J. Phys. Chem. B 2007, 111, 3977-3981
3977
Theoretical Assessment of Naphazoline Redoxchemistry and Photochemistry Klefah A. K. Musa and Leif A. Eriksson* Department of Natural Sciences and O ¨ rebro Life Science Center, O ¨ rebro UniVersity, 701 82 O ¨ rebro, Sweden ReceiVed: January 10, 2007; In Final Form: February 16, 2007
The imidazoline derivative naphazoline (2-(1-naphtylmethyl)-2-imidazoline) is an R2-adrenergic agonist used as non-prescription eye and nasal preparations. Besides its functionality in generating vascoconstriction and decongestion in the patient, the toxicity, ROS generating capability, and recently also possible antioxidant capacity of the compound have been reported in the literature. In the current work the structural and electronic features of the drug are explored, using computational chemical tools. Electron affinities, ionization potentials, and excitation energies are reported, as well as charge and spin distributions of various forms of the drug. The difference in photochemical behavior between the protonated and unprotonated (basic) species is explained by the molecular orbital distributions, allowing for efficient excitation quenching in the basic structure but clear naphthalene to imidazolene charge transfer upon HOMOf LUMO excitation in the protonated form, enabling larger intersystem crossing capability to the imidazole localized excited triplet and a resulting higher singlet oxygen quantum yield.
Introduction The imidazoline derivative naphazoline (NP) [2-(1-naphtylmethyl)-2-imidazoline] is a sympathomimetic drug,1 used in over-the-counter eye and nasal preparations. The compound has vasoconstrictive and decongestive properties2,3 and acts on R-adrenergic receptors in the arterioles of the conjunctiva to produce vasoconstriction, resulting in decreased conjunctival congestion. It is thus widely used to relieve redness due to minor eye irritation. There are, however, some reports in the literature concerning the in vivo toxicity of naphazoline.4-6 A number of studies have been reported for the study of the physiochemical properties of NP including micellar electrokinetic chromatography,7 phosphorimetry,8,9 and spectrophotometry.10 The potential phototoxic activity of naphazoline has been addressed by studying its photoreactivity toward DNA. Photocleavage studies combined with laser flash photolysis experiments provide clear evidence that the transient species produced under NP photolysis react with DNA, thereby promoting its breakage under both aerobic and anaerobic conditions.11 The higher photocleavage quantum yield found under anaerobic conditions was attributed mainly to the direct involvement of hydrated electrons and, partially, to the nitrogen-centered radicals produced after NP photoionization in the reaction with DNA. Redox reactions involving these transient species and the DNA nucleobases are believed to be responsible for the DNA breakage. Nanosecond laser flash photolysis measurements provided clear evidence of reactions between these NP intermediates and DNA. Under aerobic conditions, hydroxyl radicals formed by the Haber-Weiss reaction were shown to initiate DNA photocleavage. The interaction between the photosensitizer and DNA played a key role in the photosensitization mechanism, enhancing the reactions involving the nitrogen-centered radicals. In addition, the formation of two NP-DNA complexes, characterized by triplet states scarcely quenchable by oxygen, were concluded to reduce the contribution of DNA oxidation pro* Address correspondence to this author. E-mail nat.oru.se.
leif.eriksson@
cesses mediated by singlet oxygen.11,12 The results obtained demonstrate the potential for NP to act as both a type I and a type II photosensitizer.12 Many drugs belonging to different pharmacological classes are characterized by one or more protonation sites and, as a consequence of the relative prototropic equilibria, different forms can be present at different pH values. The different prototropic species of a drug can in turn display different abilities to interact with their biological targets. The consequence of different drugbiomolecule binding modes can furthermore be important in determining the efficiency of the photosensitizing activity of the drug. NP, with a pKa value just above 10, has in this context been examined in aqueous solution by combining steady state and time-resolved spectroscopic experiments.13 These studies suggest that an increase in pH does not change the nature of the photochemical process but only reduces its efficiency; protonation and deprotonation of the imidazoline ring of NP play a key role in the efficiency of the photoinduced deactivation pathways of the drug, thereby modulating its potential properties as type I and type II photosensitizers. The neutral form of NP is characterized not only by a higher photostability but also by a lower efficiency as a singlet oxygen generator. Interestingly, in a recent study it was pointed out for the first time that NP may also act as an antioxidant, thus preventing the deleterious effects of γ-radiation.14 Moreover, this effect can be increased by the positive charge of NP at physiological pH, which facilitates interactions with cell membranes and anionic proteins in the serum. The radioprotective effect of NP was observed in γ-ray irradiated free DNA and in mice. Moreover, the ability of NP to inhibit the deleterious effect of OH radicals on DNA was investigated. For this purpose Fenton reactions were performed, and the results strongly suggest that NP acts as an •OH radical scavenger. This property may hence partly contribute to the inhibition of the indirect effect of γ-rays albeit the detailed mechanisms for the radioprotective property are still unknown. The aim of the present work is to use computational quantum chemistry methods to gain more insight into the overall redox properties and photochemical behavior of NP. More detailed
10.1021/jp070207f CCC: $37.00 © 2007 American Chemical Society Published on Web 03/24/2007
3978 J. Phys. Chem. B, Vol. 111, No. 15, 2007
Figure 1. Naphazoline (A), its tautomer (B), and protonated form (C). For B the atomic numbering is displayed.
knowledge may in turn enable improved drug photostability and reduced drug phototoxicity. Methodology All geometries of naphazoline (A), its tautomer (B), and its protonated form (C) shown in Figure 1 were optimized at the B3LYP/6-31G(d,p) level of theory, in their lowest singlet and triplet states, as well as their reduced or oxidized forms. Frequency calculations were performed on the optimized geometries at the same level of theory, to ensure the systems to be local minima (no imaginary vibration frequencies), and to extract zero-point vibrational energies (ZPE) and thermal corrections to the Gibbs free energies at 298 K. Solvent effects were taken into consideration implicitly, through single point calculations on the optimized geometries at the same level of theory, including the integral equation formulation of the polarized continuum model (IEFPCM).15-17 Water was used as solvent, through the value 78.31 for the dielectric constant in the IEFPCM calculations. For a less polar environment (e.g., when bound to a receptor active site), a smaller value of the dielectric constant could in principle be employed. On the other hand, the difference in effect between ) 18 (generally assumed for active sites) and 78 is normally very small due to the rapid decay of the associated function. The vertical electron affinities (VEA) and ionization potentials (VIP) were calculated as the energy difference between the neutral species and the anionic/ cationic species at the geometry of the neutral molecule. Singlet and triplet excitation energies were determined at the same levels of theory, using time-dependent (TD) DFT calculations.18,19 On the basis of its chemical structure, NP can be considered as a biochromophore formed by an electron (energy) donor subunit (2-methyl-2-imidazoline, IM) and an acceptor moiety (naphthalene, NA). To understand possible excitation energy transfer and intramolecular interactions between these, the separate subunits were also optimized in gas and aqueous phases in their singlet and triplet states, using the B3LYP/6-31G(d,p) level of theory. The lowest lying singlet and triplet excited states were also obtained from TD-DFT calculations. The numbering scheme of the atoms, used throughout the study, is given in Figure 1. All calculations were performed with the Gaussian 03 program package.20 For all closed shell species, the restricted formalism is employed, whereas all open-shell compounds (doublets and triplets) are treated at the unrestricted SCF level. Results and Discussion The three species in their different redox and electronic states were studied in gas and aqueous phases. The absolute and relative zero-point corrected energies in the gas phase, and absolute and relative Gibbs free energies in aqueous solution are presented in Table 1. The most stable neutral tautomer is A, located 5.5 kcal/mol below B. Assuming a Boltzmann distribution this energy difference will correspond to a relative distribution of less than 1/100 of a percent of B compared to A, at room temperature. The proton affinity leading to C is 249.5
Musa and Eriksson kcal/mol in the gas phase, and 296.2 kcal/mol in aqueous solution (due to the stabilizing nature of the polar solvent on charged species). Given the estimated free energy of a solvated proton in aqueous solution, -268.68 kcal/mol,21 the considerably higher proton affinity of the species agrees with the high pKa value, and ensures that at physioplogical pH the system will essentially only be present in the protonated form C. The neutral (basic) form B has the lowest singlet-triplet energy gap in both gas and aqueous phases (computed values 48.1 and 44.3 kcal/ mol, respectively), compared with ∼58 kcal/mol for both A and C. It should be noted that formation of the cationic species and the excitation to the triplet state results in a change in relative stabilities of tautomers A and B. Under extremely alkaline conditions, it is hence possible that the differences in relative energies could serve as a diriving force for tautomerization between the two neutral forms, which in turn may also influence the photochemical properties of the drug at very high pH. In Tables 2 and 3, we list the Mulliken atomic charges and spin densities, respectively, on selected atoms for species A, B, and C in their different forms, as well as for the two chromophore subunits (NA and IM). The most negative atomic charges are located to N1 and N3 of the imidazoline ring (-0.429 to -0.568) and to a less extent C6 (-0.178 to -0.341) on the methylene linker. The main positive charges are found on C2 (0.342-0.649), H7 for B and C, and H8 on all forms (0.2350.328), i.e. the largest atomic charges are for all species localized mainly on the 2-methyl-2-imidazoline moiety. To understand the possible energy or charge transfer from one subunit to another, the total charges on each subunit are also summarized in Table 2. They show that in the neutral form A, the charge is evenly distributed between the donor and acceptor for both the singlet and triplet states whereas in its radical anion or cation form, the charge is mainly localized on the NA subunit. For tautomer B, similar charge distribution is noted, except that in the radical cation more of the positive charge is found on the imidazoline unit. For the protonated form C, the charge is in both the singlet and triplet states mainly found on the 2-methyl-2-imidazoline. Reduction of C+ occurs almost exclusively on the imidazoline, whereas oxidation (to C•2+) takes place from the naphthalene unit. The Mulliken atomic spin densities for all the open shell forms of the different species show that these are localized mainly on the naphthalene subunit atoms C1′, C4′, C5′, and C8′ (cf. Figure 1 and Table 3). To a less extent, spin is also found at N1 and N3 for form A and for form B on C2. In the reduced protonated species (C•), however, all spin is localized to atom C2 on the imidazoline unit. In Table 3, we also list the total spin densities on the two subunits. These results are fully consistent with the observed localization of charge upon reduction of the different parent compounds, as discussed above. This hence indicates that a charge transfer within the protonated form C would occur from NA to IM. For the neutral form A, the NA subunit would instead be the electron acceptor, whereas the donation occurs distributed over both units. This can be compared with energetic data for the two subunits considered separately. The computed electron affinity and ionization potential for naphthalene is 33.5 and 131 kcal/mol, respectively, in aqueous solution, which in both cases is less than that for the protonated 2-methyl-2-imidazoline: 38.7 and 159.5 kcal/mol, respectively. This also implies that at physiological pH reduction occurs primarily at the IM subunit whereas oxidation occurs most readily from NA. The data for the individual subunits agree well with those for reduction/ oxidation of the protonated NP: EA 41.3 kcal/mol and IP 131.1
Naphazoline Redoxchemistry and Photochemistry
J. Phys. Chem. B, Vol. 111, No. 15, 2007 3979
TABLE 1: B3LYP/6-31G(d,p) Zero-Point Energy Corrected Electronic Energies in the Gas Phase, and IEFPCM-B3LYP/ 6-31G(d,p) Gibbs Free Energies in Aqueous Solutiona A
X (singlet) X•- (doublet) X•+(doublet) 3X (triplet)
B
C
gas phase E(ZPE)
solvent phase ∆Gaq298
gas phase E(ZPE)
solvent phase ∆Gaq298
gas phase E(ZPE)
solvent phase ∆Gaq298
-651.197057 -651.187226 -650.940256 -651.105710
-651.257198 -651.311748 -651.065545 -651.164566
absolute energies (au) -651.188238 -651.165308 -650.974604 -651.111669
-651.246519 -651.294328 -651.098093 -651.175894
-651.594592 -651.736787 -651.208050 -651.501279
-651.729167 -651.795826 -651.517019 -651.638080
X (singlet) X•- (doublet)b X•+(doublet) 3X (triplet)
0.0 -6.2 161.2 57.3
relative energies (kcal/mol) 0.0 -14.4 134.1 48.1
0.0 34.2 120.3 58.1
0.0 30.0 93.1 44.3
0.0 89.2 242.6 58.6
0.0 41.3 131.1 57.2
singlet exc states
λ (nm)
(f)
λ (nm)
(f)
λ(nm)
(f)
1st 2nd 3rd 4th 5th 6th 7th 8th 9th 10th 11th 12th 13th 14th 15th
298.32 282.71 277.09 247.68 236.10 218.86 208.16 206.87 204.25 204.04 201.41 199.49 197.31 193.46 189.75
0.0092 0.0829 0.0015 0.0107 0.0001 0.0018 0.8542 0.2396 0.0210 0.0055 0.0646 0.1832 0.0414 0.0071 0.0125
366.43 313.34 264.88 255.58 253.92 219.56 218.68 208.98 207.51 205.11 197.91 195.83 193.74 191.09 188.96
0.2380 0.0247 0.0346 0.0363 0.3381 0.0151 0.0091 0.4399 0.1753 0.0948 0.1191 0.2849 0.0972 0.0046 0.0468
356.01 293.69 286.85 276.14 235.70 217.76 211.46 205.53 198.78 196.65 195.23 192.39 191.26 189.70 187.01
0.0018 0.0013 0.0981 0.0039 0.0062 0.0052 0.4851 0.3979 0.2629 0.0790 0.1182 0.0049 0.0203 0.0121 0.0028
a Absolute energies in au, relative energies in kcal/mol, and the absorbance of singlet excited states in nm and their oscillator strengths (f). b A positive electron affinity is defined as an exothermic process (the reduced species lies energetically lower than the parent compound).
TABLE 2: Mulliken Atomic Charges (B3LYP/6-31G(d,p) Level) for Selected Atoms and the Two Subunits NA and IM system
N1
N3
C2
C6
A A•A•+ 3A B B•B•+ 3B C+ C• C•2+ 3C+
-0.533 -0.529 -0.523 -0.545 -0.569 -0.568 -0.555 -0.569 -0.528 -0.531 -0.530 -0.529
-0.478 -0.513 -0.429 -0.477 -0.584 -0.566 -0.582 -0.554 -0.549 -0.545 -0.530 -0.547
0.443 0.451 0.540 0.476 0.522 0.406 0.614 0.469 0.649 0.342 0.602 0.645
-0.273 -0.248 -0.341 -0.307 -0.248 -0.220 -0.178 -0.194 -0.338 -0.289 -0.322 -0.340
H7
H8
NA
IM
0.265 0.251 0.301 0.273 0.316 0.243 0.328 0.315
0.253 0.263 0.295 0.257 0.260 0.235 0.304 0.266 0.319 0.257 0.328 0.321
0.022 -0.824 0.644 0.041 -0.071 -0.800 0.422 -0.146 0.183 0.016 1.047 0.185
-0.022 -0.176 0.356 -0.041 0.071 -0.200 0.578 0.146 0.817 -0.016 0.953 0.815
TABLE 3: Main Components of Atomic Spin Densities (B3LYP/6-31G(d,p) Level) for the Open Shell Species system
C1′
A•A•+ a 3A B•B•+ 3B C• C•2+ 3C+
0.268 0.140 0.555 0.246
C2′
C4′
0.281 0.178 0.552 0.263 0.155 0.307 0.344 0.320 0.597 0.268 0.508
C5′
C8′
0.246 0.167 0.464 0.216
0.231 0.138 0.458 0.184
C2
C6
NA
0.997 0.602 1.991 0.887 0.155 0.454 0.217 0.346 1.415 0.746 0.022 0.216 0.291 0.294 0.995 0.529 0.496 0.474 1.990
IM 0.003 0.398 0.009 0.113 0.546 0.585 0.978 0.005 0.010
a For this system N1 and N3 have atomic spin densities of 0.155 and 0.259, respectively.
kcal/mol, in line with the above conclusions regarding oxidation of NA and reduction of the IM subunit. The singlet-triplet gap of free naphthalene is also less than that in 2-methyl-2-imidazoline. The first singlet (triplet) excited state for naphthalene is at the TD-B3LYP/6-311+G(2df,2p)
level of theory obtained at 96.8 (62.3) kcal/mol, in good agreement with the experimental value of 92 (61) kcal/mol.13,22 From previous work, the first singlet (triplet) excited states for 2-methyl-2-imidazoline were determined to lie 98.1 (68.2) kcal/ mol above the S0 state.13 This implies that it requires less energy to excite the NA subunit, whereas if a slightly shorter wavelength is employed, the IM unit can be excited. In the latter case, provided fluorescence is low, a singlet-singlet energy transfer mechanism between the two chromophores may yield excited NA followed by intersystem crossing (ISC) to the NA subunit in its excited T1 state. In addition, once the imidazoline localized singlet is formed, it can in turn also undergo fast ISC followed by triplet-triplet energy transfer. These findings agree well with previous studies,13 except we do not see any energetic rational for the proposed S1(NA) f S1(IM) excitation energy transfer. The triplet excited NA subunit of naphazoline may then react with molecular oxygen leading to the formation of singlet oxygen (excitation energy 23 kcal/mol23), which participates in the phototoxicity pathway and reacts with biomolecules including DNA.11 In fact, based on the energies of the optimized triplets listed in Table 1, all three forms considered here will be able to excite molecular oxygen from 3Σg- to the reactive 1∆ state. Due to the high electron affinity of NP (C), the EA g of the excited triplet is close to 100 kcal/mol: >4.3 eV. It can hence be expected that the excited compound will relatively easily be reduced. The ionization potential of the reduced drug is about 1.8 eV, and can hence also serve as a source for superoxide radical anions (calculated adiabatic electron affinity in water, 3.91 eV24). The difference in photochemistry between the three species A, B, and C can be further rationalized by looking at their orbitals, displayed in Figure 2. The highest occupied molecular
3980 J. Phys. Chem. B, Vol. 111, No. 15, 2007
Musa and Eriksson
Figure 3. Computed UV spectra of neutral form (A), its tautomer (B), and cationic form (C) at the TD-B3LYP/6-31G(d,p) level.
Figure 2. B3LYP/6-31G(d,p) computed orbitals for the different forms of NP.
orbital (HOMO) of the neutral (basic) form A is localized on both the naphthalene and the imidazoline rings, whereas its lowest unoccupied molecular orbital (LUMO) is mainly localized on naphthalene. Oxidation (from HOMO) or reduction (to LUMO) hence follows the patterns in charge redistribution as described in connection with Table 2. For the tautomeric form B, the HOMO and HOMO-1 (and to some extent also in LUMO and LUMO+2) orbitals are slightly modified compared to those of the more stable tautomer A. This is a consequence of the extended conjugation of the NA subunit in this species, through the inclusion of the linker moiety in the π system as seen in Figure 1. The cationic (acidic) form C, on the other hand, differs from the other two in that the HOMO is mainly localized on the naphthalene moiety whereas the LUMO is localized on the imidazoline subunit. Both LUMO+1 and LUMO+2 are localized on the naphthalene subunit. It is thus primarily the nature of the LUMO that gives the different behavior for C compared with A and B. HOMO f LUMO excitation in C is a clear charge-transfer excitation, whereas in A the CT component is much reduced. The computed absorption spectra of A, B, and C are shown in Figures 3 and 4. From the computed spectra, two immediate observations can be made. First of all, all three species exhibit very strong absorption at λ ) 210 nm (136 kcal/mol)s excitations that probably degrade the compound due to the high energy involved. We furthermore note that compounds A and C display very similar spectra, whereas B has a very different absorption pattern, with a strong, low-lying excitation at 366 nm, caused by the increased conjugation resulting from the unsaturated linker carbon. An additional clear absorption peak is found at 254 nm for B. This is attributed to the increased conjugated system via the linker atom, as discussed above. The spectroscopically interesting region (the “fingerprint” of the molecule) is that between 270 and 300 nm, in which both
Figure 4. Enlargement of the central portion (225-325 nm) of the computed UV spectra shown in Figure 3.
A and C display several low-intensity peaks; in good agreement with previous experimental studies.13,25 Besides three broad low-intensity absorptions at 272, 280, and 290 nm (and a very small shoulder at 260 nm), the full spectrum in the 200320 nm range also displays two peaks at 218 and 225 nm, with absorptions of the latter two being a factor 5-6 stronger than those in the 270-300 nm area.25 For explicit details on the calculated excitations and their respective oscillator strengths, see Table 1. From the computed absorption spectra, we conclude that a change in pH value (i.e., changing between compounds A and C) does not in a considerable way change the spectrum or energetic properties, but rather in the charge distributions within the molecule upon oxidation/reduction or excitation. For the neutral species, excitation has the character of partial CT from IM to NA, and de-excitation is enhanced due to the HOMO/LUMO orbital overlap. In the protonated form, existing at physiological pH, excitation occurs as charge transfer from NA to IM, and the HOMO/LUMO overlap is essentially zero. This allows for the system to reside a longer time in the excited singlet state, and thus also more triplet formation. These differences account for the higher photostability of the neutral species, and its lower singlet oxygen yield. Concluding Remarks The sympathiomimetic drug naphazoline (NP) is studied with use of computational quantum chemical methodology. The compound can be described as a dual chromophore represented
Naphazoline Redoxchemistry and Photochemistry by the naphthalene and imidazoline moieties, respectively. The computed energetic data for the overall compound match very well those found for the individual chromophores, and show that the protonated species (C, Figure 1) when reduced will add an electron to the imidazoline unit (highest EA), whereas ionization will preferentially occur from the naphthalene (lowest IP). In the basic counterpart (A), the situation is more complex, and can instead be described as reduction of the naphthalene and ionization from both units. The data are explained in full upon assessment of the molecular orbitals involved. The distribution of the MO’s furthermore explains the different photochemical behavior of the protonated vs neutral species. The computed UV-absorption spectra of the two compounds are very similar. Experimentally, it is however noted that the protonated form gives a high yield of triplet excited species, and a resulting high singlet oxygen yield, whereas the neutral species is far more photostable, and with a considerably lower formation of singlet oxygen.13 From the orbital analysis we can conclude that for the neutral form, efficient singlet quenching is possible due to the orbital overlap, whereas for the protonated form, singlet excitation to the imidazoline unit will provide a strong charge separation and very low orbital overlap between HOMO and LUMO. This promotes the probability for intersystem crossing to the imidazoline localized triplet, a triplet-triplet exchange to the naphthalene moiety, and excitation energy transfer to molecular oxygen. The possibility of singlet energy transfer from naphthalene to imidazoline is however ruled out on the basis of the computed energetics. The possibility of superoxide formation is also explored, and it is concluded that the electron affinity of the protonated species in its excited triplet state is rather high (more than 4.3 eV). Provided no oxygen is in the near vicinity, reduction of the excited compound should thus be possible, followed by electron transfer to molecular oxygen at a subsequent stage. The tautomeric neutral form B, on the other hand, is more than 5 kcal/mol less stable than form A, and displays a much different absorption spectrum, caused by the conjugated linker between the naphthalene and imidazolene units. The presence of the conjugated tautomer should hence be very straightforward to detect, on the basis of its different absorption characteristics. The considerably lower stability of the species will however render this to be present in very small amounts. In addition, the high proton affinity (corresponding to the high pKa value) ensures that the protonated form C is the only one present at physiological pH. Acknowledgment. The MENA programme (K.A.K.M.) and the Swedish Science Research Council (L.A.E.) are gratefully acknowledged for financial support. We also acknowledge generous grants of computing time at the National Supercomputing Center (NSC) in Linko¨ping.
J. Phys. Chem. B, Vol. 111, No. 15, 2007 3981 References and Notes (1) Goodman-Hillman, A.; Rall, T.; Nier, A.; Taylor, P. The Pharmacological Basis of Therapeutics; McGraw-Hill: New York, 1996. (2) Musshoff, F.; Gerschlauer, A.; Madea, B. Forensic Sci. Int. 2003, 134, 234. (3) Casado-Terrones, S.; Fernandez-Sanchez, J. F.; Diaz, B. C.; Carretero, A. S.; Fernandez-Gutierrez, A. J. Pharm. Biomed. Anal. 2005, 38, 785. (4) Degeville, C.; Joly, E.; Spreux, A.; Berard, E. Therapie 1995, 50, 472. (5) Claudet, I.; Fries, F. Arch. Pediatr. 1997, 4, 538. (6) Soparkar, C. N. S.; Wilhelmus, K. R.; Koch, O. D.; Wallage, G. W. Arch. Ophthalmol. 1997, 115, 34. (7) Gallego, J. M. L.; Arroyo, J. P. J. Sep. Sci. 2003, 26, 947. (8) Carretero, A. S.; Blanco, C. C.; Diaz, B. C.; Gutierrez, A. F. Analyst 1998, 123, 1069. (9) Salinas-Castillo, A.; Carretero, A. S.; Fernandez-Gutierrez, A. Anal. Bioanal. Chem. 2003, 376, 1111. (10) Goicoechea, H. C.; Olivieri, A. C. Analyst 2001, 126, 1105. (11) Sortino, S.; Giuffrida, S.; Scaiano, J. C. Chem. Res. Toxicol. 1999, 12, 971. (12) Sortino, S.; Scaiano, J. C. Photochem. Photobiol. 1999, 70, 590. (13) Sortino, S.; Cosa, G.; Scaiano, J. C. New J. Chem. 2000, 24, 159. (14) Prouillac, C.; Celaries, B.; Vicendo, P.; Rima, G. C. R. Biol. 2006, 329, 196. (15) Cance`s, M. T.; Mennucci, B.; Tomasi, J. J. Chem. Phys. 1997, 107, 3032. (16) Mennucci, B.; Tomasi, J. J. Chem. Phys. 1997, 106, 5151. (17) Cossi, M.; Scalmani, G.; Rega, N.; Barone, V. J. Chem. Phys. 2002, 117, 43. (18) Casida, M. E.; Jamorski, C.; Casida, K. C.; Salahub, D. R. J. Chem. Phys. 1998, 108, 4439. (19) Stratmann, R. E.; Scuseria, G. E.; Frisch, M. J. J. Chem. Phys. 1998, 109, 8218. (20) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Montgomery, J. A., Jr.; Vreven, T.; Kudin, K. N.; Burant, J. C.; Millam, J. M.; Iyengar, S. S.; Tomasi, J.; Barone, V.; Mennucci, B.; Cossi, M.; Scalmani, G.; Rega, N.; Petersson, G. A.; Nakatsuji, H.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Klene, M.; Li, X.; Knox, J. E.; Hratchian, H. P.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Ayala, P. Y.; Morokuma, K.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Zakrzewski, V. G.; Dapprich, S.; Daniels, A. D.; Strain, M. C.; Farkas, O.; Malick, D. K.; Rabuck, A. D.; Raghavachari, K.; Foresman, J. B.; Ortiz, J. V.; Cui, Q.; Baboul, A. G.; Clifford, S.; Cioslowski, J.; Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.; Komaromi, I.; Martin, R. L.; Fox, D. J.; Keith, T.; Al-Laham, M. A.; Peng, C. Y.; Nanayakkara, A.; Challacombe, M.; Gill, P. M. W.; Johnson, B.; Chen, W.; Wong, M. W.; Gonzalez, C.; Pople, J. A. Gaussian 03, Revision B.02; Gaussian, Inc.: Wallingford, CT, 2004. (21) Llano, J.; Eriksson, L. A. J. Chem. Phys. 2002, 117, 10193. (22) Turro, N. J. Modern Molecular Photochemistry; University Science Books: Sausalito, CA, 1991; p 352. (23) Huber, K. P.; Herzberg, G. Molecular Spectra and Molecular Structure. Part IV. Constants of Diatomic Molecules; Van Nostrand: New York, 1979. (24) Llano, J.; Raber, J.; Eriksson, L. A. J. Photochem. Photobiol., A 2003, 154, 235. (25) Hemmateenejad, B.; Ghavami, R.; Miri, R.; Shamsipur, M. Talanta 2006, 68, 1222.
