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1,8-Naphthalimide-based dual-response fluorescent probe for highly discriminating detection of cys and H2S
Dyes and Pigments 173 (2020) 107918
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1,8-Naphthalimide-based dual-response fluorescent probe for highly discriminating detection of cys and H2S Kai-Bin Li a, *, Wang-Bo Qu a, Qiongxia Shen a, Siqi Zhang a, Wei Shi a, Lei Dong b, **, De-Man Han a, *** a
Department of Chemistry, Taizhou University, Jiaojiang, 318000, PR China Intitut de Chimie et Biochimie Mol�eculaires et Supramol�eculaires, Laboratoire de Chimie Organique 2-Glycochimie, UMR 5246, CNRS and Universit�e Claude Bernard Lyon 1, Universit�e de Lyon, 1 Rue Victor Grignard, F-69622, Villeurbanne, France
b
A R T I C L E I N F O
A B S T R A C T
Keywords: Fluorescent probe Cysteine Hydrogen sulfide Image analysis
Hydrogen sulfide (H2S) and cysteine (Cys) are ubiquitous biological thiols in physiological and pathological processes of living systems. Their aberrant concentration levels are associated with many diseases. Hence, we developed a water-soluble fluorescent (FL) probe for H2S and Cys response. The probe was synthesized by two steps of reaction coupling between a triethylene glycol naphthalimide dye and 7-chlorobenzofurazan-4-sulfonyl chloride (CBD) dye linked by a rigid piperazine group. The probe displayed dual fluorescence emission for Cys (λem ¼ 571 nm) and H2S (λem ¼ 535 nm) detection, accompanied by different absorbance changes for Cys (λex ¼ 405 nm) and H2S (λex ¼ 486 nm). The probe could distinguish Cys and H2S from other biological thiols (GSH, Hcy) with longer wavelength or stronger fluorescence emission. Moreover, the probe was allowed to sense the exogenous or endogenous H2S and Cys with dual-emission signal (blue and red channel) in living MCF-7 cells.
1. Introduction Intracellular biothiols, including hydrogen sulfide (H2S), cysteine (Cys), homocysteine (Hcy) and glutathione (GSH), are ubiquitous and play an essential role in physiological and pathological processes [1–5]. Cysteine (Cys), as a thiol-amino acid, widely participates in many bio logical processes such as protein synthesis, cellular detoxification and signal transduction [6,7]. The aberrant concentration level of Cys is associated with many human diseases such as retardation of growth, edema, hair depigmentation, cardiovascular complications, Parkinson’s disease [8,9]. Hydrogen sulfide (H2S) is an important endogenous gasotransmitter in live systems, not only famous for its pungent smell and noxious nature. H2S is mainly produced from Cys and Hcy via enzyme [10]. The abnormal level in biological systems usually result in series of deleterious effects including Down’s syndrome, Alzheimer’s disease, liver cirrhosis and diabetes [11]. Hence, detecting and imaging Cys and H2S in living cells are urgent demands for disease diagnosis and therapy. Compared with a number of analysis methods [12–14] to thiol
response, hence fluorescence analysis has high selectivity, simplicity and non-invasiveness [15–19]. Numerous fluorescent probes with near-infrared emission, high quantum yield, sensitive cells imaging for thiols have been reported [20–26]. However, the probe distinguishing Cys from Hcy and GSH was still hampered for their similar structure and reactivity [27,28]. Besides, high intracellular level of GSH (1–10 mM) [29] would also interfere the H2S detection in cells [30]. At present, fewer probes were reported about dual or multiple response and dif ferentiation of Cys and H2S from GSH, Hcy or others biothiols by a single probe [31–33]. For the favorable properties and simple modification, some 1,8naphthalimides-based fluorescent probes for biomolecule detection have been reported in our previous work [34,35]. Therefore, we deco rated 7-chlorobenzofurazan-4-sulfonyl moiety (CBD) on 1,8-naphthali mides for selective detection of Cys an H2S with dual fluorescence channel. The active H2S enabled to thiolysize the sulfonamide bond between fluorophore and benzofurazan moiety during 10 min, followed by clear fluorescence enhancements (λex ¼ 535 nm). Meanwhile, the probe could distinguish Cys (λex ¼ 585 nm) from GSH and Hcy
* Corresponding author. ** Corresponding author. *** Corresponding author. E-mail addresses: [email protected] (K.-B. Li), [email protected] (L. Dong), [email protected] (D.-M. Han). https://doi.org/10.1016/j.dyepig.2019.107918 Received 3 September 2019; Received in revised form 20 September 2019; Accepted 21 September 2019 Available online 23 September 2019 0143-7208/© 2019 Elsevier Ltd. All rights reserved.
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Dyes and Pigments 173 (2020) 107918
(Trp), S2O23 , SO24 , SH , H2O2, HClO, NO2 , NO3 , Ac , Kþ, Naþ, Ca2þ, Mg2þ, Zn2þ, Cu2þ, Fe2þ, Fe3þ were all prepared in deionized water. The FL measurements were carried out with a path length of 10 mm and an excitation wavelength at 400 nm by scanning the spectra between 430 nm and 750 nm. The bandwidth for both excitation and emission spectra were 5 nm. Unless otherwise mentioned, All FLs were excited at 400 nm and acquired in 10 mM PBS buffer (pH 7.4) at 25 � C.
(λex ¼ 535 nm) after incubation 40 min. The dual-response fluorescent probe was available to detect exogenous and endogenous H2S and Cys in live MCF-7 cells. 2. Materials and methods 2.1. General All purchased chemicals and reagents are of analytical grade. Sol vents were purified by standard procedures. Reactions were monitored by TLC using E-Merck aluminum precoated plates of Silica Gel. 1H and 13 C NMR spectra were recorded on a Bruker AM-400 spectrometer using tetramethylsilane (TMS) as the internal standard. High resolution mass spectra were recorded on a Waters LCT Premier XE spectrometer using standard conditions (ESI, 70 eV). All absorption spectral were measured on a Cary 60 UV visible spectrophotometer. All fluorescence spectra were measured on a Varian Cary Eclipse Fluorescence spectrophotometer.
2.5. Cell imaging assay MCF-7 cells were cultured in DMEM supplemented with 10% FBS. Cells (1.5 � 104/well) were seeded on a black 24-well microplate with optically clear bottom overnight. For endogenous Cys and H2S labeling, the cells were incubated with 10 μM 1 for 30 min at 37 � C. For exogenous reactive sulfur species imaging, the three group of cells were preteated with 0.5 mM of N-methylmaleimide (NEM). After 30 min, 10 μM of 1 was added and incubated for anothor 30 min. Then 0.5 mM of Cys,H2S, GSH were added to the above group of cells respectively and were incubated for another 30 min. For the thiol-consumed group, the cells were incubated with 0.5 mM NEM for 30 min and then treated with the same procedure. After three rinses in phosphatic buffer solution (PBS), the fluorescence was eventually detected and photographed with a confocal laser scanning microscopy.
2.2. Synthesis of compound 2-(2-(2-(2-hydroxyethoxy)ethoxy)ethyl)-6(piperazin-1-yl)- 1,8-naphthalimides (4) Compound 2 [36] (407 mg, 1.00 mmol) and piperazine (190 mg, 2.20 mmol) were dissolved in ethylene glycol monomethyl ether (20 mL). The resulting mixture was refluxed for 3 h under nitrogen protection. Then the mixture was concentrated and washed with water and brine, and extracted with CH2Cl2. The combined organic layer was dried over anhydrous Na2SO4, filtered and concentrated in vacuum. The resulting residue was purified by column chromatography on silica gel (CH2Cl2/MeOH ¼ 20 : 1, V/V) to afford a yellow solid (377 mg, 91.5%); mp ¼ 221–222 � C; 1H NMR (400 MHz, DMSO‑d6) δ 8.48 (d, J ¼ 8.0 Hz, 2 H), 8.40 (d, J ¼ 8.0 Hz, 1 H), 7.82 (t, J ¼ 8.0 Hz, 1 H), 7.36 (d, J ¼ 8.0 Hz, 1 H), 4.22 (t, J ¼ 4.0 Hz, 2 H), 3.65–3.17 (m, 20 H); 13C NMR (100 MHz, DMSO‑d6) δ 164.0, 163.5, 155.9, 132.7, 131.2, 131.1, 129.6, 126.6, 125.8, 122.9, 116.3, 115.7, 72.8, 70.1, 70.0, 67.4, 60.6, 60.5, 52.4, 44.9; HR-ESI-MS m/z: [MþNa]þ calcd. for 436.1848 found 436.1841.
2.6. Cell viability assay Cells were plated overnight on 24-well plates at 5000 cells per well in growth medium. After seeding, cells were maintained in growth media treated at increasing concentrations (12.5 μM, 25 μM, 50 μM, 100 μM, 200 μM) of 1 (dissolved in DMSO, final concentration) for 72 h 20 μL of MTS (Promega Corp) solution (2 mg/mL) was added to each well for 2 h at 37 � C, and then the absorbance was measured on a SpectraMax 340 microplate reader (Molecular Devices, USA) at 490 nm with a reference at 690 nm. The optical density of the result in 3-(4,5-Dime thylthiazol-2-yl)-5-(3-Carboxymethoxyphenyl)-2-(4-Sulfophenyl)-2HTetrazolium (MTS) assay was directly proportional to the number of viable cells. Each experiment was done in triplicate.
2.3. Synthesis of compound 6-(4-(7-chlorobenzofurazan-4-sulfonyl) piperazin-1-yl)-2-(2-(2-(2-hydroxyethoxy)ethoxy)ethyl)- 1,8-naphthali mides (1)
3. Results and discussion 1,8-Naphthalimide, as fluorescent reporter, was decorated with 7chlorobenzofurazan-4-sulfonyl group (CBD) to obtain the target probe 1 (Scheme 1). Firstly, the 4-bromo-1,8-naphthalimide (compound 2) was substituted by piperazine (compound 3) to afford compound 4 with desirable yield. The triethylene glycol linker would improve the watersolubility to hamper intermolecular aggregation quenching the fluo rescence emission. Then, 7-chlorobenzofurazan-4-sulfonyl chloride (compound 5) was decorated on the piperazine moiety in CH2Cl2 solu tion mixed with Et3N, which could quench the inherent emission of probe 1. Next, we measured the absorption variation of probe 1 (5 μM) after incubating H2S, Cys, Hcy and GSH in 10 mM PBS buffer (pH 7.4). The probe 1 has two absorption peaks at 340 nm and 400 nm originally. After responding to analytes during different time, the absorption dis played variant changes for H2S and other three biothiols. As Fig. 1 shown, a novel absorption peak at 500 nm was generated after responding to H2S during 10 min, followed by the absorption intensity decrease at 350 nm. However, the absorption of probe 1 showed a ratiometric changes in presence of Cys, Hcy, and GSH after 40 min. These results illustrated that the probe 1 could respond to these four analytes, but have higher reactivity for H2S than Cys, Hcy and GSH. Then, we further explored the fluorescence performance of probe 1 for H2S, Cys, Hcy, and GSH. The probe 1 have low initial fluorescence emission (Φ ¼ 0.015) influenced by the electron-poor chlorobenzofur azan group. After incubating H2S during 10 min, the probe exhibited 8fold fluorescence enhancement at 535 nm (Fig. 2a, blue line). Compared
To a solution of 4 (260 mg, 0.63 mmol) and 5 (155 mg, 0.63 mmol) in CH2Cl2 (20 mL) were added triethylamine (101 mg, 1.00 mmol). The resulting mixture was stirred for 1 h at room temperature. Then the mixture was concentrated and washed with brine, and extracted with CH2Cl2. The combined organic layer was dried over Na2SO4, filtered and concentrated in vacuum. The resulting residue was purified by column chromatography on silica gel (CH2Cl2/MeOH ¼ 20 : 1, V/V) to afford a yellow solid (376 mg, 95%); mp ¼ 170–171 � C; 1H NMR (400 MHz, DMSO‑d6) δ 8.43 (d, J ¼ 8.0 Hz, 1 H), 8.37 (d, J ¼ 8.0 Hz, 1 H), 8.30 (d, J ¼ 8.0 Hz, 1 H), 8.11 (d, J ¼ 8.0 Hz, 1 H), 7.99 (d, J ¼ 8.0 Hz, 1 H), 7.74 (t, J ¼ 8.0 Hz, 1 H), 7.34 (d, J ¼ 8.0 Hz, 1 H), 4.56 (t, J ¼ 4.0 Hz, 1 H), 4.20 (t, J ¼ 4.0 Hz, 2 H), 3.62 (t, J ¼ 4.0 Hz, 2 H), 3.55–3.30 (m, 16 H); 13 C NMR (100 MHz, DMSO‑d6) δ 163.9, 163.4, 155.3, 149.6, 146.7, 136.8, 132.5, 131.2, 131.1, 130.9, 129.4, 126.7, 126.6, 125.8, 124.6, 122.9, 116.6, 116.2, 72.8, 70.1, 70.0, 67.4, 60.6, 52.5, 46.1, 39.0; HRESI-MS m/z: [MþNa]þ calcd. for 652.1245 found 652.1236. 2.4. Spectroscopic measurements Stock solution of 1 (5 mM) was prepared in DMSO. Stock solutions of 5 mM of amino acids and other competing analytes such as Cys, Hcy, GSH, histidine (His), threonine (Thr), glycine (Gly), leucine (Leu), alanine (Ala), proline (Pro), arginine (Arg), serine (Ser), phenylalanine (Phe), vitamin C (Vc), aspartic acid (Asp), methionine (Met), tryptophan 2
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Dyes and Pigments 173 (2020) 107918
Scheme 1. Synthetic procedures for compound 1.
and Cys in different pH solution. Probe 1 could maintain stable fluo rescence-off alone in different pH solution. After incubating with H2S or Cys, probe 1 could display a distinct fluorescence enhancement in neutral or alkalescent environments (pH 7–11) due to the presence and stability of thiolate (RS ) in this condition (Fig. 2d). To evaluate the relationship between the fluorescence intensity and concentration of analytes, the fluorescence signal changes at 535 nm for H2S and at 571 nm for Cys were measured respectively. The probe 1 exhibited homogenous fluorescence enhancement upon addition of H2S and stirred during 10 min (Fig. 2e). The linear relationship between fluorescence increase and concentration of H2S was calculated with desirable limit of detection (LOD ¼ 3σ/k) as low as 11.5 nM (Fig. S1). With addition of Cys, probe 1 similarly performed linear fluorescence enhancements, followed by emission wavelength red-shifted from 531 nm to 571 nm (Fig. 2F). The low LOD (16.7 nM) was sufficient to detect Cys existence in vitro or in vivo (Fig. S1). The two different mechanism of fluorescence enhancements for H2S and Cys was proposed as follows (Scheme 2): (a) The negative thio phenol (HS ) ion was nucleophilic enough to substitute chloride. Cleavage of sulfonamide bond and separation of bezofuranzan gener ated 1,8-naphthalimides with strong fluorescence at 535 nm. (b) The response processes for Cys, Hcy and GSH was different from H2S. After thiolate (RS ) replacing the chloride and combining on the benzofur azan moiety, the electron-rich sulfur atom of GSH or Hcy would slightly recover ICT effect to promote probe 1 emitting weak fluorescence at 535 nm. However, the intramolecular rearrangement promoted amino group of Cys replacing the sulfur atom and switching-on the fluorescence of benzofurazan at 571 nm. The results of mass spectrometry proved our suppositions. After probe 1 sensing analytes, the mass peaks of corre sponding fragments were found at m/z ¼ 414.2033 for H2S, m/ z ¼ 713.1700 for Cys, m/z ¼ 751.1826 for Hcy and m/z ¼ 899.2410 for GSH (Figs. S2–S5). Based on the above results, we investigated the ability of probe 1 to image the endogenous or exogenous H2S and Cys in vivo. Compared with dark horizons after incubation of NEM (cleaning endogenous biothiols) and probe 1 (Fig. 3, the second column), the MCF-7 cells incubated with probe 1 alone were observed dual-fluorescence signals in blue and red channels, which illustrated the sensitivity of probe 1 for endogenous H2S and Cys imaging (Fig. 3, the first column). Then, we respectively incu bated H2S, Cys and GSH with MCF-7 cells after incubating NEM. Probe 1 only released blue fluorescence after responding to H2S and GSH (Fig. 3, the third and fifth column). The stronger emission intensity for H2S response certified probe 1 have higher reactivity for H2S than GSH.
Fig. 1. Time-dependent absorption spectra changes of 1 (5 μM) in the presence of 50 μM of H2S (a), Cys (b), Hcy (c), GSH (d), respectively.
the weak fluorescence response for Hcy (Fig. 2a, pink line) and GSH (Fig. 2a, green line) at 535 nm, Cys was allowed to increase 11-fold fluorescence during 40 min incubation, followed by slightly redshifted emission at 571 nm. These results indicated that probe 1 has higher reactivity of H2S and Cys with dual-emission signals. Hence, we tested the time-dependent fluorescence increase of probe 1 in presence of H2S, Cys, Hcy and GSH. Probe 1 performed highest reactivity for H2S, which reached to the fluorescence maximum during 10 min. While longer response time (40 min) was required to react with other three biothiols completely, probe 1 can provide different fluorescent signal to distinguish Cys from Hcy and GSH (Fig. 2b). To test the selectivity of probe 1 toward H2S, Cys, GSH, Hcy and the possible interference from other analytes, the probe was incubated with a number of aliphatic thiols, amino acids, familiar reactive oxidant species and potential interfering ions (From left to right in Fig. 2c: Cys, H2S, Hcy, GSH, His, Thr, Gly, Leu, Ala, Pro, Arg, Ser, Phe, Vc, Asp, Met, Trp, S2O23 , SO24 , H2O2, HClO, NO2 , NO3 , Ac , Kþ, Naþ, Ca2þ, Mg2þ, Zn2þ, Cu2þ, Fe2þ, Fe3þ). The fluorescence signal increase obviously only in presence of Cys or H2S. Other species were shown to not react with probe 1, or weak fluorescence changes, hence would not interfere with the sensing system (Fig. 2c). Next, we investigated the sensing performances of probe 1 for H2S 3
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Dyes and Pigments 173 (2020) 107918
Fig. 2. (a) Fluorescence spectra of 1 (5 μM) in the presence of 25 μM of Cys, H2S, Hcy, GSH as well as the corresponding time-dependent fluorescence intensity changes (b) of 1. (c) Selectivity of 1 (5 μM) in the presence of Cys, H2S and competing analytes (50 μM). (d) pH titration of 1 (5 μM), 1 þ Cys and 1 þ H2S, modulated by utilizing aqueous hydrochloric acid (1 N) and sodium hydroxide solution (1 N). Fluorescence spectra changes of 1 (5 μM) as a function of H2S (e) and Cys (f) concentrations (0–25 μM). The FLs of Fig. 2e were acquired after incubation for 10 min while the FLs of Fig. 2f were acquired after incubation for 40 min.
Scheme 2. Proposed mechanism for discriminative detection of Cys, H2S and Hcy/GSH.
Besides, observing red emission signal indicated that probe 1 could image the exogenous Cys in living cells (Fig. 3, the fourth column). Eventually, a cytotoxicity assay has been tested with the concentrations of 12.5, 25, 50, 100, 200 μM, suggesting that probe 1 has almost no cytotoxicity (Fig. S6). These results suggest that probe 1 is cell perme able and can be used for detecting endogenous H2S and Cys in living cells.
fluorescent probe for selective detection of H2S and cysteine (Cys) with dual-fluorescence enhancement. In wide pH range of aqueous solution, the probe could specifically distinguish H2S (λex ¼ 535 nm) and Cys (λex ¼ 571 nm) from homocysteine (Hcy), glutathione (GSH) and others biothiols after response during different time. The corresponding response mechanisms were verified by mass spectrometry analysis. Moreover, probe 1 was allowed to sense the exogenous or endogenous H2S and Cys with dual-emission signal (blue and red channel) in living MCF-7 cells.
4. Conclusion In summary, we designed and synthesized a 1,8-naphthalimide based 4
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Dyes and Pigments 173 (2020) 107918
Fig. 3. Fluorescent imaging of (a) 10 μM of 1 and (b) 10 μM of 1 pretreated with 0.5 mM of NEM for 30 min. (c) 10 μM of 1 pretreated with 0.5 mM of NEM for 30 min, and further incubated with 0.5 mM of H2S, (d) Cys and (e) GSH under the same condition in live MCF-7 cells. Blue and red chan nels correspond to the emission windows of 420–470 and 650–700 nm. Excitation at 400 nm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Conflicts of interest
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Dyes and Pigments journal homepage: http://www.elsevier.com/locate/dyepig
1,8-Naphthalimide-based dual-response fluorescent probe for highly discriminating detection of cys and H2S Kai-Bin Li a, *, Wang-Bo Qu a, Qiongxia Shen a, Siqi Zhang a, Wei Shi a, Lei Dong b, **, De-Man Han a, *** a
Department of Chemistry, Taizhou University, Jiaojiang, 318000, PR China Intitut de Chimie et Biochimie Mol�eculaires et Supramol�eculaires, Laboratoire de Chimie Organique 2-Glycochimie, UMR 5246, CNRS and Universit�e Claude Bernard Lyon 1, Universit�e de Lyon, 1 Rue Victor Grignard, F-69622, Villeurbanne, France
b
A R T I C L E I N F O
A B S T R A C T
Keywords: Fluorescent probe Cysteine Hydrogen sulfide Image analysis
Hydrogen sulfide (H2S) and cysteine (Cys) are ubiquitous biological thiols in physiological and pathological processes of living systems. Their aberrant concentration levels are associated with many diseases. Hence, we developed a water-soluble fluorescent (FL) probe for H2S and Cys response. The probe was synthesized by two steps of reaction coupling between a triethylene glycol naphthalimide dye and 7-chlorobenzofurazan-4-sulfonyl chloride (CBD) dye linked by a rigid piperazine group. The probe displayed dual fluorescence emission for Cys (λem ¼ 571 nm) and H2S (λem ¼ 535 nm) detection, accompanied by different absorbance changes for Cys (λex ¼ 405 nm) and H2S (λex ¼ 486 nm). The probe could distinguish Cys and H2S from other biological thiols (GSH, Hcy) with longer wavelength or stronger fluorescence emission. Moreover, the probe was allowed to sense the exogenous or endogenous H2S and Cys with dual-emission signal (blue and red channel) in living MCF-7 cells.
1. Introduction Intracellular biothiols, including hydrogen sulfide (H2S), cysteine (Cys), homocysteine (Hcy) and glutathione (GSH), are ubiquitous and play an essential role in physiological and pathological processes [1–5]. Cysteine (Cys), as a thiol-amino acid, widely participates in many bio logical processes such as protein synthesis, cellular detoxification and signal transduction [6,7]. The aberrant concentration level of Cys is associated with many human diseases such as retardation of growth, edema, hair depigmentation, cardiovascular complications, Parkinson’s disease [8,9]. Hydrogen sulfide (H2S) is an important endogenous gasotransmitter in live systems, not only famous for its pungent smell and noxious nature. H2S is mainly produced from Cys and Hcy via enzyme [10]. The abnormal level in biological systems usually result in series of deleterious effects including Down’s syndrome, Alzheimer’s disease, liver cirrhosis and diabetes [11]. Hence, detecting and imaging Cys and H2S in living cells are urgent demands for disease diagnosis and therapy. Compared with a number of analysis methods [12–14] to thiol
response, hence fluorescence analysis has high selectivity, simplicity and non-invasiveness [15–19]. Numerous fluorescent probes with near-infrared emission, high quantum yield, sensitive cells imaging for thiols have been reported [20–26]. However, the probe distinguishing Cys from Hcy and GSH was still hampered for their similar structure and reactivity [27,28]. Besides, high intracellular level of GSH (1–10 mM) [29] would also interfere the H2S detection in cells [30]. At present, fewer probes were reported about dual or multiple response and dif ferentiation of Cys and H2S from GSH, Hcy or others biothiols by a single probe [31–33]. For the favorable properties and simple modification, some 1,8naphthalimides-based fluorescent probes for biomolecule detection have been reported in our previous work [34,35]. Therefore, we deco rated 7-chlorobenzofurazan-4-sulfonyl moiety (CBD) on 1,8-naphthali mides for selective detection of Cys an H2S with dual fluorescence channel. The active H2S enabled to thiolysize the sulfonamide bond between fluorophore and benzofurazan moiety during 10 min, followed by clear fluorescence enhancements (λex ¼ 535 nm). Meanwhile, the probe could distinguish Cys (λex ¼ 585 nm) from GSH and Hcy
* Corresponding author. ** Corresponding author. *** Corresponding author. E-mail addresses: [email protected] (K.-B. Li), [email protected] (L. Dong), [email protected] (D.-M. Han). https://doi.org/10.1016/j.dyepig.2019.107918 Received 3 September 2019; Received in revised form 20 September 2019; Accepted 21 September 2019 Available online 23 September 2019 0143-7208/© 2019 Elsevier Ltd. All rights reserved.
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Dyes and Pigments 173 (2020) 107918
(Trp), S2O23 , SO24 , SH , H2O2, HClO, NO2 , NO3 , Ac , Kþ, Naþ, Ca2þ, Mg2þ, Zn2þ, Cu2þ, Fe2þ, Fe3þ were all prepared in deionized water. The FL measurements were carried out with a path length of 10 mm and an excitation wavelength at 400 nm by scanning the spectra between 430 nm and 750 nm. The bandwidth for both excitation and emission spectra were 5 nm. Unless otherwise mentioned, All FLs were excited at 400 nm and acquired in 10 mM PBS buffer (pH 7.4) at 25 � C.
(λex ¼ 535 nm) after incubation 40 min. The dual-response fluorescent probe was available to detect exogenous and endogenous H2S and Cys in live MCF-7 cells. 2. Materials and methods 2.1. General All purchased chemicals and reagents are of analytical grade. Sol vents were purified by standard procedures. Reactions were monitored by TLC using E-Merck aluminum precoated plates of Silica Gel. 1H and 13 C NMR spectra were recorded on a Bruker AM-400 spectrometer using tetramethylsilane (TMS) as the internal standard. High resolution mass spectra were recorded on a Waters LCT Premier XE spectrometer using standard conditions (ESI, 70 eV). All absorption spectral were measured on a Cary 60 UV visible spectrophotometer. All fluorescence spectra were measured on a Varian Cary Eclipse Fluorescence spectrophotometer.
2.5. Cell imaging assay MCF-7 cells were cultured in DMEM supplemented with 10% FBS. Cells (1.5 � 104/well) were seeded on a black 24-well microplate with optically clear bottom overnight. For endogenous Cys and H2S labeling, the cells were incubated with 10 μM 1 for 30 min at 37 � C. For exogenous reactive sulfur species imaging, the three group of cells were preteated with 0.5 mM of N-methylmaleimide (NEM). After 30 min, 10 μM of 1 was added and incubated for anothor 30 min. Then 0.5 mM of Cys,H2S, GSH were added to the above group of cells respectively and were incubated for another 30 min. For the thiol-consumed group, the cells were incubated with 0.5 mM NEM for 30 min and then treated with the same procedure. After three rinses in phosphatic buffer solution (PBS), the fluorescence was eventually detected and photographed with a confocal laser scanning microscopy.
2.2. Synthesis of compound 2-(2-(2-(2-hydroxyethoxy)ethoxy)ethyl)-6(piperazin-1-yl)- 1,8-naphthalimides (4) Compound 2 [36] (407 mg, 1.00 mmol) and piperazine (190 mg, 2.20 mmol) were dissolved in ethylene glycol monomethyl ether (20 mL). The resulting mixture was refluxed for 3 h under nitrogen protection. Then the mixture was concentrated and washed with water and brine, and extracted with CH2Cl2. The combined organic layer was dried over anhydrous Na2SO4, filtered and concentrated in vacuum. The resulting residue was purified by column chromatography on silica gel (CH2Cl2/MeOH ¼ 20 : 1, V/V) to afford a yellow solid (377 mg, 91.5%); mp ¼ 221–222 � C; 1H NMR (400 MHz, DMSO‑d6) δ 8.48 (d, J ¼ 8.0 Hz, 2 H), 8.40 (d, J ¼ 8.0 Hz, 1 H), 7.82 (t, J ¼ 8.0 Hz, 1 H), 7.36 (d, J ¼ 8.0 Hz, 1 H), 4.22 (t, J ¼ 4.0 Hz, 2 H), 3.65–3.17 (m, 20 H); 13C NMR (100 MHz, DMSO‑d6) δ 164.0, 163.5, 155.9, 132.7, 131.2, 131.1, 129.6, 126.6, 125.8, 122.9, 116.3, 115.7, 72.8, 70.1, 70.0, 67.4, 60.6, 60.5, 52.4, 44.9; HR-ESI-MS m/z: [MþNa]þ calcd. for 436.1848 found 436.1841.
2.6. Cell viability assay Cells were plated overnight on 24-well plates at 5000 cells per well in growth medium. After seeding, cells were maintained in growth media treated at increasing concentrations (12.5 μM, 25 μM, 50 μM, 100 μM, 200 μM) of 1 (dissolved in DMSO, final concentration) for 72 h 20 μL of MTS (Promega Corp) solution (2 mg/mL) was added to each well for 2 h at 37 � C, and then the absorbance was measured on a SpectraMax 340 microplate reader (Molecular Devices, USA) at 490 nm with a reference at 690 nm. The optical density of the result in 3-(4,5-Dime thylthiazol-2-yl)-5-(3-Carboxymethoxyphenyl)-2-(4-Sulfophenyl)-2HTetrazolium (MTS) assay was directly proportional to the number of viable cells. Each experiment was done in triplicate.
2.3. Synthesis of compound 6-(4-(7-chlorobenzofurazan-4-sulfonyl) piperazin-1-yl)-2-(2-(2-(2-hydroxyethoxy)ethoxy)ethyl)- 1,8-naphthali mides (1)
3. Results and discussion 1,8-Naphthalimide, as fluorescent reporter, was decorated with 7chlorobenzofurazan-4-sulfonyl group (CBD) to obtain the target probe 1 (Scheme 1). Firstly, the 4-bromo-1,8-naphthalimide (compound 2) was substituted by piperazine (compound 3) to afford compound 4 with desirable yield. The triethylene glycol linker would improve the watersolubility to hamper intermolecular aggregation quenching the fluo rescence emission. Then, 7-chlorobenzofurazan-4-sulfonyl chloride (compound 5) was decorated on the piperazine moiety in CH2Cl2 solu tion mixed with Et3N, which could quench the inherent emission of probe 1. Next, we measured the absorption variation of probe 1 (5 μM) after incubating H2S, Cys, Hcy and GSH in 10 mM PBS buffer (pH 7.4). The probe 1 has two absorption peaks at 340 nm and 400 nm originally. After responding to analytes during different time, the absorption dis played variant changes for H2S and other three biothiols. As Fig. 1 shown, a novel absorption peak at 500 nm was generated after responding to H2S during 10 min, followed by the absorption intensity decrease at 350 nm. However, the absorption of probe 1 showed a ratiometric changes in presence of Cys, Hcy, and GSH after 40 min. These results illustrated that the probe 1 could respond to these four analytes, but have higher reactivity for H2S than Cys, Hcy and GSH. Then, we further explored the fluorescence performance of probe 1 for H2S, Cys, Hcy, and GSH. The probe 1 have low initial fluorescence emission (Φ ¼ 0.015) influenced by the electron-poor chlorobenzofur azan group. After incubating H2S during 10 min, the probe exhibited 8fold fluorescence enhancement at 535 nm (Fig. 2a, blue line). Compared
To a solution of 4 (260 mg, 0.63 mmol) and 5 (155 mg, 0.63 mmol) in CH2Cl2 (20 mL) were added triethylamine (101 mg, 1.00 mmol). The resulting mixture was stirred for 1 h at room temperature. Then the mixture was concentrated and washed with brine, and extracted with CH2Cl2. The combined organic layer was dried over Na2SO4, filtered and concentrated in vacuum. The resulting residue was purified by column chromatography on silica gel (CH2Cl2/MeOH ¼ 20 : 1, V/V) to afford a yellow solid (376 mg, 95%); mp ¼ 170–171 � C; 1H NMR (400 MHz, DMSO‑d6) δ 8.43 (d, J ¼ 8.0 Hz, 1 H), 8.37 (d, J ¼ 8.0 Hz, 1 H), 8.30 (d, J ¼ 8.0 Hz, 1 H), 8.11 (d, J ¼ 8.0 Hz, 1 H), 7.99 (d, J ¼ 8.0 Hz, 1 H), 7.74 (t, J ¼ 8.0 Hz, 1 H), 7.34 (d, J ¼ 8.0 Hz, 1 H), 4.56 (t, J ¼ 4.0 Hz, 1 H), 4.20 (t, J ¼ 4.0 Hz, 2 H), 3.62 (t, J ¼ 4.0 Hz, 2 H), 3.55–3.30 (m, 16 H); 13 C NMR (100 MHz, DMSO‑d6) δ 163.9, 163.4, 155.3, 149.6, 146.7, 136.8, 132.5, 131.2, 131.1, 130.9, 129.4, 126.7, 126.6, 125.8, 124.6, 122.9, 116.6, 116.2, 72.8, 70.1, 70.0, 67.4, 60.6, 52.5, 46.1, 39.0; HRESI-MS m/z: [MþNa]þ calcd. for 652.1245 found 652.1236. 2.4. Spectroscopic measurements Stock solution of 1 (5 mM) was prepared in DMSO. Stock solutions of 5 mM of amino acids and other competing analytes such as Cys, Hcy, GSH, histidine (His), threonine (Thr), glycine (Gly), leucine (Leu), alanine (Ala), proline (Pro), arginine (Arg), serine (Ser), phenylalanine (Phe), vitamin C (Vc), aspartic acid (Asp), methionine (Met), tryptophan 2
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Scheme 1. Synthetic procedures for compound 1.
and Cys in different pH solution. Probe 1 could maintain stable fluo rescence-off alone in different pH solution. After incubating with H2S or Cys, probe 1 could display a distinct fluorescence enhancement in neutral or alkalescent environments (pH 7–11) due to the presence and stability of thiolate (RS ) in this condition (Fig. 2d). To evaluate the relationship between the fluorescence intensity and concentration of analytes, the fluorescence signal changes at 535 nm for H2S and at 571 nm for Cys were measured respectively. The probe 1 exhibited homogenous fluorescence enhancement upon addition of H2S and stirred during 10 min (Fig. 2e). The linear relationship between fluorescence increase and concentration of H2S was calculated with desirable limit of detection (LOD ¼ 3σ/k) as low as 11.5 nM (Fig. S1). With addition of Cys, probe 1 similarly performed linear fluorescence enhancements, followed by emission wavelength red-shifted from 531 nm to 571 nm (Fig. 2F). The low LOD (16.7 nM) was sufficient to detect Cys existence in vitro or in vivo (Fig. S1). The two different mechanism of fluorescence enhancements for H2S and Cys was proposed as follows (Scheme 2): (a) The negative thio phenol (HS ) ion was nucleophilic enough to substitute chloride. Cleavage of sulfonamide bond and separation of bezofuranzan gener ated 1,8-naphthalimides with strong fluorescence at 535 nm. (b) The response processes for Cys, Hcy and GSH was different from H2S. After thiolate (RS ) replacing the chloride and combining on the benzofur azan moiety, the electron-rich sulfur atom of GSH or Hcy would slightly recover ICT effect to promote probe 1 emitting weak fluorescence at 535 nm. However, the intramolecular rearrangement promoted amino group of Cys replacing the sulfur atom and switching-on the fluorescence of benzofurazan at 571 nm. The results of mass spectrometry proved our suppositions. After probe 1 sensing analytes, the mass peaks of corre sponding fragments were found at m/z ¼ 414.2033 for H2S, m/ z ¼ 713.1700 for Cys, m/z ¼ 751.1826 for Hcy and m/z ¼ 899.2410 for GSH (Figs. S2–S5). Based on the above results, we investigated the ability of probe 1 to image the endogenous or exogenous H2S and Cys in vivo. Compared with dark horizons after incubation of NEM (cleaning endogenous biothiols) and probe 1 (Fig. 3, the second column), the MCF-7 cells incubated with probe 1 alone were observed dual-fluorescence signals in blue and red channels, which illustrated the sensitivity of probe 1 for endogenous H2S and Cys imaging (Fig. 3, the first column). Then, we respectively incu bated H2S, Cys and GSH with MCF-7 cells after incubating NEM. Probe 1 only released blue fluorescence after responding to H2S and GSH (Fig. 3, the third and fifth column). The stronger emission intensity for H2S response certified probe 1 have higher reactivity for H2S than GSH.
Fig. 1. Time-dependent absorption spectra changes of 1 (5 μM) in the presence of 50 μM of H2S (a), Cys (b), Hcy (c), GSH (d), respectively.
the weak fluorescence response for Hcy (Fig. 2a, pink line) and GSH (Fig. 2a, green line) at 535 nm, Cys was allowed to increase 11-fold fluorescence during 40 min incubation, followed by slightly redshifted emission at 571 nm. These results indicated that probe 1 has higher reactivity of H2S and Cys with dual-emission signals. Hence, we tested the time-dependent fluorescence increase of probe 1 in presence of H2S, Cys, Hcy and GSH. Probe 1 performed highest reactivity for H2S, which reached to the fluorescence maximum during 10 min. While longer response time (40 min) was required to react with other three biothiols completely, probe 1 can provide different fluorescent signal to distinguish Cys from Hcy and GSH (Fig. 2b). To test the selectivity of probe 1 toward H2S, Cys, GSH, Hcy and the possible interference from other analytes, the probe was incubated with a number of aliphatic thiols, amino acids, familiar reactive oxidant species and potential interfering ions (From left to right in Fig. 2c: Cys, H2S, Hcy, GSH, His, Thr, Gly, Leu, Ala, Pro, Arg, Ser, Phe, Vc, Asp, Met, Trp, S2O23 , SO24 , H2O2, HClO, NO2 , NO3 , Ac , Kþ, Naþ, Ca2þ, Mg2þ, Zn2þ, Cu2þ, Fe2þ, Fe3þ). The fluorescence signal increase obviously only in presence of Cys or H2S. Other species were shown to not react with probe 1, or weak fluorescence changes, hence would not interfere with the sensing system (Fig. 2c). Next, we investigated the sensing performances of probe 1 for H2S 3
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Fig. 2. (a) Fluorescence spectra of 1 (5 μM) in the presence of 25 μM of Cys, H2S, Hcy, GSH as well as the corresponding time-dependent fluorescence intensity changes (b) of 1. (c) Selectivity of 1 (5 μM) in the presence of Cys, H2S and competing analytes (50 μM). (d) pH titration of 1 (5 μM), 1 þ Cys and 1 þ H2S, modulated by utilizing aqueous hydrochloric acid (1 N) and sodium hydroxide solution (1 N). Fluorescence spectra changes of 1 (5 μM) as a function of H2S (e) and Cys (f) concentrations (0–25 μM). The FLs of Fig. 2e were acquired after incubation for 10 min while the FLs of Fig. 2f were acquired after incubation for 40 min.
Scheme 2. Proposed mechanism for discriminative detection of Cys, H2S and Hcy/GSH.
Besides, observing red emission signal indicated that probe 1 could image the exogenous Cys in living cells (Fig. 3, the fourth column). Eventually, a cytotoxicity assay has been tested with the concentrations of 12.5, 25, 50, 100, 200 μM, suggesting that probe 1 has almost no cytotoxicity (Fig. S6). These results suggest that probe 1 is cell perme able and can be used for detecting endogenous H2S and Cys in living cells.
fluorescent probe for selective detection of H2S and cysteine (Cys) with dual-fluorescence enhancement. In wide pH range of aqueous solution, the probe could specifically distinguish H2S (λex ¼ 535 nm) and Cys (λex ¼ 571 nm) from homocysteine (Hcy), glutathione (GSH) and others biothiols after response during different time. The corresponding response mechanisms were verified by mass spectrometry analysis. Moreover, probe 1 was allowed to sense the exogenous or endogenous H2S and Cys with dual-emission signal (blue and red channel) in living MCF-7 cells.
4. Conclusion In summary, we designed and synthesized a 1,8-naphthalimide based 4
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Dyes and Pigments 173 (2020) 107918
Fig. 3. Fluorescent imaging of (a) 10 μM of 1 and (b) 10 μM of 1 pretreated with 0.5 mM of NEM for 30 min. (c) 10 μM of 1 pretreated with 0.5 mM of NEM for 30 min, and further incubated with 0.5 mM of H2S, (d) Cys and (e) GSH under the same condition in live MCF-7 cells. Blue and red chan nels correspond to the emission windows of 420–470 and 650–700 nm. Excitation at 400 nm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Conflicts of interest
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The authors declared that they have no conflicts of interest to this work. We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted. Acknowledgement We thank the National Natural Science Foundation of China (21804097, 21808150, 21904093), Natural Science Foundation of Zhejiang province (LQ18B050001), China Postdoctoral Science Foun dation (2018M642402), Zhejiang Post-Doctoral Preferential Funding Project (zj20180082), the China Scholarship Council for a PhD stipend to Lei Dong. (No. 201606740066) and the Outstanding youth program of Taizhou University (2018YQ002). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.dyepig.2019.107918. References [1] Mancardi D, Penna C, Merlino A, Del Soldato P, Wink DA, Pagliaro P. Physiological and pharmacological features of the novel gasotransmitter: hydrogen sulfide. Biochim Biophys Acta Bioenerg 2009;1787(7):864–72. [2] Liu Y, Lv X, Hou M, Shi Y, Guo W. Selective fluorescence detection of cysteine over homocysteine and glutathione based on a cysteine-triggered dual michael addition/retro-aza-aldol cascade reaction. Anal Chem 2015;87(22):11475–83. [3] Gupta SC, Kim JH, Prasad S, Aggarwal BB. Regulation of survival, proliferation, invasion, angiogenesis, and metastasis of tumor cells through modulation of inflammatory pathways by nutraceuticals. Cancer Metastasis Rev 2010;29(3): 405–34. [4] Townsend DM, Tew KD, Tapiero H. The importance of glutathione in human disease. Biomed Pharmacother 2003;57(3–4):145–55.
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