Basic Research on the Regulation of Specific Degradation Reaction due to Hydrogen Radical

在本研究中，我们进行了氢辅助声解肽键的实验，得到的序列反映了带有脯氨酸信息的肽产物，并且没有任何副反应。超声作用于分析肽（ACTH18-39）的水溶液，使氧化产物M+nO （n=1-6）生成完整的肽分子M。产物由氢自由基和酸水解产生的峰对应，产物具有质量差为1 Da的酰咪酯和羧酸的c端结构。结果表明，在水溶液中，超声对肽键C-N和n- c - α键的水解和氢辅助裂解是相互竞争的。为了提高氢自由基声解产物的产率，在分析肽的水溶液中加入酚类化合物作为氢供体。在ACTH18-39的水溶液中加入1,2-苯二醇，可显著提高肽键声解水解产物的产率。结果表明，加入1,2-苯二醇作为氢供体并没有增强氢自由基引发的n - c - α键的裂解，而是增强了肽键的声解水解。相邻峰之间的质量差等于肽主链上氨基酸残基- nh - c - α h (R) - co -的质量，因此可以很容易地从光谱中确定序列。脱水、氧化、还原等副反应引起的干扰峰很少观察到。

In the present study, we performed the experiment of hydrogen assisted sonolytic hydrolysis of the peptide bond, which resulted in the sequence reflected peptide products with proline information and without any side reactions. The application of ultrasound to an aqueous solution of analyte peptide (ACTH18-39) resulted in the oxidation products M+nO (n=1-6) to an intact peptide molecule M. The peaks corresponding to the products caused by hydrogen radical and acid hydrolysis were observed, and the products were characterized by the C-terminal structures of imidate and carboxylic acid with the mass difference of 1 Da. The result obtained indicates that a hydrolytic sonolysis at peptide bond C-N and hydrogen-assisted cleavage at N-Cα bond occurs competitively by ultrasound in aqueous solutions. In order to enhance the yield of sonolytic products with hydrogen radical, phenolic compounds as hydrogen donor were added to the aqueous solution of the analyte peptide. The addition of 1,2-benzenediol to the aqueous solution of ACTH18-39 brought about a considerable enhancement of the yield of product originated from the sonolytic hydrolysis at peptide bonds. It was clear that the addition of 1,2-benzenediol as a hydrogen donor did not enhance the hydrogen radical-initiated cleavage at N-Cα bond, but the sonolytic hydrolysis of peptide bonds. The mass difference between adjacent peaks was equal to the mass of amino acid residues, -NH-CαH (R) -CO-, on the peptide backbone, so that sequence could be easily determined from the spectrum. Any interfering peaks originated from side reactions such as dehydration, oxidation and reduction were little observed.