Peptide synthesis

The synthesis of IN analogs was performed according to the solid phase approach using standard Fmoc methodology in a manual reaction vessel and automated microwave synthesizer (Wang et al., 1989; Malik et al., 2010). The first amino acid, N^α-Fmoc-Xaa-OH (N^α-Fmoc-Asp(OtBu)-OH, N^α-Fmoc-Glu(OtBu)-OH, N^α-Fmoc-His(N(_im)trityl(Trt))-OH, N^α-Fmoc-Trp(Boc)-OH, N^α-Fmoc-Ala-OH, N^α-Fmoc-Leu-OH, N^α-Fmoc-Val-OH, N^α-Fmoc-Lys(Boc)-OH, N^α-Fmoc-Cys(Trt)-OH, N^α-Fmoc-Met-OH, N^α-Fmoc-Ser(tBu)-OH, was linked on to the Rink resin (100–200 mesh, 1% DVB, 0.59 mmol/g) previously deprotected by a 25% piperidine solution in DMF for 30 min. The following protected amino acids were then added stepwise. Each coupling reaction was accomplished using a three-fold excess of amino acid with HBTU (3 eq.) and HOBt (3 eq.) in the presence of DIEA (6 eq.). The N^α-Fmoc protecting groups were removed by treating the protected peptide resin with a 25% solution of piperidine in DMF (1 × 5 min and 1 × 25 min). The peptide resin was washed three times with DMF and the next coupling step was initiated in a stepwise manner. The peptide resin was washed with DCM (3 ×), DMF (3 ×), and DCM (3 ×), and the deprotection protocol was repeated after each coupling step. In addition, after each step of deprotection and after each coupling step, Kaiser test was performed to confirm the complete removal of the Fmoc protecting group, respectively, and to verify that complete coupling has occurred on all the free amines on the resin. The N-terminal Fmoc group was removed as described above, and the peptides were acetylated adding a solution of Ac₂O/DCM (1:3) shaking for 30 min. Finally the peptides were released from the resin with TFA/iPr₃SiH/H₂O (90:5:5) for 3 h. The resin was removed by filtration, and the crude peptide was recovered by precipitation with cold anhydrous ethyl ether to give a white powder and then lyophilized.