4.2. General Procedure for the Solid-Phase Synthesis of Peptides

Peptides were synthesized manually by the solid-phase method using a standard Fmoc/^tBu strategy. A Fmoc-Rink-MBHA resin (0.55 mmol/g) was used for the synthesis of BP16, a Fmoc-Rink-ChemMatrix resin (0.66 mmol/g) for the synthesis of the conjugate BP358 (flg15-BP16), and a PAC-ChemMatrix resin (0.66 mmol/g) for the synthesis of flg15 and the conjugate BP359 (BP16-flg15) (Company, City, State Abbrev. if USA or Canada, Country). Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Phe-OH, Fmoc-Ile-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Gln(Trt)-OH, Fmoc-Arg(Pmc)-OH, Fmoc-Ser(^tBu)-OH, Fmoc-Asp(O^tBu)-OH, and Fmoc-Asn(Trt)-OH were used as amino acid derivatives. The coupling of the first amino acid (5 equiv.) onto the PAC-derivatized resins was performed in the presence of N,N’-diisopropylcarbodiimide (DIC) (5 equiv.), 4-dimethylaminopyridine (DMAP) (0.5 equiv.), and N,N’-diisopropylethylamine (DIEA) (1 equiv.) in N,N-dimethylformamide (DMF) at room temperature for 2 h while stirring. This treatment was repeated twice and, then the resin was washed with DMF (6 × 1 min) and CH₂Cl₂ (3 × 1 min) and dried with diethyl ether (3 × 2 min). The completion of the coupling was checked using a Fmoc test. Then, the resin was acetylated with acetic anhydride/pyridine/CH₂Cl₂ (1.35:1.35:18, 2 × 30 min) followed by washes with CH₂Cl₂ (3 × 2 min), DMF (3 × 2 min), MeOH (2 × 2 min), CH₂Cl₂ (2 × 2 min), and DMF (6 × 1 min). Peptide elongation was carried out through sequential Fmoc removal and coupling steps of the corresponding Fmoc-protected amino acid. Fmoc group removal was performed with piperidine/DMF (3:7, 2 + 10 min). Couplings of the Fmoc-amino acids (4 equiv.) were mediated by ethyl 2-ciano-2-(hydroxyimino)acetate (Oxyma) (4 equiv.), and DIC (4 equiv.) in DMF at room temperature for 1 h under stirring. The completion of the couplings was checked using the Kaiser test [59]. After each coupling and deprotection step, the resin was washed with DMF (6 × 1 min) and CH₂Cl₂ (2 × 1 min). Once the peptidyl sequence was completed, the resin was treated with piperidine/N-methyl-2-pyrrolidinone (NMP) (3:7, 2 + 10 + 10 min), washed with NMP (6 × 1 min), CH₂Cl₂ (3 × 1 min), and diethyl ether (3 × 2 min), and air-dried. Finally, the resulting resin was treated with TFA/H₂O/triisopropylsilane (TIS) (95:2.5:2.5) for 2 h at room temperature. Following TFA evaporation and diethyl ether extraction, the crude peptide was dissolved in H₂O, lyophilized, purified with a CombiFlash, analyzed by HPLC, and characterized by ESI-MS and HRMS.