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This is an Accepted Manuscript, which has been through the Royal Society of Chemistry peer review process and has been accepted for publication. Accepted Manuscripts are published online shortly after acceptance, before technical editing, formatting and proof reading. Using this free service, authors can make their results available to the community, in citable form, before we publish the edited article. We will replace this Accepted Manuscript with the edited and formatted Advance Article as soon as it is available. You can find more information about Accepted Manuscripts in the Information for Authors. Please note that technical editing may introduce minor changes to the text and/or graphics, which may alter content. The journal’s standard Terms & Conditions and the Ethical guidelines still apply. In no event shall the Royal Society of Chemistry be held responsible for any errors or omissions in this Accepted Manuscript or any consequences arising from the use of any information it contains.
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A highly selective and sensitive fluorescent thiol probe through dualreactive and dual-quenching groups
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Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX DOI: 10.1039/b000000x A new fluorescent probe installed with dual-reactive and dual-quenching groups was rationally designed for highly selective and sensitive sensing of biothiols. Sensitivity of the probe toward thiols was significantly improved by dualquenching effects. Furthermore selectivity of the probe was also greatly enhanced by installation of dual-reactive groups.
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Small-molecule thiols are essential for maintaining cellular redox environment and play important roles in regulating various cellular functions.1 Abnormal thiol concentration has been implicated with the formation of a number of important diseases. For instance, thiol deficiency is associated with liver damage, AIDS, etc.2 High concentrations of homocysteine (Hcy) are linked with cardiovascular disease and Alzheimer’s disease.3 Because altered levels of thiols in various physiological media have been linked to specific pathological conditions, researchers have shown keen interest to develop methods to detect thiols quantitatively and selectively in living organisms.4 In recent years, fluorescence-based method has become as a popular approach for detection of biothiols due to its high sensitivity, easy implementation and non-invasiveness.5 A number of organic reactions have been utilized to design various fluorescent thiol probes, including cyclization reactions between aldehyde and aminothiols,6 Michael addition reactions,7 cleavage reactions of 2,4-dinitrobenzenesulfonyl (DNBS) with thiols,8 nucleophilic substitution,9 disulfide exchange reaction,10 and others.11 These work greatly advanced the research on the fluorescent detection of biothiols; however, many of them suffer from relatively low sensitivity due to the inefficiency of the quenching group6a,7e,7f,9d,10a,10e or inadequate selectivity due to the cross reactivity of the reactive unit utilized.6b-6d,8d,9g,11d Thus, new thiol probes with improved properties are still needed to be developed. To obtain highly selective and sensitive fluorescent probes for biothiols, we herein introduce a novel design strategy through installation of dual-quenching and dual-reactive groups onto a fluorophore. The designing principle is illustrated in Scheme 1. A fluorophore is attached with two reactive groups, DNBS and a highly activated Michael acceptor. DNBS has been reported as a useful reactive unit for thiol detection in previous study,8 and the DNBS moiety can quench the fluorescence via intramolecular charge transfer (ICT) effect. On the other hand, the Michael acceptor can quench the fluorescence via a photoinduced electron transfer (PET) effect.7b We envisage that the fluorescence will be heavily quenched through combined usage of PET-ICT dualThis journal is © The Royal Society of Chemistry [year]
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quenching effects. The background fluorescence will be minimized and as a result, the fluorescent turn-on fold can be significantly increased upon reaction with thiols. This will finally lead to the enhancement of the sensitivity of the probe. On the other hand, we also envisage that selectivity of the probe will be greatly increased due to different reaction mechanisms (nucleophilic substitution and Michael addition) toward thiols. A single-reactive substance can only react with one of the reactive (quenching) groups, and another reactive (quenching) group can still partially quench the fluorescence (Scheme 1A). In another word, the fluorescence will only gain maximal turn-on effect with the molecules that can undergo two different reaction events to release both quenching groups. Such a strategy should greatly improve the selectivity of the fluorescent probe. Based on our design strategy (Scheme 1), we hypothesize that both the sensitivity and selectivity of the dual-quenching probe (DQprobe) will be improved. To our delight, herein we successfully developed such a fluorescent probe to detect biothiols.
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Scheme 1. (A) Schematic illustration of a design strategy for dualquenching probe (DQ-probe) based on the combination of two monoquenching probes (MQ-probe). ICT, intramolecular charge transfer; PET, photoinduced electron transfer. (B) Chemical structures of fluorescent probes used in this work.
As a proof-of-concept study, we chose 3-amino-7-hydroxylcoumarin as a fluorophore due to its high fluorescence intensity, excellent solubility, good cell permeability, and ease-of[journal], [year], [vol], 00–00 | 1
ChemComm Accepted Manuscript
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Huatang Zhang,‡a Changyu Zhang,‡b Ruochuan Liu,a Long Yi*,b,c and Hongyan Sun*,a
ChemComm
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DOI: 10.1039/C4CC08156K
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Fig. 1 The spectra response of fluorescent probes toward Cys. Absorption spectra of 10 µM 1 (A) or 2 (B) in the absence and presence of Cys (100 µM) in PBS buffer (10 mM, pH 7.4). The emission spectra of 1 µM 1 (C) or 2 (D) in the absence and presence of Cys in PBS. 75 35
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Encouraged by these preliminary results, we moved forward to study the reaction kinetics of the probes (Fig. 2). Probe 1 or 2 (1 µM) was incubated with 10 µM Cys in PBS buffer at 37 ºC. Both probes were shown with fast fluorescent increase at the beginning. Subsequently, the fluorescence intensity went through a gradual increase and finally reached a plateau. Both 1 and 2 showed fairly fast reaction rate. A control experiment with only 1 or 2 was incubated in PBS buffer for the stability test. The results indicated that 2 is prone to slow hydrolysis at pH 7.4 (Fig. 2B), which is similar with that of 3 in the previous report.7b However, the fluorescence increase due to hydrolysis in 1 can be almost negligible (Fig 2A) because of the dual-quenching and dualreactive effects.
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Fig. 2 Time-course experiments of 1 µM DQ-probe 1 (A) or MQ-probe 2 (B) with Cys (10 µM) in PBS buffer (10 mM, pH 7.4).
To gain detailed information about the sensitivity of DQ-probe 1, the fluorescence intensity change was closely monitored by addition of various concentrations of Cys into the probe (Fig. 3, S1-S4). The fluorescence intensity was linearly related to the concentration of Cys from 0 to 1 µM (Fig. 3B). Specifically, the detection limit of 1 was determined as low as 20 nM based on 3σ/slope method.7g These results clearly indicated that DQ-probe 1 has shown significantly improved sensitivity compared to MQprobe 2 due to the installation of dual-quenching groups.
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Fig. 3 Fluorescence response of DQ-probe 1 (A) and MQ-probe 2 (C) with different concentration of Cys (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 µM) in PBS buffer and linear relationship between fluorescence intensity (1 at 470 nm and 2 at 440 nm) and Cys concentration for 1 (B) and 2 (D).
In order to examine whether DQ-probe 1 possess better selectivity than MQ-probe 2, they were incubated with 20 different amino acids in PBS buffer and the fluorescent intensity was measured accordingly (Fig. 4, S5-S8). The fluorescent intensity of 1 with addition of different thiol species (Cys, homocysteine, reduced glutathione (GSH)) could result in more than 400-fold increase. For 2, only 12-fold fluorescent increase was observed under the same conditions. The other 19 amino acids samples exhibited no noticeable increase of fluorescence signal. These experimental results implied that the DQ-probe 1 is highly selective toward thiol group but not toward other biologically relevant nucleophiles such as amino and imidazole group. In addition, we also performed competitive selectivity studies with some representative amino acids (glycine, arginine, lysine and histidine). The results indicated that 1 or 2 can selectively detect thiols even though the concentration of other competitive amino acids is 100 equiv. higher than thiol’s concentration. It is noted that the selectivity of DQ-probe 1 toward thiols is more than 200 fold compared to other amino This journal is © The Royal Society of Chemistry [year]
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synthesis. The chemical structures of DQ-probe 1 and its control mono-quenching probes (MQ-probe 2 and 3) were shown in Scheme 1B. All probes were well characterized by 1H NMR, 13C NMR and HR-MS (see ESI). We firstly examined the absorption spectra of probes in PBS buffer (10 mM, pH 7.4). As shown in Fig 1A and 1B, 1 and 2 displayed a maximum absorbance at 330 nm and 295 nm, respectively. After reacting with cysteine, the maximum absorbance was shifted to 355 and 400 nm for 1 and 2, respectively. Thiol-mediated cleavage of the electronwithdrawing DNBS group releases oxygen donor at pH 7.4, increasing the push-pull character of the dye and resulting in large bathochromic shifts in the absorption. The fluorescent experiments were carried out in PBS buffer (10 mM, pH 7.4). Probe 1 showed almost no fluorescence (nearly the same as that of buffer only) in PBS buffer without cysteine (Fig. 1C), while 2 and 3 showed weak background fluorescence under similar condition (Fig 1D).7b These results are consistent with our design that the coumarin fluorescence in 1 is heavily quenched through combined usage of PET-ICT dual-quenching effects. After reacting with Cys, a significant fluorescent increase was observed for both 1 and 2, with the maximum emission at 473 nm and 440 nm, respectively. The increase of maximum fluorescent intensity is more than 400-fold for 1 and about 13fold for 2. From these results, we can conclude that the fluorescence turn-on fold upon reacting with thiols for DQ-probe 1 is indeed greatly increased, which agrees well with our initial hypothesis.
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Fig. 4 Selectivity and competitive selectivity studies of fluorescent probes toward different species in PBS buffer. Relative fluorescence intensity at 470 nm for 1 µM 1 (A) and at 440 nm for 1 µM 2 (B) assayed with 19 natural amino acids (1 mM), Cys (10 µM), GSH (10 µM) and Hcy (10 µM); Relative fluorescence intensity at 470 nm for 1 µM 1 (C) and at 440 nm for 1 µM 2 (D) with Cys (10 µM) in the presence of several represented amino acids (1 mM).
Finally, we examined whether DQ-probe 1 can be used to detect intracellular thiols in living cells. As shown in Fig. 5, after incubating HeLa cells with 1 for 20 min, strong blue fluorescence could be readily observed inside the cells under confocal fluorescence microscope (Fig. 5A-5C). The control experiments were performed by preincubating cells with N-ethylmaleimide (NEM) and subsequently adding of 1. N-ethylmaleimide is commonly used as a thiol-depleting agent for cells. No significant fluorescence signal was observed under similar experimental conditions (Fig. 5D-5F). These results suggest that DQ-probe 1 is cell-permeable and can react with intracellular thiols efficiently and selectively. Considering the relationship between the cytosolic biothiols level with many diseases,2 this probe may offer a simple and viable way to monitor the cytosolic thiol level. In summary, we report a novel strategy for rational design of highly selective and sensitive fluorescent thiol probe, namely, a fluorescent probe installed with dual-reactive and dual-quenching groups. The principle of our strategy has two aspects: 1) based on the dual-quenching effects, the fluorescence of the probe can be maximally inhibited, and the turn-on response upon
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thiol activation is much larger which leads to high sensitivity of the probe toward thiols; 2) based on the dual-reactive groups, the selectivity of the probe toward thiols is greatly improved, because the thiol detection can only be obviously interfered by molecules that can react with both different reactive groups. As a proof-ofconcept study, we prepared a new DQ-probe 1, which was shown to detect thiols with high sensitivity with 20 nM detection limit. The selectivity of DQ-probe 1 is also superior to many existing thiol probes and MQ-probes (2 and 3).6-11 Furthermore, our novel design strategy could be employed as a general method for preparation of other kinds of fluorescent probes. Progress for highly sensitive and selective fluorescent probes for endogenous H2S14 based on dual-quenching and dual-reactive effects will be reported recently.
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Sun and Yi thank Prof. Zhen Xi at Nankai University for general support. We would like to thank the financial support of City University of Hong Kong Grant (7004025, 9667091), NSFC (21402007), the Fundamental Research Funds for the Central Universities of BUCT (YS1401).
Notes and references a
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Department of Biology and Chemistry, City University of Hong Kong, 83 Tat Chee Avenue, Kowloon, Hong Kong, P. R. China. Fax: (852) 34420522; Tel: (852) 3442 9537; E-mail: [email protected] b State Key Laboratory of Organic-Inorganic Composites and Beijing University of Chemical Technology, Beijing University of Chemical Technology, 15 Beisanhuan East Road, Chaoyang District, Beijing 100029, China. E-mail: [email protected] c Collaborative Innovation Center of Chemical Science and Engineering (Tianjin) † Electronic Supplementary Information (ESI) available: [details of any supplementary information available should be included here]. See DOI: 10.1039/b000000x/ ‡ The authors pay equal contributions to this work. 1
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Fig. 5 Confocal bioimages of living HeLa cells. Fluorescence (A), brightfield (B) and overlay (C) images of HeLa cells incubated with 1 (1 µM) for 20 min; fluorescence (D), brightfield (E) and overlay (F) images of HeLa cells which were pre-incubated with 2 mM N-ethylmaleimide for 30 min and then treated with 1 µM of 1 for 20 min.
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(a) G. I. Giles, Curr. Pharm. Des., 2006, 34, 4427; (b) N. Brandes, S. Schmitt and U. Jakob, Antioxid. Redox. Signal., 2009, 11, 997; (c) C. K. Sen, Curr. Top. Cell. Regul., 2000, 36, 1; (d) T. P. Dalton, H. G. Shertzer and A. Puga, Annu. Rev. Pharmacol. Toxicol., 1999, 39, 67;
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acids, while that of MQ-probes 2 and 3 is lower than 10 and 60 fold,7b respectively. Taken together, the selectivity of DQ-probe is indeed greatly increased due to the usage of dual-reactive and dual-quenching groups. This can be explained by that the fluorescence of a competitive substance will only gain maximal turn-on effect when it can undergo two different reaction events, i.e. both nucleophilic substitution and Michael addition. H2S was recently identified as an important signaling molecule,12 and its reactivity is similar to that of thiols.13 We also tested DQ-probe 1 towards H2S, and results indicated that our probe showed high fluorescence signal after addition of H2S (Fig. S9).
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A.K. Mustafa, W. Mu, S. Zhang, S.H. Snyder and R. Wang, Science, 2008, 322, 587; (b) A. Papapetropoulos, A. Pyriochou, Z. Altaany, G. D. Yang, A. Marazioti, Z. M. Zhou, M. G. Jeschke, L. K. Branski, D. N. Herndon, R. Wang and C. Szabo, Proc. Natl. Acad. Sci. USA, 2009, 106, 21972; (c) K. H. Kulkarni, E. M. Monjok, R. Zeyssig, G. Kouamou, O. N. Bongmba, C. A. Opere, Y. F. Njie and S. E. Ohia, Neurochem. Res., 2009, 34, 400-406. 13 For recent reviews: (a) H. Peng, W. Chen, Y. Cheng, L. Hakuna, R. Strongin and B. Wang, Sensors, 2012, 12, 15907; (b) V. S. Lin and C. J. Chang, Curr. Opin. Chem. Biol. 2012, 16, 595; (c) Z. Q. Guo, S. Park, J. Yoon and I. Shin, Chem. Soc. Rev., 2014, 43, 16. 14 (a) L. Wei, L. Yi, F. Song, C. Wei, B.-F. Wang and Z. Xi, Sci. Rep., 2014, 4, 4521; (b) C. Wei, R. Wang, L. Wei, L. Cheng, Z. Li, Z. Xi and L. Yi, Chem. Asian J., 2014, DOI: 10.1002/asia.201402808.
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(e) S. M. Kanzok, R. H. Schirmer, I. Türbachova, R. Iozef and K. Becker, J. Biol. Chem., 2000, 275, 40180. (a) D. P. Jones, J. L. Carlson, V. C. Mody, J. Cai, M. J. Lynn and P. Sternberg, Free Radic. Biol. Med., 2000, 28, 625; (b) M. Kemp, Y. M. Go and D. P. Jones, Free Radic. Biol. Med., 2008, 44, 921; (c) T. Finkel and N. J. Holbrook, Nature, 2000, 408, 239. (a) A. S. Wierzbicki, Diab. Vasc. Dis. Res., 2007, 4, 143; (b) M. S. Morris, Lancet Neurol., 2003, 2, 425. (a) X. Li, X, Gao, W. Shi and H. Ma, Chem. Rev., 2014, 114, 590; (b) H. Peng, W. Chen, Y. Cheng, L. Hakuna, R. Strongin and B. Wang, Sensors, 2012, 12, 15907. For recent reviews: (a) X. Chen, Y. Zhou, X. Peng and J. Yoon, Chem. Soc. Rev., 2010, 39, 2120; (b) Y. Zhou and J. Yoon, Chem. Soc. Rev., 2012, 41, 52; (c) H. S. Jung, X. Chen, J. S. Kim and J. Yoon, Chem. Soc. Rev., 2013, 42, 6019. (a) H. Lin, J. Fan, J. Wang, M. Tian, J. Du, S. Sun, P. Sun and X. Peng, Chem. Commun., 2009, 45, 5904; (b) Z. Yao, X. Feng, C. Li and G. Shi, Chem. Commun., 2009, 45, 5886; (c) J. Mei, J. Tong, J. Wang, A. Qin, J. Z. Sun and B. Z. Tang, J. Mater. Chem., 2012, 22, 17063; (d) Z. Yang, N. Zhao, Y. Sun, F. Miao, Y. Liu, X. Liu, Y. Zhang, W. Ai, G. Song, X. Shen, X. Yu, J. Sun and W.-Y. Wong, Chem. Commun., 2012, 48, 3442. (a) V. Hong, A. A. Kislukhin and M. G. Finn, J. Am. Chem. Soc., 2009, 131, 9986; (b) L. Yi, H. Li, L. Sun, L. Liu, C. Zhang and Z. Xi, Angew. Chem. Int. Ed., 2009, 48, 4034; (c) W. Lin, L. Yuan, Z. Cao, Y. Feng and L. Long, Chem.-Eur. J., 2009, 15, 5096; (d) X. Chen, S. Ko, M. J. Kim, I. Shin and J. Yoon, Chem. Commun., 2010, 46, 2751; (e) S. Lim, J. O. Escobedo, M. Lowry, X. Xu and R. Strongin, Chem. Commun., 2010, 46, 5707; (f) Y.-Q. Sun, M. Chen, J. Liu, X. Lv, J.F. Li and W. Guo, Chem. Commun., 2011, 47, 11029; (g) H. Zhang, P. Wang, Y. Yang and H. Sun, Chem. Commun., 2012, 48, 10672. (a) H. Maeda, H. Matsuno, M. Ushida, K. Katayama, K. Saeki and N. Itoh, Angew. Chem. Int. Ed., 2005, 44, 2922; (b) J. Bouffard, Y. Kim, T. M. Swager, R. Weissleder and S. A. Hilderbrand, Org. Lett., 2008, 10, 37; (c) S. Wang, W. Deng, D. Sun, M. Yan, H. Zheng, J. Xu, Org. Biomol. Chem., 2009, 7, 4017; (d) X. Li, S. Qian, Q. He, B. Yang, J. Li, Y. Hu, Org. Biomol. Chem., 2010, 8, 3627; (e) S. Ji, H. Guo, X. Yuan, X. Li, H. Ding, P. Gao, C. Zhao, W. Wu, W. Wu and J. Zhao, Org. Lett., 2010, 12, 2876. (a) 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; (b) 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; (c) S.-Y. Lim, K.-H. Hong, D. I. Kim, H. Kwon and H.-J. Kim, J. Am. Chem. Soc., 2014, 136, 7018; (d) Y.-S. Guan, L.-Y. Niu, Y.-Z. Chen, L.-Z. Wu, C.-H. Tung and Q.-Z. Yang, RSC Adv., 2014, 4, 8360; (e) H. Lv, X.-F. Yang, Y. Zhong, Y. Guo, Z. Li and H. Li, Anal. Chem., 2014, 86, 1800; (f) J. Liu, Y.-Q. Sun, H. Zhang, Y. Huo, Y. Shi and W. Guo, Chem. Sci., 2014, 5, 3183; (g) B. Tang, Y. Xing, P. Li, N. Zhang, F. Yu and G. Yang, J. Am. Chem. Soc., 2007, 129, 11666. (a) J. H. Lee, C. S. Lim, Y. S. Tian, J. H. Han and B. R. Cho, J. Am. Chem. Soc., 2010, 132, 1216; (b) C. S. Lim, G. Masanta, H. J. Kim, J. H. Han, H. M. Kim and B. R. Cho, J. Am. Chem. Soc., 2011, 133, 11132; (c) M. H. Lee, J. H. Han, P.-S. Kwon, S. Bhuniya, J. Y. Kim, J. L. Sessler, C. Kang and J. S. Kim, J. Am. Chem. Soc., 2012, 134, 1316; (d) X. Cao, W. Lin and Q. Yu, J. Org. Chem., 2011, 76, 7423; (e) D. Jung, S. Maiti, J. H. Lee, J. H. Lee and J. S. Kim, Chem. Commun., 2014, 50, 3044. (a) R. Wang, L. Chen, P. Liu, Q. Zhang and Y. Wang, Chem. – Eur. J., 2012, 18, 11343; (b) H. S. Jung, J. H. Han, Y. Habata, C. Kang and J. S. Kim, Chem. Commun., 2011, 47, 5142; (c) Y. Bao, Q. Li, B. Liu, F. Du, J. Tian, H. Wang, Y. Wang and R. Bai, Chem. Commun., 2012, 48, 118; (d) P. Wang, J. Liu, X. Lv, Y. Liu, Y. Zhao and W. Guo, Org. Lett., 2012, 14, 520; (e) S. R. Malwal, A. Labade, A. S. Andhalkar, K. Sengupta and H. Chakrapani, Chem. Commun., 2014, 50, 11533. (a) G. Yang, L. Wu, B. Jiang, W. Yang, J. Qi, K. Cao, Q. Meng,
This journal is © The Royal Society of Chemistry [year]
ChemComm Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: H. Sun, L. Yi, H. Zhang, C. Zhang and R. Liu, Chem. Commun., 2014, DOI: 10.1039/C4CC08156K.
This is an Accepted Manuscript, which has been through the Royal Society of Chemistry peer review process and has been accepted for publication. Accepted Manuscripts are published online shortly after acceptance, before technical editing, formatting and proof reading. Using this free service, authors can make their results available to the community, in citable form, before we publish the edited article. We will replace this Accepted Manuscript with the edited and formatted Advance Article as soon as it is available. You can find more information about Accepted Manuscripts in the Information for Authors. Please note that technical editing may introduce minor changes to the text and/or graphics, which may alter content. The journal’s standard Terms & Conditions and the Ethical guidelines still apply. In no event shall the Royal Society of Chemistry be held responsible for any errors or omissions in this Accepted Manuscript or any consequences arising from the use of any information it contains.
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A highly selective and sensitive fluorescent thiol probe through dualreactive and dual-quenching groups
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Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX DOI: 10.1039/b000000x A new fluorescent probe installed with dual-reactive and dual-quenching groups was rationally designed for highly selective and sensitive sensing of biothiols. Sensitivity of the probe toward thiols was significantly improved by dualquenching effects. Furthermore selectivity of the probe was also greatly enhanced by installation of dual-reactive groups.
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Small-molecule thiols are essential for maintaining cellular redox environment and play important roles in regulating various cellular functions.1 Abnormal thiol concentration has been implicated with the formation of a number of important diseases. For instance, thiol deficiency is associated with liver damage, AIDS, etc.2 High concentrations of homocysteine (Hcy) are linked with cardiovascular disease and Alzheimer’s disease.3 Because altered levels of thiols in various physiological media have been linked to specific pathological conditions, researchers have shown keen interest to develop methods to detect thiols quantitatively and selectively in living organisms.4 In recent years, fluorescence-based method has become as a popular approach for detection of biothiols due to its high sensitivity, easy implementation and non-invasiveness.5 A number of organic reactions have been utilized to design various fluorescent thiol probes, including cyclization reactions between aldehyde and aminothiols,6 Michael addition reactions,7 cleavage reactions of 2,4-dinitrobenzenesulfonyl (DNBS) with thiols,8 nucleophilic substitution,9 disulfide exchange reaction,10 and others.11 These work greatly advanced the research on the fluorescent detection of biothiols; however, many of them suffer from relatively low sensitivity due to the inefficiency of the quenching group6a,7e,7f,9d,10a,10e or inadequate selectivity due to the cross reactivity of the reactive unit utilized.6b-6d,8d,9g,11d Thus, new thiol probes with improved properties are still needed to be developed. To obtain highly selective and sensitive fluorescent probes for biothiols, we herein introduce a novel design strategy through installation of dual-quenching and dual-reactive groups onto a fluorophore. The designing principle is illustrated in Scheme 1. A fluorophore is attached with two reactive groups, DNBS and a highly activated Michael acceptor. DNBS has been reported as a useful reactive unit for thiol detection in previous study,8 and the DNBS moiety can quench the fluorescence via intramolecular charge transfer (ICT) effect. On the other hand, the Michael acceptor can quench the fluorescence via a photoinduced electron transfer (PET) effect.7b We envisage that the fluorescence will be heavily quenched through combined usage of PET-ICT dualThis journal is © The Royal Society of Chemistry [year]
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quenching effects. The background fluorescence will be minimized and as a result, the fluorescent turn-on fold can be significantly increased upon reaction with thiols. This will finally lead to the enhancement of the sensitivity of the probe. On the other hand, we also envisage that selectivity of the probe will be greatly increased due to different reaction mechanisms (nucleophilic substitution and Michael addition) toward thiols. A single-reactive substance can only react with one of the reactive (quenching) groups, and another reactive (quenching) group can still partially quench the fluorescence (Scheme 1A). In another word, the fluorescence will only gain maximal turn-on effect with the molecules that can undergo two different reaction events to release both quenching groups. Such a strategy should greatly improve the selectivity of the fluorescent probe. Based on our design strategy (Scheme 1), we hypothesize that both the sensitivity and selectivity of the dual-quenching probe (DQprobe) will be improved. To our delight, herein we successfully developed such a fluorescent probe to detect biothiols.
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Scheme 1. (A) Schematic illustration of a design strategy for dualquenching probe (DQ-probe) based on the combination of two monoquenching probes (MQ-probe). ICT, intramolecular charge transfer; PET, photoinduced electron transfer. (B) Chemical structures of fluorescent probes used in this work.
As a proof-of-concept study, we chose 3-amino-7-hydroxylcoumarin as a fluorophore due to its high fluorescence intensity, excellent solubility, good cell permeability, and ease-of[journal], [year], [vol], 00–00 | 1
ChemComm Accepted Manuscript
Published on 21 November 2014. Downloaded by York University on 23/11/2014 11:31:42.
Huatang Zhang,‡a Changyu Zhang,‡b Ruochuan Liu,a Long Yi*,b,c and Hongyan Sun*,a
ChemComm
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DOI: 10.1039/C4CC08156K
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Fig. 1 The spectra response of fluorescent probes toward Cys. Absorption spectra of 10 µM 1 (A) or 2 (B) in the absence and presence of Cys (100 µM) in PBS buffer (10 mM, pH 7.4). The emission spectra of 1 µM 1 (C) or 2 (D) in the absence and presence of Cys in PBS. 75 35
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Encouraged by these preliminary results, we moved forward to study the reaction kinetics of the probes (Fig. 2). Probe 1 or 2 (1 µM) was incubated with 10 µM Cys in PBS buffer at 37 ºC. Both probes were shown with fast fluorescent increase at the beginning. Subsequently, the fluorescence intensity went through a gradual increase and finally reached a plateau. Both 1 and 2 showed fairly fast reaction rate. A control experiment with only 1 or 2 was incubated in PBS buffer for the stability test. The results indicated that 2 is prone to slow hydrolysis at pH 7.4 (Fig. 2B), which is similar with that of 3 in the previous report.7b However, the fluorescence increase due to hydrolysis in 1 can be almost negligible (Fig 2A) because of the dual-quenching and dualreactive effects.
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Fig. 2 Time-course experiments of 1 µM DQ-probe 1 (A) or MQ-probe 2 (B) with Cys (10 µM) in PBS buffer (10 mM, pH 7.4).
To gain detailed information about the sensitivity of DQ-probe 1, the fluorescence intensity change was closely monitored by addition of various concentrations of Cys into the probe (Fig. 3, S1-S4). The fluorescence intensity was linearly related to the concentration of Cys from 0 to 1 µM (Fig. 3B). Specifically, the detection limit of 1 was determined as low as 20 nM based on 3σ/slope method.7g These results clearly indicated that DQ-probe 1 has shown significantly improved sensitivity compared to MQprobe 2 due to the installation of dual-quenching groups.
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Fig. 3 Fluorescence response of DQ-probe 1 (A) and MQ-probe 2 (C) with different concentration of Cys (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 µM) in PBS buffer and linear relationship between fluorescence intensity (1 at 470 nm and 2 at 440 nm) and Cys concentration for 1 (B) and 2 (D).
In order to examine whether DQ-probe 1 possess better selectivity than MQ-probe 2, they were incubated with 20 different amino acids in PBS buffer and the fluorescent intensity was measured accordingly (Fig. 4, S5-S8). The fluorescent intensity of 1 with addition of different thiol species (Cys, homocysteine, reduced glutathione (GSH)) could result in more than 400-fold increase. For 2, only 12-fold fluorescent increase was observed under the same conditions. The other 19 amino acids samples exhibited no noticeable increase of fluorescence signal. These experimental results implied that the DQ-probe 1 is highly selective toward thiol group but not toward other biologically relevant nucleophiles such as amino and imidazole group. In addition, we also performed competitive selectivity studies with some representative amino acids (glycine, arginine, lysine and histidine). The results indicated that 1 or 2 can selectively detect thiols even though the concentration of other competitive amino acids is 100 equiv. higher than thiol’s concentration. It is noted that the selectivity of DQ-probe 1 toward thiols is more than 200 fold compared to other amino This journal is © The Royal Society of Chemistry [year]
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synthesis. The chemical structures of DQ-probe 1 and its control mono-quenching probes (MQ-probe 2 and 3) were shown in Scheme 1B. All probes were well characterized by 1H NMR, 13C NMR and HR-MS (see ESI). We firstly examined the absorption spectra of probes in PBS buffer (10 mM, pH 7.4). As shown in Fig 1A and 1B, 1 and 2 displayed a maximum absorbance at 330 nm and 295 nm, respectively. After reacting with cysteine, the maximum absorbance was shifted to 355 and 400 nm for 1 and 2, respectively. Thiol-mediated cleavage of the electronwithdrawing DNBS group releases oxygen donor at pH 7.4, increasing the push-pull character of the dye and resulting in large bathochromic shifts in the absorption. The fluorescent experiments were carried out in PBS buffer (10 mM, pH 7.4). Probe 1 showed almost no fluorescence (nearly the same as that of buffer only) in PBS buffer without cysteine (Fig. 1C), while 2 and 3 showed weak background fluorescence under similar condition (Fig 1D).7b These results are consistent with our design that the coumarin fluorescence in 1 is heavily quenched through combined usage of PET-ICT dual-quenching effects. After reacting with Cys, a significant fluorescent increase was observed for both 1 and 2, with the maximum emission at 473 nm and 440 nm, respectively. The increase of maximum fluorescent intensity is more than 400-fold for 1 and about 13fold for 2. From these results, we can conclude that the fluorescence turn-on fold upon reacting with thiols for DQ-probe 1 is indeed greatly increased, which agrees well with our initial hypothesis.
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ChemComm View Article Online
DOI: 10.1039/C4CC08156K
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Fig. 4 Selectivity and competitive selectivity studies of fluorescent probes toward different species in PBS buffer. Relative fluorescence intensity at 470 nm for 1 µM 1 (A) and at 440 nm for 1 µM 2 (B) assayed with 19 natural amino acids (1 mM), Cys (10 µM), GSH (10 µM) and Hcy (10 µM); Relative fluorescence intensity at 470 nm for 1 µM 1 (C) and at 440 nm for 1 µM 2 (D) with Cys (10 µM) in the presence of several represented amino acids (1 mM).
Finally, we examined whether DQ-probe 1 can be used to detect intracellular thiols in living cells. As shown in Fig. 5, after incubating HeLa cells with 1 for 20 min, strong blue fluorescence could be readily observed inside the cells under confocal fluorescence microscope (Fig. 5A-5C). The control experiments were performed by preincubating cells with N-ethylmaleimide (NEM) and subsequently adding of 1. N-ethylmaleimide is commonly used as a thiol-depleting agent for cells. No significant fluorescence signal was observed under similar experimental conditions (Fig. 5D-5F). These results suggest that DQ-probe 1 is cell-permeable and can react with intracellular thiols efficiently and selectively. Considering the relationship between the cytosolic biothiols level with many diseases,2 this probe may offer a simple and viable way to monitor the cytosolic thiol level. In summary, we report a novel strategy for rational design of highly selective and sensitive fluorescent thiol probe, namely, a fluorescent probe installed with dual-reactive and dual-quenching groups. The principle of our strategy has two aspects: 1) based on the dual-quenching effects, the fluorescence of the probe can be maximally inhibited, and the turn-on response upon
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thiol activation is much larger which leads to high sensitivity of the probe toward thiols; 2) based on the dual-reactive groups, the selectivity of the probe toward thiols is greatly improved, because the thiol detection can only be obviously interfered by molecules that can react with both different reactive groups. As a proof-ofconcept study, we prepared a new DQ-probe 1, which was shown to detect thiols with high sensitivity with 20 nM detection limit. The selectivity of DQ-probe 1 is also superior to many existing thiol probes and MQ-probes (2 and 3).6-11 Furthermore, our novel design strategy could be employed as a general method for preparation of other kinds of fluorescent probes. Progress for highly sensitive and selective fluorescent probes for endogenous H2S14 based on dual-quenching and dual-reactive effects will be reported recently.
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Sun and Yi thank Prof. Zhen Xi at Nankai University for general support. We would like to thank the financial support of City University of Hong Kong Grant (7004025, 9667091), NSFC (21402007), the Fundamental Research Funds for the Central Universities of BUCT (YS1401).
Notes and references a
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Department of Biology and Chemistry, City University of Hong Kong, 83 Tat Chee Avenue, Kowloon, Hong Kong, P. R. China. Fax: (852) 34420522; Tel: (852) 3442 9537; E-mail: [email protected] b State Key Laboratory of Organic-Inorganic Composites and Beijing University of Chemical Technology, Beijing University of Chemical Technology, 15 Beisanhuan East Road, Chaoyang District, Beijing 100029, China. E-mail: [email protected] c Collaborative Innovation Center of Chemical Science and Engineering (Tianjin) † Electronic Supplementary Information (ESI) available: [details of any supplementary information available should be included here]. See DOI: 10.1039/b000000x/ ‡ The authors pay equal contributions to this work. 1
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Fig. 5 Confocal bioimages of living HeLa cells. Fluorescence (A), brightfield (B) and overlay (C) images of HeLa cells incubated with 1 (1 µM) for 20 min; fluorescence (D), brightfield (E) and overlay (F) images of HeLa cells which were pre-incubated with 2 mM N-ethylmaleimide for 30 min and then treated with 1 µM of 1 for 20 min.
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(a) G. I. Giles, Curr. Pharm. Des., 2006, 34, 4427; (b) N. Brandes, S. Schmitt and U. Jakob, Antioxid. Redox. Signal., 2009, 11, 997; (c) C. K. Sen, Curr. Top. Cell. Regul., 2000, 36, 1; (d) T. P. Dalton, H. G. Shertzer and A. Puga, Annu. Rev. Pharmacol. Toxicol., 1999, 39, 67;
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acids, while that of MQ-probes 2 and 3 is lower than 10 and 60 fold,7b respectively. Taken together, the selectivity of DQ-probe is indeed greatly increased due to the usage of dual-reactive and dual-quenching groups. This can be explained by that the fluorescence of a competitive substance will only gain maximal turn-on effect when it can undergo two different reaction events, i.e. both nucleophilic substitution and Michael addition. H2S was recently identified as an important signaling molecule,12 and its reactivity is similar to that of thiols.13 We also tested DQ-probe 1 towards H2S, and results indicated that our probe showed high fluorescence signal after addition of H2S (Fig. S9).
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DOI: 10.1039/C4CC08156K
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