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Cite this: DOI: 10.1039/c5an00549c Received 19th March 2015, Accepted 1st May 2015 DOI: 10.1039/c5an00549c
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Heavy atom quenched coumarin probes for sensitive and selective detection of biothiols in living cells† Wengang Ji,a,b Yuzhuo Ji,a Qingqing Jin,a Qingxiao Tong*b and Xinjing Tang*a
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A series of turn-on fluorescent probes with halogen acetyl amide at the 3-position of coumarin derivatives were synthesized. Fluorescence of these probes was efficiently quenched by heavy halogen atoms (Br and I, not Cl), which could be successfully used for selective detection of biothiols with the sensitivity of Cys > GSH > Hcy and much higher than thiol containing proteins. These represent the smallest fluorescence quenchers in designing fluorescent probes for detecting both endogenous and exogenous biothiols in living cells.
Introduction Cysteine (Cys), homocysteine (Hcy), and glutathione (GSH) are biologically relevant reactive thiol species (biothiols) that are tightly associated with the life of living cells. For instance, Cys is versatile in protein synthesis and functions.1 As is reported, Hcy is a crucial intermediate generated during the biosynthesis of Cys. An elevated Hcy level often causes some cardiovascular disease,2 diabetes,3 renal disease4 and Alzheimer’s disease.5 The tripeptide GSH is present as the most abundant (0.1–10 mM) biothiol in living cells.6 In addition, biothiols are ideal nucleophiles in enzyme functions, excellent regulators and mild buffering molecules for maintaining the balance of the cellular redox environment. However, abnormal levels of these species in living organisms often indicate or even lead to various lesions. Due to the convenience of fluorescence detection, many fluorescent probes have been developed to detect biothiols.7 As we know, the mercapto group has both high nucleophilicity and strong reducibility.8 Thus, most of the fluorescent probes a State Key Laboratory of Natural and Biomimetic Drugs, The School of Pharmaceutical Sciences, Peking University, Beijing 100191, China. E-mail: [email protected] b Department of Chemistry, Shantou University, Guangdong, 515063, China. E-mail: [email protected] † Electronic supplementary information (ESI) available: Detailed synthesis, characterization (NMR, MS etc.) of the probes; model reaction, characterization, cell viability etc. See DOI: 10.1039/c5an00549c
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were designed based on thiol reactions, such as Michael addition,9 nucleophilic addition,10 disulphide bond cleavage,11 metal–thiol interactions,12 native chemical ligation,13,14 redox reaction,15 nucleophilic substitution,16 and combined multiple modes.13,17,18 For the design concepts of these probes, almost all of them were based on the classically certified mechanisms, such as ICT, PET and FRET. However, large moieties, such as 2,4-dinitrobenzenesulfonyl, maleamate and 4-methoxythiophenol, have been chosen as fluorescence quenchers.17,19 In this paper, we designed and synthesized a group of fluorescent coumarin probes whose fluorescence was quenched by single atoms through the heavy atom effect. This represents the smallest fluorescence quencher in designing fluorescent probes for biothiols. These probes could selectively detect endogenous and exogenous biothiols in living cells.
Results and discussion Synthesis and evaluation of fluorescence chemosensors for biothiols Based on high fluorescence quantum yield, nice water solubility and good cell permeability of the coumarinyl moiety, a series of 7-diethylamine coumarin derivatives (Cou-Cl, Cou-Br, Cou-I, and Cou-H) with halogen acetyl and acetyl amide at the 3-position were synthesized (Schemes 1 and S1, 2†). These coumarin derivatives (Cou-Br and Cou-I) with bromo and iodo modifications emitted very weak fluorescence (fluorescence
Scheme 1
The synthesis of coumarin probes for biothiol detection.
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quantum yields: 0.066 and 0.073, respectively). Cou-Cl has the same fluorescence emission wavelength as that of Cou-H but showed a much lower fluorescence emission intensity than that of Cou-H (fluorescence quantum yield: 0.15 for Cou-Cl and 0.63 for Cou-H, ESI Fig. S1–2 and Table S1†). Heavy elements, such as I−, have previously been used as anion quenchers in steady-state emission quenching experiments.20 The attachment of heavy atoms, bromo and iodo, directly on coumarin caused the significant loss of their fluorescence emission of Cou-Br and Cou-I, which is ascribed to the intramolecular heavy atom effect. These represent the smallest fluorescence quenchers in designing fluorescent probes. Currently, no literature studies have been reported to apply a heavy atom (like Br and I) as a quencher in the rational design of fluorescence sensors. To evaluate the possible use of bromo and iodo as heavy atom quenchers for turn-on fluorescence sensors, we incubated three halogen acetyl coumarin probes (Cou-Cl, Cou-Br and Cou-I) with biothiol species, such as Cys, GSH and Hcy. As shown in Fig. 1, the addition of Cys quickly triggered the nonfluorescent Cou-Br and Cou-I to high fluorescent species and reached their plateaus in about 40 min, while almost no increase in the fluorescence intensity was observed for Cou-Cl. The fluorescence turn-on rate of Cou-I ( pseudo-first-order kinetic rate constant: k′ = 10.3 × 10−2 min−1, Fig. S3†) was also a little bit larger than that of Cou-Br (k′ = 8.11 × 10−2 min−1, Fig. S3†), which is consistent with the nucleophilic substitution ability of bromo and iodo toward thiol species. To further confirm the heavy atom quenching mechanism, the reaction mixture of Cou-Br and Cys was evaluated by MS analysis and the result indicated the replacement of Br with Cys (Fig. S4†). However, to make sure whether there exists further intramolecular replacement of the aromatic amine with the primary amine of Cys, we carried out a model reaction of cysteamine and Cou-Br (ESI Scheme S3 and Fig. S5–7†). The data of MS, NMR and HMBC indicated that only nucleophilic replacement of bromo with mercaptan thiol took place without further substitution of the amide moiety of the probe by amine moiety of cysteamine. In addition, we also compared the fluorescence spectra of the product of Cou-Br and Cys reactions with Cou-NH2 and Cou-H (Fig. S2†). The turn-on fluorescence spectra of Cou-Br were the same as those of Cou-H, and both fluorescence peaks were blue-shifted compared to the starting material Cou-NH2. These results were consistent with the model experiment, which indicated that only the substitution of heavy atoms took place. Evaluation of sensitivity and selectivity Since the inability of Cou-Cl to sense thiol species, only Cou-Br and Cou-I were evaluated in further experiments. The fluorescence emission spectra of Cou-Br and Cou-I were measured with various concentrations of Cys. Fig. 2 and S8† show that the addition of Cys (0–500 μM) to Cou-Br (5 μM) or Cou-I
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Fig. 1 Fluorescence spectra of Cou-Br (a) and Cou-I (b) (5 μM, respectively) obtained upon addition of 500 μM Cys in PBS. The time interval is 5 min in each scan. (c) Kinetics of turn-on fluorescence of Cou-Cl, CouBr and Cou-I (5 μM for each probe) in PBS upon addition of 500 μM Cys. (d) Fluorescence increase with or without 500 μM Cys in 60 min. The PBS buffer was 25 mM PBS ( pH = 7.4) containing 10% CH3CN. λex = 400 nm, λem = 495 nm.
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Fig. 2 (a) Fluorescence spectra of Cou-Br (5 μM) obtained upon addition of various concentrations (0–500 μM) of Cys in PBS buffer in 45 min. (b) The linear relationship between fluorescence intensity (495 nm) of probe Cou-Br solution and Cys concentrations. The PBS buffer was 25 mM PBS (pH = 7.4) containing 10% CH3CN. λex = 400 nm, λem = 495 nm.
(5 μM) led to a concentration dependent increase in the fluorescence intensity at 495 nm. The standard curve for CouBr and Cou-I in degassed PBS was obtained between the fluorescence intensity at 495 nm and the concentrations (μM) of Cys in 45 min incubation. The regression equations were FEx/Em = 0.356 [Cys] + 6.88 and FEx/Em = 0.320 [Cys] + 8.66 with R2 = 0.999 and R2 = 0.996 for Cou-Br and Cou-I, respectively. The data suggested good linear relationships between the turn-on fluorescence intensity of Cou-Br and Cou-I at 495 nm and Cys concentrations in degassed PBS buffer. Furthermore, to explore the selectivity of Cou-Br and Cou-I, the probes were also treated with other common biologically amino acid species and Vc, such as Trp, His, Ala, Lys, Phe, Glu, Arg, Val, Try, Asp, Pro, Hcy, and GSH. It was found that almost no triggered fluorescence responses of Cou-Br solution were observed for the various common amino acids and Vc, except for GSH and Hcy with the thiol moiety (Fig. 3). However, reaction dynamics of Cou-Br to GSH and Hcy was slower than Cys in the order of Cys > GSH > Hcy, which was confirmed by their second rate constants (7.8 × 10−4 M−1 min−1 for Cys, 2.4 × 10−4 M−1 min−1 for GSH and 1.5 × 10−4 M−1 min−1 for Hcy) after fitting the first rate constants with the pseudo second dynamic equation (Fig. S3, S9 and Table S2†). For Cou-I, we observed the similar selectivity of biothiols over other amino acid species (Fig. S10†). In
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Fig. 3 (a) Fluorescence spectra of Cou-Br (5 μM), Cys (500 μM) and other analytes (500 μM Hcy and GSH, and 2 mM amino acids and Vc, and 1 mg ml−1 BSA) in PBS buffer. (b) Fluorescence responses of Cou-Br to other analytes (500 μM Hcy and GSH and 2 mM amino acids and Vc) without (black column) or with (red column) 500 μM Cys. The PBS buffer was 25 mM PBS ( pH = 7.4) containing 10% CH3CN. λex = 400 nm, λem = 495 nm.
addition, the fluorescence detection of Cys with the probe Cou-Br was also achievable over up to 1 mg mL−1 of the thiol containing proteins, such as bovine serum albumin (BSA) (Fig. S11†). To study the pH effect on fluorescence detection of biothiols, the probes, Cou-Br and Cou-I, were also evaluated for the detection of Cys in buffers with pH values from 4.4 to 10. As shown in Fig. 4 and S14,† these two probes were both effective for detecting Cys in buffers of all pH environment, however, higher fluorescence enhancement was observed under neutral and basic conditions. The lower sensitivity in low pH buffers was due to the protonation of the aromatic amine which affected the push–pull electronic system of the coumarin moiety.21 Cell studies Cell viability indicated that no obvious toxicity was observed for Cou-Br and Cou-I (up to 50 μM for 24 h) by the SRB method (Fig. S15†). To further evaluate the feasibility for
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eties in cells were consumed. Due to a low biothiol level in NEM-treated cells, we only observed very weak fluorescence emission. However, after the NEM treated cells were first washed and incubated with Cys again, high fluorescence emission in cells was fully recovered upon the addition of the probe Cou-Br. These results confirmed that the probe Cou-Br can be used to monitor both endogenous and exogenous biothiols in living cells.
Conclusions A series of coumarin derivatives were synthesized and used as turn-on fluorescent probes for biothiols. The fluorescence of these coumarin probes were efficiently quenched by a single heavy atom (such Br or I) through the heavy atom effect, which represents the smallest fluorescence quencher in the literature. Upon substitution of heavy atoms with the thiol group, the turn-on fluorescence of coumarin was quickly observed. These probes showed high selectivity toward biothiol species with the sensitivity of Cys > GSH > Hcy. Linear relationships between the turn-on fluorescence intensity and the concentrations of biothiols were obeyed. In addition, these coumarin probes with the attachment of heavy atoms were also able to detect both the endogenous and exogenous biothiols in living cells. Fig. 4 Fluorescence responses of Cou-Br (a) and Cou-I (b) (5 μM) with or without 500 μM Cys in different pH values of PBS (4.4, 5.0, 6.0, 7.0, 7.4, 7.9, 8.9, 10.0) containing 10% CH3CN, λex = 400 nm, λem = 495 nm.
biothiol detection in living cells, HeLa cells were chosen and treated with 5 μM Cou-Br for 50 min. The cells were then washed to remove extracellular reagents and then visualized using High Content Screening (HCS). As displayed in Fig. 5a–f, cells incubated with Cou-Br show strong fluorescence emission due to high concentrations of endogenous biothiols in living cells. In contrast, when N-ethylmaleimide (NEM, 1 mM, a thiol inhibitor) was added to the cell medium, most of thiol moi-
Fig. 5 (a) and (b) were images of HeLa cells incubated with 5 μM Cys-Br for 50 min. (c) and (d) were images of HeLa cells incubated with 5 μM Cys-Br for 50 min after the cells were treated with 1 mM NEM for 30 min. (e) and (f ) were images of HeLa cells treated with 1 mM NEM for 30 min first and then incubated with 5 μM Cys-Br for 50 min after the incubation with 100 μM Cys for 30 min.
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Acknowledgements This work was financially supported by the National Basic Research Program of China (‘973’ Program: 2013CB933800, 2012CB720600, and 2013CB834803), and the National Natural Science Foundation of China (no. 21422201 and 51273108).
Notes and references 1 S. A. Lipton, Y.-B. Choi, H. Takahashi, D. Zhang, W. Li, A. Godzik and L. A. Bankston, Trends Neurosci., 2002, 25, 474–480. 2 A. S. Wierzbicki, Diab. Vasc. Dis. Res., 2007, 4, 143–150. 3 E. P. Wijekoon, M. E. Brosnan and J. T. Brosnan1, Biochem. Soc. Trans., 2007, 35, 1175–1179. 4 C. v. Guldener, Nephrol. Dial. Transplant, 2006, 21, 1161–1166. 5 H. Peng, Y. Cheng, C. Dai, A. L. King, B. L. Predmore, D. J. Lefer and B. Wang, Angew. Chem., Int. Ed., 2011, 50, 9672–9675. 6 A. Pastore, G. Federici, E. Bertini and F. Piemonte, Clin. Chim. Acta, 2003, 333, 19–39. 7 X. Chen, Y. Zhou, X. Peng and J. Yoon, Chem. Soc. Rev., 2010, 39, 2120–2135; H. S. Jung, X. Chen, J. S. Kim and J. Yoon, Chem. Soc. Rev., 2013, 42, 6019–6031; H. Peng, W. Chen, Y. Cheng, L. Hakuna, R. Strongin and B. Wang, Sensors, 2012, 12, 15907–15946. 8 K. Wang, H. Peng and B. Wang, J. Cell. Biochem., 2014, 115, 1007–1022.
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9 Y. Zhang, J. H. Wang, W. J. Zheng, T. F. Chen, Q. X. Tong and D. Li, J. Mater. Chem. B, 2014, 2, 4159–4166; H. Wang, G. Zhou, H. Gai and X. Chen, Chem. Commun., 2012, 48, 8341–8343; X. Yang, Y. Guo and R. M. Strongin, Angew. Chem., Int. Ed., 2011, 50, 10690–10693; Q. Zhang, D. Yu, S. Ding and G. Feng, Chem. Commun., 2014, 50, 14002– 14005; D. Yu, Q. Zhang, S. Ding and G. Feng, RSC Adv., 2014, 4, 46561–46567. 10 A. Barve, M. Lowry, J. O. Escobedo, K. T. Huynh, L. Hakuna and R. M. Strongin, Chem. Commun., 2014, 50, 8219–8222; H. Li, J. Fan, J. Wang, M. Tian, J. Du, S. Sun, P. Sun and X. Peng, Chem. Commun., 2009, 5904–5906; K. S. Lee, T. K. Kim, J. H. Lee, H. J. Kim and J. I. Hong, Chem. Commun., 2008, 6173–6175; F. Tanaka, N. Mase and C. F. Barbas III, Chem. Commun., 2004, 1762–1763. 11 L. Rong, C. Zhang, Q. Lei, H.-L. Sun, S.-Y. Qin, J. Feng and X.-Z. Zhang, Chem. Commun., 2014, 2, 388–390; D. Jung, S. Maiti, J. H. Lee, J. H. Lee and J. S. Kim, Chem. Commun., 2014, 50, 3044–3047. 12 U. R. G. H. Agarwalla, N. Taye, S. Ghorai, S. Chattopadhyay and A. Das, Chem. Commun., 2014, 69, 9899–9902; Y. K. Yang, S. Shim and J. Tae, Chem. Commun., 2010, 46, 7766–7768. 13 X. F. Yang, Q. Huang, Y. Zhong, Z. Li, H. Li, M. Lowry, J. O. Escobedo and R. M. Strongin, Chem. Sci., 2014, 5, 2177–2183.
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14 L. Long, W. Lin, B. Chen, W. Gao and L. Yuan, Chem. Commun., 2011, 47, 893–895. 15 H. Peng, K. Wang, C. Dai, S. Williamson and B. Wang, Chem. Commun., 2014, 50, 13668–13671. 16 F. Wang, Z. Guo, X. Li, X. Li and C. Zhao, Chem. – Eur. J., 2014, 20, 11471–11478; M. Li, X. Wu, Y. Wang, Y. Li, W. Zhu and T. D. James, Chem. Commun., 2014, 50, 1751– 1753; Y. Kim, M. Choi, S. Seo, S. T. Manjare, S. Jon and D. G. Churchill, RSC Adv., 2014, 4, 64183–64186; L. Y. Niu, Y. S. Guan, Y. Z. Chen, L. Z. Wu, C. H. Tung and Q. Z. Yang, J. Am. Chem. Soc., 2012, 134, 18928–18931. 17 H. Zhang, C. Zhang, R. Liu, L. Yi and H. Sun, Chem. Commun., 2015, 51, 2029–2032. 18 J. Liu, Y. Q. Sun, Y. Huo, H. Zhang, L. Wang, P. Zhang, D. Song, Y. Shi and W. Guo, J. Am. Chem. Soc., 2014, 136, 574–577. 19 J. Liu, Y.-Q. Sun, H. Zhang, Y. Huo, Y. Shi and W. Guo, Chem. Sci., 2014, 5, 3183–3188; L. Yi, H. Li, L. Sun, L. Liu, C. Zhang and Z. Xi, Angew. Chem., Int. Ed., 2009, 48, 4034– 4037. 20 K. Kikuchi, M. Hoshi, T. Niwa, Y. Takahashi and T. Miyashi, J. Phys. Chem., 1991, 95, 38–42; I. B. Berlman, J. Phys. Chem., 1973, 77, 562–567. 21 J. Li, C. F. Zhang, S. H. Yang, W. C. Yang and G. F. Yang, Anal. Chem., 2014, 86, 3037–3042.
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Cite this: DOI: 10.1039/c5an00549c Received 19th March 2015, Accepted 1st May 2015 DOI: 10.1039/c5an00549c
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Heavy atom quenched coumarin probes for sensitive and selective detection of biothiols in living cells† Wengang Ji,a,b Yuzhuo Ji,a Qingqing Jin,a Qingxiao Tong*b and Xinjing Tang*a
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A series of turn-on fluorescent probes with halogen acetyl amide at the 3-position of coumarin derivatives were synthesized. Fluorescence of these probes was efficiently quenched by heavy halogen atoms (Br and I, not Cl), which could be successfully used for selective detection of biothiols with the sensitivity of Cys > GSH > Hcy and much higher than thiol containing proteins. These represent the smallest fluorescence quenchers in designing fluorescent probes for detecting both endogenous and exogenous biothiols in living cells.
Introduction Cysteine (Cys), homocysteine (Hcy), and glutathione (GSH) are biologically relevant reactive thiol species (biothiols) that are tightly associated with the life of living cells. For instance, Cys is versatile in protein synthesis and functions.1 As is reported, Hcy is a crucial intermediate generated during the biosynthesis of Cys. An elevated Hcy level often causes some cardiovascular disease,2 diabetes,3 renal disease4 and Alzheimer’s disease.5 The tripeptide GSH is present as the most abundant (0.1–10 mM) biothiol in living cells.6 In addition, biothiols are ideal nucleophiles in enzyme functions, excellent regulators and mild buffering molecules for maintaining the balance of the cellular redox environment. However, abnormal levels of these species in living organisms often indicate or even lead to various lesions. Due to the convenience of fluorescence detection, many fluorescent probes have been developed to detect biothiols.7 As we know, the mercapto group has both high nucleophilicity and strong reducibility.8 Thus, most of the fluorescent probes a State Key Laboratory of Natural and Biomimetic Drugs, The School of Pharmaceutical Sciences, Peking University, Beijing 100191, China. E-mail: [email protected] b Department of Chemistry, Shantou University, Guangdong, 515063, China. E-mail: [email protected] † Electronic supplementary information (ESI) available: Detailed synthesis, characterization (NMR, MS etc.) of the probes; model reaction, characterization, cell viability etc. See DOI: 10.1039/c5an00549c
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were designed based on thiol reactions, such as Michael addition,9 nucleophilic addition,10 disulphide bond cleavage,11 metal–thiol interactions,12 native chemical ligation,13,14 redox reaction,15 nucleophilic substitution,16 and combined multiple modes.13,17,18 For the design concepts of these probes, almost all of them were based on the classically certified mechanisms, such as ICT, PET and FRET. However, large moieties, such as 2,4-dinitrobenzenesulfonyl, maleamate and 4-methoxythiophenol, have been chosen as fluorescence quenchers.17,19 In this paper, we designed and synthesized a group of fluorescent coumarin probes whose fluorescence was quenched by single atoms through the heavy atom effect. This represents the smallest fluorescence quencher in designing fluorescent probes for biothiols. These probes could selectively detect endogenous and exogenous biothiols in living cells.
Results and discussion Synthesis and evaluation of fluorescence chemosensors for biothiols Based on high fluorescence quantum yield, nice water solubility and good cell permeability of the coumarinyl moiety, a series of 7-diethylamine coumarin derivatives (Cou-Cl, Cou-Br, Cou-I, and Cou-H) with halogen acetyl and acetyl amide at the 3-position were synthesized (Schemes 1 and S1, 2†). These coumarin derivatives (Cou-Br and Cou-I) with bromo and iodo modifications emitted very weak fluorescence (fluorescence
Scheme 1
The synthesis of coumarin probes for biothiol detection.
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quantum yields: 0.066 and 0.073, respectively). Cou-Cl has the same fluorescence emission wavelength as that of Cou-H but showed a much lower fluorescence emission intensity than that of Cou-H (fluorescence quantum yield: 0.15 for Cou-Cl and 0.63 for Cou-H, ESI Fig. S1–2 and Table S1†). Heavy elements, such as I−, have previously been used as anion quenchers in steady-state emission quenching experiments.20 The attachment of heavy atoms, bromo and iodo, directly on coumarin caused the significant loss of their fluorescence emission of Cou-Br and Cou-I, which is ascribed to the intramolecular heavy atom effect. These represent the smallest fluorescence quenchers in designing fluorescent probes. Currently, no literature studies have been reported to apply a heavy atom (like Br and I) as a quencher in the rational design of fluorescence sensors. To evaluate the possible use of bromo and iodo as heavy atom quenchers for turn-on fluorescence sensors, we incubated three halogen acetyl coumarin probes (Cou-Cl, Cou-Br and Cou-I) with biothiol species, such as Cys, GSH and Hcy. As shown in Fig. 1, the addition of Cys quickly triggered the nonfluorescent Cou-Br and Cou-I to high fluorescent species and reached their plateaus in about 40 min, while almost no increase in the fluorescence intensity was observed for Cou-Cl. The fluorescence turn-on rate of Cou-I ( pseudo-first-order kinetic rate constant: k′ = 10.3 × 10−2 min−1, Fig. S3†) was also a little bit larger than that of Cou-Br (k′ = 8.11 × 10−2 min−1, Fig. S3†), which is consistent with the nucleophilic substitution ability of bromo and iodo toward thiol species. To further confirm the heavy atom quenching mechanism, the reaction mixture of Cou-Br and Cys was evaluated by MS analysis and the result indicated the replacement of Br with Cys (Fig. S4†). However, to make sure whether there exists further intramolecular replacement of the aromatic amine with the primary amine of Cys, we carried out a model reaction of cysteamine and Cou-Br (ESI Scheme S3 and Fig. S5–7†). The data of MS, NMR and HMBC indicated that only nucleophilic replacement of bromo with mercaptan thiol took place without further substitution of the amide moiety of the probe by amine moiety of cysteamine. In addition, we also compared the fluorescence spectra of the product of Cou-Br and Cys reactions with Cou-NH2 and Cou-H (Fig. S2†). The turn-on fluorescence spectra of Cou-Br were the same as those of Cou-H, and both fluorescence peaks were blue-shifted compared to the starting material Cou-NH2. These results were consistent with the model experiment, which indicated that only the substitution of heavy atoms took place. Evaluation of sensitivity and selectivity Since the inability of Cou-Cl to sense thiol species, only Cou-Br and Cou-I were evaluated in further experiments. The fluorescence emission spectra of Cou-Br and Cou-I were measured with various concentrations of Cys. Fig. 2 and S8† show that the addition of Cys (0–500 μM) to Cou-Br (5 μM) or Cou-I
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Fig. 1 Fluorescence spectra of Cou-Br (a) and Cou-I (b) (5 μM, respectively) obtained upon addition of 500 μM Cys in PBS. The time interval is 5 min in each scan. (c) Kinetics of turn-on fluorescence of Cou-Cl, CouBr and Cou-I (5 μM for each probe) in PBS upon addition of 500 μM Cys. (d) Fluorescence increase with or without 500 μM Cys in 60 min. The PBS buffer was 25 mM PBS ( pH = 7.4) containing 10% CH3CN. λex = 400 nm, λem = 495 nm.
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Fig. 2 (a) Fluorescence spectra of Cou-Br (5 μM) obtained upon addition of various concentrations (0–500 μM) of Cys in PBS buffer in 45 min. (b) The linear relationship between fluorescence intensity (495 nm) of probe Cou-Br solution and Cys concentrations. The PBS buffer was 25 mM PBS (pH = 7.4) containing 10% CH3CN. λex = 400 nm, λem = 495 nm.
(5 μM) led to a concentration dependent increase in the fluorescence intensity at 495 nm. The standard curve for CouBr and Cou-I in degassed PBS was obtained between the fluorescence intensity at 495 nm and the concentrations (μM) of Cys in 45 min incubation. The regression equations were FEx/Em = 0.356 [Cys] + 6.88 and FEx/Em = 0.320 [Cys] + 8.66 with R2 = 0.999 and R2 = 0.996 for Cou-Br and Cou-I, respectively. The data suggested good linear relationships between the turn-on fluorescence intensity of Cou-Br and Cou-I at 495 nm and Cys concentrations in degassed PBS buffer. Furthermore, to explore the selectivity of Cou-Br and Cou-I, the probes were also treated with other common biologically amino acid species and Vc, such as Trp, His, Ala, Lys, Phe, Glu, Arg, Val, Try, Asp, Pro, Hcy, and GSH. It was found that almost no triggered fluorescence responses of Cou-Br solution were observed for the various common amino acids and Vc, except for GSH and Hcy with the thiol moiety (Fig. 3). However, reaction dynamics of Cou-Br to GSH and Hcy was slower than Cys in the order of Cys > GSH > Hcy, which was confirmed by their second rate constants (7.8 × 10−4 M−1 min−1 for Cys, 2.4 × 10−4 M−1 min−1 for GSH and 1.5 × 10−4 M−1 min−1 for Hcy) after fitting the first rate constants with the pseudo second dynamic equation (Fig. S3, S9 and Table S2†). For Cou-I, we observed the similar selectivity of biothiols over other amino acid species (Fig. S10†). In
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Fig. 3 (a) Fluorescence spectra of Cou-Br (5 μM), Cys (500 μM) and other analytes (500 μM Hcy and GSH, and 2 mM amino acids and Vc, and 1 mg ml−1 BSA) in PBS buffer. (b) Fluorescence responses of Cou-Br to other analytes (500 μM Hcy and GSH and 2 mM amino acids and Vc) without (black column) or with (red column) 500 μM Cys. The PBS buffer was 25 mM PBS ( pH = 7.4) containing 10% CH3CN. λex = 400 nm, λem = 495 nm.
addition, the fluorescence detection of Cys with the probe Cou-Br was also achievable over up to 1 mg mL−1 of the thiol containing proteins, such as bovine serum albumin (BSA) (Fig. S11†). To study the pH effect on fluorescence detection of biothiols, the probes, Cou-Br and Cou-I, were also evaluated for the detection of Cys in buffers with pH values from 4.4 to 10. As shown in Fig. 4 and S14,† these two probes were both effective for detecting Cys in buffers of all pH environment, however, higher fluorescence enhancement was observed under neutral and basic conditions. The lower sensitivity in low pH buffers was due to the protonation of the aromatic amine which affected the push–pull electronic system of the coumarin moiety.21 Cell studies Cell viability indicated that no obvious toxicity was observed for Cou-Br and Cou-I (up to 50 μM for 24 h) by the SRB method (Fig. S15†). To further evaluate the feasibility for
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eties in cells were consumed. Due to a low biothiol level in NEM-treated cells, we only observed very weak fluorescence emission. However, after the NEM treated cells were first washed and incubated with Cys again, high fluorescence emission in cells was fully recovered upon the addition of the probe Cou-Br. These results confirmed that the probe Cou-Br can be used to monitor both endogenous and exogenous biothiols in living cells.
Conclusions A series of coumarin derivatives were synthesized and used as turn-on fluorescent probes for biothiols. The fluorescence of these coumarin probes were efficiently quenched by a single heavy atom (such Br or I) through the heavy atom effect, which represents the smallest fluorescence quencher in the literature. Upon substitution of heavy atoms with the thiol group, the turn-on fluorescence of coumarin was quickly observed. These probes showed high selectivity toward biothiol species with the sensitivity of Cys > GSH > Hcy. Linear relationships between the turn-on fluorescence intensity and the concentrations of biothiols were obeyed. In addition, these coumarin probes with the attachment of heavy atoms were also able to detect both the endogenous and exogenous biothiols in living cells. Fig. 4 Fluorescence responses of Cou-Br (a) and Cou-I (b) (5 μM) with or without 500 μM Cys in different pH values of PBS (4.4, 5.0, 6.0, 7.0, 7.4, 7.9, 8.9, 10.0) containing 10% CH3CN, λex = 400 nm, λem = 495 nm.
biothiol detection in living cells, HeLa cells were chosen and treated with 5 μM Cou-Br for 50 min. The cells were then washed to remove extracellular reagents and then visualized using High Content Screening (HCS). As displayed in Fig. 5a–f, cells incubated with Cou-Br show strong fluorescence emission due to high concentrations of endogenous biothiols in living cells. In contrast, when N-ethylmaleimide (NEM, 1 mM, a thiol inhibitor) was added to the cell medium, most of thiol moi-
Fig. 5 (a) and (b) were images of HeLa cells incubated with 5 μM Cys-Br for 50 min. (c) and (d) were images of HeLa cells incubated with 5 μM Cys-Br for 50 min after the cells were treated with 1 mM NEM for 30 min. (e) and (f ) were images of HeLa cells treated with 1 mM NEM for 30 min first and then incubated with 5 μM Cys-Br for 50 min after the incubation with 100 μM Cys for 30 min.
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Acknowledgements This work was financially supported by the National Basic Research Program of China (‘973’ Program: 2013CB933800, 2012CB720600, and 2013CB834803), and the National Natural Science Foundation of China (no. 21422201 and 51273108).
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