• drug-polymer interaction
• diffusion
• chiral recognition
• relaxation
• resorcin[4]arenes

Molecular recognition phenomena involving the interaction between small to medium sized substrates and polymeric or macrocyclic receptors were investigated by exploiting the wide potentialities of Nuclear Magnetic Resonance spectroscopy.
In the field of protein receptors, the affinity of two different derivatives, named GANT61 and GlaB (Figure I), proposed as inhibitors for Gli1 protein, which is the downstream effector in the Hedgehog pathway often leading to tumorigenesis, was compared. Since the conformation of Gli1 protein depends on the presence of zinc inside its five zinc-finger domains, protein affinity in the presence and in the absence of the metal was also evaluated. For this study proton selective relaxation rates measurements were exploited, which are NMR parameters highly responsive to the slowing down of molecular motion due to drug-protein interaction processes. The molecular portions of GANT61 and GlaB mainly involved in the interaction with the protein were ascertained: the interaction sites of GANT61 are strongly dependent from the presence of the metal, being shifted from the dihydropyridinyl moiety in the absence of the metal to the phenyl group when the metal is present. Relatively to GlaB, the analysis of relaxation rates allowed to establish that a non-selective interaction occurs when the protein is expressed without zinc, whereas for Gli1 protein expressed with the metal the interaction becomes selective and mainly addressed to its B ring. Being known that serine residues are relevant for the interaction, some serine residues of the protein were substituted with alanine, and an NMR study was performed in mixtures containing GlaB and mutated protein. Data demonstrated that only one serine residue is relevant for the selectivity of the interaction with GlaB.
In the field of natural polymers addressed to biotechnological applications, derivatized chitosans receptors were evaluated as controlled release systems for hydrophobic and hydrophilic drugs. Quaternary ammonium chitosan conjugates, thiolated and non-thiolated, were synthesized in order to overcome the insolubility issues of natural product. Synthesized polymers were then employed as starting materials for the synthesis of nanoparticles, whose affinity towards hydrophobic and hydrophilic drugs (Dexamethasone 21-phosphate and [5-Methionine]Enkephalin, respectively) was evaluated and compared to corresponding polymers, in order to underline the role of supramolecular assembly in affinity processes. Also in this case, the spectroscopic investigation required an extensive use of mono-selective relaxation parameters, which allowed to establish that derivatized chitosan polymers have great propensity to interact with hydrophobic drugs but not with hydrophilic ones. The nanoparticulate polymer aggregation favors the interaction also with [5-Methionine]Enkephalin. Interestingly, a preference of thiolated nanoparticles for the hydrophilic drug and of non-thiolated nanoparticles for the hydrophobic one was pointed out. Also the mucoadhesivity of the two different polymers and nanoparticles was evaluated by performing NMR experiments on mixtures containing bovine mucin in addition to polymeric materials. In each case, it was pointed out that mucoadhesive properties of both polymers and nanoparticles are strongly dependent from the nature of the drug (hydrophobic vs hydrophilic).
In the field of polymeric materials employed as effectors of the aggregation of small molecules for photovoltaic applications, the affinity of perovskite precursors for five different polymers (Figure II) was evaluated. Diffusion coefficients were fundamental for evaluating the self-aggregation of perovskite precursors, which has a strong impact for material performances, whereas relaxation parameters, and particularly spin-spin relaxation times, proved to be very sensitive to the presence of the polymers, thus allowing to individuate the polymers able to provoke the higher variations in the relaxation parameters of perovskite precursors. NMR results were in good agreement with film performances as evaluated by means of other techniques.
Another important class of synthetic polymers is that of nanosponges, a novel class of colloidal structures which are based on hyper-crosslinked polymers with a three-dimensional scaffold possessing a long length polymer backbone. There are several kinds of nanosponges, some of which have been employed as sustained release carriers for drug delivery in pharmaceutical field, thanks to the fact that they are suitable for solving issues related to stability, solubility and delayed release. Here, the chirality of cyclodextrin-based nanosponges, which are usually employed for decontamination of water, was exploited for enantioselective absorption experiments on selected classes of chiral substrates (mandelic acid, phenylalanine, methyl 2-chloropropionate and tryptophan). This preliminary study indicated that the enantioselective absorption is generally low, but it can be improved by optimization of experimental conditions.
Regarding chiral recognition phenomena, bridged-basket resorcin[4]arenes were exploited as chiral solvating agents for NMR spectroscopy, whose enantiodiscriminating efficiency was evaluated and compared by choosing mandelic acid as probe of the interaction. The importance of the cooperation between lateral chains and resorcin[4]arenic core was ascertained by comparison with the corresponding diamides, whereas the importance of the sizes of lateral wings was evaluated by comparison with a larger resorcin[4]arene. The spectroscopic study was performed by employing diffusion and dipole-dipole interaction measurements, which clearly indicated that the presence of amide moieties is the pre-requisite for thermodynamic stabilization of the diastereomeric solvates, even if only the cooperation of polar groups with the resorcin[4]arene core makes possible an efficient enantiodiscrimination. Diffusion measurements indicated that the enantiodiscrimination is due to stereochemical differentiation rather that thermodynamic one, whereas ROE measurements allowed to obtain an interaction model. Finally, the importance of the cooperation between the lateral baskets and the resorcin[4]arene core in the enantiodiscrimination was pointed out by comparison with the larger resorcin[4]arene. In this last case, the greater flexibility of lateral baskets is responsible of the minor stereochemical differentiation between diastereomeric pairs.
Preliminarly to the application of highly apolar resorcin[4]arene receptors, an accurate NMR protocol was developed for the quantitative analysis and the full characterization of macrocyclic compounds obtained by performing a metathesis reaction on resorcin[4]arene substrates. This protocol allowed to individuate the experimental parameters which influence the nature of the products.