Unpaired cysteine insertions favor transmembrane dimerization and induce ligand-independent constitutive cytokine receptor signaling.

Naturally occurring gain-of-function (GOF) mutants have been identified in patients for a variety of cytokine receptors. Although this constitutive activation of cytokine receptors is strongly associated with malignant disorders, ligand-independent receptor activation is also a useful tool in synthetic biology e.g. to improve adoptive cellular therapies with genetically modified T-cells. Balanced Interleukin (IL-)7 signaling via a heterodimer of IL-7 receptor (IL-7Rα) and the common γ-chain (γc) controls T- and B-cell development and expansion, whereas uncontrolled IL-7 signaling can drive acute lymphoid leukemia (ALL) development. The ALL-driver mutation PPCL in the transmembrane domain of IL-7Rα is a mutational insertion of the four amino acids proline-proline-cysteine-leucine and leads to ligand-independent receptor dimerization and constitutive activation. We showed here in the cytokine-dependent pre-B-cell line Ba/F3 that the PPCL-insertion in a synthetic version of the IL-7Rα induced γc-independent STAT5 and ERK phosphorylation and also proliferation of the cells and that booster-stimulation by arteficial ligands additionally generated non-canonical STAT3 phosphorylation via the synthetic IL-7Rα-PPCL-receptors. Transfer of the IL-7Rα transmembrane domain with the PPCL insertion into natural and synthetic cytokine receptor chains of the IL-6, IL-12 and Interferon families also resulted in constitutive receptor signaling. In conclusion, our data suggested that the insertion of the mutated PPCL IL-7Rα transmembrane domain is an universal approach to generate ligand-independent, constitutively active cytokine receptors.

A Macrocyclic Tweezers-Shaped Receptor for the Biomimetic Recognition of the Gal(α1-3)Gal Disaccharide of the α-Gal Antigen.

The Gal(α1-3)Gal is the terminal disaccharide unit of the α-Gal epitope [Gal(α1-3)Gal(ß1-4)GlcNAc], an exogenous antigenic determinant with several clinical implications, found in all non-primate mammals and in several dangerous pathogens, including certain protozoa and mycobacteria. Its absence in humans makes the α-Gal epitope an interesting target for several infectious diseases. Here we present the development of a macrocyclic tweezers-shaped receptor, resulting from the combination of the structural features of two predecessors belonging to the family of diaminocarbazole receptors, which exhibits binding properties in the low millimolar range toward the Gal(α1-3)Gal disaccharide of the α-Gal antigen.

Synthetic Genes For Dynamic Regulation Of DNA-Based Receptors.

We present a strategy to control dynamically the loading and release of molecular ligands from synthetic nucleic acid receptors using in vitro transcription. We demonstrate this by engineering three model synthetic DNA-based receptors: a triplex-forming DNA complex, an ATP-binding aptamer, and a hairpin strand, whose ability to bind their specific ligands can be cotranscriptionally regulated (activated or inhibited) through specific RNA molecules produced by rationally designed synthetic genes. The kinetics of our DNA sensors and their genetically generated inputs can be captured using differential equation models, corroborating the predictability of the approach used. This approach shows that highly programmable nucleic acid receptors can be controlled with molecular instructions provided by dynamic transcriptional systems, illustrating their promise in the context of coupling DNA nanotechnology with biological signaling.

Vapor-Phase Synthesis of Molecularly Imprinted Polymers on Nanostructured Materials at Room-Temperature.

Molecularly imprinted polymers (MIPs) have recently emerged as robust and versatile artificial receptors. MIP synthesis is carried out in liquid phase and optimized on planar surfaces. Application of MIPs to nanostructured materials is challenging due to diffusion-limited transport of monomers within the nanomaterial recesses, especially when the aspect ratio is >10. Here, the room temperature vapor-phase synthesis of MIPs in nanostructured materials is reported. The vapor phase synthesis leverages a >1000-fold increase in the diffusion coefficient of monomers in vapor phase, compared to liquid phase, to relax diffusion-limited transport and enable the controlled synthesis of MIPs also in nanostructures with high aspect ratio. As proof-of-concept application, pyrrole is used as the functional monomer thanks to its large exploitation in MIP preparation; nanostructured porous silicon oxide (PSiO2 ) is chosen to assess the vapor-phase deposition of PPy-based MIP in nanostructures with aspect ratio >100; human hemoglobin (HHb) is selected as the target molecule for the preparation of a MIP-based PSiO2 optical sensor. High sensitivity and selectivity, low detection limit, high stability and reusability are achieved in label-free optical detection of HHb, also in human plasma and artificial serum. The proposed vapor-phase synthesis of MIPs is immediately transferable to other nanomaterials, transducers, and proteins.

Transitioning from Supramolecular Chemistry to Molecularly Imprinted Polymers in Chemical Sensing.

This perspective article focuses on the overwhelming significance of molecular recognition in biological processes and its emulation in synthetic molecules and polymers for chemical sensing. The historical journey, from early investigations into enzyme catalysis and antibody-antigen interactions to Nobel Prize-winning breakthroughs in supramolecular chemistry, emphasizes the development of tailored molecular recognition materials. The discovery of supramolecular chemistry and molecular imprinting, as a versatile method for mimicking biological recognition, is discussed. The ability of supramolecular structures to develop selective host-guest interactions and the flexible design of molecularly imprinted polymers (MIPs) are highlighted, discussing their applications in chemical sensing. MIPs, mimicking the selectivity of natural receptors, offer advantages like rapid synthesis and cost-effectiveness. Finally, addressing major challenges in the field, this article summarizes the advancement of molecular recognition-based systems for chemical sensing and their transformative potential.

Macrocycle with Equatorial Coordination Sites Provides New Opportunity for Structure-Diverse Metallacages.

Reported here is the synthesis of a macrocycle with equatorial coordination sites for the construction of self-assembled metallacages. The macrocycle is prepared via a post-modification on the equator of biphen[n]arene. Utilizing this macrocycle as a ligand, three prismatic cages and one octahedral cage were synthesized by regulating the geometric structures and coordination number of metal acceptors. The multi-cavity configuration of prismatic cage was revealed by single-crystal structure. We prove that a macrocycle with equatorial coordination sites can be an excellent building block for synthesizing structure-diverse metallacages. Our results provide a typical example and a general method for the design and synthesis of metallacages.

Precise Recognition in Water by an Endo-Functionalized Cavity: Tuning the Complementarity of Binding Sites.

Precise binding towards structurally similar substrates is a common feature of biomolecular recognition. However, achieving such selectivity-especially in distinguishing subtle differences in substrates-with synthetic hosts can be quite challenging. Herein, we report a novel design strategy involving the combination of different rigid skeletons to adjust the distance between recognition sites within the cavity, which allows for the highly selective recognition of hydrogen-bonding complementary substrates, such as 4-chromanone. X-ray single-crystal structures and density functional theory calculations confirmed that the distance of endo-functionalized groups within the rigid cavity is crucial for achieving high binding selectivity through hydrogen bonding. The thermodynamic data and molecular dynamics simulations revealed a significant influence of the hydrophobic cavity on the binding affinity. The new receptor possesses both high selectivity and high affinity, which provide valuable insights for the design of customized receptors.

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.

Fluorescence Response and Self-Assembly of a Tweezer-Type Synthetic Receptor Triggered by Complexation with Heme and Its Catabolites.

There is increasing interest in the development and applications of synthetic receptors that recognize target biomolecules in aqueous media. We have developed a new tweezer-type synthetic receptor that gives a significant fluorescence response upon complexation with heme in aqueous solution at pH 7.4. The synthetic receptor consists of a tweezer-type heme recognition site and sulfo-Cy5 as a hydrophilic fluorophore. The receptor-heme complex exhibits a supramolecular amphiphilic character that facilitates the formation of self-assembled aggregates, and both the tweezer moiety and the sulfo-Cy5 moiety are important for this property. The synthetic receptor also exhibits significant fluorescence responses to biliverdin and bilirubin, but shows very weak fluorescence responses to flavin mononucleotide, folic acid, and nicotinamide adenine dinucleotide, which contain smaller π-scaffolds.

Recognition of Chiral Carboxylates by Synthetic Receptors.

Recognition of anionic species plays a fundamental role in many essential chemical, biological, and environmental processes. Numerous monographs and review papers on molecular recognition of anions by synthetic receptors reflect the continuing and growing interest in this area of supramolecular chemistry. However, despite the enormous progress made over the last 20 years in the design of these molecules, the design of receptors for chiral anions is much less developed. Chiral recognition is one of the most subtle types of selectivity, and it requires very precise spatial organization of the receptor framework. At the same time, this phenomenon commonly occurs in many processes present in nature, often being their fundamental step. For these reasons, research directed toward understanding the chiral anion recognition phenomenon may lead to the identification of structural patterns that enable increasingly efficient receptor design. In this review, we present the recent progress made in the area of synthetic receptors for biologically relevant chiral carboxylates.

Molecular Receptors for Recognition and Sensing of Nucleotides.

Nucleotides are constituents of nucleic acids and they have a variety of functions in cellular metabolism. Synthetic receptors and sensors are required to reveal the role of nucleotides in living organisms and mechanisms of signal transduction events. In recent years, a large number of nucleotide-selective synthetic receptors have been devised, which utilize different molecular designs and sensing mechanisms. This Minireview presents recent progress in the design of synthetic molecular receptors for selective recognition of nucleotides in aqueous solution. The binding properties of receptors and the origins of their selectivity for a particular nucleotide are discussed.

Achieving High Affinity and Selectivity for Asymmetric Dimethylarginine by Putting a Lid on a Box.

The methylation states of Lys and Arg represent a particularly challenging set of targets to distinguish selectively in water using synthetic receptors. To date, trimethyllysine (Kme3) is the only post translational modification (PTM) of the eight possible methylation states of Lys and Arg that can be recognized selectively. Here, we report the first synthetic receptor capable of selectively recognizing asymmetric dimethylarginine (Rme2a). This was achieved by using a biased dynamic combinatorial chemistry (DCC) library to generate a receptor mimicking the 5-sided box-like shape of Rme2 reader proteins, a feature that has been hypothesized to impart selectivity. Additionally, we synthesized a thioether-linked analogue of the resulting receptor to provide a novel scaffold with maintained selectivity but greater stability. This work introduces strategies that can be applied towards achieving selectivity based on subtle differences in hydrophilic guests in aqueous solutions.

Recognition and sequestration of ω-fatty acids by a cavitand receptor.

One of the largest driving forces for molecular association in aqueous solution is the hydrophobic effect, and many synthetic receptors with hydrophobic interiors have been devised for molecular recognition studies in water. Attempts to create the longer, narrower cavities appropriate for long-chain fatty acids have been thwarted by solvophobic collapse of the synthetic receptors, giving structures that have no internal spaces. The collapse generally involves the stacking of aromatic panels onto themselves. We describe here the synthesis and application of a deep cavitand receptor featuring "prestacked" aromatic panels at the upper rim of the binding pocket. The cavitand remains open and readily sequesters biologically relevant long-chain molecules-unsaturated ω-3, -6, and -9 fatty acids and derivatives such as anandamide-from aqueous media. The cavitand exists in isomeric forms with different stacking geometries and n-alkanes were used to characterize the binding modes and conformational properties. Long alkyl chains are accommodated in inverted J-shaped conformations. An analogous cavitand with electron-rich aromatic walls was prepared and comparative binding experiments indicated the role of intramolecular stacking in the binding properties of these deep container molecules.

Molecular Imprinting Technology in Quartz Crystal Microbalance (QCM) Sensors.

Molecularly imprinted polymers (MIPs) as artificial antibodies have received considerable scientific attention in the past years in the field of (bio)sensors since they have unique features that distinguish them from natural antibodies such as robustness, multiple binding sites, low cost, facile preparation and high stability under extreme operation conditions (higher pH and temperature values, etc.). On the other hand, the Quartz Crystal Microbalance (QCM) is an analytical tool based on the measurement of small mass changes on the sensor surface. QCM sensors are practical and convenient monitoring tools because of their specificity, sensitivity, high accuracy, stability and reproducibility. QCM devices are highly suitable for converting the recognition process achieved using MIP-based memories into a sensor signal. Therefore, the combination of a QCM and MIPs as synthetic receptors enhances the sensitivity through MIP process-based multiplexed binding sites using size, 3D-shape and chemical function having molecular memories of the prepared sensor system toward the target compound to be detected. This review aims to highlight and summarize the recent progress and studies in the field of (bio)sensor systems based on QCMs combined with molecular imprinting technology.

Molecular Rearrangement of an Aza-Scorpiand Macrocycle Induced by pH: A Computational Study.

Rearrangements and their control are a hot topic in supramolecular chemistry due to the possibilities that these phenomena open in the design of synthetic receptors and molecular machines. Macrocycle aza-scorpiands constitute an interesting system that can reorganize their spatial structure depending on pH variations or the presence of metal cations. In this study, the relative stabilities of these conformations were predicted computationally by semi-empirical and density functional theory approximations, and the reorganization from closed to open conformations was simulated by using the Monte Carlo multiple minimum method.

Molecularly Imprinted Polymer Coated Quantum Dots for Multiplexed Cell Targeting and Imaging.

Advanced tools for cell imaging are of great interest for the detection, localization, and quantification of molecular biomarkers of cancer or infection. We describe a novel photopolymerization method to coat quantum dots (QDs) with polymer shells, in particular, molecularly imprinted polymers (MIPs), by using the visible light emitted from QDs excited by UV light. Fluorescent core-shell particles specifically recognizing glucuronic acid (GlcA) or N-acetylneuraminic acid (NANA) were prepared. Simultaneous multiplexed labeling of human keratinocytes with green QDs conjugated with MIP-GlcA and red QDs conjugated with MIP-NANA was demonstrated by fluorescence imaging. The specificity of binding was verified with a non-imprinted control polymer and by enzymatic cleavage of the terminal GlcA and NANA moieties. The coating strategy is potentially a generic method for the functionalization of QDs to address a much wider range of biocompatibility and biorecognition issues.

Sensing Protein Surfaces with Targeted Fluorescent Receptors.

A methodology for creating fluorescent molecular sensors that respond to changes that occur on the surfaces of specific proteins is presented. This approach, which relies on binding cooperatively between a specific His-tag binder and a nonspecific protein-surface receptor, enabled the development of a sensor that can track changes on the surface of a His-tag-labeled calmodulin (His-CaM) upon interacting with metal ions, small molecules, and protein binding partners. The way this approach was used to detect dephosphorylation of an unlabeled calmodulin-dependent protein kinase II (CaMKII), and the binding of Bax BH3 to His-tagged B-cell lymphoma 2 (Bcl-2) protein is also presented.

Antiviral Activity of Synthetic Aminopyrrolic Carbohydrate Binding Agents: Targeting the Glycans of Viral gp120 to Inhibit HIV Entry.

The binding abilities of a set of structurally related aminopyrrolic synthetic receptors for mannosides, endowed with antimycotic activity against yeast and yeast-like pathogens bearing mannoproteins on their cell surface, have been investigated towards the highly mannosylated gp120 and gp41 glycoproteins of the HIV envelope. A pronounced binding interaction with both glycoproteins was observed by SPR for most of the investigated compounds. Comparison of their binding properties towards the glycoproteins with their binding affinities toward mannosides revealed a direct correlation, supporting their role as carbohydrate binding agents (CBAs). Cytostatic activity studies revealed antiproliferative activity dependent on the nature and the structure of compounds. Antiviral activity studies against a broad panel of DNA and RNA viruses showed inhibitory effect against HIV infection of the T-lymphocyte CEM cell line for two compounds, suggesting antiviral activity similar to other CBAs, such as the nonpeptidic pradimicin antibiotics.

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.

Blood Group Typing: From Classical Strategies to the Application of Synthetic Antibodies Generated by Molecular Imprinting.

Blood transfusion requires a mandatory cross-match test to examine the compatibility between donor and recipient blood groups. Generally, in all cross-match tests, a specific chemical reaction of antibodies with erythrocyte antigens is carried out to monitor agglutination. Since the visual inspection is no longer useful for obtaining precise quantitative information, therefore there is a wide variety of different technologies reported in the literature to recognize the agglutination reactions. Despite the classical methods, modern biosensors and molecular blood typing strategies have also been considered for straightforward, accurate and precise analysis. The interfacial part of a typical sensor device could range from natural antibodies to synthetic receptor materials, as designed by molecular imprinting and which is suitably integrated with the transducer surface. Herein, we present a comprehensive overview of some selected strategies extending from traditional practices to modern procedures in blood group typing, thus to highlight the most promising approach among emerging technologies.