Synthesis and Characterization of Chitosan N-Betainates Having Various Degrees of Substitution Jukka Holappa,* ,† Tapio Nevalainen, † Jouko Savolainen, † Pasi Soininen, ‡ Matti Elomaa, § Rustam Safin, † Sari Suvanto, ⊥ Tuula Pakkanen, ⊥ Ma ´ r Ma ´ sson, # Thorsteinn Loftsson, # and Tomi Ja 1 rvinen † Departments of Pharmaceutical Chemistry, Chemistry, and Pharmaceutics, University of Kuopio, P.O. Box 1627, FIN-70211 Kuopio, Finland; Department of Chemistry, University of Joensuu, P.O. Box 111, FIN-80101 Joensuu, Finland; and Faculty of Pharmacy, University of Iceland, Hofsvallagata 53, 107 Reykjavik, Iceland Received December 11, 2003; Revised Manuscript Received February 2, 2004 ABSTRACT: An efficient five-step synthetic route was developed for full N-substitution of chitosan with a quaternary betaine moiety. The developed synthetic procedure can also be controlled to produce chitosan N-betainates having lesser degrees of substitution. 6-O-Triphenylmethylchitosan, which is highly soluble in organic solvents, was used as an intermediate for N-acylation reactions. Intermediate products were characterized by 13 C CP/MAS NMR, FT-IR, and elemental analysis. The water-soluble quaternary chitosan N-betainates were thoroughly characterized by 1 H NMR and 13 C NMR and by 2D 1 H- 1 H COSY NMR and 13 H- 1 H HSQC NMR. Degrees of substitution were determined from the 1 H NMR spectra. A significant degradation of the polysaccharide backbone during the synthetic procedure was determined by GPC with a light scattering detector. Introduction Chitosan (poly-1,4--D-glucosamine) is a biocompat- ible, 1 biodegradable, 2 and mucoadhesive 3 polysaccharide that has attracted considerable attention in the phar- maceutical and biomedical fields. 4-6 Chitosan has many conventional formulation applications, mostly for its ability to form films and gels. Novel applications of chitosan are delivery of peptides, 7 vaccines, 8 and genes. 9 Biomedically, the most explored feature of chitosan is its antimicrobial property. 10 The polycationic properties of chitosan are probably responsible for most of its observed activity. 4 The amino groups on chitosan are only partially protonized at physiological pH 7.4, and the major drawback of chito- san is its poor aqueous solubility, when considered as a pharmaceutical excipient. Chitosan is soluble in dilute aqueous acids due to protonation of the amino groups. Quaternary ammonium derivatives of chitosan are therefore interesting in view of pharmaceutical applica- tions. These derivatives have two major advantages over the parent chitosan: (1) they are water-soluble at physiological pH, and (2) they have a permanent posi- tive charge on the polysaccharide backbone. Quaternary chitosan derivatives have various potential pharmaceu- tical applications, e.g., as antimicrobials, 11 as perme- ation enhancers, 12 and as gene delivery systems. 13 The aim of the present study was to develop a syn- thetic route that would enable the preparation of N-betainate derivatives of chitosan having various degrees of substitution. The structurally simple qua- ternary betaine moiety was selected because it is nontoxic natural product, which could yield to more uniform quaternary chitosan derivative having physi- cochemical, biological, and pharmaceutical properties comparable to N-trimethylchitosan. It is important to study the impact of differences in substitution degrees when chitosan derivatives are tested for different ap- plications. Efforts were focused on the exact character- ization of chitosan N-betainate end products with NMR spectroscopy, which is the most reliable method for the characterization of chitosan derivatives. Aiedeh and co- workers 14 published a synthetic route for chitosan N-betaine earlier. However, they published only the preparation and characterization of a product with one degree of substitution. In addition, no NMR data or data on the synthetic procedure’s effect on molecular weight were reported. Chitosan N-betainates were characterized by 1D 1 H and 13 C NMR and also by 2D 1 H- 1 H COSY and 13 C- 1 H HSQC NMR. Degrees of substitution (ds) of the end products were determined from the 1 H NMR spectra. GPC-LS was used to analyze effects of the synthetic procedure on the molecular weight and mo- lecular weight distribution of the polymer. Structures of the intermediate products were characterized by solid-state 13 C CP/MAS NMR, FT-IR, and elemental analysis. Degrees of substitution of the intermediate products were calculated from the C/N ratios, which were obtained from the elemental analysis. Experimental Section Materials. Chitosan, used as a starting material, was donated by Primex ehf (Reykjavik, Iceland). Weight-average molar mass (Mw) 148 200 Da and number-average molar mass (Mn) 94 200 were determined by GPC-LS. The degree of deacetylation (85%) was confirmed by 1 H NMR. All other chemicals used were commercially available and used as received. Pyridine was distilled over KOH. Dialysis membrane (M w cutoff 12 kDa) was purchased from Sigma (St. Louis, MO). Characterization. 1 H and 13 C spectra were recorded on a Bruker AVANCE DRX 500, operating at 500.13 and 125.76 MHz, respectively. Samples of chitosan N-betainates (40 mg) were dissolved in 600 µL of D 2O. Chemical shifts (δ) are † Department of Pharmaceutical Chemistry, University of Kuopio. ‡ Department of Chemistry, University of Kuopio. § Department of Pharmaceutics, University of Kuopio. ⊥ University of Joensuu. # University of Iceland. * Corresponding author: Tel +358-17-163662; Fax +358-17- 162456; e-mail Jukka.Holappa@uku.fi. 2784 Macromolecules 2004, 37, 2784-2789 10.1021/ma0358780 CCC: $27.50 © 2004 American Chemical Society Published on Web 03/16/2004