The ability of compounds 3-5 and 8-10 to mediate electroneutral H + /Cl -cotransport was determined using HPTS fluorescence assays (Figure ). Initially, dose response studies were performed, followed by Hill analysis, using a NMDG-Cl HPTS assay. A lower concentration of lipids are used in these experiments (0.1 mM), meaning lower transporter concentrations are required to facilitate transport. EC50 values were calculated for the whole series of compounds 1-10 using this assay, offering a more complete picture of transport activity. Vesicles (200 nm) were prepared with an internal solution of NMDG-Cl (100 mM), buffered with HEPES (5 mM) to pH 7.0, and containing HPTS (1 mM). The LUVs were then suspended in the same external solution of NMDG-Cl which had not been treated with HPTS. An artificial pH gradient was initiated for each experiment using a base pulse of NMDG (5 mM), before the transporter was added to the suspension as a DMSO solution (5 µL to a total volume of 2.5 mL) at t = 0 s. NMDG + is a large, hydrophilic cation that cannot be transported through the bilayer; therefore, the only process possible capable of dissipating the pH gradient is H + /Cl -cotransport. Changes to the intravesicular pH induced by cotransport of HCl were followed by monitoring the change in fluorescence ratio of HPTS. Detergent was added at t = 210 s to lyse the vesicles and provide a calibration reading of 100% proton efflux. EC50 values and Hill coefficients for compounds 1-10 are displayed in Table . The transport activity of the bis-urea compounds 1-5 in this assay compared to the bis-thiourea compounds 6-10 mirrored the results of the Cl -/NO3 -ISE exchange assay. The calculated EC50 values for each of the bis-urea compound were at least 4 times smaller than their bis-thiourea analogues. Compound 3 was again the most potent transporter of the series, with an EC50 value of 0.00015 mol%, which is a factor of 10 smaller than the other anionophores tested, adding further evidence towards the effectiveness of the NO2 derivatives as anion transporters. Hill coefficients (n) were close to n = 1 in all cases aside from compound 10, where the value was closer to n = 2. These findings indicate a 1:1 stoichiometry is preferred for transport under the conditions of this assay.