Development of a new highly sensitive D,L-discriminating amino acid analyzer

我们致力于开发一种高灵敏度的方法，用于从肽的C-末端连续测定D，L-氨基酸。我们发展了一种以C-端肽键的选择性水解为关键步骤的方法。该方法包括：（1）C-端羧酸的乙酯化，（2）酯的NaBH_4还原，（3）在酸性条件下通过N*O酰基迁移选择性水解末端酰胺键，（4）从肽和单-N-MNB羰基化中分离氨基醇，（5）单-N-MNB羰基化衍生物的HPLC分析。所谓的“新一代试剂”（^+）-MNB羧酸在氨基醇的D，L-分析中比“老”（^+）-TBMB羧酸更上级。然而，单-N-MNB-羰基化方法需要真实样品进行明确的鉴别。因此，我们接下来研究了通过N，O-二-O-（S）-TBMB-羰基化氨基醇的激子-CD监测方法，通过正相柱色谱法清楚地分离，但它们的立体化学不能通过CD监测确定，因为它们没有显示特定立体异构体的特征激子CD光谱。最后，我们发现，通过N-邻苯二甲酰-O-（S）-TBMB-羰基化反应，氨基醇可以用在线HPLC-激子CD分析法进行测定。因此，我们建立了一种新的灵敏度和选择性的测定氨基酸D，L的方法。

We have focused on the development of a highly sensitive method for the sequential determination of D,L-amino acid from the C-terminus of peptides. We have developed a method which involves the selective hydrolysis of C-terminal psptide bond as the key step. The method is as follows, (1) ethyl esterification of the C-terminal carboxylic acid, (2) NaBH_4 reduction of the ester, (3) selective hydrolysis of the terminal amide bond under acidic conditions via N*O acyl migration, (4) separation of the resulting amino alcohol from peptide and mono-N-MNB carbonylation, (5) HPLC analysis of the mono-N-MNB carbonylated derivatives.The so-called "new generation reagent" (^+)-MNB carboxylic acid was superior in the D,L-analysis of amino alcohols than the "old" (^+)-TBMB carboxylic acid. However, the mono-N-MNB-carbonylation method needed the authentic samples for unequivocal identification. Therefore, we next studied the exciton-CD monitoring method by N,O-di-O- (S)-TBMB-carbonylated amino alcohols were clearly separated by normal phase column chromatography but their stereochemistry could not be determined by CD monitoring because they did not show the characteristic exciton CD spectra for the specific stereoisomer. Finally, we have found that the amino alcohols can be determined by the on-line HPLC-exciton CD analysis without reference asmples by N-phthaloyl-O-(S)-TBMB-carbonylation. Thus, we have developed a new sensitive and selective method for the determination of D,L of amino acids.