Dipartimento Di Chimica E Tecnologie Del Farmaco N-Linked
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Dipartimento di Chimica e Tecnologie del Farmaco XXIV Ciclo di Dottorato in Scienze Farmaceutiche N-linked peptidoresorc[4]arene-based receptors designed for protein surface recognition. NMR analysis of Italian virgin olive oils and structural analysis of polysaccharides. Supervisor: PhD Student: Prof. Bruno Botta Antonella Cerreto Academic year 2011-2012 Alla mia famiglia Index Contents Pag. Overview 1 Part I: N-linked peptidoresorc[4]arenes receptors designed for protein surface recognition 5 1. Introduction 6 1.1 Supramolecular chemistry 6 1.2 Molecular Recognition and Host- Guest Chemistry 8 1.3 Preorganisation and Macrocyclic Effect 11 1.4 Interactions Playing Role in Supramolecular Chemistry 16 1.5 Supramolecular chemistry and the proteins 24 1.6 Calixarenes as Artificial Receptors 28 2. Results and discussion 33 2.1 Receptors Design and Synthesis 32 2.2 NMR Analysis of N-Linked Peptidoresorc[4]arenes 38 2.3 Physical Characterization of N-Linked Peptidoresorc-[4]arenes 43 2.4 Initial Screening for Inhibition Activity 43 3. Conclusions 56 4. Experimental section 58 5. References 66 Part II: NMR characterization of Italian virgin olive oils 72 1. Introduction 73 1.1 Definitions 73 1.2 Olive oil chemical composition 74 1.3 Classification and conventional analysis of the olive oil 80 1.4 Olive oil in Italy 93 2. Food analysis with NMR 95 Index 2.1 Generalities 95 2.2 Studies of characterization, classification and identification of adulterated vegetable products 96 3. Statistical analysis of the data 100 3.1 Introduction 100 3.2 Organization of the data 100 3.3 Explorative analysis of the data 102 3.4 Analysis of the Variance 108 3.5 Principal component analysis 109 3.6 Clustering analysis 113 3.7 Linear discriminant analysis 116 4. Materials and methods 120 4.1 Sample preservation 120 4.2 Instrument and solvents 120 4.3 Protonic NMR spectrum (1H-NMR) 121 4.4 Statistical analysis applied to the signals of the 1H-NMR spectra 135 5. Results and discussion 139 5.1 Characterization of Italian olive oils 141 5.1.1 Peninsula vs islands 141 5.1.2 North, Centre, South, Islands 147 5.1.3 Single regions and single harvesting years 149 5.2 Characterization of olive oils from Sicily 151 5.2.1 Statistical analysis of the olive oils of the first year 153 5.2.2 Statistical analysis of the olive oils of the second year 157 5.2.3 Comparison between olive oils of the two harvesting years 158 5.3 Characterization of olive oils from Sardinia 160 5.3.1 Statistical analysis of the olive oils of the first year 162 5.3.2 Statistical analysis of the olive oils of second year 163 5.3.3 Comparison between olive oils of the two harvesting years 164 5.4 Characterization of olive oils from Calabria 166 5.4.1 Statistical analysis of the olive oils of the first year 168 5.4.2 Statistical analysis of the olive oils of the second year 171 Index 5.4.3 Comparison between olive oils of the two harvesting years 174 5.5 Characterization of olive oils from Apulia 175 5.5.1 Statistical analysis of the olive oils of the first year 178 5.5.2 Statistical analysis of the olive oils of the second year 183 5.5.3 Comparison between olive oils of the two harvesting years 184 5.6 Characterization of olive oils from Molise 186 5.6.1 Statistical analysis of the olive oils of the first year 188 5.6.2 Statistical analysis of the olive oils of the second year 193 5.6.3 Comparison between olive oils of the two harvesting years 196 5.7 Characterization of olive oils from Latium 197 5.7.1 Statistical analysis of the olive oils of the first year 199 5.7.2 Statistical analysis of the olive oils of the second year 205 5.7.3 Comparison between olive oils of the two harvesting years 208 5.8 Characterization of olive oils from Tuscany 210 5.8.1 Statistical analysis of the olive oils of the first year 211 5.8.2 Statistical analysis of the olive oils of the second year 212 5.8.3 Comparison between olive oils of the two harvesting years 213 5.9 Characterization of olive oils from Liguria 214 5.9.1 Statistical analysis of the olive oils of the second year 215 5.10 Characterization of olive oils from Lombardy 218 5.10.1 Statistical analysis of the olive oils of the first year 219 5.10.2 Statistical analysis of the olive oils of the second year 219 6. Conclusions 223 7. References 226 Part III: NMR structural analysis of polysaccharides 232 1. Introduction 233 1.1 Carbohydrates structure determination 233 1.2 Structural information from 1D NMR spectra 234 1.2.1 Number of sugar residues 234 Index 1.2.2 Anomeric configuration 234 1.2.3 Linkage and sequence 235 1.2.4 Position of appended groups 236 1.2.5 Structural information from 13C NMR data 236 1.2.6 Classical NMR Methods 236 1.3 Structural information from 2D NMR spectra 237 2. Structural investigation of Scleroglucan and its derivative Scl-CM-80 240 2.1 Introduction 240 2.2 Materials and methods 243 2.2.1 Preparation of the samples 243 2.2.2 NMR Spectra acquisition 243 2.2.3 NMR Spectra processing 245 2.3 Results and discussion 246 2.3.1 NMR characterization of the polysaccharides 246 2.3.2 NMR characterization of Scleroglucan 248 2.3.3 NMR characterization of Scleroglucan-CM-80 251 2.4 Conclusions 259 3. Structural investigation of polysaccharides from Hericium erinaceum 260 3.1 Introduction 260 3.2 Materials and methods 266 3.2.1 Preparation of the samples 266 3.2.2 NMR Spectra acquisition 266 3.2.3 NMR Spectra processing 269 3.3 Results and discussion 270 3.4 Conclusions 298 4. References 299 Regards 307 Overview Overview During the PhD course, the research activity focused on three main topics. N-linked peptideresorcarenes. [Part I] An important class of protein surface receptors based on the attachment of four cyclic peptides to a calix[4]arene scaffold was developed by Hamilton and targeted to the serine protease α-chymotrypsin (ChT). Moving from Hamilton’s results, we performed preliminary molecular modelling studies suggesting the design of tetra-anionically functionalized receptors that might target the predominantly cationic region of ChT surrounding the active site. We have previously designed valyl-leucine and leucyl-valine peptidoresorc[4]arenes, inspired by the evidence that N- or C-linked peptides endow calixarenes with novel properties, such as recognition of protein surfaces and carbohydrates, formation of stable inclusion complexes, and self-assembly phenomena. By varying sequence, nature, and stereochemistry of the chains we prepared anionically functionalized N- linked peptidoresorc[4]arenes by hydrogenation of their precursor benzyl esters. From this family of receptors we have identified noncompetitive inhibitors of ChT, which function by binding to the surface of the enzyme in the neighbourhood of the active site cleft (Ki values ranging from 13 to 0.8 µM). All the obtained results have been published in an article in JOC (see hereinafter). NMR characterization of Italian virgin olive oils. [Part II] Following the experimental protocol established in the Annalaura Segre NMR Laboratory (CMI, CNR in Montelibretti, Rome), based on 1H-NMR and statistical multivariate analysis of the data (ANOVA, PCA and LDA), nearly 300 Italian virgin olive oil samples, produced in two harvesting years, 1 Overview 2009/10 and 2010/11, by certified chains from Lombardy, Tuscany, Sardinia, Puglia, Calabria, Sicily, Lazio, Molise and Liguria, have been studied. The NMR results confirm that the chemical composition of olive oils, with regards to minor components, is influenced by geographical, ecological and genetic factors and depends also on the harvesting year. We could also identify some oil components that remain stable in different harvesting years, that may represent seasonal-independent characteristics of the oil produced in each region. NMR structural analysis of polysaccharides. [Part III] A contemporary investigation, carried out at CNR, was focused on structural analysis of polysaccharides. Specificaly, we performed NMR characterization of a carboxymethyl derivative of scleroglucan, a polysaccharide produced by fungi of the Sclerotium genus, to identify the positions involved in the carboxymethylation; furthermore, a preliminary structural study has been performed on a set of polysaccharides obtained from the cultures of Hericium erinaceus, with the aim of identifying the configuration and the connections between the monosaccharide units. 2 Overview The results of the research were object of the following communications: FULL PAPERS o D’Acquarica I., Cerreto A., Delle Monache G., Subrizi F., Boffi A., Tafi A., Forli S., and Botta B. J. Org. Chem., 2011, 76 (11), pp 4396–4407 “N-Linked Peptidoresorc[4]arene-Based Receptors as Noncompetitive Inhibitors for α-Chymotrypsin” ORAL COMUNICATIONS o A. Cerreto, “Peptidoresorcin[4]areni: synthesis and surface interaction studies”, Convegno unità operative PROGETTO FIRB PIATTAFORME-RETI e PRIN “ANGIOCHEMISTRY”, Certosa di Pontignano (Si), 22-23/01/2009; o A. Cerreto, F. Subrizi, B. Botta, I. D’Acquarica: “Sintesi di recettori artificiali per la ricognizione superficiale di proteine”, 10° S.A.Y.C.S., Pesaro 18-20/10/2010; o A. Cerreto, F. Subrizi, B. Botta, I. D’Acquarica “N-linked Peptidoresorc[4]arene-based Receptors for Protein Surface Recognition”, 11th International Conference on Calixarenes (Calix 11), Tarragona (Spagna), 26-29/06/2011 (Short Oral Presentation) POSTER COMUNICATIONS o A. Cerreto, F. Subrizzi, B. Botta, I. D’Acquarica: “Designed resorc[4]arene-based receptors for protein surface recognition” XXXIV Corso Estivo in Sintesi Organica "Attilio Corbella", Gargnano (Bs) 22-26/6/2009 o A.