Reactivity modulation of benzopyran-coumarin platform by introducing

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Reactivity modulation of benzopyran-coumarin platform by introducing electron-withdrawing groups: specific detection of biothiols and peroxynitrite Yong Li, Zhiwen Zhao, Yongsheng Xiao, Xu Wang, Xiaoyun Jiao, Xilei Xie, Jian Zhang, and Bo Tang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b00636 • Publication Date (Web): 10 Apr 2019 Downloaded from http://pubs.acs.org on April 11, 2019

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Analytical Chemistry

Reactivity modulation of benzopyran-coumarin platform by introducing electron-withdrawing groups: specific detection of biothiols and peroxynitrite Yong Li,† Zhiwen Zhao,† Yongsheng Xiao, Xu Wang,* Xiaoyun Jiao, Xilei Xie, Jian Zhang and Bo Tang* College of Chemistry, Chemical Engineering and Materials Science, Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in Universities of Shandong, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Shandong Provincial Key Laboratory of Clean Production of Fine Chemicals, Shandong Normal University, Jinan 250014, P. R. China ABSTRACT: A variety of fluorophores have been designed and created to fabricate organic fluorescent probes. Among these fluorophores, benzopyran-coumarin (BC) based fluorescent platform has attracted increasing attention as it shows multiple appropriate fluorescent and imaging capacities. Nevertheless, the analytical potential of BC is still urgently needed to be further excavated as its detection performance is hindered by the inherent drawbacks of current BC skeleton, that is, limited number of reactive sites. As such, in this work, by simply introducing electron-withdrawing (EW) substituent groups, we reconstructed BC skeleton to afford two fluorescent probes, BCB (–Br substitued) and BCN (−NO2 substitued), both of which featured two highly reactive sites. These two probes were capable of detecting peroxynitrite (ONOO−) and biothiols (hydrogen sulfide, glutathione, cysteine, and homocysteine) through naked eye and UV-vis absorption analysis in buffer solution. In addition, BCB was able to specifically sense biothiols with fluorescent analysis while BCN, with −NO2 instead of −Br, displayed more prominent fluorescent specificity towards ONOO−. This work provided a new strategy for the reactivity regulation of fluorophore through EW group introduction, as well as an alternative approach and method for the construction of fluorescent probes for other important biological species.

Organic fluorescent probes have attracted increasing attention in biological research as they hold a variety of appealing imaging properties in terms of high spatiotemporal contrast and dynamic real-time observation.1-3 Therefore, various fluorophores have been designed and developed to fabricate fluorescent probes for the analyte of interest.4-6 Among these fluorophores, the hybrid of benzopyran and coumarin (BC) has proved to be an appropriate fluorescent platform due to its attractive fluorescent and imaging capacities, including high extinction coefficient, large fluorescence quantum yield, two-photon excitation, and so on.7-12 Nevertheless, there are still some inherent shortcomings in this structure, such as limited number of reactive sites of the skeleton. As such, in order to improve the analytical performance of BC platform, it is of great significance to reconstruct BC skeleton so as to endow it with more reactive sites. Peroxynitrite (ONOO−), a highly reactive nitrogen specie featuring powerful oxidative and nitrative capability, plays a crucial role in various physiological and pathological processes.13-17 To date, a growing number of evidence suggests that ONOO− can generate a toxic effect on biomolecules and has been regarded as a culprit in

various disease processes.18-21 As such, strategies for tracing intracellular ONOO− levels are of considerable significance to adequately address its physiopathologic effect. On the other hand, biothiols, normally include hydrogen sulfide (H2S), glutathione (GSH), cysteine (Cys), and homocysteine (Hcy), are key contributors to the intracellular reductive status, and play an essential role in the antioxidant processes.22-28 Considering that the biothiols are preferential scavengers of ONOO− in intracellular redox signal transduction,29 the sense of ONOO− and biothiols in living system is undoubtedly very important. Herein, for the first time, through simply introducing electron-withdrawing (EW) group into coumarin moiety of BC platform, we fabricated and synthesized two new fluorescent probes, BCB (bromine atom substituted, −Br) and BCN (nitro group substituted, −NO2) (Scheme 1). Due to EW group introduction, each probe provided two reaction sites towards ONOO− and biothiols, respectively. The results showed that BCB and BCN were capable of detecting ONOO− and biothiols with colorimetric mode in PBS buffer solution. BCB was able to specifically sense biothiols with fluorescent analysis while BCN, with −NO2 instead of −Br, displayed more prominent fluorescent

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specificity towards ONOO−. Therefore, through conjugating different EW groups (−NO2 and –Br), BC platform was endowed with two reactive sites, providing a new strategy for the reactivity regulation of fluorophore, as well as ideas for fluorescent probe designing.

EXPERIMENTAL SECTION Materials and Instruments. Unless otherwise stated, all reagents for synthesis were purchased from commercial suppliers and were used without further purification. Male Kunming mice (20g) were purchased from the School of Medicine at Shandong University. All of the animal experiments were in agreement with the guidelines of the Institutional Animal Care and Use Committee. pH measurements were performed with a pH-3c digital pH-meter (Shanghai Lei Ci Device Works, Shanghai, China). Absorption spectra were recorded on a UV-1700 spectrophotometer (Shimadzu, Japan). Fluorescence spectra were obtained with a FLS-980 fluorescence spectrometer (Edinburgh Instruments Ltd., England). 1H NMR and 13C NMR spectra were taken on a 400 MHz spectrometer (Bruker Co., Ltd., Germany), δ values are in ppm relative to TMS. HRMS spectra were obtained on a maxis ultra-high resolution-TOF MS system (Bruker Co., Ltd., Germany). MTT assay was performed using a TRITURUS microplate reader. The two-photon fluorescence images were taken using a LSM 880 confocal laser scanning microscopy (Zeiss Co., Ltd. Germany). Synthesis of Compound BCB. The solution of 2-(4diethylamino-2-hydroxybenzoyl)benzoic acid (313.1 mg, 1 mmol) and 3-acetyl-6-bromochromen-2-one (266.0 mg, 1 mmol) in conc. H2SO4 (5 mL) was stirred at 90 °C for 12 h. After cooling to room temperature, the reaction mixture was poured into ice (50 g) and followed by the addition of perchloric acid (70%, 0.5 mL). The resulting purple residue was collected by filtration, washed with cold water and further purified by column chromatography on silica gel (CH2Cl2/MeOH = 10:1, v/v) to afford BCB as a purple solid (450.2 mg, 83% yield). 1H NMR (400 MHz, CDCl3) δ 8.45 (s, 1H), 7.97 (d, J = 8 Hz, 1H), 7.88 (s, 1H), 7.67−7.64 (m, 2H), 7.58 (t, J = 8 Hz, 1H), 7.28−7.23 (m, 2H), 6.77 (s, 1H), 6.54 (d, J = 8 Hz, 1H), 6.45 (s, 1H), 6.35 (d, J = 8 Hz, 1H), 3.37 (q, J = 8 Hz, 4H), 1.19 (t, J = 8 Hz, 6H), 13C NMR (100 MHz, CDCl3) δ 169.57, 157.25, 153.37, 152.44, 152.10, 149.39, 145.41, 138.39, 135.17, 134.56, 131.00, 129.51, 128.57, 126.50, 125.07, 123.88, 120.26, 119.98, 118.08, 117.28, 109.32, 104.48, 97.18, 83.29, 44.47, 12.53. HRMS-ESI(m/z): calculated for C29H22BrNO5 [M+H]+ 546.0735, found 546.0678. Synthesis of Compound BCN. The mixture of 5nitrosalicylaldehyde (167.1 mg, 1.0 mmol), ethyl acetoacetate (130.1 mg, 1.0 mmol), and piperidine (85.1 mg, 1.0 mmol) in 5 mL of anhydrous ethanol was stirred at 80 °C for 12 h. After cooling to room temperature, the solution was concentrated under vacuum to afford compound 1 which was utilized directly without further purification. After that, to a solution of compound 1 (230.3 mg) in 5 mL of conc. H2SO4 was added 2-(4-diethylamino2-hydroxybenzoyl)benzoic acid (313.1 mg, 1.0 mmol), and

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then the mixture was stirred at 90 °C for 12 h. After the reaction completed, the solution was cooled to room temperature and poured into ice (50 g) followed by the addition of perchloric acid (70%, 0.5 mL). The resulting purple residue was collected through filtration, washed with cold water and further purified by silica chromatography eluted with CH2Cl2/MeOH (10:1, v/v) to afford BCN as a purple solid (423.1 mg, 82.7% yield).1H NMR (400 MHz, CDCl3) δ 8.67 (s, 1H), 8.60 (s, 1H), 8.43 (d, J = 8 Hz, 1H), 7.98 (d, J = 8 Hz, 1H), 7.67 (t, J = 8 Hz, 1H), 7.59 (t, J = 8 Hz, 1H), 7.28 (s, 2H), 6.77 (s, 1H), 6.22 (d, J = 8 Hz, 1H), 6.47 (s, 1H), 6.35 (d, J = 8 Hz, 1H), 3.38 (q, J = 8 Hz, 4H), 1.20 (t, J = 8 Hz, 6H), 13C NMR (100 MHz, CDCl3) δ 169.51, 156.48, 153.27, 152.33, 149.43, 144.86, 144.26, 138.21, 134.64, 129.61, 128.55, 126.94, 126.42, 125.14, 124.60, 123.82, 120.98, 118.78, 117.54, 109.39, 105.29, 104.40, 97.14, 82.74, 44.46, 12.54. HRMS-ESI(m/z): calculated for C29H22N2O7 [M+H]+ 511.1500, found 511.1499. Synthesis of Compound C1. The solution of 2-(4Diethylamino-2-hydroxybenzoyl)benzoic acid (313.1 mg, 1 mmol) and diethyl malonate (320.1 mg, 2 mmol) in methanesulfonic acid (10 mL) was stirred at 90 oC for 12 h. After cooling to room temperature, the solution was poured into ice water (50 g). After the mixture was neutralize to pH= 7 with NaOH solution (40%), the precipitate was filtered and washed with water to afford crude product 2, which was directly used without further purification. Then, the solution of compound 2, 5 mL of concentrated HCl, and 5 mL of glacial acetic acid was stirred for 12 hours under reflux conditions. After the reaction completed, the solution was cooled to room temperature and poured into 50 mL of ice water. NaOH solution (40%) was added dropwise to modulate pH of the solution to 7, and a pale precipitate formed immediately. After stirring for 30 min, the precipitate was filtered, washed with water, then purified by silica chromatography eluted with CH2Cl2/MeOH (20:1, v/v) to afford C1 as yellow solid (182.6 mg, 54.2% yield). 1H NMR (400 MHz, CD3OD): δ 7.85 (d, J = 8.6 Hz 1H), 7.51 (d, J = 7.9 Hz 2H), 7.28 (q, J = 4.1 Hz, 1H), 7.02 (d, J = 8.8 Hz, 1H), 6.60 (q, J = 3.9 Hz, 1H), 6.56 (s, 1H), 5.59 (s, 1H) 3.44 (q, J = 7.1 Hz, 4H), 1.18 (t, J = 7.0 Hz, 6H),13C NMR (100 MHz, CD3OD) δ 163.60, 159.27, 156.04, 150.78, 135.01, 130.97, 129.76, 129.33, 128.90, 128.71, 128.50, 127.86, 109.03, 108.83, 106.61, 96.65, 44.21, 11.36. HRMS-ESI(m/z): calculated for C20H19NO4 [M−H]− 336.1230, found 336.1207. MTT assay. The MTT assays were conducted to evaluate the cytotoxicity of BCB and BCN . HepG2 or PC12 cells were replanted in the 96-well micro plates to a total volume of 200 µL well−1. The plates were maintained at 37°C, 5% CO2/95% air incubator for 24 hours. Then, the cells were incubated with different concentrations BCB or BCN (0, 10, 20, 50, 100 µM) for another 12 hours. Subsequently, the culture medium was removed and MTT solution (5.0 mg ml−1) was added to each well. After 4 hours, the remaining MTT solution was removed and 150 µL DMSO was added to dissolve the formazan crystals. The absorbance of solution was measured at 490 nm with


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Analytical Chemistry 5 min gentle agitation using a TRITURUS microplate reader. Two-photon fluorescence confocal imaging of mice tissues. Kunming mice (~20 g) were fasted for 12 h to avoid the possible food fluorescence interference. The mice were divided into four groups randomly and intraperitoneally injected with the corresponding dosage of AA (0, 25, 50, 100 mg kg−1 day−1). After three days of AA administration, BCN (2.5 mmol kg−1) was loaded via tail vein injection. As to the scavenging group, the mice were pretreated with UA (300 mg kg−1) via intraperitoneal injection before BCN loading. After anesthesia and anatomy, the livers and kidneys were exposed for imaging directly. The brains were incubated with 10 μM BCN (15 min) before imaged. The images were obtained with 800 nm excitation and 500–600 nm emission collection.

RESULTS AND DISCUSSION Design, reactivity, and spectral properties of BCB. As illustrated in Scheme 1a, BCB was consisted of benzopyran moiety and bromo-substituted coumarin moiety. We envisioned that BCB itself should display non-fluorescent because of the discontinuous conjugated system of benzopyran moiety caused by spirocyclic lactone form and the heavy atom effect of Br atom on coumarin, thus resulting low fluorescent background. It was reported the site 1 in benzopyran moiety was liable to be attacked by nucleophilic reagent,30 thus we anticipated that the fluorescence of BCB solution might be turned on by strong nucleophilic ONOO−. Meanwhile, in view of the fact that common biological biothiols are prone to take place nucleophilic addition,31 BCB might provide a potential reactive site (site 2) towards biothiols, which would unlock the lactone ring, and increase the length of resonance system as well as relieves the heavy atom effect, thus inducing efficient fluorescence enhancement. (a)

O COOH

RSH

SR N

N

Br

O

site 2

O Br

O

The EW group

BCB

(b)

O

N

NO2

O

site 2

N

NO2

O O

O

O

site 1

BCN non-fluorescent RSH= H2S, GSH, Hcy, Cys

C2

COOH

ONOO-

RSH

SR

O

non-fluorescent

fluorescent

O

COOH

Br

O

C1

non-fluorescent

fluorescent

HO

+ O

O

O

site 1

O

ONOON

O

O

COOH

+ N

O

O

HO O

NO2 O

O

The stronger EW group

non-fluorescent

C3

C1 fluorescent

non-fluorescent

EW=electron-withdrawing

Scheme 1. The chemical structure of BCB (a) and BCN (b) and the corresponding proposed response mechanism.

BCB was readily synthesized in one step and its structure was well-characterized with high-resolution mass spectrometry (HRMS, SI) and nuclear magnetic resonance (NMR, SI). With BCB in hand, its recognition capability towards ONOO− and biothiols was evaluated. As can be seen in Figure 1a and S1, the obvious colorimetric changes exhibited BCB possessed naked-eye response character towards ONOO− and biothiols. The kinetic investigation dispalyed that BCB was capable of capturing ONOO− and biothiols instantaneously (Figure

S2, S3). The treatment of BCB with ONOO− presented a major absorption band centered at 400 nm and a corresponding fluorescence maximum at 520 nm (Figure 1b,c), while the apparent spectral bathochromic shift of BCB with biothiols (λmaxex/em = 480/580 nm, Figure 1b,d) was observed. In addition, excellent liner relationship between BCB and biothiols were obtained (Figure S4). However, the H2S reaction product can be also excited at 400 nm and the emission spectra overlapped the half that of ONOO− (Figure S5), hinting that the influence of H2S on ONOO− fluorescently tracing was non-negligible in biological system in view of the fact that H2S and ONOO− normally coexist in biological specimens. The same was true of GSH, Cys and Hcy. Satisfactorily, other common biological molecules induced insignificant fluorescent enhancement with 400 or 480 nm excitation (Figure S6, S7). Taking together, through EW group introduction, BCB was capable of tracing ONOO− and biothiols with colorimetric analysis and meanwhile, was able to specifically detect biothiols with fluorescent mode under 480 nm excitation.

Figure 1. The response capability of BCB towards ONOO− and biothiol (H2S, GSH, Hcy, and Cys). (a) The colorimetric and fluorescent variation of 50 μM BCB in the presence of various analytes: (I) BCB only as control group; (II) ONOO− (100 μM); (III) H2S (100 μM); (IV) GSH (1 mM); (V) Hcy (1 mM); (VI) Cys (1 mM). The fluorescent image was obtained under UV light (365 nm). (b) The absorbance variation of 20 μM BCB before and after treated with various substances. The concentrations of these substances are the same as those in (a). (c) The fluorescent changes (λex = 400 nm) of 20 μM BCB in the presence of ONOO− (40 μM). (d) The fluorescent changes (λex = 480 nm) of 20 μM BCB in the presence of H2S (100 μM), GSH (1 mM), Hcy (1 mM), and Cys (1 mM).

The proposed mechanism for sensing of biothiols and ONOO−. The ONOO− reaction products compound C1 and C2 were confirmed with fluorescent and HRMS analysis ((Figure S8, S9 ). The corresponding reaction mechanism was revealed in Scheme 1a and Figure S10. It can be seen that, as a result of EW introduction, the ONOO− response mechanism of BCB was entirely different from that shown by the benzopyran-based ONOO− fluorescent probe reported previously.14 Meanwhile, the reaction process of BCB with biothiols was verified with 1H NMR and HRMS analysis. As



illustrated in Figure S11, the proton of BCB at 8.84 ppm disappeared upon the addition of 2-mercaptoethanol, an analogue of biothiol, verifying the site 2 for nucleophilic addition reaction. In addition, the prominent mass spectra peaks of biothiols addition products were clearly presented (Figure S12), thus strongly supporting the proposed sensing mechanism shown in Scheme 1a. Intracellular basal biothiols imaging of BCB. Encouraged by the fluorescent response properties of BCB towards biothiols, its feasibility of mapping intracellular biothiols level was investigated. As can be seen in Figure S14, upon BCB loading, the observable fluorescent signal was presented in both rat pheochromocytoma cells (PC12) and hepatocellular carcinoma cells (HepG2). Meanwhile, the fluorescence can be effectively attenuated in the presence of Nethylmaleimide (NEM), a widely used biothiols scavenger. Accordingly, the outcome confirmed that BCB can be employed to trace intracellular basal biothiols. Design, reactivity, and spectral properties of BCN. An attempted was made to improve BCB detection performance in order to obviate the interference of biothiols during ONOO− detection while maintain its colorimetric responsive capacity. As shown in Scheme 1b, the BC skeleton was refabricated with another EW group, nitro group (−NO2), to afford probe BCN. Similar to probe BCB, the proposed BCN still had two reactive sites because of EW effect of −NO2. However, the strong fluorescence quenching effect of −NO2 would keep the fluorescence of biothiol addition products silent. On the contrary, upon ONOO− exposure, obvious fluorescent increment of BCN could still be observed because of the fluorescence of product compound C1, which was free from −NO2 influence. Thus, we reasoned that BCN would be capable of tracing ONOO− via fluorescent analysis without biothiols interference in living system, while maintaining its colorimetric responsiveness towards ONOO− and biothiols in buffer solution. Then the optical properties of BCN were assessed. As shown in Figure 2a and S1, similar to BCB, the significant color changes of BCN solution were observed upon ONOO− and biothiols treatment, respectively. However, as expected, only ONOO− can trigger the fluorescent increment of BCN solution while biothiols not. Correspondingly, upon ONOO− treatment, BCN solution exhibited a maximal absorption peak at 400 nm and a distinctly fluorescence enhancement at 520 nm (Figure 2b,c), while the incubation of BCN with biothiols displayed obvious absorption enhancement at around 480 nm (Figure 2b) but no discernible fluorescent signal increment at 580 nm (Figure S15). The reaction products of BCN with ONOO− and biothiols were confirmed by HRMS analysis (Figure S16, S17). Therefore, it was credible to draw the conclusion that, by replacing bromine atom with nitro group, probe BCN held improved fluorescent analytical capacity of ONOO−. That is, by introducing different EW groups into BC skeleton, it was feasible to purposefully improve probe’s response activity and

control fluorescent property of corresponding reaction products so as to optimize the specificity of probe.

Figure 2. The response capability of BCN towards ONOO− and biothiol. (a) The colorimetric and fluorescent variation of 50 μM BCN in the presence of various analytes: (I) BCN only as control group; (II) ONOO− (100 μM); (III) H2S (100 μM); (IV) GSH (1 mM); (V) Hcy (1 mM); (VI) Cys (1 mM). The fluorescent image was obtained under UV light (365 nm). (b) The absorbance variation of 20 μM BCN before and after treated with various substances. The concentrations of these substances are the same as those in (a). (c) The fluorescent changes (λex = 400 nm) of 10 μM BCN upon the addition of various concentrations of ONOO− (0−40 μM).

Subsequently, the fluorescent titration of BCN with ONOO− was investigated in detail. A gradual fluorescence increasing was obtained upon different concentrations of ONOO− incubation and the limit of detection (LOD) was calculated as low as 1.2 nM (Figure 2c,d), thus hinting BCN was sensitive enough for mapping of biological ONOO− fluctuation. The results of fluorescent selective test manifested highly fluorescent specificity of BCN to ONOO− (Figure S18). Meanwhile, the results of pH interference assays displayed that BCN was suitable for sensing ONOO− in physiological conditions (Figure S19). In addition, the investigation of the dependence of BCN fluorescence upon reaction time demonstrated that BCN had a high capturing speed towards ONOO− (Figure S20). Taken together, these results suggested that BCN held promising analytical potential of ONOO− in the biological application. Exogenous ONOO− visualizing of BCN in live cells. The imaging capability of BCN was explored. The MTT assays revealed that BCN had low cytotoxicity under experimental concentration (10 μM) (Figure S21). Utilizing 3-morpholino-sydnonimine (SIN-1) as the ONOO− donor, exogenous ONOO− imaging was carried out. As can be seen in Figure 3, upon SIN-1 administration, a notably brighter fluorescence was obtained in PC12 cells than that observed in control group. In addition, this obvious fluorescent increment can be effectively attenuated by uric acid (UA), an ONOO− scavenger. Similar results were acquired in HepG2 cells. The results revealed that BCN can be employed as a reliable ONOO− imaging indicator in living cells.


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Analytical Chemistry up-regulated ONOO− levels in liver, brain, and kidney tissues upon AA administration.

Figure 3. Exogenous ONOO− imaging in PC-12 and HepG2 cells. (a) and (e) The cells were incubated with BCN only (10 μM, 15 min). (b) and (f) The cells were treated with SIN-1 (1.0 mM, 30 min) before BCN loading. (c) and (g) The cells were exposed to SIN-1 (1.0 mM, 30 min) then treated with UA (100 μM, 3 h), followed by BCN staining. (d) The relative fluorescence intensity of (a–c). (h) The relative fluorescent intensity of (e–g). The images were recorded with 800 nm excitation and 500–600 nm collection. The values are the mean ± s.d. for n = 3, ***p < 0.001. Scale bar = 20 μm.

ONOO−

Acrylamide-induced fluctuation mapping in cells and tissues. Acrylamide (AA), a hazardous compound widely existing in heat-processing foods, can distribute in various tissues after being ingested and induce multiple toxic effects on humans, such as neurotoxicity, hepatotoxicity, renal toxicity, and so on.32-34 Oxidative stress is considered to be one of the main contributors associated with toxicity effects of AA.35-37 However, as one of the highly reactive species, endogenous ONOO− fluctuation is much less clear during AA-induced oxidative injury. Hence, with the aid of BCN, the AA-induced ONOO− fluctuation was explored at the cellular and organ levels. As displayed in Figure 4, upon various dosages of AA (250, 500, 1000 μM) treatment, both PC-12 and HepG2 cells exhibited increased fluorescent signals. Meanwhile, positive correlation between fluorescence intensity and AA dosage was detected. In addition, this robust fluorescent enhancement can be effectively blocked by UA. Consequently, these results demonstrated that BCN was capable of mapping endogenous ONOO− fluctuation in vitro, and that the intracellular ONOO− levels were upregulated in PC-12 and HepG2 cells after AA exposure. Afterwards, ONOO− fluctuation in mice upon AA administration was evaluated. Kuming mice were intraperitoneally injected with various dosages of AA followed by BCN loading via tail vein injection. As to the scavenging group, the mice were pretreated with UA via intraperitoneal injection before BCN loading. As can be seen in Figure 5a-d, the dose-dependent fluorescent emission enhancement was observed in livers, and this fluorescent augmentation can be diminished by UA (Figure 5e), thus indicating increased concentration of ONOO− upon AA exposure in livers. Meanwhile, upregulated ONOO− levels in brains after AA administration were also presented (Figure 5f-i). In addition, the same observation was gained in kidneys (Figure S22). Taken together, these results indicated that the mice presented

Figure 4. AA-induced ONOO- fluctuation imaging in PC-12 and HepG2 cells. The cells were exposed to various concentrations of AA (0, 250, 500, 1000 μM) for 1 h and then treated with BCN (10 μM, 15 min). For the scavenging group (e and j), the cells were treated with AA (1.0 mM, 1 h) and then incubated with UA (100 μM, 3 h), followed by BCN staining. (k) The relative fluorescence intensity of (a–e). (l) The relative fluorescent intensity of (f–j). The images were recorded with 800 nm excitation and 500–600 nm collection. The values are the mean ± s.d. for n = 3, **p < 0.01, ***p < 0.001. Scale bar = 20 μm.

Figure 5. AA-induced ONOO− fluctuation imaging in liver (a-e) and brain (f-j) tissues. (k) The relative fluorescent intensity of liver tissues in (a-e). (l) The relative fluorescent intensity of brain tissues in (f-j). The mice were injected intraperitoneally with various concentrations of AA (mg kg−1 day−1). After three days of AA administration, BCN (2.5 mmol kg−1) was loaded via tail vein injection. For scavenging group (e and j), the mice were injected intraperitoneally with UA (300 mg kg−1) before BCN loading. The images were obtained with 800 nm excitation and 500–600 nm collection. The values are the mean ± s.d. for n = 3, ***p < 0.001. Scale bar = 100 μm.

CONCLUSION ACS Paragon Plus Environment


In summary, by introducing two EW groups (−Br and −NO2), we reconstructed the BC fluorescent platform to afford two novel fluorescent probes (BCB and BCN). Each of them can be employed to implement ONOO− and biothiols sensing through naked eye and UV-vis absorption analysis. And both two probes possessed new chemical molecule recognition and fluorescent response mechanism towards ONOO−, among which BCB was able to specifically detecting biothiols by fluorescent mode with 480 nm excitation while BCN showed better specificity towards ONOO−. The intracellular basal biothiols level was successfully imaged with the support of BCB. With the aid of BCN, ONOO− fluctuation in both cells and tissues was mapped. The results exhibited that the reactivity and selectivity of BC platform-based probe can be regulated by introducing appropriate EW groups (−Br and −NO2), endowing the probe with good analytical performance. This also provided an alternative approach and method for the construction of fluorescent probes for other important biological species.

ASSOCIATED CONTENT Supporting Information Mass spectra, Figures, Tables and NMR spectra. This material is available free of charge on the ACS Publications website.

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected], [email protected]. † These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by National Natural Science Foundation of China (21535004, 91753111, 21775093, 21877076) and the Key Research and Development Program of Shandong Province (2018YFJH0502).

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