Solid Phase Synthesis Of Thymosin α1

Solid Phase Peptide Synthesis(SPPS) was a key technology of modern protein chemistry,and SPPS was of great influence and significance on the molecule biochemistry and gene engineering． The article firstly made a brief introduction of SPPS．Fmoc quantitation method , Salicylaldehyde method and IR method had been firstly studied and compared．Finally,we choosed Fmoc quantitation method for the quantitation of the synthesis efficiency between C-terminal amino acid and HMP resin．Thymosinα1 was firstly isolated from calf thymus by Goldatein et al in 1977．It was an acidic peptide with an isoelectric point of 4.2,molecular weight of 3108,its sequence was:Ac-Ser-Asp-Ala-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-AsnThymosinα1 had been demonstrated to be a potent immunologically active thymix polypeptide．Now clinically,it was mainly used for Heptitis B and had a broad market in the world． Through the comparison between several productive methods of Thymosinα1,we choosed　HMP resin and Fmoc-amino acids to synthesis Thymosinα1 on ABI433A peptide synthesizer．The synthesis included four stages:1．Synthesis of Thymosinα1-HMP resinThymosinα1-HMP resin was synthesized on ABI433A peptide synthesizer using FastMoc 1.0mmol chemistry．According to the mass increase of HMP resin,the synthesis efficiency reached 81.9%．2．Cleavage of Thymosinα1-HMP resinThe below cleavage cocktail:95% TFA/2.5% TES/2.5% H₂O(V/V/V) could got the best result．The cleavage could finished in 1.5h under 20℃．3．Purification of Thymosinα1The purification of Thymosinα1 was carried on AKTA Explore 100 ,with C18 as the purification column,Eluent A, 25mmol NaAc in water ,pH 7.1,Eluent B,acetonitrile,detection at 220nm．After purification,the purity of Thymosinα1 reached 95% ．4．Characterization of Thymosinα1The purity of the last Thymosinα1 reached 95.53% by HPLC．The mass spectrum gived the triple charge (M+3)3+ 1037.8 and double charge (M+2)2+ 1555.0．Result of amino acid analysis was the same to the theoretical value．...