Mitochondria-targeting turn-on fluorescent probe for HClO detection and imaging in living cells

SAA-117825; No of Pages 10 Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy xxx (xxxx) xxx

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Mitochondria-targeting turn-on fluorescent probe for HClO detection and imaging in living cells Qing-Ming Wang a,⁎, Lei Jin a, Zhe-Yu Shen a, Jia-Hao Xu a, Li-Qiang Sheng a, Hui Bai b,⁎ a b

School of Pharmacy, Yancheng Teachers' University, Yancheng, Jiangsu 224051, People's Republic of China Key Laboratory of Coal Science and Technology of Ministry of Education and Shanxi Province, Taiyuan University of Technology, Taiyuan 030024, Shanxi, China

a r t i c l e

i n f o

Article history: Received 11 June 2019 Received in revised form 13 September 2019 Accepted 18 November 2019 Available online xxxx Keywords: Coumarin-based Mitochondria-targeting DFT study Fluorescence probe of HClO/ClO− Recognize mechanism

a b s t r a c t Hypochlorous/hypochlorite (HClO/ClO−), one of the most important signal molecule, plays a crucial role in many cellular signaling pathways. It is reported that the HClO/ClO− level in mitochondria is important to maintain the normal mitochondrial function. Herein, we present two simple fluorescent probes BAC and mitochondriatargeting fluorescent probe TACB for the detection of ClO−. Probes BAC & TACB could be sensitively and selectivity detecting ClO− at the nanomolar levels with the detection limit of 1.64 × 10−9 M and 9.86 × 10−8 M, respectively. Additionally, probes BAC & TACB with the response unit of C\\O moiety could selectively detect ClO− over other various analytes such as anions, metal ions and •OH, 1O2, H2O2. The response time of probe TACB for ClO− (b20 s), implying that it could offer a real-time analytical assay of ClO−. Finally, probe BAC was used for monitoring the ClO− in HEK293T cells and probe TACB could be utilized to track the fluctuations of exogenous ClO− levels in the mitochondria of Hela cells. © 2019 Published by Elsevier B.V.

1. Introduction Hypochlorous/hypochlorite (HClO/ClO−), as one of the famous bactericide and oxidizing agent, plays a vital role in water disinfection [1,2]. Research showed that there are many HClO/ClO− in biologically which could be produced by the reaction of H2O2 and Cl−, with the enzyme myeloperoxidase (MPO) as the catalyst [3–5]. Although HClO/ClO− could shoot the harmful bacteria and pathogens and take as a microbicidal mediator in human immune defense process [6,7], excess of HClO/ClO− not only pollute the water, but also bring about oxidative stress tissue and along with a number of serious diseases [8–15]. Therefore, the design and development of the convenient and sensitive methods for the detection of HClO/ClO− is of vital significance. Traditionally, colorimetry, electrochemical assay, and gas chromatography are commonly used in detection of HClO/ClO− [16–18]. Fluorescence probe became a promising tool and attracted much more attention for the detection of HClO/ClO− because of its great temporal and spatial sampling capability as well as high sensitivity [19–23]. To date, many fluorescence probes have been designed for HClO/ClO− detection and imaging in living cells [24–33]. It is reported that the HClO/ ClO− level in the organelles is tightly related to the redox balance in ⁎ Corresponding authors. E-mail addresses: [email protected] (Q.-M. Wang), [email protected] (H. Bai).

mitochondria which is important for maintaining the normal mitochondrial function [34]. So the detection of HClO/ClO− in mitochondria is very necessary. However, research on the detection of intracellular HClO/ClO− in mitochondria/lipid droplets have been rarely reported [35–41]. Thus, it is still challenged to develop organelle-specific fluorescent probes to help understand the detailed network of HClO/ClO− biology at subcellular levels. Herein, compound 3-(2-bromoacetyl)-2H-chromen-2-one (BAC) with coumarin was synthesized and the C\\O moiety of coumarin lactone functioned as the receptor. The results showed that BAC could recognize ClO− selectively and sensitively with the mechanism was that ClO− oxidative cleavage C\\O. The mechanism was different from those references reported by Prof. Wang [42] (they reported coumarin-based probe could recognize ClO− by oxidizing C_O) and Prof. Karcz [43] (they reported three coumarin-based probes response to ClO− by a chlorination of the coumarin-based). To achieve the effect of target mitochondria, a classic subcellular locating group triphenylphosphine unit was introduced to BAC. Then probe (TACB) was synthesized. The selectivity, sensitivity and optical properties of TACB towards various anions, metal ions and oxidative species have been investigated. The results showed that TACB was selective and sensitive to ClO−, with obvious colorimetric and fluorescence responses. The Hela cell imaging experiment verified that TACB is suitable for fluorescence imaging of ClO− in mitochondria.

https://doi.org/10.1016/j.saa.2019.117825 1386-1425/© 2019 Published by Elsevier B.V.

Please cite this article as: Q.-M. Wang, L. Jin, Z.-Y. Shen, et al., Mitochondria-targeting turn-on fluorescent probe for HClO detection and imaging in living cells, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, https://doi.org/10.1016/j.saa.2019.117825

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2. Experimental

125.91 (t, J = 7.4 Hz), 125.52 (s), 121.81 (d, J = 10.6 Hz), 118.76–118.27 (m), 117.18–116.44 (m), and 56.08 (s).

2.1. Materials and instruments 3. Results and discussion All the reagents were purchased from Shanghai Experiment Reagent Co., Ltd. (Shanghai, China) and used without further purification. The anions came from their sodium salts or potassium salts. The cations came from their chloride or nitrate. All of the reagents were of analytical grade. 3-acetyl coumarin (ACO) [44] was synthesized following our early reported method with some small modify. Bruker DRX 400 spectrometer with TMS as the internal standard was applied for NMR spectra. NICOLET380 FT-IR spectrometer in KBr disks were used to obtain FTIR Spectra (4000–400 cm−1). VARI-EL Elemental Analyzer was applied to record Elemental Analyses (EAs). Triple TOF TM 5600+ system was used to measure High Resolution Mass Spectra (HR-MS). RF-5301 fluorescence spectrophotometer was applied to obtain Fluorescence Spectral data. UV-1800 ENG 240 V was used to perform Ultraviolet Spectrum. Confocal laser scanning microscope with model LSM-880 was used to measure Confocal Images. Cell imaging of TACB with ClO− in mitochondria was obtained on Nikon A1R confocal with MitoTracker® Red CMXRos as the fluorescence dye.

3.1. Synthesis and structural characterization of BAC & TACB In our recent study, a series of coumarin based fluorescence probes were reported [44,45]. The results showed that the coumarin lactone seem to be the ideal reaction site for ClO−. Moreover, triphenylphosphine is a target group for the mitochondrial compartment. So as showed in Scheme 1, firstly, 3-bromoacetylcoumarin (BAC) was obtained as white shiny needles in 48.2% yield after recrystallized in glacial acetic acid by the reaction of 3-acetyl coumarin (ACO) with bromine in CHCl3. Then 3-((tripheyl-phosphinyl)acetyl) coumarin bromide (TACB) was synthesized as a yellow crystalline solid at a 90% yield by the reaction of BAC with triphenylphosphine in CH2Cl2. Structures of BAC & TACB were confirmed by IR, 1H NMR, 13C NMR and HRMS, and showed in the Supporting Information (Figs. S1– S8). 3.2. Selectivity of BAC & TACB to anions

2.2. Synthesis of the BAC & TACB 2.2.1. Synthesis of BAC BAC was synthesized by the reported method [44–48]. 3-Acetyl coumarin (ACO) (10 mmol) was dissolved in alcohol-free chloroform (30 mL) and a solution of bromine (10 mmol) in alcohol free chloroform (5 mL) was added dropwise from an equilibrating funnel, with constant stirring at 0–5 °C. After 4 h, the reaction mixture was heated for 15 min and CHCl3 was removed by rotary evaporator. The solid obtained was washed by ether. Purification by recrystallization from glacial acetic acid gave 3-bromoacetylcoumarin as white shiny needles in good yields (Yield, 48.2%). FT-IR (KBr, cm−1): 3026 (Ar\\H), 2961 (C\\H), 1725 (C_O), 1616, 1564, 1449 (Ar, C_C), 1185, 1146 (C\\O\\C), C\\Br (541). Exp. 320.8684. Elemental analysis (calcd. %) for C11H7BrO3·0.5H2O: C, 47.85; H, 2.92; Found: C, 47.84; H, 3.38. Exact mass for BAC: 265.9579, HR-MS (positive mode) [BAC + Na+]+ (m/z, cal. 288.9476; Exp. 288.8521), [BAC + H+ + 3H2O]+ (m/z, Cal. 320.9974. 1H NMR (400 MHz, CDCl3) δ 8.65 (s, 1H), 7.71 (ddd, J = 7.4, 4.2, 1.3 Hz, 2H), 7.56 (s, 1H), 7.51–7.35 (m, 2H), 4.76 (s, 1H). 13C NMR (101 MHz, CDCl3) δ 188.89 (s), 159.10 (d, J = 14.3 Hz), 155.34 (s), 149.52 (s), 135.17 (s), 130.47 (s), 125.34 (s), 118.14 (s), 117.01 (s), and 35.71 (s). 2.2.2. Synthesis of TACB Following the reported method to prepare the TACB [45–47], 3((triphenylphosphinyl)acetyl)coumarin bromide (TACB) was synthesized from 3-(bromoacetyl)coumarin (2 mmol) in 5 mL of CH2Cl2 and triphenylphosphine (2 mmol). The mixture was stirred for 1.5 h at room temperature. The volatiles were removed under reducing pressure. The crude product was triturated with diethyl ether, filtered, washed again with diethyl ether and dried in vacuo to obtain a yellow crystalline solid (90% yield). FT-IR (KBr, cm−1): 3064 (Ar\\H), 2807 (C\\H), 1739 (C_O), 1609, 1558, 1487, 1429 (Ar, C_C), 1183, 1133 (C\\O\\C), C\\P (979). Elemental analysis (calcd. %) for C29H22O3PBr ({[TACB]+Br−}): C, 65.80; H, 4.19; Found: C, 65.80; H, 4.22. Exact mass for TACB: 449.1301, HR-MS (positive mode) [TACB]+ (m/z, cal. 449.0301; Exp. 448.9849). 1H NMR (400 MHz, DMSO) δ 9.46 (s, 1H), 8.64 (s, 1H), 7.98–7.90 (m, 2H), 7.76–7.63 (m, 6H), 7.54 (td, J = 7.6, 3.3 Hz, 3H), 7.41 (d, J = 3.9 Hz, 1H), 7.39 (d, J = 2.5 Hz, 1H), 7.36 (d, J = 3.4 Hz, 1H), 7.33 (s, 1H), 7.31 (d, J = 2.2 Hz, 1H), 7.29 (s, 1H), 7.27 (s, 1H), 6.46 (s, 1H), 6.43 (s, 1H). 13C NMR (101 MHz, DMSO) δ 190.44 (s), 188.69 (s), 187.75 (s), 159.46 (s), 159.06 (s), 155.57–155.17 (m), 154.80 (s), 151.05 (s), 150.32 (s), 150.09 (s), 147.80 (s), 136.48 (s), 136.05 (s), 132.10 (s), 131.80 (s), 131.60 (s),

Fluorescence spectral properties of BAC & TACB in the presence of − 2− different anions (such as ClO−, NO− 2 , H2PO4 , HPO4 ) were analyzed in DMF. As showed in Fig. 1A, for BAC, there was no obvious fluorescence emission signal was found when excited at 412 nm, which indicated that the coumarin lactone of BAC was in off state. A significant increase in fluorescence emission signal at 480 nm was observed when NaClO (100 μM) was in the presence of BAC in DMF. 100 μM of − 2− the other tested anions (such as ClO−, NO− 2 , H2PO4 , HPO4 ) exhibited nearly no change on the fluorescence emission signal at 480 nm. The fluorescence emission peak of TACB (5 μM) was hardly observed when excited at 418 nm. But the fluorescent intensity of TACB at 480 nm was significantly increased after the addition of 100 μM of NaClO (Fig. 1B). The same as BAC, the fluorescence emission signal of TACB almost didn't exhibit changes when other tested anions were added. It revealed that there were 12-fold and 39-fold increasing in fluorescence signal at 480 nm for BAC & TACB, respectively. These suggested that BAC & TACB could be served as a probe to detect ClO− through their significant fluorescence ‘off-on’ responses. And it is more likely to generate a new compound between BAC & TACB and ClO− due to ring opening from the coumarin lactone. There is nonfluorescent and colorless for coumarin derivatives generally. However, when the coumarin lactone is opened, strong fluorescence will be released and the color changes will be visible by the naked eye. The combination of a suitable group and open the coumarin lactone could lead to the change in the color of the naked eye and the production of the fluorescent signal. Jiuyan Li et al. in 2003 reported that coumarin connected with malononitrile could induce the red shift emission and a large stokes shift [47]. The excellent response time of BAC & TACB to ClO− was studied. From Fig. S9, fluorescence intensity at 480 nm was increased dramatically when 100 μM of ClO− was present and the fluorescence signals were able to reach a maximum value in about 20 s and kept this steady state for 5 min, it is suggested that the formation of new compounds between BAC & TACB and ClO− were achieved within 20 s. So 20 s was used for the fluorescence and UV–vis spectroscopic experiments. 3.3. UV–vis absorption spectra of BAC & TACB towards ClO− The UV–vis absorption spectra of BAC & TACB with various ClO− concentrations were investigated in DMF and showed in Fig. 2. With the gradually increasing the of ClO− from 0 to 100 μM at room temperature, absorption peaks of BAC & TACB at 400 nm were gradually increased, and the absorption band at 290–330 nm was gradually

Please cite this article as: Q.-M. Wang, L. Jin, Z.-Y. Shen, et al., Mitochondria-targeting turn-on fluorescent probe for HClO detection and imaging in living cells, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, https://doi.org/10.1016/j.saa.2019.117825

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Scheme 1. Design and synthesis of the BAC & TACB.

decreased. One well-defined isosbestic point was noted at 354 nm, indicating a new compound generated. Accordingly, the visible color changed from colorless to golden yellow, which could be respected by the naked eye. The absorbance peak at 354 nm was no longer changed when the concentration of ClO− increased to 100 μM, implicating that the binding interaction between BAC & TACB and ClO− was reached saturation. A linear relationship was carried out between the absorbance intensity and the concentration of ClO− with a correlation coefficient of R2 = 0.9883 and R2 = 0.9726 for BAC & TACB, respectively. (Fig. 2 inset). The above results indicated that BAC & TACB were potentially applicable for qualitative analysis rather than quantitative analysis of ClO−. 3.4. Fluorescent sensitivity of BAC & TACB to ClO− Fluorescent titration experiment was carried out to investigate the sensitivity of BAC & TACB to ClO−. As showed in Fig. 3A, the fluorescence intensity increasing gradually was achieved for BAC (5 μM) towards ClO− at 480 nm in DMF. With the increasing of ClO− (0–120 μM), the fluorescence intensity was gradually increased, and a blue- fluorescence appeared which could be seen visually under the UV 365 nm light (Fig. 3A inset). Similar to BAC, as showed in Fig. 3B, the fluorescence intensity of TACB (5 μM) at 480 nm (λex = 418 nm) increased with the addition of ClO− (0–120 μM) in DMF. A linear relationship was carried out between the fluorescence signal and concentration of ClO− (BAC, 0–100 μM; TACB, 0–85 μM) with a correlation coefficient of R2 = 0.994 and R2 = 0.992, respectively (Fig. S10 in Supplement File). The detection limits of BAC & TACB were found to be 1.64 × 10−9 M and 9.86 × 10−8 M, which were lower enough for detection of ClO− in real water samples. During the detection test, the results showed that BAC & TACB are potentially applied to detect ClO− quantitatively. Besides, there is also meant that BAC & TACB were highly sensitive probes for ClO−. To verify the color of the systems of BAC-ClO− & TACB-ClO−, Commission Internationale de L'Eclairage (CIE) chromaticity was used to calculate the emission spectrum [49]. Each point in the CIE chromaticity

diagrams on behalf of a certain color. The CIE chromaticity values of BAC-ClO− & TACB-ClO− were measured (0.1531, 0.3457) and (0.1599, 0.3461), respectively (showed in Figs. S11 & S12).

3.5. Effect of co-existing anions To confirm that BAC & TACB were highly selective to ClO−, it is necessary to conduct the competition experiments under the co-existence of ClO− with other anions. From Fig. 1, we were found that the other anions did have no influence on the measurement of the fluorescence signal of BAC & TACB towards ClO−. From Fig. 4, we could find that after adding ClO− into BAC & TACB in the presence of various anions under the same system, obvious fluorescence enhancement was found for BAC & TACB. It is gratifying to state that all the competitive anions have no interference towards ClO− measurement, implied that BAC & TACB could be used for detecting ClO− effectively in a competitive system.

3.6. Selectivity and competitive over metal ions Furthermore, many metal ions in biological and environment systems may also be interference or competitive with ClO−, so it is very essential to test the metal ions (such as Na+, Mg2+, Ni2+, Ba2+, Co2+, Fe3 + ) in DMF. As showed in Fig. S13, the tested metal ions should not cause the fluorescence enhancement of BAC & TACB (5.0 μM) in DMF. Only the introduction of ClO− into BAC & TACB (5.0 μM) solution could induce obvious fluorescence color change. The competition experiments with the co-existence metal ions and ClO− were tested (Fig. S14). Under the same experimental conditions, the results indicated that a significant fluorescence response was appeared after ClO− was added into BAC & TACB (5.0 μM) solution with the metal ions present. The results stated clearly that the competition metal ions have no interference on ClO− measurement, implying that BAC & TACB could be applied to measure ClO− effectively in a competitive environment.

Fig. 1. The fluorescence spectra of BAC & TACB at 480 nm in the present or absence of different anions (100 μM) in DMF. A) BAC (5.0 μM) (λex = 412 nm, λem = 480 nm slits: 3 nm/3 nm). B) TACB (5.0 μM) (λex = 418 nm, λem = 480 nm slits: 3 nm/5 nm). Inset: a color change photograph for ClO− and the other anions under illumination with a 365 nm UV lamp.

Please cite this article as: Q.-M. Wang, L. Jin, Z.-Y. Shen, et al., Mitochondria-targeting turn-on fluorescent probe for HClO detection and imaging in living cells, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, https://doi.org/10.1016/j.saa.2019.117825

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Fig. 2. Absorption titration spectrum of BAC & TACB (20 μM) at increasing concentrations of ClO− (0–100 μM) in DMF, inset: plot of absorption signal against the concentration of ClO− and color changes. A) BAC; B) TACB.

Fig. 3. A) Fluorescence titration of BAC (5.0 μM) in response to ClO− (0–120 μM) in DMF solution. (λex = 412 nm, slits: 3 nm/3 nm.) B) Fluorescence titration of TACB (5.0 μM) in response to ClO− (0–120 μM) in DMF. (λex = 418 nm, slits: 3 nm/5 nm.) Inset: A visual fluorescence color change photographs for ClO− under illumination with a 365 nm UV lamp.

3.7. Proposed mechanism of BAC & TACB response to ClO− Electrospray ionization mass spectrometry (ESI-MS) [50–52] was used to confirm the compounds formed between BAC & TACB and

ClO− in solution. There is a peak at m/z = 334.1663, corresponding to [BACA + CH3OH + H2O] (Cal. 334.1401), showed in Fig. 5. Followed, procedure Mass was used to practice the theoretical and experimental value, and the result revealed that the theoretical and experimental

Fig. 4. The influence of co-existing anions on BAC-ClO− or TACB-ClO− system. Fluorescent change of BAC or TACB (5 μM) towards ClO− (100 μM) over various competitive anions (100 μM) in DMF. A) for BAC, λex = 412 nm, B) for TACB, λex = 418 nm. (Digital 1–23 respectively represents different anions: 1), probe BAC or TACB; 2), Cl−; 3), Br−; 4), I−; 5), − − − − 3− − − − − 2− 2− 2− 4− 2− − − SO2− 4 ; 6), PO4 ; 7), H2PO4 ; 8), OH ; 9), HCO3 , 10), NO3 ; 11), NO2 ; 12), C2O4 ; 13), F ; 14), HPO4 ; 15), S2O3 ; 16), SCN ; 17), Ac ; 18), P2O7 ; 19), B4O7 ; 20), CO3 ; 21), HSO3 ; − 22), SO2− 3 ; and 23), ClO ).

Please cite this article as: Q.-M. Wang, L. Jin, Z.-Y. Shen, et al., Mitochondria-targeting turn-on fluorescent probe for HClO detection and imaging in living cells, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, https://doi.org/10.1016/j.saa.2019.117825

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Fig. 5. ESI mass spectra of BAC upon addition of excess ClO−.

Fig. 6. ESI mass spectra of TACB upon addition of excess ClO−.

values were agreed with each other (Fig. 5 inset). The ESI-MS result could also indicate that the oxidation reaction between the C\\O of coumarin lactone and ClO− to give a new compound BACA. As the same, in Fig. 6, a peak at m/z = 578.9752, corresponding to [TACBA2H + 2Na + CH3OH + 2H2O]+ (Cal. 579.0098). The theoretical and experimental values also agree with each other well (Fig. 6 inset). Besides, there are two peaks at 3427 cm−1 and 3466 cm−1 in FT-IR, which could

be assigned to –OH stretching vibration for BACA and TACBA, respectively (as showed in Figs. S15 & S16). Depending on the reported studies, a plausible recognition mechanism of BAC & TACB for ClO− was proposed in Scheme 2. In the absence of ClO−, the coumarin lactone of BAC & TACB was in the state of typical closed ring and exhibited nonfluorescent and colorless. While the ClO− was added to BAC & TACB, the coumarin lactone was cleaved to give the compound BACA &

Scheme 2. The proposed sensing mechanism of probe BAC & TACB for ClO−.

Please cite this article as: Q.-M. Wang, L. Jin, Z.-Y. Shen, et al., Mitochondria-targeting turn-on fluorescent probe for HClO detection and imaging in living cells, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, https://doi.org/10.1016/j.saa.2019.117825

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Fig. 7. HOMO and LUMO orbitals of BAC and BACA.

TCABA by the oxidation hydrolysis of ClO−, in this process, ClO− was restored to Cl−, the H2O was oxidized to H2O2. The fluorescence enhancement was appeared with coumarin lactone ring was opened. To confirm the proposed mechanism for BAC & TACB response to ClO−, further investigations were performed by DFT. The Gaussian 09 program were performed with B3LYP/6-31G(d) function to calculate the compounds BAC & TACB and the products between BAC & TACB and ClO−. As early reports [53,54], the highest occupied molecular orbitals (HOMO) and the lowest unoccupied molecular orbitals (LUMO) of BAC & TACB and the products between BAC & TACB and ClO− were calculated and were listed in Figs. 7 and 8. As showed in Figs. 7 and 8, the HOMO of BAC & TACB (the energy were −7.0011 eV and −9.0174 eV, respectively) were distributed over the whole molecules, while the LUMO were mainly delocalized on the coumarin lactone group. However, when BAC & TACB (the energy were −2.8176 eV and −5.2006 eV, respectively) were converted to products by the oxidation of ClO−, the HOMO of BACA was mainly delocalized on the whole molecules expect Br atom, while the LUMO was delocalized on the benzene part (the energy was changed from −6.4616 eV to −2.2680 eV), the HOMO of TACBA was mainly delocalized on the whole molecules expect triphenylphosphine group, while the LUMO was delocalized on the benzene part (the energy was changed from −8.5556 eV to −4.9111 eV). Besides, a intramolecular hydrogen bond between the carboxyl oxygen (donor) and benzene (C\\H, receptor) was formed in BACA & TACBA. The energy gap of BAC & TACB converted to BACA & TACBA by adding

ClO− were changed from 4.184 eV and 3.817 eV to 4.194 eV and 3.645 eV, respectively. The results were consistent with the early reports. 3.8. Competition experiment over ROS The sensing mechanism between TACB and ClO− was informed that the coumarin lactone was cleaved by the oxidation of ClO−. So there are also some other reactive oxygen species such as •OH, 1O2, H2O2 could be oxidation of coumarin lactone. From Fig. 9, 100 μM of the reactive oxygen species •OH, 1O2, and H2O2 showed no obvious change on fluorescence intensity in DMF. While the addition of ClO− (100 μM) to the solution of TACB with the ROS in present, we could find a significantly enhanced fluorescence at 480 nm. The results revealed that the reactive oxygen species •OH, 1O2, and H2O2 could not get involved with the detection of TACB towards ClO−. 3.9. Fluorescent imaging of BAC & TACB in living cell Recently, cell imaging is part of the most popular biological analysis. It is useful for tracking and analyzing biological molecules in living systems. Based on eminent properties of BAC & TACB in recognizing ClO−, the potential value of BAC & TACB towards ClO− in living cells were further measured by a fluorescence Inverted microscope. The MTT assay was used to verify whether BAC and TCAB were toxic to cells or not.

Please cite this article as: Q.-M. Wang, L. Jin, Z.-Y. Shen, et al., Mitochondria-targeting turn-on fluorescent probe for HClO detection and imaging in living cells, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, https://doi.org/10.1016/j.saa.2019.117825

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Fig. 8. HOMO and LUMO orbitals of TACB and TACBA.

As showed in Fig. S17, the cells were minimally damaged by BAC and TCAB. As presented, the HEK293T cells were first incubated with BAC or TACB (10 μM) for 30 min and no fluorescence signal were showed (showed in Figs. 10b & 11b). After treated with ClO− (40 μM) and incubated for another 30 min, the bright blue fluorescence images were observed for both BAC and TACB (10 μM) by the fluorescent microscope (showed in Figs. 10f & 11f). The quantification of fluorescence intensity was calculated from the fluorescence images are 24.37 and 18.54, respectively (Figs. 10d & 11d). Compared with the blanks, there are

about 14.8-fold and 7.88-fold enhancement for BAC and TACB, respectively. These suggested that BAC & TACB could be used for biological applications, which are able to serve as a potential probe for detecting ClO− in living cells. To confirm the expected effect of TACB on the recognition of ClO− in mitochondria, we made use of MitoTracker® Red CMXRos as a mitochondrial specific fluorescence dye to study the fluorescence colocalization of TACB. Hela cells were incubated with 500 nM of MitoTracker® Red CMXRos for 20 min at 37 °C, from Fig. 12b, a strong red fluorescence and clear mitochondrial outlines was observed clearly in red channel. Then 20 μM of TACB and 20 μM of ClO− were added and co-incubated for another 30 min at 37 °C, we could find a strong blue fluorescence, as showed in Fig. 12c. The results indicated that TACB could target mitochondrial for recognizing ClO− [51,55]. 3.10. Comparison with other probes As showed in Table 1, some different probes for ClO− were summarized. Compared to these methods, BAC & TACB in this paper had fast reaction time and lower LOD. Although two probes were reported with lower LOD, the reaction time was not considered [32,36]. Three methods had wide linear range, but the response time remains to be further improved [28,30,35]. It indicated that our proposed probe could be used as a rapid analytical method for detecting ClO−. 4. Conclusions

Fig. 9. Fluorescence response of TACB (5.0 μM) to NaClO (100 μM) in DMF with the competition analytes.

In conclusion, to recognize the signaling molecule ClO− in biological, we have rationally designed and synthesized two simple probes BAC & TACB. Two probes display excellent fluorescence “turn-on” effect by

Please cite this article as: Q.-M. Wang, L. Jin, Z.-Y. Shen, et al., Mitochondria-targeting turn-on fluorescent probe for HClO detection and imaging in living cells, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, https://doi.org/10.1016/j.saa.2019.117825

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Fig. 10. Fluorescence microscopy images of HEK293T cells. Images of HEK293T cells incubated with probe BAC (10 μM) for 30 min ((a) at bright-field; (b) at fluorescence field); images of HEK293T cells incubated with probe BAC (10 μM) for 30 min; then incubated with ClO− (40 μM) for another 40 min ((e) at bright-field, (f) at fluorescence field); Image J 3D surface plot analysis of the fluorescence images ((c) BAC; (g) BAC treated with ClO−); quantification of fluorescence intensity collected from the fluorescence images ((d) BAC; (h) BAC treated with ClO−).

Fig. 11. Fluorescence microscopy images of HEK293T cells. Images of HEK293T cells incubated with probe TACB (10 μM) for 30 min ((a) at bright-field; (b) at fluorescence field); Images of HEK293T cells incubated with probe TACB (10 μM) for 30 min; then incubated with ClO− (40 μM) for another 40 min ((e) at Bright-field, (f) at fluorescence field); Image J 3D surface plot analysis of the fluorescence images ((c) TACB; (g) TACB treated with ClO−); Quantification of fluorescence intensity collected from the fluorescence images ((d) TACB; (h) TACB treated with ClO−).

Fig. 12. Hela cells co-incubated with TACB (20 μM) and MitoTracker® Red CMXRos (500 nM) at 37 °C for 20 min followed by treatment with 20 μM ClO− for 30 min (a) bright field image; (B) red channel image (emission of MitoTracker® Red CMXRos); (c) blue channel (emission of TACB-ClO−); (d) superimposed images of (b) and (c).

Please cite this article as: Q.-M. Wang, L. Jin, Z.-Y. Shen, et al., Mitochondria-targeting turn-on fluorescent probe for HClO detection and imaging in living cells, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, https://doi.org/10.1016/j.saa.2019.117825

Q.-M. Wang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy xxx (xxxx) xxx

9

Table 1 Comparison of BAC & TACB with other reported probes. LOD/M

Linear range/μM

Response time/s

Locating group

Ref.

4.3 × 10−7 4.3 × 10−7 1.95 × 10−6 7.9 × 10−7 9 × 10−9 1.8 × 10−7 1.1 × 10−7 1.79 × 10−10 1.64 × 10−9 9.86 × 10−8

0–22 0–25 0–100 0–250 0–35 2.4 × 10−8 0–100 0–80 0–120 5–94

30 – 1 50 – 30 – – – 20

– Triphenylphosphine – Quaternized pyridine moiety Triphenylphosphine Quaternized pyridine moiety Triphenylphosphine Quaternized pyridine moiety – Triphenylphosphine

[25] [26] [28] [30] [32] [33] [34] [35] This work This work

ClO− promoted the coumarin lactone ring opening process. For the determination of ClO− in mitochondria, triphenylphosphine group was introduced to form probe TACB. TACB exhibits as low toxic and highly mitochondria-targetable fluorescence probe for exogenous and realtime imaging of mitochondrial ClO− by fluorescence microscopy. The results revealed that TACB offers the opportunity to monitor the level of endogenous or exogenous ClO− in mitochondria. Declaration of competing interest There are no conflicts to declare. Acknowledgements This work are financially supported by the Six Talent Peak Project in Jiangsu Province (SWYY-063) and sponsored by Qing Lan Project of Jiangsu Province. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.saa.2019.117825.

References [1] J.D. Lambeth, Nox enzymes, ROS, and chronic disease: an example of antagonistic pleiotropy, Free Radic. Biol. Med. 43 (2007) 332–337. [2] Y.N. Xiao, R. Zhang, Z.Q. Ye, Z.C. Dai, H.Y. An, J.L. Yuan, Lanthanide complex-based luminescent probes for highly sensitive time-gated luminescence detection of hypochlorous acid, Anal. Chem. 84 (2012) 10785–10792. [3] C.J. Hu, J.P. Li, L.Q. Yan, A fluorescent probe for hypochlorite with colorimetric and fluorometric characteristics and imaging in living cells, Anal. Chem. 566 (2019) 32–36. [4] B. D’Autréaux, M.B. Toledano, ROS as signalling molecules: mechanisms that generate specificity in ROS homeostasis, Nat. Rev. Mol. Cell Biol. 8 (2007) 813–824. [5] J.E. Harrison, J. Schultz, Studies on the chlorinating activity of myeloperoxidase, J. Biol. Chem. 251 (1976) 1371–1374. [6] M.T. Sun, H. Yu, H.J. Zhu, F. Ma, S. Zhang, D.J. Huang, S.H. Wang, Oxidative cleavagebased near-infrared fluorescent probe for hypochlorous acid detection and myeloperoxidase activity evaluation, Anal. Chem. 86 (2014) 671–677. [7] Z.M. Prokopowicz, F. Arce, R. Biedron, M. Ciszek, D.R. Katz, M. Nowakowska, S. Zapotoczny, J. Marcinkiewicz, B.M. Chain, Hypochlorous acid: a natural adjuvant that facilitates antigen processing, cross-priming, and the induction of adaptive immunity, J. Immunol. 184 (2010) 824–835. [8] E. Niedzielska, I. Smaga, M. Gawlik, A. Moniczewski, P. Stankowicz, J. Pera, M. Filip, Oxidative stress in neurodegenerative diseases, Mol. Neurobiol. 53 (2016) 4094–4125. [9] C.H. Sam, H.K. Lu, The role of hypochlorous acid as one of the reactive oxygen species in periodontal disease, J. Dent. Sci. 4 (2009) 45–54. [10] Y.W. Yap, M. Whiteman, N.S. Cheung, Chlorinative stress: an under appreciated mediator of neurodegeneration, Cell. Signal. 19 (2007) 219–228. [11] M. Valko, J. Moncol, M. Izakovic, M. Mazur, Free radicals, metals and antioxidants in oxidative stress-induced cancer, Chem. Biol. Interact. 160 (2006) 1–40. [12] F.J. Huo, J.J. Zhang, Y.T. Yang, J.B. Chao, C.X. Yin, Y.B. Zhang, A fluorescein-based highly specific colorimetric and fluorescent probe for hypochlorites in aqueous solution and its application in tap water, Sens. Actuators B-Chem. 166 (2012) 44–49. [13] H. Zhu, J.L. Fan, J.Y. Wang, H.Y. Mu, X.J. Peng, An enhanced PET-based fluorescent probe with ultrasensitivity for imaging basal and elesclomol-induced HClO in cancer cells, J. Am. Chem. Soc. 136 (2014) 12820–12823.

[14] Y. Yang, H. L, W. Wang, Selective and absolute quantification of endogenous hypochlorous acid with quantum-dot conjugated microbeads, Anal. Chem. 83 (2011) 8267–8272. [15] J.M. McCord, Free radicals and inflammation: protection of synovial fluid by superoxide dismutase, Science 185 (1974) 529–531. [16] J. Zhang, X. Yang, Colorimetric determination of hypochlorite with unmodified gold nanoparticles through the oxidation of a stabilizer thiol compound, Analyst 137 (2012) 2806–2818. [17] J.G. March, M. Gual, B.M. Simonet, Determination of residual chlorine in greywater using o-tolidine, Talanta 58 (2002) 995–1001. [18] A. Gallina, P. Pastore, F. Magno, The use of nitrite ion in the chromatographic determination of large amounts of hypochlorite ion and of traces of chlorite and chlorate ions, Analyst 124 (1999) 1439–1442. [19] B.C. Zhu, M. Zhang, L. Wu, Z.Y. Zhao, C. Liu, Z. Wang, Q. Duan, Y. Wang, P. Jia, A highly specific far-red fluorescent probe for imaging endogenous peroxynitrite in the mitochondria of living cells, Sens. Actuators B-Chem. 257 (2018) 436–441. [20] X. Jiao, Y. Li, J. Niu, X. Xie, X. Wang, B. Tang, Small-molecule fluorescent probes for imaging and detection of reactive oxygen nitrogen, and sulfur species in biological systems, Anal. Chem. 90 (2018) 533–555. [21] Z. Takats, K.J. Koch, R.G. Cooks, Organic chloramine analysis and free chlorine quantification by electrospray and atmospheric pressure chemical ionization tandem mass spectrometry, Anal. Chem. 73 (2001) 4522–4529. [22] D.T. Quang, Fluoro- and chromogenic chemodosimeters for heavy metal ion detection in solution and biospecimens, Chem. Rev. 110 (2010) 6280–6301. [23] X.J. Jiao, C. Liu, Q. Wang, K. Huang, S. He, L.C. Zhao, Fluorescence probe for hypochlorous acid in water and its applications for highly lysosome-targetable live cell imaging, Anal. Chim. Acta 969 (2017) 49–56. [24] Y. You, W. Nam, Designing photoluminescent molecular probes for singlet oxygen, hydroxyl radical, and iron-oxygen species, Chem. Sci. 5 (2014) 4123–4135. [25] J.J. Hu, M.Y. Lu, X. Chen, S. Ye, A.Q. Zhao, P. Gao, J. Shen, D. Yang, HKOCl-3: a fluorescent hypochlorous acid probe for live-cell and in vivo imaging and quantitative application in flow cytometry and a 96-well microplate assay, Chem. Sci. 7 (2016) 2094–2099. [26] J. Liu, H.X. Zhang, Y.Y. Huo, Y.W. Shi, W. Guo, Simultaneous fluorescent imaging of Cys/Hcy and GSH from different emission channels, Chem. Sci. 5 (2014) 3183–3188. [27] X.Z. Wang, L. Zhou, F. Qiang, F.Y. Wang, R. Wang, C.C. Zhao, Development of a BODIPY-based ratiometric fluorescent probe for hypochlorous acid and its application in living cells, Anal. Chim. Acta 911 (2016) 114–120. [28] H. Xiao, J. Zhao, G. Yin, Y. Quan, J. Wang, R. Wang, A colorimetric and ratiometric fluorescent probe for ClO− targeting on mitochondria and the application in vivo, J. Mater. Chem. B-Chem. 3 (2015) 1633–1638. [29] L. Yuan, Y. Xie, B. Chen, J. Song, Fluorescent detection of hypochlorous acid from turn-on to FRET-based ratiometry by a HClO-mediated cyclization reaction, Chem.-Eur. J. 18 (2012) 2700–2706. [30] J. Fan, H. Zhu, J. Du, N. Jiang, J. Wang, X. Peng, Recognition of HClO in live cells with separate signals using a ratiometric fluorescent sensor with fast response, Ind. Eng. Chem. Res. 54 (2015) 8842–8846. [31] Y. Koide, K. Hanaoka, T. Terai, T. Nagano, Development of an Si-Rhodamine-based far-red to near-infrared fluorescence probe selective for hypochlorous acid and its applications for biological imaging, J. Am. Chem. Soc. 133 (2011) 5680–5682. [32] D. Li, J. Lin, M. Chen, S. Wang, X. Wang, H. Sheng, Z. Shao, M. Zhu, X. Meng, A mitochondria-targeted two-photon fluorescent probe for highly selective and rapid detection of hypochlorite and its bio-imaging in living cells, Sens. Actuators B-Chem. 222 (2016) 483–491. [33] Y.H. Yan, H.L. Ma, J.Y. Miao, B.X. Zhao, Z.M. Lin, A ratiometric fluorescence probe based on a novel recognition mechanism for monitoring endogenous hypochlorite in living cells, Analytical Chimica Acta 1064 (2019) 87–93. [34] J. Hou, J. Yang, K. Yu, Y. Liao, Y. Ran, Y. Liu, X. Zhou, X. Yu, A ratiometric fluorescent probe for in situ quantification of basal mitochondrial hypochlorite in cancer cells, Chem. Commun. 51 (2015) 6781–6784. [35] J. Zhou, W. Shi, H. Ma, HClO can appear in the mitochondria of macrophages during bacterial infection as revealed by a sensitive mitochondrial-targeting fluorescent probe, Chem. Sci. 6 (2015) 4884–4888. [36] Y.H. Chen, Z.J. Zhang, W. Zhang, J. Lv, T.T. Chen, B. Chi, F. Wang, X.Q. Chen, A mitochondria-targeted fluorescent probe for ratiometric detection of hypochlorite in living cells, Chinese Chem Lett 28 (2017) 1957–1960. [37] J.T. Hou, K. Li, J. Yang, K.K. Yu, Y.M. Xie, X.Q. Yu, Mitochondria-targeted colorimetric and fluorescent probes for hypochlorite and their applications for in vivo imaging, Chem. Commun. 50 (2014) 8640–8643.

Please cite this article as: Q.-M. Wang, L. Jin, Z.-Y. Shen, et al., Mitochondria-targeting turn-on fluorescent probe for HClO detection and imaging in living cells, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, https://doi.org/10.1016/j.saa.2019.117825

10

Q.-M. Wang et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy xxx (xxxx) xxx

[38] H. Xiao, H. Dou, G. Yin, Y. Quan, R. Wang, A fast-responsive mitochondria-targeted fluorescent probe detecting endogenous hypochlorite in living RAW 264.7 cells and nude mouse, Chem. Commun. 51 (2015) 1442–1445. [39] S. Shen, X. Zhang, X. Liu, H. Wang, Y. Dai, J. Miao, B. Zhao, A mitochondria-targeted ratiometric fluorescent probe for hypochlorite and its applications in bioimaging, J. Mater. Chem. B 5 (2017) 289–295. [40] W. Wu, L. Xi, M. Huang, W. Zeng, J. Miao, B. Zhao, A mitochondria-targeted fluorescence probe for ratiometric detection of endogenous hypochlorite in the living cells, Anal. Chim. Acta 950 (2017) 178–183. [41] W.L. Wu, H.L. Ma, L.L. Xi, M.F. Huang, K.M. Wang, J.Y. Miao, B.X. Zhao, A novel lipid droplets-targeting ratiometric fluorescence probe for hypochlorous acid in living cells, Talanta 194 (2019) 308–313. [42] Y. Tang, Y.Y. Li, J. Han, Y.L. Mao, L. Ni, Y. Wang, A coumarin based fluorescent probe for rapidly distinguishing of hypochlorite and copper (II) ion in organisms, Spectrochim. Acta A 208 (2019) 299–308. [43] K. Starzak, A. Matwijczuk, B. Creaven, A. Matwijczuk, S. Wybraniec, D. Karcz, Fluorescence quenching-based mechanism for the determination of hypochlorite by coumarin-derived sensors, Int. J. Mol. Sci. 20 (2019) 281. [44] Q.M. Wang, L. Jin, W.L. Wang, L.H. Dai, X.X. Tan, C. Zhao, Two coumarin-based turnon fluorescent probes based on for hypochlorous acid detection and imaging in living cells, Spectrochim. Acta A 211 (2019) 239–245. [45] C. Pardin, W.D. Lubell, J.W. Keillor, Two coumarin-based turn-on fluorescent probes based on for hypochlorous acid detection and imaging in living cells, J. Org. Chem. 73 (2008) 5766–5775. [46] S. KhanYusufzai, M.S. Khan, S. Mohamad, Design, characterization, in vitro antibacterial, antitubercular evaluation and structure-activity relationships of new hydrazinyl thiazolyl coumarin derivatives, Med. Chem. Res. 26 (2017) 1139–1148. [47] C. Pardin, J.N. Pelletier, W.D. Lubell, J.W. Keillor, Cinnamoyl inhibitors of tissue transglutaminase, Org. Chem. 73 (15) (2008) 5766–5775.

[48] J.Y. Li, D. Liu, Z.R. Hong, S.W. Tong, P.F. Wang, C. Ma, O. Lengyel, C.S. Lee, H.L. Kwong, S. Lee, A new family of isophorone-based dopants for red organic electroluminescent devices, Chem. Mater. 15 (2003) 1486–1490. [49] L. Jin, X.X. Tan, C. Zhao, Q.M. Wang, Highly sensitive and selective of two coumarinbased fluorometric probes for detection of ClO− and cell imaging, Anal. Methods 11 (2019) 1916–1922. [50] M. Kumar, A. Kumar, M.S.H. Faizi, S. Kumar, M.K. Singh, S.K. Sahu, S. Kishor, R.P. John, A selective ‘turn-on’ fluorescent chemosensor for detection of Al3+ in aqueous medium: experimental and theoretical studies, Sensor Actuat B- Chem 260 (2018) 888–899. [51] L.J. Tang, D. Xu, M.Y. Tian, A mitochondria-targetable far-red emissive fluorescence probe for highly selective detection of cysteine with a large Stokes shift, J. Lumin. 208 (2019) 502–508. [52] S.V. Mulay, Y. Kim, M. Choi, D.Y. Lee, J. Choi, Y. Lee, S. Jon, D.G. Churchill, Enhanced doubly activated dual emission fluorescence probes for selective imaging of glutathione or cysteine in living systems, Anal. Chem. 90 (2018) 2648–2654. [53] Z.L. Wang, Y. Zhang, J. Song, Y.Q. Yang, X. Xu, M.X. Li, H.J. Xu, S.F. Wang, A novel isolongifolanone based fluorescent probe with super selectivity and sensitivity for hypochlorite and its application in bioimaging, Anal. Chim. Acta 1051 (2019) 169–178. [54] Y. Kim, S.V. Mulay, M. Choi, S.B. Yu, S. Jonc, D.G. Churchill, Exceptional time response, stability and selectivity in doubly-activated phenyl selenium-based glutathioneselective platform, Chem. Sci. 6 (2015) 5435–5439. [55] L.J.P. He, X.M. Yan, A mitochondria-targetable fluorescent probe for ratiometric detection of SO2 derivatives and its application in live cell imaging, Sensor Actuat BChem 247 (2017) 421–427.

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