A highly selective fluorescence turn-on sensor for cysteine/homocysteine and its application in bioimaging

Article abstract of DOI:10.1021/ja073140i

We demonstrated a highly electron-deficient system 1 as a turn-on fluorescence sensor for intracellular imaging of Cys/Hcy in biological samples. On the basis of the specific nucleophilic ability of thiols of Cys and Hcy, 1 exhibited high selectivity and sensitivity for Cys/Hcy over other amino acids and thiol biomolecules. This probe is the first Cys/Hcy sensor with excitation in the visible region and turn-on (75-fold) fluorescence emission and also gives a significant increase in two-photon excited fluorescence for sensing Cys/Hcy. Moreover, confocal laser scanning microscopy and two-photon laser scanning microscopy experiments further established that 1 can be used for sensing Cys/Hcy within biological samples. Copyright

Full text of DOI:10.1021/ja073140i

Published on Web 08/02/2007
A Highly Selective Fluorescence Turn-on Sensor for Cysteine/Homocysteine
and Its Application in Bioimaging
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†
,†
‡
§
⊥
†
Meng Zhang, Mengxiao Yu, Fuyou Li,* Minwei Zhu, Manyu Li, Yanhong Gao, Lei Li,
†
§
†
†
,†
Zhiqiang Liu, Jianping Zhang, Dengqing Zhang, Tao Yi, and Chunhui Huang*
Department of Chemistry & Laboratory of AdVanced Materials, and Hospital of Obstetrics and Gynecology, Fudan
UniVersity, Shanghai, 200433, P. R. China; State Key Laboratory for Structural Chemistry of Unstable and Stable
Species, Institute of Chemistry, Chinese Academy of Sciences, Beijing, 100080, P. R. China; and Hospital of
Xinhua, Medicine School of Shanghai Jiaotong UniVersity, Shanghai, 200092, P. R. China
Received May 14, 2007; E-mail: fyli@fudan.edu.cn; chhuang@pku.edu.cn
For their participation in the process of reversible redox reactions,
cysteine (Cys) and homocysteine (Hcy) play crucial roles in
1
physiological matrices. Deficiency of Cys would lead to many
diseases, such as hematopoiesis decrease, leucocyte loss, psoriasis,
etc.2 Hcy is a risk factor for cardiovascular and Alzheimer’s
3
4
disease. Some Cys and Hcy analyses have been developed in
5
5
5
conjunction with HPLC, capillary electrophoresis, immunoassay,
6
colorimetric and fluorescence detection, etc.
Recently, a great effort has gone into the development of selective
fluorescence bioimaging for highly sensitive, high-speed spatial
7
analysis of cells. To image the distribution of Cys/Hcy in cellular
processes, suitable turn-on fluorescence chemosensors for Cys/Hcy
should be developed. Up to now, only Tanaka et al.6 have described
a fluorescence turn-on sensor for Cys; however, its maximum
emission wavelength was only 380 nm. So the lack of suitable Cys/
Hcy-sensitive sensors with turn-on fluorescence within the visible
region is obvious. We are interested in developing a fluorescence
probe that exhibits an increasing visible fluorescence emission upon
addition of Cys/Hcy to other amino acids and thiol biomacromol-
ecules. Herein, we describe a significant emission-enhancing probe
c
1
(Scheme 1) for Cys/Hcy and demonstrate its utility in bioimaging.
Qian et al. reported that the reaction of 1 with n-dodecyl thiol
8
required drastic reaction conditions (reflux with large excess of
n-dodecyl thiol in acetonitrile for 3 days). Herein, by using
methanol as the reaction solvent instead of acetonitrile, we easily
obtained compound 2 (Scheme 1) by the reaction of 1 with
Figure 1. (a) Absorption spectral changes of 1 (10 µM) upon addition of
Cys (0-330 µM). Inset shows the color changes of 1 (10 µM) in the
presence and absence of amino acids and peptide. From left to right, then
bottom to top: 1 (10 µM) and 1 with Ala, Cys, GSH, Hcy, Glu, Try, Arg,
Iso, Asp, Ser, Gly, Val, Leu, Asn, Tyr, Met, Pro, Lys, Thr, and His,
respectively. (b) Fluorescence spectral changes of 1 (5 µM) upon addition
of Hcy (0-200 µM) (λex ) 465 nm). Inset shows the two-photon absorption
spectrum of 1 upon addition of 40 equiv Hcy. Condition: in methanol-
HEPES solution (7:3, v/v, pH 7). Each spectrum is acquired 10 min after
Hcy (or Cys) addition.
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3-thiopropionic acid under mild conditions (Supporting Informa-
tion). Interestingly, 2 exhibits brilliant red-orange color and intense
orange-red fluorescence in comparison to 1. We reasoned that the
reaction of 1 with Cys or Hcy would readily promote a fluorescence
response, because Cys and Hcy are similar in chemical structure
to 3-thiopropionic acid.
In the light of the dependence of steric hindrance on the reaction
H
velocity, mild conditions should be chosen to reduce the S
N
Ar
Scheme 1. Reaction of 1 with 3-Thiopropionic Acid
reaction velocity and to enhance selectivity. Herein, the recognition
of Cys/Hcy with 1 was investigated in a mixture of methanol and
HEPES (7:3, v/v) solution at pH 7 by UV-vis absorption and
fluorescence techniques. Notably, upon addition of Cys, the
absorption band of 1 centered at 430 nm gradually decreased and
a new absorption band centered at 580 nm appeared with a distinct
isosbestic point at 465 nm (Figure 1a), corresponding to an apparent
color change from yellow-green to red-orange (Figure 1a inset),
indicating the reaction of 1 with Cys. Similar spectral features were
observed for Cys and Hcy in the absorption and fluorescence
titrations of 1. As shown in Figure 1b, the emission peak of 1 at
588 nm (λex ) 465 nm) increased rapidly upon addition of Hcy.
When 40 equiv Hcy was added to the solution of 1 (Φ ) 0.008),
F
a 75-fold increase in fluorescence intensity at 588 nm was observed,
corresponding to intense orange-red fluorescence. These facts
indicate that 1 can serve as a “naked-eye” probe for Cys/Hcy.
Fluorescence responses of 1 (5 µM) to various amino acids and
thiol biomacromolecules were also investigated. No obvious
changes of 1 were observed upon addition of other natural amino
†
Department of Chemistry, Fudan University.
‡
Hospital of Obstetrics and Gynecology, Fudan University.
§
Chinese Academy of Sciences.
⊥
Medicine School of Shanghai Jiaotong University.
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J. AM. CHEM. SOC. 2007, 129, 10322-10323
10.1021/ja073140i CCC: $37.00 © 2007 American Chemical Society

C O M M U N I C A T I O N S
interestingly, the two-photon fluorescence signals of HeLa cells
stained with 1 were located diffusely over the cytosol and nucleus
(Figure 3b), suggesting a more homogeneous distribution of
intracellular Cys/Hcy in comparison with PANC cells. The differ-
ence in fluorescence images of PANC and HeLa cells implied the
diversity of Cys/Hcy metabolism in the different type of cells.
Furthermore, we also investigated the living cell bioimaging of
Cys/Hcy, by using DMSO-PBS (1:49, v/v, pH 7) as a staining
medium. Living Caov-3 ovarian carcinoma cells incubated with 1
showed an intense intracellular fluorescence. However, after
pretreatment with N-ethylmaleimide (as a thio-reactive compound)
for 2 h, no obvious fluorescence was observed for 1-loaded cells
Figure 2. Confocal fluorescence and brightfield images of ACCM cells:
a) fluorescence image of cells incubated with 10 µM 1 for 10 min at
(
2
5 °C (λex ) 543 nm); (b) brightfield image of cells shown in panel a. The
overlay image of panels a and b is shown in panel c.
(Supporting Information), suggesting the specific reaction of 1 with
Cys/Hcy. Therefore, these data establish that 1 could be used as a
fluorescence turn-on probe for bioimaging Cys/Hcy.
In summary, we have demonstrated a new fluorescence probe 1
for imaging Cys/Hcy in living and fixed cells. This is the first Cys/
Hcy sensor with excitation in the visible region and 75-fold turn-
on fluorescence emission and features excellent selectivity for Cys/
Hcy over other amino acids and GSH. Moreover, confocal laser
scanning microscopy and two-photon laser scanning microscopy
experiments indicate that 1 can be used for bioimaging of Cys/
Hcy. We anticipate that this probe will be of great benefit to
biomedical researchers for studying the effects of Cys/Hcy in
biological systems.
Figure 3. Two-photon fluorescence images of PANC (a) and HeLa (b)
cells incubated with 10 µM 1 for 10 min at 25 °C (λex ) 880 nm).
acids (Supporting Information). Importantly, as a structurally related
thiol biomolecule, reduced glutathione (GSH) exhibited low reaction
activity to 1 within 1 h, and other thiol biomacromolecules hardly
reacted with 1 under these conditions, which may be due to their
steric hindrance. Furthermore, analysis of fluorescence spectra of
1
with other amino acids and peptides suggested recognition ability
Acknowledgment. The authors thank NSFC (Grant Nos.
20490210 and 20501006), NHTPC (Grant No. 2006AA03Z318)
and Shanghai Sci. Tech. Comm. (Grant Nos. 05DJ14004 and
06QH14002) for financial support. We also thank Dr. J. Z. Xi and
Dr. Y. Y. Huang for helpful discussion.
in the following order: Cys/Hcy . GSH > other amino acids.
The competition experiments were tested in the presence of Cys
(or Hcy), as well as in a mixture of Cys (or Hcy) and 25 equiv of
other amino acids or GSH. No significant variation in fluorescence
intensity was found in comparison with experiments containing only
Cys or Hcy. This indicates that 1 displays a high selectivity for
Cys/Hcy.
Supporting Information Available: Synthetic and experimental
details. This material is available free of charge via the Internet at http://
pubs.acs.org.
A practical application of 1 toward Cys and Hcy in biological
samples was developed by confocal laser scanning microscopy
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-
50
two-photon absorption cross-section of 11 GM (1 GM ) 10
4
-1
cm ‚s‚photon ) whereas 1 is not TPEF-active, indicating that 1 is
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(
(
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(
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