title: Application of proteases to the identification of chiral modifications in synthetic peptides.
abstract: Racemization of amino acids during solid-phase synthesis of peptides leads to the formation of side products that are chirally modified peptides. The chiral specificity of enzymes can be exploited to identify the sites of the modifications in these impurities. One such impurity, designated X5, was isolated from the target peptide, Fel-1, and demonstrated to be an optical isomer of Fel-1 by N-terminal sequencing and mass spectrometry. A chymotryptic digest was done on the isolated X5 and Fel-1. The fragments were separated on reversed-phase high-performance liquid chromatography (HPLC). Mass spectral data on the fragment from X5, with a different retention time from the analogous fragment of Fel-1, suggested that the modification was in the N-terminal portion of the peptide. Enzymatic digestion by Asp-N protease followed by HPLC of the fragments and mass spectral analysis provided evidence that an aspartic acid at position 5 was a D-amino acid in X5, because that position was not cleaved. These results contributed to the identification of X5 as an optical isomer of Fel-1, with a D-aspartic acid replacing an L-aspartic acid normally present at position 5.
authors.affiliation: ImmuLogic Pharmaceutical Corporation,Waltham, MA, USA.
authors.forename: K M
authors.initials: KM
authors.lastname: Keating