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Synthesis of o-Nitrobenzyl Amine/Alcohol Derivatives for Solid-phase Organic Synthesis and Cell Membrane Protein Isolation : 고체상 유기 합성과 세포막 단백질의 분리를 위한 오쏘-나이트로벤질 아민/알코올 유도체들의 합성
DC Field | Value | Language |
---|---|---|
dc.contributor.advisor | 이윤식 | - |
dc.contributor.author | 김재희 | - |
dc.date.accessioned | 2017-07-13T08:38:15Z | - |
dc.date.available | 2017-07-13T08:38:15Z | - |
dc.date.issued | 2014-08 | - |
dc.identifier.other | 000000022269 | - |
dc.identifier.uri | https://hdl.handle.net/10371/119717 | - |
dc.description | 학위논문 (박사)-- 서울대학교 대학원 : 화학생물공학부, 2014. 8. 이윤식. | - |
dc.description.abstract | Photochemistry is one of the unique and useful subdiscipline of chemistry. Photoreactive molecules, the core compounds in photochemistry, are undergone chemical reactions by irradiation of light, while remaining stable under various conditions such as acidic or basic reaction conditions. With such characteristic features, photoreactive molecules have been used as a linker for solid-phase organic synthesis or a photolabile protecting groups. Furthermore, the light with a wavelength above 315 nm does not give severe damages to biomolecules, and thus, the related photochemistry has been applied to chemical biology field.
In this thesis, o-nitrobenzyl amine/alcohol derivatives which can absorb UVA light (365 nm) well are synthesized for solid-phase organic synthesis and isolation cell membrane proteins. In the first part, a novel photocleavable linker which contains o-nitrobenzyl amine moiety was effectively synthesized in six steps with 33 % of synthetic yield, without any complex purification steps. Synthesized photocleavable linker could absorb UVA light with wavelength of 330 to 370 nm well, and showed similar or better photocleavage kinetics compared with established photolinkers. Based on these results, synthesized photocleavable linker was successfully applied to solid-phase organic synthesis. Leu-enkephalin amide (H-YGGFL-NH2) was synthesized by using photolinker-coupled polymer supports, with high purity. Acyl-phenylhydrazone of peptide C-terminus was oxidized by photo-oxidation of photocleavable linker, and peptide acid and ester were synthesized by addition of nucleophiles to acyl-phenyldiazene. Glycopeptides-immobilized polymer supports which can be applied to bioassays were prepared by imine formation reaction between glycans with peptides which coupled with polymer supports via photocleavable linker, and the glycopeptides were analyzed by mass spectroscopy analysis after photocleavage. The synthesized peptides on the photocleavable linker coupled polymer supports were analyzed by laser desorption-ionization mass spectroscopy method without using any additional cleavage steps and matrices. In the second part of thesis, isolation method of the cell membrane protein by using o-nitrobenzyl alcohol moiety containing linkers is described. For the isolation of cell membrane protein, linkers which consisted of amine catchable part, photoreactive part, hydrophilic spacer, and tethering part for immobilization were synthesized. As functional group for tethering, azide which can undergo the copper assisted azide-alkyne cycloaddition reaction and biotin which has high affinity with streptavidin were selected. The reactivity of synthesized molecules toward amine and alkyne was confirmed by a model reaction with amino acids and 4-pentynoic acid in solution phase. Copper assisted azide-alkyne reaction underwent between azide labeled lysine with 4-pentynoic acid in solution-phase | - |
dc.description.abstract | however, it did not undergo between azide-labeled bovine serum albumin with 4-pentynoic coupled polymer supports. Instead of azide-containing molecule, biotin-containing molecule was used for the isolation of proteins. Bovine serum albumin was labeled with biotin-containing molecule, and this labeled protein was bound with streptavidin which immobilized on the beads. And also, cell membrane proteins of Escherichia coli which were labeled with biotin-containing molecule were bound with streptavidin-coated beads. Bound proteins were released from the beads by irradiation of UVA light. Isolated proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and compared with the results of authentic proteins. | - |
dc.description.tableofcontents | ABSTRACT i
TABLE OF CONTENTS iv LIST OF TABLES x LIST OF FIGURES xi LIST OF SCHEMES xiv LIST OF ABBREVIATIONS xv I. Introduction 2 I. 1. Basics of Photochemistry 3 I. 1. 1. Principles of photochemistry 3 I. 1. 2. Photoreactive molecules 8 I. 2. Photoreactive Linkers and Protecting Groups 13 I. 2. 1. Photoreactive linkers 13 I. 2. 2. Photoreactive protecting groups 18 I. 3. Applications of Photochemistry 23 I. 3. 1. Photochemistry in solid-phase peptide synthesis 23 I. 3. 2. Photochemistry in chemical biology 30 I. 4. Research Objectives 36 II. Experimental Section 38 II. 1. General 39 II. 1. 1. Materials 39 II. 1. 2. Apparatus 41 II. 1. 3. Ninhydrin Color Test (Kaiser Test) 41 II. 1. 4. Fmoc Quantitation 42 II. 2. Synthesis and Applications of Fmoc-3-amino-3-(4,5-dimethoxy-2-nitrophenyl)propionic Acid (Fmoc-PCA Linker) 44 II. 2. 1. Synthesis of Fmoc-PCA linker 44 Synthesis of 3-amino-3-(3,4-dimethoxyphenyl)propionic acid 44 Synthesis of 1-(3,4-dimethoxyphenyl)-3-methoxy-3-oxopropan-1-aminium chloride 45 Synthesis of methyl 3-(3,4-dimethoxyphenyl)-3-(2,2,2-trifluoroacetamido)propanoate 45 Synthesis methyl 3-(4,5-dimethoxy-2-nitrophenyl)-3-(2,2,2-trifluoroacetamido)propanoate 46 Synthesis of Fmoc-PCA linker 47 II. 2. 2. Applications of Fmoc-PCA linker 49 Preparation of Acetylated PCA linker for the measurement of UV-Vis absorbency of PCA linker 49 Photocleavage of Fmoc-Phe-NH2 49 Synthesis of peptide amide with Fmoc-PCA linker coupled resins 50 Synthesis of peptide acid and peptide methyl ester with phenylhydrazine-PCA coupled resins 51 Preparation of H-β-Ala-ε-ACA-β-Ala-ε-ACA-PCA-HiCore for glycan immobilization 53 Immobilization of lactose and 3ʹ-sialyllactose to prepared resins 53 Preparation of additive solutions for direct on-bead laser desorption/ionization-time of flight (DOLDI-TOF) method 54 Loading of peptide-anchored resins onto MALDI plate 55 Analysis of peptide with DOLDI-TOF method 55 Analysis of peptide with DOLDI-TOF method by using various salt additives 56 II. 3. Synthesis and Application of Protein Fishing Molecule (ProFiM) 57 II. 3. 1. Synthesis of ProFiM 57 Synthesis of ethyl 4-(4-acetyl-2-methoxyphenoxy)butanoate 57 Synthesis of ethyl 4-(4-(1-hydroxyethyl)-2-methoxy-5-nitrophen-oxy)butanoate 58 Synthesis of 4-(4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy)-butanoic acid 59 Synthesis of ProFiM-azide and ProFiM-biotin 60 II. 3. 2. Applications of ProFiM for isolation of cell membrane proteins 62 Reactivity test of ProFiM-azide toward amine and alkyne with model amino acids 62 Preparation of alkyne-coupled polymer support for click chemistry 62 Capture-and-release performance of ProFiM-azide with model protein 63 Capture-and-release performance of ProFiM-biotin with model protein 64 Isolation of outer cell membrane proteins of E. coli with ProFiM-biotin 65 III. Results and Discussion 66 III. 1. Synthesis and Applications of Fmoc-PCA Linker 67 III. 1. 1. Synthesis of Fmoc-PCA linker 67 III. 1. 2. UV-Vis absorption spectra and photocleavage kinetics of Fmoc-PCA Linker 70 III. 1. 3. Synthesis of peptide amide with Fmoc-PCA linker coupled resins 73 III. 1. 4. Synthesis of peptide acid and peptide methyl ester with phenylhydrazine-PCA coupled resins 78 III. 1. 5. Immobilization of glycans onto PCA linker coupled resins for on-bead assays 84 III. 1. 6. Mass analysis of peptides with Direct On-bead Laser Desorption/Ionization method 86 III. 2. Synthesis and Application of Protein Fishing Molecule (ProFiM) 94 III. 2. 1. Synthesis of ProFiM 96 III. 2. 2. Reactivity test of ProFiM-azide toward amine and alkyne 103 III. 2. 3. Capture-and-release performance of ProFiM-azide and ProFiM-biotin with model protein 107 III. 2. 4. Isolation of cell membrane proteins of E. coli with ProFiM-biotin 111 IV. Conclusions 113 References 116 Abstract in Korean 133 | - |
dc.format | application/pdf | - |
dc.format.extent | 3037195 bytes | - |
dc.format.medium | application/pdf | - |
dc.language.iso | en | - |
dc.publisher | 서울대학교 대학원 | - |
dc.subject | Photochemistry | - |
dc.subject | o-Nitrobenzyl amine/alcohol derivatives | - |
dc.subject | Solid-phase organic synthesis | - |
dc.subject | Cell membrane protein | - |
dc.subject.ddc | 660 | - |
dc.title | Synthesis of o-Nitrobenzyl Amine/Alcohol Derivatives for Solid-phase Organic Synthesis and Cell Membrane Protein Isolation | - |
dc.title.alternative | 고체상 유기 합성과 세포막 단백질의 분리를 위한 오쏘-나이트로벤질 아민/알코올 유도체들의 합성 | - |
dc.type | Thesis | - |
dc.contributor.AlternativeAuthor | Jaehi Kim | - |
dc.description.degree | Doctor | - |
dc.citation.pages | xvi, 134 | - |
dc.contributor.affiliation | 공과대학 화학생물공학부 | - |
dc.date.awarded | 2014-08 | - |
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