Rational design of a highly selective and sensitive fluorescent PET probe for discrimination of thiophenols and aliphatic thiols

Article abstract of DOI:10.1039/b926070f

A novel highly sensitive and selective 'off-on' fluorescent probe for thiophenols has been developed by a PET mechanism through a rational design. The Royal Society of Chemistry 2010.

Full text of DOI:10.1039/b926070f

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Rational design of a highly selective and sensitive fluorescent PET probe
for discrimination of thiophenols and aliphatic thiolsw
Wei Jiang, Yanting Cao, Yuan Liu and Wei Wang*
Received (in Austin, TX, USA) 10th December 2009, Accepted 26th January 2010
First published as an Advance Article on the web 13th February 2010
DOI: 10.1039/b926070f
A novel highly sensitive and selective ‘off-on’ fluorescent probe
for thiophenols has been developed by a PET mechanism
through a rational design.
enhancement; (3) higher sensitivity (0.2 mM detection limit vs.
2.0 mM for probe 1).
In the design of a novel PET based fluorescent probe10 for
thiophenols, we envision that the selection of 2,4-dinitro-
benzenesulfonyl group as the critical component can kill two
birds with one stone. On one hand, it serves as a recognition
moiety due to its unique and high reactivity towards thiolate
anions. The masked sulfonamide moiety can be facilely
removed by a highly nucleophilic thiolate anion through a
SNAr process (Scheme 1).11 Taking advantage of the difference
in the acidity of thiophenols and aliphatic thiols, we envisioned
that under the physiological pH, a thiophenol could quickly
cleave the sulfonamide group since its corresponding more
nuleophilic thiolate is the dominant species owning to its pKa
(ca. 6.5). Nevertheless, under the same reaction conditions, the
aliphatic thiols with higher pKa (ca. 8.5) remain largely in the
less reactive neutral form and thus the reaction is very slow.
The nitro moieties of the 2,4-dinitrobenzenesulfonamide are
not only crucial for thiolates as essential nucleophiles in the
SNAr reaction, but also serve as an effective quencher in the
newly designed PET fluorescent probe. A benzoxazole is
selected as the fluorophore12 since it exhibits a high quantum
yield in water and can effectively participate in a PET process.
Sensitivity and selectivity are the most critical issues in the
design of any artificial receptor. In addition, the binding event
should be conveniently monitored in an ‘off-on’ signal output
manner. It is extremely challenging to develop a sensor or
probe capable of discriminating interesting analytes with close
physical and chemical properties. Thiols are such a class of
molecules. Aliphatic thiols such as cysteine,1 homocysteine2
and glutathione,3 play important roles in biological systems.
Nevertheless, thiophenols, in spite of their broad synthetic
utility,4 are highly toxic and pollutant compounds with LC50
(50% lethal concentration, e.g., a dose required to kill half
the members of a tested population) 0.01–0.4 mM for fish5
and much more toxic than aliphatic ones.6,7 Therefore, a
detection technique that can selectively differentiate toxic
thiophenols and biologically important aliphatic thiols is of
considerable significance in the fields of chemical, biological
and environmental sciences.
Recently, significant efforts have been directed toward
the development of fluorescent sensors or probes for thiols.8
It is noted that they are mainly designed for cysteine and
homocysteine. However, in general they exhibit poor selectivity
for aliphatic thiols and thiophenols. A sensor capable of
distinguishing these substances has not been realized until
recently when we developed the first highly selective fluores-
cent probe 1 for thiophenols based on an intramolecular charge
transfer (ICT) mechanism (Scheme 1, eqn (1)).9 Nevertheless,
it has the drawbacks of the relatively weak fluorescence
intensity of product 2 in aqueous solution as a result of its
low quantum yield (F = 0.02) and relatively low sensitivity
(detection limit at 2 mM). A ‘‘brighter’’ and more sensitive
sensor for thiophenols is highly desirable from the practical
application standpoint of view. Towards this end, in this
communication, we wish to disclose a new mechanistically
different, PET (photoinduced electron transfer)-based fluorescent
probe 3 (eqn (2)) with several notable features in addition
to high specificity to thiophenols: (1)
a much higher
quantum yield (F = 0.39) in an aqueous buffer; (2)
4100-fold (vs. 450 folds for probe 1) fluorescence intensity
Department of Chemistry and Chemical Biology,
University of New Mexico, MSC03 2060, Albuquerque,
NM 87131-0001, USA. E-mail: wwang@unm.edu;
Fax: (+1) 505-277-2609
w Electronic supplementary information (ESI) available: Experimental
details and spectroscopic data for the compounds 4a–4s. See DOI:
10.1039/b926070f
Scheme 1 Fluorescent probes for thiophenols.
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It is expected that the designed probe 3 will display weak
fluorescence. However, when it reacts with a thiophenol to
yield 4, a strong fluorescent signal should be generated.
To prove the concept, we developed a synthetic route for the
preparation of the newly designed PET probe 3 (see ESIw) and
then carried out fluorescence studies. We first examined its
fluorescence response toward thiophenol as a representative
and meanwhile aimed at establishing the optimal measurement
conditions. Its good water solubility enabled us to perform the
investigation in an aqueous phosphate buffer (0.01 M, pH 7.3)
at a concentration of 2.0 Â 10À6 M. As designed, the probe 3
displayed almost no fluorescence in the absence of a thiol at
lex = 335 nm due to the efficient quenching by the nitro
groups (e = 0.6 Â 104 MÀ1 cmÀ1, F o 0.01) (Fig. 1).13 Upon
the addition of thiophenol (4.0 Â 10À6 M, 2 equiv.), a dramatic
fluorescence intensity increase was observed in a few seconds
centered at 403 nm. After about 20 min, the fluorescence
intensity reached the maximum and no significant change
was seen in an extended reaction time. Notably, probe 3
exhibited a much stronger fluorescence response than previous
probe 1 though its concentration is 10 times lower, and
notably a 4100 fold fluorescence intensity increase was
obtained (Fig. 1). The reaction product was monitored and
confirmed by a comparison study with a standard pure
Fig. 2 Effect of thiophenol concentration on the fluorescence emission
of probe 3. Probe 3 (2 Â 10À6 M) was studied in a phosphate buffer
(pH 7.3, 0.01M) at room temperature in the absence and presence of a
thiophenol at different concentrations. After 10 min, the reaction
solution was sampled for emission measurement (lex = 335 nm,
fluorescence intensity at lex = 403 nm is plotted vs. concentration).
shift in fluorescence spectra and almost no change absorption
spectra (see Fig. S2, ESIw) were observed, which was in
agreement with a typical PET process. Higher concentrations
of thiophenol afforded a quicker and more dramatic response.
For example, when 4 equiv. of thiophenol (8 Â 10À6 M) was
used, the enhancement of fluorescence intensity reached the
maximum in 10 min. Further increase of thiophenol con-
centration did not result in additional enhancement of fluores-
1
compound 4 through H NMR analysis. The quantum yield
of fluorescence for the product 4 was determined to be 0.39
(e = 1.2 Â 104 MÀ1 cmÀ1).13
In a control study, we synthesized compound 5, bearing a
p-methylbenzensulfonyl (Ts) group instead of a 2,4-dinitro-
benzenesulfonyl moiety. Under the same conditions, it showed
very strong fluorescence in the presence and absence of
thiophenol and no response to thiophenol (see Fig. S1, ESIw).
These results support our working hypothesis that the two
nitro groups are essential for the high reactivity of the thiolate
mediated SNAr reaction and serve as an effective quencher of
the PET sensor 3.
cence intensity (Fig. 2, inset). Notably, even at 2 Â 10À7
M
concentration, a pronounced fluorescence signal change was
observed, indicative of its higher sensitivity than that of probe
1 (detection limit: 2 Â 10À6 M).
A survey of relevant aliphatic thiols and other common
nucleophiles revealed that probe 3 displayed excellent selectivity
towards thiophenol (Fig. 3). Aliphatic thiols tested including
2-methyl-2-propanethiol, cysteine and glutathione did not lead
to a fluorescent response to the probe 3 20 min after addition
of the thiol agents. A similar trend was observed for other
nucleophiles such as CNÀ, IÀ, PhOH, and PhNH2. Moreover,
in the presence of other nucleophiles such as a mixture of
cysteine, glutathione, CNÀ, IÀ, and BnNH2, a similar fluores-
cence intensity increase was observed to that of a pure
thiophenol, indicating that the probe 3 is particularly selective
to thiophenols without interference.
Next we examined the sensitivity of the new probe 3 at
2 Â 10À6 M for thiophenol under the same reaction conditions
(Fig. 2). The fluorescence intensity increase displayed in a
concentration dependent fashion and no obvious emission
It is expected that the pH of the tested buffer solution would
affect the fluorescence intensity and the reactivity of probe 3.
The probe itself was inert to pH change in a wide range (from
4 to 12) and no pH-dependent fluorescence change was
obtained. However, as expected, in the presence of thiophenol,
the fluorescence intensity alternation of probe 3 was pH
dependent. At low pH (o5), almost no fluorescence was
observed due to the very slow cleavage of the sulfonamide
group by the weakly nucleophilic neutral thiophenol. However,
under neutral to weakly basic conditions (pH 7 to 9),
probe 3 exhibited the most sensitive response with significant
enhancements of fluorescence with thiophenols as a result of
the strong ionization of thiophenol.
Fig. 1 Fluorescence emission time profile of probe 3 towards
thiophenol. Probe 3 (2 Â 10À6 M), prepared from a 1.0 mM stock
solution in DMF, was studied in a phosphate buffer (pH 7.3, 0.01M)
at room temperature in the absence and presence of thiophenol
(2.0 equivalents). The reaction solution was sampled for fluorescence
measurement at lex = 335 nm at the specified time periods.
In conclusion, taking advantage of the unique property and
reactivity of 2,4-dinitrobenzenesulfonyl moiety, we have
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Fig. 3 The selectivity of probe 3 toward thiols and other nucleo-
philes. Gray bar: the fluorescence intensity of only a single analyte at
10 mM with the probe (2 mM), black bar: the fluorescence intensity of a
mixture of a nucleophilic reagent at 10 mM and PhSH at 4 mM with the
probe (2 mM). (1) probe 3 only, (2) PhSH, (3) cysteine, (4) (CH3)3CSH,
(5) gluotathione, (6) glycine, (7) KCN, (8) KI, (9) PhOH, (10) PhNH2.
All data (lem = 403 nm) were acquired at 20 min after addition of
analyte(s) in a phosphate buffer (pH 7.3, 0.01 M) at room temperature
with lex = 335 nm.
developed a novel fluorescence probe 3 for the detection of
thiophenols in aqueous solution with excellent specificity. The
probe was rationally designed based on the PET mechanism,
which is different from probe 1 relying on the ICT pathway.
Importantly, the sensitivity has improved significantly
with a much higher quantum yield (F = 0.39) and 4100-fold
fluorescence intensity enhancement. These features of probe 3
mean it has great application potential for the detection and
quantification of highly toxic thiophenols in environmental
science.
Financial support of this work by the University of
New Mexico is gratefully acknowledged.
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This journal is The Royal Society of Chemistry 2010
1946 | Chem. Commun., 2010, 46, 1944–1946