A fluorescent probe for discrimination of cysteine/homocysteine, glutathione and hydrogen polysulfides

Article abstract of DOI:10.1007/s11164-020-04320-7

Biothiols of cysteine (Cys), homocysteine (Hcy), glutathione (GSH) and hydrogen polysulfides (H₂S_n, n > 1) play vital roles in physiological and pathological processes. In this manuscript, a fluorescent probe (compound 1) for simultaneous detection and discrimination of Cys/Hcy, GSH and H₂S_n was developed, employing fluorophore of 1-(benzo[d]thiazol-2-yl)naphthalen-2-oxy linked with 7-nitrobenzo-2-oxa-1,3-diazole (NBD) as a response unit and a quencher. Compound 1 exhibited different ratiometric colorimetric responses toward GSH at 430?nm and 375?nm from Cys/Hcy at 480?nm and 375?nm, while it displayed a different colorimetric response at 561?nm toward H₂S_n. Meanwhile it could act as a naked-eye probe toward GSH, Cys/Hcy and H₂S_n with corresponding solution color of light yellow, orange and purple, respectively. Upon excitation at 370?nm, compound 1 responded to all these biothiols with 'turn-on' blue fluorescence, whereas upon excitation at 470?nm, it could respond to Cys/Hcy with 'turn-on' green fluorescence. Data of mass spectra, lifetimes and ¹H NMR spectra interpreted the aromatic nucleophilic substitutions of compound 1 by GSH, Cys/Hcy and H₂S_n to produce fluorophore1-(benzo[d]thiazol-2-yl)naphthalen-2-ol (compound 2) and corresponding NBD derivatives which could be discriminated through their absorption and fluorescence signals as well as solution colors. Furthermore, compound 1 was applied to image Cys/Hcy and H₂S_n/GSH in 786-O cells.

Full text of DOI:10.1007/s11164-020-04320-7

Research on Chemical Intermediates
https://doi.org/10.1007/s11164-020-04320-7
A fuorescent probe ꢀor discrimination oꢀ cysteine/
homocysteine, glutathione and hydrogen polysulꢁdes
Puhui Xie1 · Yanru Zhu · Yanliang Wang · Guangqin Gao · Song Jiang ·
1
2
1
1
Fengqi Guo3
Received: 28 August 2020 / Accepted: 31 October 2020
©
Springer Nature B.V. 2020
Abstract
Biothiols of cysteine (Cys), homocysteine (Hcy), glutathione (GSH) and hydro-
gen polysulꢀdes (H S , n>1) play vital roles in physiological and pathological
2
n
processes. In this manuscript, a ꢁuorescent probe (compound 1) for simultaneous
detection and discrimination of Cys/Hcy, GSH and H S was developed, employing
2
n
ꢁ
uorophore of 1-(benzo[d]thiazol-2-yl)naphthalen-2-oxy linked with 7-nitrobenzo-
2
-oxa-1,3-diazole (NBD) as a response unit and a quencher. Compound 1 exhibited
diꢂerent ratiometric colorimetric responses toward GSH at 430 nm and 375 nm from
Cys/Hcy at 480 nm and 375 nm, while it displayed a diꢂerent colorimetric response
at 561 nm toward H S . Meanwhile it could act as a naked-eye probe toward GSH,
2
n
Cys/Hcy and H S with corresponding solution color of light yellow, orange and
2
n
purple, respectively. Upon excitation at 370 nm, compound 1 responded to all these
biothiols with ’turn-on’ blue ꢁuorescence, whereas upon excitation at 470 nm, it
could respond to Cys/Hcy with ’turn-on’ green ꢁuorescence. Data of mass spectra,
1
lifetimes and H NMR spectra interpreted the aromatic nucleophilic substitutions
of compound 1 by GSH, Cys/Hcy and H S to produce ꢁuorophore1-(benzo[d]thi-
2
n
azol-2-yl)naphthalen-2-ol (compound 2) and corresponding NBD derivatives which
could be discriminated through their absorption and ꢁuorescence signals as well as
solution colors. Furthermore, compound 1 was applied to image Cys/Hcy and H S /
2
n
GSH in 786-O cells.
Keywords Fluorescent probe · Biothiols · Discrimination · Ratiometric colorimetric
responses · Aromatic nucleophilic substitutions
Electronic supplementary material The online version of this article (doi:https://doi.org/10.1007/
s11164-020-04320-7) contains supplementary material, which is available to authorized users.
*
*
Puhui Xie
phxie2013@163.com
Fengqi Guo
fqguo@zzu.edu.cn
Extended author information available on the last page of the article
Vol.:(012
1
3456789)
3

P. Xie et al.
Introduction
Reactive sulfur species (RSS) are essential in many respects of biological redox
homeostasis, cell signaling and the regulation of metabolism. Important RSS
include cysteine (Cys), homocysteine (Hcy), glutathione (GSH), hydrogen sulꢀde
(
H S), and hydrogen polysulꢀdes (H S , n > 1) et al. [1–3], endogenously dis-
2
2 n
tributed in living cells. Among these RSS, H S has been recognized as the third
2
critical gaseous molecule besides carbon monoxide (CO) and nitrogen (II) oxide
(
NO) in biological systems. Explorations in recent years have demonstrated that
both the endogenously produced H S and the exogenously administrated H S
2
2
would cause protective eꢂects during the course of many pathologies [4–6]. H S
2
n
(
n > 1) have recently aroused a lot of interest, because they were found to act as
the real regulators rather than H S in cellular signaling transduction and H S may
2
2
be the ꢀnal product of H S in physiological activity [7–10]. H S and H S are
2
n
2
n
2
redox partners and possibly coexist in biological systems, and they work together
to regulate sulfur redox balance [11]. An active species of H S was reported to
2
n
be hydrogen disulꢀde (H S ) [12].
2
2
Thus, the development of novel techniques to monitor H S in living systems is
2
n
essential for good investigation of their contribution to physiology and pathology.
The usage of ꢁuorescence spectroscopy in the analysis of H S is interesting to
2
n
analyze their pathological/toxicological eꢂects in living systems [13]. Based on
the high nucleophilicity and reducing properties of H S , many research groups
2
2
n
2
−
have developed ꢁuorescent selective probes for H S /S in recent years [14–22].
2
2
RSS of Cys and Hcy are well-known precursors of H S, which can be endog-
2
enously produced by enzymes such as cystathionine β-synthase and cystathionine
γ-lyase, respectively, exhibiting vital functions in the regulation of matrix deg-
radation and cell motility [23–25]. Peptides such as GSH play important roles in
combating oxidative stress and defending against free radicals to protect thiol pro-
teins and enzymes from oxidation in biological systems [26, 27]. Due to similar
chemical structures and reaction activities of these biothiols, it is very challeng-
ing to distinguish Cys/Hcy, GSH and H S from one another. Although ꢁuores-
2
n
cent probes for selectively detecting Cys/Hcy, GSH or H S have been developed,
2
n
till now, there are rare reports about distinguishing Cys/Hcy, GSH and H S from
2
n
one another by use of a single ꢁuorescent probe [28–30].
Several reported ꢁuorescent probes that could distinguish Cys/Hcy from GSH
or H S were ꢁuorophores linked with 7-nitrobenzo-2-oxa-1,3-diazole (NBD) ether
2
moities as electrophilic reactive sites for Cys/Hcy, GSH or H S to generate diꢂerent
2
NBD-biothiols and leaving groups in the thiol-induced aromatic nucleophilic sub-
stitution as well as ꢁuorescence quenchers for ꢁuorophores to provide a low ꢁuores-
cence background with the photo-induced electron transfer (PET) process [31–42].
However, detecting for hydrogen polysulꢀdes were not involved in these NBD ether-
based ꢁuorescent probes. In this manuscript, a ꢁuorescent probe (compound 1) for
simultaneous detection and discrimination of Cys/Hcy, GSH and H S was devel-
2
n
oped by introducing a ꢁuorophore of 1-(benzo[d]thiazol-2-yl)naphthalen-2-ol (com-
pound 2) with a NBD moiety via an ether linker (Scheme 1).
1
3

A fuorescent probe ꢀor discrimination oꢀ…
Cl
N
N
N
O
S
NH2
O
N
NO2
OHC
N
N
SH
HO
S
O
HO
NO2
2
1
Scheme 1 Synthesis route of probe 1
Experimental
Reagents and apparatus
Dimethyl sulfoxide (DMSO) of spectroscopic grade and deionized water (distilled)
were used throughout the experiment. All chemicals of analytical grade for syn-
theses were purchased from commercial suppliers and were used without further
1
13
puriꢀcation. H NMR and CNMR spectra were recorded with a 400 MHz Var-
ian spectrometer. Mass spectra were measured on a Waters SYNAPT G2-Si mass
spectrometer with ion source of MALDI and α-cyano-4-hydroxycinnamic acid as
the main matrix. Absorption spectra were obtained on a TU1901 Ultraviolet–visible
spectrophotometer. The ꢁuorescence spectra were measured with a Cary Eclipse ꢁu-
orescence spectrometer. The absolute ꢁuorescence quantum yields were measured
with a Hamamatsu Absolute PL Quantum Yield Spectrometer C11347. The life-
times were recorded with a Hamamatsu Compact ꢁuorescence lifetime spectrometer
C11367. Cell imaging experiments were conducted on an Olympus FV3000 laser
scanning confocal microscope. Measurements of pH were made on a pH S-3C pH
meter.
Syntheses
1
-(Benzo[d]thiazol-2-yl)naphthalen-2-ol (compound 2) was synthesized according
to the reported literature [43]. Compound 2 (0.1390 g, 0.5 mmol), NBD-Cl (0.12 g,
0
.6 mmol) and two drops of Et N in CH Cl (20 ml) were stirred at room tempera-
3 2 2
ture for 24 h. Then, the mixture solution was evaporated under reduced pressure and
puriꢀed by silica chromatography (eluted with petroleum/ethyl acetate=4/1, v/v) to
1
aꢂord compound 1 (0.088 g, 40%). H NMR (CDCl , δ, ppm), 8.34 (d, J=8.0 Hz,
3
1
H), 8.17 (d, J=4.0 Hz, 1H), 8.13 (d, J=4.0 Hz, 1H), 8.01–8.04 (m, 2H), 7.88 (d,
J=4.0 Hz, 1H), 7.62–7.66 (m, 2H), 7.51(t, J=4.0 Hz, 1H), 7.42–7.46 (m, 2H), 6.60
1
3
(
d, J=8.0 Hz, 1H) (see the Supporting information, Fig. S1). CNMR (CDCl ,
3
δ, ppm), 160.15, 153.46, 153.03, 148.44, 144.84, 144.05, 136.05, 133.41, 132.96,
32.29, 131.10, 128.71, 128.44, 126.43, 125.87, 123.68, 123.34, 121.57, 119.57,
1
1
3

P. Xie et al.
1
08.90 (Fig. S2). HR-ESI–MS, calculated for C H N O S: 440.0579, found:
2
3
12
4
4
+
+
4
41.0657 [M+H ], 463.0476 [M+Na ] (Fig. S3).
Preparation of solutions
Sodium polysulꢀde solution was prepared from sodium sulꢀde and sulfur in deion-
ized water based on the literature [44].
2
−
−
2−
−
2−
2−
The stock solutions of each anion (5 mM) of S , SO , HSO , S O , SO ,
4
3
3
2
5
−
−
−
−
−
2−
−
2−
−
2−
HSO , NO , F , Cl , I , CO , HCO , OAc , HPO , NO and S
were
4
3
3
3
4
2
2
prepared in ultrapure water from their sodium or potassium salts. Amino acid solu-
tions (5 mM) of alanine (Ala), arginine (Arg), cysteine (Cys), glutamic acid (Glu),
glutamine (Gln), glutathione (GSH), glycine (Gly), histidine (His), homocysteine
(
Hcy), lysine (Lys), methionine (Met), phenylalanine (Phe), serine (Ser) and tyros-
ine (Tyr) were prepared in ultrapure water. Stock solution of compound 1 (1 mM)
was prepared in DMSO. 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid
(
HEPES) buꢂer solutions (10 mM) were prepared by dissolving HEPES in deion-
ized water, and the value of pH of the solution was adjusted by NaOH or HCl. For a
typical optical measurement, a solution of 1 (20 μM) was prepared in HEPES buꢂer
(
10 mM, pH 7.0, with 30% DMSO, v/v). Then 3 mL of this solution was put into a
quartz cuvette with an optical path of 1 cm. The stock solution of each analyte was
added into the quartz cuvette via a microsyringe, and the solution was mixed before
measurement of the spectra. All the measurement experiments were performed at
2
98 K.
Cell culture and ꢀuorescence imaging experiments
7
86-O cells were incubated in 90% of RPMI-1640 (GIBCO, stock No. 31800022)
supplemented with 10% of fetal bovine serum at 37 °C in an atmosphere of 5%
CO and 95% air. For cell imaging, cells were pre-treated with probe 1 (10 μM) for
2
3
0 min and then with 100 μM analytes for another 30 min. And cells incubated only
with probe 1 (10 μM) for 30 min were used as the control experiment. All the ꢁuo-
rescence images were collected after cells were washed twice with PBS buꢂer.
Results and discussion
Rational design of compound 1
Due to the strong electron-withdrawing ability of nitro group on NBD moiety, the
PET process in compound 1 could be prohibited, resulting in no emission [45].
Interaction of compound 1 with Cys/Hcy may break the ether bond based on aro-
matic thiolysis of NBD, releasing the ꢁuorophore 1-(benzo[d]thiazol-2-yl)naphtha-
len-2-ol (compound 2) with the excited-state intramolecular proton transfer (ESIPT)
properties and NBD-S-Cys/NBD-S-Hcy, followed by amino-substituted NBD
chromophores NBD-N-Cys/NBD-N-Hcy after sequential nucleophilic substitution
1
3

A fuorescent probe ꢀor discrimination oꢀ…
and intramolecular rearrangement, which will lead to ꢁuorescence ’turn-on’ in blue
and green spectral region, respectively, upon exciting with diꢂerent wavelengths.
The thiol group in GSH could induce the break of ether group and form compound
2
too and NBD-S-GSH. However, NBD-S-GSH would not undergo intramolecular
rearrangement and not emit ꢁuorescence. H S (n>1) could also react with com-
2
n
pound 1 due to its strong nucleophilicity. The ether bond of compound 1 would
also be broken to form a NBD-(S) H chromophore with a visible purple color of
n
the solution, which is non-emissive, and a free 1-(benzo[d]thiazol-2-yl)naphthalen-
2
-ol (compound 2) with an emission band in blue region. Thus, compound 1 could
simultaneously discriminate Cys/Hcy, GSH with diꢂerent excitation wavelengths
and H S with well-deꢀned spectral signals.
2
n
Spectral responses of compound 1 toward Cys/Hcy, GSH and H2Sn
The absorption responses of compound 1 (20 μM) to various anions (100 μM) and
amino acids (100 μM) (10 mM, pH 7.0, with 30% DMSO, v/v) were measured
(
Fig. 1 and Fig. S4). Compound 1 showed a main absorption peak at 375 nm in
HEPES buꢂer, attributing to the absorption from the 1-(benzo[d]thiazol-2-yl)naph-
thalen-2-oxy chromophore. In the presence of H S (5 eq.), a new strong absorp-
2
n
tion band around 561 nm could be observed, accompanied by a color change of the
2
−
solution from colorless to purple (Fig. 2). However, in the presence of S (5 eq.),
there was a much weak absorption band around 561 nm. And addition of other ani-
ons, respectively, to compound 1 caused little change in the absorption spectra (Fig.
S4a). Besides, the absorption spectra of compound 1 in the presence of Cys/Hcy
exhibited a strong peak around 480 nm (Fig. 1), corresponding to a color change
from colorless to orange, which is much diꢂerent from the solution of 1—H S
2
n
(
Fig. 2). The absorption band of compound 1 in the presence of GSH is rather weak
0.6
0.5
0.4
0.3
0.2
0.1
0.0
1
+Na2S
2
1
+Cys
1
+Hcy
1
1
+GSH
1
+Na S
2
3
00
400
500
600
700
Wavelength/nm
Fig. 1 Absorption spectra of 1 (20 μM) in the absence and presence of Cys, Hcy, GSH, Na S , Na S
2
2
2
(
100 μM) in HEPES buꢂer (10 mM, pH 7.0, with 30% DMSO, v/v). (Color ꢀgure online)
1
3

P. Xie et al.
Cys HCO3- Tyr Na2S Arg
Hcy H PO Na2Sn
2 4
-
1
GSH
Ser Phe
Fig. 2 Photograph of compound 1 in the presence of indicated analytes. (Color ꢀgure online)
around 430 nm, with a color change from colorless to yellow. However, addition
of other amino acids, respectively, to compound 1 changed little of the absorption
spectra (Fig. S4b). As diꢂerent absorption peaks suggested the formation of diꢂer-
ent products after the reaction of 1 with these biothiols, the results of absorption
measurements indicated that compound 1 could colorimetrically discriminate H S ,
2
n
Cys/Hcy and GSH from other amino acids.
To test the sensing ability of probe 1 toward H S , Cys/Hcy and GSH, diꢂerent
2
n
concentrations of these analytes were introduced and the response was evaluated by
absorption spectra, respectively. As shown in Fig. 3, there were no absorption bands
in 400–700 nm for free 1 in HEPES buꢂer (10 mM, pH 7.0, with 30% DMSO, v/v).
The new absorption peak at 561 nm appeared and the absorbance increased with the
gradual addition of Na S (Fig. 3a). It was shown that the absorbance at 561 nm was
2
2
2
linearly proportional (R =0.9933, inset in Fig. 3a) to the concentrations of Na S
2
2
concentration (0–24 μM). The detection limit (3σ/K) was 1.1 μM. Upon titration
with Cys/Hcy, the absorption of probe 1 displayed a decrease at 375 nm accompa-
nied by a drastic increase at 480 nm, with three isobestic pints 310 nm, 360 nm and
4
10 nm (Fig. 3b and c). The absorbance ratios at 480 nm and 375 nm (A /A
)
4
80 375
exhibited a drastic change from 0.30 to 1.56 in the presence of 1 eq. Cys (or from
0
.30 to 1.45 in the presence of 1 eq. Hcy). Ratiometric responses of A /A to the
480 375
concentration of Cys/Hcy were observed, which exhibited good linear relationships
2
between A /A and the concentration of Cys (0 – 20 μM, R =0.9951) or Hcy (0
4
80 375
2
–
0
30 μM, R =0.9955), respectively, and the detection limit was 0.56 μM (Cys) and
.85 μM (Hcy), respectively. Upon titration with GSH, the absorbance of probe 1
displayed a decrease at 375 nm accompanying with an increase at 430 nm, with two
isobestic points at 347 nm and 397 nm (Fig. 3d). The absorbance ratios at 430 nm
and 375 nm (A /A ) exhibited a moderate change from 0.30 to 1.10 in the pres-
4
30 375
ence of 1 eq. GSH. The linear relationship between A /A and GSH concentra-
4
30 375
2
tion (0 – 24 μM, R =0.9977) was observed, and a detection limit of 0.61 μM was
obtained. Thus, the change in absorption bands and solution colors after the interac-
tion of compound 1 with these biothiols make it possible to discern Cys/Hcy from
H S and GSH with quantitative determination.
2
n
The ꢁuorescence responses of compound 1 (20 μM) to anions (100 μM) and
amino acids (100 μM) in HEPES buꢂer (10 mM, pH 7.0, with 30% DMSO, v/v)
were studied upon exciting at 370 nm and 470 nm (Fig. 4 and Fig. S5), respec-
tively. Compound 1 was almost nonꢁuorescent, and the absolute ꢁuorescence quan-
tum yield (Φ) is less than 0.001, indicating that the ꢁuorescence of the ꢁuorophore
1
-(benzo[d]thiazol-2-yl)naphthalen-2-oxy was quenched by the linking NBD moi-
ety. Upon addition of Na S , Cys, Hcy and GSH, respectively, to the solution of
2
2
1
3

A fuorescent probe ꢀor discrimination oꢀ…
a 0.6
b
0.5
0.4
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0
.5
A= 19491 C+ 0.06468
561
A
R
/ A375 = 0.3228 + 82934 C
= 0.9951
2
42 80
0.4 R = 0.9933
0
.5
.4
.3
.2
.1
0.3
Na2S
2
0.2
0
- 32 µM
Cys
- 26 µM
0
0.1
0
0
.3
.2
.1
300
0
.0
0
.00000
0.00001 0.00002
2 2
Na S ] / M
0.0000000.0000050.0000100.000015
Cys] / M
0
[
[
0
0
0
0
3
00
400
500
600
700
400
500
600
700
Wavelength/nm
Wavelength / nm
c
d
0
.6
.5
.4
.3
.2
.1
0.6
0.5
0.4
0.3
0.2
0.1
1
.6
.2
.8
0.4
1
1
.2
.0
1
0
Hcy
0.8
0.6
0.4
0
0
- 30 µM
A480/A375 = 0.2853 + 56280 C
R
GSH
0
2
A
R
430 / A375 = 0.3950 + 36247 C
= 0.9977
= 0.9955
0 - 30 µM
2
0
.2
0
.0
0
.00000
0.00001
[Hcy] / M
0.00002
0.0
0
0.00000
0.00001
0.00002
[
GSH] / M
0
0
300
400
500
600
700
300
400
500
Wavelength / nm
600
700
Wavelength/nm
Fig. 3 Concentration-dependent absorption spectra of compound 1 (20 μM) upon titration with Na S (a,
2
2
inserted the linear relationship between absorbance at 561 nm and Na S concentration), Cys (b, inserted
2
2
the linear relationship between ratio of absorbance at 480 nm to absorbance at 375 nm and Cys concen-
tration), Hcy (c, inserted the linear relationship between ratio of absorbance at 480 nm to absorbance at
3
4
7
75 nm and Hcy concentration), GSH (d, inserted the linear relationship between ratio of absorbance at
30 nm to absorbance at 375 nm and GSH concentration), respectively, in HEPES buꢂer (10 mM, pH
.0, with 30% DMSO, v/v). (Color ꢀgure online)
compound 1, when excited at 370 nm, very strong ꢁuorescence in blue channel
appeared at 468 nm (Fig. 4a, b), with the absolute ꢁuorescence quantum yields of
0
.060, 0.043, 0.048, and 0.053, respectively, indicating the cleavage of the ether
linkage of probe 1 in the presence of these biothiols and producing of ꢁuorophore
-(benzo[d]thiazol-2-yl)naphthalen-2-ol (compound 2). The emission shoulders
1
near 418 nm and main peaks at 468 nm were attributed to the enol and keto form
of compound 2 due to its ESIPT process. Besides, very strong ꢁuorescence in green
channel of probe 1 at 548 nm (excited at 470 nm) in the presence of Cys and Hcy
was observed (Fig. 4c), with the absolute ꢁuorescence quantum yields of 0.127
and 0.138, respectively. Thus, the emission shoulders in Fig. 4b near 540 nm for 1-
Cys and 1-Hcy system could be the combination of emissions from two emissions.
In contrast, when excited at 470 nm, there was very weak ꢁuorescence of probe 1
upon interaction with GSH (Φ=0.011), and almost no ꢁuorescence change upon
its interaction with Na S (Φ=0.002) or with Na S (Φ=0.001). The presence of
2
2
2
other amino acids and anions caused almost no increase in ꢁuorescence intensity of
1
3

P. Xie et al.
a
700
b
350
300
250
200
150
100
50
0
1
+ Na S2
2
600
500
400
300
200
100
0
1
+ Hcy
1
+ Cys
1
+ GSH
1
+ Na S
2
1
+ anions
1 + other aminoacids
400
450
500
550
600
650
700
400
450
500
550
600
650
700
Wavelength / nm
Wavelength/nm
c
700
600
500
400
300
200
100
0
d
1
+ Hcy
1
+ Cys
1
1+
1+
1+
1+
Na2S GSH Hcy Cys
2
1
+ GSH
1
+ other aminoacids
550
500
600
650
700
Wavelength/nm
Fig. 4 Fluorescence spectra of 1 (20 μM) in the absence and presence of analytes (100 μM) in HEPES
buꢂer (10 mM, pH 7.0, with 30% DMSO, v/v). a Anions excited at 370 nm. b amino acids, excited at
3
70 nm. c amino acids, excited at 470 nm. d Photograph of compound 1 in the presence of indicated ana-
lytes (λ =370 nm). (Color ꢀgure online)
ex
1
when excited at either 370 nm or 470 nm. Therefore, the change in ꢁuorescence
bands after the interaction of compound 1 with these biothiols makes it possible to
discern Cys/Hcy from H S and GSH under diꢂerent excitation wavelengths.
2
n
The ꢁuorescence of compound 1 was measured by the ꢁuorescence titration
experiment in order to identify its performance. As shown in Fig. 5, in a certain
range, the ꢁuorescence intensity of 1 increased gradually with increased concentra-
tions of Cys/Hcy, GSH and H S , respectively, in the blue channel (Fig. 5a, c, e and
2
n
f) when excited at 370 nm. Good linear relationships were obtained between the ꢁu-
orescence intensities of compound 1 (20 μM) and the concentration of these thiols
within a certain range (10 μM for Cys, 16 μM for Hcy, 25 μM for GSH, and 16 μM
for Na S ). The detection limits were 0.17 μM for Cys, 0.30 μM for Hcy, 0.32 μM
2
n
for GSH, and 0.30 μM for H S when excited at 370 nm, calculated based on the
2
n
equation DL=3σ/K, where σ is the standard deviation of a blank measurement
and K is the slope between ꢁuorescence intensity at 468 nm and analyte concentra-
tion. Besides, the ꢁuorescence intensity of 1 increased indicating gradually follow-
ing increased concentrations of Cys/Hcy in the green channel (Fig. 5b and d) when
1
3

A fuorescent probe ꢀor discrimination oꢀ…
a
350
b
500
200
1
+ Cys
1
+ Cys
300
250
y = 55.7586 + 1.1212E7 *
R = 0.99266
2
x
160
3
2
2
1
1
00
50
00
50
00
y
R
2
=52.2587
=0.9905
+
2.5441E7
*
x
400
120
80
40
0
200
150
100
300
200
50
0.000000
0.000004
0.000008
/ M
0.000012
0
[Cys]
0
.000000 0.000002 0.000004 0.000006 0.000008 0.000010
[Cys]
/ M
100
5
0
0
0
4
00
450
500
550
600
650
700
500
550
600
650
700
wavelength/nm
Wavelength / nm
c
d
4
00
50
00
50
00
50
00
1 + Hcy
1
+ Hcy
300
500
400
300
500
Y = 56.5383 + 1.7033E7 * X
= 0.9994
3
3
2
2
1
1
250
Y
R
2= 47.5668+1.6388E7X
0.9955
400
R
2
=
2
1
00
50
300
200
100
100
5
0
0
0
0
.000000
0.000004
0.000008
-1
0.000012
0.000016
0.00000
0.00001
0.00002
0.00003
[
Hcy]/
M
[Hcy] / M-1
200
5
0
100
0
0
400
450
500
550
600
650
700
500
550
600
650
700
Wavelength/nm
Wavelength / nm
e
f
4
3
3
2
2
1
1
00
50
00
50
00
50
00
400
300
200
100
0
1
+Na S2
350
300
1
+ GSH
2
3
50
Y
=
37.9352
2
R =0.9932
+
1.5143E7
*
X
Y =34.3701+1.8676E7 * X
R =0.9909
300
2
250
200
150
100
250
200
150
100
50
0
50
0
.000000 0.000005 0.000010 0.000015 0.000020 0.000025
0
[GSH]
/
M
0
.000000
0.000004
0.000008
0.000012
2 2
S ] / M
0.000016
[Na
5
0
0
400
450
500
550
600
650
700
400
450
500
550
600
650
700
Wavelength/nm
Wavelength/nm
Fig. 5 Concentration-dependent ꢁuorescence of 1 (20 μM) with Cys (a), Hcy (c), GSH (e), H S (f) upon
2
n
exciting at 370 nm, and with Cys (b), Hcy (d) upon exciting at 470 nm. Insets: plot of the linear relation-
ship between the ꢁuorescence intensity at respective wavelengths (468 nm, 548 nm) and the low concen-
tration of biothiols. (Color ꢀgure online)
excited at 470 nm. Good linear relationships were obtained between the ꢁuorescence
intensities of probe 1 and the concentration of these thiols within 12 μM. The results
indicated that probe 1 could quantitatively detect these thiols at low concentrations.
The time- and pH-dependent experiments of the probe 1 to H S , Cys/Hcy and
2
n
GSH, respectively, were carried out. The kinetic studies indicated that in the pres-
ence of Cys or H S , the ꢁuorescence intensity of 1 at 468 nm showed the fastest
2
n
growth rate and reached a plateau within 15 min (Fig. S5). In the ꢀrst 15 min inter-
val, the relative emission intensities (calculated according to the equation: Relative
1
3

P. Xie et al.
Emission Intensity=F / F , where F was Emission intensity λ=468 nm after addi-
0
tion of analyte, F =Emission intensity of the probe 1 at λ=468 nm without the
0
addition of any analyte) were 9.98 and 7.72 for 1-Na S and 1-Cys systems, respec-
2
n
tively, whereas the relative emission intensities increased 2.33 and 1.80 for 1-Hcy
and 1-GSH systems, respectively. The response rate of 1 toward Cys, H S was
2
n
faster than Hcy, GSH. The nucleophilicities of the biothiols could be attributed to
diꢂerent response rates [45]. The lowest pK value of H S (pK =5.0) among these
a
2
2
a
−
analytes rendered H S highly nucleophilic as a thiolate anion (HS ) under neutral
2
2
2
condition [46]. In addition, in the presence and absence of H S , Cys/Hcy and GSH,
2
n
the ꢁuorescence intensity at 468 nm remained stable over a pH range from 4.5 to 8.0
Fig. S6), indicating that compound 1 could detect these thiols in the physiological
pH range.
(
The ꢁuorescence intensity at 468 nm of compound 1 with the addition of anions,
Cys/Hcy, GSH, other amino acids and were monitored upon exciting at 370 nm in
order to investigate the selectivity of compound 1 toward H S . As shown in Fig. 6,
2
n
introduction of 1 equivalent of H S , Cys/Hcy and GSH to a solution of 1 resulted
2
n
in an increase in ꢁuorescence intensity, with about sixfold enhancements. Anions of
2
−
−
sulꢀtes (SO
and HSO ) and pyrosulꢀtes interfere the ꢁuorescence response of
3
2
3
1
toward H S , possibly due to oxidation–reduction reactions of these anions with
n
polysulꢀdes. The ꢁuorescent intensity of 1 toward other anions or amino acids was
much lower than those of H S , Cys/Hcy and GSH, and showed almost no interfere
2
n
upon detecting these biothiols. The presence of other amino acids would not inter-
fere the detection of Hcy either (Fig. S7).
Possible interaction mechanisms
As shown in Scheme 2, electrophilic NBD derivative of compound 1 underwent
nucleophilic substitution with biothiols Cys/Hcy/GSH to produce compound
2
(which can lead to the ꢁuorescence enhancement at 468 nm upon excitation at
3
70 nm) and non-ꢁuorescent thiolate derivatives (such as NBD-S-Cys, NBD-S-
Hcy, or NBD-S-GSH, respectively), and then the amino moiety in NBD-S-Cys,
a450
b450
1
1
+ anions + H Sn
+ anions
1
1
+ amino acids + H Sn
+ amino acids
2
2
4
3
3
2
2
1
1
00
50
00
50
00
50
00
400
350
300
250
200
1
50
00
1
50
5
0
0
2-
0
-
Sn Glu Met Gln Gly Phe His Ser Arg Ala Tyr Lys Cys GSH Hcy
2-
-
2-
-
-
-
-
-
-
-
2-
2-
2
-
4
l
4
-
C
4
2
3
2
-
O
3
3
2-
3
5
2
S
O
C
P
H
O
F
A
O
C
lO
O
3
N
O
O
I
C
H
S
H
O
S
O
O
2
S
O
S
H
N
S
C
S
Fig. 6 The ꢁuorescence intensity at 468 nm of 1 (20 μM) toward H S (20 μM) in the presence of anions
2
n
(
20 μM) (a) and amino acids (20 μM) (b), respectively. (Color ꢀgure online)
1
3

A fuorescent probe ꢀor discrimination oꢀ…
Scheme 2 Schematic illustration of the reaction of compound 1 with Cys/ Hcy, GSH and H2Sn
NBD-S-Hcy can easily replace the thiolate via intramolecular nucleophilic aro-
matic (S Ar) substitution reactions (Smiles rearrangement) to convert to ꢁuores-
N
cent amino-substituted NBD derivatives (NBD-N-Cys and NBD-N-Hcy) through a
ꢀ
ve- or six-membered ring intermediate. Fluorescence eꢃciency of 4-substituted-
7
-nitro-2,1,3-benzoxadiazoles (NBD-X) markedly depends on the electron-donating
ability of X through eꢂective ICT process (e.g., relative ꢁuorescence quantum yields
in methanol: X=NHCH , 62.7; SCH , 0.6; OCH , 0.0) [47]. Due to the strong
3
3
3
push–pull electron eꢂects between amino and nitro groups of benzoxadiazole, NBD
derivatives of NBD-N-Cys and NBD-N-Hcy could emit at 550 nm [48–50], whereas
NBD-S-GSH could not further react due to the long distance of NH group toward
2
the thioether group within the molecules. H S could also nucleophilically attack the
2
n
ether oxygen atom of compound 1 as Cys /Hcy and GSH did to generate compound
2
and derivatives of NBD-S H. NBD-S H could not emit but absorbed at 561 nm, a
n n
much diꢂerent wavelength from NBD-N-Cys, NBD-N-Hcy and NBD-S-GSH. Kand
et al. applied time-dependent density functional theory (TDDFT) calculations at the
B3LYP/6-311G (d,p) level on optimized geometries in predicting the ꢁuorescence
properties of NBD-S-Cys and NBD-N-Cys, and the TDDFT calculations for NBD-
N-Cys conꢀrmed an allowed S →S transition and S as the emissive state, whereas
0
1
1
the presence of a dark S state and allowed S →S transition conꢀrmed NBD-S-Cys
1
0
2
as a nonꢁuorescent species [51].
The sensing mechanism could be proved by mass spectrometry analysis of the
reaction mixture of compound 1 with these biothiols. Peaks at m/z=278.0 in the
ESI–MS spectra of 1 in the presence of Cys, Hcy, GSH and H S , respectively (Fig.
2
n
+
S8~Fig. S11), corresponded to [M+H] of compound 2. Peaks at m/z=284.3
(
Fig. S8), 299.0 (Fig. S9), 471.0 (Fig. S10) and 316.0 (Fig. S11), corresponding
1
3

P. Xie et al.
+
+
+
+
to [NBD-Cys] , [NBD-Hcy] , [NBD-GSH] and [NBD-S H+Na] , respectively,
4
were observed in the ESI–MS spectra, conꢀrming the mechanisms proposed in
Scheme 2. The assignment from the mass spectra was in accordance with the ꢁuo-
rescence spectra, where the probe with addition of all these biothiols provided a ꢁu-
orescence band at 468 nm corresponding to compound 2 and Cys/Hcy led to a new
ꢁ
uorescence band peak at 550 nm corresponding to NBD-N-Cys and NBD-N-Hcy.
These results support our proposed sensing mechanism, which is in good agreement
with that reported in the literature [47–50].
In the same media, the lifetime of compound 2 was determined and compared
with lifetimes of compound 1 in the presence of Cys, Hcy, GSH and H S , respec-
2
n
tively (Fig. S12 and Table S1). The lifetime of compound 2 was found to be 4.33 ns
when monitored at 468 nm. The solution of 1 displayed lifetimes of 4.34 ns moni-
tored at 468 nm, 5.24 ns monitored at 550 nm for Cys, and 4.32 ns monitored at
4
68 nm, 4.72 ns monitored at 550 nm for Hcy. The mixture of 1 with GSH or H S
2
n
showed a single lifetime monitored at 468 nm (τ=4.38 ns for GSH, τ=4.35 ns for
H S ). As the lifetimes measured at 468 nm of 1 in the presence of Cys, Hcy, GSH
2
n
and H S were very close to the lifetime of compound 2, the results proved that the
2
n
reaction of 1 with Cys, Hcy, GSH and H S generated the same product of com-
2
n
pound 2.
1
H NMR comparison of 1 in DMSO-d in the presence of Cys, Hcy, GSH and
6
H S with compound 2 displayed that the signals belonging to the aromatic protons
2
n
of compound 1 were shifted and most of the signals were close to signals of com-
pound 2 (Fig. 7 and Fig. S13), indicating the reaction of 1 with these biothiols and
1
Fig. 7 H NMR spectra of 1 in dmso-d before and after addition of Cys (1 eq) and GSH (1 eq), respec-
6
tively. (Color ꢀgure online)
1
3

A fuorescent probe ꢀor discrimination oꢀ…
the release of 2. The peak at δ 6.50 ppm in the 1+Cys mixture indicated the forma-
tion of NBD-N-Cys [50]. The rather weak peak at δ 6.36 ppm in the 1+Hcy mix-
ture indicated the slower conversion to NBD-N-Hcy, whereas no peaks appeared in
this area in the 1+GSH indicated no conversion to NBD-N-GSH from NBD-S-GSH
(
Fig. S13).
Fig. 8 Confocal microscopic images of compound 1 in presence of biothiols in living cells. 786-O cells
incubated with compound 1 (10 μM) for 30 min (A1-A3). 786-O cells pre-incubated with compound 1
(
10 μM) for 30 min and then, respectively, with 100 μM of Hcy (B1-B3), Cys (C1-C3), GSH (D1-D3),
H S (E1-E3) for 30 min. Excitation wavelengths were 370 nm; emissions were collected at 450–490 nm
2
n
for blue channels, and excitation wavelengths were 480 nm; emissions were collected at 530–560 nm for
green channels. (Color ꢀgure online)
1
3

P. Xie et al.
Cell imaging
As shown in Fig. 8, after the 786-O cells were pre-incubated with compound 1
(
10 μM) for 30 min, very weak ꢁuorescence appeared in blue and green channels,
indicating compound 1 was slightly responsive to intracellular biothiols. When the
pre-treated cells with compound 1 were incubated with 100 μM diꢂerent analytes for
another 30 min, much stronger ꢁuorescence were observed in blue and green chan-
nels for Cys/Hcy, whereas for GSH/H S , only signiꢀcant ꢁuorescence increases in
2
n
blue channels were observed. These results indicated that compound 1 was cell per-
meable and could be used for sensing intracellular Cys/Hcy and GSH/H S , simulta-
2
n
neously from diꢂerent emission channels.
Conclusions
In conclusion, a simple ꢁuorescent probe (compound 1) bearing two ꢁuorophores
for simultaneous detection and discrimination of Cys/Hcy, GSH and H S was
2
n
developed. The probe could selectively detect Cys/Hcy, GSH and H S from other
2
n
amino acids by naked eyes. The interaction of compound 1 with these biothiols
would form the ꢁuorophore compound 2, with turn on ꢁuorescence at 468 nm, and
form diꢂerent NBD derivatives with diꢂerent spectroscopic properties. The reac-
tion of 1 with Cys/Hcy Probe 1 could be used to discriminate Cys/Hcy from GSH at
excitation wavelength of 470 nm. Therefore, compound 1 could simultaneously dis-
criminate Cys/Hcy, GSH with diꢂerent excitation wavelengths and H S with well-
2
n
deꢀned absorption spectral signals. Preliminary cell imaging experiments indicated
that compound 1 could simultaneously monitor intracellular Cys/Hcy and GSH/
H S , from diꢂerent emission channels in living 786-O cells.
2
n
Acknowledgements The authors thank the National Natural Science Foundation of China (21702047,
8
1700578), Scientiꢀc and Technological Project of the Henan Province of China (192102110053), and
Henan International Joint Laboratory of Laser Technology in Agricultural Sciences for Financial Support.
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and institutional aꢃliations.
1
3

P. Xie et al.
Aꢀliations
Puhui Xie1 · Yanru Zhu · Yanliang Wang · Guangqin Gao · Song Jiang ·
1
2
1
1
Fengqi Guo3
1
College of Sciences, Henan Agricultural University, Zhengzhou 450002,
People’s Republic of China
2
Department of Nephrology, Henan Provincial Key Laboratory of Kidney Disease
and Immunology, Henan Provincial People’s Hospital (Zhengzhou University People’s
Hospital), Zhengzhou 450003, Henan, People’s Republic of China
3
Henan Institute of Advanced Technology, Zhengzhou University, Zhengzhou 450001,
People’s Republic of China
1
3