Halogen bonding relay and mobile anion transporters with kinetically controlled chloride selectivity

Selective transmembrane transport of chloride over competing proton or hydroxide transport is key for the therapeutic application of anionophores, but remains a significant challenge. Current approaches rely on enhancing chloride anion encapsulation within synthetic anionophores. Here we report the first example of a halogen bonding ion relay in which transport is facilitated by the exchange of ions between lipid-anchored receptors on opposite sides of the membrane. The system exhibits non-protonophoric chloride selectivity, uniquely arising from the lower kinetic barrier to chloride exchange between transporters within the membrane, compared to hydroxide, with selectivity maintained across membranes with different hydrophobic thicknesses. In contrast, we demonstrate that for a range of mobile carriers with known high chloride over hydroxide/proton selectivity, the discrimination is strongly dependent on membrane thickness. These results demonstrate that the selectivity of non-protonophoric mobile carriers does not arise from ion binding discrimination at the interface, but rather through a kinetic bias in transport rates, arising from differing membrane translocation rates of the anion–transporter complexes.


1 Materials and methods
All reagents and solvents were purchased from commercial sources and used without further purification. Lipids were purchased from Avanti polar lipids and used without further purification. Where necessary, solvents were dried by passing through an MBraun MPSP-800 column and degassed with nitrogen. Triethylamine was distilled from and stored over potassium hydroxide. Normal phase silica gel flash column chromatography was performed manually using Merck® silica gel 60 under a positive pressure of nitrogen. Where mixtures of solvents were used, ratios are reported by volume. NMR spectra were recorded on a Bruker AVIII 400, Bruker AVII 500 (with cryoprobe) and Bruker AVIII 500 spectrometers. Chemical shifts are reported as δ values in ppm. Mass spectra were carried out on a Waters Micromass LCT and Bruker microTOF spectrometers. Fluorescence spectroscopic data were recorded using a Horiba Duetta fluorescence spectrophotometer, equipped with a Peltier temperature controller and stirrer. UV-Vis spectra were recorded on a V-770 UV-Visible/NIR Spectrophotometer equipped with a Peltier temperature controller and stirrer, using quartz cuvettes of 1 cm path length. Experiments were conducted at 25 °C unless otherwise stated. Vesicles were prepared as described below using Avestin "LiposoFast" extruder apparatus, equipped with polycarbonate membranes with 200 nm pores. GPC purification of vesicles was carried out using GE Healthcare PD-10 desalting columns prepacked with Sephadex G 25 medium. Compounds S1·HB 2 , S1·XB 2 , S3 1 , 3·HB 3 , 4·HB, 4 5·XB 5 and 5·ChB 5 were prepared according to literature procedures.

Determination of incorporation efficiency
UV-vis analysis was employed to determine the incorporation efficiency of the relay transporter 1·XB into the membrane of pre-formed 200 nm POPC LUVs. First, the solution phase UV-vis of 1·XB was measured in DMSO. The following procedure was then carried out to determine the incorporation efficiency of 1·XB:

Vesicle Preparation
A thin film of lipid (2.1875 µmol) and transporter in various ratios was formed by evaporating a chloroform solution under reduced pressure on a rotary evaporator (20 °C) and then under high vacuum for 6 hours. The lipid film was hydrated by vortexing with the prepared buffer (1 mL, 100 mM NaCl, 10 mM HEPES, 1 mM 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (HPTS), pH 7.0). The lipid suspension was then subjected to 5 freeze-thaw cycles using liquid nitrogen and a water bath (40°C) followed by extrusion 19 times through a polycarbonate membrane (pore size 200 nm). Extrusion was performed at 50°C in the case of DPPC lipids. Extra-vesicular components were removed by size exclusion chromatography on a Sephadex G-25 column eluted with 100 mM NaCl, 10 mM HEPES, pH 7.0. Final conditions: LUVs (0.625 mM lipid, 3.5 mL vesicle suspension); inside 100 mM NaCl, 10 mM HEPES, 1 mM HPTS, pH 7.0; outside: 100 mM NaCl, 10 mM HEPES, pH 7.0. Vesicles for the sodium gluconate assay were prepared by the same procedure, substituting NaCl for NaGluconate in the buffer solution.

Transport assays with HPTS
In a typical experiment, the LUVs containing HPTS with transporter pre-incorporated (100 L, final lipid concentration 31.25 M) were added to buffer (1880 L of 100 mM NaCl, 10 mM HEPES, pH 7.0) at 25°C under gentle stirring. A pulse of NaOH (20 L of 0.5 M solution, final concentration 5 mM) was added at 10 s to initiate the experiment. After 210 s, detergent (25 L of 11% Triton X-100 in 7:1 (v/v) H2O-DMSO) was added to lyse the vesicles and calibrate the assay. The fluorescence emission was monitored at λem = 510 nm (λex = 405/460 nm). The fractional fluorescence intensity (Irel) was calculated from Equation S1, where Rt is the fluorescence ratio at time t, (ratio of intensities from 460 nm / 405 nm excitation) R0 is the fluorescence ratio at time 0 s immediately before the base pulse, and Rd is the fluorescence ratio at time 255 s, after the addition of detergent. For each compound, each individual concentration was repeated at least three times and averaged; error bars represent standard deviations.
The fractional fluorescence intensity (Irel) at 205 s just prior to lysis, defined as the fractional activity y, was plotted as a function of the ionophore concentration (x / µM). Hill coefficients (n) and EC50 values were calculated by fitting to the Hill equation (S2): The kini values were calculated by fitting the transport kinetics curves (Irel vs t) with the exponential function Irel = a -exp(-kt/b) using Origin 17. kini (the initial rate of transport at t = 10) is then given by differentiation as kini = 1/b (s -1 ).
Experiments with DPPC lipids were conducted in the same way. For elevated temperature studies, the buffer was equilibrated at 45°C for 5 minutes prior to initiating the experiment.
Experiments in the presence of protonophore trifluoromethoxy carbonylcyanide phenylhydrazone (FCCP) were carried out using the above procedure, except that a DMSO solution of FCCP (5 µL of 100 µM solution, final concentration 0.25 µM/0.8 mol%) was added to the vesicle suspension prior to the addition of the NaOH pulse. At this concentration, FCCP does not cause appreciable dissipation of the transmembrane pH gradient alone.