Comprehensive Investigation of the Antioxidant and Pro-oxidant

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Comprehensive Investigation on the Antioxidant and ProOxidant Effects of Phenolic Compounds: A DoubleEdged Sword in the Context of Oxidative Stress? Romina Castañeda-Arriaga, Adriana Pérez-Gonzalez, Miguel Reina, Juan Raul Alvarez-Idaboy, and Annia Galano J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b03500 • Publication Date (Web): 17 May 2018 Downloaded from http://pubs.acs.org on May 17, 2018

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The Journal of Physical Chemistry

Comprehensive Investigation on the Antioxidant and Prooxidant Effects of Phenolic Compounds: A Double-Edged Sword in the Context of Oxidative Stress? Romina Castañeda-Arriaga,1 Adriana Pérez-González,2 Miguel Reina,1 J. Raúl AlvarezIdaboy,3 Annia Galano,1* 1

Departamento de Química. Universidad Autónoma Metropolitana-Iztapalapa. San Rafael Atlixco 186, Col. Vicentina. Iztapalapa. C. P. 09340. México D. F. México.

2

CONACYT - Universidad Autónoma Metropolitana - Iztapalapa. San Rafael Atlixco 186, Col. Vicentina. Iztapalapa. C. P. 09340. México D. F. México. 3

Facultad de Química, Departamento de Física y Química Teórica, Universidad Nacional Autónoma de México, México DF 04510, México.

*

E-mail: [email protected], [email protected]

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Abstract Oxidative stress (OS) is a health-threatening process that is involved, at least partially, in the development of several diseases. Although antioxidants can be used as a chemical defense against OS, they might also exhibit pro-oxidant effects, depending on environmental conditions. In this work, such a dual behavior was investigated for phenolic compounds (PhCs), within the framework of the density functional theory, and based on kinetic data. Multiple reaction mechanisms were considered in both cases. The presence of redox metals, the pH, and the possibility that PhCs might be transformed into benzoquinones were identified as key aspects in the antioxidant versus pro-oxidant effects of these compounds. The main virtues of PhCs as antioxidants are their radical trapping activity, their regeneration under physiological conditions, and their behavior as OHinactivating ligands. The main risks of PhCs as pro-oxidants are predicted to be the role of phenolate ions in the reduction of metal ions, which can promote Fenton-like reactions; and the formation of benzoquinones that might cause protein arylation at cysteine sites. Although the benefits seem to overcome the hazards, to properly design chemical strategies against OS using PhCs, it is highly recommended to carefully explore their duality in this context. Keywords: kinetics; rate constants; electron transfer, hydrogen transfer, Haber-Weiss, scavenging; metal chelation.


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1. Introduction Oxidative stress (OS) is defined as “the imbalance between biochemical processes leading to the production of reactive oxygen species (ROS) and those responsible for the removal of ROS”.1 It represents a serious hazard to molecules of crucial biological importance including lipids, proteins and DNA. OS is considered as a contributing factor to numerous diseases such as cancer;2-4 rheumatoid arthritis;5-7 pulmonary8-10 and renal diverse cardiovascular

diseases

including heart

failure,

atherosclerosis, cardiac hypertrophy and cardiomyopathy;

11-13

hypertension, 14-17

failures;

ischemia,

as well as neuro-

degeneration. The later leads to Alzheimer’s, Parkinson’s, memory loss, multiple sclerosis, depression, etc.18-24 Therefore, attenuating OS-induced molecular damage is essential to preserve human health. In addition to enzymatic defense systems, chemical antioxidants are a viable tool to achieve that purpose. Phenolic compounds (PhCs) are ubiquitous in nature, abundant in our diet, and -arguably- the most paradigmatic and known example of antioxidants. The role of PhCs as protectors against OS has been profusely investigated and reviewed.25-33 In fact, only last year there were more than 5,000 publications on this subject, and the trend on the number of related researches is still up (Figure 1).

Figure 1. Publications, in the last two decades, with the words phenol and antioxidant or pro-oxidant (in the title, abstract or keywords). The data was obtained from the Scopus database on May 16th, 2018. At the same time, there is rather convincing evidence that PhCs can exhibit pro-oxidant effects (Figure 1). The dual anti / pro-oxidant behavior of this kind of compounds seems to 3 ACS Paragon Plus Environment


be concentration-dependent

34-37

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and modulated by environmental factors such as the pH

and the presence of transition metals.34, 36-42 Antioxidants are, by definition, good reductant chemical agents, and PhCs are not the exception. Thus, there is a viable hypothesis that their pro-oxidant behavior arises from their ability to reduce metal ions, particularly Cu(II),36 making Cu(I) available to be involved in Fenton-like reactions, i.e., promoting the formation of ROS, in particular the very reactive and harmful hydroxyl radical (•OH).40, 43, 44

This, however, does not rule out other that other chemical processes might be associated

with the pro-oxidant behavior of PhCs. The pro-oxidant behavior of this family of compounds has numerous implications in the context of OS and its related health risks. For example, it accelerates lipid peroxidation and induces DNA damage.45 It has also been held responsible for the deterioration of axonal membranes.46 At the same time, the pro-oxidant behavior of PhCs, and other chemical compounds, can be beneficial for certain purposes including apoptosis of cancer cells45 and protection against infection.47 The cleavage DNA, resulting from the ROS generation associated with the redox cycling of copper, was proposed as a possible mechanism for the anticancer effects of some antioxidants including PhCs.48, 49 The data gathered so far, strongly indicate that the role of PhCs, in the OS context, is complex and needs to be fully understood to take advantage of their chemical behavior for protecting human health. In particular their abilities to scavenge free radicals, to act as •OH inactivating ligands (OILs) and to reduce Cu(II) seem to be key factors in this regard. Therefore, the present investigation aims to make quantitative estimations that allow to put into perspective the antioxidant versus pro-oxidant behavior of PhCs. In addition, the influence of pH on this dual behavior has also been explored. Hopefully, the results presented here might contribute to gain a deeper understanding on the chemistry of PhCs involved in OS.

2. Computational Details The Gaussian 09 package of programs50 was used for all the electronic calculations. The geometries of the stationary points were fully optimized within the frame of the Density Functional Theory (DFT). In particular, the M05 and M05-2X functionals51 were chosen for systems with and without Cu, respectively. Frequency calculations were done at the 4 ACS Paragon Plus Environment

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same level of theory in each case. The 6-311+G(d,p) basis set and the “solvation model based on density” (SMD),52 with water as solvent, were used in all the calculations. The number of imaginary frequencies were used to identify local minima and transition states. Intrinsic reaction coordinate (IRC) calculations were used to confirm that the transition states correspond to the appropriate reactants and products. All the reported data was obtained at 298.15 K. The M05 and M05-2X functionals were chosen for the present investigation because they are recommended as methods with broad applicability by their developers.51 In particular they have a high performance in “thermochemistry, kinetics, and non-covalent interactions” involving metal (M05) and non-metal (M05-2X) elements. Their reliability has been extensively corroborated by other authors.53-56 In addition, M05-2X has been identified as one of the best performing method for calculating energies of reactions that involve free radicals,57 as well as for kinetics, in solution.58 SMD was chosen for simulating the solvent based on previous evidence suggesting that it is adequate for charged or non-charged solutes, in quite diverse solvents or liquid media.52 In addition, it was proven to be adequate for mixed models and for geometry optimization and vibrational calculations in solution, instead of using thermodynamic cycles.59 2.1. Investigated phenolic compounds (PhC) Six representative PhCs were modeled (Scheme 1). They include different number of OH groups, as well as substituents of different chemical nature regarding their electron donor and electron withdrawing character.

Scheme 1. Phenolic compounds (PhC) investigated in this work, and their site numbering.



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2.2. Copper ions For the calculations of “free” Cu(II) ions, 4 water molecules were included in the coordination sphere, in an almost planar-square arrangement. This configuration corresponds to that accepted as the most probable for Cu(II), in water.60, 61 Considering that charged species (in the aqueous phase) are probably hydrated, this model constitutes a more appropriate representation of “free” copper ions, than bare species under physiological conditions. For consistency purposes, the same amount of water molecules were included in the Cu(I) models, even though the linear two-coordinate arrangement is preferred in this case.62-64 As a result, Cu(I) is in fact coordinated to only 2 water molecules (the other 2 are in the solvation sphere). 2.3. Kinetics The “quantum mechanism-based test for overall free radical scavenging activity” (QMORSA) protocol65 was used to obtain the kinetic data. This protocol was specifically designed for the calculation of rate constants, in solution. It was previously demonstrated that the QM-ORSA uncertainties are similar to those arising from experiments.65 Using this protocol implies considering all possible reaction mechanisms. In this study they are: “single electron transfer” (SET), “formal hydrogen atom transfer” (f-HAT), “radical adduct formation” (RAF), and “sequential proton loss electron transfer” (SPLET). The conventional transition state theory (TST)66-68 was used to compute the rate constants, together with the 1M standard state. For the SET reactions, the Gibbs energies of activation were estimated using the Marcus theory.69 For high rate constants that correspond to diffusion-limited reactions, the Collins-Kimball theory was used to include the diffusion control,70 in combination with the Smoluchowski71steady-state for irreversible bimolecular reactions, and the Stokes–Einstein72,

73

approach for the diffusion coefficients. More

detailed information on this protocol can be found elsewhere.65, 74 2.4. Deprotonation Routes For the investigated PhCs that are polyprotic molecules, it was necessary to elucidate the most likely deprotonation pathway. To that purpose, the relative energies of every possible species with the same deprotonation degree were calculated (at 298.15 K and using the 1M standard state). The deprotonation route with the lowest Gibbs free energy was assumed as


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the most likely one. This procedure is in line with previous strategies successfully used to identify the deprotonation routes of poliprotic species.75-80 2.5. Chelation Routes All the possible chelation sites (N and O atoms) in the investigated PhCs were explored (Scheme 1). In addition, when possible, their roles as mono-dentate and bi-dentate ligands have both been considered. Five chemical routes yielding Cu(II) chelates were explored: Route (I): Direct chelation by neutral phenols (HnA): Cu(H2O)42+ + HnA [Cu(H2O)4-j(HnA)]2+ + jH2O Route (II): Coupled deprotonation-chelation involving neutral phenols (HnA): Cu(H2O)42+ + HnA [Cu(H2O)4-j(Hn-1A)]+ + jH2O + H+ Route (III): Direct chelation by anionic phenols (Hn-1A−): Cu(H2O)42+ + Hn-1A− [Cu(H2O)4-j(Hn-1A)]+ + jH2O Route (IV): Coupled deprotonation-chelation involving anionic phenols (Hn-1A−): Cu(H2O)42+ + Hn-1A− [Cu(H2O)4-j(Hn-2A)] + jH2O + H+ Route (V): Direct chelation by di-anionic phenols (Hn-2A2−): Cu(H2O)42+ + Hn-2A2− [Cu(H2O)4-j(Hn-2A)] + jH2O 2.6. Damage to biomolecules Three different kinds of biological targets were considered here, namely: lipids, proteins and DNA. The models used to represent them are shown in Scheme 2. The lipid model (LM) represents unsaturated fatty acids and is a simplified model of linoleic acid, which maintains its most important chemical feature (2 allylic H atoms). The validity of using this model has been previously proven.81 The model used to represent amino acid residues in proteins is commonly referred to as a realistic model and has been successfully used for investigating protein reactions at specific sites.82-93 The residues considered here were chosen based on previous reports showing that leucine (Leu), cysteine (Cys), methionine (Met), tyrosine (Tyr), histidine (His) and tryptophan (Trp) are particularly susceptible to OS.84,

94-99

Their reactions with oxidants

may involve different mechanisms including SET (Tyr, Trp), f-HAT transfer (Cys, Leu, Met, His), RAF (Tyr, Trp, His), and protein arylation (Cys). 7 ACS Paragon Plus Environment


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Scheme 2. Models used to represent the investigated biological targets. For modeling oxidative damage to DNA, guanine (G) and 2’-deoxyguanosine (2dG) were chosen since G is the most easily oxidized of the nucleobases.100-103 This explains why oxidation of DNA, via SET, mainly involve G sites.104 Consequently, if a chemical agent oxidizes 2dG it can also cause oxidative damage to DNA. In contrast, if such an agent does not oxidize 2dG it is expected to be innocuous to DNA. A three-unit double-stranded oligomer was also used to test DNA oxidation. In this particular case the calculations were performed with the ONIOM method with a two-layer approach. The high level layer includes the central 2dG-2dC pair, and was treated at M05-2X/6-311+G(d,p) level of theory. On the other hand, the low layer was modeled with the semi-empirical method PM6. It is among the most accurate semi-empirical methods, in particular for energies and geometries of systems that involve hydrogen bonding.105 This model has been successfully used before.106


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3. Results and Discussion 3.1. Acid-Base Behaviour Since phenolate anions are expected to be better reductants than neutral PhCs, the acid-base equilibria may strongly influence both the antioxidant and the pro-oxidant behavior of this kind of compounds. In addition, the pH may also be an important environmental factor in this regard, since its relationship with the pKas of PhCs would determine the proportion of neutral and deprotonated molar fractions (Mf). The experimental pKa values of the investigated phenols, together with the Mf of each acid-base species at physiological pH, are presented in Table 1. The most likely deprotonation route for the investigated PhCs that are polyprotic species, with ambiguous mono-anions (Hn-1A−), is shown in Scheme 3. Table 1. Experimental pKa values of the investigated PhCs and the molar fractions (Mf) of the different acid-base species, at physiological pH. M

pKas pKa1 phenol catechol pyrogallol p-hydroxybenzoic acid p-nitrophenol p-aminophenol a Ref. 107, b Ref. 108

pKa2

pKa3

9.997 a 9.45

a

8.96

a

4.58

a

7.15

a

13.3

a

11.0

a

9.46

a

14.0

a

10.30 b

f (pH=7.4)

HnA

Hn-1A−

Hn-2A−2

0.997

0.003

0.991

0.009

~0.0

0.973

0.027

~0.0

0.001

0.990

0.009

0.360

0.640

0.999

0.001

At pH = 7.4 the dominant form of the most of the investigated phenols is the neutral species (HnA). The exceptions are hydroxybenzoic acid and nitrophenol, for which Hn-1A− is the most abundant species at this pH. On the other hand, the fractions of di-anions for catechol, pyrogallol and hydroxybenzoic acid are almost negligible. However, it should be noted that such low proportion does not necessarily means that these species are unimportant for the antioxidant, or pro-oxidant activity of PhCs. Which would really matter would be the product of Mf by the rate corresponding rate constant.109-111



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Scheme 3. Most likely deprotonation routes for polyprotic PhCs, with different possible mono-anionic species.

3.2. In the Absence of Redox Metals 3.2.1. Radical Trapping The free radical scavenging activity of the investigated phenols was evaluated considering their reactions with the hydroperoxy radical (HOO•). This choice was made based on the following facts: -

Peroxy radicals (ROO•) are biologically-relevant species that can be successfully trapped to inhibit OS.112 This is because they have long enough half-lives to allow antioxidants to timely intercept them.113

-

The reactivity of ROO• is low to moderate, which is a convenient feature for establishing trends in radical scavenging activities.114,

115

On the contrary, the

•

reactions of highly-reactive radicals, like OH, usually occur at diffusion-limited rates. Therefore, kinetic data for the reactions between •OH and a wide variety of chemical compounds might lead to miss-conclude that most of them have similar antioxidant effects, when they have not. -

ROO• have been proposed, in the OS context, as the most important reaction partners for phenolic antioxidants.113 Moreover, it was proposed that the most important function of phenolic compounds, as antioxidants, is to scavenge peroxy radicals.116


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-

HOO• is the smallest member of the ROO• family. It plays a crucial role in the toxic side effects inherent to aerobic respiration. In addition, more information is still needed on its reactivity.117

-

HOO• can be used also as a model for the reactions of non-halogenated aliphatic ROO•. Using HOO• as a model for this type of ROO• was recently validated by comparisons with experimental data.118

Some comments on the last point seems worthwhile. The difference of reactivity between HOO• and non-halogenated aliphatic ROO•, for example CH3OO•, is a consequence of the HOO• acidity in aqueous solution (pKa = 4.8).117 Consequently, ignoring the molar fraction of HOO•, at the pH of interest, allows to use HOO• as an adequate model to mimic the reactions of ROO•, under the same conditions.118 Using phenol to illustrate that, the corresponding overall rate constants would be calculated as: •

M

TST , H n A f ( H n A)ktotal +

•

M

ROO f HOO• koverall ( )

ROO koverall =

HOO koverall =

M

TST , H n −1 A f H A− ktotal ( n −1 )

−

•

The Gibbs energies of the reactions between HOO• and the investigated PhCs are shown in Table 2. Formal hydrogen transfer (f-HAT) from the phenolic OH groups in both neutral and anionic catechol and pyrogallol were found to be exergonic, as well as that involving paminophenol. The reactions with neutral phenol and p-hydroxybenzoic acid are almost isoergonic, and all the rest are endergonic. This indicates that OH groups in ortho position and electrodonating groups promote the reactivity of PhCs towards peroxy radicals. In addition, for PhCs with more than one OH groups (catechol and pyrogallol in this work), the thermochemical viability of f-HAT from one OH is increased when the phenolate ion is formed. Regarding the SET mechanism, only the reaction involving the anion of paminophenol is exergonic.



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Table 2. Gibbs energies of the reactions (∆G, kcal/mol) between HOO• and the investigated PhCs, at 298.15K.

phenol

catechol

pyrogallol

p-hydroxybenzoic acid

p-nitrophenol

p-aminophenol

Product label*

Mechanism

Species / Site

∆G

Ph-R1

f-HAT

HnA / OH(1)

0.93

Ph-RC

SET

HnA

35.55

Ph-R1

SET

Hn-1A−

4.59

Cat-R1

f-HAT

HnA / OH(1)

-5.22

CatA-R2

f-HAT

Hn-1A− / OH (2)

-13.33

Cat-RC

SET

HnA

29.63

Cat-R1

SET

Hn-1A−

2.14

Pyr-R1

f-HAT

HnA / OH(1)

-8.51

Pyr-R2

f-HAT

HnA / OH(2)

-3.77

PyrA-R2

f-HAT

Hn-1A− / OH(2)

-14.14

Pyr-RC

SET

HnA

29.27

Pyr-R1

SET

Hn-1A

0.73

pHB-R1

f-HAT

HnA / OH(1)

5.08

pHBA-R1

f-HAT

Hn-1A− / OH(1)

1.23

pHB-RC

SET

HnA

44.07

pHB-R4

SET

Hn-1A−

35.88

pHBA-R1

SET

Hn-2A2−

6.26

pNO2-R1

f-HAT

HnA / OH(1)

8.23

pNO2-RC

SET

HnA

50.33

pNO2-R1

SET

Hn-1A−

20.86

pNH2-R1

f-HAT

HnA / OH(1)

-12.18

pNH2-RC

SET

HnA

10.97

pNH2-R1 *

−

SET

−

Hn-1A

-10.96

R and RC stand for radical and radical cation, respectively.

The reaction pathways found to be endergonic are likely to be significantly reversible, i.e., the amounts of products formed through them are expected to be negligible. In other words, 12 ACS Paragon Plus Environment

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they are assumed to have negligible contributions to the overall peroxy scavenging activity of PhCs. However, they were considered in the kinetic calculations for illustrative purposes, regarding structure-activity relationships. In addition, for the particular case of SET reactions with positive, but small, values of ∆G might be significant. The located transition states (TS) for the f-HAT pathways are shown in Figure 2. Those involving the mono-anionic species of catechol and pyrogallol were found to be barrierless, thus no TS structures were located in these two cases. To further prove this partial optimizations were carried out, with frozen O---H and H---OOH bond distances. This procedure led to structures with only one imaginary frequency. However, when these distances were unfrozen, during a saddle point optimization, H----OH increased while the imaginary frequency and the corresponding gradient decreased, leading to the formation of the reactants. Decreasing the H---OH distance, through a relaxed scan, produced a similar result, i.e., the energy continuously decreased up to the point when the H atom is completely transferred. This demonstrates that the reaction pathway is barrier-less, and would be controlled by diffusion.

Figure 2. Optimized geometries of the located f-HAT transition states. Color code: red = O, gray = C, blue = N and white = H. In general, the located TS become earlier as the number of OH groups in the PhCs increases, and also with the electron-donor ability of the substituents. In addition, the 13 ACS Paragon Plus Environment


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finding that the reactions between anionic catechol and pyrogallol are barrier-less indicates that deprotonation increases the reactivity of these compounds towards free radicals. The reaction barriers and rate constants for all the modeled reaction pathways are provided as Supporting Information, together with the imaginary frequencies of the corresponding TS (Table S1). The reorganization energies, reaction barriers and rate constants for the different SET reaction pathways are also provides as Supporting Information (Table S2). The SET reactions from the anions actually correspond to the SPLET mechanism. The overall rate constants corresponding to the peroxy radical scavenging activity of the investigated PhCs, at pH=7.4, are reported in Table 3. They were calculated as previously explained. In the same table the total rate constants for each acid-base species are also reported. They were calculated, at the same pH, as: TST , H n A TST , H n A TST , H n A ktotal = k HAT + k SET −

−

TST , H n −1 A TST , H n −1 A TST , H n −1 A ktotal = k HAT + k SET

−

Table 3. Overall rate constants corresponding to the HOO• and ROO• scavenging activity HOO • ROO • of the investigated PhCs at pH=7.4 and 298.15 K ( k overall and k overall , pH = 7.4 , pH = 7.4 , respectively), and total rate constants for each acid-base fraction, under the same conditions. All expressed in units of M-1 s-1. Hn A ktotal

−

H n −1 A ktotal

−2

H n −2 A ktotal

•

•

ROO koverall , pH =7.4

HOO koverall , pH =7.4

phenol

6.76E+02

2.05E+05

2.06E+05

5.15E+02

catechol

9.65E+04

7.66E+07

7.66E+07

1.92E+05

pyrogallol

4.18E+05

2.15E+08

2.16E+08

5.40E+05


4.24E-03

3.37E+02

1.54E+05

3.85E+02

p-nitrophenol

3.51E-02

1.74E-07

3.51E-02

8.80E-05

p-aminophenol

7.77E+06

1.90E+07

2.68E+07

6.71E+04

1.53E+05

It was found that all the investigated phenols are capable of efficiently scavenging peroxy radicals, except p-nitrophenol. This exception can be attributed to the strong electron withdrawing effects of the NO2 group. In addition, all the other PhCs are expected to be better scavengers than Trolox, since they react faster with peroxy radicals than this 14 ACS Paragon Plus Environment

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reference antioxidant.119 The branching ratios, measuring the contributions of each acidbase species to the overall scavenging activity of PhCs towards peroxy radical, are reported in Table 4. Table 4. Branching ratios (Γ, in %), considering each acid-base species separately. Γ(HnA)

Γ(Hn-1A−)

phenol

0.33

99.67

catechol

0.13

99.87

pyrogallol

0.19

99.81


0.00

0.22

p-nitrophenol

100.0

0.00

p-aminophenol

28.97

71.03

Γ(Hn-2A−2)

99.78

In general, at physiological pH, the most important species in this context are the phenolate anions, even though they are in relative low proportion at this pH. This supports the previous proposal that the important quantity to identify the species with the highest contributions to the overall free radical scavenging activity of antioxidant in general, and of PhCs in particular, is the product

M

q

f

(H j A ) q

H A k totalj . Being such a product as important as it is to

the antioxidant protection exerted by phenols, there -logically- would be a significant influence of the pH on their protective effects. This is clearly shown in Figures S1 and S2 (Supporting Information). In addition, comparing the data reported in tables S1 and S2 (Supporting Information), it can be concluded that the single electron transfer reaction, from the phenolate anions to the free radicals is faster than the f-HAT processes (for each PhC). Thus, the SPLET mechanism is the dominant in, or at least it significantly contributes to, the peroxy radical scavenging activity of PhCs. Pyrogallol is the only exception to this trend, for which SPLET is more than two orders of magnitude slower than the “sequential proton loss H atom transfer” (SPLHAT). However, the first step in both chemical routes is a deprotonation from the phenolic moieties. In other words, the more reductant is the PhC the



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more effective it would be in deactivating peroxy radicals, which makes phenolate anions the key species in this context. 3.2.2. Regeneration There are other aspects that might influence the balance between antioxidant and prooxidant effects of PhCs. For example, it was previously proposed that this kind of compounds can be repaired by stronger reductants, such as O2•−,120-124 that are already present in biological systems. If that were the case, the involved virtuous circle would promote the ability of PhCs to scavenge multiple free radical equivalents, potentiating their antioxidant behavior. Therefore this aspect was also explored here. The involved process is presented in Scheme 4, using phenol to exemplify the chemical route, and the relevant values estimated for all the investigated PhCs are reported in Table 5. Table 5. Gibbs energies of the reactions (∆G, kcal/mol), reorganization energies (λ, kcal/mol), Gibbs free energies of activation (∆G≠, kcal/mol) and rate constants (k, M-1 s-1), at 298.15 K, for the regeneration reactions. ∆G

λ

∆G≠

k

Ph-R1

-18.16

30.46

1.24

7.92E+09

Cat-R1

-15.71

32.98

2.26

7.56E+09

5.29

34.05

11.36

2.90E+04

-14.31

33.10

2.67

7.17E+09

PyrA-R2

4.62

34.39

11.06

4.81E+04

pHB-R1

-27.91

29.90

0.03

7.99E+09

pHBA-R1

-34.16

45.10

0.66

7.97E+09

pNO2-R1

-34.44

29.98

0.17

7.99E+09

pNH2-R1

-2.60

36.59

7.89

1.01E+07

CatA-R2 Pyr-R1


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Scheme 4. Chemical route of the free radical (•R) trapping, followed by regeneration. It was found that all the investigated compounds can be regenerated by O2•−, via SET, after deactivating the first free radical. Moreover, most of the investigated reactions are predicted to take place at diffusion-controlled rates (rate constants around 109 M-1 s-1). The only two exceptions are CarA-R2 and PyrA-R2. In this case the rate constants for the regeneration process were found to be ~104 M-1 s-1. However, these two species are yielded only by fHAT from the anionic species, while Car-R1 and Pyr-R1 are expected to be most abundant, since they are yielded by f-HAT from the neutral species and by SET from the anions (SPLET). In addition, CarA-R2 and PyrA-R2 are connected to CarA-R1 and PyrA-R1, which at physiological pH would favor the latter. Accordingly, it is expected that in the aqueous phase, under physiological conditions, all the investigated PhCs would be able of trapping several free radical equivalents, two per cycle (one HOO• and one O2•−). This kind of “recycling” is expected to continue until the phenolic species are consumed through reactions with other species. Based on the variety of phenolic compounds investigated here it might be anticipated the same behavior for most PhCs. 3.2.3. Pro-Oxidant Effects of Phenoxy Radicals and Benzoquinones The phenoxy radicals included in this part of the study are those formed to the highest extension (Table 4), and that yielded from phenol, which can be considered isoergonic 17 ACS Paragon Plus Environment


Page 18 of 41

(considering 1 kcal/mol as the chemical accuracy). In addition, the possibility that 1,2benzoquinones (BQs) may be formed from catechol (Cat-BQ) and pyrogallol (Pyr-BQ) was also taken into account, since they might be involved in protein arylation (PAryl). For that purpose all different C-sites in the PhCs were considered, i.e., sites 3 and 4 in Cat-BQ and sites 3, 4 and 5 in Pyr-BQ. According to the Gibbs free energies of reaction (∆G, Table 6), all the investigated phenoxy radicals can cause damage to unsaturated fatty acids. On the contrary, they are unable to oxidize DNA. It seems worthwhile mentioning that the results obtained when using 2dG are very similar to those derived from the three-unit double-stranded oligomer (DNA model). Those obtained using G, on the other hand, are quite different. Thus, it seems that 2dG is good enough to model site reactivity in DNA, while G is not. Table 6. Gibbs free energies of reaction (∆G, kcal/mol, at 298.15 K) for damage to target

molecules induced by radicals and benzoquinones derived from phenolic compounds. Target molecules

Ph-R1 Cat-R1 CatA-R2 Pyr-R1

Pyr-R2 PyrA-R2

LM

-16.02

-9.88

-1.76

-6.59

-11.32

-0.95

-2.91

DNA model

19.88

22.33

43.33

23.73

18.99

42.66

35.44

2dG

24.58

27.04

48.04

28.44

23.70

47.37

40.15

G

6.89

12.59

20.66

13.06

11.56

20.98

19.04

NF-Leu (γ site)

4.35

10.49

18.61

13.78

9.05

19.42

17.46

NF-Cys (SH site)

-4.18

1.96

10.08

5.26

0.52

10.89

8.93

NF-Tyr (OH site)

-0.38

5.76

13.88

9.06

4.32

14.69

12.73

NF-Tyr (SET)

30.00

32.45

53.45

33.85

29.11

52.78

45.56

NF-Trp (SET)

16.98

19.43

40.44

20.83

16.10

39.77

32.54

NF-Met (γ site)

3.78

9.93

18.04

13.22

8.48

18.85

16.89

NF-His (β site)

-0.11

6.03

14.15

9.32

4.59

14.96

13.00

Cat-BQ

Pyr-BQ

NF-Cys (PAryl, site3)

-14.81

-17.48

NF-Cys (PAryl, site 4)

-15.51

-16.31

NF-Cys (PAryl, site 5)

pNH2-R1

-17.05


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For protein residues the viability of the damage mainly depends on the particular PhCs involved, albeit the nature of the affected residue also modulates the corresponding ∆G value. Among the radicals investigated here only that derived from phenol (Ph-R1) leads to exergonic reactions with amino-acid residues. However, not all the residues seems to be equally vulnerable to such damage. Only the reactions with Cys, Tyr and His were found to be exergonic, and only when they take place via f-HAT. Regarding protein arylation, the reactions of both Cat-BQ and Pyr-BQ with Cys residues are significantly exergonic, for any of the investigated reaction site in these BQs. Kinetic data was also calculated for the reactions identified as exergonic (Table 7). The corresponding transition states are showed in Figures 3-5. For the damage induced by the different phenoxy radicals to the modeled unsaturated fatty acids, it was found that most of the reaction (albeit thermochemically viable) are very slow. Thus, these radicals do not represent a substantial risk to the integrity of lipids. The only exception was the reaction of Ph-R1, which has a rate constant of 5.7 × 103 M-1 s-1. Table 7. Gibbs free energies of activation (∆G≠, kcal/mol) and rate constants (k, M-1 s-1) for damage to target molecules, at 298.15 K. ∆G≠

k

Lipid, f-HAT Ph-R1

14.70

5.68E+03

Cat-R1

18.60

2.15E+01

CatA-R2

42.18

2.55E-14

Pyr-R1

21.69

1.58E-01

Pyr-R2

20.78

5.27E-01

PyrA-R2

42.88

9.53E-15

pNH2-R1

25.91

4.51E-03

Protein residues, f-HAT Ph-R1 + NF-Cys (SH site)

12.70

1.57E+04

Ph-R1 + NF-Tyr (OH site)

12.68

3.13E+03

Ph-R1 + NF-His (β site)

19.72

Protein arylation (Cys) Cat-BQ, site 3 + NF-Cys

7.15E-01 *

0.95

9.26E+07 19

ACS Paragon Plus Environment


Page 20 of 41

Cat-BQ, site 4 + NF-Cys

5.89

2.39E+07

Pyr-BQ, site 3 + NF-Cys

2.37

9.22E+07


6.10

1.01E+07


8.26

2.93E+05

Considering pKa(Cys) = 8.64 and the mechanism proposed in ref. 81

*

Figure 3. Transition states, and their imaginary frequencies (iF, cm-1), for the damage to lipids

(LM) by phenoxy radicals. Color code: red = O, gray = C, blue = N and white = H.

Figure 4. Transition states, and their imaginary frequencies (iF, cm-1), for the damage to proteins, via f-HAT, by phenoxy radicals. Color code: red = O, gray = C, blue = N, yellow =S and

white = H. 20 ACS Paragon Plus Environment

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Figure 5. Transition states, and their imaginary frequencies (iF, cm-1), for the damage to proteins, via Cys arylation, by benzoquinones. Color code: red = O, gray = C, blue = N, yellow =S and

white = H. Regarding the damage of phenoxy radicals to amino-acid residues, two of the three pathways, previously identified as exergonic, have rate constants higher than 103 M-1 s-1. They are the f-HAT from Cys (SH site) and Tyr (OH site) to Ph-R1. Therefore, these two pathways may be considered as a risk to proteins integrity. However, these rate constants are lower (by several orders of magnitude) than those corresponding to the regeneration via reaction with O2•−, present in relatively large concentrations in biological media. On the other hand, protein arylation (at Cys residues) seems to be the major hazard posed by PhCs that may be transformed in BQs. All the arylation pathways investigated here are not only significantly exergonic, but also take place very fast. This means that one of the key features to consider regarding the safety of PhCs is their susceptibility to be converted into BQs, under physiological conditions. However, it is important to consider that the conversion of phenoxy radicals to the corresponding BQ is mediated by the oxidation of these radicals. This would be a parallel reaction to regeneration and both may be diffusioncontrolled. Therefore, the balance between arylation and regeneration would depend on the [HOO•]/[O2•−] ratio, which is much lower than one under usual physiological conditions.



Page 22 of 41

In addition, it should be noted that in the calculations of the rate constants for protein arylation (Table 7) the proportion of PhCs transformed into BQs was not included. For other phenolic compounds it has been estimated to be around 5% to 10% of the total phenolic amount.125 Thus the apparent rate constants reported in table 7 can be considered as upper-limits, while the actual values might be about 20 to 10 times lower. 3.3. In the Presence of Redox Metals 3.3.1. Pro-Oxidant Effects by Cu(II) Reduction As previously discussed, the reductant capability of phenolate ions is a crucial feature to their antioxidant protection. However, at the same time it may become an oxidative risk provided that such ions are also capable of reducing redox-active transition metals, such as Cu(II). In such a case they may promote the formation of ROS, via Fenton-like reactions. Therefore, such scenario was also explored in this work, considering both PhCs and their corresponding anions. To put the calculated data into some perspective, the capability of the investigated PhCs to reduce Cu(II) was compared with those of the superoxide radical anion (O2•−) and the ascorbate anion (Asc−). The first one is the reducing agent in metalcatalyzed Haber-Weiss recombination (MC-HWR) processes, and the second one is present in the coper-ascorbate mixture frequently used in experiments to induce oxidative conditions. The calculated data for the reduction of Cu(II), is provided in Table S3 (Supporting Information). The reported rate constants were calculated including the molar fraction of the reductants (PhCs, O2•− and Asc−) at the pH of interest. The reliability of the used approach was tested by comparisons with experimental data. The rate constant calculated here for the reaction between free Cu(II) and O2•− was found to be only 1.9 times lower than the experimentally measured one (8.1×109 M-1s-1)126. Such a good agreement supports the reliability of the kinetic data reported here. At physiological pH (Table S3, Supporting Information) the anionic PhCs are predicted to reduce Cu(II) at significant rates, except that corresponding to p-nitrophenol. The high electron-withdrawing effects of the NO2 group is held responsible for this behavior. For all the investigated compounds only the phenolate anions are strong enough reductants to 22 ACS Paragon Plus Environment

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convert Cu(II) into Cu(I). Thus neutral phenols are not expected to exhibit pro-oxidant effects in this way. The only exception is p-aminophenol, for which the neutral species is also effective for reducing Cu(II). The pyrogallol anion was found to be the strongest reductant, among the investigated ones, with a rate constant for the Cu(II) reduction, at pH=7.4, very similar to that of Asc−. The anions of catechol and p-aminophenol, in that order, are next in this trend. The influence of the pH on the reductant ability of the anionic species of PhCs is shown in Figure 6. At basic pHs (8 to 10) the reductant ability of the investigated species, except for phenol, become intermediate with respect to those of O2•− and Asc−, while at higher pHs some of them are as good reductant as O2•−. This justifies the experimental evidences regarding the pH influence on the pro-oxidant effects of PhCs, and indicates that this environmental factor might rule -to a significant extent- the dual (antioxidant vs. prooxidant) behavior of this kind of compounds.

Figure 6. Influence of the pH on the kinetics of Cu(II) reduction induced by the anionic species of PhCs. The data corresponding to O2•− and Asc− is presented to facilitate comparisons. Trying to get a more complete picture on this chemical dichotomy, a comparison on the peroxy radical scavenging activity of PhCs, and their Cu(II) reduction ability has been performed in terms of kinetics, and considering the pH (Figure S3, Supporting Information). At physiological pH, both processes are similarly faster for catechol, pyrogallol and p-aminophenol, while for phenol, p-nitrophenol and p-hydroxybenzoic acid 23 ACS Paragon Plus Environment


Page 24 of 41

the pro-oxidant effects are predicted to surpass the antioxidant activity. In general, at acid pHs (lower than 4) the investigated compounds are predicted to be better anti-oxidants than pro-oxidants. This is rationalized based on the fact that at such pHs the fractions of the anionic species become very small. 3.3.2. OIL behavior To fully account for the chemical behavior of PhCs, in the presence of redox metal ions, the possibility that PhCs behave as •OH-inactivating ligand (OIL)127,

128

also deserves to be

explored. Such behavior can be exhibited in two different ways:129 - OIL-1: by impeding the reduction of metal ions, - OIL-2: by scavenging •OH yielded through Fenton-like reactions. In both cases OIL molecules should act as metal chelating agents. When they behave as OIL-1, the metal center in the yielded complex is protected from reduction, and the consequent •OH production, via the Fenton reaction, is inhibited. On the other hand, when a molecule behave as OIL-2, the metal in the complex is reduced. Thus, it may partake in the production of •OH, via Fenton reaction. However, being the ligand the molecular frame closer to the site of •OH formation, it would act as a sacrifice target, preventing this radical from reaching molecules that are of vital importance for human beings, such as DNA, proteins and lipids. Since chelation is a necessary step in both cases, this was the first aspect explored here. For the sake of simplicity only those complexes representing ≥ 5% population, according to the Maxwell-Boltzmann distribution, are presented here (Tables 8 and 9); while information on the rest is provided in Tables S4 and S5 (Supporting Information). Because the CDCM mechanism (routes II and IV) comprises two simultaneous processes: deprotonation from the reactive site in the ligand and Cu(II) chelation, the corresponding Gibbs energies and equilibrium constant explicitly depend on the pH. Thus, this effect has been considered here, and the reported data correspond to pH=7.4. More information on the CDCM mechanism, and particular details on the procedure to calculate conditional magnitudes (at specific pHs) can be found elsewhere.118, 130-132


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Table 8. Acronyms and structures of the PhCs-Cu(II) complexes that are in a proportion larger than 5%, according to the of Maxwell-Boltzmann distribution. Acronym

3D View (a)

Structure (H 2O)3Cu

+ O

PhA-C1

Cu(H 2O) 2

O

CatA2-C2

O

Cu(H 2O) 2

O

PyrA2-C2

HO

O

O

pHBA2-C2 O

O Cu (H 2O) 2

(H 2O)3Cu

+ O

pNO2A-C1 O (H 2O)3Cu

N

O +

O

pNH2A-C1 NH 2 (a)

Color code: red = O, gray = C, blue = N, white = H and orange = Cu. 25 ACS Paragon Plus Environment


Page 26 of 41

Table 9. Chelation routes (ChR), chelation sites (ChS) and conditional Gibbs free energies of reaction (∆G’, at pH=7.4, in kcal/mol) for the chelation pathways yielding complexes that that are in a proportion larger than 5%, according to the Maxwell-Boltzmann distribution (%MB). Phenol

ChS

ChR

∆G

PhA-C1

O1

CatA2-C2

%MB

II

-6.84

III

-15.16

O1,O2

IV

-23.24

~100

PyrA2-C2

O1,O2

IV

-22.71

~100

pHBA2-C2

O4,O4

V

-19.83

95.2

pNO2A-C1

O1

II

-10.50

III

-8.86

pNH2A-C1

O1

II

-8.82

III

-19.89

~100

~100 ~100

The values in Table 9 show that Cu(II) chelation, by all the investigated PhCs, involves at least one significantly exergonic pathway. In addition, for all of them only one main product is expected, with contributions larger than 95%. They were the ones used to explore the potential role of the PhCs as OIL-1. The reductants included in this part of the investigation are O2•− and Asc−. The first one is the reductant involved in the metalcatalyzed Haber-Weiss recombination (MC-HWR) and it is a potent reductant. Asc− can be considered as a moderate reductant, although it is strong enough to reduce Cu(II) to Cu(I), promoting the production of •OH, through the Fenton reaction. To identify the most likely reduction site within the chelates, their lowest unoccupied orbital (LUMO) were inspected (Figure S4). In all the cases the LUMO was located around the metal center, which indicates that the reduction should mainly affect Cu(II). This was corroborated by energy calculations (Table S6). It was found that Cu(II) quelation, by any of the investigated PhCs, inhibits the reduction of this metal to some extent, compared to “free” copper (Table 10, Table S7). However, some of these compounds are actually very good as OIL-1, while others are not. The effects of phenol and p-nitrophenol on this context is only minor. On the contrary catechol and pyrogallol are capable of fully inhibiting the Cu(II) reduction by both O2•− and Asc−. Thus, they are expected to be very efficient as OIL26 ACS Paragon Plus Environment

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1 antioxidants. The rest of the investigated PhCs exhibit an intermediate behavior, turning off the reduction by Asc−, and slowing down the reduction by O2•−, but without fully inhibiting it. Table 10. Gibbs free energies of reaction (∆G, kcal/mol), and rate constants (k, M-1 s-1), for the reactions of the PhCs-Cu(II) complexes with the reductants O2•− and Asc−, in aqueous solution at 298.15 K and pH=7.4. O•̶2

Asc-

∆G

k

∆G

k

CuII (H2O)4

-21.86

4.18E+09

-1.53

5.51E+07

PhA-C1

-18.00

5.11E+07

1.34

7.72E+04

CatA2-C2

1.09

3.85E+02

20.42

4.13E-04

PyrA2-C2

2.39

5.22E+02

21.72

1.69E-04

pHBA2-C2

-14.60

3.09E+07

4.73

1.84E+04

pNO2A-C1

-20.76

4.93E+08

-1.43

1.93E+06

pNH2A-C1

-12.60

4.33E+07

6.74

1.33E+04

According to the previously discussed results, PhCs-Cu(II) can participate in the production of ·OH, via MC-HWR, except when PhCs = catechol or pyrogallol. Therefore, the OIL-2 behavior was also analyzed. Catechol and pyrogallol chelates were included in this part of the investigation to test if, even though they do not contribute to the ·OH production, they can scavenge ·OH radicals formed otherwise. The same mechanisms considering for radical trapping (section 3.2.1) were investigated here, i.e., SET, f-HAT and RAF (Scheme 5), for the chelates identified as the most abundant ones.



Page 28 of 41

Scheme 5. Mechanisms explored to analyze the •OH-inactivating ligand type 2 (OIL-type 2) of phenolic complexes.

Table 11. Gibbs free energies of reaction (∆G, kcal/mol), reorganization energies (λ, kcal/mol), Gibbs free energies of activation (∆G≠, kcal/mol) and rate constants (k, M-1 s-1); for the SET reactions between PhCs and •OH. ∆G°

λ

∆G≠

kapp

PhA-C1

-22.04

16.35

0.49

7.43 x 109

CatA2-C2

-38.98

17.58

6.51

1.03 x 108

PyrA2-C2

-38.07

16.39

7.17

3.42 x 107

pHBA2-C2

-27.03

16.36

1.74

7.30 x 109

pNO2A-C1

-8.25

16.26

0.99

7.42 x 109

pNH2A-C1

-35.38

23.38

1.54

7.37 x 109

Because of the high electronegativity of •OH, the only expected chemical route, via SET, would be with the electron being transferred from the complexes to the radical. In this case the location of the “highest occupied molecular orbital” (HOMO) of the PhCs-Cu(II) might be used as an indicator of the site involved in the SET process. It was found that in all cases the ligand is the fragment acting as the electron donor (Figure S5). These SET reactions were all identified as exergonic (Table 11) and predicted to occur rapidly, with rate 28 ACS Paragon Plus Environment

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constants controlled by diffusion. For comparison purposes the equivalent reaction was modeled for “free” copper, i.e., Cu(II)·4H2O. It was found to be highly endergonic (73.85 kcal/mol), contrary to the results for the PhCs-Cu(II) chelates. These results suggest that the presence of PhCs in the coordination sphere of Cu(II) is a key factor in the deactivation of •

OH via SET.

Regarding the f-HAT mechanism there are different sites in the PhCs-Cu(II) chelates that may act as H donors. They are: (i) Phenolic OH groups, if there is still any not involved in the chelation (only for pyrogallol in this work). (ii) A water molecule coordinated to Cu(II) in the coordination site next to the ligand. (iii)

A water molecule in the coordination site opposed to the ligand, for monodentate complexes.

All the modeled f-HAT pathways are highly exergonic, regardless of the phenolic ligand, and of the reaction site (Table 12). In addition they were identified as barrier-less (Figure S6). Thus the f-HAT reactions between PhCs-Cu(II) and •OH are predicted to be diffusioncontrolled. Being that the case for routes (ii) and (iii), which involve water molecules in the coordination sphere of Cu(II), a logical question arises: would it be possible that hydrated Cu(II) act as an •OH scavenger via f-HAT? Thus, such a possibility was also tested. It was found that f-HAT from water ligands in Cu(II)·4H2O is endergonic by 1.6 kcal/mol, i.e., “free” copper cannot not immediately deactivate OH radicals, leaving them available to damage biological targets. In other words, water as a Cu(II) ligand is not capable of acting as an H donor, unless there is another ligand (phenolic in the present study) modifying its chemical environment. Considering these results and those for f-HAT, combined, it can be stated that PhCs are efficient OIL-2 antioxidants. Table 12. Gibbs free energies of reaction (∆G, kcal/mol) and diffusion rate constants (kD, M-1 s-1) for the reactions of PhCs-Cu(II) with •OH, via f-HAT, at 298.15 K.

PhA-C1 CatA2-C2

Site

∆G°

(ii)

-34.48

(iii)

-33.98

(ii)

-48.52

KD 2.26E+09 2.28E+09 29

ACS Paragon Plus Environment


PyrA2-C2 pHBA2-C2 pNO2A-C1 pNH2A-C1

(i)

-51.01

(ii)

-48.64

(ii)

-38.04

(ii)

-21.19

(iii)

-22.85

(ii)

-46.58

(iii)

-45.99

Page 30 of 41

2.26E+09 2.23E+09 2.24E+09 2.21E+09

Fort the RAF mechanism all the possible sites in the phenolic rings were considered. The first assumption here was that the RAF reactions would take place starting at the isolated PhCs and •OH. Within that assumption all the modeled reaction pathways were exergonic (Table S8). In addition, many of these RAF barriers have negative values, which frequently indicates that a more complex mechanism is taking place. After performing the corresponding IRC analyses it became evident that the formation of the adducts would take place subsequently to the f-HAT processes. Figure S7 shows, as an example to illustrate this point, the energy surface for the reaction between •OH and PhA-C1 (site 2 in the phenolic ligand) and the structures of the involved species in the relevant points. Therefore, the Gibbs energies (∆G) for the RAF reactions were recalculated considering the f-HAT products as the RAF reactants. Within this chemical route all the RAF pathways were identified as endergonic and very slow (Table S9). Thus, the RAF mechanism was ruled out as a possible contributor to the OIL-2 behavior of PhCs. 3.4. Some Generalizations Living systems are very complex and comprise a wide variety of chemical species, many of which may participate in OS-related processes. Therefore, chemicals in the biological environment may modulate both antioxidant and pro-oxidant effects of any particular compound. On the other hand, antioxidants are usually good reductants, which in this context might become a double-edged sword. In the particular case of PhCs, their reductant capability is significantly increased upon deprotonation. In the absence of metal ions, PhCs are very efficient radical scavengers, significantly surpassing Trolox in this role except for those having groups with strong electron 30 ACS Paragon Plus Environment

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withdrawing effects. The deprotonated species have a preponderant role in the overall radical trapping activity of PhCs, thus pH is expected to significantly influence such activity. Under physiological conditions, these compounds can be regenerated, which is expected to allow PhCs to trap several free radical equivalents. The products yield during the free radical scavenging activity of phenolic antioxidants can be considered rather innocuous to DNA, lipids and proteins. On the contrary, PhCs might represent a risk to the chemical integrity of proteins, due to arylation of Cys residues, when they are susceptible to be transformed in BQs (metabolically or otherwise). This seems to be a feature that deserves careful attention. Most PhCs are expected to reduce Cu(II), when present as phenolate anions, yielding Cu(I) which in turn may produce •OH, via the Fenton reaction. Therefore, pH might also influence the pro-oxidant behavior of PhCs. At physiological pH, deactivation of peroxy radicals and Cu(II) reduction were found to be similarly faster for catechol, pyrogallol and p-aminophenol, while for phenol, p-nitrophenol and p-hydroxybenzoic acid the pro-oxidant effects are predicted to surpass the antioxidant activity. However, at the same time, through their chelation capability, catechol and pyrogallol are capable of fully inhibiting the Cu(II) reduction by both O2•− and Asc−. Thus, they are expected to be very efficient as OIL-1 antioxidants. The rest of the investigated PhCs exhibit an intermediate behavior, disabling the Cu(II) reduction by Asc−, and slowing down the reduction by O2•−, without fully inhibiting it. In addition, PhCs were found to be very efficient as OIL-2 antioxidants, capable of deactivating •OH, immediately after formation, through SET and f-HAT pathways. In addition, it should be noted that the oxidation of phenolate species by Cu(II), or other species (including peroxy radicals), may start chemical routes leading to the formation of BQs. Therefore, the possible pro-oxidant effects of PhCs (with multiple OH groups), in the presence of metal ions, may involve not only the formation of Cu(I), but also the formation of BQs. Cu(I) is then ready to participate in Fenton-like reactions, while BQs may cause protein arylation at Cys sites.



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4. Conclusions Oxidative stress (OS) is a health-threatening process that is involved, at least partially, in the development of several diseases. Although antioxidants can be used as a chemical defense against OS, they might exhibit a dual behavior, depending on environmental conditions. Anticipating the behavior of PhCs as protectors against OS considering only their freeradical scavenging activity in model systems (theoretical or experimental) might not be enough. Some explorations regarding their pro-oxidant behavior are recommended. Most of the PhCs with known antioxidant effects have multiple phenolic OH groups and are involved in acid-base equilibria. Considering these features, together with the results from this work, at least three factors should be explored to predict the role of PhCs in the OS context: the influence of pH, the presence of redox metals, and the possibility of transformation into benzoquinones (metabolically or otherwise). Since antioxidant vs. pro-oxidant behavior would determine the overall effects of any particular compounds, regarding OS, this duality must be carefully explore to properly design efficient strategies against OS. Such a knowledge would be equally important for appropriately choosing a possible anticancer therapy based on pro-oxidant effects.

Supporting Information Reaction barriers, imaginary frequencies of the transition states and rate constants for the f‐HAT pathways. Reorganization energies, reaction barriers and rate constants for the SET pathways. Thermochemical and kinetic data on the Cu(II) reduction reactions. Information on the complexes with less than 5% population. Thermochemical and kinetic data on the reactions of PhCs‐Cu(II) complexes with reductants and •OH. Influence of the pH on the HOO• and ROO• scavenging activity of the investigated PhCs. Influence of the pH on the dual antioxidant / prooxidant behavior. Lowest unoccupied and highest occupied molecular orbitals for the chelates. Energy scans of the f‐HAT reactions between PhCs‐Cu(II) and • OH. IRC for the RAF reaction between •OH and PhA‐C1.


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Acknowledgements We thank the Laboratorio de Visualización y Cómputo Paralelo at Universidad Autónoma Metropolitana-Iztapalapa and project LANCAD-UNAM-DGTIC-192 of DGTIC at Universidad Nacional Autónoma de México, for access to computing resourses. A. P.-G. acknowledges the support of the Programa de Cátedras – CONACYT (ID-Investigador 435) from CONACyT - UAMI (2015-2025).

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Comprehensive Investigation of the Antioxidant and Pro-oxidant

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