Is Vitamin A an Antioxidant or a Pro-oxidant? - The Journal of Physical

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Is Vitamin A an Antioxidant or a Prooxidant? Duy Quang Dao, Thi Chinh Ngo, Nguyen Minh Thong, and Pham Cam Nam J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.7b07065 • Publication Date (Web): 22 Sep 2017 Downloaded from http://pubs.acs.org on September 23, 2017

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The Journal of Physical Chemistry

Is Vitamin A an Antioxidant or a Prooxidant? Duy Quang Dao1,*, Thi Chinh Ngo1, Nguyen Minh Thong2, Pham Cam Nam3,* 1

Institute of Research and Development, Duy Tan University, 03 Quang Trung, Danang, 550000, Viet Nam

2

Campus in Kon Tum, The University of Danang, 704 Phan Dinh Phung, Kon Tum, 580000, Viet Nam

3

Department of Chemistry, The University of Da Nang – University of Science and Technology, 54 Nguyen Luong Bang, Danang, 550000, Viet Nam.

ACS Paragon Plus Environment

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ABSTRACT

Antioxidant efficiency of all-trans-retinol has been studied on the basis of characteristic thermochemical properties using density functional theory. The influence of the solvent polarity has also been evaluated. It is found that retinol may act in parallel as an effective antioxidant via Hatom donating as well as a prooxidant in yielding reactive hydroxyl radical. In fact, the lowest values of bond dissociation enthalpy were found at C18−H and C18−OH positions. Retinol was also determined as good electron donor but bad acceptor in the single electron transfer (ET) reaction with hydroperoxyl (HOO●) radical. In addition, potential energy surfaces of H-atom transfer (HAT) and radical adduct formation (RAF) reactions between retinol and HOO● radical were also investigated in the gas phase and in the solvent. The results demonstrated that RAF mechanism was generally more predominant than ET and HAT ones. The most favored radical addition position was found at C2=C3 double bond in cyclohexenyl ring. Moreover, the radical scavenging reactivity via RAF reactions was strongly exergonic and thermodynamically feasible while the ET one was endergonic. Natural bond orbital analysis showed that the lone pairs of electron on oxygen atom of the HOO• radical were donated to unoccupied antibonding orbital of transferred H-atom in HAT reactions. In contrast, in the case of RAF reactions, strong interactions between 2p orbitals on oxygen atoms of the radical and π−orbital of double bond on retinol molecule were recognized. The results obtained in this work were in agreement with previous experimental observations.


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INTRODUCTION Vitamin A (retinol) is a generic term referring to a form of retinoids (group of fat-soluble unsaturated hydrocarbons) including retinol, retinal and retinoic acid. By using in vitro peroxidation system, Das and corworkers has demonstrated that antioxidant activities of retinoids have been ranked as retinol ≥ retinal > retinoic acid.1,2 The main sources of vitamin A in food are provitamin A carotenoids from vegetables and retinyl esters from animal tissues.3,4 Vitamin A are important regulators of embryogenesis, cell growth and differentiation, vision and reproduction.5 Deficiency of vitamin A leads to well-described defects in vision, fertility and, in animals, increased susceptibility to carcinogenesis.6 One of the most active forms of vitamin A is all-trans-retinol.7 The biological metabolites of retinol are unique. They contain five conjugated double bonds located at their cyclohexenyl ring as well as isoform specific side chains.8 This interesting structure has a significant effect on their reactivity like antioxidant actions. According to the literature, all-trans-retinol can act as chain-breaking antioxidant in effectively scavenging lypoperoxyl radical in solution.7 Additionally, the radical adduct formation (RAF) reaction to cyclohexenyl ring on retinol has been experimentally indicated to be more predominant than hydrogen atom transfer (HAT) ones.9,10 The obtained radical product may undergo decomposition reaction to form an alkoxyl radical (LO•) from the lipoperoxyl one (LOO•), and 2,3retinoid epoxide compound (see Figure 1 for numbering of atomic sites). Another reaction pathway is that the RAF product radical reacts with singlet oxygen (O2) to form retinoid-derivative peroxyl radical (RetOO•); or it may undergo a second addition of lipoperoxyl radical.10 It can be seen that the epoxide formation in consuming LOO• radical generates another radical, i.e. LO• radical, thus,


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this does not tend to net radical scavenging. Vitamin A like retinol can act as radical trapping antioxidant in adding a maximum of six LOO• radicals to its conjugated system. On the other hand, RetOO• radical could cause a self-oxidation by reacting with other retinoid compounds, or could become a prooxidant for lipid system.10 Several computational studies on the provitamin A β-carotene suggest that the abstraction reaction is more predominantly to occur from the C4 position (as numbered in Figure 1) of the cyclohexenyl ring.11,12 As a result, the H-atom abstraction from C4 position results in highly stabilized radical and unpaired electron is delocalized over the polyene chain. Chen et al investigated the reaction between β−carotene and HO•, HOO• radicals13 as well as antioxidative activity of carotenes against the peroxidation of lipids initiated by nitrogen dioxide.14 In their works, hydrogen-atom transfer (HAT), radical adduct formation (RAF) and electron transfer (ET) mechanisms were investigated. Among these mechanisms, HAT and RAF were found to be thermodynamically more favorable than SET one. Moreover, antioxidant action via single electron transfer (SET) mechanism of vitamin A has also been studied in calculating its ionization energy (IE) quantity. Katsumata et al studied experimentally the electronic structure of all-trans-retinol by means of the gas phase HeI photoelectron spectroscopy and the authors calculated ionization energy (IE) of each compounds by using HF/6-311G level of theory.15 As a result, experimental and calculated adiabatic IE values of vitamin A are of 6.73 and 7.13 eV, respectively. The large deviation is due to insufficiently high computational level of theory used. Similarly, Martinez et al also calculated at the BPW91/D95V model chemistries the vertical IE and vertical EA values of the vitamin A equal to 6.22 and 0.54 eV, respectively.16 Recently, Abyar and Farrokhpour updated IE values and the main electronic


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configurations of the vitamin A ion states and the ones of its derivatives by performing calculations using direct SAC-C-CI theory considering electron correlations.17 The authors find that the IE value is around 7.11 eV. Thus, retinol may act as a strong antioxidant via SET mechanism in comparing to IE value of phenol (i.e. 8.49 eV or 195.79 kcal/mol18) which is a referenced phenolic antioxidant.

On the basis of above resumes, it is evident that the vitamin A plays an important role for human health, thus it is important to analyze its viable antioxidant mechanism especially on the fact of free radical scavenging properties. Although different experimental data concerning the antioxidant activity of the vitamin A as well as separated computational results related to its thermochemical parameters, there are no systematical theoretical works which determine all characterizing parameters of its free radical scavenging capacity as well as calculate the possible antioxidant reaction mechanisms. Regarding to this purpose, the main goal of this work is to systematically evaluate five different mechanisms of antioxidant action including hydrogen atom transfer (HAT) / proton coupled electron transfer (PCET), radical adduct formation (RAF), single electron transfer followed by proton transfer (SETPT) and sequential proton loss electron transfer (SPLET). Several thermochemical properties characterizing these mechanisms have been firstly calculated in the gas phase, chloroform and diethyl ether solvents: bond dissociation enthalpy (BDE), ionization energy (IE), electron affinity (EA), proton dissociation enthalpy (PDE), proton affinity (PA) and electron transfer enthalpy (ETE). Secondly, in order to provide more insight into the competition between HAT and RAF mechanisms, potential energy surface of the reactions between all-trans-retinol and hydroperoxyl (HOO•) radical on all possible sites was also performed. Moreover, the hydroxyl


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group (−OH) at the chain end of retinol molecule which is affected by five conjugated double bonds could be dissociated to generate hydroxyl radical (HO•). The prooxidant action of retinol was also elucidated. All calculations have been carried out at (RO)BMK/6-311++G(2d,2p)//BMK/6-31G model chemistries.

COMPUTATIONAL DETAILS Gaussian 09 Rev. E.01 program package was used.19 All calculations were performed by using BMK functional (Boese and Martin’s τ-dependent hybrid functional).20 The optimized geometries of neutral molecules, respective radicals, radical anions and cations and the vibrational frequencies of these species were calculated at the 6-31G basis set. Enthalpy values were evaluated from single point calculations at the basis set of the 6-311++G(2d,2p) and taking into account thermal corrections to enthalpy from the respective 6-31G results. The restricted open shell formalism was used for the radicals. This computational scheme was chosen as the most reliable one based on the comparison of measured and computed BDE values of different small species which have reasonably similar molecular structure with a part of the vitamin A (see Figure S1 and Table S1 of Supporting information). Moreover, theoretical calculations have also indicated that the BMK functional signiﬁcantly outperformed the other popular density functional theory methods for the calculation of bond dissociation enthalpies (BDEs). The accurate predictions depend on the type of the bonds. For example, BMK could reliably predict the C−H BDEs with a mean deviation of ca. 0.5 kcal/mol21, +0.3 kcal/mol for C−C bond22 and +1.8 kcal/mol for C−H bond of N, O, Scontaining mono-heterocyclic compounds. 23


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Three most common antioxidant mechanisms including HAT, SETPT and SPLET which have been widely accepted were investigated in terms of their characteristic thermochemical properties24–26: + Hydrogen atom transfer (HAT) A−H → A• + H • , (BDE)

(R1)

+ Single electron transfer followed by proton transfer (SETPT) A−H → AH • + e , (IE)

(R2)

AH • → A• + H , (PDE)

(R3)

+ Sequential proton loss electron transfer (SPLET) A−H → A + H , (PA)

(R4)

A → A• + e , (ETE)

(R5)

The corresponding thermochemical properties which are characterized the above mechanisms at 298.15 K and 1 atm are calculated as follow27,28: BDEA−H = HA• + HH • − HA−H

(Eq.1)

IE = HAH • + He − HA−H

(Eq.2)

PDE = HA• + HH − HAH •

(Eq.3)

PA = HA + HH − HA−H

(Eq.4)

ETE = HA• + He − HA

(Eq.5)


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Where H is the total enthalpy of the studied species at T = 298.15 K and usually estimated from following expression: H = E0 + ZPE + Htrans + Hrot + Hvib + RT

(Eq.6)

The Htrans, Hrot, and Hvib are the translational, rotational, and vibrational contributions to the enthalpy, respectively. E0 is the total energy at 0 K and ZPE is the zero-point vibrational energy. The influence of the solvents on the thermochemical properties was also studied. The calculation in solvents is based on integral equation formalism of polarizable continuum model (IEF-PCM) at the same level of theory as in the gas phase.29 The enthalpy value for the hydrogen atom (H•) was calculated at the same level of theory. The enthalpies in the gas phase of proton (H+) being 1.4811 kcal/mol (5/2RT, the value of an ideal gas) and of electron (e−) being 0.7519 kcal/mol are commonly accepted.30 Solvation enthalpies of proton and electron in the DEE solvent was calculated using a recent approach31,32 in which it is proposed that a proton or an electron when surrounded by another solvent molecules will bind to a solvent molecule (DEEsol) to yield a charged particle DEE or . And these particles are embedded in a dielectric continuum. Cartesian coordinates of DEE

neutral and charged particles (e.g. anion and cation forms) of diethyl ether (DEE) molecule in the same solvent optimized at the BMK/6-31G level of theory are shown in Table S2 of Supporting information. Furthermore, potential energy surface (PES) of reaction between retinol and HOO• radical was investigated in the gas phase at the (RO)BMK/6-311++G(2d,2p)//BMK/6-31G model chemistries in order to evaluate the HAT and RAF mechanisms. Natural bond orbital (NBO)33 analyses were also


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performed at transition states (TS) to provide more insights into the electron transfer between retinol and the free radical.

RESULTS AND DISCUSSION 1 Optimized molecular structure of retinol Geometry of all-trans-retinol was firstly optimized in the gas phase at the BMK/6-31G level of theory. Figure 1 displays the optimized geometry with the numbered atomic sites of all-transretinol calculated in gas phase at the BMK/6-31G model chemistry. As observed in Figure 1, there is generally a chain of five π(C=C) orbitals in the molecular structure of retinol in which four of them are located in polyene chain (i.e. C10=C11; C12=C13; C14=C15 and C16=C17) with bond lengths of around 1.36 to 1.37 Å, and one is found in the cyclohexenyl ring (i.e. C2=C3 having bond length of 1.36 Å). Moreover, the lengths of five single C–C bonds of the cyclohexenyl ring vary from 1.52 to 1.55 Å. These bond distances around 1.52–1.55 Å show that all the carbon atoms C1, C6, C5 and C4 are in typical sp3 hybridization. And the C2=C3 equilibrium distance (i.e. 1.36 Å) implies that there is a formation of localized double bond between these two atoms. The bond angles of C2–C1–C6 (110.9o), C1–C6–C5 (112.3o), C6–C5–C4 (109.3o) and C5–C4–C3 (113.7o) are slightly different.


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Figure 1: Optimized geometry with the numbered atomic sites of all-trans-retinol calculated at the BMK/6-31G level of theory.

Moreover, the dihedral angle between C2=C3 and C10=C11 is found to be 42.6o. This shows that the polyene chain that contains 4 π(C=C) orbitals is inclined against the plan containing the cyclohexenyl ring and leads to a bent conformation. Thus, the π(C=C) orbitals of the polyene chain and the one of cyclohexenyl ring are not completely conjugated. Moreover, the –OH group of the all-trans-retinol structure is turned down and creates a C17–C18–O–H dihedral angle of −56.6o (Figure 1). And the C18–OH bond length of the conformer 1 is 1.45 Å.

2 Characteristic thermochemical properties for antioxidant activity In order to evaluate the free radical-scavenging activity of retinol via H-atom transfer (HAT) mechanism and its possible pro-oxidant property, bond dissociation enthalpies (BDE) were calculated for either C−H bonds or C−OH, O–H and C–CH3 ones. It was well known that vitamin A is preferably soluble in non-polarized solvent. Indeed, Wilkie extracted vitamin A using chloroform (ε=4.7113) and diethyl ether (ε=4.2400) as solvents.34 In this experiment, the latter was found to have an appreciably higher absorption value than the former. Thus, in the actual study diethyl ether


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(DEE) was evidently chosen to evaluate the influence of solvent on thermochemical properties including BDE. The results of all the thermochemical properties (i.e. BDE, PDE, ETE and PA) calculated in both the chloroform and diethyl ether solvent are resumed in Table S3 of Supporting information.

Table 1: Thermochemical properties (BDE, PDE, PA, ETE) of the retinol in the gas phase (GAS) and in diethyl ether (DEE) solvent calculated at the (RO)BMK/6-311++G(2d,2p)//BMK/6-31G model chemistries, (unit in kcal/mol).

Bonds

PA(a) GAS DEE 353.9 91.5 407.6 138.8 402.5 136.8 401.4 130.5 360.9 96.9 382.7 117.6 374.9 112.6 380.5 117.4 379.0 114.9 386.8 118.0 387.7 117.6 349.4 85.9 368.7 101.3 380.2 111.1 366.9 90.9

ETE(b) GAS DEE 37.3 55.3 4.1 36.8 10.3 37.8 14.0 38.4 35.0 61.3 28.3 55.9 33.5 57.9 29.8 58.3 30.1 56.4 37.2 68.3 37.0 68.8 34.5 59.9 33.3 62.2 25.5 59.9 48.9 87.8

PDE(c) GAS DEE 61.5 −200.6 260.3 −179.9 261.4 −178.9 264.0 −176.2 244.6 −195.4 259.6 −180.1 257.0 −183.1 259.0 −177.8 257.8 −182.3 272.7 −167.2 273.3 −167.2 232.6 −207.7 250.6 −190.1 254.4 −185.7 264.4 −174.9

C4−H C5−H C6−H C7−H C9−H C10−H C11−H C13−H C14−H C15−H C17−H C18−H C19−H C20−H O−H C18−OH C3−CH3 C1−CH3 (a) BDEA−H = HA• + HH • − HA−H (b) PDE = HA• + HH − HAH • (c) PA = HA + HH − HA−H (d) ETE = HA• + He − HA

BDE(d) GAS DEE 77.2 76.7 97.7 97.4 98.8 98.4 101.4 101.1 82.0 81.9 97.0 97.2 94.4 94.2 96.3 99.5 95.2 95.0 110.1 110.1 110.7 110.1 70.0 69.6 88.0 87.2 91.7 91.6 101.8 102.4 69.4 68.1 97.5 97.0 73.8 73.5


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Calculated BDE values in the gas phase as well as in DEE are presented in Table 1. As can be seen, antioxidant capacity via HAT mechanism of retinol is completely decided by C18–H donating site with BDE(C18–H) equal to 70.0 kcal/mol in the gas phase. Thus, retinol may display as a strong antioxidant via HAT mechanism with ∆BDE of −18.3 kcal/mol35 in comparison with the experimental BDE(O−H) value of phenol (i.e. 88.3 ± 0.8 kcal/mol36), a referenced phenolic antioxidant. The other H-donating sites of retinol are located at the C4−H, C9−H and C19−H positions with BDE equal to 77.2, 82.0 and 88.0 kcal/mol in the gas phase, respectively. The lower BDE values of C4−H, C9−H and C18−H bonds result from electron-withdrawing effect of hydroxyl –OH group and the conjugated π−system of polyene chain. It is interesting to note that BDE(O–H) being of 101.8 kcal/mol is much higher than the one of almost BDE(C–H). So, O–H bond cannot contribute to the H–donating capacity of retinol. Furthermore, C1–CH3 bond also shows a significantly low BDE of 73.8 kcal/mol in the gas phase, and this leads to a high possibility to form CH3• free radical from retinol. On the other hand, the lowest bond dissociation enthalpy in the entire retinol molecule is found at C18–OH bond position. Indeed, BDE(C18–OH) is equal to 69.4 kcal/mol in the gas phase. This notably low BDE value can be explained by the negative inductive effect (−I) of four double bonds that are regularly distributed along the long polyene chain. The strong polarization of C18–OH bond may result in the generation of a reactive •OH radicals that can consequently react with biological molecules such as DNAs, lipids and proteins, and causing the oxidative stress.37 Regarding the effect of solvent, it is observed that the low polarity of DEE solvent tends to inconsiderable effect on BDE value. In fact, BDE value is varied about 0.1 to 3.2 kcal/mol in DEE by compared to the one in the gas phase (Table 1). For example, BDE(C18−H) is of 70.0 and 69.6


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kcal/mol in the gas phase and DEE solvent, while BDE(O−H) is equal to 101.8 and 102.4 kcal/mol, respectively. So, the order of bond dissociation enthalpies can be classified as following: BDE (C18–OH) ≈ BDE (C18−H) < BDE (C1–CH3) < BDE (C4–H) < BDE (C9–H). The low BDE(C4−H) value which indicates a strong H-atom donating capacity at C4 position is quite coherent with the previous studies on the provitamin A β-carotene.11,12 On the basis of BDE evaluation, it is generally found that retinol can act in parallel as a strong antioxidant with high H-atom donor capacity, and a strong reactive oxygen species donor (i.e. a prooxidant) in generating OH• reactive free radical.

Electron transfer corresponds to the first step of single electron transfer followed by proton transfer (SETPT) mechanism that is one of the most important actions of antioxidant.27,28 Ionization energy (IE) is the required energy quantity that an isolated atom or molecule in the ground electronic state must absorb to discharge an electron in forming a cation. This quantity characterizes the electron donor capacity of antioxidant molecule. The lower IE value represents the higher antioxidant activity. The adiabatic IE values in the gas phase and DEE solvent were calculated by applying the equation (Eq.2). As a result, adiabatic IE in the gas phase is equal to 6.41 eV (or 147.81 kcal/mol). The error margin is of 0.32 eV in comparison with the experimental IE reported by Katsumata and Ikehata (i.e. 6.73 eV).15 Otherwise, the calculation of IE in DEE at the same level of theory shows a lower value of 5.28 eV due to the reason that the low polarity of DEE facilitates electron-releasing process in forming radical cation. On the other hand, by comparing to the standard IE value of phenol in the gas phase (i.e. 8.49 eV or 195.79 kcal/mol18), the absolute difference (∆IE) is equal to – 47.98


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kcal/mol. Thus, retinol may be a strong antioxidant via SETPT mechanism based on its electrondonating capacity with the ∆IE around −45 kcal/mol.35 The electron transfer mechanism is evaluated not only based on its electron-donor capacity but also electron-acceptor one which is characterized by electron affinity (EA) quantity.38 The higher EA value represents the stronger electron acceptor capacity. Consequently, adiabatic EA values calculated in the gas phase and the DEE solvent at the (RO)BMK/6-311++G(2d,2p) model chemistries are equal to 0.61 and 1.76 eV, respectively. These results are coherent with the ones obtained by Martinez et al16. Moreover, the adiabatic IE and EA of HOO• radical, one of representative reactive free radical, are also evaluated. As a result, the IE and EA values in the gas phase are 12.05 and 0.90 eV, respectively. Similarly, IE and EA in DEE solvent are equal to 9.53 and 3.43 eV, respectively. In applying full electron donator acceptor map (FEDAM)38,39 which allows for a qualitative identification of the relative electron-donor / -acceptor capability among antioxidant molecule and free radical, it can be seen that retinol seems to be a good electron donator but bad electron acceptor. In fact, retinol has a tendency to transfer one electron to the HOO• radical in both the gas phase and the DEE solvent. The FEDAM of retinol and HOO• radical can be found in Figure S2 of Supporting information. The second step of SETPT mechanism consists in deprotonation of the formed radical cation which is described by proton dissociation enthalpy (PDE). The PDE quantity shows thermodynamically preferred C−H or O−H site of the radical cation for deprotonation. The PDEs calculated in the gas phase and in the DEE solvents by BMK method are reported in Table 1. As a result, the most favorable deprotonation position are found at C18, C4, C9 and C20 atoms with PDE value of 232.6, 239.8, 244.6 and 254.4 kcal/mol, respectively. The order is in line with the one of BDE as shown


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previously. Moreover, the results demonstrated that PDEs calculated in the solvent are significantly lower than the corresponding values in the gas phase. For example, PDE value at C18 reduces from 232.6 kcal/mol in the gas phase to −207.7 kcal/mol in DEE. The lower PDEs in the DEE solvent may result from high solvation enthalpy of proton (i.e. −235.7 kcal/mol) in such non-polar solvent like DEE. The similar results are obtained with the chloroform solvent as presented in Table S4 of Supporting information. This phenomenon was also confirmed by different works in literature.28,40 Thus, it indicates that the deprotonation ability of retinol is more preferable in solvent than in the gas phase.

The sequential proton loss electron transfer (SPLET) also displays as an important antioxidant mechanism which is initiated by the first step of proton loss in forming retinol anion (R4). This important step is characterized by proton affinity (PA) value (Eq.4). The lower PA value is, the higher antioxidant activity via SPLET mechanism is. The PAs at different carbon atoms calculated in the gas phase and the DEE solvent are resumed in Table 1. It is observed that the PAs at different carbon positions follow an increasing order: C18−H < C4−H < C9−H < O−H < C19−H with values equal to 349.4, 353.9, 360.9, 366.9 and 368.7 kcal/mol, respectively. This trend is quite coherent with the one of BDE. Additionally, a remarkable effect of the solvent on PAs is noted due to the higher solvation enthalpy of proton in solvent compared to that in the gas phase. In fact, PAs at C18−H position reduce significantly from 349.4 in the gas phase to 85.9 kcal/mol in DEE. In comparing PAs with IE and BDEs in both phases, it is clearly observed that the BDEs are always lower than corresponding values of PAs and IE. This indicates that HAT mechanism is more preferred than both SPLET and SETPT ones.


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Single electron releasing process from the retinol anion which is symbolized by electron transfer enthalpies (ETE) (Eq.5) consists in the second step of SPLET mechanism. Noting that in both the gas phase and the DEE, ETE values are always lower than that of IE. For example, the lowest ETEs obtained in the gas phase and the DEE are 4.1 and 36.8 kcal/mol, respectively, while the IEs are equal to 6.41 eV (148.8 kcal/mol) and 5.28 eV (121.8 kcal/mol). It means that single electron donation from the anionic form is more preferable than from its neutral one. Moreover, the solvent effect tends to a significant increase of ETE which indicates that the DEE solvent is unfavorable for the electron transfer process. Thus, on the basis of all the thermochemical properties considered in this section, HAT mechanism is thermodynamically preferred in both gas phase and solvent media. The most favorable H-atom donor sites are located at C18, C4 and C9 carbon atoms. However, the antioxidant capacity via HAT and the prooxidant capacities of retinol in yielding HO• radical may be in concurrence owing to the quasi-equal values of BDE (C18–OH) (69.4 kcal/mol) and BDE (C18−H) (70.0 kcal/mol).

3 Interaction of retinol with HOO• radical In order to provide more insight into the free radical quenching capacity of retinol, its interaction with HOO• radical which consists in a representative reactive radical is evaluated in the gas phase. Three types of reaction are considered: electron transfer (ET), H-atom transfer (HAT) and radical adduct formation (RAF). The relative importance of these reaction pathways depends on several factors such as: nature of free radical, structural features of antioxidant molecule, and in biological systems, its locations and orientations with membrane.41


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Potential energy surface (PES), transition state structures and singly occupied molecular orbital (SOMO) were systematically investigated for HAT and RAF mechanisms. Natural bond orbital (NBO) analyses realized at transition states (TSs) were also performed to evaluate electron transfer mechanism during these reactions. 3.1 Electron transfer reactions Adiabatic reaction enthalpies (∆H) and Gibbs free energies (∆G) of the single electron transfer reaction between retinol and HOO• radical are considered in order to study the associated energy evolution. The electron-donating/-accepting process between retinol (Ret) and HOO• radical may occur as follow38: Ret + HOO• → Ret • + HOO ,

(R6)

Ret + HOO• → Ret • + HOO ,

(R7)

For reaction (R6), the adiabatic reaction enthalpy and Gibbs energy are calculated as follow: ∆H = HRet • + HHOO − HRet + HHOO• ,

(Eq.7)

∆G = GRet • + GHOO − GRet + GHOO•

(Eq.8)

Whereas for reaction (R7), the calculations correspond to: ∆H !!"#$ = HRet • + HHOO − HRet + HHOO• ,

(Eq.9)

∆G!!"#$ = GRet • + GHOO − GRet + GHOO• ,

(Eq.10)


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As a result, ∆H and ∆G values of the electron-donnating process in the gas phase are

equal to 125.7 and 125.5 kcal/mol, respectively. These values in the DEE solvent are of 46.2 and 61.9 kcal/mol, respectively. In addition, ∆H !!"#$ and ∆G!!"#$ values of the electron-accepting process in the gas phase are 264.0 and 266.3 kcal/mol, respectively, while these values in the DEE solvent are 178.6 and 195.2 kcal/mol, in turn. On the basis of these results, it can be noted that (i) the electron-donating process is thermodynamically more favorable than the electron-accepting one in both media with two times lower values of ∆H and ∆G; (ii) the electron-donating and -accepting processes are more feasible in the DEE solvent than in the gas phase; and (iii) the electron transfer processes are strongly endogenic with high positive values of reaction enthalpies and Gibbs energies.

3.2 Hydrogen Atom Transfer reactions On the basis of BDE calculations, it is observed that the three weakest C−H bonds are found at the position of C4−H (BDE=77.2 kcal/mol), C9−H (BDE=82.0 kcal/mol) and C18−H (BDE=70.0 kcal/mol). Thus, the reactions of retinol with HOO• radical were investigated at these three positions to evaluate H-atom donation process. All structural optimizations were firstly performed in the gas phase at the BMK/6-31G level of theory and single point energy calculations were both obtained in the gas phase and the DEE solvent at the (RO)BMK/6-311++G(2d,2p). The PES of these reactions in the gas phase is displayed in Figure 2. Cartesian coordinates and molecular enthalpies of all transition states of HAT reactions are presented in Table S4 of Supporting


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information. And IRC plots for these transition states at the BMK/6-31G level of theory are also shown in Figure S3 of Supporting information. In general, the reaction occurs as follow (R8): Ret(−H) + HOO• Ret⋅⋅⋅H⋅⋅⋅OOH Ret• + HOOH

(R8)

Figure 2: PES of HAT reactions between retinol and HOO• radical calculated in the gas phase at the (RO)BMK/6-311++G(2d,2p)//BMK/6-31G model chemistries

At the initiation step of H−atom abstraction reaction, the O2- atom of HOO• free radical approaches the H- atom of the weak C−H bonds of retinol to form a hydrogen bond in a reactant intermediate


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(INT-1) with energy lying below the separated reactants by −0.30, −4.42 and −7.21 kcal/mol corresponding to C4−H, C9−H and C18−H, respectively. Then, the weak H−atom is separated from retinol molecule in forming chemical bond with HOO• radical via transition state (TS) lying energy barrier above reactants by 12.94, 13.38 and 9.16 kcal/mol for reaction at C4−H, C9−H and C18−H positions, respectively. The studied C−H bonds are elongated in this state with the bond distance of 1.27, 1.28 and 1.26 % assigning to C4⋅⋅⋅H, C9⋅⋅⋅H and C18⋅⋅⋅H bond, respectively (Figure 3). At the same time, the O2⋅⋅⋅H bond length is equal to 1.31, 1.30 and 1.32 %, in sequence.

Figure 3: Optimized TS structure and SOMO distribution of TSs of H-atom abstraction reactions between retinol and HOO• radical


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Passing through the transition states, the formation of HOOH molecule and of Ret• radical is recognized in a product intermediate (INT-2) (Figure 2) with relative energy of −9.42, −5.25 and −16.90 kcal/mol below the reactants for C4−H, C9−H and C18−H, respectively. Finally, these product intermediates are taken away to form separated products (PROD) with relative energies equal to −7.87, −3.48 and −14.83 kcal/mol below the reactants, respectively.

Table 2 summarizes reaction enthalpies and Gibbs energies (in kcal/mol, at 298 K) for HAT and RAF reactions of the retinol with HOO• radical in the gas phase and the DEE solvent. It is shown that all the HAT reactions are exergonic and thermodynamically favorable. Indeed, Gibbs energy (∆G) of HAT reactions at C4−H, C9−H and C18−H positions are of −7.67, −3.86 and −14.54 kcal/mol, respectively (Table 2). Thus, retinol can scavenge HOO• radical through this mechanism with an increasing reactivity as follow: C9−H < C4−H < C18−H. The non-polar diethyl ether solvent slightly lowers the reaction enthalpies and Gibbs free energies of the HAT reactions (Table 2). Relative energies of all the reaction states during HAT reactions calculated in the gas phase and the DEE solvent are resumed in Table S5 of Supporting information. As can be seen in Table 2, the relative enthalpies (∆H) of the HAT reactions are slightly lowered in the DEE solvent. For example, the ∆H value for the reaction at C4−H is reduced from −7.87 to −8.61 kcal/mol in the gas phase and the DEE solvent, respectively. Moreover, the HAT reactions are more exergonic in the DEE than in the gas phase. The ∆G value for reaction at C4−H is decreased from −7.67 to −8.41 kcal/mol, in sequence.


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Table 2: Relative reaction enthalpies (∆H) and relative Gibbs free energies (∆G) (at T = 298 K) for HAT and RAF of the retinol with HOO• radical in the gas phase and the DEE solvent.

Reactions HAT reactions C4−H + OOH C9−H + OOH C18−H + OOH RAF reactions C2=C3 + OOH C10=C11 +OOH C12=C13 + OOH C14=C15 +OOH C16=C17 +OOH

∆H, kcal/mol DEE GAS

∆G, kcal/mol DEE GAS

−7.87 −3.48 −14.83

−8.61 −4.01 −15.77

−7.67 −3.86 −14.54

−8.41 −4.39 −15.48

−27.89 −15.40 −17.00 −16.97 −21.78

−27.91 −15.49 −17.68 −17.30 −22.33

−24.46 −13.72 −14.54 −14.68 −19.14

−24.75 −13.95 −15.39 −15.18 −19.91

3.3 Radical Adduct Formation reactions The addition reaction of the HOO• radical to C=C double bond on the polyene chain of the retinol occurs generally through two steps (R9) and (R10):

In the first step, HOO• free radical is added to α or β position of double C=C bond to form C−OOH bond. The epoxidation reaction then takes place in the second step in which the oxygen atom closes


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the epoxide ring in separating HO• radical. So, the whole reaction process takes place via two transition states (TS-1 and TS-2) and three intermediate states (INT-1, INT-2 and INT-3) before arriving the product (PROD) (Figure 4). Figure 4 displays PES of all RAF reactions between HOO• radical and the retinol calculated in the gas phase at the (RO)BMK/6-311++G(2d,2p)//BMK/6-31G model chemistries. Cartesian coordinates and molecular enthalpies of all transition states of RAF reactions are resumed in Table S7 of Supporting information. IRC plots for these transition states at the BMK/6-31G level of theory in the gas phase are also displayed in Figure S4 of Supporting information. As can be seen, the reaction pathways are initiated by an approach of the free radical to carbon center of the retinol to form the first intermediate complex (INT-1) with energy lying below the separated reactants from −2.41 kcal/mol for reaction at C14=C15 to −6.62 kcal/mol in case of C16=C17. The HOO• radical then forms a chemical bond with the carbon center of the retinol via the first transition state (TS-1) which is characterized by an energy barrier varied from 7.12 to 15.49 kcal/mol for the reaction at C16=C17 and C10=C11 bonds, respectively (Figure 4). And C⋅⋅⋅O2 bond distance is equal to 2.12 and 2.03%, respectively, whereas the bond angle ∠C16-C17-O2 is of 99.1o and ∠C10-C11-O2 equal to 98.8o (Figure 5). The molecular systems are then stabilized by passing the second intermediate state (INT-2) with relative energy lying by −21.27 to −2.77 kcal/mol below the initial reactants for reaction at C2=C3 and C10=C11 bonds, respectively.


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Figure 4: PES of addition reactions of HOO• radical to π-bonds on retinol molecule calculated in the gas phase at the (RO)BMK/6-311++G(2d,2p)//BMK/6-31G model chemistries


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Figure 5: Optimized TS structures of all RAF reactions between the retinol and HOO• radical calculated in the gas phase at the BMK/6-31G level of theory

In the second transition state of the reaction pathway (TS-2), the O2 atom tends to form chemical bond with the remained carbon center of C=C double bond in establishing an epoxide bridge. The TS-2 states are described by energy barriers of 0.45 to 13.55 kcal/mol for the reactions at C16=C17 and C10=C11 bond, respectively. And the bond distances of C16⋅⋅⋅O2 and C10⋅⋅⋅O2 are equal to


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1.93 and 1.96%, while bond angles ∠C16-C17-O2 and ∠C10-C11-O2 are now reduced to 82.0 and 83.8o, respectively. Moreover, the O2⋅⋅⋅O3 bond of HOO• radical is elongated and HO• radical is released at the same time with the closure of epoxide ring. The molecular system is stabilized at the third intermediate state (INT-3) before passing the final separated products (PROD) including the retinol epoxide and the hydroxyl radical. The INT-3 state is characterized by relative energies lying from −32.76 to −19.29 kcal/mol underneath reactants for the reactions at C2=C3 and C10=C11 bond, respectively. Similarly, the relative energy of products is equal to −27.89 to −15.41 kcal/mol. It is noted that the HOO• radical addition to C2=C3 double bond on the cyclohexenyl ring of the retinol is the most favorable with the lowest values of ∆H and ∆G, i.e. −27.89 and −24.46 kcal/mol, respectively (Table 2). This theoretical observation is quite coherent with the one experimentally obtained on β-carotene.9 Moreover, as observed for the HAT process, all the RAF reactions are slightly more feasible and exergonic in the DEE solvent than in the gas phase with the lower ∆H and ∆G values. Furthermore, the RAF reactions are more strongly exergonic than the HAT ones (Table 2) with relative Gibbs free energy (∆G) in the gas phase varies from −24.46 to −13.72 kcal/mol for the reactions at C2=C3 and C10=C11 bond, respectively. Whereas, the ∆G values calculated in the DEE solvent varies from −24.75 to −13.95 kcal/mol for the similar reactions. This leads to conclude that the free radical scavenging activity of the retinol based on the RAF reactions is thermodynamically more feasible. 3.4 SOMO and NBO analyses of HAT and RAF reactions In this section, singly occupied molecular orbital (SOMO) and natural bond orbital (NBO) analyses are investigated to provide more insight into mechanism of HAT and RAF reactions between the


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retinol and HOO• radical. Figure 3 and 6 display SOMO density surfaces of HAT and RAF transition states, respectively. Table 3 resumes NBO analysis results of the transition states for both types of reaction.

Figure 6: SOMO distribution of TSs of all RAF reactions between the retinol and HOO• radical


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It is widely accepted that the hydrogen atom transfer (HAT) and the proton coupled electron transfer (PCET) consist in two distinct mechanisms in which an electron and a proton are transferred in a single step.42 The electron and the proton are transferred together in HAT mechanism as a hydrogen atom, while they are transferred between different sets of orbitals in PCET mechanism.43 It is predicted that PCET reactions can occur in a pre-reaction hydrogenbonded complex, but also in lone pair–π and π – π interactions.43–46 Since reactants and products of both the reactions are similar, analysis of their transition state is necessary to distinct between them. Martinez et al proposed that the analysis of SOMO of TS seems to be a reliable criterion to differentiate between HAT and PCET processes.47 It is observed that SOMO of HAT TS has significant atomic orbitals density that is distributed along, or nearly along H-transition vector. Conversely, SOMO of PCET TS involves p orbitals that are orthogonal to the transition vector.43

Table 3: NBO analysis of transition states of reactions between retinol and HOO• radical Reactions C4−H + HOO•

C9−H + HOO•

C18−H + HOO•

C2=C3 + HOO• (TS-1)

Donor NBO (i) LP*(1) C4 LP (2) O2 LP (3) O2 LP*(1) C9 LP (3) O2 LP (2) O2 LP*(1) C18 LP (3) O2 LP (2) O2 σ(1) C3 - O2 LP*(1) C2 LP(2) O3

Acceptor NBO (j) LP*(1) H LP*(1) H LP*(1) H LP*(1) H LP*(1) H LP*(1) H LP*(1) H LP*(1) H LP*(1) H LP*(1) C2 σ*(1) C3 - O2 σ*(1) C3 - O2

E(2), kcal/mol 313.78 23.31 126.16 307.30 133.09 23.10 345.14 132.07 21.63 42.25 38.94 14.09


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C2=C3 + HOO• (TS-2) C10=C11 + HOO• (TS-1)

C10=C11 + HOO• (TS-2) C12=C13 + HOO• (TS-1)

C12=C13 + HOO• (TS-2)

C14=C15 + HOO• (TS-1)

C14=C15 + HOO• (TS-2)

C16=C17 + HOO• (TS-1)

C16=C17 + HOO• (TS-2)

LP*(1) C2 σ(1) O2 - O3 σ(1) C11 - O2 LP*(1) C10 LP (2) O3 LP*(1) C10 LP (2) O2 σ (1) C13 - O2 LP*(1) C12 LP (2) O3 LP*(1) C12 σ(1) O2 - O3 σ(1) C13- O2 LP(2) O3 σ (2) C12 - C13 LP*(1) C14 LP (1) C15 LP (1) C15 LP (2) O2 LP (1) O2 σ (1) O2 - O3 LP (2) O3 LP*(1) C16 σ*(1) C17 - O2 LP*(1) C16 σ (1) O2 - O3 σ (1) C17 - O2 LP (2) O2 LP (1) O2

σ*(1) O2 - O3 LP*(1) C2 LP*(1) C10 σ*(1) C11 - O2 σ*(1) C11 - O2 σ*(1) O2 - O3 LP*(1) C10 LP*(1) C12 σ*(1) C13 - O2 σ*(1) C13 - O2 σ*(1) O2 - O3 LP*(1) C12 LP*(1) C12 LP*(3) O2 LP*(1) C14 σ*(2) C12 - C13 LP*(1) C14 σ*(2) C16 - C17 LP*(1) C14 LP*(1) C14 LP*(1) C14 σ*(1) C17 - O2 σ*(1) C17 - O2 LP*(1) C16 σ *(1) O2 - O3 LP*(1) C16 LP*(1) C16 LP*(1) C16 LP*(1) C16

10.11 6.90 36.79 26.47 13.08 7.08 7.68 39.13 29.58 12.54 8.54 8.33 9.98 31.04 37.58 16.95 244.47 14.96 5.87 5.04 11.12 17.20 49.60 43.06 9.04 11.96 9.87 4.37 5.32

As can be seen in Figure 3, the SOMO in all transition states has a node plane at shifting H-atom and it is mostly localized at C⋅⋅⋅H⋅⋅⋅O2 transition vectors which is corresponding to HAT transition states. NBO analysis at TS indicates that two lone pairs of electron on O2 atom of HOO• radical are transferred to the vacant orbital on donated H-atom during HAT process. For example, the second and third lone pairs of electron on O2 atom, LP(2) O2 and LP(3) O2, are transferred to the


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unoccupied antibonding orbital on H-atom, LP*(1) H, with stabilization energies of 21.63 and 132.07 kcal/mol, respectively, during the reaction of HOO• radical with the retinol at C18−H position. On the reverse trend, the unoccupied antibonding orbital on C18, LP*(1) C18, interacts with the one on H-atom, LP*(1) H, with stabilization energy equal to 345.14 kcal/mol. This indicates a degeneration of H-atom from C18−H bond toward O2 atom to form new O2−H bond. The orbital interaction is quite similar to the other studied HAT reactions. Regarding RAF reactions, the observation on SOMO shows that 2p orbitals on O2 and O3 atoms have high tendency to overlap with π−orbital on the C2=C3 double bond. NBO analysis demonstrates that the σ bonding orbital of formed C3−O2 bond, σ(1) C3-O2, donates electrons to the unoccupied antibonding orbital on C2 atom, LP*(1) C2, with stabilization energy equal to 42.25 kcal/mol. Moreover, electron is also transferred from the second lone pair of electron on O3 atom of the radical, LP(2) O3, to the σ antibonding orbital on C3−O2 bond, σ*(1) C3-O2, with stabilization energy of 14.09 kcal/mol. And the interaction between two unoccupied orbitals, LP*(1) C2 and σ*(1) C3-O2, is also recognized with stabilization energy of 38.94 kcal/mol (Table 3). Thus, the electron transfer between 2p orbital on O2 and O3 atoms with π-orbital of the C2=C3 double bond tends to form a new C3−O2 σ-bond after passing the first transition state TS-1. At the second TS of the reaction (TS-2) between HOO• radical with retinol at the C2=C3 bond, an overlap between σorbital of O2−O3, σ(1) O2-O3, with unoccupied orbital on C2 atom, LP*(1) C2, is observed with stabilization energy of 6.90 kcal/mol. Furthermore, the σ antibonding orbital of O2−O3, σ*(1) O2O3, is also overlapped with LP*(1) C2 with an energy of 10.11 kcal/mol (Table 3). This process can also be observed on SOMO density surfaces (Figure 6).


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It is the overlapping between the orbital on O2−O3 with the unoccupied one on C2 atom that results in formation of new bond C2−O2 to complete the epoxidation process as the second step of RAF reactions. The reactions at other π-bonds on the polyene chain of retinol take place with quite similar manner.

CONCLUDING REMARKS The radical scavenging properties of all-trans-retinol have been evaluated in the gas phase and diethyl ether solvent. The different thermochemical properties (BDE, IE, EA, PDE, PA and ETE) which characterize the antioxidant mechanisms (HAT, SETPT, and SPLET) were calculated. The potential energy surfaces of HAT and RAF reactions between retinol and hydroperoxyl radical were also investigated. Several findings can be noted. HAT mechanism is more predominant than SETPT and SPLET ones in both the gas phase and the DEE solvent. The H-atom donor capacity of retinol is the most preferred at C18−H, C4−H and C9−H positions with the BDEs of 70.0, 77.2 and 82.0 kcal/mol in the gas phase, in turn. In addition to be a strong antioxidant via HAT, retinol also display as a prooxidant in yielding hydroxyl radical owing to the lowest BDE of C18−OH bond being 69.4 kcal/mol in the gas phase. The non-polar DEE solvent slightly lowers the BDEs of the retinol. Retinol acts as good electron donor but bad electron acceptor in interacting with HOO• radical. The electron-donating and -accepting processes are more preferred in the DEE solvent than in the gas phase. And the electron transfer (ET) processes are strongly endogenic with highly positive values of reaction enthalpy and Gibbs free energy.


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The PA evaluated along the retinol molecule follows the same tendency with BDE with an increasing order C18−H, C4−H and C9−H. It means that at these carbon positions, retinol may act as antioxidant by donating H-atom (HAT) or proton (SPLET). All the HAT reactions of the retinol and HOO• radical are exergonic and thermodynamically favorable with negative Gibbs free energy. This type of reactions is more exergonic in the DEE than in the gas phase. The singly occupied molecular orbitals evaluated at TSs confirm that the H-atom transfer process really takes place. The addition reaction of HOO• radical to double bonds in the cyclohexenyl ring (at the C2=C3 position) is the most favored and this is coherent with the result reported in litterature9. The RAF reaction at C16=C17 position being nearby HO group at the chain end is also preferred. Moreover, RAF mechanism is more predominant than ET and HAT ones, and all the radical scavenging reactivity via RAF reactions is strongly exergonic and thermodynamically feasible. NBO analyses at TSs shows that different lone pairs of electron on O2 atom of HOO• radical are donated to the unoccupied antibonding orbital of transferred H-atom during HAT process. Additionally, the strong overlapping between 2p orbitals on O atoms of the radical with π−orbital on double bond of retinol is recognized during RAF reactions. The obtained results contribute to a better understanding of the antioxidant actions of all-transretinol which might potentially allow preventing oxidative damage in living systems.


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ASSOCIATED CONTENT Supporting Information. Validation of the used DFT/BMK methods; Cartesian coordinates of neutral, anion and cations forms of DEE in the same solvent; thermochemical properties of the retinol in CHLO, DEE and in the gas phase; FEDAM of the retinol and HOO• radical; Cartesian coordinates, enthalpies and IRC of the TS of all HAT reactions and RAF ones.

List of Supporting Information Figure S1. Molecular structure of small species used for validating the computational methods. Table S1. Comparisons of experimental and calculated BDE(C–C), BDE(C–H), BDE(C–OH) and BDE(O–H) values of small species using different DFT methods at 6-311++G(2d,2p) basis set. Table S2. Cartesian coordinates of neutral, anion and cation forms of diethyl ether (DEE) in the same solvent optimized at BMK/6-31G level of theory. Table S3. Thermochemical properties (BDE, PDE, PA, ETE) of the retinol in the gas phase (GAS), chloroform (CHLO) and diethyl ether (DEE) solvent calculated at (RO)BMK/6311++G(2d,2p)//BMK/6-31G model chemistries, (unit in kcal/mol) Figure S2. FEDAM of retinol and HOO• radical in the gas phase and DEE solvent Table S4. Cartesian coordinates and molecular enthalpies of all transition states of HAT reactions optimized at RO(BMK)/6-311++G(2d,2p)// BMK/6-31G level of theory. Figure S3. IRC plots for all transition states of HAT reactions of HOO• radical with retinol at BMK/6-31G level of theory in the gas phase Table S5. Relative energies (in kcal/mol) of H-atom transfer and radical adduct formation reactions between the vitamin A and HOO• radical in the gas phase (GAS) and diethyl ether (DEE) solvent at (RO)BMK/6-311++G(2d,2p)//BMK/6-31G model chemistries Figure S4. IRC plots for all transition states of RAF reactions of HOO• radical with retinol at BMK/6-31G level of theory in the gas phase


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AUTHOR INFORMATION *Corresponding authors: [email protected] (Duy Quang Dao); [email protected] (PCN)

ACKNOWLEDGMENT This research is funded by Vietnam National Foundation for Science and Technology Development (NAFOSTED) under grant number 104.06-2015.09.

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Is Vitamin A an Antioxidant or a Pro-oxidant? - The Journal of Physical

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