Y.-H. Sun, H.-H. Han, J.-M. Huang et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 261 (2021) 120055
on CARY Eclipse spectrophotometer and Varian Cary 500 spec-
trophotometer, respectively. 1H NMR and 13C NMR spectra were
obtained by Bruker AM-400 and Ascend 600 spectrometer. Waters
LCT Permier XE spectrometer was used to measure high-resolution
mass spectra, and a pH-10C digital pH meter was used to measure
pH.
1. Introduction
Cysteine (Cys) and glutathione (GSH) are important biothiols
implicated in a number of biological processes [1,2]. They are
responsible for balancing intracellular redox processes, modulating
cell metabolism and detoxification, and facilitating protein synthe-
sis [3–5]. While the lack of Cys can lead to skin lesions, neurotox-
icity, edema and liver damage, the excess of which may cause
cardiovascular and Parkinson’s disease [6–8]. In the meanwhile,
GSH, which is consisted of glutamic acid, cysteine and glycine,
[9] contributes to the redox balance of cells by eliminating intra-
cellular reactive oxygen species (ROS). The decrease of GSH is an
early activation signal of cell apoptosis [10]. Intracellular Cys and
GSH concentration is closely related to biosynthesis and a variety
of human diseases [11]. Recent studies suggest that lysosomal
Cys and GSH play an important role in digesting and metabolizing
intracellular substances [12,13]. As a result, sensitive analytical
tools that can effectively detect and quantify Cys and GSH at the
subcellular level is of significance for basic biomedical research
and disease diagnosis [14,15].
Fluorescent probes, owing to their synthetic simplicity, highly
tunable photophysical properties and superior sensitivity to many
other existing analytical agents, have become the technique of
choice for biosensing and bioimaging of biothiols [16,17]. The sens-
ing mechanisms include cyclization with aldehydes, [18–20] conju-
gate addition-cyclization with acrylates, [21–23] Michael addition,
[24–26] aromatic substitution-rearrangement reaction, [27,28]
cleavage of sulfonamide, [29–31] sulfonate ester [32,33] and super-
imposed applications of the above methods [34,35]. Among them,
acrylate reacts quickly and selectively with biothiols, and has
become a popular reactive group for the construction of biothiol
probes [36–40]. However, problems remain for the majority of the
reported biothiol probes in terms of a short emission wavelength
(<600 nm), small Stokes shift (<100 nm), relatively long response
time (>10 min), and the inability to precisely localize biothiols at
the subcellular level [41–43]. For lysosome localization, alkane-
linked morpholine groups are frequently used, whereas the use of
aromatic amines for lysosome targeting has been much less
explored [44–46]. With the development of detection technology,
some lysosomal localization probes with morpholine have been
able to achieve near-infrared emission (650 nm ~ 800 nm) [21,47]
The limit of detection has reached the nM level (5 nM ~ 200 nM)
[27,48] and the response time is also accelerating
(5 min ~ 15 min) [22,36]. Probes with these properties have better
development potential in the field of biological detection. (Table S1)
Here, we synthesized a simple fluorescent probe, DCIMA, for
lysosome-targeted imaging of Cys and GSH in live cells. Dicyanoi-
sophorone with a long conjugated double bond was constructed to
endow DCIMA with a long-wavelength emission and large Stokes
shift. Morpholine was used to serve as both a lysosome-targeting
2.2. Synthesis
Synthesis of compound 1 and 2 was described in supporting
information (Fig. S1, S2).
Synthesis of DCIMH: Compound 2 (1 mmol., 0.207 g) and dicya-
noisophrone (1 equiv, 0.186 g) were mixed in MeCN (10 mL) in a
round bottom flask with rigorous stirring. The reaction solution
was refluxed for 12 h, and then the solvent was removed under
reduced vacuum. The residue was purified by column chromatog-
raphy (CH2Cl2) to produce a deep-red solid (DCIMH) (0.150 g,
yield: 40%). 1H NMR (400 MHz, Chloroform-d)
d 7.42 (d,
J = 8.8 Hz, 1H), 7.33 (d, J = 16.1 Hz, 1H), 6.97 (d, J = 16.1 Hz, 1H),
6.75 (s, 1H), 6.50 (dd, J = 8.8, 2.2 Hz, 1H), 6.30 (d, J = 2.1 Hz, 1H),
3.86–3.83 (t, J = 4.8 Hz, 4H), 3.25–3.20 (t, J = 4.8 Hz, 4H), 2.56 (s,
2H), 2.47 (s, 2H), 1.06 (s, 6H). 13C NMR (151 MHz, CDCl3) d
187.85, 169.43, 160.93, 156.39, 155.88, 132.70, 129.04, 126.14,
121.75, 107.96, 66.59, 47.89, 46.93, 40.72, 26.60, 24.72. HRMS
(ESI): m/z Calcd. for C23H26N3O2 ([M + H]+): 376.2025; Found
376.2029 (Fig. S3-5).
Synthesis of DCIMA. DCIMH (1 mmol, 0.375 g) and Et3N (2
equiv., 0.200 g) were mixed in dry CH2Cl2 (10 mL) in a round bot-
tom flask. Then, acryloyl chloride (2 equiv., 0.180 g) was added at
0 °C dropwise, and the reaction was proceeded at room tempera-
ture. After stirring for 6 h, the mixture was poured into ice water.
The resulting crude product was extracted with CH2Cl2
(20 mL Â 3), and solvent was removed by vacuum evaporation.
The residue was purified by column chromatography (CH2Cl2) to
produce a dark-orange solid (DCIMA) (0.197 g, yield: 46%). 1H
NMR (600 MHz, DMSO d6) d 7.83 (d, J = 8.7 Hz, 1H), 7.24 (d,
J = 16.0 Hz, 1H), 7.00 (d, J = 16.1 Hz, 1H), 6.94 (d, J = 8.2 Hz, 1H),
6.80 (s, 1H), 6.76 (s, 1H), 6.61 (d, J = 17.2 Hz, 1H), 6.55–6.47 (m,
1H), 6.22 (d, J = 10.1 Hz, 1H), 3.72 (s, 4H), 3.26 (s, 4H), 2.58 (s,
2H), 2.42 (s, 2H), 0.99 (s, 6H).13C NMR (151 MHz, CDCl3) d
169.16, 164.31, 154.32, 152.73, 150.29, 133.53, 130.09, 128.00,
127.70, 127.45, 122.86, 118.84, 113.77, 112.98, 112.83, 108.20,
66.52, 47.75, 43.01, 39.01, 31.99, 28.04. HRMS (ESI): m/z Calcd.
for
(Fig. S6-S8).
C26H27N3O3Na ([M +
Na]+): 452.1950; Found 452.1948
2.3. Spectroscopic properties test
Stock solution of DCIMA (1.0 mM) was prepared in DMSO. Bio-
molecules including Cysteine (Cys), homocysteine (Hcy), glu-
tathione (GSH), alanine (Ala), Arginine (Arg), asparagine (Asn),
glutamine (Gln), glutamic acid (Glu), glycine (Gly), histidine
(His), lysine (Lys), methionine (Met), phenylalanine (Phe), proline
(Pro), threonine (Thr), tyrosine (Tyr), valine (Val) and anions
(S2O32–, HSO3–, HS–, SO24–) were prepared in distilled water at
and electron-donating group to enable
a ‘‘push–pull” effect,
enhancing the emission wavelength of the probe by the
intramolecular charge transfer (ICT) mechanism. The response
time of DCIMA was determined to be within 5 min to Cys and
GSH with a large Stokes shift (180 nm), which is beneficial for
bioimaging applications. The probe also showed excellent lysoso-
mal localization effect and low detection limits (35.2 nM for GSH
and 34.8 nM for Cys, respectively).
10 mM. DCIMA (10
lM) were prepared by stock solution
(1.0 mM) and DMSO/PBS (1:4, v/v, pH 7.4), and then different ana-
lyte stock solutions (10 mM) were added to determine the respon-
siveness of DCIMA. The limit of detection was calculated by 3
r/k,
2. Experiment sections
Cys or GSH stock solutions were added to DCIMA (10 M) incre-
l
mentally for quantitative detection, and k was obtained by calcu-
2.1. Materials and instruments
lating the slope of linear fitting. For interference study, DCIMA
(10
tions (50
the anti-interference ability. All spectroscopic measurements were
l
M) was incubated with 15
l
L competing species stock solu-
All reagents and solvents used in this work were purchased
commercially. All UV–vis and fluorescence spectra were measured
lM) for 10 min. Then Cys (50
lM) were added to confirm
2