Synthesis, Characterization, Critical Micelle Concentration and Antimicrobial Activity of Two-headed Amphiphiles

Abstract

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.