Article pubs.acs.org/ac
Oxidative Cleavage-Based Near-Infrared Fluorescent Probe for Hypochlorous Acid Detection and Myeloperoxidase Activity Evaluation Mingtai Sun,† Huan Yu,† Houjuan Zhu,†,§ Fang Ma,†,§ Shan Zhang,†,§ Dejian Huang,‡ and Suhua Wang*,†,§ †
Institute of Intelligent Machines, Chinese Academy of Sciences, Hefei, Anhui 230031, People’s Republic of China Food Science and Technology Program, Department of Chemistry, National University of Singapore, 3 Science Drive 3, Singapore 117543 § Department of Chemistry, University of Science & Technology of China, Hefei, Anhui 230026, People’s Republic of China ‡
S Supporting Information *
ABSTRACT: A near-infrared (NIR) fluorescent probe was synthesized and demonstrated to be highly selective in reaction with hypochlorous acid (HOCl), an endogenous reactive oxygen species (ROS) produced by myeloperoxidase in neutrophils. The reaction with HOCl resulted in the NIR fluorescence quenching at 774 nm and the absorbance decreasing at 710 nm, accompanied by the appearance of a new absorption band at 520 nm. The reaction mechanism was carefully examined and proposed to proceed by initial formation of chlorohydrins and subsequent degradation. This NIR fluorescent probe was successfully applied as a selective and sensitive indicator for HOCl on the basis of either colorimetry or fluorometry, which showed detection limits of 0.13 and 0.70 μM, respectively. In addition, the molecular probe was further demonstrated for NIR fluorescence imaging of HOCl in cells and for evaluating the enzymatic activity of myeloperoxidase in the HOCl generation by measuring absorbance change.
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Apak et al. reported a spectrofluorometric probe for HOCl based on the chlorination of resorcinol and successfully developed a method for the measurement of HOCl scavenging activity.14 Here, we report a novel NIR fluorescent probe (CY-FPA) by covalently linking an electron-donating group to the cyanine backbone, which greatly increased the fluorescence quantum yield, broadened the excitation wavelength range, and tuned the reactivity for HOCl. Upon reaction to HOCl, the probe exhibited sensitive change of color and fluorescence intensity, which could be used as a colorimetric or fluorometric indicator for HOCl. The NIR fluorescence probe has been demonstrated for the determination of enzymatic activity of myeloperoxidase under physiological conditions. This molecular probe has a relatively narrow (full width at half-maximum = 60 nm) and symmetric NIR fluorescence band centered at 774 nm, which specifically and sensitively responses to HOCl, showing the potential for in vivo imaging of hypochlorous acid.
ypochlorous acid can be produced from peroxidation of chloride ions under the catalysis of heme enzyme myeloperoxidase (MPO) in neutrophils,1 and it is involved in immune defense and inflammation.2 Most developed methods for HOCl detection such as iodometric, colorimetric, and polarographic methods have limitations for living organisms.3 Thus, there is a demand to develop novel fluorescence probes for selective and sensitive detection of HOCl due to their superior sensitivity and spatiotemporal resolution4 for bioanalysis and labeling.5,6 NIR fluorescence in the 650−900 nm region are more favorable for in vivo imaging because of their deep tissue penetration, less photodamage, less light scattering, and low autofluorescence background.7 Cyanine-based compounds with spectral properties in the NIR region can be employed for designing NIR fluorescent probes.8−10 Currently, reports on efficient NIR fluorescent probes based on the cyanine fluorophore are relatively limited because of their intrinsically low photostability in aqueous environment.11 Thus, great effort has been devoted into improving the photostability and photochemical properties of these existing NIR cyanine dyes. Koshinsky et al. reported a rapid nucleic acid detection method based on the accelerated photobleaching of the lightsensitive cyanine dye.12 Nagano et al. presented a well-designed NIR fluorescent probe for reactive oxygen species (ROS) based on differential reactivity of linked cyanine dyes.13 Recently, © XXXX American Chemical Society
Received: September 22, 2013 Accepted: December 5, 2013
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NMR (CDCl3, 400 MHz): δ (ppm) 1.40 (6H, t, J = 7.2 Hz), 1.48 (12H, s), 1.95 (2H, q, J = 6.4 Hz), 2.61 (4H, t, J = 6.4 Hz), 4.05 (4H, q, J = 7.2 Hz), 4.64 (2H, s), 4.67 (2H, s), 5.92 (1H, s), 5.96 (1H, s), 6.42 (1H, d, J = 3.2 Hz), 6.48 (1H, d, J = 3.2 Hz), 7.03 (2H, d, J = 7.6 Hz), 7.16 (2H, t, J = 7.2 Hz), 7.29− 7.39 (6H, m), 7.50 (1H, d, J = 1.6 Hz), 7.55 (1H, s), 7.58 (1H, s), 7.85 (1H, td, J1 = 7.6 Hz, J2 = 1.6 Hz), 8.72 (1H, d, J = 4.8 Hz). Density Functional Theory (DFT) Calculations. To understand the difference in spectral and chemical properties between the new functionalized compound CY-FPA and the initial compound CY-Cl, the calculations have been performed on them using the Gaussian 09 program package. The groundstate geometries were optimized with B3LYP/6-31+G (d). The HOMO (highest occupied molecular orbital) and LUMO (lowest unoccupied molecular orbital) of the molecules were visualized with Gauss View 5.0. Procedures for Sensing HOCl and Other Reactive Species. Generally, 6 μL of stock solution of probe CY-FPA (1 × 10−3 M) in ethanol was diluted in 2.0 mL of PBS (pH 7.4, 50 mM PB with 10% ethanol, 0.1 M KCl) to obtain a final concentration of 3 μM. tert-Butylhydroperoxide (TBHP), sodium hypochlorite, and potassium superoxide were freshly prepared with a stock solution concentration of 3.0 mM. Hydrogen peroxide was prepared with a stock concentration of 0.1 M. Hydroxyl radical was generated in situ either by adding ferrous sulfate (9 μM) in the presence of H2O2 (180 μM)3,5,6,10,13 or by irradiating H2O2 (9 μM) at 254 nm according to the literature.15 tert-Butyl peroxyl radical (TBO·) was generated in situ by addition of ferrous sulfate (9 μM) in the presence of TBHP (180 μM). Concentrations of H2O2, NaClO, and KO2 in DMSO were determined by measuring their UV absorbance immediately before use. The fluorescence intensities were recorded before and 5 min after the addition of ROS. Other ions or anions were prepared with a stock solution concentration of 3.0 mM. These species were added into the PBS solution followed by recording the fluorescence spectra. All fluorescence spectra were recorded in the range from 710 to 870 nm using a 700 nm excitation wavelength and a 500 nm/ min scan rate. Myeloperoxidase (MPO) Activity Measurement. MPO solution was prepared by dissolving 5 units of MPO in 200 μL of water (containing 20% of glycerol). The solution was stored at 4 °C and used within 2 weeks. For monitoring the generation of HOCl by MPO in PBS, the probe solution (2 mL of PBS) was incubated with 1 μL of MPO solution in the presence or absence of H2O2, and the fluorescence intensity was measured at 37 °C and recorded for 5 min. For monitoring the generation of HOCl by MPO in PB, the probe solution was incubated with 1 μL of MPO solution and 10 μM of H2O2 in the presence or absence of Cl−. For the MPO biological activity assay by the UV−vis absorption technique, to the probe solution (8 μM of CY-FPA in 2 mL of PBS) incubated with 2 mM of Cl− and 5− 40 μM H2O2 was added 0.025 units of MPO. After 5 min, 0.1 M NaN3 was added to terminate the reaction. Then, the absorption spectra were measured at room temperature. Various control experiments were performed to validate the results of the above measurements. Fluorescence Imaging of Living Cells. The A549 cells were obtained from the hospital of the Hefei Institute of Physical Sciences and cultured in Roswell Park Memorial Institute (RPMI) medium supplemented with 10% fetal bovine serum in an atmosphere of 5% CO2 and 95% air at 37 °C. The
EXPERIMENTAL SECTION Materials. The chemicals and solvents were obtained from commercial sources (Sigma Aldrich or Aladdin) and were used directly without further purification unless specified. The solvents N,N-dimethylformamide and acetonitrile were further purified before use by distillation using all-glass stills and were dried over molecular sieves before use. The pH values of the solution were adjusted to 5.86, 6.3, 6.78, 7.40, 7.77, 8.07, 8.64, and 9.18 by phosphate-buffered saline, which was prepared by disodium hydrogen phosphate dodecahydrate and sodium dihydrogen phosphate dihydrate. Aqueous solutions were all prepared using ultrapure water (18.2 MΩ·cm) from a Millipore water purification system, and all glassware was cleaned with ultrapure water and then dried before use. The concentration of sodium hypochlorite (5.5% aqueous solution) has been verified by iodometry before use. MPO was obtained from Sigma Aldrich. Instrumentation and Methods. Fluorescence measurement was recorded on a Perkin-Elmer LS-55 luminescence spectrometer (Liantriant, U.K.) equipped with a plotter unit and a quartz cell (1 cm × 1 cm). UV−vis absorption was recorded at room temperature on a Shimadzu UV-2550 spectrometer. The molar absorption coefficient of CY-FPA in PBS was determined on the basis of the Beer−Lambert Law and the absorbance of the freshly prepared probe solutions with concentrations at 1, 2, 3, 4, 5, 6, and 8 μM. The fluorescence quantum yield was measured using the compound CY-Cl in ethanol (Φ = 0.085) as a standard. Photographs were taken with a canon 350D digital camera. 1H NMR spectra were measured using a Varian Mercury-400 NMR spectrometer, and mass spectrometry was performed on a Thermo Proteome XLTQ MS mass spectrometer in ES positive mode. Silica gel-60 (230−400 mesh) were used as the solid phases for column chromatography. Thin-layer chromatography (TLC) was performed by using Merck F254 silica gel-60 plates. TLC plates were viewed with UV light or after developing with ninhydrin stain. Synthesis of 1-(Furan-2-yl)-N-(pyridin-2-ylmethyl)methanamine (FPA). A portion (270 mg, 2.5 mmol) of 2aminomethylpyridine was mixed with furaldehyde (200 mg, 2.0 mmol) in 6 mL of EtOAc to yield a brown solution, which was stirred at room temperature for 10 h. The solvent was removed by evaporation to give the crude intermediate imine, which was dissolved in 5 mL of MeOH and mixed with NaBH4 (150 mg, 4.0 mmol). The mixture was stirred for 2.5 h, the reaction was quenched with 10 mL of saturated brine, and the brine was extracted with dichloromethane (3 × 10 mL). The combined organic layers were dried over anhydrous Na2SO4, and the solvent was evaporated to yield brown oil. TLC analysis showed enough purity (>90%) for the next reaction. Synthesis of 1-Ethyl-2-((E)-2-((E)-3-((E)-2-(1-ethyl-3,3dimethylindolin-2-ylidene)ethylidene)-2-((furan-2ylmethyl)(pyridin-2-ylmethyl)amino)cyclohex-1-en-1yl)vinyl)-3,3-dimethyl-3H-indol-1-ium iodide (CY-FPA). One hundred twenty-eight milligrams (0.2 mmol) of compound CY-Cl and 150 mg (0.8 mmol) of FPA were dissolved in 5 mL of anhydrous DMF in a 25 mL round-bottom flask. The mixture was stirred at 80 °C under nitrogen for 24 h. Then, the mixture was evaporated to dryness on a rotary evaporator. The solid was purified on silica gel chromatography eluted with dichloromethane/methanol (20:1 v/v, Rf = 0.22 by TLC). Yield (82 mg, 51.8%). ESI-MS: 663.4 (M − I−). 1H B
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solubility in water and is resistant to photobleaching (Figure S4). These properties made it a suitable fluorescent probe for biological analysis. Compared with unmodified CY-Cl, the introduction of a moderate electron-donating group increased the electron density on the polymethine chain of CY-FPA (theoretical calculation, Figure S-5). Thus, we can assume that the CY-FPA molecule possesses remarkable potential to be oxidized in the presence of reactive oxygen species such as HOCl, which is evidenced by the experimental results. For practical application, the pH effect on the fluorescence of CY-FPA in the absence and presence of HOCl/ClO− was examined (Figure S-6). The variation of pH does not significantly affect the fluorescence of the probe in the absence of hypochlorite. However, the fluorescence is quenched almost completely by hypochlorite at pH values lower than the pKa of HOCl (pKa = 7.46), whereas the fluorescence quenching efficiency decreases gradually as the pH value increased higher than the pKa. The results suggest that it is hypochlorous acid, not the hypochlorite anion, that quenches the fluorescence of CY-FPA. Therefore, it is appropriate to carry out all the experiments in physiological conditions in PBS buffer with pH 7.4, and under these conditions, the reactive species hypochlorous acid has sufficient concentration. To explore its reaction behavior with HOCl, the probe CYFPA was treated with hypochlorous acid at ambient temperature in an aqueous environment (Figure 1). The fluorescence
cells were grown on uncoated 35 mm diameter glass-bottomed dishes. The confocal fluorescence images were acquired through a Carl Zeiss microscopy (LSM710) equipped with a cooled CCD camera using 663 nm as excitation wavelength. In the control experiment, the cells were incubated with 10 μM solution of probe CY-FPA for 10 min at room temperature, washed with 1 mL of PBS three times, and then the fluorescence images were obtained at 0, 1, and 4 days, respectively. For detection of HOCl in vivo, the cells were incubated with a 10 μM solution of probe CY-FPA for 10 min at room temperature. The cells were washed with 1 mL PBS three times before 50 μM of HOCl was added to the mixture. The mixture was mounted on a microscope stage to give fluorescence image immediately at 1 and 12 h. Statistical Evaluation. Descriptive statistical analyses were performed using Origin 8.0 for calculating the means and the standard error of the mean. Results were expressed as the mean ± standard deviation (SD).14
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RESULTS AND DISCUSSION The target NIR fluorescent probe was synthesized by modifying the polymethine group with a moderate electron-donating group, which tuned the reactivity of one of the double bond to be selective for hypochlorous acid, as shown in Scheme 1. The Scheme 1. Synthesis of the Probe CY-FPA and Its Reaction Properties with HOCl. (a) FPA, DMF, 80 °C; FPA: 1(Furan-2-yl)-N-(pyridin-2-ylmethyl)methanamine. The Polymethine Group of CY-Cl Was Modified with FPA by Substitution Reaction To Give the NIR Fluorescent Probe CY-FPA, Which Tuned the Reactivity of Bonds To Be Selective for HOCl
Figure 1. Fluorescence quenching (λex = 700 nm) of CY-FPA (3 μM) upon addition of HOCl (0−9 μM) at pH 7.4 in 50 mM PBS/EtOH (9:1). I and I0 are the fluorescence intensity of CY-FPA in the presence and absence of HOCl, respectively.
structure of the probe was confirmed by ESI-MS and 1H NMR (Figure S-1). Compound CY-FPA displays absorption and emission maxima in the NIR region at 700 and 774 nm, respectively, with a relatively large Stokes shift (74 nm). The molar absorption coefficient of CY-FPA was measured as ε = 1.72 × 105 M−1 cm−1 using the Beer−Lambert Law, within which the relative standard deviation (RSD) was 1.6% (Figure S-2). CY-FPA also has better fluorescent quantum yield (Φ = 0.177 in ethanol) and broader excitation spectrum than the unmodified indocyanine dye CY-Cl (Φ = 0.085 in ethanol16) (Figure S-3). For comparison, the absorption and emission profiles of CY-FPA in different solvents including water, acetonitrile, methanol, acetone, ethanol, and 2-propanol are compiled in Table S-1. It can be seen that the maximum absorption slightly red-shifts when the solvent dielectric constants decrease. As the solvent changes from water to ethanol, the solvent polarity decreases and a 10 nm red-shift of the absorption maximum is observed, indicating an intramolecular charge-transfer state.17 The probe CY-FPA has good
intensity of CY-FPA dispersed in PBS decreased gradually upon addition of HOCl with a good linear relationship (R2 = 0.995), which can be used for the quantification of HOCl. There is no new emission band generated after the addition of HOCl, which indicates that the CY-FPA has been oxidized to nonfluorescent species by HOCl. The limit of detection (LOD) and limit of quantification (LOQ) for HOCl were calculated using the equations: LOD = 3 σ/k and LOQ = 10 σ/ k, respectively, where σ is the standard deviation of a blank and k is the slope of the calibration line. The LOD and LOQ for HOCl were found to be 0.70 and 2.33 μM, respectively, which is comparable to that of the resorcinol probe for HOCl.14 Unlike CY-FPA, the fluorescence of CY-Cl neither can be quenched by excess amount of HOCl nor is the absorption affected by HOCl (Figure S-7). The result suggested that the C
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Figure 2b represents the distinct color change of CY-FPA solution from blue to purple and then to pink as the concentration of HOCl increased, which could be used for ratiometric detection. Other reactive oxygen species such as H2O2, ·O2−, TBO·, and TBHP showed no apparent affect on the absorption of the probe (Figure S-11), except for ·OH, which resulted in a slightly decrease of the absorption band at 710 nm (Figure S-12). The result illustrates that the probe CYFPA has good selectivity to HOCl and can be conveniently used for its detection by colorimetric method. To examine the reaction mechanism of CY-FPA with HOCl, the intermediates and product were carefully analyzed by mass spectra (Figure 3). The reaction of CY-FPA with 1 equiv of HOCl first produced three new peaks, which belong to intermediate chlorohydrin CY-1, epoxide CY-2, and glycol CY3. Upon addition of 2 equiv of HOCl, one more new peak at m/z 506.28 was detected in addition to CY-2 and CY-3. Particularly, CY-2 was the main product of the reaction. Upon addition of excess HOCl (5 equiv), the ESI-MS spectra gave three peaks at m/z = 679.40, 506.28, and 212.10, which were assigned to epoxides and oxidative decomposition products. These results suggested that CY-FPA experienced an electrophilic addition on one of the double bonds to form chlorohydrin CY-1 with m/z = 715.41 and then quickly generated epoxide CY-2 through dehydrochlorination. Part of the epoxide CY-2 was hydrolyzed to form glycol CY-3, which was subsequently cleaved to produce CY-4 and CY-5 by HOCl. The reaction mechanism and reactive position are apparently different from those of other cyanine dyes by photoinduced decomposition reported by Chang et al.19 From the production of CY-4 and CY-5, we can affirm that the electrophilic addition of a double bond with HOCl occurred at the side of the uncharged 3,3-bimethylindolinene framework. This could be attributed to both the steric hindrance of the FPA group and the higher electron density of the double bond at the uncharged side. It has been reported that chlorohydrins, epoxides, and glycol intermediates were produced when HOCl reacted with an unsaturated bond of cholesterol, lipids, phosphatidylcholines, and so forth.20 This is in agreement with our observation that when CY-FPA reacted with HOCl, nonfluorescent epoxides and glycol intermediates were obtained and both the fluorescence and maximum absorption decreased, accompanied by the appearance of a new absorption band. The reaction of CY-FPA with excess HOCl within 30 min gave other new peaks. These peaks corresponded to continuous oxidation product of the epoxide (Figure S-13). These results suggested that CY-FPA rapidly reacted with HOCl to form the epoxide intermediates, followed by slow hydrolysis to glycol. The glycol intermediates can be further oxidized to aldehyde and carboxyl compounds by HOCl. Myeloperoxidase (MPO) mediates the peroxidation of chloride ions by H2O2 in physiological conditions to produce hypochlorous acid. So, the probe could be used to monitor the production of HOCl. Figure 4a shows the fluorescence intensity of CY-FPA with H2O2 in PBS 7.4 against the addition of MPO. Negative control experiments were conducted to rule out the direct quenching effect of MPO, which showed no obvious fluorescence quenching in the absence of either MPO or H2O2. These observations strongly suggest that it is the HOCl generated by H2O2 and Cl− in the presence of MPO that oxidizes CY-FPA. The fluorescence time dependence was also consistent with those reported for known myeloperoxidasemediated hypochlorous acid production.5 More control experi-
initial indocyanine CY-Cl is stable against HOCl, and the introduction of the FPA group to the cyanine dye increases the reactivity by activating one of the double bonds of the polymethine group, which enhances the fluorescence response of CY-FPA. The responses of the probe to other reactive species including hydrogen peroxide (H2O2), superoxide anions (·O2−), tert-butyl peroxyl radical (TBO·), tert-butylhydroperoxide (TBHP), and hydroxyl radical (·OH) were carefully examined at the same conditions as HOCl (Figure S-8). Clearly, the fluorescence was greatly quenched by hypochlorous acid and only slightly quenched by hydroxyl radicals generated from either Fenton reaction or UV light irradiation of hydrogen peroxide. However, other ROS showed no apparent fluorescence quenching effect. The results indicate that the CY-FPA does not react with these ROS or that the reaction rate is very slow. This could be explained in that HOCl not only has the highest standard redox potential (1490 mV) in comparison with H2O2 (320 mV), ·O2− (940 mV), TBHP (−900 mV), and TBO· (1000 mV)18 but also has a partial positively charged chlorine atom which readily initiates electrophilic addition to the double bond. In addition, none of the biological relevant metal ions or anions such as Zn2+, Cu2+, Fe3+, Ca2+, Mg2+, NO2−, NO3− resulted in obvious fluorescence responses (Figure S-9). These results suggested that the fluorescence probe CY-FPA exhibits good selectivity toward hypochlorous acid in physiological conditions. Furthermore, the reaction of CY-FPA with hypochlorous acid results in a new absorption band at 520 nm accompanied by gradually decreasing of the original absorption peak at 710 nm, leading to the formation of an isosbestic point at 570 nm (Figure 2a). A good linear relationship between the absorbance
Figure 2. (a) UV−vis absorption spectral responses of a 3 μM solution of CY-FPA in aqueous solution upon the addition of HOCl. Absorption band at 710 nm gradually decreased, and a new absorption band emerged at 520 nm as the amount of HOCl increased. (b) Dependence of the color change of probe CY-FPA (3 μM) on the amount of HOCl (1−9 μM) added in 50 mM PBS (pH 7.4, 100 mM KCl). Photos were taken under daylight.
ratio of A520/A710 and the added amount of HOCl was obtained (Figure S-10). The LOD based on the colorimetry was thus measured to be 0.13 μM, which was slightly lower than those of the CY-FPA NIR probe (0.70 μM) and the resorcinol probe (0.48 μM).14 The higher sensitivity of the colorimetry may be attributed to the probe’s high molar absorption coefficient. D
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Figure 3. Possible reaction scheme of CY-FPA with HOCl, and the corresponding evidence of ESI-MS spectra. (a) CY-FPA before the addition of HOCl; (b) CY-FPA plus 1 equiv of HOCl; (c) CY-FPA plus 2 equiv of HOCl ; and (d) CY-FPA plus more excessive HOCl (5 equiv).
added, the quenching effect became apparent. The fluorescence quenching efficiency was greatly increased as higher concentration of chloride ions (2 mM) was added, which is in the physiological range of intracellular chloride ion concentration.21 These results clearly indicated that the probe is also applicable to evaluate the enzymatic MPO system. The absorption spectral responses of the probe to MPO are consistent with the fluorescence responses, further implying that MPO catalyzes the reaction between H2O2 and Cl− in aqueous solution to produce HOCl. HOCl quickly reacts with the probe and rapidly reaches equilibrium (Figure S-14), whereas the MPO−H2O2−Cl system reacts with the probe within 10 min due to the diffusion of generated HOCl (Figure S-15). When the inhibitor of MPO, NaN3, was added into the mixture at 3 min, both the decreasing of absorbance at 710 nm and the increasing of absorbance at 520 nm were terminated. The absorbance at the two wavelengths remained constant with further increasing reaction time, suggesting that the catalytic activity of MPO is completely inhibited by NaN3.3 In addition, the new absorbance at 520 nm was linearly dependent on the amount of HOCl added, with a correlation coefficient of 0.998 (Figure 5a). Thus, the enzymatic activity of MPO in the MPO−H2O2−Cl system can be measured using the absorbance at 520 nm. Figure 5b represents the dependence of absorbance at 520 nm on the amounts of MPO in the presence of fixed and sufficient concentrations of H2O2 (200 μM) and Cl− (2 mM). It can be seen that the absorbance increased linearly with correlation coefficient of 0.998. The enzymatic activity can thus be expressed as MPO amount ∝ A−A0 or MPO amount ∝ nHOCl, where A−A0 is the increased absorbance at 520 nm; nHOCl is added HOCl or produced HOCl in the reaction system. This method takes advantage of the absorption change of CY-FPA upon the exposure to HOCl to achieve the measurement of MPO activity. The application of the NIR fluorescent probe for live cell imaging was preliminarily demonstrated. A549 cells were first incubated with 10 μM of CY-FPA for 10 min, and the stained cells were still alive and showed strong fluorescence after incubation for 4 days (Figure 6a, d−f). These results suggested that the probe is cell-membrane permeable and shows low cytotoxicity to living cells, which is a good fluorescence probe
Figure 4. (a) Time dependence of fluorescence intensity of CY-FPA (3 μM) upon the addition of MPO and H2O2 in PBS 7.4. [MPO] = 0.025 units, [H2O2] = 10 μM; (b) Time dependence of fluorescence intensity of CY-FPA (3 μM) upon the addition of MPO, H2O2, and Cl− in PB 7.4. [MPO] = 0.025 units, [H2O2] = 10 μM.
ments were carried out to further confirm the fluorescence quenching effect of HOCl generated by MPO catalysis. The experiments for the confirmation of the role of chloride ion in the generation of HOCl were done in PB 7.4 without chloride ions (Figure 4b). Clearly, the fluorescence intensity of CY-FPA was nearly constant in the presence of both MPO and the mixture of MPO−H2O2, indicating very slow oxidation of the probe without chloride ion. When 10 μM of chloride ions was E
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CY-FPA was appropriate for cell imaging and responded to HOCl by fluorescence quenching in the living cells.
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CONCLUSIONS In summary, we have demonstrated a novel dual functional NIR fluorescent probe capable of ultrasensitive and selective detection of hypochlorous acid, monitoring the HOCl generation by MPO, and detecting the existence of HOCl by the naked eye. The probe was synthesized by activating the polymethine chain of a cyanine dye to be selectively reactive to hypochlorous acid. The reaction mechanism between the probe and HOCl was confirmed by mass spectra as electrophilic addition to the polymethine chain, followed by oxidation cleavage. The NIR probe shows good cell-membrane permeability and low cytotoxicity to living cells, which is demonstrated as a new fluorescent probe for HOCl imaging in living cells. In addition, the enzymatic activity of MPO can be evaluated on the basis of a colorimetric method.
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ASSOCIATED CONTENT
* Supporting Information S
Additional information as noted in the text (Figures S1−S15). This material is available free of charge via the Internet at http://pubs.acs.org.
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Figure 5. (a) Increased absorbance of CY-FPA (8 μM) at 520 nm upon the addition of HOCl (0−24 μM) in PBS 7.4. (b) Standard curves for detection of MPO using the CY-FPA assay. The horizontal axis represents the amounts of MPO added into the mixture of H2O2 (200 μM) and Cl− (2 mM) in the presence of the CY-FPA probe (8 μM). Vertical axis represents the increased absorbance at 520 nm after addition of MPO.
AUTHOR INFORMATION
Corresponding Author
*E-mail: [email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the National Basic Research Program of China (2011CB933700), the Natural Science Foundation of China (Nos. 21077108, 21302187, 21205120), and the Innovation Project of Chinese Academy of Sciences (KJCX2-YW-H29).
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REFERENCES
(1) Pattison, D. I.; Davies, M. J. Biochemistry 2006, 45, 8152−8162. (2) (a) Roos, D.; Winterbourn, C. C. Science 2002, 296, 669−671. (b) Fang, F. C. Nat. Rev. Microbiol. 2004, 2, 820−832. (3) (a) Yan, Y.; Wang, S.; Liu, Z.; Wang, H.; Huang, D. Anal. Chem. 2010, 82, 9775−9781. (b) Yang, Y.; Lu, H.; Wang, W.; Liau, I. Anal. Chem. 2011, 83, 8267−8272. (4) (a) Chan, J.; Dodani, S. C.; Chang, C. J. Nat. Chem. 2012, 4, 973−984. (b) Yang, Y.; Zhao, Q.; Feng, W.; Li, F. Chem. Rev. 2013, 113, 192−270. (c) Schäferling, M. Angew. Chem., Int. Ed. 2012, 51, 3532−3554. (5) Figueiredo, J. L.; Aikawa, E.; McCarthy, J.; Weissleder, R.; Hilderbrand, S. A. J. Am. Chem. Soc. 2009, 131, 15739−15744. (6) (a) Kenmoku, S.; Urano, Y.; Kojima, H.; Nagano, T. J. Am. Chem. Soc. 2007, 129, 7313−7318. (b) Yuan, L.; Lin, W.; Xie, Y.; Chen, B.; Song, J. Chem.Eur. J. 2012, 18, 2700−2706. (c) Sun, Y.-Q.; Liu, J.; Lv, X.; Liu, Y.; Zhao, Y.; Guo, W. Angew. Chem., Int. Ed. 2012, 51, 7634−7636. (7) (a) Achilefu, S. Angew. Chem., Int. Ed. 2010, 49, 9816−9818. (b) Ntziachristos, V.; Tung, C. H.; Bremer, C.; Weissleder, R. Nat. Med. 2002, 8, 757−760. (c) Yuan, L.; Lin, W.; Zheng, K.; He, L.; Huang, W. Chem. Soc. Rev. 2013, 42, 622−661. (d) Weissleder, R.; Ntziachristos, V. Nat. Med. 2003, 9, 123−128. (e) Leevy, W. M.; Gammon, S. T.; Jiang, H.; Johnson, J. R.; Maxwell, D. J.; Jackson, E. N.; Marquez, M.; Worms, D. P.; Smith, B. D. J. Am. Chem. Soc. 2006, 128, 16476−16477.
Figure 6. Fluorescence images of cell incubated with 10 μM of CYFPA (a) and further treated with HOCl (50 μM) for 1 h (b), 12 h (c). Fluorescence image of A549 cells incubated with only CY-FPA for 1 h (d), 12 h (e), and 4 days (f).
for cell imaging. Specifically, the probe penetrated the cell membrane very fast but could not be phagocytized by cell nucleus. When the living A549 cells loaded with CY-FPA were then treated with HOCl, the fluorescence was gradually decreased and finally disappeared. Figure 6b,c show the quenching effect of HOCl on the intracellular CY-FPA (upper panel). It can be seen that the fluorescence of CYFPA was completely quenched after treatment with HOCl (50 μM) for 12 h. In contrast, no apparent quenching effect was observed without HOCl treatment (lower panel) even for a longer incubation time. These results indicate that the probe F
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(8) (a) Qian, G.; Wang, Z. Y. Chem.Asian J. 2010, 5, 1006−1029. (b) Luo, S.; Zhang, E.; Su, Y.; Cheng, T.; Shi, C. Biomaterials 2011, 32, 7127−7138. (c) Kiyose, K.; Kojima, H.; Nagano, T. Chem.Asian J. 2008, 3, 506−515. (9) (a) Sasaki, E.; Kojima, H.; Nishimatsu, H.; Urano, Y.; Kikuchi, K.; Hirata, Y.; Nagano, T. J. Am. Chem. Soc. 2005, 127, 3684−3685. (b) Kiyose, K.; Kojima, H.; Urano, Y.; Nagano, T. J. Am. Chem. Soc. 2006, 128, 6548−6549. (10) Kundu, K.; Knight, S. F.; Willett, N.; Lee, S.; Taylor, W. R.; Murthy, N. Angew. Chem., Int. Ed. 2009, 48, 299−303. (11) Engel, E.; Schraml, R.; Maisch, T.; Kobuch, K.; Konig, B.; Szeimies, R. M.; Hillenkamp, J.; Baumler, W.; Vasold, R. Invest. Ophthalmol. Visual Sci. 2008, 49, 1777−1783. (12) Wang, M.; Davis, R. H.; Rafinski, Z.; Jedrzejewska, B.; Choi, K. Y.; Zwick, M.; Bupp, C.; Izmailov, A.; Paczkowski, J.; Warner, B.; Koshinsky, H. Anal. Chem. 2009, 81, 2043−2052. (13) Oushiki, D.; Kojima, H.; Terai, T.; Arita, M.; Hanaoka, K.; Urano, Y.; Nagano, T. J. Am. Chem. Soc. 2010, 132, 2795−2801. (14) Ö zyürek, M.; Bekdeşer, B.; Gücļ ü, K.; Apek, R. Anal. Chem. 2012, 84, 9529−9536. (15) (a) Baxendale, J. H.; Wilson, J. A. Trans. Faraday Soc. 1957, 53, 344−356. (b) Suzuki, T.; Moriwaki, N.; Kurokawa, K.; Inukai, M. Bioorg. Med. Chem. Lett. 2009, 19, 3217−3219. (16) Chen, G.; Song, F.; Wang, J.; Yang, Z.; Sun, S.; Fan, J.; Qiang, X.; Wang, X.; Dou, B.; Peng, X. Chem. Commun. 2012, 48, 2949− 2951. (17) Peng, X.; Song, F.; Lu, E.; Wang, Y.; Zhou, W.; Fan, J.; Gao, Y. J. Am. Chem. Soc. 2005, 127, 4170−4171. (18) Buettner, G. R. Arch. Biochem. Biophys. 1993, 300, 535−543. (19) Samanta, A.; Vendrell, M.; Dasa, R.; Chang, Y. T. Chem. Commun. 2010, 46, 7406−7408. (20) Panasenko, O. M.; Osipov, A. N.; Schiller, J.; Arnhold, J. Biochemistry 2002, 67, 889−900. (21) Friedel, P.; Bregestovski, P.; Medina, I. Front. Mol. Neurosci. 2013, DOI: 10.3389/fnmol.2013.00007.
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dx.doi.org/10.1021/ac403603r | Anal. Chem. XXXX, XXX, XXX−XXX
Oxidative Cleavage-Based Near-Infrared Fluorescent Probe for Hypochlorous Acid Detection and Myeloperoxidase Activity Evaluation Mingtai Sun,† Huan Yu,† Houjuan Zhu,†,§ Fang Ma,†,§ Shan Zhang,†,§ Dejian Huang,‡ and Suhua Wang*,†,§ †
Institute of Intelligent Machines, Chinese Academy of Sciences, Hefei, Anhui 230031, People’s Republic of China Food Science and Technology Program, Department of Chemistry, National University of Singapore, 3 Science Drive 3, Singapore 117543 § Department of Chemistry, University of Science & Technology of China, Hefei, Anhui 230026, People’s Republic of China ‡
S Supporting Information *
ABSTRACT: A near-infrared (NIR) fluorescent probe was synthesized and demonstrated to be highly selective in reaction with hypochlorous acid (HOCl), an endogenous reactive oxygen species (ROS) produced by myeloperoxidase in neutrophils. The reaction with HOCl resulted in the NIR fluorescence quenching at 774 nm and the absorbance decreasing at 710 nm, accompanied by the appearance of a new absorption band at 520 nm. The reaction mechanism was carefully examined and proposed to proceed by initial formation of chlorohydrins and subsequent degradation. This NIR fluorescent probe was successfully applied as a selective and sensitive indicator for HOCl on the basis of either colorimetry or fluorometry, which showed detection limits of 0.13 and 0.70 μM, respectively. In addition, the molecular probe was further demonstrated for NIR fluorescence imaging of HOCl in cells and for evaluating the enzymatic activity of myeloperoxidase in the HOCl generation by measuring absorbance change.
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Apak et al. reported a spectrofluorometric probe for HOCl based on the chlorination of resorcinol and successfully developed a method for the measurement of HOCl scavenging activity.14 Here, we report a novel NIR fluorescent probe (CY-FPA) by covalently linking an electron-donating group to the cyanine backbone, which greatly increased the fluorescence quantum yield, broadened the excitation wavelength range, and tuned the reactivity for HOCl. Upon reaction to HOCl, the probe exhibited sensitive change of color and fluorescence intensity, which could be used as a colorimetric or fluorometric indicator for HOCl. The NIR fluorescence probe has been demonstrated for the determination of enzymatic activity of myeloperoxidase under physiological conditions. This molecular probe has a relatively narrow (full width at half-maximum = 60 nm) and symmetric NIR fluorescence band centered at 774 nm, which specifically and sensitively responses to HOCl, showing the potential for in vivo imaging of hypochlorous acid.
ypochlorous acid can be produced from peroxidation of chloride ions under the catalysis of heme enzyme myeloperoxidase (MPO) in neutrophils,1 and it is involved in immune defense and inflammation.2 Most developed methods for HOCl detection such as iodometric, colorimetric, and polarographic methods have limitations for living organisms.3 Thus, there is a demand to develop novel fluorescence probes for selective and sensitive detection of HOCl due to their superior sensitivity and spatiotemporal resolution4 for bioanalysis and labeling.5,6 NIR fluorescence in the 650−900 nm region are more favorable for in vivo imaging because of their deep tissue penetration, less photodamage, less light scattering, and low autofluorescence background.7 Cyanine-based compounds with spectral properties in the NIR region can be employed for designing NIR fluorescent probes.8−10 Currently, reports on efficient NIR fluorescent probes based on the cyanine fluorophore are relatively limited because of their intrinsically low photostability in aqueous environment.11 Thus, great effort has been devoted into improving the photostability and photochemical properties of these existing NIR cyanine dyes. Koshinsky et al. reported a rapid nucleic acid detection method based on the accelerated photobleaching of the lightsensitive cyanine dye.12 Nagano et al. presented a well-designed NIR fluorescent probe for reactive oxygen species (ROS) based on differential reactivity of linked cyanine dyes.13 Recently, © XXXX American Chemical Society
Received: September 22, 2013 Accepted: December 5, 2013
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NMR (CDCl3, 400 MHz): δ (ppm) 1.40 (6H, t, J = 7.2 Hz), 1.48 (12H, s), 1.95 (2H, q, J = 6.4 Hz), 2.61 (4H, t, J = 6.4 Hz), 4.05 (4H, q, J = 7.2 Hz), 4.64 (2H, s), 4.67 (2H, s), 5.92 (1H, s), 5.96 (1H, s), 6.42 (1H, d, J = 3.2 Hz), 6.48 (1H, d, J = 3.2 Hz), 7.03 (2H, d, J = 7.6 Hz), 7.16 (2H, t, J = 7.2 Hz), 7.29− 7.39 (6H, m), 7.50 (1H, d, J = 1.6 Hz), 7.55 (1H, s), 7.58 (1H, s), 7.85 (1H, td, J1 = 7.6 Hz, J2 = 1.6 Hz), 8.72 (1H, d, J = 4.8 Hz). Density Functional Theory (DFT) Calculations. To understand the difference in spectral and chemical properties between the new functionalized compound CY-FPA and the initial compound CY-Cl, the calculations have been performed on them using the Gaussian 09 program package. The groundstate geometries were optimized with B3LYP/6-31+G (d). The HOMO (highest occupied molecular orbital) and LUMO (lowest unoccupied molecular orbital) of the molecules were visualized with Gauss View 5.0. Procedures for Sensing HOCl and Other Reactive Species. Generally, 6 μL of stock solution of probe CY-FPA (1 × 10−3 M) in ethanol was diluted in 2.0 mL of PBS (pH 7.4, 50 mM PB with 10% ethanol, 0.1 M KCl) to obtain a final concentration of 3 μM. tert-Butylhydroperoxide (TBHP), sodium hypochlorite, and potassium superoxide were freshly prepared with a stock solution concentration of 3.0 mM. Hydrogen peroxide was prepared with a stock concentration of 0.1 M. Hydroxyl radical was generated in situ either by adding ferrous sulfate (9 μM) in the presence of H2O2 (180 μM)3,5,6,10,13 or by irradiating H2O2 (9 μM) at 254 nm according to the literature.15 tert-Butyl peroxyl radical (TBO·) was generated in situ by addition of ferrous sulfate (9 μM) in the presence of TBHP (180 μM). Concentrations of H2O2, NaClO, and KO2 in DMSO were determined by measuring their UV absorbance immediately before use. The fluorescence intensities were recorded before and 5 min after the addition of ROS. Other ions or anions were prepared with a stock solution concentration of 3.0 mM. These species were added into the PBS solution followed by recording the fluorescence spectra. All fluorescence spectra were recorded in the range from 710 to 870 nm using a 700 nm excitation wavelength and a 500 nm/ min scan rate. Myeloperoxidase (MPO) Activity Measurement. MPO solution was prepared by dissolving 5 units of MPO in 200 μL of water (containing 20% of glycerol). The solution was stored at 4 °C and used within 2 weeks. For monitoring the generation of HOCl by MPO in PBS, the probe solution (2 mL of PBS) was incubated with 1 μL of MPO solution in the presence or absence of H2O2, and the fluorescence intensity was measured at 37 °C and recorded for 5 min. For monitoring the generation of HOCl by MPO in PB, the probe solution was incubated with 1 μL of MPO solution and 10 μM of H2O2 in the presence or absence of Cl−. For the MPO biological activity assay by the UV−vis absorption technique, to the probe solution (8 μM of CY-FPA in 2 mL of PBS) incubated with 2 mM of Cl− and 5− 40 μM H2O2 was added 0.025 units of MPO. After 5 min, 0.1 M NaN3 was added to terminate the reaction. Then, the absorption spectra were measured at room temperature. Various control experiments were performed to validate the results of the above measurements. Fluorescence Imaging of Living Cells. The A549 cells were obtained from the hospital of the Hefei Institute of Physical Sciences and cultured in Roswell Park Memorial Institute (RPMI) medium supplemented with 10% fetal bovine serum in an atmosphere of 5% CO2 and 95% air at 37 °C. The
EXPERIMENTAL SECTION Materials. The chemicals and solvents were obtained from commercial sources (Sigma Aldrich or Aladdin) and were used directly without further purification unless specified. The solvents N,N-dimethylformamide and acetonitrile were further purified before use by distillation using all-glass stills and were dried over molecular sieves before use. The pH values of the solution were adjusted to 5.86, 6.3, 6.78, 7.40, 7.77, 8.07, 8.64, and 9.18 by phosphate-buffered saline, which was prepared by disodium hydrogen phosphate dodecahydrate and sodium dihydrogen phosphate dihydrate. Aqueous solutions were all prepared using ultrapure water (18.2 MΩ·cm) from a Millipore water purification system, and all glassware was cleaned with ultrapure water and then dried before use. The concentration of sodium hypochlorite (5.5% aqueous solution) has been verified by iodometry before use. MPO was obtained from Sigma Aldrich. Instrumentation and Methods. Fluorescence measurement was recorded on a Perkin-Elmer LS-55 luminescence spectrometer (Liantriant, U.K.) equipped with a plotter unit and a quartz cell (1 cm × 1 cm). UV−vis absorption was recorded at room temperature on a Shimadzu UV-2550 spectrometer. The molar absorption coefficient of CY-FPA in PBS was determined on the basis of the Beer−Lambert Law and the absorbance of the freshly prepared probe solutions with concentrations at 1, 2, 3, 4, 5, 6, and 8 μM. The fluorescence quantum yield was measured using the compound CY-Cl in ethanol (Φ = 0.085) as a standard. Photographs were taken with a canon 350D digital camera. 1H NMR spectra were measured using a Varian Mercury-400 NMR spectrometer, and mass spectrometry was performed on a Thermo Proteome XLTQ MS mass spectrometer in ES positive mode. Silica gel-60 (230−400 mesh) were used as the solid phases for column chromatography. Thin-layer chromatography (TLC) was performed by using Merck F254 silica gel-60 plates. TLC plates were viewed with UV light or after developing with ninhydrin stain. Synthesis of 1-(Furan-2-yl)-N-(pyridin-2-ylmethyl)methanamine (FPA). A portion (270 mg, 2.5 mmol) of 2aminomethylpyridine was mixed with furaldehyde (200 mg, 2.0 mmol) in 6 mL of EtOAc to yield a brown solution, which was stirred at room temperature for 10 h. The solvent was removed by evaporation to give the crude intermediate imine, which was dissolved in 5 mL of MeOH and mixed with NaBH4 (150 mg, 4.0 mmol). The mixture was stirred for 2.5 h, the reaction was quenched with 10 mL of saturated brine, and the brine was extracted with dichloromethane (3 × 10 mL). The combined organic layers were dried over anhydrous Na2SO4, and the solvent was evaporated to yield brown oil. TLC analysis showed enough purity (>90%) for the next reaction. Synthesis of 1-Ethyl-2-((E)-2-((E)-3-((E)-2-(1-ethyl-3,3dimethylindolin-2-ylidene)ethylidene)-2-((furan-2ylmethyl)(pyridin-2-ylmethyl)amino)cyclohex-1-en-1yl)vinyl)-3,3-dimethyl-3H-indol-1-ium iodide (CY-FPA). One hundred twenty-eight milligrams (0.2 mmol) of compound CY-Cl and 150 mg (0.8 mmol) of FPA were dissolved in 5 mL of anhydrous DMF in a 25 mL round-bottom flask. The mixture was stirred at 80 °C under nitrogen for 24 h. Then, the mixture was evaporated to dryness on a rotary evaporator. The solid was purified on silica gel chromatography eluted with dichloromethane/methanol (20:1 v/v, Rf = 0.22 by TLC). Yield (82 mg, 51.8%). ESI-MS: 663.4 (M − I−). 1H B
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solubility in water and is resistant to photobleaching (Figure S4). These properties made it a suitable fluorescent probe for biological analysis. Compared with unmodified CY-Cl, the introduction of a moderate electron-donating group increased the electron density on the polymethine chain of CY-FPA (theoretical calculation, Figure S-5). Thus, we can assume that the CY-FPA molecule possesses remarkable potential to be oxidized in the presence of reactive oxygen species such as HOCl, which is evidenced by the experimental results. For practical application, the pH effect on the fluorescence of CY-FPA in the absence and presence of HOCl/ClO− was examined (Figure S-6). The variation of pH does not significantly affect the fluorescence of the probe in the absence of hypochlorite. However, the fluorescence is quenched almost completely by hypochlorite at pH values lower than the pKa of HOCl (pKa = 7.46), whereas the fluorescence quenching efficiency decreases gradually as the pH value increased higher than the pKa. The results suggest that it is hypochlorous acid, not the hypochlorite anion, that quenches the fluorescence of CY-FPA. Therefore, it is appropriate to carry out all the experiments in physiological conditions in PBS buffer with pH 7.4, and under these conditions, the reactive species hypochlorous acid has sufficient concentration. To explore its reaction behavior with HOCl, the probe CYFPA was treated with hypochlorous acid at ambient temperature in an aqueous environment (Figure 1). The fluorescence
cells were grown on uncoated 35 mm diameter glass-bottomed dishes. The confocal fluorescence images were acquired through a Carl Zeiss microscopy (LSM710) equipped with a cooled CCD camera using 663 nm as excitation wavelength. In the control experiment, the cells were incubated with 10 μM solution of probe CY-FPA for 10 min at room temperature, washed with 1 mL of PBS three times, and then the fluorescence images were obtained at 0, 1, and 4 days, respectively. For detection of HOCl in vivo, the cells were incubated with a 10 μM solution of probe CY-FPA for 10 min at room temperature. The cells were washed with 1 mL PBS three times before 50 μM of HOCl was added to the mixture. The mixture was mounted on a microscope stage to give fluorescence image immediately at 1 and 12 h. Statistical Evaluation. Descriptive statistical analyses were performed using Origin 8.0 for calculating the means and the standard error of the mean. Results were expressed as the mean ± standard deviation (SD).14
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RESULTS AND DISCUSSION The target NIR fluorescent probe was synthesized by modifying the polymethine group with a moderate electron-donating group, which tuned the reactivity of one of the double bond to be selective for hypochlorous acid, as shown in Scheme 1. The Scheme 1. Synthesis of the Probe CY-FPA and Its Reaction Properties with HOCl. (a) FPA, DMF, 80 °C; FPA: 1(Furan-2-yl)-N-(pyridin-2-ylmethyl)methanamine. The Polymethine Group of CY-Cl Was Modified with FPA by Substitution Reaction To Give the NIR Fluorescent Probe CY-FPA, Which Tuned the Reactivity of Bonds To Be Selective for HOCl
Figure 1. Fluorescence quenching (λex = 700 nm) of CY-FPA (3 μM) upon addition of HOCl (0−9 μM) at pH 7.4 in 50 mM PBS/EtOH (9:1). I and I0 are the fluorescence intensity of CY-FPA in the presence and absence of HOCl, respectively.
structure of the probe was confirmed by ESI-MS and 1H NMR (Figure S-1). Compound CY-FPA displays absorption and emission maxima in the NIR region at 700 and 774 nm, respectively, with a relatively large Stokes shift (74 nm). The molar absorption coefficient of CY-FPA was measured as ε = 1.72 × 105 M−1 cm−1 using the Beer−Lambert Law, within which the relative standard deviation (RSD) was 1.6% (Figure S-2). CY-FPA also has better fluorescent quantum yield (Φ = 0.177 in ethanol) and broader excitation spectrum than the unmodified indocyanine dye CY-Cl (Φ = 0.085 in ethanol16) (Figure S-3). For comparison, the absorption and emission profiles of CY-FPA in different solvents including water, acetonitrile, methanol, acetone, ethanol, and 2-propanol are compiled in Table S-1. It can be seen that the maximum absorption slightly red-shifts when the solvent dielectric constants decrease. As the solvent changes from water to ethanol, the solvent polarity decreases and a 10 nm red-shift of the absorption maximum is observed, indicating an intramolecular charge-transfer state.17 The probe CY-FPA has good
intensity of CY-FPA dispersed in PBS decreased gradually upon addition of HOCl with a good linear relationship (R2 = 0.995), which can be used for the quantification of HOCl. There is no new emission band generated after the addition of HOCl, which indicates that the CY-FPA has been oxidized to nonfluorescent species by HOCl. The limit of detection (LOD) and limit of quantification (LOQ) for HOCl were calculated using the equations: LOD = 3 σ/k and LOQ = 10 σ/ k, respectively, where σ is the standard deviation of a blank and k is the slope of the calibration line. The LOD and LOQ for HOCl were found to be 0.70 and 2.33 μM, respectively, which is comparable to that of the resorcinol probe for HOCl.14 Unlike CY-FPA, the fluorescence of CY-Cl neither can be quenched by excess amount of HOCl nor is the absorption affected by HOCl (Figure S-7). The result suggested that the C
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Figure 2b represents the distinct color change of CY-FPA solution from blue to purple and then to pink as the concentration of HOCl increased, which could be used for ratiometric detection. Other reactive oxygen species such as H2O2, ·O2−, TBO·, and TBHP showed no apparent affect on the absorption of the probe (Figure S-11), except for ·OH, which resulted in a slightly decrease of the absorption band at 710 nm (Figure S-12). The result illustrates that the probe CYFPA has good selectivity to HOCl and can be conveniently used for its detection by colorimetric method. To examine the reaction mechanism of CY-FPA with HOCl, the intermediates and product were carefully analyzed by mass spectra (Figure 3). The reaction of CY-FPA with 1 equiv of HOCl first produced three new peaks, which belong to intermediate chlorohydrin CY-1, epoxide CY-2, and glycol CY3. Upon addition of 2 equiv of HOCl, one more new peak at m/z 506.28 was detected in addition to CY-2 and CY-3. Particularly, CY-2 was the main product of the reaction. Upon addition of excess HOCl (5 equiv), the ESI-MS spectra gave three peaks at m/z = 679.40, 506.28, and 212.10, which were assigned to epoxides and oxidative decomposition products. These results suggested that CY-FPA experienced an electrophilic addition on one of the double bonds to form chlorohydrin CY-1 with m/z = 715.41 and then quickly generated epoxide CY-2 through dehydrochlorination. Part of the epoxide CY-2 was hydrolyzed to form glycol CY-3, which was subsequently cleaved to produce CY-4 and CY-5 by HOCl. The reaction mechanism and reactive position are apparently different from those of other cyanine dyes by photoinduced decomposition reported by Chang et al.19 From the production of CY-4 and CY-5, we can affirm that the electrophilic addition of a double bond with HOCl occurred at the side of the uncharged 3,3-bimethylindolinene framework. This could be attributed to both the steric hindrance of the FPA group and the higher electron density of the double bond at the uncharged side. It has been reported that chlorohydrins, epoxides, and glycol intermediates were produced when HOCl reacted with an unsaturated bond of cholesterol, lipids, phosphatidylcholines, and so forth.20 This is in agreement with our observation that when CY-FPA reacted with HOCl, nonfluorescent epoxides and glycol intermediates were obtained and both the fluorescence and maximum absorption decreased, accompanied by the appearance of a new absorption band. The reaction of CY-FPA with excess HOCl within 30 min gave other new peaks. These peaks corresponded to continuous oxidation product of the epoxide (Figure S-13). These results suggested that CY-FPA rapidly reacted with HOCl to form the epoxide intermediates, followed by slow hydrolysis to glycol. The glycol intermediates can be further oxidized to aldehyde and carboxyl compounds by HOCl. Myeloperoxidase (MPO) mediates the peroxidation of chloride ions by H2O2 in physiological conditions to produce hypochlorous acid. So, the probe could be used to monitor the production of HOCl. Figure 4a shows the fluorescence intensity of CY-FPA with H2O2 in PBS 7.4 against the addition of MPO. Negative control experiments were conducted to rule out the direct quenching effect of MPO, which showed no obvious fluorescence quenching in the absence of either MPO or H2O2. These observations strongly suggest that it is the HOCl generated by H2O2 and Cl− in the presence of MPO that oxidizes CY-FPA. The fluorescence time dependence was also consistent with those reported for known myeloperoxidasemediated hypochlorous acid production.5 More control experi-
initial indocyanine CY-Cl is stable against HOCl, and the introduction of the FPA group to the cyanine dye increases the reactivity by activating one of the double bonds of the polymethine group, which enhances the fluorescence response of CY-FPA. The responses of the probe to other reactive species including hydrogen peroxide (H2O2), superoxide anions (·O2−), tert-butyl peroxyl radical (TBO·), tert-butylhydroperoxide (TBHP), and hydroxyl radical (·OH) were carefully examined at the same conditions as HOCl (Figure S-8). Clearly, the fluorescence was greatly quenched by hypochlorous acid and only slightly quenched by hydroxyl radicals generated from either Fenton reaction or UV light irradiation of hydrogen peroxide. However, other ROS showed no apparent fluorescence quenching effect. The results indicate that the CY-FPA does not react with these ROS or that the reaction rate is very slow. This could be explained in that HOCl not only has the highest standard redox potential (1490 mV) in comparison with H2O2 (320 mV), ·O2− (940 mV), TBHP (−900 mV), and TBO· (1000 mV)18 but also has a partial positively charged chlorine atom which readily initiates electrophilic addition to the double bond. In addition, none of the biological relevant metal ions or anions such as Zn2+, Cu2+, Fe3+, Ca2+, Mg2+, NO2−, NO3− resulted in obvious fluorescence responses (Figure S-9). These results suggested that the fluorescence probe CY-FPA exhibits good selectivity toward hypochlorous acid in physiological conditions. Furthermore, the reaction of CY-FPA with hypochlorous acid results in a new absorption band at 520 nm accompanied by gradually decreasing of the original absorption peak at 710 nm, leading to the formation of an isosbestic point at 570 nm (Figure 2a). A good linear relationship between the absorbance
Figure 2. (a) UV−vis absorption spectral responses of a 3 μM solution of CY-FPA in aqueous solution upon the addition of HOCl. Absorption band at 710 nm gradually decreased, and a new absorption band emerged at 520 nm as the amount of HOCl increased. (b) Dependence of the color change of probe CY-FPA (3 μM) on the amount of HOCl (1−9 μM) added in 50 mM PBS (pH 7.4, 100 mM KCl). Photos were taken under daylight.
ratio of A520/A710 and the added amount of HOCl was obtained (Figure S-10). The LOD based on the colorimetry was thus measured to be 0.13 μM, which was slightly lower than those of the CY-FPA NIR probe (0.70 μM) and the resorcinol probe (0.48 μM).14 The higher sensitivity of the colorimetry may be attributed to the probe’s high molar absorption coefficient. D
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Figure 3. Possible reaction scheme of CY-FPA with HOCl, and the corresponding evidence of ESI-MS spectra. (a) CY-FPA before the addition of HOCl; (b) CY-FPA plus 1 equiv of HOCl; (c) CY-FPA plus 2 equiv of HOCl ; and (d) CY-FPA plus more excessive HOCl (5 equiv).
added, the quenching effect became apparent. The fluorescence quenching efficiency was greatly increased as higher concentration of chloride ions (2 mM) was added, which is in the physiological range of intracellular chloride ion concentration.21 These results clearly indicated that the probe is also applicable to evaluate the enzymatic MPO system. The absorption spectral responses of the probe to MPO are consistent with the fluorescence responses, further implying that MPO catalyzes the reaction between H2O2 and Cl− in aqueous solution to produce HOCl. HOCl quickly reacts with the probe and rapidly reaches equilibrium (Figure S-14), whereas the MPO−H2O2−Cl system reacts with the probe within 10 min due to the diffusion of generated HOCl (Figure S-15). When the inhibitor of MPO, NaN3, was added into the mixture at 3 min, both the decreasing of absorbance at 710 nm and the increasing of absorbance at 520 nm were terminated. The absorbance at the two wavelengths remained constant with further increasing reaction time, suggesting that the catalytic activity of MPO is completely inhibited by NaN3.3 In addition, the new absorbance at 520 nm was linearly dependent on the amount of HOCl added, with a correlation coefficient of 0.998 (Figure 5a). Thus, the enzymatic activity of MPO in the MPO−H2O2−Cl system can be measured using the absorbance at 520 nm. Figure 5b represents the dependence of absorbance at 520 nm on the amounts of MPO in the presence of fixed and sufficient concentrations of H2O2 (200 μM) and Cl− (2 mM). It can be seen that the absorbance increased linearly with correlation coefficient of 0.998. The enzymatic activity can thus be expressed as MPO amount ∝ A−A0 or MPO amount ∝ nHOCl, where A−A0 is the increased absorbance at 520 nm; nHOCl is added HOCl or produced HOCl in the reaction system. This method takes advantage of the absorption change of CY-FPA upon the exposure to HOCl to achieve the measurement of MPO activity. The application of the NIR fluorescent probe for live cell imaging was preliminarily demonstrated. A549 cells were first incubated with 10 μM of CY-FPA for 10 min, and the stained cells were still alive and showed strong fluorescence after incubation for 4 days (Figure 6a, d−f). These results suggested that the probe is cell-membrane permeable and shows low cytotoxicity to living cells, which is a good fluorescence probe
Figure 4. (a) Time dependence of fluorescence intensity of CY-FPA (3 μM) upon the addition of MPO and H2O2 in PBS 7.4. [MPO] = 0.025 units, [H2O2] = 10 μM; (b) Time dependence of fluorescence intensity of CY-FPA (3 μM) upon the addition of MPO, H2O2, and Cl− in PB 7.4. [MPO] = 0.025 units, [H2O2] = 10 μM.
ments were carried out to further confirm the fluorescence quenching effect of HOCl generated by MPO catalysis. The experiments for the confirmation of the role of chloride ion in the generation of HOCl were done in PB 7.4 without chloride ions (Figure 4b). Clearly, the fluorescence intensity of CY-FPA was nearly constant in the presence of both MPO and the mixture of MPO−H2O2, indicating very slow oxidation of the probe without chloride ion. When 10 μM of chloride ions was E
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CY-FPA was appropriate for cell imaging and responded to HOCl by fluorescence quenching in the living cells.
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CONCLUSIONS In summary, we have demonstrated a novel dual functional NIR fluorescent probe capable of ultrasensitive and selective detection of hypochlorous acid, monitoring the HOCl generation by MPO, and detecting the existence of HOCl by the naked eye. The probe was synthesized by activating the polymethine chain of a cyanine dye to be selectively reactive to hypochlorous acid. The reaction mechanism between the probe and HOCl was confirmed by mass spectra as electrophilic addition to the polymethine chain, followed by oxidation cleavage. The NIR probe shows good cell-membrane permeability and low cytotoxicity to living cells, which is demonstrated as a new fluorescent probe for HOCl imaging in living cells. In addition, the enzymatic activity of MPO can be evaluated on the basis of a colorimetric method.
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ASSOCIATED CONTENT
* Supporting Information S
Additional information as noted in the text (Figures S1−S15). This material is available free of charge via the Internet at http://pubs.acs.org.
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Figure 5. (a) Increased absorbance of CY-FPA (8 μM) at 520 nm upon the addition of HOCl (0−24 μM) in PBS 7.4. (b) Standard curves for detection of MPO using the CY-FPA assay. The horizontal axis represents the amounts of MPO added into the mixture of H2O2 (200 μM) and Cl− (2 mM) in the presence of the CY-FPA probe (8 μM). Vertical axis represents the increased absorbance at 520 nm after addition of MPO.
AUTHOR INFORMATION
Corresponding Author
*E-mail: [email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the National Basic Research Program of China (2011CB933700), the Natural Science Foundation of China (Nos. 21077108, 21302187, 21205120), and the Innovation Project of Chinese Academy of Sciences (KJCX2-YW-H29).
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REFERENCES
(1) Pattison, D. I.; Davies, M. J. Biochemistry 2006, 45, 8152−8162. (2) (a) Roos, D.; Winterbourn, C. C. Science 2002, 296, 669−671. (b) Fang, F. C. Nat. Rev. Microbiol. 2004, 2, 820−832. (3) (a) Yan, Y.; Wang, S.; Liu, Z.; Wang, H.; Huang, D. Anal. Chem. 2010, 82, 9775−9781. (b) Yang, Y.; Lu, H.; Wang, W.; Liau, I. Anal. Chem. 2011, 83, 8267−8272. (4) (a) Chan, J.; Dodani, S. C.; Chang, C. J. Nat. Chem. 2012, 4, 973−984. (b) Yang, Y.; Zhao, Q.; Feng, W.; Li, F. Chem. Rev. 2013, 113, 192−270. (c) Schäferling, M. Angew. Chem., Int. Ed. 2012, 51, 3532−3554. (5) Figueiredo, J. L.; Aikawa, E.; McCarthy, J.; Weissleder, R.; Hilderbrand, S. A. J. Am. Chem. Soc. 2009, 131, 15739−15744. (6) (a) Kenmoku, S.; Urano, Y.; Kojima, H.; Nagano, T. J. Am. Chem. Soc. 2007, 129, 7313−7318. (b) Yuan, L.; Lin, W.; Xie, Y.; Chen, B.; Song, J. Chem.Eur. J. 2012, 18, 2700−2706. (c) Sun, Y.-Q.; Liu, J.; Lv, X.; Liu, Y.; Zhao, Y.; Guo, W. Angew. Chem., Int. Ed. 2012, 51, 7634−7636. (7) (a) Achilefu, S. Angew. Chem., Int. Ed. 2010, 49, 9816−9818. (b) Ntziachristos, V.; Tung, C. H.; Bremer, C.; Weissleder, R. Nat. Med. 2002, 8, 757−760. (c) Yuan, L.; Lin, W.; Zheng, K.; He, L.; Huang, W. Chem. Soc. Rev. 2013, 42, 622−661. (d) Weissleder, R.; Ntziachristos, V. Nat. Med. 2003, 9, 123−128. (e) Leevy, W. M.; Gammon, S. T.; Jiang, H.; Johnson, J. R.; Maxwell, D. J.; Jackson, E. N.; Marquez, M.; Worms, D. P.; Smith, B. D. J. Am. Chem. Soc. 2006, 128, 16476−16477.
Figure 6. Fluorescence images of cell incubated with 10 μM of CYFPA (a) and further treated with HOCl (50 μM) for 1 h (b), 12 h (c). Fluorescence image of A549 cells incubated with only CY-FPA for 1 h (d), 12 h (e), and 4 days (f).
for cell imaging. Specifically, the probe penetrated the cell membrane very fast but could not be phagocytized by cell nucleus. When the living A549 cells loaded with CY-FPA were then treated with HOCl, the fluorescence was gradually decreased and finally disappeared. Figure 6b,c show the quenching effect of HOCl on the intracellular CY-FPA (upper panel). It can be seen that the fluorescence of CYFPA was completely quenched after treatment with HOCl (50 μM) for 12 h. In contrast, no apparent quenching effect was observed without HOCl treatment (lower panel) even for a longer incubation time. These results indicate that the probe F
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Article
(8) (a) Qian, G.; Wang, Z. Y. Chem.Asian J. 2010, 5, 1006−1029. (b) Luo, S.; Zhang, E.; Su, Y.; Cheng, T.; Shi, C. Biomaterials 2011, 32, 7127−7138. (c) Kiyose, K.; Kojima, H.; Nagano, T. Chem.Asian J. 2008, 3, 506−515. (9) (a) Sasaki, E.; Kojima, H.; Nishimatsu, H.; Urano, Y.; Kikuchi, K.; Hirata, Y.; Nagano, T. J. Am. Chem. Soc. 2005, 127, 3684−3685. (b) Kiyose, K.; Kojima, H.; Urano, Y.; Nagano, T. J. Am. Chem. Soc. 2006, 128, 6548−6549. (10) Kundu, K.; Knight, S. F.; Willett, N.; Lee, S.; Taylor, W. R.; Murthy, N. Angew. Chem., Int. Ed. 2009, 48, 299−303. (11) Engel, E.; Schraml, R.; Maisch, T.; Kobuch, K.; Konig, B.; Szeimies, R. M.; Hillenkamp, J.; Baumler, W.; Vasold, R. Invest. Ophthalmol. Visual Sci. 2008, 49, 1777−1783. (12) Wang, M.; Davis, R. H.; Rafinski, Z.; Jedrzejewska, B.; Choi, K. Y.; Zwick, M.; Bupp, C.; Izmailov, A.; Paczkowski, J.; Warner, B.; Koshinsky, H. Anal. Chem. 2009, 81, 2043−2052. (13) Oushiki, D.; Kojima, H.; Terai, T.; Arita, M.; Hanaoka, K.; Urano, Y.; Nagano, T. J. Am. Chem. Soc. 2010, 132, 2795−2801. (14) Ö zyürek, M.; Bekdeşer, B.; Gücļ ü, K.; Apek, R. Anal. Chem. 2012, 84, 9529−9536. (15) (a) Baxendale, J. H.; Wilson, J. A. Trans. Faraday Soc. 1957, 53, 344−356. (b) Suzuki, T.; Moriwaki, N.; Kurokawa, K.; Inukai, M. Bioorg. Med. Chem. Lett. 2009, 19, 3217−3219. (16) Chen, G.; Song, F.; Wang, J.; Yang, Z.; Sun, S.; Fan, J.; Qiang, X.; Wang, X.; Dou, B.; Peng, X. Chem. Commun. 2012, 48, 2949− 2951. (17) Peng, X.; Song, F.; Lu, E.; Wang, Y.; Zhou, W.; Fan, J.; Gao, Y. J. Am. Chem. Soc. 2005, 127, 4170−4171. (18) Buettner, G. R. Arch. Biochem. Biophys. 1993, 300, 535−543. (19) Samanta, A.; Vendrell, M.; Dasa, R.; Chang, Y. T. Chem. Commun. 2010, 46, 7406−7408. (20) Panasenko, O. M.; Osipov, A. N.; Schiller, J.; Arnhold, J. Biochemistry 2002, 67, 889−900. (21) Friedel, P.; Bregestovski, P.; Medina, I. Front. Mol. Neurosci. 2013, DOI: 10.3389/fnmol.2013.00007.
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