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Probing the Structure of Acetylcholinesterase Inhibitors in Their Binding Site Using Solid State Nuclear Magnetic Resonance

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Probing the Structure of Acetylcholinesterase Inhibitors in their Binding Site using Solid State Nuclear Magnetic Resonance Scott Goodall Submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy Christ Church, Oxford Trinity 2002 Every attempt to employ mathematical methods in the study of chemical questions must be considered profoundly irrational and contrary to the spirit of chemistry … if mathematical analysis should ever hold a prominent place in chemistry, an aberration which is happily almost impossible, it would occasion a rapid and widespread degeneration of that science. Auguste Comte (1798-1857) Abstract Probing the Structure of Acetylcholinesterase Inhibitors in their Binding Site using Solid State Nuclear Magnetic Resonance Scott Goodall, DPhil Thesis Christ Church, Trinity 2002 Inhibitors of acetylcholinesterase are of commercial and medical interest as pesticides and as therapeutics in the treatment of Alzheimer’s Disease. An understanding of the conformation of inhibitors in the binding site enables the rational design of novel inhibitors with increased potency and specificity. Solid state NMR is a novel approach to the investigation of acetylcholinesterase and the binding of acetylcholinesterase inhibitors. Two compounds, 4- amino-5-fluoro-2-methyl-3-(3-trifluoroacetylbenzyl-oxymethyl)quinoline (R414425) and 4- amino-2-methyl-3-(3-trifluoroacetylbenzyl-oxymethyl)quinoline (R414983) were selected for study from a series of structures developed by Syngenta during research into novel pesticides. The series, based around a chimera of tacrine and m-(N,N,N-trimethylammonio)-2,2,2- trifluoroacetophenone, are ideal candidates for initial studies using solid state NMR since they demonstrate a high potency, have a large degree of conformational freedom and bind covalently to the enzyme at the active-site. Both compounds, R414425 and R414983 were successfully synthesised with the incorporation of 13C isotopic labels which, in conjunction with the fluorine atoms already present in their structures, provided the potential for homonuclear and heteronuclear distance measurements. 13 13 13 The rotational resonance technique was used to determine C- C distances in C5-R414983 bound to Electrophorus electricus AChE. The reliability of distance measurements in the presence of broad spectral linewidths was first examined through the measurement of two rigid internuclear distances, indicating that an accuracy of 0.5 Å could be attained using linewidth based estimation of zero-quantum relaxation parameters. Subsequently, the internuclear distance for the benzyl methylene/2-methyl spin pair was determined to be 3.9 ± 0.5 Å and that for the benzyl methylene/quinoline C2 pair to be 3.5 ± 0.5 Å. The use of a rotational resonance echo showed a low level of zero-quantum relaxation under rotational resonance for 13 13 19 one example pair of spins from C5-R414983 bound to Electrophorus electricus AChE. C- F REDOR experiments performed on a dilute rigid standard indicated that a carbon observed scheme where dephasing pulses were also applied to the carbon channel produced significantly more accurate results than similar schemes with dephasing pulses in the fluorine channel. 13C- 19F REDOR was then utilised to quantify the separation of the trifluoromethyl function and 13 13 the C atom at the 2-methyl position in C5-R414983 bound to Electrophorus electricus AChE, providing a distance of 7.1 ± 0.5 Å. The error level for each of the distance measurements is unfortunately high. However, modelling suggests that the determination of a single conformation for the bound inhibitor is restricted by the number of distance measurements presently available rather than the precision of the current values. Significant progress has been made towards elucidation of the first structure for an inhibitor bound to Electrophorus electricus acetylcholinesterase and, for the first time, determination of the conformation of an inhibitor bound to acetylcholinesterase through a means other than x-ray crystallography. Acknowledgements A great number of individuals have contributed to this work in some way or another. The quick mention of these people, carefully tailored to fit in a single side of A4, doesn’t really do justice to them. A reference to ten men in a boat comes to mind, but few people would understand that. Financial support was provided in the form of a CASE studentship from the Engineering and Physical Sciences Research Council in collaboration with Syngenta. Terry Lewis, Peter Howe, John Taylor and Caroline Personeni from Syngenta supplied me with support and technical advice at various points, for which I am very grateful. In addition to the opportunity and the infrastructure necessary to complete this work Tony has frequently exceeded his duties as a supervisor, supporting both my interests outside the lab and those of my wife. A special thanks to those who provided advice on particular areas – Ian, Carsten, Rene, Vincent and Stephan. Paul has helped with multiple sections and has always given freely of his time, while making me do everything the long, hard, correct way. Other members of the lab, past and present, provided helpful discussions on many occasions. Lastly, a big thanks to my family and friends, especially my parents who put in a lot of hard work over the last 27 years to get me to this point and my wife who has probably suffered more than me over the last few months. - iv - Contents Abstract............................................................................................................................ iii Acknowledgements ......................................................................................................... iv Abbreviations and Symbols ............................................................................................. ix Chapter 1 Introduction.............................................................................. 1 1.1 Biological Membranes..................................................................................................1 1.2 Acetylcholine.................................................................................................................3 1.2.1 Nerve Signalling ..............................................................................................3 1.2.2 Cholinergic Signalling.....................................................................................5 1.3 Acetylcholinesterase ...................................................................................................10 1.3.1 Nomenclature................................................................................................10 1.3.2 Macromolecular Structure and Cellular Localisation...............................11 1.3.3 Structure of Catalytic Subunits....................................................................13 1.3.4 Non-cholinergic Roles of Acetylcholinesterase........................................19 1.3.5 Acetylcholinesterase Inhibition...................................................................19 1.4 Nuclear Magnetic Resonance....................................................................................21 1.4.1 The NMR Spectrum.....................................................................................21 1.4.2 Solid State NMR ...........................................................................................25 1.5 Solid State NMR and Acetylcholinesterase.............................................................29 1.6 Project Aims................................................................................................................36 Chapter 2 Synthesis of Labelled Inhibitors............................................. 38 2.1 Overview......................................................................................................................38 2.2 Introduction ................................................................................................................39 - v - 2.2.1 Synthetic Route .............................................................................................39 2.2.2 Labelling Schemes.........................................................................................40 2.2.3 Internuclear Distance Constraints..............................................................42 2.3 Synthesis.......................................................................................................................43 2.3.1 General ...........................................................................................................43 2.3.2 Synthesis of 4-amino-5-fluoro-2-methyl-3-(3-trifluoroacetylbenzyl- oxymethyl)quinoline (R414425)..................................................................43 13 2.3.3 Synthesis of C5-4-amino-5-fluoro-2-methyl-3-(3-trifluoroacetylbenzyl- 13 oxymethyl)quinoline ( C5-R414425)..........................................................48 2.3.4 Synthesis of 4-amino-2-methyl-3-(3-trifluoroacetylbenzyl- oxymethyl)quinoline (R414983)..................................................................52 13 2.3.5 Synthesis of C5-4-amino-2-methyl-3-(3-trifluoroacetylbenzyl- 13 oxymethyl)quinoline ( C5-R414983)..........................................................56 2.4 Discussion....................................................................................................................61 Chapter 3 Characterisation of Inhibitors ................................................ 63 3.1 Overview......................................................................................................................63
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