An iminocoumarin-Cu(ii) ensemble-based chemodosimeter toward thiols

Article abstract of DOI:10.1039/c1cc10672d

An 'ensemble'-based fluoregenic chemodosimeter 1-Cu(ii) for detection of thiols is reported. Complex 1-Cu(ii) sensitively senses thiols followed by hydrolysis to give a marked fluorescence enhancement over other amino acids at pH 7.4 under aqueous media.

Full text of DOI:10.1039/c1cc10672d

Chemical Communications
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Volume 47 | Number 18 | 14 May 2011 | Pages 5085–5344
ISSN 1359-7345
COMMUNICATION
Chulhun Kang, Jong Seung Kim et al.
An iminocoumarin–Cu(II) ensemble-based chemodosimeter toward thiols

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Cite this: Chem. Commun., 2011, 47, 5142–5144
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An iminocoumarin–Cu(II) ensemble-based chemodosimeter toward thiolsw
Hyo Sung Jung,a Ji Hye Han,b Yoichi Habata,c Chulhun Kang*b and Jong Seung Kim*a
Received 3rd February 2011, Accepted 23rd February 2011
DOI: 10.1039/c1cc10672d
An ‘ensemble’-based fluoregenic chemodosimeter 1–Cu(II) for
detection of thiols is reported. Complex 1–Cu(II) sensitively
senses thiols followed by hydrolysis to give a marked fluores-
cence enhancement over other amino acids at pH 7.4 under
aqueous media. Confocal microscopic imaging of complex
1–Cu(II) is also herewith demonstrated for cellular thiol detection
in HepG2 cells.
various fluorescent chemodosimeters. In addition, the ensemble-
based detection method would have a critical advantage of
water solubility in biological applications because the metal
ionic pairs in the probe itself may contribute to its water
solubility in aqueous medium.
Compound 1 was synthesized by condensation of coumarin-
aldehyde and 2-aminophenol. The metal complex, 1–Cu(II),
was obtained by mixing 1 with Cu(ClO4)2 in DMSO solution
(Scheme S1, ESIw). To demonstrate the role of phenolic OH in
formation of the metal complex, 2 was prepared by synthetic
modification of 1 for comparison. Detailed characterizations
of the prepared compounds are provided in ESI.w In
Schiff bases, the CQN bond is known to be labile in aqueous
media and is easily hydrolysed.11 Fig. S1 (ESIw) shows the
time-dependent fluorescence changes of 1 and 2 in the absence
or presence of the Cu(II) ion under aqueous environment
(10 mM PBS buffer, pH 7.4, 1.0% DMSO).
Biological thiols play important roles in the cellular anti-
oxidant defense system.1 Glutathione (GSH) is the most abundant
cellular thiol whose concentration ranges from 1.0 to 15 mM.2
It serves many cellular functions, including maintenance of
intracellular redox activities, xenobiotic metabolism, intra-
cellular signal transduction, and gene regulation.3 Moreover,
abnormal levels of GSH are known to be associated with
human diseases such as leucocyte loss, psoriasis, liver damage,
cancer, and AIDS.4
In the absence of the Cu(II) ion, both 1 and 2 were easily
hydrolyzed to produce strongly fluorescent 3. The NMR
spectrum of the hydrolysis product 1 is identical (Fig. S2, ESIw)
to that of 3 showing an absorption band at 468 nm and an
emission band at 514 nm (Ff = 0.67) (Fig. S1 and Table S1, ESIw).
The time course of the fluorescence enhancement using
each compound apparently follows first order rate kinetics.
Interestingly, we found that in the presence of the Cu(II) ion
the fluorescence intensity of 1 was quenched in a similar
condition but that of 2 was not. Unlike 2, the o-OH unit of
1 contributes to its complex formation with Cu(II) (Scheme 1)
which is resistant to further hydrolysis. The crystal structure of
1–Cu(II) (Fig. 2) confirms that the o-OH group in 1 serves as
an additional binding site for the Cu(II) ion coordination to
provide a stable complex which gives a strongly reduced
fluorescence presumably due to the MLCT5b,12-based heavy
Fluorescent probes possess innate advantages over the probes
of other types because of their high sensitivity, specificity,
simplicity of implementation, and fast response times, offering
application methods not only for in vitro assays but also for
in vivo imaging studies.5 Accordingly, considerable efforts
have been made to develop them for many applications
including detection of thiol species such as GSH in biological
samples.6–10 The reported fluorescent probes for thiols are
mostly based on Michael addition,6 cyclization with aldehyde,7
cleavage of disulfide8 and sulfonamide9 by thiols, and demetali-
zation from Cu-complex.10
Although thiol-facilitated fluorescence enhancement from
a nonfluorescent copper complex has been introduced as a
chemosensing ‘ensemble’ system,10 its chemodosimetric version
has never been explored yet for thiol detection. Such detection
may be achievable in such a way of copper coordinated Schiff
base followed by demetalization and hydrolytic cleavage of the
Schiff base to generate fluorescence (Scheme 2). This approach
may improve the sensitivity due to a thiol specific affinity of a
copper ion and also broaden the methodologies for designing
a Department of Chemistry, Korea University, Seoul 136-701, Korea.
E-mail: jongskim@korea.ac.kr; Fax: +82 2-3290-3121
b The School of East-West Medical Science, Kyung Hee University,
Yongin 446-701, Korea. E-mail: kangch@khu.ac.kr
c Department of Chemistry, Toho University, Chiba, 274-8510, Japan
w Electronic supplementary information (ESI) available: Experimental
details; synthesis; spectroscopic data; crystal data for the X-ray diffrac-
tion studies. CCDC [CCDC NUMBER(S)]. For ESI and crystallo-
graphic data in CIF or other electronic format see DOI: 10.1039/
c1cc10672d
Scheme 1 Schematic illustration of fluorescence changes of 1 and 2
upon addition of various metals in aqueous media.
c
5142 Chem. Commun., 2011, 47, 5142–5144
This journal is The Royal Society of Chemistry 2011

Scheme 2 Schematic illustration of the thiol detecting chemodosimetric
mechanism of 1–Cu(II) in aqueous media.
Fig. 3 Fluorescence spectra (2.0 mM) of 1–Cu(II) in aqueous solution
(10 mM PBS buffer, pH 7.4, 1% DMSO) upon addition of (A) various
amino acids (GSH, Cys, Hcy, Ala, Arg, Asn, Asp, Gln, Gluc, Glu,
Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Tau, Thr, Trp, Tyr, Val)
after 30 min and (B) the variable concentrations of GSH (0–14 eq. to
[1–Cu(II)]) after 5 min. Excitation at 479 nm (slit = 1.5/1.5). Inset: the
plot of emission intensity at 514 nm vs. [GSH]/[1–Cu(II)].
Fig. 1 (A) UV-Vis (5.0 mM) and (B) fluorescence (2.0 mM) spectra of
1 in aqueous solution (10 mM PBS buffer, pH 7.4, 1.0% DMSO) upon
addition of various concentrations of Cu(II) (0–1.2 eq.). Excitation at
479 nm after 5 min (slit = 1.5/1.5). Inset: the plot of emission intensity
at 514 nm vs. [Cu(II)]/[1].
Fig. 4 (a) Color and (b) fluorescence photographs of 1–Cu(II) (2.0 mM)
in the absence (left) and presence (middle) of GSH (10 eq.) or (right)
EDTA (100 eq.) in aqueous solution (10 mM PBS buffer, pH 7.4,
1.0% DMSO). Excitation at 365 nm using a UV lamp after 10 min.
concentration of GSH up to 10 eq. Consistently, the visual
emission color of 1–Cu(II) solution turned from dark to green
(Fig. 4b) as well.
Fig. 2 Crystal structure of 1–Cu(II). Thermal ellipsoids are shown at
the 50% probability level. Hydrogen atoms are omitted for clarity.
metal ion effect. Absorption and emission changes of 1 are
saturated with addition of only 1.0 equivalents of Cu(II). The
ESI-MS spectrum (Fig. S3, ESIw) of the 1–Cu(II) complex is
indicative of the 1 : 1 complex of 1 for the Cu(II) ion.
Competitive experiments (Fig. S4, ESIw) also confirm that
1–Cu(II) shows a selective response to thiols even in the
presence of other amino acids. Naked eyed turn-on sensing
for GSH using 1–Cu(II) is also demonstrated as shown in
Fig. 4a. For the detection limit, chemodosimetric reaction of
1–Cu(II) with GSH is linear and sensitive enough to detect
10À8 M GSH in aqueous solutions (Fig. S5 and S6, ESIw). In
addition, the fluorescence change of 1–Cu(II) with GSH is
independent of pH in the range 5 to 10 (Fig. S7, ESIw).
Therefore, the complex 1–Cu(II) in our study is desirable as
a chemodosimetric fluorescence probe toward thiols in aspect
of a simple and reliable sensing assay in biological systems.
Since it is known that the GSH may reduce Cu(II) to Cu(I),13
it deserves to be considered a possibility involving the putative
reduction of Cu(II) in this study. So, we have tested fluores-
cence changes of compound using Cu(I) upon addition of
thiol. In a similar manner with Cu(II), fluorescence of the
1–Cu(I) complex was found to be enhanced with addition of
thiols as indicated in Fig. S9 (ESIw) as well. It is therefore
notable that regardless of oxidation states of the copper ion,
a copper complex with 1 is well formed, and demetalized
by thiols followed by hydrolysis to give rise to an enhanced
fluorescence.
From the results in Fig. 1 and 2, and Fig. S1 (ESIw), it could
be inferred that removal of Cu(II) from the 1–Cu(II) complex
followed by hydrolysis of its Schiff base (namely, ensemble
system) by water provides a strong fluorescence because the
coumarinaldehyde (3) is produced. Thereby, it is interesting to
suggest that selective removal of the Cu(II) ion by target
analytes such as thiols could play an important role in the
‘fluorescence Off–On’ analyte detection system. To the best of our
knowledge, this ‘ensemble’ system in regard of the chemo-
dosimetric thiol detection has not been exploited yet.
To investigate the ability of 1–Cu(II) as an ensemble-based
chemodosimetric probe for thiols in biological media, absorp-
tion and emission changes of aqueous solution of 1–Cu(II)
(10 mM PBS buffer, pH 7.4, 1.0% DMSO) were examined
with addition of various amino acids. Indeed, as shown in
Fig. 3A, 1–Cu(II) revealed remarkably enhanced emission with
only addition of thiols whereas no change was observed with
other amino acids (Ala, Arg, Asn, Asp, Gln, Gluc, Glu, Gly,
His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Tau, Thr, Trp, Tyr, Val).
We found that emission spectrum of a mixture of 1–Cu(II)
and thiols is identical to that of 3 (Fig. S2 and S10, ESIw). As
seen in Fig. 3B, when 1–Cu(II) was titrated with GSH, the
fluorescence intensity of the solution was proportional to the
In order to further demonstrate that 1–Cu(II) can detect
intracellular thiols, confocal microscopic experiments were
conducted. Fig. 5 shows fluorescence images of the HepG2
cell line, a human hepatoma cell line, regarding the presence of
c
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Chem. Commun., 2011, 47, 5142–5144 5143

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