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. 1H 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 1H NMR Spectroscopy .................................................................. 7

2.2 UV-Visible Spectroscopy ........................................................................ 7 2.3 1H 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 4.2 4-Sulfonatocalix[6]arene ....................................................................... 11 4.3 4-Sulfonatocalix[8]arene ....................................................................... 12 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 1H NMR spectroscopy (400 Hz)..................16 Table 3.2 Chemical shifts for SCX[4] using 1H NMR spectroscopy (400 Hz)..................16 Table 3.3 Chemical shifts for SCX[4] using 1H NMR spectroscopy (400 Hz)..................17 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

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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 1H 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. 1H 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 1H 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 1H 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 1H 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, 1H 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, 1H, 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. 1H 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 1H 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 1H 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 1H NMR spectrometer. A stacked 1H 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 1H NMR spectrometer. This concentration range was used also as a general estimate for the 400 Hz SCX[6] and SCX[8] titrations. 400 Hz 1H NMR Spectra A stacked 1H 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.

3.2 SCX[6]:Guest Complexes 400 Hz 1H NMR Spectra A stacked 1H NMR plot (Appendix 7.6) demonstrated shift changes as the host 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]. The change in chemical shift (Δδobs) of the peaks was plotted against the SCX[6] concentration, with the concentration values adjusted according to the integration 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.

3.3 SCX[8]:Guest Complexes 400 Hz 1H NMR Spectra A stacked 1H NMR plot (Appendix 7.8) demonstrated shift changes as the host 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. The change in chemical shift (Δδobs) of the peaks was plotted against the SCX[8] concentration, with the concentration values adjusted according to the integration 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 1H 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).

4.2 4-Sulfonatocalix[6]arene The spectral shifts and integration values provided by the 400 Hz 1H 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.

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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.

4.3 4-Sulfonatocalix[8]arene The spectral shifts and integration values provided by the 400 Hz 1H 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

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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 1H 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 1H 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

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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 1H 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.

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7.0 Appendix

Supplemental Tables and Figures Table 3.1 Chemical shifts for SCX[4] using 1H 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 Table 3.2 Chemical shifts for SCX[6] using 1H NMR spectroscopy (400 Hz)

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

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Table 3.3 Chemical shifts for SCX[8] using 1H NMR spectroscopy (400 Hz) 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)

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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)

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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

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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

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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]

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