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

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.