Chemical Modification and Structural Characterization of Lactosilated Hyaluronic Acid

Hyaluronic acid (HA) is a polysaccharide which exhibits anti-inflammatory and anti-angiogenic properties, it’s involved in cellular activity and promotes tissue reconstruction[1]. HA structure allows its functionalization, thus increasing its potential applications[2]. The modification of HA with an amino-lactitol derivative (LAC-NH2) produces a new biopolymer named HYLACH® [3]. Accordingly, the newly introduced β-galactose residues provide potential molecular probes for interaction with Galectins. Galectin3 is found to be overly expressed in fibrotic diseases [4]. The aim of functionalizing HA with β-galactose residues is to couple the biocompatible and anti-inflammatory properties of HA with the capability of subtracting Gal3 from the pro-fibrotic environment. For HYLACH® synthesis, LAC-NH2 is covalently grafted to HA. The lactosylated HA are characterized by NMR, Size exclusion chromatography and their stability towards enzymatic activity is investigated. HYLACH® molecules showed molecular weights in the range of 80-280kDa, and degree of substitution (DS) up to 45%, according to synthetic conditions. HA. Interestingly, the DS determination by heteronuclear 2D-NMR is performed both on hydrolysed and un-hydrolysed compounds and the results compared and investigated. Increase of stability towards enzyme degradation is observed in HYLACH® respect to native. The conformational and structural behaviour is evaluated by molecular dynamics simulation, where simulation predicts for HYLACH® a less extended helical conformation than HA, and that the β-galactose is accessible to molecular recognition by proteins. Interactions with Galectin3 (Gal3), studied by isothermal titration calorimetry resulted to be entropically and exothermic favoured with affinity in the range of µM. References 1. Fraser, J.; Laurent, T.; U. Laurent, U.; Journal of Internal Medicine, 1997, 242, 27. 2. Schanté C. E;, Zubera, G.; Herlin, C.; Vandammea, T.F.; Carbohdrate Polymers, 2011, 85, 469. 3. Donati, I.; Borgogna, M.; Turello, E.; Cesaro, A.; Paoletti, S.; Biomacromolecules 2007, 8, 1471 4. . Sindrewicz, P.;Yates, A. E.;Turnbull, J.L.; Lian L.Y.; Biochem Biophys Res Commun, 2020, 532 (2), 336.