Metallocalix[4]arene Polymers for Gravimetric Detection of N-Nitrosodialkylamines.

N-Nitrosamines are found in food, drugs, air, water, and soil. They pose a significant risk to human health because of their carcinogenicity; consequently, materials that can be used to selectively and sensitively detect nitrosamines are needed. In this work, we designed and synthesized two polymers bearing calix[4]arene or 4-tert-butylcalix[4]arene tungsten-imido complexes (PCalixH and PCalixtBu) as N-nitrosodimethylamine (NDMA) receptors. The interaction between metallocalix[4]arene monomers/polymers and NDMA was confirmed by 1H NMR and IR spectroscopy. Single-crystal X-ray analysis further revealed that the host-guest interaction is based on binding of the terminal oxygen of NDMA to tungsten within the calixarene cavity. Gravimetric detection of NDMA was performed on a quartz crystal microbalance (QCM) in air. Both polymers show responses to NDMA, with PCalixtBu exhibiting a low theoretical limit of detection of 5 ppb for NDMA. The sensor also shows high selectivity toward NDMA and moderate humidity tolerance. This work provides a sensitive sensor for detection of NDMA and also offers a class of new, selective, and efficient NDMA receptors for the future design of NDMA sensors and NDMA extraction materials.

Flexible Synthetic Carbohydrate Receptors as Inhibitors of Viral Attachment.

Carbohydrate-receptor interactions are often involved in the docking of viruses to host cells, and this docking is a necessary step in the virus life cycle that precedes infection and, ultimately, replication. Despite the conserved structures of the glycans involved in docking, they are still considered "undruggable", meaning these glycans are beyond the scope of conventional pharmacological strategies. Recent advances in the development of synthetic carbohydrate receptors (SCRs), small molecules that bind carbohydrates, could bring carbohydrate-receptor interactions within the purview of druggable targets. Here we discuss the role of carbohydrate-receptor interactions in viral infection, the evolution of SCRs, and recent results demonstrating their ability to prevent viral infections in vitro. Common SCR design strategies based on boronic ester formation, metal chelation, and noncovalent interactions are discussed. The benefits of incorporating the idiosyncrasies of natural glycan-binding proteins-including flexibility, cooperativity, and multivalency-into SCR design to achieve nonglucosidic specificity are shown. These studies into SCR design and binding could lead to new strategies for mitigating the grave threat to human health posed by enveloped viruses, which are heavily glycosylated viroids that are the cause of some of the most pressing and untreatable diseases, including HIV, Dengue, Zika, influenza, and SARS-CoV-2.

Highly Efficient, Tripodal Ion-Pair Receptors for Switching Selectivity between Acetates and Sulfates Using Solid-Liquid and Liquid-Liquid Extractions.

A tripodal, squaramide-based ion-pair receptor 1 was synthesized in a modular fashion, and 1H NMR and UV-vis studies revealed its ability to interact more efficiently with anions with the assistance of cations. The reference tripodal anion receptor 2, lacking a crown ether unit, was found to lose the enhancement in anion binding induced by presence of cations. Besides the ability to bind anions in enhanced manner by the "single armed" ion-pair receptor 3, the lack of multiple and prearranged binding sites resulted in its much lower affinity towards anions than in the case of tripodal receptors. Unlike with receptors 2 or 3, the high affinity of 1 towards salts opens up the possibility of extracting extremely hydrophilic sulfate anions from aqueous to organic phase. The disparity in receptor 1 binding modes towards monovalent anions and divalent sulfates assures its selectivity towards sulfates over other lipophilic salts upon liquid-liquid extraction (LLE) and enables the Hofmeister bias to be overcome. By changing the extraction conditions from LLE to SLE (solid-liquid extraction), a switch of selectivity from sulfates to acetates was achieved. X-ray measurements support the ability of anion binding by cooperation of the arms of receptor 1 together with simultaneous binding of cations.

Multimerization strategies for efficient production and purification of highly active synthetic cytokine receptor ligands.

Cytokine signaling is transmitted by cell surface receptors which act as natural biological switches to control cellular functions such as immune reactions. Recently, we have designed synthetic cytokine receptors (SyCyRs) consisting of green fluorescent protein (GFP)- and mCherry-nanobodies fused to the transmembrane and intracellular domains of cytokine receptors. Following stimulation with homo- and heterodimeric GFP-mCherry fusion proteins, the resulting receptors phenocopied signaling induced by physiologically occurring cytokines. GFP and mCherry fusion proteins were produced in E. coli or CHO-K1 cells, but the overall yield and stability was low. Therefore, we applied two alternative multimerization strategies and achieved immunoglobulin Fc-mediated dimeric and coiled-coil GCN4pII-mediated trimeric assemblies. GFP- and/or mCherry-Fc homodimers activated synthetic gp130 cytokine receptors, which naturally respond to Interleukin 6 family cytokines. Activation of these synthetic gp130 receptors resulted in STAT3 and ERK phosphorylation and subsequent proliferation of Ba/F3-gp130 cells. Half-maximal effective concentrations (EC50) of 8.1 ng/ml and 0.64 ng/ml were determined for dimeric GFP-Fc and mCherry-Fc, respectively. This is well within the expected EC50 range of the native cytokines. Moreover, we generated tetrameric and hexameric GFP-mCherry-Fc fusion proteins, which were also biologically active. This highlighted the importance of close juxtaposition of two cytokine receptors for efficient receptor activation. Finally, we used a trimeric GCN4pII motif to generate homo-trimeric GFP and mCherry complexes. These synthetic cytokines showed improved EC50 values (GFP3: 0.58 ng/ml; mCherrry3: 0.37 ng/ml), over dimeric Fc fused variants. In conclusion, we successfully generated highly effective and stable multimeric synthetic cytokine receptor ligands for activation of synthetic cytokine receptors.

The Effect of Substitution Pattern on Binding Ability in Regioisomeric Ion Pair Receptors Based on an Aminobenzoic Platform.

A series of ditopic ion pair receptors equipped with 4-nitrophenylurea and 1-aza-18-crown-6-ether linked by ortho-(1), meta-(2), and para-(3) substituted benzoic acid were readily synthesized in three steps from commercially available materials. The binding properties of these regioisomeric receptors were determined using UV-vis and 1H NMR spectroscopy in MeCN and in the solid state by single-crystal X-ray diffraction crystallography. The solution studies revealed that, apart from carboxylates, all the anions tested formed stronger complexes in the presence of sodium cations. Receptors 2 and 3 were found to interact with ion pairs with remarkably higher affinity than ortho-substituted 1. 1H NMR titration experiments showed that both urea NH protons interacted with anions with comparable strength in the case of receptors 2 and 3, but only one of the NHs was effective in anion binding in the case of receptor 1. X-ray analysis of the crystal structure of receptor 1 and 1·NaPF6 complex showed that binding was hampered due to the formation of an intramolecular hydrogen bond. Analysis of the crystal structures of 2·NaBr and 3·NaBr complexes revealed that proper mutual orientation of binding domains was responsible for the improved binding of the sodium salts.

Counteracting in Vitro Toxicity of the Ionophoric Mycotoxin Beauvericin-Synthetic Receptors to the Rescue.

Beauvericin (BEA) and enniatins are toxic ionophoric cyclodepsipeptides that mainly occur in grains. As such, their presence in food commodities poses a concern for public health. To date, despite recent European Food Safety Authority emphasis on the need for more data to evaluate long-term toxicity effects, no suitable affinity reagents are available to detect the presence of BEA and derivatives in food samples. We here report on the synthesis of a small library of artificial receptors with varying cavity sizes and different hydrophobic building blocks. Immobilization of one of the receptors on solid support resulted in a strong retention of beauvericin, thus revealing promising properties as solid-phase extraction material for sample pretreatment. Furthermore, treatment of HepG2 cells with the most promising receptor markedly reduced beauvericin-induced cytotoxicity, hinting toward the possibility of using synthetic receptors as antidotes against ionophoric toxins.

Porphyrin cage compounds based on glycoluril - from enzyme mimics to functional molecular machines.

The study of artificial receptor molecules with the intention to mimic enzyme-substrate binding processes and catalysis in nature has always been a traditional area of research in supramolecular chemistry. Along this line, our group has developed a family of porphyrin cage compounds based on glycoluril and employed these in host-guest binding studies, as components of allosterically controlled self-assembled processes, in which structural changes in the cage upon complexation of a guest or a ligand change binding equilibria, and as enzyme mimics in supramolecular catalysis. In a recently started research program aimed at developing a new molecular approach to long-term data storage, porphyrin cage compounds are studied as molecular machines to encode information into synthetic polymer chains. In this Feature Article we will give an overview of the above aspects of our porphyrin cage compounds and place them in the context of related systems reported in the literature.

Multisite Binding of Drugs and Natural Products in an Entropically Favorable, Heteroleptic Receptor.

The cavities of artificial receptors are defined by how their components fit together. The encapsulation of specific molecules can thus be engineered by considering geometric principles; however, intermolecular interactions and steric fit scale with receptor size, such that the ability to bind multiple guests from a specific class of compounds remains a current challenge. By employing metal-organic self-assembly, we have prepared a triangular prism from two different ligands that is capable of binding more than 20 different natural products, drugs, and steroid derivatives within its prolate cavity. Encapsulation inflates the host, enhancing its ability to bind other guests in peripheral pockets and thus enabling our system to bind combinations of different drug and natural product cargoes in different locations simultaneously. This new mode of entropically favorable self-assembly thus enables central encapsulation to amplify guest-binding events around the periphery of an artificial receptor.

Anti-Zika Activity of a Library of Synthetic Carbohydrate Receptors.

Zika virus (ZIKV), a mosquito-borne flavivirus, is a global health concern because of its association with severe neurological disorders. Currently, there are no antiviral therapies that have been specifically approved to treat ZIKV, and there is an urgent need to develop effective anti-ZIKV agents. Here, we report anti-ZIKV activity of 16 synthetic carbohydrate receptors (SCRs) that inhibit ZIKV infection in Vero and HeLa cells. Using a ZIKV reporter virus particle-based infection assay, our data demonstrates these SCRs are highly potent with IC50s as low as 0.16 µM and negligible toxicity at several-fold higher concentrations. Time-of-addition studies showed that these SCRs inhibit the early stages of the virus infection, which is consistent with the proposed mode of action, where the SCRs likely inhibit binding between the virus and cell-surface glycans, thereby preventing viral entry into the cells and, as such, this study demonstrates a potential new strategy against ZIKV.

Facile Preparation of a Bacteria Imprinted Artificial Receptor for Highly Selective Bacterial Recognition and Label-Free Impedimetric Detection.

The effective identification and quantification of pathogenic bacteria is essential for addressing serious public health issues. Here, we demonstrate a simple and universal impedimetric sensor for highly selective and sensitive detection of pathogenic bacteria based on the recognition by a bacteria-imprinted polypyrrole (BIP) film. The BIP film was facilely prepared via one-step electro-polymerization followed by in situ removal of the bacterial template. The film structure is novel with noncavity-like imprinted sites situated at the surface of the polypyrrole (PPy) matrix, which are more accessible for the target bacteria and should enhance the mass transfer and the binding kinetics. A limit of quantitation low to 103 CFU/mL was achieved within 1 h for the detection of E. coli O157:H7, which is comparable to the antibody-based assays. Moreover, the sensor displayed remarkable selectivity, especially regarding the specific identification of bacterial serotypes. When employed to analyze E. coli O157:H7 in real drinking water, apple juice, and milk samples, the sensor showed recoveries from 96.0% to 107.9% with relative standard derivations (RSDs) less than 4%. The BIP-based sensing strategy provides a universal approach for specific, selective, and rapid detection of pathogenic bacteria. As compared to conventional biosensors based on biomolecular recognition, this sensor shows clear advantages including easy-of-preparation, robustness, and low cost, which may hold great potential in fields of food/public safety monitoring.

A Biomimetic Synthetic Receptor Selectively Recognising Fucose in Water.

Carbohydrate recognition in water by biomimetic receptors is an attractive, but very challenging goal. Despite advances achieved in glucose recognition, little or no success has been obtained in the recognition of other saccharidic epitopes of paramount importance in biological processes. Herein, the unprecedented recognition of fucose in water by an artificial receptor that shows affinities closely comparable to those of several lectins is reported. The receptor has been constructed by assembling a hydrogen-bonding element (carbazole), a hydrophobic aromatic moiety (anthracene), and a water-solubilising function (phosphonate) into a macrocyclic structure to provide the appropriate binding geometry. The described receptor binds fucose with sub-millimolar affinity in water at physiological pH; this shows that enthalpic binding can be ascribed to hydrogen bonding to saccharidic hydroxy groups and to CH-π interactions between the sugar backbone and aromatic moieties. Experimental NOE contacts coupled to conformational search calculations return a picture of a binding site in which fucose assumes a staggered orientation reminiscent of that shown by fucose when bound to the Ralstonia solanacearum lectin (RSL).

Modulation of the Selectivity in Anions Recognition Processes by Combining Hydrogen- and Halogen-Bonding Interactions.

Most of the halogen bonding receptors for anions described use halogen bonding binding sites solely in the anion recognition process; only a few examples report the study of anion receptors in which the halogen bonding interaction has been used in combination with any other non-covalent interaction. With the aims to extend the knowledge in the behaviour of this kind of mixed receptors, we report here the synthesis and the anion recognition and sensing properties of a new halogen- and hydrogen- bonding receptor which binds anions by the cooperation of both non-covalent interactions. Fluorescence studies showed that the behaviour observed in the anion recognition sensing is similar to the one previously described for the halogen analogue and is quite different to the hydrogen one. On the other hand, the association constants obtained by ¹H-NMR data demonstrate that the mixed halogen- and hydrogen-bonding receptor is more selective for SO42- anion than the halogen or hydrogen analogues.

Hybrid Synthetic Receptors on MOSFET Devices for Detection of Prostate Specific Antigen in Human Plasma.

The study reports the use of extended gate field-effect transistors (FET) for the label-free and sensitive detection of prostate cancer (PCa) biomarkers in human plasma. The approach integrates for the first time hybrid synthetic receptors comprising of highly selective aptamer-lined pockets (apta-MIP) with FETs for sensitive detection of prostate specific antigen (PSA) at clinically relevant concentrations. The hybrid synthetic receptors were constructed by immobilizing an aptamer-PSA complex on gold and subjecting it to 13 cycles of dopamine electropolymerization. The polymerization resulted in the creation of highly selective polymeric cavities that retained the ability to recognize PSA post removal of the protein. The hybrid synthetic receptors were subsequently used in an extended gate FET setup for electrochemical detection of PSA. The sensor was reported to have a limit of detection of 0.1 pg/mL with a linear detection range from 0.1 pg/mL to 1 ng/mL PSA. Detection of 1-10 pg/mL PSA was also achieved in diluted human plasma. The present apta-MIP sensor developed in conjunction with FET devices demonstrates the potential for clinical application of synthetic hybrid receptors for the detection of clinically relevant biomarkers in complex samples.

Computational approaches in the design of synthetic receptors - A review.

The rational design of molecularly imprinted polymers (MIPs) has been a major contributor to their reputation as "plastic antibodies" - high affinity robust synthetic receptors which can be optimally designed, and produced for a much reduced cost than their biological equivalents. Computational design has become a routine procedure in the production of MIPs, and has led to major advances in functional monomer screening, selection of cross-linker and solvent, optimisation of monomer(s)-template ratio and selectivity analysis. In this review the various computational methods will be discussed with reference to all the published relevant literature since the end of 2013, with each article described by the target molecule, the computational approach applied (whether molecular mechanics/molecular dynamics, semi-empirical quantum mechanics, ab initio quantum mechanics (Hartree-Fock, Møller-Plesset, etc.) or DFT) and the purpose for which they were used. Detailed analysis is given to novel techniques including analysis of polymer binding sites, the use of novel screening programs and simulations of MIP polymerisation reaction. The further advances in molecular modelling and computational design of synthetic receptors in particular will have serious impact on the future of nanotechnology and biotechnology, permitting the further translation of MIPs into the realms of analytics and medical technology.

A high-yield synthesis of [m]biphenyl-extended pillar[n]arenes for an efficient selective inclusion of toluene and m-xylene in the solid state.

[m]Bp-ExPn with a rigid and nanometer-sized cavity, as an extended version of pillar[n]arene by replacing 1,4-dimethoxybenzene monomers with biphenyl entities, was synthesized for the first time. Intriguingly, toluene and m-xylene can be stably included within the cavity of [2]Bp-ExP6, which endows these newly developed synthetic receptors with great potential in the purification of petrochemicals.

A threading receptor for polysaccharides.

Cellulose, chitin and related polysaccharides are key renewable sources of organic molecules and materials. However, poor solubility tends to hamper their exploitation. Synthetic receptors could aid dissolution provided they are capable of cooperative action, for example by multiple threading on a single polysaccharide molecule. Here we report a synthetic receptor designed to form threaded complexes (polypseudorotaxanes) with these natural polymers. The receptor binds fragments of the polysaccharides in aqueous solution with high affinities (K(a) up to 19,000 M(-1)), and is shown--by nuclear Overhauser effect spectroscopy--to adopt the threading geometry. Evidence from induced circular dichroism and atomic force microscopy implies that the receptor also forms polypseudorotaxanes with cellulose and its polycationic analogue chitosan. The results hold promise for polysaccharide solubilization under mild conditions, as well as for new approaches to the design of biologically active molecules.

Secondary Binding Interactions in a Synthetic Receptor for Trimethyllysine.

We have systematically studied how secondary interactions with neighboring lysine (Lys) and arginine (Arg) residues influence the binding and selectivity of the synthetic receptor A2 N for trimethyllysine (Kme3 ). Multiple secondary binding sites on A2 N are formed by carboxylates rigidly positioned over aromatic rings, a motif that has been shown to stabilize salt bridges. We varied the spacing between KmeX (X=0, 3) and an ancillary Lys or Arg and measured binding by isothermal titration calorimetry (ITC). These studies revealed that both neighboring residues improve the binding of A2 N to KmeX by approximately 1 kcal mol(-1) , with little influence of the spacing. Nonetheless, the improvement in affinity caused by Arg is enthalpically driven, while for Lys it is entropically driven, suggesting different mechanisms by which the residues interact with the secondary binding site.

Structurally Flexible C3-Symmetric Receptors for Molecular Recognition and Their Self-Assembly Properties.

The bioinspired design and synthesis of building blocks and their assemblies by the supramolecular approach has ever fascinated scientists to utilize such artificial systems for numerous purposes. Flexibility is a basic feature of natural systems. However, in artificial systems this is difficult to control, especially if there is no preorganization of the component(s) of a system. We have designed and synthesized a series of C3 -symmetric N-bridged flexible receptors and successfully utilized them to selectively entrap the notorious and toxic nitrate anion in aqueous medium. This was the first report of highest binding affinity for the nitrate anion in aqueous medium. An impressive self-sorting phenomenon of reversibly formed hydrogen-bonded capsules, which self-assembled from flexible tripodal receptors having branches of similar size and bearing the same amide functionality, has been disclosed. Encapsulated nitrate anion has been further utilized for the photochemical [2+2] cycloaddition reaction for the synthesis of strained four-membered ring structures through dynamic self-assembly. In this Personal Account, we summarize these results showing the utility of naturally inspired flexibility in artificial systems.

Post-imprinting and In-Cavity Functionalization.

Molecularly imprinted polymers (MIPs) are artificial materials capable of molecular recognition for target molecules. Currently MIPs have been prepared without further modification after polymerization, and used for predetermined single purposes. Post-imprinting modifications (PIMs) presented here can provide site-specific modifications within the molecularly imprinted binding cavities after polymerization, enabling MIPs to become more complex functional materials as were the cases of naturally occurring conjugated proteins. We present an overview of the research on MIPs involving PIMs, including transformation of binding sites, on/off switching of binding activity, introduction of desirable functions such as fluorescent signalling functions, catalytic activity, and so on. The combination of PIMs with molecular imprinting appears to be a powerful tool for preparing a diverse range of biomimetic functional materials.

A thermodynamic insight into the recognition of hydrophilic and hydrophobic amino acids in pure water by aza-scorpiand type receptors.

Interactions of different hydrophilic (His, Asp, Glu,) and hydrophobic (Ala, Phe, Tyr, Trp) amino acids in water with a scorpiand aza-macrocycle (L1) containing a pyridine group in the ring and its derivative (L2) bearing a naphthalene group in the tail have been analysed by potentiometric and calorimetric measurements. Theoretical calculations corroborate that major attractive forces that hold the adduct together are hydrogen bonds and salt-bridges, even though other interactions such as π-stacking or NH(+)⋯π may contribute in the case of hydrophobic amino acids and L2. Calorimetric measurements indicate that the interactions between L1 and the different amino acids are principally driven by entropy, often associated with solvation/desolvation processes.