Biosensors and Bioelectronics 56 (2014) 58–63
Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
A water-soluble BODIPY derivative as a highly selective “Turn-On” fluorescent sensor for H2O2 sensing in vivo Jian Xu a,b, Qian Li a, Ying Yue a, Yong Guo a, Shijun Shao a,n a Key Laboratory of Chemistry of Northwestern Plant Resources and Key Laboratory for Natural Medicine of Gansu Province, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Lanzhou 730000, PR China b University of Chinese Academy of Sciences, Beijing 100039, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 26 November 2013 Received in revised form 25 December 2013 Accepted 27 December 2013 Available online 9 January 2014
A type of BODIPY derivatives was designed and synthesized by the N-alkylation reaction of meso-(4pyridinyl)-substituted BODIPY. The water-solubility of entire molecule was improved to a large extent as a result of the formation of cationic quaternary ammonium salt, while the strong fluorescence inherent to the BODIPY dye fragment is extinguished on alkylation of the pyridine N atom due to the photoinduced electron transfer (PET) process. The N-alkylated BODIPY derivative 4, as a novel water-soluble “Turn-On” fluorescent probe for the discrimination of H2O2, was constructed by incorporating 4(Bromomethyl)benzeneboronic acid pinacol ester moiety, which showed highly selective fluorescent response to H2O2 against other interferences of ROS and RNS species under physiological conditions, and the reaction mechanism of boronate oxidation was confirmed by 1H NMR, mass spectrum and optical spectroscopy analysis. As a biocompatible probe in biological systems, probe 4 was successfully applied for monitoring and imaging of H2O2 both in vitro and in vivo using HepG2/LO2 cells and angelfish. & 2014 Elsevier B.V. All rights reserved.
Keywords: N-alkylpyridinium substituted BODIPY derivatives Water-soluble fluorescent probe Fluorescent sensor H2O2 In vitro and in vivo imaging
1. Introduction Hydrogen peroxide (H2O2), one of the important products of oxygen metabolism, acts as a signaling molecule in a wide variety of signaling transduction processes. Mounting evidence supports a physiological role for H2O2 as an oxidative stress marker and a defense agent in response to pathogen invasion (Alamiry et al., 2008; Lippert et al., 2011; Yuan et al., 2012). H2O2 bursts can trigger several classes of essential signaling proteins that affect cell proliferation and thus lead to various diseases including cancer, diabetes, and cardiovascular and neurodegenerative disorders (D’Autréaux and Toledano, 2007; Miller et al., 2010; Paulsen and Carroll, 2009; Rhee, 1999, 2006; Winterbourn, 2008). In bioanalytical chemistry, many efforts have been devoted to the determination of H2O2 (Chen et al., 2012; Fähnrich et al., 2001; Nogueira et al., 2005). The development of fluorescent probe for discriminating H2O2 offers a promising method for monitoring this dynamic change (Abo et al., 2011; Alamiry et al., 2008; Maeda et al., 2004; Onoda et al., 2003; Ulrich and Ziessel, 2004; Wolfbeis et al., 2002; Xu et al., 2005). However, the most important challenge is the transient nature of H2O2, which makes it highly difficult to adopt a traditional “lock-and-key” molecular recognition approach. In this context, Chang and coworkers recently developed a highly efficient reaction-based approach by utilizing the unique
n
Corresponding author. Tel.: þ 86 931 4968209; fax: þ 86 931 8277088. E-mail address: [email protected] (S. Shao).
0956-5663/$ - see front matter & 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bios.2013.12.065
reactivity between H2O2 and boronate moiety; a series of fluorescence probes have been devised and some of them have been used for biological applications (Albers et al., 2006; Chang et al., 2004; Dickinson and Chang, 2008; Lippert et al., 2011; Miller et al., 2005). Furthermore, for the biochemical importance of H2O2, the suitable design of water-soluble fluorescence probes for selectively sensing H2O2 especially for sensing in vivo is still a challenge for chemists. Significantly, the properties of the fluorescent probe heavily rely on the emissive core, which should embrace excellent optical features, easy modification, good water-solubility, insensitivity to pH and solvent polarity, excellent thermal and photochemical stabilities, as well as low biological toxicity (Loudet and Burgess, 2007; Ulrich et al., 2008). Along this line, BODIPY (4,4-Difluoro-4bora-3a,4a-diaza-s-indacene) fluorescence dyes meet most of the requirements mentioned above, and widely used as fluorescent switches, chemosensors, laser dyes and especially as labeling reagents in proteins and DNA research fields (Cheng et al., 2008; Hong et al., 2006; Karolin et al., 1994). However, the low hydrophilicity of BODIPY dyes restricted their applications in environmental and biological fields, and the effort to improve the water solubility of BODIPY derivatives still remains challenging. Most of the reported strategies involve introduction of oligo (ethylene glycol) chains, α-galactosylceramide, N,N-bis(2-hydroxyethyl) amine, nucleotides, carbohydrates, carboxylates, sulfonates, or ammonium groups to BODIPY skeleton (Brellier et al., 2010; Bura and Ziessel, 2011; Dodani et al., 2009; Jiao et al., 2010; Li et al., 2008; Niu et al., 2009; Vo‐Hoang et al., 2003; Zhu et al., 2010). For most of them,
J. Xu et al. / Biosensors and Bioelectronics 56 (2014) 58–63
synthetic methodologies remain elusive and purification procedures are extremely tedious. Recently, a few BODIPY derivatives bearing a quaternarized pyridine moiety in their meso-position have been reported as functional dyes for various applications (Alamiry et al., 2008; Harriman et al., 2007; Jiang et al., 2013; Ulrich and Ziessel, 2004; Zhang et al., 2013), which showed good solubility in water because of the cationic pyridinium unit on the head moiety (Wang et al., 2009). It is noteworthy that, for the quaternarized 4-pyridinylsubstituted BODIPY derivative, fluorescence from the dye fragment was extensively quenched due to the onset of a light-induced charge-shift reaction ( Harriman et al., 2007). We decided to further explore its application in the development of watersoluble “Turn-On” fluorescent sensors. Herein, we disclosed a new approach toward this goal. Several new water-soluble BODIPY derivatives were synthesized by the N-alkylation reaction of 4-pyridyl substituted BODIPY. Especially, the N-alkylated BODIPY derivative by incorporating 4-(Bromomethyl)benzeneboronic acid pinacol ester moiety was constructed as a water-soluble “Turn-On” fluorescent probe for the discrimination of H2O2, and highly selective fluorescent response to H2O2 under physiological conditions was demonstrated. As a biocompatible probe in biological systems, the probe was successfully applied for both in vitro and in vivo imaging of H2O2 using HepG2/LO2 cells and angelfish.
2. Experimental 2.1. Materials and measurements. All chemicals were purchased from Sigma-Aldrich reagent company without further purification except especial instruction. All the organic solvents were of analytical grade. Water was purified by a Milli-Q system. Melting points were determined on a PHMKG 05 (Germany) apparatus. 1H and 13C NMR spectra were measured on a Varian INOVA 400 M spectrometer. ESI mass spectra were recorded on an Agilent 1100 series LC/MSO Trap of MS spectrometer. HepG2 and LO2 cells were purchased from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences. All spectroscopic measurements were performed in 10 mM phosphate buffer (pH 7.4) at room temperature. All pH measurements were performed with a Sartorius basic pH-Meter. UV– visible spectral studies were performed on a Perkin-Elmer Lambda-35 UV–visible double beam scanning spectrophotometer. Solution fluorescence spectra were measured on a Perkin-Elmer LS 55 scanning spectrofluorometer equipped with a Xenon flash lamp. Samples for absorption and fluorescence measurements were contained in 1 cm 1 cm quartz cuvettes.
59
400 MHz): δ ¼14.6, 121.8, 123.3, 130.3, 137.6, 142.6, 143.6, 150.6, 156.4. MS(ESI): Calcd for C18H18BF2N3: 325.2, found: m/z 326.2 (M þH) þ . Compound 2: Compound 1 (0.09 mmol, 0.03 g) and methyl iodide (0.9 mmol, 60 μL) were dissolved in toluene, and then the mixture was refluxed at 110 1C for 12 h. The solvent was removed and purified by silica gel column chromatography (CH2Cl2/methanol, 10/1, v/v, as eluent) to afford the desired compound 2 as deep red crystals (0.011 g, 35%). m.p. 228–230 1C. 1H NMR (CDCl3, 400 MHz): δ ¼7.70 (d, 2H, pyridine H), 7.52 (d, 2H, pyridine H), 6.03 (s, 2H, pyrrole H), 4.28 (s, 3H, CH3), 2.60 (s, 6H, CH3), 1.23 (s, 6H, CH3). 13C NMR (CDCl3, 400 MHz): δ ¼13.7, 19.2, 24.2, 29.7, 30.5, 31.0, 65.6, 128.8, 130.9, 132.2, 167.8. MS(ESI): Calcd for [C19H21BF2N3] þ : 340.2, found: m/z 340.2 (M) þ . Compound 3: Compound 1 (0.06 mmol, 0.02 g) and benzyl bromide (0.6 mmol, 0.10 g) were dissolved in toluene, and then the mixture was refluxed at 110 1C for 12 h. After filtration, the precipitate was filtered, washed with toluene and dried in vacuo to afford pure compound 3 (0.018 g, 62%). m.p. 165–168 1C. 1H NMR (CDCl3, 400 MHz): δ ¼9.85 (d, 2H, pyridine H), 7.91 (d, 2H, pyridine H), 7.64 (d, 2H, ArH), 7.33 (d, 3H, ArH), 6.45 (s, 2H, CH2), 5.97 (s, 2H, pyrrole H), 2.54 (s, 6H, CH3), 1.37 (s, 6H, CH3). 13C NMR (CDCl3, 400 MHz): δ ¼ 14.7, 15.5, 64.4, 122.8, 128.3, 129.2, 129.3, 129.7, 130.1, 132.6, 133.0, 141.9, 146.3, 153.1, 158.3. MS(ESI): Calcd for [C25H25BF2N3] þ : 416.2, found: m/z 416.3 (M) þ . Compound 4: Compound 1 (1.2 mmol, 0.40 g) and 4-(Bromomethyl)- benzeneboronic acid pinacol ester (2.4 mmol, 0.71 g) were dissolved in toluene, and then the mixture was refluxed at 110 1C for 12 h. The obtained orange powdery solid was filtered, washed with toluene and dried in vacuo to afford pure compound 4 (0.18 g, 24%). m.p. 258–260 1C. 1H NMR (CDCl3, 400 MHz): δ ¼9.82 (d, 2H, pyridine H), 7.96 (d, 2H, pyridine H), 7.83 (d, 2H, ArH), 7.57 (d, 2H, ArH), 6.55(s, 2H, CH2), 6.02 (s, 2H, pyrrole H), 2.54 (s, 6H, CH3), 1.37 (s, 6H, CH3), 1.34(s, 12H, CH3). 13 C NMR (CDCl3, 400 MHz): δ ¼ 14.8, 16.5, 24.9, 64.7, 84.2, 122.9, 128.2, 128.5, 129.3, 132.5, 135.6, 136.1, 141.9, 146.3, 153.3, 158.4. MS (ESI): Calcd for [C31H36B2F2N3O2] þ : 542.2, found: m/z 542.3 (M) þ . 2.3. Measurement of fluorescence quantum yields For determination of the fluorescence quantum yields (Φfl), we used a Perkin-Elmer LS 55 instrument, with fluorescein in 0.1 M NaOH as a fluorescence standard. Fluorescence quantum yields (Φfl) were obtained by the following equation (F denotes fluorescence intensity at each wavelength and Σ[F] was calculated by summation of fluorescence intensity):
Φfl sample ¼ Φfl standard Absstandard Σ[Fsample] / Abssample Σ[Fstandard]
2.2. Synthesis of BODIPY derivatives 1–4.
3. Results and discussions
Compound 1: 4-Pyridinecarboxaldehyde (9.0 mmol, 0.96 g) was stirred with 2,4-dimethylpyrrole (19.4 mmol, 1.85 g) in deoxygenated CH2Cl2 (150 mL). One drop of TFA was added and the mixture was stirred overnight under N2 at room temperature. The red solution was treated with TCBQ (9.0 mmol, 2.21 g), stirring was continued for 30 min followed by the addition of Et3N (15 mL). After 15 min, BF3 Et2O (15 mL) was added at 0 1C, and the mixture was stirred at room temperature for further 3 h. After washing with saturated aqueous NaHCO3, the organic phase was separated, dried with MgSO4, filtered, and concentrated. The residue was purified by silica gel column chromatography (CH2Cl2/ethyl acetate, 1/1, v/v, as eluent) to afford the desired compound 1 as red powder (0.35 g, 12%). m.p. 241–243 1C. 1H NMR (CDCl3, 400 MHz): δ ¼8.75 (d, 2H, pyridine H), 7.28 (d, 2H, pyridine H), 5.99 (s, 2H, pyrrole H), 2.54 (s, 6H, CH3), 1.38 (s, 6H, CH3). 13C NMR (CDCl3,
3.1. Spectroscopic property of probe 4 and its response to H2O2 The synthetic routes of the N-alkylated BODIPY derivatives are depicted in Scheme 1. According to a procedure described previously (Harriman et al., 2007; Ulrich and Ziessel, 2004), we firstly synthesized 4-pyridinyl-substituted BODIPY precursor 1 which showed poor water solubility. When the pyridine group of 1 was alkylated to form the N-methyl-substituted derivative 2 or N-benzyl-substituted derivative 3, as expected, the watersolubility of entire molecule was improved to a large extent due to the newly formed cationic quaternary ammonium salt (Scheme 1). More interestingly, the process of N-alkylated derivatives can simultaneously exterminate the fluorescence of the BODIPY dyes. The BODIPY dye 1 showed strong green fluorescence (λem ¼520 nm) with high fluorescence quantum yield (Φfl ¼0.78).
60
J. Xu et al. / Biosensors and Bioelectronics 56 (2014) 58–63
Scheme 1. Synthesis of the N-alkylpyridinium substituted BODIPY derivatives 2, 3 and 4.
Comparatively, the quantum yield for the N-alkylated compounds 2 (Φfl ¼0.005) and 3 (Φfl ¼0.003) is much weaker (Table S1 in ESI†), which is attributed to the emission quenching due to the photo-induced electron transfer (PET) process. Encouraged by these findings, we envisioned that “Turn-On” fluorescence probe for sensing H2O2 could be constructed based on this novel water soluble N-alkylated BODIPY by incorporating boronate moiety. To demonstrate that, fluorescent probe 4 was designed, where p-pinacolborylbenzyl unit attached through C–N linkage to the meso-pyridine group of BODIPY. As expected, probe 4 has a very low fluorescence quantum efficiency (Φfl ¼0.002) due to the PET process. Incubation of probe 4 with H2O2 would release fluorophore 1, leading to turning-on of the fluorescence (Scheme 1). The entirely charged ionic structure of probe 4 can not only improve its water-solubility but also make it easier to penetrate cytomembrane for intracellular and in vivo sensing. Firstly, the spectroscopic property of probe 4 and its response to H2O2 were evaluated under physiological conditions (10 mM PBS, pH 7.4). Probe 4 features a maximum absorbance at 508 nm and a very weak emission at 520 nm. In the presence of H2O2, the absorption at 508 nm of probe 4 decreased and a new blue-shift band at 500 nm evolved (Figs. S2 and S3 in ESI†), which was reasonably attributed to the H2O2-induced oxidation of probe 4 to release BODIPY fluorophore 1. As shown in Fig. 1, treatment of probe 4 with H2O2 triggers a dramatic increase of fluorescence intensity at 520 nm, which ramps up to the maximum in ca. 120 min. Correspondingly, an obvious bright green-colored fluorescence was clearly observed after H2O2 addition. 58-fold increase in its fluorescence intensity suggested that probe 4 is one of the most sensitive probes for the detection of H2O2 in abiotic systems (Dickinson et al., 2010; Lippert et al., 2010). The detailed emission titration experiments of probe 4 with various concentrations of H2O2 were also carried out in 10 mM PBS (pH 7.4) buffer solution. There was a good linearity between the fluorescence intensity and the concentrations of H2O2 in the range of 0.1–40 mM (Fig. S1 in ESI†) with a detection limit of 0.03 mM (see ESI†). 3.2. Stability under different pH and the selectivity test We next investigated the pH-dependence of probe 4 in the detection of H2O2. As shown in Fig. 2A, the fluorescence intensity dramatically increased when the pH value was higher than 7.0 and reached a peak value about 10. It is mainly because arylboronic acids can only react with H2O2 under mild alkaline conditions to generate phenols and the phenomenon has been demonstrated by other groups (Lo and Chu, 2003). We also evaluated the potential interfering effect of various reactive oxygen species (ROS) and reactive nitrogen species (RNS) on the reaction of 4 with H2O2. The changes in fluorescence intensity before and after the addition of interferent (Fig. 2B) indicated that fluorescence augmentation occurred only upon reaction with H2O2 and probe 4 exhibits excellent selectivity toward H2O2 among the various ROS/ RNS and biologically
Fig. 1. Fluorescence turn-on response of 5 μM probe 4 to 100 μM H2O2. Spectra shown were acquired before H2O2 addition and 5, 15, 30, 45, 60, 90 and 120 min after H2O2 was added. Inset: fluorescence photograph of 5 μM probe 4 upon incubation with 100 μM H2O2 for 120 min. All measurements were acquired at 25 1C in 10 mM PBS, pH 7.4, with excitation at 480 nm.
relevant species in abiotic systems. The excellent selectivity is ascribed to the H2O2-specific boronate deprotection reaction and the ambiphilic properties of H2O2 as a two-electron transferred oxidant (Lippert et al., 2011). 3.3. Proposed reaction mechanism and NMR analysis The “Turn-On” fluorescence sensing mechanism for probe 4 toward H2O2 was proposed in Fig. 3A. Triggered by the oxidation of the C–B bond by the nucleophilic attack of H2O2 (4 to 5), the emissive fluorogen 1 is released for realizing the sensing purpose (Lo and Chu, 2003). The oxidation process of 4 by H2O2 and subsequent release of 1 were confirmed by 1H NMR analysis. When the reaction of 4 with H2O2 was carried out in CD3CN/D2O (1/1, v/v), the intermediate 5 was observed (Fig. 3). Firstly, compound 4 was transformed to compound 5 by 52% after 5 min according to the ratio of integral areas (Fig. 3B-b), as evidenced by the presence of H80 (δ ¼8.94, doublet), H90 (δ ¼8.15, doublet), H20 (δ ¼7.38, doublet), H30 (δ ¼ 6.93, doublet) of compound 5 and the gradual disappearance of H8 (δ ¼9.06, doublet), H9 (δ ¼ 8.21, doublet), H2 (δ ¼7.91, doublet), H3 (δ ¼7.46, doublet) of compound 4. Subsequently, 1,6-benzyl elimination of 5 took place leading to fluorogen 1 and quinone methide which spontaneously reacted with H2O to form compound 6. This was evidenced by the appearance of H2″ (δ ¼7.22, doublet), H3″ (δ ¼ 6.82, doublet) and H7″ (δ ¼5.78, singlet) of compound 6 (Fig. 3B-b and B-c). Fluorogen 1 was released in about 32% within 5 min and 49% within 3 h, as shown by the appearance of H8″ (δ ¼ 8.76, doublet),
J. Xu et al. / Biosensors and Bioelectronics 56 (2014) 58–63
61
Fig. 2. (A) Fluorescence intensity of 5 μM probe 4 with the addition of 10 mM H2O2 at various pH values. (B) Fluorescence responses of 5 μM probe 4 to 100 μM various reactive oxygen species (ROS) and reactive nitrogen species (RNS). All measurements were acquired at 25 1C in 10 mM PBS, pH 7.4, with excitation at 480 nm. Every data point was the mean of three measurements. The error bars represent the standard deviation.
Fig. 3. (A) Proposed reaction mechanism between probe 4 and H2O2. (B) 1H NMR analysis of the reaction between probe 4 and H2O2 in a mixture of D2O/CD3CN (1/1, v/v): (a) probe 4 only; (b) 5 min after addition of H2O2; (c) 3 h after addition of H2O2; (d) 1H NMR of 1 in a mixture of D2O/CD3CN (1/1, v/v).
H9″ (δ ¼7.48 doublet) and H13″ (δ ¼ 6.17, singlet) (Fig. 3B-c). These data are consistent with the authentic sample of fluorogen 1 (Fig. 3B-d). Moreover, formation of fluorogen 1 was further confirmed by the UV spectrum analysis and ESI-MS measurement (Figs. S3 and S12 in ESI†).
intracellular regions. These data establish that probe 4 is capable of live-cell imaging of H2O2 at natural immune response levels. In addition, experiments in LO2 cells give similar results (Fig. 4g-l).
3.5. In vivo imaging of H2O2 using angelfish 3.4. Fluorescence imaging of H2O2 in live cells Further, probe 4 was applied for fluorescence imaging of H2O2 in live biological samples. Firstly, HepG2 cells were selected as a model to test the sensing ability of probe 4 (10 mM) for exogenous H2O2. We observed no obvious fluorescence in the cells prior to treatment with the stimulants (Fig. 4a). Treatment of probe-loaded cells with 100 mM H2O2 for 90 min triggers a striking bright-green fluorescence increase, consistent with H2O2-mediated boronate cleavage occurring within these cells (Fig. 4c). The overlapped brightfield image confirmed that the cells are viable throughout the imaging experiments (Fig. 4d). Because exposure of cells to stimuli such as lipopolysaccharide (LPS) and phorbol myristate acetate (PMA) will activate the generation of ROS (Karton-Lifshin et al., 2011; Srikun et al., 2008), in this experiment, we then use PMA (1 μg/mL) to test the ability of 4 for detecting endogenous bursts of H2O2 produced within living cells. The image of Fig. 4e showed clear increases in green emission localized within entire
The cell experiments confirmed that probe 4 can serve as a biocompatible probe in live biological systems, which encouraged us to evaluate the feasibility for the application of probe 4 to in vivo imaging. The young gray angelfish (Pterophyllum scalare), due to its transparent body, is selected as an ideal model for H2O2 sensing in living organisms. Twenty-day-old angelfish was treated with 10 μM 4 in E3 embryo media (Greene and Tischler, 1976; Lo and Chu, 2003) for 30 min at 25 1C, washed with PBS to remove the remaining chemosensor, and incubated in a solution containing 100 μM H2O2 for 120 min. Fluorescent images obtained are shown in Fig. 5. When angelfish were incubated with 4 alone, weak fluorescence was observed (Fig. 5a). When angelfish was first exposed to 4 and later incubated with H2O2, strong fluorescence was observed all over its body (Fig. 5c), indicating that the probe might have penetrated into the angelfish through its cuticular layer and reacted with H2O2 inside body. These results suggest that probe 4 was highly suitable for sensing H2O2 in vivo.
62
J. Xu et al. / Biosensors and Bioelectronics 56 (2014) 58–63
Fig. 4. Fluorescence images of HepG2 cells incubated with 10 μM probe 4 for 30 min at 37 1C (a) with a brightfield overlay (b) to confirm viability. Probe-stained HepG2 cells treated with 100 μM H2O2 for 90 min at 37 1C (c) with a brightfield overlay (d). Probe-stained HepG2 cells stimulated with 1 μg/mL PMA for 90 min at 37 1C (e) with a brightfield overlay (f). Fluorescence images of LO2 cells incubated with 10 μM probe 4 for 30 min at 37 1C (g) with a brightfield overlay (h). Probe-stained LO2 cells treated with 100 μM H2O2 for 90 min at 37 1C (i) with a brightfield overlay (j). Probe-stained LO2 cells stimulated with 1 μg/mL PMA for 90 min at 37 1C (k) with a brightfield overlay (l).
Fig. 5. In vivo fluorescence images of 20-day-old angelfish treated with 10 μM probe 4 for 30 min at 25 1C (a) with a brightfield image (b). Probe-stained angelfish treated with 100 μM H2O2 for 120 min at 25 1C (c) with a brightfield image (d).
4. Conclusions On the basis of the N-alkylation reaction of meso-(4-pyridinyl)substituted BODIPY, we developed several water-soluble BODIPY derivatives. By combining a 4-(Bromomethyl)benzeneboronic acid pinacol ester moiety and pyridinyl-substituted BODIPY chromophore, a novel water-soluble BODIPY-based fluorescent probe was constructed, which has realized a complete selectivity in “Turn-
On” fluorescent response to H2O2 against other interferences of ROS and RNS species under physiological conditions. Such selectivity results from the unique boronate oxidation mode of the p-pinacolborylbenzyl unit toward H2O2, which leads to release BODIPY fluorophore. Further, the probe was successfully applied for both in vitro and in vivo imaging of H2O2 using HepG2/LO2 cells and angelfish. This work provides a novel approach to design water-soluble BODIPY-based fluorescent probe for biological
J. Xu et al. / Biosensors and Bioelectronics 56 (2014) 58–63
detection, demonstrating its potential applicability to develop a “Turn-On” fluorescent material for monitoring and imaging both in vitro and in vivo. Acknowledgments This work was supported by the National Natural Science Foundation of China (20972170 and 21275150), the Funds for Distinguished Young Scientists of Gansu (1210RJDA013) and the Natural Science Foundation of Gansu province (1107RJYA069). The authors gratefully acknowledge Dr. Xiaoming He (currently at University of Calgary) for helpful discussion. Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2013.12.065. References Abo, M., Urano, Y., Hanaoka, K., Terai, T., Komatsu, T., Nagano, T., 2011. J. Am. Chem. Soc. 133 (27), 10629–10637. Alamiry, M.A.H., Harriman, A., Mallon, L.J., Ulrich, G., Ziessel, R., 2008. Eur. J. Org. Chem. 2008 (16), 2774–2782. Albers, A.E., Okreglak, V.S., Chang, C.J., 2006. J. Am. Chem. Soc. 128 (30), 9640–9641. Brellier, M., Duportail, G., Baati, R., 2010. Tetrahedron Lett. 51 (9), 1269–1272. Bura, T., Ziessel, R., 2011. Org. Lett. 13 (12), 3072–3075. Chang, M.C., Pralle, A., Isacoff, E.Y., Chang, C.J., 2004. J. Am. Chem. Soc. 126 (47), 15392–15393. Chen, W., Cai, S., Ren, Q.Q., Wen, W., Zhao, Y.D., 2012. Analyst 137 (1), 49–58. Cheng, T., Xu, Y., Zhang, S., Zhu, W., Qian, X., Duan, L., 2008. J. Am. Chem. Soc. 130 (48), 16160–16161. D’Autréaux, B., Toledano, M.B., 2007. Nat. Rev. Mol. Cell Biol. 8 (10), 813–824. Dickinson, B.C., Chang, C.J., 2008. J. Am. Chem. Soc. 130 (30), 9638–9639. Dickinson, B.C., Huynh, C., Chang, C.J., 2010. J. Am. Chem. Soc. 132 (16), 5906–5915. Dodani, S.C., He, Q., Chang, C.J., 2009. J. Am. Chem. Soc. 131 (50), 18020–18021. Fähnrich, K.A., Pravda, M., Guilbault, G.G., 2001. Talanta 54 (4), 531–559. Greene, L.A., Tischler, A.S., 1976. Proc. Natl. Acad. Sci. USA 73 (7), 2424–2428. Harriman, A., Mallon, L.J., Ulrich, G., Ziessel, R., 2007. ChemPhysChem 8 (8), 1207–1214.
63
Hong, R., Han, G., Fernández, J.M., Kim, B.-j., Forbes, N.S., Rotello, V.M., 2006. J. Am. Chem. Soc. 128 (4), 1078–1079. Jiang, N., Fan, J., Liu, T., Cao, J., Qiao, B., Wang, J., Gao, P., Peng, X., 2013. Chem. Commun. 49 (90), 10620–10622. Jiao, L., Yu, C., Liu, M., Wu, Y., Cong, K., Meng, T., Wang, Y., Hao, E., 2010. J. Org. Chem. 75 (17), 6035–6038. Karolin, J., Johansson, L.B.-A., Strandberg, L., Ny, T., 1994. J. Am. Chem. Soc. 116 (17), 7801–7806. Karton-Lifshin, N., Segal, E., Omer, L., Portnoy, M., Satchi-Fainaro, R., Shabat, D., 2011. J. Am. Chem. Soc. 133 (28), 10960–10965. Li, L., Han, J., Nguyen, B., Burgess, K., 2008. J. Org. Chem. 73 (5), 1963–1970. Lippert, A.R., De Bittner, G.C.V., Chang, C.J., 2011. Acc. Chem. Res. 44 (9), 793–804. Lippert, A.R., Gschneidtner, T., Chang, C.J., 2010. Chem. commun. 46 (40), 7510–7512. Lo, L.-C., Chu, C.-Y., 2003. Chem. Commun. 21, 2728. Loudet, A., Burgess, K., 2007. Chem. Rev. 107 (11), 4891–4932. Maeda, H., Fukuyasu, Y., Yoshida, S., Fukuda, M., Saeki, K., Matsuno, H., Yamauchi, Y., Yoshida, K., Hirata, K., Miyamoto, K., 2004. Angew. Chem. 43 (18), 2389–2391. Miller, E.W., Albers, A.E., Pralle, A., Isacoff, E.Y., Chang, C.J., 2005. J. Am. Chem. Soc. 127 (47), 16652–16659. Miller, E.W., Dickinson, B.C., Chang, C.J., 2010. Proc. Natl. Acad. Sci. USA 107 (36), 15681–15686. Niu, S.L., Ulrich, G., Ziessel, R., Kiss, A., Renard, P.-Y., Romieu, A., 2009. Org. Lett. 11 (10), 2049–2052. Nogueira, R.F., Oliveira, M.C., Paterlini, W.C., 2005. Talanta 66 (1), 86–91. Onoda, M., Uchiyama, S., Endo, A., Tokuyama, H., Santa, T., Imai, K., 2003. Org. Lett. 5 (9), 1459–1461. Paulsen, C.E., Carroll, K.S., 2009. ACS Chem. Biol. 5 (1), 47–62. Rhee, S.G., 1999. Exp. Mol. Med. 31 (2), 53–59. Rhee, S.G., 2006. Science 312, 1882–1883. Srikun, D., Miller, E.W., Domaille, D.W., Chang, C.J., 2008. J. Am. Chem. Soc. 130 (14), 4596–4597. Ulrich, G., Ziessel, R., 2004. J. Org. Chem. 69 (6), 2070–2083. Ulrich, G., Ziessel, R., Harriman, A., 2008. Angew. Chem. 47 (7), 1184–1201. Vo‐Hoang, Y., Micouin, L., Ronet, C., Gachelin, G., Bonin, M., 2003. ChemBioChem 4 (1), 27–33. Wang, D., Miyamoto, R., Shiraishi, Y., Hirai, T., 2009. Langmuir 25 (22), 13176–13182. Winterbourn, C.C., 2008. Nat. Chem. Biol. 4 (5), 278–286. Wolfbeis, O.S., Dürkop, A., Wu, M., Lin, Z., 2002. Angew. Chem. Int. Ed. 41 (23), 4495–4498. Xu, K., Tang, B., Huang, H., Yang, G., Chen, Z., Li, P., An, L., 2005. Chem. Commun. 48, 5974–5976. Yuan, L., Lin, W., Xie, Y., Chen, B., Zhu, S., 2012. J. Am. Chem. Soc. 134 (2), 1305–1315. Zhang, S., Wu, T., Fan, J., Li, Z., Jiang, N., Wang, J., Dou, B., Sun, S., Song, F., Peng, X., 2013. Org. Biomol. Chem. 11 (4), 555–558. Zhu, S., Zhang, J., Vegesna, G., Luo, F.-T., Green, S.A., Liu, H., 2010. Organ. Lett. 13 (3), 438–441.
Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
A water-soluble BODIPY derivative as a highly selective “Turn-On” fluorescent sensor for H2O2 sensing in vivo Jian Xu a,b, Qian Li a, Ying Yue a, Yong Guo a, Shijun Shao a,n a Key Laboratory of Chemistry of Northwestern Plant Resources and Key Laboratory for Natural Medicine of Gansu Province, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Lanzhou 730000, PR China b University of Chinese Academy of Sciences, Beijing 100039, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 26 November 2013 Received in revised form 25 December 2013 Accepted 27 December 2013 Available online 9 January 2014
A type of BODIPY derivatives was designed and synthesized by the N-alkylation reaction of meso-(4pyridinyl)-substituted BODIPY. The water-solubility of entire molecule was improved to a large extent as a result of the formation of cationic quaternary ammonium salt, while the strong fluorescence inherent to the BODIPY dye fragment is extinguished on alkylation of the pyridine N atom due to the photoinduced electron transfer (PET) process. The N-alkylated BODIPY derivative 4, as a novel water-soluble “Turn-On” fluorescent probe for the discrimination of H2O2, was constructed by incorporating 4(Bromomethyl)benzeneboronic acid pinacol ester moiety, which showed highly selective fluorescent response to H2O2 against other interferences of ROS and RNS species under physiological conditions, and the reaction mechanism of boronate oxidation was confirmed by 1H NMR, mass spectrum and optical spectroscopy analysis. As a biocompatible probe in biological systems, probe 4 was successfully applied for monitoring and imaging of H2O2 both in vitro and in vivo using HepG2/LO2 cells and angelfish. & 2014 Elsevier B.V. All rights reserved.
Keywords: N-alkylpyridinium substituted BODIPY derivatives Water-soluble fluorescent probe Fluorescent sensor H2O2 In vitro and in vivo imaging
1. Introduction Hydrogen peroxide (H2O2), one of the important products of oxygen metabolism, acts as a signaling molecule in a wide variety of signaling transduction processes. Mounting evidence supports a physiological role for H2O2 as an oxidative stress marker and a defense agent in response to pathogen invasion (Alamiry et al., 2008; Lippert et al., 2011; Yuan et al., 2012). H2O2 bursts can trigger several classes of essential signaling proteins that affect cell proliferation and thus lead to various diseases including cancer, diabetes, and cardiovascular and neurodegenerative disorders (D’Autréaux and Toledano, 2007; Miller et al., 2010; Paulsen and Carroll, 2009; Rhee, 1999, 2006; Winterbourn, 2008). In bioanalytical chemistry, many efforts have been devoted to the determination of H2O2 (Chen et al., 2012; Fähnrich et al., 2001; Nogueira et al., 2005). The development of fluorescent probe for discriminating H2O2 offers a promising method for monitoring this dynamic change (Abo et al., 2011; Alamiry et al., 2008; Maeda et al., 2004; Onoda et al., 2003; Ulrich and Ziessel, 2004; Wolfbeis et al., 2002; Xu et al., 2005). However, the most important challenge is the transient nature of H2O2, which makes it highly difficult to adopt a traditional “lock-and-key” molecular recognition approach. In this context, Chang and coworkers recently developed a highly efficient reaction-based approach by utilizing the unique
n
Corresponding author. Tel.: þ 86 931 4968209; fax: þ 86 931 8277088. E-mail address: [email protected] (S. Shao).
0956-5663/$ - see front matter & 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bios.2013.12.065
reactivity between H2O2 and boronate moiety; a series of fluorescence probes have been devised and some of them have been used for biological applications (Albers et al., 2006; Chang et al., 2004; Dickinson and Chang, 2008; Lippert et al., 2011; Miller et al., 2005). Furthermore, for the biochemical importance of H2O2, the suitable design of water-soluble fluorescence probes for selectively sensing H2O2 especially for sensing in vivo is still a challenge for chemists. Significantly, the properties of the fluorescent probe heavily rely on the emissive core, which should embrace excellent optical features, easy modification, good water-solubility, insensitivity to pH and solvent polarity, excellent thermal and photochemical stabilities, as well as low biological toxicity (Loudet and Burgess, 2007; Ulrich et al., 2008). Along this line, BODIPY (4,4-Difluoro-4bora-3a,4a-diaza-s-indacene) fluorescence dyes meet most of the requirements mentioned above, and widely used as fluorescent switches, chemosensors, laser dyes and especially as labeling reagents in proteins and DNA research fields (Cheng et al., 2008; Hong et al., 2006; Karolin et al., 1994). However, the low hydrophilicity of BODIPY dyes restricted their applications in environmental and biological fields, and the effort to improve the water solubility of BODIPY derivatives still remains challenging. Most of the reported strategies involve introduction of oligo (ethylene glycol) chains, α-galactosylceramide, N,N-bis(2-hydroxyethyl) amine, nucleotides, carbohydrates, carboxylates, sulfonates, or ammonium groups to BODIPY skeleton (Brellier et al., 2010; Bura and Ziessel, 2011; Dodani et al., 2009; Jiao et al., 2010; Li et al., 2008; Niu et al., 2009; Vo‐Hoang et al., 2003; Zhu et al., 2010). For most of them,
J. Xu et al. / Biosensors and Bioelectronics 56 (2014) 58–63
synthetic methodologies remain elusive and purification procedures are extremely tedious. Recently, a few BODIPY derivatives bearing a quaternarized pyridine moiety in their meso-position have been reported as functional dyes for various applications (Alamiry et al., 2008; Harriman et al., 2007; Jiang et al., 2013; Ulrich and Ziessel, 2004; Zhang et al., 2013), which showed good solubility in water because of the cationic pyridinium unit on the head moiety (Wang et al., 2009). It is noteworthy that, for the quaternarized 4-pyridinylsubstituted BODIPY derivative, fluorescence from the dye fragment was extensively quenched due to the onset of a light-induced charge-shift reaction ( Harriman et al., 2007). We decided to further explore its application in the development of watersoluble “Turn-On” fluorescent sensors. Herein, we disclosed a new approach toward this goal. Several new water-soluble BODIPY derivatives were synthesized by the N-alkylation reaction of 4-pyridyl substituted BODIPY. Especially, the N-alkylated BODIPY derivative by incorporating 4-(Bromomethyl)benzeneboronic acid pinacol ester moiety was constructed as a water-soluble “Turn-On” fluorescent probe for the discrimination of H2O2, and highly selective fluorescent response to H2O2 under physiological conditions was demonstrated. As a biocompatible probe in biological systems, the probe was successfully applied for both in vitro and in vivo imaging of H2O2 using HepG2/LO2 cells and angelfish.
2. Experimental 2.1. Materials and measurements. All chemicals were purchased from Sigma-Aldrich reagent company without further purification except especial instruction. All the organic solvents were of analytical grade. Water was purified by a Milli-Q system. Melting points were determined on a PHMKG 05 (Germany) apparatus. 1H and 13C NMR spectra were measured on a Varian INOVA 400 M spectrometer. ESI mass spectra were recorded on an Agilent 1100 series LC/MSO Trap of MS spectrometer. HepG2 and LO2 cells were purchased from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences. All spectroscopic measurements were performed in 10 mM phosphate buffer (pH 7.4) at room temperature. All pH measurements were performed with a Sartorius basic pH-Meter. UV– visible spectral studies were performed on a Perkin-Elmer Lambda-35 UV–visible double beam scanning spectrophotometer. Solution fluorescence spectra were measured on a Perkin-Elmer LS 55 scanning spectrofluorometer equipped with a Xenon flash lamp. Samples for absorption and fluorescence measurements were contained in 1 cm 1 cm quartz cuvettes.
59
400 MHz): δ ¼14.6, 121.8, 123.3, 130.3, 137.6, 142.6, 143.6, 150.6, 156.4. MS(ESI): Calcd for C18H18BF2N3: 325.2, found: m/z 326.2 (M þH) þ . Compound 2: Compound 1 (0.09 mmol, 0.03 g) and methyl iodide (0.9 mmol, 60 μL) were dissolved in toluene, and then the mixture was refluxed at 110 1C for 12 h. The solvent was removed and purified by silica gel column chromatography (CH2Cl2/methanol, 10/1, v/v, as eluent) to afford the desired compound 2 as deep red crystals (0.011 g, 35%). m.p. 228–230 1C. 1H NMR (CDCl3, 400 MHz): δ ¼7.70 (d, 2H, pyridine H), 7.52 (d, 2H, pyridine H), 6.03 (s, 2H, pyrrole H), 4.28 (s, 3H, CH3), 2.60 (s, 6H, CH3), 1.23 (s, 6H, CH3). 13C NMR (CDCl3, 400 MHz): δ ¼13.7, 19.2, 24.2, 29.7, 30.5, 31.0, 65.6, 128.8, 130.9, 132.2, 167.8. MS(ESI): Calcd for [C19H21BF2N3] þ : 340.2, found: m/z 340.2 (M) þ . Compound 3: Compound 1 (0.06 mmol, 0.02 g) and benzyl bromide (0.6 mmol, 0.10 g) were dissolved in toluene, and then the mixture was refluxed at 110 1C for 12 h. After filtration, the precipitate was filtered, washed with toluene and dried in vacuo to afford pure compound 3 (0.018 g, 62%). m.p. 165–168 1C. 1H NMR (CDCl3, 400 MHz): δ ¼9.85 (d, 2H, pyridine H), 7.91 (d, 2H, pyridine H), 7.64 (d, 2H, ArH), 7.33 (d, 3H, ArH), 6.45 (s, 2H, CH2), 5.97 (s, 2H, pyrrole H), 2.54 (s, 6H, CH3), 1.37 (s, 6H, CH3). 13C NMR (CDCl3, 400 MHz): δ ¼ 14.7, 15.5, 64.4, 122.8, 128.3, 129.2, 129.3, 129.7, 130.1, 132.6, 133.0, 141.9, 146.3, 153.1, 158.3. MS(ESI): Calcd for [C25H25BF2N3] þ : 416.2, found: m/z 416.3 (M) þ . Compound 4: Compound 1 (1.2 mmol, 0.40 g) and 4-(Bromomethyl)- benzeneboronic acid pinacol ester (2.4 mmol, 0.71 g) were dissolved in toluene, and then the mixture was refluxed at 110 1C for 12 h. The obtained orange powdery solid was filtered, washed with toluene and dried in vacuo to afford pure compound 4 (0.18 g, 24%). m.p. 258–260 1C. 1H NMR (CDCl3, 400 MHz): δ ¼9.82 (d, 2H, pyridine H), 7.96 (d, 2H, pyridine H), 7.83 (d, 2H, ArH), 7.57 (d, 2H, ArH), 6.55(s, 2H, CH2), 6.02 (s, 2H, pyrrole H), 2.54 (s, 6H, CH3), 1.37 (s, 6H, CH3), 1.34(s, 12H, CH3). 13 C NMR (CDCl3, 400 MHz): δ ¼ 14.8, 16.5, 24.9, 64.7, 84.2, 122.9, 128.2, 128.5, 129.3, 132.5, 135.6, 136.1, 141.9, 146.3, 153.3, 158.4. MS (ESI): Calcd for [C31H36B2F2N3O2] þ : 542.2, found: m/z 542.3 (M) þ . 2.3. Measurement of fluorescence quantum yields For determination of the fluorescence quantum yields (Φfl), we used a Perkin-Elmer LS 55 instrument, with fluorescein in 0.1 M NaOH as a fluorescence standard. Fluorescence quantum yields (Φfl) were obtained by the following equation (F denotes fluorescence intensity at each wavelength and Σ[F] was calculated by summation of fluorescence intensity):
Φfl sample ¼ Φfl standard Absstandard Σ[Fsample] / Abssample Σ[Fstandard]
2.2. Synthesis of BODIPY derivatives 1–4.
3. Results and discussions
Compound 1: 4-Pyridinecarboxaldehyde (9.0 mmol, 0.96 g) was stirred with 2,4-dimethylpyrrole (19.4 mmol, 1.85 g) in deoxygenated CH2Cl2 (150 mL). One drop of TFA was added and the mixture was stirred overnight under N2 at room temperature. The red solution was treated with TCBQ (9.0 mmol, 2.21 g), stirring was continued for 30 min followed by the addition of Et3N (15 mL). After 15 min, BF3 Et2O (15 mL) was added at 0 1C, and the mixture was stirred at room temperature for further 3 h. After washing with saturated aqueous NaHCO3, the organic phase was separated, dried with MgSO4, filtered, and concentrated. The residue was purified by silica gel column chromatography (CH2Cl2/ethyl acetate, 1/1, v/v, as eluent) to afford the desired compound 1 as red powder (0.35 g, 12%). m.p. 241–243 1C. 1H NMR (CDCl3, 400 MHz): δ ¼8.75 (d, 2H, pyridine H), 7.28 (d, 2H, pyridine H), 5.99 (s, 2H, pyrrole H), 2.54 (s, 6H, CH3), 1.38 (s, 6H, CH3). 13C NMR (CDCl3,
3.1. Spectroscopic property of probe 4 and its response to H2O2 The synthetic routes of the N-alkylated BODIPY derivatives are depicted in Scheme 1. According to a procedure described previously (Harriman et al., 2007; Ulrich and Ziessel, 2004), we firstly synthesized 4-pyridinyl-substituted BODIPY precursor 1 which showed poor water solubility. When the pyridine group of 1 was alkylated to form the N-methyl-substituted derivative 2 or N-benzyl-substituted derivative 3, as expected, the watersolubility of entire molecule was improved to a large extent due to the newly formed cationic quaternary ammonium salt (Scheme 1). More interestingly, the process of N-alkylated derivatives can simultaneously exterminate the fluorescence of the BODIPY dyes. The BODIPY dye 1 showed strong green fluorescence (λem ¼520 nm) with high fluorescence quantum yield (Φfl ¼0.78).
60
J. Xu et al. / Biosensors and Bioelectronics 56 (2014) 58–63
Scheme 1. Synthesis of the N-alkylpyridinium substituted BODIPY derivatives 2, 3 and 4.
Comparatively, the quantum yield for the N-alkylated compounds 2 (Φfl ¼0.005) and 3 (Φfl ¼0.003) is much weaker (Table S1 in ESI†), which is attributed to the emission quenching due to the photo-induced electron transfer (PET) process. Encouraged by these findings, we envisioned that “Turn-On” fluorescence probe for sensing H2O2 could be constructed based on this novel water soluble N-alkylated BODIPY by incorporating boronate moiety. To demonstrate that, fluorescent probe 4 was designed, where p-pinacolborylbenzyl unit attached through C–N linkage to the meso-pyridine group of BODIPY. As expected, probe 4 has a very low fluorescence quantum efficiency (Φfl ¼0.002) due to the PET process. Incubation of probe 4 with H2O2 would release fluorophore 1, leading to turning-on of the fluorescence (Scheme 1). The entirely charged ionic structure of probe 4 can not only improve its water-solubility but also make it easier to penetrate cytomembrane for intracellular and in vivo sensing. Firstly, the spectroscopic property of probe 4 and its response to H2O2 were evaluated under physiological conditions (10 mM PBS, pH 7.4). Probe 4 features a maximum absorbance at 508 nm and a very weak emission at 520 nm. In the presence of H2O2, the absorption at 508 nm of probe 4 decreased and a new blue-shift band at 500 nm evolved (Figs. S2 and S3 in ESI†), which was reasonably attributed to the H2O2-induced oxidation of probe 4 to release BODIPY fluorophore 1. As shown in Fig. 1, treatment of probe 4 with H2O2 triggers a dramatic increase of fluorescence intensity at 520 nm, which ramps up to the maximum in ca. 120 min. Correspondingly, an obvious bright green-colored fluorescence was clearly observed after H2O2 addition. 58-fold increase in its fluorescence intensity suggested that probe 4 is one of the most sensitive probes for the detection of H2O2 in abiotic systems (Dickinson et al., 2010; Lippert et al., 2010). The detailed emission titration experiments of probe 4 with various concentrations of H2O2 were also carried out in 10 mM PBS (pH 7.4) buffer solution. There was a good linearity between the fluorescence intensity and the concentrations of H2O2 in the range of 0.1–40 mM (Fig. S1 in ESI†) with a detection limit of 0.03 mM (see ESI†). 3.2. Stability under different pH and the selectivity test We next investigated the pH-dependence of probe 4 in the detection of H2O2. As shown in Fig. 2A, the fluorescence intensity dramatically increased when the pH value was higher than 7.0 and reached a peak value about 10. It is mainly because arylboronic acids can only react with H2O2 under mild alkaline conditions to generate phenols and the phenomenon has been demonstrated by other groups (Lo and Chu, 2003). We also evaluated the potential interfering effect of various reactive oxygen species (ROS) and reactive nitrogen species (RNS) on the reaction of 4 with H2O2. The changes in fluorescence intensity before and after the addition of interferent (Fig. 2B) indicated that fluorescence augmentation occurred only upon reaction with H2O2 and probe 4 exhibits excellent selectivity toward H2O2 among the various ROS/ RNS and biologically
Fig. 1. Fluorescence turn-on response of 5 μM probe 4 to 100 μM H2O2. Spectra shown were acquired before H2O2 addition and 5, 15, 30, 45, 60, 90 and 120 min after H2O2 was added. Inset: fluorescence photograph of 5 μM probe 4 upon incubation with 100 μM H2O2 for 120 min. All measurements were acquired at 25 1C in 10 mM PBS, pH 7.4, with excitation at 480 nm.
relevant species in abiotic systems. The excellent selectivity is ascribed to the H2O2-specific boronate deprotection reaction and the ambiphilic properties of H2O2 as a two-electron transferred oxidant (Lippert et al., 2011). 3.3. Proposed reaction mechanism and NMR analysis The “Turn-On” fluorescence sensing mechanism for probe 4 toward H2O2 was proposed in Fig. 3A. Triggered by the oxidation of the C–B bond by the nucleophilic attack of H2O2 (4 to 5), the emissive fluorogen 1 is released for realizing the sensing purpose (Lo and Chu, 2003). The oxidation process of 4 by H2O2 and subsequent release of 1 were confirmed by 1H NMR analysis. When the reaction of 4 with H2O2 was carried out in CD3CN/D2O (1/1, v/v), the intermediate 5 was observed (Fig. 3). Firstly, compound 4 was transformed to compound 5 by 52% after 5 min according to the ratio of integral areas (Fig. 3B-b), as evidenced by the presence of H80 (δ ¼8.94, doublet), H90 (δ ¼8.15, doublet), H20 (δ ¼7.38, doublet), H30 (δ ¼ 6.93, doublet) of compound 5 and the gradual disappearance of H8 (δ ¼9.06, doublet), H9 (δ ¼ 8.21, doublet), H2 (δ ¼7.91, doublet), H3 (δ ¼7.46, doublet) of compound 4. Subsequently, 1,6-benzyl elimination of 5 took place leading to fluorogen 1 and quinone methide which spontaneously reacted with H2O to form compound 6. This was evidenced by the appearance of H2″ (δ ¼7.22, doublet), H3″ (δ ¼ 6.82, doublet) and H7″ (δ ¼5.78, singlet) of compound 6 (Fig. 3B-b and B-c). Fluorogen 1 was released in about 32% within 5 min and 49% within 3 h, as shown by the appearance of H8″ (δ ¼ 8.76, doublet),
J. Xu et al. / Biosensors and Bioelectronics 56 (2014) 58–63
61
Fig. 2. (A) Fluorescence intensity of 5 μM probe 4 with the addition of 10 mM H2O2 at various pH values. (B) Fluorescence responses of 5 μM probe 4 to 100 μM various reactive oxygen species (ROS) and reactive nitrogen species (RNS). All measurements were acquired at 25 1C in 10 mM PBS, pH 7.4, with excitation at 480 nm. Every data point was the mean of three measurements. The error bars represent the standard deviation.
Fig. 3. (A) Proposed reaction mechanism between probe 4 and H2O2. (B) 1H NMR analysis of the reaction between probe 4 and H2O2 in a mixture of D2O/CD3CN (1/1, v/v): (a) probe 4 only; (b) 5 min after addition of H2O2; (c) 3 h after addition of H2O2; (d) 1H NMR of 1 in a mixture of D2O/CD3CN (1/1, v/v).
H9″ (δ ¼7.48 doublet) and H13″ (δ ¼ 6.17, singlet) (Fig. 3B-c). These data are consistent with the authentic sample of fluorogen 1 (Fig. 3B-d). Moreover, formation of fluorogen 1 was further confirmed by the UV spectrum analysis and ESI-MS measurement (Figs. S3 and S12 in ESI†).
intracellular regions. These data establish that probe 4 is capable of live-cell imaging of H2O2 at natural immune response levels. In addition, experiments in LO2 cells give similar results (Fig. 4g-l).
3.5. In vivo imaging of H2O2 using angelfish 3.4. Fluorescence imaging of H2O2 in live cells Further, probe 4 was applied for fluorescence imaging of H2O2 in live biological samples. Firstly, HepG2 cells were selected as a model to test the sensing ability of probe 4 (10 mM) for exogenous H2O2. We observed no obvious fluorescence in the cells prior to treatment with the stimulants (Fig. 4a). Treatment of probe-loaded cells with 100 mM H2O2 for 90 min triggers a striking bright-green fluorescence increase, consistent with H2O2-mediated boronate cleavage occurring within these cells (Fig. 4c). The overlapped brightfield image confirmed that the cells are viable throughout the imaging experiments (Fig. 4d). Because exposure of cells to stimuli such as lipopolysaccharide (LPS) and phorbol myristate acetate (PMA) will activate the generation of ROS (Karton-Lifshin et al., 2011; Srikun et al., 2008), in this experiment, we then use PMA (1 μg/mL) to test the ability of 4 for detecting endogenous bursts of H2O2 produced within living cells. The image of Fig. 4e showed clear increases in green emission localized within entire
The cell experiments confirmed that probe 4 can serve as a biocompatible probe in live biological systems, which encouraged us to evaluate the feasibility for the application of probe 4 to in vivo imaging. The young gray angelfish (Pterophyllum scalare), due to its transparent body, is selected as an ideal model for H2O2 sensing in living organisms. Twenty-day-old angelfish was treated with 10 μM 4 in E3 embryo media (Greene and Tischler, 1976; Lo and Chu, 2003) for 30 min at 25 1C, washed with PBS to remove the remaining chemosensor, and incubated in a solution containing 100 μM H2O2 for 120 min. Fluorescent images obtained are shown in Fig. 5. When angelfish were incubated with 4 alone, weak fluorescence was observed (Fig. 5a). When angelfish was first exposed to 4 and later incubated with H2O2, strong fluorescence was observed all over its body (Fig. 5c), indicating that the probe might have penetrated into the angelfish through its cuticular layer and reacted with H2O2 inside body. These results suggest that probe 4 was highly suitable for sensing H2O2 in vivo.
62
J. Xu et al. / Biosensors and Bioelectronics 56 (2014) 58–63
Fig. 4. Fluorescence images of HepG2 cells incubated with 10 μM probe 4 for 30 min at 37 1C (a) with a brightfield overlay (b) to confirm viability. Probe-stained HepG2 cells treated with 100 μM H2O2 for 90 min at 37 1C (c) with a brightfield overlay (d). Probe-stained HepG2 cells stimulated with 1 μg/mL PMA for 90 min at 37 1C (e) with a brightfield overlay (f). Fluorescence images of LO2 cells incubated with 10 μM probe 4 for 30 min at 37 1C (g) with a brightfield overlay (h). Probe-stained LO2 cells treated with 100 μM H2O2 for 90 min at 37 1C (i) with a brightfield overlay (j). Probe-stained LO2 cells stimulated with 1 μg/mL PMA for 90 min at 37 1C (k) with a brightfield overlay (l).
Fig. 5. In vivo fluorescence images of 20-day-old angelfish treated with 10 μM probe 4 for 30 min at 25 1C (a) with a brightfield image (b). Probe-stained angelfish treated with 100 μM H2O2 for 120 min at 25 1C (c) with a brightfield image (d).
4. Conclusions On the basis of the N-alkylation reaction of meso-(4-pyridinyl)substituted BODIPY, we developed several water-soluble BODIPY derivatives. By combining a 4-(Bromomethyl)benzeneboronic acid pinacol ester moiety and pyridinyl-substituted BODIPY chromophore, a novel water-soluble BODIPY-based fluorescent probe was constructed, which has realized a complete selectivity in “Turn-
On” fluorescent response to H2O2 against other interferences of ROS and RNS species under physiological conditions. Such selectivity results from the unique boronate oxidation mode of the p-pinacolborylbenzyl unit toward H2O2, which leads to release BODIPY fluorophore. Further, the probe was successfully applied for both in vitro and in vivo imaging of H2O2 using HepG2/LO2 cells and angelfish. This work provides a novel approach to design water-soluble BODIPY-based fluorescent probe for biological
J. Xu et al. / Biosensors and Bioelectronics 56 (2014) 58–63
detection, demonstrating its potential applicability to develop a “Turn-On” fluorescent material for monitoring and imaging both in vitro and in vivo. Acknowledgments This work was supported by the National Natural Science Foundation of China (20972170 and 21275150), the Funds for Distinguished Young Scientists of Gansu (1210RJDA013) and the Natural Science Foundation of Gansu province (1107RJYA069). The authors gratefully acknowledge Dr. Xiaoming He (currently at University of Calgary) for helpful discussion. Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2013.12.065. References Abo, M., Urano, Y., Hanaoka, K., Terai, T., Komatsu, T., Nagano, T., 2011. J. Am. Chem. Soc. 133 (27), 10629–10637. Alamiry, M.A.H., Harriman, A., Mallon, L.J., Ulrich, G., Ziessel, R., 2008. Eur. J. Org. Chem. 2008 (16), 2774–2782. Albers, A.E., Okreglak, V.S., Chang, C.J., 2006. J. Am. Chem. Soc. 128 (30), 9640–9641. Brellier, M., Duportail, G., Baati, R., 2010. Tetrahedron Lett. 51 (9), 1269–1272. Bura, T., Ziessel, R., 2011. Org. Lett. 13 (12), 3072–3075. Chang, M.C., Pralle, A., Isacoff, E.Y., Chang, C.J., 2004. J. Am. Chem. Soc. 126 (47), 15392–15393. Chen, W., Cai, S., Ren, Q.Q., Wen, W., Zhao, Y.D., 2012. Analyst 137 (1), 49–58. Cheng, T., Xu, Y., Zhang, S., Zhu, W., Qian, X., Duan, L., 2008. J. Am. Chem. Soc. 130 (48), 16160–16161. D’Autréaux, B., Toledano, M.B., 2007. Nat. Rev. Mol. Cell Biol. 8 (10), 813–824. Dickinson, B.C., Chang, C.J., 2008. J. Am. Chem. Soc. 130 (30), 9638–9639. Dickinson, B.C., Huynh, C., Chang, C.J., 2010. J. Am. Chem. Soc. 132 (16), 5906–5915. Dodani, S.C., He, Q., Chang, C.J., 2009. J. Am. Chem. Soc. 131 (50), 18020–18021. Fähnrich, K.A., Pravda, M., Guilbault, G.G., 2001. Talanta 54 (4), 531–559. Greene, L.A., Tischler, A.S., 1976. Proc. Natl. Acad. Sci. USA 73 (7), 2424–2428. Harriman, A., Mallon, L.J., Ulrich, G., Ziessel, R., 2007. ChemPhysChem 8 (8), 1207–1214.
63
Hong, R., Han, G., Fernández, J.M., Kim, B.-j., Forbes, N.S., Rotello, V.M., 2006. J. Am. Chem. Soc. 128 (4), 1078–1079. Jiang, N., Fan, J., Liu, T., Cao, J., Qiao, B., Wang, J., Gao, P., Peng, X., 2013. Chem. Commun. 49 (90), 10620–10622. Jiao, L., Yu, C., Liu, M., Wu, Y., Cong, K., Meng, T., Wang, Y., Hao, E., 2010. J. Org. Chem. 75 (17), 6035–6038. Karolin, J., Johansson, L.B.-A., Strandberg, L., Ny, T., 1994. J. Am. Chem. Soc. 116 (17), 7801–7806. Karton-Lifshin, N., Segal, E., Omer, L., Portnoy, M., Satchi-Fainaro, R., Shabat, D., 2011. J. Am. Chem. Soc. 133 (28), 10960–10965. Li, L., Han, J., Nguyen, B., Burgess, K., 2008. J. Org. Chem. 73 (5), 1963–1970. Lippert, A.R., De Bittner, G.C.V., Chang, C.J., 2011. Acc. Chem. Res. 44 (9), 793–804. Lippert, A.R., Gschneidtner, T., Chang, C.J., 2010. Chem. commun. 46 (40), 7510–7512. Lo, L.-C., Chu, C.-Y., 2003. Chem. Commun. 21, 2728. Loudet, A., Burgess, K., 2007. Chem. Rev. 107 (11), 4891–4932. Maeda, H., Fukuyasu, Y., Yoshida, S., Fukuda, M., Saeki, K., Matsuno, H., Yamauchi, Y., Yoshida, K., Hirata, K., Miyamoto, K., 2004. Angew. Chem. 43 (18), 2389–2391. Miller, E.W., Albers, A.E., Pralle, A., Isacoff, E.Y., Chang, C.J., 2005. J. Am. Chem. Soc. 127 (47), 16652–16659. Miller, E.W., Dickinson, B.C., Chang, C.J., 2010. Proc. Natl. Acad. Sci. USA 107 (36), 15681–15686. Niu, S.L., Ulrich, G., Ziessel, R., Kiss, A., Renard, P.-Y., Romieu, A., 2009. Org. Lett. 11 (10), 2049–2052. Nogueira, R.F., Oliveira, M.C., Paterlini, W.C., 2005. Talanta 66 (1), 86–91. Onoda, M., Uchiyama, S., Endo, A., Tokuyama, H., Santa, T., Imai, K., 2003. Org. Lett. 5 (9), 1459–1461. Paulsen, C.E., Carroll, K.S., 2009. ACS Chem. Biol. 5 (1), 47–62. Rhee, S.G., 1999. Exp. Mol. Med. 31 (2), 53–59. Rhee, S.G., 2006. Science 312, 1882–1883. Srikun, D., Miller, E.W., Domaille, D.W., Chang, C.J., 2008. J. Am. Chem. Soc. 130 (14), 4596–4597. Ulrich, G., Ziessel, R., 2004. J. Org. Chem. 69 (6), 2070–2083. Ulrich, G., Ziessel, R., Harriman, A., 2008. Angew. Chem. 47 (7), 1184–1201. Vo‐Hoang, Y., Micouin, L., Ronet, C., Gachelin, G., Bonin, M., 2003. ChemBioChem 4 (1), 27–33. Wang, D., Miyamoto, R., Shiraishi, Y., Hirai, T., 2009. Langmuir 25 (22), 13176–13182. Winterbourn, C.C., 2008. Nat. Chem. Biol. 4 (5), 278–286. Wolfbeis, O.S., Dürkop, A., Wu, M., Lin, Z., 2002. Angew. Chem. Int. Ed. 41 (23), 4495–4498. Xu, K., Tang, B., Huang, H., Yang, G., Chen, Z., Li, P., An, L., 2005. Chem. Commun. 48, 5974–5976. Yuan, L., Lin, W., Xie, Y., Chen, B., Zhu, S., 2012. J. Am. Chem. Soc. 134 (2), 1305–1315. Zhang, S., Wu, T., Fan, J., Li, Z., Jiang, N., Wang, J., Dou, B., Sun, S., Song, F., Peng, X., 2013. Org. Biomol. Chem. 11 (4), 555–558. Zhu, S., Zhang, J., Vegesna, G., Luo, F.-T., Green, S.A., Liu, H., 2010. Organ. Lett. 13 (3), 438–441.
