Ab initio molecular dynamics of the reaction of quercetin with superoxide radical

Chemical Physics 475 (2016) 32–38

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Ab initio molecular dynamics of the reaction of quercetin with superoxide radical Laure Lespade Institut des Sciences Moléculaires, UMR 5255, Univ. Bordeaux, 351 crs de la Libération, 33400 Talence, France

a r t i c l e

i n f o

Article history: Received 8 April 2016 In final form 11 June 2016 Available online 14 June 2016 Keywords: CPMD ab initio molecular dynamics Flavonoids Superoxide Solvation

a b s t r a c t Superoxide plays an important role in biology but in unregulated concentrations it is implicated in a lot of diseases such as cancer or atherosclerosis. Antioxidants like flavonoids are abundant in plant and are good scavengers of superoxide radical. The modeling of superoxide scavenging by flavonoids from the diet still remains a challenge. In this study, ab initio molecular dynamics of the reaction of the flavonoid quercetin toward superoxide radical has been carried out using Car–Parrinello density functional theory. The study has proven different reactant solvation by modifying the number of water molecules surrounding superoxide. The reaction consists in the gift of a hydrogen atom of one of the hydroxyl groups of quercetin to the radical. When it occurs, it is relatively fast, lower than 100 fs. Calculations show that it depends largely on the environment of the hydroxyl group giving its hydrogen atom, the geometry of the first water layer and the presence of a certain number of water molecules in the second layer, indicating a great influence of the solvent on the reactivity. Ó 2016 Elsevier B.V. All rights reserved.

1. Introduction Superoxide radical anion has a great importance in biochemical processes [1] and atmospheric chemistry [2]. For a long time, literature has focused on the deleterious character of reactive species derived from oxygen (ROS) since oxidative damage contributes to the pathology of a lot of human diseases such as cancer and atherosclerosis. But, nowadays, it is admitted that the field is far more complex than that [3]. Free radicals and, more specifically, superoxide radical, are derived either from normal essential processes in the human body or from external sources such as exposure to ozone, cigarette smoking, pollutants . . . [4]. The main internal sources of superoxide are mitochondria and immune defense system. The essential role of some ROS in life survival is better and better documented: apart from its importance in the defense immune system, superoxide radical also plays a role of signaling molecule in a lot of biochemical processes [5]. Scientists think that evolution has taken advantage of the deleterious properties of superoxide radical and its presence in the body in small quantities is important. However, an excess of ROS accompanies a lot of chronic diseases. The balance between the formation of ROS and the antioxidant defense is essential in maintaining good health. This balance is assured by

E-mail address: [email protected] http://dx.doi.org/10.1016/j.chemphys.2016.06.006 0301-0104/Ó 2016 Elsevier B.V. All rights reserved.

several mechanisms that produce antioxidants ‘‘in situ” (endogenous antioxidants) or by exogenous antioxidants supplied through foods. It exists an abundant literature on the antioxidant properties of polyphenols from the diet, the main part of exogenous antioxidants. In particular, a class of molecules has dealt a lot of attention because of their abundance and properties: the flavonoids. One aspect of their antioxidant properties consists in their radical scavenging abilities: they can give hydrogen radical or electron to the reactive species. In theoretical chemistry, the enthalpies corresponding to the two types of reactions, bond dissociation enthalpies (BDE) and electron transfer enthalpies (ETE), can be calculated with a good accuracy [6]. Different chemical assay have been imagined to measure the radical scavenging abilities of plant polyphenols [7,8]. They use relatively stable radicals as ABTS+ ((2,20 -azinobis-(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt) radical cation) or DDPH (2,2-diphenyl-1-picryl-hydrazyl). The experimental results of such assays on flavonoids roughly correlate with calculated BDE [9]. Other experiments have directly measured the reactivity of flavonoids with superoxide anions, produced either enzymatically through xanthine oxidase [10–12] or non-enzymatically [13–14]. The two protocols do not give the same results. The experiments with xanthine oxidase are difficult to read since the inhibition of superoxide radical is a consequence of three simultaneous reactions: inhibition of xanthine oxidase by flavonoids, reduction of

L. Lespade / Chemical Physics 475 (2016) 32–38

the enzyme and scavenging of superoxide radical [15]. However, the non-enzymatic experiments sometimes give opposite results showing the difficulties of such assays. Moreover, the correlation between superoxide scavenging activity and other antioxidant measurements is poor [16]. Theoretical chemistry may help in understanding these discrepancies. In this paper, the dynamics of the reaction of superoxide anion with one flavonoid, quercetin, is investigated. Quercetin displays good antioxidant abilities as measured by DPPH or ABTS+. Dhaouadi et al. have theoretically investigated its reactivity toward superoxide radical in gas phase [17]. This study has shown that, contrary to what happen in water solvent, in gas phase, quercetin gives a proton to superoxide. There is no radical scavenging. In water solution, modeled by a cavity of overlapping spheres [18], the reactants are calculated at lower free energy than the products. It is necessary to introduce explicit water molecules in the system to model the reaction of polyphenols toward superoxide radical [19] in a realistic way. Preliminary calculations have shown that at least eight to ten water molecules were necessary to lower the product free energy at the same level than the reactants. With so many molecules the system is very flexible and it is difficult to explore all the possible conformations. It is necessary to perform dynamics calculations.

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2. Methods and computational details Quercetin possesses five hydroxyl groups at positions, 30 , 40 , 3, 5 and 7. It undergoes eight configurations with non-negligible concentration (Fig. 1). Its pKa is 6.5 [20]. Thus, quercetin essentially exists in deprotonated form at physiological pH. It is generally admitted that the deprotonation occurs at position 7. However, theoretical calculations indicate that the energy to deprotonation is not identical for all the conformations. When the hydroxyl group at position 40 points in the opposite side to position 30 , the enthalpy to deprotonation of position 7 and 40 are close together and the two corresponding anions are likely to coexist. Thus, five cases will be considered in this study: the reactivity of superoxide radical with the hydroxyl groups 3, 30 , and 40 of quercetin anion deprotonated at position 7 and the reactivity of superoxide radical near the position 3 and 7 of quercetin anion deprotonated at 40 . The ab initio molecular dynamics simulations are carried out using the Car–Parrinello method [21] and CPMD code [22]. The system consists in quercetin anion with superoxide radical surrounded by different amounts of water molecules ranging from 8 to 50. Geometry optimization of the cluster is initially performed with Gaussian package [23] and density functional theory (DFT),

a

b

0,3 kcal

0,2 kcal

c

d

0,1 kcal

e

f

0,1 kcal

0,1 kcal

g

h

0,3 kcal

0,2 kcal

Fig. 1. Different conformations of quercetin with non-negligible probabilities. The difference in enthalpies is indicated for each conformation in kcal/mol. The calculations have been made with Gaussian 09 software, and DFT method with B3LYP functional and 6-311+G(d,p) basis set.

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with B3LYP functional and 6-31+G(d) basis set. Then a further geometry optimization is performed using CPMD with BLYP functional and norm-conserving ultrasoft Vanderbilt pseudopotentials. The valence electronic wave function is described with a plane wave basis with an energy cutoff of 50 Ry. The local spin density (LSD) approximation is employed to account for the unpaired electron at superoxide radical. The system is isolated in a box the dimension of which depends on the number of water molecules. The optimization is stopped when the maximum force gradient is less than 104 for the nuclei. The dynamics simulations are carried out with a fictitious mass of 600 au and a simulation temperature of 300 K. The fictitious electron kinetic energy and the dynamics are controlled by a chain of three Nose–Hoover thermostats [24] operating at characteristic frequencies of 15,000 cm1 and 3000 cm1 respectively. The average fictitious kinetic energies depend on the size of the system, ranging from 0.02 to 0.06 Ha. The time step is of 0.1 fs and the total simulation time 5 ps. The molecular orbitals of the reactants or products are calculated with Gaussian package with the geometry supplied by the ab initio dynamics simulations. BDE of quercetin anions used in the study have also been calculated with Gaussian package using the procedure of reference [8]. 3. Results 3.1. Importance of the site The simulations have been performed with initial conditions positioning superoxide anion near the hydroxyl groups at position 3, 30 , 40 and 7. Even with a large amount of water molecules, the hydroxyl groups at positions 3 and 30 do not react with superoxide. The superoxide position near position 3 is not stable since the radical flees away. This is not the case for position 30 , superoxide stays in the site at least three picoseconds but does not react. In contrast, the reactivity is instantaneous at position 40 and 7 when the number of water molecules excesses 20 (two layers around superoxide). Quercetin gives a hydrogen atom to superoxide and some water molecule gives a proton to form hydrogen peroxide. It is necessary to fix constraints on hydrogen atom during equilibration to avoid the reaction. When constraints are removed, the hydrogen radical transfer occurs in less than 100 fs. Fig. 2 gives examples of the OO or OH distance variations during the dynamics simulation: the higher green curve displays the variation of the distance between the oxygen atom of the hydroxyl group at position 40 (upper figure) or 7 (lower figure) and one oxygen atom of the superoxide radical. In every case, with reaction or not, this distance undergoes fluctuations around 2.5 and 3 Å. The reactivity always occurs when this distance approaches 2.5 Å. The two other dotted curves display the distance between the hydrogen atom and the oxygen atoms of superoxide or hydroxyl group. The exchange is clear and abrupt. The red solid line represents the OO bond distance of superoxide radical. This distance increases during the reaction since the OO bond of hydrogen peroxide is larger than superoxide one. One can see that all these distances are slightly overestimated with Vanderbilt pseudopotentials. 3.2. Importance of water molecules When the total number of water molecules is higher than 20, at least three water molecules must be directly linked to superoxide to observe a reaction in position 7. Indeed, it is sufficient that superoxide hydration shell possesses three water molecules in the configuration displayed in Fig. 3: superoxide, the three water molecules and quercetin are displayed in ball and sticks. They

Fig. 2. Time evolution of distances between hydroxyl and superoxide atoms at sites 40 (A) and 7 (B) with more than 20 water molecules surrounding superoxide. The green dotted curve represents the distance between the oxygen atoms of hydroxyl and superoxide. The red line represents the OO distance of superoxide bond. The black dotted line represents the OH distance of hydroxyl and the blue dotted line the OH distance of hydrogen peroxide. The distances are given in Å. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

are roughly situated in a plane perpendicular to the quercetin plane. For a better visualization, the molecular model is given in Supplementary Materials. With this configuration, the hydrogen transfer is immediate. In position 40 , there is no reactivity if only three water molecules are bonded to superoxide. One extra molecule, perpendicular to the above mentioned plane is needed. It may be linked either to the higher oxygen of superoxide or to the lower. When the number of water molecules decreases, these minimalist configurations are no longer sufficient. Table 1 summarizes the dependence of the required configurations for reactivity in function of the number of water molecules around superoxide. At position 7, when the number of water molecules is comprised between 17 and 20, the reaction can occur with four water molecules linked to superoxide. In clusters with less water molecules, there is no longer reactivity. With 17 water molecules, the reaction occurs with another configuration: the superoxide is situ-

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Fig. 3. Positions of superoxide, hydroxyl and three water molecules before reaction at site 7. One can observe the plane of quercetin. Superoxide is roughly perpendicular to that plane. Oxygen atoms are colored in red (or pink for the three water molecules) and carbon in blue. Other water are represented by sticks. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

ated in the plane of quercetin instead of being perpendicular. When superoxide is perpendicular, hydrogen transfer can occur but it is not stable. At position 40 , when the cluster possesses 14 water molecules, one can still have a reaction with only four water linked to superoxide but it is reversible as pictured in Fig. 4: at 1.1 ps the black and blue curves displaying OH bonds cross: there is a transfer of the hydrogen atom from the hydroxyl to superoxide. The OO bond distance of superoxide undergoes large fluctuations but does not increase and the two former curves cross again with the transfer of hydrogen from superoxide to hydroxyl. One picosecond later, another cross of the curves indicates another transfer of hydrogen that remains linked to superoxide. In the two cases, the configurations of surrounding water molecules are not the same. In the first case, there are four water molecules linked to superoxide by hydrogen bonds, in the second case, there are five (Fig. 5 and Supplementary Materials). But the number of water molecules linked to superoxide is not sufficient to initiate the reaction since at the beginning of the simulation, there were also five molecules linked to superoxide. Their respecting positions are also important. When the clusters possess less than 12 water molecules no reaction is observed at site 40 . 3.3. Characterization of the molecular orbitals In order to go further in the analysis of the conditions necessary to initiate reactivity, the molecular orbitals of the different clusters have been visualized.

Fig. 4. Time evolution of distances between hydroxyl and superoxide atoms at site 40 for a cluster possessing 14 water molecules around superoxide. The upper green dotted curve represents the OO distance between superoxide and hydroxyl. The red full line curve represents the OO bond distance of superoxide. Black and blue dotted curves represent OH bond distances of hydroxyl and hydrogen peroxide respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Position 7: in Fig. 6, the highest occupied molecular orbital (HOMO) of the reactants of a cluster with more than 20 water molecules is displayed at the beginning of the simulation run and after 40 fs when hydrogen transfer starts. The four atoms of hydroxyl and superoxide are colored in blue. At the beginning of the run, the HOMO (in green and pink) is essentially localized on quercetin. Action contours mark out a precise delimitation of the molecular orbitals in the plane containing the four atoms, COH of hydroxyl and the oxygen atom of superoxide that is involved in the reaction. In the line formed by the two oxygen atoms of hydroxyl and superoxide (21 and 25) there is a sign change of the molecular orbital. The two atoms are situated near two separate lobes with opposite sign. Hydrogen atom is situated in a green lobe near the oxygen of hydroxyl group. At 40 fs, the distance between the two oxygen atoms 21 and 25 has decreased. The HOMO is more delocalized on the two molecules with four lobes on superoxide. Their orientations have changed and the green lobe of superoxide goes continuously toward the oxygen 21 on the line between 21 and 25. There is no more a sign change perpendicular to the line. The electron can move along the line. A few steps further, at 60 fs, the hydrogen atom is inside the pink lobe of superoxide and there is again a sign change in the molecular orbital along

Table 1 Abstract of different cases that have been tested. The two conformers differ by a rotation of 180° of the B ring of quercetin. It has been tested that there is no influence on the conformation on reactivity in the cases with large number of water only. Number of waters surrounding superoxide

Number of waters linked to superoxide

Position 3

Position 30

Position 40

Position 7

50 > waters > 20

3

No reactivity Two conformers No reactivity Two conformers No reactivity No reactivity No reactivity No reactivity No reactivity

No reactivity Two conformers No reactivity Two conformers No reactivity No reactivity No reactivity No reactivity No reactivity

No reactivity Two conformers Reactivity Two conformers No reactivity Reactivity Reactivity Reactivity No reactivity

Reactivity Two conformers Reactivity Two conformers No reactivity Reactivity No reactivity No reactivity No reactivity

4 20 P waters P 17 16 P waters P 15 14 P waters P 12 12 > waters

3 4 4 5 5

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distance between the two oxygen atoms of both hydroxyl and superoxide is sufficiently low. When there are less water molecules, some extra water molecule directly linked to superoxide is needed to rotate the position of the lobes.

Fig. 5. Configuration of the cluster before the two hydrogen transfers illustrated in Fig. 4. Superoxide, quercetin, and water molecules linked to superoxide by hydrogen bonds are represented by balls. On top, which corresponds to the reversible transfer at 1.1 ps there are four water molecules. The configuration at bottom corresponds to the geometry before hydrogen transfer at 2.2 ps. There are five molecules directly linked to superoxide.

the line 21–25. The electron of hydrogen atom cannot move anymore near the oxygen atom of hydroxyl group. The configuration is the same for all the clusters possessing more than 16 water molecules. During the hydrogen transfer, there is continuity of lobes between the oxygen atoms of hydroxyl and superoxide. If continuity does not exist, transfer can occur if the lobe near hydroxyl is long enough but the atom does not remain attached to superoxide. Position 40 : the same observations are made for position 40 . The positions of the lobes are somewhat different but hydrogen transfer always occurs when there is a junction of the lobes situated near superoxide and hydroxyl oxygen atoms positions. When there are sufficiently water molecules, atoms are linked by several hydrogen bonds forming chains. They slightly modify the positions of HOMO lobes favoring this type of configuration when the

" Fig. 6. HOMO of the cluster during hydrogen transfer. The hydroxyl atoms (21 and 113) and superoxide atoms (24, 25) are colored in blue. The HOMO is depicted in pink and green. Pink and green lines indicate the contours actions in the plane containing the four atoms: COH of hydroxyl and the oxygen atom of superoxide involved in the reaction. (A) Before hydrogen transfer, HOMO is essentially localized on quercetin. However, pink and green lines around atom 25 of superoxide indicate a low electronic presence probability. (B) When the distance between superoxide and hydroxyl decreases, the HOMO localization on superoxide increases. There is also a rotation of the lobes and a junction of the green lobes situated near the atoms 21 and 25. The probability of electronic presence never cancels on the line between the two atoms. (C) After hydrogen atom transfer, the atoms 21 and 113 are situated near to two separates lobes. The transfer is complete.

L. Lespade / Chemical Physics 475 (2016) 32–38 Table 2 BDE of quercetin anions used in the simulation (in kcal/mol).

Deprotonated at 7 Deprotonated at 40

Position 3

Position 30

Position 40

74 70

76

70

Position 7 74

4. Discussion Table 2 gives the BDE of hydroxyl groups tested in the simulations. The BDE value is not sensitive to molecular configurations, except when there is a modification of hydrogen bonds. But hydroxyl groups tested in simulations are not involved in hydrogen bonds. When quercetin is deprotonated at position 7, the lowest BDE belongs to position 40 . This is the only site where reaction occurs. But when quercetin is deprotonated at position 40 , the site 3, which possesses the lower BDE, does not react. This result explains the differences in the various experimental assays findings. There are many negative charges next to the position 3. Superoxide does not remain in the vicinity of the hydroxyl group. On the contrary, radicals such as ABTS+ or DPPH can approach the position. Thus, the reactivity of hydroxyl groups toward superoxide does not depend only on the value of the BDE but also on the environment around the reaction site. The lack of reactivity at position 3 is coherent with some experimental results. It has been evidenced that a glycosylation of the hydroxyl at position 3 was not associated with a significant change in the reaction kinetics [25]. Moreover, galanguin, a flavonoid with only hydroxyl groups at position 3 and 5 is not a radical scavenger of superoxide even though it possesses a moderate reactivity with DPPH or ABTS+. The hydroxyl group at position 7 gives its hydrogen atom in configurations with only three water molecules linked to superoxide. This configuration corresponds to a superoxide tetrahydrate complex that is very stable in water [26]. The dynamics of the reaction is fast at this site. Reaction at position 40 needs a more complex configuration since another water must be linked to the radical. This configuration is less stable and its occurrence is lower. But, when it is reached, the hydrogen atom transfer occurs in less than 100 fs. The dynamics of reaction seems to be faster at position 7 in large clusters of water. In small clusters, this is no more the case. Reaction occurs in smaller clusters at position 40 than at position 7. This is certainly linked to the lower BDE at position 40 . 5. Conclusion This study has explored the ab initio dynamic reactivity of quercetin anion at different sites with low BDE. It has shown that the reaction is very rapid at two sites, 7 and 40 and that superoxide does not react with the hydroxyl group at position 3, which is in accordance with experimental results. Dynamic simulations have been performed with different initial conditions varying the total number of solvent molecules and the number of water directly linked to superoxide radical. They have shown that reactivity depends on different factors: – Electrostatic environment of the hydroxyl. Superoxide radical is an anion with a small size; it does not go in the vicinity of negative charges. This explains the discrepancies between the results of experimental assays using neutral or positively charged radicals and superoxide. – Total number of water molecules in the cluster. The presence of surrounding water molecules stabilizes the products.

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– Junction of HOMO lobes. The reaction occurs when there is a junction of the two lobes surrounding the oxygen atoms of hydroxyl and superoxide, permitting a tunneling of the electron from quercetin to superoxide. This junction is allowed when adequate positions of surrounding water molecules are achieved. The present work could provide a basis to a more general study of the superoxide radical scavenging properties of polyphenols and endogenous antioxidants in solution. It could be interesting to investigate if the present findings can be generalized to neutral flavonoids or other antioxidants. Acknowledgments The calculations have been made with an SGI computer and a cluster IBM P5-575 purchased with the funds of the Région Aquitaine, France. These computational facilities are provided by the ‘‘Pôle Modélisation” of the ISM and the MCIA (Mesocentre de Calcul Intensif Aquitain) of the University of Bordeaux. The Conseil Regional d’Aquitaine and the French Ministry of Research and Technology fund the two structures. The author also thanks P. Aurel for helpful assistance. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.chemphys.2016. 06.006. References [1] D. Salvemini, Z. Wang, J.L. Zweiner, Science 286 (1999) 304. [2] R.P. Wayne, Chemistry of Atmospheres: An Introduction to the Chemistry of Atmospheres of Earth, The Planets, and their Satellites, second ed., Clarendon Press: Oxford University Press, Oxford, England, 1991. [3] B. Halliwell, Trends Pharmacol. Sci. 32 (2011) 125–130. [4] O.I. Aruoma, in: Mutat. Res. 532 (2003) 9–20. [5] J.M. McCord, Am. J. Med. 108 (2000) 652–659. [6] J.S. Wright, E.R. Johnson, G.A. Di Labio, J. Am, Chem. Soc. 123 (2001) 1173– 1183. [7] N.J. Miller, C.A. Rice-Evans, Redox Rep. 2 (1996) 161–171. [8] W. Brand-Williams, M.E. Cuvelier, C. Berset, Food Sci. Technol. 28 (1995) 25– 30. [9] L. Lespade, S. Bercion, Free Radical Res. 46 (2012) 346–358. [10] J. Robak, R. Gryglewsky, Biochem. Pharmacol. 37 (1988) 837–841. [11] H. Matsuda, T. Wang, H. Managi, M. Yosikawa, Bioorg. Med. Chem. 11 (2003) 5317–5323. [12] P. Cos, L. Ying, M. Calomme, J.P. Hu, K. Cimanga, B. Van Poel, L. Pieters, A. Vlietinck, D. Vanden Berghe, J. Nat. Prod. 61 (1998) 71. [13] A.I. Huguet, S. Manez, M.J. Alcaraz, Z. Naturforsch. C 45 (1990) 19–24. [14] D. Taubert, T. Breitenbach, A. Lazar, P. Censarek, S. Harlfinger, R. Berkels, W. Klaus, R. Roesen, Free Radic. Biol. Med. 35 (2003) 1599–1607. [15] N. Masuoka, M. Matsuda, I. Kubo, Food Chem. 131 (2012) 541–545. [16] L. Lespade, S. Bercion, J. Phys. Chem. 114 (2010) 921–928. [17] Z. Dhaouadi, M. Nsangou, N. Garrab, E.H. Anouar, K. Marakchi, S. Lahmar, J. Mol. Struct. Theochem. 904 (2009) 35. [18] S. Miertus, E. Scrocco, J. Tomasi, Chem. Phys. 55 (1981) 117. [19] L. Lespade, J. Theoret. Chem. (2014), http://dx.doi.org/10.1155/2014/740205. [20] Drugbank, . [21] R. Carr, M. Parrinello, Phys. Rev. Lett. 55 (1985) 2471. [22] CPMD, IBM Corp.: Armonk, NY, 2006; MPI für Festkörperforshung: Stuttgart, Germany, 2001. [23] M.J. Frisch, G.W. Trucks, H.B. Schlegel, G.E. Scuseria, M.A. Robb, J.R. Cheeseman, J.A. Montgomery Jr., T. Vreven, K.N. Kudin, J.C. Burant, J.M. Millam, S.S. Iyengar, J. Tomasi, V. Barone, B. Mennucci, M. Cossi, G. Scalmani, N. Rega, G.A. Petersson, H. Nakatsuji, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, M. Klene, X. Li, J.E. Knox, H. P. Hratchian, J.B. Cross, C. Adamo, J. Jaramillo, R. Gomperts, R.E. Stratmann, O. Yazyev, A.J. Austin, R. Cammi, C. Pomelli, J.W. Ochterski, P.Y. Ayala, K. Morokuma, G.A. Voth, P. Salvador, J.J. Dannenberg, V.G. Zakrzewski, S. Dapprich, A.D. Daniels, M.C. Strain, O. Farkas, D.K. Malick, A.D. Rabuck, K. Raghavachari, J.B. Foresman, J.V. Ortiz, Q. Cui, A.G. Baboul, S. Cliford, J. Cioslowski, B.B. Stefanov, G. Liu, A. Liashenko, P. Piskorz, I. Komaromi, R.L. Martin, D.J. Fox, T. Keith, M.A. Al-Laham, C.Y. Peng, A. Nanayakkara, M.

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