Recently, we have reported the suitability of 'anion gradient' assays for determining the selectivity of a transporter towards chloride over other anions (Figure ). Vesicles were prepared in a solution of NaCl (100 mM) and HPTS (1 mM), buffered to pH 7.0 with HEPES (10 mM). Following this procedure, the vesicles were suspended in external solutions of NaX (100mM), also buffered to pH 7.0, where X -= Cl -, Br -, NO3 -, I -, or ClO4 -. No base pulse was added, and the experiment was initiated by the addition of a transporter as a DMSO solution (5 µL to a total volume of 2.5 mL) at t = 0 s. Selectivity is determined by observing the maximum ΔpH values reached during each experiment. If the rate of HCl efflux exceeds the rate of HX influx, then the intravesicular solution is basified, and ΔpH will be a positive value. If the rate of HX influx is the faster process then protons will enter the vesicles at a quicker rate than protons leaving through HCl efflux, and therefore the ΔpH value should be negative. To exhibit chloride-selective transport, a compound must return a positive ΔpH value against the other anions tested. The transport selectivity for compounds 1-10 generally followed the order I -> Br -> Cl -> NO3 -> ClO4 -. The trend for the halide anions follows the sequence predicted by the Hofmeister series, with the transport of more hydrophobic anions facilitated more efficiently. Interestingly, all compounds transported chloride at a faster rate than NO3 -and ClO4 -, despite the higher lipophilicity of the two anions. It is likely that the preorganised, convergent bis-(thio)urea binding site is a better geometrical match for spherical halide anions over the more complex geometries of NO3 -and ClO4 -. A similar Cl -> NO3 - transport selectivity was previously observed for tren-based thiourea compounds, and therefore the transport of NO3 -is the rate-limiting step in the Cl -/NO3 -assay for these compounds. Therefore, the true Cl -transport activity achieved by compounds 3-5 and 8-10 may have been underestimated due to poor transport of NO3 -. Notably, compound 4 exhibited minimal transport selectivity for Br -over Cl -, and the negative ΔpH value afforded by an external solution of HI was also small (Figure ). By comparison, the bis-thiourea analogue 9 showed considerable I -> Cl -selectivity (Figure ). The tight, planar binding site afforded by the bis-urea groups, coupled with the Please do not adjust margins Please do not adjust margins steric bulk of the 3,5-bis-CF3 substituents, creates the most Cl - size-selective binding site of the series.