Oligo(ethylene glycol)-Functionalized Ratiometric Fluorescent Probe

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Oligo (ethylene glycol)-Functionalized Ratiometric Fluorescent Probe for the Detection of Hydrazine in Vitro and Vivo Jun Li, Yuanchao Cui, Chenxi Bi, Shaoqiong Feng, Fengzhen Yu, En Yuan, Shenzhen Xu, zhe hu, Qi Sun, Dengguo Wei, and Juyoung Yoon Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b01223 • Publication Date (Web): 07 May 2019 Downloaded from http://pubs.acs.org on May 7, 2019

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Analytical Chemistry

Oligo (ethylene glycol)-Functionalized Ratiometric Fluorescent Probe for the Detection of Hydrazine in Vitro and Vivo Jun Li, 1, ‡ Yuanchao Cui, 1, ‡ Chenxi Bi, 1 Shaoqiong Feng, 1 Fengzhen Yu, 1 En Yuan, 1 Shenzhen Xu, 1 Zhe Hu, 2 Qi Sun, 4,* Dengguo Wei, 1, 2 * Juyoung Yoon 3,* 1.

Department of Chemistry, College of Science, Huazhong Agricultural University, Wuhan 430070, P. R. China.

2.

State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, P. R. China.

3.

Department of Chemistry and Nano Science, Ewha Womans University, Seoul 120-750, Korea.

4.

Key Laboratory for Green Chemical Process of Ministry of Education and School of Chemistry and Environmental Engineering, Wuhan Institute of Technology, Wuhan 430205, P. R. China.

ABSTRACT: Hydrazine induced toxicity causes serious harms to the health of humans. The detection of N2H4 in vitro and vivo has been attracted great attention, especially in the context of fluorescent probes. Although some fluorescent N2H4 probes have been reported, only a few operate in purely aqueous media and, as a result, require the use of organic cosolvents which hinders their use in analysis of real samples. In addition, most of the current N2H4 probes are either “off-on” or “onoff” types, in which it is difficult to eliminate interference from background fluorescence commonly occurring in vitro and in vivo systems. Furthermore, some probes are unable to differentiate hydrazine form other organic amines. To address above problems, we develop a novel oligo (ethylene glycol)-functionalized fluorescent probe for the detection of N2H4. The probe, which has a donor–π-acceptor (D–π–A)-type structure, is water soluble, and it can be utilized to selectively detect N2H4 in both colorimetric and ratiometric mode. Furthermore, the probe is able to differentiate hydrazine form other organic amines and can be used to detect hydrazine gas, and for imaging A549 cells and zebrafish.

poly (ethylene glycol) (PEG) or oligo (ethylene glycol) (OEG) into drug molecules can enhance their water solubility and improve their biocompatibility.32-37 Very recently, Wu and Zeng’s group developed a water soluble fluorescent probe for the detection of leucine aminopeptidase (LAP) using above strategy.32 We also envisioned that introducing a OEG group, which compared with PEG group is more synthetically feasible, into a N2H4 probe would also promote the same effects. Molecules having D–π–A type structures are wildly used in photoelectric functional materials that possess good light absorption and emission properties.38 We have used these properties to design a new hydrazine probe that incorporates a donor–π-acceptor (D–π–A) type fluorophore containing a coumarin core (Figure 1), linked to an electron donating N, N-diethylamine group at the 7-position and conjugated cyano and a OEG tethered pyridinium electron withdrawing groups at the 3-position. We surmised that the presence of this strong electron push-pull system would enable the probe to fluoresce at wavelengths in the far-red region. Furthermore, owing to the electrophilic nature of the moiety at C-3, this substance we anticipated that it would be highly reactive with the potently nucleophilic hydrazine bringing about removal of the C-3 electron withdrawing moiety. The results of this effort described below showed that the probe has good water solubility and

INTRODUCTION Hydrazine (N2H4) induced toxicity (including hepatotoxicity, neurotoxicity and mutagenicity) causes serious harms to the health of humans. Owing to these effects, N2H4 is classified as a human carcinogen with a low threshold limit value (TLV) of 10 ppb.1, 2 Despite its high toxicity, N2H4 is widely used as a reagent in organic synthesis and as a component of rocket fuel.3-5 Thus, the detection of N2H4 in vitro and vivo has been attracted great attention, especially in the context of fluorescent probes which have advantageous features including high sensitivity, simplicity of operation and capability of visualization.6-13 Although fluorescent N2H4 probes that display high selectivity and sensitivity have been devised,14-31 only a few operate in purely aqueous media and, as a result, require the use of organic cosolvents which hinders their use in analysis of real samples. In addition, most of the current N2H4 probes are either “off-on” or “onoff” types, in which it is difficult to eliminate interference from background fluorescence commonly occurring in vitro and in vivo systems. Furthermore, some probes are unable to differentiate hydrazine form other organic amines. Thus, an urgent need exists to develop water soluble and ratiometric type fluorescent probes for the detection of N2H4. It is well-known that incorporation of hydrophilic 1

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were incubated with N2H4 (500 μM) for different times (30, 60, 90 min) at 37 °C. At last, the A549 cells were image din laser scanning confocal microscopy (Olympus FV1000MP, Japan).

that it can be used to detect N2H4 with colorimetric and ratiometric response in 99.9% PBS solution; in addition, the probe displayed good selectivity that is able to differentiate hydrazine form other organic amines. Furthermore, it can be successfully used for N2H4 gas detection, cells and zebrafish imaging.

Zebrafish imaging Zebrafishes post-fertilization were

obtained from Eze-Rinka Company (Nanjing, China). All zebrafishes were cultured in 50 mL medium which including1-phenyl-2-thiourea (PTU) in a Beaker for 72 h at 30 °C. Zebrafishes were then incubated with a solution of the probe (10 μM) for 30 min. After washing the medium by using PBS buffer three times, zebrafishes were imaged in red and blue channels. Then, the zebrafishes were treated with 100 μM N2H4, and their fluorescence images were recorded after different times (30, 60 and 90 min).

Figure 1 Design and sensing mechanism of new N2H4 probe

EXPERIMENTAL SECTION Materials and Chemicals All chemical reagents were commercially available and treated with standard methods before use unless otherwise noted. Silica gel column chromatography (CC) (silica gel 200-300 mesh) was obtained from Qingdao Makall Group Co., Ltd, Qingdao, China. 1H NMR and 13C NMR spectra were recorded in DMSO-d6 on a 600 MHz Varian VNMRS. Spectrometer and resonances were given in ppm relative to tetramethylsilane (TMS). The following abbreviations were used to designate chemical shift multiplicities: s = singlet, d = doublet, t = triplet, m = multiplet, br = broad. Highresolution mass spectra (HRMS) were acquired in positive mode on a MALDI SYNAPT G2 high-definition mass spectrometer (Waters, Milford, MA).

MTT assay The A549 cells were seeded in a 96-well plate

for 24 h and then treated with different concentrations of the probe (0, 5, 10, 20, 50 μM) in the culture medium for 24 h at 37 °C. After removing the culture medium, the A549 cells were incubated with 3-(4, 5-dimethylthiazol-2yl)-3, 5-diphenytetrazoliumbromide (MTT) solution for 4 h. Then, 200 μL DMSO was added to the cell sample after removal of the MTT solution. The absorbance was measured at 495 nm with a microplate reader (SpectraMax M5, Molecular Devices).

RESULT AND DISCUSSION Synthesis The probe was synthesized by using 5 step’s reactions that take place in moderate yields. The final probe was purified by using silica column chromatography and characterized by using 1H NMR and 13C NMR spectroscopy, and high-resolution mass spectrometry. The spectrums were attached in Figure S8 –S14, and the synthetic route is shown in Scheme 1. Compound 1, 2 and 3 were prepared using the methods described in reference 41.

Determination of Detection Limit The detection limit (DL) was calculated according to below formula： Sδ

DL = Nk (1) S/N is signal to noise ratio, δ is standard deviation of the blank, k is the slope of the regression line.39

CHO N

Determination of Quantum yield The Quantum yield was

OH

O

O O

HCl

O

O EtOH, reflux

N

O

Reflux

O

CHO

POCl3, DMF N

1

O

O

N

60oC

2

O

O

3

calculated according to below formula：

N EtOH CN

Fs Ac Φs = Φc (2) Fc As

N

N

O

O

+

O

O

O

O

CN

N2H4 Probe

Fs and Fc are fluorescence integral area of sample and reference substance respectively. As and Ac are absorbance of sample and reference substance respectively. Φc is the

O

O

ACN, reflux

N Br N

O

O

CN

4

Scheme 1 Synthetic route of N2H4 probe

Synthesis of 3 Anhydrous POCl3 (5 mL) and DMF (5 mL) were mixed in 50 mL round-bottomed flask and stirred for 30 min at room temperature under the protection of N2, subsequently, a solution of compound 2 (1.57g, 7mmol) were dropped to above mixture, and stirred at 60 oC overnight. After that, the reaction mixture was poured into ice water and kept stirring, and NaHCO3 powder was used to adjust the pH to 7. The precipitated solid was filtered and dried under vacuum to get compound 3 as a yellow solid (0.83 g, 49%).41 1H NMR (600 MHz, DMSO-d6) δ: 9.90 (s, 1H), 8.39 (s, 1H), 7.66 (d, J = 9.0 Hz, 1H), 6.836.81 (dd, J = 3.0 Hz, J = 9.6 Hz, 1H), 6.59 (d, J = 2.4 Hz,

quantum yield of reference substance (using fluorescein (Φ= 0.98, 0.1 M NaOH) as a reference).40 Cell Culture and imaging A549 cells were cultured in

DMEM medium supplemented with 10% (v/v) fetal bovine serum and 1% (v/v) penicillin/streptomycin on a cell culture flask in the atmosphere of 5% CO2 at 37 °C. The A549 cells were seeded in a 6-well plate for 18 h. After that, cells were incubated with a solution of the probe (10 μM) for 30 min. The A549 cells were imaged in both red channel and green channel after washing three times with PBS to remove the remaining probe. Then, the A549 cells 2


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1H), 3.43 (q, J = 7.2 Hz, 4H), 1.16 (t, J = 7.2 Hz, 6H). GCMS calc: 245.28, found: 245.44.

350

(b)

300 250 F.I.

Synthesis of 4 To a solution of 3 (6 mmol, 1.48 g) and 2(pyridin-4-yl) acetonitrile (7 mmol, 0.83 g) in 20 mL anhydrous EtOH were added 10 drops of piperidine. The mixture was kept at 60 oC for 6 h. The formed precipitate was separated by filtration and washed with cold EtOH to yiled 4 as brown solid (1.43 g, yield 68%). The solid is not fully soluble in CDCl3, DMSO, CD3CN and MeOH, thus it cannot be characterized using NMR and was directly used for the next step. GC-MS calc: 345.40, found: 345.53. IR (neat): 2973, 2214, 1707, 1610, 1560, 1507, 1350, 1260, 1131 cm-1.

600

0 eq.

UV and Fluorescence Response of Probe to N2H4 A solution of the probe (10 μM) in pure PBS (10 mM, pH 7.4) displays a UV-visible absorption maximum at 560 nm (Figure S1). Addition of increasing concentrations of N2H4 (0-100 μM) causes a decrease in the intensity of the absorption maximum at 560 nm with concomitant formation of a new peak at 450 nm (Figure S1b). In contrast, other substances including a diverse array of amines (methylamine ammonia, morpholine, N, Ndiisopropylethylamine (DIPEA), aniline, isoniazid and urea), biothiols (Cys, Hcy and GSH) and metal ions (Zn2+, Cd2+ and Hg2+) did not promote observable changes in the absorption spectrum of the probe except H2S (Figure S1a). The light absorption changes caused by addition of N2H4 can be clearly observed in the form of a pink to yellow color change by using the naked (Figure S3).

Next, the fluorescence response of probe (10 μM in 10 mM PBS, pH 7.4) to N2H4 (0-100 μM) was assessed. As can be seen by viewing the emission spectra shown in Figure 2a and 2b, the intensity of the fluorescence maximum of the probe at 640 nm (excitation at 560 nm, red channel) was decreased gradually after addition of N2H4 (0-100 μM). At the same time, a new emission band arises at 510 nm (Φ: 0.012 to 0.24) when 450 nm excitation (green channel) is utilized. Addition of other substances, except H2S, does not induce fluorescent changes in both of red and green signal channels (Figure S2a and S2b). It is worth noting that H2S induced fluorescence quenching in red channel (Figure S2a), however, no fluorescence enhancement was observed in green channel compared with hydrazine (Figure S2b). Thus, this probe still displays high selectivity to hydrazine between the ratio of fluorescence intensity at 510 and 640 nm (Figure 3). Competition experiment was performed, and the results indicate the ratio of fluorescence intensity at 510 and 640 nm (I510/I640) can increase obviously after addition of hydrazine when coexisting with other analytes, comparing with them, aniline, H2S and Hg2+ induced lower enhancement than other analytes (Figure S4). Moreover, the results of a time course study show that both channels can reach the platform within 1 hour while the probe is stable in aqueous solution and no fluorescence change was observed in both channels (Figure S6).

(a)

0 eq.

10 eq.

100 0

150

0 460 480 500 520 540 560 580 600 Wavelength/nm Figure 2 Fluorescence response (a and b) of the probe (10 μM, in 10 mM PBS, pH 7.4) to N2H4 (0-100 μM). Red Channel (a): λex = 560 nm, λem = 640 nm, slit width: 5, 5 nm. Green Channel (b): λex = 450 nm, λem = 510 nm, slit width: 1.5, 3 nm.

300 200

200

50

500 400

10 eq.

100

Synthesis of the N2H4 probe A solution of 4 (1.2mmol, 0.42 g) and 1-bromo-2-(2-(2-methoxyethoxy) ethoxy) ethane (3.6 mmol, 0.82 g) in 20 mL acetonitrile was stirred at reflux overnight. The solvent was removed under reduced pressure, giving a residue that was subjected to silica column chromatography to yield the probe as fuchsia solid (157 mg, yield 23%). 1H NMR (600 MHz, DMSO-d6) δ: 8.98 (d, J = 6.6 Hz, 2H), 8.86 (s, 1H), 8.41 (s, 1H), 8.32 (d, J = 7.2 Hz, 2H), 7.67 (d, J = 9.0 Hz, 1H), 6.90-6.88 (dd, J = 2.4 Hz, J = 9.0 Hz, 1H), 6.69 (d, J = 2.4 Hz, 1H), 4.78 (t, J = 4.2 Hz, 2H), 3.91 (t, J = 4.8 Hz, 2H), 3.58-3.55 (m, 6H), 3.48-3.44 (m, 4H), 3.41-3.39 (m, 2H), 3.24 (s, 3H), 1.18 (t, J = 6.6 Hz, 6H). 13C NMR (150 MHz, DMSO-d6) δ: 160.52, 158.11, 154.21, 150.22, 145.68, 145.32, 144.81, 133.08, 122.73, 116.96, 111.57, 111.19, 108.86, 101.98, 97.25, 71.75, 70.13, 70.01, 69.99, 69.10, 59.88, 58.58, 45.27, 12.94. HRMS [M+H+] calc: 492.2493, found 492.24888.

F.I.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

600 620 640 660 680 700 720 Wavelength (nm)

3


Analytical Chemistry The observations described above show that the probe displays good selectivity and sensitivity in response to N2H4. A good linear relationship (R2 = 0.989) was found to exist between the ratio of fluorescence intensity at 510 and 640 nm (I510/I640) and hydrazine concentrations in the range of 0-35 μM (Figure S5) and the probe has a detection limit of 0.38 μM (S/N = 3, Figure 4). The effect of pH on operation of the probe was also investigated (Figure S7). The results demonstrate that the probe is stable in the pH range of 4-8. Because base enhances the rates of reactions in which N2H4 serves as a nucleophile, the I510/I640 ratio was observed to increase. Figure 5 NMR titration of probe with N2H4

Hydrazine gas detection Because hydrazine in its gaseous form is dangerous to humans, the ability of the probe for N2H4 gas detection was evaluated. For this purpose, TLC plates were soaked in a stock solution of the probe (1.0 mM) and dried. The plates were then placed on the top of 10 mL glass bottles, containing 1 mL of different concentrations of N2H4, for 15 min at room temperature. As can be seen by viewing the images shown in Figure 6, color and fluorescence changes can be clearly observed using the naked eye and a hand held UV lamp (λex= 365 nm) even when the probe impregnated plate is exposed to hydrazine vapor arising from a solution at the low concentration of 0.1%. Notably, no color or fluorescence change occurs when a probe containing plate is exposed to air and water vapor.

12 10 I510/I640

8 6 4 2 0

0 1 2 3 4 5 6 7 8 9 10111213141516 Analytes

Figure 3 Selectivity of probe (10 μM) after addition of different analytes (100 μM); 1. Probe; 2. N2H4; 3. CH3NH2; 4. NH4OH; 5. Morpholine; 6. DIPEA; 7. Aniline; 8. Isoniazid; 9. Cys; 10. Hcy; 11. GSH; 12. H2S; 13. Urea; 14. Zn2+; 15. Hg2+; 16. Cd2+. 0.12

Equation

y = a + b*x

Adj. R-Squar

0.98911 Value

0.09 I510/I640

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Standard Erro

B

Intercept

0.0324

0.00106

B

Slope

0.0129

4.75455E-4

0.06

Figure 6 Visual and fluorescence changes of probe (1.0 mM)impregnated TLC plates after exposure hydrazine gas under UV lamp (λ = 365 nm)

0.03 0.00

0

2 4 Concentrations (10-6 M)

Bioimaging applications. An evaluation of the ability to utilize the probe to image N2H4 in A549 cell was carried out by using a fluorescence confocal microscope. In the experiments, the cells were stained using a 10 μM solution of the probe for 30 min, washed with PBS, and then exposed to 500 μM N2H4. Inspection of the microscope images displayed in Figures 7, show the cells exhibit strong red fluorescence (λem = 570-625 nm) in red channel (λex = 559 nm) and very week fluorescence (λem = 500-545 nm) in green channel (λex = 488 nm). However, after 30-90 min incubation, the intensity of fluorescence in red channel emanating from the cells decreases, while emission in green channel is enhanced can be observed. Moreover, a standard 3-(4, 5-dimethylthiazol-2yl)-2, 5diphenyltetrazolium bromide (MTT) assay was conducted with A549 cells treated with different concentrations of the probe (5, 10, 20 and 50 μM) (Figure S16). The results show that, even after incubation with 50 μM probe for 24 h, more

6

Figure 4 Linear relationship between the fluorescence intensity ratio of I510/I640 and low N2H4 concentration range (0-5.0 μM)

Sensing mechanism In order to confirm the sensing mechanism, the NMR titration was performed. After addition of 20 equivalences of hydrazine, the chemical shift of proton 3 at high filed disappeared, and a new single peak at lower filed (7.81 ppm) produced indicates the formation of imine compound (proton 3’). Furthermore, the highresolution mass spectrum of a mixture of the probe and N2H4, incubated for 1 h, contained a strong peak at m/z 260.14002 (Figure S15). This mass matches the molecular weight of the product (calcd for M+H+: 260.13935) expected to form in reaction of the probe with N2H4 shown in Figure 1. 4


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Analytical Chemistry than 90% of the cells survive, indicating that the probe has a low cytotoxicity. It is well known that N2H4 is a serious water pollutant that threatens fish life. In order to determine if the probe can be used in a real-life application, it was employed to image N2H4 in zebrafish. As the images in Figure 8 show, red fluorescence arises from zebrafish after they are incubated a solution of the probe (10 μM) and then treated with 100 μM of N2H4. Green fluorescence from the zebrafish appears within 30 min following incubation while the red fluorescence diminishes gradually. The ratiometric changes in both red and green channels can be clearly observed after 90 min using a fluorescence microscope. The results demonstrate clearly that the new probe can be utilized for real-time and ratiometric imaging N2H4 in cells and zebrafish.

Figure 8 The fluorescence images of probe in zebrafish following addition of N2H4

CONCLUSION In the effort described above, we designed and synthesized a novel OEG containing fluorescent probe for the detection of hydrazine. The probe displayed below superiorities over the majority of reported fluorescent N2H4 probe: (ⅰ) the probe can detect N2H4 in aqueous solution (99.9% PBS) without the use of large amount of organic cosolvent. (ⅱ) It possesses good selectivity to N2H4, other organic amines (including methylamine, ammonia, morpholine, N, Ndiisopropylethylamine (DIPEA), aniline) do not cause any UV or fluorescence response. (ⅲ) The ratiometric response can overcome the drawbacks of the most of current N2H4 probes which are either “off-on” or “on-off” types (Table S-1). Furthermore, the successful images of probe in A549 cells and zebrafish indicate its potential values of further study in various biological samples. ASSOCIATED CONTENT AUTHOR INFORMATION

Figure 7 Fluorescence images of A549 cells incubated with probe in the presence and absence of N2H4 in the red channel (λex = 559 nm, λem = 570-625 nm) and green channel (λex = 488 nm, λem = 500-545nm). Scale bar, 30 μm.

Corresponding Author * [email protected]; fax: 82-2-3277-2385 [email protected]; fax: 86-27-87283690 [email protected]; fax: 86-27-87283690

Author Contributions 5



The manuscript was written through contributions of all authors. / All authors have given approval to the final version of the manuscript. / ‡These authors contributed equally. Li, J. and Cui, Y. synthesized the probe and determined the UV/Vis and fluorescence properties in aqueous solution. Bi, C.; Feng, S.; Yu, F. and Yuan, E. repeated the experiment. Sun, Q. and Hu, Z. performed cells and zebrafish experiment. Li, J. wrote the paper, Yoon. J and Wei. D guided the project and revised the paper.

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Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT Yoon, J. thanks the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (No. 2012R1A3A2048814). Li, J. thanks the National Natural Science Foundation of China (No. 21806048) and the Fundamental Research Funds for the Central Universities (No. 2662018QD009); Wei, D. thanks the National Natural Science Foundation of China (No. 21502060, 31672558, and 21732002), Huazhong Agricultural University Scientific & Technological Self-innovation Foundation (No. 2015RC013, 2662017PY113, and 2662015PY208), Open fund of The State Key Laboratory of Bio-organic and Natural Products Chemistry, CAS (SKLBNPC16343), Open fund of Beijing National Laboratory. Sun, Q. thanks the National Natural Science Foundation of China (No. 21804102) and Provincial Natural Science Foundation of China (No. 2017CFB222）

Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Including 1H NMR, 13C NMR, IR, GC-MS and HRMS spectrums, UV absorbance and fluorescence spectrums, time and pH dependence, MTT assay and table S-1. The Supporting Information is available free of charge on the ACS Publications website.

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Oligo(ethylene glycol)-Functionalized Ratiometric Fluorescent Probe

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