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Keywords:

  • cleavage;
  • diketopiperazines;
  • N-methylamino acids;
  • N-methylated peptides;
  • N-methylpeptides;
  • solid-phase synthesis

Abstract:  A library of peptides required for a project investigating the factors relevant for blood–brain barrier transport was synthesized on solid phase. As a result of the high N-methylamino acid content in the peptides, their syntheses were challenging and form the basis of the work presented here. The coupling of protected N-methylamino acids with N-methylamino acids generally occurs in low yield. (7-azabenzotriazol-1-yloxy)-tris(pyrrolidino)phosphonium hexafluorophosphate (PyAOP) or PyBOP/1-hydroxy-7-azabenzotriazole (HOAt), are the most promising coupling reagents for these couplings. When a peptide contains an acetylated N-methylamino acid at the N-terminal position, loss of Ac-N-methylamino acid occurs during trifluoroacetic acid (TFA) cleavage of the peptide from the resin. Other side reactions resulting from acidic cleavage are described here, including fragmentation between consecutive N-methylamino acids and formation of diketopiperazines (DKPs). The time of cleavage is shown to greatly influence synthetic results. Finally, high-performance liquid chromatography (HPLC) profiles of N-methyl-rich peptides show multiple peaks because of slow conversion between conformers.