Protein ion channels form an integral part of every living cell. Their mimics play an essential role in investigating the functions of these ion channels and provide an insight into the related biological events. Misregulation of transport of highly biologically abundant ions like Cl ─ , HCO3 -, phosphate, and SO4 results in channelopathies. Phosphate and phosphorylated molecules serve as the building block for various essential substances used by the cell for important biological purposes. Sodium-dependent phosphate transporters such as type II (SLCA34) and type III (SLC20) are primarily accountable for the influx of extracellular phosphate transport. However, disruption of the natural protein machinery responsible for the transport of phosphate ions causes phosphate imbalance in the body, resulting in hyperphosphatemia or hypophosphatemia. Recent studies also revealed that chloride channel proteins are overexpressed in cancer cells and promote proliferation by effluxing Cl -ions. Synthetic Cl -ion transporters showed anticancer activities. Therefore, synthetic transporters capable of transporting phosphate and Cl -ions via antiport pathway are highly beneficial for anticancer activities. Recently, several synthetic ion transporters like prodigiosin, calix [4]pyrroles, and others have been developed to mimic natural protein machinery's functions and to induce apoptosis in cancer cells. However, ion transport-promoted unwanted death of normal cells remains a major concern for ion therapy. So, to minimize the apoptosis of the normal cells and selectively target the cancer cells, biomimetic stimuli-responsive systems are being used, which are triggered in the presence of any external or natural stimuli like a ligand, mechanical force, and light, specifically in the cancer cells. The use of phototriggered ion transporters can give site-selective precise control over the functioning of the ion transporters. Lately, "optochemical genetics" has integrated natural protein channels with various photoswitches to achieve optical control. Therefore, a strategically placed photocleavable or photoisomerisable moiety tethered to the main anionophore can serve the purpose well. Herein, we synthesized C2 symmetric guanidinium-based macrocycles. The ion binding scaffold and the hydrophobic domain impart lipophilicity to transport the ions. To further explore the concept of a photocleavable proanionophore, we attached the o-nitrobenzyl (ONB) group with the guanidinium-N moiety. On shining light (365 nm), the ONB group easily cleaves off, paving the way for the anionophore to carry its ion transport function (Fig. ). The ONB group has been immensely exploited in numerous applications to cleave under photoirradiation. However, in ion transport, the use of the ONB group has been rare except for a few examples. These modular macrocyclic compounds were synthesized in two steps (Scheme S1). Initially, thiourea-based macrocyclic intermediate was synthesized, and then desulfurization of thiourea moiety with alkyl amines resulted in the expected guanidinium-based macrocyclic compounds. To investigate the role of hydrophobicity in ion transportation, alkyl amines with different chain lengths (n = 4, 8, and 10) were used. The presence of guanidinium moieties in all synthesized compounds prompted us to measure their phosphate ion binding properties by UV absorption-based titrations assay. The binding analysis showed compound 2 has a higher binding affinity for phosphate ions than the other synthesized compounds (Fig. and Table ). Hence compound 2 was selected for further studies. In addition, compound 2 also showed a similar binding affinity for Cl -ions. The cyclic bisguanidinium moiety could bind with the Cl -and phosphate ions. Meanwhile, the binding of compound 2 with NO3 -ions couldn't be calculated due to insignificant spectral changes. In prospect of the phosphate transport by these compounds, we explored their ion transport properties in large unilamellar vesicles (LUVs) using the fluorescence assay with bis-Nmethylacridinium nitrate (lucigenin) dye (Fig. ). Lucigenin fluorescence intensity is sensitive to halides and various oxyanions (Fig. ). Hence, it has been used as a tool to monitor the transmembrane transport of phosphate ions. The egg-yolk phosphatidylcholine (EYPC) and cholesterol (CHOL) in 8:2 molar ratios were used to prepare the model LUVs. As the NO3 -ion binding by compound 2 is underwhelming, hence transport properties of the compounds were investigated by the lucigenin-based fluorescence assay as described (Fig. ). The LUVs were dispersed in a 225 mM aqueous solution of NaH2PO4. This time we found an increase in the fluorescence intensity of the lucigenin dye after adding the compound, evincing the transport of Cl -and phosphate ions in an antiport fashion. We also investigated the fluorescence assay using LUVs (prepared using 225 mM NaH2PO4) dispersed in a 225 mM aqueous solution of NaCl. A similar transport property of compound 2 was observed (Fig. ). The decrease in lucigenin fluorescence intensity also demonstrates the transport of Cl - and phosphate ions in an antiport fashion. The outcome is in accordance with the UV-based titration methods. Hence, all transport-related experiments were carried out by preparing the LUVs in a 225 mM aqueous solution of NaCl. The ion transport activities of the compounds 1-4 were also investigated with the lucigenin-based fluorescence assay by dispersing the LUVs in an aqueous solution of 225 mM of NaH2PO4 and adding the pulse of a compound at t = 50 s. The phosphate transport efficacy of compound 2 was higher than the compounds 1, 3, and 4 (Fig. ). The higher transport proficiency of compound 2 could be attributed to its subtle balance of hydrophilicity and membrane residence aptitude. Compound 4 exhibited very low phosphate transport efficacy. In comparison, compound 4 with ONB moiety can be reckoned as a bowl with a lid. The presence of a plug (ONB moiety) could shield the ion binding face of compound 4, making the recognition and transport of ions inconvenient. Consequently, compound 2 was selected to carry out further biophysical studies. The anion selectivity of compound 2 was performed by lucigenin assay dispersing the LUVs in an aqueous of 225 mM NaxAy salt (where NaxAy = NaH2PO4, Na2SO4, NaOAc, NaClO4, and NaNO3). The kinetics was commenced by adding a pulse of the compound. The selectivity studies revealed that compound 2 is more selective towards phosphate ions than the other tested oxyanions (Fig. ). The dose-dependent lucigenin assay was further performed to investigate the phosphate ion transport efficacy of the compounds. The calculated half-maximal transport efficiency (EC50) values revealed that compound 2 has superior transport activity among all the anionophores (Fig. and Table ). In addition, chloride-ion selective electrode (Cl-ISE)-based transport assay was performed (Fig. ). The LUVs (20 mM HEPES buffer containing 225 mM of NaCl, pH 7.2) were dispersed in an isotonic aqueous solution of NaH2PO4, and the kinetics was started at t = 50 s by adding the pulse of compound 2. The Cl-ISE-based assay showed compound 2 mediated transport of phosphate/Cl ─ ions across the lipid bilayers. To comprehend the mechanistic pathway, the cation selectivity of compound 2 was checked in the presence of Na + and K + ions (Fig. ). To further confirm the absence of any cation selectivity, we have performed the assay with EYPC/CHOL-LUVs⊃lucigenin containing 225 mM Na2SO4, dispersed in an isotonic solution of NaH2PO4 (Fig. ). A similar change in fluorescence intensity was observed for both the salts. This leads to the deduction that metal ions are not involved in the pathway. No change of fluorescence intensity was observed after the addition of the compound, which may be due to the inability of compound 2 to facilitate the SO4 2─ /phosphate antiport process as the selectivity for SO4 2─ is too low for compound 2. This result reveals the independent nature of the transport mechanism with respect to the cations. The observed transport of oxyanion could also be due to the compoundmediated movement of H + /OH ─ across the lipid bilayer. In this regard, a set of U-tube assays was performed, keeping 225 mM of NaH2PO4 in the source end and isotonic NaCl solution in the receiver end (Fig. ). Both the left and right arms of the Utube were monitored periodically. Chloride concentration in the left arm was monitored with the Cl-ISE, and it showed a steady increase in Cl - concentration with time. The spectrophotometric and ammonium molybdate-based assays were performed to examine the phosphate ions concentration in the right arm. The non-involvement of H + in the mechanistic pathway could be assured using HPTS-based fluorescence assay in the absence and presence of FCCP (transporter of H + ions) (Fig. ). In this assay, the increase in fluorescence intensity upon the addition of FCCP along with compound 2 ensured that the H + is not involved in the transport process. Hence, the possibility of phosphate/Cl -symport for compound 2 can be precluded. The only persisting pathway is the antiport mechanism which can be inferred from the lucigenin-based fluorescence experiments. Due to the addition of various anions in the external solution, the fluorescence intensity of lucigenin changes, insinuating the antiport pathway. Additionally, the U-tube assay also propounds a similar result. Likewise, to investigate whether compound 2 behaves as a carrier or forms a channel within the lipid bilayer, we performed the temperature-dependent DPPC assay using DPPC-LUV⊃lucigenin. Increment in phosphate/Cl - antiport at T = 50 °C evinces the carrier-mediated phosphate/Cl - antiport pathway mediated by compound 2 (Fig. ). The Utube assay also validated the carrier-mediated pathway as transport from one side to the other side of the U-tube through the organic layer containing compound 2 can only be effectuated by a carrier molecule. The transport activities across the Tb(III)-complex-based fluorescent chemosensor (of phosphate ion) LUVs also demonstrated the phosphate transport activities of compound 2 (Fig. ). We also examined the transmembrane transport of phosphate ions in the presence of compound 2 using giant unilamellar vesicles (GUVs). The GUVs were prepared in 100 mM HEPES buffer containing 200 mM sucrose, 225 mM NaH2PO4, and 1 mM lucigenin. The GUVs were dispersed in 100 mM HEPES buffer containing 200 mM glucose, 225 mM NaCl and transferred to the microscope chamber. A significant quenching of lucigenin fluorescence was observed after the addition of compound 2 (45 µM) to the extravesicular solution of the GUVs (Fig. ). A similar GUV experiment was deployed, having the reverse gradient of anions as in the previous case. A significant enhancement of lucigenin fluorescence was observed after the addition of compound 2 and NaH2PO4 to the extravesicular solution (Fig. ). These changes in lucigenin fluorescence intensity across the bilayers of GUVs could be due to the transport of Cl -and phosphate ions via the antiport pathway. Additionally, we performed 31 P NMR experiment to provide indisputable proof of the phosphate/Cl -antiport process carried by compound 2. The 31 P NMR spectra of NaH2PO4 encapsulated vesicles provided a distinguishable peak of 31 P (Fig. ). The addition of Mn 2+ , a paramagnetic substance, did not affect the intensity of 31 P. However, due to the addition of compound 2, the 31 P signal decreased after 40 mins incubation and finally disappeared after 2 hours of incubation (Fig. and). The interaction of phosphate ions with the paramagnetic Mn 2+ ions after the efflux of the phosphate ions led to the disappearance of the intensity of the 31 P signal. The molecular dynamics (MD) simulation studies with compound and phosphate ion embedded inside a preequilibrated dipalmitoyl phosphocholine (DPPC)/water lipid bilayer system (Fig. ) studies revealed the retention of the compound within the bilayer and expulsion of phosphate ion from the complex suggesting the successful transport of the phosphate ion by the compound. Meanwhile, the optimized gas-phase structure showed the interaction of phosphate ions with the guanidinium moieties of compound 2 (Fig. ).