Selective Fluorescence Detection of Cysteine over Homocysteine and

Article pubs.acs.org/ac

Selective Fluorescence Detection of Cysteine over Homocysteine and Glutathione Based on a Cysteine-Triggered Dual Michael Addition/ Retro-aza-aldol Cascade Reaction Yawei Liu,† Xin Lv,† Min Hou,† Yawei Shi,‡ and Wei Guo*,† †

School of Chemistry and Chemical Engineering and ‡Institute of Biotechnology, Shanxi University, Taiyuan, Shanxi 030006, China

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S Supporting Information *

ABSTRACT: In this work, a cysteine (Cys)-triggered dual Michael addition/retro-aza-aldol cascade reaction has been exploited and utilized to construct a fluorescent probe for Cys for the first time. The resulting fluorescent probe 8alkynylBodipy 1 contains an activated alkynyl unit as Michael receptor and a Bodipy dye as fluorescence reporter and can highly selectively detect Cys over homocysteine (Hcy)/ glutathione (GSH) as well as other amino acids with a significant fluorescence off−on response (∼4500-fold) and an ultralow detection limit (0.38 nM). The high selectivity of 1 for Cys could be attributed to a kinetically favored five-membered cyclic intermediate produced by the dual Michael addition of Cys with the activated alkynyl unit of 1. The big fluorescence off− on response is due to the subsequent retro-aza-aldol reaction of the five-membered cyclic intermediate that results in the release of a highly fluorescent 8-methylBodipy dye 2. The probe has been successfully used to detect and image Cys in serum and cells, respectively.

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highly valuable for understanding their respective molecular mechanism of action, but considerably challenging due to the similar structures and reactivity of Cys/Hcy/GSH. Thus, in the past few years, many efforts have been devoted to address the concerns.35 As far as Cys is concerned, the pioneering works should be attributed to Strongin and his co-workers on the cyclization of aldehyde or acrylate with Cys/Hcy,36−38 by which the GSH-induced fluorescence signal could be largely precluded. For the same reason, the Cys/Hcy-triggered native chemical ligation with thioester or condensation reaction with cyano group were also reported.39−42 Further, for precluding the interference of Hcy, some new strategies were exploited on the basis of the extended version of the cyclization strategies,43−56 or GSH-protected silver nanoclusters,57 or Michael addition combined with steric, electrostatic, and hydrogen binding interactions.58−62 Besides, by utilizing the Cys-induced SNAr substitution-rearrangement reaction coupled with the different photophysical properties of amino- and thiosubstituted dyes, Cys could also be successfully discriminated from Hcy and GSH.63−68 These excellent works not only provide the important tools for studying Cys-relevant biological functions but also greatly stimulate research interest in the development of the new strategy for discriminating Cys from its competitive species, especially Hcy and GSH. In this work, a Cys-triggered dual Michael addition/retroaza-aldol cascade reaction was exploited and utilized to

iothiols, such as cysteine (Cys), homocysteine (Hcy), and glutathione (GSH), play the crucial roles in various physiological processes.1−4 Among them, Cys is an essential amino acid, involved in protein synthesis, detoxification, metal binding, post-translational modification, and metabolism.5,6 Cys deficiency is implicated in many syndromes, such as the slowed growth rate, skin lesions, lethargy, mentation, edema, and muscle and fat loss, as well as liver damage.7 The elevated levels of Cys could induce severe neurotoxicity and cardiovascular diseases.8 Therefore, an efficient method for selectively probing Cys in biological systems is of great importance for understanding its biological functions and even for early diagnosis of some diseases. Since the fluorescent indicators for calcium ion were reported by Tsien and co-workers in the early 1980s,9,10 fluorescent probes have been recognized as the efficient molecular tools to map the spatial and temporal distribution of biological molecules in biological systems due to their sensitivity, visualization, and noninvasive detection.11−21 To date, a large number of fluorescent probes have been developed for biothiols, and the corresponding design strategies are generally based on the strong nucleophilicity of thiol group combined with various reaction mechanisms such as Michael addition, cyclization, displacement of coordination, cleavage reactions, and others.22−34 From the selectivity point of view, the early stage design strategies are mainly focused on distinguishing Cys/Hcy/GSH from thiol-free amino acids. Because Cys, Hcy, and GSH levels are related to different physiological processes and diseases, the development of fluorescent probes that could discriminate between them is © 2015 American Chemical Society

Received: August 20, 2015 Accepted: October 19, 2015 Published: October 19, 2015 11475

DOI: 10.1021/acs.analchem.5b03286 Anal. Chem. 2015, 87, 11475−11483

Article

Analytical Chemistry

Scheme 1. (A) Reaction Mechanisms of an 8-AkenylBodipy Dye with Thiol and Morpholine, Respectively;a (B) Proposed Reaction Mechanism of Probe 1 with Cys in This Work

a

Reported by Tang and co-workers.75

construct Cys fluorescent probe for the first time. The resultant fluorescent probe 8-alkynylBodipy 1, containing an activated alkynyl unit as Michael receptor and a Bodipy dye as fluorescence reporter, could react with Cys more efficiently than with Hcy or GSH to release a highly fluorescent 8methylBodipy dye 2, enabling the highly selective and sensitive detection of Cys over Hcy and GSH as well as other amino acids. The potentials of the probe for detecting and imaging Cys in serum and cells, respectively, have successfully been demonstrated.

Synthesis of Probe 1. Probe 1 was synthesized by Sonogashira coupling of 8-chloroBodipy and phenylacetylene according to a reported method,69 and the detailed procedures were shown in the Supporting Information. Procedures for Biothiols Sensing. The solutions of amino acids and reduced glutathione (GSH) were prepared in deionized water. A stock solution of 1 (1 mM) was prepared in CH3CN. The stock solution of 1 was then diluted to the concentration of 5 μM with PBS (pH 7.4, 20 mM, containing 10% CH3CN) at 25 °C. Spectra data were recorded at an indicated time after the addition of biothiols and various species. Detection of Cys in Reduced Human Serum Samples. 100 μL of human serum sample was thawed at room temperature and transferred to a 1.5 mL centrifuge tube. Then, 15 μL of a 66.7 g/L tris(2-carboxyethyl)phosphine solution (TCEP, reducing reagent) at nearly neutral pH (ca. pH 6) was added to the sample. The resulting mixture was vigorously vortex-mixed at intervals and incubated for 30 min at room temperature. Afterward, 500 μL of acetonitrile was introduced to precipitate the proteins. The obtained slurry was blow-dried by a pure N2 flow. Then, 500 μL of a 0.125 M borate buffer solution (pH 9.5, with 4 mM EDTA) was added to the tube, vortex-mixed, and centrifuged at 15 000g for 5 min. For fluorescence analysis, 20 μL of the supernatant was carefully taken out and mixed with 2 mL of PBS (pH 7.4, 20 mM, containing 10% CH3CN) containing 5 μM of probe 1, and 10 min later, fluorescence signal was recorded. For HPLC analysis, 180 μL of the supernatant was carefully taken out and

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EXPERIMENTAL SECTION Materials and Instruments. All reagents and solvents were purchased from commercial sources and were of the highest grade. Solvents were dried according to standard procedures. All reactions were magnetically stirred and monitored by thin-layer chromatography (TLC). Flash chromatography was performed using silica gel 60 (200−300 mesh). Absorption spectra were taken on Varian Carry 4000 spectrophotometer. Fluorescence spectra were taken on Hitachi F-7000 fluorescence spectrometer. The 1H NMR and 13C NMR spectra were taken on a Bruker spectrometer and recorded at 600 and 150 MHz, respectively. The following abbreviations were used to explain the multiplicities: s = singlet; d = doublet; t = triplet; q = quartet; m = multiplet; br = broad. High resolution mass spectra were obtained on a Varian QFTESI mass spectrometer. The fluorescence images were acquired through an Olympus FluoView FV1000 confocal microscope. 11476


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Figure 1. Time-dependent absorption spectra changes of 1 (5 μM) in the presence of 5 equiv of Cys (A), Hcy (B), and GSH (C), respectively, as well as the corresponding absorption spectra at the time point of 20 min (D) in PBS (pH 7.4, 20 mM, containing 10% CH3CN) at 25 °C.

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mixed with 20 μL of a 5 g/L ammonium 7-fluorobenzo-2-oxa1,3-diazole-4-sulfonic acid (SBD-F) solution for the derivatization reaction. The reaction was performed at 60 °C in a water bath for 1 h. The final solution was filtered with a 0.22 μm Millipore membrane and kept at 4 °C for further HPLC analysis. The determination of Cys in human serum was performed using a Waters UPLC system coupled with a Phenomenex C18 column (4.6 × 250 mm, 4 μm particles) and a fluorescence detector (Ex: 385 nm; Em: 515 nm). The mobile phases and the detection procedures are consistent with those reported in the literature.68 Cell Culture and Fluorescence Imaging. HeLa cells were provided by Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education (China). Cells were grown in Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with 10% FBS (Fetal Bovine Serum) and 1% antibiotics at 37 °C in humidified environment of 5% CO2. Cells were plated on 6-well plate and allowed to adhere for 12 h. Before the experiments, cells were washed with PBS 3 times. Then, the cells were incubated with 1 (5 μM) or pretreated with NEM (1 mM, 40 min) and then Cys, Hcy, and GSH (500 μM for all, 30 min) in DMEM medium at 37 °C. After each treatment, the cells were washed with PBS 3 times. Fluorescence imaging was performed with a Olympus FluoView FV1000 confocal microscope. Emission was collected at 495−540 nm (λex: 488 nm). Cyotoxicity Assays. HeLa cells were grown in Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with 10% FBS (fetal bovine serum) and 1% antibiotics at 37 °C in a humidified environment of 5% CO2. Immediately before the experiment, the cells well placed in a 96-well plate, followed by addition of increasing concentrations of probe 1. The final concentrations of the probe were kept from 0 to 6 μM. The cells were then incubated at 37 °C in an atmosphere of 5% CO2 and 95% air for 24 h, followed by MTT assays (n = 6). The untreated assay with DMEM medium (n = 6) was also conducted under the same conditions.

RESULTS AND DISCUSSION Design Rationale. First reported in 1968,70 Bodipy dyes have become increasingly valuable fluorophores due to their Scheme 2. Model Reaction of 1 with 2-Aminoethanethiol (an Analog of Cys)

excellent characteristics, including high fluorescence quantum yields, narrow absorption and emission bands, high thermal and photochemical stability, and easy functionalization.71−74 We noted that, in 2011, Tang and co-workers synthesized a series of 8-alkenylBodipy dyes via Liebeskind-Srogl cross-coupling starting from 8-thiomethyl-substituted Bodipy.75 It was found that the double bonds at the 8-position of these Bodipy dyes are highly activated in analogy to those of α,β-unsaturated carbonyl compounds due to the strong electron-withdrawing Bodipy nucleus. Thus, these 8-alkenylBodipy dyes display Michael acceptor-like reactivity and could react with thiol to form thiol adducts under mild conditions. Interestingly, instead of forming the expected adduct, one of these dyes reacts with morpholine to give an unexpected 8-methylBodipy product in high yield (Scheme 1A). The reaction mechanism was proposed to be a Michael addition-triggered retro-aza-aldol type process by Tang and co-workers.75 Inspired by these findings, we envisioned that it is possible to utilize the abovementioned two types of reactions to construct a reaction-based fluorescent probe for Cys, providing that an alkynyl group could be installed at Bodipy 8-position. With the above consideration in mind, we designed a 8alkynylBodipy 1 by introducing an ethynylphenyl moiety into 11477


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Scheme 3. (A) Proposed Reaction Mechanism of Probe 1 with Hcy or GSH; (B) The Model Reaction of 1 with Ethanethiol

Figure 2. Time-dependent fluorescence spectra of 1 (5 μM) in the presence of 5 equiv Cys (A), Hcy (B), and GSH (C) as well as the corresponding time-dependent fluorescence intensity changes (D) at 505 nm. Conditions: PBS (pH 7.4, 20 mM, containing 10% CH3CN) at 25 °C; λex = 480 nm; λem = 505 nm; slits: 5/5 nm.

byproduct 3 (Scheme 1B). If so, it is promising for 1 to act as a Cys fluorescent probe given the distinct chemical structures and thus different photophysical properties between 1 and 2. Theoretically, it is also possible for Hcy or GSH to elicit such a cascade reaction because the two species both contain nucleophilic thiol and amino groups as Cys does. However, considering the kinetically unfavored six-membered or tenmembered cyclic transition state in the initial dual Michael addition reaction stage, it appears to be difficult for Hcy or GSH to elicit the cascade reaction. Given these, compound 1

Bodipy 8-position (Scheme 1B). Similar to the alkenyl group in 8-alkenylBodipy dyes,75 the alkynyl group in 1 should also be activated by the electron-withdrawing Bodipy moiety. Thus, it was envisioned that the dual Michael additions of Cys, containing the nucleophilic thiol and amino groups, with the activated alkynyl group would produce a five-membered cyclic intermediate M2 via M1, which is, in structure, similar to the morpholine adduct shown in Scheme 1A, and thus would very likely perform a similar retro-aza-aldol reaction to lead to the highly fluorescent 8-methylBodipy 2 as well as a thiazoline 11478


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Figure 1D, revealed that the reaction route of 1 with Cys is different from that with Hcy or GSH, and the latter two may share the same reaction mechanism. To explain the absorption spectra changes of 1 treated Cys, we performed a synthetic experiment independently. TLC analysis suggested a clean chemical transformation, and a new species with a bright green emission was observed. The species was then successfully separated and unambiguously confirmed to be 8-methylBodipy 2 by 1H NMR, 13C NMR, and HRMS spectra (Supporting Information). Moreover, the absorption and emission maxima of the synthetic product matched well with those of 1 treated with Cys (Figure S2). Thus, the absorption peak at 489 nm observed in Figure 1A should be attributed to 8-methylBodipy 2. In addition, the HRMS assay of 1 treated with Cys provided two main peaks at m/z 229.0716 and 208.0423 (Figure S3) corresponding to [2 + Na]+ and [3 + H]+, respectively, suggesting that the reaction provides not only 8-methylBodipy 2 but also thiazoline 3. Although it was difficult for us to isolate 3 from the reaction mixture due to its strong polarity, the model reaction of 1 with 2-aminoethanethiol (an analog of Cys) provided both 8-methylBodipy 2 and a control compound thiazoline 4 (Scheme 2), which has been fully identified by 1H NMR, 13C NMR, and HRMS (Supporting Information). Obviously, the results strongly support our proposed reaction mechanism of 1 with Cys. As for Hcy and GSH, on the basis of the produced absorption peaks around 506 nm longer than that of 8methylBodipy 2 (489 nm) (Figure 1D), it was expected that the resulting products should possess a bigger π-conjugation than that of 2. Thus, we speculated that the reaction of 1 with Hcy or GSH mainly stays at the stage of the initial Michael addition, leading to the corresponding products 1-Hcy and 1GSH both possessing a bigger π-conjugation due to the conjugated CC bond (Scheme 3A). The speculation was supported by HRMS assays of 1 treated with Hcy and GSH, where the main peaks at m/z 428.1408 and m/z 600.1900, respectively, corresponding to [1-Hcy + H]+ and [1-GSH + H]+, could clearly be observed (Figure S4). Further, we also synthesized a control compound 5 by the model reaction of 1 with ethanethiol (Scheme 3B). The well-matched absorption spectrum of 5 with that of 1 treated with Hcy or GSH further supported our speculation (Figure S5). Although 1-Hcy and 1GSH both have the nucleophilic amino group, it is indeed difficult for them to perform the subsequent intramolecular

Figure 3. Fluorescence intensities of 1 (5 μM) treated with 5 equiv of various amino acids in PBS (pH 7.4, 20 mM, containing 10% CH3CN) at 25 °C. (1) 1 only, (2) Cys, (3) Hcy, (4) GSH, (5) His, (6) Glu, (7) Asp, (8) Val, (9) Phe, (10) Tyr, (11) Ala, (12) Ser, (13) Leu, (14) Arg, (15) Pro, (16) Thr, (17) L-Glu, (18) Trp, (19) Ile, and (20) Lys. The data were obtained at the time point of 10 min. λex = 480 nm; λem = 505 nm; slits: 5/5 nm.

was greatly expected to be a selective fluorescent probe for Cys over Hcy and GSH. Reactivity of 1 toward Cys/Hcy/GSH. In order to support our speculation, we tested the fluorescence color changes of 1 treated with Cys, Hcy, and GSH, respectively, in phosphatebuffered saline (PBS) (pH 7.4, 20 mM, containing 10% CH3CN) with the help of a 365 UV lamp. As shown in Figure S1, probe 1 itself displayed a bright yellow fluorescence (Φ = 0.66); upon being treated with Cys, a dramatic fluorescence color change from yellow to green was observed. By comparison, Hcy only elicited a very slight green fluorescence, and no any fluorescence was observed when GSH was used. The preliminary assays verified the feasibility of our strategy and also suggested the potential of 1 as a selective fluorescent probe for Cys over Hcy and GSH. Subsequently, we carefully examined the reactivity of 1 with Cys, Hcy, and GSH, respectively, through UV−vis spectra in the same condition. As shown in Figure 1A, probe 1 showed two main absorption bands at 410 and 538 nm, respectively; upon addition of Cys, both the two absorption bands decreased gradually, accompanied by the emergence of a new blue-shifted band centered at 489 nm. Surprisingly, although a blue-shifted band was also observed when Hcy or GSH was used, their absorption maxima were found around 506 nm (Figures 1B,C), distinct from that of Cys. These results, as clearly shown in

Figure 4. (A) Fluorescence spectra changes of 1 (5 μM) as a function of Cys concentrations (0−32.5 μM) in PBS (pH 7.4, 20 mM, containing 10% CH3CN) at 25 °C. (B) The corresponding fluorescence intensity changes. The data were obtained after 10 min. λex = 480 nm; λem = 505 nm; slits: 5/5 nm. 11479


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Figure 5. Confocal fluorescence images of HeLa cells recorded at different time points after the addition of 1 (5 μM). Emission was collected at 495−540 nm (excited at 488 nm). Scale bar: 20 μm.

Figure 6. Fluorescence imaging of Cys in HeLa cells using 1. (A) HeLa cells only; (B) HeLa cells incubated with 1 (5 μM, 30 min); (C) HeLa cells preincubated with Cys (200 μM, 30 min) and then treated with 1 (5 μM, 30 min); (D) HeLa cells preincubated with NEM (1 mM, 40 min) and then treated with 1 (5 μM, 30 min); (E−H) the corresponding bright-field images. Emission was collected at 495−540 nm (excited at 488 nm). Scale bar: 20 μm.

fluorescence intensity (from 1.39 to 6244) was observed. Such a significant fluorescence of f−on response could be attributed to not only the almost negligible background fluorescence of 1 at 505 nm but also the high fluorescence quantum yield of the produced 8-methylBodipy 2 (Φ = 0.87).76 By comparison, Hcy only elicited a slight fluorescence enhancement at 505 nm (Figure 2B). Considering the obvious absorption spectra change of 1 treated with Hcy, it is clear that the reaction of 1 with Hcy mainly produces 1-Hcy, but also a small number of 8-methylBodipy 2 via the dual Michael addition/retro-aza-aldol reaction (Scheme 3A). In sharp contrast, GSH only resulted in a decrease in fluorescence intensity of 1 at 560 nm and did not elicit any fluorescence enhancement at 505 nm at all (Figure 2C), indicating the reaction solely stays at the stage of 1-GSH. Notably, both 1-Hcy and 1-GSH, structurally similar to the nonfluorescent 8-alkenylBodipy dyes,75 exhibited no fluorescence even when excited at their absorption maximum of 506 nm (Figure S6) presumably due to the CC bond isomerization induced fluorescence quenching.77 Figure 2D

cyclization due to the kinetically unfavored six- and tenmembered cyclic transition states. In addition, it should be mentioned that, except for the main product 1-Hcy, the reaction of 1 with Hcy also resulted in a small amount of 2 via the dual Michael addition/retro-aza-aldol cascade reaction similar to that of 1 treated with Cys, as indicated by the slight enhancement of absorption intensity at 489 nm in the late stage of the reaction (Figure 1B) as well as the fluorescence studies (see below). Fluorescence Responses of 1 toward Cys, Hcy, and GSH. Subsequently, we examined emission behaviors of 1 in the presence of Cys, Hcy, and GSH, respectively, in the same condition. Although 1 is highly fluorescent around 560 nm when excited at its absorption maximum,69 it only showed a poor emission at the wavelength when excitation was set at 480 nm (near the absorption maximum of 2). However, upon treatment of 1 with Cys, a blue-shift new peak appeared at 505 nm and gradually reached equilibrium within 10 min (Figure 2A), and in this case, an approximate ∼4500-fold increase in 11480


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Figure 7. Fluorescence imaging of Cys in Hela cells using probe 1. (A) HeLa cells only; (B) HeLa cells treated in sequence with NEM (1 mM, 40 min), Cys (500 μM, 30 min), and 1 (5 μM, 30 min); (C) HeLa cells treated in sequence with NEM (1 mM, 40 min), Hcy (500 μM, 30 min), and 1 (5 μM, 30 min); (D) HeLa cells treated in sequence with NEM (1 mM, 40 min), GSH (500 μM, 30 min), and 1 (5 μM, 30 min); (E−H) the corresponding bright-field images. Emission was collected at 495−540 nm (excited at 488 nm). Scale bar: 20 μm.

method was first used for the determination of Cys in the serum samples from three healthy volunteers, and as a control, the same serum samples were also analyzed by a standard HPLC method. The blood serum samples were treated by reference of a reported procedure,68 and the details were shown in the Experimental Section. As shown in Table S1, by the standard addition method using Cys as the standard, the unknown concentrations of Cys in the three human serum samples were determined to be 280, 204, and 248 μM, respectively, which were in close agreement with HPLC results as well as those reported in the literature.78,79 Selectively Imaging Cys in Living Cells. MTT assays were first conducted to assess the cytotoxicity of 1. As shown in Figure S10, more than 80% of HeLa cells survive after a 24 h incubation with 1 at a concentration range of 0−6 μM, indicative of the low cytotoxicity of 1. Given that the intracellular concentration of Cys ranges from 30 to 200 μM,81,82 we subsequently evaluated whether or not probe 1 is cell-permeable and could image Cys by confocal microscopy experiments. Notably, the high concentrations of GSH could consume probe 1 (Figure S11) by production of the nonfluorescent 1-GSH adduct; thus, in the cell imaging experiment, a relatively high probe loading (5 μM) is necessary to obtain the satisfying results. In this study, HeLa cells were incubated with 1, and then, the fluorescence images were recorded at different time points for 60 min. As shown in Figure 5, the fluorescence intensity increased significantly over time and remained steady after 20 min, indicating that probe 1 is cell-permeable and could likely image Cys in the cells. Further, we performed a “NEM (N-ethylmaleimide, a trapping reagent of thiol species) experiment” to evaluate whether or not the green fluorescence is derived from intracellular biothiol, such as Cys. As shown in Figure 6A, HeLa cells were found to have almost no fluorescence when excited at 488 nm; when the cells were incubated with 1 (5 μM), an obvious fluorescence in green channel was observed (Figure 6B); when the cells were pretreated with 0.2 mM Cys and then incubated with 1 (5 μM), they gave the stronger fluorescence (Figure 6C); when the cells were pretreated with NEM (1.0 mM) and then incubated with 1 (5 μM), almost no

showed a comparison of the time-dependent fluorescence intensity changes of 1 treated with the three biothiols, respectively, from which the high selectivity of 1 for Cys over Hcy and GSH could be clearly observed. Further, we examined the fluorescence responses of 1 treated with various thiol-free natural amino acids including His, Glu, Asp, Val, Phe, Tyr, Ala, Ser, Leu, Arg, Pro, Thr, L-Glu, Trp, Ile, and Lys. As shown in Figure 3, these amino acids did not lead to any significant fluorescence enhancement of 1 at 505 nm, revealing the importance of the thiol group for triggering the dual Michael addition/retro-aza-aldol cascade reaction. Also, probe 1 was fluorescently silent toward some biologically relevant cations and anions, such as Na+, Ca2+, Fe3+, Zn2+, K+, HCO3−, CO32−, F−, I−, Br−, and SO42− (Figure S7A). Even in the presence of the above-mentioned amino acids, cations, and anions, probe 1 still displayed a similar fluorescence enhancement as that only treated with Cys (Figures S7B and S8). Although Hcy or GSH could consume the probe and result in a decrease of the fluorescence intensity of 1 treated with Cys to some extent due to producing nonfluorescent 1-Hcy or 1-GSH adduct (Figure S8), the practical application of 1 for detection of Cys in human serum is still feasible in view of the higher concentration of Cys (165−335 μM)78,79 than that of Hcy80 or GSH (ca. 12 μM)78,79 in human serum samples. Next, the fluorescence titration experiment was conducted to examine the sensitivity of 1 for Cys. In fact, the probe was considerably sensitive for Cys, and even 0.2 μM Cys could elicit a 110-fold fluorescence enhancement at 505 nm (Figure 4A). A linear calibration graph of the fluorescent intensities (I505) to Cys concentrations from 0 to 8 μM was obtained (R2 = 0.9982) (Figure 4B), and the detection limit was estimated to be 0.38 nM based on S/N = 3, which is lower than some Cys-sensitive fluorescent probes (Table S2). In addition, probe 1 was stable in a wide pH range of 2−12 and displayed the best response for Cys in the region of 7−8 (Figure S9). Thus, the probe is compatible with physiological pH. Detecting Cys in the Reduced Human Serum. It is known that in the reduced human serum the concentration of Cys (165−335 μM)78,79 is much higher than those of Hcy (9− 13 μM)80 and GSH (14 ± 7 μM).78,79 Thus, the proposed 11481


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Analytical Chemistry fluorescence was observed (Figure 6D). These results confirmed that the green fluorescence in the cells is due to biothiol, and probe 1 could respond to intracellular Cys. Finally, we performed the control experiments to prove the selectivity of 1 for Cys over Hcy and GSH in the cell environment. First, HeLa cells were preincubated with 1 mM NEM to remove the endogenous biothiols. Subsequently, the NEM-treated cells were incubated with 0.5 mM of Cys, Hcy, and GSH, respectively, and then with probe 1 (5 μM). As can be seen in Figure 7B−D, only Cys-treated cells gave rise to fluorescence in the green channel, confirming that the observed green fluorescence in HeLa cells is due to the reaction of probe 1 with Cys rather than with Hcy or GSH.

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CONCLUSIONS In summary, a new fluorescence probe 8-alkynylBodipy 1 has been developed, which could selectively detect Cys over Hcy/ GSH as well as other amino acids based on a Cys-triggered dual Michael addition/retro-aza-aldol cascade reaction that leads to release of a highly fluorescent 8-methylBodipy dye 2. The potentials of the probe for detecting and imaging Cys in serum and cell environments, respectively, have been demonstrated. Studies are underway either to optimize the sensing method by modifying the reaction group and fluorophore or to exploit the new application of the intriguing cascade reaction.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.5b03286. Additional spectra data as noted in the text, and 1H NMR, 13C NMR, and HRMS data. (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: +86 351 7010588. Fax: +86 351 7011688. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge the Natural Science Foundation of China (Nos. 21172137 and 21302114), Specialized Research Fund for the Doctoral Program of Higher Education (SRFDP, 20101401110010), and Program for New Century Excellent Talents in University (NCET-11-1034) for support of this work.

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Selective Fluorescence Detection of Cysteine over Homocysteine and

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