4th International Congress of Serbian Society of Mechanics 4-7th June, 2013, Vrnjačka Banja

Minisymposium Computational

Bioengeering

Minisymposium Organizers: Nenad Filipović

Miloš Kojić Zoran Marković

Fourth Serbian (29th Yu) Congress on Theoretical and Applied Mechanics Vrnjačka Banja, Serbia, 4-7 June 2013

PREFACE

The major advancements in understanding of biological and biomedical processes can only be made when we can integrate the work from researchers in different scientific fields. Computational Bioengineering is a specific discipline which uses computer simulations for the study and prediction of chemical, biological and biomedical processes. Particularly it helps to understand complex dynamic, such as chemical, biological, and biomedical processes,. Computer simulations are indispensable in testing hypotheses and putting information in a quantitative context.

The purpose of Minisymposium Computational Bioengineering is to attract physicians, bioengineers, mechanical and biomechanical engineers, clinicians in cardiology and other cardiovascular specialties, vascular surgeons, radiologists, biologists, biochemists, chemists, biophysicists together on the same place in order to find fresh ideas for understanding behavior of human organism. Main topics of this minisymposim are:

• Biomechanics • Nanomedicine • Cardiovascular Mechanics • Tissue Engineering • Dental Biomechanics • Bone Mechanics • Musculoskeletal Mechanics • Sport Biomechanics • Cellular and Molecular Mechanics • Medical Image Computing • Computational Chemistry

The Minisymposium Computational Bioengineering contains a number of papers related to tissue engineering, blood flow through arteries, medical image computing, electrostimulation, clinical cardiology, diffusion, electromyography, bones, plaque development, stent fracture analysis, dental mechanics, neural modeling, sports biomechanics, etc. A special section is devoted to Computational Chemistry where a few international scientists are invited to present the latest and most relevant topics in this interdisciplinary field. This Minisymposium will provide an exceptional opportunity to meet and network with large numbers of computational bioengineer scientists in Serbia and Europe.

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Fourth Serbian (29th Yu) Congress on Theoretical and Applied Mechanics Vrnjačka Banja, Serbia, 4-7 June 2013

We want to thank all colleagues for the effort they have devoted for preparing this minisymposium, that also serves as a significant support for the development of computational bioengineering research and practice in Serbia.

On Behalf of Organization Committee

Nenad Filipović Miloš Kojić Zoran Marković

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Fourth Serbian (29th Yu) Congress on Theoretical and Applied Mechanics Vrnjačka Banja, Serbia, 4-7 June 2013 M1-20

DFT INVESTIGATION OF THE REACTION OF BAICALEIN WITH HYDROXY RADICAL

Dejan Milenković1, ZoranMarković1,2, Jasmina Dimitrić Marković3, Bono Lučić

4

1

34000 Kragujevac, Republic of Serbia Bioengineering Research and Development Center , Prvoslava Stojanovića 6

e-mail: deki82@kg.ac.rs 2

State University of Novi Pazar, Vuka Karadžića bb, 36300 Novi Pazar, Republic of Serbia Department of Chemical-Technological Sciences

e-mail: zmarkovic@np.ac.rs 3

e-mail: Faculty of Physical Chemistry, University of Belgrade, Studentski trg 12-16, 11000

Belgrade, Republic of Serbia markovich@ffh.bg.ac.rs

4

e-mail: NMR Center, Rudjer Bošković Institute, P.O. Box 180, HR-10002 Zagreb, Croatia

lucic@irb.hr

Abstract. Examination of reaction of baicalein with hydroxy radical, using the M052X/6-311++G(d,p) level of theory, was performed. The applied method successfully reproduces the bond dissociation enthalpy, and reveals that the reaction of baicalein with the hydroxy radical is governed by a hydrogen atom transfer mechanism.

1. Introduction The flavonoid family is a vast and major group among the phenolics with more than several thousand known compounds. Numerous investigations provided some circumstantial evidence that flavonoids exhibit a variety of beneficial actions generally related to their pronounced antioxidant activity, which operates at different levels in the oxidative process. Baicalein (5,6,7-trihydroxy-2-phenyl-4H-chromen-4-one) is one of the major bioactive compounds found in the traditional Chinese medicinal herb Baikal skullcap (Scutellaria baicalensis Georgi). It is widely used in the treatment of copious and disease-related symptoms such as insomnia, fever, and perspiration. Baicalein was also the subject of numerous studies which gave promising results in different areas, such as inhibition of iron-induced lipid peroxidation, anticancer, anti-inflammatory, and antioxidant activities. This paper addresses the DFT investigation of the reaction of baicalein and hydroxyl radical. The reaction is quantified in terms of BDE, IP and PDE values. 1.1. Mechanisms of antioxidant activity

of flavonoids

There are three possible reaction paths by which flavonoids and other phenolic compounds (ArOH) can achieve the antioxidant activity

:

1) Transfer of the hydrogen atoms (Hydrogen Atom Transfer) - HAT mechanism [3]

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Fourth Serbian (29th Yu) Congress on Theoretical and Applied Mechanics Vrnjačka Banja, Serbia, 4-7 June 2013 M1-20

R• + ArOH → ArO• + RH

2) Transfer of a single electron and proton transfer (Single Electron Transfer - Proton Transfer) - SET-PT mechanism [4]

.

R• + ArOH → R- +ArO•+ → RH + ArO

•

3) Sequential loss of proton and electron transfer (Sequential Proton Loss Electron Transfer) - SPLET mechanism [4]

.

ArOH → ArO- + HArO

+ - + R• → ArO• + RR

- - + H+

→ RH

Important thermodynamic parameters that determine the mechanism by which the flavonoids "scavenge" free radicals are: 1) Bond Dissociation Enthalpy (BDE) of molecule ArOH for HAT mechanisam 2) Ionization Potential (IP) of ArOH molecule and Proton Dissociation Enthalphy of radical cation ARO+• for SET-PT mechanisam 3)Proton Affinity (PA) of molecule ArOH and Electron Energy Transfer-ETE corresponding to anion ARO − for

SPLET mechanisam

2. Methodology

2.1. Antioxidant activity by HAT mechanism

baicalein

The majority of theoretical investigation of baicalein is focused on the A ring, where OH groups are located. Geometric optimization, first and second derivative of energy for all stationary points are calculated using a new DFT method M052X, which has been developed recently by Truhlar’s group [5]. The 6-311+ +

G (d, p) basis set, implemented in GAUSSIAN 09 program package [6], was used. The vibrational frequencies were obtained from diagonalization of the corresponding M05-2X Hessian matrices. The nature of the stationary points was determined by analyzing the number of imaginary frequencies: 0 for minimum and 1 for transition state. Relative enthalpies were calculated at 298 K.

3. Results Reaction enthalpies, using DFT methods, were calculated for all three antioxidant mechanisms of baicalein. The obtained values are shown in Table

1:

Gas-phase Water HAT SET-PT SPLET HAT SET-PT SPLET

BDE IP PDE PA ETE BDE IP PDE PA ETE 728 341 5 390 984 1418 294 361 20 123 238 6 332 926 1401 253 317 -24 107 210 7 379 973 1391 310 363 22 101 261

Table 1. Reaction enthalpy values for all three antioxidant mechanisms of baicalein (kJ/mol) in different environments

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Fourth Serbian (29th Yu) Congress on Theoretical and Applied Mechanics Vrnjačka Banja, Serbia, 4-7 June 2013 M1-20

As can be seen from Table 1, the preferred mechanism of action in the gas-phase and a non-polar solvent benzene is HAT, because the BDE values of OH groups of baicalein are lower than the corresponding IP and PA values. On the basis of obtained values for BDE and PDE, it is clear that 6-OH group should be more reactive OH group of baicalein. In water PAs are significantly lower than corresponding BDE values. This indicates that SPLET mechanism thermodynamically represents the most probable reaction pathway in polar solvent. On the other hand, if we compare BDE to the sums of (PA + ETE) (SPLET), and (IP + PDE) (SET-PT), it is clear that these values are mutually very similar. One can conclude, on the basis of these facts, that all three mechanisms are competitive in the aqueous solution. The reactions were studied in all 3 positions. On the basis of the obtained BDE values it was possible to get insight into the reactivity of baicalein’s OH groups. The potential antioxidant activity of baicalein, for each reactive site (OH group), is simulated in the reaction with hydroxyl radical. Hydroxyl radical is chosen for a number of oxygen compounds similar in structure, so that the obtained results can show their actions. The results show that the separation of hydrogen can be made from all three OH positions. Each reaction takes place through a transition state and through an intermediate (appropriate form of radicals). All three possible reaction mechanisms of the hydroxyl radical- baicalein reaction are shown in Figure 1:

Figure1. Three possible reaction pathways for the reaction of baicalein with hydroxy radical

Rate constants for the hydroxyl radical-baicalein reaction were calculated using conventional transition state theory (TST) [7], implemented in TheRate program [8], according to the following 1 M standard state equation (1):

Bk Tkh

σκ= exp(-∆G‡

/RT) (1)

where kB and h stand for the Boltzman and Planck constants, ΔG‡ is the free activation energy, σ represents the reaction path degeneracy caculated for the number of equivalent reaction paths, and κ accounts for tunneling corrections. The tunneling corrections, defined

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Fourth Serbian (29th Yu) Congress on Theoretical and Applied Mechanics Vrnjačka Banja, Serbia, 4-7 June 2013 M1-20

as the Boltzman average of the ratio between the quantum and classical probabilities, were calculated using the zero-curvature tunneling (ZCT) approach. Using the equation (1), free activation energies ‡∆ Ga and the rate constants kTST and kZCT, were calculated for all hydroxyl radical-baicalein reactions. The activation energies and rate constants at 298 K, as well as the reaction free energies for all hydrogen atom transfer reactions are presented in Table 2. With increasing the temperature this effect rapidly decreases.

These findings underline tunneling effects as those responsible for making the reaction between quercetin and hydroxy radical faster. This behavior is to be expected, since the abstraction reaction involves the motion of a light particle (hydrogen atom) that can easily tunnel through the reaction barrier. As also expected, the reactions are distinctly exothermic, while the activation energies are low and corresponding rate constants high. The O6-H6 homolytic bond cleavage requires the lowest activation energy (and shows the highest rate constant values), which can be attributed to the weakness of the O6-H6 bond, due to the involvement of O6 in the relatively strong hydrogen bonds with H5 and H7. In addition, this reaction path yields the most stable PC. The optimized geometries of RC, TS, and PC, for the most favorable hydrogen atom transfer reaction, are depicted in Fig. 2.

Site ΔG‡

(kJ/mol) a k

(MTST

-1 s-1k

) (MZCT -1 s-1

ΔG) (kJ/mol)

r

5 52.39 4.19x10 3.64x104 -95.69 8 6 18.76 3.48x10 1.26x109 -141.37 11 7 31.09 3.47x10 5.47x108 -101.88 9

Table 2. The calculated values of thermochemical parameters for the reaction of baicalein and hydroxyl radicals. ΔG‡

a , kTST, kZCT, and ΔGr

denote activation free energy, rate constants, and reaction free energy, respectively

Based on Table 2, it is possible to conclude that the C6-OH position of baicalein is the best for the reaction with hydroxyl radical. The reaction time is shown in Figure 2. The distances between CO, OH and H-O11 bonds are given in pm.

Figure 2. Reaction path for the H-atom transfer from the C6-OH position of baicalein to the hydroxyl radical

4. Conclusion From the obtained results it can be concluded that the investigated hydroxyl radical-baicalein

reaction is conducted by HAT mechanism. The calculated activation energies show that the reaction is fastest in position C6-OH, while the slowest in C5-OH. The reaction is highly exothermic with the low activation energies and high corresponding reaction rate.

Acknowledgement. This work was supported by the Ministry of Science of the Republic of Serbia (Project Nos.: 172015, 174028, 69-00-74/2010-02, and Serbia–Croatia Bilateral

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Fourth Serbian (29th Yu) Congress on Theoretical and Applied Mechanics Vrnjačka Banja, Serbia, 4-7 June 2013 M1-20

agreement 2011–2012), and by the Ministry of Science, Education and Sports of the Republic of Croatia (Projects Nos.: 079-0000000-3211, 098-1770495-2919, 098-0982464-2511, and Croatia–Serbia Bilateral agreement 2011–2012).

5. References

[1] Li H B, Chen F (2005) Isolation and purification of baicalein, wogonin and oroxylin A from the medicinal plant Scutellaria baicalensis by high-speed counter-current chromatography, Journal of Chromatography A, 1074, pp. 107-110.

[2] Cai Y Z,

[3] Marković Z S, Dimitrić-Marković J M, Doličanin Ć B (2010) Mechanistic pathways for the reaction of quercetin with hydroperoxy radical,

Sun M, Xing J, Luo Q, Corke H (2006) Structure–radical scavenging activity relationships of phenolic compounds from traditional Chinese medicinal plants, Life Sciences, 78, pp. 2872-2888.

Theoretical Chemistry Accounts, 127, pp. 69-80 [4] Rimarčik J, Lukeš V, Klein E, Ilčin M (2010) Study of the solvent effect on the enthalpies of homolytic and

heterolitic N–H bond cleavage in p-phenylenediamine and tetracyano-p-phenylenediamine Journal of Molecular Structure (Theochem), 952, pp. 25-30.

[5] Zhao Y, Schultz N E, Truhlar D G (2006) Design of density functionals by combining the method of constraint satisfaction with parametrization for thermochemistry, thermochemical kinetics, and noncovalent interactions, Journal Chemical Theory and Computation, 2, pp. 364-382.

[6] Frisch M J, Trucks G W, Schlegel H B, et al (2009) Gaussian 09, revision A.1-SMP.Wallingford, CT: Gaussian, Inc.

[7] Duncan W T, Bell R L, Truong T N (1998) TheRate: Program for ab initio direct dynamics calculations of thermal and vibrational-state-selected rate constants.

[8] Truhlar D G, Hase W L, Hynes J T (1983) Current Status of Transition-State Theory. Journal of Physical Chemistry, 87, pp. 2664-2682.

Journal of Computational Chemistry, 19, pp. 1039-1052.

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