Expanding the π-system of Fatty Acid-Anion Transporter Conjugates Modulates Their Mechanism of Proton Transport and Mitochondrial Uncoupling Activity.

Mitochondrial uncoupling by small molecule protonophores is a promising strategy for developing novel anticancer agents. Recently, aryl urea substituted fatty acids (aryl ureas) were identified as a new class of protonophoric anticancer agents. To mediate proton transport these molecules self-assemble into membrane-permeable anionic dimers in which intermolecular hydrogen bonds between the carboxylate and aryl-urea anion receptor delocalise the negative charge across the aromatic π-system. In this work, we extend the aromatic π-system by introducing a second phenyl substituent to the aryl urea scaffold and compare the proton transport mechanisms and mitochondrial uncoupling actions of these compounds to their monoaryl analogues. It was found that incorporation of meta-linked phenyl substituents into the aryl urea scaffold enhanced proton transport in vesicles and demonstrated superior capacity to depolarise mitochondria, inhibit ATP production and reduce the viability of MDA-MB-231 breast cancer cells. In contrast, diphenyl ureas linked through a 1,4-distribution across the phenyl ring displayed diminished proton transport activity, despite both diphenyl urea isomers possessing similar binding affinities for carboxylates. Mechanistic studies suggest that inclusion of a second aryl ring changes the proton transport mechanism, presumably due to steric factors that impose higher energy penalties for dimer formation.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Development of a Library of Thiophene-Based Drug-Like Lego Molecules: Evaluation of Their Anion Binding, Transport Properties, and Cytotoxicity.

The anion-binding and transport properties of an extensive library of thiophene-based molecules are reported. Seventeen bis-urea positional isomers, with different binding conformations and lipophilicities, have been synthesized by appending α- or ß-thiophene or α-, ß-, or γ-benzo[b]thiophene moieties to an ortho-phenylenediamine central core, yielding six subsets of positional isomers. Through 1 H NMR, X-ray crystallography, molecular modelling, and anion efflux studies, it is demonstrated that the most active transporters adopt a pre-organized binding conformation capable of promoting the recognition of chloride, using urea and C-H binding groups in a cooperative fashion. Additional large unilamellar vesicle-based assays, carried out under electroneutral and electrogenic conditions, together with N-methyl-d-glucamine chloride assays, have indicated that anion efflux occurs mainly through an H+ /Cl- symport mechanism. On the other hand, the most efficient anion transporter displays cytotoxicity against tumor cell lines, while having no effects on a cystic fibrosis cell line.

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.

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.

Fatty Acid Fueled Transmembrane Chloride Transport.

Generation of chemical gradients across biological membranes of cellular compartments is a hallmark of all living systems. Here we report a proof-of-concept prototype transmembrane pumping system in liposomes. The pump uses fatty acid to fuel chloride transport, thus generating a transmembrane chloride gradient. Addition of fatty acid to phospholipid vesicles generates a transmembrane pH gradient (pHin < pHout), and this electrochemical H+ potential is harnessed by an anionophore to drive chloride efflux via H+/Cl- cotransport. Further addition of fatty acid efficiently fuels the system to continuously drive chloride transport against the concentration gradient, up to [Cl-]in 65 mM | [Cl-]out 100 mM, and is 1400 times more efficient than using an external fuel. Based on our findings from dissecting the H+/Cl- flux process with the use of different liposomal fluorescence assays, and supported by additional liposome-based 13C NMR and DLS studies; we proposed that the presence of an anionophore can induce asymmetric distribution of fatty acid, and contribute to another Cl- flux mechanism in this system.

Supramolecular Transmembrane Anion Transport: New Assays and Insights.

Transmembrane anion transport has been the focus of a number of supramolecular chemistry research groups for a number of years. Much of this research is driven by the biological relevance of anion transport and the search to find new treatments for diseases such as cystic fibrosis, which is caused by genetic problems leading to faulty cystic fibrosis transmembrane conductance regulator (CFTR) channels, which in turn lead to reduced chloride and bicarbonate transport through epithelial cell membranes. Considerable effort has been devoted to the development of new transporters, and our group along with others have been searching for combinations of organic scaffolds and anion binding groups that produce highly effective transporters that work at low concentration. These compounds may be used in the future as "channel replacement therapies", restoring the flux of anions through epithelial cell membranes and ameliorating the symptoms of cystic fibrosis. Less effort has been put into gaining a fundamental understanding of anion transport processes. Over the last 3 years, our group has developed a number of new transport assays that allow anion transport mechanisms to be determined. This Account covers the latest developments in this area, providing a concise review of the new techniques we can use to study anion transport processes individually without resorting to measurement of exchange processes and the new insights that these assays provide. The Account provides an overview of the effects of anion transporters on cells and an explanation of why many systems perturb pH gradients within cells in addition to transporting chloride. We discuss assays to determine whether anionophores facilitate chloride or HCl transport and how this latter assay can be modified to determine chloride versus proton selectivity in small-molecule anion receptors. We show how molecular design can be used to produce receptors that are capable of transporting chloride without perturbing pH gradients. We cover the role that anion transporters in the presence of fatty acids play in dissipating pH gradients across lipid bilayer membranes and the effect that this process has on chloride-selective transport. We also discuss how coupling of anion transport to cation transport by natural cationophores can be used to determine whether anion transport is electrogenic or electroneutral. In addition, we compare these new assays to the previously used chloride/nitrate exchange assay and show how this exchange assay can underestimate the chloride transport ability of certain receptors that are rate-limited by nitrate transport.

Design of Chiral Supramolecular Polymers Exhibiting a Negative Nonlinear Response.

Many synthetic and supramolecular chiral polymeric systems are known to exhibit the "majority rules effect" (MRE), a positive nonlinear response in which a small enantiomeric excess (ee) of the chiral building blocks leads to unproportionally large chiroptical signals near zero ee. In contrast, the opposite "racemate rules effect" (RRE), a negative nonlinear response in which the chiroptical signals are flat near zero ee, while giving large nonlinear chiroptical responses to ee at high values, has only been occasionally observed. The origin of this unusual ee dependence remains elusive largely because few systems have been established that exhibit this effect. Herein, we present a design approach that enables the development of chiral supramolecular polymers with a pronounced negative nonlinear response akin to RRE. This is achieved by in situ generating a bidentate inducer for supramolecular polymerization that exists in both meso- and homochiral forms upon reacting with chiral guests. The presence of the meso-inducer creates an aggregate structure that has a little response in the circular dichroism (CD) spectra as a function of ee at a particular wavelength, but a homochiral inducer gives large changes in response to ee at this wavelength. This allowed for an RRE-like response to be observed when the CD intensity of the supramolecular polymers was plotted against the ee of the chiral guests that generate the meso- and homochiral inducers without the necessity of the racemic guest preferentially being incorporated into the polymer.

Investigating the Influence of Steric Hindrance on Selective Anion Transport.

A series of symmetrical and unsymmetrical alkyl tren based tris-thiourea anion transporters were synthesised and their anion binding and transport properties studied. Overall, increasing the steric bulk of the substituents resulted in improved chloride binding and transport abilities. Including a macrocycle in the scaffold enhanced the selectivity of chloride transport in the presence of fatty acids, by reducing the undesired H⁺ flux facilitated by fatty acid flip-flop. This study demonstrates the benefit of including enforced steric hindrance and encapsulation in the design of more selective anion receptors.

Voltage-Switchable HCl Transport Enabled by Lipid Headgroup-Transporter Interactions.

Synthetic anion transporters that facilitate transmembrane H+ /Cl- symport (cotransport) have anti-cancer potential due to their ability to neutralize pH gradients and inhibit autophagy in cells. However, compared to the natural product prodigiosin, synthetic anion transporters have low-to-modest H+ /Cl- symport activity and their mechanism of action remains less well understood. We report a chloride-selective tetraurea macrocycle that has a record-high H+ /Cl- symport activity similar to that of prodigiosin and most importantly demonstrates unprecedented voltage-switchable transport properties that are linked to the lack of uniport activity. By studying the anion binding affinity and transport mechanisms of four other anion transporters, we show that the lack of uniport and voltage-dependent H+ /Cl- symport originate from strong binding to phospholipid headgroups that hampers the diffusion of the free transporters through the membrane, leading to an unusual H+ /Cl- symport mechanism that involves only charged species. Our work provides important mechanistic insights into different classes of anion transporters and a new approach to achieve voltage-switchability in artificial membrane transport systems.

New Insights into the Anion Transport Selectivity and Mechanism of Tren-based Tris-(thio)ureas.

The anion transport properties of a series of previously reported tren-based anionophores have been revisited using new assays designed to measure anion uniport. This study provides new insights into the transport mechanism and selectivity of this important class of transporters. Specifically, we report the chloride and nitrate transport selectivity of these systems and quantify sulfate transport to determine EC50 values for sulfate transport for the first time. Two new assays were developed to study bicarbonate transport allowing accurate quantification of chloride/bicarbonate exchange.