A dual-response naphthofluorescein-based fluorescent probe for multiple-channel imaging of cysteine/homocysteine in living cells

Article abstract of DOI:10.1016/j.tetlet.2018.04.017

A naphthofluorescein-based fluorescent probe with two independent reaction sites (nitro-2,1,3-benzoxadiazole and acrylate moiety) was developed. Integrating these two reaction sites into a single molecule not only can guarantee the selective detection of Cys/Hcy in an elegant fashion, but also can enable Cys/Hcy detection in a multiple-channel responsive manner.

Full text of DOI:10.1016/j.tetlet.2018.04.017

Accepted Manuscript
A dual-response naphthofluorescein-based fluorescent probe for multiple-chan-
nel imaging of cysteine/homocysteine in living cells
Xiuxiu Yue, Wenxiu Li, Wenqiang Chen, Liangliang Zhang, Guofang Li,
Jiarong Sheng
PII:
S0040-4039(18)30460-X
https://doi.org/10.1016/j.tetlet.2018.04.017
TETL 49880
DOI:
Reference:
Tetrahedron Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
24 January 2018
2 April 2018
10 April 2018
Please cite this article as: Yue, X., Li, W., Chen, W., Zhang, L., Li, G., Sheng, J., A dual-response
naphthofluorescein-based fluorescent probe for multiple-channel imaging of cysteine/homocysteine in living cells,
Tetrahedron Letters (2018), doi: https://doi.org/10.1016/j.tetlet.2018.04.017
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A dual-response naphthofluorescein-based
fluorescent probe for multiple-channel
imaging of cysteine/homocysteine in living
cells
Leave this area blank for abstract info.
Xiuxiu Yue,†a,b Wenxiu Li,†c Wenqiang Chen,  a,b Liangliang Zhang,*c Guofang Li,a,b Jiarong Sheng*a,b

1
Tetrahedron Letters
A dual-response naphthofluorescein-based fluorescent probe for multiple-channel
imaging of cysteine/homocysteine in living cells
Xiuxiu Yue,†a,b Wenxiu Li,†c Wenqiang Chen, a,b Liangliang Zhang,*c Guofang Li,a,b Jiarong Sheng*a,b
a College of Chemistry and Materials Science, Guangxi Teachers Education University, 530001 Nanning, Guangxi, P. R. China.
b China.Guangxi Key Laboratory of Natural Polymer Chemistry and Physics (Guangxi Teachers Education University), Nanning, 530001, P. R. China.
c State Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources of Education Ministry, Guangxi Normal University, 541004 Guilin,
Guangxi, P. R.
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
A naphthofluorescein-based fluorescent probe with two independent reaction sites (nitro-2,1,3-
benzoxadiazole and acrylate moiety) was developed. Integrating these two reaction sites into a
single molecule not only can guarantee the selective detection of Cys/Hcy in an elegant fashion,
but also can enable Cys/Hcy detection in a multiple-channel responsive manner.
Available online
2009 Elsevier Ltd. All rights reserved.
Keywords:
fluorescent probe
naphthofluorescein-based
Cysteine (Cys)
homocysteine (Hcy)
Introduction
samples, deep tissue penetration and negligible interference from
background.7b Unfortunately, NIR fluorescent probes for
Cysteine (Cys) and homocysteine (Hcy) play crucial roles in
numerous physiological processes1 and are closely related to a
variety of health disorders including Alzheimer’s disease,2
cardiovascular disease,3 osteoporosis,4 skin lesions,5 liver damage
and edema.6 As a consequence, the assessment of abnormal
levels of Cys/Hcy in living systems may aid early diagnosis of
some diseases. Given its advantages of excellent sensitivity and
high spatiotemporal resolution, fluorescent probes now have been
recognized as the effective molecular tools that can help to detect
or visualize biologically active molecules in living cells or
tissues.7 However, although these probes can highly selectively
distinguish these biothiols from other amino acids, most of them
cannot distinguish Cys/Hcy/GSH from each other due to the
similar structures and reactivity of these biothiols. In fact, the
discrimination between them has been a focal point and also a
tough challenge for researchers, albeit some advances have been
obtained.
Cys/Hcy are rare, to the best of our knowledge, only two NIR
fluorescent probes for Cys/Hcy have been reported.10 Moreover,
almost all of the reported fluorescent probes for Cys/Hcy exhibit
fluorescence signal variations only in one channel, the variations
in sample environment, probe distribution and the instrument
factors may be problematic for utilization in quantitative
measurements. In fact, to eliminate the false positive that arise
from environmental factors, the development of small molecule
fluorescent probe that can sense Cys/Hcy in multiple channels is
highly valuable11 but even more challenging. Herein, we have
developed a multiple-channel responsive fluorescent probe for
Cys/Hcy by incorporation of two potential reaction sites into a
NIR naphthofluorescein dye. The probe not only can distinguish
Cys/Hcy from GSH but also display obvious turn-on
fluorescence signals in green, yellow and NIR emission channels,
thereby holding great potential in biological applications.
The design rational is depicted in Scheme 1 and illustrated as
follows. Naphthofluorescein (dye 4) was prudentially selected as
the fluorophore based on the following considerations: (1)
naphthofluorescein is a bright NIR emitter;12 (2) like the
fluorescein, the photophysical properties of naphthofluorescein
can be easily tuned through the protection/de-protection of its
hydroxyl group;13 (3) naphthofluorescein contains two hydroxyl
groups which is well suited for integrating two independent
reaction sites into a single fluorescent probe. Next, 7-nitro-2,1,3-
benzoxadiazole (NBD) was chosen as the first reaction site (Site
Pioneered by Strongin et al., the selective detection of
Cys/Hcy over GSH was realized for the first time by taking
advantage of the unique cyclization of Cys/Hcy with aldehydes.8
From then on, a large numbers of fluorescent probes for Cys/Hcy
were constructed based on the further extension of this strategy.9
However, most of the reported probes for Cys/Hcy emit in the
visible range. It is well known that fluorescent probes that emit in
near-infrared (NIR) regions are more suitable for biological
imaging applications due to minimum photodamage to biological
———
 Corresponding author. e-mail: chenwq@csu.edu.cn (W.Chen).

2
Tetrahedron
1) because it functions not only as a leaving group in the
Scheme 1. The chemical structure of Probe 1 and the proposed
sensing mechanism of Probe 1 to Cys/Hcy over GSH
reaction of thiol-mediated SNAr substitution, but also as an
effective fluorescence quencher to ensure a low background
emission of the probe. We speculated that the 4-aryloxy-7-nitro-
2,1,3-benzoxadiazoles (NBD-OAr) moiety could initially be
cleaved by Cys (or Hcy) to produce thioNBD 2a (or 2b), and the
subsequent intramolecular Smiles rearrangement involving 5 (or
6) membered ring would ultimately lead to strongly emissive
aminoNBD 3a (or 3b). However, in the case of GSH, non-
fluorescent thioNBD 2c instead of aminoNDB 3c was expected
to be the desired product due to the kinetically disfavored
formation of macrocyclic transition state.14 As a result, only
Cys/Hcy could trigger significant fluorescence enhancement in
the visible region due to the formation of highly emissive
aminoNBD 3a/3b. Now, let us turn our attention to the
fluorescence signal in the near-infrared region. If the NBD
moiety is the only reaction site of this probe, it appears to be
difficult to discriminate Cys/Hcy from GSH in the NIR region
due to the similar reactivity of Cys, Hcy and GSH, thereby
necessitating an additional regulatory factor, here is an acrylate
moiety (Site 2), a classical Michael receptor. We anticipate that
Cys, Hcy and GSH would react with unsaturated double bond in
acrylate moiety to produce the corresponding thioethers through
Michael addition reaction. In the case of Cys/Hcy, the
corresponding thioethers 3d/3e could rapidly undergo a further
intermolecular cyclization15 to release the NIR fluorophore dye 4,
thereby triggering significant fluorescence enhancement in NIR
range. As for GSH, the initially produced thioether 3f could not
undergo the subsequent cyclization due to the kinetically
disfavored 12-member cyclic transition formation, therefore
resulted the failure of the release of dye 4. Overall, if our
working hypothesis is rational, the selective detection of Cys/Hcy
in a multiple-channel responsive manner might be realized.
As a proof-of-concept, a dual-response fluorescent probe,
Probe 1, was synthesized in two steps (Scheme 2). Moreover, to
help unravel the mechanism of Probe 1 with biothiols, the
reference compounds aminoNBD 6 and thioNBD 7 were
synthesized (Scheme 2) according to our previously reported
method.14a The chemical structure of Probe 1 was fully
characterized by 1H NMR, 13C NMR and HRMS (see supporting
information). With Probe 1 in hand, we first examined its
reactivity toward Cys/Hcy/GSH in PBS buffer (pH = 7.4, 10 mM,
containing 1 mM cetyl trimethylammonium bromide (CTAB))
through UV-vis absorption spectra. As shown in Figure S1,
Probe 1 alone showed a main absorption peak at 380 nm. Upon
addition of Cys/Hcy, the initial absorption peak decreased, alone
with the appearance of two new peaks at 474 nm and 632 nm.
According the aforementioned speculation, the absorption peak at
474 nm should be assigned to the corresponding products 3a/3b,
which was strongly supported by the control compound
aminoNBD 6, whose main absorption peak was found at around
479 nm under the same condition (Figure S1-S2). Moreover, the
absorption peak around 612 nm coincides with the hallmarks of
dye 4, indicating the release of dye 4. Next, let us turn our
attention to GSH. Addition of GSH to the solution of Probe 1
also results the disappearance of the initial absorption peak and
the concomitant appearance of a new peak at 431 nm should be
assigned to the thioNBD 2c, which was supported by control
compound thioNBD 7 (Figure S3). Noteworthy, a slight increase
of the absorption peak at 612 nm can also be observed in this
case, suggesting a small amount of dye 4 released during the
reaction process. The GSH-triggered release of dye 4 might be
explained by the CTAB promoted macrocyclic (12-membered
ring) transition formation, which could be supported by some
recent publications on large ring products.16
Scheme 2. The reference compounds aminoNBD 6 and thioNBD 7.
Figure 1. Fluorescence spectral changes of Probe 1 (10 µM) upon
addition of Cys (0-20 equiv.) (a, b) or Hcy (0-15 equiv.) (c, d) in
PBS buffer (pH = 7.4, 10 mM, containing 1 mM CTAB). λex = 470
nm for (a) and (c); λex = 620 nm for (b) and (d).

3
Subsequently, we studied the emission behaves of Probe 1
upon addition of Cys/Hcy through time-dependent fluorescence
spectra in PBS buffer (pH = 7.4, 10 mM, containing 1 mM
CTAB) by using of two different excitations at 470 nm and 620
nm, near the absorption maximum of aminoNBD 3a and dye 4.
In fact, regardless of excitation wavelength, free Probe 1 is
essentially non-emissive due to the formation of non-conjugated
spirocyclic structure. First, the 470 nm excitation wavelength was
selected to probe Cys/Hcy (Figure S4). Upon excitation, addition
of 20 equiv. of Cys to the solution of Probe 1, the fluorescence
intensity at 557 nm increased immediately and got saturation
within 60 min. A similar observation was made for Hcy. By
contrast, addition of 20 equiv. of GSH elicited negligible
emission at 557 nm. Therefore, Probe 1 could selectively detect
Cys/Hcy from the visible emission channel. Second, the 620 nm
excitation wavelength was selected to probe Cys/Hcy in the NIR
range (Figure S5). Similarly, addition of 20 equiv. of Cys or Hcy
caused significant fluorescence enhancement (84 fold for Cys; 52
fold for Hcy) at 693 nm. In contrast, addition of 20 equiv. of
selective to Cys/Hcy when excited at 470 nm. Similarly, with
the excitation at 620 nm, only GSH produced a slight
interference for Cys/Hcy detection, other competitive species
triggered almost no fluorescence intensity changes in the spectra
of Probe 1 (Figure 2b). These results suggest that Probe 1 is high
selective for Cys/Hcy over other competitive species.
To test whether Probe 1 functions well under physiological
environment and to evaluate the limitations of its use in aqueous
solution, the corresponding fluorescence spectra were measured
at a variety of different pH values. As seen in Figure 3, changing
the pH from 1 to 11 has little effect on igniting the fluorescence
signals at 557 nm or 696 nm, suggesting this probe is quite
suitable for applications in biological samples. However, in
strongly basic media (pH > 11), the solution of Probe 1 displays a
slight fluorescence enhancement at 557 nm and a significant
fluorescence enhancement at 696 nm, which was likely ascribed
the decomposition of Probe 1 under these conditions. In an
additional set of experiments, the pH-dependent fluorescence
response of Probe 1 toward Cys (or Hcy) was further investigated.
As shown in Figure 3, significant fluorescence enhancements at
557 nm or 696 nm were observed in the pH range of 7.0-9.0 upon
addition of 20 equiv. of Cys or Hcy, suggesting Probe 1 can
function under physiological conditions. Noteworthy, upon
addition of 20 equiv. of Cys or Hcy, the fluorescence intensity at
696 nm was increased with the enhancement of pH value form
6.0-11.0. This phenomenon may attribute to the formation of
phenolate form of the released dye 4 under basic conditions. The
obvious fluorescence decrease at pH > 11.0 was presumably due
to the formation of disulfide, thereby diminishing the
concentration of Cys or Hcy.
GSH only elicited
a
slight fluorescence enhancement
(approximate 15 fold) at 693 nm, indicating the potential ability
of Probe 1 to selective detect Cys/Hcy in the NIR emission
channel.
Figure 2. Fluorescence spectra of Probe 1 (10 µM) in response to
various relevant species in PBS buffer (pH = 7.4, 10 mM, containing
1 mM CTAB). (a) λex = 470 nm; (b) λex = 620 nm. These species
included Cys, Hcy, His, Glu, Asp, Val, Phe, Tyr, Ala, Ser, Leu, Arg,
-
Pro, Thr, Gln, Try, Lys, DTT and GSH, S2-, S2O32-, HSO3 , SO42- and
NO3-, Ca2+, K+, Mg2+ and Zn2+, ClO- and H2O2.
Next, we performed fluorescence titration studies of Probe 1
for Cys/Hcy at a time point of 30 min after incubation in PBS
buffer (pH = 7.4, 10 mM, containing 1 mM CTAB). With the
incremental addition of Cys/Hcy, both the emission bands in
visible (centered at 557 nm) and NIR (centered at 693 nm) ranges
were increased dramatically. Moreover, a good linear relation
was obtained (Figure S6-S7). For Cys, the fluorescence intensity
at 557 nm increased linearly with the concentrations of Cys from
0 to 60 µM. The fluorescence intensity at 693 nm also increased
linearly with Cys in the concentrations range from 0 to 50 µM.
The detection limits were calculated to be 1.5×10-7 M and
4.5×10-7 M (based S/N=3) for Cys versus the corresponding
emission at 557 and 693 nm, respectively. In the case of Hcy,
both fluorescence intensity at 557 nm and 693 nm increased
linearly with the concentrations of Hcy from 0 to 120 µM (Figure
S8-S9), and the corresponding detection limits were calculated to
be 1.1×10-7 M and 1.4×10-6 M, respectively.
Figure 3. Fluorescence intensity of Probe 1 (10 µM) at different pH
values in the absence/presence of Cys or Hcy. (a) Fluorescence
intensity at 557 nm (λex = 470 nm); (b) Fluorescence intensity at 696
nm (λex = 620 nm).
Encouraged by the aforementioned results, we sought out to
investigate the capability of Probe 1 to visualize intracellular Cys
or Hcy in a multi-channel manner. Initially, we conducted MTT
assays on HeLa cells to evaluate the cytotoxicity of Probe 1. The
results indicated that this probe exhibited low cytotoxicity for
living cells (Figure S10). When HeLa cells were incubated with
Probe 1 (10 µM), a relatively weak fluorescence in the green,
yellow and red channels can be observed (Figure 4 A1-A3). For
the control experiments, HeLa cells were pre-treated with 1 mM
Cys, Hcy or GSH and, after changing the growth medium, were
further incubated with Probe 1 (10 µM). As shown in Figure 4
B1-B3 and C1-C3, pre-treated with 1 mM Cys or Hcy, then
incubated with Probe 1 (10 µM), strong fluorescence in green,
yellow and red channels were observed. In sharp contrast, no
obvious fluorescence changes in green, yellow and red channels
were observed when cells pre-treated with 1 mM GSH then
incubated with Probe 1 (10 µM) (Figure 4 D1-D3). As a control,
when HeLa cells were pre-treated with 1 mM N-ethylmaleimide
(a general scavenger of thiols17), then further incubated with
Probe 1 (10 µM), cells displayed non-noticeable fluorescence in
green, yellow and red channels (Figure 4 E1-E3). Taken together,
these experiments demonstrated that Probe 1 has the potential to
Further, we investigated the selectivity of Probe 1 toward
other biologically related species, including various common
amine acids (His, Glu, Asp, Val, Phe, Tyr, Ala, Ser, Leu, Arg,
Pro, Thr, Gln, Lys as well as with DTT, NAC and GSH),
-
-
2-
-
representative anions (S2 , S2O32-, HSO3 , SO4 and NO3 ) and
cations (Ca2+, K+, Mg2+ and Zn2+) and biologically reactive
oxygen species (ClO- and H2O2). As shown in Figure 2a, only
Cys/Hcy induced a significant fluorescence enhancement at 557
nm, other species, including GSH, DTT and NAC, caused
negligible fluorescence variations. Thus, Probe 1 was highly

4
Tetrahedron
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Figure 4. Confocal fluorescence images of Cys/Hcy in HeLa cells.
(A1-A4) HeLa cells incubated with 10 µM Probe 1; (B1-B4) HeLa
cells pre-treated with 1 mM Cys and then incubated with 10 µM
Probe 1; (C1-C4) HeLa cells pre-treated with 1 mM Hcy and then
incubated with 10 µM Probe 1; (D1-D4) HeLa cells pre-treated with
1 mM GSH and then incubated with 10 µM Probe 1; (E1-E4) HeLa
cells pre-treated with 1 mM NEM and then incubated with 10 µM
Probe 1. λex = 488 nm, emission was collected at 500-530 nm for
green channel; λex = 488 nm, emission was collected at 550-570 nm
for yellow channel; λex = 633 nm, emission was collected at 660-750
nm for red channel. Scale bar: 10 µm.
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Supplementary Material
Conclusions
In conclusion, we have developed
a
dual-response
naphthofluorescein based fluorescent probe that can selectively
sense Cys/Hcy in multiple emission channels. Significantly, we
have further demonstrated that Probe 1 is capable of visualizing
intracellular Cys/Hcy in three different emission channels. We
hope this work could open a new avenue for the exploration of
fluorescent probes that can sense Cys/Hcy in a multiple-channel
responsive manner.
Acknowledgments
This work was supported by the Guangxi Natural Science
Foundation (2016GXNSFBA380229,) and “BAGUI Scholar”
program of Guangxi Province of China.
References and notes
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Engl. J. Med.2004, 350, 2033.
5. Shahrokhian, S. Anal Chem. 2001, 73, 5972.

5
Highlights



Two independent moieties were
integrated into a naphthafluorescein dye.
This probe can detect of Cys/Hcy in a
multiple-channel responsive manner.
This probe was capable of visualizing
Cys/Hcy in three different emission
channels.

6
Tetrahedron
A dual-response naphthofluorescein-based
fluorescent probe that can selectively sense
Cys/Hcy in multiple emission channels was
reported in this work.

7