Rebecca Pierce Thesis

Departments of Chemistry and Engineering Chemistry
Queen’s University, Kingston, ON
Supramolecular Drug Reversal using 4-
Sulfonatocalix[n]arenes
Rebecca Pierce
March 31, 2015
Supervisor: Dr. Donal Macartney
Examiner: Dr. Simon Hesp
Abstract
The binding ability of 4-sulfonatocalix[n]arenes (n=4,6,8) as a host molecule was
studied with an acetylcholinesterase inhibitor, 1,5-Bis(4-
allyldimethylammoniumphenyl)pentan-3-one dibromide, in order to ascertain its potential
as a drug reversal agent. 1
H NMR and UV-Visible spectroscopy were used to
measure chemical shifts with increasing host concentration and determine the
mechanism of binding, as well as the final conformation of the complex. Results
show that increasing the number of aromatic groups in the host (N=4,6,8) would
allow for different 1:1 conformations of host and guest in the bound complex, and
that by increasing the host molar ratio (in the case of SCX[4]), 2:1 complexes may
also be formed. While 4-Sulfonatocalix[n]arenes were not confirmed to be suitable
for drug reversal, the results suggested favorable properties that recommend them for
further research in this area.

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Table of Contents
1.0 Introduction ................................................................................. 3
1.1 Acetylcholinesterase Inhibitors................................................................ 3
1.2 Guest Molecule: BW284c51 .................................................................... 4
1.3 Host Molecules: (4-Sulfonatocalix[N]arene)................................................... 5
1.3.1 Literature Review: Cucurbit[7]uril Macrocycles..................................................5
2.0 Experimental ................................................................................ 6
2.1 Preparation of Stock Solutions ................................................................ 6
2.1.1 Titrations for UV-Visible Spectroscopy...............................................................6
2.1.1 Titrations for 1
H NMR Spectroscopy..................................................................7
2.2 UV-Visible Spectroscopy........................................................................ 7
2.3 1
H Nuclear Magnetic Resonance Spectroscopy............................................. 8
3.0 Results........................................................................................ 8
3.1 SCX[4]:Guest Complexes ...................................................................... 9
3.2 SCX[6]:Guest Complexes .....................................................................10
3.3 SCX[8]:Guest Complexes .....................................................................10
4.0 Discussion ..................................................................................11
4.1 4-Sulfonatocalix[4]arene.......................................................................11
4.4 Assessment of Experiment.....................................................................12
4.5 Potential Errors..................................................................................13
4.6 Future Work......................................................................................13
5.0 Conclusions ................................................................................14
6.0 References ..................................................................................15
7.0 Appendix....................................................................................16
Supplemental Tables and Figures ....................................................................16
Figure 4.1 SCX[4] and guest complex conformations..................................................17
Figure 4.2 SCX[6] and guest complex conformations..................................................18
Figure 4.3 SCX[8] and guest complex conformations ................................................18
7.0 Labeled Molecular Diagram ..................................................................19
7.1 UV-Vis spectra ...................................................................................19
7.2 UV-Vis plot.......................................................................................20
7.4 400 Hz stacked plot SCX[4] ...................................................................21
7.5 SCX[4] shift graph ..............................................................................21
7.6 400 Hz stacked plot SCX[6] ...................................................................22
7.7 SCX[6] shift graph ..............................................................................22
7.8 300 Hz stacked SCX[8].........................................................................23
7.9 SCX[8] shift graph ..............................................................................23

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List of Tables and Figures
Figure 1.1 Acetylcholine neurotransmitter molecule (ACh)………………………..…………2
Figure 1.2 Guest molecule, 1,5-bis(4-allyldimethylammoniumphenyl)pentan-3-one
dibromide………….………………………………………………………………….…………..4
Figure 1.3 3D Conformational Diagram of Host Molecules: SCX[4], SCX[6], and
SCX[8]............... …………………………………………………………….…………….….4
Figure 1.3.1 Cucurbit[7]uril structure……………………………………….…………….......5
Table 3.1 Chemical shifts for SCX[4] using 1
H NMR spectroscopy (400 Hz)..................16
Figure 4.1 SCX[4] and guest complex conformations………………………………...........17

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1.0 Introduction
The ability to selectively bind and release drug molecules through intermolecular
forces and the creation of a host/guest complex are processes that are often used in
supramolecular chemistry. Two or more molecules are associated through van der
Waals forces, polar attractive forces, hydrogen bonding, and hydrophobic effects to
self-assemble into an ordered complex with different properties than the original
molecules. These properties can be studied and controlled, allowing for manipulation
of the complex and of the molecule inside.1
Macrocyclic host molecules have been extensively researched in terms of drug
delivery and controlled release; however, there is a paucity of information on their
potentials as drug reversal agents. To improve upon gap in knowledge, the host-guest
complexation abilities of a macrocyclic molecule and a neuromuscular blocking
agent are investigated. Some of the common categories of these macrocycles used for
selective recognition of molecules in water include cyclodextrins, cucurbiturils, and
4-sulfonatocalixarenes - the latter of these is studied more extensively in this project.
The host molecule is the drug 1,5-bis(4-allyldimethylammoniumphenyl)pentan-3-one
dibromide - an acetylcholinesterase inhibitor that is used in research as a steroidal
neuromuscular blocking agent. The host molecules used are a series of 4-
sulfonatocalixarenes that vary in the number of aromatic rings: 4-
Sulfonatocalix[4]arene, 4-Sulfonatocalix[6]arene, and 4-Sulfonatocalix[8]arene. In this
study, the complexation abilities of these two molecules in an aqueous solution will
be examined using UV-visible and 1
H NMR spectroscopy. Background information
for each of these species will be provided in the following sections.
1.1 Acetylcholinesterase Inhibitors
Acetylcholine (ACh) is a neurotransmitter in the autonomous nervous system that is
responsible for a variety of functions in the central and peripheral nervous systems
(Figure 1). A neurotransmitter is a chemical signal that is emitted from the terminal
end of a nerve to bind to specific receptors on targeted postsynaptic neurons.2
It is
especially eminent in the visceral motor system, and is involved in many involuntary
muscle functions in smooth and cardiac muscle fibers, as well as in some glands.3
Figure 1.1: Acetylcholine neurotransmitter molecule (ACh)
Acetylcholine is degraded through hydrolysis by the enzyme acetylcholinesterase
(AChE), which can in turn be repressed by an acetylcholinesterase inhibitor. The
deactivation of the enzyme would lead to an increase in acetylcholine activity in the
nicotinic and muscarinic receptors of the nervous system. For this reason,
acetylcholinesterase inhibitors can be considered either a drug or a toxin, according
to its application. Reversible inhibitors are typically associated with medicinal

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applications, such as the inhibitor used in this experiment, whereas irreversible
inhibitors are often regarded as toxic.4
Acetylcholinesterase inhibitors have been used in the medical field to reverse the
effects of neuromuscular blockers (muscle relaxants), and to treat diseases such as
Parkinson’s and Alzheimer’s. Alzheimer’s disease in particular is associated with a
deficit of acetylcholine and a decrease in the amount of cholinergic neurons in the
brain, and many attempts to treat the disease target the inhibition of the
acetylcholinesterase enzyme.5
However, negative side effects of AChE inhibitors
(hypotension, muscle contraction, weight loss, etc.) can occur. Due to this, and due
to the toxicity of irreversible inhibitors, additional research into the controlled
binding and release of the enzyme inhibitors is becoming increasingly necessary.
Past research has already been conducted that has established the ability of
cucurbit[7]uril to selectively bind to acetylcholinesterase inhibitors.
Sulfonatocalixarenes are similar in reactivity and structure to cucurbiturils,
suggesting that they might have similar binding abilities. For this reason, it was
hypothesized that sulfonatocalixarenes of various sizes could complex to the
acetylcholinesterase inhibitor, 1,5-bis(4-allyldimethylammoniumphenyl)pentan-3-one
dibromide.
1.2 Guest Molecule: BW284c51
(1,5-bis(4-allyldimethylammoniumphenyl)pentan-3-one dibromide)
The guest molecule that is being examined for its binding properties, 1,5-bis(4-
allyldimethylammoniumphenyl)pentan-3-one dibromide (BW284c51), is one of the most
selective acetylcholinesterase inhibitors currently known (Figure 1.2). It used in
research, but not for drug or clinical use. Its mechanisms for blocking the nicotinic
ACh receptors are not completely understood, but it is known that it affects them
noncompetitively and reversibly by blocking receptor channels.6
In this way, the
acetylcholinesterase inhibitor can serve as an indirect reversal agent for
neuromuscular blockers by increasing the competitive ability of ACh to bind to the
available active sites.
Figure 1.2 Guest molecule, 1,5-bis(4-allyldimethylammoniumphenyl)pentan-3-one dibromide

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BW284c51 is a bis-quaternary ammonium compound with the molecular formula
C27H38H2OBr2, as seen in Figure 2.7
When the 4-sulfonatocalixarene host molecule
interacts with it, it is hypothesized that it will recognize BW284c51’s cationic
ammonium groups through hydrophobic effects and non-covalent ion/dipole
interactions, and will bind to them.
1.3 Host Molecules: (4-Sulfonatocalix[N]arene)
4-Sulfonatocalixarenes are a class of macrocycles that have hydrophobic cavities
capable of containing smaller molecules and ions. Three sizes of 4-
sulfonatocalixarenes are investigated in this study- 4-sulfonatocalix[4]arene, 4-
sulfonatocalix[6]arene, and 4-sulfonatocalix[8]arene – have four, six, and eight aromatic
rings in their cycles, respectively (Figure 1.3).
Figure 1.3 3D Conformational Diagram of Host Molecules: SCX[4], SCX[6], and SCX[8], respectively
These molecules are multifunctional and can form various supramolecular
aggregations. They possess very hydrophilic upper and lower rims and hydrophobic
core, and are easily affected by exterior physical and chemical interactions.8
Water-
soluble calixarenes are becoming more prominent in supramolecular chemistry and
engineering, but their selectivity is not well understood due to the unpredictable and
case-by-case nature of their reaction pathways. It is for this reason 4-
sulfonatocalixarenes require more research on their binding and complexation
capabilities, and the results of the experiment will be compared to previous studies
using a similar macrocycles such as cucurbit[7]uril.
1.3.1 Literature Review: Cucurbit[7]uril Macrocycles
The ability of cucurbit[7]uril macrocycles to bind to amphiphilic cations has
been examined in previous studies, using NMR and ESI-MS spectroscopy to
find binding sites and strength.

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Figure 1.3.1 Cucurbit[7]uril structure (varying views)
Cucurbit[7]uril was found to recognize the cationic quaternary ammonium or
iminium groups on a guest molecule and was proven through NMR and ESI-
MS spectroscopy to bind fairly well to AChE inhibitors.9
Binding constants
have been measured in the range of 103
-106
M-1
for various drug molecules
(also AChE inhibitors), showing good complexation, but likely not strong
enough for biological applications.9
4-Sulfonatocalix[N]arenes are of similar
shape and function to cucurbit[N]urils, suggesting that a similar binding
mechanism may take place, and allowing for comparison of the complexes.
2.0 Experimental
Materials: NaCl, distilled water, D2O, 4-sulfonatocalix[N]arene (N=4,6,8) (MW
744.72 g/mol, 117.09 g/mol, and 1489.45 g/mol, respectively), 5-bis(4-
allyldimethylammoniumphenyl)pentan-3-one dibromide (BW284c51) (MW 566.4
g/mol).
Measurements: UV-Visible spectra were obtained using water as a solvent. 1
H NMR
spectra were obtained in 300 MHz and 400 MHz instruments using D2O as a solvent.
2.1 Preparation of Stock Solutions
A variety of titrations were prepared for UV-Visible spectroscopy and 1
H NMR
spectroscopy. UV-Visible spectroscopy is more sensitive than NMR, requiring
smaller concentrations of magnitude 10-4
or 10-5
M (versus approx. 10-3
M for NMR).
The solutions for UV-Visible spectroscopy only involved the guest molecule and 4-
sulfonatocalix[4]arene as the host, whereas NMR spectra from all three sizes of host
molecules were examined. Solutions were made in distilled water or D2O and salt,
using a constant concentration of guest molecule, BW284c51 (1,5-bis(4-
allyldimethylammoniumphenyl)pentan-3-one dibromide), and increasing concentrations of
the 4-sulfonatocalix[N]arenes, SCX[4], SCX[6], and SCX[8].
2.1.1 Titrations for UV-Visible Spectroscopy
NaCl (29.30mg) was added to 10mL distilled water and stirred to make a
0.050 M solution. Powdered BW284c51 (0.58mg) was added to the 10mL to

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make a 10-4
M guest solution. Powdered SCX[4] (3.81mg) was added to 5mL
of this solution to make a 10-3
M host-guest solution. The host-guest solution
was titrated into the guest solution to make solutions at increasing host
concentrations, from 0.0 mM to 0.9 mM host in 0.1 mM guest solution.
2.1.1 Titrations for 1
H NMR Spectroscopy
SCX[4] (300 Hz): NaCl (148.60mg) was added to 50mL D2O and stirred to
make a 0.05 M solution. Powdered BW284c51 (5.66mg) was added to 10mL
of the salt and D2O to make a 1 mM guest solution. Powdered SCX[4]
(7.60mg) was added to 5mL of this solution to make a 2 mM host-guest
solution. The host-guest solution was titrated into the guest to make solutions
at a 0:1, 1:1, 5:1, and 10:1 concentrations of host to guest for NMR tubes used
for the preliminary examination of the spectra.
SCX[4], SCX[6], SCX[8] (400 Hz): BW284c51 (5.66mg) was added to 10mL
NaCl D2O solution to make 1 mM guest solution. SCX[4] (18.6mg) was
added to 5mL of this solution to make a 5 mM host-guest solution. Starting
with pure 0.5mL guest solution, a series of ten NMR tubes were prepared by
adding increasing amounts of host-guest solution in 0.1mL increments to
make ten solutions from concentrations of 0:1 to 1:1 host to guest.
SCX[6] and SCX[8] have larger molecular weights that must be accounted
for, and so the technique differs for each only by adding SCX[6] (27.93mg) to
the 5mL guest solution, or SCX[8] (37.24mg) to solution.
2.2 UV-Visible Spectroscopy
UV-Visible spectroscopy is an important method that assesses a sample’s absorbed or
reflected light in the ultraviolet-visible spectral region. In this spectral region
(wavelength λ~190-750nm), ultraviolet and visible radiation causes electrons in
molecules to transition from their ground state to a high-energy state and directly
affects how the colour of the species is perceived. UV-Visible spectroscopy is useful
technique for investigating host-guest complexes - measuring the alteration of the
guest’s spectrum upon gradual addition of host molecule allows for determination of
the binding constant and the nature of the complexation. One major constraint is
that the species being absorbed must absorb light in this range to be observed, and
both guest and host molecules meet this criterion.
The absorbance of UV-visible light can be measured by relating wavelength with
concentration, as shown by Beer’s law in Equation (1).
𝐴 = 𝜀𝑐𝑙 (1)
Where ε is the molar absorptivity coefficient (L/mol cm), c is the concentration
(mol/L), and l is the path length (cm). The maximum absorbance measured is
examined in detail as it is affected by the changing conditions of the experiment. The
increase in the maximum absorbance peak demonstrates interactions occurring
between the host and guest molecules, and fitting data of the absorbance at this peak

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against the host concentration and accounting for integration values will provide the
ratio of host that binds to the guest molecule.
2.3 1
H Nuclear Magnetic Resonance Spectroscopy
Nuclear magnetic resonance (NMR) spectroscopy is a research method that uses the
ability of certain atomic nuclei to exhibit magnetic properties in order to provide
insight on the structure and chemical environment of a molecule. In this application,
1
H NMR, or Proton NMR, spectroscopy is used, which measures the changes in
resonance frequency in the intramolecular field of protium – the most common
isotope of hydrogen, 1
H, that consists only of a proton in the nucleus. The resonance
frequency is interpreted on the spectra as the chemical shift, where the location and
amount of shifts determine the molecule’s structure. Additionally, the integration
curve of a peak shows the relative abundance of protons in a specific chemical
environment.
1
H NMR can measure the dynamics and reaction state in species, and so is another
useful technique for analyzing the host-guest behaviour. Upon interaction with the
host molecule, the protons on the guest molecule will shift upfield (Δδ < 0ppm) or
downfield (Δδ u 0 ppm). Protons in the hydrophobic cavity of the guest molecule will
be more shielded, so their resonances will move upfield, whereas protons outside the
cavity and near the carbonyl group will be deshielded and will shift downfield. For
host-guest chemistry, the limiting chemical shift change induced by formation of the
complex can be shown by Equation (2).
δ!"# = δ!"#$% − δ!"## (2)
Where δbound (ppm) is the shift associated with the guest proton in the presence of the
host molecule, and δfree (ppm) is the chemical shift of the free guest proton with no bound
host. This provides information on which guest protons are located within the host’s
hydrophobic cavity to show where binding is taking place, and expresses whether more
than one SCX molecule can bind to the guest molecule. It is initially assumed the
complexation involves non-competitive binding, and that the host and guest bind at a one
to one ratio. The binding constant for the species can be then be found by fitting the curve
of a plot of chemical shift change versus host concentration.
3.0 Results
For clarification, the host (SCX[4,6,8]) molecules and guest molecule were presented
in 2-D conformation, with labeling to account for the relevant protons in 1
H NMR
analysis (Appendix 7.0). Table 3.0 gives an overview of the maximum chemical NMR
shifts for the protons on the guest molecule as it complexes to different sizes of host,
with shading on the most relevant shifts.

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Table 3.0 Maximum Δδ (ppm) for Guest Molecule Protons (1H NMR)
Host
α
β
γ
α'
Aromatic
[1]
[2]

SCX[4]
-‐
-‐0.22
-‐
-‐0.08
-‐0.08
-‐0.02
-‐0.02

SCX[6]
-‐
-‐0.53
-‐0.5
3.87
-‐0.42
-‐0.06
-‐0.48

SCX[8]
-‐0.09
-‐0.13
-‐
-‐0.01
-‐1.99
-‐0.43
-‐0.43

Additionally, each section has a table outlining the relevant chemical shift changes
for each host molecule that can be seen in the supplemental figures section of the
appendix.
3.1 SCX[4]:Guest Complexes
UV-Visible Spectra
A stacked plot of the UV-Visible spectra obtained over increasing SCX[4]
concentrations (Appendix 7.1) shows the maximum absorbance peak to occur at
282nm. This peak was examined in greater detail, with its increase in absorbance
plotted against the host concentration (Appendix 7.2) and fitted to demonstrate the
binding constant at a value of 125 M-1
. As this was the first test conducted, it was a
high enough value to suggest adequate binding and encourage further trials.
300 Hz 1
H NMR Spectra
Preliminary NMR spectra were obtained to approximate the binding behaviour of
the host and guest complex, using SCX[4], the guest molecule, and a 300 Hz 1
H
NMR spectrometer. A stacked 1
H NMR plot exhibits the shift changes as the host to
guest proportion increases to a 10:1 ratio of host to guest concentration (Appendix
7.3). Upfield shifts can be seen to occur at the α, β, and γ protons of the guest
molecule (Δδα=-0.732ppm, Δδβ=-1.588ppm, and Δδγ=-0.801ppm) as well as on the
methyl group (α’) on the ammonium group (Δδα’=-0.388ppm), indicating internal
binding of the guest within the host cavity. Downfield shifts occur at the aromatic
protons on the host molecule (Δδβ=+0.062ppm), indicating binding outside the
cavity.
The data from the 300 Hz spectrometer suggested that most of the binding activity
occurred while titrating at lower concentrations (~3mM host molecule). To examine
the binding mechanism more closely, a more intensive series of titrations was
conducted at lower concentrations and analyzed using a 400 Hz 1
H NMR
spectrometer. This concentration range was used also as a general estimate for the
400 Hz SCX[6] and SCX[8] titrations.
400 Hz 1
H NMR Spectra
A stacked 1
H NMR plot (Appendix 7.4) demonstrated shift changes as the host
concentration increased. The upfield shifts occurred on the guest molecule protons:
with the most significant at the β protons (Δδβ=-0.22ppm), some minor shifts at the
guest aromatic protons and α’ protons (ΔδGA=-0.08ppm, Δδα’=-0.08ppm), and
minimal shifts of protons [1] and [2] (Δδ1,2=-0.02ppm). A downfield shift was
observed at the peak of the host aromatic protons (ΔδHA=+0.08ppm). Table 3.1 in the

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supplementary figures section of the appendix shows the relevant changes in
chemical shifts for SCX[4].
The change in chemical shift (Δδobs) of the peaks was plotted against the SCX[4]
concentration, with the concentration values adjusted according to the integration
values on the spectra. A fit of the data (Appendix 7.5) for the most relevant peak, β,
suggested that the host molecule initially binds at a 1:1 ratio of host to guest, then
binds at a 2:1 ratio as the concentration increases.
400 Hz 1
H NMR Spectra
A stacked 1
concentration increased. The behaviour of the complexes can be visibly observed to
differ from that of SCX[4], such as the splitting of a single peak for the [1] and [2]
protons into separate doublets. Large upfield shifts occurred on the α’ guest molecule
protons and the host aliphatic protons (Δδα’=+3.87ppm, ΔδHAl=+3.87ppm). Major
downfield shifts included the β, γ, [2], and aromatic guest protons (Δδβ=-0.53ppm,
Δδγ=-0.50ppm, Δδ[2]=-0.48ppm, and ΔδGA=-0.42ppm), with minor downfield shifts at
the[1] guest protons and host aromatic protons (Δδ[1]=-0.06ppm, ΔδHA=-0.06ppm.
Table 3.2 in the appendix shows the relevant changes in chemical shifts for SCX[6].
values on the spectra. A fit of the data (Appendix 7.7) for the most relevant peak, α’,
suggested that the host molecule binds at a 1:1 ratio of host to guest.
400 Hz 1
H NMR Spectra
A stacked 1
concentration increased. Significant downfield shifts occurred on the guest aromatic
protons (ΔδGA=-1.99ppm), as well as on protons [1] and [2] on the guest molecule
((Δδ1,2=-0.43ppm), and a minor upfield shift of the guest α proton (Δδα’=-0.10ppm).
Additionally, there was a slight downfield movement of the host aromatic peak
(ΔδHA=+0.07ppm). Table 3.3 shows the relevant changes in chemical shifts for
SCX[8] in the supplemental figures section of the appendix.
values on the spectra. A fit of the data (Appendix 7.9) for the most relevant peak – the
[1] and [2] protons - suggested that the host molecule binds at a 1:1 ratio of host to
guest.

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4.0 Discussion
The spectral data for each size of 4-sulfonatocalix[N]arene showed strong binding to the
guest molecule, 1,5-bis(4-allyldimethylammoniumphenyl)pentan-3-one dibromide, through
the large change in chemical shift that occurred upon addition of host molecule
solution. A fit of UV-visible spectral data for SCX[4] showed an estimate of a
binding constant at approximately 125 M-1
, although more data should be collected
before assuming this as a correct value.
4.1 4-Sulfonatocalix[4]arene
The spectral shifts and integration values provided by both 300 Hz and 400 Hz 1
H
NMR spectrometers suggest that SCX[4] binds by engulfing the guest molecule at
both terminals near the cation groups, as there is a large upfield shift at the β protons
that signifies binding at that location within the host cavity A ChemDraw structure
depicts the two binding mechanisms of the SCX[4] molecules to the guest molecule
in the supplemental figures section of the appendix in Figure 4.1.
The fitted data of Δδβ vs. host concentration (Appendix 7.5) also supports this theory
of binding. The host molecule is shown to initially bind at a 1:1 ratio, with one host
molecule engulfing the tail end of the guest molecule near the β protons. As the
concentration increases, another host molecule engulfs the other tail end of the guest
molecule (2:1 ratio). Upon addition, this second host molecule’s anionic groups will
repel the initial host molecule, making the binding of the second host molecule
weaker than the first. This mechanism is represented by the host’s location of binding
near the β protons at the far end of the chain, rather than closer to the cationic group
(shown by the large Δδβ).
The spectral shifts and integration values provided by the 400 Hz 1
H NMR
spectrometer suggest that SCX[6] binds by engulfing the guest molecule such that the
macrocycle surrounds the area near the carbonyl group at the center the guest
molecule. This theory is supported by the large upfield shift at the [1] and [2] protons
next to the carbonyl group, indicating that these protons interact the most with the
host cavity. Additionally, it can be observed that there was a single peak for the [1]
and [2] protons for SCX[4], but the peaks separate in the SCX[6] spectra. The
separation of peaks indicates different chemical environments, which would occur in
this conformation due to host molecule having anionic sulfonated groups on one
side, and anionic hydroxyl groups on the opposite side. If the guest molecule was
threaded through the host cavity it would reflect these chemical differences, as is
shown in the SCX[6] spectra. Another possible conformation that could cause these
spectral shifts would be if the guest molecule loosely lay on top of the host
macrocycle, with the carbonyl group (represented by the [1] and [2] protons) within
the cavity. A ChemDraw 3D structure depicts these binding mechanisms of the
SCX[6] molecule to the guest molecule in the appendix in Figure 4.2.

12
The fitted data of Δδβ vs. host concentration (Appendix 7.7) also supports these
theories by showing that the host molecule binds at a 1:1 ratio.
The spectral shifts and integration values provided by the 400 Hz 1
H NMR
spectrometers suggest that guest molecule binds to SCX[8] by resting on top of the
host cavity, with interactions between the host and guest aromatic groups. While it is
also possible that the guest molecule could thread through the host macrocycle, this
theory is supported by the large upfield shift of the guest aromatic protons, indicating
that this area is what has the most interactions with the ring structure of the host
molecule. Another possible conformation would be if the guest molecule threaded
through the host macrocycle, similar to the complexation of SCX[6]. The guest
molecule is flexible enough that the [1] and [2] protons on the carbonyl group would
be within the cavity, with the aromatic groups bending such that they interact with
the aromatic groups on the host molecule. A ChemDraw 3D structure in the
appendix depicts this binding mechanism of the SCX[8] molecules to the guest
molecule (Figure 4.3). The upfield shifts of the [1], [2], α, and β protons on the guest
molecules show that a majority of the guest molecule is within the host cavity,
suggesting that the first structure is more probable. The fitted data of Δδβ vs. host
concentration (Appendix 7.9) also determines that the host molecule binds at a 1:1
ratio.
4.4 Assessment of Experiment
When considering the size of each host molecule, the binding mechanisms can be
more easily understood. The smallest host molecule, SCX[4], is only large enough to
just fit on the very ends of the guest molecule, before it encounters too much
electrostatic repulsion. The medium host molecule, SCX[6], is larger and therefore
less inhibited by these repulsion. This allows the host to enclose the molecule at the
guest’s center, at the carbonyl group, and be stabilized on either side by the guest’s
cationic groups. A secondary SCX[6] complex that could occur involves the guest
molecule loosely resting on top of the host cavity, with weak interactions at the
aromatic groups; however, this would require slightly more energy for the guest
molecule to bend to fit the SCX[6] ring. The largest host molecule, SCX[8], allows
the entire guest chain to rest on top of the host macrocycle. This occurs because the
guest molecule does not need to overly constrict itself when it binds its ends to the
aromatic macrocycle on the host molecule, nor does it need to overcome the
electrostatic repulsions of passing through the macrocycle. It can also thread the
guest molecule through the host cavity - this would involve overcoming steric
repulsion and electrostatic repulsion in the binding mechanism, and therefore is less
likely.
The arrangement of the SCX[4] and guest complex is similar to that found in
previous studies of the host molecule, cucurbit[7]uril, and guest, 1,8-bis(p-
aminobenzamidine)octane, where the host binds at either a 1:1 or 2:1 ratio at the ends
of the guest molecular chain. The SCX[6] conformation also matches that found in
previous studies of the cucurbit[7]uril, and the AChE inhibitor Nafamostat, in which

13
the guest molecule is threaded through the hydrophobic cavity of the host.9
Additionally, previous studies on the structure on p-sulfonatocalix[4]arenes have
shown the conformations that occur in both SCX[4] and SCX[8] complexes to occur
during binding.10
This supports the theory that host molecules of this similar
structure are capable of binding to AChE inhibitors through predictable
conformations.
4.5 Potential Errors
The qualitative information that this experiment provides for the binding mechanism
of the host to guest molecule has enough support to be assumed accurate; however,
the project could be better understood if more 1
H NMR spectra was provided at a
lower concentration of host and guest complex. There is enough data to deduce the
binding complex and mechanism, but a quantitative value could be determined for
the binding constant if there was more data for the start of the binding reaction. The
binding of the host and guest may be strong, but in a biological environment there
are numerous compounds that could out-compete the 4-sulfonatocalix[N]arene.
Therefore, it is necessary to predict the binding constant to see if it can limit the
competitive processes.
Accuracy was also lost in the fitted plots of Δδβ vs. host concentration: the data
points could vary by + 0.01ppm through spectrometer error, and not enough data
points were available to confidently find the binding constant. Understanding of the
complexation of SCX[4] was imprecise, as the data in the fitted plot of Δδβ vs. host
concentration for this host molecule was slightly irregular. This was due to the
complicated nature of having two equilibrium reactions - a 1:1 complexation reaction
at lower concentrations, and a 2:1 complexation at higher concentrations.
Additionally, the molecular weights for the host molecules were rough estimates
based on the anhydride form of the molecule, and may be inaccurate. Overall, the
mechanism could be better investigated by gaining more spectral data, and by the
additional methods suggested below.
4.6 Future Work
To further investigate the binding mechanisms, more 1
H NMR spectra should be
obtained for host and guest concentrations that are even lower than in this study - the
spectral shifts at these concentrations would demonstrate more of the activity
occurring during binding. This would provide the necessary data that is currently
needed in the plots of Δδobs against the SCX concentration, which could be then be fit
to calculate the binding constant, K. Also, more information on the nature of the
molecules and their chemical reactivity when binding could be gained by using UV-
visible spectroscopy for all three SCX complexations, rather than just for preliminary
observation. Mass spectrometry could also be useful in measuring the mass-to-charge
ratio of the molecules. This will improve the accuracy of the available data and can
be used to better understand the exact nature of the binding mechanism.
It is also recommended that this experiment be replicated using the 4-
sulfonatocalix[N]arene molecules (N=4,6,8) and different AChE inhibitors, rather

14
than just 1,5-bis(4-allyldimethylammoniumphenyl)pentan-3-one dibromide. As this
molecule is used only for research and not for clinical or drug use, the information
determined in this study on the binding of acetylcholinesterase inhibitors can only be
applied to laboratory settings. In order to establish if the 4-sulfonatocalix[N]arenes
can be used as a drug reversal agent, the host-guest binding behaviour should be
tested with AChE inhibitors that are used in clinical applications such as Rocuronium,
Vecuronium, and Pancuronium. It is already known that the length of the guest
molecular chain affects the binding mechanism of 4-sulfonatocalix[N]arenes, making
this research critical.14
To model binding in the various conditions that could be
encountered in a biological environment, the experiment could also be run in
conditions of varying acidity, with different types of salts used (rather than just
NaCl).
Alternatively, 4-sulfonatocalix[N]arenes could be researched for their application
selective binders in non-clinical applications and their potential as biosensors, due to
their ability to absorb light in the UV-visible range wavelength (λ~190-750nm).
5.0 Conclusions
The experiment successfully determined that 4-sulfonatocalix[N]arenes can bind to
an AChE inhibitor guest molecule, 1,5-bis(4-allyldimethylammoniumphenyl)pentan-
3-one dibromide, at a 1:1 and 2:1 ratio, and explained the varying binding
mechanisms of the complexes formed. It was proved that increasing the number of
aromatic groups in the host (N=4,6,8) would allow for different conformations in the
bound complex, and that by increasing host molar ratio, (in the case of SCX[4]) 2:1
complexes can be formed.
The data gathered through this experiment provides information on a new type of
host molecule that can substantially bind to an AChE inhibitor in a variety of
arrangments, according to the size of the host molecule. A large binding constant is
needed to out perform biological competitors; for drug reversal, however, the guest
molecule must not bind so strongly that it cannot be released in specific conditions.
4-Sulfonatocalix[N]arenes are useful because their macrocycle size can be easily
altered to fit a specific guest molecule by changing the number of aromatic groups,
and they possess unique reactivity due to their their sulfonated and hydroxylated
anionic side chains. They can also form either 1:1 or 2:1 complexes with the guest
molecule, and can bind at multiple locations in various conformations.
Although it was not confirmed if 4-sulfonatocalix[N]arenes are suitable drug reversal
agents for acetylcholinesterase inhibitors, the information presented recommends
them for future research. Further 1
H NMR, UV-visible, and mass spectrometry
experimentation with these molecules at lower concentrations would shed light on
their qualitative binding constants; research into 4-sulfonatocalix[N]arenes’ binding
abilities with various clinical AChE inhibitors in a biological environment would
confirm if these host molecules are suitable for medical applications.

15
6.0 References
(1) Lehn, J.M. Supramolecular Chemistry – Scope and Perspective Molecules,
Supermolecules, and Molecular Devices. Angew. Chem. Int. Ed. Engl. 1988, 27, 89-
112.
(2) Purves, D., Augustine, G.J., Fitzpatrick, D., et al. Neuroscience, 2nd ed.; Sinauer
Associates: Sunderland, 2001.
(3) Ard, M.D. Fundamental Neuroscience, 2nd ed.; Sinauer Associates: Philadelphia,
2002.
(4) Colovic, M.B., Drstic, D.Z., Lazarevic-Pasti, T.D., et al. Acetylocholinesterase
Inhibitors: Pharmacology and Toxicology. Curr. Neuropharmacol. [Online] 2013.
11, 3. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3648782 (accessed Mar
19, 2015).
(5) Lane, R.M., Potkin, S.G., and Enz, A. Targeting Acetylcholinesterase and
Butyrylcholinesterase in Dementia. Nerusopyschoph. [Online] 2006, 91, 101-124.
http://www.ncbi.nlm.nih.gov/pubmed/16083515 (accessed Mar 19, 2015).
(6) Olivera-Bravo, S., Ivorra, I., and Morales, A. The Acetylcholinesterase Inhibitor
BW284c51 is a Potent Blocker of Torpedo Nicotinic AchRs Incorporated into the
Xenopus Oocyte Membrane. Br. J. Pharmacol. [Online] 2005, 144, 88-97.
http://www.ncbi.nlm.nih.gov/pubmed/15644872 (accessed Mar 21, 2015).
(7) Rang, H.P., Dale, M.M., and Ritter, J.M. Pharmacology, 3rd ed.; Harcourt
Publishers, Ltd.: Edinburgh, 2001; pp 19-46.
(8) Rong-Guang, L., La-Sheng, L., Rong-Bin, H., et al. pH-Controlled Formation of
4-Sulfocalix[4]arene-based 1D and 2D coordination polymers. Inorg. Chem.
Comm. [Online] 2007, 10, 1257-1261.
http://www.sciencedirect.com/science/article/pii/S1387700307002833
(accessed Mar 19, 2015).
(9) Henderson, K. B.Sc. Thesis, Queen’s University, 2014.
(10) Francisco, V., and Garcio-Rio, L. Interaction of Bolaform Surfactants with p-
Sulfonatocalix[4]arene: The Role of Two Positive Charges in the Binding.
Langmuir 2014, 30, 6748-6755.
(11) Wyman, I.W., and Macartney, D.H. Host-Guest Complexes and
Pseudorotaxanes of Cucurbit[7]uril with Acetylcholinesterase Inhibitors. J. Org.
Chem. 2009, 74, 8031-8038.

16
7.0 Appendix
Supplemental Tables and Figures
H NMR spectroscopy (400 Hz)
Host
α
β
γ
α'
Aromatic
[1]
[2]

0
N/A
5.43
5.47
2.73
7.36
2.81
2.81

0.26
N/A
4.98
5.06
2.73
6.95
2.68
2.46

0.445714286
N/A
4.92
5
2.76
-‐
2.54
2.38

0.585
N/A
4.94
5.01
2.76
6.99
2.59
2.41

0.693333333
N/A
4.91
4.99
2.77
6.94
2.5
2.36

0.78
N/A
4.91
4.99
2.78
7.01
2.49
2.36

0.850909091
N/A
4.9
4.99
2.78
7.01
2.49
2.36

0.91
N/A
4.9
4.99
2.79
7.01
2.48
2.35

0.96
N/A
4.9
4.99
2.79
7.01
2.46
2.34

1.002857143
N/A
4.92
4.97
2.79
7.01
2.45
2.33

Max.
Δδ
-‐
-‐0.53
-‐0.5
3.87
-‐0.42
-‐0.06
-‐0.48

Host
α
β
γ
α'
Aromatic
[1]
[2]

0
N/A
5.44
5.42
0
7.57
0
0

0.7475
N/A
5.42
5.4
0
7.57
0
0

1.281428571
N/A
5.41
5.39
-‐0.01
7.54
0
0

1.681875
N/A
5.38
5.36
-‐0.03
7.55
-‐0.01
-‐0.01

1.993333333
N/A
5.37
5.35
-‐0.04
7.55
-‐0.01
-‐0.01

2.2425
N/A
5.34
5.32
-‐0.04
7.55
-‐0.01
-‐0.01

2.446363636
N/A
5.31
5.29
-‐0.05
7.54
-‐0.01
-‐0.01

2.61625
N/A
5.3
5.28
-‐0.05
7.54
-‐0.01
-‐0.01

2.76
N/A
5.28
5.26
-‐0.08
7.53
-‐0.01
-‐0.01

2.883214286
N/A
5.22
5.2
-‐0.08
7.53
-‐0.02
-‐0.02

2.99
N/A
5.25
5.22
-‐0.07
7.53
-‐0.01
-‐0.01

Max.
Δδ
-‐
-‐0.22
ppm
-‐0.21
ppm
-‐0.08
ppm
-‐0.08
ppm
-‐0.02
ppm
-‐0.02
ppm

17
Host
α
β
γ
α'
Aromatic
[1]
[2]

0
4.47
5.43
N/A
2.73
7.36
2.81
2.81

0.341666667
4.43
5.3
N/A
2.73
6.56
2.47
2.47

0.585714286
4.43
5.31
N/A
2.76
6.17
2.38
2.38

0.76875
4.35
5.31
N/A
2.76
6.17
2.38
2.38

0.911111111
4.37
5.31
N/A
2.77
6.17
2.38
2.38

1.025
4.4
5.31
N/A
2.78
6.17
2.38
2.38

1.118181818
4.38
5.3
N/A
2.78
6.17
2.38
2.38

Max.
Δδ
-‐0.09
-‐0.13
-‐
-‐0.01
-‐1.99
-‐0.43
-‐0.43

Figure 4.1 SCX[4] and guest complex conformations
Figure 4.1 Binding conformations for SCX[4]. 1:1 binding complex at low concentrations (left)
and 2:1 binding complex at higher concentrations (right)

18
Figure 4.2 Binding conformations for SCX[6]. Complex with guest molecular chain threaded through host cavity
(left) and complex with guest resting on top of host molecule (right)
Figure 4.3 Binding conformations for SCX[8]. Complex with majority of guest within host cavity (top), and
complex with guest molecular chain threaded through host cavity (bottom)

19
7.0 Labeled Guest Molecular Diagram
Appendix 7.0 Labeled Guest Molecular Diagram
7.1 UV-Visible SCX[4] spectra
Appendix 7.1 UV-Vis Spectra
aromatics
α'
α'
α
β
γ
1
2

20
7.2 Fitted UV-Vis Data
Appendix 7.2 Fitted UV-Vis Data
7.3 300 Hz SCX[4] Stacked Plot
Appendix 7.3 300 Hz Stacked Plot
0
0.5
1
1.5
2
2.5
0 0.2 0.4 0.6 0.8 1
Absorbance
Host Concentration (mM)
Increase in Absorbance at 282nm over
Increasing Host Concentrations

21
7.4 400 Hz stacked plot SCX[4]
Appendix 7.4 400 Hz Stacked Plot SCX[4]
7.5 SCX[4] shift graph
Appendix 7.5 Shift Graph SCX[4]
-0.25
-0.2
-0.15
-0.1
-0.05
0
0 0.5 1 1.5 2 2.5 3 3.5
Deltaδ(ppm)
[H]/[G]
SCX[4] Δδ ppm with Beta

22
7.6 400 Hz stacked plot SCX[6]
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
0 0.5 1 1.5 2 2.5 3 3.5
deltaδ(ppm)
[H]/[G]
SCX[4] Δδ ppm with Beta

23
7.8 400 Hz Stacked Plot SCX[8]
-1.4
-1.2
-1
-0.8
-0.6
-0.4
-0.2
0
0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2
Δδppm
[H]/[G]
SCX8 Δδ ppm (Aromatics)

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