Vessella, Giulia (2021) Development of site-selective procedures towards biomedically relevant semi-synthetic polysaccharides. [Tesi di dottorato]

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Polysaccharides are highly abundant biopolymers, possessing enormous structural diversity and functional versatility. They naturally possess outstanding applications and uses, but their chemical manipulation can improve the given features and open the way to the development of new products and materials. An important group of natural polysaccharides comprises the sulfated ones, which perform key biological functions. Among them, sulfated GAGs are the most abundant in animal species, where they play crucial roles in many physio-pathological processes. Thanks to their bioproperties, GAGs are currently used as therapeutics and biomaterials, however for many applications native GAGs may exhibit limitations linked to production cost, batch standardization, immunogenicity, degradability, or other aspects. The biological functions of sulfated polysaccharides often depend on their selective binding to proteins, and it has been suggested —for GAGs primarily— that the sulfation pattern sequence might be able to encode functional information to this aim. The obtainment of engineered sulfated polysaccharides (ESPs), by chemical or enzymatic modification of natural ones or by chemical synthesis of oligo- or polysaccharides, can lead to well-defined structures with a precise sulfation pattern, thus helping the elucidation of the effects of their structural parameters on the exhibited functions. Besides, ESPs can exhibit new and enhanced biological properties compared to their unmodified counterparts and can also be produced in large quantities at low costs from renewable raw materials or from microbial production, being an easier alternative to isolation of GAGs from animal tissues. In this work, I investigated the possibility to achieve regioselective modifications of naturally occurring polysaccharides, exploiting suitably tailored multi-step sequences, often embedding cyclic protecting groups for vicinal diols, to obtain GAG analogues or mimics. I first focused the attention on the development of semi-synthetic pathways to structurally well-defined chondrotin sulfate (CS) and fucosylated chondroitin sulfate (fCS) —two GAG family members— polysaccharides. Starting from a microbial sourced unsulfated chondroitin and employing a modular approach, comprising regioselective steps together with sulfation and final deprotection, a library of twelve (semi)-homogeneous CSs, possessing almost all the possible sulfation patterns on the polymeric chain, was afforded. A library of ten fCS structures, with a strict regiocontrol of both fucose branch position and sulfation pattern, was obtained by chemical glycosylation of suitably protected chondroitin polysaccharides, having selectively free hydroxyl position(s), with properly prepared L-fucose donors, followed by four additional steps (acetylation, selective cleavage of orthogonal protecting groups, sulfation, global deprotection). A study for regioselective sulfation of other naturally occurring polysaccharides was also carried out. Three polymers were considered: (1) curdlan, a β-1→3-glucan produced, for commercial purposes, from mutant Agrobacterium strains; (2) an alginate polysaccharide, a copolymer of 1→4-linked β-D-ManA (M) and α-L-GulA, commercially available as an M-rich alginic acid; (3) a marine-sourced bacterial exopolysaccharide, resembling GAG structure due to the presence, in its backbone, of aminosugars and uronic acids. Differently sulfated species, some of them showing well-defined and unprecedented sulfation patterns, were obtained both by direct regioselective sulfation/desulfation reactions and by multi-step procedures, also including the use of cycling protecting groups.

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