Dottorato in SCIENZE FARMACEUTICHE

Titolo della tesi: Synthesis of Resorc[4]arene modifiers and Azobenzene photoswitches for biosensing and drug delivery application

PART A: In recent years, several efforts have been made to develop selective, sensitive, fast response, and miniaturized immunosensors with improved performance. However, one of the main technical challenges in developing immunosensors is overcoming the complexity of binding antibodies to the sensor surface. Most immobilizing approaches lead to a random orientation of antibodies, resulting in lower binding site density and immunoaffinity. In this context, supramolecular chemistry has recently emerged as a suitable surface modification strategy for obtaining pre-organized and ordered surfaces, in which the bioreceptors are guided to assume an oriented arrangement (end-on). Among the large pool of macrocycles available, resorc[4]arenes turned out to be a good option for the construction of highly sensitive immunosensors. In this project, the resorc[4]arene architecture has been widely exploited for the functionalization of several electrode surfaces. In particular, the first resorc[4]arene presented, RW, was functionalized at the lower rim with long thioether alkyl chains to covalently install the artificial linkers on gold-coated magnetic nanoparticles while the upper rim was decorated with eight hydrophilic carboxylate groups, to tailor its recognition properties toward the Fc portion of Abs and to promote solubility in biocompatible and nontoxic aqueous solvent, avoiding the SPE degradation. The second resorc[4]arene presented, RN2 +, was rationally designed to extend the application of the resorc[4]arene-based sensor strategy to a wide range of electrode surfaces, including carbon materials, metals, and polymers, which can be functionalized through electrografting technique. Notably, the upper rim is decorated with eight hydrophilic carboxylate groups, similarly to RW, while the lower rim is characterized by aryl diazonium salts, whose great versatility and reactivity will allow the functionalization of several different electrode surfaces. PART B: Antibiotic resistance is recognized as a major problem worldwide; hence, developing new strategies to counteract antimicrobial resistance has been the focus of scientific research over the last years. One of these strategies consists in limiting the release of active antibiotics into the environment: in this context, photopharmacology represents a promising approach, allowing to achieve a control over drug release and activation and offering new insights for targeted and personalized treatments. Accordingly, light can be used as a valuable tool to modulate the properties, and consequently the activity, of drugs at will. Recently, Just-Baringo et al. have demonstrated how the introduction of a tetra-ortho-chlorinated azobenzene photoswitch can allow control over the activity of antimicrobial peptides using visible light. Nevertheless, this previous work was limited by the reactivity of tetra-ortho-chlorinated azobenzenes in the presence of piperidine, which is the reagent of choice for Fmoc deprotection during solid-phase peptide synthesis (SPPS). Methoxylated analogues represent a much more appealing alternative for SPPS as they would prevent substitutions of the ortho substituents in the presence of nucleophiles while maintaining the possibility to be isomerized in the red-shifted region of visible light. However, since methods reported in the literature had extremely low yields and limitations in functionalization, to date the synthesis of tetra-ortho-methoxylated azobenzenes is highly limited. On this regard, a two-step approach to synthetize a broad range of tetra-ortho-methoxyazobenzenes with good overall yields has been successfully developed. A test to demonstrate the robustness of this scaffold for Solid Phase Synthesis applications when compared to its chlorinated analogues has been carried out, showcasing their potential for use in the synthesis of red-light-operated peptides.