Comparison of reaction rates of NHS esters of mPEG with amino and alcohol group. To test reaction with amino group at 25°C, final ethanolamine concentration in DMF was 0.83 mmol/L. After the reaction for an indicated period, residual ethanolamine was quantified by withdrawing 50 μL sample for reaction with TNBS in borate buffer of 1.0 mL at 55°C for 15 min. The reaction mixture was cooled with tap water before assay of absorbance. The percentages of residual NHS esters are calculated and plotted versus reaction time. To test reaction with alcohol group at 25°C, final ethanol was 0.83 mmol/L in a total volume of 0.95 mL DMF containing an indicated NHS ester; after reaction for an indicated period, 50 μL diluted ethanolamine solution in DMF was added for final 0.83 mmol/L and 15-min reaction at 25°C. The residual ethanolamine was determined as described above. The percentages of residual NHS esters are calculated and plotted versus reaction time. A: NHS-SC-mPEG5k with ethanolamine; B: NHS-SC-mPEG5k with ethanol; C: NHS-CB-mPEG5k with ethanolamine; D: NHS-CB-mPEG5k with ethanol.