Analysis of racemization during “Standard” solid phase peptide synthesis: a multicenter study

Abstract

Publisher Summary Evaluation of the degree to which racemization occurs in peptides produced by core laboratories is very appropriate. The Peptide Synthesis Research Committee of the Association of Biomolecular Resource Facilities (ABRF) conducts anonymous studies to evaluate the ability of ABRF member laboratories to synthesize and characterize test peptides. The committee prepared for the 1996 study by designing and testing appropriate short peptides that should be straightforward to synthesize. 48 laboratories participated in this study, submitting 53 samples for analysis. In addition to the results of this multicenter study, this chapter also discusses a summary of the committee's preparatory experiments. During the preparatory phase of this study, it was found that handling and analysis of a hexapeptide with two potential sites of racemization were too problematic to be useful as a model for this multicenter study. A hexapeptide susceptible to racemization at a single His residue was subsequently designed, tested, and found suitable for the study. This synthetic peptide was requested from each of the core laboratories for evaluation by the committee. Coded samples of unpurified peptides were characterized by amino acid analysis (AAA), AAA following reaction with Marfey's reagent, high pressure liquid chromatography (HPLC), electrospray ionization mass spectrometry (ESI-MS), matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), and enzymatic digestion by carboxypeptidase A (CPA) followed by MALDI-MS. The overall results from this study indicate that racemization during peptide assembly is not a serious problem in most participating laboratories. Nevertheless, it is evident that laboratories can produce peptides that have predominantly composed of the desired product, yet exhibit unacceptably high levels of racemization.