Development of a D–π–A pyrazinium photosensitizer possessing singlet oxygen generation

(D–p–)2A pyrazinium dyes (OEJ-1 and OEJ-2) bearing a counter anion (X 1⁄4 Br or I ) have been newly developed as a photosensitizer possessing singlet oxygen (O2) generation. The two dyes show specific solvatochromism, leading to a large bathochromic shift of the photoabsorption band in halogenated solvents, compared to polar and non-polar solvents. The effects of the counter anion and solvents on the O2 generation efficiency such as FD and the rate constant (Kobs) have been investigated. It was revealed that OEJ-2 (X 1⁄4 I ) exhibits a higher O2 quantum yield (FD) than OEJ-1 (X 1⁄4 Br ). This result indicates that the (D–p–)2A pyrazinium dyes possess the ability to generate O2 under visible light irradiation, due to the effective intersystem crossing (ISC) from the singlet excited state of the photosensitizer (S*) to the triplet excited state (S*) by the superior heavy-atom effect of I ion as the counter anion. Moreover, it was found that THF and dichloromethane are favorable solvents for the (D–p–)2A pyrazinium dyes to efficiently generate O2 compared with the polar solvents such as acetonitrile and DMSO. On the basis of the O2 quantum yield, the rate constant for O2 generation, the HOMO and LUMO energy levels of OEJ-1 and OEJ-2, and density functional theory (DFT) calculation, the photoabsorption and O2 generation properties of the D–p–A pyrazinium dyes are discussed.


Introduction
Photosensitizers possessing the ability to generate singlet oxygen ( 1 O 2 ) have received considerable attention in recent years from the viewpoint of not only fundamental study in photochemistry and photophysics, but also their potential applications in photodynamic therapy (PDT). 1-4 1O 2 generally occurs through the following processes: initially the photosensitizer absorbs light (hn) to generate the singlet excited state of the photosensitizer ( 1 S*), then the photoexcited dye ( 1 S*) undergoes intersystem crossing (ISC) to generate the triplet excited state ( 3 S*).Subsequent energy transfer from the photoexcited dye ( 3 S*) to triplet oxygen ( 3 O 2 ) produces 1 O 2 .Thus, to enhance ISC efficiency is one of the most effective strategies to generate high 1 O 2 quantum yield.3][4]17 However, there have been few efforts to develop new organic photosensitizers possessing the ability to generate 1 O 2 . 18hus, in this work, to gain insight into a direction in molecular design toward creating new photosensitizer family possessing 1 O 2 generation, we have developed (D-p-) 2 A pyrazinium dyes (OEJ-1 and OEJ-2) bearing bromide ion (Br À ) or iodide ion (I À ) as a counter anion (Scheme 1).The heavy atoms such as bromine and iodine would be expected to facilitate ISC by the heavy-atom effect.Moreover, D-p-A pyrazinium dyes have an advantage over the conventional photosensitizers in carrying out the fundamental study on 1 O 2 generation, that is, we can obtain a great deal of useful knowledge for the relationship between the molecular structures and 1 O 2 generation efficiency, by easily exchanging the counter ion.Interestingly, it was found that the (D-p-) 2 A pyrazinium dyes show specic solvatochromism, leading to a large bathochromic shi of absorption band in halogenated solvents, compared to polar and non-polar solvents.Therefore, the effects of the counter anion and solvents on the 1 O 2 generation efficiency have been investigated.On the basis of 1 O 2 quantum yield (F D ), rate constant (K obs ) for 1 O 2 generation, the HOMO and LUMO energy levels of OEJ-1 and OEJ-2, and density functional theory (DFT) calculation, the photoabsorption and 1 O 2 generation properties of the (D-p-) 2 A pyrazinium dyes are discussed.

Photoabsorption properties
The photoabsorption spectra of OEJ-1 and OEJ-2 in various solvents (THF, acetonitrile, DMSO and dichloromethane) are shown in Fig. 1 and their optical data are summarized in Table 1.The two dyes show a broad absorption band (l abs ) at around 500-700 nm, which is assigned to the intramolecular chargetransfer (ICT) excitation from electron donor moiety (diphenylamino group) to electron acceptor moiety (pyrazinium group).In all the four solvents, the l abs for ICT band of OEJ-2 occurs at a longer wavelength than of OEJ-1.Interestingly, the two dyes showed the specic solvatochromism as with the previously reported D-p-A pyridinium dyes, 20 leading to a large bathochromic shi of absorption band in halogenated solvent such as dichloromethane, compared with that in polar and non-polar solvents; the l abs for ICT bands of OEJ-1 and OEJ-2 in dichloromethane occurs at a longer wavelength by ca. 30 nm and ca.70 nm, respectively, than those in acetonitrile.It is worthy to note here that the specic solvatochromism depends on the counter anion of the (D-p-) 2 A pyrazinium dyes, that is, the bathochromic shis of ICT band for OEJ-2 bearing I À ion is larger than that of OEJ-1 bearing Br À ion.

Electrochemical properties
The electrochemical properties of OEJ-1 and OEJ-2 were determined by cyclic voltammetry (CV) in acetonitrile containing 0.1 M tetrabutylammonium perchlorate (Bu 4 NClO 4 ).The potentials were referred to ferrocene/ferrocenium (Fc/Fc + ) as the internal reference (Fig. 2).For OEJ-2, the oxidation wave for the iodide counter ion was observed at around 0.05 V.The oxidation waves were observed at 0.43 V for OEJ-1 and 0.35 V for OEJ-2, respectively, vs. Fc/Fc + (Table 1).The corresponding reduction waves appeared at 0.36 V for OEJ-1 and 0.18 V for OEJ-2, respectively.However, the oxidation and corresponding reduction waves are reversible for OEJ-1, but irreversible for OEJ-2.In fact, at second cycle OEJ-1 also showed the reversible oxidation wave, but OEJ-2 showed cathodic shi by ca.0.1 V for the oxidation wave as well as disappearance of the oxidation wave for the iodide counter ion (Fig. S3 †).The HOMO energy level vs. vacuum level is À5.20 eV for OEJ-1 and À5.07 eV for OEJ-2, respectively, which was evaluated through equation À[E ox 1/2 + 4.8]eV from the half-wave potential for oxidation (E ox 1/2 ¼ 0.40 V vs. Fc/Fc + for OEJ-1 and 0.27 V vs. Fc/Fc + for OEJ-2).On the other hand, the LUMO energy level is À3.20 eV for OEJ-1 and À3.07 eV for OEJ-2, respectively, which was estimated from the HOMO and the onset of photoabsorption spectra (620 nm; 2.0 eV for both OEJ-1 and OEJ-2) in acetonitrile.

Theoretical calculations
In order to examine the HOMO and LUMO of OEJ-1 and OEJ-2, the molecular orbitals of the (D-p-) 2 A pyrazinium dye cation (OEJ) was calculated using density functional theory (DFT) at the B3LYP/6-31G(d,p) level (Fig. 3). 21The DFT calculation for the dye cation indicates that the HOMO is mostly localized on the diphenylamine-carbazole moiety containing a thiophene ring.On the other hand, the LUMO is mainly concentrated on pyrazinium moiety.Accordingly, the DFT calculations reveal that excitation of the dye upon light irradiation induces a strong ICT from the diphenylamine-carbazole moiety to the pyrazinium moiety.
spectral change of the known 1 O 2 scavenger 1,3-diphenylisobenzofuran (DPBF) accompanied by the reaction of DPBF with the generated 1 O 2 , that is, DPBF can trap 1 O 2 through its photooxidation. 22All the solvents were bubbled with air for 15 min.The air-saturated solution containing the dye (OEJ-1 or OEJ-2) and DPBF was irradiated with 509 nm (160 mW cm À2 , see Table 1 for 3/M À1 cm À1 @l abs ¼ 509 nm) obtained by passage of xenon light through monochromator.The absorption band of DPBF at around 410 nm decreased with the increase in the photoirradiation time (Fig. 4 and 5), which indicate the reaction of DPBF with 1 O 2 generated upon the excitation of (D-p-) 2 A pyrazinium dyes.To gain insight into the effect of the solvent and the counter anion on the efficiency of DPBF photooxidation, the changes in optical density (DOD) of DPBF are plotted against the photoirradiation time (Fig. 6), and the slope (m sl ) is used to estimate the 1 O 2 quantum yield (F D ) for OEJ-1 and OEJ-2.It was revealed that the m sl value for OEJ-2 becomes steeper in the following order: DMSO (À0.4 Â 10 À3 ) < acetonitrile (À0.5 Â 10 À3 ) < dichloromethane (À0.8 Â 10 À3 ) < THF (À2.6 Â 10 À3 ), that is, the m sl value in THF is larger than those in the other solvents.It was also found that the m sl value of OEJ-2 is larger than that of OEJ-1 (À2.0 Â 10 À3 in THF).Consequently, this result indicates that THF is a favorable solvent for the (D-p-) 2 A pyrazinium dyes to present a high DPBF-oxidation efficiency compared with the other solvents.Moreover, the plots demonstrate that OUJ-2 bearing I À ion exhibits higher DPBF-oxidation O 2 quantum yield (relative decomposition rate of DPBF), with Rose Bengal (RB) as standard (F D ¼ 0.80 in methanol, 15 see Fig. S4) and 1,3diphenylisobenzofuran (DPBF) as 1 O 2 scavenger.These values were estimated under an assumption that the reactivity of singlet oxygen is independent of the kind of solvents.b First-order rate constant for the reaction of DPBF with 1 O 2 generated upon photoexcitation of OEJ-1 or OEJ-2.The K obs for RB is 0.250 min À1 (see Fig. S5).c Too low.d Estimated from the slope for the range of 5-10 min in Fig. 9b.efficiency than OUJ-1 bearing Br À ion.Thus, the F D values of OEJ-1 and OEJ-2 were estimated by the relative method using Rose Bengal (RB) (F D ¼ 0.80) in methanol as the standard (Table 1).The F D value of OEJ-2 is 0.03, 0.05, 0.07 and 0.22 in DMSO, acetonitrile, dichloromethane and THF, respectively, which is in good agreement with the m sl value.A higher F D value in THF is ascribable to that as for the (D-p-) 2 A pyrazinium dyes the ISC from 1 S* to the 3 S* may be facilitated by THF, although further study for the solvent effects on 1 O 2 generation is necessary to ensure the hypothesis.It is worth noting that the F D values of OEJ-2 in all the four solvents are higher than those of OEJ-1.Therefore, the high F D value of OEJ-2 relative to OEJ-1 is attributed to the fact that I À ion possesses superior heavy-atom effect rather than Br À ion, resulting in the facilitation of the ISC.
In order to evaluate the photosensitizing ability of the (D-p-) 2 A pyrazinium dyes, the ln(C t /C 0 ) is plotted against the photoirradiation time, where C t is a concentration of DPBF at the reaction time (t) and C 0 is the initial concentration of DPBF before photoirradiation (Fig. 9).All the four solvents (THF, acetonitrile, DMSO and dichloromethane) were bubbled with air for 15 min.The air-saturated solution containing the dye (OEJ-1 or OEJ-2) and DPBF was irradiated with visible light (>510 nm, 6 mW cm À2 ) obtained by passage of xenon light through a 510 nm long path lter.The photoabsorption spectral changes for the photooxidation of DPBF using OEJ-1 and OEJ-2 under photoirradiation with the visible light in the four solvents are shown in Fig. 7 and 8, respectively.The ln(C t /C 0 ) decreased almost linearly with the increase in the photoirradiation time, although the linear relationship for the ln(C t /C 0 ) for OEJ-2 in dichloromethane become steeper aer the photoirradiation for 5 min under this photoirradiation.Thus, this result indicates the ln(C t /C 0 ) bears a linear relationship with the photoirradiation time to provide the rst-order rate constants (K obs ) for the photooxidation of DPBF using OEJ-1 and OEJ-2 as the photosensitizer (Table 1).The K obs values for OEJ-2 are greater than those of OEJ-1, although the K obs value for OEJ-2 in THF is lower than that of OEJ-1 due to the low photoabsorption property of OEJ-2 in THF.Interestingly, the plot of OEJ-2 in dichloromethane show a non-linear relationship, but the slope for OEJ-2 become steeper aer 5 min of photoirradiation and the K obs value for OEJ-2 in dichloromethane is greater than those in the other solvents.This interesting observation may be attributed to not only the bathochromic shi and broadening of absorption but also the enhancement of heavy-atom effect with the increase in the photoirradiation time in dichloromethane, that is, the signicant specic solvatochromic behavior of the   (D-p-) 2 A pyrazinium dye bearing I À ion.Therefore, this result demonstrates that OEJ-2 exhibits more efficient photosensitizing ability compared to OEJ-1, due to a superior heavy-atom effect of I À ion.
In addition, we performed an electron paramagnetic resonance (EPR) method with 2,2,6,6-tetramethyl-4-piperidone (4oxo-TEMP) as the spin-trapping agent, which can react with 1 O 2 to produce 4-oxo-TEMPO as a stable nitroxide radical. 14,23When the air-saturated solution containing OEJ-2 and 4-oxo-TEMP was irradiated with visible light (>510 nm, 14 mW cm À2 ) obtained by passage of xenon light through a 510 nm long path lter, the ESR spectrum of 4-oxo-TEMPO was clearly observed as a characteristic 1:1:1 triplet (Fig. S6 †).Moreover, to obtain the direct evidence of 1 O 2 generation by (D-p-) 2 A pyrazinium dyes, phosphorescence spectrum of 1 O 2 was measured in airsaturated THF solution of OEJ-2.The phosphorescence maximum of 1 O 2 produced upon the excitation of OEJ-2 at 467 nm was clearly observed at around 1270 nm (Fig. S7 †).6b, 14,24 Consequently, this work demonstrated that (D-p-) 2 A pyrazinium dyes possess the ability to generate 1 O 2 under visible light irradiation.

Conclusions
(D-p-) 2 A pyrazinium dyes bearing a counter anion (X À ¼ Br À or I À ) which show specic solvatochromic behavior leading to the bathochromic shi of photoabsorption band in halogenated solvents, have been designed and developed as a photosensitizer possessing singlet oxygen ( 1 O 2 ) generation.This work demonstrated that the (D-p-) 2 A pyrazinium dyes possess the ability to generate 1 O 2 under visible light irradiation, due to the effective intersystem crossing (ISC) from the singlet excited state of the photosensitizer ( 1 S*) to the triplet excited state ( 3 S*) by the heavy-atom effect of the counter anion.It was found that the 1 O 2 quantum yield (F D ) of OEJ-2 bearing I À ion is higher than that of OEJ-1 bearing Br À ion.Consequently, this result indicates that the high F D value of OEJ-2 relative to OEJ-1 is attributed to the fact that I À ion possesses superior heavy-atom effect rather than Br À ion, resulting in the facilitation of the ISC.Moreover, it was found that THF is a favorable solvent for the (D-p-) 2 A pyrazinium dyes to provide higher F D value compared with the polar solvents such as acetonitrile and DMSO.Thus, this result suggests that as for (D-p-) 2 A pyrazinium the ISC from 1 S* to the 3 S* may be facilitated by THF, although much effort for the solvent effects on 1 O 2 generation is necessary to ensure the hypothesis.Interestingly, the rst-order rate constants (K obs ) for the photooxidation of DPBF using OEJ-2 in dichloromethane is greater than those in the other solvents, which is attributed to the bathochromic shi and broadening of absorption in dichloromethane, that is, the signicant specic solvatochromic behavior of the (D-p-) 2 A pyrazinium dye bearing I À ion.Further study to gain greater insight into the effects of the molecular structure of pyrazinium dyes on the 1 O 2 generation efficiency is now in progress by developing the (D-p-) 2 A pyrazinium photosensitizers possessing strong photoabsorption property in body therapeutic window (650-900 nm) and water solubility.

Fig. 2
Fig. 2 Cyclic voltammograms of (a) OEJ-1 and (b) OEJ-2 in acetonitrile containing 0.1 M Bu 4 NClO 4 at the first cycle.The arrow denotes the direction of the potential scan.
Photosensitizing abilityPhotosensitizing ability of the (D-p-) 2 A pyrazinium dyes (OEJ-1 and OEJ-2) in various solvents (THF, acetonitrile, DMSO and dichloromethane) was evaluated by plotting the ln(C t /C 0 ) against the photoirradiation time, where C t is a concentration of DPBF at the reaction time (t) and C 0 is the initial concentration of DPBF before photoirradiation.All the solvents were bubbled with air for 15 min.The air-saturated solution containing the photosensitizer (1 Â 10 À5 M for OEJ-1 and OEJ-2, 1 Â 10 À6 M for RB) and DPBF (5 Â 10 À5 M) was irradiated with visible light (>510 nm, 6 mW cm À2 ) obtained by passage of xenon light through a 510 nm long path lter.The absorbance of DPBF was adjusted to around 1.0 in air-saturated solvent.The photooxidation of DPBF with the photoirradiation was monitored by following the decrease in the photoabsorption at around 410 nm with an interval of 20 s up to 10 min.The concentration (C t ) of DPBF at the reaction time (t) was calculated based on Lambert-Beer law (A DPBF ¼ 3cl).The ln(C t /C 0 ) decreased almost linearly with the increase in the photoirradiation time due to the photooxidation of DPBF, that is, the slope was used to estimate the rate constants (K obs ).

Table 1
Optical data of OEJ-1 and OEJ-2, and 1 O 2 quantum yield (F D ) and first-order rate constant (K obs ) for the photooxidation of DPBF using OEJ-1 and OEJ-2 as photosensitizer Dye Solvent l abs /nm for ICT band 3/M À1 cm À1 @l abs ¼ 509 nm F D 1O 2 scavenger 1,3-diphenylisobenzofuran (DPBF) accompanied by the reaction of DPBF with the generated 1 O 2 , that is, DPBF can trap 1 O 2 through its photooxidation.All the solvents were bubbled with air for 15 min.The absorbance of DPBF was adjusted to around 1.0 in air-saturated solvent.Concentration of OEJ-1 or OEJ-2 was adjusted with an absorbance of 0.2-0.3 at the irradiation wavelength (509 nm).The airsaturated solution containing the photosensitizer (OEJ-1 or OEJ-2) and DPBF was irradiated with 509 nm (160 mW cm À2 ) obtained by passage of xenon light through monochromator.The photoabsorption spectral change of DPBF with the photoirradiation was monitored with an interval of 5 s up to 40 s.The absorption band of DPBF at around 410 nm decreased with the increase in the photoirradiation time.The changes in optical density (DOD) of DPBF are plotted against the photoirradiation time, and the slope is used to estimate the F D of OEJ-1 and OEJ-2.The F D of OEJ-1 and OEJ-2 was estimated by the relative method using Rose Bengal (RB) (F D ¼ 0.80) in methanol as the standard.Therefore, the 1 F D values were calculated according to the following eqn (1): D ) for singlet oxygen ( 1 O 2 ) generation by (Dp-) 2 A pyrazinium dyes (OEJ-1 and OEJ-2) in various solvents (THF, acetonitrile, DMSO and dichloromethane) were evaluated by monitoring the photoabsorption spectral change of the known