A Sensitive and Selective Fluorescent Probe for Selenol in Living

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A Sensitive and Selective Fluorescent Probe for Selenol in Living Cells Designed via a pKa Shift Strategy Shengrui Zhang, Qin Wang, Xiawei Liu, jianjian zhang, Xiaofeng Yang, Zheng Li, and Hua Li Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b00066 • Publication Date (Web): 22 Feb 2018 Downloaded from http://pubs.acs.org on February 22, 2018

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Analytical Chemistry

A Sensitive and Selective Fluorescent Probe for Selenol in Living Cells Designed via a pKa Shift Strategy Shengrui Zhang,†,‡ Qin Wang,†,‡ Xiawei Liu,§ Jianjian Zhang,† Xiao-Feng Yang,*,† Zheng Li,§ and Hua Li*,∥ †

Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of Ministry of Education, College of Chemistry and Materials Science, Northwest University, Xi’an, Shaanxi 710127, P. R. China. Fax: (+) 86-29-81535026, E-mail: [email protected] ‡ Shaanxi Key Laboratory of Catalysis, School of Chemistry and Environment Science, Shaanxi University of Technology, Hanzhong, Shaanxi 723000, P. R. China § College of Life Sciences, Northwest University, Xi’an, Shaanxi 710069, P. R. China ∥ College of Chemistry and Chemical Engineering, Xi’an Shiyou University, Xi’an, Shaanxi 710065, P. R. China. Fax: (+) 86-29-88382217, E-mail: [email protected] ABSTRACT: Selenocysteine (Sec) is a primary kind of reactive selenium species in cells and its vital roles in physiological processes have been featured. Thus, the development of highly sensitive and selective methods for the sensing of Sec is of great significance. This work reports a turn-on fluorescent probe for selenol based on the unique fluorescence OFF-ON switching between the Schiff base (SB) and its complementary protonated Schiff base (PSB) form of merocyanine dyes. The probe consists of a merocyanine Schiff base fluorophore and a 2,4-dinitrobenzenesulfonamide moiety that reacts with selenol specially. The fluorescence turn-on response of MC-Sec is realized via the selective removal of the strong electron-withdrawing 2, 4dinitrobenzenesulfonyl group by Sec, leading to the pKa of the imine nitrogen of the probe shifts from 6.40 to 9.04, and thus significantly increases the population of the fluorescent PSB form of the dye at physiological pH. MC-Sec shows good selectivity and sensitivity for Sec and has been applied in the imaging of exogenous and endogenous selenol in living cells by confocal fluorescence microscopy. The proposed mechanism would be useful for developing future probes directed to other target molecules by employing this simple but effective pKa shift strategy.

As an fundamental dietary micronutrient, Selenium is related to human and other mammals health.1,2 Although different metabolites of selenium exist, selenocysteine (Sec), seems to be the major form of biological seleniums.3,4 Sec is a cysteine analogue and the 21st amino acid in ribosomemediated protein synthesis.5 It exists naturally in all kingdoms of life and is a necessary building block for selenoproteins (SePs), which have biological functions in redox signaling, antioxidant defense, thyroid hormone metabolism and immune responses.6 In addition, SePs have been believed to be closely linked with several human diseases, such as cancer, Keshan disease, virus infections, male infertility, and abnormalities in immune responses and thyroid hormone function.6-9 Considering the vital roles of Sec in biological systems, the development of effective detection techniques for Sec in realtime is propitious to elucidate the function of the various Secontaining species in living systems.10-12 Fluorescence probes are powerful tools in the field of bioimaging analysis owing to their versatile advantages such as high specificity and sensitivity, less invasiveness, rapid response, high spatial and temporal resolution and good biological compatibility. Recently, a few fluorescent probes have been developed to detect selenol in living systems,13-26

most of which rely on the strong nucleophilicity of the selenol group. These probes are generally constructed via the protection of the functional groups of fluorophore with different electrophilic recognition units including 2,42,4dinitrobenzenesulfonamide (or sulfonate),14-21 22,23 dinitrobenzenoxy, 2,1,3-benzoselenadiazole (BS),24 and acrylate.25 The protective group can be readily removed by selenol, affording turn-on fluorescence emission. In fact, the protection/deprotection a conjugated functional group of the fluorophore represents a new research focus for developing selective probes. The masking of the functional group reduces the conjugated π-electron system of the dye and thus turns off the fluorescence of the probe. Removal of the trigger restores the conjugation of the functional group with the π-electrons of the dye and thereby affords turn-on fluorescence output. In fact, significant advances have been made in the field.27 However, this protection/deprotection strategy cannot be readily coupled for use in the π-electron system without a conjugated functional group (such as cyanine dyes).28,29 To install such a turn-on sensing mechanism in these dyes, a new strategy should be exploited. In recent years, it was reported that merocyanine Schiff base provides an ideal platform for switching on and off an

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observable spectroscopic signal. The imine (Schiff base, SB) usually absorbs at short wavelength and is essentially nonfluorescent, whereas the iminium (protonated Schiff base, PSB) gives rise to long wavelength absorption and strong fluorescence (Figure S1a).30,31 In fact, non-N-alkylated cyanine (one of the indole nitrogen atoms on a cyanine dye is not alkylated) has a protonable amino group within the chromophore core and shows similar spectroscopic changes by protonation/ deprotonation of the indole nitrogen atom of the dye (Figure S1b).32-39 The intense absorption and emission properties of the iminium (or non-N-alkylated cyanine dyes) are largely due to a resonance effect between the nitrogen atoms at two ends of the polyene via the conjugated pentamethine bridge. Thus, regulation of the equilibrium between SB and PSB forms of merocyanine Schiff base would provide an alternative strategy to modulate the optical properties of the dye. On the other hand, it was reported that the pKa values of the imine nitrogen of merocyanine Schiff base will dependent on the electron-withdrawing ability of the substituent proximal to the imine group, the stronger the electron-withdrawing ability of the substituent, the lower pKa value of the imine nitrogen.4042 Apparently, the pH region between two pKa values that is formed by structural change of an imine-containing probe would be a suitable sensing window with a notable change in the spectral characteristics of the dye. Thus, if a specific reaction can remove the electron-withdrawing substituent adjacent to the imine group of the probe, it would elicit an upward pKa shift of the merocyanine Schiff base due to the variations in the electronic properties, which in turn leads to marked changes in optical properties of the dye. We envisioned that such a pKa shift strategy may serve as the underlying principle to develop fluorescent probes for the target molecule.

absorbs at a relatively short wavelength and is essentially nonfluorescent. While in the presence of Sec, its 2, 4dinitrobenzenesulfonamide group was readily cleaved via a nucleophilic reaction, leading to the generation of free MCP. Due to the removable of strong electron-withdrawing substituent adjacent to the imine, the pKa of MCP shifts to 9.04. Therefore, MCP is predominantly present as the PSB form at pH 7.4, which leads to a bathochromically shifted chromophore and a significant increase in fluorescence intensity simultaneously (Scheme 1). The probe shows high sensitivity and specificity toward Sec over biological thiols under physiological conditions and is able to monitor the changes in the selenol level in living cells. So far as we know, this is the first example of turn-on fluorescent probe for biological species that utilizes the large and tunable pKa shift of imines.

Experimental Section Reagents and Apparatus. All commercial chemicals were used without further purification. Deionized water (18.4 MΩ•cm) from a Milli-Q reference system (Millipore) was used in all experiments. Fluorescence spectra were acquired on a RF-5301PC fluorescence spectrometer. A UV-Vis spectrophotometer (UV-2550, Shimadzu) was used to record the absorbance spectrum. NMR spectra were recorded on an INOVA-400 spectrometer (Varian Unity). Chemicals shifts (δ) were specified in ppm relative to solvent residual peaks as internal standards. High-resolution electrospray ionization mass spectra were obtained on a Bruker micrOTOF-Q II mass spectrometer. Fluorescent images of cells were obtained by a confocal laser-scanning microscope (Olympus FV1000). The pH measurements were carried out with a Sartorius PB-10 pH meter. HPLC analysis was performed on a Shimadzu HPLC system (Kyoto, Japan) consisting of two LC-20AT pumps and a SPD-20A UV–Vis detector. The analytical column (150 mm× 4.6 mm I.D., TSKgel ODS-100Z, 5 µm) was obtained from Tosoh Co. (Kyoto, Japan). The column was maintained at 25 °C and the flow rate was 1.0 mL min-1. The injection volume was 20 µL. Cell Cultures and Imaging Experiments. SMMC-7721 cells were grown at 37 °C using RPMI-1640 medium supplemented with 10% fetal bovine serum and penicillinstreptomycin (both 100 U mL-1) in an atmosphere of air with 5% CO2 and constant humidity. Cells were passaged at about 80 % cell confluency using a 0.25 % trypsin solution. Immediately before the experiments, the cells were washed with phosphate buffer saline (PBS) (pH 7.4) three times, and then incubated with MC-Sec (20 µM) for 15 min. After washing with PBS, the cells were incubated with Sec (20 or 50 µM) for 15 min. Next, the cells were stained with DAPI (1.0 µg mL-1) for 10 min, and washed with PBS three times. Fluoresce images of the cells was then immediately captured by an Olympus fluorescence microscope. In order to induce endogenous Sec, the cells were exposed to sodium selenite (0, 10 or 20 µM) for 12 h. Then the cells were incubated with MC-Sec (20 µM) and DAPI (1.0 µg mL-1) for 15 min. After removal of the staining solution and washed with PBS, the fluorescence images were acquired immediately. Excitation wavelength was 543 nm for MC-Sec and 405 nm for DAPI. Emission collection was at 555-655 nm for MC-Sec and 425475 nm for DAPI.

Scheme 1. Schematic illustration of sensing process of MCSec toward Sec based on a pKa shift strategy. Herein, we report our progress toward the development of a merocyanine-based fluorescent probe MC-Sec for Sec based on a pKa shift mechanism. In our design system, 2, 4dinitrobenzenesulfonamide group was selected as a recognition unit, and merocyanine dye was employed as a fluorophore. The pKa value of MC-Sec is around 6.40, which exists mainly as the SB form under physiological pH conditions (pH 7.4). Due to the absence of the iminium, it


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Analytical Chemistry 127.35, 123.56, 123.40, 122.14, 120.45, 120.12, 119.39, 106.93, 96.37, 64.55, 45.78, 44.53, 36.50, 33.13, 28.28, 24.46, 11.49. HRMS (ESI): m/z calcd for C29H33N5O6S [M+H]+: 580.2230; found: 580.2261. Synthesis of MCP. Compound MCP was prepared via the reaction of compound 7 with 4-aminopiperidine using the procedure described for the synthesis of MC-Sec. Yield: 86 %. HRMS (ESI): m/z calcd for C23H31N3 [M+H]+: 350.2590; found: 350.2587.

Synthesis Synthesis of compound 3. A solution of compound 1 (400 mg, 2.0 mmol) in 10 mL of CH2Cl2 and Et3N (3 equiv) were combined. The solution of compound 2 (532 mg, 2.0 mmol) in CH2Cl2 (5 mL) was then added dropwise to the above solution at 0 oC. After 10 min, the ice-bath was removed and the mixture was stirred for 6 h at ambient temperature. The reaction mixture was diluted with CH2Cl2, washed with water (15 mL × 3), dried over Na2SO4 and then evaporated to give the crude material, which was further subjected to flash chromatography (silica gel, CH2Cl2/MeCN = 20:1, v/v) to afford compound 3 as a white solid (0.79 g, 92% yield). Synthesis of compound 4. Compound 3 (430 mg, 1.0 mmol) was dissolved in CH2Cl2 (8 mL), and then trifluoroacetic acid (8 mL) was added. The mixture solution was stirred at room temperature overnight. The solvent was removed by evaporation and the resulting reside was subjected to flash column chromatography (silica gel, CH2Cl2/MeOH = 10:1, v/v) to give compound 4 (0.31 g, 94% yield). 1H NMR (400 MHz, DMSO-d6), δ (ppm): 9.03 (s, 1H), 8.60 (d, J =8.0 Hz, 1H), 8.30, (d, J =8.0 Hz, 1H), 8.00 (s, 3H), 3.82 (d, J =12.0 Hz, 2H), 3.19 (s, 1H), 2.96 (t, J = 12.0 Hz, 2H), 1.98 (d, J =12.0 Hz, 2H), 1.49 (q, J =12.0 Hz, 2H). 13C NMR (100 MHz, DMSO-d6), δ (ppm): 150.59, 135.34, 132.67, 127.42, 120.47, 46.74, 44.40, 40.37, 29.49. HRMS (ESI): m/z calcd for C11H14N4O6S [M+H]+: 331.0712; found: 331.0714. Synthesis of compound 7. Compound 5 was prepared according to the literature procedure.43 Compound 6 (0.57 g, 2.0 mmol) and sodium acetate (360 mg) were added to a solution of compound 5 (0.63 g, 2.0 mmol) in acetic anhydride (24 mL). The mixture was stirred for 1h at ambient temperature, and then poured into 120 mL of Et2O. The precipitate was filtered and then isolated by flash column chromatography (silica gel, CH2Cl2/MeOH = 50:1, v/v) to afford compound 7 as a brown solid (0.82 g, 80% yield). 1H NMR (400 MHz, CDCl3), δ (ppm): 8.15 (d, J = 14.0 Hz, 1 H), 7.89 (dd, J = 14.4, 11.6 Hz， 1 H), 7.59-7.45 (m, 9 H), 7.14 (d, J =6.8 Hz, 2 H), 6.97-6.90 (m, 1 H), 5.41-5.35 (m, 1 H), 4.75 (q, J =7.2 Hz, 2 H), 1.95 (s, 3 H), 1.71 (s, 6 H), 1.50 (t, J = 7.2 Hz, 3 H). 13C NMR (100 MHz, CDCl3), δ (ppm): 179.73, 169.51, 156.34, 151.81, 143.04, 141.23, 140.49, 137.94, 130.81, 130.23, 130.01, 129.56, 128.91, 128.16, 122.73, 113.85, 113.64, 113.26, 51.51, 43.14, 27.26, 23.48, 14.04. HRMS (ESI): m/z calcd for C26H29N2O+ [M]+ 385.2280; found: 385.2270. Synthesis of MC-Sec. To a stirred solution of compound 7 (510 mg, 1.0 mmol) in ethanol (20 mL) was added compound 4 (330 mg, 1.0 mmol). The mixture was stirred for 3 h atambient temperature, and then the solvent was removed under reduced pressure. Purification of the residue by flash column chromatography (silica gel, CH2Cl2/MeOH = 30:1, v/v) provided pure MC-Sec as a dark purple solid (0.46 g, 79% yield). 1H NMR (400 MHz, DMSO-d6), δ (ppm): 9.01 (s, 1H), 8.59 (d, J =8.0 Hz, 1H), 8.31 (d, J =6 Hz, 1H), 7.92 (d, J =4.0 Hz, 1H), 7.25 (d, J =4.0 Hz, 1H), 7.16 (m, 2H), 6,94 (m, 1H), 6.84 (m, 2H), 6.10 (t, J =12.0 Hz, 1H), 6,05 (m, 1H), 3.67 (m, 4H), 3.18(s, 1H), 3.05 (t, J =12.0 Hz 2H), 1.75 (m, 2H), 1.56 (m, 2H), 1.50 (s, 6H), 1.09 (t, J =8.0 Hz, 3H). 13C NMR (100 MHz, DMSO-d6), δ (ppm): 162.32, 150.56, 148.17, 144.07, 139.78, 139.13, 135.20, 132.63, 129.11, 128.22,

Scheme 2. Synthesis of MC-Sec and MCP. Reagents and conditions: (a) CH2Cl2, Et3N, room temperature, 6 h; (b) CH2Cl2, trifluoroacetic acid, room temperature, overnight; (c) sodium acetate, acetic anhydride, room temperature, 1 h; (d) EtOH, compound 4 or 4-aminopiperidine, room temperature, 3 h.

Results and Discussion Probe Design and Synthesis. In our newly developed sensing system, mecocyanine Schiff base MCP was chosen as a scaffold to construct fluorescent probes because its SB and PSB form have completely different spectral properties.30 In addition, a 2, 4-dinitrobenzenesulfonamide moiety was selected as the specific trigger moiety for Sec because it is a good reaction site for Sec but not for biothiols under physiological conditions.14 Moreover, its strong electronwithdrawing ability favors lowering the pKa value of imino nitrogen of the probe. With the reasoning described above, we prepared MC-Sec in four steps according to the synthetic route shown in Scheme 2. Meanwhile, a control compound MCP without a 2, 4-dinitrobenzenesulfonamide unit was also synthesized for comparison. All the intermediates and desirable products were characterized by NMR and HRMS. To understand the pKa shift sensing mechanism, we initially investigated the effect of pH on the optical properties of MCSec and MCP, respectively. As shown in Figure 1a, the pHdependent UV-visible spectral changes of MC-Sec were observed. It can be seen that as the solution pH is adjusted from acidic (pH 2.4) to basic conditions (pH 10.5), the characteristic absorption maximum for the PSB form of MCSec at 566 nm is greatly reduced as a new peak evolves at 427 nm. The absorption peak at 427 nm is due to the increased presence of the SB form of the probe. This phenomenon can



be attributed to the iminium cation of the dye has been changed to its corresponding imine, and as a result, it absorbs at short wavelength. The inset of Figure 1a shows the absorbance changes of MC-Sec at 566 nm as a function of pH, it can be seen that a regular sigmoidal response is observed for this probe in response to pH. The pKa of the transition between SB and PSB forms of MC-Sec was calculated to be 6.40 by using the Hasselbach-type mass action equation44 (for the detailed calculation, please see the Supporting Information).

Figure 2. Photographs of the solutions of MC-Sec (a) and MCP (b) at different pH values taken under ambient light.

Next, the pH titrations of MCP were carried out and it shows similar spectral variations compared with that of MCSe (Figure 1c and d). However, its pKa value was determined as 9.04, which is much higher (upward shift by 2.64 units) than that of MC-Sec. Their significant difference in pKa values is apparently due to the strong electron-withdrawing effect of 2, 4-dinitrobenzenesulfonamide moiety in MC-Sec. Therefore, at the physiological pH of 7.4, MC-Sec is present in an equilibrium between a nonfluorescent SB form (~90.9%) and a fluorescent PSB form (9.1%). while MCP is predominately present as a fluorescent PSB form (~97.8% abundance). This difference can be reflected by their drastic distinctions in the absorption and emission spectra at pH 7.4 (Figure S2), as well as the pH-dependent color changes of their respective solutions (Figure 2 and Figure S3). Such a large and finely tunable pKa shift between MC-Sec and MCP provides a new avenue for fluorescent sensing of Sec via the selective removal of the electron-withdrawing 2,4dinitrobenzenesulfonyl group.

Figure 1. Absorption and fluorescence spectra of MC-Sec and MCP (both 10.0 µM) at various pH values. (a) and (c) show the pH-dependent absorption spectra of MC-Sec and MCP, respectively. (b) and (d) show the pH-dependent fluorescence spectra (λex = 550 nm) of MC-Sec and MCP, respectively. Inset: (a) and (b) show the pH-dependent absorbance (λabs = 566 nm) or fluorescence intensity (λem = 593 nm) changes of MC-Sec; (c) and (d) show the pH-dependent absorbance (λabs = 572 nm) or fluorescence intensity (λem = 600 nm) changes of MCP.

We then examined the fluorescence profiles of MC-Sec at different pH values (Figure 1b). Upon increasing pH from 2.4 to 10.5, it can be observed that MC-Sec loses almost all of its characteristic emission at 593 nm. This is apparently due to the conversion from PSB to SB occurs with decreasing the acidity of the solution. Therefore, MC-Sec exists in two forms in aqueous solution, as either the fluorescent PSB form or the complementary non-fluorescent SB form (Figure S1a), and the population of them will depend on the solution pH. The significant difference in the absorption and fluorescence spectra between the SB and PSB forms of MC-Sec can be explained as follows. The PSB form of MC-Sec features one nitrogen atom with an electron pair as a donor moiety (the indole ring) and a second positively charged nitrogen atom as an acceptor (the iminium cation), bridged by the conjugated pentamethine chain. This push−pull π-electron system has similar conjugation pattern to that of Cy5 (Figure S1), thus affording cyanine-like optical properties. Abstraction of a proton from this system (to form its SB form) interrupts the resonance effect between the two nitrogen atoms of the dye, and as result, it absorbs at short wavelength and has no fluorescence emission.33 This embodies a relatively facile means to modulate the spectroscopic characteristic of a dye without the need to cage its optically tunable functional group.

Figure 3. Absorption (a) and fluorescence spectra (λex = 550 nm) of MC-Sec (10 µM) upon addition of Sec (0-130 µM) in phosphate buffer (20 mM, pH 7.4, 1% acetone) for 5 min. Inset of Figure 3b shows the emission intensity at 593 nm as a function of Sec concentration.

Optical Response of MC-Sec to Sec. The optical response of MC-Sec toward Sec was then investigated by UV-vis absorption and fluorescence spectroscopy in phosphate buffer (pH 7.4, 20 mM). As Sec is not stable, we generated it in situ by mixing tris(2-carboxyethyl) phosphine hydrochloride (TCEP) and selenocystine ((SeC)2) according to the reported method.45 The UV-vis spectrum of MC-Sec exhibits two absorption bands centered at 423 and 566 nm, which was assigned to its SB and PSB form, respectively. With the introduction of Sec (0-130 µM) to the solution of MC-Sec, the absorption peak at 423 nm declines in the dark with concomitant increase in the absorption at 566 nm (Figure 3a). This conversion exhibits an isosbestic point at ~488 nm, which indicates the generation of a well-defined new product. The


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Analytical Chemistry above spectral changes are apparently due to the pKa difference between MC-Sec and MCP. Simultaneously, the fluorescence response of MC-Sec toward Sec was also systematically investigated. As anticipated, MC-Sec was weakly emissive under this condition. Upon treating with increasing concentrations of Sec (0-130 µM), the maximum emission of the solution at 593 nm gradually increases, and the final enhancement factor is over 21-fold (Figure 3b). Moreover, the fluorescence intensities were linearly proportional to Sec at the concentration range of 0-70 µM (r = 0.9989, Figure 3b, inset). The detection limit of MC-Sec for Sec was calculated to be 68 nM (3σ/slope method) under the test conditions. It should be noted that the pKa value of 6.40 measured for MC-Sec is not sufficiently low to maintain its nonfluorescent SB form be predominated at physiological pH, which would inevitably leads to a background fluorescence signal. This should be addressed in further studies by optimizing the probe’s structure. The above sensing mechanism of MC-Sec toward Sec has been proved by HRMS and HPLC analyses. Mass spectrometric analysis of the product generated from the incubation of MC-Sec with Sec in acetone/phosphate buffer (2 : 1, v/v, pH 7.4) solution confirms the formation of MCP, and an intense peak at m/z 350.2567 corresponding to [MCP + H]+ (calcd. 350.2590 for C27H31N2O+) is clearly observed in the HRMS data (Figure S4). To further validate this reaction mechanism, the reaction solution of MC-Sec and Sec was subjected to HPLC. As shown in Figure S5, authentic solutions of MC-Sec and MCP display a single peak with a retention time of 37.8 and 8.0 min, respectively. When the solution of MC-Sec was treated with Sec, a new peak correlated well with that of MCP is clearly observed. These data confirms that MC-Sec can react with Sec to afford MCP, which is responsible for the fluorescence signal observed in the sensing system. As MC-Sec contains an electron-deficient 2,4-dinitrobenzyl moiety, the intramolecular photo-induced electron (PET) process from the excited fluorophore to the electron-deficient 2,4-dinitrobenzyl moiety might occur, thus quenching the fluorescence of MC-Sec.46 To ascertain whether the PET process was involved, the fluorescence behavior of MC-Sec and MCP were compared under acid conditions (pH = 4.0). In this condition, MC-Sec and MCP are predominantly present as their fluorescent PSB form, and their fluorescence quantum yields were determined to be 0.050 and 0.092, respectively, with rhodamine B (φF = 0.66, in ethanol) as reference.47 Generally, nitro-containing dyes are almost nonfluorescent due to quenching of the singlet excited state by intramolecular PET process. However, in this case, it is evident that the PET from the mecocyanine dye to the 2, 4-dinitrobenzyl group within MC-Sec is less favorable. Thus, we can conclude that the weak emission of MC-Sec under neutral conditions is the combined effects of PET and the disruption of the push-pull πconjugation system of the probe via proton abstraction. However, the latter contributes to a much greater extent than the former, which can be evidenced from the absorption and fluorescence spectral changes of MC-Sec upon treating with Sec (Figure 3). The time-course fluorescence response of MC-Sec toward Sec was investigated. As shown in Figure 4, the fluorescence

intensity of MC-Sec (10 µM) in the presence of Sec increased and reached a plateau at about 3 min, whereas the free probe exhibited no noticeable changes in the emission intensity. This fast and distinct fluorescence signal change indicates that MCSec might be suitable for real-time sensing and imaging of intracellular Sec. In the case of other reactive sulfur species (Cys, GSH and NaHS), no significant fluorescence increase is observed under identical conditions. The pseudo-first-order rate constants for Sec and GSH were determined as 0.793 and 0.0042 min-1, respectively (Figure S6).48. The observed rate constant for Sec is ~189-fold faster than that for GSH, which are informative for the underlying reasons of the selectivity of the probe to Sec. All these initial results suggest that MC-Sec can be used as a fluorescent turn-on probe for rapid detection of Sec under mild conditions.

Figure 4. Time-dependent fluorescence intensity changes of MCSec (10 µM) in the presence of Sec (40 µM) or other reactive sulfur species (Cys, 300 µM; GSH, 1 mM; NaHS,100 µM) in phosphate buffer (20 mM, pH 7.4, 1% acetone). λex/λem = 550/593 nm.

Selectivity Studies. To test the selectivity of MC-Sec toward Sec, the probe was incubated with other biologically relevant species in phosphate buffer (20 mM, pH 7.4, 1% acetone). As displayed in Figure 5, only Sec induced an obvious absorption variation and fluorescence enhancement, while other reactive sulfur species (NaHS, HSO3-, Cys, GSH), biologically related ROS (ClO-, H2O2), reducing agents (ascorbic acid, TCEP), glucose, Na2SeO3, (SeC)2, amino acids (Val, Leu, Ile, Pro, Phe, Tyr, Trp, Ser, Thr, Met, Asn, Gln, Asp, Glu, Lys, Arg, His, Gly, Ala), metal ions (Cd2+, Mg2+, Mn2+, Al3+, Pb2+, Fe3+, Cu2+, Co2+, Ca2+), exhibited no or negligible emission intensity changes (Figure S7). The high selectivity of MC-Sec toward Sec can also be easily perceived with the naked eye, and only Sec induces an obvious color change under ambient light or UV light at 365 nm (Figure 5, inset). This selectivity is due to the relatively lower pKa (SeH) of Sec and higher nucleophilicity of selenolate (R-Se-).21 Thus, these results demonstrate that MC-Sec can selectively report Sec without the interference from biologically relevant analytes under physiological conditions.



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by MC-Sec afforded almost no fluorescence signal, indicating the species in living cells including biothiols do not interfere with the sensing of Sec when using MC-Sec. However, when the cells were treated with MC-Sec, and further incubated with Sec (20 and 50 µM), a concentration-dependent red fluorescence was observed. These results clearly indicate that MC-Sec is cell permeable and can be used to detect exogenous Sec in living cells. The above results motivate us to further sense the endogenous Sec in living cells. As Na2SeO3 can be metabolized to selenol through a series of physiological processes,49,50 the cells were firstly stimulated with Na2SeO3 (two different concentrations: 10 and 20 µM) for 12 h, and then incubated with MC-Sec. As expected, a concentrationdependent fluorescence enhancement was clearly observed (Figure 7). Furthermore, the semiquantitation of the averaged fluorescence intensity in Figure 6 and 7 was conducted. It was observed the emission intensity of the cells shows a dosedependent fluorescence increase with different concentrations of Sec or Na2SeO3 (Figure S10). Taken together, the above cell imaging experiments demonstrate that MC-Sec can report both exogenous and endogenous selenol in living cells.

Figure 5. Absorption spectral (a) and fluorescence intensity changes (b) of MC-Sec (10 µM) upon incubation with Sec and other biologically relevant species in phosphate buffer (20 mM, pH 7.4, 1% acetone) for 5 min. λex/λem = 550/593 nm. 1, Blank; 2, 100 µM NaHS; 3, 1 mM HSO3-; 4, 300 µM Cys; 5, 1 mM GSH; 6, 100 µM ClO-; 7, 100 µM H2O2; 8, 500 µM ascorbic acid; 9, 1 mM TCEP; 10, 300 µM Glucose; 11, 500 µM Na2SeO3; 12, 500 µM (SeC)2; 13, 40 µM Sec. Data were expressed as mean ± standard deviation (SD) of three experiments. Inset of Figure 5a and 5b are the photographs of MC-Sec solution in the presence of Sec and other reactive sulfur species exposed to ambient light or a UV lamp at 365 nm, respectively.

Figure 6. Fluorescence imaging of Sec in SMMC-7721 cells stained with MC-Sec. First row: cells were incubated with probe MC-Sec (20.0 µM) for 15 min. Second and third: cells were pretreated with Sec (20 or 50 µM) for 15 min and then incubated with MC-Sec (20.0 µM) for 15 min. DAPI (1.0 µg mL-1) was used to track the cell nuclei. Scale bars represent 20 µm.

Application of MC-Sec in Biological Systems. Encouraged by the above-mentioned outcome, we further investigated the potential application of MC-Sec for detection of Sec in biological fluids. Fetal bovine serum (FBS) samples were diluted 10-fold with phosphate buffer (20 mM, pH 7.4) and then spiked with different amounts of Sec (0-100 µM). The above solutions were then incubated with MC-Sec for 5 min and subjected to fluorescence measurements. As shown in Figure S8, a concentration-dependent fluorescence increment was acquired in these serum solutions, which confirms that MC-Sec can be used for the selective detection Sec in complex biological system such as serum. Furthermore, the capability of MC-Sec for tracking Sec in living cells was evaluate using SMMC-7721 as a model cell line. Before cell imaging applications, the cytotoxicity of MCSec in SMMC-7721 cells was investigated by a conventional MTT assay. The results indicate that MC-Sec has very low cytotoxicity to the cells below 50 µM (Figure S9). Fluorescent imaging of Sec with MC-Sec in living cells was then investigated. As shown in Figure 6, SMMC-7721 cells stained

Figure 7. Confocal fluorescence images of endogenously produced selenol through Na2SeO3 stimulation in SMMC-7721 cells. First row: cells were incubated with MC-Sec (20.0 µM) for 15 min. Second and third row: cells were pretreated with Na2SeO3 (10 or 20 µM) for 12 h and then incubated with MC-Sec (20.0 µM)


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Analytical Chemistry for 15 min. DAPI (1.0 µg mL-1) was used to track the cell nuclei. Scale bars represent 20 µm.

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CONCLUSION In conclusion, we report in this study a merocyanine-based fluorescent probe for Sec by exploiting the unique fluorescence OFF-ON switching between the SB and its complementary PSB form of the merocyanine dyes. The fluorescence turn-on response of MC-Sec is realized via the selective removal of the strong electron-withdrawing 2, 4dinitrobenzenesulfonyl group by Sec, leading to the pKa shift of the imine nitrogen of the probe from 6.40 to 9.04, and thus significantly increases the population of the fluorescent PSB form of the dye at physiological pH. In addition, MC-Sec has been demonstrated to be capable of imaging both exogenous and endogenous Sec in living cells. To our best knowledge, this is the first example of fluorescent probe for biological species designed via the large and tunable pKa shift of imines. The proposed mechanism is quite general, and therefore, we can reasonably expect to develop future probes directed to other target molecules by employing this simple but effective design strategy.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Experimental methods, NMR and mass spectra data of probes, and more experimental results and figures as noted in the text.

AUTHOR INFORMATION Corresponding Author *Fax: (+) 86-29-81535026. E-mail: [email protected]. *Fax: (+) 86-29-88382217. E-mail: [email protected].

ORCID Xiao-Feng Yang: 0000-0003-3640-2741

Notes Any additional relevant notes should be placed here.

ACKNOWLEDGMENT We acknowledge the National Natural Science Foundation of China (Nos. 21475105, 21675123) for financial support.

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Analytical Chemistry

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A Sensitive and Selective Fluorescent Probe for Selenol in Living

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