Cyanidin, as one important plant pigment, was theoretically (at M05-2X/6-311+G(d,p) level of theory) investigated for its ability to scavenge potentially, highly damaging hydroxyl radical. Free radical scavenging of cyanidin was studied through electron transfer mechanism – ET (the second step in SPLET mechanism) in water and ethanol, as solvents. Examination was performed using density functional theory (DFT) and Marcus theory. Based on the thermochemical and kinetic data, it is clear that O‒H group of cyanidin in position 3` is the most suitable for reaction with hydroxyl radical through mentioned antioxidant mechanism

Dimitric Markovic, Jasmina

Djorovic, Jelena

Jeremic, Svetlana

Markovic, Zoran

Milenkovic, Dejan

English

CaSA NaRA

“XXI  SAVETOVANJE  O  BIOTEHNOLOGIJI”                      Zbornik radova, Vol. 21.(24), 2016. 781  EXAMINATION OF ELECTRON TRANSFER MECHANISM OF CYANIDIN   Zoran Marković1, Dejan Milenković2, Svetlana Jeremić1, Jelena Đorović2,  Jasmina Dimitrić Marković3  Abstract: Cyanidin, as one important plant pigment, was theoretically (at M05-2X/6-311+G(d,p) level of theory) investigated for its ability to scavenge potentially, highly damaging hydroxyl radical. Free radical scavenging of cyanidin was studied through electron transfer mechanism – ET (the second step in SPLET mechanism) in water and ethanol, as solvents. Examination was performed using density functional theory (DFT) and Marcus theory. Based on the thermochemical and kinetic data, it is clear that O‒H group of cyanidin in position 3` is the most suitable for reaction with hydroxyl radical through mentioned antioxidant mechanism.   Key words: cyanidin, hyrdoxyl radical, ET and SPLET mechanisms  Introduction  Anthocyanins are natural pigments widely distributed in nature (Delgado-Vargas & Paredes-López, 2003). These compounds are flavonoids that belong to the family of polyphenols. Cyanidin (Cy) (2-(3,4-dihydroxyphenyl) chromenylium-3,5,7-triol) is one of the major water-soluble anthocyanidins.  This paper addresses the DFT investigation of the reaction of cyanidin and hydroxyl radical. The reaction is quantified in terms of values of thermodynamic and kinetic parameters.  Material and methods  The majority of theoretical investigation of Cy is focused on the all rings, where OH groups are located. Geometry optimizations for all species involved in radical scavenging mechanisms have been carried out using density functional method (M05-2X), developed by the Truhlar group (Zhao & Truhlar, 2008) and 6-311+G(d,p) basis set implemented in the Gaussian 09 package (Frisch et al., 2009). The influence of water and ethanol as solvents was approximated by (SMD) solvation model (Marenich et al., 2009).                                                        1Department of Chemical-Technological Sciences, State  University of  Novi Pazar, Vuka Karadžića bb, 36300 Novi Pazar, Republic of Serbia (zmarkovic@kg.ac.rs); 2Bioengineering Research and Development Center , Prvoslava Stojanovića 6, 34000 Kragujevac, Republic of Serbia; 3Faculty of Physical Chemistry, University of Belgrade, Studentski trg 12-16, 11000 Belgrade, Republic of Serbia. “XXI  SAVETOVANJE  O  BIOTEHNOLOGIJI”                      Zbornik radova, Vol. 21.(24), 2016. 782   Slika 1. Hemijska struktura cijanidina Figure 1. Chemical structures of cyanidin.  Results and discussion  Thermodynamic parameters: Scavenging properties of Cy are related to their ability to transfer H atom to a free radical specie. In order to examine the influence of radical specie to an antiradical mechanism of Cy (Fig.1), the reactive particle HO• were used. In the present paper, the equations for calculation of the thermodynamic parameters were taken from Dimitrić-Marković and co-workers (2014). The potential antiradical activity of Cy, for each reactive site (OH group), is simulated in the reactions with hydroxyl (OH) radical. The reaction enthalpies for the reaction of Cy with selected radical were calculated using M05-2X/6-311+G(d,p) model. Calculations were performed in water and ethanol as solvents. The preferred mechanism of antiradical activity of Cy can be estimated from ΔHBDE, ΔHIP, and ΔHPA values. The calculated reactions enthalpies are presented in Table 1.  Tabela 1. Izračunate reakcione entalpije (kJ/mol) za reakciju cijanidina sa hidroksi radikalom Table 1. Calculated reaction enthalpies (kJ/mol) for the reactions of Cy with hydroxyl radical M05-2X/6-311+G(d,p)  Water ε=78.35  Ethanol ε=24.85 cyanidin HAT SET-PT SPLET  HAT SET-PT SPLET  ΔHBDE ΔHIP ΔHPDE ΔHPA ΔHETE  ΔHBDE ΔHIP ΔHPDE ΔHPA ΔHETE   100      120    CyOH-3+.OH -131  -231 -145 14  -129  -249 -150 22 CyOH-3`+.OH -136  -236 -130 -7  -134  -254 -133 -1 CyOH-4`+.OH -140  -240 -156 16  -138  -259 -163 25 CyOH-5+.OH -119  -219 -151 32  -115  -236 -157 42 CyOH-7+.OH -114  -214 -151 37  -110  -230 -158 48  Which of the mechanisms is preferred can be estimated from the lowest value of calculated thermodynamic parameters. On the basis of thermodynamically values from “XXI  SAVETOVANJE  O  BIOTEHNOLOGIJI”                      Zbornik radova, Vol. 21.(24), 2016. 783  Table 1, it is clear that only HAT and SPLET are operative radical scavenging mechanisms of Cy in all solvents under investigations. On the basis of obtained ΔHBDE values, it is clear that 4`−OH group should be more reactive OH group of Cy. The 4`−OH group has the lowest ΔHBDE value in all solvents. ΔHPAs values of all present OH groups of Cy indicating proton transfer from C4’ group is easier comparing to other OH groups. In all solvents ΔHPAs are significantly lower than corresponding ΔHBDE values. This indicates that SPLET mechanism thermodynamically represents the more probable reaction pathway in both solvents. Based on these results further was mechanistic investigated the second step of SPLET mechanism. Kinetic parameters: One of the viable mechanisms to scavenge free radicals is electron transfer (ET), the second step in SPLET mechanism (Burton & Ingold,1984):  Cy–O HO Cy–O HO                                              (1)  In reaction (1) cyanidin anion acts as the free radical scavenger. Transition states are necessary for calculating the G term in Eq. (6) for HAT reactions. However, for electron transfer reaction, transition state cannot be located using electronic structure methods, as it is not possible to describe mechanistic pathway of electron motion. To estimate the reaction barrier (the ΔG≠ term) in such cases, the Marcus theory was used (Marcus, 1997). Within this transition-state formalism, the SPLET activation barrier (ΔG≠SPLET) is defined in terms of the free energy of reaction (ΔG0SPLET) and the nuclear reorganization energy (λ):      20SPLETSPLET = 14GG                                                  (2)  is the energy associated with the nuclear rearrangement involved in the formation of products in an ET reaction, which implies not only the nuclei of the reacting species but also those of the surrounding solvent. For  calculation, a very simple approximation was used:         0SPLETE G                                                             (3) ΔE is the nonadiabatic energy difference between reactants and vertical products, that is, Cy‒O• and HO- in geometries of Cy‒O- and HO•:     SPLET = (Cy–O ) (HO )  (Cy–O ) (HO )E E E E E                      (4) The adiabatic Gibbs free energies of reaction were calculated as:  0SPLET =[ (Cy–O ) (HO ) ] [ (Cy–O ) (HO )]G G G G G                   (5) This approach is similar to that which was used by Nelsen and co-workers (1987) for a large set of self-exchange reactions. The rate constants were calculated using transition state theory (TST) as implemented in The Rate program (Duncan et al.,1998) and 1 M standard state using equation (6):   B expk T Gkh RT                                                    (6) “XXI  SAVETOVANJE  O  BIOTEHNOLOGIJI”                      Zbornik radova, Vol. 21.(24), 2016. 784  where Bk  and h stand for the Boltzman and Planck constants. In this equation G is the free activation energy, which is calculated as the energy difference between transition state and reactants.  represents degeneracy accounting for the number of equivalent reaction paths, and  accounts for tunneling corrections (Marcus, 1997). If calculated rate constant is close to the diffusion limit, appropriate corrections are considered (the Collins–Kimball theory) as proposed by Galano and Alvarez-Idaboy (2013). The apparent rate constant (kapp) cannot be directly obtained from TST calculations. The Collins–Kimball (1949) theory is used to correct the rate constant, and kapp is calculated as:           dappdk kkk k                                                       (7) where k is the thermal rate constant, obtained from TST calculations. This constant, kd, for an irreversible bimolecular diffusion-controlled reaction, can be calculated with following equation:               d AB Ak RD N                                                      (8)  where R denotes the reaction distance, NA is the Avogadro number, and DAB is the mutual diffusion coefficient of the reactants A (free radical (HO•) and B (Cy‒O-)). DAB were calculated from DA and DB according to Truhlar (1985). DA and DB were estimated from the Stokes–Einstein approach (1903):                                  6 αBk TD                                                          (9) where   denotes the viscosity of the solvents, in our case water (η= 8.9x10-4 Pa s) and ethanol (η=1.1x10-3 Pa s) and α  is the radius of the solute. On the basis of the obtained the ΔHETE values (Table 1), the reaction between HO• and Cy‒O- were studied only in positions C3, C3` and C4`. The kinetic parameters of the reactions were analyzed in terms of their Gibbs free energies (Table 2).   Tabela 2. DFT izračunate konstante brzine povezane sa drugim korakom SPLET mehanizma Table 2. DFT calculations of rate constants related to second step of SPLET mechanisms Cyanidin ΔG‡SPLET (kJ/mol) ΔG0SPLET (kJ/mol) λ (kJ/mol) kd (M-1 s-1) kapp (M-1 s-1) Water  ε=78.35   3+•OH 21.1 15.0 50.0 4.2x109 9.5x108 3`+.•OH 9.8 -5.7 49.8 8.3x109 7.8 x109 4`+.•OH 24.0 18.3 52.9 4.0x108 3.4x108 Ethanol ε=24.85 3+•OH 22.0 21.3 30.5 3.4x109 6.9x108 3`+•OH 8.1 -0.2 30.2 6.8x109 6.6x109 4`+•OH 27.9 27.4 35.4 6.9x109 8.0x107 “XXI  SAVETOVANJE  O  BIOTEHNOLOGIJI”                      Zbornik radova, Vol. 21.(24), 2016. 785  As can be seen from Table 2, the 3`‒OH group of Cy‒O- has the lowest values of activation energy (corresponding values of rate constants are higher and possible reaction is faster) in reaction with hydroxyl radical in both solvents, implying that is most favorable position for the reaction via electron transfer mechanism (ET). These results are in according with the results presented in Table 1.  Conclusion  Reaction enthalpies (ΔHBDE and ΔHPA) are calculated using the M052X/6-311+G(d,p) level of theory. The obtained results show that cyanidin reacts with hydroxyl radical via both, HAT and SPLET mechanisms, in water and ethanol. The C4’−OH group of Cy is the most favored site for homolytic and heterolytic O−H breaking in all solvents. The ΔHETE values shows that 3`‒OH group of Cy‒O- is favored for reaction with •OH via ET mechanism.  Mechanistic investigations of antioxidative action of Cy were studied via second step of SPLET mechanism, electron transfer. Simulation of the reaction of anion of cyanidin with the hydroxyl radical confirmed that position 3` of Cy‒O- is the most suitable for reaction with •OH through electron transfer mechanism (ET).   Acknowledgment  The authors acknowledge financial support of the Ministry of Science of the Republic of Serbia, grants No. 172015 and 174028.        References  Delgado-Vargas F., Paredes-López O. (2003). Natural Colorants for Food and Nutraceutical Uses, CRC Press, Boca Raton, FL. Frisch M. J., Trucks G. W., Schlegel H. B., et al. (2010). Gaussian 09, Revision C.01, Gaussian, Inc.,  Wallingford, CT, USA. Marenich A.V., Cramer C.J.,Truhlar D.G. (2009).Universal Solvation Model Based on Solute Electron Density and on a Continuum Model of the Solvent Defined by the Bulk Dielectric Constant and Atomic Surface Tensions. Journal of Physical Chemistry B. 113: 6378-6396. Dimitrić Marković J.M., Milenković D., Amić D., Mojović M., Pašti I., Marković Z.S. (2014). The preferred radical scavenging mechanisms of fisetin and baicalein towards oxygen-centred radicals in polar protic and polar aprotic solvents. RSC Advances, 4: 32228-32236. Burton G. W., Ingold K. U. (1984). Beta-Carotene: an usual type of lipid antioxidant. Science, 224: 569-573. Marcus R. A. (1997). Electron transfer reactions in chemistry. Theory and experiment. Pure & Appl. 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Examination of Electron Transfer Mechanism of Cyanidin

Marković, Zoran

Milenković, Dejan

Jeremić, Svetlana

Djorović, Jelena

Dimitrić-Marković, Jasmina

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Examination of Electron Transfer Mechanism of Cyanidin

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