The Preparation Process And Hypolipidemic Activities Of Small Glycopeptides

With the development of the lifestyle and eating habits,the trend of incidence of hyperglycemia has a significant growth.Epidemiological studies have shown that dietary intervention is the basis treatment of metabolic disease.Therefore,nutritional products play an important role in regulating lipid metabolism and lower total serum cholesterol levels.Bioactive peptide is a kind of nutritional products which commonly used in the research and development.Through the role of cytokine network control the body's immune system,metabolic system and nerve system.Glycopeptide is an important model on the study of glycoproteins,different glycopeptide have various characteristics of chemistry,biology and medicine.The chemical synthesis and biological activity of small molecule glycopeptide(cyclic dipeptide)are rarely reported.We predict the hypolipidemic activity of cyclic dipeptide and glycopeptide by molecular docking;Molecular dynamics was used to study the structure stability and space conformation for identified for further research of compound;the liquid-phase synthesis method was used for preparing the cyclic dipeptide and glycopeptide;At last,the isolated pure compounds would be studied on the hypolipidemic biological activity.We have the following conclusions:(1)Based on the crystal structure of hHMGR and using surflex-dock program including SYBYL8.1,were the virtual screening model of hHMGR inhibitors established.The reliability of the proposed model was tested and verified by repeating the crystal structure,comparing docking score and analyzing compound.The results showed that model's docking results were stable and consistented with the actual test.Data indicated that the model had high credibility and could be used in virtual screening.Using this model for predicting the hypolipidemic activities of four different compounds,the results indicated that there was a good combining ability between cyclic dipeptide and hHMGR,and Glycopeptide had significantly improved the ability of combination between the small molecule and the target.(2)The results of conformational analysis of four small molecules,under vacuum and periodic boundary condition,indicated that no racemization cyclic dipeptide and glycopeptide conformation were stable.Glycopeptide had higher energy and lower conformation stable than corresponding cyclic dipeptides,then still had lower energy and maintain good structure stability.It is well known that cP-YG energy was lower than cF-HPG and has higher structural stability;the difference between the two cyclic dipeptides was smaller.Form molecular dynamics simulation results,it could be seen that molecules structure was very stable,and less dynamics differences between the four compounds and keep energy stability under the periodic boundary condition.The results show that the conformation was stable and had high structure stability under the periodic boundary condition.(3)We prepared the target cyclic dipeptide cP-Y by liquid-phase synthesis method.The purity of the final products was 98.06%by HPLC determination.It means that compound has a high chromatographic purity.The structure characteristics of cP-Y were identified by ESI MS/MS,FT-IR,GC-MS,1H-NMR and 13C-NMR respectively.The compound was confirmed to be the target product by the attribution fragments of mass spectrometry,peaks of spectrum.In a word,the proposed method could be applicable to synthesis of series cyclic peptide and have higher yield and purity.(4)The target glycopeptide cP-YG was achieved using liquid-phase synthesis method.The most difficult step in this experiment is the synthesis of glycosidic linkage.We selected BF3·Et2O as a catalyst,took ammonia catalytic alcoholysis reaction of acetyl ester,and then got the final products.The chromatographic purity of the material identificated by HPLC,data indicated that there was no obvious impurity peak which shown that the compound had a high chromatographic purity.Using ESI MS/MS,FT-IR and GC-MS analyzes the structure characteristics,and attribution fragments of mass spectrometry and spectrum of the compound respectively.The result shows that the substance is the target product and the glycosidic bond is beta type.While the yield is only 8%,in order to obtain high yield that a further study to optimize synthesis processing is needed.(5)The cell experiment results indicated that,cP-Y had no significant effect on the cytotoxicity and anti-proliferative in HepG2 cells in the range of 20-200?M concentration.It means that the cP-Y with low toxicity do not have significantly influence on the growth of cells.But in Western blot test,the results show that the cP-Y have significantly effect on the protein expression of LDLR,FAS and SREBP1 in dose-response.Meanwhile lower FAS and SREBP1 protein expression as a dose-effect of linear correlation.Therefore,through regulating metabolism of fatty acid and cholesterol metabolism,cP-Y has significant effects on the cholesterol-lowering,and also shows that the cP-Y is a small molecule which is commonly regulating lipid metabolism by multiple targets.