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Publicly Available Published by De Gruyter April 18, 2019

Phenolic betalain as antioxidants: meta means more

Letícia C. P. Gonçalves, Nathana B. Lopes, Felipe A. Augusto, Renan M. Pioli, Caroline O. Machado, Barbara C. Freitas-Dörr, Hugo B. Suffredini and Erick L. Bastos ORCID logo

Abstract

Betalains are phytochemicals of nutraceutical importance that emerged as potent antioxidants, preventing radical chain propagation and the deleterious health effects of oxidative stress. However, despite the wide application of betalains as color additives in products for human consumption, little is known about the relationship between their structure and antioxidant potential. Here we investigate the mechanism of antioxidant action of three regioisomeric phenolic betalains and show that the meta isomer has higher antiradical capacity than most natural betalains, anthocyanins and flavonoids. Structural and pH effects on redox and antiradical properties were investigated and the results are rationalized in light of quantum chemical calculations. Our results demonstrate that hydrogen atom transfer/proton-coupled electron transfer or sequential proton loss electron transfer mechanisms are plausible to explain the radical chain breaking properties of phenolic betalains in water. Furthermore, mesomeric effects are responsible for the stabilization of the resulting radical phenolic betalains. These findings are useful for the design of biocompatible antioxidants and for the development of novel additives for functional foods and cosmetics with high antioxidant potential.

Introduction

Radical and closed-shell reactive oxidant species (hereafter called ROS) are involved in key processes for life maintenance such as cell respiration, cell-cell signaling and immune response [1]. However, physiological homeostasis depends, among other factors, on the capacity of endogenous antioxidants and enzymes to detoxify these ROS and to repair the oxidative damage caused by their overproduction [2], [3]. The judicious intake of foods rich in antioxidants, either naturally-occurring or supplemented, has been reported to contribute for the redox balance in vivo [4]. In particular, many phytochemicals of nutraceutical importance have been used to prevent and treat diseases related to oxidative stress such as atherosclerosis, Alzheimer’s and Parkinson’s diseases, and cancer [5], [6], [7], [8].

Betalains are chiral diazapolymethine chromoalkaloids found in plants and fungi, e.g. bougainvillea, amaranth, beetroots and fly agaric, that originate from the spontaneous coupling between amines or amino acids and betalamic acid [9], [10], [11]. The best-known betalain is betanin (Bn), a FDA-approved color additive [12] that emerged as a potent non-toxic antioxidant [13], [14], [15], an anti-inflammatory agent [16], [17], an electroactive component for solar cells [18], [19], [20], and a mediator for hydrogen production [21], [22]. Bn can scavenge more radicals, i.e. has higher antiradical capacity, than its non-phenolic analog indicaxantin (BtP). The antioxidant performance of Bn, BtP, and betalamic acid (Fig. 1a) is higher than that of several flavonoids, ascorbic acid, and tocopherols [14], [23], [24].

Fig. 1: Natural and non-natural betalains and their common precursor. (a) Betanin (betanidin-5-O-β-glucoside, Bn), indicaxanthin (L-proline betalain, BtP) and betalamic acid (HBt). (b) Phenolic betalains (OH-pBeets).

Fig. 1:

Natural and non-natural betalains and their common precursor. (a) Betanin (betanidin-5-O-β-glucoside, Bn), indicaxanthin (L-proline betalain, BtP) and betalamic acid (HBt). (b) Phenolic betalains (OH-pBeets).

Gandía-Herrero and coworkers investigated the role of the structure on the antioxidant potential and photophysical properties of betalains and betalamic acid and found that phenolic betalains show higher antioxidant capacity and lower fluorescence compared to non-phenolic derivatives [25], [26], [27], [28]. However, knowledge on structure-properties relationships of betalains is still modest compared to other important classes of plant pigments, such as anthocyanins and carotenoids, and information on the relative effect of hydroxyl groups in position 5 and 6 of the cyclo-DOPA portion of betanin is scarcely available [14]. Here we analyze the effect of the position of the hydroxyl group on the antioxidant capacity and redox behavior of prototypical betalains bearing a conjugated phenol moiety. Three regioisomers of OH-pBeets (Fig. 1b) were obtained by partial chemical synthesis (semisynthesis), characterized and the dependence of their antioxidant capacity and voltammetric potentials on the pH was determined. The mechanism of antiradical action of these compounds was rationalized in terms of their electronic properties and quantum chemical calculations.

Results and discussion

Semisynthesis and electronic properties of OH-pBeets

Three non-natural regioisomeric phenolic betalains (OH-pBeets) were semisynthesized by coupling betalamic acid (1) with the appropriate aminophenol 2 in water (Fig. 2a), according to the procedure described by Schliemann and co-authors [29]. 1H NMR analysis reveals that the position of the hydroxyl group has a minor effect on the chemical shifts of hydrogen atoms of OH-pBeets. However, meta substitution deshields the H5 and H2 and slightly shields the H8 compared to the ortho and para regioisomers (Fig. 2b), indicating that the N1 atom of the 2-piperideine ring of m-OH-pBeet has larger positive charge density. Calculated partial charge density show that electron donation by the phenolic ring reduces electron delocalization through the diazapolymethine system and that intramolecular hydrogen bonding stabilize the positive charge at the N9 of o-OH-pBeet (Fig. 2c). Color analysis from the absorption spectra show that the three regioisomers are light red in aqueous solution (CIELa*b* around 90; 25; 4, Illuminant D65, 10°, Fig. 2d). The molar absorption coefficients (ε) were measured using an endpoint method (Fig. S1, Table S1) [27], viz., 50 000 L mol−1 cm−1 for o-OH-pBeet and 63 000 for both m- and p-OH-pBeet. The fluorescence quantum yields (ΦFL) of OH-pBeets are roughly one order of magnitude lower than that of several natural betalains in water, i.e. o-OH-pBeet (2.6×10−4), m-OH-pBeet (6.7×10−4), and p-OH-pBeet (1.8×10−4) vs. indicaxanthin (4.6×10−3) [30], vulgaxanthin II (7.3×10−3) [31], and miraxanthin V (3.0×10−3) [32], but similar to that of betanin (7.0×10−4) [33] possibly due to a higher conformational/torsional freedom of electronically-excited betalains containing electron-rich diazapolymethine systems [27], [30], [32], [33], [34].

Fig. 2: Semisynthesis and spectroscopic data of OH-pBeets. (a) Acid-catalyzed coupling of betalamic acid (1) and aminophenols 2 in water. The product is presented in its ammonium salt form (see Experimental part) and atom numbers are shown in red. (b) 1H NMR spectra of OH-pBeets (70 μmol L−1, 500 MHz, D2O at 293K). (c) Geometries of OH-pBeets optimized at the SMD(water)/M06-2X/6-311++G(d,p) level and partial charges of the nitrogen atoms according to the Merz-Kollman-Singh (MKS) scheme constrained to reproduce the dipole moment (μ). (d) Normalized UV-vis () and fluorescence (---/filled) spectra of OH-pBeets in water.

Fig. 2:

Semisynthesis and spectroscopic data of OH-pBeets. (a) Acid-catalyzed coupling of betalamic acid (1) and aminophenols 2 in water. The product is presented in its ammonium salt form (see Experimental part) and atom numbers are shown in red. (b) 1H NMR spectra of OH-pBeets (70 μmol L−1, 500 MHz, D2O at 293K). (c) Geometries of OH-pBeets optimized at the SMD(water)/M06-2X/6-311++G(d,p) level and partial charges of the nitrogen atoms according to the Merz-Kollman-Singh (MKS) scheme constrained to reproduce the dipole moment (μ). (d) Normalized UV-vis (

) and fluorescence (---/filled) spectra of OH-pBeets in water.

Radical scavenging properties

The antioxidant capacity of aminophenols and OH-pBeets were determined using the Trolox Equivalent Antioxidant Capacity (TEAC)/ABTS+˙ colorimetric assay [35], which allows the study of both hydrogen atom transfer (HAT) and single electron transfer (SET) processes, the two main classes of mechanisms of free radical scavenging by chain-breaking antioxidants [36], [37], [38], [39]. This assay has been used for the quantification of several antioxidants and can be performed over a wide pH range, being suitable for comparison purposes [38], [40]. At pH 7.4, the TEAC value of m-OH-pBeet (5.1±0.3) is roughly twice as high as that of the other regioisomers and the precursor aminophenols, and four-fold higher than that of ascorbic acid (AscH, TEAC=1.2±0.1) (Figs. 3a and S2). Noteworthy, despite the fact that m-OH-pBeet has a single phenolic hydroxyl group, its antiradical capacity is slightly higher than that of betanin (4.7±0.3), and the flavonoids quercetin (TEAC=4.9±0.4) [41] and epicatechin gallate (TEAC=4.8 [42], [43]; 4.9 [44]). The TEAC of m-OH-pBeet increases as the medium pH increases from 4 to 7, reaching a plateau at pH 6 (Fig. 3b). This behavior contrasts with that of o-OH-pBeet and p-OH-pBeet whose TEAC is not affected by the pH, but agrees with that of betanin, which is the most efficient antioxidant betalain.

Fig. 3: Antioxidant capacity and square-wave voltammograms of OH-pBeets. (a) TEAC of aminophenols 2 and OH-pBeet in phosphate buffer, pH 7.4. Asterisks indicate significant differences according to the analysis of variance (ANOVA, p<0.05) and comparison of the groups using the Tukey’s test; **compared to unmarked columns, ***compared to o-OH-pBeet. The raw data used to calculate the TEAC are presented in Fig. S2. (b) Effect of pH on the TEAC of OH-pBeets and betanin (see SI). (c) Dependence of the contour of normalized currents vs. applied potential (E vs. Ag/AgCl, KCl 3 mol L−1) of the aminophenols 2 and OH-pBeet on the pH. Conditions: [2]=10 μmol L−1; [OH-pBeets]=10 μmol L−1, 25°C.

Fig. 3:

Antioxidant capacity and square-wave voltammograms of OH-pBeets. (a) TEAC of aminophenols 2 and OH-pBeet in phosphate buffer, pH 7.4. Asterisks indicate significant differences according to the analysis of variance (ANOVA, p<0.05) and comparison of the groups using the Tukey’s test; **compared to unmarked columns, ***compared to o-OH-pBeet. The raw data used to calculate the TEAC are presented in Fig. S2. (b) Effect of pH on the TEAC of OH-pBeets and betanin (see SI). (c) Dependence of the contour of normalized currents vs. applied potential (E vs. Ag/AgCl, KCl 3 mol L−1) of the aminophenols 2 and OH-pBeet on the pH. Conditions: [2]=10 μmol L−1; [OH-pBeets]=10 μmol L−1, 25°C.

The comparison of voltammetric potentials provide insight on the effect of the structure on the redox properties of antioxidants [45]. Therefore, we determined the influence of the pH on the square-wave peak potentials (Eps) of OH-pBeets and the precursor aminophenols. Overall, the behavior of each OH-pBeets is qualitatively similar to that of the corresponding aminophenol (Figs. 3c and S3). The Eps of the ortho and para-substituted aminophenols and OH-pBeet decrease as the pH increases, possibly due to the formation of the corresponding semi-iminoquinone (−1e) or iminoquinone (−2e). Within the pH range 4–8, the oxidation of m-OH-pBeet occurs at a higher Ep (876 mV–767 mV vs. Ag/AgCl) compared to o- (501 mV–417 mV) and p-OH-pBeet (494 mV–264 mV) (Fig. S4). The voltammetric peak of m-2 (764 mV–730 mV) corresponds to a reversible oxidation that occurs at a slightly lower Ep compared to the irreversible oxidation of m-OH-pBeet (876 mV–767 mV). Betalamic acid is irreversibly oxidized when a similar potential is applied (Ep=849 mV, pH 9); therefore, the Eps corresponding to the oxidation of the phenolic moiety and the diazapolymethinic system of m-OH-pBeet are expected to overlap, explaining the single voltammetric peak observed in m-OH-pBeet oxidation.

The structures of the most stable conformers of each OH-pBeet in four protonation forms (Fig. 4, forms A-D) were optimized in water in order to calculate thermodynamic parameters that can contribute to the rationalization of the experimental results [46]. The acidic dissociation constants of betalains are scarcely described [47], [48], and we were unable to measure the pKaH of the iminium group or the pKa of the OH group due to the occurrence of hydrolysis (Fig. S9). Therefore, we calculated the change in Gibbs energy for acid-base equilibria of OH-pBeets and concluded that the form A of the three regioisomers is the major species in neutral water (Fig. 4, Table S4).

Fig. 4: More O’Ferrall-Jencks diagrams for the ionization and oxidation of (a) o-OH-pBeet, (b) m-OH-pBeet and (c) p-OH-pBeet in different protonation states; compounds are presented as carboxylates since this is the expected major form in water at pH higher than 4. Values refer to enthalpy changes between states. Spin density surfaces (isocontour=0.004 a.u., positive/orange and negative/white) and chemical structure indicating the delocalization of the unpaired electron; formal charges are kept localized for clarity; gray regions indicate higher spin density.

Fig. 4:

More O’Ferrall-Jencks diagrams for the ionization and oxidation of (a) o-OH-pBeet, (b) m-OH-pBeet and (c) p-OH-pBeet in different protonation states; compounds are presented as carboxylates since this is the expected major form in water at pH higher than 4. Values refer to enthalpy changes between states. Spin density surfaces (isocontour=0.004 a.u., positive/orange and negative/white) and chemical structure indicating the delocalization of the unpaired electron; formal charges are kept localized for clarity; gray regions indicate higher spin density.

The ionization potentials (IP) and bond dissociation energies (BDE) of phenols are affected by mesomeric effects and by the planarity of the resulting radical. IP and BDE have been compared to determine whether hydrogen atom transfer/proton-coupled electron transfer (HAT/PCTE) or single electron transfer (SET) mechanism predominates in the reaction between phenols and radicals [49]. Phenolic antioxidants acting by H˙ donation usually have low BDE [49], [50]. Indeed, the BDE for the formation of [OH-pBeets]˙ from OH-pBeets is lower that both the IP of OH-pBeets to produce [OH-pBeets]+˙ and the electron transfer enthalpy (ETE) related to the formation of [OH-pBeets]˙ from [OH-pBeets] (Table 1). Therefore, the occurrence of single electron transfer followed by proton transfer (SET-PT) is energetically less probable than the first step of the sequential proton loss electron transfer (SPLET) and HAT/PCET mechanisms [40], [51].

Table 1:

Thermodynamic parametersa for the three most common antiradical mechanisms for the OH-pBeetsb (in kJ mol−1).

Isomer Site HAT/PCET
SET-PT
SPLET
ΔH° ΔEiso
BDE IP PDE PA ETE
ortho 451
−ArOH (B) 332 67 140 378 518 −32
=N+HAr (C)c 350 86 141 395 536 −13
meta 459
−ArOH 369 96 154 401 555 5
=N+HAr 352 79 140 398 538 −12
para 442
−ArOH 332 76 155 363 518 −32
=N+HAr 345 89 139 392 531 −19

  1. aEqs. 1–5, see methods. bForm A of the OH-pBeet (charge −1, Fig. 4). cThe deprotonation (−H+, PA) and oxidation (−H˙, BDE) of N1 instead of N9 are less favorable. PAs: (o-) 163 kJ mol−1, (m-) 162 kJ mol−1, (p-) 163 kJ mol−1; BDEs: (o-) 378 kJ mol−1, (m-) 382 kJ mol−1 and (p-) 373 kJ mol−1.

According to the ΔEiso parameter (Eq. 6 in methods) [52], the phenoxyl radicals of o- and p-OH-pBeet (form A) are more stable than the diazapolymethine radical by ca. 10 kJ mol−1 (Table 1). Conversely, the diazapolymethine radical of m-OH-pBeet is 17 kJ mol−1 more stable than the phenoxyl radical. BDEs follow the same trend. Thus, in case HAT/PCET takes place the oxidation of m-OH-pBeet in water is more favorable via homolysis of the N–H bond instead of the phenolic O–H bond. The deprotonation of [OH-pBeets]+˙ to produce [OH-pBeets]˙ can be more favorable in the OH or the NH sites depending on the regioisomer. For o- and p-OH-pBeets, deprotonation of the phenolic OH group are, respectively, 19 and 13 kJ mol−1 more favorable than the NHAr moiety, but the deprotonation of the NHAr moiety is 17 kJ mol−1 more favorable for m-OH-pBeet. For comparison, the homolytic breaking of the phenolic OH bond of catecholamines and dopamine derivatives requires 40–80 kJ mol−1 less energy than breaking the NH bonds [46].

The stability of radicals can be evaluated from their spin density profiles since spin delocalization improves stability. Analysis of the spin density surfaces of the species involved in the 1H+ deprotonation and 1e oxidation of OH-pBeet show that the unpaired electron can be delocalized over the phenol and/or the diazapolymethine moieties (Fig. 4). Spin densities at the phenol groups of both [o-OH-pBeet]+˙ and [p-OH-pBeet]+˙ (forms A˙) are higher compared to the meta regioisomer. The deprotonation of the phenolic group followed by oxidation (SPLET mechanism), results in phenoxyl-centered radicals (form B˙). The o- and p-OH-pBeets show some degree of radical delocalization upon the diazapolymethine system that is absent in the meta regioisomer. The deprotonation of the diazapolymethine system followed by oxidation leads to species C˙ whose spin density is delocalized upon the diazapolymethine system for all regioisomers.

Thermodynamic data (Table 1) shows that the preferred pathway for the antiradical action of the ortho and para regiosiomers results in phenoxyl radicals (form B˙), whereas for the m-OH-pBeet, the formation of the diazapolymethine radical (form C˙) is more favorable. In case the form C of OH-pBeets is the main species in water, species D˙ are formed, whose spin density is highly delocalized throughout the whole conjugated system for o-OH-pBeet and p-OH-pBeet, but distributed in the diazapolymethine system for the meta regioisomer (Fig. 4).

The oxidation of the N–H and O–H deprotonated forms of OH-pBeets are less thermodynamically favorable than the direct homolytic bond breaking and the preferred site is the O–H group for the ortho and para regioisomers and the N–H group for the m-OH-pBeet (Fig. 4, Table 1). However, one cannot unequivocally discern between sequential proton loss electron transfer (SPLET) and HAT/PCET mechanisms in the oxidation of OH-pBeet by ABTS+˙ without proper kinetic analysis [36], [40], [53].

The BDE, IP and ETE of OH-pBeets show the same overall trend of the voltammetric potential, viz.: following the sequence, p-OH-pBeet<o-OH-pBeet<m-OH-pBeet. The TEAC assay quantify the total number of ABTS˙+ quenched by the antioxidant/antiradical compound and no correlation between the redox potential and TEAC values have been found for several phenolic compounds [40]. The 2e oxidation of the phenol moiety and the diazapolymethine system of o- and p-OH-pBeet leads to a closed shell product, whereas a diradical is formed in the case of m-OH-pBeet due to the lack of conjugation between the OH phenolic group and the diazapolymethine system. Therefore, additional oxidation of the meta regioisomer is required to reach a closed shell product, resulting in higher value of TEAC as observed experimentally (Fig. 3a and b).

Conclusions

The study of the antiradical capacity of non-natural phenolic betalains contributed to the overall understanding of the antioxidant properties of betalains, in particular betanin. The antiradical capacity follows the order: m-OH-pBeet>o-OH-pBeet>p-OH-pBeet. The meta regioisomer is able to quench more ABTS˙+ compared to natural products such as betanin and most flavonoids and carotenoids, possibly because the phenolic group is not conjugated with the diazapolymethine system, but both groups can stabilize radicals by resonance and are prone to further oxidation. Furthermore, the N–H iminic bond of the meta regioisomer is the preferred site for oxidation, whereas the 1e oxidation of the phenolic groups of o- and p-OH-pBeet results in a stable semi-quinone, leading to lower values of Ep compared to m-OH-pBeet. The high IP of OH-pBeets indicate that the occurrence of SET-PT radical scavenging mechanism is unlikely. The occurrence of SPLET and HAT/PCTE mechanism cannot be discriminated because the difference between the values of BDE and ETE are around 30 kJ mol−1. However, SPLET could be favored in water and other polar solvents due to charge stabilization. These findings explain the high antiradical capacity of the natural pigment betanin and show that betalains can be tailored in order to enhance their radical scavenging/antioxidant properties.

Methods

General

All chemicals were purchased from Sigma-Aldrich and used without further purification, except as otherwise stated. Solutions were prepared using deionized water (18.2 MΩ cm at 25°C, TOC≤4 ppb, Milli-Q, Millipore).

Semisynthesis of phenolic betalains

o-, m- and p-OH-pBeets were semisynthesized using betalamic acid according to a procedure adapted from Schliemann and coauthors [29]. In a 5-mL three-necked round-bottom flask protected from light and equipped with magnetic stirring and a pH electrode were placed a solution of betalamic acid (0.7 mg, 3.2 μmol) in aqueous ammonium hydroxide (2.0 mL, pH 11) and solid aminophenol (32 μmol, 10 equiv.). After 30 min at 25°C, the reaction was cooled to 5±2°C, and HOAc (conc.) was added slowly until pH 5 was reached. Next, the cooling bath was removed, and the solution was kept under stirring for 30 min. Products were purified through reversed phase column chromatography (Silicagel 90 C18 percolated with MeOH, mobile phase: water/MeOH gradient until reach 10% v/v MeOH). Red colored fractions were combined, the methanol was evaporated under reduced pressure (20 mbar, 25°C) and the resulting aqueous solution was lyophilized. Raw products were kept in the dark at −20°C and purified by reversed-phase flash column chromatography immediately before use. Products were characterized by NMR spectroscopy (Fig. S5–S7) and HPLC-DAD-HRMS/MS (Fig. S8).

o-OH-pBeet

Yield: 40% from HBt.

1H NMR (D2O, 500 MHz) δ 8.38 (brs, 1H), δ 7.44 (d, J=7.7 Hz, 1H), δ 7.18 (t, J=7.7 Hz, 1H), δ 7.09–6.97 (m, 2H), δ 6.28 (brs, 1H), δ 6.23 (s, 1H), δ 4.33 (t, J=7.6 Hz, 1H), δ 3.32 (dd, J=17.2, 7.6 Hz, 1H), δ 3.18 (dd, J=17.2, 7.6 Hz, 1H) (Fig. S5).

HRMS: calculated for C15H15N2O5+ (m/z) [M+H]+=303.0975, found=303.0983; diff: 2.6 ppm (Fig. S8).

UV-Vis/Fl: λabsmax=510 nm (water), λFlmax=567 nm (water, λexc=500 nm), ε510 nm=5.0×104 L mol−1 cm−1 (water), ΦFl=2.6×10−4 (water).

m-OH-pBeet

Yield: 60% from HBt.

1H NMR (D2O, 500 MHz) δ 8.32 (brs, 1H), δ 7.32 (t, J=8.0 Hz, 1H), δ 6.89 (dd, J=8.0, 1.4 Hz, 1H), δ 6.82 (s, 1H), δ 6.75 (d, J=8.0, 1H), δ 6.26 (s, 1H), δ 6.20 (brs, 1H), δ 4.34 (t, J=7.6 Hz, 1H), δ 3.33 (dd, J=17.3, 7.6 Hz, 1H), δ 3.18 (dd, J=17.3, 7.6 Hz, 1H) (Fig. S6).

HRMS: calculated for C15H15N2O5+ (m/z) [M+H]+=303.0975, found=303.0976; diff: 0.3 ppm (Fig. S8).

UV-Vis/Fl: λabsmax=510 nm (water), λFlmax=561 nm (water, λexc=500 nm), ε510 nm=6.2×104 L mol−1 cm−1 (water), ΦFl=6.7×10−4 (water).

p-OH-pBeet

Yield: 65% from HBt.

1H NMR (D2O, 500 MHz) δ 8.33 (brs, 1H), δ 7.27 (d, J=8.6 Hz, 2H), δ 6.96 (d, J=8.6 Hz, 2H), δ 6.21 (d, J=11.2 Hz, 1H), δ 6.16 (s, 1H), δ 4.30 (t, J=7.4 Hz, 1H), δ 3.32–3.27 (m, 1H), δ 3.16 (dd, J=17.0, 7.4 Hz, 1H) (Fig. S7).

HRMS: calculated for C15H15N2O5+ (m/z) [M+H]+=303.0975, found=303.0983; diff: 2.6 ppm (Fig. S8).

UV-Vis/Fl: λabsmax=513 nm (water), λFlmax=577 nm (water, λexc=500 nm), ε510 nm=6.3×104 L mol−1 cm−1 (water), ΦFl=1.8×10−4 (water).

Antiradical capacity

The antiradical capacity was determined by the TEAC/ABTS˙+ assay using the protocol developed by Re and coauthors [35]. Briefly, a stock solution of ABTS˙+/ABTS in water was prepared via partial oxidation of ABTS [2,2′-azinobis-(3-ethyl-benzothiazoline-6-sulfonic acid), 7 mmol L−1] by potassium persulfate (2.45 mmol L−1) in the dark at room temperature for 16 h. The stock solution of ABTS˙+/ABTS was diluted in phosphate buffer at the different pHs (0.1 mol L−1) to an absorbance of 0.7 (46.7 μmol L−1 ABTS˙+) at 734 nm. The kinetic of bleaching of ABTS˙+ after addition of antioxidant (20–60 μL, final concentration within the μmol L−1 range) was monitored by change in the absorption at 734 nm over 6 min (ΔA). The slope of the linear correlation between ΔA and the concentration of antioxidant, α, is a measure of antioxidant capacity. Consequently, the αsampleTrolox ratio is the Trolox Equivalent Antioxidant Capacity, TEAC.

Square-wave voltammetry

Square-wave voltammetry (SWV) experiments were carried out at room temperature using a Metrohm-μautolab potentiostat/galvanostat system operated with the GPES 4.9 software. A three-electrode Pyrex cell equipped with a boron-doped diamond working electrode (BDD electrode, 0.28 cm2), a Pt foil (0.72 cm2) auxiliary electrode, and a Ag/AgCl (3.0 mol L−1 KCl) reference electrode was used. The working electrode was cleaned before each measurement by application of +3.0 V potential in a 0.5 mol L−1 H2SO4 solution. Baseline-corrected voltammograms were used to determine peak potentials (Ep) and peak currents (Ip). Analite solutions were prepared using Britton-Robinson buffer (0.4 mol L−1, pH range: 4–8) and experimental conditions were set as follows: pulse amplitude: 50 mV, pulse frequency: 10 Hz, and scan range: −0.2 V to 1.6 V vs. Ag/AgCl.

Computational methods

The equilibrium geometries of OH-pBeets as well as their radicals, radical cations, and anions, were optimized at the SMD/M06-2X/6-311++G(d,p) level [54], [55], which has been widely used for the calculation of antiradical parameters [46]. Stationary points were characterized as minima based on vibrational analysis; the coordinates of the optimized structures are provided in the SI. All reported energies include zero-point energy (ZPE) as well as thermal corrections (T=298.15 K) from frequency calculations. All calculations were carried out using the Gaussian09 rev. D.01 program suite [56].

The thermodynamic parameters governing the radical scavenging mechanism were calculated from the standard enthalpies of the appropriate species according to Eqs. 1–5 [57].

(1) (Bond dissociation energy, BDE, BetH)=H(Bet)+H(H)H(BetH)
(2) (Ionization potential, IP, BetH)=H(BetH+)+H(e)H(BetH)
(3) (Proton dissociation enthalpy, PDE, BetH+)=H(Bet)+H(H+)H(BetH+)
(4) (Proton affinity, PA, BetH)=H(Bet)+H(H+)H(BetH)
(5) (Electron transfer enthalpy, ETE, Bet)=H(Bet)+H(e)H(Bet)

The enthalpies for the proton 6.197 kJ mol−1), electron (3.146 kJ mol−1) and hydrogen atom (−1306 kJ mol−1), as well as the solvation enthalpies for proton (−1055.7 kJ mol−1), electrons (−77.5 kJ mol−1) and hydrogen atom (−4 kJ mol−1) in water were taken from the literature [58], [59]. The radical stability (ΔEiso) was calculated using phenol and the phenoxyl radical as reference compounds (Eq. 6) [52].

(6) ΔEiso=H(Bet)+H(PhOH)H(BetH)H(PhO)

Article note:

A collection of invited papers based on presentations at the 4th International Conference on Bioinspired and Biobased Chemistry & Materials (NICE-2018), Nice, France, 14–17 October 2018.


Acknowledgements

We thank the São Paulo Research Foundation – FAPESP (ELB, 2014/22136-4 and 2016/21445-9; LCPG, 2007/59407-1; COM, 2015/24760-0), the Brazilian National Council for Scientific and Technological Development – CNPq (ELB, 303341/2016-5), and the Coordination for the Improvement of Higher Education Personnel – CAPES (NBL, 33002010191P0; RMP) for financial support. We thank Dr. Guilherme S. Buzzo for the fabrication of the BDD electrode.

  1. Author contributions: E.L.B. conceived the study; E.L.B., H.B.S., and L.C.P.G. designed the experiments; L.C.P.G., N.B.L., R.M.P., C.O.M., and B.F.–D. performed experimental work; E.L.B. and F.A.A. carried out theoretical calculations. E.L.B. and L.C.P.G. interpreted results and wrote the paper.

  2. Conflicts of interest: There are no conflicts to declare.

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Supplementary Material

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Published Online: 2019-04-18
Published in Print: 2020-02-25

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