Development of water-soluble phenazine-2,3-diol-based photosensitizers for singlet oxygen generation

Abstract

Phenazine-2,3-diol-based dyes, KY-1Na and KY-2Na bearing one and two carboxylic acid sodium salts, respectively, have been newly developed as water-soluble photosensitizers (PSs) possessing the ability to generate singlet oxygen (¹O₂). In order to evaluate the solubility of KY-1Na and KY-2Na in water, the hydrophobicity/hydrophilicity of the two PSs was investigated by experimental measurement of the logarithms (log P_o/w) of the 1-octanol/water partition coefficient (P_o/w) for the PS. The log P_o/w values of both KY-1Na and KY-2Na were determined to be −0.9, indicating that both the PSs are more hydrophilic than Rose Bengal (−0.6) and have hydrophilicity equivalent to methylene blue (−0.9). Both the PSs in water show a broad photoabsorption band in the range of 500 to 600 nm. Thus, we estimated the ¹O₂ quantum yields (Φ_Δ) of KY-1Na and KY-2Na in water by using 9,10-anthracenediyl-bis(methylene)dimalonic acid (ABDA) as a water-soluble ¹O₂ scavenger. It was found that in water the Φ_Δ value (0.19) of KY-2Na is higher than that of KY-1Na (0.06). Density functional theory (DFT) calculations suggested that the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) distributions for the molecular structure of KY-2Na are adequately separated, leading to a decrease in the energy gap (ΔE_ST) between the singlet state (S₁) and the triplet state (T₁) that causes efficient intersystem crossing (ISC), compared to that for the molecular structure of KY-1Na. Indeed, time-dependent DFT (TD-DFT) calculations demonstrated that the ΔE_ST(S₁–T₁) value (0.82 eV) of KY-2Na is smaller than that (0.98 eV) of KY-1Na, resulting in a relatively high Φ_Δ value of KY-2Na. Consequently, we demonstrate that phenazine-2,3-diol-based PSs bearing carboxylic acid salts possess high solubility and moderate ¹O₂ generation ability in water.

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Introduction

Singlet oxygen (¹O₂) generated by light irradiation to photosensitizers (PSs),¹ which is one of the reactive oxygen species (ROS), has gained more and more attention as an effective and convenient oxidant of alkenes and dienes for Schenck–ene reactions and [2 + 2]- and [4 + 2]-cycloadditions in organic synthesis,^2–5 degradation of water pollutants in environmental and quality control monitoring systems,⁶ inactivation of microbiological contamination in point-of-use water disinfection systems,⁷ and cancer treatment in photodynamic therapy (PDT).⁸¹O₂ is generally produced through the following processes: initially, the PS (¹PS) absorbs light (hν) to generate the singlet excited state (¹PS*), and then the ¹PS* undergoes intersystem crossing (ISC) to generate the triplet excited state (³PS*). Subsequent energy transfer from the photoexcited PS (³PS*) to triplet oxygen (³O₂) produces ¹O₂. Thus, in order to obtain a high ¹O₂ quantum yield (Φ_Δ), it is necessary to enhance the ISC efficiency of PSs. Moreover, for use in water purification systems and PDT, the PSs are required to possess high solubility in water as well as efficient ISC. Methylene blue (MB),⁹ Rose Bengal (RB),¹⁰ and hydrophobically modified porphyrin derivatives¹¹ have been used as water-soluble PSs with high ¹O₂Φ_Δ values (0.50–0.60 for MB,^9b,c 0.70–0.80 for RB,^9b,10b and 0.50–0.60 for free base 5,10,15,20-tetrakis(4-carboxylatophenyl)porphyrin,^11a,b respectively). Meanwhile, halogen atoms such as iodide and bromide atoms are often introduced into chromophores (e.g.RB) to promote ISC based on spin–orbit coupling,¹¹ but they cause dark cytotoxicity.¹² Indeed, MB as a halogen atom-free-heteroanthracene-based PS¹³ is used as not only a reference water-soluble PS for the evaluation of Φ_Δ,^9b,c but also a fungicide and antidote in aquaculture.^9f,g

Thus, in our previous work, we have designed and developed phenazine-2,3-diol-based dyes (KO-0-3)¹⁴ as halogen atom-free-heteroanthracene-based PSs and demonstrated that the modification of the phenazine-2,3-diol chromophore with formyl groups promotes ISC, leading to efficient ¹O₂ generation (Fig. 1a). According to the El-Sayed rule, formyl and carbonyl substituents in a chromophore skeleton facilitate the ISC from the singlet (S_n) to triplet (T_n) states, that is, by the change in the molecular orbital type such as transitions of ¹(nπ*) to ³(ππ*) and ¹(ππ*) to ³(nπ*). In fact, KO-1-3 exhibited moderate ¹O₂ generation ability (Φ_Δ = 0.22–0.41) in THF.

Fig. 1 Chemical structures of phenazine-2,3-diol-based PSs, (a) KO-0-3 in our previous study and (b) KY-1, KY-2, KY-1Na, and KY-2Na in this study.

In this work, in order to attain the solubility of phenazine-2,3-diol-based PSs in water, we have developed KY-1Na and KY-2Na bearing one and two carboxylic acid sodium salts, respectively, as the water-soluble derivatives of KO-2 exhibiting a moderate Φ_Δ value (0.17) in DMSO (Fig. 1b). Actually, in order to evaluate the solubility of KY-1Na and KY-2Na in water, the hydrophobicity/hydrophilicity of the two PSs was investigated by experimental measurement of the logarithms (log P_o/w) of the 1-octanol/water partition coefficient (P_o/w) for the PS. Both the PSs in water show a broad photoabsorption band in the range of 500 to 600 nm. It was found that in water KY-2Na exhibits a moderate Φ_Δ value, which is higher than that of KY-1Na. Density functional theory (DFT) and time-dependent DFT (TD-DFT) calculations were performed to gain insight into the ¹O₂ generation properties of KY-1Na and KY-2Na based on the ISC efficiency. Herein we report that the introduction of carboxylic acid salts into the phenazine-2,3-diol chromophore is effective in yielding solubility in water and a small energy gap (ΔE_ST) between the S₁ state and the T₁ state for efficient ISC, leading to the development of water-soluble halogen-atom-free-heteroanthracene-based PSs.

Results and discussion

Synthesis

Phenazine-2,3-diol-based PSs, KY-1 and KY-2 bearing one and two carboxylic acids, respectively, and their sodium salt derivatives KY-1Na and KY-2Na, were synthesized by a stepwise synthetic protocol (Scheme 1). We prepared compounds 1 and 2,¹⁵ respectively, for the construction of the phenazine skeleton and the introduction of the formylthiophene unit on the chromophore by the Stille coupling (see the ESI†). Phenazine-2,3-diol chromophores 3 and 4 were prepared by the cyclodehydration of 2,5-dibromo-3,6-dihydroxy-p-quinone with methyl 3,4-diaminobenzoate or compound 1, respectively, and then converted into methoxymethyl (MOM)-protected compounds 5 and 6, respectively. The Stille coupling reaction of 5 or 6 with compound 2 gave compounds 7 and 8, respectively. Compounds 7 and 8 were hydrolyzed by treatment with a base to generate compounds 9 and 10, respectively, and then the hydrolysis of 9 and 10 with an acid gave the carboxylic acids KY-1 and KY-2, respectively. Finally, KY-1Na and KY-2Na were obtained by treatment of KY-1 and KY-2, respectively, with a base.

Scheme 1 Synthetic route to phenazine-2,3-diol-based PSs KY-1, KY-2, KY-1Na, and KY-2Na.

In order to evaluate the hydrophobicity/hydrophilicity of KY-1Na and KY-2Na, the logarithms (log P_o/w)¹⁶ of the 1-octanol/water partition coefficient (P_o/w) for the PS were measured experimentally. The log P_o/w values of both KY-1Na and KY-2Na were determined to be −0.9, indicating that both the PSs are more hydrophilic than RB (−0.6)^10c and have hydrophilicity equivalent to MB (−0.9).^9d,e Incidentally, KO-2 exhibits hydrophobicity with a log P_o/w value of 1.3. Thus, this fact indicates that KY-1Na and KY-2Na possess high solubility in water.

Optical properties

The photoabsorption and fluorescence spectra of KY-1Na and KY-2Na in DMSO and in water (D₂O) are shown in Fig. 2 with those of KO-2, KY-1, and KY-2 in DMSO as references for the characterization of the photoabsorption properties of KY-1Na and KY-2Na, and their optical data are summarized in Table 1. All the five dyes show an intense photoabsorption maximum (λ^abs_max) at around 400–450 nm with a relatively high molar extinction coefficient (ε_max = 25 000–30 000 M⁻¹ cm⁻¹), which can be assigned to the π → π* transition of the phenazine skeleton containing two formylthiophene units.¹⁴ In addition, for KO-2, KY-1, and KY-2, a broad photoabsorption band (λ^abs_max = 520–535 nm) with a moderate ε_max value (6000–12 000) was also observed in the range of 500 to 650 nm, which is ascribable to the formation of phenoxide ions by the partial deprotonation of the hydroxyl groups.¹⁴ On the other hand, for KY-1Na and KY-2Na both in DMSO and in water, the photoabsorption spectra in the range of 500 to 600 nm are significantly broadened, and exhibit a hypsochromic shift and become a shoulder band, compared to those of KO-2, KY-1, and KY-2 in DMSO. This fact indicates that KY-1Na and KY-2Na existed as carboxylate anion (–COO⁻) species in DMSO as well as in water.¹⁷ In the corresponding fluorescence spectra, all the five dyes show a feeble fluorescence band with a fluorescence maximum wavelength (λ^fl_max) at 600–635 nm. In fact, their fluorescence quantum yields (Φ_fl) were significantly low (≤0.03).

Fig. 2 (a) Photoabsorption and fluorescence spectra (λ^ex = 532 nm) of KO-2, KY-1, KY-2, KY-1Na, and KY-2Na (5.0 × 10⁻⁵ M) in DMSO. (b) Photoabsorption and fluorescence spectra (λ^ex = 532 nm) of KY-1Na and KY-2Na (5.0 × 10⁻⁵ M) in D₂O.

Table 1 Optical data and ¹O₂ quantum yields (Φ_Δ) of KO-2, KY-1, KY-2, KY-1Na and KY-2Na

Dye	λ ^absmax/nm (εmax/M^−1 cm^−1)	ε/M^−1 cm^−1@λ^abs = 532 nm	λ ^flmax/nm (Φfl)^c	Φ Δ
a In DMSO. b In D2O. c Fluorescence quantum yields (Φfl) were determined using a calibrated integrating sphere system (λ^ex = 532 nm). d ^1O2 quantum yields (ΦΔ) based on the relative decomposition rate of DPBF using RB as the standard PS (ΦΔ = 0.76 in DMSO,^9a see Fig. S44a†) and DPBF as the ^1O2 scavenger under irradiation with monochromatic light (532 nm, 300 μW cm^−2) in DMSO. e Φ Δ based on the relative decomposition rate of AMDA using RB as the standard PS (ΦΔ = 0.75 in water,^9b see Fig. S44b†) and AMDA as the ^1O2 scavenger under irradiation with monochromatic light (532 nm, 300 μW cm^−2) in D2O/DMSO (99 : 1, v/v).
KO-2	522 (11 500)^a	11 300^a	634 (0.03)^a	0.17^d
KY-1	534 (7700)^a	7700^a	625 (<0.02)^a	0.14^d
KY-2	534 (6000)^a	6000^a	631 (<0.02)^a	0.17^d
KY-1Na	486^shoulder (21 500)^a	9600^a	611 (0.02)^a	0.13^d
KY-2Na	486^shoulder (15 900)^a	7700^a	603 (0.02)^a	0.32^d
KY-1Na	500^shoulder (8100)^b	6100^b	611 (<0.02)^b	0.06^e
KY-2Na	500^shoulder (7100)^b	4500^b	611 (<0.02)^b	0.19^e

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¹O₂ generation

We evaluated the ¹O₂ generation ability of the phenazine-2,3-diol-based PSs KO-2, KY-1, KY-2, KY-1Na, and KY-2Na by using an ¹O₂ scavenger. It is well known that 1,3-diphenylisobenzofuran (DPBF) acts as an efficient ¹O₂ scavenger in organic solvents to produce its oxidized product, o-dibenzoylbenzene.^18a,b Meanwhile, 9,10-anthracenediyl-bis(methylene)dimalonic acid (ABDA), which can react rapidly and irreversibly with ¹O₂ to produce the corresponding endoperoxide (endoperoxideABDA), is widely used as a water-soluble ¹O₂ scavenger.^18b,c Therefore, ¹O₂ generation by the five PSs was investigated by monitoring the changes in the photoabsorption spectra of DPBF in the DMSO solution of each PS and ABDA in the aqueous solution (D₂O/DMSO 99 : 1, v/v) of KY-1Na and KY-2Na under photoirradiation (Fig. 3a–d for KY-1Na and KY-2Na, and Fig. S45† for KO-2, KY-1, KY-2, and RB in DMSO). DMSO and D₂O/DMSO were bubbled with air for 15 min prior to preparing solutions. Air-saturated DMSO solutions containing DPBF or aqueous solutions containing ABDA and each PS were irradiated with monochromatic light at 532 nm (300 μW cm⁻²) that was obtained by the passage of a xenon light source through a monochromator. In all the DMSO solutions, the photoabsorption of DPBF at around 415 nm decreased with the increase in the photoirradiation time (Fig. 3a, b and S45†), which indicates that DPBF reacted with ¹O₂ generated by the photosensitization of the PSs. In order to make clear the difference in ¹O₂ generation ability between the five PSs, the changes in the optical density (ΔOD) at around 415 nm of DPBF are plotted against the photoirradiation time (Fig. 3e), and the slope (m_sam) is used to estimate the ¹O₂ quantum yield (Φ_Δ): the Φ_Δ values for PSs were determined by the relative method using the slope value (m_ref = −0.0100) of the plot for RB (Φ_Δ = 0.76 in DMSO)^9a as a standard (Table 1). Indeed, the correlation coefficient (R²) values for the calibration curves of the five PSs are 0.860–0.996, which indicates good linearity, and the m_sam values become steeper in the following order: KY-1Na (−0.0018) < KY-1 (−0.0033) ≈ KY-2 (−0.0037) < KO-2 (−0.0048) < KY-2Na (−0.0058). It was found that the Φ_Δ values (0.13–0.17) of KY-1, KY-2, and KY-1Na are equivalent to that of KO-2 (Φ_Δ = 0.17), and surprisingly KY-2Na exhibited the highest ¹O₂ generation ability (Φ_Δ = 0.32). Moreover, for the aqueous solutions of KY-1Na and KY-2Na, the photoabsorption of ABDA at around 400 nm decreased with the increase in the photoirradiation time (Fig. 3c and d), indicating ABDA reacted with ¹O₂ generated by the photosensitization of the PSs. The plots of ΔOD at around 400 nm of ABDA versus the photoirradiation time revealed that the calibration curve for KY-2Na shows good linearity with an R² value of 0.956, but that for KY-1Na has a mediocre R² value (0.756) which may be due to a small change in the ΔOD (Fig. 3f). In fact, the m_sam value (−0.00043) for KY-2Na is larger than that (−0.00007) for KY-1Na. Thus, the Φ_Δ values for PSs were determined by the relative method using the slope value (m_ref = −0.00084) of the plot for RB (Φ_Δ = 0.75 in water)^9b as a standard. It is worth noting here that in water the Φ_Δ value (0.19) of KY-2Na is much higher than that of KY-1Na (0.06), indicating that KY-2Na bearing two carboxylic acid sodium salts is superior to KY-1Na bearing one carboxylic acid sodium salt for ¹O₂ generation. Meanwhile, both the Φ_fl and Φ_Δ values for phenazine-2,3-diol-based photosensitizers are quite low which indicates a large loss of the excited energy. Thus, we performed time-resolved fluorescence spectroscopy to determine the fluorescence lifetime (τ_fl), radiative rate constant (k_r = Φ_fl/τ_fl), and nonradiative rate constant (k_nr = (1 − Φ_fl)/τ_fl) for KY-2Na in DMSO. The τ_fl, k_r, and k_nr values for KY-2Na are 2.17 ns, 9.20 × 10⁶ s⁻¹, and 4.51 × 10⁸ s⁻¹, respectively. The ratio of the nonradiative constant to radiative constant (k_nr/k_r) is estimated to be 49, indicating that the k_nr value is significantly larger than the k_r value. Thus, this result suggests that for the phenazine-2,3-diol-based photosensitizers, the accelerated nonradiative decay of the excited state may be the main reason for the large loss of the excited energy. Nevertheless, the photodynamic activities of KY-1Na and KY-2Na for PDT in murine colon carcinoma cells (Colon26)¹⁹ under visible light irradiation (>510 nm) were investigated. Colon26 cells were incubated with KY-1Na or KY-2Na for 24 h. The cells were irradiated for 30 min (9 mW), and cell viability was determined after 24 h of incubation using a WST-8 assay. Both under dark and irradiation conditions, unfortunately, the cell viability changed little despite the increasing concentration of KY-1Na and KY-2Na (Fig. S46†). In order to investigate the degree of cellular uptake of KY-2Na in Colon26 cells, we performed confocal laser scanning microscopy (CLSM) for live cell fluorescence imaging. As is expected, the fluorescence image originating from the photosensitizer KY-2Na was not observed (Fig. S47†). These low photodynamic activities indicate that KY-1Na and KY-2Na with high solubility in water (log P_o/w = –0.9) are impermeable to the cell membrane of Colon26.²⁰ Consequently, these results suggested that the ¹O₂ generation ability of phenazine-2,3-diol-based PSs is attributed to the facilitated ISC from the singlet to triplet states by the carbonyl groups based on El-Sayed's rule which allows the change in the molecular orbital type such as transitions of ¹(nπ*) to ³(ππ*) and ¹(ππ*) to ³(nπ*). Moreover, it was found that KY-2Na possesses moderate ¹O₂ generation ability not only in DMSO but also in water, which is attributed to the efficient ISC characteristics, as discussed later in the next section.

Fig. 3 Photoabsorption spectra for the photooxidation of DPBF (5.0 × 10⁻⁵ M) in the presence of (a) KY-1Na and (b) KY-2Na under photoirradiation with 532 nm (300 μW cm⁻²) in DMSO. Insets are magnifications of maxima in the spectra at around 415 nm. Photoabsorption spectra for the photooxidation of ABDA (5.0 × 10⁻⁵ M) in the presence of (c) KY-1Na and (d) KY-2Na under photoirradiation with 532 nm (300 μW cm⁻²) in D₂O/DMSO (99 : 1, v/v). Insets are magnifications of maxima in the spectra at around 400 nm. (e) Plots of ΔOD at around 415 nm for the photooxidation of DPBF in the presence of KO-2, KY-1, KY-2, KY-1Na, KY-2Na, and RB against the photoirradiation time (532 nm, 300 μW cm⁻²) in DMSO. (f) Plots of ΔOD at around 400 nm for the photooxidation of ABDA in the presence of KY-1Na, KY-2Na, and RB against the photoirradiation time (532 nm, 300 μW cm⁻²) in D₂O/DMSO (99 : 1, v/v).

Theoretical calculations

Density functional theory (DFT) and time-dependent DFT (TD-DFT) calculations²¹ at the B3LYP/6-311G(d,p)/DMSO-IEFPCM level for KO-2, KY-1, and KY-2, the B3LYP/6-311+G(d,p)/DMSO-IEFPCM level for KY-1Na and KY-2Na in carboxylate anion forms, and the B3LYP/6-311+G(d,p)/Water-IEFPCM level for KY-1Na and KY-2Na in carboxylate anion forms were performed to gain insight into the ¹O₂ generation properties of these PSs based on the ISC efficiency (Fig. 4). There is little difference in the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) energy levels among the five PSs. Moreover, the DFT calculations indicate that for KO-2, KY-1, KY-2, and KY-1Na, the HOMOs are delocalized over the phenazine-2,3-diol skeleton and formylthiophene units. On the other hand, for KY-2Na, the HOMO is delocalized on the phenazine-2,3-diol skeleton containing the carboxylate anion. Meanwhile, the LUMOs for KO-2, KY-1Na, and KY-2Na are delocalized over the phenazine-2,3-diol skeleton and formylthiophene units, but those for KY-1 and KY-2 are delocalized mainly on the phenazine-2,3-diol skeleton and localized partially on formylthiophene units. Thus, DFT calculations suggested that the HOMO and LUMO distributions for the molecular structure of KY-2Na are adequately separated, leading to a decrease in the energy gap (ΔE_ST) between the S₁ state and the T₁ state that causes efficient ISC,^22,23 compared to that for the molecular structures of KO-2, KY-1, KY-2, and KY-1Na. Indeed, the TD-DFT calculations demonstrated that the ΔE_ST (S₁–T₁) values (0.80 eV in DMSO and 0.82 eV in water) of KY-2Na are smaller than those (0.96 eV in DMSO and 0.98 eV in water) of KY-1Na, that (0.85 eV) of KY-1 and that of (0.81 eV) of KY-2 as well as that (0.93 eV) of KO-2, resulting in a relatively high Φ_Δ value of KY-2Na (Table S7, ESI†). Consequently, we demonstrated that phenazine-2,3-diol chromophore-bearing carboxylic acid salts can act as water-soluble halogen-atom-free-heteroanthracene-based PSs for ¹O₂ generation.

Fig. 4 Energy level diagram, HOMO, and LUMO of (a) KO-2, KY-1, and KY-2 at the B3LYP/6-311G(d,p)/DMSO-IEFPCM level, KY-1Na and KY-2Na as carboxylate anions at the B3LYP/6-311+G(d,p)/DMSO-IEFPCM level, and (b) KY-1Na and KY-2Na at the B3LYP/6-311+G(d,p)/Water-IEFPCM level using DFT calculations.

Conclusions

We have designed and developed phenazine-2,3-diol-based dyes, KY-1Na and KY-2Na bearing one and two carboxylic acid sodium salts, respectively, as water-soluble halogen-atom-free-heteroanthracene-based photosensitizers (PSs) possessing the ability to generate ¹O₂. The logarithms (log P_o/w) of the 1-octanol/water partition coefficient (P_o/w) demonstrated that both KY-1Na and KY-2Na are more hydrophilic than Rose Bengal and have hydrophilicity equivalent to methylene blue. Both the PSs in water show a broad photoabsorption band in the range from 500 to 600 nm. It was found that in water KY-2Na exhibits a moderate ¹O₂ quantum yield (Φ_Δ = 0.19) value, which is higher than that (0.06) of KY-1Na. Density functional theory (DFT) calculations suggested that the HOMO and LUMO distributions for the molecular structure of KY-2Na are adequately separated, leading to a decrease in the energy gap (ΔE_ST) between the S₁ state and the T₁ state that causes efficient intersystem crossing (ISC), compared to that for the molecular structure of KY-1Na. Indeed, time-dependent DFT (TD-DFT) calculations demonstrated that the ΔE_ST (S₁–T₁) value (0.82 eV) of KY-2Na is smaller than that (0.98 eV) of KY-1Na, resulting in a relatively high Φ_Δ value of KY-2Na. Consequently, we propose that phenazine-2,3-diol chromophore-bearing carboxylic acid salts are one of the promising water-soluble halogen-atom-free-heteroanthracene-based PSs for ¹O₂ generation. Further studies on adjustment in the hydrophobicity/hydrophilicity of phenazine-2,3-diol-based PSs suitable for the light-induced cytotoxicity assessment are now in progress to gain insight into the ¹O₂ generation under physiological conditions.

Experimental

General

All solvents and reagents were used as received unless otherwise noted. Rose Bengal (RB) was purchased from Sigma Aldrich and recrystallized from methanol twice for the use in the photoabsorption spectral measurement. 1,3-Diphenylisobenzofuran (DPBF) was purchased from Tokyo Chemical Industry and recrystallized from a mixture of dichloromethane and methanol for use in the photoabsorption spectral measurement. High-grade 9,10-anthracenediyl-bis(methylene)dimalonic acid (ABDA) was purchased from Funakoshi Co., Ltd. ¹H NMR and ¹³C NMR spectra were recorded using a Varian-500 (500 MHz) FT NMR spectrometer. FT-IR spectra were recorded using a Shimadzu IRTracer-100 instrument. High-resolution mass spectral data were acquired using a Thermo Fisher Scientific LTQ Orbitrap XL mass spectrometer. Photoabsorption spectra were recorded using Shimadzu and UV-3600-plus spectrophotometers. Fluorescence spectra were measured using a Hitachi F-4500 spectrophotometer. The fluorescence quantum yields (Φ_fl) were determined with a Hamamatsu C9920-01 instrument equipped with a CCD using a calibrated integrating sphere system. Fluorescence decay measurement was performed on a HORIBA DeltaFlex modular fluorescence lifetime system, using a Nano LED pulsed diode excitation source (451 nm). The irradiance of monochromatic and continuous light for photosensitizing reactions was adjusted using a Newport 1918-C optical power meter.

Synthesis

Methyl 6,9-dibromo-7,8-dihydroxyphenazine-2-carboxylate (3). A solution of bromanilic acid (2.98 g, 10.0 mmol) and methyl 3,4-diaminobenzoate (1.66 g, 10.0 mmol) in ethanol (500 mL) was stirred at 80 °C overnight. After the disappearance of the reactants, the reaction mixture was cooled to room temperature, and the precipitate was filtered and washed with a small amount of ethanol to obtain 3 as a red solid (3.80 g, 89% yield); m.p. over 300 °C; IR (ATR): [small nu, Greek, tilde] = 3204, 2957, 1709, 1624, 1609, 1572, 1551 cm⁻¹; ¹H NMR (500 MHz, DMSO-d₆): δ = 8.61 (br, 1H, aromatic), 8.20 (d, J = 8.60 Hz, 1H, aromatic), 8.12 (d, J = 8.70 Hz, 1H, aromatic) ppm; the ¹³C NMR spectrum could not be obtained because of the low solubility in any solvents; HRMS (APCI): m/z found 424.87924 [M − H]⁻, calculated for C₁₄H₇O₄N₂Br₂ [M − H]⁻: 424.87671.

Dimethyl 6,9-dibromo-7,8-dihydroxyphenazine-2,3-dicarboxylate (4). A solution of bromanilic acid (1.28 g, 4.29 mmol) and 1 (0.961 g, 4.29 mmol) in ethanol (350 mL) was stirred at 80 °C overnight. After the disappearance of the reactants, the reaction mixture was cooled to room temperature, and the precipitate was filtered and washed with a small amount of ethanol to obtain 4 as a red solid (1.59 g, 76% yield); m.p. over 300 °C; IR (ATR): [small nu, Greek, tilde] = 3204, 2951, 1701, 1649, 1622, 1611, 1587, 1572, 1557, 1508 cm⁻¹; ¹H NMR (500 MHz, DMSO-d₆): δ = 8.36 (br, 2H, aromatic), 3.89 (s, 6H, CH₃) ppm; the ¹³C NMR spectrum could not be obtained because of the low solubility in any solvents; HRMS (APCI): m/z found 484.89825 [M + H]⁺, calculated for C₁₆H₁₁O₆N₂Br₂ [M + H]⁺: 484.89784.

Methyl 6,9-dibromo-7,8-bis(methoxymethoxy)phenazine-2-carboxylate (5). A solution of sodium hydride abt. 60% oil suspension (328 mg, 20 eq.) and 3 (172 mg, 0.404 mmol) in THF (100 mL) was stirred at 0 °C for 30 min. Then, chloromethyl methyl ether (608 μL, 8.08 mmol) was added to the solution, and the mixture was stirred at 0 °C overnight. After concentrating under reduced pressure, the residue was chromatographed on silica gel (ethyl acetate/dichloromethane = 1/3 as an eluent) to obtain 5 as a yellow solid (155 mg, 75% yield); m.p. 160–161 °C; IR (ATR): [small nu, Greek, tilde] = 2924, 2839, 1709, 1591 cm⁻¹; ¹H NMR (500 MHz, CDCl₃): δ = 9.11 (s, 1H, aromatic), 8.45–8.39 (m, 2H, aromatic), 5.45–5.44 (s, 4H, CH₂), 4.05 (s, 3H, CH₃), 3.75 (s, 6H, CH₃) ppm; the ¹³C NMR spectrum could not be obtained because of the low stability in CDCl₃ and acetone-d₆; HRMS (APCI): m/z found 536.92700 [M + Na]⁺, calculated for C₁₈H₁₆O₆N₂Br₂Na [M + Na]⁺: 536.92673.

Dimethyl 6,9-dibromo-7,8-bis(methoxymethoxy)phenazine-2,3-dicarboxylate (6). A solution of sodium hydride abt. 60% oil suspension (307 mg, 15 eq.) and 4 (250 mg, 0.514 mmol) in dry DMF (250 mL) was stirred at 0 °C for 20 min under a N₂ atmosphere. Then, bromomethyl methyl ether (610 μL, 7.71 mmol) was added to the solution, and the mixture was stirred at 0 °C for 90 min. The reaction was quenched with water, and then the solution was extracted with ethyl acetate and hexane (=1/4). After concentrating under reduced pressure, the residue was chromatographed on silica gel (ethyl acetate/dichloromethane = 1/3 as an eluent) to obtain 6 as a yellow solid (123 mg, 42% yield); m.p. 166–168 °C; IR (ATR): [small nu, Greek, tilde] = 2953, 1722 cm⁻¹; ¹H NMR (500 MHz, CDCl₃): δ = 8.78 (s, 2H, aromatic), 5.46 (s, 4H, CH₂), 4.02 (s, 6H, CH₃), 3.75 (s, 6H, CH₃) ppm; the ¹³C NMR (125 MHz, CDCl₃) δ = 166.96, 153.54, 142.60, 141.08, 133.27, 131.80, 117.03, 100.47, 58.96, 53.25 ppm; HRMS (ESI): m/z found 572.95044 [M + H]⁺, calculated for C₂₀H₁₉O₈N₂Br₂ [M + H]⁺: 572.95027.

Methyl 7,8-bis(methoxymethoxy)-6,9-bis(5-(4,4,5,5-tetramethyl-1,3-dioxolan-2-yl)thiophen-2-yl)phenazine-2-carboxylate (7). A solution of 5 (235 mg, 0.455 mmol), 2 (567 mg, 1.51 mmol), and Pd(PPh₃)₄ (89 mg, 0.0770 mmol) in toluene (110 mL) was stirred at 110 °C for 24 h. After concentrating under reduced pressure, the residue was chromatographed on silica gel (ethyl acetate/hexane = 1/3 as an eluent) to obtain 7 as a red solid (292 mg, 82% yield); m.p. 104–106 °C; IR (ATR): [small nu, Greek, tilde] = 2955, 2872, 1726, 1622, 1580, 1537 cm⁻¹; ¹H NMR (500 MHz, CDCl₃): δ = 8.87 (s, 1H, aromatic), 8.38 (d, J = 1.90 Hz, 1H, aromatic), 8.30 (d, J = 1.90 Hz, 1H, aromatic), 7.81(d, J = 3.75 Hz, 1H, aromatic), 7.78 (d, J = 3.75 Hz, 1H, aromatic), 7.37–7.20 (m, 2H, aromatic), 6.31 (s, 1H, CH), 6.30 (s, 1H, CH), 5.30 (s, 4H, CH₂), 4.03 (s, 3H, CH₃), 3.30 (s, 6H, CH₃), 1.36–1.33 (m, 24H, CH₃) ppm; ¹³C NMR (125 MHz, CDCl₃): δ = 166.63, 151.96, 151.27, 146.58, 146.50, 143.18, 141.24, 140.96, 140.83, 133.88, 133.85, 133.03, 131.31, 131.28, 131.20, 129.97, 129.09, 125.26, 125.22, 125.05, 124.83, 99.80, 99.76, 97.20, 97.18, 83.28, 83.26, 58.06, 58.05, 52.74, 24.37, 24.34, 22.28, 22.27 ppm; HRMS (ESI): m/z found 801.24866 [M + Na]⁺, calculated for C₄₀H₄₆O₁₀N₂NaS₂ [M + Na]⁺: 801.24861.

Dimethyl 7,8-bis(methoxymethoxy)-6,9-bis(5-(4,4,5,5-tetramethyl-1,3-dioxolan-2-yl)thiophen-2-yl)phenazine-2,3-dicarboxylate (8). A solution of 6 (55.0 mg, 0.0960 mmol), 2 (144 mg, 0.380 mmol), and Pd(PPh₃)₄ (50 mg, 0.0428 mmol) in toluene (50 mL) was stirred at 110 °C for 24 h. After concentrating under reduced pressure, the residue was chromatographed on silica gel (ethyl acetate/dichloromethane = 1/3 as an eluent) to obtain 8 as a red solid (71.0 mg, 89% yield); m.p. 61–63 °C; IR (ATR): [small nu, Greek, tilde] = 2978, 1728 cm⁻¹; ¹H NMR (500 MHz, acetone-d₆): δ = 8.56 (s, 2H, aromatic), 7.81–7.75 (m, 2H, aromatic), 7.35–7.29 (m, 2H, aromatic), 6.31 (s, 2H, CH), 5.31 (s, 4H, CH₂), 3.98 (s, 6H, CH₃), 3.30 (s, 6H, CH₃), 1.34 (m, 24H, CH₃) ppm; ¹³C NMR (125 MHz, acetone-d₆): δ = 167.61, 153.49, 148.20, 142.63, 142.05, 134.31, 133.61, 132.28, 132.21, 126.01, 125.72, 100.77, 97.81, 83.75, 58.18, 53.39, 24.70, 22.53 ppm; HRMS (APCI): m/z found 837.27246 [M + H]⁺, calculated for C₄₂H₄₉O₁₂N₂S₂ [M + H]⁺: 837.27214.

7,8-Bis(methoxymethoxy)-6,9-bis(5-(4,4,5,5-tetramethyl-1,3-dioxolan-2-yl)thiophen-2-yl)phenazine-2-carboxylic acid (9). A solution of 9 (106 mg, 0.136 mmol) and NaOH (100 mg) in the mixture of methanol (85 mL), water (17 mL), and THF (10 mL) was stirred at 40 °C for 3 h. After concentrating under reduced pressure, the residue was neutralized with 1 N HCl aq. and extracted into dichloromethane to obtain 9 as a red solid (97 mg, 93% yield); m.p. 211–212 °C; IR (ATR): [small nu, Greek, tilde] = 2976, 2926, 2851, 1717, 1699, 1653, 1558, 1541, 1506 cm⁻¹; ¹H NMR (500 MHz, acetone-d₆): δ = 8.91 (dd, J = 1.90 Hz and 0.55 Hz, 1H, aromatic), 8.42 (dd, J = 9.00 Hz and 1.90 Hz, 1H, aromatic), 8.31 (dd, J = 9.00 Hz and 0.61 Hz, 1H, aromatic), 7.82 (d, J = 3.70 Hz, 1H, aromatic), 7.78 (d, J = 3.70 Hz, 1H, aromatic), 7.34–7.27 (m, 2H, aromatic), 6.31 (s, 1H, CH), 6.30 (s, 1H, CH), 5.30 (s, 4H, CH₂), 3.31 (s, 6H, CH₃), 1.38–1.29 (m, 24H, CH₃) ppm; the ¹³C NMR spectrum could not be obtained because of the low solubility in any solvents; HRMS (ESI): m/z found 765.25153 [M + H]⁺, calculated for C₃₉H₄₅O₁₀N₂S₂ [M + H]⁺: 765.25101.

7,8-Bis(methoxymethoxy)-6,9-bis(5-(4,4,5,5-tetramethyl-1,3-dioxolan-2-yl)thiophen-2-yl)phenazine-2,3-dicarboxylic acid (10). A solution of 8 (110 mg, mmol) and NaOH (100 mg) in the mixture of methanol (100 mL), water (20 mL), and THF (10 mL) was stirred at 40 °C for 6 h. After concentrating under reduced pressure, the residue was neutralized with 1 N HCl aq. and extracted into dichloromethane to obtain 10 as a red solid (63 mg, 59% yield); m.p. over 300 °C; IR (ATR): [small nu, Greek, tilde] = 2976, 2924, 2850, 1728, 1605, 1541, 1508 cm⁻¹; ¹H NMR (500 MHz, acetone-d₆): δ = 9.24 (s, 2H, aromatic), 7.81 (d, J = 3.75 Hz, 2H, aromatic), 7.30 (d, J = 3.70 Hz, 2H, aromatic), 6.30 (s, 2H, CH), 5.29 (s, 4H, CH₂), 3.31 (s, 6H, CH₃), 1.37–1.29 (m, 24H, CH₃) ppm; the ¹³C NMR spectrum could not be obtained because of the low solubility in any solvents; HRMS (ESI): m/z found 807.22552 [M − H]⁻, calculated for C₄₀H₄₃O₁₂N₂S₂ [M − H]⁻: 807.22519.

6,9-Bis(5-formylthiophen-2-yl)-7,8-dihydroxyphenazine-2-carboxylic acid (KY-1). A solution of 9 (175 mg) and 1 N HCl aq. (0.5 mL) in the mixture of THF (100 mL) and water (100 mL) was stirred at 70 °C overnight. The precipitate was filtered and washed with hexane to obtain KY-1 as a black solid (70 mg, 64% yield); m.p. over 300 °C; IR (ATR): [small nu, Greek, tilde] = 3505, 3159, 2961, 2799, 2737, 2349, 2085, 1996, 1989, 1703, 1643, 1614, 1557, 1520, 1508 cm⁻¹; ¹H NMR (500 MHz, DMSO-d₆): δ = 9.97 (s, 1H, CHO), 9.93 (s, 1H, CHO), 8.96 (s, 1H, aromatic), 8.76 (d, J = 3,20 Hz, aromatic), 8.71–8.70 (m, 1H, aromatic), 8.19–8.16 (m, 2H, aromatic), 8.04–8.00 (m, 2H, aromatic) ppm; the ¹³C NMR spectrum could not be obtained because of the low solubility in any solvents; HRMS (APCI): m/z found 477.02100 [M + H]⁺, calculated for C₂₃H₁₃O₆N₂S₂ [M + H]⁺: 477.02095.

6,9-Bis(5-formylthiophen-2-yl)-7,8-dihydroxyphenazine-2,3-dicarboxylic acid (KY-2). A solution of 10 (63.0 mg, mmol) and 1 N HCl aq. (2 mL) in the mixture of THF (50 mL) and water (50 mL) was stirred at 70 °C overnight. The precipitate was filtered and washed with hexane to obtain KY-2 as a black solid (8.00 mg, 20% yield); m.p. over 300 °C; IR (ATR): [small nu, Greek, tilde] = 3198, 2376, 2347, 2160, 1726, 1701, 1697, 1678, 1655, 1612, 1595, 1535 cm⁻¹; ¹H NMR (500 MHz, DMSO-d₆): δ = 9.95 (s, 2H, CHO), 8.91–8.87 (m, 2H, aromatic), 8.34 (s, 2H, aromatic), 8.01 (d, J = 4.25 Hz, 2H, aromatic) ppm; ¹³C NMR spectrum could not be obtained because of the low solubility in any solvents; HRMS (ESI): m/z found 518.99603 [M − H]⁻, calculated for C₂₄H₁₁O₈N₂S₂ [M − H]⁻: 518.99513.

Sodium 6,9-bis(5-formylthiophen-2-yl)-7,8-dihydroxyphenazine-2-carboxylate (KY-1Na). A solution of KY-1 (42 mg, 0.881 mmol) and 1 N NaOH aq. (70 μL) in water (100 mL) was stirred at room temperature for 10 min and concentrated to obtain KY-1Na as a red solid (46% yield); m.p. over 300 °C; IR (ATR): [small nu, Greek, tilde] = 3200, 2303, 2110, 1994, 1846, 1578 cm⁻¹; ¹H NMR (500 MHz, D₂O): δ = 9.51 (s, 1H, CHO), 9.36 (s, 1H, CHO), 8.71 (s, 1H, aromatic), 8.59 (s, 1H, aromatic), 8.43 (s, 1H, aromatic), 8.00 (d, J = 8.62 Hz, 1H, aromatic), 7.93 (d, J = 8.50 Hz, 1H, aromatic), 7.84 (s, 1H, aromatic), 7.68 (s, 1H, aromatic) ppm; the ¹³C NMR spectrum could not be obtained because of the low solubility in any solvents; HRMS (ESI): m/z found 475.00620 [M − Na]⁻, calculated for C₂₃H₁₁O₆N₂S₂ [M − Na]⁻: 475.00530.

Sodium 6,9-bis(5-formylthiophen-2-yl)-7,8-dihydroxyphenazine-2,3-dicarboxylate (KY-2Na). A solution of KY-2 (8.00 mg, 0.0154 mmol) and 1 N NaOH aq. (31.0 μL) in water (50 mL) was stirred at room temperature for 10 min and concentrated to obtain KY-2Na as a red solid (10 mg, quant.); m.p. over 300 °C; IR (ATR): [small nu, Greek, tilde] = 3318, 1560 cm⁻¹; ¹H NMR (500 MHz, D₂O): δ = 9.84 (s, 1H, CHO), 9.78 (s, 1H, CHO), 8.45 (m, 1H, aromatic), 8.39 (m, 1H, aromatic), 8.27 (s, 1H, aromatic), 8.14 (s, 1H, aromatic), 8.03 (s, 2H, aromatic) ppm; the ¹³C NMR spectrum could not be obtained because of the low solubility in any solvents; HRMS (APCI): m/z found 518.99615 [M + H − 2Na]⁻, calculated for C₂₄H₁₁O₈N₂S₂ [M + H − 2Na]⁻: 518.99513.

n-Octanol/water partition coefficient

The log P_o/w values were determined from the concentration of PSs partitioned into water (100 mL) and n-octanol (100 mL) and the concentration of PSs partitioned into each phase.

Evaluation of the ¹O₂ quantum yield

¹O₂ generation by phenazine-2,3-diol-based PSs KO-2, KY-1, KY-2, KY-1Na, and KY-2Na was investigated by monitoring the changes in the photoabsorption spectra of DPBF in the DMSO solution of each PS and ABDA in aqueous solution (D₂O/DMSO 99 : 1, v/v) of KY-1Na and KY-2Na under photoirradiation. DMSO and water were bubbled with air for 15 min prior to preparing solutions. Air-saturated DMSO containing DPBF or aqueous solutions containing ABDA and each PS were irradiated with monochromatic light at 532 nm (300 μW cm⁻²) that was obtained by passage of a xenon light source (HAL-320, Asahi Spectra) through a monochromator (CMS-100, Asahi Spectra). The concentration of DPBF and ABDA was 5 × 10⁻⁵ M in air-saturated DMSO or aqueous solutions. The concentration of the PSs and RB was adjusted so that the absorbance was ca. 0.03 at an irradiation wavelength of 532 nm. Except for nonirradiated solutions, each spectrum was measured immediately after photoirradiation for 1 min. The procedure using DPBF and ABDA was promptly repeated until the total photoirradiation time reached 10 min and 60 min, respectively. The changes in the optical density (ΔOD) of DPBF and ABDA were plotted against the photoirradiation time to obtain the slopes (m). The Φ_Δ values of KO-2, KY-1, KY-2, KY-1Na, and KY-2Na were determined using the relative method using RB (Φ_Δ = 0.76 in DMSO^9a and 0.75 in water^9b) as a standard according to the following equation:

Φ_Δsam = Φ_Δref × [(m_sam/m_ref) × (L_ref/L_sam)]

where Φ_Δsam and Φ_Δref are the ¹O₂ quantum yields of phenazine-2,3-diol-based PSs and RB, respectively, m_sam and m_ref are slopes in the plots of ΔOD at the photoabsorption maximum wavelength of DPBF (415 nm) or ABDA (400 nm) against the photoirradiation time, and L_sam and L_ref are light harvesting efficiencies, which are given by L = 1–10^−A (“A” is the absorbance at the photoirradiation wavelength).

Evaluation of the cellular uptake of KY-2Na

Colon26 cells were seeded on glass bottom dishes at a density of 1 × 10⁵ cells per well and incubated overnight. The cells were treated with KY-2Na for 24 h. The medium was collected and the fluorescence spectrum was recorded. Then, the cells were observed under a confocal laser scanning microscope (LSM700, Carl Zeiss, Germany).

Photodynamic activity against cancer cell lines

Colon26 cells were seeded on 48-well plates (Thermo Fischer Science) at 1.71 × 10⁴ cells (N = 3) and incubated overnight (approximately 18 h). The cells were exposed to KY-1Na or KY-2Na at varying concentrations. After 24 h of incubation, the cells were washed with PBS thrice, and photoirradiation (>510 nm, 9 mW cm⁻²) was carried out for 30 min. Afterward, the cells were incubated for 24 h, and the Cell Counting Kit-8 solution was added to the cells. After 30 min of incubation, the absorbance at 450 nm was measured using a microplate reader.

Theoretical calculations

The Gaussian 16 program²¹ was used for density functional theory (DFT) calculations and time-dependent DFT (TD-DFT) calculations. DFT and TD-DFT calculations at the B3LYP/6-311G(d,p)/DMSO-IEFPCM level for KO-2, KY-1, and KY-2, the B3LYP/6-311+G(d,p)/DMSO-IEFPCM level for KY-1Na and KY-2Na in carboxylate anion forms, and the B3LYP/6-311+G(d,p)/Water-IEFPCM level for KY-1Na and KY-2Na in carboxylate anion forms were performed. Geometrical optimizations of the S₀ state at each calculation level were performed with frequency calculations. There are no imaginary frequencies for all optimized structures. The TD-DFT calculations of both excited singlet and triplet states were performed using optimized S₀ geometries.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This work was supported by Grants-in-Aid for Scientific Research (B) from the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Number 22H02123 and by The Futaba Foundation.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d3ob00491k

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