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

Autor:	AMRITA, KUMARI
Jazyk:	angličtina
Rok vydání:	2017
Předmět:	Multivalent Biosensors Lectins Surface plasmon resonance Carbohydrates Tryptophan Biotin Glycoclusters Ligand Avidin Photoluminescence Analyte
Zdroj:	博士論文（埼玉大学大学院理工学研究科（博士後期課程））.
Popis:	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. List of Figures, Schemes, Tables and Equations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vAbbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ixAbstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiiCHAPTER-11. INTRODUCTION1.1 Carbohydrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.1.1 N-acetyl-D-glucosamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31.2 Lectins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41.2.1 Animal lectins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .71.2.2 C-type lectins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81.2.3 Plant lectins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91.2.4 L-type lectin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111.2.5 Wheat germ agglutinin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .131.3 Carbohydrate-Lectin interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .151.4 Bioprobes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .171.5 Glycocluster effect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .201.6 Methods for measuring protein-carbohydrate interactions . . . . . . . . . . . . . . . 221.7 Photoluminescence spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231.7.1 PL Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231.7.2 Hill`s Equation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241.7.3 Tryptophan fluorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .241.8 Surface Plasmon Resonance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271.8.1 SPR Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281.8.2 SPR instrumentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 311.8.3 Sensor chip . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .311.9 Aim of the present study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34CHAPTER-22. CARBOHYDRATE SYNTHESIS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 352.1 Synthesis of GlcNAc derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 352.1.1 acetamido-tetra-O-acetyl-α-D-glucosamine(2) . . . . . . . . . . . . . . . . . . . . . . . . .372.1.2 Oxazoline derivative(3) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 382.1.3 6-Chloro-2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranoside(4) . .382.1.4 6-Azidohexyl-2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranoside(5)402.1.5 6-Azidohexyl-2-Acetamido -2-deoxy-β-D- glucopyranoside (6) . . . . . . . . . . . . 412.1.6 6-Aminohexyl-2-Acetamido -2-deoxy-β-D- glucopyranoside (7) . . . . . . . . . . . . 422.2 Synthesis of Biotin-GlcNAc conjugate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 432.2.1 DMAP-DIC Coupling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 432.2.2 DMT-MM Coupling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .432.2.3 6-(Biotynylamido)hexyl-2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranoside(9) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .452.2.4 6-(Biotynylamido)hexyl-2-Acetamido-2-deoxy-β-D-glucopyranoside (10) . . . . .462.3 Physical & Chemical properties of compound 9 & 10 . . . . . . . . . . . . . . . . . . . . . . .48CHAPTER-33. ANALYTICAL TOOLS TO EXPLAIN CARBOHYDRATE STRUCTURES . . . . . 493.1 Nuclear magnetic resonance (NMR) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .493.2 Infrared spectroscopy (IR) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .523.3 Mass spectroscopy (MS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54CHAPTER-44. SYNTHESIS OF ABG COMPLEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .564.1 Avidin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 564.2 Biotin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 574.3 Avidin-Biotin system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 574.4 ABG tetrameric complex synthesis & purification . . . . . . . . . . . . . . . . . . . . . . . . .584.5 Absorbance concentration analysis of ABG complex . . . . . . . . . . . . . . . . . . . . . . .60CHAPTER-55. BIOLOGICAL EVALUATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 615.1 Fluorometric assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 625.1.1 Preparation of WGA solution & sampling to measure PL . . . . . . . . . . . . . . . . . .625.1.2 PL analysis for GlcNAc only . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 635.1.3 PL analysis for compound 10 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .655.1.4 PL analysis for ABG complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .675.1.5 Kinetic affinity results for PL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .69CHAPTER-66. BIOSENSOR ASSAY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 706.1 Surface Plasmon Resonance (SPR) experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . 716.1.1 pH Scouting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 716.1.2 Immobilization of WGA to CM5 chip . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 736.1.3 Analytes used for kinetic analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 766.1.4 Kinetic analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 786.1.4.1 Regeneration of the sensor chip surface . . . . . . . . . . . . . . . . . . . . . . . . 806.1.4.2 Kinetic analysis for ABG complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 826.1.4.3 Kinetic analysis for glycopolymer 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 846.1.4.4 Kinetic analysis for glycopolymer 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 866.1.4.5 Kinetic analysis for glycopolymer 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 886.2 Kinetic parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .90CHAPTER-77. COMPARATIVE STUDY BETWEEN SPR & PL . . . . . . . . . . . . . . . . . . . . . . . . . . . .91CHAPTER-88. CONCLUSIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .92CHAPTER-99. GENERAL METHODS & MATERIALS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .94CHAPTER-1010. SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105List of conferences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119List of publications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120Awards and Honors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122Declaration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124Thesis dedicated to . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126 主指導教員 : 松岡浩司
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