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A Far-Red Fluorescent Probe for Imaging of Vicinal DithiolContaining Proteins in Living Cells Based on a pKa Shift Mechanism Shengrui Zhang, Guojun Chen, Yuanyuan Wang, Qin Wang, Yaogang Zhong, Xiaofeng Yang, Zheng Li, and Hua Li Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b05429 • Publication Date (Web): 19 Jan 2018 Downloaded from http://pubs.acs.org on January 19, 2018

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Analytical Chemistry

A Far-Red Fluorescent Probe for Imaging of Vicinal DithiolContaining Proteins in Living Cells Based on a pKa Shift Mechanism Shengrui Zhang,†,‡ Guojun Chen,† Yuanyuan Wang,† Qin Wang,†,‡ Yaogang Zhong,§ 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: Vicinal dithiol-containing proteins (VDPs) play fundamental roles in intracellular redox homeostasis and are responsible for many diseases. In this work, we report a far-red fluorescence turn-on probe MCAs for VDPs exploiting the pKa shift of the imine functionality of the probe. MCAs is composed of a merocyanine Schiff base as the fluorescent reporter and a cyclic 1, 3, 2-dithiarsenolane as the specific ligand for VDPs. The imine pKa of MCAs is 4.8, and it exists predominantly in the Schiff base (SB) form at physiological pH. Due to the absence of a resonating positive charge, it absorbs at a relatively short wavelength and is essentially nonfluorescent. Upon selective binding to reduced bovine serum albumin (rBSA, selected as the model protein), MCAs was brought from aqueous media to the binding pockets of the protein, causing a large increase in pKa value of MCAs (pKa = 7.1). As a result, an increase in the protonated Schiff base (PSB) form of MCAs was observed at the physiological pH conditions, which in turn leads to a bathochromically shifted chromophore (λabs = 634 nm) and a significant increase in fluorescence intensity (λem = 657 nm) simultaneously. Furthermore, molecular dynamics simulations indicate that the salt bridges formed between the iminium in MCAs and the residues D72 and D517 in rBSA resist the dissociation of proton from the probe, thus inducing an increase of the pKa value. The proposed probe shows excellent sensitivity and specificity toward VDPs over other proteins and biologically relevant species and has been successfully applied for imaging of VDPs in living cells. We believe that the present pKa shift switching strategy may facilitate the development of new fluorescent probes that are useful for a wide range of applications.

Vicinal dithiol-containing proteins (VDPs) are proteins that contain two space-closed thiol groups. These proteins are attracting more and more attention as the interconversion between protein vicinal dithiols and disulfides plays an particularly important role in many biological processes, such as the maintenance of intracellular redox homeostasis, protein synthesis and post translational modification.1, 2 In addition, VDPs hold a particularly prominent position in formation and stabilization of protein structures, and are responsible for many diseases such as cancer,3 diabetes,4 stroke,5 and neurodegeneration.6 Recent studies indicate that VDPs are overexpressed in aggressive tumors and the tumor cells appear more dependent on these VDPs for DNA synthesis.7 Thus, it is important to develop an efficient method for the detection and quantification of VDPs under physiological conditions. Fluorescent probes are well-suited for monitoring intracellular VDPs. In recent years, several fluorescent probes for VDPs have been developed based on different mechanism. Huang et al. first developed a series of 1, 3, 2-dithiarsenolanetethered fluorophores as VDP-specific fluorescent probes.8-10

Unfortunately, as the free and VDP-binding probes show almost no difference in emission intensity, a tedious washing procedure is required to remove the unbound probes in order to reduce the background signals, which may restrict their applications in real-time imaging of VDPs in living systems. Later, they developed a ratiometric fluorescent probe for VDPs based on fluorescence resonance energy transfer mechanism;11 however, the proposed probe exhibits only moderate fluorescence variations (ca. 6-fold) upon reaction with VDPs. Alternatively, several turn-on fluorescent probes for VDPs have been developed by using two space-closed maleimide groups as the recognition unit.12-14 Unfortunately, this kind of probe can also react with millimolar concentrations of biothiols in cells to afford a fluorescence enhancement, which will impose serious interference in fluorescence imaging of intracellular VDPs. In order to address these issues, we recently constructed two fluorescent probes for VDPs by using polarity-sensitive fluorophores as the fluorescent reporter and 1, 3, 2-dithiarsenolane as VDPspecific ligand.15, 16 However, one major concern for environment-sensitive probes is their potential nonspecific

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interactions with serum proteins and lipid membranes, causing high background fluorescence signals.17, 18 To date, only a few of them have been successfully applied in imaging of target proteins in living cells.19-21 Clearly, the establishment of a new sensing strategy to construct turn-on fluorescent probes for VDPs in living systems is highly desirable. In recent years, it was reported that retinal Schiff base generated from cellular retinoid-binding proteins and retinal provides an ideal platform for switching on and off an observable spectroscopic signal.22 The imine (Schiff base, SB) usually absorbs at short wavelength, whereas the iminium (protonated Schiff base, PSB) causes a bathochromic shift in the absorption maximum that is dependent on the protein environment (see Figure S1, Support Information).23-28 The bathochroism in absorption for the iminium is probably due to the more delocalized nature of the cationic charge along the poylene. Later, this system was extended to merocyanine Schiff base because its polyene tail structurally resembles the retinal derivatives.29-31 In addition to the above red-shifted absorption maxima, the conversion of the SB to the PSB leads to a dramatic fluorescence enhancement.30 Thus, regulation of the equilibrium between SB and PSB forms of merocyanine Schiff base would yield turn-on fluorescent probes for the target molecule. On the other hand, encapsulation of chromophoric dyes in the cages of supramolecular assemblies such as cyclodextrins,32, 33 calixarenes,34 and cucurbiturils35-39 is a wellrecognized strategy to modify the protonation–deprotonation equilibria (pKa values) of the dyes through a complexationinduced pKa shift method. Moreover, fluorescent dyes often respond strikingly to such complexation-induced supramolecular pKa shifts because many of their physicochemical properties are intrinsically related to their chemical identity.40 Thus, we envisioned that the pKa of the imine nitrogen of merocyanine Schiff base should be sensitive to its microenvironment, and the biomolecular pockets of protein might have pronounced effect on the prototropic equilibrium of the dye, which in turn leads to large spectral changes as well as dramatic fluorescence enhancement. These changes can be used to detect a target protein by attaching the dyes to an “affinity reagent” that binds only to the target protein.

In this study, we report MCAs as a far-red fluorescence turn-on probe for VDPs exploiting the pKa shift of the imine functionality of the probe. Our rationale is depicted in Scheme 1. MCAs is predominantly present as the SB form under physiological pH conditions. Due to the absence of a resonating positive charge, it absorbs at a relatively short wavelength and is essentially nonfluorescent. Upon selective binding of VDPs, MCAs was brought from aqueous media to the binding pockets of the protein, and the microenvironment provided by the protein has considerable impact on the electronic charge distribution of the probe, which in turn leads to an increase in the pKa value of MCAs. As a result, an increase in the PSB form of MCAs was observed at physiological pH, which leads to a bathochromically shifted chromophore and a significant increase in fluorescence intensity simultaneously. The proposed probe shows excellent sensitivity and specificity toward VDPs over other proteins and biological thiols and has been successfully applied for imaging of VDPs in living cells. To the best of our knowledge, this is the first example of implementing protein-induced pKa shift concept in the selective detection of VDPs.

EXPERIMENTAL SECTION Materials, Apparatus and General Experimental Methods. For details, see Supporting Information.

Scheme 2. Synthesis of MCAs and MCSB. Reagents and conditions: (a) sodium acetate, acetic anhydride, room temperature, 60 min; (b) EtOH, room temperature, 30 min. Fluorescence imaging of VDPs in living cells. Before experiments, SMMC-7721 cells were seeded in 6-well culture plates containing sterile coverslips and allowed to adhere for 24 h. The medium was removed and the cells were incubated with MCAs (5.0 µM) in RPMI medium at 37 °C for 30 min. The cells were further incubated with nucleus staining dye DAPI (1.0 µg mL-1) in the same medium for 10 min. After that, the staining solution was replaced with fresh PBS to remove the remaining free dye. Cell imaging was then acquired with an Olympus FV1000 confocal laser scanning microscope immediately. Excitation wavelength for MCAs: 635 nm; Emission collection: 655-755 nm; Excitation wavelength for DAPI: 405 nm; Emission collection: 425-475 nm. In the redox stimulus experiments, the cells were pretreated with H2O2 (100 µM) or DTT (10 mM) for 30 min. After washing with PBS three times, the cells were further treated with MCAs and imaged using the conditions described above.

Scheme 1. Schematic illustration the fluorescence turn-on response of MCAs toward VDPs.


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Analytical Chemistry transition between SB and PSB forms in MCAs was calculated to be 4.7 by using the Hasselbach-type mass action equation41 (for the detailed calculation, please see Supporting Information). Next, pH-dependent fluorescence spectral changes of MCAs were investigated. As the SB form of MCAs is essentially nonfluorescent, the absorption wavelength corresponding to the PSB form (590 nm) was selected as the excitation wavelength. It was observed that the solution of MCAs gives weak emission under neutral to basic conditions. Upon decreasing pH from 10.5 to 2.3, the fluorescence intensity of the solution of MCAs undergoes a ca. 139-fold increase and the emission maximum shifts from 644 to 650 nm (Figure 1c), indicating that the conversion from SB to PSB occurs with increasing the acidity of the solution. Figure 1d shows the pH-dependent fluorescence intensity changes of MCAS at 650 nm, exhibiting a pKa of 4.8, which was very close to that from the absorption measurements. Meanwhile, the pH titrations of MCSB were carried out under identical conditions and it shows similar spectral changes compared with that of MCAs (Figure S2). However, its pKa value (7.5) is substantially higher (upward shift by 2.7 units) than that of MCAs. Their significant difference in pKa values is apparently due to the electron-withdrawing effect of cyclic dithiaarsane in MCAs. Therefore, at physiological pH of 7.4, MCAs exists predominantly in the nonfluorescent SB form (~ 99.7% abundance), while MCSB is present in equilibrium between the SB (44.3 %) and PSB form (55.7 %). This difference can be reflected by their drastic distinctions in the absorption and emission spectra (Figure S3), as well as the pH-dependent color changes of their respective solutions (Figure S4). The above results also indicate that the absorption and emission spectra of MCAs can be regulated by shifting its pKa value to a higher pH region. The significant difference in the absorption and fluorescence spectra between the SB and PSB form of MCAs can be explained as follows. The PSB form features one nitrogen atom with an electron pair as a donor moiety and a second positively charged nitrogen atom as an acceptor, bridged by a polymethine π-electron chain. This push−pull conjugative backbone has similar pattern to that of Cy5 (see Figure S5, Supporting Information), thus affording cyaninelike optical properties. In the case of its SB form, however, the iminium cation of the dye has been changed to the corresponding imine nitrogen, which disrupts the push-pull πconjugation system of the dye, and as a result, it absorbs at short wavelength and has negligible fluorescence emission. Optical response of MCAs to VDPs. Before carrying out the measurements, the stability of MCAs in aqueous solution was initially investigated as the Schiff base is susceptible to hydrolysis.42 To our delight, there was virtually no discernible changes in the absorption and fluorescence spectra of MCAs within 1 h, indicating that MCAs is remarkably stable in aqueous medium (Figure S6 and S7). Furthermore, the photostability of MCAs in aqueous solution was investigated. As shown in Figure S8, the photostability of MCAs is higher than that of Cy5 under identical conditions. After 90 min continuous irradiation, about 90% of the initial probe was still retained, indicating that MCAs has sufficient photostability

RESULTS AND DISCUSSION Design and synthesis of MCAs for VDPs. In our newly developed sensing system, merocyanine Schiff base was selected as the signaling unit because its SB and PSB forms afford completely different spectral properties. 2-(4Aminophenyl)-1, 3, 2-dithiarsolane (PAO-EDT) was selected to construct the probe on the basis of the following considerations: (i) its As(III) center can selectively discriminate vicinal dithiols from other forms of thiols through the interchange of 1, 2-ethanedithiol (EDT) in cyclic dithiaarsanes with vicinal dithiols in proteins;8 (ii) its amino group facilitates the formation of Schiff base with merocyanine aldehyde. With the reasoning described above, we designed probe MCAs by coupling merocyanine aldehyde with PAO-EDT. Meanwhile, a control probe (MCSB) without a cyclic dithiaarsane was also synthesized (Scheme 2). The spectroscopic characterizations of these compounds are provided in Supporting Information.

Figure 1. Absorption (a) and fluorescence spectra (c, λex = 590 nm) of MCAs (9.0 µM for absorption and 3.0 µM for fluorescence spectra) in various pH values (2.3-10.5). (b) and (d) show the pH-dependent absorbance (λabs = 617 nm) or fluorescence intensity (λem = 650 nm) changes of MCAs. e) Photograph of MCAs at different pH values under ambient light.

With the probe MCAs in hand, we initially explored its optical properties in aqueous medium as a function of pH. Expectedly, the optical properties of MCAs are pH-sensitive. Figure 1a depicts the absorption spectra of MCAs at different pH values (2.3 – 10.5). It was observed that the SB form of MCAs dominates when pH ≥ 7.0, showing its absorption peak at 473 nm. On the other hand, acidification of the solution of MCAs results in a large red shift in the absorption spectrum (λabs = 617 nm) and a prominent color change from brown to blue simultaneously (Figure 1e), consistent with protonation of the imine nitrogen of MCAs to give its PSB form. A single isosbestic point at 519 nm was observed for this conversion, and the two forms of MCAs exist in a pH-dependent equilibrium (Figure 1a). Figure 1b shows the pH-dependent the absorbance changes of MCAs at 617 nm. The pKa of the



physiological pH owing to its SB form prevails. The addition of rBSA to the solution of MCAs leads to a significant, rBSA concentration-dependent fluorescence turn-on response, accompanied by a bathochromic shift of emission maxima from 643 to 657 nm. A maximum fluorescence intensity (ca. 27-fold increase) is reached when the amount of rBSA is more than 2.5 µM (Figure 2b). The optical response of MCAs toward rBSA can also be observed by the naked eye, and an obvious color change under visible or UV light was observed (Figure 2, inset figures). Furthermore, the fluorescence intensity at 657 nm is linearly dependent on the concentration of rBSA in the range of 0.06-1.05 µM (Figure 3). The detection limit of MCAs for rBSA was calculated to be 15 nM based on 3δ/k. These results demonstrate that MCAs can quantitatively detect rBSA with high sensitivity. In addition, other reduced forms of proteins (lysozyme, trypsin, αchymotrypsin, ovalbumin, γ-globulin and human serum albumin) were examined under the same conditions, and it was observed that reduced casein, γ-globulin and albumins afford fluorescence increments, while reduced lysozyme and trypsin afford almost no changes in emission intensity (Figure S9). The possible explanation is that lysozyme and trypsin contain almost no favorable local protein environment as in rBSA, thus failing to regulate the pKa value of MCAs.

for biological applications. Next, we evaluated the absorbance and fluorescence responses of MCAs toward VDPs in phosphate buffer (20 mM, pH 7.4) at 37 °C. Freshly prepared reduced bovine serum albumin (rBSA) was used as the model protein because it contains eight vicinal cysteine (Cys) pairs. The free probe shows a major absorption band at 473 nm in neutral solution, indicating its SB form dominates. However, upon treating with increasing concentrations of rBSA, the absorption of MCAs at 473 nm gradually decreased; simultaneously, a new absorption band centered at 634 nm emerged (Figure 2a). The variations in the absorption spectra indicate that the PSB form increased in the solution, suggesting a significant increase in the pKa value of MCAs upon binding to rBSA. Moreover, it was observed that the PSB absorption of MCAs-rBSA complex affords a bathochromic shift of ∼17 nm in comparison with that of free MCAs. This is apparently due to the change of the environment of the probe from bulky water to the binding pockets of the protein.

Figure 3. The linear relationship between the fluorescence intensity and the concentrations of rBSA (0.06-1.05 µM). λex/λem = 590/657 nm. The inset shows the emission intensity at 657 nm as a function of rBSA concentration.

Sensing Mechanism. Some experiments were carried out to decipher the underlying mechanism responsible for the fluorogenic response of MCAs toward VDPs. First, the reference compound MCSB was treated with rBSA; however, it shows a negligible fluorescence increase at 643 nm (Figure S10). The above experiments provide strong evidence that the cyclic 1, 3, 2-dithiarsenolane in MCAs is involved in the protein recognition and the fluorescence enhancement is indeed due to the selective binding of MCAs with vicinal dithiols in proteins. Next, the pKa value of MCAs-rBSA complex was calculated from its pH-dependent fluorescence intensity changes at 657 nm (Figure S11 and S12). The pKa value of MCAs-rBSA complex was determined to be 7.1, which affords a large upward pKa shift of ~ 2.3 units from its free probe value (Figure 4). Crucially, this rBSA-induced pKa shift significantly increases the PSB form of MCAs (from 0.3 % to

Figure 2. Absorption (a) and Fluorescence spectral (λex = 590 nm) changes of MCAs (3.0 µM) upon treating with increasing concentrations of rBSA (0-3.0 µM) in phosphate buffer (20 mM, pH 7.4, 1% acetone) for 25 min. The inset shows the photographs of MCAs solution upon addition of rBSA under ambient light (a) or UV light at 365 nm (b). Subsequently, the fluorescence sensing behavior of MCAs toward rBSA was examined under the same conditions. As shown in Figure 2b, the free MCAs is almost nonfluorescent at


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residue pair was calculated by the STRIDE program.45 As shown in Table S1, the Cys residues in the pairs of C75-C91 and C513-C558 have large values of SASA, indicating that these residues are largely exposed to aqueous medium. Accordingly, these two pairs are selected as active binding sites in simulations. For simplicity, the sites involving the pair C75-C91 and C513-C558 are referred to as pocket 1 and pocket 2, respectively.

33.4%) at pH 7.4, which is responsible for the absorption spectral changes as well as fluorescence enhancement of the sensing system. Furthermore, the fluorescence intensity of the acid solutions of MCAs and MCAs-rBSA complex were compared (Figure 1d and Figure S12), and it was observed that the fluorescence intensity of the latter was about 1.44-fold compared to the former despite the fact that both of them exist exclusively in the fluorescent PSB form. This discrepancy in fluorescence intensity is putatively due to the binding of the probe with rBSA, which could rigidify the backbone of the fluorophore, thus leading to an additional fluorescence enhancement. The above results also suggest that the fluorescence increase of the present system is mainly contributed by the binding-induced upward pKa shift of the imine nitrogen in MCAs. 1.00

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Figure 5. The typical snapshots of MCAs binding in the pocket 1(a) and pocket 2 (b) of rBSA, selected from the time evolutions of binding in equilibration. The pocket 1 is composed of 19 residues: E63, K64, S65, T68, L69, G71, D72, E73, L74, C75, K76, L80, A88, C91, E92, K93, Q94, E95, and R98. The 20 residues, viz., L178, P179, E182, T183, E186, E399, K431, T434, R435, F508, C513, D517, K520, Q521, K524, F553, V554, D555, C557, and C558, constitute the pocket 2. (c) and (d), magnified representation of MCAs binding in the pocket 1 and pocket 2 in (a) and (b), respectively. The salt bridge formed between MCAs and rBSA is represented by a red dotted line.

pH Figure 4. pKa shift of MCAs (3.0 µM) upon binding to rBSA (3.0 µM) monitored through fluorescence intensity change upon pH variation for the free probe MCAs and MCAs-rBSA complex.

As the hydrophobic cavity of BSA is generally in a lowpolar environment, we further investigate the effect of solution polarity on the fluorescence emission of MCAs. As shown in Figure S13, the fluorescence intensity of MCAs showed no discernible changes in a set of dielectric constant values ranging from 2.21 (1,4-dioxane) to 78.36 (H2O).43 It is worth noting that the polarity of the hydrophobic cavity of BSA is similar to that of acetone,44 and the fluorescence emission of MCAs in acetone are identical with that of in water, which proves that the fluorescence enhancement of the present sensing system is irrelevant to the microenvironmental polarity in the hydrophobic domain of rBSA. Thus, we assumed that the electrostatic environment in the vicinity of MCAs plays a key role in shifting the pKa value of the probe.30 Molecular Modeling. To gain insight into the pKa shift mechanism, the molecular dynamics simulations were performed for the binding of MCAs on rBSA (see Models and simulation models in Supporting Information). In the simulation, the vicinal Cys residue pairs on the surface of the protein are considered to be active binding sites, since the arsenic group can form strong covalent bonds with the thiols of vicinal Cys residue pair in the protein.8 To locate the vicinal Cys residue pairs on the surface of the protein, the disulfide bonds were identified from the native PDB structure of BSA, then the solvent accessible surface area (SASA) for each Cys

Figure 6. The time evolutions of salt bridges formed between MCAs and rBSA. (a) and (b) are the electrostatic interactions between the iminium in MCAs and D72 in the pocket 1, and D517 in the pocket 2 along the time evolution of binding,


Analytical Chemistry respectively. (c) and (d) are the number of hydrogen bonds formed between the iminium in MCAs and D72 in the pocket 1, and D517 in the pocket 2 as a function of binding time, respectively. The red curve in (a) and (b) is a line smoothed by adjacent-averaging algorithm.

SB form ((λabs = 473 nm) (Figure S16). This further proves that the fluorescence enhancement is indeed due to the formation of the PSB form of MCAs. In the case of BSA, it induced almost no absorption spectra and fluorescence intensity changes under identical conditions (Figure S17), indicating the high selectivity of MCAs toward vicinal dithiols in proteins.

The van der Waals interactions between MCAs and rBSA along the time evolution were calculated to check the equilibration of the system and the extent of the packing between the probe and protein. As shown in Figure S15, after the first 4 ns, the van der Waals interactions become stable in the pocket 1 and the corresponding energy Evdw is about -125 kJ mol-1; In the case of pocket 2, the van der Waals interactions attain equilibration upon the first 3 ns and the Evdw is around -175 kJ mol-1. The above results indicate that MCAs in pocket 2 has a better packing than that in pocket 1. The representative configurations of the probe binding in pockets 1 and 2 of the protein are displayed in Figure 5a and b, respectively. In the binding pocket 1, a salt bridge is formed between the probe and protein, since the distance between the hydrogen atom in the iminium of MCAs and the oxygen atom in the carboxylate of D72 is 1.9 Å (Figure 5c). In the binding pocket 2, the distance between the hydrogen atom in the iminium and the oxygen atom in the carboxylate of D517 is only 1.7 Å, indicating the presence of one salt bridge between the two groups as well. The salt bridge formed between MCAs and rBSA can increase the binding affinity. To maintain the salt bridge, the imine in MCAs would be kept in its protonated form as long as possible, which leads to an increase in the pKa value of the probe. To examine the robustness of the salt bridges, we calculated the electrostatic interactions and the number of hydrogen bonds between the iminium in MCAs and the carboxylates of D72 and D517 in protein along the time evolution of the binding (Figure 6). Upon the first 3 ns, the electrostatic interactions between the two groups are stabilized, the corresponding energy Eele is ~ 60 kJ mol-1 for the two binding pockets (Figure 6a and b); the number of hydrogen bonds remains to be 1 (Figure 6c and d), confirming that the salt bridges between MCAs and the two binding pockets of protein exist continuously. In addition, much weaker electrostatic interactions between the iminium in MCAs and residues, E73, E95, E182, and E399, in pockets 1 and 2 were identified during the binding, which might contribute to the shifting of the pKa value insignificantly. Thus, our study indicates that the salt bridges formed between the iminium in MCAs and the specific residues in rBSA resist the dissociation of proton from the probe, thus inducing an increase of the pKa value. Kinetic studies. The time-dependent fluorescence intensity changes of MCAs in the presence of rBSA were initially studied. As shown in Figure 7, the free MCAs exhibited almost no observable changes in the emission intensities at 657 nm. Upon addition of rBSA to the solution of MCAs, however, a significant fluorescence enhancement was immediately observed and it took about 20 min to attain an equilibrium state, indicative of a rapid response to rBSA, which makes it suitable for monitoring the dynamic changes of VDPs in situ. In addition, the mixture of MCAs and rBSA affords a time-dependent variation in the absorption spectra; that is, the absorption of the PSB form ((λabs = 634 nm) increases at the expense of the absorption characteristics of the

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Selectivity Studies. We then examined the specificity of MCAs towards VDPs under the same analytical conditions as previously noted. As shown in Figure 8, only rBSA promoted a significant fluorescence enhancement, while other common amino acids (Arg, Lys, His), biothiols (Cys, Hcy, GSH), tris(2-carboxyethyl) phosphine (TCEP), vitamin C (Vc), dithiothreitol (DTT) and proteins (lysozyme, trypsin, chymotrypsin, ovalbumin, HSA, BSA) displayed a negligible response. In addition, H2O2 and some common metal ions (Fe3+, Fe2+, Cu2+ and Zn2+) have negligible effects on the fluorescence emission of MCAs (Figure S18). We further examined the sensing behavior of MCAs toward rBSA in the presence of a large amount of biothiols (GSH, Cys and Hcy). As shown in Figure S19, these species exhibited virtually no interference in the detection of rBSA, which further confirms the specificity of MCAs towards VDPs over other cellular biothiols. The specificity of MCAs toward VDPs was then verified by polyacrylamide gel electrophoresis (PAGE). It can be observed from Figure S20, a fluorescence band was observed in the lane loaded with rBSA and MCAs, whereas the lane loaded with BSA and MCAs exhibited no fluorescence signal at the same conditions. In contrast, BSA and rBSA were both clearly observed in the gel after silver staining, demonstrating that the fluorescence band was indeed due to the formation of MCAs-rBSA complex. Moreover, the CD spectra of BSA and rBSA with the introduction of MCAs were recorded (Figure S21). It was observed that the introduction of MCAs to BSA solution affords negligible changes in the CD data, while the ellipticity of rBSA solution at 220 nm decreases by ~ 25% when MCAs was added. This can be explained by the fact that the arsenic group of MCAs can form strong covalent bonds with the thiols of vicinal Cys residue pair in the protein, thus the secondary structure of rBSA being partially perturbed. The above results further


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demonstrate the selective binding of rBSA with MCAs. In view of the above results, we can conclude that MCAs possesses excellent selectivity toward VDPs over other biologically relevant species.

determination clearly demonstrated that the fluorescence intensity decreases by 55% relative to control (Figure S25), indicating that levels of VDPs decreased owning to the formation of disulfide under the oxidant stimulus. Furthermore, addition of 10 mM DTT (a reductant stimulation that can lead to an increase in the levels of endogenous VDPs11) to the cell culture prior to incubation with MCAs resulted in intense fluorescence signal, and the fluorescence intensity leads to a ~1.47-fold increase compared to DTT untreated cells (Figure S24). In addition, nuclear staining with DAPI confirms that SMMC-7721 cells are viable under redox reagents stimulus. These results suggest that MCAs is amenable for monitoring the changes of VDPs under different cellular redox states in living cells.

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300 200 100 0 Bla nk Arg Ly s His Cy s Hc y GS TC H EP Vc D Ca TT γ -G s e i n l Lys obulin ozy m a -C hym Tryps e otr in Ov ypsi alb n um in HS A BS A rBS A

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Figure 8. Fluorescence intensity of MCAs (3.0 µM) upon mixing with different species in phosphate buffer (20 mM, pH 7.4, 0.3% acetone) for 25 min. λex/λem = 590/657 nm. Arg, Lys, His, Cys, Hcy and GSH, 1 mM; TCEP, Vc and DTT, 0.3 mM; casein, 0.2 mg/mL; γ-globulin, lysozyme, trypsin, α-chymotrypsin, ovalbumin, HSA, BSA and rBSA, 3.0 µM.

To further evaluate the feasibility of MCAs in cellular studies, the possible nonspecific interactions of the probe with serum proteins and lipid members were investigated by using BSA (the key component of serum) and lipid vesicles (the models of biological membranes) as the models.46 As shown in Figure S22 and Figure S23, MCAs displayed negligible fluorescence enhancement with the addition of increasing amount of BSA and large unilamellar vesicles (composed of dioleoylphosphatidylcholine, DOPC) to its aqueous solution, indicating that nonspecific interactions of MCAs with serum proteins or lipids would not affect the selective detection of VDPs. This can overcome the relatively high nonspecific background fluorescence signals of the previously reported environment-sensitive probes, which is crucial for vivo imaging studies. Fluorescence imaging of VDPs in living cells. Having established that MCAs can selectively respond to VDPs in aqueous solution, we further sought to apply this probe in cellular bioimaging applications using SMMC-7721 as a model cell line. Before cell imaging experiments, MTT assays were carried out to evaluate the cytotoxicity of MCAs on SMMC-7721 cells. The results indicate that MCAs (up to 30 µM) presents minimal toxic effects on cell viability (Figure S24). The application of MCAs for imaging of VDPs in SMMC-7721 cells was then carried out. As show in Figure 9, upon loading MCAs (5.0 µM) at 37 °C for 30 min, the cells displayed a bright red fluorescence image. We next tested whether this probe could respond to dynamic changes of VDPs under a redox stimulus in living cells. First, the cells were preincubated with 100 µM H2O2 (H2O2 is a commonly used thiol oxidant that can oxidize protein vicinal thiols to disulfide47) for 30 min to globally deplete intracellular VDPs levels, and further stained with MCAs and imaged by confocal microscopy. As shown in Figure 9, a significant decrease in fluorescence intensity was observed. Semiquantitative

Figure 9. Confocal fluorescence images of VDPs in SMMC-7721 cells by MCAs. First row: cells were incubated with probe MCAs (5.0 µM). Second row: cells were pretreated with H2O2 (100 µM) and then incubated with MCAs (5.0 µM). Third row: cells were pretreated with DTT (10 mM) and then incubated with MCAs (5.0 µM). DAPI (1.0 µg mL-1) was used to track the cell nuclei. Excitation wavelength for MCAs: 635 nm; Emission collection: 655-755 nm. Excitation wavelength for DAPI: 405 nm; Emission collection: 425-475 nm. Scale bars represent 30 µm.

CONCLUSION In conclusion, we have developed a merocyanine-based farred fluorescence turn-on probe for VDPs based on a pKa shift mechanism. The method employs the selective binding of VDPs to MCAs to increase the pKa value of its imine unit, causing an increase in the PSB form of MCAs at physiological pH conditions, which in turn leads to a bathochromic shift in the absorption spectrum and a significant fluorescence enhancement simultaneously. The proposed probe features high selectivity and sensitivity toward VDPs over biothiols and other proteins and has been demonstrated to be capable of imaging endogenous VDPs in living cells. So far as we know, this is the first example of target protein sensing system that utilizes the large pKa shift of imines at physiological pH. We believe that the present approach may facilitate the development of new fluorescent probes that are useful for a wide range of applications.



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ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Additional text, experimental methods, supplementary photophysical characterization 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: (+) 86-29-81535026. E-mail: [email protected]. ORCID Xiao-Feng Yang: 0000-0003-3640-2741 Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This research was supported by the National Natural Science Foundation of China (Nos. 21475105, 21675123). G. C. acknowledges the National Training Programs of Innovation and Entrepreneurship for Undergraduates (201710697015).

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