Figure 1. The reaction scheme of TCS synthesis, One-Step Reaction and The first step and The second step of the new method. Covalent attachment was achieved by the formation of amide bonds between the primary amino groups of CS and the carboxylic acid groups of TGA mediated by a carbodiimide (EDAC) and NHS. One-Step Reaction used water as reaction medium, while The first step of the new method used DMF as reaction medium to avoid the unstable O-acylisourea ester hydrolyzing in water, and obtained much higher thiol groups substituted in TCS, at 1411.01±4.02 versus 231.51±15.52 μmol/g.