Fluorescein-Based Chromogenic and Ratiometric Fluorescence Probe

Subscriber access provided by GAZI UNIV

Article

A New Fluorescein-based Chromogenic and Ratiometric Fluorescence Probe for Highly Selective Detection of Cysteine and Its Application in Bioimaging Zhen-Hai Fu, Xiao Han, Yongliang Shao, Jianguo Fang, Zhihong Zhang, Ya-Wen Wang, and Yu Peng Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b04431 • Publication Date (Web): 23 Dec 2016 Downloaded from http://pubs.acs.org on December 25, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Analytical Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 8

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

A New Fluorescein-based Chromogenic and Ratiometric Fluorescence Probe for Highly Selective Detection of Cysteine and Its Application in Bioimaging Zhen-Hai Fu,†‡ Xiao Han,† Yongliang Shao,† Jianguo Fang,† Zhi-Hong Zhang,‡ Ya-Wen Wang,*,† and Yu Peng*,† （[email protected]; [email protected]. Fax: +86-931-8912582） †State Key Laboratory of Applied Organic Chemistry, Lanzhou University, Lanzhou, 730000, People’s Republic of China ‡Key Laboratory of Salt Lakes Resources and Chemistry, Qinghai Institute of Salt Lakes, Chinese Academy of Sciences, Xining, 810008, People’s Republic of China ABSTRACT: A dual mode fluorescent probe, which is based on an integration of fluorescein and coumarin fluorophores, was developed for the discrimination of Cys from Hcy and GSH. This probe (2) shows the advantage of quick reaction (5 min) with Cys, resulting in a strong fluorescence turn-on response when excited at 450 nm. Notably, it also demonstrates the ratiometric fluorescence property while excited by a shorter wavelength (332 nm). All of results suggest probe 2 has a high selectivity toward Cys even in the presence of other amino acids, cations and anions. The detection limit of Cys was calculated as 0.084 µM, which was much lower than the intracellular concentration. 1H NMR, MS and DFT calculation were used to reveal the detection mechanism further. Finally, this low cytotoxic probe was successfully applied in bioimaging within HepG2 cells.

Cysteine (Cys), homocysteine (Hcy), and glutathione (GSH), are main thiol-containing amino acids, which play essential roles in many biological processes such as the growth of tissues and the cellular antioxidant defense.1–4 Abnormal levels of intracellular thiols are also associated with many diseases. For example, Cys deficiency can result in lethargy, slow growth, liver damage and AIDS.5–7 Higher levels of Hcy may induce Alzheimer’s disease, neural tube defect, cardiovascular disease and coronary heart disease.8–10 Some other diseases such as cancer are attributed to the abnormal amount of GSH as well.11,12 Due to their importance in physiology, a series of work for detecting them including electrochemical assays, immune assays, high performance liquid chromatography methods, and optical techniques and so on, have been reported in recent years.13 Among them, optical techniques especially the fluorescence approach has attracted a great deal of attention owing to the advantages of real-time, simplicity, low detection limits, low cost and the ability to be applied in bioimaging.14 Nevertheless, it is still a challenge to discriminate these biothiols from each other. Currently, considerable attentions have been devoted to develop such fluorescent probes for selectively detecting Cys over Hcy and GSH.15 The nucleophilicity and binding affinity with metal ions of thiols are often involved in the sensing mechanism of these probes, such as the Michael addition,16–18 cleavage reactions,19–21 cyclization with aldehydes,22–24 nucleophilic substitution,25–27 conjugated addition-cyclization,28,29 metal complex-displace coordination and others.30–32 As shown in Figure 1a, a cyclization reaction of Cys with acrylates has also been used:33–41 cyclic amides were formed after the conjugate addition of Cys (or Hcy) with acrylates, but the formation of eight-membered ring structure (n =2) resulting from

Hcy is kinetically disfavored, thus providing the possibility for selective detection of Cys.

Figure 1. (a) Conjugated addition-cyclization mechanism. (b) Our fluorescence probe.

With the above analysis in mind, studies on the preparation of such probes based on various fluorophores including the fluorescein have attracted attentions in recent years. Fluorescein was often selected as the fluorophore due to its advantages of fluorescence, solubility, and modifiability.42,43 Up to now, some fluorescein-based probes (A~E) have been reported and summarized in Table S1. Chen group reported probes A and B,44 which were directly synthesized by unmodified fluorescein and acryloyl chloride. The probes are highly selective toward Cys than Hcy (or GSH) in ethanol-phosphate buffer solution (pH = 7.4, 2/8, v/v) after 15 min or 10 min. Yang and Strongin developed a seminaphthofluorescein-based probe C for selective detection of Cys within 25 min,45 providing a fluorescence enhancement at a higher wavelength compared

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

with fluorescein. Recently, a colorimetric and near-infrared fluorescent probe D was also reported.46 Upon the addition of Cys, it shows fluorescence enhancement at 690 nm in DMSOPBS buffer (pH = 7.4, 4/6, v/v) when excited at 612 nm within 10 min. Kim and co-workers also reported a fluorescent probe E for Cys in HEPES (pH = 7.4) within 400 min.47 Although Cys fluorescence probes based on this strategy have appeared,

Page 2 of 8

most of them show some disadvantages, such as long reaction time,29,33,38 high detection limit,38,47 no color change,38,39 or only single output fluorescence signal.28,29,38,39,44–47 Herein, we reported a new probe 2 (Figure 1b) based on a hybrid of fluorescein and coumarin fluorophore, and it demonstrated very fast, highly selective, chromogenic and ratiometric detection of Cys over Hcy and GSH in aqueous solution.

Scheme 1. Syntheses of Probes

Figure 2. The solid state images of compounds 1−4 under visiblelight and UV light: (a), (e) for 1; (b), (f) for 2; (c), (g) for 3; and (d), (h) for 4. Irradiated at 365 nm by use a handheld UV-lamp.

EXPERIMENT SECTION Design and Syntheses of Probes. The rational design of a chromogenic and ratiometric fluorescent probe for the detection of Cys is based on the mentioned-above reaction with acrylate moiety. In our research the probe should satisfy the following characteristics: (1) probe itself has fluorescence character; (2) a new emission at a different wavelength can achieve after the reaction of the probe with Cys; (3) there is an overlap between two fluorescence emissions; (4) after the recognition of Cys, the absorption in UV−vis spectra should be different, thus resulting in the color change. With this concept in mind, fluorescein monoaldehyde was obtained through a Reimer−Tieman reaction, which can be further used to prepare probes 2 and 4. The synthetic route of probe 2 is shown in Scheme 1. First, an aldehyde-functionalized fluorescein was prepared according to previous reports (see the Supporting Information).48,49 The further reaction of fluorescein monoaldehyde with diethyl malonate afforded a coumarin hybrid 1,50 which can be treated with acryloyl chloride to modify another hydroxyl group under basic condition,51 eventually resulting in the formation of probe 2 (see the Experiment Section for details). For a control experiment, we also prepared the compounds 3 and 4 by the similar way. The single crystal structures of probes 2 and 4 were obtained and shown in Scheme 1. All compounds were characterized by 1H, 13C NMR and mass spectra (Figure S1−S15). Figure 2 shows their physical status and the solid

state fluorescence images. Among the four compounds only probe 2 shows blue fluorescence when irradiated at 365 nm by using a handheld UV-lamp. Meanwhile, the fluorescence responses of probe 2 are better than the controlled substrate 4 in different solvents (Figure S16 and S17). So the following experiments were carried out using probe 2 only. Synthesis and Characterization of Compound 1. To a solution of monoaldehyde-functionalized fluorescein (360 mg, 1.0 mmol) and diethyl malonate (182 µL, 1.2 mmol) in dry EtOH (20 mL) was added piperidine (3 drops) and glacial acetic acid (1 drop) at room temperature. The resulting mixture was stirred for 30 min, and then allowed to heat gradually up to 80 °C and reflux for 20 h. The above mixture was cooled to room temperature, followed by filter and crystallization. The product was then washed with EtOH (20 mL) and dried in vacuum to afford the desired compound 1 as a solid (311 mg, 68%). m.p. >295 °C. 1H NMR (DMSO-d6, 400 MHz) δ = 10.39 (s, 1H), 9.01 (s, 1H), 8.04 (d, J = 7.6 Hz, 1H), 7.81 (td, J = 7.2, 0.8 Hz, 1H), 7.75 (td, J = 8.0, 0.8 Hz, 1H), 7.31 (d, J = 7.6 Hz, 1H), 7.14 (dd, J = 13.6, 9.2 Hz, 2H), 6.96 (t, J = 1.2 Hz, 1H), 6.65 (d, J = 1.2 Hz, 2H), 4.35 (q, J = 7.2 Hz, 2H), 1.36 (t, J = 7.2 Hz, 3H) ppm. 13C NMR (DMSO-d6, 100 MHz) δ = 168.5, 162.5, 159.9, 155.6, 155.4, 152.3, 150.8, 147.7, 141.7, 135.9, 133.8, 130.5, 129.1, 125.7, 124.9, 124.1, 117.6, 114.3, 113.8, 112.2, 108.9, 107.4, 102.7, 81.5, 61.6, 14.1 ppm. ESI−MS: m/z 457.3 [M + H]+. Synthesis and Characterization of Probe 2. Acryloyl chloride (80 µL, 1.0 mmol) was added dropwise to a solution of 1 (149 mg, 0.33 mmol) and Et3N (69 µL, 0.50 mmol) in anhydrous CH2Cl2 (10 mL) at 0 oC. Kept stirring at this temperature for half an hour, and then the resulting mixture was further stirred for 10 h at room temperature. Water (10 mL) was added to the mixture, followed by extraction with CH2Cl2 (15 mL × 3). Combined organic phase was dried by anhydrous Na2SO4. The solvent was removed by evaporation, and the residue was purified by flash column chromatography (petroleum ether/EtOAc = 1:1) on silica gel to afford the probe 2 as a white solid (133 mg, 79 %). m.p. 168-170 °C. 1H NMR (CDCl3, 400 MHz) δ = 9.10 (s, 1H), 8.08 (d, J = 6.8 Hz, 1H), 7.75−7.66 (m, 2H), 7.36 (d, J = 2.0 Hz, 1H), 7.17 (d, J = 7.2 Hz, 1H), 7.06 (s, 2H), 6.95 (dd, J = 8.8, 2.4 Hz, 1H), 6.89 (d, J = 8.4 Hz, 1H), 6.66 (dd, J = 17.2, 0.8 Hz, 1H), 6.34 (dd, J = 17.2, 10.4 Hz, 1H), 6.09 (dd, J = 10.4, 0.8 Hz, 1H), 4.48 (q, J = 7.2 Hz, 2H), 1.46 (t, J = 7.2 Hz, 3H) ppm. 13C NMR (CDCl3,


Page 3 of 8

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


100 MHz) δ = 168.7, 163.9, 162.8, 156.3, 155.9, 152.4, 152.2, 150.6, 148.0, 142.4, 135.7, 133.8, 133.7, 130.6, 129.2, 127.3, 126.0, 125.5, 123.9, 118.8, 118.0, 116.4, 114.5, 112.7, 110.6, 107.9, 80.8, 62.2, 14.3 ppm. ESI−MS: m/z 511.9 [M + H]+. Synthesis and Characterization of Compound 3. To a solution of monoaldehyde-functionalized fluorescein (360 mg, 1.0 mmol) and ethyl acetoacetate (152 µL, 1.2 mmol) in dry EtOH (10 mL) was added piperidine (3 drops) and glacial acetic acid (1 drop) at room temperature. The resulting mixture was stirred for 30 min, and then allowed to warm gradually to 80 °C and reflux for 20 h. The above mixture was cooled to room temperature, followed by filter and crystallization. The product was then washed with EtOH (20 mL), and dried in vacuum to afford the desired compound 3 as a solid (146 mg, 34%). m.p. > 275 °C. 1H NMR (DMSO-d6, 400 MHz) δ = 10.36 (s, 1H), 8.89 (s, 1H), 8.04 (d, J = 7.6 Hz, 1H), 7.81 (td, J =7.6, 1.2 Hz, 1H), 7.75 (td, J = 7.6, 0.8 Hz, 1H), 7.32 (d, J = 7.6 Hz, 1H), 7.14 (dd, J = 15.6, 9.2 Hz, 2H), 6.94 (t, J =1.2, Hz, 1H), 6.65 (d, J = 0.8 Hz, 2H), 2.62 (s, 3H) ppm. 13C NMR (DMSO-d6, 100 MHz) δ = 194.9, 168.6, 159.9, 158.0, 155.8, 152.4, 150.9, 148.1, 140.1, 136.0, 133.9, 130.6, 129.3, 125.7, 125.0, 124.2, 124.1, 114.4, 113.9, 112.2, 109.0, 107.9, 102.8, 81.5, 30.2 ppm. ESI−MS: m/z 427.3 [M + H]+. Synthesis and Characterization of Control Substrate 4. Acryloyl chloride (83 µL, 1.02 mmol) was added dropwise to a solution of 3 (146 mg, 0.34 mmol) and Et3N (94 µL, 0.68 mmol) in anhydrous CH2Cl2 (10 mL) at 0 oC. Kept stirring at this temperature for half an hour, and then the resulting mixture was further stirred at room temperature for 10 h. Water (10 mL) was added to the mixture, followed by extraction with CH2Cl2 (15 mL × 3). Combined organic phase was dried by anhydrous Na2SO4. The solvent was removed by evaporation, and the residue was purified by flash column chromatography (petroleum ether/EtOAc = 2:1) on silica gel to afford the compound 4 as a white solid (135 mg, 92 %). m.p. 262.7-265.4 °C. 1H NMR (CDCl3, 400 MHz) δ = 9.11 (s, 1H), 8.08 (dd, J = 6.8, 0.8 Hz, 1H), 7.74-7.66 (m, 2H), 7.36 (d, J = 2.4 Hz, 1H), 7.17 (d, J = 7.2 Hz, 1H), 7.07 (s, 2H), 6.96 (dd, J = 8.8, 2.4 Hz, 1H), 6.89 (d, J = 8.4 Hz,1H), 6.66 (dd, J = 17.6, 0.8 Hz, 1H), 6.34 (dd, J = 17.6, 10.4 Hz, 1H), 6.09 (dd, J = 10.4, 0.8 Hz, 1H), 2.77 (s, 3H) ppm. 13C NMR (CDCl3, 100 MHz) δ = 195.0, 168.8, 163.9, 158.6, 156.6, 152.6, 152.3, 150.7, 148.7, 141.6, 135.7, 133.9, 133.8, 130.6, 129.3, 127.4, 126.1, 125.6, 124.1, 124.0, 119.0, 116.4, 114.7, 112.7, 110.8, 108.4, 80.9, 30.7 ppm. ESI−MS: m/z 481.6 [M + H]+.

UV-vis and Fluorescence Spectra of Probe 2. Probe 2 exhibits good stability in pH ranging from 1.0 to 10.0 (Figure S18). Upon the addition of Cys, probe 2 shows good fluorescence response for Cys in pH range of 6.0 to 11.0. Considering the applications in biological system, pH 7.4 was selected for the following experiments. In order to find spectroscopic response of probe 2 to Cys, all the measurements were carried out in DMSO−PBS buffer solution (10.0 mM, pH = 7.4, 3:7, v/v) at 25 oC. As shown in Figure 3a, probe 2 exhibits a maximum absorption peak at 332 nm. Upon the addition of Cys to the solution of probe 2, the intensity at 332 nm increased slightly and a new strong absorption peak appeared at about 450 nm, resulting in the color change from colorless to yellow. These results indicated ringopening of fluorescein skeleton,52 that is, ring-opening structure of compound 1 was formed, which can be supported by UV−vis spectra (Figure S19). Accordingly, the fluorescence spectra response of probe 2 toward Cys was carried out at two excitation wavelengths (450 nm and 332 nm), respectively. First, excitation at 450 nm was chose to study Cys sensing, as shown in Figure 3b. Probe 2 has almost no fluorescence emission (Φ = 0.007), and a significant enhancement of fluorescence intensity was observed at about 540 nm (Φ = 0.020), after the addition of Cys. The fluorescence intensity increased 62.4-fold than that of probe 2 within 5 min. In addition, the fluorescence responses for Hcy and GSH were also investigated in the same condition. The fluorescence intensity increased 10.9-fold for Hcy (Φ = 0.009) and 4.1-fold for GSH (Φ = 0.008), respectively (Figure S20−S22). These results suggested that the selectivity of probe 2 for Cys is better than those of Hcy and GSH. Further, a smaller wavelength (332 nm) was selected for excitation. The probe 2 exhibits a strong fluorescence emission peak at about 472 nm (Figure 3c), which can be attributed to the monomer of coumarin.53,54 Notably, upon the addition of Cys, the fluorescence intensity at 472 nm disappeared, and a new emission peak was observed at 540 nm, which can be attributed to the emission of ring-opening structure of fluorescein. These results suggested that probe 2 has potential to become a ratiometric fluorescence probe for the detection of Cys. Notably, the fluorescence response of probe 2 for Hcy and GSH are almost negligible compared to that of Cys (Figure 3d and Figures S23−S24). Therefore, probe 2 can discriminate Cys over Hcy and GSH.

RESULTS and DISCUSSION



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 8

Figure 3. (a) UV−vis spectra of the probe 2 (10.0 µM) with 125.0 µM of Cys in DMSO-PBS buffer (10.0 mM, pH = 7.4, 3:7, v/v). (b) Time-dependent fluorescence spectra of the probe 2 (10.0 µM) with 125.0 µM of Cys in DMSO-PBS buffer (c) Time-dependent fluorescence spectra of the probe 2 (10.0 µM) with 125.0 µM of Cys in DMSO-PBS buffer. (d) Fluorescence responses of probe 2 (10.0 µM) with 125.0 µM of Hcy and GSH in DMSO-PBS buffer (b: λex = 450 nm. c, d: λex = 332 nm. slits: 5 nm/5 nm). The kinetic studies were conducted by monitoring the fluorescence intensity changes of probe 2 at 540 nm in the presence of 12.5 equivalent of Cys. It shows that the reaction between probe 2 and Cys was completed within 5 minutes (Figure 3b, inset) and the observed rate constant (Figure S25) was -1 calculated to be 1.095 min .55 Consequently, probe 2 shows fast fluorescence response for Cys detection. To get insight into fluorescence intensity changes with increas of Cys concentration, the fluorescence spectra changes of probe 2 (10.0 µM) toward Cys (0-125.0 µM) were evaluated in DMSO-PBS buffer (10.0 mM, pH = 7.4, 3:7, v/v). As shown in Figure 4a, the fluorescence intensity of probe 2 at 540 nm enhanced with increasing the amount of Cys in solution, when excited at 450 nm. And a good linear relationship between fluorescence intensity and the amount of Cys was observed in the range of 0-10.0 µM (Figure S26). According to the reported definition (S/N = 3),56 the detection limit was estimated to be 0.084 µM that is much lower than 30-200 µM of the intracellular Cys concentration.57 The results proved that probe 2 has a higher sensitivity toward Cys than some reported Cys probes.28,29,33,38,39,41,44−47 Furthermore, to confirm the ratiometric character of probe 2 toward Cys, the fluorescence changes of probe 2 with different concentration of Cys were displayed in Figure 4b. Upon the addition of Cys, the fluorescence intensity at 472 decreased gradually, and the significant enhancement of fluorescence appeared at 540 nm when excited at 332 nm. Subsequently, a good linear relationship between the ratio of fluorescence intensity at 540 nm/472 nm and Cys concentration (0-10.0 µM) was observed. Consequently, probe 2 also successfully served as a ratiometric fluorescent probe for Cys, which is superior to the reported work with single fluorescence output only.28,29,39,44−47 Fluorescence Selectivity of Probe 2. To further demonstrate the selectivity of probe 2 toward different analytes, The UV−vis and fluorescence spectra of probe 2 in the presence of various amino acids (His, Asp, Arp, Phe, Ala, Gly, Gln, Glu, Lys, Leu, Met, Asn, Ser, Tyr, Thr, Lle, Trp, Pro, Val, Hcy, GSH) were investigated in DMSO-PBS buffer (10.0 mM, pH = 7.4, 3:7, v/v). As shown in Figure S19, after the addition of various amino acids to the system, only Cys resulted in an obvious absorption at 450 nm in the UV−vis spectra, others have almost no influence. In fluorescence studies, no matter what excitation wavelength was used, only Cys caused notable

fluorescence enhancement at 540 nm (Figure 5 and Figure S27). The fluorescence spectra of probe 2 with different cations (Li+, Na+, K+, Mg2+, Ca2+, Sr2+, Ba2+, Hg2+, Fe2+, Mn2+, Fe3+, Cr3+, Al3+) and anions (AcO−, BF4−, Br−, Cl−, ClO4−, F−, H2PO4−, HSO4−, I−, NO3−, SCN−) were also investigated, no response was observed (Figure S29, S31, S33, S35). Moreover, the competitive experiments confirmed that there was no influence of other analytes on the fluorescence response of probe 2 toward Cys (Figure S28, S30, S32, S34, S36).


Page 5 of 8

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 4. Fluorescence spectral changes of probe 2 (10.0 µM) with the addition of Cys (0−125.0 µM) in DMSO-PBS buffer (10.0 mM, pH = 7.4, 3:7, v/v). (a) Inset: the fluorescence intensity changes at 540 nm upon the addition of Cys (λex = 450 nm). (b) Inset: the fluorescence intensity of I540/I472 changes upon the addition of Cys (λex = 332 nm). All spectra were recorded after 5 min.

Proposed Mechanism. Based on the above results, 1H NMR titration experiments were conducted to account for the behavior of probe 2 upon the addition of Cys in a mixture of DMSO-d6 and D2O (10/1, v/v) solution. As shown in Figure 6, Ha, Hb and Hc belong to the proton signals of acrylate moiety in probe 2. Notably, these signals disappeared after the addition of Cys (Figure 6c), and instead both proton signals of quinone form of compound 1 and Cys were observed. Meanwhile, the proton signals of a released lactam with sevenmembered ring was also observed in 1H NMR (Figure S37).33 In mass spectra, a peak at m/z 455.0903 was assigned to 1, and a peak at m/z 174.0275 was assigned to the seven-membered lactam (Figure S38). Accordingly, the detection mechanism was described as follows: when Cys was added into the solution of probe 2, the conjugate addition reaction occurred, followed by the formation of the lactam, and the “ring-opening” form of 1.58,59 The strong fluorescence at 540 nm in DMSOPBS buffer (10.0 mM, pH = 7.4, 3:7, v/v) was attributed to this quinone structure.

(10.0 µM) (λex = 450 nm). The black bars represent the emission intensity of probe 2 in the presence of other amino acids (125.0 µM), the red bars represent the emission intensity that occurs upon the subsequent addition of 125.0 µM of Cys to the above solution. From 0 to 21: none, His, Asp, Arp, Phe, Ala, Gly, Gln, Glu, Lys, Leu, Met, Asn, Ser, Tyr, Thr, Lle, Trp, Pro, Val, Hcy, GSH. All spectra were recorded after 5 min.

Figure 6. Proposed mechanism of probe 2 with Cys and 1H NMR spectra in DMSO-d6/D2O (10/1, v/v): (a) quinone form of 1. (b) probe 2 only. (c) probe 2 + Cys, and (d) Cys only. The signals of D2O have been omitted for clarity.

Computational Studies. To better understand the behavior of probe 2 toward Cys, we carried out computational studies60 by Gaussian 09 program. The B3LYP/6-31G (d, p) basis set was first used for optimizing structure, and TDDFT//B3LYP/6-31G (d, p) was then employed for study the photophysical properties of probe 2 and the control substrate 4 (Figures S39 and S40).

Figure 7. The optimized structures, HOMO−LUMO energy levels and the molecular orbital plots of compound 1 and probe 2.

Figure 5. (a) Fluorescence responses of the probe 2 (10.0 µM) with 125.0 µM of different amino acids in DMSO-PBS buffer (10.0 mM, pH = 7.4, 3:7, v/v). (b) The selectivity of the probe 2

As shown in Figure 7, for probe 2, the LUMO is mainly distributed at the coumarin moiety, which suggested the probe itself exhibits fluorescence properties. However, when the reaction between Cys and probe 2 was completed, the LUMO of generated quinone form of 1, is distributed at the large π system and therefore shown strong fluorescence at a longer excited wavelength because of the small energy gap than that of probe 2. Furthermore, the time-dependent functional theory (TD-DFT) 61 calculations further confirmed probe 2 has better



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

fluorescence character than control substrate 4 (Figure S41 and Table S4). The main allowed transition is HOMO-1→ LUMO transition (S0 → S3) for both of them (Oscillator Strength, f = 0.2483 for probe 2; f = 0.1911 for 4). In addition, the transition HOMO→LUMO also contributed to the probe 2. As a result, probe 2 is better than 4 for the detection of Cys. Bioimaging of Probe 2. Since the probe 2 is highly selective for Cys over other amino acids including Hcy and GSH under physiological conditions (pH = 7.4), it was next applied for bioimaging in living cells (see the Supporting Information for details). First, cytotoxicity effect of probe 2 was carried out in HepG2 cells by the MTT assay according to our previous work.62 HepG2 cells were incubated with 0, 5.0, 10.0, 20.0 and 40.0 µM of probe 2 for 12 h. As shown in Figure S42, probe 2 almost showed no influence on the cells viability compared with HepG2 cells. The results suggested that probe 2 was low cytotoxic to HepG2 cells and could be applied in imaging.

Page 6 of 8

Subsequently, the fluorescence imaging of probe 2 in HepG2 cells was carried out (Figure 8). Upon the addition of Cys to HepG2 cells, the strong green fluorescence was observed (Figure 8f) compared with HepG2 cells only (Figure S43), which is attributed to the reaction of probe 2 with intracellular Cys. For a control experiment, NEM (Nethymaleimide), a well-known regent for decreasing thiols,63 was used to treat HepG2 cells firstly. As a result, the fluorescence of HepG2 cells became very weak after the treatment with NEM (Figure 8g), which means intracellular thiols was blocked successfully. Then these cells were incubated with Cys, Hcy and GSH, respectively. As shown in Figure 8h−8j, with the incubation of probe 2, only the group coped with Cys resulted in a significant fluorescence emission, while the group of Hcy and GSH have almost no fluorescence response. These results indicate that probe 2 is highly selective for the detection of Cys in living cells. The similar fluorescence images were also obtained in blue channel (Figure S44).

Figure 8. HepG2 cell images: (a) bright field and (f) fluorescence images of HepG2 cells with probe 2 (10.0 µM) for 20 min; (b) bright field and (g) fluorescence images of HepG2 cells incubated with NEM (50.0 µM) for 30 min, and then incubated with probe 2 (10.0 µM) for 20 min; (c) (d) (e) bright field and (h) (i) (j) fluorescence images of HepG2 cells incubated with NEM (50.0 µM) for 30 min, then incubated with Cys, Hcy and GSH (100.0 µM) for 30 min, and finally incubated with probe 2 (10.0 µM) for 20 min.

Supporting Information

CONCLUSION In summary, a chromogenic and ratiometric probe 2 based on the integration of two fluorophores was successfully developed according to our rational design strategy. Among the amino acids including Hcy and GSH, this probe is highly selective for the detection of Cys. With the treatment of Cys, the turn-on fluorescence responses were observed at 540 nm by using two different excited wavelengths, especially accompanied by color change from colorless to yellow. The kinetic studies indicated that probe 2 reacts with Cys within 5 min and the observed rate constants was calculated as 1.095 min−1. All of fluorescence results indicate that this probe is highly selective for Cys over other amino acids, cations and anions. The detection limit is calculated as 0.084 µM. 1H NMR, MS and computational calculation further supported our design concept. Moreover, probe 2 shows good cell permeability and low cytotoxicity to HepG2 cells, and was therefore successfully applied in bioimaging. We hope this strategy can be used to develop fluorescence probes for different analytes.

Spectra data, copies of 1H/13C NMR, ESI-MS and other materials. 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].

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS We thank the National Natural Science Foundation of China (Nos. 21572091 and 21472075), Program for Changjiang Scholars and Innovative Research Team in University (PCSIRT-15R28), and the Fundamental Research Funds for the Central Universities (Nos. lzujbky-2016-ct02 and lzujbky-2016-51) of Ministry of Education.

REFERENCES ASSOCIATED CONTENT ACS Paragon Plus Environment

Page 7 of 8

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(1) Liu, Y.-W.; Lv, X.; Hou, M.; Shi, Y.-W.; Guo, W. Anal. Chem. 2015, 87, 11475–11483. (2) Kim, Y.; Mulay, S. V.; Choi, M.; Yu, S. B.; Jon, S.; Churchill, D. G. Chem. Sci. 2015, 6, 5435–5439. (3) Long, L.; Zhou, L.; Wang, L.; Meng, S.; Gong, A.; Du, F.; Zhang, C. Org. Biomol. Chem. 2013, 11, 8214–8220. (4) Zhang, X.; Ren, X.; Xu, Q.-H.; Loh, K. P.; Chen, Z.-K. Org. Lett. 2009, 11, 1257–1260. (5) Zhang, W.; Yin, C.; Zhang, Y.; Chao, J.; Huo, F. Sens. Actuators, B. 2016, 233, 307–313. (6) Ding, S.; Feng, G. Sens. Actuators, B. 2016, 235, 691–697. (7) Zhou, X.; Jin, X.; Sun, G.; Wu, X. Chem.—Eur. J. 2013, 19, 7817–7824. (8) Hao, W.; Mcbride, A.; Mcbride, S.; Jian, P. G.; Zhi, Y. W. J. Mater. Chem. 2011, 21, 1040–1048. (9) Wang, Y.-W.; Liu, S.-B.; Ling, W.-J.; Peng, Y. Chem. Commun. 2016, 52, 827–830. (10) Lv, H.; Yang, X.-F.; Zhong, Y.; Guo, Y.; Li, Z.; Li, H. Anal. Chem. 2014, 86, 1800–1807. (11) Lee, D.; Kim, G.; Yin, J.; Yoon, J. Chem. Commun. 2015, 51, 6518–6520. (12) Zhang, Y.; Shao, X.; Wang, Y.; Pan, F.; Kang, R.; Peng, F.; Huang, Z.; Zhang, W.; Zhao, W. Chem. Commun. 2015, 51, 4245– 4248. (13) Zhang, M.; Yu, M.; Li, F.; Zhu, M.; Li, M.; Gao, Y.; Li, L.; Liu, Z.; Zhang, J.; Zhang, D.; Yi, T.; Huang, C. J. Am. Chem. Soc. 2007, 129, 10322–10323. (14) Mei, J.; Tong, J.; Wang, J.; Qin, A.; Sun, J. Z.; Tang, B. Z. J. Mater. Chem. 2012, 22, 17063–17070. (15) Jung, H. S.; Chen, X.; Kim, J. S.; Yoon, J. Chem. Soc. Rev. 2013, 42, 6019–6031. (16) Li, X.; Zheng, Y.; Tong, H.; Qian, R.; Zhou, L.; Liu, G.; Tang, Y.; Li, H.; Lou, K.; Wang, W. Chem.—Eur. J. 2016, 22, 9247–9256. (17) Ha, H.-J.; Yoon, D.-H.; Park, S.; Kim, H.-J. Tetrahedron 2011, 67, 7759–7762. (18) Yang, Y.; Huo, F.; Yin, C.; Chao, J.; Zhang, Y. Dyes Pigm. 2015, 114, 105–109. (19) Gao, X.; Li, X.; Li, L.; Zhou, J.; Ma, H. Chem. Commun. 2015, 51, 9388–9390. (20) Maeda, H.; Matsuno, H.; Ushida, M.; Katayama, K.; Saeki, K.; Itoh, N. Angew. Chem., Int. Ed. 2005, 44, 2922–2925. (21) Yang, Y.-L.; Zhang, F.-M.; Wang, Y.-W.; Zhang, B.-X.; Fang, R.; Fang, J.-G.; Peng, Y. Chem.—Asian J. 2015, 10, 422–426. (22) Yang, C.; Wang, X.; Shen, L.; Deng, W.; Liu, H.; Ge, S.; Yan, M.; Song, X. Biosens. Bioelectron. 2016, 80, 17–23. (23) Tian, M.; Guo, F.; Sun, Y.; Zhang, W.; Miao, F.; Liu, Y.; Song, G.; Ho, C.-L.; Yu, X.; Sun, J. Z.; Wong, W.-Y. Org. Biomol. Chem. 2014, 12, 6128–6133. (24) Meng, X.; Ye, W.; Wang, S.; Feng, Y.; Chen, M.; Zhu, M.; Guo, Q. Sens. Actuators. B. 2014, 201, 520–525. (25) Xu, C.; Li, H.; Yin, B. Biosens. Bioelectron. 2015, 72, 275–281. (26) Wang, F.; Guo, Z.; Li, X.; Li, X.; Zhao, C. Chem.—Eur. J. 2014, 20, 11471–11478. (27) Wang, F.; An, J.; Zhang, L.; Zhao, C. RSC Adv. 2014, 4, 53437– 53441. (28) Zhang, J.; Wang, J.; Liu, J.; Ning, L.; Zhu, X.; Yu, B.; Liu, X.; Yao, X.; Zhang, H. Anal. Chem. 2015, 87, 4856–4863. (29) Wang, J.; Li, B.; Zhao, W.; Zhang, X.; Luo, X.; Corkins, M. E.; Cole, S. L.; Wang, C.; Xiao, Y.; Bi, X.; Pang, Y.; Mcelroy, C. A.; Bird, A. J.; Dong, Y. ACS Sens. 2016, 1, 882–887. (30) Liu, Y.; Meng, F.; Lin, W. RSC Adv. 2016, 6, 30951–30955. (31) Pathak, R. K.; Hinge, V. K.; Mahesh, K.; Rai, A.; Panda, D.; Rao, C. P. Anal. Chem. 2012, 84, 6907–6913. (32) Miao, F.; Zhan, J.; Zou, Z.; Tian, D.; Li, H. Tetrahedron. 2012, 68, 2409–2413.

(33) Yang, X.; Guo, Y.; Strongin, R. M. Angew. Chem., Int. Ed. 2011, 50, 10690–10693. (34) Niu, W.; Guo, L.; Li, Y.; Shuang, S.; Dong, C.; Wong, M. S. Anal. Chem. 2016, 88, 1908–1914. (35) Zhu, B.; Guo, B.; Zhao, Y.; Zhang, B.; Du, B. Biosens. Bioelectron. 2014, 55, 72–75. (36) Yu, D.; Zhang, Q.; Ding, S.; Feng, G. RSC Adv. 2014, 4, 46561– 46567. (37) Guo, Z.; Nam, S. W.; Park, S.; Yoon, J. Chem. Sci. 2012, 3, 2760–2765. (38) Dai, X.; Zhang, T.; Liu, Y.-Z.; Yan, T.; Li, Y.; Miao, J.-Y.; Zhao, B.-X. Sens. Actuators. B. 2015, 207, 872–877. (39) Lv, H.-M.; Yuan, D.-H.; Liu, W.; Chen, Y.; Au, C.-T.; Yin, S.-F. Sens. Actuators. B. 2016, 233, 173–179. (40) Zheng, M.; Huang, H.; Zhou, M.; Wang, Y.; Zhang, Y.; Ye, D.; Chen, H.-Y. Chem.—Eur. J. 2015, 21, 10506–10512. (41) Liu, Y.; Yu, D.; Ding, S.; Xiao, Q.; Guo, J.; Feng, G. ACS Appl. Mater. Interfaces. 2014, 6, 17543–17550. (42) Zheng, H.; Zhan, X.-Q.; Bian, Q.-N.; Zhang, X.-J. Chem. Commun. 2013, 49, 429–447. (43) Abebe, F. A.; Sinn, E. Tetrahedron Lett. 2011, 52, 5234–5237. (44) Wang, H.; Zhou, G.; Gai, H.; Chen, X. Chem. Commun. 2012, 48, 8341–8343. (45) Yang, X.; Guo, Y.; Strongin, R. M. Org. Biomol. Chem. 2012, 10, 2739–2741. (46) Xue, S.; Ding, S.; Zhai, Q.; Zhang, H.; Feng, G. Biosens. Bioelectron. 2015, 68, 316–321. (47) Lim, S.-Y.; Yoon, D.-H.; Ha, D. Y.; Ahn, J. M.; Kim, D. I.; Kown, H.; Ha, H.-J.; Kim, H.-J. Sens. Actuators. B. 2013, 188, 111– 116. (48) Wang, W.; Rusin, O.; Xu, X.; Kim, K. K.; Escobedo, J. O.; Fakayode, S. O.; Fletcher, K. A.; Lowry, M.; Schowalter, C. M.; Lawrence, C. M.; Fronczek, F. R.; Warner, I. M.; Strongin, R. M. J. Am. Chem. Soc. 2005, 127, 15949–15958. (49) Lee, H.; Kim, H.-J. Org. Biomol. Chem. 2013, 11, 5012–5016. (50) Garino, C.; Tomita, T.; Pietrancosta, N.; Laras, Y.; Rosas, R.; Herbette, G.; Maigret, B.; Quéléver, G.; Iwatsubo, T.; Kraus, J.-L. J. Med. Chem. 2006, 49, 4275–4285. (51) Li, H.; Jin, L.; Kan, Y.; Yin, B. Sens. Actuators. B. 2014, 196, 546–554. (52) Klonis, N.; Sawyer, W. H. J. Fluoresc. 1996, 6, 147–157. (53) Murata, C.; Masuda, T.; Kamochi, Y.; Todoroki, K.; Yoshida, H.; Nohta, H.; Yamaguchi, M.; Takadate, A. Chem. Pharm. Bull. 2005, 53, 750–758. (54) Kim, D.; Na, S.-Y.; Kim, H.-J. Sens. Actuators. B. 2016, 226, 227–231. (55) Lee, D.; Kim, G.; Yin, J.; Yoon, J. Chem. Commun. 2015, 51, 6518–6520. (56) For the method employed, see: Sun, Y.-Q.; Chen, M.; Liu, J.; Lv, X.; Li, J.-F.; Guo, W. Chem. Commun 2011, 47, 11029-11031. (57) Wang, J.; Liao, Y.; Shao, S. Chem. Lett. 2015, 44, 1437–1439. (58) Ali, M.; Dutta, P.; Pandey, S. J. Phys. Chem. B. 2010, 114, 15042–15051. (59) Panchompoo, J.; Aldous, L.; Matthew, Baker.; Wallace, M. I.; Compton, R. G. Analyst 2012, 137, 2054–2062. (60) Peng, Y.; Dong, Y.-M.; Dong, M.; Wang, Y.-W. J. Org. Chem. 2012, 77, 9072–9080, and references cited therein. (61) Kand, D.; Kallb, A. M.; Talukdar, P. Org. Biomol. Chem. 2013, 11, 1691–1701. (62) Zhang, B.; Ge, C.; Yao, J.; Liu, Y.; Xie, H.; Fang, J. J. Am. Chem. Soc. 2015, 137, 757–769. (63) Kong, F.; Liu, R.; Chu, R.; Wang, X.; Xu, K.; Tang, B. Chem. Commun. 2013, 49, 9176–9178.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 8

for TOC only

8


Fluorescein-Based Chromogenic and Ratiometric Fluorescence Probe

Recommend Documents