The Labeling Of α₁-Adrenergic Receptor Agonist And Antagonist And The Research Of Their Preliminary Application

This thesis includes three parts: I .Preface. II. The synthesis of biotinylated α₁-adrenergic receptor agonist and antagonist. III. The application of ai-adrenergic receptor antagonist labeled QDs .In Chaper I .of this thesis, first of all, the researchs of single living cell were introduced briefly. Secondly, the emphases of this chaper is ai-adrenergic receptor and its agonist and antagonist. Thirdly, the review of fluorescent probes were delivered, which included GFP, Alexa fluorescent dyes and QDs. Fourthly, the QDs labeling of ligands was introduced, which included the methods of QDs labeling Hgands and the preparations of biotinylated derivatives.In Chaper II., the methods for the synthesis of biotinylated ai-adrenergic receptor agonist and antagonist were developed. In this part, We have prepared five new biotinylated ligands that may be attached to streptavidin conjugated quantum dots, which might be a promising and powerful tools for investigating interactions between receptors and ligands and dynamic behaviors of ai-adrenergic receptor in living cell.The first part is the preparation of biotinylated prazosin. Our biotinylated ligand was 2-(1-Piperazinyl)-4-amino-6,7-dimethoxyquinazol. Initially, piperazine was reacted with 48% aqueous HBr at room temperature, then the mixture was warmed to 60°C and 2-chloro-4-amino-6,7-dimethoxyquinazol was added over a 1h period. The resulting mixture was heated at reflux for 4 h to give2-(1-Piperazinyl)-4-amino-6,7-dimethoxyquinazol in a 94.6% yield and this was coupled to biotin using biotinylated NHS in acetonitrile by stirring at room temperature to stay overnight, resulting in biotinylated prazosin in a 56.4% yield. The target compound's structure were confirmed by ¹HNMR and ¹³CNMR.The second part is the preparation of biotinylated phenylephrine. We thought that the short linker arm between biotin and phenylephrine may reduce the biological activity of the biotinylated derivative when it was bound to QDs. There may be considerable steric interactions between the surface of the dot and the ai-adrenergic receptor. In order not to affect the biological activity of the biotinylated derivative, we synthesized different length of the linker arms between biotin and phenylephrine. First, phenylephrine was reacted with the derivative of ethylene or diglycol's sulfonylation, then the product after aminolysis was coupled to biotin using biotinylated NHS in acetonitrile by stirring at 38℃ to stay 16h, resulting in biotinylated phenylephrine in a 45% yield. The target compound's structure were confirmed by ¹HNMR and ¹³CNMR.In Chaper III., The application of ai-adrenergic receptor antagonist labeled QDs was researched preliminarily with total internal reflection fluorescence microscopy, streptavidin conjugated quantum dots and the biotinylated prazosin which was prepared in the chapter two. The method for location of ai-adrenergic receptor on living HEK293A cell member was developed on the single cell level. Several parameters including, incubating time, incubating temperature, prazosin-QD concentration was optimized. The following conditions were suitable for the determination of ai-adrenergic receptor on cell member: incubating time: 1h; incubating temperature: 37℃; prazosin-QD concentration: 5×10^-10 mol/L.And we also demonstrated specific of prazosin-QD binding to ai-adrenergic receptors.