Hardeman K, Samyn B, Van der Eycken J, Van Beeumen J
University of Gent, Department of Biochemistry, Physiology and Microbiology, Belgium.
Protein Sci. 1998 Jul;7(7):1593-602. doi: 10.1002/pro.5560070713.
An improved chemical method, capable of derivatizing all natural amino acids to their corresponding thiohydantoins, is described. This involves activation by acetyl chloride in TFA followed by derivatization with ammonium thiocyanate. Possible interference of reactive side chains was investigated by reacting N-acetylamino acids as well as several peptides with propionyl chloride instead of acetyl chloride. The products were characterized by PDMS mass spectrometry and 1H-NMR. This chemical method allows, for the first time, complete derivatization of N-acetylproline to proline thiohydantoin. Applying this chemistry to peptides with a C-terminal proline, the yields for formation of proline thiohydantoin were found to be up to 60%, depending on the peptide sequence. The previous inability to derivatize C-terminal proline to thiohydantoin was thought to stem from the fact that proline cannot form the oxazolonium ion required for efficient reaction with the thiocyanate ion. However, we have found mass spectrometric evidence for the existence of a proline oxazolonium ion, under basic as well as under acidic conditions. This improvement in derivatization of C-terminal amino acids including proline is a major step forward in the development of a general chemical C-terminal sequencing method that permits the C-terminal sequence analysis of proteins of any amino acid composition.
本文描述了一种改进的化学方法,该方法能够将所有天然氨基酸衍生化为相应的硫代乙内酰脲。这包括在三氟乙酸中用乙酰氯进行活化,然后用硫氰酸铵进行衍生化。通过使N - 乙酰氨基酸以及几种肽与丙酰氯而非乙酰氯反应,研究了反应性侧链可能产生的干扰。产物通过聚二甲基硅氧烷质谱法和1H - NMR进行表征。这种化学方法首次实现了将N - 乙酰脯氨酸完全衍生化为脯氨酸硫代乙内酰脲。将这种化学方法应用于具有C末端脯氨酸的肽时,发现脯氨酸硫代乙内酰脲的形成产率高达60%,具体产率取决于肽序列。以前无法将C末端脯氨酸衍生化为硫代乙内酰脲被认为是由于脯氨酸不能形成与硫氰酸根离子有效反应所需的恶唑鎓离子。然而,我们已经发现了在碱性和酸性条件下都存在脯氨酸恶唑鎓离子的质谱证据。包括脯氨酸在内的C末端氨基酸衍生化方面的这一改进是通用化学C末端测序方法发展中的一个重大进展,该方法允许对任何氨基酸组成的蛋白质进行C末端序列分析。