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Title: Biomolecules as recognition elements for bioactive polyphenols in coffee
Other Titles: Biomolecole come elementi di riconoscimento per polifenoli del caffè
Authors: Sinisi, Valentina
Supervisore/Tutore: Berti, Federico
Cosupervisore: Forzato, Cristina
Issue Date: 7-Apr-2014
Publisher: Università degli studi di Trieste
Coffee is a worldwide diffused beverage and it has a very high impact on the global economy. The success of coffee is due to the perfect combination between the overall involvement of our senses and the well-known stimulating effect.
During the years, many studies focused on its volatile fraction, but our knowledge about the taste responsible compounds is still nowadays scarce. Among such compounds present in the coffee beans, chlorogenic acids (CGAs), that belong to the polyphenols family, acquire more and more importance, due to their key-role in determining the coffee flavor, particularly its bitterness, their beneficial health properties, and their possible use as marker to control the industrial process. The roasting of green coffee beans causes a partial loss of CGAs due to the formation of degradation products, such as the corresponding lactones, whose amount depends on the roasting degree; the identification and the quantification of acids and lactones in coffee could be therefore a useful parameter to evaluate the final quality of the beverage.
This PhD project arises from the interest in searching new methodologies for the selective analysis of the lactone fraction, exploiting the recognition properties of biomolecules, proteins or peptides, or of designed sensing elements with high affinity for such lactonic compounds.
The chosen molecules, not commercially available, were first synthesized and fully characterized, namely: 3,4-O-dicaffeoyl-1,5-gamma-quinide, 3-O-[3,4-(dimethoxy)cinnamoyl]-1,5-gamma-quinide, 3,4-O-bis[3,4-(dimethoxy)cinnamoyl]-1,5-gamma-quinide, and 1,3,4-O-tris[3,4-(dimethoxy)cinnamoyl]-1,5-gamma-quinide. A direct synthesis of the tri-substituted quinide was also tuned up, starting from D-(-)-quinic acid and 3,4-dimethoxycinnamoyl chloride. Considering the potential biological activities of polyphenols, the antiviral properties of these compounds against many viruses have been also evaluated.
A possible approach to develop a selective biosensor is to use natural peptide scaffolds, with stable and highly organized conformations, and reduce its dimensions down to the limit of receptor stability, exploiting even the randomization of the aminoacid within the binding site to improve its ligand properties. In this perspective, the specific binding constants to Human Serum Albumin (HSA), more exactly to its Sudlow site I, of caffeic acid, ferulic acid, 3,4-dimethoxycinnamic acid, 5-O-caffeoyl quinic acid and of the four synthesized quinides were measured in physiological conditions by fluorescence spectroscopy, reaching promising KD values, in the micromolar range; moreover 3,4-O-dicaffeoyl-1,5-gamma-quinide gave a peculiar result, showing a very interesting double binding in the same active site of the protein.
The two diester quinides were also used to test, always by means of fluorescence spectroscopy, the binding ability of a functional 100 aminoacids fragment that replicates the binding site I of the whole protein: this peptide, called HSA100, has been obtained by our research group; even some mutants of HSA100 have been prepared and four of them, randomly chosen, were used in the fluorescence assays. The obtained binding constants with HSA100 and its four mutants remain in the micromolar range, close to those measured with the whole protein, suggesting that proper mutations could lead to a selective biosensor with high affinity for the quinides.
The Surface Plasmon Resonance (SPR) technique could offer a way for the fast screening of the binding properties. In the perspective of setting up a SPR method to quickly select the hits of the mutants libraries, a linker-equipped quinide was synthesized and immobilized on a gold chip, then the binding with HSA was tested in this way.
Considering the well-known interaction between polyphenols compounds, such as chlorogenic acid, and caffeine in aqueous solution, NMR titrations were performed to study the behavior of caffeine with the synthesized quinides. This kind of binding may be exploited to develop a selective biosensor for quinides by introducing several molecules of caffeine or other xanthines on a suitable scaffold.
Ciclo di dottorato: XXVI Ciclo
Keywords: coffee
chlorogenic acids
miniaturized receptors
Language: en
Type: Doctoral Thesis
Settore scientifico-disciplinare: CHIM/06 CHIMICA ORGANICA
NBN: urn:nbn:it:units-12564
Appears in Collections:Scienze chimiche

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