In a fritted syringe, the resin (125 mg, 200 µmol, 1.00 equiv.) was swollen in 2 mL of methylene chloride by shaking at 21 °C for at least 30 min. The solvent was removed. (2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-[4-[(2-methylpropan-2-yl)oxy]phenyl]propanoic acid (368 mg, 800 μmol, 4.00 equiv) and N-ethyl-N-propan-2-ylpropan-2-amine (103 mg, 139 μL, 800 μmol, 4.00 equiv) were dissolved in 2 mL of 1-methylpyrrolidin-2-one and added to the resin. After shaking for at least 4 h, the solvent was removed and the resin was washed with dimethylformamide, methanol and methylene chloride. Deprotection of the Fmoc-protecting group was carried out using a solution of 20% piperidine in dimethylformamide. 3 mL of this mixture were added to the resin and shaken for at least 5 min. The deprotection cocktail was removed and the procedure was repeated twice. The resin was washed as described. Subsequently, (2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-5-[(2-methylpropan-2-yl)oxy]-5-oxidanylidene-pentanoic acid (340 mg, 800 μmol, 4.00 equiv) and 1-hydroxybenzotriazole;hydrate (123 mg, 800 μmol, 4.00 equiv) were dissolved in 2 mL of 1-methylpyrrolidin-2-one. N,N'-di(propan-2-yl)methanediimine (101 mg, 125 μL, 800 μmol, 4.00 equiv) was added. The reaction mixture was directly added to the resin and shaken for at least 4 h. The solvent was removed and the resin was washed as described. Deprotection and coupling with (2R)-1-(9H-fluoren-9-ylmethoxycarbonyl)proline (270 mg, 2.00 mL, 800 μmol, 0.400M, 4.00 equiv) and (2S)-2-(9H-fluoren-9-ylmethoxycarbonylamino)-3-methylpentanoic acid (283 mg, 800 μmol, 4.00 equiv) were repeated. The resin was washed with methylene chloride and 2.5 mL of a solution of 33% 1,1,1,3,3,3-hexafluoro-2-propanol in methylene chloride were added. The resin was incubated with the cleavage cocktail for 16 h. The solvent was collected and the resin was flushed with methylene chloride. The solvent was removed under air stream resulting in the crude linear precursor (2S)-2-((2S)-2-((2R)-1-((2S)-2-amino-3-methylpentanoyl)pyrrolidine-2-carboxamido)-5-(tert-butoxy)-5-oxopentanamido)-3-(4-(tert-butoxy)phenyl)propanoic acid (110 mg, 174 μmol, 87% yield). The linear tetramer was cyclized without further purification (see reaction: https://www.chemotion-repository.net/pid/13201).
