A dansyl-based fluorescent probe for the highly selective detection of cysteine based on a d-PeT switching mechanism

Article abstract of DOI:10.1039/c7ra00212b

A novel fluorescent probe, DN-C, for detection of cysteine (Cys) based on a d-PeT switching mechanism is reported. In the presence of Cys, the probe exhibits a turn-on fluorescence signal and nearly 28-fold fluorescence intensity enhancement. The cellular imaging experiment indicated the DN-C possess desirable cell permeability for biological applications.

Full text of DOI:10.1039/c7ra00212b

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A dansyl-based fluorescent probe for the highly
selective detection of cysteine based on a d-PeT
switching mechanism†
Cite this: RSC Adv., 2017, 7, 21050
a
a
Yudong Xiao,b Yujin Guo,a Ruili Dang,a Xin Yan,c Pengfei Xu
*
*
and Pei Jiang
Received 6th January 2017
Accepted 3rd April 2017
A novel fluorescent probe, DN-C, for detection of cysteine (Cys) based on a d-PeT switching mechanism is
reported. In the presence of Cys, the probe exhibits a turn-on fluorescence signal and nearly 28-fold
fluorescence intensity enhancement. The cellular imaging experiment indicated the DN-C possess
desirable cell permeability for biological applications.
DOI: 10.1039/c7ra00212b
rsc.li/rsc-advances
As an important amino acid, cysteine (Cys) plays a crucial role in broad functional properties of Cys within complex biological
protein synthesis, detoxication, and metabolism.1,2 Cys is systems.
a precursor amino acid of glutathione (GSH) and both are taken
Dansyl uorophore is widely used in amino acids modica-
in by the human body through food or are formed as a meta- tion, protein sequencing and amino analysis, because of its
bolic product of homocysteine (Hcy). Deciency of Cys is asso- high uorescence quantum yields and large Stokes shi. To
ciated with many syndromes, including slowed growth, hair date, a number of dansyl based probes for the detection of
depigmentation, lethargy, liver damage, muscle and fat loss, metal ion have been reported, mainly based on the electron
skin lesions, and weakness.3 Its signicant biological role transfer effect (ET) between metal ion and the excited dansyl
explains the considerable contemporary effort devoted to the uorophore in the coordination state.17–21 Obviously, this
development of an efficient method for the detection and strategy isn't suit for the design of probes for biological mole-
quantication of Cys under physiological conditions.
cules such as Cys. To the best of our knowledge, dansyl based
A number of analytical methods for the detection of Cys have uorescent probes for Cys are rare. In the fact, there is only one
been developed using high-performance liquid chromatog- reported dansyl based uorescent probe for Cys by Wang
raphy (HPLC), capillary electrophoresis, electrochemical assay, group.22 In this work, we introduced a new strategy to design
UV/Vis, FTIR, mass, and uorescence spectroscopy.4–10 Among dansyl based probes and realized the detection of Cys in vivo.
these methods, uorescence probes are more desirable due to
Our strategy to design a dansyl based uorescence probe for
its high selectivity, low detection limit, fast response and great cellular Cys relies on modulating donor-excited photoinduced
potential for bioimaging.11 Probes rely on uorescence electron transfer (d-PeT) process from the excited uorophore
quenching suffer from inherent drawbacks including low to a strong electron-withdrawing group, which can switch the
signal-to-noise ratio and nonspecic quenching, so that “turn- emission signal.23,24 Thus, the core of the strategy is depending
on” type uorescence probes are preferred.12,13 To date, various on the carefully choosing a Cys responding group, which can
types of organic reactions, such as cyclization reaction and simultaneously the PET acceptor. On other hand, it has been
Michael addition, have been employed to design uorescence- known for several years that the conjugate addition of Cys to
based probes. Nevertheless, only a few probes are able to acrylates will generate the corresponding thioether, which can
discriminate between Cys, Hcy, and GSH.14–16 Thus, further further undergo an intramolecular cyclization to yield 3-
development of synthetically simple and practical probes for carboxy-5-oxoperhydro-1,4-thiazepine. Meanwhile, acryloyl
selective detection of Cys is still highly required because of the group has low LUMO energy level and can serve as the PET
acceptor. Therefore, a,b-unsaturated carbonyl moiety was
selected as the responding group and signal modulator for
aInstitute of Clinical Pharmacy & Pharmacology, Jining First People's Hospital, Jining probe DN-C.
Medical University, Jining 272000, China. E-mail: jiangpeicsu@sina.com;
The synthetic route for DN-C was shown in Scheme 1. DN-C
was readily prepared in two convenient steps under facile
pengfeixucsu@outlook.com
bDepartment of Radiology, The Second Xiangya Hospital of Central South University,
conditions with high yield. The DN was prepared starting from
Changsha 410010, China
dansyl chloride and 3-aminophenol in acetonitrile at room
temperature. The reaction of DN and acryloyl chloride in
anhydrous acetonitrile afforded DN-C with a high yield of 87%.
cResearch Center for Drug Discovery, School of Pharmaceutical Sciences, Sun Yat-sen
University, 132 East Circle at University City, Guangzhou, 510006, China
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c7ra00212b
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Scheme 1 The synthetic route of DN-C.
1
The structure of DN-C was fully characterized by H NMR, 13C
NMR and MS.
Fig. 2 Frontier molecular orbitals (MOs) of dansyl group and acrylate
group. In the ball-and-stick model, carbon, oxygen, sulfur and
nitrogen atoms are colored in gray, red, yellow and blue, respectively.
Spectral properties of DN and DN-C were rst examined in
a mixed solution of CH3CN : PBS (50 : 50, v/v, pH ¼ 7.4, 10 mM).
As shown in Fig. 1, DN exhibits a robust emission peak with
maximum at 545 nm. However, the emission intensity of DN-C
decline markedly. The suppression of uorescence emission
may attribute to the occurrence of donor-excited photoinduced
electron transfer (d-PeT) process. The process of d-PeT involves
transfer of one electron from the excited donor unit to the
lowest unoccupied molecular orbital (LUMO) of the electron-
decient unit. To verify this hypothesis, the HOMO and
LUMO energy levels of dansyl group and acryloyl group were
calculated by suite of Gaussian 09 programs [density functional
theory (DFT) time-dependent density function theory (TD-DFT)]
at B3LYP/6-31G basis sets. The calculation result indicated that
the LUMO energy level of acryloyl group is in the range of
between the HOMO and LUMO energy levels of dansyl group,
which is appreciate for d-PeT process (Fig. 2). The electron
transfer from the dansyl framework (PET donor) to the acryloyl
group (PET acceptor) weakened the uorescence of the
primordial uorophore. Thus, the dansyl uorophore will be in
the off state. Once removal of the d acryloyl group, like the
structure of DN, the d-PeT progress will disappear and the
uorescence will restore.
100 mM) were recorded. As shown in Fig. 3, the free DN-C dis-
played quite weak uorescence. With the addition of Cys, the
uorescence intensity of DN-C increased signicantly at 545 nm
due to specically trigger the cleavage of acryloyl group by Cys.
As shown in Fig. 4, the emission intensities of 545 nm increased
by nearly 28-fold. In addition, the increase in uorescence
intensity is in a concentration dependent manner and exhibits
good linear correlation with the amount of Cys (0–30 mM, R2 ¼
0.98412). A maximal uorescent signal is achieved in the pres-
ence of only 5 equiv. of Cys and the detection limit of DN-C for
Cys was determined as 13 nM, indicating that the probe is
sensitive enough to Cys and might be suitable for detecting
endogenous Cys in biological samples.
To evaluate the selectivity of DN-C for Cys, we measured the
uorescence intensity changes for various analytes upon addi-
tion of excess guests (Fig. 5). The uorescence intensity of DN-C
was highly enhanced only by the addition of Cys. Other amino
acids, such as Hcy, GSH, Gly, Phe, Ser, Glu, Lys, Arg, His, Ala,
Gln, Met, and Tyr, did not cause any signicant changes in the
uorescence emission intensity of 545 nm. This result indicated
that probe DN-C exhibited remarkably higher selectivity for Cys
over Hcy, GSH and other amino acids at neutral conditions.
Furthermore, the practical utilities of DN-C was investigated
for uorescent imaging of intracellular Cys in living T-47D
Subsequently, we performed the uorescence titration
studies of DN-C towards Cys in a mixed solution of CH3CN : PBS
(50 : 50, v/v, pH ¼ 7.4, 10 mM). The absorption and uorescence
spectra of the solution of DN-C treated with a series of Cys (0 to
Fig. 1 Fluorescence spectra of DN and DN-C (10 mM). Inset: fluores- Fig. 3 Emission spectra of DN-C (10 mM) in a mixed solution of
cence changes of probe DN-C upon the addition of Cys (100 mM) CH3CN : PBS (50 : 50, v/v, pH ¼ 7.4, 10 mM) upon addition of Cys (0–
under excitation with UV light (365 nm).
100 mM) with lex ¼ 380 nm.
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demonstrated that DN-C possesses good membrane perme-
ability and is able to detect intracellular Cys in living cells.
Conclusions
In conclusion, we have designed and synthesized selective and
sensitive uorescent probe DN-C for Cys based on dansyl
chromophore. The probe exhibits a turn-on uorescence signal
for responding Cys via d-PeT switching mechanism. It has
a selectivity and sensitivity for in vitro Cys over other various
biologically relevant species, and could detect Cys in living cells.
Therefore, we anticipate that the superior properties of DN-C
will make it of potential use in real-time Cys monitoring, and as
an efficient research tool for the study on Cys related molecular
events in biological systems.
Fig. 4 Plot of emission intensity at 545 nm as a function of Cys
concentration when using DN-C (10 mM) in a mixed solution of CH3-
CN : PBS (50 : 50, v/v, pH ¼ 7.4, 10 mM) with lex ¼ 380 nm.
Acknowledgements
The study was supported by the National Natural Science
Foundation of China (No. 81602846) and Natural Science
Foundation of Shandong Province (No. ZR2016HQ21).
Fig. 5 Fluorescence responses of DN-C (10 mM) to various analytes in
a mixed solution of CH3CN : PBS (50 : 50, v/v, pH ¼ 7.4, 10 mM) with
lex ¼ 380 nm.
References
1 K. G. Reddie and K. S. Carroll, Curr. Opin. Chem. Biol., 2008,
12, 746–754.
2 C. E. Paulsen and K. S. Carroll, Chem. Rev., 2013, 113, 4633–
4679.
3 S. Shahrokhian, Anal. Chem., 2001, 73, 5972–5978.
4 N. Ercal, P. Yang and N. Aykin, J. Chromatogr. B: Biomed. Sci.
Appl., 2001, 753, 287–292.
5 P. Ryant, E. Dolezelova, I. Fabrik, J. Baloun, V. Adam,
P. Babula and R. Kizek, Sensors, 2008, 8, 3165–3182.
6 O. Rusin, N. N. St 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, 438–439.
human breast duct carcinoma cells (T-47D human breast duct
carcinoma cells were purchased from Abcam). As shown in
Fig. 6, in the control experiment, when N-ethylmaleimide
(NEM), a well know thiol trapping reagent, was added to the cell
culture prior to the addition DN-C (10 mM), faint uorescence
was observed in the green channel (b). However, when T-47D
cells were only incubated with DN-C (10 mM) for 30 min,
bright green uorescence could be observed (d). These results
7 X. Guan, B. Hoffman, C. Dwivedi and D. P. Matthees, J.
Pharm. Biomed. Anal., 2003, 31, 251–261.
8 D. Potesil, J. Petrlova, V. Adam, J. Vacek, B. Klejdus,
J. Zehnalek, L. Trnkova, L. Havel and R. Kizek, J.
Chromatogr. A, 2005, 1084, 134–144.
9 Y. Sato, T. Iwata, S. Tokutomi and H. Kandori, J. Am. Chem.
Soc., 2005, 127, 1088–1089.
10 Y. Zhou and J. Yoon, Chem. Soc. Rev., 2012, 41, 52–67.
11 X. Chen, Y. Zhou, X. Peng and J. Yoon, Chem. Soc. Rev., 2010,
39, 2120–2135.
12 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, 1498–1501.
13 X. Zhang, X. Ren, Q. H. Xu, K. P. Loh and Z. K. Chen, Org.
Lett., 2009, 11, 1257–1260.
14 H. Wang, G. Zhou, H. Gai and X. Chen, Chem. Commun.,
2012, 48, 8341–8343.
15 X. Yang, Y. Guo and R. M. Strongin, Org. Biomol. Chem.,
2012, 10, 2739–2741.
16 Z. Guo, S. W. Nam, S. Park and J. Yoon, Chem. Sci., 2012, 3,
2760–2765.
Fig. 6 Top: T-47D human breast duct carcinoma cells were pre-
incubated with 500 mM NEM for 50 min and then treated with 10 mM
of DN-C for 30 min; bottom: T-47D human breast duct carcinoma
cells were treated with 10 mM of DN-C for 30 min ((a and c), bright-field
image; (b and d), fluorescence images. The excitation wavelength is
330–400 nm and the emission was collected in green channel).
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17 K. H. Jung, E. T. Oh, H. J. Park and K. H. Lee, Biosens. 21 J. Piao, J. Lv, X. Zhou, T. Zhao and X. Wu, Spectrochim. Acta,
Bioelectron., 2016, 85, 437–444. Part A, 2014, 128, 475–480.
18 P. Xie, F. Guo, L. Wang, S. Yang, D. Yao and G. Yang, J. 22 H. Zhang, P. Wang, Y. Yang and H. Sun, Chem. Commun.,
Fluoresc., 2015, 25, 319–325. 2012, 48, 10672–10674.
19 C. Rivas, G. J. Stasiuk, M. Sae-Heng and N. J. Long, Dalton 23 X. Zhang, L. Chi, S. Ji, Y. Wu, P. Song, K. Han, H. Guo,
Trans., 2015, 44, 4976–4985.
20 L. J. Ma, J. Liu, L. Deng, M. Zhao, Z. Deng, X. Li, J. Tang and
T. D. James and J. Zhao, J. Am. Chem. Soc., 2009, 131,
17452–17463.
L. Yang, Official Journal of the European Photochemistry 24 S. Ji, J. Yang, Q. Yang, S. Liu, M. Chen and J. Zhao, J. Org.
Association and the European Society for Photobiology,
Chem., 2009, 74, 4855–4865.
Photochem. Photobiol. Sci., 2014, 13, 1521–1528.
This journal is © The Royal Society of Chemistry 2017
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