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Apr 27, 2015 - Department of Inorganic Technology, Faculty of Chemical and Food Technology, Slovak University of Technology in Bratislava,. Radlinské...
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Electrochemistry and Spectroelectrochemistry of Bioactive Hydroxyquinolines: A Mechanistic Study Romana Sokolova, Jacek E. Nycz, Sarka Ramesova, Jan Fiedler, Ilaria Degano, Marcin Szala, Viliam Kolivoska, and Miroslav Gal J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.5b00098 • Publication Date (Web): 27 Apr 2015 Downloaded from http://pubs.acs.org on May 3, 2015

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The Journal of Physical Chemistry B is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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The Journal of Physical Chemistry

Electrochemistry and spectroelectrochemistry of bioactive hydroxyquinolines: a mechanistic study Romana Sokolová,* † Jacek E. Nycz, ‡ Šárka Ramešová, † Jan Fiedler, † Ilaria Degano, § Marcin Szala, ‡ Viliam Kolivoška, † Miroslav Gál, †,†† † J. Heyrovský Institute of Physical Chemistry, v.v.i., The Czech Academy of Sciences, Dolejškova 3, 18223 Prague, Czech Republic ‡ Institute of Chemistry, University of Silesia, Szkolna 9; PL-40006 Katowice, Poland § Department of Chemistry and Industrial Chemistry, University of Pisa, Via Moruzzi 3, 56124 Pisa, Italy †† Department of Inorganic Technology, Faculty of Chemical and Food Technology, Slovak University of Technology in Bratislava, Radlinského 9, 81237 Bratislava, Slovakia

Corresponding Author *Email: [email protected]; Tel.: +420266053525; Fax: +42028658 2307 (R. Sokolová)

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ABSTRACT. The oxidation mechanism of selected hydroxyquinoline carboxylic acids such as 8-hydroxyquinoline-7-carboxylic acid (1), the two positional isomers 2-methyl-8hydroxyquinoline-7-carboxylic acid (3), 2-methyl-5-hydroxyquinoline-6-carboxylic acid (4) as well as other hydroxyquinolines were studied in aprotic environment using cyclic voltammetry, controlled potential electrolysis, in-situ UV-Vis and IR spectroelectrochemistry and HPLCMS/MS techniques. IR spectroelectrochemistry showed that oxidation unexpectedly proceeds together with protonation of the starting compound. We proved that the nitrogen atom in the heterocycle of hydroxyquinolines is protonated during the apparent 0.7 electron oxidation process. This was rationalized by the auto-deprotonation reaction by another two starting molecules of hydroxyquinoline, so that the overall oxidation mechanism involves two electrons and three starting molecules. Both, the electrochemical and spectroelectrochemical results showed that the oxidation mechanism is not influenced by the presence of carboxylic group in the chemical structure of hydroxyquinolines, as results from oxidation of 2,7-dimethyl-5hydroxyquinoline (6). In the presence of a strong proton acceptor such as pyridine, the oxidation ECEC process involves two electrons and two protons per one molecule of the hydroxyquinoline derivative. The electron transfer efficiency of hydroxyquinolines in biosystems may be related to protonation of biocompounds containing nitrogen bases. Molecular orbital calculations support the experimental findings. TOC GRAPHICS

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KEYWORDS Electron transfer, Oxidation mechanism, Spectroelectrochemistry, Proton coupled electron transfer, Hydroxyquinoline 1. INTRODUCTION Hydroxyquinoline

carboxylic

acids

such

as

(E)-8-hydroxy-2-[2-(4,5-dihydroxy-3-

methoxyphenyl)-ethenyl]-quinoline-7-carboxylic acid, are promising integrase inhibitors, which block the replication of HIV-1 virus in cell cultures at nontoxic concentrations.1 In vitro experiments and ex vivo assays suggested that their biological activity is closely related to the presence of a carboxyl group at C-7 and a hydroxyl group at C-8 of the quinoline core, respectively.1 Although the importance of these functional groups is known with respect to their biological function, their electrochemical behaviour and/or mode of action are not clear. Since electron and proton transfers play a crucial role in biological systems, the clarification of the electrochemical behaviour or possible intramolecular proton coupled electron transfer of bioactive hydroxyquinolines is highly demanded. Since the drug mentioned above contains a hydroxyquinoline moiety in its chemical structure, the paper is focused on the electrochemical activity of simple hydroxyquinolines (Figure 1). The understanding of a detailed electron transfer mechanism coupled with proton transfer requires a study of positional isomers and derivatives. The redox behaviour of organic compounds containing hydroxyl group is often strongly dependent on the presence of proton donors or acceptors in solution.2-5 Nitrogen heterocyclic

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organic compounds such as pyridine or flavines are known to be easily protonated.6-8 Proton coupled electron transfer (PCET) in compounds including both these functionalities (hydroxyl groups and nitrogen heteroatom) can thus involve intramolecular or intermolecular proton transfer between oxygen and nitrogen atoms.9-11 Detailed electrochemical study of such effects in compounds containing a quinoline unit has not been performed yet. Some hydroxyquinoline carboxylic acids and their conjugate bases were characterized by 13C and 15N NMR spectroscopy in a solution and in a solid state.12 The crystals contain, not surprisingly, zwitterions of hydroxyquinoline carboxylic acid formed by protonation of the nitrogen atom in the quinoline unit by the carboxyl group. The hydroxyquinoline derivatives were fully characterized by spectroscopic methods including multinuclear NMR, UV-Vis, IR, MS, high resolution mass spectroscopy (HRMS) and some of them by single crystal X-ray analysis.13-15 The aim of this work is the explanation of the electrochemical behavior and elucidation of the redox mechanism of hydroxyquinoline compounds 1 - 6, which has never been performed. The relationship between molecular structure and electron and proton transfer processes is proposed.

2. EXPERIMENTAL METHODS Reagents. Compounds 1 - 6 were synthesized according to the literature.14-16 Acetonitrile (anhydrous, 99.8%), dimethylsulfoxide (DMSO, content of H2O < 0.005%) and pyridine (anhydrous, 99.8%) were purchased from Sigma-Aldrich, Germany. Tetrabutylammonium hexafluorophosphate (TBAPF6), which was used as supporting electrolyte, was obtained from Sigma and was dried before use. Acetonitrile used for HPLC (Carlo Erba, Milan, Italy) was of the HPLC grade. Predried tetrahydrofurane (THF) was heated under reflux with benzophenone and sodium until a deep violet color appeared; it was then distilled under argon.

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Electrochemical setup. Electrochemical measurements were carried out in 0.1 M TBAPF6 in acetonitrile. Cyclic voltammetry as well as exhaustive electrolysis were performed using a PGSTAT 12 AUTOLAB potentiostat (Metrohm Autolab, The Netherlands). A three-electrode electrochemical cell was used with an Ag|AgCl|1M LiCl reference electrode separated from the test solution by a salt bridge (0.44 V against Fc/Fc+). The working electrode was a glassy carbon electrode (diameter 0.7 mm). The auxiliary electrode was a platinum net. Oxygen was removed from the solution by passing a stream of argon (99.998 %, Messer). The oxidation products of 3 were prepared by exhaustive electrolysis on carbon paste electrode and identified by analytical separation techniques. UV-VIS and IR spectroelectrochemistry. Spectroelectrochemistry was performed using an optically transparent thin-layer electrode (OTTLE) cell17 with a three electrode system (Ag/AgCl reference electrode) mounted in a thin layer (thickness 0.18 mm) between optical windows (CaF2). Sufficiently optically transparent platinum gauze (80 mesh) of the size 5x5 mm served as the working electrode. The Ag-wire served as quasireference electrode. The geometry of the cell allows completing the electrolysis within time of several tens of seconds (20 s when tested with ferrocene in acetonitrile). The potential scan rate was 5 mV·s-1. Spectral changes in the course of electrolysis were registered using Agilent 8453 diode-array UV-Vis spectrometer. Spectral changes at the IR range 4000 – 1100 cm-1 during the electrolysis in OTTLE cell were recorded by Nicolet iS50 FTIR spectrometer. Additionally, the 1.0 cm quartz cuvettes were used for recording the absorption spectra when testing the stability of compound exposed to the atmospheric oxygen. High-pressure liquid chromatography with electro-spray ionization tandem mass spectrometer (HPLC-ESI-MS/MS). HPLC-ESI-MS/MS was carried out using a 1200 Infinity

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HPLC (Agilent Technologies, USA), coupled to a Jet Stream ESI interface (Agilent) with a Quadrupole-Time of Flight tandem mass spectrometer 6530 Infinity Q-TOF (Agilent Technologies). The chromatographic separation took place at 30 °C and was performed on an analytical reverse phase C-18 column (C18-extended 1.8 µm, 50 x 2.1 mm, Agilent Technologies, USA) connected to a C-18 pre-column (TC-C18 (2) 5 µm, 12.5 x 2.1 mm, Agilent Technologies, USA). The eluents were: A, 98% aqueous solution of 1% formic acid, and B, 2% solution of 1% formic acid in acetonitrile, and the flow rate was 0.2 mL·min-1. The programme was: 98% A and 2% B for 4 minutes; then to 49% A and 51% B in 7 minutes; then to 30% A to 70% B in 3 minutes; then to 10% A to 90% B in 4 minutes, hold for 4 minutes. Conditioning takes 5 minutes. Injection volume was 2 µL. The ESI operating conditions were: drying gas (N2, purity >98%): 350 °C and 10 L/min; capillary voltage 4.5 kV; nebulizer gas 35 psig; sheath gas (N2, purity >98%): 375 °C and 11 L/min. The collision gas was nitrogen (purity 99.999%). The fragmentor potential was 175 V. High resolution MS and MS/MS spectra were achieved in negative mode in the range 10-500 m/z; the mass axis was calibrated using the Agilent tuning mix HP0321 (Agilent Technologies) prepared in acetonitrile and water. Theoretical calculations. Molecular orbital calculations were performed using the density functional theory (DFT) calculations employing B3LYP functional and 6-31G* basis set in the environment of Spartan ’10, v.1.1.0. (Wavefunction, Inc.). Distribution of HOMO (highest occupied molecular orbital) and electrostatic potentials (ESP) and bond orders were calculated for molecules 1 – 6 in vacuum. The DFT calculations employing EDF2 functional and 6-31G* basis set were used for calculations of IR (Table S1) and UV-Vis spectra (not shown).

3. RESULTS AND DISCUSSION

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The chemical structures of all the studied compounds (1 to 6) are shown in Figure 1, alongside their cyclic voltammograms (CVs), acquired within the electrochemical window reaching up to 1.75 V against Ag/AgCl/1M LiCl reference electrode. The obtained redox potentials of the compounds are listed in Table 1, together with theoretically obtained highest occupied orbital (HOMO) energy.

Table 1. Oxidation potentials measured against Ag/AgCl/1M LiCl reference electrode, variation of peak potential with scan rate (δEp/δlog v), number of consumed electrons (based on exhaustive electrolysis), the peak width, charge-transfer coefficient α and calculated values of HOMO energies of studied compounds using the density functional theory (DFT) calculations with B3LYP functional and 6-31G* basis set.

Ep/V

δEp/δlog v /mV

1

1.15

36

0.7

2

1.14

36

3

1.06

4

α

EHOMO/eV

0.105

0.45

-6.44

0.6

0.125

0.38

-6.41

34

0.7

0.123

0.39

-6.35

1.05

37

0.5

0.114

0.42

-6.09

5

0.96

38

-

0.145

0.33

-6.16

6

0.90

34

0.7

0.103

0.46

-5.49

Compound

Ep-Ep/2/V

n

α is calculated from α = 1.857 R T /(F (|Ep – Ep/2|))21 The potential of the first oxidation wave decreases in the series from compound 1 to 6 (Table 1, Figures 1, 2, S2). This trend is in good agreement with that of the calculated energies for HOMO of the six compounds (Figure 2). The positional isomers 3 and 4 that differ from each other only in the position of carboxylic and hydroxyl group in their chemical structure show only a slight difference in their first oxidation potential. The lowest oxidation potential was found for

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compound 6 which differs from the other investigated hydroxyquinolines due to the methyl group at position C7 instead of carboxylic group. The low Eox and EHOMO are due to the positive induction effect of the methyl group.

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Figure 1. Cyclic voltammogram of 0.2 mM (A) 1, (B) 2, (C) 3, (D) 4, (E) 5 and (F) 6 in acetonitrile containing 0.1 M TBAPF6 on glassy carbon electrode. Scan rate was 0.1 V·s-1. Dotted lines represent the CVs registered by reversing the scan polarity upon reaching the first oxidation wave.

Figure 2. The correlation of EHOMO of compounds 1 – 6 calculated in vacuum with their experimental oxidation potential (Table 1). The interpretation of the oxidation mechanism of the studied compounds is not simple, as their electrochemical response is strongly dependent on the basicity of solvent, and on the presence of dissociated forms in the solution. Under experimental conditions selected in this study, all six compounds show two irreversible oxidation waves up to the potential 1.75 V. Compound 5 yields one additional oxidation wave at 1.6 V. Out of all compounds, only compound 6 shows a corresponding counterpeak appearing at 0.55 V, demonstrating that the first oxidation process is quasireversible (Figures 1F and S2, dotted line). Figure 3 shows the corresponding electrode

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polarization rate dependence. The anodic peak current in the cyclic voltammograms is directly proportional to the square root of the electrode polarization rate (also for all other studied compounds), thus suggesting that oxidation is diffusion controlled (Figure 3B). The analyses of cyclic voltammograms suggest the occurrence of a fast chemical reaction, which follows the electron transfer: This is confirmed both by the independence of Ip/v1/2 on electrode polarization rate, and the shift of the oxidation potential value with the electrode polarization rate δEp/δlog v = 34 mV (Figure 3A). Similar values of δEp/δlog v were found for all studied compounds 1 – 6 (Table 1).

Figure 3. Selected cyclic voltammograms of 0.2 mM 6 in 0.1 M TBAPF6 in acetonitrile on glassy carbon electrode at different electrode polarization rates: (a) 0.032, (b) 0.064, (c) 0.100, (d) 0.125 and (e) 0.250 V·s-1. Inset A: a dependence of anodic peak potential on the scan rate. Inset B: a dependence of anodic peak current on the square root of scan rate. The electron transfer may generally be coupled with several kinds of chemical reactions such as proton transfer, hydroxylation or dimerization. They are known to take place upon oxidation of hydroxy compounds.18-20 The dimerization as a possible coupled chemical reaction can be

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excluded, as the diagnostic criteria δEp/δlog v and δEp/δlog c are not met (they both do not give the theoretical value 19 mV).21 Proton transfer appears as a plausible candidate for the occurring chemical reaction, considering that the compounds 1 - 5 are substituted carboxylic acids, and thus a proton dissociation from carboxylic group may occur in solutions depending on the polarity of the solvent and basicity of the solution. Moreover, the nitrogen atom in the quinoline heterocycle can be easily protonated.14,22 The dissociation and the proton-transfer have several consequences to the electrochemical behavior of the investigated species. For instance, the height of the first oxidation wave of the two positional isomers 3 and 4 differs (Figure 1). This finding is in the agreement with Gudat et al,12 who described that the rate of proton transfer between the carboxylic group and the basic nitrogen atom in the quinoline unit in 4 is significantly lower than in 3. This is ascribed to another chemical reaction participating in the overall oxidation mechanism, auto-deprotonation (explained further). In order to clarify the effect of the proton transfer, further investigations focused on the oxidation mechanism in the presence of pyridine as a strong proton acceptor, to evaluate the role of nitrogen present in the quinoline heterocycle of the studied compounds. Proton abstraction by bases is a common reaction of electrogenerated cation radicals, giving rise to ECE-DISP (electrochemical-chemical-electrochemical mechanism-disproportionation reaction) two-electron mechanism.21 A typical example of such processes is the oxidation of synthetic analogues of NADH.21 Panel A in Figure 4 shows the cyclic voltammograms of 3 in acetonitrile at different concentrations of pyridine. The height of the oxidation wave significantly increases as the concentration of pyridine in the solution is elevated, gradually reaching a constant value (Figure 4B). The height of the resulting

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oxidation wave corresponds to two electron oxidation process, based on the comparison to the cyclic voltammogram of ferrocene/ferrocenium couple considering a correction for different diffusion coefficients. The number of electrons involved in oxidation was also confirmed by exhaustive electrolysis. It is necessary to note that number of electrons per molecule were evaluated assuming that all the molecules of 3 were oxidized (as it will be shown later, only a fraction of the molecules is oxidized in a two electron process). The peak potential of the first oxidation wave is shifted towards lower values as the concentration of pyridine increases (Figure 4C). The linear regression of the peak potential dependence on the pyridine concentration gives two slopes for two different cPy ranges, namely δEp/δlog cPy = 34 mV and 59 mV, indicating the participation of protons in the oxidation reaction. It is proposed that hydroxyl group is involved in oxidation. This finding is confirmed by the distribution of calculated HOMO, depicted for all compounds in the supporting information (Figure S1). The redox processes involving a proton and electron transfer are characterized by the shift of redox potential according to the basicity of solution. The number of protons (p) can be determined from the equation: δ Ep/δ ln[H] = p·RT/nF.23 Since Py serves as a proton acceptor, the slope δEp/δlog cPy = 59 mV suggests an equal number of protons p and electrons n.

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Figure 4. Cyclic voltammogram of 0.34 mM 3 at different concentration of pyridine: a) 0, b) 0.04, c) 0.08, d) 0.15, e) 0.30, f) 0.56, g) 3.9, h) 13.9, i) 27, j) 53, k) 200 mM (A) and 0.31 mM 6 at different concentration of pyridine: a) 0, b) 0.038, c) 0.075, d) 0.19, e) 0.91, f) 1.42, g) 16, h) 47, i) 66 mM (D) in 0.1 M TBAPF6 in acetonitrile on glassy carbon electrode. Scan rate was 0.1

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V·s-1. Anodic peak current (B,E) and anodic peak potential (C,F) dependences on concentration of pyridine are shown.

Scheme 1. The oxidation mechanism of compounds 3 and 6. Symbols for starting compound, its phenoxy cation and resulting product used in ECEC mechanism are indicated below their chemical formulas. Eqs. (1 – 5) and (6 - 10) show the oxidation mechanism in the presence and absence of pyridine Py, respectively.

Experimentally obtained slope δEp/δlog cPy = 59 mV, obtained for the compound 3 in solution with the concentration of pyridine higher than 0.17 mM indicates that two electrons and two protons participate in the overall oxidation mechanism (Scheme 1, eq. 4). The rate determining step of the whole process is a chemical reaction which involves a deprotonation step (Scheme 1,

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eq. 4). These findings also exclude a possible intramolecular PCET (proton coupled electron transfer), described in previous studies of compounds with an intramolecular O-H…N hydrogen bond (see Scheme S1 in Supporting Information).24-26 The irreversible two-electron process (Scheme 1) shows O-H bond dissociation and generation of a short-living phenoxyl radical and phenoxonium ion ArO+N followed by a subsequent chemical reaction, hydroxylation. Moreover, PCET is not favoured in the compounds under study due to the lack of an appropriate geometry, as the distance between H of the hydroxyl group and nitrogen atom in the quinoline core is too large to form a hydrogen bond. The influence of strong proton donors or acceptors in the solution on the electron transfer processes is well known.2-4,10,11 A base-dependent oxidation mechanism of ferrocenyl catechols in the presence of imidazole was described recently.5 When no proton acceptor is present in the solution, the consumption of 0.7 electrons was obtained in the first oxidation step of the neutral form of 3. It is likely that the nitrogen atom of the heterocycle serves as a proton acceptor during the oxidation process and the proton transfer proceeds between two or more different molecules of 3. The height of the first oxidation peak of 1 – 6 depends linearly on the concentration, the peak width of the first oxidation peak |Ep – Ep/2| = 123 mV gives the value of transfer coefficient 0.39 for compound 3. This implies that the chemical reaction, e.g. cleavage of proton (Scheme 1, Eq. 7), is fast or even a concerted proton electron transfer occurs.21 Such the behavior was found for all studied compounds (Table 1). In this case, the chemical reaction (Scheme 1, Eq. 9), which involves the participation of the other starting molecule 3, is the rate determining step. In summary, the overall oxidation mechanism involves 2 electrons and 3 starting molecules (Scheme 1, Eq. 10, where Eq. 10 is the sum of Eqs. 6 - 9). This is in agreement with the consumed charge corresponding to 0.7 electrons. The participation of the other starting molecule of 3 in the proposed mechanism is supported also by

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the fact that the oxidation mechanism of 4 proceeds at almost the same oxidation potential as 3 (Table 1). The chemical structure of 4 does not allow the participation of intramolecular proton transfer at all. It is evident, that all compounds 1 – 6 behave similarly (compare cyclic voltammograms in Figure 1 and characteristics in Table 1) and this is supported also by a very good relation of their oxidation potentials with calculated EHOMO (Figure 2). A kinetic character of their currents and similar number of electrons per molecule obtained from the consumed charge in the course of exhaustive electrolyses imply that auto-deprotonation influence their oxidation mechanism. In addition, compound 3 is known to occur as two forms, i.e. the neutral and the zwitterionic forms. Gudat et al.12 found by NMR shifts investigation that the ratio between these two forms of 3 in the solution of dimethyl sulfoxide is approximately one. To get a better insight into this behaviour, compound 6, lacking a carboxyl group in its chemical structure, was investigated at different concentrations of pyridine (Figure 4D). As it is depicted in panels E and F of Figure 4, the obtained results are similar to those obtained in the study of the cyclic voltammetry of 3. The height of the oxidation peak reaches the limiting current at a concentration ratio of neutral form of 6 to pyridine 1:2 and remained constant further (Figure 4E). At low concentration of pyridine, no change of potential value occurred; compound 6 acts as a sufficiently strong proton acceptor at this concentration of Py (Figure 4F). At higher pyridine concentrations, the oxidation potential is linearly dependent on the cPy value, with a slope of δEp/δlog cPy = 60 mV indicating the participation of two electrons and two protons in the oxidation process. All these findings imply that in the presence of a sufficient concentration of a proton acceptor, the mechanism involves two electrons and two protons. The controlled potential coulometry in the presence of a strong

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proton acceptor Py confirms that two electrons are involved in the oxidation process (Scheme 1, Eq. 5, where Eq. 5 is the sum of Eqs. 1 - 4). To further support the hypothesis of the auto-deprotonation mechanism, we performed the exhaustive electrolysis at the potential pertaining to the diffusion limiting current of the first oxidation wave, corresponding to oxidation of the neutral compound 3. During the electrolysis, both cyclic voltammograms (inset in Figure 5) and high pressure liquid chromatograms were recorded. The first oxidation wave decreased while the second oxidation wave increased. It is likely that the second oxidation wave increases due to the formation of the protonated starting molecules. Since the cyclic voltammogram of all measured compounds 1 – 6 contain two wellresolved oxidation waves belonging to oxidation of two different forms of molecules present in the solution, this finding is in the agreement with results found in literature about the presence of both zwitterionic and neutral form in the solution.12 The oxidation waves of zwitterionic and protonated forms may appear at close values of potential at about 1.6 V. The ratio of the height of the first oxidation wave of 3 before the electrolysis (at 1.06 V, curve a in the inset of Figure 5) and the second oxidation wave of protonated molecule of 3 recorded after the electrolysis (at 1.6 V, curve c in the inset of Figure 5) is approximately 1:2, according to Eq. 10 in Scheme 1.

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Figure 5. UV-Vis spectroelectrochemistry of 0.34 mM 3 in 0.1 M TBAPF6 in acetonitrile at the first oxidation wave. The inset: Cyclic voltammogram of 0.57 mM 3 in 0.1 M TBAPF6 in acetonitrile on a glassy carbon electrode (a) before electrolysis, (b) after consumption of 0.4 and (c) after consumption of 0.6 electron per molecule. Scan rate was 0.1 V·s-1. The arrow indicates the potential of electrolysis. Chemical structures of neutral and protonated starting molecule of 3 are shown.

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Figure 6. IR spectroelectrochemistry of 3 in 0.1 M TBAPF6 in tetrahydrofuran (A) and 6 in 0.1 M TBAPF6 in acetonitrile (B). For comparison, dashed curves are IR spectra of 3 and 6 in the presence of triflic acid used as a proton donor.

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UV-Vis spectra during oxidation of 3 were obtained using spectroelectrochemistry in an optically transparent thin-layer electrode (OTTLE) cell. The spectra obtained at the potential of the first oxidation wave show a decrease of the absorption bands at 205 nm and 260 nm, and an increase of the absorption band at 287 nm and 447 nm; the absorption band at 340 nm slightly shifts to higher wavelength (349 nm) (Figure 5). The increase in the range around 450 nm is +

consistent with the generation of the protonated molecule Ar(OH)NH .26 A short-living oneelectron product such as a phenoxy radical formed as intermediate was not stable enough to be visible at 450 – 500 nm under these conditions.27,28 The subsequent hydroxylation results in 5,8dihydroxy-2-methylquinoline-7-carboxylic acid (Ar(OH)2N, Scheme 1).15,26,29 The product of oxidation Ar(OH)2N was identified by HPLC-MS/MS (Figure S3). The high resolution ESI-QToF mass spectrum of 3 is characterized by the [M-H]- pseudomolecular ion at 202.055 m/z. Moreover, a neutral loss of CO2 typical of benzoic acids occurs in source and produces a fragment ion at 158.064 m/z ([M-CO2-H]-). The high resolution mass spectrum of the oxidation product Ar(OH)2N is characterized by its pseudomolecular ion at 218.042 m/z ([M-H]-) and also in this case a neutral loss occurs in source, producing a fragment ion at 174.061 m/z ([M-CO2-H]-). The identification of the hydroxylated product was performed by the identification of the raw formula (C11H9NO4) by high resolution spectrum analysis and interpretation of the in-source fragmentation. Moreover, a hydroxylated product elutes at shorter retention times with respect to 3, thus confirming its higher polarity (Figure S4 shows its ESI-QToF MS). The in situ IR spectroelectrochemistry was performed both in acetonitrile and tetrahydrofuran. Figure 6A shows the IR spectrum of the compound 3 in THF with characteristic bands of the molecule at 1669 cm-1 (assigned to C=O stretches of carboxylic group), 1627 cm-1, 1610 cm-1

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(the C=N and C=C stretching vibration bands in aromatic ring) and 1559 cm-1. The band of hydroxyl groups of COOH and hydroxyl group C8-OH found in powder of 3 (ATR-FTIR measurement) at 3403 and 3280 cm-1, respectively, are hidden in the measurement performed in the solution. The C=O stretching band at 1669 cm-1 decreases and shifts to 1682 cm-1 during oxidation, the bands 1627 and 1610 cm-1 are intensified and slightly shifted to 1621 and 1604 cm-1 (Fig. 6A). Simultaneously a broad band appears at higher frequency region, around 3100 cm-1, obscured partially by solvent absorption. Growing of all these bands is particularly observed in the later stage of the electrolysis at more positive potentials and it is obviously related to a protonation reaction as evident from the comparison to the spectrum of the compound 3 in presence of triflic acid, which was used to protonate molecule 3. The comparative spectrum of 3 in the presence of triflic acid exhibits an analogous band at 3120 cm-1 and resembles to the final spectrum after oxidation of 3 also in the range of C=O and ring vibrations (Fig. 6A). This finding points to formation of Ar(OH)NH+ (protonated 3) during the electrolysis at the first oxidation peak. Equation 10 in the Scheme 1 predicts Ar(OH)NH+ as a prevailing product; IR spectroelectrochemical results are thus in agreement with the proposed oxidation mechanism. The IR spectrum of the compound 6 in acetonitrile shows characteristic bands of the molecule at 1604 cm-1, 1580 cm-1, 1570 cm-1 and 1513 cm-1 (C=N and C=C vibration bands in aromatic ring).30 A development of the band 1639 cm-1 and a slight shift to 1644 cm-1 during oxidative electrolysis as well as the increase of absorption at 1590 cm-1 are related to the protonation of molecule, as follows from the comparison with the spectrum of 6 in presence of an excess of triflic acid (Figure 6B). The formation of Ar(OH)NH+ (protonated 6) is indicated also by increasing absorption in the range 3000 – 3400 cm-1, which can be ascribed to N-H stretching,31-

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however the spectra are obscured due to a strong absorption of the solvent. Appearance of a

new vibration band at 1550 cm-1 development (while bands at 1580 cm-1 and 1570 cm-1 decrease) also supports the formation of Ar(OH)NH+ during the electrolysis at the first oxidation peak. The increase of the absorbance at 1635 – 1618 cm-1 can be attributed to the formation of the oxidation product Ar(OH)2N. Experimental data and frequencies calculated employing EDF2 functional and 6-31G* basis set for molecules 3 and 6 in vacuum are summarized in Table S1. The in situ IR spectroelectrochemistry of both compounds is in accordance with the proposed oxidation mechanism in Scheme 1.

4. CONCLUSION The coupling of proton transfer with electron transfer was found to play an important role in the oxidation mechanism of hydroxyquinoline compounds. Auto-deprotonation takes part in their oxidation mechanism due to the nitrogen atom present in the chemical structure. Three starting molecules participate in oxidation of one molecule if no strong proton acceptor is present in the solution: two molecules serve as proton acceptors on account of the basicity of nitrogen heteroatom in the quinoline structure. Both, the electrochemical and spectroelectrochemical results showed that the oxidation mechanism is not influenced by the presence of carboxylic group in the chemical structure of hydroxyquinolines. The oxidation of hydroxyquinolines proceeds quantitatively, when a strong proton acceptor is present in the solution. Pyridine was chosen as strong proton acceptor in this study, to mimic possible nitrogen containing heterocycles present in vivo. Our findings imply that in biosystems the electron transfer could proceed easily by the interactions with purine or pyrimidine bases. The complete understanding of this effect can help to explain the bio efficiency of hydroxyquinolines in vivo.

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ASSOCIATED CONTENT Supporting Information. Cyclic voltammograms at higher concentration, molecular calculations results, experimental and calculated values of vibration frequencies and ESI-Q-ToF spectra. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *Email: [email protected]; Tel.: +420266053525; Fax: +42028658 2307 (R. Sokolová) Notes The authors declare no competing financial interests. ACKNOWLEDGMENT This work was supported by The Czech Academy of Sciences (M200401201). ABBREVIATIONS UV-Vis, ultraviolet-visible; IR, infrared; HPLC, high pressure liquid chromatography; MS, mass spectrometry; ECEC, electrochemical-chemical-electrochemical-chemical; PCET, proton coupled electron transfer; NMR, nuclear magnetic resonance; HRMS, high resolution mass spectroscopy; OTTLE, optically transparent thin-layer electrode; DFT, density functional theory; B3LYP, Becke-3-Lee-Yang-Parr; HOMO, highest occupied molecular orbital; DISP, disproportionation reaction

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