Solid phase peptide synthesis

Peptides were synthesized on Rink Amide-ChemMatrix resin (PCAS Biomatrix), either using manual addition of the reagents (using a stream of dry N2 to agitate the reaction mixture) or on a Liberty Peptide Synthesizer equipped with a Discovery microwave module (CEM).

For manual solid phase synthesis, typical cycles were: (a) Fmoc group deprotection with 3 mL of 20% piperidine in DMF (1 x 1 min, 2 x 8 min) and (b) coupling of 5 eq. of amino acid to the growing peptide chain with 4.9 eq. of either HATU or HOBt/HBTU and 10 eq. of DIPEA for 20 – 45 min. Double couplings were used when necessary to ensure complete acylation. Between each step, the resin was thoroughly washed with DMF. Upon completion of the synthesis, peptides were cleaved from the resin with 95% TFA, 2.5% TIS and 2.5% H₂O. The crude peptide was then precipitated with diethyl ether, dissolved in water with 0.1% TFA and analyzed via RP-HPLC. Subsequently, the peptide was purified by preparative RP-HPLC, lyophilized and stored at −20 °C.