Selective fluorometric detection of aromatic thiols by a chemosensor containing two electrophilic sites with different local softness

Article abstract of DOI:10.1039/c3cc47220e

A resorufin-dinitrophenyl ether conjugate (1) shows emission enhancement for aromatic thiols in aqueous media with a neutral-basic pH, while being nonemissive for aliphatic thiols. This is achieved by two electrophilic sites with different local softness on compound 1; the respective sites selectively react with aromatic or aliphatic thiolate anions.

Full text of DOI:10.1039/c3cc47220e

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Selective fluorometric detection of aromatic thiols by a
chemosensor containing two electrophilic sites with
different local softness†
Cite this: Chem. Commun., 2013,
49, 11680
Received 21st September 2013,
Accepted 22nd October 2013
Yasuhiro Shiraishi,* Kohei Yamamoto, Shigehiro Sumiya and Takayuki Hirai
DOI: 10.1039/c3cc47220e
www.rsc.org/chemcomm
A resorufin–dinitrophenyl ether conjugate (1) shows emission enhance-
Our strategy for selective detection of aromatic thiols is based on
ment for aromatic thiols in aqueous media with a neutral–basic pH, while the different nucleophilic reactivity of aromatic and aliphatic
being nonemissive for aliphatic thiols. This is achieved by two electro- thiolates. On the basis of the local hard–soft acid–base (HSAB)
philic sites with different local softness on compound 1; the respective principle,5 nucleophilic reactivity of thiolate depends on the local
sites selectively react with aromatic or aliphatic thiolate anions.
softness of the sulfur atom (ssulfurÀ) and thÀe electrophilic site
(selectrophile+).6,7 The thiolate with larger ssulfur reacts preferably
Thiols are an important class of molecules in biological systems. with the electrophilic site with larger selectrophile+, whereas the
Aliphatic thiols are contained in several molecules including cysteine, thiolate with smaller ssulfur reacts preferably with the smaller
À
+
À
homocysteine, and glutathione, which are associated with a variety of selectrophile site.8 The ssulfur value is derived from the Fukui
biologically important functions.1 In contrast, aromatic thiols are very function (fsulfurÀ),9,10 which is calculated by Mulliken electronic
toxic to the human body. Exposure to the thiols induces serious population analysis of thiolate and defined as the change in
damage to the central nervous system and other related pathologies electron density of the sulfur atom, when the thiolate provides an
including increased respiration, muscular weakness, paralysis of electron to the electrophilic site during nucleophilic reaction.6,9
limbs and coma,2 although they are versatile intermediates for the
À
fsulfur = qsulfur(N) À qsulfur(N À 1)
(1)
synthesis of pesticides, polymers, and pharmaceuticals.3 The design
of chemosensors that facilitate selective and quantitative detection of
aromatic thiols by simple spectroanalysis in the environmental and
biological samples is, therefore, currently the focus of attention.
A number of fluorescent chemosensors for thiols have been
proposed; however, there are only a few reports of chemosensors
that allow discrimination of aromatic thiols over aliphatic ones.4 All
of these sensors are based on nucleophilic reaction with a thiolate
anion (–SÀ). Aromatic thiols have pKa values (ca. 6.5) lower than
those of aliphatic ones (ca. 8.5); therefore, at a neutral pH, aromatic
thiols exist mainly in a thiolate form while aliphatic ones are
protonated. A chemosensor, when added to a solution with a
neutral pH, undergoes selective nucleophilic reaction with aromatic
thiolate, resulting in selective fluorescence enhancement. However,
at a basic pH, aliphatic thiols also exist in a thiolate form and
qsulfur(N) and qsulfur(N À 1) are the electron density of the sulfur
atom before and after electron donation, respectively. ssulfurÀ is
calculated using the global softness (S) of the molecule.6,9,10
À
À
ssulfur = fsulfur  S
(2)
+
In contrast, the Fukui function of an electrophilic site (felectrophile
)
is defined as the change in electronic density of the electro-
philic atom when a molecule receives an electron.6,9 felectrophile+ and
selectrophile+ for an electrophilic site can be expressed as follows:6,9,10
+
felectrophile = qelectrophile(N + 1) À qelectrophile(N)
(3)
(4)
+
+
selectrophile = felectrophile  S
As reported,11 ssulfur of aliphatic thiolate is larger than that
À
also show fluorescence enhancement. A new strategy is therefore of the aromatic one. This implies that two electrophilic sites with
+
necessary to create the sensors that facilitate selective detection different selectrophile values, if created on the sensor molecule,
of aromatic thiols at a wide pH range.
might react selectively with aromatic or aliphatic thiolates.
Based on the above scenario, we synthesized a chemosensor
containing two electrophilic sites with different selectrophile
values (Scheme 1). The resorufin–dinitrophenyl ether conjugate (1)
contains electron-deficient ether (C*) and olefinic (C**) carbons as
the electrophilic sites. Aromatic thiolates with smaller local softness
react selectively with the C* atom with smaller local softness,
producing a fluorescent resorufin (2). In contrast, aliphatic thiolates
+
Research Center for Solar Energy Chemistry, and Division of Chemical Engineering,
Graduate School of Engineering Science, Osaka University, Toyonaka 560-8531,
Japan. E-mail: shiraish@cheng.es.osaka-u.ac.jp; Fax: +81 6 6850 6273;
Tel: +81 6 6850 6271
† Electronic supplementary information (ESI) available: Experimental methods
and supplementary data (Tables S1 and S2, Fig. S1–S16, and Cartesian coordi-
nates of compounds). See DOI: 10.1039/c3cc47220e
c
11680 Chem. Commun., 2013, 49, 11680--11682
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Scheme 1 Different nucleophilic reactions promoted by aromatic or aliphatic
thiolates on compound 1.
with larger softness react selectively with the C** atom with larger
softness and produce a nonfluorescent adduct (3). The different
reactions promoted by aromatic and aliphatic thiolates facilitate
selective fluorometric detection of aromatic thiols even at basic pH.
Compound 1 was synthesized by condensation of resorufin and
2,4-dinitrofluorobenzene with 88% yield (ESI†). Its purity was
Fig. 2 Fluorescence titration (lex = 545 nm) of 1 (5 mM) with 4a in a buffered
MeCN–water mixture (1/1 v/v; HEPES 100 mM, pH 7.0). The respective spectra
were obtained after stirring the solution for 30 min. The inset shows the change
in the intensity of emission at 587 nm obtained by the titration with 4a or 5a.
Absorption titration data are summarized in Fig. S5 (ESI†).
1
confirmed by H, 13C NMR, and EI-MS analysis (Fig. S1–S3, ESI†).
Ab initio calculation based on the density functional theory (DFT)
performed using the Gaussian 03 program (Table S1, ESI†) revealed
that the local softness of the ether carbon (sC*+) and olefinic carbon
(sC**+) of 1 is 0.216 and 0.340, respectively. This suggests that the
C** atom is a more soft electrophilic site and might react preferably
with the thiolate with larger local softness (ssulfurÀ).
Fig. 1a shows the fluorescence spectra of 1 (5 mM) measured in a
buffered MeCN–water mixture (1/1 v/v) at a basic pH (10.0). 1 shows
almost no fluorescence, where the fluorescence quantum yield (FF)
is o0.001. Addition of 15 equiv. of benzenethiol (4a), however,
creates strong orange emission at 587 nm (FF = 0.38), where the
emission increase terminates within 10 min (Fig. S4, ESI†). In
contrast, addition of 1-propanethiol (5a) shows almost no emission
(FF o 0.001), although aliphatic thiol also exists in a thiolate form at
this basic pH. As shown in Fig. 2, stepwise addition of 4a to the
solution containing 1 increases the emission at 587 nm, whereas the
addition of 5a is silent. As shown in Fig. 3, substituted benzenethiols
(4b–d) also show the emission at 587 nm, whereas aliphatic thiols
(5b–d) do not. In addition, oÀther nucleophiles such as aniline,
phenol, IÀ, CNÀ, S2À, and SO3 do not show the emission. These
emission behaviors are similar to those observed at a neutral pH
(Fig. S6, ESI†). These suggest that 1 allows selective fluorometric
detection of aromatic thiolates even at basic pH; this is the first
Fig. 3 Fluorescence intensity (lex = 545 nm; lem = 587 nm) of a buffered MeCN–
water mixture (1/1 v/v; CHES 100 mM, pH 10.0) containing 1 (5 mM), measured
after stirring for 30 min with respective nucleophiles (15 equiv.). The detailed
spectra are shown in Fig. S7 (ESI†).
chemosensor that facilitates discrimination of aromatic thiolates
over aliphatic ones at a basic pH.4
As shown in Scheme 1, the emission enhancement of 1 upon
addition of aromatic thiolate is due to the formation of free resorufin
(2), via the ether bond cleavage by nucleophilic reaction between the
sulfur and C* atoms. NMR and EI-MS spectra of the product
recovered after reaction of 1 with 4a (Fig. S8–S10, ESI†) are similar
to those of standard compound 2, clearly indicative of the ether
cleavage. In contrast, almost no emission enhancement of 1 upon
addition of aliphatic thiolate is due to the formation of a nonemissive
adduct (3) by nucleophilic reaction between the sulfur and C** atoms
(Scheme 1). This is also confirmed by NMR and EI-MS analysis of the
product recovered after reaction of 1 with 5a (Fig. S11–S13, ESI†).
These data clearly suggest that aromatic or aliphatic thiolate under-
goes nucleophilic reaction at the different electrophilic sites of 1.
À
This difference is due to the different local softness (ssulfur
)
values of aromatic and aÀliphatic thiolates. As summarized in
Table S2 (ESI†), the ssulfur values for aromatic thiolates (4a–d)
determined by DFT calculation are 4.04, 3.75, 3.84, and 5.01,
respectively, whereas those of aliphatic thiolates (5a–d) are 6.11,
5.71, 6.00, and 6.05, respectively. This suggests that the sulfur atoms
for aromatic thiolates have smaller ssulfurÀ. As a result of this,
Fig. 1 (a) Fluorescence (lex = 545 nm) and (b) absorption spectra of 1 (5 mM)
measured in a buffered MeCN–water mixture (1/1 v/v; CHES 100 mM, pH 10.0), after
stirring for 30 min with 15 equiv. of benzenethiol (4a) or 1-propanethiol (5a).
À
aromatic thiolates with smaller ssulfur react selectively with the
ether carbon (C*) with smaller local softness, whereas aliphatic
c
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Fig. 5 Fluorescence titration (lex = 545 nm, lem = 587 nm) of 1 (100 mM) in a
buffered MeCN–water mixture (1/1 v/v; CHES 100 mM, pH 10.0) with 4a in the
absence (K) or presence (J) of 5a (5 mM). Measurements were carried out after
stirring the solutions for 30 min.
Fig. 4 Relationship between the local softness (ssulfurÀ) of respective thiolates
(4a–d and 5a–d) and the first-order rate constant for (a) ether cleavage or (b)
substitution reaction.
when performed with 100 mM of 1, with 4a in the absence and
presence of 5a (5 mM). A linear relationship is observed in the range
of 0.07–5.0 mM in the absence or presence of 5a. The detection limit,
0.07 mM, is similar to that obtained by the previously reported
sensors,4a–d although these sensors are unable to detect aromatic thiols
selectively at basic pH. These data indicate that accurate quantification
of a very small amount of aromatic thiols is facilitated when using an
excess amount of 1 even in the presence of aliphatic thiols.
In summary, we found that compound 1 containing two electro-
philic sites with different local softness facilitates selective fluoro-
metric detection of aromatic thiols even at basic pH. There are still
several problems that must be addressed by further study. Never-
theless, the molecular design presented here, based on the creation
of two electrophilic sites with different local softness, may contribute
to the design of more efficient chemosensors for accurate and
selective quantification of a very small amount of aromatic thiols
in aqueous media with a broad pH range.
thiolates with larger ssulfurÀ react selectively with the olefinic carbon
(C**) with larger local softness. The results are consistent with the
local HSAB principle.5 These imply that the respective electrophilic
sites with different local softness of 1 selectively react with aromatic
or aliphatic thiolates, resulting in selective emission enhancement
for aromatic thiolates.
Fig. 1b shows the absorption spectra of 1 measured after the
reaction with 4a or 5a. Reaction with 4a creates new absorption at
578 nm, assigned to free resorufin (2). In contrast, the reaction with
5a creates a band at 439 nm assigned to the adduct product (3).
Kinetic absorption analysis after addition of respective thiols was
performed to further clarify the reactivity of respective thiols with C*
and C** atoms (Fig. S14, ESI†). Fig. 4 summarizes the relationship
À
between the ssulfur values of respective thiolates and the first-order
rate constant for the ether cleavage (kC*) and the substitution to
olefinic carbon (kC**). As shown in Fig. 4a, the ether bond cleavage is
promoted only by aromatic thiolates and the rate constants (kC*)
increase with a decrease in the ssulfurÀ value of thiolates. In contrast,
as shown in Fig. 4b, the nucleophilic substitution to the olefinic
carbon is promoted only by aliphatic thiolates and the rate constants
(kC**) increase with an increase in the ssulfurÀ value of thiolates. The
clear ssulfurÀ-dependence of the respective reactions indicates that
the difference in local softness for two electrophilic sites of 1 indeed
promotes selective reaction with aromatic or aliphatic thiolates.
There are two problems with chemosensor 1 that must be
addressed. The first is that, as shown in Fig. 3, the aromatic
thiolate with an electron-withdrawing group such as –NO2 (4d)
shows very low emission enhancement. This is due to the low activity of
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suggests that fluorometric detection of aromatic thiolates with an
electron-withdrawing group is difficult. The second problem is that
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simultaneous addition of 4a (75 mM) and 5a–d (75 mM, respectively)
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c
11682 Chem. Commun., 2013, 49, 11680--11682
This journal is The Royal Society of Chemistry 2013