Subscriber access provided by CORNELL UNIVERSITY LIBRARY
Article
A Near-infrared Fluorescent Probe with High Quantum Yield and Its Application in the Selective Detection of Glutathione in Living Cells and Tissues Jun-Ying Xie, Chunyan Li, Yongfei Li, Junjie FEI, Fen Xu, Juan Ou-Yang, and Juan Liu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b02646 • Publication Date (Web): 08 Sep 2016 Downloaded from http://pubs.acs.org on September 9, 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 Near-infrared Fluorescent Probe with High Quantum Yield and Its Application in the Selective Detection of Glutathione in Living Cells and Tissues Jun-Ying Xie,† Chun-Yan Li,*,†,§ Yong-Fei Li,‡ Junjie Fei†, Fen Xu†, Juan Ou-Yang† and Juan Liu† †
Key Laboratory of Environmentally Friendly Chemistry and Applications of Ministry of Education, College of Chemistry, Xiangtan University, Xiangtan, 411105, PR China. ‡ College of Chemical Engineering, Xiangtan University, Xiangtan, 411105, PR China. § State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry & Chemical Engineering, Hunan University, Changsha, 410082, PR China. ABSTRACT: Glutathione (GSH), cysteine (Cys) and homocysteine (Hcy) are small-molecular biothiols that play key roles in various biological systems. Among these biothiols, GSH is the most abundant intracellular thiol. Until now, a small number of the nearinfrared (NIR) fluorescent probes have been designed for the detection of GSH. Unfortunately, most of these NIR probes are based on cyanine dyes, which generally suffer low fluorescence quantum yield (Φ < 0.25), which are not suitable for bio-imaging. In addition, some probes are difficult to effectively distinguish GSH from Cys and Hcy. In this work, NIR fluorescent probe with high fluorescence quantum yield is developed by introducing rigid coplanar structure such as rhodamine dyes. And the NIR probe (CyR) with spirolactam structure is firstly synthesized and used to recognize GSH. The characteristics of this NIR probe are as following: (1) probe CyR exhibits high fluorescence quantum yield (Φ = 0.43) after the addition of GSH and high sensitivity toward GSH with 75-fold fluorescence enhancement. (2) The probe is highly selective, which will not interfere with the other biological thiols (Cys, Hcy) and amino acids. (3) A possible reaction mechanism of the NIR probe CyR and GSH (Cys, Hcy) can be proposed and proved by 1H NMR, 13C NMR and MS (mass spectra). (4) The NIR probe displays selective detection of GSH in biological samples such as living cells and tissues.
Biothiols, such as glutathione (GSH), cysteine (Cys), and homocysteine (Hcy) (Figure S1), play crucial roles in many physiological processes and pathological processes. Among these biothiols, GSH is the most abundant intracellular thiol and its concentration is in the millimolar range.1,2 It has been proved that the abnormal levels of GSH are associated with many diseases, including cancer, liver damage, AIDS, neurodegenerative diseases, and diseases caused by aging.3,4 Because of its biological and clinical significance, detection of GSH in biological samples is essential. Among the various detection methods reported, fluorescent probes have attracted increasing attention due to their great temporal and spatial sampling capability as well as high sensitivity.5-9 And several excellent fluorescent probes for the detection of GSH have been developed.10-15 However, most of these probes show emission and absorption within ultraviolet or visible range (300-650 nm), which will hinder the applications in biological systems. Near-infrared (NIR) fluorescent probes are highly needful in vivo imaging studies, because light in the NIR region (650-900 nm) possesses several merits, such as less damage to living cells, better tissue penetration, and minimum interference from background auto-fluorescence by biomolecules in the living systems.16-23 Until now, a small number of the NIR fluorescent probes have been designed for the detection of GSH.24-34 Unfortunately, most of these NIR probes are based on cyanine dyes (Cy7, Scheme 1),24-31 which generally suffer low fluorescence quantum yield (Φ < 0.25)
(Table S1), which are not suitable for bio-imaging. In addition, some probes are difficult to effectively distinguish GSH from Cys and Hcy, owing to the fact that Cys and Hcy possess similar molecular backbone and reactivity with GSH.24-26, 32-33 Therefore, it is necessary to develop a NIR fluorescent probe with high quantum yield to detect GSH selectively. As we all know, rhodamine dyes have high fluorescence quantum yield (Φ > 0.80) because of their rigid coplanar structure.35-36 Moreover, the equilibrium between the non-fluorescent spirolactam form and the strong fluorescent ring-opened form provides an ideal model for the construction of turn-on probes. So, numerous probes based on rhodamine spirolactam have been developed and their main application are in the detection of various metal cations.37-41 However, it is pity that emission of rhodamine dyes is in the visible region that is not beneficial for their application in biological samples. In order to shift the wavelength of emission from visible region to nearinfrared region, the chemical structure of rhodamine dyes is required to modify. Recently, Lin’s group designed a class of NIR fluorescent dyes by using modified rhodamine dyes and one of the dyes was employed as fluorescent probe to detect hypochlorous acid (HClO) in living cells and mice.42 Subsequently, Tiwari’ group and Zhao’ group developed NIR fluorescent probes based on same chromophore for detection of lysosomal pH inside living cells and monitoring methylmercury in vivo, respective-
ACS Paragon Plus Environment
Analytical Chemistry
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
ly.43,44 Nevertheless, there is no NIR fluorescent probe based on modified rhodamine dyes for GSH. Herein, a NIR fluorescent probe with high fluorescence quantum yield is developed by introducing rigid coplanar structure such as rhodamine dyes. Initially, compound 1 (Scheme 1) is synthesized by attaching rhodamine to the cyanine skeleton and exhibits high fluorescence quantum yield (Φ = 0.43). And then probe CyR with spirolactam structure is firstly synthesized (Scheme 1) and shows a high sensitive and selective detection of GSH. Scheme 1. The design of the probe CyR. rigid coplanar structure N
R N+
O N+
N
HOOC
Cy7
1
low quantum yield
high quantum yield
N
O N
N N
GSH reaction site
O
O
CyR
EXPERIMENTAL SECTION Reagents. Glutathione (GSH), cysteine (Cys), and homocysteine (Hcy) were purchased from Sigma-Aldrich and their chemical structures were shown in Figure S1. Hydrazine hydrate (85%), cyclohexanone, perchloric acid (70%), 2-(4Diethylamino-2-hydroxybenzoyl) benzoic acid, concentrated sulfuric acid, fisher aldehyde, BOP reagent, indocyanine green (ICG) and glyoxal were also purchased from Sigma-Aldrich. All other chemicals were acquired from qualified reagent supplies with analytical reagent grade. Apparatus. NMR spectra were taken on a Bruker Avance II 400 spectrometer. The mass spectra were carried out on a Bruker Autoflex MALDI-TOF mass spectrometer. Element analysis was obtained on Perkin Elmer 2400 elemental analyzer. Fluorescence spectra were obtained by a Perkin Elmer LS 55 fluorescence spectrometer at the slits of 5.0/5.0 nm. Absorption spectra were performed with a Perkin Elmer Lambda 25 spectrophotometer. The pH measurements were recorded on a Mettler-Toledo Delta 320 pH meter. Fluorescence imaging experiments were recorded in biological samples by an Olympus FV1000 fluorescence microscope with excitation at 635 nm. Synthesis. Compound CyR was synthesized conveniently following the synthetic route described in Scheme 2 (1H NMR, 13 C NMR and MS shown in Figure S2 – S7). Compound 3: Cyclohexanone (12.74 mmol, 1.32 mL) was added dropwise to concentrated H2SO4 (14.0 mL) and cooled down to 0 °C. Then, 2-(4-Diethylamino-2-hydroxybenzoyl) benzoic acid (6.4 mmol, 2.00 g) was added in portions with
Page 2 of 8
vigorous stirring. The reaction mixture was heated at 90 °C for 2 h, cooled down, and poured onto ice (100.0 g). Perchloric acid (70%, 1.4 mL) was then added, and the resulting precipitate was filtered off and washed with cold water (50.0 mL). Compound 3 was gotten as a red solid. Yield: 2.21 g (92 %). 1 H NMR (400 MHz, CDCl3): δ 8.30 (d, J = 8.0 Hz, 1H), 7.78 (t, J = 7.2 Hz 1H), 7.69 (t, J = 7.6 Hz, 1H), 7.22 (d, J = 7.5 Hz, 1H), 7.06-7.13 (q, 2H), 6.89 (s, 1H), 3.61-3.66 (q, 4H), 3.073.18 (m, 2H), 2.29-2.31 (m, 2H), 1.99 (s, 2H), 1.79 (s, 2H), 1.34 (t, J = 6.6 Hz, 6H). MS (TOF, m/z): Calcd for C24H26NO3+ [M]+, 376.2; Found 376.2. Compound 1: Compound 3 (4.2 mmol, 1.58 g) and Fisher aldehyde (4.4 mmol, 0.88 g) were dissolved in acetic anhydride (25.0 mL), and the mixture was stirred at 50 °C for 1.5 h. Then, water (25.0 mL) was added to the reaction mixture to quench the reaction. The solvent was evaporated and the crude product was purified via chromatography (silica gel, CH2Cl2:CH3CH2OH = 20:1) to get a green solid product. Yield: 0.59 g (25 %). 1H NMR (400 MHz, CDCl3): δ 8.50 (d, J = 12.0 Hz, 1H), 8.19 (d, J = 8.0 Hz, 1H), 7.93 (d, J = 7.6 Hz, 1H), 7.55-7.62 (m, 3H),7.16 (s, 1H), 7.06 (q, 2H), 6.48-6.60 (m, 3H), 5.92 (d, J=8.0 Hz, 1H), 3.57 (s, 3H), 3.48 (q, 4H), 2.59 (t, J= 8.0 Hz, 2H), 2.45 (t, J = 8.0 Hz, 2H), 1.76 (8H), 1.13 (t, J = 8.0 Hz, 6H). 13C NMR (100 MHz, CDCl3): δ 174.81, 163.52, 156.16, 153.05, 143.10, 140.34, 135.35, 132.93, 131.57, 131.46, 129.16, 129.00, 128.94, 125.30, 122.14, 121.27, 114.13, 112.55, 110.02, 109.28, 98.27, 95.54, 48.74, 45.25, 31.21, 28.53, 27.53, 26.48, 24.27, 12.51. MS (TOF, m/z): Calcd for C37H39N2O3+ [M]+, 559.3; Found 559.4. Compound 4: Compound 1 (0.76 mmol, 0.55 g), NH2NH2·H2O (7.6 mmol, 0.5 mL) and BOP reagent (0.80 mmol, 0.36 g) were dissolved in CH2Cl2 (25.0 mL). The mixture was stirred at room temperature for 2 h. The solvent was evaporated and the crude product was purified via chromatography (silica gel, CH2Cl2:CH3CH2OH = 25:1) to obtain a yellow solid product. Yield: 0.32 g (74 %). 1H NMR (400 MHz, CDCl3): δ 7.81 (d, J = 8.8 Hz, 1H), 7.34-7.43 (m, 3H), 7.077.13 (m, 3H), 6.76 (d, J = 7.2 Hz, 1H), 6.54 (d, J = 7.6 Hz, 1H), 6.29 (d, J = 8.8 Hz, 2H), 6.20 (s, 1H), 5.30 (d, J = 8.0 Hz, 1H), 3.28 (q, 4H), 3.07 (s, 3H), 2.50 (2H), 1.63-1.65 (10H), 1.10 (t, J = 8.0 Hz, 6H). MS (TOF, m/z): Calcd for C37H40N4O2 [M+H]+, 573.3; Found 573.3. Compound CyR: Compound 4 (0.5 mmol, 0.29 g) and glyoxal (0.058 g, 1.00 mmol) were dissolved in anhydrous methanol (25.0 mL). The reaction mixture was stirred at room temperature for 6 h. The solvent was evaporated and the crude product was purified via chromatography (silica gel, CH2Cl2:CH3CH2OH = 28:1) to obtain a yellow solid product. Yield: 0.26 g (86 %). 1H NMR (400 MHz, CDCl3): δ 9.44 (d, J = 6.8 Hz, 1H), 7.82 (d, J = 7.2 Hz, 1H), 7.35-7.43 (m, 4H), 7.08-7.13 (m, 3H), 6.76 (d, J = 8.0 Hz, 1H), 6.53 (d, J = 7.6 Hz, 1H), 6.20-6.29 (m, 3H), 5.29 (d, J = 8.0 Hz, 1H), 3.27 (q, 4H), 3.07 (s, 3H), 2.25 (2H), 1.63-1.65 (10H), 1.10 (t, J = 8.0 Hz, 6H). 13C NMR (100 MHz, CDCl3): δ 192.67, 168.21, 156.15, 153.04, 152.15, 149.59, 143.07, 140.68, 140.33, 134.16, 132.92, 131.47, 129.16, 128.82, 128.26, 125.29, 124.53, 123.80, 122.15, 121.26, 110.03, 109.27, 106.22, 105.06, 97.07, 95.53, 68.24, 48.24, 44.55, 31.21, 29.68, 28.56, 24.27, 22.19, 12.51. Anal. calcd. for C39H40N4O3 (CyR): C, 76.44; H, 6.58; N, 9.14. Found: C, 75.43; H, 6.60; N, 9.35. MS
2
ACS Paragon Plus Environment
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
Analytical Chemistry
(TOF, m/z): Calcd for C39H40N4O3 [M+H]+, 613.3; Found 613.4. Scheme 2. Synthetic Route for Compound CyR. N
O
N O
N
N O+
HO
O
Ac2O
H2SO4 , HClO4
O HOOC
N+
HOOC
HOOC
1
3
N
N
NH2NH2 H2O ,BOP
O
O O
CH2Cl2
O CH3OH
N
N
N H2N
N N
O
O
O
4
CyR
General Fluorescence Measurements. The fluorescence measurements were obtained by using excitation at 698 nm and emission at 720-800 nm. A stock solution of CyR (1.0×10-4 M) was prepared by dissolving CyR in solution (H2O:EtOH = 9:1). A stock solution of GSH (1.0×10-2 M) was prepared by dissolving GSH in water and the solution was further diluted to 1.0×10-3-1.0×10-7 M stepwise. The solution of CyR with GSH was prepared by the using the stock solutions and diluting with phosphate-buffered saline (PBS) buffer solution. In the solutions thus obtained, the concentrations of CyR were 1.0×10-5 M and GSH were 1.0×10-3 -1.0×10-8 M (H2O:EtOH = 99:1). Determination of Detection Limit. The detection limit was calculated according to the fluorescence titration. The emission intensity of CyR without GSH was measured three times and the standard deviation of blank measurements was determined. Three independent duplication measurements of emission intensity were performed in the presence of GSH and each average value of the intensities was plotted as a concentration of GSH for determining the slope. The detection limit is then calculated with equation (1): detection limit =3σ/k (1) where σ is the standard deviation of the emission intensity of CyR, and k is the slope between the emission intensity vs concentration. Determination of the Fluorescence Quantum Yield. The quantum yield of the probe was determined according to equation (2). Φ = Φ
(
)
(2)
where Φst is the quantum yield of the standard, D is the area under the emission spectra, A is the absorbance at the excitation wavelength, and η is the refractive index of the solvent used. x subscript denotes unknown, and st means standard. We chose ICG (Φf = 0.13 in DMSO) as the standard. Cell Incubation and Imaging. HeLa cells were purchased by XiangYa Central Experiment Laboratory of Central South University (Changsha, China). HeLa cells were treated in DMEM (Dulbecco’s modified Eagle’s medium) supplemented with FBS (10% fetal bovine serum) in an atmosphere of 5%
CO2 and 95% air at 37 °C. And then the cells were plated on 35 mm culture dish and allowed to adhere for 24 h. Firstly, the cells were incubated with 10 µM probe CyR for 30 min at 37 °C after they were washed with PBS buffer solution for three times. Secondly, the Hela cells were pretreated with 100 µM NEM in culture medium at 37 °C for 30 min, washed with PBS for three times, then incubated with 10 µM probe CyR in culture medium at 37 °C for 30 min. Finally, after pretreated with NEM and incubated with 10 µM probe CyR at the same condition, 100 µM GSH, Cys or Hcy was added at 37 °C for another 30 min, separately. Preparation and Staining of Rat Liver Tissue Slices. Tissue slices were prepared from rat liver. A side of the tissue was cut flat using a vibrating-blade microtome. One tissue slice was cultured with 10 µM CyR in an incubator at 37°C for 60 min, followed by washing three times with PBS before imaging. The other tissue slice was cultured with 100 µM NEM and then 10 µM CyR in an incubator at 37°C for 60 min, followed by washing three times with PBS before imaging.
RESULTS AND DISCUSSION Spectroscopic Properties of Probe CyR toward GSH. The UV-visible spectra of probe CyR in the absence of GSH and in the presence of GSH were recorded in Figure S8. Probe CyR exhibited almost no characteristic absorption in the absence of GSH. While, in the presence of GSH, an absorption band at 698 nm accompanied by color change from yellow to green was found. To investigate the sensitivity of probe CyR to GSH, its emission spectra variation was monitored during titration experiments with various concentrations of GSH (Figure 1). For free CyR, no obvious emission was noticed, demonstrating the probe was at spirolactam form. The fluorescence quantum yield of free CyR is less than 0.01 using indocyanine green (ICG) as standard. Upon the addition of GSH, a significant fluorescence turn-on response was observed at 735 nm, which showed that the spirolactam ring was opened. The fluorescence intensity of the probe gradually increased with increasing GSH concentration. When 25 µM GSH were added, the fluorescence intensity considerably increased around 75-fold. The quantum yield reached 0.43, which is higher than that of the NIR probe for GSH reported in the literatures (Table S1). As the inset of Fig. 2 shows, the linear range of probe CyR for GSH covers from 0.5 to 25 µM with a detection limit of 0.15 µM (3σ/k). Moreover, pH fluorescent titration experiments of 1 in the presence and absence of GSH were also carried out (Figure S9). The results show that probe CyR possesses excellent fluorescence response to GSH in pH range of 6.0-8.0, which is suitable for the detecting GSH under the physiological pH conditions. In addition, the kinetics of the fluorescence of CyR upon the addition of GSH was studied by time-dependent fluorescence spectroscopy (Figure S10). From Figure S10, we can see that stable readings can be acquired in less than 3 min. Once a plateau is reached, the fluorescence intensity of the probe stay relatively stable for the remainder of the measurement, indicating that it is photostable. All these results further prove that the NIR probe CyR has the ability to sense GSH.
3
ACS Paragon Plus Environment
Analytical Chemistry
Fluorescence Intensity
700
Fluorescence Intensity
600 500 400
700 600 500 400 300 200 100 0 0
5
300 GSH
10 15 20 25 30
[GSH] (µM)
200 100 0 720
740
760
780
800
Wavelength (nm)
Figure 1. Fluorescence spectra of CyR (10 µM) after the addition of GSH (0, 0.5, 2.0, 4.0, 6.0, 8.0, 10.0, 12.0, 14.0, 16.0, 18.0, 20.0, 22.0, 25.0 µM) in PBS buffer solution (pH = 7.4). Excitation is 698 nm. Inset: plot of fluorescence intensity versus GSH concentrations.
700 600
Fluorescence Intensity
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
GSH
Page 4 of 8
CyR was first mixed with GSH, and then other biological thiols and amino acids were added, respectively (Figure S13). The probe showed almost unchanged response to GSH before and after the addition of the competing analytes. All these results clearly indicate that probe CyR exhibits an unexpectedly high selectivity for GSH. Response Mechanism. Based on the evidence from fluorescence spectra, a possible reaction mechanism of the NIR probe CyR and GSH could be proposed in Figure 3 (details in Figure S14). In the presence of GSH, the SH residue in GSH and the aldehyde group in CyR could undergo the addition reaction, followed by the formation of an unstable intermediate. Once the intermediate is formed, the distance between spirolactam (probe CyR) and negatively charged carboxylate groups (GSH) becomes closer and the acid microenvironment is therefore generated. Then, the spirolactam may transform into the open-ring form in such acid conditions. Finally, a hydrolysis reaction happens that leads to the formation of 1 and induces strong fluorescence. Similarly, Asp and Glu, containing two carboxylate groups resulting in weak acid environment as well as GSH, showed a slight emission enhancement. By contrast, in the presence of Cys or Hcy, the probe could initially undergo the addition reaction to form an unstable intermediate. Then the following rearrangement and cyclization would lead to the production of the stable five- or sixring heterocycles (2a or 2b). It is found that 2a and 2b are non-fluorescence because they are still in spirolactam ring form.
500 N
400 O N+
300 200 100
1 N N
Asp Glu
N
N
0 740
O
Cys O
720
HOOC
GSH
Blank, Cys, Hcy, Ala, ,Ile, Leu, Met, Phe, Pro, Trp, Val, Asn, Gln, Ser, Thr, Tyr, Arg, His, Lys
760
780
800 O
b S
a
CyR
Wavelength (nm)
N N O
N N O
Figure 2. Fluorescence spectra of CyR (10 µM) in the presence of GSH (25.0 µM), other biological thiols (1 mM) or amino acids (1 mM) in PBS buffer solution (pH = 7.4). Selectivity of Probe CyR toward GSH. To evaluate the selectivity of NIR probe CyR for GSH, a range of biological thiols and amino acids were examined. As shown in Figure 2 and Figure S11, remarkable fluorescent enhancement was observed upon the addition of GSH (25 µM). While, the other biological thiols (Cys, Hcy) and 17 amino acids (Ala, Ile, Leu, Met, Phe, Pro, Trp, Val, Asn, Gln, Gly, Ser, Thr, Tyr, Arg, His, Lys), even at high concentration (1 mM), showed almost no effect on the fluorescence of the probe. Interesting, two other amino acids (Asp, Glu) showed a slight emission enhancement, but the change was to a much lesser extent. In addition, other sulfydryl compounds such as dithiothreitol (DTT) and N-acetylcysteine (NAC) had no interference effect on the fluorescence of the probe (Figure S12). The results indicate that CyR has high selectivity towards GSH. It is worth mentioning that the probe can discriminate GSH from Cys and Hcy, which have similar molecular structure with GSH. Competition experiments were also conducted in which
NH c
e d COOH 2a
Hcy
N
O N
N N O
b S
NH c
COOH f ed
2b
4
ACS Paragon Plus Environment
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
Analytical Chemistry
Figure 3. Proposed reaction mechanisms and 1H NMR spectra of probe CyR with GSH, Cys and Hcy. 1 H NMR, 13C NMR and MS (mass spectra) were employed to prove the proposed mechanisms. In 1H NMR experiments (Figure 3), the characteristic proton signal of the aldehyde moiety at 9.44 ppm of CyR disappeared after mixing with GSH. And there was neglectable change on the other proton signals. These experimental results prove the formation of 1. In contrast, upon the addition of Cys (or Hcy), the proton signals of the aldehyde moiety disappeared and the new peaks at 2.61 ppm, 2.75 ppm, 3.63 ppm and 4.19 ppm (or 2.48 ppm, 2.61 ppm, 2.75 ppm, 3.63 ppm and 4.19 ppm) were distinctly observed. These phenomena indicate that the five- or six- ring heterocycle (2a or 2b) forms. More direct evidences were obtained by comparing the 13C NMR data (Figure S15). For CyR, 13 C chemical shift of quaternary-C was 68.2 ppm. While, in the presence of GSH, it was shifted to the aryl region, demonstrating that spirolactam ring was opened. The quaternary-C was still existed with the addition of Cys (or Hcy), showing that the structure was in spirolactam ring form. In MS, the peak at m/z = 559.4 corresponding to 1 occurred upon the addition of GSH (Figure S16). By contrast, upon the addition of Cys (or Hcy), the peak at m/z = 716.3 (or m/z = 730.4) corresponding to 2a (or 2b) appeared (Figure S17 and S18). These data are in good agreement with the proposed mechanism.
Figure 4. (a) The DFT optimized structure of 1. (b) Molecular orbitals (LUMO and HOMO) and HOMO/LUMO energy gaps of 1. To understand the optical properties of 1, the density functional theory (DFT) calculation was conducted at the B3LYP/6-31G* level using Gaussian 09 programs. Figure 4 shows the optimized structures and molecular orbitals (HOMO and LUMO) of 1. The HOMO/LUMO energy gaps of 1 is 2.270 eV. As a control, the optimized structures and molecular orbitals (HOMO and LUMO) of rhodamine B was conducted and the results were shown in Figure S19. The HOMO/LUMO energy gaps of rhodamine B is 2.780 eV, which is bigger than that of 1. And this is in good agreement with that the measured emission wavelengths of 1 (735 nm) is longer than that of rhodamine B (about 580 nm). Therefore, these data can indicate that 1 is a NIR dye.
Figure 5. Fluorescence images of HeLa cells. (a) Cells were incubated with probe CyR (10 µM). (b) Cells were pretreated with NEM (100 µM), and then incubated with probe CyR (10 µM). (c-e) Cells were pretreated with 100 µM NEM, treated with probe CyR (10 µM) and then incubated with 100 µM (c) GSH, (d) Cys, (e) Hcy. Fluorescence Imaging in Live Cells. The practical utility of probe CyR for selective fluorescent imaging of GSH in living cells was studied (Figure 5). A significant bright NIR fluorescence was found when HeLa cells were incubated with probe CyR (Figure 5a). The result indicates that probe CyR is capable of permeating into cells and reacting with endogenous GSH to generate NIR fluorescence. When the HeLa cells were pre-treated with N-ethylmaleimide (NEM), a well-known thiol-blocking agent for the depletion of intracellular thiol species, before an incubation with CyR, there was no emission detected (Figure 5b), indicating that thiol species are completely reacted by NEM. Upon the addition of GSH, Cys or Hcy to the NEM-pre-treated HeLa cells followed by incubating with CyR, NIR fluorescence was only found in living cells with the treatment of GSH (Figure 5c). On the contrary, the treatment of Cys or Hcy could not induce any NIR fluorescence at all (Figure 5d, 5e). Such findings are consistent with the fact that CyR shows selective GSH-induced fluorescence responses in living cells. The cytotoxicity of CyR in HeLa cells by MTT assays was investigated (Figure S20). After incubation with 0 - 20 µM CyR, the cellular viability was more than 97%, indicating that CyR possesses low cytotoxicity to the cultured cells and has great potential for biological applications. Fluorescence Imaging in Tissues. To further show the advantage of NIR fluorescence probe (CyR), fluorescence images of rat liver tissue slices were carried out (Figure 6). One tissue slice treated only with probe CyR showed noticeable NIR fluorescence signal (Figure 6a). The other tissue was pretreated with NEM and then probe CyR, and no fluorescence was observed (Figure 6b). The fluorescence signal of tissue slice with probe CyR at different tissue depths was collected in the Z-scan mode (Figure 6c). Probe CyR is successfully applied for imaging in tissue, with imaging depths of 40−120 µm. All these results indicate that probe CyR has outstanding tissue penetrating and staining ability.
5
ACS Paragon Plus Environment
Analytical Chemistry
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 6 of 8
Chemometrics Foundation (2015007) and Scientific Research Fund of Hunan Provincial Education Department (15K125).
REFERENCES
Figure 6. Fluorescence images of tissues. (a) Tissue slice was incubated with probe CyR (10 µM). (b) Tissue slice was pretreated with NEM (100 µM), and then incubated with probe CyR (10 µM). (c) The confocal z-scan imaging sections at different depths for 0, 20, 40, 60, 80, 100, 120, 140 µm.
CONCLUSIONS In summary, we have developed a NIR fluorescent probe with high fluorescence quantum yield by introducing rigid coplanar structure such as rhodamine dyes. And the NIR probe (CyR) with spirolactam structure is firstly synthesized and used to recognize GSH. Probe CyR exhibits high fluorescence quantum yield (Φ = 0.43) after the addition of GSH and high sensitivity toward GSH with 75-fold fluorescence enhancement. Furthermore, the probe is highly selective, which will not interfere with the other biological thiols (Cys, Hcy) and amino acids. In particular, the NIR probe displays selective detection of GSH in biological samples such as living cells and tissues. It is expected that the NIR fluorescent probe could be widely applied to detect various biological analytes by changing recognition sites.
ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Comparison of the probes, chemical structures of biological thiols, 1 H NMR, 13C NMR and MS spectra, and additional spectroscopic data.
AUTHOR INFORMATION Corresponding Author * E-mail:
[email protected]. Tel: +86-731-58292205. Fax: +86-731-58292477.
Notes The authors declare no competing financial interest.
ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (21005068, 21475114), Hunan Provincial Natural Science Foundation of China (13JJ6039, 2015JJ6104), China Postdoctoral Science Foundation funded project (2014M552140, 2015T80876), State Key Laboratory of Chemo/Biosensing and
(1) Meister, A.; Anderson, M. E. Ann. Rev. Biochem. 1983, 52, 711760. (2) Coles, B.; Ketterer, B. Biochem. Mol. Biol. 1990, 25, 47-70. (3) Estrela, J. M.; Ortega, A.; Obrador, E. Crit. Rev. Clin. Lab. Sci. 2006, 43, 143-181. (4) Herzenberg, L. A.; De Rosa, S. C.; Dubs, J. G.; Roederer, M.; Anderson, M. T.; Ela, S. W.; Deresinski, S. C.; Herzenberg, L. A. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 1967-1972. (5) Quang, D. T.; Kim, J. S. Chem. Rev. 2010, 110, 6280-6301. (6) Yang, Y.; Zhao, Q.; Feng, W.; Li, F. Chem. Rev. 2012, 113, 192270. (7) Yin, J.; Hu, Y.; Yoon, J. Chem. Soc. Rev. 2015, 44, 4619-4644. (8) Sharma, S. K. D.; Bera, R. K.; Crisponi, G.; Callan, J. F. Chem. Soc. Rev. 2012, 41, 7195-7227. (9) Sahana, A.; Banerjee, A.; Lohar, S.; Panja, S.; Mukhopadhyay, S. K.; Matalobos, J. S.; Das, D. Chem. Commun. 2013, 49, 7231-7233. (10) Tang, B.; Xing, Y.; Li, P.; Zhang, N.; Yu, F.; Yang, G. J. Am. Chem. Soc. 2007, 129, 11666-11667. (11) Wang, L.; Chen, H.; Wang, H.; Wang, F.; Kambam, S.; Wang, Y.; Zhao, W.; Chen, X. Sens. Actuators B. 2014, 192, 708-713. (12) Kim, G. J.; Lee, K.; Kwon, H.; Kim, H. J. Org. Lett. 2011, 13, 2799-2801. (13) Jia, M. Y.; Niu, L. Y.; Zhang, Y.; Yang, Q. Z.; Tung, C. H.; Guan, Y. F.; Feng, L. ACS Appl. Mater. Interfaces. 2015, 7, 59075914. (14) Cao, M.; Chen, H.; Chen, D.; Xu, Z.; Liu, S.; Chen, X.; Yin, J. Chem. Commun. 2016, 52, 721-724. (15) McMahon, B. K.; Gunnlaugsson, T. J. Am. Chem. Soc. 2012, 134, 10725-10728. (16) Zhu, W.; Huang, X.; Guo, Z.; Wu, X.; Yu, H.; Tian, H. Chem. Commun. 2012, 48, 1784-1786. (17) Yuan, L.; Lin, W.; Zhao, S.; Gao, W.; Chen, B.; He, L.; Zhu, S. J. Am. Chem. Soc. 2012, 134, 13510-13523. (18) Guo, Z.; Park, S.; Yoon, J.; Shin, I. Chem. Soc. Rev. 2014, 43,1629. (19) Li, Y.; Wang, Y.; Yang, S.; Zhao, Y.; Yuan, L.; Zheng, J.; Yang, R. Anal. Chem. 2015, 87, 2495-2503. (20) Han, X.; Yu, F.; Song, X.; Chen, L. Chem. Sci. 2016, 7, 50985107. (21) Huang, Y.; Yu, F.; Wang, J.; Chen, L. Anal. Chem. 2016, 88, 4122-4129. (22)Yu, F.; Gao, M., Li, M., Chen, L. Biomaterials. 2015, 63, 93-101. (23) Gao, M.; Yu, F.; Chen, H.; Chen, L. Anal. Chem. 2015, 87, 36313638. (24) Yu, F.; Li, P.; Song, P.; Wang, B.; Zhao, J.; Han, K. Chem. Commun. 2012, 48, 4980-4982. (25) Maity, D.; Govindaraju, T. Org. Biomol. Chem. 2013, 11, 20982104. (26) Xu, K.; Qiang, M.; Gao, W.; Su, R.; Li, N.; Gao, Y.; Xie, Y.; Kong, F.; Tang, B. Chem. Sci. 2013, 4, 1079-1086. (27) Lim, S. Y.; Hong, K. H.; Kim, D. I.; Kwon, H.; Kim, H. J. J. Am. Chem. Soc. 2014, 136, 7018-7025. (28) Hu, Q.; Yu, C.; Xia, X.; Zeng, F.; Wu, S. Biosens. Bioelectron. 2016, 81, 341-348. (29) Yin, J.; Kwon, Y.; Kim, D.; Lee, D.; Kim, G.; Hu, Y.; Yoon, J. J. Am. Chem. Soc. 2014, 136, 5351-5358. (30) Yu, F.; Li, P.; Li, G.; Zhao, G.; Chu, T.; Han, K. J. Am. Chem. Soc. 2011, 133, 11030-11033. (31) Yu, F.; Li, P.; Wang, B.; Han, K. J. Am. Chem. Soc. 2013, 135, 7674-7680. (32) Li, M.; Wu, X.; Wang, Y.; Li, Y.; Zhu, W.; James, T. D. Chem. Commun. 2014, 50, 1751-1753. (33) Bao, B.; Liu, M.; Liu, Y.; Zhang, X.; Zang, Y.; Li, J.; Lu, W. Tetrahedron. 2015, 71, 7865-7868.
6
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
Analytical Chemistry
(34) Xu, Y.; Li, B.; Han, P.; Sun, S.; Pang, Y. Analyst. 2013, 138, 1004-1007. (35) Beija, M.; Afonso, C. A. M.; Martinho, J. M. G. Chem. Soc. Rev. 2009, 38, 2410-2433. (36) Chen, X.; Pradhan, T.; Wang, F.; Kim, J. S.; Yoon, J. Chem. Rev. 2012, 112, 1910-1956. (37) Kim, H. N.; Lee, M. H.; Kim, H. J.; Kim, J. S.; Yoon, J. Chem. Soc. Rev. 2008, 37, 1465-1472. (38) Li, X.; Gao, X.; Shi, W.; Ma, H. Chem. Rev. 2013, 114, 590-659. (39) Zhao, Y.; Zhang, X. B.; Han, Z. X.; Qiao, L.; Li, C. Y.; Jian, L. X.; Shen, G. L.; Yu, R. Q. Anal. Chem. 2009, 81, 7022-7030. (40) Tang, J. L.; Li, C. Y.; Li, Y. F.; Lu, X.; Qi, H. R. Anal. Chim. Acta 2015, 888, 155-161. (41) Li, D.; Li, C. Y.; Li, Y. F.; Li, Z.; Xu, F. Anal. Chim. Acta 2016, 934, 218-225. (42) Yuan, L.; Lin, W.; Yang, Y.; Chen, H. J. Am. Chem. Soc. 2012, 134, 1200-1211. (43) Vegesna, G. K.; Janjanam, J.; Bi, J.; Luo, F. T.; Zhang, J.; Olds, C.; Tiwari, A.; Liu, H. J. Mater. Chem. B. 2014, 2, 4500-4508. (44) Yang, H.; Han, C.; Zhu, X.; Liu, Y.; Zhang, K. Y.; Liu, S.; Zhao, Q.; Li, F.; Huang, W. Adv. Funct. Mater. 2016, 26, 1945-1953.
7
ACS Paragon Plus Environment
Analytical Chemistry
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
ACS Paragon Plus Environment
