A highly selective fluorescent probe for thiophenols

Article abstract of DOI:10.1002/anie.200702271

A rapid response to thiophenols is obtained with probe 1 (see scheme), which induces a significant (> 50-fold) fluorescence enhancement as a result of cleavage of the electron-withdrawing moiety, thus generating strongly fluorescent molecule 2. No fluores

Full text of DOI:10.1002/anie.200702271

Angewandte
Chemie
DOI: 10.1002/anie.200702271
Fluorescent Probes
A Highly Selective Fluorescent Probe for Thiophenols**
Wei Jiang, Qingquan Fu, Hongyou Fan, Joe Ho, and Wei Wang*
The development of highly sensitive and selective detection
techniques for the discrimination of relevant biologically
active and toxic molecules is of considerable importance in
the fields of chemical, biological, and environmental sciences.
Thiols are an important class of molecules in biological
systems and chemical science. Aliphatic thiols are found in
several biologically important molecules including cysteine,[1]
homocysteine,[2] and glutathione,[3] which are associated with
a wide range of biological functions, while thiophenols, in
spite of their broad synthetic utility,[4] are a class of highly
toxic and pollutant compounds.
The study of the toxicity of thiols in fish reveals that the
median lethal dose (LC50) values range from 0.01 to 0.4 mm.[5]
Generally, thiophenols are more toxic than aliphatic alco-
hols.[6] Symptoms of exposure include a burning sensation,
coughing, wheezing, laryngitis, shortness of breath, headache,
nausea, and vomiting through targeting of the central nervous
system, kidney, and liver.[7] In a worst-case scenario, they can
result in death. The main sources of the production of toxic
and pollutant thiophenols include oil and coal refineries,[8] the
plastics and rubber industry,[9] and waste-deposit landfills.[10]
A variety of detection methods,[11] including sensitive
fluorescent probes, have been reported.[12–14] However, the
examination of these probes reveals that most of them suffer
from poor selectivity toward aliphatic thiols and thiophenols.
Accordingly, a fluorescent reagent that enables thiophenols
and aliphatic thiols to be selectively differentiated is needed.
It is fully realized that the design of such fluorescent reagents
is a challenging task because of the similar chemical profiles
of aliphatic thiols and thiophenols. To the best of our
knowledge, such a probe has not been described before.
Herein, we report a novel, sensitive fluorescent probe for the
highly selective detection of thiophenols. The studies dem-
onstrated that a > 50-fold fluorescence intensity enhance-
ment is observed for thiophenols, but there is no gain for
aliphatic thiols under neutral aqueous conditions.
Manipulation of the electronic features of the substituents
of a fluorophore may lead to a change in fluorescence
emission profile, through either intramolecular charge trans-
fer (ICT) or photoinduced electron transfer (PET) path-
ways.[15] The design of the PET-based fluorescent sensor is
relatively easier because the efficiency of the PET process can
be predicted. Nevertheless, the ICT type of fluorescent
reagent can afford high sensitivity as it has a very low
intrinsic fluorescence. A typical example is the nonfluores-
cent probe 4-chloro-7-nitro-2,1,3-benzoxadiazole (NBD-Cl)
as a result of two electron-withdrawing groups (EWGs; 4-
chloro- and 7-nitro), which block the ICT process.[16] How-
ever, the substitution of the Cl atom by an amino moiety as
electron-donating group (EDG) results in a dramatic increase
in fluorescence. Based on this observation, we hypothesized
that masking the amino group by an EWG would give rise to a
nonfluorescent molecule. Removal of the protecting moiety
of the amino group should lead to a highly fluorescent
compound.
It is known that the strongly electron-withdrawing 2,4-
dinitrobenzenesulfonyl group has been used for the protec-
tion of an amino group.[17] The resulting sulfonamide can be
readily cleaved by a thiolate anion, derived from a thiol under
basic conditions through an SNAr process (Scheme 1). This
[*] W. Jiang,Dr. Q. Fu,Dr. J. Ho,Prof. Dr. W. Wang
Department of Chemistry and Chemical Biology
University of New Mexico,MSC03 2060,Albuquerque,NM 87131-
0001 (USA)
Scheme 1. Design of a fluorescent probe for thiophenols.
Fax: (+1)505-277-2609
E-mail: wwang@unm.edu
Homepage: http://www.unm.edu/~wwang/
Dr. H. Fan
Ceramic Processing and Inorganic Materials Department
01815 Sandia National Laboratories,Albuquerque,NM 87185
(USA)
mechanism underlines the importance of the nucleophilic
thiolate, which is an essentially reactive form for the reaction.
The pKa value of thiophenols is around 6.5, whereas that of
aliphatic thiols is about 8.5. In a neutral reaction medium (for
example, pH 7.3), the high degree of dissociation of thiophe-
nols results in the predominant generation of the correspond-
ing thiolate, which can effectively react with 2,4-dinitroben-
zenesulfonamide (Scheme 1). However, under the same
reaction conditions, the aliphatic thiols remain as a less
[**] Financial support of this research by the University of New Mexico
(Start-up and the RAC fund) and the Sandia National Laboratories
through the SURP program is gratefully acknowledged.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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reactive neutral form and thus the cleavage of the sulfona-
mide is very slow.
Therefore, it is possible to design a fluorescent probe for
the discrimination of aliphatic thiols and thiophenols under
neutral, physiological conditions. By taking advantage of the
unique reactivity profile and strong EWG capacity of the 2,4-
dinitrobenzenesulfonamide, we envisioned that masking the
amino group in the fluorescence-active 4-amino-7-nitro-2,1,3-
benzoxadiazole (2) by the 2,4-dinitrobenzenesulfonyl moiety
could generate a probe 1 for thiophenols (Scheme 1). It is
expected that the designed probe 1 will not be fluorescent.
However, when it reacts with a thiophenol to yield 2, strong
fluorescence should be observed.
Figure 1. Reaction–time profile of probe 1 and thiophenol. Probe 1
(2”10À5 m),prepared from a stock solution (10 m m) in EtOH,was
studied in a phosphate buffer (pH 7.3,0.01 m) at room temperature in
the absence and presence of thiophenol (2.0 equiv). The reaction
solution was sampled for fluorescence measurement at lex =465 nm
after the specified time periods. If: Fluorescence intensity.
To demonstrate the working hypothesis, we first synthe-
sized the probe 1. Its synthesis is straightforward in two steps
(Scheme 2). Reaction of NBD-Cl with ammonium hydroxide
philes, such as NaCN and benzylamine (BnNH2). As shown in
Figure 2, probe 1 was highly selective to thiophenols. Signifi-
cant fluorescence intensity enhancement was observed for 4-
Scheme 2. Synthesis of probe 1.
in methanol gave 4-amino-7-nitro-2,1,3-benzoxadiazole (2) in
60% yield. Introduction of the sulfonyl moiety into the amino
group was achieved by reacting 2 with 2,4-dinitrobenzene-
sulfonyl chloride in the presence of NaH in THF to afford the
target molecule 1 in 62% yield.
With probe 1 in hand, we first examined its fluorescence
property in the absence and presence of a thiol and
established the optimal measurement conditions. Notably,
compound 1 had good solubility in water. Accordingly, the
experiment was performed in an aqueous phosphate buffer
(0.01m, pH 7.3) containing 1 at a concentration of 2.0 ” 10À5 m.
As designed, probe 1 exhibited almost no fluorescence in the
absence of a thiol at lex = 465 nm (Figure 1). When thiophe-
nol (4.0 ” 10À5 m, 2.0 equiv) was added, a significant increase
in fluorescence intensity (> 50 times) was observed.
Figure 2. The selectivity of probe 1 toward thiols and other nucleo-
philes. Probe 1 (2”10À5 m) was studied in a phosphate buffer (pH 7.3,
0.01m) at room temperature in the absence and presence of a thiol
(2.0 equiv). After 10 min,the reaction solution was sampled for
fluorescence measurement at lex =465 nm. The fluorescence intensity
at lem =555 nm is plotted versus analytes. *A mixture of PhSH,
cysteine,KCN,and BnNH 2,each with a concentration of 4”10 À5 m.
The reaction product 2 was monitored and confirmed by a
comparison study with a standard pure compound 2 through
1H NMR analysis. Remarkably, probe 1 showed a quick
response toward thiophenol based on the study of reaction
time. A pronounced intensity increase was obtained even
after 5 min. The reaction reached completion after around
10 min. A limited change of fluorescence intensity was
observed when longer reaction times were examined. There-
fore, a reaction time of 10 min was selected to explore the
selectivity of probe 1 toward thiols.
chloro, 4-methoxy-, and 4-methylthiophenols. An increase for
4-nitrothiophenol, which has a strong nitro EWG, was also
obtained, but the magnitude was much smaller than those for
the other thiophenols tested. A plausible reason is that the
nitro group in 4-nitrothiophenol significantly decreases the
nucleophilicity of the thiol, thus reducing its reactivity for the
SNAr reaction. Remarkably, as expected, no fluorescence
intensity change was seen for molecules possessing aliphatic
thiol moieties, such as t-butyl thioalcohol, cysteine, gluta-
thione, NaCN, and BnNH2. More significantly, in the presence
A variety of thiols were probed, including thiophenols,
thioalcohols, cysteine, and glutathione, and other nucleo-
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of other nucleophiles, such as cysteine, glutathione, NaCN,
and BnNH2, a similar fluorescence intensity increase was
observed to that of a pure thiophenol, which indicates that
probe 1 is particularly selective toward thiophenols without
interference.
times. However, no fluorescence is obtained with aliphatic
thiols, including biologically interesting cysteine and gluta-
thione, and other nucleophiles. Therefore, probe 1 can be
used for the detection and quantification of highly toxic
thiophenols in environmental science.
The sensitivity of the chemical reagent 1 at 2 ” 10À5 m was
examined next by using thiophenol as an analyte with a
concentration ranging from 0.2 to 4.0 ” 10À5 m under the same
reaction conditions described above (Figure 3). The increase
Received: May 22, 2007
Revised: August 24, 2007
Published online: September 28, 2007
Keywords: analytical methods · environmental chemistry ·
.
fluorescent probes · fluorophores · thiophenols
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