Dipea

4-Dimethylaminoacetophenone, 4-dimethylaminobenzaldehyde, 2-acetylthiophene, thiophene-2-carboxaldehyde, diethylamine (DEA), potassium hydroxide, nitromethane, potassium carbonate, NH₄OAc, n-BuOH were purchased from commercial suppliers in China. BF₃·OEt₂, TFA, TEA, NH₄OAc, sodium sulfate anhydrous, sodium chloride, potassium hydrogen phthalate, potassium hydrogen phosphate, ethyl actetate, DMSO and DIPEA were purchased from Sigma Aldrich (St. Louis, MO, USA) and used as received. CH₂Cl₂, ethanol (EtOH), methanol, potassium chloride and hydrochloric acid were purchased from Minema (Johannesburg, South Africa). Sodium acetate was purchased from Saarchem (Johannesburg, South Africa), and acetic acid was purchased from B&M Scientific (Cape Town, South Africa). All chemicals were analytically pure and were used as received. Acetone-d₆ and CDCl₃ for ¹H NMR spectroscopy, and spectroscopic grade solvents for optical spectroscopy were purchased from Sigma-Aldrich and Merck (Darmstadt, Germany), respectively.

The deferoxamine mesylate salt and the 8-arm-PEG-SG with different molecular weights (20 kDa and 40 kDa) were dissolved in anhydrous dimethyl sulfoxide. The DIPEA (Sigma, Aldrich) was added into the solution under magnetic stirring. The reaction was carried out under nitrogen at 25 °C for 24 h. The purification process was carried out by dialyzing against deionized water at 25 °C for another 24 h. The solution was freeze-dried to obtain the final products.

MSNs were synthesized using the sol–gel emulsion described in [9 (link)]. In short, cetyltrimethylammonium bromide (CTAB, ≥99%, Sigma-Aldrich, Shanghai, China) served as a mesoporous template, followed by in situ polymerization using tetraethyl orthosilicate (TEOS, ≥99%, Sigma-Aldrich). After two hours, the surfactant CTAB was removed, and the resulting MSNs were filtered, washed, and dried under a high vacuum. Surface amine grafting was achieved through overnight refluxing by dried toluene (>99.3%, Honeywell Fluka, Seelze, Germany) and aminopropyl triethoxysilane (APTES, ≥99%, Sigma-Aldrich). Finally, the mixture was filtered and dried under vacuum to obtain MSN-NH₂. Successful synthesis of the MNSs was assessed transmission electron microscopy as described in [9 (link)]. To protect the conformation of the peptide, interactions between the peptide and the MSNs were inhibited by modifying the MSNs with PEG₄-N₃. As described in [34 (link)], N₃-PEG₄-COOH (1 eq., >97%, Biomatrik, Jiaxing, Zhejiang, China), HATU (2 eq., 99%, Alfa Aesar, Kendel, Germany), and DIPEA, (3 eq., ≥99%, Sigma-Aldrich) were added to a suspension of MSN-NH₂ in DMF (≥99.8%, Biosolve Chimie, Dieuze, France) and stirred overnight at room temperature, after which the MSNs were filtered and washed with water and ethanol to obtain MSN-PEG₄-N₃.

Inspired by Yang et al. (2009) (link). Briefly, in a round-bottom flask (R)-1-phenylprop-2-ylamine hydrochloride salt (103 mg, 0.6 mmol, 2 equiv., Asiba Pharmatec.) was placed under inert atmosphere. A solution of methyl-PEG-NHS-ester reagent (1.1 mL, 100 mg, 0.39 mmol, 1 equiv., Thermo Scientific) in DMSO was then added, followed by the addition of diisopropylethylamine (DIPEA, 105 μL, 0.6 mmol, 2 eq, Sigma-Aldrich). The reaction was stirred at room temperature for 46 h, after which a multiple extraction with water/ethyl acetate was performed to remove the product from DMSO. Then preparative chromatography (5% EtOAc in MeOH, v/v) was performed to isolate compound PEGyAMPH in 98% yield (0.1 g). Characterization: ¹H NMR (300 MHz, CDCl₃) δ 7.25 – 7.11 (m, 5H), 6.53 – 6.26 (m, 1H), 4.19 (p, J = 6.8 Hz, 1H), 3.63 – 3.47 (m, 14H), 3.32 (s, 3H), 2.79 (dd, J = 13.5, 6.1 Hz, 1H), 2.65 (dd, J = 13.5, 7.1 Hz, 1H), 2.37 (t, J = 6.4 Hz, 2H), 1.06 (d, J = 6.6 Hz, 3H) ppm. ¹³C NMR (75 MHz, CDCl₃) δ 170.92, 138.38, 129.55, 128.36, 126.40, 72.01, 70.70, 70.60, 70.46, 70.34, 67.43, 59.11, 46.02, 42.60, 37.21 ppm. HRMS: [M+H]⁺_calc = 354.22750; [M+H]⁺_real = 354.22783 (error –0.9 ppm). Scale-up of the reaction for the chronic in vivo treatments was reproduced by Wuxi AppTec.