Spectroscopic Properties and Conformational Analysis of Methyl Ester

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Spectroscopic Properties and Conformational Analysis of Methyl Ester of Sinapic Acid in Various Environments Bogdan Smyk, Grzegorz M#dza, Adam Kasparek, Maciej Pyrka, Ignacy Gryczynski, and Maciej Maciejczyk J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.7b05508 • Publication Date (Web): 10 Jul 2017 Downloaded from http://pubs.acs.org on July 16, 2017

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The Journal of Physical Chemistry

Spectroscopic Properties and Conformational Analysis of Methyl Ester of Sinapic Acid in Various Environments B. Smyk1*, G. Mędza1, A. Kasparek1, M. Pyrka1, I. Gryczynski2 and M. Maciejczyk1* 1

Department of Physics and Biophysics, University of Warmia and Mazury, Oczapowskiego 4, 10-719 Olsztyn, Poland. 2

Department of Cell Biology and Immunology, Center for Fluorescence Technologies and Nanomedicine, University of North Texas Health Science Center, Fort Worth, TX 76107, USA.

First Corresponding Author: Bogdan Smyk Department of Physics and Biophysics, University of Warmia and Mazury Oczapowskiego 4 10-719 Olsztyn Poland. E-mail: [email protected] Tel. +4889 5233556 Second Corresponding Author: Maciej Maciejczyk Department of Physics and Biophysics, University of Warmia and Mazury Oczapowskiego 4 10-719 Olsztyn Poland. E-mail: [email protected] Tel. +4889 5233234

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Abstract A methyl ester of sinapic acid (MESA) has recently attracted attention due to its antioxidant action. This paper presents results of a study on the spectral and physicochemical properties of MESA, using quantum chemistry (QC), steady-state (absorption and fluorescence) and timeresolved fluorescence techniques (TCSPC). The pKa of the phenol group in the ground state was determined (8.6). The pKa* values in the excited state calculated from the Förster cycle (1.9) and from fluorescence spectra (8.5) differed significantly but the experimental data suggested that the first was the more probable one. Quantum yields (QYs) for both forms have been determined. The QYs were very low (0.0017 and 0.0007) for non-dissociated and dissociated forms, respectively and lifetimes were very short ≤ 10 ps for both forms. The differences in the probability of H-bond formation in the ground and the excited states were estimated by the application of the SdP polarity scale. Dipole moments in the ground state were calculated using QC. The ratio between dipole moments in the ground and the excited state for free molecule was obtained from Bilot-Kawski (B-K) method. Analysis of all collected results suggests that radical route (through hydrogen atom abstraction) of antioxidant activity of MESA is the more probable one in a water environment at pH below 6.


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1. Introduction Sinapic acid (SA) is one of the four most widespread hydroxycinnamic acids in the plant kingdom. It can be found in plants belonging to the Brassicaceae family and is present in various edible sources: fruits, vegetables, cereal grains, herbs, and spices.1,2 SA shows antioxidant, antimicrobial, anti-inflammatory, anticancer, and anti-anxiety activities3. The consumption of plant foods is implicated to decrease the risk of oxidative stress related to such diseases as cancer, cardiovascular diseases, stroke, and neurodegenerative disorders.3 SA can be found either as a free acid or as a bound acid in the form of various types of esters. A methyl ester of sinapic acid (MESA) is one of them. It has been found a major constituent of methanolic extracts from radish sprout (Raphanus sativus L.) and brown mustard (Brassica nigra).3,4 Chung and others4 reported that MESA was the best scavenger of the OH• radical in the methanolic extract from brown mustard (Brassica nigra). The scavenging effect was decreasing in the following order: MESA > SA > 3,4,5-trimethoxycinnamic acid methyl ester.4 The esterification of SA enhances solubility of hydrophilic phenolic antioxidants in the apolar media and it has been shown5 that the increased lipophilicity of MESA significantly improves the antioxidant activity of protocatechuic acid alkyl esters. To determine the influence of esterification on the antioxidant efficiency of sinapic acid, Gaspar and others6 investigated the antioxidant activities of methyl, ethyl, propyl, and butyl sinapates. The esters were synthesized and their antioxidant activities were evaluated using 2,2diphenyl-1-picrylhydrazyl (DPPH), ferric reducing antioxidant power (FRAP UV-vis), and differential scanning calorimetry methods. From the overall results, it was concluded that various linear alkyl ester sinapates had almost the same antioxidant activity and reducing capacity. Furthermore, the addition of an alkyl ester group increases the partition coefficient of sinapic acid between different media in a more lipophilic medium. The DPPH• radical can react with phenols 3 ACS Paragon Plus Environment


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(Ph) in two ways: (a) a direct abstraction of phenol H-atom by the radical, and (b) an electrontransfer process from PhOH or its phenoxide anion (PhO-) to the radical.7 In the apolar solvents, the (a) mechanism is predominant, but with strongly oxidizing radicals such as Cl3COO•, the mechanism of electron transfer can be the preferential route even in these media.8 It has been demonstrated by Foti et al.,7 that in methanol and ethanol the electron transfer mechanism was predominating and that the reaction rate was higher for methyl esters of caffeic and sinapic acids than for free acids. Nenadis et al.,9 reported on differences in the antioxidant activity of some biosynthetically-related ferulic acid (FA) derivatives induced by the presence of characteristic groups (-COOH, -CHO, -CH2OH, -CH3, and -COOC2H5) at the end of their carbon side chain. Results were obtained using both experimental and computational methods. The most effective action has been observed for methyl and ethyl ferulates. Phenolic acids: caffeic, ferulic, gallic, phydroxycinnamic, sinapic and their ethyl esters have been used to study the inhibition of coppercatalyzed low-density lipoprotein oxidation, the radical attack of erythrocyte membranes by 2,2′azobis(2-aminopropane) dihydrochloride, and the scavenger potency using the DPPH• test.10 These analyses demonstrated that ethyl esterification increased the lipophilicity of the five acids and that caffeate, sinapate, and ferulate ethyl esters were more potent inhibitors of apoprotein oxidation and of hydroperoxide formation than their corresponding acid forms. The phenolic acids were effective in scavenging the DPPH radical, but only the p-hydroxycinnamic acid presented a higher scavenging activity after ethyl esterification. The other field of interest is lipophilization of phenolic acids by lipases. Lipophilization of phenolic acids with fatty alcohols can be used as a tool to modify phenolics solubility in oil-based formulas and emulsions. These new amphiphilic antioxidant molecules could be used as multifunctional emulsifiers in the food, cosmetic, and pharmaceutical industries, as they should preserve their other functional properties (UV A and UV B filters, antimicrobial, antiviral, 4 ACS Paragon Plus Environment

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bacteriostatic, etc.).11 Serum albumin is responsible for a variety of physiological functions involving the binding, transport, and deposition of many endogenous and exogenous compounds/drugs present in blood circulation. Hydroxycinnamic acid derivatives (HCAs), as mentioned above, are a group of naturally-occurring phenolics which display various pharmacological activities. Therefore, phenolic acids (PAs) interaction with albumins has been studied in different aspects. One of them was to evaluate the hypochlorite (ClO−) scavenging properties of hydroxycinnamic acids (HCAs), based on their ability to inhibit the formation of protein chloramines and carbonyls due to the oxidation of albumin by ClO−.12 Zou and others13 concluded that sinapic acid suppressed the formation of ONOO− through an electron donation mechanism. Several ligand-albumin binding studies have been done with HCAs. Binding affinities of eight HCAs to BSA were investigated under physiological conditions by Trnková and others14 who applied the UV-Vis absorption spectroscopy and tryptophan fluorescence quenching method. They showed that HCAs could be transported in blood by serum albumin due to their high binding affinity. The interaction between SA and BSA in a buffer at three pH values due to pKa values of carboxyl and hydroxyl groups of SA was investigated by Smyk15 with the spectroscopic methods. The interaction leading to the complex formation was not observed at pH 2.0 and 10.5. Only a weak complex was formed at pH 6.4. The NMR technique, fluorescence and molecular docking models were used to describe the binding of HCAs to BSA.16 They allowed deducing that chlorogenic acid, FA, m-coumaric acid, and p-coumaric acid revealed similar binding modes and orientation, in which the phenyl ring was in close contact with the protein surface, whereas carboxyl group stuck outside of the protein surface. However, FA and SA showed slightly different binding modes, due to the steric hindrance of methoxy-substituents on the phenyl ring and PAs with methoxy-substituents on the phenyl ring. It has been shown17 that at physiological pH (7.4), the 5 ACS Paragon Plus Environment


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ionic form of SA has to be considered due to its pKa = 4.47 value18 of carboxylic group to properly estimate the antioxidant activity of SA in an aqueous solution, whereas its neutral form should be used for the study in the non-polar medium. It has been found that environment polarity plays an important role in the relative efficiency of these compounds as peroxyl scavengers.19 It was stated17 that in the aqueous solution the pH was a key factor for the overall reactivity of hydroxycinnamic acid derivatives towards peroxyl radicals, for their relative antioxidant capacity, and for the relative importance of the different mechanisms of reaction. The main goals of this work were to determine the pKa values of MESA in the ground and excited states and to describe other spectroscopic properties in water and other environments. These goals were achieved through the application of the absorption, steady-state and time-resolved fluorescence techniques combined with QC computational methods. The dipole moments in the ground state were computed with QC methods and then used in the Bilot-Kawski (B-K) method for computation of dipole moments in the excited states.20-22 It has been shown that QC methods can be successfully applied to various biochemical phenomena concerning biological compounds, e.g., chemical reaction,23,24 excited state prediction,25,26 electronic charge distribution27, and tautomerization.28,29 In the future, the results presented in this publication will be used to set up the investigation of mechanisms of MESA interaction with serum albumins. 2. Materials and Methods 2.1 Materials SA was purchased from Sigma-Aldrich (Poland). MESA was prepared from sinapic acid according to the procedure described by Fujita et al.30 After double recrystallization, the compound was subjected to flash chromatography (with silica gel (70 – 230 mesh), pore size 60 Å, column 4 x 20 cm, elution with AcOEt/n-hexane 1:4). Purity (>98%) was determined on 6 ACS Paragon Plus Environment

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UFLC (Shimadzu, Japan), C-8 reverse-phase with Kromasil column, dimensions: 250 x 4,6 mm, eluent: linear gradient from 0 to 80% aqueous MeCN over 50 minutes, flow: 1 mL/min., UV detection at 230 nm, 254 nm and 320 nm. 1H NMR (500 MHz, CDCl3, 300K) 3.78 (s, 3H, OCH3), 3.90 (s, 6H, OCH3), 5.74 (s*, 1H, OH), 6.28 (d, J = 15.90 Hz, 1H, CH), 6.75 (s, 2H, ArCH), and 7.58 (s, J = 15.90 Hz, 1H, CH). A stock solution of MESA (1 mM) for pH dependence study was prepared by diluting MESA in water from the Millipore Simplicity 185 Personal Ultrapure Water System and then diluted (3:100 v/v) with phosphate (0.1 M) and carbonate (0.1 M) buffers. The pH of the solutions was measured with a pH meter (Jenway 3030, UK) at 25 °C. The concentration of MESA in organic solvents was kept constant at 30 µM. The following solvents were used: n-hexane purchased from Merck (Germany), methanol and MeCN from Sigma-Aldrich (Poland), methylene chloride, dimethyl sulfoxide, ethanol, chloroform, ethyl acetate from POCH (Poland), and cyclohexane from Scharlau (Spain). All solvents were of spectroscopic or analytic grade and were all used as received. 2.2 Methods 2.2.1 Apparatus Absorption spectra were measured using a Cary 5000 (Agilent, Australia) spectrometer in 1 cm quartz cells. Fluorescence was measured on a Cary Eclipse (Agilent, Australia) fluorimeter in a 1 cm quartz cell using right angle geometry. Excitation and emission slits were set at 10 nm. Both instruments were equipped with a Peltier accessory. Temperature was stabilized at 25 °C. Fluorescence spectra of each form of MESA were measured using an appropriate wavelength of excitation: at the maximum and at both sides of the band. Every fluorescence spectrum was corrected for wavelength-dependent instrument sensitivity and for inner filter effects I and II. 7 ACS Paragon Plus Environment


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Rayleigh and Raman scatterings were subtracted using Grams Ai Spectroscopy Software (Thermo Fisher Scientific, USA). All other calculations were done using Graphpad Prism 6.0v. (Graphpad software, USA) and Matlab v.2014 (MathWorks, USA). Quantum yield (QY) was estimated using a standard compound - quinine sulfate QY(QS) in 0.1 N H2SO4 = 0.54.31 The QY was calculated using refractive indexes and area under fluorescence spectra of both MESA and QY(QS) and taking advantage of the plot of QY(QS) fluorescence as a function of absorbance at a given wavelength of excitation. Fluorescence lifetimes were measured using a FluoTime 200 spectrometer (PicoQuant, Germany) with a TCSPC module and an MCP PMT detector. Sources of excitation were SuperK EXTREME EXW-20 (High Power Systems) with a SuperK EXTEND-DUV supercontinuum extension unit (265 - 345 nm, 3 – 30 µW) from NKT Photonics (Denmark) and a laser diode 375 nm (PicoQuant – Germany). The FWHM of the NKT Photonics laser depends on the percent of power consumption. Cell holder with right-angle geometry and 4 ps resolution was applied. The count rate per second at the detector was kept below 1 % of the laser replication rate to avoid pulse-pileup. The excitation and observation was set at the maximum of absorption and fluorescence spectrum and for some wavelengths on both sides of the band. An additional excitation wavelength was also chosen in the same manner. Lifetimes were calculated using normal and global analysis option. Data was analyzed with FluoFit 4.6.6 version software (PicoQuant, Germany) using the multiexponential intensity decay model as follows: ௧

௡

‫ܫ‬ሺ‫ݐ‬ሻ = න ‫ ܨܴܫ‬ሺ‫ ݐ‬ሻ ෍ ߙ௜ ݁ ᇱ

௜ୀଵ

ିஶ

ି

௧ି௧ ᇲ ఛ೔ ݀‫ ݐ‬ᇱ

where: IRF (t’) is the instrument response function at time t’, αi is the amplitude of the decay of the i-th component at time t, and τi is the lifetime of the i-th component. 8 ACS Paragon Plus Environment

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Goodness of fit was estimated by calculating χ2, using plane error analysis (PEA) and autocorrelation function implemented in FluoFit program. 2.2.2 Computational details All ab initio calculations were carried out in Gaussian03 package with a higher level DFTBHandHLYP (or Time Dependent-DFT for excited states) method32,33 and aug-cc-pVDZ basis set.34 This level of theory was previously applied to FA,35 which is structurally familiar to MESA. It was also shown that employment of the above-mentioned basis set is appropriate for dipole moment calculations,36 which were performed on optimized ground and excited state structures. To confirm achievement of stationary points, vibrational analyses were executed. The optimized ground-state geometries were used as the initial one for electronic excitation computations. 2.2.3 Calculation of dipole moments Calculation of ratio between dipole moments in the ground and the excited state for free molecule was done according to the B-K method20-22 – assuming that polarizability of a solute can be neglected. To calculate this ratio from Stokes shifts, it is needed to define following functions: ∆ν = νamax – νfmax = m1 f (εr, n) + const

(1)

Σν = νamax + νfmax = - m2 (f(εr,n)+2g(n)) + const

(2)

where: ݉ଵ =

ሬԦ೐ ିሬሬሬԦ ଶሺఓ ఓ೒ ሻమ ௛௖௔య

, ݉ଶ =

మሻ ሬԦ೐మ ିఓ ሬԦ೒ ଶሺఓ

௛௖௔య

, ݂ሺε௥ , ݊ሻ =

ଶ௡మ ାଵ கೝ ିଵ ௡మ ି ଵ ሺ − ሻ, మ ௡ ାଶ கೝ ାଶ ௡మ ା ଶ

ଷ ௡ర ିଵ

݃ሺ݊ሻ = ଶ ሺ௡మ ାଶሻమ ;

a – Onsager radius, ߤԦ௚ - dipole moment in the ground state, ߤԦ௘ - dipole moment in the excited state, c - speed of light, and h - Planck constant.



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Parameters m1 can be determined from the slope of Stokes shift versus f (εr, n). Parameters m2 can be determined from the slope on the graph νamax + νfmax versus f(εr, n)+2g(n). If ߤԦ௘ and ߤԦ௚ are ఓ

parallel to each other, the value of ఓ ೐ can be calculated from the expression: ೒

ఓ೐

ఓ೒

= ቚቀ

௠మ ା௠భ ௠మ ି௠భ

ቁቚ

(3)

2.2.4 Calculation of natural fluorescence lifetime To calculate the natural lifetime of fluorescence τn, the Strickler–Berg relation was used37: ଵ

ఛ೙

∫ ிೡ ሺ௩೑ ሻௗ௩೑

= 2,88 × 10ିଽ × ݊ଶ × ∫ ௩షయ ி ሺ௩ ೑

ೡ

೑ ሻௗ௩೑

×∫

ɛሺ௩ೌ ሻௗ௩ೌ

(4)

௩ೌ

where: n - refractive index, ε - molar absorption coefficient, and Fv - normalized fluorescence intensity per frequency interval. 3. Results and Discussion 3.1 Water environment The hydroxyl group of MESA is titrable and changing pH of water solution can lead to protonated/deprotonated structures both in the ground and the excited state. On the other hand, the electronic excitation of MESA can initiate intramolecular charge transfer from electron-donor groups (hydroxyl and metoxyl) to electron-withdrawing group (carbonyl) in the side chain of molecule. OCH3 O

OH

H3CO

OCH3

Scheme 1. Molecular structure of MESA.


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Before pH analysis, the Lambert-Beer law has been checked. No deviation from linearity was found in the range of the studied concentrations. The pKa of (-OH) group in MESA should be about 9, therefore a series of solutions were prepared starting from pH = 5.9 in a phosphate buffer and ending at pH 10.7 in a carbonate buffer. Because of low stability at alkaline solutions, pH values higher than 10.7 were not applied. The spectroscopic method for pKa determination was chosen and absorption spectrum was measured for each solution (Fig. 1).

Figure 1. Absorption spectra of MESA versus pH. Concentration 30 µM, path length 1 cm.

At the same time as pH was growing, the corresponding band of non-dissociated form (MESA(OH)) with the maximum at 322 nm was disappearing and a new absorption band of the -

anionic form (MESA(O )) with the maximum at 383 nm was formed. Consequently, isosbestic points appeared, which strongly suggested that the equilibrium between the two forms of MESA existed. Based on absorption spectra, pKa and the spectrum of each form of MESA was calculated using a method described earlier38. The collected results were presented in Tab. 1 and spectra of each form in Fig. S1. To verify pKa in the ground state excitation spectra observed at 460 and 500 nm corrected for inner filter effects were used in calculation of pKa (fl exc) and were



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shown in Fig. S2. The value obtained for both observations was close to that obtained from absorption and was equal to 8.5 (Tab. 1).

Table 1. Properties of MESA in water environment. pKa

pKa*

MESA (OH)

8.6 ± 0.2 8.5 (fl exc) MESA (O-)

λamax (nm)

ε (λamax) (mol-1 l cm-1)

λfmax (nm)

QY x103

τn (ns)

τ (ps)

µg (D)

322

18630 ± 150

464

1.7

6.47

≤ 10

3.02

500

0.7

6.91

≤ 10a

9.94

1.9 8.5 (fl)

383

24600 ± 200

a - laser 375 nm. pKa* (pKa in the excited state), QY – quantum yield of fluorescence, τn, τ – calculated (natural) and measured lifetimes respectively. µg - calculated dipole moments of the ground state.

The value of pKa (8.6) was close to that obtained for SA18 and sinapine – a choline ester of sinapic acid (SNP).39 These results indicated that esterification was not strongly influenced on the pKa values of these phenolics and could not affect their antioxidative effects. Antioxidant properties of MESA in the water environment should be better at pH lower than 6, because the main mechanism of such an action is H-abstraction reaction.8 Electron transfer from a molecule to radicals after proton dissociations should dominate at pH higher than 6. It is important to know whether excitation to the higher singlet state (S1) changes the pKa values.


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Figure 2. Corrected fluorescence spectra of MESA versus pH. Concentration 30 µM, λex = 320 nm, quartz cell 1 cm, right angle geometry.

In other words, is the MESA in the excited state a stronger or a weaker acid than in the ground state? To acquire this information, fluorescence spectra were measured and Förster cycle40 was applied. To obtain pKa* in the excited state, a wavenumber value of 0-0 transitions or absorption and fluorescence maxima for each form were needed. Therefore, the fluorescence spectra excited at: 300, 320, 340 and 375 nm were registered. Examples for λex = 320 nm were shown in Fig. 2 and for λex = 340 nm in Fig. S3. Fluorescence spectrum at pH 5.9 with the maximum at 464 nm shifted to 500 nm at pH 10.7, with a simultaneous decrease in the intensity. Spectra at these pHs may be considered as the spectra of the dissociated and non-dissociated form of MESA respectively, since less than 1 % of each form of MESA was present in these solutions. To calculate pKa* at the excited state from the Förster cycle, entropy changes in the ground and excited states were necessary. It was assumed that they were equal. The desired wavenumbers for 0-0 transition were taken at the maximum of the absorption and fluorescence spectrum of each MESA form.41 Based on this data and data from Tab. 1, pKa* was calculated. The obtained value 13 ACS Paragon Plus Environment


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was much smaller than in the ground state, which indicated that MESA in the excited state was a stronger acid than in the ground state. The electronically excited MESA in water solution of pH 5.9, contrary to the ground state molecule, should exist almost solely in the dissociated form and the deprotonation ought to proceed more readily in the excited state. The average dipole moment of MESA molecules in the acid-base equilibrium in the excited state should be much bigger than the dipole moment in the ground state and practically almost the same as a dipole moment of the electronically excited MESA in alkaline pH (both in neutral and alkaline pH the population of protonated electronically excited MESA is negligible). Dipole moments in the ground state were obtained using QC calculations (Tab. 1). Their values were typical and not similar for both forms, due to proton dissociation in the ground state in alkaline pH. Charge distribution, calculated with QC methods, in the ground and not relaxed excited states (Fig. S4) suggested only a tendency for -OH bond weakening in the excited state. Electron density on the O atom in the -OH group decreased by about 0.05 unit compared to the ground state with a simultaneously increasing positive charge on the benzene ring, and increasing electron density on the O atom in the -CO group in alkyl chain, which confirmed all above-mentioned observations and suggested that the photoinduced intramolecular charge transfer took place. To verify the pKa* value, corrected fluorescence spectra were applied. Correction for inner filter effect I and II allows us to calculate pKa* for different wavelengths of excitation, not only at the isosbestic point. Applying the sigmoidal dose-response (variable slope) function in the program to the total fluorescence (area under spectrum), the average value of LogEC50 (inflection point) = pKa* (fl) was calculated. This value was 8.5 (Tab. 1) and not about 2. Respective plots for the three wavelengths of excitation are shown in Fig. 3. Fluorescence spectra measured in 0.1 N and 1.0 N HCL and 0.1 H2SO4 gave the same spectrum as at pH 5.9. It also suggested that the spectrum at pH 5.9 was the spectrum of the undissociated form. This observation suggested that fluorescence occurred before 14 ACS Paragon Plus Environment

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deprotonation and steady-state fluorescence was not appropriate method for pKa* determination of MESA.

Figure 3. Relative normalized fluorescence intensities (area under spectrum) of MESA versus pH. Concentration 30 µM, λex = 320 nm, quartz cell 1 cm, right angle geometry.

Measurements of lifetimes for both forms gave very small values (Tab. 1) and for this reason these measurements were very difficult. For the acidic form, observation was provided from 450 nm to 500 nm and for the basic form - from 460 to 510 nm applying the global analysis in calculation. Examples of such decays at pH 5.9 were shown in Fig. 4a. Mainly acidic form was present in the solution at this pH, therefore λex = 322 nm (100% of power, 8.64 MHz, FWHM about 90 ps) was used to excite MESA, whereas in the alkaline pH above 10 mainly the dissociated form occurred with the absorption maximum at 383 nm, therefore, a 375 nm laser (FWHM about 64 ps) was used to excite the molecules (Fig. S5). Proton pumping effect to the solvent in the excited state was not observed at pH 5.9, however lifetimes at pH 5.9 was too short to observe this effect. Unexpected results from fluorescence strongly suggested that at pH 5.9, the non-ionized form of MESA is emitted. Steady-state and time-resolved results suggested that pKa* obtained from Förster cycle is better and rate constant for deprotonating or protonating process 15 ACS Paragon Plus Environment


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can be a lower compared to the radiative rate constant causing faster de-excitation than proton transfer. In such a case, in the excited state, the thermodynamics from the ground state will play the crucial role. This hypothesis was supported by a very short lifetime. Using absorption and fluorescence spectra one can calculate QY versus pH (Fig. S6).

Figure 4. Fluorescence intensity decays of MESA: a) pH 5.9, λex = 322 nm, b) Green – methanol (λex = 327) nm, blue – DMSO (λex = 320 nm). Red - IRF on both plots.

The QY was very low (the order of 10-3) and for the acidic form it was about two times higher than for the basic one (Tab.1). Values of QY of each form (Tab.1) were achieved as the mean 16 ACS Paragon Plus Environment

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value of “bottom” and “top” parameters in the sigmoidal dose-response (variable slope) function in Fig S6. The MESA at acidic and alkaline pH has one exponential decay. To check such short decays, the natural lifetime τn was calculated based on formula (4). The τn equaled 6.47 ns for the non-dissociated form and 6.91 ns for the dissociated form. Multiplying these values by QY, we obtained 11 ps and 4.8 ps for lifetimes for each form, respectively. These results are in relatively good agreement with the measured lifetimes shown in Tab. 1. Lifetimes reaching few picoseconds are comparable with vibrational relaxation time, but big Stokes shift suggested that the emission is from the relaxed state at both pH values. A big difference between the natural (τn) and the measured lifetimes (τ) may result from the interactions with the solvent, and from a change in the excited-state geometry.41 Having QY and lifetimes for both forms of MESA, the rate constants for radiative (kr = QY / τ) and non-radiative (knr = (1 - QY) / τ) processes of energy de-excitation can be calculated.41 Assuming τ = 10 ps, knr was 1 x 1011 s-1 and 1.3 x 1011 s-1, kr was 1.7 x 108 s-1 and 0.9 x 108 s-1 for MESA(OH) and MESA(O-) forms, respectively. These values indicated that the excitation energy was very quickly transferred to the triplet state or was converted into heat. Further investigations made in non-polar environments implied that the second possibility was more likely. 3.2 Non-water environment Absorption and fluorescence spectra of MESA were measured in the following solvents: nhexane, cyclohexane, chloroform, ethyl acetate, methylene chloride, ethanol, methanol, MeCN, and dimethyl sulfoxide (DMSO). Wavelengths of absorption maximum (λamax) were between 318 nm (n-hexane) and 334 nm (DMSO) (Fig. 5). Generally, the dependence of the



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Figure 5. Absorption spectra of MESA in different solvents. Concentration 30 µM, path length 1 cm.

maximum of the absorption spectra on the basic solvent properties (polar protic, polar aprotic, apolar-aprotic) cannot be explained by any heuristic model, which depends only on the dielectric constant of the solvent (Tab. 2). For example, λamax shifted from 322 nm in water (pH 5.9) through 327 nm in methanol to 329 nm in ethanol. This is the opposite direction often observed for n - π* transition, but the registered bands of MESA were π – π* transition, due to a high molar absorption coefficient which was about 2 x 104 mol-1 L cm-1. The same behavior was observed for MeCN and DMSO and there was no regular shifting between MESA bands in alcohols and MeCN and DMSO. Maxima of absorption spectra for the some apolar-aprotic solvents also shifted to longer wavelengths compared to water. Only spectra in n-hexane and cyclohexane shifted towards the shorter wavelengths. The absorption coefficient was at a similar level for each solvent – the highest for ethanol, and the lowest for methylene chloride (Tab. 2), which is in good agreement with dipole moments value (Tab. 2.). Such sets of absorption data were related to the molecular structure of MESA which is neither polar nor apolar in nature. Consequently, this compound is well soluble in all solvents, however better in organic one than in water where the 18 ACS Paragon Plus Environment

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highest concentration could be about 4 mM. Such solubility makes MESA applicable as an antioxidant in many environments. The wavelength of fluorescence maximum (λfmax) was between 362 nm (n-hexane and cyclohexane) and 463 nm in water (Fig. 6 and 7, Tab. 2). Stokes shift in apolar-aprotic solvents is much smaller than in the polar one. This suggests that the interaction with solvent molecules has a great impact on fluorescence properties. Fluorescence spectra, in contrast to the absorption spectra, regularly shifted towards the shorter wavelength when dielectric constants of solvents (εr) decreased (Fig. 6). This is very well evident in Fig. 7 depicting normalized spectra. Intensities of fluorescence spectra (Fig.6) may be compared between themselves because the dependence of fluorescence intensity from the absorbance is linear after inner filters correction. The QY of MESA was calculated for each solvent using the



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Table 2. Properties of MESA in non-water environment. Solvent

εr

QY

τn [ns]

QY x τn [ps]

τ ± PEA [ps]

kr x 10-8 [s-1]

knr x 10-11 [s-1]

λamax [nm]

λfmax [nm]

νamax [cm-1]

νfmax [cm-1]

εmax (L mol-1 cm-1)

∆ν [cm-1]

µg [D]

n-hexane

1.89

0.0012

3.54

4.2

≤ 10

~ 1.9

~ 1.6

318

362

31447

27624

19900

3822

2.95

cyclohexane

2.02

0.0014

2.67

3.7

≤ 10

~ 2.2

~ 1.5

319

364

31348

27473

20460

3875

2.97

chloroform

4.81

0.0018

3.68

6.6

≤ 10

~ 1.8

~ 1.0

323

392

30960

25510

19100

5450

2.97

ethyl acetate

6.02

0.0032

4.42

14.1

≤ 10

~ 3.2

~ 1.0

324

392

30864

25510

19160

5354

2.96

methylene chloride

9.08

0.0027

3.82

10.3

21.6 ± 4.8 40.4 ± 1.5

1.3 6.7

0.46 0.25

322

397

31056

25189

19800

5867

2.68

ethanol

24.30

0.0047

4.12

19.3

22.7 ± 0.3

2.1

0.44

329

426

30395

23474

21780

6921

2.99

methanol

32.63

0.0041

5.26

21.6

20.7 ± 0.3

2.0

0.48

326

434

30675

23041

19860

7633

3.17

acetonitrile

36.64

0.0044

4.90

21.6

14.7 ± 0.2

3.0

0.68

322

413

31056

24213

18520

6843

3.00

dimethyl sulfoxide

47.24

0.0124

4.19

52.0

52.7 ± 0.3

2.4

0.19

334

435

29940

22989

19540

6952

3.02

εr - dielectric constant,42 QY - quantum yield, τn – calculated natural fluorescence lifetime, τ – measured lifetime, kr, knr – radiation and not radiation rate constants, λamax and λfmax - wavelength of absorption and fluorescence maximum, νamax and νfmax – wavenumbers of absorption and fluorescence maximum, ∆ν - Stokes shift, µg - calculated dipole moment (QC-method) in the ground state. kr and knr for the first four solvents were calculated assuming τ = 10 ps. 20 ACS Paragon Plus Environment

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Figure 6. Corrected fluorescence spectra of MESA in different solvents. Concentration 30 µM, λex = 320nm, quartz cell 1 cm, right angle geometry.

Figure 7. Normalized fluorescence spectra from Fig. 6.

same procedure like for the two forms of MESA in buffers. Results were shown in Table 2. The QY was practically low in all solvents except for DMSO. In this solvent, it was about 10 times higher than the lowest value in n-hexane and water solutions. Lifetimes of MESA in methanol and DMSO were measured using different wavelengths of excitation, which was



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feasible by using SuperK EXTREME high Power Systems with DUV extension (60% of power, 8.64 MHz, FWHM 88 – 120 ps). Both, excitation and observation wavelength were established due to the maxima of absorption and fluorescence spectra. Lifetimes of MESA in methanol and DMSO were presented in Fig. 4b. Natural fluorescence lifetime τn was calculated for MESA in each solvent using formula (4). The results were shown in Table 2. As one can see, there were no big differences between all the lifetimes. The lowest τn was for cyclohexane (3.19 ns), and the highest one for DMSO (4.76 ns). The τn of MESA was clearly dependent on dielectric constant – the higher the dielectric constant, the higher the τn value. To compare the measured and calculated lifetimes, τn was multiplied by appropriate QY of MESA in each solvent. There was relatively good compatibility between dielectric constants and lifetimes, like in water. To investigate dependence of absorption and fluorescence maxima on solvent polarity and its ability to hydrogen bonds formation, plots of νamax and νfmax versus SdP polarity scale parameters was made according to Catalan.43 The following formulas were used: νamax = 33479 – 2959SP – 14SdP – 340SA - 1523SB [cm-1] νfmax = 31872 – 6219SP – 1964SdP – 3807SA - 2084SB [cm-1] where: SP - the measure of polarizability effect of the solvent, SdP - the measure of dipolarity effect of the solvent, SA – the index of solvent acidity, ability to act as a hydrogen bond donor, and SB – the index of solvent basicity, ability to act as a hydrogen bond acceptor. Goodness of fit to νamax and νfmax was determined at a level of R2 = 0.967 and R2 = 0.996, respectively. Such high level of R2 means that the absorption and fluorescence maxima of MESA in various solvents can be predicted with high accuracy. The absorption maxima of MESA, as shown in Fig. S7, are well reproduced by the heuristic model based on SdP scale and for fluorescence maxima (Fig. S8) the reproduction is almost perfect. According to the obtained fits, MESA in the ground state should acted as the donor of protons (SB / SA ≈ 4.5) forming hydrogen bonds. However, in the excited 22 ACS Paragon Plus Environment

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state, a different situation occurred (SB / SA ≈ 0.55) - MESA acted slightly better as an acceptor than as a donor of protons. Simultaneously it was a better acceptor (3807 / 340 ≈ 11) in the excited state than in the ground state. Five oxygen atoms of MESA can act as proton acceptors. The fraction of (3807 + 2084) / (340 +1523) = 3.16, which is three times higher in the excited than in the ground state, suggests that the process of formation of the hydrogen bonds was enhanced in the excited state. It is possible that one of the reasons of such low values of QY is the fact that energy of excitation may escape to the triplet state due to intersystem crossing when hydrogen bonds are formed, but data for the measured and calculated lifetimes for n-hexane and cyclohexane (Tab. 2.) did not confirm this hypothesis, because the lifetimes and QY are comparable with those obtained in polar solvents, hence it seems that the excitation energy was mainly converted to the heat. Our results confirmed that MESA is stabilized in the ground and the excited state by polarizability. Low dipolarity in the ground state suggests that in the ground state dipole moments are solvent independent – which is confirmed by calculated dipole moments in the ground state. Dipolarity of the solvent is much higher in the excited state (about 140 times), which strongly suggests that solvent effect and dipole moment is much higher in the excited state and that photoinduced intramolecular charge transfer occurs. This is partially confirmed by calculation of charge distribution of MESA molecule in both states (Fig. S4). Two mechanisms of antioxidant action: dehydrogenation or electron transfer reaction, were shown by Foti et al.7 The intermolecularly hydrogen-bonded phenol group -OH is essentially unreactive to all radicals (due to steric protection of the -OH group by the solvent), with only the “free”, non-hydrogen-bonded -OH group being reactive.44 3.3 Computation of dipole moments in the ground state MESA molecule in the protonated form is shown in Fig. 8. The molecule has four rotatable bonds, marked by thick lines in Fig. 8, and at the room temperature it can exist in an equilibrium 23 ACS Paragon Plus Environment


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of many conformations. In the protonated form, one of the metoxyl groups can rotate (α dihedral angle) and reach three energy minimum positions (checked by lower-level QC calculations – data not shown), but the rotation of the second metoxyl (β dihedral angle) group is blocked by a steric clash with proton of the hydroxyl group.

Figure 8. The structure of the protonated MESA molecule with the assignment of atom numbers. Four rotatable bonds are marked with thick lines and they are defined as follows: α = C4 - C5 - O9 - C11, β = C2 C1 - O10 - C15, γ = C2 - C3 - C21 – C22, δ = C21 – C22 – C23 – O25. In the protonated MESA, these dihedral angles can assume following conformations: α = {00, 1200, -1200}; β = {00}; γ = {00, 1800}; δ = {00, 1800}. All possible conformations related to changes of these dihedral angles are listed in Table T1 of SI. In the deprotonated MESA, the flexibility of β dihedral angle is increased and, similarly to α dihedral, it can assume three conformations β = {00, 1200, -1200}.

Therefore, there are three possible conformations of metoxyl groups attached to the ring – one planar (in which both methyl carbons lie in the plane of the ring, ߙ = 0௢ ) and two non-planar (in which methyl groups are either below or above the plane of the ring, ߙ = ሼ−120௢ , 120௢ ሽ). The conformational flexibility of the tail of the molecule is defined by the values of two dihedral angles (γ and δ), which can assume either cis (0o) or trans (180o) conformation. Therefore, the tail 24 ACS Paragon Plus Environment

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of MESA can exist in four possible conformations and consequently the whole protonated MESA molecule can assume 12 distinct configurations – four planars and eight non-planars (each of the degenerated twice). Eight configurations (four off-planar symmetric configurations are not shown) of protonated MESA are presented in Fig. 9.



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Figure 9. Eight conformers of the protonated MESA molecule with their respective populations. The dominant planar conformations are shown in the left column (a-d) and four off-planar configurations are shown in the right column (e-h). There are four symmetric (equal free energy) off-planar configurations which are not shown. Off-planar populations include both conformations shown in the figure and its mirror image. Dipole moment of conformer b) is very small (0.3 D) so it is not visible in the figure. Note that conformational changes lead to large changes of the dipole moment of MESA. Conformers with methyl group and proton of the hydroxyl group placed on the same side of C3-C5 axis (a, d, e, h) have significantly larger dipole moment than the other four conformers (b, c, f, g).

For deprotonated MESA, both methoxyl groups can assume three possible configurations, which combined with four available configurations of the tail leads to the total of 36 possible conformations. Some of them overlap and detailed analysis shows that there are total of 20 distinct configurations of deprotonated MESA. All possible configurations of protonated and deprotonated forms of MESA are defined in Table S1. Electric dipole moments for all possible configurations of protonated MESA immersed in various solvents were computed with QC methods. It should be stressed here that our computations show that dipole moment of this molecule strongly depends on its conformation, as can be seen in Fig. 9, in which dipole moments for protonated MESA in water changes its value from 0.3 D to 7.5 D. Moreover, several conformational states are energetically very close, therefore the total dipole moment of the molecule in the solvent must be computed as a Boltzmann average and the approximation of the total dipole moment as the one computed in the global energy minimum is not valid for this molecule. Two lowest-energy planar conformations are almost equally populated, but have vastly different electric dipole moments (see Fig. 9). The free energy difference between planar and non-planar configurations is only 0.6 kcal / mol. Boltzmann-averaged electric dipole moments are collected in Table 2. Interestingly, the value of the total dipole moment does not significantly depend on the environment as it is the lowest for methylene chloride (2.68 D) and the highest for methanol (3.17 D). The dipole moment of 26 ACS Paragon Plus Environment

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deprotonated MESA in water is much higher (9.94 D) than the one of protonated MESA (3.02 D), as it can be seen in Table 1. It also does not significantly depend on the conformation of deprotonated MESA molecule. The conformational equilibrium significantly changes after dissociation of proton and the equilibrium between planar and non-planar configurations is shifted towards the latter one by 0.74 kcal / mol. Therefore, it should be expected that changes in pH of the buffer cause not only protonation / deprotonation of MESA but also a significant change in the geometry of the molecule. Total dipole moments collected in Tab 2 were used to compute the excited-state dipole moments using the B-K method and the data obtained from spectroscopic measurements. 3.4 Computation of dipole moments in the excited state To calculate ratio between dipole moments in the ground and the excited state for free molecule, two parameters m1 and m2 must be determined. With this purpose, two plots were created - one according to Eq. 1 (Fig. 10a) and the second one to Eq. 2 (Fig. 10b). Then, ratio between dipole moments in the ground and the excited state for free molecule were calculated from Eq. 3. The calculated ratio was at level

ఓ೐

ఓ೒

= 4.39. This result was obtained assuming that ߤԦ௘ and ߤԦ௚ are

parallel to each other in the B-K method. According to data from the plot presented in Fig. 10a, Stokes shifts of MESA both in protic and aprotic solvents (with single exception of water) were roughly the same. The calculated dipole moments in both ground and excited states for the protic and aprotic solvent were almost equal, but these calculations were done without considering hydrogen bonds formation.



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Figure 10. B-K graphs. a) Stokes shift versus f (ε, n) (Eq. 1). Parameter m1 = 3811 [cm-1], R2 = 0.96. b) Σν versus f (ε, n) + 2g(n) (Eq. 2). Parameter m2 = 6056 [cm-1], R2 = 0.92.

4. Conclusions The pKa obtained in the water environment suggest that a good antioxidant activity of MESA through radical route should be expected at pH below 6, where MESA exists mainly in the nondissociated form, and hydrogen can be transferred from the phenol group of MESA to another species. In alkaline pH, due to a lack of proton at the phenol group, only the electron can be transferred from anion of MESA to the radical. One of the possible explanations of significant difference between pKa* values obtained from Förster cycle and from fluorescence spectra is that the rate constant for protonation/deprotonation must be lower than rate constant of fluorescence. Therefore, in the excited state the equilibrium cannot be achieved and fluorescence occurs before 28 ACS Paragon Plus Environment

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deprotonation. QC calculations showed that in the ground state MESA exists in complicated conformational equilibrium. This equilibrium shifts towards nonplanar conformation as a result of deprotonation of MESA. Most probably the system exists in complicated conformational equilibrium also in the excited state, although this equilibrium might be different than the one observed for the ground state. Therefore, the entropy changes between ground and the excited states might be different, while in Förster cycle assumption is made that they are equal, although this hypothesis should be verified by QC calculations of conformational equilibrium in the relaxed excited state. MESA showed very low QY and lifetime values in all solvents. There are two possible explanations of these phenomena - the filling of a triplet state due to intersystem crossing (ISC), which can be enhanced by hydrogen bond formation in the excited state and internal conversion of the excitation energy into the heat. The analysis of experimental data in the context of SdP - scale suggests that the second explanation is more likely. Results of SdP - scale analysis show also that in polar solvents the efficiency of ISC processes increases, but lifetimes and QY remain low. Therefore, conversion of excitation energy into the heat seems to be the main route for de-excitation. Nevertheless, further investigations with photo-acoustic technics and phosphorescence should explain a big loss of the energy in the excited state. Supporting Information The Supporting Information is available free of charge on the ACS Publications website at… Figures S1 - S8 and Table S1. Acknowledgements The authors express their gratitude to NKT Photonics (Denmark) and INTERLAB (Poland) for lending SuperK EXTREME EXW-20 (High Power Systems) with SuperK EXTEND-DUV supercontinuum extension unit. We would like to acknowledge the support by the project No. 29 ACS Paragon Plus Environment


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17.610.011-300. All QC calculations were performed in Wrocław Centre for Networking and Supercomputing (http://wcss.pl, Grant No. 349), Regional Computer Center in Olsztyn, Academic Computer Center in Gdansk and ICM KDM in Warsaw (Grant No. G59-14), Poland. References (1) Niiforovi, N.; Abramovi, H. Compr. Sinapic Acid and Its Derivatives: Natural Sources and Bioactivity. Rev. Food Sci. Food Saf. 2014, 13, 34-51. (2) 2. Manach, C.; Scalbert, A.; Morand, C.; Rémésy, C., Jimenez, L.; Polyphenols: Food Sources and Bioavailability. Am. J. Clin. Nutr. 2004, 79, 727–747. (3) Takaya, Y.; Kondo, Y.; Furukawa, T.; Niwa, M. Antioxidant Constituents of Radish Sprout (Kaiware-daikon) Raphanus Sativus L. J. Agric. Food Chem. 2003, 51, 8061–8066. (4) Chung, S.-K.; Osawa, T.; Kawakishi, S. Hydroxyl Radical-Scavenging Effects of Spices and Scavengers from Brown Mustard (Brasica nigra). Biosci. Biotechnol. Biochem. 1997, 61, 118-123. (5) Reis, B.; Martins, M.; Barreto, B.; Milhazes, N.; Garrido, E. M.; Silva, P.; Garrido, J.; Borges, F. Structure-Property-Activity Relationship of Phenolic Acids and Derivatives. Protocatechuic Acid Alkyl Esters. J. Agric. Food Chem. 2010, 58, 6986–6993. (6) Gaspar, A.; Martins, M.; Silva, P.; Garrid, E. M.; Garrido, J.; Firuzi, O.; Miri, R.; Saso, L.; Borges, F. Dietary Phenolic Acids and Derivatives. Evaluation of the Antioxidant Activity of Sinapic Acid and Its Alkyl Esters. J. Agric. Food Chem. 2010, 58, 11273–11280. (7) Foti, M.C.; Daquino, C.; Geraci, C. Electron-Transfer Reaction of Cinnamic Acids and Their Methyl Esters with the DPPH• Radical in Alcoholic Solutions. J. Org. Chem., 2004, 69, 23092314.


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(8) Alfassi, Z. B.; Huie, R. E.; Neta, P. In Peroxyl Radicals, Alfassi, Z. B., Ed., Wiley: New York, 1997, Chapter 9. (9) Nenadis, N.; Zhang, H-Y.; Tsimidou, M.Z. Structure-Antioxidant Activity Relationship of Ferulic Acid Derivatives: Effect of Carbon Side Chain Characteristic Groups. J. Agric. Food Chem. 2003, 51, 1874−1879. (10) Frankel, E. N.; Meyer, A. S. The Problems of Using One-Dimensional Methods to Evaluate Multifunctional Food and Biological Antioxidants. J. Sci. Food Agric. 2000, 80, 1925-1941. (11) Figueroa-Espinoza, M-C.; Villeneuve, P. Phenolic Acids Enzymatic Lipophilization. J. Agric. Food Chem. 2005, 53, 2779−2787. (12) Firuzi, O.; Giansanti, L.; Vento, R.; Seibert, C.; Petrucci, R.; Marrosu, G.; Agostino, R.; Saso, L. Hypochlorite Scavenging Activity of Hydroxycinnamic Acids Evaluated by a Rapid Microplate Method Based on the Measurement of Chloramines. J. Pharm. Pharmacol. 2003, 55, 1021–1027. (13) Zou, Y.; Kim, A. R.; Kim, J. E.; Choi, J. S.; Chung, H. Y. Peroxynitrite Scavenging Activity of Sinapic Acid (3,5-Dimethoxy-4-Hydroxycinnamic Acid) Isolated from Brassica Juncea. J. Agric. Food Chem. 2002, 50, 5884–90. (14) Trnková, L.; Boušová, I.; Kubίček, V.; Dršata, J. Binding of Naturally Occurring Hydroxycinnamic Acids to Bovine Serum Albumin. Nat. Sci. 2010, 2, 563–570. (15) Smyk, B. Fluorescence Study of Sinapic Acid Interaction with Bovine Serum Albumin and Egg Albumin. J. Fluoresc. 2003, 13, 349-356. (16) Jin, X. L.; Wei, X.; Qi, F. M.; Yu, S. S.; Zhou, B.; Bai, S. Characterization of Hydroxycinnamic Acid Derivatives Binding to Bovine Serum Albumin. Org. Biomol. Chem. 2012, 10, 3424–3431.



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(17) Galano, A.; Francisco-Marquez, M.; Alvarez-Idaboy, J. R. Mechanism and Kinetics Studies on the Antioxidant Activity of Sinapinic Acid. Phys. Chem. Chem. Phys. 2011, 13, 11199– 11205. (18) Smyk, B.; Drabent, R. Spectroscopic Investigation of the Equilibria of the Ionic Forms of Sinapic Acid. Analyst. 1989, 114, 723-726. (19) Leόn-Carmona, J. R.; Alvarez-Idaboyb, J. R.; Galano, A. On the Peroxyl Scavenging Activity of Hydroxycinnamic Acid Derivatives: Mechanisms, Kinetics, and Importance of the Acid–Base Equilibrium. Phys. Chem. Chem. Phys. 2012, 14, 12534-12543. (20) Bilot, L.; Kawski, A. Zur Theorie des Einflusses von Lösungsmitteln auf die Elektronenspektren der Moleküle. Z. Naturforsch. 1962, 17A, 621-627. (21) Kawski, A. Der Wellenzahl von Elecktronenbanden Lumineszie-Renden Moleküle. Acta Phys. Polon. 1966, 29, 507-518. (22) Kawski, A. On the Estimation of Excited-State Dipole Moments from Solvatochromic Shifts of Absorption and Fluorescence Spectra. Z. Naturforsch. 2002, 57A, 255-262. (23) Jena, N. R.; Mishra, P. C. Mechanisms of Formation of 8-Oxoguanine Due To Reactions of One and Two OH• Radicals and the H2O2 Molecule with Guanine: A Quantum Computational Study. J. Chem. Phys. B 2005, 109, 14205-14218. (24) Parks, J. M.; Ford, G. P.; Cramer, C. J. Quantum Chemical Characterization of the Reactions of Guanine with the Phenylnitrenium Ion. J. Org. Chem. 2001, 66, 8997-9004. (25) Černý, J.; Špirko, V.; Mons, M.; Hobza, P.; Nachtigallova, D. Theoretical Study of the Ground and Excited States of 7-Methyl Guanine and 9-Methyl Guanine: Comparison with Experiment. Phys. Chem. Chem. Phys. 2006, 8, 3059-3065. (26) Shukla, M. K.; Mishra, P. C. A Gas Phase Ab Initio Excited State Geometry Optimization Study of Thymine, Cytosine and Uracil. Chem. Phys. 1999, 240, 319-329. 32 ACS Paragon Plus Environment

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