A lysosome-targetable fluorescent probe for the simultaneous sensing of Cys/Hcy and GSH from different emission channels

Article abstract of DOI:10.1039/C9RA00210C

A lysosome-specific fluorescent probe, Lyso-AC, for biothiols was developed by incorporation of a 4-nitrophenol moiety into a coumarin dye. The Cys/Hcy-triggered substitution-rearrangement cascade, and GSH-induced substitution reaction lead to the corresp

Full text of DOI:10.1039/C9RA00210C

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A lysosome-targetable fluorescent probe for the
simultaneous sensing of Cys/Hcy and GSH from
different emission channels†
Cite this: RSC Adv., 2019, 9, 7955
Hui Zhang,‡ab Lizhen Xu,‡a Wenxiu Li,c Wenqiang Chen,
a Qi Xiao, a Jun Huang,a
*
b
a
a
*
Chusheng Huang, Jiarong Sheng and Xiangzhi Song
A lysosome-specific fluorescent probe, Lyso-AC, for biothiols was developed by incorporation of a 4-
nitrophenol moiety into a coumarin dye. The Cys/Hcy-triggered substitution-rearrangement cascade,
and GSH-induced substitution reaction lead to the corresponding blue emissive amino-coumarin and
yellow emissive thiol-coumarin, thereby enabling Cys/Hcy and GSH detection from distinct emissions.
Received 10th January 2019
Accepted 25th February 2019
Moreover, this probe displayed an excellent lysosome targeting property with
a 0.92 Pearson's
colocalization coefficient by using Neutral Red as a reference. Significantly, biological experiments
indicated Lyso-AC has the potential to monitor lysosome Cys/Hcy and GSH simultaneously in living
HeLa cells from distinct emissions.
DOI: 10.1039/c9ra00210c
rsc.li/rsc-advances
different biothiols are rare.18,19 In fact, growing evidence
suggests Cys, Hcy and GSH levels in biological systems are
interrelated.20 To elucidate the complicated molecular mecha-
nism by which Cys, Hcy and GSH involved in physiological
processes or diseases, uorescent probes that can detect two or
all three of them from distinct signals at the same time are
urgently needed.
Introduction
Given that intracellular low-molecular-weight thiols (such as
cysteine (Cys), homocysteine (Hcy) and glutathione (GSH)), are
involved in vital pathways in human physiology,1,2 disen-
tangling the chemical biology of these species has become
a current intense interest. Biothiols play central roles in main-
taining cellular redox homeostasis or combating oxidative
stress and the abnormal levels of these species are closely
related to critical illnesses, such as AD, psoriasis, AIDS, liver
damage, cardiovascular diseases, and even cancer.3–5 As a result,
precise determinations of biothiols in biological samples are
meaningful in the clinical diagnosis of many diseases.
Fluorescence sensing is a powerful technique that has been
extensively used to detect or visualize a wide variety of bioactive
molecules by virtue of its high sensitivity and non-invasive
operation as well as excellent spatiotemporal resolution.6–8 In
recent years, a large numbers of uorescent probe for biothiols
have been developed by taking advantage of various mecha-
nisms.9–17 However, due to the intrinsic similarities exist among
these species, uorescent probes that could distinguish
Contributed by Strongin and co-workers, simultaneous
detection of Cys and Hcy was realized by virtue of the distinct
ring-formation kinetics between Cys/Hcy and acrylates.21 From
then on, some more specic probes for Cys and GSH22 or Cys/
Hcy and GSH were constructed.23–26 Recently, the differentia-
tion of Cys, Hcy and GSH was also achieved by Yin's group
through a simple molecule.27 While the above examples repre-
sent the new frontier in this eld, it is noteworthy that almost all
these probes only functioned at cellular level. Fluorescent
probes for biothiols differentiation at subcellular level are rare,
but even more valuable.
Lysosome is a membrane-bound organelle found in nearly
all animal cells, it providing various hydrolases for degradation
and recycling of biomacromolecules.28 Besides, lysosome also
play signicant roles in many cellular functions, including
plasma membrane repair, cell signaling, secretion and energy
metabolism.29 In lysosome, biothiols are mainly produced
through the proteolysis.30 It was reported that GSH is involved
in maintaining the lysosome membranes stabilization.31 Re-
ported evidences indicating Cys and GSH are capable of
promoting the intralysosomal proteolysis. For example, Cys, but
not GSH, is an effective simulator of formaldehyde-treated
albumin degradation in liver lysosomes; however, GSH should
be a more effective stimulant of proteolysis than Cys in kidney
lysosomes.32 To better understand the actual roles of each
aCollege of Chemistry and Materials Science, Guangxi Key Laboratry of Natural
Polymer Chemistry and Physics, Nanning Normal University, Nanning 530001, P. R.
China. E-mail: chenwq@csu.edu.cn; Fax: +86 771 3908065; Tel: +86 771 3908065
bCollege of Chemistry & Chemical Engineering, Central South University, Changsha,
Hunan 410083, P. R. China
cState Key Laboratory for the Chemistry and Molecular Engineering of Medicinal
Resources of Education Ministry, Guangxi Normal University, 541004 Guilin,
Guangxi, P. R. China
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c9ra00210c
‡ These authors contributed equally.
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lysosomal thiols, the development of a lysosome-targetable residue was used directly for next step. Mp: 184–187 ꢁC. 1H NMR
uorescent probe for sensing Cys/Hcy and GSH simulta- (300 MHz, DMSO-d6) d 10.18 (s, 1H), 7.52 (t, J ¼ 7.8 Hz, 1H), 7.44
neously is highly desirable. However, such a uorescent probe (dd, J ¼ 5.7, 3.6 Hz, 2H), 7.21 (m, 1H), 2.89 (s, 4H). 13C NMR (75
has not been reported so far.
MHz, DMSO-d6) d 173.24, 170.80, 162.22, 158.40, 131.22, 126.03,
Herein, we have developed a lysosome targetable uorescent 123.13, 121.08, 116.41, 26.01.
probe Lyso-AC, which can simultaneously detect Cys/Hcy and
GSH from blue and yellow emission channels with excellent Synthesis of compound 3
selectivity. Signicantly, simultaneously sensing of lysosomal
Compound 2 (529 mg, 2 mmol) and N-(3-aminopropyl) mor-
Cys/Hcy and GSH from two distinct emissions through Lyso-AC
pholine (288 mg, 2 mmol) were dissolved in 10 mL CH2Cl2. The
mixture was allowed to stir for 1.5 hours at room temperature
before 100 mL ice water was added. The resulting solution was
extracted with CH2Cl2 twice (2 ꢂ 50 mL), and the organic layer
was separated and dried over MgSO4. Aer removal the solvent
in vacuum, the resultant residue was further puried by silica
gel chromatography to afford the desired product as a white
solid (423 mg, 80%). Mp: 130–133 ꢁC. 1H NMR (300 MHz,
CDCl3) d 8.51 (s, 1H), 8.11 (s, 1H), 7.46 (s, 1H), 7.20 (s, 2H), 6.98
(d, J ¼ 3.4 Hz, 1H), 3.69 (s, 4H), 3.49 (d, J ¼ 5.3 Hz, 2H), 1.80–
1.76 (m, 2H). 13C NMR (75 MHz, CDCl3) d 168.19, 157.44, 135.62,
129.65, 119.11, 117.76, 114.74, 66.47, 57.90, 53.53, 40.05, 24.17.
was also realized.
Experimental section
Materials and methods
All reagents used in this work were in AR grade, which was
obtained from Aladdin and J&K without additional purication.
All solvents were used in HPLC grade. The NMR spectra were
tested on a BRUKER 300 MHz NMR instrument (USA) with
0.03% TMS as an internal standard. The HRMS spectra were
measured on a Waters Xevo G2 QTof MS (USA). A Shimadzu UV
2450 spectrophotometer was used for absorption spectra
measuring. Fluorescence spectra were acquired on a HITACHI
F-4600 spectrouorometer. Cell images were obtained on
a Zeiss LSM 710 confocal microscope (Germany).
Synthesis of compound 5
Compound 3 (26 mg, 0.1 mmol) was combined with Et3N (420
mL, 3 mmol) in 3 mL dry CH2Cl2 and the mixture was chill to
0
ꢁC through an ice bath. Then a portion of compound 4
General procedure for spectral measurements
(0.1 mmol, dissolved in 2 mL dry CH2Cl2) was added dropwise
to the above solution. The resulting reaction mixture was
allowed to stir in the ice bath for additional 2 hours. The solvent
was removed under reduced pressure and the resultant residue
was further puried by silica gel chromatography to give
compound 5 as a yellow powder (27 mg, 50%). Mp: 122–125 ꢁC.
1H NMR (300 MHz, CDCl3) d 8.06 (s, 1H), 7.75 (m, 1H), 7.70 (d, J
¼ 9.1 Hz, 2H), 7.45–7.39 (m, 2H), 6.68 (dd, J ¼ 9.3, 2.4 Hz, 1H),
6.47 (d, J ¼ 2.4 Hz, 1H), 3.76–3.66 (m, 4H), 3.57 (d, J ¼ 11.4,
5.7 Hz, 2H), 3.46 (q, J ¼ 7.1 Hz, 4H), 2.56 (dd, J ¼ 12.3, 6.0 Hz,
6H), 1.87–1.75 (m, 2H), 1.24 (t, J ¼ 7.1 Hz, 6H).$13C NMR (75
MHz, CDCl3) d 166.36, 161.91, 157.65, 155.44, 152.71, 150.62,
149.36, 136.54, 129.69, 127.90, 124.98, 124.67, 120.31, 111.75,
109.97, 106.17, 96.73, 66.78, 58.34, 53.76, 45.10, 40.35, 24.22,
12.40. HRMS (ESI) m/z: calculated for C28H33ClN3O6 [M + H]+:
542.2052; found 542.2052.
Lyso-AC was prepared at 10ꢀ3 M in DMSO as a stock solution.
Aqueous solutions of cysteine (Cys), homocysteine (Hcy),
glutathione (GSH), isoleucine (Ile), valine (Val), alanine (Ala),
phenylalanine (Phe), serine (Ser), glutamine (Gln), histidine
(His), threonine (Thr), lysine (Lys), proline (Pro), arginine (Arg),
KCl, CaCl2, MgCl2, ZnCl2, NaCl, Na2S, H2O2 were newly
prepared in ultrapure water. Leucine (Leu), tyrosine (Tyr),
aspartic acid (Asp), glutamate (Glu) were freshly prepared in
PBS buffer (10 mM, pH ¼ 7.4). Typically, a test solution (2.0 mL)
was prepared in a quartz cuvette by adding 0.02 mL of Lyso-AC
(1 mM), 0.38 mL of DMSO, and an appropriate volume of ana-
lyte into apposite amount of PBS buffer. The solution was well
mixed and then incubated at 25 ꢁC for 60 min before measuring
its optical spectra.
Cell culture and uorescence imaging procedure
HeLa cells were obtained from the rst affiliated hospital of
guangxi medical university and cultured in DMEM medium
(GIBCO) supplemented with 10% s and 100 mg mLꢀ1 peni-
cillin. The cells were seeded in 24-well plates and allowed to
adhere for 24 h at 37 ꢁC in a humidied atmosphere containing
5% CO2. The cytotoxic effect of Lyso-AC was evaluated using
classical MTT assay. lex ¼ 405 nm. Blue channel: at 450–490 nm;
yellow channel: emissions were collected at 540–580 nm.
Synthesis of Lyso-AC
To a solution of compound 5 (54 mg, 0.1 mmol) and p-nitro-
phenol (139 mg, 1 mmol) in 5 mL dry CH3CN was added Et3N
(140 mL, 1 mmol). The mixture was warmed to reux for 2 hours.
Aer removal of the solvent in vacuo, the resultant residue was
further puried by silica gel chromatography to give Lyso-AC as
an orange powder (55 mg, 85%). Mp: 163–166 ꢁC. 1H NMR (300
MHz, CDCl3) d 8.25 (d, J ¼ 9.2 Hz, 2H), 7.91 (s, 1H), 7.63 (d, J ¼
7.8 Hz, 1H), 7.42 (s, 1H), 7.35 (dd, J ¼ 7.8, 3.6 Hz, 2H), 7.19 (d, J
¼ 9.2 Hz, 2H), 7.04 (dd, J ¼ 8.1, 1.5 Hz, 1H), 6.61–6.52 (m, 2H),
Synthesis of compound 2
3-Hydroxybenzoic acid (138 mg, 1 mmol), N-hydroxy succini- 3.68 (d, J ¼ 6.0 Hz, 4H), 3.53 (dd, J ¼ 11.4, 6.0 Hz, 2H), 3.46 (q, J
mide (115 mg, 1 mmol), EDCI (192 mg, 1 mmol) and DMAP (5 ¼ 7.2 Hz, 4H), 2.56 (dd, J ¼ 12.3, 6.0 Hz, 6H), 1.83–1.73 (m, 2H),
mg) were dissolved in anhydrous CH3CN (6 mL). The mixture 1.24 (t, J ¼ 7.1 Hz, 6H). 13C NMR (75 MHz, CDCl3) d 166.13,
was stirred at 25 ꢁC for 2 hours. Aer removal of the solvent, the 164.11, 161.43, 160.95, 159.39, 157.24, 153.18, 150.40, 143.54,
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136.45, 129.49, 126.20, 124.53, 124.24, 120.24, 116.60, 109.91,
102.76, 102.45, 97.14, 66.84, 58.50, 53.81, 45.18, 40.46, 24.20,
12.39. HRMS (ESI) m/z: calculated for C34H36N4NaO9 [M + Na]+:
667.2374; found 667.2375.
Results and discussion
Design and synthesize
Motivated by the above-mentioned concerns, we invested
effort into exploiting a lysosome-targetable uorescent probe
that could simultaneously sense Cys/Hcy and GSH from
distinct emissions. As shown in Scheme 1, the probe, Lyso-AC,
was designed based on the following concerns: (1) 7-dieth-
ylaminocoumarin was selected as the uorophore due to its
outstanding optical properties.33 Moreover, the photophysical
properties of 7-diethylaminocoumarin is adjustable, which
can be easily tuned through proper structure modication. For
instance, in 2013 Guo's group reported several amino- and
thiol-modied diethylaminocoumarin dyes, and the absorp-
tion of 4-amino-7-diethylaminocoumarin peaks at about
360 nm whereas the 4-thio-7-diethylaminocoumarin absorbs
at about 385 nm.19 We speculated that coupling the remark-
able photophysical differences of amino- and thio-
diethylaminocoumarin with the unique Cys/Hcy-triggered
substitution-rearrangement cascade would serve as an effec-
tive approach to sense Cys/Hcy and GSH simultaneously. (2) 4-
Nitrophenol moiety was chosen as the biothiols recognition
group. First, it can function as an effective leaving group.
Second, it also can act as a quencher to minimize the back-
ground uorescence signal of the probe via the efficient d-PET
process. Furthermore, the 4-nitrophenol moiety should be
linked to the 4 position of the coumarin, so that it can be
activated by two nearby carbonyls.34 (3) A morpholine moiety
was employed as the lysosome targeting group due to the
effective lysosome-targetable feature of this moiety.35–42
Scheme 2 Synthetic route for Lyso-AC and the chemical structures of
reference dyes A1 and A2.
In the case of GSH, Lyso-AC-S-GSH is expected to generate.
However the following intermolecular Smiles rearrangement of
Lyso-AC-S-GSH is difficult to undergo due to the fact that an
unstable transition state would be involved in during this
process. Based on the distinct emission wavelengths of the 4-
amino- and 4-thio-diethylaminocoumarin, simultaneous
detection of lysosomal Cys/Hcy and GSH from distinct emission
signals would likely to be realized.
As a proof of concept, Lyso-AC was synthesized through the
corresponding synthetic route in Scheme 2 (details were
shown in the Experimental section). The structure of Lyso-AC
was fully identied by HRMS spectrum and NMR spectra (see
in ESI†).
UV-vis spectra studies
First, the reactivity of Lyso-AC toward Cys/Hcy/GSH was exam-
ined through time-dependent UV-vis spectra in PBS buffer
(10 mM, pH 7.4, containing 20% DMSO). Free Lyso-AC exhibited
a major absorption band centred at 430 nm (3 ¼ 4.34 ꢂ 104 L
molꢀ1 cmꢀ1). As expected, upon treatment of 10 equivalents of
Cys to Lyso-AC, the initial peak at 430 nm decreased gradually,
along with simultaneous occurrence of a new absorption peak
at 383 nm (Fig. 1A and B). The absorption peak at 383 nm
belongs to Lyso-AC-N-Cys, which was further demonstrated by
the reference dye A1, whose absorption peak was determined to
be around 383 nm (Fig. S1†). Similar results can be observed
when addition of 10 equivalents of Hcy to Lyso-AC (Fig. 1C, D
and Fig. S2†). By contrast, upon treatment 10 equivalents of
GSH to Lyso-AC, the absorption peak at 430 nm decreased and
slightly blue-shied to 426 nm. Unlike the case of Cys or Hcy, no
absorption peak at 383 nm in this case can be seen, indicating
the failure of the subsequent intramolecular rearrangement in
this case (Fig. 1E and F). The new slightly blue-shied peak at
423 nm belongs to Lyso-AC-S-GSH, which was further supported
by the reference dye A2 (Fig. S3†). In addition, all adducts (Lyso-
AC-N-Cys, Lyso-AC-N-Hcy and Lyso-AC-S-GSH) were detectable
in the corresponding HRMS data (Fig. S4–S6†). All to all, the
above traits in good agreement with the mechanism proposed
in Scheme 2. Moreover, the photostability of Lyso-AC has been
studied. As show in Fig. S7,† it appears a favourable photo-
stability of Lyso-AC.
Finally, by considering all above issues, Lyso-AC was
designed by integrating a 4-nitrophenol moiety and a morpho-
line moiety into a diethylaminocoumarin dye (Scheme 1). We
speculated that the addition of Cys (or Hcy) to Lyso-AC would
initially result the corresponding SNAr substituted product Lyso-
AC-S-Cys (or Lyso-AC-S-Hcy) and the subsequent Smiles rear-
rangement would ultimately generate the Lyso-AC-N-Cys (or
Lyso-AC-N-Hcy) via a ve (or six)-membered cyclic intermediate.
Scheme 1 Proposed sensing mechanism of Lyso-AC for Cys/Hcy and
GSH.
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Fig. 2 Time-dependent fluorescence response of Lyso-AC (10 mM)
toward 10 equiv. of Cys, Hcy and GSH excited at 383 nm (A1–A3).
Time-dependent fluorescence intensity changes toward 10 equiv. of
Cys, Hcy and GSH excited at 425 nm (B1–B3). Condition: slits, 5/5 nm;
PBS buffer (10 mM, pH 7.4, containing 20% DMSO) at 25 ꢁC.
Fig. 1 Time-dependent absorption spectra of Lyso-AC (10 mM) in the
presence of 10 equiv. of Cys (A), Hcy (C) or GSH (E) in PBS buffer (pH
7.4, 10 mM, containing 20% DMSO, v/v) at room temperature. Time-
dependent absorption intensity changes toward 10 equiv. of Cys (B),
Hcy (D) or GSH (F) at 425 nm and 383 nm in PBS buffer (pH 7.4, 10 mM,
containing 20% DMSO, v/v) at room temperature.
introduction of 10 equivalents of Cys or Hcy did not elicit
remarkable uorescence at 550 nm (Fig. 2B1 and B2). The cor-
responding uorescence increments at 550 nm for GSH, Cys
and Hcy were 29.7-, 2.7- and 2.4-fold respectively. These results
indicated that Lyso-AC was capable of differentiate GSH from
Cys/Hcy when excited at 425 nm. Notably, the reaction between
Lyso-AC and GSH also obeys a classic pseudo-rst-order and the
corresponding Kobs was determined to be 0.0506 minꢀ1
(Fig. S10†).
To evaluate the sensitivity of Lyso-AC, uorescence titration
experiments were conducted. Upon addition of increasing
amounts of Cys (or Hcy), the uorescence intensity of Lyso-AC at
485 nm increased gradually. Moreover, as shown in Fig. S11 and
S12,† a good linearity between uorescence intensity at 485 nm
and concentrations of Cys (0–60 mM) or Hcy (0–45 mM) was
observed. The detection limits for Cys and Hcy were calculated
to be 0.86 mM and 1.52 mM, respectively. As for GSH, the uo-
rescence at 550 nm increases with the incremental GSH added
and a good linearity between F550 and concentrations of GSH in
the range of 0–40 mM was observed (Fig. S13†). And the corre-
sponding detection limit was determined to be 1.75 mM.
Fluorescence spectra studies
The emission behaviours of Lyso-AC upon addition of Cys, Hcy
and GSH were further evaluated. Initially, time-dependent
uorescence spectra of Lyso-AC with respective biothiols were
studied. The corresponding uorescence spectra were rst
measured with an excitation wavelength of 383 nm. Free Lyso-
AC was essentially non-uorescence due to the efficient d-PET
process. However, upon treatment of 10 equivalents of Cys to
Lyso-AC, a remarkable uorescence enhancement (19.5-fold) at
485 nm can be observed due to the production of Lyso-AC-N-Cys
(Fig. 2A1). A similar observation was made in the case of Hcy
(about 16.5-fold enhancement at 485 nm) (Fig. 2A2). However,
in comparison with Cys/Hcy, GSH elicited only a slight uo-
rescence enhancement (3.6-fold) at 485 nm (Fig. 2A3). These
results demonstrated that Lyso-AC can be used to discriminate
Cys/Hcy from GSH when excited at 383 nm. Moreover, the
kinetic studies revealed that Lyso-AC respond to Cys/Hcy obey
classic pseudo-rst-order reaction, and the corresponding rate
constants kobs determined to be about 0.0727 minꢀ1 for Cys
(Fig. S8†) and 0.0931 minꢀ1 for Hcy (Fig. S9†).
Selectivity studies and pH effect
To examine the selectivity of Lyso-AC toward Cys/Hcy and GSH,
the uorescence behaviour of Lyso-AC in response to various
biologically related species, including some essential amino
acids (Tyr, Val, Gly, Ala, Asp, Arg, Iso, Lys, Met, His, Phe, Thr,
Next, the uorescence response of Lyso-AC toward Cys/Hcy/
GSH was further investigated by using excitation at 425 nm.
Similarly, Lyso-AC displayed weak uorescence under this
excitation wavelength. Upon introduction of 10 equivalents of
GSH to Lyso-AC, uorescence at 550 nm increased rapidly and
then got saturation within 60 min (Fig. 2B3). On the other hand,
Ser, Pro and Glu, 0.5 mM for each), biological metal ions (Ca2+
,
Na+, K+, Zn2+ and, Mg2+ 0.5 mM for each), hydrogen sulphide
(H2S, 0.5 mM) as well as reactive oxygen species (H2O2, 0.1 mM)
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Fig. 3 Fluorescence intensity of Lyso-AC (10 mM) at 485 nm (A) and
550 nm (B) upon addition of various species in PBS buffer (pH 7.4,
10 mM, containing 20% DMSO) at 25 ꢁC. Each spectrum recorded at
60 min after addition of the corresponding species.
were investigated, The uorescence intensities were then
measured at 485 nm and 550 nm, respectively. As illustrated in
Fig. 3A, only Cys/Hcy induced a signicant uorescence
enhancement at 485 nm whereas other analytes, including the
GSH and H2S, gave marginal increments. On the other hand,
with the excitation of 425 nm, all these interfering species
caused insignicant uorescence variations, and only GSH
caused noticeable uorescence enhancement at 550 nm
(Fig. 3B). These results suggesting Lyso-AC is highly selective for
Cys/Hcy or GSH. In addition, the effect of pH values were also
examined, and the results indicating Lyso-AC was quite stable
over a wide pH range (1–10), and displayed noticeable uores-
cence enhancements upon treatment of Cys, Hcy or GSH in the
region of 4–10 (Fig. S14†). Therefore, Lyso-AC is suitable to be
used at physiological pH range.
Fig. 4 Confocal microscopic images of biothiols in living HeLa cells.
(A1–A4) HeLa cells pretreated with incubated with 2 mM Lyso-AC for
30 min; HeLa cells pretreated with 1 mM NEM for 15 min then 1 mM
Cys (B1–B4), Hcy (C1–C4), or GSH (D1–D4) for 15 min, finally incu-
bated with 2 mM Lyso-AC for additional 30 min; (E1–E4) HeLa cells
pretreated with 1 mM NEM for 15 min and then incubated with 2 mM
Lyso-AC for 30 min. Excitation wavelength; 405 nm. Emissions were
collected at 450–490 nm for blue channel and 540–580 nm for
yellow channel. Scale bar: 10 mm.
Cell imaging
On the basis of the above excellent sensing traits of Lyso-AC, we
sought to further investigate the capability of Lyso-AC to selec-
tively detect intracellular Cys/Hcy and GSH at the same time.
When HeLa cells were treated with 2 mM Lyso-AC, a strong
uorescence in yellow channel was observed, implying the
reaction of intracellular GSH with the probe (Fig. 4A2). When
HeLa cells were successive treated with 1 mM NEM and 1 mM
Cys (or Hcy), then further treated with 2 mM Lyso-AC, a strong
uorescence in blue channel (Fig. 4B1 or Fig. 4C1) and an
insignicant uorescence in yellow channel (Fig. 4B2 or
Fig. 4C2) were taking place. When HeLa cells were successive
treated with 1 mM NEM and 1 mM GSH, then further treated
with 2 mM Lyso-AC, an obvious uorescence in yellow channel
(Fig. 4D2) and negligible emission in blue channel (Fig. 4D1)
were observed. For comparison, HeLa cells were successively
introduced with 1 mM NEM and 2 mM Lyso-AC. As a result,
negligible emissions can be observed both in blue channel and
yellow channel (Fig. 4E1–E4). Moreover, to verify Lyso-AC is
lysosome targetable, colocalization experiments were per-
formed by using Lyso-AC and a commercial lysosome sensor
Neutral Red (NR) (Fig. 5). As shown in Fig. 5C, the ne merged
images strongly convinced that Lyso-AC ts well with lysosomes
inside the cells with a Pearson's coefficient of 0.92. These
experiments demonstrated that Lyso-AC can specically localize
Fig. 5 The images of living HeLa cells co-stained with Lyso-AC (2 mM)
and Lyso-Tracker NR (2 mM) for 30 min (a–c). (a) Blue channel (lex
¼
405 nm, lem ¼ 450–490 nm); (b) red channel image (lex ¼ 559 nm, lem
¼ 586–610 nm); (c) bright field; (d) merged images of (a–c); (e)
intensity profile of regions of interest across HeLa cells; (f) Intensity
correlation plot of Lyso-AC and NR. Scale bar: 10 mm.
minimal cytotoxicity, which was supported by corresponding
MTT assay (Fig. S15†).
Conclusions
in lysosomes and be used to detect Cys/Hcy and GSH in lyso- In conclusion, we have developed a novel lysosome targetable
somes of living HeLa cells. In addition, Lyso-AC exhibited uorescent probe, Lyso-AC, which can selectively detect
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lysosomal Cys/Hcy and GSH from distinct emission signals. 18 C. X. Yin, K. M. Xiong, F. J. Huo, J. C. Salamanca and
Preliminary cell imaging studies have revealed that Lyso-AC was
R. M. Strongin, Angew. Chem., Int. Ed., 2017, 56, 13188–
capable of sensing Cys/Hcy and GSH in living HeLa cells at the
13198.
same time. We hope this probe can become a robust tool for 19 J. Liu, Y. Q. Sun, Y. Huo, H. Zhang, L. Wang, P. Zhang,
lysosomal biothiols investigation.
D. Song, Y. Shi and W. Guo, J. Am. Chem. Soc., 2014, 136,
574–577.
20 H. Kimura, Amino Acids, 2011, 41, 113–121.
21 O. Rusin, N. N. S. Luce, R. A. Agbaria, J. O. Escobedo, S. Jiang,
I. M. Warner, F. B. Dawan, K. Lian and R. M. Strongin, J. Am.
Chem. Soc., 2004, 126, 438–439.
Conflicts of interest
There are no conicts to declare.
22 J. Liu, Y. Q. Sun, Y. Huo, H. Zhang, L. Wang, P. Zhang,
D. Song, Y. Shi and W. Guo, J. Am. Chem. Soc., 2014, 136,
574–577.
23 X. F. Yang, Q. Huang, Y. G. Zhong, Z. Li, H. Li, M. Lowry,
J. O. Escobedo and R. M. Strongin, Chem. Sci., 2014, 5,
2177–2183.
24 J. Liu, Y. Q. Sun, H. X. Zhang, Y. Y. Huo, Y. W. Shi and
W. Guo, Chem. Sci., 2014, 5, 3183–3188.
25 F. Y. Wang, L. Zhou, C. C. Zhao, R. Wang, Q. Fei, S. H. Luo,
Z. Q. Guo, H. Tian and W. H. Zhu, Chem. Sci., 2015, 6, 2584–
2589.
26 W. Q. Chen, H. C. Luo, X. J. Liu, W. F. James and X. Z. Song,
Anal. Chem., 2016, 88, 3638–3646.
27 G. X. Yin, T. T. Niu, Y. B. Gan, T. Yu, P. Yin, H. M. Chen,
Y. Y. Zhang, H. T. Li and S. Z. Yao, Angew. Chem., Int. Ed.,
2018, 57, 4991–4994.
Acknowledgements
This work was supported by the Guangxi Natural Science
Foundation
(2016GXNSFBA380229,
2017GXNSFA198342),
Guangxi Scientic and Technological Development Projects
(AD17195081), “BAGUI Scholar” program of Guangxi Province
of China, and the Fundamental Research Funds for the Central
Universities of Central South University (2018zzts109).
Notes and references
1 N. Brandes, S. Schmitt and U. Jakob, Antioxid. Redox
Signaling, 2009, 11, 997–1014.
2 D. M. Townsend, K. D. Tew and H. Tapiero, Biomed.
Pharmacother., 2003, 57, 145–155.
3 M. T. Heaeld, S. Fearn, G. B. Steventon, R. H. Waring, 28 K. Surendran, S. P. Vitiello and D. A. Pearce, Pediatr.
A. C. Williams and S. G. Sturman, Neurosci. Lett., 1990,
110, 216–220.
4 P. Sachdev, J. Neurol. Sci., 2004, 226, 81–87.
Nephrol., 2014, 29, 2253–2261.
29 S. Carmine, F. Alessandro, D. L. Medina and B. Andrea, Nat.
Rev. Mol. Cell Biol., 2013, 14, 283–296.
5 Z. A. Wood, E. Schroder, J. H. Robin and L. B. Poole, Trends 30 D. R. Balce, E. R. O. Allan, N. McKenna and R. M. Yates, J.
Biochem. Sci., 2003, 28, 32–40. Biol. Chem., 2014, 289, 31891–31904.
6 L. Yuan, W. Lin, K. Zheng, L. He and W. Huang, Chem. Soc. 31 Q. K. Gao, W. Z. Zhang, B. Song, R. Zhang, W. H. Guo and
Rev., 2013, 42, 622–661.
J. L. Yuan, Anal. Chem., 2017, 89, 4517–4524.
7 M. Fernandez-Suarez and A. Y. Ting, Nat. Rev. Mol. Cell Biol., 32 J. L. Mego, J. Biol. Chem., 1984, 218, 775–783.
2008, 9, 929–943.
8 J. Chan, S. C. Dodani and C. J. Chang, Nat. Chem., 2012, 4,
973–977.
9 X. Chen, Y. Zhou, X. Peng and J. Yoon, Chem. Soc. Rev., 2010,
39, 2120–2135.
10 Y. Zhou and J. Yoon, Chem. Soc. Rev., 2011, 41, 52–67.
33 S. Sanghi, D. Mohan and R. D. Singh, Asian J. Phys., 1995, 4,
283–288.
34 Y. Kim, S. V. Mulay, M. Choi, S. B. Yu, S. Jon and
D. G. Churchill, Chem. Sci., 2015, 6, 5435–5439.
35 M. G. Ren, Z. H. Li, J. Nie, L. Wang and W. Y. Lin, Chem.
Commun., 2018, 54, 9238–9241.
11 L. Y. Niu, Y. Z. Chen, H. R. Zheng, L. Z. Wu, C. H. T and 36 Y. Liu, F. F. Meng, L. W. He, K. Y. Liu and W. Y. Lin, Chem.
Q. Z. Yang, Chem. Soc. Rev., 2015, 44, 6143–6160.
Commun., 2016, 52, 7016–7019.
12 Y. M. Yang, Q. Zhao, W. Feng and F. Y. Li, Chem. Rev., 2013, 37 B. L Dong, X. Z. Song, C. Wang, X. Q. Kong, Y. H. Tang and
113, 192–270.
W. Y. Lin, Anal. Chem., 2016, 88, 4085–4091.
13 Y. Li, W. M. Liu, P. P. Zhang, H. Y. Zhang, J. S. Wu, J. C. Ge 38 M. G. Ren, B. B. Deng, J. Y. Wang, X. Q. Kong, Z. R. Liu,
and P. F. Wang, Biosens. Bioelectron., 2017, 90, 117–124.
14 X. F. Yang, W. Y. Liu, J. Tang, P. Li, H. B. Weng, Y. Ye,
K. Zhou, L. W. He and W. Y. Lin, Biosens. Bioelectron.,
2016, 79, 237–243.
M. Xian, B. Tang and Y. F. Zhao, Chem. Commun., 2018, 39 H. B. Yu, Y. Xiao and L. J. Jin, J. Am. Chem. Soc., 2012, 134,
54, 11387–11390.
17486–17489.
15 D. Qiao, T. L. Shen, M. Y. Zhu, X. Liang, L. Zhang, Z. Yin, 40 X. T. Jing, F. B. Yu and L. X. Chen, Chem. Commun., 2014, 50,
B. H. Wang and L. Q. Shang, Chem. Commun., 2018, 54,
13252–13255.
16 C. Y. Zhang, S. Wu, Z. Xi and L. Yi, Tetrahedron, 2017, 73,
6651–6656.
17 L. Sun, Y. Q. Jiang, C. Y. Zhang, X. R. Ji, D. Q. Lv, Z. Xi and
L. Yi, New J. Chem., 2018, 42, 15277–15283.
14253–14256.
41 T. Y. Liu, Z. C. Xu, D. R. Spring and J. N. Cui, Org. Lett., 2013,
15, 2310–2313.
42 M. G. Ren, B. B. Deng, J. Y. Wang, X. Q. Kong, Z. R. Liu,
K. Zhou, L. W. He and W. Y. Lin, Biosens. Bioelectron.,
2016, 79, 237–243.
7960 | RSC Adv., 2019, 9, 7955–7960
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