theoretical investigation of the formation of a new series of antioxidant depsides from the...

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Theoretical Investigation of the Formation of a New Series of Antioxidant Depsidesfrom the Radiolysis of Flavonoid CompoundsAuthor(s): David Kozlowski, Philippe Marsal, Michelle Steel, Redouane Mokrini, Jean-Luc Duroux,Roberto Lazzaroni, and Patrick TrouillasSource: Radiation Research, 168(2):243-252. 2007.Published By: Radiation Research SocietyDOI: http://dx.doi.org/10.1667/RR0824.1URL: http://www.bioone.org/doi/full/10.1667/RR0824.1

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243

RADIATION RESEARCH 168, 243–252 (2007)0033-7587/07 $15.00� 2007 by Radiation Research Society.All rights of reproduction in any form reserved.

Theoretical Investigation of the Formation of a New Series ofAntioxidant Depsides from the Radiolysis of Flavonoid Compounds

David Kozlowski,a Philippe Marsal,b Michelle Steel,a Redouane Mokrini,a Jean-Luc Duroux,a Roberto Lazzaronib andPatrick Trouillasa,1

a EA 4021 ‘‘Biomolecules et therapies anti-tumorales’’, Universite de Limoges, 87025 Limoges Cedex, France; andb Service de Chimie des Materiaux Nouveaux, Universite de Mons-Hainaut, Mons, Belgium

Kozlowski, D., Marsal, P., Steel, M., Mokrini, R., Duroux,J-L., Lazzaroni, R. and Trouillas, P. Theoretical Investigationof the Formation of a New Series of Antioxidant Depsidesfrom the Radiolysis of Flavonoid Compounds. Radiat. Res.168, 243–252 (2007).

This paper deals with the formation of a series of antioxi-dant depsides obtained from flavonoid solutions irradiatedwith � rays. These reactions take place in radiolyzed alcoholsolutions, a medium that is very rich in many different highlyreactive species and that hosts specific reactions. We focus onthe first step of those reactions, i.e., reactivity of the solute(flavonoid) with the alkoxy radicals CH3O• and CH3CH2O•

formed in methanol and ethanol, respectively, and their car-bon-centered isomers: the 1-hydroxy-methyl (•CH2OH) andthe 1-hydroxy-ethyl (CH3

•CHOH) radicals. Among the differ-ent flavonoid groups of molecules, only flavonols are trans-formed. To establish the structure–reactivity relationship thatexplains why the radiolytic transformation occurs only forthose compounds, the process is rationalized theoretically,with Density Functional Theory calculations, taking into ac-count the solvent effects by a Polarizable Continuum Modeland a microhydrated environment (one or two water mole-cules surrounding the active center). The first redox reaction,occurring between the flavonol and the reactive speciesformed upon irradiation of the solvent, is studied in terms of(1) the O-H bond dissociation enthalpy of each OH group ofthe flavonoids and (2) electron abstraction from the molecule.We conclude that the reaction, initiated preferentially by thealkoxy radicals, first occurs at the 3-OH group of the flavonol.It is then followed by the formation of a peroxyl radical (aftermolecular oxygen or superoxide addition). The different cas-cades of reactions, which lead to the formation of depsides viaC-ring opening, are discussed on the basis of the correspond-ing calculated energetic schemes. � 2007 by Radiation Research Society

INTRODUCTION

The flavonoid family is a group of natural polyphenolswidely distributed in human food (fruit, vegetables, bev-

1 Address for correspondence: EA4021, Faculte de Pharmacie, 2 ruedu Dr. Marcland, 87025 Limoges Cedex, France; e-mail: [email protected].

erages, etc.). Over the past 30 years, numerous studies havedemonstrated the biological activities of these compounds,including inhibition of cancer cell proliferation, enzyme in-hibition, and antiviral and antibacterial activities. Epide-miological studies established their role in protectionagainst cardiovascular diseases and cancers (1, 2). Thosecompounds are antioxidants; they act as free radical scav-engers, lipid peroxidation inhibitors, iron chelators, or xan-thine oxidase inhibitors (3–5). Antoxidant activity is attrib-uted in part to their strong redox capacities and is oftencorrelated with the other biological activities.

We previously investigated the reactivity of various fla-vonoids with the radicals produced in radiolyzed alcohol(methanol and ethanol) solutions (6–10). Among the com-pounds selected from representative flavonoid families (Fig.1), only the flavonols (i.e., the compounds with a doublebond between the C-2 and C-3 atoms of the C-ring and anOH group on C-3 position) are degraded into radiolyticcompounds, while the other flavonoids are not, even at highdoses (up to 20 kGy). The radiolysis of flavonols (querce-tin, kaempferol, galangin, morin) in solution produces re-actions leading to a new series of polyphenols belongingto the depside group of molecules (Fig. 2) (6–10). It isimportant to note that the same compounds are obtained inmethanol and ethanol, except for the substitution at C8: Themethoxy-depsides are obtained in methanol and the ethoxy-depsides in ethanol. Compared to the parent molecule (i.e.,the flavonol), the A-ring and the B-ring are kept intact inthe depsides. Those experimental observations thus indicatethat the flavonoid reaction and the depside formation re-quire the concomitant presence of the 3-OH group and the2,3-double bond, i.e., the structural features typical of fla-vonols.

The aim of the present work was to gain insight, fromtheoretical calculations, into the reaction mechanism thatoccurs specifically for flavonols in radiolysis solutions.

Irradiation of flavonoid solutions produces chemical re-actions between the solute and reactive species (radicals,ions, reactive molecules) formed from the radiolyzed sol-vent. Thus one can suspect that the transformation of theflavonoids that can occur in such conditions is essentially

244 KOZLOWSKI ET AL.

FIG. 1. Chemical structures of the 11 flavonoids considered in this study.

FIG 2. Chemical structures of the depsides formed.

related to the redox capacity of these compounds, at leastin the first steps of the transformation mechanism.

Radiolyzed solutions are very rich in many differenthighly reactive species. As a consequence, many differentradical reactions can occur. This paper deals with the roleof the oxygen-centered radicals (CH3O• and CH3CH2O• formethanol and ethanol, respectively) and their carbon-cen-tered isomers (•CH2OH and CH3

•CHOH for methanol andethanol, respectively), which are major components in theradiolyzed alcohol solutions.

First, Density Functional Theory (DFT) calculations ofthe redox properties of flavonoids [O-H bond dissociationenthalpies (BDE) and electron transfer capacity] are pre-sented. The reactivity with oxygen- and carbon-centeredradicals is then discussed, supported by the theoreticalstudy. Along with theoretical data available in the literature(11–13), our theoretical results allow for an understandingof the general trends in flavonoid reactivity and the specificevents occurring in radiolyzed solutions. On the basis ofnew experimental data and a theoretical kinetic study, re-actions with molecular oxygen and formation of peroxylradicals are then discussed. Finally, the following steps arerationalized theoretically, identifying the different possiblereaction sequences that lead to the C-ring opening and tothe formation of the observed depsides.

METHODS

Experimental Procedures

HPLC-grade methanol (99.8%) from SdS (Peypin, France) and aceticacid (�99.8%) from Merck (Darmstadt, Germany) were used. Quercetinwas purchased from Sigma (St. Louis, MO).

Quercetin was dissolved in methanol (5 � 10�3 M) and was irradiatedin aliquots of 1 ml with doses ranging from 0.2 to 20 kGy at a dose rateof 0.22 Gy/s in a 60Co source carrier type Oris experimental irradiator.Methanol solutions of quercetin either were used as prepared or weredeareated with helium to investigate the role of molecular oxygen duringthe irradiation.

Quercetin solutions (irradiated or not) were injected into an analyticalWaters HPLC system consisting of a 600 model pump, a variable-wave-length photodiode array detector (PDA 996), and a 600 model controller.The column used was a 250 � 4.6-mm, 10-�m �Bondapak C18 cartridge(Waters). The mobile phase consisted of a mixture of methanol and 1%aqueous acetic acid. Analyses were performed using a linear gradientfrom 20% methanol to 80% methanol over 40 min at a flow rate of 1ml/min.

245THEORETICAL STUDY OF FLAVONOID RADIOLYSIS

The chemical structures of the radiolytic products have been confirmedby NMR and mass spectroscopy. All details concerning the identificationprocedure are available in our previous papers (6, 7).

Theoretical Methodology

A number of theoretical studies have been carried out over the last 5years with the prospect of studying the antioxidant properties of phenoliccompounds. Semi-empirical calculations of the O-H bond dissociationenthalpy have been employed extensively and provided a satisfactoryqualitative interpretation of the redox capacity of flavonoids (14–18).More recently, theoretical Density Functional Theory (DFT)-based cal-culations, which include electron correlation, have contributed to the un-derstanding of the experimental structure–activity relationship of phenoliccompounds as antioxidants (11–13, 19–24). We have recently shown thatan unrestricted (25) DFT approach using the B3P86 functional (26, 27),with an adapted 6-311�G(d,p) basis set, gives bond dissociation enthalpy(BDE) values for phenol and catechol very close to the experimentalvalues in the gas phase (with an accuracy better than 1 kcal/mol) (19).

(U)B3P86/6-311�G(d,p) full geometry optimizations were carried outon the following molecules: quercetin, kaempferol, morin, galangin, lu-teolin, apigenin, chrysin, (�)-taxifolin, (�)-naringenin, (�)-eriodictyol,(–)-epicatechin (Fig. 1). Geometry optimizations of the radicals were thenperformed starting from the optimized structure of the parent molecule,after the H-atom was removed from the 3, 5, 7, 2�, 3� or 4� positions.The BDE is calculated for each OH group as follows:

• •BDE E(Flav-OH) � E(Flav-O ) � E(H ), (1)

in which Flav-OH is the flavonoid and Flav-O• is the phenoxy radicalformed after H abstraction.

For quercetin, the temperature-dependent corrections [zero point en-ergy (ZPE), translational, rotational and vibrational energies at 298 K]are 8.4, 7.9, 7.9, 9.1, and 8.6 kcal/mol for 3-OH, 3�-OH, 4�-OH, 5-OHand 7-OH, respectively (19). For taxifolin they are 9.3, 7.9, 7.9, 8.9 and9.0 kcal/mol for 3-OH, 3�-OH, 4�-OH, 5-OH and 7-OH, respectively (19).Because the difference between the corrections for a given OH site onthe two molecules is less than 1.0 kcal/mol, it is quite reasonable toextend the values of the corrections to the other flavonoids. The mostreasonable scheme is then to attribute the following corrections: (1) 8.4kcal/mol for the 3-OH groups of all the flavonols, (2) 7.9 kcal/mol forthe OH groups of the B-ring, and (3) 9.0 kcal/mol for the A-ring for allthe flavonoids.

The ionization potentials are calculated as the difference in energybetween the neutral molecule and the cation at the B3P86/6-311�G(d,p)level.

The solvent effect has been taken into account initially by using a purePolarizable Continuum Model (Integral-Equation-Formalism-PCM) (28–30) as implemented in Gaussian 03 (31), which considers that the mol-ecule under study is embedded in a polarizable medium representing thesolvent, with no explicit solvent molecules included. Such an approachhas been used successfully previously for various phenolic compounds(20). Nonetheless, taking explicit solvent molecules into account couldbe of great importance for flavonoids due to the presence of large dipolemoments and H bonding interactions. Guerra and coworkers recently in-vestigated the interaction of the water molecules with the OH group ofphenol (32). Including a large number of explicit solvent molecules foreach OH group of the 11 flavonoids would have been intractable. Thus,in a second step, we used a hybrid PCM model, in which the first-sol-vation-shell effect is approximated considering the influence of one ortwo discrete water molecules placed in the vicinity of each OH group.The long-range solvent effects of bulk water were included by use of thePCM model. Hanus and coworkers (33) discussed the reliability of sucha hybrid model especially when the solute dipole moment becomes large.Calculations with a hybrid PCM model have thus been performed forquercetin, considered as a good flavonol model. For each OH group,numerous positions of the water molecules have been tested and the cor-responding clusters (Flav-OH—water) were optimized. The conforma-

tions of the most stable clusters obtained for each OH group, which favorsthe interaction between the water molecule and the OH group, are re-ported in the supplementary materials. The same procedure has been usedfor the phenoxy radicals obtained after H-abstraction, and the correspond-ing BDEs were calculated as follows:

• •BDE E(Flav-OH—water) � [E(H ) � E(Flav-O —water)], (1a)

in which E(Flav-OH—water) and E(Flav-O•—water) are the hybrid PCM-DFT energies of the microhydrated flavonoid and phenoxy radical, re-spectively. Here we used water molecules rather than alcohol moleculesto mimic the strongest polarization and H-bonding effects that could existin solution.

To study the different steps of the proposed chemical pathways, gradualbond elongations to bond cleavage of interest have been carried out. Theenergy maximum has been taken as the starting point to calculate thetransition states (TS). It has been confirmed that (1) the normal modecorresponding to the imaginary frequency of TS corresponds to the mo-tion that tends to deform the transition structure as expected (i.e., bondbreaking or bond formation in our case) and (2) TS leads to the initialand the final products by use of the Intrinsic Reaction Coordinate (IRC)procedure.

All calculations were carried out with the Gaussian03 software (31).

RESULTS AND DISCUSSION

Because of the low solute concentration used to observethe flavonoid transformation (�7 � 10�3 M) and due to theshape of the dilution curves (6–10), the reactions occurringas a result of the direct effect of the radiation are minorand essentially occur as indirect effects between the solute(i.e., the flavonoid) and reactive species formed in the ir-radiated solvent (34).

To understand the first step of the degradation mecha-nism, one must know (1) all the species that could react inthose solutions and (2) the redox properties of flavonoids.

Reactive Species Present in Irradiated Alcohol Solutions

The radiolysis of alcohols has been studied extensively,and an exhaustive list of the reactive species produced isavailable in the literature (35–38). The free radicals formedin the early stages of the irradiation process are the H• atom,the oxygen-centered radicals (CH3O• and CH3CH2O• formethanol and ethanol, respectively), and solvated electrons( ). The alkoxy radicals (RO•), which are powerful oxi-�esolv

dants, are equivalent to the •OH in the water radiolysis.Contrary to the case for water, those radicals can react rap-idly with the solvent to yield the carbon-centered radicals,as follows (for methanol):

• •CH O � CH OH → CH OH � CH OH3 3 3 2 (2)5 �1 �1(k 2.6 � 10 M s ; i.e., t 108 ns) (37)1/2

The possibility of direct isomerization (between CH3O•

and •CH2OH) has been studied theoretically, and it cannotoccur due to the corresponding relatively high barrier (39).However, according to this study, this process can best beviewed as the acid-catalyzed rearrangement CH3O•/

→ •CH2OH/ . The alkoxy radicals exhibit� �CH OH CH OH3 2 3 2

oxidative properties while their carbon-centered counter-parts have reducing properties. The latter can also act as


TABLE 1Gas-Phase BDE298K(kcal/mol) and IP (eV) for the 11 Flavonoids

3-OH 2�-OH 3�-OH 4�-OH 5-OH 7-OH IP

Flavonols Kaempferol 83.7 83.8 99.0 88.4 7.88Galangine 84.8 99.0 89.2 8.14Morin 83.2 87.0 86.1 99.7 88.6 7.87Quercetin 83.7 77.0 74.6 99.6 88.3 7.81

Flavones Luteolin 77.9 76.4 103.9 89.1 8.22Apigenin 86.1 103.7 89.4 8.31Chrysin 103.6 89.8 8.24

Dihydro-Flavonol Taxifoline 108.1 76.1 76.1 99.8 92.2 8.21Flavanones Eriodyctiol 76.5 76.3 102.0 91.1 8.25

Naringenin 86.1 101.9 104.7 8.40Flavan-3-ol Catechin 103.8 77.3 76.6 83.1 85.2 8.01

H-atom abstractors (40) and add on double bonds (41–43).Obviously both radicals could also enter radical-radical re-actions.

Among the radical species that are known to react withthe solute, •CH2OH for methanol and CH3

•CHOH for eth-anol are of major importance. They are often the speciesmost involved in redox reactions onto the solute, due totheir high radiolytic rates and their longer half-life timecompared to the alkoxy radicals (36). Nonetheless, we willshow in our case (i.e., reaction with flavonoid antioxidants)that the oxidant capacity of CH3O• and CH3CH2O• alsomakes those radicals good candidates for understanding thefirst redox attack on flavonoids.

In the presence of molecular oxygen, the solvated elec-trons and the H• atoms react rapidly as follows:

� •�e � O → O (3)solv 2 2

• •H � O → HO (4)2 2

The pKa of (4.8) is such that is the predomi-• •HO HO2 2

nant form below pH 4.5 while predominates above•�O2

about pH 5 (as in our case). The superoxide radical •�O2

acts as an oxidizing or reducing agent depending on thesolute.

It should be noted that the alkoxy radicals (CH3O• andCH3CH2O•) do not react with O2 while hydroxyalkyl radi-cals (•CH2OH and CH3

•CHOH) could react to form peroxylradicals by a process which is usually irreversible (44).

Redox Properties of Flavonoids: A Theoretical Approach

The first step of the flavonoid reaction in a radiolyticsolution must be envisioned by regarding flavonoids as phe-nolic antioxidants. It is well known that those compoundscan react via two possible pathways:

• •Flav-OH � R → Flav-O � RH (5)• •� � •Flav-OH � R → Flav-OH � R Flav-O � RH (6)

•(E ) [E(Flav-O ) � E(RH)]•1 R

•� [E(Flav-OH) � E(R )] (5b)•� �(E ) [E(Flav-OH ) � E(R )]•2 R

•� [E(Flav-OH) � E(R )] (6b)

R• corresponds to the radical species formed in the ra-diolytic solutions and is involved in the primary reactions:CH3O• and CH3CH2O• or their carbon-centered isomers(•CH2OH, CH3

•CHOH) for methanol and ethanol, respec-tively. RH is thus CH3OH or CH3CH2OH.

Reaction (5) is a homolytic dissociation governed by theO-H bond dissociation enthalpy (BDE). Reaction (6) is anoxidation reaction to form a cation, which can rapidly dis-sociate by heterolytic dissociation, in the radiolyzed solu-tion. The first step in this reaction is governed by the ion-ization potential of Flav-OH and the energy. The(E ) •2 R

final compounds are identical in the two schemes but thechemical pathways are different.

In any case, the radical (Flav-O•) formed must be rela-tively stable, so that reactions (5) and (6) are thermody-namically favorable (in the sense that it is easier to removea hydrogen atom from Flav-OH than from CH3OH orCH3CH2OH).

Flavonoids are polyphenolic compounds; therefore, re-actions (5) and (6), which were initially proposed for mono-phenolic compounds, can in principle take place on eachOH group of the flavonoid. The difference between mono-and polyphenolic compounds can originate from (1) the ex-tension of �-electron delocalization, (2) the stabilizing ef-fects of H-bonds between neighboring OH groups, and (3)the electronic effects due to the presence of the other OHsubstituents.

The theoretical gas-phase BDEs of the 11 flavonoids arereported in Table 1; and are reported in the(E ) (E )• •1 R 2 R

supplementary materials for the 11 flavonoids. In agree-ment with other theoretical studies on the same type ofcompounds (12, 13, 19, 20, 45), the lowest BDEs are foundfor the OH groups on the B-ring, especially in the presenceof the catechol moiety, and the highest ones for those onthe A-ring. This is in agreement with the well-known struc-ture activity relationship [see ref. (3) for a review].

The 3-OH group exhibits a special feature depending onthe presence of the 2,3-double bond: In the presence of thisdouble bond (i.e., in flavonols), the BDEs are about 84 kcal/mol, close to those of the B-ring without the catechol moi-ety. In contrast, for dihydroflavonols and flavan-3-ols


TABLE 2BDE298K (kcal/mol) of Quercetin without Taking the Solvent into Account (gas phase),with a Pure PCM Solvent Model (PCM water and PCM ethanol), and with a Hybrid

PCM Model (microhydrated PCM water)

3-OH 3�-OH 4�-OH 5-OH 7-OH

Gas phase 83.7 77.0 74.6 99.5 88.2PCM water 79.2 82.0 79.4 91.4 89.5PCM ethanol 79.4 81.7 79.0 92.1 89.4microhydrated-PCM water 79.7 84.2 81.8 89.3 92.2

(which have no C2-C3 double bond), the 3-OH BDEs arevery high, indicating that H-transfer from this group is anunfavorable thermodynamic event. The 3-OH BDEs arevery similar for all the flavonols, suggesting that these lowBDEs are essentially due to the �-electron conjugation be-tween the 3-position and the B-ring, which is not stronglymodified by the substitution pattern of the B-ring.

A very interesting feature is pointed out by the PCMcalculations for quercetin (Table 2). The change in BDEdue to the solvent is different for the 3-OH group, com-pared to the OH groups of the B-ring. The BDE is de-creased by about 5 kcal/mol for the 3-OH group, whereasit is increased by about 5 kcal/mol for 3�-OH and 4�-OH.As a result, the 3-OH BDE of quercetin is very close tothose of 3�-OH and 4�-OH, which clearly points to the im-portant role of this group in the molecule reactivity. Thehierarchy in H-transfer capacity is thus 3-OH � 4�-OH �3�-OH � 5-OH � 7-OH. The two last groups exhibit rel-atively high BDEs, indicating their minor role in the redoxreactions that could occur in radiolyzed solutions.

Those differences in the effect of the solvent on the BDEvalues can probably be attributed to a competition effectexisting between intramolecular and intermolecular inter-actions. Here intermolecular H-bonding (i.e., H-bondingbetween the OH groups of the flavonoid and the solventmolecules) is taken into account in an indirect mannerthrough the electrostatic perturbation due the implicit sol-vent (PCM calculations). In the case of the 3-OH group,such a perturbation weakens the intramolecular interactionwith the carbonyl group. Indeed, in quercetin, the distancebetween the H-atom of the 3-OH group and the O-atom ofthe carbonyl group is 1.98 A and 2.13 A in the gas phaseand in the presence of the PCM solvent, respectively. As aconsequence, the solvent effect leads to an easier O-H bondscission, and hence to a lower BDE.

The hybrid model including one explicit water moleculein the vicinity of each OH group gives similar results (Table2), leading to the same hierarchy between active OH groups(3-OH � 4�-OH � 3�-OH). The BDEs of the other twogroups remain relatively high (around 90 kcal/mol) so thatthe 5-OH and 7-OH groups are still not expected to partic-ipate in the redox reactivity. The use of two water mole-cules does not strongly influence those results (i.e., 79.7kcal/mol and 80.7 kcal/mol for the 3-OH group with oneand two explicit water molecules, respectively).

First Step of the Degradation Mechanism

The (5b) values are reported in the supplementary(E ) •1 R

materials for all the OH groups of the eleven flavonoids.For the 3-OH group of quercetin and for the radicalsformed for methanol (CH3O• and •CH2OH), reaction (5) isexothermic [i.e., the energy of the products is lower thanthat of the reactants, �18.5 kcal/mol and �11.2(E ) •1 R

kcal/mol for CH3O• and •CH2OH, respectively]. This isfound for all the radicals R• considered (•CH2OH,CH3

•CHOH, CH3O• and CH3CH2O•), except in the case ofthe 5-OH group (gas-phase calculations). Nonetheless, thereaction with •CH2OH requires an activation barrier ofabout 9.9 kcal/mol, while the barrier of the reaction withCH3O• is of about 4.2 kcal/mol. It is interesting to note thatthe energy barrier of reaction (2) is about 5.1 kcal/mol (ourB3P86 calculations). This suggests that the reaction ofCH3O• with the solute (flavonol) is faster than with meth-anol (or at least has the same reaction rate), indicating that,when it is formed by the irradiation process in the vicinityof the flavonol, the alkoxy radical could react before beingconverted into •CH2OH (i.e., reaction with methanol mol-ecules).

For quercetin, the values of ( are calculated to beE ) •2 R

very positive in the absence of the solvent: e.g., �134.3kcal/mol and �175.5 kcal/mol for CH3O• and •CH2OH, re-spectively (see supplementary materials). This implies thatthe first step in reaction (2) is highly unfavorable in the gasphase. In contrast, the second step, i.e., the heterolytic dis-sociation from the cation to the flavonoxy radical, is, asexpected, very favorable thermodynamically. However, alarge decrease in E2 is observed when the solvent is takeninto account: It is decreased from 134.3 kcal/mol to 25.3kcal/mol and from 175.5 kcal/mol to 78.3 kcal/mol forCH3O• and •CH2OH, respectively, due to the stabilization ofthe ArOH•� cation by the solvent. Those results confirm thestronger oxidant character of the alkoxy radical comparedto the hydroxyalkyl radical. Nonetheless, E2 still remainsrelatively high (25.3 kcal/mol with CH3O•); such an energydifference cannot be overcome even under the experimentalconditions occurring in radiolytic solutions, where all re-actions are favored and where the temperature is slightlyincreased.

Therefore, these calculations point to the predominanceof reaction (5) (H-atom abstraction) compared to reaction


FIG. 3. Variation in quercetin concentration as a function of dose, withand without oxygen. The initial quercetin concentration is 5 � 10�3 M.

(6) (electron transfer) as the most likely mechanism, andthe BDE calculations show that the predominant sites arethe 3-OH group and the OH groups of the B-ring. Thus themost probable reaction is the reaction of the alkoxy radicals(more probable than the hydroxyalkyl radicals) with thoseOH groups to form the flavonoxy radicals Flav-O• (e.g., Q•

for quercetin) by H abstraction.

The Flavonoxy Radicals Formed after H Abstraction

All the experimental evidence (6–10) indicates that thedegradation process requires the concomitant presence ofthe 3-OH group and the 2,3-double bond (i.e., the flavonolstructure), while the B-ring is kept intact. To understandwhy the degradation process is observed only for flavonols,what is important is not only the redox capacity of the OHgroups but also the reactions that could occur after this firstreaction step. The spin density of the radicals formed afterH transfer from the OH groups of the B-ring has been com-puted for all systems. For flavonols it is delocalized fromthe B-ring to the 2,3-double bond, indicating a relativelysmall reactivity for these intermediate species (because ofthe absence of high spin density on a given site). This resultis in good agreement with the presence of stabilized qui-nones, which has been observed after H-abstraction fromthe B-ring of flavonoids in radiolytic solutions (46).

In contrast, the calculated spin density of the Q• issuedfrom the 3-OH group is substantially localized on two sites:the O-3 atom and, most important, the C-2 atom (0.44 forquercetin). This observation is of major interest because itshows that C-2 becomes a very reactive site for a secondredox attack and for further reaction, exclusively in flavo-nols.

The Role of Molecular Oxygen

In aerated solutions, molecular oxygen is well known toparticipate in the radiolytic reactions of the solute (36, 44,47, 48). We propose that the second step of the reactionmechanism of flavonoids in radiolysis solutions is the ad-dition of molecular oxygen at C-2 of Q•. To confirm thisassumption, we irradiated flavonols in deaerated solutions.In Fig. 3, the quercetin concentration is plotted as a func-tion of dose in the presence or absence of molecular oxy-gen. While the reaction is complete at 15 kGy in the pres-ence of oxygen, no reaction is observed in the absence ofoxygen, even for doses higher than 20 kGy. This demon-strates that molecular oxygen indeed participates in thetransformation of flavonols, probably at the second step ofthe mechanism, through addition at the C-2 atom of Q• toform an intermediate peroxyl radical QOO•,

• •Q � O → QOO2 (7a)

Our calculations give an energy barrier for this additionreaction of 3.3 kcal/mol.

Such addition of oxygen at the C-2 atom after H transferfrom the 3-OH group has previously been suggested in the

metabolization of quercetin by quercetin 2,3-dioxygenase(49, 50). Quercetin 2,3-dioxygenase is a copper-containingenzyme, and the substrate (quercetin) is known to bind tothe metal atom at O-3 (51); Balogh-Hergovich and co-workers suggested that this binding is followed by additionof oxygen molecules that bind at C-2 (49).

The Alkoxy Radical Formation

After the oxygen addition step, which leads to the for-mation of the QOO• peroxyl radical, different possible path-ways can be considered.

1. Mechanism 1

The first mechanism has previously been proposed forexplaining the degradation of quercetin by quercetin 2,3-dioxygenase (49). This mechanism has recently been in-vestigated theoretically and confirmed by two groups (51,52). Different chemical pathways were explored, consid-ering the QOO• formation (after addition of oxygen at C-2) as the starting point. Following the hypothesis of Bal-ogh-Hergovich and coworkers (49), those pathways lead tocyclization via binding between the terminal O atom of theperoxyl group and either C-4 (1,3-cycloaddition) or C-3(1,2-cycloaddition) (Fig. 1). The former cycloaddition leadsto a classical depside formation by CO release, as observedin the metabolization of quercetin (49, 50). This mechanismmost probably is not at work in our case because we didnot observe that depside. In contrast, 1,2 cycloadditionleads to the type of depside we found in radiolysis solu-tions, without CO release. Indeed, this could be one pos-sible mechanism for explaining what we observed. On thebasis of B3LYP/6-31�G(d) energies, Fiorucci and cowork-ers (53) concluded that the formation of the bridging C-Obond with C-3 is the rate-limiting step of this sequence.The free-energy barrier is calculated to be 16.4 kcal/molfor the 1,2-cycloaddition. To compare with the other pos-sible mechanisms (described below), we calculated thatbarrier at the B3P86/6-311�G(d,p) level, and we obtained


15.1 kcal/mol and 14.3 kcal/mol in the gas phase and inthe presence of the PCM solvent, respectively. The follow-ing steps, leading to the C-ring opening, are found to beenergetically favorable (53).

Such a mechanism (peroxyl radical formation followedby cycloaddition) appears to provide a reasonable expla-nation for the C-ring opening. However, it is important tonote that this mechanism requires the addition of •CH3, or•CH3CH2 at the last step (51, 52). The •CH3 and •CH3CH2

radicals are formed in radiolyzed alcohol solutions but onlywith very low radiolytic rates (35, 36). The formation ofthe depsides we have observed can therefore hardly be ex-plained by such mechanism. The chemical evolution of thesystem can therefore be drastically different here with re-spect to what is occurring during the metabolization ofquercetin in a biological environment.

2. Mechanism 2

After addition of molecular oxygen at C-2 and QOO•

formation, a hydroperoxide could be formed by addition ofan H atom. Addition of H• is obviously a very favorablethermodynamic event. However, in the presence of molec-ular oxygen, the H• atoms formed in the radiolyzed solutionreact rapidly according to reaction (4).

Thus in this mechanism the H atom could come fromthe solvent molecules according to the following reactions(in methanol and ethanol, respectively):

• •QOO � CH OH → QOOH � CH O3 3

•(or CH OH) (8a)2

• •QOO � CH (CH )OH → QOOH � CH (CH )O3 2 3 2

•(or CH CHOH) (8b)3

The calculated energy differences show that those reac-tions are endothermic with the alkoxy radicals as well aswith the hydroxyethyl radicals. The transition state of theH transfer has been obtained, and the energy barrier is 13.9kcal/mol and 9.4 kcal/mol with CH3O• and •CH2OH, re-spectively.

Biradical reactions are sometimes involved (54) betweenQOO• and any radical species present in the radiolyzed so-lution (QOO• itself, / , for example). Such processes• •�HO O2 2

lead to an electron transfer, from a reducing agent ( , for•�O2

example) to QOO• to form a peroxyl anion QOO�. To formQOOH, the QOO� base must be strong enough to depro-tonate a solvent alcohol molecule for which the pKa isabout 15.7 (as for water).

�QOO � CH (CH )OH3 2 (9)�→ QOOH � CH (CH )O3 2

After hydroperoxide (QOOH) formation, one can considerthe cleavage of the O-O bond. The B3P86 calculations givea very low BDE value of 41.0 kcal/mol, indicating that the

O-O bond can be broken easily after hydroperoxide for-mation, to form a QO alkoxy radical.

3. Mechanism 3

In radiolyzed solutions, molecular oxygen could also re-act with solvated electrons to form superoxide radicals, ac-cording to reaction (3). Thus one can also suspect additionof superoxide rather than molecular oxygen to form a per-oxyl anion QOO�,

• •� �Q � O → QOO2 (7b)

In such a case QOO� is then able to deprotonate accord-ing to reaction (9).

The following steps in such a mechanism are the sameas for Mechanism 2. We calculated the energy barrier ofthe superoxide addition and obtained a value of about 10.4kcal/mol. Due to this relatively high barrier compared tothat obtained for molecular oxygen addition, and due to thelower concentration of the superoxide radicals compared tomolecular oxygen in the solutions, Mechanism 2 appearsto be more probable than Mechanism 3.

4. Mechanism 4

Peroxyl radicals could also undergo bimolecular reac-tions (head-to-head termination reaction) to form interme-diate tetroxides (ROOOOR) (44).

•2 � QOO → QOOOOQ (10)

For quercetin the formation of the tetroxide is stronglyfavored (32.6 kcal/mol) and possesses a very low barrierof about 1 kcal/mol (gas-phase calculation). The most sta-ble conformer exhibits stabilization effects due to � stack-ing between the A-rings of the two monomers. ROO• andROOOOR compounds are usually in equilibrium, but thetetroxide could also dissociate (44).

The best-known decomposition reaction is the Russellmechanism, in which molecular oxygen is released and anH atom is transferred from one R group to the other one(55). For quercetin, due to the absence of H atoms in thevicinity of the O-O bonds in QOOOOQ, this route is notpossible. We propose that the decomposition process couldoccur as follows:

•QO-OO-OQ → 2 � QO � O2 (11)

We calculated the energy barrier of the decompositionprocess by symmetrically elongating the two O-O bondsand identifying the transition state between the singlet state(QOOOOQ) and the final state (2 � QO• � O2), for whichthe spin multiplicity is 5. The calculated energy barrier(about 30 kcal/mol) is high, indicating that this decompo-sition reaction is improbable. This is in due part to thestabilizing effects that exist for QOOOOQ.

Experimental evidence suggests that tetroxide formationis not predominant because QOOOOQ is not a major ob-served radiolytic compound. This is probably due to a low


FIG. 4. Final steps of the chemical pathways for explaining the radiolytic transformation of flavonols into depsides.

probability of interaction between two QOO• monomers inthe radiolytic solution. Nonetheless, this compound couldexist as minor undetected product.

The Final Steps: C-Ring Opening and Depside Formation

At this stage, all the possible mechanisms except the firstone led to the alkoxy radical QO•. The next step we inves-tigated is the C-ring opening (Fig. 4), by C2-C3 bond dis-sociation. Interestingly, this cleavage is a thermodynami-cally favorable event, with a very low energy barrier, lessthan 1 kcal/mol. Further rearrangements of torsion angles(C1-C7-C8-R and C2-O-C-C1� in Fig. 2) in the open-QO•

lead to a more stable conformer, and the energy differencebetween that stable conformation of the dioxo radical (theopen-QO•) and the initial QO• compound then becomes 21.2kcal/mol, in favor of the C-ring opening.

At this stage, the open-QO• radical is likely to recombinewith radicals present in the radiolysis solution, especiallyalkoxy radicals including CH3O• and CH3CH2O• in metha-nol and ethanol, respectively. Such addition reactions arestrongly exothermic (�92.2 and �91.5 kcal/mol, for CH3O•

and CH3CH2O•, respectively), with a very low barrier of 0.3kcal/mol.

CONCLUSION

The formation mechanism of a new series of depsides inradiolyzed alcohol solutions of flavonoids is proposed here,based on the combination of experimental data and DFTcalculations. In this study it has been demonstrated that thealkoxy radical species issued from the radiolysis of alcoholprobably attack the 3-OH groups of flavonols. This mech-anism most probably occurs by H atom transfer rather thanelectron transfer. Attack of the 3-OH group is the mostfavorable in flavonols (lowest BDE) and leads to the Q•

formation for quercetin. Q• has a high spin density at C-2,which allows molecular oxygen addition and formation ofthe QOO•. At this stage the feasibility of different mecha-nisms leading to the formation of the QO• alkoxy radicalhas been investigated and discussed. Mechanism 2 (QOOHformation by H abstraction from the solvent molecules) ex-hibits the lowest energy barrier and involves species presentin high concentration in the radiolytic solutions. QO• canthen easily reorganize by C-ring opening. Finally, addition

of alkoxy radicals (formed by the radiolysis of the solvent)yields the depsides found as the major radiolytic productof the flavonols.

Some of the depsides formed exhibit good antioxidantactivities (8, 10). Those new antioxidants could be of greatinterest due to the presence of an open C-ring, due to goodredox capacity and better flexibility in enzymes. Furtherinvestigations of those compounds are in progress to eval-uate their biological activities.

SUPPLEMENTARY MATERIALS

Tables I-a, -b, -c and -d: and for all the(E ) (E )• •1 R 2 R

eleven flavonoids studied in this paper, and for R• CH3O•,•CH2OH, CH3CH2O• and : http://dx.doi.org/10.•CH CHOH3

1667/RR0824.1.s1.Figure A: Most stable conformers obtained for the Flav-

OH–water clusters and for each OH group of quercetin:http://dx.doi.org/10.1667/RR0824.1.s2.

ACKNOWLEDGMENTS

The authors thank the ‘‘Conseil Regional du Limousin’’ for financialsupport and IDRIS (Institut du Developpement et des Ressources Infor-matiques Scientifiques, Orsay, Paris) for computing facilities. The workin Mons is partly supported by the Belgian Science Policy InterUniversityAttraction Pole Program (Project 5/3) and the Belgian National ScienceFoundation (FNRS). The authors are grateful to Monique Gardes-Albert,Fabrice Collin and Dominique Bonnefont-Rousselot (University ofParisV) for stimulating and helpful discussions on radiolysis and hydro-peroxide formation.

Received: September 8, 2006; accepted: February 12, 2007

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