Regioselective modifications of natural polysaccharides

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

Elastin-Hyaluronan Bioconjugate as Bioactive Component in Electrospun Scaffolds

Hyaluronic acid or hyaluronan (HA) and elastin‐inspired peptides (EL) have been widely recognized as bioinspired materials useful in biomedical applications. The aim of the present work is the production of electrospun scaffolds as wound dressing materials which would benefit from synergic action of the bioactivity of elastin peptides and the regenerative properties of hyaluronic acid. Taking advantage of thiol‐ene chemistry, a bioactive elastin peptide was successfully conjugated to methacrylated hyaluronic acid (MAHA) and electrospun together with poly‐d,l‐lactide (PDLLA). To the best of our knowledge, limited reports on peptide‐conjugated hyaluronic acid were described in literature, and none of these was employed for the production of electrospun scaffolds. The conformational studies carried out by Circular Dichroism (CD) on the bioconjugated compound confirmed the preservation of secondary structure of the peptide after conjugation while Scanning Electron Microscopy (SEM) revealed the supramolecular structure of the electrospun scaffolds. Overall, the study demonstrates that the bioconjugation of hyaluronic acid with the elastin peptide improved the electrospinning processability with improved characteristics in terms of morphology of the final scaffolds

“Tuning aggregative versus non-aggregative lectin binding with glycosylated nanoparticles by the nature of the polymer ligand”

Glycan–lectin interactions drive a diverse range of biological signaling and recognition processes. The display of glycans in multivalent format enables their intrinsically weak binding affinity to lectins to be overcome by the cluster glycoside effect, which results in a non-linear increase in binding affinity. As many lectins have multiple binding sites, upon interaction with glycosylated nanomaterials either aggregation or surface binding without aggregation can occur. Depending on the application area, either one of these responses are desirable (or undesirable) but methods to tune the aggregation state, independently from the overall extent/affinity of binding are currently missing. Herein, we use gold nanoparticles decorated with galactose-terminated polymer ligands, obtained by photo-initiated RAFT polymerization to ensure high end-group fidelity, to show the dramatic impact on agglutination behaviour due to the chemistry of the polymer linker. Poly(N-hydroxyethyl acrylamide) (PHEA)-coated gold nanoparticles, a polymer widely used as a non-ionic stabilizer, showed preference for aggregation with lectins compared to poly(N-(2-hydroxypropyl)methacrylamide) (PHPMA)-coated nanoparticles which retained colloidal stability, across a wide range of polymer lengths and particle core sizes. Using biolayer interferometry, it was observed that both coatings gave rise to similar binding affinity and hence provided conclusive evidence that aggregation rate alone cannot be used to measure affinity between nanoparticle systems with different stabilizing linkers. This is significant, as turbidimetry is widely used to demonstrate glycomaterial activity, although this work shows the most aggregating may not be the most avid, when comparing different polymer backbones/coating. Overall, our findings underline the potential of PHPMA as the coating of choice for applications where aggregation upon lectin binding would be problematic, such as in vivo imaging or drug delivery

Mn-Doped Glass–Ceramic Bioactive (Mn-BG) Thin Film to Selectively Enhance the Bioactivity of Electrospun Fibrous Polymeric Scaffolds

In recent years, significant progress has been made in the development of new technologies to meet the demand for engineered interfaces with appropriate properties for osteochondral unit repair and regeneration. In this context, we combined two methodologies that have emerged as powerful approaches for tissue engineering application: electrospinning to fabricate a nanofibrous polymeric scaffold and pulsed laser deposition to tune and control the composition and morphology of the scaffold surface. A multi-component scaffold composed of synthetic and natural polymers was proposed to combine the biocompatibility and suitable mechanical properties of poly(D,L-lactic acid) with the hydrophilicity and cellular affinity of gelatin. As part of a biomimetic strategy for the generation of bi-functional scaffolds, we coated the electrospun fibers with a thin film of a bioactive glass–ceramic material supplemented with manganese ions. The physico-chemical properties and composition of the bi-layered scaffold were investigated, and its bioactivity, in terms of induced mineralization, was tested by incubation in a simulated body fluid buffer. The processes of the inorganic film dissolution and the calcium phosphate phases growth were followed by microscopic and spectroscopic techniques, confirming that a combination of bioactive glass–ceramics and nanofibrous scaffolds has promising potential in the regeneration of osteochondral tissue due to its ability to induce mineralization in connective tissues. © 2022 by the authors

Domains 12 to 16 of tropoelastin promote cell attachment and spreading through interactions with glycosaminoglycan and integrins alphaV and alpha5beta1

Elastin is an extracellular matrix component with key structural and biological roles in elastic tissues. Interactions between resident cells and tropoelastin, the monomer of elastin, underpin elastin’s regulation of cellular processes. However, the nature of tropoelastin–cell interactions and the contributions of individual tropoelastin domains to these interactions are only partly elucidated. In this study, we identified and characterized novel cell-adhesive sites in the tropoelastin N-terminal region between domains 12 and 16. We found that this region interacts with aV and a5b1 integrin receptors, which mediate cell attachment and spreading. A peptide sequence from within this region, spanning domains 14 to mid-domain 16, binds heparan sulfate through electrostatic interactions with peptide lysine residues and induces conformational ordering of the peptide. We propose that domains 14–16 direct initial cell attachment through cell-surface heparan sulfate glycosaminoglycans, followed by aV and a5b1 integrin-promoted attachment and spreading on domains 12–16 of tropoelastin. These findings advance our mechanistic understanding of elastin matrix biology, with the potential to enhance tissue regenerative outcomes of elastin-based materials

Protecting group free synthesis of glyconanoparticles using amino-oxy-terminated polymer ligands

Glycomaterials display enhanced binding affinity to carbohydrate-binding proteins due to the nonlinear enhancement associated with the cluster glycoside effect. Gold nanoparticles bearing glycans have attracted significant interest in particular. This is due to their versatility, their highly tunable gold cores (size and shape), and their application in biosensors and diagnostic tools. However, conjugating glycans onto these materials can be challenging, necessitating either multiple protecting group manipulations or the use of only simple glycans. This results in limited structural diversity compared to glycoarrays which can include hundreds of glycans. Here we report a method to generate glyconanoparticles from unprotected glycans by conjugation to polymer tethers bearing terminal amino-oxy groups, which are then immobilized onto gold nanoparticles. Using an isotope-labeled glycan, the efficiency of this reaction was probed in detail to confirm conjugation, with 25% of end-groups being functionalized, predominantly in the ring-closed form. Facile post-glycosylation purification is achieved by simple centrifugation/washing cycles to remove excess glycan and polymer. This streamlined synthetic approach may be particularly useful for the preparation of glyconanoparticle libraries using automation, to identify hits to be taken forward using more conventional synthetic methods. Exemplar lectin-binding studies were undertaken to confirm the availability of the glycans for binding and show this is a powerful tool for rapid assessment of multivalent glycan binding

A review of chemical methods for the selective sulfation and desulfation of polysaccharides

Sulfated polysaccharides are known to possess several biological activities, with their sulfation pattern acting as a code able to transmit functional information. Due to their high biological and biomedical importance, in the last two decades many reports on the chemical modification of their sulfate distribution as well as on the regioselective insertion of sulfate groups on non-sulfated polysaccharides appeared in literature. In this Review we have for the first time collected these reports together, categorizing them into three different classes: i) regioselective sulfation reactions, ii) regioselective desulfation reactions, iii) regioselective insertion of sulfate groups through multi-step strategies, and discussing their scope and limitations

Inter vs. intraglycosidic acetal linkages control sulfation pattern in semi-synthetic chondroitin sulfate

tMicrobial-sourced unsulfated chondroitin could be converted into chondroitin sulfate (CS) polysaccha-ride by a multi-step strategy relying upon benzylidenation and acetylation reactions as key-steps for itsregioselective protection. By conducting the two reactions one- or two-pots, CSs with different sulfationpatterns could be obtained at the end of the semi-synthesis. In particular, a CS polysaccharide possess-ing sulfate groups randomly distributed between positions 4 and 6 of N-acetyl-galactosamine (GalNAc)units could be obtained through the two-pots route, whereas the one-pot pathway allowed an additionalsulfation at position 3 of some glucuronic acid (GlcA) units. This difference was ascribed to the stabi-lization of a labile interglycosidic benzylidene acetal involving positions O-3 and O-6 of some GlcA andGalNAc, respectively, when the benzylidene-acetylation reactions were conducted in a one-pot fashion.Isolation and characterization of a polysaccharide intermediate showing interglycosidic acetal moietieswas accomplished