| Abstract: | The sequence Arg-Gly-Asp (RGD) is present in fibrinogen, which is a protein of blood that can be selectively bound to the main glycoprotein receptor of the platelet surface (GPIIb/IIIa) promoting platelet aggregation. Competitive binding of RGD containing peptides and fibrinogen to the platelet receptor GPIIb/IIIa has triggered the interest of many researchers in their attempts to find new, more potent anticoagulant agents. On the other hand, the fact that Southern French people have very low incidence of coronary heart disease despite their high fat diet and heavy smoking habits, which is also known as “French Paradox”, has strongly been related to moderate red wine consumption. In particular, the beneficial properties of red wine have been attributed to the presence of resveratrol (3,4΄,5-trihydroxy-trans-stilbene), a molecule that exerts a broad range of biological activities, among which is its ability to inhibit platelet aggregation. In the present thesis the synthesis of linear Arg-Gly-Asp (RGD) analogs and derivatives is reported. In particular, the basic amino acid arginine was substituted by lysine or histidine, while derivatives of the Leu-Asp dipeptide were also synthesized. Peptides with free carboxylic group on their C-termini were synthesized, while the β-carboxylic group of the aspartic acid was either free or protected by the benzyl group. Peptide amides with free β-carboxylic group of the aspartic residue were also synthesized, while in all the above cases the synthesis of the corresponding peptide derivatives with acetylated α-amino group of the N-terminal amino acid occurred. Finally, the conjugation of peptide derivatives with resveratrol took place. The peptide synthesis was carried out by the solid phase technique according to the Fmoc/tBu strategy using the 2-chlorotrityl chloride resin as solid support, for the synthesis of peptides with free carboxylic group in their C-termini, while for the synthesis of peptide amides the Rink Amide MBHA resin was used. The coupling reactions took place via the method of active esters (DIC/HOBt). The conjugation of the peptide derivatives with resveratrol took place in solution using DCC as coupling reagent and DMAP as catalyst, at room temperature, protecting the reaction mixture from light exposure. All the synthesized compounds were purified by reversed phase high performance liquid chromatography (RP-HPLC) and identified by mass spectrometry (ESI-MS). The synthesized compounds were tested for inhibitory activity on human platelet aggregation in vitro, by adding common aggregation agonists to citrated platelet rich plasma (PRP). The aggregation was determined using a four channel aggregometer by recording the light transmission. In vitro studies on the influence of the peptides and their conjugation products with resveratrol on human platelet aggregation have demonstrated that peptide-amides are active against platelet aggregation triggered by ADP (10µM) or collagen (2µg/ml). As a matter of fact, the platelet amides Ac-Arg-Gly-Asp-NH2 and Arg-Gly-Asp-NH2 inhibited platelet aggregation significantly. Regarding the conjugation products of peptide derivatives with resveratrol, they also present inhibitory activity on platelet aggregation induced by ADP (10µM), though the quantitative determination of the biological activity of resveratrol derivatives was restricted due to their limited solubility in aqueous solutions. Finally, an attempt was made to molecularly imprint the Arg-Gly-Asp-OH (RGD) tripeptide in polymers, in order to determine whether these MIPs can be farther used in the selective separation of RGD derivatives. In particular, six molecular imprinted polymers were synthesized combining two different monomers, such as methacrylic acid and acrylamide, with three cross-linkers (EGDMA, TRIM and Ν,Ν΄-methylene-bis-acrylamide), while azobis-cycloexanecarbonitrile was used as polymerization initiator. The polymer produced by the co-polymerization of MAA and TRIM and to a lesser extend the polymer synthesized by the combination of acrylamide and EGDMA were found to exert greater RGD rebinding efficiency than the rest, and also greater selectivity for RGD against other peptides (CCK-3, CCK-5, Gramicidin). These results are considered to be very interesting, since it is proved not only that the molecular imprinting technique can be used for the production of polymers recognizing the RGD sequence, but also that these polymers could be possibly applied in the purification of several RGD derivatives and their conjugation products |
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