Anion binding and transport with meso-alkyl substituted two-armed calix[4]pyrroles bearing urea and hydroxyl groups.

Calix[4]pyrroles bearing hydroxyl (1) or urea (3) groups attached to the meso-positions with propyl linkers were synthesized as cis- and trans-isomers. The anion binding properties of cis-1 and cis-3 were screened with ion-mobility mass spectrometry, where cis-1 formed complexes with Cl-, Br- and H2PO4-, whereas cis-3 formed complexes with most of the investigated anions, including Cl-, Br-, I-, NO3-, ClO4-, OTf-, SCN- and PF6-. The structures of the chloride complexes were further elucidated with density functional theory calculations and a crystal structure obtained for cis-1. In solution, chloride and dihydrogenphosphate anion binding with cis-1 and cis-3 were compared using 1H NMR titrations. To assess the suitability of two-armed calix[4]pyrroles as anion transporters, chloride transport studies of cis-1, cis-3 and trans-3 were performed using large unilamellar vesicles. The results revealed that cis-3 had the highest activity among the investigated calix[4]pyrroles, which was related to the improved affinity and isolation of chloride inside the binding cavity of cis-3 in comparison to cis-1. The results indicate that appending calix[4]pyrroles with two hydrogen bonding arms is a feasible strategy to obtain anion transporters and receptors with high anion affinity.

Chemically Fueled Communication Along a Scaffolded Nanoscale Array of Squaramides.

Relaying conformational change over several nanometers is central to the function of allosterically regulated proteins. Replicating this mechanism artificially would provide important communication tools, but requires nanometer-sized molecules that reversibly switch between defined shapes in response to signaling molecules. In this work, 1.8 nm long rigid rod oligo(phenylene-ethynylene)s are scaffolds for switchable multi-squaramide hydrogen-bond relays. Each relay can adopt either a parallel or an antiparallel orientation relative to the scaffold; the preferred orientation is dictated by a director group at one end. An amine director responded to proton signals, with acid-base cycles producing multiple reversible changes in relay orientation that were reported by a terminal NH, which is 1.8 nm distant. Moreover, a chemical fuel acted as a dissipative signal. As the fuel was consumed, the relay reverted to its original orientation, illustrating how information from out-of-equilibrium molecular signals can be communicated to a distant site.

Electrochemical assessment of a tripodal thiourea-based anion receptor at the liquid|liquid interface.

Thiourea-based receptors for anions have been widely studied due to their ability to transport anions across phospholipid bilayers. The binding affinity of a tripodal thiourea-based receptor for anions was assessed at the aqueous|organic interface using electrochemical measurements. A 1 : 1 stoichiometry was determined for the complexation of most anions, with a higher stoichiometry found in the presence of excess Cl- and Br- anions. High stability constants were estimated for the formation of the complexes at the aqueous|1,2-dichlorobenzene (DCB) interface. When compared with an organic solvent of higher polarity, nitrobenzene (NB), the high stability constants observed in DCB are believed to be due to the less competitive environment of the less polar solvent. Protonation of the receptor at the bridgehead tertiary amine was also inferred from the potential-dependent voltammetric measurements that are not related to anion:receptor complexation. The inherent advantages of the electrochemical method with the use of low polarity solvents are expected to provide new insights into the binding and transport of newly-developed neutral receptors.

Light-Driven Photoconversion of Squaramides with Implications in Anion Transport.

Simple, clean and fast photoconversion of aniline-derived squaramides was achieved by flashlight illumination. UV irradiation enabled the photochemical squaramide ring-opening to generate 1,2-bisketenes, which DMSO trapped as the nucleophilic oxidant. The only photoproducts isolated were 3,4-arylamino maleic anhydrides, which present conformational preferences very different from those of their parent squaramides. Similar photoconversion was achieved in MeOH. The UV-mediated time-dependent anion transport inhibition was demonstrated, establishing a new approach for modulating the transport abilities of AD-squaramides.

Transmembrane Transport of Bicarbonate by Anion Receptors.

The development of synthetic anion transporters is motivated by their potential application as treatment for diseases that originate from deficient anion transport by natural proteins. Transport of bicarbonate is important for crucial biological functions such as respiration and digestion. Despite this biological relevance, bicarbonate transport has not been as widely studied as chloride transport. Herein we present an overview of the synthetic receptors that have been studied as bicarbonate transporters, together with the different assays used to perform transport studies in large unilamellar vesicles. We highlight the most active transporters and comment on the nature of the functional groups present in active and inactive compounds. We also address recent mechanistic studies that have revealed different processes that can lead to net transport of bicarbonate, as well as studies reported in cells and tissues, and comment on the key challenges for the further development of bicarbonate transporters.

Monofunctionalized Fluorinated Bambusurils and Their Conjugates for Anion Transport and Extraction.

Bambusurils are macrocyclic molecules that are known for their high binding affinity and selectivity toward anions. Here, we present the preparation of two bambusurils bearing fluorinated substituents and one carboxylic function. These monofunctionalized bambusurils were conjugated with crown ether and cholesterol units. The resulting conjugates were successfully tested in liquid-liquid extraction of inorganic salts and chloride/bicarbonate transport across lipid bilayers.

Calix[6]arenes with halogen bond donor groups as selective and efficient anion transporters.

Here we present the anion binding and anion transport properties of a series of calix[6]arenes decorated on their small rim with either halogen bond or hydrogen bond donating groups. We show that the halogen bond donating iodotriazole groups enable highly selective transport of chloride and nitrate anions, without transport of protons or hydroxide, at rates similar to those observed with thiourea or squaramide groups.

Hydrazones in anion transporters: the detrimental effect of a second binding site.

Synthetic anion transporters can be developed using anion receptors that are able to bind the anion and stabilize it in the lipophilic interior of a bilayer membrane, and they usually contain functional groups with acidic NHs, such as ureas, thioureas and squaramides. To assess the suitability of acylhydrazones as a new functional group for the preparation of anion transporters, we have studied a family of thioureas functionalized with these and related functional groups. 1H NMR titrations and DFT calculations indicate that the thioureas bearing acylhydrazone groups behave as chloride receptors with two separate binding sites, of which the acylhydrazone binds weaker than the thiourea. Chloride transport studies show that the additional binding site has a detrimental effect on thiourea-based transporters, and this phenomenon is also observed for bis(thio)ureas with two separate binding sites. We propose that the presence of a second anion binding unit hinders the transport activity of the thiourea due to additional interactions with the phospholipids of the membrane. In agreement with this hypothesis, extensive molecular dynamics simulations suggest that the molecules will tend to be positioned in the water/lipid interface, driven by the interaction of the NHs of the thiourea and of the acylhydrazone groups with the POPC polar head groups and water molecules. Moreover, the interaction energies show that the poorest transporters have indeed the strongest interactions with the membrane phospholipids, inhibiting chloride transport. This detrimental effect of additional functional groups on transport activity should be considered when designing new ion transporters, unless these groups cooperatively promote anion recognition and transmembrane transport.

Transmembrane Transport of Bicarbonate Unravelled.

Invited for the cover of this issue are Dr. Stephen Butler, Dr. Hennie Valkenier and co-workers at Université Libre de Bruxelles, Loughborough University, Masaryk University, and the University of Bristol. The image depicts the transport of bicarbonate anions versus the spontaneous diffusion of CO2 across the lipid bilayer of a liposome. Read the full text of the article at 10.1002/chem.202100491.

Transmembrane Transport of Bicarbonate Unravelled*.

Anion receptors can be used to transport ions across lipid bilayers, which has potential for therapeutic applications. Synthetic bicarbonate transporters are of particular interest, as defects in transmembrane transport of bicarbonate are associated with various diseases. However, no convenient method exists to directly observe bicarbonate transport and study the mechanisms involved. Here, an assay is presented that allows the kinetics of bicarbonate transport into liposomes to be monitored directly and with great sensitivity. The assay utilises an encapsulated europium(III) complex, which exhibits a large increase in emission intensity upon binding bicarbonate. Mechanisms involving CO2 diffusion and the dissipation of a pH gradient are shown to be able to lead to an increase in bicarbonate concentration within liposomes, without transport of the anion occurring at all. By distinguishing these alternative mechanisms from actual bicarbonate transport, this assay will inform the future development of bicarbonate transporters.

Introducing a squaramide-based self-immolative spacer for controlled drug release.

Herein we report the design, synthesis and assessment of the first example of a squaramide-based self-immolative system triggered by an enzymatic reduction. We have proved that the release of the alkylating agent N',N'-(bis(2-chloroethyl)benzene)-1,4-diamine (ANM) provokes a dramatic reduction of the survival factor in glioblastoma cells, evidencing the suitability of the squaramide-based spacer for drug delivery applications.

Influence of the Insertion Method of Aryl-Extended Calix[4]pyrroles into Liposomal Membranes on Their Properties as Anion Carriers.

We disclose the results of our investigations on the influence that the insertion method of aryl-extended calix[4]pyrrole into liposomal membranes exerts on their properties as anion carriers. We use the standard HPTS assay to assess the transport properties of the carriers. We show that the post-insertion of the carrier, as DMSO solution, assigns better transport activities to the "two-wall" α,α-aryl-extended calix[4]pyrrole 1 compared to the "four-wall" α,α,α,α-counterpart 2. Notably, opposite results were obtained when the carriers were pre-inserted into the liposomal membranes. We assign this difference to an improved incorporation of carrier 2 into the membrane when delivered by the pre-insertion method. On the other hand, carrier 1 shows comparable levels of transport independently of the method used for its incorporation. Thus, an accurate comparison of the chloride transport activities featured by these two carriers demands their pre-incorporation in the liposomal membranes. In contrast, using the lucigenin assay with the pre-insertion method both carriers displayed similar transport efficiencies.

The Role of N-Methyl Squaramides in a Hydrogen-Bonding Strategy to Fold Peptidomimetic Compounds.

Small peptides and peptomimetic compounds are valuable tools to probe and study biological systems. Small synthetic peptide analogues adopt a given secondary structure driven by structural modules that organize the compound architecture. Among them, ß- and α-turn mimetics are widely used. This work reports SQ4 and SQ5 squaramido-based turn modules that combine tertiary and secondary squaramide bonds in their structure to control their conformational properties. The efficacy of this combination has been evaluated to promote folding in peptide-like compounds to obtain parallel and antiparallel-hairpin model compounds in hydrogen-bonding competitive media. Crystallographic structures of model compounds and conformational studies based on NMR spectroscopic analysis of the squaramido-peptides confirm that secondary-tertiary squaramides are more prone to adopt the E,Z-conformation than di-secondary squaramides, and consequently are more suitable to gain conformational control over foldable peptidomimetic compounds.