Peroxide-responsive boronate ester-coupled turn-on fluorogenic probes: Direct linkers supersede self-immolative linkers for sensing peroxides

Article abstract of DOI:10.1016/j.dyepig.2021.109363

Turn-on fluorogenic probes are commonly utilized for an efficient estimation of reactive oxygen species such as H₂O₂. In the present study, three different sets of turn-on fluorogenic probes for sensing H₂O₂ wer

Full text of DOI:10.1016/j.dyepig.2021.109363

Dyes and Pigments 191 (2021) 109363
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Dyes and Pigments
journal homepage: http://www.elsevier.com/locate/dyepig
Peroxide-responsive boronate ester-coupled turn-on fluorogenic probes:
Direct linkers supersede self-immolative linkers for sensing peroxides
a
a,
b
a
a,b,*
Abu Sufian , Debojit Bhattacherjee , Tripti Mishra , Krishna P. Bhabak
a
Department of Chemistry, Indian Institute of Technology Guwahati, Guwahati, 781039, Assam, India
b
Centre for the Environment, Indian Institute of Technology Guwahati, Guwahati, 781039, Assam, India
A R T I C L E I N F O
A B S T R A C T
Keywords:
Turn-on fluorogenic probes are commonly utilized for an efficient estimation of reactive oxygen species such as
. In the present study, three different sets of turn-on fluorogenic probes for sensing H were rationally
Boronate ester
ROS
H
2
O
2
2 2
O
designed by coupling boronate ester group with three important fluorescent dyes either by linking directly or via
self-immolative linkers. Interestingly, our results reveal that directly-linked boronate ester probes are superior
and sensitive than the self-immolative linker-containing probes in detecting traces of exogenous and endogenous
Peroxide
Sensor
Self-immolative linker
Turn-on fluorescence
levels of H
used. Considering these aspects, our detailed studies reveal that the directly-linked boronate ester-based fluo-
rescein probe 19 is the best probe for efficiently sensing H both in aqueous as well as cellular medium with
2 2
O . The difference in probe efficiency was also dependent on the nature of fluorophore unit being
2 2
O
very high level of sensitivity. The present observation would be useful while designing the fluorogenic sensors for
ROS as well as during their utilization in ROS-associated pathologies.
1
. Introduction
probes are being reported by them or by other research groups by
coupling the boronate ester group with different fluorescent dyes
Hydrogen peroxide (H
2
O
2
) is an important non-radical oxidant
(Table S1, Supplementary information section). In most of the turn-on
fluorogenic probes for sensing H or H -meidated drug delivery,
among reactive oxygen species (ROS), which is produced enzymatically
in the mammalian system by the metabolism of molecular oxygen [1].
2
O
2
2 2
O
the boronate ester group was linked to the fluorophore unit using two
main strategies namely-i) direct installation on to the aromatic nuclei in
place of –OH group (probes 1, 2, 7 and 13) [14–17]; ii) coupling of
2 2
An overproduction of H O has been implicated as the hallmark of
oxidative stress in several pathological conditions as well as disease
states and therefore, it is extremely important to estimate the endoge-
self-immolative boronate benzyl group with the –OH or –NH
2
group on
nous level of H
of turn-on fluorogenic probes containing H
been shown as very useful non-invasive and sensitive tools for moni-
toring the endogenous level of H in living systems and for
2
O
2
under normal and pathological conditions [2–6]. Use
aromatic nuclei via an ether (probes 5, 6, 9–11 and 14) [18–23] or
carbonate/carbamate (probes 3, 4, 8 and 12) [24–27] linkage (Scheme
1).
2 2
O
-reactive group(s) has
2
O
2
The active fluorophore is released in the second strategy upon the
ROS-triggered drug delivery under a particular disease condition [7].
Despite the use of various ROS-sensitive groups in ROS-sensors or
ROS-mediated drug delivery vehicles, boronate esters are utilized
extensively since last two decades for its enhanced selectivity towards
2 2
reaction with H O followed by self-immolation process via quinone
methide (QM) intermediates as shown in Scheme 1A. The first ether-
linked turn-on probe 5 was reported by Cohen and co-workers in
2013, considering the synthetic ease and aqueous stability of the probe
H
2
O
2
over the other oxidants in ROS family [8–12]. First boronate
[18] and the strategy was used further for H
2 2 2 2
O sensing and H O -me-
ester-coupled turn-on fluorogenic probe with a self-immolative carba-
mate linkage to aminocoumarin was developed by Lo and co-workers in
diated drug delivery. Considering the H -responsive directly-linked or
2 2
O
self-immolative boronate ester-containing probes reported till date,
there are few observations-i) coupling strategies (direct vs
self-immolative) were mostly made randomly or by considering syn-
thetic ease and aqueous stability without analysing their sensitivity
2
003 [13]. In 2004, Chang’s group reported the first directly-linked
boronate ester probe by utilizing both the hydroxyl groups in fluores-
cein (probe 1, Table S1, ESI) [14]. Since then a number of H -sensitive
2 2
O
*
Corresponding author. Department of Chemistry, Indian Institute of Technology Guwahati, Guwahati, 781039, Assam, India.
E-mail address: kbhabak@iitg.ac.in (K.P. Bhabak).
https://doi.org/10.1016/j.dyepig.2021.109363
Received 29 January 2021; Received in revised form 24 March 2021; Accepted 29 March 2021
Available online 3 April 2021
0
143-7208/© 2021 Elsevier Ltd. All rights reserved.

A. Sufian et al.
Dyes and Pigments 191 (2021) 109363
Scheme 1. (A) Reported schemes for H
2
O
2
-sensing and H
2
O
2
-mediated drug delivery. (B) Chemical structures of the designed turn-on fluorogenic probes with three
2 2
different types of boronate ester linkers and the schematic representation for fluorophore release in the presence of H O .
towards the analyte; ii) absence of comparative data while reporting a
new probe with different coupling strategies; iii) incidentally, several of
the reported probes are not sensitive enough to detect the endogenous
2. Experimental section
2.1. Materials and methods
2 2 2 2
level of H O in cellular medium and thus external addition of H O or
H
2
O
2
-inducer were necessary for fluorescence turn-on [28,29]. How-
All the chemicals and solvents were purchased either from Sigma
Aldrich, Merck or from reputed local suppliers. All the solvents were
either distilled or dried following standard method before using in re-
actions and chemicals were used without further purification. Thin layer
chromatographic (TLC) analyses were carried out on pre-coated silica
gel on aluminium sheets and the compounds were visualized by irradi-
ation with UV (254 nm) or fluorescent light (366 nm) and stained by
iodine. Organic solvents used for chromatographic separations were
distilled before use. Melting point of the synthesized compounds were
recorded in a Büchi B540 melting point apparatus and the values are
uncorrected. The NMR spectra were recorded with a Bruker Ascend ™
400 MHz and 600 MHz NMR Spectrometers. Chemical shifts are cited
ever, a proper rationalization is essential not only for the synthetic ease
and stability of the probe, but for the sensitivity and reactivity of the
probe towards the analyte of interest (H
above drawbacks and to understand and identify the suitable coupling
strategies of boronate ester groups with fluorophore units in H -re-
2 2
O ). Therefore, concerning the
2 2
O
sponsive probes, we have rationally designed possible combination of
boronate ester-based probes using three different and well-known flu-
orophores (22–24) covering a wide emission spectral range (Scheme 1).
Herein, we report for the first time that, directly-linked boronate
2 2
ester probes are significantly more sensitive and reactive towards H O
than the probes with self-immolative linkers under both of aqueous and
cellular environments, making them as best candidates for sensing traces
4
with respect to Me Si as internal standard. Mass spectra were obtained
of exogenous and endogenous H
2
O
2
. Furthermore, the marked selec-
using an Agilent 6520 Accurate- Mass Quadrupole Time-of-Flight (Q-
TOF) LC/MS spectrometer. The compounds 23 and 24 were synthesized
following the reported procedures [30,31].
tivity of directly-linked probes over the self-immolative probes was also
dependent on the nature of fluorophore used and probably associated
with their relative reactivities.
2
.2. Synthesis of the probes
Synthesis of compound 15 [32]: To a stirred solution of compound
2

A. Sufian et al.
Dyes and Pigments 191 (2021) 109363
3
3 (0.40 g, 1.29 mmol) and bis(pinacolato)diboron (0.60 g, 2.59 mmol)
in 1,4-dioxane (20 mL) was sequentially added Pd(dppf)Cl (0.10 g,
.13 mmol) and KOAc (0.40 g, 3.89 mmol) under argon atmosphere at
The combined organic layer was washed with water and brine (3 × 20
mL) and dried over anhydrous sodium sulfate. The solvent was evapo-
rated under reduced pressure to afford the crude product, which was
purified by flash chromatography using 10% ethyl acetate in pet ether to
2
0
◦
room temperature. The reaction mixture was stirred at 85 C for 12 h.
Upon completion, the solvent was evaporated under reduced pressure
and the residue was diluted with ethyl acetate (20 mL). The reaction
mixture was filtered through Celite bed to remove undesired materials
and washed with ethyl acetate (3 × 20 mL). The combined organic layer
was washed with water and brine (3 × 20 mL). The organic layer was
dried over anhydrous sodium sulfate and concentrated under reduced
pressure to afford the crude compound. The crude product was purified
by silica gel column chromatography using 20% ethyl acetate in pet
afford the pure product 17 as white solid. R
pet ether). Yield: 0.11 g (38%); M.P. = 115–117 C. H-NMR (CDCl
400 MHz): δ (ppm): 9.11 (dd, J = 8.5 Hz, J = 1.0 Hz, 1H), 8.60 (dd, J
= 7.2 Hz, J = 1.0 Hz, 1H), 8.56 (d, J = 7.3 Hz, 1H), 8.29 (d, J = 7.3 Hz,
1H), 7.78 (dd, J = 7.3 Hz, , 1H), 4.21-4.15 (m, 2H), 1.78-
= 8.4 Hz, J
1.68 (m, 2H), 1.52-1.39 (m, 2H), 1.45 (s, 12H), 0.98 (t, J = 7.4 Hz, 3H).
f
= 0.5 (10% ethyl acetate in
◦
1
3
,
1
2
1
2
1
2
1
3
3
C NMR (100 MHz, CDCl ) δ (ppm): 164.4, 164.4, 135.8, 135.3, 134.9,
130.8, 129.7, 127.8, 127.0, 124.8, 122.6, 84.6, 40.3, 30.2, 25.0, 20.4,
+
ether to afford the pure product 15 as white solid. R
f
= 0.5 (25% ethyl
13.9. ESI-MS (+ve) m/z calcd for C22
H26BNO
4
[M+H] : 380.2030;
◦
1
acetate in pet ether). Yield: 0.38 g (89%); M.P. = 145–147 C. H-NMR
found: 380.2030.
(
(
CDCl
3
, 400 MHz): δ (ppm) = 7.74 (s, 1H), 7.69 (d, J = 7.8 Hz, 1H), 7.58
Synthesis of compound 6 [19]: To a mixture of compound 23 (30
mg, 0.11 mmol) and compound 31 (40 mg, 0.13 mmol) in anhydrous
d, J = 7.8 Hz, 1H), 6.33 (d, J = 1.1 Hz, 1H), 2.45 (d, J = 1.1 Hz, 3H),
1
1
.37 (s, 12H). 13C NMR (100 MHz, CDCl
3
) δ (ppm): 160.8, 152.9, 152.0,
acetonitrile (2 mL) was added anhydrous K
2
CO
3
(23 mg, 0.17 mmol)
◦
30.0, 123.7, 123.0, 122.0, 116.1, 84.4, 24.9, 18.6. ESI-MS (+ve) m/z
under argon atmosphere at 0 C. After 30 min, the reaction mixture was
allowed to attain room temperature and stirred for overnight. Upon
completion of the reaction, solvent was evaporated under reduced
pressure and the residue was diluted with ethyl acetate (50 mL). The
mixture was washed with water and brine and the organic layer was
dried over anhydrous sodium sulfate and evaporated under reduced
pressure. The crude residue was purified by flash chromatography using
20% ethyl acetate in pet ether to afford the pure product 6 as bright
+
calcd for C16
H
19BO
4
[M + H] : 287.1455; found: 287.1455.
Synthesis of compound 16: To a stirred solution of compound 22
(
0.20 g, 1.13 mmol) in anhydrous DMF (0.5 ml) was added K
2
CO
3
(0.10
◦
g, 1.70 mmol) under argon atmosphere at 0 C and the mixture was
stirred for 1 h. A solution of compound 31 (0.20 g, 1.36 mmol) in
anhydrous DMF (0.5 mL) was added dropwise to the reaction mixture
and the mixture was stirred at room temperature for 12 h. Upon
completion, the reaction mixture was diluted with ethyl acetate (50 mL)
and washed with water and brine (3 × 10 mL). The organic layer was
dried over anhydrous sodium sulfate and concentrated under reduced
pressure to afford the crude residue. The crude product was purified by
flash chromatography using 20% ethyl acetate in pet ether to afford the
green solid. R
(55%); M.P. = 166–168 C. H-NMR (CDCl
(m, 2H), 8.52 (dd, J = 1.5 Hz, 1H), 7.89 (d, J = 7.9 Hz, 2H),
= 8.2 Hz, J
7.71 (m, 1H), 7.52 (d, J = 7.8 Hz, 2H), 7.09 (dd, J = 8.3 Hz, J = 1.0 Hz,
1H), 5.39 (s, 2H), 4.17 (m, 2H), 1.71 (m, 2H), 1.45 (m, 2H), 1.36 (s,
f
= 0.5 (20% ethyl acetate in pet ether); Yield: 60 mg
◦
1
3
, 600 MHz): δ (ppm): 8.61
1
2
1
2
1
3
pure product 16 as white solid. R
Yield: 0.19 g (85%); M.P. = 192–194 C. H-NMR (CDCl
ppm): 7.84 (d, J = 7.9 Hz, 2H), 7.49 (d, J = 8.8 Hz, 1H), 7.43 (d, J = 7.8
Hz, 2H), 6.93 (dd, J
f
= 0.5 (20% ethyl acetate in pet ether).
12H), 0.97 (t, J = 7.4 Hz, 3H). C NMR (150 MHz, CDCl
3
) δ (ppm):
◦
1
3
, 400 MHz): δ
164.5, 164.0, 159.7, 138.5, 135.3, 133.3, 131.6, 129.4, 128.7, 126.7,
(
126.0, 123.6, 122.5, 115.4, 106.5, 84.0, 70.8, 40.1, 30.3, 24.9, 20.4,
+
1
= 8.8 Hz, J
2
= 2.4 Hz, 1H), 6.87 (d, J = 2.4 Hz, 1H),
13.9. ESI-MS (+ve) m/z calcd for C29
H32BNO
5
[M+H] : 486.2452;
1
3
6
.14 (s, 1H), 5.15 (s, 2H), 2.39 (s, 3H), 1.35 (s, 12H). C NMR (100
found: 486.2483.
MHz, CDCl
3
) δ (ppm): 161.6, 161.2, 155.2, 152.5, 139.0, 135.2, 126.6,
Synthesis of compound 18: To a stirred solution of triphosgene
(0.13 g, 0.42 mmol) in anhydrous DCM (2 mL) was added pyridine (0.07
ml, 0.85 mmol) dropwise at ice-cold condition under argon atmosphere.
The reaction mixture was stirred at room temperature for 30 min, then
1
25.6, 113.8, 112.9, 112.1, 102.0, 83.9, 70.4, 24.8, 18.6. ESI-MS (+ve)
+
m/z calcd for C23
H
25BO
5
[M + H] : 393.1873; found: 393.1880.
Synthesis of compound 12: To a stirred solution of compound 22
◦
(
0.13 g, 0.75 mmol) and triethylamine (0.20 mL, 1.50 mmol) in anhy-
kept at ꢀ 70 C. To this solution, the compound 30 (0.20 g, 0.85 mmol)
drous DCM (5 mL), the solution of compound 32 (0.20 g, 0.50 mmol) in
anhydrous DCM (3 mL) was added in a dropwise manner at ice-cold
condition under argon atmosphere and the reaction mixture was
allowed to attain room temperature and stirred for 24 h. Upon
completion, the reaction mixture was diluted with DCM (50 mL) and
in dry DCM (2 mL) was added dropwise at that condition. Progress of the
reaction was monitored by TLC analysis. Upon consumption of com-
pound 30 completely, the mixture was added dropwise into the solution
of compound 23 (0.11 g, 0.42 mmol) and triethylamine (0.06 mL, 0.42
◦
mmol) in anhydrous DCM (5 mL) at ꢀ 70 C. Slowly the reaction mixture
sequentially washed with saturated NaHCO
3
solution, water and brine
was warmed to room temperature and kept stirring for 2–3 h. Progress of
the reaction was monitored by TLC analysis. Upon completion of the
reaction, the excess triphosgene was quenched with water, extracted by
DCM (3 × 20 mL). The combined organic layer was washed with brine
(2 × 10 mL) and dried over anhydrous sodium sulfate. The solvent was
evaporated under reduced pressure to afford the crude residue, which
was purified by flash chromatography using 15% ethyl acetate in pet
solution (3 × 10 mL). The organic layer dried over sodium sulfate and
evaporated under reduced pressure to afford the crude product, which
was purified by flash chromatography using 10% ethyl acetate in pet
ether to afford the pure product 12 as white solid. R
acetate in pet ether). Yield: 0.11 g (64%); M.P. = 140–141 C. H-NMR
CDCl
, 600 MHz): δ (ppm): 7.85 (d, J = 7.9 Hz, 2H), 7.61 (d, J = 8.7 Hz,
H), 7.45 (d, J = 7.9 Hz, 2H), 7.23 (d, J = 2.3 Hz, 1H), 7.17 (dd, J = 8.7
Hz, J
= 2.3 Hz, 1H), 6.28 (d, J = 0.9 Hz, 1H), 5.31 (s, 2H), 2.44 (d, J =
f
= 0.5 (20% ethyl
◦
1
(
3
1
1
ether to afford the pure product 18 as white solid. R
acetate in pet ether). Yield: 60 mg (32%); M.P. = 131–132 C. H-NMR
(CDCl = 8.4 Hz, J = 1.0
, 400 MHz): δ (ppm): 8.62 (m, 2H), 8.32 (dd, J
= 8 Hz, J = 7.4 Hz, 1H),
f
= 0.5 (20% ethyl
◦
1
2
1
3
1
1
1
.0 Hz, 3H), 1.36 (s, 12H). C NMR (100 MHz, CDCl
3
) δ (ppm): 160.4,
3
1
2
54.1, 153.2, 152.8, 151.9, 137.2, 135.1, 127.6, 125.5, 118.0, 117.3,
Hz, 1H), 7.87 (d, J = 8.0 Hz, 2H), 7.77 (dd, J
1
2
14.7, 110.0, 84.0, 70.7, 24.9, 18.7. ESI-MS (+ve) m/z calcd for
7.67 (d, J = 8.1 Hz, 1H), 7.47 (d, J = 8.0 Hz, 2H), 5.37 (s, 2H), 4.18 (t, J
= 8.0 Hz, 2H), 1.70 (m, 2H), 1.45 (m, 2H), 1.36 (s, 12H), 0.98 (t, J = 7.4
+
C
24
H
25BO
7
[M + H] : 437.1772; found: 437.1772.
Synthesis of compound 17: To a mixture of bis(pinacolato)diboron
0.60 g, 2.25 mmol) and compound 34 (0.25 g, 0.75 mmol) in anhydrous
,4-dioxane (20 mL) was added Pd(dppf)Cl (55 mg, 0.07 mmol) and
Hz, 3H). 13C NMR (150 MHz, CDCl
) δ (ppm): 164.0, 163.4, 152.6,
3
(
151.4, 137.1, 135.2, 131.8, 131.7, 129.3, 127.7, 127.6, 127.4, 124.8,
1
2
122.9, 120.6, 118.6, 84.0, 71.0, 40.3, 30.2, 24.9, 20.4, 13.9. ESI-MS
+
KOAc (0.18 g, 2.25 mmol) under argon atmosphere at room tempera-
(+ve) m/z calcd for C30
H32BNO
7
[M + H] : 530.2350; found: 530.2360.
◦
ture. The reaction mixture was stirred at 90 C for overnight and upon
Synthesis of compound 19 [33]: To a solution of bis(pinacolato)
diboron (0.53 g, 2.09 mmol), compound 35 (0.50 g, 1.04 mmol), and
KOAc (0.20 g, 3.12 mmol) in anhydrous 1, 4-dioxane (20 mL) was added
completion, the solvent was evaporated under reduced pressure. The
residue was diluted with ethyl acetate (20 mL) and the precipitate was
filtered through Celite bed and washed with ethyl acetate (3 × 20 mL).
2
Pd(dppf)Cl (70 mg, 0.10 mmol) under argon atmosphere at room
3

A. Sufian et al.
Dyes and Pigments 191 (2021) 109363
◦
temperature. The reaction mixture was stirred at 90 C for 12 h. Upon
completion, the solvent was evaporated under reduced pressure and the
residue was diluted with ethyl acetate (20 mL). The mixture was filtered
through Celite pad and washed with ethyl acetate (3 × 20 mL). The
combined organic layer was washed with water and brine (3 × 20 mL)
and the organic layer was dried over anhydrous sodium sulfate. The
solvent was evaporated under reduced pressure to afford the crude
product, which was purified by flash chromatography using 17% ethyl
2.3. UV–visible and fluorescence spectroscopic studies
All the stock solution of probes and the released fluorophores were
prepared in spectroscopy grade DMSO (note: 20% THF was added with
DMSO for the naphthalimide-based probes 17, 6 and 18 to dissolve them
completely) and the stock solution of hydrogen peroxide was prepared
freshly in mili-Q water. All the spectroscopic studies were performed
under physiological conditions (phosphate buffer, 20 mM, pH = 7.5).
Buffers with higher pH (8, 9 and 10) were prepared by adding 1 N NaOH
solution to the buffer. Samples for absorption and emission spectro-
scopic measurements were carried out in quartz cuvettes (1.0 ml).
UV–Vis spectroscopic analyses were performed on a Lambda 45 UV–Vis
spectrophotometer and fluorescence emission spectra were recorded on
acetate in pet ether to afford the pure product 19 as white solid. R
f
= 0.5
(
20% ethyl acetate in pet ether); Yield: 0.38 g (58%); M.P. = 166–168
◦
1
C. H-NMR (CDCl
3
, 600 MHz): δ (ppm): 8.03 (d, J = 7.4 Hz, 1H), 7.74(s,
H), 7.63 (m, 2H), 7.43 (dd, J = 0.7 Hz, 1H), 7.12 (d, J =
= 7.8 Hz, J
.5 Hz, 1H), 6.81 (d, J = 7.8 Hz, 1H), 6.77 (d, J = 2.5 Hz, 1H), 6.72 (d, J
8.8 Hz, 1H), 6.62 (dd, J = 2.5 Hz, 1H), 3.84 (s, 3H), 1.35
= 8.8 Hz, J
s, 12H). 13C NMR (150 MHz, CDCl
) δ (ppm): 169.5, 161.4, 153.5,
52.4, 150.7, 135.1, 129.7, 129.4, 129.0, 127.2, 126.2, 125.1, 123.8,
1
7
1
2
=
1
2
a Fluoromax-4 spectrophotometer (FluoroMax-4, HORIBA). For
(
3
coumarin-based probes, λex = 322 nm and λem = 447 nm with slit width
= 3/3 nm, for naphthalimide-based probes, λex = 442 nm and λem = 550
nm with slit width = 5/5 nm and for fluorescein-based probes, λex = 454
nm and λem = 511 nm with slit width = 3/3 nm were used in the fluo-
rescence emission spectroscopic studies. The selectivity of the directly-
1
1
23.5, 121.5, 111.7, 110.9100.9, 84.2, 82.7, 55.6, 24.9. ESI-MS (+ve)
+
m/z calcd. for C27
H25BO
6
[M+H] : 457.1822; found: 457.1841.
Synthesis of compound 20: The compound was prepared following
the reported method with modifications [34]. To a stirred solution of
compound 24 (0.20 g, 0.57 mmol) and compound 31 in anhydrous
toluene (10 mL) was added silver oxide (0.20 g, 0.86 mmol) under inert
atmosphere at room temperature. The reaction mixture was refluxed for
linked probe 19 (10
μ
M) towards H
2
O
2
(200 M) was carried out over
μ
different analytes such as tert-butyl hydroperoxide (tBu-OOH), cumene
ꢀ
hydroperoxide (Cum-OOH), potassium superoxide (KO
2
, O
2
), reduced
glutathione (GSH), L-cysteine (CYS), N-acetyl cysteine (NAC), sodium
5
h. Progress of the reaction was monitored by TLC analysis. Upon
chloride (NaCl) and potassium chloride (KCl). The probe was initially
completion, the solvent was evaporated under reduced pressure and the
residue was diluted with dichloromethane (20 mL). The mixture was
filtered through Celite pad and washed with DCM (3 × 10 mL). The
combined organic layer was dried over sodium sulfate and the solvent
was evaporated to afford the crude product. The crude product was
purified by flash chromatography using 20% ethyl acetate in pet ether to
incubated with the analytes (200
μ
M) for 30 min and the emission in-
(200 M) was added to
tensity was measured. An equal amount of H
2
O
2
μ
the resulting solution and the emission was measured after incubation of
another 30 min.
2.4. Determination of limit of detection (LOD) of the probes
afford the pure product 20 as yellow solid. R
f
= 0.5 (20% ethyl acetate in
◦
1
pet ether) Yield: 0.12 g (37%); M.P. = 98–100 C. H-NMR (CDCl
3
, 600
The limit of detection (LOD) of all the fluorescein-based probes 19,
20 and 21 were determined using the fluorescence titration. The
MHz): δ (ppm): 8.02 (d, J = 7.6 Hz, 1H), 7.83 (d, J = 7.8 Hz, 2H), 7.66 (t,
J = 7.2 Hz, 1H), 7.61 (t, J = 7.3 Hz, 1H), 7.43 (d, J = 7.7 Hz, 2H), 7.15
detection limit was calculated using the equation, LOD = 3
σ
/K, where
σ
(
d, J = 7.5 Hz, 1H), 6.82 (s, 1H), 6.76 (d, J = 2.2 Hz, 1H), 6.68 (m, 3H),
is the standard deviation of blank measurements (without H O ) and K is
2
2
6
1
1
1
5
.60 (dd, J
1
= 8.7 Hz, J
2
= 2.4 Hz, 1H), 5.12 (s, 2H), 3.83 (s, 3H), 1.34 (s,
) δ (ppm): 169.4, 161.3, 160.3, 153.2,
the slope of emission intensity versus H O2 concentration. To get the
2
1
3
2H). C NMR (150 MHz, CDCl
3
standard deviation of the blank measurements, the emission intensity of
52.5, 152.4, 139.4, 135.1, 135.0, 129.7, 129.2, 129.1, 126.5, 126.5,
all the probes (10
For the determination of slopes, the emission spectra were measured at
different concentrations of H O (2–50 M) after incubating each reac-
2 2
μM) were measured 10 times in the absence of H O .
25.0, 124.0, 112.3, 111.7, 111.5, 111.2, 101.9, 100.8, 83.9, 83.2, 70.1,
+
5.6, 24.9. ESI-MS (+ve) m/z calcd for C34
H
31BO
7
[M + H] : 563.2241;
μ
2
2
found: 563.2275.
tion mixture for 30 min.
Synthesis of compound 21: To a stirred solution of compound 24
(
0.20 g, 0.34 mmol) and triethylamine (0.14 mL, 1.04 mmol) in anhy-
2.5. Analysis of reaction kinetics using reverse phase HPLC method
◦
drous DCM (8 mL) at 0 C under argon atmosphere was dropwise added
a solution of compound 32 (0.35 g, 0.86 mmol) in anhydrous DCM (2
mL). The mixture was then allowed to attain room temperature and
stirred for 48 h. Progress of the reaction was monitored by TLC analysis.
Upon completion, the reaction mixture was diluted with DCM (50 mL)
Purity of the synthesized compounds were analyzed by analytical
high performance liquid chromatography (HPLC), Agilent 1220 infinity
II LC system using reverse phase C18 column (Luna®, 250 × 4.6 mm, 5
μ
m). HPLC Chromatogram of the compounds 19, 20 and 21 were
and washed with water followed by saturated NaHCO
3
solution. The
detected by PDA detector at 254 nm and 454 nm at room temperature
using a flow rate of 1.0 ml/min over 15 min. Concentrations of the pure
organic layer was washed with brine (3 × 10 mL) and dried over sodium
sulfate. The solvent was evaporated under reduced pressure to afford the
crude residue, which was purified by flash chromatography using 30%
ethyl acetate in pet ether to afford the pure product 21 as off-white solid.
probes and the released fluorophores were 100
μ
M and in the reaction
mixture, 100 equivalents of H O (10 mM) was added. The reactions of
2
2
probes with H O were carried out in HPLC grade acetonitrile and water
2
2
R
f
= 0.5 (30% ethyl acetate in pet ether). Yield: 80 mg (23%); M.P. =
system (3:1) at room temperature and the samples were injected into the
system at different time intervals to monitor the progress of the reaction.
◦
1
1
1
1
2
00–102 C. H-NMR (CDCl
H), 7.84 (d, J = 7.9 Hz, 2H), 7.68 (t, J = 7.1 Hz, 1H), 7.63 (t, J = 7.3 Hz,
H), 7.44 (d, J = 7.9 Hz, 2H), 7.16 (m, 2H), 6.88 (dd, J = 8.7 Hz, J
.3 Hz, 1H), 6.81 (d, J = 8.7 Hz, 1H), 6.78 (d, J = 2.4 Hz, 1H), 6.70 (d, J
8.8 Hz, 1H), 6.63 (dd, J = 2.5 Hz, 1H), 5.29 (s, 2H), 3.84
= 8.8 Hz, J
s, 3H), 1.35 (s, 12H). 13C NMR (150 MHz, CDCl
) δ (ppm): 169.3, 161.5,
53.0, 152.2, 152.1, 151.8, 137.4, 135.2, 135.1, 129.9, 129.2, 129.0,
27.6, 126.4, 125.1, 124.0, 117.0, 116.9, 112.0, 110.8, 109.8, 100.8,
3
, 600 MHz): δ (ppm): 8.03 (d, J = 7.6 Hz,
1
2
=
2.6. Detection of endogenous level of H O2 in HeLa cells by cellular
2
imaging
=
1
2
(
3
HeLa cells were cultured in high glucose Dulbecco’s modified Eagle’s
1
1
8
medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and
◦
1% penicillin/streptomycin at 37 C under 5% CO2 atmosphere. Cells
4
4.0, 82.4, 75.0, 70.5, 55.6, 24.9. ESI-MS (+ve) m/z calcd for C35
H31BO
9
were then plated (0.5 × 10 cells/plate) in 35 mm cell culture petri
+
◦
[
M+H] : 607.2139; found: 607.2187.
dishes containing 2.0 mL of DMEM and incubated at 37 C under 5%
CO
2
for 24 h. The confluent cells were washed with DPBS and finally
◦
incubated with the fluorescein-based probes (19–21) (5.0
μ
M) at 37 C
4

A. Sufian et al.
Dyes and Pigments 191 (2021) 109363
Fig. 1. Absorption (A–C) and emission (D–F) spectra of directly-linked probes 15, 17 and 19 (10
μ
M) in the absence and presence of H
2
O
2
(200 M) along with the
μ
spectra of pure released fluorophores (22–24, 10
μ
2 2
M) in phosphate buffer of pH 7.5. The final spectra of the probes with H O were recorded after 90 min.
under 5% CO
2
for 1 h. After washing with DPBS (5 times), cellular
using a microplate reader (Multiskan Go microplate reader, Thermo
Scientific). In experimental set, similar MTT treatment protocol was
followed only after 48 h. The mean ΔOD values were calculated by the
subtraction of mean OD values of 0 h plate (control) from the mean OD
values of identical wells at 48 h plate (experimental) and the percentage
proliferation was calculated keeping the mean ΔOD of untreated control
as 100%.
morphology was carefully observed and imaged in a Bio-Rad ZOE™
fluorescent cell imager under bright field and suitable fluorescent
colored filters. Additionally, the control experiments were designed and
performed with the pre-treatment of NAC (N-acetyl cysteine) (2.0 mM)
to the HeLa cells to affirm the quenching of endogenous ROS or
hydrogen peroxide level. The cells were washed with DPBS and treated
further with the fluorogenic probes and incubated for 1 h. Finally, the
treated cells were washed with DPBS (5 times) and imaged in a Bio-Rad
ZOE™ fluorescent cell imager to understand the reactivity of hydrogen
peroxide with fluorescent probes. Further, to confirm the sensitivity of
3. Results and discussion
3.1. Design and synthesis of the probes
the boronate ester-based probes towards hydrogen peroxide, exogenous
◦
H
2
O
2
(200
μ
M) was added to the cells and incubated at 37 C under 5%
In the present study, three different sets of turn-on fluorescent probes
were developed considering fluorophores 22–24 (experimental section
and Schemes S1–S4, supplementary information section). In the first set,
boronic ester group was incorporated directly to the aromatic nuclei
with the replacement of hydroxyl group (probes 15, 17 and 19). The
boronic ester group was installed by the Pd-catalyzed transmetalation
reaction of triflate/bromide derivative of the fluorophores with bis
CO
2
for 1 h. The cells were washed with DPBS and probes (19–21) (5.0
M) were added and incubated as discussed above. Finally, after
μ
sequential washing with DPBS, treated cells were imaged under Bio-Rad
ZOE™ fluorescent cell imager.
2
.7. Cell culture and cellular viability studies
(
pinacolato)diboron under inert conditions. In the second set, the bor-
onate benzyl alcohol moiety was installed on the fluorophores via ether
linkage (probes 16, 6 and 20). These probes were synthesized by the
coupling of fluorophore alcohols with boronate benzyl bromide in the
Human cervical cancer cell line (HeLa) was obtained from the Na-
tional Centre for Cell Science (NCCS), Pune, India. HeLa cells were
cultured in DMEM medium (Gibco) supplemented with 10% (v/v) FBS
presence of Cs
benzyl alcohol was coupled to the fluorophores via carbonate linkage
probes 12, 18 and 21). These probes were achieved either by the
2 3 2 3
CO or K CO . Whereas, in the third set, the boronate
(
Gibco) and 1% Pen-Strep (Gibco). Cells were cultured as a monolayer in
◦
a humidified incubator at 37 C in the presence of 5% CO₂ level. All the
coumarin-based, naphthalimide-based and fluorescein-based turn-on
fluorescent probes along with the corresponding released fluorophores
(
coupling of fluorophore alcohols with 4-nitrophenyl carbonate adduct of
boronate benzyl alcohol or by the conventional coupling using tri-
phosgene. All the final boronate ester-based probes, obtained in
reasonably good yields, were purified and characterized by analytical
methods before the planned spectroscopic studies.
(
22–24) were screened for their anti-proliferative activities using the
conventional MTT assay. All the test compounds were dissolved in
DMSO (HiMedia) except naphthalimide-based probes (stock solution
was prepared in THF and diluted further using media with final THF
concentration kept below 0.05%). HeLa cells were seeded in 96-well
4
culture plates at a density of 1 × 10 cells/100
μ
l/well and treated
M) for 0 h
control) and 48 h (experimental). At the end of treatment period, 10.0
3.2. Absorption and emission spectroscopic studies
with the freshly prepared test compounds (5.0, 10.0 and 25.0
μ
(
The initial UV–Vis spectroscopic studies were carried out with the
directly-linked probes (15, 17 and 19) with and without H O to iden-
2 2
μ
L of 5.0 mg/mL of MTT was added to the plate (control) and incubated
for 4 h. Following the 4 h incubation, the culture media from the plate
was removed and the purple formazan crystals were dissolved using 100
tify the absorption maxima of the intact probes and the released fluo-
rophores. As shown in Fig. 1 (A-C), a significant red-shift was observed
μ
L of DMSO (HiMedia) and the absorbance at 570 nm was measured
2 2
upon their reactions with H O and the spectra overlapped well with
5

A. Sufian et al.
Dyes and Pigments 191 (2021) 109363
Fig. 2. Emission spectra of (A) coumarin-, (B) naphthalimide- and (C) fluorescein-based probes (10
μ
M) with and without H
2
O
2
(200
μ
M) over 90 min in phosphate
buffer (20 mM) of pH 7.5. (D) Emission spectra of self-immolative probes (6, 18, 20 and 21) (10
μ
M) in the presence of H
2
O
2
(200 M) over a longer time (180 min)
μ
under identical condition.
that of pure fluorophores (22–24). For the probe 19, very weak ab-
sorption band above 300 nm was probably due to the protection of both
hydroxyl groups in fluorescein. Knowing the absorption maxima of all
fluorophores 22–24, the turn-on fluorescence efficacy of the probes 15,
3.3. Kinetic studies using fluorescence spectroscopy
With the preliminary results in hand, we carried out kinetic experi-
ments to understand the relative reactivity of all the boronate ester
1
7 and 19 were evaluated using fluorescence spectroscopy. As shown in
Fig. 1 (D-F), highly intense emission response was observed for all the
probes in the presence of H and the emission pattern matched well
with that of the respective released fluorophores, indicating the H
triggered turn-on fluorescence. The absorption and emission spectra of
linked probes in the presence of H
Fig. 2A, a clear difference in the relative reactivity/sensitivity among the
coumarin-based probes (15, 16 and 12) towards H is observed. A
2
O
2
up to 90–180 min. As shown in
2
O
2
2 2
O
2
O
2
-
significantly higher reactivity is noted for the directly-linked probe 15
and the lowest reactivity was noted for the ether-linked probe 16. While
other probes with and without H
2
O
2
are shown in the supplementary
2 2
probe 15 was highly reactive towards H O and the reaction was almost
information (Figs. S1–S2, ESI).
saturated within 30 min, the saturation time was found to be relatively
longer for the corresponding self-immolative probes. Interestingly, a
Fig. 3. (A) Relative initial rates for the turn-on fluorescence of carbonate-linked (green) and directly-linked (red) probes w.r.t ether-linked (black) probes of a
particular fluorophore in their reactions with H M) in the presence of variable concentrations of
. (B) Emission spectra of fluorescein-based probes (19–21, 10
(0–500 M) with a fixed incubation time of 30 min.
2
O
2
μ
H
2
O
2
μ
6

A. Sufian et al.
Dyes and Pigments 191 (2021) 109363
Scheme 2. Generalized and most plausible mechanistic pathways for the reactions of fluorescein-based directly-linked probe 19 (A) and ether-linked self-immolative
probe 20 towards H for the release of fluorophore 24.
2 2
O
similar but more pronounced effect is observed for naphthalimide- and
fluorescein-containing probes. For example, the initial saturation time
was much longer for the directly-linked probes 17 and 19 (~50 min) as
compared to 15 (~30 min). This indicates the higher reactivity of
coumarin-based directly-linked boronate ester probes than the corre-
sponding naphthalimide- and fluorescein-based probes. Furthermore,
the self-immolative probes corresponding to naphthalimide (6 and 18)
and fluorescein (20 and 21) fluorophores were found to be much less
reactive than the corresponding directly-linked probes 17 and 19
directly-linked boronate ester probes for sensing traces of H
ever, the ether- or carbonate-based self-immolative probes might be
suitable for H -triggered drug delivery applications, often requiring
2 2
O . How-
2 2
O
sustained drug release profiles [23,35,36].
While both of naphthalimide and fluorescein fluorophores (23 and
24) have almost similar emission ranges (yellow color, naked eye), the
turn-on fluorescence emission intensity from the fluorescein-based
probes (19–21) were higher than that from the naphthalimide-based
probes (17, 6 and 18) under identical condition at a fixed concentra-
(
Fig. 2B and C). Between, two types of self-immolative probes, the
tion (10
μM) in the presence of H
2
O
2
(200 M). Furthermore, the solu-
μ
reactivity of the ether-linked probes were found to be lower than the
carbonate-linked probes in general. Furthermore, it should be noted
here that, like coumarin-based probes, typical saturation kinetics was
not observed for naphthalimide- and fluorescein-based self-immolative
probes till 90 min and therefore, the kinetic experiments were extended
up to 180 min to understand their nature of kinetic pattern (Fig. 2D).
Interestingly, while some level of saturation kinetics was observed with
relatively higher emission intensity for the carbonate-linked probes (18
and 21), both the ether-linked probes (6 and 20) reflected lower and
almost steep increasing intensity pattern without saturation up to 180
min (Fig. 2D). These results clearly indicate the higher reactivity of
directly-linked boronate ester probes than the corresponding self-
immolative linker-based probes.
bility profiles of naphthalimide-based probes were much narrower than
that of fluorescein-based probes. Naphthalimide-based probes were
soluble only in THF and not completely soluble in other polar solvents
such as DMSO, DMF, ACN and water. However, fluorescein-based
probes were soluble in a wider ranges of polar organic solvents
including the solvents mentioned above except water. Due to these
reasons, fluorescein-based probes (19–21) were chosen for further
studies. A significant difference in kinetic pattern of probes 19–21 (10
μM) with 200
μM H
2
O
2
(20-fold) inspired us to understand their
M) at a fixed
behavior with a varied concentration of peroxide (0–500
μ
incubation time (30 min). Interestingly, as shown in Fig. 3B, a clear
difference in emission intensity was observed at lower concentration
and the difference increased gradually with H
porting our views that directly-linked probe 19 is much efficient in
sensing H than probes 20 and 21.
2 2
O concentration, sup-
2 2
O
3
.4. Nature of fluorophore unit on probe efficiency
The fluorophore dependency of the relative rate of fluorescence turn-
3.5. Sensitivity of the probes
on from three types of boronate ester-linked probes was evidenced in
Fig. 3. As shown in Fig. 3A, the difference in relative initial rate of re-
action between two different kinds of self-immolative probes was not
significant for all three fluorophores and in general, the carbonate-
linked probes were slightly more reactive than the corresponding
ether-linked probes. However, a significantly higher difference in rela-
tive rate is observed between the directly-linked probes and the ether-
To further understand the relative sensitivity of fluorescein-based
probes 19–21 towards H
lated as per standard method using fluorescence titration [29]. The
emission intensity of the probes (10 M) was measured in the absence of
and at a lower variable concentration range (2–50 M) of H
Interestingly, a significant difference in the sensitivity of probes 19–21
towards was observed. For example, while the LOD of
2 2
O , the limit of detection (LOD) was calcu-
μ
H
2
O
2
μ
2 2
O .
linked probes towards H
2
O
2
. Furthermore, this difference was found
2 2
H O
to be dependent on the nature of fluorophore unit being used. For
example, the difference was 14.6-fold, 9.1-fold and 1.7-fold for fluo-
rescein-, naphthalimide- and coumarin-based probes, respectively
directly-linked boronate ester probe 19 was 33 nM, the LOD of ether-
and carbonate-linked probes (20 and 21) were significantly higher (1.3
and 0.37 M, respectively), indicating much higher sensitivity of 19
μ
(
Fig. 3A). The above difference in turn-on response of directly-linked
than 20 and 21 (Fig. S3, Table S2, ESI). It should be noted here that the
result on their relative sensitivity matches well with their fluorescence
turn-on response over time and analyte concentration (Figs. 2C and 3B).
and self-immolative-linked probes form various fluorophores appears
to be inversely proportional to the reactivity of directly-linked probes of
various fluorophores towards H
2
O
2
. For example, as the coumarin-based
than the naphthalimide- and
probes react much faster with H
2
O
2
3.6. Mechanistic insights for turn-on fluorescence process
fluorescein-based probes, the difference in fluorescence response of
directly-linked and self-immolative-linked probes is less pronounced for
coumarin-based probes than two other fluorophore-based probes.
Overall, the kinetic experiments clearly reveal the better suitability of
To understand possible reasons for the relatively slower reactivity of
self-immolative probes over the directly-linked probes, the underlying
stepwise mechanistic pathways were proposed depicting the reaction of
7

A. Sufian et al.
Dyes and Pigments 191 (2021) 109363
Fig. 4. (A) Emission spectra of probes 19–21 (3
μ
M) in the absence and presence of H
M) + H
+ NaOH (25 mM). The chromatograms L-1 to L-7 were visualized at 254 nm and L-8 was visualized at
54 nm to monitor the released fluorophore 24. Peak at 2.46 min appears from the stabilizers in commercial H sample. (C) Vials under visible light; (D) under UV
(vials 2, 4 and 7); probe 20 + H + NaOH (vial 5).
M) followed by the treatment with H (200 M). The probe was initially
(200 M) to the resulting
2
O
2
(200
μ
M) at variable pH of the medium. (B) HPLC chromatogram of pure
probes 19–21, released fluorophore (24) and the reaction mixtures of probes (100
μ
2
O
2
(10 mM) after 30 min. L-1: pure 24; L-2 to L-4: pure probes 19–21; L-
5
4
: 19 + H
2
O
2
; L-6: 20 + H
2
O
2
; L-7: 21 + H
2
O
2
; L-8: 20 + H
2 2
O
2 2
O
light (254 nm); (E) under fluorescent light (366 nm); Pure probes 19–21 (vials 1, 3 and 6); probes 19–21 + H
F) Reactivity of the directly-linked probe 19 (10 M) towards different analytes (200
2
O
2
2 2
O
(
μ
μ
2
O
2
μ
incubated with the analytes for 30 min and the emission intensity was measured. Final response was measured upon the addition of H
solution followed by the incubation for another 30 min.
2
O
2
μ
ꢀ
representative fluorescein-based probes 19 and 20 towards H
Scheme 2). The plausible reaction pathways for the reaction of boro-
nate ester group with H is well-established and is used extensively in
2
O
2
hydroperoxide ion (HOO ; pKa of H
2
O
2
: 11.6). Furthermore, the self-
(
immolation process via QM intermediate would also be enhanced by
the predominant existence of phenoxide ion in the intermediate 29
(Scheme 2B) at higher pH ranges (pKa of phenols: ~10). Additionally,
the experiment further evidenced the overall stability of probes 19–21
2 2
O
turn-on fluorescence processes [37,38]. The initial nucleophilic reaction
of hydroperoxide anion at the electrophilic boron-center of the
directly-linked probe 19 followed by the rearrangement and subsequent
nucleophilic attack of hydroxyl anion leads to the release of fluorescein
fluorophore 24 with turn-on fluorescence (Scheme 2A). However, a
similar process in probe 20 would generate the intermediate 29. From
Scheme 2B, it is evident that, unlike probe 19, the fluorescence will be
turned-on from 20 only after the self-immolation of benzyl group in the
intermediate 29 via quinone methide (QM) species, generation of which
is quite well-established for the self-immolative fluorogenic probes (Step
2 2
in the absence of H O over a wide pH range of the medium indicating
their compatibility under normal physiological as well as pathological
conditions.
3
.8. Kinetic studies using reverse phase HPLC method
The relative reactivity of the boronate ester groups in probes 19–21
towards H O was studied by kinetic experiments using reverse phase
2 2
4
, Scheme 2B) [18,25].
HPLC method (Fig. 4B). Interestingly, while a clean conversion of the
directly-linked probe 19 to the fluorophore 24 was observed in the
3
.7. pH responsiveness of the probes
2 2
presence of H O with time (L-5, Fig. 4B), the self-immolative probe 20
produced the intermediate phenolic compound 29, which was quite
persistent under HPLC condition in acetonitrile and water (3:1) mixture
with traces of fluorophore (24) release (L-6, Fig. 4B). However, the in-
termediate 29 could be rapidly converted to fluorophore 24 upon the
treatment of NaOH solution (L-8). The formation of 29 was also evi-
denced in ESI-MS analysis (Fig. S8, ESI). The carbonate-linked probe 21
rather reacted rapidly with the release of fluorophore 24 and probably
with very traces of intermediate (L-7, Fig. 4B, Fig. S6, ESI). The peak at
around 2.46 min with almost fixed intensity appears from the sample of
To understand the feasibility of turn-on fluorogenic process and the
stability of the probes over a wider range of pH, the emission studies
were carried out with variable pH (6.0–10.0) of the medium. Interest-
ingly, a steady increased emission pattern was observed for the probe 19
with increasing pH of the medium from 6 to 8 and the emission almost
saturates at pH 9. In contrast, steep increase in emission intensity was
observed for both the self-immolative probes 20 and 21 with increasing
pH of the medium and eventually that crossed the emission intensity
from the probe 19 (Fig. 4A). This is not surprising as increasing pH
commercially available H
2
O
2
(Merck, India), which is probably origi-
. The relative formation of
would facilitate the reaction by partial conversion of
2
H O
2
to
nated from the stabilizers used in H O
2 2
8

A. Sufian et al.
Dyes and Pigments 191 (2021) 109363
Fig. 5. Fluorescence microscopy images (bright field, green channel and overlay) of HeLa cells in the presence of fluorescein-based probes 19–21 for the detection of
endogenous level of H
2 2
O .
released fluorophore 24 from probes 19–21 in the presence of H
2
O
2
over
cylinders, Fig. 4F) following the similar protocol. As shown in Fig. 4F, a
significant enhancement of the emission intensity was observed upon
6
0 min was also estimated and the results show significantly higher
release of 24 from the directly-linked probe 19 over other two self-
immolative linker-based probes 20–21 (Fig. S7, ESI).
2 2
the addition of H O in the presence of all the analytes studied herein.
We like to mention here that the directly-linked probe was reported by
Chang and co-workers in 2010 along with few other directly-linked
probes and showed the enhanced selectivity of the probes towards
The difference in fluorescence turn-on process for the fluorescein-
based probes 19–21 can be clearly visualized with naked eyes
(
Fig. 4C) and also upon irradiation with UV (Fig. 4D) and fluorescence
2 2
H O over other relevant oxidants and radicals [33].
light (Fig. 4E). These observations indicate that the boronate ester
cleavage process by H is reasonably fast for all the probes and the
2 2
O
3
2 2
.10. Detection of the endogenous level of H O in HeLa cells
difference in fluorescence emission response from the directly-linked
and self-immolative linker-based probes is mainly due to the relatively
slower rate of self-immolative processes via QM intermediate particu-
larly for the ether-linked probes. Our observation with significantly
higher fluorescence-response for the directly linked boronate ester
probes than self-immolative probes is found to be in contrast to the
behavior and reactivity of non-fluorogenic peroxide-sensitive methyl
salicylate-based inhibitors of metalloproteinase and the 2,4-dinitro-
phenyl group-based NIR probes for thiophenol sensing [39,40].
To understand the compatibility of the probes in cellular medium for
the detection of endogenous level of H , the probes (5.0 M) were
2
O
2
μ
treated to human cervical cancer cells (HeLa) and the emission was
measured after 60 min (Fig. 5). Interestingly, a significantly higher
emission response under green channel was observed for the probe 19 as
compared to 20 and 21. The emission response was much weaker for the
ether-linked probe 20 under the identical condition, indicating much
lesser sensitivity of 20 in detecting the endogenous level of H
cells. To understand the selectivity of the directly-linked probe 19 to-
wards the endogenous level of H , HeLa cells were pre-treated with N-
acetyl cysteine (NAC, 2.0 mM) that scavenges the endogenous H
2 2
O in HeLa
3
.9. Selectivity of the directly-linked probe 19 towards H
2
O
2
2 2
O
2 2
O .
Considering the evidences that directly-linked probe 19 exhibited
significantly higher reactivity towards H than that of the corre-
sponding self-immolative probes 20 and 21, the selectivity of the probe
towards H was studied. Some of the oxidizing agents, reducing
agents and inorganic salts were chosen to see their relative impacts on
Interestingly, no emissive response in the NAC pre-treated cells served as
the negative control and also evidenced the lack of any background
emission response of the probe 19 (Fig. S10, ESI). This also proved the
stability of probe 19 under cellular environment. Positive control ex-
2 2
O
2 2
O
periments were also carried out upon the exogenous addition of H
the cells followed by the treatment of probe 19. Interestingly, enhanced
intensity from all the probes upon exogenous treatment of H to the
2 2
O to
the selectivity of probe 19 towards H
incubated with various analytes (200
2
O
2
. The probe (10
μ
M) was first
μM) and the emission intensity
2 2
O
was measured (red cylinders, Fig. 4F). To the solution mixture, H
2
O
2
cells further confirms the peroxide-responsive fluorescence turn-on
process (Fig. S11, ESI) under cellular environment and the suitability
(
200
μM) was added and the final emission was measured (blue
9

A. Sufian et al.
Dyes and Pigments 191 (2021) 109363
Fig. 6. Cellular viability of HeLa cells in the presence of probes 19–21 at variable doses (5.0, 10.0 and 25.0
μM). The cells were incubated with the probes for 48 h at
◦
3
7
C in humidified incubator in the presence of 5% CO
2
level.
of these probes for sensing the endogenous level of H
response of the naphthalimide-based probes were also studied for the
detection of endogenous level of H in HeLa cells under identical
condition, however, the images had background noises (Fig. S9, ESI).
2
O
2
. Emission
CRediT authorship contribution statement
2
O
2
Abu Sufian: optimized the experimental condition. Debojit Bhat-
tacherjee: carried out cellular experiments and fluorescent microscopic
experiments. Tripti Mishra: compounds and intermediates and char-
acterized the products. Krishna P. Bhabak: Supervision, Formal
analysis.
3
.11. Toxicity profiles of the probes
To understand the compatibility of the turn-on fluorogenic probes in
cellular medium, their toxicity profiles were studied in the same cell line
HeLa) using conventional MTT assay. While imaging studies were
carried out with 5.0 M of the probes, toxicity profiles of the probes were
evaluated at 5.0 M and at two higher concentration levels (10.0 and
5.0 M) and incubated for a longer time (48 h). Interestingly, all the
probes were found to be non-toxic at 5.0 and 10.0 M concentrations
Declaration of competing interest
(
μ
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
μ
2
μ
μ
indicating the feasibility of their applications in cellular medium (Fig. 6
and Fig. S12, ESI). However, few of them exhibited around 20% cellular
Acknowledgements
toxicity at a 5-fold higher concentration (25.0
μ
M). Although, the
K.P.B. acknowledges SERB for the research grant (Ref: EEQ/2016/
directly-linked boronate ester probes were found to be significantly
more sensitive than the self-immolative linker-based probes of naph-
0
00090). K.P.B. thanks DST, India for the INSPIRE Faculty Award. We
acknowledge DST-FIST program, CoE-FAST and IIT Guwahati for
infrastructural facilities. A.S. and D.B. thank IIT Guwahati for their
fellowships.
2 2
thalimide and fluorescein fluorophores for sensing H O , further anal-
ysis might be necessary considering a wider ranges of fluorophore units
for a more generalized understanding.
Appendix A. Supplementary data
4
. Conclusions
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.dyepig.2021.109363.
In summary, we highlight for the first time that direct coupling of the
boronate ester group to the fluorophore unit in peroxide-responsive
turn-on fluorogenic probes is strategically superior than the coupling
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and based on several control experiments, fluorescein-based directly-
linked probe 19 was found to be the best candidate among all the probes
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