A probe with double acetoxyl moieties for hydrazine and its application in living cells

Accepted Manuscript A probe with double acetoxyl moieties for hydrazine and its application in living cells

Xinrong Shi, Caixia Yin, Ying Wen, Yongbin Zhang, Fangjun Huo PII: DOI: Reference:

S1386-1425(18)30512-2 doi:10.1016/j.saa.2018.05.112 SAA 16146

To appear in:

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy

Received date: Revised date: Accepted date:

18 April 2018 22 May 2018 28 May 2018

Please cite this article as: Xinrong Shi, Caixia Yin, Ying Wen, Yongbin Zhang, Fangjun Huo , A probe with double acetoxyl moieties for hydrazine and its application in living cells. Saa (2017), doi:10.1016/j.saa.2018.05.112

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A Probe with Double Acetoxyl Moieties for Hydrazine and its Application in Living Cells Xinrong Shi,a Caixia Yin,*,a YingWen,b Yongbin Zhang, Fangjun Huo**,b a

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Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Key Laboratory of Materials for Energy Conversion and Storage of Shanxi Province, Institute of Molecular Science, Shanxi University, Taiyuan 030006, China. b Research Institute of Applied Chemistry, Shanxi University, Taiyuan, 030006, China. *Corresponding author: F. J. Huo, E-mail: [email protected]； C. X. Yin, E-mail: [email protected].

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Abstract:

As a common chemical reductant, hydrazine has been widely used in various

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fields. However, its high toxicity to human and environment have also attracted

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people’s attention. In this work, a new fluorescence “turn-on” probe based on coumarin for hydrazine was successfully synthesized. The probe with double acetoxyl

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moieties as the reaction sites can obtain the detection limit as low as 2.98 nM for the

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detection of hydrazine in distilled water, which was lower than the U.S. Environmental Protection Agency standard (10 ppb). In addition, it also responded

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obvious fluorescence enhancement and high selectivity to hydrazine over other

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molecules. Furthermore, this probe could visualize the hydrazine in living cells. Keywords: Coumarin; Fluorescent probe; Hydrazine; Detection; Bioimaging

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1. Introduction Hydrazine (NH2-NH2) is a simple diamine and powerful reducing agent [1] that plays a crucial role in various chemical industries, such as pharmaceuticals, photography, chemicals, emulsifiers, dyes and corrosion inhibitors [2]. It also can be

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used for large boiler water because of its oxygen removal performance [3]. In addition,

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due to its high enthalpy of combustion, hydrazine is often employed as fuel for missile

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and rocket propulsion systems as well as electrochemical fuel cells [4]. As consequence, its widespread usage inevitably causes the release of hydrazine into the environment

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which can bring serious environmental health problems [5]. The U.S. Environmental

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Protection Agency (EPA) identified hydrazine as a potential carcinogen with a threshold limit of 10 ppb [6]. Besides, hydrazine is highly toxic, it may cause gene mutation, cancer

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and other adverse health effects for human beings through skin absorption or respiration

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[7]. Therefore, it is very necessary to design a simple, sensitive and rapid analytical method for the determination of hydrazine.

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Several methods can be used to efficiently determine hydrazine have been reported

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[8], such as HPLC [9], capillary electrophoresis [10] gas chromatography [11, 12], chemiluminescence [13, 14], spectrophotometry [15], electrochemistry [16 17, 18], titrimetry [19] and surface-enhanced Raman spectroscopy [20]. Recently, fluorescent probe have became a powerful tool for monitoring the biologically relevant species and to understand their functions due to its high sensitivity, high selectivity and rapid response time, which made it possible to overcome the conventional method’s faults such as high requirement for facility and complex operation [21]. Moreover, compared with

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fluorescence quenching probes, fluorescence “turn-on” probes are preferred due to their larger signal-to-noise ratio and less interference from the nonspecific quenching by factors other than the analytes [22-25]. Of course, a lot of fluorescent probes have been designed and synthesised for

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hydrazine detection using various dyes including BODIPY [26, 27], coumarin [28-31],

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naphthalimide [32, 33], resorufin [34, 35], benzothiazole [36-39], heptamethine cyanine

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and hemicyanine [40-42], fluorescein and rhodamine [43, 44]. Among these reported, several prosthetic groups have been explored including malononitrile [45-48], aldehyde

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group [49], phthalimide group [50-52], allyl carbonate-Pd2+ group [53], aliphatic group

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[54-57], ect [58-60] were often utilized as sensing moiety to detect hydrazine. However, most of these hydrazine probes were single response points, and few were reported with

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double response points [61-64]. Herein, we developed a new fluorescent probe based on

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coumarin for hydrazine recognition with two distinct reaction units. The addition of hydrazine cause the probe 1 to display a stronger blue fluorescence enhancement

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response and that function in pure aqueous media were scarce. Moreover, the probe have

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low toxicity, and the application of the probe 1 for selective detection hydrazine in living cells has been successfully demonstrated. 2. Materials and Methods 2.1. Materials All chemicals were purchased from commercial suppliers and used without further purification. All solvents for analytical texts were analytical grade also without further purification. Distilled water was used to prepare all aqueous solutions.

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2.2. Instruments A pH meter (Mettler Toledo, Switzerland) was used to determine the pH of buffer solution. Ultraviolet-Visible (UV-Vis) spectra and fluorescence spectra were measured on a Cary 50 Bio UV-Vis spectrophotometer and F-7000 FL fluorescence

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spectrophotometer, respectively. A PO-120 quartz cuvette (10 mm) was purchased

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from Shanghai Huamei Experiment Instrument Plants, China. 1H NMR and 13C NMR

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spectra were recorded on a Bruker AVANCE III-600 MHz and 150 MHz NMR spectrometer (Bruker, Billerica, MA) respectively. Chemical shifts were internally

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referenced to tetramethylsilane. ESI-MS was carried out on AB Triple TOF 5600plus

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System (AB SCIEX, Framingham, USA). The ability of probe 1 reacting to hydrazine in the living cells was evaluated by using Nikon Eclipse Ti Microsystems.

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2.3. Preparation and Characterization of Probe 1

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2.3.1 Preparation of Compound 1

Phloroglucinol dihydrous (10 mmol, 1.621 g) was added to absolute ethyl

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alcohol (8 mL) and stirred to dissolve it. After sometime, acetoacetate (11 mmol,

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1.432 g), 2 mL H2SO4 and 1 mL triethylamine was added, respectively. The mixture was refluxed at 90 °C for 3 h. After reaction, the solvent was removed under reduced pressure, and the crude product was purified by column chromatography (silica gel, EtOAc: MSO: DCM = 1: 1: 2) to afford compound 1 as a light yellow solid (0.719 g, 40.4%). 1H NMR (600 MHz, DMSO-d6): δ (ppm): 10.51 (s, 1H), 10.29 (s, 1H), 6.25 (s, 1H), 6.14 (d, J = 24.5 Hz, 1H), 5.84 (s, 1H), 2.48 (s, 3H).

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C NMR (151 MHz,

DMSO-d6): δ: (ppm) 161.5, 160.6, 158.4, 157.0, 155.4, 109.3, 102.6, 99.5, 95.0, 23.9.

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HR-MS of the compound 1: m/z: [compound 1 + H]+ Calcd for C10H8O4 193.0423, Found 193.0490 (Fig. S1). 2.3.2. Preparation and Characterization of Probe 1 Compound 1 (0.267 g, 1.4 mmol), adequate cetic anhydride (0.4 mL, 4 mmol),

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and pyridine (0.2 mL) were dissolved in dichloromethane. The mixture was stirred for

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12 h at room temperature. After reaction, the solvent was removed under reduced

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pressure, and the crude product was purified by column chromatography (silica gel, EtOAc: MSO = 1: 1) to afford probe 1 as a white solid (0.239 g, 62.1%). 1H NMR

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(600 MHz, CDCl3): δ (ppm): 7.06 (s, 1H), 6.87 (s, 1H), 6.20 (s, 1H), 2.49 (s, 1H),

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2.37 (s, 2H), 2.32 (s, 1H). 13C NMR (151 MHz, CDCl3): δ (ppm): 116.3, 113.6, 108.6, 77.2, 77.0, 76.8, 22.8, 21.4, 21.1. HR-MS of the probe 1: m/z: [probe 1 + H]+ Calcd

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for C14H12O6 277.0634, Found 277.0711. (Fig. S2)

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2.4. General UV–Vis and Fluorescence Spectra Measurements

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The stock solution of probe 1 (0.2 mM) was prepared in CH3CN, hydrazine and

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other analytes (20 mM) were prepared in distilled water. Before test, probe 1 (5 μL) was diluted into distilled water (2 mL) with the final concentration of 0.5 μM. At the same time, all fluorescence spectra were recorded using a fluorescence spectrophotometer (λex = 360 nm, λem = 452 nm, slit widths: 5 nm/5 nm) within 15 min at room temperature. 2.5. Cytotoxicity Assay HepG-2 cells were also used to study the cytotoxicity of probe 1. The cell

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viability assay was assessed by Cell Counting Kit-8 (CCK-8), and the absorbance at 450 nm was measured to explicate the cells viability. HepG-2 cells were seeded on a 96-well microtiter to a total volume of 100 μL/well, then the cells were incubated at 37 °C in a 5 % CO2 incubator for 24 h. Different concentrations of probe 1 (0 μM, 1

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μM, 2.5 μM, 5 μM, 10 μM, 20 μM, 30 μM, 50 μM) were then added to the wells.

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After incubation for 5 h or 10 h, CCK-8 (10 % in serum free culture medium) was

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added to each well, and the plate was incubated for another 1 h. The absorbance of each well was measured at 450 nm on a microplate reader.

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2.6. Cell Culture and Fluorescence Imaging

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HepG-2 cells were cultured in 1 × SPP medium (1 % proteose peptone, 0.2 % glucose, 0.1 % yeast extract, 0.003 % EDTA ferricsodium salt) at 37 °C. The cells

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were plated on 6-well plates and allowed to adhere for 12 h. Then, cells were washed

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with PBS butter to remove the culture solution and incubated with probe 1 (0.5 μM). After incubating for 15 min, excess probe 1 was gently washed with PBS butter two

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times and incubated with hydrazine (20 μM) for further 15 min at indoor temperature.

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Cell imaging was then carried out after washing cells with PBS buffer two times. Besides, fluorescence images were recorded at blue channel. 3. Results and Discussion 3.1. Absorption and Emission Spectroscopic of Probe 1 The optical properties of the probe 1 investigated by the UV-Vis absorption and fluorescence spectra were both carried out. As shown in Fig. 1 (a), the probe 1 (30 μM) in distilled water (2 mL) exhibited an absorption at 285 nm, with the addition of

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hydrazine (0-40 equiv.), the peak at 285 nm decreased along with the peak at 360 nm appearanced and increased gradually. In Fig. 2 (b), the probe 1 (0.5 μM) exhibited an extremely weak fluorescence. By the reaction with different concentrations of hydrazine within 15 min, the fluorescence intensity at 452 nm increased dramatically

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and became saturated with 40 equivalents of hydrazine (λex = 360 nm, slit widths: 5

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nm/5 nm). With the addition of hydrazine, the solution of probe 1 turned from

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non-fluorescence to bright blue fluorescence.

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3.2. The Detection Limit of Probe 1 for Hydrazine

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Whereafter, we investigated the detection limit of the probe 1 for hydrazine. Probe 1 (0.5 μM) was treated with various concentrations of hydrazine within 15 min

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in distilled water. From the fluorescence intensity of probe 1 at 452 nm was plotted as

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a function of the hydrazine concentration, we draw the standard curve (Fig. 2), it was showed a good linear relationship (R = 0.98796) between F452nm-F0 and hydrazine

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concentration in the concentration range of 0 to 20 μM, and based on the definition by

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IUPAC (CDL=3 Sb/m) [65], the detection limit on fluorescence response of the probe 1 can be as low as 2.98 nM (﹤10 ppb), implied that hydrazine can be quantitatively detected in a wide concentration range. 3.3. pH Dependent of Probe 1 The pH is an important factor affecting the reaction of probe 1 with hydrazine. We investigated the fluorescence properties of probe 1 (0.5 μM) and probe 1 (0.5

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μM)-hydrazine (20 μM) under different pH values and distilled water within 15 min, respectively (λex = 360 nm, λem = 452 nm, slit widths: 5 nm/5 nm). As shown in Figure 3, probe 1 was stable in the pH range from 2.0 to 6.0 and began to decompose in strong base environment (pH = 11.0-13.0). With the addition of hydrazine the

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fluorescence intensity have no change from 2.0 to 6.0 and the fluorescence enhanced

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from 7.4 to 10.0, it showed that in acid environment, the probe 1 and hydrazine

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almost did not react, and they worked well during this pH range from 7.4-10.0. However, the probe 1 began to decompose not obviously from 7.4 to 10.0 and

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extremely unstable under strong alkaline environment. But it was found that the probe

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1 was stable and worked very well with hydrazine in distilled water. Thus, the distilled water can be selected for the further research.

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3.4. Time-Dependence in the Detection Process of Hydrazine Time-dependent modulation in the fluorescence spectra of probe 1 (0.5 μM) was

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monitored in the presence of 30.0 μM of hydrazine in distilled water. The kinetic

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study showed that the fluorescence emission at 452 nm was immediately initiated and leveled off within 10 min, displaying a rapid response between probe 1 and hydrazine under the selected experimental conditions (Fig. 4). 3.5. The Selective Response of Probe 1 to Hydrazine

It is well known that one of the most significant behavior of a probe is the high selectivity towards the analyte. In order to evaluate the selectivity of the probe 1 for

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hydrazine, the fluorescence response of probe 1 to hydrazine (40 equiv.) and various interferences (200 equiv.) were investigated. As shown in Fig. 5, the free probe 1 exhibited almost no fluorescence intensity at 452 nm, with the addition of 40 equivalents of hydrazine and 200 equivalents various ions within 15 min, respectively,

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the solution containing hydrazine showed remarkable changed, whereas other

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interferents did not cause any appreciable change to the fluorescence of probe 1.

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Furthermore, we also examined several common amine-containing species, such as phenylamine, n-butylamine, hexamethylenediamine, propylamine, hydroxylamine

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hydrochloride, ethanediamine, methanamide, thiol and so on. As shown in figure S3,

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it was found that the fluorescence intensity of probe 1 (0.5 μM) was enhanced by the addition of 40 equivalents of hydrazine and 200 equivalents of several amine

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compounds (n-butylamine, propylamine, ethanediamine, methanamide). Hcy induced

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a slight red shift of probe 1. These result indicated that probe 1 possessed excellent selectivity toward hydrazine (λex = 360 nm, λem = 452 nm, slit widths: 5 nm/5 nm).

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3.6. Proposed Mechanism To confirm the mechanism, the reaction solution of probe 1 and hydrazine were identified by 1H NMR titration and MS analysis (Figure S4 and S5). In figure S4, the result showed that when excessive amounts of hydrazine was added to the solution of probe 1 in DMSO-d6, the characteristic methyl protons (~e and ~f) of acetyl group in probe 1 disappeared in [probe 1 + hydrazine], and the methyl protons (~h) of acetylhydrazine was clearly found. In addition, the hydroxyl protons (~g1/g2) was also

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clearly appeared. Furthermore, MS spectrometry analysis was also used to understand the mechanism, the reaction product of probe with hydrazine displayed a peak m/z = 193.0501(Figure S5). Based our results and reported literatures [66, 67], the reaction of probe 1 with

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hydrazine was produced compound 1, these finding elucidated the mechanism of the

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new hydroxyl group had replaced the ester (Scheme 2).

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fluorescence enhancement that N2H4 had cleaved the phenolic ester linkage, and a



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3.6. Cytotoxicity Assay

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We investigated the potential toxicity of probe 1 to HepG-2 cells was obtained through CKK-8 method. The living cells were incubated with probe 1 of various

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concentrations (0 -50 μM) for 5 h and 10 h, respectively. As shown in Figure 6, when

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the concentration of probe 1 was 50 μM, the cell viability decreased by only 20%, supporting the ability of the probe 1 for living cells imaging at a working



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concentration below 50 μM.

3.7. Imaging of Living Cell In order to evaluate the cell permeability and reaction of probe 1 to hydrazine, cellular imaging studies were also carried out (Fig. 7). As shown in Figure 7b, HepG-2 cells incubated with 0.5 μM of probe 1 for 15 min at room temperature showed nearly non fluorescence. In a further experiment it was found that HepG-2 cells displayed bright blue fluorescence when the cells were first incubated with 0.5

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μM of probe 1 for 15 min and then incubated with 20 μM hydrazine for 15 min at room temperature (Fig. 7d). These imaging experiments indicated that probe 1 can be used to detect hydrazine in imaging modality.

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4. Conclusions

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In this work, a new “turn-on” fluorescence probe based on coumarin derivative

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and acetic anhydride was designed and synthesized. The probe 1 with double acetoxyl moieties as the reaction sites can make the detection limit as low as 2.98 nM for the

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detection of hydrazine in distilled water. In addition, it also responded obviously

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fluorescence enhancement and highly selective to hydrazine over other molecules. Moreover, the probe 1 showed a rapid detection process (10 min). The fluorescence

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scanning microscopic experiments demonstrated that the probe 1 can be used for

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Acknowledgments

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detecting hydrazine in living HepG-2 cells.

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We thank the National Natural Science Foundation of China (No. 21672131, 21775096, 21705102), Talents Support Program of Shanxi Province (2014401), Shanxi Province Foundation for Returness (2017-026), the Shanxi Province Science Foundation for Youths (No. 201701D221061) and Scientific Instrument Center of Shanxi University.

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Figure captions Scheme 1 The synthesis of the compound 1 and probe 1. Fig. 1 (a) UV-Vis spectra of the probe 1 (30 µM) on the addition of hydrazine (0-40 equiv.) in distilled water. (b) Fluorescence spectral change of the probe 1 (0.5 µM)

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upon addition of hydrazine (0-20 µM) in distilled water (λex = 360 nm, λem = 452 nm,

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slit widths: 5 nm/5 nm).

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Fig. 2 Fluorescence intensities of the mixed solution of probe 1 (0.5 µM) showed

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linear response to add hydrazine in a range of 0-20 µM with a correlation coefficient of R = 0.98796. Inset shows the plot of fluorescence intensity of probe 1 (λex = 360

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nm, λem = 452 nm, slit widths: 5 nm/5 nm) as a function of added hydrazine from 0 to

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Fig. 3 Reaction time profiles of probe 1 (0.5 μM) with hydrazine (30 μM) at 452 nm

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Fig. 4 Fluorescence emission at 452 nm of probe 1 (0.5 μM) at different pH values and distill water in the absence or presence of hydrazine (20 μM).

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Fig. 5 Optical density three-dimensional graph of emission at 452 nm of the probe 1 (0.5 µM) in the presence of 20.0 µM within 15 min in the distilled water (λex = 360nm, λem =452 nm, slit: 5 nm /5 nm) (1. Probe, 2. F-, 3. Cl-, 4. Br-, 5. I-, 6. S2-, 7.ClO-, 8. CN-, 9. HSO3- 10. BO3- 11. K+ 12. Na+ 13. N2H4). Scheme 2 Proposed detection mechanism of the probe 1 to hydrazine. Fig. 6 Cytotoxicity of probe 1. Cell viability of HepG-2 cells incubated with probe 1 (0-5 mM) for 5h and 10 h.

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Fig. 7 Fluorescence images of probe 1 in HepG-2 cells: (a) Bright field image of the cells treated with probe 1; (b) Fluorescence images of probe 1 (0.5 µM; blue channel); (c) Bright field image of the cells treated with probe 1 and hydrazine (20 µM); (d)

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Fluorescence images of the cells treated with probe 1 and hydrazine.

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Scheme 1

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Figure 1

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(b)

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Figure 2

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Figure 3

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Figure 4

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Figure 5

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Scheme 2

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Figure 6

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Graphical abstract The Ttitle

A Probe with Double Acetoxyl Moieties for Hydrazine and Its Application in

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Living Cells

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The statement:

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Highlight 1. The probe with double acetoxyl moieties as a “turn-on” probe for detecting hydrazine in aqueous. 2.

Thes system has the detection limit as lower as 2.98 nM for The application of the probe for selective detection hydrazine in

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HepG-2 cells has been successfully demonstrated.

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3.

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hydrazine.