5] Trifluoroacetic acid cleavage and deprotection of resin-bound peptides following synthesis by Fmoc chemistry

Abstract

The successful assembly of a peptide sequence represents only half the challenge of solid-phase peptide synthesis (SPPS). The side-chain protecting groups and linkers designed for fluorenylmethyloxycarbonyl (Fmoc) protocols are labile to trifluoroacetic acid (TFA). The procedure described is suitable for the various linkers cleaved by TFA, whether producing C-terminal acids or amides. Commercially available and suggested linker-resins for generating peptide acids are 4-alkoxybenzyl alcohol, 4-hydroxymethylphenoxyacetic acid (HMPA/PAB), and 2 chlorotrityl chloride. Under certain circumstances, TFA can cleave both at the linker-peptide bond and at the attachment point of the linker to the resin. Consequently, two events may occur. The free carbonium ion of the linker may alkylate Trp, with the resulting peptide exhibiting a more hydrophobic high-performance liquid chromatography (HPLC) elution behavior and a higher mass than the desired peptide. In addition, the cleavage yield may be noticeably reduced due to a reaction between a Trp or Cys residue and the linker ion while still attached to the resin. In a study of the correlation between the proximity of Trp to Arg in a sequence and extent of modification of Trp, it was found that this side reaction is greatest when Trp and Arg are separated by one residue.