Detection of thiophenol in buffer, in serum, on filter paper strip, and in living cells using a red-emitting amino phenothiazine boranil based fluorescent probe with a large Stokes shift

Article abstract of DOI:10.1016/j.tet.2017.06.011

A novel red-emitting dye, PB-NH₂, was synthesized by incorporation of an electron rich phenothiazine moiety to classical boranil dye. PB-NH₂ displayed excellent photophysical properties, such as long emission wavelength, large Stokes

Full text of DOI:10.1016/j.tet.2017.06.011

Accepted Manuscript
Detection of thiophenol in buffer, in serum, on filter paper strip, and in living cells
using a red-emitting amino phenothiazine boranil based fluorescent probe with a large
Stokes shift
Wenqiang Chen, Linlin Zhu, Yuanqiang Hao, Xiuxiu Yue, Jinyan Gai, Qi Xiao, Shan
Huang, Jiarong Sheng, Xiangzhi Song
PII:
S0040-4020(17)30630-0
10.1016/j.tet.2017.06.011
TET 28776
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 8 April 2017
Revised Date: 1 June 2017
Accepted Date: 2 June 2017
Please cite this article as: Chen W, Zhu L, Hao Y, Yue X, Gai J, Xiao Q, Huang S, Sheng J, Song X,
Detection of thiophenol in buffer, in serum, on filter paper strip, and in living cells using a red-emitting
amino phenothiazine boranil based fluorescent probe with a large Stokes shift, Tetrahedron (2017), doi:
10.1016/j.tet.2017.06.011.
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ACCEPTED MANUSCRIPT
Novel phenothiazine boranil dye was utilized for thiophenol fluorescent probe
development. Significantly, Probe 1 showed great potential for practical applications
in real water samples, in serum, on filter paper strips as well as in living cells.

ACCEPTED MANUSCRIPT
Detection of thiophenol in buffer, in serum, on filter paper
strip, and in living cells using a red-emitting amino
phenothiazine boranil based fluorescent probe with a large
Stokes shift
a*
a
b
a
a
a
Wenqiang Chen, Linlin Zhu, Yuanqiang Hao, Xiuxiu Yue, Jinyan Gai, Qi Xiao,
a
a*
c
Shan Huang, Jiarong Sheng and Xiangzhi Song
a
College of Chemistry and Materials Science, Guangxi Teachers Education University, 530001
Nanning, Guangxi, P. R. China.
b
Henan Key Laboratory of Biomolecular Recognition and Sensing, College of Chemistry and
Chemical Engineering, Shangqiu Normal University, 476000 Shangqiu, Henan, P. R. China.
c
College of Chemistry & Chemical Engineering, Central South University, Changsha, Hunan
Province 410083, P. R. China.
*
Corresponding author E-mail: chenwq@csu.edu.cn ; Fax: +86 771 3908065; Tel: +86 771
3908065; E-mail: shengjiarong99@163.com; Fax: +86 771 3908018; Tel: +86 771 3908018.
1

ACCEPTED MANUSCRIPT
Abstract
A novel red-emitting dye, PB-NH , was synthesized by incorporation of an electron
2
rich phenothiazine moiety to classical boranil dye. PB-NH displayed excellent
2
photophysical properties, such as long emission wavelength, large Stokes shift, strong
emission both in solution and in solid state. Based on this attractive platform, Probe 1,
was constructed for selective detection of thiophenol. Notably, Probe 1 was
ultrasensitive in response to thiophenol, and the corresponding detection limits for
thiophenol in theoretical and in experimental were determined to be 1.4 and 10 nM,
respectively. Significantly, Probe 1 showed great potential for practical applications,
and the applications in real water samples, in serum, on filter paper strips and in living
cells were successfully demonstrated.
Key words: phenothiazine boranil; fluorescent probe; red-emitting; large Stokes shift;
thiophenol detection
2

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1
. Introduction
Thiophenols have broad synthetic utility and they are widely used in
chemical industry, such as the preparation of pesticides, medicine and various
1-3
industrial products. However, despite their great practical value, thiophenols
have been listed as one of the prioritized pollutants by the United States
4
Environmental Protection Agency (USEPA) due to the highly toxic property.
Long-term exposure of thiophenols liquid or vapor is extremely detrimental to
human health, which may induce severe central nervous system damage and
5
other related system injuries, even death. Toxicological studies revealed that
thiophenols process the median lethal concentration (LC50) values of 0.01-0.4
6-7
mM in fish and a median lethal dose (LD50) value of 46.2 mg/kg in mouse.
Therefore, the development of highly sensitive and selective methodologies for
the detection of thiophenols is of considerable significance in the fields of
chemical, biological and environmental science. Owing to the advantages of
simplicity and high sensitivity as well as non-invasiveness and high
spatiotemporal resolution, fluorescent methods have been recognized as the
effective tools that can help monitor and visualize various cations, anions, and
8
biomolecules both in vitro and in vivo. In the past decade, great efforts have
been devoted to the development of fluorescent probe for thiophenols over
aliphatic thiols. Pioneered by Wang’s group, the selective detection of
thiophenols was realized by taking advantage of the thiophenols-mediated
9
cleavage of sulphonamide. From then on, a variety of fluorescent probes
capable of detecting thiophenols selectively had been reported based on the
10-34
extended version of the above-mentioned strategy.
Despite the significant
advances have obtained so far, it is noteworthy that most of the reported probes
suffer from short emission wavelength and relatively small Stokes shift. It is
well known that fluorescent probes with emission in red or near-infrared range
are optimal for biological imaging applications due to the deep tissue
penetration, decreased light scattering, reduced autofluorescence and minimum
3

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35
photodamage. Moreover, fluorophores with large Stokes shift are quite
suitable for fluorescence microscopy studies, because the large Stokes shift
enables the clearly separated excitation and emission bands, which can
effectively minimize the interferences caused by self-absorption or
10
auto-fluorescence. Unfortunately, red or near-infrared fluorescent probes for
thiophenol with large Stokes shift are rare up until now. Among the reported
thiophenol probes, three fluorescent probes emit in red or near-infrared region
(
λem ≥ 600 nm) as well as display large Stokes shift (≥ 120 nm); they could
detect thiophenol in solutions, but not suitable for thiophenol detection in solid
state due to the severe ACQ (aggregation-caused quenching) process (see Table
S1). Fluorophores that exhibit large Stokes shift and strong red or near-infrared
fluorescence both in solution and in solid state are highly desirable for
thiophenol probes development, from the stand point of practical application.
In this work, we have exploited a unique amino phenothiazine boranil dye
PB-NH2, which displays strong red emission not only in solutions but also in
solid state. More importantly, the Stokes shift of PB-NH2 could reach to
incredibly 172 nm in EtOH/PBS buffer (v/v=1:1) solution. Thereby, PB-NH2
offers a robust platform upon which novel fluorescent probe for thiophenol was
based. Herein, we synthesized a novel fluorescent Probe 1 for thiophenol by
incorporation of a dinitrobenzene sulfonate (DNBS) moiety to PB-NH2, which
could detect thiophenol both in aqueous solutions and in solid state.
2
. Results and discussion
3
.1 Design rational
In recent years, heteroatom-modified organic dyes, such as BODIPYs have
received considerable attention due to their outstanding optical characteristics,
such as relatively narrow absorption and emission band, high molar absorption
coefficient, intense fluorescence quantum yield, satisfactory photostability and
chemostability, and exceptional insensitivity to the polarity of solvents and to
36
pH.
However, classical BODIPYs suffered from severe fluorescence
4

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quenching in solid state due to the tight intermolecular π-π stacking. Moreover,
most BODIPYs exhibit very small Stokes shift (< 20 nm, in most cases), which
inevitably results in self-quenching and in measurement errors by excitation
41
and scattering light. Therefore, the development of novel red emissive boron
difluoride complexes with large Stokes shift is of great importance. In fact, this
also represents the current state-of-art in the field of novel BODIPY analogues
exploitation.
38
Phenothiazine, which was first synthesized by Bernthsen in 1883, has been
widely used in a variety of industries including the manufacturing of dyes and
39
pigments, drugs, dye-sensitized solar cells and photocopying materials.
Phenothiazine contains electron-rich nitrogen and sulfur heteroatoms in a
heterocyclic structure with strong electron-donating ability. We anticipate that
the introduction of a phenothiazine moiety to BODIPY analogues may serve as
a robust strategy for developing red-emitting fluorescent dyes with large Stokes
shift. Moreover, the nonplanar butterfly-like molecular conformation of
40
phenothiazine can effectively preclude the intermolecular π-π stacking,
thereby leading to strong solid fluorescence.
As a proof-of concept, we designed novel amino phenothiazine boranil dye,
PB-NH2, by incorporation of a phenothazine moiety to classical amino boranil
dye, and readily synthesized it from commercial available compound
steps (Scheme 1). Condensation of compound with n-bromobutane to form
compound , the subsequent Vilsmeier acylation with POCl /DMF obtained
phenothiazine-2-methoxy-3-aldehyde (compound
compound by AlI (prepared in suit) afforded the key intermediate
phenothiazine salicylaldehyde
1 in five
1
2
3
3
). Demethylation of
3
3
4
,
which was then treated with
p
-phenylenediamine formed Schiff base . Finally, the BF complexation of
5
2
compound
5 and BF ·Et O in 1,2-dichloroethane generated the target dye
3 2
1
PB-NH2. The chemical structures of all compounds were confirmed by H
13
NMR, C NMR and HRMS (see supporting information).
5

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Scheme 1. Synthetic route of PB-NH . Reagents and conditions are as follows: (a) n-bromobutane,
2
NaOH, KI, DMSO, 100°C, Ar, 6 h; (b) POCl /DMF, 0°C, Ar, 15 min, then compound 2, DMF,
3
6
3
2
0°C, 4 h; (c) Aluminium powder, iodine, CH CN, room temperature, Ar, 30 min, then compound
3
, CH CN, 80°C, 6 h; (d) p-phenylenediamine, ethanol, room temperature. (e) BF ·Et O, DIEA, 1,
3
3
2
-dichloroethane, reflux, 5 h.
With dye PB-NH2 in hand, we were eager to investigate its optical properties
in different solvents. The absorption and emission spectra of PB-NH2 in
different solvents (dichloromethane, EtOH, pH 7.4 PBS buffer/EtOH (v/v=1:1)
and CH CN) are shown in Figure S1, and the corresponding photophysical data
3
are summarized in Table 1. As designed, PB-NH2 displays emission peaks well
into the red region, with maximum at 605-626 in different solvents. Moreover,
PB-NH2 has the fluorescence quantum yields of 0.08-0.28 in different solvents,
which are great values for red-emitting dyes with large Stokes shift.
Remarkably, PB-NH2 exhibits large Stokes shifts of 154-172 nm in different
solvents, which are much superior to those of classical BODIPYs (< 20 nm in
most cases). In addition, PB-NH2 is a new solid-emissive fluorophore, which
shows strong red fluorescence in powder state and on test paper strip (Figure 1)
Table 1. Photophysical data of PB-NH in different solvents
2
a
b
c
d
Solvent
λ /nm
λ /nm
Stokes
shift
161
160
154
Фf
τ /ns
abs
em
dichloromethane
460
453
451
454
621
613
605
0.28
0.20
0.19
0.08
3.68
2.81
2.62
1.16
CH CN
3
EtOH
EtOH/PBS (1:1)
626
172
a
b
c
The maximal absorption of the PB-NH . The maximal emission of the PB-NH . Ф is the
2
2
f
6

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relative fluorescence quantum yield estimated by using fluorescein (Ф = 0.79 in ethanol) as a
f
d
standard. Fluorescence lifetime.
Figure 1. (a) Fluorescence spectrum of PB-NH powder. Insert: photograph of PB-NH powder
2
2
under a UV 365 lamp; (b) Fluorescence spectrum of PB-NH coated filter paper strip. Insert:
2
photograph of PB-NH coated filter paper strip under a UV 365 lamp.
2
Inspired by these encouragine findings, we anticipate that PB-NH2 could be
employed as an attractive platform upon which a novel fluorescent probe, that
allows thiophenol detection both in aqueous solutions and in solid state, is
based. With all these in mind, we designed and synthesized Probe 1 by
incorporation of a DNBS moiety to PB-NH2 (Scheme 2) The chemical
1
13
structure of Probe 1 was fully confirmed by H NMR, C NMR and HRMS
(
see supporting information). The design rationale is also depicted in Scheme 2
and illustrated as follows. The DNBS moiety was selected because it functions
not only as a recognition group for thiophenol, but also as an effective
fluorescence quencher via the photo-induced electron transfer (PET) process to
ensure a low fluorescence background. Upon addition of thiophenol, the
masked DNBS moiety can be removed smoothly through the S Ar process, to
N
release dye PB-NH2, thereby resulting in a strong, time-dependent red
fluorescence signal.
7

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Scheme 2. Synthesis of Probe 1 and the proposed response mechanism of Probe 1 to thiophenol
3
.2 Sensing properties of Probe 1 to thiophenol
The sensing ability of Probe 1 in response to thiophenol was investigated in
PBS buffer (10 mM, pH = 7.4, containing 1 mM CTAB). As shown in Figure
S2, free Probe 1 displayed an absorption band centered at 465 nm. However,
upon addition of the incremental amounts of thiophenol to the solution of
Probe 1, the absorption peak at 465 nm was shifted to 445 nm and concomitant
appearance of a new peak at 340 nm. The absorption peaks at 445 nm and 340
nm could be assigned to PB-NH2 and compound 6, respectively, which were
supported by the corresponding absorption comparison in Figure S3. Moreover,
free Probe 1 was essentially non-fluorescent, addition of thiophenol to the
solution of Probe 1 induced significant fluorescence enhancement at 621 nm
(
Figure S4). Importantly, Probe 1 displayed a large Stokes shift of about 158
nm in response to thiophenol which can effectively reduce the interference from
self-absorption and therefore can greatly improve the sensitivity in the
fluorescence detection. To gain insight into the sensitivity of Probe 1 in PBS
buffer (10 mM, pH = 7.4, containing 1 mM CTAB), the fluorescence intensity
of Probe 1 in response to the incremental amounts of thiophenol was
investigated. As expected, the fluorescence intensity increased gradually with
incremental addition of thiophenol. When 1.4 equivalent of thiophenol was
added to the solution of Probe 1, the fluorescence intensity reached a plateau
and the enhancement was up to 104-fold (Figure 2). In addition, further study
suggested a plot of fluorescence intensity at 621 nm versus the concentrations
8

ACCEPTED MANUSCRIPT
of thiophenol exhibited good linearity (R = 0.9949) in the range of 0-12 µM. In
addition, the theoretical detection limit was calculated to be 1.4 nM (based on
S/N = 3) (Figure S5). Notably, the experimental detection limit is 10 nM, which
displays an approximately 1.2-fold increase of the fluorescence intensity with
good reproducibility (Figure S6). To the best of our knowledge, it was the
lowest experimental detection limit ever reported. All these traits suggested that
Probe 1 was indeed a sensitive detector for quantitative analysis of thiophenol
in aqueous media.
Figure 2. Fluorescence spectra of Probe 1 (10 µM) in the presence of thiophenol (0-16 µM) in
PBS buffer (10 mM, pH=7.4, containing 1 mM CTAB). Excited at 470 nm.
3
.3 Kinetic studies
Time-dependent fluorescence arising from the interaction of aqueous Probe
1
(10 µM) with 0.5 and 1.0 equivalent of thiophenol were investigated by
monitoring the fluorescence intensity changes at 621 nm as a function of time.
As shown in Figure 3, upon addition of thiophenol to the solution of Probe 1
,
9

ACCEPTED MANUSCRIPT
the fluorescence intensity at 621 nm increased immediately and reached a stable
signal plateau within 5 min, indicating that Probe 1 could respond to
thiophenol rapidly. Moreover, this reaction obeys a typical pseudo-first-order
-1
with rate constant kobs determined to be 0.7727 min (Figure S7). In addition,
in the absence of thiophenol, the solution of Probe 1 displayed negligible
changes in the fluorescence intensity at 621 nm during the identical interval
suggesting that free Probe 1 was highly stable in aqueous solution.
Figure 3. Kinetics of fluorescence enhancement rate at 621 nm for Probe 1 (10 ꢀM) in the
absence (black line) and presence of 5 ꢀM (red line) and 10 ꢀM (blue line) thiophenol in PBS
buffer (10 mM, pH=7.4, containing 1 mM CTAB).
3
.4 Mechanism studies
The optical changes of Probe 1 in response to thiophenol indicated the
releasing of PB-NH2. The proposed sensing mechanism is shown in Scheme 2.
1
To further confirm the sensing mechanism, H NMR titration was conducted
for a mixture of Probe 1 and thiophenol in DMSO-d6. As shown in Figure 4,
With the addition of incremental amounts of thiophenol to Probe 1, the signal
peaks of Probe 1, especially those marked with (■), disappeared gradually,
along with the concomitant appearance the signal peaks of PB-NH2 (peaks
marked with (*), indicating the cleavage of 2,4-dinitrophenolate moiety in
1
0

ACCEPTED MANUSCRIPT
Probe 1 and leading to the release of PB-NH2. It is worthy to note that no
obvious signal peak that assigned to the protons of amino group in PB-NH2
(
peak marked with (★)) was observed after addition of thiophenol to the
solution of Probe 1. We reasoned that this phenomenon may explained by the
substitution of hydrogen atoms on amino group by deuterium atoms in DMSO-
d6. Moreover, all signal hallmarks of the reference compound
with (▼)) can be observed in the mixture of Probe 1 and thiophenol, indicating
the formation of compound during the reaction process. Similarly, the HRMS
6 (peaks marked
6
spectrum of the reaction mixture of Probe 1 and thiophenol displayed a main
peak at m/z=460.1439 (Figure S8) which was nearly identical to the exact
+
molecular weight of PB-NH2 ([M+Na ]=460.1437). Additionally, HPLC
analysis for Probe 1, PB-NH2 and the reaction mixture of Probe 1 with
thiophenol in PBS buffer (10 mM, pH 7.4, containing 1 mM CTAB and 5%
DMF) was carried out (Figure S9). Probe 1 displayed a single peak with a
retention time at 2.88 min. Upon addition of 1.0 equiv. of thiophenol, the peak
of Probe 1 decreased dramatically and a new peak was emerged with a
retention time 2.24 min, which was assigned to PB-NH2. Taking together, all
these results provide strong support for the proposed sensing mechanism in
Scheme 2.
1
1

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1
Figure 4. H NMR spectral comparison of Probe 1
, Probe 1 with incremental amounts of
thiophenol, PB-NH2 and compound . All spectra were obtained in DMSO-d6
6
.
3
.5 Selectivity and effect of pH
Another important parameter to evaluate the performance of a fluorescent
probe is the selectivity. To gain insight into the selectivity of Probe 1 toward
thiophenol, the fluorescence behaviours of Probe 1 in response to various
potential interfering species, including aliphatic thiols (2-aminoethanethiol,
DTT, Cys and Hcy, 0.1 mM for each, and 1 mM for GSH), some essential
amino acids (Lys, Gly, Gln, Phe, Met, Tyr, Thr, Ser, His, Ile, Pro, Ala, Asp Arg
-
2-
3
-
3
and Val, 0.1 mM for each), representative anions (CH COO , S O , HSO ,
3
2
2-
4
3-
4
-
2
-
3
2-
SO , PO ,NO , NO , and S , 0.1 mM for each) and biologically abundant
+
2+
2+
2+
metal ions (K , Ca , Mg and Zn , 0.1 mM for each) as well as reactive
-
oxygen species (H O and ClO , 0.1 mM for each) were investigated. The result
2
2
showed in Figure 5 only thiophenol induced a drastic fluorescence
enhancement, no significant fluorescence changes were observed upon addition
of other analytes, including the aliphatic thiols (1-mercaptopane, cysteamine,
1
2

ACCEPTED MANUSCRIPT
DTT, Cys, Hcy and GSH). The drastic differences in fluorescence spectra
allowed the naked eye differentiation of thiophenol from other analytes under a
UV 365 nm lamp irradiation (Figure 5). Moreover, competition experiments
were carried out by adding thiophenol (16 µM) to the solution of Probe 1 (10
µM) in the presence of 100 µM of other interfering species. As shown in Figure
S10, strong fluorescence was observed when Probe 1 was exposed to
thiophenol, which was not disturbed by other interfering species. Therefore, it
can be concluded that Probe 1 exhibits high selectivity toward thiophenol.
Figure 5. Selectivity of Probe 1 (10 µM) toward thiophenol and other analytes in PBS
3
-
buffer (10 mM, pH=7.4, containing 1 mM CTAB) for 30 min. Legend: (1) PO , (2)
4
-
2-
-
2-
-
-
2-
+
2+
CH COO , (3) S O , (4) HSO , (5) SO , (6) NO , (7) NO , (8) S , (9) K , (10) Ca ,
3
2
3
3
4
2
3
2
+
2+
-
(
11) Mg , (12) Zn , (13) H O , (14)ClO , (15) Lys, (16) Gly, (17) Gln, (18) Phe, (19)
2 2
Met, (20) Tyr, (21) Thr, (22) Ser, (23) His (24) Ile, (25) Pro, (26) Ala, (27) Asp, (28) Arg,
29) Val, (30) Cys, (31) Hcy, (32) 2-aminoethanethiol, (33) DTT, 0.1 mM for each, (34)
(
GSH, 1mM, (35) blank and (36) PhSH, 16 µM. Insert: Emission images of Probe 1 with
different analytes under UV 365 lamp.
To evaluate the limitations of Probe 1 in aqueous solution, the effect of pH
on the its fluorescence intensity in the absence and presence of thiophenol was
investigated (Figure S11). Free Probe 1 exhibited negligible fluorescence in the
pH range from 1 to 12, suggesting that Probe 1 was pH insensitive. As
1
3

ACCEPTED MANUSCRIPT
expected, in the presence of 16 µM thiophenol, the fluorescence intensity
increased with the pH range from 2.0 to 12.0, and the preferred pH range was
6
.0-11.0. The weaker fluorescence response at pH ≤ 5.0 may attributed to the
reduced nucleophilicity of thiophenol under these conditions. The obvious
fluorescence decrease at pH > 11.0 was presumably due to the formation of
12
disulfide, thereby diminishing the concentration of thiophenol. The above
studies demonstrated that Probe 1 can detect thiophenol in environmental and
biological studies.
3
.6 Application potential of Probe 1
Encouraged by the above-mentioned sensing performance of Probe 1, its
practical applications including the detection of thiophenol in real-water
samples, in serum, and on filter paper strips as well as in living cells were
further demonstrated.
Detection of thiophenol in real-water samples. To verify the practicality of
Probe 1 in the environmental science, we applied Probe 1 to quantify
thiophenol concentrations in real water samples with the standard addition
37
method. The crude water samples form tap water and South Lake in Nanning
city were passed through a microfiltration membrane (0.22 µm) before use. The
pH value of the water samples were adjusted to pH 7.4 initially, and then the
aliquots of the water samples were spiked with different concentrations of
thiophenol (0, 1, 5 and 12 µM). The results were summarized in Table 2, and
the resultant thiophenol recoveries were not less than 96%. These data led us to
conclude that Probe 1 has the potential for quantitative detection of thiophenol
in real water samples.
Table 2. Determination of thiophenol concentration in water samples
Sample
Thiophenol spiked
Thiophenol recovered
(µM)
Recovery
(%)
(µM)
South
0
Not detected
Lake water
1
4

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1
5
1.09±0.15
5.32±0.10
11.76±0.08
Not detected
0.99±0.03
5.40±0.18
11.58±0.11
109
106
98
1
2
Tap water
0
1
5
99
108
96
1
2
Detection of thiophenol by Probe 1 coated filter paper strips. Motivated by the
strong fluorescence of PB-NH2 in solid state, the detection of thiophenol using
Probe 1 coated filter paper was carried out. Free Probe 1 coated filter paper
was essentially non-fluorescent. Upon addition of incremental concentrations of
thiophenol, strong red fluorescence can be observed and the corresponding
-2
fluorescence enhancement at 621 nm can reach to 40 folds when 10 M
thiophenol was added (Figure 6). Moreover, the fluorescence intensity at 621
nm was fairly linear in the thiphenol concentration range of 0.01-10 mM
(
Figure S12). To the best of our knowledge, this was the first paper strip that
could be used for thiophenol detection, and this technology may provide a
convenient method for large screening of environmental pollution samples.
1
5

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Figure 6. (a) Photograph of the corresponding samples on filter paper strip under UV 365 nm
lamp; (b) Fluorescence spectra obtained in the titration of Probe 1 with different equivalents of
thiophenol on the filter paper strip.
Detection of thiophenol in Serum. To validate the biological applicability of
Probe 1, the fluorescence titrations in the presence of fetal bovine serum (0.5
mL + 2.0 mL PBS buffer (10 mM, pH=7.4, containing 1 mM CTAB)) was
carried out. Free Probe 1 shows weak fluorescence in serum. Upon addition of
thiophenol, strong red fluorescence peaked at 607 nm can be observed (Figure
7
). The Probe 1 shows a 225 fold enhancement of fluorescence intensity as a
function of the concentration of added thiophenol and the corresponding
1
6

ACCEPTED MANUSCRIPT
detection limit was calculate to be 5.9 nM (Figure S13). Thus, Probe 1 is
sensitive enough to detect thiophenol in biological fluids. It is worth noting that
the fluorescence spectra induced by thiophenol in serum is slightly blue shifted
(
about 14 nm) when comparing the fluorescence spectra in PBS buffer (621 nm
in PBS buffer vs. 607 nm in serum). This phenomenon might be explained by
the accumulation of Probe 1 into the hydrophobic cavities of the folding
proteins in serum. Upon addition of thiophenol, the released dye PB-NH2
would stay in a relatively hydrophobic environment, as a result the
non-radiative relaxation of PB-NH2 should be suppressed in some degree,
thereby provoking an enhancement of the fluorescence intensity and a blue shift
of the emission spectra. In addition, the above-mentioned speculation was
further supported by the reaction kinetic study of Probe 1 and thiophenol in
serum (Figure S14-S15). The corresponding pseudo-first-order rate constant
-1
k ’ was determined to be 0.0617 min , which was much slower than the rate
obs
-1
constant in PBS buffer (kobs = 0.7727 min ), presumably due to the fact that the
rigid barrel pocket of the protein in serum can preclude the effective collision
between Probe 1 and thiophenol.
Figure 7. Fluorescence spectra of Probe 1 upon addition of incremental amounts of thiophenol
(0-40 µM) in the medium of fetal bovine serum in PBS buffer. Insert: photograph of Probe 1 in
1
7

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the absence and presence of thiophenol in the medium of serum in PBS buffer under the
illumination by a UV 365 lamp.
Detection of thiophenol in living cells. Inspired by the excellent performance in
aqueous solution, we sought out to investigate the capability of Probe 1 to
visualize thiophenol in living cells. Initially, we conducted MTT assays on
HeLa cells to evaluate the cytotoxicity of Probe 1. The results suggested that
this probe exhibited low cytotoxicity for living cells (Figure S16). When HeLa
cells were pre-treated with 20 µM thiophenol for 20 min at 37°C then incubated
with 10 µM Probe 1 and 1 mM CTAB for additional 10 min, strong red
fluorescence in the cell cytoplasm was observed (Figure 8c). In contrast, cells
displayed non-noticeable fluorescence when cells only incubated with 10 µM
Probe 1 and 1 mM CTAB for 10 min (Figure 8a). This result indicated that
Probe 1 is cell-permeable and can be used for intracellular thiophenol
detection.
Figure 8. Confocal microscopic images of living HeLa cells. (a-b) HeLa cells incubated
with 10 µM Probe 1 and 1 mM CTAB for 15min; (c-d) HeLa cells pre-treated with 20 µM
1
8

ACCEPTED MANUSCRIPT
thiophenol for 10 min then 10 µM Probe 1 and 1 mM CTAB for additional 15 min. (a, c)
fluorescence images; (b, d) Bright field images. Emission was collected at 600-650 nm
window upon excited at 488 nm. Scale bar: 10 µm.
3
. Experimental Section
2
.1 Materials and Instruments
Unless otherwise noted, all reagents were obtained from commercial
suppliers and used without further purification. Solvents were purified by
standard methods prior to use. Twice-distilled water was used throughout all
experiments. NMR spectra were recorded on a BRUKER 300 spectrometer
with TMS as an internal standard. All accurate mass spectrometric experiments
were performed on a Xevo G2 QTof MS (Waters, USA). UV-Vis absorption
spectra were recorded on a TU-1901 (Puxi, P.R. China) spectrophotometer.
Fluorescence spectra were recorded at room temperature using a HITACHI
F-4600 fluorescence spectrophotometer with both the excitation and emission
slit widths set at 5.0 nm. TLC analysis was performed on silica gel plates and
column chromatography was conducted using silica gel (mesh 200-300), both
of which were obtained from Qingdao Ocean Chemicals, China. HPLC analysis
was performed on a Waters Acquity UPLC H-Class system (Milford, MA,
USA) equipped with a quaternary solvent delivery system, a column oven, an
auto sampler, and a photodiode array detector.
2
.2 General Procedure for Spectral Measurements
A stock solution of Probe 1 was prepared at 1 mM in DMF. Solutions of
various testing species were prepared from Lys, Gln, Phe, His, Ala, Asp, Gly,
Thr, Tyr, Met, Cys, Ser, Ile, Arg, Pro, Val, GSH, DTT, NaOAc, Na SO ,
2
4
Na S O , NaHSO , NaNO , NaNO , Na PO , Na S, MgCl , ZnCl , CaCl , KCl,
2
2
3
3
2
3
3
4
2
2
2
2
H O , NaClO in twice-distilled water. Hcy was prepared in PBS buffer (10
2
2
mM, pH =7.4). 2-aminoethanethiol was prepared in ethanol. A typical test
solution (10.0 mL) was prepared by placing 0.05 mL of Probe 1 (1 mM), 5.0
1
9

ACCEPTED MANUSCRIPT
mL of PBS buffer (20 mM, pH = 7.4, containing 2 mM Cetrimonium Bromide
(
CTAB)), and an appropriate aliquot of each analyte stock solution into an
appropriate amount of twice-distilled water. The resulting solution was shaken
well and kept at room temperature (25°C) for 20 min before recording its
absorption and fluorescence spectra.
2
.3 Determination of the fluorescence quantum yield
Fluorescence quantum yield of PB-NH2 was determined by using fluorescein
42
(
Ф = 0.79 in ethanol) as a fluorescence standard. The quantum yield was
f
calculated using the following formula (1):
2
Ф =Ф (A F /A F )(n /n )
(1)
F(x)
F(s)
S
X
X
S
X
S
Where Φ is the fluorescence quantum yield, A is the absorbance at the
F
excitation wavelength, F is the area under the corrected emission curve, and n is
the refractive index of the solvents used. Subscripts and refer to the standard
S
X
and the unknown, respectively.
.4 Prepare the test paper strips
Probe 1 (2 mg) was dissolved in 10 mL dichloromethane. Filter paper was
2
immersed in the solution and then taken out to dry in air. Finally, the paper
coated with Probe 1 was cut into strips as the test paper for thiophenol
detection.
2
.5 Cell Culture and Fluorescence Imaging
HeLa cells were seeded in a 6-well plate in Dulbecco’s modified Eagle’s
medium (DMEM) supplemented with 10% fetal bovine serum and 1%
penicillin. The cells were incubated under an atmosphere of 5% CO and 95%
2
air at 37°C for 24 h. Cell imaging was performed with a Zeiss LSM 710 laser
scanning confocal microscope. Before each experiment, cells were washed with
PBS buffer 3 times. The cells were then incubated with Probe 1 (10 µM), or
pretreated with thiophenol (20 µM, 20 min) and further incubated with Probe 1
(
10 µM) at 37 °C (for 10 min). The samples were excited at 488 nm. Emission
was collected at 600-650 nm.
2
0

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2
.6 Synthesis of compound 2
In a 100 mL round-bottom flask, compound
1
(2.29 g, 10 mmol),
n-bromobutane (13.6 g, 100 mmol), NaOH (0.8g, 20 mmol) and a catalytic
amount of KI were dissolved in anhydrous DMSO (25 mL). The resulting
mixture was stirred at 100 °C for 6 h under an argon atmosphere. After cooling
to room temperature, the reaction mixture was poured into 300 mL water and
extracted with dichloromethane (3×100 mL). The organic layer was separated
and washed successively with brine and water, and dried over anhydrous
Na SO . After removal of the solvent, the residue was further purified by silica
2
4
gel flash chromatography using dichloromethane as an eluent to afford pure
1
compound
2
(2.7g, 95% yield) as a colorless oil. H NMR (300 MHz, CDCl ) δ
3
7
2
7
1
.25-7.12 (m, 2H), 7.06 (d, J = 8.2 Hz, 1H), 6.92 (d, J = 8.1 Hz, 2H), 6.54 (s,
H), 3.81 (d, J = 4.4 Hz, 5H), 1.89-1.79 (m, 2H), 1.56-1.46 (m, 2H), 0.99 (t, J =
13
.2 Hz, 3H). C NMR (75 MHz, CDCl ) δ 159.8, 146.6, 145.0, 127.6, 127.3,
3
27.0, 125.5, 122.4, 116.0, 115.6, 106.7, 103.4, 55.5, 47.3, 29.0, 20.2, 13.8.
2
.7 Synthesis of compound 3
Dimethylformamide (0.17 mL, 2.4 mmol) was added to phosphorous
oxychloride (0.2 mL, 2.4 mmol) at 0°C under argon atmosphere. The resulting
mixture was allowed to stirring at this temperature for additional 15 min. Then
a portion of compound
DMF) was added to the cooled reagent with stirring. The mixture warmed to
0°C and stirred for 4 h, then poured into ice water (100 mL). The clear
2 (570 mg, 2 mmol) (dissolved in 2 mL anhydrous
6
solution obtained was neutralized by NaHCO solution (10%). The resulting
3
sticky mass was extracted with dichloromethane (3×100 mL). The organic
layers were separated, combined and washed successively with brine and water,
dried over anhydrous Na SO and vacuum evaporated. The residue was further
2
4
purified by silica gel chromatography using petroleum ether/ethyl acetate (v/v,
4
:1) as an eluent to give pure compound 3 (470 mg, 76% yield) as yellow
1
powder. Mp: 81-82°C. H NMR (300 MHz, CDCl ) δ 10.20 (s, 1H), 7.53 (s,
3
2
1

ACCEPTED MANUSCRIPT
1
5
H), 7.13 (dd, J = 16.8, 7.7 Hz, 2H), 7.01-6.85 (m, 2H), 6.37 (s, 1H), 3.90 (s,
13
H), 1.84 (m, 2H), 1.48 (m, 2H), 0.97 (t, J = 7.1 Hz, 3H). C NMR (75 MHz,
CDCl ) δ 187.2, 162.6, 152.4, 142.9, 127.4, 127.3, 126.7, 124.5, 123.5, 119.5,
3
1
16.0, 115.9, 98.6, 55.7, 47.8, 28.9, 20.1, 13.8. HRMS (ESI) m/z: calcd for
+
C H NO SNa [M+Na] , 336.1029; found 336.1028.
18
19
2
2
.8 Synthesis of compound 4
Aluminium powder (67.5 mg, 2.5 mmol) was added to anhydrous acetonitrile
(
5 mL) and stirred at room temperature for 5 min. To the slurry, iodine (394
mg, 1.55 mmol) was added in small portions and stirred under nitrogen
atmosphere till the colour changed to yellow. Compound (313 mg, 1 mmol)
3
was dissolved in anhydrous acetonitrile (4 mL) and added to the slurry
dropwise. The reaction mixture was then gently refluxed for 6 h, cooled to
room temperature and poured into ice water (80 mL). The mixture was
extracted with ethyl acetate (3×60 mL). Combined ethyl acetate extracts were
washed with water, dried over anhydrous sodium sulphate. After removal of the
solvent, the residue was further purified by silica gel chromatography using
petroleum ether/dichloromethane (v/v, 4:1) as an eluent to afford compound 4
1
(
162 mg, 54% yield) as a yellow oil. H NMR (300 MHz, CDCl ) δ 11.40 (s,
3
1
H), 9.62 (s, 1H), 7.23-7.10 (m, 3H), 7.02-6.91 (m, 2H), 6.40 (s, 1H), 3.99-3.78
13
(
m, 2H), 1.88-1.78 (m, 2H), 1.51-1.44 (m, 2H), 0.98 (t, J = 7.4 Hz, 3H). C
NMR (75 MHz, CDCl ) δ 193.3, 163.1, 153.2, 142.5, 130.9, 127.4, 124.0,
3
1
23.7, 116.2, 115.9, 114.4, 103.0, 48.0, 28.8, 20.0, 13.7. HRMS (ESI) m/z:
+
calcd for C H NO SNa [M+Na] , 322.0872; found 322.0873.
17
17
2
2
.9 Synthesis of compound 5
P-phenylenediamine (173 mg, 1.6 mmol) was dissolved in 12 mL absolute
ethanol, and then a solution of compound (100 mg, 0.33 mmol) in 5 mL
4
ethanol was added dropwise. The resulting mixture was stirred at room
temperature under an argon atmosphere and TLC was used to monitor the
reaction. After completion, the precipitation was filtrated, washed by cool
2
2

ACCEPTED MANUSCRIPT
ethanol and dried to afford compound (95 mg, 74% yield) as red powder
5
1
solids. Mp: 194-195°C. H NMR (300 MHz, DMSO-d6) δ 8.78 (s, 1H), 7.56 (d,
J = 8.8 Hz, 1H), 7.28 (d, J = 8.5 Hz, 1H), 7.14 (s, 1H), 7.22-7.14 (m, 2H),
7
1
2
1
5
.07-6.96 (m, 2H), 6.79 (d, J = 8.8 Hz, 1H), 6.64 (d, J = 8.7 Hz, 1H), 5.53 (s,
H), 3.93 (t, J = 6.9 Hz, 2H), 1.78–1.51 (m, 2H), 1.42 (dd, J = 14.9, 7.4 Hz,
13
H),0.90 (t, J = 7.3 Hz,3H). C NMR (75 MHz, DMSO-d6) δ 159.8, 152.8,
49.8, 142.9, 132.6, 131.2, 128.3, 127.8, 124.3, 122.5, 117.1, 114.0, 109.5,
6.4, 47.5, 29.0, 19.6, 19.0, 14.0. HRMS (ESI) m/z: calcd for C H N OSNa
23
23
3
+
[
M+Na] , 412.1460; found 412.1461.
2
.10 Synthesis of PB-NH2
In a 25 mL round-bottom flask, compound
5 (0.136g, 0.825 mmol) and
DIEA (0.208 mL, 1.65 mmol) were dissolved in 4 mL 1,2-dichloroethane. The
mixture was stirred at 80°C for 10 min, and then BF ·Et O (0.208 mL, 1.65
3
2
mmol) was added and the reaction mixture was left stirring at refluxing
condition for 5h. After cooling to room temperature, the reaction mixture was
diluted with 30 mL dichloromethane. The organic phase was washed
successively with NaHCO and brine water, dried over anhydrous sodium
3
sulfate. After removal of the solvent, the residue was further purified by silica
gel flash chromatography using petroleum ether/ethyl acetate (v/v, 1:5) as an
eluent to give pure PB-NH2 (73 mg, 42% yield) as yellow powder. Mp:
1
9
8-100°C. H NMR (300 MHz, CD CN) δ 8.33 (s, 1H),7.30-7.23 (m, 4H), 7.18
3
(
dd, J = 7.6, 1.4 Hz, 1H),7.11-7.01 (m, 2H), 6.73 (d, J = 8.8 Hz, 1H), 6.58 (s,
1
1
1
1
H), 4.46 (s, 1H) ,3.98 (t, J =7.1 Hz, 2H), 1.90 – 1.71 (m, 2H) ,1.47 (dd, J =
13
5.0, 7.4 Hz, 2H), 0.95 (t, J=7.4 Hz, 3H). C NMR (75 MHz, CD CN) δ 160.1,
3
59.2, 154.3, 148.6, 142.2, 132.6, 129.0, 127.7, 127.1, 124.1, 123.8, 123.4,
15.1, 114.4, 103.8, 47.6, 28.3, 19.5, 12.9. HRMS (ESI) m/z: calcd for
+
C H BF N OS [M+H] , 438.1617; found 438.1620.
23
23
2
3
2
.11 Synthesis of Probe 1
In a 10 mL round-bottom flask, PB-NH (50 mg, 0.12 mmol), pyridine (0.018 mL,
2
2
3

ACCEPTED MANUSCRIPT
0
.23 mmol) were dissolved in 2 mL dichloromethane. The mixture was stirred at 0°C
for 5 minute under an argon atmosphere, and then a solution of
,4-dinitrobenzenesulfonyl chloride (61 mg, 0.23 mmol) in 2 mL dichloromethane
2
was added. The resulting mixture was warmed to room temperature and stirred
overnight. The reaction mixture was poured into water, and extracted with
dichloromethane (2×30 mL). Organic layers were combined, dried over anhydrous
sodium sulfate. After removal of the solvent, the residue was further purified by silica
gel flash chromatography using petroleum ether/dichloromethane (v/v, 1:5) as an
eluent to give pure compound Probe 1 (71 mg, 93% yield) as red powder. Mp:
1
1
99-200°C. H NMR (300 MHz, DMSO-d ) δ 11.38 (s, 1H), 8.90 (d, J = 2.2 Hz, 1H),
6
8
.77 (s, 1H), 8.60 (dd, J = 8.7, 2.2 Hz, 1H), 8.28 (d, J = 8.7 Hz, 1H), 7.51 (d, J = 8.7
Hz, 2H), 7.43 (s, 1H), 7.26-7.22(m, 3H), 7.16 (dd, J = 18.6, 7.9 Hz, 2H), 7.03 (d, J =
7
1
1
1
1
6
.5 Hz, 1H) ,6.63 (s, 1H), 3.98 (t, J = 7.0 Hz, 1H), 1.75–1.55 (m, 2H), 1.39 (dd, J =
4.9, 7.4 Hz, 2H), 0.87 (t, J = 7.4 Hz, 3H). 13C NMR (75 MHz, DMSO-d ) δ 161.7,
6
59.9, 154.2, 150.0, 147.7, 141.3, 138.9, 136.0, 135.9, 131.5, 129.7, 127.8, 127.2,
27.0, 124.2, 123.8, 122.3, 121.1, 120.3, 117.0, 114.1, 110.9, 103.3, 47.0, 27.9, 19.1,
+
3.4. HRMS (ESI) m/z: calcd for C H BF N O S Na [M+Na] , 692.1232; found
2
9
24
2
5
7 2
92.1237.
4
. Conclusions
In summary, by introducing electron-rich phenothiazine moiety to the boranil
dye, we have successfully developed a novel boron difluoride complex
PB-NH2 PB-NH2 displays much superior photophysical properties to those of
.
classical BODIPYs, such as deep red emission, large Stokes shift and strong
emission in solid state. By taking advantage of this robust platform, a novel
fluorescent probe 1 was designed and synthesized. Notably, Probe 1 was
ultrasensitive in response to thiophenol, the theoretical detection limit was
calculated to be 1.4 nM and the experimental detection limit was 10 nM, to the
best of our knowledge, it was the lowest experimental detection limit that has
2
4

ACCEPTED MANUSCRIPT
ever been reported. More importantly, Probe 1 was successfully applied to
detect thiophenol in real-water samples, in serum, on filter paper strip and in
living HeLa cells, suggesting that Probe 1 has great potential for practical
applications both in environmental and biological systems.
Acknowledgements
This work was supported by the Guangxi Natural Science Foundation
(
2016GXNSFBA380229) and “BAGUI Scholar” program of Guangxi Province
of China.
Appendix A. Supplementary data
Electronic Supplementary Information (†ESI) available in the online version at
http://dx.doi.org/xxx/xxx.
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●
●
●
●
Novel amino phenothiazine boranil based fluorescent probe (Probe 1) for
thiophenol was constructed.
Probe 1 features significant red fluorescence enhancement and remarkable Stokes
shift in response to thiophenol in aqueous solution.
Probe 1 was ultrasensitive in response to thiophenol with a detection limit of 1.4
nM.
Probe 1 was successfully applied to detect thiophenol in real water samples, in
serum, on filter paper strips and in living cells.