Synthesis And Bioactivity Study On The Site-Specific PEGylated Thymopentin(10KD)

The first part of this paper conludes the exploration and study of new synthetic methods of siite-directed pegylation of thymopentin(mPEG(10KD)-TP5) on the basis of previous research and enhance the synthesis yield of site-specific pegylated thymopentin (mPEG (10kD)-TP5).To remain its bioactivity, Lys side chain amino (s-NH2) of TP5was attached to methoxy polyethylene glycol succinimidyl ester(mPEG-SPA,10kDa).All the amino acids were step-wise assembled on Fmoc-Tyr(tBu)-Wang resin by Fmoc/t-Butyl strategy. Then the wang resin were cleaved and the tBu and Boc were removed from the Tyr(tBu) and Lys(Boc) by treatment with trifluoracetic acid.In alkalescent condition, the Mono-PEGylation was targeted to the s-amine group of the lysine.The pegylated thymopentin was made by pd/c catalytic hydrogenation.purification was made by dialysis and reversed-phase high performance liquid chromatography.The target compound structure identification was made by1H-NMR spectra, MOLD-TOF-MS mass spectrometry and amino acid sequencing.The study achieved the designated pegylated thymopentin successfully and the total yield was20%, which was improving for10times in comparison with the yield reported by us.The second part of this paper conludes mouse spleen T lymphocyte proliferation induced by ConA and anti-enzymatic stability in plasma in vitro and the drug safety evaluation.Mouse spleen T lymphocyte proliferation induced by ConA in vitro was studied with different molecular weights and the different dose of pegylated thymopentin (5kDa、10kDa)in vitro.Enzymatic degradation and drug safety evaluation were also explored. Splenocyte proliferation was evaluated by MTT method.RP-HPLC was employed to evaluate the plasma half-life of TP5and peglated TP5analogues and Conventional test tube methodin vitro was employed to evaluate the hemolytic of the target product. The results showed that, splenocyte proliferation and the plasma half-life of PEGylated TP5analogues were increased in various degrees. The solution of the target product has no hemolytic and cohesion in3hours.This method is easily handing with high yield and low cost for preparing pegylated TP5. The pegylated analogues kept the biological activities of thymopentin in vitro and its proteolytic stabilities was significantly extended and the target product solutions does not occur hemolysis and cohesion within three hours.This study provides a the site-specific modification of peptide drugs orthogonal protection with solid phase synthesis with greatly improving the synthetic yield of site-specific modification.The study provides a research base for the larger molecular weight polyethylene glycol.