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A Ratiometric Fluorescent Probe for Vicinal Dithiol-Containing Proteins in Living Cells Designed via Modulating the ICT–TICT Conversion Process Yuanyuan Wang, Yaogang Zhong, Qin Wang, Xiaofeng Yang, Zheng Li, and Hua Li Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b02923 • Publication Date (Web): 20 Sep 2016 Downloaded from http://pubs.acs.org on September 20, 2016

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Analytical Chemistry

A Ratiometric Fluorescent Probe for Vicinal Dithiol-Containing Proteins in Living Cells Designed via Modulating the ICT–TICT Conversion Process Yuanyuan Wang,a † Yaogang Zhong,b † Qin Wang,a Xiao-Feng Yang,*,a Zheng Li,b Hua Li*,a, c a

Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of Ministry of Education, College of Chemistry & Materials Science, Northwest University, Xi’an 710127, P. R. China. Fax: (+) 86-29-81535026, E-mail: [email protected] b College of Life Sciences, Northwest University, Xi'an 710069, P. R. China c College of Chemistry and Chemical Engineering, Xi'an Shiyou University, Xi'an 710065, P. R. China. ABSTRACT: Vicinal dithiol-containing proteins (VDPs) play a significant role in maintaining the cellular redox homeostasis and are implicated in many diseases. To provide new chemical tools for VDPs imaging, we report here a ratiometric fluorescent probe CAsH2 for VDPs using 7-diethylaminiocoumarin as the fluorescent reporter and cyclic 1,3,2-dithiarsenolane as the specific ligand. CAsH2 shows peculiar dual fluorescence emission from the excited intramolecular charge transfer (ICT) and twisted intramolecular charge transfer (TICT) states in aqueous media. However, upon selective binding of protein vicinal dithiols to the trivalent arsenical of CAsH2, the probe was brought from the polar water media into the hydrophobic protein domain, causing the excited state ICT to TICT conversion being restricted; as a result, an increase from the ICT emission band and a decrease from the TICT emission band were observed simultaneously. The designed probe shows high selectivity toward VDPs over other proteins and biological thiols. Preliminary experiments show that CAsH2 can be used for the ratiometric imaging of endogenous VDPs in living cells. So far as we know, this is a rare example of ratiometric fluorescent probe designed via modulating the ICT–TICT conversion process, which provides a new way to construct various protein-specific ratiometric fluorescent probes.

leading to a nonspecific fluorescent labeling reaction.17 Another method adopts 1,3,2-dithiarsenolane as the binding site.20-22 This approach was initially developed by Griffin et al to image proteins in living cells based on the specific binding of two appropriately spaced arsenics within a bisarsenical fluorescent probe to two pairs of vicinal thiols in tetracysteine motifs (CCXXCC, where C and X represent Cys and any amino acid, respectively) that were genetically fused to the target protein.23 Later, this strategy was employed by Huang et al to develop fluorescent probes for VDPs in living cells.20,21 Unfortunately, unbound probes remaining inside cells will cause an increase in the background fluorescence and thus hinder the identification of labeled proteins. To address this issue, extensive washing of cells has to be carried out to remove free probes, which are not readily amenable to realtime monitoring of VDPs in living cells. Recently, fluorogenic probes have been developed to circumvent this problem.24,25 Generally, these probes exhibit low background fluorescence in a free state and glow only upon association with a prescribed protein. Because the removal of the unbound probes is unnecessary, real-time measurements and monitoring of molecular events in living cells is possible. In our earlier investigation, we created a light-up fluorescent probe for VDPs by adopting a fluorogenic mechanism based on an environment-sensitive fluorophore.26 Despite advances in the light-up fluorescent probes, they tend to be influenced by a variety of factors such as fluctuations in

Introduction Native proteins with cysteine-sulfhydryls in proximity that can undergo reversible dithiol/disulfide conversions are referred to as vicinal dithiol-containing proteins (VDPs).1 VDPs are recognized as key components involved in many biological processes through a fine balance between protein vicinal dithiols and disulfides, and play fundamental roles in the maintenance of redox homeostasis of living systems.2,3 VDPs are also associated with the formation and stabilization of protein structures during the protein post-translational modification.4 Apart from that, VDPs are involved in protein synthesis and functions, and are responsible for many diseases such as cancer,5,6 stroke, 7 and human immunodeficiency virus type1(HIV-1)8 and neurological disorders.9-11 Therefore, the development of fluorescence probes for VDPs in biological systems is of significant importance. Over the past few years, numerous fluorescent probes for thiol-containing compounds have been developed.12-16 However, probes that can discriminate VDPs from biothiols are sparsely reported.17-22 Currently, two strategies have been reported for constructing fluorescent probes for VDPs. One method employs two space-closed maleimide groups as the recognition unit, which quenches the probe’s fluorescence until they both undergo specific thiol addition reaction.17-19 However, their application in intracellular labeling is interfered by millimolar concentrations of glutathione (GSH) in cells, which can undergo a similar addition reaction, thus

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excitation intensity, environmental conditions, and the probe concentration. Comparatively, ratiometric fluorescent probes can reduce the above-mentioned errors by outputting a ratiometric signal of the fluorescence intensities at two emission bands. So far, only one ratiometric fluorescent probe for imaging VDPs have been developed based on the fluorescence resonance energy transfer (FRET) mechanism.27 Unfortunately, FRET-based probe is constrained by the requirement a substantial spectral overlap and favorable orientations of the transition dipoles for the donor emission and acceptor absorption bands.28 Obviously, these prerequisite would restrict the choice of dye pair, thus making the probe design complicated and lacking versatility. Clearly, the establishment of new switching strategies for VDP-specific ratiometric fluorescent probes is highly desired. In this study, we describe the rational design of CAsH2 as a new ratiometric fluorescent probe for VDPs based on the modulation of ICT to TICT conversion process (Scheme 1). In the proposed sensing system, 7-diethylamincoumarin was selected as the fluorescence reporter and cyclic dithiaarsane as the specific ligand for VDPs. In aqueous media, the proposed probe exists both as ICT and TICT excited states upon photoexcitation, thus a dual fluorescence emission being observed. Upon selective binding of protein vicinal dithiols to the trivalent arsenical of CAsH2, the probe was brought from the polar water media into the hydrophobic protein domain, which is generally in a low-polar environment. With the decrease of environmental polarity around CAsH2, the formation energy barrier of TICT increases, causing the ICTTICT conversion being restricted. Hence, an increase in the ICT emission band and a decrease in the TICT emission band should be observed in the fluorescence spectra simultaneously. Because of the separate emission bands from the ICT and TICT states, the above fluorescence changes enable us to develop a ratiometric fluorescent probe for VDPs with a simple structure. Although TICT-based probes have been developed for different analytes,29-32 most of them are operated on the fluorescence turn–on mode. To the best of our knowledge, this is the first time that a ratiometric fluorescent probe with high selectivity for VDPs was designed via modulating the ICT–TICT state conversion process. The proposed probe has been successfully applied for visualization of VDPs in living cells. Scheme 1. Proposed mechanism for the ratiometric fluorescent sensing of VDPs with CAsH2

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Experimental Section Materials and instruments. All chemicals and reagents were used directly as obtained commercially unless otherwise stated. Solvents were dried by standard procedures before use. Double distilled water was used throughout the experiments. Flash chromatography was performed using Qingdao Ocean silica gel (200 - 300 mesh). Human hepatocellular carcinoma SMMC-7721 cells and murine fibrosarcoma L929 cells were obtained from Cell Engineering Research Centre and Department of Cell Biology of Fourth Military Medical University (China). CAsH1, CAsH2, CAsH3 and compound 6 were dissolved in DMSO as stock solution. Reduced bovine serum albumin (rBSA) and other reduced form proteins were prepared and stored according to the literature.17 Fluorescence measurements were performed on a Perkin Elmer LS-55 fluorescence spectrometer in 10 × 10 mm quartz cells. Unless specific noted, the PMT voltage was set at 750 V. UV-visible spectra were recorded in 1 cm quartz cells with a Shimadzu UV-2550 spectrophotometer. 1H NMR and 13C NMR spectra were recorded at 400 and 100 MHz on an INOVA-400 spectrometer (Varian Unity), respectively. Chemical shifts are reported in ppm (δ) and are referenced to residual protic peaks. High-resolution mass spectra were acquired with a Bruker micrOTOF-Q II mass spectrometer (Bruker Daltonics Corp., USA) in electrospray ionization (ESI) mode. Fluorescence imaging was performed on an Olympus FV1000 confocal laser scanning microscope (Japan). Cell Cultures and fluorescence imaging. L929 cells were seeded in 6-well culture plates containing sterile coverslips and were cultured in RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum (FBS), 100 U mL-1 penicillin and 100 µg mL-1 streptomycin at 37 oC in a humidity atmosphere under 5% CO2 for 24 h. The medium was removed and the adherent cells were washed with PBS buffer (pH 7.4) three times. After the cells were incubated with 5 µM of CAsH2 (or compound 6) in serum-free RPMI medium at 37 °C for 20 min, the staining solution was replaced with fresh PBS to remove the remaining free probe. Cell imaging was then performed by an Olympus FV1000 confocal laser scanning microscope immediately. Emission was collected at 430–490 nm for the cyan channel and at 530–590 nm for the yellow channel (λex = 405 nm). For the control experiment, the cells were pretreated with dithiothreitol (DTT, 10 mM) for 30 min. After washing with PBS three times, the cells were further treated with CAsH2 and imaged using the conditions described above. To test colocalization with the mitochondria, SMMC-7721 cells (which were cultured using the same procedure for L929 cells) were co-stained with 2 µM of CAsH2 and 0.5 µM of Mito-Tracker deep red for 30 min and then fluorescence image were acquired by confocal microscopy. Emissions were collected at 430–490 nm for CAsH2 (λex = 405 nm) and at 655–755 nm for Mito-Tracker deep red (λex = 640 nm). ImageJ software was used for analysis of the images.

Results and Discussion Probe design. In our continuing efforts to develop fluorescent probes for VDPs, we have developed a fluorogenic

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Scheme 2. Synthesis of probe CAsH1, CAsH2, CAsH3 and compound 6 a

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O N

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CAsH1

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CAsH3

NH2

g 2

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+ O

O 5 N H

6 a

Reagents and conditions: (a) EDC, HOBt, CH2Cl2, room temperature, 4 h; (b) 6-aminohexanoic acid, DSC, DMAP, DMF, room temperature, 4 h; (c) PAO-EDT, HATU, DIPEA, DMF, room temperature, overnight; (d) diethyl malonate, piperidine, C2H5OH, reflux, 5 h; (e) 6-aminohexanoic acid, DSC, DMAP, DMF, room temperature, 4 h; (f) PAO-EDT, HATU, DIPEA, DMF, room temperature, overnight; (g) HATU, DIPEA, DMF, room temperature, overnight. probe for specific detection of VDPs in living cells. However, the intensity-based probe is inferior to ratiometric one, as the former is vulnerable to variations in the assay environment, thus posing potential problems for their use in quantitative measurements of VDPs in biological systems. Therefore, ratiometric fluorescent probes for VDPs are highly desired. It is well-known that 7-dialkylaminiocoumarins are solvatochromic dyes, which exhibit dim fluorescence in polar solvents but become brightly fluorescent in low-polar solvents.33 The marked change of the emission yields of these coumarin derivatives is interpreted in terms of a charge stabilizing influence by solvent or the microenvironment of the dye, which tends to affect the energy barrier for torsional motion leading to the TICT state.34-36 Such control on the charge distribution in the coumarins and hence their optical properties have been widely studied.37-40 It is thus expected that solvatochromic coumarins will be ideal fluorophores to probe the target proteins by incorporating with a proteinspecific ligand. On the other hand, 2-(4-aminophenyl)-1,3,2dithiarsolane (PAO-EDT) was selected as the recognition unit

because it can discriminate VDPs from monothiols proteins through the specific interchange of 1,2-ethanedithiol (EDT) in cyclic dithiaarsanes with vicinal dithiols in proteins.20 With the reasoning described above, we designed probe CAsH1 and CAsH2 by coupling 7-diethylaminocoumarin-3carboxylic acid with 2-(4-aminophenyl)-1,3,2-dithiarsolane (PAO-EDT). We envisioned that the binding of the probe to VDPs would bring it into the hydrophobic pocket of the protein, which is generally less polar than the bulk aqueous phase and would greatly affect the ICT and TICT emission, thus resulting in ratiometric fluorescent signals output. Scheme 2 shows the synthesis routes of CAsH1 and CAsH2, as well as its control probe CAsH3 and compound 6. The detailed synthetic procedures and spectroscopic characterization of these compounds are provided in the Supporting Information. With the probe in hand, we initially study its optical properties in aqueous media. Surprisingly, compared with common aminocoumarin dyes, it was observed that CAsH2 features a broad emission peak at 550 nm with a blue-shifted

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Analytical Chemistry shoulder at 475 nm (Figure 1). This dual emission can be rationalized by the well-accepted ICT–TICT conversion process in aminocoumarins.41,42 Upon photoexcitation, CAsH2 initially forms an ICT state with a partial charge transfer. In highly polar media, the diethylamino group subsequently undergoes twisting which makes the donor orbital perpendicular to the acceptor orbital. This results in a full charge separation and in the formation of the TICT state. Based on the above facts, it is assumed that the observed dual emission of CAsH2 in aqueous solution is attributed to the two excited states ICT and TICT, respectively. To prove the above hypothesis, we studied the optical characteristics of CAsH2 by measuring its absorption and emission spectra in the mixture solutions of water and 1,4dioxane with different polarities. As the proportion 1,4dioxane of in the binary mixture (1,4-dioxane – water) increased from 0% to 99%, corresponding to a decrease in solution polarity (∆f: 0.32 → 0.08),43 the absorption maxima of CAsH2 shifted from 430 to 408 nm, and only small changes were observed in the absorbance in all of these solutions with different polarities (Figure S1). In contrast, the fluorescence band of CAsH2 at 475 nm was found to shift to 440 nm, concomitant with a significantly increased fluorescence intensity (110-fold increase). Obviously, this emission band originates from the ICT process, which generally shows a positive response to the environmental polarity similarly to classical solvatochromic dyes. Meanwhile, it was observed that the longer emission band at 550 nm is very sensitive to the solution polarity and the emission band completely vanished even in 10% 1,4-dioxane aqueous solution (Figure 1), which indicates that this emission band is indeed contributed by the TICT state. The quenching of TICT emission can be explained as follows: with the decrease of solution polarity, the formation energy barrier of TICT state increases; as a result, TICT process is prohibited. To get more information about the effect of solution polarity on the emission behavior of CAsH2, we further test the fluorescence spectra of CAsH2 in the mixture solutions of water and ethanol with different polarities. Because the polarity of ethanol is higher than that of 1,4-dioxane (dielectric constant at 20 oC: ethanol, ɛ = 25.3; 1,4-dioxane, ɛ = 2.219),44 it was observed that when the ethanol fraction (fe, by volume%) in the H2O/ethanol mixture is increased from 5 to 30%, the intensity of the TICT emission band of CAsH2 at 550 nm decreases markedly, while the ICT band at 475 nm undergoes a distinct increment simultaneously. When fe is 30%, the TICT peak (550 nm) in the longer wavelength region disappears, and the ICT emission (475 nm) becomes the only peak observed in the fluorescence spectrum (Figure S2). As a result, the ratio of ICT and TICT emission intensities (IICT/ITICT) can be used as a polarity indicator. It was also observed from Figure S2 that the emission peak at 475 nm further increased with decreasing the polarity of the media (fe > 30%), which is caused by reducing the charge transfer between the fluorophore and its surrounding media.

Fluorescence Intensity (a.u.)

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fd(vol%) 0 10 20 30 40 50 60 70 80 90 99

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Wavelength (nm) Figure 1. Fluorescence spectra (λex = 415 nm) of CAsH2 (1 µM) in water/1,4-dioxane mixtures with different volume fractions of 1,4-dioxane (fd). HV = 650 V.

Next, to confirm the longer emission band of CAsH2 is from the TICT state, we prepared probe CAsH3, where unlike the probe CAsH2, the N,N-diethylamino group is substituted by a bridged julolidinyl structure which makes the rotation of the diethylamino group with respect to the coumarin skeleton unavailable; as a result, TICT state formation would be absolutely forbidden. The emission spectra of CAsH2 and CAsH3 in aqueous media were shown Figure S3. Contrary to the dual emission of CASH2, CAsH3 exhibits one major emission band from the ICT state (λem = 495 nm). It is thus evident that the second red-shifted emission band (λem = 550 nm) of CAsH2 is attributed to the TICT state, since no such emission is observed for CAsH3. Despite the wide acceptance of TICT process in aminocoumarin derivatives, fluorescence emission from the TICT states in these dyes are scarcely observed. To further ascertain the contribution of the emissive TICT in CAsH2, compound 6 was prepared and compared its optical properties with that of CAsH2. The molecular structures of CAsH2 and compound 6 differ in that the former bears a cyclic dithiaarsane (Scheme 2). As shown in Figure S4, the fluorescence spectra of compound 6 showed a maximum emission at 475 nm. In the case of CAsH2, however, a redshifted new emission band (λmax = 550 nm) was observed, which has been proved to be contributed by the TICT state. Thus, it can be concluded that the 5-membered dithiarsolane ring is exclusively responsible for the emissive TICT state of CAsH2. However, the complexity of the photophysical pattern requires further investigation to provide a detailed explanation. Based on the above results, it is shown that CAsH2 affords peculiar dual fluorescence emissions from the ICT and TICT excited states in highly polar solvent, their populations depending on the environmental polarity. Moreover, the ICT and TICT emissions of CAsH2 show completely opposite polarity-dependence, and a hypochromatic shift of ~ 110 nm was observed with decreasing the polarity of the media. The above characteristic makes CAsH2 favorable for the dualemission ratiometric sensing the polarity of its immediate environment. Thus, we assumed that CAsH2 would serve as a ratiometric fluorescent probe for VDPs. The selective binding of protein vicinal dithiols to the trivalent arsenical of CAsH2 would bring the probe from the polar water media to the

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o C. Inset: time course of fluorescence intensity of CAsH2 in the presence of rBSA.

hydrophobic protein domains with low polarity, which would result in the enhancement of ICT emission channel at the expense of TICT emission band. Therefore, ratiometric sensing of VDPs might be realized. Optical response of CAsH2 toward VDPs. Inspired by the ratiometric fluorescence response of CAsH2 toward the environmental polarity, we then examined the sensing of probe CAsH2 toward VDPs in phosphate buffer solution (20 mM, pH 7.4, containing 1% DMSO). We selected rBSA as the model protein because it contains eight vicinal Cys pairs after BSA is reduced with tris(2-carboxyethyl)phosphine (TCEP).17 In our initial attempt to develop ratiometric fluorescent probe for VDPs, we constructed CAsH1 and examined its sensing behavior toward rBSA (Figure S5). Although CAsH1 shows a desirable ratiometric fluorescence response toward rBSA, its slow binding kinetics (> 60 min) makes its unsuitable for monitoring the dynamic changes of VDPs inside living cells. We envisioned that the slow response of CAsH1 toward rBSA might be due to the steric hindrance arising from the fluorophore adjacent the active site. In an advance, this issue was addressed by placing a flexible linker (6-aminocaproic acid) between the coumarin fluorophore and PAO-EDT (to form CAsH2). The time course of CAsH2 in the presence of rBSA was then examined by fluorescence spectroscopy. It can be observed from Figure 2 that the fluorescence emission band at 468 nm increases dramatically upon addition of rBSA, concomitant with a decrease of TICT emission band at 550 nm. The emission ratio (I468/I550) of the sensing system affords an initial fast, followed by a gradual increase in the sensing process, while the free probe exhibited no noticeable emission ratio changes under identical conditions (Figure S6). The emission ratio of the sensing system essentially reached a maximum after 20 min, and thereafter remained almost unchanged. Compared with CAsH1 and the previous reported probe, the present probe CAsH2 affords a fast response to rBSA, which makes it suitable for monitoring the dynamic changes of VDPs in situ. In addition, the reversibility of the present fluorescent sensing system was also proved by adding a large amount of EDT to the solution of CAsH2-rBSA complex (Figure S7).20

750

Next, the sensing behavior of CAsH2 toward rBSA was examined with absorption and fluorescence spectra. As shown in Figure S8, the absorption spectrum of CAsH2 displays a strong ICT band at 440 nm. Upon incremental addition of rBSA, this band blue-shifted to 428 nm, concomitant with a gradual increase in absorbance signal. The above results are consistent with the solvatochromic properties of environmentsensitive dyes. Furthermore, free CAsH2 (1 µM) shows two emission bands centered at 475 and 550 nm (λex = 415 nm), which can be assigned as the ICT and TICT emission bands, respectively. When increasing concentrations of rBSA (0 – 1.8 µM) were introduced, the fluorescent spectrum of CAsH2 exhibited significant changes (Figure 3). It was observed that the emission band at 475 nm undergoes a distinct increase and the emission maximum blue-shifted to 468 nm, while the band at 550 nm exhibits an obvious decrease simultaneously. Meanwhile, it was observed that the emission color of CAsH2 turned from pale green to cyan in the presence of rBSA when illuminated by a hand-held UV lamp at 365 nm (inset of Figure 3). The above changes in emission spectra suggest that location of the probe was changed from the polar aqueous environment to the low-polar protein interior. Another possible explanation is that the interior of protein might be too rigid to twist the probe in the excited state, thus the relaxation from the highly fluorescent ICT state to a weakly fluorescent TICT state being hampered. The ratio of emission intensities at 468 and 550 nm (I468/I550) with the addition of rBSA varies from 0.33 to 24.5, a ca. 74-fold emission ratio change. Furthermore, the emission ratios (I468/I550) were plotted as a function of rBSA concentration and a typical calibration graph was obtained as shown in Figure 4. The I468/I550 value was linearly related to rBSA concentration up to 1.2 µM with a detection limit of 2.6 nM (3δ). These results demonstrate that CAsH2 can detect rBSA quantitatively. 600

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Wavelength (nm) Figure 3. Fluorescence spectra of CAsH2 (1 µM) upon addition of increasing concentrations of rBSA (0 – 1.8 µM) in phosphate buffer (pH 7.4, 20 mM, containing 1% DMSO) for 20 min at 37 o C. λex = 415 nm. HV = 700 V. Inset: images of CAsH2 alone and in the presence of rBSA exposed to a UV lamp at 365 nm.

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Figure 2. Time-dependent fluorescence spectral (λex = 415 nm) changes of CAsH2 (1 µM) in the presence of rBSA (0.6 µM) in phosphate buffer (20 mM, pH 7.4, containing 1% DMSO) at 37

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Y = 7.474X + 0.121 r = 0.9953

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Figure 4. Linear plot of the emission ratio (I468/I550) against rBSA concentration when using CAsH2 (1 µM).

(b)

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Furthermore, to test the generalization of the probe, other reduced forms of proteins (human serum albumin, ovalbumin, lysozyme, pepsin and trypsin) were screened under the same conditions, and it was observed that reduced human serum albumin (rHSA) affords a fluorescence response similar to rBSA, while reduced forms of ovalbumin and lysozyme affords a relatively small fluorescence changes (Figure S9). This is apparently due to different VDPs having different reactivities with CAsH2. It was further observed that reduced pepsin and trypsin afford almost no changes in emission ratio. This can be explained by the fact that pepsin and trypsin contain almost no hydrophobic pockets as in rBSA.45 To verify our hypothesis that the fluorescence change comes from the binding reaction between CAsH2 and VDPs and provide a better understanding of the above detecting mechanism, the following experiments were carried out. First, the reference compound 6 was treated with rBSA, however, it shows negligible fluorescence increase (increased by 26%) at 480 nm (Figure S10). The above experiments prove that the 5membered dithiarsolane ring in CAsH2 is involved in the binding reaction and the fluorescence change is indeed due to the selective binding of CAsH2 with vicinal dithiols in proteins, while not the nonspecific interaction between the fluorophore and proteins. Next, the sensing behavior of CAsH3 toward rBSA was examined. As shown in Figure S11, CAsH3 displays a moderate increase (~ 3-fold increase) in fluorescence intensity with the addition of rBSA, accompanied by a blue-shift of emission maxima from 495 to 488 nm. The above variations in fluorescence spectrum are analogous to that of CAsH2 at its ICT emission band. As the diethylamino group is rigidized by the julolidinyl structure, the fluorescence enhancement of CAsH3 is apparently ascribed to the lowpolar environment of protein interior, which reduces the charge transfer between the fluorophore and polar media, thus exhibiting a pronounced fluorescence enhancement at ICT band. However, due to a slight blue-shift in emission maxima (~ 7 nm), only fluorescence ‘turn-on” mode is applicable for rBSA detection in terms of CAsH3. The data convincingly demonstrates the participation of TICT in the signaling event of CAsH2 toward rBSA.

I468/I550

3 2 1

Hc Cy y sti ne as co DT rb T ic a ly cid so z ov ym alb e um in pe ps in a -c try hy ps m in ot ry ps in BS A rB SA

0 C A m sH et hi 2 on in e Cy s G SH

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Fluorescence intensity (a.u.)


Figure 5. (a) Fluorescence spectra (λex = 415 nm) of CAsH2 (1 µM) with the addition of various species in phosphate buffer (pH 7.4, 20 mM, containing 1% DMSO) for 20 min at 37 oC. The testing species are methionine, Cys, GSH, Hcy, cystine, DTT ascorbic acid (1 mM of each), lysozyme, ovabumin, pepsin, trypsin, α-chymotrypsin, BSA, rBSA (0.6 µM of each), respectively. (b) Fluorescence intensity ratio (I468/I550) of CAsH2 with the addition of various species. Selectivity studies. The selectivity of CAsH2 was studied by incubating the probe with some biologically relevant species in phosphate buffer. As shown in Figure 5, no significant fluorescence changes were observed when CAsH2 was mixed with some amino acids (methionine, Cys, homocysteine (Hcy), cystine), GSH, ascorbic acid and DTT (1 mM of each), proteins (lysozyme, ovabumin, pepsin, trypsin, α-chymotrypsin, BSA, 0.6 µM of each). In contrast, a dramatic ratiometric fluorescence change was obtained upon the addition of rBSA. It is worth noting that BSA (monothiol protein) affords a very small fluorescence change under identical conditions. This can be explained by the fact that monothiol arsines formed between BSA and CAsH2 are acyclic compounds which are unstable and spontaneously decompose to the starting materials.46,47 Thus, only VDPs can bind to CAsH2 with high affinity. It the case of DTT, although it contains vicinal dithiols, it induces no fluorescence changes under identical conditions. This is apparently because DTT is a small molecule and contains no hydrophobic pocket in its structure. The above results suggest the pronounced specificity of the probe toward VDPs.

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Analytical Chemistry channel (430–490 nm); middle: the emission was collected at yellow channel (530–590 nm); right: bright-field image. λex = 405 nm. Scale bar: 40 µm.

Imaging of endogenous VDPs in live cells. Encouraged by the above-mentioned outcome, we then examined the capability of CAsH2 for VDPs ratiometric fluorescence imaging in living cells. In our experiments, L929 cells were selected to demonstrate the application of live cell imaging of VDPs with CAsH2. Initially, the cytotoxicity of CAsH2 was evaluated by a standard MTT assay. Though inorganic arsenic is associated with high cytotoxicity, organic arsenicals are reported to be more stable and less toxic owing to enhanced rates of excretion.48 As shown in Figure S12, CAsH2 has no marked toxicity to L929 cells below 10 µM. Therefore, 5 µM of CAsH2 was selected for further imaging experiments in live cells. The dual-channel fluorescence images recorded at 430-490 nm and 530-590 nm with excitation at 405 nm were shown in Figure 6. Incubation of L929 cells with CAsH2 for 30 min provided a strong fluorescence in the cyan channel (Figure 6a) and a weak fluorescence in the yellow channel (Figure 6b). By contrast, in a control experiment, compound 6 was used for cell stain and it affords almost no fluorescence emission at the two emission bands (Figure 6 d and e). The above results indicate the selective binding of CAsH2 to endogenous VDPs inside living cells. Furthermore, the cells were pretreated with DTT (DTT is introduced as a reductant stimulation and can lead to an increase in the levels of endogenous VDPs22) for 30 min, and further incubated with CAsH2 for 30 min, eliciting an obvious fluorescence increase in the cyan channel (Figure 6g) but essentially no fluorescence in the yellow channel (Figure 6h). The semiquantitative calculation of ratio of cyan channel to yellow channel was further conducted, and it was observed that the ratio of DTTtreated cells is higher than that of the control cells (Figure S13). The above variations are consistent with the VDPsinduced fluorescence spectral changes of the probe. Thus, these data demonstrate that CAsH2 can be applied for ratiometric tracking of endogenous VDPs levels in living cells, which is of great importance to clarify the physiological roles of VDPs in living systems.

It was reported that there are abundant VDPs distributed in the mitochondria of cells, which make a significant contribution to the responses of mitochondria to both oxidative damage and redox signaling.49 Therefore, we further investigate the subcellular localization of VDPs in SMMC7721 cells by fluorescence colocalization experiments. The cells were incubated with CAsH2 and Mito-Tracker Deep Red (a commercially available mitochondrial localizing dye) simultaneously for 30 min, and then the cells were imaged by confocal microscopy. As shown in Figure 7, there is an obvious overlap between fluorescence signal from CASH2 and Mito-Tracker Deep Red (the Pearson’s correlation coefficient between cyan and red fluorescence images was calculated to 0.82), implying that CAsH2-labeled VDPs are mainly distributed in the mitochondria of these live cells. The above experiments prove that there are abundant VDPs within mitochondria, which is in accordance with the previous studies.22,49

a

b

c

Figure 7. Colocalization experiments of CAsH2 and Mito-Tracker Deep Red in SMMC-7721 cells. (a) CAsH2 (2 µM) stain, λex = 405 nm, collected 430- 490 nm. (b) Mito-Tracker Deep Red (0.5 µM) stain, λex = 635 nm, collected 655- 755 nm. (c) Merged image of (a) and (b). Scale bar, 10 µm.

Conclusion In summary, CAsH2 has been designed as a ratiometric fluorescent probe for VDPs in living cells, where 1,3,2dithiarsenolane group functions not only as a recognition unit but also as an initiator of TICT emission. Comparison of structural analogues of CAsH2 in their fluorescent properties suggests that the sensing mechanism for VDPs occurs through the modulation of ICT-TICT conversion process. The designed probe shows high selectivity toward VDPs over other proteins and biological thiols and proves to be able to imaging of VDPs in living cells. Compared with the existing FRETbased probe, the present probe requires no dye pair in proximity to provide a signal and can be prepared via simple synthetic steps. As far as we know, this is a rare example of ICT-TICT based ratiometric fluorescence sensing system, and we believe that the present study paves the way to construct new protein-specific ratiometric fluorescent probes. Figure 6. Confocal fluorescence images of L929 cells treated with CAsH2 or compound 6 (both 5 µM). (a – c), The cells was stained by CAsH2; (d – f), the cells was stained by compound 6; (g – i), the cells were preincubated with DTT (10 mM) followed by incubation with CAsH2. Left: the emission was collected at cyan

ASSOCIATED CONTENT Supporting Information Experimental supplementary methods for chemical synthesis and characterization of compounds, more experimental results and

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figures as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]; [email protected];

Author Contributions Y.W. and Y.Z. contributed equally to the present work.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This research was supported by the National Natural Science Foundation of China (nos. 21475105, 21275117, 21375105) and the Education Department (no. 12JK0581) of Shaanxi Province of China.

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