Carbohydrate Polymers 86 (2011) 747–752 Contents lists available at ScienceDirect Carbohydrate Polymers jo u rn al hom epa ge: www.elsevier.com/locate/carbpol Synthesis of N-alanyl-hyaluronamide with high degree of substitution for enhanced resistance to hyaluronidase-mediated digestion Carole Schanté a,b,* , Guy Zuber a , Corinne Herlin b , Thierry F. Vandamme a a Laboratoire de Conception et Application de Molécules Bioactives, Université de Strasbourg, CNRS UMR7199, Faculté de Pharmacie, 74 route du Rhin, 67400 Illkirch, France b Laboratoire IDENOV, 8 rue de Reims, 67000 Strasbourg, France a r t i c l e i n f o Article history: Received 2 February 2011 Accepted 16 May 2011 Available online 23 May 2011 Keywords: Hyaluronic acid modification Amidation SEC-MALS Viscosity Enzymatic degradation a b s t r a c t In order to increase the resistance of hyaluronic acid towards enzymatic digestion, we prepared N-alanyl-hyaluronamide derivatives using different amidation methods performed either in water, water/acetonitrile mixture or anhydrous dimethylformamide. Our results indicate that amidation of hyaluronic acid in an anhydrous solvent is effective and led to a degree of substitution of the carboxylic groups up to 100%. We also demonstrated the N-alanyl-hyaluronamides present enhanced resistance towards enzymatic digestion while forming solutions with similar viscosities than solutions of hyaluronic acids of similar lengths. © 2011 Elsevier Ltd. All rights reserved. 1. Introduction Hyaluronic acid (HA), also called hyaluronan (Meyer & Palmer, 1934), is a high molecular weight polymer with repeating dis- accharide units of N-acetyl-d-glucosamine and d-glucuronic acid, linked by alternating glycosidic bonds -(1,4) and -(1,3) (Fig. 1) (Weissmann & Meyer, 1954). It is a major component of the extra- cellular matrix of vertebrates and is found in high concentrations in synovial fluid, vitreous body and skin (Fraser, Laurent, & Laurent, 1997). Its main function is to serve as a lubricant, shock absorber and matrix (Baumann, 2004; Laurent, Laurent, & Fraser, 1995), properties which are strongly related to HA’s ability to form shear- dependant and highly viscous solutions (Laurent & Fraser, 1992). HA can be easily obtained in large quantity from animal tissues such as rooster comb or more recently from recombinant bacte- ria (Shiedlin et al., 2004). This has allowed extensive use of HA and gels of HA for visco-supplementation by injection into joints to treat arthritis, for ophthalmic surgery and for tissue augmenta- tion in cosmetic surgery (Kogan, Soltés, Stern, & Gemeiner, 2007). More recent applications include drug delivery and wound healing (Chen & Abatangelo, 1999; Esposito, Menegatti, & Cortesi, 2005). However, repeated injections of HA are often necessary to compen- sate for its naturally occurring degradation (Stern, Kogan, Jedrzejas, * Corresponding author at: Laboratoire de Conception et Application de Molécules Bioactives, Université de Strasbourg, CNRS UMR 7199, Faculté de Pharmacie, 74 route du Rhin, 67400 Illkirch, France. Tel.: +33 3 68 85 41 06. E-mail address: carole.schante@etu.unistra.fr (C. Schanté). & ˇ Soltés, 2007). The hyaluronidase enzymes and the CD44 (Clus- ter of Differentiation-44) cell surface receptors are responsible for HA degradation (Aruffo, Stamenkovic, Melnick, Underhill, & Seed, 1990). Molecular modeling based on crystallographic studies indi- cated that the recognition sites of the Hyal-2 enzymes and CD44 receptors are the carboxylate groups of HA (Banerji et al., 2007). Its chemical modification may hence diminish enzymatic recognition and enhances the stability of HA towards hyaluronidase-mediated hydrolysis. In here, we evaluated the effect of grafting alanine onto HA to mask the HA backbone for enzymatic recognition by hyaluronidase while maintaining the overall anionic density of the polymer. For integral masking of the HA, the ideal is to obtain the high- est achievable degree of modification and we therefore assayed various amidation methods. The effect of the chemical process on the physicochemical behavior of the Ala-HA and stability to hyaluronidase digestion was also reported. 2. Materials and methods 2.1. Materials 1-Ethyl-3-[3-(dimethylamino)-propyl]-carbodiimide (EDC), N- hydroxysuccinimide (NHS), l-alanine ethyl ester HCl (AlaOEt-HCl), sodium hydroxide (NaOH), tetrabutylammonium (TBA) hydroxide solution (∼40% or 1.5 M in water), 2-chloro-1-methylpyridinium iodide (CMPI), triethylamine, acetonitrile, N-methylmorpholine (NMM), 2-chloro-dimethoxy-1,3,5-triazine (CDMT) were pur- 0144-8617/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.carbpol.2011.05.017