Synthesis of Peptide-Modified PEG Compounds

For the synthesis of peptide-modified PEG-Peptide compounds, the Michael addition reaction according to Guo et al.^{49 (link)} was used, whereby peptides were irreversibly bonded to PEG(Mal)₄ moieties (Fig. 1E). Briefly, 1 g of PEG(Mal)₄ was dissolved in 3 ml DPBS, followed by the addition of the desired amount of peptide dissolved in 4 ml DBPS under vigorous stirring and the exclusion of light. After 3 h, the reaction was stopped, the solution was transferred into dialysis tubes (molecular weight cut off: 6–8 kDa) and dialyzed for 24 h against 5 l of ultra-pure water with daily water changes. After dialysis, all compounds were frozen at −20 °C and then lyophilised, respectively. Table 1 depicts the concentrations of Peptide A, Peptide B and Peptide C yielding in a theoretical single-arm substitution of PEG(Mal)₄ for PEG-Peptide A, PEG-Peptide B and PEG-Peptide C. In a last approach, all three peptides were bonded to one corresponding PEG(Mal)₄ compound by the addition of Peptide A, Peptide B and Peptide C in the same concentration occupying theoretically 3 of 4 arms of PEG(Mal)₄.