WEKO3
アイテム
{"_buckets": {"deposit": "1ffd0d2c-ed81-41e7-9d9e-074c2438f7ec"}, "_deposit": {"created_by": 15, "id": "17933", "owners": [15], "pid": {"revision_id": 0, "type": "depid", "value": "17933"}, "status": "published"}, "_oai": {"id": "oai:sucra.repo.nii.ac.jp:00017933", "sets": ["858"]}, "author_link": [], "item_113_alternative_title_1": {"attribute_name": "タイトル(別言語)", "attribute_value_mlt": [{"subitem_alternative_title": "魅力的なバイオプローブとしてのABG (avidin-biotin-GlcNAc) 糖鎖クラスターの合成とPL法とSPR法によるWGAとの多価認識"}]}, "item_113_biblio_info_9": {"attribute_name": "書誌情報", "attribute_value_mlt": [{"bibliographicIssueDates": {"bibliographicIssueDate": "2017", "bibliographicIssueDateType": "Issued"}}]}, "item_113_date_35": {"attribute_name": "作成日", "attribute_value_mlt": [{"subitem_date_issued_datetime": "2018-01-15", "subitem_date_issued_type": "Created"}]}, "item_113_date_granted_20": {"attribute_name": "学位授与年月日", "attribute_value_mlt": [{"subitem_dategranted": "2017-03-22"}]}, "item_113_degree_grantor_22": {"attribute_name": "学位授与機関", "attribute_value_mlt": [{"subitem_degreegrantor": [{"subitem_degreegrantor_name": "埼玉大学"}], "subitem_degreegrantor_identifier": [{"subitem_degreegrantor_identifier_name": "12401", "subitem_degreegrantor_identifier_scheme": "kakenhi"}]}]}, "item_113_degree_name_21": {"attribute_name": "学位名", "attribute_value_mlt": [{"subitem_degreename": "博士(学術)"}]}, "item_113_description_13": {"attribute_name": "形態", "attribute_value_mlt": [{"subitem_description": "xiii, 126 p.", "subitem_description_type": "Other"}]}, "item_113_description_23": {"attribute_name": "抄録", "attribute_value_mlt": [{"subitem_description": "Interactions between carbohydrates and proteins are progressively accelerating to a greater extent and being recognized as crucial in many biological processes, such as cellular adhesion, cell signaling, immune responses, fertilization, cancer metastasis and communication. The concernment of these natural events at the cell membrane surface provided the motivation for their study in a biomimetic environment. In order to investigate the interactions of carbohydrates and proteins, the development of efficient analytic technologies, as well as novel strategies for the synthesis of carbohydrates, have to be explored.\nIn this research a tetravalent GlcNAc pendant glycocluster was fruitfully constructed with a linker of C6 length consisting of biotin. Carbohydrate-protein interactions are especially well suited for multivalency, therefore in this research we are using such reaction system to synthesize a glycopolymer of tetrameric structure using N-acetyl-D-glucosamine (GlcNAc) as a target carbohydrate conjugated with biotin via DMT-MM as coupling reagent, followed by biotin-avidin interaction leading to the formation of glycocluster of avidin-biotin-GlcNAc conjugate (ABG complex). To check the binding affinity of GlcNAc conjugate with a lectin WGA (Wheat Germ Agglutinin) we used fluorometric assay by means of specific excitation of tryptophan at λex 295 nm and it was found to be very high in case of ABG complex as compared to GlcNAc only with the phenomenon proven to be due to glycocluster effect. Another analytical biosensing method Surface plasmon resonance (SPR) was implied to give the detailed information of binding kinetics between ABG and WGA compared to three linear-type GlcNAc polymers binding with WGA. WGA used as a ligand and immobilized on sensor surface via amine coupling method. Artificial glycopolymers of GlcNAc with polystyrene based polymeric backbone of acrylamide (linear-type polymers) was used as control against ABG complex. Kinetic analysis was performed by separated numerical integration of the association and dissociation phases by using a Langmuir (1:1) model. The efficiency of the method was exhibited in the analysis of the interactions that covered a high affinity range; namely the strong binding (KA ~ 107 M-1) for ABG compared with the control polymers, which show the binding of (KA ~ 105 M-1). Both the techniques PL and SPR shown to be very rapid and powerful techniques, which supervise the mechanistic studies of carbohydrate-protein interactions at interfaces in real time and quantitative manner.\nThese methodologies have been used to probe carbohydrate-lectin-interactions for a cereal lectin named WGA and the usefulness of these synthetic glycoconjugates as tools in the study of sugar-lectin interactions has been proved due to glycocluster effect. SPR and PL analysis can afford data with desirable reproducibility and therefore offers the possibility of a detailed computational analysis.", "subitem_description_type": "Abstract"}]}, "item_113_description_24": {"attribute_name": "目次", "attribute_value_mlt": [{"subitem_description": "List of Figures, Schemes, Tables and Equations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v\nAbbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix\nAbstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xii\nCHAPTER-1\n1. INTRODUCTION\n1.1 Carbohydrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1\n1.1.1 N-acetyl-D-glucosamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3\n1.2 Lectins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4\n1.2.1 Animal lectins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7\n1.2.2 C-type lectins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8\n1.2.3 Plant lectins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9\n1.2.4 L-type lectin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11\n1.2.5 Wheat germ agglutinin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .13\n1.3 Carbohydrate-Lectin interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .15\n1.4 Bioprobes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .17\n1.5 Glycocluster effect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .20\n1.6 Methods for measuring protein-carbohydrate interactions . . . . . . . . . . . . . . . 22\n1.7 Photoluminescence spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23\n1.7.1 PL Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23\n1.7.2 Hill`s Equation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24\n1.7.3 Tryptophan fluorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .24\n1.8 Surface Plasmon Resonance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27\n1.8.1 SPR Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28\n1.8.2 SPR instrumentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31\n1.8.3 Sensor chip . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .31\n1.9 Aim of the present study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34\nCHAPTER-2\n2. CARBOHYDRATE SYNTHESIS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35\n2.1 Synthesis of GlcNAc derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35\n2.1.1 acetamido-tetra-O-acetyl-α-D-glucosamine(2) . . . . . . . . . . . . . . . . . . . . . . . . .37\n2.1.2 Oxazoline derivative(3) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38\n2.1.3 6-Chloro-2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranoside(4) . .38\n2.1.4 6-Azidohexyl-2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranoside(5)40\n2.1.5 6-Azidohexyl-2-Acetamido -2-deoxy-β-D- glucopyranoside (6) . . . . . . . . . . . . 41\n2.1.6 6-Aminohexyl-2-Acetamido -2-deoxy-β-D- glucopyranoside (7) . . . . . . . . . . . . 42\n2.2 Synthesis of Biotin-GlcNAc conjugate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43\n2.2.1 DMAP-DIC Coupling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43\n2.2.2 DMT-MM Coupling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .43\n2.2.3 6-(Biotynylamido)hexyl-2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranoside\n(9) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .45\n2.2.4 6-(Biotynylamido)hexyl-2-Acetamido-2-deoxy-β-D-glucopyranoside (10) . . . . .46\n2.3 Physical \u0026 Chemical properties of compound 9 \u0026 10 . . . . . . . . . . . . . . . . . . . . . . .48\nCHAPTER-3\n3. ANALYTICAL TOOLS TO EXPLAIN CARBOHYDRATE STRUCTURES . . . . . 49\n3.1 Nuclear magnetic resonance (NMR) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .49\n3.2 Infrared spectroscopy (IR) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .52\n3.3 Mass spectroscopy (MS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54\nCHAPTER-4\n4. SYNTHESIS OF ABG COMPLEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .56\n4.1 Avidin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56\n4.2 Biotin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57\n4.3 Avidin-Biotin system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57\n4.4 ABG tetrameric complex synthesis \u0026 purification . . . . . . . . . . . . . . . . . . . . . . . . .58\n4.5 Absorbance concentration analysis of ABG complex . . . . . . . . . . . . . . . . . . . . . . .60\nCHAPTER-5\n5. BIOLOGICAL EVALUATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61\n5.1 Fluorometric assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62\n5.1.1 Preparation of WGA solution \u0026 sampling to measure PL . . . . . . . . . . . . . . . . . .62\n5.1.2 PL analysis for GlcNAc only . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63\n5.1.3 PL analysis for compound 10 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .65\n5.1.4 PL analysis for ABG complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .67\n5.1.5 Kinetic affinity results for PL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .69\nCHAPTER-6\n6. BIOSENSOR ASSAY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70\n6.1 Surface Plasmon Resonance (SPR) experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . 71\n6.1.1 pH Scouting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71\n6.1.2 Immobilization of WGA to CM5 chip . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73\n6.1.3 Analytes used for kinetic analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76\n6.1.4 Kinetic analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78\n6.1.4.1 Regeneration of the sensor chip surface . . . . . . . . . . . . . . . . . . . . . . . . 80\n6.1.4.2 Kinetic analysis for ABG complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82\n6.1.4.3 Kinetic analysis for glycopolymer 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84\n6.1.4.4 Kinetic analysis for glycopolymer 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86\n6.1.4.5 Kinetic analysis for glycopolymer 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88\n6.2 Kinetic parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .90\nCHAPTER-7\n7. COMPARATIVE STUDY BETWEEN SPR \u0026 PL . . . . . . . . . . . . . . . . . . . . . . . . . . . .91\nCHAPTER-8\n8. CONCLUSIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .92\nCHAPTER-9\n9. GENERAL METHODS \u0026 MATERIALS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .94\nCHAPTER-10\n10. SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99\nReferences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105\nList of conferences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119\nList of publications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120\nAwards and Honors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121\nAppendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122\nDeclaration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123\nAcknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124\nThesis dedicated to . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126", "subitem_description_type": "Other"}]}, "item_113_description_25": {"attribute_name": "注記", "attribute_value_mlt": [{"subitem_description": "主指導教員 : 松岡浩司", "subitem_description_type": "Other"}]}, "item_113_description_33": {"attribute_name": "資源タイプ", "attribute_value_mlt": [{"subitem_description": "text", "subitem_description_type": "Other"}]}, "item_113_description_34": {"attribute_name": "フォーマット", "attribute_value_mlt": [{"subitem_description": "application/pdf", "subitem_description_type": "Other"}]}, "item_113_dissertation_number_19": {"attribute_name": "学位授与番号", "attribute_value_mlt": [{"subitem_dissertationnumber": "甲第1067号"}]}, "item_113_identifier_registration": {"attribute_name": "ID登録", "attribute_value_mlt": [{"subitem_identifier_reg_text": "10.24561/00017927", "subitem_identifier_reg_type": "JaLC"}]}, "item_113_publisher_11": {"attribute_name": "出版者名", "attribute_value_mlt": [{"subitem_publisher": "埼玉大学大学院理工学研究科"}]}, "item_113_publisher_12": {"attribute_name": "出版者名(別言語)", "attribute_value_mlt": [{"subitem_publisher": "Graduate School of Science and Engineering, Saitama University"}]}, "item_113_record_name_8": {"attribute_name": "書誌", "attribute_value_mlt": [{"subitem_record_name": "博士論文(埼玉大学大学院理工学研究科(博士後期課程))"}]}, "item_113_text_31": {"attribute_name": "版", "attribute_value_mlt": [{"subitem_text_value": "[出版社版]"}]}, "item_113_text_36": {"attribute_name": "アイテムID", "attribute_value_mlt": [{"subitem_text_value": "GD0000903"}]}, "item_113_text_4": {"attribute_name": "著者 所属", "attribute_value_mlt": [{"subitem_text_value": "埼玉大学大学院理工学研究科(博士後期課程)理工学専攻"}]}, "item_113_text_5": {"attribute_name": "著者 所属(別言語)", "attribute_value_mlt": [{"subitem_text_value": "Graduate School of Science and Engineering, Saitama University"}]}, "item_113_version_type_32": {"attribute_name": "著者版フラグ", "attribute_value_mlt": [{"subitem_version_resource": "http://purl.org/coar/version/c_970fb48d4fbd8a85", "subitem_version_type": "VoR"}]}, "item_access_right": {"attribute_name": "アクセス権", "attribute_value_mlt": [{"subitem_access_right": "open access", "subitem_access_right_uri": "http://purl.org/coar/access_right/c_abf2"}]}, "item_creator": {"attribute_name": "著者", "attribute_type": "creator", "attribute_value_mlt": [{"creatorNames": [{"creatorName": "AMRITA, KUMARI", "creatorNameLang": "en"}, {"creatorName": "アモリタ, クマリ", "creatorNameLang": "ja-Kana"}]}]}, "item_files": {"attribute_name": "ファイル情報", "attribute_type": "file", "attribute_value_mlt": [{"accessrole": "open_date", "date": [{"dateType": "Available", "dateValue": "2018-02-05"}], "displaytype": "detail", "download_preview_message": "", "file_order": 0, "filename": "GD0000903.pdf", "filesize": [{"value": "10.8 MB"}], "format": "application/pdf", "future_date_message": "", "is_thumbnail": false, "licensetype": "license_note", "mimetype": "application/pdf", "size": 10800000.0, "url": {"label": "GD0000903.pdf", "objectType": "fulltext", "url": "https://sucra.repo.nii.ac.jp/record/17933/files/GD0000903.pdf"}, "version_id": "3f4e0b33-60ae-43b5-ae06-78a6041dcce6"}]}, "item_keyword": {"attribute_name": "キーワード", "attribute_value_mlt": [{"subitem_subject": "Lectins", "subitem_subject_scheme": "Other"}, {"subitem_subject": "Carbohydrates", "subitem_subject_scheme": "Other"}, {"subitem_subject": "Avidin", "subitem_subject_scheme": "Other"}, {"subitem_subject": "Biotin", "subitem_subject_scheme": "Other"}, {"subitem_subject": "Biosensors", "subitem_subject_scheme": "Other"}, {"subitem_subject": "Photoluminescence", "subitem_subject_scheme": "Other"}, {"subitem_subject": "Surface plasmon resonance", "subitem_subject_scheme": "Other"}, {"subitem_subject": "Tryptophan", "subitem_subject_scheme": "Other"}, {"subitem_subject": "Glycoclusters", "subitem_subject_scheme": "Other"}, {"subitem_subject": "Ligand", "subitem_subject_scheme": "Other"}, {"subitem_subject": "Analyte", "subitem_subject_scheme": "Other"}, {"subitem_subject": "Multivalent", "subitem_subject_scheme": "Other"}]}, "item_language": {"attribute_name": "言語", "attribute_value_mlt": [{"subitem_language": "eng"}]}, "item_resource_type": {"attribute_name": "資源タイプ", "attribute_value_mlt": [{"resourcetype": "doctoral thesis", "resourceuri": "http://purl.org/coar/resource_type/c_db06"}]}, "item_title": "Synthesis of ABG (avidin-biotin-GlcNAc) glycocluster as an attractive bioprobe \u0026 its multivalent recognition with WGA by PL \u0026 SPR methods", "item_titles": {"attribute_name": "タイトル", "attribute_value_mlt": [{"subitem_title": "Synthesis of ABG (avidin-biotin-GlcNAc) glycocluster as an attractive bioprobe \u0026 its multivalent recognition with WGA by PL \u0026 SPR methods", "subitem_title_language": "en"}]}, "item_type_id": "113", "owner": "15", "path": ["858"], "permalink_uri": "https://doi.org/10.24561/00017927", "pubdate": {"attribute_name": "PubDate", "attribute_value": "2018-01-15"}, "publish_date": "2018-01-15", "publish_status": "0", "recid": "17933", "relation": {}, "relation_version_is_last": true, "title": ["Synthesis of ABG (avidin-biotin-GlcNAc) glycocluster as an attractive bioprobe \u0026 its multivalent recognition with WGA by PL \u0026 SPR methods"], "weko_shared_id": -1}
Synthesis of ABG (avidin-biotin-GlcNAc) glycocluster as an attractive bioprobe & its multivalent recognition with WGA by PL & SPR methods
https://doi.org/10.24561/00017927
https://doi.org/10.24561/00017927e1147c9f-1e30-40c5-b3e5-8e939b312f48
名前 / ファイル | ライセンス | アクション |
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GD0000903.pdf (10.8 MB)
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Item type | 学位論文 / Thesis or Dissertation(1) | |||||||||
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公開日 | 2018-01-15 | |||||||||
タイトル | ||||||||||
言語 | en | |||||||||
タイトル | Synthesis of ABG (avidin-biotin-GlcNAc) glycocluster as an attractive bioprobe & its multivalent recognition with WGA by PL & SPR methods | |||||||||
言語 | ||||||||||
言語 | eng | |||||||||
キーワード | ||||||||||
主題Scheme | Other | |||||||||
主題 | Lectins | |||||||||
キーワード | ||||||||||
主題Scheme | Other | |||||||||
主題 | Carbohydrates | |||||||||
キーワード | ||||||||||
主題Scheme | Other | |||||||||
主題 | Avidin | |||||||||
キーワード | ||||||||||
主題Scheme | Other | |||||||||
主題 | Biotin | |||||||||
キーワード | ||||||||||
主題Scheme | Other | |||||||||
主題 | Biosensors | |||||||||
キーワード | ||||||||||
主題Scheme | Other | |||||||||
主題 | Photoluminescence | |||||||||
キーワード | ||||||||||
主題Scheme | Other | |||||||||
主題 | Surface plasmon resonance | |||||||||
キーワード | ||||||||||
主題Scheme | Other | |||||||||
主題 | Tryptophan | |||||||||
キーワード | ||||||||||
主題Scheme | Other | |||||||||
主題 | Glycoclusters | |||||||||
キーワード | ||||||||||
主題Scheme | Other | |||||||||
主題 | Ligand | |||||||||
キーワード | ||||||||||
主題Scheme | Other | |||||||||
主題 | Analyte | |||||||||
キーワード | ||||||||||
主題Scheme | Other | |||||||||
主題 | Multivalent | |||||||||
資源タイプ | ||||||||||
資源タイプ識別子 | http://purl.org/coar/resource_type/c_db06 | |||||||||
資源タイプ | doctoral thesis | |||||||||
ID登録 | ||||||||||
ID登録 | 10.24561/00017927 | |||||||||
ID登録タイプ | JaLC | |||||||||
アクセス権 | ||||||||||
アクセス権 | open access | |||||||||
アクセス権URI | http://purl.org/coar/access_right/c_abf2 | |||||||||
タイトル(別言語) | ||||||||||
その他のタイトル | 魅力的なバイオプローブとしてのABG (avidin-biotin-GlcNAc) 糖鎖クラスターの合成とPL法とSPR法によるWGAとの多価認識 | |||||||||
著者 |
AMRITA, KUMARI
× AMRITA, KUMARI
|
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著者 所属 | ||||||||||
埼玉大学大学院理工学研究科(博士後期課程)理工学専攻 | ||||||||||
著者 所属(別言語) | ||||||||||
Graduate School of Science and Engineering, Saitama University | ||||||||||
書誌 | ||||||||||
収録物名 | 博士論文(埼玉大学大学院理工学研究科(博士後期課程)) | |||||||||
書誌情報 |
発行日 2017 |
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出版者名 | ||||||||||
出版者 | 埼玉大学大学院理工学研究科 | |||||||||
出版者名(別言語) | ||||||||||
出版者 | Graduate School of Science and Engineering, Saitama University | |||||||||
形態 | ||||||||||
内容記述タイプ | Other | |||||||||
内容記述 | xiii, 126 p. | |||||||||
学位授与番号 | ||||||||||
学位授与番号 | 甲第1067号 | |||||||||
学位授与年月日 | ||||||||||
学位授与年月日 | 2017-03-22 | |||||||||
学位名 | ||||||||||
学位名 | 博士(学術) | |||||||||
学位授与機関 | ||||||||||
学位授与機関識別子Scheme | kakenhi | |||||||||
学位授与機関識別子 | 12401 | |||||||||
学位授与機関名 | 埼玉大学 | |||||||||
抄録 | ||||||||||
内容記述タイプ | Abstract | |||||||||
内容記述 | Interactions between carbohydrates and proteins are progressively accelerating to a greater extent and being recognized as crucial in many biological processes, such as cellular adhesion, cell signaling, immune responses, fertilization, cancer metastasis and communication. The concernment of these natural events at the cell membrane surface provided the motivation for their study in a biomimetic environment. In order to investigate the interactions of carbohydrates and proteins, the development of efficient analytic technologies, as well as novel strategies for the synthesis of carbohydrates, have to be explored. In this research a tetravalent GlcNAc pendant glycocluster was fruitfully constructed with a linker of C6 length consisting of biotin. Carbohydrate-protein interactions are especially well suited for multivalency, therefore in this research we are using such reaction system to synthesize a glycopolymer of tetrameric structure using N-acetyl-D-glucosamine (GlcNAc) as a target carbohydrate conjugated with biotin via DMT-MM as coupling reagent, followed by biotin-avidin interaction leading to the formation of glycocluster of avidin-biotin-GlcNAc conjugate (ABG complex). To check the binding affinity of GlcNAc conjugate with a lectin WGA (Wheat Germ Agglutinin) we used fluorometric assay by means of specific excitation of tryptophan at λex 295 nm and it was found to be very high in case of ABG complex as compared to GlcNAc only with the phenomenon proven to be due to glycocluster effect. Another analytical biosensing method Surface plasmon resonance (SPR) was implied to give the detailed information of binding kinetics between ABG and WGA compared to three linear-type GlcNAc polymers binding with WGA. WGA used as a ligand and immobilized on sensor surface via amine coupling method. Artificial glycopolymers of GlcNAc with polystyrene based polymeric backbone of acrylamide (linear-type polymers) was used as control against ABG complex. Kinetic analysis was performed by separated numerical integration of the association and dissociation phases by using a Langmuir (1:1) model. The efficiency of the method was exhibited in the analysis of the interactions that covered a high affinity range; namely the strong binding (KA ~ 107 M-1) for ABG compared with the control polymers, which show the binding of (KA ~ 105 M-1). Both the techniques PL and SPR shown to be very rapid and powerful techniques, which supervise the mechanistic studies of carbohydrate-protein interactions at interfaces in real time and quantitative manner. These methodologies have been used to probe carbohydrate-lectin-interactions for a cereal lectin named WGA and the usefulness of these synthetic glycoconjugates as tools in the study of sugar-lectin interactions has been proved due to glycocluster effect. SPR and PL analysis can afford data with desirable reproducibility and therefore offers the possibility of a detailed computational analysis. |
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目次 | ||||||||||
内容記述タイプ | Other | |||||||||
内容記述 | List of Figures, Schemes, Tables and Equations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xii CHAPTER-1 1. INTRODUCTION 1.1 Carbohydrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1.1.1 N-acetyl-D-glucosamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 1.2 Lectins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 1.2.1 Animal lectins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7 1.2.2 C-type lectins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 1.2.3 Plant lectins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 1.2.4 L-type lectin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 1.2.5 Wheat germ agglutinin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .13 1.3 Carbohydrate-Lectin interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .15 1.4 Bioprobes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .17 1.5 Glycocluster effect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .20 1.6 Methods for measuring protein-carbohydrate interactions . . . . . . . . . . . . . . . 22 1.7 Photoluminescence spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 1.7.1 PL Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 1.7.2 Hill`s Equation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 1.7.3 Tryptophan fluorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .24 1.8 Surface Plasmon Resonance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 1.8.1 SPR Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 1.8.2 SPR instrumentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 1.8.3 Sensor chip . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .31 1.9 Aim of the present study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 CHAPTER-2 2. CARBOHYDRATE SYNTHESIS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 2.1 Synthesis of GlcNAc derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 2.1.1 acetamido-tetra-O-acetyl-α-D-glucosamine(2) . . . . . . . . . . . . . . . . . . . . . . . . .37 2.1.2 Oxazoline derivative(3) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 2.1.3 6-Chloro-2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranoside(4) . .38 2.1.4 6-Azidohexyl-2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranoside(5)40 2.1.5 6-Azidohexyl-2-Acetamido -2-deoxy-β-D- glucopyranoside (6) . . . . . . . . . . . . 41 2.1.6 6-Aminohexyl-2-Acetamido -2-deoxy-β-D- glucopyranoside (7) . . . . . . . . . . . . 42 2.2 Synthesis of Biotin-GlcNAc conjugate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 2.2.1 DMAP-DIC Coupling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 2.2.2 DMT-MM Coupling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .43 2.2.3 6-(Biotynylamido)hexyl-2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranoside (9) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .45 2.2.4 6-(Biotynylamido)hexyl-2-Acetamido-2-deoxy-β-D-glucopyranoside (10) . . . . .46 2.3 Physical & Chemical properties of compound 9 & 10 . . . . . . . . . . . . . . . . . . . . . . .48 CHAPTER-3 3. ANALYTICAL TOOLS TO EXPLAIN CARBOHYDRATE STRUCTURES . . . . . 49 3.1 Nuclear magnetic resonance (NMR) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .49 3.2 Infrared spectroscopy (IR) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .52 3.3 Mass spectroscopy (MS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54 CHAPTER-4 4. SYNTHESIS OF ABG COMPLEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .56 4.1 Avidin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56 4.2 Biotin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 4.3 Avidin-Biotin system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 4.4 ABG tetrameric complex synthesis & purification . . . . . . . . . . . . . . . . . . . . . . . . .58 4.5 Absorbance concentration analysis of ABG complex . . . . . . . . . . . . . . . . . . . . . . .60 CHAPTER-5 5. BIOLOGICAL EVALUATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 5.1 Fluorometric assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62 5.1.1 Preparation of WGA solution & sampling to measure PL . . . . . . . . . . . . . . . . . .62 5.1.2 PL analysis for GlcNAc only . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63 5.1.3 PL analysis for compound 10 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .65 5.1.4 PL analysis for ABG complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .67 5.1.5 Kinetic affinity results for PL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .69 CHAPTER-6 6. BIOSENSOR ASSAY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70 6.1 Surface Plasmon Resonance (SPR) experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . 71 6.1.1 pH Scouting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71 6.1.2 Immobilization of WGA to CM5 chip . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73 6.1.3 Analytes used for kinetic analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76 6.1.4 Kinetic analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78 6.1.4.1 Regeneration of the sensor chip surface . . . . . . . . . . . . . . . . . . . . . . . . 80 6.1.4.2 Kinetic analysis for ABG complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82 6.1.4.3 Kinetic analysis for glycopolymer 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84 6.1.4.4 Kinetic analysis for glycopolymer 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86 6.1.4.5 Kinetic analysis for glycopolymer 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88 6.2 Kinetic parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .90 CHAPTER-7 7. COMPARATIVE STUDY BETWEEN SPR & PL . . . . . . . . . . . . . . . . . . . . . . . . . . . .91 CHAPTER-8 8. CONCLUSIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .92 CHAPTER-9 9. GENERAL METHODS & MATERIALS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .94 CHAPTER-10 10. SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105 List of conferences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119 List of publications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120 Awards and Honors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121 Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122 Declaration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123 Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124 Thesis dedicated to . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126 |
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注記 | ||||||||||
内容記述タイプ | Other | |||||||||
内容記述 | 主指導教員 : 松岡浩司 | |||||||||
版 | ||||||||||
[出版社版] | ||||||||||
著者版フラグ | ||||||||||
出版タイプ | VoR | |||||||||
出版タイプResource | http://purl.org/coar/version/c_970fb48d4fbd8a85 | |||||||||
資源タイプ | ||||||||||
内容記述タイプ | Other | |||||||||
内容記述 | text | |||||||||
フォーマット | ||||||||||
内容記述タイプ | Other | |||||||||
内容記述 | application/pdf | |||||||||
作成日 | ||||||||||
日付 | 2018-01-15 | |||||||||
日付タイプ | Created | |||||||||
アイテムID | ||||||||||
GD0000903 |