Peptide Conjugation to Alginate and ADA

For the irreversible coupling of the peptides to ALG and ADA, the well-known carbodiimide reaction was used, as described by Rowley et al.^{48 (link)}. In this regard, peptides were covalently coupled to ALG by reacting their amine groups with the carboxylic acid groups of ALG or ADA, which results in an irreversible amide bond formation (Fig. 1B and C). In general, a monomer activation of 5% was used, which corresponds to a ratio of 1 : 20 EDC to ALG monomers. EDC·HCl was stabilized with Sulfo-NHS in a ratio of 2 : 1. ALG-Peptide A, ALG-Peptide B, ALG-Peptide C and ALG-Peptide ABC with a theoretical degree of substitution of 0.25% was synthesized using Peptide A (M_W: 810.92 g mol⁻¹), Peptide B (M_W: 1105.22 g mol⁻¹) Peptide C (M_W: 744.95 g mol⁻¹) shown in Table 1. For the preparation of ALG-Peptide ABC, each peptide was coupled separately to ALG by adding a Peptide A, Peptide B and Peptide C containing solution subsequently after each other to the ALG solution aiming for a final peptide substitution degree of 0.75% (3 × 0.25% per peptide). Briefly, 1 g ALG was dissolved in 100 ml 0.1 M MES/0.3 M NaCl buffer at pH 6.5.
This buffer was produced by adding 21.33 g MES monohydrate and 17.53 g NaCl to 1 l ultra-pure water. The pH value was adjusted using a 1 M NaOH solution. To the dissolved ALG solution, 48.14 mg EDC·HCl and 24.07 mg Sulfo-NHS dissolved each in 1 ml MES buffer were added. The solution was allowed to stir for 15 min. Then, the desired amount of peptide dissolved in 1 ml buffer solution was added (see Table 1) and the solution was stirred for a further 24 h. Then, all products were filled into dialysis tubes (molecular weight cut off: 6 kDa–8 kDa) and dialyzed for 3 days against 6 l of ultra-pure water with daily water changes. After dialysis, all compounds were frozen at −20 °C and then lyophilized, respectively. The same procedure was applied for ADA which has a lower molecular weight compared to ALG, because the sodium ions being present in ALG were removed during the purification step of ADA. Therefore, different quantities of the reactants were needed: For 1 g ADA, 54.42 mg EDC·HCl and 30.82 mg Sulfo-NHS were used for the synthesis of ADA-Peptide A, ADA-Peptide B, ADA-Peptide C and ADA-Peptide ABC with a theoretical degree of substitution of 0.25% using the same method and the Peptide A, Peptide B and Peptide C (see Table 1).
For ALG-Peptide ABC and ADA-Peptide ABC all three peptides with a respective degree of substitution of 0.25% for each peptide and a final peptide concentration of 0.75% per gram ADA were used.