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An AND-logic based fluorescent probe for selective detection of lysosomal bisulfite in living cells Ji-Zhen Li, Yi-Hang Sun, Chengyun Wang, Zhiqian Guo, Yongjia Shen, and Wei-Hong Zhu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b02749 • Publication Date (Web): 19 Aug 2019 Downloaded from pubs.acs.org on August 19, 2019

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Analytical Chemistry

An AND-logic based fluorescent probe for selective detection of lysosomal bisulfite in living cells Ji-Zhen Li, Yi-Hang Sun, Cheng-Yun Wang *, Zhi-Qian Guo *, Yong-Jia Shen and Wei-Hong Zhu * Key Laboratory for Advanced Materials and Institute of Fine Chemicals, School of Chemistry and Molecular Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, PR China ABSTRACT: Sulfur dioxide (SO2) plays significant roles in regulating cell apotosis and inflammation. However, there are complex interactions between small biomolecules in cells, and the identification of these coexisting biomarkers remains a challenge. Herein, we report a AND logic gate based fluorescent probe (NY-Lyso), operating by responding to pH differences between organelles in cell and selectively reacting with bisulfite (HSO3-). This approach allows the fluorescence of the probe to remain silent under neutral or alkaline conditions, notably, is activated by co-stimulation of lower pH and bisulfite. Furthermore, it was confirmed to be biocompatible and could be employed to monitor HSO3- in lysosomes of living cells. The proposed method demonstrated more practical and outstanding capabilities in targeted and real-time monitoring, providing an effective optical tool for biomarker sensing.

INTRODUCTION Sulfur dioxide (SO2), existing in the form of bisulfite (HSO3-) and sulfite (SO32-) in organism and neutral solutions, is considered as one of the most menacing air pollutants 1-4. However, they are also the most commonly used preservatives and antioxidants in foods, pharmaceuticals and other fields 5, 6. In addition, in biological systems, they can be produced via the oxidation of some sulfur-containing species (e.g., amino acids) 7, 8. Plentiful studies confirm that SO2 participates actively in equilibrating the redox status as an antioxidant against reactive oxygen species (ROS) 9-11. Lysosome, as the major digestive compartment for the degradation of cell constituents (proteins, nucleic acids, carbohydrates and lipids, etc.), is considered as the key regulators of cellular homeostasis 12-14. In particular, the interior of lysosome exhibits an acidic pH range of 4.5-5.5, which provides a favourable environment for digestive enzymes (such as acidic hydrolases, cathepsins, nucleases, lipases) to degrade cellular components. There has been increasing evidence indicating that lysosomal dysfunction was closely related to the diseases such as silicosis, rheumatoid arthritis (RA), neurodegenerative disorders and even cancer 15, 16. However, abnormal SO2 levels could affect the lysosome enzymes amounts and activities in macrophages, resulting in decreased number and viability of alveolar macrophages, reduced macrophage adhesion, and further impaired local immune function 17. Therefore, monitoring the SO2 levels of acid lysosome in the biological system is of great significance in the field of disease diagnosis and therapy. The development of smart probes and imaging instruments provide great opportunities for both environmental and bioimaging applications 18-20. But it is

still a challenge to distinguish the coexistence biomarkers, such as ions, neutral species, pH, temperature and viscosity. Since the prominent work by de Silva and coworkers in 1980s, many multi-stimulus-responsive fluorescent probes based on molecular logic gates have been developed 21-24. In these systems, the analytes and corresponding signal changes could be served as the inputs and outputs, respectively. It would be very valuable when they correlated with Boolean logic ideas 25-28. Inspired by these, we hypothesize that the fluorescence response of the developed probe could be combined with the concentrations of bisulfite and acidity in lysosomes, then produces specific output signal, which is in fact very consistent with the concept of an AND logic gate. Until now, many researchers have devoted considerable enthusiasm to the development of SO2 bioprobes 29-37, but an AND logic-based, pH-activatable probe for specific detection of bisulfite has not been reported. Herein we present a new AND logic-based fluorescence probe (NY-Lyso), consisting of morpholine unit, semicyanine, and 1, 8-naphthalimide chromophore, was developed for specific detection of bisulfite in lysosomes (Scheme 1). The smart probe is composed of two functional components: the morpholine group of NY-Lyso produces a sensitive response to pH differences between acidic lysosomes (pH 4.5-5.5) and other organelles in cell 38-40, and the C=C bond between thiophene and cyanine is selectively reactive with HSO3- through nucleophilic addition 41-43. The morpholine and semi-cyanine moieties are both independently capable of quenching the emission via the photoinduced electron transfer (PET) and

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Scheme 1. Chemical Structure of NY-LYso and Proposed Sensing Mechanism to HSO3-.

intramolecular charge transfer (ICT) mechanisms, respectively 44, 45, and thus the free probe showed no fluorescence. At the simultaneous presence of SO2 and H+, the probe exhibited strong fluorescence emission at 524 nm. That is, only when the SO2 and pH are simultaneously considered as “inputs” will the emission intensity “output”. Notably, the probe based on this AND logic approach not only shows high selectivity, fast response (less than 200s), very low detection limit (LOD= 20.7 nM) and excellent lysosomal targeting properties, but overcomes the limitations of detecting HSO3- in complex cellular pH environments. Compared to previously reported bioprobes for SO2 derivatives, the AND logic-based fluorescence probe shows distinct advantages both in performances and applications (Supporting Information Table S1).

EXPERIMENTAL SECTION Synthesis of Probe NY-LYso. Compound 2 and 3 were synthesized according to the previously reported methods 46, 47. A mixture of Compound 2b (105 mg, 0.25 mmol) and Compound 2 (138 mg, 0.5 mmol) were added to a 50mL round-bottom flask and dissolved with 15 mL of anhydrous ethanol. The mixture was reacted at 80 oC for 12h. After Compound 2b was consumed, the solvent was then evaporated in vacuo. The crude product was further purified by silica-gel column with CH2Cl2/MeOH (v/v, 80:1 to 20:1) as the eluent to afford a deep red solid of NY-Lyso. Yield: 85%. 1H NMR (400 MHz, DMSO-d6), δ (ppm): 8.83 (d, J=16.0 Hz, 1H), 8.68 (d, J=8.8 Hz, 1H), 8.58 (t, J=7.6 Hz, 2H), 8.44 (d, J=4.0 Hz, 1H), 8.06 (d, J=7.6 Hz, 1H), 8.00-7.91 (m, 3H), 7.83 (d, J=4.0 Hz, 1H), 7.65 (m, 2H), 7.51 (d, J=15.6 Hz, 1H), 4.72 (m, 2H), 4.20 (t, J=6.8 Hz, 2H), 3.54 (t, J= 4.0 Hz, 4H), 2.60 (t, J=6.8 Hz, 2H), 2.48 (br, 4H), 1.84 (s, 6H), 1.47 (t, J=7.2 Hz, 3H). 13C NMR (600 MHz, DMSO-d6, ppm) δ: 181.11, 163.67, 163.38, 148.71, 146.10, 144.42, 142.39, 140.95, 138.45, 137.23, 132.41, 132.00, 131.72, 130.75, 129.85, 129.66, 129.43, 129.16, 128.85, 128.56, 123.62, 123.06, 123.00, 115.52, 111.72, 70.25, 66.68, 60.23, 55.95, 53.86, 52.63, 26.00, 14.25. HR-MS (m/z): [M-Br-]+ Calcd. for [C36H36N3O3S]+: 590.2472, found: 590.2474. Materials and Instruments. Unless otherwise stated, all reagents and solvents (analytical grade) in this work

were purchased from commercial suppliers. The experiments were monitored by thin-layer chromatography (TLC), and the target product and intermediates were obtained by column chromatography. The UV–vis spectra and fluorescence spectra were recorded on Varian Cary 500 spectrophotometer and CARY Eclipse Spectrophotometer, respectively. 1H NMR and 13C NMR spectra were recorded on a Bruker AM-400 spectrometer. High resolution mass spectra were recorded on a Waters LCT Permier XE spectrometer. The fluorescence quantum yields were determined with quinine sulfate (Φf = 0.55 in 0.05 M sulfuric acid) as reference and corrected for index differences between water and tetrahydrofuran. The measurements of pH were done using a pH-10C digital pH meter. General Procedures for Spectral Measurements. The stock solutions of probe NY-Lyso (1.0 mM) were prepared in the DMSO solution. The various analytes stock solutions (F-, Cl-, Br-, I-, ACO-, H2PO4-, SO42-, S2O32-, S2O82-, HS-, S2-, Cys, Hcy, GSH, H2O2, ClO-, Ascorbic acid) were dissolved in double-distilled water at 10 mM, and the standard solutions of NaHSO3 (10 mM) were freshly prepared by double-distilled water. The test solutions were obtained by placing 30 μL of probe stock solution (the final concentration was 10 μM) and the necessary amount of each analyte in PBS buffer solution (10 mM, 5% DMSO, v/v). Cell Culture and Fluorescence Imaging. HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) blended with 10% FBS (Fetal Bovine Serum) and antibiotics (100 mg/mL penicillin and 100 mg/ mL streptomycin) at 37 °C under an atmosphere of 5% CO2 and 95% air, then the cells were seeded into CoverglassBottom confocal dishes with a density of about 1×10-5 cells per dish and incubated for 24 h at 37 oC before the imaging experiments. Then the cells were washed three times with PBS buffer (pH 7.4) and divided into several groups. The fluorescence imaging of NY-Lyso in HeLa cells were carried out on a Nikon A1R confocal laser scanning microscope. Co-location Studies. For co-location experiments, the cells were co-incubated with 10 μM of NY-Lyso, 20 μM of HSO3- and 0.5 μM of commercial biomarker (Lyso-Tracker Red C1046) for 30 min at room temperature. After washing three times with PBS buffer solution (pH= 7.4), the cells were observed under the confocal microscope (NY-Lyso channel: λex = 400 nm, λem = 500-550 nm; LysoTracker Red C1046 channel: λex =577 nm, λem=580-600 nm). Cell Cytotoxicity Evaluated by MTT Assay. HeLa cells were seeded into 96-well plates at a concentration of 5000 cells per well in 100 μL of DMEM medium with 10% FBS. Then different concentrations of NY-Lyso (0-50 μM) were added into the 96-well plates and the plates were maintained at 37 ℃ under an atmosphere of 5% CO2 and 95% air incubator for 24 h. After the supernatant was removed, 100 μL of MTT solution (0.5 mg/ mL in PBS) was added ion each well and treated for another 4 h.


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Analytical Chemistry Subsequently, the remaining MTT solution was removed and replaced by 150 μL of DMSO. After shaking for 10 min, the absorbance of each well was acquired by using the microplate reader at 400 nm. Each experiment was performed at least 3 times. The relative cell viability was calculated by the equation: survival viability (%) = (ODtreated/ODcontrol) × 100%. Visualization of Intracellular SO2. The HeLa cells were pretreated with different-pH high K+ buffer (0.5 mM MgSO4, 1 mM CaCl2, 1 mM NaH2PO4, 5 mM glucose, 30 mM NaCl, 120 mM KCl, and 20 mM HEPES) in the presence of nigericin (10 μM). The first group of the cells was incubated with NY-Lyso (10 μM) for 30 min at pH=5.5. In the second and third groups, the cells stained NY-Lyso were further incubated with HSO3- (50 μM) for 30 min at pH= 7.4 and 5.5, respectively.

RESULTS AND DISCUSSION Design and Synthesis of the Probe NY-Lyso. As shown in Scheme 1, it can be seen that the probe is composed of two functional components: a lysosome-targeted group morpholine and a specific binding site for bisulfite. This design strategy allows the probe to have two response sites simultaneously, controlling the fluorescence of naphthalimide fluorophore by PET and ICT, respectively. In the presence of bisulfite, the conjugated structure of semi-cyanine moiety is interrupted, which results in a partial enhancement of the fluorescence intensity through inhibition of the ICT process. When exposing the SO2-probe complex to an acidic environment, such as lysosomes, the morpholine group will be protonated, and then leads to a remarkable increase in emission intensity since the PET process is also blocked. Scheme 2 shows the synthetic route of our probe, as well as naphthalimide models 2a, 2b which can serve as the specific data support for mechanistic studies. The related compounds were determined by 1H NMR, 13C NMR and HR-MS (Supporting Information Figure S1- Figure S11). Scheme 2. The Synthetic Routes of Naphthalimide Models 2a, 2b and Probe NY-Lyso.

Figure 1. (a) UV−vis spectra of NY-Lyso (10 μM) upon addition of HSO3- (0−100 μM) in PBS solution (10 mM, 5% DMSO, pH= 5.5). (b) Fluorescence spectra changes of NY-Lyso (10 μM) in the presence of different concentrations of HSO3(0−100 μM) in PBS solution (10 mM, 5% DMSO, pH= 5.5). Inset: photograph of free NY-Lyso (left) and in the presence of HSO3- (right) under visible light and UV lamp (λex = 380 nm).

The Photophysical Properties of NY-Lyso. Since the lysosomal pH values range from 4.5 to 5.5, we first examine the influence of pH on the photophysical properties of probe NY-Lyso in the presence and absence of HSO3-. As expected, the results show that pH behaves vitally to the probe (Figure S12). At pH=7.0, NY-Lyso displays no fluorescence via the PET and ICT processes in the absence of HSO3-. However, when HSO3- is added, the emission intensity remains at a low value, indicating that the PET process is still working. When the pH is decreased to 6.0, the protonation of the N atom within the morpholine group blocks the PET process, and then leads to a remarkable emission enhancement at 524 nm (Φf = 0.49). As the acidity of the system increases, HSO3- will be preferentially captured by H+, causing the nucleophilic addition process to be blocked and the ICT process to be reactivated. To verify this mechanism, we tested the photophysical properties of a series of naphthalimide models. The results of all investigated compounds were summarized in Table S2. Considering the solvation effect, we chose THF as the test solvent. As envisioned, the addition of 1equiv of H+ yielded a distinct fluorescence enhancement of Compound 2b (Φf = 0.16). Additionally, the model Compound 2a in which morpholine was substituted with n-butyl group was not affected by PET and exhibited stable fluorescence emission (λem = 458 nm for 2a and 450 nm for 2aH+; Φf = 0.036 and 0.035, respectively). Thus, these results suggest that NY-Lyso could be served as a pH-activatable fluorescence probe for the detection of HSO3-, which keeps silent under neutral and alkaline conditions but performs selective fluorescence activation in slightly acidic condition. Subsequently, the optical response of NY-Lyso to HSO3was examined by measuring its absorption and fluorescence spectra changes in PBS solution (10 mM, 5% DMSO, pH= 5.5). As shown in Figure 1a, the free probe NYLyso (10 μM) showed a main absorption band at 475 nm. Upon the addition of HSO3- (0-10 eq), the maximum peak

Reagents and conditions: (A) Pd(PPh3)4, K2CO3, THF/H2O=3:1, 70 oC. (B) EtOH, 80 oC.



Figure 2. 1H NMR titration spectra of probe NY-Lyso without and with10.0 eq. HSO3- in DMSO-d6.

at 475 nm gradually decreased, along with a distinct color change from pale yellow to colorless. These changes could be attributed to the interruption of the conjugated structure of semi-cyanine moiety by HSO3-, and thus blocked the ICT pathway, so the absorption spectrum of the probe gave significantly blue-shifted. On the other hand, the free NY-Lyso (10 μM) exhibited almost no fluorescence emission (Figure 1b). As the concentration of HSO3- increased, a distinct fluorescence turn-on was observed at 524 nm when excited at 380 nm. When 10 eq. of HSO3- was added, the fluorescence intensity reached the maximum and showed a good linearity to the concentrations of HSO3- with a detection limit (3σ/K) of 20.7 nM (Figure S13). Furthermore, by analysing the fluorescence intensity at 524 nm, we investigated the reaction kinetics of the probe. At various concentrations of HSO3- (0-60 μM), the fluorescence intensity of the probe gradually increased with the reaction time and reached equilibrium within 200 s, and remained stable over 60 min (Figure S14). The above experiments show that the probe responds fast to SO2 and can be used for instant imaging of SO2. Selectivity and Interference. To determine the selectivity of NY-Lyso toward HSO3- over other species, including ions (F-, Cl-, Br-, I-, ACO-, H2PO4-, SO42-, S2O32-, S2O82-, HS-, S2-), bio-thiols (Cys, Hcy, GSH), oxidation and reducing substances (H2O2, ClO- and Ascorbic acid), we examined the fluorescence spectra of the probe in PBS buffer solution (10 mM, 5% DMSO, v/v, pH=5.5). As shown in Figure S15, no obvious fluorescence increase could be observed except for HSO3-, resulting in a 12-fold enhancement of fluorescence intensity. Furthermore, the anti-interference experiments confirmed that the coexistence of these species did not cause substantial influence in HSO3- detection (Figure S16). Thus, these results demonstrated that the probe has high selectivity toward HSO3- over other anions and biological species, making it suitable for HSO3- studies in complex cellular samples. Sensing Mechanism of the Probe with Bisulfite. The sensing mechanism of probe NY-Lyso for HSO3- was

determined by the ESI-MS spectra and 1H NMR titration experiments. As shown in Figure 2, after addition of HSO3-, the proton signals assigned to the double bond of the probe NY-Lyso (δ 8.65 for Hb and δ 8.44 for Ha) shift to δ 5.15 (Hb´) and δ 4.85 (Ha´), respectively. The proton signal at δ 1.85 (Hc) of the two methyl groups also shifted forward and split into two signals (Hc´ at δ 1.33 and 1.52). In addition, a new weak proton signal appeared at δ 9.98 (Hd), which might be assigned to HSO3- after undergoing the nucleophilic addition to the alkene moiety. Moreover, the 1, 4-addition product was also characterized by ESI-MS (Figure S11), where the peak at m/z value of 670.2056 (calca. = 670.2046) corresponds to [M´-H]-. Therefore, taking above results into consideration, the 1, 4-addition reaction mechanism of the probe toward HSO3- was proposed in Scheme 1. Logic Behavior of the Probe toward H+ and Bisulfite. According to the results presented above, the AND logic characteristics of probe were determined in PBS buffer solution (10 mM, 5% DMSO, v/v) by recording the fluorescence spectra under four possible input conditions (Figure 3). Specifically, H+ and HSO3- were used as inputs and the emission intensity at 524 nm of the system as output. For inputs, the presence and absence of H+ or HSO3were defined as “1” and “0”, respectively. For output, the fluorescence “ON” and “OFF” were defined as “1” and “0”, respectively. In the presence of the two inputs (1/1), the fluorescence intensity increased sharply, exerting an output signal of “1”. Otherwise, in other cases (no inputs or input alone), the fluorescence output was “0” because of PET process occurring from the morpholine group to the fluorophore or ICT process occurring from the fluorophore to cyanine moiety. Fluorescence Imaging of Bisulfite in Living Cells. Encouraged by the excellent photophysical properties and response performances of NY-Lyso to bisulfite under acidic conditions outside of the cell, we continued to explore the capabilities of NY-Lyso to image bisulfite in the lysosomes of living cells. Initially, we performed the cytotoxic assay of the probe, as shown in Figure S17, the


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Analytical Chemistry probe exhibited low cytotoxicity to HeLa cells, indicated that NY-Lyso was suitable for imaging in living cells.

Figure 3. (a) Logic scheme and (b) truth table for the “AND” logic gate with two inputs.

Similar to other lysosomes-targetable probes with morpholine group, the introduction of the morpholine groups significantly increase the possibility of probe enrichment in lysosomes. In order to examine the lysosome-targeted property of NY-Lyso, the co-localization experiments were performed. The HeLa cells were coincubated with probe NY-Lyso (10 μM), HSO3- (20 μM) and Lyso-Tracker (0.5 μM) for 30 min at room temperature. As shown in Figure 4, the cells showed green fluorescent channel from NY-Lyso under a microscope (Figure 4a), and red fluorescent channel from the commercial LysoTracker (Figure 4b). The merged image (Figure 4c) indicated that hat the two channels overlaid very well, and the Pearson’s co-localization coefficients was calculated to be 0.85, confirming that NY-Lyso can specifically localize in the lysosomes of living cells.

Figure 4. Probe NY-Lyso (10 μM) and LysoTracker Red (0.5 μM) colocalization imaging in HeLa cells: (a)Green emission of NY-Lyso (λem = 500-550 nm); (b) Red emission of LysoTracker Red (λem = 580-600 nm); (c) Merged images of (a) and (b); (d) brightfield image of HeLa cells; (e) intensity scatter plot of the green and red channels, R=0.85; (f) Intensity profiles of linear region of part (c) across the HeLa cell, Ch1 represents the intensity of NY-lyso, Ch2 represents the intensity of the LysoTracker Red. Excitation wavelength of NY-Lyso and LysoTracker Red was 400 nm and 577 nm, respectively. Scale bar: 20 μM.

Subsequently, the ability of the probe for intracellular HSO3- detection and imaging were investigated. In order to test the AND logic characteristics of the probe in cells, we must first regulate the pH values internal and external the cells. A common method is to change the external pH of the cells to a fixed value using a high K+ buffer, and then impose the inside of the cells to the same pH using the nigericin, a K+/H+ ionophore [48-50]. In addition, the buffers with pH at 5.5 and 7.4 are obtained by using 20 mM citric acid and 20 mM HEPES, respectively. Then, for H+, the pH at 5.5 and 7.4 could be defined as “1” and “0”, respectively. The presence and absence of HSO3- are also defined as “1” and “0”, respectively. As shown in Figure 5, we divided the cells into three groups, which were (1, 0), (0, 1) and (1, 1). However, in case of untreated cells (Figure 5a) and those incubated with HSO3- only (Figure 5b), no fluorescence was observable on the cells. In contrast, the cells that were pretreated with the buffer at pH 5.5 successfully observed the strong green fluorescence at 524 nm after incubating with 50 μM of HSO3- (Figure 5c). Comparing with the results of extracellular experiments, it can be concluded that we have successfully developed a AND logic-based fluorescent probe for selective detection of HSO3- in living cells.

Figure 5. The AND logic behavior of NY-Lyso (10 μM) for imaging HSO3- in HeLa cells at different pH values. HeLa cells were incubated with NY-Lyso and high K+ buffer at pH 5.5 (a); NY-Lyso, HSO3- and high K+ buffer at pH 7.4 (b); NY-Lyso, HSO3- and high K+ buffer at pH 5.5 (c). Left to right: bright-field images; fluorescence images at green channel (λem = 500−550 nm, λex = 400 nm); and merged images. Scale bar: 20 μM.

CONCLUSIONS In summary, we developed a AND logic-based fluorescent probe NY-Lyso for detecting HSO3- via the regulation of PET and ICT mechanisms. The accessible probe displayed high selectivity and sensitivity (LOD= 20.7 nM) toward HSO3-. The



AND logic behaviour of the probe allows its fluorescence to remain silent under neutral or alkaline conditions but is activated by co-stimulation of lower pH and HSO3-. In addition, it was confirmed to be biocompatible and could be employed to monitor lysosomal HSO3- in living cells. Comparing to the classical probes, this AND logic-based probe avoids the complex and undesirable interactions in the cellular environment, providing better selectivity and higher sensitivity. This method demonstrated more practicable and outstanding capacity in targetable and real-time monitoring.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at http://pubs.acs.org. Figures showing NMR and HRMS characterization data for Compounds interest and targeted NY-Lyso, UV-vis and fluorescence spectral data, time-dependent fluorescent spectra of NY-Lyso toward HSO3-, cytotoxicity of NY-Lyso.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected], (C. Y. Wang) *E-mail: [email protected], (Z. Q. Guo) *E-mail: [email protected], (W. H. Zhu)

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by the Natural Science Foundation of Shanghai (16ZR1408000), and the National Key Program of China (2016YFA0200302).

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for TOC only


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