Double functional group transformations for fluorescent probe construction: A fluorescence turn-on probe for thioureas

Article abstract of DOI:10.1002/chem.201000244

[Figure Presented] Double up: We have developed the first fluorescence turn-on probe for thioureas by the double functional group transformation strategy (see graphic). The probe is highly selective for thioureas over other structurally and chemically related species, which demonstrates the advantage of the double functional group transformation approach. Furthermore, we have shown that the probe could be employed to monitor thiourea in both water and soil samples.

Full text of DOI:10.1002/chem.201000244

DOI: 10.1002/chem.201000244
Double Functional Group Transformations for Fluorescent Probe
Construction: A Fluorescence Turn-On Probe for Thioureas
Weiying Lin,* Xiaowei Cao, Lin Yuan, and Yundi Ding[a]
The development of fluorescent probes has attracted con-
tinuing attention due to the simplicity and high sensitivity of
fluorescence detection. Recently, analyte-mediated organic
reactions have been extensively employed to design a wide
variety of fluorescent probes. In many reaction-based fluo-
rescent probes, the fluorescence properties of the dyes are
regulated by a single functional group. Thus, only a single
functional group transformation is involved in the analyte-
mediated reactions for the fluorescence response (Figure 1,
top). However, the development of fluorescent probes
based on a single functional group transformation with high
selectivity is still very challenging, since structurally and
chemically related analytes may compete with the same key
functional group in the probes. For instance, a fluorescent
probe with an electrophilic carbonyl group is known to react
with strong nucleophiles, such as Cys and CNÀ.[1] Conse-
quently, the probe has poor selectivity for these distinct ana-
lytes. On the other hand, fluorescence turn-on probes may
be designed based on the double functional group transfor-
mation strategy provided that the fluorescence intensity of a
dye is suppressed by two different functional groups
(Figure 1, bottom). Obviously, the intense fluorescence is re-
stored only after both functional groups are transformed by
the target analyte. However, if neither, or only one, func-
tional group is transformed by other analytes, the fluores-
cence of the dye may remain very dim. Thus, a double func-
tional group transformation strategy may allow the probe to
show selectivity for the target analyte over other potentially
competing species that can incite no, or only single, func-
tional group transformation on the probe. This design ap-
proach, in principle, may be favorable for improvement of
selectivity, as double and single functional group transforma-
tion products may have discrete emission profiles.
Thioureas have been widely used as fungicides[2] or accel-
erators of sprouting in dormant tubers[2a] in agriculture and
as vulcanization accelerators in industry.[3] However, the tox-
icity of thioureas is associated with diseases, such as derma-
titis, pulmonary edema, chronic goitrogenic difficulties, and
thyroid and liver tumors.[4] Thus, it is critical to detect thio-
ureas. To the best of our knowledge, fluorescence turn-on
probes for thioureas that can operate in aqueous solution
have not been previously developed.[5]
In this contribution, we report the development of the
first fluorescence turn-on thiourea probe 1 (Scheme 1) by
the double functional group transformation strategy. Probe
1 is composed of a coumarin dye, a carbonyl group, and a
bromide group. The choice of the carbonyl and bromide
groups is based on the following considerations: 1) The car-
bonyl group may significantly decrease the fluorescence of
the coumarin dye due to the intersystem crossing from sin-
glet (n, p*) to triplet state (p, p*).[6] In addition, the bro-
mide moiety is also a well-known fluorescence quencher in
light of its heavy atom effect. Thus, these two fluorescence
Figure 1. Top: development of reaction-based fluorescence turn-on
probes by the single functional group transformation approach. Bottom:
development of reaction-based fluorescence turn-on probes by the
double functional group transformation strategy.
[a] Prof. W. Lin, X. Cao, L. Yuan, Y. Ding
State Key Laboratory of Chemo/Biosensing and Chemometrics
College of Chemistry and Chemical Engineering, Hunan University
Changsha, Hunan 410082 (P.R. China)
Fax : (+86)731-88821464
E-mail: weiyinglin@hnu.cn
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201000244.
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COMMUNICATION
Scheme 1. Design of fluorescence turn-on thiourea probe 1.
quenching factors should render probe 1 essentially non-
fluorescent. 2) When treated with thioureas, these two func-
tional groups may undergo double functional group transfor-
mations to become the thiazole compounds 5 by the
Hantzsch reaction.[7] Thus, we envisioned that the chemical
transformation of the a-bromoketone to thiazole structure
should inhibit both the fluorescence quenching processes
and, thus, restore the intense fluorescence of the coumarin
dye. In other words, we should observe a pronounced fluo-
rescence turn-on signal, if probe 1 can be converted to thia-
zole compounds 5 by thioureas. Notably, the Hantzsch reac-
tion has not been previously exploited to construct fluores-
cent probes for thioureas.
Figure 2. Emission spectra of probe 1 (10 mm) in HEPES buffer pH 7.4/
DMF (1:1) in the presence of increasing concentrations of thiourea (0–
1.0ꢁ10À3 m). The inset shows the changes in the fluorescence intensity of
probe 1 (10 mm) at 494 nm in the presence of increasing concentrations of
thiourea (0–1.0ꢁ10À3 m).
porting Information), which further supports the fluores-
cence amplified response. The fluorescent intensities at
494 nm were plotted as a function of the thiourea concentra-
tion to obtain a linear calibration graph ranging from 5ꢁ
10À7 to 1.5ꢁ10À4 m (Figure S2, see the Supporting Informa-
tion), indicating that probe 1 is potentially useful for the
quantitative determination of thiourea concentrations with a
large dynamic range. We examined the kinetic profiles of
the reaction under the pseudo-first-order conditions[9] with a
large excess of thiourea (1.5 mm) over probe 1 (10 mm) in
HEPES buffer pH 7.4/DMF (1:1) at 258C. The pseudo-first-
order rate constant for the reaction was obtained, k’=4.92ꢁ
10À2 sÀ1 (Figure S3a and its inset, see the Supporting Infor-
mation).By varying the concentrations of thiourea (from 0.1
to 1.5 mm), the second-order rate constant for the reaction
Compound 1 was readily synthesized in one step by reac-
1
tion of ketocoumarin 2 with CuBr2 in EtOH (Scheme 2). H
and 13C NMR spectroscopy, and ESI-MS were employed to
of probe
1 (10 mm) and thiourea was calculated, k=
3.35mÀ1 sÀ1 (Figure S3b, see the Supporting Information.). In
addition, the probe showed high sensitivity to thiourea with
a detection limit of 2.8ꢁ10À7 m under the experimental con-
ditions (Figure S4, see the Supporting Information).
Scheme 2. Synthesis of compound 1 and the structures of reference com-
pounds 3 and 4.
characterize the structure of the product. Reference coumar-
in 3 is highly fluorescent (Ff =0.556, see the Supporting In-
formation)[8] in DMF. By contrast, ketocoumarin 2 has much
weaker fluorescence (Ff =0.035, see the Supporting Infor-
mation) attributed to the aforementioned intersystem cross-
ing. Indeed, introduction of a bromide group on the keto-
coumarin scaffold further depresses the fluorescence of a-
bromoketo coumarin 1 (Ff =0.004). Thereby, compound 1
seems promising as a fluorescence turn-on probe for thio-
The selectivity of probe 1 was then examined. As antici-
pated, the addition of thiourea and its derivatives, such as
phenylthiourea (PTU) and a-naphthylthiourea (ANTU)
caused a significant enhancement in the fluorescent intensity
around 494 nm in HEPES buffer pH 7.4/DMF (1:1)
(Figure 3). In contrast, only a minimal fluorescence response
was noted upon introduction of the representative species
including glucose, CS2, urea, phenol, aniline, DMSO, and ar-
ginine. Remarkably, the probe exhibited a high selectivity
for thiourea over structurally related ureas, since a-bromo-
keto compounds do not react with ureas and no functional
group transformation on probe 1 occurs. By contrast, the re-
ported fluorescence quenching thiourea probe has poor se-
lectivity for thiourea over urea.[5] Furthermore, the visual re-
sponse of probe 1 to the various species (Figure S1, see the
Supporting Information) indicates that probe 1 can be em-
ployed conveniently for detection of thioureas by simple
ACHTUNGTRENNUNGureas provided that the fluorescence quenching groups, car-
bonyl and bromide, can be transformed by thioureas.
Although free probe 1 is almost nonfluorescent, introduc-
tion of thiourea elicited a dramatic increase in the emission
around 494 nm in 2-[4-(2-hydroxyethyl)piperazin-1-yl]eth-
ACHTUNGTRENNUNGanesulfonic acid (HEPES) buffer pH 7.4/DMF (1:1)
(Figure 2). Furthermore, the addition of thiourea immedi-
ately turned the visual emission color of the solution of
probe 1 from dark to bright green (Figure S1 see the Sup-
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W. Lin et al.
Figure 3. The fluorescence spectra of probe 1 (10 mm) in the presence of
various relevant analytes (70 equiv) in HEPES buffer pH 7.4/DMF (1:1):
Thiourea, phenylthiourea (PTU), a-naphthylthiourea (ANTU), glucose,
CS2, urea, phenol, aniline, DMSO, and arginine.
visual inspection. Additionally, other species have negligible
interference with the fluorescence response of the probe to
thiourea (Figure S5, see the Supporting Information), rein-
forcing the high selectivity of the probe.
To shed light on the functional role of the carbonyl and
bromide groups of probe 1, we investigated the fluorescence
response of control compounds 2 and 4 to thiourea. As
shown in Figure 4a, control compound 2, which contains
only a carbonyl group has almost no response to thiourea.
Similarly, control compound 4, which possesses only a bro-
mide group showed a minimal response to thiourea (Fig-
ure 4b). Thus, these data clearly demonstrate that both the
carbonyl and bromide functional groups of probe 1 are re-
quired for the fluorescence response to thioureas.
To verify that the fluorescence sensing response of the
probe to thioureas is indeed due to the double functional
group transformation of probe 1 to the corresponding thia-
zole compounds 5a–5c, the reaction products of probe 1
with thioureas were isolated by column chromatography.
Studies by 1H NMR spectroscopy and mass spectrometry
confirmed that probe 1 reacted with thioureas to form the
corresponding thiazole compounds 5a–5c (Figures S6–11 in
the Supporting Information). This is further supported by
the observation that the absorption and emission spectra of
the isolated products were identical to those of the corre-
sponding standard compounds 5a–5c (Figures S12–14 in the
Supporting Information). The photophysical data of the
novel compounds 5a–5c are compiled in Table 1.
Figure 4. a) The emission spectra of control compound 2 (10 mm) in the
~
*
absence ( ) or presence of 70 equiv of thiourea ( ) in DMF; b) The
*
emission spectra of control compound 4 (10 mm) in the absence ( ) or
~
presence of 70 equiv of thiourea ( ) in DMF; c) The emission spectra of
&
*
probe 1 in the absence ( ) or presence of 70 equiv of thiophenol ( ) in
DMF. For comparison, the emission spectrum of probe 1 in the presence
~
of 70 equiv of thiourea ( ) in DMF was also shown.
Table 1. Photophysical data of compounds 5a–5c and 6.
labs [nm][a]
Log emax
lem [nm][b]
Ff[c]
5a
5b
5c
6
428
432
426
451
4.67
4.60
4.58
4.80
494
498
495
498
0.498
0.490
0.439
0.034
When a strong nucleophile, such as thiophenol, was added
to the solution of probe 1, almost no fluorescence enhance-
ment was observed (Figure 4c), although it was anticipated
that thiophenol could react with probe 1 by a single func-
tional group transformation. To confirm this, the reaction
product of probe 1 with thiophenol was isolated and charac-
[a] The maximal absorption of the coumarins; [b] The maximal emission
of the coumarins; [c] Fluorescence quantum yield was determined in
spectroscopic DMF by using quinine sulfate (Ff =0.546) as a standard.[8]
1
terized as compound 6 (Scheme 3) by H NMR spectroscopy
and mass spectrometry (Figures S15 and 16 in the Support-
ing Information). Compound 6 still possesses the ketocou-
marin scaffold and consequently exhibits very weak fluores-
cence (Table 1). Thereby, double functional group transfor-
Scheme 3. Reaction of probe 1 with thiophenol to form compound 6.
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Fluorescence Probe for Thioureas
COMMUNICATION
mations between probe 1 and thiourea allow the probe to
distinguish thiourea over thiophenol, which can only render
single functional group transformation on the probe.
ple functional group transformation approach can be ex-
tended to construct other fluorescent probes.
It is important to determine the level of thioureas in the
water samples because thioureas are extensively used as fun-
gicides in agriculture and may contaminate the water sour-
ces. Probe 1 was preliminarily employed to measure the con-
centrations of thiourea in the water samples of YueLu
spring and Xiang River (see the Supporting Information).
Added thiourea in the water samples could be accurately
determined with good recovery (Table S1, see the Support-
ing Information), suggesting that probe 1 is effective for
quantitative detection of thiourea in water samples.
Acknowledgements
Funding was partially provided by NSFC (20872032, 20972044), NCET
(08-0175), and the Key Project of the Chinese Ministry of Education
(No. 108167).
Keywords: coumarins
·
dyes/pigments
·
fluorescence
spectroscopy · fluorescent probes · thioureas
The widespread use of thioureas in agriculture may also
lead to contamination in soil. To examine whether probe 1
could be applied to detect thiourea in soil samples, we con-
ducted a prototypic experiment. The soil sample containing
different levels of thiourea could be readily detected with a
large fluorescence signal (Figure 5a). This indicates that the
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fluorescent probe operates well in crude soil samples. The
detection of thiourea in soil samples can be conveniently vi-
sualized (Figure 5b). Furthermore, the probe could also be
employed to quantitatively detect thiourea in soil samples
(Table S2, see the Supporting Information), indicating the
potential usefulness of the fluorescence detection system.
In conclusion, we have developed the first fluorescence
turn-on probe for thioureas by the double functional group
transformation strategy. The probe exhibited high sensitivity
with a detection limit of 2.8ꢁ10À7 m. Remarkably, the probe
is highly selective for thioureas over other structurally and
chemically related species, including urea and thiophenol,
demonstrating the advantage of the double functional group
transformation approach, since urea and thiophenol can
induce no or only single functional group transformations
on the probe, respectively. Furthermore, we have shown that
the probe could be employed to monitor thiourea in both
water and soil samples. We believe that the double or multi-
[9] a) T. J. Dale, J. Rebek Jr. , J. Am. Chem. Soc. 2006, 128, 4500–4501.
Received: January 28, 2010
Published online: May 19, 2010
Chem. Eur. J. 2010, 16, 6454 – 6457
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