Screening and investigation of a cyanine fluorescent probe for simultaneous sensing of glutathione and cysteine under single excitation

Article abstract of DOI:10.1039/c4cc06637e

Here we report the synthesis and screening of Cy-3-NO₂ that showed simultaneous fluorescence sensing ability towards glutathione and cysteine under single excitation, and its application in living cell imaging.

Full text of DOI:10.1039/c4cc06637e

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Screening and investigation of a cyanine fluorescent
probe for simultaneous sensing of glutathione and
cysteine under single excitation†
Cite this:DOI: 10.1039/c4cc06637e
Received 23rd August 2014,
Accepted 16th October 2014
Xu Wang, Jianzheng Lv, Xueying Yao, Yong Li, Fang Huang, Mengmeng Li,
Jie Yang, Xiuyun Ruan and Bo Tang*
DOI: 10.1039/c4cc06637e
www.rsc.org/chemcomm
Here we report the synthesis and screening of Cy-3-NO2 that showed systems. Therefore, to expound the mutual transformation between
simultaneous fluorescence sensing ability towards glutathione and GSH and Cys and its effect on cell function, a fluorescent probe
cysteine under single excitation, and its application in living cell imaging. characterized of single excitation and different emission regions is
indispensable.
Fluorescent probes that exhibit distinct signals in response to
The mechanism of the intramolecular rearrangement cascade
intracellular biothiols (Scheme S1, ESI†) simultaneously are reaction induced by biothiols on electrophilic sites of fluorophores
highly desirable, since the clarification of the complicated has paved a way for single biothiol detection (such as for GSH8a or
processes involving biothiols is important for the treatment Cys8b). We envisioned that besides the difference in the second-
and diagnosis of the related diseases.1 Glutathione (GSH) and nucleophilic properties of biothiols, the electrophilic nature of the
cysteine (Cys) are two essential low-molecular-weight thiol central carbon (the activatable site) on heptamethine cyanine that
compounds in biological systems.2,3 It is important that there could be delicately modulated by variation of substitution groups, as
is significant correlation between GSH and Cys levels. It was well as the resultant spatial steric hindrance effect, may integratively
found that the decreased concentration of GSH in plasma of influence the reaction process, which may lead to NIR dyes that can
HIV-infected patients could be due to the decreased availability discriminate between GSH and Cys simultaneously. Inspired by this,
of Cys, which is rate-limiting in glutathione synthesis.1c,4 On we synthesized eight heptamethine cyanine dyes (Fig. 1) with N-, O-,
the other hand, GSH may also serve as an effective reservoir of and S-aryl substituents in the central position.
Cys in the liver or in the central nervous system.5
First, we investigated and summarized the photophysical
A large number of fluorescent probes have been proposed for properties of the synthesized dyes and the reacting products,
detection of biothiols based on different mechanisms.6 However, namely Cy–Cys, Cy–Hcy, and Cy–GSH (Table 1). As can be seen
the probes that can indeed discriminate and simultaneously from Table 1, for the dyes with the same linking atom, the Ffl of
detect GSH and Cys with different emission regions and single nitro-containing ones are lower than the ones without nitro
excitation have not been reported yet. Although the probes for groups, which is due to the d-PET effect from the heptamethine
the discrimination of GSH and Cys/Hcy have been proposed,7 cyanine moiety to the benzene ring. Among the nitro-containing
the response to Hcy cannot be excluded and actually the total dyes, the 3-position substitution (Cy-3-NO2) induces a stronger d-PET
amount of Cys and Hcy is detected. In addition, requirement of a effect than the substitution at the 4-position (Cy-4-NO2),
surfactant,6g short excitation (in the ultraviolet region),6g,7a and whereas simultaneous substitution at both the 2- and 4-positions
two sets of excitation/emission wavelengths in the cellular
imaging6g,7 partially limit their practical application in biological
College of Chemistry, Chemical Engineering and Materials Science,
Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in
Universities of Shandong, Key Laboratory of Molecular and Nano Probes,
Ministry of Education, Shandong Provincial Key Laboratory of Clean Production of
Fine Chemicals, Shandong Normal University, Jinan 250014, P. R. China.
E-mail: tangb@sdnu.edu.cn; Fax: +86-531-8618-0017
† Electronic supplementary information (ESI) available: Experimental details,
including experimental procedures, synthesis and characterization, DFT calcula-
tions, fluorescence spectra, high resolution mass spectra, and additional confocal
Fig. 1 Molecular structures of synthesized dyes.
microscopy images. See DOI: 10.1039/c4cc06637e
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Table 1 Photophysical properties of the synthesized dyes
Extinction coefficienta/
104 MÀ1 cmÀ1
b
Dyes
Absmaxa/nm
Exmaxa/nm
Emmaxa/nm
Stokes shifta/nm
Ffl (Â102)
Cy–O–Ph
Cy-3-NO2
Cy-4-NO2
Cy-2,4-NO2
Cy–Ph–Ph
Cy–S–Ph
Cy–S–NO2
Cy–N–Ph
Cy–Cys
760
765
765
763
710, 783
783
788
739
788
764
763
765
740
763
780
785
735
734
773
780
786
786
788
760
783
805
806
780
754
797
806
7.39
5.66
7.85
3.90
3.68
6.89
4.02
2.40
1.54
1.76
10.1
22
23
23
20
20
25
21
44
20
24
26
10.3
0.41
2.0
1.60
0.03
0.76
0.36
1.50
2.60
0.45
4.30
Cy–Hcy
Cy–GSH
787
782
a
b
Measured in 50 mM phosphate buffer (pH 7.4, 1% CH3CN).
a fluorescence standard.
F
fl is the relative fluorescence quantum yield estimated by using ICG (F = 0.139) as
(Cy-2,4-NO2) triggers a medium d-PET effect. When the 4-position
nitro group is replaced by a benzamide (Cy–Ph–Ph), the lowest Ffl
was observed because of the strongest d-PET effect induced by
N-(4-hydroxy-3-nitrophenyl)benzamide. For the dyes with the same
functional group but different linking atoms, Ffl shows apparent
dependence on the nature of the linking atoms which increases in
the order of Cy–S–Ph, Cy–N–Ph, and Cy–O–Ph. The Emmax also
shows dependence on the linking atoms. The dye with the linking
atom N exhibits Emmax at 780 nm, while those with O and S exhibit
Emmax red-shifted to 780–790 and 805 nm. DFT calculations were
performed to provide the bond length, bond order, and net charge
of the central carbon (Table S1, ESI†), as well as the optimized
molecular structure of the dyes (Fig. S1, ESI†).
Fig. 2 FS variation of Cy-3-NO2 (10 mM) in the presence of GSH. (a) The
FS recorded after Cy-3-NO2 reacted with 0, 2.5, 5, 10, 20, 30, 40, 50, 60,
70, 80, 90, 100, 200, 300, 400, 500, 700, 900, and 1000 mM GSH for 2 h.
(b) The FS recorded after Cy-3-NO2 reacted with GSH (500 mM) for 0, 2.5,
5, 10, 15, 20, 25, 30, 45, 60, 70, 90, 100, 110, and 120 min. lex = 710 nm,
50 mM PBS buffer (pH = 7.4, 1% CH3CN), 37 1C.
The photophysical properties of the synthesized dyes in the
presence of biothiols (Fig. S2 and Table S2, ESI†) showed that
the reaction with Cys was accompanied by an apparent Emmax
blue shift, while that with GSH exhibited an apparent red shift.
Reaction with Hcy induced a blue or red Emmax shift, depending on
the original Emmax. This is similar to the Emmax of the reaction
products of Cy–Cys (754 nm), Cy–Hcy (797 nm), and Cy–GSH
(806 nm) as shown in Table 1. Cy–Ph–Ph showed apparent
fluorescence enhancement and a red shift in the presence of
GSH, whereas the variation caused by Cys and Hcy was negligible
(the detailed investigation of its response characters and reaction
mechanism are provided in the ESI,† part II). Most excitingly,
Cy-3-NO2 was found to exhibit simultaneous response to GSH
and Cys under single excitation with good spectral separation
and fluorescence enhancement.
Fig. 3 FS variation of Cy-3-NO2 (10 mM) in the presence of Cys. (a) The FS
recorded after Cy-3-NO2 reacted with 0, 5, 10, 15, 20, 30, 40, 50, 60, 80,
100, 150, 200, 300, 400, 500, 600, 700, 800, 900, and 1000 mM Cys for
2 h. (b) The FS recorded after Cy-3-NO2 reacted with Cys (500 mM) for 0, 5,
10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, and 120 min. lex = 710 nm,
50 mM PBS buffer (pH = 7.4, 1% CH3CN), 37 1C.
Cy-3-NO2 itself displays an Emmax at 786 nm (Fig. 2). The
fluorescence analysis exhibited that the fluorescence intensity Cy-3-NO2 remained unchanged during the first 30 min, and
(FI) of Cy-3-NO2 increased with the increasing level of GSH up then a new peak at 755 nm appeared and increased with the
to B700 mM, accompanied by a red shift of Emmax to 805 nm. reaction time (B120 min, Fig. 3b). The effect of Hcy on FS of
When more GSH was added, the FI leveled off and reached a Cy-3-NO2 (Fig. S5 and S6, ESI†) was negligible since the Ffl
plateau (Fig. 2a and Fig. S3a, ESI†). The dependence of fluores- values of Cy-3-NO2 and Cy–Hcy were almost equal (0.41 vs. 0.45,
cence spectra (FS) of Cy-3-NO2 on reaction times showed a rapid Table 1).
increase of FI in the first 10 min, and then increased steadily
with time up to 90 min with a red shift of Emmax to 805 nm. respectively, in the presence of GSH, Cys, and Hcy at different
After that, the FS remained unchanged (Fig. 2b). With regard to incubation times (Fig. 4). As can be seen in Fig. 4a, with lex/em
The FI of Cy-3-NO2 at 805 nm and 755 nm was recorded,
=
Cys, the FS of Cy-3-NO2 increased with increasing Cys concen- 710/805 nm, Cy-3-NO2 possessed the highest S/N ratio for GSH
tration (0–1000 mM), accompanied by an emergence of a new and the influence from Cys and Hcy was insignificant. Mean-
peak at 755 nm (Fig. 3a). In the dynamic examination, the FS of while, Cy-3-NO2 showed the highest S/N ratio for Cys through
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Fig. 4 The dependence of FI of Cy-3-NO2 (10 mM) in the presence of
biological thiols (500 mM each) on reaction times. (a) lex/lem = 710/805 nm.
(b) lex/lem = 710/755 nm. 50 mM PBS buffer (pH = 7.4, 1% CH3CN), 37 1C.
Fig. 5 The proposed reaction mechanism of Cy-3-NO2 towards biothiols.
lex/em = 710/755 nm with tiny influence from GSH and Hcy
(Fig. 4b). The results suggested that Cy-3-NO2 would be an
imaging probe with dual emission for intracellular GSH and Therefore, based on FS, NMR, and HRMS results, the proposed
Cys simultaneously under single excitation. reaction mechanism of Cy-3-NO2 with control compounds is
From the DFT calculations shown in Table S1 (ESI†), it can be shown in Fig. S12 (ESI†).
seen that the bond length (1.392 Å vs. 1.394 Å) and bond order
The mass spectra of Cy-3-NO2 in the presence of Cys, Hcy, and
(0.9230 vs. 0.9190) of C–O (the central carbon of heptamethine GSH (Fig. S13 and Table S4, ESI†) showed that both Cys and Hcy
cyanine and its linking oxygen atom) in Cy-3-NO2 and Cy-4-NO2 resulted in the bridging compounds (5a/5b, the numbered com-
are almost equal, as well as the net charge of the central carbon pounds are shown in Fig. 5) besides the thiol/amino substituted
(0.368 vs. 0.366), indicating almost equal electrophilicity of the products (2a/4a and 2b/4b). Only the thiol-substituted product (1)
central carbon to biothiols. But these two showed very different was found in the presence of GSH. These results suggest that the
response behaviours towards GSH and Cys. We think that the nitro bridging product of (Cy)2-thiol commonly exists in the presence of
group in the meta-position (Cy-3-NO2) inclines to form a hydrogen Cys or Hcy even the amounts of Cys and Hcy are higher than the
bond with amide of GSH and carboxyl of Cys, reducing the spatial dyes, which is determined by the thermodynamically favored intra-
steric hindrance and facilitating a suitable conformation, and finally molecular rearrangement cascade reaction and the subsequent
favoring the nucleophilic attack of sulfhydryl. The bond order of intermolecular nucleophilic coupling (Fig. 5). Although the exact
Cy–Ph–Ph (0.9146) is apparently smaller than that of Cy-3-NO2 contents or proportion of 5a/5b could not be obtained, they are
(0.9230), indicating that sulfhydryl can attack it more easily. But supposed to account for only a small part of the final products since
Cy–Ph–Ph only showed unique specificity to GSH. We attribute the tested biothiols are excessively large compared with the probe.
this to the formation of multiple intermolecular hydrogen bonds Therefore, the effect of 5a/5b on FS can be neglected. The bulky
between the benzamide moiety of Cy–Ph–Ph and the amide and molecular structure of GSH induces higher spatial steric hindrance,
amine moieties of GSH, resulting in a closer distance of the two preventing the second-attack by amino and formation of the brid-
reactive sites (SH group and C–O group, Fig. S21, ESI†).
ging product.
The investigation on pH (Fig. S7, ESI†) showed that Cy-3-NO2
Based on the above investigations, the reaction mechanism
is stable enough in a relatively wide pH range of 6.5–9.0. The of Cy-3-NO2 and the synthesized dyes towards biothiols are shown
selectivity studies (Fig. S8, ESI†) showed that the fluorescence in Fig. 5 and Fig. S14 (ESI†), respectively. As shown in Fig. S14
of Cy-3-NO2 apparently enhanced upon reaction with GSH or (ESI†), the insusceptibility of Cy–N–Ph to biothiols indicates that
Cys in the respective detection channels.
the C–N bond is relatively stable to biothiols under the present
The FS of Cy-3-NO2 in the presence of control compounds conditions. But when the linking atoms change to ‘‘S’’ or ‘‘O’’, and
(Scheme S1, ESI†) showed that b-ME and NAC (GSH analogues) especially when electron-withdrawing groups are introduced in the
resulted in an increase in FI and a red shift of Emmax (Fig. S9a aromatic ring, the intramolecular rearrangement cascade reaction
and b, ESI†), which is similar to the situation upon GSH occurs in the presence of Cys or Hcy.9a Unavoidably, a small part of
addition (Fig. 2). However, the clear distinction caused by the intramolecular rearrangement product will undergo an inter-
cysteamine (analogue of Cys), which has both sulfhydryl and molecular nucleophilic coupling to form a bridging compound.
amino groups, first resulted in a FI decrease (Fig. S9b, ESI†) and GSH exerts influence on FS only by its free sulfhydryl because of its
then a dramatic blue shift of FS to 755 nm after 24 h (Fig. S9c, bulky molecule and the relatively long spacing distance between
ESI†), which is similar to the situation with Cy–Cys (Table 1). sulfhydryl and amino groups. The specificity and dynamics are
1
The H NMR spectral analysis (Fig. S10, ESI†) exhibited the ready closely related to the eletrophilicity of the central carbon and the
substitution with a thiol group and release of the m-nitrophenol spatial steric hindrance is determined by the exact leaving groups.
moiety. The HRMS spectra (Fig. S11 and Table S3, ESI†) of Cy-3-NO2 Combined with the original Ffl and Emmax of the dyes, the specific
in the presence of b-ME or NAC indicated the existence of thiol- detection of one certain biothiol or two biothiols can be tuned and
substituted products 6/7 (Fig. S12, ESI†), while that of cyste- achieved. In the case of Cy-3-NO2 (Fig. 5), the effect of Hcy is really
amine indicated the coexistence of the thiol/amino substituted excluded due to its slow intramolecular rearrangement and the
product (8/9) and the bridging product (Cy)2-cysteamine (10). fluorescence character of Cy–Hcy, endowing the simultaneous
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were designed and synthesized. It is exciting and delightful that
Cy-3-NO2 was found to simultaneously detect GSH and Cys
under single excitation. The sensing mechanism was investigated
and expounded. Finally, Cy-3-NO2 was successfully used to image
GSH and Cys in Hela cells. The results proposed here not only
provide a NIR probe that is capable of sensing GSH and Cys
simultaneously, but construct a strategy that is suitable for multi-
component analysis in the living systems.
This work was supported by the 973 Program (2013CB933800),
the National Natural Science Foundation of China (21227005,
21390411, 91313302, 21035003, 21375080) and the Program for
Changjiang Scholars and Innovative Research Team in University.
Fig. 6 Confocal fluorescence images of GSH and Cys in Hela cells by
Cy-3-NO2. (a)–(d), images obtained with the band path of 700–740 nm
(blue fluorescence, pseudo color, assigned to Cys). (e)–(h), images
obtained with the band path of 760–800 nm (red fluorescence, assigned
to GSH). (a), (e), and (i), without the probe. Cells in (b), (f), and (j) were
incubated with 5 mM Cy-3-NO2 for 15 min. Cells in (c), (g), and (k) were
incubated with 5 mM Cy-3-NO2 for 60 min. Cells in (d), (h), and (l) were first
incubated with NEM for 30 min, then incubated with 5 mM Cy-3-NO2 for
15 min. 37 1C, scale bar = 25 mm, 633 nm laser excitation.
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