ESIPT triggered swift determination of cysteine in HeLa cell line during redox imbalance

Article abstract of DOI:10.1016/j.jphotochem.2020.112875

Detection of cysteine is crucial as its deviation from normal concentration leads to various diseases. In this work, benzothiazole based chemodosimeter was synthesized to discriminate cysteine rapidly in the presence of other competing interferences. The

Full text of DOI:10.1016/j.jphotochem.2020.112875

Journal of Photochemistry & Photobiology, A: Chemistry 403 (2020) 112875
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Journal of Photochemistry & Photobiology, A: Chemistry
journal homepage: www.elsevier.com/locate/jphotochem
ESIPT triggered swift determination of cysteine in HeLa cell line during
redox imbalance
a
b
a,
Kathiresan Anusuyadevi , Shu Pao Wu , Sivan Velmathi *
a
Organic and Polymer Synthesis Laboratory, Department of Chemistry, National Institute of Technology, Tiruchirappalli, 620015, India
b
Bioinorganic Lab, Department of Applied Chemistry, National Chiao Tung University, 300, Taiwan
A R T I C L E I N F O
A B S T R A C T
Keywords:
Detection of cysteine is crucial as its deviation from normal concentration leads to various diseases. In this work,
benzothiazole based chemodosimeter was synthesized to discriminate cysteine rapidly in the presence of other
competing interferences. The probe exhibit ESIPT governed colorimetric and fluoremetric response on addition
of cysteine via conjugate addition-cyclization reaction and it is confirmed by NMR and HRMS. The probe can
ESIPT
Swift response
Biothiols
Discrimination
Redox imbalance
detect lower concentration of cysteine with limit of detection 0.07 M in 2 min that assist the system to work well
μ
in biological systems. Hence the sensor was profitably employed in imaging of cysteine in HeLa cell line and
variance in the redox state was also investigated.
1
. Introduction
detection of cysteine via ESIPT [17]. Even though various reports are
available for detection of cysteine, they are associated with certain
Cysteine plays an incredible role in biological systems due to its
versatile contributions in bioactivities [1,2]. The aberrant increase of
cysteine from its optimum concentration (30–200 M) leads to oxidative
stress which is associated with liver injury, Parkinson disease & Arthritis
whereas its deficiency causes retarded growth and skin lesions [3–5].
Thus it’s of high demand to develop sensors to monitor the level of
cysteine in biological systems.
limitations such as poor selectivity towards biothiols discrimination
[18–25], Long detection time [26–36], weak emission [37], small Stokes
shift (less than 100 nm), high cytotoxicity, less cellular uptake which
leads to lack of in vivo imaging [15,16]. Thus it’s imperative to fabricate
a sensor which vanquish the inadequacy of available reports. Few re-
ports from literatures are presented in Table S2 depicts the molecular
probes which discriminates cysteine selectively but with prolonged re-
action time. For example, Huang et al. and Dai et al. have developed
chromone based fluorophore for the selective detection of cysteine
which requires reaction time of about 50 and 40 min respectively [26,
33]. Chen et al. and Dong et al. designed a probe which detects cysteine
based on conjugate addition cyclization within 80 and 20 min respec-
tively [28,30]. Liu et al. prepared coumarin derivative to detect cysteine
via nucleophilic aromatic substitution which takes 60 min of response
time [27]. Hence it is obligatory to furnish faster detection of cysteine to
achieve impressive biological applications.
μ
High cost, complex operational techniques and limitation in per-
forming in vivo studies makes traditional methods (HPLC & mass
spectrometry) unsuitable for the detection of cysteine [6–8]. Relatively,
fluorimetric detection is more advantageous as it affords outstanding
selectivity, sensitivity and in vivo imaging [9,10]. Hence, various fluo-
rescent probes were developed to detect cysteine chiefly through certain
reactions such as Michael addition, Cyclization with aldehyde, Conju-
gate addition-cyclization reaction, Nucleophilic aromatic substitution
and rearrangement [11–14] etc. ICT, PET and ESIPT are commonly
encountered detection mechanism behind these reactions based probes.
Among those, ESIPT probes are considered to be more beneficial because
of its striking qualities to furnish large Stokes shift which hinders
auto-fluorescence in imaging and can render extremely fast reaction rate
In this work an ESIPT active 1-(benzo[d]thiazol-2-yl)naphthalen-2-
ol (NTZ) fluorophore was functionalized with acrylate moiety (NTZ-AC)
as a reaction site for cysteine detection. This functionalized probe
demonstrates admirable sensing properties such as i) highly selective
detection of cysteine at pH 7.4 over other amino acids includes homo-
cysteine and glutathione. ii) Swift determination of cysteine in 2 min
with bright cyan emission and Stokes shift more than 100 nm. iii)
[
[
15]. Huace sheng et al. have reported an ESIPT probe based on imidazo
1,5
α
] pyridine sensor platform for rapid cysteine identification [16]. Yu
′
et al. reported (4 -dimethylamino-3-hydroxyflavone) for selective
*
Corresponding author.
E-mail address: velmathis@nitt.edu (S. Velmathi).
https://doi.org/10.1016/j.jphotochem.2020.112875
Received 8 May 2020; Received in revised form 16 August 2020; Accepted 26 August 2020
Available online 28 August 2020
1
010-6030/© 2020 Elsevier B.V. All rights reserved.

K. Anusuyadevi et al.
Journal of Photochemistry & Photobiology, A: Chemistry 403 (2020) 112875
Excellent imaging in HeLa cells and redox disparity inside cells were
investigated efficiently.
reacted with acryloyl chloride to produce NTZ-AC which was charac-
terized by NMR and Mass spectrometry.
Electronic spectra of NTZ-AC (50
0 mM PBS buffer) shows a small hump around 400 nm whereas an
increase in absorption was observed due to the visible colour change to
pale yellow on addition of cysteine (100 M) which accounts for the
formation of NTZ [38] (Fig. 1). NTZ-AC (50 M, DMSO-PBS 1:1 v/v,
0 mM PBS buffer at pH 7.4,) was non-emissive upon excitation at
70 nm due to the presence of acrylate moiety which impedes ESIPT.
μ
M, DMSO-PBS 1:1 v/v at pH 7.4,
1
1
.1. Synthesis of NTZ
μ
NTZ was synthesized by a modified procedure of reported literature
μ
[
38,39].The ethanolic solution of 2-Hydroxy naphthaldehyde (3 mmol,
.516 g) and aminothiophenol (3 mmol, 313 L) was refluxed for 8 h
with catalytic amount of conc. H SO (Scheme 1). The reaction
1
3
0
μ
2
4
But there was an instant increase in fluorescence (cyan emission) was
observed on cysteine addition (100 M) with maximum emission at
completion was monitored by TLC. Then the crude obtained after sol-
μ
vent evaporation was purified by column chromatography employing
DCM as an eluent. The obtained yield was 62 % (0.515 g). 1H NMR
478 nm that is owing to the cleavage of acrylate moiety thereby ESIPT
was restored [38] (Fig. 2). In order to understand the reactivity of probe
further in detail, the response of NTZ-AC towards different concentra-
tion of cysteine was verified with addition of increasing concentration of
cysteine. Initially the probe was non emissive due to detained ESIPT
whereas on addition of increasing concentration of cysteine, the peak at
(
DMSO-d
6
) δ 11.04 (s, 1 H), 8.24 (d, J =8.6 Hz, 1 H), 8.15 (dd, J = 14.6,
8
.0 Hz, 2 H), 7.99 (d, J =8.9 Hz, 1 H), 7.90 (d, J =8.0 Hz, 1 H), 7.58 (t, J
1
3
=
8.2 Hz, 1 H), 7.53 – 7.46 (m, 2 H), 7.36 (dd, J = 19.8, 8.0 Hz, 2 H).
C
NMR (DMSO-d
6
) δ 164.19, 155.26,152.69, 135.84, 132.88, 132.81,
1
1
28.83, 128.31, 128.12, 126.55, 125.66, 124.38, 123.87, 123.12,
4
78 nm slowly increases which denotes the progress of conjugate
addition-cyclization reaction [31,32,16]. Saturation was accomplished
at 60 M addition of cysteine which implies the completion of the re-
22.29, 118.72, 112.54. The spectra are displayed in Figure S1 and S2.
μ
1
.2. Synthesis of NTZ-AC
action to produce NTZ (ESIPT regenerated) (Fig. 3). The LOD and LOQ
was calculated from the linear plot between concentration and intensity
NTZ (1 mmol, 0.277 g) was taken in dry DCM which was followed by
at 478 nm as 0.07
μ
M and 0.2 M respectively which is well below the
μ
consecutive addition of acryloyl chloride (5 mmol, 404
μ
L) and trie-
normal level of cysteine in biological systems (inset of Fig. 4) Thus it is
the superior probe to monitor cysteine even at lower concentration
under physiological condition.
thylamine (5 mmol, 696 L) under nitrogen atmosphere was stirred at
μ
room temperature for three hours. The solvent was evaporated and the
crude was extracted with DCM. The organic phase was concentrated
under reduced pressure after dried with anhydrous magnesium sulfate.
The resultant was purified by column chromatography using DCM to
achieve desired product NTZ-AC (0.197 g, 60 % yield) Scheme 1.
2.2. Detection time
1
H NMR (Chloroform-d) δ 8.19 (dd, J = 8.2, 1.1, 0.6 Hz, 1 H), 8.03
Reaction time is one of the crucial parameters while developing a
sensor. From table S1, it is understood that the probe requires minimum
of 10 min and maximum of one hour for the detection of cysteine by
conjugate addition cyclization reaction. Thus developing a sensor which
detects cysteine with short reaction time is highly appreciable. Firstly,
(
d, J =8.9 Hz, 1 H), 7.99 – 7.94 (m, 2 H), 7.92 (dd, J = 6.9, 2.5 Hz, 1 H),
7
6
.59 – 7.44 (m, 4 H), 7.40 (d, J =8.9 Hz, 1 H), 6.48 (d, J =18.5 Hz, 1 H),
13
.19 (dd, J = 17.3, 10.5 Hz 1 H), 5.91 (d, J =11.7 Hz, 1 H). C NMR
(
Chloroform-d) δ 164.48, 161.79, 153.31, 146.91, 136.29, 133.41,
1
32.65, 131.79, 131.71, 128.21, 127.79, 127.29, 126.26, 126.19,
NTZ-AC (50
μ
M) was treated with cysteine (100 M) then the intensity
μ
1
25.50, 125.48, 123.76, 122.52, 121.51, 121.46. Spectra are given in
was monitored with respect to time. The intensity increased with time
and attains maximum intensity in two minutes (Fig. 5). Thus all the
spectra were taken immediately after the addition of cysteine. This swift
response makes it as an efficient probe for in vivo imaging.
+
Figure S3 and S4. HR-MS: Calculated for C20
found 331.0666 in Figure S5.
H13NO
2
S [M] 331.06670,
2
. Results and discussion
.1. Sensing studies of NTZ-AC towards cysteine
The sensor was synthesized via two steps reaction. In first step,
2
.3. Effect of pH
2
In order to carry over the application towards imaging in live cells it
is unavoidable to examine the effect of pH in detecting cysteine. NTZ-AC
highly emissive NTZ was synthesized from the reaction between 2-hy-
droxy naphthaldehyde and 2- aminothiophenol. NTZ was further
Fig. 1. Absorption spectrum of NTZ-AC (50
cysteine (100 M) (red) to NTZ-AC in DMSO-PBS (1:1 v/v, 10 mM PBS buffer at
pH 7.4). The inset demonstrates naked eye colour change.
μ
M) (black) and addition of
μ
Scheme 1. Synthesis of NTZ-AC.
2

K. Anusuyadevi et al.
Journal of Photochemistry & Photobiology, A: Chemistry 403 (2020) 112875
Fig. 2. Emission spectrum at 370 nm excitation wavelength of NTZ-AC (50 M)
μ
(
black) and addition of cysteine (100
μ
M) (red) to NTZ-AC in DMSO-PBS (1:1 v/
Fig. 5. The intensity change on addition of 2 eq. of cysteine to 1 eq. of NTZ-AC
v, 10 mM PBS buffer at pH 7.4). The inset is fluorescent change on addition
of cysteine.
with respect to time.
(
50
marked change in intensity was noted. Then the influence of pH after
addition of cysteine (100 M) to NTZ-AC (50 M) was analysed fluo-
μ
M) was subjected to different pH from 3 to 10. There was no
μ
μ
remetrically. No prominent change in intensity at 3–5 pH was observed
whereas the intensity increases dramatically while moving towards
basic pH 6–10 (Fig. 6). The possible explanation for the observed phe-
nomena is as follows: As the mode of detection utilizes the nucleophilic
amino and thiol groups of cysteine, it is necessary to retain the nucle-
ophilicity of cysteine. In acidic pH those groups lose its nucleophilicity
because of protonation, the addition reaction is seized. Thus NTZ-AC
doesn’t detect cysteine in acidic pH. While in basic medium, the thiol
group is deprotonated which makes the addition reaction facile and
easier. Hence the probe detects cysteine efficiently with less reaction
time on moving towards basic medium. All the other experiments were
carried out at pH 7.4 as it’s suitable for biological studies.
2
.4. Selectivity and sensitivity
NTZ-AC rapidly recognize the presence of cysteine effectively with
Fig. 3. Fluorescence intensity of NTZ-AC (50
tration of cysteine from 0 M - 60 M.
μ
M) on increasing the concen-
μ
μ
slight colour change and bright cyan emission at physiological pH 7.4.
Thus it’s indispensable to enquire about selectivity and sensitivity of the
probe to enhance its efficiency. As the detection is based on the nucle-
ꢀ
ꢀ
ophilicity of cysteine it is necessary to study the effect of anions (F , Cl ,
Fig. 4. The plot of concentration vs change in fluorescence intensity at λ max
78 nm. The inset shows the linear plot of concentration vs Intensity.
4
Fig. 6. Variation in intensity of NTZ-AC and NTZ-AC þ 2 eq. of cysteine at
different pH from 3-10.
3

K. Anusuyadevi et al.
2-
Journal of Photochemistry & Photobiology, A: Chemistry 403 (2020) 112875
ꢀ
ꢀ
ꢀ
2-
2 2 3
Br , I , OH , S , H O , SO ) and other amino acids (Homocysteine,
Glutathione, Methionine, Histidine, Aspartic acid, Glutamic acid,
Alanine, Phenyl alanine, Arginine, Serine, Proline, Lysine and Isoleu-
cine). In absorption spectrum, hyperchromic absorption was observed at
4
00 nm for NTZ-AC (50
μ
M) þ Cys (100
μ
M) due to the slight change in
colour from colourless to yellow while no characteristic change in
absorbance was observed for other aminoacids and anions (Fig. 7). In
emission spectrum, a turn-on response was observed on addition of 2 eq.
of cysteine (100 M) to NTC-AC under 370 nm excitation (Fig. 8). But
μ
only trivial change in intensity and absorption was observed when 2 eq.
of other anions and amino acids were added to NTZ-AC under same
excitation which clearly depicts the conjugate addition cyclization re-
action is facile only with cysteine (Fig. 7 & 8). It is notable that NTZ-AC
doesn’t respond to potent interfering amino acids such as homocysteine
and glutathione which can be explained by the concept of kinetics. The
reaction of NTZ-AC is prompt with cysteine whereas it is sluggish with
homocysteine (Fig. 5) due to the increase in number of carbon atoms and
the reaction is not feasible with GSH. Thus the probe NTZ-AC manifests
exquisite detection of cysteine.
Fig. 8. Emission spectrum for the selective detection of cysteine (100
NTZ-AC (50 M) over other interferences (100 M).
μM) by
μ
μ
The effectiveness of the probe in revealing the existence of cysteine
even in the presence of its potential interferences, competitive studies
were performed. The fluorescence intensity of NTZ-AC was examined in
the presence of above indicated interferences and only insignificant
change in intensity was observed (Fig. 9 & 10 ). Thus it is inferred that
NTZ-AC is an outstanding probe in discriminating cysteine with high
capability among other dynamic nucleophilic interferents.
2
.5. Proposed mechanism of detection
Detection of cysteine by NTZ-AC is triggered by addition of thiol to
,β- unsaturated carbonyl then succeeded by the cyclization of amino
α
group which cleaves the acrylate moiety as thioether and produce NTZ
(
Scheme 2). Therefore, ESIPT was reactivated (effortless proton transfer
between hydroxyl and nitrogen of thiazole groups) and the turn-on
response was observed. Strongin et al. have reported the mechanism
behind the detection of cysteine based on cleavage of acrylate moiety
[
23,40]. The progress of the cleavage of acrylate unit was investigated
through NMR titration as there is a distinct change in chemical shift of
Fig. 9. Interference studies of NTZ-AC (50
μ
M) demonstrates the sensitive
M).
alkene protons of acrylate moiety (Fig. 11 (a)-(d)).
1
detection of cysteine (100 M) in presence of amino acids (100
μ
μ
6
H NMR of NTZ-AC was taken in DMSO- d which exhibit distinct
peaks at chemical shift 6.0, 6.2 and 6.4 corresponds to protons of
acrylate moiety Fig. 11 (a). On addition of 0.5 eq. to 1 eq. of cysteine
2
dissolved in D O the intensity of peaks at those regions were decreased
due to the reaction between NTZ-AC and cysteine Fig. 11 (b)-(c) While 2
eq. addition of cysteine vanished those peaks corresponds to acrylate
Fig. 10. Interference studies of NTZ-AC (50
μ
M) demonstrates the sensitive
detection of cysteine (100 M) in presence of anions (100
μ
μ
M).
Fig. 7. Absorption spectrum for the selective detection of cysteine (100
NTZ-AC (50 M) over other anions and amino acids (100 M).
μM) by
μ
μ
4

K. Anusuyadevi et al.
Journal of Photochemistry & Photobiology, A: Chemistry 403 (2020) 112875
diphenyltetrazolium bromide) assay before conducting imaging ex-
periments to study the survival efficiency of the cells under different
concentration of NTZ-AC (Figure S6).
The cells exhibited low cytotoxicity on increasing the concentration
◦
of NTZ-AC from 0 to 40
μ
M inside HeLa cells at 37 C for 24 h (Fig-
ure S6). Thus MTT assay reveals the less toxicity of NTZ-AC towards
HeLa cells.
The potential of NTZ-AC in live cell imaging of cysteine was scruti-
nized. 10 M NTZ-AC was treated with HeLa cells for 20 min, strong
μ
cyan emission was observed Fig. 13 (A) clearly due to permeability of
NTZ-AC into the cells and reacts with the endogenous cysteine. In order
to study the specificity of the probe towards cysteine, cells were treated
with 1 mM of notorious thiol blocking agent N- Ethylmaleimide (NEM)
followed by the incubation of NTZ-AC for 20 min, only weak emission
was noticed Fig.13 (B) which indicates the cysteine was removed by
NEM. However, when the cells were treated with NEM (1 mM) for
Scheme 2. Conjugate addition cyclization mechanism of NTZ-AC.
2
0 min followed by the incubation of cysteine (200 M) strong cyan
μ
emission was restored Fig.13 (C). This depicts the high specificity of the
probe NTZ-AC towards cysteine.
Disparity in the redox state is mainly due to either surplus production
of Reactive Oxygen Species or deficiency in antioxidants which can
cause cell death [30]. As cysteine acts as a potential endogenous anti-
oxidant, redox balance was altered by hydrogen peroxide and the
change in fluorescence of NTZ-AC inside HeLa cells were investigated.
HeLa cells on treatment with NTZ-AC (20
emission due to the presence of endogenous cysteine Fig. 14 (D). When
the cells were treated with hydrogen peroxide (200 M and 400 M)
prior to the incubation of NTZ-AC (20 M) decrease in fluorescence
μM) yields bright cyan
μ
μ
μ
intensity was observed in Fig. 14 (E) & (F). This is due to the antioxidant
ability of cysteine which involves in the neutralization of oxidative stress
induced by hydrogen peroxide. As NTZ-AC can detect cysteine in two
minutes it is highly efficient probe to study the redox imbalance inside
the cells.
1
4. Conclusion
Fig. 11. NMR Titration: (a) H NMR spectrum of NTZ-AC (5 mM) in DMSO-d
b), (c) & (d) are the spectra corresponds to the addition of 0.5 eq.- 2 eq. of
cysteine in D O to NTZ-AC in DMSO-d
6
(
In summary, a turn-on ESIPT probe NTZ-AC was synthesized for
rapid detection of cysteine utilizing acrylate moiety as a recognition
unit. NTZ-AC exhibits an instant colorimetric and fluorimetric response
on addition of cysteine via conjugate addition cyclization mechanism
with exceptional selectivity and sensitivity among other amino acids and
2
6
.
moiety that evidenced the completion of the reaction to produce NTZ
Fig. 11 (d)). However, the hydroxyl group of NTZ doesn’t occur which
attributes to exchange with deuterium in D O.
(
2
1
anions. This reaction mechanism was studied and confirmed by H NMR
The reaction mechanism was further confirmed by HRMS. The mass
+
titration and HRMS. In addition, NTZ-AC also display low cytotoxicity
corresponds to NTZ-AC is [M] 331.0666 (calculated 331.0667). On
+
addition of Cysteine to NTZ-AC shows an abundant peak at [M]
2
77.0553 which corresponds to NTZ (calculated 277.0561) (Fig. 12).
This demonstrates that the cleavage of acrylate moiety leads to the
formation of NTZ which is highly emissive due to ESIPT.
3
. Bio imaging
We explore the practical applicability of NTZ-AC to image cysteine in
HeLa cells. Therefore cytotoxicity of NTZ-AC was estimated by MTT (3-
(
4,5-dimethylthiazol-2-yl)-2,5
Fig. 13. Confocal microscopy images (A) denotes 20 min incubation of HeLa
cells with 10 M NTZ-AC. (B) Cells were treated with 1 mM NEM for 20 min
prior to the incubation with 10 M NTZ-AC. (C) Cells were pre-treated with
1 mM NEM and 200 M cysteine followed by the incubation of 10 M NTZ-AC.
μ
μ
Fig. 12. High Resolution Mass spectrum corresponds to NTZ-AC þ Cys.
μ
μ
5

K. Anusuyadevi et al.
Journal of Photochemistry & Photobiology, A: Chemistry 403 (2020) 112875
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