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Cite this: Org. Biomol. Chem., 2013, 11, 8166 Received 16th September 2013, Accepted 22nd October 2013 DOI: 10.1039/c3ob41884g
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A colorimetric and fluorometric BODIPY probe for rapid, selective detection of H2S and its application in live cell imaging† Tanmoy Saha,‡ Dnyaneshwar Kand‡ and Pinaki Talukdar*
www.rsc.org/obc
A BODIPY-azide based colorimetric and fluorescence turn-ON probe for rapid, selective and sensitive detection of H2S is reported. The probe displayed a fast response time (10 min in HEPES and 30 s in serum albumin), 28-fold fluorescence enhancement and low detection limit up to 259 nM. The application of the probe to the estimation of H2S in live cells was demonstrated.
Hydrogen sulfide (H2S) is the third most important gasotransmitter,1 produced endogenously in the cytosol and mitrochondria2 of mammalian cells from cysteine by the reaction catalysed by two pyridoxal 5′-phosphate dependent enzymes, i.e., cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE).3 Along with nitric oxide (NO) and carbon monoxide (CO), it acts as a gaseous mediator for regulating neuronal, cardiovascular, endocrine, immune and gastrointestinal systems.1a,4 Unlike CO and NO, H2S does not produce reactive oxygen species (ROS); rather it acts as a scavenger of ROS.5 Under stress conditions, H2S acts as a regulator of energy production in the mitochondria of mammalian cells.6 Low level of H2S in the body can be elevated by the use of H2S releasing prodrugs, particularly in the treatment of cardiovascular and inflammatory diseases.7 On the other hand, overexpression of H2S producing enzymes8 and exposure to the environmentally/ industrially produced H2S gas result in high levels of H2S in biological systems leading to a variety of diseases e.g. Alzheimer’s disease,9 Down’s syndrome,10 diabetes,11 and liver cirrhosis.12 Hence the quantitative estimation of H2S present in biological as well as in environmental systems is of great importance. Such an approach is also helpful for understanding the physiological and pathological functions of H2S in various biological systems. However, rapid, selective, highly sensitive and reliable detection of the species is necessary due
Department of Chemistry, Mendeleev Block, Indian Institute of Science Education and Research, Pune, India. E-mail: [email protected]; Fax: +91 20 2589 9790; Tel: +91 20 2590 8001 † Electronic supplementary information (ESI) available: Experimental procedures, supplemental data, and the 1H-, 13C-NMR spectrum. See DOI: 10.1039/c3ob41884g ‡ Equal contributions of both authors.
8166 | Org. Biomol. Chem., 2013, 11, 8166–8170
to its rapid catabolism in endogenous systems, resulting in high fluctuations of the concentration. Extensive studies for the selective detection of H2S included colorimetric,13 electrochemical14 and gas chromatographic (GC)15 techniques. However, most of these methods are not applicable to biological systems. Selective determination of H2S by a fluorescence probe is beneficial for its in vitro as well as in vivo detection and also offers rapid, accurate and real time analysis. The pioneering approach involving H2S mediated reduction of azide to amine in fluorescent probes was reported independently by Chang16 and Wang5 et al. at nearly the same time. Since then, several fluorescent probes including 1–3 have been reported based on this concept by changing the fluorophores.1c,17 Probe 1 displayed a good response time of 3 minutes and a detection limit of 680 nM but it provided only a 16-fold sensitivity (Table 1). Although improved sensitivities were reported for probes 2 and 3, slow response times and high detection limits can be considered to be disadvantages of these probes. Alternate strategies including thiolysis of 2,4-dinitro phenyl ether,1b,18 nucleophilic attack of H2S on an aldehyde functionality,19 and nucleophilic substitution on a 2,4-dinitro sulfonyl group20 were also reported. Some of these probes suffer from drawbacks such as excitation in the UV region,17f long response times (1–2 h),17b, f,19,21 an inability to detect endogenous/intracellular H2S,5,16 etc. Hence, H2S selective fluorescent probes with fast response times, high sensitivities and low detection limits are essential for applications in vitro and in vivo (Fig. 1).
Table 1
Properties of reported fluorescence probes 1–3
Probe
λex (nm)
Response time
Detection limit
Fold enhancement
117d 217f 317e
474 340 435
3 min 60 min 45 min
680 nM N/A 1–5 μM
16 36 60
N/A = not available.
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Fig. 2 Effect of different solvents and buffers on the reaction of probe 4 (10 μM) with Na2S (200 μM). Normalised fluorescent intensity of probe 4 before (front row) and after addition of Na2S (back row).
Fig. 1 Schematic diagram illustrating the fluorescence turn-ON mechanism for H2S sensing based on the reduction of azide to amine (A); structures of reported fluorescent probes 1–3 and a new probe 4 (B).
Scheme 1
Synthesis of probe 4 and amine 5.
Herein, we report on the design and synthesis of a new BODIPY-azide 4 and evaluate the H2S sensing ability of the probe using UV-visible and fluorescence spectroscopy and live cell imaging. Recently, we have reported strong fluorescence properties of the corresponding BODIPY-amine 5 (Scheme 1).22 The intense absorption in visible light, relatively high molar extinction coefficient (ε), strong fluorescence and high quantum yield of 5 prompted us to select the BODIPYamine 5 as the active fluorophore. Turned-OFF fluorescence in azide 4 was expected due to electron-rich α-nitrogen of the azido group23 which would favour the electron transfer to the BODIPY core resulting in quenching of fluorescence.24 The fluorescence properties of probe 4 could be restored with the H2S mediated reduction of azide to amine 5. Probe 4 was synthesised by the SNAr reaction of 6 with NaN3 in 84% yield. Similarly, synthesis of amine 5 was carried out by using aqueous NH3 following a reported procedure.22 To validate the OFF–ON nature of sensing, the photophysical properties of compounds 4 and 5 were investigated. The absorption spectrum of probe 4 exhibited λmax = 515 nm in HEPES buffer (10 mM, pH = 7.4) with a molar extinction coefficient ε = 23 000 M−1 cm−1 (see Fig. S1†) but no significant fluorescence was observed when excited at λex = 515 nm (under identical conditions). In contrast, the amine 5 exhibited an absorption band centred at λmax = 444 nm in HEPES buffer
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(10 mM, pH = 7.4) with a molar extinction coefficient ε = 21 192 M−1 cm−1.22 The fluorescence spectra acquired for 5 indicate a strong fluorescence emission at λem = 520 nm when excited at 444 nm (see Fig. S2†). These photophysical data satisfy the criteria of probe 4 to act as an efficient turn ON fluorometric chemodosimeter for H2S. In the next stage of the study, solvent compatibility of probe 4 was evaluated in various solvents and buffers. In each case, fluorescence emission spectra were recorded for probe 4 (10 μM) before and after treatment with Na2S (200 μM). It was observed that in the H2S mediated reductions, higher fluorescence enhancements can be achieved in methanol–water (1 : 1) and acetonitrile–water (1 : 1) compared to that of 100% water or organic solvents (Fig. 2). As there is a considerable effect of buffer solutions in the detection of H2S,25 different buffers were screened. Probe 4 (10 μM) was treated with Na2S (200 μM) in either PBS (10 mM, pH = 7.4), Tris-base (10 mM, pH = 7.4) or HEPES (10 mM, pH = 7.4) buffer for 10 min at room temperature. Maximum enhancement in fluorescence intensity was observed in HEPES buffer. In order to evaluate the response time of probe 4 towards H2S detection the fluorescence spectra were acquired with time. Probe 4 (10 μM) was treated with Na2S (200 μM) in HEPES buffer (10 mM, pH = 7.4) and fluorescence spectra were recorded after 0.5, 1.5, 3.0, 4.5, 6.5, 8.0, 10.0 and 12.0 min. A strong fluorescence increment was observed within 3 min and the reaction was completed within 10 min (Fig. 3). When similar experiments were carried out for methanol–water (1 : 1) and acetonitrile–water (1 : 1) solvents, faster rates of reactions in these solvents were observed compared to that in HEPES (10 mM, pH = 7.4) and fluorescence intensities were comparable after 12 minutes (Fig. 3, inset ). Hence, on the basis of biological relevance, HEPES (10 mM, pH = 7.4) buffer was selected for further studies. The selectivity and sensitivity of probe 4 towards H2S in the presence of various analytes were examined. In each case, probe 4 (10 μM) was treated separately with 200 μM of each analyte (either NaCl, NaBr, NaI, NaF, Na2SO3, Na2SO4, Na2S2O3, NaSCN, cysteine, homocysteine, glutathione, NaOH, NaNO2, NaNO3 or H2O2) in HEPES buffer (10 mM, pH = 7.4) and fluorescence spectra were recorded after 10 minutes at room temperature (Fig. 4). However, no significant
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Fig. 3 Fluorescence responses of probe 4 (10 μM) to Na2S (200 μM) after 0.5, 1.5, 3.0, 4.5, 6.5, 8.0, 10.0 and 12.0 minutes. Inset: time dependence of fluorescence intensity, recorded at 520 nm in HEPES (10 mM, pH = 7.4), CH3CN–H2O (1 : 1) and CH3OH–H2O (1 : 1).
Organic & Biomolecular Chemistry
Fig. 5 UV-visible spectra of probe 4 (10 μM) with increasing concentration (0 to 200 μM) of Na2S in HEPES buffer. Inset: cuvette images of probe 4 before and after addition of Na2S taken under ambient light.
Fig. 4 Relative fluorescence intensity enhancements [(I − Io)/Io] at 520 nm for probe 4 (10 μM) towards Na2S (200 μM) in HEPES buffer. Front row: changes in intensities in the presence of various analytes (200 μM); back row: Na2S was added in the presence of the respective analyte.
fluorescence enhancements (at λem = 520 nm) were observed for all analytes (front row, Fig. 4). The sensitivity of probe 4 towards H2S was evaluated by addition of Na2S (200 μM) in the presence of individual competing analytes. Fluorescence spectra were recorded after 10 min at room temperature in each case. Significant fluorescence enhancements with a ∼28fold increase in most cases confirm the potential of probe 4 to detect H2S in the presence of various competing analytes (back row, Fig. 4). The decrease in the fluorescence increment only in the case of H2O2 can be attributed to the oxidation of H2S in the presence of H2O2.26 In the next stage the quantitative response of probe 4 towards H2S was evaluated by UV-visible spectroscopy. Upon the addition of Na2S (0 to 200 μM) to probe 4 (10 μM) in HEPES (10 mM, pH = 7.4) in a concentration dependent manner, the absorption maxima at λ = 373 and 515 nm (characteristic of probe 4) displayed a sharp decrease with a new absorption band emerging at 444 nm which corresponds to amine 5 (Fig. 5). The resulting blue shift (from λ = 515 nm to λ = 444 nm) is consistent with our initial comparison of UVvisible spectra of 4 and 5 (Fig. S1†). The blue shifted λmax value is also attributed to the visible color change from pink to yellow which can be confirmed by naked eye detection (Fig. 5,
8168 | Org. Biomol. Chem., 2013, 11, 8166–8170
Fig. 6 Fluorescence spectra of probe 4 (10 μM) with increasing concentrations (0 to 200 μM) of Na2S in HEPES buffer (10 mM, pH = 7.4). Inset: cuvette images of probe 4 before and after addition of Na2S taken under a hand held UV-lamp (λex = 365 nm).
inset ). Selectivity towards H2S of probe 4 could also be confirmed by a shift in λmax value in UV-visible spectra as well as by naked eye detection (Fig. S4 and S5†). We further examined the quantitative response of probe 4 towards H2S by fluorometric titrations. Sharp enhancements in fluorescence (λex = 444 nm) were observed (Fig. 6A) when titrations were carried out by addition of increasing concentrations of Na2S (0 to 200 μM) in the probe 4 (10 μM) in HEPES buffer (10 mM, pH = 7.4). When fluorescence intensities at 520 nm were plotted against the concentrations of H2S, excellent linear correlation (regression factor, R = 0.996) was observed up to one equivalent of the H2S added (Fig. S5†). A detection limit of 259 nM was calculated for the probe 4 in HEPES buffer (10 mM, pH = 7.4) during the detection of H2S, based on the signal to noise ratio, S/N = 3 (Fig. 7A). As shown in Fig. 6 (inset ), fluorescence turn-ON response by probe 4 upon sensing of H2S was also confirmed by the appearance of green fluorescence when cuvette images were taken under a
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Linear relationship between fluorescence intensity at 520 nm versus the concentration of Na2S added in HEPES buffer (A) and in BSA solution (B).
hand held UV-lamp (λex = 365 nm). The sensing of H2S by probe 4 was associated with a 40-fold enhancement in quantum yield, Φ (from 0.0119 to 0.4744; standard: Rhodamine-G in water; Φ = 0.76).27 In order to examine the ability of probe 4 to detect H2S in the biological samples, commercially available bovine serum albumin (BSA), a major component of human blood plasma, was used. When probe 4 (10 μM) was reacted with Na2S (20 μM) in BSA solution (50 mg mL−1), a sharp enhancement (∼29 fold) of fluorescence intensity at 520 nm was observed within 30 seconds which shows the fast response of probe 4 towards H2S (Fig. S7†). The detection limit of 265 nM for probe 4 in BSA solution was also determined using the same protocol as that in the case of HEPES (Fig. 7B). Interestingly, the detection limits in HEPES buffer and BSA solutions were also comparable. The detection limits for probe 4 lie well below the reported ranges of biological H2S such as endogenous (10–300 μM),28 blood (5–100 μM) and in brain homogenates (50–160 μM).29 Detection limits in nanomolar concentrations and faster response times in BSA solution as well as in HEPES buffer indicate the practical application of probe 4 in endogenous H2S detection. In order to evaluate the cell permeability and capability of probe 4 to selectively detect intracellular H2S, live-cell imaging studies were carried out. A human cervical cancer cell line (HeLa) was used for cell imaging studies. A significant fluorescence intensity was observed when HeLa cells were incubated with only probe 4 (5 μM in 1 : 100 DMSO–DNEM v/v, pH = 7.4) at 37 °C for 30 min (Fig. 8B). Then the same HeLa cells ( pre-incubated with probe 4) were incubated with Na2S (100 μM in 1 : 100 H2O–DMEM, pH = 7.4) at 37 °C for 30 min, and strong fluorescence inside the cell was observed (Fig. 8E). The appearance of fluorescence upon treating HeLa cells with probe 4 (Fig. 8B) confirms its reaction with intracellular H2S already present in these cells. Finally, we determined the quantitative change of the fluorescence intensity in HeLa cells with time. The pixel intensity obtained from the selected region of interest (ROI) versus time was plotted (Fig. 9 and Fig. S11†). Initially the HeLa cells were incubated with only probe 4 (5 μM) and the intensity was obtained from ROI which was low (Fig. 9, red bar). A gradual enhancement of the intensity of ROI was observed after incubation of the same cells with Na2S (100 μM). Each image was
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Fig. 8 Image of HeLa cells: brightfield (A), fluorescence (B), and overlay (C), incubated with probe 4 (5 μM) for 30 min. (D–F) are the respective brightfield, fluorescence and overlay images of HeLa cells pre-incubated with probe 4 followed by Na2S (100 μM) incubation for 30 min.
Fig. 9 Fluorescence images of HeLa cells incubated with probe 4 (5 μM) followed by incubation with Na2S (100 μM), acquired at different time intervals (0, 5, 10 and 15 min). Bar diagram of the average intensity of ROI versus time.
taken after 5 min intervals and the increment in the intensities saturated after 10 min. From these data it was confirmed that the increment in the fluorescence intensity was due to the increase in the concentration of H2S in the cells. These livecell studies confirm that probe 4 could be a good candidate for monitoring intracellular H2S levels. In summary, we have developed a BODIPY-based colorimetric and fluorescence turn-ON chemodosimeter for a rapid, selective and sensitive detection of H2S. The probe is capable of discriminating biological thiols (such as Cys and GSH), various biologically relevant nucleophiles, reducing agents and ROS from H2S. Since the catabolism of H2S is very fast, the fast response times (10 min in HEPES, 30 seconds in BSA), low
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Notes and references 1 (a) H. Kimura, Amino Acids, 2011, 41, 113; (b) T. Liu, Z. Xu, D. R. Spring and J. Cui, Org. Lett., 2013, 15, 2310; (c) S. Chen, Z.-J. Chen, W. Ren and H.-W. Ai, J. Am. Chem. Soc., 2012, 134, 9589. 2 P. Kamoun, Amino Acids, 2004, 26, 243. 3 D. Boehning and S. H. Snyder, Annu. Rev. Neurosci., 2003, 26, 105. 4 (a) L. Li, P. Rose and P. K. Moore, Annu. Rev. Pharmacol. Toxicol., 2011, 51, 169; (b) C. Peers, C. C. Bauer, J. P. Boyle, J. L. Scragg and M. L. Dallas, Antioxid. Redox Signaling, 2012, 17, 95. 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. 6 (a) M. Fu, W. Zhang, L. Wu, G. Yang, H. Li and R. Wang, Proc. Natl. Acad. Sci. U. S. A., 2012, 109, 2943; (b) P. Nicholls, Biochem. Soc. Trans., 1975, 3, 316. 7 J. L. Wallace, Trends Pharmacol. Sci., 2007, 28, 501. 8 (a) K. Wang, S. Ahmad, M. Cai, J. Rennie, T. Fujisawa, F. Crispi, J. Baily, M. R. Miller, M. Cudmore, P. W. F. Hadoke, R. Wang, E. Gratacos, I. A. Buhimschi, C. S. Buhimschi and A. Ahmed, Circulation, 2013, 127, 2514; (b) C. Szabo, C. Coletta, C. Chao, K. Módis, B. Szczesny, A. Papapetropoulos and M. R. Hellmich, Proc. Natl. Acad. Sci. U. S. A., 2013, 110, 12474. 9 K. Eto, T. Asada, K. Arima, T. Makifuchi and H. Kimura, Biochem. Biophys. Res. Commun., 2002, 293, 1485. 10 P. Kamoun, M.-C. Belardinelli, A. Chabli, K. Lallouchi and B. Chadefaux-Vekemans, Am. J. Med. Genet. A, 2003, 116A, 310. 11 W. Yang, G. Yang, X. Jia, L. Wu and R. Wang, J. Physiol., 2005, 569, 519. 12 S. Fiorucci, E. Antonelli, A. Mencarelli, S. Orlandi, B. Renga, G. Rizzo, E. Distrutti, V. Shah and A. Morelli, Hepatology, 2005, 42, 539. 13 (a) W. Lei and P. K. Dasgupta, Anal. Chim. Acta, 1989, 226, 165; (b) D. Jimenez, R. Martinez-Manez, F. Sancenon,
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18 19
20 21
22 23 24 25 26 27 28 29
J. V. Ros-Lis, A. Benito and J. Soto, J. Am. Chem. Soc., 2003, 125, 9000. D. G. Searcy and M. A. Peterson, Anal. Biochem., 2004, 324, 269. P. R. Berube, P. D. Parkinson and E. R. Hall, J. Chromatogr., A, 1999, 830, 485. A. R. Lippert, E. J. New and C. J. Chang, J. Am. Chem. Soc., 2011, 133, 10078. (a) S. K. Das, C. S. Lim, S. Y. Yang, J. H. Han and B. R. Cho, Chem. Commun., 2012, 48, 8395; (b) L. A. Montoya and M. D. Pluth, Chem. Commun., 2012, 48, 4767; (c) Q. Wan, Y. Song, Z. Li, X. Gao and H. Ma, Chem. Commun., 2013, 49, 502; (d) G. Zhou, H. Wang, Y. Ma and X. Chen, Tetrahedron, 2013, 69, 867; (e) L. A. Montoya and M. D. Pluth, Chem. Commun., 2012, 48, 4767; (f ) B. Chen, W. Li, C. Lv, M. Zhao, H. Jin, H. Jin, J. Du, L. Zhang and X. Tang, Analyst, 2013, 138, 946; (g) C. Yu, X. Li, F. Zeng, F. Zheng and S. Wu, Chem. Commun., 2013, 49, 403. X. Cao, W. Lin, K. Zheng and L. He, Chem. Commun., 2012, 48, 10529. Y. Qian, J. Karpus, O. Kabil, S.-Y. Zhang, H.-L. Zhu, R. Banerjee, J. Zhao and C. He, Nat. Commun., 2011, 2, 495. X.-F. Yang, L. Wang, H. Xu and M. Zhao, Anal. Chim. Acta, 2009, 631, 91. C. Liu, J. Pan, S. Li, Y. Zhao, L. Y. Wu, C. E. Berkman, A. R. Whorton and M. Xian, Angew. Chem., Int. Ed., 2011, 50, 10327. D. Kand, P. K. Mishra, T. Saha, M. Lahiri and P. Talukdar, Analyst, 2012, 137, 3921. K. Sivakumar, F. Xie, B. M. Cash, S. Long, H. N. Barnhill and Q. Wang, Org. Lett., 2004, 6, 4603. C. Wang, F. Xie, N. Suthiwangcharoen, J. Sun and Q. Wang, Sci. China Chem., 2012, 55, 125. X. Wang, J. Sun, W. Zhang, X. Ma, J. Lv and B. Tang, Chem. Sci., 2013, 4, 2551. M. R. Hoffmann, Environ. Sci. Technol., 1977, 11, 61. J. Olmsted, J. Phys. Chem., 1979, 83, 2581. N. L. Whitfield, E. L. Kreimier, F. C. Verdial, N. Skovgaard and K. R. Olson, Am. J. Physiol., 2008, 294, R1930. (a) M. Yusuf, B. T. K. Huat, A. Hsu, M. Whiteman, M. Bhatia and P. K. Moore, Biochem. Biophys. Res. Commun., 2005, 333, 1146; (b) C. J. Richardson, E. A. M. Magee and J. H. Cummings, Clin. Chim. Acta, 2000, 293, 115; (c) K. Abe and H. Kimura, J. Neurosci., 1996, 16, 1066; (d) L. R. Goodwin, D. Francom, F. P. Dieken, J. D. Taylor, M. W. Warenycia, R. J. Reiffenstein and G. Dowling, J. Anal. Toxicol., 1989, 13, 105.
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Cite this: Org. Biomol. Chem., 2013, 11, 8166 Received 16th September 2013, Accepted 22nd October 2013 DOI: 10.1039/c3ob41884g
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A colorimetric and fluorometric BODIPY probe for rapid, selective detection of H2S and its application in live cell imaging† Tanmoy Saha,‡ Dnyaneshwar Kand‡ and Pinaki Talukdar*
www.rsc.org/obc
A BODIPY-azide based colorimetric and fluorescence turn-ON probe for rapid, selective and sensitive detection of H2S is reported. The probe displayed a fast response time (10 min in HEPES and 30 s in serum albumin), 28-fold fluorescence enhancement and low detection limit up to 259 nM. The application of the probe to the estimation of H2S in live cells was demonstrated.
Hydrogen sulfide (H2S) is the third most important gasotransmitter,1 produced endogenously in the cytosol and mitrochondria2 of mammalian cells from cysteine by the reaction catalysed by two pyridoxal 5′-phosphate dependent enzymes, i.e., cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE).3 Along with nitric oxide (NO) and carbon monoxide (CO), it acts as a gaseous mediator for regulating neuronal, cardiovascular, endocrine, immune and gastrointestinal systems.1a,4 Unlike CO and NO, H2S does not produce reactive oxygen species (ROS); rather it acts as a scavenger of ROS.5 Under stress conditions, H2S acts as a regulator of energy production in the mitochondria of mammalian cells.6 Low level of H2S in the body can be elevated by the use of H2S releasing prodrugs, particularly in the treatment of cardiovascular and inflammatory diseases.7 On the other hand, overexpression of H2S producing enzymes8 and exposure to the environmentally/ industrially produced H2S gas result in high levels of H2S in biological systems leading to a variety of diseases e.g. Alzheimer’s disease,9 Down’s syndrome,10 diabetes,11 and liver cirrhosis.12 Hence the quantitative estimation of H2S present in biological as well as in environmental systems is of great importance. Such an approach is also helpful for understanding the physiological and pathological functions of H2S in various biological systems. However, rapid, selective, highly sensitive and reliable detection of the species is necessary due
Department of Chemistry, Mendeleev Block, Indian Institute of Science Education and Research, Pune, India. E-mail: [email protected]; Fax: +91 20 2589 9790; Tel: +91 20 2590 8001 † Electronic supplementary information (ESI) available: Experimental procedures, supplemental data, and the 1H-, 13C-NMR spectrum. See DOI: 10.1039/c3ob41884g ‡ Equal contributions of both authors.
8166 | Org. Biomol. Chem., 2013, 11, 8166–8170
to its rapid catabolism in endogenous systems, resulting in high fluctuations of the concentration. Extensive studies for the selective detection of H2S included colorimetric,13 electrochemical14 and gas chromatographic (GC)15 techniques. However, most of these methods are not applicable to biological systems. Selective determination of H2S by a fluorescence probe is beneficial for its in vitro as well as in vivo detection and also offers rapid, accurate and real time analysis. The pioneering approach involving H2S mediated reduction of azide to amine in fluorescent probes was reported independently by Chang16 and Wang5 et al. at nearly the same time. Since then, several fluorescent probes including 1–3 have been reported based on this concept by changing the fluorophores.1c,17 Probe 1 displayed a good response time of 3 minutes and a detection limit of 680 nM but it provided only a 16-fold sensitivity (Table 1). Although improved sensitivities were reported for probes 2 and 3, slow response times and high detection limits can be considered to be disadvantages of these probes. Alternate strategies including thiolysis of 2,4-dinitro phenyl ether,1b,18 nucleophilic attack of H2S on an aldehyde functionality,19 and nucleophilic substitution on a 2,4-dinitro sulfonyl group20 were also reported. Some of these probes suffer from drawbacks such as excitation in the UV region,17f long response times (1–2 h),17b, f,19,21 an inability to detect endogenous/intracellular H2S,5,16 etc. Hence, H2S selective fluorescent probes with fast response times, high sensitivities and low detection limits are essential for applications in vitro and in vivo (Fig. 1).
Table 1
Properties of reported fluorescence probes 1–3
Probe
λex (nm)
Response time
Detection limit
Fold enhancement
117d 217f 317e
474 340 435
3 min 60 min 45 min
680 nM N/A 1–5 μM
16 36 60
N/A = not available.
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Fig. 2 Effect of different solvents and buffers on the reaction of probe 4 (10 μM) with Na2S (200 μM). Normalised fluorescent intensity of probe 4 before (front row) and after addition of Na2S (back row).
Fig. 1 Schematic diagram illustrating the fluorescence turn-ON mechanism for H2S sensing based on the reduction of azide to amine (A); structures of reported fluorescent probes 1–3 and a new probe 4 (B).
Scheme 1
Synthesis of probe 4 and amine 5.
Herein, we report on the design and synthesis of a new BODIPY-azide 4 and evaluate the H2S sensing ability of the probe using UV-visible and fluorescence spectroscopy and live cell imaging. Recently, we have reported strong fluorescence properties of the corresponding BODIPY-amine 5 (Scheme 1).22 The intense absorption in visible light, relatively high molar extinction coefficient (ε), strong fluorescence and high quantum yield of 5 prompted us to select the BODIPYamine 5 as the active fluorophore. Turned-OFF fluorescence in azide 4 was expected due to electron-rich α-nitrogen of the azido group23 which would favour the electron transfer to the BODIPY core resulting in quenching of fluorescence.24 The fluorescence properties of probe 4 could be restored with the H2S mediated reduction of azide to amine 5. Probe 4 was synthesised by the SNAr reaction of 6 with NaN3 in 84% yield. Similarly, synthesis of amine 5 was carried out by using aqueous NH3 following a reported procedure.22 To validate the OFF–ON nature of sensing, the photophysical properties of compounds 4 and 5 were investigated. The absorption spectrum of probe 4 exhibited λmax = 515 nm in HEPES buffer (10 mM, pH = 7.4) with a molar extinction coefficient ε = 23 000 M−1 cm−1 (see Fig. S1†) but no significant fluorescence was observed when excited at λex = 515 nm (under identical conditions). In contrast, the amine 5 exhibited an absorption band centred at λmax = 444 nm in HEPES buffer
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(10 mM, pH = 7.4) with a molar extinction coefficient ε = 21 192 M−1 cm−1.22 The fluorescence spectra acquired for 5 indicate a strong fluorescence emission at λem = 520 nm when excited at 444 nm (see Fig. S2†). These photophysical data satisfy the criteria of probe 4 to act as an efficient turn ON fluorometric chemodosimeter for H2S. In the next stage of the study, solvent compatibility of probe 4 was evaluated in various solvents and buffers. In each case, fluorescence emission spectra were recorded for probe 4 (10 μM) before and after treatment with Na2S (200 μM). It was observed that in the H2S mediated reductions, higher fluorescence enhancements can be achieved in methanol–water (1 : 1) and acetonitrile–water (1 : 1) compared to that of 100% water or organic solvents (Fig. 2). As there is a considerable effect of buffer solutions in the detection of H2S,25 different buffers were screened. Probe 4 (10 μM) was treated with Na2S (200 μM) in either PBS (10 mM, pH = 7.4), Tris-base (10 mM, pH = 7.4) or HEPES (10 mM, pH = 7.4) buffer for 10 min at room temperature. Maximum enhancement in fluorescence intensity was observed in HEPES buffer. In order to evaluate the response time of probe 4 towards H2S detection the fluorescence spectra were acquired with time. Probe 4 (10 μM) was treated with Na2S (200 μM) in HEPES buffer (10 mM, pH = 7.4) and fluorescence spectra were recorded after 0.5, 1.5, 3.0, 4.5, 6.5, 8.0, 10.0 and 12.0 min. A strong fluorescence increment was observed within 3 min and the reaction was completed within 10 min (Fig. 3). When similar experiments were carried out for methanol–water (1 : 1) and acetonitrile–water (1 : 1) solvents, faster rates of reactions in these solvents were observed compared to that in HEPES (10 mM, pH = 7.4) and fluorescence intensities were comparable after 12 minutes (Fig. 3, inset ). Hence, on the basis of biological relevance, HEPES (10 mM, pH = 7.4) buffer was selected for further studies. The selectivity and sensitivity of probe 4 towards H2S in the presence of various analytes were examined. In each case, probe 4 (10 μM) was treated separately with 200 μM of each analyte (either NaCl, NaBr, NaI, NaF, Na2SO3, Na2SO4, Na2S2O3, NaSCN, cysteine, homocysteine, glutathione, NaOH, NaNO2, NaNO3 or H2O2) in HEPES buffer (10 mM, pH = 7.4) and fluorescence spectra were recorded after 10 minutes at room temperature (Fig. 4). However, no significant
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Fig. 3 Fluorescence responses of probe 4 (10 μM) to Na2S (200 μM) after 0.5, 1.5, 3.0, 4.5, 6.5, 8.0, 10.0 and 12.0 minutes. Inset: time dependence of fluorescence intensity, recorded at 520 nm in HEPES (10 mM, pH = 7.4), CH3CN–H2O (1 : 1) and CH3OH–H2O (1 : 1).
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Fig. 5 UV-visible spectra of probe 4 (10 μM) with increasing concentration (0 to 200 μM) of Na2S in HEPES buffer. Inset: cuvette images of probe 4 before and after addition of Na2S taken under ambient light.
Fig. 4 Relative fluorescence intensity enhancements [(I − Io)/Io] at 520 nm for probe 4 (10 μM) towards Na2S (200 μM) in HEPES buffer. Front row: changes in intensities in the presence of various analytes (200 μM); back row: Na2S was added in the presence of the respective analyte.
fluorescence enhancements (at λem = 520 nm) were observed for all analytes (front row, Fig. 4). The sensitivity of probe 4 towards H2S was evaluated by addition of Na2S (200 μM) in the presence of individual competing analytes. Fluorescence spectra were recorded after 10 min at room temperature in each case. Significant fluorescence enhancements with a ∼28fold increase in most cases confirm the potential of probe 4 to detect H2S in the presence of various competing analytes (back row, Fig. 4). The decrease in the fluorescence increment only in the case of H2O2 can be attributed to the oxidation of H2S in the presence of H2O2.26 In the next stage the quantitative response of probe 4 towards H2S was evaluated by UV-visible spectroscopy. Upon the addition of Na2S (0 to 200 μM) to probe 4 (10 μM) in HEPES (10 mM, pH = 7.4) in a concentration dependent manner, the absorption maxima at λ = 373 and 515 nm (characteristic of probe 4) displayed a sharp decrease with a new absorption band emerging at 444 nm which corresponds to amine 5 (Fig. 5). The resulting blue shift (from λ = 515 nm to λ = 444 nm) is consistent with our initial comparison of UVvisible spectra of 4 and 5 (Fig. S1†). The blue shifted λmax value is also attributed to the visible color change from pink to yellow which can be confirmed by naked eye detection (Fig. 5,
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Fig. 6 Fluorescence spectra of probe 4 (10 μM) with increasing concentrations (0 to 200 μM) of Na2S in HEPES buffer (10 mM, pH = 7.4). Inset: cuvette images of probe 4 before and after addition of Na2S taken under a hand held UV-lamp (λex = 365 nm).
inset ). Selectivity towards H2S of probe 4 could also be confirmed by a shift in λmax value in UV-visible spectra as well as by naked eye detection (Fig. S4 and S5†). We further examined the quantitative response of probe 4 towards H2S by fluorometric titrations. Sharp enhancements in fluorescence (λex = 444 nm) were observed (Fig. 6A) when titrations were carried out by addition of increasing concentrations of Na2S (0 to 200 μM) in the probe 4 (10 μM) in HEPES buffer (10 mM, pH = 7.4). When fluorescence intensities at 520 nm were plotted against the concentrations of H2S, excellent linear correlation (regression factor, R = 0.996) was observed up to one equivalent of the H2S added (Fig. S5†). A detection limit of 259 nM was calculated for the probe 4 in HEPES buffer (10 mM, pH = 7.4) during the detection of H2S, based on the signal to noise ratio, S/N = 3 (Fig. 7A). As shown in Fig. 6 (inset ), fluorescence turn-ON response by probe 4 upon sensing of H2S was also confirmed by the appearance of green fluorescence when cuvette images were taken under a
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Fig. 7
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Linear relationship between fluorescence intensity at 520 nm versus the concentration of Na2S added in HEPES buffer (A) and in BSA solution (B).
hand held UV-lamp (λex = 365 nm). The sensing of H2S by probe 4 was associated with a 40-fold enhancement in quantum yield, Φ (from 0.0119 to 0.4744; standard: Rhodamine-G in water; Φ = 0.76).27 In order to examine the ability of probe 4 to detect H2S in the biological samples, commercially available bovine serum albumin (BSA), a major component of human blood plasma, was used. When probe 4 (10 μM) was reacted with Na2S (20 μM) in BSA solution (50 mg mL−1), a sharp enhancement (∼29 fold) of fluorescence intensity at 520 nm was observed within 30 seconds which shows the fast response of probe 4 towards H2S (Fig. S7†). The detection limit of 265 nM for probe 4 in BSA solution was also determined using the same protocol as that in the case of HEPES (Fig. 7B). Interestingly, the detection limits in HEPES buffer and BSA solutions were also comparable. The detection limits for probe 4 lie well below the reported ranges of biological H2S such as endogenous (10–300 μM),28 blood (5–100 μM) and in brain homogenates (50–160 μM).29 Detection limits in nanomolar concentrations and faster response times in BSA solution as well as in HEPES buffer indicate the practical application of probe 4 in endogenous H2S detection. In order to evaluate the cell permeability and capability of probe 4 to selectively detect intracellular H2S, live-cell imaging studies were carried out. A human cervical cancer cell line (HeLa) was used for cell imaging studies. A significant fluorescence intensity was observed when HeLa cells were incubated with only probe 4 (5 μM in 1 : 100 DMSO–DNEM v/v, pH = 7.4) at 37 °C for 30 min (Fig. 8B). Then the same HeLa cells ( pre-incubated with probe 4) were incubated with Na2S (100 μM in 1 : 100 H2O–DMEM, pH = 7.4) at 37 °C for 30 min, and strong fluorescence inside the cell was observed (Fig. 8E). The appearance of fluorescence upon treating HeLa cells with probe 4 (Fig. 8B) confirms its reaction with intracellular H2S already present in these cells. Finally, we determined the quantitative change of the fluorescence intensity in HeLa cells with time. The pixel intensity obtained from the selected region of interest (ROI) versus time was plotted (Fig. 9 and Fig. S11†). Initially the HeLa cells were incubated with only probe 4 (5 μM) and the intensity was obtained from ROI which was low (Fig. 9, red bar). A gradual enhancement of the intensity of ROI was observed after incubation of the same cells with Na2S (100 μM). Each image was
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Fig. 8 Image of HeLa cells: brightfield (A), fluorescence (B), and overlay (C), incubated with probe 4 (5 μM) for 30 min. (D–F) are the respective brightfield, fluorescence and overlay images of HeLa cells pre-incubated with probe 4 followed by Na2S (100 μM) incubation for 30 min.
Fig. 9 Fluorescence images of HeLa cells incubated with probe 4 (5 μM) followed by incubation with Na2S (100 μM), acquired at different time intervals (0, 5, 10 and 15 min). Bar diagram of the average intensity of ROI versus time.
taken after 5 min intervals and the increment in the intensities saturated after 10 min. From these data it was confirmed that the increment in the fluorescence intensity was due to the increase in the concentration of H2S in the cells. These livecell studies confirm that probe 4 could be a good candidate for monitoring intracellular H2S levels. In summary, we have developed a BODIPY-based colorimetric and fluorescence turn-ON chemodosimeter for a rapid, selective and sensitive detection of H2S. The probe is capable of discriminating biological thiols (such as Cys and GSH), various biologically relevant nucleophiles, reducing agents and ROS from H2S. Since the catabolism of H2S is very fast, the fast response times (10 min in HEPES, 30 seconds in BSA), low
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Notes and references 1 (a) H. Kimura, Amino Acids, 2011, 41, 113; (b) T. Liu, Z. Xu, D. R. Spring and J. Cui, Org. Lett., 2013, 15, 2310; (c) S. Chen, Z.-J. Chen, W. Ren and H.-W. Ai, J. Am. Chem. Soc., 2012, 134, 9589. 2 P. Kamoun, Amino Acids, 2004, 26, 243. 3 D. Boehning and S. H. Snyder, Annu. Rev. Neurosci., 2003, 26, 105. 4 (a) L. Li, P. Rose and P. K. Moore, Annu. Rev. Pharmacol. Toxicol., 2011, 51, 169; (b) C. Peers, C. C. Bauer, J. P. Boyle, J. L. Scragg and M. L. Dallas, Antioxid. Redox Signaling, 2012, 17, 95. 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. 6 (a) M. Fu, W. Zhang, L. Wu, G. Yang, H. Li and R. Wang, Proc. Natl. Acad. Sci. U. S. A., 2012, 109, 2943; (b) P. Nicholls, Biochem. Soc. Trans., 1975, 3, 316. 7 J. L. Wallace, Trends Pharmacol. Sci., 2007, 28, 501. 8 (a) K. Wang, S. Ahmad, M. Cai, J. Rennie, T. Fujisawa, F. Crispi, J. Baily, M. R. Miller, M. Cudmore, P. W. F. Hadoke, R. Wang, E. Gratacos, I. A. Buhimschi, C. S. Buhimschi and A. Ahmed, Circulation, 2013, 127, 2514; (b) C. Szabo, C. Coletta, C. Chao, K. Módis, B. Szczesny, A. Papapetropoulos and M. R. Hellmich, Proc. Natl. Acad. Sci. U. S. A., 2013, 110, 12474. 9 K. Eto, T. Asada, K. Arima, T. Makifuchi and H. Kimura, Biochem. Biophys. Res. Commun., 2002, 293, 1485. 10 P. Kamoun, M.-C. Belardinelli, A. Chabli, K. Lallouchi and B. Chadefaux-Vekemans, Am. J. Med. Genet. A, 2003, 116A, 310. 11 W. Yang, G. Yang, X. Jia, L. Wu and R. Wang, J. Physiol., 2005, 569, 519. 12 S. Fiorucci, E. Antonelli, A. Mencarelli, S. Orlandi, B. Renga, G. Rizzo, E. Distrutti, V. Shah and A. Morelli, Hepatology, 2005, 42, 539. 13 (a) W. Lei and P. K. Dasgupta, Anal. Chim. Acta, 1989, 226, 165; (b) D. Jimenez, R. Martinez-Manez, F. Sancenon,
8170 | Org. Biomol. Chem., 2013, 11, 8166–8170
18 19
20 21
22 23 24 25 26 27 28 29
J. V. Ros-Lis, A. Benito and J. Soto, J. Am. Chem. Soc., 2003, 125, 9000. D. G. Searcy and M. A. Peterson, Anal. Biochem., 2004, 324, 269. P. R. Berube, P. D. Parkinson and E. R. Hall, J. Chromatogr., A, 1999, 830, 485. A. R. Lippert, E. J. New and C. J. Chang, J. Am. Chem. Soc., 2011, 133, 10078. (a) S. K. Das, C. S. Lim, S. Y. Yang, J. H. Han and B. R. Cho, Chem. Commun., 2012, 48, 8395; (b) L. A. Montoya and M. D. Pluth, Chem. Commun., 2012, 48, 4767; (c) Q. Wan, Y. Song, Z. Li, X. Gao and H. Ma, Chem. Commun., 2013, 49, 502; (d) G. Zhou, H. Wang, Y. Ma and X. Chen, Tetrahedron, 2013, 69, 867; (e) L. A. Montoya and M. D. Pluth, Chem. Commun., 2012, 48, 4767; (f ) B. Chen, W. Li, C. Lv, M. Zhao, H. Jin, H. Jin, J. Du, L. Zhang and X. Tang, Analyst, 2013, 138, 946; (g) C. Yu, X. Li, F. Zeng, F. Zheng and S. Wu, Chem. Commun., 2013, 49, 403. X. Cao, W. Lin, K. Zheng and L. He, Chem. Commun., 2012, 48, 10529. Y. Qian, J. Karpus, O. Kabil, S.-Y. Zhang, H.-L. Zhu, R. Banerjee, J. Zhao and C. He, Nat. Commun., 2011, 2, 495. X.-F. Yang, L. Wang, H. Xu and M. Zhao, Anal. Chim. Acta, 2009, 631, 91. C. Liu, J. Pan, S. Li, Y. Zhao, L. Y. Wu, C. E. Berkman, A. R. Whorton and M. Xian, Angew. Chem., Int. Ed., 2011, 50, 10327. D. Kand, P. K. Mishra, T. Saha, M. Lahiri and P. Talukdar, Analyst, 2012, 137, 3921. K. Sivakumar, F. Xie, B. M. Cash, S. Long, H. N. Barnhill and Q. Wang, Org. Lett., 2004, 6, 4603. C. Wang, F. Xie, N. Suthiwangcharoen, J. Sun and Q. Wang, Sci. China Chem., 2012, 55, 125. X. Wang, J. Sun, W. Zhang, X. Ma, J. Lv and B. Tang, Chem. Sci., 2013, 4, 2551. M. R. Hoffmann, Environ. Sci. Technol., 1977, 11, 61. J. Olmsted, J. Phys. Chem., 1979, 83, 2581. N. L. Whitfield, E. L. Kreimier, F. C. Verdial, N. Skovgaard and K. R. Olson, Am. J. Physiol., 2008, 294, R1930. (a) M. Yusuf, B. T. K. Huat, A. Hsu, M. Whiteman, M. Bhatia and P. K. Moore, Biochem. Biophys. Res. Commun., 2005, 333, 1146; (b) C. J. Richardson, E. A. M. Magee and J. H. Cummings, Clin. Chim. Acta, 2000, 293, 115; (c) K. Abe and H. Kimura, J. Neurosci., 1996, 16, 1066; (d) L. R. Goodwin, D. Francom, F. P. Dieken, J. D. Taylor, M. W. Warenycia, R. J. Reiffenstein and G. Dowling, J. Anal. Toxicol., 1989, 13, 105.
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