Blood Peptide X And Its Derivatives, Purification, Activity Assay And Quality Research

In this paper the base labile 9-fluorenylmethoxycarbony (Fmoc) was used to protect the alpha-amino group of amino acid. In order to avoid side reaction we used t-butyl to protect the side chain functional group. Four analytical methods including thin -layer chromatography (TLC), reversed phase high performance liquid chromatography (RP-HPLC), melting point and UV spectroscopy were used to investigate the purity of amino acid derivatives.After the attachment of the first Fmoc protected amino acid to 2-Chlorotrityl chloride resin (CLTR), the short peptide X and its derivatives G, E and H were successfully synthesized according to the strategy of stepwise solid phase peptide synthesis by manual and automated peptide synthesis instrument. The results showed that the CLTR resin was better than that of Wang resin. The reaction condition was optimized through orthogonal designed experiments.The peptides were successfully purified by preparative reversed phase medium performance liquid chromatography (MPLC) and freeze-dried with the help of analytical reverse-phase HPLC, LC-MS and freeze-drier in combination.Five methods were used independently to assess the characteristics, including analytical reversed-phase HPLC and capillary electrophoresis (CE) to determine the purity; amino acid analysis (AAA) to quantitate the amount and the ratio of the amino acids; Edman degradation sequence analysis and electrospray ionization mass spectrometry (ESI-MS) to corroborate the molecular weight and the covalent structure.The oxygen utilization of hypoxic tissues was examined by Warburg's test. The activity of the synthetic peptide X and its derivatives were different. The results showed that peptide X and its derivative H (5mg/ml) had the same activity as that of Actovegin.In order to establish a satisfactory RP-HPLC assay method, factors of effecting separation, such as the mobile phase, pH values were studied. With the help of LC-MS and the reducing agent DTT, the disulfide formation is corroborated.According to the results, the synthetic peptide X and its derivative H may become the substitutes of Actovegin...