Highly selective fluorescent and colorimetric probe for live-cell monitoring of sulphide based on bioorthogonal reaction

H2S is the third endogenously generated gaseous signaling compound and has also been known to involve a variety of physiological processes. To better understand its physiological and pathological functions, efficient methods for monitoring of H2S are desired. Azide fluorogenic probes are popular because they can take place bioorthogonal reactions. In this work, by employing a fluorescein derivative as the fluorophore and an azide group as the recognition unit, we reported a new probe 5-azidofluorescein for H2S with improved sensitivity and selectivety. The probe shows very low background fluorescence in the absence of H2S. In the presence of H2S, however, a significant enhancement for excited fluorescence were observed, resulting in a high sensitivity to H2S in buffered (10 mmol/L HEPES, pH 7.0) aqueous acetonitrile solution (H2O/CH3CN = 1:3, v/v) with a detection limit of 0.035 μmol/L observed, much lower than the previously reported probes. All these features are favorable for direct monitoring of H2S with satisfactory sensitivity, demonstrating its value of practical application.

H 2 S is the third endogenously generated gaseous signaling compound and has also been known to involve a variety of physiological processes. To better understand its physiological and pathological functions, efficient methods for monitoring of H 2 S are desired. Azide fluorogenic probes are popular because they can take place bioorthogonal reactions. In this work, by employing a fluorescein derivative as the fluorophore and an azide group as the recognition unit, we reported a new probe 5-azidofluorescein for H 2 S with improved sensitivity and selectivety. The probe shows very low background fluorescence in the absence of H 2 S. In the presence of H 2 S, however, a significant enhancement for excited fluorescence were observed, resulting in a high sensitivity to H 2 S in buffered (10 mmol/L HEPES, pH 7.0) aqueous acetonitrile solution (H 2 O/CH 3 CN 5 153, v/v) with a detection limit of 0.035 mmol/L observed, much lower than the previously reported probes. All these features are favorable for direct monitoring of H 2 S with satisfactory sensitivity, demonstrating its value of practical application. F luorogenic probes activated by bioorthogonal chemical reactions can enable biomolecule imaging in situations where it is not possible to wash away unbound probe 1 . Much work has been devoted to expanding the toolbox of bioorthogonal reactions, and these efforts can be complemented by the development of fluorogenic probes 2 . Such probes are typically endowed with a functionality that suppresses fluorescence. Its transformation during the reaction creates a new functionality that no longer quenches the fluorescence of the underlying system, resulting in a fluorescence enhancement. Such probes offer significant advantages for imaging studies in which it is not possible to wash away unreacted probe, such as real-time imaging of dynamic processes in cells or visualization of molecules in live organisms.
One of the most widely used bioorthogonal reactions is the azide2alkyne [3 1 2] cycloaddition to form a triazole 3,4 . This reaction has enabled the selective visualization of azide-or alkyne-labeled proteins, glycans, nucleic acids, and lipids 4,5 . Several azide-6-9 fluorogenic probes have been reported, largely based on coumarins 6,10 , naphthalimides 8 , and other systems that require UV excitation and emit blue light 7,11,12 . Such wavelengths are not ideal for biological imaging because of high levels of autofluorescence and poor tissue penetrance 13 .
An obvious improvement upon these designs would be the development of azido fluorogenic probes with longer excitation and emission wavelengths. Some attempts at achieving this goal have been made 8,12,14 . The utility of azide pairs in biological settings remains unclear. Thus, fluorogenic azido probes that perform well as cellimaging reagents remain an important goal. Bertozzi reported the rational design and experimental validation of azide-functionalized fluorogenic probes based on the widely used blue-excitation/green-emission fluorescein scaffold 15 . In their work, they have prepared a series of azidefluorescein compounds under NaNO 2 /NaN 3 condition, and the azidefluorescein was used to biological imaging in Chinese hamster ovary (CHO) cells labled with alkynylsialic acid nor H 2 S.
It is well known that H 2 S have been demonstrated to exert protective effects in many pathologies and physiologies [16][17][18][19][20][21][22][23][24][25][26] . So the discovery of these emerging biological roles of H 2 S has resulted in rising interest in H 2 S research. Accordingly, rapid, accurate and reliable methods for H 2 S detection are in high demand, as they have potential to provide useful information for better understanding its biological functions 27 . And simple, specific, and real-time analytic methods/sensors are highly desirable for H 2 S in biological systems. In fact, it is a good choice to introduce an azido group into probes to be reduced by H 2 S due to the simple synthesis, relatively good selectivity, suitable reaction time, and non-cell toxicity [28][29][30][31][32][33][34] .
With these considerations in mind, we also prepared 5-azidefluorescein from 5-aminofluorescein under NaNO 2 /NaN 3 condition according to literature 15 (Fig. 1) and tried to use this compound to detect H 2 S. It is delightful that we obtained the crystal of 5-aminofluorescein and found probe can be used as a high selective and sensitive fluorescent probe for H 2 S firstly. Furthermore, the probe also was applied in cell imaging.
5-aminofluorescein (0.35 g, 1 mmol), a deep-red solid, was dissolved in 10 mL 251 AcOH/H 2 O and cooled to 0uC. To this deep red solution was added NaNO 2 , a white powder (0.10 g, 1.5 mmol). After stirring for 15 minutes, the solution had turned to a light red color. NaN 3 (0.10 g, 1.5 mmol) was then carefully added (caution: gas evolution!), turning the solution to a yellow slurry. The reaction was stirred for 2 hr at 0uC. The slurry was filtered over vacuum and the solid washed with 20 mL 2 mol/L HCl and 100 mL H 2 O, yielding 5-azidofluoresceinquinone (0.30 g, 80%) as a yellow solid after further drying in vacuo and characterized by NMR, ESI-MS, elemental analysis, X-ray crystal diffractometer (see Figure S1).
Reaction of probe (1 mmol/L) with Na 2 S (2 mmol/L) as an aqueous sulphide source at room temperature in buffered (10 mmol/L HEPES, pH 7.0) aqueous acetonitrile solution (H 2 O/CH 3 CN 5 153, v/v) yielded a time-dependent fluorescence increase, which was completed within 5 s (Supplementary Fig. S2). DF . 50-fold increase in the fluorescence intensity accompanied (W 5 0.35) with a green emission at 531 nm. However, the analytes without hydrogen sulfide induced no changes in the fluorescence emission properties under the same conditions (Fig. 2a). The competing experiments indicated other analytes did not disturb the determination for sulphide (Fig. 2b). It is noted that the unprecedented speed of this probe's response and high selectivity compared with other probes [35][36][37][38] suggests the possibility of quantitative detection without the need for sample pretreatment. The results reason that H 2 S-mediated reduction of azides to amines would generate highly fluorescent products (Fig. 3) 39 . H 2 S-induced product was confirmed its molecular formula by electrospray ionization mass spectrometry (ESI-MS). The peak at m/z 346.42 corresponding to [5-aminofluorescein-H] 1 , was clearly observed ( Supplementary Fig. S3). Further 1 HNMR spectroscopic analysis also provided the evidence for the product of 5-aminofluorescein. With addition of 2 equiv. of Na 2 S (containing crystal water) to probe in DMSO-d 6 (Fig. S4), the resonance of the original proton (azidebenzene CH) at 7.28 and 7.49 ppm all shifted to upfield owing to presence of electron-pushing group NH 2 (Supplementary Fig. S4) and appeared at 6.65 , 6.76 ppm.
Next, varying concentrations of Na 2 S (0-2.0 mmol/L) were added to the test reaction solution. The fluorescence intensity increased linearly with the concentration of Na 2 S up to 2.0 mmol/L, and, thereafter, reached a steady state (Fig. 4). The detection limit, based on the definition by IUPAC (C DL 5 3 S b /m) 40 , was found to be 0.035 mmol/ L from 10 blank solutions ( Supplementary Fig. S5). This probe therefore shows a high sensitivity toward sodium sulfide comparable to that of other reported S 2 2chemosensors 35-38 (Table 1).
We also performed absorption spectral experiments in the buffered (10 mmol/L HEPES, pH 7.0) aqueous acetonitrile solution   of H 2 S. The probe has no absorbance at UV-Vis area, immediately there generated an absorbance at 510 nm and the absorbance intensity enhanced with increased H 2 S corresponding solution color change from colorless to yellow. The notable variation was ended after about 4 equiv. of H 2 S added, relating to the H 2 S-mediated reduction of 5-azidefluoresceinquinone to 5-aminofluorescein (ring-open).
Most publications suggest that the average endogenous H 2 S level is in the mmol/L range 31,32,41 , Since the detection limit of this probe was found to be 0.035 mmol/L, thus it become possible that the probe can detect H 2 S level in tissue imaging. The ability of probe to detect sulphide within living cells was also evaluated by laser confocal fluorescence imaging using a Leica TCS SP5 laser scanning microscope. Imaging of sulphide substrates in HeLa cells after 30 min incubation using probe (2 mmol/L) showed weak green fluorescence (Fig. 5b).
HepG2 cells incubated with 2 mmol/L probe for 30 min at 37uC, and with 4 mmol/L exogenous H 2 S for another 30 min at 37uC, showed green fluorescence (Fig. 5c) (it is noted that 30 min was usually selected in cell imaging experiment). We also carried out time course experiment in the cell. Fig. 6 (left) indicated that a 15 min is enough for cell permeability (Fig. 6h) reaction and the cell can survive even if in a 45 min after H 2 S was added (Fig. 6i). In addition, according to the Qian's method 42 , we employed sodium nitroprusside (SNP, a NO donor) to stimulate the production of endogenous H 2 S in cells 43 . With the addition of probe into the culture of the SNP (100 mmol/ L or 200 mmol/L)-loaded cells for 20 min, a drastic increase of emission intensity (Fig. 6l, 6m), indicating the generation of endogenous H 2 S within the cells. These results demonstrate that this probe is selective for sulphide and amenable for live-cell imaging.
The development of innovative fluorescent imaging probes has revolutionized cell biology, allowing localization and dynamic monitoring of cellular metabolite and inorganic ion pools [43][44][45] . Recently, fluorescence and/or colorimetric chemosensors for H 2 S/aqueous sulphide based on some reaction mechanisms between probes and H 2 S have been reported. These include the cleavage of alcoxyl(R-O) bond [45][46][47] , the cleavage of S-O bond [47][48][49] , copper displacement approach 49-51 , nucleophilic addition approach 5,38,52,53 . A significant bottleneck in the above emerging field of H 2 S/aqueous sulphide signalling is the absence of technology for effective in vivo detection and imaging. And this problem is exacerbated by fact that similar substances such as sulphide which contain SH group may mislead the detection of intracellular thiol concentration. In this study, we have successfully developed an azide-to-amine reduction chemical strategy for selective sulphide detection, which can be used to monitor sulphide generation in live cells in the presence of large excess of thiols. We show that the same chemistry can be readily adapted to different fluorescent templates for sulphide detection and imaging.
The same chemistry will lead to new probes with faster response, which may help to monitor fluctuations of H 2 S in situ. Further optimization and utilization of this strategy and this class of probes should dramatically accelerate future studies of H 2 S in biology.