4-Nitroimidazole-3-hydroxyflavone conjugate as a fluorescent probe for hypoxic cells

Article abstract of DOI:10.1016/j.dyepig.2016.03.019

A novel off-on fluorescent probe for selective detection hypoxia or nitroreductase (NTR), 2-(4-(dimethylamino)phenyl)-3-((1-methyl-4-nitro-1H-imidazol-5-yl)methoxy)-6,8-bis(morpholinomethyl)-4H-chromen-4-one (3-HF-NO₂), was developed using 4-ni

Full text of DOI:10.1016/j.dyepig.2016.03.019

Accepted Manuscript
4-Nitroimidazole-3-hydroxyflavone conjugate as a fluorescent probe for hypoxic cells
Weipei Feng, Yuechai Wang, Shizhu Chen, Chao Wang, Shuxiang Wang, Shenghui
Li, Hongyan Li, Guoqiang Zhou, Jinchao Zhang
PII:
S0143-7208(16)30079-1
10.1016/j.dyepig.2016.03.019
DYPI 5144
DOI:
Reference:
To appear in: Dyes and Pigments
Received Date: 29 January 2016
Revised Date: 6 March 2016
Accepted Date: 11 March 2016
Please cite this article as: Feng W, Wang Y, Chen S, Wang C, Wang S, Li S, Li H, Zhou G, Zhang J, 4-
Nitroimidazole-3-hydroxyflavone conjugate as a fluorescent probe for hypoxic cells, Dyes and Pigments
(2016), doi: 10.1016/j.dyepig.2016.03.019.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
4-Nitroimidazole-3-hydroxyflavone conjugate as a fluorescent probe for
hypoxic cells
Weipei Fenga, Yuechai Wanga, Shizhu Chena, Chao Wanga, Shuxiang Wanga,b , Shenghui Lia,b,
Hongyan Lib, Guoqiang Zhoua,b, and Jinchao Zhanga,b
aKey Laboratory of Chemical Biology of Hebei province, College of Chemistry and Environmental Science, Hebei University, Baoding
071002, China
bKey Laboratory of Medicinal Chemistry and Molecular Diagnosis of Ministry of Education, Hebei University, Baoding 071002, China
ABSTRCT
A novel off-on fluorescent probe for selective detection hypoxia or nitroreductase (NTR),
2-(4-(dimethylamino)phenyl)-3-((1-methyl-4-nitro-1H-imidazol-5-yl)methoxy)-6,8-bis(morpholino
methyl)-4H-chromen-4-one (3-HF-NO2), was developed using 4-nitroimidazole moiety as a
hypoxic trigger and introducing two morpholine groups into 3-HF scaffold at 6,8-position. The
design was based on a NTR-catalyzed reduction of the 4-nitroimidazole moiety in the presence of
reduced nicotinamide adenine dinucleotide (NADH) as an electron donor and followed by the
1,6-rearrangement-elimination and the release of free 3-HF dye. The detection limit (S/N = 3) for
NTR was 63 ng/mL. This probe displayed desired properties such as high selectivity, "Turn-On"
fluorescence response with suitable sensitivity, no cytotoxicity, large Stokes’ shift, and dual
emission. This probe was successfully applied for imaging the hypoxic status of tumor cells (e.g.
HeLa cells). We hope to apply this novel probe in the biomedical research fields for the imaging of
disease-relevant hypoxia.
Key words: 4-nitroimidazole, hypoxic trigger, 3-HF-NO2, off-on fluorescent probe, monitor hypoxic
level
1. Introduction
Hypoxia is an important character of many solid tumors and influences malignant disease
progression, development of metastases, clinical behavior, and response to conventional
treatments [1,2]. The Hypoxic status has been considered an indicator of an adverse prognosis for
solid tumors because it indicates tumor progression toward a more malignant phenotype with
increased metastatic potential and resistance to treatment. Therefore, there has been increasing
interest in developing clinical method which can rapidly, selectively and sensitively detect
characterize the hypoxic tumor cells [3]. To date, many approaches have been developed to
detect hypoxia, including immunostaining [4,5], positron emission tomography [6,7],
single-photon emission computed tomography [8,9], magnetic resonance imaging [10,11],
doppler optical coherence tomography [12], phosphorescence imaging [13,14], and fluorescence
imaging [15~19]. Among these approaches, fluorescence imaging method offers various
advantages, including high sensitivity, high resolution, non-invasiveness, and simple operation.
Corresponding author. Tel/fax: +86 312 507 9482. E-mail: jczhang6970@163.com (J. Zhang).
Corresponding author. Tel/fax: +86 312 507 7373. Email: wsx@hbu.cn (S. Wang).

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In solid tumor, nitroreductase (NTR) is over expressed, and the NTR level is directly
correlated with the Hypoxic status [20~22]. Thus, the detection of the NTR level has been used to
investigate the hypoxic status of a tumor, and some fluorescent probes were developed to detect
hypoxia [23~26]. These fluorescent probes usually utilize nitro aromatics (e.g. pimonidazole,
nitrofuran, nitrobenzene, and 2-nitroimidazole) as exogenous trigger for detection hypoxia
[27~29]. The trigger is a key factor when design an optical probe to detect hypoxia. However, so
far, the hypoxic trigger for the detection of hypoxia is still lacking. Therefore, it is necessary to
develop a hypoxic trigger and fluorescent probe for the detection of hypoxia.
3-Hydroxyflavone (3-HF) derivatives, based on excited-state intramolecular photon-transfer
(ESIPT) process, exhibited excellent optical properties such as a large Stokes shift, dual emission,
good photostability, and reasonable fluorescent quantum yield, which has been used in the study
of molecular interactions in solution or biological systems [30~35]. Modification of the hydroxyl
group of 3-HF can block the ESIPT process and results in quenching of the emission. Conversely,
the regeneration of the free hydroxyl group leads to restore the emission of 3-HF. In view of their
optical properties, some 3-HF derivatives have been applied for the detection of fluoride anion
and cysteine anion [36~38].
In
this
study,
we
developed
an
off-on
fluorescent
probe
2-(4-(dimethylamino)phenyl)-3-((1-methyl-4-nitro-1H-imidazol-5-yl)methoxy)-6,8-bis(morpholino
methyl)-4H-chromen-4-one (3-HF-NO2) using 4-nitroimidazole moiety as a hypoxic trigger for
detecting hypoxic status or NTR level of tumor cells. Under hypoxic conditions, 4-nitroimidazole
moiety is reduced, 3-HF is released by means of rearrangement-elimination, and fluorescence is
restored concomitantly. As we expected, 3-HF-NO2 displayed a high selectivity, sensitivity, quickly
response, no cytotoxicity for the detection of hypoxic status, and could be applied to monitor
hypoxic status in living cells.
2. Experimental section
2.1. Instrumentation and materials
NMR spectra were measured on a Bruker AVIII 600 NMR spectrometer in DMSO-d6.
Electrospray ionization mass spectra (ESI-MS) were recorded in negative mode with a HRMS apex
ultra 7.0T US+. The IR spectra were recorded on a Perkin-Elmer Model-683 spectrophotometer
using KBr pellets. Fluorescence measurements were performed on a Hitachi F-7000 luminescence
spectrophotometer in 10×10 mm quartz cells. A liquid chromatography system from Agilent
Technologies (Agilent, American) was applied to all chromatography tests. The optical density (OD)
was measured on a microplate spectrophotometer (Bio-Rad Model 680, USA). The absorbance
for MTT analysis was recorded on a microplate spectrophotometer (MolecularDevices, VersaMax,
USA). Fluorescence imaging experiments were performed on confocal microscope (Olympus, IX81,
JPN) at λex = 405 nm.
NTR (≥100 units/mg) and reduced nicotinamide adenine dinucleotide (NADH) were purchased
from Sigma-Aldrich. The lyophilized powder of NTR was dissolved in pure water, and the solution
was divided into 20 parts as suitable amounts for daily experiments. The enzyme solutions were
frozen immediately at −20 °C for storage and allowed to thaw before use, which results in no
change of the enzyme activity [29]. Dulbecco's modified Eagle's medium (DMEM), fetal bovine
serum (FBS), trypsin and phosphate-buffer saline (PBS; 137 mmol/L NaCl, 2.7 mmol/L KCl, 4.3

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mmol/L Na2HPO4, 1.4 mmol/L KH2PO4, pH 7.4) were obtained from Gibco (Grand Island, NY, USA).
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), benzyl penicillin and
streptomycin were obtained from Sigma. All other chemicals used were local products of
analytical grade. A stock solution of 3-HF-NO2 (1 mmol/L) was prepared by dissolving an
appropriate amount of 3-HF-NO2 in DMSO. Ultrapure water (over 18 MΩ/cm) was used.
The reagents and solvents were purchased as reagent grade and used without purification. All
reactions were performed under nitrogen or argon and monitored by TLC. The products were
purified by column chromatography on silica gel (200~300 mesh).
2.2. Synthesis and characterization
2.2.1. 3,5-bis(chloromethyl)-2-hydroxyl acetophenone (1)
To a 50 mL round-bottom flask equipped with a magnetic stirrer, 1.24 g paraformaldehyde,
1.16 g 2-hydroxyacetophenone (8.5 mmol) and concentrated hydrochloric acid (10 mL) were
added and heated at 50 ℃ for 10 h. After cooling to room temperature, the mixture was
extracted with dichloromethane. The organic phase was washed with saturated sodium
bicarbonate solution and dried over anhydrous Na2SO4 and filtered. The solvent was removed
under reduced pressure. The precipitate was purified by recrystallization in mixed solvent (carbon
tetrachloride: n-hexane = 1:1) to afford 1. Light yellow solid, yield 65%; mp: 83.2-84.6 oC.
2.2.2. 2-(4-(dimethylamino)phenyl)-6,8-bis(ethoxymethyl)-3-hydroxyflavone (2)
To a 50 mL round-bottom flask equipped with a magnetic stirrer, 15 mL solution of 703 mg
4-(dimethylamino)benzaldehyde (6 mmol) in EtOH, 1.399 g compound 1 (6 mmol) and 30% NaOH
(6 mL) were added and stirred at room temperature for 24 h. Then, 7 mL of 30% H2O2 solution
was added drop wise to the reaction mixture in an ice-water bath, and the resulting mixture was
stirred at 45 ℃ for 3 h. After cooling to room temperature, the reaction mixture was neutralized
with 2 M HCl solution to pH 7 and extracted with dichloromethane. The organic phase was dried
over Na2SO4 and filtered. The solvent was removed under reduced pressure. The crude product
was purified by column chromatography on silica gel to give 2. Brown red solid, yield 41%; mp:
1
170.5- 171.3 ℃; H NMR (DMSO-d6, 600 MHz) 9.25 (s, 1H), 8.12 (d, 2H, J = 7.8 Hz), 7.96 (s, 1H),
7.70 (s, 1H), 6.87 (d, 2H, J = 7.8 Hz), 4.87 (s, 2H), 4.59 (s, 2H), 3.64 (q, 2H, J = 6.6 Hz), 3.54 (q, 2H, J
= 6.6 Hz), 3.03 (s, 6H), 1.24~1.18 (m, 6H); 13C NMR (CDCl3, 150 MHz) δ (ppm): 171.86, 151.20,
150.99, 146.42, 137.24, 134.40, 131.13, 128.78, 127.88, 122.11, 121.05, 118.07, 111.42, 70.79,
66.23, 65.65, 65.11, 15.10; HRMS (ESI) m/z calcd. for C23H28NO5: 398.19620 [M+H]+, found
398.19586.
2.2.3. 2-(4-(dimethylamino)phenyl)-6,8-bis(morpholinomethyl)-3-hydroxyflavone (3)
A mixture of 557 mg compound 2 (1.4 mmol), and 18 mL hydrobromic acid were heated at
100 ℃ for 4 h. After cooling, the reaction mixture was treated with saturated sodium
bicarbonate solution until pH was
7
with the gradual formation of precipitation
The collected
(6,8-bis(bromomethyl)-2-(4-(dimethylamino)phenyl)-3-hydroxyflavone).
precipitation was dried and the crude product was used for the next step without further
purification.
To a round-bottom flask (50 mL) equipped with a magnetic stirrer, the crude product of
6,8-bis(bromomethyl)-2-(4-(dimethylamino)phenyl)-3-hydroxyflavone dissolved in 30 mL of THF,
and 870 mg morpholine (10 mmol) were added and stirred at room temperature for 10 h. The

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solvent was removed under reduced pressure. Then the crude product was purified by column
chromatography on silica gel to give 3 as a pure compound. Yellow solid, yield 51%; mp:
o
1
207.5-208.6 C; H NMR (DMSO-d6, 600 MHz) 9.22 (s, 1H), 8.14 (d, 2H, J = 9.0 Hz), 7.89 (s, 1H),
7.67 (s, 1H), 6.86 (d, 2H, J = 9.0 Hz), 3.84 (s, 2H), 3.65~3.54 (m, 10H), 3.01 (s, 6H), 2.56~2.31 (m,
8H); 13C NMR (DMSO-d6, 150 MHz) δ (ppm): 172.46, 151.49, 140.18, 137.67, 134.96, 133.93,
130.52, 130.12, 129.36, 127.29, 123.91, 118.70, 111.97, 111.13, 66.68, 62.08, 59.51, 56.56, 53.83,
53.56; HRMS (ESI) m/z calcd for C27H34N3O5: 480.24930 [M+H]+, found: 480.24869.
2.2.4.
2-(4-(dimethylamino)phenyl)-3-((1-methyl-4-nitro-1H-imidazol-5-yl)methoxy)-6,8-bis
(morpholinomethyl)-4H-chromen-4-one (3-HF-NO2)
To a round-bottom flask (50 mL) equipped with a magnetic stirrer, 240 mg compound 3 (0.5
mmol) dissolved in 3 mL of dry DMF and 138 mg potassium carbonate (1 mmol) were added and
stirred at room temperature for 30 min, followed by addition of 110 mg
5-(bromomethyl)-1-methyl-4-nitro-1H-imidazole (0.5 mmol). The mixture was stirred room
temperature for 12 h. Then the reaction mixture was extracted with ethyl acetate (15 mL×3).
The organic phase was washed with water twice, dried over Na2SO4. After filtration and
concentration in vacuum, the crude product was purified by column chromatography on silica gel
to give probe 3-HF-NO2. Orange solid, yield 38%; mp: 182-184 oC; 1H NMR (DMSO-d6, 600 MHz, )
δ (ppm): 8.17 (d, J = 9.0 Hz, 2H), 8.09 (s, 1H); 8.04 (d, J = 9.0 Hz, 2H), 7.71 (s, 1H), 7.63 (s, J = 8.4
Hz, 2H), 6.73 (d, J = 8.4 Hz, 2H), 5.22 (s, 2H), 3.85 (s, 2H), 3.75 (t, J = 4.8 Hz, 4H), 3.72 (t, J = 4.8 Hz,
4H), 3.60 (s, 2H), 3.09 (s, 6H), 2.57 (s, 4H), 2.47 (s, 4H); 13C NMR (DMSO-d6, 150 MHz, ) δ (ppm):
173.70, 156.93, 152.60, 152.19, 145.36, 137.92, 137.48, 135.49, 134.58, 129.99, 128.62, 127.41,
124.11, 123.75, 116.66, 66.68, 61.98, 60.68, 56.51, 53.75, 53.56, 33.05; HRMS (ESI) m/z calcd for
C32H39N6O7: 619.28747 [M+H]+, found 619.28759.
2.3. NTR assay
All the fluorescence measurements were carried out according to the following procedure. In a
5 mL tube, 2 mL of PBS (10 mmol/L, pH = 7.4), and 2.5 mL 10 μmol/L 3-HF-NO2 were mixed,
followed by addition of NADH (final concentration, 1 mmol/L), and an appropriate volume of NTR
solution. The final volume was adjusted to 5 mL with PBS; the reaction solution was mixed rapidly
o
and degassed with nitrogen for 30 min to remove oxygen. After incubation at 37 C for 15 min,
the spectra were measured (λex/em = 412/560 nm) with 10/10 nm slit widths. In the meantime, a
blank solution containing no NTR (control) was prepared and measured under the same
conditions for comparison.
2.4 HPLC for the determination of reduction assay of NM-3-HF
A liquid chromatography system from Agilent Technologies (Agilent, American) was applied to
all chromatography tests. The HPLC system was performed with a quaternary G1311C pump, a
G1314F UV-vis detector. The data acquisition and processing was performed throughout a LC
solution chromatographic workstation (Agilent, Japan). The analytes column was a Agilent
HC-C18 column (250 mm × 4.6 mm, 5 um) from Bonna-Agela Technologies. The optimized mobile
phase consisted of acetonitrile-0.01M K2HPO4 solution (20:80, V/V) and the flow rate was
maintained at 0.5 mL/min. The UV detector was set at 254 nm and injected aliquots of 10 μL
sample into the column.
The solution of NM-3-HF (0.5 mM) in DMSO and H2O (1:1, V/V) was added Na2S2O4 (10 mg)

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o
and then incubated for 4 min in 25 C. The mixture solution was filtered and then detected
through HPLC.
2.5. MTT Assays
HeLa cells were cultured under normoxic condition (5% CO2 at 37 oC) in a humidified incubator
o
(SANYO, MCO-20AIC, JPN) or under hypoxic condition (2% O2, 5% CO2, and 93% N2 at 37 C) in a
humidified incubator (Heal Force, HF 100, CHN). The effect of the compounds on cells viability
was determined by MTT method [39]. In brief, the HeLa cells harvested from the flask at
subconfluent stage were seeded in 96 well culture plates at density of 2×104 cells per well and
incubated overnight. Then 3-HF-NO2 was added to the wells to achieve final concentrations.
Control wells were prepared by addition of culture medium. Wells containing culture medium
without cells were used as blanks. At the end of incubation, 10 μL of MTT (5.0 mg/mL) was added
into each well and incubated for 4 h. Then, the supernatant was removed and 100 μL DMSO was
added to each well for dissolve the MTT formazan. The OD of formazan solutions were recorded
on a microplate spectrophotometer (Molecular Devices, Versa Max, USA) at 570 nm. The cell
viability was presented as the fold over the control group and was calculated according to
formula: cell viability (%) = (ODsample-ODblank)/(ODcontrol-ODblank×100).
2.6. Hypoxic conditions in cell imaging
HeLa cells were cultured under normoxic condition (5% CO2 at 37 oC) in a humidified incubator
o
(SANYO, MCO-20AIC, JPN) or under hypoxic condition (2% O2, 5% CO2, and 93% N2 at 37 C) in a
humidified incubator (Heal Force, HF 100, CHN). After treatment of 3-HF-NO2 at certain
concentration (5 μmol/L) for different time, then the cells were washed with PBS twice. The
fluorescence imaging experiments were performed on a confocal laser scanning microscope with
FV5-LAMAR for excitation at 405 nm and variable band pass emission filter set to 545-645 nm
through a 60×1.42 NA objective. Optical section was acquired at 0.8 um.
3. Results and discussion
3.1. Synthesis and structural characterization
The
synthetic
route
of
3-HF-NO2
is
depicted
in
Scheme
1.
5-(Bromomethyl)-1-methyl-4-nitro-1H-imidazole was prepared via the reported method [40].
3-HF-NO2 was synthesized from o-hydroxyl acetophenone, 4-(dimethylamino)cinnamaldehyde
and 5-(bromomethyl)-1-methyl-4-nitro-1H-imidazole by Blanc chloromethylation, Aldol
condensation, Algar-Flynn-Oyamada (AFO) reaction, bromination reaction, and substitution
reaction. Their structures were characterized by NMR and HRMS (Fig. S3-S11, ESI†). By modifying
the structure of flavonoids with two morpholine groups at 6,8-positions, the water-solubility and
cell permeability of 3-HF-NO2 were improved. Thus, we selected a pH 7.4 PBS buffer solution
containing 0.5% (v/v) DMSO for further experiments. In contrast, compound 4 (unmodified by
morpholine group, Fig. 1) and 5 (using a p-nitrobenzyl in place of 4-nitroimidazole moiety, Fig. 1)
were precipitated out in MTT assays at reasonable concentration.
Scheme 1
Fig. 1
3.2. Photophysical properties of 3-HF-NO2 and compound 3

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UV-Vis absorption and emission spectra of 3-HF-NO2 (5 μmol/L) and compound 3 (5 μmol/L) in
in 10 mmol/L PBS were presented in Fig. 2. The UV spectra of compound 3 exhibited absorption
at λmax 412 nm, 3-HF-NO2 exhibited two bands at λmax 314 (attributed to 4-nitroimidazole moiety)
and 402 nm (Fig. 2a). The blue shift (10 nm) is due to the modification of the hydroxyl group of 3
by 4-nitroimidazole moiety. The fluorescence spectra of 3 and 3-HF-NO2 were shown in Fig. 2b.
Compound 3 exhibited a relatively strong fluorescence emission at 560 nm (excitation at 412 nm),
whereas 3-HF-NO2 displayed no fluorescence due to the combined effects of the photo-induced
electron-transfer (PET) process and the blocking ESIPT process by 4-nitroimidazole moiety.
However, upon incubation of 3-HF-NO2 with NTR and NADH in PBS buffer (pH 7.4, 0.01 mol/L,
o
0.5% DMSO) at 37 C, the reaction mixture produces a remarkable fluorescence change from
almost non-fluoresce to strong green fluorescence (λex/em= 412/560 nm, Fig. 2b). The absorption
at 412 nm and obvious fluorescence enhancement at 560 nm suggested that the 4-nitroimidazole
moiety of 3-HF-NO2 was reduced by NTR, thus eliminating the possibility of PET-induced
quenching, and concomitantly allowing fluorescence to occur. In addition, the above data
suggested that the hypoxic probe has a larger stokes shift (148 nm).
Fig. 2
3.3 Response towards NTR or hypoxia
Next, the fluorescence response of 3-HF-NO2 to NTR at varied concentrations in the presence
of NADH was also tested. As shown in Fig. 3, the fluorescence intensity of the reaction solution
increased gradually with the increase of NTR concentration, and a good linearity is obtained in
the concentration range of 1-4 μg/mL NTR, with the regression equation y = 51.7952x+12.7415
(R2 = 0.997). The detection limit is 63 ng/mL NTR. Thus, 3-HF-NO2 exhibits high sensitivity to NTR
and hypoxia.
Fig. 3
3.4. Selective response of probe 3-HF-NO2 to NTR
To evaluate the effect of 3-HF-NO2 selective response to NTR, considering the complexity of the
intracellular environment, we investigated the influence of various analytes, including reductants
(VC, glutathione), biothiols (cysteine, homocysteine, and dithiothreitol), inorganic salts (CaCl2,
MgCl2), saccharide (glucose), amino acid (arginine), and reactive oxygen species (H2O2, NaOCl) on
the fluorescence behaviour of 3-HF-NO2 in PBS buffer. As shown in Fig. 4, 3-HF-NO2 uniquely
responds to NTR to produce a prominent fluorescence enhancement at 560 nm, whereas has
nearly no fluorescence responses to other reductants, biothreitol, saccharide, amino acids, metal
salts, and reactive oxygen species under same conditions. The above results demonstrated that
the fluorescent change of 3-HF-NO2 was induced by NTR in presence of NADH. Therefore, it can
be concluded that 3-HF-NO2 displays extremely high selectivity for sensing NTR.
Fig. 4
3.5. The effect of pH on the reaction between 3-HF-NO2 and NTR
The fluorescent response of 3-HF-NO2 toward NTR in PBS buffer (10 mmol/L, 0.5% DMSO) at
different pH conditions were further investigated in biological systems. As shown in Fig. S2 (ESI†),
the probe 3-HF-NO2 response to NTR displayed pH-dependent. The fluorescence intensity of the
reaction mixture increased obviously when pH is increased from 5.0 to 9.0. This trend is

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consistent with the fluorescence intensity change of compound 3 along with pH increasing from
5.0 to 9.0 (Fig. S1, ESI†). The stable optical property of this probe reveals that it functions well
under physiological conditions.
3.6 The cytotoxicity of 3-HF-NO2
In order to evaluate the cytotoxicity of 3-HF-NO2, MTT assay was performed in HeLa cells under
normoxic (20% O2) and hypoxic conditions (2% O2) at 37 °C for 24 h. As shown in Fig. 5, the HeLa
cell viability is found to be upon 90% when the concentration of 3-HF-NO2 is lesser than 10
μmol/L under normoxic/hypoxic condition. The results showed that 3-HF-NO2 exhibited low
cytotoxicity and good biocompatibility under experimental conditions.
Fig. 5
3.7 The mechanism of 3-HF-NO2 to specifically recognize NTR
4-Nitroimidazole moiety is not only a receptor for hypoxia/NTR but also an excellent
fluorescence-quench moiety. 3-HF-NO2 displays an extremely low background signal due to the
PET process based on electron withdrawing property of nitro group, combined with the effects of
blocking ESIPT process. Under hypoxic condition, the 4-nitroimidazole moiety is reduced via a
series of one-electron reduction processes to form an unstable 4-aminomidazol derivative, which
leads to rearrangement-elimination. Thus, it is expected that 3-HF which exhibits distinct
photophysical property is released, which concomitantly cause the fluorescence to be restored
(as shown in Scheme 2). The reduction of 3-HF-NO2 was suppressed by molecular oxygen under
aerobic conditions. Hence, the fluorescent response to hypoxia/NTR with high selectivity is
expected using this probe.
Scheme 2
To examine this plausible mechanism of 3-HF-NO2 to specifically recognize NTR or hypoxia, we
determined the reduction product by HPLC and MS. First, Na2S2O4 was used as the reductant to
convert 3-HF-NO2 to fluorophore 3 as expected. The formation of 3 was confirmed by HPLC
analysis (Fig. 6). Then, the reaction of 3-HF-NO2 with NTR in the presence of NADH under hypoxia
was investigated and determined by MS analysis. After being separated and purified, the HRMS of
the product showed a peak at 480.24945 attributing to compound 3 (same to calcd for
C27H34N3O5 480.24869 [M+H]+, Fig. S12, ESI†). This is responsible for the observed enhancement
of fluorescence emission. A signal of 149.1 (Fig. S13, ESI†) is responsible for 7 (calcd for C5H6N2O
149.0 [M+K]+) obtained from the hydrolysis reaction from the immediate 6 in PBS buffer. Under
hypoxia (or in the presence of NTR and NADH), the 4-nitroimidazole moiety was reduced via a
series of one-electron reduction processes to form the amino intermediates that leads to the
rearrangement-elimination.
Fig. 6
3.8 Cell imaging
To further evaluate the potential of 3-HF-NO2 for practical application, HeLa cell was selected
as cell model for cellular fluorescence imaging. HeLa cells were first incubated under normoxic
(20% O2) and hypoxic conditions (10%, 5%, and 2% O2) at 37 °C for 16 h, and then incubated with
5 μmol/L 3-HF-NO2. In order to determine the reduction rates of 3-HF-NO2 under different

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hypoxic conditions, HeLa cells were incubated with 3-HF-NO2 for 20 and 40 min under the
respective conditions. As expected, incubated with 3-HF-NO2 under normoxic conditions for
different times (20 and 40 min), no considerable fluorescence was observed (Fig. 7a, g). A
significant green fluorescence was observed under hypoxic conditions with the aid of a confocal
fluorescence microscope (Fig. 7d, j). The reduction of 4-nitroimidazole moiety of 3-HF-NO2 under
hypoxic conditions leads to the establishment of a sensitive and selective method for imaging the
hypoxic cells. The results showed that cells under normoxic and hypoxic conditions could be
differentiated successfully by this method.
In addition, HeLa cells rapidly respond to 3-HF-NO2 under hypoxic conditions (less than 20 min).
More importantly, the fluorescence intensity increases in the cells was observed with the
decrease of the O2 concentration from 10% to 2% (Fig. 7m~u). This implied that 3-HF-NO2 can
indicate the hypoxic status in the cells rapidly.
Fig. 7
4. Conclusions
In summary, by taking advantage of an efficient hypoxic marker 4-nitroimidazole moiety, a
novel fluorescent probe 3-HF-NO2 for hypoxia detection and imaging hypoxic status in tumor cells
was synthesized. 3-HF-NO2 is quenched by 4-nitroimidazole moiety. Under hypoxia or in the
presence of NTR with NADH, 3-HF-NO2 generated highly fluorescent 3-HF via the selective
reduction and rearrangement-elimination. The probe displayed excellent properties such as high
selectivity, sensitivity, no cytotoxicity, large Stokes’ shift, and low detection limit. This probe was
successfully applied in imaging of hypoxic HeLa cells. Thus, the conjugation of a 4-nitroimidazole
moiety to a fluorophore is a useful strategy for designing hypoxia-sensitive probe. The simple
chemical structures, large Stokes’ shift and dual emission makes 3-HF-NO2 a practically useful tool
to investigate a variety of hypoxia-related biological phenomena.
Acknowledgments
This work was supported by the Key Basic Research Special Foundation of Science Technology
Ministry of Hebei Province (12966418D), the Key Research Project Foundation of Department of
Education of Hebei Province (Grant No. ZH2012041), the Technology Research and Development
Foundation of Hebei Province (No. 11276431).
References
[1] He F, Deng X, Wen B, Liu Y, Sun X, Xing L, Minami A, Huang Y, Chen Q, Zanzonico PB, Ling CC, Li
GC. Noninvasive molecular imaging of hypoxia in human xenografts: comparing
hypoxia-induced gene expression with endogenous and exogenous hypoxia markers. Cancer
Res 2008;68:8597-606.
[2] Wilson WR, Hay MP. Targeting hypoxia in cancer therapy. Nat Rev Cancer 2011;11:393-410.
[3] Kiyose K, Hanaoka K, Oushiki D, Nakamura T, Kajimura M, Suematsu M, Nishimatsu H, Yamane
T, Terai T, Hirata Y, Nagano T. Hypoxia-sensitive fluorescent probes for in vivo real-time
fluorescence imaging of acute ischemia. J Am Chem Soc 2010;132:15846-8.
[4] Varia MA, Calkins-Adams DP, Rinker LH, Kennedy AS, Novotny DB, Fowler WC, Raleigh JA.
Pimonidazole: a novel hypoxia marker for complementary study of tumor hypoxia and cell

ACCEPTED MANUSCRIPT
proliferation in cervical carcinoma. Gynecol Oncol 1998;71:270-7.
[5] Kaanders JHAM, Wijffels KIEM, Marres HAM, Ljungkvist ASE, Pop LAM, van den Hoogen FJA,
de Wilde PCM, Bussink J, Raleigh JA, van der Kogel AJ. Pimonidazole binding and tumor
vascularity predict for treatment outcome in head and neck cancer. Cancer Res
2002;62:7066-74.
[6] Yang DJ, Wallace S, Cherif A, Li C, Gretzer MB, Kim EE, Podoloff DA. Development of
F-18-labeled fluoroerythronitroimidazole as a PET agent for imaging tumor hypoxia.
Radiology 1995;194: 795-800.
[7] Dearling JL, Lewis JS, Mullen GE, Welch MJ, Blower PJ. Copper bis(thiosemicarbazone)
complexes as hypoxia imaging agents: structure-activity relationships. J Biol Inorg Chem
2002;7:249-59.
[8] de Barros ALB, Mota LG, Ferreira CA, de Oliverira MC, de Góes AM, Cardoso VN. Bombesin
derivative radiolabeled with technetium-99m as agent for tumor identification. Bioorg Med
Chem Lett 2010;20:6182-4.
[9] Sakr TM, Motaleb MA, Ibrahim IT. 99mTc–meropenem as a potential SPECT imaging probe for
tumor hypoxia. J Radioanal Nucl Chem 2012;292:705-10.
[10] Pacheco-Torres J, Lopeź-Larrubia P, Ballesteros P, Cerdán S. Imaging tumor hypoxia by
magnetic resonance methods. NMR Biomed 2011;24:1-16.
[11] Iwaki S, Hanaoka K, Piao W, Komatsu T, Ueno T, Terai T, Nagano T. Development of
hypoxia-sensitive Gd3+-based MRI contrast agents. Bioorg Med Chem Lett 2012;22:2798-892.
[12] Dai C, Liu X, Zhang HF, Puliafito CA, Jiao S. Absolute retinal blood flow measurement with a
dual-beam Doppler optical coherence tomography. Invest Ophthalmol Vis Sci
2013;54:7998-8003.
[13] Zhang S, Hosaka M, Yoshihara T, Negishi K, Iida Y, Tobita S, Takeuchi T. Phosphorescent
light-emitting iridium complexes serve as a hypoxia-sensing probe for tumor imaging in living
animals. Cancer Res 2010;70:4490-8.
[14] Sakadžić S, Roussakis E, Yaseen MA, Mandeville ET, Srinivasan VJ, Arai K, Ruvinskaya S, Devor
A, Lo EH, Vinogradov SA, Boas DA. Two-photon high-resolution measurement of partial
pressure of oxygen in cerebral vasculature and tissue. Nat Methods 2010;7:755-9.
[15] Sun X, Niu G, Chan N, Shen B, Chen X. Tumor hypoxia imaging. Mol Imaging Biol.
2011;13:399-410.
[16] Takahashi S, Piao W, Matsumura Y, Komatsu T, Ueno T, Terai T, Kamachi T, Kohno M, Nagano T,
Hanaoka K. Reversible off-on fluorescence probe for hypoxia and imaging of
hypoxia-normoxia cycles in live cells. J Am Chem Soc 2012;134:19588-91.
[17] Myochin T, Hanaoka K, Komatsu T, Terai T, Nagano T. Design strategy for a near-infrared
fluorescence probe for matrix metalloproteinase utilizing highly cell permeable boron
dipyrromethene. J Am Chem Soc 2012;134:13730-7.
[18] Piao W, Tsuda S, Tanaka Y, Maeda S, Liu F, Takahashi S, Kushida Y, Komatsu T, Ueno T, Terai T,
Nakazawa T, Uchiyama M, Morokuma K, Nagano T, Hanaoka K. Development of Azo-based
fluorescent probes to detect different levels of hypoxia. Angew Chem Int Ed
2013;52:13028-32.
[19] Uddin MI, Evans SM, Craft JR, Marnett LJ, Uddin MJ, Jayagopal A. Applications of azo-based
probes for imaging retinal hypoxia. ACS Med Chem Lett 2015;6:445-9.
[20] Cui L, Zhong Y, Zhu W, Xu Y, Du Q, Wang X, Qian X, Xiao Y. A new prodrug-derived ratiometric

ACCEPTED MANUSCRIPT
fluorescent probe for hypoxia: High selectivity of nitroreductase and imaging in tumor Cell.
Org Lett 2011;13:928-31.
[21] Napp J, Behnke T, Fischer L, Wurth C, Wottawa M, Katschinski DM, Alves F, Resch-Genger U,
Schaeferling M. Targeted luminescent near-infrared polymer-nanoprobes for in vivo imaging
of tumor hypoxia. Anal Chem 2011;83:9039-46.
[22] Huang HC, Wang KL, Huang ST, Lin HY, Lin CM. Development of a sensitive long-wavelength
fluorogenic probe for nitroreductase: A new fluorimetric indictor for analyte determination
by dehydrogenase-coupled biosensors. Biosens Bioelectron 2011;26:3511-6.
[23] Zhu R, Liu MC, Luo MZ, Penketh PG, Baumann RP, Shyam K, Sartorelli AC.
4-Nitrobenzyloxycarbonyl derivatives of O(6)-benzylguanine as hypoxia-activated prodrug
inhibitors of O(6)-alkylguanine-DNA alkyltransferase (AGT), which produces resistance to
agents targeting the O-6 position of DNA guanine. J Med Chem 2011;54:7720-8.
[24] Baumann RP, Ishiguro K, Penketh PG, Shyam K, Zhu R, Sartorelli AC. KS900: A
hypoxia-directed, reductively activated methylating antitumor prodrug that selectively
ablates O(6)-alkylguanine-DNA alkyltransferase in neoplastic cells. Biochemical Pharmacol.
2011; 81:1201-10.
[25] Kizaka-Kondoh S, Konse-Nagasawa H. Significance of nitroimidazole compounds and
hypoxia-inducible factor-1 for imaging tumor hypoxia. Cancer Sci 2009;100:1366-73.
[26] Guo T, Cui L, Shen JN, Zhu WP, Xu YF, XH Qian Highly Sensitive Long-wavelength Fluorescence
Probe for Nitroreductase and Hypoxia: Selective Detection and Quantification
. Chem.
Commun. 2013; 49:10820-2.
[27] Okuda K, Okabe Y, Kadonosono T, Ueno T, Youssif BGM, Kizaka-Kondoh S, Nagasawa H.
2-Nitroimidazole-tricarbocyanine conjugate as a near-infrared fluorescent probe for in vivo
imaging of tumor hypoxia. Bioconjugate Chem 2012;23:324-9.
[28] Li Z, Li X, Gao X, Zhang Y, Shi W, Ma H. Nitroreductase detection and hypoxic tumor cell
imaging
by
a
designed
sensitive
and
selective
fluorescent
probe,
7-[(5-nitrofuran-2-yl)methoxy]-3H-phenoxazin-3-one. Anal Chem 2013;85:3926-32.
[29] Xu K, Wang F, Pan X, Liu R, Ma J, Kong F, Tang B. High selectivity imaging of nitroreductase
using a near-infrared fluorescence probe in hypoxic tumor. Chem Commun 2013,49:2554-6.
[30] Cellier M, Fabrega OJ, Fazackerley E, James AL, Orenga S, Perry JD, Salwatura VL, Stanforth SP.
2-Arylbenzothiazole, benzoxazole and benzimidazole derivatives as fluorogenic substrates for
the detection of nitroreductase and aminopeptidase activity in clinically important bacteria.
Bioorg Med Chem 2011;19:2903-10.
[31] Darwich Z, Kucherak OA, Kreder R, Richert L, Vauchelles R, Mely Y, Klymchenko AS. Rational
design of fluorescent membrane probes for apoptosis based on 3-hydroxyflavone. Methods
Appl Fluoresc 2013;1:250-4.
[32] Klymchenko AS, Demchenko AP. 3-Hydroxychromone dyes exhibiting excited-state
intramolecular proton transfer in water with efficient two-band fluorescence. New J Chem
2004;28:687-92.
[33] Chen S, Hou P, Zhou B, Song X, Wu J, Zhang H, Foley JW. A red fluorescent probe for thiols
based on 3-hydroxyflavone and its application in living cell imaging. RSC Adv 2013;3:11543-6.
[34] Lin B, Wang J, Zhang G, Bai R, Pang Y. Flavone-based ESIPT ratiometric chemodosimeter for
detection of cysteine in living cells. ACS Appl Mater Interfaces 2014;6:4402-7.
[35] Kim TI, Kang HJ, Han G, Chung SJ, Kim Y. Highly selective fluorescent ESIPT probe for the dual

ACCEPTED MANUSCRIPT
specificity phosphatase MKP-6. Chem. Commun. 2009;45:5895-7.
[36] Wang F, Wu J, Zhuang X, Zhang W, Liu W, Wang P, Wu S. A highly selective fluorescent sensor
for fluoride in aqueous solution based on the inhibition of excited-state intramolecular
proton transfer. Sens Actuators B: Chem. 2010;146: 260-5.
[37] Hu R. Feng J. Hu D, Wang S, Li S, Li Y, Yang G. A rapid aqueous fluoride ion sensor with dual
output modes. Angew Chem Int Ed 2010;49:4915-8.
[38] Santra M, Roy B, Ahn KH. A “reactive” ratiometric fluorescent probe for mercury species. Org
Lett 2011;13:3422-5.
[39] Mosmann T. Rapid colorimetric assay for cellular growth and survival: Application to
proliferation and cytotoxicity assays. J Immunol Methods 1983;65:55-63.
[40] Makosza M, Bialecki M. Synthesis of (nitroaryl)chloromethanes via vicarious nucleophilic
substitution of hydrogen. Synlett 1991;1991:181-2.

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Legends to Tables and Figures
Scheme 1 Synthetic route for 3-HF-NO2
Scheme 2 Proposed mechanism of 3-HF-NO2 detection NTR or hypoxia
Fig. 1 The structure of compounds 4 and 5
Fig. 2 UV-Vis absorption spectra and fluorescence spectra of 3 and 3-HF-NO2
a: UV-Vis absorption spectra of 3 (5 μmol/L) and 3-HF-NO2 (5 μmol/L)
b: Fluorescence spectra of 3 (5 μmol/L), 3-HF-NO2 (5 μmol/L), and 3-HF-NO2 (5 μmol/L) treated
with NTR (3 μg/mL) in presence of NADH (1 mmol/L) for 0.5 h at λex=412 nm
Fig. 3 Fluorescence response of 3-HF-NO2 (5 μmol/L) to different concentrations of NTR at λex/em
=
412/560 nm
Fig. 4 Fluorescence responses of 3-HF-NO2 (5 μmol/L) to various species (λex/em=412/560 nm)
1: CaCl2 (2.5 mmol/L); 2: MgCl2 (2.5 mmol/L); 3: H2O2 (10 μmol/L); 4: NaOCl (10 μmol/L); 5: Vc (1
mmol/L); 6: glutathione (DTT, 1 mmol/L); 7: cysteine (GSH, 2 mmol/L); 8: homocysteine (1
mmol/L); 9: dithiothreitol (1 mmol/L); 10: glucose (10 mmol/L); 11: arginine (Arg, 1 mmol/L); 12:
NTR (3 μg/mL) with NADH (1 mmol/L)
Fig. 5 Cell viability assays of 3-HF-NO2 under normoxic and hypoxic conditions
Fig. 6 HPLC profiles of a) 3; b) 3-HF-NO2; c) mixture of 3 and 3-HF-NO2; d) solution of 3-HF-NO2
incubated with Na2S2O4
Fig. 7 Confocal fluorescence imaging of HeLa cells incubated with 3-HF-NO2
(a-l) Incubated with 3-HF-NO2 for 20 min (a, b, and c: 20%O2; d, e, and f: 2%O2) and 40 min (g, h,
and i: 20%O2, j, k, and l: 2%O2); (m-u) Incubated with 3-HF-NO2 under different hypoxic
conditions: 10% O2 (m, n, and o); 5% O2 (p, q, and r); 2% O2 (s, t, and u)

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Fig 1

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Fig. 2
0.10
700
b
a
3
600
500
400
300
200
100
0
3-HF-NO
3
2
3-HF-NO
0.08
0.06
0.04
0.02
0.00
2
3-HF-NO + NTR
2
300 350 400 450 500 550 600
Wavelength (nm)
400 450 500 550 600 650 700
Wavelenghth (nm)

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Fig. 3
240
0
µ
g/mL
a
250
200
150
100
50
0.5 µg/mL
b
220
200
180
160
140
120
100
80
1
2
3
µg/mL
µg/mL
µg/mL
3.5 µg/mL
µg/mL
4
y = a + b*x
87.3107
Equation
Residual Sum
of Squares
60
0.99731
Adj. R-Square
40
Value
Standard Error
2.70402
20
B
B
Intercept
Slope
12.7415
51.7952
0
1.0974
0
480 500 520 540 560 580 600 620 640 660
0
1
2
3
4
Concentrations of NTR (µg/mL
)
Wavelength (nm)

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Fig 4

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Scheme 1

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Scheme 2

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Highlights
•4-Nitroimidazole moiety is an efficient hypoxic trigger.
•A novel probe 3-HF-NO2 for the selective detection hypoxia or NTR was developed.
•3-HF-NO2 showed excellent selectivity, sensitivity, and low detection limit.
•3-HF-NO2 was successfully used for monitoring hypoxic status in HeLa cells.