Yuanyuan
Wang
a,
Xiao-Feng
Yang
*a,
Yaogang
Zhong
b,
Xueyun
Gong
a,
Zheng
Li
b and
Hua
Li
*ac
aKey Laboratory of Synthetic and Natural Functional Molecule Chemistry of Ministry of Education, College of Chemistry & Materials Science, Northwest University, Xi'an 710069, P. R. China. E-mail: xfyang@nwu.edu.cn
bCollege of Life Sciences, Northwest University, Xi'an 710069, P. R. China
cCollege of Chemistry and Chemical Engineering, Xi'an Shiyou University, Xi'an 710065, P. R. China. E-mail: huali@nwu.edu.cn
First published on 23rd October 2015
Vicinal dithiol-containing proteins (VDPs) play a key role in cellular redox homeostasis and are responsible for many diseases. Here, we develop a red fluorescent light-up probe FAsH for the highly selective and sensitive detection of VDPs using the environment-sensitive 2-(4-dimethylaminophenyl)-4-(2-carboxyphenyl)-7-diethylamino-1-benzopyrylium (F1) as the fluorescent reporter and cyclic dithiaarsane as the targeting unit. FAsH is almost nonfluorescent in aqueous solution. However, it exhibits intense fluorescence emission upon binding to reduced bovine serum albumin (rBSA, selected as the model protein). The fluorescence intensity of FAsH is directly proportional to the concentration of rBSA over the range of 0.06–0.9 μM, with a detection limit (3δ) of 0.015 μM. Importantly, the fast kinetics of binding between FAsH and VDPs (∼2.5 min) enables the dynamic tracing of VDPs in biological systems. Preliminary experiments show that FAsH can be used for the no-wash imaging of endogenous VDPs in living cells. In addition, our study shows that F1 presents both high environment-sensitivity and good fluorescence properties, and is promising for the development of no-wash fluorescent light-up probes for target-specific proteins in living cells.
In recent years, two strategies have been adopted for the development of fluorescent probes for VDPs. One method employs two maleimide groups as the recognition unit, which quenches the probe's fluorescence until they both undergo thiol addition during the labeling reaction. These fluorogenic probes have been used to label vicinal thiol-containing peptides/proteins.7 However, intracellular labeling still remains a challenge for these probes since intracellular glutathione (GSH) (1–10 mM) can undergo a similar addition reaction, thus leading to a nonspecific fluorescent labeling reaction.7b Alternatively, 1,3,2-dithiarsenolane was incorporated into a variety of fluorophores to develop fluorescent probes for VDPs which have been used to identify VDPs in living cells.8 The method employs the fact that trivalent arsenicals can bind to vicinal thiol proteins with high affinities,9 whereas proteins with thiols that are not vicinal (referred to as monothiols) interact weakly with arsenicals.10 However, a potential drawback of these fluorescent probes is the strong background fluorescence from the unreacted probes inside cells which hinders the identification of labeled proteins.8a–c To circumvent this problem, a tedious washing procedure (>15 min) is required to remove the unbound probes in order to reduce the background fluorescence, which will inevitably delay the acquisition of microscopic data and thus makes the measurements prone to artifacts. To overcome this deficiency, a ratiometric fluorescent probe for VDPs was recently developed by Huang et al. based on the fluorescence resonance energy transfer (FRET) mechanism.8d Although ratiometric probes can overcome the influence of a variety of factors such as instrumental efficiency, environmental conditions and the probe concentration, the proposed probe exhibits only moderate fluorescence variations (ca. 6-fold) upon binding to VDPs. Furthermore, the probe shows a sluggish response to VDPs (>60 min), which makes it unsuitable for the real time monitoring of VDPs inside living cells. Therefore, it is highly desirable to develop fluorescence turn-on probes for intracellular VDPs that have the combination of high selectivity, fast labeling rates, and high fluorescent turn-on ratios.
Recently, a simple strategy for designing fluorescence turn-on probes for selective protein detection has been developed by taking advantage of environment-sensitive fluorophores,11 which generally involves the incorporation of an environment-sensitive fluorophore into a ligand specific to the target protein. Typically, these probes exhibit very weak fluorescence in polar and protic environments, while the fluorescence is enhanced when the environment becomes hydrophobic or less polar. They could provide a fluorogenic response to their immediate environment, resulting in a variety of applications in bioanalytical chemistry. This light-up strategy has paved a new way for the detection of targeting proteins with high sensitivity and selectivity.
Although several environment-sensitive fluorophores have been reported,12 they show some crucial drawbacks. First, most of those used for light-up probe design are limited to those with blue or green emission, whereas far-red and near-infrared (NIR) dyes are advantageous for cellular studies due to lower photodamage, light scattering, and autofluorescence in living systems.13 Second, sensitivity to solvent polarity of these dyes is frequently not enough to detect subtle changes in the environment of the biomolecule of interest. Finally, polarity-sensitive dyes with red emission generally exhibit relatively lower sensitivity to polarity compared to blue dyes.12b Therefore, currently intensive research is focusing on the design of environment-sensitive fluorophores with red-shifted emission.
Herein, we report the design and synthesis of a red fluorescent light-up probe for the rapid detection of VDPs both in vitro and in vivo with excellent sensitivity and specificity. In the proposed sensing system, 2-(4-dimethylaminophenyl)-4-(2-carboxyphenyl)-7-diethylamino-1-benzopyrylium (F1) was selected as the environment-sensitive fluorescence reporter and cyclic dithiaarsane as the specific ligand for VDPs. Our rationale is depicted in Scheme 1. We envisioned that the selective binding of protein vicinal dithiols to the trivalent arsenical of FAsH would bring the fluorophore into the hydrophobic protein domain, and the hydrophobic environment would cause the fluorophore to emit strong fluorescence. In contrast, in the absence of the target protein, the probe would remain in aqueous solution and should emit only weak fluorescence. Based on the above mechanism, we created a selective fluorescence turn-on probe toward VDPs inside living cells with no-wash procedures. Compared with the reported fluorescent probes, FAsH is cell-permeable and shows a rapid response toward VDPs with high sensitivity. Moreover, the proposed probe can operate in the red region, which is favorable for biological applications in vitro and in vivo. The proposed probe has been used for rapid no-wash imaging of VDPs in living cells.
Scheme 1 (a) Proposed reaction between FAsH and rBSA; (b) schematic illustration of the fluorescence turn-on mechanism for rBSA detection with FAsH. |
Fig. 1 Schematic illustrating the rotational freedom and the electron donor–acceptor–donor (D1–A–D2) system in F1. |
We then studied the solvatochromic properties of F1 by measuring its absorption and emission spectra in different proportions of water and 1,4-dioxane with different polarities. As shown in Fig. S1 (ESI†), all the absorption spectra have maxima at about 598 nm, and there are no significant changes observed in the solutions with different polarities. In contrast, solvent polarity had a dramatic effect on the emission spectra of F1. When the orientation polarizability (Δf) of the solution decreased from 0.32 (99% water) to 0.292 (20% water),16 the maximum emission wavelength of F1 shifted from 646 to 635 nm, concomitant with a gradual increase in fluorescence intensity (Fig. 2a). The fluorescence intensity of F1 at 635 nm increased by a factor of 14.2. The above results reveal that F1 is a polar-sensitive (solvatochromic) fluorescent dye.
Moreover, the multiple electron-donating groups in F1 are linked to the benzopyrylium unit via a single bond. Thus, it features high rotational flexibility and possesses two different twisted intramolecular charge transfer (TICT) channels within the whole molecule (involving twisting of the dimethylaminophenyl and diethylamino groups, respectively, as shown in Fig. 1).17 These intramolecular rotations lead to the nonradiative deactivation of the fluorescent excited state, which might be another cause for the fluorescence quenching of F1 in aqueous solution. To test this assumption, we checked the effect of solvent viscosity on F1 emission. The intramolecular rotation process is reported to be influenced by the viscosity of the medium: the higher the viscosity of the medium, the slower the intramolecular rotation and hence the stronger the F1 emission.18 We then evaluated the viscosity effect on the emission behavior of F1 in methanol/glycerol mixtures with different fractions of glycerol (fg). As shown in Fig. 2b, the emission intensity of F1 is greatly enhanced as the solvent viscosity increases from 0.60 (methanol) to 950 cP (99% glycerol) at room temperature (25 °C),19 which is typically observed for molecular rotors. These experimental results support the fact that F1 is a viscosity-sensitive dye.
Since F1 features a dual dependency of emission intensity on both solvent polarity and viscosity, we thus expect that it would hold great promise in the development of environment-sensitive fluorescent probes compared to the traditional solvatochromic fluorescent dyes. Next, we checked the possible nonspecific interactions of F1 with serum proteins by introducing bovine serum albumin (BSA, 1.0 mg mL−1) to the aqueous solution of F1. As shown in Fig. S2 (ESI†), only negligible changes in the emission intensity (2.6-fold increase) of F1 were observed, suggesting there are few nonspecific interactions between F1 and BSA. This is crucial for the development of probes for in vivo imaging, as a high nonspecific background signal is the main reason for their failure. Finally, F1 contains a carboxylic acid group, which enables facile attachment of the recognition moiety. On the basis of the aforementioned results, F1 was selected as the fluorophore to construct the probe.
Additionally, we selected 2-(4-aminophenyl)-1,3,2-dithiarsolane (PAO-EDT) as the recognition unit because 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.20 In addition, its 5-membered dithiarsolane ring is a more stable complex compared with the 6- and 7-membered ones.21 In view of the above mentioned results, we rationally designed a red fluorescence turn-on probe FAsH for VDPs in living cells. The detailed synthetic procedures and characterization of FAsH are shown in Scheme 2. Meanwhile, F4 which lacks the 5-membered dithiarsolane ring in its structure was also prepared for comparison purposes.
The fluorescence response of FAsH toward rBSA was examined by introducing increasing concentrations of rBSA (0–2.4 μM) to the solution of FAsH. As shown in Fig. 4, the free probe gives extremely weak fluorescence in aqueous solution (φf = 0.006, using F1 in CH2Cl2 as a reference).23 However, the addition of an increasing amount of rBSA to the solution of FAsH elicits a gradual increase in the fluorescence intensity and the final enhancement factor is over 70-fold (φf = 0.21). This intensity increase was also accompanied by a hypsochromic shift in the emission spectra from 658 to 651 nm during the titration. The increase in the fluorescence intensity and the hypsochromic shift of the fluorescence emission maxima may be attributed to the binding of FAsH to the hydrophobic domain of rBSA. The fluorescence intensity at 651 nm as a function of rBSA concentration was recorded, and a nearly linear relationship over the range of 0.06–0.9 μM was obtained (Fig. S6, ESI†). The detection limit (3δ) for rBSA was calculated to be 0.015 μM. These results demonstrate that FAsH can detect rBSA with high sensitivity. Furthermore, to test the sensing behavior of FAsH toward different VDPs, we examined other reduced forms of proteins (human serum albumin, ovalbumin and lysozome) and found that reduced human serum albumin (rHSA) also induces a dramatic increment in emission intensity, while reduced ovalbumin affords a moderate fluorescence enhancement. In the case of reduced lysozome, a very small increment in emission intensity is observed (Fig. S7, ESI†). This is apparently due to different VDPs having different reactivities with FAsH. Thus, FAsH is unsuitable for the quantitative determination of VDP content in complicated biological systems because different VDPs will afford different increments in emission intensity.
Fig. 6 The selective binding of FAsH to VDPs was verified by SDS-PAGE. “+”: the compound was present in the detection system; “−”: the compound was absent in the detection system. |
Fig. 7 Absorption spectra of FAsH (10 μM) in the presence of rBSA or BSA (both 0.3 μM) in phosphate buffer (20 mM, pH 7.4, containing 1% acetone as cosolvent). |
Furthermore, to prove the fluorescence enhancement of the sensing process is caused by the hydrophobic pocket of rBSA, guanidine hydrochloride (GdnHCl), a strong protein denaturant, was introduced into the solution of the FAsH–rBSA complex, and a significant decrease in emission intensity was observed (Fig. S11, ESI†). This is apparently due to the unfolding of rBSA and the hydrophobic pocket in rBSA is destroyed.27 As a result, the probe gets more exposed to the polar environment, which is undesirable for fluorescence emission. The above results reveal the essential role of the hydrophobic cavities of the protein folding structure in the present sensing system. Collectively, we can conclude that the fluorescence enhancement of the present sensing system is achieved by reducing the charge transfer between the fluorophore and the polar media and restricting the intramolecular rotations via aggregation simultaneously (Scheme 1b).
Next, some experiments were performed to evaluate FAsH in live-cell imaging assays using human hepatoma cells (SMMC-7721) as a model cell line. Initially, the cytotoxicity of FAsH was evaluated using a standard MTT assay. Although PAO is quite toxic, the results showed that FAsH has minimal cytotoxicity at concentrations of 2–20 μM (Fig. S13, ESI†). This is apparently due to the EDT caging unit, which prevents FAsH from exerting acutely toxic effects.20 Next, SMMC-7721 cells were incubated with FAsH for 20 min in PBS, and a strong fluorescence signal was observed. Furthermore, in a control experiment, the cells were pretreated with 30 μM PAO (a selective VDP binding reagent) to reduce the amount of intracellular free VDPs prior to incubation with FAsH. A pronounced fluorescence quenching was observed (Fig. S14, ESI†), which reveals that the above fluorescence emission (Fig. 8) is indeed induced by VDPs. By contrast, F4 was used for cell staining and it affords negligible fluorescence emission under the same conditions (Fig. 8). The semiquantitative calculation of the averaged fluorescence intensity was further conducted. The emission intensity of FAsH-stained cells is about 19-fold higher than that of F4-treated cells (Fig. S15, ESI†). The significant difference in emission intensity indicates the selective binding of FAsH to endogenous VDPs inside live cells, and this selective binding is apparently due to the 5-membered dithiarsolane ring. These results demonstrate the capacity of FAsH for in situ imaging of VDPs in living cells.
To further investigate the subcellular localization of VDPs, a commercially available mitochondrial tracker (rhodamine 123) was used for a colocalization study with confocal microscopy. As displayed in Fig. 9, the observed fluorescence signal from FAsH extensively overlaps with that of rhodamine 123, implying that FAsH-labeled VDPs are mainly localized to the mitochondria of these live cells. Furthermore, nuclear staining with DAPI indicates that the cells are viable throughout the imaging experiments. The above experiments prove that there is an abundance of VDPs distributed in the mitochondria of SMMC-7721 cells, which is consistent with previous studies.8a,28
Footnote |
† Electronic supplementary information (ESI) available: Experimental procedures, characterization data, and additional spectra. See DOI: 10.1039/c5sc02824h |
This journal is © The Royal Society of Chemistry 2016 |