Vydac c18 analytical column

Buffered aerial oxidation of peptide 3 was carried out according to a procedure described by Clark and colleagues [56 (link)]. Crude peptide 3 (84.5 mg, 46.8 μmol) in H₂O:MeCN 9:1 (30 mL) was added to a stirred solution of 0.1 M NH₄HCO₃ pH 8.5 (300 mL) at room temperature under a constant stream of air. Reaction progress was monitored by RP-HPLC (Grace, Columbia, MD, USA, Vydac C18 analytical column, 5–35% buffer B over 30 min) and mass spectral analysis. After 22 h, LC and MS analysis confirmed the formation of the desired peptide 4. RP-HPLC (Grace, Columbia, MD, USA, Vydac C18 analytical column, 5–35% buffer B over 30 min): t_R = 16.1 min. Mass spectrum (ESI⁺, MeCN, H₂O, TFA): m/z 903.4 [M + 2H]²⁺, ½(C₇₃H₁₁₁N₂₃O₂₅S₃) theoretical 902.9; m/z 914.4 [M + H + Na]²⁺, ½(C₇₃H₁₁₀NaN₂₃O₂₅S₃) theoretical 913.9. The reaction mixture was then acidified to pH 3 with glacial AcOH and purified by preparative RP-HPLC (Grace, Columbia, MD, USA, Vydac C18 preparative column, 5–35% buffer B over 45 min, t_R = 25.8 min). Selected fractions were combined and lyophilised to give the target peptide 4 a colourless solid (19.4 mg, >99% purity). RP-HPLC (Grace, Columbia, MD, USA, Vydac C18 analytical column, 5–35% buffer B over 30 min): t_R = 16.1 min.

Dicarba peptide 5 (0.3 mg, 170 nmol) was dissolved in TFA (0.5 mL). To the peptide solution, 2-methyl-2-butene (11 µL, 103 µmol) and bromotrimethylsilane were added (26 µL, 197 µmol). The reaction was shaken at room temperature for 15 min before removing TFA under flow of N₂. The remaining residue was taken up in ice-cold Et₂O (1 mL), and the precipitated peptide was collected by centrifugation (1 × 1 min). RP-HPLC and mass spectral analysis of the peptide supported the formation of the methionine-containing dicarba peptide 6 in quantitative conversion as a mixture of E- and Z-isomers. RP-HPLC (Grace, Columbia, MD, USA, Vydac C18 analytical column, 5–35% buffer B over 30 min): t_R = 11.4 and 11.6 min. Mass spectrum (ESI⁺, MeCN:H₂O:TFA): m/z 875.3 [M + 2H]²⁺, (C₆₉H₁₀₃N₂₃O₂₅S₃) theoretical 874.8. The reaction mixture was purified by analytical RP-HPLC (Grace, Columbia, MD, USA, Vydac C18 column, 5–35% buffer B over 30 min, t_R = 11.4 and 11.6 min). Coeluting isomer fractions were combined and lyophilised to give the target peptide 6 a colourless solid (0.1 mg, 95% purity).