Research Article On the Importance of Water Molecules in...

Research ArticleOn the Importance of Water Molecules in the Theoretical Studyof Polyphenols Reactivity toward Superoxide Anion

Laure Lespade

Institut des Sciences Moleculaires UMR 5255 Universite de Bordeaux 351 cours de la Liberation 33400 Talence France

Correspondence should be addressed to Laure Lespade llespadeismu-bordeaux1fr

Received 9 June 2014 Accepted 24 July 2014 Published 14 September 2014

Academic Editor John R Sabin

Copyright copy 2014 Laure LespadeThis is an open access article distributed under theCreativeCommonsAttribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Numerous studies have shown the benefits of a diet rich in fruits and vegetables These benefits are partly due to the radicalscavenging properties of polyphenols contained in fruits and vegetables since polyphenols can fight against an excess of radicalswhich goes along inflammation in a certain number of diseases This pathological state called oxidative stress results from theaerobic condition of human organism when OH radical hydrogen peroxide superoxide anion or peroxynitrite is produced inexcess If hydrogen peroxide is easily handled by human defense against radicals the other radicals can cause damage to biologicalconstituents like lipids cell membranes and other biomolecules This paper is devoted to the theoretical study of the interactionof superoxide anion (O

2

∙minus) with a very potent radical scavenger 12468-pentahydroxynaphthalene The importance of hydrationof superoxide radical for the reactivity is analyzed Potential energy surfaces (PES) are calculated for different number of watermolecules around the radical and it is shown that the transition barrier vanishes when complete hydration with six water moleculesis explicitly handled The nature of the reactivity is determined by using the natural bond orbital (NBO) analysis

1 Introduction

The production of radicals in the body is a consequenceof the aerobic metabolism of the organism [1] Free radicalreactive oxygen species (ROS) can be produced duringmitochondrial dysfunction or in pathophysiological condi-tions Superoxide anion is one of the most important andbiologically relevant ROS radicals in living organisms It isformed from one-electron reduction of oxygen It is muchless reactive and much more selective than hydroxyl radicalwhich reacts with most biomolecules at a nearly diffusion-controlled rate (1010Mminus1sminus1) The lifetime of superoxide inbiological systems is typically a few seconds It can reactwith another superoxide anion to give hydrogen peroxide orwith nitric oxide to form a very potent oxidant peroxynitriteSuperoxide anion is also produced by an enzyme NADPH inphagocytes to kill invading pathogens Although O

2

∙minus is nota strong microbicidal [1] it is essential for bacterial killingIt is a source of hydrogen peroxide H

2O2 which in presence

of released iron could form hydroxyl radical via the Fentonmechanism

Fe2+ +H2O2997888rarr Fe3+ +OH∙ +OHminus (1)

or the Haber-Weiss mechanism

O2

∙minus+H2O2

Fe3+997888997888997888rarr OH∙ +OHminus +O

2(2)

There are several pathological cases where overproduction ofsuperoxide leads to tissue damage in particular in ischemicepisodes [2] The deprivation of oxygen a consequence ofsevere restriction in blood flow leads to the conversion ofxanthine dehydrogenase an enzyme which participates inthe degradation of purines in another form of enzymexanthine oxidase During the degradation of xanthine inuric acid xanthine dehydrogenase uses a cofactor NADP+as an electron acceptor In xanthine oxidase some sulphurbridges are broken and the cavity whereNADPH should reactis partly obstructed Only small molecules like oxygen canenter and capture electrons to form superoxide and hydrogenperoxide During the reperfusion after an ischemic episodexanthine oxidase transforms oxygen into superoxide whichleads to oxidative stress and tissues damage One way toprotect against this damage is to use scavengers of peroxideand inhibitors of the enzyme

Oxidative stress is also the consequence of depletion ofdietary antioxidants (vitamins A C and D flavonoids and

Hindawi Publishing CorporationJournal of eoretical ChemistryVolume 2014 Article ID 740205 9 pageshttpdxdoiorg1011552014740205

2 Journal of Theoretical Chemistry

carotenoids) and micronutrients indispensable to the properfunctioning of antioxidants enzymes A lot of studies havetried to characterize the antioxidant properties of naturalcompoundsTheflavonoid family has been particularly inves-tigated These compounds can scavenge radicals either byhydrogen atom transfer or by charge transfer

FOH + R∙ 997888rarr FO∙ + RH (3)

FOminus + R∙ 997888rarr FO∙ + Rminus (4)

In the first case the reactivity is governed by the OH bonddissociation enthalpy (BDE) If the flavonoid BDE is lowerthan the BDE of RH the reaction is favored

In the second case the thermodynamic parameter whichdescribes the reaction is the electron transfer enthalpy (ETE)[3]

The calculated BDE of flavonoids correlates very wellwith the inhibition of low-density lipoprotein oxidationindicating that lipid peroxide scavenging [4] is done byhydrogen atom transfer with no barrier On the contrarysuperoxide anion scavenging by these natural moleculesseems more complex Experimental results [5ndash8] sometimesgive opposite results even in nonenzymatic assays wherethere is no competition with enzyme inhibition Theoreticalcalculations can be of some help in the study of the conditionsof reactivity Dhaouadi et al [9] have performed a DFTstudy of the reaction of quercetin with superoxide radicalTheir calculations were done in gas phase The reactionhad no barrier but was characteristic of a proton transferinstead of hydrogen atom transfer as expected Due to theanionic character of superoxide radical one may questionthe importance of superoxide hydration in the reactivityThis is the purpose of this paper studying the influenceof explicit hydration on the reactivity of polyphenols withsuperoxide anion In order to simplify the study the reactionhas been analyzed with one of the best known radicalscavengers 12468-pentahydroxynaphthalene (PNH) Thiscompound has been designed [10] for its very low BDE of57 kcalmol (vitamin C 68 kcalmol quercetin 72 kcalmol)It is a weak acid the deprotonated form is preponderant atphysiological pH The study will be limited to the reaction atthe most labile hydroxyl group of PNH It is divided into twoparts first the electronic and free energies of reactants andproducts are calculated with an increasing number of watermolecules around superoxide radical The reaction consistsin a stretching of the OO bond of superoxide and a largeincrease of the OH bond length of the hydroxyl group Tocharacterize the reaction potential energy surfaces (PES)have been constructed in function of these two coordinatesThen in a second part analysis of natural bond orbitals triesto give a better insight into the conditions of spontaneousreactions

2 Methods

The attack of superoxide anion on 12468-pentahydroxyn-aphthalene (PHN) has been studied only on the most labilehydrogen on position 4 [10] by modifying the solvation of

superoxide anion with explicit water molecules In additionthe effect of the bulk solvent has been taken into accountthrough the field polarized continuum model (PCM) whichmimics the bulk solvent by creating a solute cavity via a setof overlapping spheres [11 12] The importance of the watermolecules surrounding superoxide anion has been studiedby varying their number and positions It was impossibleto investigate all the possible configurations for the watermolecules Two different configurations among the moststable ones have been chosen in order to determine theimportance of geometry of the reactants

This study has been carried out by using Gaussian 09package [13] within density functional theory Two function-als have been tested cam-B3lyp developed by Yanai et al[14] which contains long range corrections and wB97XD[15] which includes empirical dispersion cam-B3lyp has theadvantage to lead to a better localization of the charges whichcan avoid artifacts in calculations with superoxide anions Inthe reactivity studied in this paper the two reactants havea negative charge and there is no possibility of a chargetransfer artifact This is why it has been possible to test alsothe wB97XD functional to evaluate the possible dispersioneffects All the calculations have been performed by using the6-311+G(dp) basis set

Harmonic vibrational frequencies of reactants and prod-ucts were calculated with the same functional and basis set inorder to verify that they are true minima

The potential energy surfaces (PES) describing the reac-tivity of 12468-pentahydroxynaphthalene with superoxideanion solvated by a certain number of water molecules havebeen obtained by calculating the electronic energy of theadducts in function of the two coordinates involved in thereaction the OH bond length of the hydroxyl bond and theOO bond length of superoxide anionThe electronic energieshave been obtained by optimizing the geometry with thesetwo fixed coordinates The OO bond was varied from 131 A(or 132 A depending on adduct initial geometry) to 147 Aby step of 003 A The OH bond was varied from 099 A to18 A by step of 01 A Near the transition state the steps werelowered to 005 A

In order to characterize precisely the transition statethe different PES have been parameterized with the Surfersoftware [16] by using the local polynomial gridding methodwith polynomial of order 3

3 Results

31 Effect of Explicit Hydration of Superoxide Anion on theBarrier to Reaction Table 1 displays the electronic energiesdifferences between the reactants and the transition states orthe products Even if there are some discrepancies betweenDFT functional results or water molecules conformationsa general trend emerges the addition of water moleculesstabilizes the products and decreases the barrier to reactionWith six or more molecules there is no more barrier thereaction is barrierless

No Water Molecule Figure 1 displays the geometry of reac-tants andproductswith nowatermoleculeThe conformation

Journal of Theoretical Chemistry 3

Table 1 Electronic energies differences between the reactants and transition state or products For two to four water molecules twoconformations have been tested The free energies are given relative to the most stable reactants

Number of water molecules Transition state Products Relative reactants free energy Relative products free energy

0 in cam-B3lyp 15 kcalmol minus4 kcalmol minus34 kcalmolin wB97XD 15 kcalmol minus4 kcalmol minus31 kcalmol

1in cam-B3lyp 8 kcalmol minus9 kcalmol 07 kcalmol minus87 kcalmol

4 kcalmol minus10 kcalmol minus10 kcalmol

in wB97XD 8 kcal mol minus9 kcalmol minus81 kcalmol6 kcalmol minus9 kcalmol 06 kcalmol minus88 kcalmol

2in cam-B3lyp +8 kcalmol minus17 kcalmol 18 kcalmol minus112 kcalmol

+4 kcalmol minus14 kcalmol minus139 kcalmol

in wB97XD +9 kcalmol minus16 kcalmol 11 kcalmol minus132 kcalmol+7 kcalmol minus11 kcalmol minus144 kcalmol







6in cam-B3lyp No barrier minus24 kcalmol

minus20 kcalmol

in wB97XD minus23 kcalmolminus21 kcalmol

(a) (b)

Figure 1 Optimized geometry of reactants (a) and products (b) with no explicit water molecules

of PNH depicted in Figure 1 corresponds to the first stepof acid-base proton exchange with water This conformationmay be followed by a rotation of the hydroxyl group on posi-tion 1 which leads to the more stable conformation studiedin [10] The choice of conformer has few effects on reactivityThe reactants adducts have the same geometry with theexception of the hydroxyl position In the reactants adductthe superoxide anion is more or less situated in the plane ofthe substituted naphthalene The addition of the hydrogenatom to the radical modifies the dihedral angle between theOO bond and the PHN plane which becomes 70 degrees Toattain the products it is necessary to stretch bothOO andOHbonds The OO bond length varies from 132 to 1479 A and

the OH bond length from 1 to 1837 A The transition stategeometry is analogous to that of products with a OO bondalmost perpendicular to the PHNplaneHowever the lengthsof OO and OH bonds are intermediate respectively 137and 142 A (Table 2) Without water molecule the reactioncannot be complete since it lacks a proton in order to obtainhydrogen peroxide However the products HOOminus and PHNradicals are more stable by 3 kcalmol than the reactantsfor the PNH conformer depicted in Figure 1 For the othermore stable conformer the difference in free energy is lower1 kcalmolThis discrepancy in the energy difference betweenthe two possible conformers decreases with the number ofwater molecules It is negligible with six water molecules


Table 2 Geometry of transition state

Number of water molecules OO bond OH bond

0 in cam-b3lyp 137 142in wB97xd

1in cam-b3lyp 138 121

136 112

in wb97xd 137 123140 109


136 109

in wb97xd 138 142137 115


138 116

in wb97xd 137 143137 137


140 103

in wb97xd 137 105136 128

One Water Molecule The most stable geometry of the reac-tants with one water molecule is different according to thefunctional used for calculations However in all cases thefree energy difference is low (between 06 and 07 kcalmol)In the most stable conformation for wB97XD the watermolecule forms a bridge between the two other molecules(Figure 2(a)) The superoxide anion and water molecule arein a perpendicular plane with respect to PNH The productskeep approximately the same pattern with the watermoleculemore inclined The water does not give a proton to OOHminusanion but its OH bond is slightly stretched with a length of102 A In the transition state conformation (Figure 2(b)) thepattern is conserved with the hydrogen atom approximatelyin the middle of the two oxygen atoms This transition statecorresponds to a saddle with a OO bond length of 138 Aand OH bond length near 121 A for cam-b3lyp functionalThe other stable conformation with the cam-b3lyp functional(Figure 2(c)) has the water molecule at the opposite side ofsuperoxide anion The free energies of the two configura-tions are similar but their electronic energies are differentby approximately 4 kcalmol This configuration leads to amore exothermic reaction (Table 1) with a lower barrierThe transition state geometry of this configuration is moresimilar to that of the reactants since the OH bond length isshorter by 08 A Thus from the comparison between thesetwo configurations it can be concluded that the position ofthe water molecule at the end of the superoxide anion isimportant in lowering the barrier height However it is notsufficient to complete the reaction since in the productsthe water does not give a proton to the OOHminus moleculeHowever the bond length of the hydroxyl group situatednear the OOHminus anion is greater than usual 106 A insteadof 102 A As in the former case with no water molecule theconformation of PNH has few effects on reactivity with the

most stable conformation only a slightly lower differencebetween the electronic energies is calculated

Two Water Molecules Adducts with two water molecules arenumerous and it was not possible to study all the possibilitiesHowever two configurations similar to the former with onewater molecule have been chosen in order to verify if thegeometry influence on barrier height was still effective In thefirst configuration the two water molecules formed a bridgebetween PNH and superoxide one is in the plane of PNHand the other is perpendicular However after the reactionhas occurred the bridge in the PNH plane no longer exitsand the conformation of the reactants corresponding to theproduct geometry (Figure 3(a)) is slightly more stable thanthe geometry with the two bridges Thus the reactivity wasstudied with this conformation as initial state The additionof a water molecule forming a hydrogen bond on the oxygenatom of reactive hydroxyl group leads to a better stabilisationof the products The barrier height remains of the sameorder of magnitude The second studied configuration stillpossesses a water molecule forming a bridge between thereactants The second water is situated at the end of super-oxide (Figure 3(b)) Contrary to what has been calculatedfor adducts with one water molecule this configuration islargely favoured since it possesses a lower free energy by18 kcalmol The comparison of the results with one or twowater molecules shows that addition of the water moleculedoes not modify the barrier height in the two configurationsHowever the position of the saddle varies differently In thefirst configuration (Figure 3(a)) the saddle point is furtherfrom initial state with OO and OH bonds length of 137and 133 A respectively In the second configuration it is theother way round the bridge formed by the water moleculediminishes the OH bond distance of the saddle to 109 A Onehas to notice that positioning the water molecule toward thelower oxygen atom of superoxide on the opposite side of thehydroxyl group leads to similar results

Three Water Molecules From Table 1 it can be observed thatthe addition of a thirdmolecule around superoxide anion andthe hydroxyl group of PNH does not modify fundamentallythe transition state barrier height or the stabilisation of theproducts The two configurations that have been chosen aredepicted in Figure 4 In the two cases the upper oxygen atomof superoxide is linked to two water molecules The loweroxygen atom is linked to the hydroxyl PNH group only inthe first configuration It is approached by another watermolecule in the second This second configuration is morestable by 19 kcal in cam-b3lyp

FourWaterMoleculesThe two configurations with four watermolecules were built by adding water at the lower oxygenatom of superoxide in the configurations with three watermolecules (Figure 5) As a consequence the difference infree energy between the two configurations decreases to06 kcalmol There are a further stabilization of the productsand a lowering of the barrier height in the first configurationWith the cam-b3lyp functional in the transition state thehydroxyl OH bond has a short length close to that of to theinitial state one


(a) (b)

(c)

Figure 2 (a) Optimized geometry of the first conformation of reactants with one water molecule (b) transition state conformationcorresponding to the configuration (a) (c) optimized geometry of the second conformation of reactants with one water molecule

(a) (b)

Figure 3 Optimized geometries with two water molecules (a) optimized geometry of the products in the first configuration (b) optimizedgeometry of the reactants in the second tested configuration

(a) (b)

Figure 4 Optimized geometry of reactants in the two tested configurations with three water molecules

(a) (b)

Figure 5 Optimized geometry of reactants in the two tested configurations with four water molecules


(a) (b)

0

minus10

minus20

18

17

16

15

14

13

12

11

1 135

145

OO bond distance

OH bond distance

(c)

Figure 6 Optimized geometries with six water molecules (a) Reactant configuration which leads to a reactivity with a low barrier (b)reactant configurationwhich leads to spontaneous reactivity (c) PES for the first configurationThe ordinates correspond to electronic energydifference (in kcalmol) The axis is given in A

Six Water Molecules With six water molecules (Figure 6)there are no more barriers for two configurations Thereaction is spontaneous and the reactants geometry does notcorrespond to a minimum It has been calculated by freezingthe two OO and OH bonds In these two configurations theupper oxygen atom of superoxide anion is approached byfour water molecules In the first case one water moleculeis linked to the lower oxygen atom in the second case twowater molecules form hydrogen bonds in addition to thehydroxyl group of PNH (Figure 6(b)) In a third configura-tion (Figure 6(a)) the water molecule is displaced from thelower oxygen atom of superoxide versus the oxygen atomof the hydroxyl group In that case the reaction is no morespontaneous but has a very low barrier Thus the number ofwater molecules pointing toward the oxygen of superoxidehas some importance in the spontaneous character of thereactivity However if dynamic effects had been included inthe model the reaction would have taken place also in thethird configuration since the barrier is very low (Figure 6(c))

Thus the hydration of superoxide modifies its reactivitywith PNH This study could not be done thoroughly withMP2 method but it has been verified that this result was notdependent on the method of calculation In MP2 also withone or two water molecules the reaction passes through abarrier It is spontaneous with six water molecules

It has been shown that the first layer of water moleculesaround superoxide radical indirectly participates in the reac-tion What about the molecules around PNH Calculationshave been done with a complete layer of water molecules

around the reactant For these calculations the two setsof water molecules around superoxide and around PNHhave been described with two basis sets Reactants and sixwater molecules around superoxide were described with the6-311+G(dp) basis set The other 43 water molecules weredescribed with the low basis set 3-21+G In this case also thereactivity is spontaneous The difference in electronic energybetween reactants and products still increases

32 Study of Frontier Molecular Orbitals

321 Frontier Molecular Orbitals of the Reactants In order tounderstand the effect of hydration on reactivity the frontiermolecular orbital occupancies of the reactants have beenpictured The natural orbital occupancies of the reactantswith no water molecule can be divided into two typesFor some electronic levels electrons are only localized onPNH in molecular orbitals similar to PNH ones They aredelocalized on all the heavy atoms of the molecule Thecorresponding alpha and beta electronic states have almostthe same energy Other molecular orbitals correspond tothe orbitals of superoxide Their energies are indicated initalic numbers in Table 3 This is the case of HOMO-1 whichis localized on both superoxide and the hydroxyl groupof PNH In this case the alpha and beta electronic levelenergies are different The SOMO has a relatively low energynext to HOMO-4 As a consequence there is a mixing ofthe occupancies of the alpha electrons in the two levels(Figure 7(a)) Addition of a small number of water molecules


(a) (b)

Figure 7 (a) SOMO of reactants with no water molecule (b) HOMO alpha of reactants with six water molecules

does not modify thoroughly the localization of the molecularorbital occupancies localized on PNH It stretches on oxygenatom of bounded water molecules but in a very limitedmanner On the contrary superoxide orbitals are largelydelocalized on bonded water moleculesThemixing betweenSOMO and alpha HOMO-4 becomes less important whenincreasing the number of water molecules since the energydifference between the levels increases With four moleculesthe SOMO is almost pureHowever there is amixing betweenthe three lowest alpha electronic levels of Table 3 For thesethree levels the molecular orbital occupancies are extendedon both superoxide and PNH

With six water molecules for the case with a verylow barrier the pattern continues the molecular orbitaloccupancies are principally localized either on PHN andbounded water molecules or hydrated superoxide There isno delocalization due to resonances between levels But in thetwo other cases the delocalization due to resonance is movedto the two highest occupied orbitals of alpha electrons Theiroccupancies are extended on all the reactants (Figure 7(b))Moreover the corresponding alpha and beta electrons arenot localized on the same sites There is no more similitudebetween alpha and beta level energies The spontaneousreactivity is correlated with this high delocalization of thehighest occupied levels

It is also possible to characterize the molecular orbitaloccupancies of the two reactants the hydrated superoxideand PNH The occupied frontier orbitals of the hydratedsuperoxide in the frozen geometry corresponding to reactantare delocalized also on water molecules directly bounded tothe anion This is not the case of the LUMO With one ortwo water molecules this molecular orbital is only localizedon superoxide For three or four molecules delocalizationbegins but on water situated orthogonally to the OHsdot sdot sdotOaxis With six molecules the delocalization occurs on thewater molecules parallel to the OHsdot sdot sdotO axis in the con-figuration with a low barrier When the number of watermolecules directly bounded to the superoxide increases thedelocalization progressively extends to all water moleculesMoreover addition of water molecules around the anion

Figure 8 SOMO of the products with four water molecules

lowers the energies In particular the LUMO beta has apositive or slightly negative value for all the configurationswith a barrier It becomes frankly negative in the two otherconfigurations with no barrier (Table 3) Thus the existenceof a the first layer of water molecules around superoxide leadsto a decrease of the electronic level energies in particular thebeta LUMO and facilitates its interaction with the HOMOof PNH (minus53 eV) As a consequence the reactants highestoccupied orbitals are largely delocalized

322 Characterization of the Products The SOMO of theproducts is depicted in Figure 8 It has the same localizationwhatever the number of water molecules Its large delocaliza-tion on PNH is an indication of the relatively good stabilityof PNH radicalThus the reaction corresponds to a hydrogenatom transfer as expected The second part of the reactionproton transfer from water molecule to OOHminus is neverachieved even with six water molecules around OOHminus It iswell known that water is a locally structured medium andproton transfer a collective motion Thus one layer of watermolecules around superoxide is not sufficient for achieving


Table 3 (a) Energies of occupied natural orbitals of reactants (in eV) (b) Energies of frontier orbitals of hydrated superoxide with the frozengeometry corresponding to the most stable reactants (in eV)

(a)

Number of water molecules 0 1 2 3 4 6 6 without barrier

Alpha

minus505562 minus514922 minus517616 minus526704 minus580906 minus531466 minus614293minus673175 minus710344 minus730725 minus735405 minus790831 minus73886 minus62349minus724929 minus728874 minus748602 minus761064 minus823483 minus780682 minus637993minus767676 minus772002 minus77358 minus778124 minus833633 minus786777 minus806994minus770506 minus777199 minus779757 minus784355 minus835429 minus803892 minus846394

minus892134

Beta


minus892842SOMO minus778396 minus808355 minus839592 minus849578 minus904488 minus900515

(b)

0 1 2 3 4 6 6 without barrier 6 without barrierAlpha

HOMO minus616089 minus654183 minus696059 minus706725 minus732466 minus740466 minus880897 minus918256LUMO 340479 239584 208973 163587 14005 110445 139043 128513

BetaHOMO minus570322 minus608987 minus651326 minus66221 minus68844 minus695569 minus841224 minus880135LUMO 055944 029632 minus00068 minus019428 minus03072 minus063263 minus214823 minus268644

SOMO minus729201 minus759159 minus793525 minus806804 minus824 minus850585 minus981655 minus994662

the proton transfer It needs at least two supplementary layersfor the stable position of the proton to be nearer to OOHminusthan to the water molecule

4 Conclusion

This study has evidenced the importance of solvent watermolecules in the reactivity of superoxide radical withpolyphenols Both barriers to reaction and reaction freeenergies depend on the number of explicit water moleculesaround superoxide radical PNH possesses a sufficientlylow BDE for the reaction to be energetically favored evenwith no water molecule But in this case the calculatedreaction free energy is low It is multiplied by a factor fiveto six between the two cases no or four water moleculesThe first layer of surrounding water molecules is essentialfor the reactivity it lowers the energies of the electronicexcited states of hydrated superoxide anion and facilitates thereaction Thus the reactivity seems to depend on at least twoimportant factors the BDE of the polyphenol hydroxyl groupand the environment of that hydroxyl group Indeed thisenvironment can thoroughlymodify the number of hydratingwater molecules A better understanding of the second pointis essential It will be the starting point for a further upcomingstudy

Conflict of Interests

The author declares that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

The calculations have been made with computers purchasedwith the funds of the Region Aquitaine France

References

[1] B Kalyanaraman ldquoTeaching the basics of redox biology tomedical and graduate students oxidants antioxidants anddisease mechanismsrdquo Redox Biology vol 1 no 1 pp 244ndash2572013

[2] J M McCord ldquoOxygen-derived free radicals in postischemictissue injuryrdquoTheNew England Journal of Medicine vol 312 no3 pp 159ndash163 1985

[3] G Litwinienko and K U Ingold ldquoSolvent effects on the ratesand mechanisms of reaction of phenols with free radicalsrdquoAccounts of Chemical Research vol 40 no 3 pp 222ndash230 2007

[4] J Vaya S Mahmood A Goldblum et al ldquoInhibition of LDLoxidation by flavonoids in relation to their structure andcalculated enthalpyrdquo Phytochemistry vol 62 no 1 pp 89ndash992003


[5] D Taubert T Breitenbach A Lazar et al ldquoReaction rateconstants of superoxide scavenging by plant antioxidantsrdquo FreeRadical Biology and Medicine vol 35 no 12 pp 1599ndash16072003

[6] K Furuno T Akasako and N Sugihara ldquoThe contributionof the pyrogallol moiety to the superoxide radical scavengingactivity of flavonoidsrdquo Biological amp Pharmaceutical Bulletin vol25 no 1 pp 19ndash23 2002

[7] P Cos L Ying M Calomme et al ldquoStructure-activity relation-ship and classification of flavonoids as inhibitors of xanthineoxidase and superoxide scavengersrdquo Journal of Natural Prod-ucts vol 61 no 1 pp 71ndash76 1998

[8] C Xu S Liu Z Liu and F Song ldquoSuperoxide generatedby pyrogallol reduces highly water-soluble tetrazolium salt toproduce a soluble formazan a simple assay for measuringsuperoxide anion radical scavenging activities of biological andabiological samplesrdquo Analytica Chimica Acta vol 793 pp 53ndash60 2013

[9] Z Dhaouadi M Nsangou N Garrab E H Anouar KMarakchi and S Lahmar ldquoDFT study of the reaction ofquercetin with Ominus

2and OH radicalsrdquo Journal of Molecular

Structure Theochem vol 904 pp 35ndash42 2009[10] L Lespade ldquoTheoretical design of new very potent free radical

scavengersrdquo Computational and Theoretical Chemistry vol1009 pp 108ndash114 2013

[11] S Mierts E Scrocco and J Tomasi ldquoElectrostatic interactionof a solute with a continuum A direct utilizaion of ABinitio molecular potentials for the prevision of solvent effectsrdquoChemical Physics vol 55 no 1 pp 117ndash129 1981

[12] M Cossi V Barone R Cammi and J Tomasi ldquoAb initio studyof solvated molecules anew implementation of the polarizablecontinuum modelrdquo Chemical Physics Letters vol 255 no 4ndash6pp 327ndash335 1996

[13] M J Frisch G W Trucks H B Schlegel et al Gaussian 03Gaussian Inc Pittsburgh Pa USA 2009

[14] T Yanai D P Tew and N C Handy ldquoA new hybrid exchange-correlation functional using the Coulomb-attenuating method(CAM-B3LYP)rdquo Chemical Physics Letters vol 393 no 1ndash3 pp51ndash57 2004

[15] J D Chai and M Head-Gordon ldquoLong-range corrected hybriddensity functionals with damped atom-atom dispersion correc-tionsrdquo Physical Chemistry Chemical Physics vol 10 no 44 pp6615ndash6620 2008

[16] Surfer 11 Golden Software Inc Golden Colo USA

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carotenoids) and micronutrients indispensable to the properfunctioning of antioxidants enzymes A lot of studies havetried to characterize the antioxidant properties of naturalcompoundsTheflavonoid family has been particularly inves-tigated These compounds can scavenge radicals either byhydrogen atom transfer or by charge transfer

FOH + R∙ 997888rarr FO∙ + RH (3)

FOminus + R∙ 997888rarr FO∙ + Rminus (4)

In the first case the reactivity is governed by the OH bonddissociation enthalpy (BDE) If the flavonoid BDE is lowerthan the BDE of RH the reaction is favored

In the second case the thermodynamic parameter whichdescribes the reaction is the electron transfer enthalpy (ETE)[3]

The calculated BDE of flavonoids correlates very wellwith the inhibition of low-density lipoprotein oxidationindicating that lipid peroxide scavenging [4] is done byhydrogen atom transfer with no barrier On the contrarysuperoxide anion scavenging by these natural moleculesseems more complex Experimental results [5ndash8] sometimesgive opposite results even in nonenzymatic assays wherethere is no competition with enzyme inhibition Theoreticalcalculations can be of some help in the study of the conditionsof reactivity Dhaouadi et al [9] have performed a DFTstudy of the reaction of quercetin with superoxide radicalTheir calculations were done in gas phase The reactionhad no barrier but was characteristic of a proton transferinstead of hydrogen atom transfer as expected Due to theanionic character of superoxide radical one may questionthe importance of superoxide hydration in the reactivityThis is the purpose of this paper studying the influenceof explicit hydration on the reactivity of polyphenols withsuperoxide anion In order to simplify the study the reactionhas been analyzed with one of the best known radicalscavengers 12468-pentahydroxynaphthalene (PNH) Thiscompound has been designed [10] for its very low BDE of57 kcalmol (vitamin C 68 kcalmol quercetin 72 kcalmol)It is a weak acid the deprotonated form is preponderant atphysiological pH The study will be limited to the reaction atthe most labile hydroxyl group of PNH It is divided into twoparts first the electronic and free energies of reactants andproducts are calculated with an increasing number of watermolecules around superoxide radical The reaction consistsin a stretching of the OO bond of superoxide and a largeincrease of the OH bond length of the hydroxyl group Tocharacterize the reaction potential energy surfaces (PES)have been constructed in function of these two coordinatesThen in a second part analysis of natural bond orbitals triesto give a better insight into the conditions of spontaneousreactions

2 Methods

The attack of superoxide anion on 12468-pentahydroxyn-aphthalene (PHN) has been studied only on the most labilehydrogen on position 4 [10] by modifying the solvation of

superoxide anion with explicit water molecules In additionthe effect of the bulk solvent has been taken into accountthrough the field polarized continuum model (PCM) whichmimics the bulk solvent by creating a solute cavity via a setof overlapping spheres [11 12] The importance of the watermolecules surrounding superoxide anion has been studiedby varying their number and positions It was impossibleto investigate all the possible configurations for the watermolecules Two different configurations among the moststable ones have been chosen in order to determine theimportance of geometry of the reactants

This study has been carried out by using Gaussian 09package [13] within density functional theory Two function-als have been tested cam-B3lyp developed by Yanai et al[14] which contains long range corrections and wB97XD[15] which includes empirical dispersion cam-B3lyp has theadvantage to lead to a better localization of the charges whichcan avoid artifacts in calculations with superoxide anions Inthe reactivity studied in this paper the two reactants havea negative charge and there is no possibility of a chargetransfer artifact This is why it has been possible to test alsothe wB97XD functional to evaluate the possible dispersioneffects All the calculations have been performed by using the6-311+G(dp) basis set

Harmonic vibrational frequencies of reactants and prod-ucts were calculated with the same functional and basis set inorder to verify that they are true minima

The potential energy surfaces (PES) describing the reac-tivity of 12468-pentahydroxynaphthalene with superoxideanion solvated by a certain number of water molecules havebeen obtained by calculating the electronic energy of theadducts in function of the two coordinates involved in thereaction the OH bond length of the hydroxyl bond and theOO bond length of superoxide anionThe electronic energieshave been obtained by optimizing the geometry with thesetwo fixed coordinates The OO bond was varied from 131 A(or 132 A depending on adduct initial geometry) to 147 Aby step of 003 A The OH bond was varied from 099 A to18 A by step of 01 A Near the transition state the steps werelowered to 005 A

In order to characterize precisely the transition statethe different PES have been parameterized with the Surfersoftware [16] by using the local polynomial gridding methodwith polynomial of order 3

3 Results

31 Effect of Explicit Hydration of Superoxide Anion on theBarrier to Reaction Table 1 displays the electronic energiesdifferences between the reactants and the transition states orthe products Even if there are some discrepancies betweenDFT functional results or water molecules conformationsa general trend emerges the addition of water moleculesstabilizes the products and decreases the barrier to reactionWith six or more molecules there is no more barrier thereaction is barrierless

No Water Molecule Figure 1 displays the geometry of reac-tants andproductswith nowatermoleculeThe conformation


















minus20 kcalmol


(a) (b)









136 112

in wb97xd 137 123140 109


136 109

in wb97xd 138 142137 115


138 116

in wb97xd 137 143137 137


140 103

in wb97xd 137 105136 128







(a) (b)

(c)


(a) (b)


(a) (b)


(a) (b)



(a) (b)

0

minus10

minus20

18

17

16

15

14

13

12

11

1 135

145

OO bond distance

OH bond distance

(c)









(a) (b)










(a)


Alpha


minus892134

Beta



(b)






4 Conclusion




Acknowledgment


References





























Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


















minus20 kcalmol


(a) (b)









136 112

in wb97xd 137 123140 109


136 109

in wb97xd 138 142137 115


138 116

in wb97xd 137 143137 137


140 103

in wb97xd 137 105136 128







(a) (b)

(c)


(a) (b)


(a) (b)


(a) (b)



(a) (b)

0

minus10

minus20

18

17

16

15

14

13

12

11

1 135

145

OO bond distance

OH bond distance

(c)









(a) (b)










(a)


Alpha


minus892134

Beta



(b)






4 Conclusion




Acknowledgment


References





























Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of






136 112

in wb97xd 137 123140 109


136 109

in wb97xd 138 142137 115


138 116

in wb97xd 137 143137 137


140 103

in wb97xd 137 105136 128







(a) (b)

(c)


(a) (b)


(a) (b)


(a) (b)



(a) (b)

0

minus10

minus20

18

17

16

15

14

13

12

11

1 135

145

OO bond distance

OH bond distance

(c)









(a) (b)










(a)


Alpha


minus892134

Beta



(b)






4 Conclusion




Acknowledgment


References





























Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


(a) (b)

(c)


(a) (b)


(a) (b)


(a) (b)



(a) (b)

0

minus10

minus20

18

17

16

15

14

13

12

11

1 135

145

OO bond distance

OH bond distance

(c)









(a) (b)










(a)


Alpha


minus892134

Beta



(b)






4 Conclusion




Acknowledgment


References





























Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


(a) (b)

0

minus10

minus20

18

17

16

15

14

13

12

11

1 135

145

OO bond distance

OH bond distance

(c)









(a) (b)










(a)


Alpha


minus892134

Beta



(b)






4 Conclusion




Acknowledgment


References





























Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of


(a) (b)










(a)


Alpha


minus892134

Beta



(b)






4 Conclusion




Acknowledgment


References





























Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of



(a)


Alpha


minus892134

Beta



(b)






4 Conclusion




Acknowledgment


References





























Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

























Journal of

Chemistry


Advances in

Physical Chemistry



Journal of

Volume 2014














Journal of

Spectroscopy



Journal of


Quantum Chemistry






CatalystsJournal of

Research Article On the Importance of Water Molecules in...

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