Mechanism of Action of Sulforaphane as a Superoxide Radical Anion

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Mechanism of Action of Sulforaphane as a Superoxide Radical Anion and Hydrogen Peroxide Scavenger by Double Hydrogen Transfer: A Model for Iron Superoxide Dismutase Ajit Kumar Prasad, and Phool Chand Mishra J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.5b01496 • Publication Date (Web): 28 May 2015 Downloaded from http://pubs.acs.org on June 3, 2015

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Mechanism of Action of Sulforaphane as a Superoxide Radical Anion and Hydrogen Peroxide Scavenger by Double Hydrogen Transfer: A Model for Iron Superoxide Dismutase Ajit Kumar Prasad and P.C. Mishra* Department of Physics Banaras Hindu University Varanasi – 221 005 (India)

Abstract The mechanism of action of sulforaphane as a scavenger of superoxide radical anion (O2•‾) and hydrogen peroxide (H2O2) has been investigated using density functional theory in both gas phase and aqueous media. Iron-superoxide dismutase (Fe-SOD) involved in scavenging superoxide radical anion from biological media was modeled by a complex consisting of the ferric ion (Fe3+) attached to three histidine rings. Reactions related to scavenging of superoxide radical anion by sulforaphane were studied using density functional theory (DFT) in the presence and absence of Fe-SOD represented by this model in both gas phase and aqueous media. The scavenging action of sulforaphane towards both superoxide radical anion and hydrogen peroxide was found to involve the unusual mechanism of double hydrogen transfer. It was found that sulforaphane alone, without FeSOD, cannot scavenge superoxide radical anion in gas phase or aqueous media efficiently as the corresponding reaction barriers are very high. However, in the presence of Fe-SOD represented by the above mentioned model, the scavenging reactions become barrierless and so sulforaphane scavenges superoxide radical anion by converting it to hydrogen peroxide efficiently. Further, sulforaphane has been found to scavenge hydrogen peroxide also very efficiently by converting it into water. Thus the mechanism of action of sulforaphane as an excellent anti-oxidant has been unravelled. *NASI Senior Scientist. Corresponding author: E-mail [email protected]. Keywords: Sulforaphane, Anti-oxidant, O2•‾ scavenger, H2O2 scavenger, DFT calculation.

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INTRODUCTION Reactive oxygen species (ROS) and reactive nitrogen oxide species (RNOS) are mostly free radicals that are produced in biological systems during metabolic activities, and these can also be formed due to exposure of biological systems to radiation or pollution. Due to their high chemical reactivities, ROS and RNOS can cause oxidation and nitration of DNA, proteins and enzymes and thus give rise to several diseases including cancer.1-4 Superoxide radical anion (O2•), hydroxyl radical (OH•), hydrogen peroxide (H2O2), nitrogen dioxide (NO2•), peroxynitrite (ONOO-) etc. belong to the ROS or RNOS family.5 The superoxide radical anion is intrinsically poorly reactive but in the presence of other chemical species, it can produce other highly reactive agents. For example, its reaction with nitric oxide (NO•) produces peroxynitrite (ONOO-), both these species being poorly reactive, but when this reaction occurs in the presence of CO2, the complex nitrosoperoxycarbonate is formed which dissociates rapidly into two highly reactive species i.e. nitrogen dioxide (NO2•) and carbonate radical anion (CO3.-).6 Thus, superoxide radical anion can play crucial roles in causing DNA damage, depolymerize polysaccharides, peroxidize lipids, kill mammalian cells and inactivate enzymes.7-14 Generation of superoxide radical anion signals the first sign of oxidative burst.15 Superoxide radical anion is produced in the human body by one electron reduction of molecular oxygen through the involvement of various oxidative enzymes.16,17 The enzyme xanthine oxidase catalyses production of superoxide radical anion18 while the enzyme superoxide dismutase (SOD) catalyses its conversion to H2O2 .19,20 SOD can have different forms depending on the presence of cation of iron, zinc, manganese or copper in it. The enzymes glutathione peroxidase and catalase serve as strong endogenous anti-oxidants as the former catalyses conversion of H2O2 into water while the latter catalyses its conversion into water and oxygen.21-24

Certain non-enzymatic exogenous antioxidants which can be taken as components of diet include ascorbic acid, α-tocopherol, vitamin A, lycopene etc.25 Epidemiological studies have shown that an increased dietary intake of fruits and vegetables strongly decreases the risk of several chronic diseases such as cardiovascular diseases, neurological disorders, diabetes, cancer etc.26,27 Several plant metabolites including polyphenolics, glucosinolates and allyl sulfides play crucial roles in the prevention of several diseases.28,29 Sulphoraphane is also a naturally occurring sulfur containing compound that is present in certain vegetables such as broccoli sprouts, cabbage,


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Brussels sprouts, cauliflower, bok choy, collards, mustard, turnip etc.30 Several studies have revealed that sulforaphane possesses strong anti-oxidant and anti-cancer properties. It has been shown that sulforaphane inhibits histone deacetylase (HDAC) activity in human colon, prostate, breast, cervical and ovarian cancers as also in human leukemia cells.31-33 In other studies, combinations of sulforaphane with cytotoxic drugs e.g. cisplatin, gemcitabine, doxorubicin etc. have been found to enhance effectiveness of treatment of cancer.34 Sulforaphane is highly effective in reducing the androgen receptor (AR) protein level by decreasing secretion of prostate specific antigen (PSA) which is an AR regulated gene product in human prostate cancer cells.35 Sulforaphane has numerous other highly useful medical applications.36-41

Electronic and vibrational properties of sulforaphane have been studied using a semiempirical method.42 Yuan et al43 have shown experimentally that sulforaphane can scavenge both superoxide radical and hydroxyl radical. Niu et al44 have shown that benzynes are capable of removal of two vicinal hydrogen atoms from a hydrocarbon in a concerted manner which causes alkane to alkene conversion. To the best of our knowledge, double hydrogen abstraction by superoxide radical anion or hydrogen peroxide from sulforaphane or any other anti-oxidant has not yet been studied theoretically. The purpose of the present study is to show that both superoxide radical anion and hydrogen peroxide can be scavenged through double hydrogen abstraction from sulforaphane, the reactions in the former case being catalyzed by a SOD. Other anti-oxidants scavenge other ROS through single hydrogen abstraction or addition reaction mechanisms.45 The novelty of the present work is that it is the first theoretical study having the following three important features: (i) it explains the mechanism of scavenging action of sulforaphane towards superoxide radical anion and hydrogen peroxide, (ii) it shows double hydrogen transfer from an anti-oxidant i.e. sulforaphane to both superoxide radical anion and hydrogen peroxide, and (iii) it presents a simple model for iron superoxide dismutase (Fe-SOD) that explains well the catalytic activity of the enzyme. 2. COMPUTATIONAL DETAILS All the calculations reported here were performed at the B3LYP/6-311+G(d) and M06-2X/6311+G(d) levels of theory. Geometries of all the reactant molecules, reactant complexes (RCs), transition states (TSs) and product complexes (PCs) were optimized in gas phase at the



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B3LYP/6-311+G(d) and M06-2X/6-311+G(d) levels of density functional theory.46-50 Solvent effect of aqueous media was treated by single point energy calculations using the gas phase optimized geometries and employing the integral equation formalism of the polarizable continuum model (IEF-PCM)51,52 at the same level of theory at which the gas phase result was obtained. However, there were certain exceptions as follows: (i) In certain cases, geometry optimization calculations at the M06-2X/6-311+G(d) level could not be completed due to internal errors or lack of convergence. In these cases, as indicated in the corresponding sections, single point energy calculations were performed at the M06-2X/6-311+G(d) level using the geometries optimized at the B3LYP/6-311+G(d) level. And, (ii) In the study of reactions involving sulforaphane and hydrogen peroxide, geometries of the RCs, TSs and PCs were optimized in both gas phase and aqueous media at both the B3LYP/6-311+G(d) and M06-2X/6311+G(d) levels of density functional theory. Reactions were considered to initiate from optimized geometries of RCs. Intrinsic reaction coordinate (IRC) calculations53 were performed using optimized TSs to confirm that hydrogen transfer actually took place from the considered sites of sulforaphane. Forward Gibbs barrier energies were calculated as differences of Gibbs free energies of the corresponding TSs and RCs while reverse Gibbs barrier energies were obtained as differences of Gibbs free energies of the corresponding TSs and PCs at 298.15K. Vibrational frequency analysis was carried out for each optimized geometry. TSs were found to be associated with one imaginary vibrational frequency each whereas all RCs and PCs were characterized by all real vibrational frequencies. Genuineness of the calculated transition states was confirmed by visually examining the vibrational modes related to the imaginary frequencies and applying the condition that these connected the corresponding reactant and product complexes properly. Rate constants were calculated using the transition states theory.54-56 The Windows version of the Gaussian 09 suite of programs (G09W, ver. B.01) was used for all the calculations.57 The optimized geometries and vibrational modes were visualized using the GaussView program (ver. 5.0).58 3. RESULTS AND DISCUSSION 3.1 Scavenging superoxide radical anions (O2•‾) (i) Without iron superoxide dismutase (Fe-SOD)


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Superoxide radical anion reacts with other molecules to form hydrogen peroxide.59,60 Our calculations indicated that double hydrogen abstraction from sulforaphane by superoxide radical anion can occur much more easily than single hydrogen abstraction, and therefore we have investigated only the former mechanism. For example, when a transition state search was made by placing superoxide radical anion near the (C7,C9) pair of carbon atoms (Fig. 1) such that one of its oxygen atoms was nearer to one of the hydrogen atoms of the methyl (C9H3) group and the initial C9H and OH distances were ~1.3 Å each. When the transition state search was continued for 10 steps, the H atom that was to be abstracted from the C9 site came back near it so that the C9H and OH distances changed to ~1.11 and 1.66 Å. Thus the transition state search involving a single hydrogen abstraction from the C9 site (Fig. 1) did not succeed. Similar observations were made in other cases involving single hydrogen abstraction by superoxide radical anion also. By double hydrogen abstraction from an alkyl chain, an organic molecule would get converted from the alkane to the alkene form. This transformation is an interesting aspect of chemical synthesis.44 Double hydrogen transfer occurring from sulforaphane can be represented as follows

S-Hn + O2•‾

∆Gnb

S-Hn-2 + H2O2

(1)

where ∆Gnb is the forward Gibbs barrier energy of the reaction, S-Hn is the sulforaphane molecule with n hydrogen atoms and S-Hn-2 is a form of the molecule with (n-2) hydrogen atoms, the two hydrogen atoms having been abstracted from two neighboring carbon sites of sulforaphane, and superoxide radical anion (O2•‾) is converted to hydrogen peroxide (H2O2) due to the reaction. Pairs of hydrogen atoms can be abstracted from the pairs of neighboring sites of sulforaphane i.e. (C4, C5), (C5, C6), (C6, C7) and (C7, C9) (Fig.1). Gibbs free energies of RCs, TSs and PCs corresponding to double hydrogen abstraction by superoxide radical anion from different pairs of neighboring carbon sites of sulforaphane obtained using B3LYP and M06-2X functionals along with the 6-311+G(d) basis set in gas phase and aqueous media are presented in Table S1. The calculated Gibbs interaction energies of RCs as well as forward and reverse Gibbs barrier energies corresponding to double hydrogen abstraction by superoxide radical anion from sulforaphane in gas and aqueous media are



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presented in Table 1. The gas phase barrier energies for double hydrogen abstraction from the (C6,C7) sites given in this table were obtained by geometry optimization at both the B3LYP/6311+G(d) and M06-2X/6-311+G(d) levels while those from the other sites were obtained by single point energy calculations at the M06-2X/6-311+G(d) level using the geometries optimized at the B3LYP/6-311+G(d) level. The Gibbs barrier energies in aqueous media were obtained by single point energy calculations at the same levels at which the gas phase barrier energies were obtained. The Gibbs interaction energies of RCs (defined in each case as Gibbs free energy of the complex – sum of Gibbs free energies of the individual reactants) in gas phase and aqueous media obtained at both the levels of theory mentioned above and included in Table 1 reveal the following information. The calculated gas phase Gibbs interaction energies are all negative while those in aqueous media are all positive. When superoxide radical anion is solvated in aqueous media, it gets highly stabilized or trapped due to the negative charge and associated polarization of the aqueous medium. Due to this reason, the second term in the above definition of Gibbs interaction energy in aqueous media becomes highly negative making the corresponding Gibbs interaction energies positive. Positive Gibbs interaction energies imply that the reactants under ambient conditions would not be likely to make stable complexes due to which the probability of reactions would be highly reduced. The optimized structures of reactant complexes (RC1, RC2, RC3 and RC4) and transition states (TS1, TS2, TS3 and TS4) involved in double hydrogen transfer from sulforaphane to superoxide radical anion are shown in Figs. 2(a,b,c,d) and Figs. 3(a,b,c,d) respectively. In these figures, the corresponding imaginary vibrational frequencies (ν) are also presented. The optimized structures of product complexes (PC1, PC2, PC3 and PC4) formed by double hydrogen abstraction from sulforaphane by superoxide radical anion are presented in Fig. 4(a,b,c,d). Certain optimized geometrical parameters at the M06-2X/6-311+G(d) level of theory are also given in Figs. 2(a,b,c,d), 3(a,b,c,d) and 4(a,b,c,d). It is noted that in one case i.e. in the product complex PC2 corresponding to double hydrogen abstraction from the (C5, C6) pair of carbon sites (Fig. 4(b)), the OSCH3 portion of sulforaphane gets dissociated from the rest of the molecule. The forward Gibbs barrier energies for double hydrogen abstraction by superoxide radical anion from the different pairs of carbon sites of sulforaphane in gas phase and aqueous media


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presented in Table 1 and obtained at the B3LYP/6-311+G(d) or M06-2X/6-311+G(d) level of theory lie in the range between ~35 and ~50 kcal/mol. All these calculated Gibbs forward barrier energies in both gas phase and aqueous media are very high (Table 1). The calculated reverse barrier energies for double hydrogen abstraction presented in Table 1 are quite different for the different pairs of carbon sites. These barrier energies corresponding to double hydrogen abstraction from the (C4,C5) and (C5,C6) pairs of sites in gas phase and aqueous media obtained at both the levels of theory mentioned above lie in the range ~12 to ~21 kcal/mol (Table 1). The corresponding reverse Gibbs barrier energies for the reaction from the (C6, C7) pair of sites lie in the range ~2.9 to ~6.5 kcal/mol while those for the reaction involving the (C6, C7) pair of sites lie in the range -1.44 to 2.1 kcal/mol (Table 1). The two negative reverse Gibbs barrier energies (Table 1) imply that the energies under consideration lie above the Gibbs barrier due to which the reaction cannot occur. All other reverse Gibbs barrier energies are positive but appreciably or much smaller than the corresponding forward Gibbs barrier energies due to which the product complexes would be appreciably or highly endothermic. The calculated rate constants for double hydrogen abstraction from the different pairs of sites of sulforaphane at the B3LYP/6-311+G(d) and M06-2X/6-311+G(d) levels of theory using the forward Gibbs barrier energies given in Table 1 (not given) have very small values (

Mechanism of Action of Sulforaphane as a Superoxide Radical Anion

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