Lability of N-alkylated peptides towards TFA cleavage

Trifluoroacetic acid (TFA) is a common reagent in both solid-phase and solution peptide synthesis. It is used for the deprotection and/or cleavage of the synthesized peptide from the resin. The use of TFA under these standardized conditions is thought to be sufficiently mild, thereby preventing degradation of the desired product. However, peptides of the general structure R1-(N-alkyl X1)-X2-R2 are hydrolyzed by standard TFA solid-phase peptide synthesis (SPPS) cleavage/deprotection conditions providing fragments R1-(N-alkyl X1)-OH and H-X2-R2. The fragmentation is observed during a TFA cleavage both from the resin and in solution. The hydrolysis is proposed to proceed via an oxazolone-like intermediate in which equilibration of the chiral center of the N-alkylated residue occurs. This mechanism is supported by H/D exchange as observed MS and NMR in conjunction with HPLC.