Scheme 3. Schematic of synthetic route for chitosan-SH. Scheme 3 was employed to produce and characterize chitosan-SH, a chitosan derivative containing thiol groups. The synthesis involved dissolving chitosan (MW = 100 kDa) in DI water (5 mg/mL) and mixing it with three molar equivalents of cystamine dihydrochloride at pH 4.8. The carboxyl group of chitosan was activated using EDC and HOBT (at 3 equivalents) for two hours, and the conjugation process took place overnight. The resulting product was purified using dialysis tubing with a molecular weight cutoff of 10,000 kDa against DI water for three days. To break the disulfide linkage of the cystamine component, the solution was treated with TCEP at 5 equivalents and stirred for two hours. The pH was then adjusted to 3.5 with HCl, and chitosan-SH was precipitated in ethanol, re-dissolved in DI water, lyophilized, and stored at -20 ºC.