Supramolecular fluorescent probes for investigating dynamic processes at the molecular level

This thesis aims to explore the use of novel fluorescent probes for monitoring three distinct dynamic processes in biological systems and synthetic materials: cell contraction, protein aggregation, and polymer elongation. A range of fluorogenic compounds, from small molecules to more complex calixarene-based bichromophoric systems, have been designed, synthesized and studied, each tailored to probe these processes through innovative sensing mechanisms. The thesis is organized in four chapters. After a first introductory chapter on the use of linear and nonlinear fluorescence techniques for optical sensing, the second chapter is devoted to the development of fluorescent probes designed to image the contraction mechanism in cardiac cells using nonlinear optical methods. Two distinct approaches are explored, both based on the incorporation of the probes within the cell membrane. The first strategy uses small fluorescent quadrupolar dyes capable of responding to the electrical changes occurring during cell contraction through electrochromism and second harmonic generation. The second approach involves calixarene-based bichromophoric systems designed to undergo conformational changes in response to myocardial contraction, triggering an optical response. This last approach is based on the hypothesis that the lateral mechanical forces, generated within the cell membrane, could alter the conformation of the scaffold, thereby modulating the intramolecular interactions between the dyes, and inducing a change in the spectroscopic properties of the probe. In addition to the synthesis and the spectroscopic characterization of the dyes, the chapter reports the preliminary results obtained from experiments where cardiomyocytes were incubated with the dyes and analysed using multiphoton microscopy, in collaboration with Prof. Michele Miragoli and Prof. Francesca Terenziani at the Dipartimento di Medicina e Chirurgia and Dipartimento di Scienze Chimiche, della Vita e della Sostenibilità Ambientale, respectively, of the University of Parma. The third chapter focuses on the design and synthesis of calixarene-based mechanophores for the development of mechanoresponsive luminescent polymeric materials. This research was partially conducted at the Institut Català d’Investigació Química (ICIQ) in Tarragona (ES), under the supervision of Dr. José Augusto Berrocal. The chapter details the synthesis of calix[4]arenes derivatives functionalized with two pyrene units and investigates the intramolecular dimerization between the two chromophores, a process facilitated by the flexibility of the macrocyclic structure. Subsequently, the calixarene-based systems were covalently incorporated into polymeric matrices to confer mechanoresponsive properties. It was demonstrated that the application of mechanical tensile forces to the polymer films could physically dissociate the pyrene dimer, resulting in a measurable change in the fluorescence properties of the material. The fourth chapter aims to study the dynamics of protein aggregation using two-dimensional electronic spectroscopy. This technique exploits nonlinear spectroscopy to achieve an ultrafast temporal resolution, allowing the investigation of rapid molecular events. The method relies on labelling the aggregating proteins with fluorescent probes capable of undergoing energy transfer processes. Indeed, by monitoring variations in energy transfer efficiency, it becomes possible to measure distances between the proteins and track the aggregation process in real time. The chapter describes the synthesis of a BODIPY-based donor-acceptor pair specifically designed to label the cysteine residues in β-lactoglobulin proteins. In collaboration with the research group of Prof. Andrea Lapini at the European Laboratory for Non-Linear Spectroscopy (LENS) in Florence, early studies on protein aggregation were performed using pump-probe experiments and 2D electronic spectroscopy.