叔丁氧羰基保护的环状氨基酸的合成及其在多肽固相合成中的应用

Synthesis of t-butyloxy carbonyl protected cyclic amino acids and its application in peptides solid synthesis

目的合成叔丁氧羰基(Boc)保护的环状氨基酸Acc5、Acc6,并将这类构型限制型氨基酸用于多肽的固相合成,考察环状氨基酸在多肽固相合成中的应用以及在强酸条件下的稳定性。方法环状氨基酸通过相应的环酮与KCN和铵盐反应得到含有氰基的环状化合物,再通过氰基的酸性水解而得到,最后在弱碱性条件下与二碳酸二叔丁酯(Boc2O)反应,得到Boc保护的环状氨基酸;通过1H NMR和ESI-MS表征其结构;采用对甲基二苯甲基氨基树脂,通过Boc、芴甲氧羰基(Fmoc)的氨基保护策略,对肽序列进行固相合成,最后在强酸HF条件下裂解,通过HPLC对粗肽进行纯度分析,ESI-MS表征其结构。结果1H NMR和ESI-MS图谱显示环状氨基酸Acc5和Acc6均为目标化合物。HPLC结果显示,得到的2个促性腺激素释放激素类似物(GnRH),粗肽纯度良好,且易于纯化;ESI-MS图谱确证肽序列结构正确。结论环状氨基酸在固相合成工艺中有很好的缩合效率,并且能够在强酸裂解条件下保持稳定。

Objective To synthesize the t-butyloxy carbonyl（Boc） protected cyclic amino acids Boc-Acc5-OH and Boc-Acc6- OH, and then use this type of conformational constrained amino acids in the solid synthesis of peptides in order to investigate their ap- plication in the solid synthesis and stability in strong acid environment. Methods With corresponding cyclic ketones reacting with KCN and ammonium salt, cyclic compounds with cyano group were obtained. Then cyclic amino acids were achieved through the acidic hydrolysis of the cyano group. By reacting with Boc20 in the weak base solution, we obtained the Boc protected cyclic amino acids which were characterized in term of relative molecular mass and structure using ESI-MS and 1 H NMR. We used solid-phase synthesis, p-methylbenzhydrylamine resin, Boc and 9-fiuorenylmethoxycarbonyl（Fmoc） synthesis strategy to prepare the peptides, and analyzed them by HPLC and ESI-MS. Results The cyclic amino acids were identified by 1H NMR and ESI-MS. Besides, we obtained two gon- adotropin-releasing hormone analogues which were characterized by ESI-MS. The crude peptides had a relatively high purity by HPLC and were easy to purify. Conclusion The cyclic amino acids have a favorable condensation efficiency in the solid synthesis technolo- gy, moreover, they can keep stable in strong acid condition.