芋螺多肽Eb1.6的制备工艺研究

Preparation method of conopeptide Eb1.6

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 probe a method for large-scale synthesis of α-conopeptide Eb1.6 in order to provide a large quantity of α-conopeptide Eb1.6 for preclinical trial.Methods Starting with Rink resins and Fmoc-protected amino acids,linear Eb1.6 was synthesized by solid-phase peptide synthesis method（SPPS） in which DIC/HOBt was used as a coupling agent.The linear peptide（0.2 mg/ml） was folded in 0.1 mol/L NH4HCO3 buffer at room temperature for 16 to 24 h.The folded peptide was then absorbed by the Amberlite XAD16 resin,and desorbed by alcohol.Finally,the crude Eb1.6 was concentrated by rotary evaporation and purified by reversed-phase HPLC.Results The crude yield of linear peptide was 94.6%,and the recovery rate of folded Eb1.6 by Amberlite XAD16 was 82.3%.Eb1.6 with a purity of more than 98.5% was obtained after the purification of RP-HPLC.Conclusion The purity and yield of Eb1.6 are high when DIC/HOBt is used as a coupling agent.The absorption of Eb1.6 with Amberlite XAD16 resin and desorption by alcohol are a highly efficient method for concentration of folded Eb1.6 at a low cost.