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A Two-Input Fluorescent Probe for Thiols and Hydrogen Sulfide Chemosensing and Live Cell Imaging Chun-Guang Dai, Xiu-Ling Liu, Xiao-Jiao Du, Yan Zhang, and Qin-Hua Song ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.6b00291 • Publication Date (Web): 01 Jun 2016 Downloaded from http://pubs.acs.org on June 3, 2016
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ACS Sensors
A Two-Input Fluorescent Probe for Thiols and Hydrogen Sulfide Chemosensing and Live Cell Imaging Chun-Guang Dai,†a Xiu-Ling Liu,† a Xiao-Jiao Du,b Yan Zhang,c and Qin-Hua Song*a a
b
Department of Chemistry, University of Science and Technology of China, Hefei 230026, P. R. China. School of c Life Sciences, University of Science and Technology of China, Hefei 230027, P. R. China. State Key Laboratory of Analytical Chemistry for Life Science, Institute of Chemistry & BioMedical Sciences, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, 210093, P. R. China. KEYWORDS: Fluorescent probes, Recognition, Thiols and H2S, Two-photon imaging, Molecular logic gate, Chemical inputs
ABSTRACT: Development of multi-input fluorescent probes would facilitate the monitoring of multiple biomolecular events leading to a common disease pathology. Here, we reported a two-input fluorescent probe (QME-N3) based on quinoline scaffold with two distinct reactive sites, the α,β-unsaturated carbonyl at 2-site and 6-azido group, which were specific acceptors of RSH and H2S respectively. The turn-on fluorescent probe could react with the two molecules via the Michael addition at the double bond for RSH and the reduction of the azide by H2S without mutual interference, to generate the intensively fluorescent product, which could act as a two-input AND logic gate. Furthermore, QME-N3 has very low cytotoxicity and excellent stability, and the resulting sensing product has a high two-photon absorption cross section. Consequently, detection of intracellular thiols and H2S can be achieved by cell fluorescence imaging under both one- and two-photon excitations.
H
ydrogen sulfide (H2S) and thiols are belong to important members of reactive sulfur species (RSS).1-3 H2S is considered as the third most important gasotransmitter following nitric oxide (NO) and carbon monoxide (CO),4-5 and generated from cysteine (Cys) with three enzymes, cystathionine β-synthase (CBS), cystathionine γlyase (CSE), and 3-mercaptopyruvate sulfur-transferase in the cytosols and mitochondria of mammalian cells in relatively high concentration (10-100 µM).6-11 The significance of endogenous H2S has been recognized in a number of physiological and pathological processes.6, 7, 12, 13 For example, H2S is a physiologic vasodilator and regulator of blood pressure.14 Similarly, intracellar thiols (RSH) such as Cys, homocysteine (Hcy) and glutathione (GSH) play significant roles in many physiological processes.15, 16 Abnormal levels of cellular thiols are associated with many human diseases.17-21 Therefore, the detection of cellular thiols and H2S is of growing importance. Among the various detection methods, optical approach based on synthetic colorimetic and fluorescent molecular probes has attracted increasing interest due to their simplicity, inexpensiveness, sensitivity and selectivity during the last decade. A variety of colorimetic and fluorescent probes for thiols22-25 or H2S26-29 have been constructed by exploiting their nucleophilicity, high transi-
tion metal affinity or reducibility to achieve a specific reaction between the probe and thiols or H2S. The vast majority of those fluorescent molecules behave as singleinput sensors, where only one input (H2S or RSH) is required to interact with the probe, and generate a fluorescent output (i.e., a YES logic gate). Recently, fluorescent probes for dual- or multi-analyte detection have emerged as valuable and powerful analytical tools for (bio)sensing and bioimaging applications.30-38 Thereinto, two applications highlighted are fluorescent molecular logic gates, and “smart” medical diagnostics relying on the simultaneous quantitative detection of several analytes to one pathology.30 The multi-input fluorescent probes mean that two- and three- targeted (bio)analytes must work in tandem or in a sequential manner to convert the probe into a luminescent product (“turn-on” fluorescence response) or to induce a dramatic shift in its excitation/emission profiles (ratiomatic response). The introduction of multiple inputs into the sensing paradigm endows the fluorescent signal with higher information content than single-input systems and could be useful for sophisticated imaging experiments, therapy or molecular computation.30, 31, 39-41 In past decade years, two- and three-input reactive fluorescent probes have constructed by two main strategies: 1) the conver-
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sion of convertional fluorogenic dyes into profluorophores with two and three distinct reaction sites; and 2) the internal construction of a bioluminophore /fluorophore scaffold controlled by the target (bio)analytes. The reactive molecules require the convergence of multiple inputs to yield a fluorescent output (i.e, an AND logic gate).42 Compared to the single analyte sensing, multi-input sensing systems are higher selectivity and sensitivity for the simultaneous detection of several (bio)analytes at the nano-scale and within the sample or biological medium. The majority of inputs in cases reported are H+, metal cations, anions and one biorelevant molecule of inputs.30-38 Among these reports, dualenzyme responsive fluorescent probes have also attracted considerable attention, and only few two-input fluorescent fluorescent probes have been developed for two biorelevant neutral molecules. However, there is no fluorescent probe constructed for the simultaneous detection of RSH and H2S In this work, we have developed a novel fluorescent probe based on quinolone scaffold with two distinct reactive sites (Scheme 1), which can give large fluorescent signal only in response to two important bioanalytes, H2S and RSH, and act as a dual-input “AND” fluorogenic logic gate. More importantly, the probe can detect simultaneously the two molecules in live cells by fluorescence imaging under both one- and two-photon excitations. N3
CO 2Et N
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Scheme 1. The proposed sensing mechanism for the probe QME-N3.
■ EXPERIMENTAL SECTION Instrumentation and Methods. 1H and 13C NMR spectra were recorded on a NMR spectrometer operating at 400 MHz or 300 MHz and 100 MHz, respectively. FT-IR spectra were carried out with an infrared spectrometer. High-resolution mass spectrometry data were obtained with a FTMS spectrometer. UV-Vis absorption spectra and fluorescence spectra were recorded at room temperature on a UV/Vis spectrometer and a sprectrofluorophotometer, respectively. Sample Preparation. Sample solutions (0.01 M pH 7.4 phosphate buffer/EtOH = 3:1) were prepared in quartz cuvette (1 cm × 1 cm). Various 250 mM analytes were prepared in pH 7.4 phosphate buffer, and a 10 L of the analyte was added to the sample solution. All pH values were measured on a pH meter. All measurements were performed at room temperature. Measurement of Two-photon Cross Section (δ) Two-photon excitation fluorescence (TPEF) spectra were measured using femtosecond laser pulse and Ti: sapphire system (680–1080 nm, 80 MHz, 140 fs, Chameleon II) as the light source. All measurements were carried out in air
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at room temperature. Two-photon absorption cross sections were measured using the two-photon-induced fluorescence measurement technique. The two-photon absorption cross sections (δ) were determined by comparing their TPEF to that of fluorescein, according to the following equation: δ = δ ref
Φref cref nref F ΦcnF ref
In the equation, the subscript ref stands for the reference molecule. δ is the two-photon adsorption crosssection value, c is the concentration of solution, n is the refractive index of the solution, F is the TPEF integral intensities of the solution emitted at the exciting wavelength, and Φ is the fluorescence quantum yield. The δref value of reference was taken from the literature.43 Cell Culture and MTT Assay. MCF-7 cells were seeded in DMEM (Dulbecco’s modified Eagle’s medium) supplemented with 10% FBS (fetal bovine serum) in an atmosphere of 5% CO2 and 95% air at 37 °C. Before the experiment, the cells well placed in a 96-well plate, followed by addition of various concentrations of probe. The final concentrations of probe varied from 0 to 50 μM. The cells were then incubated at 37 °C in an atmosphere of 5% CO2 and 95% air for 24 h, after which the culture medium was removed and 100 L of 1 mg mL−1 MTT reagent in PBS was added to each well. Thereafter, it was incubated for 4 h; during this period active mitochondria of viable cells reduce MTT to purple formazan. Unreduced MTT was then discarded and DMSO (100 μL) was added into each well to dissolve the formazan precipitate, which was then measured spectrophotometrically using a microplate reader (Biorad, USA) at 570 nm. The cytotoxic effect of each treatment was expressed as percentage of cell viability relative to the untreated control cells. Cell Imaging Experiments. MCF-7 cells cultured under above condition were employed for cell imaging experiments. The imaging of MCF-7 cells was performed by laser scanning confocal fluorescence microscope (Zeiss LSM 510 Meta NLO). The cells were treated initially with 20 M QME-N3 for 0.5 h. Then, 2 mM Na2S was added to the incubation medium and the cells were incubated for 1 h. Also, the cells were incubated with 200 M Cys for 2h after the addition of QME-N3. The excitation wavelengthes were 405 nm and 700 nm for one-photon and twophoton assays, respectively. Fluorescence signals were collected from the green channel (450–550 nm). ■ RESULTS AND DISCUSSION Synthesis of Related Compounds. The synthesis routes of related compounds are outlined in Scheme 2. The probe, diethyl 2-((6-azidoquinolin-2-yl)methylene) malonate, QME-N3, was prepared with p-nitroaniline as a starting material. First, 2-methyl-6-nitroquinoline (1) and 2-methyl-6-aminoquinoline (2) were synthesized according to the literature.44 Next, in the presence of HCl/NaNO2 and NaN3, compound 2 was converted into 6-
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azido-2-methylquinoline (3), which was oxidized with SeO2 to form the aldehyde 4. Finally, the target product QME-N3 was obtained via a Knoevenagel condensation of 4 with diethyl malonate. As a reference, the azidoreduced product of QME-N3, QME-NH2 was prepared with 2 as a starting material. Except the protection (5,45 645 and 7) and deprotection of the amino group, the synO2N
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thetic procedures were similar to those of QME-N3. In addition, another reference QME-NH2-SPr as the final sensing product was prepared by the Michael addition of QME-NH2 with n-propanethiol. All new compounds were well characterized by 1H NMR, 13C NMR, IR spectra and high resolution mass spectra (HRMS). N3
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Scheme 2. Synthetic routes for QME-N3, QME-NH2 and QME-NH2-SPr. (a) SnCl2∙2H2O, HCl, CH3CH2OH, r.t., 30 min; (b) NaNO2/HCl, NaN3, 0°C, 2 h; (c) SeO2, 1,4-dioxane, 60°C, 6 h; (d) diethyl malonate, piperidine, CH3CH2OH, 50°C, 4 h; (e) (BOC)2O, DMAP, THF, 0~5 °C, 6 h; (f) CF3COOH, CH2Cl2, r. t., overnight. (g) propanethiol, r. t., overnight.
subsequent addition of Na2S lead to a large fluorescence increment. 400
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Spectral Response of QME-N3 to Cys and H2S. In our previous papers,46-48 the α, β-unsaturated carbonyls of 2(quinolin-2-ylmethylene)malonic acids and their esters were the Michael reaction sites as thiol probes. The azido group was employed as the reduction site of H2S fluorescent probes.49-53 When the two reactive sites were combined into one fluorescent quinoline scaffold to construct a two-analyte fluorescent probe QME-N3, we wondered if it could sense specifically the corresponding molecule (RSH/H2S). Hence, measurements involving sensing behavior of the probe were performed in the following addition manners of two analytes (RSH/H2S). Herein, Cys was employed as a representative of RSH. First Cys then H2S. Upon addition of Cys into the solution of QME-N3, the UV/Vis spectra displayed a decrease in absorption in the region near two peaks at 277 nm and 360 nm (Fig. 1a). The fluorescence spectra showed a bit fluorescent increment (2-fold at 455 nm) during 10 min (Fig. 1b). The final spectra were similar to those of compound 3 (Fig. S1 provided as Supporting Information). This implies that the Michael addition of thiols to QMEN3 occurs, and the photophysical property of the resulting product is similar to that of compound 3. Subsequently, Na2S was added into the solution above. The absorption in the region of 350-450 nm increased gradually, and the fluorescence increased dramatically in the region of 400– 600 nm with a peak at 460 nm (22-fold increment) within 40 min (Fig.1b). The final spectra were similar with those of QME-NH2-SPr (Fig. S1), implying the azido group can be reduced by H2S into amino group. Obviously, there was only weak fluorescence response to Cys alone, and
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Figure 1. (a) UV/vis absorption and (b) fluorescence spectra of QME-N3 (20 µM) with Cys (10 equiv.) recorded for 10 min and subsequent addition of Na2S (100 equiv.) for additional 40 min in PBS (pH 7.4) buffered water–ethanol (v/v, 3:1), λex = 320 nm.
First H2S then Cys. The time-dependent spectra of QMEN3 after sequential addition of H2S and Cys were shown in Fig. 2. The absorption spectra underwent a contrary change with the former sequential manner, first increased in the region of 350-460 nm, and then decreased in two regions near two peaks at 277 nm and 360 nm (Figure 2a). However, the fluorescence spectra showed a similar change, small increase in the region of 420-600 nm and then large increase near the fluorescence peak of 460 nm (Figure 2b). Similarly, the solution system of QME-N3 exhibited a slight fluorescence increment after adding the first analyte H2S, and a dramatic increment for subsequent addition of the second analyte Cys.
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Figure 2. (a) UV/vis absorption and (b) fluorescence spectra of QME-N3 (20 µM) in present of Na2S (100 equiv.) for 40 min and subsequently addition of Cys (10 equiv.) for 10 min in PBS (pH 7.4) buffered water–ethanol (v/v, 3:1). λex = 350 nm.
H2S and Cys together. When two analytes RSH and H2S were added into the solution of the probe simultaneously, spectral changes of the reaction solution were shown in Fig. 3. In the absorption spectra, the absorption peaks at 277 nm and 360 nm fast disappeared resulting from the Michael addition of Cys at the double bond. Sequentially, there was a slow increase in the long-band region of 350450 nm, ascribing to the reduction of azido group by H2S. In contrast, fluorescence with a peak of 460 nm enhanced gradually and reached to maximum within 35 min.
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cent probe while no intensive fluorescence response to single analyte, RSH or H2S. Confirmation of the Sensing Mechanism. To confirm the proposed sensing mechanism, the reactions of QME-N3 with Cys or H2S in DMSO-d6 were tracked by means of NMR spectroscopy. After addition of Cys, the vinylic proton (Ha at 7.87 ppm) of QME-N3 disappeared gradually, with the concomitant appearance of new two sets of two pairs of double peaks around 4.54 and 4.80 ppm (Fig. 4), which were assigned to two protons (Ha’, Hb’) of the adduct product QME-N3-Cys, which is a pair of diastereomers, similar to our earlier reports.46-48 The peaks of neat QME-N3 around at 8.5~7.5 ppm shifted to high field upon the addition of Cys. As shown in partial 1H NMR spectra, the reaction of QME-N3 with Cys should undergo a Michael addition with a high yield. After addition of Na2S, a characteristic single peak at 7.87 ppm of the double-bond proton shifted to 7.72 ppm still as a single peak in the 1H NMR spectra, and a newmigrated single peak appeared and two new sets of peaks around at 4~5 ppm (Fig. S4). It is shown that the sensing reaction of QME-N3 with H2S is the reduction of azido to amino rather than a Michael addition at the double bond.
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Figure 3. UV/vis absorption (a) and fluorescence spectra (b) of QME-N3 (20 µM) in the presence of Cys (10 equiv.) and Na2S (100 equiv.) recorded for 35 min in the buffered water– ethanol (v/v, 3:1, pH 7.4). λex = 320 nm.
All three manners show that the Michael addition of Cys is much faster than the reduction of H2S. As shown in Insets of Fig.1-3, the changes in absorption and fluorescence increments clearly exhibit the sensing behavior of QME-N3, which gives the excellent turn-on fluorescence response in the presence of both analytes, but a weak fluorescence response to single analyte, RSH or H2S. Furthermore, compound 3 without the double bond as a reference of a Michael adduct of QME-N3 with a thiol exhibited a specific reduction by H2S, and QME-NH2 as the reduction product of QME-N3 by H2S can react only with thiols via the Michael addition. Upon addition of H2S, compound 3 displayed a large fluorescence increment, 29fold, and no fluorescence change to Cys (Fig. S2). QMENH2 gave a remarkable absorption change and large fluorescence increment to Cys, and no significant change to H2S (Fig. S3). These results further demonstrate that two reactive sites of QME-N3 are specific acceptors for RSH and H2S respectively, and QME-N3 is a two-input fluores-
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Figure 4. Partial H NMR spectra of QME-N3 before (a) and after addition (b, c, d, e) of Cys•HCl recorded for four times in DMSO-d6.
In addition, the high-resolution mass spectra of solution mixtures of the probe QME-N3 with Cys and H2S showed a dominant peak at 484.1270 and 315.1347 in accordance with the adduct ([M+Na+]: 484.1267) and the reduction product (([M+H+]: 315.1347)), respectively (Figure S5). Selectivity. The two reactive sites of QME-N3 are undependent to sense the corresponding target molecule RSH or H2S. Moreover, the selectivity of probe towards RSH/H2S over other relveant analytes was examined by the UV/Vis absorption and fluorescence spectra (Fig. 5). In the presence of thiols, the absorption spectra of QMEN3 exhibited large changes, and didn‘t cause significant change for other analytes including aniline, phenol and other representative amio acids (Ser, Met and Asn) (Fig. 5a). After incubated with 10 equiv. Cys, the solutions of QME-N3 were further incubated with 100 equiv. Na2S or other reducing reagents, sulfite (SO32-), bisulfite (HSO3−),
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thiosulfate (S2O32−), dithionite (S2O42−) and ascorbic acid (Vc) (Fig. 5b). As shown in Figure 5b, only Na2S caused a large fluorescence enhancement while no significant change for other analytes. Therefore, the probe QME-N3 is a two-input fluorescent probe with high selectivity for thiols and H2S.
Pluth.54 The latter has been unambiguously proved by pH titration in our previous papers.46,48 N3
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Figure 5. (a) UV/vis absorption of 20 µM QME-N3 (ethanol/PBS, v/v 1:3, pH 7.4) incubation with 10 equiv. analytes for 10 min. Analytes: 1, blank; 2, n-propylthiol; 3, Cys, 4, Hcy, 5, GSH; 6, Methyl thioglycolate; 7, Na2S; 8, NaHSO3; 9, Na2S2O3, 10, Aniline; 11, Phenol; 12, Serine; 13, Methionine; 14, Asparagine. (b) fluorescence spectra of 20 µM QME-N3 incubated with first 10 equiv. Cys and then 100 equiv. analytes: blank, Na2S and others inculding NaHSO3, Na2SO3, Na2S2O3, Na2S2O4, ascorbic acid, NaCl, NaBr, NaI, NaNO2, NaNO3 and NaHCO3.
In addition, the selectivity could be observed by naked eyes. As shown in Fig. 6, green fluorescence was observed from the solutions of QME-N3 only in the presence of thiols-Na2S (2-4), and no observable fluorescence for the solutions of QME-N3 with other relevant analytes (6, 7, 9, 11, 12) or a single analyte either one biothiol (5) or Na2S (8).
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Scheme 3. Sensing mechanisms of QME-N3 response to thiols and H2S.
For this reason, the sensing behavior of QME-N3 toward a thiol and H2S can act as an AND logic gate. Fig. 7 shows the fluorescence intensities at 460 nm of QME-N3 without and with Cys/H2S, and there are four possible input combinations, no input (0, 0), single Cys input (1, 0), single H2S input (0, 1) and two inputs both Cys and H2S (1, 1). Using the intensity (I) = 50 as the ON/OFF threshold value, their outputs were 0 for the former three inputs, (0, 0), (1, 0), (1, 0).When both Cys and H2S inputs were carried out, the system gave intensive fluorescence as the output signal of 1 (I >>50). The fluorescent increment as the output of 1 was a very large value, more than 7-fold related to the threshold value (50), 10-fold compared to that of a single-analyte input, and much higher than those of the most fluorescent probes as AND logic gates.30, 33-38
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Figure 6. Photograph for the fluorescence change of QMEN3 (20 µM) solutions in the presence of various analytes (0.2 mM thiols, and 2 mM for others) in the solution (ethanol/PBS, v/v 1:3, pH 7.4). 1, blank; 2, Cys + Na2S; 3, Hcy + Na2S, 4, GSH + Na2S; 5, Cys; 6, Cys + Ascorbic Acid; 7, Cys + Na2SO3; 8, Na2S; 9, Na2SO3; 10, Na2S2O3; 11, NaHSO3; 12, Ascorbic Acid.
Therefore, above results show clearly that the sensing reactions of QME-N3 with RSH and H2S undergo a Michael addition at the double bond and reduction of the azido to amino, respectively, and generate a fluorescent QME-NH2-SR (Scheme 3). As the two reactions occur, the system exhibits a large fluorescence increment as an output, and only weak fluorescence response to one single input RSH or H2S. It is emphasized that HS− and RS− are the real reactive species in the reduction of the azido group54 and the Michael addition,46,48 respectively. The sensing mechanism or the former has been demonstrated by Henthorn and
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Figure 7. Relative fluorescence intensities and truth table for QME-N3, a two-input “AND” fluorescent molecular logic gate. Samples containing 20 µM QME-N3 in the buffered EtOHPBS (1:3, v/v, pH 7.4) with or without 10 equiv. Cys/100 equiv. H2S.
As we know, two-photon or near-IR excitation can avoid to several disadvantages such as photobleaching, photodamage and cellular auto-fluorescence from onephoton excitation.55-57 In contrast to QME-N3, QME-NH2 and the adduct QME-NH2-SPr could undergo an ICT process with a 6-amino as a donor when they are excited, thereby, the two-photon excitation could be achieved. For
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this reason, two-photon absorption cross-sections (δ) were obtained by determining the two-photon excitation fluorescence (TPEF) spectra of QME-NH2 solutions before and after treatment with Cys as well as a reference adduct QME-NH2-SPr. As shown in Fig. 8, the two-photon crosssections of QME-NH2 were higher due to its stronger electron acceptor, and a maximum value of 1811 GM at 690 nm. QME-NH2-SPr and the system of QME-NH2 with Cys had similar two-photon cross-sections, 594 GM and 496 GM at 700 nm, respectively. Therefore, QME-N3 could detect biothiols/H2S in living cells by cell fluorescence imaging under two-photon excitation.
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Probe /[µM]
Figure 8. Left: Two-photon excitation fluorescence spectra of QME-NH2 (○), QME-NH2 with Cys (▲) and the adduct QME-NH2-SPr (∆). Right: MTT assay of MCF-7 cells in the presence of different concentrations of the probe QME-N3.
Figure 9. Confocal fluorescence images of MCF-7 cells. (a–c) Cells in control. (d–f) Cells incubated with QME-N3 for 30 min. (g–i) Cells incubated with NEM for 1 h then incubated with QME-N3 for 30 min. (j–l)/ (p–r) Cells incubated with Na2S for another 1h after the incubation of QME-N3. (m–o) Cells incubated with QME-N3 for 30 min then incubated with Cys for 2h. Fluorescence images of one-photon excitation at 405 nm (a, d, g, j, m), and two-photon excitation at 700 nm (p), bright field images (b, e, h, k, n, q), and overlay images of fluorescence images and the corresponding bright field images (c, f, i, l, o, r).
Fluorescence Imaging of Living Cells. The most important advantage of fluorescent probes would be intracellular detection. In order to detect thiols in living MCF-7 cells, a MTT assay is carried out to assess the cytotoxicity of the probe QME-N3. In the MTT assay, MCF-7 cells are dealt with the probe in different concentrations from 10 to 50 µM for 24 h. The results show that the cell viabilities are more than 91% even at concentration of 50 µM. It is shown that the probe has very low toxicity to culture cells under the experimental conditions (Fig. 8 right). Moreover, the probe revealed excellent photostability and thermo-stability. The solutions of the probe with and without Cys/H2S were irradiated by continuous monochromatic light from a fluorophotometer with a xenon lamp (150 W) for 120 min, and didn’t exhibit significant change in absorption and fluorescence spectra (Fig. S6 and S7). Hence, these results showed that the intracellular thiols and H2S could be detected by cell fluorescence imaging. Finally, fluorescence microscopic imaging of intracellular thiols and H2S in MCF-7 cells was performed, shown in Fig. 9. MCF-7 cells were incubated with QME-N3 for 30 min and washed with PBS for three times, then imaged by confocal fluorescence microscopy. As shown in Fig. 9, the
weak fluorescence emission shows that the probe QMEN3 is cell-permeable. As the concentration of intracellular thiols is high, the weak fluorescence emission implies a low concentration of H2S in normal MCF-7 cells. To examine selectivity of the probe for thiols, the control assay of cell imaging with a thiol quencher (N-ethylmaleimide, NEM) was performed. After incubation with 500 μM of NEM for 1 h, the cells were further stained with probe QME-N3 under the same conditions. No fluorescence could be observed from their fluorescence imaging (Fig. 9g-i). When incubated with QME-N3 for 30 min and subsequent incubation with Na2S for another 1 h, the cells emited much bright green fluorescence under excitation at 405 nm (Fig. 9j-l). The cell imaging result was obtained from exogenous H2S. Furthermore, the capability of the probe to detect endogenous H2S was examined through incubating the cells with probe QME-N3 for 0.5 h and then with Cys for 1 h, as H2S can be synthesized from cysteine. Bright green fluorescence was observed from the cells. This implies that cell imaging from endogenous H2S can be achieved with the probe QME-N3. Meanwhile, under two-photon excitation at 700 nm, the cells displayed also bright green fluorescence. These results demonstrate
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that the probe QME-N3 can detect intracellular thiols and H2S by cell fluorescence imaging under one- and twophoton excitations. ■ CONCLUSION In summary, we have developed a two-input fluorescent probe QME-N3 with two distinct reaction sites, which can sense thiols and H2S based on the Michael addition at the double bond and the reduction of azido to amino, respectively. The sensing reactions of two reactive sites are independent and without mutual interference. Thus, the two targeted analytes (RSH/H2S) can work in tandem manner to convert the probe into the luminescent product, with a turn-on fluorescent response. The two-input fluorescent probe can detect RSH/H2S with a high selectivity over single analyte of either RSH or H2S or other relevant analytes. For this reason, it can act as a two-input AND fluorescent logic gate. After two inputs both RSH and H2S react with the probe, the resulting product emits intensively fluorescence as output signal. Furthermore, the probe can detect intracellular thiols and H2S by cell fluorescence imaging under both one- and two-photon excitations. The two-input fluorescent probe can be helpful for the detection of multiple biomolecular events leading to a common disease pathology.
ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website UV/vis absorption and fluorescence spectra of related compounds, spectral response of compound 3 and QME1 NH2 to H2S and Cys, partial H NMR spectra of QME-N3 with H2S, HRMS confirmation of QME-N3 the sensing for Cys and H2S, photostability and thermostability of QMEN3,synthesis and characterization data of related compounds, copies of NMR spectra of related compounds. (PDF)
AUTHOR INFORMATION Corresponding Author * E-mail address:
[email protected]; 63607992; Fax: +86 551 63601592
Tel:
+86
551
Author Contributions †
These authors contributed equally.
Funding Sources National Natural Science Foundation of China (Grant No. 21272224).
ACKNOWLEDGMENT We are grateful for financial support from the National Natural Science Foundation of China (Grant No. 21272224), and the facility support from Prof. Jun Wang's Lab at USTC for cell culture and Prof. Hongping Zhou of Anhui University for measurements of two-photon excitation fluorescence spectra of three compounds.
REFERENCES (1) Sies, H. Glutathione and Its Role in Cellular Functions. Free Radical Biol. Med. 1999, 27, 916-921. (2) Estrela, J. M.; Ortega, A.; Obrador, E. Glutathione in Cancer Biology and Therapy. Crit. Rev. Clin. Lab. Sci. 2006, 43, 143181. (3) Shen, X.; Peter, E. A.; Bir, S.; Wang, R.; Kevil, C. G. Analytical Measurement of Discrete Hydrogen Sulfide Pools in Biological Specimens. Free Radic. Biol. Med. 2012, 52, 2276–2283. (4) Boehning, D.; Snyder, S. H. Novel Neural Modulators. Annu. Rev. Nurosci. 2003, 26, 105-131. (5) Szabó, C. Hydrogen Sulphide and Its Therapeutic Potential. Nat. Rev. Drug Discov. 2007, 6, 917-935. (6) Li, L.; Rose, P.; Moore, P. K. Hydrogen Sulfide and Cell Signaling. Annu. Rev. Pharmacol. Toxicol. 2011, 51, 169-187. (7) Krishnan, N.; Fu, C.; Pappin, D.; Tonks, N. K. H2S-Induced Sulfhydration of PTP1B and Its Role in the Endoplasmic Reticulum Stress Response. Sci. Signal. 2011, 4, ra86. (8) Ishigami, M.; Hiraki, K.; Umemura, K.; Ogasawara, Y.; Ishii, K.; Kimura, H. A Source of Hydrogen Sulfide and a Mechanism of Its Release in the Brain. Antioxid Redox Signal. 2009, 11, 205-214. (9) Benavides, G. A.; Squadrito, G. L.; Mills, R. W.; Patel, H. D.; Isbell, T. S.; Patel, R. P.; Darley-Usmar, V. M.; Doeller, J. E.; Kraus, D. W. Hydrogen Sulfide Mediates the Vasoactivity of Garlic. Proc Natl. Acad. Sci. USA. 2007, 104, 17977-17982. (10) Singh, S.; Banerjee, R. PLP-Dependent H2S Biogenesis. Biochim. Biophys. Acta. 2011, 1814, 1518-1527. (11) Ida, T.; Sawa, T.; Ihara, H.; Tsuchiya, Y.; Watanabe, Y.; Kumagai, Y.; Suematsu, M.; Motohashi, H.; Fujii, S.; Matsunaga, T.; Yamamoto, M.; Ono, K.; Devarie-Baez, N. O.; Xian, M.; Fukuto, J. M.; Akaike, T. Reactive Cysteine Persulfides and SPolythiolation Regulate Oxidative Stress and Redox Signaling. Proc. Natl. Acad. Sci. 2014, 111, 7606-7611. (12) Kamoun, P. Endogenous Production of Hydrogen Sulfide in Mammals. Amino Acids. 2004, 26, 243-254. (13) Kabil, O.; Banerjee, R. Redox Biochemistry of Hydrogen Sulfide. J. Biol. Chem. 2010, 285, 21903-21907. (14) Yang, G.; Wu, L.; Jiang, B.; Yang, W.; Qi, J.; Cao, K.; Meng, Q.; Mustafa, A. K.; Mu, W.; Zhang, S.; Snyder, S. H.; Wang, R. H2S as a Physiologic Vasorelaxant: Hypertension in Mice with Deletion of Cystathionine -Lyase. Science 2008, 322, 587-590. (15) Zhang, S.-Y.; Ong, C.-N.; Shen, H.-M. Critical Roles of Intracellular Thiols and Calcium in Parthenolide-Induced Apoptosis in Human Colorectal Cancer Cells. Cancer Lett. 2004, 208, 143-153. (16) Ball, R. O.; Courtney-Martin, G.; Pencharz, P. B. The in Vivo Sparing of Methionine by Cysteine in Sulfur Amino Acid Requirements in Animal Models and Adult Humans. J. Nutr. 2006, 136, 1682S-1693S. (17) Shahrokhian, S. Lead Phthalocyanine as a Selective Carrier for Preparation of a Cysteine-Selective Electrode. Anal. Chem. 2001, 73, 5972-5978. (18) Refsum, H.; Ueland, P. M.; Nygard, O.; Vollset, S. E. Homocysteine and Cardiovascular Disease. Annu. Rev. Med. 1998, 49, 31-62. (19) Seshadri, S.; Beiser, A.; Selhub, J.; Jacques, P. F.; Rosenberg, I. H.; D'Agostino, R. B.; Wilson, P. W. F.; Wolf, P. A. Plasma Homocysteine as a Risk Factor for Dementia and Alzheimer's Disease. N. Engl. J. Med. 2002, 346, 476-483. (20) van Meurs, J. B. J.; Dhonukshe-Rutten, R. A. M.; Pluijm, S. M. F.; van der Klift, M.; de Jonge, R.; Lindemans, J.; de Groot, L. C. P. G. M.; Hofman, A.; Witteman, J. C. M.; van Leeuwen, J. P. T. M., Breteler, M. M. B.; Lips, P.; Pols, H. A. P.; Uitterlinden, A. G.
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Homocysteine Levels and the Risk of Osteoporotic Fracture. N. Engl. J. Med. 2004, 350, 2033-2041. (21) Wu, G.; Fang, Y.-Z.; Yang, S.; Lupton, J. R.; Turner, N. D. Glutathione Metabolism and Its Implications for Health. J. Nutr. 2004, 134, 489-492; (22) Chen, X.; Zhou, Y.; Peng, X.; Yoon, J. Fluorescent and Colorimetric Probes for Detection of Thiols. Chem. Soc. Rev. 2010, 39, 2120-2135. (23) Jung, H. S.; Chen, X.; Kim, J. S.; Yoon, J. Recent Progress in Luminescent and Colorimetric Chemosensors for Detection of Thiols. Chem. Soc. Rev. 2013, 42, 6019-6031. (24) Yin, C.; Huo, F.; Zhang, J.; Martínez-Máñez, R.; Yang, Y.; Lv, H.; Li, S. Thiol-Addition Reactions and Their Applications in Thiol Recognition. Chem. Soc. Rev. 2013, 42, 6032-6059. (25) Niu, L.-Y.; Chen, Y.-Z.; Zheng, H.-R.; Wu, L.-Z.; Tung, C.H.; Yang, Q.-Z. Design Strategies of Fluorescent Probes for Selective Detection among Biothiols. Chem. Soc. Rev. 2015, 44, 61436160. (26) Lin, V. S.; Chang, C. J. Fluorescent Probes for Sensing and Imaging Biological Hydrogen Sulfide. Curr. Opin. Chem. Biol. 2012, 16, 595-601. (27) Peng, H.; Chen, W.; Burroughs, S.; Wang, B. Recent Advances in Fluorescent Probes for the Detection of Hydrogen Sulfide. Curr. Org. Chem. 2013, 17, 641-653. (28) Yu, F.; Han, X.; Chen, L. Fluorescent Probes for Hydrogen Sulfide Detection and Bioimaging. Chem. Commun. 2014, 50, 12234-12249. (29)Lin, V. S.; Chen, W.; Xian, M.; Chang, C. J. Chemical Probes for Molecular Imaging and Detection of Hydrogen Sulfide and Reactive Sulfur Species in Biological Systems, Chem. Soc. Rev. 2015, 44, 4596-4618. (30) Romieu, A. “AND” Luminescent “Reactive” Molecular Logic Gates: a Gateway to Multi-Analyte Bioimaging and Biosensing. Org. Biomol. Chem. 2015, 13, 1294-1306, and references cited therein. (31) Yu, L.; Wang, S.; Huang, K.; Liu, Z.; Gao, F.; Zeng, W. Fluorescent Probes for Dual and Multi Analyte Detection. Tetrahedron 2015, 71, 4679-4706, and references cited therein. (32) Georgiev, N. I.; Sakra, A. R.; Bojinov, V. B. Design and Synthesis of a Novel PET and ICT Based 1,8-Naphthalimide FRET Bichromophore as a Four-Input Disabled–Enabled-OR Logic Gate. Sens. Actuators, B 2015, 221, 625-634. (33) Balamurugan, A.; Lee, H.-I. Aldoxime-Derived WaterSoluble Polymer for the Multiple Analyte Sensing: Consecutive and Selective Detection of Hg2+, Ag+, ClO−, and Cysteine in Aqueous Media. Macromolecules 2015, 48, 3934-3940. (34) Chen, Y.; Song, Y.; Wu, F.; Liu, W.; Fu, B.; Feng, B.; Zhou X. A DNA Logic Gate Based on Strand Displacement Reaction and Rolling Circle Amplification, Responding to Multiple LowAbundance DNA Fragment Input Signals, and Its Application in Detecting miRNAs. Chem. Commum. 2015, 51, 6980-6983. (35) Debieu, S.; Romieu, A. Dual Enzyme-Responsive “TurnOn” Fluorescence Sensing Systems Based on in Situ Formation of 7-Hydroxy-2-Iminocoumarin Scaffolds. Org. Biomol. Chem. 2015, 13, 10348-10361. (36) Bradberry, S. J.; Byrne, J. P.; McCoy, C. P.; Gunnlaugsson, T. Lanthanide Luminescent Logic Gate Mimics in Soft Matter: [H+] and [F-] Dual-Input Device in a Polymer Gel with Potential for Selective Component Release. Chem. Commun. 2015, 51, 16565-16568. (37) Li, S.-Y.; Liu, L.-H.; Cheng, H.; Li, B.; Qiu, W.-X.; Zhang, X.-Z. A Dual-FRET-Based Fluorescence Probe for the Sequential Detection of MMP-2 and Caspase-3. Chem. Commun. 2015, 51, 14520-14523.
Page 8 of 9
(38) Grimm, J. B.; Gruber, T. D.; Ortiz, G.; Brown, T. A.; Lavis, L. D. Virginia Orange: A Versatile, Red-Shifted Fluorescein Scaffold for Single- and Dual-Input Fluorogenic Probes. Bioconjugate Chem. 2016, 27, 474-480. (39)de Silva, P. A.; Gunaratne, H. Q. N.; Gunnlaugsson, T.; Huxley, A. J. M.; McCoy, C. P.; Rademacher, J. T.; Rice, T. E. Signaling recognition events with fluorescent sensors and switches. Chem. Rev. 1997, 97, 1515−1566. (40) De Silva, A. P.; Uchiyama, S. Molecular Logic and Computing. Nat. Nanotechnol. 2007, 2, 399−410. (41) Wu, J.; Kwon, B.; Liu, W.; Anslyn, E. V.; Wang, P.; Kim, J. S. Chromogenic/Fluorogenic Ensemble Chemosensing Systems. Chem. Rev. 2015, 115, 7893−7943. (42) This isn’t the same concept with dual- or multi-analyte fluorescent probes recently reviewed by Zeng et al, see: Ref. 31. (43) Xu, C.; Webb, W. W. Measurement of Two-Photon Excitation Cross Sections of Molecular Fluorophores with Data from 690 to 1050 nm. J. Opt. Soc. Am. B 1996, 13, 481-491. (44) Saravanan, M.; Satyanarayana, B.; Reddy, P. P. New and Practical Synthesis of Montelukast Sodium, an Antiasthmatic Drug. Syn. Commun. 2013, 43, 2050-2056. (45) Butler, S. J. Ratiometric Detection of Adenosine Triphosphate (ATP) in Water and Real-Time Monitoring of Apyrase Activity with a Tripodal Zinc Complex. Chem.-Eur. J. 2014, 20, 15768-15774. (46) Song, Q.-H.; Wu, Q.-Q.; Liu, C.-H.; Du, X.-J.; Guo, Q.-X. A Novel Fluorescent Probe for Selective Detection of Thiols in Acidic Solutions and Labeling of Acidic Organelles in Live Cells. J. Mater. Chem. B 2013, 1, 438-442. (47) Wu, Q.-Q.; Xiao, Z.-F.; Du, X.-J.; Song Q.-H. A Novel Ratiometric Two-Photon Fluorescent Probe for the Detection of Biothiols in Solution and Imaging of Living Cells. Chem. Asian J. 2013, 8, 2564-2568. (48) Dai, C.-G.; Du, X.-J.; Song, Q.-H. Acid-Activatable Michael-Type Fluorescent Probes for Thiols and for Labeling Lysosomes in Live Cells. J. Org. Chem. 2015, 80, 12088-12099. (49) Lippert, A. R.; New, E. J.; Chang, C. J. Reaction-Based Fluorescent Probes for Selective Imaging of Hydrogen Sulfide in Living Cells. J. Am. Chem. Soc. 2011, 133, 10078-10080. (50) Peng, H.; Cheng, Y.; Dai, C.; King, A. L.; Predemore, B. L.; Lefer. D. J.; Wang, B. A Fluorescent Probe for Fast and Quantitative Detection of Hydrogen Sulfide in Blood. Angew. Chem., Int. Ed. 2011, 50, 9672-9675. (51) Chen, S.; Chen, Z.-J.; Ren, W.; Ai, H.-W. Reaction-Based Genetically Encoded Fluorescent Hydrogen Sulfide Sensors. J. Am. Chem. Soc. 2012, 134, 9589-9592. (52) Bae, S. K.; Heo, C. H.; Choi, D. J.; Sen, D.; Joe, E.-H.; Cho B. R.; Kim, H. M. A Ratiometric Two-Photon Fluorescent Probe Reveals Reduction in Mitochondrial H2S Production in Parkinson’s Disease Gene Knockout Astrocytes. J. Am. Chem. Soc. 2013, 135, 9915-9923. (53) Liu, X.-L.; Du, X.-J.; Dai, C.-G.; Song, Q.-H. Ratiometric Two-Photon Fluorescent Probes for Mitochondrial Hydrogen Sulfide in Living Cells. J. Org. Chem. 2014, 79, 9481-9489. (54) Henthorn, H. A.; Pluth, M. D. Mechanistic Insights into the H2S-Mediated Reduction of Aryl Azides Commonly Used in H2S Detection, J. Am. Chem. Soc. 2015, 137, 15330−15336. (55) Zipfel, W. R.; Williams, R. M.; Webb, W. W. Nonlinear Magic: Multiphoton Microscopy in the Biosciences. Nat. Biotechnol. 2003, 21, 1369-1377. (56) Helmchen, F.; Denk, W. Deep Tissue Two-Photon Microscopy. Nat. Methods 2005, 2, 932-940.
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(57) Kim, H. M.; Cho, B. R. Two-Photon Probes for Intracellular Free Metal Ions, Acidic Vesicles, and Lipid Rafts in Live Tis-
sues. Acc. Chem. Res. 2009, 42, 863-872.
Graphic for manuscript
RSH
Fluor
H2S N3
CO2Et N
CO2Et
RSH, H2S
H2N
CO2Et N
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CO2Et SR
