N-Methylation

Amino acid derivatives were N-methylated using the method described by Miller et al.⁴⁷. This process can be divided into three steps: protection and activation with o-nitrobenzensulfonyl chloride (o-NBS), deprotonation and methylation, and o-NBS removal. This method is not suitable for the N-alkylation of the first amino acid anchored onto the resin. Hence, the commercially available Fmoc-(Nα-Me)-L-His(Trt)-OH was employed as first coupled amino acid for the synthesis of H-HA(NMe)I(NMe)YPR(NMe)H-NH₂ and Cf-HA(NMe)I(NMe)YPR(NMe)H-NH₂. To perform the protection, o-NBS (3 eq.) and collidine (5 eq.) in DCM were added to the resin and left for 60 min. This step was repeated once and the completion of the reaction was monitored by the ninhydrin test. Methyl p-nitrobenzensulfonate (4 eq.) and 7-methyl-1,5,7-triazabicyclo[4.4.0]dec-5-ene (3 eq.) in DMF were added to the resin and left for 30 min. This step was repeated once. To remove o-NBS, β-mercaptoethanol (10 eq.) and DBU (5 eq.) in DMF were added to the resin and left to react for 10 min under N₂ atmosphere. This operation was repeated once for 40 min.