Umeno Tomohiro, Fujihara Moeka, Matsumoto Shota, Iizuka Naoko, Usui Kazuteru, Karasawa Satoru
Faculty of Pharmaceutical Sciences, Showa Pharmaceutical University, 3-3165 Higashi-Tamagawagakuen, Machida 194-8543, Japan.
Anal Chem. 2023 Oct 24;95(42):15803-15809. doi: 10.1021/acs.analchem.3c03350. Epub 2023 Oct 13.
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 p value was carefully tuned, was deprotonated by amines, allowing the appearance of a yellow color. A good linear relationship ( = 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.
固相肽合成（SPPS）是肽合成的一项重要技术。半个世纪以来，人们开发了许多胺检测方法来监测SPPS过程中的偶联反应。尽管做出了这些努力，但据我们所知，尚未开发出一种无损且定量的比色法。在此，我们基于电子供体-受体型染料的盐酸盐与树脂上氨基之间的酸碱反应，开发了第一种定量且无损的比色胺检测方法。在该方法中，由精心调节p值的染料组成的无色盐酸盐溶液被胺去质子化，从而呈现出黄色。证实了有色溶液的吸光度与胺基数量之间具有良好的线性关系（ = 0.999）。对于所有测试的蛋白质ogenic和非蛋白质ogenic氨基酸，我们实现了定量比色分析，相对标准偏差较小（RSD < 3.6%）。此外，在使用凯氏试验实际合成含有不可检测氨基酸的八肽过程中，我们的胺检测能够详细监测偶联反应，使得粗品形式的肽比使用凯氏试验获得的肽纯度显著更高。
