The primary objective of the proposed research is to synthesize new compounds that can be used to control lipid-mediated membrane fusion. An interdisciplinary project is proposed involving synthetic chemistry, theory, biophysics, and cell biology aimed at expanding the range of materials that are currently available for accelerating this fundamentally important process. The proposed materials will be incorporated within guest membrane vesicles as masked, nonfusogenic compounds that will become fusogenic upon localized chemical triggering by exposure to low pH or oxidative environment--an unmasking process that is analogous to influenza hemagglutinin and other viral protein-based membrane fusion pathways. Preliminary computational experiments have been used to design a set of synthetic target molecules, containing vinyl ether bonding motifs at optimal strategic loci, that will efficiently promote membrane fusion after degradation of this linkage has been triggered. Synthetic methodology developed by Run & Thompson [J. Org. Chem. 1994 59 5758; Chem. Eur.J. 1996 2 1505] will be used to install the labile vinyl ether linkages in the proposed series of masked amphiphilic fusogens. The resulting compounds will then be tested for their ability to promote membrane fusion in model membrane systems under experimental conditions capable of deploying the fusogen via vinyl ether bond cleavage. HPLC analysis and membrane fusion fluorescence assays will be used to monitor the rates of fusogen unmasking, vesicle lipid mixing, and vesicle contents mixing after chemical activation has occurred; these results will be compared with the predictions made by mean-field single chain theory. Physical characterization of the membrane structures, before and after triggering, will also be performed using 31P NMR, freeze-fracture electron microscopy, and x-ray scattering techniques. The most efficient fusogens in the model membrane experiments will be assayed for their efficacy in promoting cytoplasmic release from KB cell endosomal compartments targeted via folate-conjugated DSPE-PEG. Cytoplasmic membrane fusion efficiency, in the absence of endosomal uptake, will also be determined using flow cytometry and laser confocal microscopy techniques to evaluate the utility of these materials for intracellular delivery hydrophilic reagents that typically experience slow rates of membrane translocation (e.g., peptides, antisense oligonucleotides, and plasmids).
