Dottorato in MOLECULAR DESIGN AND CHARACTERIZATION FOR THE
PROMOTION OF HEALTH AND WELL-BEING: FROM DRUG TO FOOD

Titolo della tesi: The multifaceted role of chemistry to success RNA therapeutics: from photoremovable protecting groups to modified nucleosides

Therapeutic oligonucleotides (TOs) hold substantial potential for precise cancer treatment by modulating cellular pathways; however, effective delivery to cancer cells and their inner stability in physiological environments remain significant challenges. Chemical modifications play a pivotal role in overcoming biological barriers and enhancing the pharmacokinetic and pharmacodynamic properties of TOs. This Ph.D. thesis focuses on two main topics and is divided into two sections (Part A and Part B). In part A, a modular quinoline-based photolabile protective group was designed and synthesized to allow the caging of therapeutic oligonucleotides, increasing their stability while realizing spatial-temporal control over their release. We optimized an efficient synthetic strategy for a quinoline-based photocaging compound consisting of a two-photon excitable system (2PE). Two synthetic strategies were developed: a linear approach and a convergent one. The linear strategy was initially designed to characterize the final compound and conduct bioconjugation trials, while the convergent strategy became necessary to find a more efficient method to produce the final compound in sufficient quantities, which was crucial for the caging purposes.Based on a quinoline moiety, this system can cage hypermodified therapeutic RNAs, such as small interfering RNAs (siRNAs) and antisense oligonucleotides (ASOs), enabling controlled release upon activation. Furthermore, the potential for tissue selectivity within our modular system will be explored through the covalent linkage of the quinoline scaffold to a thiol-selective targeting unit, such as an antibody. Nucleosides and their corresponding phosphate forms (nucleotides) are the building blocks of nucleic acids and are implicated in all biological functions of cellular life, including metabolic regulation, catalysis, and energy transfer. Given their broad and vital roles, the chemistry of these molecular families is a key research area in bioorganic and medicinal chemistry, particularly in nucleic acid chemistry, where there is an increasing demand for more efficient and stable nucleoside analogues. In part B, to enhance the stability and efficacy of our caged oligonucleotides, we developed a novel and more efficient synthetic approach to produce 4'-fluorouridine (F-uridine), a nucleoside analogue that retains the 2'-hydroxyl (OH) group. The proposed approach scales up the previously reported synthesis leveraging the strategy to a multi-gram scale, while reducing the number of steps involved, enhancing its potential applications and increasing the versatility of its production. This analogue preserves the stereoelectronic properties of 2'-fluorinated compounds while maintaining the functional benefits of the 2'-OH group. Additionally, since the C4' position is part of the sugar-phosphate backbone and typically interacts with nucleic acid-binding proteins, 4'-fluoro-RNA emerges as a promising candidate for the development of oligonucleotide therapeutics based on nucleoside analogues. Ultimately, this innovative approach synergizes optochemical biology strategies with chemical modifications to advance targeted oligonucleotide-based therapy, offering promising improvements in stability for the clinical translation of RNA-based therapeutics.