Fluorescent chemodosimeter for Cys/Hcy with a large absorption shift and imaging in living cells

Article abstract of DOI:10.1039/c0ob00957a

A novel molecule T1 with efficient intramolecular charge transfer was designed as a fluorescent chemodosimeter for cysteine (Cys) and homocysteine (Hcy). Upon addition of Cys/Hcy, T1 exhibited greatly enhanced fluorescence intensity as well as a large absorption peak shift (70 nm), and can be used for bioimaging of Cys/Hcy in living cells and detection in human plasma by visual color change. The detection mechanism was proved by ¹H NMR, mass spectrometry analysis and Gaussian calculations.

Full text of DOI:10.1039/c0ob00957a

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Organic &
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Fluorescent chemodosimeter for Cys/Hcy with a large absorption shift and
imaging in living cells†
Mingming Hu,a Jiangli Fan,*a Honglin Li,a Kedong Song,b Song Wang,a Guanghui Chenga and Xiaojun Peng*a
Received 30th October 2010, Accepted 24th November 2010
DOI: 10.1039/c0ob00957a
A novel molecule T1 with efficient intramolecular charge
transfer was designed as a fluorescent chemodosimeter for
cysteine (Cys) and homocysteine (Hcy). Upon addition of
Cys/Hcy, T1 exhibited greatly enhanced fluorescence intensity
as well as a large absorption peak shift (70 nm), and can be
used for bioimaging of Cys/Hcy in living cells and detection
in human plasma by visual color change. The detection
mechanism was proved by 1H NMR, mass spectrometry
analysis and Gaussian calculations.
mental effects and improve the detection sensitivity.52–55 Thus,
a chemodosimeter exhibiting greatly changed emission intensity
as well as a large absorption peak shift is ideal for sensing.
After Rusin and Strongin et al. designed the first fluorescent
chemodosimeter for Cys/Hcy by colorimetric and fluorometric
responses with excitation in the visible region,26 some relative
works have been reported.29,30,33,36 However, they have not achieved
intracellular detection of Cys/Hcy yet. It still leaves significant
work to explore novel ratiometric chemodosimeters in the visible
region with appropriate membrane permeability.
In our previous study of a novel type of squarylium infrared
absorbing dye,56 we found that, part of the dye, 7-dimethylamino-
1,4-benzoxazin-2-one showed good spectral properties. This small
molecule is not symmetrical, and the dipole moment in the
excited state is much larger than in the ground state.57 The slight
structure differences between coumarin and 7-dimethylamino-
1,4-benzoxazin-2-one, make the latter have a longer wavelength.
Therefore, we introduced a simple aldehyde group to this
molecule and developed a novel fluorescent chemodosimeter
T1 which displayed absorption ratiometric and fluorescence
enhanced responses to Cys/Hcy based on cyclization of mercapto
biomolecules with aldehydes.39 (Fig. 1) In this molecule, the
first singlet state offers an internal charge transfer, owing to
conjugation between the electron-donor amino substituent in the
7-position (Scheme S1†), and the electron withdrawing carbonyl
group of the heterocycle. Consequently, a large Stokes shift
between absorbance and fluorescence maxima is expected.58 To the
best of our knowledge, it is the first fluorescent chemodosimeter
based on the 7-dimethylamino-1,4-benzoxazin-2-one dyes.
For participation in the process of reversible redox reaction, mer-
capto biomolecules play a crucial role in maintaining biological
redox homeostasis,1 in which cysteine (Cys) and homocysteine
(Hcy) are essential biological molecules relevant to the growth
of cells and tissues in living systems.2 Cys deficiency is involved
in many syndromes, for instances, slow growth in children, liver
damage, skin lesions and weakness.3 Hcy has also been linked to
increased risk of Alzheimer’s disease,4 inflammatory bowel disease
and osteoporosis.5
Recently, a lot of effort has gone into the development of
selective fluorescent bioimaging for highly sensitive, high-speed
spatial analysis of cells. Chemodosimeters, which are based on
an irreversible or essentially irreversible reaction, have attracted
much interest from researchers. Compared with chemosensors,
chemodosimeters have advantages in terms of selectivity and
sensitivity, and their accumulative effect plays an important role
in detection of analytes.6 To image the distribution of Cys/Hcy
in cellular processes, some suitable turn-on fluorescent chemod-
osimeters for Cys/Hcy have been developed.7 For example, works
based on either the nucleophilic addition of thiol,8–25 cyclization
with aldehydes,26–41 or cleavage by thiols,42–51 are all significant for
the detection of mercapto biomolecules.
Ratiometric probes allow measurement at two different wave-
lengths, which could provide a built-in correction for environ-
aState Key Laboratory of Fine Chemicals, Dalian University of Technology,
Dalian 116012, China. E-mail: pengxj@dlut.edu.cn, fanjl@dlut.edu.cn;
Fax: +86-411-3989-3906; Tel: +86-411-3989-3899
bDalian R&D Center for Stem Cell and Tissue Engineering, Dalian
University of Technology, Dalian, 116023, China
Fig. 1 Reaction of aldehyde with N-terminal Cys residue to form
thiazolidine.
† Electronic supplementary information (ESI) available: Synthetic pro-
cedure, 1H NMR, 13C NMR and data of T1. 1H NMR and TOF-
MS of products of reaction of T1 with Cys. Fluorescence spectra and
calculation in the assay. Cell incubation and fluorescence imaging. See
DOI: 10.1039/c0ob00957a
Herein, the recognition of Cys/Hcy with T1 was investigated
in a mixture of acetonitrile and HEPES (3 : 7, v/v) solution
at pH 7.4 by UV–vis absorption and fluorescence techniques.
980 | Org. Biomol. Chem., 2011, 9, 980–983
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The pH independent spectra show that T1 can work in a wide
physiological pH range (Figure S1†). All the tests were taken 1 h
later after addition of the Cys/Hcy to ensure the reaction reaching
equilibrium (Figure S2, S3†).
The free T1 is weakly fluorescent in pH 3–8, a wide physiological
pH range. Upon addition of Cys, the fluorescence increased
rapidly, peaking at lem = 560 nm. (Fig. 2a) After 50 equiv Cys
were added to the solution of T1, about 5-fold enhancement in
fluorescence was observed at room temperature in acetonitrile–
HEPES buffer (20 mM, pH = 7.4) solution (3 : 7, v/v). The fluo-
rescence intensities of T1 at 560 nm show a good linear relationship
with the concentration of Cys, using physiological levels ranging
from 0 to 500 mM58,59 (the total concentration of Cys in healthy
plasma is in the range of 240 mM–360 mM) (Fig. 2b). The intensity
is also linear in response to lower concentrations of Cys/Hcy
-7
(Figure S4, S5†). Its detection limit for Cys is 6.8 ¥ 10
M
in acetonitrile–HEPES.
Fig. 3 (a). Absorption spectral changes of T1 (10 mM) upon addition of
Cys (0–2500 mM). Each spectrum was recorded after 60 min. Inset shows
the color changes of T1 (10 mM) in the absence of Cys (left) and presence
of 500 mM Cys (right). (b) Plot of the absorption intensity ratios at 500 nm
and 430 nm of T1 (10 mM) upon addition of Cys (0–500 mM). Conditions:
acetonitrile–HEPES buffer (20 mM, pH = 7.4) solution (3 : 7, v/v, rt).
which means it can detect the normal human physiological Cys
concentration.4,59 An obvious color change was also similarly seen
upon addition of Hcy (Figure S7†)
The fluorescence responses of T1 (10 mM) to various amino
acids and thiol biomacromolecules (50 equiv.) were also inves-
tigated, such as glycine: Gly, alanine: Ala, valine: Val, leucine:
Leu, isoleucine: Ile, and proline: Pro, polar groups (serine: Ser,
threonine: Thr) including glutathione (GSH), and acidic (glutamic
acid: Glu) and basic (arginine: Arg, lysine: Lys) side chains.
None of the amino acids except Hcy and Cys induced any
fluorescence intensity enhancement. (Fig. 4) Importantly, as a
structurally related thiol biomolecule, reduced glutathione (GSH)
hardly reacted with T1 under these conditions and did not exhibit
any absorption and fluorescence signal changes, which may be due
to its steric hindrance.
To confirm the formation of thiazolidine after addition of
Cys/Hcy to T1, the partial 1H NMR spectra of the reaction “T1-
p” of T1 with Cys are shown in Fig. 5. The resonance signal
corresponding to the aldehyde proton at 9.83 ppm disappeared;
however, concomitantly, two new peaks at 5.81 and 5.64 ppm
assigned to the methine proton of the thiazolidine diastereomer
emerged. This formation was further characterized by mass spec-
trometry analysis (Figure S12†). Q-TOF MS:[T1-p-H]- 320.0705;
found 320.0713.
Fig. 2 (a). Fluorescence spectral changes of T1 (10 mM) upon addition of
Cys (0–3000 mM). Each spectrum was recorded after 60 min. Inset shows
the fluorescence changes of T1 (10 mM) in the absence of Cys (left) and
presence of 500 mM Cys (right). (b) Fluorescence intensity at 560 nm of
T1 (10 mM) upon addition of Cys (0–500 mM). Inset shows the detection
limit for Cys. Conditions: acetonitrile–HEPES buffer (20 mM, pH = 7.4)
solution (3 : 7, v/v, rt), lex = 430 nm.
As displayed in Fig. 3a, upon addition of Cys, the absorption
band of T1 centered at 500 nm gradually decreased and a new
absorption band centered at 430 nm appeared with a distinct
isosbestic point at 452 nm, corresponding to an apparent color
change from orange to yellow (Figure S7†). This fact indicates
that T1 can serve as a “naked-eye” chemodosimeter for Cys/Hcy.
A typical calibration graph of the absorption response of T1 to
Cys under the optimum experimental conditions was obtained as
shown in Fig. 3b. The absorbance ratios at 500 nm and 430 nm
are linearly related to the Cys concentration between 0–500 mM,
Meanwhile, the calculation based on density functional theory
(DFT) for T1 and the product T1-p was performed to further
understand the dependence of the CHO group on photophysical
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The detection of GSH in human plasma without prior depro-
teinization is an important practical application of T1 for Cys/Hcy
(Figure S8†). In our tests, commercial native human blood plasma
samples were simply reconstituted with HEPES buffer (pH 7.4).
The samples were spiked with Hcy, Cys, and GSH respectively. In
the presence of T1, upon adding trifluoroacetic acid to precipitate
protein, the samples afforded selective yellow color formation in
the presence of Cys and Hcy, while the plasma with GSH still
showed the orange color of T1. This result is completely consistent
with our prior results in buffer solutions. The results in native
plasma solutions show its potential to be used in a physiological
environment.12,26,60
To test the capability of T1 to image Cys in living cells, we
further applied T1 to osteoblast cells. After incubation with 10 mM
T1 in culture medium at 37 ◦C for 20 min, osteoblast cells showed
marked intracellular fluorescence (Fig. 7a, 7b). In contrast, when
cells were not incubated with T1, there was no intracellular
background fluorescence under the same bioimaging conditions
(Fig. 7c, 7d). The results suggest that T1 can easily penetrate cell
membranes and make fast fluorescent labeling. Considering the
relationship between Cys levels and many diseases, this probe may
offer a simple and visible way to detect Cys in vivo at real time.
Fig. 4 Fluorescence responses of T1 (10 mM) to various bioanalytes
at 560 nm. Each spectrum was recorded after 60 min. Conditions:
acetonitrile–HEPES buffer (20 mM, pH = 7.4) solution (3 : 7, v/v, rt),
lex = 430 nm. Black bars represent the addition of various bioanalytes to
T1 solution. White bars represent the addition of Cys (500 mM) to the
above solutions, respectively. lex = 430 nm. 1. Cys, 2. Hcy, 3. Ala, 4. Ser, 5.
Trp, 6. Met, 7. Gln, 8. Val, 9. Arg, 10. Phe, 11. Pro, 12. Thr, 13. Gly, 14.
Lys, 15. His, 16. Asp, 17. Ile, 18. Asn, 19. Leu, 20. Glu, 21. cystine, 22. Tyr,
23. GSH.
Fig. 7 Fluorescence images of osteoblasts cells incubated with (a, b) or
without (c, d) 10 mM T1 at 37 ◦C. After 20 min incubation: (a) brightfield
image of cells; and (b) excited in 365 nm; (c) scanning was taken on
brightfield; (d) excited at 365 nm, no obvious fluorescence was observed.
Fig. 5 1H NMR (400 MHz) spectra of sensor T1 in DMSO-d6 (a) and
T1-p in DMSO-d6 (b).
In summary, we have developed a specific chemodosimeter T1
which can be used for selective bioimaging of Cys/Hcy over other
amino acids in living cells and detection in human plasma by
visual color change. To the best of our knowledge, it is the first
fluorescent chemodosimeter based on the 7-dimethylamino-1,4-
benzoxazin-2-one dyes.
properties. Comparing the levelchanges ofthe HOMO(the highest
occupied molecular orbitals) and LUMO (the lowest unoccupied
molecular orbitals) in T1 and T1-p, respectively, (Fig. 6) due to the
ICT effect, after reaction with Cys/Hcy both of them increased.
As the LUMOs increased much more, the HOMO–LUMO energy
gaps were calculated as 3.35 eV and 3.39 eV for T1 and T1-
p, respectively, which is in agreement with the remarkable blue-
shift in the absorption spectra. These results from the spectra and
the Gaussian calculation suggest that the rational design of this
fluorophore can be used for designing new fluorescent probes.
Acknowledgements
This work was supported by NSF of China (21076032, 20725621
and 20923006), National Basic Research Program of China
(2009CB724706), Ministry of Education of China (Program for
Changjiang Scholars and Innovative Research Team in Univer-
sity, IRT0711; and Cultivation Fund of the Key Scientific and
Technical Innovation Project, 707016).
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