Synthesis and Conjugation of Peptide Intermediate

Synthesis of 6 was performed by elongation of 5 (0.75 mmol, 1 equiv.) according to Kuo et al. [13 (link)]. Conjugation of each Fmoc-protected amino acid was achieved by adding a solution of the amino acid (3 mmol, 4 equiv.) in DMF (10 ml) with HBTU (O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate; 1.15 g, 2.94 mmol, 3.9 equiv.) and DIPEA (1.04 ml, 6 mmol, 8 equiv.). The resin was agitated for 2 h. Fmoc-groups were deprotected as previously described with a 20% solution of 4-methyl piperidine in DMF. Incomplete steps were repeated. Fmoc-2-Nal-OH, Fmoc-tranexamic acid, Fmoc-Lys(ivDde)-OH, Fmoc-Glu(tBu)-OH and 4-(p-iodophenyl)butyric acid were coupled using this protocol. Deprotection of the ivDde (1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl) group with a 5% hydrazine solution in DMF provided the key intermediate 6, which was directly used without further purification.