Research article Received: 25 October 2014,
Revised: 15 January 2015,
Accepted: 03 March 2015
Published online in Wiley Online Library: 29 April 2015
(wileyonlinelibrary.com) DOI 10.1002/bio.2912
A coumarin-based fluorescent turn-on probe for detection of biothiols in vitro Mengqiang Liu,a Qian Jiang,a Zhiyun Lu,a Yan Huang,a Yanfei Tanb* and Qing Jiangb* ABSTRACT: A novel fluorescent probe (CA-N) was designed and synthesized for detection of biothiols. CA-N displayed a strong fluorescence in the presence of biothiols with high sensitivity, and the mechanism for detection biothiols was based on the Michael addition reaction of a thiol group to α,β-unsaturated ketones. CA-N showed low detection limit for cysteine (Cys), homocysteine (Hcy), and glutathione (GSH), which were calculated as 3.16, 0.19 and 5.15 μM, respectively. At the same time, CA-N exhibited high selectivity toward biothiols compared with other biological amino acids. In vitro cell experiments proved that CA-N had no cytotoxicity, high cell permeability and could be employed in living cell imaging for biothiols. Copyright © 2015 John Wiley & Sons, Ltd. Additional supporting information may be found in the online version of this article at the publisher’s website. Keywords: fluorescent probe; Michael addition; coumarin; bioimaging
Introduction
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Experimental Materials and methods Unless otherwise indicated, all materials and solvents were obtained from commercial suppliers and used without further purification. The exception was spectrographically pure dimethylsulfoxide (DMSO) used for the biological evaluation. Double-distilled water was used in the buffer solutions. 1H NMR and 13C NMR spectra were measured using a Bruker AVANCE ΙΙ-400 MHz spectrometer and TMS as an internal standard.
* Correspondence to: Y. Tan and Q. Jiang, National Engineering Research Center for Biomaterials, Sichuan University, Chengdu, 610064, China. E-mail: [email protected]; [email protected] a
Key Laboratory of Green Chemistry and Technology (Ministry of Education), College of Chemistry, Sichuan University, Chengdu, 610064, China
b
National Engineering Research Center for Biomaterials, Sichuan University, Chengdu, 610064, China
Copyright © 2015 John Wiley & Sons, Ltd.
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Biological thiols, such as cysteine (Cys), homocysteine (Hcy) and glutathione (GSH), play a key role in many physiological processes, and connections have been found between abnormal levels of thiols and a number of diseases. A decrease in the level of Cys may lead to many health problems, such as liver damage, slow growth, skin lesions, AIDS and oxidative damage (1–4). An abnormal level of Hcy in human plasma has been shown to be correlated with Alzheimer’s disease, osteoporosis and cardiovascular disease (5–7). GSH is the most abundant (1–10 mM) intracellular thiol and has many cellular functions (8). Changes in cellular GSH levels can also lead to health problems, such as leucocyte loss, heart problems and cancer (9–11). Therefore, the detection and quantification of biothiols in biological systems is of great importance. In the past few years, a number of techniques have been exploited to detect these biological thiols, such as highperformance liquid chromatography (HPLC) (12,13), Fourier transform infrared (FTIR) spectroscopy (14), mass spectrometry (MS) (15,16), surface-enhanced Raman scattering (17), capillary electrophoresis (18,19), electrochemistry assay (20,21), UV/vis spectroscopy (22,23) and fluorescence spectroscopy. Among these various strategies, fluorescent probes for the detection of biothiols have been developed quickly because of the method’s ease of operation, high selectivity, sensitivity, low cost, low detection limits and potential for in vivo imaging (24–26). Therefore, various fluorescent probes for biothiols based on different fluorophores have been constructed, including rhodamine (27–29), naphthalimide (30), BODIPY (31,32), fluorescein (33–36), cyanine (37–39), flavone (40,41) and coumarin (42–50). Of these, coumarin dye is one of the molecules used most widely in fluorescent probes owing to its high photostability, large Stokes shift, high cell permeability, visible emission wavelength and high fluorescence quantum yield (51,52). However, many of these probes suffer from some limitations, including high-pH aqueous solution, high background fluorescence, low sensitivity in vitro and poor biocompatibility. Thus,
there remains an urgent need for a highly sensitively probe that could be used in the further application of biotechnology. Here, we reported a novel coumarin-based derivative fluorescent probe, namely (E)-7-(diethylamino)-3-(3-(4-nitrophenyl)acryloyl)-2Hbenzo- pyran-2-one (CA-N). CA-N was constructed based on the Michael addition reaction of thiol to α,β-unsaturated ketones. The quencher group of nitrobenzene led to the non-fluorescence of CA-N, although a strong green fluorescence was detected once the probe reacted with a thiol group. Furthermore, CA-N had high sensitivity and good biocompatibility, which could be successfully applied to living cell imaging in vitro. In addition, CA-N could detect qualitative changes in thiol concentrations in living cells.
M. Liu et al. Fluorescent emission spectra were obtained using a Perkin–Elmer LS55 fluorescence spectrophotometer. UV/vis absorption spectra were performed on a Perkin–Elmer Lambda 950 UV/VIS Spectrometer. High-resolution MS were obtained using a Shimadzu LCMSIT-TOF. pH was measured with a PHS-3E pH meter. Fluorescence microscopic images were obtained from laser scanning confocal microscopy (CLSM; TCS-SP5, Leica, Germany).
Synthesis 3-Acetyl-7-(diethylamino)-2H-benzopyran-2-one (2). Compound 2 was synthesized according to a previously reported procedure (53). 4-Diethylamino salicylaldehyde (10 g, 51.7 mmol) and ethyl acetonacetate (13.5 g, 100 mmol) were dissolved in absolute ethanol (100 mL), and piperidine (0.2 mL) was added as a catalyst. After the mixture was refluxed under argon atmosphere for 6 h, a yellow solid was obtained and collected by filter. The solid was washed with cool absolute ethanol, crude product was recrystallized from the absolute ethanol, and a yellow solid was collected (10.6 g, yield: 79%). 1 H NMR (400 MHz, CDCl3) δ (ppm): 8.44 (s, 1H), 7.41 (d, J = 8.8 Hz, 1H), 6.64 (dd, J = 8.8 Hz, J = 2.0 Hz, 1H), 6.48 (d, J = 1.6 Hz, 1H), 3.49 (q, 4H), 2.68 (s, 3H), 1.26 (t, J = 7.2 Hz, 6H). m.p. 151–153 °C.
Detection limit studies The detection limit was calculated based on a method described previously (54,55). The fluorescence intensity of CA-N alone was measured seven times and the standard deviation of the blank measurement was obtained. To improve sensitivity, 10 μM CA-N was employed. Under this condition, a good linear working range between the emission intensity and the Cys concentration with the range 10–100 μM was observed. The detection limit was determined as 3σ/k where σ is the standard deviation of the blank measurement and k is the slope of a plot of fluorescence intensity against sample concentration.
Kinetic analysis The time-dependent response of CA-N (10 μM) with Cys, Hcy and GSH (100 eq.) in DMSO/phosphate buffer (1: 1 v/v, pH 7.4) exhibited pseudo-first-order reaction conditions at room temperature, and the fluorescence intensity at 494 nm was recorded. As described in the literature (56,57), the pseudo-first-order rate constant for the reaction was determined by fitting the fluorescence intensities of CA-N to following the pseudo-first-order equation: . ln ðF max F t Þ
F max
(E)-7-(diethylamino)-3-(3-(4-nitrophenyl)acryloyl)-2H-benzopyran-2one (CA-N). Compound 2 (100 mg, 0.39 mmol) and 4-
nitrobenzaldehyde (89.2 mg, 0.59 mmol) were dissolved in absolute ethanol (10 mL) and piperidine (0.1 mL) was added as a catalyst. After the mixture had been refluxed under argon atmosphere for 24 h, the solvents were removed by distillation under reduced pressure and the red solid was collected. The crude solid was extracted with dichloromethane and the solvent was then removed. The crude solid was further purified by column chromatography on silica gel to give a red solid. After recrystallization in absolute ethanol, and drying under vacuum, a red solid was acquired (45.9 mg, yield: 30%). 1 H NMR (400 MHz, CDCl3) δ (ppm): 8.58 (s, 1H), 8.31 (d, J = 16.0 Hz, 2H), 8.26 (d, J = 8.8 Hz, 1H), 7.82 (d, J = 8.8 Hz, 2H), 7.81 (d, J = 16.0 Hz, 1H), 7.46 (d, J = 9.2 Hz, 1H), 6.67–6.64 (dd, J = 8.8 Hz, 2.4 Hz, 1H), 6.51 (d, J = 2.0 Hz, 1H), 3.51 (q, 4H), 1.29 (t, J = 7.2 Hz, 6H). 13C NMR (100 MHz, CDCl3) δ (ppm): 184.9, 160.0, 157.9, 152.4, 148.2, 147.3, 140.6, 138.60, 131.1, 128.1, 128.0, 123.0, 114.9, 109.1, 107.7, 95.7, 44.2, 11.5. HR-MS (ESI): m/z [M + Na]+ 415.1265, calcd.: 415.1264. m.p. 227–228 °C.
¼ k’ t
Where Ft and Fmax are the fluorescence intensities at 494 nm at time t and the maximum value obtained after the reaction was completed. k’ is the pseudo-first-order rate constant.
Cell experiments For the cell cytotoxicity assay, MG63 cells were seeded in 96-well plates at a concentration of 1 × 104 cells/well for 24 h, then different concentrations of CA-N (0, 5, 10 and 20 μM final concentration) were added to the wells and the cells were cultured for another 24 h. Cytotoxicity was determined by MTT colorimetric method using five parallel wells at each time point. Cells were incubated with 5 mg/mL of MTT for 4 h at 37 °C, the supernatant was then discarded, and DMSO (200 μL/well) was added to dissolve the formazan; the absorbance was then measured at 570 nm with a microplate reader (BioRad, model 550). Cytotoxicity assay.
Bioimaging for biothiols in living cells Preparation of solutions for fluorescence and UV/vis absorption
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A stock solution of CA-N (1.5 mM) was prepared in DMSO. Stock solution of Cys, Hcy, GSH (30 mM) and other amino acids (10 mM) including Ala, Arg, Asp, Glu, Gly, His, Leu, Lys, Phe, Pro, Ser, Tle, Thr, Trp, Tyr and Val were dissolved in double-distilled water, and solutions (DMSO/aqueous buffer, 1: 1 v/v, pH 1.0–11.0) with different pH values were prepared. In a typical experiment, 20 μL of the CA-N stock solution was added to 3 mL DMSO/phosphate buffer (1: 1 v/v, pH 7.4) and then an appropriate buffer of each amino acids was added. Fluorescence and absorption spectra were recorded after the addition of amino acids at room temperature.
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MG63 cells were seeded on 35 mm glass-bottomed dishes at a density of 1 × 105 cells/dish in culture medium and incubated overnight in preparation for live cell imaging. The cells were divided into four groups: (1) no added dye or biothiols; (2) treatment with 10 μM of CA-N in a serum-free medium for 30 min and washed twice with prewarmed PBS; (3) treatment with 1.0 mM Nethylmaleimide (NEM, a thiol-blocking reagent) for 30 min to decrease the concentration of cellular biothiols, then washed twice with prewarmed PBS buffer, followed by addition of CA-N for 30 min; (4) pretreatment with NEM for 30 min, followed by addition of Cys (100 μM) and CA-N for 30 min and washed twice with prewarmed PBS. The four groups were observed under CLSM using the excitation channel (λex = 405 nm).
Copyright © 2015 John Wiley & Sons, Ltd.
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Coumarin-based probe for the detection of biothiols
Results and discussion Design and synthesis of CA-N To obtain a fluorescent probe that selectively detected thiols for potential biological applications, the α,β-unsaturated ketones moiety was chosen as the Michael addition reaction recognition unit, and diethylaminocoumarin was selected as the fluorophore. The synthetic route for probe CA-N is shown in Scheme 1. Compound 2 was synthesized and used as a reference compound to reveal the Michael addition reaction mechanism. The structure of CA-N was confirmed by 1H NMR, 13C NMR and high-resolution MS.
UV/vis absorption and fluorescence studies with thiols The absorption and fluorescence emission spectra of CA-N in the absence and presence of Cys in DMSO/phosphate buffer (1: 1 v/v, pH 7.4) at room temperature were examined and are recorded in Fig. 1. Because of use of the nitrobenzene group as the quencher moiety, CA-N alone did not exhibit fluorescence, although upon addition of Cys (100 eq.), the fluorescence spectra changed dramatically, and a strong emission peak was observed at 494 nm. Furthermore, in the absorption spectrum, addition of Cys caused a clear hypsochromic shift (~20 nm) from 472 to 452 nm. The changes in the absorption spectrum and fluorescence intensity of CA-N in the presence of Cys were attributed to the conjugate addition of thiol to a carbon–carbon double bond through Michael addition. When CA-N reacted with thiols, it was able to break the carbon–carbon double bond, the quencher was broken and the fluorescence recovered (Scheme 2). Thus, CA-N could be used as a fluorescent turn-on probe for thiols.
Detection limit studies To evaluate the detection limit of CA-N for Cys, Hcy and GSH in solution, CA-N was treated with various concentrations of Cys, Hcy or GSH in DMSO/phosphate buffer (1: 1 v/v, pH 7.4) at room temperature. The results show that the fluorescence intensity of CA-N gradually increased with increasing of Cys concentration (0–100 eq.) (Fig. 2). Simultaneously, under the same conditions, when Hcy or GSH (0–100 eq.) was added to CA-N solution, similar results were obtained (Fig. S1). The results of the titration experiment indicated a good linear relationship between the fluorescence intensity and Cys concentration within the range 10–100 μM (Fig. 3). Under these conditions, the detection limit for Cys was calculated as 3.16 μM. Under the same conditions, the detection limits for Hcy and GSH were 0.19 and 5.15 μM, respectively (Table 1 and Fig. S2). The low detection limit showed that CA-N was highly sensitive to
Figure 1. Normalized absorption spectrum of CA-N (■) and upon addition of 100 eq. Cys (●). Normalized fluorescence spectra of CA-N (▲) and upon addition of 100 eq. Cys (▼). Conditions: CA-N in the absence and presence of Cys in DMSO/phosphate buffer (1: 1 v/v, pH 7.4) at room temperature (λex = 400 nm).
biological thiols. Meanwhile, these data suggested that CA-N could be applied to the quantitative detection of biothiols.
pH-dependent studies In order to understand the pH-dependent effects of physiological media, CA-N solutions at different pH values (1.0–11.0) in the presence and absence of Cys, Hcy and GSH were investigated. As shown in Fig. 4, CA-N alone showed weak fluorescence only, its intensity did not change at different pH values (1.0–11.0), which suggested that CA-N was not affected by pH. By contrast, in the presence of Cys, the fluorescence intensity showed a large enhancement when the pH was > 5.0, and the fluorescence intensity reached a maximum at pH 8.5. At the same time, in the presence of Hcy or GSH, the fluorescence intensity reached a maximum value at pH 8.5 and 9.0, respectively. These results might be due to the influence of the pKa value of aliphatic thiols on the reaction. The pKa value of aliphatic thiols was ~ 8.5 (58,59)._ENREF_47 These results indicate that CA-N was sensitive enough to detect thiols under neutral to alkaline conditions.
Kinetic analysis The time-dependent fluorescence intensity of CA-N (10 μM) with Cys, Hcy and GSH (100 eq.) in DMSO/phosphate buffer (1: 1 v/v, pH 7.4) was recorded. As shown in Fig. 5, the fluorescence intensity reached a maximum after ~ 35 min in the presence Cys and Hcy. However, in the presence of GSH, the fluorescence intensity
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Scheme 1. Synthetic route for probe CA-N.
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Scheme 2. The design concept of a fluorescent turn-on probe (CA-N) for thiols.
Figure 2. Fluorescent titrations of probe CA-N (10 μM) upon addition of various concentrations of Cys (0–100 eq.) in DMSO/phosphate buffer (1: 1 v/v, pH 7.4) after 35 min at room temperature (λex = 400 nm).
Figure 3. Linear plot of CA-N (10 μM) in the presence of Cys (10–100 μM) against fluorescence intensity at 494 nm (λex = 400 nm).
Figure 4. Fluorescence intensity of CA-N (10 μM) at 494 nm before and after addition of Cys, Hcy and GSH (0–100 eq.) at different pH values in DMSO/phosphate buffer for 35 min at room temperature (λex = 400 nm).
Figure 5. Time-dependent fluorescence intensity of CA-N (10 μM) at 494 nm upon addition of Cys, Hcy and GSH (100 eq.) in DMSO/phosphate buffer (1: 1 v/v, pH 7.4) at room temperature (λex = 400 nm).
Table 2. Pseudo-first-order rate constants and lifetime of CA-N to Cys, Hcy, and GSH Table 1. Detection limit of the probe to Cys, Hcy and GSH
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Number
Thiol
σ
k
Detection limit (μM)
1 2 3
Cys Hcy GSH
0.7157 0.7157 0.7157
6.79 × 105 1.14 × 106 4.17 × 105
3.16 0.19 5.15
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Number
Thiol
Kinetic constant; k′ (min–1)
Life time; t1/2 (min)
1 2 3
Cys Hcy GSH
0.106 0.114 0.020
6.54 6.10 34.55
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Coumarin-based probe for the detection of biothiols fluorescence intensity at 494 nm was recorded and the results are shown in Fig. 6. We found that CA-N displayed weak fluorescence in the presence of Ala, Arg, Asp, Glu, Gly, His, Leu, Lys, Phe, Pro, Ser, Tle, Thr, Trp, Tyr and Val. However, under the same conditions, when thiol-containing analytes (Cys, Hcy and GSH) were added to CA-N, a great enhancement of the fluorescence intensity was observed. The data suggest that CA-N had a high selectivity for thiol-containing molecules. Meanwhile, to further test the Cys selectivity of CA-N in the presence of other analytes, CA-N (10 μM) was added to Cys (10 eq.) in the presence of various amino acids (10 eq.) in DMSO/phosphate buffer. None of analytes measured had an effect on the fluorescence detection of Cys (Fig. S7). Therefore, at physiological pH, CA-N could be highly selective for biothiols, even in the presence of these relevant amino acids in a biological system. Figure 6. Fluorescence intensity of CA-N (10 μM) at 494 nm in the presence of various amino acids (10 eq.) in DMSO/phosphate buffer (1: 1 v/v, pH 7.4) for 35 min at room temperature (λex = 400 nm). 1, Cys; 2, Hcy; 3, GSH; 4, Ala; 5, Arg; 6, Asp; 7, Glu; 8, Gly; 9, His; 10, Leu; 11, Lys; 12, Phe; 13, Pro; 14, Ser; 15, Tle; 16, Thr; 17, Trp; 18, Tyr; 19, Val; 20, blank.
reached a maximum after ~ 90 min (Fig. S3). CA-N exhibited much higher reactivity toward Cys and Hcy than towards GSH, in agreement previous reports (55,60). This may be explained by steric hindrance effects on the thiol 1,4-addition reaction. Meanwhile, the kinetic profiles of the CA-N reaction with Cys, Hcy and GSH were calculated from the fluorescence intensity at 494 nm. The pseudo-first-order rate constant k’ was determined following the pseudo-first-order equation. The pseudo-first-order rate constants for Cys, Hcy and GSH were determined as k’ = 0.106, 0.114 and 0.020 min-1, respectively; the corresponding half-life (t1/2) values were 6.45, 6.10 and 34.55 min, respectively (Table 2 and Figs. S4–6). These results further demonstrated that CA-N had high reactivity for Cys and Hcy compared with GSH. Selective studies In order to check the selective response of CA-N toward biothiols over other amino acids, various amino acids were separately added to CA-N in DMSO/phosphate buffer for 35 min, and the
Mechanism of CA-N for detecting Cys From the fluorescence spectral studies, CA-N shows a selective response to biothiols. CA-N was able to react through conjugate addition with a thiol group of Cys and form a CA-N–Cys adduct. In order to provide evidence of the reaction mechanism, the fluorescence spectra of adduct CA-N–Cys and compound 2 in DMSO/phosphate buffer at room temperature were recorded. Compound 2 had a similar structure to the diethylaminocoumarin fluorophore with CA-N–Cys adduct and served as a reference compound. We found that CA-N and compound 2 had almost the same fluorescence spectra (Fig. S8). Meanwhile, 1H NMR spectroscopy studies also provided evidence of CA-N in the absence or presence of Cys. As shown in Fig. 7, the alkene proton at around 8.08 ppm (Ha) disappeared, and a new peak emerged at around 4.58 ppm (Ha′); there was another alkene proton at around 7.76 ppm (Hb), and in the presence of Cys, two new peaks emerged at 3.71 ppm (Hb′) and 3.32 ppm (Hb′′). These results proved that CA-N could react with the thiol group of Cys to form a carbon–carbon double bond via Michael addition reaction. Furthermore, MS analysis of CA-N with Cys revealed formation of a CA-N–Cys adduct. The MS displayed peaks at m/z 415.1254 and 536.1466, corresponding to [CA-N + Na] and [CA-N–Cys + Na], respectively (Fig. S9). Taken together, these results consistently
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Figure 7. (Upper) A proposed mechanism of the reaction between CA-N and Cys. (Lower) H NMR spectra changed of CA-N in the absence (A) or presence (B) of Cys in DMSO-d6/ D2O (4: 1, v/v) at room temperature.
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Figure 8. Confocal Laser microscope images of MG63 cells. (A–C) Control group. (D–F) MG63 cells incubated with CA-N (10 μM) for 30 min at 37 °C. (G– I) Cells pretreated with NEM (1.0 mM) for 30 min and then incubated with CA-N (10 μM) for 30 min at 37 °C. ( J–L) Cells pretreated with NEM (1.0 mM) for 30 min and tzhen added to Cys (100 μM) and incubated with CA-N (10 μM) for 30 min at 37 °C. (Left) Bright channel, (middle) fluorescence channel (right) overlay. (M) Fluorescence intensity of each group.
revealed the reaction of CA-N with Cys and the formation of the CA-N–Cys adduct.
these results proved that CA-N could be used as an excellent fluorescent probe for biothiol imaging in living cells, and the alteration of fluorescence intensity indicates changes in the concentration of cellular biothiols.
Fluorescent imaging in MG63 cells
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To investigate the permeability of cells and clarify whether CA-N was sensitive enough to detect biothiols in living cells, the cytotoxicity of CA-N was evaluated using MTT assays. The results showed that cells grew very well even with 20 μM CA-N at 37 °C for 24 h and no significant difference was observed between the groups. The results indicated no cytotoxicity of CA-N (Fig. S10). Furthermore, cell-imaging experiments with CA-N were carried out and observed under CLSM. As shown in Fig. 8, MG63 cells without added CA-N did not show any fluorescence (Fig. 8B), whereas after incubation with CA-N at 37 °C for 30 min, significant green fluorescence was observed in the cytoplasm of these cells (Fig. 8E), which proved that CA-N was capable of permeating cells and reacting with intracellular biothiols. To further determine whether CA-N could highlight changes in the concentration of biothiols in living cells, one group was pretreated with 1.0 mM NEM for 30 min to eliminate any biothiols in the cells; only weak fluorescence was observed in these cells after incubation with CA-N (Fig. 8H). However, strong green fluorescence was again observed, when NEM-treated cells were added with Cys (100 μM) and incubated with CA-N for another 30 min (Fig. 8K). According to Image J™ software, the fluorescence intensity value of each group was obtained (Fig. 8M). Taken together,
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Conclusions We designed and synthesized a novel coumarin-based fluorescent probe CA-N, which exhibited high sensitivity and selectivity toward biothiols (Cys, Hcy, GSH) over other natural amino acids. Fluorescence spectra, 1H NMR and MS studies demonstrated the mechanism by which thiols combined with the α,β-unsaturated ketone of CA-N via the Michael addition reaction. In addition, in vitro cell imaging found that CA-N could easily penetrate cell membranes, react with biothiols in the cytoplasm, and show strong green fluorescence. Our results prove that CA-N is useful for imaging biothiols in living cells.
Acknowledgements The authors acknowledge the financial support for this work by Research Fund for the Doctoral Program of Higher Education of China (20130181110089), the National Natural Science Foundation of China (NSFC)(Grants No.21372168) and National Key Technology R&D Program (2012BAI42G01). Electronic Supplementary Information (ESI) are available.
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Luminescence 2015; 30: 1395–1402
Coumarin-based probe for the detection of biothiols
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Supporting information Additional supporting information may be found in the online version of this article at the publisher’s website.
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Luminescence 2015; 30: 1395–1402
Revised: 15 January 2015,
Accepted: 03 March 2015
Published online in Wiley Online Library: 29 April 2015
(wileyonlinelibrary.com) DOI 10.1002/bio.2912
A coumarin-based fluorescent turn-on probe for detection of biothiols in vitro Mengqiang Liu,a Qian Jiang,a Zhiyun Lu,a Yan Huang,a Yanfei Tanb* and Qing Jiangb* ABSTRACT: A novel fluorescent probe (CA-N) was designed and synthesized for detection of biothiols. CA-N displayed a strong fluorescence in the presence of biothiols with high sensitivity, and the mechanism for detection biothiols was based on the Michael addition reaction of a thiol group to α,β-unsaturated ketones. CA-N showed low detection limit for cysteine (Cys), homocysteine (Hcy), and glutathione (GSH), which were calculated as 3.16, 0.19 and 5.15 μM, respectively. At the same time, CA-N exhibited high selectivity toward biothiols compared with other biological amino acids. In vitro cell experiments proved that CA-N had no cytotoxicity, high cell permeability and could be employed in living cell imaging for biothiols. Copyright © 2015 John Wiley & Sons, Ltd. Additional supporting information may be found in the online version of this article at the publisher’s website. Keywords: fluorescent probe; Michael addition; coumarin; bioimaging
Introduction
Luminescence 2015; 30: 1395–1402
Experimental Materials and methods Unless otherwise indicated, all materials and solvents were obtained from commercial suppliers and used without further purification. The exception was spectrographically pure dimethylsulfoxide (DMSO) used for the biological evaluation. Double-distilled water was used in the buffer solutions. 1H NMR and 13C NMR spectra were measured using a Bruker AVANCE ΙΙ-400 MHz spectrometer and TMS as an internal standard.
* Correspondence to: Y. Tan and Q. Jiang, National Engineering Research Center for Biomaterials, Sichuan University, Chengdu, 610064, China. E-mail: [email protected]; [email protected] a
Key Laboratory of Green Chemistry and Technology (Ministry of Education), College of Chemistry, Sichuan University, Chengdu, 610064, China
b
National Engineering Research Center for Biomaterials, Sichuan University, Chengdu, 610064, China
Copyright © 2015 John Wiley & Sons, Ltd.
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Biological thiols, such as cysteine (Cys), homocysteine (Hcy) and glutathione (GSH), play a key role in many physiological processes, and connections have been found between abnormal levels of thiols and a number of diseases. A decrease in the level of Cys may lead to many health problems, such as liver damage, slow growth, skin lesions, AIDS and oxidative damage (1–4). An abnormal level of Hcy in human plasma has been shown to be correlated with Alzheimer’s disease, osteoporosis and cardiovascular disease (5–7). GSH is the most abundant (1–10 mM) intracellular thiol and has many cellular functions (8). Changes in cellular GSH levels can also lead to health problems, such as leucocyte loss, heart problems and cancer (9–11). Therefore, the detection and quantification of biothiols in biological systems is of great importance. In the past few years, a number of techniques have been exploited to detect these biological thiols, such as highperformance liquid chromatography (HPLC) (12,13), Fourier transform infrared (FTIR) spectroscopy (14), mass spectrometry (MS) (15,16), surface-enhanced Raman scattering (17), capillary electrophoresis (18,19), electrochemistry assay (20,21), UV/vis spectroscopy (22,23) and fluorescence spectroscopy. Among these various strategies, fluorescent probes for the detection of biothiols have been developed quickly because of the method’s ease of operation, high selectivity, sensitivity, low cost, low detection limits and potential for in vivo imaging (24–26). Therefore, various fluorescent probes for biothiols based on different fluorophores have been constructed, including rhodamine (27–29), naphthalimide (30), BODIPY (31,32), fluorescein (33–36), cyanine (37–39), flavone (40,41) and coumarin (42–50). Of these, coumarin dye is one of the molecules used most widely in fluorescent probes owing to its high photostability, large Stokes shift, high cell permeability, visible emission wavelength and high fluorescence quantum yield (51,52). However, many of these probes suffer from some limitations, including high-pH aqueous solution, high background fluorescence, low sensitivity in vitro and poor biocompatibility. Thus,
there remains an urgent need for a highly sensitively probe that could be used in the further application of biotechnology. Here, we reported a novel coumarin-based derivative fluorescent probe, namely (E)-7-(diethylamino)-3-(3-(4-nitrophenyl)acryloyl)-2Hbenzo- pyran-2-one (CA-N). CA-N was constructed based on the Michael addition reaction of thiol to α,β-unsaturated ketones. The quencher group of nitrobenzene led to the non-fluorescence of CA-N, although a strong green fluorescence was detected once the probe reacted with a thiol group. Furthermore, CA-N had high sensitivity and good biocompatibility, which could be successfully applied to living cell imaging in vitro. In addition, CA-N could detect qualitative changes in thiol concentrations in living cells.
M. Liu et al. Fluorescent emission spectra were obtained using a Perkin–Elmer LS55 fluorescence spectrophotometer. UV/vis absorption spectra were performed on a Perkin–Elmer Lambda 950 UV/VIS Spectrometer. High-resolution MS were obtained using a Shimadzu LCMSIT-TOF. pH was measured with a PHS-3E pH meter. Fluorescence microscopic images were obtained from laser scanning confocal microscopy (CLSM; TCS-SP5, Leica, Germany).
Synthesis 3-Acetyl-7-(diethylamino)-2H-benzopyran-2-one (2). Compound 2 was synthesized according to a previously reported procedure (53). 4-Diethylamino salicylaldehyde (10 g, 51.7 mmol) and ethyl acetonacetate (13.5 g, 100 mmol) were dissolved in absolute ethanol (100 mL), and piperidine (0.2 mL) was added as a catalyst. After the mixture was refluxed under argon atmosphere for 6 h, a yellow solid was obtained and collected by filter. The solid was washed with cool absolute ethanol, crude product was recrystallized from the absolute ethanol, and a yellow solid was collected (10.6 g, yield: 79%). 1 H NMR (400 MHz, CDCl3) δ (ppm): 8.44 (s, 1H), 7.41 (d, J = 8.8 Hz, 1H), 6.64 (dd, J = 8.8 Hz, J = 2.0 Hz, 1H), 6.48 (d, J = 1.6 Hz, 1H), 3.49 (q, 4H), 2.68 (s, 3H), 1.26 (t, J = 7.2 Hz, 6H). m.p. 151–153 °C.
Detection limit studies The detection limit was calculated based on a method described previously (54,55). The fluorescence intensity of CA-N alone was measured seven times and the standard deviation of the blank measurement was obtained. To improve sensitivity, 10 μM CA-N was employed. Under this condition, a good linear working range between the emission intensity and the Cys concentration with the range 10–100 μM was observed. The detection limit was determined as 3σ/k where σ is the standard deviation of the blank measurement and k is the slope of a plot of fluorescence intensity against sample concentration.
Kinetic analysis The time-dependent response of CA-N (10 μM) with Cys, Hcy and GSH (100 eq.) in DMSO/phosphate buffer (1: 1 v/v, pH 7.4) exhibited pseudo-first-order reaction conditions at room temperature, and the fluorescence intensity at 494 nm was recorded. As described in the literature (56,57), the pseudo-first-order rate constant for the reaction was determined by fitting the fluorescence intensities of CA-N to following the pseudo-first-order equation: . ln ðF max F t Þ
F max
(E)-7-(diethylamino)-3-(3-(4-nitrophenyl)acryloyl)-2H-benzopyran-2one (CA-N). Compound 2 (100 mg, 0.39 mmol) and 4-
nitrobenzaldehyde (89.2 mg, 0.59 mmol) were dissolved in absolute ethanol (10 mL) and piperidine (0.1 mL) was added as a catalyst. After the mixture had been refluxed under argon atmosphere for 24 h, the solvents were removed by distillation under reduced pressure and the red solid was collected. The crude solid was extracted with dichloromethane and the solvent was then removed. The crude solid was further purified by column chromatography on silica gel to give a red solid. After recrystallization in absolute ethanol, and drying under vacuum, a red solid was acquired (45.9 mg, yield: 30%). 1 H NMR (400 MHz, CDCl3) δ (ppm): 8.58 (s, 1H), 8.31 (d, J = 16.0 Hz, 2H), 8.26 (d, J = 8.8 Hz, 1H), 7.82 (d, J = 8.8 Hz, 2H), 7.81 (d, J = 16.0 Hz, 1H), 7.46 (d, J = 9.2 Hz, 1H), 6.67–6.64 (dd, J = 8.8 Hz, 2.4 Hz, 1H), 6.51 (d, J = 2.0 Hz, 1H), 3.51 (q, 4H), 1.29 (t, J = 7.2 Hz, 6H). 13C NMR (100 MHz, CDCl3) δ (ppm): 184.9, 160.0, 157.9, 152.4, 148.2, 147.3, 140.6, 138.60, 131.1, 128.1, 128.0, 123.0, 114.9, 109.1, 107.7, 95.7, 44.2, 11.5. HR-MS (ESI): m/z [M + Na]+ 415.1265, calcd.: 415.1264. m.p. 227–228 °C.
¼ k’ t
Where Ft and Fmax are the fluorescence intensities at 494 nm at time t and the maximum value obtained after the reaction was completed. k’ is the pseudo-first-order rate constant.
Cell experiments For the cell cytotoxicity assay, MG63 cells were seeded in 96-well plates at a concentration of 1 × 104 cells/well for 24 h, then different concentrations of CA-N (0, 5, 10 and 20 μM final concentration) were added to the wells and the cells were cultured for another 24 h. Cytotoxicity was determined by MTT colorimetric method using five parallel wells at each time point. Cells were incubated with 5 mg/mL of MTT for 4 h at 37 °C, the supernatant was then discarded, and DMSO (200 μL/well) was added to dissolve the formazan; the absorbance was then measured at 570 nm with a microplate reader (BioRad, model 550). Cytotoxicity assay.
Bioimaging for biothiols in living cells Preparation of solutions for fluorescence and UV/vis absorption
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A stock solution of CA-N (1.5 mM) was prepared in DMSO. Stock solution of Cys, Hcy, GSH (30 mM) and other amino acids (10 mM) including Ala, Arg, Asp, Glu, Gly, His, Leu, Lys, Phe, Pro, Ser, Tle, Thr, Trp, Tyr and Val were dissolved in double-distilled water, and solutions (DMSO/aqueous buffer, 1: 1 v/v, pH 1.0–11.0) with different pH values were prepared. In a typical experiment, 20 μL of the CA-N stock solution was added to 3 mL DMSO/phosphate buffer (1: 1 v/v, pH 7.4) and then an appropriate buffer of each amino acids was added. Fluorescence and absorption spectra were recorded after the addition of amino acids at room temperature.
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MG63 cells were seeded on 35 mm glass-bottomed dishes at a density of 1 × 105 cells/dish in culture medium and incubated overnight in preparation for live cell imaging. The cells were divided into four groups: (1) no added dye or biothiols; (2) treatment with 10 μM of CA-N in a serum-free medium for 30 min and washed twice with prewarmed PBS; (3) treatment with 1.0 mM Nethylmaleimide (NEM, a thiol-blocking reagent) for 30 min to decrease the concentration of cellular biothiols, then washed twice with prewarmed PBS buffer, followed by addition of CA-N for 30 min; (4) pretreatment with NEM for 30 min, followed by addition of Cys (100 μM) and CA-N for 30 min and washed twice with prewarmed PBS. The four groups were observed under CLSM using the excitation channel (λex = 405 nm).
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Luminescence 2015; 30: 1395–1402
Coumarin-based probe for the detection of biothiols
Results and discussion Design and synthesis of CA-N To obtain a fluorescent probe that selectively detected thiols for potential biological applications, the α,β-unsaturated ketones moiety was chosen as the Michael addition reaction recognition unit, and diethylaminocoumarin was selected as the fluorophore. The synthetic route for probe CA-N is shown in Scheme 1. Compound 2 was synthesized and used as a reference compound to reveal the Michael addition reaction mechanism. The structure of CA-N was confirmed by 1H NMR, 13C NMR and high-resolution MS.
UV/vis absorption and fluorescence studies with thiols The absorption and fluorescence emission spectra of CA-N in the absence and presence of Cys in DMSO/phosphate buffer (1: 1 v/v, pH 7.4) at room temperature were examined and are recorded in Fig. 1. Because of use of the nitrobenzene group as the quencher moiety, CA-N alone did not exhibit fluorescence, although upon addition of Cys (100 eq.), the fluorescence spectra changed dramatically, and a strong emission peak was observed at 494 nm. Furthermore, in the absorption spectrum, addition of Cys caused a clear hypsochromic shift (~20 nm) from 472 to 452 nm. The changes in the absorption spectrum and fluorescence intensity of CA-N in the presence of Cys were attributed to the conjugate addition of thiol to a carbon–carbon double bond through Michael addition. When CA-N reacted with thiols, it was able to break the carbon–carbon double bond, the quencher was broken and the fluorescence recovered (Scheme 2). Thus, CA-N could be used as a fluorescent turn-on probe for thiols.
Detection limit studies To evaluate the detection limit of CA-N for Cys, Hcy and GSH in solution, CA-N was treated with various concentrations of Cys, Hcy or GSH in DMSO/phosphate buffer (1: 1 v/v, pH 7.4) at room temperature. The results show that the fluorescence intensity of CA-N gradually increased with increasing of Cys concentration (0–100 eq.) (Fig. 2). Simultaneously, under the same conditions, when Hcy or GSH (0–100 eq.) was added to CA-N solution, similar results were obtained (Fig. S1). The results of the titration experiment indicated a good linear relationship between the fluorescence intensity and Cys concentration within the range 10–100 μM (Fig. 3). Under these conditions, the detection limit for Cys was calculated as 3.16 μM. Under the same conditions, the detection limits for Hcy and GSH were 0.19 and 5.15 μM, respectively (Table 1 and Fig. S2). The low detection limit showed that CA-N was highly sensitive to
Figure 1. Normalized absorption spectrum of CA-N (■) and upon addition of 100 eq. Cys (●). Normalized fluorescence spectra of CA-N (▲) and upon addition of 100 eq. Cys (▼). Conditions: CA-N in the absence and presence of Cys in DMSO/phosphate buffer (1: 1 v/v, pH 7.4) at room temperature (λex = 400 nm).
biological thiols. Meanwhile, these data suggested that CA-N could be applied to the quantitative detection of biothiols.
pH-dependent studies In order to understand the pH-dependent effects of physiological media, CA-N solutions at different pH values (1.0–11.0) in the presence and absence of Cys, Hcy and GSH were investigated. As shown in Fig. 4, CA-N alone showed weak fluorescence only, its intensity did not change at different pH values (1.0–11.0), which suggested that CA-N was not affected by pH. By contrast, in the presence of Cys, the fluorescence intensity showed a large enhancement when the pH was > 5.0, and the fluorescence intensity reached a maximum at pH 8.5. At the same time, in the presence of Hcy or GSH, the fluorescence intensity reached a maximum value at pH 8.5 and 9.0, respectively. These results might be due to the influence of the pKa value of aliphatic thiols on the reaction. The pKa value of aliphatic thiols was ~ 8.5 (58,59)._ENREF_47 These results indicate that CA-N was sensitive enough to detect thiols under neutral to alkaline conditions.
Kinetic analysis The time-dependent fluorescence intensity of CA-N (10 μM) with Cys, Hcy and GSH (100 eq.) in DMSO/phosphate buffer (1: 1 v/v, pH 7.4) was recorded. As shown in Fig. 5, the fluorescence intensity reached a maximum after ~ 35 min in the presence Cys and Hcy. However, in the presence of GSH, the fluorescence intensity
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Scheme 1. Synthetic route for probe CA-N.
Luminescence 2015; 30: 1395–1402
Copyright © 2015 John Wiley & Sons, Ltd.
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M. Liu et al.
Scheme 2. The design concept of a fluorescent turn-on probe (CA-N) for thiols.
Figure 2. Fluorescent titrations of probe CA-N (10 μM) upon addition of various concentrations of Cys (0–100 eq.) in DMSO/phosphate buffer (1: 1 v/v, pH 7.4) after 35 min at room temperature (λex = 400 nm).
Figure 3. Linear plot of CA-N (10 μM) in the presence of Cys (10–100 μM) against fluorescence intensity at 494 nm (λex = 400 nm).
Figure 4. Fluorescence intensity of CA-N (10 μM) at 494 nm before and after addition of Cys, Hcy and GSH (0–100 eq.) at different pH values in DMSO/phosphate buffer for 35 min at room temperature (λex = 400 nm).
Figure 5. Time-dependent fluorescence intensity of CA-N (10 μM) at 494 nm upon addition of Cys, Hcy and GSH (100 eq.) in DMSO/phosphate buffer (1: 1 v/v, pH 7.4) at room temperature (λex = 400 nm).
Table 2. Pseudo-first-order rate constants and lifetime of CA-N to Cys, Hcy, and GSH Table 1. Detection limit of the probe to Cys, Hcy and GSH
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Number
Thiol
σ
k
Detection limit (μM)
1 2 3
Cys Hcy GSH
0.7157 0.7157 0.7157
6.79 × 105 1.14 × 106 4.17 × 105
3.16 0.19 5.15
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Number
Thiol
Kinetic constant; k′ (min–1)
Life time; t1/2 (min)
1 2 3
Cys Hcy GSH
0.106 0.114 0.020
6.54 6.10 34.55
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Coumarin-based probe for the detection of biothiols fluorescence intensity at 494 nm was recorded and the results are shown in Fig. 6. We found that CA-N displayed weak fluorescence in the presence of Ala, Arg, Asp, Glu, Gly, His, Leu, Lys, Phe, Pro, Ser, Tle, Thr, Trp, Tyr and Val. However, under the same conditions, when thiol-containing analytes (Cys, Hcy and GSH) were added to CA-N, a great enhancement of the fluorescence intensity was observed. The data suggest that CA-N had a high selectivity for thiol-containing molecules. Meanwhile, to further test the Cys selectivity of CA-N in the presence of other analytes, CA-N (10 μM) was added to Cys (10 eq.) in the presence of various amino acids (10 eq.) in DMSO/phosphate buffer. None of analytes measured had an effect on the fluorescence detection of Cys (Fig. S7). Therefore, at physiological pH, CA-N could be highly selective for biothiols, even in the presence of these relevant amino acids in a biological system. Figure 6. Fluorescence intensity of CA-N (10 μM) at 494 nm in the presence of various amino acids (10 eq.) in DMSO/phosphate buffer (1: 1 v/v, pH 7.4) for 35 min at room temperature (λex = 400 nm). 1, Cys; 2, Hcy; 3, GSH; 4, Ala; 5, Arg; 6, Asp; 7, Glu; 8, Gly; 9, His; 10, Leu; 11, Lys; 12, Phe; 13, Pro; 14, Ser; 15, Tle; 16, Thr; 17, Trp; 18, Tyr; 19, Val; 20, blank.
reached a maximum after ~ 90 min (Fig. S3). CA-N exhibited much higher reactivity toward Cys and Hcy than towards GSH, in agreement previous reports (55,60). This may be explained by steric hindrance effects on the thiol 1,4-addition reaction. Meanwhile, the kinetic profiles of the CA-N reaction with Cys, Hcy and GSH were calculated from the fluorescence intensity at 494 nm. The pseudo-first-order rate constant k’ was determined following the pseudo-first-order equation. The pseudo-first-order rate constants for Cys, Hcy and GSH were determined as k’ = 0.106, 0.114 and 0.020 min-1, respectively; the corresponding half-life (t1/2) values were 6.45, 6.10 and 34.55 min, respectively (Table 2 and Figs. S4–6). These results further demonstrated that CA-N had high reactivity for Cys and Hcy compared with GSH. Selective studies In order to check the selective response of CA-N toward biothiols over other amino acids, various amino acids were separately added to CA-N in DMSO/phosphate buffer for 35 min, and the
Mechanism of CA-N for detecting Cys From the fluorescence spectral studies, CA-N shows a selective response to biothiols. CA-N was able to react through conjugate addition with a thiol group of Cys and form a CA-N–Cys adduct. In order to provide evidence of the reaction mechanism, the fluorescence spectra of adduct CA-N–Cys and compound 2 in DMSO/phosphate buffer at room temperature were recorded. Compound 2 had a similar structure to the diethylaminocoumarin fluorophore with CA-N–Cys adduct and served as a reference compound. We found that CA-N and compound 2 had almost the same fluorescence spectra (Fig. S8). Meanwhile, 1H NMR spectroscopy studies also provided evidence of CA-N in the absence or presence of Cys. As shown in Fig. 7, the alkene proton at around 8.08 ppm (Ha) disappeared, and a new peak emerged at around 4.58 ppm (Ha′); there was another alkene proton at around 7.76 ppm (Hb), and in the presence of Cys, two new peaks emerged at 3.71 ppm (Hb′) and 3.32 ppm (Hb′′). These results proved that CA-N could react with the thiol group of Cys to form a carbon–carbon double bond via Michael addition reaction. Furthermore, MS analysis of CA-N with Cys revealed formation of a CA-N–Cys adduct. The MS displayed peaks at m/z 415.1254 and 536.1466, corresponding to [CA-N + Na] and [CA-N–Cys + Na], respectively (Fig. S9). Taken together, these results consistently
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Figure 7. (Upper) A proposed mechanism of the reaction between CA-N and Cys. (Lower) H NMR spectra changed of CA-N in the absence (A) or presence (B) of Cys in DMSO-d6/ D2O (4: 1, v/v) at room temperature.
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Figure 8. Confocal Laser microscope images of MG63 cells. (A–C) Control group. (D–F) MG63 cells incubated with CA-N (10 μM) for 30 min at 37 °C. (G– I) Cells pretreated with NEM (1.0 mM) for 30 min and then incubated with CA-N (10 μM) for 30 min at 37 °C. ( J–L) Cells pretreated with NEM (1.0 mM) for 30 min and tzhen added to Cys (100 μM) and incubated with CA-N (10 μM) for 30 min at 37 °C. (Left) Bright channel, (middle) fluorescence channel (right) overlay. (M) Fluorescence intensity of each group.
revealed the reaction of CA-N with Cys and the formation of the CA-N–Cys adduct.
these results proved that CA-N could be used as an excellent fluorescent probe for biothiol imaging in living cells, and the alteration of fluorescence intensity indicates changes in the concentration of cellular biothiols.
Fluorescent imaging in MG63 cells
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To investigate the permeability of cells and clarify whether CA-N was sensitive enough to detect biothiols in living cells, the cytotoxicity of CA-N was evaluated using MTT assays. The results showed that cells grew very well even with 20 μM CA-N at 37 °C for 24 h and no significant difference was observed between the groups. The results indicated no cytotoxicity of CA-N (Fig. S10). Furthermore, cell-imaging experiments with CA-N were carried out and observed under CLSM. As shown in Fig. 8, MG63 cells without added CA-N did not show any fluorescence (Fig. 8B), whereas after incubation with CA-N at 37 °C for 30 min, significant green fluorescence was observed in the cytoplasm of these cells (Fig. 8E), which proved that CA-N was capable of permeating cells and reacting with intracellular biothiols. To further determine whether CA-N could highlight changes in the concentration of biothiols in living cells, one group was pretreated with 1.0 mM NEM for 30 min to eliminate any biothiols in the cells; only weak fluorescence was observed in these cells after incubation with CA-N (Fig. 8H). However, strong green fluorescence was again observed, when NEM-treated cells were added with Cys (100 μM) and incubated with CA-N for another 30 min (Fig. 8K). According to Image J™ software, the fluorescence intensity value of each group was obtained (Fig. 8M). Taken together,
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Conclusions We designed and synthesized a novel coumarin-based fluorescent probe CA-N, which exhibited high sensitivity and selectivity toward biothiols (Cys, Hcy, GSH) over other natural amino acids. Fluorescence spectra, 1H NMR and MS studies demonstrated the mechanism by which thiols combined with the α,β-unsaturated ketone of CA-N via the Michael addition reaction. In addition, in vitro cell imaging found that CA-N could easily penetrate cell membranes, react with biothiols in the cytoplasm, and show strong green fluorescence. Our results prove that CA-N is useful for imaging biothiols in living cells.
Acknowledgements The authors acknowledge the financial support for this work by Research Fund for the Doctoral Program of Higher Education of China (20130181110089), the National Natural Science Foundation of China (NSFC)(Grants No.21372168) and National Key Technology R&D Program (2012BAI42G01). Electronic Supplementary Information (ESI) are available.
Copyright © 2015 John Wiley & Sons, Ltd.
Luminescence 2015; 30: 1395–1402
Coumarin-based probe for the detection of biothiols
References
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