UNIVERSITÀ DEGLI STUDI DI TRIESTE XXVI CICLO DEL DOTTORATO DI RICERCA IN SCIENZA, TECNOLOGIA ED ECONOMIA NELL’INDUSTRIA DEL CAFFÈ BIOMOLECULES AS RECOGNITION ELEMENTS FOR BIOACTIVE POLYPHENOLS IN COFFEE CHIM/06 Ph.D. Student Valentina Sinisi Ph.D. program Coordinator and Thesis Supervisor Dr. Federico Berti Thesis Co-supervisor Dr. Cristina Forzato ANNO ACCADEMICO 2012/2013 Odi et amo. Quare id faciam, fortasse requiris. Nescio, sed fieri sentio et excrucior.* Catullus, carmen 85 * I hate and I love. Wherefore would I do this, perhaps you ask? I do not know. But I feel that it happens and I am tormented. Table of contents Table of contents Table of contents .............................................................................................................................. i Abstract .............................................................................................................................................. v List of abbreviations ..................................................................................................................... xii Chapter 1 - Introduction ................................................................................................................. 1 1.1 Coffee polyphenols ................................................................................................................... 3 1.1.1 Coffee: a successful beverage ............................................................................................. 3 1.1.2 The composition of coffee .................................................................................................. 3 1.1.3 The aroma of coffee ............................................................................................................. 5 1.1.4 The taste of coffee ................................................................................................................ 5 1.1.5 Phenolic compounds ........................................................................................................... 6 1.1.5.1 Chlorogenic acids (CGAs) ............................................................................................... 7 1.1.5.2 Thermal transformation of CGAs ................................................................................... 9 1.1.5.3 Chlorogenic lactones (CGLs) ......................................................................................... 10 1.1.5.4 Chlorogenic compounds are absorbed in humans ......................................................... 12 1.2 Recognition and sensing elements ....................................................................................... 19 1.2.1 Sensors: biomolecules and their mimics ........................................................................ 19 1.2.2 Molecular recognition elements ...................................................................................... 20 1.2.2.1 Antibodies ...................................................................................................................... 20 1.2.2.2 Receptors ....................................................................................................................... 21 1.2.2.3 Lectins ........................................................................................................................... 21 1.2.2.4 Short peptides ............................................................................................................... 21 1.2.2.5 Aptamers ....................................................................................................................... 24 1.2.2.6 Peptide Nucleic Acids ................................................................................................... 25 1.3 Human Serum Albumin ......................................................................................................... 31 1.3.1 Human Serum Albumin as a starting point .................................................................. 31 1.3.2 HSA structure and properties .......................................................................................... 31 1.3.3 Binding sites ....................................................................................................................... 32 1.3.3.1 Site I .............................................................................................................................. 34 i Table of contents 1.3.3.2 Site II ............................................................................................................................. 36 1.3.4 Influence of endogenous ligands on HSA binding properties .................................... 37 1.4 HSA100 and mutant libraries ................................................................................................ 41 1.4.1 Directed evolution strategy .............................................................................................. 41 1.4.2 HSA100 ............................................................................................................................... 41 1.4.2.1 HSA100 identification and optimization ...................................................................... 41 1.4.2.2 Expression of HSA100 .................................................................................................. 42 1.4.3 HSA100 mutant libraries .................................................................................................. 44 1.4.3.1 Structural analysis ........................................................................................................ 44 1.4.3.2 HSA100 site-directed mutagenesis ............................................................................... 45 Chapter 2 - Aim of research ......................................................................................................... 47 2. Aim of research .......................................................................................................................... 49 Chapter 3 - Results and discussion ........................................................................................... 51 3.1 Quinides synthesis .................................................................................................................. 53 3.1.1 Synthesis of 3,4-O-dicaffeoyl-1,5--quinide (7) .............................................................. 53 3.1.2 Synthesis of 1,3,4-O-tris[3,4-(dimethoxy)cinnamoyl]-1,5--quinide (10), 3,4-O- bis[3,4-(dimethoxy)cinnamoyl]-1,5--quinide (11), and 3-O-[3,4-(dimethoxy)cinnamoyl]- 1,5--quinide (12) ........................................................................................................................ 55 3.1.3 Synthesis of 3-O-[4-(aminobutanamidoethoxy)feruloyl]-4-O-[3,4- (dimethoxy)cinnamoyl]-1,5--quinide (28) ............................................................................. 59 3.2 HSA, HSA100 and its mutants: interaction studies .......................................................... 65 3.2.1 Human serum albumin (HSA) ........................................................................................ 65 3.2.1.1 The choice of fluorescence spectroscopy ......................................................................... 65 3.2.1.2 Titration assays ............................................................................................................. 66 3.2.1.3 HSA fluorescence quenching ......................................................................................... 67 3.2.1.4 Stern-Volmer analysis ................................................................................................... 69 3.2.1.5 Dissociation constants and binding sites ...................................................................... 71 3.2.2 HSA100 and its mutants ................................................................................................... 77 3.2.2.1 Binding biomolecules and titration assays .................................................................... 77 3.2.2.2 Fluorescence quenching and dissociation constants ..................................................... 79 3.3 Surface Plasmon Resonance and Biacore assay ................................................................. 83 3.3.1 Surface Plasmon Resonance ............................................................................................. 83 3.3.2 Evaluation of the association constant ........................................................................... 85 3.3.3 The experiment .................................................................................................................. 86 ii Table of contents 3.4 NMR investigation on the caffeine-quinides interaction ................................................ 91 3.4.1 Caffeine interacts with polyphenols ............................................................................... 91 3.4.2 Study of the interaction between caffeine and the synthesized quinides ................. 91 3.4.2.1 Titrations of 7, 10, 11 and 12 with caffeine in DMSO-d6/D2O 75-25% ...................... 92 3.4.2.2 Titrations of 7 with caffeine in D2O/DMSO-d6 75-25% .............................................. 96 3.5 Antiviral assays ...................................................................................................................... 103 3.5.1 Further investigations of the bioactivity of the chlorogenic compounds ................ 103 3.5.2 Influenza virus ................................................................................................................