Racemization of Amino Acids in Solid-Phase Peptide Synthesis Investigated by Capillary Electrophoresis

Abstract

The rate of racemization during solid-phase peptide synthesis was studied using capillary electrophoresis and 18-crown-6 tetracarboxylic acid as chiral selector. For this purpose, the tripeptide d-Tyr-l-Lys-l-Trp as a model compound was synthesized by solid-phase peptide synthesis. A separation method based on capillary electrophoresis was developed which allowed all eight optical isomers of the tripeptide to be separated in a single run. The separation method was validated and was found to be well suited for purity analysis, with a limit of detection of 0.05% of the major compound. The method was revealed to be highly sensitive even to small variations in the buffer pH. Capillary electrophoresis was also employed to prove the enantiomeric purity of the Fmoc-protected amino acids used for peptide synthesis. A separation method based on micellar electrokinetic chromatography and γ-cyclodextrin was developed for this purpose. The formation of optical isomers during peptide synthesis was investigated in the final product without hydrolyzing the tripeptide. This strategy allowed the rate of racemization to be determined by activation of amino acids in coupling cycles and cleavage of the peptides from the resin and from side-chain protecting groups. The formation of stereoisomers could be verified and was 0.4% or less per synthesis cycle. The experimental data agreed well with theoretical considerations, showing that racemization takes place mainly at the carboxy-activated amino acid during coupling.