Accepted Manuscript Title: A ratiometric fluorescent probe for gasotransmitter hydrogen sulfide based on a coumarin-benzopyrylium platform Author: Yu-Wei Duan Xiao-Feng Yang Yaogang Zhong Yuan Guo Zheng Li Hua Lia PII: DOI: Reference:
S0003-2670(15)00002-1 http://dx.doi.org/doi:10.1016/j.aca.2014.12.054 ACA 233660
To appear in:
Analytica Chimica Acta
Received date: Revised date: Accepted date:
15-10-2014 22-12-2014 31-12-2014
Please cite this article as: Yu-Wei Duan, Xiao-Feng Yang, Yaogang Zhong, Yuan Guo, Zheng Li, Hua Lia, A ratiometric fluorescent probe for gasotransmitter hydrogen sulfide based on a coumarin-benzopyrylium platform, Analytica Chimica Acta http://dx.doi.org/10.1016/j.aca.2014.12.054 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
A ratiometric fluorescent probe for gasotransmitter hydrogen sulfide based on a coumarin-benzopyrylium platform Yu-Wei Duan,a Xiao-Feng Yang, *a Yaogang Zhong,b Yuan Guo,*a Zheng Li, b and Hua Lia a
Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of the Ministry of Education, College
of Chemistry and Materials Science, Northwest University, Xi’an 710069, P. R. China ； E-mail: [email protected]; [email protected] College of Life Sciences, Northwest University, Xi'an 710069, China.
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b
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ratiometric
fluorescent
probe
for
has
been
developed
based
on
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coumarin-benzopyrylium platform.
H 2S
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Highlights
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The ratiometric sensing is realized by coupling the azide-based strategy with the
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intramolecular spirolactamization reaction.
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The proposed probe utilizes the acyl azide as the recognition moiety and exhibits a rapid
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response towards H2S (ca. 1 min).
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Abstract
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Preliminary experiments show that the proposed probe has potential to track H2S in live cells.
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A ratiometric fluorescent probe for H2S was developed based on a coumarin- benzopyrylium
platform. The ratiometric sensing is realized by a selective conversion of acyl azide to the corresponding amide, which subsequently undergoes an intramolecular spirocyclization to alter the large π-conjugated system of CB fluorophore. Compared with the traditional azide-based H2S
probes, the proposed probe utilizes the acyl azide as the recognition moiety and exhibits a rapid response (~ 1 min) towards H2S, which is superior to most of the azide-based H2S probes. Preliminary fluorescence imaging experiments show that probe 1 has potential to track H2S in living cells.
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Keywords: Hydrogen sulfide, benzopyrylium, spirocyclization, acyl azide, ratiometric
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1. Introduction
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Hydrogen sulfide (H2S) is an endogenous gaseous transmitter and can be produced from cystathionine-β-synthase, cystathionine-γ-lyase, and cysteine (Cys) amino transferase in
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combination with 3-mercaptosulfurtransferase [1-3]. H2S has also been known to be involved
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in a variety of physiological processes, such as angiogenesis, vasodilation, cardioprotection,
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inflammation and neuromodulation [4-7]. On the other hand, the abnormal H2S level is correlated to diseases such as Alzheimer’s disease, Down’s syndrome, diabetes and liver
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cirrhosis [8-11]. Therefore, in order to understand the physiological and pathological functions
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specimens is crucial.
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of H2S, selective tracking and quantifying of this small molecule within living biological
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Fluorescence imaging through staining with a smaller probe has now been recognized as the most attractive molecular imaging techniques for in vivo monitoring and visualizing of
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biomolecules by virtue of its high sensitivity/selectivity, high spatiotemporal resolution and non-destructive advantage. In recent years, a number of fluorescent probes have been developed for detection and imaging of H2S based on different sensing mechanisms [12-13], including reduction of azido [14-24] and nitro groups [25-26], unique dual nucleophilic reaction [27-34],
high binding affinity with Cu2+ complexes [35-37], thiolysis of dinitrophenyl ether [38-40], as well as specific addition reaction towards unsaturated double bonds [41]. Among these, the strategy based on the conversion of azides to amines is particularly attractive from the selectivity point of view, as azides are widely valued for their inert chemical behavior in biological specimens such as abundant cellular thiols (glutathione and Cys), amino acids, reducing species and extracellular fluids. Unfortunately, most of azide-functionalized probes display a delayed response time [14, 17, 18, 22, 24] -except for dansyl azide [15], and thus are not suitable
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further efforts to innovate the azide-based design strategy are still needed.
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for real-time imaging of H2S due to its rapid metabolism in endogenous systems. In view of these,
2-(7-diethylamino-2-oxo-2H-1-benzopyran-3-yl)-4-(2carboxyphenyl)-7-diethylamino-1-benzop
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yrylium (CB), a hybrid fluorophore of coumarin and benzopyrylium, shows a near-infrared (NIR)
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absorbance and fluorescence in neutral conditions and has been reported as laser dyes previously
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[42]. Recent studies show that CB has the carboxylic-acid-regulated fluorescence switching
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mechanism by intramolecular spirocyclization [43], which is quite similar to those of rhodamine
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derivatives. More significantly, unlike traditional rhodamine derivatives, which are colorless and
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nonfluorescent in the spirocyclic form [44], CB contains a 7-diethylaminecoumarin moiety and
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features coumarin spectral properties even in its spirocyclic form. Therefore, CB has become a
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robust platform for the development of ratiometric fluorescent probes based on altering the π-conjugation of CB derivatives by spirocyclic/ring-open switching mechanism, and fluorescent
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probes for Hg2+ [43] and Cys/Homocysteine (Hcy) have been reported [45].
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Motivated by the above facts, we design and synthesize a new NIR fluorescent probe 1 for the ratiometric sensing of H2S by employing CB as the dye scaffold and acyl azide as the recognition unit. The design rationale is depicted in Scheme 1. We reasoned that HS-, the main stable form of H2S in the physiological condition, can selectively react with the acyl azide of 1 to afford the corresponding amide 4. At the physiological pH, the resulting N atom of the amide group may
further attack the benzopyrylium C-4 atom to undergo an intramolecular spirocyclization to give 5. As a result, the π-conjugation system of 1 is interrupted, thereby leading to the decrease of the NIR emission. On the other hand, since 1 contains a 7-diethylaminecoumarin moiety, it can afford coumarin emission even in its spirolactam form. Therefore, two well-resolved emission peaks before and after adding H2S could be obtained due to the distinct emission between CB and the produced coumarin fluorophore. Based on the above strategy, ratiometric sensing of H2S can be realized. In addition, acyl azide, while not aryl or aliphatic azides, was selected as the recognition
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unit, as its structure is similar to that of sulfonyl azide (both possessing adjacent oxygen atom),
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thus expecting to display a very quick response to H2S.
To test the above-mentioned possibilities, we decided to synthesize and examine the spectra
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profiles of 1. Compound 1 was easily prepared by the reaction of CB with POCl3 followed without 13
C NMR and
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purification by sodium azide, and its structure was characterized by IR, 1H NMR,
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HRMS spectra.
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2. Materials and methods
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2.1 Synthesis of 1
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Compound CB was synthesized according to the reported procedure [45]. A solution of CB
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(0.537 g, 1.0 mmol) in 1,2-dichloroethane (6 mL) was stirred and POCl3 (0.3 mL) was added
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dropwise over 5 min. The solution was refluxed for 4 h. After cooling to room temperature, the reaction mixture was evaporated in vacuo to give CB acid chloride. The crude acid chloride was
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dissolved in CH3CN (12 mL), to which NaN3 (0.35 g) was added and the reaction solution was stirred at 0-5 °C overnight. The reaction mixture was washed with water and dried in vacuum to afford the crude product, which was then purified by silica gel flash chromatography using CH2Cl2/CH3OH (50:1, v/v) as eluent to afford probe 1 as a dark green solid (227.4 mg, 46% yield) (Scheme 2). FI-IR (ATR, cm-1): 2967.62, 2931.30, 2888.92, 2139.12, 1724.72, 1619.77, 1434.16,
1333.17, 1257.34, 1175.14, 1077.21, 990.60, 769.07. 1H NMR (400 MHz, CDCl3): δ (ppm) 9.53 (s, 1H), 8.27 (s, 1H), 8.20-8.16 (m, 2H), 7.78 (t, J = 3.6 Hz, 1H), 7.71-7.65 (m, 2H), 7.36 (d, J = 7.2 Hz, 1H), 7.12 (d, J = 9.2 Hz, 1H), 6.92 (d, J = 9.6 Hz, 1H), 6.78 (dd, J1 = 2.0 Hz, J2 = 9.2 Hz 1H), 6.45 (bs, 1H), 3.73 (q, J = 4 Hz, 4H), 3.53 (q, J = 6.4 Hz, 4H), 1.34 (t, J = 7.2 Hz, 6H), 1.28 (t, J = 6.8 Hz, 6H).
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C NMR (DMSO-d6, 100 MHz): 167.03, 158.03, 157.91, 157.83, 154.79,
154.49, 145.59, 132.89, 130.59, 130.28, 129.49, 129.33, 117.03, 115.18, 111.83, 111.07, 109.50, 105.27, 96.32, 96.08, 45.43, 45.07, 12.57. ESI-HRMS: [1]+ m/z 562.2482, calcd for C33H32N5O4+
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562.2449.
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2.2 Materials and Instruments
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All reagents and solvents were purchased from commercial sources and were of the highest grade. Solvents were purified and dried by standard methods prior to use. Flash chromatography
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was performed using silica gel (200-300 mesh) obtained from the Qingdao Ocean Chemicals.
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Double-distilled water was used throughout the experiments. The stock solution of probe 1 (1.0
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mM) were prepared in CH2Cl2 and were stored at refrigerator (4 oC) [46]. Solutions of various species (2.0 mM) were prepared from NaCl, KBr, KI, KSCN, NaNO3, NaNO2, NaOAc, NaClO,
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Na2CO3, NaHCO3, Na2S2O3·5H2O, Na2SO3, NaHSO3, glutathione, cysteine, homocysteine, ascorbic acid and NaHS.
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The fluorescence spectra and relative fluorescence intensity were measured with a Shimadzu
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RF-5301 spectrofluorimeter with a 10 mm quartz cuvette. Absorption spectra were recorded using a Shimadzu UV-2550 spectrophometer. IR spectra were recorded on a Bruker Vertex 70 FT-IR
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spectrometer using a diamond ATR attachment. High-resolution mass spectra were collected using a Bruker micrOTOF-Q II mass spectrometer (Bruker Daltonics Corp, USA) in electrospray ionization (ESI) mode. 1H and
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C NMR spectra were recorded on an INOVA-400 spectrometer
(Varian Unity), using tetramethylsilane (TMS) as the internal standard. The pH measurements were carried out on a Sartorius PB-10 pH meter.
2.3 Cell cultures and fluorescence imaging Human hepatic stellate cells were obtained from Cell Engineering Research Centre and Department of Cell Biology, Fourth Military Medical University (Xi’an, China). 2×105 human hepatic stellate cells were seeded in 6-well culture plates containing sterile cover slips and were cultured in RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum (FBS), penicillin (100 U mL-1), and streptomycin (100 µg mL-1) at 37 °C in a 95% humidity atmosphere under 5%
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CO2 for two days. Before imaging, the cells were washed with PBS three times and then incubated with probe 1 (10.0 µM) in a DMSO/PBS (5: 95, v/v) solution at 37 °C for 30 min. Then, the
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samples were rinsed with PBS three times to remove the remaining probe and observed under an
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Olympus BX 61 fluorescence microscope. Experiments to assess H2S uptake were performed in the same medium supplemented with NaHS (200 µM) for 20 min at 37 oC. The fluorescence
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images were acquired after the cells had been rinsed with PBS.
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2.4 General procedure for the spectral measurements
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Test solutions were prepared by placing 100 µL of probe 1 (1.0 mM), 3.0 mL of acetone, 1.0
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mL of phosphate buffer (10 mM, pH 7.4), and appropriate aliquot of each analyte stock solution
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into a 10 mL volumetric flask, and diluting the solution to 10 mL with water. The resulting
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solution was kept at room temperature (25 °C) for 2 min, and then the absorption or fluorescence spectra were recorded. The fluorescence ratio of I478/I702 was measured with the excitation and
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emission wavelengths at 410 nm and 478/702 nm.
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2.5 Cytotoxicity assays Human hepatic stellate cells were seeded in 96-well plates and incubated with different
concentrations of probe 1 (0-20 µM) (n = 5) in an atmosphere of 5% CO2 and 95% air at 37 °C for 6 h. Then, 20 µL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 5.0 mg mL-1) was added to each well, followed by further incubation at 37 °C for 4 h. After that, 100 µL
of the supernatant was removed, and 150 µL of DMSO was added to each well to dissolve the reddish-blue crystals. The plate was shaken for 10 min, and then the absorbance was measured at 570 nm with a microplate reader (ELX 800 UV, BIO-TEK Instruments Inc.). The relative cell viability (VR) was calculated using the following equation: VR = A/A0 × 100%, where A and A0 are the absorbance of the experimental group and control group, respectively. 2.6 Theoretical Methods
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Density functional theory calculations at the B3LYP/6-311++G** level of Gaussian 09 program were carried out to optimize geometries for all of the species studied here. In the DFT
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calculations, Ph-N(H)N2S and Ph-C(O)N(H)N2S were used to model the real reactions. With a
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continuum medium, we have calculated the single point solvation energy for all the species, using the conductor polarizable continuum model (CPCM). In the CPCM calculations, H2O was used as
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the solvent, corresponding to the experimental conditions.
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3. Results and discussion
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3.1 Absorption and fluorescent spectra of probe 1
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With compounds 1 in hand, we studied its spectral properties in CH2Cl2 and observed that its absorption and fluorescence emission maximum display bathochromic shift when compared to CB
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(from 658 to 668 nm in absorption spectra and from 692 to 703 nm in emission spectra) (Fig. S1,
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Supplemental Material). More significantly, unlike the common azide-based fluorescent probes, which are generally weakly fluorescent due to a favorable electron transfer from the electron-rich
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α-nitrogen of the azide group to the excited fluorophore [47], the fluorescence quantum yield of 1 is calculated to be 0.56 in CH2Cl2, which is slightly lower than that of CB (φF = 0.84). This can be explained by the fact that almost all NIR fluorescence dyes have relatively high-lying highest occupied molecular orbital (HOMO) energy levels, and thus intramolecular photo-induced electron fluorescence quenching is difficult to occur within these dyes [48].
3.2 Sensing response of probe 1 to H2S The free probe 1 is stable at room temperature (25 oC) and only displays the characteristic peaks of CB dye at around 660 nm. With increasing NaHS concentration (0-30 equiv), the absorption peak at 660 nm decreased, while the peak at 423 nm increased accordingly (Fig. 1a). In addition, such a large blue-shift of 237 nm in the absorption behavior changes the color of the resultant solution from blue into pink, allowing “naked-eye” detection (Fig. 1a, inset). Furthermore,
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fluorescence titration studies of 1 towards H2S were examined under the same conditions. Upon excitation at 410 nm, the free probe 1 exhibits two well-resolved emission peaks centered at 478
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and 702 nm, which can be assigned as the emission band of coumarin and CB, respectively.
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However, fluorescence titration of the solution of 1 with NaHS (0-30 equiv) induces a marked decrease of emission band at 702 nm and concurrently a dramatic enhancement of the band at 478
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nm (Fig. 1b). Such a large hypochromatic shift of 224 nm makes this probe favorable for the dual
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emission ratiometric sensing owing to the minimum overlap between the two emission bands. The
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fluorescence intensity ratios at 478 nm and 702 nm (I478/I702) increase from 0.39 to 19.46 upon NaHS treatment, and the final enhancement factor is about 50-fold. In addition, the intensity ratios
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(I478/I702) were plotted as a function of HS- concentration and a typical calibration graph was obtained (Fig. S2, Supplemental Material). The I478/I702 value is linearly related to NaHS
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concentration in the range of 2 to 100 µM with a detection limit of 0.22 µM (3δ). This result
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suggests that the present probe is favourable for direct imaging of intracellular H2S whose concentrations are usually in a micromolar range [35]. As indicated in Table S1
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(Supplemental Material), probe 1 shows excellent analytical performance compared to the most previously reported azide-based fluorescent probes for H2S. According to the well established reaction mechanism, the spectra changes in the sensing process could be assigned to the formation of compound 5, which was also verified by mass spectrometry analysis of the mixture of probe 1 and NaHS, and two predominant peaks at m/z
536.2554 and 558.2372, corresponding to [5 + H]+ (calcd 536.2549 for C33H34N3O4+) and [5 + Na]+ (calcd 558.2369 for C33H33N3O4Na+) were shown in the HRMS data (Fig. S3, Supplemental Material). In addition, the absorption and emission spectra of compound 5 are highly similar to those of coumarin 7 (Fig. S4, Supplemental Material). This is in accordance with the fact that 5 and 7 have almost the same coumarin fluorophore, which further proves the disruption of the π-electron conjugation in probe 1 at the bezopyrylium moiety. Collectively, these results provide strong support for the sensing mechanism outlined in Scheme 1.
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3.3 Kinetic studies
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The time course of the emission ratio (I478/I702) of probe 1 (10 µM) in the absence and presence
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of HS- (20 equiv) was studied, and the results are shown in Fig. 2 and Fig. S5 (Supplemental Material). Notably, probe 1 displayed a rapid response to HS- and the sensing reaction almost
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terminated within 1 min. The observed rate constant was then determined to be 0.015 ± 0.0005 s-1
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under the pseudo-first-order conditions (Fig. S6, Supplemental Material). In fact, probe 1 is able to
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detect H2S more rapidly than most of the reported azide-based probes, and comparable to that of arylsulfonyl azides. This favors the real-time intracellular imaging, when considering the variable
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nature and quick metabolism of endogenous H2S in biological systems. Despite the similar structures of acyl and aryl azides, acylazide-based fluorescent probes showed
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better kinetic feature than those of aryl ones. We envisioned that the reaction of HS- with 1
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proceeds by the nucleophilic attack of HS- at the terminal azide nitrogen of 1 to afford a free SH containing intermediate 2 [49], which subsequently transforms to thiolate intermediate 3, and 3 is
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readily decomposed to give amide 4 and dinitrogen sulfide (N2S) [50-51]. In the case of aryl azides, the analogous reaction can occur to afford the corresponding amine (Scheme S1, Supplemental Material). To elucidate the significant difference in reaction kinetics of above azide-based probes, benzoyl and phenyl azides were used to model the real reactions. The energy profiles for the pathway of
leading to dissociation of the thiolate intermediates (Ph-N(H)N2S- and Ph-C(O)N(H)N2S-) were calculated using density functional theory (DFT) in combination with solvent effect within the Gaussian 09 program [52]. The energy profiles of the two reactions were illustrated in Fig. 3. The calculated results showed that the free energy barriers for the dissociation of Ph-C(O)N(H)N2S- to produce the corresponding amide was found to be 9.3 kcal/mol, significantly lower than that found for Ph-N(H)N2S- (16.6 kcal/mol). The above results indicate that the decomposition of Ph-N(H)N2S- is much slower and less efficient than that of Ph-C(O)N(H)N2S-, which accounts for
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the significant differences in reaction rates for acyl and aryl azides upon treating with H2S.
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3.4 Selectivity studies
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To assess the selectivity of probe 1 towards H2S, changes in the fluorescence spectra 1 caused by various species, including common anions (HS-, F-, Cl-, Br-, I-, SCN-, NO3-, NO2-, AcO-, ClO-,
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SO42-, S2O42-, SO32-, S2O32-, HCO3-, biological thiols (GSH, Cys, Hcy) and ascorbic acid were
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measured (Fig. 4 and Fig. S7, Supplemental Material). As can be seen from Fig. 4 and Table S2
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(Supplemental Material), only HS- induces a dramatic increment of the emission ratio (I478/I702), while other species, especially cellular sulfhydryl-containing compounds (GSH, Hcy and Cys)
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virtually caused negligible changes under the same conditions. Furthermore, probe 1 can still retain its sensing response to H2S even in the presence of a large amount of biothiols (GSH, Cys and
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Hcy) (Fig. S8, Supplemental material), which further confirms the high selectivity of probe 1
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towards H2S over other cellular sulfhydryl-containing compounds. Finally, the high selectivity of probe 1 towards H2S is readily detectable visually, and only NaHS gives rise to an obvious color
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change from dark green to light yellow (Fig. S9, Supplemental Material). In fact, the selectivity observed for H2S over other biological relevant species is remarkably
high. Based on the above facts, we speculated that the reaction between azide and HS- is not simply a redox process, since other reducing reagents such as ascorbic acid, SO32- and S2O32induce almost no reaction under identical conditions, even their concentrations are much higher
than that of HS-. This suggests that the nucleophilic addition of HS- to the azide moiety of probe 1 indeed occurs as depicted in Scheme 1, which further explains why common reducing compounds elicit almost no response to probe 1 under identical conditions. In the case of cellular biothiols, such as GSH, Cys and Hcy, although their thiol groups are nucleophilic, they possess higher pKa values (ca. 8.5) than that of H2S (ca. 6.9) [53, 54]. Thus, based on the marked distinctions in terms of size and pKa values, the nucleophilic attack of azide may be chemoselective for H2S over these biothiols in aqueous media at physiological pH. In addition, the negligible reactivity of biothiols,
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which cannot form intermediate 3 with a thiolate group, provides additional support for the
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mechanism shown in Scheme 1.
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3.5 Bioimaging and cytotoxicity studies
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The capability of probe 1 to sense H2S in live cells was evaluated. Human hepatic stellate cells
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stained with probe 1 displayed a strong emission in the red channel (Fig. 5c) and very dim
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fluorescence in the blue emission (Fig. 5b). However, when the cells were pretreated with probe 1
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(10 µM), and further incubated NaHS (200 µM) for 20 min, a decrease in the red emission (Fig. 5f)
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and a dramatic enhancement in the blue emission (Fig. 5e) were observed simultaneously. The
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above image variations are in accordance with the proposed mechanism shown in Scheme 1. In addition, the MTT assays demonstrate that probe 1 at a concentration of 15 µM shows minimal
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cytotoxicity to the cells (Fig. S10, Supplemental Material), which are comparable to the previous
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reported H2S probes [55]. The above results demonstrate that 1 is suitable for ratiometric
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fluorescence imaging of H2S in live cells.
4. Conclusions
In summary, we have developed a new ratiometric fluorescent probe for H2S by a coumarin-benzopyrylium platform. By combining the selective acyl azide/amide conversion with
intramolecular spirocyclization reaction, the proposed probe affords a high sensitivity and ratiometric response toward H2S. Compared with the traditional azide-based H2S probes, the proposed probe utilizes the acyl azide as the recognition unit and shows a quick response towards H2S (ca. 1 min), thus they favour to use the real-time intracellular H2S imaging. Preliminary fluorescence imaging experiments showed that probe 1 has potential to track H2S in living human
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hepatic stellate cells. The superior properties of 1 make it of great potential use in other biological
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systems.
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Acknowledgments
This research was supported by the Natural Science Foundation of China (No. 21275117,
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21475105, 21175106), the Science & Technology Department (No. 2012JM2004) and the
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Education Department (No. 12JK0518) of Shaanxi Province of China.
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[22] C.S. Lim, S.K. Das, S.Y. Yang, E.S. Kim, H.J. Chun, S.R. Cho, Quantitative estimation of the total sulfide concentration in live tissues by two-photon microscopy, Anal. Chem. 85 (2013) 9288-9295. [23] J.Y. Zhang, W. Guo, A new fluorescent probe for gasotransmitter H2S: high sensitivity, excellent selectivity, and a significant fluorescence off-on response, Chem. Commun. 50 (2014) 4214-4217. [24] G.J. Mao, T.T. Wei, X.X. Wang, S.Y. Huan, D.Q. Lu, J. Zhang, X.B. Zhang, W.H. Tan, G.L.
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Chem. Sci. 3 (2012) 2920-2923.
[30] Z. Xu, L. Xu, J. Zhou, Y.F. Xu, W.P. Zhu, X.H. Qian, A highly selective fluorescent probe for fast detection of hydrogen sulfide in aqueous solution and living cells, Chem. Commun. 48 (2012) 10871-10873.
[31] C.R. Liu, B. Peng, S. Li, C.M. Park, A.R. Whorton, M. Xian, Reaction based fluorescent probes for hydrogen sulphide, Org. Lett. 14 (2012) 2184-2187. [32] J.Y. Zhang, Y.Q. Sun, J. Liu, Y.W. Shi, W. Guo, A fluorescent probe for the biological signaling molecule H2S based on a specific H2S trap group, Chem. Commun. 49 (2013) 11305-11307. [33] X. Wang, J. Sun, W.H. Zhang, X.X. Ma, J.Z. Lv, B. Tang, A near-infrared ratiomateric fluorescent probe for rapid and highly sensitive imaging of endogenous hydrogen sulfide in
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living cells, Chem. Sci. 4 (2013) 2551-2556.
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sulfide and imaging in living cells, Inorg. Chem. 51 (2012) 2454-2460.
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[37] M.Q. Wang, K. Li, J.T. Hou, M.Y. Wu, Z. Huang, Z.Q. Yu, BINOL-based fluorescent sensor for recognition of Cu(II) and sulfide anion in water, J. Org. Chem. 77 (2012) 8350-8354.
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[38] X.W. Cao, W.Y. Lin, K.B. Zheng, L.W. He, A near-infrared fluorescent turn-on probe for
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[39] J.L. Wang, W.Y. Lin, W.L. Li, Three-channel fluorescent sensing via organic white light-emitting dyes for detection of hydrogen sulfide in living cells, Biomaterials 34 (2013) 7429-7436.
[40] T. Liu, Z. Xu, D.R. Spring, J. Cui, A lysosome-targetable fluorescent probe for imaging hydrogen sulfide in living cells, Org. Lett. 15 (2013) 2310-2313. [41] Y. Chen, C. Zhu, Z. Yang, J. Chen, Y. He, Y. Jiao, W. He, L. Qiu, J. Cen, Z. Guo, A Ratiometric fluorescent probe for rapid detection of hydrogen sulfide in mitochondria, Angew. Chem. Int. Ed. 52 (2013) 1688-1691. [42] P. Czerney, G. Graneβ, E. Birckner, F. Vollmer, W. Rettig, Molecular engineering of cyanine-type fluorescent and laser dyes, J. Photochem. Photobiol. A: Chem. 89 (1995) 31-36.
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[43] J. Liu, Y.Q. Sun, P. Wang, J.Y. Zhang, W. Guo, Construction of NIR and ratiometric
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spiroring-opening of xanthenes and related derivatives, Chem. Rev. 112 (2012) 1910-1956.
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[45] H.M. Lv, X.F. Yang, Y.G. Zhong, Y. Guo, Z. Li, H. Li, Native Chemical ligation combined
A
with spirocyclization of benzopyrylium dyes for the ratiometric and selective fluorescence
M
detection of cysteine and homocysteine, Anal. Chem. 86 (2014) 1800-1807.
TE
D
[46] The solution of probe 1 showed no significant changes at least for 7 days when it was stored at refrigerator (4 oC). However, a Curtius rearrangement reaction would occur at higher
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temperatures, leading probe 1 to its corresponding isocyanate.
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[47] F. Xie, K. Sivakumar, Q.B. Zeng, M.A. Bruckman, B. Hodges, Q. Wang, A fluorogenic ‘click’ reaction of azidoanthracene derivatives, Tetrahedron 64 (2008) 2906-2914.
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[48] K. Kiyose, S. Aizawa, E. Sasaki, H. Kojima, K. Hanaoka, T. Terai, Y. Urano, T. Nagano, Molecular design strategies for near-infrared ratiometric fluorescent probes based on the unique spectral properties of aminocyanines, Chem. Eur. J. 15 (2009) 9191-9200.
[49] C.S. Lim, S.K. Das, S.Y. Yang, E.S. Kim, H.J. Chun, B.R. Cho, Quantitative estimation of the total sulfide concentration in live tissues by two-photon microscopy, Anal. Chem. 85 (2013) 9288-9295. [50] J.L. Norcliffe, L.P. Conway, D.R.W. Hodgson, Reduction of alkyl and aryl azides with sodium thiophosphate in aqueous solutions, Tetrahedron Lett. 52 (2011) 2730-2732. [51] R.V. Kolakowski, N. Shangguan, R.R. Sauers, L.J. Williams, Mechanism of thio acid/azide amidation, J. Am. Chem. Soc. 128 (2006) 5695-5702.
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[52] M.J. Frisch, et al. Gaussian 09, Revision C.01 (2009); Gaussian, Inc., Wallingford CT.
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[53] M.P. Lutolf, N. Tirelli, S. Cerritelli, L. Cavalli, J.A. Hubbell, Systematic modulation of michael-type reactivity of thiols through the use of charged amino acids, Bioconjugate Chem.
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12 (2001) 1051-1056.
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[54] C.N. Salinas, K.S. Anseth, Mixed mode thiol-acrylate photopolymerizations for the synthesis
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of PEG-peptide hydrogels, Macromolecules 41 (2008) 6019-6026.
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[55] J. Liu, Y.-Q. Sun, J. Zhang, T. Yang, J. Cao, L. Zhang, W.Guo, A ratiometric fluorescent
M
probe for biological signaling molecule H2S: fast eesponse and high selectivity, Chem. Eur. J.
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EP
TE
D
19 (2013) 4717-4722.
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Figure Captions Scheme 1. The proposed sensing mechanism of probe 1 for H2S. Scheme 2. Synthesis of probe 1. Fig. 1 Absorption (a) and fluorescence (b, λex = 410 nm) spectra of probe 1 (10 µM) upon addition of increasing concentrations of NaHS (0-300 µM) in acetone/phosphate buffer (3:7 v/v, 10 mM, pH 7.4). Inset: (a) color changes of 1 (10 µM) upon addition of NaHS (10 equiv); (b) fluorescence
intensity ratio (I478/I702) changes of probe 1 (10 µM) with the amount of NaHS. Each spectrum was recorded after incubation of the probe with NaHS for 2 min at room temperature (25 °C). Fig. 2 Time-dependent changes the emission ratio (I478/I702) of probe 1 (10 µM) in the presence of NaHS (20 equiv) in acetone/phosphate buffer (3:7 v/v, 10 mM, pH 7.4). Fig. 3 The potential energy profile calculated for the dissociation of Ph-N(H)N2S- (a) and Ph-C(O)N(H)N2S- (b). The calculated relative free energies are given in kcal/mol. Fig. 4 The emission ratio (I478/I702) of probe 1 (10 µM) in the presence of various species in
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acetone/phosphate buffer (3:7 v/v, 10 mM, pH 7.4) for 2 min. a) Blank, b) HS-, c) F-, d) Cl-, e) Br-,
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f) I-, g) SCN-, h) NO3-, i) NO2-, j) AcO-, k) ClO-, l) SO42-, m) S2O42-, n) SO32-, o) S2O32-, p) HCO3(20 equiv of each), q) Cys (5 mM), r) GSH (5 mM), s) Hcy (5 mM), t) ascorbic acid (10 mM).
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Fig. 5 Fluorescence images of exogenous H2S in human hepatic stellate cells with probe 1. (a)
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Bright field image of cells incubated with 1 (10 µM) for 30 min; (b) fluorescence image of (a)
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from blue channel; (c) fluorescence image of (a) from red channel. (d) Bright field image of cells
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incubated with probe 1 (10 µM) for 30 min and further incubation with NaHS (200 µM) for 20
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min; (e) fluorescence image of (d) from blue channel; (f) fluorescence image of (d) from red
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channel.
O N N N
NH
O N
H2S N
O O
O
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5
1
Spirolactam form, λem = 478 nm
Open form, λem = 702 nm
H+
HS-
OH
-
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Scheme 1
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Reflux
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POCl3
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NaN3 o
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Scheme 2
O
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0.7
a) Absorbance
H2S
Blank
0.6 0.5 0.4 0.3 0.2
360
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12.5
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Fluorescence intensity (a.u.)
Wavelength / nm
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Wavelength / nm Fig. 1
700
750
25 1 only 1 + NaHS
I478/I702
20
15
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0 30
60
90
120
180
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graphic abstract .
S0003-2670(15)00002-1 http://dx.doi.org/doi:10.1016/j.aca.2014.12.054 ACA 233660
To appear in:
Analytica Chimica Acta
Received date: Revised date: Accepted date:
15-10-2014 22-12-2014 31-12-2014
Please cite this article as: Yu-Wei Duan, Xiao-Feng Yang, Yaogang Zhong, Yuan Guo, Zheng Li, Hua Lia, A ratiometric fluorescent probe for gasotransmitter hydrogen sulfide based on a coumarin-benzopyrylium platform, Analytica Chimica Acta http://dx.doi.org/10.1016/j.aca.2014.12.054 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
A ratiometric fluorescent probe for gasotransmitter hydrogen sulfide based on a coumarin-benzopyrylium platform Yu-Wei Duan,a Xiao-Feng Yang, *a Yaogang Zhong,b Yuan Guo,*a Zheng Li, b and Hua Lia a
Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of the Ministry of Education, College
of Chemistry and Materials Science, Northwest University, Xi’an 710069, P. R. China ； E-mail: [email protected]; [email protected] College of Life Sciences, Northwest University, Xi'an 710069, China.
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b
A
ratiometric
fluorescent
probe
for
has
been
developed
based
on
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coumarin-benzopyrylium platform.
H 2S
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Highlights
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The ratiometric sensing is realized by coupling the azide-based strategy with the
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intramolecular spirolactamization reaction.
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The proposed probe utilizes the acyl azide as the recognition moiety and exhibits a rapid
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response towards H2S (ca. 1 min).
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Abstract
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Preliminary experiments show that the proposed probe has potential to track H2S in live cells.
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A ratiometric fluorescent probe for H2S was developed based on a coumarin- benzopyrylium
platform. The ratiometric sensing is realized by a selective conversion of acyl azide to the corresponding amide, which subsequently undergoes an intramolecular spirocyclization to alter the large π-conjugated system of CB fluorophore. Compared with the traditional azide-based H2S
probes, the proposed probe utilizes the acyl azide as the recognition moiety and exhibits a rapid response (~ 1 min) towards H2S, which is superior to most of the azide-based H2S probes. Preliminary fluorescence imaging experiments show that probe 1 has potential to track H2S in living cells.
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Keywords: Hydrogen sulfide, benzopyrylium, spirocyclization, acyl azide, ratiometric
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1. Introduction
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Hydrogen sulfide (H2S) is an endogenous gaseous transmitter and can be produced from cystathionine-β-synthase, cystathionine-γ-lyase, and cysteine (Cys) amino transferase in
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combination with 3-mercaptosulfurtransferase [1-3]. H2S has also been known to be involved
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in a variety of physiological processes, such as angiogenesis, vasodilation, cardioprotection,
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inflammation and neuromodulation [4-7]. On the other hand, the abnormal H2S level is correlated to diseases such as Alzheimer’s disease, Down’s syndrome, diabetes and liver
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cirrhosis [8-11]. Therefore, in order to understand the physiological and pathological functions
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specimens is crucial.
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of H2S, selective tracking and quantifying of this small molecule within living biological
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Fluorescence imaging through staining with a smaller probe has now been recognized as the most attractive molecular imaging techniques for in vivo monitoring and visualizing of
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biomolecules by virtue of its high sensitivity/selectivity, high spatiotemporal resolution and non-destructive advantage. In recent years, a number of fluorescent probes have been developed for detection and imaging of H2S based on different sensing mechanisms [12-13], including reduction of azido [14-24] and nitro groups [25-26], unique dual nucleophilic reaction [27-34],
high binding affinity with Cu2+ complexes [35-37], thiolysis of dinitrophenyl ether [38-40], as well as specific addition reaction towards unsaturated double bonds [41]. Among these, the strategy based on the conversion of azides to amines is particularly attractive from the selectivity point of view, as azides are widely valued for their inert chemical behavior in biological specimens such as abundant cellular thiols (glutathione and Cys), amino acids, reducing species and extracellular fluids. Unfortunately, most of azide-functionalized probes display a delayed response time [14, 17, 18, 22, 24] -except for dansyl azide [15], and thus are not suitable
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further efforts to innovate the azide-based design strategy are still needed.
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for real-time imaging of H2S due to its rapid metabolism in endogenous systems. In view of these,
2-(7-diethylamino-2-oxo-2H-1-benzopyran-3-yl)-4-(2carboxyphenyl)-7-diethylamino-1-benzop
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yrylium (CB), a hybrid fluorophore of coumarin and benzopyrylium, shows a near-infrared (NIR)
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absorbance and fluorescence in neutral conditions and has been reported as laser dyes previously
N
[42]. Recent studies show that CB has the carboxylic-acid-regulated fluorescence switching
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mechanism by intramolecular spirocyclization [43], which is quite similar to those of rhodamine
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derivatives. More significantly, unlike traditional rhodamine derivatives, which are colorless and
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nonfluorescent in the spirocyclic form [44], CB contains a 7-diethylaminecoumarin moiety and
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features coumarin spectral properties even in its spirocyclic form. Therefore, CB has become a
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robust platform for the development of ratiometric fluorescent probes based on altering the π-conjugation of CB derivatives by spirocyclic/ring-open switching mechanism, and fluorescent
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probes for Hg2+ [43] and Cys/Homocysteine (Hcy) have been reported [45].
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Motivated by the above facts, we design and synthesize a new NIR fluorescent probe 1 for the ratiometric sensing of H2S by employing CB as the dye scaffold and acyl azide as the recognition unit. The design rationale is depicted in Scheme 1. We reasoned that HS-, the main stable form of H2S in the physiological condition, can selectively react with the acyl azide of 1 to afford the corresponding amide 4. At the physiological pH, the resulting N atom of the amide group may
further attack the benzopyrylium C-4 atom to undergo an intramolecular spirocyclization to give 5. As a result, the π-conjugation system of 1 is interrupted, thereby leading to the decrease of the NIR emission. On the other hand, since 1 contains a 7-diethylaminecoumarin moiety, it can afford coumarin emission even in its spirolactam form. Therefore, two well-resolved emission peaks before and after adding H2S could be obtained due to the distinct emission between CB and the produced coumarin fluorophore. Based on the above strategy, ratiometric sensing of H2S can be realized. In addition, acyl azide, while not aryl or aliphatic azides, was selected as the recognition
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unit, as its structure is similar to that of sulfonyl azide (both possessing adjacent oxygen atom),
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thus expecting to display a very quick response to H2S.
To test the above-mentioned possibilities, we decided to synthesize and examine the spectra
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profiles of 1. Compound 1 was easily prepared by the reaction of CB with POCl3 followed without 13
C NMR and
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purification by sodium azide, and its structure was characterized by IR, 1H NMR,
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HRMS spectra.
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2. Materials and methods
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2.1 Synthesis of 1
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Compound CB was synthesized according to the reported procedure [45]. A solution of CB
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(0.537 g, 1.0 mmol) in 1,2-dichloroethane (6 mL) was stirred and POCl3 (0.3 mL) was added
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dropwise over 5 min. The solution was refluxed for 4 h. After cooling to room temperature, the reaction mixture was evaporated in vacuo to give CB acid chloride. The crude acid chloride was
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dissolved in CH3CN (12 mL), to which NaN3 (0.35 g) was added and the reaction solution was stirred at 0-5 °C overnight. The reaction mixture was washed with water and dried in vacuum to afford the crude product, which was then purified by silica gel flash chromatography using CH2Cl2/CH3OH (50:1, v/v) as eluent to afford probe 1 as a dark green solid (227.4 mg, 46% yield) (Scheme 2). FI-IR (ATR, cm-1): 2967.62, 2931.30, 2888.92, 2139.12, 1724.72, 1619.77, 1434.16,
1333.17, 1257.34, 1175.14, 1077.21, 990.60, 769.07. 1H NMR (400 MHz, CDCl3): δ (ppm) 9.53 (s, 1H), 8.27 (s, 1H), 8.20-8.16 (m, 2H), 7.78 (t, J = 3.6 Hz, 1H), 7.71-7.65 (m, 2H), 7.36 (d, J = 7.2 Hz, 1H), 7.12 (d, J = 9.2 Hz, 1H), 6.92 (d, J = 9.6 Hz, 1H), 6.78 (dd, J1 = 2.0 Hz, J2 = 9.2 Hz 1H), 6.45 (bs, 1H), 3.73 (q, J = 4 Hz, 4H), 3.53 (q, J = 6.4 Hz, 4H), 1.34 (t, J = 7.2 Hz, 6H), 1.28 (t, J = 6.8 Hz, 6H).
13
C NMR (DMSO-d6, 100 MHz): 167.03, 158.03, 157.91, 157.83, 154.79,
154.49, 145.59, 132.89, 130.59, 130.28, 129.49, 129.33, 117.03, 115.18, 111.83, 111.07, 109.50, 105.27, 96.32, 96.08, 45.43, 45.07, 12.57. ESI-HRMS: [1]+ m/z 562.2482, calcd for C33H32N5O4+
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562.2449.
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2.2 Materials and Instruments
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All reagents and solvents were purchased from commercial sources and were of the highest grade. Solvents were purified and dried by standard methods prior to use. Flash chromatography
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was performed using silica gel (200-300 mesh) obtained from the Qingdao Ocean Chemicals.
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Double-distilled water was used throughout the experiments. The stock solution of probe 1 (1.0
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mM) were prepared in CH2Cl2 and were stored at refrigerator (4 oC) [46]. Solutions of various species (2.0 mM) were prepared from NaCl, KBr, KI, KSCN, NaNO3, NaNO2, NaOAc, NaClO,
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Na2CO3, NaHCO3, Na2S2O3·5H2O, Na2SO3, NaHSO3, glutathione, cysteine, homocysteine, ascorbic acid and NaHS.
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The fluorescence spectra and relative fluorescence intensity were measured with a Shimadzu
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RF-5301 spectrofluorimeter with a 10 mm quartz cuvette. Absorption spectra were recorded using a Shimadzu UV-2550 spectrophometer. IR spectra were recorded on a Bruker Vertex 70 FT-IR
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spectrometer using a diamond ATR attachment. High-resolution mass spectra were collected using a Bruker micrOTOF-Q II mass spectrometer (Bruker Daltonics Corp, USA) in electrospray ionization (ESI) mode. 1H and
13
C NMR spectra were recorded on an INOVA-400 spectrometer
(Varian Unity), using tetramethylsilane (TMS) as the internal standard. The pH measurements were carried out on a Sartorius PB-10 pH meter.
2.3 Cell cultures and fluorescence imaging Human hepatic stellate cells were obtained from Cell Engineering Research Centre and Department of Cell Biology, Fourth Military Medical University (Xi’an, China). 2×105 human hepatic stellate cells were seeded in 6-well culture plates containing sterile cover slips and were cultured in RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum (FBS), penicillin (100 U mL-1), and streptomycin (100 µg mL-1) at 37 °C in a 95% humidity atmosphere under 5%
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CO2 for two days. Before imaging, the cells were washed with PBS three times and then incubated with probe 1 (10.0 µM) in a DMSO/PBS (5: 95, v/v) solution at 37 °C for 30 min. Then, the
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samples were rinsed with PBS three times to remove the remaining probe and observed under an
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Olympus BX 61 fluorescence microscope. Experiments to assess H2S uptake were performed in the same medium supplemented with NaHS (200 µM) for 20 min at 37 oC. The fluorescence
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images were acquired after the cells had been rinsed with PBS.
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2.4 General procedure for the spectral measurements
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Test solutions were prepared by placing 100 µL of probe 1 (1.0 mM), 3.0 mL of acetone, 1.0
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mL of phosphate buffer (10 mM, pH 7.4), and appropriate aliquot of each analyte stock solution
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into a 10 mL volumetric flask, and diluting the solution to 10 mL with water. The resulting
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solution was kept at room temperature (25 °C) for 2 min, and then the absorption or fluorescence spectra were recorded. The fluorescence ratio of I478/I702 was measured with the excitation and
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emission wavelengths at 410 nm and 478/702 nm.
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2.5 Cytotoxicity assays Human hepatic stellate cells were seeded in 96-well plates and incubated with different
concentrations of probe 1 (0-20 µM) (n = 5) in an atmosphere of 5% CO2 and 95% air at 37 °C for 6 h. Then, 20 µL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 5.0 mg mL-1) was added to each well, followed by further incubation at 37 °C for 4 h. After that, 100 µL
of the supernatant was removed, and 150 µL of DMSO was added to each well to dissolve the reddish-blue crystals. The plate was shaken for 10 min, and then the absorbance was measured at 570 nm with a microplate reader (ELX 800 UV, BIO-TEK Instruments Inc.). The relative cell viability (VR) was calculated using the following equation: VR = A/A0 × 100%, where A and A0 are the absorbance of the experimental group and control group, respectively. 2.6 Theoretical Methods
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Density functional theory calculations at the B3LYP/6-311++G** level of Gaussian 09 program were carried out to optimize geometries for all of the species studied here. In the DFT
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calculations, Ph-N(H)N2S and Ph-C(O)N(H)N2S were used to model the real reactions. With a
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continuum medium, we have calculated the single point solvation energy for all the species, using the conductor polarizable continuum model (CPCM). In the CPCM calculations, H2O was used as
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the solvent, corresponding to the experimental conditions.
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3. Results and discussion
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3.1 Absorption and fluorescent spectra of probe 1
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With compounds 1 in hand, we studied its spectral properties in CH2Cl2 and observed that its absorption and fluorescence emission maximum display bathochromic shift when compared to CB
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(from 658 to 668 nm in absorption spectra and from 692 to 703 nm in emission spectra) (Fig. S1,
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Supplemental Material). More significantly, unlike the common azide-based fluorescent probes, which are generally weakly fluorescent due to a favorable electron transfer from the electron-rich
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α-nitrogen of the azide group to the excited fluorophore [47], the fluorescence quantum yield of 1 is calculated to be 0.56 in CH2Cl2, which is slightly lower than that of CB (φF = 0.84). This can be explained by the fact that almost all NIR fluorescence dyes have relatively high-lying highest occupied molecular orbital (HOMO) energy levels, and thus intramolecular photo-induced electron fluorescence quenching is difficult to occur within these dyes [48].
3.2 Sensing response of probe 1 to H2S The free probe 1 is stable at room temperature (25 oC) and only displays the characteristic peaks of CB dye at around 660 nm. With increasing NaHS concentration (0-30 equiv), the absorption peak at 660 nm decreased, while the peak at 423 nm increased accordingly (Fig. 1a). In addition, such a large blue-shift of 237 nm in the absorption behavior changes the color of the resultant solution from blue into pink, allowing “naked-eye” detection (Fig. 1a, inset). Furthermore,
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fluorescence titration studies of 1 towards H2S were examined under the same conditions. Upon excitation at 410 nm, the free probe 1 exhibits two well-resolved emission peaks centered at 478
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and 702 nm, which can be assigned as the emission band of coumarin and CB, respectively.
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However, fluorescence titration of the solution of 1 with NaHS (0-30 equiv) induces a marked decrease of emission band at 702 nm and concurrently a dramatic enhancement of the band at 478
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nm (Fig. 1b). Such a large hypochromatic shift of 224 nm makes this probe favorable for the dual
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emission ratiometric sensing owing to the minimum overlap between the two emission bands. The
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fluorescence intensity ratios at 478 nm and 702 nm (I478/I702) increase from 0.39 to 19.46 upon NaHS treatment, and the final enhancement factor is about 50-fold. In addition, the intensity ratios
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(I478/I702) were plotted as a function of HS- concentration and a typical calibration graph was obtained (Fig. S2, Supplemental Material). The I478/I702 value is linearly related to NaHS
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concentration in the range of 2 to 100 µM with a detection limit of 0.22 µM (3δ). This result
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suggests that the present probe is favourable for direct imaging of intracellular H2S whose concentrations are usually in a micromolar range [35]. As indicated in Table S1
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(Supplemental Material), probe 1 shows excellent analytical performance compared to the most previously reported azide-based fluorescent probes for H2S. According to the well established reaction mechanism, the spectra changes in the sensing process could be assigned to the formation of compound 5, which was also verified by mass spectrometry analysis of the mixture of probe 1 and NaHS, and two predominant peaks at m/z
536.2554 and 558.2372, corresponding to [5 + H]+ (calcd 536.2549 for C33H34N3O4+) and [5 + Na]+ (calcd 558.2369 for C33H33N3O4Na+) were shown in the HRMS data (Fig. S3, Supplemental Material). In addition, the absorption and emission spectra of compound 5 are highly similar to those of coumarin 7 (Fig. S4, Supplemental Material). This is in accordance with the fact that 5 and 7 have almost the same coumarin fluorophore, which further proves the disruption of the π-electron conjugation in probe 1 at the bezopyrylium moiety. Collectively, these results provide strong support for the sensing mechanism outlined in Scheme 1.
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3.3 Kinetic studies
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The time course of the emission ratio (I478/I702) of probe 1 (10 µM) in the absence and presence
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of HS- (20 equiv) was studied, and the results are shown in Fig. 2 and Fig. S5 (Supplemental Material). Notably, probe 1 displayed a rapid response to HS- and the sensing reaction almost
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terminated within 1 min. The observed rate constant was then determined to be 0.015 ± 0.0005 s-1
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under the pseudo-first-order conditions (Fig. S6, Supplemental Material). In fact, probe 1 is able to
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detect H2S more rapidly than most of the reported azide-based probes, and comparable to that of arylsulfonyl azides. This favors the real-time intracellular imaging, when considering the variable
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nature and quick metabolism of endogenous H2S in biological systems. Despite the similar structures of acyl and aryl azides, acylazide-based fluorescent probes showed
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better kinetic feature than those of aryl ones. We envisioned that the reaction of HS- with 1
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proceeds by the nucleophilic attack of HS- at the terminal azide nitrogen of 1 to afford a free SH containing intermediate 2 [49], which subsequently transforms to thiolate intermediate 3, and 3 is
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readily decomposed to give amide 4 and dinitrogen sulfide (N2S) [50-51]. In the case of aryl azides, the analogous reaction can occur to afford the corresponding amine (Scheme S1, Supplemental Material). To elucidate the significant difference in reaction kinetics of above azide-based probes, benzoyl and phenyl azides were used to model the real reactions. The energy profiles for the pathway of
leading to dissociation of the thiolate intermediates (Ph-N(H)N2S- and Ph-C(O)N(H)N2S-) were calculated using density functional theory (DFT) in combination with solvent effect within the Gaussian 09 program [52]. The energy profiles of the two reactions were illustrated in Fig. 3. The calculated results showed that the free energy barriers for the dissociation of Ph-C(O)N(H)N2S- to produce the corresponding amide was found to be 9.3 kcal/mol, significantly lower than that found for Ph-N(H)N2S- (16.6 kcal/mol). The above results indicate that the decomposition of Ph-N(H)N2S- is much slower and less efficient than that of Ph-C(O)N(H)N2S-, which accounts for
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the significant differences in reaction rates for acyl and aryl azides upon treating with H2S.
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3.4 Selectivity studies
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To assess the selectivity of probe 1 towards H2S, changes in the fluorescence spectra 1 caused by various species, including common anions (HS-, F-, Cl-, Br-, I-, SCN-, NO3-, NO2-, AcO-, ClO-,
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SO42-, S2O42-, SO32-, S2O32-, HCO3-, biological thiols (GSH, Cys, Hcy) and ascorbic acid were
A
measured (Fig. 4 and Fig. S7, Supplemental Material). As can be seen from Fig. 4 and Table S2
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(Supplemental Material), only HS- induces a dramatic increment of the emission ratio (I478/I702), while other species, especially cellular sulfhydryl-containing compounds (GSH, Hcy and Cys)
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virtually caused negligible changes under the same conditions. Furthermore, probe 1 can still retain its sensing response to H2S even in the presence of a large amount of biothiols (GSH, Cys and
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Hcy) (Fig. S8, Supplemental material), which further confirms the high selectivity of probe 1
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towards H2S over other cellular sulfhydryl-containing compounds. Finally, the high selectivity of probe 1 towards H2S is readily detectable visually, and only NaHS gives rise to an obvious color
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change from dark green to light yellow (Fig. S9, Supplemental Material). In fact, the selectivity observed for H2S over other biological relevant species is remarkably
high. Based on the above facts, we speculated that the reaction between azide and HS- is not simply a redox process, since other reducing reagents such as ascorbic acid, SO32- and S2O32induce almost no reaction under identical conditions, even their concentrations are much higher
than that of HS-. This suggests that the nucleophilic addition of HS- to the azide moiety of probe 1 indeed occurs as depicted in Scheme 1, which further explains why common reducing compounds elicit almost no response to probe 1 under identical conditions. In the case of cellular biothiols, such as GSH, Cys and Hcy, although their thiol groups are nucleophilic, they possess higher pKa values (ca. 8.5) than that of H2S (ca. 6.9) [53, 54]. Thus, based on the marked distinctions in terms of size and pKa values, the nucleophilic attack of azide may be chemoselective for H2S over these biothiols in aqueous media at physiological pH. In addition, the negligible reactivity of biothiols,
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which cannot form intermediate 3 with a thiolate group, provides additional support for the
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mechanism shown in Scheme 1.
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3.5 Bioimaging and cytotoxicity studies
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The capability of probe 1 to sense H2S in live cells was evaluated. Human hepatic stellate cells
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stained with probe 1 displayed a strong emission in the red channel (Fig. 5c) and very dim
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fluorescence in the blue emission (Fig. 5b). However, when the cells were pretreated with probe 1
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(10 µM), and further incubated NaHS (200 µM) for 20 min, a decrease in the red emission (Fig. 5f)
D
and a dramatic enhancement in the blue emission (Fig. 5e) were observed simultaneously. The
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above image variations are in accordance with the proposed mechanism shown in Scheme 1. In addition, the MTT assays demonstrate that probe 1 at a concentration of 15 µM shows minimal
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cytotoxicity to the cells (Fig. S10, Supplemental Material), which are comparable to the previous
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reported H2S probes [55]. The above results demonstrate that 1 is suitable for ratiometric
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fluorescence imaging of H2S in live cells.
4. Conclusions
In summary, we have developed a new ratiometric fluorescent probe for H2S by a coumarin-benzopyrylium platform. By combining the selective acyl azide/amide conversion with
intramolecular spirocyclization reaction, the proposed probe affords a high sensitivity and ratiometric response toward H2S. Compared with the traditional azide-based H2S probes, the proposed probe utilizes the acyl azide as the recognition unit and shows a quick response towards H2S (ca. 1 min), thus they favour to use the real-time intracellular H2S imaging. Preliminary fluorescence imaging experiments showed that probe 1 has potential to track H2S in living human
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hepatic stellate cells. The superior properties of 1 make it of great potential use in other biological
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systems.
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Acknowledgments
This research was supported by the Natural Science Foundation of China (No. 21275117,
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21475105, 21175106), the Science & Technology Department (No. 2012JM2004) and the
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Education Department (No. 12JK0518) of Shaanxi Province of China.
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[46] The solution of probe 1 showed no significant changes at least for 7 days when it was stored at refrigerator (4 oC). However, a Curtius rearrangement reaction would occur at higher
EP
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Figure Captions Scheme 1. The proposed sensing mechanism of probe 1 for H2S. Scheme 2. Synthesis of probe 1. Fig. 1 Absorption (a) and fluorescence (b, λex = 410 nm) spectra of probe 1 (10 µM) upon addition of increasing concentrations of NaHS (0-300 µM) in acetone/phosphate buffer (3:7 v/v, 10 mM, pH 7.4). Inset: (a) color changes of 1 (10 µM) upon addition of NaHS (10 equiv); (b) fluorescence
intensity ratio (I478/I702) changes of probe 1 (10 µM) with the amount of NaHS. Each spectrum was recorded after incubation of the probe with NaHS for 2 min at room temperature (25 °C). Fig. 2 Time-dependent changes the emission ratio (I478/I702) of probe 1 (10 µM) in the presence of NaHS (20 equiv) in acetone/phosphate buffer (3:7 v/v, 10 mM, pH 7.4). Fig. 3 The potential energy profile calculated for the dissociation of Ph-N(H)N2S- (a) and Ph-C(O)N(H)N2S- (b). The calculated relative free energies are given in kcal/mol. Fig. 4 The emission ratio (I478/I702) of probe 1 (10 µM) in the presence of various species in
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acetone/phosphate buffer (3:7 v/v, 10 mM, pH 7.4) for 2 min. a) Blank, b) HS-, c) F-, d) Cl-, e) Br-,
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f) I-, g) SCN-, h) NO3-, i) NO2-, j) AcO-, k) ClO-, l) SO42-, m) S2O42-, n) SO32-, o) S2O32-, p) HCO3(20 equiv of each), q) Cys (5 mM), r) GSH (5 mM), s) Hcy (5 mM), t) ascorbic acid (10 mM).
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Fig. 5 Fluorescence images of exogenous H2S in human hepatic stellate cells with probe 1. (a)
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Bright field image of cells incubated with 1 (10 µM) for 30 min; (b) fluorescence image of (a)
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from blue channel; (c) fluorescence image of (a) from red channel. (d) Bright field image of cells
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incubated with probe 1 (10 µM) for 30 min and further incubation with NaHS (200 µM) for 20
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min; (e) fluorescence image of (d) from blue channel; (f) fluorescence image of (d) from red
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channel.
O N N N
NH
O N
H2S N
O O
O
O O
N
O
N
5
1
Spirolactam form, λem = 478 nm
Open form, λem = 702 nm
H+
HS-
OH
-
O N
N
N
HN
SH
O O
N O
O
NH2
S
O
- N2S
N
O
N
O
2
O
O O
N
O
N
4
3
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N
N
N
M
A
N
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Scheme 1
O
O
N
+
O
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6
N
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OH
D
COOH
CC A
Reflux
1) H2SO4, 90 oC 2) 70% HClO4
O
N
O O
7
CB
O
O
Cl
POCl3
COOH
O
N
N3
NaN3 o
N
0-5 C
O O
O
N
8
N
O O 1
Scheme 2
O
N
0.7
a) Absorbance
H2S
Blank
0.6 0.5 0.4 0.3 0.2
360
420
480
540
600
660
15.0
I478/I702
10.0 7.5
A
400
N
12.5
500
RI
SC
b)
720
U
600
M
5.0
0.0
0
50
100
150
200
250
NaHS concentration/µM
300
TE
200
2.5
D
300
100
EP
Fluorescence intensity (a.u.)
Wavelength / nm
PT
0.1
A
CC
0
450
500
550
600
650
Wavelength / nm Fig. 1
700
750
25 1 only 1 + NaHS
I478/I702
20
15
10
0 30
60
90
120
180
SC
Time / s
150
RI
0
PT
5
M
A
N
U
Fig. 2
TE
D
16.6
0.0
EP
H N
N
N
S
NHH N N
N
S-
N 2S
NH-
+
-9.5
-
(a) 9.3
CC A
+
O C O
0.0
C
O C
N H
N
N N H
N S-
(b)
N S-
-18.7
N 2S
Fig. 3
PT
15
RI
9
SC
I478/I702
12
N
U
6
M
a b c d e f g h i j k l m n o p q r s t
A
CC
EP
TE
D
0
A
3
Fig. 4
TE
EP
CC
A D
PT
RI
SC
U
N
A
M Fig. 5
TE
EP
CC
A D
PT
RI
SC
.
U
N
A
M
Fig 2
TE
EP
CC
A D
PT
RI
SC
.
U
N
A
M
Fig 3
TE
EP
CC
A D
PT
RI
SC
.
U
N
A
M
Fig 4
TE
EP
CC
A D
PT
RI
SC
.
U
N
A
M
Fig 5
TE
EP
CC
A D
.
PT
RI
SC
U
N
A
M
Fig1-a
TE
EP
CC
A D
PT
RI
SC
.
U
N
A
M
Fig1-b
TE
EP
CC
A D
PT
RI
SC
U
N
A
M
graphic abstract .
