Synthesis and characterization of Tetra-PEG-DBCO

The preloaded precursor Tetra-PEG-DBCO was synthesized by a one-step amidation reaction between DBCO acid and Tetra-PEG-amine⁴² (Supplementary Fig. ¹). DBCO acid (171 mg, 0.56 mmol), HATU (213 mg, 0.56 mmol), and 3 ml anhydrous DMF were added into a dry flask to give a light-yellow solution under stirring. Tetra-PEG-amine (M_n = 40 kDa, 2.8 g, 0.07 mmol) in 12 ml of anhydrous DMF was then added at 0 °C. After the addition of 200 μLof anhydrous DIEA, the reaction mixture was stirred at 25 °C for 36 h. Afterward, the mixture was dropwise added into cold Et₂O with stirring to afford a white solid powder. The product was further washed by Et₂O for three times and dried under vacuum to give a light-yellow powder (10.5 g, yield 91.3%).

The structure of the obtained compound was confirmed by proton nuclear magnetic resonance (¹H NMR) spectrum (Bruker Avance 500 spectrometer). Chemical shifts (δ) were expressed in ppm. According to 1H NMR (Supplementary Fig. ²), the end-functionalization was 91.2%. ¹H NMR (400 MHz, CDCl₃): 7.67–7.32 (m, 32H), 6.19 (br, 4H), 5.17 (d, J = 13.6 Hz, 4H), 3.86–3.31 (m, 3678H), 2.80–1.93 (m, 16H) ppm.