On the Scavenging Ability of Homogentisic Acid and Ergosterol

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B: Biophysics; Physical Chemistry of Biological Systems and Biomolecules

On the Scavenging Ability of Homogentisic Acid and Ergosterol towards Free Radicals Derived from Ethanol Consumption Manuel Eusebio Medina, Annia Galano, and Angel Trigos J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b04619 • Publication Date (Web): 09 Jul 2018 Downloaded from http://pubs.acs.org on July 10, 2018

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The Journal of Physical Chemistry

On the Scavenging Ability of Homogentisic Acid and Ergosterol towards Free Radicals Derived from Ethanol Consumption Manuel E. Medina,*,† Annia Galano‡ and Ángel Trigos§ †

Centro de Investigaciones Biomédicas, Universidad Veracruzana, Av. Luis Castelazo s/n, Col.

Industrial Animas, Xalapa Veracruz 91190, México. ‡

Departamento de Química, División de Ciencias Básica e Ingeniería, Universidad Autónoma

Metropolitana-Iztapalapa, Av. San Rafael Atlixco No. 186, Col. Vicentina, México D. F. 09340, México. §

Laboratorio de Alta Tecnología de Xalapa, Universidad Veracruzana, Calle Médicos 5, Col.

Unidad del Bosque, Xalapa Veracruz 91010, México.

ABSTRACT.

*

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

ACS Paragon Plus Environment

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Acute, or chronic, ethanol consumption leads to the formation of free radicals in the liver, which is related to hepatic damage. Among these radicals 1-hydroxyethyl, •CH(OH)CH3, is the most abundant one. Thus, efficient •CH(OH)CH3 scavengers are likely candidates to offer liver protection after ethanol consumption. In the present work ergosterol and homogentisic acid (HGA), which are found in edible mushrooms, were investigated as potential candidates to that purpose. The investigation was carried out following the QM-ORSA protocol, and using the density functional theory (DFT). The overall rate constants calculated for the •CH(OH)CH3 radical scavenging activity of ergosterol in lipid and ethanol media are 1.34 x 107 and 1.86 x 107 M-1 s-1, respectively. For homogentisic acid the overall rate constant in lipid, ethanol and aqueous media are 4.33 x 108, 2.74 x 106 and 3.62 x 107 M-1 s-1, respectively. Accordingly, both compounds are predicted to efficiently scavenge the •CH(OH)CH3 radical. Thus, the results from this investigation supports the antioxidant capability of edible mushrooms, their potential beneficial effects against ethanol hepatotoxicity, and the nutraceuticals properties of ergosterol and homogentisic acid.

INTRODUCTION The increase in free radical amounts, within living organisms, arises as a consequence of the imbalance between their generation and consumption. This causes an oxidative stress condition that has been related to several pathological states, widely distributed amongst human population.1,2 The largest damage, at molecular level, due to the oxidation of cellular components by free radicals is lipid peroxidation, where the lipid part of cell membranes is oxidized, inducing deformation, function loss and finally the death of the cell.3


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Probably the most known free radicals are those considered as reactive oxygen species (ROS) and reactive nitrogen species (RNS), which are O-centered and N-centered radicals, respectively.4 However, there are also other kind of radicals, such as the S-centered (RSS) and the C-centered (RCS) that also represent a threat to the chemical integrity of biological molecules. The 1-hydroxyethyl radical, •CH(OH)CH3, which is an ethanol derivative yielded by the liver metabolism, belongs to the RCS family.5 The role of free radicals in ethanol toxic effects has been well documented. Acute, as well as chronic, ethanol consumption leads to the formation of free radicals in the liver, which are related to hepatic damage through lipid peroxidation. In addition, it has been reported that the overall radical production in chronic alcohol administration is enhanced by high levels of dietary fat5 or iron overload.6 The data gathered so far suggest that acute and chronic ethanol ingestion induces a decrease in the hepatic GSH/GSSG (reduced and oxidized forms of glutathione) ratio and increases the lipid peroxidation in the liver of animals and humans.7 In addition, it has been proposed that enhanced generation of reactive oxygen intermediates by microsomes may contribute to the hepatotoxic effects of ethanol.8 It has been reported that 25 to 50% of the acetaldehyde formed during microsomal ethanol oxidation, under different experimental conditions, could arise via the

•

CH(OH)CH3

intermediate.9 It has also been suggested that dietary and/or pharmacological agents (such as GSH, ascorbate and α-tocoferol) that are capable to prevent the ethanol-induced oxidative stress, may reduce the incidence of ethanol toxicity in humans.10,11 Therefore, chemical species that may act as efficient •CH(OH)CH3 scavengers are promising candidates to counteract such toxicity.


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The present study is focused on two compounds present in edible mushrooms: ergosterol and homogentisic acid (HGA). Ergosterol is an essential component of cell membranes, specific to fungi. It has similar functions as cholesterol in vertebrates and phytosterols in plants, including permeability, fluidity, microdomain formation, protein functionality and membrane activities.1217

. It has also been proposed that ergosterol exhibits a protective role against mechanical and

oxidative stress,18 and is a precursor of vitamin D2. Since sterols are the most abundant components of cell membranes,18,19,20 their susceptibility to oxidative damage has been the focus of several investigations. Based on bond dissociation energies (BDE) calculations, it has been proposed that oxidation is more likely to occur in the sterol nuclei than in the side chain, in particular at site 14 (Scheme 1).21 The rate constant of the reaction between ergosterol and •CH(OH)CH3 was experimentally measured (5.2 ± 0.1 x 107 M-1 s-1), and characterized as a second-order rate constant.22 In addition, site 14 in ergosterol was confirmed as the most susceptible to the attack of this free radical. On the other hand, in a recent study concerning the oxidative damage to ergosterol by peroxyl radicals, it was concluded that hydrogen transfer from site 9 to •OOH is faster than the equivalent reaction at site 14. In that report, the kinetic selectivity was attributed to an attractive interaction, in the transition state, between the two double bonds in ergosterol and the hydrogen atom in the •OOH radical.23 HGA is another interesting molecule in the oxidative stress context which has been identified as the main antioxidant in some edible mushrooms such as Agaricus bisporus, Boletus edulis, Calocybe gambosa, Cantharellus cibarius, Craterellus cornucopioides, Hygrophorus marzuolus, Lactarius deliciosus and Pleurotus ostreatus.24,25 HGA can scavenge intracellular ROS, and 1,1diphenyl-2-picrylhydrazyl (DPPH) radicals, preventing lipid peroxidation in human fibroblast


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(WI 38) cells.26 Its antioxidant activity has been proposed to surpass that of the reference antioxidant α-tocopherol.27 The antioxidant activity of HGA has also been predicted based on BDE calculations, and attributed to the presence of the semiquinone structure.28 It has also been reported that HGA exhibits protective effect against thermal-cholesterol degradation. In addition, pre-treatment with HGA was found to preserve liposomes and low density lipoproteins from Cu(II)-induced oxidative damage.29 Based on the data gathered so far, it seems that both ergosterol and HGA are relevant molecules within the context of oxidative stress. Moreover, they might play significant roles on the protection exerted by edible mushrooms against this dangerous chemical stress. In particular, these compounds may help preventing ethanol-induced damage, and decreasing the incidence of ethanol toxicity in humans. To gain further knowledge on such possibilities, the •CH(OH)CH3 radical scavenging activity of ergosterol and homogentisic acid, in ethanol, aqueous solution and lipid media, was investigated in this work using the density functional theory (DFT). Three different reactions mechanisms were considered: single electron transfer (SET), hydrogen transfer (HT) and radical adduct formation (RAF). All possible sites of reaction were taken into account. The apparent reaction rate constants were calculated, as well as the branching ratios.

COMPUTATIONAL DETAILS Geometry optimization and frequency calculations were carried out by using the Gaussian 09 package of programs,30 employing the M06-2X31 functional with the 6-31+G(d,p) basis-set. The reaction was modeled in ethanol (ε=24.85), water (ε=78.35) and pentylethanoate (ε=4.73) media using the continuum solvation model density (SMD). Ethanol was chosen to facilitate


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comparisons with the available experimental data, while pentylethanoate and water were used to mimic the physiological environment of the cell membrane. SMD is a solvation model that can be applied to any charged or uncharged solute in a liquid medium for which few key descriptors are known.32 The M06-2X was recommended by its developers31 as one of the best performing functionals for calculating reaction energies involving free radicals and for kinetic calculations in solutions.33,34 It has also been successfully used by independent authors for this purpose.35,36 Local minimal and transition states were identified by the number of imaginary frequencies (0 and 1, respectively); intrinsic reaction coordinate calculations were performed to confirm that the transition states properly connect with the reactants and products. For the transition states, it was confirmed that the imaginary frequency corresponds to the expected motion along the reaction coordinate. The energy values were improved by single point calculation at M06-2X/cc-pVDZ level. The relative energies including thermodynamic corrections at 298.15 K were calculated with respect to the sum of the isolated reactants. They all correspond to the 1M standard state. In addition, the solvent cage effects have been included according to the correction proposed by Okuno,37 taking into account the free volume theory.38 The rate constants (k) were calculated using the conventional transition state theory (TST):39,40,41 =

/

(1)

Where the σ represents the reaction path degeneracy, accounting for the number of equivalents path; κ accounts for tunneling corrections; kB and h are the Boltzmann and Plank constant; ∆G≠ is the Gibbs free energy of activation; R and T are the gas constant and temperature. The tunneling corrections are defined as the Boltzmann average of the ratio of the quantum and classical probabilities and was calculated using the zero-curvature tunneling correction.42


6

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Even though the endergonic reaction path might take place at a significant reaction rate, in the kinetic study they were not included because such reactions would be reversible to such an extent that the corresponding products would not be observed. In the present investigation, a methodology was employed that is in line with the quantum mechanics-based test for overall free radical scavenging activity (QM-ORSA), which has been validated by comparison with experimental results and its uncertainties have been proven to be no larger than those arising from experiments.43

RESULTS AND DISCUSSION •

CH(OH)CH3 radical scavenging activity of ergosterol in ethanol and lipid media

Ethanol media. In the study of •CH(OH)CH3 radical scavenging activity of ergosterol in ethanol media was carried out considering the following reaction mechanisms: •

Hydrogen transfer (HT) Ergosterol + •CH(OH)CH3 → [Ergosterol(-H)] • + CH3CH2OH

•

Radical adduct formation (RAF) Ergosterol + •CH(OH)CH3 → [Ergosterol-CH(OH)CH3] •

All possible reaction sites in ergosterol, susceptible to be involved in HT processes, where taken into account. They are 1-4, 9, 11, 12, 14-21, 24-28 and OH sites of reactions; while for the RAF mechanism all of the sp2 carbon atoms in ergosterol were considered (Scheme 1). The results of the thermodynamic study on the •CH(OH)CH3 radical scavenging capacity of ergosterol are shown in Table 1. According to the thermodynamic results, reaction at sites 4, 9,


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14, 20 and 24 are exergonic. These results are in line with the relative large stability of radical centers at allylic position, which can be explained based on the possibility of carrying out the conjugation of the two double bonds with the radical formed. All these reaction sites may be important in the oxidative damage of ergosterol caused by •CH(OH)CH3 in ethanol. All other modeled HT reaction pathway were found to be endergonic and therefore, the possibility of HT reaction involving these positions, was ruled out. In other words, they are not expected to contribute to the •CH(OH)CH3 radical scavenging capacity of ergosterol in ethanol, to a significant extent. For the RAF mechanism, all reaction positions were considered where the carbon atom has the sp2 hybridization and in this case, all the reaction pathway were exergonic to yield the radical adduct. Therefore, they may be relevant to the •CH(OH)CH3 radical scavenging activity of ergosterol in ethanol.


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Table 1. M06-2X/cc-pVDZ//M06-2X/6-31+G(d,p) Gibbs free energy of reaction (∆G, kcal/mol) for the pathway involved in the •CH(OH)CH3 radical scavenging capacity of ergosterol in ethanol media, at 298 K. RS*

∆G HT

RS*

∆G

20

-12.06

1

3.06

21

5.08

2

4.47

24

-10.20

3

0.11

25

1.56

OH

11.53

26

7.07

4

-16.07

27

6.23

9

-19.23

28

5.86

11

0.01

12

3.86

5

-13.47

14

-22.30

6

-7.64

15

3.59

7

-6.77

16

1.77

8

-5.00

17

1.60

22

-5.12

18

4.66

23

-1.61

19

4.19

RAF

* RS = reaction site The above-discussed results, were used to assess the potential viability of the different reaction pathway. Since the endergonic ones are reversible, favoring the reactants population, the corresponding products are not expected to be observed, even if they take place at a significant rate. For that reason, they have been ruled out as viable and, consequently are not included in the kinetic study. The results of the kinetic study concerning to •CH(OH)CH3 radical scavenging activity of ergosterol, in ethanol media, are shown in Table 2. It was found that the HT mechanism in the


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allylic positions of ergosterol has the lowest Gibbs free energies of activation. Thus, these reactions pathways have the highest reaction rate constants. The two reaction sites identified as the most reactive are the 9 and 14 sites, based on the finding that they have the highest reaction rate constants. These results obey the fact that both tertiaries carbons can be conjugated with the double bond at positions 5 and 7 of ergosterol. However, the reaction at site 9 is predicted to be about twice as fast as the reaction at site 14, indicating that the contribution of HT from site 9 to the overall reactivity of ergosterol towards •CH(OH)CH3 are larger than those of site 14. The reaction sites that do not correspond to allylic position have the highest Gibbs free energy of activation and, hence, their reaction rate constant are significantly lower. The results of the •CH(OH)CH3 radical scavenging capacity of ergosterol in ethanol media through the RAF mechanism, show that the reaction sites 5, 6, and 7 have the lowest Gibbs free energies of activation. Therefore, the corresponding reaction rate constants were the highest, with values of 7.24 x 104, 4.66 x 103 and 6.05 x 104 M-1 s-1, respectively. On the contrary, the 8, 22 and 23 positions have the highest Gibbs free energies of activation and hence, the lowest reaction rates constant. To assess the relative importance of the HT and RAF mechanism in the •CH(OH)CH3 scavenging activity of ergosterol, the rate constants for the total HT (() ) and RAF (() ) processes in ethanol media were calculated from the reaction rate constants

obtained for each reaction pathway: () = ∑ [()]

(2)

() = ∑ [()]

(3)


10

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Table 2. M06-2X/cc-pVDZ//M06-2X/6-31+G(d,p) Gibbs free energy of activation (∆G≠, kcal/mol), tunnel correction (κ), reaction rate constant (k, M-1 s-1) and branching ratios (Γ, %) of •

CH(OH)CH3 radical scavenging capacity of ergosterol in ethanol media, at 298 K. ∆G≠

RS*

k

κ

Γ

HT 4α

15.52

4β

10.83

9

9.53

14

10.46

69.00

1.81 x 103

32.64

6

12.6

7

70.8

6

15.9

-1

0.0

20.39 22.33

2.33 x 10 1.31 x 10

2.96 x 10

0.0

20

19.93

62.82

9.56 x 10

24

17.81

31.49

1.74 x 101

0.0

1.84 x 107

99.3

#$ (

!")

RAF 5

10.98

1.30

7.24 x 104

0.4

6

12.62

1.33

4.66 x 103

0.0

7

11.10

1.34

6.05 x 104

0.3

8

21.33

1.3

1.85 x 10-3

0.0

22

20.06

1.33

1.63 x 10-2

0.0

23

20.88

1.36

4.21 x 10-3

0.0

1.37 x 105

0.7

#$ #%&(

!")

#$ "'()*++(

!")

1.86 x 107

* RS = reaction site

The overall reaction rate constant (,-./00() ) for •CH(OH)CH3 radical scavenging activity

of ergosterol was calculated as:


11


= () + () ,-./00()

Page 12 of 29

(4)

The () and () values are 1.84 x 107 and 1.37 x 105 M-1 s-1 (Table 2),

respectively, which correspond to HT and RAF branching ratios of 99.3 and 0.7 %. Accordingly, the HT mechanism is predicted to be the key chemical route for the •CH(OH)CH3 radical scavenging capacity of ergosterol, in ethanol media, with the contribution of site 9 and 14 being 70.8 and 15.9, respectively. Therefore, both reactions sites are important in this context, albeit the relative importance of site 9 is higher. The overall reaction rate constant for the scavenging activity of ergosterol toward CH(OH)CH3 was of 1.86 x 107 M-1 s-1, in ethanol media and this result is in agreement with the experiment.22 At this point it seems worthwhile to comment on the fact that the most exergonic pathway is that involving site 14, which is in agreement with a previous estimation.22 However, the most exergonic channel is not necessarily the most important one. Thus, to properly quantified site selectivity, the kinetics must be taken into account. The optimized geometries of the transition states (TSs) corresponding to HT and RAF mechanisms, are shown in Figures S1 and S2 (supporting information). They show that the earliest HT transition state is that corresponding to position 9, which is the reaction site involved in the pathway with the highest reaction rate constant. Thus, these results are in line with the Hammond postulate. On the contrary, for the RAF reaction pathways this is not the case. The TS corresponding to the reaction at site 8 was found to be the earliest while this pathway is not the fastest one.


12

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Lipid media (pentylethanoate media). Based on the results already discussed for ethanol media, for the reaction between •CH(OH)CH3 and ergosterol, in lipid media, only the most reactive sites (9 and 14) were considered. The obtained results are shown in Table 3 and Figure S3 (supporting information). It was found that, in this case, site 14 is more reactive toward the target radical than site 9, being their contributions to the overall reactivity of ergosterol equal to 75.3 and 24.7%, respectively. Therefore, it seems that the polarity of the environment influences the site reactivity of ergosterol.

Table 3. M06-2X/cc-pVDZ//M06-2X/6-31+G(d,p) Gibbs free energy of reaction and activation (∆G , ∆G≠, kcal/mol), tunnel correction (κ), reaction rate constant (k, M-1 s-1) and branching ratios (Γ, %) of •CH(OH)CH3 radical scavenging capacity of ergosterol in lipid media, at 298 K. ∆G

∆G≠

κ

k

Γ

9

-18.50

9.62

6.05

3.31 x 106

24.7

14

-22.20

9.14

8.19

1.01 x 107

75.3

RS*

1.34 x 107

#$ 2!*+(345)


The overall rate constant in lipid media (,-./00(67) ) was calculated as:

,-./00(67) = 8(67) + 9:(67)

(5)


13


Page 14 of 29

It was found to be 1.34 x 107 M-1 s-1, i.e., about 1.4 times lower than in ethanol media. Thus the polarity of the solvent also seems to influence the overall scavenging activity of ergosterol towards the •CH(OH)CH3 radical.

•

CH(OH)CH3 radical scavenging activity of homogentisic acid in ethanol, lipid and aqueous

media Ethanol and lipid media. The •CH(OH)CH3 radical scavenging of HGA was first investigated in the same solvent used for ergosterol, i.e., ethanol and lipid media. In this case three reactions mechanism were considered: HT, RAF and single electron transfer (SET): •

Single Electron Transfer (SET): HGA + •CH(OH)CH3 → HGA+• + ‒CH(OH)CH3

The SET mechanism has been included in this case, since HGA is a phenolic compound, while ergosterol is not, and there is previous evidence that SET may be important for this kind of compounds.44,45 However, the SET mechanism was found to be significantly endergonic in ethanol media by about 83.53 kcal/mol, and even more endergonic (106.05 kcal/mol) in lipid media (Tabla 4). This is a logical finding since the later, and other non-polar aprotic solvents cannot provide the necessary solvation to stabilize the ionic species yielded by SET. According to these results, the SET mechanism was ruled out as important in the •CH(OH)CH3 scavenging activity of HGA, when it take place in these two media. Regarding the HT mechanism, it was found that the reaction pathways involving sites 2 and 5 routes are exergonic by -11.66 and -10.70 kcal/mol in ethanol media and by -10.66 and -11.61


14

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kcal/mol in lipid media, respectively (Table 4). Thus, they may be relevant to the •CH(OH)CH3 radical scavenging capacity of HGA in ethanol and lipid. On the other hand, the RAF reaction pathways were all found to be endergonic, except for that involving site 3 in ethanol and the sites 4 and 6 in the pentylethanoate.

Table 4. M06-2X/cc-pVDZ//M06-2X/6-31+G(d,p) Gibbs free energy of reaction (∆G, kcal/mol) of •CH(OH)CH3 radical scavenging activity of homogentisic acid in ethanol and lipid media, at 298 K. RS*

∆G (ethanol)

∆G (pentylethanoate)

SET

83.53

106.05

2

-11.66

-10.66

5

-10.70

-11.61

1

2.71

3.31

2

5.25

4.36

3

-0.83

1.81

4

1.60

-2.10

5

5.35

4.89

6

3.41

-0.75

8

2.25

23.72

HT

RAF

* RS = reaction sites

The kinetic data for the reaction pathways identified as the thermochemically viable is reported in Table 5. The overall rate constants were calculated as:


15


= ;() + ) = ;(07>) + ) + :(07>) + ?(07>)

Page 16 of 29

(6)

(6)

The optimized geometries of the TSs corresponding to the different reactions pathways investigated for the reaction between •CH(OH)CH3 and HGA are provided in Figure S4 and S5 (supporting information). The earliest of the transition states was identified to be that involved in the HT from site 2 in ethanol media and correspond with the fastest one, these results were in agreement with the Hamond postulate; in the case of the lipid media, the transition state earliest was HT from site 5 of HGA, however this route has the highest Gibbs free energy and therefore, this route was the slowest of HT mechanisms; hence, in this case, the result was not according to the Hammond postulate. This, again, indicates the importance of kinetics for assessing the relative importance of different pathways. According to the estimated branching ratios the HT mechanism is responsible for almost the whole •CH(OH)CH3 scavenging activity of HGA. The contribution of the RAF mechanism to such activity is predicted to be small and negligible in ethanol and pentylethanoate, respectively. It was found that, regardless of the solvent polarity, the pathway contributing the most to the overall •CH(OH)CH3 radical scavenging activity of HGA is the HT from site 2 (Table 5), with is relative importance being larger in lipid media than in ethanol. Similarly to what was found for ergosterol, the reaction of HGA with •CH(OH)CH3 in pentylethanoate is faster than in ethanol. However, for HGA the difference is larger, i.e., about 158 times, instead of 1.4. Comparing the reactivity of both compounds towards •CH(OH)CH3 of both compounds, it was found that the rate constant of the reaction involving ergosterol is about 25 times larger than that involving HGA, in ethanol. On the contrary, in lipid media the HGA reacts about twice as fast as


16

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ergosterol. Thus, for scavenging •CH(OH)CH3 ergosterol is predicted to be better than HGA in ethanol, while HGA is predicted to be better than ergosterol in lipid media.

Table 5. M06-2X/cc-pVDZ//M06-2X/6-31+G(d,p) Gibbs free energy of activation (∆G≠, kcal/mol), tunnel effect correction (κ), reaction rate constant (k, M-1 s-1) and branching ratios (Γ, %) of •CH(OH)CH3 radical scavenging activity of homogentisic acid in ethanol and lipid media, at 298 K. ∆G≠

RS*

κ

k

Γ

Ethanol media HT 2

10.63

16.84

1.69 x 106

61.7

5

11.75

26.18

3.95 x 105

14.4

2.09 x 106

76.1

6.54 x 105

23.9

6.54 x 105

23.9

$% ( !") RAF 3

9.65

$% #%&(

1.24

!")

% "'()*++(

2.74 x 106

!")

Lipid media HT 2

8.54

5

9.55

97.21

3.31 x 108

76.3

163.7

8

23.5

8

4.32 x 10

99.8

9.71 x 105

0.2

3

0.0

5

0.2

$% (3454@)

1.02 x 10

RAF 4

9.44

6

12.58

$% #%&(3454@) % "'()*++(3454@)

1.3 1.29

4.82 x 10

9.71 x 10

8

4.33 x 10



17


Page 18 of 29

Homogentisic acid, aqueous media. While ergosterol is hydrophobic, HGA is expected to be significantly soluble in water. Therefore, this solvent was also considered for the •CH(OH)CH3 scavenging activity of HGA. In addition, HGA is a polyprotic molecule, susceptible to be involved in three acid-base equilibria: one involving the carboxyl group and two involving the phenolic OH groups. The first pKa has been reported to be equal to 4.4 and corresponds to the carboxylic group.46 However, to our best knowledge, there are no reports on the second and third pKas of HGA. Therefore, they have been estimated here using the fitting parameters approach. This method allows fast and reliable pKa calculation using only the Gibbs free energy difference between an acid and its conjugate base in aqueous solution.47 According to the above, to apply this methodology in the pKa calculations of HGA, the molecules in basal state were optimized with the functional M06-2X/6-311++G(d,p) and the results shown that the pKa for 2 and 5 positions were 11.35 and 10.71 respectively. According to the pKa values, under the physiological conditions (pH=7.4), the neutral species and the dianion are predicted to be in negligible amounts (0.1% and 0.05%, respectively, of the total HGA population). Therefore, they were not taken into account in this study. The thermodynamic results corresponding to the •CH(OH)CH3 scavenging activity of HGA in a aqueous media are reported in Table 6. These results show that the SET mechanism is endergonic, hence this mechanism is not expected to be important in the •CH(OH)CH3 scavenging activity of HGA in physiological aqueous media. The thermodynamic results also show that HT from position 2 and 5 are exergonic by -6.80 y -16.56 kcal/mol. Accordingly the HT mechanism may be important in the •CH(OH)CH3 scavenging activity of HGA in


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physiological aqueous media. The thermodynamic results for the RAF mechanisms indicate that the reaction pathways involving sites 3 and 5 routes are exergonic by -2.33 and -0.15 kcal/mol. Thus, these reactions pathways may also be important to the •CH(OH)CH3 radical scavenging activity of HGA. The other position considered in the RAF mechanisms lead to endergonic reactions and therefore, they are not expected to contribute to the •CH(OH)CH3 scavenging activity of HGA in physiological aqueous media.

Table 6. M06-2X/cc-pVDZ//M06-2X/6-31+G(d,p) Gibbs free energy of reaction (∆G, kcal/mol) for the SET, HT and RAF mechanism in the reaction between HGA and •CH(OH)CH3 in aqueous media, at 298 K. RS*

∆G

SET

24.76

HT

Entry

∆G

2

2.77

3

-2.33

2

-6.80

4

0.06

5

-16.56

5

-0.15

6

0.05

8

0.06

RAF 1

5.02


The results of the kinetic study for the •CH(OH)CH3 scavenging activity of HGA in physiological aqueous media are shown in Table 7. In this case, both thermochemical and kinetics analysis indicate that HT is the most important mechanism, with site 5 in HGA being the most reactive towards •CH(OH)CH3. The Gibbs free energy of activation for the HT mechanism


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involving site 5 is 2.46 kcal/mol lower than that corresponding to site 2. The HT from site 5 is predicted to have a rate constant of 3.48 x 107 M-1 s-1 and the about 25 times than HT from site 2. Consecuently, the pathway contributing the most (96.1%) to the overall reactivity of HGA towards •CH(OH)CH3 is the HT from site 5. Regarding the RAF mechanism, the Gibbs free energy of activation for routes 3 and 5 were estimated to be 11.60 and 17.11 kcal/mol, and the rate constants 2.51 x 104 and 2.53 M-1 s-1, respectively. The contributions of both RAF pathways to the overall •CH(OH)CH3 scavenging activity of HGA were found to be almost negligible (0.10%).

Table 7. M06-2X/cc-pVDZ//M06-2X/6-31+G(d,p) Gibbs free energy of activation (∆G, kcal/mol), tunnel effect correction (κ), reaction rate constant (k, M-1 s-1) and branching ratios (Γ, %) of •CH(OH)CH3 radical scavenging activity of homogentisic acid in aqueous media, at 298 K. RS*

∆G≠

κ

k

Γ

HT 2

12.32

239.11

1.38 x 106

3.8

5

9.86

94.91

3.48 x 107

96.1

3.61 x 107

$% (A ") RAF 3

11.60

1.28

2.51 x 104

0.1

5

17.11

1.41

2.53

0.0

$% #%&(A ")

2.51 x 104

% "'()*++(A ")

3.62 x 107



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The geometries of transition states of •CH(OH)CH3 scavenging activity of HGA are shown in Figures S6 and S7 (supporting information). The geometrical features of the HT transition states are in line with the above discussed data. The TS corresponding to HT from position 5 was earlier than that corresponding to HT from site 2. This agrees with the Hammond postulate. The overall reaction rate constant (,-./00( ) for the •CH(OH)CH3 scavenging activity of B )

HGA was obtained from the individual rate constants of the thermochemically viable pathways, as: ,-./00( = ;(;) +

On the Scavenging Ability of Homogentisic Acid and Ergosterol

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