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Visible Light-Controlled Nitric Oxide Release from Hindered Nitrobenzene Derivatives for Specific Modulation of Mitochondrial Dynamics Kai Kitamura, Mitsuyasu Kawaguchi, Naoya Ieda, Naoki Miyata, and Hidehiko Nakagawa ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.5b00962 • Publication Date (Web): 16 Feb 2016 Downloaded from http://pubs.acs.org on February 19, 2016

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ACS Chemical Biology

Visible Light-Controlled Nitric Oxide Release from Hindered Nitrobenzene Derivatives for Specific Modulation of Mitochondrial Dynamics Kai Kitamura, Mitsuyasu Kawaguchi, Naoya Ieda, Naoki Miyata, and Hidehiko Nakagawa∗ Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya, Aichi 467-8603, Japan KEYWORDS: Nitric Oxide, Caged Compound, Visible Light-control, Mitochondrial Fragmentation, Drp1. Abstract Nitric oxide (NO) is a physiological signaling molecule, whose biological production is precisely regulated at the subcellular level. Here, we describe the design, synthesis and evaluation of novel mitochondria-targeted NO releasers, Rol-DNB-mor and Rol-DNB-pyr, that are photocontrollable not only in the UV wavelength range but also in the biologically favorable visible wavelength range (530–590 nm). These caged NO compounds consist of a hindered nitrobenzene as the NO-releasing moiety and a rhodamine chromophore. Their NO-release properties were characterized by an electron spin resonance (ESR) spin trapping method and fluorometric analysis using NO probes, and their mitochondrial localization in live cells was confirmed by co-staining. Furthermore, we demonstrated visible light-control of mitochondrial fragmentation via activation of dynamin-related protein 1 (Drp1) by means of precisely controlled NO delivery into mitochondria of cultured HEK293 cells, utilizing Rol-DNB-pyr.

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INTRODUCTION Nitric oxide (NO) was characterized as an endothelium-derived relaxing factor (EDFR) in blood vessels in 1987,1 and has since been shown to have various bioactivities, including neuronal effects,2,3 biophylaxis,4,5 and regulation of gene expression.6,7 Dysfunction of NO dynamics is involved in diseases such as cancer,8 Alzheimer's disease9 and schizophrenia.10 Recent studies suggested that microenvironmental modulation by endogenous NO plays important roles in physiological processes. For example, the effect of NO in cancer seems to depend on the concentration and duration of NO exposure, as well as the localization of nitric oxide synthase (NOS) isoforms,11,12 and it is suggested that NOS in mitochondria is involved in mitochondrial biogenesis and morphological regulation.13,14 Because NO is a highly reactive gas under ambient conditions, biological research requires the use of donor molecules to generate NO in situ, and many NO donors have been reported.15 Among them, photo-controllable NO donors,16 or caged NOs, are of particular interest, because they enable precise spatiotemporal control of NO delivery. We have previously reported some 2,6-dimethylnitrobenzene (DNB) based caged NOs,17-20 because of its stability, which permits precise on/off switching of NO release without spontaneous NO release or reaction with biomolecules. The NO-releasing reaction of DNB is expected to proceed as shown in Figure 1: the twisted nitro group is isomerized to phenyl nitrite via a roaming transition state, followed by homolytic dissociation to afford NO and a sterically stabilized phenoxyl radical, resembling vitamin E radical.17-21 We had developed a mitochondria-targeting NO donor, RpNO which localized to mitochondria due to its cationic rhodamine moiety and release NO in response to UV-light.19 Since then, we have synthesized and evaluated some NO donors tethered to rhodamine chromophores. Among them, we discovered Rol-DNB-mor (1) and Rol-DNB-pyr (2) which could release NO with not only UV light but also non-cytotoxic green-yellow light. Furthermore, we confirmed its suitability for precisely controlled NO delivery to manipulate mitochondrial dynamics. RESULTS AND DISCUSSION Design and synthesized of a novel light-controllable NO donor Our design of novel caged NOs, Rol-DNB-mor and Rol-DNB-pyr, is shown in Figure 2. A hindered nitrobenzene, 2,6-dimethylnitrobenzene (DNB) was directly tethered to a rhodamine chromophores in expectation of some kind of interactions between the DNB moiety and rhodamine moiety. These compounds were synthesized as shown in Scheme 1. Briefly, terminal alkyne was introduced to nitrobenzene derivative 319 by Sonogashira-Hagihara coupling and deprotection of TMS to afford ethynyl nitrobenzene 5, which was converted to boronate ester compound 6 by hydroboration using Schwartz’s reagent.22 Suzuki-Miyaura coupling of prepared compounds 6 and 723 gave compound 8 in good yield. Acetyl groups of 8 were then converted to triflyl groups, and Buchwald-Hartwig catalytic amination of triflyl compound 9 afforded rhodamine compound, Rol-DNB-mor and Rol-DNB-pyr, in accordance with the method of rhodamine synthesis reported by


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Lavis et al.24 Compounds having substructures of Rol-DNB-mor and Rol-DNB-pyr, rhodamines S1, S2 and stilbene S3, were also synthesized by Lavis’ method or a method shown in supplementary information (shown in Figure S1). The ultraviolet-visible absorption and fluorescence spectra of Rol-DNB-mor and Rol-DNB-pyr were measured and compared with those of the substructural components (Figure S2). Rol-DNB-mor and Rol-DNB-pyr showed two absorption maxima at around 300 nm and 550 nm; the shorter-wavelength one was assigned to stilbene structure and the other to rhodamine. The molar extinction coefficient (ε) around 500–600 nm was larger for Rol-DNB-pyr than for Rol-DNB-mor. This difference seems to depend on the open/closed equilibrium state of xanthene structure. Morpholine, contained in Rol-DNB-mor, is a more electron-deficient amine than pyrrolidine in Rol-DNB-pyr due to its oxygen atom, which favors formation of the 5-membered lactone (closed form). This idea is supported by a measurement of pH-dependent absorption spectra changes of NO-releasers i.e., pH-dependent absorbance increment at acidic conditions (pH 2~3) was observed in Rol-DNB-mor, in contrast, not in Rol-DNB-pyr (Figure S3).

Detection of photoinduced NO release To examine photoinduced NO release from Rol-DNB-mor and Rol-DNB-pyr, an ESR spin-trapping method with ferrous N-methylglucamine dithiocarbamate complex (Fe-MGD2)25 was adopted first. Fe-MGD2 traps NO to form a stable NO-Fe-MGD2 complex, which exhibits a typical triplet signal at around 330 mT in 1 GHz ESR spectroscopy (Figure 3). The typical triplet signal was observed after UV irradiation (325–385 nm) to a solution of Rol-DNB-pyr or Rol-DNB-mor containing Fe-MGD2 complex (Figure 3A, B). Interestingly, after green-yellow (530–590 nm) light irradiation of a solution of Rol-DNB-pyr or Rol-DNB-mor, the triplet signal was also observed, while no ESR signal was observed in the absence of the compounds (Figure 3C–E). These results suggested that Rol-DNB-mor and Rol-DNB-pyr release NO radical upon exposure to green-yellow light (530–590 nm). In the case of RpNO, a previously reported DNB-type NO donor tethered to rhodamine chromophore, the signal was not observed after visible light irradiation (data not shown). Next, to examine the importance of each substructure of Rol-DNB-mor and Rol-DNB-pyr in more detail, we performed fluorometric analysis of NO production using DAF-FM,26 a green fluorescence probe for NO (Figure S4). A time-dependent increase of green fluorescence (ex. 500 nm/ em. 515 nm) was observed in 7 µM DAF-FM solution containing Rol-DNB-mor or Rol-DNB-pyr, while only a weak increase or no increase of the fluorescence was observed in solutions containing combinations of the substructure compounds, rhodamine S1 and stilbene S3, or rhodamine S2 and stilbene S3, respectively. These results confirmed the importance of both directly linked moieties, namely, it is indispensable for visible light-induced NO release that the NO-releasing nitrobenzene and the photoabsorbing rhodamine are combined in a single molecule. We also confirmed that Rol-DNB-mor and Rol-DNB-pyr both released NO in a photoirradiation time-dependent manner,



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and that 10 µM of Rol-DNB-pyr released 0.91 µM of NO based on a fluorescence standard curve obtained with DAF-FM and 1-hydroxy-2-oxo-3-(N-methyl-3-aminopropyl)-3-methyl-1-triazene (NOC-7).27 Furthermore, the quantum yield of photodecomposition, ΦPD, of Rol-DNB-pyr was determined with a potassium ferrioxalate actinometer as described previously.28 The value of ΦPD was found to be 0.0023 ± 0.0005 at 550 nm. The ESR signal and fluorescence increment of Rol-DNB-pyr was stronger than those of Rol-DNB-mor, and their NO releasing capability apparently seemed to depend on their absorption coefficient around 530–590 nm. These results implied that the energy absorbed by the rhodamine moiety was utilized for isomerization and homolysis reaction of the nitrobenzene moiety via some kind of relaxation processes. Site-specific NO release control in cell. The novel visible-light controllable NO releasers in hand, we next applied them to live cells. Intracellular localization of these compounds was investigated by co-staining with MitoTracker Green FM, a selective fluorescent stain for mitochondria. As shown in Figure S5, red fluorescence of Rol-DNB-mor (Figure S5A) and Rol-DNB-pyr (Figure S5B) coincided with green fluorescence of MitoTracker in living HCT116 cells (human colon carcinoma cell line). This highly matched co-localization was confirmed by calculation of Manders' overlap coefficients,29,30 which were 0.956 for Rol-DNB-mor and 0.956 for Rol-DNB-pyr. Thus, these compounds are highly accumulated in mitochondria in live cells. Next, we employed confocal microscopy to evaluate the controllability of NO delivery at the cellular level by measuring intracellular NO release with DAF-FM DA,26 a cell-permeable green fluorescence probe for NO (Figure 4). We used a 562 nm diode laser to irradiate living HCT116 cells treated with the NO releasers and DAF-FM DA within a specified area (circle) and for the indicated time. In the cells treated with Rol-DNB-mor (Figure 4A) or Rol-DNB-pyr (Figure 4B), green fluorescence derived from the probe was specifically increased in the irradiated region, while the no increase was observed in the absence of the NO releasers. Rol-DNB-pyr seemed to generate a fluorescence increase more quickly (within 30 seconds) than Rol-DNB-mor, which is consistent with the results of the in vitro fluorometric analysis (Figure S4). This result may reflect the difference of molar extinction coefficient (ε) between Rol-DNB-mor and Rol-DNB-pyr. In summary, by using the NO releasers, we were able to achieve precisely controlled NO delivery at the subcellular organelle level via focused stimulation with a 562 nm diode laser, which is a relatively conventional and non-cytotoxic light source. Specific modulation of mitochondrial dynamics. Finally, we tried to regulate mitochondrial dynamics via precisely controlled NO delivery to mitochondria; in other words, biomimetic production of mitochondrial NO by visible light (Figure 5). Mitochondria are known to fuse and divide constantly, and this property appears to be important for


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their functional regulation.31 Dynamin-related protein 1 (Drp1) is a GTPase that regulates mitochondrial division. It is suggested to be activated by S-nitrosylation, an NO-dependent posttranslational modification, and appears to be involved in neuronal disorders.8,32,33 However, this remains controversial, because Bossy-Wetzel et al. indicated that S-nitrosylation of Drp1 did not affect the enzymatic activity, but activation of Drp1 in cells occurred via other mechanisms.34 Thus, the mechanism of NO-mediated mitochondrial fragmentation is unclear. In order to examine NO-mediated fragmentation of mitochondria, we applied Rol-DNB-pyr, a more efficient NO releaser, to HEK293 (human embryonic kidney 293) cells, whose mitochondria were made visible by transfection of a baculovirus vector expressing emGFP fused to the leader sequence of E1 alpha pyruvate dehydrogenase (CellLight Mitochondria-GFP, BacMam 2.0). The cells were exposed to green-yellow (530–590 nm) light for 3 minutes, and the dynamic changes of the mitochondria were chased by confocal fluorescence microscopy. The mitochondria were apparently fragmented upon visible light exposure (Figure 5A, D), and Mdivi-1,35,36 a Drp1-selective inhibitor, suppressed the fragmentation (Figure 5B, D). This result indicated that the division might be induced via Drp1 activation by mitochondrial NO production from Rol-DNB-pyr. The mitochondria of HEK293 cells treated with the rhodamine S2 were never shed (Figure 5C, D). The above interpretation was supported by calculation of the aspect ratio (major axis/minor axis), the shape factor most commonly used for evaluation of mitochondrial morphology (Figure 5D). Mitochondrial fragmentation was induced within one hour after uncaging of NO, in accordance with previous reports.8,32,33 Interestingly, treatment with only 1 µM Rol-DNB-pyr and brief visible light irradiation (3 min) sufficiently induced mitochondrial fragmentation, whereas over 50 µM S-nitrosocysteine (SNOC), a spontaneous NO donor with a half-life of 0.52±0.07 hour in cell-culture medium,37 was required in previous work.8,32,33 The difference might be at least partly due to differences of NO concentration in the mitochondrial microenvironment. Thus, two explanations can be considered: one is that mitochondrial NO production by Rol-DNB-pyr efficiently S-nitrosylates Drp1, leading to mitochondrial shedding, and the other is that not S-nitrosylation of the protein, but some other mechanism related to production of NO radical in mitochondria might be deeply involved in mitochondrial fragmentation via unrevealed signaling. Meanwhile, NO radical production at mitochondria also affected cell morphology (Figure 5A), and this change was not suppressed by Mdivi-1 treatment (Figure 5B). This observation suggests that the change of cell morphology occurred independently of mitochondrial fragmentation in this case. These findings suggest that Rol-DNB-pyr, a green-yellow (530–590 nm) light-controllable mitochondria-targeted NO releaser, is an effective tool for visible light-manipulation of mitochondrial dynamics via strictly localized NO radical production at mitochondria. This probe should be useful to resolve conflicting data on the role of NO in mitochondrial dynamics. In conclusion, we have synthesized two caged NOs, Rol-DNB-mor and Rol-DNB-pyr, which are mitochondria-targeted and controllable with green-yellow (530–590 nm) light, e.g., from a 562 nm diode laser. These caged NO compounds consist of a hindered nitrobenzene as the



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NO-releasing moiety and a rhodamine chromophore. In most cases, Rol-DNB-pyr exhibited more efficient NO releasing capability in response to visible light than Rol-DNB-mor. Although we have no conclusive evidence about the mechanism of NO release by our NO-releasers, Rol-DNB-mor and Rol-DNB-pyr, yet, we propose two hypotheses based on our present results. The one is that the head-to-tail stacking complex of two or more NO releaser molecules are formed in aqueous buffer. Therefore, when the rhodamine fluorophore is excited with visible light, the interacting DNB moiety is partially excited through the stacking interaction. A homolytic dissociation and NO release occur at the excited DNB moiety. This hypothesis is partially supported by our data shown in Figure S4, i.e., small amount of NO was detected from just mixed solution of rhodamine S2 and stilbene S3 by photoirradiation. The other is that after rhodamine fluorophore is excited with visible light, the excited energy of rhodamine is converted to vibration energy of the DNB moiety through non-irradiative relaxation process. A homolytic dissociation and NO release occur at the non-excited but high vibration level of the DNB moiety. It may be possible because fluorescence quantum yields of NO releasers are very low, therefore, non-irradiative relaxation is dominant. The isomerization and homolytic dissociation is considered to be possible according to the calculation reported previously.38 We are now conducting experiments to examine the above hypotheses.

Mitochondrial localization of Rol-DNB-mor and Rol-DNB-pyr was confirmed by a co-staining study with MitoTracker. The rhodamine moiety was found to be very effective for the targeting to mitochondria in our case. We also observed mitochondrial fragmentation via activation of dynamin-related protein 1 (Drp1) in response to precisely controlled NO delivery into mitochondria of cultured HEK293 cells, utilizing Rol-DNB-pyr. These results indicated that the specific treatment with NO by the compounds in combination with visible-light irradiation efficiently induced biological actions at relatively small amount. In other words, our compounds enabled fine control of NO delivery to mitochondria for precise regulation of biological actions. In addition, these compounds might be candidates for novel photodynamic therapeutic agents, and should also be useful tools for biomedical research into the mitochondrial functions of NO.

MATERIALS AND METHODS General information Melting point was determined using a Yanaco micromelting point apparatus or a Büchi 545 melting point apparatus. Proton nuclear magnetic resonance spectra (1H-NMR) and carbon nuclear magnetic resonance spectra (13C-NMR) were recorded on a Varian VNMRS 500 spectrometer in the indicated solvent. Chemical shifts (δ) are reported in parts per million relative to the internal standard, tetramethylsilane (TMS). Elemental analysis was performed with a Yanaco CHN CORDER NT-5 analyzer, and all values were within ±0.4% of the calculated values. Electrospray ionization (ESI) mass spectra were recorded on an ion trap mass spectrometer (Quattro Premier XE, Waters Co.) equipped with a


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nanospray ion source. Ultraviolet-visible-light absorption spectra were recorded on an Agilent 8453 spectrophotometer. ESR spectra were taken on a JES-RE2X spectrometer (JEOL Co. Ltd.). MGD (N-(dithiocarbamoyl)-N-methyl-D-glucamine, sodium salt) was obtained from Dojindo Laboratories. All other reagents and solvents were purchased from Sigma-Aldrich, Tokyo Chemical Industry Co., Ltd. (TCI), Wako Pure Chemical Industries, Nacalai Tesque, Kanto Kagaku, or Kishida Kagaku, and used without purification. Flash column chromatography was performed using silica gel 60 (particle size 0.032–0.075) supplied by Taikoh-shoji. Synthesis of 1,3-dimethyl-2-nitro-5-[2-(trimethylsilyl)ethenyl]benzene (4) Pd(PPh3)4 (169 mg, 0.146 mmol, 0.01 equiv.) and CuI (68 mg, 0.36 mmol, 0.02 equiv.) were placed in a dried flask and the flask was flushed with nitrogen. A solution of 3 (3.98 g, 14.4 mmol) and 2.10 mL (1.61 g, 15.9 mmol, 1.1 equiv.) of Et3N in 15 mL of anhydrous THF was added, and the flask was cooled to 0 °C. To the cooled and stirred mixture was slowly added TMS acetylene (2.25 mL, 1.56 g, 15.9 mmol, 1.1 equiv.) in 5 mL of anhydrous THF. The reaction mixture was stirred at room temperature for 23 hours, and then filtered through Celite. The Celite pad was washed with THF. The filtrate was mixed with silica gel (about 10 g) and concentrated in vacuo to dryness. Purification of the residue by silica gel flash column chromatography (dry load with silica gel, toluene/n-hexane = 1/3 to 1/2) afforded 3.11 g (12.6 mmol, 87%) of 4 as pale yellow crystals: 1H-NMR (500 MHz, CDCl3, δ; ppm) 7.23 (2H, s), 2.28 (6H, s), 0.25 (9H, s). Synthesis of 1,3-dimethyl-5-ethynyl-2-nitrobenzene (5) To a suspension of K2CO3 (7.15 g, 51.7 mmol, 4.1 equiv.) in 30 mL of MeOH was added a solution of 4 (3.10 g, 12.5 mmol) in 15 mL of THF. The mixture was stirred at room temperature for 1.5 hours, and then concentrated in vacuo. The residue was poured into water and extracted with CHCl3. The combined organic layer was washed with brine and dried over Na2SO4. Filtration, evaporation and purification of the residue by silica gel flash column chromatography (CHCl3/n-hexane = 1/2) afforded 2.06 g (11.8 mmol, 94%) of 5 as a slightly yellow powder: 1H-NMR (500 MHz, CDCl3, δ; ppm) 7.25 (2H, s), 3.14 (1H, s), 2.29 (6H, s). Synthesis of 2-[(E)-2-(3,5-dimethyl-4-nitrophenyl)ethenyl]-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (6) To a solution of 5 (1.38 g, 7.89 mmol) in 30 mL of anhydrous CH2Cl2 was added pinacol borane (5.00 mL, 4.41 g, 34.5 mmol, 4.4 equiv.) under nitrogen gas. The mixture was stirred at room temperature for 30 minutes and transferred to another nitrogen-flushed flask containing Cp2ZrHCl (2.03 g, 7.87 mmol, 1.0 equiv.) at 0 °C. The first flask was washed with 20 mL of anhydrous CH2Cl2 and the washing was also transferred to the above flask at 0 °C. The reaction mixture was stirred at room temperature for 16 hours, then poured into water, and extracted with CHCl3. The combined organic layer was washed with brine and dried over Na2SO4. Filtration, evaporation and purification of the residue by silica gel flash column chromatography (toluene only) afforded 871 mg (2.87 mmol, 36%) of 6 as orange crystals: 1H-NMR (500 MHz, CDCl3, δ; ppm) 7.29 (1H, d, J = 18.0 Hz), 7.21 (2H, s), 6.18 (1H, d, J = 18.0 Hz), 2.31 (6H, s), 1.31 (12H, s).



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Synthesis of 6’-acetyloxy-5-[(E)-2-(3,5-dimethyl-4-nitrophenyl)ethenyl]-3-oxo-3Hspiro[2-benzofuran-1,9’-xanthene]-3’-yl acetate (8) Compound 6 (440 mg, 1.45 mmol), 7 (876 mg, 1.60 mmol, 1.1 equiv.), cesium fluoride (888 mg, 5.85 mmol, 4.0 equiv.) and Pd(PPh3)4 (180 mg, 0.156 mmol, 0.11 equiv.) were placed in a dried flask, and the flask was flushed with argon. To the flask was added 8 mL of 1,2-dimethoxymethane (DME). The reaction mixture was refluxed for 16 hours and then poured into water, and extracted with AcOEt. The combined AcOEt layer was washed with brine and dried over Na2SO4. Filtration, evaporation and purification by silica gel flash column chromatography (AcOEt/n-hexane = 1/2) gave 778 mg (1.32 mmol, 91%) of 8 as a yellow solid: 1

H-NMR (500 MHz, CDCl3, δ; ppm) 8.15 (1H, s), 7.79 (1H, dd, J = 6.7 Hz), 7.30 (2H, s), 7.22 (1H, d, J = 16.5 Hz), 7.19 (1H, d, J = 7.9 Hz), 7.18 (1H, d, J = 16.4 Hz), 7.11 (2H, d, J = 2.2 Hz), 6.87 (2H, d, J = 8.7 Hz), 6.83 (2H, dd, J1 = 8.7 Hz, J2 = 2.2 Hz), 2.37 (6H, s), 2.32 (6H, s). Synthesis of 5-[(E)-(3,5-dimethyl-4-nitrophenyl)ethenyl]-3-oxo-6’-(trifluoromethane)sulfonyloxy-3H-spiro[2 -benzofuran-1,9’-xanthene]-3’-yl trifluoromethanesulfonate (9) To a solution of 8 (305 mg, 0.516 mmol), in THF/MeOH (1/1, 10 mL) was added 1.17 mL (1.17 mmol, 2.3 equiv.) of 1 N aqueous NaOH. The reaction mixture was stirred at room temperature for 1 hour and then concentrated in vacuo to provide a red solid. The solid was suspended in 7 mL of anhydrous CH2Cl2 and to the suspension was added 640 µL (602 mg, 7.61 mmol, 15 equiv.) of anhydrous pyridine. The mixture was cooled to 0 °C on ice, and 640 µL (1.07 g, 3.80 mmol, 7.4 equiv.) of trifluoroacetic anhydride was added dropwise to it. The ice-water bath was removed, and the reaction mixture was stirred at room temperature for 4 hours, then poured into water, and extracted with CH2Cl2. The combined organic extracts were washed with brine and dried over Na2SO4. Filtration, evaporation and purification by silica gel flash column chromatography (load with toluene, AcOEt/n-hexane = 1/5) gave 170 mg (0.221 mmol, 44%) of 9 as a white solid: 1H-NMR (500 MHz, CDCl3, δ; ppm) 8.18 (1H, s), 7.83 (1H, dd, J1 = 8.1 Hz, J2 = 1.6 Hz), 7.31 (2H, d, J = 2.3 Hz), 7.30 (2H, s), 7.24 (1H, d, J = 16.5 Hz), 7.19 (1H, d, J = 16.3 Hz), 7.19 (1H, d, J = 8.0 Hz), 7.05 (2H, dd, J1 = 8.9 Hz, J2 = 2.4 Hz), 7.01 (2H, J = 8.8 Hz), 2.37 (6H, s). Synthesis of 4-(9-{2-carboxylato-4-[(E)-2-(3,5-dimethyl-4-nitrophenyl)ethenyl]phenyl}-6-(morpholin-4-yl)-3 H-xanthen-3-ylidene)-1,4-morpholin-4-ylium (Rol-DNB-mor (1)) In a dried flask were placed Pd(OAc)2 (10 mg, 0.0440 mmol, 0.20 equiv.), 2,2'-bis(diphenyl phosphino)-1,1'-binaphthyl ((±)-BINAP) (42 mg, 0.055 mmol, 0.32 equiv.) and Cs2CO3 (222 mg, 0.681 mmol, 3.2 equiv.), and the flask was flushed with argon. To the flask was added a solution of 9 (166 mg, 0.216 mmol) and morpholine (46 µL, 46 mg, 0.53 mmol, 2.5 equiv.) in 4.0 mL of anhydrous toluene. The reaction mixture was stirred at 100 °C for 22 hours, then cooled to room temperature, and diluted with MeOH. Silica gel (about 3 g) was added and the mixture was concentrated to dryness in vacuo. Purification by silica gel flash column chromatography (dry load with silica gel, 0–7 % MeOH in CHCl3, linear gradient) afforded 81 mg (0.125 mmol, 58%) of a crude pink solid, which was purified by


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recrystallization from CHCl3/n-hexane to give 50 mg (0.0771 mmol, 36%) of Rol-DNB-mor (1) as a pink powder: mp > 300 °C; 1H-NMR (500 MHz, CDCl3, δ; ppm) 8.13 (1H, s), 7.76 (1H, d, J = 7.0 Hz), 7.29 (2H, s), 7.2–7.3 (1H, m), 7.1–7.2 (2H, m), 6.65-6.75 (4H, m), 6.60 (2H, dd, J1 = 8.9 Hz, J2 = 2.3 Hz), 3.85 (8H, t, J = 4,8 Hz), 3.21 (8H, t, J = 7.8 Hz), 2.37 (6H, s); 13C-NMR (125 MHz, CDCl3, δ; ppm) 169.32, 152.99, 152.62, 151.19, 138.57, 138.26, 133.35, 130.43, 129.21, 129.18, 128.81, 127.97, 127.07, 125.85, 124.33, 122.31, 111.41, 109.56, 101.79, 83.90, 66.67, 48.33, 17.75; MS (ESI) m/z 646 (MH+), 668 (MNa+); Anal. Calcd. for C38H35N3O7+H2O: C, 68.77; H, 5.62; N, 6.33. Found: C, 69.01; H, 5.45; N, 6.12. Synthesis of 4-(9-{2-carboxylato-4-[(E)-2-(3,5-dimethyl-4-nitrophenyl)ethenyl]phenyl}-6-(pyrrolidine-4-yl)-3 H-xanthen-3-ylidene)-1,4-pyrrolidin-4-ylium (Rol-DNB-pyr (2)) In a dried flask were placed Pd(OAc)2 (9 mg, 0.04 mmol, 0.2 equiv.), (±)-2,2'-bis(diphenylphosphino)-1,1'-binaphthyl (37 mg, 0.059 mmol, 0.34 equiv.) and Cs2CO3 (179 mg, 0.549 mmol, 3.2 equiv.), and the flask was flushed with nitrogen. To the flask was added a solution of 9 (134 mg, 0.174 mmol) and pyrrolidine (40 µL, 35 mg, 0.48 mmol, 2.8 equiv.) in 4 mL of anhydrous toluene. The reaction mixture was stirred at 100 °C for 19.5 hours, then cooled to room temperature, and diluted with MeOH. Silica gel (about 4 g) was added and the mixture was concentrated to dryness in vacuo. Purification by silica gel flash column chromatography (dry load with silica gel, 0–15% MeOH in CHCl3, linear gradient) afforded 45 mg (0.073 mmol, 42%) of a crude purple solid, which was purified by recrystallization from CHCl3/n-hexane to give 23 mg (0.037 mmol, 22%) of Rol-DNB-pyr (2) as a purple powder: mp > 230 °C (unmeasuralble due to gradual decomposition); 1H-NMR (600 MHz, CDCl3, δ; ppm) 8.16 (1H, s), 7.73 (1H, d, J = 7.8 Hz), 7.29 (2H, s), 7.23 (1H, d, J = 16.2 Hz), 7.1–7.2 (2H, m), 6.69 (1H, d, J = 7.2 Hz), 6.37 (2H, s), 6.30 (2H, d, J = 7.8 Hz), 3.32 (8H, m), 2.37 (6H, s), 2.02 (8H, m); 13

C-NMR (150 MHz, CDCl3, δ; ppm) 169.60, 153.16, 151.06, 149.63, 138.42, 138.13, 132.88, 130.39, 129.48, 128.97, 128.77, 127.02, 127.96, 124.63, 122.43, 122.30, 108.57, 106.09, 97.78, 70.58, 47.67, 25.47, 17.77; MS (ESI) m/z 614 (MH+), 636 (MNa+); Anal. Calcd. for C38H35N3O5+5/2H2O: C, 69.28; H, 6.12; N, 6.38. Found: C, 69.11; H, 5.81; N, 6.36. ESR Analysis Using Iron Dithiocarbamate Complex The Fe2+ complex of N-(dithiocarbamoyl)-N-methyl-D-glucamine (MGD) [Fe2+-MGD2, (Fe-MGD2)] was used to trap NO. Fresh stock solution of 3 mM Fe-MGD2 (Fe2+:MGD = 1:4) complex was prepared by adding 2.5 µL of 60 mM aqueous ferrous sulfate, 5 µL of 240 mM aqueous MGD and 2.5 µL of 200 mM potassium phosphate buffer (pH 7.4) to 140 mL of Milli-Q water. To the solution of Fe-MGD2 (1:4) was added 50 µL of a DMSO solution of 4 mM Rol-DNB-mor (1) or 4 mM Rol-DNB-pyr (2) to prepare a sample solution (total volume 200 µL, 1 mM compound, 1.5 mM Fe-MGD2 (Fe2+:MGD = 1:4), 2.5 mM potassium phosphate buffer (pH 7.4), 25% DMSO), which was subsequently introduced into a flat quartz cuvette. ESR spectra were recorded with a JES-RE 2X spectrometer (JEOL Co. Ltd.) after photoirradiation at 300–350 nm (15 min, 25 mW cm-2 at 330 nm) or 530–590 nm (15 min, 80 mW cm-2 at 560 nm). Photoirradiation was performed with a MAX-302 light source (300 W, Xe lamp,



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Asahi Spectra). Spectrometer settings were modulation frequency, 100 kHz; modulation amplitude, 1.25 G; scan time, 4 min; microwave power, 10 mW; and microwave frequency, 9.42 GHz. Detection of cellular NO with DAF-FM DA Human colon carcinoma (HCT116) cells were placed on 3.5 cm glass-bottomed dishes (Poly-L-Lysine coated, Matsunami Glass Ind., Ltd.) at 1×105 cells/dish with 2 mL of McCoy’s 5A culture medium containing penicillin and streptomycin supplemented with fetal bovine serum, according to the manufacturer’s instructions. The cells were incubated at 37 °C in a humidified 5% (v/v) CO2 incubator for a day, and the medium was replaced with 2 mL of McCoy’s 5A culture medium containing penicillin, streptomycin and 1 mM NG-nitro-L-arginine (L-NNA). The cells were then treated with or without 10 µM Rol-DNB-mor (1) or Rol-DNB-pyr (2) and 10 µM DAF-FM DA (1% DMSO) for an hour in a humidified 5% (v/v) CO2 incubator. The medium was replaced with 2 mL of Dulbecco’s phosphate-buffered saline (D-PBS) containing CaCl2 and MgCl2, and then the cells were subjected to confocal fluorescence microscopy (A1RMP, Nikon Instech Co., Ltd.) with a 562 nm diode laser. Photoirradiation was performed by the 562 nm diode laser only within the indicated irradiation circle for the indicated time. F. Visible Light-manipulation of Mitochondrial Fragmentation Human embryonic kidney 293 (HEK293) cells were placed on 3.5 cm glass-bottomed dishes (Poly-L-Lysine coated, Matsunami Glass Ind., Ltd.) at 1×104 cells/dish with 1 mL of Dulbecco's modified Eagle’s medium (DMEM) containing penicillin and streptomycin supplemented with fetal bovine serum, according to the manufacturer’s instructions. The cells were treated with 30 particles per cell of CellLight® Mitochondria-GFP, BacMam 2.0 (LifeTechnologies Corporation) and incubated at 37 °C in a humidified 5% (v/v) CO2 incubator for more than 46 hours. The medium was replaced with the DMEM containing 1 µM Rol-DNB-pyr with or without 50 µM Mdivi-1, a cell-permeable selective inhibitor of mitochondrial division dynamin-related GTPase (DRP1) (1% DMSO), and incubation was continued for 1 hour in a humidified 5% (v/v) CO2 incubator. The medium was replaced with 2 mL of Hank's Balanced Salt Solution (HBSS) containing calcium, magnesium, but no phenol red. The cells were subjected to confocal fluorescence microscopy (LSM510META with a 63x oil-immersion objective lens, Carl Zeiss Japan Co. Ltd.) within 30 minutes. Photoirradiation was performed on the stage of the microscope by using a MAX-302 light source (300 W, Xe lamp, Asahi Spectra) with a 530–590 nm bandpass filter at the indicated light intensity for 3 minutes. Aspect ratio was calculated by using ImageJ. Associated Content Supporting Information Supporting Information Available: This material is available free of charge via the Internet. DOI: 10.1021/acschem-bio. Additional Figures S1−S5, Scheme S1, synthetic details, chemical and cellular methods (Word file).


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Author Information Corresponding author *E-mail: [email protected] Notes The authors declare no competing financial interest.

Acknowledgements This work was supported by the JST PRESTO program (H.N.) from Japan Science and Technology Agency, as well as by a JSPS KAKENHI Grant Number 25293028 (H.N.) and Hoansha Foundation (H.N). References 1. Ignarro, L. J., Buga, G. M., Wood, K. S., Byrns, R. E., and Chaudhuri, G. (1987) Endothelium-derived relaxing factor produced and released from artery and vein is nitric oxide, Proc. Natl. Acad. Sci. U.S.A. 84, 9265–9269 2. Bredt, D. S. and Snyder, S. H. (1994) Transient nitric oxide synthase neurons in embryonic cerebral cortical plate, sensory ganglia, and olfactory epithelium, Neuron 13, 301–313. 3. Bohme, G. A., Bon, C., Lemaire, M., Reibaud, M., Piot, O., Stutzmann, J. -M., Doble, A., and Blanchar, J. -C. (1993) Altered synaptic plasticity and memory formation in nitric oxide synthase inhibitor-treated rats, Proc. Natl. Acad. Sci. U.S.A. 90, 9191–9194. 4. Laubach, V. E., Shesely, E. G., Smithies, O., and Sherman, P. A. (1995) Mice lacking inducible nitric oxide synthase are not resistant to lipopolysaccharide-induced death, Proc. Natl. Acad. Sci. U.S.A. 92, 10688–10692. 5. Bogdan, C. (2001) Nitric oxide and the immune response, Nat. Immunol. 2, 907–916. 6. Nott, A., Watson, P. M., Robinson, J. D., Crepaldi, L., and Riccio, A. (2008) S-Nitrosylation of histone deacetylase 2 induces chromatin remodelling in neurons, Nature 455, 411–415. 7.

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Figure and Scheme

Figure 1 Plausible mechanism of NO release from the hindered nitrobenzene

Figure 2 Molecular design of novel caged NO releasers. Rol-DNB-mor (1) and Rol-DNB-pyr (2), which are visible light-controllable and mitochondria-targeted.


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Figure 3 ESR spin-trapping detection of NO radical released from Rol-DNB-mor or Rol-DNB-pyr; ESR spectrum of a 1.5 mM Fe-MGD2 solution in 2.5 mM potassium phosphate buffer (pH 7.3, 25% DMSO) containing (A, C) 1 mM Rol-DNB-mor, (B, D) 1 mM Rol-DNB-pyr or (E) none (control) were recorded after irradiation with UV light for (A) and (B) (300 W Xe lamp, 300–350 nm, 25 mW cm-2) or green-yellow light for (C), (D) and (E) (300 W Xe lamp, 530–590 nm, 80–100 mW cm-2 at 560 nm) for 15 min.



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Figure 4 Fluorescence images before and after focused 562 nm diode laser irradiation; HCT116 cells were treated with (A) Rol-DNB-pyr or (B) Rol-DNB-mor or (C) none (blank), and 7 µM DAF-FM DA (1% DMSO) for an hour in a humidified 5% (v/v) CO2 incubator. The cells were subjected to confocal fluorescence microscopy (A1RMP, Nikon Instech Co., Ltd.) with a 562 nm diode laser. Photoirradiation was performed within the indicated irradiation circle for the indicated time (under each image).


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Figure 5 Mitochondria-visualized HEK293 cells were treated with (A) 1 µM Rol-DNB-pyr, (B) 1 µM Rol-DNB-pyr and 50 µM Mdivi-1, or (C) rhodamine S2 and then subjected to confocal fluorescence microscopy. Green: Mitochondria-GFP, Red: Rol-DNB-pyr. (D) Statistical evaluation of mitochondrial morphology in terms of aspect ratio. Photoirradiation was performed at 530–590 nm (300 W Xe lamp, 10–40 mW cm-2) for 3 minute. Aspect ratio (major axis/minor axis) was calculated by using ImageJ. Error bars represent S.E.M., **P < 0.01, *P < 0.05, ns means no significant difference (Welch's t test, n > 8).



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Scheme 1 Synthesis of Rol-DNB-mor and Rol-DNB-pyr

Reagents and conditions: (a) TMS-acetylene, Pd(PPh3)4, CuI, Et3N, THF, 0 °C to r.t., 87%; (b) K2CO3, MeOH, THF, q.y.; (c) Cp2ZrHCl, CH2Cl2, 0 °C to r.t., 36%; (d) 7, Pd(PPh3)4, CsF, DME, reflux, 91%; (e) (1) 1M NaOH aq., MeOH/THF (1:1), (2) Tf2O, pyridine, CH2Cl2, 0 °C to r.t., 44%; (f) morpholine or pyrrolidine, Pd(OAc)2, BINAP, Cs2CO3, toluene, 100 °C (1: 36%, 2: 22%). TMS = trimethylsilyl, THF = tetrahydrofuran, Cp = cyclopentadienyl, Ac = acetyl, DME = 1,2-dimethoxyethane, Tf = triflyl, BINAP = (±)-2,2'-bis(diphenylphosphino)-1,1'-binaphthyl, aq. = aqueous solution, q. y. = quantitative yield, r.t. = room temperature.


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Graphical Abstract 508x381mm (72 x 72 DPI)


Visible Light-Controlled Nitric Oxide Release from ... - ACS Publications

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