Semisynthesis of Aminomethyl-Insulin: An Atom-Economic Strategy to Increase the Affinity and Selectivity of a Protein for Recognition by a Synthetic Receptor.

Advancements in the molecular recognition of insulin by nonantibody-based means would facilitate the development of methodology for the continuous detection of insulin for the management of diabetes mellitus. Herein, we report a novel insulin derivative that binds to the synthetic receptor cucurbit[7]uril (Q7) at a single site and with high nanomolar affinity. The insulin derivative was prepared by a four-step protein semisynthetic method to present a 4-aminomethyl group on the side chain of the PheB1 position. The resulting aminomethyl insulin binds to Q7 with an equilibrium dissociation constant value of 99 nM in neutral phosphate buffer, as determined by isothermal titration calorimetry. This 6.8-fold enhancement in affinity versus native insulin was gained by an atom-economical modification (-CH2NH2). To the best of our knowledge, this is the highest reported binding affinity for an insulin derivative by a synthetic receptor. This strategy for engineering protein affinity tags induces minimal change to the protein structure while increasing affinity and selectivity for a synthetic receptor.

Molecular Recognition in Water Using Macrocyclic Synthetic Receptors.

Molecular recognition in water using macrocyclic synthetic receptors constitutes a vibrant and timely research area of supramolecular chemistry. Pioneering examples on the topic date back to the 1980s. The investigated model systems and the results derived from them are key for furthering our understanding of the remarkable properties exhibited by proteins: high binding affinity, superior binding selectivity, and extreme catalytic performance. Dissecting the different effects contributing to the proteins' properties is severely limited owing to its complex nature. Molecular recognition in water is also involved in other appreciated areas such as self-assembly, drug discovery, and supramolecular catalysis. The development of all these research areas entails a deep understanding of the molecular recognition events occurring in aqueous media. In this review, we cover the past three decades of molecular recognition studies of neutral and charged, polar and nonpolar organic substrates and ions using selected artificial receptors soluble in water. We briefly discuss the intermolecular forces involved in the reversible binding of the substrates, as well as the hydrophobic and Hofmeister effects operating in aqueous solution. We examine, from an interdisciplinary perspective, the design and development of effective water-soluble synthetic receptors based on cyclic, oligo-cyclic, and concave-shaped architectures. We also include selected examples of self-assembled water-soluble synthetic receptors. The catalytic performance of some of the presented receptors is also described. The latter process also deals with molecular recognition and energetic stabilization, but instead of binding ground-state species, the targets become elusive counterparts: transition states and other high-energy intermediates.

Synthetic Receptors Based on Abiotic Cyclo(pseudo)peptides.

Work on the use of cyclic peptides or pseudopeptides as synthetic receptors started even before the field of supramolecular chemistry was firmly established. Research initially focused on the development of synthetic ionophores and involved the use of macrocycles with a repeating sequence of subunits along the ring to facilitate the correlation between structure, conformation, and binding properties. Later, nonnatural amino acids as building blocks were also considered. With growing research in this area, cyclopeptides and related macrocycles developed into an important and structurally diverse receptor family. This review provides an overview of these developments, starting from the early years. The presented systems are classified according to characteristic structural elements present along the ring. Wherever possible, structural aspects are correlated with binding properties to illustrate how natural or nonnatural amino acids affect binding properties.

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.

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.

Tailoring minimal synthetic receptors to reconstitute signaling properties through multiple tyrosine motifs.

As intracellular signal transduction is important for determining cell fate, artificial control of signaling properties through engineered receptors is attractive in the fields of synthetic biology and cell therapy. In this study, we tailored minimal synthetic receptors to reconstitute signaling properties by incorporating multiple tyrosine motifs. The size of molecular parts including the linker between the tyrosine motifs was minimized as much as possible to create the minimal synthetic receptors. By combining the membrane localization signal sequence, a mutant of FK506-binding protein, a JAK-binding domain, tyrosine motifs, and linkers, we successfully reconstituted simple receptor chains that were activated by dimerization via a synthetic small-molecule ligand capable of membrane permeation. Furthermore, up to four signaling molecules of interest were able to be recruited and activated by the minimal synthetic receptors. Thus, the tailored minimal synthetic receptors could be utilized to analyze the role of specific signaling molecules/pathways in controlling cell fate and to efficiently induce specific cell fate for therapeutic applications in the future.

Biomimetic carbohydrate recognition.

Carbohydrates are important but challenging targets for supramolecular chemists. They possess complex, irregular and variable structures, and are strongly attracted to water, their natural environment. This tutorial review describes work on synthetic receptors which bind carbohydrates through non-covalent interactions, mimicking the strategies used in biology. Emphasis is placed on systems which operate in purely aqueous solution, without involvement of organic solvents. Although the problem is difficult, the careful design of complementary cavities can lead to surprisingly good results. In particular, a receptor for glucose has achieved performance which generally matches biology, and augurs well for real-world applications.

Cholesteric Molecular Tweezer Artificial Receptor for Rapid and Highly Selective Detection of Ag+ in Food Samples.

Chiral cholesteric molecular tweezer 7a was synthesized, and its recognition properties for Ag+, Al3+, Ca2+ etc., were investigated by UV and fluorescence spectra. The results showed that in ethanol/Tris (1/1, v/v, pH 7.0) buffer solution, the host molecular tweezer 7a had a specific recognition ability for Ag+, the detection limit was up to 1 × 10-6 mol/L, and other metal ions had little effect on Ag+ recognition. At the same time, the naked-eye detection of Ag+ was realized by the light red color of the complex solution. Furthermore, the mechanism of recognition of Ag+ by molecular tweezer 7a was studied by a nuclear magnetic titration test and computer molecular simulation, and a rapid detection method of Ag+ using host molecular tweezer 7a was established. Through the determination of Ag+ in milk powder, quinoa and other food samples, it was proved that this novel method had a good application prospect for the detection of Ag+ in food.

Molecular Recognition and Cocrystallization of Methylated and Halogenated Fragments of Danicalipin A by Enantiopure Alleno-Acetylenic Cage Receptors.

Enantiopure (P)4- and (M)4-configured alleno-acetylenic cage (AAC) receptors offer a highly defined interior for the complexation and structure elucidation of small molecule fragments of the stereochemically complex chlorosulfolipid danicalipin A. Solution (NMR), solid state (X-ray), and theoretical investigations of the formed host-guest complexes provide insight into the conformational preferences of 14 achiral and chiral derivatives of the danicalipin A chlorohydrin core in a confined, mostly hydrophobic environment, extending previously reported studies in polar solvents. The conserved binding mode of the guests permits deciphering the effect of functional group replacements on Gibbs binding energies ΔG. A strong contribution of conformational energies toward the binding affinities is revealed, which explains why the denser packing of larger apolar domains of the guests does not necessarily lead to higher association. Enantioselective binding of chiral guests, with energetic differences ΔΔG293 K up to 0.7 kcal mol-1 between diastereoisomeric complexes, is explained by hydrogen- and halogen-bonding, as well as dispersion interactions. Calorimetric studies (ITC) show that the stronger binding of one enantiomer is accompanied by an increased gain in enthalpy ΔH but at the cost of a larger entropic penalty TΔS stemming from tighter binding.

Diverse Properties of Guanidiniocarbonyl Pyrrole-Based Molecules: Artificial Analogues of Arginine.

The guanidinium moiety, which is present in active sites of many enzymes, plays an important role in the binding of anionic substrates. In addition, it was also found to be an excellent binding motif for supramolecular chemistry. Inspired by Nature, scientists have developed artificial receptors containing guanidinium scaffolds that bind to a variety of oxoanions through hydrogen bonding and charge pairing interactions. However, the majority of binding studies is restricted to organic solvents. Polyguanidinium based molecules can form efficient complexes in aqueous solvents due to strong electrostatic interactions. However, they only have moderate association constants, which are significantly decreased in the presence of competing anions and salts. Hence, to improve the binding affinity of the guanidinium moiety, our group developed the cationic guanidiniocarbonyl pyrrole (GCP) moiety. This rigid planar analogue binds efficiently to oxoanions, like carboxylates even in aqueous solvents. The lower p Ka value (7-8) of GCP compared to guanidinium derivatives (p Ka 13) favors the formation of strong, hydrogen bonded ion pairs. In addition, carboxylate binding is further enhanced by additional amide hydrogen bond donors located at the five position of the pyrrole core. Moreover, the design has allowed for introducing secondary interactions between receptor side chains and guest molecules, which allows for optimizing binding specificity and selectivity. The spectroscopic data confirmed stabilization of guanidiniocarbonyl pyrrole/oxoanion complexes through a combination of ion pairing and multiple hydrogen bonding interactions. The key role of the ionic interaction in a polar solvent, is demonstrated by a zwitterion derivative of the guanidiniocarbonyl pyrrole, which self-assembles in both dimethyl sulfoxide and pure water with association constants of K > 1010 M-1 and K = 170 M-1, respectively. In this Account, we discuss strategies for making GCP functionalized compounds, in order to boost their ability to bind oxoanions. Then we explore how these building blocks have been incorporated into different synthetic molecules and peptide sequences, highlighting examples that demonstrated the versatility of this binding scaffold. For instance, the high oxoanion binding property of GCP-based compounds was exploited to generate a detectable signal for sensing applications, thus improving selectivity and sensitivity in aqueous solution. Moreover, peptides and molecules containing GCP have shown excellent gene transfections properties. Furthermore, the self-assembly and zwitterionic behavior of zwitterionic GCP analogues was used to develop variety of supramolecular architectures such as stable supramolecular ß-helix structure, linear supramolecular oligomers, one-dimensional rods or two-dimension sheets, fibers, vesicles, soft nanospheres, as well as stimuli responsive supramolecular gels.

Synthesis and Binding of Mannose-Specific Synthetic Carbohydrate Receptors.

Synthetic carbohydrate receptors (SCRs) that selectively recognize cell-surface glycans could be used for detection, drug delivery, or as therapeutics. Here we report the synthesis of seven new C2h symmetric tetrapodal SCRs. The structures of these SCRs possess a conserved biaryl core, and they vary in the four heterocyclic binding groups that are linked to the biaryl core via secondary amines. Supramolecular association between these SCRs and five biologically relevant C1 -O-octyloxy glycans, α/ß-glucoside (α/ß-Glc), α/ß-mannoside (α/ß-Man), and ß-galactoside (ß-Gal), was studied by mass spectrometry, 1 H NMR titrations, and molecular modeling. These studies revealed that selectivity can be achieved in these tetrapodal SCRs by varying the heterocyclic binding group. We found that SCR017 (3-pyrrole), SCR021 (3-pyridine), and SCR022 (2-phenol) bind only to ß-Glc. SCR019 (3-indole) binds only to ß-Man. SCR020 (2-pyridine) binds ß-Man and α-Man with a preference to the latter. SCR018 (2-indole) binds α-Man and ß-Gal with a preference to the former. The glycan guests bound within their SCR hosts in one of three supramolecular geometries: center-parallel, center-perpendicular, and off-center. Many host-guest combinations formed higher stoichiometry complexes, 2:1 glycan⋅SCR or 1:2 glycan⋅SCR, where the former are driven by positive allosteric cooperativity induced by glycan-glycan contacts.

A Preorganized Hydrogen-Bonding Motif for the Molecular Recognition of Carbohydrates.

The choice between adaptive and preorganized architectures, or of the most effective hydrogen bonding groups to be selected, are dilemmas that supramolecular chemists must address in designing synthetic receptors for such a challenging guest as carbohydrates. In this paper, structurally related architectures featuring two alternative hydrogen bonding motifs were compared to ascertain the structural and functional origin of their binding differences and the advantages that can be expected in monosaccharide recognition. A set of structurally related macrocyclic receptors were prepared, and their binding properties were measured by NMR and ITC techniques in chloroform vs a common saccharidic target, namely, the ß-octyl glycoside of D-glucose. Results showed that the diaminocarbazolic motif, recently reported as the constituting unit of highly effective receptors for saccharides in water, is a superior hydrogen bonding motif compared to the previously described diaminopyrrolic motif, which was successfully employed in molecular recognition of carbohydrates in polar organic solvents, due to intrinsic structural and functional factors, rather than to hydrophobic contributions. In addition, the occurrence of a rare example of a thermodynamic template effect exerted by the beta-glucoside has been ascertained, enhancing the synthesis outcome of the otherwise low yielding preparation of the described macrocyclic receptors.

A fluorescent artificial receptor with specific imprinted cavities to selectively detect colistin.

A novel and facile fluorescent artificial receptor on the basis of the molecularly imprinted polymer-coated graphene quantum dots was engineered successfully to detect colistin. The colistin imprinted graphene quantum dots (CMIP-GQDs) was synthesized by vinyl-based radical polymerization between functional monomers and crosslinker at around the template molecule on the surface of graphene quantum dots. The size of bare, CNIP-GQDs, and CMIP-GQDs was about 4.8 ± 0.6 nm, 18.4 ± 1.7 nm, and 19.7 ± 1.3 nm, respectively. The CMIP-GQDs, which showed the strong fluorescence emission at 440 nm with the excitation wavelength fixed at 380 nm, had excellent selectivity and specificity to rapidly recognize and detect colistin. The linear range of fluorescence quenching of this fluorescent artificial receptor for detection colistin was 0.016-2.0 µg mL-1 with a correlation coefficient (R2) of 0.99919, and the detection limit was 7.3 ng mL-1 in human serum samples. The designed receptor was successfully applied to detect colistin in human serum samples and it achieved excellent recoveries shifted from 93.8 to 105%. Graphical abstract.

The challenges of glycan recognition with natural and artificial receptors.

Glycans - simple or complex carbohydrates - play key roles as recognition determinants and modulators of numerous physiological and pathological processes. Thus, many biotechnological, diagnostic and therapeutic opportunities abound for molecular recognition entities that can bind glycans with high selectivity and affinity. This review begins with an overview of the current biologically and synthetically derived glycan-binding scaffolds that include antibodies, lectins, aptamers and boronic acid-based entities. It is followed by a more detailed discussion on various aspects of their generation, structure and recognition properties. It serves as the basis for highlighting recent key developments and technical challenges that must be overcome in order to fully deal with the specific recognition of a highly diverse and complex range of glycan structures.

The Power of Assemblies at Interfaces: Nanosensor Platforms Based on Synthetic Receptor Membranes.

Synthetic sensing materials (artificial receptors) are some of the most attractive components of chemical/biosensors because of their long-term stability and low cost of production. However, the strategy for the practical design of these materials toward specific molecular recognition in water is not established yet. For the construction of artificial material-based chemical/biosensors, the bottom-up assembly of these materials is one of the effective methods. This is because the driving forces of molecular recognition on the receptors could be enhanced by the integration of such kinds of materials at the 'interfaces', such as the boundary portion between the liquid and solid phases. Additionally, the molecular assembly of such self-assembled monolayers (SAMs) can easily be installed in transducer devices. Thus, we believe that nanosensor platforms that consist of synthetic receptor membranes on the transducer surfaces can be applied to powerful tools for high-throughput analyses of the required targets. In this review, we briefly summarize a comprehensive overview that includes the preparation techniques for molecular assemblies, the characterization methods of the interfaces, and a few examples of receptor assembly-based chemical/biosensing platforms on each transduction mechanism.

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.

Selective Recognition of Amino Acids and Peptides by Small Supramolecular Receptors.

To this day, the recognition and high affinity binding of biomolecules in water by synthetic receptors remains challenging, while the necessity for systems for their sensing, transport and modulation persists. This problematic is prevalent for the recognition of peptides, which not only have key roles in many biochemical pathways, as well as having pharmacological and biotechnological applications, but also frequently serve as models for the study of proteins. Taking inspiration in nature and on the interactions that occur between several receptors and peptide sequences, many researchers have developed and applied a variety of different synthetic receptors, as is the case of macrocyclic compounds, molecular imprinted polymers, organometallic cages, among others, to bind amino acids, small peptides and proteins. In this critical review, we present and discuss selected examples of synthetic receptors for amino acids and peptides, with a greater focus on supramolecular receptors, which show great promise for the selective recognition of these biomolecules in physiological conditions. We decided to focus preferentially on small synthetic receptors (leaving out of this review high molecular weight polymeric systems) for which more detailed and accurate molecular level information regarding the main structural and thermodynamic features of the receptor biomolecule assemblies is available.

Highly Sensitive and Selective Fluorescent Probes for Cu(II) Detection Based on Calix[4]arene-Oxacyclophane Architectures.

A new topological design of fluorescent probes for sensing copper ion is disclosed. The calix[4]arene-oxacyclophane (Calix-OCP) receptor, either wired-in-series in arylene-alt-ethynylene conjugated polymers or standing alone as a sole molecular probe, display a remarkable affinity and selectivity for Cu(II). The unique recognition properties of Calix-OCP system toward copper cation stem from its pre-organised cyclic array of O-ligands at the calixarene narrow rim, which is kept in a conformational rigid arrangement by a tethered oxacyclophane sub-unit. The magnitude of the binding constants (Ka = 5.30 - 8.52 × 104 M-1) and the free energy changes for the inclusion complexation (-ΔG = 27.0 - 28.1 kJmol-1), retrieved from fluorimetric titration experiments, revealed a high sensitivity of Calix-OCP architectures for Cu(II) species. Formation of supramolecular inclusion complexes was evidenced from UV-Vis spectroscopy. The new Calix-OCP-conjugated polymers (polymers 4 and 5), synthesized in good yields by Sonogashira-Hagihara methodologies, exhibit high fluorescence quantum yields (ΦF = 0.59 - 0.65). Density functional theory (DFT) calculations were used to support the experimental findings. The fluorescence on-off behaviour of the sensing systems is tentatively explained by a photoinduced electron transfer mechanism.

Recognition and Stabilization of Unsaturated Fatty Acids by a Polyaromatic Receptor.

Selective recognition of natural fatty acids is intrinsically difficult owing to the long, flexible, and poorly interactive hydrocarbon chains. Inspired by biological recognition systems, we herein demonstrate the exclusive binding of a monounsaturated fatty acid by an artificial polyaromatic receptor from a mixture of the unsaturated and corresponding saturated substrates (i.e., oleic and stearic acids) in water. The selectivity stems from multiple CH-π/π-π interactions between the host framework and the guest in its roughly coiled conformation. Moreover, competitive binding experiments elucidate higher binding affinities of the receptor for oligo- and polyunsaturated fatty acids (e.g., α-linolenic acid and EPA). Within the receptor, the biosubstrates are remarkably stabilized against air, light, and heat owing to the polyaromatic shielding effect.

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.