| Summary: | 碩士 === 國立臺灣大學 === 化學研究所 === 87 === The solid phase peptide synthesis method was proposed by R. B. Merrfield in 1963. During these years, this technology was not only used to make peptide synthesis faster and more simply, but also applied to many fields, such as glycoprotein、lipoprotein、polysaccharides and combinatorial library, etc.
This thesis is composed of two parts:
Part I The Application of Microwave Irradiation on Peptide Synthesis
Recently the use of microwave irradiation in organic reactions is an area of growing interest. Microwave irradiation provides heating effects on molecules that have dipole or ion conductivity. Many papers have demonstrated that microwave enhanced the rate of organic reactions.
In our experiments, a microwave oven was combined with an automated peptide synthesizer. And UV detector was used to monitor the concentration of released Fmoc in the deprotection step. Using the data obtained from UV detector, the effects of microwave irradiation on coupling efficiency was studied. In peptide synthesis, the problem of incomplete coupling results in the low yields. It is mainly due to the structure or so called "Difficult Sequences". Until now, there is still no efficient and convenient method to solve this problem. Here, we hope that the application of microwave irradiation on the peptide synthesizer can not only shorten the coupling reaction time, but also solve the problem of incomplete coupling.
Three different sequences were studied. The results showed that microwave cut down the coupling reaction time to be 5 minutes while the coupling efficiency was improved. Therefore, when microwave irradiation combines with peptide synthesis, the advantages of convenience and automation in solid peptide synthesis can be maintained. Besides, both efficiency and percentage of yields can be improved.
Part II The Application of Solid Phase Synthesis Method on Glycopeptide Synthesis
There are a large number of asialoglycoprotein receptors (ASGP-r) on the cell membrance of hepatocytes in mammalian species. More than 80% of ASGP-r in the body are distributed on liver cells. ASGP-r recognizes specifically the ligands which have terminal galactose (Gal) or N-acetylgalactose (GalNAc) residues, and the affinity depends on the valency of Gal / GalNAc and the three-dimensional arrangement.
YEE(GalNAcAH)3 reported by R. T. Lee, and Y. C. Lee in 1987 is a synthetic ligand for ASGP-r. However, according to the published synthesis methods, the low yield of YEE(GalNAcAH)3 synthesis was due to its solubility and complicated procedures.
Here, we use N-Fmoc aminohexanol glycoside and Fmoc (a-GalNAcAc3AH) Glu as building blocks in solid phase peptide synthesis. The preparations of YEE(GalNAcAH)3 and fluorescein labeled YEE(GalNAcAH)3 are fast and simple. The yields of product are over 80 %. While only using Fmoc (a-GalNAcAc3AH) Glu as a building block, we design YEEE(GalNAcAH)3, an analogue of YEE(GalNAcAH)3. By solid phase peptide synthesis method, YEEE(GalNAcAH)3 and its fluorescien labeled derivative were also synthesized with high yields.
Using HepG2 cells as bioassay models, the binding abilities of fluorescein labeled YEE(GalNAcAH)3 or YEEE(GalNAcAH)3 to ASGP-r were studied. The endocytosis phenomenon was also investigated. The binding of synthetic fluorescein labaled ligands to the ASGP-r was demonstrated by fluorescent microscope. And then the internalization of ligands mediated by ASGP-r was observed by confocal microscope. Additionally, both the competitive binding studies using ligands with or without fluorescein labeled and specificity studies were presented in current paper.
From the results of above experiments, both YEE(GalNAcAH)3 and YEEE(GalNAcAH)3 were synthesized successfully by solid phase peptide synthesis method with suitable building blocks. Therefore, the synthesis and the utilization of these two ligands as a carrier for drug delivery specifically to liver cells become a novel tool for future clinical studies.
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