Development of an Azo-Based Photosensitizer Activated under Mild

Article pubs.acs.org/JACS

Development of an Azo-Based Photosensitizer Activated under Mild Hypoxia for Photodynamic Therapy Wen Piao,† Kenjiro Hanaoka,*,† Tomotsumi Fujisawa,‡ Satoshi Takeuchi,‡,§ Toru Komatsu,†,¶ Tasuku Ueno,† Takuya Terai,†,∇ Tahei Tahara,‡,§ Tetsuo Nagano,⊥ and Yasuteru Urano*,†,∥,# †

Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan Molecular Spectroscopy Laboratory, RIKEN, 2-1 Hirosawa, Wako 351-0198, Japan § Ultrafast Spectroscopy Research Team, RIKEN Center for Advanced Photonics (RAP), RIKEN, 2-1 Hirosawa, Wako 351-0198, Japan ¶ PRESTO, Japan Science and Technology Agency, Saitama 332-0012, Japan ⊥ Drug Discovery Initiative, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan ∥ Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan # AMED CREST (Japan) Agency for Medical Research and Development, 1-7-1 Otemachi, Chiyoda-ku, Tokyo 100-0004, Japan ‡

S Supporting Information *

ABSTRACT: Photodynamic therapy (PDT) utilizes photoirradiation in the presence of photosensitizers to ablate cancer cells via generation of singlet oxygen (1O2), but it is important to minimize concomitant injury to normal tissues. One approach for achieving this is to use activatable photosensitizers that can generate 1O2 only under specific conditions. Here, we report a novel photosensitizer that is selectively activated under hypoxia, a common condition in solid tumors. We found that introducing an azo moiety into the conjugated system of a seleno-rosamine dye effectively hinders the intersystem crossing process that leads to 1O2 generation. We show that the azo group is reductively cleaved in cells under hypoxia, enabling production of 1O2 to occur. In PDT in vitro, cells under mild hypoxia, within the range typically found in solid tumors (up to about 5% O2), were selectively ablated, leaving adjacent normoxic cells intact. This simple and practical azobased strategy should be widely applicable to design a range of activatable photosensitizers.

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cancer cells.5 The design strategies for activatable photosensitizers can be classified into three categories, based on hindering (1) the photoexcitation process, (2) the intersystem crossing process, or (3) the 1O2 conversion process.1 Among them, the second approach is challenging, because intersystem crossing proceeds within picoseconds after photoexcitation, being much faster than typical fluorescence emission, which usually occurs on a nanosecond time scale. Therefore, ultrafast deactivation of the excited state after photoexcitation is required to prevent intersystem crossing. Recently we and other groups have reported the development of hypoxia-sensitive fluorescence probes, and we proposed a new design strategy for controlling the fluorescence emission process by introducing an azo moiety into the conjugated system of rhodamine fluorophores (Figure 1a).6 This caused strong suppression of fluorescence and proved to be an effective strategy for obtaining hypoxia-sensitive fluorescence probes. This design strategy is useful not only for rhodamine derivatives but also for other fluorophores.6f,g

INTRODUCTION Photosensitizers are chemicals that readily undergo intersystem crossing after excitation to afford the triplet state, from which energy is transferred to triplet oxygen, resulting in the formation of highly reactive singlet oxygen, 1O2 (defined as a type II process).1 Therefore, they are used in photodynamic therapy (PDT) to generate oxidant stress for ablation of cells under photoirradiation, e.g., for treatment of cancer.2 But, although photosensitizers are considered to be safe, it is important to take into account the potential for injury to normal tissues. Various approaches have been taken to address this concern. For example, conjugation of a kind of photosensitizer to appropriate antibodies can increase the selectivity; this is known as photoimmunotherapy (PIT).3 However, the efficacy of PIT is highly dependent on the characteristics of the antibodies and corresponding antigens, so most research has focused on improvement of the antibodies or on conjugation chemistry, rather than on the photosensitizers. Another practical strategy is to utilize activatable photosensitizers, which generate 1O2 only under specific conditions.4 For example, several groups have developed “smart” photosensitizers to target the high concentration of glutathione in © 2017 American Chemical Society

Received: May 15, 2017 Published: September 5, 2017 13713

DOI: 10.1021/jacs.7b05019 J. Am. Chem. Soc. 2017, 139, 13713−13719

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Journal of the American Chemical Society

azobenzene moiety is also important for reductive cleavage of the azo bond.7 Conjugation of the azo group was achieved by an azo coupling reaction,6a,7 yielding the desired photosensitizer, azoSeR (Figure 1b). Compared to SeR, azoSeR showed a red-shifted and broader absorption spectrum, and no emission was observed, as we expected (Figure 2a, Table 1).

Figure 1. (a, b) Design strategy and chemical structures of (a) a “smart” fluorescence probe and (b) an activatable photosensitizer. (c) Proposed photophysical process underlying the activatable photosensitizer. ISC: intersystem crossing. Figure 2. (a) Absorption spectra of 1 μM SeR and azoSeR in PBS at pH 7.4 containing 0.1% DMF for SeR and 1% DMF for azoSeR as a cosolvent. (b) Emission of 1O2 in MeOH upon excitation of 0.1 μM SeR or 1 μM azoSeR at 532 nm. (c) Time-dependent spectral change of azoSeR (1 μM) before and after addition of NADPH (50 μM) under normoxia or hypoxia in the presence of rat liver microsomes in 100 mM phosphate buffer (pH 7.4; 3 mL) at 37 °C. Pictures of reaction mixtures under normoxia and hypoxia are also shown.

Importantly, we found that the S1 lifetime of an azo-based fluorescence probe was in the picosecond range (less than 5 ps).7 Therefore, we hypothesized that this design strategy could also be utilized to obtain activatable photosensitizers. In other words, the very fast conformational change around the NN bond after excitation may block the intersystem crossing process and thereby prevent 1O2 generation (Figure 1b,c). Another feature of our previous work that encouraged us to target hypoxia-activated photosensitizers for PDT was that our hypoxia-sensitive fluorescence probe showed high sensitivity to hypoxia, working at oxygen concentrations up to about 5%.6a This is an important result, because it has been shown that the efficiency of PDT at 5% oxygen concentration remains similar to that at ambient oxygen concentration,8 so we anticipated that cell ablation via 1O2 generation could be successfully achieved under mildly hypoxic conditions. Furthermore, mildly hypoxic environments are thought to be a key niche for acquisition of stemness by cancer cells.9 Thus, cancer cells in such environments are an especially important target. Therefore, we expected that our azo-based strategy would be well suited to the design of practical hypoxia-sensitive photosensitizers.

Table 1. Photophysical Properties of SeR and azoSeR dye SeR azoSeR

λabsa (nm) 533 630

λfla (nm)

ε (M−1 cm−1)

Φflb

Φ(1O2)c

557 N.D.d

5.9 × 10 3.6 × 104

0.006 0.001

0.56 0.03

4

a

Photophysical properties in PBS at pH 7.4. bFor determination of the fluorescence quantum efficiency (Φfl), Rhodamine B in EtOH (Φfl = 0.65) was used as a fluorescence standard. cRelative Φ(1O2) was determined by comparison with the initial rate of 1,3-diphenylisobenzofuran (DPBF) consumption (i.e., the slope) calibrated in terms of relative number of absorbed photons at the maximum absorbance of each dye. All dyes were dissolved in a 1:1 (v/v) mixture of sodium phosphate buffer at pH 7.4 (0.1 M) and MeOH containing 20 μM DPBF and 0.1% DMF as a cosolvent. Rose bengal (Φ(1O2) = 0.75 in H2O/MeOH = 1:1) was used as a standard. dN.D. = not detectable.

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RESULTS AND DISCUSSION Design, Synthesis, and Photochemical Properties of azoSeR. For the photosensitizer scaffold, we utilized a selenorosamine-based dye, SeR (Figure 1b). Various chromophores containing a selenium atom have been utilized for fluorescence probes and photosensitizers.10 Especially, Detty and co-workers have intensively developed selenium-containing rosamine dyes and evaluated their photophysical properties as photosensitizers.11 SeR contains a selenium atom at the 10-position of the xanthene moiety, and this results in efficient intersystem crossing due to the internal heavy atom effect.12 To prevent possible attack of nucleophiles at the 9-position of the xanthene moiety, a 2′,6′-dimethylbenzene moiety was introduced to provide steric hindrance.13 The N(Me)2 group on the

To compare the 1O2 production efficiency of azoSeR with that of SeR, we conducted three experiments. First, we observed emission from 1O2 after excitation of azoSeR or SeR. 1O2 exhibits emission around 1270 nm when it converts to the ground state. As expected, SeR dissolved in MeOH showed strong emission at 1268 nm upon irradiation at 532 nm. On the other hand, azoSeR exhibited very weak emission, even at higher concentration (Figure 2b). This result indicates that 1O2 production was well suppressed in azoSeR. Next, for quantitative evaluation, relative 1O2 yield (Φ(1O2)) was determined with an 1O2 chemical trap, 1,3-diphenylisobenzofuran (DPBF) (Figure S1). After irradiation of an aqueous solution of azoSeR or SeR in the presence of DPBF, a 13714


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We found that the absorbance spectrum was hardly changed in the presence of GSH at concentrations up to 10 mM (Figure S7). These results indicate that SeR is selectively generated from azoSeR under hypoxic conditions. Next, we examined whether azoSeR works in live cells. Human lung cancer-derived A549 cells were incubated with azoSeR under either normoxia or hypoxia. Then, the cells were subjected to light irradiation at 535 nm under normoxia (21% O2). After the photoirradiation, cells were incubated under normoxia for another 14 h, and the cell viability was assessed using a LIVE/DEAD cell viability assay with ethidium homodimer (EthD) and Calcein AM (Figure 3a). Only the

significant decrease of absorbance at 410 nm was observed only in SeR solution, indicating the formation of o-dibenzoylbenzene through [4+2] cycloaddition with 1O2 (Figure S1a). Using Rose bengal (Φ(1O2) = 0.75) as a standard,14 the Φ(1O2) values of SeR and azoSeR were calculated as 0.56 and 0.03, respectively (Table 1). Finally, to confirm that suppression of 1 O2 generation is due to hindrance of intersystem crossing by the azo group, we performed femtosecond time-resolved absorption measurements to analyze the relaxation process of each dye. After photoexcitation, SeR initially exhibited a sharp transient absorption band due to the excited singlet (S1) state at around 420 nm, and then a long-lived flat transient absorption appeared in the 380−1000 nm region as the S1 absorption vanished (Figure S2, yellow line). Since SeR has high Φ(1O2) and low fluorescence quantum yield, this spectral change is assignable to the intersystem crossing process, and the longlived transient absorption is attributed to the triplet state. Kinetic analysis at four representative wavelengths indicated that the lifetime of the S1 state was 21 ps in CHCl3 (Figure S3). In sharp contrast, time-resolved absorption spectra of azoSeR showed that the S1 state of azoSeR decays much faster, generating only very weak transient absorption due to the triplet state. More importantly, the ground-state bleaching largely recovers with the decay of the S1 state (Figure S4a,b). These observations directly indicate that the S1 state is predominantly relaxed to the S0 state and the intersystem crossing quantum yield of azoSeR is low. Analysis of the decay curve of stimulated emission (SE) at 823 nm yielded a lifetime of the S1 state of 4.5 ps (Figure S4c,d, Table 2). The time-

Figure 3. (a) Schematic illustration of cell ablation with azoSeR. (b) Fluorescence confocal microscopy images of A549 cells 14 h after treatment. Cells were incubated with 1 μM azoSeR containing 0.1% DMF as a cosolvent under normoxia or hypoxia (0.1% O2) for 6 h. Then, the medium was replaced with phenol red-free DMEM, and the cells were subjected to photoirradiation (535 nm, 28 mW/cm2, 3 min) under normoxia. After treatment, the cells were incubated under normal culture conditions for 14 h and then stained with 2 μM Calcein AM (live staining, green) and 4 μM EthD (dead staining, red) for 30 to 60 min.

Table 2. Time Constants of SeR and azoSeR in CHCl3 dye

S1 lifetime (ps)

vibrational cooling (ps): See Figure S4

SeR azoSeR

21 4.5

− 13

resolved absorption data of azoSeR clearly show that introducing the azo group into the conjugation system of the photosensitizer hinders the intersystem crossing process by accelerating internal conversion to the S0 state. We note that the rate of the ultrafast internal conversion due to the azo moiety is insensitive to the polarity of the solvent, as we revealed previously.7 Thus, all the data support our hypothesis that azo coupling to the conjugated system of the xanthene moiety can reduce 1O2 generation by hindering intersystem crossing in the photoexcited dye (Figure S5). Applications of azoSeR to Biological Assay. On the basis of these results, we examined whether or not azoSeR can be activated selectively under hypoxia. First, we conducted an in vitro assay using rat liver microsomes. It is known that enzymatic reduction proceeds under hypoxia, and azo compounds can be readily reduced under such conditions.6a,15 When azoSeR was added to microsomes, no change in absorption spectrum was detected, under either normoxia or hypoxia (Figure 2c). In contrast, when NADPH (50 equiv) was added as an electron donor, the absorption of azoSeR immediately disappeared, concomitantly with the appearance of a new absorbance peak, seen only under hypoxia (Figure 2c). The reaction reached a plateau within 20 min (Figure S6a), and generation of SeR was confirmed by HPLC analysis (Figure S6b). Since intracellular biomolecules may also reduce the azo group, we examined the reactivity of azoSeR with reduced glutathione (GSH), which is a major biological reducing agent.

cells treated with azoSeR under hypoxia were predominantly stained with EthD, indicating selective induction of cell death (Figure 3b). Cell viability was positively correlated with light irradiation dose (Figure S8), but little cytotoxicity was seen among cells not exposed to light irradiation or cells under normoxia (Figure 3b). Addition of sodium azide, a 1O2 scavenger,16 to the medium resulted in recovery of the viability of cells treated with azoSeR under hypoxia, supporting the view that 1O2 is responsible for the cell damage in this experimental system (Figure S9). We then examined whether or not azoSeR can selectively ablate cells subjected to hypoxia. To test this, we used a system in which cells under normoxia and cells subjected to hypoxia coexisted on the same cell culture plate. A thin cover glass was placed over half of the cell culture plate to block oxygen supply to the cells beneath it, resulting in a decrease of oxygen concentration (Figure 4a).6a,17 Then, the cover glass was removed and the whole plate was irradiated. Live/dead staining clearly showed that cells that had been under the cover glass were killed, while cells that had not been under the cover glass appeared to suffer little damage (Figure 4b). This result indicates that azoSeR could selectively ablate hypoxic cells, while leaving adjacent normoxic cells intact. 13715


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Figure 5. (a) Construction of a mildly hypoxic environment (∼8% O2) with an AnaeroPouch-MicroAero. (b) Live/Dead staining images of A549 cells. Cells were photoirradiated under normoxia or hypoxia (535 nm, 22 mW/cm2, 2 min). Values are represented as mean ± SD (n = 3). * indicates p < 0.05 by Student’s t test.

This effectively blocks the intersystem crossing process, which would otherwise lead to 1O2 generation. The azo group is reductively cleaved in cells under hypoxia (even at 5% oxygen concentration), enabling production of 1O2 to occur. Since low oxygen concentration impairs PDT efficiency, neither azoSeR nor other photosensitizers, such as the commonly used porphyrins, can work in anoxic environments.2c,8 However, PDT is still effective for treatment of tumors under mildly hypoxic conditions (around 5% oxygen concentration).8 Therefore, we anticipate that azoSeR, which is reductively activated specifically in cells under mild hypoxia, leading to 1O2 production, would have potential clinical application for tumortargeted PDT with reduced side effects. In other words, azoSeR exhibits high selectivity for cells under hypoxia and could ablate mildly hypoxic cells, leaving adjacent normoxic cells intact. Although oxygen concentrations differ in various tissues inside the body9 and azoSeR may show cytotoxicity in tissues under low oxygen concentration, highly hypoxic environments (up to about 5% oxygen) are mostly found in solid tumors.18 We believe this straightforward azo-based strategy may be generally applicable to design a range of activatable photosensitizers for cancer therapy.

Figure 4. (a) Schematic image of cellular hypoxia induced by a cover glass. (b) Live/Dead staining images of A549 cells. A549 cells were stained with 2 μM Calcein AM (live, green) and 4 μM EthD (dead, red) for 30 to 60 min. n = 4 for irradiation+, 3 for irradiation−. Representative figures are shown.

Finally, we wanted to establish the range of oxygen concentration over which azoSeR would be able to ablate cells. First, we examined the sensitivity of azoSeR by photoirradiating azoSeR-loaded cells exposed to various oxygen concentrations. A549 cells were incubated with azoSeR at various oxygen concentrations and then returned to normoxia and subjected to photoirradiation. We observed a high level of cytotoxicity even at an oxygen concentration as high as 5%, along with a fluorescence increase derived from SeR (Figure S10). This was a promising result since tumor hypoxic microenvironments exhibit a wide range of oxygen concentration in the range of 0 to 5%,18 and PDT is effective even at 5% oxygen concentration. These results suggest that azoSeR would be practically useful to ablate cells even in mildly hypoxic environments. Furthermore, we found that SeR, the product formed from azoSeR under hypoxia, was localized mainly in mitochondria (Figure S11). It is reported that mitochondria are an effective organelle target for PDT even under hypoxia, probably because they have a higher local oxygen concentration than other organelles.19 Therefore, it can be expected that azoSeR would be effective in solid tumors. To further confirm this, we photoirradiated cells either under normoxia or under mild hypoxia (oxygen concentration ∼8%) generated with an AnaeroPouch in a clear container (Figure 5a). The hypoxic cells showed a significant decrease of cell viability compared with normoxic cells, indicating that azoSeR can induce cell damage even at ∼8% oxygen concentration (Figure 5b).

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EXPERIMENTAL SECTION

General Procedures and Materials. Reagents and solvents were of the best grade available, purchased from Tokyo Chemical Industries, Wako Pure Chemical, Aldrich Chemical Co., and Invitrogen, and were used without further purification. Dimethylformamide (DMF, fluorometric grade) used for stock solutions was purchased from Dojindo. Reactions were monitored by means of TLC and ESI mass spectrometry. All compounds were purified on a silica gel column and by preparative HPLC. Compounds were characterized by means of 1H NMR, 13C NMR, ESI-MS (Figure S12), and IR. Instruments. 1H and 13C NMR spectra were recorded on a JEOL JNM-LA300 or a JMN-AL400 spectrometer. Mass spectra were measured with a JEOL JMS-T100LC mass spectrometer (ESI+). HPLC purifications were performed on a Jasco PU-2080 system fitted with a reversed-phase column (GL Sciences, Tokyo, Japan), Inertsil ODS-3 10 mm × 250 mm, using eluent A (H2O containing 0.1% trifluoroacetic acid (TFA, (v/v)) and eluent B (CH3CN with 20% H2O containing 0.1% TFA (v/v)), at a flow rate of 5 mL/min. HPLC analyses were performed on a system composed of a pump (PU-2080, JASCO) and a detector (MD-2015, JASCO), fitted with a reversedphase column (Inertsil ODS-3 4.6 mm × 250 mm (GL Sciences, Tokyo, Japan)), using eluent A and eluent B at a flow rate of 1 mL/

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CONCLUSIONS In conclusion, we have developed a photosensitizer that is activated under hypoxic conditions, by introducing an azo moiety into the conjugated system of a seleno-rosamine dye. 13716


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20% was controlled with a multi gas incubator (Panasonic) by means of N2 substitution. PDT Assays with A549 Cells Stimulated with a Cover Glass. 1.5 × 105 A549 cells were seeded on 35 mm poly-L-lysine-coated glassbottomed dishes (Matsunami Glass Ind., Ltd.) and cultured for 2 days before assay. Cells were washed with PBS, then incubated in 1 mL of DMEM containing 1 μM azoSeR and 0.1% DMF as a cosolvent. After incubation for 5.5 h, a 13 mm diameter cover glass cut in half (Matsunami Glass Ind., Ltd.) was gently placed on top of them. Cells were cultured for 2 h under standard conditions. Then, the cover glass was gently removed, and the medium was replaced with phenol-redfree DMEM. The cells were irradiated with light (535 nm, 28 mW/ cm2, 3 min) and incubated under standard conditions for 24 h; then the medium was replaced with DMEM containing 2 μM Calcein AM and 4 μM EthD. Singlet Oxygen Detection by Using 1,3-Diphenylisobenzofuran. Photosensitizer (1 μM) and DPBF (20 μM) were dissolved in a 1:1 (v/v) mixed solution of sodium phosphate buffer at pH 7.4 (100 mM) and MeOH containing 0.1% DMF as a cosolvent. The solution was irradiated with a 300 W Xe light source (MAX-300-S, Asahi Spectra, Japan), which was filtered around the maximum absorption wavelength of the photosensitizers (1.5 mW/cm2; 535 ± 25 nm for SeR, 546 ± 5 nm for Rose bengal, 620 ± 10 nm for azoSeR). The rate of DPBF consumption at the initial stage (i.e., the slope) was calibrated in terms of the relative number of absorbed photons at the maximum absorbance of each dye, and this calculated value corresponds to the relative efficiency of 1O2 generation by the irradiated photosensitizer.22 Rose bengal (Φ(1O2) = 0.75 in H2O/ MeOH = 1:1) was used as a standard.23 PDT Assays with A549 Cells under Various Oxygen Concentrations. A549 cells were incubated with 1 μM azoSeR containing 0.1% DMF as a cosolvent under various oxygen concentrations. After hypoxic stimulation, cells were returned to normoxia and the medium was changed to phenol-red-free DMEM. Then, the cells were irradiated at 535 nm. The irradiated cells were incubated under standard conditions for 14 or 24 h, and the medium was replaced with DMEM containing 2 μM Calcein AM and 4 μM EthD. Cell viability was calculated as follows: Cell viability = Calcein AM-positive cells/(Calcein AM-positive cells + EthD-positive cells). PDT Assays with AnaeroPouch. 3 × 104 A549 cells were seeded on eight-chamber plates (NUNC) and cultured for 1 day before the assay. Cells were washed with PBS once and then incubated in 200 μL of phenol-red-free DMEM containing 1 μM azoSeR and 0.1% DMF as a cosolvent. The cells were then incubated under normoxia or hypoxia in an Anaero Pouch-MicroAero (Mitsubishi Gas Chemical Company, Inc.) in a thin-type jar for 6 h. The cells were irradiated at 535 nm (22 mW/cm2, 2 min) and removed from the jar. The DMEM was changed, and the irradiated cells were incubated under standard conditions for 24 h; then the medium was replaced with DMEM containing 2 μM Calcein AM and 4 μM EthD. Femtosecond Time-Resolved Absorption Measurements. The design of the femtosecond time-resolved absorption setup has been described elsewhere.24 Briefly, the light source was a Ti:sapphire oscillator/regenerative amplifier system (Legend Elite Duo, Coherent) that generates 7.9 mJ pulses at 800 nm with a duration of ∼80 fs at 1 kHz. A ∼4 mJ portion of the 800 nm output was used to pump an optical parametric amplifier (TOPAS, Light Conversion) to produce near-infrared pulses at 2120 or 2400 nm (idler component). The idler pulse was frequency-quadrupled to produce the pump pulse for exciting the samples (pulse energy: ∼300 nJ). Then, the chloroform solutions of SeR and azoSeR were photoexcited at 530 and 600 nm, respectively. The time-resolved absorption spectra after photoexcitation were measured in the visible to near-infrared wavelength regions. The white-light continuum pulse (350−750 nm) to observe the visible region was obtained by focusing ∼0.2% of the output from the light source on a slowly translating CaF2 crystal, whereas the continuum pulse covering the near-infrared region (600−1100 nm) was obtained by focusing the idler component from TOPAS on a sapphire plate. The continuum pulses were divided into probe and reference pulses. After the probe pulse was introduced into the excited

min. LC-MS analyses were performed on a reversed-phase column (Inertsil ODS-3 3.0 mm × 250 mm (GL Sciences (Tokyo, Japan)) using eluent A (H2O containing 0.1% formic acid (v/v)) and eluent B (CH3CN with 20% H2O containing 0.1% formic acid (v/v)) at the flow rate of 0.5 mL/min, fitted on an Agilent Technologies 1200 HPLC system equipped with an Agilent Technologies 6130 mass spectrometer (ESI+, ESI−). Absorption spectra were obtained with Shimadzu UV-1650 and UV-2550 instruments (Tokyo, Japan). Fluorescence spectroscopic studies were performed with a Hitachi F7000 spectrometer (Tokyo, Japan). The excitation and emission slit widths were 5 nm. The photomultiplier voltages were 700 V. The photoirradiation assay was performed with a 300 W Xe light source (MAX-300-S, Asahi Spectra, Japan). Fluorescence confocal microscopic images were acquired by using a Leica Application Suite Advanced Fluorescence (LAS-AF) instrument with a TCS SP5. The light source was a white-light laser. The excitation and emission wavelengths for SeR were 535 nm and 550−650 nm, respectively. Singlet oxygen was detected by using a near-infrared emission spectrometer equipped with a DPSS laser (C8232TR3-45, Hamamatsu, Japan). IR spectra were measured with an FT-IR/ATR (FT/IR4100, JASCO). Optical Properties and Relative Fluorescence Quantum Efficiency. Molecular weight of SeR or azoSeR was taken to be that of the TFA salt (dye: CF3COO− = 1:1), and each compound was dissolved in DMF to prepare the stock solution. Optical properties of dyes were examined in phosphate-buffered saline (PBS) or CHCl3 containing 0.1% (v/v) DMF as a cosolvent. For determination of the fluorescence quantum efficiency (Φfl), Rhodamine B in ethanol (Φfl = 0.65)20 was used as a standard. Fluorescence quantum efficiencies were calculated according to the following equation.

Φx /Φst = [A st /A x ][n x 2 /n st 2][Dx /Dst] where st is standard; x is sample; A is absorbance at the excitation wavelength; n is refractive index; and D is area under the fluorescence spectra on an energy scale. Preparation of Rat Liver Microsomes. All animal experiments were conducted in accordance with institutional guidelines. Rats (Wistar, 6−7 weeks old) were purchased from CLEA Japan. Rats received intraperitoneal injection of 60 mg/kg sodium phenobarbital once daily for 3 days, then were fasted overnight and sacrificed by exsanguination from the abdominal aorta. The liver containing 0.15 M KCl (pH 7.4) was homogenized in 3 volumes of the same buffer. Microsomes were prepared according to the method of Omura and Sato.21 Microsomes contained 67.8 mg protein/mL and 1.82 nmol P450/mg protein. They were diluted with 0.1 M potassium phosphate buffer at pH 7.4 for assay, and the final concentration was 226 μg/3 mL. In Vitro Assay with Rat Liver Microsomes. The hypoxic condition in vitro (enzyme assay in cuvette) was prepared by bubbling argon gas into the reaction solution (0.1 M potassium phosphate buffer, pH 7.4) for 30 min. Rat liver microsomes (226 μg/3 mL) were preincubated at 37 °C for 5 min, and then a 1 μM probe containing 0.1% DMF as a cosolvent was added. As a cofactor for reductases, 50 μM nicotinamide adenine dinucleotide phosphate (NADPH) was added at 5 min. Cell Lines and Culture Conditions. Human lung carcinoma cell line A549 was purchased from RIKEN Bioresource Center cell bank (Tsukuba, Japan). A549 cells were cultured in DMEM (Dulbecco’s modified Eagle’s medium) (Invitrogen) containing 10% fetal bovine serum (Invitrogen) and 1% penicillin−streptomycin (Invitrogen). All cell lines were maintained at 37 °C under 5% CO2 in air (the standard conditions). Hypoxic Conditions for Live Cell Fluorescence Imaging. An O2 concentration of 0.1% was generated with an Anaero Pack (Mitsubishi Gas Chemical Company, Inc.) and a 2.5 L rectangular jar (Mitsubishi Gas Chemical Company, Inc.), while an O2 concentration of 8% was generated with an Anaero Pouch-Micro Aero (Mitsubishi Gas Chemical Company, Inc.) and a 0.4 L rectangular jar (Mitsubishi Gas Chemical Company, Inc.). O2 concentration in the range of 1− 13717


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volume of the sample solution, the probe pulse and the reference pulses were simultaneously dispersed in a 32 cm polychromator (iHR320, Horiba) and detected by a CCD (ProEM, Princeton Instruments) using the different vertical positions. The probe and reference spectra of every five laser shots were read out at 100 Hz from the CCD, which was synchronized with the laser system. The reference spectrum was used for correction of spectral fluctuations of the probe pulse. The relative polarization of the pump and probe pulses was set at the magic angle (54.7°). To calibrate the chirp of the probe pulse, we recorded the wavelength dependence of OKE (optical Kerr effect) signals of the solvent. The time-zero dispersion curve was obtained, and the time origin of the time-resolved absorption data was corrected accordingly. The effective time resolution after this time-zero correction was ∼150 fs.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/jacs.7b05019. Synthesis, experimental details, femtosecond timeresolved absorption measurements, and in vitro analysis of azoSeR (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*[email protected] *[email protected] ORCID

Kenjiro Hanaoka: 0000-0003-0797-4038 Tomotsumi Fujisawa: 0000-0002-3282-6814 Toru Komatsu: 0000-0002-9268-6964 Takuya Terai: 0000-0002-3425-3589 Tahei Tahara: 0000-0001-8421-242X Yasuteru Urano: 0000-0002-1220-6327 Present Address ∇

Graduate School of Science and Engineering, Saitama University, 255 Shimo-Okubo, Sakura-ku, Saitama City, Saitama 338-8570, Japan. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported in part by JSPS KAKENHI Grant Numbers 26104509, 16H00823, and 16H05099 to K.H., JP 25104005 to T.Tahara, JP16H04102 to S.T., JP16K17859 to T.F., 16H06574 to T.U., and SENTAN, JST to K.H., and grants to K.H. from Mochida Memorial Foundation for Medical and Pharmaceutical Research. P.W. was supported by a Grant-inAid for JSPS Fellows.

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REFERENCES

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Development of an Azo-Based Photosensitizer Activated under Mild

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