Here, the CAC is defined as the concentration at which any additional SSA added to a solution will result in the production of high-order self-associated species, such as the spherical aggregates previously described. However, it does not mean that these larger self-associated aggregates do not exist in solution below the CAC, these species will instead exist in equilibria with those species present at the solution interface. To define CAC, we used the pendant drop method to elucidate the surface tension of H2O 5% EtOH solutions of 1, 3, 5, 7, 9 and 1:1 SSA enantiomeric mixtures at 291 K (Table ). Although the Q 1 H NMR spectroscopy, DLS and zeta potential measurements confirmed the presence of higher order aggregated species at 5.56 mM, and a decrease in surface tension with respect to increasing SSA concentration confirmed surfactant properties in all cases, a CAC could not be determined for solutions of 3, 5, 7 or 9 due to compound solubility. Biological activity: SSAs have been previously reported to exhibit weak antimicrobial properties against both Gram-positive clinically relevant S. aureus -MRSA USA300 and Gram-negative E. coli DH10B bacteria. As previously discussed, next generation SSAs 3, 5, 7 and 9 have been specifically designed to increase the activity of SSAs against both drug susceptible and resistant S. aureus and ovarian cancer cells. Figure reports the MIC values obtained for previously reported 1 and novel SSAs 3, 5, 7 and 9, provided as single agents or 1:1 enantiomeric mixture against four different strains of S. aureus. These strains include: (i) USA300, a common clinically relevant, methicillin resistant strain; (ii) ATCC 9144, a fully genome sequenced, β-lactamase negative and penicillin susceptible strain; (iii) NCTC 13616, a clinically relevant methicillin resistant strain; 49 (iv) 1199B, which over expresses the NorA efflux pump that has a wide range of substrates including fluoroquinolone antibiotics, antiseptics, and quaternary ammonium compounds, causing this strain to be classed as multidrug resistant. . Previous to this study, the lowest MIC50 (the minimum amount of a compound required to inhibit bacterial growth by 50 %) reported for an SSA against MRSA USA300 was 0.25 mM. Here, 7, 9 and the 1:1 enantiomeric mixture of 7 and 9 exhibited an MIC (a complete absence of any detectable bacterial growth) of 87 µM, against this same strain of S. aureus, making these phenylalanine incorporated SSAs by far the most active SSA antimicrobial agents reported against MRSA USA300 to date. When compared to 1 (the glycine appended SSA where R = H), the substitution of this R-group for either an isobutane (3 and 5, leucine) or a benzyl (7 and 9, phenylalanine) derivative resulted in an overall enhancement of antimicrobial activity. This activity is hypothesised to be due to the additional hydrophobicity and/or π-π stacking interactions provided by these functionalities, increasing the membrane permeability properties of these agents, alongside alteration in the strength of molecular self-association interactions (3 and 5, Table ), in particular the formation of hydrogen bonded SSA anion complexes as previously described. Interestingly, the presence of unnatural D-amino acids within the SSA structure (3 and 7), led to an enhanced activity against the methicillin susceptible ATCC 9144 strain, over their Lsubstituted counterparts ( and). MIC values determined for the different SSA enantiomers support the hypothesis that these SSAs are interacting with a chiral target (e.g. specific phospholipid headgroups). An enhancement in activity where there is a 1:1 ratio of enantiomers present suggests that an alteration in SSA selfassociative complex and/or high-order aggregate formation (see Figure and Tables and) also contributes to enhanced antimicrobial activity, e.g. through stabilisation of cell membrane permeation events. This is exemplified when comparing the antimicrobial activity of the 1:1 enantiomeric mixture of 3 and 5 against USA300 (MIC = 174 µM), to that of either 3 or 5 alone (MIC = 10 mM). Finally, the antimicrobial activity of 3, 7 and 9 against 1199B, provides supporting evidence towards the hypothesis that the SSA anion is integral to the activity of these SSAs, as this strain of S. aureus overexpresses the NorA efflux pump, which specifically targets quaternary ammonium species, such as TBA, the SSA counter cation. This hypothesis is further supported through the change in antimicrobial efficacy of 1, 3, 5, 7 and 9, which is a direct result of the modification in structure of the SSA anionic component.