Part A. Thermodynamics of radical formation
The first part of our study aims to obtain de values for free energies of reaction of the antioxidants with HO•, HOO•, CH3O• e DPPH●.
Stability of the tested free radicals
The radicalar stability (RS) was calculated by the difference of energy between free energies of formation of free radical •R and the molecule formed by capture of atomic hydrogen from antioxidant (HR) as describe by Equation 8.
RS = G°(R●) – G°(HR) (8)
The lower the value of RS, the more stable the free radical and the more selective the hydrogen abstraction it will be. The values of RS are shown in the second column of Table 1. To facilitate the comparison of stability, the values of RS were normalized. For this calculation the value of RS for DPPH● was considered as zero Then was obtaining a relative stability value of radical (ΔRS) through Equation 9.
ΔRS = RS – RS(DPPH) (9)
Table 1. Reactivity and relative reactivity for attacking radicals
Radical
|
RS
/kcal·mol-1
|
ΔRS /kcal·mol-1
|
HO●
|
427.7
|
39.1
|
HOO●
|
395.7
|
7.1
|
CH3O●
|
412.7
|
24.1
|
DPPH●
|
388.6
|
0.0
|
These values show HO● the most unstable free radical. Both HOO● and CH3O● are less reactive than hydroxyl radical due to hyperconjugation effect. Table 2 show the Wiberg analysis of bond order (WBO) for molecules and its respective free radicals.
Table 2. Wiberg bond order for H2O2, CH3OH and its radicals
Bond
|
HR
|
WBO
|
R●
|
WBO
|
O – O
|
HOOH
|
1.0130
|
HOO●
|
1.2031
|
C – O
|
HOCH3
|
0.9483
|
CH3O●
|
1.0649
|
The increasing of WBO of O – O bond from 1.0130 to 1.2031 shows the higher stabilization of HOO● by hyperconjugation what implies in a less reactivity of this radical compared to CH3O●. The DPPH● is the most stable free radical due to mesomeric effect through conjugation of the nitrogen radical with the tri-nitrated ring.
Selectivity of antioxidant sites. The selectivity of antioxidants sites was measured in Table 3. Values of ΔrG° for reactions between eugenol and its derivatives with HO●, HOO●, CH3O● and DPPH●. These results show the following trends for hydrogen loss of studied functional groups: Benzyl ~ Allyl > Phenoxyl > Methoxyl > Acetyl.
The higher selectivity of the allylic and benzyl radicals is due to the isoenergetic resonance of the electron unpaired with the π* orbitals of allyl and aromatic ring, that allows a better spreading of the unpaired electron density by the structure leading to a more stable radical.
Table 3. Gibbs Free Energies of Reaction (ΔrG°) for First Hydrogen Transfer, at 298.15 in kcal·mol-1, with Respect to the Isolated Reactants.
COMPOUND
SITE
|
HO●
|
HOO●
|
CH3O●
|
DPPH●
|
E1
Phenoxyl
Benzyl
Metoxyl
E2
Acetyl
Allyl
Methoxyl
E3
Acetyl
Benzyl
Methoxyl
E4
Phenoxyl
Benzyl
Methoxyl
E5
Phenoxyl(1)
Phenoxyl(2)
Benzyl
E6
Acetyl
Benzyl
Methoxyl
|
–30.6
–38.9
–13.7
–19.4
–33.5
–20.7
–19.4
–38.0
–20.5
–26.3
–37.9
–19.6
–27.5
–26.8
–37.9
–18.7
–37.6
–20.4
|
1.4
–6.9
18.3
12.6
–1.5
11.3
12.6
–6.0
–11.5
5.7
–5.9
12.4
4.5
5.1
–5.9
13.3
–5.6
11.5
|
–15.5
–23.9
1.2
–4.4
–18.4
–5.7
–4.3
–22.9
–5.6
–11.3
–22.9
–4.7
–12.4
–22.9
–11.9
–3.7
–22.6
–5.5
|
8.5
0.2
25.4
19.7
5.7
18.4
19.8
1.1
18.6
12.8
1.2
19.6
11.6
1.2
12.3
20.4
1.5
18.7
|
Among the antioxidants evaluated, eugenol proved to be the most favorable molecule to transfer atomic H in the first step of reaction showing more negative ΔRG° of -38.9·kcal·mol-1 with the radical HO●.
The E4 and E5 structures have stronger hydrogen bond O-H... O-N compared to the present in eugenol, O-H... OCH3, due to the greater electrostatic attraction between phenolic OH and the nitro group. This stronger bond increases ΔrG° from 4.0·kcal·mol-1 to E4 and 3.1·kcal·mol-1 for E5 for phenol site compared to eugenol. The presence of the nitro group also shows influence in the formation of the benzyl radical (BR), increasing ΔrG° in approximately 1.0·kcal·mol-1 for E4, E5 and E6. Acetylation and the formation of E2 also showed to difficult the H transfer in 1.0·kcal·mol-1.
Selectivity to free radicals.
The reactions with the radical HO● showed negative values of free energy of reaction, showing that any H atom of these evaluated sites can be spontaneously transferred to this radical. The CH3O● showed be able to accept hydrogen of almost all sites evaluated, except for formation of Eugenol methoxyl radical (MR), which showed a positive reaction-free energy value of 1.2·kcal·mol-1. The radical HOO● showed be very selective reacting only with allylic and benzyl sites due its less reactivity. None of the sites showed spontaneity of reaction with the DPPH●. The literature shows that the DPPH reaction with eugenol follows a 1:2 reaction stoichiometry, reinforced by the metabolic study of eugenol in the body, which shows the elimination of a methide quinone through the urine.
Part B. Kinetics of radical formation.
Influence of the attacker radical stability. The Table 4 show the free energies of activation and rates constants as well.
Table 4. Gibbs Free Energies of Activation (Δ‡G°) in kcal·mol-1 and Rate Constants for First Hydrogen Atom Transfer, in M–1·s–1, at 298.15 K
COMP.
SITE
|
HO●
|
HOO●
|
CH3O●
|
Δ‡G°
|
k
|
Δ‡G°
|
k
|
Δ‡G°
|
k
|
E1
Phen.
Benz.
Met.
|
6.3
6.4
7.2
|
1.5·108
1.2·108
3.5·107
|
17.1
20.0
25.0
|
1.8
1.4·10–2
2.9·10–6
|
10.6
12.5
14.2
|
1.1·105
4.1·103
2.4·102
|
E2
Acet.
All.
Met
|
9.2
8.3
7.8
|
1.1·106
5.3·106
1.3·107
|
28.4
23.1
21.3
|
1.0·10–8
7.2·10–5
1.5·10–3
|
18.2
13.8
15.8
|
3.0·10–1
5.0·102
1.6·101
|
E3
Acet.
Benz.
Met.
|
10.7
7.5
7.9
|
9.6·104
2.0·107
1.1·107
|
28.6
20.9
24.7
|
7.0·10–9
3.0·10–3
5.1·10–6
|
18.7
12.8
15.4
|
1.3·10–1
2.4·103
3.3·101
|
E4
Phen.
Benz.
Met.
|
11.0
7.5
8.0
|
5.1·104
1.9·107
9.1·106
|
19.0
20.6
24.8
|
7.2·10–2
5.0·10–3
4.3·10–6
|
14.8
12.8
15.5
|
8.5·101
2.5·103
2.8·101
|
E5
Phen.1
Phen.2
Benz.
|
12.5
8.2
8.4
|
4.2·103
6.3·103
4.6·106
|
24.6
23.5
21.3
|
6.2·10–6
3.4·10–5
1.4·10–3
|
15.6
13.9
14.2
|
2.3·101
3.8·102
2.6·102
|
E6
Acet.
Benz.
Met.
|
10.5
5.7
2.9
|
1.2·105
3.9·108
4.6·1010
|
32.8
20.4
24.1
|
5·10–12
6.6·10–3
1.2·10–5
|
18.2
12.3
10.6
|
2.7·10–1
5.5·103
1.1·105
|
In general, the less stable is the attacking radical, the lower the activation free energy. In all evaluated sites, the HO● showed to be the fastest to abstract the atomic H, showing lower values of Δ‡G°.
The only exception found was the formation of the BR in E4, where the smallest energy barrier found was for the attack of the radical CH3O●. The presence of the nitro group does not allow the methoxyl group to be in the same plane of the ring which assists the formation of a stable intermediate of 6 membered ring.
Influence of the attacker radical reactivity. The eugenol molecule presented Δ‡G° of 6.3·kcal·mol-1 for the formation of the phenoxyl radical (PR) and 6.4·kcal·mol-1 for the formation of BR in the reaction with HO•. In the reaction with HOO● and CH3O●, as the radical becomes more stable, eugenol shows a kinetic preference to oxidize the phenol than the benzyl group.
The nitro group in E4 and E5 shows increasing the free energy of activation for PR formation due to the stronger hydrogen bond that difficult the transfer of H process and BR due to the meta-position substituent effect that destabilizes the formed radical. In E4 the most kinetically favorable site is benzyl hydrogen with value of Δ‡G° of 7.5·kcal·mol-1, whereas in E5, is phenoxyl-2 hydrogen with 8.2·kcal·mol-1.
Separately, both thermodynamic and kinetic data for a single atomic hydrogen transferred show that eugenol is expected to have a better antiradical activity than its derivatives, which is inconsistent with literature data where E5 has a higher capacity antioxidant that eugenol and E4 has no activity in DPPH assay [12].
Part C. Thermodynamics of quinone formation
The first transfer of H did not bring satisfactory answers to justify the reactivity or not of eugenol and its derivatives. Moreover, none showed to be spontaneous with the DPPH radical in a stoichiometry of 1:1. This encouraged us to investigate the second abstraction to explain the facts present in the literature. Only the E1, E4 and E5 structures are able to form methide-quinones, and only E5 can form an ortho-quinone, as shown in the Figure 2.
The thermodynamics of formation of quinones was evaluated by comparation of ΔrG° for reaction between ArXH and two equivalents of R●. Table 5 shows the values of ΔrG° with free radicals for two consecutive steps of HAT.
Table 5. Gibbs Free Energies of Reaction (ΔRG°), at 298.15 in kcal·mol-1 for Formation of Quinones, with Respect to the Isolated Reactants.
COMPOUND
Quinone(QN)
|
HO●
|
HOO●
|
CH3O●
|
DPPH●
|
E1
MQ
E4
MQ
E5
MQ
OQ
|
–91.4
–85.0
–86.9
–69.9
|
–27.5
–21.1
–23.0
–6.0
|
–61.4
–54.9
–56.9
–39.9
|
–13.2
–6.8
–8.7
–8.3
|
The results show that the less stable the attacker free radical, the more spontaneous the formation of the quinones. The formation of methide quinones showed be more spontaneous than ortho-quinone, probably due to electrostatic repulsion among lonely electron pairs of the three oxygen centers presents in the last one.
Among the structures that show the highest tendency to form methide quinone is E1, with -91.4·kcal·mol-1, followed by E5 with -86.9·kcal·mol-1. The reaction of E4 quinone is only 0.3·kcal·mol-1 less stable than that of E5.
For the three structures analyzed, the formation of the methide quinones (MQ) showed spontaneous reaction with DPPH●. The formation of ortho-quinone (OQ) presented ΔrG° of 8.3·kcal·mol-1, suggesting that this mechanistic route is the least favorable to occur. The structural inability to form MQ may explain the negative result of antioxidant activity of E2, E3 and E6 derivatives in DPPH assay, but the experimental data for E4 still unclear.
Part D. Kinetics of quinone formation
The achieving of the transition state for the formation of quinones was performed from the most stable radical of each structure. For E1, phenoxyl radical was used, while for E4 and E5 were used its respective benzyl radicals. Table 6 shows the values of Δ‡G° and k for formation of quinones.
Table 6. Gibbs Free Energies of Activation (Δ‡G°) in kcal·mol-1 and Rate Constants for Quinone Formation in M–1·s–1, at 298.15 K, with Respect to the Isolated Reactants
COMP.
SITE
|
HO●
|
HOO●
|
CH3O●
|
Δ‡G°
|
k
|
Δ‡G°
|
K°
|
Δ‡G°
|
k
|
E1
MQ
|
7.3
|
2.9·107
|
18.6
|
1.5·10–1
|
11.5
|
2.3·104
|
E4
MQ
|
–3.3
|
1.6·1015
|
24.9
|
3.3·10–6
|
10.0
|
2.7·105
|
E5
MQ
OQ
|
–1.7
5.5
|
1.2·1014
5.9·108
|
28.1
38.0
|
1.4·10–8
8·10–16
|
7.3
19.5
|
2.9·107
3.1·10–2
|
Similarly, to the transition state of the formation of radicals, the free energy of activation is less, the less stable the attacker radical. The Δ‡G is 6.6·kcal·mol-1 in the E1 reaction with HO● and shows only the transfer of hydrogen, as expected. However, the second transfer for E4 and E5 occurs in a different way.
First occurs the addition of radical to 1-carbon of the aromatic ring. This intermediary is so stabilized by hydrogen bonding that when the reaction with HO●, that Δ‡G° computed directly from ArX● and R● show negative values: –3.3·kcal·mol-1 for E4 and –1.7·kcal·mol-1 for E5. The IRC calculation shows that in the next step evolves in a concerted mechanism with the effective transfer of phenolic hydrogen and elimination of water. Figure 3 shows a scheme of this quinone formation path.
The same effect was seen when were used HOO● and CH3O●, forming
4-membered and 5-membered rings, respectively. The formation of these intermediaries is responsible for decreasing of free energy of transition state.
Contrary to what is observed for reactions with other radicals, the energy barrier for the reaction with HOO● is higher for nitrated derivatives in relation to activation-free energy compared to the reaction of this radical with eugenol. These results suggest that the more reactive the radical, the greater its selectivity to attack nitrated derivatives for the formation of methide quinones.
Since the DPPH● is more stable than HOO●, is expected that it is more susceptible to the formation of quinone from eugenol, which still did not explain well the fact that E5 presents antioxidant activity superior to eugenol in the test.
Part E. Statistical analysis of structure and reactivity
Based on the data obtained, a statistical study was carried out in three parts: (a) structural aspects, (b) reaction-free energy and (c) reaction kinetics.
Structural Aspects.
In the structural study, a multiple linear regression was performed using the following factors: presence of phenolic (PR1 and PR2), benzyl sites (BR) and ability to form methyl quinone (MQ) and orthoquinone (OQ). PR1 was defined as original phenolic group and PR2, as the phenolic group produced by demethylation of the methoxy group in E5. The response variable was defined as ln(Co/Ct) values of the antioxidant capacity in the DPPH test present in the work of Hidalgo and in this work it was called antioxidant activity (AA) [12].
In case of the presence of the antioxidant site was interpreted as 1 and the absence was represented by 0. The ability to form quinone of molecule was interpreted as 1 (e.g. E1, E4 and E5) and the inability to form, as 0 (e.g. E2, E3 and E6). In the model the term B was considered being equal to 0, because in the absence of specific functional groups, the molecule should not present any antioxidant activity. Table 7 show the results for structural analysis.
Table 7. Strucutral Analysis of Antioxidant Profile
Compound
|
PR1
|
PR2
|
BR
|
MQ
|
OQ
|
AA
|
E1
|
1
|
0
|
1
|
1
|
0
|
0.71
|
E2
|
0
|
0
|
0
|
0
|
0
|
0.00
|
E3
|
0
|
0
|
1
|
0
|
0
|
0.00
|
E4
|
1
|
0
|
1
|
1
|
0
|
0.00
|
E5
|
1
|
1
|
1
|
1
|
1
|
1.85
|
E6
|
0
|
0
|
1
|
0
|
0
|
0.00
|
The multiple linear regression of above data showed a relation of the activity with the Equation 10.
AA = 0.71 × (P.R.1) + 1.14 × (P.R.2) (10)
This result shows the dependence of the antioxidant activity with the DPPH with the presence of phenolic sites. An explanation for this may be the presence of a hydrogen bond of type N... H-O between DPPH and antioxidant in the H-transfer through phenolic sites. The presence of a second phenolic sites shows to be responsible by 61.6% of antioxidant activity.
Free Energy of Reaction.
The second study was based on the free energy data of the formation of phenolic and benzyl radicals as the quinones, maintaining the equal response variable. The Table 8 shows the data for ΔrG° for the main reactive sites. In the absence of the site, the free energy value was defined as being equal to zero.
The evaluated data set formed the following relationship with the antioxidant activity represented by the Equation 11.
AA = -0.356 × ΔrG°(RF1) + 0.111 × ΔrG°(QM) - 0.0249 × ΔRG°(OQ) (11)
According to this equation, the more negative the reaction-free energy for the formation of the phenoxyl radical-1 and ortho-quinone, the greater the anti-radicalar activity. The positive coefficient of ΔRG° (QM) may suggest that greater stability of MQ may decrease the antioxidant activity in the DPPH test. This can be explained because there would be a side reaction where two PR can disproportionate into a methide quinone and regenerating the original antioxidant molecule. This reaction competes with the reaction with the DPPH radical which could slow down the change in the absorption band of the DPPH at 515 nm.
Table 8. Free Energies of Reactions and Antioxidant Profile
Compound
|
PR1
|
PR2
|
BR1
|
BR2
|
MQ
|
OQ
|
AA
|
E1
|
-30.6
|
0
|
-38.9
|
-38.9
|
-91.4
|
0
|
0.71
|
E2
|
0
|
0
|
0
|
0
|
0
|
0
|
0.00
|
E3
|
0
|
0
|
-38.0
|
-38.0
|
0
|
0
|
0.00
|
E4
|
-26.6
|
0
|
-37.9
|
-37.9
|
-85.0
|
0
|
0.00
|
E5
|
-27.5
|
-26.8
|
-37.8
|
-37.9
|
-86.9
|
-69.9
|
1.85
|
E6
|
0
|
0
|
-37.6
|
-33.1
|
0
|
0
|
0.00
|
Reaction Kinetics.
The third statistical study was based on the constants of bimolecular reaction rates obtained by the Eyring equation. Table 9 show values of k for formation of PR1, PR2, BR, QM and OQ, using as y the results in DPPH test [12].
Table 9. Kinetics Analysis of Antioxidant Profile
Compound
|
PR1
|
PR2
|
BR
|
MQ
|
OQ
|
AA
|
E1
|
6.3
|
0
|
6.4
|
7.3
|
0
|
0.71
|
E2
|
0
|
0
|
0
|
0
|
0
|
0.00
|
E3
|
0
|
0
|
7.5
|
0
|
0
|
0.00
|
E4
|
11.0
|
0
|
7.5
|
-3.1
|
0
|
0.00
|
E5
|
12.5
|
8.2
|
8.4
|
-1.7
|
5.5
|
1.85
|
E6
|
0
|
0
|
5.7
|
0
|
0
|
0.00
|
From data analysis of Table 9, was obtained the Equation 12, correlating kinetics data with antioxidant activity.
AA = 0.0220 × k(PR1) + 0.2082 × k(PR2) + 0.0782 × k(QM) (12)
This equation showed that the activity is influenced by the activation barriers for the formation of PR1, QM and mainly PR2. These results suggest that the presence of PR2 assists the reaction kinetics, while the thermodynamic of antioxidant activity depends on the ΔRG°(PR1). Nevertheless, only possession of these data is impossible to suggest the chemical explanation for the improvement of the antioxidant profile by PR2.
Part F. Rates of Reaction With DPPH Radical.
In order to understand the lack of activity of E4 and the greater reactivity of E5 in relation to E1, the transition states were obtained for the reaction of these three antioxidants with the DPPH●. They were used as phenolic sites for the transfer of H, since the statistical study disposes the influence of the allyl and benzyl sites in the test.
The Table 10 show the value of ΔG‡ and k for these reactions. These results show perhaps one of the best results of this work: the stereoselectivity of the DPPH to E1 and E5. The data of free energies of activation with DPPH● show values of 20.6 kcal·mol-1 for the PR and 24.0 kcal·mol-1 for the BR of E1, following the trend seen that the greater the stability of the radical, the greater the preference of reaction with the phenolic site.
Table 10. Kinetics data for reaction of sites with DPPH•
Compound
|
Site
|
ΔG‡
kcal·mol-1
|
k
L·mol-1·s-1
|
E1
|
PR
|
20.6
|
4.9 × 10-3
|
|
BR
|
24.0
|
1.6 × 10-5
|
E4
|
PR
|
29.4
|
1.6 × 10-9
|
E5
|
PR1
|
22.6
|
1.6 × 10-4
|
|
PR2
|
21.1
|
2.2 × 10-3
|
This difference in the energy barrier suggests that the reaction is approximately 300 times faster with the phenolic site, which would justify that E2, E3 and E6, acetylated derivatives, without the presence of this functional group, do not showing anti-radicalar activity during analysis time in the DPPH assay.
Despite E4 be very reactive with the radicals HO●, HOO● and CH3O●, the phenol site of this nitro-derivative presents greater difficulty to access the H acceptor site of the DPPH due to the volume of the methoxyl group and the electrostatic repulsion between the nitro groups of both species. The free energy of activation for phenolic site of E1 is the lowest which makes the reaction faster than its nitro-derivatives.
The ΔG‡ for reaction with E4 is 29.4·kcal·mol-1 what makes this reaction be almost 106 slower than with E1. This could explain the reason of E4 did not show antioxidant behaviour over time of DPPH test. Figure 4 show the transitions states of hydrogen transfer from phenolic sites to DPPH of E1, E4 and E5.
The arrangement almost parallel of the aromatic rings suggests that the reaction appears should be facilitated by an π-stacking interaction. The presence of the nitro group gives rise to a repulsion force that prevents the stacking of the aromatic rings. Only E1 and PR2 of E5 be able to reach this arrangement, due to the absence of nitro group in E1 and to considerable distance between this group in meta to second phenoxyl group.
From this perspective, the second phenol group (PR2) in the structure should act in two different ways: as an amplifier of the reactive surface area, because the two sites of E5 have close activation free energies; or intramolecular catalyst as shown in Figure 5. The reaction in only one step is about ten times slower.
The PR1 formation indirect route has 3 steps: first, the H-transfer from E5 to DPPH producing PR2 by a faster step (b-I; followed by rotation of HOCC torsion making a hydrogen bonding O-H…•O (b-II), ending with the internal transfer of H producing PR1 (b-III). The PR1 formation indirect route has 3 steps: first, the H-transfer from E5 to DPPH producing PR2 by a faster step (b-I; followed by rotation of HOCC torsion making a hydrogen bonding O-H…•O (b-II), ending with the internal transfer of H producing PR1 (b-III).