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THE BINDING, DELIVERY, REVERSAL, AND STABILIZATION OF DRUG MOLECULES BY REBECCA PIERCE SUPERVISOR: DR. DONAL MACARTNEY A STUDY ON THE BINDING ABILITIES OF A GUEST MOLECULE, 4-SULFOCALIX[4]ARENE , TO AN ACETYLCHOLINESTERASE INHIBITOR Acetylcholinesterase inhibitors (AChEI) are chemicals that restrain the enzyme acetylcholinesterase (AChE) from decomposing the neurotransmitter acetylcholine (ACh). While they can naturally exist as venoms, AChEI are also used in a variety of medical applications for conditions including Alzheimer’s & Parkinson’s diseases, glaucoma, and cognitive impairments in patients with schizophrenia. They can serve as indirect reversal agents for neuromuscular blockers by increasing the competitive ability of ACh to bind to the available binding sites. Successful research has already been conducted on the binding ability of host molecule Cucurbit[7]uril to acetylcholinesterase inhibitors. For this study, a molecule similar in structure to Cucurbit[7]uril will be used as the host molecule: 4-Sulfocalix[4]arene ( SCX[4] ). SCX[4]’s ability to selectively bind to the acetylcholinesterase inhibitor 1,5-Bis(4-allyldimethylammoniumphenyl)pentan-3-one dibromide will be analyzed through the 1 [H] NMR spectra (300 Hz) and UV-Vis spectra of increasing concentrations of host molecule in a constant concentration of guest solution. 0 0.5 1 1.5 2 2.5 3 200 206 212 218 224 230 236 242 248 254 260 266 272 278 284 290 296 302 308 314 320 326 332 338 344 350 Absorbance Wavelength (nm) Formation of Peaks over Titration of Increasing Host Concentration (0.000 mM -> 0.909 mM) 0.000 mM 0.099 mM 0.196 mM 0.291 mM 0.385 mM 0.476 mM 0.566 mM 0.654 mM 0.741 mM 0.826 mM 0.909 mM 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 However, many of these substances have the potential to produce negative side effects in patients, leading to the necessity of research into the controlled binding and release of acetylcholinesterase inhibitors. A host molecule can be used that recognizes the cationic quaternary ammonium or iminium groups on the guest molecule through hydrophobic effects and non-covalent ion/dipole interactions, and binds to it. The UV-Vis spectra can be seen for concentrations of SCX[4] from 0.000 mM to 0.909 mM, exhibiting a shift in peaks as the amount of host molecule increased (Figure 1). The peak at 282nm was examined in detail at the varying concentrations, and graphed (Figure 2) to show the increase in absorbance as the host molecule is added. The peak shifts demonstrate the interactions occurring between the host and guest molecule, and a fit of data in Figure 2 produced a binding constant of 125 M -1 . Figure 1 : UV-Vis spectra over increasing SCX[4] concentrations Figure 2 : Absorbance at 282nm Acetylcholine (ACh) is broken down by… Acetylcholinesterase (AChE) which is restricted by… Acetylcholinesterase inhibitor (AChEI) which can be controllably bound & released by… A selective host molecule, 4- Sulfocalix[4] arene Guest Molecule 1,5-Bis(4-allyldimethylammoniumphenyl)pentan-3-one dibromide Host Molecule 4-Sulfocalix[4]arene SCX[4] A series of titrations was also conducted for use in 1 [H] NMR spectroscopy (300 MHz). Figure 3 shows four different spectra at increasing concentrations of host molecule: 1. Pure guest molecule 2. 1:1: concentration of host to guest 3. 5:1 concentration of host to guest 4. 10:1 concentration of host to guest ppm Figure 3 : [1] H NMR (300 Hz) spectra over increasing SCX[4] concentrations Significant data from the [1] H NMR spectra includes: • Upfield shift of α, β, γ carbons on Guest molecule • Δδ α = -0.732 ppm • Δδ β = -1.588 ppm • Δδ γ = - 0.801 pp • Upfield shift of methyl groups (1) attached to the quaternary ammonium group on Guest molecule • Δδ 1 = - 0.388 ppm • Downfield shift of aromatic protons on the Host molecule • Δδ Host aromatic = + 0.062 ppm • The large upfield shifts show that the SCX[4] host is likely binding by engulfing both sides of the guest molecule near the ammonium groups. The downfield shift represents minor binding activity at the outside of the guest, near the carbonyl group. 3 2 γ α β γ α β γ γ γ α α α β β β 2 & 3 1 1 1 1 1 2 & 3 2 & 3 2 & 3 --water-- Host aromatic Host aromatic Host aromatic l l --acetone-- (contaminant)
