Quantitative and Nondestructive Colorimetric Amine Detection Method for the Solid-Phase Peptide Synthesis as an Alternative to the Kaiser Test

Abstract

Solid-phase peptide synthesis (SPPS) is an essential technique for the synthesis of peptide. For half a century, many amine detection methods have been developed to monitor coupling reactions during SPPS. Despite such efforts, to the best of our knowledge, a nondestructive and quantitative colorimetric method has not been developed. Here, we developed the first quantitative and nondestructive colorimetric amine detection method based on an acid−base reaction between HCl salt of electron donor−acceptor type dyes and amino groups on the resin. In this method, a noncolored solution of HCl salt consisting of dyes, whose pK BH+ value was carefully tuned, was deprotonated by amines, allowing the appearance of a yellow color. A good linear relationship (R 2 = 0.999) between the absorption of the colored solution and the amine group quantities was confirmed. For all tested proteinogenic and nonproteinogenic amino acids, we achieved quantitative colorimetric analysis with a small relative standard deviation (RSD < 3.6%). Furthermore, during the practical synthesis of an octapeptide containing undetectable amino acids with the Kaiser test, our amine detection allowed for detailed monitoring of the coupling reaction, resulting in a significantly purer peptide in the crude form than that obtained using the Kaiser test.