Having found that TT is not ac7ve as a transporter, while TA is able to perform anion transport, we set out to prepare TA in situ in the liposomal membrane by DCvC. Not knowing the 7mescale required for the azine metathesis reac7on inside the membrane, we replaced the fluorescent probe lucigenin (N,N'-dimethyl-9,9'-biacridinium dinitrate) by its more hydrophilic analogue SPBA (N,N'-bis(3sulphonatopropyl)-9,9'-biacridinium), which is less prone to leak out of the liposomes over 7me. 39 Before star7ng the DCvC experiments, we first needed to ensure that the azine TT is stable in the membrane and does not hydrolyse over 7me to release aldehyde T, as this is an ac7ve anion transporter. We have preincorporated TT, again at 1 mol%, in liposomes with SPBA encapsulated and for the rest iden7cal condi7ons to those described above. Transport was tested directly afer prepara7on of the liposomes with TT (black curve in Fig. ) and again afer 24h (red curve). No significant transport was observed for both cases, indica7ng that there is no significant hydrolysis of TT taking place in the lipid membranes, highligh7ng again the remarkable stability of azines compared to imines. This control experiment paved the way for DCvC experiments, which were performed in glass vials containing 3 mL of liposome solu7on (0.4 mM lipids, 12 nmol of TT, 1 equiv.) at room temperature with con7nuous s7rring. The symmetrical azine AA (12 µL, 5.0 mM in MeCN, 5 equiv.) was added to the liposomes and transport runs were recorded at different intervals afer the addi7on of azine AA. A very clear transport ac7vity was observed, even afer 1 hour (Fig. ). This shows that the post-inserted azine AA was effec7vely delivered into the membrane where the azine metathesis between preincorporated TT and AA occurred, resul7ng in the forma7on of the ac7ve anion transporter TA. Control experiments in which AA was added to liposomes without TT did not show any transport (Fig. ). The transport ac7vity in the DCvC transport experiments increased from 1h to 2h, while hardly any further improvement was observed afer 24h, indica7ng that the thermodynamic equilibrium of the exchange reac7on is reached afer ~2h. A concentra7on of TA of 1.67 mol% is then expected sta7s7cally (see Table ).