Preparation and characterization of branched chitosans

Fully de-N-acetylated chito-oligomers were prepared by de-amination of chitosan using nitrous acid (HNO2). The dimer to hexamer fractions of chitosan oligomers were fractionated with preparative size-exclusion chromatography (SEC) and characterized using 1H NMR spectroscopy. The purified oligomers were polymerized by reductive N-alkylation by the action of NaCNBH3, in a reaction between the terminal 2,5-anhydro-d-mannofuranose (M-unit) and the primary amines of the repeating units (1 → 4) bound β-d-glucosamine (D-unit) resulting in a covalent bond and formation of secondary amines. The polymerized products were studied by SEC-RI and SEC-MALLS-RI and were confirmed to be branched reaching molecular weights of up to 10 000 g/mol. Analysis by 1H NMR spectroscopy confirmed that the chitosan oligomers had been branched by formation of secondary amines. de-N-acetylated chitosan (FA < 0.001) was degraded to various extents using nitrous acid resulting in degrees of scission (αs) in the range 0.01–0.10. The resulting chitosans, with the reactive M-unit on the reducing end, were branched by reductive N-alkylation using NaCNBH3. Analysis by SEC-MALLS-RI-visc showed that the weight-average molecular weight (Mw) and polydispersity (Mw/Mn) had increased, with a degree of branching in the range of 1.2–2.1. The structure in solution had become more compact compared to linear chitosan of the same molecular weight as demonstrated by a significant reduction of the radius of gyration and the intrinsic viscosity, with a corresponding change in the calculated viscosity contraction factor (g′) and the geometric contraction factor (g), respectively.