Nickel(II) dithiocarbamate complexes containing sulforhodamine B as fluorescent probes for selective detection of nitrogen dioxide

Article abstract of DOI:10.1021/ja401555y

We synthesized complexes of Ni(II) with dithiocarbamate ligands derived from the ortho and para isomers of sulforhodamine B fluorophores and demonstrated they are highly selective in reactions with nitrogen dioxide (NO₂). Compared with the para isomer, the ortho isomer showed a much greater fluorescence increase upon reaction with NO₂, which led to oxidation and decomplexation of the dithiocarbamate ligand from Ni(II). We applied this probe for visual detection of 1 ppm NO₂ in the gas phase and fluorescence imaging of NO₂ in macrophage cells treated with a nitrogen dioxide donor.

Full text of DOI:10.1021/ja401555y

Communication
pubs.acs.org/JACS
Nickel(II) Dithiocarbamate Complexes Containing Sulforhodamine B
as Fluorescent Probes for Selective Detection of Nitrogen Dioxide
Yan Yan,† Saarangan Krishnakumar,† Huan Yu,‡ Srinivas Ramishetti,§ Lih-Wen Deng,∥ Suhua Wang,*,‡
Leaf Huang,§ and Dejian Huang*,†
†Department of Chemistry, National University of Singapore, 3 Science Drive 3, Singapore 117543, Singapore
‡Institute of Intelligent Machines, Chinese Academy of Sciences, Hefei, Anhui 230031, China
∥Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, National University Health
System, 8 Medical Drive, Singapore 117597, Singapore
§Division of Molecular Pharmaceutics, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill,
North Carolina 27599, United States
S
* Supporting Information
Scheme 1
ABSTRACT: We synthesized complexes of Ni(II) with
dithiocarbamate ligands derived from the ortho and para
isomers of sulforhodamine B fluorophores and demon-
strated they are highly selective in reactions with nitrogen
dioxide (NO2). Compared with the para isomer, the ortho
isomer showed a much greater fluorescence increase upon
reaction with NO2, which led to oxidation and
decomplexation of the dithiocarbamate ligand from Ni(II).
We applied this probe for visual detection of 1 ppm NO2
in the gas phase and fluorescence imaging of NO2 in
macrophage cells treated with a nitrogen dioxide donor.
s a dominant component in the NOx (x = 1, 2) family,
A
nitrogen dioxide (NO2) is a major air pollutant generated
from high-temperature oxidation of N2 in combustion.1
Biologically, NO2 has been implicated as the root cause of
toxic effects of reactive nitrogen species derived from oxidation
of nitric oxide (NO) by oxygen in cells2 and from one-electron
reduction of nitrite by metalloenzymes.3 As an air-stable and
strong lipophilic oxidant, NO2 can trigger lipid auto-oxidation4
and oxidative nitration of aromatic amino acids, particularly
tyrosine.5 However, there is a lack of selective fluorescent
probes for convenient detection of NO2. Most of the reported
fluorescent probes are for NO detection. While probes based
on transition-metal complexes contain a NO-reactive metal as a
fluorescence quencher,6−10 organic-dye-based probes detect
NO indirectly through oxidation of aromatic amines by
N2O3,11,12 which is produced via oxidation of NO by O2.13,14
We herein report the first fluorescent probe for selective
detection of NO2, which contains Ni(II) as a fluorescence
quencher and NO2 reaction center.
The reaction of sulforhodamine with oxalyl chloride yielded a
mixture of two isomeric compounds having the −SO2Cl group
at the para or ortho position of the phenyl ring.15 The mixture
was treated directly with piperazine, and the two isomers
produced were separated readily by column chromatography to
give the para isomer, 1, and the ortho isomer, 2 (Scheme 1).
Because of their similarity, it was difficult to distinguish their
structures unambiguously on the basis of NMR and mass
spectra alone. Hence, the structures were determined by single-
crystal X-ray diffraction [Figure S1 in the Supporting
Information (SI)], and accordingly, the 1H NMR spectral
data were assigned with certainty. In the presence of sodium
hydroxide, the two isomers reacted readily with carbon disulfide
to produce the sodium dithiocarbamate salts 3 and 4,
respectively. Dithiocarbamate is a bidentate ligand that
promptly forms complexes with many transition metals, leading
to quenching of the ligand fluorescence. It has been reported
that Ni(II) bis(dithiocarbamate) complexes, Ni(RNCS2)2, react
immediately with NO2 to yield the oxidatively dimerized ligand,
(RNCS2)2,16 which can be fluorescent if R is a fluorophore.
Hence, to prepare Ni(II)-based turn-on fluorescent probes for
NO2, we mixed nickel(II) nitrate with 2 equiv of 3 and 4, which
afforded 5 and 6, respectively (Scheme 1). The structures of 5
1
and 6 were characterized by H NMR spectroscopy, and the
Received: February 12, 2013
Published: March 26, 2013
© 2013 American Chemical Society
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results were in agreement with a diamagnetic nickel complex
having a square-planar configuration. Additionally, their
MALDI-TOF mass spectra showed isotope distribution
patterns matching the expected molecular formula (Figure S2).
To illustrate the effect of the two isomers on fluorescence
quenching, the fluorescence quantum yield (QY) of each
compound shown in Scheme 1 was measured (Table S1 in the
SI). The presence of the piperazine group at the ortho position
in 2 (QY = 0.21) reduced the QY relative to the parent
compound sulforhodamine B (QY = 0.34) more significantly
than having this group at the para position in 1 (QY = 0.28).
The addition of dithiocarbamate groups further reduced the
quantum yield a little bit. The ortho isomer of the Ni(II)
complex, 6, had the lowest QY, which was 70 times lower than
that of ligand 4 alone, while the QY of para isomer 5 was only 5
times than that of the corresponding ligand 3. Energy transfer is
not likely, as there is only a small overlap of the fluorophore
emission and quencher absorption bands (Figure S3).
The mechanism behind the fluorescence quenching probably
involves photoinduced electron transfer (PET) from the
electron donor, Ni(II), to the sulforhodamine B excited state.
The energies of the highest-occupied molecular orbital
(HOMO) and lowest-unoccupied MO (LUMO) of Ni-
(Me2NCS2)2 were calculated to be −4.09 and −1.03 eV,
respectively, and dithiocarbamate ligands are known to stabilize
Ni(III).17 The HOMO and LUMO energies of rhodamine B
are −8.058 and −5.266 eV, respectively,18 and therefore, PET
from Ni to the rhodamine B chromophore is energetically
favored. Because PET is highly sensitive to the distance
between the fluorophore and the quencher, the shorter distance
from Ni to the center of the fluorophore in 6 is likely to result
in a much higher quenching efficiency than for 5. From the
crystal structures of 1 and 2, the distances from the NH
nitrogen in the piperazine group to the center of the
fluorophore were calculated to be 9.4 and 7.4 Å, respectively.
Although we failed to obtain single-crystal structures of the Ni
complexes, it is reasonable to assume that the distance from the
Ni to the rhodamine B chromophore would be shorter in 6
than in 5. It should be borne in mind that in solution, molecular
motions will have a significant effect on the distances.
We evaluated the selectivity of the ortho isomer 6 toward
common reactive oxygen species (ROS) in methanol (Figure
S5). Only NO2 could increase the fluorescence intensity more
than 12-fold, whereas other biological relevant ROS could not
do so. Neither nitrite nor nitrate could switch on the
fluorescence. Moreover, addition of the NO donor diethyl-
amine NONOate (DEANO) rapidly turned on the fluores-
cence 12-fold within minutes, and there was an excellent dose−
response relationship between the fluorescence intensity and
the DEANO concentration (Figure S7). In comparison, when
the para isomer 5 was used, the magnitude of the fluorescence
increase was only 1.9-fold (Figure S6). Although there is a
sulfonate group on the fluorophore of probe 6, its water
solubility is extremely poor. In aqueous media (water and
buffer), there was no fluorescence intensity gain after addition
of NO2. It turned out that the reaction products precipitated
out even at the micromolar concentration level used in the
experiment. Addition of a surfactant (Tween 20) to the
reaction mixture before addition of NO2 led to fluorescence
turn-on. Therefore, to introduce the probe under physiological
conditions, we applied 1,2-dioleoyl-3-trimethylammoniumpro-
pane chloride salt (DOTAP) liposomes as a delivery vehicle for
our probe. It was found that probe 6 delivered using DOTAP
demonstrated excellent selectivity for NO2 (Figure 1) and a
good dose−response relationship to DEANO (Figure 2) in
buffered aqueous media to simulate physiological conditions.
Figure 1. Ortho isomer 6 is selective toward common ROS under
physiological conditions. (A) Fluorescence responses obtained upon
addition of various ROS (1 μM) to 6 (0.2 μM) delivered using 20
equiv of DOTAP in 10 mM PBS (pH 7.4) at 37 °C (λex = 540 nm, λem
= 590 nm). (B) Digital photos showing that NO2 turns on the
fluorescence but other ROS do not under illumination by a 365 nm
UV lamp 5 min after addition of ROS (5 μM) to 6 (1 μM) delivered
using 20 equiv of DOTAP in 10 mM PBS (pH 7.4).
Figure 2. Responses of ortho isomer 6 to DEANO at different
concentrations under physiological conditions. (A) Increments of
fluorescence intensity of 6 (0.2 μM) delivered using 20 equiv of
DOTAP against time after the addition of DEANO at different
concentrations in 10 mM PBS (pH 7.4) at 37 °C (λex = 540 nm, λem
=
590 nm). (B) Dose−response curve for the fluorescence intensity of 6
(0.2 μM) delivered using 20 equiv of DOTAP obtained 3.5 min after
the addition of DEANO in 10 mM PBS (pH 7.4) at 37 °C (λex = 540
nm, λem = 590 nm).
As a further test of whether the probe is sensitive to NO or
NO2, we carried out the reaction in the absence of oxygen by
degassing the probe solutions and DEANO solutions before
mixing them under nitrogen, and we failed to observe any
fluorescence turn-on event. This observation showed that the
fluorescence turn-on by DEANO required oxygen, suggesting
that it is NO2 that reacts with the probe as NO generated from
DEANO is rapidly oxidized to NO2 by oxygen present in the
solution.19 This was confirmed by a separate test in which the
responses of the probe to NO2 formed by mixing NO and air
and to commercial NO2 gas were examined. Indeed, the
fluorescence was turned on. To verify that the products of the
reactions of 6 with DEANO in the presence of air and with
NO2 gas are identical, the reaction products were analyzed
using HPLC and LC−MS. The results suggested that the same
major products with fluorescence were generated by the
reactions of the probe with DEANO in the presence of air and
with NO2 gas in methanol. These results clearly show that NO2
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can turn on the fluorescence of the probe. Moreover, the para
isomer 5 also reacted with NO2 in the same way as the ortho
isomer 6, as characterized by HPLC chromatograms (Figure 3).
formed from the molecular cation through homolytic cleavage
of the S−S bond and subsequent loss of CS2 (Figure S4).
In addition, the formula of the species corresponding to
peaks A′ and B′ were confirmed by high-resolution MS (Table
S2). Our results are in agreement with literature work, which
showed that bis(diethyldithiocarbamato)nickel(II) [Ni(dtc)2]
reacts with NO2 (but not with NO) to give corresponding
paramagnetic complex NiIII(dtc)3 as an intermediate, which
decomposes to give the oxidized dithiocarbamate ligand [i.e.,
thiuram disulfide, (R2NCS2)2].16
In view of the high toxicity of NO2 in the atmosphere, the
U.K. Health and Safety Executive (HSE) has approved
occupational exposure standards of 1 ppm (8 h time-weighted
average) and 5 ppm (short-term exposure limit) for NO2.20
Convenient detection of gaseous NO2 would be ideal for
monitoring air pollution due to NOx (x = 1 or 2, with NO2
being dominant species). For this purpose, probe 6 was
successfully applied in visual detection of gaseous NO2 with a
detection limit reaching 1.0 ppm by using NO2 indicator strips
prepared by dropping a 1:1 methanol/chloroform solution of 6
(800 nM) on filter paper to give round stains ca. 5 mm in
diameter after solvent evaporation. The NO2 indicator strips
were exposed to different concentrations of NO2 gas for 10
min. Under regular laboratory light, no apparent spot could be
detected on any of the indicator strips (Figure 4 right). When
Figure 3. HPLC chromatograms of the products of the reactions of 5
and 6 with NO2, recorded at 560 nm.
The structures of the reaction products were then deduced
on the basis of LC−MSn spectra, as shown in Scheme 2.
Scheme 2
Figure 4. Visual detection of NO2 gas. NO2 concentrations of 0, 1, 10,
and 100 ppm were tested. The images on the left were obtained under
illumination by a 352 nm UV lamp in the dark. For comparison, no
color can be seen in the images on the right, which were obtained
under laboratory lighting without UV light excitation.
Apparently, NO2 oxidizes the dithiocarbamate ligand and
generates Ni(II)-free compounds that strongly fluoresce. We
propose that the reaction is initiated by oxidative formation of
an adduct of NO2 with 5/6, NiIII(R2NCS2)2(NO2). A
subsequent intramolecular electron transfer reaction forms
NiII(R2NCS2)(NO2) and a R2NCS2· radical, and these radicals
couple to give ligand dimers, which correspond to peak B/B′
(Scheme 2). The compound corresponding to peak A/A′ is
generated by a secondary oxidation reaction of (R2NCS2)2
followed by methanolysis. Consistent with the two proposed
compounds, electrospray ionization mass spectrometry (ESI-
MS) analysis of the compound corresponding to peak A/A′
gave an [M + H]+ peak at m/z 749.2 and an [M + Na]+ peak at
m/z 771.2, while ESI-MS analysis of the compound
corresponding to peak B/B′ demonstrated two dications, [M
+ 2H]2+ at m/z 702.2 and [M + 2Na]2+ at m/z 724.2. Our
assignments are supported by ESI-MS2 fragmentation patterns
of A′ and B′. The MS2 spectrum of peak A′ showed three
fragments at m/z 717.1, 669.2, and 637.0 due to [M + H −
CH3OH]+, [M + H − SO3]+, and [M + H − CH3OH − SO3]+,
respectively. The MS2 spectrum of B′ gave a peak at m/z 627.2
the strips were exposed to a UV lamp (352 nm), red
fluorescence spots could be observed visually for strips exposed
to NO2 gas at concentrations as low as 1.0 ppm, and the
fluorescence intensity increased in a dose-dependent manner at
10 and 100 ppm NO2 (Figure 4 left). These results
demonstrated that NO2 indicator strips immobilized with
probe 6 can serve as a simple, direct, and economical tool for
rapid detection of gaseous NO2 at low concentrations.
To introduce the probe into a biological system, a liposomal
carrier was applied. Cationic liposomes have been studied
extensively in gene therapy applications, and cationic lipids are
effective in vaccine formulations to treat cancers.21,22 Therefore,
we applied DOTAP liposomes as a delivery vehicle for our
probe. Although it is known that DOTAP liposomes at high
concentration are toxic to cells, our observations showed that
RAW 264.7 cells can withstand up to 100 μM DOTAP (Figure
S8). To illustrate that fluorescence from probe 6 uptaken by
cells can be turned on by addition of NO2, RAW 264.7 cells
treated with DOTAP liposomes containing 6 (5 μM) were
further treated with DEANO as a NO2 donor.19 The
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fluorescence images of the cells (Figure 5B,D) clearly illustrate
that the fluorescence intensity could be efficiently enhanced by
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In conclusion, we have demonstrated that Ni(II) complexes
of dithiocarbamate ligands containing sulforhodamine B
fluorophores are highly selective probe for detection of NO2.
To the best of our knowledge, this is the first example of live-
cell fluorescence imaging of NO2 activity and visual detection of
gaseous NO2 at low concentrations. Many third-row transition
metals are good fluorescence quenchers because their
complexes with fluorescent ligands are paramagnetic as a result
of the unpaired metal d electrons. In contrast, the Ni(II)
coordination spheres in complexes 5 and 6 adopt a square-
planar geometry and are diamagnetic. Further research to
understand the quenching mechanisms of Ni(II) and develop
even more sensitive probes by rational combination of
fluorophores and nickel complexes is warranted.
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ASSOCIATED CONTENT
* Supporting Information
■
S
Synthetic procedures and spectroscopic data for 1−6, crystal
structures of 1 and 2, and experimental procedures for cell
imaging and selectivity of the probe toward different ROS. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
shwang@iim.ac.cn; chmhdj@nus.edu.sg
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
D.H. and L.W.D. thank the Agency of Science, Technology and
Research (A*Star) of Singapore for financial support (Grant
112 177 0036). D.H. and S.W. thank the National Natural
Science Foundation of China for Grant 21228702. L.H. thanks
the National Institutes of Health for Grant CA129421.
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