An ESIPT-based fluorescent probe for sensitive and selective detection of Cys/Hcy over GSH with a red emission and a large Stokes shift

Article abstract of DOI:10.1016/j.tetlet.2017.06.082

An ESIPT-based fluorescent probe (Probe 1) using acrylate as recognition group for the selective and sensitive detection of cysteine/homocysteine (Cys/Hcy) has been developed. In the presence of Cys/Hcy, this probe was transformed into 1,3-bis(bispyridin-

Full text of DOI:10.1016/j.tetlet.2017.06.082

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Tetrahedron Letters
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An ESIPT-based fluorescent probe for sensitive and selective detection of Cys/Hcy
over GSH with a red emission and a large Stokes shift
1
, a
1, a
Xingjiang Liu , HuiHui Tian , Lei Yang , Yuanan Su , Man Guo and Xiangzhi Song
a
a
a
a, b*
a
College of Chemistry & Chemical Engineering, Central South University, Changsha, Hunan Province, P. R. China, 410083. E-mail:
song@rowland.harvard.edu
b
State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian 116024, China.
1
These authors contribute equal to this work.
ARTICLE INFO
ABSTRACT
Article history:
Received
An ESIPT-based fluorescent probe (Probe 1) using acrylate as recognition group for the
selective and sensitive detection of cysteine/homocysteine (Cys/Hcy) has been developed. In the
presence of Cys/Hcy, this probe was transformed into 1,3-bis(bispyridin-2ylimino)isoindolin-4-
ol (dye 4) which displayed red fluorescence with a large Stokes shift (217 nm) when excited.
The detection limits are as low as 5.4 nM and 7.0 nM for Cys and Hcy respectively (based on
S/N = 3). Importantly, this probe has been successfully demonstrated for the detection of
intracellular Cys/Hcy in living cells.
Received in revised form
Accepted
Available online
Keywords:
Fluorescent probe
Cysteine (Cys)
Homocysteine (Hcy)
Large Stokes shift
Red emission
2
009 Elsevier Ltd. All rights reserved.
1
. Introduction
sensing by virtue of deep tissue penetration, minimal interference
31
from background, and less photo-damage to biological samples.
Biological thiols such as cysteine (Cys), homocysteine (Hcy)
and glutathione (GSH) are of great importance in various
Also, large Stokes shift is a favorable property for fluorescent
probes, which can reduce self-absorption and minimize auto-
32-34
fluorescence, and therefore improve the detection sensitivity.
1
physiological and pathological processes. Numerous studies
-4
have shown that abnormal levels of biothiols in vivo can cause
5
many diseases. For instance, elevated level of Cys can lead to
1,3-bis(bispyridin-2ylimino)isoindolin-4-ol (shown in Scheme
1), dye 4, was a desired scaffold to construct fluorescent probes
due to the long emission wavelength (λem = 585 nm) and large
Stokes shift (217 nm) resulting from the excited-state
6
,7
neurotoxicity, while its deficiency can cause slow growth, hair
depigmentation, dropsy, somnolence, liver damage, muscle and
8
fat loss, skin lesions, and asthenia. Moreover, the level of Hcy is
considered to be closely associated with cardiovascular diseases
3
5
intramolecular photon transfer (ESIPT) process. In this study,
we developed Probe 1 by assembling an acrylate moiety into dye
4 as a sensing group for sensitive detection of Cys/Hcy over GSH
and other amino acids. The synthetic route for Probe 1 was
presented in Scheme 1. We expected that the acrylate moiety in
9
and neural tube defects. GSH has important functions in
maintaining redox activities, xenobiotic metabolism, signal
transmission, and gene regulation in cells.
the valid approaches, especially those that can differentiate these
three species, for the detection of biothiols with high sensitivity
and selectivity are highly demanded.
1
0-12
As a consequence,
Fluorescent technique has been a dominant methodology
which is extensively used for the detection of various species by
virtue of its easy operation, high sensitivity, and high spatial and
1
3-17
temporal resolution.
fluorescent probes has been reported for the detection of
In recent years, although many
1
8-25
Scheme 1 Synthetic route of Probe
1
. (a) K CO , NaNO , DMSO, 130
2
2
3
biothiols,
only a handful of them are able to discriminate
°
C, 30 min, yield 54%; (b) 2-aminopyridine, CaCl
2
,
n
-BuOH, 110 °C, 5
these three biothiols because of their similar structure and
reactivity. The assembly of an acrylate moiety into fluorescent
dyes was proved to be a valid method for discriminating Cys/Hcy
days, yield 25%; (c) acryloyl chloride, DCM, triethylamine, r.t., 10 min,
yield 81.2 %.
26-29
over GSH and other amino acids.
So far, acrylate-based
fluorescent probes with both red/NIR emission and large Stokes
Probe 1 would induce a photo-induced electron transfer (PET)
process and inhibit ESIPT process in its excited state and
3
shift for specific detection of Cys/Hcy are rare. Fluorescent
0
3
therefore would quench its fluorescence effectively. In the
0
probes with long wavelength emission is preferred in fluorescent
presence of Cys/Hcy, Probe 1 underwent a conjugate addition

2
3
6,37
reaction with Cys/Hcy to generate a thioether,
which was
mM CTAB). Spectra were obtained after incubation for 20 min and the
excitation wavelength was 368 nm.
further transformed into dye 4 through an intramolecular
cyclization reaction (shown in Scheme 2). As for GSH, the
corresponding intermediate thioether could not undergo
intramolecular cyclization reaction due to the kinetically
unfavourable ten-membered ring formation (shown in Scheme
3
8-40
S1).
As a consequence, Probe 1 would be able to specifically
detect Cys/Hcy against GSH and other amino acids with a red
emission and a large Stokes shift.
Fig. 2 Plot of fluorescence intensity at 585 nm of Probe 1 (5.0 µM) vs Cys
concentration. Inset: a linear correlation between the fluorescence intensity at
5
85 nm and concentration of Cys. Data were obtained after incubation for 20
min and excitation wavelength was 368 nm.
The UV-vis absorption and fluorescence studies on Probe 1
suggested the transformation process from this probe to dye 4
(
shown in Fig. S1 and S2). To further confirm the sensing
mechanism, high performance liquid chromatography (HPLC)
analysis was adopted to monitor the interaction of Probe 1 with
Cys. The reference dye 4 with a retention time at 24.6 min was
used for observing the generation of dye 4 from the reaction of
Probe 1 with Cys. The solution of Probe 1 displayed a single
peak at 15.0 min. Upon the treatment with 0.5 equiv. of Cys, the
signal for Probe 1 was decreased and a new peak at 24.6 min
occurred. When treated with 5.0 equiv. of Cys, the solution of
Probe 1 only exhibited a single peak at 24.6 min (Fig. 3c). The
HPLC analysis for the interaction of this probe with GSH was
Scheme 2 Proposed reaction mechanism of Probe
1 with Cys/Hcy.
2
. Results and discussion
The sensitivity of Probe 1 was investigated by incubating this
probe with different concentrations of Cys/Hcy in PBS buffer
10.0 mM, pH 7.4, 1.0 mM CTAB). As we expected, the solution
(
of Probe 1 had no fluorescence. However, the addition of Cys to
the solution of Probe 1 resulted in a remarkable fluorescence
enhancement with a maximum at 585 nm. Moreover, the
fluorescence intensity was gradually increased along with the
added amount of Cys. When 5.0 equiv. of Cys was added to the
solution of Probe 1, a maximized fluorescence enhancement (86-
fold) was obtained. The plot of fluorescent intensity at 585 nm
was linear with the concentration of Cys in the range of 0.0 to 7.0
µM. Based on S/N = 3, the calculated detection limit was 5.4 nM.
As for Hcy, similar results were obtained and the detection limit
was as low as 7.0 nM (shown in Fig. S3 and S4). These results
indicated that Probe 1 was highly sensitive to Cys/Hcy in
aqueous media.
1
13
depicted in Fig. S13. In addition, H NMR, C NMR and HRMS
spectra of the reaction product of Probe 1 with excessive Cys
were nearly identical to that for dye 4. These results strongly
confirmed the proposed sensing mechanism shown in Scheme 2.
Fig. 3 HPLC chromatograms: (a) Probe
1
(50.0
M) treated with 0.5 equiv. of Cys; (c) Probe
.0 equiv. of Cys; (d) dye (50.0 M). The data was recorded after
µ
M); (b) Probe
1 (50.0
µ
1
(50.0 M) treated with
µ
5
4
µ
incubation for 20 min in PBS buffer (10.0 mM, pH 7.4, 1.0 mM CTAB).
Condition: eluent, H O/CH CN (v/v, 3/7); flow rate, 1.0 mL/min;
temperature, 25 °C; detection wavelength, 370 nm; injection volume,
0.0 L.
2
3
Fig. 1 Fluorescent spectra of Probe 1 (5.0 µM) treated with different
2
µ
concentrations of Cys (0.0-5.0 equiv.) in PBS buffer (10.0 mM, pH 7.4, 1.0

Next, we investigated the selectivity of Probe 1 toward
physiologically relevant amino acids, including Asp, Ala, Val,
Phe, His, Leu, Ser, IIe, Trp, Lys, Arg, Pro, Gly, Met, Tyr, Glu,
Thr, Cys, Hcy and GSH. As shown in Fig. 4, a remarkable
fluorescence enhancement was observed after Cys/Hcy was
added to the solution of Probe 1. To our delight, GSH gave rise
to a little fluorescence enhancement, and negligible fluorescence
was induced by other amino acids. In addition, anti-interference
ability of Probe 1 toward Cys was determined by investigating its
fluorescence performance with the co-existence of other relevant
amino acids. It’s seen in Fig. 5 that the presence of other
interfering analytes had little impact on the sensing properties of
Probe 1. These results implied that Probe 1 was able to recognize
Cys/Hcy with high selectivity and good anti-interference ability.
10 min and 20 min for Cys and Hcy, respectively. The
observed first order rate constant for Probe
1, kobs, was
-1
-
1
determined to be 0.2023 min and 0.1494 min when this
probe reacted with Cys and Hcy respectively. On the
contrary, Probe with GSH exhibited a little fluorescence
enhancement. In contrast, negligible fluorescence change
was observed for free Probe during the same interval.
1
1
Fig. 6 Time-dependent fluorescence change of Probe 1 (5.0 µM) after the
addition of Cys (5.0 equiv., black), Hcy (5.0 equiv., red) and GSH (5.0 equiv.,
blue) in PBS buffer (10.0 mM, pH 7.4, 1.0 mM CTAB). Data were obtained
after incubation for 20 min and excitation wavelength was 368 nm.
We investigated the pH effect on the fluorescence behavior of
Probe 1 toward Cys/Hcy in aqueous media, shown in Fig. 7. It’s
found that free Probe 1 show little fluorescence between pH 2.0
and 12.0. Between pH 6.0 and 10.0, the solution of Probe 1 with
Cys/Hcy displayed strong fluorescence signals. Therefore, it can
Fig. 4 Fluorescence spectra of Probe 1 (5.0 µM) in response to various amino
acids (Asp, Ala, Val, Phe, His, Leu, Ser, IIe, Trp, Lys, Arg, Pro, Gly, Met,
Tyr, Glu, GSH, Hcy and Cys, 25.0 µM for each) in PBS buffer (10.0 mM,
pH 7.4, 1.0 mM CTAB). Data were obtained after incubation for 20 min and
excitation wavelength was 368 nm.
be concluded that Probe
physiological conditions.
1 could function well under
Fig. 7 Fluorescence changes of Probe 1 (5.0 µM) in the absence/presence of
Cys/Hcy (5.0 equiv.) in the pH range from 2.0 to 13.0. Data were obtained
after incubation for 20 min and excitation wavelength was 368 nm.
Fig. 5 Fluorescence response of Probe
the co-existence of other amino acids (25.0 µM for each) in PBS buffer
10.0 mM, pH 7.4, 1.0 mM CTAB). Data were obtained after incubation for
0 min and excitation wavelength was 368 nm. (1) Cys, (2) Asp, (3) Ala,
4) Val, (5) Phe, (6) His, (7) Leu, (8) Ser, (9) IIe, (10) Trp, (11) Lys, (12)
1 (5.0 µM) to Cys (5.0 equiv.) in
(
2
Encouraged by the excellent properties of Probe 1 in aqueous
media, we used it to visualize intracellular Cys/Hcy in living 4T1
cells. When cells were incubated with Probe 1 (10.0 µM) at 37 °C
for 30 min, the endogenous Cys/Hcy in living cells reacted with
Probe 1 to result in strong red fluorescence (Fig. 8-B1),
indicating that this probe was able to permeate cell membrane
and to detect intracellular Cys and Hcy in living cells. When the
cells were treated with Cys/Hcy (1.0 mM) for 30 min at 37 °C
and subsequently incubated with Probe 1 (10.0 µM) for another
(
Arg, (13) Pro, (14) Gly, (15) Met, (16) Tyr, (17) Glu, (18) Thr, (19) GSH.
Data were obtained after incubation for 20 min and excitation wavelength
was 368 nm.
Kinetic studies were carried out by recording the
fluorescence intensity at 585 nm along with the reaction
time after the addition of Cys, Hcy and GSH (5.0 equiv. for
each) to the solution of Probe
, the addition of Cys/Hcy induced rapid and remarkable
fluorescence enhancements which were maximized within
1 (5.0 µM). As shown in Fig.
6
3
0 min at 37 °C, stronger red fluorescence signals were observed
in consequence of the higher concentrations of Cys/Hcy built up

4
exogenously (Fig. 8-C1 and Fig. 8-D1). For the control
experiment, cells were pre-treated with N-ethylmaleimide (1.0
mM), a thiol-blocking reagent, at 37 °C for 30 min and then
incubated with Probe 1 (10.0 µM) at 37 °C for 30 min. Since
biothiols in living cells were blocked to prevent reacting with
Probe 1, very weak fluorescence signal could be seen (Fig. 8-A1).
These results indicated that this probe could detect Cys/Hcy in
living cells.
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at

Graphical Abstract
An ESIPT-based fluorescent probe for
sensitive and selective detection of Cys/Hcy
over GSH with a red emission and a large
Stokes shift
Leave this area blank for abstract info.
Xingjiang Liu, HuiHui Tian, Lei Yang, Yuanan Su, Man Guo and Xiangzhi Song*

6
Research Highlights
This probe can successfully discriminate Cys/Hcy
from GSH and other amino acids.
This probe exhibited a red emission (λem = 585 nm)
and a large Stokes shift (217 nm).
The detection limits were 5.4 nM and 7.0 nM for
Cys and Hcy respectively.