10 kDa PEG-octa-hydrazine (8-H) was synthesized by dissolving tri-Boc-hydrazinoacetic acid (1.372 g, 3.52 mmol, 2.1 equiv. per amine) in anhydrous DMF (10 mL) and was activated with HATU (1.216 g, 3.20 mmol, 2.0 equiv.) and N-methylmorpholine (0.792 mL, 7.2 mmol, 4.5 equiv.). The reaction was stirred for 5 minutes, and then the 4-arm 20 kDa (8.000 g, 0.4 mmol) PEG was added, and the reaction was allowed to proceed overnight at room temperature. The product was precipitated in ice-cold diethyl ether, dried, treated with a solution of 50 : 50 DCM–TFA for 4 hours to remove the Boc group. The resulting compound was precipitated in ether, dissolved in DI water, dialyzed (2 000 MWCO) against DI water for 24 hours, and lyophilized, after which it was used for experimentation. 1.25 kDa PEG-mono-hydrazine, used for the toxicity experiments, was synthesized in an identical manner except the 2 000 MWCO dialysis tube was replaced with a 500 Da MWCO tube.
8-H 1H NMR (D2O, 400 MHz): δ = 3.59 (s, PEG).
8-H 1H NMR(DMSO-d6, 400 MHz): δ = 8.1 (t, J = 4 Hz, 1H), δ = 4.45 (m, 2H), δ = 3.51 (s, PEG), δ = 3.2 (m, 1H), δ = 2.96 (m, 2H).
10 kDa PEG-octa-aldehyde (8-AA) was synthesized using a Swern oxidation.32 Oxalyl chloride (1.5 mL, 17.6 mmol, 11 equiv. per hydroxyl) was dissolved in anhydrous DCM (20 mL) in a flame-dried flask purged with argon, and the reaction flask was cooled in an acetone/dry ice bath. DMSO (1.3 mL, 18.5 mmol, 11.5 equiv.) diluted 1 : 5 in anhydrous DCM was added dropwise over the course of 5 minutes. The reaction was allowed to proceed for 10 minutes to ensure formation of the alkoxysulfonium ion intermediate. 8-arm 10 kDa PEG-OH (2 g, 0.2 mmol) or 4-arm 10 kDa PEG-OH (4 g, 0.4 mmol) was dissolved in anhydrous DCM (5 mL) and added dropwise over 10 minutes and allowed to react for 2 hours. Triethylamine (5.6 mL, 40 mmol, 25 equiv.) was added dropwise over 10 minutes and given 20 minutes to react. Finally, the reaction was allowed to warm to room temperature, and the product was precipitated in ether and dialyzed as previously described.
8-AA 1H NMR (D2O, 400 MHz): δ = 5.04 (t, J = 6 Hz, 1H), δ = 3.76 (t, J = 4 Hz, 2H), δ = 3.59 (s, PEG). Aldehyde exists in the diol form in D2O.
8-AA 1H NMR (DMSO-d6, 400 MHz): δ = 9.61 (s, 1H), δ = 4.23 (s, 2H), δ = 3.54 (s, PEG). 8-AA gels in organic solvents; therefore, NMR peaks were significantly broadened.
8-H 1H NMR (D2O, 400 MHz): δ = 3.59 (s, PEG).
8-H 1H NMR(DMSO-d6, 400 MHz): δ = 8.1 (t, J = 4 Hz, 1H), δ = 4.45 (m, 2H), δ = 3.51 (s, PEG), δ = 3.2 (m, 1H), δ = 2.96 (m, 2H).
10 kDa PEG-octa-aldehyde (8-AA) was synthesized using a Swern oxidation.32 Oxalyl chloride (1.5 mL, 17.6 mmol, 11 equiv. per hydroxyl) was dissolved in anhydrous DCM (20 mL) in a flame-dried flask purged with argon, and the reaction flask was cooled in an acetone/dry ice bath. DMSO (1.3 mL, 18.5 mmol, 11.5 equiv.) diluted 1 : 5 in anhydrous DCM was added dropwise over the course of 5 minutes. The reaction was allowed to proceed for 10 minutes to ensure formation of the alkoxysulfonium ion intermediate. 8-arm 10 kDa PEG-OH (2 g, 0.2 mmol) or 4-arm 10 kDa PEG-OH (4 g, 0.4 mmol) was dissolved in anhydrous DCM (5 mL) and added dropwise over 10 minutes and allowed to react for 2 hours. Triethylamine (5.6 mL, 40 mmol, 25 equiv.) was added dropwise over 10 minutes and given 20 minutes to react. Finally, the reaction was allowed to warm to room temperature, and the product was precipitated in ether and dialyzed as previously described.
8-AA 1H NMR (D2O, 400 MHz): δ = 5.04 (t, J = 6 Hz, 1H), δ = 3.76 (t, J = 4 Hz, 2H), δ = 3.59 (s, PEG). Aldehyde exists in the diol form in D2O.
8-AA 1H NMR (DMSO-d6, 400 MHz): δ = 9.61 (s, 1H), δ = 4.23 (s, 2H), δ = 3.54 (s, PEG). 8-AA gels in organic solvents; therefore, NMR peaks were significantly broadened.
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