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Mitochondrial Specific H2Sn Fluorogenic Probe for Live Cell Imaging by Rational Utilization of A Dual-Functional-Photocage Group Linqi Han, Riri Shi, Chenqi Xin, Qiaoqiao Ci, Jingyan Ge, Jinhua Liu, Qiong Wu, Cheng-wu Zhang, Lin Li, and Wei Huang ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.8b00456 • Publication Date (Web): 27 Aug 2018 Downloaded from http://pubs.acs.org on August 27, 2018
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Mitochondrial Specific H2Sn Fluorogenic Probe for Live Cell Imaging by Rational Utilization of A Dual-Functional-Photocage Group Linqi Han†#, Riri Shi†#, Chenqi Xin†, Qiaoqiao Ci†, Jingyan Ge‡*, Jinhua Liu†, Qiong Wu†, Chengwu Zhang†*, Lin Li†* and Wei Huang†, § †
Key Laboratory of Flexible Electronics (KLOFE) & Institute of Advanced Materials (IAM), Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), Nanjing Tech University (NanjingTech), 30 South Puzhu Road, Nanjing, 211816, P. R. China ‡
Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Chaowang Road 18, Hangzhou 310014, P. R. China §
Shaanxi Institute of Flexible Electronics (SIFE), Northwestern Polytechnical University, 127 West Youyi Road, Xi'an 710072, P. R. China Supporting Information Placeholder ABSTRACT: Reactive sulfur species play a very important role in modulating neural signal transmission. Hydrogen polysulfides (H2Sn, n > 1) are recently suggested to be the actual signaling molecules. There are still few spatiotemporal controllable-based probes to detect H2Sn. In this work, for the first time, we proposed the photocleavage product of the common photoremovable protecting group (2-nitrophenyl moiety) capable of trapping H2Sn. Taking this advantage, we constructed the probe H1 containing a photocontrollable group, a mitochondrial directing unit and a signal reporter fluorescein dye. H1 exhibited excellent fluorescence enhancement (50 folds) in response to H2Sn under the aqueous buffer only after UV irradiation. H1 was also shown high selectivity and sensitivity for H2Sn over other reactive sulfur species, reactive oxygen species and other analytes, especially biothoils including hydrogen sulfide, cysteine, homocysteine and glutathione. We showed the utility of H1 to image H2Sn in living cells with high spatiotemporal resolution. Keywords: Hydrogen polysulfide, Fluorogenic probe, Photocontrollable group, light control, Mitochondria, Live cell imaging
Reactive sulfur species (RSS) play a vital role in physiological and pathological processes ranging from modulation of cardiovascular to CNS functions. [1] Among them, hydrogen sulfide (H2S), as the third gas transmitter after NO and CO[2], has multiple functions, including protecting the neurons from oxidative stress by facilitating glutathione generation and acting as an antioxidant by scavenging reactive oxygen species (ROS) in mitochondria.[3] H2S could be oxidized to be more stable forms, such as hydrogen polysulfides (H2Sn, n>1).[4] H2Sn is known to compensate for the production of H2S when the latter is insufficient.[4a] Recent studies reported that H2Sn might be a potential signaling molecule that modulate the activity of receptors, enzymes and ion channels with higher effective than H2S.[5] For example, H2Sn are approximately 300
times potent in inducing Ca2+ influx by activating transient receptor potential A1 channels in astrocytes.[5a, 5b] H2S biosynthesis enzymes located in the mitochondria, such as cystathionine γ-lyase and cystathionine-β-synthase, were also found to produce persulfides (RSSH), which could further be converted to H2Sn.[6] Therefore, to deeply understand the roles of H2Sn, it is urgent to develop screening tools to detect fluctuation of H2Sn in biological systems, especially in a specific organelle like mitochondria. Fluorescent probes have been reported in monitoring H2Sn with high sensitivity and selectivity[7]. Pioneered by Xian’s group[8], smart fluorogenic H2Sn probes were reported, including utilization of 2-fluorobenzoiate[8a] and phenyl 2-(benzoylthio)benzoate[8b] group by reacting with H2Sn to generate a cyclized mediated benzodithiolone product formation. These H2Sn reactive groups exhibited high sensitivity and selectivity with neglectable H2S response. However, probes monitoring H2Sn spatiotemporally in living cell systems are still very rare. In order to design a new H2Sn probe with temporal control[9], we noticed that the product of 2-nitrophenyl group after UV irradiation could generate an aldehyde/ketone [10] which might have a nucleophilic reaction with H2Sn. Hence, as shown in Figure 1, taking this advantage, a benzyl linked 2-nitrophenyl group could been adopted into the fluorophore through an ester bond to serve as a dual-functional group, including not only a photocleavage protecting moiety with a spatial and temporal controllable manner but also an effective H2Sn reactive group after photocleavage. Herein, we report the design, synthesis, evaluation of a novel H2Sn probe, H1, which exclusively detects H2Sn at mitochondria in a spatial and temporal manner. The structure of H1 was shown in Figure 1. It was constructed with 1) a mitochondrial localized group-the lipophilic triphenylphosphonium (TPP) cation[11]; 2) a fluorophore-fluorescein with easy modification, good photophysical properties and good solubility; 3) 2-nitrophenzyl based photocleavage moiety and 4) a simple benzyl masking group. The full working strategy was described in Figure 1. H1 will quickly enter and accumulate in
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Page 2 of 6 Our design:
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Figure 1 Structure, working strategy and response mechanism of H1 toward H2Sn in living cell systems. Our designed probe contained mito-directing (Red), fluorophore (Green) and dual-functional photocleavable groups (Blue). also synthesized by clicking 4 and TPP-N3 together. The detailed synthetic procedures and characterizations of compounds were enclosed in the supporting information.
Scheme 1 The synthetic route of H1 and H2. mitochondria. Without UV irradiation, H1 will not react with H2Sn. After UV irradiation, the benzyl group is released. 2-Nitrophenyl moiety is further activated to become a aldehyde product I1 which is a proposed polysulfide recognition site and attacked by H2Sn. The corresponding persulfide intermediate I2 is underwent a cyclization followed by the breakage of the ester bond to form benzodithiolone product I3. Concurrently, the phenol of fluorescein is exposed with liberation of fluorescence. Therefore, this strategy would allow us to monitor H2Sn in a specific location without responding H2Sn during the transit of probes. The synthetic route of H1 was shown in Scheme 1, starting from a commercially available fluorescein. The dual functional moiety 3 was synthesized from several steps with quantitative yields according the literatures [12]. The alkyne moiety was first attached with fluorescein through alkylation to form compound 4. Subsequently, 3 was directly coupled with 4 to form an ester bond as compound H2, which served as a control probe without a mitochondrial directing group. In the end, TPP-N3 (full structure shown in Scheme S1) was clicked with H2 to obtain the final probe H1 under CuI catalysis[13]. Furthermore, a control probe H3, as shown in Scheme S1, was
With these probes in hand, we first tested their absorbance and fluorescence responses in the presence and absence of H2Sn before and after UV irradiation. Na2S2 is known to be a H2Sn donor which is rapidly generated H2Sn in the buffer, hence Na2S2 solution was freshly prepared for every experiment. All the reactions were carried out in a 25 mM PBS buffer, pH 7.35. Results were shown in Figure 2A and 2B. In the absence of Na2S2, H1 has very low absorption and fluorescence as it is still in a closed-ring state. Almost no increasement in fluorescence was observed when adding different concentrations of Na2S2 (Figure 2B and S2). Irradiation of the probes in the absence of H2Sn also did not cause increasement in fluorescence and absorption either. According to our previous works[10a, 10b], 150 s UV irradiation (365 nm, 4 W) could fully make the cleavage occur (Figure S1). As expected, upon 150 s UV irradiation, when 100 μM Na2S2 was added, a huge enhancement (around 50-folds) in absorption and fluorescence intensity of 10 μM H1 was observed. The spectroscopic property (λabs = 458 nm, λex = 460 nm and λem = 525 nm) was the same as the fluorescein, indicating H1 was resulted into an open-loop state to achieve a “turn-on” affect. The fluorometric titration of H1 toward various concentrations of Na2S2 was carried out to evaluate the efficiency of H1. As shown in Figure 2C, varying concentrations of Na2S2 (0-200 μM) were added with H1 (10 μM) before UV irradiation. After UV irradiation, the fluorescent intensity at 520 nm gradually increased upon the treatment with increasing amount of Na2S2 until up to 20 equiv. There was good linearity between the relative fluorescent intensity and the concentrations of Na2S2 in the range of 0-30 μM. The detection limit was calculated to be around 150 nM, indicating H1 was highly sensitive to quantify trace concentrations of H2Sn. We then proceeded to investigate the time course of H1 (10 μM) reacting with 100 μM Na2S2. In Figure 2D, the maximum emission intensity of H1 was reached in less than 1 hour, which is slower than 2-fluorobenzoiate reactive group. It is possible that the aromatic substitution between fluorobenzoiates and H2Sn reacts faster than the
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Figure 2 (A) Absorption and (B) fluorescent emission spectra of H1/ H1 + UV/H1 + Na2S2/H1 (10 μM) + Na2S2 (100 μM) + UV (irradiation for 150 s with 365 nm hand-handle 4 W UV lamp) in 25 mM PBS buffer, pH 7.35. (C) Fluorescence emission spectra of H1 with varied concentrations of Na2S2 (0, 2, 4, 6, 8, 10, 15, 20, 25, 30, 35, 40, 60, 80, 100, 120 μM, respectively) after 150 s UV irradiation. Reactions were carried out for 1 hour at room temperature. (D) Time-dependent fluorescence emission spectra of 10 μM H1 in the presence of 100 μM Na2S2 with 150 s irradiation at room temperature (Recorded at 0, 15, 20, 25, 30, 35, 40, 50 min time points). Spectra were acquired with 460 nm excitation.
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Figure 3 (A) Time-dependent fluorescent intensity changes of H1 (10 μM) with Na2S2 (100 μM, red) or Na2S (100 μM, black) with 150 s UV irradiation. (B) Fluorescence response of H1 (10 μM) to various RSS (100 μM) in the absence ((2)-(11)) or absence ((12)-(21)) of Na2S2 after 150 s UV irradiation. (1) H1 alone (c) Fluorescence changes of H1 in the presence of various other biological related species. (1)-(13) 100 μM different cations and ions; 500 μM (14)-(31) amino acids. (D) Some representative photos of colour changes of H1 solution with treatment of the above analytes. Reactions were carried out for 50 min at room temperature. The excitation wavelength was 458 nm. Error bars represent s.e.m., n = 3.
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To evaluate the selectivity of H1 toward Na2S2, H1 was first treated with a series of RSS and ROS including S2-, SO32-, SO42, S8, D/L-Cys, Hcy, GSH, H2O2 and ClO-. As shown in Figure 3A, after UV irradiation, the addition of Na2S2 resulted in a 20fold enhancement of the fluorescence intensity compared with the addition of Na2S. In Figure 3B, no significant fluorescence increase was observed for any of these compounds. Only Na2S2 addition induced stronger fluorescence increase. Moreover, we recorded fluorescence signal of H1 (10 μM) with 150 s UV irradiation in the presence of various biological species, including inorganic reagents, amino acids, biological related hydropersulfides and hydropolysulfides. No apparent response was observed upon treatment of different analytes (Figure 3C and Figure S5). The competitive experiments confirmed that there was no influence of other analytes on the fluorescence response of H1 toward Na2S2 (Figure S4 and S5). Furthermore, the fluorescent enhancement was negligible when H1 was treated with cell lysate and medium (Figure S6). Thus, these data demonstrate that H1 has high selective sensing behaviour for H2Sn over other biological species under their biologically relevant concentrations, indicating that H1 is compatible for studies of H2Sn in the complex living cellular systems.
H1 and H2 was used to fluorescent image of H2S2, as a model of H2Sn, in HepG2 cells. Cells were first treated with probes (10 μM) for 1 hour and washed twice with DMEM at 37 oC before the addition of 100 μM Na2S2. Upon 150 s UV irradiation, cells were further incubated for another 1 hour. The probe channel was recorded at the excitation of 488 nm. As shown in Figure 4, cells treated with H1 and H2 did not show fluorescence if no UV irradiation was applied (Figure. 4(1) and (7)) or no Na2S2 was added (Figure. 4(2) and (8)). By contrast, after UV irradiation, large fluorescence enhancement was observed (Figure. 4(3) and (9)). This clearly validates the temporal control of H1 and H2 in “turning-on” the detection only when needed. Moreover, co-staining with a commercial available Mito tracker (Invitrogen), was conducted. It is showed that the signal of H1 was merged well with that of mito-tracker (Figure. 4(5)), implying a preferential distribution of H1 in mitochondria. The intensity profiles of the linear regions (Yellow line in the figures) of interest across cells co-stained with H1 and tracker were changing in close synchrony (Figure. 4(6)).
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out in human hepatocellular carcinoma (HepG2) cells using the XTT assay. As shown in Figure S7, there is no obvious cell toxicity to cells when probes were incubated with cells for 24 hours. Over 95% cells survived even the concentrations of H1 up to 50 μM, suggesting that probes were suitable to do cellular imaging. Moreover, UV irradiation itself, which activated H1 by photocleavage did not cause detectable cell death (Figure S8). Encouraged by these results, including high selectivity and low cell toxicity of H1, we next evaluated that the capability of H1 to selectively image H2Sn at mitochondria in live cells system with the aid of confocal microscopy.
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Figure 4 Confocal microscopy images of live HepG2 cells. Cells was co-stained with H1/H2 and MitoTracker® Red CMXRos. Cells were first incubated with H1 or H2, together with 50 nM Mito-tracker for 30 min, then washed, and subjected to 100 μM Na2S2 addition. Cells were continued to incubate for 50 min with/without 150 s UV irradiation. Finally, after twice washing with medium, fluorescent confocal microscopic images were taken. The excitation wavelength and PMT range were 488 nm and 500– 550 nm for H1/H2 channels (1)/(2)/(3)/(7)/(8)/(9). Insets in (1)/(2)/(3)/(7)/(8)/(9) are the differential interference contrast images (DIC), while those in (4)/(10) are related Mito-tracker channel images. (5) is merged images of (3) and (4). (11) is merged images of (9) and (10). (6) and (12) are the intensity profile of regions of interest (Yellow arrow in panel (3)/(4) and (9)/(10)). Scale bar =15 μm. In addition, the Pearson’s colocalization coefficient of H1 and tracker was Rr = 0.72. However, H2 without TPP mitochondria-targeting moiety showed much lower overlap ratio (Figure. 4(11), Rr = 0.4). Taken together, the fluorescence imaging study convinced that H1 and H2 are capable of visualizing H2Sn inside living cells with temporal resolution. Especially, H1 can specifically monitor mitochondrial H2Sn level, which is of great importance to clarify the physiological roles of H2Sn in the organelle. In conclusion, we have successfully designed and synthesized a photocontrollable and fluorogenic H2Sn probe that could detect mitochondrial H2Sn with high spatiotemporal resolution. We also expanded the utilization of photocleavage group 2-nitrophenyl group. By adding an ester linkage, the photocleavaged product aldehyde-benzoiate could be further carried on H2Sn-mediated benzodithiolone-ring formation. It serves not only as a photolabile group but also a H2Sn sensitive moiety, to provide controllable and reactive manners. Moreover, H1 behaves high selectivity and sensitivity for H2Sn with a detention limit of 150 nM. Co-localization studies of H1 with Mito tracker confirmed the precise localization in Mitochondrial organelles. The probe is a promising fluorescent tool for monitoring H2Sn in cells. Based on this design, the localization moiety can easily be tuned through the clickable handle to develop more other specific organelles and diseases targeting H2Sn probes.
ASSOCIATED CONTENT Supporting Information Supporting Information Available: The following file is available free of charge on the ACS Publications website at DOI: Experimental details, synthesis, NMR and additional spectroscopic data as noted in text.
AUTHOR INFORMATION
Corresponding Author * E-mail:
[email protected] * E-mail:
[email protected] * E-mail:
[email protected] Author Contributions #Students
(Linqi Han and Riri Shi) contributed equally.
ACKNOWLEDGMENT This work was financially supported by the National Natural Science Foundation of China (81672508, 61505076, 21708034, 21877100), Jiangsu Provincial Foundation for Distinguished Young Scholars (BK20170041), Natural Science Foundation of Zhejiang Province (LQ16B020003), China-Sweden Joint Mobility Project (51661145021), Key University Science Research Project of Jiangsu Province (Grant 16KJA180004), and SICAM Fellowship & Scholarship by Jiangsu National Synergetic Innovation Center for Advanced Materials.
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