New NBD-based fluorescent probes for biological thiols

Tetrahedron Letters 56 (2015) 3909–3912

Contents lists available at ScienceDirect

Tetrahedron Letters journal homepage: www.elsevier.com/locate/tetlet

New NBD-based fluorescent probes for biological thiols Zhentao Zhu a, Wei Liu b,⇑, Longhuai Cheng c, Zhifei Li a, Zhen Xi c, Long Yi a,c,⇑ a

State Key Laboratory of Organic-Inorganic Composite, Beijing University of Chemical Technology, Beijing 100029, China Medical College, NanTong University, NanTong 226001, China c Collaborative Innovation Center of Chemical Science and Engineering, Nankai University, Tianjin 300071, China b

a r t i c l e

i n f o

Article history: Received 22 March 2015 Revised 26 April 2015 Accepted 28 April 2015 Available online 9 May 2015 Keywords: Biological thiols Fluorescence probe Cellular bioimaging

a b s t r a c t Biothiols including cysteine (Cys), reduced glutathione (GSH), and hydrogen sulfide (H2S) play critical roles in living organisms. New reaction-based fluorescent turn-on probes based on fast thiolysis of NBD (7-nitro-1,2,3-benzoxadiazole) ether were explored for sensing of biothiols in aqueous buffer. The probes showed fast response toward biothiols with more than 100-fold off–on signal enhancement. Moreover, the probes were highly selective toward thiols over other amino acids and could be used for bioimaging in mammalian cells. Ó 2015 Elsevier Ltd. All rights reserved.

Introduction Small-molecule biothiols are essential for maintaining cellular redox environment and play important roles in regulating various cellular functions.1 Malfunction of thiol concentration has been implicated with the formation of a number of important diseases. 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 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 thiolysis of 2,4-dinitrobenzenesulfonyl (DNBS) esters,8 nucleophilic substitution,9 disulfide exchange,10 and others.11,12 These work greatly advanced the research on the fluorescent detection of biothiols. However, some of the probes showed slow reaction rate and the synthesis of probes sometimes was relatively complicated and laborious. Furthermore, in order to develop highly sensitive and selective fluorescent probes, we reported the dual-reactive and dual-quenching strategy as a general method for the synthesis

⇑ Corresponding authors. Tel./fax: +86 22 23504782. E-mail addresses: [email protected] (W. Liu), [email protected] (L. Yi). http://dx.doi.org/10.1016/j.tetlet.2015.04.117 0040-4039/Ó 2015 Elsevier Ltd. All rights reserved.

of reactive-based thiol probes.11f Therefore, new thiol probes with advance properties are still needed to be developed. Recently, we discovered a FRET-based H2S probe based on thiolysis of NBD (7-nitro-1,2,3-benzoxadiazole) amine,12a which showed excellent selectivity for H2S over cysteine (Cys). We proposed that fluorescent probes based on thiolysis of NBD ether could be used to detect H2S with improving reactive rate,12b but these NBD ether probes showed lowering selectivity between H2S and Cys. We therefore hypothesized that the fast thiolysis of NBD ether could be used for design of thiol probes. Herein, we employed the excited-state intramolecular proton transfer (ESIPT) fluorophores13 and NBD ether as the reactive moiety for construction of fast-response thiol probes. As shown in Schemes 1 and 2-(20 -hydroxyphenyl)benzothiazole (HBT) and 2-(20 -hydroxy-30 -methoxyphenyl)benzothiazole (HMBT) were chosen as the ESIPT fluorophores. ESIPT is a photochemical process occurring in the excited singlet state of a molecular with intramolecular hydrogen bond. Probes 1 and 2 can react with RSH efficiently by thiolysis of NBD ether to give NBD-thiol additive product12 and the HBT or HMBT fluorophore. As a result, strong fluorescence of the fluorophores through the ESIPT effect can be observed during the reaction (Scheme 1). Probes 1 and 2 could be conveniently prepared by two-step syntheses from commercial chemicals (see Experimental part). Reaction of NBD-Cl and HBT or HMBT under the basic conditions produced the target probes (Scheme 2).The facile and economic synthesis is important for the wide use of such type of probes. The structural characterizations of probes 1 and 2 were confirmed by 1H NMR, 13C NMR, and high resolution mass spectra (see SI).

3910

Z. Zhu et al. / Tetrahedron Letters 56 (2015) 3909–3912

Scheme 1. Structures of probes 1 and 2 and their reaction with thiol (RSH).

Scheme 2. Synthesis of probes 1 and 2.

We have found that the NBD-based ether could be selectively thiolylated by H2S in aqueous solution.12b Increased reaction rate of thiolysis for the NBD-based ether could be employed for design of thiol probes, because the pKa of H2S (6.9) is near the typical free thiols (about 8.5).14 Herein we successfully obtained such kind of thiol probes 1 and 2. We first examined the absorption spectra of the probes in PBS buffer (50 mM, PH 7.4, containing 30% DMSO as co-solvent). As shown in Figure 1a and b, 1 and 2 displayed a maximum absorbance at 380 nm, and did not exhibit noticeable absorbance in the range of 450-600 nm. After reaction with Cys, the maximum absorbance of the probe solution was shifted to 477 nm and 480 nm for 1 and 2, respectively, indicating the efficient reaction of probes with Cys. We also observed the concentration-dependent absorbance spectrum changes through treatment of the probe solution with different concentrations of Cys in PBS buffer (data not shown). It is noted that both probes could react with H2S to give significant absorbance peak at 534 nm (Figs. S1 and S2), which is attributed to the production of nitrobenzofurazan thiol (NBD-SH).15 Such unique absorbance can be used for selective detection of H2S.12b The fluorescence tests were carried out in PBS buffer containing 10% DMSO as co-solvent. Both 1 and 2 did not display noticeable fluorescence of enol-like emission of HBT or HMBT (Fig. 1c and d). The fluorescence quenching may occur attributed to energy transfer from the ESIPT fluorophores to NBD, while the NBD fluorophores lay out low fluorescence in aqueous solution.12b As a result, both probes 1 and 2 showed much weak fluorescence in aqueous buffer. After reacting with Cys, a significant fluorescence increase was observed for both 1 and 2 with the maximum emission at 460 nm and 485 nm, respectively. The increase of maximum fluorescence intensity is about 115-fold for 1 and 33-fold for 2. As expected, the excitation and emission spectra of probes 1 and 2 after treatment with Cys are similar to those of HBT and HMBT, respectively. These preliminary results encouraged us to further check the reactivity and selectivity of the probes. The time-dependent emission of 1 or 2 upon treatment with Cys in PBS buffer (pH 7.4) is shown in Figures 2, S3 and S4. The results indicated that the fluorescence intensity could reach the steady state within less than 10 min at 25 °C. The reaction kinetics of a probe and Cys is an important parameter to examine its biological applicability for real-time detection.7 To obtain the kinetic parameters, the fluorescence signal at 460 nm or 485 nm for 1 or 2, respectively, was plotted as a function of time for data

Figure 1. Spectral response of fluorescent probes toward Cys. Absorbance spectra of 10 lM 1 (a) or 5 lM 2 (b) in the absence and presence of Cys (100 lM) in PBS buffer (50 mM, pH 7.4, containing 30% DMSO as co-solvent). The emission spectra (excitation at 305 nm) of 1 lM 1 (c) or 1 lM 2 (d) in the absence and presence of Cys (100 lM) in PBS buffer (50 mM, pH 7.4, containing 10% DMSO as co-solvent). Slit width: 2.5/5.0 nm for 1, 5.0/5.0 nm for 2. All reactions were performed at 25 °C for 10 min.

Figure 2. Time-dependent emission intensity of 1 lM 1 (j) or 2 (N) upon reaction with Cys (100 lM) at 25 °C. The solid lines represent the best fitting with singleexponential function. Excitation 305 nm, emission 460 nm for 1 (slit width: 2.5/ 5.0 nm); emission 485 nm for 2 (slit width: 5.0/5.0 nm).

analysis (Fig. 2). The pseudo-first-order rate, kobs, was found to be 2.24 and 2.20 min1 for 1 and 2, respectively, by fitting the fluorescence intensity data with single exponential function. The reaction rate (k2) for 1 was calculated as 74.4 M1 s1, which is among the reported fast-response thiol probes.5 The reaction between probes and H2S (Figs. S1–S4) was slightly more efficient than that of Cys, which is due to stronger nucleophilic ability of H2S.14 Because the turn-on response of 1 upon treatment with thiols is larger than that of 2, we only chose probe 1 in the following studies. We further checked the fluorescent signal change of probe 1 with various concentrations (0-9 lM) of Cys (Fig. 3). As expected, a strong emission peak at 460 nm could be detected when the concentrations of Cys were increased. Further data analysis revealed an excellent linear relationship (r = 0.996) between the fluorescence signal at 460 nm and the concentration of Cys (0–7 lM).The detection limit was determined to be 0.31 lM through the method of 3 r / k. The data suggested probe 1 could react with Cys both sensitively and quantitatively. A major challenge of biothiols’ detection in biological systems is to develop highly selective probes that exhibit notably distinctive response to biothiols over other cellular molecules. To investigate

Z. Zhu et al. / Tetrahedron Letters 56 (2015) 3909–3912

3911

Figure 3. (a) Concentration-dependent fluorescence spectra of probe 1 (1 lM) upon reaction with Cys (0–9 lM) at 25 °C. (b) The relationship between fluorescence intensity at 460 nm and Cys concentration in PBS (pH 7.4).

the selectivity of 1, various amino acids were incubated with probe 1 in PBS buffer and their fluorescence responses were tested (Figs. 4, S5). The fluorescent intensity of 1 with addition of different thiol species (Cys, GSH, H2S) could result in more than 100-fold increase, while the other amino acids samples exhibited no obvious increase of fluorescence signal. These experimental results implied that 1 is highly selective toward thiols but not toward other biologically relevant nucleophiles such as amino and imidazole group. Furthermore, the fluorescent turn-on response could be observed by naked eye under a UV lamp to give strong blue fluorescence (Fig. 4, inset), implying that 1 could be used to visualize and detect biothiols. As evidenced by these results, probe 1 is highly selective to biothiols over other biologically amine acids. To study the pH effect on fluorescence detection of biothiols, probe 1 was also evaluated for the detection of Cys in buffers with pH values from 6.0 to 8.5. As shown in Figure 5, probe 1 was effective for detection of Cys in buffers of the tested pH values, and higher fluorescence enhancement was observed in neutral and basic conditions. These results demonstrated that probe 1 could be used to detect Cys under physiological pH range. Finally, we examined whether 1 can be used to detect intracellular thiols in living cells. As shown in Figure 6, after incubating cells with 1 for 30 min, a strong fluorescence could be readily observed inside the cells under fluorescence microscope (Fig. 6a). The control experiments were performed by preincubating cells

Figure 5. Emission at 460 nm of probe 1 (1 lM) with Cys (100 lM) at different pH values at 25 °C for 12 min. Excitation at 305 nm with slit width: 2.5/5.0 nm.

Figure 6. Microscopy images of HEK293 cells using 1. Fluorescence (a) and brightfield (b) images of cells incubated with 1 (1 lM) for 30 min; fluorescence (c) and brightfield (d) images of cells that were pre-incubated with 50 lM 4dimethylaminophenylazophenyl-40 -maleimide (DABMI) for 30 min and then treated with 1 lM 1 for 30 min.

with 4-dimethylaminophenylazophenyl-40 -maleimide (DABMI) and subsequently adding 1. No significant fluorescence signal was observed under similar experimental conditions (Fig. 6c). These preliminary studies suggest that 1 is cell-permeable and can react with intracellular thiols efficiently and selectively. In summary, two new NBD-based fluorescent turn-on probes were rationally designed and synthesized for sensing of biothiols in physiological buffer. The probes are based on the fast thiolysis of the NBD ether bond of ESIPT fluorophores, which could be a novel and general design strategy for the design of thiols’ probes. The fluorescence enhancement of probe 1 upon thiols treatment could reach 115-fold at pH 7.4. The probe is highly selective to thiols over other amino acids. The probe also has good cell-permeability and can be used for imaging of thiols in HEK293 cells. Acknowledgements This work was supported by the MOST (2010CB126102), NSFC (21332004, 21402007), 111 project (B14004) and the Fundamental Research Funds for the Central Universities (YS1401). Figure 4. Selectivity of 1 (1 lM) in PBS buffer (50 mM, pH = 7.4, containing 10% DMSO as a co-solvent). All reactions were incubated for 5 min and fluorescence intensity at 460 nm was recorded (excitation 305 nm, slit width: 2.5/5.0 nm). The reaction species of amino acids (1 mM) and GSH (100 lM), Cys (100 lM), H2S (100 lM) were indicated below bars. Inset, visual fluorescence color of 1 (1 lM) in the presence of various species (1 mM) in PBS buffer under a handheld UV lamp (365 nm). Vials 1–7: probe only, Lys, Arg, Pro, Cys, GSH, H2S, respectively.

Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.tetlet.2015.04. 117.

3912

Z. Zhu et al. / Tetrahedron Letters 56 (2015) 3909–3912

References and notes 1. (a) Giles, G. I. Curr. Pharm. Des. 2006, 34, 4427; (b) Brandes, N.; Schmitt, S.; Jakob, U. Antioxid. Redox Signal. 2009, 11, 997; (c) Sen, C. K. Curr. Top. Cell. Regul. 2000, 36, 1; (d) Dalton, T. P.; Shertzer, H. G.; Puga, A. Annu. Rev. Pharmacol. Toxicol. 1999, 39, 67; (e) Kanzok, S. M.; Schirmer, R. H.; Türbachova, I.; Iozef, R.; Becker, K. J. Biol. Chem. 2000, 275, 40180. 2. Jones, D. P.; Carlson, J. L.; Mody, V. C.; Cai, J.; Lynn, M. J.; Sternberg, P. Free Radic. Biol. Med. 2000, 28, 625; (a) Kemp, M.; Go, Y. M.; Jones, D. P. Free Radic. Biol. Med. 2008, 44, 921; (b) Finkel, T.; Holbrook, N. J. Nature 2000, 408, 239. 3. Wierzbicki, A. S. Diabetes Vasc. Dis. Res. 2007, 4, 143; (a) Morris, M. S. Lancet Neurol. 2003, 2, 425. 4. Li, X.; Gao, X.; Shi, W.; Ma, H. Chem. Rev. 2014, 114, 590; (a) Peng, H.; Chen, W.; Cheng, Y.; Hakuna, L.; Strongin, R.; Wang, B. Sensors 2012, 12, 15907. 5. For recent reviews: (a) Chen, X.; Zhou, Y.; Peng, X.; Yoon, J. Chem. Soc. Rev. 2010, 39, 2120; (b) Zhou, Y.; Yoon, J. Chem. Soc. Rev. 2012, 41, 52; (c) Jung, H. S.; Chen, X.; Kim, J. S.; Yoon, J. Chem. Soc. Rev. 2013, 42, 6019. 6. Lin, H.; Fan, J.; Wang, J.; Tian, M.; Du, J.; Sun, S.; Sun, P.; Peng, X. Chem. Commun. 2009, 5904; (a) Yao, Z.; Feng, X.; Li, C.; Shi, G. Chem. Commun. 2009, 5886; (b) Mei, J.; Tong, J.; Wang, J.; Qin, A.; Sun, J. Z.; Tang, B. Z. J. Mater. Chem. 2012, 22. 7. Hong, V.; Kislukhin, A. A.; Finn, M. G. J. Am. Chem. Soc. 2009, 131, 9986; (a) Yi, L.; Li, H.; Sun, L.; Liu, L.; Zhang, C.; Xi, Z. Angew. Chem., Int. Ed. 2009, 48, 4034; (b) Lin, W.; Yuan, L.; Cao, Z.; Feng, Y.; Long, L. Chem.-Eur. J. 2009, 15, 5096; (c) Chen, X.; Ko, S.; Kim, M. J.; Shin, I.; Yoon, J. Chem. Commun. 2010, 2751; (d) Lim, S.; Escobedo, J. O.; Lowry, M.; Xu, X.; Strongin, R. Chem. Commun. 2010, 5707; (e) Sun, Y.-Q.; Chen, M.; Liu, J.; Lv, X.; Li, J.-F.; Guo, W. Chem. Commun. 2011, 11029; (f) Zhang, H.; Wang, P.; Yang, Y.; Sun, H. Chem. Commun. 2012, 10672. 8. Maeda, H.; Matsuno, H.; Ushida, M.; Katayama, K.; Saeki, K.; Itoh, N. Angew. Chem., Int. Ed. 2005, 44, 2922; (a) Bouffard, J.; Kim, Y.; Swager, T. M.; Weissleder, R.; Hilderbrand, S. A. Org. Lett. 2008, 10, 37; (b) Wang, S.; Deng, W.; Sun, D.; Yan, M.; Zheng, H.; Xu, J. Org. Biomol. Chem. 2009, 7, 4017; (c) Li, X.; Qian, S.; He, Q.; Yang, B.; Li, J.; Hu, Y. Org. Biomol. Chem. 2010, 8, 3627; (d) Ji, S.; Guo, H.; Yuan, X.; Li, X.; Ding, H.; Gao, P.; Zhao, C.; Wu, W.; Wu, W.; Zhao, J. Org. Lett. 2010, 12, 2876.

9. Niu, L.-Y.; Guan, Y.-S.; Chen, Y.-Z.; Wu, L.-Z.; Tung, C.-H.; Yang, Q.-Z. J. Am. Chem. Soc. 2012, 134, 18928; (b) Yang, X.-F.; Huang, Q.; Zhong, Y.; Li, Z.; Li, H.; Lowry, M.; Escobedo, J. O.; Strongin, R. M. Chem. Sci. 2014, 5, 2177; (c) Lim, S.Y.; Hong, K.-H.; Kim, D. I.; Kwon, H.; Kim, H.-J. J. Am. Chem. Soc. 2014, 136, 7018; (d) Guan, Y.-S.; Niu, L.-Y.; Chen, Y.-Z.; Wu, L.-Z.; Tung, C.-H.; Yang, Q.-Z. RSC Adv. 2014, 4, 8360; (e) Lv, H.; Yang, X.-F.; Zhong, Y.; Guo, Y.; Li, Z.; Li, H. Anal. Chem. 1800, 2014, 86; (f) Liu, J.; Sun, Y.-Q.; Zhang, H.; Huo, Y.; Shi, Y.; Guo, W. Chem. Sci. 2014, 5, 3183; (g) Tang, B.; Xing, Y.; Li, P.; Zhang, N.; Yu, F.; Yang, G. J. Am. Chem. Soc. 2007, 129, 11666; (h) Bae, Jihee.; Choi, Jiyoung.; Tae Jung Park; Suk-Kyu Chang Tetrahedron Lett. 2014, 55, 1171. 10. Lee, J. H.; Lim, C. S.; Tian, Y. S.; Han, J. H.; Cho, B. R. J. Am. Chem. Soc. 2010, 132, 1216; (a) Lim, C. S.; Masanta, G.; Kim, H. J.; Han, J. H.; Kim, H. M.; Cho, B. R. J. Am. Chem. Soc. 2011, 133, 11132; (b) Lee, M. H.; Han, J. H.; Kwon, P.-S.; Bhuniya, S.; Kim, J. Y.; Sessler, J. L.; Kang, C.; Kim, J. S. J. Am. Chem. Soc. 2012, 134, 1316; (c) Cao, X.; Lin, W.; Yu, Q. J. Org. Chem. 2011, 76, 7423; (d) Jung, D.; Maiti, S.; Lee, J. H.; Lee, J. H.; Kim, J. S. Chem. Commun. 2014, 3044. 11. Wang, R.; Chen, L.; Liu, P.; Zhang, Q.; Wang, Y. Chem. -Eur. J. 2012, 18, 11343; (b) Jung, H. S.; Han, J. H.; Habata, Y.; Kang, C.; Kim, J. S. Chem. Commun. 2011, 5142; (c) Bao, Y.; Li, Q.; Liu, B.; Du, F.; Tian, J.; Wang, H.; Wang, Y.; Bai, R. Chem. Commun. 2012, 118; (d) Wang, P.; Liu, J.; Lv, X.; Liu, Y.; Zhao, Y.; Guo, W. Org. Lett. 2012, 14, 520; (e) Malwal, S. R.; Labade, A.; Andhalkar, A. S.; Sengupta, K.; Chakrapani, H. Chem. Commun. 2014, 11533; (f) Zhang, H.; Zhang, C.; Liu, R.; Yi, L.; Sun, H. Chem. Commun. 2015, 2029. 12. Wei, C.; Wei, L.; Xi, Z.; Yi, L. Tetrahedron Lett. 2013, 54, 6937; (a) Wei, C.; Zhu, Q.; Liu, W.; Chen, W.; Xi, Z.; Yi, L. Org. Biomol. Chem. 2014, 12, 479; (b) Wei, C.; Wang, R.; Wei, L.; Cheng, L.; Li, Z.; Xi, Z.; Yi, L. Chem. Asian J. 2014, 9, 3586; (c) Wei, L.; Yi, L.; Song, F.; Wei, C.; Wang, B.; Xi, Z. Sci. Rep. 2014, 4, 4521; (d) Zhang, C.; Wei, L.; Wei, C.; Zhang, J.; Wang, R.; Xi, Z.; Yi, L. Chem. Commun. 2015, 7505. 13. Zhang, J.; Guo, W. Chem. Commun. 2014, 4214. 14. Mathai, J. C.; Missner, A.; Kügler, P.; Saparov, S. M.; Zeidel, M. L.; Lee, J. K.; Pohl, P. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 16633. 15. Montoya, L. A.; Pearce, T. F.; Hansen, R. J.; Zakharov, L. N.; Pluth, M. D. J. Org. Chem. 2013, 78, 6550.