Test for Racemization in Model Peptide Synthesis by Direct Chromatographic Separation of Diastereomers of the Tetrapeptide Leucylalanylglycylvaline

The peptide Leu-Ala-Gly-Val has been developed as a test for racemization in model peptide synthesis. The single D-amino acid diastereomers, L-Leu-D-Ala-Gly-L-Val and D-Leu-L-Ala-Gly-L-Val, were separated from one another and from the all L-amino acid tetrapeptide on the standard amino acid analyzer 0.9 X 58 cm column of sulfonated polystyrene resin with 0.2 N sodium citrate buffers. Chromatography at pH 3.49 was chosen as the standard condition for separating the diastereomers. The color yields of the diastereomers were similar to that of valine. The determination of the D-amino acid-containing diastereomers was accurate above 0.1% for a standard load of 4 μmol of the tetrapeptide. The limit of detection was less than 0.01% for a 12-μmol load. The analysis was applied to the crude products of stepwise solid phase syntheses; no D-amino acid-containing diastereomers were detected (<0.02%).