Laezza, Antonio (2017) Regioselective modifications of natural polysaccharides. [Tesi di dottorato]


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Item Type: Tesi di dottorato
Lingua: English
Title: Regioselective modifications of natural polysaccharides
Date: 10 April 2017
Number of Pages: 246
Institution: Università degli Studi di Napoli Federico II
Department: Scienze Chimiche
Dottorato: Scienze chimiche
Ciclo di dottorato: 29
Coordinatore del Corso di dottorato:
Bedini, EmilianoUNSPECIFIED
Date: 10 April 2017
Number of Pages: 246
Uncontrolled Keywords: Regioselectivity, polysaccharides, synthesis
Settori scientifico-disciplinari del MIUR: Area 03 - Scienze chimiche > CHIM/06 - Chimica organica
Date Deposited: 03 May 2017 17:23
Last Modified: 14 Mar 2018 10:05
DOI: 10.6093/UNINA/FEDOA/11761


Polysaccharides are polymeric carbohydrates, usually formed of repeating units (either mono-, or higher oligosaccharides) joined together by glycosidic bonds. Some of these macromolecules are characterized by high natural availability (starch, cellulose, glycogen and chitin among others) and they have also a great biological importance, since they can be a source of energy for animal species. Moreover, they are structural elements of cellular walls and identification sites of cellular surfaces. An important class of polysaccharides is that of glycosaminoglycans animal sourced biomacromolecules that play a pivotal role in several biological processes. CS is included into the family of sulfated GAGs and is involved in the treatment of osteoarthritis and osteoarthrosis. From the structural point of view it is composed of a disaccharide repeating unit containing GlcA and GalNAc linked together through β-(1-->3) and β-(1-->4) glycosidic bonds, and displaying different sulfation patterns after in vivo polymerization. Indeed, depending on the position of sulfate groups, different disaccharide subunits could be described. Nonetheless, the low abundance of raw material, the labourious downstream purification and the growing application of this polysaccharide as a drug, led to development of a non-animal derived CS with a well-defined sulfation pattern, starting from Escherichia coli O5:K4:H4 sourced unsulfated chondroitin, through the optimization of a suitable sequenece of regioselective steps for its structural modification. This was based on the selective protection of O-4,6-GalNAc diol with a cyclic group (beznylidene), followed by acylation of O-2,3-GlcA diol on the polysaccharide backbone. By conducting benzylidenation and acetylation reactions one- or two pots, CSs with different sulfation patterns were obtained. In particular, sulfate groups randomly distributed either at position O-4 or at position O-6 of GalNAc units (CS-A,C) were obtained through the two-pots strategy, whereas the presence of additional sulfate groups was found at position O-3 of GlcA units when the protection reactions were conducted in one-pot fashion. This difference was ascribed to the formation of interglycosidic acetals during the insertion of benzylidene ring on O-4,6-GalNAc diol. These unusual acetals were rather acid-labile and could be not conserved after reaction work-up, thus, at the end of the semi-synthetic strategy, a chondroitin polysaccharide bearing sulfate groups exclusively on GalNAc units was afforded. Differently, stabilization in alkaline environment of the labile interglycosidic acetals by the two-pots strategy and their following oxidative cleavage allowed the semi-synthesis of CS species possessing sulfate groups not only on GalNAc units but also at position O-3 of some GlcA ones. It is worth noting that the detailed understanding of the factors influencing finely tailored chemical modifications on microbial sourced chondroitin is rather valuable because it allows the preparation of biologically relevant CSs from non-animal sources and with different, but highly controlled sulfation patterns. Indeed, CS-A,C is employed for several biomedical applications, as well as CSs possessing GlcA units decorated at O-3 position with sulfate groups are interesting for their neurite outgrowth promotion in the central nervous system. To GAGs family belongs also fCS. It is a glycosaminoglycan extracted from sea cucumbers (Echinodermata) and composed of a chondroitin sulfate backbone, substituted at position O-3 of GlcA units with heavily sulfated L-fucose side branches. fCS shows several biological properties, above all anticoagulant and antithrombotic activities that are tied to the branches of sulfated fucose on CS backbone. As heparin, fCS exerts these two activities by a serpin-dependent mechanism, in which thrombin inhibition is mediated by AT and HC-II. Importantly, and in contrast to heparin, fCS inhibits Xase factor and furthermore the Xa itself, through a serpin-independent mechanism too. These peculiar properties position fCS to potentially substitute heparin as anticoagulant and antithrombotic agent; indeed, fCS is currently under investigation in clinical trials as a new antithrombotic drug. In order to overcome the serious downsides of using animal-sourced polysaccharides for therapeutic purposes, such as ethical problems, contamination risks and discrepancies in composition, a regioselective modification of a chondroitin polysaccharide, obtained by fed-batch fermentation of E. coli O5:K4:H4, was developed, with the final aim to produce a safer and highly controllable fCS-based drug candidate. Derivatization started by esterification (either methylation or n-dodecylation) of carboxylic acid of GlcA subunits, to make chondroitin more soluble in aprotic solvents, then O-4,6 diol of GalNAc was protected by introduction of a benzylidene ring. The obtained derivatives were used as polysaccharide acceptors for glycosylation reactions, by coupling with suitable per-O-benzylated fucosyl donors under several conditions, trying to achieve a regiochemical and stereochemical control of glycosidic bond formation. Fucosylated products were further modified, obtaining at the end of semi-synthetic route fCS polysaccharides bearing persulfated Fuc branches. In order to obtain different sulfation patterns on Fuc units, the semi-synthetic strategy was upgraded, with the synthesis of new suitably protected fucosyl donors, for achieving polysaccharides with a even higher control of regio- and stereoselectivity of Fuc branching and sulfation pattern on the chondroitin backbone. Moreover, modification on polysaccharide backbone afforded a different glycosyl acceptor, useful to further enlarge the library of the semi-synthesized fucosylated chondroitin sulfate and chondroitin sulfates (fC and fCS, respectively) polysaccharides for future detailed structure-activity relationship investigations. They were preliminarily assayed for anticoagulant activity, displaying an AT-dependent activity against factor Xa in the same range of low molecular mass fCS species obtained by partial depolymerization of natural polysaccharides. For HC-II mediated factor IIa activity, data were very close to heparin for fCSs with Fuc branches on the GlcA units, regardless of their sulfation pattern, whereas two of the three fCSs with Fuc branches on the GalNAc units, as well as unsulfated polysaccharides, displayed a much reduced anticoagulant activity. Among biological properties of fCS polysaccharides, it is worth noting that the inhibition of P- and L-selectin interaction with sialyl Lewis(x), is stronger than the heparin one. Interestingly, oligosaccharides prepared by depolymerization of fCS from Holoturia forskali still maintained a high affinity for P- and L-selectins, but displaying a lower adverse effects than native polysaccharide. In order to evaluate the same inhibition activity of depolymerized fucosylated chondroitin sulfate (dfCS) from natural sources, a semi-synthetic fCS polysaccharide was submitted to β-eliminative depolymerization to give a oligosaccharide to be tested for its interaction with P- and L- selectins by STD-NMR techniques, displaying a slightly minor affinity with respect to that obtained from the natural one. Chondroitin polysaccharide obtained from the fed-batch fermentation of E.coli O5:K4:H4 is, from a structural point of view, similar to the backbone of Colwellia psychrerythraea 34H capsular polysaccharide (CPS) displaying an unprecedented cryoprotectant function, and consisting of a tetrasaccharide repeating units composed of two aminosugars and two uronic acids, with one of the two latter bearing a L-threonine as substituent. In order to better understand the structure-cryoprotectant function relationship of this polysaccharide, microbial sourced chondroitin was coupled with L-threonine under several conditions, producing a semi-synthetic derivative that displayed a ice recrystallization inhibition much lower than the C. psychrerythraea CPS. A combined NMR-molecular dynamic study of its 3D structure showed a rather far arrangement between the two polysaccharides, thus demonstrating that threonine decoration of biomacroolecules is not a sufficient element for gaining ice ricrystallization inhibition in spite of several examples of Thr-rich (glycol)-proteins and polysaccharides with cryoprotectant activity in Nature. Another polysaccharide that was subjected to regioselective modifications is alginate, that consists of 1-->4-linked β-D-mannuronic acid (M) and its C-5 epimer α-L-guluronic acid (G) units. This natural copolymer is an important component of algae such kelp, and is also an exopolysaccharide of bacteria including Pseudomonas aeruginosa. Alginates are widely used in food, cosmetic and pharmaceutical industry. The sulfation of these polysaccharides exhibits compounds with carboxylic and sulfate groups close to each others as in heparin ones. Randomly sulfated alginates show anticoagulant activity, so regioselective modification of the polysaccharide backbone may help to understand the relationship between structure and properties in alginate sulfates. Indeed, a semi-synthetic sulfated alginate derivative (propylene glycol alginate sodium sulfate, PSS), has been employed as anti-cardiovascular disease drug in China, without control of degree of sulfation. Due to incomplete solubility and highly heterogeneous structure of natural alginic acids the strategy to obtain a regioselectively sulfated alginate polysaccharide was applied to β-D-polymannuronic acid, that is the simplest polysaccharide possessing the most homogeneous structure of all alginic acids. It was protected at O-2,3 diol by either application of an orthoester or benzylidene ring and in the latter case, the polymannuronic acid was derivatized at carboxylic function too in order to enhance its solubility in aprotic solvent. At the end of the semi-synthetic route compounds with different sulfation pattern were obtained, but with unclear and probably not complete regioselectivity. Therefore, further optimization on semi-synthetic strategy is needed for the production of regioselectively sulfated alginates and for the evaluation of their structure-activity relationships.

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