Toward the development of the direct and selective detection of nitrates by a bioinspired Mo-Cu system

Article abstract of DOI:10.1021/ol2022627

The development of a new platform for the direct and selective detection of nitrates is described. Two thioether-based chemosensors and the corresponding sulfoxides and sulfones were prepared, and their photophysical properties were evaluated. Upon select

Full text of DOI:10.1021/ol2022627

ORGANIC
LETTERS
2011
Vol. 13, No. 20
5532–5535
Toward the Development of the Direct and
Selective Detection of Nitrates by a
Bioinspired MoÀCu System
Hanit Marom, Yanay Popowski, Svetlana Antonov, and Michael Gozin*
Department of Chemistry, Raymond and Beverly Sackler Faculty of Exact Science,
Tel-Aviv University, Tel Aviv 69978, Israel
cogozin@mgchem.tau.ac.il
Received August 21, 2011
ABSTRACT
The development of a new platform for the direct and selective detection of nitrates is described. Two thioether-based chemosensors and the
corresponding sulfoxides and sulfones were prepared, and their photophysical properties were evaluated. Upon selective sulfoxidation of these
thioethers with nitrates via an oxygen-transfer reaction promoted by a bioinspired MoÀCu system, significant fluorescence shifts were measured.
A selective response of these systems, discriminating between nitrate salts and H2O2, was also shown.
Nitrates are the world’s most widespread groundwater
contaminant, mainly resulting from crop fertilization, live-
stock wastes, and organic wastes (nitrates that are formed
from organic nitrogen-containing compounds found in
manure).1 Excessive consumption of nitrates can lead to
a fatal medical condition called methemoglobinemia and
other problems, such as spontaneous abortion and birth
defects in the central nervous system.2
spectrophotometry,3 ion chromatography,4 flow-injection
analysis,5 electrochemistry,6 and capillary electrophoresis.7
Spectroscopic methods are the most commonly used be-
cause of their low detection limits and convenient sample-
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techniques (in many cases in conjunction with suitable
chemical reactions) are applicable, including UVÀvis,8
chemoluminescence,9 fluorescence,10 IR,11 Raman,12 and
molecular-cavity emission spectroscopies.13 However, these
A variety of analytical methods have been developed for
the determination of nitrates. Most of them are based on
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10.1021/ol2022627
Published on Web 09/29/2011
2011 American Chemical Society

chemical reactions for the detection of nitrate (including
the most popular, the Griess assay14) are indirect and rely
on the detection of the more reactive nitrite, which is
produced by chemical reduction of nitrate.15 The concen-
tration of the latter anion is analyzed by measurements of
concentration of compounds formed in a reaction between
suitablechromogenicor fluorogenicprecursorsandnitrite.
More recent chemical detection methodologies utilize the
reaction of nitrite with 4-(N-methylhydrazino)-7-nitro-2,1,
3-benzooxadiazole (MNBDH),16 2,3-diaminonaphthalene,17
or functionalized gold nanoparticles.18 To the best of our
knowledge, no direct detection of nitrates by colorimetric
or fluorometric methods has been reported in the literature
to date, and the development of chemical systems for the
direct detection of nitrates still presents a significant
challenge. Reliabletechnologiesforsimple-to-usechemical
sensors could be very important for the monitoring quality
of water sources, soils, and foods. No less important is the
issue of field detection of nitrate-containing explosives.19
In our previous work, we reported the selective oxida-
tion of thioethers to the corresponding sulfoxides through
the oxygen-atom-transfer (OAT) reaction with the use of a
bioinspired molybdenumÀcopper catalytic system (see
Scheme 2) and nitrate salts as oxidants.20 With the use of
a thioether-based chemosensor21 in the latter chemical
transformation, one can envision a novel platform for
the development of a direct nitrate detection system.
Here we report a new strategy for the selective detection
of nitrate salts by fluorescence spectroscopy. Moreover,
our methodology allows the construction of chemosensors
capable of discriminating between nitrates and other oxi-
dants such as peroxides. We designed, synthesized, and
evaluated the performance of compound 3 as a nitrate
chemosensor (Scheme 1). The structure of 3 is based on
the frame of the 1,8-naphthalimide fluorophore, as 1,
8-naphthalimide derivatives are conveniently accessible
and their spectral properties can be readily fine-tuned.
Two possible functionalization sites are available on the
1,8-naphthalimide frame: the naphthalic aromatic moiety
and the imide moiety. Certain derivatives substituted at the
aromatic C-4 position, have an extended π-conjugated
system. These compounds frequently exhibit a bathochro-
mic shift in their absorption and fluorescence spectra
(vs the parent chromophore), especially in cases of electron-
donating substituents.22
The preparation of 3 included two synthetic steps
(Scheme 1). 6-Bromobenzo[de]isochromene-1,3-dione (1)
was treated with 2,6-diisopropylaniline in a mixture of
N-methylpyrrolidine (NMP) and acetic acid at 120 °C to
produce compound 2 in 90% yield. The yield could be
further improved to quantitative by using an excess of 2,
6-diisopropylaniline and conducting the reaction for a
prolonged period oftime. The synthesisof3 was completed
in 87% yield by Suzuki coupling of 2 with 4-(methylthio)-
benzeneboronic acid in a mixture of toluene and etha-
nol in the presence of Pd(PPh3)4 catalyst under an inert
atmosphere.
Prior to testing the performance of chemosensor 3, we
prepared the corresponding sulfoxide 4 by oxidation of 3
with 1 equiv of H2O2. The absorbance and fluorescence
spectra of 4 were measured, revealing a strong absorption
peak at λmax = 350 nm (vs λmax= 370 nm for 3) and a high-
intensity emission peak at λmax =425nm(vsλmax =540nm
for 3), thus strongly indicating the potential of 3 to func-
tion as a chemosensor in our system [see the Supporting
Information (SI) and Figure 1]. Optimization of the
response of chemosensor 3 required the determination
of the concentration at which compound 4 would give
the strongest fluorescence signal. By preparing and
measuring the fluorescence of a series of CH3CN solu-
tions containing compound 4 (at concentrations be-
tween 6.7 Â 10À9 and 6.7 Â 10À4 M), we found that the
strongest fluorescence was observed at approximately
3 Â 10À5 M. At higher concentrations, reduction of the
fluorescence signal intensity could be explained by self-
quenching.
Scheme 1. Preparation of 3 and 6
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The linear range of the fluorescence response of chemo-
sensor 3 (the range over which the correlation between the
concentration and the fluorescence intensity of 4 is linear)
was found to be 5.5 Â 10À7 to 8.3 Â 10À6 M; thus, the
(20) Marom, H.; Antonov, S.; Popowski, Y.; Gozin, M. J. Org.
Chem. 2011, 76, 5240.
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Org. Lett., Vol. 13, No. 20, 2011
5533

amount of nitrate could be determined quantitatively over
this range.
fluorescence signal of the final reaction mixture. This
mixture contained the MoÀCu promoting system, com-
pounds 3 and 4 (at various ratios), tetrabutylammonium
nitrate (TBAN), and tetrabutylammonium nitrite. We
found that these mixtures did not affect the intensity of
the fluorescence signal of compound 4 in comparison to
the same measurements of 4 alone in CH3CN.
Following the control experiments, the nitrate analysis
was carried out using a detection mixture that contained
the MoÀCu promoting system (0.7 mM), 4MTA (0.7 mM),
and chemosensor 3 (2.1 mM) in CH3CN. In a typical
detection experiment in which TBAN was used as the
target analyte (7 mM), we monitored the progress of the
reaction (at 60 °C) by fluorescence spectroscopy. During
the course of this reaction (the time of the complete
conversion of 3 to 4 was about 5 h), 10 μL samples were
withdrawn from the reaction mixture every 30 min, diluted
1000-fold (in order to avoid photomultiplier saturation
and self-quenching), and analyzed. The results obtained
(see Figure 2 and the SI) clearly showed the rise of a strong
emission peak at λmax = 425 nm (assigned to 4), with the
concomitant reduction of the original peak at λmax =540nm
(assigned to 3). The corresponding changes in the color
of the visible emission from yellow to blue are shown in
Figure 2a.
The detection process was also tested on additional
nitrate-containing analytes, including KNO3 and urea
nitrate. In both cases, results identical to those for TBAN
were obtained, showing very good response of the
detection system toward different nitrate salts. No re-
sponse was obtained upon application of our detection
methodology to various nitrite salts or to common
explosives such as TNT and RDX, indicating high selec-
tivity of the MoÀCu/thioether system to the nature of the
analyte.
Figure 1. Determination of the optimal concentration of 4 based
on fluorescence spectroscopy; inset: a correlation between the
concentration of 4 and the intensity of the corresponding
fluorescence.
In the previously reported MoÀCu catalytic system for
the sulfoxidation of thioethers, the first step of the OAT
catalytic cycle is the oxidation of the thioether substrate.20
These conditions would be unsuitable for the purpose of
nitrate detection, as such an approach would generate a
false-positive response (1 equiv of the catalyst would lead
to the oxidation of 1 equiv of chemosensor 3 without the
presence of nitrate). Therefore, we needed to invert the
starting point of the reaction by converting the dioxo MoVI
complex to the reduced mono-oxo MoIV complex so that
the conversion of nitrate to nitrite would initiate the
detection process. Our solution to this problem was the
addition of an equimolar amount (with respect to the
dioxo MoVI complex) of 4-methoxythioanisole (4MTA)
“reducing agent” prior to the addition of chemosensor 3 to
the detection mixture (Scheme 2). Also, it was necessary to
use CuCl2 instead of Cu(NO3)2 as a source of the Cu
copromoter, as nitrate ions are the target analyte.
Additional tests that preceded our nitrate detection
experiments included reference measurements of the
Scheme 2. Detection Process
Figure 2. (a) Changes in the color of the fluorescence during the
oxidation of 3 to 4. (b) Changes in the fluorescence spectra
during the oxidation of 3 to 4, showing the concomitant rise in
the fluorescence corresponding to 4 at 425 nm with the decay in
the fluorescence at 540 nm corresponding to 3.
5534
Org. Lett., Vol. 13, No. 20, 2011

In addition, the detection process was evaluated with
H2O2 as another representative oxidant. Under the detec-
tion conditions developed for nitrate, upon reaction with
H2O2, 3 was completely converted to the corresponding
sulfone 5, as was found by chromatography, mass spectro-
scopy, and NMR analyses. The latter compound exhibited
a lower fluorescence intensity than the sulfoxide 4, while
the emission of 5 at λmax = 411 nm closely overlapped the
emission of 4 at λmax = 425 nm (Figure 3).
of the formed compound. The measured red shift was from
max = 425 nm (assigned to 6) to λmax = 492 nm (assigned
λ
to 7), in contrast to the blue-shift response observed in the
sulfoxidation of 3. On the other hand, oxidation of 6 to
sulfone 8 using H2O2 resulted in a major reduction in the
fluorescence intensity and was accompanied by a blue shift
of 30 nm to λmax = 395 nm. It should be mentioned that
under the reaction conditions in which nitrate was used as
the oxidant, the formation of sulfoxide 7 proceeded at a
lower rate than the formation of 4.
Figure 3. Fluorescence spectra of 3 (ΦF = 0.26), 4 (ΦF = 0.11),
and 5.
Figure 4. Fluorescence spectra of 6, 7, and 8.
In conclusion, we have developed a new platform for the
direct and selective detection of nitrate salts. In the present
work, the first examples of thioether-based chemosensors,
namely, compounds 3 and 6, have been synthesized and
evaluated in a bioinspired OAT detection process pro-
moted by a MoÀCu system. Sulfoxidation of 3 resulted in
a large fluorescent hypsochromic shift (115 nm), while its
isomer 6 was found to be capable of producing an un-
precedented discriminating response upon its exposure to
nitrate or H2O2. We are currently conducting further
studies to improve the performance of our methodology
via preparation of more efficient promoting systems and
development of chemosensors and conditions for shorter
reaction times.
These results provide a demonstration of the plausible
selectivity of our detection methodology, which is poten-
tially capable of distinguishing between nitrate salts and
H2O2-containing formulations. Therefore, a redesign of 3
was required in order to accommodate the demands for
significant and visually observable differences among the
emission properties of the thioether chemosensor and its
sulfoxide and sulfone derivatives. Most reported structural
modifications of the C-4-substituted 1,8-naphthalimides
have been carried out by replacing the attached substituent
with another one having a different electronic nature. These
substitutions typically result in significant changes in the
photophysical properties of the fluorophore.21 However,
such an approach was found to be unsuitable in our case,
since the presence of the thioether functional group is crucial
to the detection chemistry. Our solution to this problem was
to modify the structure of the fluorophore by altering the
position of the thioether substituent on the aromatic ring
attached to the naphthalimide frame. A precedent for a
similar approach was reported by Huang, Lu, and co-
workers.23 An isomer of 3that exhibited the required spectro-
scopic properties was found to be compound 6 (Figure 4).
Chemosensor 6 was prepared in 80% overall yield by a
route similar to that for 3, with the use of 2-(methylthio)-
benzeneboronic acid instead of 4-(methylthio)benzene-
boronic acid in the last synthesis step (Scheme 1). Remark-
ably, the nitrate-based oxidation of 6 to sulfoxide 7
revealed a significant red shift in the emission spectrum
Acknowledgment. We are grateful to Prof. A. Amirav
(School of Chemistry, Tel Aviv University) for the Super-
sonic GCÀMS analyses and to Dr. S. Hauptman and
Dr. N. Tal (both from the School of Chemistry, Tel Aviv
University) for the HRMS analyses. We thank Dr. S. Cohen
(The Institute of Chemistry, The Hebrew University,
Jerusalem) for the X-ray crystallography. We thank the
Israel Ministry of Science and Technology, the Marian
Gertner Institute for Medical Nanosystems, and Tel Aviv
University for financial support.
Supporting Information Available. Experimental pro-
cedures; synthesis of substrates; and 1H NMR, 13C NMR,
MS, FT-IR, UVÀvis, and fluorescence spectra. This
material is available free of charge via the Internet at
http://pubs.acs.org.
(23) Wang, Y.; Zhang, X.; Han, B.; Peng, J.; Hou, S.; Huang, Y.; Sun,
H.; Xie, M.; Lu, Z. Dyes Pigm. 2010, 86, 190.
Org. Lett., Vol. 13, No. 20, 2011
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