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Effects of nitro- and amino-group on the antioxidant activity of genistein: A theoretical study
Accepted Manuscript Effects of nitro- and amino-group on the antioxidant activity of genistein: a theoretical study Lingling Wang, Fengjian Yang, Xiuhua Zhao, Yuanzuo Li PII: DOI: Reference:
S0308-8146(18)31679-0 https://doi.org/10.1016/j.foodchem.2018.09.108 FOCH 23595
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Food Chemistry
Received Date: Revised Date: Accepted Date:
17 May 2018 15 September 2018 18 September 2018
Please cite this article as: Wang, L., Yang, F., Zhao, X., Li, Y., Effects of nitro- and amino-group on the antioxidant activity of genistein: a theoretical study, Food Chemistry (2018), doi: https://doi.org/10.1016/j.foodchem. 2018.09.108
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Effects of nitro- and amino-group on the antioxidant activity of genistein: a theoretical study
Lingling Wanga, Fengjian Yanga, Xiuhua Zhaoa,*, Yuanzuo Lib,* a
Key Laboratory of Forest Plant Ecology, Ministry of Education, Northeast Forestry
University, Harbin, Heilongjiang 150040,China b
College of Science, Northeast Forestry University, Harbin, Heilongjiang 150040,
China
*Corresponding authors: Xiuhua Zhao and Yuanzuo Li E-mail addresses: [email protected]; [email protected]
1
Abstract Five novel compounds (Gen-NO2, Gen-2NO2, Gen-NH2, Gen-2NH2 and Gen-6NH2) have been designed via introducing an electron-withdrawing group –NO2 and an electron-donating group –NH2 into the structure of genistein. The effects of –NO2 and –NH2 groups on the antioxidant ability of genistein were investigated via quantum chemistry method in gas and methanol phases. The crucial parameters related to three antioxidant mechanisms were calculated. Moreover, the frontier molecular orbital, natural bond orbital and global descriptive parameters were calculated to evaluate the reactivity of genistein and its derivatives. Calculated results indicate the antioxidant process of genistein and its derivatives inclines to the hydrogen atom transfer (HAT) and sequential proton loss electron transfer (SPLET) mechanisms in gas and methanol phases, respectively. Moreover, introducing –NH2 group into genistein can improve its antioxidant activity owing to the outstanding activities of amino-substituents of genistein, which will provide valuable guidance for the synthesis of new antioxidants experimentally.
Keywords: Genistein; Structural modification; Antioxidant mechanism; Bond dissociation enthalpy; Density functional theory
2
1. Introduction Recently, the researches on antioxidants have become more and more concerned in the field of medicine, due to such as cancer, arteriosclerosis, diabetes, cataract, cardiovascular disease, Alzheimer's disease, arthritis and so on, being considered to be related to the free radicals (Bielli, Scioli, Mazzaglia, Doldo, & Orlandi, 2015; Bjorklund & Chirumbolo, 2017). The relationship between the chemical structure and the activity of antioxidants is also an important problem that researchers have been exploring (Farhoosh, Johnny, Asnaashari, Molaahmadibahraseman, & Sharif, 2016; Cai, Chen, Xie, Zhang, & Hou, 2014). In recent decades, the theoretical method based on quantum chemical calculation has been adopted to study the relationship between the structure and activity of antioxidants, owing to its efficiency and convenience (Leopoldini, Russo, & Toscano, 2011; Zheng, Wu, Yang, Zhang, Zhang, & Wu, 2017). Genistein (Gen), also known as 4',5,7-trihydroxyisoflavone and the isoflavone constituent of Onodis spinosae radix, is an effective anticancer compound (Crupi, Majolino, Paciaroni, Rossi, Stancanelli, Venuti, et al., 2010; Sekine, Vongsvivut, Robertson, Spiccia, & McNaughton, 2011), and exhibits good antioxidant activity due to the high reactivity of its phenolic hydroxyl groups and the ability to scavenge free radicals (Valsecchi, Franchi, Panerai, Sacerdote, Trovato, & Colleoni, 2008; Sekine, Robertson, & McNaughton, 2011). The chemical structure of genistein is shown in Figure 1. In recent years, many studies have been done on its antioxidant activity experimentally (Buddhiranon, DeFine, Alexander, & Kyu, 2013; Choi, Ryu, Chung, Park, Hwang, Shin, et al., 2005; Seol, Kim, Yi, & Lee, 2014). Choi et al. (Choi, et al., 3
2005) investigated the reactivity of genistein towards stable radical and reactive oxygen species, and the effects were compared with other isoflavonoids and antioxidants, which indicated that genistein was more effective in scavenging hypochlorous acid than superoxide and hydrogen peroxide. Seol and co-workers (Seol, Kim, Yi, & Lee, 2014) determined the radical scavenging activities of mixtures of genistein and riboflavin using DPPH and ABTS assays, and the results shown that the radical scavenging abilities of photo-sensitized genistein derivatives were significantly increased compared to samples without visible light irradiation for 100 min of photosensitization. Jurzak et al. (Jurzak, Ramos, & Pilawa, 2017) investigated the effect of ultraviolet irradiation on the interaction between genistein and free radicals (DPPH), suggesting that the interaction of genistein with DPPH free radicals in the absence of ultraviolet irradiation was shown to be slow; however, this interaction was much faster under ultraviolet irradiation. In addition to the experimental research on the antioxidant activity of genistein, the studies on the structure-activity relationship of genistein based on density functional theory (DFT) have also been reported (Lengyel, Rimarcik, Vaganek, & Klein, 2013; Machado, de Carvalho, Otero, & Marques, 2013). Zhang et al. (Zhang, Wang, & Sun, 2003) investigated the structure–activity relationship for genistein to scavenge peroxyl radical by DFT calculations using the B3LYP/6-31G(d,p) method, and the calculated results indicated that the conjugation of an electron-withdrawing 1,4-pyrone group with A-ring of genistein was not beneficial to enhancing the radical-scavenging activities. Zhou and co-workers (Zhang, Du, Peng, Lu, Gao, & Zhou, 2010) studied 4
the structural and electronic properties of daidzein, genistein, formononetin, biochanin A and their radicals by DFT method, which revealed that B-ring of isoflavonoids was the active center, and the hydrogen atom transfer (HAT) appeared as a major mechanism in antioxidants action. Because the hydroxyl in B-ring was the active center of genistein to scavenge peroxyl radical (Zhang, Wang, & Sun, 2003), we introduced an electron-withdrawing group –NO2 and an electron-donating group –NH2 into the 2`- and 3`-position in B-ring of genistein (named as Gen-NO2, Gen-2NO2, Gen-NH2 and Gen-2NH2, respectively, see Figure 1) to investigate the influence of these two groups on the antioxidant activity of genistein. The parameters, containing bond dissociation enthalpy (BDE), ionization potential (IP), proton dissociation enthalpy (PDE), proton affinity (PA) and electron transfer enthalpy (ETE), related to three antioxidant mechanisms: hydrogen atom transfer (HAT), single electron transfer followed by proton transfer (SET-PT) and sequential proton loss electron transfer (SPLET), were systematically calculated via DFT method. Moreover, the frontier molecular orbital energies and natural bond orbital (NBO) analysis were executed to evaluate the free radical scavenging ability of genistein and its derivatives. In addition, the space non-covalent interaction existing in the molecules was taken into account by using the reduced density gradient (RDG) method to study the effect of weak interaction on the activity of phenolic hydroxyl group. The calculated results show that Gen-NH2 and Gen-2NH2 exhibits the stronger free radical scavenging ability. In view of this, the –NH2 group was introduced into the 6-position of A-ring in genistein (entitled as 5
Gen-6NH2) to explore the effects of –NH2 group on the activity of 5- and 7-phenolic hydroxyls.
2. Computational methods According to the previous literatures (Leopoldini, Russo, & Toscano, 2011; Zheng, Deng, Chen, Liang, Guo, & Fu, 2018), the phenolic antioxidants take effect mainly through three possible mechanisms in the process of antioxidation, which contain hydrogen atom transfer (HAT), single electron transfer followed by proton transfer (SET-PT) and sequential proton loss electron transfer (SPLET). The expressions of these three mechanisms are as follows: HAT: R∙ + ArOH → RH + ArO∙
(1)
SET-PT: R∙ + ArOH → R− + ArOH +∙ → RH + ArO∙ SPLET: ArOH → ArO− + H + + R∙ → ArO∙ + R− + H + → RH + ArO∙
(2) (3)
For the first mechanism (HAT), the antioxidants react with the free radicals by transferring a hydrogen atom to the free radical, in which the hydrogen atom is derived from the cleavage of phenolic hydroxyl group. In this mechanism, the activity of antioxidants can be estimated by the BDE value of the hydroxyl group, in which the lower the BDE value is, the higher the antioxidant activity is. Moreover, the second mechanism (SET-PT) consists of two processes: the electron is first separated from the antioxidant molecule, followed by the transfer of proton from the antioxidant cation. The values of IP and PDE are used to measure the ability of antioxidant to scavenge free radical in this case, in which the lower IP and PDE indicate the higher 6
activity of antioxidant. For the last mechanism (SPLET), the values of PA and ETE determine the activity of antioxidant, and the lower PA and ETE imply the higher antioxidant activity. The values of BDE, IP, PDE, PA and ETE are obtained via the following equations (Zheng, Deng, Chen, Liang, Guo, & Fu, 2018): BDE = H(ArO∙ ) + H(H ∙ ) − H(ArOH) IP = H(ArOH +∙ ) + H(e− ) − H(ArOH) PDE = H(ArO∙ ) + H(H + ) − H(ArOH +∙ )
(4) (5) (6)
PA = H(ArO− ) + H(H + ) − H(ArOH)
(7)
ETE = H(ArO∙ ) + H(e− ) − H(ArO− )
(8)
where, H(ArO∙ ), H(ArOH +∙ ) and H(ArO− ) represent the enthalpy of the radical, cation radical and anion radical, respectively. All the calculations in this work were completed by using Gaussian 09 software (Frisch, Trucks, Schlegel, Scuseria, Robb, Cheeseman, et al., 2009). The geometric configurations of genistein and its derivatives were optimized in the framework of DFT (Hohenberg & Kohn, 1964), with B3LYP (Becke, 1988; Lee, Yang, & Parr, 1988) functional (unrestricted B3LYP for the radicals) at 6-311++G(2d,2p) level, and then the related enthalpies of molecules were calculated by using the same method. The enthalpy of hydrogen atom in gas phase adopted the value in the work of Nenadis et al. (Nenadis & Tsimidou, 2012), and the enthalpy of proton and electron were employed the value of 1.483 kcal/mol and 0.752 kcal/mol, respectively (Bartmess, 1994). The methanol (MeOH) was selected as the solvent on account of the polarizable continuum model (PCM) with the integral equation formalism variant (IEFPCM) 7
(Cossi, Scalmani, Rega, & Barone, 2002), and the enthalpy of hydrogen atom, proton and electron were used the value in the previous literature (Rimarčík, Lukeš, Klein, & Ilčin, 2010). In addition, the electron density distribution of the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) for genistein and its derivatives were obtained based on the optimized geometric configurations by using DFT//B3LYP/6-31G(d) method. The natural bond orbital (NBO) analysis for the studied molecules was completed by adopted the NBO program embedded in Gaussian 09 software (Foster & Weinhold, 1980). The reduced density gradient (RDG) analysis for the space non-covalent interaction existing in the molecules was carried out by using Multiwfn 3.3.7 software (Lu & Chen, 2012) and VMD 1.9.3 program (Humphrey, Dalke, & Schulten, 1996). The non-covalent interaction can be described vividly by the RDG method, which originates from electron density as a dimensionless quantity and the first gradient of RDG was developed
by
Yang
and
co-workers
(Johnson,
Keinan,
Mori-Sánchez,
Contreras-García, Cohen, & Yang, 2010): RDG(r) =
1
|∇2 ρ(r)|
1 2(3π2) ⁄3
4 ρ(r) ⁄3
(9)
The graph of ρ(r) against sign λ2 can help to understand the nature and intensity of interactions, and the second largest eigenvalue of Hessian matrix of electron density (called λ2) plays a role in defining the nature of interaction, in which the interaction shows attractive if λ2 is less than zero and the interaction presents repulsive if λ2 exceeds zero (Chithiraikumar, Gandhimathi, & Neelakantan, 2017). In addition, the global descriptive parameters containing vertical ionization potential 8
(VIP), vertical electron affinity (VEA), chemical potential (μ), chemical hardness (η) and global electrophilicity (ω) were calculated according to the previous literatures (Obot, Macdonald, & Gasem, 2015; Ngo, Dao, Thong, & Nam, 2016).
3. Results and discussion 3.1. Optimized geometric configurations The calculation for the structural parameters of genistein and its derivatives is conducive to understanding the antioxidant activity of molecules. The geometric configurations of genistein and its derivatives were optimized by using DFT//B3LYP/6-311++G(2d, 2p) method, and the selected bond lengths and dihedral angles for the neutral forms, radicals, cation radicals and anion radicals of each molecules are listed in Table S1-S4 (see Supporting materials Table S1-S4). As listed in Table S1-S4, it can be found that the selected bond lengths have no obvious change when the neutral molecules turn to the phenoxy (ArO∙), cation radicals (ArOH +∙ ) and the anion (ArO− ) forms. While, for the dihedral angle D(2,3,1`,2`), it can be seen in Table S1 and Table S2 that when the neutral molecules lose a hydrogen atom translating the radical forms, especially for the 4`-OH, the distortion degree of dihedral angles D(2,3,1`,2`) for the six molecules becomes smaller. For instance, the dihedral angles D(2,3,1`,2`) of Gen and Gen-r4` are -42.845° and -33.681°, respectively. That is to say, the C-ring and B-ring in the genistein and its derivatives tend to be planarity when the phenol hydroxyl on the B ring loses a hydrogen atom. For the cation forms, the dihedral angles D(2,3,1`,2`) are closer to planarization 9
compared with that of the corresponding neutral forms (see Table S1 and Table S3, the maximum difference is 19.628° for Gen and its cation form among the six molecules). As listed in Table S4, the dihedral angles D(2,3,1`,2`) of anions originated from the 4`-OH of the six molecules show the lower values, while the other anions present the larger dihedral angles. The 5-OH in genistein can form an intramolecular hydrogen bond (IHB) with the 4-carbonylic oxygen (Whaley, Rummel, & Kastrapeli, 2006). The RDG charts showing interactions in genistein and its derivatives are presented in Figure 2, in which the RDG analysis was executed with the isosurface value of 0.5. As shown, the elliptical slabs between 5-OH and the adjacent 4-carbonylic oxygen of genistein and its derivatives all show navy blue color, indicating that there is a strong intramolecular hydrogen bond (IHB) interaction between 5-OH and the adjacent 4-carbonylic oxygen. In addition, there also exists a navy blue color between the 4`-OH and the oxygen in nitro-group of Gen-NO2, which shows that there is a strong IHB interaction between them, thus affecting the reactivity of 4`-OH in Gen-NO2.
3.2. HAT mechanism The calculated bond dissociation enthalpy (BDE) values of genistein and its derivatives in gas and MeOH phases are listed in Table 1. It can be found from Table 1 that the BDE of 4`-OH in genistein shows the lowest value compared with that of 5-OH and 7-OH, implying that the 4`-OH in B-ring of genistein exhibits the greatest activity among the three phenolic hydroxyls. The relevant literature has shown that 10
the B ring is the active center of genistein (Zhang, Wang, & Sun, 2003), which also illustrates the reliability of our calculated results. It is worth noting that the BDE values for the 5-OH of the six molecules in gas and MeOH phases are greater than the other two phenolic hydroxyls (4`-OH and 7-OH), which attributes to the IHB formed between the 5-phenolic hydroxyl and 4-carbonylic oxygen in the molecules (see Figure 2). Taking away the hydrogen atom from the 5-OH will destroy the IHB, which causes the molecules to show a greater BDE value. Besides, the BDE of 5-OH in Gen-NO2 shows the significant maximum among the six values in gas and MeOH phases, which may be due to the strong IHB interaction between the 4`-OH and the oxygen in nitro-group. However, the BDE of 5-OH in Gen-2NO2 has declined compared with that in Gen-NO2, which due to the negligible intramolecular interaction between the 4`-OH and the oxygen atom in nitro-group. Because genistein and its derivatives have three phenolic hydroxyls in their structures, the lowest BDE values of the phenolic hydroxyl for the investigated molecules were used to weigh up their ability to scavenge free radicals. As listed in Table 1, the lowest BDE values in gas phase for Gen, Gen-NO2, Gen-2NO2, Gen-NH2 and Gen-2NH2 follow the order of Gen-NH2 (72.0 kcal/mol) Gen-2NH2> Gen> Gen-2NO2> Gen-NO2. Similarly, the ability to scavenge free radicals of the five molecules in MeOH phase follows the order of Gen-NH2> Gen> Gen-2NH2 > Gen-2NO2> 11
Gen-NO2. The results suggest that introducing an electron-donating unit –NH2 into the 2`- and 3`-position of B-ring in genistein will increase the antioxidant activity of the molecule; while the introduction of electron withdrawing unit -NO2 will play the opposite role. In addition, it can be found from Table 1 that the introduction of –NH2 or -NO2 into the 2`- and 3`-positions of B-ring in genistein exhibits no significant effect on the BDE values of 5-OH and 7-OH in gas and MeOH phases. Considering the positive effect of –NH2 group on the antioxidant activity of molecule, we have introduced the –NH2 group into the 6-position of A-ring in genistein to explore the effects of –NH2 group on the activity of 5- and 7-phenolic hydroxyl. As presented in Table 1, though the lowest BDE values of Gen-6NH2 in gas and MeOH are greater than that of Gen-NH2, it is still lower than that of Gen, implying that introducing a –NH2 group into the 6-position of A-ring in genistein can also improve its antioxidant activity. In gas and MeOH phases, the BDE values of 5-OH and 7-OH in Gen-6NH2 all are lower than those of in Gen; while the BDEs of 4`-OH in Gen-6NH2 is almost equal to that in Gen. This indicates that the introduction of –NH2 group into the 6-position of A-ring in genistein obviously improve the activity of A-ring of genistein. According to the results obtained above, Gen-NH2, Gen-2NH2 and Gen-6NH2 both show the higher ability to scavenge free radicals, which is well worth further research in the experiment.
3.3. SET-PT mechanism 12
Although phenolic antioxidants are usually by means of hydrogen abstraction to achieve antioxidation, we have also considered the SET-PT mechanism because some studies have reported the reaction with the DPPH radical by this mechanism (Bamonti, Hosoya, Pirker, Bohmdorfer, Mazzini, Galli, et al., 2013; Wang, Xue, An, Zheng, Dou, Zhang, et al., 2015). The calculated ionization potential (IP) and proton dissociation enthalpy (PDE) values of genistein and its derivatives in gas and MeOH phases are listed in Table 2. As listed in Table 2, it can be found that in gas phase, the IPs of Gen, Gen-NO2, Gen-2NO2, Gen-NH2 and Gen-2NH2 are in the order of Gen-2NH2 (163.9 kcal/mol)
the BDE value being affected by the local change caused by the substituent group, and the IP value being influenced by the whole molecular structure (Xue, Zheng, An, Dou, & Liu, 2014). Moreover, it can be found from Table 2 that the PDE values of the investigated molecules in MeOH phase are lower than that in gas phase. For the 4`-OH, the PDE values of the investigated molecules in gas and MeOH phases are in the order of Gen-2NO2< Gen-NH2
antioxidant process tends to the SET-PT mechanism.
3.4. SPLET mechanism The calculated proton affinity (PA) and electron transfer enthalpy (ETE) values of genistein and its derivatives in gas and MeOH phases are listed in Table 3. As listed in Table 3, it can be seen that all the PA values of 4`-OH for Gen, Gen-NO2, Gen-2NO2, Gen-NH2 and Gen-2NH2 in gas follow the order of Gen-2NO2< Gen-NO2< Gen-NH2
genistein can be contributed to the decrease of the ETE values of 5-OH and 7-OH for Gen. As mentioned above, which mechanism the molecule tends to is mainly determined by the values of BDE, IP and PA, in which the values of IP and PA are related to the first step of SET-PT and SPLET mechanisms, respectively. From the obtained results we can see that in gas phase, the lowest IP and PA values for genistein and its derivatives are all higher than the lowest BDE values corresponding to the six molecules, illustrating that the antioxidant mechanism of these four molecules is more prone to the HAT mechanism in gas phase. By contrast, genistein and its derivatives have the lowest PA values in MeOH phase compared with their BDE and IP values, which indicate the antioxidant process tends to the SPLET mechanism. In addition, introducing the –NH2 group into genistein can effectively improve the antioxidant activity of the molecule owing to the outstanding activities of amino-substituents of genistein.
3.5. Frontier molecular orbitals According to the literature (Xue, Zheng, An, Dou, & Liu, 2014), the HOMO energy is another important parameter to predict the free radical scavenging ability of molecules, and the molecules with higher HOMO energy will show the stronger electron donating ability. The frontier molecular orbital energies containing HOMO and LUMO energies and the electron density distribution for those orbitals are presented in Figure 3. It can be found from Figure 3 that the HOMO energies of 16
genistein and its derivatives are following the order of Gen-2NH2 (-5.38 eV)>Gen-NH2 (-5.59 eV)> Gen-6NH2 (-5.70 eV)> Gen (-5.71 eV) > Gen-2NO2 (-6.11 eV)> Gen-NO2 (-6.22 eV), indicating that Gen-2NH2 would exhibit the strongest electron donating ability. As shown in Figure 3, the electron density distribution for the LUMO orbitals of Gen, Gen-NH2, Gen-2NH2 and Gen-6NH2 are distributed on the A- and C-ring; while for Gen-NO2 and Gen-2NO2, the electron density of LUMO orbital are located at the B-ring. In terms of the HOMO orbitals, the electron density distribution for the HOMO orbitals of the six molecules are distributed on the whole molecule, in which the electron density distributed on the B-ring of Gen, Gen-NH2, Gen-2NH2 and Gen-6NH2 show little more than that on the B-ring of Gen-NO2 and Gen-2NO2. This indicates the reactivity of the B-ring of Gen-NO2 and Gen-2NO2 will be reduced compared with the other four molecules.
3.6. Natural bond orbital analysis The hydrogen atoms with more positive charge in the phenolic hydroxyls are prone to be attacked by the oxygen free radicals with negative charge in vivo. That is to say, the more positive charges of hydrogen atoms on phenolic hydroxyl groups are, the easier it is to react with oxygen free radicals. In view of this, the natural bond orbital (NBO) charges on the hydrogen atoms of phenolic hydroxyls for genistein and its derivatives have been calculated and the detailed results are listed in Table S5. As listed, it can be found that for Gen, Gen-NO2, Gen-2NO2, Gen-NH2 and Gen-2NH2, the charges on the hydrogen atoms of 4`-OH follow the order of Gen-NO2> 17
Gen-2NO2 >Gen-NH2> Gen-2NH2> Gen, indicating that the activities of the 4`-OH for Gen-NO2, Gen-2NO2, Gen-NH2 and Gen-2NH2 is higher than that for Gen. Comparing the charges on the hydrogen atoms of 5-OH and 7-OH of Gen with that of Gen-6NH2, we found that the charges on the hydrogen atoms of 5-OH and 7-OH of Gen-6NH2 all are greater than that of corresponding phenolic hydroxyl of Gen. This results illustrate that introducing a -NH2 group into the 6-position of A-ring in genistein can improve the activity of 5-OH and 7-OH.
3.7. Global descriptive parameters According to the work of Rajan et al. (Rajan & Muraleedharan, 2017), the global and local descriptive parameters can provide useful references for predicting the chemical activity of molecules. The calculated global descriptive parameters of genistein and its derivatives, including vertical ionization potential (VIP), vertical electron affinity (VEA), chemical potential (μ), chemical hardness (η) and electrophilicity (ω), are listed in Table S6. The chemical hardness indicates the resistance of cloud polarization or deformation of chemical species, and the molecule with the lower value of chemical hardness implies that it would exhibit the higher reactivity (Ngo, Dao, Thong, & Nam, 2016). It can be found from Table S6 that the chemical hardness of genistein and its derivatives are in the order of Gen-6NH2< Gen-2NH2< Gen-NH2< Gen< Gen-2NO2< Gen-NO2, suggesting that the chemical activity gradually increases in the following order: Gen-NO2< Gen-2NO2< Gen< Gen-NH2< Gen-2NH2< Gen-6NH2. The above results illustrate that the introduction 18
of –NH2 into the structure of genistein will improve its reactivity, while the introduction of –NO2 will play an inhibitory role. Taking the electrophilicity into account, it stands for the capacity of a system to acquire an electron, which can also be used to evaluate the chemical activity of molecule (Parr, Szentpály, & Liu, 1999). The molecule with a larger electrophilicity would show the higher chemical activity. As listed in Table S6, Gen exhibits the largest electrophilicity among the six molecules, and Gen-NH2 and Gen-2NH2 are immediately behind it. It is worth noting that Gen-NO2 has the lowest electrophilicity among the six molecules, implying that introducing the –NO2 group into the structure of genistein would obviously restrain its reactivity.
4. Conclusion Five novel compounds (Gen-NO2, Gen-2NO2, Gen-NH2, Gen-2NH2 and Gen-6NH2) have been designed via introducing an electron-withdrawing group –NO2 and an electron-donating group –NH2 into the molecular structure of genistein. The effects of –NO2 and –NH2 groups on the free radical scavenging ability of genistein were investigated by using density functional theory (DFT) method. The crucial parameters related to three antioxidant mechanisms, containing bond dissociation enthalpy (BDE), ionization potential (IP), proton dissociation enthalpy (PDE), proton affinity (PA) and electron transfer enthalpy (ETE) were calculated. Moreover, the frontier molecular orbital energies, natural bond orbital (NBO) and reduced density gradient (RDG) analysis, global descriptive parameters like chemical potential (μ), 19
chemical hardness (η), and global electrophilicity (ω) were calculated to evaluate the reactivity and stability of genistein and its derivatives. The following conclusions can be drawn from the calculated results: (a) the RDG analysis indicates that there exists a strong intramolecular hydrogen bond (IHB) interaction between 5-OH and the adjacent 4-carbonylic oxygen, as well as the 4`-OH and the oxygen in nitro-group of Gen-NO2 of genistein and its derivatives, which will affect the reactivity of the phenolic hydroxyls; (b) the antioxidant mechanisms of genistein and its derivatives incline to the HAT and SPLET mechanisms in gas and MeOH phases, respectively, due to the lowest BDE and PA values corresponding to the six molecules in gas and MeOH phases; (c) introducing the –NH2 group into genistein can effectively improve its antioxidant activity owing to the higher HOMO energies, and the lower BDE and chemical hardness values of Gen-NH2, Gen-2NH2 and Gen-6NH2. The above results will provide valuable guidance for synthesizing novel antioxidants in experiment.
Acknowledgements The authors would like to acknowledge financial support from the National key research and development program (2017YFD060070601).
Conflict of interest Authors declare no conflict of interest.
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Journal of Computational Chemistry, 33(5), 580-592. Machado, N. F. L., de Carvalho, L., Otero, J. C., & Marques, M. P. M. (2013). A conformational study of hydroxylated isoflavones by vibrational spectroscopy coupled with DFT calculations. Vibrational Spectroscopy, 68, 257-265. Nenadis, N., & Tsimidou, M. Z. (2012). Contribution of DFT computed molecular descriptors in the study of radical scavenging activity trend of natural hydroxybenzaldehydes and corresponding acids. Food Research International, 48(2), 538-543. Ngo, T. C., Dao, D. Q., Thong, N. M., & Nam, P. C. (2016). Insight into the antioxidant properties of non-phenolic terpenoids contained in essential oils extracted from the buds of Cleistocalyx operculatus: a DFT study. RSC Advances, 6(37), 30824-30834. Obot, I. B., Macdonald, D. D., & Gasem, Z. M. (2015). Density functional theory (DFT) as a powerful tool for designing new organic corrosion inhibitors. Part 1: An overview. Corrosion Science, 99, 1-30. Parr, R. G., Szentpály, L. v., & Liu, S. (1999). Electrophilicity Index. Journal of the American Chemical Society, 121(9), 1922-1924. Rajan, V. K., & Muraleedharan, K. (2017). A computational investigation on the structure, global parameters and antioxidant capacity of a polyphenol, Gallic acid. Food Chemistry, 220, 93-99. Rimarčík, J., Lukeš, V., Klein, E., & Ilčin, M. (2010). Study of the solvent effect on the enthalpies of homolytic and heterolytic N–H bond cleavage in 24
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Xue, Y., Zheng, Y., An, L., Dou, Y., & Liu, Y. (2014). Density functional theory study of the structure–antioxidant activity of polyphenolic deoxybenzoins. Food Chemistry, 151, 198-206. Zhang, H.-Y., Wang, L.-F., & Sun, Y.-M. (2003). Why B-ring is the active center for genistein to scavenge peroxyl radical: A DFT study. Bioorganic & Medicinal Chemistry Letters, 13(5), 909-911. Zhang, J., Du, F., Peng, B., Lu, R., Gao, H., & Zhou, Z. (2010). Structure, electronic properties, and radical scavenging mechanisms of daidzein, genistein, formononetin, and biochanin A: A density functional study. Journal of Molecular Structure: THEOCHEM, 955(1), 1-6. Zheng, W., Wu, Y. P., Yang, W., Zhang, Z., Zhang, L. Q., & Wu, S. Z. (2017). A Combined Experimental and Molecular Simulation Study of Factors Influencing the Selection of Antioxidants in Butadiene Rubber. Journal of Physical Chemistry B, 121(6), 1413-1425. Zheng, Y. Z., Deng, G., Chen, D. F., Liang, Q., Guo, R., & Fu, Z. M. (2018). Theoretical studies on the antioxidant activity of pinobanksin and its ester derivatives: Effects of the chain length and solvent. Food Chemistry, 240, 323-329.
26
Figure captions: Figure 1. The molecular structures of genistein (Gen) and its derivatives. Figure 2. Reduced density gradient (RDG) charts showing interactions in genistein and its derivatives, in which the blue, green and red region represent the strong attraction, Van der Waals interaction and strong repulsion, respectively. Figure 3. The electron density distribution and energies of HOMO and LUMO for genistein (Gen) and its derivatives calculated at the B3LYP/6-31G(d) level in gas phase.
27
Figure 1. The molecular structures of genistein (Gen) and its derivatives.
28
Figure 2. Reduced density gradient (RDG) charts showing interactions in genistein and its derivatives, in which the blue, green and red region represent the strong attraction, Van der Waals interaction and strong repulsion, respectively.
29
Figure 3. The electron density distribution and energies of HOMO and LUMO for genistein (Gen) and its derivatives calculated at the B3LYP/6-31G(d) level in gas phase.
30
Table 1. Calculated bond dissociation enthalpy (BDE) values of genistein and its derivatives in gas and MeOH phases (unit: kcal/mol).
Gas
MeOH
BDE
Gen
Gen-NO2 Gen-2NO2 Gen-NH2 Gen-2NH2 Gen-6NH2
4`-OH
82.9
95.0
85.7
72.0
82.8
82.8
5-OH
100.1
100.7
100.1
100.0
100.2
91.1
7-OH
89.4
90.0
88.7
89.4
89.3
75.3
4`-OH
396.0
404.8
401.0
384.5
396.7
395.9
5-OH
408.6
409.1
407.7
408.5
409.8
397.2
7-OH
402.8
403.6
402.7
402.7
403.9
387.4
31
Table 2. Calculated ionization potential (IP) and proton dissociation enthalpy (PDE) values of genistein and its derivatives in gas and MeOH phases (unit: kcal/mol). Gas
MeOH
4`-OH 5-OH 7-OH IP
PDE
4`-OH 5-OH 7-OH
Gen
170.8
115.2
Gen-NO2
181.6
123.1
Gen-2NO2 180.0
124.3
Gen-NH2
165.9
106.5
Gen-2NH2 163.6
107.3
Gen-6NH2 159.0
101.3
Gen
227.2
244.4 233.8
10.9
23.5
17.8
Gen-NO2
228.7
234.3 223.6
11.9
16.2
10.7
Gen-2NO2
220.9
235.2 223.9
6.8
13.6
8.5
Gen-NH2
221.3
249.3 238.7
8.1
32.1
26.4
Gen-2NH2
234.4
251.8 240.9
19.6
32.7
26.7
Gen-6NH2
239.0
247.3 231.6
24.8
26.0
16.3
32
Table 3. Calculated proton affinity (PA) and electron transfer enthalpy (ETE) values of genistein and its derivatives in and MeOH phases (unit: kcal/mol). Gas
MeOH
4`-OH
5-OH
7-OH
4`-OH
5-OH
7-OH
Gen
337.6
347.0
329.2
44.5
49.5
37.8
Gen-NO2
331.1
341.1
323.2
38.5
48.5
37.0
Gen-2NO2
326.7
343.7
326.8
38.8
48.7
37.4
Gen-NH2
334.9
346.9
329.1
43.1
49.6
37.8
Gen-2NH2
339.8
346.9
329.0
47.2
48.7
37.3
Gen-6NH2
337.8
344.3
328.6
44.6
49.2
36.8
Gen
60.5
68.2
75.4
81.6
89.2
95.2
Gen-NO2
79.2
74.8
82.0
96.5
90.8
96.8
Gen-2NO2
74.2
71.6
77.1
92.4
89.2
95.4
Gen-NH2
52.2
68.3
75.5
71.6
89.1
95.1
Gen-2NH2
58.2
68.6
75.5
79.7
91.3
96.7
Gen-6NH2
60.2
61.9
61.9
81.5
78.2
80.7
PA
ETE
Highlights >Five novel kinds of nitro- and amino-substituted genistein molecules were designed. >Antioxidant activity of genistein and the substituents were studied in theory. >Effects of solvent on the antioxidant mechanisms were considered. >Two different mechanisms tend to occur in gas and methanol phases, respectively. >Introducing amino-group into genistein can enhance its antioxidant activity. 33
34
S0308-8146(18)31679-0 https://doi.org/10.1016/j.foodchem.2018.09.108 FOCH 23595
To appear in:
Food Chemistry
Received Date: Revised Date: Accepted Date:
17 May 2018 15 September 2018 18 September 2018
Please cite this article as: Wang, L., Yang, F., Zhao, X., Li, Y., Effects of nitro- and amino-group on the antioxidant activity of genistein: a theoretical study, Food Chemistry (2018), doi: https://doi.org/10.1016/j.foodchem. 2018.09.108
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Effects of nitro- and amino-group on the antioxidant activity of genistein: a theoretical study
Lingling Wanga, Fengjian Yanga, Xiuhua Zhaoa,*, Yuanzuo Lib,* a
Key Laboratory of Forest Plant Ecology, Ministry of Education, Northeast Forestry
University, Harbin, Heilongjiang 150040,China b
College of Science, Northeast Forestry University, Harbin, Heilongjiang 150040,
China
*Corresponding authors: Xiuhua Zhao and Yuanzuo Li E-mail addresses: [email protected]; [email protected]
1
Abstract Five novel compounds (Gen-NO2, Gen-2NO2, Gen-NH2, Gen-2NH2 and Gen-6NH2) have been designed via introducing an electron-withdrawing group –NO2 and an electron-donating group –NH2 into the structure of genistein. The effects of –NO2 and –NH2 groups on the antioxidant ability of genistein were investigated via quantum chemistry method in gas and methanol phases. The crucial parameters related to three antioxidant mechanisms were calculated. Moreover, the frontier molecular orbital, natural bond orbital and global descriptive parameters were calculated to evaluate the reactivity of genistein and its derivatives. Calculated results indicate the antioxidant process of genistein and its derivatives inclines to the hydrogen atom transfer (HAT) and sequential proton loss electron transfer (SPLET) mechanisms in gas and methanol phases, respectively. Moreover, introducing –NH2 group into genistein can improve its antioxidant activity owing to the outstanding activities of amino-substituents of genistein, which will provide valuable guidance for the synthesis of new antioxidants experimentally.
Keywords: Genistein; Structural modification; Antioxidant mechanism; Bond dissociation enthalpy; Density functional theory
2
1. Introduction Recently, the researches on antioxidants have become more and more concerned in the field of medicine, due to such as cancer, arteriosclerosis, diabetes, cataract, cardiovascular disease, Alzheimer's disease, arthritis and so on, being considered to be related to the free radicals (Bielli, Scioli, Mazzaglia, Doldo, & Orlandi, 2015; Bjorklund & Chirumbolo, 2017). The relationship between the chemical structure and the activity of antioxidants is also an important problem that researchers have been exploring (Farhoosh, Johnny, Asnaashari, Molaahmadibahraseman, & Sharif, 2016; Cai, Chen, Xie, Zhang, & Hou, 2014). In recent decades, the theoretical method based on quantum chemical calculation has been adopted to study the relationship between the structure and activity of antioxidants, owing to its efficiency and convenience (Leopoldini, Russo, & Toscano, 2011; Zheng, Wu, Yang, Zhang, Zhang, & Wu, 2017). Genistein (Gen), also known as 4',5,7-trihydroxyisoflavone and the isoflavone constituent of Onodis spinosae radix, is an effective anticancer compound (Crupi, Majolino, Paciaroni, Rossi, Stancanelli, Venuti, et al., 2010; Sekine, Vongsvivut, Robertson, Spiccia, & McNaughton, 2011), and exhibits good antioxidant activity due to the high reactivity of its phenolic hydroxyl groups and the ability to scavenge free radicals (Valsecchi, Franchi, Panerai, Sacerdote, Trovato, & Colleoni, 2008; Sekine, Robertson, & McNaughton, 2011). The chemical structure of genistein is shown in Figure 1. In recent years, many studies have been done on its antioxidant activity experimentally (Buddhiranon, DeFine, Alexander, & Kyu, 2013; Choi, Ryu, Chung, Park, Hwang, Shin, et al., 2005; Seol, Kim, Yi, & Lee, 2014). Choi et al. (Choi, et al., 3
2005) investigated the reactivity of genistein towards stable radical and reactive oxygen species, and the effects were compared with other isoflavonoids and antioxidants, which indicated that genistein was more effective in scavenging hypochlorous acid than superoxide and hydrogen peroxide. Seol and co-workers (Seol, Kim, Yi, & Lee, 2014) determined the radical scavenging activities of mixtures of genistein and riboflavin using DPPH and ABTS assays, and the results shown that the radical scavenging abilities of photo-sensitized genistein derivatives were significantly increased compared to samples without visible light irradiation for 100 min of photosensitization. Jurzak et al. (Jurzak, Ramos, & Pilawa, 2017) investigated the effect of ultraviolet irradiation on the interaction between genistein and free radicals (DPPH), suggesting that the interaction of genistein with DPPH free radicals in the absence of ultraviolet irradiation was shown to be slow; however, this interaction was much faster under ultraviolet irradiation. In addition to the experimental research on the antioxidant activity of genistein, the studies on the structure-activity relationship of genistein based on density functional theory (DFT) have also been reported (Lengyel, Rimarcik, Vaganek, & Klein, 2013; Machado, de Carvalho, Otero, & Marques, 2013). Zhang et al. (Zhang, Wang, & Sun, 2003) investigated the structure–activity relationship for genistein to scavenge peroxyl radical by DFT calculations using the B3LYP/6-31G(d,p) method, and the calculated results indicated that the conjugation of an electron-withdrawing 1,4-pyrone group with A-ring of genistein was not beneficial to enhancing the radical-scavenging activities. Zhou and co-workers (Zhang, Du, Peng, Lu, Gao, & Zhou, 2010) studied 4
the structural and electronic properties of daidzein, genistein, formononetin, biochanin A and their radicals by DFT method, which revealed that B-ring of isoflavonoids was the active center, and the hydrogen atom transfer (HAT) appeared as a major mechanism in antioxidants action. Because the hydroxyl in B-ring was the active center of genistein to scavenge peroxyl radical (Zhang, Wang, & Sun, 2003), we introduced an electron-withdrawing group –NO2 and an electron-donating group –NH2 into the 2`- and 3`-position in B-ring of genistein (named as Gen-NO2, Gen-2NO2, Gen-NH2 and Gen-2NH2, respectively, see Figure 1) to investigate the influence of these two groups on the antioxidant activity of genistein. The parameters, containing bond dissociation enthalpy (BDE), ionization potential (IP), proton dissociation enthalpy (PDE), proton affinity (PA) and electron transfer enthalpy (ETE), related to three antioxidant mechanisms: hydrogen atom transfer (HAT), single electron transfer followed by proton transfer (SET-PT) and sequential proton loss electron transfer (SPLET), were systematically calculated via DFT method. Moreover, the frontier molecular orbital energies and natural bond orbital (NBO) analysis were executed to evaluate the free radical scavenging ability of genistein and its derivatives. In addition, the space non-covalent interaction existing in the molecules was taken into account by using the reduced density gradient (RDG) method to study the effect of weak interaction on the activity of phenolic hydroxyl group. The calculated results show that Gen-NH2 and Gen-2NH2 exhibits the stronger free radical scavenging ability. In view of this, the –NH2 group was introduced into the 6-position of A-ring in genistein (entitled as 5
Gen-6NH2) to explore the effects of –NH2 group on the activity of 5- and 7-phenolic hydroxyls.
2. Computational methods According to the previous literatures (Leopoldini, Russo, & Toscano, 2011; Zheng, Deng, Chen, Liang, Guo, & Fu, 2018), the phenolic antioxidants take effect mainly through three possible mechanisms in the process of antioxidation, which contain hydrogen atom transfer (HAT), single electron transfer followed by proton transfer (SET-PT) and sequential proton loss electron transfer (SPLET). The expressions of these three mechanisms are as follows: HAT: R∙ + ArOH → RH + ArO∙
(1)
SET-PT: R∙ + ArOH → R− + ArOH +∙ → RH + ArO∙ SPLET: ArOH → ArO− + H + + R∙ → ArO∙ + R− + H + → RH + ArO∙
(2) (3)
For the first mechanism (HAT), the antioxidants react with the free radicals by transferring a hydrogen atom to the free radical, in which the hydrogen atom is derived from the cleavage of phenolic hydroxyl group. In this mechanism, the activity of antioxidants can be estimated by the BDE value of the hydroxyl group, in which the lower the BDE value is, the higher the antioxidant activity is. Moreover, the second mechanism (SET-PT) consists of two processes: the electron is first separated from the antioxidant molecule, followed by the transfer of proton from the antioxidant cation. The values of IP and PDE are used to measure the ability of antioxidant to scavenge free radical in this case, in which the lower IP and PDE indicate the higher 6
activity of antioxidant. For the last mechanism (SPLET), the values of PA and ETE determine the activity of antioxidant, and the lower PA and ETE imply the higher antioxidant activity. The values of BDE, IP, PDE, PA and ETE are obtained via the following equations (Zheng, Deng, Chen, Liang, Guo, & Fu, 2018): BDE = H(ArO∙ ) + H(H ∙ ) − H(ArOH) IP = H(ArOH +∙ ) + H(e− ) − H(ArOH) PDE = H(ArO∙ ) + H(H + ) − H(ArOH +∙ )
(4) (5) (6)
PA = H(ArO− ) + H(H + ) − H(ArOH)
(7)
ETE = H(ArO∙ ) + H(e− ) − H(ArO− )
(8)
where, H(ArO∙ ), H(ArOH +∙ ) and H(ArO− ) represent the enthalpy of the radical, cation radical and anion radical, respectively. All the calculations in this work were completed by using Gaussian 09 software (Frisch, Trucks, Schlegel, Scuseria, Robb, Cheeseman, et al., 2009). The geometric configurations of genistein and its derivatives were optimized in the framework of DFT (Hohenberg & Kohn, 1964), with B3LYP (Becke, 1988; Lee, Yang, & Parr, 1988) functional (unrestricted B3LYP for the radicals) at 6-311++G(2d,2p) level, and then the related enthalpies of molecules were calculated by using the same method. The enthalpy of hydrogen atom in gas phase adopted the value in the work of Nenadis et al. (Nenadis & Tsimidou, 2012), and the enthalpy of proton and electron were employed the value of 1.483 kcal/mol and 0.752 kcal/mol, respectively (Bartmess, 1994). The methanol (MeOH) was selected as the solvent on account of the polarizable continuum model (PCM) with the integral equation formalism variant (IEFPCM) 7
(Cossi, Scalmani, Rega, & Barone, 2002), and the enthalpy of hydrogen atom, proton and electron were used the value in the previous literature (Rimarčík, Lukeš, Klein, & Ilčin, 2010). In addition, the electron density distribution of the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) for genistein and its derivatives were obtained based on the optimized geometric configurations by using DFT//B3LYP/6-31G(d) method. The natural bond orbital (NBO) analysis for the studied molecules was completed by adopted the NBO program embedded in Gaussian 09 software (Foster & Weinhold, 1980). The reduced density gradient (RDG) analysis for the space non-covalent interaction existing in the molecules was carried out by using Multiwfn 3.3.7 software (Lu & Chen, 2012) and VMD 1.9.3 program (Humphrey, Dalke, & Schulten, 1996). The non-covalent interaction can be described vividly by the RDG method, which originates from electron density as a dimensionless quantity and the first gradient of RDG was developed
by
Yang
and
co-workers
(Johnson,
Keinan,
Mori-Sánchez,
Contreras-García, Cohen, & Yang, 2010): RDG(r) =
1
|∇2 ρ(r)|
1 2(3π2) ⁄3
4 ρ(r) ⁄3
(9)
The graph of ρ(r) against sign λ2 can help to understand the nature and intensity of interactions, and the second largest eigenvalue of Hessian matrix of electron density (called λ2) plays a role in defining the nature of interaction, in which the interaction shows attractive if λ2 is less than zero and the interaction presents repulsive if λ2 exceeds zero (Chithiraikumar, Gandhimathi, & Neelakantan, 2017). In addition, the global descriptive parameters containing vertical ionization potential 8
(VIP), vertical electron affinity (VEA), chemical potential (μ), chemical hardness (η) and global electrophilicity (ω) were calculated according to the previous literatures (Obot, Macdonald, & Gasem, 2015; Ngo, Dao, Thong, & Nam, 2016).
3. Results and discussion 3.1. Optimized geometric configurations The calculation for the structural parameters of genistein and its derivatives is conducive to understanding the antioxidant activity of molecules. The geometric configurations of genistein and its derivatives were optimized by using DFT//B3LYP/6-311++G(2d, 2p) method, and the selected bond lengths and dihedral angles for the neutral forms, radicals, cation radicals and anion radicals of each molecules are listed in Table S1-S4 (see Supporting materials Table S1-S4). As listed in Table S1-S4, it can be found that the selected bond lengths have no obvious change when the neutral molecules turn to the phenoxy (ArO∙), cation radicals (ArOH +∙ ) and the anion (ArO− ) forms. While, for the dihedral angle D(2,3,1`,2`), it can be seen in Table S1 and Table S2 that when the neutral molecules lose a hydrogen atom translating the radical forms, especially for the 4`-OH, the distortion degree of dihedral angles D(2,3,1`,2`) for the six molecules becomes smaller. For instance, the dihedral angles D(2,3,1`,2`) of Gen and Gen-r4` are -42.845° and -33.681°, respectively. That is to say, the C-ring and B-ring in the genistein and its derivatives tend to be planarity when the phenol hydroxyl on the B ring loses a hydrogen atom. For the cation forms, the dihedral angles D(2,3,1`,2`) are closer to planarization 9
compared with that of the corresponding neutral forms (see Table S1 and Table S3, the maximum difference is 19.628° for Gen and its cation form among the six molecules). As listed in Table S4, the dihedral angles D(2,3,1`,2`) of anions originated from the 4`-OH of the six molecules show the lower values, while the other anions present the larger dihedral angles. The 5-OH in genistein can form an intramolecular hydrogen bond (IHB) with the 4-carbonylic oxygen (Whaley, Rummel, & Kastrapeli, 2006). The RDG charts showing interactions in genistein and its derivatives are presented in Figure 2, in which the RDG analysis was executed with the isosurface value of 0.5. As shown, the elliptical slabs between 5-OH and the adjacent 4-carbonylic oxygen of genistein and its derivatives all show navy blue color, indicating that there is a strong intramolecular hydrogen bond (IHB) interaction between 5-OH and the adjacent 4-carbonylic oxygen. In addition, there also exists a navy blue color between the 4`-OH and the oxygen in nitro-group of Gen-NO2, which shows that there is a strong IHB interaction between them, thus affecting the reactivity of 4`-OH in Gen-NO2.
3.2. HAT mechanism The calculated bond dissociation enthalpy (BDE) values of genistein and its derivatives in gas and MeOH phases are listed in Table 1. It can be found from Table 1 that the BDE of 4`-OH in genistein shows the lowest value compared with that of 5-OH and 7-OH, implying that the 4`-OH in B-ring of genistein exhibits the greatest activity among the three phenolic hydroxyls. The relevant literature has shown that 10
the B ring is the active center of genistein (Zhang, Wang, & Sun, 2003), which also illustrates the reliability of our calculated results. It is worth noting that the BDE values for the 5-OH of the six molecules in gas and MeOH phases are greater than the other two phenolic hydroxyls (4`-OH and 7-OH), which attributes to the IHB formed between the 5-phenolic hydroxyl and 4-carbonylic oxygen in the molecules (see Figure 2). Taking away the hydrogen atom from the 5-OH will destroy the IHB, which causes the molecules to show a greater BDE value. Besides, the BDE of 5-OH in Gen-NO2 shows the significant maximum among the six values in gas and MeOH phases, which may be due to the strong IHB interaction between the 4`-OH and the oxygen in nitro-group. However, the BDE of 5-OH in Gen-2NO2 has declined compared with that in Gen-NO2, which due to the negligible intramolecular interaction between the 4`-OH and the oxygen atom in nitro-group. Because genistein and its derivatives have three phenolic hydroxyls in their structures, the lowest BDE values of the phenolic hydroxyl for the investigated molecules were used to weigh up their ability to scavenge free radicals. As listed in Table 1, the lowest BDE values in gas phase for Gen, Gen-NO2, Gen-2NO2, Gen-NH2 and Gen-2NH2 follow the order of Gen-NH2 (72.0 kcal/mol)
Gen-NO2. The results suggest that introducing an electron-donating unit –NH2 into the 2`- and 3`-position of B-ring in genistein will increase the antioxidant activity of the molecule; while the introduction of electron withdrawing unit -NO2 will play the opposite role. In addition, it can be found from Table 1 that the introduction of –NH2 or -NO2 into the 2`- and 3`-positions of B-ring in genistein exhibits no significant effect on the BDE values of 5-OH and 7-OH in gas and MeOH phases. Considering the positive effect of –NH2 group on the antioxidant activity of molecule, we have introduced the –NH2 group into the 6-position of A-ring in genistein to explore the effects of –NH2 group on the activity of 5- and 7-phenolic hydroxyl. As presented in Table 1, though the lowest BDE values of Gen-6NH2 in gas and MeOH are greater than that of Gen-NH2, it is still lower than that of Gen, implying that introducing a –NH2 group into the 6-position of A-ring in genistein can also improve its antioxidant activity. In gas and MeOH phases, the BDE values of 5-OH and 7-OH in Gen-6NH2 all are lower than those of in Gen; while the BDEs of 4`-OH in Gen-6NH2 is almost equal to that in Gen. This indicates that the introduction of –NH2 group into the 6-position of A-ring in genistein obviously improve the activity of A-ring of genistein. According to the results obtained above, Gen-NH2, Gen-2NH2 and Gen-6NH2 both show the higher ability to scavenge free radicals, which is well worth further research in the experiment.
3.3. SET-PT mechanism 12
Although phenolic antioxidants are usually by means of hydrogen abstraction to achieve antioxidation, we have also considered the SET-PT mechanism because some studies have reported the reaction with the DPPH radical by this mechanism (Bamonti, Hosoya, Pirker, Bohmdorfer, Mazzini, Galli, et al., 2013; Wang, Xue, An, Zheng, Dou, Zhang, et al., 2015). The calculated ionization potential (IP) and proton dissociation enthalpy (PDE) values of genistein and its derivatives in gas and MeOH phases are listed in Table 2. As listed in Table 2, it can be found that in gas phase, the IPs of Gen, Gen-NO2, Gen-2NO2, Gen-NH2 and Gen-2NH2 are in the order of Gen-2NH2 (163.9 kcal/mol)
the BDE value being affected by the local change caused by the substituent group, and the IP value being influenced by the whole molecular structure (Xue, Zheng, An, Dou, & Liu, 2014). Moreover, it can be found from Table 2 that the PDE values of the investigated molecules in MeOH phase are lower than that in gas phase. For the 4`-OH, the PDE values of the investigated molecules in gas and MeOH phases are in the order of Gen-2NO2< Gen-NH2
antioxidant process tends to the SET-PT mechanism.
3.4. SPLET mechanism The calculated proton affinity (PA) and electron transfer enthalpy (ETE) values of genistein and its derivatives in gas and MeOH phases are listed in Table 3. As listed in Table 3, it can be seen that all the PA values of 4`-OH for Gen, Gen-NO2, Gen-2NO2, Gen-NH2 and Gen-2NH2 in gas follow the order of Gen-2NO2< Gen-NO2< Gen-NH2
genistein can be contributed to the decrease of the ETE values of 5-OH and 7-OH for Gen. As mentioned above, which mechanism the molecule tends to is mainly determined by the values of BDE, IP and PA, in which the values of IP and PA are related to the first step of SET-PT and SPLET mechanisms, respectively. From the obtained results we can see that in gas phase, the lowest IP and PA values for genistein and its derivatives are all higher than the lowest BDE values corresponding to the six molecules, illustrating that the antioxidant mechanism of these four molecules is more prone to the HAT mechanism in gas phase. By contrast, genistein and its derivatives have the lowest PA values in MeOH phase compared with their BDE and IP values, which indicate the antioxidant process tends to the SPLET mechanism. In addition, introducing the –NH2 group into genistein can effectively improve the antioxidant activity of the molecule owing to the outstanding activities of amino-substituents of genistein.
3.5. Frontier molecular orbitals According to the literature (Xue, Zheng, An, Dou, & Liu, 2014), the HOMO energy is another important parameter to predict the free radical scavenging ability of molecules, and the molecules with higher HOMO energy will show the stronger electron donating ability. The frontier molecular orbital energies containing HOMO and LUMO energies and the electron density distribution for those orbitals are presented in Figure 3. It can be found from Figure 3 that the HOMO energies of 16
genistein and its derivatives are following the order of Gen-2NH2 (-5.38 eV)>Gen-NH2 (-5.59 eV)> Gen-6NH2 (-5.70 eV)> Gen (-5.71 eV) > Gen-2NO2 (-6.11 eV)> Gen-NO2 (-6.22 eV), indicating that Gen-2NH2 would exhibit the strongest electron donating ability. As shown in Figure 3, the electron density distribution for the LUMO orbitals of Gen, Gen-NH2, Gen-2NH2 and Gen-6NH2 are distributed on the A- and C-ring; while for Gen-NO2 and Gen-2NO2, the electron density of LUMO orbital are located at the B-ring. In terms of the HOMO orbitals, the electron density distribution for the HOMO orbitals of the six molecules are distributed on the whole molecule, in which the electron density distributed on the B-ring of Gen, Gen-NH2, Gen-2NH2 and Gen-6NH2 show little more than that on the B-ring of Gen-NO2 and Gen-2NO2. This indicates the reactivity of the B-ring of Gen-NO2 and Gen-2NO2 will be reduced compared with the other four molecules.
3.6. Natural bond orbital analysis The hydrogen atoms with more positive charge in the phenolic hydroxyls are prone to be attacked by the oxygen free radicals with negative charge in vivo. That is to say, the more positive charges of hydrogen atoms on phenolic hydroxyl groups are, the easier it is to react with oxygen free radicals. In view of this, the natural bond orbital (NBO) charges on the hydrogen atoms of phenolic hydroxyls for genistein and its derivatives have been calculated and the detailed results are listed in Table S5. As listed, it can be found that for Gen, Gen-NO2, Gen-2NO2, Gen-NH2 and Gen-2NH2, the charges on the hydrogen atoms of 4`-OH follow the order of Gen-NO2> 17
Gen-2NO2 >Gen-NH2> Gen-2NH2> Gen, indicating that the activities of the 4`-OH for Gen-NO2, Gen-2NO2, Gen-NH2 and Gen-2NH2 is higher than that for Gen. Comparing the charges on the hydrogen atoms of 5-OH and 7-OH of Gen with that of Gen-6NH2, we found that the charges on the hydrogen atoms of 5-OH and 7-OH of Gen-6NH2 all are greater than that of corresponding phenolic hydroxyl of Gen. This results illustrate that introducing a -NH2 group into the 6-position of A-ring in genistein can improve the activity of 5-OH and 7-OH.
3.7. Global descriptive parameters According to the work of Rajan et al. (Rajan & Muraleedharan, 2017), the global and local descriptive parameters can provide useful references for predicting the chemical activity of molecules. The calculated global descriptive parameters of genistein and its derivatives, including vertical ionization potential (VIP), vertical electron affinity (VEA), chemical potential (μ), chemical hardness (η) and electrophilicity (ω), are listed in Table S6. The chemical hardness indicates the resistance of cloud polarization or deformation of chemical species, and the molecule with the lower value of chemical hardness implies that it would exhibit the higher reactivity (Ngo, Dao, Thong, & Nam, 2016). It can be found from Table S6 that the chemical hardness of genistein and its derivatives are in the order of Gen-6NH2< Gen-2NH2< Gen-NH2< Gen< Gen-2NO2< Gen-NO2, suggesting that the chemical activity gradually increases in the following order: Gen-NO2< Gen-2NO2< Gen< Gen-NH2< Gen-2NH2< Gen-6NH2. The above results illustrate that the introduction 18
of –NH2 into the structure of genistein will improve its reactivity, while the introduction of –NO2 will play an inhibitory role. Taking the electrophilicity into account, it stands for the capacity of a system to acquire an electron, which can also be used to evaluate the chemical activity of molecule (Parr, Szentpály, & Liu, 1999). The molecule with a larger electrophilicity would show the higher chemical activity. As listed in Table S6, Gen exhibits the largest electrophilicity among the six molecules, and Gen-NH2 and Gen-2NH2 are immediately behind it. It is worth noting that Gen-NO2 has the lowest electrophilicity among the six molecules, implying that introducing the –NO2 group into the structure of genistein would obviously restrain its reactivity.
4. Conclusion Five novel compounds (Gen-NO2, Gen-2NO2, Gen-NH2, Gen-2NH2 and Gen-6NH2) have been designed via introducing an electron-withdrawing group –NO2 and an electron-donating group –NH2 into the molecular structure of genistein. The effects of –NO2 and –NH2 groups on the free radical scavenging ability of genistein were investigated by using density functional theory (DFT) method. The crucial parameters related to three antioxidant mechanisms, containing bond dissociation enthalpy (BDE), ionization potential (IP), proton dissociation enthalpy (PDE), proton affinity (PA) and electron transfer enthalpy (ETE) were calculated. Moreover, the frontier molecular orbital energies, natural bond orbital (NBO) and reduced density gradient (RDG) analysis, global descriptive parameters like chemical potential (μ), 19
chemical hardness (η), and global electrophilicity (ω) were calculated to evaluate the reactivity and stability of genistein and its derivatives. The following conclusions can be drawn from the calculated results: (a) the RDG analysis indicates that there exists a strong intramolecular hydrogen bond (IHB) interaction between 5-OH and the adjacent 4-carbonylic oxygen, as well as the 4`-OH and the oxygen in nitro-group of Gen-NO2 of genistein and its derivatives, which will affect the reactivity of the phenolic hydroxyls; (b) the antioxidant mechanisms of genistein and its derivatives incline to the HAT and SPLET mechanisms in gas and MeOH phases, respectively, due to the lowest BDE and PA values corresponding to the six molecules in gas and MeOH phases; (c) introducing the –NH2 group into genistein can effectively improve its antioxidant activity owing to the higher HOMO energies, and the lower BDE and chemical hardness values of Gen-NH2, Gen-2NH2 and Gen-6NH2. The above results will provide valuable guidance for synthesizing novel antioxidants in experiment.
Acknowledgements The authors would like to acknowledge financial support from the National key research and development program (2017YFD060070601).
Conflict of interest Authors declare no conflict of interest.
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Figure captions: Figure 1. The molecular structures of genistein (Gen) and its derivatives. Figure 2. Reduced density gradient (RDG) charts showing interactions in genistein and its derivatives, in which the blue, green and red region represent the strong attraction, Van der Waals interaction and strong repulsion, respectively. Figure 3. The electron density distribution and energies of HOMO and LUMO for genistein (Gen) and its derivatives calculated at the B3LYP/6-31G(d) level in gas phase.
27
Figure 1. The molecular structures of genistein (Gen) and its derivatives.
28
Figure 2. Reduced density gradient (RDG) charts showing interactions in genistein and its derivatives, in which the blue, green and red region represent the strong attraction, Van der Waals interaction and strong repulsion, respectively.
29
Figure 3. The electron density distribution and energies of HOMO and LUMO for genistein (Gen) and its derivatives calculated at the B3LYP/6-31G(d) level in gas phase.
30
Table 1. Calculated bond dissociation enthalpy (BDE) values of genistein and its derivatives in gas and MeOH phases (unit: kcal/mol).
Gas
MeOH
BDE
Gen
Gen-NO2 Gen-2NO2 Gen-NH2 Gen-2NH2 Gen-6NH2
4`-OH
82.9
95.0
85.7
72.0
82.8
82.8
5-OH
100.1
100.7
100.1
100.0
100.2
91.1
7-OH
89.4
90.0
88.7
89.4
89.3
75.3
4`-OH
396.0
404.8
401.0
384.5
396.7
395.9
5-OH
408.6
409.1
407.7
408.5
409.8
397.2
7-OH
402.8
403.6
402.7
402.7
403.9
387.4
31
Table 2. Calculated ionization potential (IP) and proton dissociation enthalpy (PDE) values of genistein and its derivatives in gas and MeOH phases (unit: kcal/mol). Gas
MeOH
4`-OH 5-OH 7-OH IP
PDE
4`-OH 5-OH 7-OH
Gen
170.8
115.2
Gen-NO2
181.6
123.1
Gen-2NO2 180.0
124.3
Gen-NH2
165.9
106.5
Gen-2NH2 163.6
107.3
Gen-6NH2 159.0
101.3
Gen
227.2
244.4 233.8
10.9
23.5
17.8
Gen-NO2
228.7
234.3 223.6
11.9
16.2
10.7
Gen-2NO2
220.9
235.2 223.9
6.8
13.6
8.5
Gen-NH2
221.3
249.3 238.7
8.1
32.1
26.4
Gen-2NH2
234.4
251.8 240.9
19.6
32.7
26.7
Gen-6NH2
239.0
247.3 231.6
24.8
26.0
16.3
32
Table 3. Calculated proton affinity (PA) and electron transfer enthalpy (ETE) values of genistein and its derivatives in and MeOH phases (unit: kcal/mol). Gas
MeOH
4`-OH
5-OH
7-OH
4`-OH
5-OH
7-OH
Gen
337.6
347.0
329.2
44.5
49.5
37.8
Gen-NO2
331.1
341.1
323.2
38.5
48.5
37.0
Gen-2NO2
326.7
343.7
326.8
38.8
48.7
37.4
Gen-NH2
334.9
346.9
329.1
43.1
49.6
37.8
Gen-2NH2
339.8
346.9
329.0
47.2
48.7
37.3
Gen-6NH2
337.8
344.3
328.6
44.6
49.2
36.8
Gen
60.5
68.2
75.4
81.6
89.2
95.2
Gen-NO2
79.2
74.8
82.0
96.5
90.8
96.8
Gen-2NO2
74.2
71.6
77.1
92.4
89.2
95.4
Gen-NH2
52.2
68.3
75.5
71.6
89.1
95.1
Gen-2NH2
58.2
68.6
75.5
79.7
91.3
96.7
Gen-6NH2
60.2
61.9
61.9
81.5
78.2
80.7
PA
ETE
Highlights >Five novel kinds of nitro- and amino-substituted genistein molecules were designed. >Antioxidant activity of genistein and the substituents were studied in theory. >Effects of solvent on the antioxidant mechanisms were considered. >Two different mechanisms tend to occur in gas and methanol phases, respectively. >Introducing amino-group into genistein can enhance its antioxidant activity. 33
34