LIST OF CONTENTS ................................................ I
LIST OF TABLES ................................................. IV
LIST OF FIGURES ................................................. V
LIST OF ABBREVIATIONS ......................................... VII
1 INTRODUCTION ................................................. 1
2 BACKGROUND ................................................... 3
2.1 Maillard reaction in food ............................... 3
2.1.1 Melanoidins ...................................... 8
2.2 Coffee ................................................. 11
2.2.1 General aspects ................................. 11
2.2.1.1 Coffee production ........................... 12
2.2.1.2 General chemical composition ................ 14
2.2.1.3 Coffee and health ........................... 20
2.2.2 Coffee melanoidins .............................. 24
2.2.2.1 Chemistry of coffee melanoidins ............. 24
2.2.2.2 Properties of coffee melanoidins ............ 29
2.3 Zinc metallopeptidases ................................. 32
2.3.1 Matrix metalloproteinases (MMPs) ................. 33
2.3.1.1 Functions of MMPs ........................... 35
2.3.1.2 Structure of MMPs ........................... 37
2.3.1.3 Inhibition of MMPs .......................... 39
2.3.2 Clostridium histolyticum collagenase (ChC) ...... 43
2.3.2.1 Functions of ChC ............................ 43
2.3.2.2 Structure of ChC ............................ 43
2.3.2.3 Inhibition of ChC ........................... 44
2.3.3 Agiotensin converting enzyme (ACE) .............. 45
2.3.3.1 Functions of ACE ............................ 45
2.3.3.2 Structure of ACE ............................ 46
2.3.3.3 Inhibition of ACE ........................... 48
3 EXPERIMENTAL SECTION ........................................ 50
3.1 Chemicals, materials and equipment ...................... 50
3.1.1 Chemicals ....................................... 50
3.1.2 Material ........................................ 52
3.1.3 Equipment ....................................... 52
3.1.4 Solutions ....................................... 54
3.2 Synthesis of Nα-carboxyalkylated peptides .............. 55
3.2.1 Nα-carboxyalkylation of GP, LL, IA, GA, GL,
AP, IP and IPP by reductive alkylation .......... 55
3.2.2 Nα-carboxyalkylation of IW using sodium
cyanoborohydride ................................ 56
3.3 Purification ........................................... 57
3.3.1 Ion Exchange Chromatographic purification ....... 57
3.3.1.1 Spotting test ............................... 58
3.3.2 HPLC purification of CM-IW ...................... 58
3.3.3 Overview of the synthesis and elution
conditions ...................................... 59
3.4 Characterization of carboxyalkylated peptides .......... 61
3.4.1 Mass spectrometry ............................... 61
3.4.2 Elemental Analysis .............................. 61
3.4.3 Analytical characteristics of carboxyalkylated
peptides ........................................ 62
3.5 Preparation of coffee fractions ........................ 65
3.5.1 Roasting conditions ............................. 65
3.5.2 Fractionation of coffee samples: Isolation of
coffee melanoidins .............................. 67
3.6 Structural studies ..................................... 69
3.6.1 Estimation of the molecular weight .............. 69
3.6.2 C/N ratio ....................................... 70
3.6.3 Amino acid analysis ............................. 70
3.6.3.1 Acid hydrolysis ............................. 70
3.6.3.2 General amino acid analysis ................. 70
3.6.3.3 Lysinoalanine ............................... 71
3.6.3.4 Pentosidine ................................. 72
3.6.4 Total phenols ................................... 74
3.6.5 Raman spectroscopy .............................. 74
3.7 Study on inhibition of zinc metalloproteases ........... 75
3.7.1 Inhibition of ACE ................................ 75
3.3.7.1 General enzymatic assay ..................... 75
3.3.7.2 Quantification .............................. 78
3.7.3 Inhibition of MMP-1, -2 and -9 .................. 79
3.7.2.1 General enzymatic assay ..................... 80
3.7.2.2 Effect of zinc addition on the inhibition
of MMP-1 by melanoidins ..................... 81
3.7.3 Inhibition of ChC ............................... 82
3.7.3.1 General enzymatic assay ..................... 82
3.7.3.2 Quantification .............................. 84
3.7.4 Calculation of IC50 ............................. 84
4 RESULTS AND DISCUSSION ...................................... 86
4.1 Coffee melanoidins ..................................... 86
4.1.1 Isolation of coffee fractions ................... 86
4.2 Inhibition of zinc-dependent peptidases by coffee
fractions .............................................. 89
4.2.1 Inhibition of MMPs .............................. 89
4.2.2 Inhibition of other zinc metalloproteases ....... 98
4.2.3 General considerations ........................... 99
4.3 Structural studies on coffee melanoidins .............. 101
4.3.1 Gel permeation chromatography .................. 102
4.3.2 Elemental analysis: C/N ratio .................. 113
4.3.3 Amino acid analysis ............................ 116
4.3.4 Total phenolics ................................ 120
4.3.5 Correlation between total phenols content and
C/N ratio in coffee melanoidins ................ 123
4.3.6 Raman spectroscopy ............................. 124
4.4 Derivatization of peptides ............................ 129
4.4.1 Nα-carboxyalkylation of peptides by reductive
alkylation ..................................... 130
4.5 Preliminary investigations on the inhibitory
potential of Nα-carboxyalkyl derivatives of peptides
against metalloproteases .............................. 133
4.5.1 Inhibition against ACE ......................... 134
4.5.2 Inhibition against other zinc
metalloproteases ............................... 138
5 SUMMARY 1 .................................................. 141
6 REFERENCES ................................................. 145
LIST OF PUBLICATIONS AND CONFERENCE CONTRIBUTIONS ............. 168
AKNOWLEDGMENTS ................................................ 169
ERKLÄRUNG ..................................................... 170
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