N,N'-Dimethylsquaramide as a central scaffold for anionophore design.

The N,N'-dimethylation of a diphenylsquaramide induces a conformational change in the orientation of the phenyl rings. This has been exploited to create a series of bis-urea, -thiourea and -squaramide anionophores. The compounds were shown to bind to Cl- using proton NMR titration techniques and to transport H+/Cl- through the lipid bilayers, whereas a non-methylated analogue displayed limited transport activity. Despite their potency in transport studies, the series had a negligible impact on cancer cell viability.

Fatty Acid-Activated Proton Transport by Bisaryl Anion Transporters Depolarises Mitochondria and Reduces the Viability of MDA-MB-231 Breast Cancer Cells.

In respiring mitochondria, the proton gradient across the inner mitochondrial membrane is used to drive ATP production. Mitochondrial uncouplers, which are typically weak acid protonophores, can disrupt this process to induce mitochondrial dysfunction and apoptosis in cancer cells. We have shown that bisaryl urea-based anion transporters can also mediate mitochondrial uncoupling through a novel fatty acid-activated proton transport mechanism, where the bisaryl urea promotes the transbilayer movement of deprotonated fatty acids and proton transport. In this paper, we investigated the impact of replacing the urea group with squaramide, amide and diurea anion binding motifs. Bisaryl squaramides were found to depolarise mitochondria and reduce MDA-MB-231 breast cancer cell viability to similar extents as their urea counterpart. Bisaryl amides and diureas were less active and required higher concentrations to produce these effects. For all scaffolds, the substitution of the bisaryl rings with lipophilic electron-withdrawing groups was required for activity. An investigation of the proton transport mechanism in vesicles showed that active compounds participate in fatty acid-activated proton transport, except for a squaramide analogue, which was sufficiently acidic to act as a classical protonophore and transport protons in the absence of free fatty acids.

A potent fluorescent transmembrane HCl transporter perturbs cellular pH and promotes cancer cell death.

A series of fluorescent coumarin bis-ureas 1-4 have been synthesised, and their anion transport properties studied. The compounds function as highly potent HCl co-transport agents in lipid bilayer membranes. Single crystal X-ray diffraction of compound 1 showed antiparallel stacking of the coumarin rings, stabilised by hydrogen bonds. Binding studies, using 1H-NMR titration, showed moderate chloride binding in DMSO-d6/0.5% with 1 : 1 binding mode (for transporter 1) and 1 : 2 binding mode (host: guest, for transporters 2-4). We examined the cytotoxicity of compounds 1-4 against three cancer cell lines, lung adenocarcinoma (A549), colon adenocarcinoma (SW620) and breast adenocarcinoma (MCF-7). The most lipophilic transporter, 4 showed a cytotoxic effect against all three cancer cell lines. Cellular fluorescence studies showed compound 4 crossed the plasma membrane and localised in the cytoplasm after a short time. Interestingly, compound 4, lacking any lysosome targeting groups, was co-localised with LysoTracker Red at 4 and 8 h in the lysosome. Cellular anion transport of compound 4 was assessed by measuring intracellular pH and showed a decrease in cellular pH, which may be due to the capacity of transporter 4 to co-transport HCl across biological membranes, as evidenced by the liposomal studies.

Fluorinated tetrapodal anion transporters.

Synthetic anion transporters show potential in treating life-threatening diseases like cystic fibrosis and cancer. However, with increasingly complex transporter architectures designed to control anion binding and transport, it is important to consider solubility and deliverability during transporter design. The fluorination of synthetic anion transporters has been shown to tune the transporter lipophilicity, transport rates, and binding strength. In this work, we expand on our previously reported tetrapodal (thio)urea transporters with a series of fluorinated tetrapodal anion transporters. The effects of fluorination on tuning the lipophilicity, solubility, deliverability, and anion transport selectivity of the tetrapodal scaffold were investigated using anion-binding and transport assays. The primary mode of anion transport was H+/X- cotransport, with the most fluorinated tetrathiourea (8) displaying the highest transport activity in the 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) assay. Intriguingly, inversion of the transmembrane Cl- vs NO3 - transport selectivity compared with previously reported tripodal (thio)urea transporters was observed under a modified HPTS assay.

Protonophoric and mitochondrial uncoupling activity of aryl-carbamate substituted fatty acids.

Aryl-urea substituted fatty acids are protonophores and mitochondrial uncouplers that utilise a urea-based synthetic anion transport moiety to carry out the protonophoric cycle. Herein we show that replacement of the urea group with carbamate, a functional group not previously reported to possess anion transport activity, produces analogues that retain the activity of their urea counterparts. Thus, the aryl-carbamate substituted fatty acids uncouple oxidative phosphorylation and inhibit ATP production by collapsing the mitochondrial proton gradient. Proton transport proceeds via self-assembly of the deprotonated aryl-carbamates into membrane permeable dimeric species, formed by intermolecular binding of the carboxylate group to the carbamate moiety. These results highlight the anion transport capacity of the carbamate functional group.

Measuring anion binding at biomembrane interfaces.

The quantification of anion binding by molecular receptors within lipid bilayers remains challenging. Here we measure anion binding in lipid bilayers by creating a fluorescent macrocycle featuring a strong sulfate affinity. We find the determinants of anion binding in lipid bilayers to be different from those expected that govern anion binding in solution. Charge-dense anions H2PO4- and Cl- that prevail in dimethyl sulfoxide fail to bind to the macrocycle in lipids. In stark contrast, ClO4- and I- that hardly bind in dimethyl sulfoxide show surprisingly significant affinities for the macrocycle in lipids. We reveal a lipid bilayer anion binding principle that depends on anion polarisability and bilayer penetration depth of complexes leading to unexpected advantages of charge-diffuse anions. These insights enhance our understanding of how biological systems select anions and guide the design of functional molecular systems operating at biomembrane interfaces.

Structure-Activity Relationship and Mechanistic Studies of Bisaryl Urea Anticancer Agents Indicate Mitochondrial Uncoupling by a Fatty Acid-Activated Mechanism.

Targeting the cancer cell mitochondrion is a promising approach for developing novel anticancer agents. The experimental anticancer agent N,N'-bis(3,5-dichlorophenyl)urea (SR4) induces apoptotic cell death in several cancer cell lines by uncoupling mitochondrial oxidative phosphorylation (OxPhos) using a protein-free mechanism. However, the precise mechanism by which SR4 depolarizes mitochondria is unclear because SR4 lacks an acidic functional group typically found in protein-independent uncouplers. Recently, it was shown that structurally related thioureas can facilitate proton transport across lipid bilayers by a fatty acid-activated mechanism, in which the fatty acid acts as the site of protonation/deprotonation and the thiourea acts as an anion transporter that shuttles deprotonated fatty acids across the phospholipid bilayer to enable proton leak. In this paper, we show that SR4-mediated proton transport is enhanced by the presence of free fatty acids in the lipid bilayer, indicating that SR4 uncouples mitochondria through the fatty acid-activated mechanism. This mechanistic insight was used to develop a library of substituted bisaryl ureas for structure-activity relationship studies and subsequent cell testing. It was found that lipophilic electron-withdrawing groups on bisaryl ureas enhanced electrogenic proton transport via the fatty acid-activated mechanism and had the capacity to depolarize mitochondria and reduce the viability of MDA-MB-231 breast cancer cells. The most active compound in the series reduced cell viability with greater potency than SR4 and was more effective at inhibiting adenosine triphosphate production.

Organoplatinum Compounds as Anion-Tuneable Uphill Hydroxide Transporters.

Active transport of ions uphill, creating a concentration gradient across a cell membrane, is essential for life. It remains a significant challenge to develop synthetic systems that allow active uphill transport. Here, a transport process fuelled by organometallic compounds is reported that creates a pH gradient. The hydrolysis reaction of PtII complexes results in the formation of aqua complexes that established rapid transmembrane movement ("flip-flop") of neutral Pt-OH species, leading to protonation of the OH group in the inner leaflet, generating OH- ions, and so increasing the pH in the intravesicular solution. The organoplatinum complex effectively transports bound hydroxide ions across the membrane in a neutral complex. The initial net flow of the PtII complex into the vesicles generates a positive electric potential that can further drive uphill transport because the electric potential is opposed to the chemical potential of OH- . The OH- ions equilibrate with this transmembrane electric potential but cannot remove it due to the relatively low permeability of the charged species. As a result, effective hydroxide transport against its concentration gradient can be achieved, and multiple additions can continuously drive the generation of OH- against its concentration gradient up to ΔpH>2. Moreover, the external addition of different anions can control the generation of OH- depending on their anion binding affinity. When anions displayed very high binding affinities towards PtII compounds, such as halides, the external anions could dissipate the pH gradient. In contrast, a further pH increase was observed for weak binding anions, such as sulfate, due to the increase of positive electric potential.

Photomodulation of Transmembrane Transport and Potential by Stiff-Stilbene Based Bis(thio)ureas.

Membrane transport proteins fulfill important regulatory functions in biology with a common trait being their ability to respond to stimuli in the environment. Various small-molecule receptors, capable of mediating transmembrane transport, have been successfully developed. However, to confer stimuli-responsiveness on them poses a fundamental challenge. Here we demonstrate photocontrol of transmembrane transport and electric potential using bis(thio)ureas derived from stiff-stilbene. UV-vis and 1H NMR spectroscopy are used to monitor E-Z photoisomerization of these bis(thio)ureas and 1H NMR titrations reveal stronger binding of chloride to the (Z)-form than to the (E)-form. Additional insight into the binding properties is provided by single crystal X-ray crystallographic analysis and DFT geometry optimization. Importantly, the (Z)-isomers are much more active in transmembrane transport than the respective (E)-isomers as shown through various assays. As a result, both membrane transport and depolarization can be modulated upon irradiation, opening up new prospects toward light-based therapeutics as well as physiological and optopharmacological tools for studying anion transport-associated diseases and to stimulate neuronal activity, respectively.

Halide-selective, proton-coupled anion transport by phenylthiosemicarbazones.

Phenylthiosemicarbazones (PTSCs) are proton-coupled anion transporters with pH-switchable behaviour known to be regulated by an imine protonation equilibrium. Previously, chloride/nitrate exchange by PTSCs was found to be inactive at pH 7.2 due to locking of the thiourea anion binding site by an intramolecular hydrogen bond, and switched ON upon imine protonation at pH 4.5. The rate-determining process of the pH switch, however, was not examined. We here develop a new series of PTSCs and demonstrate their conformational behaviour by X-ray crystallographic analysis and pH-switchable anion transport properties by liposomal assays. We report the surprising finding that the protonated PTSCs are extremely selective for halides over oxyanions in membrane transport. Owing to the high chloride over nitrate selectivity, the pH-dependent chloride/nitrate exchange of PTSCs originates from the rate-limiting nitrate transport process being inhibited at neutral pH.

Delivering anion transporters to lipid bilayers in water.

Cyclodextrins have been employed as delivery agents for lipophilic anion transporters, which allow their incorporation into lipid bilayers without using an organic solvent or pre-incorporation.

Acridinone-based anion transporters.

The arrangement of hydrogen bond donors around a central lipophilic scaffold has proven to be a successful strategy in the development of potent chloride transporters. In this work, we revisit an acridinone 1,9-bis(thio)urea motif which had previously shown promise as an anion sensor and expand the series of compounds by appending a variety of electron-withdrawing groups to the peripheral phenyl moieties. High levels of activity were achieved by the most effective compounds in the series, which facilitated strictly electroneutral transport.

A Two-Dimensional Metallacycle Cross-Linked Switchable Polymer for Fast and Highly Efficient Phosphorylated Peptide Enrichment.

The selective and efficient capture of phosphopeptides is critical for comprehensive and in-depth phosphoproteome analysis. Here we report a new switchable two-dimensional (2D) supramolecular polymer that serves as an ideal platform for the enrichment of phosphopeptides. A well-defined, positively charged metallacycle incorporated into the polymer endows the resultant polymer with a high affinity for phosphopeptides. Importantly, the stimuli-responsive nature of the polymer facilitates switchable binding affinity of phosphopeptides, thus resulting in an excellent performance in phosphopeptide enrichment and separation from model proteins. The polymer has a high enrichment capacity (165 mg/g) and detection sensitivity (2 fmol), high enrichment recovery (88%), excellent specificity, and rapid enrichment and separation properties. Additionally, we have demonstrated the capture of phosphopeptides from the tryptic digest of real biosamples, thus illustrating the potential of this polymeric material in phosphoproteomic studies.

Measuring anion transport selectivity: a cautionary tale.

pH-dependent liposomal assays are often used to determine anion selectivity in transmembrane anion transport experiments. We discuss the validity and limitations of these assays, and provide guidelines for their use to avoid misleading results.

Advances in applied supramolecular technologies.

Supramolecular chemistry is a comparatively young field that to date has mainly been focused on building a foundation of fundamental understanding. With much progress in this area, researchers are seeking to apply this knowledge to the development of commercially viable products. In this review we seek to outline historical and recent developments within the field of supramolecular chemistry that have made the transition from laboratory to market, and to bring to light those technologies that we believe have commercial potential. In doing so we hope we may illuminate pathways to market for research currently being conducted.

Synthetic Na+/K+ exchangers promote apoptosis by disturbing cellular cation homeostasis.

A number of artificial cation ionophores (or transporters) have been developed for basic research and biomedical applications. However, their mechanisms of action and the putative correlations between changes in intracellular cation concentrations and induced cell death remain poorly understood. Here, we show that three hemispherand-strapped calix[4]pyrrole-based ion-pair receptors act as efficient Na+/K+ exchangers in the presence of Cl- in liposomal models and promote Na+ influx and K+ efflux (Na+/K+ exchange) in cancer cells to induce apoptosis. Mechanistic studies reveal that these cation exchangers induce endoplasmic reticulum (ER) stress in cancer cells by perturbing intracellular cation homeostasis, promote generation of reactive oxygen species, and eventually enhance mitochondria-mediated apoptosis. However, they neither induce osmotic stress nor affect autophagy. This study provides support for the notion that synthetic receptors, which perturb cellular cation homeostasis, may provide new small molecules with potentially useful apoptotic activity.

Tetrapodal Anion Transporters.

Synthetic anion transporters that facilitate chloride transport are promising candidates for channelopathy treatments. However, most anion transporters exhibit an undesired side effect of facilitating proton transport via interacting with fatty acids present in the membrane. To address the limitation, we here report the use of a new tetrapodal scaffold to maximize the selective interaction with spherical chloride over binding the carboxylate headgroup of fatty acids. One of the new transporters demonstrated a high selectivity for chloride uniport over fatty acid-induced proton transport while being >10 times more active in chloride uniport than strapped calixpyrroles that were previously the only class of compounds known to possess similar selectivity properties.

Advances in anion transport and supramolecular medicinal chemistry.

Advances in anion transport by synthetic supramolecular systems are discussed in this article. Developments in the design of discrete molecular carriers for anions and supramolecular anion channels are reviewed followed by an overview of the use of these systems in biological systems as putative treatments for diseases such as cystic fibrosis and cancer.

Stimuli-Responsive Cycloaurated "OFF-ON" Switchable Anion Transporters.

Anion transporters have shown potential application as anti-cancer agents that function by disrupting homeostasis and triggering cell death. In this research article we report switchable anion transport by gold complexes of anion transporters that are "switched on" in situ in the presence of the reducing agent GSH by decomplexation of gold. GSH is found in higher concentrations in tumors than in healthy tissue and hence this approach offers a strategy to target these systems to tumors.