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目的研究α-芋螺多肽Eb1.6的制备工艺,为其临床前实验奠定基础。方法采用Fmoc保护氨基酸,以Rink树脂为固相载体,N,N-二异丙基碳二酰亚胺(DIC)/1-羟基苯并三唑(HOBt)为缩合剂,手工固相法规模合成目标肽。线性肽折叠采用空气氧化法,将线性肽按0.2 mg/ml的浓度溶于0.1 mol/L NH4HCO3缓冲液中,室温下搅拌16～24 h。折叠结束后,加入Amberlite XAD16大孔吸附树脂富集,树脂用乙醇浸洗,旋转蒸发浓缩得折叠后粗品,然后进行反相纯化。结果 Eb1.6线性肽的粗产率为94.6%,Eb1.6折叠肽粗品的回收率为82.3%,粗品经C18反相纯化后纯度高于98.5%。结论以DIC/HOBt为缩合剂合成Eb1.6的纯度及产率均较高,Eb1.6折叠后用吸附树脂富集,乙醇浸泡再释放多肽,可实现缓冲体系及树脂的反复循环,降低了成本。
Objective To study the preparation of α-taro polypeptide Eb1.6 and lay a foundation for its preclinical experiments. Methods Fmoc was used to protect the amino acids. Rink resin was used as the solid phase carrier and N, N-diisopropylcarbodiimide (DIC) / 1-hydroxybenzotriazole (HOBt) The target peptide is synthesized. Linear peptide folding using air oxidation, the linear peptide at a concentration of 0.2 mg / ml was dissolved in 0.1 mol / L NH4HCO3 buffer, stirring at room temperature for 16 ~ 24 h. After folding, Amberlite XAD16 macroporous adsorption resin was added, the resin was washed with ethanol, concentrated by rotary evaporation to give a folded product, and then subjected to reverse phase purification. Results The crude yield of Eb1.6 linear peptide was 94.6%, the recovery of crude product of Eb1.6 folded peptide was 82.3%, and the purity of crude product was higher than 98.5% after reversed-phase C18 purification. Conclusion The purity and yield of Eb1.6 synthesized by DIC / HOBt as condensing agent are both high. Eb1.6 is folded and adsorbed on resin, then ethanol is soaked to release the polypeptide, which can reduce the repetition of buffer system and resin. cost.