Ratiometric Fluorescent Probe for Lysosomal pH Measurement and

Article

A Ratiometric Fluorescent Probe for Lysosomal pH Measurement and Imaging in Living Cells Using Single-Wavelength Excitation Xingjiang Liu, Yuanan Su, Huihui Tian, Lei Yang, Hongyan Zhang, Xiangzhi Song, and James Walter Foley Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 29 May 2017 Downloaded from http://pubs.acs.org on May 29, 2017

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Analytical Chemistry

A Ratiometric Fluorescent Probe for Lysosomal pH Measurement and Imaging in Living Cells Using Single-Wavelength Excitation Xingjiang Liu a, Yuanan Su a, Huihui Tian a, Lei Yang a, Hongyan Zhang b*, Xiangzhi Song a,c* and James W. Foley d a

College of Chemistry & Chemical Engineering, Central South University, Changsha 410083, China. Email: [email protected]. b Key Laboratory of Photochemical Conversion and Optoelectronic Materials, Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing 100190, China. Email: [email protected]. c State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian 116024, China. d Rowland Institute at Harvard, Harvard University, Cambridge MA 02142, USA. ABSTRACT: A novel lysosome-targeting ratiometric fluorescent probe (CQ-Lyso) based on the chromenoquinoline chromorphore has been developed for the selective and sensitive detection of intracellular pH in living cells. In acidic media, the protonation of the quinoline ring of CQ-Lyso induces an enhanced intramolecular charge transfer (ICT) process, which results in large red-shifts in both the absorption (104 nm) and emission (53 nm) spectra which forms the basis of a new ratiometric fluorescence pH sensor. This probe efficiently stains lysosomes with high Pearson’s colocalization coefficients using LysoTracker®Deep Red (0.97) and LysoTracker®Blue DND-22 (0.95) as references. Importantly, we show that CQ-Lyso quantitatively measures and images lysosomal pH values in a ratiometric manner using single wavelength excitation.

Intracellular pH values play vital roles in mediating many physiological processes such as cell metabolism, enzyme activity and immunization.1-4 Lysosomes are acidic organelles (pH 4.0-5.5) in eukaryotic cells that maintain their pH value by the inward pumping of protons (H+ ions) from the cytosol across the cell membrane via proton pumps. Low pH is necessary for the hydrolytic enzymes and proteins contained in lysosome to function at their optimum activity.5-8 For example, digestive enzymes in lysosomes require an acidic environment in the range of pH 4.0-5.5 to degrade ingested proteins, DNA, polysaccharides, lipids, viruses, RNA and bacteria.9-11 Abnormal pH values in lysosomes can result in cellular dysfunction and consequently lead to many severe lysosomal diseases such as lipid storage disorders and mucolipidoses.12-13 Because of the critical importance of pH for lysosomes to function properly, it is essential for health professional to have simple and effective methodologies for the rapid, selective and sensitive determination of lysosomal pH values in biological specimens. Due to their high sensitivity, easy operation, real-time and noninvasive imaging properties, fluorescent probes have been useful tools for sensing and imaging a variety of cations, anions and biomolecules in living cells and tissues.14-18 They are especially useful when they are able to become predominantly localized in a single cellular component because they provide accurate and reliable information about the micro-environment within the targeted organelle.19-26 In recent years, a number of fluorescence based pH probes have been reported.27 However, few of them are selective pH indicator stains for lysosomes,24,27-33 and fewer still specifically determine pH in lysosomes in a ratiometric manner.28-35 It’s known that ratiometric fluorescent probe can avoid data distortions caused by

variable probe concentration, instrument sensitivity and environmental conditions through the self-calibration of two emission bands.36 Among the previously reported ratiometric fluorescent probes for lysosomal pH, noteworthy are the works of Kim and colleagues who have developed two-photon ratiometric fluorescent probes,29 Ma’s group who have developed a near-infrared ratiometric fluorescent probe based on the semicyanine skeleton in 2014,32 and, most recently, Lin et. al.’s cleverly designed probe which integrated ICT, PET and FRET in their ratiometric fluorescent pH probe.34 The importance of these contributions is that each incorporates an aspect of fluorescent probe design that is generally prized by practicing lifescientists. These include, in addition to each being ratiometric, at least one of the following: (1) The ability to utilize a single excitation wavelength for cell imaging because this significantly simplifies data acquisition, minimizes background noise associated with multiple wavelength excitations, and reduces auto-fluorescence interference from cellular organelles.37-38 (2) Emission in the red or near infrared (NIR) spectral region because longer wavelength photons have superior tissue penetration and have minimal interference from auto-fluorescence emitted by cellular components.39 For lysosome-targeting fluorescent probes, a high Pearson’s coefficient is mandatory for specific staining, which is measured by colocolization experiments.40-41 To date, with the exception of Ma et. al.’s work, all reported ratiometric fluorescent probes emit at wavelength shorter than 600 nm. As a result, lysosomotropic pH fluorescent probes simultaneously having a high Pearson’s coefficient in addition to the above mentioned desiderata remain in a high demand. The scarcity of few such materials was the inspiration for the work reported herein (shown in Scheme S1, ESI†).

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Chromenoquinoline derivatives are mainly used as estrogenic agents42 and, with the exception of Talukdar’s43 and Lin’s44 works, have seldom been subjected to photophysical property studies. In this report, we designed and synthesized a lysosome-targeting chromenoquinoline (CQ-Lyso) that is a fluorescent probe for measuring the pH of lysosomes in living cells using only single wavelength excitation (molecular structure and synthesis shown in Scheme 1). A morpholine moiety was introduced in CQ-Lyso for lysosome-targeting purposes. Although this probe exhibits a strong yellow florescence in neural/basic media, when treated with an increasing gradient of acidity, the quinoline moiety in CQ-Lyso would be expected to proportionally become protonated (shown in Scheme 2). Based on the previous report,31, 45 we expected that such protonation would promote an enhanced intramolecular charge transfer (ICT) process which would concomitantly induce significant red-shifts in both its absorption and emission spectra. In this manner the pH-sensitive emission spectra of CQLyso would serve as a ratiometric fluorescent probe for lysosomal pH measurements. Scheme 1. Synthetic route of CQ-Lyso.

(a) 1,4-dibromobutane, acetone, K2CO3, 56 °C, 48 h; (b) morpholine, DMF, K2CO3, KI, 80 °C, 12 h; (c) Pd/C, H2, CH3OH, 65 °C, 12 h; (d) 3-bromoprop-1-yne, acetone, K2CO3, 56 °C, 12 h; (e) CuI, DMF, 110 °C, 4 h. Scheme 2. The proposed mechanism of CQ-Lyso for pH measurements in aqueous media or lysosome.

EXPERIMENTAL SECTION Materials and Instruments. All commercial reagents were used without further purification. Twice-distilled water was used throughout all the experiments. Bruker 400 and 500 spectrometers were used to record 1H and 13C NMR spectra with tetramethylsilane (TMS) as an internal standard for chemical shift. A LCT Premier XE mass spectrometer (Waters) was used to obtain high-resolution mass spectra. Absorption and fluorescence spectra were respectively measured on a UV2450 spectrophotometer and a F-280 spectrometer (Tianjin Gangdong Sci. & Tech. Development Co. Ltd). A Leici PHS3C meter was used for pH measurements. Fluorescence imaging experiments were performed on an Olympus FV1000 and a Nikon ARsiMP confocal microscopes. TLC silica gel plates and silica gel (mesh 200-300) for column

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chromatography were supplied by Qingdao Ocean Chemicals (China). Theoretical Calculations. Density functional theory (DFT) calculations with Becke’s three parameterized Lee-Yang-Parr (B3LYP) exchange functional were performed for geometry optimization of CQ-Lyso in the absence/presence of H+ using a Gaussian 09 program.45-46 The basis set was 6-31G (d, p). The energy levels of the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) were obtained using time-dependent density functional theory with the same basis sets as mentioned above. Spectral Measurements. The absorption and emission spectra were measured in Britton-Robinson (B-R) buffer solution (40.0 mM) with 30% CH3CN (v/v). A stock solution of CQLyso was prepared at 1.0 mM in CH3CN. The BrittonRobinson buffers with pH values at 2.0, 3.0, 4.0, 4.2, 4.4, 4.6, 4.8, 5.0, 5.2, 5.4, 5.6, 5.8, 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, 7.6, 7.8, 8.0 and 9.0 were prepared according to the standard procedures. Solutions for testing the effect of possible interfering species were prepared by dissolving Al(NO3)3, CdCl2, CoCl2, CrCl3, FeCl3, NiCl2, Pb(NO3)2, ZnCl2, FeCl2, CuSO4, BaCl2, MnCl2, MgCl2, CaCl2, NaCl, KCl, glucose, GSH, Cys, Hcy, Gly, Glu, Val, Arg, Lys, Try, Thr, Asp, H2O2, NaHS, NaNO3, Na3PO4, Na2S2O3, NaSCN, Na2SO3, Na2SO4 and NaClO in twice-distilled water, respectively. ONOO- was prepared according to literature method.47-48 For titration experiments, a test solution of CQ-Lyso (5.0 µM) was prepared by placing 15.0 µL of CQ-Lyso (1.0 mM) into a mixture of B-R buffer (2.1 mL) and CH3CN (0.885 mL) in a quartz optical cell with 1.0 cm optical path length. For selectivity experiments, test solutions were prepared by placing the stock solution of CQ-Lyso into the mixture of B-R buffer and CH3CN, and then adding the appropriate amount of the stock solution containing the corresponding analyte. NaOH (1.0 M) or HCl (1.0 M) were used to adjust the pH of the test solutions.33,49 All the resulting solutions were shaken well and incubated at 25 °C for 15 min before absorption and fluorescence spectral measurements. Cell Culture and Fluorescence Imaging. The culture of HeLa cells was conducted in DMEM (Dulbecco’s modified Eagle’s medium) at 37 °C with 10% fetal bovine serum and 1% penicillin as supplement in atmosphere of 5% CO2 and 95% air.50 When the adherent cells with stable growth in culture flasks reached 85%-95%, cells were seeded in glass dishes and allowed to adhere for 24 hours. Then, discard the culture fluid and wash cells three times with PBS buffer before the experiments. LysoTracker®Deep Red and LysoTracker®Blue DND-22 were used as the colocolization agents. Chloroquine was used as the stimulant to induce pH increase of lysosome. Synthesis of 1-(4-bromobutoxy)-4-nitrobenzene (1). This compound was prepared according to a literature method.51 To a solution of 4-nitrophenol (0.5760 g, 4.0 mmol) and 1,4dibromobutane (1.3281 g, 8.0 mmol) in acetone (6.0 mL) was added potassium carbonate (0.8482 g, 8.0 mmol). The resulting mixture was stirred at 56 °C for 48 h. After being cooled to room temperature, the reaction mixture was filtered and the filtrate was concentrated in vacuum to give an oil residue which was purified by flash silica gel chromatography (dichloromethane/petroleum ether as eluent, v/v = 1:2) to afford compound 1 as a white solid (0.6012 g, 54.5% yield). 1 H NMR (400 MHz, CDCl3) δH 8.18 (d, J = 9.2 Hz, 2H), 6.95


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(d, J = 9.3 Hz, 2H), 4.10 (t, J = 5.9 Hz, 2H), 3.51 (t, J = 6.4 Hz, 2H), 2.40-1.81 (m, 4H). 13C NMR (100 MHz, CDCl3) δC 163.9, 141.5, 125.9, 114.4, 67.8, 33.2, 29.2, 27.6. Synthesis of 4-(4-(4-nitrophenoxy)butyl)morpholine (2). To a solution of compound 1 (1.0963 g, 4.0 mmol) and morpholine (0.4482 g, 5.2 mmol) in DMF (10.0 mL) was added potassium iodide (0.1713 g, 1.0 mmol) and potassium carbonate (0.5531 g, 5.2 mmol). The resulting mixture was stirred at 80 °C for 12 h. After being cooled to room temperature, the reaction mixture was poured into 40.0 mL of cold water. Then, the mixture was extracted with dichloromethane (30.0 mL × 3), and the organic layers were combined and dried over anhydrous sodium sulfate. After removal of the solvent, compound 2 was obtained as a yellow solid (1.1012 g, 98.2% yield). HRMS (ESI) m/z: calcd for C14H21N2O4 [M+1]+, 281.1501; found, 281.1512. 1H NMR (400 MHz, CDCl3) δH 8.21 (d, J = 9.3 Hz, 2H), 6.95 (d, J = 9.3 Hz, 2H), 4.10 (t, J = 6.3 Hz, 2H), 3.89-3.58 (m, 4H), 2.482.41(m, 6H), 1.92-1.85 (m, 2H), 1.74-1.67 (m, 2H). 13C NMR (100 MHz, CDCl3) δC 164.1, 141.4, 125.9, 114.4, 68.5, 67.0, 58.5, 53.7,26.9, 22.9. Synthesis of 4-(4-morpholinobutoxy)aniline (3). Compound 2 (0.8402 g, 3.0 mmol) was hydrogenated at slightly above the atmosphere pressure in methanol (20.0 mL) at 65 °C for 12 h with 10% Pd/C (0.1803 g) as a catalyst. Next, the reaction mixture was filtered through celite to remove the catalyst. Filtrate was concentrated under vacuum to yield compound 3 as a brown solid (0.6604 g, 88.0% yield). HRMS (ESI) m/z: calcd for C14H23N2O2 [M+1]+, 251.1760; found, 251.1760. 1H NMR (400 MHz, CDCl3) δH 6.73 (d, J = 8.8 Hz, 2H), 6.63 (d, J = 8.8 Hz, 2H), 3.90 (t, J = 6.3 Hz, 2H), 3.73-3.71 (m, 4H), 3.37 (s, 2H), 2.45-2.38 (m, 6H), 1.87-1.72 (m, 2H), 1.71-1.56 (m, 2H). 13C NMR (100 MHz, CDCl3) δC 152.1, 140.0, 116.4, 115.6, 68.3, 67.0, 58.7, 53.7, 27.3, 23.1. Synthesis of 8-hydroxy-1,2,3,5,6,7-hexahydropyrido [3,2,1ij]quinoline-9-carbaldehyde (4). Compound 4 was synthesized according to a literature procedure.46 Synthesis of 8-(prop-2-ynyloxy)-1,2,3,5,6,7hexahydropyrido[3,2,1-ij]quinoline-9-carbaldehyde (5). To a solution of compound 4 (0.2173 g, 1.0 mmol) and 3bromoprop-1-yne (0.2380 g, 2.0 mmol) in acetone (10.0 mL) was added potassium carbonate (0.2764 g, 2.0 mmol). The resulting suspension was refluxed for 12 h. The reaction mixture was filtered and the filtrate was concentrated under vacuum to give a crude product, which was further purified using flash silica gel chromatography (ethyl acetate/petroleum ether as eluent, v/v = 1:3) to afford compound 5 as a yellow solid (0.2002 g, 78.4% yield). HRMS (ESI) m/z: calcd for C16H18NO2 [M+1]+, 256.1338; found, 256.1337. 1H NMR (500 MHz, CDCl3) δH 10.03 (s, 1H), 7.34 (s, 1H), 4.63 (d, J = 2.4 Hz, 2H), 3.52-3.05 (m, 4H), 2.81 (t, J = 6.3 Hz, 2H), 2.73 (t, J = 6.3 Hz, 2H), 2.55 (t, J = 2.4 Hz, 1H), 2.01-1.82 (m, 4H). 13C NMR (125 MHz, CDCl3) δC 187.8, 157.8, 148.8, 127.5, 117.5, 117.0, 112.6, 78.7, 76.1, 62.1, 50.0, 49.7, 27.3, 21.4, 21.3, 20.7. Synthesis of CQ-Lyso. Compound 3 (0.2503 g, 1.0 mmol), cuprous iodide (0.0573 g, 0.3 mmol) and compound 5 (0.2662 g, 1.0 mmol) were placed in 5.0 mL dry DMF. The resulting solution was stirred at 110 °C for 4 h. After being cooled to room temperature, the mixture was poured into 40.0 mL of cold water and extracted with ethyl acetate (40.0 mL × 4). The organic layers were combined, washed with water and dried

over anhydrous sodium sulfate. Following removal of the solvent, the obtained crude product was further purified by flash silica gel chromatography (ethyl acetate/petroleum ether as eluent, v/v = 1:1) to give CQ-Lyso as a yellow solid (0.0902 g, 18.5% yield). HRMS (ESI) m/z: calcd for C30H36N3O3 [M+1]+, 486.2757; found, 486.2756. 1H NMR (400 MHz, DMSO-d6) δH 7.89 (s, 1H), 7.81 (d, J = 9.1 Hz, 1H), 7.67 (s, 1H), 7.29 (dd, J = 9.1, 2.8 Hz, 1H), 7.25 (d, J = 2.8 Hz, 1H), 5.24 (s, 2H), 4.10 (t, J = 6.5 Hz, 2H), 3.62-3.49 (m, 4H), 3.20-3.16 (m, 4H), 2.75 (t, J = 6.3 Hz, 2H), 2.63 (t, J = 6.5 Hz, 2H), 2.36-2.32 (m, 6H), 1.91-1.88 (m, 4H), 1.83-1.77 (m, 2H), 1.65-1.58 (m, 2H). 13 C NMR (100 MHz, CDCl3) δC 156.1, 153.7，148.4, 145.6, 144.4, 130.1, 129.0, 127.6, 125.1, 122.5, 121.5, 116.1, 111.0, 107.9, 106.4, 68.6, 67.9, 67.0, 58.6, 53.7, 50.1, 49.6, 27.4, 27.2, 23.1, 22.1, 21.3, 21.0.

RESULTS AND DISCUSSION Sensing properties of probe CQ-Lyso to pH. The optical properties of CQ-Lyso (5.0 µM) were studied in BrittonRobinson buffer with 30% CH3CN as a co-solvent at different pH values. The probe featured an intense absorption band between 350-470 nm with a maximum at 397 nm in neutral/basic media. When the pH was made more acidic, the 397 nm band gradually disappeared and a new band in the range 410-580 nm (λabsmax = 501 nm) emerged and the color visually changed from pale yellow to red (shown in Figure S1, ESI†). This result is the basis upon which CQ-Lyso could be used to measure pH via spectroscopic absorptivity analysis or, more crudely as a naked-eye pH detector. When excited at 470 nm, the probe displayed a strong yellow florescence with λemmax = 560 nm in neutral/basic media (Φ = 0.26, using coumarin 102 in ethanol at 25 °C as a standard, Φ = 0.93).52 In contrast, when the pH of the solution was down-regulated (shown in Figure 1) to more acidic values, the fluorescence gradually changed from yellow to red (λemmax = 613 nm, Φ = 0.12, using fluorescein in 0.1 M NaOH at 25 °C as a standard, Φ = 0.92) under the same excitation wavelength.53 Thus, increasing acidity was accompanied by red-shifts for both absorption and emission spectra of 104 nm and 53 nm, respectively, when the pH changed from neutral to acidic. It was found that the ratio between the fluorescence intensities at 613 nm and 560 nm (I613 nm/I560 nm) displayed a 10-fold enhancement (from 0.524 at pH 7.4 to 5.586 at pH 4.0,

Figure 1. Fluorescence spectra of CQ-Lyso (5.0 µM) in B-R buffers with 30% CH3CN at various pH values. Excitation wavelength was 470 nm. Inset: photographs of CQ-Lyso in B-



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R buffer with 30% CH3CN at pH 7.4 (left) and pH 4.0 (right) under 365 nm irradiation. Figure 2). Based on the Henderson-Hasselbalch equation: log[(Imax-I)/(I-Imin)] = pH - pKa, the pKa value of CQ-Lyso was calculated to be 5.0.49,54 Notably, a good linearity (R = 0.99) between the ratio and pH in the range of 4.0 to 6.0 was obtained, suggesting that this probe had the potential to detect lysosomal pH (4.0-5.5) in a ratiometric manner (shown in Figure 2, inset). It’s noted that the single-wavelength excitation for this ratiometric fluorescent probe simplified the experimental measurements and improved the data accuracy in fluorescence detection.

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Fe2+, (15) Cu2+, (16) Ba2+, (17) Mn2+, (18) glucose, (19) GSH, (20) Cys, (21) Hcy, (22) H2O2, (23) Gly, (24) Glu, (25) Val, (26) Arg, (27) Lys, (28) Try, (29) Thr, (30) Asp, (31) ClO-, (32) HS-, (33) NO3-, (34) ONOO-, (35) PO43-, (36) S2O32-, (37) SCN-, (38) SO32-, (39) SO42-. Selectivity studies. In order to evaluate the selectivity of CQLyso, we investigated the fluorescence spectra of CQ-Lyso (5.0 µM) in response to relevant interfering species including cations (Al3+, Cd2+, Co2+, Cr3+, Fe3+, Ni2+, Pb2+, Zn2+, Fe2+, Cu2+, Ba2+ and Mn2+, 0.05 mM for each; Mg2+ and Ca2+, 0.1 mM for each; Na+ and K+, 1.0 mM for each), anions (HS-, NO3-, PO43-, S2O32-, SCN-, SO32-, SO42-, 0.5 mM for each), and small biomolecules (glucose, GSH, Cys, Hcy, Gly, Glu, Val, Arg, Lys, Try, Thr and Asp, 1.0 mM for each) as well as reactive oxygen species (H2O2, 1.0 mM; ONOO-, 0.5 mM; ClO-, 0.1 mM). As shown in Figure 3, little or no interfering effects were observed on the ratio (I613 nm/I560 nm) of CQ-Lyso in the presence of all the above species with the exception of ClO- at pH 4.0. Under acidic condition, the strong oxidative ability of ClO- likely destroys the probe which leads to its failure in the determination of pH (shown in Figure S10).55 These results supports the probe has excellent selectivity for pH measurement.

Figure 2. Ratio of fluorescence intensity (I613 nm/I560 nm) of CQLyso (5.0 µM) in B-R buffers with 30% CH3CN at different pH values. Inset: the linear relationship between I613 nm/I560 nm and pH value (4.0 to 6.0).

Figure 4. The time course of the fluorescence intensity ratios (I613 nm/I560 nm) of CQ-Lyso (5.0 µM) in B-R buffer with 30% CH3CN at pH 4.0 and pH 7.4.

Figure 3. Fluorescence intensity ratio (I613 nm/I560 nm) of CQLyso (5.0 µM) in the presence of relevant species in B-R buffers with 30% CH3CN at pH 4.0 (a) and pH 7.4 (b). (1) Blank, (2) Al3+, (3) Ca2+, (4) Cd2+, (5) Co2+, (6) Cr3+, (7) Fe3+, (8) K+, (9) Mg2+, (10) Na+, (11) Ni2+, (12) Pb2+, (13) Zn2+, (14)

Figure 5. The fluorescence reversibility of CQ-Lyso (5.0 µM) in B-R buffer with 30% CH3CN between pH 4.0 and pH 7.4. Kinetic and reversible studies. The time-dependent fluorescence of CQ-Lyso (5.0 µM) at pH 4.0 and 7.4 was


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investigated by monitoring the fluorescence intensity ratio at 613 nm and 560 nm (I613 nm/I560 nm), respectively, as a function of time. As shown in Figure 4, after the addition of CQ-Lyso to B-R buffer solutions at pH 4.0 and 7.4, respectively, the intensity ratio (I613 nm/I560 nm) of this probe rapidly reached an equilibrium and remained unchanged for at least 10 min. In addition, the reversibility of this probe was also studied in B-R buffer at pH 7.4 and 4.0. As shown in Figure 5, this probe functions well even after 6 cycles. From these results, it can be concluded that this probe can server as a real-time, reversible monitor for pH measurement. Mechanism studies The optical performance of CQ-Lyso induced by pH changes is consistent with the occurrence of the protonation process depicted in Scheme 2. To support this proposed mechanism, 1 H NMR spectral analysis was performed on CQ-Lyso by titration studies using trifluoroacetic acid (TFA) in DMSOd6.31 As illustrated in Figure 6, the addition of TFA induced downfield shifts of the quinolinic protons: H2, H3, H4 and H5 in CQ-Lyso by 0.13, 0.11, 0.12 and 0.04, respectively. The occurrence of a new peak at 9.90 was assigned to proton H6’, suggesting the protonation of the nitrogen atom in the chromenoquinolin moiety. Furthermore, the line shape of proton H1 changed from a single sharp peak at 7.89 to a broad peak at 8.10 due to the protonation process of proton H6’. In order to better understand the photophysical properties of CQLyso and its protonated form CQ-Lyso+2H+, the timedependent density functional theory (TD-DFT) calculations were performed. As shown in Figure 7, the highest occupied molecular orbitals (HOMO) of CQ-Lyso and CQ-Lyso+2H+ were essentially located on the entire molecular skeleton, and the lowest unoccupied molecular orbitals (LUMO) were mainly distributed on the quinoline moiety. The lowest energy transition of CQ-Lyso was the same with that of CQLyso+2H+ from HOMO to LUMO, and the calculated excited wavelengths are 403 nm and 490 nm respectively, which were in good agreement with the experimental results (shown in table S2). It’s found that the HOMO-LUMO energy gap of CQ-Lyso+2H+ (2.6201 eV) was smaller than that of CQ-Lyso (3.4410 eV), indicating the enhancement of ICT process induced by the protonation of nitrogen atom of chromenoquinoline ring.46 Therefore, optical studies, 1H NMR analysis and theoretical calculation strongly support the mechanism shown in Scheme 2.

Figure 6. Partial 1H NMR spectra of CQ-Lyso before (a) and after (b) the addition of two equivalents of trifluoroacetic acid in DMSO-d6 solution.

Figure 7. The HOMO-LUMO energy gaps and the interfacial plots of the orbitals for CQ-Lyso and CQ-Lyso+2H+. Fluorescence Imaging in Living Cells. Encouraged by the excellent properties of CQ-Lyso in aqueous media, we evaluated the ability of this probe to visualize intracellular pH in living cells. First, an MTT assay was performed on CQLyso, the result of which showed that this probe was non-toxic for living cells below 10.0 µM (shown in Figure S2, ESI†). Next, the selectivity of CQ-Lyso for lysosomes was determined with a colocalization experiment in living HeLa cells using LysoTracker®Deep Red (1.0 µM) as a reference. HeLa cells were incubated with CQ-Lyso (5.0 µM) at 37 °C for 15 min, and followed by incubation with LysoTracker®Deep Red (1.0 µM) at 37 °C for another 15 min. Three color channels were used for these experiments: Red fluorescence channel (570-620 nm, pseud-colored as yellow for clarity) and green fluorescence channel (515-550 nm) for CQ-Lyso, deep red/infrared channel (663-738 nm) for LysoTracker®Deep Red. As shown in Figure 8, the cells exhibited both green and red fluorescence emitted by CQLyso under a 488 nm excitation. When the cells excited at 640 nm, LysoTracker®Deep Red was observed to give deep

Figure 8. Images of living HeLa cells co-stained with CQLyso (5.0 µM) and LysoTracker®Deep Red (1.0 µM). (a) Bright field; (b) green fluorescence (channel 515-550 nm for CQ-Lyso with excitation at 488 nm); (c) pseudo-yellow fluorescence (red channel 570-620 nm for CQ-Lyso with excitation at 488 nm, pseudo-colored as yellow color for clarity); (d) deep red fluorescence (deep red channel 663-738 nm for LysoTracker®Deep Red with excitation at 640 nm); (e) overlay of panels a-d; (f) the correlation of CQ-Lyso (green channel) and LysoTracker®Deep Red (deep red channel).



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Figure 9 (a) Fluorescence images of CQ-Lyso (5.0 µM) in living Hela cells at various pH. Top row: bright field images; second and third row: fluorescence images from green channel and red channel with excitation at 488 nm; fourth row: merged images of the first, second and third row; bottom row: ratio images (Representing the ratio of fluorescence intensity between green channel and red channel). (b) Ratios of Ired/Igreen at different pH.

Figure 10 (a) Fluorescence images of CQ-Lyso (5.0 µM) in living Hela cells with chloroquine as a stimulant (Top row: 0.0 µM, middle row: 100.0 µM, bottom row: 200.0 µM). First column: bright field images; second column: fluorescence images from green channel with excitation at 488 nm; third column: fluorescence images from red channel with excitation at 488 nm; fourth row: overlayed images of the first, second and third column; fifth column: ratio images (Representing the ratio of fluorescence intensity between green channel and red channel). (b) Average fluorescence intensity ratios (Ired/Igreen) between red channel and green channel. red/near infrared fluorescence signal. A Pearson’s correlation established. Cells were firstly incubated with CQ-Lyso (5.0 coefficient between the green/red channel (CQ-Lyso) and µM) in PBS buffer (pH 7.4) for 15 min at 37 °C and then deep red/near infrared channel (LysoTracker®Deep Red) incubated in buffers with various pH values (3.0-8.0) in the was calculated to be 0.97, which is an unusually high value as presence of 10.0 µM of nigericin for 30 min at 37 °C. As shown in Figure 9a, when the pH changed from 3.0 to 6.0, the compared to previously reported lysosome-targeted probes (shown in Figures 8f, S4 and S5).24,28-35,49,54,56-60 In addition, green fluorescence became stronger (second row) while the additional co-localization experiment was performed using red fluorescence signal decreased (third row). The ratio of the LysoTracker®Blue DND-22 to confirm the above findings. average fluorescence intensities from these two channels As can be seen in Figures S13 and S14, the results recorded (Ired/Igreen) was dependent of the pH value of cells: 6.31 (pH from this material corresponded exactly to those found for 3.0), 5.45 (pH 4.0), 4.96 (pH 5.0) and 3.79 (pH 6.0), which LysoTracker®Deep Red. These results provided additional was consistent with the fluorescence behavior of CQ-Lyso in strong support that CQ-Lyso was able to selectively stain aqueous media. These results support our contention that CQlysosomes in living cells with high selectivity. Lyso has the potential to serve as a sensitive detector to To investigate the performance of CQ-Lyso for quantitative measure intracellular pH in a ratiometric manner. detection of intracellular pH values, the intracellular pH In addition, the ability of CQ-Lyso to monitor dynamic pH changes in living HeLa cells was evaluated. It’s known that calibration profile of CQ-Lyso in living HeLa cells was


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the antimalarial drug chloroquine, a cell-permeable base, can stimulate living cells to increase lysosomal pH.26, 49 Initially, we examined the effect of chloroquine (100.0 and 200.0 µM) on the fluorescence behavior of the probe at pH 7.4 and 4.0 (shown in Figure S7) in solution. We found that in solution the presence of chloroquine at these concentrations had little impact on the fluorescence behavior of this probe. This positive finding allows us to study the effect of chloroquine in HeLa cells. HeLa cells were initially incubated with CQ-Lyso (5.0 µM) at 37 °C for 15 min and then incubated with varying concentrations of chloroquine (0.0 µM, 100.0 µM and 200.0 µM, respectively) at 37 °C for 30 min. As can be seen in Figure 10 that the chloroquine-treated cells showed an enhanced green fluorescence (green channel) and a decreased red fluorescence (red channel), indicating the increase of pH in lysosomes. The intensity ratios of the green and red fluorescence signals (Ired/Igreen) in the living cells were calculated to be 4.38 (no chloroquine), 3.50 (100.0 µM chloroquine) and 3.25 (200.0 µM chloroquine). Based on the intracellular pH calibration profile of CQ-Lyso in living HeLa cells, the pH values of chloroquine-treated cells were determined to be 5.4 (0.0 µM), 6.5 (100.0 µM) and 6.8 (200.0 µM), respectively. These results demonstrated that CQ-Lyso could indeed visualize the dynamic pH changes in lysosomes.

CONCLUSION In conclusion, we have developed a chromenoquinoline-based ratiometric fluorescent probe, CQ-Lyso, for lysosomal pH detection and imaging having excellent selectivity, high sensitivity and long wavelength emission. This probe can selectively stain lysosomes with an unusually high Pearson’s correlation coefficient (using LysoTracker®Deep Red (0.97) and LysoTracker®Blue DND-22 (0.95) as references). Importantly, this probe was successfully applied to quantitatively detect lysosomal pH values in a ratiometric manner under single wavelength excitation in living cells.

ASSOCIATED CONTENT Supporting Information Chemical structures of compounds, additional spectral data, confocal microscopy images, 1H NMR, 13C NMR and HRMS spectra of compounds. The Supporting Information is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * Fax: +86-731-88836954; Tel: +86-731-88836954; E-mail: [email protected] *[email protected].

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (No. U1608222) and the State Key Laboratory of Fine Chemicals (KF1606). We thank Modern Analysis and Testing Center of Central South University for NMR spectral data.

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Ratiometric Fluorescent Probe for Lysosomal pH Measurement and

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