An indocyanine-based turn-on fluorescent probe for specific detection of biothiols

Article abstract of DOI:10.3987/COM-18-13878

A novel heptamethine cyanine-based turn-on fluorescent probe Cy-DNBS was designed and synthesized, which was used successfully to detect biothiols with high selectivity and sensitivity. With the addition of biothiols, the probe displayed ~30-fold fluorescence enhancement at 625 nm. Meanwhile, the solution color changed remarkably from green to red, which provides a method to recognize biothiols by the ‘naked eye’. The detection limit of Cys, GSH and Hcy are 2.24 nM, 1.99 nM and 4.46 nM, respectively.

Full text of DOI:10.3987/COM-18-13878

HETEROCYCLES, Vol. 96, No. 4, 2018
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HETEROCYCLES, Vol. 96, No. 4, 2018, pp. 707 - 715. © 2018 The Japan Institute of Heterocyclic Chemistry
Received, 15th February, 2018, Accepted, 16th March, 2018, Published online, 20th March, 2018
DOI: 10.3987/COM-18-13878
AN INDOCYANINE-BASED TURN-ON FLUORESCENT PROBE FOR
SPECIFIC DETECTION OF BIOTHIOLS
Xiaojing Wang, Di Jin, Xue Li, Xilong Yan,* and Bowei Wang*
School of Chemical Engineering and Technology, Tianjin University, Yaguan
Road 135#, Jinnan District 300350, Tianjin, China. Email: yan@tju.edu.cn;
bwwang@tju.edu.cn
Abstract – A novel heptamethine cyanine-based turn-on fluorescent probe
Cy-DNBS was designed and synthesized, which was used successfully to detect
biothiols with high selectivity and sensitivity. With the addition of biothiols, the
probe displayed ~30-fold fluorescence enhancement at 625 nm. Meanwhile, the
solution color changed remarkably from green to red, which provides a method to
recognize biothiols by the ‘naked eye’. The detection limit of Cys, GSH and Hcy
are 2.24 nM, 1.99 nM and 4.46 nM, respectively.
INTRODUCTION
Biothiols such as cysteine (Cys), homocysteine (Hcy) and glutathione (GSH) play significant roles in
1
maintaining the redox balance during many physiological and pathological processes. In fact, a number
of works have shown that abnormal level of thiols can result in some diseases,2,3 such as the deficiency of
Cys is related to liver damage, slow growth, skin lesions and hair depigmentation,4,5 while high level of
Hcy will increase the risk of Alzheimer’s disease and cardiovascular.6,7 GSH, the most abundant
intracellular nonprotein thiol, is important in controlling oxidative stress, and the alteration of its level is
connected with metabolism, stress responses, immune responses.8,9 Therefore, developing an analytical
method that can effectively detect biothiols is meaningful.
Over the past decades, many methods had been developed for biothiols’ detection, such as
electrochemical method, high performance liquid chromatography (HPLC) and mass spectrometry
(
MS).10-12 Nonetheless, most of them require expensive instruments and complex operations. Compared
with the above methods, fluorescence detection has been widely concerned by virtue of sensor simplicity,
high sensitivity, good selectivity, noninvasiveness and convenient operation even in live cell and in vivo.
In recent years, plenty of optical sensors have been developed to detect biothiols by several sensing
strategies including Michael addition,13,14 cleavage reactions,8,15-18 cyclization reaction and others.20-25
19

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Despite all recent progess, it’s still a challenge to develop novel simple probes, which can be synthesized
easily and possess high sensitivity and selectivity.
Among well-known fluorophores, cyanine dyes are characteristic of high absorption coefficient, relatively
long absorption and emission wavelength. Cyanine-ketone (CyK) derivatives are attractive dye because
the polymethine π-conjugated system can be rationally modulated by introducing specific group on the
2
6
meso-oxygen atom of the structure. Several cyanine dyes functionalized at the meso-oxygen have been
reported for detection of hydrazine and H
S.28,29
2
7
2
With these considerations in mind, a novel probe based on cyanine was designed and synthesized for the
recognition of biothiols in PBS. The probe Cy-DNBS employed CyK as the fluorophore and
2
,4-dinitrobenzenesulfonyl (DNBS) group as a recognition unit. The probe showed good sensitivity and
selectivity to biothiols over other amino acids. Moreover, the solution color change provided a simple
way for naked eye recognition of biothiols.
Scheme 1. The design and structure of probe Cy-DNBS
RESULTS AND DISCUSSION
The response time is an important parameter to evaluate a probe, therefore the time-dependent absorption
and fluorescence intensity of probe Cy-DNBS towards thiols were investigated first. As shown in Figure
1
A, in the prescence of 1 equiv. Cys, the absorption peak at 790 nm decreased significantly accompanied
by the emergency of a new absorption peak at 544 nm, and the sensing process could reach to a plateau
within 35 min. Meanwhile, an obvious isosbestic point at 610 nm was also observed, which distinctly
demonstrated that a new species came into being upon the treatment of the probe with Cys. In addition,
the fluorescence spectra displayed the same tendency with the absorption spectra upon addition of Cys.
Probe Cy-DNBS is almost non-fluorescence originally. However, its fluorescence intensity increased
significantly upon addition of 1 equiv. Cys and achieved the balance within 25 min (Figure 1B).
Moreover, the reaction towards GSH and Hcy show similar results and changes of spectra can be

HETEROCYCLES, Vol. 96, No. 4, 2018
709
completely finished within 45 min (Figures S7, S8). Therefore, all of the following tests were performed
after 45 min.
To investigate the sensitivity of probe Cy-DNBS to biothiols, fluorescence titration was carried out. As
shown in Figure 2, the fluorescence intensity at 625 nm increased gradually with the increase of Cys
concentration. And a good linear relationship between fluorescence intensity and the concentration of Cys
was observed in the range of 0-4 μM with a correlation coefficient of 0.9896, displayed in the inset of
Figure 2B. The detection limit for Cys was calculated to be 2.24 nM based on the signal to noise ratio,
S/N=3. In addition, when Hcy and GSH were carried out the same experiments with the probe Cy-DNBS,
similar phenomenon were observed (Figures S9, S10), and the detection limits for GSH and Hcy were
evaluated to be 1.99 nM and 4.46 nM, respectively. Compared with the reported biothiols’ probes,
especially fluorescent probes having a DNBS group,1,7,15,16 our probe Cy-DNBS presented a relatively
low detection limit, which indicating that probe Cy-DNBS possesses high sensitivity.
Figure 1. (A) Time-dependent absorption spectra of probe Cy-DNBS (10 μM) after adding 1 equiv. Cys.
Inset: the absorbance at 544 nm along with time. (B) Time-dependent fluorescence spectra of probe
Cy-DNBS (10 μM) after adding 1 equiv. Cys. Inset: the fluorescence intensity at 625 nm along with time.
Figure 2. (A) Fluorescence spectra of probe Cy-DNBS (10 μM) treated with Cys, (B) Fluorescence
intensity at 625 nm for probe Cy-DNBS (10 μM) as the function of the concentration of Cys.

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It’s significant that a probe has good selectivity towards specific analyte. Therefore the selectivity of
probe Cy-DNBS for biothiols over other amino acids was investigated. As shown in Figure 3A, there was
no significant change in absorption spectra with the addition of 10 equiv. other amino acids. However, the
absorption peak at 790 nm decreased obviously and a new absorption peak at 544 nm arose dramatically
with the addition of 1 equiv. Cys, Hcy, GSH. In the emission spectra, only biothiols induced a remarkable
fluorescence enhancement with an excitation at 544 nm, while almost no change in fluorescence spectra
was observed upon the treatment with the other amino acids over an hour (Figure 3B). Furthermore, the
color of the solution changed distinctly from green to red, which can be identified by naked eye (Figure
3
0
3
C). In addition, DNBS group has also been reported to be reacted with hydrogen sulfide (H
2
S), thus the
reactivity of Cy-DNBS with H S was also examined. It can be seen from Figure 3, the absorption and
2
-
fluorescence spectra only exhibited slight changes after addition of 10 equiv. HS , which can be neglected
when compared with the changes caused by biothiols.
Figure 3. (A) Absorption spectra of probe (10 μM) in the presence of different analytes. (B) Fluorescence
spectra of probe (10 μM) in the presence of different analytes. (C) Visual color changes of probe upon
addition of various analytes. (D) Fluorescence intensity at 625 nm of probe in the presence of different-
analytes (black bars), and upon addition of 1 equiv. of Cys (red bars) (The other amino acids and HS
were 10 equiv. except biothiols were 1 equiv).

HETEROCYCLES, Vol. 96, No. 4, 2018
711
In addition, to further explore the selectivity of the probe to biothiols in the presence of other nature
-
amino acids and HS , interference experiments were conducted as follows: probe Cy-DNBS was first
-
mixed with 10 equiv. other amino acids and HS , and 1 equiv. Cys was then added to the mixture. It can
be seen from Figure 3D that upon the addition of 1 equiv. Cys, fluorescence intensity of the mixture was
apparently enhanced, which demonstrated that probe Cy-DNBS possesses high selectivity toward
biothiols even if it coexists with 10-fold concentration of other amino acids and HS-.
For the sake of wide applications, the effect of pH on the fluorescence properties of probe Cy-DNBS was
tested. First, the fluorescence intensity at 625 nm of probe Cy-DNBS at different pH was examined. The
pH range from 2 to 12 was adjusted by 0.1 M hydrochloric acid solution and 0.1 M sodium hydroxide
solution. It can be seen from Figure 4 that there was no significant change for the probe Cy-DNBS’s
fluorescence intensity with the change of pH value, which indicated that the probe was unchangeable in a
wide pH range from 2 to 12. Subsequently, the response of the probe Cy-DNBS towards Cys at different
pH was also investigated. After addition of 1 equiv. Cys, the fluorescence intensity was obviously
enhanced in the pH range of 5-11, which illustrated that the probe can be used for the detection of
biothiols in a wide pH range and has potential applications in physiological conditions. In the acidic
conditions (pH 2-4), Cy-DNBS did not react with Cys because the detection of thiols is a process of
nucleophilic substitution and the nucleophilic reaction is prevented under acidic conditions.1
Figure 4. pH effect on the fluorescence properties of probe Cy-DNBS and the response of the probe
towards Cys. The spectroscopic properties of probe Cy-DNBS was tested in ethanol-water (3:7, v/v).

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Scheme 2. Proposed responding mechanism of probe Cy-DNBS toward biothiols
In order to better understanding the interaction of the probe and biothiols, the response mechanism was
studied. Several probes1,15,16,31 with sulfonate ester linkage have been reported to selectively recognizing
biothiols by a process of nucleophilic aromatic substitution. Therefore, we speculated that probe
Cy-DNBS may also detect biothiols through the process of nucleophilic substitution. To confirm our
assumption, the comparative experiments on absorption spectra and fluorescence spectra of CyK, probe
Cy-DNBS and the reaction product of probe Cy-DNBS and biothiols were carried out. As shown in
Figure S11, after treated with 1 equiv. thiol, probe Cy-DNBS exhibited an absorption peak shift from 790
nm to 544 nm, and a ~30-fold fluorescence response at 625 nm, which was in accordance with the
absorption and fluorescence emission spectra of CyK. Furthermore, the results of mass spectrometry of
probe Cy-DNBS treated with 1 equiv. Cys also indicated the generation of desired compound CyK (m/z
-
4
63.2725, for [M-H] ) (Figure S12). These results suggest that the compound CyK was obtained indeed
after treated with thiols. Thus, the response mechanism of probe Cy-DNBS toward biothiols was based on
the nucleophilic substitution reactions as shown in Scheme 2.
In conclusion, a novel turn-on fluorescent probe for biothiols was successfully designed and synthesized
based on heptamethine cyanine. The probe presents remarkable color changes upon the treatment with
biothiols, which provides a simple way to detect thiols by naked eyes. Besides, the probe demonstrated
good selectivity toward biothiols over other amino acids and the detection limit for Cys, GSH, Hcy was
2
.24 nM, 1.99 nM and 4.46 nM, respectively. These results prove it to be an excellent probe for the
selective detection of biothiols.

HETEROCYCLES, Vol. 96, No. 4, 2018
713
EXPERIMENTAL
The synthetic route of probe Cy-DNBS is illustrated in Scheme 3.
Cl
O
OH
Cl
3
H
O
AcOH
H2SO4
MeI
N
NH2
N
N
N
N
MeCN
EtOH, reflux, 3 h
1
2
NO2
O
Cl
S
O
O
NO2
AcONa
DMF, 80 oC, 3 h
O
S
O2N
NO2
N
N
O
O
N
N
CH2Cl2, TEA, rt
CyK
Probe Cy-DNBS
Scheme 3. The synthetic route of probe Cy-DNBS
Synthesis of CyK: This compound 1, compound 2 and compound 3 were prepared according to our
previously reported method.32,33 A mixture of compound 3 (305 mg, 0.50 mmol) and sodium acetate (123
o
mg, 1.50 mmol) in anhydrous DMF (10 mL) was stirred at 90 C for 3 h under nitrogen atmosphere. After
cooling to room temperature, the solvent was removed and the crude product was purified by
chromatography on silica gel (petroleum ether/EtOAc = 3:1, v/v) to afford compound CyK as a red solid
1
(
151 mg, 65% yield). H NMR (400 MHz, CDCl
3
) δ (ppm): 8.19 (d, J = 13.2 Hz, 2H), 7.19 (d, J = 7.6 Hz,
4
5
1
H), 6.91 (t, J = 7.6 Hz, 2H), 6.70 (d, J = 8.0 Hz, 2H), 5.43 (d, J = 13.2 Hz, 2H), 3.21 (s, 6H), 2.62 (t, J =
13
.6 Hz, 4H), 1.91-1.86 (m, 2H), 1.67 (s, 12H). C NMR (100 MHz, CDCl3) δ (ppm): 186.42, 163.17,
44.57, 139.57, 132.73, 127.61, 126.80, 121.70, 120.45, 106.40, 92.51, 46.40, 29.26, 28.67, 25.82, 22.51.
+
HRMS (ESI Positive) calc. for C32
yield: 10%.
H
36
N
2
O, [M+H] 465.2900, found 465.2908. Fluorescence quantum
Synthesis of probe Cy-DNBS: Compound CyK (464 mg, 1 mmol) was dissolved in 15 mL CH
2
Cl and
2
stirred under the ice bath. Then triethylamine (101 mg, 1 mmol) was added to the solution and the
mixture was stirred for 15 min. The solution of 2,4-dinitrobenzenesulfonyl chloride (798 mg, 3 mmol) in
CH
stirring in ice bath for another 30 min. After that the mixture was warmed naturally to room temperature
and stirred for 1 h. Then the mixture was poured into water (50 mL) and extracted with CH Cl (25 mL ×
). The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated to yield a black
2
Cl
2
(5 mL) was added dropwise within 30 min under nitrogen protection. The reaction mixture kept
2
2
2
solid. The crude product was purified by column chromatography on silica gel (CH
2
Cl /MeOH = 20:1,
2
1
v/v) to afford a green solid (353 mg, 28% yield). H NMR (400 MHz, CDCl
3
) δ (ppm): 8.38 (d, J = 8.5
Hz, 1H), 8.31 – 8.25 (m, 2H), 7.73 (d, J = 14.0 Hz, 2H), 7.37 (t, J = 7.7 Hz, 2H), 7.29 (d, J = 7.2 Hz, 2H),

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HETEROCYCLES, Vol. 96, No. 4, 2018
7
1
1
1
.22 (t, J = 7.4 Hz, 2H), 7.14 (d, J = 7.9 Hz, 2H), 6.16 (d, J = 14.0 Hz, 2H), 3.67 (s, 6H), 2.72 (m, 4H),
.99 (m, 2H), 1.44 (s, 12H). 13C NMR (100 MHz, CDCl
) δ (ppm): 172.65, 156.46, 150.96, 148.83,
3
48.26, 147.42, 145.53, 142.66, 140.92, 140.12, 134.59, 134.26, 131.90, 128.81, 128.36, 125.59, 125.29,
23.88, 122.10, 120.47, 118.27, 110.93, 102.02, 53.51, 49.10, 31.90, 27.25, 25.67, 20.33. HRMS (ESI
+
Positive) calc. for C38
39
H N
4
O
7
S, [M] 695.2539, found 695.2529. Fluorescence quantum yield: 9%.
ACKNOWLEDGEMENTS
We are grateful for the financial support from the National Natural Science Foundation of China
21576194).
(
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