Problems on Identification of Impurities

A similar problem associated with synthetic peptides of chain length 5 amino acid residues is the identification of side reactions. Those involve substitutions on imidazole-, phenol- and indole moieties of histidine, tyrosine, and tryptophane as well as conversions, transamidation, and cyclizations of aspartic and glutamic side chains. As long as structural variations are stable under the reaction conditions of an Edman degradation, they can be detected from the proper phenylthiohydantoines in combination with H-NMR- and mass spectrometry. Quantitative amino acid analyses of impure peptides after acidic total hydrolysis do not indicate those structural deviations between main product and contaminations. 

Because of this uncertainly, we try, first, to control the course of the synthesis as completely as possible during its processing. Second, we try to determine the quality of the synthetic end product on polymer by the aid of Edman degradation with quantitative exploitation of the phenylthiohydantoines obtained [161,164]. In this way contaminations of the product by false sequences can be detected in relative amounts of as small as 0.1% of the main chain. Generally we experienced purer peptides synthesized than liberated from polymer by any detachment reaction. This can be demonstrated qualitatively by the aid of thin layer chromatograms of the crude peptide products released from the support after the cleavage reaction and by end-group determinations before and after peptide detachment. 

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