In this project, we synthesized a new homologous series of five long-chain, two-headed amphiphiles [2CAm13, 2CAm15, 2CAm17, 2CAm19, 2CAm21; CH3(CH2)n-1CONHC(CH3)(CH2CH2COOH)2, n = 13, 15, 17, 19, 21]. The synthesis of the 2CAmn series was accomplished in four steps. The first step involves a reaction of nitroethane and two equivalents of tert-butyl acrylate to create the nitrodiester synthon [O2NC(CH3)(CH2CH2COOtBu)2] by successive Michael additions. The second step in the synthesis consists of a reduction of nitrodiester with H2 and Raney nickel to give the diesteramine [H2NC(CH3)(CH2CH2COOtBu)2]. The third step is the condensation of an acid chloride with diesteramine to give an alkanamido diester [2EAmn; CH3(CH2)n-1CONHC(CH3)(CH2CH2COOtBu)2, n = 13, 15, 17, 19, 21]. The final step is the removal of the tert-butyl protecting groups to give 2CAmn. Critical micelle concentration measurements were collected by the pendant drop method for measuring surface tension for a series of triethanolamine/2CAmn solutions to establish the concentration required for detergency. The CMCs for the 2CAmn series were found to decrease in value from 3.0 Ã 10â 2 M (2CAm13) to 1.7 Ã 10â 4 M (2CAm21) in a linear fashion [log CMC = (â 0.28 ± 0.01)n + (2.2 ± 0.1)]. The CMCs for the 2CAmn series falls in between the CMCs for three series of homologues three-headed amphiphiles (3CAmn, 3CCbn, 3CUrn) and the CMCs for fatty acids, with fatty acids having the lowest CMCs. Antibacterial activity (minimal inhibitory concentrations, MICs) for a series of homologous dendritic two-headed amphiphiles and three series of homologous, three-headed amphiphiles against Staphylococcus aureus and methicillin-resistent S. aureus (MRSA) were measured by broth microdilution to compare the effect of chain length and, hence, hydrophobicity. Inoculum density affected antibacterial activity of the 2CAmn series against both S. aureus and MRSA. MIC measurements at different cell densities showed that activity decreased with higher cell densities. For all four series, the MICs were relatively flat at low inoculum densities. This flat region defines the intrinsic activity, MIC0. The MIC0 results revealed that inoculum density, chain-length, and hydrophobicity all influenced antibacterial activity and that activity correlates strongly with clogp, an established measure of hydrophobicity. The most hydrophobic members from each homologous series exhibited antibacterial activity. The most active homologue of the 2CAmn series was 2CAm21 with MIC0 of 2.0 ± 1.0 and 3.2 ± 1.0 μM against S. aureus and MRSA, respectively. The CMCs and MIC0s of the two- and three-headed amphiphiles were compared for both S. aureus and MRSA to gauge the effect that micelles may have on activity. Amphiphile 2CAm19 has the largest ratio between CMC and MIC0 (CMC/MIC0 = 205) against S. aureus and 3CUr20 has the largest ratio (CMC/MIC0 = 339) against MRSA. These ratios suggest that micelle formation is not a mechanism of action for anti-Staphylococcal activity. / Master of Science

This project is about the synthesis of homologous series of two-headed, long-chain amphiphiles (the 2CCbn series, where n = 16, 18, 20, 22, 30, 5α-cholestan-3Ã -ol). The 2CCbn series was synthesized in five steps. The first step involves a reaction of nitroethane and two equivalents of tert-butyl acrylate to form nitrodiester by successive Michael addition reaction. The second step is the reduction of nitrodiester with Raney nickel to form aminodiester. The third step involves a reaction of aminodiester with di-tert-butyl dicarbonate [(Boc)2O] to form isocyanatediester. The fourth step is addition of iscocyanatediester with aliphatic alcohol to give alkyl carbamate diester (2ECbn) series. The fifth step is the removal of the tert-butyl protecting group to give the 2CCbn series. The critical micelle concentrations (CMC) were measured by the pyrene-based fluorescent probe method. The pyrene excited at 345 nm and fluoresces with maxima at 374 nm (I1) and 385 nm (I3). The stock solution and the dilution series for each amphiphiles were made in 0.9% triethanolamine solution. The CMCs were measured at two pH ~9.2 and 7.4. The CMCs were determined by plotting I1/I3 vs. concentrations. The CMCs were decreasing with increasing chain length. The CMCs for the 2CCbn series are lower than the 3CCbn series but higher than the fatty acids. The minimal inhibitory concentrations were measured against Staphylococcus aureus and methicillin-resistant Staphylococcus aureus. These strains were grown on BHIB+S with 5% triethanolamine. The MICs of the 2CCbn series amphiphiles were measured by using microtiter plate reader and by looking turbidity. The cutoff effect was found for the 2CCbn series. The MIC decreased up to C20 chain length and started rising for C22. The 2CCb18 (MICâ 2.2 µg/mL) of the 2CCbn series was the most effective amphiphile against S. aureus and MRSA. The CMC/MIC ratio was used to determine the safety of an amphiphile as a drug use. The amphiphile 2CCb18 has given the largest safety ratio (CMC/MIC = 273) against S. aureus and MRSA. It suggests that micelle formation is not a mechanism of action for anti-Staphylococcal activity. / Master of Science