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Solid-phase Synthesis And Purification For Fusukang

Posted on:2007-03-03Degree:MasterType:Thesis
Country:ChinaCandidate:J ZhaoFull Text:PDF
GTID:2121360212480387Subject:Biochemical Engineering
Abstract/Summary:PDF Full Text Request
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%.
Keywords/Search Tags:solid-phase peptide synthesis (SPPS), Fusukang, convergent synthesis, ion-exchange chromatography (IEC), reversed-phase high performance chromatography (RP-HPLC)
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