A fluorogenic and red-shifted diphenyl phosphinate-based probe for selective peroxynitrite detection as demonstrated in fixed cells

Article abstract of DOI:10.1039/c7nj02530k

The development of NIR fluorescent probes for biomedical applications has outstanding potential because of the lower background fluorescence present involving less scattering and deeper tissue penetration, resulting in limited damage to living cells. Recently, dicyanomethylene-4H-pyran systems have been utilized as an NIR fluorophore for the detection of various analytes and in cell imaging studies. These efforts have involved single- as well as two-photon microscopy. As part of an effort to generate novel probes that can allow biologists/neurobiologists better visualization/quantification of the role of reactive oxygen/nitrogen species in the living system, a new dicyanomethylene-4H-pyran-based fluorescent probe DCPO-DP has been designed, synthesized and characterized. The fluorescence of the probe is pre-quenched by hydroxyl protection (diphenyl phosphinate protecting group). A selective "TURN-ON" fluorescence response involving an ～120-fold emission intensity enhancement was observed upon reaction with ONOO^-, compared with emission signals arising from other ROS/RNS. The reaction was completed within 20 min. The detection limit was determined to be 4.62 μM. Fixed cells were used in imaging experiments and helped demonstrate this novel chemosensing strategy for the detection of ONOO^- in living systems with phosphinates.

Full text of DOI:10.1039/c7nj02530k

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Jason B. Benedict et al.
The role of atropisomers on the photo-reactivity and fatigue of
diarylethene-based metal–organic frameworks
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Fluorogenic and red-shifted diphenyl phosphinate-based probe
for selective peroxynitrite detection as demonstrated in fixed cells
Received 00th January 20xx,
Accepted 00th January 20xx
Sandip V. Mulay,[a,b] Youngsam Kim, [b,a] Kyung Jin Lee,[c] Tesla Yudhistira,[b] Hee-Sung Park,[c] and
David G. Churchill [b,a]
*
DOI: 10.1039/x0xx00000x
www.rsc.org/
The development of NIR fluorerscent probes for biomedical applications has outstanding potential because of the lower
background fluorescence present involving less scattering, deeper tissue penetration, resulting in limited damage to living
cells. Recently, dicyanomethylene-4H-pyran systems have been utilized as an NIR fluorophore for the detection of various
analytes and cell imaging studies. These efforts have involved single- as well as two-photon microscopy. As part of an
effort to generate novel probes that can allow biologists/neurobiologists better visualization/quantification of the role of
reactive oxygen/nitrogen species in the living system, a new dicyanomethylene-4H-pyran-based fluorescent probe DCPO-
DP has been designed, synthesized and characterized. The fluorescence of the probe is pre-quenched by hydroxyl
protection (diphenyl phosphinate protecting group). A selective “TURN-ON” fluorescence response involving a ~120-fold
emission intensity enhancement was observed upon reaction with ONOO−, compared with emission signals arising from
other ROS/RNS. The reaction was completed within 20 min. The detection limit was determined to be 4.62 μM. Fixed cells
were used in imaging experiments and helped demonstrate this novel chemosensing strategy for detection of ONOO− in
living systems with phosphinates.
neurodegenerative disease states (e.g., Alzheimer’s disease),
cancer, arthritis, autoimmune and inflammatory diseases as well as
other diseases/disorders.1c, 5 Similar to other ROS/RNS, ONOO− is
Introduction
Peroxynitrite (ONOO−) is a short-lived and highly reactive species
generated by a diffusion-controlled reaction between nitric oxide
and superoxide; ONOO− acts as a powerful oxidizing and nitrating
species in biology and is of great interest to study in vivo.1 ONOO− in
living systems is formed intracellularly, mainly in the
mitochondria;1c, 2 ONOO− has the ability to interact/react with other
biological species in the human body such as proteins, nucleic acids,
lipids and other related and essential biological species.1c, 3 The
highly reactive nature of ONOO− towards biological species is due to
its strong nucleophilicity. ONOO− is capable of generating reactive
secondary radicals together with the hydroxyl radical (•OH),
nitrogen dioxide (•NO2) and carbonate radicals (CO3•−). These
newly-formed secondary radicals further interact with bioactive
species (in the human body) that lead to the damage of cells and
cell death.4 Concentrations of ONOO− in many pathogenic processes
also playing an important role in health: the immune system and in
signal transduction pathways in living systems.4, 6 To understand
more details about the mechanism of ONOO− in biology, novel
systems with enhanced properties are required. The development
of next-generation selective, sensitive, and rapid responsive
detection techniques are necessary and may take on a multitude of
designs. In recent years, fluorescent probes have been used
extensively for important biological interrogations; they are
sensitive and efficient analytical tools marked by simplicity, give
high temporal and spatial resolutions in imaging, possess non-
invasive characteristics and can be used for real-time detection and
imaging in living systems of a myriad of analytes, many of which are
related (ROS/RNS, biothiols, metal ions etc).7
In particular, a number of small-molecular fluorescent probes
have been designed and tested to-date for the detection of ONOO−,
based on peroxynitrite-triggered reactions such as the nitration of
aromatic [C-H] positions,8 oxidation of organoselenium/tellurium
compounds,9 oxidative cleavage,10 formations of dioxirane with
activated ketones11 and oxidative N-dearylation reactions.12 Yang
and co-workers have reported the novel fluorescent probe for
selective detection of ONOO− from other competing ROS, such as
HOCl and H2O2, based on the chemical oxidizing ability of ONOO−.13
The phosphyl/phosphinate functional group [-P(O)Ph2], therefore,
was explored for the selective detection of superoxide over other
ROS by simple and rational detachment via hydrolytic cleavage
through an addition-elimination pathway.14
correlates with
a
number of human diseases such as
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Recently, dicyanomethylene-4H-pyran-based moieties have been Synthetic procedures
utilized as a module for intended NIR fluorophore detection of
2-(2-Methyl-4H-chromen-4-ylidene)malononitrile (2): Compound
1 was synthesized by a known literature procedure.15a 2-Methyl-4H-
chromen-4-one 1 (1.60 g, 10.0 mmol) and malononitrile (0.86 g, 1.3
mmol) were dissolved in 10 mL acetic anhydride. The solution was
refluxed for 15 h; the solvent was then evaporated under reduced
pressure. Deionized water (35 mL) was added to the residue and
the mixture was refluxed for another 0.5 h, followed by extraction
with CH2Cl2. The combined organic layers were dried over Na2SO4,
filtered, and concentrated under reduced pressure. The obtained
crude product was purified by silica gel column chromatography
using hexane and EtOAc (5:1 to 3:1, v/v) to afford 2 as an orange
solid (0.94 g, 45 %). mp = 187–188 °C; IR (CHCl3) νmax = 3038 (w),
2961 (w), 2918 (w), 2212 (m), 1635 (s), 1509 (m), 1459 (w), 1397
(w), 1323 (w), 1270 (w), 1226 (w), 1142 (w), 1033 (w), 975 (w), 852
(m), 771 (m) cm-1; 1H NMR spectroscopy (600 MHz, CDCl3/TMS): δ =
2.43 (s, 3H, H14), 6.68 (s, 1H, H3), 7.43 (t, 3JH-H = 7.8 Hz, 1H, H7), 7.46
(d, 3JH-H = 8.5 Hz, 1H, H5), 7.71 (t, 3JH-H = 7.8 Hz, 1H, H6), 8.86 (d, 3JH-H
= 8.4 Hz, 1H, H8); 13C NMR (150 MHz, CDCl3): δ = 20.6 (C14), 62.3
(C11), 105.5 (C3), 115.5 (C13), 116.7 (C12), 117.6 (C10), 118.8 (C5),
125.8 (C8), 126.1 (C7), 134.7 (C6), 152.9 (C4), 153.3 (C9), 161.9 (C2);
HR-MS (ESI): calcd for C13H8N2O + Na: 231.0534, found: m/z
231.0506 (M + Na)+.
various analytes and cell imaging studies using single-, as well as
two-photon microscopy.15 Due to the excellent photophysical
properties imparted by this probe class, ((i) large Stokes’ shift, (ii)
comparable photostability e.g. to the squaraine and cyanine class,
(iii) emission wavelength at the near infrared region, etc)
dicyanomethylene-4H-pyran-based
molecular
probes
are
potentially very suitable for applications in biological systems; such
probes (i) produce lower background fluorescence with less
scattering, (ii) deeper tissue penetration and (iii) cause less damage
to living cells, all constituting important “upsides” to this
approach.16 Strategies utilized for these reports are based on simple
protection of a phenol or amine moiety with reactive functional
groups to make the chromophore/fluorophore non-fluorescent.
Herein, we have designed and synthesized DCPO-DP (masking of
the DCPO chromophore with diphenyl phosphinate group to disable
fluorescence) for the selective turn-on response with the ONOO−
analyte under a range of bioanalytic conditions (Scheme 1). The
strong nucleophilic propensity of ONOO− was explored for the
intended goal of selective detection over other reactive oxygen/
nitrogen species and biothiols (Cys, Hcy and GSH). In addition, to
demonstrate this strategy, cell imaging experiments were carried
out with fixed Hela cells.10d, 10f, 17 Thus, in this paper, a simple
protection/deprotection strategy for selective detection of ONOO−
was carried out.
(E)-2-(2-(4-Hydroxystyryl)-4H-chromen-4-ylidene)malononitrile
(DCPO): Compound
2
(0.90 g, 4.3 mmol) and 4-
hydroxybenzaldehyde (0.53 g, 4.3 mmol) were dissolved in
anhydrous toluene (20 mL) whereupon piperidine (1.3 mL, 12.9
mmol, 3.0 equiv.) and acetic acid (0.75 mL, 12.9 mmol, 3.0 equiv.)
were added. The reaction mixture was stirred under reflux for 12.0
h. After completion of the reaction (as monitored by TLC), the
reaction mixture was cooled to room temperature and then
concentrated under reduced pressure. The crude residue was
purified by silica gel chromatography using hexane and EtOAc (4:1
to 2:1, v/v) to give DCPO as a red solid (0.55 g, 41%). mp = 148–149
°C; IR (CHCl3) νmax = 3337 (br), 2939 (w), 2208 (m), 1628 (m), 1596
(s), 1556 (s), 1500 (m), 1456 (w), 1404 (w), 1266 (w), 1198 (m), 1168
(w), 1023 (w), 971 (w), 821 (w), 778 (w) cm-1; 1H NMR spectroscopy
(600 MHz, DMSO-d6): δ = 6.76 (s, 1H, H3), 6.80 (d, 3JH-H = 8.3 Hz, 2H,
H18), 7.08 (d, 3JH-H = 16.0 Hz, 1H, H15), 7.48 (t, 3JH-H = 7.8 Hz, 1H, H7),
7.51 (d, 3JH-H = 17.0 Hz, 1H, H14), 7.52 (d, 3JH-H = 6.5 Hz, 2H, H17), 7.62
(d, 3JH-H = 8.3 Hz, 1H, H5), 7.80 (t, 3JH-H = 7.7 Hz, 1H, H6), 8.59 (d, 3JH-H
= 8.3 Hz, 1H, H8), 10.11 (s, 1H, OH, H19); 13C NMR spectroscopy (150
MHz, DMSO-d6): δ = 59.2 (C11), 105.6 (C3), 115.7 (C15), 115.9 (C13),
116.0 (2C18), 117.0 (C12), 117.3 (C10), 118.8 (C5), 124.5 (C8), 125.9
(C16), 126.0 (C7), 130.3 (2C17), 135.1 (C6), 139.1 (C14), 151.8 (C9),
152.5 (C4), 158.7 (C2), 160.0 (C19); HRMS (ESI): calcd for C20H10N2O2 +
Na: 335.0796, found: m / z 335.0776 (M + Na)+.
Scheme 1. Sensing mechanism of DCPO-DP for ONOO− anion.
Experimental
Materials and methods: All chemicals used herein were used as
received from commercial suppliers (Aldrich, Tokyo Chemical
Industry). Thin layer chromatography (TLC) was performed on DC
Kieselgel 60 F254 silica gel plates. The spots were visualized by
staining with KMnO4 or by using a bench-top UV lamp. Column
chromatographic purification was carried out on silica gel (230–400
mesh ASTM). 1H, 13C, and 31P NMR spectra were acquired using a
Bruker Avance 400 and Agilent-NMR-vnmrs 600 MHz spectrometer.
TMS was used as an external standard. Infrared (IR) spectra were
acquired on Bruker Alpha ATR FTIR spectrometer. Frequencies are
given in reciprocal centimeters (cm-1). Bands are characterized as
broad (br), strong (s), medium (m), or weak (w). ESI-mass
spectrometry was performed on a microTOF-Q II type instrument
by BRUKER; these experiments were carried out by the research
support staff at KAIST. A Time-of-Flight mass spectrometer was
operated at a resolution of 20,000. Absorption spectra were
Synthesis of Probe (DCPO-DP): A solution of DCPO (312 mg, 1.0
mmol) in anhydrous THF (30 mL) was prepared and triethylamine
(0.21 mL, 1.5 mmol, 1.5 equiv) and Ph2P(=O)Cl (0.19 mL, 1.0 mmol,
1.0 equiv) were added dropwise at room temperature. The reaction
mixture was refluxed for 45 min. After cooling to room
temperature, water (30.0 mL) was added to the reaction mixture;
the mixture was repeatedly extracted with EtOAc (3 × 30 mL). The
combined organic layers were washed with brine, dried over
Na2SO4, filtered and concentrated under reduced pressure. The
crude product was purified by silica gel column chromatography
using hexane and EtOAc (5:1 to 3:1, v/v) to give DCPO-DP as a red
solid (420 mg, 82%). mp = 219–220 °C; IR (CHCl3) νmax = 3055 (w),
2979 (w), 2210 (m), 1630 (m), 1595 (s), 1557 (m), 1501 (s), 1481
(m), 1405 (m), 1334 (w), 1231 (m), 1203 (m), 1169 (m), 1130 (w),
measured using
a JASCO V–530 UV-Vis spectrophotometer.
Fluorescence measurements were carried out with a Shimadzu RF–
5301pc spectrofluorophotometer.
2 | J. Name., 2012, 00, 1-3
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977 (w), 910 (m), 747 (m) cm-1; 1H NMR spectroscopy (600 MHz, incubated further for 24 h. Subsequent washing with PSBS (2×) and
3
CDCl3/TMS): δ = 6.65 (d, JH-H = 15.9 Hz, 1H, H15), 6.79 (s, 1H, H3), treatment with the proliferation reagent WST-1 (10 μL) to each well
3
3
7.29 (d, JH-H = 8.3 Hz, 1H, H18), 7.40 (t, JH-H = 7.8 Hz, 1H, H7), 7.46 was carried out; the reagent-applied cells were incubated for 2 h at
3
(d, JH-H = 8.7 Hz, 2H, H17), 7.47−7.50 (m, 5H, H14, 22, 25), 7.51 (d, 3JH-H 37°C, 5% CO2. Absorbances were measured using a Varioskan®
= 6.9 Hz, 1H, H5), 7.56 (t, 3JH-H = 7.5 Hz, 2H, H23,27), 7.70 (t, 3JH-H = 7.8 FLASH (Thermoscientific) at 450 nm and the reference wavelength
3
3
Hz, 1H, H6), 7.89 (d, JH-H = 8.2 Hz, 2H, H21), 7.91 (d, JH-H = 8.0 Hz, was 590 nm.
2H, H25), 8.86 (d, JH-H = 8.3 Hz, 1H, H8); 13C NMR spectroscopy (150
3
MHz, CDCl3): δ = 62.9 (C11), 106.9 (C3), 115.7 (C13), 116.8 (C12), 117.8
(C10), 118.4 (C15), 118.7 (C5), 121.5 (C18), 121.6 (C18′), 125.8 (C8),
126.1 (C7), 128.8 (2C22), 128.9 (2C26), 129.5 (2C17), 130.2 (C16), 131.1
(C20), 131.2 (C24), 131.8 (2C21), 131.9 (2C25), 132.8 (C23), 132.9 (C27),
134.8 (C6), 137.9 (C14), 152.3 (C9), 152.7 (C4), 152.8 (C19), 157.4 (C2);
31P NMR spectroscopy (162 MHz, CDCl3): δ = 32.0; HRMS (ESI): calcd
for C32H21N2O3P + Na: 535.1187, found: m/z 535.1176 (M + Na)+.
NC
CN
O
O
OH
OHC
NC
CN
Piperidine, acetic acid
toluene, reflux, 12 h,
41%
Acetic anhydride,
reflux, 15 h, 45%
O
2
1
NC
CN
NC
CN
Ph2P(O)Cl, Et3N
THF, rt, 30 min,
82%
O
O
O
P
O
OH
DCPO
DCPO-DP
Scheme 2. Synthesis of Probe DCPO-DP.
DFT Calculations:
Geometry optimized structures and HOMO–LUMO distributions of
the states were estimated by calculational methods specifically
using DFT/TDDFT calculations and using the Gaussian 09 program.
The B3LYP functional with a 6–31g* basis set was used. All
calculations were performed in the gas phase. Configuration
interaction (CI) coefficients were obtained by square of the values.
Figure 1. (a) Absorption spectra of DCPO-DP (10.0 µM) with 10.0 equiv. of
•−
ROS/RNS (ONOO−, O2 , H2O2, NO, NaOCl, •OH, tBuO•, and tBuO2H) in solution
(DMSO / 10 mM PBS, pH 7.4, 1:1 v/v) and incubated for 20.0 min at RT; (b)
Plot of relative fluorescence intensity maxima of ONOO−, O2•−, H2O2, NO,
t
t
•
NaOCl, OH, BuO•, and BuO2H (10.0 μM) trials upon addition of ROS/RNS in
solution (DMSO / 10 mM PBS, pH 7.4, 1:1 v/v), incubation time: 20.0 min,
λex: 555 nm, λem: 690 nm, slit width 5 nm / 5 nm, (Data are expressed as
mean of three experiments accompanied by a ± SD value). Inset: Selectivity
of DCPO-DP with ROS/RNS; λex: 555.
Cell maintenance and fluorescence imaging:
HeLa cells were cultured in an Eagle's minimum essential medium
(MEM, Gibco) supplemented with 10 % (v/v) fetal bovine serum
(Gibco) and 1% penicillin/streptomycin (100 U/mL, Gibco). The HeLa
cells were seeded at a density of 0.4 × 105 cells on sterilized μ-slides
(Ibidi, München, Germany). After 24 h, 40 μM DCPO-DP were added
to the cells and the cells were incubated for 30 min at 37 °C. Next,
cells were washed with D-PBS 2 times fixation with 4% (w/v)
paraformaldehyde for 30 min at room temperature was carried out;
washing two times was then performed and before their being
treated with ROS/RNS. Fluorescent images were acquired on Zeiss
LSM 780 (Carl Zeiss, Hena, Germany) laser scanning confocal
microscope and a 40× and 63× objective lens were used. The
excitation wavelengths were 405 nm, 561 nm; detection
wavelengths were 410–498 nm for DAPI, 630-700 nm for DCPO-DP
and DCPO, respectively.
Results and discussion
The synthesis of the molecular probe central to our contribution
and discussed within this paper is depicted in Scheme 2. The
synthesis of compound DCPO was achieved by a known literature
procedure.15a In brief, the reaction of chromone
1
with
dicyanomalonate gave compound 2; then, the Knoevenagel
condensation of with 4-hydroxybenzaldehyde under reflux
2
condition afforded DCPO as a red solid. The hydroxyl group of DCPO
was protected with diphenyl phosphinate [P(O)Ph2] in the presence
of trimethylamine; this reaction sequence provided probe DCPO-DP
in high yield. The structure of 2, DCPO, and DCPO-DP were
characterized by spectroscopic techniques (multinuclear NMR
spectral data, as well as mass spectrometric data; Figs. S1-18,
Supporting Information).
Cell viability assays:
The proliferation of HeLa cells was determined by a WST-1 assay kit
(Roche) using the manufacturer’s protocols. Briefly, the HeLa cells
were seeded at a density of 1 × 104 cells into a 96-well plate (n = 4).
Next, cells were pretreated with the DCPO-DP at various
concentrations (10, 20, 30, 40, 50 μM) for 30 min and then
It has been reported that when the phenolic hydroxyl group of the
DCPO chromophore is protected, the chromophore becomes non-
emissive. Initially, the response of DCPO-DP (15.0 µM in the
solution of DMSO/10 mM PBS, pH = 7.4, 1:1 v/v) with ROS/RNS
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were examined using UV-vis absorption spectroscopy. UV-vis for ONOO−. This results shows that the current probe could be
absorption spectra of DCPO-DP with other ROS/RNS including useful for the detection of ONOO− at physiological pH.
ONOO− are shown in Figure 1a; DCPO-DP shows two maxima in the
To measure the sensitivity of the probe, titration experiments of
DCPO-DP were carried out at various concentrations of ONOO− (0.0
UV-vis absorption spectra at ~415 and ~440 nm. However, upon the
addition of ONOO− (10.0 equiv) to the DCPO-DP the absorbance
to 20.0 equiv); these results are depicted in Figure 2. Upon
increasing the concentration of ONOO−, absorption peaks at 415
causes the peak to undergo a red shift whereupon a maximum
at ~555 nm is produced.
and 440 nm were found to decrease; simultaneously new red-
Upon addition of other ROS/RNS, there were no significant shifted single peak at 570 nm with concomitant increases in
observed changes of any sort in the absorption spectra of intensity were observed (Figure 2a). Fluorimetric titration results
DCPO-DP. Moreover, the absorption spectral features of revealed that the emission intensity gradually increased upon the
DCPO-DP after treatment with ONOO− were matched with the addition of ONOO− from 0 to 20.0 equiv; it was found to be linearly
spectra of DCPO under similar conditions. (Figures S19-S20, proportional to the concentration of ONOO− (Figure 2b, Figure S25,
Supporting Information). Next, selectivity experiments of the Supporting Information). The DCPO-DP reached emission intensity
DCPO-DP (15.0 μM) with ROS/RNS were performed using saturation after the addition of 18.0 equiv of ONOO−. Further, to
fluorescence emission spectroscopy (Figure 2b). Excellent determine the limit of detection, a linearity curve was prepared,
selectivity of the probe towards ONOO− (20.0 equiv) was plotted from 0.0 to 10.0 equiv of ONOO−. The limit of the detection
observed; there were no observable changes in emission for DCPO-DP (3σ/k, where σ represents the standard deviation of
intensity with other ROS/RNS including competitive species 10 blank samples measurements; k is the slope of a linear equation)
such as superoxide (O2•−) at higher concentration (up to 50.0 were determined to be 4.62 µM (Figure S25, Supporting
equiv). Importantly, probe DCPO-DP is virtually non- Information).
fluorescent (very low background fluorescence); after the
addition of ONOO− analyte to the probe solution, it shows a
“turn-on” response with a maximum emission intensity at 690
nm; the enhancement in fluorescence intensity was found to
be ~120-fold. It is well-known that, DMSO is unstable to some
oxidizing agents, such as ClO− and •OH. To demonstrate the
“TURN-ON“response is only due to the reaction of probe with
ONOO−, we performed the selectivity experiment in an
alternative solution (CH3CN/10 mM PBS, pH = 7.4, 1:1 v/v); the
obtained results showed selectivity with only ONOO− (Figures
S21, Supporting Information). The presence of metal ions in
solution of DCPO-DP were thought to be potential interefering
species; they may favorably bind the phosphyl/phosphinate
group. This may cause interference for the reaction of the
probe with ONOO−. For this, we then added portions of various
metal ions into the solution of DCPO-DP (15.0 µM) then
followed by addition of ONOO− (20.0 equiv). Through these
experiments we determined that metal ion interference is not
brought to bear in the emission properties of DCPO-DP and
ONOO− reactions (Figure. S22, Supporting Information). Also,
the combination of superoxide and nitric oxide does not show
any sort of “TURN-ON“ response with DCPO-DP under similar
conditions (Figure. S22, Supporting Information).
After the UV-vis absorbance study was complete, time-
dependent experiments of the DCPO-DP (15.0 μM) were
carried out; this involved the addition of 30.0 equiv of ONOO−
Figure 2. (a) Absorption spectral changes of DCPO-DP (15.0 µM) with
addition of ONOO− at various concentrations (0.0 to 20.0 equiv.) in solution
(DMSO / 10 mM PBS, pH 7.4, 1:1 v/v) incubated for 30 min at RT; (b)
Fluorescence emission spectral changes of DCPO-DP (15.0 µM) with
various concentrations of ONOO− (0 – 20.0 equiv) in solution (DMSO / 10
mM PBS, pH 7.4, 1:1 v/v) incubated for 30.0 min at RT, λex = 555 nm, slit
width 5 nm/5 nm.
It is known that ONOO− act as a nucleophile in biological systems.4
To understand the proposed sensing mechanism (nucleophilic
attack of the ONOO− to phosphinate group to give fluorescent
DCPO, Scheme 1), the reaction of ONOO− with DCPO-DP was
carried out under similar conditions (DMSO and 10 mM PBS, pH 7.4,
1:1 v/v). The reaction product was isolated; then 1H NMR, HRMS
spectral data on the isolated product were recorded (Figs. S26-S27,
Supporting Information). The 1H NMR spectrum of the isolated
product showed a similar pattern to that for DCPO. HRMS data of
(Figure S23, Supporting Information). The maximum
fluorescence intensity was attained and saturated within ~20
min; this result suggests that the complete cleavage of the
phosphinate group takes around 20 min. Next, and
importantly, to demonstrate the effect of pH on the DCPO-DP
probe, the fluorescence emission of the probe was tested with
a wide range of pH (pH 4 to 12) and interrogated at the
wavelength λex = 555 nm. The results show that under acidic
media, DCPO-DP is stable, whereas under basic media
(concentration of OH-) it undergoes cleavage of the
phosphinate group. Futhermore, ONOO− (20.0 equiv.) was
added to the solutions of DCPO-DP (15.0 µM) at different specific
pH values; then the fluorescence emission intensity values were
acquired and analyzed (Figure S24, Supporting Information). In the
pH 6-12 regime, DCPO-DP showed a strong fluorescence intensity
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the isolated product shows m/z peak at 335.0775 (found) is
consistent with that for the calcuated value for DCPO [335.0796
(calc.) [M + H]+ (Figure S27, Supporting Information). The GC-MS
spectrum of the crude product from the reaction of DCPO-DP and
ONOO− showed a m/z data peak at 217.1 (Figure S28, Supporting
Information); the m/z peak at 217.1 might be the species 3 (for
structure 3, Please see Supporting Information, Figure S28). These
NMR, Mass and GC-MS results strongly support that the ONOO−
analyte cleaves the C-O phosphinate bond in DCPO-DP and affords
the fluorescent DCPO compound as shown in Scheme 1.
Finally, to support the photomechanism and enhancement of the
strong red fluorescence after cleavage of phosphinate ester, density
functional theory (DFT) and time-dependent density functional
theory (TD-DFT) were exploited in tandem with the understanding
of the experimental results. The structures of DCPO and DCPO-DP
were optimized with the B3LYP/6-31g* basis set; electronic
distributions were obtained from the optimized structures. The
electronic distributions of the highest occupied molecular orbital
(HOMO) and the lowest unoccupied molecular orbital (LUMO) in
DCPO are populated on the fluorophore. Moreover, the largest
intense transition of DCPO is from HOMO to LUMO with f = 1.0601
(CI = 89.6 %) (Table S1). These results support that DCPO provides
strong fluorescence. On the other hand, DCPO-DP showed two
strong intense transitions represented by f = 0.3771 and 0.6474.
The dominant transition of DCPO-DP is from HOMO-2 to LUMO
with CI = 83.2 %. Considering the electronic distributions between
HOMO-2 and LUMO in DCPO-DP (Figure S29, Supporting
Information), they do not overlap in the molecule: this makes
DCPO-DP non-fluorescent. These data assist in supporting the
proposed mechanism in which the “turn-on” fluorescence
originates from the cleavage of phosphinate ester group.
Figure 4. Confocal fluorescence images of HeLa cells; a) cells pretreated with
DCPO-DP (40 μM) for 30 min; b) cells were treated with DCPO-DP (40 μM)
for 30 min washed by D-PBS after fixing and followed by incubation with
ONOO− (2 mM) for 1 min; c) cells treated with DCPO (40 μM) for 30 min.
Furthermore, to support these results that the fluorescence
response is selectively created by the reaction of ONOO− and DCPO-
DP, confocal fluorescence microscopy in living cells (HeLa cells) was
undertaken. HeLa cells were treated with DCPO-DP for 30 min
followed by incubation with 400 μM (10.0 equiv) and 2 mM (50.0
equiv) and of peroxynitrite (ONOO−) for 1 min. The confocal
fluorescence images are shown in Figure S30 and Figure 4. Control
cells (cells treated with DCPO-DP) were almost non-fluorescent
(Figure 4a). Cells pre-treated with DCPO-DP and then with ONOO−,
however, showed strong red fluorescence (Figure 4b). Moreover,
the red fluorescence observed in Figure 4b show the same
fluorescence when HeLa cells were treated with DCPO only (Figure
4c). Also, to confirm that observed red fluorescence in HeLa cells is
Finally, to demonstrate biological applications of the DCPO-DP in
live cells, HeLa cells were chosen as a biological medium for these
chemosensing experiments. To confirm the biocompatibility
of DCPO-DP, the first point of study involved cell viability testing.
HeLa cells were pre-incubated with various concentrations of the
probe (10, 20, 30, 40, and 50 μM). Then, WST-1 cell proliferation
assays were carried out after cells were pre-incubated in DCPO-DP
for 30 min. As shown in Figure 3, there was retention of cell viability
values after treatment with the probe. These results revealed no
significant cytotoxicity of the DCPO-DP at concentrations of up to
50.0 μM; this helps gain a better understanding for potential
biological applications.
only due to the cleavage of phosphinate with ONOO−, control
•−
)
experiments with other closely-related ROS (H2O2, NaOCl, and O2
were carried out (Figure S31, Supporting information). From these
results, we believe that the red fluorescence in cells is due to
nucleophilic displacement of the phosphinate group with ONOO− so
to generate highly fluorescent DCPO in cellular media.
Conclusions
Herein, we have reported the design and development of a
simple “TURN-ON” fluorescent chemosensing system for the
selective detection of ONOO− based on the nucleophilic
cleavage of diphenyl phosphinate. The probe displays excellent
selectivity, and sensitivity. Short time responses (< few
minutes) were also noted for the detection of peroxynitrite.
The probe showed fluorescence enhancement of up to ~120-
fold and afforded detection limits of 4.62 μM. It was found
that in the DCPO-DP, the diphenyl phosphinate blocks the ICT
process; therefore, in the absence of ONOO−, the probe shows
negligible fluorescence and a severe decrease in fluorescence
quantum yield. The selective cleavage of the phosphinate
with ONOO− allowed for the recovery of the ICT process; this
gives the turn-on fluorescence response. To the best of our
knowledge, this is the first nucleophilic cleavage-based probe
for the selective detection of ONOO− involving the phosphinate
group. Selective detection of peroxynitrite with a “turn-on”
Figure 3. Concentration-dependent cell viability assays. HeLa cells were pre-
incubated for 30 min with various concentrations (10, 20, 30, 40 and 50 μM)
of DCPO-DP. Cell viability was measured after 24 h with WST-1, a cell
proliferation reagent (data expressed as mean ±SD of four experiments).
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response in fixed HeLa cells was confirmed by confocal 9. (a) K. Xu, H. Chen, J. Tian, B. Ding, Y. Xie, M. Qiang and B. Tang,
microscopy imaging. This strategy could be useful for the
design of fluorescent probe for selective detection of ONOO−
based on the cleavage of a phosphinate group.
Chem. Commun., 2011, 47, 9468-9470; (b) B. Wang, F. Yu, P. Li,
X. Sun and K. Han, Dyes and Pigments, 2013, 96, 383-390; (c) F.
Yu, P. Li, G. Li, G. Zhao, T. Chu and K. Han, J. Am. Chem. Soc.,
2011, 133, 11030-11033; (d) F. Yu, P. Li, B. Wang and K. Han, J.
Am. Chem. Soc., 2013, 135, 7674-7680.
10. (a) J. Kim, J. Park, H. Lee, Y. Choi and Y. Kim, Chem. Commun.,
2014, 50, 9353-9356; (b) X. Jia, Q. Chen, Y. Yang, Y. Tang, R.
Wang, Y. Xu, W. Zhu and X. Qian, J. Am. Chem. Soc., 2016, 138,
10778-10781; (c) D. Cheng, Y. Pan, L. Wang, Z. Zeng, L. Yuan, X.
Zhang and Y.-T. Chang, J. Am. Chem. Soc., 2017, 139, 285-292;
(d) A. C. Sedgwick, X. Sun, G. Kim, J. Yoon, S. D. Bull and T. D.
James, Chem. Commun., 2016, 52, 12350-12352; (e) J. Zhang, Y.
Li and W. Guo, Anal. Methods, 2015, 7, 4885-4888; (f) X. Sun, Q.
Xu, G. Kim, S. E. Flower, J. P. Lowe, J. Yoon, J. S. Fossey, X. Qian,
S. D. Bull and T. D. James, Chem. Sci., 2014, 5, 3368-3373; (g) F.
Yu, P. Song, P. Li, B. Wang and K. Han, Analyst, 2012, 137, 3740-
3749.
Acknowledgements
The Molecular Logic Gate Laboratory operated by Prof. D. G.
Churchill acknowledges support from the Mid-Career
Researcher Program through the NRF (National Research
Foundation) of Korea (NRF-2014R1A2A1A11052980) funded by
MEST and the Institute for Basic Science (IBS-R010) for
financial support. Dr. S. V. Mulay and Y. Kim both acknowledge
IBS research fellowships and KAIST for providing research
facilities. The research support staff at KAIST facilitated the
acquisition of MS data.
11. (a) D. Yang, H.-L. Wang, Z.-N. Sun, N.-W. Chung and J.-G. Shen, J.
Am. Chem. Soc., 2006, 128, 6004-6005; (b) Z.-N. Sun, H.-L.
Wang, F.-Q. Liu, Y. Chen, P. K. H. Tam and D. Yang, Org. Lett.,
2009, 11, 1887-1890; (c) T. Peng and D. Yang, Org. Lett., 2010,
12, 4932-4935.
Notes and references
12. (a) T. Peng, N.-K. Wong, X. Chen, Y.-K. Chan, D. H.-H. Ho, Z. Sun,
J. J. Hu, J. Shen, H. El-Nezami and D. Yang, J. Am. Chem. Soc.,
2014, 136, 11728-11734; (b) H. Zhang, J. Liu, Y.-Q. Sun, Y. Huo,
Y. Li, W. Liu, X. Wu, N. Zhu, Y. Shi and W. Guo, Chem. Commun.,
2015, 51, 2721-2724.
13. Q. Zhang, Z. Zhu, Y. Zheng, J. Cheng, N. Zhang, Y.-T. Long, J.
Zheng, X. Qian and Y. Yang, J. Am. Chem. Soc., 2012, 134, 18479-
18482.
1. (a) R. J. Gryglewski, R. M. J. Palmer and S. Moncada, Nature,
1986, 320, 454-456; (b) J. S. Beckman, T. W. Beckman, J. Chen,
P. A. Marshall and B. A. Freeman, Proc. Natl. Acad. Sci., 1990,
87, 1620-1624; (c) G. Ferrer-Sueta and R. Radi, ACS Chem. Bio.,
2009, 4, 161-177.
2. R. Radi, A. Cassina, R. Hodara, C. Quijano and L. Castro, Free
Radic. Bio. Med., 2002, 33, 1451-1464.
3. (a) H. Ischiropoulos and A. B. Al-Mehdi, FEBS Letters, 1995, 364,
279-282; (b) B. Alvarez and R. Radi, Amino Acids, 2003, 25, 295-
311; (c) C. Szabó and H. Ohshima, Nitric Oxide, 1997, 1, 373-385;
(d) R. P. Patel, J. McAndrew, H. Sellak, C. R. White, H. Jo, B. A.
Freeman and V. M. Darley-Usmar, Biochim. Biophys. Acta, 1999,
1411, 385-400; (e) R. G. Brannan and E. A. Decker, J. Agri. Food
Chem., 2001, 49, 3074-3079.
4. L. Liaudet, G. Vassalli and P. Pacher, Front. Biosci., 2009, 14,
4809-4814.
5. (a) C. Szabo, H. Ischiropoulos and R. Radi, Nat. Rev. Drug.
Discov., 2007, 6, 662-680; (b) P. Pacher, J. S. Beckman and L.
Liaudet, Physiol. Rev., 2007, 87, 315-424; (c) T. Nagano, J. Clin.
Biochem. Nutr., 2009, 45, 111-124.
6. (a) R. G. Allen, W. P. Lafuse, N. D. Powell, J. I. Webster
Marketon, L. M. Stiner-Jones, J. F. Sheridan and M. T. Bailey,
Infect. Immun., 2012, 80, 3429-3437; (b) P. A. Darrah, M. K.
Hondalus, Q. Chen, H. Ischiropoulos and D. M. Mosser, Infect.
Immun., 2000, 68, 3587-3593.
7. (a) L. Basabe-Desmonts, D. N. Reinhoudt and M. Crego-Calama,
Chem. Soc. Rev., 2007, 36, 993-1017; (b) M. Fernandez-Suarez
and A. Y. Ting, Nat. Rev. Mol. Cell. Biol., 2008, 9, 929-943; (c) J.
Chan, S. C. Dodani and C. J. Chang, Nat. Chem., 2012, 4, 973-
984; (d) K. P. Carter, A. M. Young and A. E. Palmer, Chem. Rev.,
2014, 114, 4564-4601; (e) S. T. Manjare, Y. Kim and D. G.
Churchill, Acc. Chem. Res., 2014, 47, 2985-2998; (f) Z. Lou, P. Li
and K. Han, Acc. Chem. Res., 2015, 48, 1358-1368; (g) D. Wu, L.
Chen, N. Kwon and J. Yoon, Chem, 2016, 1, 674-698; (h) X. Chen,
F. Wang, J. Y. Hyun, T. Wei, J. Qiang, X. Ren, I. Shin and J. Yoon,
Chem. Soc. Rev., 2016, 45, 2976-3016.
14. (a) D. P. Murale, H. Kim, W. S. Choi and D. G. Churchill, Org.
Lett., 2013, 15, 3946-3949; (b) J. Zhang, C. Li, R. Zhang, F. Zhang,
W. Liu, X. Liu, S. M.-Y. Lee and H. Zhang, Chem. Commun., 2016,
52, 2679-2682; (c) K. Xu, X. Liu, B. Tang, G. Yang, Y. Yang and L.
An, Chem. Eur. J., 2007, 13, 1411-1416; (d) K. Xu, X. Liu and B.
Tang, ChemBioChem, 2007, 8, 453-458.
15. (a) M. Li, X. Wu, Y. Wang, Y. Li, W. Zhu and T. D. James, Chem.
Commun., 2014, 50, 1751-1753; (b) D. Yu, Q. Zhang, S. Ding and
G. Feng, RSC Adv., 2014, 4, 46561-46567; (c) P. Wang, J. Xia and
Y. Gu, Tetrahedron Lett., 2015, 56, 6491-6494; (d) K. Gu, Y. Xu,
H. Li, Z. Guo, S. Zhu, S. Zhu, P. Shi, T. D. James, H. Tian and W.-H.
Zhu, J. Am. Chem. Soc., 2016, 138, 5334-5340; (e) J. Wang, B. Li,
W. Zhao, X. Zhang, X. Luo, M. E. Corkins, S. L. Cole, C. Wang, Y.
Xiao, X. Bi, Y. Pang, C. A. McElroy, A. J. Bird and Y. Dong, ACS
Sensors, 2016, 1, 882-887; (f) J. Ma, J. Fan, H. Li, Q. Yao, J. Xia, J.
Wang and X. Peng, Dyes and Pigments, 2017, 138, 39-46; (g) B.
Bao, M. Liu, Y. Liu, X. Zhang, Y. Zang, J. Li and W. Lu,
Tetrahedron, 2015, 71, 7865-7868; (h) X. Zhang, L. Zhang, Y. Liu,
B. Bao, Y. Zang, J. Li and W. Lu, Tetrahedron, 2015, 71, 4842-
4845.
16. (a) Z. Guo, W. Zhu and H. Tian, Chem. Commun., 2012, 48, 6073-
6084; (b) W. Zhu, X. Huang, Z. Guo, X. Wu, H. Yu and H. Tian,
Chem. Commun., 2012, 48, 1784-1786.
17. T. Yudhistira, S. V. Mulay, K. J. Lee, Y. Kim, H.-S. Park and D. G.
Churchill, Chem. Asian J., DOI: 10.1002/asia.201700527.
8. T. Ueno, Y. Urano, H. Kojima and T. Nagano, J. Am. Chem. Soc.,
2006, 128, 10640-10641.
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