Synthesis, structural analysis, and characterization of various glycosides using glycosyltransferases from Leoconostoc mesenteroides = Leoconostoc mesenteroides 유래의 glycosyltransferase들을 이용한 다양한 배당체 합성, 구조분석 및 특성 연구

국문 초록 (Abstract)

Leuconostoc mesenteroides B-1299CB와 512FMC M1FT로부터 얻은 당전이 효소를 이용하여 EGCG, quercetin, arbutin 및 acarbose 배당체 화합물들을 합성하였다. 이들 화합물들은 각각 epigallocatechin gallate 7-O-a-D-glucopyranoside, epigallocatechin gallate 7,4"-O-a-D-glucopyranoside, epigallocatechin gallate 7,4'-O-a-D-glucopyranoside, epigallocatechin gallate 4"-O-a-D-glucopyranoside, epigallocatechin gallate 4'-O-a-D-glucopyranoside, quercetin-3-O--D-glucopyranoside, quercetin-4-O--D-glucopyranoside, 4-hydroxyphenyl β-isomaltoside와 4-hydroxyphenyl β-isomaltotrioside, 1І-β-D-fructofuranosyl-α-acarbose으로 구조해석하였다. 이들 배당체 화합물들은 항산화 활성에 있어서는 수용체 화합물보다는 낮았지만, 물에 대한 용해도, 항갈변 효과가 크게 증가되었으며, tyrosinase 와 당분해 관련 효소들의 저해 활성은 그대로 유지되고, 생체내 gluconeogenesis와 antioxidant 활성에 있어서는 수용체 보다 전반적으로 좋은 효과를 보였다. 앞으로 glcosyltransferases와 이들 산물들은 식품, 화장품 및 의약 산업에 응용될 수 있을 것으로 사료된다.

다국어 초록 (Multilingual Abstract)

Various glycosides were synthesized by the reaction of acceptor reaction with sucrose and glycansucrases from Leuconostoc mesenteroides. Their structures were assigned as epigallocatechin gallate 7-O-a-D-glucopyranoside, epigallocatechin gallate 7,4"-O-a-D-glucopyranoside, epigallocatechin gallate 7,4'-O-a-D-glucopyranoside, epigallocatechin gallate 4"-O-a-D-glucopyranoside, epigallocatechin gallate 4'-O-a-D-glucopyranoside, epigallocatechin gallate 4¢,4"-O-a-D-glucopyranoside, quercetin-4-O--D-glucopyranoside, quercetin-3-O--D-glucopyranoside, 4-hydroxyphenyl β-isomaltoside, 4-hydroxyphenyl β-isomaltotrioside, 1І-β-D-fructofuranosyl-α-acarbose after 1H, 13C, HSQC, H-H COSY, HMBC analyses.
Epigallocatechin gallate glucosides showed a different antioxidant effects according to their structures and showed the strong stability in a browning resistance than epigallocatechin gallate. Furthermore, the water solubility of the glucosides was 50-120 times higher than epigallocatechin gallate. Quercetin-4-O--D-glucopyranoside evidenced slower effects on DPPH radical scavenging activity (SC50 = 25.2 μM) than quercetin (SC50 = 6.5 μM). The water solubility was 12.7 mM, whereas the quercetin was barely soluble in water. The Ki value of quercetin-4-O--D-glucopyranoside (674.5 μM) was almost identical to that of quercetin (673.3 μM) with regard to tyrosinase inhibition effects. 4-Hydroxyphenyl β-isomaltoside exhibited slower effects on DPPH radical scavenging effects than arbutin and almost similar effects on tyrosinase inhibition effects. However, the glucoside showed better inhibitory effect than arbutin on MMP-1 production induced by UVB. 1І-β-D-fructofuranosyl-α-acarbose showed stronger effects on inhibition effect against α-amylase (porcin pancreatics) and amyloglucosidse (aspergillus niger), not α-glucosidase (baker’s yeast) and CGTase (Bacillus marcerans), comparing with that of acarbose.

목차 (Table of Contents)

• Ⅰ. Introduction = 11
• Ⅱ. Literature review = 14
• 1. Glycosyltranferase = 14
• A. Glucansucrases = 14
• B. Levansucrase = 18
• C. Acceptor reaction mechanism of glycansucrase = 21
• 2. Various acceptors and their glycosylation = 28
• 3. Objectives of this study = 33
• Ⅲ. Synthesis, structural analysis and characterization of epigallocatechin gallate glucosides using glucansucrase = 34
• 1. Introduction = 34
• 2. Materials and methods = 36
• A. Materials = 36
• B. Preparation of purified glucansucrase = 36
• C. Glucansucrase activity assays = 36
• D. Synthesis of EGCG glucosides = 37
• E. Sephadex LH-20 column chromatography = 37
• F. High performance liquid chromatography = 38
• G. Matrix-assisted laser desorption ionization-time of flight analysis = 38
• H. Structure determination using nuclear magnetic resonance = 39
• I. DPPH radical scavenging effects = 39
• J. Browning resistance effect = 40
• K. Water solubility = 40
• L. Animals and treatments = 41
• M. Blood and tissue sampling = 41
• N. Primer extension and real-time reverse transcription-PCR analysis = 42
• O. Statistical analysis = 43
• 3. Results and discussion = 45
• A. Synthesis and purification of EGCG glucosides = 45
• B. Structural determination of EGCG glucosides = 48
• C. DPPH radical scavenging effects = 64
• D. Browning resistant effects = 66
• E. Water solubility = 66
• F. Biochemical changes in rats = 67
• G. Comparison of gluconeogenesis genes (PEPCK, FBP, and G6P) expression = 67
• H. Comparison of antioxidant genes (CAT, SOD, and GSH) expression = 71
• Ⅳ. Synthesis, structural analysis and characterization of quercetin glucosides using glucansucrase = 76
• 1. Introduction = 76
• 2. Materials and methods = 77
• A. Materials = 77
• B. Preparation of purified glucansucrase = 77
• C. Synthesis of quercetin glucosides = 78
• D. Purification by organic solvent partition = 78
• E. High performance liquid chromatography = 79
• F. Matrix-assisted laser desorption ionization-time of flight analysis = 79
• G. Structure determination using nuclear magnetic resonance = 80
• H. Antioxidant effects = 80
• I. Water solubility = 81
• J. Tyrosinase inhibition effect = 81
• 3. Results and discussion = 83
• A. Synthesis and purification of quercetin glucosides = 83
• B. Structural determination of quercetin glucosides = 83
• C. DPPH radical scavenging effects = 88
• D. Tyrosinase inhibition effects = 90
• E. Water solubility = 91
• Ⅴ. Synthesis, structural analysis and characterization of arbutin glucosides using glucansucrase = 94
• 1. Introduction = 94
• 2. Materials and methods = 96
• A. Materials = 96
• B. Preparation of purified glucansucrase = 96
• C. Synthesis of arbutin glucosides = 96
• D. Bio gel P-2 column chromatography = 97
• E. High performance liquid chromatography = 97
• F. Matrix-assisted laser desorption ionization-time of flight analysis = 98
• G. Structure determination using nuclear magnetic resonance = 98
• H. Antioxidant effects = 98
• I. Tyrosinase inhibitory effect = 99
• J. UV irradiation and MMP-1 production test = 99
• K. Enzyme-linked immunosolvent assay = 100
• 3. Results and discussion = 101
• A. Synthesis and purification of arbutin glucosides = 101
• B. Structural determination of arbutin glucosides = 101
• C. Antioxidant effects = 108
• D. Tyrosinase Inhibitory Effect = 108
• E. UV irradiation and MMP-1 production test = 112
• Ⅵ. Synthesis, structural analysis and characterization of acarbose fructosides using levansucrase = 114
• 1. Introduction = 114
• 2. Materials and methods = 116
• A. Materials = 116
• B. Preparation of purified levansucrase = 116
• C. Synthesis of acarbose fructosides = 117
• D. Bio gel P-2 column chromatography = 117
• E. High performance liquid chromatography = 118
• F. Matrix-assisted laser desorption ionization-time of flight analysis = 118
• G. Structure determination using nuclear magnetic resonance = 118
• H. Inhibition effects on diabetes-related enzymes = 119
• 3. Results and discussion = 120
• A. Synthesis and purification of acarbose fructosides = 120
• B. Structural determination of acarbose fructosides = 120
• C. Inhibition effects on diabetes enzymes = 127
• Ⅶ. Conclusions = 132
• Ⅷ. References = 135
• Abbreviation = 151
• 국문초록 = 153
• 감사의 글 = 155