1. ELECTROCHEMISTRY OF QUERCETIN ON GOLD ELECTRODES MODIFIED WITH THIOLATED CYCKLODEXTRINS MONOLAYERS Anna Huszał b), Sylwester Huszał a), Andrzej Temeriusz* a), Jerzy Golimowski a) a) Faculty of Chemistry, Warsaw University; Pasteura 1, PL-02-093 Warsaw, Poland; * ) Email: atemer@alfa.chem.uw.edu.pl b) Oil and Gas Institute, Warsaw Brench; Kasprzaka 25, 01-224 Warsaw, Poland; Email: huszal@inig.pl Cyclodextrins (CDs) are torus-shaped cyclic oligosaccharides, made up of 6(α), 7(β), or 8(γ) α(1 4) linked D- glucose units, which have a relatively hydrophobic cavity for guest binding 1. These compounds are therefore potent hosts for guest molecules capable of entering the cavity and thus forming noncovalent host-guest inclusion complexes. The versatility of CDs as hosts is related to different cavity sizes and the possibility of one or many alcohol moieties functionalization 2. Cyclodextrins substituted with thiol groups have been widely used to modify the surface of electrodes. Their molecules are spontaneously chemisorbed on Au surface to give highly organised self-assembly monolayer 3. This work describes a chemically modified electrodes (CMEs) based on suitable cyclodextrin derivatives in the form of self-assembled monolayer (SAM). Our work regards the synthesis of thiolated cyclodextrins, the preparation of CME based on SAM formation on gold electrode, their electrochemical characterisation and potent analytical applications. Thiolated cyclodextrin derivatives were prepared according to the procedure reported by Stoddart et al. 4. Per-(6- deoxy-6 thio-2,3-di-O-methyl)-α- and β-cyclodextrins (SH-αCD and SH-βCD, respectively; Figure 1) were prepared in four steps from native species. The synthesis involved the following sequence of reactions: protection of the primary side by silyl groups, exhaustive methylation of the secondary rim, removing of the protection of primary hydroxyl groups with simultaneously conversion to the bromide and finally into thiol functionalities. The monolayer of the particular cyclodextrin derivative was formed by immersion of a well cleaned gold electrode into a deoxygenated 1mM solutions of SH-αCD and SH-βCD, respectively, in DMF overnight. The properties of the obtained SAMs did not significantly improve by changing the formation time or the solution concentration. The SAM makes up an array of ultramicroelectrodes, which capture electroactive molecules such as those of flavonoids when the SAM-modified electrode is exposed to their solutions. Flavonoids are benzo-γ-pyrone derivatives widely distributed in nature. In this family, polyhydroxyflavones such as quercetin (Figure 2) are the subgroup of well-known natural antioxidant molecules 5. Quercetin with the catechol group on the B-ring (3,4-dihydroxyl) is the most representative dietary flavonoid and the potent antioxidant. Due to the 3,4-dihydroxy substitution patterns it is one of the most easily oxidised flavonoids. In our studies quercetin was found to form supramolecular complexes with both investigated cyclodextrin derivatives immobilised on the gold surface. The voltammograms of the cyclodextrin modified electrodes recorded for appropriate quercetin solution reveal the presence of its molecules in the cyclodextrin cavities. The aim of this work was to prepare and investigate the electrochemical behaviour and application of the gold electrode modified with monolayers of thiolated cyclodextrins. In α- and β-cyclodextrin molecules, the six and seven primary hydroxyl groups respectively have been replaced by thiol groups. The electrochemical data presented here indicate that these receptors chemisorb strongly and readily on the gold surface through the formation of thiolate-gold bonds. The resulting monolayers of α- and β-cyclodextrins exhibit excellent binding ability toward appropriate species in the contacting solution. Our voltammetric data demonstrate that quercetin molecules are included in the cavities of the interfacial receptors. Modified electrodes showed effective binding properties when immersed in aqueous solution containing low concentrations (10 -6 M for SH- αCD and 510 -7 M for SH-βCD) of quercetin. Their voltammetric response exhibited the waves anticipated for the reversible oxidation of the surface-confined (cyclodextrin-bound) quercetin molecules. Hendrickson and co-workers 5 reported the electrochemistry of quercetin at a glassy carbon electrode. According to them the response of 3, 4-adjacent hydroxyl groups is a two-electron and two- proton electrode reaction. We obtained similar results using gold electrodes. This oxidation is a chemically reversible process. The oxidation peak (E pa = + 0.140 V) corresponds to the oxidation of the 3, 4-dihydroxy substituents on the B-ring. A reduction peak (E pc =+ 0.100 V) appeared on the negative scan, indicating that the electrochemical oxidation product was easily reduced. The reversibility is highly scan rate dependent, indicating an electrochemical-chemical reaction mechanism. The effect of scan rate on the reversibility of the I a /I c couple observed by cyclic voltammetry for quercetin is shown in Figure 5. The peak currents of quercetin are linearly dependent on the scan rate. This suggests that the electrode reaction of this molecule is a reversible surface electrochemical reaction, with both the reactant and product strongly adsorbed on the electrode surface. We observed the electrochemical oxidation of quercetin for gold electrodes modified with monolayers of thiolated cyclodextrins, too. It allowed us to quantitatively determine very low amounts of the electroactive compounds in its methanolic solutions. Figure 1. Synthesis of thiolated cyclodextrins. Figure 2. Proposed mechanism of electrochemical oxidation of quercetin. Figure 5. Cyclic voltammetric peak current ratio as a function of scan rate in sodium phosphate buffer. Figure 3. Cyclic voltammograms for the Au electrode coated with a monolayer of SH- CD (green line), SH- CD (blue line), SH- CD (violet line) and for a bare Au electrode (red line) in sodium phosphate buffer (v=0.1 V/s). Figure 6. Cyclic voltammograms for the Au electrode coated by a monolayer of SH- CD recorded in the presence of 10 -6, 210 -6, 410 -6, 10 -5, 210 -5, 410 -5, 10 -4 M of quercetin in sodium phosphate buffer (v=0.1 V/s). Figure 4. Calibration curve for quercetin determination recorded at bare Au electrode in sodium phosphate buffer (v=0.1 V/s). Figure 7. Calibration curve for quercetin determination recorded at Au electrode coated by a monolayer of SH- CD in sodium phosphate buffer (v=0.1 V/s). Figure 8. Cyclic voltammograms for the Au electrode coated by a monolayer of SH- CD recorded in the presence of 10 -6, 510 -6, 10 -5, 210 -5, 510 -5, 10 -4 M of quercetin in sodium phosphate buffer (v=0.1 V/s). Figure 9. Calibration curve for quercetin determination recorded at Au electrode coated by a monolayer of SH- CD in sodium phosphate buffer (v=0.1 V/s). References: 1) J.Szejtli, Chem. Rev. 98, 1743 (1998). 2) e.g. G. Wenz, Angew. Chem. Int. Ed. Engl. 33, 803 (1994). 3) A. E. Kaifer, M. Gómez-Kaifer, Supramolecular Electrochemistry, Wiley-VCH, Weinheim (1999). 4) M. T. Rojas, R. Königer, J. F. Stoddart, A. E. Kaifer, J. Am. Chem. Soc. 117, 336 (1995). 5) H. P. Hendrickson, A. D. Kaufman, C. E. Lunte, J. Pharm. Biomed. Anal. 12, 325 (1994). PPh 3 /Br 2 CH 2 Cl 2 (H 2 N) 2 C=S KOH DMF Cyclic voltammetry measurements were performed in a three-electrode arrangement with Ag/AgCl electrode as the reference, platinum wire as the counter, and gold electrode as the working electrode. A supporting electrolyte used in all experiments was 0.05 M sodium phosphate buffer (pH=7) with 0.5 M potassium nitrate in 50% methanol. Acknowledgment Financial support from the Warsaw University is gratefully acknowledged.