A novel HPQ-based turn-on fluorescent probe for detection of fluoride ions in living cells

Article abstract of DOI:10.1016/j.tetlet.2017.09.049

2-(2′-Hydroxyphenyl)-4(3H)-quinazolinone (HPQ) has been reported as a precipitating fluorescent molecule with excellent optical properties, such as large Stokes shift and strong fluorescence intensity. HPQF, a novel HPQ-based turn-on probe for localizable detection of fluoride ions, was designed, synthesized and fully characterized by ¹H NMR, ¹³C NMR and HRMS. As a chemogenic fluoride probe, the tert-butyldiphenylsilane moiety of HPQF can be easily cleaved by fluoride. After spontaneous 1,6-elimination, HPQ molecule was generated to emit fluorescence under the excitation light. Further study shows that HPQF exhibited high selectivity and sensitivity for detection of fluoride. In addition, HPQF was utilized for the detection of fluoride in living cells.

Full text of DOI:10.1016/j.tetlet.2017.09.049

Accepted Manuscript
A Novel HPQ-based Turn-on Fluorescent Probe for Detection of Fluoride Ions
in Living Cells
Zhou Zhao, Xinzhou Bi, Wuxiang Mao, Xiaowei Xu
PII:
S0040-4039(17)31176-0
DOI:
http://dx.doi.org/10.1016/j.tetlet.2017.09.049
TETL 49319
Reference:
Tetrahedron Letters
To appear in:
Received Date:
Accepted Date:
13 September 2017
18 September 2017
Please cite this article as: Zhao, Z., Bi, X., Mao, W., Xu, X., A Novel HPQ-based Turn-on Fluorescent Probe for
Detection of Fluoride Ions in Living Cells, Tetrahedron Letters (2017), doi: http://dx.doi.org/10.1016/j.tetlet.
2017.09.049
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Tetrahedron Letters
A Novel HPQ-based Turn-on Fluorescent Probe for Detection of Fluoride Ions in
Living Cells
Zhou Zhaoa, Xinzhou Bib, Wuxiang Maoc, and Xiaowei Xua
a State Key Laboratory of Natural Medicines, Key Lab of Drug Metabolism and Pharmacokinetics, China Pharmaceutical University, 210009, Nanjing, China
b State Key Laboratory of Natural Medicines, Center of Drug Discovery, China Pharmaceutical University, 210009, Nanjing, China
c Hubei Collaborative Innovation Center for Green Transformation of Bio-resources, College of Life Sciences, Hubei University, Wuhan, Hubei 430062, China
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
2-(2’-hydroxyphenyl)-4(3H)-quinazolinone (HPQ) has been reported as a precipitating
fluorescent molecule with excellent optical properties, such as large Stokes shift and strong
fluorescence intensity. HPQF, a novel HPQ-based turn-on probe for localizable detection of
fluoride ions, was designed, synthesized and fully characterized by 1H NMR, 13C NMR and
HRMS. As a chemogenic fluoride probe, the tert-butyldiphenylsilane moiety of HPQF can be
easily cleaved by fluoride. After spontaneous 1, 6-elimination, HPQ molecule was generated to
emit fluorescence under the excitation light. Further study shows that HPQF exhibited high
selectivity and sensitivity for detection of fluoride. In addition, HPQF was utilized for the
detection of fluoride in living cells.
Available online
Keywords:
HPQ
2009 Elsevier Ltd. All rights reserved.
fluoride
fluorescence probe
———
 Corresponding author. e-mail: xw@cpu.edu.cn
Telephone:+86-025-83271179; Fax:+86-025-83271060

Tetrahedron Letters
Fluoride ions detection attracts many chemists’ interest
2
All the chemicals were purchased from Adamas-Beta
(Shanghai, China). CDCl3 was purchased from Cambridge
Isotope Laboratory. Milli-Q water was supplied by a Milli-Q Plus
System (Millipore Corp., Breford, USA). All fluorescence
measurements were carried out on a Shimadzu RF-5301
fluorescence spectrometer with both excitation and emission slits
owing to its association with plenty of environmental, biological
and chemical processes.1 For example, excessive intake of
fluoride could lead to many diseases such as fluorosis,
urolithiasis, acute gastric, kidney injuries and even cancer.2
However, fluoride ions also play an important role in dental
health and has potential use for osteoporosis treatment.3-5 In
addition, fluoride could even be used in military such as nerve
gas monitor6 and nuclear weapon manufacture.7 Therein many
literatures have been reported for fluoride ions detection based on
different mechanisms and technology over the past decades.
Among them colorimetric and fluorescence sensors have
attracted the greatest interest as they have high sensitivities and
low limit of detection and are easy to performed even in live cell
and in vivo.8, 9 Enormous amounts of fluoride ions probes have
been developed and the sensing strategies included reaction-
based chemosensors,10-12 fluoride-boron interaction-based
chemosensors13, hydrogen bond or π-π interaction based
chemosensors14, metal ions binding based chemosensors and
nanoparticles based sensors.15-18 For chemogenic fluoride probes,
desilylation of Si-O bond was applied frequently due to its high
sensitivity and selectivity.
Herein we developed a novel fluorescent probe based on the
structure 2-(2’-hydroxyphenyl)-4(3H)-quinazolinone (HPQ),
which is a solid-state fluorophore and has two-photon properties.
HPQ was completely insoluble in aqueous media but could emit
20
strong fluorescence in solid state.19,
HPQ possessed many
set at 5.0 nm.
optical properties, such as large Stokes shift, intense
luminescence and excellent photostability.19 As known, poor
solubility of HPQ leads to the precipitating fluorescent feature
through excited state intramolecular proton transfer (ESIPT).21
Blockade of phenolic group of HPQ could dismiss the ESIPT
mechanism to result in turn-on probes. Given these advantages,
HPQ was employed for further modifications to imaging certain
enzymes, such as phosphatases, esterases, lipases, glucuronidase,
lactamases and peptidases through corresponding trigger
reactions.22-25 Most recently, Tan and his co-workers designed
Scheme 1 Synthetic route to HPQF. (a) TBDPSCl, imidazole, DMF, r.t; (b)
NaBH4, MeOH; (c) PBr3, dichloromethane; (d) K2CO3, 2-
hydroxybenzaldehyde, r.t; (e) 2-aminobenzamide, p-TSA, DDQ.
4-hydroxybenzaldehyde(2.45 g, 20 mmol) was dissolved in
dichloromethane (30 mL) followed by imidazole (1.31 g, 19.2
mmol) added at room temperature for 15 min. tert-
butylchlorodiphenylsilane (TBDPSCl, 5.2 mL) was added and
stirred overnight. Washed by water (100 mL×3) then dried over
anhydrous sodium sulfate before being filtered and the volatiles
removed under vacuum. The residue was purified by a silica gel
column chromatography to give compound 1 as a white solid
(3.97 g, 54.6%).30 1H NMR (300 MHz, DMSO-d6) δ (ppm) :
9.80(s, 1H), 7.75 ~ 7.44 (m, 12H), 6.91 (d, J = 8.4 Hz, 2H), 1.06
(s, 9H). ESI-MS C23H24O2Si [M+Na]+ calcd 383.1, found 383.1.
and synthesized
a HPQ-based probe for localizable and
photoactivatable bioimaging with spatiotemporal and two-photon
properties.21 Blocked by the 2-nitrobenzyl group, a photolabile
group which can be removed by ultra-violet light, HPQ cannot
emit fluorescence any more. When the phenolic group was
released by the light, precipitating fluorescence occurred. With a
certain quaternary ammonium moiety which could target
mitochondria, localizable imaging was realized in living cells. In
addition, two tautomers of HPQ have different fluorescence. This
feature is similar with some other fluorescent probes, especially
acid-responsive probes.26-29
To a solution of compound 1 (2.67 g, 7.4 mmol) in methanol
(100 mL) in ice bath, sodium borohydride (627 mg, 16.5 mmol)
was added slowly. After being stirred for 3 h, the reaction
mixture was poured into water to quench the rest sodium
borohydride. The volatiles were removed to minimum volume
and extracted with ethyl ether (100 mL×3) and dried over
anhydrous sodium sulfate. The solvent was removed in vacuum
to afford the compound 2 as colorless oil (2.67 g, 99.4%).30 1H
NMR (300 MHz, DMSO-d6) δ (ppm) : 7.68 ~ 7.44 (m, 12H),
7.07 (d, J = 8.4 Hz, 2H), 6.68 (d, J = 8.5 Hz, 2H), 1.03 (s, 9H).
ESI-MS C23H26O2Si [M+Na]+ calcd 385.2, found 385.2.
To a solution of compound 2 in dichloromethane (1.83 g),
phosphorus tribromide (0.5 mL) was added slowly at 0 ℃ and
stirred overnight. The mixture was poured into aqueous sodium
bicarbonate, extracted with dichloromethane (50 mL×3) and
dried over anhydrous sodium sulfate. The solvent was removed
to give title compound as colorless oil (1.07 g, 49.8 %).30 1H
NMR (300 MHz, DMSO-d6) δ (ppm) : 7.66 (d, J = 7.0 Hz, 4H),
7.49 ~ 7.42 (m, 6H), 7.23 (d, J = 8.0 Hz, 2H), 6.69 (d, J = 8.1 Hz,
Fig. 1 Schematic illustration of the design of HPQF.
Inspired by this work, we attempted to introduce the tert-
butyldiphenylsilane (TBDPS) group into HPQ (Fig. 1). As shown
in Scheme 1. HPQF can be synthesized through five steps with
satisfied yield.

2H), 4.60 (s, 2H), 1.03 (s, 9H). ESI-MS C23H25BrOSi [M-81Br]+,
[M-79Br]+, calcd 347.2, 345.2 found 347.2, 345.2.
The response of HPQF to fluoride ions was investigated.
Since HPQ is insoluble in aqueous media, HPQF exhibited quite
poor solubility due to its hydrophobic TBDPS group. Therefore,
we attempted to use organic solvents to make HPQF dissolved.
Enlightened by Tan and his co-workers21, tetrahydrofuran (THF)
was chosen to be the solvent to conduct the consequent reaction.
The Si-O bond of the HPQF can be easily cleaved by fluoride
ions to give the fluorescent product HPQ via spontaneously 1, 6-
elimination (Fig. 2). Once the phenolic hydroxyl group was
uncaged, the fluorescence can be excited by UV light. To
confirm the desilylation mechanism, LC-MS method was used to
give convinced evidence. HPQ was also synthesized to be a
reference compound. As shown in the liquid choromatography
(Fig. S4), the retention time of HPQ and HPQF are 3.36 min and
5.76 min, respectively. After the reaction conducted, the peak of
HPQF decreased significantly and the peak of HPQ arose
consequently, even though the baselines of the chromatograms
were not smooth enough. Obviously, the mass spectra results of
the corresponding time are consistent with the molecular weight
of HPQ and HPQF (Fig. S5 & S6).
2-hydroxybenzaldehyde (273.0 mg, 2.2 mmol)was dissolved
in acetonitrile (4 mL), then potassium carbonate (655.2 mg, 4.7
mmol) was added and stirred at room temperature for 10 min.
Compound 3 (850.6 mg, 2.0 mmol) was added and stirred
overnight. The solvent was removed, washed with water and
extracted with ethyl acetate. After filtering, the volatiles removed
and the residue was purified on a silica chromatography to give
1
the title compound as yellowish solid (625.2 mg, 67.1 %). H
NMR (300 MHz, CDCl3) δ (ppm) : 10.48 (s, 1H), 7.84 ~ 7.82 (m,
1H), 7.71 (d, J = 6.4 Hz, 4H), 7.53 ~ 7.35 (m, 7H), 7.16 (d, J =
8.3 Hz, 2H), 7.05 ~ 7.00 (m, 2H), 6.78 (d, J = 8.5 Hz, 2H), 5.03
(s, 2H), 1.10 (s, 9H). 13C NMR (75 MHz, CDCl3) δ (ppm) :
189.75, 135.73, 135.47, 129.91, 128.70, 128.31, 127.75, 120.85,
119.89, 113.12, 70.40, 26.47ESI-MS C30H30O3Si [M+Na]+ calcd
489.2 found 489.2.
Compound 4 (550.6 mg, 1.18 mmol) and 2-aminobenzamide
(177.3 mg, 1.3 mmol) were dissolved in ethanol (40 mL) and
stirred at 0 ℃ for 10 min to form precipitation. The resulting
mixture was refluxed for 30 min. p-TSA (20 mg) was added and
the mixture was refluxed for 1 h. After cooled to room
temperature, 2, 3-dichloro-5,6-dicyano-1,4-benzoquinone (270.8
mg, 1.2 mmol) was added and stirred overnight with exposed to
the air. The precipitation was filtered out, washed with cold
ethanol and dried to give the HPQF (300.2 mg, 43.7 %). 1H NMR
(300 MHz, CDCl3) δ (ppm) : 10.96 (s, 1H), 8.53 (d, J = 6.99 Hz,
1H), 8.29 (d, J = 7.86 Hz, 1H), 7.77 ~ 7.70 (m, 6H), 7.48 ~ 7.35
(m, 8H), 7.20 ~ 7.13 (m, 3H), 7.07 (d, J = 8.31 Hz, 1H), 6.81 (d,
J = 8.37 Hz, 2H), 5.19 (s, 2H), 1.11 (s, 9H). 13C NMR (75 MHz,
CDCl3) δ (ppm) : 161.69, 157.05, 156.05, 150.88, 149.43,
135.60, 134.45, 133.08, 132.73, 131.58, 130.03, 128.92, 127.90,
127.65, 126.48, 126.47, 122.14, 121.36, 120.54, 120.34, 113.91,
71.55, 26.57. ESI-HRMS C37H34N2O3Si [M+Na]+ calcd
605.22309 found 605.22552.
Fig. 3 (A) Fluorescence spectra of 200 μM HPQF response to different
concentration (0 – 2000 μM) TBAF, inset was the reaction mixture (left) and
HPQF solution (right); (B) Selectivity experiments of HPQF against a cast of
anions. TBAF was dissolved in THF and the reaction was conducted in THF
after adding HPQF. Other reactions were all carried on in aqueous media; (C)
Kinetic curve of HPQF with TBAF in THF solution. Excitation wavelength
350 nm.
Immediately prior to the imaging experiments, the cells were
washed with phosphate-buffered solution (PBS), incubated with
HPQF stock solution (final concentration 20 μM) for 60 min at
37 °C, then washed with PBS for three times. After incubated
with 100 μM TBAF for another 0.5 h at 37 °C, the HepG2 cells
were washed with PBS three times and imaging. Confocal
fluorescence imaging were observed under an LSM700 confocal
microscope (Zeiss, Oberkochen, Germany). Excitation
wavelength of the laser was 405 nm. Emissions were set at 470–
530 nm.
Since the mechanism of sensing fluoride had been elucidated,
we put our attention on investigating the response of HPQF to
fluoride. TBAF was dissolved in THF and diluted into different
concentration (0, 10, 20, 50, 100, 200, 500, 1000 and 2000 μM).
HPQF was dissolved in DMSO to give 20 mM stock solution. All
the reaction was conducted at room temperature for 30 min and
measured using Shimadzu RF5301 spectrometer.
Over the past decades, the strategy that desilylation of Si-O
bond was utilized frequently due to the high sensitivity and
selectivity. Si-O bond can be attacked by nucleophilic fluoride
ions easily, then the HPQ can be released through 1, 6-
elimination. As shown in Fig. 1 and Scheme 1, HPQF was
As shown in Fig. 3A
& B, HPQF exhibited good
concentration-dependent fluorescent properties and excellent
selectivity towards fluoride ions. Fluoride can be detected very
quickly by HPQF in a few seconds (Fig. 3C). Interestingly, the
reaction conducted in THF resulted in blue fluorescence which
was emitted by the keto form of HPQ molecule. We calculate the
limit of detection out as 140 nM.
1
designed, synthesized and fully characterized by H, 13C NMR
and HRMS (Detailed spectra can be found in Supplementary
Materials).
Since fluoride ions have multiple functions in physical and
pathological conditions, image fluoride ions in living cells could
provide clinicians more information for clinical diagnosis.
HepG2 cells were incubated with HPQF stock solution (final
concentration 20 μM) for 60 min and then exited under laser
confocal microscopy to detect fluoride in living cells (Fig. 4).
The cells was incubated with fluorides ions for 4 hours before the
HPQF was added. And from the left picture below we could see
Fig. 2 Proposed mechanism of the turn-on detection of fluoride by HPQF.

Tetrahedron Letters
4
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2
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HPQF still showed good response to fluoride.
A
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Fig. 4 Confocal imaging of fluoride (20μM) in HepG2 cells. (A) Bright
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incubation. λex: 405nm. Scale bar: 10 μm.
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HPQ has two photon properties, two photon imaging will be
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Acknowledgments
This work was supported by the National Natural Science
Foundation of China (No. 21602254) and the Natural Science
Foundation of Jiangsu province, China (BK20160767).
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1. HPQF, a novel HPQ-based turn-on probe for localizable
detection of fluoride ions, was designed, synthesized and fully
characterized by 1H NMR, 13C NMR and HRMS.
2. HPQF exhibited high selectivity and sensitivity for
detection of fluoride.
3. HPQF was utilized for the detection of fluoride in living
cells.
Graphical Abstract
Leave this area blank for abstract info.
A Novel HPQ-based Turn-on Fluorescent
Probe for Detection of Fluoride Ions in Living Cells
Zhou Zhaoa, Xinzhou Bib, Wuxiang Maoc, and Xiaowei Xua
a State Key Laboratory of Natural Medicines, Key Lab of Drug Metabolism and Pharmacokinetics, China Pharmaceutical University, 210009,
Nanjing, China
b State Key Laboratory of Natural Medicines, Center of Drug Discovery, China Pharmaceutical University, 210009, Nanjing, China
c Hubei Collaborative Innovation Center for Green Transformation of Bio-resources, College of Life Sciences, Hubei University, Wuhan, Hubei
430062, China