Density functional theory study of the role of benzylic hydrogen atoms in the antioxidant properties of lignans

Antioxidants are a diverse group of chemicals with proven health benefits and thus potential preventive medicine and therapeutic applications. While most of these compounds are natural products, determining their mechanism of radical scavenging and common motifs that contribute to antioxidant activity would allow the rational design of novel antioxidants. Here the origins of the antioxidant properties of ten natural products of the lignan family were studied in silico by calculating their thermochemical properties by using ROB3LYP/6-311++G(2df,2p)//B3LYP/6-311G(d,p) model chemistry. Three conditions were modelled: gas phase, ethanol and water solvents. The results allowed assigning the antioxidant activity to specific moieties and structural features of these compounds. It was found that the benzylic hydrogen atoms are the most likely to be abstracted to form radicals and hence define antioxidant properties in most of the studied compounds. The results also suggested that the most likely mechanism of HOO• radical scavenging differs by the key moiety: it is hydrogen atom transfer in case the benzylic C-H bonds, however it is proton coupled electron transfer in case of the compounds where O-H bonds are responsible for radical scavenging.

It is clear from former experimental and computational studies that the ability of phenolic compounds to scavenge free radicals is due to the presence of phenolic hydroxyl groups, and that it follows either the hydrogen atom transfer (HAT) or the single electron transfer followed by proton transfer (SETPT) mechanism 8,[30][31][32][33][34] 25 . Moreover, the anti-oxidative activity of (−)-eudesmin (64.1%), which was formed by the methoxylation of pinoresinol, is nearly as high as that of vitamin C (76.4%) in the 2,2′-azinobis(3-e thylbenzothiazoline-6-sulfonic acid test, despite of the fact that there are no hydroxyl groups in this compound 39 . Recent studies showed that the lowest BDE values are not always found for the phenolic hydroxyl groups but for the benzylic C-H 40 . Hence, in predicting antioxidant activity of lignans, the contributions of the benzylic hydrogen atoms should be also investigated. The purpose of this study is to evaluate the antioxidant capacity of ten phenolic compounds ( Fig. 1) of the lignan families identified in the extracts of the Abies, Larix, Picea, Pinus and Tsuga genera 25 ; here antioxidant capacity is defined by the ability of electron transfer and/or H-donation to the free radicals, where H-donating ability is determined to all hydrogen containing moieties. The mechanistic pathway of the radical scavenging activity will be also investigated based on the calculation of thermodynamic parameters. Finally, potential energy surfaces (PES) and natural bond orbital (NBO) will be calculated for evaluating of the mechanism of reactions between the typical antioxidants and the HOO • radical.

Results and Discussions
Bond dissociation enthalpies. Previous studies have shown that the BDE value is an important factor to evaluate the antioxidant activity of compounds as it characterizes the ability of donating a hydrogen radical and forming a stable radical of the donor 8,41 . In order to qualitatively identify the lowest BDE values, the BDEs of all possible X-H (X=C, O) bonds breaking in the studied compounds were calculated by the B3LYP/6-31G(d,p) method and presented in Table S1 (SI). With the purpose of accurate prediction, the lowest BDEs of X-H (X=C, O) were then calculated using ROB3LYP/6-311++G (2df,2p)//B3LYP/6-311G(d,p) model chemistry. It was found that, while all the weak O-H bonds are found at the C3 or C4 of the aromatic rings, the lowest BDEs of C sp3 -H bonds are mostly predicted for the C7′-H (benzylic hydrogen atoms).
Based on the prediction of the weakest bonds for each compound in the gas phase, further BDE calculations have been performed in specific solvents; the other thermochemical parameters and PES have also been calculated for these reactive centers.
The hydrogen atom transfer (HAT) mechanism. It is well known that HAT mechanism is characterized by the BDE values that correspond to the ability of a XH (X=C, O) moiety to donate its hydrogen atom and consequently form a radical. The lower the BDE of the relevant X-H (X=C, O) bond, the higher antioxidant potency. Thus in this section, the BDE values of the weakest X-H (X=C, O) bonds of each compound were computed in the study environment and shown in Table 1 42 . It is clear from the Table 1 25 . Therefore, we should consider the effect of C7′-H (benzylic) group in studying the antioxidant mechanism for hydroxymatairesinol 5 rather than only focus on phenolic hydroxyl group O4′-H 43 .
Typically, the O-H bond that is easiest to dissociate is found at the C4 of the aromatic rings whereas the weakest C-H bond is identified at the C7′-H of the lignans. This can be explained with the electron-withdrawing conjugation effect of the π delocalization in the aromatic the rings. An electron is released from a lone pair of the O atom of the O-H bond into the aromatic ring, leading to increased polarization of the O-H bonds. Upon bond dissociation, the single electron at the O or C atom of the radical is released to the aromatic rings by resonance, i.e. the formed radical is stabilized. This favors X-H (X=C, O) bond breaking, as reflected in the lower BDEs of these bonds compared to other ones ( Fig. 1).
It is clear that the hydrogen-donating ability is affected by the polarity of solvents because of the change of BDE values. Therefore, in this study ethanol and water solvents were used as the environment for calculating of BDEs. Ethanol was chosen because the experimental studies of the antioxidant activity and radical trapping   The sequential electron transfer proton transfer (SETPT) mechanism. Ionization energies.
According to SETPT mechanism, ionization of the antioxidant molecule is the first step, and thus IE is used to describe the electron donor ability. The lower the IE value, the easier the electron transfer and the higher the antioxidant activity. The adiabatic IE values were calculated using the same model: ROB3LYP/6-311++G (2df,2p)// B3LYP/6-311G(d,p) in gas phase. The results are shown in Table 2. The sequence of vertical IE values in gas phase Proton dissociation enthalpies. The last step of the SETPT mechanism is the loss of a proton from the cation radical formed in the first step, thus it is characterized by the PDE that determines the thermodynamically preferred X-H (X=C, O) group for deprotonation. The calculated results are given in Table 2.
The easiest deprotonation generally is assigned to the X-H (X=C, O) bond with the lowest BDE that is most likely to break. Among the studied compounds, the lowest PDEs are approximately 230 kcal.mol −1 that were calculated for 4, 8 and 9. Assuming SETPT mechanism, the antioxidant potential is defined by the combination of PDE and IE; Table 2 shows that, according to this mechanism, compounds 1, 2, 6, 7, 9 should be the most potential antioxidants, of which compound 9 has the lowest PDE + IE value at 392.8 kcal.mol −1 . Hence this compound would be the best antioxidant irrespective of whether the reaction follows the HAT or the SETPT mechanism. Comparison with the calculated BDE and IE of phenol (87.7 kcal.mol −1 and 196.9 kcal.mol −1 , respectively) at ROB3LYP/6-311++G(2df,2p)//B3LYP/6-311G(d,p), the ΔBDEs and ΔIEs of the studied compounds are in the range of −9.3 to 0.3 kcal.mol −1 and −24.4 to −39.0 kcal.mol −1 , respectively. Thus the HAT mechanism appears to be the main pathway for the lignans 44 .
The sequential proton loss electron transfer (SPLET) mechanism. Proton affinities. The sequential proton loss electron transfer (SPLET) mechanism starts with the dissociation of the acidic moiety, which can be characterized by the proton affinity; this is followed by an electron transfer to the free radical, at a cost of the electron transfer energy. Lower PA is characteristic of higher antioxidant capacity via this mechanism.
As described above, the PAs values of the compounds were first evaluated by the B3LYP/6-31G(d, p) method (Table S2, SI), then the lowest PAs of X-H (X=C, O) were calculated using ROB3LYP/6-311++G (2df,2p)// B3LYP/6-311G(d,p) model chemistry. The results are shown in Table 3. As can be seen from the Table 3  Electron transfer enthalpies. The ETE is a key descriptor of the last step of the SPLET mechanism. It is clear from Table 3 that the IEs (data in Table 2) are much higher than the ETE values in the gas. Therefore, the single electron transfer process from the neutral form is not as preferable as from the anionic form. This result agrees with the previous studies 45, [49][50][51][52] . It was found that the following electron transfer process does not favor in the polar solvents. In fact, there is a significant increase in ETE values in the studied solvents (both water and ethanol) compared with those obtained in the gas phase. On the calculated PA and ETE values of both step of the SPLET mechanism, 9 has the highest antioxidant activity with the total PA + ETE value at around 393 kcal.mol −1 in all of the studied environment. That agrees with our results in HAT and SETPT mechanism investigations.
The reaction with HOO• radical: potential energy surfaces (PES). The antioxidant capacity of the phenolic compounds with the lowest BDE can be expressed by constructing the PES of their reaction with a ROO • radical. Thus the PESs of the reactions of the most potential antioxidant compounds 9 and the lowest BDE(C-H) compounds 1, 7 with HOO • radical were investigated. The optimized structures and the energies of the reactants (R), the transition states (TS), the intermediates (Int) and the products (P) of each compound were calculated using the same computational method. The IRCs were calculated to ensure that each transition state connects to the expected reactant and product and shown in Fig. S1 (SI). All optimized TS structures were shown in Fig. S2 (SI) and the PESs were built and displayed in Fig. 2. Based on the calculations, the reaction path for the hydrogen abstraction can be described as follows:  (Fig. S2, SI). The energies of the studied TSs are only −0.4 and −3.7 kcal.mol −1 for 1-O4-H-OOH and 9-O4′-H-OOH, respectively, while for others are in the range of 2.6-6.2 kcal.mol −1 (Fig. 2). The results suggest that compound 9 has the highest antioxidant activity due to the lowest TS energy, in a good agreement with the above-described results. These phenolic compounds can easily transfer the hydrogens of either of the benzylic groups or phenyl hydroxyl groups to the peroxide radical species, forming the neutral compound (HOOH) and an unreactive radical (ArX • ). On the basis of the calculated Gibbs free energies given in the last column of Fig. 2, these hydrogen abstraction reactions will be spontaneous. Noticing that, Gibbs free energies of the reactions of the benzylic hydrogen at 7 are more negative than these for the hydrogen atom of phenolic hydroxyl group (ΔG (7-C2-H + HOO • ) = −5.91 kcal. mol −1 compared to −0.04 kcal.mol −1 for the 7-O4′-H + HOO • reaction). Thus the hydrogen abstraction reaction of the 7-C2-H has a higher priority than that of 7-O4′-H.  Natural bond orbital (NBO). In order to gain further insights of the antioxidant mechanism, natural bond population (NBP) charge, the atomic spin densities (ASD) and singly-occupied molecular orbitals (SOMO) of the transition states of the studied compounds were also determined as shown in Table 4 (Table 4). Thus it suggests that the HAT mechanism is most likely for the H-atom abstraction of the C-H bonds, while the PCET mechanism is favored for the H-atom abstraction of O-H bonds.
Theoretical and computational methods. In this work, all computational calculations were performed using the Gaussian 09 suite of programs 55 . The B3LYP/6-311G(d,p) level of theory was used for optimizing the geometry and determining the vibrational frequencies of each neutral compound and the related radicals, cationic radicals and anions 56,57 . The ROB3LYP/6-311++G(2df,2p) method was then used to calculate the single point electronic energies 42 . For the species that have multiple conformers, all of these were investigated and the conformer with the lowest electronic energy and possible intermolecular hydrogen bonds was included in the analysis [58][59][60] .The potential energy surfaces of the reaction between the selected lignans and the HOO • radical were investigated based on the calculation of the transition states, intermediates, products and the intrinsic   In order to determine the mechanistic pathway of the radical scavenging process, thermochemical properties were used, by assessing the energetics of the determining step of each pathway. The literature recognizes three common mechanisms of antioxidant activity 8,41 . In the hydrogen atom transfer (HAT) mechanism, the first step is the homolytic bond breakage in an appropriate moiety to yield a hydrogen radical, which then reacts with the free radical species; here the bond dissociation energy of the R-H moiety determines the enthalpy of the first step.
In the "Single electron transfer followed by proton transfer" (SETPT) mechanism the first step is electron loss to form a radical cation, characterized by the ionization energy, followed by a deprotonation step that is described with the proton dissociation energy.
The third mechanism, "Sequential proton loss electron transfer" (SPLET), starts with the dissociation of the acidic moiety, which can be characterized by the proton affinity; this is followed by an electron transfer to the free radical, at a cost of the electron transfer energy Thus the reaction enthalpies of the individual steps in the above described mechanisms of antioxidant activity in gas phase (at 298.15 K and 1 atm) are calculated as follows 41,46,49 :  In the gas phase, the enthalpy of hydrogen atom was of −0.5 Hartree and for other environment, the enthalpy of hydrogen atom was calculated at the same method. The calculated enthalpies of the electron (e − ) and proton (H + ) were taken from the literature 41,63-65 . Vibrational frequencies obtained at the B3LYP/6-31G(d,p) and B3LYP/6-311G(d,p) levels were scaled by a factor of 0.9611 and 0.9669, respectively 66,67 .

Conclusions
The antioxidant activities of ten lignans in gas phase, ethanol and water solvents has been successfully evaluated via their thermochemical properties by using the ROB3LYP/6-311++G(2df,2p)//B3LYP/6-311G(d,p) calculation method. The BDE values of the X-H (X=C, O) moieties of the studied compounds were all found in the range of 78.4-88.0 kcal.mol −1 . Cyclolariciresinol, secoisolariciresinol, nortrachelogenin, pinoresinol and lariciresinol are predicted to be the most potential antioxidants, especially lariciresinol that was found to be the best antioxidant compound under all of the studied conditions. The results suggest that the benzylic hydrogen atoms play an important role in antioxidant properties of lignans alongside the hydrogen atoms of phenolic hydroxyl groups. Modelling the HOO • radical scavenging mechanism of cyclolariciresinol, pinoresinol and lariciresinol suggests that the hydrogen atom transfer mechanism is most likely for the H-atom abstraction of the C-H bonds, while the proton coupled electron transfer mechanism is favored for the H-atom abstraction of O-H bonds.