Synthesis, Characterization, Critical Micelle Concentration and Biological Activity of two-Headed Amphiphiles

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.