Amino acid coupling at room temperature.

Four equivalents of Fmoc-Xaa, 5 eq of DIPEA, and 4 eq of O-benzotriazol-1-yl-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU) were added to the resin in DMF. The reaction mixture was agitated via shaking for 45 min and then drained. The resin was washed with DMF (3 times with 3 ml each) and DCM (3 times with 3 ml each). The reaction was monitored by liquid chromatography-mass spectrometry (LC-MS) and repeated until the starting material was no longer observed. For microwave conditions, a solution of 4 eq of Fmoc-Xaa, 4 eq of HBTU, and 6 eq of DIPEA in DMF was allowed to prereact for 5 min. This solution was added to the deprotected peptide on-resin and allowed to react for 10 min at 50°C under microwave heating. The solution was drained, and the resin was washed with DMF (3 times with 3 ml each) and DCM (3 times with 3 ml each). The reaction was monitored by LC-MS and repeated until the starting material was no longer observed.