Assessing the Protective Activity of a Recently Discovered Phenolic

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Assessing the Protective Activity of a Recently Discovered Phenolic Compound against Oxidative Stress using Computational Chemistry Yenny Villuendas-Rey, Juan Raul Alvarez-Idaboy, and Annia Galano J. Chem. Inf. Model., Just Accepted Manuscript • DOI: 10.1021/acs.jcim.5b00513 • Publication Date (Web): 01 Dec 2015 Downloaded from http://pubs.acs.org on December 12, 2015

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Journal of Chemical Information and Modeling

Assessing the Protective Activity of a Recently Discovered Phenolic Compound against Oxidative Stress using Computational Chemistry Yenny Villuendas-Rey,1 Juan Raul Alvarez-Idaboy,2 Annia Galano1∗ 1

Departamento de Química. Universidad Autónoma Metropolitana-Iztapalapa. San Rafael Atlixco 186, Col. Vicentina. Iztapalapa. C. P. 09340. México D. F. México. 2

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

Abstract The protection exerted by 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA), a phenolic compound recently isolated from the Pacific oyster, against oxidative stress (OS) is investigated using the density functional theory. Our results indicate that DHMBA is an outstanding peroxyl radical scavenger, being about 15 times and four orders of magnitude better than Trolox for that purpose in lipid and aqueous media, respectively. It was also found to react faster with HOO• than other known antioxidants such as resveratrol and ascorbic acid. DHMBA is also predicted to be able of sequestering Cu(II) ions, consequently inhibiting the OS induced by Cu(II)-ascorbate mixtures, and downgrading the •

OH production via Haber-Weiss reaction. However, it is proposed that DHMBA is more

efficient as a primary antioxidant (free radical scavenger), than as a secondary antioxidant (metal ion chelator). In addition, it was found that DHMBA can be efficiently regenerated in aqueous solution, at physiological pH. Such regeneration is expected to contribute to increase the antioxidant protection exerted by DHMBA. These results suggests that probably synthetic routes for this compound should be pursued, because albeit its abundance in nature is rather low, its antioxidant activity is exceptional. Keywords: antioxidant, free radical scavenger, metal chelation, reaction mechanisms, kinetics

∗

To whom correspondence should be addressed. E-mail: [email protected]; [email protected]

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TOC


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Introduction Phenolic compounds are ubiquitous and versatile substances widely distributed in nature and common in the human diet. They can be found in a diversity of foods and beverages including coffee, tea, chocolate, vegetables, fruits, and wine.1 It has been proposed that polyphenols can play multiple biological roles that make them suitable for different therapeutic purposes. Some of them are the prevention and treatment of different kinds of cancer,2-6 cardiovascular7-9 and neurodegenerative diseases,10-12 as well as the inhibition of skin damage,13,14 cataracts,15,16 inflammation,17-19 diabetes,20-22 atherosclerosis,23,24 and arthritis.25-27 Polyphenols are also known for their excellent antioxidant activity,28-30 which is believed to be responsible, at least in part, for many of their health benefits. It seems interesting to note that, probably because of their relative abundance in nature, polyphenols are better known as antioxidants than other phenolic compounds. However, according to several studies at molecular level, usually one of the phenolic rings is responsible for the overall activity of polyphenols.31-33 Moreover, some phenols that are well known for their excellent antioxidant activity, such as caffeic and gallic acids,34,35 present only one phenolic ring. Recently, 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA, Scheme 1) was isolated from the Pacific oyster (Crassostrea Gigas).36 Despite of being a rather simple phenolic compound, it was proposed as the major non-enzymatic antioxidant in this kind of oysters. It was demonstrated that DHMBA provides two-fold antioxidant activity: by scavenging free radicals and by retarding the copper-mediated oxidation of low-density lipoproteins. In another study,37 it was found that DHMBA is a stronger antioxidant than chlorogenic and L-ascorbic acids, when using diphenyl-1-pyrenylphosphine (DPPP) as a fluorescent probe. Moreover, because of its amphiphilicity, it was suggested that DHMBA may serve as an effective antioxidant in cells, with low cytotoxicity, which is expected to be quickly eliminated from cells. In a following study,38 the antioxidant and hepatoprotective properties of DHMBA were confirmed. It was proposed that this compound is a potent antioxidant, able of effectively protecting cultured hepatocytes from the apoptosis and necrosis caused by oxidative stress.



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Scheme 1. Structure and site numbering of DHMBA. Probably because the antioxidant effects of DHMBA has been discovered only recently, further studies on this particular activity are still needed. For example there is no information on the kinetic data relevant to the chemical reactions involved in the free radical scavenging activity of this compound. There is also a lack of information on the main reaction mechanism (or mechanisms) involved in the DHMBA antioxidant activity and, consequently on the main products yielded during the associated chemical processes. The influence of the environment (polarity of the solvent, pH in aqueous solution, etc.) on its antioxidant protection has not been assessed yet. The possible interactions with metal ions, copper in particular, have not been characterized. The possibility that DMHBA might be able of regeneration, thus scavenging several free radical equivalents, has not been explored either. Accordingly, it is the main goal of the present study to provide quantitative information on those aspects and, hopefully, contributing to gain deeper understanding on the physicochemical processes involved in the protection exerted by DMHBA against oxidative stress. The hydroperoxyl radical (•OOH) has been chosen to explore the free radical scavenging activity of DHMBA. This choice has been made taken into consideration that •OOH is the smallest member of the peroxyl radicals (ROO•). It has been proposed to play an essential role in the toxic side effects associated with aerobic respiration,39 and it has been pointed out that more information is still needed on the reactivity of this particular radical. Peroxyl radicals are relevant within the context of antioxidant activity since they are among those radicals that can be successfully scavenged to retard oxidative stress (OS).40 This is because their half-lives are not too short, thus they can be efficiently intercepted by phenolic compounds.41 Moreover, ROO• have been proposed as the most important reaction partners 4 ACS Paragon Plus Environment

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for phenolic compounds in oxidative stress related chemical processes.41 In fact, it has been suggested that the key antioxidant function of phenols is just to deactivate peroxyl radicals.42,43 In addition, ROO• have low to moderate reactivity, which is considered as a desirable characteristic for studying trends in free radical scavenging activities.44,45 This is because highly reactive radicals are typically involved in diffusion-limited reactions, thus using the kinetic data of these reactions as a comparative criterion might lead to missconclude that all the studied compounds have similar antioxidant capacity. Regarding the interactions with metal ions, they are important since they may contribute to inhibit •OH production. This radical is highly reactive and among the most damaging oxidative species that can be found in biological systems. •OH is so reactive that it is capable of immediately reacting, after formation, with almost any molecule in its vicinity with little selectivity toward the various possible sites of attack. Moreover, it has been estimated that •OH is responsible for 60%-70% of the tissue damage caused by ionizing radiations,46 and for the most important oxidative damage to DNA.47 Therefore inhibiting •

OH formation is expected to be an important way to reduce oxidative stress.

The main intracellular sources of •OH probably are the Fenton reaction and the metal catalyzed Haber-Weiss recombination. The most likely metal ions involved in the later are iron and copper, but it has been reported that under identical experimental conditions the toxicity of Cu(II), in term of oxidative damage, is larger than that of Fe(III).48,49 In addition, Cu-ascorbate mixtures are frequently used for triggering oxidative stress in in vitro investigations. Thus, we have chosen this metal for the present study. In addition, Cu(II) is the most abundant and stable oxidative state of copper, so it is more likely that this ion is involved in a catalyzed Haber-Weiss reaction, than Cu(I) in a direct Fenton reaction. Therefore, chelating agents able of decreasing the viability of the reactions yielding Cu(I) from Cu(II) are expected to be effective for preventing, or inhibiting, the •OH production, and consequently oxidative stress. Accordingly, the potential role of DHMBA as Cu(II) chelator, and the consequent inhibition of •OH production, is studied here in detail.



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Computational Details The Gaussian 09 package of programs50 was used for all the electronic structure simulations. Geometry optimizations and frequency calculations were performed with the 6-311+G(d,p) basis set, i.e. a Pople’s split-valence triple-zeta basis set,51 with polarization52 and diffuse53 functions. The solvation model based on density (SMD)54 was used, in all the cases, in conjunction with the M06-2X and M06 functionals55 for the systems without and with Cu, respectively. All the calculations necessary for modeling the free radical scavenging activity were performed using pentyl ethanoate and water as solvents to mimic lipid and aqueous environments, respectively. On the other hand, the metal chelation study was carried out only in aqueous solution, because this is the phase were ions are expected to be found. The M06-2X functional has been chosen because their developers recommend it for kinetic calculations,55 and also because several independent authors have used it successfully for that purpose.56-64 In addition, it has been identified among the best performing functionals for modeling chemical reactions involving free radicals,65 as well as for kinetic calculations in solution.66 The M06 functional was chosen to study the Cu involving systems because it was parameterized including both transition metals and nonmetals, while M06-2X was parameterized only for nonmetals. Thus, M06 is recommended for organometallic systems.67 SMD has been chosen for mimicking the solvent effects because it can be safely used for estimating solvation free energies for any charged or uncharged solute with relatively low errors.54 The number of imaginary frequencies (0 or 1) was used to identify local minima and transition states, respectively. In addition, intrinsic coordinate calculations (IRC) were carried out to verify that the imaginary frequency of the transition states actually corresponds to the expected motion along the reaction coordinate. Unrestricted calculations were used for open shell systems. Relative energies were calculated including thermodynamic corrections (rigid rotor model / harmonic oscillator approximation / no frequency scaling factor) at 298.15 K. The conventional transition state theory (TST)68-70 and the 1M standard state, were used to calculate the rate constants (k). These computational details are in line with the quantum mechanics based test for overall free radical scavenging activity (QM-ORSA) protocol.71 It was validated by comparisons with


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experimental results, and proven to produce uncertainties no larger than those arising from experiments.71

Results and Discussion Acid-Base equilibria in aqueous solution Acid-base equilibria are crucial for chemical species under physiological conditions, in particular in the aqueous phase. Such equilibria are ruled by the acid dissociation constants of the investigated chemical compound, which are usually expressed as pKa values. However, to our best knowledge there are no previous reports on the pKa of DHMBA. Thus, it has been estimated here for the first time. To that purpose the proton exchange method, also known as the isodesmic method, or the relative method,72 has been used. To that purpose the following reaction scheme is used:

HA + Ref − ↔ A− + HRef where HRef-Ref- is the acid-base pair of a reference compound that should be structurally similar to the system of interest. It represents a proton exchange equilibrium between the acid of interest (DHMBA in this case), and the conjugated base of the reference acid. Within this approach the pKa is calculated as:

pKa ( HA) =

∆Gs + pKa ( HRef ) RT ln (10 )

We have used resorcinol and guaiacol as HRef. The experimentally measured pKa values for these compounds are 9.1573 and 9.93,74 respectively. Using these values, the calculated pKa of DHMBA is 7.24 or 7.59, depending on the used HRef. The average value is then 7.41, which is the proposed pKa for DHMBA, and corresponds to the deprotonation of one of the phenolic OH groups (which are symmetrically equivalent). Using this pKa value the estimated molar fractions of the neutral and mono-anionic species of DHMBA, at pH=7.4, are 0.508 and 0.492, respectively. Therefore, under physiological conditions in aqueous solution both species are present in similar amounts, i.e., both of them may be involved in the antioxidant activity of DHMBA. That is why they are both considered in the present work when investigating reactions in aqueous phase. In addition, the calculated pKa 7 ACS Paragon Plus Environment


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indicates that a significant proportion of DHMBA would correspond to the neutral species (50.8%), at physiological pH, thus it is expected to be able of crossing biological barriers.

Free radical scavenging activity Different reaction mechanisms may be involved in the free radical scavenging activity of chemical compounds. Those included in the present investigation are: single electron transfer (SET), sequential proton loss electron transfer (SPLET), hydrogen transfer (HT), and radical adduct formation (RAF). The Gibbs free energies of reaction (∆G) for all the investigated reaction paths are provided in Table 1. In both tested solvents, and regardless of the acid-base species in aqueous solution, the RAF pathways are all predicted to be endergonic. Therefore, this mechanism has been ruled out as feasible for the peroxyl radical scavenging activity of DHMBA.

Table 1. Gibbs free energies of reaction (∆G, kcal/mol) at 298.15 K.

SET(a) HT site 1a site 2a(b) site 6a(b) site 7 site 7a RAF site 1 site 2(b) site 3 site 4 site 5 site 6(b) (a)

Neutral lipid 78.31

Neutral water 24.41

Anion water 6.33

12.36 -1.38 -1.38 -2.97 16.52

10.45 -1.94 -1.94 -6.25 47.72

10.95 -4.25

7.38 15.61 13.76 17.13 13.41 15.61

7.38 13.31 12.80 14.94 12.41 13.31

3.06 14.01 9.80 16.24 8.05 14.01

-3.11 13.11

SET from the anion corresponds to the second elementary step in the SPLET mechanism. (b) Sites 2 and 6 are symmetrically equivalent in neutral DHMBA. 8 ACS Paragon Plus Environment

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All the reaction pathways involving electron transfers were also found to be endergonic. However, for the SET reaction from the anionic species, in aqueous solution, the endergonicity is rather low. It corresponds to the second elementary step of the SPLET mechanism, and can still be relevant to the antioxidant activity of DHMBA provided that the yielded products are involved in some further fast reactions. On the other hand, the SET processes from neutral DHMBA are not expected to contribute to the peroxyl scavenging activity of this compound, since the corresponding ∆G values are rather high (78.3 and 24.4 kcal/mol in lipid and aqueous solution, respectively). The reaction pathways predicted to be significantly endergonic are not included in the kinetic study because they would be thermodynamically unfeasible. It is expected that the products yielded through such reaction pathways will not be experimentally observed, even if they might take place at a significant rate. On the contrary, moderately endergonic processes might still represent significant pathways, provided that the products evolve into other species through fast enough reactions. Their contributions to the overall reactivity of a particular compound would be particularly relevant when the following reactions are significantly exergonic and with rather low reaction barriers. These features are in line with the SPLET mechanism, because it yields radical anions, which are expected to be highly reactive. Thus, this mechanism was also included in the kinetic analyses, despite of being moderately endergonic (∆G=6.3 kcal/mol). The fully optimized geometries of the transition states (TS) are shown in Figure 1, together with the corresponding imaginary frequencies. The corresponding Cartesian coordinates are provided as Supporting Information. The HT reactions from the phenolic groups in the anionic species, in aqueous solution, are a particular case. Reaction pathway 2a is not available since this phenolic OH is deprotonated. On the other hand, it was not possible to locate the TSs corresponding to the HT pathway from site 6a. Using partial optimizations with frozen O---H and H---OH bond distances, we obtain partially optimized geometries with a single imaginary frequency corresponding to the looked-for transition vector. However, when these two distances are unfrozen, in a saddle point optimization, such calculation invariably led to an increase of the H----OH distance, and to the decrease of the corresponding imaginary frequency and gradient, i.e., it yield the separated reactants. A relaxed scan, computed by decreasing the H---OH distance, produces similar results; i.e., 9 ACS Paragon Plus Environment


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the energy decreases until the H atom is completely transferred. This indicates that this reaction is strictly barrier-less and diffusion-controlled, meaning that every encounter is effective and leads to the conversion of reactants into products.

Figure 1. Optimized geometries of the transition states, and their imaginary frequencies (if, cm-1). Distances are reported in Å. It was found that the TS systematically become more reactant like, i.e., earlier, as the polarity of the solvent increases. In addition, in aqueous solution, the TS corresponding to HT from site 7 is early for the anionic species than for neutral DHMBA. These findings suggest that environmental conditions would influence the peroxyl radical scavenging activity of this phenolic compound. It was also found that TS 2a/6a (as mentioned before they are symmetrically equivalent) for neutral DHMBA present a H bond like interaction involving the H atom in the OOH radical and the O atom in the methoxy group of DHMBA; while the TS corresponding to HT from site 7 does not present this feature. This 10 ACS Paragon Plus Environment

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kind of attractive interaction is expected to lower the energy barriers, thus contributing to increase the reactivity of site 2a/6a when compared with that of site 7. It seems also interesting to note that changing the solvent’s polarity has an effect on the imaginary frequencies (if) of the TS. As the polarity increases so does the if of the TS corresponding to HT from phenolic sites, contrary to what happens with the TS for site 7. Since the if values are inversely proportional to the barrier width, it is expected that as the polarity of the solvent increases the tunneling effects would also increase for TS 2a/6a, while decrease for TS 7. Therefore, the geometrical features of the transition states, considered altogether, indicate that increasing the polarity of the environment should promote the reactivity of DHMBA towards peroxyl radicals. The rate constants for each reaction pathway, as well as the total (ktotal) and overall (koverall) rate coefficients are reported in Table 2, together with the Gibbs free energies of activation (∆G≠). The ktotal values were calculated as the sum of the rate constants of each individual pathway, while koverall in aqueous solution was obtained taking into account the molar fractions of the different acid-base species, at the pH of interest (physiological pH, 7.4): n

ktotPEal = ∑ kineutral DHMBA i =1

n W neutral DHMBA ktotal , neutral = ∑ ki i =1

n

k

W total , anion

= ∑ kianionic DHMBA + k SPLET i =1

n

n

W , pH = 7.4 pH = 7.4 pH = 7.4 koverall = mf ( neutral k neutral , DHMBA + mf ( an k anionic DHMBA , DHMBA) ∑ i ionic DHMBA ) ∑ i i =1

i =1

where PE = pentyl ethanoate (lipid solution), and W = water (aqueous solution). The values of koverall obtained this way are expected to be directly comparable with the experimental data for the reactions of interest. The tunneling corrections used to obtain the rate constant of each individual reaction path are provided as Supporting Information (Table S1). According to the calculated kinetic data, as the polarity of the solvent increases so does the rate at which DHMBA reacts with peroxyl radicals, which is in line with the structural features of the TS, above-described. The rate constant for the reaction between •OOH and 11 ACS Paragon Plus Environment


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neutral DHMBA was found to be 4.1 times faster in aqueous than in lipid solution. In addition, anionic DHMBA reacts about four orders of magnitude faster than the neutral species, in aqueous solution, which strongly suggests that deprotonation significantly promotes the free radical scavenging activity of this compounds. The fastest reaction pathway systematically is the HT from the phenolic sites, regardless of the polarity of the environment and of the acid-base species involved in the scavenging process. This is a rather unusual behavior for phenolic compounds, for which the SPLET mechanism is usually faster than HT. The only other phenolic compound that has been identified so far to present this peculiarity is the propyl gallate.75 It seems important to note that DHMBA and propyl gallate are structurally similar, the only difference between them (regarding the phenolic ring) is that DHMBA presents a methoxy group in site 1a, while propyl gallate has an hydroxy group in this site. Table 2. Gibbs free energies of activation (∆G≠, kcal/mol), rate constants of each individual pathway, total and overall rate coefficients (k, M-1 s-1), at 298.15 K. Neutral (lipid) k ∆G≠ SPLET HT, site 2a(a) HT, site 6a(a) HT, site 7 total Overall (a)

14.75 14.75 17.28

2.58×104 2.58×104 6.97×101 5.18×104

Neutral (water) k ∆G≠ 15.88 15.88 16.25

1.06×105 1.06×105 2.18×102 2.13×105

Anion (water) k ∆G≠ 10.51 1.24×105 0.0 2.73×109 16.88

4.91×101 2.73×109 1.34×109

Sites 2 and 6 are symmetrically equivalent in neutral DHMBA.

The total rate in both the studied solvents are larger than that associated with the HOO• damage to polyunsaturated fatty acids (1.18-3.05 × 103 M-1 s-1).39 Therefore, the kinetic data indicate that DHMBA is efficient for scavenging peroxyl radicals, offering protection against oxidative stress in this manner. Albeit the reference rate constant corresponds to polyunsaturated fatty acids, the reactivity of other biological targets such as proteins and DNA is lower than that of bis-allylic hydrogens in polyunsaturated acids. Accordingly, their rate coefficients when reacting with HOO• are lower than the above-mentioned threshold.76 Thus, DHMBA would also react faster than proteins and DNA with peroxyl 12 ACS Paragon Plus Environment

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radicals, which makes DHMBA suitable for protecting also these molecules against oxidation induced by peroxyl radicals. In order to put the obtained data in perspective, it has been compared with those obtained in a similar way for other antioxidants. It was found that, in non-polar (lipid) media, DHMBA reacts with HOO• about 15 and times faster than Trolox,77 which is commonly used as a reference antioxidant. It was also found to be a better scavenger than other known antioxidants such as caffeic acid,78 propyl gallate,75 resveratrol,32 ascorbic acid,71 and melatonin;79 while its protection is predicted to be slightly lower than those of gallic acid80 and piceatannol.32 On the other hand, in aqueous solution, at physiological pH, DHMBA is predicted to be a more efficient peroxyl scavenger than any of the above mentioned compounds. Moreover, it was found to react about four orders of magnitude faster than Trolox under such conditions. This can be attributed to two characteristics of DHMBA, its relatively low pKa and the high reactivity of its site 2a, via HT, which is diffusion controlled. The excellent activity of DHMBA as free radical scavenger makes this compound an interesting candidate for treating health disorders related to oxidative stress. In particular, it would be very interesting to see further investigations regarding its potential role for prevention, or treatment, of neurodegenerative disorders. It has been proposed that one of the possible routes leading to neurodegeneration is through the free radical induced chemical damage to the species constituting the cellular and mitochondrial membranes.81 This is supported by other studies indicating that dietary components with scavenging activity may influence some of the biochemical events associated with Parkinson’s disease pathology.82 Therefore, considering that DHMBA is among the best free radical scavengers identified so far, it seems to be promising in this context. On the other hand, there are previous studies showing that some phenolic compounds can exhibit increased reactivity toward carcinogens and, at the same time, controlled reactivity towards DNA.83,84 These aspects deserve further investigation for DHMBA. To quantify the contributions of the different mechanisms and reaction pathways to the overall HOO• scavenging activity of DHMBA the corresponding branching ratios (Γ) have been estimated: 13 ACS Paragon Plus Environment


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Γ iPE =

ki k total

ΓWi ,pH =7.4 =

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× 100

(3)

mf i pH =7.4 ki × 100 W ,pH = 7.4 koverall

(4)

where i represents each individual reaction pathway. The values obtained that way are presented in Table 3. Table 3. Branching ratios (%), at 298.15 K. Neutral (lipid) SPLET HT, site 2a HT, site 6a HT, site 7

Neutral (water)

49.9 49.9 0.1

∼0.0 ∼0.0 ∼0.0

Anion (water) ∼0.0 ∼100.0 ∼0.0

It was confirmed that the HT from the phenolic sites is systematically the main reaction channel involved in the peroxyl radical scavenging activity of DHMBA. This is the case in both modeled solvents, and also for both acid-base species in aqueous solution. The contributions of HT from site 7, and SPLET pathways were found to be negligible for the reaction of DHMBA with HOO•. However, it might be possible that for larger free radicals, leading to steric hindrance in the transition states, the relative contributions of the SPLET mechanism become larger. Inhibition of •OH production by copper chelation To achieve such a goal it is essential that chelation yield stable complexes, i.e. the chelation reactions must be exergonic. Thus, the thermochemical viability of the Cu(II) chelation by DHMBA has been first explored. To that purpose both the neutral and anionic forms of this compound have been considered. This part of the investigation has been carried out only for aqueous solution because this is the phase where ionic species are expected to be found. To represent “free” copper ions, they were modeled coordinated to water molecules. This model is more adequate than the naked ions, because they are expected to be hydrated in the aqueous phase, i.e. in biological systems. Four water molecules were chosen, based on 14 ACS Paragon Plus Environment

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previous investigations showing that the most likely configuration of Cu(II) water complexes, in the aqueous phase, corresponds to an almost square-planar four-coordinate geometry.85,86 For consistency purposes, the hydrated Cu(I) ions were also modeled with four water molecules. However, in this case the linear two-coordinate configuration is preferred, i.e., Cu(I) is coordinated only to two water molecules, and the other two are solvating the system. This linear, two-coordinate, structure is consistent with previous experimental evidences.87-89 All the O atoms in DHMBA (Scheme 1) have been considered as potential chelation sites. In addition, two different chelation routes have been investigated, the direct chelation (DCM) and the coupled-deprotonation-chelation (CDCM) mechanisms. However, the later is only possible when the chelation site is acidic, thus it can deprotonate at the same time that the chelation process occurs. The optimized geometries of the chelates are provided in Figure 2, while their corresponding cartesian coordinates are provided as Supporting Information. The Gibbs free energies (∆G) of reaction for all the studied chelation pathways are reported in Table 4. It seems worthwhile to call attention to the fact that CDCM routes are affected by the pH, while DCM routes are not. Details on these mechanisms, and on the calculations of the associated ∆G values can be found elsewhere.90,91



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Figure 2. Fully optimized geometries of the DHMBA-Cu(II) complexes. Distances are reported in Å.

Table 4. Gibbs free energies of reaction (kcal/mol), of the Cu(II) chelation by DHMBA, at 298.15 K. Site

DCM

O1a O2a (a) O7a O1a,O2a (a)

-0.03 4.33 0.51 5.54

(a)

CDCM (pH=7.4) -6.31 0.35 -8.55

Sites 2 and 6 are symmetrically equivalent


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It was found that via DCM only two pathways (O1a and O7a) are predicted to be feasible, being practically isoergonic, while the rest are endergonic. On the other hand, via CDCM pathways O2a and O1aO2a are significantly exergonic, while path O7a remains isoergonic, at physiological pH (pH=7.4). Accordingly, it is predicted that the thermochemical viability of the Cu(II) chelation by DHMBA is increased by the deprotonation of the chelating site. Therefore, increasing the pH would favor Cu(II) to be sequestered by this compound. Moreover, under physiological conditions CDCM is expected to be the main Cu(II) chelation mechanism for DHMBA. Regarding the Cu(II) reduction, it was found (Table 5) that it is fully turned off by the DHMBA chelation process when the reductant agent is the ascorbate ion (Asc−). Thus, this would be the expected outcome for experiments using Cu-ascorbate mixture as oxidant. On the other hand, the Cu(II) reduction by the superoxide radical anion (O2•-) is predicted to be inhibited but only partially. Accordingly, it is expected that DHMBA may downgraded the production of •OH in biological systems, but not completely inhibit it.

Table 5. Gibbs free energies of reaction (kcal/mol, at 298.15K) for the reduction of DHMBA - Cu(II) complexes, by O2•- and Asc-. Site Cu(II) DCM O1a O7a CDCM O2a (a) O7a O1a,O2a (a) (a)

O2•− -26.19

Asc− -2.24

-19.94 -24.05

4.01 -0.11

-18.11 -15.83 -17.73

5.84 8.11 6.22

Sites 2 and 6 are symmetrically equivalent



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Analyzing these results together with those corresponding to the peroxyl radical scavenging activity of DHMBA, above-discussed, it is proposed that DHMBA is more efficient as a primary antioxidant (free radical scavenger), than as a secondary antioxidant (metal ion chelator). In other words, the free radical scavenging activity of DHMBA contributes to a larger extent to the protection against oxidative stress than metal chelation in biological systems, albeit the contributions of the later are still expected to be significant. This statement is based on the assumption that in biological systems the most important reductant partner for Cu(II) would be O2•-.

Regeneration Taking into account that the main antioxidant action of DHMBA is predicted to take place by scavenging free radicals, and that it has been demonstrated that other phenolic compounds75,92-96 are able of regeneration (in aqueous solution, at physiological pH), thus been able of scavenging more than one radical equivalent, such a possibility has been considered (Scheme 2). This regeneration route involves two steps after the scavenging of the first radical took place. They are: (i) an electron transfer from O2•- to the DHMBA radical product − yielding the corresponding anion −, and (ii) the protonation of this anion from the environment. Albeit the regeneration route is shown in Scheme for neutral DHMBA as initial reactant, a similar route can be established also for its anion.

Scheme 2. Mechanism of the free radical scavenging activity and regeneration of DHMBA.

Step (i) is predicted to easily take place under physiological conditions for both anionic and neutral DHMBA. It was found to be exergonic in both cases (Table 6), and very fast with 18 ACS Paragon Plus Environment

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rate constants within the diffusion limit regime (∼ 8.0×109 M-1s-1). Step (ii) is pH dependent, so the corresponding Gibbs energy of reaction has been calculated including this fact. Further details on such calculations can be found elsewhere.92 This step was also found to be exergonic at the pH of interest (pH=7.4) for both species, neutral and anionic. These results supports the feasibility of the regeneration process, which is expected to take place very fast, in a cascade way, under physiological conditions.

Table 6. Gibbs energies of reaction (kcal/mol), at 298.15 K, for steps (i) and (ii) in the regeneration route of DHMBA, in aqueous solution at pH=7.4.

neutral DHMBA anionic DHMBA

Step (i) -20.43 -16.62

Step (ii) -9.81 -24.94

According to the proposed regeneration route, it is predicted that DHMBA can be efficiently regenerated at the same time that it scavenges two free radicals (one HOO• and one O2•-). This virtuous cycle may continue unless some of the intermediates are consumed through reactions with other species in the environment. Thus, the regeneration process may contribute to increase the antioxidant protection of DHMBA. This is an advantageous ability because in the regions where free radical are generated, within biological systems, the concentration of this −or any other− antioxidant is expected to be relatively low.

Conclusions The protection exerted by DHMBA, phenolic compound recently isolated from the Pacific oyster, against oxidative stress has been investigated using the density functional theory. Its potential capability as primary (radical scavenger) and secondary (metal chelator) antioxidant have been considered. It was found that DHMBA is an exceptional peroxyl radical scavenger, being about 15 and four orders of magnitude better than Trolox for that purpose in lipid and aqueous media, respectively. It was also found to react faster with HOO• than other known antioxidants such as resveratrol and ascorbic acid. In fact, in aqueous solution it is proposed as the most 19 ACS Paragon Plus Environment


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efficient peroxyl radical scavenger identified so far. Such protection is predicted to be increased by the polarity of the solvent, and by deprotonation in aqueous solution. Albeit, hydrogen transfers from the phenolic sites in DHMBA are proposed as the main reaction pathway when reacting with HOO•, it might be possible that the relative contributions of the SPLET mechanism increase for larger free radicals, leading to steric hindrance in the transition states. On the other hand, DHMBA is predicted to be able of sequestering Cu(II) ions mainly by the CDCM chelation route. This ability would lead to fully inhibit the oxidative stress induced by Cu(II)-ascorbate mixtures, and to downgrade the •OH production via HaberWeiss reaction. However, it is proposed that DHMBA is more efficient as a primary antioxidant (free radical scavenger), than as a secondary antioxidant (metal ion chelator). In addition, it is proposed that DHMBA can be efficiently regenerated after scavenging the first free radical in such a way that it would deactivate s two free radicals (one HOO• and one O2•-) per cycle. Thus, the regeneration process may contribute to increase the antioxidant protection of DHMBA. DHMBA is a natural product, which so far has only been identified in the Pacific oysters, thus it is not highly abundant in nature. On the contrary it has an exceptional antioxidant activity, when acting as a free radical scavenger, especially in aqueous solution. Therefore it is likely that synthetic routes for DHMBA should be pursued, so it can be used as an additive in foods.

Acknowledgements We gratefully acknowledge the Laboratorio de Visualización y Cómputo Paralelo at Universidad Autónoma Metropolitana-Iztapalapa and DGTIC, UNAM for computing time. This work was partially supported by projects SEP-CONACyT 167491 and 167430, and DGAPA PAPIIT- IN220215. Y. V.-R. acknowledges the economic support provided by project SEP-CONACyT 167491 during her postdoctoral scholarship.


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Supporting Information: Tunneling corrections. Cartesian coordinates of the optimized geometries of the transition states and of the DHMBA-Cu(II) complexes. This material is available free of charge via the Internet at http://pubs.acs.org.

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Perez-Gonzalez, A.; Muñoz-Rugeles, L.; Alvarez-Idaboy, J. R., Tryptophan: Antioxidant or Target of Oxidative Stress? A Quantum Chemistry Elucidation. RSC Adv. 2014, 4, 56128-56131. 77

Alberto, M. E.; Russo, N.; Grand, A.; Galano, A., A Physicochemical Examination of the Free Radical Scavenging Activity of Trolox: Mechanism, Kinetics and Influence of the Environment. Phys. Chem. Chem. Phys. 2013, 15, 4642-4650. 78

León-Carmona, J. R.; Alvarez-Idaboy, J. R.; Galano, A., On the Peroxyl Scavenging Activity of Hydroxycinnamic Acid Derivatives: Mechanisms, Kinetics, and Importance of the Acid-Base Equilibrium. Phys. Chem. Chem. Phys. 2012, 14, 12534-12543.

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Galano, A., On the Direct Scavenging Activity of Melatonin Towards Hydroxyl and a Series of Peroxyl Radicals. Phys. Chem. Chem. Phys. 2011, 13, 7178-7188. 80

Marino, T.; Galano, A.; Russo, N., Radical Scavenging Ability of Gallic Acid toward OH and OOH Radicals. Reaction Mechanism and Rate Constants from the Density Functional Theory. J. Phys. Chem. B 2014, 118, 10380-10389. 81

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Galano, A.; Medina, M. E.; Tan, D. X.; Reiter, R. J., Melatonin and its Metabolites as Copper Chelating Agents and their Role in Inhibiting Oxidative Stress: A Physicochemical Analysis. J. Pineal Res. 2015, 58, 107-116. 91

Álvarez-Diduk, R.; Galano, A., Adrenaline and Noradrenaline: Protectors against Oxidative Stress or Molecular Targets? J. Phys. Chem. B 2015, 119, 3479-3491. 92

Galano, A.; Pérez-González, A., On the Free Radical Scavenging Mechanism of Protocatechuic Acid, Regeneration of the Catechol Group in Aqueous Solution. Theor. Chem. Acc. 2012, 131, 113. 93

Galano, A.; Francisco Marquez, M.; Pérez-González, A., Ellagic acid: An Unusually Versatile Protector against Oxidative Stress. Chem. Res. Toxicol. 2014, 27, 904-918.

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Caicedo, C.; Iuga, C.; Castañeda-Arriaga, R.; Alvarez-Idaboy, J. R., Antioxidant Activity of Selected Natural Polyphenolic Compounds from Soybean via Peroxyl Radical Scavenging. RSC Adv. 2014, 4, 38918-38930.



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Assessing the Protective Activity of a Recently Discovered Phenolic

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