Endoplasmic Reticulum-Localized Two-Photon-Absorbing Boron

Article Cite This: J. Med. Chem. 2018, 61, 3952−3961

pubs.acs.org/jmc

Endoplasmic Reticulum-Localized Two-Photon-Absorbing Boron Dipyrromethenes as Advanced Photosensitizers for Photodynamic Therapy Yimin Zhou,† Ying-Kit Cheung,‡ Chao Ma,§ Shirui Zhao,† Di Gao,∥ Pui-Chi Lo,*,∥ Wing-Ping Fong,‡ Kam Sing Wong,§ and Dennis K. P. Ng*,† †

Department of Chemistry, The Chinese University of Hong Kong, Shatin, N. T., Hong Kong, China School of Life Sciences, The Chinese University of Hong Kong, Shatin, N. T., Hong Kong, China § Department of Physics, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China ∥ Department of Biomedical Sciences, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong, China ‡

S Supporting Information *

ABSTRACT: Two advanced boron dipyrromethene (BODIPY) based photosensitizers have been synthesized and characterized. With a glibenclamide analogous moiety, these compounds can localize in the endoplasmic reticulum (ER) of HeLa human cervical carcinoma cells and HepG2 human hepatocarcinoma cells. The BODIPY π skeleton is conjugated with two styryl or carbazolylethenyl groups, which can substantially red-shift the Q-band absorption and fluorescence emission and impart two-photon absorption (TPA) property to the chromophores. The TPA cross section of the carbazolecontaining analogue reaches a value of 453 GM at 1010 nm. These compounds also behave as singlet oxygen generators with high photostability. Upon irradiation at λ > 610 nm, these photosensitizers cause photocytotoxicity to these two cell lines with IC50 values down to 0.09 μM, for which the cell death is triggered mainly by ER stress. The two-photon photodynamic activity of the distyryl derivative upon excitation at λ = 800 nm has also been demonstrated.

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and Ca2+ signaling mechanism.12−14 It is believed that the ROS generated at the ER during PDT can induce ER stress, and such photosensitizers can enhance the anticancer efficacy through multiple cell-killing pathways. We report herein two novel boron dipyrromethene (BODIPY)-based photosensitizers, which can localize in the ER and exhibit substantial twophoton absorption (TPA). BODIPY derivatives have been widely used for bioimaging due to their excellent spectroscopic and photophysical properties, as well as high environmental stability.15,16 By introducing two heavy halogen atoms at the meso positions, BODIPYs can generate singlet oxygen effectively through the heavy-atom effect and therefore function as efficient photosensitizers for PDT.17 With a view to targeting the ER of cancer cells, we have introduced a glibenclamiderelated moiety to the BODIPY core to bind with the ATPsensitive K+ channels, which are widely found in the ER.18 The TPA property enables the compounds to be excited at longer wavelengths, which can facilitate the light penetration into tissues.19,20 With these desirable features, these compounds

INTRODUCTION

Photodynamic therapy (PDT) has been regarded as a promising treatment option for various kinds of cancers.1 It is based on light-induced activation of photosensitizers to generate cytotoxic reactive oxygen species (ROS), leading to cell death and tumor ablation. The photosensitizers thus play a key role in determining the therapeutic outcome. In particular, their tumor selectivity and ROS generation efficiency in the tumor microenvironment are two of the most important characteristics. In addition, it has been revealed that the subcellular localization of photosensitizers has profound influence on the signaling pathways and mode of cell death initiated by PDT.2 Mitochondria have long been recognized as important subcellular targets in PDT to induce intrinsic and extrinsic cell death.3,4 By contrast, despite endoplasmic reticulum (ER) plays an important role in the assembly and transport of proteins and the accumulation of unfolded proteins in the ER would trigger ER stress response, which could be a target for the development of chemotherapeutic agents,5−7 PDT with ER-localized photosensitizers remains little studied.8−11 In fact, many stimuli can induce stress in the ER resulting in apoptosis through the unfolded protein response © 2018 American Chemical Society

Received: December 25, 2017 Published: April 22, 2018 3952

DOI: 10.1021/acs.jmedchem.7b01907 J. Med. Chem. 2018, 61, 3952−3961

Journal of Medicinal Chemistry

Article

corresponding condensed products 5 and 6, respectively. The triethylene glycol chain was introduced to enhance the aqueous solubility, biocompatibility, and cellular uptake of the dyes, while the electron-donating carbazole group was added with a view to improving the TPA property.24 These compounds were then hydrolyzed with NaOH in a mixture of tetrahydrofuran (THF) and water to give the carboxy BODIPYs 7 and 8, and subsequently coupled with 4-(2-aminoethyl)-N(cyclohexylcarbamoyl)benzenesulfonamide (9),25 which is a glibenclamide analogue, in the presence of 1-ethyl-3-(3(dimethylamino)propyl)carbodiimide (EDCl) and N,N-dimethylaminopyridine (DMAP) to give the target compounds 10 and 11. Both BODIPY derivatives were highly soluble in common organic solvents and could be purified readily by column chromatography on silica gel. All the new compounds were characterized with various spectroscopic methods. The electronic absorption spectra of these two compounds were recorded in phosphate buffered saline (PBS) in the presence of a small amount of Tween 80 (0.3% v/v) and N,Ndimethylformamide (DMF) (1% v/v) (Figure 1a), and the data are compiled in Table 1. The Q-band of 11 was significantly

could serve as multifunctional photosensitizers, which are highly promising for advanced PDT.

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RESULTS AND DISCUSSION The synthetic scheme used to prepare these compounds is shown in Scheme 1. Bromination of the previously described Scheme 1. Synthesis of the ER-Localized BODIPYs 10 and 11

Table 1. One-Photon and Two-Photon Absorption and Fluorescence Emission Data for BODIPYs 10 and 11 BODIPY

λabs (nm) (log ε)a

10

316 (4.56), 382 (4.76), 442 (4.28), 617 (4.66), 669 (5.06) 336 (4.77), 405 (4.48), 513 (4.45), 708 (4.94)

11

λem (nm)a,b

Φfa,c

ΦΔd,e

σ2 (GM)max [λex (nm)]a,f

692

0.32

0.11

373 [800]

753

0.17

0.11

453 [1010]

a

In PBS with 0.3% v/v Tween 80 and 1% v/v DMF. bExcited at 610 nm. cFluorescence quantum yield (ΦF) with reference to ZnPc (ΦF = 0.28 in DMF). dIn DMF. eSinglet oxygen quantum yield (ΦΔ) with reference to ZnPc (ΦΔ = 0.56 in DMF). fGM = 10−50 cm4 s photon−1 molecule−1.

red-shifted (by 39 nm) compared with that of 10, which can be attributed to the strong intramolecular charge transfer effect arising from the two electron-donating carbazole units. Compound 11 also displayed an additional band at 513 nm assignable to these moieties.24 Upon excitation at 610 nm, both compounds showed a fluorescence emission (Figure 1b). Compound 11 exhibited a larger Stokes shift (844 cm−1 vs 497 cm−1 for 10), giving the emission maximum at 753 nm, but its fluorescence quantum yield (Φf = 0.17) was significantly lower than that of 10 (Φf = 0.32). The TPA properties of these two

BODIPY 121 with N-bromosuccinimide (NBS) gave the dibromo BODIPY 2, which underwent Knoevenagel condensation with the aryl aldehyde 322 or 423 to afford the

Figure 1. Normalized (a) electronic absorption and (b) fluorescence spectra of 10 and 11 in PBS (pH = 7.4 with 0.3% v/v Tween 80 and 1% v/v DMF). 3953



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compounds were then studied using a two-photon excited fluorescence (TPEF) method in PBS in the range of 800−1100 nm with Rhodamine B as reference. As shown in Figure 2, the

Figure 2. TPA spectra of 10 and 11 in PBS (pH = 7.4 with 0.3% v/v Tween 80 and 1% v/v DMF).

carbazole-conjugated BODIPY 11 exhibited a higher TPA cross section across virtually the whole region. The maximum values were calculated to be 373 GM (at 800 nm) for 10 and 453 GM (at 1010 nm) for 11, which were significantly higher than those of other BODIPY-based TPA dyes (45−300 GM).26−28 All these spectral data are also summarized in Table 1. The singlet oxygen generation efficiency of these compounds was also studied in DMF and in PBS, as reflected by the rate of decay of the singlet-oxygen quencher 1,3-diphenylisobenzofuran (DPBF). As shown in Figure S1 in the Supporting Information, both compounds could induce photodegradation of DPBF with a comparable efficiency in DMF, but the efficiency was significantly lower than that of the unsubstituted zinc(II) phthalocyanine (ZnPc) used as the reference. Their singlet oxygen quantum yields (ΦΔ) were calculated to be 0.11 with reference to ZnPc (ΦΔ = 0.56) (Table 1). Their efficiency was also comparable in PBS, which was higher than that of methylene blue used as the reference (Figure S2 in the Supporting Information). As the singlet oxygen quantum yield of methylene blue in this solvent system has not been reported, the corresponding values for 10 and 11 were not determined. The in vitro photodynamic activity of 10 and 11 was then investigated against HeLa human cervical carcinoma cells and HepG2 human hepatocarcinoma cells. The light source consisted of a 300 W halogen lamp, a water tank for cooling, and a color glass filter cut-on at λ = 610 nm. Although the Qband absorptions of 10 and 11 appear at different positions (Table 1), they are embedded by the spectrum of the light source (Figure S3 in the Supporting Information). Hence, both compounds should be excited by the very similar number of photons. Upon irradiation with this source (λ > 610 nm), which has a fluence rate of 40 mW cm−2, for 20 min giving a total fluence of 48 J cm−2, the effect of drug dose was determined as shown in Figure 3. The IC50 values, defined as the dye concentrations required to kill 50% of the cells, are summarized in Table 2. It can be seen that both compounds are essentially noncytotoxic in the absence of light. However, upon illumination with this light source, they become cytotoxic, and 10 is much more potent than 11 by ca. 35-fold. The IC50 values of 10 are comparable with those of the tris(triethylene glycol)substituted analogue reported by us previously,29 showing that the glibenclamide analogous moiety does not significantly affect the photocytotoxicity. As both compounds have similar singlet

Figure 3. Cytotoxicities of (a) 10 and (b) 11 against HeLa (squares) and HepG2 (circles) cells in the absence (closed symbols) and presence (open symbols) of light (λ > 610 nm, 40 mW cm−2, 48 J cm−2). Data are expressed as mean value ± standard error of the mean value (SEM) of three independent experiments, each performed in quadruplicate.

Table 2. IC50 (μM) Values of BODIPYs 10 and 11 BODIPY

for HeLa cells

for HepG2 cells

10 11

0.09 ± 0.01 3.2 ± 0.3

0.16 ± 0.01 5.2 ± 0.5

oxygen generation efficiency, their difference in IC50 values could be attributed to their difference in cellular uptake. To provide evidence to support this hypothesis, the intracellular fluorescence intensities of these two compounds in HeLa and HepG2 cells were measured and compared using flow cytometry, which could shed light on their cellular uptake. As shown in Figure 4, the fluorescence intensity of 10 was significantly higher than that of 11 (12.8-fold for HeLa cells and 4.5-fold for HepG2 cells) despite a shorter incubation time, which indicated that 10 was taken up preferentially by these two cell lines compared with 11. It seems that the extended π skeleton constructed by the larger carbazole units of 11 hinders the uptake process. To reveal if these photosensitizers were stable during the photodynamic treatment, we monitored their absorption spectra in PBS under the same conditions over 25 min. It was found that the spectra remained essentially unchanged, and the Q-band absorbance did not change with time (Figures S4 and S5 in the Supporting Information). The results clearly showed that both 10 and 11 were stable even upon illumination. It is worth noting that the absorbance at 417 nm also did not vary along with time, showing that in the study 3954



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were imaged. This method is commonly used to demonstrate the in vitro potency of two-photon-activated photosensitizers.30,31 It can be seen clearly in Figure 5 that the cells after two-photon PDT treatment showed a red fluorescence signal in the PI channel, indicating the occurrence of cell death. Without light irradiation, the fluorescence signal due to PI could hardly be observed. Moreover, the bright field image also showed significant cell shrinkage upon treatment with 10 and irradiation. After two-photon irradiation, the fluorescence intensity of 10 was diminished as the dye leaked out from the cells when the cells were dying. As mentioned above, this compound could generate singlet oxygen upon one-photon excitation. It is believed that this ROS was also generated under a two-photon excitation condition as the same excited states were involved, and this ROS was responsible for the observed cellular damage. The subcellular localization of these compounds was further examined with confocal microscopy. As shown in Figures 6 and 7, the merged images in panels (d) and (h) with ER-Tracker Green indicate selective localization of 10 and 11 at the ER in both the cell lines. The studies of subcellular localization using Mito-Tracker Green and Lyso-Tracker Green showed no obvious mitochondrial and lysosomal localization (Figures S7− S10 in the Supporting Information). BODIPY 10 showed a much higher fluorescence intensity in both the cell lines compared with 11, which could be attributed to its higher fluorescence quantum yield (Table 1) and cellular uptake (Figure 4). To reveal the role of the glibenclamide-derived moiety, the subcellular localization of the nonglibenclamidecontaining analogues 5 and 6 was also studied under the same conditions. It was found that they did not exhibit preferential localization in any of the above subcellular organelles (Figures S11−S14 in the Supporting Information). The results clearly indicated that the ER-targeting effect of 10 and 11 should be due to the glibenclamide-derived moiety. Interestingly, the fluorescence images of the cells could also be obtained under two-photon excitation (at 800 nm), though the intensity was weaker compared to the images obtained upon one-photon

Figure 4. Comparison of the relative intracellular fluorescence intensities of 10 and 11 in HeLa and HepG2 cells as determined by flow cytometry. The incubation time was 2 h for 10 and 4 h for 11. Data are expressed as mean value ± standard deviation of three independent experiments.

of singlet oxygen generation, the decrease in absorbance at that position was solely due to the decay of DPBF. Apart from the effect of drug dose, we also examined the effect of light dose on the cell viability for 10 against HepG2 cells. At a concentration of 0.19 μM, the cell viability decreased almost monotonically with the irradiation time and reached a value of ca. 50% after irradiation for 20 min (with a total fluence of 48 J cm−2) (Figure S6 in the Supporting Information). The results are in good agreement with those of the drug-dose study as described above (Figure 3a). Apparently, there was not a threshold level of light required for the photocytotoxicity. The two-photon photodynamic activity of 10 was also examined using a 800 nm laser equipped in a Zeiss laser scanning microscope. After the HeLa cells were incubated with 10 for 2 h followed by two-photon irradiation for 10 min, they were kept in a chamber with 5% CO2 at 37 °C for 3 h to allow the occurrence of cell death. After 3 h postirradiation, a PBS solution of propidium iodide (PI) was added to distinguish the dead and viable cells. PI cannot pass through the plasma membrane of the viable cells, while it can stain the late apoptotic and necrotic cells. After the removal of PI, the cells

Figure 5. Fluorescence images of HeLa cells after incubation with 10 (2 μM) for 2 h in the presence or absence of two-photon irradiation (800 nm, 40 mW) for 10 min. The PI was excited at 488 nm and its fluorescence was monitored at 550−700 nm, while 10 was excited at 633 nm and its fluorescence was monitored at 640−735 nm. The red color shown in the PI channel indicates the dead cells. 3955



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Figure 6. Visualization of the intracellular fluorescence of 10 (2 μM) after 2 h incubation and ER-Tracker Green (0.2 μM) after 15 min incubation in HeLa and HepG2 cells: (a,e) fluorescence of 10; (b,f) fluorescence of ER-Tracker Green; (c,g) bright field images; and (d,h) corresponding superimposed images. Scare bar = 25 mm.

Figure 7. Visualization of the intracellular fluorescence of 11 (4 μM) after 4 h incubation and ER-Tracker Green (0.2 μM) after 15 min incubation in HeLa and HepG2 cells: (a,e) fluorescence of 11; (b,f) fluorescence of ER-Tracker Green; (c,g) bright field images; and (d,h) corresponding superimposed images. Scare bar = 25 mm.

excitation (at 633 nm) (see Figure S15 in the Supporting Information for the images of 10 in HeLa cells). It has been reported that subcellular localization of photosensitizers could strongly affect the cell death pathways.2 As these two BODIPYs showed high affinity to the ER, we focused on the ER stress induced by the PDT action of the more potent 10 on HepG2 cells. As ER stress is associated with the generation and accumulation of ROS, a state commonly regarded as oxidative stress,32 we measured the production of ROS inside HepG2 cells using 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) as a probe.33 While the intracellular ROS level remained low and unchanged in the absence of light, there was a trend of increase in ROS generation with increasing the concentration of 10 (Figure 8). At a concentration of 0.8 μM, the ROS level was about 9-fold higher than that of the dark control. Ca2+ in the ER lumen is either free or bound to luminal proteins such as calreticulin and calnexin. Oscillations in ER Ca2+ concentration participate in the regulation of normal cell function at multiple levels.34 Thus, the cell death induced by the photoexcited 10 via ER stress can be illustrated by the

Figure 8. Intracellular ROS production induced by 10. HepG2 cells were incubated with different concentrations of 10 (0.05, 0.1, 0.2, 0.4, and 0.8 μM) for 2 h. The intracellular ROS levels, as reflected by the fluorescence of H2DCFDA (10 μM), after the treatment with 10 with or without light illumination (λ > 610 nm) are shown as the means with SEM of three independent experiments. *p < 0.05; ***p < 0.001.

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intracellular Ca2+ levels as probed by Fluo-3 AM. As shown in Figure 9, the level of intracellular Ca2+ increased as the

Figure 10. Western blot analysis after the treatment with 10. HepG2 cells after being treated with 10 (0.15 μM) with or without illumination (λ > 610 nm) were lysed using radioimmunoprecipitation assay buffer at 4, 6, and 8 h. Equal amounts of proteins were electrophoresed on 10% polyacrylamide gels and transferred to polyvinylidene fluoride membrane. The membrane was probed with GRP78, CHOP, and Actin antibodies. The control was the cells without being treated with 10, but upon illumination.

Figure 9. Intracellular calcium level after the treatment with 10. HepG2 cells were incubated with different concentrations of 10 (0.05, 0.1, 0.2, 0.4, and 0.8 μM) for 2 h. The intracellular calcium level in cells, as reflected by the fluorescence of Fluo-3 AM (2.5 μM), after the treatment with 10 with or without light illumination (λ > 610 nm) are shown as the means with SEM of three independent experiments. *p < 0.05; ***p < 0.001.

illumination lead to cell death mainly by ER stress, which has been illustrated by a series of ER stress studies. The high TPA cross sections of these compounds in the therapeutic window are also desirable. With these advantageous features, these compounds could serve as multifunctional photosensitizers for advanced PDT.

concentration of 10 increased and reached about 3.5-fold to the baseline upon light illumination, whereas this trend could not be seen when the cells were treated with 10 alone in dark. The generation of intracellular ROS and overload of Ca2+ contribute to the decrease in viability of HepG2 cells after PDT since perturbation of the steady-state Ca2+ level in the ER as well as the acute Ca2+ release can be apoptogenic. Any insult that disturbs ER homeostasis ultimately results in ER stress as a result of the accumulation of misfolded proteins. In this process, the expression of proteins related to unfolded protein response, including chaperones (e.g., BiP/GRP78 and calreticulin) and CHOP/GADD153, a transcription factor that decreases the expression of Bcl-2 and can therefore induce apoptosis, will be on a high level.35 Therefore, the ER stress induced by 10 was also studied by measuring the expression level of the marker proteins GRP78 and CHOP via Western blot analysis. BODIPY 10 at the concentration of 0.15 μM (i.e., the IC50 value) was tested for ER stress-related protein expression. As shown in Figure 10, the expression of GRP78 and CHOP was clearly detected at 4, 6, and 8 h after PDT. The CHOP exhibited an expression pattern of an early rise followed by a decrease in CHOP with the highest expression level at 4 and 6 h, while the expression level of GRP78 was the highest at 8 h. However, both GRP78 and CHOP remained low or undetectable after irradiation treatment alone or incubation with 10 without light illumination. All these data clearly show that 10 can induce ER stress via ROS generation in ER after light illumination.

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EXPERIMENTAL SECTION

General. All the reactions were performed under an atmosphere of nitrogen. THF and toluene were distilled from sodium benzophenone ketyl and sodium, respectively. DMF was dried over barium oxide and distilled under reduced pressure. All other solvents and reagents were of reagent grade and used as received. All the reactions were monitored by thin layer chromatography (TLC) performed on Merck precoated silica gel 60 F254 plates. Chromatographic purification was performed on silica gel (Macherey-Nagel, 230−400 mesh) with the indicated eluents. Compounds 1,21 3,22 4,23 and 925 were prepared as described. 1 H and 13C{1H} NMR spectra were recorded on a Bruker Avance III 400 spectrometer (1H, 400 MHz; 13C, 100.6 MHz) or a Bruker Avance III 700 spectrometer (1H, 700 MHz; 13C, 176.0 MHz) in deuterated solvents. Spectra were referenced internally by using the residual solvent [1H, δ = 7.26 (for CDCl3), δ = 5.30 (for CD2Cl2)] or solvent [13C, δ = 77.16 (for CDCl3)] resonances relative to SiMe4. Electrospray ionization (ESI) mass spectra were recorded on a Thermo Finnigan MAT 95 XL mass spectrometer. UV−vis absorption and steady-state fluorescence spectra were taken on a Shimazu UV1800 UV−vis spectrophotometer and a Horiba FluoroMax spectrofluorometer, respectively. The purity of 10 and 11 was examined by high-performance liquid chromatography (HPLC) and found to be >95% (Figure S16 in the Supporting Information). Preparation of 2,6-Dibromo-8-(4-(ethoxycarbonylmethoxy)phenyl)-1,3,5,7-tetramethyl-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (2). A mixture of 1 (210 mg, 0.5 mmol) and NBS (177 mg, 1.0 mmol) in CH2Cl2 (20 mL) was stirred at room temperature (r.t.) for 2 h. The solvent was then evaporated under reduced pressure. The residue was purified by column chromatography on silica gel with hexane/CH2Cl2 (1:1 v/v) as eluent to afford 2 (230 mg, 79%). 1H NMR (700 MHz, CDCl3): δ 7.16 (d, J = 8.4 Hz, 2 H, ArH), 7.05 (d, J = 8.4 Hz, 2 H, ArH), 4.71 (s, 2 H, OCH2), 4.30 (q, J = 7.0 Hz, 2 H, OCH2), 2.60 (s, 6 H, CH3), 1.41 (s, 6 H, CH3), 1.31 (t, J = 7.0 Hz, 3 H, CH3). 13C{1H} NMR (176.0 MHz, CDCl3): δ 168.5, 158.9, 154.0, 141.9, 140.7, 130.8, 129.3, 127.6, 115.7, 111.9, 65.5, 61.8, 14.3, 14.0, 13.8. HRMS (ESI): m/z calcd for C23H23BBr2F2N2NaO3 [M + Na]+, 607.0014; found, 607.001.

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CONCLUSIONS In summary, we have prepared two novel BODIPY-based photosensitizers and evaluated their TPA properties and in vitro photodynamic activities. It has been found that the glibenclamide-derived moiety can selectively direct the BODIPYs to the ER of cancer cells. This strategy can be used to prepare other ER-localized theranostic agents. BODIPY 10 is highly photocytotoxic with IC50 values down to 0.09 μM. The localization of 10 in ER and the generation of ROS upon 3957



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Preparation of 2,6-Dibromo-8-(4-(ethoxycarbonylmethoxy)phenyl)-1,7-dimethyl-3,5-bis(2-(4-(3,6,9-trioxadecoxy)phenyl)ethenyl)-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (5). Dibromo BODIPY 2 (115 mg, 0.2 mmol) and triethylene glycol-substituted benzaldehyde 3 (215 mg, 0.8 mmol) were dissolved in toluene (50 mL). Acetic acid (0.3 mL) and piperidine (0.3 mL) were then added. The mixture was heated under reflux with a Dean−Stark trap. After consumption of 2 as indicated by TLC, the mixture was cooled to r.t., and the solvent was evaporated under reduced pressure. Water (150 mL) was then added to the residue, and the crude product was extracted with CH2Cl2 (100 mL × 3). The combined organic phase was dried over anhydrous Na2SO4, and the solvent was evaporated under reduced pressure. The crude product was purified by column chromatography on silica gel with CH2Cl2/CH3OH (70:1 v/v) as eluent to afford 5 as a green solid (128 mg, 58%). 1H NMR (700 MHz, CDCl3): δ 8.10 (d, J = 16.8 Hz, 2 H, CHCH), 7.59−7.62 (m, 6 H, CHCH and ArH), 7.20 (d, J = 8.4 Hz, 2 H, ArH), 7.06 (d, J = 8.4 Hz, 2 H, ArH), 6.96 (d, J = 8.4 Hz, 4 H, ArH), 4.71 (s, 2 H, OCH2), 4.31 (q, J = 7.0 Hz, 2 H, OCH2), 4.19 (t, J = 4.9 Hz, 4 H, OCH2), 3.89 (t, J = 4.9 Hz, 4 H, OCH2), 3.76 (t, J = 4.9 Hz, 4 H, OCH2), 3.70 (t, J = 4.9 Hz, 4 H, OCH2), 3.67 (t, J = 4.9 Hz, 4 H, OCH2), 3.56 (t, J = 4.9 Hz, 4 H, OCH2), 3.38 (s, 6 H, OCH3), 1.44 (s, 6 H, CH3), 1.31 (t, J = 7.0 Hz, 3 H, CH3). 13C{1H} NMR (176.0 MHz, CDCl3): δ 168.5, 160.1, 158.8, 148.5, 141.0, 138.9, 138.5, 132.4, 130.0, 129.9, 129.4, 128.1, 116.2, 115.6, 115.1, 110.2, 72.0, 71.0, 70.8, 70.7, 69.8, 67.6, 65.5, 61.7, 59.2, 14.3, 14.0. HRMS (ESI): m/z calcd for C51H59BBr2F2N2NaO11 [M + Na]+, 1107.2433; found, 1107.2437. Preparation of 2,6-Dibromo-8-(4-(ethoxycarbonylmethoxy)phenyl)-1,7-dimethyl-3,5-bis(2-(N-(3,6,9-trioxadecyl)carbazolyl)ethenyl)-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (6). A mixture of BODIPY 2 (115 mg, 0.2 mmol), benzaldehyde 4 (273 mg, 0.8 mmol), acetic acid (0.3 mL), and piperidine (0.3 mL) in toluene (50 mL) was heated under reflux with a Dean−Stark trap. After consumption of 2, the mixture was briefly cooled and evaporated under reduced pressure. The residue was washed with 150 mL of water, and the product was extracted with CH2Cl2 (100 mL × 3). The combined organic phase was dried over anhydrous Na2SO4, and the solvent was evaporated under reduced pressure. The crude product was purified by column chromatography on silica gel using CH2Cl2 as eluent. The green band was collected to give a dark green solid (106 mg, 43%). 1H NMR (700 MHz, CDCl3): δ 8.40 (d, J = 16.1 Hz, 2 H, CHCH), 8.34 (d, J = 0.7 Hz, 2 H, ArH), 8.16 (d, J = 7.7 Hz, 2 H, ArH), 7.87 (dd, J = 1.4, 8.4 Hz, 2 H, ArH), 7.83 (d, J = 16.1 Hz, 2 H, CHCH), 7.51 (d, J = 8.4 Hz, 2 H, ArH), 7.48 (d, J = 4.2 Hz, 4 H, ArH), 7.23−7.25 (m, 4 H, ArH), 7.08 (d, J = 8.4, 2 H, ArH), 4.73 (s, 2 H, OCH2), 4.53 (t, J = 6.3 Hz, 4 H, NCH2), 4.32 (q, J = 7.0 Hz, 2 H, OCH2), 3.90 (t, J = 6.3 Hz, 4 H, OCH2), 3.53−3.55 (m, 4 H, OCH2), 3.50−3.51 (m, 4 H, OCH2), 3.46−3.47 (m, 4 H, OCH2), 3.40−3.41 (m, 4 H, OCH2), 3.32 (s, 6 H, OCH3), 1.49 (s, 6 H, CH3), 1.33 (t, J = 7.0 Hz, 3 H, CH3). 13C{1H} NMR (176.0 MHz, CDCl3): δ 168.6, 158.8, 148.6, 141.7, 141.2, 140.8, 140.6, 137.7, 132.4, 130.0, 128.5, 128.3, 126.2, 125.7, 123.5, 123.1, 120.8, 120.7, 119.8, 115.6, 110.3, 109.6, 109.4, 71.9, 71.1, 70.7, 70.6, 69.4, 65.5, 61.7, 59.1, 43.5, 14.3, 14.1 (two of the aromatic signals are overlapped). HRMS (ESI): m/z calcd for C63H65BBr2F2N4NaO9 [M + Na]+, 1253.3070; found, 1253.3076. Preparation of 2,6-Dibromo-8-(4-(carboxymethoxy)phenyl)-1,7dimethyl-3,5-bis(2-(4-(3,6,9-trioxadecoxy)phenyl)ethenyl)-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (7) and 2,6-Dibromo-8-(4(carboxymethoxy)phenyl)-1,7-dimethyl-3,5-bis(2-(N-(3,6,9trioxadecyl)carbazolyl)ethenyl)-4,4-difluoro-4-bora-3a,4a-diaza-sindacene (8). To a solution of 5 (54 mg, 0.05 mmol) or 6 (65 mg, 0.05 mmol) in THF (5 mL), an aqueous solution of NaOH (4 M, 5 mL) was added. The mixture was stirred at r.t. for 3 h. The solvent was then removed under reduced pressure. The residue was dissolved in 20 mL of water and washed with CH2Cl2 (10 mL × 3). The aqueous layer was then acidified to pH 3 with 2 M hydrochloride acid. The acidic solution was extracted with CH2Cl2 (15 mL × 3). The organic phase was combined and dried over anhydrous Na2SO4, and the solvent was evaporated under reduced pressure. The crude products were used in the next step without further purification.

Preparation of 2,6-Dibromo-8-(4-(N-(4-(N-(cyclohexylcarbamoyl)sulfamoyl)phenylethyl)carbamoylmethoxy)phenyl)-1,7-dimethyl-3,5-bis(2-(4-(3,6,9-trioxadecoxy)phenyl)ethenyl)-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (10). To a mixture of 7 (40 mg, 38 μmol) and 9 (15.6 mg, 48 μmol) in CH2Cl2 (10 mL), EDCI (9.2 mg, 59 μmol) and DMAP (2.8 mg, 23 μmol) were added. The mixture was stirred at r.t. overnight. It was then poured into saturated aqueous NH4Cl solution (15 mL), which was extracted with CH2Cl2 (10 mL × 2). The combined organic layer was washed with saturated aqueous NaCl solution (30 mL × 2), dried over anhydrous Na2SO4, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel with CH2Cl2/CH3OH (30:1 v/ v) to give 10 as a green solid (20 mg, 37%). 1H NMR (400 MHz, CDCl3): δ 8.11 (d, J = 16.8 Hz, 2 H, CHCH), 7.86 (d, J = 8.4 Hz, 2 H, ArH), 7.58−7.63 (m, 6 H, CHCH and ArH), 7.42 (d, J = 8.4 Hz, 2 H, ArH), 7.23 (d, J = 8.4 Hz, 2 H, ArH), 7.04 (d, J = 8.8 Hz, 2 H, ArH), 6.96 (d, J = 8.8 Hz, 4 H, ArH), 6.77 (t, J = 6.0 Hz, 1 H, NH), 6.44 (d, J = 8.0 Hz, 1 H, NH), 4.57 (s, 2 H, OCH2), 4.19 (t, J = 4.8 Hz, 4 H, OCH2), 3.89 (t, J = 4.8 Hz, 4 H, OCH2), 3.75−3.77 (m, 4 H, OCH2), 3.65−3.71 (m, 10 H, OCH2 and NHCH2), 3.55−3.57 (m, 4 H, OCH2), 3.39 (s, 6 H, OCH3), 2.99 (t, J = 7.6 Hz, 2 H, CH2), 1.83− 1.86 (m, 2 H, c-hexane), 1.65−1.70 (m, 3 H, c-hexane), 1.43 (s, 6 H, CH3), 1.15−1.37 (m, 6 H, c-hexane). 13C{1H} NMR (100.6 MHz, CDCl3): δ 167.8, 160.2, 158.0, 150.2, 148.7, 145.4, 140.8, 139.2, 138.1, 138.0, 132.4, 130.3, 130.0, 129.8, 129.4, 128.8, 127.5, 116.2, 115.6, 115.1, 110.4, 72.1, 71.0, 70.8, 70.7, 69.8, 67.7, 67.4, 59.2, 49.4, 40.0, 35.9, 33.1, 25.5, 24.7, 14.1. HRMS (ESI): m/z calcd for C64H76BBr2F2N5NaO13S [M + Na]+, 1386.3479; found, 1386.3497. Preparation of 2,6-Dibromo-8-(4-(N-(4-(N-(cyclohexylcarbamoyl)sulfamoyl)phenylethyl)carbamoylmethoxy)phenyl)-1,7-dimethyl-3,5-bis(2-(N-(3,6,9-trioxadecyl)carbazolyl)ethenyl)-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (11). According to the procedure described for 10 using 8 (48 mg, 40 μmol) as a starting material, 11 was obtained as a dark green solid (12 mg, 20%). 1H NMR (400 MHz, CD2Cl2): δ 8.42 (d, J = 16.4 Hz, 2 H, CHCH), 8.38 (d, J = 0.8 Hz, 2 H, ArH), 8.18 (d, J = 8.0 Hz, 2 H, ArH), 7.86−7.89 (m, 4 H, ArH), 7.81 (d, J = 16.4 Hz, 2 H, CHCH), 7.45−7.58 (m, 8 H, ArH), 7.24−7.32 (m, 4 H, ArH), 7.10 (d, J = 8.4 Hz, 2 H, ArH), 6.78 (t, J = 6.0 Hz, 1 H, NH), 6.36 (d, J = 7.2 Hz, 1 H, NH), 4.53−4.57 (m, 6 H, OCH2 and NCH2), 3.91 (t, J = 5.6 Hz, 4 H, OCH2), 3.62−3.67 (m, 2 H, NHCH2), 3.51−3.53 (m, 4 H, OCH2), 3.46−3.48 (m, 4 H, OCH2), 3.41−3.44 (m, 4 H, OCH2), 3.36−3.38 (m, 4 H, OCH2), 3.27 (s, 6 H, OCH3), 3.00 (t, J = 7.2 Hz, 2 H, CH2), 1.81−1.84 (m, 2 H, chexane), 1.65−1.70 (m, 3 H, c-hexane), 1.51 (s, 6 H, CH3), 1.15−1.38 (m, 6 H, c-hexane). 13C{1H} NMR (176.0 MHz, CDCl3): δ 167.9, 157.9, 150.1, 148.7, 145.4, 141.8, 141.2, 140.8, 140.5, 138.1, 137.2, 132.4, 130.5, 129.9, 129.0, 128.5, 127.5, 126.2, 125.7, 123.6, 123.2, 120.9, 120.8, 119.9, 115.6, 110.4, 109.7, 109.4, 72.0, 71.2, 70.8, 70.7, 69.5, 67.4, 59.1, 49.4, 43.6, 40.0, 35.9, 33.1, 25.5, 24.7, 14.2 (two of the aromatic signals are overlapped). HRMS (ESI): m/z calcd for C76H82BBr2F2N7NaO11S [M + Na]+, 1532.4116; found, 1532.4148. HPLC Analysis. Compounds 10 and 11 were first dissolved in CH2Cl2. Normal-phase HPLC analysis was performed on a Sunfire Prep silica column (5 μm, 4.6 mm × 250 mm) using a Waters 1525 binary HPLC pump with a Waters 2998 photodiode array detector. Pure CH2Cl2 was used as the mobile phase. Total elution time was 15 min. The flow rate was fixed at 1.5 mL min−1. Determination of Φf. The values of Φf were determined by the equation Φf(s) = (Fs/Fref)(Aref/As)(ns2/nref2)Φf(ref),36 where subscript s refers to the sample solutions and ref stands for the reference. F, A, and n are the measured fluorescence (area under the emission peak), the absorbance at the excitation position (610 nm), and the refractive index of the solvent, respectively. ZnPc in DMF was used as the reference [Φf(ref) = 0.28].37 To minimize reabsorption of radiation by the ground-state species, the emission spectra were obtained in very dilute solutions of which the absorbance at 610 nm was about 0.03. Determination of ΦΔ. The values of ΦΔ were calculated by using ZnPc in DMF as reference (ΦΔ = 0.56)38 and DPBF as the singlet oxygen scavenger. A solution of DPBF (30 μM) and the photosensitizer (1 μM) in DMF was irradiated with red light from a 100 W 3958



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humidified chamber for 3 h. For the dark control, the cells were not irradiated and just kept in an incubator with 5% CO2 at 37 °C for 3 h. PI (50 μg) in PBS (1 mL) was added to the cells. After 10 min, the PI solution was removed. The cells were rinsed with PBS (1 mL) and then re-fed with PBS (2 mL). The images were taken with a Zeiss laser scanning microscope (LSM 880 NLO) equipped with a 488 nm argon laser and a 633 nm laser. The PI was excited at 488 nm and monitored at 550−700 nm, while the BODIPY 10 was excited at 633 nm and monitored at 640−735 nm. Determination of Cellular Uptake. Cells seeded on 35 mm dishes were incubated with 10 (2 μM, for 2 h) or 11 (4 μM, for 4 h) in RPMI 1640 medium at 37 °C under 5% CO2. After removing the medium, the cells were rinsed with PBS (1 mL) and then harvested by 0.25% trypsin-ethylenediaminetetraacetic acid (0.2 mL). The activity of the trypsin was quenched with the medium (0.5 mL), and the mixture was centrifuged at 400g for 3 min at r.t. The pellet was washed with PBS (1 mL) and then centrifuged. The cells were suspended in Hank’s balanced salt solution (HBSS) (1 mL) and then subjected to flow cytometric analysis by using a BD FACSVerse flow cytometer (Becton Dickinson) with 104 cells counted in each sample. The compounds were excited by an argon laser at 640 nm, and the emitted fluorescence was monitored at 720−840 nm. The data collected were analyzed by using the BD FACSuite. All experiments were performed in triplicate. Study of Subcellular Localization. Cells on glass-bottom dishes of 35 mm diameter (MatTek Corporation, P35G-0-14-C) were incubated in the culture medium with 5 (2 μM) for 2 h, 6 (4 μM) for 4 h, 10 (2 μM) for 2 h, or 11 (4 μM) for 4 h at 37 °C. After being washed twice with PBS, the cells were stained in HBSS with 0.2 μM ER-tracker green (Thermo Fisher Scientific Inc., E34251), 0.2 μM Mito-tracker green (Thermo Fisher Scientific Inc., M7514), or 2 μM Lyso-tracker green (Thermo Fisher Scientific Inc., L7526) at 37 °C for 15, 15, and 30 min, respectively. After being washed twice with PBS, the cells were re-fed with HBSS and observed using a confocal microscope (Leica TCS SP8 high speed imaging system with CO2 incubator) with a 488 nm argon laser and a 633 nm laser. All the trackers were excited at 488 nm, and their fluorescence was monitored at 500−570 nm, while the BODIPYs were excited at 633 nm and their fluorescence was monitored at 650−760 nm. One-Photon and Two-Photon Cellular Imaging. Approximately 1.2 × 105 HeLa cells in 2 mL of the culture medium were incubated on 35 cm2 glass-bottom confocal dishes overnight. The cells were treated with 10 in the medium containing 0.125% v/v DMF and 0.025% v/v Tween 80 (2 μM, 2 mL) for 2 h, and then rinsed with PBS (1 mL × 2). The images were taken with a Zeiss laser scanning microscope (LSM 880 NLO) equipped with a 633 nm laser (for onephoton excitation) and a 800 nm laser (for two-photon excitation). Study of Intracellular ROS Production. Solutions of 10 in RPMI 1640 medium (100 μL) at different concentrations (0.8, 0.4, 0.2, 0.1, and 0.05 μM) were added to HepG2 cells on 96-well plates. After 2 h, the cells were washed twice with PBS, and then H2DCFDA (Thermo Fisher Scientific Inc., D399) in HBSS (10 μM, 100 μL) was added. The cells were incubated at 37 °C for 30 min in dark. After being washed twice with PBS, the cells were re-fed with HBSS and then subjected to PDT. The fluorescence signal was measured by a fluorescence plate reader using a 485 nm excitation filter and a 535 nm emission filter with a gain of 150 (Tecan Spark 10M Microplate Reader). Determination of Intracellular Calcium Level. Solutions of 10 in RPMI 1640 medium (100 μL) at different concentrations (0.8, 0.4, 0.2, 0.1, and 0.05 μM) were added to HepG2 cells on 96-well plates. After PDT, the cells were washed twice with PBS, and then Fluo-3 AM (Thermo Fisher Scientific Inc., F1242) in HBSS (2.5 μM, 100 μL) was added. The cells were incubated at 37 °C for 1 h in dark. After being washed twice with PBS, the cells were re-fed with HBSS, and the fluorescence signal was measured by a fluorescence plate reader using a 485 nm excitation filter and a 535 nm emission filter with a gain of 202 (Tecan Spark 10M Microplate Reader). Western Blotting Analysis. A solution of 10 in RPMI 1640 medium (150 nM) was added to HepG2 cells on dishes of 60 mm

halogen lamp after passing through a water tank for cooling and a color filter with cut-on wavelength at 610 nm (Newport). The absorption maximum of DPBF at 417 nm was monitored along with time. The ΦΔ values were calculated according to the equation ΦΔ(s) = ΦΔ(ref)(WsIabs(ref))/(WrefIabs(s)), where ΦΔ(ref) is the ΦΔ of ZnPc in DMF, Ws and Wref are the DPBF photobleaching rates in the presence of the photosensitizer and ZnPc, respectively, and Iabs(s) and Iabs(ref) are the rates of light absorption by the photosensitizer and ZnPc, respectively. TPA Cross Section Measurements. The TPEF method39 was applied to measure the TPA cross sections. The excitation source for two-photon excitation was a femtosecond optical parametric amplifier (Coherent OPerA Solo) pumped by an amplified Ti:sapphire system (Coherent Legend Elite system) and then detected with a spectrometer (Spex 500M) coupled to an CCD. All of the sensing experiments were carried out using freshly prepared solutions, and the concentration of samples in PBS was fixed at 20 μM. After the TPEF emission spectra of the sample and the TPA standard were captured, the TPEF and TPA cross sections were estimated using the following equations: σTPEF(s) = (Fs/Fref)(cref/cs) σTPEF(ref) and σTPA = σTPEF/ϕ, where σTPEF and σTPA are TPEF and TPA cross sections, respectively, subscript s refers to the sample solutions and ref stands for the reference, F refers to the integrated intensity of fluorescence spectra after being corrected with the instrumental spectral response function, and c stands for the molar concentration of fluorophores in the sample solutions. ϕ is one-photon excited fluorescence quantum efficiency, which is used here under the assumption that the fluorescence quantum efficiencies involved in one- and two-photon excited fluorescence are identical. Rhodamine B (40 μM) in methanol was used as the reference. Cell Lines and Culture Conditions. All cell culture reagents were purchased from Thermo Fisher Scientific Inc., Carlsbad, CA, USA, unless otherwise specified. HepG2 cells were maintained in Roswell Park Memorial Institute (RPMI) 1640 medium (with 10% fetal bovine serum, 100 unit mL−1 penicillin, and 100 μg mL−1 streptomycin) in a humidified incubator with 5% CO2 at 37 °C. HeLa cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (with 10% fetal bovine serum, 100 unit mL−1 penicillin, and 100 μg mL−1 streptomycin) in a humidified incubator with 5% CO2 at 37 °C. Study of One-Photon Photodynamic Activity. A stock solution of 10 or 11 (40 μM) was prepared by dissolving the compound (40 nmol) in DMF (25 μL), followed by the addition of Tween 80 (5 μL) and the culture medium (970 μL). The stock solution was diluted to various concentrations with the culture medium for various in vitro assays. Photodynamic treatment was carried out on cells seeded on 96well plates (2 × 105 cells mL−1) 24 h prior to the assays. The cells were incubated with the photosensitizers at different concentrations (100 μL) (0.4, 0.2, 0.1, 0.05, 0.025, and 0.0125 μM for 10; 8, 6, 4, 2, 1, and 0.5 μM for 11) in the dark for 2 h. After being washed twice with PBS and re-fed with the culture medium, the cells were illuminated with a halogen lamp (300 W) with a red glass filter (Newport, cut-on at λ = 610 nm) for 20 min at r.t. The fluence rate used was 40 mW cm−2, giving a total fluence of 48 J cm−2. For the light-dose study, HepG2 cells were incubated with 10 (0.19 μM) for 2 h. After being washed twice with PBS and re-fed with the culture medium, the cells were illuminated with the above light source for 10, 15, 20, or 25 min. A 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (3 mg mL−1 in PBS, 50 μL) was then added to the cells, which were incubated at 37 °C for 4 h followed by the addition of dimethyl sulfoxide (70 μL). Absorbance at 490 nm was measured using a plate reader (Tecan Spark 10 M Microplate Reader). Study of Two-Photon Photodynamic Activity. Approximately 1.2 × 105 HeLa cells in 2 mL of the culture medium were incubated on 35 cm2 glass-bottom confocal dishes overnight. The cells were treated with 10 in the medium containing 0.125% v/v DMF and 0.025% v/v Tween 80 (2 μM, 2 mL) for 2 h, and then were rinsed with PBS (1 mL × 2). The cells were re-fed with the culture medium and then illuminated with a 800 nm laser (400 mW) of a Zeiss laser scanning microscope (LSM 880 NLO) equipped with a humidified chamber with 5% CO2 at 37 °C for 10 min. The cells were kept in the 3959



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diameter. The cells were lysed using radioimmunoprecipitation assay buffer at 2, 4, 6, and 8 h after PDT. Equal amounts of proteins were electrophoresed on 10% polyacrylamide gels and transferred to a polyvinylidene fluoride membrane. After blocking with 5% nonfat milk for 1 h, primary antibodies (anti-Actin, 1:4000, Sigma-Aldrich, A2103; anti-CHOP, 1:2000, Abcam, ab11419; anti-GRP78, 1:400, Santa cruz, sc-13968) were bound for 1 h. After washing with Tris-buffered salineTween20 (TBS-T), membranes were probed with horseradish peroxidase (HRP)-conjugated anti-Mouse immunoglobulin G (IgG) (1:2000, Thermo Fisher Scientific Inc., 31430) or anti-Rabbit IgG (1:2000, Thermo Fisher Scientific Inc., G-21234) antibodies for 1 h. Luminata Crescendo Western HRP Substrate (Merck, WBLUR0500) was used as the substrate. Statistical Analysis. Data shown on figures were presented as the means with the SEM of three independent experiments. The data were analyzed using a student t-test with p values < 0.05 considered as significant; *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant. Statistical calculations were performed using a Microsoft Excel Spreadsheet (Microsoft Corporation, Redmond, WA, USA).

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performance liquid chromatography; HRP, horseradish peroxidase; IC50, dye concentration required to kill 50% of the cells; IgG, immunoglobulin G; MTT, 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide; NBS, N-bromosuccinimide; PBS, phosphate buffered saline; PDT, photodynamic therapy; r.t., room temperature; ROS, reactive oxygen species; RPMI, Roswell Park Memorial Institute; SEM, standard error of the mean; THF, tetrahydrofuran; TLC, thin layer chromatography; TPA, two-photon absorption; TPEF, two-photon excited fluorescence; ZnPc, unsubstituted zinc(II) phthalocyanine; ΦF, fluorescence quantum yield; ΦΔ, singlet oxygen quantum yield

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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/acs.jmedchem.7b01907. Molecular formula strings and the associated data (CSV) Photodegradation of DPBF by 10 and 11, spectral overlap between the light source and the absorption of 10 and 11, photostability of 10 and 11, light-dependent survival curve for 10 against HepG2 cells, subcellular localization of 5, 6, 10, and 11 in HeLa and HepG2 cells, two-photon fluorescence image of 10 in HeLa cells, HPLC chromatograms of 10 and 11, and 1H and 13 C{1H} NMR and ESI mass spectra of the new compounds (PDF)

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Corresponding Authors

*Phone: +852-3442-4493. Fax: +852-3442-0549. E-mail: gigi. [email protected]. *Phone: +852-3943-6375. Fax: +852-2603-5057. E-mail: [email protected]. ORCID

Yimin Zhou: 0000-0002-9683-5955 Dennis K. P. Ng: 0000-0001-9087-960X Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by a grant from the National Natural Science Foundation of China and the Research Grants Council (RGC) of the Hong Kong Special Administrative Region Joint Research Scheme (ref. No. N_CUHK443/12), and RGC Areas of Excellence Scheme (ref. No. AoE/P-02/12).

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ABBREVIATIONS USED BODIPY, boron dipyrromethene; DMAP, N,N-dimethylaminopyridine; DMEM, Dulbecco’s modified Eagle’s medium; DMF, N,N-dimethylformamide; DPBF, 1,3-diphenylisobenzofuran; EDCl, 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide; ER, endoplasmic reticulum; ESI, electrospray ionization; H2DCFDA, 2′,7′-dichlorodihydrofluorescein diacetate; HBSS, Hank’s balanced salt solution; HPLC, high3960



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Endoplasmic Reticulum-Localized Two-Photon-Absorbing Boron

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