Solid-phase Peptide Ligation And Analysis For Thymosin α₁

Solid-phase peptide ligation of thymosinα1 was studied in this thesis on the basis of solid-phase peptide synthesis(SPPS). First, protected peptide segment of thymosinα1 was synthesized by SPPS, then the effects of synthetic conditions on the yield of protected thymosinα1 segment were systemically studied. The optimal synthetic conditions were determined where DMF was used as the coupling solvent, HBTU as the coupling reagent, TFE, HOAc and DCM(1:2:7) as the cleavage condition. By using the optimal conditions peptide was synthesized at a rate of one amino acid condensed per 8 hours. Synthesized protected peptides were identified by mass spectrum(MS) and reversed-phase high performance liquid chromatography (RP-HPLC). The yield of peptides less than 11 amino acids was over 90% with the purity of over 93% and that of the longer ones was over 83% with the purity of over 79%.The synthesized peptide segments were ligated by solid-phase synthesis with different schemes. The optimal technique was obtained by studying the effects of coupling time, coupling solvents, coupling reagents and acetylation ways on solid-phase ligation yield. Using DMF and DCM(3:1) as the coupling solvent, HATU as the coupling reagent, two synthetic schemes with different acetylation way were applied in the thymosinα1 synthesis at a rate of one segment condensed per 20 hours. The yield of crude thymosinα1 was 67.9% with a purity of 60.7% identified by MS and RP-HPLC. Compared with SPPS, the thymosinα1 synthesized by solid-phase peptide ligation has great superiority in separation and purification.