
UV-Vis experimental procedure
Measurements were made on the UV-VIS-NIR Varian Cary 5000 spectrophotometer using
the Scanning Kinetics software. Depending on the rate of hydrolysis, the wavelength range
monitored was 395 – 400 nm (for the fastest reactions) to 225 – 500 nm (for the slowest
reactions). Matched quartz 1.0 cm, 0.5 cm and 0.2 cm path length cells were used with a scan rate
of 1200 nm/min and a 1.0 nm interval. All experiments were thermostatted at 298.2 K.
For the faster reactions – [potential catalyst] ≥ 250 × [ester], for which the half-life is
generally less than three minutes – only one experiment for one system was done at a time. The
reactant solutions were combined in the quartz cell and inverted once and simultaneously the
kinetics measurements were started so as to ensure the t0 scan was recorded correctly. For slower
269
reactions, multiple experiments for multiple systems were done at once whilst still ensuring the
reactant solution combination was synced with the initial t0 scan.
All reactions were monitored continuously for a minimum of the first 2.5 half-lives. For some
very slow reactions, the cell containing the reacting mixture was subsequently thermostatted at
323.2 K until reaction was complete. Another single scan was then taken to record the absorbance
at tinfinity (ie Ainfinity).
Because the activity of these potential catalysts under these conditions had not been tested
previously, the initial UV-Vis kinetics setup was determined qualitatively as the length of
experiment and frequency of scans required was initially unknown. Therefore, for each system a
mid-range catalyst concentration was tested individually first – for example [potential catalyst] =
1 × 10-3 mol dm-3. An optimised experimental setup could then be determined for each
catalyst/ester concentration ratio.
The data from each experiment was then manipulated using Excel to obtain the absorbance
variation at 405 nm with time. This data was subsequently fit using GraphPad Prism to determine
the rate and Michaelis constants for each potential catalyst species.
8.4 Experimental for Part II, Chapter 7 – stZinquin

All reagents and starting materials were obtained from Sigma Aldrich, Chem-Supply, Strem
Chemicals, Fluka or Ajax, and were used as received. Styryl-functionalised Zinquin A - 2-((E)-2-
phenyl)ethenyl-8-(N-4-methylbenzenesulfonyl)aminoquinol-6-yloxyacetic acid (stZQA) - was
provided by Dr. Bruce May and prepared according to the literature.18
8.4.1 Procedures for potentiometric titrations (Part II)

The potentiometric titration setup and solution preparation for this project was identical to
that for the experiments conducted in Chapters 3 and 4, except that the solvent system was 25%
v/v aqueous ethanol for this project in order to mimic cellular fluid. See Section 8.2.2 for the
determination of ligand pKas. Because only the ligand pKas were determined for stZQA and not
complex stability constants, only a solution of acidified stZQA was required for titration. This
270
solution ([HClO4] = 9.2 × 10-3 mol dm-3 [stZQA] = 1.0 × 10-3 mol dm-3) was titrated against the
conventional base solution ([NaOH] = 0.10 mol dm-3) which was again standardised against
potassium hydrogen phthalate solution in order to ensure a precisely known concentration.
8.4.2 UV-Visible studies (Part II)

All solutions for UV-Vis analysis were prepared in a buffer stock (I = 0.1 mol dm-3, NaClO4)
with a pH of 6.6 (NaPIPES at 1.0 × 10-3 mol dm-3) in 25% v/v aqueous ethanol. The spectral
range investigated in every case was 250 – 500 nm with a scan rate of 600 nm/min and SBW of 1
nm and using a pair of matched 1.0 cm quartz cuvettes. Similar studies have been reported in the
literature,19-24 however with little detail about the exact experimental procedure. The methods
reported here were developed for this study.
UV-Vis titration to determine stability constants for stZQA in the presence of Zn2+
For the UV-Vis titration these individual solutions contained a constant concentration of
stZQA (1.82 × 10-5 mol dm-3) and increasing Zn2+ concentrations from 0 to 1.72 × 10-4 mol dm-3.
As discussed in Section 7.3.1, all of the solutions contained a Zn2+ impurity which was quantified
as 3.9 × 10-7 mol dm-3. This amount of adventitious Zn2+ was included in the experimental data
fitting. For example, when zero Zn2+ stock solution was added, 3.9 × 10-7 mol dm-3 of Zn2+(aq)
was actually present. Consequently, the spectrum for the free stZQA ligand in the absence of
Zn2+ was obtained by adding the Zn2+ chelator EDTA to the solution ([EDTA] = 1 × 10-4 mol dm-
3
).
All spectra were obtained by a single scan for each solution over the range 250 – 500 nm and
these were used to identify isosbestic points at which more dilute solutions might be irradiated
for fluorescence investigations.
UV-Vis investigation of the photoisomerism of stZQA
To investigate the photoisomerism of the stZQA, a sample of the ligand (1.82 × 10-5 mol dm-3
in the standard PIPES buffer stock at pH 6.6) in 100% E form was monitored over time after
271
exposure to ambient light. Individual scans were run over the range 250 – 500 nm at 1 minute
intervals and in between runs the cell was left on the bench in the light.
UV-Vis investigation of the binding of stZQA to metal cations other than Zn2+
The testing of the binding of metal cations other than Zn2+ to stZQA was assessed by
preparing samples of stZQA (1.76 × 10-5 mol dm-3) and M2+ (3.63 × 10-4 mol dm-3) in the
conventional buffer stock at pH 6.6. For M2+ = Ca2+, Cd2+, Cu2+, Co2+ and Ni2+,single scan
spectra of these solutions were obtained over the range 250 – 500 nm.
8.4.3 Fluorescence studies (Part II)

Determination of adventitious Zn2+
The adventitious Zn2+ concentration was determined for a bulk buffer solution (subsequently
used for solution preparation for both fluorescence and UV-Vis titrations) by an EDTA titration.
This titration involved measuring the fluorescence (at the λmax ~488 nm) of samples containing 5
× 10-6 mol dm-3 stZQA and increasing concentrations of EDTA (0 – 1 × 10-4 mol dm-3). There is
a linear relationship between the amount of fluorescence produced by the complex formed
between stZQA and Zn2+Adventitious and the concentration of EDTA that preferentially binds this
Zn2+ impurity. Therefore a plot of [EDTA] against fluorescence yields an x-intercept equivalent
to the adventitious Zn2+ concentration in the solvent system tested. Once the adventitious Zn2+
concentration was determined it could be incorporated into the rest of the fluorescence
investigations.
Selectivity of stZQA for Zn2+
The selectively of the stZQA ligand for Zn2+ was then tested. Solutions of stZQA (1.76 × 10-6
mol dm-3) and M2+ (3.63 × 10-3 mol dm-3 – M2+ is Ca2+, Cd2+, Cu2+, Co2+ and Ni2+) prepared in
the conventional buffer system were irradiated at 336 nm and the resulting spectrum was
measured from 430 to 650 nm.
272
Fluorescence titration to determine stability constants for stZQA in the presence of Zn2+
Finally, a fluorescence titration was conducted, similar to the UV-Vis experiments. Individual
solutions containing a constant concentration of stZQA (5.56 × 10-6 mol dm-3) and increasing
Zn2+ (0 – 5 × 10-5 mol dm-3) were irradiated at 336 nm and the resulting fluorescence spectra
were measured over the range 430 – 650 nm (scan rate 600 nm/min, SBW 1 nm). This
concentration range for Zn2+ was employed as it was broad enough to cause saturation – enough
Zn2+ was added such that all he stZQA was complexed and a maximum fluorescence was
achieved.
The fluorescence variation data were fit at 0.5 nm intervals over the range 430–650 nm using
Specfit to determine both 1:1 L:M and 2:1 L:M metal complex stability constants (as described in
Section 7.3.3).
Investigation of fluorescence spectrum variation with excitation wavelength
Finally, a three-dimensional investigation of the variation of fluorescence spectrum for the

stZQA/Zn2+ system with varying excitation wavelength was undertaken. A solution prepared in
the conventional buffer system at pH 6.6 consisted of stZQA (5.0 × 10-6 mol dm-3) and Zn2+ (8.0
× 10-5 mol dm-3). It was irradiated at 1 nm intervals over the range 326 – 416 nm and at each
excitation wavelength the resulting fluorescence spectrum from 430 – 650 nm was recorded.
273
8.5 References
1997, 3157-3160.
2. H.-J. Schneider, F. Hacket, V. Ruediger and H. Ikeda, Chem. Rev., 1998, 98, 1755-1785.
3. W. L. F. Armarego and C. Chai, Purification of Laboratory Chemicals, 5th Edition,

Butterworth-Heinemann, 2003.
5. P. Gans, Protonic Software, 2 Templegate Avenue, Leeds LS15 0HD, UK
6. M. Meloun, Z. Ferencikova, H. Malkova and T. Pekarek, Cent. Eur. J. Chem., 2012, 10,
338-353.
7. E. Koort, K. Herodes, V. Pihl and I. Leito, Anal. Bioanal. Chem., 2004, 379, 720-729.
8. GraphPad Prism, Version 5.04 for Windows, GraphPad Software, San Diego California
USA, www.graphpad.com, 2010
9. Specfit/32 software for Windows, Spectrum Software Association, USA.
10. H. Gampp, M. Maeder, C. J. Meyer and A. D. Zuberbuehler, Talanta, 1986, 33, 943-951.
11. E. K. Barefield and F. Wagner, Inorg. Chem., 1973, 12, 2435-2439.
12. G. H. Searle and R. J. Geue, Aust. J. Chem., 1984, 37, 959-970.
13. S. Plush, PhD Thesis, Interactions of Proximate Amino Acid Residues in Polyaza
Macrocycles, University of Adelaide, Australia, 2004.
14. W. Nernst, Zeit. Physikal. Chem., 1889, 4, 129-181.
15. P. C. Duckworth & J. Cawthray, Personal Communication, 2007
16. L. Alderighi, P. Gans, A. Ienco, D. Peters, A. Sabatini and A. Vacca, Coord. Chem. Rev.,
1999, 184, 311-318.
274
Zinc(II) Specific Fluorophore, University of Adelaide, 1999.
46, 4336-4356.
20. M. M. Ibrahim and G. A. M. Mersal, J. Inorg. Organomet. Polym. Mater., 2009, 19, 549-
557.
J., 2009, 4, 1354-1360.
2011, 335, 222-227.
23. D. Desbouis, I. P. Troitsky, M. J. Belousoff, L. Spiccia and B. Graham, Coord. Chem.

Rev., 2012, 256, 897-937.
Chem., 2015, 396, 346-352.
275
Hilary Coleman Appendix A: Supporting figures for Chapter 2
APPENDIX A
Supporting figures for Chapter 2
276
Speciation plots for the tren- and tacn-based systems characterised by the pKa,
logK, logK’ and pKaH2O values in Tables 2.1 – 2.7.
These values were derived from the average of two titration experiments. For the speciation
simulations a total ligand concentration of 1.0 × 10-3 mol dm-3 and a total M2+ concentration of
1.0 × 10-3 mol dm-3 (for the titrations containing Zn2+, Cu2+, Ni2+ and Cd2+) were used. The
species shown in the keys denote the species that were included in the data analysis in order to
obtain each fit.
100
90
80 free L
70
LH⁺
60
50 LH₂²⁺
40
30 LH₃³⁺
20
LH₄⁴⁺
10
0
3 5 7 9 11
pH
Figure A1. Speciation plot showing the variation of the protonation of tren with pH, relative to the total
concentration of all tren and its protonated species being equivalent to 100%: trenH44+ (LH4 4+), trenH33+ (LH3 3+),
trenH22+ (LH2 2+), trenH+ (LH+) and tren (free L).
277
100
90

80 free L
70
LH⁺
60
50 LH₂²⁺
40
30 LH₃³⁺
20
LH₄⁴⁺
10
0
3 5 7 9 11
pH
Figure A2. Speciation plot showing the variation of the protonation of Me 6tren with pH, relative to the total
concentration of all tren and its protonated species being equivalent to 100%: Me6trenH44+ (LH4 4+), Me6trenH33+
(LH3 3+), Me6trenH22+ (LH2 2+), Me6 trenH+ (LH+) and Me6tren (free L).
100
90
free L
80
70 LH⁺
60 LH₂²⁺
50 LH₃³⁺
40 LH₄⁴⁺
30 [MLH]³⁺
20
[ML]²⁺
10
[M(L)(OH)]⁺
0
3 5 7 9 11
pH
Figure A3. Speciation plot showing the variation of the protonation and complexation of tren with pH in the
presence of Zn2+, relative to the total concentration of all tren and its protonated and complexed species being
equivalent to 100%: trenH44+ (LH4 4+
), trenH33+ (LH3 3+
), trenH22+ (LH2 2+
), trenH+ (LH+), tren (free L),
[Zn(trenH)] 3+([MLH] 3+), [Zn(tren)] 2+ ([ML] 2+) and [Zn(tren)(OH)] + ([M(L)(OH)] +).
278
100
90
free L

80
LH⁺
70
LH₂²⁺
60
LH₃³⁺
50
LH₄⁴⁺
40
[MLH]³⁺
30
[ML]²⁺
20
[M(L)(OH)]⁺
10
[M(L)(OH)₂]
0
3 5 7 9 11
pH
presence of Cu2+, relative to the total concentration of all tren and its protonated and complexed species being
), trenH33+ (LH3 3+
), trenH22+ (LH2 2+
[Cu(trenH)] 3+ ([MLH] 3+), [Cu(tren)] 2+ ([ML] 2+), [Cu(tren)(OH)] + ([M(L)(OH)] +) and [Cu(tren)(OH)2]
([M(L)(OH)2]).
100
90
free L
80
70 LH⁺
60 LH₂²⁺
50 LH₃³⁺
40 LH₄⁴⁺
30 [MLH]³⁺
20
[ML]²⁺
10
[M(L)(OH)]⁺
0
3 5 7 9 11
pH
presence of Ni2+, relative to the total concentration of all tren and its protonated and complexed species being
), trenH33+ (LH3 3+
), trenH22+ (LH2 2+
[Ni(trenH)] 3+ ([MLH] 3+), [Ni(tren)] 2+ ([ML] 2+) and [Ni(tren)(OH)] + ([M(L)(OH)] +).
279
100
90
free L

80
70 LH⁺
60 LH₂²⁺
50 LH₃³⁺
40 LH₄⁴⁺
30 [MLH]³⁺
20
[ML]²⁺
10
[M(L)(OH)]⁺
0
3 5 7 9 11
pH
presence of Cd2+, relative to the total concentration of all tren and its protonated and complexed species being
), trenH33+ (LH3 3+
), trenH22+ (LH2 2+
[Cd(trenH)] 3+ ([MLH] 3+), [Cd(tren)] 2+ ([ML] 2+) and [Cd(tren)(OH)] + ([M(L)(OH)] +).
100
90
free L
80
70 LH⁺
60 LH₂²⁺
50 LH₃³⁺
40 LH₄⁴⁺
30 [MLH]³⁺
20
[ML]²⁺
10
[M(L)(OH)]⁺
0
3 5 7 9 11
pH
Figure A7. Speciation plot showing the variation of the protonation and complexation of Me6tren with pH in the
presence of Zn2+, relative to the total concentration of all Me6tren and its protonated and complexed species being
equivalent to 100%: Me6trenH44+ (LH4 4+), Me6trenH33+ (LH3 3+), Me6trenH22+ (LH2 2+),Me6trenH+ (LH+), Me6tren
(free L), [Zn(Me6trenH)] 3+([MLH] 3+), [Zn(Me6tren)] 2+ ([ML] 2+) and [Zn(Me6tren)(OH)] + ([M(L)(OH)] +).
280
100
90
free L

80
70 LH⁺
60 LH₂²⁺
50 LH₃³⁺
40 LH₄⁴⁺
30 [MLH]³⁺
20
[ML]²⁺
10
[M(L)(OH)]⁺
0
3 5 7 9 11
pH
Figure A8. Speciation plot showing the variation of the protonation and complexation of Me6tren with pH in the
presence of Cu2+, relative to the total concentration of all Me6tren and its protonated and complexed species being
(free L), [Cu(Me6trenH)] 3+ ([MLH] 3+), [Cu(Me6tren)] 2+ ([ML] 2+), and [Cu(Me6tren)(OH)] + ([M(L)(OH)] +).
100
90
free L
80
70 LH⁺
60 LH₂²⁺
50 LH₃³⁺
40 LH₄⁴⁺
30 [MLH]³⁺
20
[ML]²⁺
10
[M(L)(OH)]⁺
0
3 5 7 9 11
pH
Figure A9. Speciation plot showing the variation of the protonation and complexation of Me 6tren with pH in the
presence of Ni2+, relative to the total concentration of all Me6tren and its protonated and complexed species being
(free L), [Ni(Me6trenH)] 3+ ([MLH] 3+), [Ni(Me6tren)] 2+ ([ML] 2+) and [Ni(Me6tren)(OH)] + ([M(L)(OH)] +).
281
100
90

80 free L
70 LH⁺
60
LH₂²⁺
50
LH₃³⁺
40
30 LH₄⁴⁺
20 [ML]²⁺
10
[M(L)(OH)]⁺
0
3 5 7 9 11
pH
Figure A10. Speciation plot showing the variation of the protonation and complexation of Me 6tren with pH in the
presence of Cd2+, relative to the total concentration of all Me6tren and its protonated and complexed species being
(free L), [Cd(Me6trenH)] 3+ ([MLH] 3+), [Cd(Me6tren)] 2+ ([ML] 2+) and [Cd(Me6tren)(OH)] + ([M(L)(OH)] +).
100
90
80 free L
70
60 LH⁺
50
40 LH₂²⁺
30
20 LH₃³⁺
10
0
3 5 7 9 11
pH
Figure A11. Speciation plot showing the variation of the protonation of tacn with pH, relative to the total
concentration of all tacn and its protonated species being equivalent to 100%: tacnH 33+ (LH3 3+), tacnH22+ (LH2 2+),
tacnH+ (LH+) and tacn (free L).
282
100
90

80 free L
70
60 LH⁺
50
40 LH₂²⁺
30
20 LH₃³⁺
10
0
3 5 7 9 11
pH
Figure A12. Speciation plot showing the variation of the protonation of Me 3tacn with pH, relative to the total
concentration of all tacn and its protonated species being equivalent to 100%: Me 3tacnH33+ (LH3 3+), Me3tacnH22+
(LH2 2+), Me3tacnH+ (LH+) and Me3tacn (free L).
100
90
80 free L
70 LH⁺
60
LH₂²⁺
50
LH₃³⁺
40
30 [ML]²⁺
20 [M(L)(OH)]⁺
10
[M(L)(OH)₂]
0
3 5 7 9 11
pH
Figure A13. Speciation plot showing the variation of the protonation and complexation of tacn with pH in the
presence of Zn2+, relative to the total concentration of all tacn and its protonated and complexed species being
equivalent to 100%: tacnH33+ (LH3 3+
), tacnH22+ (LH2 2+
), tacnH+ (LH+), tacn (free L), [Zn(tacn)] 2+ ([ML] 2+),
[Zn(tacn)(OH)] + ([M(L)(OH)] +) and [Zn(tacn)(OH)2] ([M(L)(OH)2]).
283
100
90

80 free L
70 LH⁺
60
LH₂²⁺
50
LH₃³⁺
40
30 [ML]²⁺
20 [M(L)(OH)]⁺
10
[M(L)(OH)₂]
0
3 5 7 9 11
pH
presence of Cu2+, relative to the total concentration of all tacn and its protonated and complexed species being
equivalent to 100%: tacnH33+ (LH3 3+
), tacnH22+ (LH2 2+
), tacnH+ (LH+), tacn (free L), [Cu(tacn)] 2+ ([ML] 2+),
[Cu(tacn)(OH)] + ([M(L)(OH)] +) and [Cu(tacn)(OH)2] ([M(L)(OH)2]).
100
90
80 free L
70
LH⁺
60
50 LH₂²⁺
40
LH₃³⁺
30
20 [ML]²⁺
10 [M(L)(OH)]⁺
0
3 5 7 9 11
pH
presence of Ni2+, relative to the total concentration of all tacn and its protonated and complexed species being
equivalent to 100%: tacnH33+ (LH3 3+), tacnH22+ (LH2 2+), tacnH+ (LH+), tacn (free L), [Ni(tacn)] 2+ ([ML] 2+), and
[Ni(tacn)(OH)] + ([M(L)(OH)]+).
284
100
90

80 free L
70
LH⁺
60
50 LH₂²⁺
40
LH₃³⁺
30
20 [ML]²⁺
10 [M(L)(OH)]⁺
0
3 5 7 9 11
pH
presence of Cd2+, relative to the total concentration of all tacn and its protonated and complexed species being
equivalent to 100%: tacnH33+ (LH3 3+), tacnH22+ (LH2 2+), tacnH+ (LH+), tacn (free L), [Cd(tacn)] 2+ ([ML] 2+), and
[Cd(tacn)(OH)] + ([M(L)(OH)] +).
100
90
80 free L
70 LH⁺
60
LH₂²⁺
50
LH₃³⁺
40
30 [ML]²⁺
20 [M(L)(OH)]⁺
10
[M(L)(OH)₂]
0
3 5 7 9 11
pH
Figure A17. Speciation plot showing the variation of the protonation and complexation of Me 3tacn with pH in the
presence of Zn2+, relative to the total concentration of all Me3tacn and its protonated and complexed species being
equivalent to 100%: Me3tacnH33+ (LH3 3+
), Me3tacnH22+ (LH2 2+
), Me3tacnH+ (LH+), Me3tacn (free L),
[Zn(Me3tacn)] 2+ ([ML] 2+), [Zn(Me3tacn)(OH)] + ([M(L)(OH)] +) and [Zn(Me3tacn)(OH)2] ([M(L)(OH)2]).
285
100
90

80 free L
70 LH⁺
60
LH₂²⁺
50
LH₃³⁺
40
30 [ML]²⁺
20 [M(L)(OH)]⁺
10
[M(L)(OH)₂]
0
3 5 7 9 11
pH
presence of Cu2+, relative to the total concentration of all Me 3tacn and its protonated and complexed species being
), Me3tacnH+ (LH+) and Me3tacn (free L),
[Cu(Me3tacn)] 2+ ([ML] 2+), [Cu(Me3tacn)(OH)] + ([M(L)(OH)] +) and [Cu(Me3tacn)(OH)2] ([M(L)(OH)2]).
100
90
80 free L
70 LH⁺
60
LH₂²⁺
50
LH₃³⁺
40
30 [ML]²⁺
20 [M(L)(OH)]⁺
10
[M(L)(OH)₂]
0
3 5 7 9 11
pH
presence of Ni2+, relative to the total concentration of all Me3tacn and its protonated and complexed species being
), Me3tacnH+ (LH+), Me3tacn (free L),
[Ni(Me3tacn)] 2+ ([ML] 2+), [Ni(Me3tacn)(OH)] + ([M(L)(OH)] +) and [Ni(Me3tacn)(OH)2] ([M(L)(OH)2]).
286
100
90

80 free L
70 LH⁺
60
LH₂²⁺
50
LH₃³⁺
40
30 [ML]²⁺
20 [M(L)(OH)]⁺
10
[M(L)(OH)₂]
0
3 5 7 9 11
pH
presence of Cd2+, relative to the total concentration of all Me 3tacn and its protonated and complexed species being
), Me3tacnH+ (LH+) and Me3tacn (free L),
[Cd(Me3tacn)] 2+ ([ML] 2+), [Cd(Me3tacn)(OH)] + ([M(L)(OH)] +) and [Cd(Me3tacn)(OH)2] ([M(L)(OH)2]).
287
Hilary Coleman Appendix B: Supporting figures for Chapter 3
APPENDIX B
288
Speciation plots for the βCDtren- and βCDtacn-based systems characterised

by the pKa, logK, logK’ and pKaH2O values in Tables 3.1 – 3.9.
These values were derived from the average of two titration experiments. For the speciation
simulations a total ligand concentration of 1.0 × 10-3 mol dm-3 and a total M2+ concentration of
1.0 × 10-3 mol dm-3 (for the titrations containing Zn2+, Cu2+, Ni2+ and Cd2+) was used. The species
shown in the keys denote the species that were included in the data analysis in order to obtain
each fit.
100
90
80 free L
70
LH⁺
60
50 LH₂²⁺
40
30 LH₃³⁺
20
LH₄⁴⁺
10
0
3 5 7 9 11
pH
Figure B1. Speciation plot showing the variation of the protonation of βCDtren with pH, relative to the total
concentration of all βCDtren and its protonated species being equivalent to 100%: βCDtrenH44+ (LH4 4+
),
βCDtrenH33+ (LH3 ), 3+
βCDtrenH22+ (LH2 ), βCDtrenH (LH ) and βCDtren (free L).
2+ + +
289
100
90

80 free L
70
LH⁺
60
50 LH₂²⁺
40
30 LH₃³⁺
20
LH₄⁴⁺
10
0
3 5 7 9 11
pH
Figure B2. Speciation plot showing the variation of the protonation of βCDMe5tren with pH, relative to the total
concentration of all βCDMe5tren and its protonated species being equivalent to 100%: βCDMe5trenH44+ (LH4 4+),
βCDMe5trenH33+ (LH3 3+), βCDMe5trenH22+ (LH2 2+), βCDMe5trenH+ (LH+) and βCDMe5tren (free L).
100
free L
90
LH⁺
80
70 LH₂²⁺
60
LH₃³⁺
50
LH₄⁴⁺
40
30 MLH⁺
20 [ML]²⁺
10
[M(L)(OH)]⁺
0
3 5 7 9 11 [M(L)(OH)₂]
pH
Figure B3. Speciation plot showing the variation of the protonation and complexation of βCDtren with pH in the
presence of Zn2+, relative to the total concentration of all βCDtren and its protonated and complexed species being
equivalent to 100%: βCDtrenH44+ (LH4 4+
), βCDtrenH33+ (LH3 3+
), βCDtrenH+ (LH+) and
βCDtren (free L), [Zn(βCDtrenH)] 3+([MLH] 3+), [Zn(βCDtren)] 2+ ([ML] 2+), [Zn(βCDtren)(OH)] + ([M(L)(OH)] +)
and [Zn(βCDtren)(OH)2] ([M(L)(OH)]).
290
100
free L
90
LH⁺

80
70 LH₂²⁺
60
LH₃³⁺
50
LH₄⁴⁺
40
30 MLH⁺
20 [ML]²⁺
10
[M(L)(OH)]⁺
0
3 5 7 9 11 [M(L)(OH)₂]
pH
presence of Cu2+, relative to the total concentration of all βCDtren and its protonated and complexed species being
), βCDtrenH22+ (LH2 ), βCDtrenH+ (LH+) and
2+
βCDtren (free L), [Cu(βCDtrenH)] 3+ ([MLH] 3+), [Cu(βCDtren)] 2+ ([ML] 2+), [Cu(βCDtren)(OH)] + ([M(L)(OH)] +)
and [Cu(βCDtren)(OH)2] ([M(L)(OH)2]).
100
free L
90
80 LH⁺
70
LH₂²⁺
60
50 LH₃³⁺
40
LH₄⁴⁺
30
20 MLH⁺
10 [ML]²⁺
0
3 5 7 9 11 [M(L)(OH)]⁺
pH
presence of Ni2+, relative to the total concentration of all βCDtren and its protonated and complexed species being
), βCDtrenH22+ (LH2 ), βCDtrenH+ (LH+) and
2+
βCDtren (free L), [Ni(βCDtrenH)] 3+ ([MLH] 3+), [Ni(βCDtren)] 2+ ([ML] 2+) and [Ni(βCDtren)(OH)] +
([M(L)(OH)] +).
291
100
free L
90
LH⁺

80
70 LH₂²⁺
60
LH₃³⁺
50
LH₄⁴⁺
40
30 MLH⁺
20 [ML]²⁺
10
[M(L)(OH)]⁺
0
3 5 7 9 11 [M(L)(OH)₂]
pH
presence of Cd2+, relative to the total concentration of all βCDtren and its protonated and complexed species being
), βCDtrenH+ (LH+) and
βCDtren (free L), [Cd(βCDtrenH)] 3+ ([MLH] 3+), [Cd(βCDtren)] 2+ ([ML] 2+), [Cd(βCDtren)(OH)] + ([M(L)(OH)] +)
and [Cd(βCDtren)(OH)2] ([M(L)(OH)2]).
100
free L
90
LH⁺
80
70 LH₂²⁺
60
LH₃³⁺
50
LH₄⁴⁺
40
30 MLH⁺
20 [ML]²⁺
10
[M(L)(OH)]⁺
0
3 5 7 9 11 [M(L)(OH)₂]
pH
Figure B7. Speciation plot showing the variation of the protonation and complexation of βCDMe5tren with pH in the
presence of Zn2+, relative to the total concentration of all βCDMe5tren and its protonated and complexed species
being equivalent to 100%: βCDMe5trenH44+ (LH4 4+
), βCDMe5trenH33+ (LH3 3+
),
βCDMe5trenH (LH ) and βCDMe5tren (free L), [Zn(βCDMe5trenH)] ([MLH] ), [Zn(βCDMe5tren)]
+ + 3+ 3+ 2+ 2+
([ML] ),
[Zn(βCDMe5tren)(OH)] + ([M(L)(OH)] +) and [Zn(βCDMe5tren)(OH)2] ([M(L)(OH)2]).
292
100
free L
90
LH⁺

80
70 LH₂²⁺
60
LH₃³⁺
50
LH₄⁴⁺
40
30 MLH⁺
20 [ML]²⁺
10
[M(L)(OH)]⁺
0
3 5 7 9 11 [M(L)(OH)₂]
pH
presence of Cu2+, relative to the total concentration of all βCDMe5tren and its protonated and complexed species
),
βCDMe5trenH+ (LH+) and βCDMe5tren (free L), [Cu(βCDMe5trenH)] 3+ ([MLH] 3+), [Cu(βCDMe5tren)] 2+ ([ML] 2+),
[Cu(βCDMe5tren)(OH)] + ([M(L)(OH)] +) and [Cu(βCDMe5tren)(OH)2] ([M(L)(OH)2]).
100
free L
90
80 LH⁺
70
LH₂²⁺
60
50 LH₃³⁺
40
LH₄⁴⁺
30
20 [ML]²⁺
10 [M(L)(OH)]⁺
0
3 5 7 9 11 [M(L)(OH)₂]
pH
presence of Ni2+, relative to the total concentration of all βCDMe5tren and its protonated and complexed species
),
βCDMe5trenH (LH ) and βCDMe5tren (free L), [Ni(Me6tren)]
+ + 2+ 2+ +
([ML] ), [Ni(βCDMe5tren)(OH)] ([M(L)(OH)] ) +
and [Ni(βCDMe5tren)(OH)2] ([M(L)(OH)2]).
293
100
free L
90
LH⁺

80
70 LH₂²⁺
60
LH₃³⁺
50
LH₄⁴⁺
40
30 MLH⁺
20 [ML]²⁺
10
[M(L)(OH)]⁺
0
3 5 7 9 11 [M(L)(OH)₂]
pH
Figure B10. Speciation plot showing the variation of the protonation and complexation of βCDMe5tren with pH in
the presence of Cd2+, relative to the total concentration of all βCDMe5tren and its protonated and complexed species
being equivalent to 100%: βCDMe5trenH44+ (LH4+), βCDMe5trenH33+ (LH3+), βCDMe5trenH22+ (LH22+),
βCDMe5trenH+ (LH+) βCDMe5tren (free L), [Cd(βCDMe5trenH)] 3+ ([MLH] 3+), [Cd(βCDMe5tren)] 2+ ([ML] 2+),
[Cd(βCDMe5tren)(OH)] + ([M(L)(OH)] +) and [Cd(βCDMe5tren)(OH)2] ([M(L)(OH)2]).
100
90
80 free L
70
60 LH⁺
50
40 LH₂²⁺
30
20 LH₃³⁺
10
0
3 5 7 9 11
pH
Figure B11. Speciation plot showing the variation of the protonation of βCDtacn with pH, relative to the total
concentration of all βCDtacn and its protonated species being equivalent to 100%: βCDtacnH33+ (LH3 3+
),
βCDtacnH22+ (LH2 2+), βCDtacnH+ (LH+) and βCDtacn (free L).
294
100
90

80 free L
70
LH⁺
60
50 LH₂²⁺
40
30 LH₃³⁺
20
LH₄⁴⁺
10
0
3 5 7 9 11
pH
Figure B12. Speciation plot showing the variation of the protonation of βCDMe2tacn with pH, relative to the total
concentration of all tacn and its protonated species being equivalent to 100%: βCDMe2tacnH33+ (LH3+),
βCDMe2tacnH22+ (LH22+), βCDMe2tacnH+ (LH+) and βCDMe2tacn (free L).
100
90
80 free L
70 LH⁺
60
LH₂²⁺
50
LH₃³⁺
40
30 [ML]²⁺
20 [M(L)(OH)]⁺
10
[M(L)(OH)₂]
0
3 5 7 9 11
pH
Figure B13. Speciation plot showing the variation of the protonation and complexation of βCDtacn with pH in the
presence of Zn2+, relative to the total concentration of all βCDtacn and its protonated and complexed species being
equivalent to 100%: βCDtacnH33+ (LH3 ), βCDtacnH22+ (LH2
3+ 2+
), βCDtacnH+ (LH+), βCDtacn (free L),
[Zn(tacn)] 2+ ([ML] 2+), [Zn(tacn)(OH)] + ([M(L)(OH)] +) and [Zn(tacn)(OH)2] ([M(L)(OH)2]).
295
100
90
free L

80
LH⁺
70
LH₂²⁺
60
LH₃³⁺
50
[MLH]³⁺
40
[ML]²⁺
30
[M(L)(OH)]⁺
20
[M(L)(OH)₂]
10
[M(L)(OH)₃]⁻
0
3 5 7 9 11
pH
presence of Cu2+, relative to the total concentration of all βCDtacn and its protonated and complexed species being
3+ 2+
[Cu(βCDtacnH)] 3+ ([MLH] 3+), [Cu(βCDtacn)] 2+ ([ML] 2+), [Cu(βCDtacn)(OH)] + ([M(L)(OH)] +), [CuβCD
(tacn)(OH)2] ([M(L)(OH)2]) and [CuβCD (tacn)(OH)3] – ([M(L)(OH)3] – ).
100
90
80 free L
70 LH⁺
60
LH₂²⁺
50
LH₃³⁺
40
30 [ML]²⁺
20 [M(L)(OH)]⁺
10
[M(L)(OH)₂]
0
3 5 7 9 11
pH
presence of Ni2+, relative to the total concentration of all βCDtacn and its protonated and complexed species being
3+ 2+
[Ni(βCDtacn)] 2+ ([ML] 2+), [Ni(βCDtacn)(OH)] + ([M(L)(OH)] +) and [Ni(βCDtacn)(OH)2] ([M(L)(OH)2]).
296
100
90
free L

80
70 LH⁺
60 LH₂²⁺
50 LH₃³⁺
40 [MLH]³⁺
30 [ML]²⁺
20
[M(L)(OH)]⁺
10
[M(L)(OH)₂]
0
3 5 7 9 11
pH
presence of Cd2+, relative to the total concentration of all βCDtacn and its protonated and complexed species being
3+ 2+
[Cd(βCDtacnH)] 3+ ([MLH]3+), [Cd(βCDtacn)] 2+ ([ML] 2+), [Cd(βCDtacn)(OH)] + ([M(L)(OH)] +) and
[Cd(βCDtacn)(OH)2] ([M(L)(OH)2]).
100
90
80 free L
70 LH⁺
60
LH₂²⁺
50
LH₃³⁺
40
30 [MLH]³⁺
20 [ML]²⁺
10
[M(L)(OH)]⁺
0
3 5 7 9 11
pH
Figure B17. Speciation plot showing the variation of the protonation and complexation of βCDMe2tacn with pH in
the presence of Zn2+, relative to the total concentration of all βCDMe2tacn and its protonated and complexed species
being equivalent to 100%: βCDMe2tacnH33+ (LH3 3+
), βCDMe2tacnH22+ (LH2 2+
), βCDMe2tacnH+ (LH+),
βCDMe2tacn (free L), [Zn(βCDMe2tacnH)] 3+ ([MLH] 3+), [Zn(βCDMe2tacn)] 2+ ([ML] 2+), and
[Zn(βCDMe2tacn)(OH)] + ([M(L)(OH)] +).
297
100
90
free L

80
LH⁺
70
LH₂²⁺
60
LH₃³⁺
50
[MLH]³⁺
40
[ML]²⁺
30
[M(L)(OH)]⁺
20
[M(L)(OH)₂]
10
[M(L)(OH)₃]⁻
0
3 5 7 9 11
pH
the presence of Cu2+, relative to the total concentration of all βCDMe2tacn and its protonated and complexed species
βCDMe2tacn (free L), [CuβCDMe2tacnH] 3+ ([MLH] 3+), [Cu(βCDMe2tacn)] 2+ ([ML] 2+), [Cu(βCDMe2tacn)(OH)] +
([M(L)(OH)] +), [Cu(βCDMe2tacn)(OH)2] ([M(L)(OH)2]) and [Cu(βCDMe2tacn)(OH)3] – ([M(L)(OH)3] –).
As noted in the text, the stability constant for the formation of [Ni(βCDMe2tacn)]2+ was likely
to be too small to be accurately measured by the techniques used in this work.
298
100
90
free L

80
LH⁺
70
LH₂²⁺
60
LH₃³⁺
50
[MLH]³⁺
40
[ML]²⁺
30
[M(L)(OH)]⁺
20
[M(L)(OH)₂]
10
[M(L)(OH)₃]⁻
0
3 5 7 9 11
pH
the presence of Cd2+, relative to the total concentration of all βCDMe2tacn and its protonated and complexed species
βCDMe2tacn (free L), [CdβCDMe2tacnH] 3+ ([MLH] 3+), [Cd(βCDMe2tacn)] 2+ ([ML] 2+), [Cd(βCDMe2tacn)(OH)] +
([M(L)(OH)] +), [Cd(βCDMe2tacn)(OH)2] ([M(L)(OH)2]) and [Cd(βCDMe2tacn)(OH)3] – ([M(L)(OH)3] –).
299
Titration curves and fits for the newly-synthesised ligands in this study:
βCDMe5tren and βCDMe2tacn pKas and M2+ complex characterisation where
M2+ is Zn2+, Cu2+, Ni2+ and Cd2+. Each experiment was conducted in duplicate.
Presented here is one example from each set. The data derived from these
titrations and fits are reported in Tables 3.1 – 3.9.
11.5
10.5
9.5
8.5
Observed
7.5
pH Fit
6.5
5.5
4.5
3.5
2.5
0.129 0.179 0.229 0.279
Titre (mL)
Figure B20. Potentiometric titration curve for the determination of the protonated βCDMe5tren ligand pKa values –
observed points (◊) and best-fit (-).
300
11.5
10.5
9.5
8.5
Observed
7.5
pH Fit
6.5
5.5
4.5
3.5
2.5
0.12 0.17 0.22 0.27 0.32
Titre (mL)
Figure B21. Potentiometric titration curve for the characterisation of the Zn2+/βCDMe5tren system – observed
points (◊) and best-fit (-).
301
11.5
10.5
9.5
8.5
Observed
7.5
pH Fit
6.5
5.5
4.5
3.5
2.5
0.143 0.193 0.243 0.293
Titre (mL)
Figure B22. Potentiometric titration curve for the characterisation of the Cu2+/βCDMe5tren system – observed
302
11.5
10.5
9.5
8.5
Observed
7.5
pH Fit
6.5
5.5
4.5
3.5
2.5
0.119 0.169 0.219 0.269 0.319
Titre (mL)
Figure B23. Potentiometric titration curve for the characterisation of the Ni2+/βCDMe5tren system – observed points
(◊) and best-fit (-). Note: as discussed in Chapter 3, this system exhibited significant slow equilibria. As is visible on
the plot, the range over which a maximum wait time was observed between titrant additions was between 0.217 and
0.271 mL. As referenced in Chapter 3, no logK1 was measured for this system as a result of this.
303
11.5
10.5
9.5
8.5
Observed
7.5
pH Fit
6.5
5.5
4.5
3.5
2.5
0.124 0.174 0.224 0.274
Titre (mL)
Figure B24. Potentiometric titration curve for the characterisation of the Cd2+/βCDMe5tren system – observed
304
11.5
10.5
9.5
8.5
Observed
7.5
pH Fit
6.5
5.5
4.5
3.5
2.5
0.141 0.161 0.181 0.201 0.221 0.241
Titre (mL)
Figure B25. Potentiometric titration curve for the determination of the protonated βCDMe2tacn ligand pKa values –
observed points (◊) and best-fit (-).
305
11.5
10.5
9.5
8.5
Observed
7.5
pH Fit
6.5
5.5
4.5
3.5
2.5
0.148 0.168 0.188 0.208 0.228 0.248 0.268 0.288
Titre (mL)
Figure B26. Potentiometric titration curve for the characterisation of the Zn2+/βCDMe2tacn system – observed
306
11.5
10.5
9.5
8.5
Observed
7.5
pH Fit
6.5
5.5
4.5
3.5
2.5
0.136 0.186 0.236 0.286
Titre (mL)
Figure B27. Potentiometric titration curve for the characterisation of the Cu2+/βCDMe2tacn system – observed
307
11.5
10.5
9.5
8.5
Observed
7.5
pH Fit
6.5
5.5
4.5
3.5
2.5
0.125 0.175 0.225 0.275
Titre (mL)
Figure B28. Potentiometric titration curve for the characterisation of the Cd2+/βCDMe2tacn system – observed
Note: as noted in Section 3.2.4, the titration for the Cd2+/βCDMe2tacn system involved slow
equilibrium processes over the titre range 0.18 – 0.21 mL which may affect the reliability of the
fitted results.
308
Hilary Coleman Appendix C: Supporting figures for Chapter 4
APPENDIX C
309
Please note: As discussed in Chapters 4 and 7, the duration of each kinetics experiment depended on the
concentration of enzyme mimic present. In order to maximise data collection, multiple experiments were run
simultaneously. Therefore, the density of data points collected was not consistent across each UV-Vis run, as
reflected in the figures throughout this appendix. However, regardless of the density of points, each plot was
satisfactorily fit as described in Section 4.1.3.
Second note: for the experiments represented below, the concentrations recorded are the amount of each
component of the enzyme mimic that was added. For example, for a free ligand experiment, this concentration
represents the amount of free ligand added, for a free metal experiment it represents the amount of free metal added,
and for a complex experiment it represents the amount of ligand and metal added. However, for these solution
compositions, the actual concentration of catalytic species – [M(L)(OH)]+ and [M(L)(OH)2] – must then be
determined using the corresponding speciation plots contained in Appendix B.
Kinetics experiments for the βCDtren/Zn2+ system that produced the data
presented in Figures 4.9 and 4.10.
0 .8
0 .6
0 .4
0 .2
0 .0
0 50000 100000 150000 200000 250000
T im e (s )
Figure C1. Monitoring of the evolution of 4-nitrophenolate over time in the absence of any enzyme mimic by
measurement of the absorbance at 400 nm using UV-Vis spectrophotometry. Aqueous NaClO4 (I = 0.10 mol dm-3),
pH 9.14 ([buffer] = 1.0 × 10-2 mol dm-3) and [S] = [4-nitrophenyl acetate] = 4 × 10-5 mol dm-3.
310
1 .0
0 .8
0 .6
0 .4
0 .2
0 .0
0 10000 20000 30000 40000
T im e (s )
Figure C2. Monitoring of the evolution of 4-nitrophenolate over time in the presence of the enzyme mimic
βCDtren/Zn2+ by measurement of the absorbance at 400 nm using UV-Vis spectrophotometry. Aqueous NaClO4 (I =
0.10 mol dm-3), pH 9.14 ([buffer] = 1.0 × 10-2 mol dm-3) and [S] = [4-nitrophenyl acetate] = 4 × 10-5 mol dm-3, [E]
= βCDtren/Zn2+ = 4 × 10-4 mol dm-3.
1 .0
0 .8
0 .6
0 .4
0 .2
0 .0
0 50000 100000 150000 200000 250000
T im e (s )
311
1 .0
0 .8
0 .6
0 .4
0 .2
0 .0
0 5000 10000
T im e (s )
= βCDtren/Zn2+ = 1.0 × 10-3 mol dm-3.
1 .5
1 .0
0 .5
0 .0
0 5000 10000
T im e (s )
= βCDtren/Zn2+ = 3.0 × 10-3 mol dm-3.
312
2 .0
1 .5
1 .0
0 .5
0 .0
0 1000 2000 3000 4000
T im e (s )
= βCDtren/Zn2+ = 4.0 × 10-3 mol dm-3.
2 .0
1 .5
1 .0
0 .5
0 .0
0 1000 2000 3000 4000
T im e (s )
= βCDtren/Zn2+ = 6.0 × 10-3 mol dm-3.
313
2 .0
1 .8
1 .6
1 .4
1 .2
1 .0
0 500 1000 1500 2000
T im e (s )
= βCDtren/Zn2+ = 1.0 × 10-2 mol dm-3.
2 .2
2 .0
1 .8
1 .6
1 .4
1 .2
1 .0
0 1000 2000 3000 4000
T im e (s )
= βCDtren/Zn2+ = 1.2 × 10-2 mol dm-3.
314
2 .6
2 .4
2 .2
2 .0
1 .8
1 .6
1 .4
0 500 1000 1500
T im e (s )
= βCDtren/Zn2+ = 1.5 × 10-2 mol dm-3.
0 .3
0 .2
0 .1
0 .0
0 10000 20000 30000 40000 50000
T im e (s )
315
0 .3
0 .2
0 .1
0 .0
0 10000 20000 30000 40000 50000
T im e (s )
0 .8
0 .6
0 .4
0 .2
0 .0
0 20000 40000 60000 80000 100000
T im e (s )
The plot for the evolution of 4-nitrophenolate in the presence of βCDtren/Zn2+ at pH 9.14 is already displayed in
Figure C2.
316
1 .0
0 .8
0 .6
0 .4
0 .2
0 .0
0 10000 20000 30000 40000 50000
T im e (s )
317
Kinetics experiments for the βCDtren/Cu2+ system that produced the data
presented in Figures 4.9 and 4.10.
1 .0
0 .8
0 .6
0 .4
0 .2
0 .0
0 50000 100000 150000
T im e (s )
Figure C15. Monitoring of the evolution of 4-nitrophenolate over time in the absence of any enzyme mimic by
pH 8.68 ([buffer] = 1.0 × 10-2 mol dm-3) and [S] = [4-nitrophenyl acetate] = 4 × 10-5 mol dm-3.
0 .8
0 .6
0 .4
0 .2
0 .0
0 10000 20000 30000 40000
T im e (s )
βCDtren/Cu2+by measurement of the absorbance at 400 nm using UV-Vis spectrophotometry. Aqueous NaClO4 (I =
= βCDtren/Cu2+ = 4 × 10-4 mol dm-3.
318
0 .8
0 .6
0 .4
0 .2
0 .0
0 10000 20000 30000 40000
T im e (s )
1 .0
0 .8
0 .6
0 .4
0 .2
0 .0
0 10000 20000 30000
T im e (s )
= βCDtren/Cu2+ = 1.0 × 10-3 mol dm-3.
319
1 .2
1 .0
0 .8
0 .6
0 .4
0 2000 4000 6000
T im e (s )
= βCDtren/Cu2+ = 4.0 × 10-3 mol dm-3.
1 .8
1 .6
1 .4
1 .2
1 .0
0 .8
0 1000 2000 3000
T im e (s )
= βCDtren/Cu2+ = 6.0 × 10-3 mol dm-3.
320
0 .4 5
0 .4 0
0 .3 5
0 .3 0
0 .2 5
0 1000 2000 3000 4000
T im e (s )
= βCDtren/Cu2+ = 1.0 × 10-2 mol dm-3.
0 .5 5
0 .5 0
0 .4 5
0 .4 0
0 .3 5
0 1000 2000 3000
T im e (s )
= βCDtren/Cu2+ = 1.5 × 10-2 mol dm-3.
321
1 .0
0 .8
0 .6
0 .4
0 .2
0 .0
0 10000 20000 30000 40000 50000
T im e (s )
1 .0
0 .8
0 .6
0 .4
0 .2
0 .0
0 10000 20000 30000 40000 50000
T im e (s )
The plot for the evolution of 4-nitrophenolate in the presence of βCDtren/Cu2+ at pH 8.68 is already displayed in
Figure C16.
322
0 .8
0 .6
0 .4
0 .2
0 .0
0 10000 20000 30000 40000
T im e (s )
0 .8
0 .6
0 .4
0 .2
0 .0
0 10000 20000 30000 40000
T im e (s )
323
Kinetics experiments for the other potential enzyme mimics presented in

Section 4.3.
Please note: For complexes that have corresponding titration experiments, the pH for these kinetics runs was
chosen as the pKaH2O of the associated water ligand as recorded in Chapters 2 and 3. The remaining experiments were
performed at pH 8.68 or 9.14 (as indicated) to facilitate easy comparison with the rest of the data presented in
Chapter 4.
S ta n d a rd is e d a b s o rb a n c e a t 4 0 0 n m 1 .0
0 .8
0 .6
0 .4
0 .2
0 .0
0 10000 20000 30000 40000 50000
T im e (s )
Figure C27. Monitoring of the evolution of 4-nitrophenolate over time in the presence of the enzyme mimic tren by
pH 8.68 ([buffer] = 1.0 × 10-2 mol dm-3) and [S] = [4-nitrophenyl acetate] = 4 × 10-5 mol dm-3, [E] = tren = 4 × 10-
4
mol dm-3.
324
1 .0
0 .8
0 .6
0 .4
0 .2
0 .0
0 200000 400000 600000 800000 1000000
T im e (s )
tren/Cu2+ by measurement of the absorbance at 400 nm using UV-Vis spectrophotometry. Aqueous NaClO4 (I = 0.10
mol dm-3), pH 8.93 ([buffer] = 1.0 × 10-2 mol dm-3) and [S] = [4-nitrophenyl acetate] = 4 × 10-5 mol dm-3, [E] =
tren/Cu2+ = 4 × 10-4 mol dm-3.
1 .5
1 .0
0 .5
0 .0
0 50000 100000 150000 200000
T im e (s )
Figure C29. Monitoring of the evolution of 4-nitrophenolate over time in the presence of the enzyme mimic Me6tren
by measurement of the absorbance at 400 nm using UV-Vis spectrophotometry. Aqueous NaClO4 (I = 0.10 mol dm-
3
), pH 8.68 ([buffer] = 1.0 × 10-2 mol dm-3) and [S] = [4-nitrophenyl acetate] = 4 × 10-5 mol dm-3, [E] = Me6tren =
4 × 10-4 mol dm-3.
325
1 .0 0
0 .7 5
0 .5 0
0 .2 5
0 .0 0
0 50000 100000 150000
T im e (s )
Figure C30. Monitoring of the evolution of 4-nitrophenolate over time in the presence of the enzyme mimic βCDtren
by measurement of the absorbance at 400 nm using UV-Vis spectrophotometry. Aqueous NaClO4 (I = 0.10 mol dm-
), pH 8.68 ([buffer] = 1.0 × 10-2 mol dm-3) and [S] = [4-nitrophenyl acetate] = 4 × 10-5 mol dm-3, [E] = βCDtren =
3
4 × 10-4 mol dm-3.
1 .0 0
0 .7 5
0 .5 0
0 .2 5
0 .0 0
0 50000 100000 150000
T im e (s )
βCDMe5tren by measurement of the absorbance at 400 nm using UV-Vis spectrophotometry. Aqueous NaClO4 (I =
= βCDMe5tren = 4 × 10-4 mol dm-3.
326
0 .6
0 .4
0 .2
0 .0
0 10000 20000 30000
T im e (s )
Figure C32. Monitoring of the evolution of 4-nitrophenolate over time in the presence of the enzyme mimic Zn2+ by
pH 9.14 ([buffer] = 1.0 × 10-2 mol dm-3) and [S] = [4-nitrophenyl acetate] = 4 × 10-5 mol dm-3, [E] = Zn2+ = 4 ×
10-4 mol dm-3. The absorbance at t∞ was not determined for this experiment.
0 .8
0 .6
0 .4
0 .2
0 .0
0 50000 100000 150000
T im e (s )
Figure C33. Monitoring of the evolution of 4-nitrophenolate over time in the presence of the enzyme mimic Cu2+ by
pH 8.68 ([buffer] = 1.0 × 10-2 mol dm-3) and [S] = [4-nitrophenyl acetate] = 4 × 10-5 mol dm-3, [E] = Cu2+ = 4 ×
10-4 mol dm-3. The absorbance at t∞ was not determined for this experiment.
327
Full derivation for the Michaelis-Menten variation presented is Section 4.1.3.

Please note: the Equation numbers are aligned with those in Section 4.1.3 for ease of
reference.
Enzyme and enzyme mimic kinetics have been analysed using the Michaelis-Menten model
for a century. The analysis is based on the following scheme:
k1 k2
E + S ⇌ E•S ⇌ E + P E
k-1 k-2
In this study, the conventional Michaelis-Menten scheme was amended to give the new
Equation 4.11:
k1 kcat(RDS)
S + E ⇌ E•S ⇌ E + P 4.11
k-1
kuncat
This overall reaction scheme recognises the two possible pathways for the ester to be
converted into the phenolate product: via catalysis with the enzyme mimic (represented by kcat) or
spontaneously by reaction with hydroxide in the bulk solution (represented by kuncat). The total
overall observed rate is therefore contributed to by both the catalysed and uncatalysed processes
and when there is no enzyme present the observed rate is entirely due to the uncatalysed reaction.
Based on the overall reaction scheme described by Equation 4.11, the following rate law is
expressed:
rate = kobs([S] + [E•S]) = kuncat[S] + kcat[E•S] 4.12
Where kobs is the experimentally determined, observed overall pseudo first order rate constant
for a given excess enzyme concentration. Just like the assumptions made for the conventional
Michaelis-Menten derivation, it is also assumed for the scheme above that the processes
described by k1 and k-1 occur instantaneously and therefore equilibrium concentrations of S, E
and [E•S] are immediately attained.
328
Again, [E•S] is difficult to determine and must be replaced by measurable quantities.

Therefore, just as the Michaelis constant (the dissociation constant for the enzyme-substrate
complex) can be calculated by the ratio of rate constants in the conventional Michaelis-Menten
derivation above, it can also be calculated in terms of reactant and product equilibrium
concentrations:
KM = [E][S]/[E•S] 4.13
Rearranging gives Equation 4.14 which describes [E•S] in terms of enzyme and substrate
concentrations:
[E•S] = [E][S]/KM 4.14
Substituting Equation 4.14 into Equation 4.12 yields:
kobs{[S] + ([E][S]/KM)} = kun[S] + kcat([E][S]/KM)
Expanding (multiply all terms by KM):
kobs{[S] + ([E][S]/KM)}KM = kun[S]KM + kcat × [E][S]
Multiply both terms in the {} brackets on the LHS by the KM on that side:
kobs{[S] × KM + [E][S]} = kun[S]KM + kcat × [E][S]
Group like terms – [S] – on both sides of the equality sign (NB all forms of [S], [E] and [E•S]
are assumed to be equilibrium concentrations):
kobs × [S]{KM + [E]} = [S](kunKM + kcat[E])
Divide both sides by [S](KM + [E]):
kobs = [S] × (kunKM + kcat[E])/[S] × (KM + [E])
Cancel [S] in the numerator and denominator on the RHS to produce the overall desired
equation for graphing:
329

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An investigation of transition metal complex chemistry:

enzyme mimicry and Zn(II) detection

Hilary Ceywood Coleman

A thesis submitted for the degree of

2.2.2 Tren and Me6tren metal complex characterisation 50

3.2.1 Determination of protonated βCDtren and βCDMe5tren pKas 80

4.1.5 Aims and methods of this study 127

5.4 Chapter 4 – Kinetic characterisation of the enzyme mimic systems 201

6.1.4 The chemistry of Zinquin 211

7.2 UV-Vis characterisation of 2-((E)-2-Phenyl)ethenyl-8-(N-4-

7.2.2 Photoisomerism of the stZQA ligand 234

7.3.1 Determination of Adventitious Zn(II) 239

7.4 Conclusions and Future Work 249

Harry Potter and the Half-Blood Prince

Ainfinity UV-Vis absorbance at an infinite reaction time

c speed of light, 3 × 108 m s-1

CHEF chelation enhanced fluorescence

E observed electrode potential (millivolts) (Part II)

E0 standard electrode potential (millivolts)

EPR electron paramagnetic resonance

E•S enzyme-substrate Michaelis complex

F Faraday constant, 9.6487 × 104 C mol-1

h Planck’s constant, 6.63 × 10-34 J s

[H+] proton concentration (mol dm-3)

I constant ionic strength (mol dm-3)

J coupling constant (context; NMR) or joules

K1 stability constant for the 1:1 (M2+:ligand) transition metal complex

Ka acid dissociation constant

L free, deprotonated ligand

LH+ free, monoprotonated ligand

LH22+ free, diprotonated ligand

LH33+ free, triprotonated ligand

LH44+ free, tetraprotonated ligand

[MLH]3+ 1:1 M2+:monoprotonated ligand complex (Part I)

[ML]2+ 1:1 M2+:deprotonated ligand (Part I)

1:1 stZQA:M2+ (Part 2)

[MLOH]+ 1:1:1 M2+:ligand:hydroxo

[ML(OH)2] 1:1:2 M2+:ligand:2×hydroxo

NMR nuclear magnetic resonance

ppm parts per million

R Ideal gas constant, 8.314 J K-1 mol-1

S substrate (in this study; ester 4-nitrophenyl acetate – Part I)

S0 ground electronic state (Part II)

S1 first excited electronic state (Part II)

T Temperature (˚C or Kelvin)

tinfinity An infinite time into a reaction

v0 ground vibrational state

v1 first excited vibrational state

Zn2+Ad adventitious Zn2+

Bicine 2-(bis(2-hydroxyethyl)amino)acetic acid (buffer)

DEPP 1,4-diethylpiperazine (buffer)

HEPES 2-[4-(2-hydroxyethyl)piperazin-a-yl]ethanesulfonic acid (buffer)

MES 2-(N-morpholino)sulfonic acid (buffer)

MOPS 3-morpholinopropane-1-sulfonic acid (buffer)

PIPES 1,4-piperazinediethanesulfonic acid (buffer)

TEEN N-N-N'-N'-tetraethylethylenediamine (buffer)

ZQA 2-methyl-8-p-toluenesulfonamido-6-quinolyloxyacetic acid (Zinquin A)

INTRODUCTION PART I: METALLO-AMINE

Nature has developed a spectacular catalogue of metalloenzymes to enhance the rates of

1.1.2 Features of metalloenzymes – Carbonic anhydrase

Metalloenzymes work as catalysts by providing an alternative reaction pathway of lower

CO2 + H2O ⇌ H2CO3 ⇌ HCO3 ̅ + H+ 1.1