【作者】 赵炯；

【导师】 甘一如；

【作者基本信息】 天津大学 ， 生物化工， 2006， 硕士

【摘要】 本文首先研究了36肽扶素康的Fmoc法固相化学合成。系统地研究了扶素康的Fmoc固相逐步化学合成和Fmoc固相片断缩合化学合成。Fmoc固相逐步化学合成的工艺条件为:以DMF为反应溶剂;HBTU/HOBt/DIEA为缩合剂;王树脂为固相载体;TFA/p-甲基苯酚/EDT/水(97/0.25/0.25/2.5,V/V)为切割条件,保护氨基酸2倍过量进行缩合反应。固相片断缩合化学合成扶素康中,建立了合成全保护多肽片段的合成方法:以2-氯三苯甲基树脂和王树脂为固相载体;HBTU/HOBt/DIEA为缩合剂;DMF为反应溶剂,HOAc/TFE/ DCM(1:2:7,体积比)为切割条件。在片段缩合中,系统研究了多肽片断的分段策略、片断缩合溶剂、片断缩合剂对片断缩合法合成扶素康质量的影响,确立了优化的片段缩合工艺条件为:将扶素康按残基1~10,11~20,21~25,26~36分作4段;以王树脂为固相载体;HATU/HOAt/DIEA为缩合剂(4倍过量);DMF/DCM(1:1)为反应溶剂;每步加入的全保护肽段为1.5倍过量;缩合及脱保护完成后以DCM为洗涤溶剂洗涤5次,进行固相片段缩合反应。该条件下合成的扶素康,粗品收率达67.31%,按标准曲线定量分析其纯度达38.75%。通过对扶素康粗品的分离纯化研究,确立了纯化工艺路线为:先用离子交换层析进行初步纯化,再以反相高效液相制备层析进行最终纯化。经离子交换层析后产品的纯度提高到78%,收率为55%;最终纯化结果纯度达95%。两步纯化的总收率为35%。更多还原

【Abstract】 In this thesis, Fmoc solid-phase synthesis of Fusukang was studied firstly, including Fmoc solid-phase synthesis by both the stepwise(SPPS) and the convergent approach. The SPPS methodology utilizes DMF as the coupling solvent, HBTU/HOBt/DIEA as the coupling reagent, Wang resin as solid supporter, TFA /p-methyl phenol/EDT/H2O (97:0.25:0.25:2.5) as the cleavage condition, with 2 equiv. molar excess of protected amino acid. In the Fmoc solid-phase convergent synthesis of Fusukang, the protected peptide segments were synthesized by solid-phase peptide methodology using 2-Chlorotrityl resin and Wang resin as the solid supporter; DMF as the coupling solvent; HBTU as the coupling reagent, HOAc/TFE/DCM (1:2:7,v/v) as the cleavage condition, with 2 fold molar excess of protected amino acid. As for the solid-phase convergent synthesis reaction, the effects of convergent protocol, coupling solvents, and coupling reagents on the yield of Fusukang were systemically studied, and the protocol of condensation process was optimized, i.e. using the sequential condensation of four protected peptide segments(1~10, 11~20,21~25,26~36) with 1.5-fold molar excess of protected peptide segments in solvent; HATU/HOAt/DIEA (4 equiv) as the coupling reagent; mixture DMF/DCM(1:1,v/v) as the coupling solvent; TFA/p-methyl phenol/EDT/H2O (97:0.25:0.25:2.5) as the cleavage condition, washing resin 5 times with DCM before and after each reaction. The yield of crude Fusukang was 67.31% , and the purity was 38.75%In addition, the isolations and purifications of crude Fusukang were studied, and the optimal technology was established: the crude product was purified by ion-exchange chromatography (IEC), and then was completely purified by reversed-phase high performance liquid chromatography (RP-HPLC). The purity after IEC was 78% and yield was 55%, and the total yield after RP-HPLC was 35% with the purity of 95%.更多还原