A rhodol-based fluorescent chemosensor for hydrazine and its application in live cell bioimaging

Article abstract of DOI:10.1016/j.saa.2017.05.058

A rhodol cinnamate fluorescent chemosensor (RC) has been developed for selective detection of hydrazine (N₂H₄). In aqueous medium, the rhodol-based probe exhibited high selectivity for hydrazine among other molecules. The addition of hydrazine triggered a fluorescence emission with 48-fold enhancement based on hydrazinolysis and a subsequent ring-opening process. The chemical probe also displayed a selective colorimetric response toward N₂H₄ from colorless solution to pink, readily observed by the naked eye. The detection limit of RC for hydrazine was calculated to be 300?nM (9.6?ppb). RC is membrane permeable and was successfully demonstrated to detect hydrazine in live HepG2 cells by confocal fluorescence microscopy.

Full text of DOI:10.1016/j.saa.2017.05.058

Accepted Manuscript
A rhodol-based fluorescent chemosensor for hydrazine and its
application in live cell bioimaging
Khomsan Tiensomjitr, Rattha Noorat, Kanokorn Wechakorn,
Samran Prabpai, Kanoknetr Suksen, Phongthon Kanjanasirirat,
Yongyut Pewkliang, Suparerk Borwornpinyo, Palangpon
Kongsaeree
PII:
DOI:
Reference:
S1386-1425(17)30441-9
doi: 10.1016/j.saa.2017.05.058
SAA 15200
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
To appear in:
Received date:
Revised date:
Accepted date:
10 February 2017
16 May 2017
28 May 2017
Please cite this article as: Khomsan Tiensomjitr, Rattha Noorat, Kanokorn Wechakorn,
Samran Prabpai, Kanoknetr Suksen, Phongthon Kanjanasirirat, Yongyut Pewkliang,
Suparerk Borwornpinyo, Palangpon Kongsaeree , A rhodol-based fluorescent
chemosensor for hydrazine and its application in live cell bioimaging, Spectrochimica Acta
Part A: Molecular and Biomolecular Spectroscopy (2017), doi: 10.1016/j.saa.2017.05.058
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ACCEPTED MANUSCRIPT
A rhodol-based fluorescent chemosensor for hydrazine and its
application in live cell bioimaging
Khomsan Tiensomjitra,b, Rattha Noorata,b, Kanokorn Wechakorna,b, Samran Prabpaia,b, Kanoknetr
Suksenc, Phongthon Kanjanasiriratd, Yongyut Pewkliangd, Suparerk Borwornpinyod,e and
Palangpon Kongsaereea,b,*
a Department of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of
Science, Mahidol University, Bangkok 10400, Thailand.
b Center for Excellence in Protein and Enzyme Technology, Faculty of Science, Mahidol University,
Bangkok 10400, Thailand
c Department of Physiology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand
d Excellent Center for Drug Discovery, Faculty of Science, Mahidol University, Bangkok 10400,
Thailand
e Department of Biotechnology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand
*Corresponding author
E-mail: palangpon.kon@mahidol.ac.th
Tel: +66-2-201-5848; Fax: +66-2-354-7151

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Graphical abstract
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Research highlights
 A rhodol-based fluorogenic probe with a cinnamate moiety was synthesized and
characterized by 1H NMR, 13C NMR and HRMS.
 The chemical probe is highly selective to hydrazine with a 48-fold fluorescence
enhancement and a detection limit of 9.6 ppb.
 The cinnamoyl rhodol chemosensor is membrane permeable and used in bioimaging of
hydrazine in HepG2 cells.
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A rhodol-based fluorescent chemosensor for hydrazine and its
application in live cell bioimaging
Abstract
A rhodol cinnamate fluorescent chemosensor (RC) has been developed for selective detection of
hydrazine (N2H4). In aqueous medium, the rhodol-based probe exhibited high selectivity for
hydrazine among other molecules. The addition of hydrazine triggered a fluorescence emission with
48-fold enhancement based on hydrazinolysis and a subsequent ring-opening process. The chemical
probe also displayed a selective colorimetric response toward N2H4 from colorless solution to pink,
readily observed by the naked eye. The detection limit of RC for hydrazine was calculated to be 300
nM (9.6 ppb). RC is membrane permeable and was successfully demonstrated to detect hydrazine in
live HepG2 cells by confocal fluorescence microscopy.
Keywords: Hydrazine-selective chemosensor; Fluorescent detection; Rhodol; Bioimaging
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1. Introduction
Hydrazine (N2H4), a colorless flammable inorganic liquid, has been used as a high
energy fuel in missile propulsion systems [1,2]. Due to its basic and reducing properties,
hydrazine is also an essential reagent in chemical, pharmaceutical, and agricultural industries
[3-5]. Because of its high degree of toxicity to human health and the environment, hydrazine
has been classified as a probable human carcinogen by the U.S. Environmental Protection
Agency (EPA) with a threshold limit value (TLV) of 10 ppb [6,7]. Hydrazine causes
neurotoxicity and mutagenic effects to liver, lung, and kidney by absorption through skin and
lung [8,9]. Several methods can be used to efficiently determine hydrazine such as HPLC
[10], gas chromatography [11], and capillary electrophoresis [12]. However, these methods
are not applicable for intracellular detection of hydrazine due to its incompatible sample
preparation. Therefore, development of chemosensors for selective recognition of hydrazine
has attracted a great deal of scientific attention in recent years. Fluorescence-based methods
are very practical in chemical and biological studies because of their advantageous high
sensitivity and selectivity as well as biocompatibility [13-15]. With a reaction-based strategy,
several naked eye and fluorescent chemosensors have been developed for hydrazine detection
using various dyes including BODIPY [16,17], coumarin [18-20], naphthalimide [7,21],
resorufin [22], benzothiazole [23], pyrazoline [24,25], and xanthene analogs [26-28]. Among
these reported hydrazine-selective chemosensors, several prosthetic groups have been
explored including malononitrile [17,24,29-31], cyanoacrylate [16], bromoacrylate
[22,23,26], acrylate [19,21,27], acetylacetonate [32,33], and trifluoroacetylacetonate [7,18]
but not cinnamate. Rhodol (or rhodafluor), a hybrid structure of rhodamine and fluorescein,
is a useful organic dye with excellent photophysical properties such as high extinction
coefficient, quantum yields, photostability, and strong fluorescence with low excitation
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wavelength [34,35]. It has been applied for fluorescence detection of various cations [36-42],
anions [36,39], NAD(P)H [43], glutathione and cysteine [44,45] but not for hydrazine.
Herein, we present a rhodol derivative appended with a cinnamate moiety as a highly
selective and sensitive turn-on fluorescent probe for hydrazine with a lower detection limit
and a faster detection time than some of previously reported probes. We envisioned that the
rhodol cinnamate conjugate would be colorless and non-emissive. A loss of the cinnamate
moiety, promoted by hydrazinolysis, would lead to a subsequent ring opening of the
spirocyclic moiety and trigger chromogenic and fluorogenic changes.
2. Materials and methods
All chemicals and solvents used in this work were of analytical grade, obtained commercially
and used without further purification. Column chromatography was performed using silica gel 60
(Merck). Other common solvents including CH2Cl2, EtOAc and hexane were distilled prior to use.
UV-vis absorption spectra were recorded on an Agilent 89090A spectrophotometer at ambient
temperature. Fluorescence measurements were performed on a Cary Eclipse fluorescence
1
13
spectrophotometer with a 10-mm quartz cuvette. H- and C-NMR spectra were obtained from a
Bruker AV-400 spectrometer in CDCl3 using tetramethylsilane (TMS) as an internal standard. ESI-
HRMS mass spectra were acquired from a Bruker MicrOTOF mass spectrometer.
2.1 Synthesis of rhodol cinnamate (RC)
Preparation of 2-(4-(N,N-diethylamino)-2-hydroxybenzoyl) benzoic acid (1)
Compound 1 was synthesized according to the previously reported procedure (Scheme 1)
[40]. A suspension of 3-diethylaminophenol (1.46 g, 10.6 mmol) and o-phthalic anhydride (1.57 g,
10.6 mmol) in toluene was refluxed for 20 h. The reaction solvent was removed under vacuo and the
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crude product was dissolved in CH2Cl2. The organic phase was washed with 1 N HCl and then dried
over anhydrous Na2SO4. After filtration and removal of the solvent, the crude product was purified
by silica gel column chromatography to give 1 as a white solid (2.08 g, 55% yield).
Scheme 1. Synthesis of rhodol cinnamate (RC).
Preparation of rhodol (2)
Rhodol (2) was obtained in excellent yield according to the reported literature procedure [40].
To a suspended solution of 1 (0.94 g, 3 mmol) in trifluoroacetic acid (TFA, 25 mL) was added
resorcinol (0.34 g, 3 mmol) and then the reaction mixture was stirred and refluxed for 12 h. The
reaction mixture was cooled to room temperature and then the solvent was evaporated under vacuo
to give the product precipitate. The crude product was further purified by recrystallization from
EtOAc to obtain rhodol 2 as a white solid (1.12 g, 3 mmol, 99 %yield).
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Preparation of rhodol cinnamate (RC)
Rhodol (2) (387 mg, 1.0 mmol) was stirred in CH2Cl2 (30 mL) and triethylamine (TEA) (5
mL) for 30 min (Scheme 1). The mixture was cooled to 0 °C and cinnamoyl chloride (330 mg, 2.2
mmol) in CH2Cl2 (10 mL) was slowly added and stirred overnight. The solvent was then removed
under reduced pressure and the obtained residue was further purified by column chromatography,
with EtOAc-hexane (40:60, v/v) as an eluent. The purified product was recrystallized in EtOH-hexane
1
to obtain the desired RC probe as colorless prism-like crystals (390 mg, 76% yield). H-NMR
(400 MHz, CDCl3) δ 8.17 (d, J = 7.5 Hz, 1H), 7.91 (d, J = 6 Hz, 1H), 7.68 (t, J = 7.4 Hz, 1H), 7.63
(t, J = 4 Hz, 1H), 7.62 (t, J = 3.5 Hz, 1H), 7.46 (t, J = 2.5 Hz, 3H), 7.26 (t, J = 9.3 Hz, 2H), 7.16 (d,
J = 1.7 Hz, 1H), 6.87 (d, J = 8.6 Hz, 1H), 6.83 (d, J = 8.6 Hz, 1H), 6.65 (d, J = 16 Hz, 1H), 3.39 (q,
J = 7.1 Hz, 4H), 1.25 (t, J = 7.1 Hz, 6H); 13C-NMR (100 MHz, CDCl3) δ 164.9, 152.9, 151.9, 147.2,
134.93, 134.0, 130.9, 129.7, 129.1, 128.4, 124.99, 124.2, 118.9, 110.3, 77.2, 12.4; HRMS (ESI) m/z
518.1966 [M+H]+, calcd for C33H28NO5 518.1962.
2.2. Spectroscopic experiments
A stock solution of RC (1 × 10-3 M) was prepared in EtOH. All stock solution (1 × 10-2 M)
of additives including cations (Na+, K+, Ag+, Ba2+, Ca2+, Mg2+, Co2+, Cu2+, Pb2+, Zn2+, Cd2+,
¯
2-
¯
3¯
¯
Fe2+, Fe3+, Pd2+, Ni2+, and Hg2+), anions (HCO3 , CO3 , NO3 , PO4 , Cl¯, I¯, Br¯, F¯, CN¯, N3 ,
-
2-
-
2-
HSO4 , SO4 , HSO3 , SO3 , SCN¯, CH3COO¯, and HS¯), and other neutral species (NH2OH,
CH3NH2, urea, thiourea, cysteine, and diaminopropane) were prepared in HEPES buffer (10 mM,
pH 7.4). All measurements were carried out in aqueous EtOH (1:1, v/v, 10 mM HEPES buffer, pH
7.4) at room temperature. The samples were excited at 520 nm with the 5-nm slit widths and the
emission spectrum was scanned from 525 nm to 700 nm.
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2.3. Cell culture and confocal fluorescence microscopy
HepG2 cells were cultured in Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12
(DMEM/F-12) with 10% FBS, 100 µg/ml streptomycin and 100 U/ml penicillin at 37 C in an
atmosphere of 5% CO2. Cells were plated on a 96-well microtiter plate and allowed to adhere for 24
h. The cultured cells (3 × 104/well) were incubated with Hoechst 33342 (20 µM) at 37 C for 10 min
and then treated with hydrazine (900 µM) in DMEM/F-12 medium at 37 C for 1 h. Treated cells
were then washed with DMEM/F-12 medium to remove excess hydrazine prior to incubation with
RC (30 µM, 0.1% DMSO) for 1 h. Cells were imaged in DMEM/F-12 medium at 37 C under 5%
CO2 with an Operetta high-content imaging system (PerkinElmer) using a 40× objective lens and the
Harmony® software. For the fluorescence imaging experiments, the emission wavelength was
collected at 560-630 nm and the excitation wavelength was set at 520-550 nm. For Hoechst-stained
nucleus, the emission wavelength was collected at 410-480 nm and the excitation wavelength was set
at 360-400 nm.
3. Results and discussion
The rhodol cinnamate probe (RC) was successfully synthesized in high yield via a
coupling reaction between rhodol and cinnamoyl chloride (Scheme 1). The structure of RC
was established by analysis of 1H-, 13C-NMR, and HR-ESIMS data. Its 1H-NMR spectral data
with the doublet signals at δ 7.91 and δ 6.65 ppm (J = 16 Hz) supported a trans-cinnamate
13
moiety. In the C-NMR data, the characteristic spirocarbon signal at δ 77.2 ppm indicated
the spirolactone ring in the closed form, and thus the compound was non-fluorescent due to
the absence of the conjugation system. Furthermore, the structure of RC was investigated by
a single-crystal X-ray crystallographic analysis and the results clearly confirmed the
spirocyclic lactone form (Fig. 1).
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Fig. 1. ORTEP plot of rhodol cinnamate (RC).
A colorless solution of RC in aqueous EtOH (1:1, v/v, 10 mM HEPES buffer, pH 7.4)
was non-emissive and showed no absorption band in the visible range (400-700 nm). Upon
the addition of hydrazine (10 equiv.), the solution of RC (10 M) turned orange with a new
absorption band centered at 520 nm and strongly emitted yellow-orange fluorescence with a
maximum emission peak at 545 nm (Fig. S1). This optical change was due to the ring-opening
process of the spirolactone that gave fluorescein-like spectroscopic properties [39,41,42,45].
To evaluate the selectivity of RC to various cations, anions and neutral species (10 equiv.)
including Na+, K+, Ag+, Ba2+, Ca2+, Mg2+, Co2+, Cu2+, Pb2+, Zn2+, Cd2+, Fe2+, Fe3+, Pd2+, Ni2+,
3-
-
2-
-
2-
-
Hg2+, HCO3-, NO3-, PO4 , Cl-, I-, Br-, F-, CN-, N3 , CO3 , HS-, SCN-, HSO4 , SO4 , HSO3 ,
2-
SO3 , CH3COO-, NH2OH, CH3NH2, urea, thiourea, cysteine, and diaminopropane, were
investigated for their ability that may affect the fluorescence change of RC compared with
hydrazine. These analytes did not trigger any fluorescence change of the probe indicating that
RC could be used as a highly selective “off-on” fluorescent chemosensor for hydrazine (Fig.
2). Interference studies also suggested that the fluorescence response of RC to hydrazine
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remained unaffected in the presence of the above-mentioned neutral and ionic additives (Fig.
S2).
Fig. 2. Fluorescence spectra of RC (10 M) in aqueous EtOH solution (1:1, v/v, 10 mM HEPES
buffer pH 7.4) upon the addition of various additives (Na+, K+, Ag+, Ba2+,
Mg2+, Co2+, Cu2+,
3-
2-
-
Pb2+, Zn2+, Cd2+, Fe2+, Fe3+, Pd2+, Ni2+, Hg2+, HCO3-, NO3-, PO4 , Cl-, I-, Br-, CO3 , CN-, N3 , F-,
2-
2-
HS-, SCN-,
SO4 , SO3 , CH3COO-, NH2OH, CH3NH2, urea, thiourea, cysteine,
and diaminopropane) and hydrazine (10 equiv.) for 30 min (λex = 520 nM). Inset: a) color change
observed in visible light and b) fluorescence emission under blue-LED illumination.
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A quantitative “off-on” response of RC was further investigated by a fluorescence
titration study. The emission spectra of RC (10 µM) solutions with increasing concentrations
of hydrazine in aqueous EtOH (1:1, v/v, 10 mM HEPES buffer, pH 7.4) were recorded (Fig.
3). Upon the addition of hydrazine (0-300 equiv.), the fluorescence intensity at 545 nm of RC
was responsive to the hydrazine concentrations with a linear response in the range of 0.25-1.5
mM. The limit of detection (3σ/slope) of RC to hydrazine, the lowest concentration of
hydrazine that the probe can reliably detect [46], was calculated to be 300 nM (9.6 ppb), below
the TLV recommended by the U.S. EPA, and lower than those of several other reported
hydrazine-sensitive probes (Table S1) [16-18,22,24]. For the kinetic of the reaction, the
fluorescence intensity of RC with hydrazine (10 and 20 equiv.) at 545 nm in aqueous EtOH
Fig. 3. Fluorescence titration of RC (10 M) in aqueous EtOH solution (1:1, v/v, 10 mM HEPES
buffer, pH 7.4) upon the addition hydrazine (0-3.0 mM) (λex = 520 nM). Inset: a linear plot of
[I/I0-1] vs. the hydrazine concentration (0.25-1.5 mM).
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(1:1, v/v, 10 mM HEPES buffer, pH 7.4) revealed that fluorescence intensity was increased
to reach a plateau within 30 min (Fig. S3), faster than some reported hydrazine probes
[7,19,22,47,48].
In order to understand the transformation of RC by hydrazine, the reaction between
RC and hydrazine (1:1 molar ratio) in DMSO-d6 (Scheme 2) was investigated with 1H NMR
and HR-ESIMS analyses. It is plausible that hydrazinolysis of RC occurred via a nucleophilic
1,4-addition of hydrazine at the β-carbon of the cinnamate moiety to give an enol which was
then tautomerized to a carbonyl group. Subsequently, the nucleophilic amine attacked the
carbonyl ester with the intramolecular cyclization then eliminated the rhodol unit and
triggered a ring-opening of spirolactone and the fluorescence emission (
4). A major molecular
ion of rhodol [M+H]+ m/z 388.1547 was detected (calcd 388.1543) along with a molecular ion
m/z 161.0686 (calcd. 161.0709) for the corresponding cyclization product (
5
) (Fig. S10). The
1
ring-closing reaction of hydrazine was similar to other reported systems [23,26]. In the H
Scheme 2. A proposed mechanism of hydrazine detection by RC.
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NMR data, the trans-Ha and Hb doublet signals of RC at
were absent upon incubation with hydrazine (Fig. 4b). The transformation was also confirmed
by a down-field shift of the doublet Hc peak of RC from 8.03 ppm (Fig. 4a) to 8.12 ppm
due to the presence of more electrophilic carboxylic group in (Fig. 4b) [40]. In addition, the
 6.91 ppm and  7.90 ppm (Fig. 4a)


4
reactivity of rhodol butanoate, a saturated analog of RC, was evaluated and it was found to be
20 times less reactive toward hydrazine than RC, supporting the significance of the cinnamate
ester (Fig. S11).
Fig. 5. Confocal fluorescence images of hydrazine detection by RC in live HepG2 cells. The
cultured cells were incubated with hydrazine (900 M) in DMEM/F-12 medium for 1 h (e-h) and
then incubated with RC (30 M) for 1 h (a-h). (a) and (e) Bright-field images; (b) and (f)
fluorescence images of nuclei counterstained with Hoechst 33342; (c) and (g) fluorescence
images; (d) and (h) merged fluorescence images of (b)-(c) and (f)-(g), respectively.
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The ability of RC to detect hydrazine in cellular environments was further investigated
in live HepG2 human liver cancer cells by confocal fluorescence microscopy (Fig. 5). HepG2
cells incubated with hydrazine (900 M) for 1 h prior to incubation with RC (30 M) for 1 h
showed a distinct intracellular fluorescence emission (Fig. 5g). For the control experiment,
the cells incubated with only RC were not emissive (Fig. 5c). The cytotoxicity was also
evaluated for RC (30 M) and for hydrazine (1500 M), showing that there was no any
adverse effect on cell viability (Fig. S12). These results suggested that RC was membrane
permeable and could be utilized to detect hydrazine in living cells.
4. Conclusion
The rhodol-cinnamate chemosensor RC was successfully developed as a hydrazine-
selective probe with a turn-on fluorescence response at 545 nm. It was demonstrated that the
cinnamate group could be used as a hydrazine-sensing unit with rhodol as a reporter group.
The chemosensor was successfully applied to monitor hydrazine in HepG2 cells by
fluorescence microscopy.
Acknowledgments
We would like to acknowledge Mahidol University, National Research University Project, the
Thailand Research Fund, and Center for Innovation in Chemistry (PERCH-CIC) for financial
support. K.T. and R.N. thank the Development and Promotion of Science and Technology Talents
Project (DPST) under the Royal Thai Government for scholarships.
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Fig. 4
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