Cleavage, Deprotection, and Isolation of Peptides after Fmoc Synthesis

Abstract

Cleavage and deprotection is one of the most crucial steps in peptide synthesis. The treatment of a peptidyl-resin with a cleavage cocktail is not one simple reaction, but a series of competing reactions. Unless suitable reagents and reaction conditions are selected, the peptide can be irreversibly modified or damaged. The goal of cleavage/deprotection is to separate the peptide from the support while removing the protecting groups from the side-chains. This should be done as quickly as possible to minimize the exposure of the peptide to the cleavage reagent. The peptide is then recovered from the reaction mixture and analyzed. This technical bulletin describes procedures for cleavage of peptides from solid supports assembled via Fmoc/tBu-based strategies. There are numerous procedures in the literature which describe a variety of cleavage and deprotection methods for peptides synthesized with an Fmoc/tBu strategy.2-4. Most of these techniques are TFA-based, and they differ primarily in the final concentration of TFA, types of scavengers used, and reaction times. Some of the variablity in methods reflects the preferences of individual labs, but it is mainly dictated by the amino acid composition of the peptide. Some amino acids have potentially reactive side-chains which generate carbonium ions and other reactive species during TFA cleavage of the peptide from the support. Therefore, the appropriate scavengers and reaction conditions must be chosen to minimize modification or destruction of the sensitive amino acids. Since prolonged treatment with the cleavage acid is needed to remove some protecting groups, scavengers must be used to protect the reactive sites of the peptide during extended reaction times. The 21 naturally-occurring Fmoc amino acids and their side-chain protecting groups are listed in Table 1.