Selective Interaction of Dopamine with the Self‐Assembled Fibrillar Network of a Molecular Hydrogel Revealed by STD‐NMR

A molecular hydrogel formed by a derivative of L ‐valine with pendant isonicotinoyl moieties interacts selectively with protonated dopamine in the presence of related compounds such as 3‐methylcatechol, and protonated or neutral phenethylamine. A two‐point interaction with the gel fibers is postulated to explain the results. The conclusions are obtained from nuclear magnetic resonance saturation transfer experiments (STD‐NMR), illustrating how this technique is perfectly suited to monitor the interaction of substrates with the fibrillar network of a molecular gel.     

Boronate‐Affinity Glycan‐Oriented Surface Imprinting: A New Strategy to Mimic Lectins for the Recognition of an Intact Glycoprotein and Its Characteristic Fragments

Lectins possess unique binding properties and are of particular value in molecular recognition. However, lectins suffer from several disadvantages, such as being hard to prepare and showing poor storage stability. Boronate‐affinity glycan‐oriented surface imprinting was developed as a new strategy for the preparation of lectin‐like molecularly imprinted polymers (MIPs). The prepared MIPs could specifically recognize an intact glycoprotein and its characteristic fragments, even within a complex sample matrix. Glycan‐imprinted MIPs could thus prove to be powerful tools for important applications such as proteomics, glycomics, and diagnostics.     

Cooperative Effect of a Classical and a Weak Hydrogen Bond for the Metal‐Induced Construction of a Self‐Assembled β‐Turn Mimic

A novel metal‐induced template for the self‐assembly of two independent phosphane ligands by means of unprecedented multiple noncovalent interactions (classical hydrogen bond, weak hydrogen bond, metal coordination, π‐stacking interaction) was developed and investigated. Our results address the importance and capability of weak hydrogen bonds (WHBs) as important attractive interactions in self‐assembling processes based on molecular recognition. Together with a classical hydrogen bond, WHBs may serve as promoters for the specific self‐assembly of complementary monomeric phosphane ligands into supramolecular hybrid structures. The formation of an intermolecular C? H???N hydrogen bond and its persistence in the solid state and in solution was studied by X‐ray crystal analysis, mass spectrometry and NMR spectroscopy analysis. Further evidence was demonstrated by DFT calculations, which gave specific geometric parameters for the proposed conformations and allowed us to estimate the energy involved in the hydrogen bonds that are responsible for the molecular recognition process. The presented template can be regarded as a new type of self‐assembled β‐turn mimic or supramolecular pseudo amino acid for the nucleation of β‐sheet structures when attached to oligopeptides.     

Replacement of Water Molecules in a Phosphate Binding Site by Furanoside‐Appended lin‐Benzoguanine Ligands of tRNA‐Guanine Transglycosylase (TGT)

The enzyme tRNA‐guanine transglycosylase has been identified as a drug target for the foodborne illness shigellosis. A key challenge in structure‐based design for this enzyme is the filling of the polar ribose‐34 pocket. Herein, we describe a novel series of ligands consisting of furanoside‐appended lin‐benzoguanines. They were designed to replace a conserved water cluster and differ by the functional groups at C(2) and C(3) of the furanosyl moiety being either OH or OMe. The unfavorable desolvation of Asp102 and Asp280, which are located close to the ribose‐34 pocket, had a significant impact on binding affinity. While the enzyme has tRNA as its natural substrate, X‐ray co‐crystal structures revealed that the furanosyl moieties of the ligands are not accommodated in the tRNA ribose‐34 site, but at the location of the adjacent phosphate group. A remarkable similarity of the position of the oxygen atoms in these two structures suggests furanosides as a potential phosphate isoster.     

Site‐Specific Investigation of the Steady‐State Kinetics and Dynamics of the Multistep Binding of Bile Acid Molecules to a Lipid Carrier Protein

The investigation of multi‐site ligand–protein binding and multi‐step mechanisms is highly demanding. In this work, advanced NMR methodologies such as 2D ¹H–¹⁵N line‐shape analysis, which allows a reliable investigation of ligand binding occurring on micro‐ to millisecond timescales, have been extended to model a two‐step binding mechanism. The molecular recognition and complex uptake mechanism of two bile salt molecules by lipid carriers is an interesting example that shows that protein dynamics has the potential to modulate the macromolecule–ligand encounter. Kinetic analysis supports a conformational selection model as the initial recognition process in which the dynamics observed in the apo form is essential for ligand uptake, leading to conformations with improved access to the binding cavity. Subsequent multi‐step events could be modelled, for several residues, with a two‐step binding mechanism. The protein in the ligand‐bound state still exhibits a conformational rearrangement that occurs on a very slow timescale, as observed for other proteins of the family. A global mechanism suggesting how bile acids access the macromolecular cavity is thus proposed.     

Detailed Characterization of the Dynamics of Ibuprofen in the Solid State by a Multi‐Technique NMR Approach

The internal rotations and interconformational jumps of ibuprofen in the solid state are fully characterized by the simultaneous analysis of a variety of low‐ and high‐resolution NMR experiments for the measurement of several ¹³C and ¹H spectral and relaxation properties, performed at different temperatures and, in some cases, frequencies. The results are first qualitatively analyzed to identify the motions of the different molecular fragments and to assign them to specific frequency ranges (slow, <10³ Hz; intermediate, 10³–10⁶ Hz; and fast, >10⁶ Hz). In a second step, a simultaneous fit of the experimental data sets most sensitive to each frequency range is performed by means of suitable motional models to obtain, for each motion, values of correlation times and activation energies. The rotations of the three methyl groups around their ternary symmetry axes, which occur in the fast regime, are characterized by slightly different activation energies. Thanks to the simultaneous analysis of ¹H and ¹³C data, the π‐flip of the dimeric structure made by the acidic groups is also identified and seen to occur in the fast regime. On the contrary, the π‐flip of the phenyl ring is found to occur in the slow motional regime, while the rotations of the isobutyl and propionic groups are frozen. The approach used appears to be of general applicability for studying the dynamics of small organic molecules.     

Gel‐to‐Sol and Sol‐to‐Gel Transitions Utilizing the Interaction of α‐Cyclodextrin with Dodecyl Side Chains Attached to a Poly(acrylic acid) Backbone

Summary: By utilizing the interaction of α‐cyclodextrin (α‐CD) with dodecyl side chains in polymers of x mol‐% dodecyl‐modified poly(acrylic acid) (p(AA/C₁₂(x))), systems that undergo gel‐to‐sol and sol‐to‐gel transitions were successfully constructed. Rheological experiments indicated that addition of α‐CD to the hydrogel of p(AA/C₁₂(5)) caused a drastic decrease in the viscosity, while addition of oligo(α‐CD) to the solution of p(AA/C₁₂(2)) led to a remarkable increase in the viscosity.

Photographs for a gel‐to‐sol transition upon addition of α‐CD to 5.0 g · L⁻¹ p(AA/C₁₂(5)).     

Light‐Triggered Capture and Release of DNA and Proteins by Host–Guest Binding and Electrostatic Interaction

The development of an effective and general delivery method that can be applied to a large variety of structurally diverse biomolecules remains a bottleneck in modern drug therapy. Herein, we present a supramolecular system for the dynamic trapping and light‐stimulated release of both DNA and proteins. Self‐assembled ternary complexes act as nanoscale carriers, comprising vesicles of amphiphilic cyclodextrin, the target biomolecules and linker molecules with an azobenzene unit and a charged functionality. The non‐covalent linker binds to the cyclodextrin by host–guest complexation with the azobenzene. Proteins or DNA are then bound to the functionalized vesicles through multivalent electrostatic attraction. The photoresponse of the host–guest complex allows a light‐induced switch from the multivalent state that can bind the biomolecules to the low‐affinity state of the free linker, thereby providing external control over the cargo release. The major advantage of this delivery approach is the wide variety of targets that can be addressed by multivalent electrostatic interaction, which we demonstrate on four types of DNA and six different proteins.     

Interaction of Carrier Protein with Potential Metallic Drug Candidate N-Glycoside ‘GATPT’: Validation by Multi-Spectroscopic and Molecular Docking Approaches

Lysozyme is often used as a model protein to study interaction with drug molecules and to understand biological processes which help in illuminating the therapeutic effectiveness of the drug. In the present work, in vitro interaction studies of 1-{(2-hydroxyethyl)amino}-2-amino-1,2-dideoxy-d-glucose triphenyl tin (IV) (GATPT) complex with lysozyme were carried out by employing various biophysical methods such as absorption, fluorescence, and circular dichroism (CD) spectroscopies. The experimental results revealed efficient binding affinity of GATPT with lysozyme with intrinsic binding (K_b) and binding constant (K) values in the order of 10⁵ M⁻¹. The number of binding sites and thermodynamic parameters ΔG, ΔH, and ΔS at four different temperatures were also calculated and the interaction of GATPT with lysozyme was found to be enthalpy and entropy driven. The CD spectra revealed alterations in the population of α–helical content within the secondary structure of lysozyme in presence of GATPT complex. The morphological analysis of the complex with lysozyme and lysozyme-DNA condensates was carried out by employing confocal and SEM studies. Furthermore, the molecular docking studies confirmed the interaction of GATPT within the larger hydrophobic pocket of the lysozyme via several non-covalent interactions.     

Deciphering the Non‐Equivalence of Serine and Threonine O‐Glycosylation Points: Implications for Molecular Recognition of the Tn Antigen by an anti‐MUC1 Antibody

The structural features of MUC1‐like glycopeptides bearing the Tn antigen (α‐O‐GalNAc‐Ser/Thr) in complex with an anti MUC‐1 antibody are reported at atomic resolution. For the α‐O‐GalNAc‐Ser derivative, the glycosidic linkage adopts a high‐energy conformation, barely populated in the free state. This unusual structure (also observed in an α‐S‐GalNAc‐Cys mimic) is stabilized by hydrogen bonds between the peptidic fragment and the sugar. The selection of a particular peptide structure by the antibody is thus propagated to the carbohydrate through carbohydrate/peptide contacts, which force a change in the orientation of the sugar moiety. This seems to be unfeasible in the α‐O‐GalNAc‐Thr glycopeptide owing to the more limited flexibility of the side chain imposed by the methyl group. Our data demonstrate the non‐equivalence of Ser and Thr O‐glycosylation points in molecular recognition processes. These features provide insight into the occurrence in nature of the APDTRP epitope for anti‐MUC1 antibodies.     

Tuning the Guest‐Binding Ability of a Helically Folded Capsule by In Situ Modification of the Aromatic Oligoamide Backbone

Starting from a previously described aromatic oligoamide helically folded capsule that binds tartaric acid with high affinity and diastereoselectivity, we demonstrate the feasibility of the direct in situ modification of the helix backbone, which results in a conformational change that reduces its affinity for guests by two orders of magnitude. Specifically, ring contraction of the central pyridazine unit into a pyrrole in the full helical sequence was investigated by using electrochemical and chemical processes. The sequence containing the pyrrole was synthesized independently in a convergent manner to ascertain its structure. The conformation of the pyrrolic folded capsule was elucidated in the solid state by X‐ray crystallography and in solution by using ¹H and ¹³C NMR spectroscopy. Solution studies revealed an unanticipated solvent‐dependent equilibrium between the anti–anti and syn–syn conformations of the pyrrole ring with respect to its two adjacent pyridine units. Titrations of the pyrrole‐containing sequence monitored by ¹H NMR spectroscopy confirmed the expected drop in affinity for tartaric acid and malic acid that arises from the conformation change in the backbone that follows the replacement of the pyridazine by a pyrrole. The reduction of the pyridazine to a pyrrole was characterized by cyclic voltammetry both on the entire sequence and on a shorter precursor. The lower cathodic potential of the precursor made its preparative‐scale electroreduction possible. Direct in situ modification of the pyridazine within the entire capsule sequence was achieved chemically by using zinc in acetic acid.     

Molecular Recognition and Visual Detection of G‐Quadruplexes by a Dicarbocyanine Dye

The interactions of a dicarbocyanine dye 3,3′‐diethylthiadicarbocyanine, DiSC₂(5) , with DNA G‐quadruplexes were studied by means of a combination of various spectroscopic techniques. Aggregation of excess dye as a result of its positive charge is promoted by the presence of the polyanionic quadruplex structure. Specific high‐affinity binding to the parallel quadruplex of the MYC promoter sequence involves stacking of DiSC₂(5) on the external G‐tetrads; the 5′‐terminal tetrad is the favored binding site. Significant energy transfer between DNA and the dye in the UV spectral region is observed upon DiSC₂(5) binding. The transfer efficiency strongly depends on the DNA secondary structure as well as on the G‐quadruplex topology. These photophysical features enable the selective detection of DNA quadruplexes through sensitized DiSC₂(5) fluorescence in the visible region.