Discriminative fluorescence detection of cysteine, homocysteine and glutathione via reaction-dependent aggregation of fluorophore-analyte adducts

Article abstract of DOI:10.1039/c2jm32892e

A novel fluorescence probe capable of discriminatively and simultaneously detecting Cys, Hcy and GSH has been developed. This specially designed probe can selectively react with Cys and Hcy to form thiazinane and thiazolidine derivatives in the presence of diverse amino acids, protected Cys and glucose and display the expected aggregation-induced emission (AIE) properties. Relying on the differences in kinetics, Cys can be easily and discriminately detected over Hcy by the observation of FL responses. GSH shows great interference with the detection of Cys and Hcy and it can be quantitatively detected by the FL spectroscopic titration method. The threshold of the FL turn-off concentration for GSH is measured to be 1 mM. This is the first report of using a single fluorescent probe to discriminately detect Cys, Hcy and GSH by FL turn-on and turn-off strategies. The discrimination relies on the reaction-dependent fluorophore aggregation, or the solubility of adducts of the probe molecule and analytes. The present strategy is intrinsically a fluorescent titration, which combines the high sensitivity of FL spectroscopy and the reliability of precipitate titration methodology. The threshold concentration of Cys (375 μM, at which the FL is turned-on) coincides with the upper margin of the deficient Cys levels in human plasma, and the primary investigation of the FL response to deproteinized human plasma indicates that this FL probe is a promising one for the discriminatory detection of Cys over Hcy and GSH on a clinical level. The Royal Society of Chemistry 2012.

Full text of DOI:10.1039/c2jm32892e

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PAPER
Discriminative fluorescence detection of cysteine, homocysteine and
glutathione via reaction-dependent aggregation of fluorophore-analyte
adducts†
a
a
a
a
a
Ju Mei, Jiaqi Tong, Jian Wang, Anjun Qin, Jing Zhi Sun and Ben Zhong Tang
ab
*
*
Received 8th May 2012, Accepted 26th June 2012
DOI: 10.1039/c2jm32892e
A novel fluorescence probe capable of discriminatively and simultaneously detecting Cys, Hcy and
GSH has been developed. This specially designed probe can selectively react with Cys and Hcy to form
thiazinane and thiazolidine derivatives in the presence of diverse amino acids, protected Cys and
glucose and display the expected aggregation-induced emission (AIE) properties. Relying on the
differences in kinetics, Cys can be easily and discriminately detected over Hcy by the observation of FL
responses. GSH shows great interference with the detection of Cys and Hcy and it can be quantitatively
detected by the FL spectroscopic titration method. The threshold of the FL turn-off concentration for
GSH is measured to be 1 mM. This is the first report of using a single fluorescent probe to
discriminately detect Cys, Hcy and GSH by FL turn-on and turn-off strategies. The discrimination
relies on the reaction-dependent fluorophore aggregation, or the solubility of adducts of the probe
molecule and analytes. The present strategy is intrinsically a fluorescent titration, which combines the
high sensitivity of FL spectroscopy and the reliability of precipitate titration methodology. The
threshold concentration of Cys (375 mM, at which the FL is turned-on) coincides with the upper margin
of the deficient Cys levels in human plasma, and the primary investigation of the FL response to
deproteinized human plasma indicates that this FL probe is a promising one for the discriminatory
detection of Cys over Hcy and GSH on a clinical level.
electrophoresis separations coupled with different detection
methods,6 electro-chemical assays,7 optical detection based on
the interplasmon coupling of Au nanorods,8 nano-particles9 and
synthetic reporters.10 Among these strategies, optical assays
based on fluorescent probes have attracted extensive interest in
the past decades owing to their high sensitivity, simplicity and
low cost.10j The fluorescence (FL) detection is the most appealing
one due to its low detection limit and potential for in vivo imaging
of living cells, whereas the visual or colorimetric protocol
possesses inherent merits, including visual detection and real-
time in situ responses, which are suitable for the in vitro analysis
of biological samples.10j–10l
Introduction
Discriminative fluorescence detection of biological thiols, such as
cysteine (Cys), homocysteine (Hcy) and glutathione (GSH) is of
great fundamental and practical significance because Cys,1 Hcy2
and GSH3 possess various important biochemical functions. So
far, several strategies have been applied to detect Cys, Hcy and
GSH, for example, high performance liquid chromatography,4
gas
chromatography-mass
spectrometry,5
capillary
aMOE Key Laboratory of Macromolecular Synthesis and
Functionalization, Department of Polymer Science and Engineering,
Zhejiang University, Hangzhou 310027, P. R. China. E-mail: sunjz@zju.
edu.cn; Fax: +86-571-87953734; Tel: +86-571-87953734
bDepartment of Chemistry, Institute for Advanced Study, Institute of
Molecular Functional Materials, State Key Laboratory of Molecular
Neuroscience, and Division of Biomedical Engineering, The Hong Kong
University of Science & Technology, Clear Water Bay, Kowloon, Hong
Kong, P. R. China. E-mail: tangbenz@ust.hk; Fax: +86-852-2358-7375;
Tel: +86-852-2358-1594
In principle, most of the reported fluorescent probes sensitive
to Cys/Hcy/GSH are based on the active thiol group on the
residue, and different mechanisms such as Michael addition11
and cleavage reactions12 have been proposed. Though these
probes show high sensitivity toward thiol-containing
compounds, the selective and simultaneous detection of
Cys/Hcy/GSH is hampered because of ‘‘cross-talking’’ between
different thiols. Since the pioneering work of Strongin and
colleagues, fluorophores bearing aldehydes have been used as
fluorescent probes for both Cys and Hcy.13 The mechanism was
confirmed to be the well-known cyclization of Cys/Hcy with
aldehydes to form thiazolidines/thiazinanes. Due to the similarity
† Electronic
supplementary
information
(ESI)
available:
Characterization data for DMBFDPS and the resultant thiazolidine
derivatives (Fig. S1 and S2), AIE properties of this probe in DMSO/10
mM HEPES (6/4 in volume, pH 7.4) (Fig. S3), absorption spectra,
emission spectra, fluorescence photographs taken under ambient and
UV light (Fig. S4–S9 and Fig. S12–14), proposed reaction mechanisms
for the interference of Hcy and GSH in the detection of Cys (Schemes
S1–S4, Fig. S10 and S11). See DOI: 10.1039/c2jm32892e
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in the reactivity of the amino-thiol moieties of Cys and Hcy
towards aldehydes, the methodology of using thiazolidine/thia-
zinane formation often renders the detection of total content of
Cys and Hcy.14 Recently, selective detection for either Cys or Hcy
has been developed by using some aldehyde-containing fluores-
cent probes. For instance, Strongin et al. reported an optical
method to discriminate Cys and Hcy from other amino acids and
thiols at physiological pH.15 The discrimination of Cys and Hcy
is attributed to different rates of intramolecular cyclizations of
their respective thioether adducts derived from 2-(20-hydroxy-
30-methoxyphenyl)-benzothiazole acrylate.16 Thus the selective
and simultaneous fluorescence detection of Cys and Hcy was
achieved. More recently, Yu and colleagues reported a carba-
zole-based fluorescent probe which is capable of selectively
detecting Cys over Hcy, but the exact mechanism has not been
clarified yet.10m This progress suggests that a huge space to
develop novel fluorescent probes for the selective and simulta-
neous detection of Cys, Hcy and GSH still exists.
Scheme 1 Mechanistic representation of the discriminative detection of
Cys and Hcy.
Herein, we demonstrate an unprecedented strategy to selec-
tively and simultaneously detect Cys, Hcy and GSH. In
comparison with the reported ones, the most significant modifi-
cation is that we have taken the advantage of the aggregation of
reaction-dependent fluorophore. The fluorophore used in the
present work shows distinctive FL behavior, which is termed
aggregation-induced emission (AIE). AIE active molecules are
non-emissive in dilute solution but become highly emissive upon
aggregate formation.17 It means that the FL turn-on relies on the
formation of aggregates. Thus it is rational to conceive that the
same aldehyde-functionalized AIE active fluorophore may react
with Cys, Hcy and GSH (analyte) in different kinetic rates and
produce different adducts of AIE-fluorophore-analyte. These
adducts may show different critical concentrations for aggregate
formation if they have different solubilities. The differences
potentially allow them to be separately and/or stepwise detected
by FL turn-on strategy. Such a process is similar to the classical,
simple and reliable precipitation titration analysis that is widely
used in the detection of different metal cations based on the
solubility parameter (Ksp) of the compounds.
be fabricated, which could increase the dynamic change of the
FL measurement.
DMBFDPS was prepared via the coupling reaction of 2-(3-
bromophenyl)-1,3-dioxolane and the key intermediate 1,1-
dimethyl-2,5-bis(zinc monochloride)-3,4-diphenylsilole, which
was derived from bis(phenylethynyl)silanes. The subsequent
deprotection reaction of the coupling resultant afforded the final
product in a moderate yield. The structures of the final resultant
DMBFDPS and the intermediates were fully characterized by
1
multiple techniques including H NMR, 13C NMR, FTIR and
element analysis (see Experimental section).
AIE property of probe and detection medium
We firstly measured the FL spectrum of DMBFDPS in dimethyl-
sulfone (DMSO), considering that DMSO is widely used in
bioassay systems as a biocompatible organic solvent. The results
shown in Fig. S1† indicate that DMBFDPS is very weakly
emissive in its 10 mM DMSO solution. To examine the possi-
bility of using DMBFDPS as a fluorescent probe in the biological
medium, we then checked its FL behavior in the mixture of
HEPES buffer/DMSO (8/2 in v/v). As shown in Fig. S3,† the
emission is considerably intense because DMBFDPS is insoluble
in the buffer and suspension rather than the solution which was
formed [Fb/F0 ¼ 65, Fb ¼ 946; where F0 and Fb are the FL
intensity recorded in DMSO and the mixture of HEPES buffer/
DMSO (8/2 in v/v), respectively]. Fig. S1† also demonstrates the
variation of FL intensity (F) with the volume fraction of HEPES
buffer (fb) respect to DMSO. When fb is lower than 50%, F is
small and remains unchanged; once fb is up to 50%, F raises
abruptly and is boosted continuously with the increase of fb.
These data indicate that DMBFDPS is a typical AIE active flu-
orophore with a threshold of fb at which aggregates generate and
FL is turned on. To ensure the biocompatibility, fb should be as
high as possible; however, to assure strong ‘‘turn-on’’ effect, fb
should be as low as possible. To achieve a balance between the
two aspects, the mixture of 10 mM HEPES buffer and DMSO
with fb ¼ 40% and pH ¼ 7.4 was chosen as the action ratio and
was subsequently used as the detection medium without specific
notation in the following works.
Results and discussion
Rational design and characterization of probe
Silacyclopentadienes (siloles) are in the list of widely explored
AIE active molecules.18 We used an aldehyde-functionalized
silole derivative [DMBFDPS, 1,1-dimethyl-2,5-bis(meso-for-
mylphenyl)-3,4-diphenylsilole] (Scheme 1) as an AIE-active
fluorescent probe. It is well-known that the cyclization reaction
of an aldehyde group with amino and thiol groups results in
thiazinane or thiazolidine; this reaction takes place in mild
conditions with high efficiency and specialty thus has been widely
used for Cys and Hcy detection.13,14 We take advantage of the
stability of thiazinane or thiazolidine to reduce the side reactions
of DMBFDPS molecules with other amino acids. In addition,
the transition of aldehyde to thiazinane/thiazolidine by cycliza-
tion reaction can change the conjugation mode of the resultant
products in comparison with DMBFDPS. It is expected that the
detection process can be monitored by the changes in both FL
intensity and color. As a result, a ratiometric probing system may
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The recognition of Cys and Hcy in different kinetics
induce the emission of DMBFDPS. As a result, an increase in
intensity of the FL band peaked at 479 nm has been observed.
When more and more resultant 1 was produced, the new emis-
sion band (424 nm) became predominant.
DMBFDPS has moderate solubility in such medium and the
solution with a concentration of 25 mM DMBFDPS displays an
FL band peaked at 479 nm at a very low intensity (Fig. 1a).
When Cys was added to the solution, it readily reacts with
DMBFDPS at room temperature (rt), leading to thiazolidine
derivative [1, Scheme 1, route (i)]. Molecule 1 should have higher
solubility in the medium than DMBFDPS, because of the
carboxylic and thiazolidine groups. But in fact, the intermolec-
ular hydrogen-bonding between these groups may help to form
aggregates thereby results in lower solubility. Since the reaction
solution turned from transparent to turbid, this process could be
observed by naked eyes. Monitoring the process with FL
revealed that the FL response started immediately after the
addition of Cys to the reaction medium and leveled off in about
100 minutes (Fig. 1b). The FL band peaked at 479 nm enhanced
gradually. In comparison with Fig. S1,† this band is assigned to
the emission of DMBFDPS and its aggregates. When Cys was
introduced to the reaction system, the solubility of DMBFDPS
was decreased and its aggregates were formed, thereby the
emission was enhanced. Meanwhile, a new band peaked at 424
nm appeared and its intensity grew quickly with the increasing
amount of the added Cys (Fig. 1a). This band is assigned to the
resultant 1 and its aggregates. The blue-shift of the emission
feature can be associated with the transformation of the aldehyde
to the thiazolidine auxochrome, which eliminates the conjuga-
tion between the aldehyde and the silole core. The precipitation
of molecule 1 may also take some DMBFDPS molecules along
due to their intrinsic hydrophobicity. According to the restricted
intramolecular rotation mechanism of AIE,17c,18a this process can
A quantitative evaluation of the changes in emission intensity
and peak wavelength is displayed in Fig. 1b. The maximum
intensity shows a drastic elevation from ꢀ7 in the absence of Cys
to ꢀ102 in the presence of Cys (100 equiv.), or about 13-fold
enhancement (for short, the emission enhancement is defined as
F/F0 ꢁ 1). Concurrently, the emission peak shows a significant
blue-shift (ꢀ55 nm), and the emission color changes from
greenish-blue to purple-blue. The emission enhancement can be
ascribed to the poor solubility (precipitate) of the resultant
thiazolidine in the buffer mixture. The precipitate is resulted
from molecular aggregation, which induced the observed emis-
sion enhancement. This observation suggests that the probe
could be employed for Cys detection by simple visual inspection.
In contrast, when Hcy was added to the DMSO/HEPES buffer
mixture under the same conditions, little change in emission
features was recorded for hours. In a long interval of 160
minutes, F/F0 ꢁ 1 just increased from 0 to 1.2 and the emission
peak showed a red-shift of about 15 nm (Fig. 1d). Three days
later, white solids came out of the transparent solution (inset of
Fig. 1c). Simultaneously, the FL peak blue-shifted from 472 to
424.5 nm and F/F0 ꢁ 1 was measured to be 31.4. These obser-
vations imply that the reaction of Hcy with the probe molecule to
produce thiazolidine is quite slow in comparison with Cys
[Scheme 1, route (ii)]. According to literature, upon reaction with
aldehyde, Cys forms the more favored 5-membered ring
heterocycle, which is beneficial to the reaction proceeding, as
compared to Hcy (6-membered ring formation).13,14 1H NMR
and KBr pellet FTIR measurements were carried out to confirm
the mechanism (Fig. S2 and S3†). It is worth noting that the
discriminative detection of Cys and Hcy can be reflected quali-
tatively and concurrently by three parameters: precipitate
formation, FL intensity enhancement and spectral shift.
The sensitivity of the probe to Cys
The sensitivity of DMBFDPS to Cys was investigated by FL
spectroscopic titration experiments. The changes in FL features
with Cys concentration in DMSO/HEPES buffer mixture are
displayed in Fig. S4a.† From 0 to 2.5 mM, FL intensity increases
slowly. The plot of F/F0 ꢁ 1 versus Cys concentration is nearly a
flat line parallel to the abscissa before the concentration of Cys
increased to 375 mM. Once the Cys concentration is above 375
mM, the plot takes off with a sharp inflexion, the FL peak starts
to blue-shift (Fig. S5†) and the clear mixture turned turbid at this
point as well. At 2.5 mM of Cys, F/F0 ꢁ 1 grows to over 8 in 1 h
and white solid is clearly visible (inset in Fig. S4b†).
Since AIE behavior depends on the formation of aggregates or
the solubility of the fluorescent species, it is expected that the FL
intensity of DMBFDPS-Cys adduct will show temperature
dependence because solubility normally decreases with the
lowering of temperature. This was confirmed by monitoring the
changes of F/F0 ꢁ 1 versus Cys concentration at different
temperatures. As shown in Fig. S6,† the FL enhancement at ꢂ18
ꢂC (lower temperature) is more pronounced than that at 36 C
(higher temperature).
Fig. 1 Time dependent FL spectra when (a) Cys or (c) Hcy was added to
the mixture of DMSO and 10 mM HEPES buffer (6/4 in volume, pH 7.4)
containing DMBFDPS; (b) and (d) are corresponding plots of the changes
in FLintensityand FLpeakfor(a)and (c), respectively. [DMBFDPS] ¼ 25
mM, [analyte] ¼ 2.5 mM, lex ¼ 356 nm. F for FL intensity in (a) and (c); Dl
for wavelength shift, F for peak FL intensity and F0 for the peak FL
intensity of DMBFDPS at t ¼ 0 min in (b) and (d). Inset of (c): FL
photographs of DMBFDPS and Hcy in the above mixture taken 3 days
later under ambient (top row) and UV-light (bottom row, lex ¼ 365 nm).
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The specificity of probe to Cys
The selectivity of the probe to Cys
In addition to sensitivity, the specificity of a fluorescent probe is a
crucial parameter for potential applications. To examine the
specificity of DMBFDPS to Cys, the responses of the probe to
other amino acids, glucose, GSH and protected Cys (Cya and
Cyt) were investigated. As shown in Fig. 2 and S7,† among all the
tested analytes, Cys gave the highest FL response. FL enhance-
ment is more than 11-fold and the blue-shift of the FL peak is
45 nm for Cys in 60 min (Fig. 2a and b). About 4, 1.7 and 1-fold
enhancement was recorded for Cyt, Hcy and Lys, respectively.
And below 0.67 was observed for the rest analytes. The order of
the sensitivity is Cys > Cyt > Hcy > Lys > other amino acids,
glucose and GSH. Although both Cys and Hcy present a similar
blue-shifted emission peak, it is easy to recognize one from the
other by checking the emission intensity and formation of white
precipitate (Fig. 2).
In practical detection, Cys may co-exist with other amino acids,
peptides and polypeptides containing free thiol group (e.g.
GSH). Examination on the selective detection of Cys in the
presence of other analytes is an essential step. When only
2.5 mM of Cys exists in the buffer solution (control sample), F/
F0 ꢁ 1 value is 11.4, and the shift of emission peak (Dl) is
about ꢁ40 nm. By measuring the FL spectra of mixtures con-
taining Cys (2.5 mM) and other analytes (2.5 mM), it was
found that most of the analytes, except GSH, Hcy and Cya,
have exerted little impact on the detection of Cys in FL spec-
troscopic titration method (Fig. 3a and b, S8 and S9†). While,
in the presence of Hcy, Cya and GSH (2.5 mM), the F/F0 ꢁ 1
values are 2.4, 2.2 and 0.4-folds, respectively, which are
evidently lower than that of the control one. Meanwhile, the
shifts of emission peak (Dl) are about ꢁ38, ꢁ1 and 25.5 nm for
Cya, GSH and Hcy, respectively. The interference responses
can be associated with the formation of different Schiff-base
intermediates and the successive competing reaction of the
Fig. 2 Ratiometric and specific response of DMBFDPS to diverse
amino acids (Ala, Arg, Asp, Cys, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,
Ser, Thr, Tyr, Val, Pro, Hcy, Pgl and GSH), glucose or Glyc and pro-
tected Cys [Fmoc-Cys(Trt)-OH, or Cyt and N-acetyl-L-Cys, or Cya] in
the mixture of DMSO and 10 mM HEPES buffer (6/4 by volume, pH
7.4), [probe] ¼ 25 mM, [analyte] ¼ 2.5 mM, lex ¼ 356 nm. All measure-
ments were conducted at rt 60 min later. The probe is DMBFDPS.
Fig. 3 FL response of DMBFDPS to the mixture of Cys in the presence
of various analytes (Ala, Arg, Asp, Cys, Glu, Gly, His, Ile, Leu, Lys, Met,
Phe, Ser, Thr, Tyr, Val, Pro, Hcy, Glyc, Cys, Cyt, Cya, GSH, and glucose
or Glyc) in the defined buffer, [DMBFDPS] ¼ 25 mM, [analyte] ¼ 2.5 m
M, lex ¼ 356 nm. All measurements were conducted at rt after incubation
for 60 min.
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different Schiff-base intermediates with the free thiol group in
Hcy, Cya and GSH as shown by the reactions in Scheme S1–
S4.† These intermediates and the final adducts themselves, on
one hand, are soluble in the reaction solution; and on the other
hand, they interrupt the production of insoluble resulting
products (aggregates). Thus the aggregation-induced emission
has not been observed. The good solubility of the products
1
allows us to measure their H NMR and IR spectra (Fig. S10
and S11†). But the data are too complex to be clearly recog-
nized and assigned, indicating the existence of multiple
components.
Fig. 4 FL spectra of DMBFDPS and Cys in the presence of different
GSH concentrations in the mixture of defined buffer. [Cys] ¼ 2.5 mM,
[DMBFDPS] ¼ 25 mM, lex ¼ 356 nm. All measurements were conducted
at rt 60 min later.
In comparison with Cya, it can be considered as strong proof
that Cyt had little interference with the FL response of Cys
(Fig. 3a and b), because the thiol group in Cyt has been protected
by a triphenylmethane group. It is noticeable that, when Hcy
(2.5 mM) co-exists with Cys (2.5 mM), the addition of
DMBFDPS (2.5 mM) to the mixture results in the formation of a
mixture of intermediates, which subsequently proceed to a
resultant mixture containing molecule 1, 2, and other derivatives
(3–10, Scheme S1†). These derivatives have higher solubility and
a lower ability to form aggregates in the buffer solution, thus
showed decreased FL intensity (Fig. 3a). As shown in Fig. 3b, the
addition of DMBFDPS to the mixture of Cys and Hcy leads to a
red-shift of the FL peak. This observation is remarkably different
from the phenomena observed for the addition of DMBFDPS to
the Cys or Hcy buffer solution (Fig. 2b). The difference in
spectral shift can be associated with the reactions shown in
Schemes 1 and S1.† In this case, the intermediates rather than the
resultant 1 are the main-products in 60 min. The intermediates
(Schiff base structure) have larger conjugate system and higher
solubility in comparison with the resultant products thus shows
red-shifted and weak FL features.
the trend of FL intensity (Fig. 4b). In addition, as shown in
Fig. S13,† the reverse detection of GSH was visually observed
by FL quenching and the turbid-to-clear transition of the
solutions.
FL response of probe to Cys in deproteinized human plasma
As mentioned above, the normal level of Cys is 240–360 mM in
human plasma, and the above results give the clue that this probe
could be applied as a potential indicator of Cys deficiency
(Fig. S4). To confirm this assumption, deproteinized human
serum was used in the following investigation. The removal of
proteins is to lessen the effect of viscosity of the fluid on the
emission behavior of the fluorescent probe according to the
mechanism of the AIE phenomenon.18a Even though the FL
intensity recorded for the contrast sample (without the probe
molecule in the deproteinized human plasma) is higher than that
recorded for the probe in HEPES buffer/DMSO solution due to
the higher viscosity of the deproteinized plasma (Fig. 5), the FL
intensity of the probe molecule in the mixture of the human
serum containing 250 mM spiked Cys (10 equiv.) is still lower
than the contrast sample and the FL response does not reverse to
positive until the amount of Cys approaches the normal level
The fluorescent response to GSH
GSH shows a significant quenching effect on the FL responses
of the probe molecule to Cys. This is rational because the
intermediates and the resultant products have good solubility
in aqueous solution and can hardly form aggregates (Schemes
S2 and S3†) and thereby have low FL intensity (Fig. 3a and b).
This observation triggered a new idea. That is to use the
mixture of Cys and DMBFDPS as a FL probe to detect GSH
in the DMSO/buffer solution via a FL turn-off mechanism,
which likes the retro-titration methodology used in analytical
chemistry. Accordingly, we monitored the changes in FL, UV-
vis absorption spectra and the appearance of the mixtures of
Cys (2.5 mM) and GSH at different concentrations (Fig. 4, S12
and S13†). The FL peak (470.5 nm) intensity for the reference
sample (25 mM free DMBFDPS in the defined buffer solution)
was measured to be 10, while the FL peak (424 nm) intensity of
mixture containing equivalent Cys and DMBFDPS without
GSH was 358. Upon adding GSH into the buffer solution
containing Cys, emission goes down rapidly. Along with the
decrease of the F/F0 ꢁ 1 value from 34.8 without GSH to
0.4 with 2.5 mM GSH, the emission peak red-shifts from 424 to
484.5 nm. The FL quenching slows down at 1 mM GSH
[40 equivalent (equiv. for short) to DMBFDPS] and the
quenching factor is about 100 for the solution without GSH.
Meanwhile, the change of FL peak is in good accordance with
Fig. 5 (a) FL spectra of DMBFDPS with different Cys concentrations
(equiv.) in the mixture of DMSO and deproteinized human plasma (6/4 in
volume, pH 7.4), [DMBFDPS] ¼ 25 mM, lex ¼ 356 nm. Inset: FL
photographs of DMBFDPS and Cys in the above mixtures, taken under
ambient (top) and UV-light (lex ¼ 365 nm, bottom). (b) F/F0 ꢁ 1 versus
Cys concentration (C) in the above mixtures. Inset: shift of FL peak
versus Cys concentration. F0 and F are the peak intensity when Cys
concentration is 0 and other values. All measurements were carried out at
rt after incubation for 3 h.
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(375 mM, or 15 equiv.). This concentration can be defined as the
threshold of FL turn-on. Coincidently, the FL peak wavelength
exhibits a monotonous blue-shift from 460 to 424 nm upon
increasing the Cys concentration. The FL enhancement with
spiked 2.5 mM (100 equiv.) Cys is about 4-fold and the blue-shift
of the emission peak is about 36 nm (inset of Fig. 5b). The cor-
responding UV-visible spectra show little changes in absorption
features (Fig. S14†). Meanwhile, the appearance change can be
easily witnessed by the naked eye (from light-yellow transparent
solution to turbid suspension) and under a UV lamp (enhanced
and apparent blue-shift FL) (inset of Fig. 5a). Hence, this probe
should be a candidate biological sensor for Cys detection and
recognition.
method. The threshold of the FL turn-off concentration for
GSH is measured to be 1 mM.
This is an unprecedented report of using a single fluorescent
probe to discriminately detect Cys, Hcy and GSH by FL turn-on
and turn-off strategies. The discrimination relies on the reaction-
dependent fluorophore aggregation, or the solubility of adducts
of the probe molecule and analytes. The present strategy is
intrinsically a fluorescent titration, which combines the high
sensitivity of FL spectroscopy and the reliability of precipitate
titration methodology. Fortunately, the threshold concentration
of Cys (375 mM, at which the FL is turned-on) coincides with the
upper margin of the deficient Cys levels in human plasma, and
the primary investigation of the FL response to deproteinized
human plasma indicates that this FL probe is a promising one for
the discriminatory detection of Cys over Hcy and GSH on a
clinical level.
Conclusions
In summary, we have developed a novel FL probe that is
capable of discriminately and simultaneously detecting Cys,
Hcy and GSH. The probe molecule is composed of two alde-
hyde groups as reactive functionalities and an AIE active
DMTPS core as the signaling moiety. Due to the aldehyde
functionalities, this specially designed probe can selectively react
with Cys and Hcy to form thiazinane and thiazolidine deriva-
tives in the presence of diverse amino acids, protected Cys and
glucose. Due to the DMTPS core, the resulted thiazinane and
thiazolidine derivatives display the expected AIE properties, or
they are non-emissive in DMSO solution but become highly
emissive in suitable mixture of DMSO and HEPES buffer.
Because the reactions of the probe molecule with Cys and Hcy
undergo distinct kinetics, which are determined by the forma-
tion of 5-membered thiazinane and 6-membered thiazolidine
rings, the time of FL turn-on for probe-Cys system (ꢀ120 min)
is much shorter than that for probe-Hcy system (ꢀ3 days).
Under the same conditions and in a limited time (e.g. 60 min),
the interaction of Cys with probe molecule can lead to an
evident blue-shift of FL peak from 479 to 424 nm and a
pronounced FL enhancement, but Hcy cannot. Relying on the
differences in kinetics, Cys can be easily and discriminately
detected over Hcy by the observation of FL responses. GSH
shows great interference with the detection of Cys and Hcy. In
the presence of a proper amount of GSH, the FL of the mixture
of Cys and probe molecule in DMSO/HEPES buffer solution is
heavily quenched. The experiment on the addition of a stoi-
chiometric amount of GSH to the prepared mixture containing
equivalent Cys and probe molecule suggests that GSH can be
quantitatively detected by the FL spectroscopic titration
Experimental
Chemicals and materials
THF was distilled from sodium benzophenone ketyl under dry
nitrogen immediately prior to use. Dimethyl sulfoxide
(DMSO) is chromatographically pure purchased from Alfa
Aesar. p-Toluene sulfonic acid and magnesium sulphate were
purchased from Sinopharm Chemical Reagent Co., Ltd. 3-
Bromobenzaldehyde was obtained from Acros Organics Co.
Ethylene glycol was purchased from Alfa Aesar. Dichloro-
(N,N,N0,N0-tetramethylenediamine)zinc
(ZnCl2-TMEDA),
HEPES, alanine, arginine, aspartic acid, cysteine (Cys), glu-
tamic acid, glycine, histidine, isoleucine, leucine, lysine,
methionine, phenylalanine, serine, threonine, tyrosine, valine,
proline, homocysteine (Hcy), phenylglycine and glutathione,
glucose and protected Cys (Fmoc-Cys(Trt)-OH and N-acetyl-
L-Cys) were purchased from Aldrich Chemical Co. Bis(-
triphenyl phosphine)-palladium(II) dichloride (Pd(II)) were
purchased from ABCR GmbH&Co.KG. All other chemicals
and reagents were commercially available and used as received
without further purification. Human plasma (EDTA as an
anticoagulant) was obtained from a local hospital and stored
at ꢁ20 ꢂC until analysis. Distilled water was used in all
experiments. Di(phenylethynyl)silanes were prepared by lith-
iation of phenylacetylene followed by reaction with
dichlorosilanes.19
General characterization
1H and 13C NMR spectra were measured on a Bruker AV 400
spectrometer in deuterated chloroform and deuterated acetone
using tetramethylsilane (TMS; d ¼ 0) as the internal reference.
FTIR spectra were recorded on a Bruker Vector 22 spectrometer.
The element analysis was carried out on Thermo Finnigan Flash
EA 1112. The melting point was determined by differential
scanning calorimetry (DSC) measurement conducted on Perkin-
Elmer DSC 7 under N2 atmosphere at a heating/cooling rate of
10 K minꢁ1. UV-visible (UV-vis) absorption spectra were
measured on a Varian CARY 100 Bio UV-visible spectropho-
tometer. Fluorescence (FL) spectra were recorded on a Perkin-
Elmer LS 55 spectrofluorometer.
Scheme 2 Synthesis of the fluorescent probe 1,1-dimethyl-2,5-bis(meso-
benzaldehyde)-3,4-diphenylsilole (DMBFDPS).
17068 | J. Mater. Chem., 2012, 22, 17063–17070
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View Article Online
Synthesis of fluorescent probe of 1,1-dimethyl-2,5-bis(meso-
formylphenyl)-3,4-diphenylsilole (DMBFDPS)
was allowed to stand at RT for 60 min and underwent the
necessary measurement.
The targeted silole derivative, i.e. DMBFDPS was prepared from
di(phenylethynyl)-silane according to the synthetic routes shown
in Scheme 2.20
Preparation of the plasma sample and the detection of Cys in the
human serum
The synthesis of DMBFDPS followed the procedure described
in literature.20 A solution of lithium naphthalenide (LiNp) was
made by stirring a mixture of naphthalene (2.10 g, 16 mmol) and
lithium granular (0.21 g, 30 mmol) in THF (20 mL) at room
temperature for 12 h under nitrogen atmosphere. The resultant
dark green mixture was transferred to a pre-deoxygenated two
necked flask via a cannula as soon as possible. A solution of
di(phenylethynyl)silane (1.04 g, 4 mmol) in THF (10 mL) was
added dropwise to the solution of LiNp, and the resultant
mixture was stirred for 3 h at room temperature. After the
solution was cooled to ꢁ10 ꢂC, ZnCl2-TMEDA (4.00 g, 16
mmol) and 40 mL of THF were added. The fine suspension was
stirred for 1 h and then 160 mg Pd (II) and 2-(3-bromophenyl)-
1,3-dioxolane (1.83 g, 8 mmol) in THF (20 mL) were added
quickly to this system. Afterward the mixture was refluxed at
85 ꢂC for 12 h. 1 M HCl aqueous solution (50 mL) was added and
then after stirring for 30 min, the mixture was extracted with
dichloromethane. The organic layer was washed successively
with brine, and dried over magnesium sulphate. After filtration,
the solvent was evaporated under reduced pressure, and the
residue was purified by silica-gel column chromatography using
petroleum ether (60–90 ꢂC)/ethyl acetate ¼ 10 : 1 (v/v) as eluent,
Rf ¼ 0.30. Pale yellow green solid of DMBFDPS was obtained in
73.4% yield (1.39 g). 1H NMR (400 MHz, CDCl3): d (TMS, ppm)
9.89–9.71 (s, 2H), 7.69–7.55 (d, 2H), 7.48–7.39 (s, 2H), 7.33–7.22
(t, 2H), 7.20–7.10 (d, 2H), 7.09–6.93 (m, 6H), 6.90–6.71 (d, 4H),
0.58–0.45 (s, 6H). 13C NMR (80 MHz, CDCl3): d (TMS, ppm)
192.7, 155.5, 141.2, 141.0, 138.0, 136.5, 135.0, 130.5, 130.4, 130.1,
129.0, 127.9, 127.0, ꢁ3.8. IR (KBr): n ¼ 3061 (w), 2959 (w), 2824
(w), 2724 (w), 1699 (s), 1586 (s), 1479 (m), 1435 (w), 1385 (w),
1302 (m), 1253 (w), 1206 (w), 1158 (m), 1081 (w), 1020 (w), 862
(w), 788 (s), 697 (s), 651 (w), 578 (w) cmꢁ1. Anal. calcd for
C32H26O2Si: C, 81.66; H, 5.57. Found: C, 81.655; H, 5.51. mp:
165 ꢂC. UV (THF): lmax (3max): 356 nm (10ꢁ5 mol Lꢁ1).
Human plasma (EDTA as an anticoagulant) was obtained from
the local hospital and was stored at ꢁ20 ꢂC until analysis. When
used, the plasma sample was thawed and deproteinized by add-
ing 9 mL of acetone to 3 mL of plasma sample. After vortex-
mixing for 30 s, the mixture was centrifuged at 8000 rpm for 8
min (Mikro 22R refrigerated centrifuge, Hettich, Germany) to
precipitate protein, and the supernatants obtained were
concentrated by evaporation to ca. 1/3 of the total volume, under
nitrogen flow. And then the solution was recentrifuged at 10 000
rpm for another 8 min, the resultant supernatants were stored at
4–8 ꢂC for further use. 1 mL of the deproteinized human plasma
was doped with Cys to afford stock solution of human plasma
with Cys solution (12.5 mM, 2.5 mM). The stock solution was
prepared to the appropriate concentration, and the detection of
Cys in the deproteinized plasma was carried out via a similar
procedure to the above mentioned synthesis of thiazolidine in the
DMSO/10 mM HEPES (6/4 in volume, pH 7.4).
Acknowledgements
This work is supported by the National Science Foundation of
China (21074113 and 20974028), the Ministry of Science and
Technology of China (2009CB623605), the Natural Science
Foundation of Zhejiang Province (Z4110056), the Research
Grants Council of Hong Kong (603509, HKUST2/CRF/10, and
604711), the SRFI grant of HKUST (SRFI11SC03PG), the
NSFC/RGC grant (N_HKUST620/11), the University Grants
Committee of Hong Kong (AoE/P-03/08). B.Z.T. thanks the
support from the Cao Guangbiao Foundation of Zhejiang
University.
Notes and references
1 (a) K. G. Reddie and K. S. Carroll, Curr. Opin. Chem. Biol., 2008, 12,
746; (b) E. Weerapana, C. Wang, G. M. Simon, F. Richter, S. Khare,
M. B. D. Dillon, D. A. Bachovchin, K. Mowen, D. Baker and
B. F. Cravatt, Nature, 2010, 468, 790; (c) S. Shahrokhian, Anal.
Chem., 2001, 73, 5972.
Preparation of the nano-aggregates
Stock solution of DMBFDPS in DMSO with a concentration of
0.25 mM was prepared respectively. Aliquots (1 mL) of the stock
solutions were transferred to 10 mL volumetric flasks. After
adding appropriate amounts of DMSO, 10 mM HEPES buffer
solution (pH 7.4) was added dropwise under vigorous stirring to
furnish 25 mM solutions with defined fractions of buffer (fb ¼ 0–
80 vol%). Spectral measurements of the resultant solutions or
aggregate suspensions were performed immediately.
ꢀ
2 (a) H. Refsum, P. M. Ueland, O. Nygard and S. E. Vollset, Annu. Rev.
Med., 1998, 49, 31; (b) S. Seshadri, A. Beiser, J. Selhub, P. F. Jacques,
I. H. Rosenberg, R. B. D’Agostino and P. W. F. Wilson, N. Engl. J.
Med., 2002, 346, 476.
3 (a) R. Dringen and J. Hirrlinger, Biol. Chem., 2003, 384, 505; (b)
R. Cruz, W. Almaguer Melian and J. A. Bergado Rosado, Rev.
Neurol., 2003, 36, 877; (c) J. Schulz, J. Lindenau, J. Seyfried and
J. Dichgans, Eur. J. Biochem., 2000, 267, 4904.
4 (a) A. R. Ivanov, I. V. Nazimov and L. Baratova, J. Chromatogr., A,
2000, 895, 157; (b) K. Amarnath, V. Amarnath, K. Amarnath,
H. L. Valentine and W. M. Valentine, Talanta, 2003, 60, 1229; (c)
X. F. Guo, H. Wang, Y. H. Guo, Z. X. Zhang and H. S. Zhang, J.
Chromatogr., A, 2009, 1216, 3874; (d) L. Lin, X. N. Cao, W. Zhang,
Y. Y. Zhou, J. H. Li and L. T. Jin, Chin. J. Anal. Chem., 2003, 31,
261; (e) O. Nekrassova, N. S. Lawrence and R. G. Compton,
Talanta, 2003, 60, 1085; (f) W. Chen, Y. Zhao, T. Seefeldt and
X. M. Guan, J. Pharm. Biomed. Anal., 2008, 48, 1375.
Detection of Cys in DMSO/10 mM HEPES (6/4 in volume, pH
7.4)
The appropriate amount of Cys was dissolved in 4 mL 10 mM
HEPES and then 5 mL chromatographically pure DMSO was
added into the mixture, finally 1 mL DMSO solution of
DMBFDPS (0.25 mM) was added under vigorous stirring to
afford the detection system with 25 mM of probe. The mixture
5 (a) M. J. MacCoss, N. K. Fukagawa and D. E. Matthews, Anal.
Chem., 1999, 71, 4527; (b) A. Windelberg, O. Arseth, G. Kvalheim
and P. M. Ueland, Clin. Chem., 2005, 51, 2103.
This journal is ª The Royal Society of Chemistry 2012
J. Mater. Chem., 2012, 22, 17063–17070 | 17069

View Article Online
6 A. Zinellu, S. Sotgia, B. Scanu, M. F. Usai, A. G. Fois, V. Spada,
A. Deledda, L. Deiana, P. Pirina and C. Carru, Amino Acids, 2009,
37, 395.
7 (a) H. Jiang and H. X. Ju, Anal. Chem., 2007, 79, 6690; (b) W. Wang,
L. Li, S. F. Liu, C. P. Ma and S. S. Zhang, J. Am. Chem. Soc., 2008,
130, 10846; (c) M. Wen, H. Q. Liu, F. Zhang, Y. Z. Zhu, D. Liu,
Y. Tian and Q. S. Wu, Chem. Commun., 2009, 4530; (d)
A. A. Ensafi, S. Dadkhah-Tehrani and H. karimi-Maleh, Anal. Sci.,
2011, 27, 409.
N. Zhang, F. B. Yu and G. W. Yang, J. Am. Chem. Soc., 2007,
129, 11666; (j) H. L. Chen, Q. Zhao, Y. B. Wu, F. Y. Li, H. Yang,
T. Yi and C. H. Huang, Inorg. Chem., 2007, 46, 11075; (k)
H. Maeda, H. Matsuno, M. Ushida, K. Katayama, K. Saeki and
N. Itoh, Angew. Chem., 2005, 117, 2982; Angew. Chem., Int. Ed.,
2005, 44, 2922.
13 O. Rusin, N. N. S. Luce, R. A. Agbaria, J. O. Escobedo, S. Jiang,
I. M. Warner, F. B. Dawan, K. Lian and R. M. Strongin, J. Am.
Chem. Soc., 2004, 126, 438.
8 P. K. Sudeep, S. T. S. Joseph and K. G. Thomas, J. Am. Chem. Soc.,
2005, 127, 6516.
14 (a) R. G. Kallen, J. Am. Chem. Soc., 1971, 6236; (b) Y.-F. Li, C.-Y. Li,
F. Xu, Y. Zhou and Q.-C. Xiao, Sens. Actuators, B, 2011, 155, 253; (c)
L. Yuan, W. Lin and Y. Yang, Chem. Commun., 2011, 47, 6275; (d)
M.-J. Li, C.-Q. Zhan, M.-J. Nie, G.-N. Chen and X. Chen, J.
Inorg.Biochem., 2011, 105, 420; (e) W. Lin, L. Long, L. Yuan,
Z. Cao, B. Chen and W. Tan, Org. Lett., 2008, 10, 5577; (f) H. Li,
J. Fan, J. Wang, M. Tian, J. Du, S. Sun, P. Sun and X. Peng,
Chem. Commun., 2009, 5904; (g) W. Wang, O. Rusin, X. Xu,
K. K. Kim, J. O. Escobedo, S. O. Fakayode, K. A. Fletcher,
M. Lowry, C. M. Schowalter, C. M. Lawrence, F. R. Fronczek,
I. M. Warner and R. M. Strongin, J. Am. Chem. Soc., 2005, 127,
15949; (h) F. Tanaka, N. Mase and C. F. Barbas III, Chem.
Commun., 2004, 1762; (i) K.-S. Lee, T.-K. Kim, J. H. lee, H.-J. Kim
and J.-I. Hong, Chem. Commun., 2008, 6173; (j) T.-K. Kim,
D.-N. Lee and H.-J. Kim, Tetrahedron Lett., 2008, 49, 4879; (k)
X. Zhang, X. Ren, Q.-H. Xu, K. P. Loh and Z.-K. Chen, Org.
Lett., 2009, 11, 1257; (l) S. Lim, J. O. Escobedo, M. Lowry, X. Xu
and R. Strongin, Chem. Commun., 2010, 46, 5707.
9 (a) W. A. Zhao, M. A. Brook and Y. F. Li, ChemBioChem, 2008, 9,
2363; (b) R. Gill, M. Zayats and I. Willner, Angew. Chem., 2008,
120, 7714; Angew. Chem., Int. Ed., 2008, 47, 7602; (c) J.-S. Lee,
P. A. Ulmann, M. S. Han and C. A. Mirkin, Nano Lett., 2008, 8,
529; (d) W. Zhong, Anal. Bioanal. Chem., 2009, 394, 47; (e)
S. Huang, Q. Xiao, R. Li, H.-L. Guan, J. Liu, X.-R. Liu, Z.-K. He
and Y. Liu, Anal. Chim. Acta, 2009, 645, 73.
10 (a) W. Wang, J. O. Escobedo, C. M. Lawrence and R. M. Strongin, J.
ꢁ
Am. Chem. Soc., 2004, 126, 3400; (b) J. V. Ros-Lis, B. Garcıa,
ꢁ
ꢁ
ꢁ~
ꢁ
D. Jimenez, R. Martınez-Manez, F. Sancenon, J. Soto, F. Gonzalvo
and M. C. Valldecabres, J. Am. Chem. Soc., 2004, 126, 4064; (c)
F. Tanaka, N. Mase and C. F. Barbas, Chem. Commun., 2004,
1762; (d) K.-S. Lee, T.-K. Kim, J. H. Lee, H.-J. Kim and
J.-I. Hong, Chem. Commun., 2008, 6173; (e) Y.-B. Ruan, A.-F. Li,
J.-S. Zhao, J.-S. Shen and Y.-B. Jiang, Chem. Commun., 2010, 46,
4938; (f) Y.-K. Yang, S. Shim and J. Tae, Chem. Commun., 2010,
46, 7766; (g) S. Sreejith, K. P. Divya and A. Ajayaghosh, Angew.
Chem., 2008, 120, 8001; Angew. Chem., Int. Ed., 2008, 47, 7883; (h)
W. Hao, A. McBride, S. McBride, J. P. Gao and Z. Y. Wang, J.
Mater. Chem., 2011, 21, 1040; (i) X. Chen, Y. Zhou, X. Peng and
J. Yoon, Chem. Soc. Rev., 2010, 39, 2120; (j) W. Wei, X. Liang,
G. Hu, Y. Guo and S. Shao, Tetrahedron Lett., 2011, 52, 1422; (k)
Z. Yan, S. Guang, H. Xu and X. Liu, Analyst, 2011, 136, 1916; (l)
Z. Yao, H. Bai, C. Li and G. Shi, Chem. Commun., 2011, 47, 7431;
(m) Z. Yang, N. Zhao, Y. Sun, F. Miao, Y. Liu, X. Liu, Y. Zhang,
W. Ai, G. Song, X. Shen, X. Yu, J. Z. Sun and W.-Y. Wong,
Chem. Commun., 2012, 48, 3442.
15 X. Yang, Y. Guo and R. M. Strongin, Angew. Chem., 2011, 123,
10878; Angew. Chem., Int. Ed., 2011, 50, 10690.
16 (a) N. J. Leonard and R. Y. Ning, J. Org. Chem., 1966, 31, 3928; (b)
P. Blondeau, R. Gauthier, C. Berse and D. Gravel, Can. J. Chem.,
1971, 49, 3866; (c) C. Galli, G. Illuminati, L. Mandolini and
P. Tamborra, J. Am. Chem. Soc., 1977, 99, 2591; (d) G. L. Khatik,
R. Kumar and A. K. Chakraborti, Org. Lett., 2006, 8, 2433.
17 (a) J. Luo, Z. Xie, J. W. Y. Lam, L. Cheng, H. Chen, C. Qiu,
H. S. Kwok, X. Zhan, Y. Liu, D. Zhu and B. Z. Tang, Chem.
Commun., 2001, 1740; (b) G. Yu, S. Yin, Y. Liu, J. Chen, X. Xu,
X. Sun, D. Ma, X. Zhan, Q. Peng, Z. Shuai, B. Z. Tang, D. Zhu,
W. Fang and Y. Luo, J. Am. Chem. Soc., 2005, 127, 6335; (c)
Y. Hong, J. W. Y. Lam and B. Z. Tang, Chem. Commun., 2009, 4332.
18 (a) J. Chen, C. C. W. Law, J. W. Y. Lam, Y. Dong, S. M. F. Lo,
I. D. Williams, D. B. Zhu and B. Z. Tang, Chem. Mater., 2003, 15,
1535; (b) H. Sohn, M. J. Sailor, D. Magde and W. C. Trogler,
J. Am. Chem. Soc., 2003, 125, 3821; (c) J. H. Lee, Q. D. Liu,
D. R. Bai, Y. J. Kang, Y. Tao and S. N. Wang, Organometallics,
2004, 23, 6205; (d) A. J. Boydston and B. L. Pagenkopf, Angew.
Chem., 2004, 116, 6496; Angew. Chem., Int. Ed., 2004, 43, 6336; (e)
L. Aubouy, P. Gerbier, N. Huby, G. Wantz, L. Vignau, L. Hirsch
and J. M. Janot, New J. Chem., 2004, 28, 1086; (f) Y. Ren,
Y. Dong, J. W. Y. Lam, B. Z. Tang and K. S. Wong, Chem. Phys.
Lett., 2005, 402, 468; (g) B. Mi, Y. Dong, Z. Li, J. W. Y. Lam,
11 (a) H. Kwon, K. Lee and H. J. Kim, Chem. Commun., 2011, 47, 1773;
(b) H. S. Jung, K. C. Ko, G. H. Kim, A. R. Lee, Y. C. Na, C. Kang,
J. Y. Lee and J. S. Kim, Org. Lett., 2011, 13, 1498; (c) Y. Liu, Y. Yu,
J. W. Y. Lam, Y. N. Hong, M. Faisal, W. Z. Yuan and B. Z. Tang,
Chem.–Eur. J., 2010, 16, 8433; (d) X. Q. Chen, S.-K. Ko, M. J. Kim,
I. Shin and J. Yoon, Chem. Commun., 2010, 46, 2751; (e) L. Yi,
H. Y. Li, L. Sun, L. L. Liu, C. H. Zhang and Z. Xi, Angew. Chem.,
2009, 121, 4094; Angew. Chem., Int. Ed., 2009, 48, 4034; (f)
W. Y. Lin, L. Yuan, Z. M. Cao, Y. M. Feng and L. L. Long,
Chem.–Eur. J., 2009, 15, 5096; (g) V. Hong, A. A. Kislukhin and
M. G. Finn, J. Am. Chem. Soc., 2009, 131, 9986; (h) W. Jiang,
Q. Q. Fu, H. Y. Fan, J. Ho and W. Wang, Angew. Chem., 2007,
119, 8597; Angew. Chem., Int. Ed., 2007, 46, 8445.
€
12 (a) L. L. Long, W. Y. Lin, B. B. Chen, W. S. Gao and L. Yuan, Chem.
Commun., 2011, 47, 893; (b) J. Y. Shao, H. M. Guo, S. M. Ji and
J. Z. Zhao, Biosens. Bioelectron., 2011, 26, 3012; (c) H. Y. Shiu,
H. C. Chong, Y. C. Leung, M. K. Wong and C. M. Che, Chem.–
Eur. J., 2010, 16, 3308; (d) X. Li, S. J. Qian, Q. J. He, B. Yang,
J. Li and Y. Z. Hu, Org. Biomol. Chem., 2010, 8, 3627; (e)
X.-F. Yang, Z. Su, C. H. Liu, H. P. Qi and M. L. Zhao, Anal.
Bioanal. Chem., 2010, 396, 2667; (f) S.-P. Wang, W.-J. Deng,
D. Sun, M. Yan, H. Zheng and J.-G. Xu, Org. Biomol. Chem.,
2009, 7, 4017; (g) B. Tang, L. L. Yin, X. Wang, Z. Z. Chen,
L. L. Tong and K. H. Xu, Chem. Commun., 2009, 5293; (h)
A. Shibata, K. Furukawa, H. Abe, S. Tsuneda and Y. Ito, Bioorg.
Med. Chem. Lett., 2008, 18, 2246; (i) B. Tang, Y. L. Xing, P. Li,
M. Haubler, H. H. Y. Sung, H. S. Kwok, Y. Dong, I. D. Williams,
Y. Liu, Y. Luo, Z. Shuai, D. Zhu and B. Z. Tang, Chem. Commun.,
2005, 3583; (h) Y. Q. Dong, J. W. Y. Lam, Z. Li, H. Tong,
Y. P. Dong, X. D. Feng and B. Z. Tang, J. Inorg. Organomet.
Polym. Mater., 2005, 15, 287; (i) L. Peng, M. Wang, G. Zhang,
D. Zhang and D. Zhu, Org. Lett., 2009, 11, 1943; (j) J. Mei,
J. Wang, A. Qin, H. Zhao, W. Yuan, Z. Zhao, H. H. Y. Sung,
C. Deng, S. Zhang, I. D. Williams, J. Z. Sun and B. Z. Tang, J.
Mater. Chem., 2012, 22, 4290.
19 S. Yamaguchi, T. Endo, M. Uchida, T. Izumizawa, K. Furukawa and
K. Tamao, Chem.–Eur. J., 2000, 6, 1683.
20 K. Tamao, M. Uchida, T. Izaumizawa, K. Furukawa and
S. Yamaguchi, J. Am. Chem. Soc., 1996, 118, 11974.
17070 | J. Mater. Chem., 2012, 22, 17063–17070
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