English

• 1.   Introduction

  As the redox-active species, reactive oxygen (ROS) and nitrogen species (RNS), which could possess an elaborate regulation system for cells to maintain their redox balance, is closely associated with some human diseases, such as cancer, neurodegenerative diseases, cardiovascular diseases, diabetes mellitus and gastrointestinal diseases.^[1-3] Among ROS and RNS, peroxynitrite (ONOO^－), as the product of the diffusion-controlled reaction of nitric oxide and superoxide radicals, has attracted special attention due to its unusually potent oxidizing ability and reactive nucleophilic character.^[4] Recently, ONOO^－ was also found as a crucial trigger of skeletal muscle hypertrophy via activation of calcium signaling. Hence, the development of effective and applicable techniques to detect ONOO^- in biological samples is of great significance.^[5] To date several approaches including UV-Vis spectroscopy, electrochemical analysis, electron spin resonance and immunohistochemistry have been developed for the detection of ONOO^－.^[6-7] A myriad of fluorescent probes have been developed for ONOO^－ detection in the past few years.^[8-9] There is no doubt that this is driven by many advantages of fluorescent probes, including their high measurement efficiency, non-invasive detection, excellent spatial and temporal resolution. Yet some problems of these probes might be encountered, such as interference from other highly reactive oxidants (especially from ClO^－ and •OH), low quantum yield and particularly low water solubility.

  Boron-dipyrromethene (BODIPY) derivatives, similar to other fluorescent dyes, have been widely studied and used as luminescent devices, chemical sensors and molecular wires due to their advantages including high fluorescence quantum efficiency, large molar extinction coefficient, fair chemical stability as well as structural features with magnificient photophysical properties.^[10-13] In the field of biological labeling, BODIPY dyes also act as promising candidates for small biological molecules detection in vivo and organism imagine.^[14-19] However, the low water solubility greatly limits high sensitivity imaging in vivo since the fluorescence of BODIPY was quenched in aqueous media.^[20-21]

  A probe in which fluorophore is nontoxic, sufficiently bright and biocompatible, inert to other ROS and RNS in the complex biological context is an ideal for ONOO^－ detection. Herein, we report two BODIPY fluorescent probes possessing enhanced solubility and high fluorescence quantum yield in aqueous media for the purpose of developing a method capable of specific detecting ONOO^－ via fluorescence change. An ester group, which could be hydrolyzed into corresponding acid and hydroxyl compound by nucleophile, is introduced into the molecule.^[22] Besides, to increase electron cloud density and the red-shifted emission, four methyl groups at 1, 3, 5, 7 positions were introduced into the BODIPY probe.^[21] Moreover, as one of the widely available and essential amino acids of organism, L-phenylalanine, possessing good water-soluble and biocompatible characters, was selected as the acid fragment which was esterified with the hydroxyl substituted BODIPY. Consequently, a water-soluble Ac-Phe-BODIPY was rationally synthesized as a fluorescent reaction probe for ONOO^- over ROS and RNS species with high sensitivity, low detection limit and excellent selectivity.

  2.   Results and discussion

  Ac-Phe-BODIPY was derived from pyrrole derivatives (BODIPY), a well-known typical high fluorescence quantum yield and easy to modify fluorophore, as a reporting group and phenol ester bond as a targeting moiety. Being a derivative of acetylated L-phenylalanine, Ac-Phe-OH has little toxicity and better biocompatibility, what is more important is that it was selectively eliminated from Ac-Phe- BODIPY by ONOO- over other ROS and RNS. BODIPY-1 was prepared using 2, 4-dimethylpyrrole and p-hydroxybenzaldehyde as the raw materials. Ac-Phe-BODIPY was readily synthesized by esterification of BODIPY-1 with Ac-Phe-OH according to Scheme 1.

  Scheme 1

  

  Scheme 1.  Synthesis of Ac-Phe-BODIPY and Ac-BODIPY

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  The UV-Vis absorption spectra and fluorescence emission spectra of Ac-Phe-BODIPY and Ac-BODIPY were investigated in phosphate buffer saline (PBS) (10 mmol/L, pH＝7.4), and showed maximal excitation/emission bands at 499/504 nm and 496 /509 nm, then their selectivity toward ONOO^－ among other ROS and RNS was tested. As expected, fluorescence of Ac-Phe-BODIPY and Ac-BODIPY were insignificantly changed in the present of NO, HNO, H₂O₂, ClO^－, ¹O₂, NO₂^－, NO₃^－ and ^•OH. By comparison, fluorescence intensity greatly decreased after adding ONOO^- (Figure 1a), and the reaction time is about 15 min. Simultaneously, the UV-Vis spectra showed that the characteristic absorption band of Ac-Phe-BODIPY and Ac-BODIPY were blue-shifted by 5 and 11 nm compared to that of before adding ONOO^－, while other RNS and ROS hardly brought about changes in the absorption spectrum. According to the results above, gradual attenuation in emission intensity during the fluorescence titration for ONOO^－ by Ac-Phe-BODIPY and Ac-BODIPY were further confirmed (Figure 1b), the detection limit were examined to be 12.6 and 6.79 nmol/L, which is lower than previously most reported probe for ONOO^－. Even more remarkably, the anti-interference about pH and common hydrolase of fluorescent probes Ac-Phe-BODIPY was tested, the experimental results showed that the pH (5.0~9.0) and common hydrolase [acetyl cholinesterase (AchE)] hardly interfered with identification process of probe Ac-Phe- BODIPY in a short time (Figure 1c). And furthermore, the difference of ONOO^－ from other ROS and RNS on Ac-Phe-BODIPY could be visually distinguished under UV light (365 nm) as reflected by the emission color of solution turned from green to purple (Figure 2). The detection procedure was also demonstrated through characterizing the mixture solution by ESI-MS, a peak with m/z 339.09 conformed to that of calculated for C₁₉H₃₀BF₂N₂O (M－H⁺) 339.15. To further certify the product of the procedure, the mixture solution was isolated by column chromatography and BODIPY-1 was confirmed again as the main constituents. All of phenomena showed that phenol ester bond was broken up by ONOO^－ and consequently led to the fluorescence quenching.^[23]

  Figure 1

  

  Figure 1.  (a) Fluorescence response of Ac-Phe-BODIPY to the addition of different ROS and RNS of NO, HNO, H₂O₂, ClO^－, ¹O₂, NO₂^－, NO₃^－, ^•OH and ONOO^－, (b) changes in fluorescence spectra of Ac-Phe-BODIPY upon incremental addition ONOO^－ (0, 1, 2, 3, 4, 5, 6 μmol/L ONOO^－) and (c) fluorescence response of Ac-Phe-BODIPY to the addition of AchE during 16 min

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  Figure 2

  

  Figure 2.  Photos of Ac-Phe-BODIPY under ultraviolet lamp (365 nm) after addition of different ROS and RNS

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  Furthermore, for the purpose of understanding the sensing mechanism and to attain a better picture of the molecular orbital framework, structural optimization of Ac-Phe- BODIPY and the BODIPY-1' was carried out using density functional theory (DFT) as implemented.^[24-26] As shown in Figure 3, the π electrons of Ac-Phe-BODIPY were mainly located on the whole π-conjugated BODIPY skeleton on both the lowest unoccupied molecular orbital (LUMO) and the highest occupied molecular orbital (HOMO) which led to fluorescence emission. While the π electrons on the HOMO of BODIPY-1' were mainly located on the BODIPY skeleton, and on the LUMO of BODIPY-1 they were mainly distributed on the phenol oxygen anion moiety, therefore the LUMO and HOMO levels supported the possible photoinduced electron transfer (PET) process in BODIPY-1'. In addition, the energy difference in the HOMO and LUMO of Ac-Phe-BODIPY and BODIPY-1' were found to be 2.99 and 1.62 eV, respectively. Hence, the theory calculations were in full accord with the experimental phenomenon of fluorescence "turn-off".

  Figure 3

  

  Figure 3.  Density functional theory (DFT) optimized structures and frontier molecular orbitals (MOs) of Ac-Phe-BODIPY and BODIPY-1'

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  Additionally, to testify the capability of being taken up by live cells of the Ac-Phe-BODIPY, HeLa cells were chosen as a biological medium for these chemosensing experiments and the first point of study involved cell viability testing. Results revealed that when the concentration of Ac-Phe- BODIPY at concentrations reached 100 μmol/L, the cell viability reached the lowest value of 75%, and high concentration of Ac-Phe-BODIPY (1000 μmol/L) was helpful for cell proliferation. Ultimately, fluorescence imaging towards ONOO^－ operated in HeLa cells was employed for the staining study to prove the biologically discernable ability of Ac-Phe-BODIPY. As shown in Figure 4 (A, B), incubated with 1 μmol/L Ac-Phe-BODIPY within 15 min, the imaged HeLa cells displayed bright green fluorescence. Next the cells were incubated with 100 μL (10 μmol/L) ONOO^- for 5 min, control cells (cells treated with Ac-Phe-BODIPY) were almost non-fluorescent (Figures 4C, 4D).

  Figure 4

  

  Figure 4.  ONOO^－-induced fluorescence response in HeLa cells (A, B) Images of cells stained with Ac-Phe-BODIPY. (C, D) Cells were incubated with Ac-Phe-BODIPY. (A, C) Bright field, (B, D) dark field. Scale bar, 200 μm

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  3.   Conclusions

  To conclude, Ac-Phe-BODIPY and Ac-BODIPY fluorescent probe were reasonably designed and synthesized. The introduction of four methyl groups enhances the emission wavelength. Moreover, esterifying Ac-Phe-OH with BODIPY-OH increased the water solubility and biocompatibility. Most importantly, owing to the especial reactivity of ester group, the probe exhibits high sensitivity and excellent selectivity toward ONOO^－ among ROS and RNS compared with previous reported probes. In addition, the relatively high fluorescence quantum efficiency and good water solubility of the probe are useful for cell imaging experiments. Finally, the identification mechanism was proved to be the cleavage of the phenol ester bond of Ac-Phe-BODIPY via ESI-MS analysis and ¹H NMR spectra study, and the theoretical calculations were performed to support the experimental observations. These results show that as-synthesized BODIPY with phenol ester bond holds potential application prospect in dye behaviors in the living organisms.

  4.   Experimental section

  4.1   Instruments and reagents

  The ¹H NMR (600 MHz) spectra and the ¹³C NMR (150 MHz) spectra were recorded at room temperature on a Agilent DD2 600 spectrometer with chemical shifts referenced to internal TMS, and chemical shifts are reported relative to the solvent residue peaks (CDCl₃, δ 7.26 for ¹H NMR, δ 77.16 for ¹³C NMR). High-resolution mass spectra (HRMS) were obtained from Agilent 6510 Accurate-Mass Q-TOF LC/MS system. UV-Vis and fluorescent spectra were carried out on a T6 and F97pro fluorescence spectrophotometer, respectively. Cell imagings were performed using a Olympus X73 Fluorescent Inverted microscope and BioRad Xmark Microplate reader was used to acquired cytotoxicity. All reagents were purchased from commercial suppliers, and used as received without further purification. Acetyl cholinesterase was extracted from fly head (200 u/g). All the solvents used in optical experiment were analytical grade. Dry dichloromethane was obtained by refluxing in calcium hydride. Aqueous solutions were freshly prepared with twice-deionized water from a water purification system. Pre-coated silica plates for thin-layer chromatography (TLC) analysis and silica gel (mesh 200~300) for column chromatography were purchased from the Qingdao Ocean Chemicals. Preparation of Various ROS and RNS can be found in the literature.^[1]

  4.2   Experimental method

  4.2.1   Synthesis of BODIPY-1

  Synthesis of BODIPY-1 was carried out in a one pot reaction according to the published method.^[24] 2, 4-Dime- thylpyrrole (0.46 mL, 4.63 mmol) and p-hydroxy benzaldehyde (0.28 g, 2.30 mmol) were added to dry dichloromethane (DCM) (100 mL). To this mixture, 3 drops of trifluoroacetic acid (TFA) were added, and the reaction was left to stir in an atmosphere of argon. Consumption of the p-hydroxy benzaldehyde was monitored by TLC. After the reaction, 2, 3-dichloro-5, 6-dicyano-1, 4-benzoquinone (DDQ) (0.52 g, 2.29 mmol) was added, and the solution was left to stir over 30 min and followed by the drop-wise addition of trimethylamine (TEA) (3.16 mL, 23 mmol) and BF₃•Et₂O (2.90 mL, 23 mmol). After being stirred for 4 h at room temperature, the reaction mixture was poured into saturated saline and extracted with DCM (200 mL). The organic phase was washed successively with H₂O (200 mL) and brine (100 mL), dried with Na₂SO₄ and concentrated under reduced pressure. Then the mixture was purified by silica gel flash column chromatography (silica gel, EtOAc/petroleum ether, V:V＝1:4) yielded red crystals of BODIPY-1 (32 mg, 41% yield). m.p. 182~183 ℃; ¹H NMR (600 MHz, CDCl₃) δ: 7.13 (d, J＝8.4 Hz, 2H), 6.95 (d, J＝8.4 Hz, 2H), 5.98 (s, 2H), 2.55 (s, 6H), 1.44 (s, 6H); ¹³C NMR (150 MHz, CDCl₃) δ: 156.4, 155.5, 143.3, 141.9, 132.0, 129.6, 127.4, 121.3, 116.3, 14.7. HRMS (ESI) calcd for C₁₉H₂₀BF₂N₂O [M+H]⁺ 339.1486, found 339.1498.

  4.2.2   Synthesis of Ac-Phe-OH

  Compound Ac-phe-OH was synthesized according to the reported method.^[28] To a 50 mL round bottom flask with stir bar, a solution of the L-phenylalanine (0.33 g, 2.00 mmol) in MeOH (10 mL) was made, and then acetic anhydride (0.66 g, 5.40 mmol) was added drop wise to the stirring amino acid solution. The reaction was allowed to reflux for 6 h (the progress of the reaction was followed by TLC). The reaction mixture was then concentrated and the resulting solid was washed with H₂O (20 mL) and dried under vacuum. Then the mixture was purified by silica gel flash column chromatography (silica gel, MeOH/EtOAc, V:V＝1:20) to yield white crystals of Ac-Phe-OH (580 mg, 70% yield). m.p. 171~172 ℃ (lit.^[28] m.p. 170~174 ℃); ¹H NMR (600 MHz, DMSO-d₆) δ: 8.17 (d, J＝7.8 Hz, 1H), 7.27 (t, J＝7.2 Hz, 2H), 7.21~7.19 (m, 3H), 4.39 (s, 1H), 3.05~3.02 (m, 1H), 2.85~2.81 (m, 1H), 1.78 (s, 3H); ¹³C NMR (150 MHz, DMSO-d₆) δ: 173.2, 169.3, 137.8, 129.1, 128.2, 126.4, 53.6, 36.8, 22.4; ESI-MS m/z: C₁₁H₁₄NO₃ [M+H]⁺ 208.08.

  4.2.3   Synthesis of Ac-Phe-BODIPY

  To a 100 mL round bottom flask, a stirring solution of BODIPY-1 (30 mg, 0.089 mmol) and Ac-Phe-OH (20.27 mg, 0.098 mmol) in dry DCM (50 mL) was made and cooled to 0 ℃ under Ar gas. Then to this mixture, 4-dimethylaminopyridine (DMAP) (11.94 mg, 0.098 mmol) and N, N'-dicyclohexylcarbodiimide (DCC) (20.17 mg, 0.098 mmol) were added in order. After being stirred for 24 h at 10 ℃, the reaction mixture was poured into saturated sodium chloride and extracted with DCM (200 mL). The organic layer was washed successively with H₂O (40 mL×3) and saturated sodium chloride (100 mL), dried with Na₂SO₄ and concentrated under reduced pressure. Then the mixture was purified by silica gel flash column chromatography (silica gel, EtOAc/petroleum ether, V: V＝1:1) to yield red crystals of Ac-Phe-BODIPY (18 mg, 58% yield). m.p. 204~205 ℃; ¹H NMR (600 MHz, CDCl₃) δ: 7.35 (t, J＝7.8 Hz, 2H), 7.31 (d, J＝7.8 Hz, 1H), 7.29 (d, J＝8.4 Hz, 2H), 7.25~7.24 (m, 2H), 7.14 (d, J＝7.8 Hz, 2H), 5.99 (s, 2H), 5.11 (q, J＝6.6 Hz 1H), 4.12 (q, J＝6.6 Hz, 1H), 3.29 (d, J＝6 Hz, 2H), 2.55 (s, 6H), 2.04 (s, 3H), 1.40 (s, 6H); ¹³C NMR (150 MHz, CDCl₃) δ: 170.3, 156.0, 151.0, 143.2, 140.5, 135.6, 133.1, 131.5, 129.5, 129.0, 128.8, 127.7, 127.3, 122.4, 121.6, 116.3, 53.6, 38.0, 14.7; HRMS (ESI) calcd for C₃₀H₃₁BF₂N₃O₃ [M+H]⁺ 530.2426, found 530.2428.

  4.2.4   Synthesis of Ac-BODIPY

  To a 100 mL round bottom flask, a stirring solution of BODIPY-1 (26 mg, 0.08 mmol) and acetic acid (5 mg, 0.08 mmol) in dry DCM (50 mL) was made and cooled to 0 ℃ under Ar gas. Then to this mixture, 4-dimethylami- nopyridine (DMAP) (9.8 mg, 0.08 mmol) and N, N'-di- cyclohexylcarbodiimide (DCC) (16.5 mg, 0.08 mmol) were added in order. After being stirred for 24 h at 10 ℃, the reaction mixture was poured into saturated sodium chloride and extracted with DCM (200 mL). The organic layer was washed successively with H₂O (40 mL×3) and saturated sodium chloride (100 mL), dried with Na₂SO₄ and concentrated under reduced pressure. Then the mixture was purified by silica gel flash column chromatography (silica gel, EtOAc/petroleum ether, V:V＝1:4) to yield red crystals of Ac-Phe-BODIPY (14 mg, 47% yield). m.p. 188~189 ℃; ¹H NMR (600 MHz, CDCl₃) δ: 7.30 (d, J＝6 Hz, 2H), 7.25 (d, J＝6 Hz, 2H), 5.99 (s, 2H), 7.25~7.24 (m, 2H), 7.14 (d, J＝7.8 Hz, 2H), 5.99 (s, 2H), 2.55 (s, 6H), 2.33 (s, 3H), 1.42 (s, 6H); ¹³C NMR (150 MHz, CDCl₃) δ: 169.1, 155.9, 151.4, 143.3, 140.8, 132.6, 131.6, 129.3, 122.6, 121.5, 14.7, 14.7; HRMS (ESI) calcd for C₂₁H₂₂BF₂N₂O₂ [M+H]⁺ 383.2115, found 383.2606.

  4.3   MTS assay for cytotoxicity

  CCK-8 assays were performed to assess the metabolic activity of Hela cells. Hela cells were cultured (37 ℃, 5% CO₂) in Dulbecco's Modified Eagle Medium (DMEM) medium containing 10% fetal bovine serum and 1% penicillin streptomycin. Cells were seeded in 96-well plates (Costar, IL, USA) at an intensity of 2×10⁴ cells/mL. After 48 h incubation, the old medium was replaced by the Ac-Phe-BODIPY solution in medium at different Ac-Phe-BODIPY concentrations, and the cells were then incubated for 23 h. After the designated time intervals, CCK-8 (10 μL) was then added into each well and the incubating was continued for 1 h. The absorbance of CCK-8 at 450 nm was monitored by the microplate reader.

  Supporting Information Spectral data and fitting results, 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay for cytotoxicity, ¹H NMR, ¹³C NMR and MS spectra of Ac-phe-BODIP and Ac-BODIPY. The Supporting Information is available free of charge via the Internet at http://sioc-journal.cn.

  
  
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  Scheme 1  Synthesis of Ac-Phe-BODIPY and Ac-BODIPY

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  Figure 1  (a) Fluorescence response of Ac-Phe-BODIPY to the addition of different ROS and RNS of NO, HNO, H₂O₂, ClO^－, ¹O₂, NO₂^－, NO₃^－, ^•OH and ONOO^－, (b) changes in fluorescence spectra of Ac-Phe-BODIPY upon incremental addition ONOO^－ (0, 1, 2, 3, 4, 5, 6 μmol/L ONOO^－) and (c) fluorescence response of Ac-Phe-BODIPY to the addition of AchE during 16 min

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  Figure 2  Photos of Ac-Phe-BODIPY under ultraviolet lamp (365 nm) after addition of different ROS and RNS

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  Figure 3  Density functional theory (DFT) optimized structures and frontier molecular orbitals (MOs) of Ac-Phe-BODIPY and BODIPY-1'

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  Figure 4  ONOO^－-induced fluorescence response in HeLa cells (A, B) Images of cells stained with Ac-Phe-BODIPY. (C, D) Cells were incubated with Ac-Phe-BODIPY. (A, C) Bright field, (B, D) dark field. Scale bar, 200 μm

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