Coupling Fmoc-His(Trt)-OH in solid-phase peptide synthesis is frequently accompanied by significant racemization. Histidine is among the most susceptible amino acid residues inclined to racemize in peptide syntheses. In this study, the His racemized impurity could not be effectively purged by the applied chromatographic purification. Consequently, a Taguchi design of experiment (DOE) was first applied to screen the critical process parameters affecting the histidine racemization. Optimization of the DOE is subsequently performed to search for the optimum. The derived DOE models reveal that the conditions of Fmoc-His(Trt)-OH carboxylate pre-activation prior to its coupling to growing peptide chains are critical for the subject histidine racemization. Intensive Fmoc-His(Trt)-OH pre-activation stimulates this side reaction. On the other hand, without the amino acid pre-activation, by adopting the in situ Fmoc-Xaa-OH activation, another side reaction, that is, peptide N^α endcapping by N,N-diisopropylcarbodiimide, is boosted. A conflicting relationship between histidine racemization and peptide N^α endcapping has been detected through the DOE investigation. Significant models are established for the histidine racemization and peptide N^α endcapping, and reconciliation to balance these two side reactions is accomplished on this basis.

中文翻译：

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通过实验设计抑制 Fmoc-His(Trt)-OH 消旋化和 Nα-DIC 封端在固相肽合成中的作用及其对肽合成中氨基酸活化策略的意义

在固相肽合成中偶联 Fmoc-His(Trt)-OH 经常伴随着显着的外消旋化。组氨酸是在肽合成中倾向于外消旋的最敏感的氨基酸残基之一。在这项研究中，His 外消旋杂质不能通过应用色谱纯化得到有效净化。因此，首先应用田口实验设计 (DOE) 来筛选影响组氨酸外消旋化的关键工艺参数。随后执行 DOE 的优化以搜索最优值。衍生的 DOE 模型表明，Fmoc-His(Trt)-OH 羧酸盐在其与生长肽链偶联之前的预活化条件对于主题组氨酸消旋化至关重要。强化 Fmoc-His(Trt)-OH 预激活会刺激这种副反应。另一方面，N ,N-二异丙基碳二亚胺的N ^α封端得到促进。通过 DOE 调查发现了组氨酸消旋化和肽N ^α封端之间的矛盾关系。建立了组氨酸消旋和肽Nα封端的重要模型，^并在此基础上完成了平衡这两个副反应的调节。