A highly sensitive and selective off–on fluorescent chemosensor for hydrazine based on coumarin β-diketone

Article abstract of DOI:10.1016/j.saa.2017.06.062

A coumarin-based sensor C1, namely 3-acetoacetylcoumarin was designed, synthesized and applied for hydrazine detection. Hydrazinolysis of the chemosensor gives a fluorescent coumarin-pyrazole product C1???N₂H₄ [3-(3-methyl-1H-pyrazol-5-yl)coumarin], and thus resulting in a prominent fluorescence off-on response toward hydrazine under physiological conditions. The probe is highly selective toward hydrazine over cations, anions and other biologically/environmentally abundant analytes. The detection limit of the probe is 3.2?ppb. The sensing mechanism was supported by ¹H NMR, IR, MS and DFT calculation. The application of the fluorescent probe in monitoring intracellular hydrazine in glioma cell line U251 was also demonstrated.

Full text of DOI:10.1016/j.saa.2017.06.062

Accepted Manuscript
A highly sensitive and selective off–on fluorescent chemosensor
for hydrazine based on coumarin β-diketone
Wei-Na Wu, Hao Wu, Yuan Wang, Xian-Jie Mao, Xiao-Lei Zhao,
Zhou-Qing Xu, Yun-Chang Fan, Zhi-Hong Xu
PII:
DOI:
Reference:
S1386-1425(17)30542-5
doi: 10.1016/j.saa.2017.06.062
SAA 15278
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
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Revised date:
Accepted date:
8 April 2017
23 June 2017
30 June 2017
Please cite this article as: Wei-Na Wu, Hao Wu, Yuan Wang, Xian-Jie Mao, Xiao-Lei
Zhao, Zhou-Qing Xu, Yun-Chang Fan, Zhi-Hong Xu , A highly sensitive and selective
off–on fluorescent chemosensor for hydrazine based on coumarin β-diketone,
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2017), doi:
10.1016/j.saa.2017.06.062
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ACCEPTED MANUSCRIPT
A highly sensitive and selective off–on fluorescent chemosensor for hydrazine
based on coumarin -diketone
Wei-Na Wua, Hao Wua, Yuan Wanga,*, Xian-Jie Maoa, Xiao-Lei Zhaoa,*, Zhou-Qing Xua, Yun-Chang
Fana, Zhi-Hong Xub,*
a College of Chemistry and Chemical Engineering, Henan Polytechnic University, Jiaozuo 454000, P.
R. China
b Key Laboratory of Chemo/Biosensing and Detection, School of Chemistry and Chemical Engineering,
Xuchang University, 461000, PR China
*Corresponding author. Tel.: +86 391 3987818; Fax: +86 391 3987811; e-mail:
wangyuan08@hpu.edu.cn
(Y.
Wang);
zhaoxiaolei@hpu.edu.cn
(X.-L.
Zhao);
xuzhihong1980@yahoo.com (Z.-H. Xu).
Abstract:
A coumarin-based sensor C1, namely 3-acetoacetylcoumarin was designed, synthesized and applied
for hydrazine detection. Hydrazinolysis of the chemosensor gives a fluorescent coumarin-pyrazole
product C1-N2H4 [3-(3-methyl-1H-pyrazol-5-yl)coumarin], and thus resulting in a prominent
fluorescence off-on response toward hydrazine under physiological conditions. The probe is highly
selective toward hydrazine over cations, anions and other biologically/environmentally abundant
1
analytes. The detection limit of the probe is 3.2 ppb. The sensing mechanism was supported by H
NMR, IR, MS and DFT calculation. The application of the fluorescent probe in monitoring intracellular
hydrazine in glioma cell line U251 was also demonstrated.
Key word: Fluorescent probe; coumarin; hydrazine; -diketone; cell imaging.
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1. Introduction
The highly reactive nature of hydrazine (N2H4) has rendered its wide use in many fields including
industry, agriculture, pharmacy, military, and aerospace [1]. However, hydrazine exhibits considerable
toxic effects for human, which include irritation to the eyes and skin, damage to the liver, kidneys, and
the central nervous system [2]. The U.S. Environmental Protection Agency (EPA) has identified
hydrazine as a probable human carcinogen with a low threshold limit value (TLV) of 10 ppb [3].
Therefore, it is highly desirable to develop reliable and sensitive analytical methods for hydrazine
detection.
Traditional quantitative detection approaches for hydrazine detection employed several analytical
techniques, such as chromatography-mass spectrometry, titrimetry and electrochemical methods, etc.
But these methods involved special equipment, complicated sample preparation, consuming time for
real-time and on-site analysis, as well as destruction of tissues or cells, which were not suitable to
detect hydrazine in living systems [4]. By contrast, fluorescence spectroscopy is the most frequently
used modes due to its high sensitivity and selectivity, spatiotemporal resolution and noninvasiveness
[5].
Generally, fluorescent probes have been designed based on the nucleophilicity of hydrazine, which
reacts with carboxylate [6-12], -ketone-carboxylate [2, 13, 14], -bromobutyrate [15-18], methylene
malononitrile [19-24], methylene ethyl cyanoacetate [25] or phthalimide [26-29] moieties of the probes,
to release fluorophore or give fluorescent substitution derivatives. However, there is still a question
concerning the selective detection of hydrazine over other competitive amines, such as ammonia,
diamines and relative bio-amines, which are crucial to effectively detect hydrazine in complex cellular
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milieus [13, 26]. Recently, it is demonstrated that trifluoroacetyl acetonate deritives could act as
fluorescent probes towards hydrazine via cyclization mechanism, without interference of amine species
[30-32]. Nevertheless, most of them suffer from several limits, such as utilizing in much higher organic
cosolvent (>70%, v:v) [30, 31] or low pH (pH<5) condition [30, 32], which would limit their
application in physiological condition. Furthermore, some of them usually require complicated
syntheses involving severe reaction conditions and expensive chemicals [30].
Therefore, we report here the potential properties as fluorescent hydrazine sensor of a coumarin
-diketone deritive C1 (Scheme 1), which could be easily prepared by one-step condensation of
salicylaldehyde and 4-hydroxy-6-methyl-2-pyrone with high yield, and most importantly, without any
further purification. The probe C1 functions by cyclization reaction to form a fluorescent
coumarin-pyrazole product C1-N2H4. In addition, the application of the fluorescent probe in monitoring
N2H4 in glioma cell line U251 has also been demonstrated.
Insert Scheme 1.
2. Experimental section
2.1. Materials and instrumentation
Solvents and starting materials for syntheses were purchased commercially and used as received.
The melting point of the probe is determined on an XT4-100X microscopic melting point apparatus
1
(made in Beijing, China). Elemental analyses were carried out on an Elemental Vario EL analyzer. H
and 13C NMR spectra is recorded on a Bruker AV400 NMR spectrometer in DMSO-d6 solution. The IR
spectra (=4000–400 cm-1) were determined by the KBr pressed disc method on a Bruker V70 FT-IR
spectrophotometer. The UV spectra were recorded on a Purkinje General TU-1800 spectrophotometer.
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Fluorescence spectra were determined on a Varian CARY Eclipse spectrophotometer, in the
measurements of emission and excitation spectra the pass width is 10 nm. ESI-MS spectra were
obtained on a Bruker Daltonics Esquire 6000 mass spectrometer. Fluorescent images were taken on
Zeiss Leica inverted epifluorescence/reflectance laser scanning confocal microscope. The X-ray
diffraction measurement for C1 was performed on a Bruker SMART APEX II CCD diffractometer
equipped with a graphite monochromatized MoK radiation (λ = 0.71073 Å) by using φ-ω scan mode.
Semi-empirical absorption correction was applied to the intensity data using the SADABS program
[33]. The structure was solved by direct methods and refined by full matrix least-square on F2 using the
SHELXTL-97 program [34]. All non-hydrogen atoms were refined anisotropically. All H atoms were
positioned geometrically and refined using a riding model.
2.2. Synthesis of C1 and C1-N2H4
Compound C1 was synthesized in high yield by facile one-step condensation of salicylaldehyde and
4-hydroxy-6-methyl-2-pyrone in ethanol according to the literature method [35]. Yellow needles of C1
suitable for X-ray diffraction analysis were obtained during the cooling process of the reaction solution.
Yield 86%. M.p. 147-148 ℃. Anal. Calc. for C13H10O4: C, 67.82; H, 4.38. Found: C, 67.66; H, 4.46%.
FT-IR (cm-1): v(C=O)
1730, v(C=O) 1609, v(C=C) 1586. 1H NMR (400 MHz, DMSO-d6) (Fig.
coumarin
S1, ESI),δ(ppm): 16.06 (s, 1H, OH), 8.84/8.77 (s, 1H, H7), 7.97-8.02 (m, 1H, H4), 7.74-7.79 (m, 1H,
H2), 7.42-7.50 (m, 2H, H1,3), 6.92/4.18 (s, 1H, H11), 2.24/2.26 (s, 3H, H13). 13C NMR (400 MHz,
DMSO-d6) (Fig. S2, ESI),δ(ppm): 203.78/199.55 (C12), 192.22/173.49 (C10), 159.28/157.85 (C9),
155.12/154.43 (C6), 148.36/146.59 (C8), 135.38/134.96 (C4), 131.56/130.86 (C2), 125.61/125.46 (C7),
123.51/120.21 (C3), 118.80/118.60 (C5), 116.64/116.54 (C1), 101.48/56.94 (C11), 30.89/27.52 (C13).
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ESI-MS: m/z = 231.0789 for [M+H]+; 253.0665 for [M+Na]+. Crystal data for C13H10O4: crystal size:
0.20×0.18×0.16 mm, triclinic, space group P-1. a = 6.766(5) Å, b = 8.886(6) Å, c = 9.994(8) Å,  =
82.316(14)°,  = 76.132(13)°,  = 71.315(12)°, V = 551.5(7) Å3, Z = 2, T = 296(2) K,  = 2.10-25.00°,
2764 reflections measured, 1911 unique (Rint= 0.0163). Final residual for 155 parameters and 1911
reflections with I>2σ(I): R1 = 0.0543, wR2= 0.1580 and GOF = 1.034 (Fig. 1). CCDC：1540246.
Insert Figure 1.
C1 (230 mg, 1 mmol) was added to ethanol (20 mL) and stirred at room temperature to dissolve it to
form a solution. After sometime, 85% hydrazine hydrate (76 mg, 1 mmol) was added to it. The mixture
was stirred for 30 mins, filtrated and the filtrate was kept for one day. The large amount of colorless
neddles of C1-N2H4 were filtered, washed with ethanol, and then dried in air. Yield 69%. M.p.
181-182 ℃. Anal. Calc. for C13H10N2O2: C, 69.02; H, 4.46; N, 12.38. Found: C, 69.13; H, 4.56; N,
1
12.19 %. FT-IR (cm-1): v(C=O)
1721, v(C=N)pyrazole 1609, v(C=C)pyrazole 1579. H NMR (400
coumarin
MHz, DMSO-d6) (Fig. S3, ESI),δ(ppm): 12.89 (s, 1H, NH), 8.58 (s, 1H, H7’), 7.86-7.89 (m, 1H, H4’),
7.61-7.65 (m, 1H, H2’), 7.38-7.47 (m, 2H, H1’, 3’), 6.72 (s, 1H, H11’), 2.32 (s, 3H, H13’). 13C NMR (400
MHz, DMSO-d6) (Fig. S4, ESI),δ(ppm): 158.95 (C9’), 152.67 (C6’), 144.49 (C7’), 139.01 (C10’),
136.47 (C12’), 131.28 (C8’), 128.58 (C2’), 124.56 (C4’), 120.52 (C3’), 119.29 (C5’), 115.79 (C1’),
104.91/99.49 (C11’), 10.34 (C13’). ESI-MS: m/z = 227.0822 for [M+H]+.
2.3. General UV–vis and fluorescence spectra measurements
The spectral analyses were accomplished in buffered EtOH/HEPES (10 mM, pH = 7.40, 1/1, v/v)
solution at room temperature. The concentration of the probe C1 for UV-vis and fluorescence
measurement was 10 M. Metal ions (as their chloride or nitrate salts), anions (as their sodium salts),
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hydrazine hydrate, ammonia, hydroxylamine (as its chloride), urea, butylamine, ethylenediamine and
other amino acids in deionized water were used for the sensing behavior experiment. UV-vis and
fluorescence spectrophotometric titrations were conducted directly in 2 mL cuvette by successive
addition of corresponding chemical reagent using a microliter syringe. Upon addition of every aliquot,
the solution was well mixed then the spectrum was measured.
3. Results and Discussions
3.1. Selectivity of C1 to hydrazine and the other analytes
The optical responses of C1 to N2H4 were exemplified through its reaction with hydrazine in
buffered EtOH/HEPES (10 mM, pH = 7.40, 1/1, v/v) solution at room temperature. The probe C1 itself
exhibited negligible fluorescence corresponding to a peak of weak emission intensity at 425 nm on
excitation at 350 nm having low fluorescence quantum yield (Φ= 0.008), using quinine sulfate as
standard (Φc=0.546 in 0.5 mol/L H2SO4) [36]. This is probably due to the strong electron-withdrawing
effect of -diketone group, which could quench the fluorescence of the coumain moiety via a
photoinduced electron transfer (PET) process [37].
Upon addition of N2H4 (5 eq.) to the receptor solution, the peak intensity enhanced dramatically at
425 nm with enhancement of quantum yield (Φ = 0.18). Actually, this large increase in fluorescence
intensity results in the ‘naked eye’ blue emission of the resulting solution under 365 nm UV lamp (Fig.
2). It is suggested that C1 reacted with N2H4 to produce C1-N2H4 (vide infra), in which the PET
process was blocked to induce fluorescence turn-on [37]. The fluorescence intensity at 425 nm of C1
also showed a good linearity as a function of the concentration of N2H4 in the range of 0 to 12.5 μM
(0–1.25 eq., linearly dependent coefficient: R = 0.99141) (Fig. 3). The detection limit of C1 for N2H4 is
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calculated to be 0.1 M (3.2 ppb) using the equation DL= 3Sbi/S (where Sbi is the standard deviation of
blank measurements and S is the slope of the intensity versus sample concentration) [16], which is
much lower than that of TLV (10 ppb) recommended by EPA and WHO [5]. This low detection limit
together with the large fluorescence enhancement and also the increase of quantum yield demonstrates
that C1 is highly sensitive to N2H4.
Insert Figure 2.
Insert Figure 3.
In addition, Job’s plots experiment evaluated from the fluorescent spectra suggests that the binding
of C1 to N2H4 is of 1:1 stoichiometry. When the molecular fraction of N2H4 was close to 0.5, the
fluorescent intensity peaked (Fig. S5, ESI). The association constant (Ka) was determined to be about
6.38×103 M-1, as obtained by fitting the data to the Benesi-Hildebrand expression (Fig. S6, ESI).
On the other hand, the tested primary amines (such as ammonia, n-butylamine, ethylenediamine,
hydroxylamine and urea), amino acids (such as Ala, Asp, Gly, His, Leu, Met, Phe, Trp and Val),
together with other environmental and/or biological abundant metal ions, namely, Ag+, Al3+, Ca2+,
Cd2+, Co2+, Cr3+, Cu2+, Fe3+, Hg2+, K+, Mg2+, Mn2+, Na+, Ni2+, Pb2+ and Zn2+ ions, as well as anions
−
−
−
−
2−
3−
such as AcO−, Br−, Cl−, ClO4 , F−, H2PO4 , HSO3 , I−, NO3 , SO4 and PO4 caused almost no
changes in the emission spectra of probe C1 (Fig. 2). Furthermore, probe C1 can respond to N2H4 in
the presence of all of these primary amines and ions in the identical system (Fig. S7, ESI). Thus, it is
corroborated that probe C1 can detect N2H4 with satisfied selectivity.
3.2. Reaction mechanism of C1 with N2H4
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To explore the reaction mechanism of C1 with N2H4, the ESI-MS analysis (Fig. 4) was employed.
As the results show, a new peak at m/z 227.0890 corresponding to the C1-N2H4 was observed upon the
addition of N2H4 to the EtOH solution of C1 (C1+N2H4). Fig. 5 shows the 1H NMR spectral of C1 with
different amount of N2H4 (0-1 eq.). Upon the gradual addition of N2H4, the changes in the chemical
shift for the protons of the coumarin core of C1 were found. Meanwhile, the main peaks of coumarin
moiety of C1-N2H4 (eg. H1’-4’, H7’, H11’) are observed when the addition of N2H4 exceeding 0.5 eq. The
IR spectrum of C1+N2H4 (Fig. 6) presents three absorption bands at 1721, 1609 and 1579 cm-1, which
should be assigned to the vibrations of C=O, C=N(pyrazole) and C=C(pyrazole) of C1-N2H4,
respectively. All findings established that there exists C1-N2H4 in the C1+N2H4 system.
On the other hand, the UV-vis absorbance (Fig. S8, ESI) and fluorescence spectra (Fig. S9, ESI) of
C1, C1+N2H4 and C1-N2H4 were also record in the optimized experimental condition. C1 and
C1-N2H4 alone exhibited strong absorption peaks at 354 and 334 nm, respectively, which should be
assigned to the coumarin moiety. After adding N2H4 to the C1 solution, the absorption peak decreased
clearly, while the absorption ratio A334/A354 increased from 0.82 to 0.90, probably due to the formation
of C1-N2H4. In addition, the fluorescent emission spectra of C1+N2H4 is quite similar as that of
C1-N2H4, which also supports the above conclusion. Therefore, it is suggested that the probe C1
functions through the cyclization mechanism, as illustrated in Scheme 2.
Insert Figure 4.
Insert Figure 5.
Insert Figure 6.
Insert Scheme 2.
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To further understand the change of the photophysical properties, density functional theory (DFT)
calculations of the energy gaps between HOMO (the highest occupied molecular orbitals) and LUMO
(the lowest unoccupied molecular orbitals) of C1 and C1-N2H4 were performed [38]. As shown in Fig.
7, in each of C1 and C1-N2H4, the HOMO are located on almost the whole molecule, indicating the
charge transfer between the -diketone/pyrazole and coumarin moieties. Comparing the HOMO of C1
and C1-N2H4, it is found that -diketone group may have higher charge transfer efficiency with
coumarin core in C1 than pyrazole ring in C1-N2H4. Therefore, electron-withdrawing-diketone group
could totally quench the fluorescence of the coumain moiety through PET process. By contrast, in the
case of C1-N2H4, the electron-donating group pyrazole inhibits such procedure, and thus recoveries the
coumarin fluorescene emission. In addition, the energy gap between HOMO and LUMO of C1-N2H4
was calculated to be 3.17 eV, which is similar as that of C1 (3.18 eV), suggesting the similar
absorption peaks of both compounds.
Insert Figure 7.
3.3. Kinetic assay and effect of environmental factors
The pH value is the primary factor that affects the response of the chemosensors [5, 16, 31]. The
spectrum response of C1 (10 M) with or without N2H4 (1 eq.) in the buffer solutions at different pH
values was evaluated at room temperature. As shown in Fig. S10, ESI, the fluorescence intensities of
both C1 and C1+N2H4 show little dependence on pH in a wide range from 4.0 to 8.0. This is the unique
feature of the probe C1 when compared to some known fluorescent N2H4 probes, which must be used
under acid condition (pH<5) [7, 30]. Therefore, the physiological pH value 7.40 was chosen as the
optimum experimental condition.
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A short response time for the chemosensor is as important as a high sensitivity and selectivity. To
monitor the target metal ions in real time, the time courses of the response to fluorescence changes
between C1 (10 M) with the amount (1 equiv.) of hydrazine were investigated. As shown in Fig. S11,
ESI, the fluorescence response of C1 with N2H4 was taken in less than 20 min, which was a
significantly faster analysis time for hydrazine detection in comparison to the flavonoid-based (about
60 mins) [8] and coumarin-benzopyrylium probes (about 120 mins) [4]. In addition, the cyclization
reaction is reasonable to be a first order process with rate constant being 6.89×10-4 s-1, since ln[F425
has a significant linear correlation with t (Fig. S12, ESI).
]
3.4. Fluorescence imaging of intercellular hydrazine
The ability of the fluorescence chemosensor C1 to detect N2H4 in glioma cell line U251 was
examined. The cells were supplemented with C1 (10 μM) in Dulbecco’s modified Eagle’s medium
(DMEM) supplemented with 10% fetal bovine serum for 30 mins at 37℃, leading to none intracellular
fluorescence as determined by laser scanning confocal microscopy (Fig. 8b), and then loaded with 10
μM N2H4 for another 30 mins at 37℃ under the same conditions, whereupon a significant increase in
the fluorescence from the intracellular area was observed (Fig. 8e). The results suggest that the probe
C1 can be used to image intracellular N2H4 in living cells.
Insert Figure 8.
Conclusion
In summary, we have developed an effective “turn-on” coumarin-based fluorescent probe for the
detection of hydrazine, which can be easily prepared by one-step condensation reaction with high yield.
The fluorescent probe performs high selectivity and excellent sensitivity toward hydrazine with a
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detection limit of 3.2 ppb and the coexisting analytes hardly affect the detection. Also, the application
of the fluorescent probe in monitoring hydrazine in glioma cell line U251 was investigated.
Acknowledgements
This work was supported in part by the National Natural Science Foundation of China
(No.21001040), the Joint Program for Fostering Talents of National Natural Science Foundation of
China and Henan Province (U1304202 and U1604124), the Science and Technology Department of
Henan Province (162300410011, 152102210343 and 152102210314), the Education Department of
Henan Province (No.12B150011, 14B150029, 15B150016, 2014GGJS-045 and 15HASTIT002).
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Figure captions
Scheme 1. Synthesis route of the probe C1.
Scheme 2. The proposed reaction mechanism of C1 with hydrazine.
Fig. 1. The crystal structure of C1.
Fig. 2. Fluorescence emission spectra (excited at 350 nm) of 10 M probe C1 in buffered
EtOH/HEPES (10 mM, pH = 7.40, 1/1, v/v) solution with 5 equiv. of hydrazine, ammonia,
n-butylamine, ethylenediamine, hydroxylamine, urea, amino acids (such as Ala, Asp, Gly, His, Leu,
Met, Phe, Trp and Val), cations (Ag+, Al3+, Ca2+, Cd2+, Co2+, Cr3+, Cu2+, Fe3+, Hg2+, K+, Mg2+, Mn2+,
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−
−
−
−
Na+, Ni2+, Pb2+ and Zn2+ ions), anions (AcO−, Br−, Cl−, ClO4 , F−, H2PO4 , HSO3 , I−, NO3 , SO42− and
PO43−) and blank. The insert shows the fluorescence color of C1 with hydrazine.
Fig. 3. Fluorescence emission spectra (excited at 350 nm) of 10 M probe C1 upon the addition of
hydrazine (0–25 equiv.) in buffered EtOH/HEPES (10 mM, pH = 7.40, 1/1, v/v) solution. The inset
shows the normalized response of fluorescence calibration value at 425 nm as a function of hydrazine
concentration (10-250 M).
Fig. 4. Partial MS spectra of C1, C1+N2H4 and C1-N2H4 in EtOH solution.
Fig. 5 Partial of 1H NMR spectra of C1, C1+N2H4 (0-1 eq.) and C1-N2H4.
Fig. 6 Partial of IR spectra of C1, C1+N2H4 and C1-N2H4.
Fig. 7. Optimized structures and HOMO/LUMO of C1 and C1–N2H4 by DFT calculation.
Fig. 8. Confocal fluorescence images of U251 cells: Brightfield (a), confocal fluorescence (b), and
overlay (c) images of U251 cells incubated with 10 M of C1 for 30mins at 37℃; Brightfield (d),
confocal fluorescence (e), and overlay (f) images of U251 cells incubated with 10 M of C1 for 30
mins at 37℃ and then incubated with 10 M N2H4 for another 5 h at 37℃.
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Fig. 1
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Fig. 2
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Fig. 3
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Fig. 4
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Fig. 5
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Fig. 6
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Fig. 7
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Fig. 8
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Scheme 1
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Scheme 2
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Graphical abstract
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Highlights
A coumarin-based probe containing acetyl acetonate moiety was synthesized.
The probe could act as a fluorescent off-on sensor toward hydrazine.
The detection limits of the probe is at the parts per billion level.
Selective fluorescence sensing of hydrazine via cyclization mechanism.
The probe has been applied in monitoring hydrazine in living cell.
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