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Scavenging of hydroxyl, methoxy, and nitrogen dioxide free radicals by some methylated isoflavones

  • Manish Kumar Tiwari
  • Phool Chand MishraEmail author
Original Paper
  • 81 Downloads
Part of the following topical collections:
  1. International Conference on Systems and Processes in Physics, Chemistry and Biology (ICSPPCB-2018) in honor of Professor Pratim K. Chattaraj on his sixtieth birthday

Abstract

Free radicals can be scavenged from biological systems by genistein, daidzein, and their methyl derivatives through hydrogen atom transfer (HAT), single-electron transfer (SET), and sequential proton-loss electron-transfer (SPLET) mechanisms. Reactions between these derivatives and the free radicals OH., OCH3., and NO2. via the HAT mechanism in the gas phase were studied using the transition state theory within the framework of DFT. Solvation of all the species and complexes involved in the HAT reactions in aqueous media was treated by performing single point energy calculations using the polarizable continuum model (PCM). The SET and SPLET mechanisms for the above reactions were also considered by applying the Marcus theory of electron transfer, and were found to be quite sensitive to geometry and solvation. Therefore, the geometries of all the species involved in the SET and SPLET mechanisms were fully optimized in aqueous media. The calculated barrier energies and rate constants of the HAT-based scavenging reactions showed that the OH group of the B ring in genistein, daidzein, and their methyl derivatives plays a major role in the scavenging of free radicals, and the role of this OH group in the HAT-based free-radical scavenging decreases in the following order: OH. > OCH3. > NO2.. The SPLET mechanism was found to be an important mechanism in these free-radical scavenging reactions, whereas the SET mechanism was not important in this context.

Keywords

Isoflavone Genistein Daidzein Methyl derivatives Antioxidant Free radicals 

Introduction

During normal cellular metabolism, various enzymatic and nonenzymatic reactions yield free radicals that are commonly known as reactive oxygen species (ROS) and reactive nitrogen oxide species (RNOS) [1, 2, 3]. The hydroxyl radical (OH.) is the most reactive ROS—it can react with any biological macromolecule [1, 2]. Hydroxyl radicals are produced during both enzymatic and nonenzymatic reactions or the radiolysis of water inside cells [1, 2]. Another oxygen-containing free radical is the methoxy radical. Methoxy radicals can be produced through several routes, such as the radiolysis of liquid methanol, the pyrolysis of dimethyl peroxide, and the photolysis of methyl acetate [4]. The nitrogen dioxide radical (NO2.), the hydroxyl radical (OH.), and the carbonate anion radical (CO3.-) are produced in the homolysis of peroxynitrite (ONOOH) and the dissociation of the nitrosoperoxycarbonate anion (ONOOCO2) [5, 6, 7]. High abundances of these free radicals inside a biological environment induces modifications to DNA bases, proteins, and lipids that hinder normal cell function, and can therefore result in serious conditions such as inflammation, premature aging, non-neurodegenerative diseases such as mutation, cancer, and cardiovascular diseases, as well as neurodegenerative diseases such as Alzheimer’s and Parkinson’s diseases [8, 9, 10, 11, 12, 13, 14, 15]. However, in biological environments, free radicals undergo enzymatic or nonenzymatic reactions with naturally occurring endogenous scavengers. Thus, the harmful effects of the free radicals are eliminated by either removing them or converting them into nonreactive forms. Diseases can develop when the concentration of endogenous antioxidants is too low to adequately to scavenge all of the free radicals generated in the biological environment. In that situation, exogenous antioxidants obtained from fruits and vegetables in the diet are very useful [16, 17, 18, 19].

Over the last few decades, studies in the field of dietary free-radical scavengers have yielded valuable results. Most of these scavengers are plant-derived polyphenolic phytochemicals. Flavonoids and isoflavonoids are families of polyphenols that include more than 4000 naturally occurring compounds [20]. Flavones and isoflavones are structurally similar to flavonoids and isoflavonoids except for the presence of a carbonyl group C=O and a C2=C3 double bond in the flavone or isoflavone structure [20, 21]. The general molecular structure of an isoflavone comprises two benzene rings A and B and a heterocyclic pyrone ring (ring C) fused to ring A [20, 21]. There is a carbonyl group attached to the C4 site of ring C, and ring B is connected to the C3 site of ring C (Fig. 1a, e) [20, 21]. Different isoflavones can be distinguished from one another by the substituents on rings A and B. Isoflavones are structurally very similar to mammalian estrogens, which explains why they are also known as phytoestrogens [22]. Phytoestrogens are also used as an alternative to hormone replacement therapy (HRT) and estrogen replacement therapy (ERT), although this can have serious side effects (e.g., induction of breast cancer [22, 23, 24, 25]). The molecular structures of two phytoestrogens, 5,7,4′-trihydroxyisoflavone (also called genistein) and 7,4′-dihydroxyisoflavone (also called daidzein), differ only in a C5–OH group that is present in the former molecule but not in the latter (Fig. 1a, e) [20, 21]. Genistein and daidzein are known to be effective drugs for ovarian disorders induced by chemotherapeutics [26, 27] and to be anticancer agents [28, 29, 30, 31, 32, 33]. They are the most abundant isoflavones in plant species of the pea family (Fabaceae, which includes the soybean), and they are also present in appreciable amounts in peas, nuts, grain products, coffee, tea sprouts, Moringa oleifera, ginkgo, and red clover. Both genistein and daidzein are phenolic and act as potent radical scavengers [34, 35].
Fig. 1

Optimized geometries of and atom numbering schemes used for genistein, daidzein, and their monomethyl and dimethyl derivatives, as obtained at the M06-2X/6-311++G(d,p) level of density functional theory in the gas phase

In previous studies, it was found that a recombinant E. coli expressing tyrosinase from Bacillus megaterium and a recombinant E. coli expressing O-methyltransferase from Streptomyces peucetius catalyze the hydroxylation and methylation of genistein and daidzein, respectively [36, 37, 38]. Thus, the presence of genetically modified microorganisms catalyzes biotransformations (hydroxylation and methylation) of soy isoflavones [36, 37, 38], which modifies the cellular environment (e.g., in terms of polarity), potentially affecting the antioxidant activities of phenolic molecules. Therefore, to understand the effects of the methylation of genistein and daidzein on their antioxidant activities, three methyl derivatives (the 7-O-methyl, 4′-O-methyl, and 7,4′-O-dimethyl derivatives) of both genistein and daidzein were investigated in the present study.

In view of the various useful pharmacological activities of isoflavones and their biotransformations, we probed the scavenging of three radicals (OH., NO2., and OCH3.) by genistein, daidzein, and some of their methyl derivatives via the hydrogen-atom transfer (HAT) mechanism and two electron-transfer mechanisms (single-electron transfer (SET) and sequential proton-loss electron transfer (SPLET)) using density functional theory, the Marcus theory of electron transfer [39, 40, 41], and transition state theory.

Hydrogen abstraction or hydrogen atom transfer (HAT) from a OH or OMe group of genistein, daidzein, or any of its three methyl derivatives (A–Hn) to a radical (R.) may be expressed by the following general equation:

$$ \mathrm{A}-{\mathrm{H}}_n+{\mathrm{R}}^{.}\kern0.5em \overset{\varDelta {G_n}^{\mathrm{b}}}{\to}\kern0.5em {\left[\mathrm{TS}\right]}_n\kern0.5em \overset{\varDelta {G_n}^{\mathrm{r}}}{\to}\kern0.5em {A}^{.}+{\mathrm{H}}_n-\mathrm{R}, $$
(1)
where A–Hn represents genistein, daidzein, or any of their three methyl derivatives; Hn is the hydrogen atom of a OH or OMe group that is bonded to the carbon atom Cn and is abstracted by a radical (OH., OCH3., or NO2.). After the necessary Gibbs activation energy (∆Gnb) has been provided, the transition-state complex [TS]n is formed. The product complex (A. + Hn–R) is then generated following the release of the Gibbs energy (∆Gnr). Each barrier energy was calculated with respect to the sum of the Gibbs free energies of the free reactants. ∆Gnb and ∆Gnr can also be considered the forward and reverse barrier energies, respectively [16, 17, 18, 19].
The single-electron transfer (SET) mechanism for the scavenging of free radicals (R.) by an antioxidant (A) can be presented as in the following general equation:
$$ \mathrm{A}+{\mathrm{R}}^{.}\kern0.5em \overset{\varDelta {G^{\mathrm{b}}}_{\mathrm{ET}}}{\to}\kern0.5em {\mathrm{A}}^{.}+{\mathrm{R}}^{-} $$
(2)
where A represents an electron donor (here, genistein, daidzein, or one of their three methyl derivatives), while R. is an electron acceptor (here, a hydroxyl radical (OH.), a methoxy radical (OCH3.), or a nitrogen dioxide radical (NO2.)) [16, 17, 18, 19].
The general two step sequential proton-loss electron-transfer (SPLET) reaction equation can be presented as follows:
$$ A\kern0.5em {\left(\mathrm{A}-\mathrm{H}\right)}^{-}+{\mathrm{H}}^{+}\to $$
(3a)
$$ {\left(\mathrm{A}-\mathrm{H}\right)}^{-}+{\mathrm{R}}^{.}\overset{\varDelta {G^{\mathrm{b}}}_{\mathrm{SPLET}}}{\to }{\left(\mathrm{A}-\mathrm{H}\right)}^{.}+{\mathrm{R}}^{-} $$
(3b)
where A is the antioxidant, (A–H) is the deprotonated antioxidant anion, R. is the radical to be scavenged, (A–H). is the radical after the loss of one hydrogen, and R is the anion of the free radical (which is less reactive than the corresponding free radical) [42, 43, 44].

Computational details

First, we searched for the most stable conformers of genistein and daidzein by considering different relative orientation of the OH groups which have to be found similar, as reported in a previous study of daidzein in particular [45]. The geometries of the various conformers of genistein and daidzein were initially optimized in the gas phase at the unrestricted M06-2X/6-31G(d,p) level of theory, after which further geometry optimization calculations were performed at the M06-2X/6-311++G(d,p) level of unrestricted density functional theory (DFT) [46, 47, 48]. The solvation of the various conformers and complexes in aqueous media was treated by implementing single point energy calculations based on the polarizable continuum model (PCM) [49, 50]. For the HAT-based scavenging reactions of genistein, daidzein, or their monomethyl and dimethyl derivatives with any of the free radicals considered here (OH., OCH3., and NO2.), transition state theory was employed to obtain barrier and released energies using full geometry optimization of transition states (TSs), free reactants, and product complexes (PCs) in the gas phase by applying the unrestricted M06-2X DFT functional along with the 6-31G(d,p) basis set. These calculations were followed by single point energy calculations using the same DFT functional along with the 6-311++G(d,p) basis set. Barrier energies for the HAT mechanism were calculated with respect to the energies of the free reactants in both the gas phase and in aqueous media. For all of the TSs and PCs, the expectation value of the spin operator <S2> was found to be close to 0.75, corresponding to doublet multiplicity. Thus, spin contamination was negligible. Vibrational frequency analysis was carried out, as were thermal energy corrections to the total energies, in order to obtain Gibbs barrier energies at 298.15 K in the gas phase. The thermal energy corrections obtained in the gas phase were also considered to be valid for aqueous media in all cases.

To study the scavenging of the three free radicals (OH., OCH3., and NO2.) through single-electron transfer (SET) and sequential proton-loss electron-transfer (SPLET) mechanisms, full geometry optimization was carried out for all the species involved in the calculations at the M06-2X/6-311++G(d,p) level of unrestricted density functional theory in aqueous media. The SET and SPLET mechanisms were studied using the Marcus theory [39, 40, 41] of electron transfer, as used by Nelsen and coworkers [51, 52]. The calculated barrier energies for different reaction mechanisms were used to calculate rate constants [53] as follows:
$$ k=\Gamma (T)\left({K}_{\mathrm{B}}T/h\right)\exp \left(-\Delta {G}^{\mathrm{b}}/ RT\right), $$
where k is the thermal rate constant, Г(T) is the quantum-mechanical tunneling factor, KB is the Boltzmann constant, T is the absolute temperature (298.15 K), h is Planck’s constant, R is the gas constant, and ∆Gb is the Gibbs barrier energy for the reaction under consideration. The quantum-mechanical tunneling factor Г(T) was computed via the asymmetric parabolic potential barrier method of Skodje and Truhlar, which has been shown to provide comparable accuracy to that given by the WKB method [54]. The Windows version of the Gaussian G09 suite of programs (G09 W) [55] was used for all calculations, while the GaussView (ver. 5.0) program was used to visualize molecular structures and vibrational modes [56].

Results and discussion

Radical scavenging mainly involves the hydrogen atom transfer (HAT), single-electron transfer (SET), and sequential proton-loss electron-transfer (SPLET) mechanisms [16, 17, 18, 19]. In previous studies, the radical scavenging abilities of two isoflavones, genistein and daidzein, were estimated theoretically by calculating the bond dissociation enthalpy (BDE), ionization potential (IP), proton affinity (PA), and electron transfer enthalpy (ETE) using the B3LYP functional along with three different basis sets (6-31G(d,p), 6-31 + G(d,p), and 6-311++G(d,p)) in the gas phase, an aqueous medium, and benzene [57, 58]. Those previous studies suggested that the minimum bond dissociation enthalpy (BDE) for the cleavage of a hydrogen atom from the hydroxyl group at the C4′ site of the B ring of the isoflavone is about ~81.0 kcal/mol [57, 58]. Therefore, hydrogen atom transfer (HAT) from the B ring must be prominently involved in the radical scavenging abilities of isoflavones. Similarly, the calculated values of IP, PA, PDE, and ETE for different kinds of isoflavones support the idea that electron transfer may also play an important role [57, 58]. In view of the abovementioned studies, we considered hydrogen atom transfer using transition state theory and electron-transfer mechanisms with Marcus theory.

Molecular structures and conformations

There are various relative orientations of the OH groups in the isoflavones of interest: genistein has eight and daidzein has four different conformations (see Fig. S1 (G1–G8, D1–D4) in the “Electronic supplementary material,” ESM). To generate the monomethyl and dimethyl derivatives of genistein and daidzein, the 7-OH group and/or 4′-OH group of genistein and daidzein were substituted with methyl groups (OMe) while retaining the relative orientations of the three rings. This allowed us to probe the effects of the mono- and dimethylation of genistein and daidzein on hydrogen abstraction from these isoflavones. The geometries of the various conformations of genistein, daidzein, and their methyl derivatives were optimized at the M06-2X/6-311++G(d,p) level of theory in the gas phase and are presented in Fig. 1a–h.

Hydrogen abstraction

The Gibbs barrier energies and released energies for the HAT reactions involved in the scavenging of the free radicals OH., OCH3., and NO2. by genistein, daidzein, and their 7-O-methyl, 4′-O-methyl, and 7,4′-O-dimethyl derivatives, as calculated using Eq. 1 at the M06-2X/6-31G(d,p) and M06-2X/6-311++G(d,p) levels of theory in the gas phase and aqueous media, are presented in Tables 1, 2, and 3. As the results obtained in aqueous media are more relevant to biological environments than those obtained in the gas phase, only the results obtained at the M06-2X/6-311++G(d,p) level of density functional theory in aqueous media are discussed below.
Table 1

Gibbs barrier energies (∆Gb, kcal/mol) and released energies (∆Gr, kcal/mol) obtained at different levels of theory in the gas phase and aqueous media at 298.15 K for HAT reactions at different sites on genistein, daidzein, and their mono- and dimethyl derivatives during the scavenging of the free radical OH. by those isoflavones

Molecules

Reaction site

Genistein and its derivatives

Daidzein and its derivatives

M06-2X/6-31G(d,p)

M06-2X/6-311++G(d,p)a

M06-2X/6-31G(d,p)

M06-2X/6-311++G(d,p)a

Nonmethylated molecule

G5b

13.2 (14.6)

15.2 (15.0)

G5r

−25.0 (−29.2)

−27.8 (−30.8)

G7b

6.5 (9.6)

8.7 (12.2)

9.7 (12.3)

13.5 (16.1)

G7r

−19.6 (−28.2)

−27.6 (−28.5)

−32.5 (−32.6)

−35.5 (−35.8)

G4′b

4.9 (7.9)

8.2 (8.5)

5.2 (8.2)

7.4 (7.8)

G4′r

−32.2 (−34.4)

−33.4 (−35.3)

−32.9 (−35.1)

−34.3 (−34.1)

7-O-methyl derivatives

G5b

16.1 (16.0)

17.9 (17.3)

G5r

−28.3 (−30.6)

−31.3 (−33.2)

G7b

8.1 (9.7)

9.3 (10.8)

8.0 (9.8)

9.7 (11.0)

G7r

−21.1 (−21.8)

−22.9 (−23.3)

−20.6 (−21.5)

−22.6 (−23.2)

G4′b

5.1 (8.4)

6.9 (9.4)

5.0 (7.8)

6.8 (9.5)

G4′r

−32.8 (−35.3)

−33.3 (−35.7)

−32.7 (−34.6)

−33.8 (−35.7)

4′-O-methyl derivatives

G5b

12.3 (13.7)

14.4 (14.6)

G5r

−23.7 (−27.9)

−26.1 (−29.4)

G7b

6.4 (9.6)

8.6 (10.2)

5.6 (9.0)

8.0 (7.6)

G7r

−26.5 (−28.5)

−28.0 (−30.6)

−28.6 (−29.5)

−30.1 (−27.5)

G4′b

9.9 (11.1)

10.3 (11.3)

7.3 (9.0)

8.4 (10.0)

G4′r

−23.8 (−24.7)

−25.2 (−25.1)

−21.1 (−22.2)

−23.0 (−3.8)

7,4′-O-dimethyl derivatives

G5b

16.3 (16.1)

18.1 (17.5)

G5r

−28.4 (−30.7)

−31.3 (−33.3)

G7b

8.1 (9.7)

9.3 (10.8)

13.1 (9.8)

9.3 (11.1)

G7r

−21.0 (−21.7)

−22.7 (−23.1)

−20.6 (−24.0)

−22.6 (−23.2)

G4′b

9.9 (11.2)

10.2 (11.3)

15.0 (11.2)

10.1 (11.3)

G4′r

−23.4 (−23.8)

−24.8 (−24.8)

−23.6 (−24.0)

−25.0 (−25.0)

Results obtained in aqueous media are given in parentheses

aObtained by performing single point energy calculations using the corresponding geometries optimized at the M06-2X/6-31G(d,p) level

Table 2

Gibbs barrier energies (∆Gb, kcal/mol) and released energies (∆Gr, kcal/mol) obtained at different levels of theory in the gas phase and aqueous media at 298.15 K for HAT reactions at different sites on genistein, daidzein, and their mono- and dimethyl derivatives during the scavenging of the free radical OCH3. by those isoflavones

Molecules

Reaction site

Genistein and its derivatives

Daidzein and its derivatives

M06-2X/6-31G(d,p)

M06-2X/6-311++G(d,p)a

M06-2X/6-31G(d,p)

M06-2X/6-311++G(d,p)a

Nonmethylated molecule

G5b

19.9 (19.3)

21.9 (21.1)

G5r

−17.7 (−20.2)

−19.2 (−21.8)

G7b

10.4 (13.5)

21.8 (16.1)

9.5 (10.9)

21.0 (15.7)

G7r

−16.5 (−19.0)

−26.6 (−20.4)

−18.0 (−18.9)

−27.6 (−19.6)

G4′b

7.8 (10.3)

9.9 (12.2)

10.2 (8.8)

10.3 (12.8)

G4′r

−21.3 (−22.9)

−21.6 (−23.1)

−23.1 (−23.5)

−22.2 (−23.7)

7-O-methyl derivatives

G5b

20.1 (19.5)

22.0 (21.3)

G5r

−18.3 (−20.7)

−19.9 (−22.2)

G7b

13.9 (15.2)

12.8 (15.1)

16.0 (17.1)

17.5 (18.8)

G7r

−12.0 (−12.9)

−12.5 (−13.6)

−14.1 (−14.3)

−15.7 (−15.8)

G4′b

7.6 (10.3)

9.7 (12.2)

7.8 (10.6)

9.9 (12.6)

G4′r

−20.6 (−22.2)

−21.0 (−22.5)

−21.7 (−23.3)

−22.0 (−23.5)

4′-O-methyl derivatives

G5b

20.3 (19.4)

22.4 (21.0)

G5r

−18.5 (−20.1)

−19.7 (−21.9)

G7b

10.7 (14.0)

12.4 (16.4)

9.2 (13.6)

13.6 (15.3)

G7r

−17.8 (−19.1)

−27.8 (−19.9)

−17.9 (−19.6)

−20.6 (−19.5)

G4′b

13.4 (14.9)

14.9 (16.6)

13.8 (15.2)

15.2 (16.9)

G4′r

−12.8 (−13.4)

−14.6 (−14.9)

−13.9 (−14.5)

−15.6 (−16.1)

7,4′-O-dimethyl derivatives

G5b

20.1 (19.5)

22.1 (21.2)

G5r

−18.1 (−20.6)

−19.8 (−22.2)

G7b

16.4 (17.2)

17.7 (18.8)

16.1 (17.2)

17.6 (18.8)

G7r

−14.5 (−13.9)

−16.0 (−16.4)

−14.8 (−14.8)

−16.4 (−16.0)

G4′b

14.6 (16.0)

16.0 (17.6)

14.1 (15.6)

15.6 (17.3)

G4′r

−13.3 (−13.9)

−15.0 (−15.4)

−13.5 (−14.1)

−15.3 (−15.7)

Results obtained in aqueous media are given in parentheses

aObtained by performing single point energy calculations using the corresponding geometries optimized at the M06-2X/6-31G(d,p) level

Table 3

Gibbs barrier energies (∆Gb, kcal/mol) and released energies (∆Gr, kcal/mol) obtained at different levels of theory in the gas phase and aqueous media at 298.15 K for HAT reactions at different sites on genistein, daidzein, and their mono- and dimethyl derivatives during the scavenging of the free radical NO2. by those isoflavones

Molecules

Reaction site

Genistein and its derivatives

Daidzein and its derivatives

M06-2X/6-31G(d,p)

M06-2X/6-311++G(d,p)a

M06-2X/6-31G(d,p)

M06-2X/6-311++G(d,p)a

Nonmethylated molecule

G5b

40.2 (42.7)

40.4 (32.0)

G5r

−17.3 (−23.8)

−17.7 (−13.6)

G7b

26.1 (32.0)

29.2 (32.4)

24.3 (28.1)

27.5 (33.3)

G7r

−10.9 (−19.0)

−11.8 (−15.4)

−9.8 (−13.0)

−7.9 (−12.5)

G4′b

20.1 (16.3)

23.0 (17.5)

29.3 (12.5)

21.3 (16.0)

G4′r

−13.6 (−10.5)

−14.3 (−9.5)

−14.9 (−12.0)

−15.2 (−14.0)

7-O-methyl derivatives

G5b

33.4 (27.3)

35.0 (27.5)

G5r

−14.3 (−11.6)

−15.2 (−11.5)

G7b

30.5 (30.2)

32.6 (32.2)

30.3 (30.3)

32.2 (32.2)

G7r

−7.9 (−7.8)

−9.1 (−8.8)

−7.5 (−7.5)

−9.7 (−8.5)

G4′b

19.7 (15.2)

22.8 (31.3)

19.0 (14.7)

22.1 (15.6)

G4′r

−14.5 (−11.2)

−15.9 (−13.0)

−13.1 (−9.2)

−13.9 (−7.8)

4′-O-methyl derivatives

G5b

34.8 (28.4)

36.5 (28.1)

G5r

−13.7 (−11.2)

−14.7 (−10.4)

G7b

24.7 (23.0)

28.1 (24.4)

30.1 (23.5)

26.9 (25.3)

G7r

−9.9 (−8.3)

−11.2 (−7.8)

−11.56 (−10.2)

−12.6 (−9.9)

G4′b

28.8 (28.4)

30.6 (30.2)

29.7 (28.5)

30.8 (30.4)

G4′r

−7.2 (−7.0)

−8.4 (−5.9)

−7.3 (−6.9)

−8.1 (−7.6)

7,4′-O-dimethyl derivatives

G5b

34.6 (28.1)

36.2 (27.5)

G5r

−14.0 (−11.2)

−15.1 (−10.3)

G7b

30.3 (30.1)

32.3 (32.0)

23.4 (30.2)

32.1 (32.1)

G7r

−7.6 (−7.5)

−8.7 (−8.4)

−11.3 (−11.1)

−8.2 (−7.95)

G4′b

29.4 (29.1)

31.3 (30.8)

28.7 (28.6)

31.7 (30.5)

G4′r

−8.0 (−7.7)

−9.1 (−8.6)

−7.2 (−7.0)

−8.6 (−7.9)

Results obtained in aqueous media are given in parentheses

aObtained by performing single point energy calculations using the corresponding geometries optimized at the M06-2X/6-31G(d,p) level

Hydrogen abstraction by the OH. radical

The transition states (TSs) involved in hydrogen abstraction by a OH. radical from a OH or OMe group bound to C5, C7, or C4′ in genistein or bound to C7 or C4′ in daidzein or the three methyl derivatives (7-O-methyl, 4′-O-methyl, and 7,4′-O-dimethyl) of genistein and daidzein, along with some important bond lengths and imaginary vibrational frequencies, were obtained at the M06-2X/6-31G(d,p) level of density functional theory in the gas phase and are shown in Figs. 2 and 3. The PCs corresponding to these transition states are presented in Figs. S2 and S3 of the ESM, respectively. The Gibbs barrier energies and released energies associated with the HAT-mechanism-based scavenging of the OH. radical by genistein, daidzein, and their methyl derivatives were calculated at the M06-2X/6-31G(d,p) and M06-2X/6-311++G(d,p) levels of theory in both gas and aqueous media and are presented in Table 1. Table 1 shows that the Gibbs barrier energy (calculated using Eq. 1) for the abstraction of the hydrogen atom from the hydroxyl group attached to C5 of the A ring in genistein (there is no hydroxyl group at the C5 site in daidzein) by the OH. radical was found to be 15.0 kcal/mol. This high barrier energy arises due to the presence of a strong intramolecular hydrogen bond between the OH group at C5 and the oxygen of the carbonyl group attached to C4 of the pyrone ring (ring C), in agreement with an earlier study [57]. The Gibbs barrier energies for the abstraction of hydrogen from the hydroxyl group at C7 in the A ring of genistein and daidzein by the OH. radical were found to be 12.2 and 16.1 kcal/mol, respectively (Table 1), whereas the corresponding values for the abstraction of hydrogen from the hydroxyl group at C4′ in the B ring were lower: 8.5 and 7.8 kcal/mol, respectively, in agreement with previous studies [57, 58]. The Gibbs released energies for the abstraction of hydrogen from the hydroxyl group at C5 in genistein and the hydroxyl groups at C7 and C4′ in genistein and daidzein are negative and much larger in magnitude than the corresponding calculated Gibbs barrier energies. Hence, all of these reactions would be exergonic, leading to the spontaneous formation of the corresponding stable product complexes. This conclusion is also supported by the results of previous studies [57, 58] based on the calculations of the smallest BDE for the hydroxyl group at C4′ in the B ring of genistein or daidzein at various levels of density functional theory in different media, including aqueous media.
Fig. 2a–l

Optimized geometries along with some important bond lengths (Å) and characteristic imaginary frequencies (cm−1) of transition states (al) involved in hydrogen abstraction from the hydroxyl groups or methyl groups at C5, C7, and C4′ in genistein and its mono- and dimethyl derivatives by a hydroxyl radical, as calculated at the M06-2X/6-31G(d,p) level of density functional theory in the gas phase

Fig. 3a–h

Optimized geometries along with some important bond lengths (Å) and characteristic imaginary frequencies (cm−1) of transition states (ah) involved in hydrogen abstraction from the hydroxyl groups or methyl groups at C7 and C4′ in daidzein and its mono- and dimethyl derivatives by a hydroxyl radical, as calculated at the M06-2X/6-31G(d,p) level of density functional theory in the gas phase

The Gibbs barrier energy for hydrogen abstraction from the hydroxyl group at C5 of the A ring in the 7-O-methyl derivative of genistein (17.3 kcal/mol) by a hydroxyl radical is moderately larger than the corresponding value for nonmethylated genistein, whereas the corresponding Gibbs barrier energies for hydrogen abstraction from the methylated site C7 in the 7-O-methyl derivatives of genistein and daidzein (10.8 and 11.0 kcal/mol, respectively) are appreciably smaller than the corresponding values for nonmethylated genistein and daidzein (Table 1). The Gibbs barrier energy for hydrogen abstraction from the hydroxyl group at C4′ of the B ring in the 7-O-methyl derivatives of genistein and daidzein by a hydroxyl radical were found to be 9.4 and 9.5 kcal/mol, respectively (Table 1). These Gibbs barrier energies are larger than the corresponding values for nonmethylated genistein and daidzein.

The Gibbs released energies for hydrogen abstraction from the hydroxyl group at C5 in the 7-O-methyl derivative of genistein and at C7 (methylated) and C4′ (nonmethylated) in the 7-O-methyl derivatives of genistein and daidzein are appreciably larger than the corresponding Gibbs barrier energies. Therefore, all of these reactions would be exergonic and thus the corresponding stable product complexes would form spontaneously. This means that methylation at the O7 site of the A ring in genistein and daidzein enhances their hydroxyl radical scavenging activities. Further, the trends in the Gibbs barrier energies for hydrogen abstraction from various sites in the 7-O-methyl derivatives of genistein and daidzein by a hydroxyl radical are similar to the barrier energy trends for the same sites in genistein and daidzein, and the derivatives present similar barrier energy values to those of genistein and daidzein.

The Gibbs barrier energy for hydrogen abstraction from the hydroxyl group at C5 of the A ring in the 4′-O-methyl derivative of genistein by a hydroxyl radical, 14.6 kcal/mol, was slightly smaller that the corresponding value for nonmethylated genistein. Hydrogen abstraction from the hydroxyl group at C7 in the 4′-O-methyl derivatives of genistein and daidzein was found to have smaller Gibbs barrier energies (10.2 and 7.6 kcal/mol, respectively) than the corresponding values for genistein, daidzein, and their 7-O-methyl derivatives. However, the Gibbs barrier energy for hydrogen abstraction from the methyl groups present at C4′ in the B ring in the 4′-O-methyl derivatives of genistein and daidzein were calculated as 11.3 and 10.0 kcal/mol, respectively. Thus, the methylation at O4′ of the B ring in genistein and daidzein reduces its hydroxyl radical scavenging activity in comparison to nonmethylated genistein and daidzein as well as their 7-O-methyl derivatives, but enhances the hydroxyl radical scavenging activity of the A ring in the 4′-O-methyl derivatives of genistein and daidzein.

The Gibbs released energies for hydrogen abstraction from the hydroxyl and methyl groups at C5 in the 4′-O-methyl derivative of genistein or at C7 (A ring) or C4′ (B ring) in the 4′-O-methyl derivatives of genistein and daidzein are much more negative than the corresponding calculated Gibbs barrier energies. Thus, all of these reactions would be exergonic, leading to the spontaneous formation of stable product complexes. However, the trends in the Gibbs barrier energies for hydrogen abstraction from various sites in the 4′-O-methyl derivatives of genistein and daidzein by a hydroxyl radical are the reverse of the trends in Gibbs barrier energy seen for the various sites in genistein and daidzein and their 7-O-methyl derivatives, even though the barrier energy values of the derivatives are fairly similar to those of genistein and daidzein.

The Gibbs barrier energy for hydrogen abstraction from the hydroxyl group at C5 of the A ring in the 7,4′-O-dimethyl derivative of genistein by a hydroxyl radical, 17.5 kcal/mol, is larger than the corresponding energies in all of the above cases. The hydrogen abstraction reactions at the methylated C7 and C4′ sites in the 7,4′-O-dimethyl derivatives of genistein and daidzein involve Gibbs barrier energies of 10.8 and 11.1 kcal/mol, respectively, for the C7 sites and 11.3 kcal/mol for the C4′ site in the B ring. These barrier energies are larger than the corresponding energies for genistein and daidzein and their 7-O-methyl and 4′-O-methyl derivatives. However, methylation at both the A and the B rings of genistein and daidzein enhances the hydroxyl radical scavenging activity of the A ring but reduces that of the B ring.

The Gibbs released energies for hydrogen abstraction from the hydroxyl group at C5 site in the 7,4′-O-dimethyl derivative of genistein or from the methyl groups at O7 and O4′ in the 7,4′-O-dimethyl derivatives of genistein and daidzein are much more negative than the corresponding Gibbs barrier energies. Therefore, all of these reactions would be exergonic and the stable product complexes would form spontaneously. However, the trends in the Gibbs barrier energies for hydrogen abstraction from various sites in the 7,4′-O-dimethyl derivatives of genistein and daidzein are similar to the barrier energy trends for the same sites in genistein and daidzein, although the derivatives present rather different barrier energy values to genistein and daidzein. Thus, methylation of the A ring in genistein and daidzein enhances the hydroxyl radical scavenging ability of that ring, whereas methylation of the B ring in genistein and daidzein reduces the hydroxyl radical scavenging ability of that ring.

Hydrogen abstraction by the OCH3 . radical

The optimized transition state (TS) geometries involved in hydrogen abstraction from hydroxyl (OH) or methyl (OMe) groups bonded to the C5, C7, and C4′ sites of genistein, the C7 and C4′ sites of daidzein, and those sites in the three methyl derivatives (7-O-methyl, 4′-O-methyl, and 7,4′-O-dimethyl) of genistein or daidzein by the methoxy radical (OCH3.), along with some important bond lengths and imaginary vibrational frequencies, were calculated at the M06-2X/6-31G(d,p) level of density functional theory in the gas phase. These are shown in Figs. 4 and 5, while the PCs for these transition states are presented in Figs. S4 and S5 of the ESM. The Gibbs barrier energies and released energies involved in the HAT-facilitated scavenging of the OCH3. radical by genistein, daidzein, and their three methyl derivatives, as calculated using Eq. 1 at the M06-2X/6-31G(d,p) and M06-2X/6-311++G(d,p) levels of density functional theory in both the gas phase and aqueous media, are presented in Table 2. This table reveals that the Gibbs barrier energy for the abstraction of hydrogen from the hydroxyl group at C5 of the A ring in genistein (there is no hydroxyl group at C5 in daidzein) by the methoxy radical is 21.1 kcal/mol, which is in good agreement with the corresponding value for the abstraction of hydrogen by the hydroxy radical. The Gibbs barrier energies for the abstraction of hydrogen from the hydroxyl group at C7 of the A ring in genistein and daidzein by the methoxy radical were found to be 16.1 and 15.7 kcal/mol, respectively, while the corresponding barrier energies for the abstraction of hydrogen from the hydroxyl group at C4′ of the B ring in genistein and daidzein were 12.2 and 12.8 kcal/mol, respectively. As the Gibbs barrier energy and the Gibbs released energy for the reaction at C5 in genistein have very similar values, the product would not be quite unstable. However, the Gibbs released energies for hydrogen abstraction at the C7 and C4′ sites in genistein and daidzein by the methoxy radical are in the ranges of −20.4 to −19.6 kcal/mol and − 23.1 to −23.7 kcal/mol, respectively. Thus, the reactions that occur at C7 and C4′ would be exergonic and their product complexes would be stable.
Fig. 4a–l

Optimized geometries along with some important bond lengths (Å) and characteristic imaginary frequencies (cm−1) of transition states (al) involved in hydrogen abstraction from the hydroxyl groups or methyl groups at C5, C7, and C4′ in genistein by a methoxy radical, as obtained at the M06-2X/6-31G(d,p) level of density functional theory in the gas phase

Fig. 5a–h

Optimized geometries along with some important bond lengths (Å) and characteristic imaginary frequencies (cm−1) of transition states (ah) involved in hydrogen abstraction from the hydroxyl groups or methyl groups at C7 and C4′ in daidzein and its mono- and dimethyl derivatives by a methoxy radical, as obtained at the M06-2X/6-31G(d,p) level of density functional theory in the gas phase

The Gibbs barrier energies and released energies for hydrogen abstraction from the hydroxyl group at C5 in all three methyl derivatives (7-O-methyl, 4′-O-methyl, and 7,4′-O-dimethyl) of genistein (there is no hydroxyl group at C5 in daidzein) by a methoxy radical were found to be in the ranges of 21.0 to 21.3 kcal/mol and − 21.9 to −22.2 kcal/mol, respectively (Table 2). As the Gibbs barrier energy is very similar in value to the Gibbs released energy for the reaction of the methoxy radical at C5 in all three methyl derivatives of genistein, the product complex would not be quite stable. The Gibbs barrier energy is also rather similar to the released energy for reactions of the methoxy radical at nonmethylated sites in derivatives of genistein and daidzein, such as C4′ in 7-O-methyl and C7 in the 4′-O-methyl derivatives (Table 2). Therefore, the C4′ site in the 7-O-methyl derivative of genistein or daidzein would yield more stable product complexes than the C7 site in the 4′-O-methyl derivative of genistein or daidzein. The Gibbs barrier energies for hydrogen abstraction from the methyl groups bonded to the O7 and O4′ sites in the 7-O-methyl, 4′-O-methyl, and 7,4′-O-dimethyl derivatives of genistein and daidzein by the methoxy radical lie in the range 15.1–18.8 kcal/mol (Table 2). However, the corresponding Gibbs released energies lie in the range of −13.6 to −16.4 kcal/mol, which indicates that methylation reduces the radical scavenging abilities of genistein and daidzein for methoxy radicals.

Hydrogen abstraction by the NO2 . radical

The optimized geometries of the transition states (TSs) involved in hydrogen abstraction from hydroxyl or methyl groups bonded to the C7 and C4′ sites of genistein, daidzein, and their methyl derivatives (the 7-O-methyl, 4′-O-methyl, and 7,4′-O-dimethyl derivatives) as well as the C5 site in genistein and its methyl derivatives by the nitrogen dioxide (NO2.) radical, along with some important bond lengths, as calculated at the M06-2X/6-31G(d,p) level of density functional theory in the gas phase, are shown in Figs. 6 and 7. The PCs for these transition states are presented in Figs. S6 and S7 of the ESM. The Gibbs barrier energies and released energies for hydrogen abstraction from genistein, daidzein, and their methyl derivatives by the NO2. radical, as calculated using Eq. 1 at the M06-2X/6-31G(d,p) and M06-2X/6-311++G(d,p) levels of density functional theory in both the gas phase and aqueous media, are presented in Table 3. The calculated Gibbs barrier energies presented in Table 3 reveal that scavenging of nitrogen dioxide (NO2.) radicals via the HAT mechanism would not take place, as the barrier energies are rather high. The corresponding released energies are very small in magnitude compared with the barrier energies, implying that the reactions would be endergonic, meaning that they would not be spontaneous. In other words, no stable product complex would form.
Fig. 6a–l

Optimized geometries along with some important bond lengths (Å) and characteristic imaginary frequencies (cm−1) of the transition states (al) involved in hydrogen abstraction from hydroxyl groups or methyl groups at C5, C7, and C4′ in genistein and its mono- and dimethyl derivatives by the nitrogen dioxide radical, as calculated at the M06-2X/6-31G(d,p) level of density functional theory in the gas phase

Fig. 7a–h

Optimized geometries along with some important bond lengths (Å) and characteristic imaginary frequencies (cm−1) of transition states (ah) involved in hydrogen abstraction from hydroxyl groups or methyl groups at C7 and C4′ in daidzein and its mono- and dimethyl derivatives by the nitrogen dioxide radical, as calculated at the M06-2X/6-31G(d,p) level of density functional theory in the gas phase

The Gibbs barrier energies and released energies at 298.15 K for hydrogen abstraction from the O4′ site in the 4′-O-methyl derivatives of genistein and daidzein by the hydroxyl radical (OH.), the methoxy radical (OCH3.), and the nitrogen dioxide radical (NO2.), as calculated by performing optimization in aqueous media and single point energy calculations using the gas-phase-optimized geometries at the M06-2X/6-31G(d,p) level of theory in aqueous media, are presented in Table S1 of the ESM. The barrier energies obtained using these two methods are observed to be very similar. This justifies our use of the gas-phase-optimized geometries to calculate single point barrier energies in aqueous media.

Single-electron transfer mechanism

Single electron transfer from an electron donor to an electron acceptor involves a barrier energy, which may be estimated using the Marcus theory of electron transfer [39, 40, 41]. The expression for estimating the electron-transfer barrier energy in terms of the Gibbs free energy of reaction (ΔG0ET) and the reorganization energy (λ) is as follows [39, 40, 41]:
$$ \varDelta {G^{\mathrm{b}}}_{\mathrm{ET}}=\frac{\lambda }{4}{\left(1+\frac{\Delta {G}_{\mathrm{ET}}^0}{\lambda}\right)}^2. $$
As discussed by Nelsen and coworkers [51, 52], the reorganization energy (λ) may be obtained using the following equation:
$$ \lambda =\Delta {E^0}_{\mathrm{ET}}-\Delta {G^0}_{\mathrm{ET}}, $$
where ΔE0ET is the vertical energy difference between the reactants and products. The rate constant can be obtained from the reaction rate equation [53]. Reorganization and barrier energies for electron transfer from genistein, daidzein, and their three methyl derivatives (the 7-O-methyl, 4′-O-methyl, and 7,4′-O-dimethyl derivatives) to the hydroxyl radical (OH.), the methoxy radical (OCH3.), and the nitrogen dioxide radical (NO2.) were calculated using Eq. 2 by performing full geometry optimization of all of the species involved at the M06-2X/6-311++G(d,p) level of density functional theory in aqueous media. These results are presented in Table 4. It should be noted that the reorganization energy (λ) is relatively insensitive to the functional and basis set used in the calculations, whereas the Gibbs barrier energy is quite sensitive to both—particularly the basis set (Table 4).
Table 4

Gibbs barrier energies (∆Gb, kcal/mol) at 298.15 K for single-electron transfer from genistein, daidzein, and their mono- and dimethyl derivatives to the free radicals OH., OCH3., and NO2., as calculated at the M062X/6-311++G(d,p) level of theory in aqueous media

Radical

Nonmethylated molecules

7-O-methyl derivatives

4′-O-methyl derivatives

7,4′-O-dimethyl derivatives

OH.

38.1 (37.0)

38.3 (37.2)

34.7 (33.4)

35.0 (33.1)

OCH3.

61.3 (60.1)

61.5 (60.4)

57.7 (56.3)

58.1 (55.9)

NO2.

24.1 (23.6)

23.9 (23.6)

22.6 (22.1)

22.6 (22.0)

Results for daidzein and its three methyl derivatives are given in parentheses

The calculated single-electron transfer barrier energies involved in electron transfer from genistein, daidzein, and their three methyl derivatives (the 7-O-methyl, 4′-O-methyl, and 7,4′-O-dimethyl derivatives) to the hydroxyl radical (OH.) and the methoxy radical (OCH3.) were found to be in the ranges 33.1–38.3 kcal/mol (for the hydroxyl radical) and 55.9–61.5 kcal/mol (for the methoxy radical; see Table 4), respectively. However, the calculated single-electron transfer barrier energies corresponding to electron transfer from genistein, daidzein, and their three methyl derivatives to the NO2. radical were found to be lie in the range 24.1–22.0 kcal/mol (Table 4). The calculated SET barrier energies presented in Table 4 reveal the following: (i) the methylation of genistein and daidzein does not make a big difference to the SET barrier energy (it changes by ≤ 4 kcal/mol); (ii) the calculated Gibbs barrier energies for SET increase in the order NO2. < OH. < OCH3.; (iii) all of the calculated SET barrier energies are very high (>22.0 kcal/mol), so the rate constants would be negligibly small; (iv) the SET mechanism would not be operational during the scavenging of hydroxyl (OH.), methoxy (OCH3.), and nitrogen dioxide (NO2.) radicals.

Sequential proton-loss electron-transfer mechanism

The Gibbs barrier energies (∆GbSPLET) for the scavenging of hydroxyl (OH.), methoxy (OCH3.), and nitrogen dioxide (NO2.) radicals via the SPLET mechanism, as calculated using Eqs. 3a and 3b, are presented in Table 5. A number of remarks can be made about the data in Table 5. (i) Electron transfer from genistein, daidzein, and their three methyl derivatives to the hydroxyl radical through the SPLET mechanism involves barrier energies in the range 2.6–11.4 kcal/mol. Therefore, the scavenging of a hydroxyl radical through the SPLET mechanism is more efficient than scavenging based on the HAT and SET mechanisms, as discussed earlier. (ii) Derivatives of genistein and daidzein with methylated A rings, as well as their 7-O-methyl and 7,4′-O-dimethyl derivatives, have barrier energies of 1.1–5.2 kcal/mol (Table 5) for the scavenging of the methoxy radical via the SPLET mechanism. However, genistein, daidzein, and their 4′-O-methyl and 7,4′-O-dimethyl derivatives (methylation mainly in the B ring) exhibit large barrier energies of 24.5–30.3 kcal/mol for the scavenging of a methoxy radical (OCH3.) using the SPLET mechanism. Therefore, methylation of the A ring enhances the efficiency with which a methoxy radical is scavenged via the SPLET mechanism. (iii) The scavenging of a nitrogen dioxide radical via the SPLET mechanism involves barrier energies of 0.3–7.6 kcal/mol (Table 5). Thus, SPLET is a more efficient mechanism for scavenging a nitrogen dioxide (NO2.) radical than the HAT and SET mechanisms are.
Table 5

Gibbs barrier (∆Gb, kcal/mol) energies at 298.15 K for sequential proton-loss electron transfer (SPLET) from genistein, daidzein, and their monomethyl and dimethyl derivatives to OH., OCH3., and NO2. radicals, as calculated at the M062X/6-311++G(d,p) level of theory in aqueous media

Radical

Nonmethylated molecules

7-O-methyl derivatives

4′-O-methyl derivatives

7,4′-O-dimethyl derivatives

OH.

6.9 (3.0)

2.6 (2.9)

7.2 (3.1)

11.3 (11.4)

OCH3.

29.7 (24.5)

5.2 (4.3)

30.2 (24.7)

1.1 (1.2)

NO2.

7.6 (1.7)

1.9 (1.7)

7.5 (6.2)

0.3 (0.4)

Results for daidzein and its three methyl derivatives are given in parentheses

Rate constants

Thermal rate constants k (M−1 s−1), including Skodje–Thrular tunneling corrections, for hydrogen atom transfer from hydroxyl (OH) or methyl (OMe) groups in genistein, daidzein, and their three methyl derivatives (the 7-O-methyl, 4′-O-methyl, and 7,4′-O-di-methyl derivatives) to OH., NO2., and OCH3. free radicals were calculated at the M06-2X/6-31G(d,p) and M06-2X/6-311++G(d,p) levels of density functional theory in both gas and aqueous media. The corresponding results obtained in aqueous media and calculated at the M06-2X/6-311++G(d,p) level of density functional theory are now discussed.

The calculated thermal rate constants for hydrogen atom transfer from hydroxyl (OH) or methyl (OMe) groups in genistein, daidzein and their three methyl derivatives to a hydroxyl radical are presented in Table 6, while the corresponding Skodje–Thrular tunneling corrections Г(T) for genistein, daidzein, and their three methyl derivatives are presented in Tables S2 and S3 of the ESM. Table 6 reveals that the calculated thermal rate constants for hydrogen abstraction from hydroxyl (OH) or methyl (OMe) groups at C7 and C4′ in genistein, daidzein, and their three methyl derivatives in aqueous media are in the range of 105 to 108 M−1 s−1, while the calculated thermal rate constants in aqueous media for hydrogen abstraction from the hydroxyl group at C5 in genistein and its three methyl derivatives by a hydroxyl radical (OH.) are in the range of 104 to 107 M−1 s−1. Using pulse radiolysis, Zielonka et al. [59] showed that the rate constant for OH. radical scavenging by genistein is ~1010 M−1 s−1 at pH 8.3 and 3.0, which is very close to the diffusion limit. Thus, the thermal rate constants of up to ~107–108 M−1 s−1 calculated in the present study are close to the corresponding experimental results.

The calculated thermal rate constants for hydrogen atom transfer from hydroxyl (OH) or methyl (OMe) groups in genistein, daidzein, and their three methyl derivatives to a methoxy radical are presented in Table S4 of the ESM, while the corresponding Skodje–Thrular tunneling corrections Г(T) for genistein and daidzein and their three methyl derivatives are presented in Tables S5 and S6 of the ESM. Table S4 of the ESM reveals that the calculated thermal rate constants for hydrogen abstraction from a hydroxyl (OH) group in genistein, daidzein, and their three methyl derivatives by a methoxy radical are in the range of 103 to 106 M−1 s−1, whereas the calculated thermal rate constants for hydrogen abstraction from a methyl (OMe) group in the 7-O-methyl, 4′-O-methyl, and 7,4′-O-dimethyl derivatives of genistein and daidzein by a methoxy radical are in the range of 101 to 104 M−1 s−1. Thus, the methylation of genistein and daidzein reduces their methoxy radical scavenging abilities.

The calculated thermal rate constants for the HAT-based scavenging of a nitrogen dioxide radical via hydrogen transfer from a hydroxyl (OH) or a methyl (OMe) group in genistein, daidzein, and their three methyl derivatives are presented in Table S7 of the ESM, while the corresponding Skodje–Thrular tunneling corrections Г(T) for genistein and daidzein and their three methyl derivatives are presented in Tables S8 and S9 of the ESM. Table S7 of the ESM reveals that the calculated thermal rate constants for hydrogen transfer from a hydroxyl group in genistein, daidzein, and their three methyl derivatives to a nitrogen dioxide radical are in the range of 100 to 103 M−1 s−1. However, when a methyl group is present at the site from which the hydrogen atom is abstracted, the calculated thermal rate constant is found to be on the order of 10−5 M−1 s−1. Therefore, methylation of genistein and daidzein markedly reduces their nitrogen dioxide radical scavenging abilities.
Table 6

Reaction rate constants (RjG or D; j  = 5, 7, or 4′) for hydrogen abstraction from the C5, C7, and C4′ sites in genistein (G), daidzein (D), and their three methyl derivatives by the hydroxy radical at 298.15 K (M−1S−1), as calculated at different levels of theory in the gas phase and aqueous media

Molecule

Reaction rate constant

Value calculated at the M06-2X/6-31G(d,p) level of theory

Value calculated at the M06-2X/6-311++G(d,p) level of theorya

Nonmethylated molecules

R 5 G

6.1 × 107 (1.6 × 107)

9.3 × 106 (1.1 × 107)

R 7 G

4.5 × 109 (7.7 × 107)

2.5 × 108 (2.4 × 106)

R 7 D

2.5 × 106 (3.2 × 104)

4.2 × 103 (5.2 × 101)

R 4′ G

1.7 × 1010 (2.0 × 108)

1.3 × 108 (8.3 × 107)

R 4′ D

1.6 × 1010 (2.4 × 108)

7.6 × 108 (4.3 × 108)

7-O-methyl derivatives

R 5 G

9.2 × 104 (1.0 × 105)

1.1 × 104 (2.2 × 104)

R 7 G

2.2 × 107 (1.4 × 106)

2.8 × 106 (2.3 × 105)

R 7 D

2.1 × 107 (9.9 × 105)

1.2 × 106 (1.3 × 105)

R 4′ G

1.4 × 1010 (1.1 × 108)

1.0 × 109 (2.4 × 107)

R 4′ D

1.2 × 1010 (1.8 × 108)

8.5 × 108 (1.3 × 107)

4′-O-methyl derivatives

R 5 G

1.1 × 108 (2.8 × 107)

1.4 × 107 (1.2 × 107)

R 7 G

5.0 × 109 (7.4 × 107)

2.8 × 108 (3.3 × 107)

R 7 D

1.0 × 1010 (9.0 × 107)

3.7 × 108 (6.4 × 108)

R 4′ G

9.8 × 106 (1.5 × 106)

5.3 × 106 (1.1 × 106)

R 4′ D

6.9 × 107 (3.9 × 106)

1.1 × 107 (7.2 × 105)

7,4′-O-dimethyl derivatives

R 5 G

6.2 × 104 (7.9 × 104)

7.1 × 103 (1.4 × 104)

R 7 G

2.3 × 107 (1.5 × 106)

3.0 × 106 (2.4 × 105)

R 7 D

3.5 × 103 (9.4 × 105)

2.2 × 106 (1.0 × 105)

R 4′ G

9.8 × 106 (1.3 × 106)

6.2 × 106 (1.1 × 106)

R 4′ D

3.1 × 103 (1.2 × 106)

6.4 × 106 (1.0 × 106)

Results obtained in aqueous media are given in parentheses; these were calculated using the barrier energies given in Table 1

aSingle point energy calculations were performed using the geometries of corresponding TSs and PCs optimized at the M06-2X/6-31G(d,p) level

As the Gibbs barrier energies involved in single-electron transfer (SET) from genistein, daidzein, and their three methyl derivatives to hydroxyl, methoxy, and nitrogen dioxide free radicals were calculated to be > 22.0 kcal/mol (Table 4), the corresponding thermal rate constants would be ≤ 10−4 M−1 s−1. Therefore, electron transfer via SET would be unlikely to occur. On the other hand, the Gibbs barrier energies for the SPLET mechanism from genistein, daidzein, and their three methyl derivatives to hydroxyl, methoxy, and nitrogen dioxide free radicals (Table 5) suggest that the SPLET mechanism would be important in this context (the corresponding thermal rate constants would be up to 1012 M−1 s−1). Table 5 also reveals that methylation of genistein and daidzein increases the efficiency of their scavenging of hydroxyl, methoxy, and nitrogen dioxide free radicals via the SPLET mechanism.

Conclusions

We can draw the following conclusions based on the results of the present study of the effects of the methylation of genistein and daidzein on their OH., OCH3., and NO2. radical scavenging abilities:
  1. 1.

    Among the various hydrogen atoms in genistein and daidzein that can potentially be abstracted by OH., OCH3., and NO2. radicals, the H4′ atom of the hydroxyl group at the C4′ site of ring B in genistein and daidzein is the one most likely to be abstracted. Also, the potential of the radicals to be scavenged via H-atom transfer decreases in the order OH. > OCH3. > NO2..

     
  2. 2.

    Methylation of genistein and daidzein does not significantly alter the scavenging potencies of these molecules towards the hydroxyl radical via the HAT mechanism, but it does moderately affect their scavenging potencies towards OCH3. and NO2. radicals. The SET and SPLET mechanisms are strongly affected by the methylation of genistein and daidzein.

     
  3. 3.

    According to our calculations, the single-electron transfer (SET) mechanism does not contribute significantly to the scavenging of hydroxyl, methoxy, and nitrogen dioxide radicals by genistein, daidzein, and their three methyl derivatives.

     
  4. 4.

    On the other hand, the sequential proton-loss electron-transfer (SPLET) mechanism does contribute significantly to the scavenging of hydroxyl, methoxy, and nitrogen dioxide radicals by genistein, daidzein, and their three methyl derivatives. The potential of the radicals to be scavenged via the SPLET mechanism decreases in the order NO2. > OCH3. > OH..

     

Notes

Acknowledgements

One of the authors (PCM) is thankful to the National Academy of Sciences, India (NASI) for the award of a Senior Scientist Fellowship along with the related financial support.

Supplementary material

894_2018_3805_MOESM1_ESM.docx (8.8 mb)
ESM 1 (DOCX 8999 kb)

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Institute of Science, Department of PhysicsBanaras Hindu UniversityVaranasiIndia

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