Synthesis of ethynylated phenothiazine based fluorescent boronic acid probes

Article abstract of DOI:10.1007/s10895-010-0791-1

Ethynylated phenothiazine based fluorescent boronic acid probes were prepared. Sonogashira coupling reaction was used to introduce substituted phenylethynylene fragments to the phenothiazine fluorophore to extend the π-conjugation and to enhance the emission property. The photophysical properties and the binding properties of these probes with hydroxyl acids were investigated. We found that the probes with significant ICT effect show emissions which are sensitive to solvent polarity. The phenothiazine moiety is proved to be electron-donating. We found the substitution profile imparts significant effect on the photophysical properties of the probes. For example, one of the probes shows d-PeT effect, whereas the regioisomer probe with similar π-conjugation fragment but different substitution profile shows the a-PeT effect. The easy derivatization of phenothiazine fluorophore, the structure-photophysical property relation and the novel d- PeT fluorescence transduction profile of the phenothiazine based probes described herein may inspire more investigation into this fascinating research area. Springer Science+Business Media, LLC 2011.

Full text of DOI:10.1007/s10895-010-0791-1

J Fluoresc (2011) 21:1143–1154
DOI 10.1007/s10895-010-0791-1
ORIGINAL PAPER
Synthesis of Ethynylated Phenothiazine Based Fluorescent
Boronic Acid Probes
Yubo Wu & Huimin Guo & Jingyin Shao & Xin Zhang &
Shaomin Ji & Jianzhang Zhao
Received: 20 September 2010 /Accepted: 9 December 2010 /Published online: 7 January 2011
#
Springer Science+Business Media, LLC 2011
Abstract Ethynylated phenothiazine based fluorescent bo-
ronic acid probes were prepared. Sonogashira coupling
reaction was used to introduce substituted phenylethyny-
lene fragments to the phenothiazine fluorophore to extend
the π-conjugation and to enhance the emission property.
The photophysical properties and the binding properties of
these probes with hydroxyl acids were investigated. We
found that the probes with significant ICT effect show
emissions which are sensitive to solvent polarity. The
phenothiazine moiety is proved to be electron-donating.
We found the substitution profile imparts significant effect
on the photophysical properties of the probes. For example,
one of the probes shows d-PeT effect, whereas the
regioisomer probe with similar π-conjugation fragment
but different substitution profile shows the a-PeT effect.
The easy derivatization of phenothiazine fluorophore, the
structure-photophysical property relation and the novel d-
PeT fluorescence transduction profile of the phenothiazine
based probes described herein may inspire more investiga-
tion into this fascinating research area.
Introduction
Fluorescent probes attract much attention due to their
versatile application in chemical, biological and envi-
ronmental science. Binding units and fluorophores are
the two critical components of the probes. Various
sensing mechanisms have been employed for probes,
such as photo-induced electron transfer (PeT) and
intramolecular charge transfer (ICT) [1–12]. However,
the binding motifs of the fluorescent probes are limited to
hydrogen bonding or dative bonding. Probes based on
hydrogen bonds are usually not effective in protic
solvents, due to the strong competition of the solvents
molecules. Concerning this aspect, the boronic acid
probes are special due to their covalent bonding nature
with saccharides or hydroxyl acids analyte [5, 9, 13–15].
Recognition of glucose or α-hydroxyl acids in aqueous
solution have been achieved with fluorescent boronic acid
probes [16–24].
We have been interested in study of boronic acid probes.
For example, chiral recognition of tartaric acids has been
observed with BINOL or anthracene based boronic acid
probes [17–20]. For these PeT boronic acid probes (a-PeT,
i.e. the fluorophore serves as the electron acceptor in the
PeT process), usually the fluorescence emission is intensive
at acidic pH, due to the protonation of the nitrogen atom
and thus the inhibition of the PeT effect (quenching effect)
[1–3]. However, the recognition of tartaric acid is not ideal
at acidic pH, due to the strong background fluorescence and
the minor fluorescence transduction upon analyte binding at
acidic pH. We found that carbazole based boronic acid
probe show the d-PeT effect, i.e. reverse PeT effect, with
fluorophore as the electron donor and protonated amine/
boronic acid group as the electron acceptor [21]. d-PeT
probes show weak emission at acidic pH compared to that
.
.
Keywords Boronic acid Fluorescent chemosensors
.
d-PeT Phenothiazine
Electronic supplementary material The online version of this article
(doi:10.1007/s10895-010-0791-1) contains supplementary material,
which is available to authorized users.
:
:
:
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Y. Wu H. Guo (*) J. Shao X. Zhang S. Ji J. Zhao (*)
State Key Laboratory of Fine Chemicals, School of Chemical
Engineering, Dalian University of Technology,
Dalian 116024, People’s Republic of China
e-mail: guohm@dlut.edu.cn
J. Zhao
e-mail: zhaojzh@dlut.edu.cn

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at neutral or basic pH. This novel fluorescence transduction
is beneficial for the fluorescent sensing of hydroxyl acids,
for which the binding at acidic pH is much stronger than
that at neutral pH.
added n-C4H9Br (8.2 g, 60 mmol). The mixture was
refluxed for 6 h. Then the solvent was removed. The
residue was extracted with dichloromethane (DCM) and
washed with water. The organic phase was dried over
anhydrous Na2SO4. The solvent was removed and the
residue was purified by column chromatography (silica
gel, DCM/petroleum ether, 1/3, V/V). 6.64 g of light
green liquid was obtained, yield: 52.0%. 1H NMR
(CDCl3, 400 MHz, TMS) δ 7.10–7.14 (m, 4 H), 6.86 (t,
2 H, J=7.2 Hz), 6.83 (d, 2 H, J=8.4 Hz), 3.80 (t, 2 H, J=
7.2 Hz), 1.73–1.80 (m, 2 H), 1.38–1.48 (m, 2 H), 0.90 (t,
3 H, J=7.8 Hz).
Herein we extend the d-PeT fluorophore from carbazole
to phenothiazine, which is a strong electron donating
fluorophore, to construct d-PeT fluorescence boronic acid
probes. As the phenothiazine itself shows very weak
emission, therefore we prepared the ethynylated derivatives
by Sonogashira coupling to extend the π-conjugation
system and thus to enhance the fluorescence emission. We
tuned the electronic feature of the phenothiazine by
introducing electron donating and withdrawing groups and
found out that the ICT effect imparts drastic influence on
the emission profile (such as sensitivity toward solvent
polarity) and the PeT effect of the probes. We found the
substitution profile imparts significant effect on the photo-
physical properties of the probes. The regioisomer of probe
5, i.e. probe 1, with similar π-conjugation fragment but
different substitution profile, shows the a-PeT effect.
Furthermore, during the synthesis of the probes, we found
a side reaction, i.e. the C≡C triple bonds of the modified
fluorophore (with electron-deficient appendents) can be
reduced by NaBH4 to C=C double bonds, whereas
NaBH3(CN) does not reduce the C≡C bonds. The feasible
derivatization of phenothiazine makes these boronic acids
interesting platform for further improvement of the selec-
tivity and the photophysical properties. These findings may
inspire new impetus in the area of fluorescent boronic acid
probes and study of the photophysical properties of
fluorescent probes.
10-butyl-10H-phenothiazine-3-carbaldehyde (7)
To stirred DMF (1.48 mL, 0.02 mol) was added dropwise
POCl3 (1.84 mL, 0.01 mol) of at 0 °C; when the solution
turned to white solid, a solution of 6 (5.1 g, 0.02 mol) in
chloroform (12 mL) was added. After the solid was
dissolved, the mixture was refluxed for 16 h and then
poured into ice water. The pH value of the mixture was
brought to 8.0 by addition of sodium bicarbonate. After
30 min of stirring, the mixture was extracted with DCM.
The organic phase was dried over anhydrous Na2SO4. The
solvent was removed and the residue was purified by
column chromatography (silica gel, DCM/petroleum ether,
2/1, V/V). 4.61 g of yellow oil was obtained, yield: 81.3%.
1H NMR (CDCl3, 400 MHz, TMS) δ 9.71 (s, 1 H), 7.51 (d,
1 H, J=8.0 Hz), 7.46 (s, 1 H), 7.08 (t, 1 H, J=8.0 Hz), 7.01
(d, 1 H, J=8.0 Hz), 6.87 (t, 1 H, J=8.0 Hz), 6.79 (d, 1 H, J=
8.0 Hz), 6.76 (d, 1 H, J=8.0 Hz), 3.75 (t, 2 H, J=8.0 Hz),
1.67–1.74 (m, 2 H), 1.36–1.42 (m, 2 H), 0.87 (t, 3 H, J=
8.0 Hz). APCI-mass: m/z calcd for C17H17NOS([M + H]+)
284.1, found 284.0.
Experimental
Materials and General Methods
7-bromo-10-butyl-10H-phenothiazine-3-carbaldehyde (8)
All the chemicals are analytical pure and were used as
received. NMR spectra were taken on a 400 MHz Varian
Unity Inova spectrophotometer. Mass spectra were recorded
with Q-TOF Micro MS spectrometer. UV–vis spectra were
taken on a HP8453 UV-visible spectrophotometer. Fluores-
cence spectra were recorded on a JASCO FP-6500 or a
Sanco 970 CRT spectrofluorometer. Fluorescence quantum
yields were measured with quinine sulfate as the standard
(Φ=0.54 in 0.05 M H2SO4). Fluorescence lifetimes were
measured on a Horiba Jobin Yvon Fluoro Max-4 (TCSPC)
instrument.
To a solution of 7 (4.0 g, 14.1 mmol) in 15 mL of glacial
acetic acid was added dropwise a solution of bromine
(0.72 mL, 14.1 mmol) in 5 mL of glacial acetic acid. The
brown mixture was stirred at room temperature for 2 d.
After addition of 150 mL of water and extracted with
diethyl ether (3×100 mL), the organic layer was dried over
MgSO4. The solvents were removed and the residue was
purified by column chromatography (silica gel, DCM/
petroleum ether, 3/1, V/V). 3.98 g of yellow solid was
1
obtained, yield: 78.0%. Mp: 78–79 °C. H NMR (CDCl3,
400 MHz, TMS) δ 9.75 (s, 1 H), 7.57 (d, 1 H, J=8.4 Hz),
7.48 (s, 1 H), 7.17 (d, 1 H, J=8.6 Hz), 7.11 (s, 1 H), 6.83
(d, 1 H, J=8.4 Hz), 6.64 (d, 1 H, J=8.6 Hz), 3.77 (t, 2 H, J=
7.2 Hz), 1.69–1.76 (m, 2 H), 1.37–1.47 (m, 2 H), 0.90 (t,
3 H, J=7.6 Hz). APCI-mass: m/z calcd for C17H17BrNOS
([M + H]+) 362.0, found 362.0.
10-butyl-10H-phenothiazine (6)
To a stirred solution of phenothiazine (9.95 g, 50 mmol),
CTAB (0.5 g) and NaOH (3.0 g) in 50 mL of acetone was

J Fluoresc (2011) 21:1143–1154
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10-butyl-7-(2-phenylethynyl)-10H-phenothiazine-3-
carbaldehyde (9)
stirring was continued for 0.5 h. The resulting mixture was
evaporated to dryness. The residue was extracted with
water and dichloromethane. The organic layer was separat-
ed and dried over MgSO4. After removing the solvent, the
residual was purified by column chromatography (silica
gel, DCM/MeOH, 40/1, V/V). 90.0 mg of yellow oil was
obtained, yield: 66.0%. 1H NMR (CDCl3, 400 MHz, TMS)
δ 7.48 (d, 1 H, J=8.0 Hz), 7.25–7.34 (m, 10 H), 7.10
(s, 2 H), 6.76–6.87 (m, 2 H), 3.80 (t, 2 H, J=8.0 Hz), 3.79
(s, 2 H), 3.69 (s, 2 H), 1.73–1.8 (m, 2 H), 1.4–1.49
(m, 2 H), 0.92 (t, 3 H, J=7.2 Hz). TOF MS: (C32H30N2S +
H+) calcd 475.2, found 475.3.
To a degassed solution of 8 (500.0 mg, 1.38 mmol) in dry
Et3N (10 mL) and THF (3 mL) were successively added Pd
(OAc)2 (15.5 mg, 0.07 mmol), PPh3 (14.5 mg, 0.06 mmol),
CuI (10.5 mg, 0.06 mmol) and phenylacetylene (211.0 mg,
2.07 mmol). The reaction mixture was refluxed under
nitrogen for 6 h. After removal of the solvent, the residue
was purified by column chromatography (silica gel, DCM/
petroleum ether, 3/1, V/V). 270 mg of yellow solid was
1
obtained, yield: 52.0%. Mp: 31–32 °C. H NMR (CDCl3,
400 MHz, TMS) δ 9.79 (s, 1 H), 7.62 (d, 1 H, J=8.4 Hz),
7.56 (s, 1 H), 7.49–7.51 (m, 2 H), 7.21–7.34 (m, 5 H),
6.88 (d, 1 H, J=8.4 Hz), 6.80 (d, 1 H, J=8.4 Hz), 3.86 (t,
2 H, J=7.2 Hz), 1.74–1.82 (m, 2 H), 1.42–1.51 (m, 2 H),
0.94 (t, 3 H, J=7.2 Hz). APCI-mass: m/z calcd for
C25H21NOS ([M + H]+) 384.1, found 384.0.
Compound (13)
13 was synthesized with similar procedure to that of 12.
Yellow oil was obtained, yield: 73.4%, H NMR (CDCl3,
1
400 MHz, TMS) δ 7.24–7.38 (m, 10 H), 7.09 (s, 1 H), 6.78
(d, 1 H, J=8.0 Hz), 6.74 (d, 1 H, J=8.0 Hz), 6.83 (d, 1 H, J=
8.8 Hz), 3.79–3.83 (m, 4 H), 2.98 (s, 6 H), 1.73–1.80 (m,
2 H), 1.40–1.49 (m, 2 H), 0.92 (t, 3 H, J=8.0 Hz). APCI-
mass: m/z calcd for C34H35N3S ([M + H]+) 518.3, found
518.3.
10-butyl-7-{2-[4-(dimethylamino)phenyl]ethynyl}-10
H-phenothiazine-3-carbaldehyde (10)
10 was synthesized with similar procedure to that of 9.
Yield: 45.0%, yellow solid. Mp: 91–92 °C. 1H NMR
(CDCl3, 400 MHz, TMS) δ 9.80 (s, 1 H), 7.63 (d, 1 H, J=
8.4 Hz), 7.58 (s, 1 H), 7.38 (d, 1 H, J=8.4 Hz), 7.27 (d, 1 H,
J=9.2 Hz), 7.24 (s, 1 H), 6.89 (d, 1 H, J=8.4 Hz), 6.80 (d,
1 H, J=8.4 Hz), 6.67 (d, 1 H, J=8.4 Hz), 3.88 (t, 2 H, J=
7.6 Hz), 3.0 (s, 6 H), 1.77–1.84 (m, 2 H), 1.44–1.53 (m,
2 H), 0.95 (t, 3 H, J=7.6 Hz). APCI-mass: m/z calcd for
C27H26N2OS ([M + H]+) 427.2, found 427.1.
Compound (14)
14 was synthesized with similar procedure to that of 12.
Yellow oil was obtained, yield: 76.6%. H NMR (CDCl3,
1
400 MHz, TMS) δ 7.59 (d, 2 H, J=8.0 Hz), 7.53 (d, 2 H, J=
8.0 Hz), 7.26 (s, 1 H), 7.23–7.33 (m, 7 H), 7.10 (d, 2 H, J=
12.0 Hz), 3.82 (t, 2 H, J=8.0 Hz), 3.79 (s, 2 H), 3.70 (s,
2 H), 1.74–1.81 (m, 2 H), 1.41–1.50 (m, 2 H), 0.92 (t,
3 H, J=8.0 Hz). TOF MS: (C33H29N3S + H+) calcd 500.2,
found 500.0.
4-[2-(10-butyl-3-formyl-10H-phenothiazin-7-yl)ethynyl]
benzonitrile (11)
11 was synthesized with similar procedure to that of 9.
Yellow solid was obtained, yield: 23.0%. Mp: 141–142 °C.
1H NMR (CDCl3, 400 MHz, TMS) δ 9.8 (s, 1 H), 7.55–
7.65 (m, 6 H), 7.31 (d, 1 H, J=8.4 Hz), 7.26 (s, 1 H), 6.91
(d, 1 H, J=8.4 Hz), 3.88 (t, 2 H, J=8.0 Hz), 1.76–1.84 (m,
2 H), 1.43–1.52 (m, 2 H), 0.95 (t, 3 H, J=8.0 Hz). APCI-
mass: m/z calcd for C26H20N2OS ([M + H]+) 409.5, found
409.0.
Probe (1)
12 (90.0 mg, 0.19 mmol), 2-(2-bromomethylphenyl)-1,3,2-
dioxaborinane (58.0 mg, 0.23 mmol) and K2CO3 (95.0 g,
0.69 mmol) were mixed in dry MeCN (5 mL), then the
mixture was refluxed for 10 h under N2. The reaction
mixture was cooled to room temperature and diluted HCl
was added, then the mixture was stirred for further 1 h. The
solvent was removed under vacuum, and DCM was added
to take up the residue. The organic layer was washed with
water and dried over anhydrous MgSO4. The solvent was
removed under vacuum and the residue was purified by
column chromatography (silica gel, DCM/MeOH, 30/1, V/
V). 40 mg of light yellow powder was obtained, yield:
34.5%. Mp: 110–111 °C. 1H NMR (CDCl3, 400 MHz,
TMS) δ 7.79 (s, 1 H), 7.48 (d, 2 H, J=8.0 Hz), 7.27–7.34
(m, 14 H), 6.97 (s, 1 H), 6.80 (m, 2 H), 3.82 (t, 2 H, J=
7.2 Hz), 3.70 (s, 2 H), 3.59 (s, 2 H), 3.38 (s, 2 H), 1.75–
N-{[10-butyl-3-(2-phenylethynyl)-10H-phenothiazin-7-yl]
methyl}phenylmethanamine (12)
To a degassed solution of 9 (110.0 mg, 0.287 mmol) in dry
THF (1.0 mL) and ethanol (5.0 mL) was added benzyl-
amine (40.0 mg, 0.373 mmol). The reaction mixture was
refluxed under nitrogen for 8 h. After the solution was
cooled to room temperature, NaBH4 (54.0 mg, 1.44 mmol)
was added in several portions to the stirred solution and

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J Fluoresc (2011) 21:1143–1154
1.82 (m, 2 H), 1.42–1.51 (m, 2 H), 0.94 (t, 3 H, J=7.2 Hz).
13C NMR (100 MHz, CDCl3/CD3OD) δ 145.1, 144.1,
141.6, 136.7, 136.0, 131.5, 131.3, 130.8, 130.3, 130.2,
130.1, 129.9, 129.0 128.9, 128.5, 128.3, 128.0, 127.7,
127.5, 127.4, 124.4, 123.9, 123.5, 117.0, 115.4, 114.5,
89.2, 88.9, 77.4, 77.1, 76.7, 61.3, 57.3, 56.4, 47.3, 28.9,
20.2, 13.8. ESI-HRMS (C39H37BN2O2 + H+) calcd
609.2747, found 609.2745.
8.0 Hz), 7.03 (d, 2 H, J=8.0 Hz), 6.97 (s, 1 H), 6,80 (d, 2 H,
J=8.0 Hz), 3.82 (t, 2 H, J=8.0 Hz), 3.69 (s, 2 H), 3.58 (s,
2 H), 3.5 (s, 2 H), 1.74–1.82 (m, 2 H), 1.42–1.51 (m, 2 H),
0.93 (t, 3 H, J=8.0 Hz). 13C NMR (100 MHz, CDCl3/
CD3OD) δ 145.9, 143.7, 141.5, 136.7, 135.9, 132.0,
131.8, 131.3, 131.1, 130.4, 129.9, 129.1, 128.9, 128.5,
128.4, 127.7, 127.5, 124.6, 123.7, 118.6, 115.8, 115.5
115.0, 111.1, 93.6, 87.8, 77.3, 77.0, 76.7, 61.4, 57.4, 56.4,
53.4, 47.4, 28.8, 20.1, 13.8. ESI-HRMS (C40H36BN3O2S +
H+) calcd 634.2700, found 634.2722.
Probe (2)
13 (72.0 mg, 0.14 mmol), 2-(2-bromomethylphenyl)-1,3,2-
dioxaborinane (43.0 mg, 0.17 mmol) and K2CO3 (69.0 g,
0.5 mmol) were mixed in dry MeCN (5 mL), the mixture
was refluxed for 10 h under N2. The reaction mixture was
cooled to room temperature and diluted HCl was added,
then the mixture was stirred for further 1 h. The solvent was
removed under vacuum, and the residue was taken up with
DCM. The organic layer was washed with water and dried
over anhydrous MgSO4. The solvent was removed under
vacuum and the residue was purified with column chroma-
tography (silica gel, DCM/MeOH, 30/1, V/V). 32 mg of
yellow powder was obtained, yield: 32.8%. Mp: 137–138 °C.
1H NMR (CDCl3, 400 MHz, TMS) δ 7.73 (s, 1 H), 7.15–
7.29 (m, 11 H), 7.08 (s, 1 H), 6.94 (d, 1 H, J=7.6 Hz), 6.88
(s, 1 H), 6.68 (d, 2 H, J=8.4 Hz), 6.56 (d, 1 H, J=8.4 Hz),
3.43–3.74 (m, 4 H), 2.89 (s, 6 H), 1.65–1.72 (m, 2 H), 1.32–
1.41 (m, 2 H), 0.84 (t, 3 H, J=7.2 Hz). 13C NMR (100 MHz,
CDCl3/CD3OD) δ 150.1, 144.5, 144.4, 141.8, 136.8, 136.1,
132.7, 131.4, 130.5, 130.4, 130.0, 129.1, 128.7, 127.8,
127.8.6, 124.4, 124.1, 118.1, 115.5, 115.1, 112.0, 110.4,
90.5, 86.8, 61.5, 57.4, 56.5, 47.4, 40.4, 29.0, 20.3, 14.0. ESI-
HRMS (C41H42BN3O2S + H+) calcd 652.3169, found
652.3169.
7-bromo-10-butyl-5-oxo-5,10-dihydro-phenothiazine-3-
carbaldehyde (15)
To a stirred solution of (1.3 g, 3.6 mmol) of 8 in 18 mL of
DCM, a solution of meta-chloroperoxybenzoic acid
(3.6 mmol, 85%) in anhydrous DCM (15 mL) was added
dropwise over 30 min at 0–5 °C. The reaction was
completed within 2–4 h (monitored by TLC). The reaction
mixture was washed with 10% potassium hydroxide
solution (2×15 mL), 5% HCl solution (10 mL), and then
saturated NaHCO3 solution (10 mL) and the organic layer
was dried over Na2SO4. After the solvent was removed, the
residue was purified by chromatography (silica gel, DCM/
MeOH, 50/1, V/V). 1.23 g of white solid was obtained,
yield: 90.0%. Mp: 171–172 °C. 1H NMR (CDCl3,
400 MHz, TMS) δ 10.0 (s, 1 H), 8.42 (s, 1 H), 8.13 (d,
1 H, J=8.4 Hz), 8.08 (s, 1 H), 7.73 (d, 1 H, J=8.0 Hz), 7.51
(d, 1 H, J=8.8 Hz), 7.36 (d, 1 H, J=8.8 Hz), 4.24 (t, 2 H, J=
6.4 Hz), 1.93–1.98 (m, 2 H), 1.52–1.62 (m, 2 H), 1.04 (t,
3 H, J=7.6 Hz). APCI-mass: m/z calcd for C17H16BrNO2S
([M + H]+) 379.3, found 379.0.
4-[2-(10-butyl-3-formyl-5-oxo-5,10-dihydro-pheno-thiazin-
7-yl)ethynyl]benzonitrile (16)
Probe (3)
To a degassed solution of 15 (757.0 mg, 2.0 mmol) in dry
Et3N (15 mL) and THF (8 mL) were successively added
Pd(PPh3)4 (69.0 mg, 0.06 mmol), PPh3 (21.0 mg,
0.08 mmol), CuI (15.0 mg 0.08 mmol) and 4-
ethynylbenzonitrile (254.0 mg 2.0 mmol). The reaction
mixture was refluxed under nitrogen for 6 h. The resulting
mixture was evaporated to dryness. The residue was
extracted with taken up with DCM and washed with
water. The organic layer was dried over MgSO4. The
solvent was removed and the residue was purified by
chromatography with silica gel (acetone/petroleum ether,
1/1, V/V). 297 mg of yellow solid was obtained, yield:
35.0%. Mp: 201–202 °C. 1H NMR (CDCl3, 400 MHz,
TMS) δ 10.02 (s, 1 H), 8.45 (s, 1 H), 8.15 (d, 2 H, J=
8.0 Hz), 7.79 (d, 1 H, J=8.0 Hz), 7.65 (d, 2 H, J=8.0 Hz),
7.61 (d, 2 H, J=8.0 Hz), 7.54 (d, 2 H, J=8.0 Hz), 7.47 (d,
14 (170.0 mg, 0.34 mmol), 2-(2-bromomethylphenyl)-
1,3,2-dioxaborinane (113.0 mg, 0.44 mmol) and K2CO3
(182.0 mg, 1.32 mmol) were mixed in dry MeCN (8.0 mL),
and the mixture was refluxed for 10 h under N2. The
reaction mixture was cooled to room temperature and
diluted HCl was added, then the mixture was stirred for
further 1 h. The solvent was removed under vacuum, and
DCM was added. The organic layer was washed with water
and dried over anhydrous MgSO4. The solvent was
removed under vacuum and the residue was purified with
column chromatography (silica gel, DCM/MeOH, 30/1, V/
V). 65 mg of yellow powder was obtained, yield: 30.2%.
Mp: 127–128 °C. 1H NMR (CDCl3, 400 MHz, TMS) δ
7.81 (d, 2 H, J=8.0 Hz), 7.60 (d, 2 H, J=8.0 Hz), 7.55 (d,
2 H, J=8.0 Hz), 7.27–7.35 (m, 9 H), 7.14 (d, 2 H, J=

J Fluoresc (2011) 21:1143–1154
1147
2 H, J=12.0 Hz), 4.28 (t, 2 H, J=8.0 Hz), 1.94–2.02 (m,
2 H), 1.55–1.65 (m, 2 H), 1.07 (t, 3 H, J=8.0 Hz). APCI-
mass: m/z calcd for C26H20N2O2S ([M + H]+) 425.5,
found 425.0.
3-bromo-10-butyl-10H-phenothiazine (18)
To a solution of 6 (1.4 g, 5.48 mmol) in chloroform
(10 mL) was added a solution of NaOH (0.66 g, 6.44 mmol)
in glacial acetic acid (40 mL), then to this solution was
added dropwise a solution of bromine (0.28 mL, 5.48 mmol)
in glacial acetic acid (6 mL) at 0 °C. The mixture was
stirred at 0–5 °C for 1 h. The solvents were removed, after
addition of 50 mL of water and 100 mL of dichloro-
methane, the organic layer was dried with MgSO4. The
solvent was removed and the residue was purified by
column chromatography (silica gel, DCM/petroleum ether,
1/3, V/V). 1.28 g of light yellow liquid was obtained, yield:
4-[2-[3-[(benzylamino)methyl]-10-butyl-5-oxo-5,
10-dihydro-phenothia-zin-7-yl]ethynyl] benzonitrile (17)
To a degassed solution of 16 (8.0 mg, 0.66 mmol) in dry THF
(2 mL) and ethanol (8 mL) was added benzylamine (92.0 mg,
0.86 mmol). The reaction mixture was refluxed under nitrogen
for 8 h. After the solution was cooled to room temperature,
(207 mg, 3.3 mmol) of NaBH4 was added in portions to the
stirred solution and the stirring was continued for 0.5 h. The
resulting mixture was evaporated to dryness. The residue was
taken up with DCM and washed with water. The organic layer
was dried over MgSO4. After removing the solvent, the
residual was purified by column chromatography (silica gel,
DCM/MeOH, 20/1, V/V). 130.0 mg of yellow solid was
obtained, yield: 38.0%. Mp: 76–77 °C. 1H NMR (CDCl3,
400 MHz, TMS) δ 8.12 (s, 1 H), 7.94 (s, 1 H), 7.70 (d, 2 H,
J=8.0 Hz), 7.60–7.66 (m, 4 H), 7.33–7.42 (m, 7 H), 4.20
(t, 2 H, J=8.0 Hz), 3.90 (s, 1 H), 3.85 (s, 1 H), 1.91–1.98
(m, 2 H), 1.53–1.62 (m, 2 H), 1.05 (t, 3 H, J=8.0 Hz). TOF
MS: calcd for C26H20N2O2S ([M + H]+) 516.2, found 516.4.
1
70.0%. H NMR (CDCl3, 400 MHz, TMS) δ 7.17 (d, 2 H,
J=8.0 Hz), 7.06–7.13 (m, 2 H), 6.86 (t, 1 H, J=8.0 Hz),
6.80 (d, 1 H, J=8.0 Hz), 6.60 (t, 1 H, J=8.0 Hz), 3.73 (t,
2 H, J=8.0 Hz), 1.66–1.75 (m, 2 H), 1.35–1.45 (m, 2 H),
0.88 (t, 3 H, J=8.0 Hz). APCI-mass: m/z calcd for
C16H16BrNS ([M + H]+) 335.3, found 335.1.
10-butyl-3-ethynyl-10H-phenothiazine (19)
To a degassed solution of 18 (1.48 g, 4.43 mmol) in dry
Et3N (8 mL) and THF (5 mL) were successively added Pd
(PPh3)2Cl2 (124.0 mg, 0.17 mmol), PPh3 (46.5 mg,
0.17 mmol), and CuI (33.7 mg, 0.17 mmol) and ethynyl-
trimethylsilane (435.0 mg, 4.43 mmol). The reaction
mixture was refluxed under nitrogen for 6 h. Then K2CO3
(1.65 g, 12 mmol) and methanol (5 mL) were added and the
solution was stirred for 1 h at room temperature. The
solvents were removed. The residue was taken up with
DCM and washed with water. The organic layer was dried
over MgSO4. After the solvent was removed, the residue
was purified by column chromatography (silica gel, DCM/
petroleum ether, 1/6, V/V). 1.04 g of yellow liquid was
obtained, yield: 84.0%. 1H NMR (CDCl3, 400 MHz, TMS)
δ 7.23 (m, 2 H), 7.09–7.16 (m, 2 H,), 6.90 (t, 1 H, J=
8.0 Hz), 6.84 (d, 1 H, J=8.0 Hz), 6.75 (d, 1 H, J=8.4 Hz),
3.81 (t, 2 H, J=7.2 Hz), 3.03 (s, 1 H), 1.73–1.80 (m, 2 H),
1.40–1.49 (m, 2 H), 0.92 (t, 3 H, J=8.4 Hz). APCI-mass:
m/z calcd for C18H17NS([M + H]+) 280.1, found 280.0.
Probe (4)
17 (100.0 mg, 0.19 mmol), 2-(2-bromomethylphenyl)-
1,3,2-dioxaborinane (63.0 mg, 0.24 mmol) and K2CO3
(100.0 g, 0.72 mmol) were mixed in dry THF (1 mL) and
MeCN (5 mL) and refluxed for 10 h under N2. The reaction
mixture was cooled to room temperature and diluted HCl
was added, then the mixture was stirred for further 1 h. The
solvent was removed under vacuum, and DCM was added
to take up the residue. The organic layer was washed with
water and then dried over anhydrous MgSO4. The solvent
was removed under vacuum and the residue was purified by
column chromatography (silica gel, DCM/MeOH, 20/1, V/
V). 38.0 mg of yellow powder was obtained, yield: 30.0%.
Mp: 151–152 °C. 1H NMR (CDCl3, 400 MHz, TMS) δ
8.14 (s, 1 H), 7.77–7.82 (m, 3 H), 7.62–7.68 (m, 4 H), 7.57
(d, 1 H, J=8.0 Hz), 7.42 (d, 2 H, J=8.0 Hz), 7.28–7.37 (m,
7 H), 7.18 (d, 2 H, J=8.0 Hz), 4.23 (t, 2 H, J=8.0 Hz),
3.63–3.77 (m, 6 H), 1.92–1.99 (m, 2 H), 1.55–1.64 (m,
2 H), 1.07 (t, 3 H, J=8.0 Hz). 13C NMR (100 MHz, CDCl3/
CD3OD) δ 141.1, 138.2, 137.2, 136.0, 135.8, 135.4, 134.9,
132.9, 132.1, 132.0, 131.2, 130.6, 130.1, 129.8, 128.6,
128.1, 127.8, 127.5, 123.4, 123.0, 118.5, 116.3, 116.1,
115.6, 111.4, 92.3, 88.6, 61.4, 57.4, 55.9, 49.6, 49.4, 49.1,
48.9, 48.7, 48.1, 28.4, 20.0, 13.7. ESI-HRMS
(C40H36BN3O3 + H+) calcd 650.2649, found 650.2629.
3-[2-(10-butyl-10H-phenothiazin-7-yl)ethynyl]
benzaldehyde (20)
To a degassed solution of 19 (0.50 g, 1.79 mmol) in dry
Et3N (5 mL) and THF (3 mL) were successively added Pd
(PPh3)4 (62.0 mg, 0.053 mmol), CuI (10.2 mg, 0.053 mmol)
and 3-bromobenzaldehyde (397 mg, 4.43 mmol). The
reaction mixture was refluxed under nitrogen for 6 h. The
solvents were removed. The residue was taken up with
DCM and washed with water. The organic layer was dried

1148
J Fluoresc (2011) 21:1143–1154
Scheme 1 Synthesis of the
6-Ethynylated Phenothiazine
Boronic acid Probes 1–3. i)
Phenothiazine, CTAB, NaOH,
n-C4H9Br, acetone, reflux, 6 h,
yield 60%; ii) POCl3, DMF,
CHCl3, reflux, 16 h, 81%; iii)
bromine, glacial acetic acid,
room temperature for 24 h; iv)
Pd(PPh3)2Cl2, CuI, Et3N, PPh3,
4-ethynyl-N,N-dimethylaniline/
phenylacetylene/4-ethynylben-
zonitrile, argon atmosphere,
reflux, 6 h, 23–52%; v) benzyl-
amine, ethanol, THF,
S
N
S
N
CHO
Br
S
N
CHO
S
(i)
(iii)
(ii)
N
H
8
6
7
R
R
S
N
S
N
CHO
N
H
(v)
(iv)
reflux, 8 h, then NaBH4 for 9/
10, NaBH3(CN) for 11, 0 °C,
30 min; vi) acetonitrile, K2CO3,
2-(2-bromomethylphenyl)-1,3,2-
dioxaborinane, reflux, 10 h,
30–35%
12: R = -H
13: R = -NMe2
14: R = -CN
9: R = -H
10: R = -NMe2
11: R = -CN
R
HO
OH
B
S
N
(vi)
N
1 R = -H
2: R = -NMe2
3: R = -CN
Scheme 2 Synthesis of the
sulfur-oxidized 6-ethynylated
Phenothiazine Boronic acid
Probes 4. i) meta-
NC
O
S
O
S
Br
S
N
CHO
Br
CHO
CHO
(ii)
(i)
chloroperoxybenzoic acid, dry
DCM; ii) Pd(PPh3)4, CuI, Et3N,
4-ethynylbenzonitrile, argon at-
mosphere, reflux, 6 h, 35%; iii)
benzylamine, ethanol, THF,
reflux, 8 h, then NaBH3(CN),
0 °C, 30 min, 38%; iv) acetoni-
trile, K2CO3, 2-(2-bromome-
thylphenyl)-1,3,2-dioxabori-
nane, reflux, 10 h, 30%
N
N
16
8
15
NC
O
S
N
(iv)
H
(iii)
N
NC
HO
OH
17
O
B
S
N
N
4

J Fluoresc (2011) 21:1143–1154
1149
Scheme 3 Synthesis of the
3-ethynylated Phenothiazine
Boronic acid Probe 5. i) bro-
mine, glacial acetic acid, 0 °C,
1 h, 70%; ii) Pd(PPh3)2Cl2, CuI,
Et3N, PPh3, ethynyltrimethylsi-
lane argon atmosphere, reflux,
6 h, then K2CO3, MeOH, 1 h,
84%; iii) Pd(PPh3)4 CuI, Et3N,
3-bromobenzaldehyde, nitrogen
atmosphere, reflux, 6 h, 64%; iv)
8, benzylamine, ethanol, THF,
reflux, 8 h, then NaBH3(CN),
0 °C, 30 min, v) acetonitrile,
K2CO3, 2-(2-bromomethyl-
phenyl)-1,3,2-dioxaborinane,
reflux, 10 h, 25%
S
N
S
N
Br
S
N
(ii)
(i)
19
18
6
H
N
CHO
S
N
(iv)
S
(iii)
N
21
20
OH
B
HO
N
(v)
S
N
5
over MgSO4. After the solvents were removed, the residue
was purified by column chromatography (silica gel, DCM/
petroleum ether, 1/1, V/V). 0.44 g of yellow oil was
obtained, yield: 64.0%. 1H NMR (CDCl3, 400 MHz, TMS)
δ 10.0 (s, 1 H), 7.98 (s, 1 H), 7.79 (d, 1 H, J=8.0 Hz), 7.71
(d, 1 H, J=7.6 Hz), 7.47 (t, 1 H, J=7.6 Hz), 7.28 (t, 2 H, J=
6.0 Hz), 7.10–7.17 (m, 2 H), 6.90 (t, 1 H, J=7.6 Hz), 6.84
(d, 1 H, J=8.0 Hz), 6.79 (d, 1 H, J=8.4 Hz), 3.83 (t, 2 H, J=
7.2 Hz), 1.75–1.82 (m, 2 H), 1.41–1.57 (m, 2 H), 0.92 (t,
3 H, J=8.0 Hz). APCI-mass: m/z calcd for C25H21NOS
([M + H]+) 384.1, found 384.0.
(209.0 mg, 1.72 mmol). The reaction mixture was refluxed
under nitrogen for 8 h. After the solution was cooled to r.t.,
NaBH3(CN) (216.0 mg, 3.45 mmol) was added in several
portions to the stirred solution and the stirring was
continued for 0.5 h. The resulting mixture was evaporated
to dryness. The residue was taken up with DCM and
washed with water. The organic layer was dried over
MgSO4. After removing the solvent the residual was
purified by column chromatography (silica gel, DCM/
MeOH, 30/1, V/V). 199.0 mg of yellow oil was obtained,
yield: 36.0%. 1H NMR (CDCl3, 400 MHz, TMS) δ 7.44 (d,
1 H, J=8.0 Hz), 7.21–7.38 (m, 10 H), 7.10–7.16 (m, 2 H),
6.89 (t, 1 H, J=8.0 Hz), 6.84 (d, 1 H, J=8.0 Hz), 6.77 (d,
1 H, J=8.0 Hz), 3.78–3.86 (m, 3 H), 3.56–3.67 (m, 2 H),
1.74–1.81 (m, 2 H), 1.42–1.50 (m, 2 H), 1.37 (d, 3 H, J=
6.8 Hz), 0.92 (t, 3 H, J=7.2 Hz). TOF MS: (C33H31N2S + H+)
calcd 489.2, found: 489.1.
N-{[3-[2-(10-butyl-10H-phenothiazin-7-yl)ethynyl]
phenyl]methyl}-1-phenylethanamine (21)
To a solution of 20 (440.0 mg, 1.15 mmol) in dry THF
(1 mL) and ethanol (5 mL) was added 1-phenylethanamine
Fig. 1 UV–vis absorption
spectra (a) and Fluorescence
1.2
1.2
5
1
2 3
4
b
a
emission spectra (b) of probes
1
2
3
4
5
1.0
0.8
0.6
0.4
0.2
0.0
1–5 (1.0×10−5 mol dm−3) in
0.9
0.05 mol dm−3 NaCl ionic
buffer (52.1% methanol in
0.6
water). pH 7.0, 25 °C
0.3
0.0
240 360 480 600 720
Wavelength/nm
360 420 480 540 600 660
Wavelength/nm

1150
J Fluoresc (2011) 21:1143–1154
Table 1 Photophysical parameters of probes 1–5
εa (M−1 cm−1
)
1abs (nm)
1em (nm)
Stokes Shift (nm)
Φb (pH 4.0)
Φb (pH 7.0)
τc (ns)
krd (107 s−1
)
knr (107 s−1
e
)
1
2
3
4
5
4.44×103
1.57×104
5.50×104
6.82×104
7.41×103
358
337
389
360
349
485
448
463
474
489
127
111
74
0.30
0.59
0.22
0.38
0.20
0.14
0.51
0.14
0.12
0.52
4.46
3.98
3.46
1.93
3.32
3.14
12.8
6.36
7.77
4.52
19.3
12.3
22.5
44.0
25.6
114
140
a In 5.0×10−2 mol dm−3 NaCl ionic buffer (52.1% methanol in water), pH 7.0; b Fluorescence quantum yields, with quinine sulfate as the standard (Φ=
0.54 in 0.05 M H2SO4); c Fluorescence lifetimes, with typical error of 0.01 ns. Concentrations of the probes are 3.0×10−5 mol dm−3 d Radiative decay rate
;
constants at pH 7.0, kr=Φ/τ; e Non-radiative decay rate constants at pH 7.0, knr=(1–Φ)/τ
Probe (5)
14 H), 6.86–6.92 (m, 2 H), 6.80 (d, 1 H, J=8.0 Hz), 4.06
(d, 1 H, J=8.0 Hz), 3.85 (s, 2 H), 3.66 (s, 2 H), 3.32–3.41
(s, 2 H), 1.75–1.82 (m, 2 H), 1.57 (d, 3 H, J=8.0 Hz), 1.43–
1.50 (m, 2 H), 0.92 (t, 3 H, J=8.0 Hz). 13C NMR
(100 MHz, CDCl3/CD3OD) δ 145.3, 144.6, 132.4, 131.2,
130.7, 130.2, 130.0, 129.9, 129.2, 129.0, 128.3, 128.2,
127.7, 127.4, 127.3, 127.2, 124.7, 124.1, 123.2, 122.6,
116.8, 115.5, 115.0, 89.0, 88.8, 58.2, 57.1, 53.4, 47.1, 41.8,
28.8, 20.0, 13.6. ESI-HRMS (C39H37BN2O2S + MeOH-
H2O + H+) calcd 623.2904, found 623.2895.
21 (199.0 mg, 0.41 mmol), 2-(2-bromomethylphenyl)-
1,3,2-dioxaborinane (125.0 mg, 0.49 mmol) and K2CO3
(170.0 mg, 1.23 mmol) were mixed in dry MeCN (5 mL),
refluxed for 10 h under N2. The reaction mixture was
cooled to room temperature and diluted HCl was added,
then the mixture was stirred for further 1 h. The solvent was
removed under vacuum, and DCM was added to take up
the residue. The organic layer was washed with water and
then dried over anhydrous MgSO4. The solvent was
removed under vacuum and the residue was purified by
column chromatography (silica gel, DCM/MeOH, 40/1, V/
V). 62.5 mg of light yellow powder was obtained, yield:
25.0%. Mp: 104–105 °C. 1H NMR (CDCl3, 400 MHz,
TMS) δ 7.79 (s, 1 H), 7.2–7.42 (m, 12 H), 7.1–7.17 (m,
Results and Discussions
Synthesis Phenothiazine is a well-known electron donor and
has been widely used in supramolecular photochemical
Fig. 2 Selected emission
400
dichloromethane
diethyl ether
spectra of (a) 1 (b) 3 (c) 4 (d) 5
b
a
600
450
300
150
0
(1.0×10−6 mol dm−3) in differ-
diethyl ether
hexane
320
acetonitrile
ent solvents. 1ex=360 nm.
methanol
buffer
0.05 mol dm−3 NaCl ionic
240
buffer (52.1% methanol in
dichloromethane
water). pH 7.0, 25 °C
160
hexane
buffer
acetonitrile
methanol
80
0
375
450
525
600
675
400
480
560
640
Wavelength/nm
Wavelength / nm
1000
375
300
225
150
75
acetonitrile
dichloromethane
buffer
dichloromethane
c
d
diethyl ether
acetonitrile
800
600
400
200
0
methanol
buffer
methanol
hexane
diethyl ether
hexane
0
450
525
600
657
375
450
525
600
675
Wavelength/nm
Wavelength/nm

J Fluoresc (2011) 21:1143–1154
1151
1.12
600
450
300
150
0
probe 4
pH 2.0
b
c
probe 5
300
240
180
120
60
a
pH 11.0
1.04
pH 11.0
0.96
0.88
0.80
pH 2.0
probe 5
probe 4
0
2
4
6
8
10 12
420 490 560 630
Wavelength/nm
420
490
560
630
pH
Wavelength/nm
Fig. 3 a Normalized fluorescence intensity-pH profile of probes 4
and 5, 1.0×10−6 mol dm−3 of probes in 0.05 mol dm−3 NaCl ionic
buffer (52.1% methanol in water); b Fluorescence spectra of probe 4
with variation of pH of the solution, λex=350 nm. c Fluorescence
spectra of probe 5 with variation of pH of the solution, λex=360 nm.
25 °C
systems, in fluorescent dyes and electro optical materials and
bioactive reagents [25–27]. However, very few examples are
reported for fluorescent molecular probes. Herein we prepared
new boronic acid probes with phenothiazine as the fluoro-
phore, with the rational the d-PeT probes may be obtained.
The design rational of the boronic acid probes lies on the
notion that the phenothiazine core is an electron donating
fluorophore, therefore potential d-PeT effect is expected. In
order to investigate the structure–property relationship of
the probes, such as the effect of the electron donating or
withdrawing ability of the substituents on the fluorescence
transduction property, substituents such as NMe2, CN, etc.,
were attached to the phenothiazine core. Phenothiazine was
used as the starting material for synthesis (Scheme 1). First
N-butylation was carried out with NaOH as the base. Then
Vilsmeier reaction was used to induce a -CHO group to the
3-position of the N-butylphenothiazine. Monobromination
was carried out to introduce bromo at the 6-position.
Phenothiazine core does not show strong emission, thus
π-conjugation extension is necessary to make the probe
emissive. Sonogashira coupling was used to extend the
π-conjugation of the fluorophore with introduce of
substituted phenylethynylene groups. It should be point-
ed out that C≡C triplet bonds, instead of C=C double
bonds, were used for extension of the π-conjugation
because it is known that C=C may quench the fluores-
cence and the C≡C bonds, as good electron communica-
tion bridge, will not quench the fluorescence [28, 29].
Reductive amination was carried out to prepare the amine
12-14, 17, 21. Interestingly, we found that the C≡C bond
can be reduced to C=C double bond with NaBH4 at
ambient temperature, especially in the case of compound
11. Therefore, a milder reagent of NaBH3(CN) was used
for the reduction. Reaction of amine 12 and 14 with 2-
(2-bromomethylphenyl)-1,3,2-dioxaborinane leads to the
final boronic acid probe 1–3. All the probes were obtained
with satisfying yields.
In order to probe the effect of electron-donating ability
of the fluorophore core on the photophysics of the probes,
we designed probe 4 as a reference (Scheme 2) [30–34]. In
probe 4, the sulfur atom was oxidized to sulfoxide. We
found drastic different photophysical properties for these
two closely related probes (vie infra). The crucial step of
synthesis of probe 4 is the selective oxidation of aldehyde
8 with m-chloroperoxybenzoic acid under mild condition.
The synthesis procedures are similar to that of the probes
1–3 (Scheme 1).
Furthermore, in order to investigate the regioisomer of the
probes 1–4, a probe with the amine/boronic moiety attached
on the phenyl end of the ethynylene bond, instead of the
phenothiazine core, was designed (probe 5, Scheme 3). The
intermediate 20 was synthesized with acetylene 19 and 3-
Fig. 4 Relative fluorescence
intensity of probe 4 and 5 vs
concentration of L-mandelic
1.44
1.26
b
a
1.35
1.20
1.14
1.08
acid and L-tartaric acid. 1.0×
10−7 mol dm−3 of probe 4 and 5
1.26
in 0.05 mol dm−3 NaCl ionic
buffer (52.1% methanol in
water). For probe 4: λex=
360 nm, λem=445 nm, pH=6.0.
For probe 5: λex=360 nm,
λem=490 nm, pH=4.0. 25 °C
1.17
1.08
0.99
5+L-tartaric acid
5+L-mandelic acid
4+L-mandelic acid
4+L-tartaric acid
1.02
pH 4.0
0.000 0.008 0.016 0.024 0.032 0.040
Concentration/mol dm-3
0.000 0.004 0.008 0.012 0.016
Concentration/mol dm-3

1152
J Fluoresc (2011) 21:1143–1154
Table 2 Stability constants (M−1) of probes 1–5 with tartaric acid and
mandelic acid
absorption at 360 nm, indicating the ICT effect is
diminished. The absorption of 2 at blue-shifted wavelength
indicates a weak ICT effect. These findings may prove
useful for future design of phenothiazine-based fluorescent
probes with red-shifted absorption at red-shifted wave-
length range.
Probes
L-tartaric acid
L-mandelic acid
1
2
3
4
5
(1.64 0.32)×102 (pH 6.0)
(3.64 0.47)×102 (pH 6.0)
(0.63 0.06)×102 (pH 6.0)
(9.77 0.79)×102 (pH 6.0)
(2.08 0.40)×103 (pH 4.0)
(2.32 0.45)×103 (pH 7.0)
(2.67 0.55)×102 (pH 6.0)
(1.42 0.22)×103 (pH 6.0)
(1.62 0.56)×102 (pH 6.0)
(3.62 0.87)×102 (pH 6.0)
(8.67 0.89)×103 (pH 4.0)
(8.02 1.63)×103 (pH 7.0)
Solvent Polarity Dependence of the Emission of the Probes The
ICT effect of the probes 1–5 was different, thus different
solvent polarity sensitivities were expected for these
probes. We investigated the emission of the ethynylated
phenothiazine fluorophore in solvents with different
polarity (Fig. 2). We found that the emission of probe 1
is not sensitive to the polarity of the solvents. For
example, the probe gives similar emission wavelength
and intensity in hexane and methanol. This result indicates
that the ICT feature of the molecule is not significant [1].
Similar profile was observed for the regioisomer of probe
1, i.e. probe 5. Probe 2 shows solvent polarity-independent
emission (see Supplementary Information). For probe 3,
however, the emission is highly sensitive to the solvent
polarity. For example, the emission wavelength red-shifted
from ca. 470 nm to ca. 600 nm with change the solvent
from hexane to menthol, concomitantly the emission
intensity decreased to 3.1% of the value in hexane. This
high sensitivity of the emission intensity to the solvent
polarity indicates that the ICT feature of the 3 is
significant [1].
bromophenylaldehyde. The preparation of 21 was carried out
by the reductive amination of 20 with NaBH3(CN).
UV–vis Absorption and Emission Spectra The excitation
and emission spectra of the probes were investigated and
the results of probes were presented in Fig. 1. The
emission band is centered at 448–489 nm. A Stokes shift
of 74–140 nm was observed. Large Stokes shift is
beneficial for analytical purpose (inner filter effect can be
eliminated) [1, 2]. Many known fluorophores are facing the
limitation of small Stokes shifts, especially for the fused aryl
fluorophores, such as BODIPY, fluorescein, xanthene dyes
such as Rhodamine and carbazole, etc. we found electron-
deficient fluorophore, such as 3, gives the emission at blue-
shifted region.
We found that the probes show moderate fluorescence
quantum yields. Furthermore, the arrangement of the
boronic acid group in the probes imparts significant effect
on the photophysical properties of the probes. For example,
probe 1 and 5 share similar π-conjugation framework,
however, the fluorescence quantum yields are different, as
well as the PeT effect. Probe 1 shows the normal a-PeT
effect, whereas 5 shows the d-PeT effect.
Interestingly, the reference of 3, i.e. probe 4, shows an
emission which is not sensitive to the polarity of the
solvents. For example, the emission of 4 in MeOH is
stronger than that in hexane. The red-shift of the emission
band is also much smaller. This result demonstrates that the
ICT effect of probe 4 is smaller than that of probe 3. This
information will be helpful for design of new phenothiazine
based fluorophores.
The photophysical properties of the 1–5 were summa-
rized in Table 1. Probe 3 shows a strong absorption band at
410 nm, which is due to the ICT effect (phenothiazine as
electron donor and cyano groups as the electron acceptor)
[1, 2, 35]. The sulfone derivative 4 shows a blue-shifted
pH Titration a-PeT vs. d-PeT effect. The emission
intensity-pH relation of the probes was studied. We found
that probes 1, 3 and 4 are normal a-PeT probes, i.e. the
Table 3 Standard deviations and limits of detection of probes 4 and 5 with tartaric acid and mandelic acid
Probe
Standard deviationa
Relative standard deviationa
analyte
limit of detectionb (mol dm−3
)
4 (pH 6.0)
0.5
0.20%
L-tartaric acid
L-mandelic acid
L-tartaric acid
L-mandelic acid
1.15×10−5
1.33×10−6
1.97×10−5
5.33×10−6
5 (pH 4.0)
1.2
0.36%
a Standard deviation of the fluorescence emission intensity of the probes alone. c=1.0×10−7 mol dm−3 in NaCl ionic buffer (52.1% methanol in water), n=
10; b The limit of detection with different analytes is obtained with analytes concentration which gives the fluorescence response equal to three times the
standard deviation

J Fluoresc (2011) 21:1143–1154
1153
emission intensity at neutral and basic pH is diminished,
while the emission is intensified at acidic pH. This is due to
the protonation of the amine N atom at acidic pH and thus
the inhibition of the photo-induced electron transfer from
the N to fluorophore [3, 13].
In summary, we prepared new ethynylated phenothiazine
based fluorescent boronic acid probes and the photo-
physical properties as well as the binding abilities of these
probes with hydroxyl acids were studied. The ICT features
of the probes were varied to study its effect on the
photophysical properties. The probes show fluorescence
emission in the range of 460–487 nm with Stokes shifts of
100–137 nm. d-PeT effect was observed for probe 5, which
shows diminished emission intensity at acidic pH but
intensified emission at neutral pH. Fluorescent recognition
of tartaric acid and mandelic acids were achieved at pH 4.0.
d-PeT effect was observed for probe 5, and normal a-PeT
effects were observed for other probes. The easy derivati-
zation of the phenothiazine core, the photophysical proper-
ties of the probes (large Stokes shift and high fluorescence
quantum yields) and the systematic study of the photo-
physical properties will be useful for design of fluorescent
probes.
Firstly the fluorescence emission intensity-pH profiles
were investigated (Fig. 3). Normal a-PeT effect was
observed for 4, i.e. the emission intensity of the probes is
higher at acidic pH than that at neutral or basic pH. This is
due to the protonation of the N atom at acidic pH, thus the
suppression of the PeT effect (quenching effect) [17–21].
Interestingly, for probe 5, d-PeT effect was observed [21,
36, 37], i.e. the fluorescence emission intensity of the probe is
intensified at neutral and basic pH, but reduced at acidic pH.
We attribute the d-PeT effect to the photo-induced electron
transfer from the fluorophore to the protonated amine/boronic
acid groups. d-PeT probes are ideal for sensing hydroxyl acids
at acidic pH with the boronic acid probes, due to the reduced
background emission of the probes, higher binding constant
compared to neutral pH and the improved fluorescence
transduction efficiency [21, 36–39].
We noticed that the amine precursor of the probes (i.e.
the amines) show much higher apparent pKa values than
the probes (c.a. pKa=5.0, Fig. 4). For example, amine 14
and 17 show apparent pKa value of 7.28 and 7.43,
respectively (see Supplementary information). The much
lower pKa values of the probe are actually not due to the
protonation of the alkyl amine moiety, rather it is due to the
perturbation of the B-N interaction (either a direct B-N
dative bond or solvent molecular inserted intramolecular
hydrogen bond form) [5, 9, 14, 40, 41].
Acknowledgements We thank the NSFC (20972024 and
21073028), the Fundamental Research Funds for the Central Univer-
sities (DUT10ZD212), Ministry of Education (SRFDP-200801410004
and NCET-08-0077), the Royal Society (UK) and NSFC (China-UK
Cost-Share Science Networks, 21011130154), State Key Laboratory
of Chemo/Biosensing and Chemometrics (2008009), the Education
Department of Liaoning Province (2009 T015), State Key Laboratory
of Fine Chemicals (KF0802) and Dalian University of Technology for
financial support.
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