Cys-loop receptors are ligand-gated ion channels that are involved in fast synaptic neurotransmission in the central and peripheral nervous system. The Cys-loop receptor RDL ('resistant to dieldrin') is a GABA-gated chloride channel from Drosophila melanogaster and is a major target site for insecticides. The aim of this dissertation was to characterise RDL receptors with particular focus on the agonist binding site. To assess the potency of a range of GABA analogues on RDL receptors, I expressed receptors in Xenopus oocytes and used voltage-clamp electrophysiology to detect receptor responses. I carried out computational modelling of these analogues to determine the dipole separation distances and atomic charges. Computational calculations and functional experiments revealed that agonists require a charged ammonium and an anionic centre, with the most potent agonists having a dipole separation distance of ~5 Å. I made a homology model of the extracellular domain of RDL and docked the active analogues into the putative binding site. I then conducted mutagenesis studies to test the accuracy of this model. Functional data from mutagenesis studies broadly support the location of GABA within this model. This model may be useful for further structure-activity studies and rational drug design. Natural compounds from the traditional Chinese medicine 'Ginkgo biloba' (ginkgolide A, ginkgolide B and bilobalide) have potent insecticidal properties and are similar in structure to picrotoxin. I tested the effect of these compounds on RDL receptor function using voltage-clamp electrophysiology. All compounds were found to inhibit RDL receptor function. I probed the binding site of these compounds using site-directed mutagenesis and electrophysiology. Mutations to the 2'A and 6'T channel-lining (M2) residues greatly reduced the potency of these compounds. I then made a homology model of the transmembrane domain of RDL and docked these compounds into the channel. Compounds docked into the channel pore close to the 2' and 6' channel-lining residues and H-bonding interactions were detected at these locations. Ginkgolides are therefore antagonists of RDL receptors, binding in the channel close to the 2' and 6' residues and this may be the mechanism underlying their potent insecticidal properties. The 5-HT3 receptor is a member of the Cys-loop receptor family and shows homology to RDL receptors. To explore different techniques for studying Cys-loop receptor function I assessed the functionality of two brain derived transcripts of the 5-HT3B subunit (Br1 and Br2) using single-channel electrophysiology and a fluorometric assay. Receptors containing Br1 were found to have a conductance identical to the 5-HT3B subunit whilst Br2 receptors were found not to be expressed. This finding has implications for 5-HT3 brain signalling, in which Br1 may play an important role. In conclusion, work here has described how agonists bind to and activate RDL GABA receptors and I have identified a candidate mechanism for the potent insecticidal properties of Ginkgo biloba extracts. I have also confirmed that 5-HT3 receptor brain transcript Br1 forms functional channels with similar properties to the 5-HT3B subunit.

Ginkgolides are naturally occurring compounds that can be extracted from the Ginkgo Biloba tree. Their synthesis remains a significant challenge for organic chemists given their complex structure and their numerous stereocenters (2 adjacent quaternary carbons and up to 12 stereocenters). Both Corey and Crimmins reported the total synthesis of (±)-ginkgolide B in 1988 and 1999 respectively. Corey also published the enantioselective formal synthesis of (±)-ginkgolide B as well as the total synthesis of (±)-ginkgolide A in 1988. However, the total synthesis of Ginkgolide C, the most oxygenated and most complex member of the family, has not yet been published. We report herein the first total synthesis of (±)-ginkgolide C as well as the formal synthesis of (±)-ginkgolide A and (±)-ginkgolide B by intercepting Corey’s intermediate (from his total synthesis of (±)-ginkgolide B). The first key step of our syntheses was the Claisen rearrangement which set the first quaternary carbon. The second key step of the syntheses was a kinetic alkylation which sets the second quaternary carbon. The third key step for our syntheses was an enyne epoxidation which enabled the formation of the E-ring. Starting from the Claisen rearrangement adduct, our target intermediate (towards ginkgolide C) was obtained in 18 steps, after which, (±)-ginkgolide C was synthesized in an additional 6 step (total of 26 linear steps). Starting again from the Claisen rearrangement adduct, Corey’s intermediate (from the total synthesis of (±)-ginkgolide B) was obtained in 15 steps which completed the formal syntheses of (±)-ginkgolide A (in an additional 10 steps) and (±)-ginkgolide B (with 6 additional steps).