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This thesis comprises three parts united by a single theme: development of flexible ditopic receptors. In part 1, two bis(crown ether)s were synthesized and their binding selectivities with alkali, alkaline earth and α,ω-primaryalkylidenediammonium cations were studied by electrospray ionization mass spectrometry (ESI-MS). First, we confirmed that the ion intensities of complexes in the gas phase are linearly related to the concentrations of complexes in solution for single crown ether dicarboxylic acid. Binding selectivities of complex bis(crown ether)s with mixtures of alkali cations and with mixtures of alkaline earth cations were then determined directly from ESI-MS spectra. The results from ESIMS are consistent with literature data if ions of like charge and similar type are compared (e. g., among the alkali metals). The stoichiometries of complexes in solution were also probed. Complexes with up to two K⁺ per crown ether were detected by ESI-MS. The research shows that ESI-MS provides an effective tool to study complexation by structurally complex molecules in solution. From the ESI-MS results, bis(crown ether) bolaamphiphiles were designed and synthesized as cation-recognition based membrane-disruption agents. Three bis(crown ether)s were obtained by capping an 18-crown-6 dicarboxylate anhydride with different lengthes of α,ω-alkanedicarboxylic acids extended as the 3-amino-1-propyl esters. Their membrane disrupting activities were explored using vesicle encapsulated 5(6)- carboxyfluorescein (CF) by a fluorescence self-quenching (FSQ) method. The membrane disrupting activity is significantly and specifically enhanced specifically by the addition Sr²⁺ or Ba²⁺ in solution. The membrane-disrupting activity is also enhanced with a increased aliphatic loop length of the starting α,ω-alkanedicarboxylic acid. Based on the mechanism studies of Regen and work conducted in this thesis, we propose that the active form for membrane-disruption is created by a U-shaped sandwich complex between Ba²⁺ and the bis(crown ether) bolaamphiphiles which interacts only with the outer leaflet of the vesicle bilayer. In part 3, a photoswitchable bis(crown ether) based on thioindigo was designed and synthesized as a cation- and photo-regulated membrane-disruption agent. The bis(crown ether) was prepared by capping an 18-crown-6 dicarboxylate anhydride with 7,7’-thioindigo dicarboxylic acid extended as the 8-amino-1-octanyl esters. There is significant difference in the membrane-disrupting activities of the cis- (U-shape) and trans- (S-shape) isomers using the vesicle entrapped CF (FSQ) method. Alkaline earth cations suppress the cis-to-trans thermal isomerization and stabilize the cis-isomers of the 7,7’- thioindigo bis(crown ether) in organic solvent. The results confirm the mechanism proposed, namely, that a U-shaped conformation is required for membrane disruption, that the bis(crown ether)s form sandwich complexes with alkaline earth metal ions. / Graduate

Thesis (Ph. D.)--Ohio State University, 2003. / Title from first page of PDF file. Document formatted into pages; contains xvii, 227 p. Includes abstract and vita. Advisor: Andrej Rotter, Dept. of Pharmacology. Includes bibliographical references (p. 201-227).

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Genetic variation within the cannabinoid 1 receptor (CB1R) locus (CNR1) has been repeatedly associated with drug addiction pathologies. Genomic annotation of CNR1 indicates the vast majority of this genetic variation likely results in altered transcriptional regulation of the CNR1 gene as a mechanistic link to the disease phenotype. There is a lack of information describing the regulation of CNR1 transcription and the potential impact of disease-associated variation within the CNR1 locus on its transcriptional regulation. This study investigates the impact of an evolutionary conserved regulatory region of CNR1, termed ECR1, and the disease-associated variation contained within, on the transcriptional activity of the cognate CNR1 promoter region. Reporter assays conducted in primary hippocampal cells demonstrate that CNR1 promoter exhibits variable transcriptional activity during periods of CB1R signalling and cell depolarisation. Coupled to allelic variants of ECR1, the CNR1 promoter shows significant changes in transcriptional activity under resting conditions indicating that disease-associated variation within ECR1 may decrease CNR1 transcription. Further, alleles of ECR1 can drive allele-specific transcriptional responses from the CNR1 promoter during periods of CB1R stimulation and cell depolarisation. The results highlight the potential for disease-associated regulatory variation of the CNR1 locus to create stratified transcriptional responses to specific cell signalling scenarios and putatively to clinical strategies employing pharmacological agents. Furthermore, investigation of DNA-protein interactions at the allelic ECR1 region demonstrate that disease-associated variation within ECR1 alters DNA-protein interactions within the nucleus consistent with a decrease in transcriptional activity in the disease-associated allele variant. Collectively the current work supports the hypothesis that disease-associated variation within the ECR1 regulatory region of the CNR1 locus has the capacity to significantly impact on CNR1 promoter transcriptional activity. It is posited that allele-specific transcriptional effects may have a major impact on the susceptibility of individuals to drug addiction or on responses to clinical pharmacological treatments.

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The implication of the transmembrane signalling Receptor Tyrosine Kinases (RTKs) in cancer has accelerated the pursuit for drugs to target these molecules. In the process our understanding of how these membrane bound molecules are entangled in cell signalling has significantly expanded. There is now evidence that RTKs can facilitate the formation of a lattice-type network of signalling molecules to elicit whole cell responses to external ligand stimuli. Although beginning to be unravelled, knowledge pertaining to the mechanisms of molecular control that initiate these signalling pathways is still in its infancy. In this thesis, a random mutagenesis approach allowed the identification of the crucial interaction surfaces between membrane-bound EphA3 and its preferential binding partner ephrinA5, that are required to induce the formation of higher-order Eph signalling complexes. Modelling and experimental dissection of this co-ordinated receptor aggregation has provided detailed insights into the molecular mechanisms of Eph receptor activation, which in some aspects may also apply to other members of the RTK family. In particular, the importance of certain molecular interfaces in determining preferential and non-preferential Eph/ephrin interactions, suggests their role in the selection of biologically important binding partners. In addition to the assignment of the ephrin-interaction surfaces, the random mutagenesis strategy also identified a continuous conformational epitope as binding site for an anti-EphA3 monoclonal antibody. Fortuitously, antibody binding to this site functionally mimics ephrin stimulation of EphA3 positive cells, and in particular together with divalent ephrinA5, yields synergistically enhanced EphA3 activation. Elucidation of the underlying mechanism has provided opportunities to develop an efficient EphA3 targeting mechanism that is based on increased affinity and accelerated ephrinA5 uptake as consequence of this unique activation mechanism. On a genetic level, novel oligonucleotide analogues known as Peptide Nucleic Acids (PNAs) were analysed for their ability to sterically inhibit EphA3 DNA transcription and suggest a dosedependent downregulation of EphA3 expression, in malignant melanoma cells. Combined, ephrinA5, the anti-EphA3 MAb (IIIA4) and PNA, offer the possibility to investigate the specific machinery involved in Eph receptor expression and signalling for the specific targeting of EphA3 expressing tumour cells.