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A New Fluorescent Probe Based on Azobenzene-Cyclopalladium for the Selective Imaging of Endogenous Carbon Monoxide under Hypoxia Conditions Yong Li, Xu Wang, Jie Yang, Xilei Xie, Mengmeng Li, Jinye Niu, Lili Tong, and Bo Tang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b03376 • Publication Date (Web): 17 Oct 2016 Downloaded from http://pubs.acs.org on October 17, 2016
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Analytical Chemistry
A New Fluorescent Probe Based on Azobenzene-Cyclopalladium for the Selective Imaging of Endogenous Carbon Monoxide under Hypoxia Conditions Yong Li,1 Xu Wang,1 Jie Yang, Xilei Xie, Mengmeng Li, Jinye Niu, Lili Tong, and Bo Tang* College of Chemistry, Chemical Engineering and Materials Science, Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in Universities of Shandong, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Shandong Provincial Key Laboratory of Clean Production of Fine Chemicals, Shandong Normal University, Jinan 250014, P. R. China ABSTRACT: Carbon monoxide (CO), a crucial gas message molecule, plays an important role in the regulation of physiological and pathological process. Hypoxia-induced CO is involved in modulating various cellular activities, including signal transduction, proliferation, and apoptosis. However, tracking CO fluctuation in the hypoxic cells is still a challenge due to lack of straight forward, visualized and non-invasive tools. In this work, based on metal palladiumcatalyzed reaction, we present the rational design, synthesis and biological utility of an azobenzene-cyclopalladiumbased fluorescent probe, ACP-2, for CO monitoring. ACP-2 exhibits capacity of detecting CO in aqueous buffer solution and live cells with high sensitivity and specificity. Utilizing ACP-2, we displayed a direct and visual evidence of endogenous CO up-regulation in live cells induced by hypoxia. Moreover, CO up-regulation during oxygen-glucose deprivation/reperfusion (OGD/R) was also imaged and certified by ACP-2.
Carbon monoxide (CO), mainly released through the catabolism of heme by heme oxygenase (HO-1) in biological systems, acts as a crucial gas message molecule in human body and attracts increasing attention for its important regulation effect on pathophysiological process.1-4 Abnormal metabolism of CO has been involved in numerous diseases, such as Alzheimer’s disease,5 hypertension,6 inflammation,7 and heart failure,8 etc. Therefore, in-depth understanding the biological and pathological behavior of CO in live system is very important. Due to the rapid and excessive using of blood supply during cancer cell growth, the oxygen concentration in portions of tumor regions is significantly lower than normal tissues, leading to a hypoxia atmosphere.9,10 Hypoxia could cause cell injury and inflammatory responses readily via the increase of intracellular reactive oxygen species (ROS) level.11 Generally, in response to hypoxia stress, HO-1 expression is highly stimulated to provide significant protection against hypoxia-induced cell damage.12 Especially, CO, one of the products of catabolism of heme by HO-1, suppresses hypoxiainduced ROS production through several signal pathways.13 Thereby, it is profound to deeply spy on the CO fluctuation in cancer cells undergoing hypoxia stress. Currently, the cellular CO production under hypoxia is assessed indirectly by evaluating the expression level of HO-1.14 Unfortunately, there is inevitable limitations exist in protein test, including extensive processing, sample destruction, and incapability of real-time detection. Although several traditional approaches, such as electrochemical analysis15 and colorimetric
detection,16 have been developed for CO-sensing, they are powerless in real-time and noninvasive detection. Taking the above situations into consideration, a new approach is desperately needed to monitor and track hypoxia-induced CO fluctuation inside live cells with a straight forward and visual mode. In recent decades, fluorescence imaging of live cells has gained much attention since it has shown outstanding properties in terms of direct visualization and satisfied temporal and spacial resolution.17-19 Fluorescence probes combined with laser scanning confocal microscope have been utilized to sense CO in cells.20-23 Although these probes are effective, some limitations also exist. The genetic encoding protein,20 possesses large molecule size, and needs extensive complex processing, resulting in potential interference to the structure or function of the protein to which it is fused.24 The organic probes21-23 show response time of 30-60 min, which is comparatively long. And most of all none of these probes are used to detect endogenous CO in live cells. Therefore, there’s still a large demand to fabricate new organic probes with improved properties including fast response, high specificity, and capability of endogenous CO detection. In the present work, we developed two new organic fluorescent probes, ACP-1 and ACP-2, which are specific for CO. The probes are constructed by combining a unique CO recognition group, azobenzenecyclopalladium, and boron-dipyrromethene (BODIPY) fluorophore. Among the two probes, ACP-2 showed much better sensitivity to CO and was selected to fluorescently monitor the fluctuation of CO in HepG2
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cells. The results showed that ACP-2 was an efficient molecular tool for endogenous CO detection. By using ACP-2, a direct and visualized evidence of apparent upregulation of intracellular CO level induced by hypoxia and oxygen-glucose deprivation/reperfusion (OGD/R) was obtained. This work not only provides a new probe for CO, but also presents information that may contribute to the intensive understanding of CO biofunction in the physiological and pathological systems, especially those in hypoxia environment.
EXPERIMENTAL SECTION Materials and Instruments. Unless otherwise stated, all reactions were performed under air, and all solvents and reagents obtained from commercial suppliers and used without further purification. DAPI was obtained from Yeasen Biotechnology Co. Ltd. (Shanghai, China). 3-(4, 5-dimethylthiazol-2-yl)-2, 5diphenyltetrazolium bromide (MTT) was purchased from Sigma Chemical Company. Dichlorotricarbonylruthenium (II) dimer (CORM-2) was bought from Sigma-Aldrich, dissolved in DMSO to release CO and used immediately. Zinc protoporphyrin IX (ZnPP) was purchased from Alfa Aesar Inc. The MGC AnaeroPouch, oxygen indicator, and anaerobic bag were purchased from Mitsubishi Gas Chemical Co. (Tokyo, Japan). HepG2 cells were purchased from Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Fluorescence spectra were scanned with a FLS-980 fluorescence spectrometer (Edinburgh Instruments Ltd., England). 1HNMR and 13CNMR spectra were taken on a 400 MHz spectrometer (Bruker Co., Ltd., Germany); δ values are in ppm relative to TMS. HRMS spectra were obtained on a maxis ultra-high resolution-TOF MS system (Bruker Co., Ltd., Germany). The fluorescence images of cells were gained on a TCS SP5 Confocal Laser Scanning Microscope (Leica Co., Ltd., Germany). MTT assay was performed using a TRITURUS microplate reader. Hypoxic condition was made using a hypoxia MCO-5M incubator (SANYO, Japan). Synthesis of Compound 1. To a solution of 4nitrobenzoly choride (140 mg, 0.76 mmol) in 5 mL CH2Cl2 was added 2, 4-dimethyl pyrrole (140 mg, 1.47 mmol). The mixture was heated at 40 oC under the flow of Ar gas for 1 h, and then cooled to room temperature. After that, triethylamine (0.35 mL) and toluene (3 mL) were added to reaction mixture. After 15 min, BF3.Et2O (0.3 mL) was added to the solution. Then the reaction was heated at 50 oC under Ar atmosphere for 3 h. The mixture was concentrated under vacuum, and the residue was purified by silica chromatography eluted with CH2Cl2/petroleum (5:1, v/v) to afford 1 as an orange solid with 30% yield. 1HNMR (400 MHz, CDCl3): δ 8.39 (d, J=8 Hz, 2H), 7.54 (d, J = 8 Hz, 2H), 6.02 (s, 2H), 2.57 (s, 6H), 1.36 (s, 6H). 13CNMR (100 MHz, CDCl3) δ 154.95, 147.08, 143.23, 142.70, 132.05, 128.95, 124.64, 120.96, 115.43, 14.67, 14.57. ESI-MS: calculated for [MH]- =368.1, found 368.1. Synthesis of Compound 2. To a solution of compound 1 (100 mg, 0.27 mmol) in 4 mL CH2Cl2 was added ammonium formate (500 mg, 0.008 mol) and Pd/C (20 mg). The mixture was stirred at room temperature for 2 h and then filtered. The filtrate was
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concentrated under vacuum and purified by silica chromatography eluted with CH2Cl2/petroleum (2:1, v/v) to afford 2 as an orange solid with 90% yield. 1HNMR (400 MHz, CDCl3): δ 7.00 (d, J= 8 Hz, 2H), 6.77 (d, J = 8 Hz, 2H), 5.96 (s, 2H), 3.84 (s, 2H), 2.54 (s, 6H), 1.49 (s, 6H). 13CNMR (100 MHz, CDCl3) δ 156.68, 148.35, 142.55, 141.94, 138.36, 130.64, 129.66, 124.37, 121.86, 14.69, 14.66. ESI-MS: calculated for [M+H]+ =340.2, found 340.2. Synthesis of Compound 3a. To a mixture of compound 2 (120 mg, 0.35 mmol) and NaNO2 (100 mg, 1.45 mmol) in 10 mL DMF was added 5 mL concentrated HCl. The reaction mixture was stirred at 0-5oC for 3 h. Then the mixture was slowly dropwised into 20 mL NaOH aqueous solution (10 %) containing phenol (100 mg, 1.06 mmol) and was stirred for another 4 h at 0-5 oC. After that, the reaction mixture was filtered, and the residue was purified by silica chromatography eluted with CH2Cl2/petroleum (1:1, v/v) to afford compound 3a as an orange solid with 10% yield. 1HNMR (400 MHz, DMSO-d6): δ 10.42 (s, 1H), 7.98 (d, J= 8 Hz, 2H), 7.86 (d, J= 8 Hz, 2H), 7.58 (d, J= 8 Hz, 2H), 6.98 (d, J=8 Hz, 2H), 6.22 (s, 2H), 2.47 (s, 6H)1.43 (s, 6H) 13CNMR (100 MHz, DMSO-d6): δ 161.85, 155.61, 152.81, 145.72, 143.15, 141.53, 136.32, 130.99, 129.64, 125.58, 123.37, 122.00, 116.52, 14.72, 14.67. ESI-MS: calculated for [M-H]=443.2, found 443.2. Synthesis of Compound 3b. 3b was synthesized according to the procedure for 3a using 3,5dimethylphenol (100 mg, 0.82 mmol) instead of phenol. 3b was afforded as an orange solid with 40% yield. 1HNMR (400 MHz, DMSO-d6): δ 10.07 (s, 1H), 7.95 (d, J= 8 Hz, 2H), 7.56 (d, J= 8 Hz, 2H), 6.63 (s, 2H), 6.21 (s, 2H), 2.47 (s, 12H), 1.44 (s, 6H). 13CNMR (100 MHz, DMSO-d6): δ 160.33, 155.56, 153.60, 143.17, 142.08, 141.70, 136.74, 135.79, 131.03, 129.56, 122.95, 121.96, 116.91, 21.18, 14.71, 14.41. ESI-MS: calculated for [M-H]=471.2, found 471.2. Synthesis of ACP-1. A mixture of compound 3a (100 mg, 0.21 mmol) and PdCl2 (35 mg, 0.20 mmol) in 5 mL CH3OH was stirred at room temperature overnight. The solution was filtered, washed with CH3OH for three times to afford an orange red solid wth 50% yield. 1HNMR (400 MHz, DMSO-d6): δ 7.99 (d, J= 8 Hz, 1H), 7.86 (d, J= 8 Hz, 2H), 7.65 (s, 1H), 7.23 (d, J= 8 Hz, 1H), 6.81 (d, J= 8 Hz, 2H), 6.21 (s, 2H), 2.46 (s, 6H), 1.49 (s, 6H). 13CNMR (100 MHz, DMSO-d6): δ 164.34, 161.59, 156.85, 155.62, 144.28, 143.16, 141.48, 135.46, 131.00, 130.10, 128.50, 126.81, 124.82, 122.00, 115.43, 14.80, 14.40. Synthesis of ACP-2. ACP-2 was synthesized according to the procedure for ACP-1 using compound 3b (100 mg, 0.21 mmol) instead of 3a. ACP-2 was afforded as an orange red solid with 60% yield. 1HNMR (400 MHz, DMSO-d6): δ 9.75 (s, 1H), 8.12 (d, J= 8 Hz, 1H), 7.56 (s, 1H), 7.35 (d, J= 8 Hz, 1H), 6.60 (s, 2H), 6.23 (s, 2H), 2.47 (s, 6H), 2.20 (s, 6H), 1.50 (s, 6H). 13CNMR (100 MHz, DMSO-d6): δ 164.33, 157.83, 155.77, 154.25, 143.01, 142.45, 141.77, 137.08, 135.09, 132.65, 130.45, 129.51, 125.83, 122.09, 114.83, 18.30, 14.78, 14.74. Confocal Imaging under Normoxia and Hypoxia Conditions. HepG2 cells were replanted on the glass-bottom dishes in high-glucose DMEM
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Analytical Chemistry
supplemented with 10% fetal bovine serum, 1% penicillin, and 1% streptomycin. Cultures were maintained in normoxia incubator (37°C, 5% CO2/95% air) one day before cell imaging. As to the hypoxia condition, the replanted cells were cultured in a hypoxia incubator (37 °C, 5%CO2/1%O2/94%N2) for 6h and 12h, respectively, before imaging. The cultured cells were washed with PBS for three times, and then co-incubated with ACP-2 and DAPI for 15 min in low-glucose DMEM. Then the medium was removed and the cells were washed with PBS for three times. The cells were excited using a 488 nm laser and the emission was collected between 500 and 550 nm. DAPI was excited at 405 nm, and the emission was collected between 430 and 480 nm. Confocal Imaging during OGD/R. HepG2 cells were washed with PBS for three times and then the culture medium was replaced with glucose-free DMEM, which had been pregassed with 5%CO2/95%N2 for 10 min to remove residual oxygen. The dishes were placed in an airtight AnaeroPouch® bag, which provides an O2 concentration
