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Reaction Kinetics of Phenolic Antioxidants Towards PhotoInduced Pyranine Free Radicals in Biological Models Alexis Aspee, Christian Aliaga, Luca Maretti, Daniel Zúñiga-Núñez, Jessica Godoy, Eduardo Pino, Gloria Ines Cardenas-Jiron, Camilo López-Alarcón, Juan C. Scaiano, and Emilio Isaac Alarcon J. Phys. Chem. B, Just Accepted Manuscript • Publication Date (Web): 09 Jun 2017 Downloaded from http://pubs.acs.org on June 10, 2017

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The Journal of Physical Chemistry

Reaction Kinetics of Phenolic Antioxidants Towards Photo-Induced Pyranine Free Radicals in Biological Models Alexis Aspée,a,* Christian Aliaga,a Luca Maretti,b Daniel Zúñiga-Núñez,a Jessica Godoy,a Eduardo Pino,a Gloria Cárdenas-Jirón,a Camilo Lopez-Alarcon,c Juan C. Scaiano,b Emilio I. Alarcon,b,d a

Facultad de Química y Biología, Universidad de Santiago de Chile, Casilla 40 Correo 33, Santiago, Chile.

b

Department of Chemistry and Biomolecular Sciences and Centre for Catalysis Research and Innovation, University of Ottawa, 10 Marie Curie, Ottawa, Ontario K1N6N5, Canada.

c

Departamento de Química Física, Facultad de Química, Pontificia Universidad Católica de Chile. C.P. 782 0436, Santiago, Chile. d

Bio-nanomaterials Chemistry and Engineering Laboratory, Division of Cardiac Surgery,

University of Ottawa Heart Institute, 40 Ruskin St., Ottawa, Ontario K1Y 4W7, Canada Corresponding Author * Alexis Aspée, Email: [email protected].

ACS Paragon Plus Environment

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ABSTRACT: 8-hydroxy-1,3,6-pyrenetrisulfonic acid (pyranine, PyOH) free radicals were induced by laser excitation at visible wavelengths (470 nm). The photochemical process involves photoelectron ejection from PyO- to produce PyO• and PyO•- with maxima absorption at 450 and 510 nm, respectively. The kinetic rate constants for phenolic antioxidants with PyO•, determined by nanosecond time resolved spectroscopy, were largely reliant on the ionic strength depending on the antioxidant phenol/phenolate dissociation constant. Further, the apparent rate constant measured in the presence of Triton X100 micelles were influenced by the antioxidant partition between the micelle and the dispersant aqueous media, but limited by its exit rates from the micelle. Similarly, the rate reaction between ascorbic acid and PyO• was markedly affected by the presence of human serum albumin responding to the dynamic of the ascorbic acid binding to the protein.


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1. INTRODUCTION Since the establishment of the oxidative stress hypothesis linked to free radical damage of biomolecules under physio-pathological conditions, and the beneficial role of antioxidants1,

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numerous experimental methodologies have been developed for determining antioxidant capacity of phenolic compounds.3,

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Most of these approaches are based on monitoring the

kinetics of antioxidant reaction with free radicals in homogeneous media by either, i) a direct evaluation of rate reaction constants by antioxidant consumption and/or product formation, or ii) indirectly using competitive reactions between a reference compound and the antioxidant against free radicals. However, in addition to absorption and bioavailability of the antioxidant, it is difficult to extrapolate that antioxidant information to biological systems; where its efficiency can be strongly affected by the environment and its compartmentalization between different cellular compartments.5-9 We have previously reported data regarding the reaction of stable free radicals such as galvinoxyl and nitroxides with antioxidants in compartmentalized systems.10-12 Those studies allowed us to identify significant accessibility and hydrophobic factors on the reaction of antioxidants with free radicals in micelles and proteins. However, that approach requires the use of stable free radicals that present low reactivity in comparison to free radicals such as peroxyl or alkoxy free radicals, biologically more significant.13-15 That can lead to underestimate or overestimate the influence of the antioxidant concentration in complex systems, depending on the location of the free radical and the antioxidants, and the dynamic of these species in compartmentalized systems. For that reason, we have lately been focused on developing photochemical techniques to produce free radicals by using laser-photolysis of coumarin dyes controlling the generation site of these species by the original location of the dye.16 That method


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has permitted us to determine kinetic rate constants for the reaction of phenolic antioxidants with highly reactive cationic coumarin free radicals. However, due to the moderate hydrophobicity of those coumarins tested it is difficult to control its location in compartmentalized systems. Recently, we have focused on the generation of free radicals derived from the hydrophilic 8hydroxy-1,3,6-pyrenetrisulfonic acid (pyranine, PyOH) by laser excitation at visible wavelength range. Pyranine is an attractive substrate due to its high solubility in aqueous media at neutral pH, as a consequence of three negative charges from three sulfonate groups (here depicted for simplicity as PyOH, see Scheme 1) that allows compartmentalizing it sidestepped from hydrophobic environments such micelles.17,

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Further, it has been detected formation of

pyranine free radical (PyO•) during PyOH oxidation mediated by peroxyl radicals using spin paramagnetic spectroscopy (EPR), exhibiting it as a relatively persistent free radical.19-21 In spite of that, competitive experiments and electrochemical studies have exposed a fast reaction of this radical towards phenol and polyphenol antioxidants, but no kinetic information has been reported.

20, 22-24

Furthermore, pyranine free radicals have been produced by direct photolysis

have been produced by direct photolysis of PyOH with UV laser excitation (354 nm).25-27 However, the use of this approach for determining kinetic rate constants between pyranine free radicals and antioxidants is limited only to substrates that do not absorb into the UV region to avoid the interference of the antioxidant absorption (UV-light) of the laser irradiation. In this work, for the first time, we generate time photo-induced pyranine free radicals by photolysis using laser irradiation in the visible range, and report kinetic rate constants for its reaction towards phenolic antioxidants. The influence of the ionic strength and pH on the rate reaction of PyO• with antioxidants is widely discussed based on the stability of the pyranine free radicals evaluated by Electron Paramagnetic Spectroscopy (EPR) and Density Functional Theory


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(DFT). Moreover, we explore the use of PyO• for studying compartmentalization and dynamic of antioxidants on micelles and in human serum albumin (HSA).

2. MATERIALS AND METHODS 2.1 Time resolved measurements. Pyranine transient absorption spectra were recorded with a LFP 111 laser flash photolysis system (Luzchem Inc., Ottawa, Canada), using a Surelite SLOPO plus laser at 470 nm, pumped by the third-harmonic output from a Nd:YAG laser as excitation source (355 nm, ~5 ns) (Surelite II, Continuum, USA). All kinetic experiments were carried our employing pyranine absorption lower than 0.5 (75 µM pyranine) at the excitation wavelength. The measurements were carried out in phosphate buffer (10 mM-100mM, pH 7.0) at room temperature in 1.0 x 1.0 cm quartz cuvettes. Transient absorption spectra were recorded using pyranine concentration range (75-120 µM) in phosphate buffer (10 mM, pH 7.0). 2.2 Electron paramagnetic resonance. Pyranine free radical was examined by electron paramagnetic resonance. Radicals were in situ produced upon irradiation of pyranine solutions in a flat cell placed into a cavity of a JEOL JES-FA 100 X-Band EPR spectrometer (JEOL USA, Peabody, MA), equipped with a JEOL ES-UCX2 cylindrical cavity. EPR spectrum was recorded on 1.2 mM pyranine in phosphate buffer (10 mM, pH 7) after laser irradiation (Lumonics EX530, 308 nm, 90 mJ per pulse) at room temperature with a 100 kHz magnetic field modulation (Frequency: 9416.174 ± 6.907514, width 0.0020 mT) at microwave output of 0.940 mW. 2.3 Computational Details. All the ground state calculations were performed using density functional theory (DFT) with the Gaussian 09 package.28 Stationary points on the potential energy surface were obtained using the B3LYP hybrid density functional.29,

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The molecular

geometry optimization was performed in the gas phase using the 6-31++G (d,p) basis set for all


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atoms.31,

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Open-shell species were calculated using the spin-unrestricted formalism. The

converged wave functions were verified by analytical computations of harmonic vibrational frequencies. Gibbs free energies in the gas phase were computed for each species within the ideal gas model, rigid rotor, and harmonic oscillator approximations at a pressure of 1 atm and a temperature of 298.15 K.33 The atomic spin densities were evaluated using the natural population analysis (NPA).34 Time-Dependent DFT (TD-DFT) was used to study the electronic absorption spectra of each radical pyranine.35,

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The vertical Franck-Condon transition was achieved

considering 30 excited states in solution phase. All the single-point calculations for spin density and TD-DFT were performed in water using a conductor-like polarizable continuum model (CPCM) with the standard parameters for water.37

3. RESULTS AND DISCUSSION Pyranine free radical formation was explored by laser irradiation at visible wavelengths for selective excitation of the pyranine phenolate form (PyO-) in equilibrium with the protonated phenol form PyOH at neutral pH (pKa =7.1).20 Thus, laser excitation using visible wavelengths took advantage of the maximum absorptions at 360 mn and 450 nm for PyOH and PyO- species, respectively. Moreover, laser flash photolysis experiments were performed using 470 nm laser irradiation, in order to maximize detection of the transients and pyranine bleaching recorded at microsecond time scales. The experiments carried out on nitrogen purged solutions showed two very long live species registered at 450 and 510 nm that can be associated with the pyranine free radicals: PyO• and PyO•-, respectively (Figure 1, scheme 1).26, 27 In addition, the ground state bleaching, and its recovery showed at 390 nm corresponds to the protonated phenol form (PyOH) in equilibrium with the phenolate form (PyO-) at pH 7.0. The spectral properties of these species, and lifetimes, were in conformity with experiments carried out by us using 355 nm after


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laser irradiation from the third harmonic of a NdYAG laser (Figure S1), as well as, with previously reported data.26, 27 Laser flash photolysis experiments in air-equilibrated solutions only showed the transient of the PyO•. The transient lifetime for this radical was not sensitive on the microsecond time scale to the presence of oxygen, in agreement with a relatively low reactivity of PyO• towards oxygen.19 The anion free radical species, PyO•-, was not observed because there is an efficient oxygen trapping of the solvated electron generating the corresponding superoxide radical anion. In fact, different quenchers of solvated electron elicited a similar behaviour; among them, N2O efficiently traps electrons at diffusion control permitting to study the chemical properties of PyO• by laser flash photolysis without the interference of superoxide free radical anion.38 The photochemistry involved in the pyranine free radical formation by visible wavelength laser irradiation is described in Scheme 1. The phenolate pyranine excitation by laser irradiation at 470 nm leads to the formation of pyranine anion singlet excited state, which, upon the absorption of a second photon produces the photoejection of an electron and PyO•. Photochemical formation of the pyranine free radicals agrees with a linear relation between radical formation and the laser energy and with the detection of solvated electron in absence of oxygen (Figure S2 and S3). At difference, pyranine photolysis using a 355 nm laser irradiation has been described by excitation of the protonated form (PyOH) rendering the singlet excited state, which is a strong acid, that fast deprotonates at neutral pH yielding the excited PyO- form. Absorption of a consecutive photon by the latter excited state species, PyO-*, induces an electron ejection with the formation of PyO• species and a solvated electron (see Scheme 1).26, 27


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Figure 1. (A) Transient spectrum recorded at 30 µs (), 150 µs (), 1 ms () and 3.8 ms () after 470 nm laser pulse excitation of 120 µM pyranine in phosphate buffer (10 mM, pH 7.0). (B) Kinetic profile recorded at 390 nm (recovery, —) and transient decays at 510 (—) and 450 nm (—). Measurements were carried out in nitrogen-purged solutions. Laser energy pulse 10 mJ. It is important to note the advantages of pyranine free radical formation by 470 nm in comparison of using 355 nm excitation laser on laser flash photolysis experiments: i) a direct excitation of the phenolate pyranine form renders to the singlet-excited state of the phenolate minimizing photodegradation of pyranine associated with laser excitation at short wavelengths, ii) it is eluded the step of fast deprotonation of the phenol form of pyranine reducing any modification of the local pH, and iii) the use of visible wavelength for laser excitation simplify


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the study of the reaction with UV-absorbers antioxidants or biological substrates avoiding filter effects and/or preventing the influence of the photochemistry of other substrates absorbing on the UV region.

Scheme 1. Proposed mechanism of pyranine free radicals formation induced by laser excitation at UV (355 nm) and visible (470 nm) wavelengths under nitrogen. The free radical characterization of the PyO• was performed by electron paramagnetic resonance spectroscopy (EPR) using a 308 nm laser for irradiation of the protonated phenol form of pyranine at pH 7.0. This laser wavelength was only used as per experimental set up of the EPR instrument. The EPR spectrum recorded showed the formation of free radical species with a high spin-density delocalization as expected for the PyO• free radical (Figure 2A). In fact, an identical EPR spectrum was observed when pyranine was incubated on 2,2’-azobis(2-


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amidinopropane) dihydrochloride (AAPH) under aerobic conditions (Figure 2B). In that case, the oxidation of pyranine is produced by peroxyl free radicals generated at a constant production rate from the thermal decomposition of AAPH. These EPR spectra were in agreement with previous results, and were analogous to the simulated spectra of PyO• recently reported. 19

Figure 2. (A) EPR spectrum after 1.2 mM pyranine irradiation by laser pulse (308 nm, 90 mJ) in phosphate buffer (20 mM, pH 7.0), nitrogen-purged solutions. (B) EPR spectrum recorded during 1.2 mM pyranine incubation in 450 mM AAPH at 25 °C in aerobic conditions. Furthermore, an identical EPR spectrum was recorded after 1.2 mM PyOH incubation on 0.4 mM DPPH free radical in (1:1) water/ethanol mixtures, or after incubation on high concentrations of the nitroxide Tempol (0.2 mM) in phosphate buffer (Figure S5 and S6). Additionally, the hyperfine structure of EPR spectrum obtained after pyranine laser irradiation under anaerobic conditions was not dependent on the pH of the media confirming the identity of the radical PyO• (Figure S7). It is important to note, that laser flash photolysis experiments using 308 nm laser irradiation showed formation of both free radicals, PyO• at 450 nm and PyO•- at 510 nm (Figure S4). Undetected EPR signal for the anionic PyO•- under the experimental


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conditions can be related with the particular spin radical density of this radical with no aromatic delocalization and a lower quantum yield than PyO• formation (vide infra). Moreover, the EPR for the PyO• detected by 308 nm irradiation only present a very slight shift on the magnetic field respect to the spectrum observed in the presence of AAPH that can be associated to the complex formed between pyranine free radical and AAPH, in resemblance to the complex observed in the ground state.39 However, a considerable rise on PyO• formation was observed on the EPR signal intensity with the increment of the pH. A similar behaviour observed during 355 nm laser irradiation was attributed to a delayed time on the PyO- excited state formation respect to the laser energy of the pulse.26 However, in our experimental conditions, 308 nm laser renders a larger excitation of the deprotonated PyO- species at high pH also due to the increase on the absorbance at 308 nm for PyO- respect to the protonated form PyOH (Figure S8). In conformity with our EPR results, spin density calculations of the PyO• radical by DFT exposed a large delocalization in the aromatic rings, involving hyperfine coupling of the six Hatoms on the free radical (Figure 3, top). This large spin delocalization also explains the reduced reactivity of PyO• toward oxygen, and coincides with the behaviour of other free radical species that has been previously reported.19, 20, 40, 41 In fact, from the analysis of the EPR spectra, it was observed that the half lifetime (t½) for the PyO• under oxygen purged solutions, is considerable shorter than that measured under nitrogen, which is in agreement with an efficient back electron transfer from superoxide radical anion (O2•-) generated under oxygen (Figure S9). In addition, the spin density of the anionic free radical, PyO•-, is highly located on antibonding molecular orbitals avoiding hydrogen hyperfine coupling. Moreover, evaluation of the electronic absorption spectra of these transient free radicals was calculated by time-dependent DFT methodology (TDDFT) matching correctly with the experimental transient spectra measured by laser flash


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spectroscopy (Figure 1 and 3). This result also confirms the spectroscopic characterization of both pyranine free radicals on the laser flash photolysis experiments.

Figure 3. Spin density distribution for pyranine free radicald PyO• (A) and PyO•- (B) at an isosurface value of 0.002 (top) and simulated absorption spectra, computed using B3LYP/631++G (d,p) and a conductor-like polarizable continuum model (C-PCM) with the standard parameters for water (bottom). Laser excitation of the pyranine in the EPR cavity carried out in the presence of antioxidants, showed in the EPR a complete disappearance of the PyO• spectrum, confirming that the reaction between antioxidants and PyO• can be associated with the electron transfer (ET) or hydrogen


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atom transfer (HAT) from the antioxidant to the pyranine free radical. Surprisingly, for caffeic acid, it was detected after the reaction an EPR signal associated to its derived free radical (Figure 4).42,

43

This is an evidence of a major persistence of the caffeic acid free radical than other

cinnamic acids, in agreement with a higher stability attributed to the intramolecular H-bond reinforced in the semiquinone radical making the radical more stable.44

Figure 4. (A) EPR spectra of PyO• generated by 308 nm laser irradiation of 1.2 mM PyOH in 20 mM phosphate at pH 10 (cyan), and EPR spectra observed at identical conditions but the presence of 0.2 mM caffeic acid (black). (B) A detail of the caffeic acid EPR radical observed after the reaction with PyO•.


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In order to evaluate the reactivity of PyO• towards antioxidants, bimolecular kinetic rate constants were determined by laser flash photolysis, using experimental conditions previously reported.16 In particular, these measurements were carried out using N2O purged-solutions to reduce any eventual influence of the solvated electrons present in nitrogen-purged solutions, or by superoxide free radical anion generated in the presence of oxygen. However, it is important to note that the use of N2O as electron-scavenging conducts to hydroxyl free radical formation.38 Conversely, hydroxyl radicals react at diffusional control in a faster time scale, not interfering on the nanosecond laser time photolysis. In fact, any reaction elicited by hydroxyl radicals is considered in the mathematical model for obtaining the kinetic rate constants.45 Table 1 summarizes second order kinetic rate constants values for the reaction of PyO• with different antioxidants evaluated by quenching experiments of the transient monitored at 450 nm. All antioxidants tested, including hydroxycinnamic acids (sinapic acid, caffeic acid, ferulic acid and p-coumaric acid) and polyphenols (quercetin, and pyrogallol red), showed relatively moderate kinetic rate constants values. The differences ranged over two or three orders of magnitude below a diffusional rate constant (kd ≈1010 M-1s-1). This behaviour is in agreement with a highly delocalized spin density free radical (PyO•), as it was developed by the EPR spectrum and spin density by DFT calculations, that contributes to a reduction on the free radical reactivity.46 However, these rate constants also suggest that there is an electrostatic effect between the pyranine free radical (three negative charged due to the deprotonated sulfonic groups at pH 7.0) and the deprotonated antioxidants (vide infra). In fact, at 10 mM phosphate buffer (Pi), a rather small value of the kinetic rate reaction constant was obtained for ascorbic acid (k 10 mM, Pi =4 x 107 M-1 s-1) than for Trolox (k 10 mM, Pi =1.8 x 108 M-1 s-1) in contrast of the redox potential or antioxidant reactivity measured using other free radical systems.11, 47 On the


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other hand, a few expected tendencies can be established on the measured kinetic rate constants considering for the reactivity pattern of the hydroxycinnamic acids and polyphenols like quercetin and pyrogallol red. Table 1. Kinetic rate constants for PyO• reaction with antioxidants. All measurements were carried out in N2O-purged solutions in phosphate buffer (Pi) at pH 7.0 at 25 ºC. Antioxidant

k, 106 M-1s-1 /10 mM Pi

k, 106 M-1s-1/100 mM Pi

Trolox

180 ± 5

240 ± 20

Quercetin

71 ± 1.2

290 ± 10

Ascorbic acid

40 ± 1.6

91 ± 2.1

Pyrogallol red

36 ± 4.9

54 ± 4.1

Fluorescein

9.6 ± 0.68

35 ± 0.5

Sinapic acid

8.8 ± 0.5

80 ± 6

Caffeic acid

3.2 ± 0.04

24 ± 5

Alizarin

2.6 ± 0.15

2.2 ± 0.4

Ferulic acid

1.7 ± 0.05

7.1 ± 0.6

p-coumaric acid

0.15 ± 0.02

0.4 ± 0.07

± Values are standard deviations calculated from three independent experiments

The relevance of the electrostatic interactions on the reaction can be recognized by comparison of kinetic rate constants at different ionic strengths. In particular, an increase on the Pi concentration from 10 to 100 mM showed consistently larger kinetic rate constants for almost all antioxidants, in reliability with a reaction that is taking place between the negatively charged PyO• and a negative charge or formal dipole on the reactive form of the antioxidant. In particular, for the case of ascorbic acid (pKa = 4.2)48 is expected a larger effect of the ionic


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strength than for the reaction with Trolox at neutral pH whose reactive phenol group is protonated (pKa = 11.9)49. However, there is no a simple correlation that can be used for explaining the increment observed from 1.5 to 9.1 amongst the different antioxidants, when the concentration of the phosphate buffer is increased from 10 mM to 100 mM (Table 1). To analyse the influence of the ionic strength in these reactions, we compared the experimental values of the kinetic rate constants at 100 mM Pi with the calculated rate constants assuming that the reaction takes place between ions like negative charge (z = -1). Under these conditions, and supposing that dilute solution is still valid in the range of 100 mM concentration, the effect of the ionic strength on the rate constant may be described by the following equation.50

, = ln + 2 ( − )

Where, k10

mM

(1)

is the experimental kinetic rate constant measured on 10 mM Pi, zA and zB

correspond to the ions charge, A is a constant equal to 0.51x 2.303 L1/2 ·mol-1/2 in aqueous solution at 25 ºC, and I is the ionic strength. Figure 5A represented the relationship between experimental kinetic rate constants with those calculated values for the reaction at 100 mM Pi, considering the ionic strength increase on the kinetic rate reaction constant at 10 mM, using equation 1. It is important to note that this equation was only used for identifying different variables taking place on this reaction. The data for the kinetic rate reaction constants measured at different phosphate buffer concentration in Table 1, showed some correlation with the calculated rate constant using eq.1 as can be seen in Figure 5. Nevertheless, such behaviour goes beyond an electrostatic effect on the ionic strength on the kinetic rate constant between charged molecules as can be observed on Figure 5A. A possible scenario considering that reaction


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between the antioxidant (ROH) with PyO• can occurs from the acid form of the OH phenolic antioxidants by hydrogen atom transfer (kROH) and/or by electron transfer from the phenolate form (kRO-) as shown in equations 3 and 4 may also have to be considered.16, 51, 52 ⇄ + !"• + → • + !" %&

!"• + '( !" + •

(2) (3) (4)

Thus, an effect of the ionic strength on the acid base equilibrium (pKa) of the antioxidant would also modify the amount of protonated and deprotonated phenolic antioxidants. In particular, the increase on the bimolecular rate constant for hydroxycinnamic acids at higher ionic strength agrees with a faster reaction between the phenolate form (in equilibrium with the phenol group) present at neutral pH with the PyO•, than for the phenol form. Thus, an increment on buffer concentration would affect more the sinapic (pKa = 9.21)22 and caffeic (pKa = 9.07)22 acids in comparison to ferulic (pKa = 9.55)22 and p-coumaric acid (pKa = 9.45)22. But the rate constant should also depend on the reactivity of the phenolate species of the hydroxycinnamic acid towards PyO• as is depicted on reactions 2-4. In general, several systems have exhibited higher reactivity for the phenolate form of antioxidants than for the protonated forms when the pH effect is considered on the rate reactions.45, 53, 54 In fact, an identical tendency is observed at the higher phosphate buffer concentration, reinforcing the influence of the reactivity and the amount of phenolate hydroxycinnamic acid conditioned by the acidity constant equilibrium. On the other hand, polyphenols such as pyrogallol red (pKa = 6.3)55, fluorescein (pKa = 6.8)56 and


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quercetin (pKa = 7.0)57 also showed a considerable increment on the rate reaction at higher ionic strength, but values for alizarin red (pKa = 5.49)58 remain practically unchanged.

Figure 5. (A) Logarithmic relationship between kinetic rate constants measured at 10 mM and 100 mM phosphate buffer (). The red line represents the calculated increment on the rate reaction at 100 mM phosphate buffer considering identical mono charged species (). Bar errors represent the standard deviation of three independent measurements. (B) Calculated ratio of the molar fraction of the phenolate species present at 100 mM to 10 mM phosphate buffer at pH 7.0, as a function of the pKa of the reactive OH group on the antioxidant.


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An evaluation of the molar fraction of the phenolate of the reactive phenol moiety present at 100 mM to 10 mM phosphate buffer [χ RO-, Pi 100 mM]/[ χ RO-, Pi 10 mM ] for the different antioxidants allows to discriminate the effect of the ionic strength on the pKa (Figure 5B). Thus, the moderate effect of the ionic strength on this ratio for Trolox and p-coumaric acid is in favour of the small effect of the ionic strength on the kinetic rate constants as a consequence of a reaction mainly between PyO• and the phenol form of the antioxidant. However, the reaction of ascorbic acid, alizarin red, and pyrogallol red, can be considered as in the other extreme being almost completely deprotonated, and then the equilibrium is little modified by the ionic strength. In that context, the reaction of the antioxidant toward the PyO• can be more likely assumed as a reaction between like charged species involving mostly the phenolate form. In such cases, the increase on the kinetic rate constants observed with the increment of the buffer ionic strength, is more related to the primary saline effect. However, the reaction cannot be so easily parameterized because even when pyranine free radical has three negative charges: It is expected that the PyO• could behave as a strong dipole considering the reaction is taking place for a phenoxy-type free radical. A similar behaviour can be expected for the phenolic antioxidants, except for those with very low pKa, and therefore the reaction is related to the phenolate species. In extension to our studies in solution, we decided to explore the use of PyO• for examining compartmentalization and dynamic of antioxidants on neutral micelles of Triton X100. In this system, the initial location of pyranine is constrained to the aqueous dispersant medium being excluded from hydrophobic micellar core due to its negative charge; in consequence, also the original location of the PyO• generated by laser flash photolysis. Moreover, PyO• can be considered as a water-soluble phenoxyl free radical, which interestingly shows kinetic rate constants with phenolic antioxidants in a similar range of magnitude that peroxyl free radicals.59


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The apparent kinetic rate constants measured for Trolox and sinapic acid in the presence of micelles were considerably lower than those measured in phosphate buffer (Table 2). This is reasonable considering that the reaction of PyO• with the antioxidant would take place in the aqueous phase. In fact, it is possible to establish a trend between the hydrophobicity of the antioxidant, expressed as Log P, and their expected partition between the micelle and the dispersant aqueous media. However, for ferulic acid, the less reactive antioxidant tested here, there was almost no difference regarding the apparent rate constant determined in presence and in absence of micelles. This behaviour goes beyond the hydrophobicity of the antioxidant in comparison with sinapic acid or caffeic acid establishing a limit on the effect observed on the apparent rate constant on Triton X-100 micelles that has to be related with the dynamic on the micelles. The dynamic of Triton X100 micelles has been characterized by a micellar diffusion of 1.15 x 106 M-1s-1, and the rate constants for the surfactant exit and re-entry of 1.1x106 s-1 and 3.7x 109 M-1 s-1 at 25 °C, respectively.60,

61

In addition, it has also to be considered the

exchanging rates of solutes from the micelle (hydrophobic environment) toward the dispersant media. In general, solute exit rates are rate-limiting steps that depend on the hydrophobicity of the solute but the entrances rates are near diffusional controlled. Thus, for 10-(4-bromo-1naphthoyl) decyltrimethyl ammonium bromide, the exits rates are found around 3 x 103 s-1, and re-entry constants are approximately 6 x 107 M-1s-1 in hexadecyltrimethylammonium chloride micelles.61 In the case of Triton X100, exit rate constants of 3.3 x 106 s-1 have been reported for dodecylpyridinium chloride.62 Taking those dynamic factors into consideration, it is possible to establish a lower limit on the value of the kinetic rate constant between PyO• and the antioxidant where it will not be possible


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to observe the influence of the micellar partition of the antioxidant. In particular, this limit will depend on a series of factors including the reactivity of the antioxidant, its partition constant and also the exit rate from the micelle. Then, as the exit rate of the antioxidant from the micelle reaches the PyO• lifetime in the experimental conditions, the micelle would act as a reservoir of antioxidant for the free radical reaction in the aqueous media, decreasing the hydrophobicity effect previously shown for the most reactive antioxidants, such as Trolox or sinapic acid. However, all cinnamic acids herein tested have similar hydrophobicities; therefore, it is clear that the reactivity is establishing a limit that expressed in the rate constant for PyO• is about k10 mM 1x106 M-1s-1. On the other side, considering the high hydrophobicity of α-tocophenol, it would be completely compartmentalized within Triton X100 micelles; however, herein it is observed a considerable fast reaction with PyO• (Table 2). That suggests that this reaction is occurring at the interface on the micelle, even the highly negative charge of the pyranine free radical that confines it at the aqueous phase, nonetheless evidently with accessibility to the micellar interface. It is important to note that; Triton X100 micelles are relatively unstructured and dynamic systems where the phenol moiety of the α-tocophenol has freedom for reaching the micellar/water interface. That has been previously observed even for more rigid systems such as liposomes, where it has been recognized accessibility of α-tocopheryl free radical to the interface in membranes, to be repaired by reaction with ascorbic acid.63 In spite of that, the observed rate constant value for α-tocophenol measured in Triton X100 (Table 2) is almost 30 times lower than the reaction with Trolox in the absence of micelles (Table 1) that mimics the phenol chromane reactive group in aqueous phase. That could be a consequence of a hindering access of the pyranine free radical toward the location of the phenol group in the micelle.


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Table 2. Observed rate constants for the reaction of PyO• with phenolic antioxidants in Triton X-100 (100 mM) in phosphate buffer (10 mM, pH 7.0). All measurements in N2O purged solutions at 25ºC. Compound

kTriton X100, 106 M-1s-1

kTriton X100/k10 mM

Log P33–35

Trolox

1.41 ± 0.10

0.008 ± 0.10

3.1

Sinapic acid

1.63 ± 0.11

0.19 ± 0.12

1.2

Caffeic acid

3.08 ± 0.14

0.96 ± 0.06

1.0

Ferulic acid

2.12 ± 0.24

1.20 ± 0.14

1.4

α-tocopherol

6.22 ± 0.52

ND

12.1

± Values are standard deviations calculated from three independent experiments

In addition to the previous studies, we also explored interaction and dynamics of PyO• with human serum albumim (HSA), probably the most relevant transporting protein in the bloodstream. It has been shown that several antioxidants and drugs can be bound to HSA in specific bonding sites denominated binding site I and site II.64-66 Interestingly, recently was also reported a strong interaction between pyranine and HSA than can be defined as a complex with an equilibrium constant Keq = 6.2 x 106 M-1.67 Spectroscopic measurements have shown a decreasing the absorbance of the PyO- form with HSA concentration that agrees with an interaction involving a hydrogen bond between pyranine in acid form (PyOH) with amino acids of the protein. However, that interaction would be heterogeneous due to possible interactions of the dye with different residues that could include both external and internal in binding sites in HSA.


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Figure 6. (A) Observed decay rate constant for PyO• measured at different concentrations of HSA. (B) Variation of the observed rate constant for the reaction of ascorbic acid with PyO• in the presence of different concentrations of HSA. Reactions carried out using PyOH 150 µM in 100 mM Pi (pH 7.0) and laser excitation 470 nm 10 mJ per pulse.

Subsequently, we evaluated the possible formation of the PyO• in the presence of HSA by exciting phenolate form of pyranine present at 470 nm. Surprisingly, the decay constant of PyO• decreased with the increase of the HSA concentration (Figure 6A). This result suggests that this increase on the lifetime of PyO• is related to stabilization by the environment on the protein. In fact, at concentration of HSA higher than 100 µM almost all pyrinine should be bound to the


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protein, but due to the heterogeneous and dynamic of this equilibrium there is still phenolate close to protein that is excited by 470 nm, generating PyO• instantaneously stabilized by hydrogen bond to the amino acid residues. In that condition, we evaluate the apparent rate constant reaction of ascorbic acid toward PyO• generated on the interaction with HSA in order to evaluated dynamic of the reaction. In particular, it has been established that ascorbic acid is bound to the binding site I in human and bovine serum albumin with a moderate association constants (Kb≈1.5 x 104 M-1),68, 69 that could give us information of the dynamic of the reaction. Kinetic measurements of the reaction of ascorbic acid with PyO• showed a drastic decrease on the apparent rate constant with the concentration of HSA, reaching a plateau at high protein concentration (Figure 6B). This behaviour can be related to different processes depending on the concentration of HSA: i) at very low concentrations of HSA, most of the PyO• would be generated by excitation of pyranine (as phenolate, PyO-) in the aqueous phase by 470 nm laser, and in consequence the decrease on the observed rate constant would be due to a decrease on the free ascorbate concentration by association to HSA, and ii) at high concentration, PyO• would be mainly associated to amino acid residues, and the observed rate constant towards ascorbate would only measure the rate of the entry or association of ascorbic acid to HSA. This kinetic analysis of the PyO• behaviour in HSA, let us to note that in the strict analysis, the value of the association constant usually determined for antioxidants would lack of meaning for reaction with free radicals without a comparison with an experimental measurement where a dynamic of the process can be developed.


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4.1 CONCLUSIONS We have efficiently generated pyranine free radicals by visible wavelength laser irradiation, in addition with a complete spectroscopic and EPR characterization well modelled by theoretical studies. The relatively low reactivity of the PyO• towards oxygen responds to a high spin delocalization but does not hinder its reaction to phenolic antioxidants. In particular, the ability of generating pyranine-derived free radicals by laser flash photolysis allow us to reveal a strong effect of the ionic strength, due to the ionic characteristics of the pyranine free radical but also attributing an ionic charged or dipole on the reactive form of the antioxidants. In addition, the extension of this methodology to study compartmentalization effects into micelles and proteins, have allowed us to evaluate the efficiency of antioxidants in terms of reactivity and also the dynamics of the antioxidant and free radical in environment more biological relevant.

ASSOCIATED CONTENT Supporting Information. Additional laser flash photolysis data and EPR experiments are included

AUTHOR INFORMATION Alexis Aspée, Email: [email protected]

ACKNOWLEDGMENTS The financial support from FONDECYT projects 3130697 and 1140240 are gratefully acknowledged. We also like to thanks to Sergei Gorelsky for preliminary calculus. Jessica Godoy (22150413) and Daniel Zúñiga (21151163) thank fellowships from CONICYT.


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