Cysteine-Containing Peptide Synthesis

MBHA resin (0.79 meq/g; Peptides International) was swelled in DMF (8–10 mL) with DIEA (650 μL). This was then coupled with Boc-Leu (2 mMol; 4 mL 0.5 M HBTU; 347 μL DIEA; 40 min.), washed with DMF then DCM. Peptidyl-resin Boc deprotection was achieved with 100% Trifluoroacetic acid (TFA; 5 min., ×2; Fisher Scientific), then flow-washed with DMF (1 min., ×2), followed by coupling with 3,3′dithiodiprionic acid (2 mMol; 8 mL of 0.5 M HBTU in DMF; 1 mL of DIEA; 40 min.). To counteract ester formation the resin-linker was washed with DMF and treated with ethanolamine (650 μL; Alfa Aesar) and DIEA (200 μL; 5 mL; in DMF; 40 min.). This was then flow-washed with DMF (1 min., ×2) and treated with 2-mercaptoethanol (Sigma-Aldrich) (650 μL; 5mL of DMF; 100μL DIEA; 60 min.). Upon peptidyl-resin reduction, the resin was flow-washed with DMF (1 min., ×2) and coupled with Boc-Cys(4-MeOBzl) (40 min.), as described above. The resulting pre-loaded Boc-Cys(4-MeOBzl) thioester linker resin (Boc-Cys(4-MeOBzl)-MPAL resin) was then used in the production of N-terminal peptide fragments for NCL at approximately 0.5 mMole scale [29 (link)].