Synthetic receptors for the detection of proteins and the enrichment of methylated and acetylated peptides

Abstract

This thesis deals with the preparation of synthetic supramolecular receptors for biological applications. After a brief introduction, in Chapter 2 the use of tetraphosphonate cavitands (Tiiii) grafted onto biocompatible ferromagnetic nanoparticles (FeNPs) for the complexation of mono-methylated lysines present as residues in histone tails is presented. The grafting process was performed through a co-precipitation method in presence of FeCl2·4H2O and FeCl3·6H2O. Before the complexation experiments, commercial animal histones (e.g., calf thymus) were subjected to proteolytic digestion as routinely performed in proteomics experiments to facilitate separation, detection via mass spectrometry and spectra identification. In addition, by using peptides as input material the enrichment is significantly more efficient, and in this way, residues otherwise hidden by protein folding are exposed to recognition by the molecular receptors. This peptide mixture was incubated with the functionalized nanoparticles to capture mainly the peptide containing the mono-methylated residues. The extracted enriched mixture was analysed via High Resolution ESI mass spectrometry in collaboration with the research group of Prof. S. Sidoli, College of Medicine (New York). In particular, we focused on H3_3_8 fragment, and the results were analyzed in term of enrichment percentage for each modification present in the fragments. The developed tool proceeds to be selective for the recognition of mono-methylated lysine residues and the results were confirmed by performing two control experiments using bare NPs and NPs functionalized with receptors not selective for the target guest. In Chapter 3, we focused on another important biomarker in epigenetics, namely acetylated lysine (Kac). The work was performed at the ICIQ institute, Tarragona (Spain), under the supervision of Prof. P. Ballester. To this aim, water-soluble calix[4]pyrroles were considered. To investigate the effect of hydrophobicity in the recognition event, calix[4]pyrroles characterized by the presence of cavities different in depth were synthetized. The performances of the obtained molecular receptors toward the recognition of Kac were tested by 1H NMR analyses. The best binding properties were displayed by the shallow receptor proving that both hydrophobicity and shape complementarity are fundamental in the binding process. Moreover, solvation effect related to the guest could represent the driving force of the binding. To move from binding studies in solution to surface, calix[4]pyrroles presenting four acidic moieties at the bottom rim were synthetized and anchored onto FeNPs via co-precipitation method. After fully characterization of the grafted system, the same will be used to recognized acetylated lysine residues in histone proteins. In Chapter 4, the design and synthesis of a host-guest templated DNA duplex exploiting the complexation properties of CB[8] are presented. The reported system, developed in collaboration with the research group of Prof. Alessandro Bertucci at University of Parma, is based on the formation of an heteroternary complex between CB[8] and two oligonucleotides derivatized with a fluorophore and a quencher, respectively. Firstly, a model system based on the functionalization of both fluorophore and quencher with a PEG-N3 chain was tested to mimic the conditions of the final system with the oligonucleotides. The anthracene-derivative fluorophore proved to be the best guest for the formation of the heteroternary complex as resulted in a variation of the fluorescence profile upon complexation with CB[8] and the quencher. Then, preliminary click reaction trials were carried out with azide-terminal oligonucleotides and analysed by ESI-MS resulting in the observation of the anthracene fluorophore-DNA product but not that viologen-DNA one. The low reactivity under CuAAC click conditions of methyl-viologen salts and derivatives and the dependence of the yield on the counterions were identified as the probable cause. Finally, in Chapter 5 the design of a fluorescent sensor for protein detection based on aptamer conjugated tetraphosphonate cavitands is reported. With this aim, a tetraphosphonate cavitand functionalized at the upper rim with four alkyl chains bearing a terminal alkyne unit was successfully synthetised. The system is based on the different affinity of the protein-hindered or unhindered receptor cavity toward a fluorescent guest, namely trans-4-[4-(dimethylamino) styryl]-1-methylpyridinium iodide (DMSI). The absence of the target allows the formation of the cavitand-DSMI complex and results in a fluorescence quenching, while in presence of the target the formation of the complex is precluded since the macrocycle cavity is sterically occluded and the fluorescence profile remain unaltered. Also in this case, a model system in which the cavitand was synthetized with four PEG-alkyne units was synthesized and tested in binding with DSMI. The formation of the complex with the unhindered receptor was confirmed through fluorescence analyses as the progressive addition of the dye resulted in the quenching of the dye emission. ITC reported an entropically and enthalpically driven 1:1 binding model with a Ka value in the order of 103 M-1. Several click reaction trials were performed with azide-functionalized DNA strands, but cavitand-DNA product formation was not observed. Future perspective will concern the performance of click reaction in different conditions.