The tetralactam series were then tested for their ability to facilitate H + /Cl -transport using the 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) transport selectivity assay under three assay conditions. Untreated vesicles were employed to analyse fatty acid flip-flop aided H + /Cl -co-transport mediated by transporters (Figure ). The second condition utilises carbonyl cyanide-mchlorophenylhydrazone (CCCP) treated vesicles, which acts as a weak acid protonophore, allowing the study of transportermediated Cl -selective uniport (Figure ). The final condition uses vesicles treated with bovine serum albumin (BSA). This protein is employed to sequester fatty acids from the lipid bilayer, enabling the study of transporter-mediated H + /Cl -co-transport in the absence of fatty acids (Figure ). The vesicles contained the pH-sensitive probe HPTS (1 mM), NaCl (100 mM), and HEPES (10 mM) and were prepared following a previously reported method. The LUVs were suspended in an external solution of NaCl (100 mM) and HEPES (10 mM), with both the internal and external solutions being buffered to pH 7. Transport was initiated when the transporters were added to the assay systems at varied concentrations as DMSO solutions, and the fractional fluorescence intensity was recorded. The data was then plotted (Figure ) before being fitted to the Hill equation (Equation ) to afford EC50 values and Hill coefficients (n). For further experimental methods, see the experimental procedures section, or for more information on experimental data and associated spectra, see Table Fitting of the data to the Hill equation (Equation ) showed that the transport facilitated by 1a-4c was again in a 1:1 host:guest stoichiometry, indicated by Hill coefficients of n ≈ 1.0 (Table ). The stoichiometries are further supported by the 1:1 host:guest binding constants and the 1:1 host:guest Hill coefficients returned by the Cl -/NO3 -exchange assay. The exception to this general observation was 2a, which yielded a Hill coefficient of n ≈ 2, which is shown to be feasible in the solid-state structure of 1b•2Cl -(Figure ) at greater Cl -concentrations (Table ). Under all three assay conditions, the 4,5-difluorophenylene-linked transporters showed the highest transport activity. Similar levels of transport activity were observed when the series was tested using CCCP-treated and untreated vesicles, indicating that negligible Cl -uniport was occurring. Also, the transport activity significantly decreased in the absence of fatty acids when BSA-treated vesicles were used. The transport activity trends indicate that the primary mechanism of transport is the electroneutral fatty acid flip-flop-aided H + /Cl -co-transport mechanism. The defined tetralactam binding cavities means the series can also bind to the carboxylate head group of fatty acids. Interestingly, the CF3-substituted transporters (3a-3c) produced lower EC50 values when fatty acids were sequestered from the lipid bilayers of the BSA-treated vesicles. This suggests that 3a-3c are capable of efficient electroneutral H + /Cl -co-transport unaided by the fatty acid flip-flop mechanism. Alternatively, the slower transport aided by fatty acids could be the result of the complex formed between 3a-3c and a fatty acid being too lipophilic, hindering membrane partitioning and slowing transport. The lipophilic balance achieved by the complexation of a fatty acid with 2a-2c allows for very efficient unhindered fatty acid flip-flop.