Multiple fluorescent behaviors of phenothiazine-based organic molecules

Article abstract of DOI:10.1016/j.dyepig.2014.06.017

We have designed a conventional one-step Suzuki coupling synthetic method to prepare 3,7-di-aryl substituted 10H-phenothiazine derivatives and investigated their optical behaviors. The compound 3,7-Bis (4-aminophenyl) phenothiazine (compound 1), substituting with electron-donating aniline, can exhibit photodamage behavior toward cancer cells. Furthermore, the compound 1 can form fluorescent organic nanoparticle (FON) in acidic aqueous whereas can emit red fluorescence in alkaline organic solvent. More importantly, compound 1 can be oxidized to manufactured a stable near-IR dye (>950 nm). Alternatively, the control compound 3,7-Bis (4-nitrophenyl) phenothiazine (compound 2) enabled us to determine that an electron-withdrawing group, when attaching on the phenothiazine, is unfavorable for molecular design to manufacture a cation form of NIR dye but is favorable to stabilize the phenothiazinate core and manufacture an anion form of NIR dye.

Full text of DOI:10.1016/j.dyepig.2014.06.017

Dyes and Pigments 112 (2015) 34e41
Contents lists available at ScienceDirect
Dyes and Pigments
journal homepage: www.elsevier.com/locate/dyepig
Multiple fluorescent behaviors of phenothiazine-based organic
molecules
*
Tung-Sheng Hsieh, Jhen-Yi Wu, Cheng-Chung Chang
Graduate Institute of Biomedical Engineering, National Chung Hsing University, Taichung, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 9 April 2014
Received in revised form
We have designed a conventional one-step Suzuki coupling synthetic method to prepare 3,7-di-aryl
substituted 10H-phenothiazine derivatives and investigated their optical behaviors. The compound 3,7-
Bis (4-aminophenyl) phenothiazine (compound 1), substituting with electron-donating aniline, can
exhibit photodamage behavior toward cancer cells. Furthermore, the compound 1 can form fluorescent
organic nanoparticle (FON) in acidic aqueous whereas can emit red fluorescence in alkaline organic
solvent. More importantly, compound 1 can be oxidized to manufactured a stable near-IR dye (>950 nm).
Alternatively, the control compound 3,7-Bis (4-nitrophenyl) phenothiazine (compound 2) enabled us to
determine that an electron-withdrawing group, when attaching on the phenothiazine, is unfavorable for
molecular design to manufacture a cation form of NIR dye but is favorable to stabilize the phenothia-
zinate core and manufacture an anion form of NIR dye.
11 June 2014
Accepted 14 June 2014
Available online 26 June 2014
Keywords:
Phenothiazine
Suzuki coupling reaction
Fluorescent organic nanoparticle
Photodamage
© 2014 Elsevier Ltd. All rights reserved.
Cancer cells
Near-IR dye
1. Introduction
are attributed to its stable radical cation heterocyclic form, which
can be used for photodynamic therapy and exhibit near infrared
The phenothiazine (PTZ) core scaffold is a class of electron-
rich tricyclic nitrogenesulfur heterocycles, which exhibits rela-
tively intense luminescence, high photoconductivities and un-
dergoes reversible oxidation processes. In general, PTZ
derivatives (PTZs) have a low oxidation potential and a high
propensity to form stable radical cations [1,2]. Moreover, the
well-defined electron-donating properties of PTZs [3e6] can be
partially associated with electrophores to alter the oxidation
potentials of PTZs [7,8], especially electronic substitutions in the
fluorescence [17e19]. In this case, the proton 10NeH of PTZ is
indispensable in the formation of a stable radical cation, but this
leads to certain limitations for structural modifications. Thus, few
reports on NIR chromophores from PTZs have been published
and most of the above applications are based on the use of a
protected PTZ structure with covalent substitutions on the ni-
trogen atom at the N-10-position.
Recently, we described the convenient preparation of divinyl-
substituted 10H-phenothiazines at the 3- or 3,7-positions without
a protecting group at the N-10 position and concluded that the
proton detachments from the 10NeH of the PTZs can be used to
construct NIR ionic sensors dyes with dual photophysical proper-
ties, red emission in DMSO solution with NIR absorptive energy and
green emission in aqueous solution [20,21]. These findings indicate
that the deprotonation of these PTZs on the nitrogen atom at the 10
position (10NeH) are critically important and that these fluo-
rophores and NIR chromophores can eventually be switched on by
the proton loss. However, we also described the aggregation-
induced emission enhancement (AIEE) properties and developed
fluorescent organic nanoparticle (FON) models of anionic and
cationic PTZs [21,22]. In particular, the AIEE properties of proton-
ated PTZs were observed for both in vitro spectral studies and
cellular staining and can eventually be used in cancer cell recog-
nizer fluorophores.
3
- or 3,7-positions. Thus, because of its ground state intra-
molecular charge transfer (ICT) and excited-state photoinduced
electron transfer (PET) properties, PTZs are widely used as
organic light-emitting diodes (OLEDs) [3,9], acidebase dyes and
pigments [10,11], semiconductors [12,13], chemical sensors [14]
or near-IR dyes [15,16]. However, most of these applications
use the protected PTZ structure with covalent substitutions on
the nitrogen atom at the 10-position (10NeH becomes 10NeR).
There is a particular examples, such as the methylene blue (MB)
derivatives, the important pharmacological applications of PTZs
*
Corresponding author. Graduate Institute of Biomedical Engineering, National
Chung Hsing University, 250 Kuo Kuang Road, Taichung 402, Taiwan. Tel.: þ886 4
2
2840734x24; fax: þ886 4 228 52422.
E-mail address: ccchang555@dragon.nchu.edu.tw (C.-C. Chang).
http://dx.doi.org/10.1016/j.dyepig.2014.06.017
143-7208/© 2014 Elsevier Ltd. All rights reserved.
0

T.-S. Hsieh et al. / Dyes and Pigments 112 (2015) 34e41
35
In the present study, we describe the convenient preparation of
diaryl-substituted 10H-phenothiazines at the 3,7-positions
compounds for 8 h, for which the DMSO stock solutions were
diluted in serum-free medium before use (1/100, v/v).
(Scheme 1). The optical properties of protonated, deprotonated
and oxidated PTZs were examined, illustrating spectral diversities
including (1) visible fluorophores and NIR chromophores switched
on by the loss of protons at 10NeH, (2) FON based on the AIEE
properties of protonated (cationic) 10H-phenothiazine derivatives,
2.4. Light-induced cytotoxicity assay
The cancer cells were examined in the assay. Varying concen-
trations of compounds were incubated with the cells in the dark for
8 h. Subsequently, the culture medium was removed and fresh
culture medium was added to each well. The plates were irradiated
using a light source, which is described in the next section, and then
(
3) a stable phenothiazinium product with an absorption maxima
of more than 950 nm after an oxidation reaction and (4) the ability
to support extra phototoxicity to cancer cells with super oxide
generation.
ꢀ
incubated overnight at 37 C. All the assays were performed in
triplicate, and the average of the three to five individual runs is
2
. Experimental
presented.
2.1. Material
2.5. Measurement
The general chemicals employed in this study were the highest
Preparation of mixed solutions: first, HCl aqueous solutions with
a pH of 2 were prepared and then directly mixed with THF at vol-
ume ratios of 0, 25, 50, 75, and 100 percent. The light source for the
measurement of reactive oxygen species (ROS) and photodamage
was a UV light (a 20-W power tunable Xenon lamp that passes
through a UV-a mirror module). The light output from the optional
grade available and were obtained from Acros Organic Co., Merck
Ltd., or Aldrich Chemical Co. and were used without further puri-
fication. All of the solvents were of spectrometric grade.
2
.2. Apparatus
2
light guide and collimator lens was an average of 3 mW/cm on the
The absorption spectra were generated using a Thermo Genesys
UVevisible spectrophotometer, and the fluorescence spectra
dish surface. The singlet oxygen yields test from the compounds
that were dissolved in DMSO was determined using a photo-
steady-state method using 1,3-diphenylisobenzofuran (DPBF) as
the scavenger. The superoxide generation from the compounds in
an aqueous solution was determined using the photo-steady-state
6
were recorded using a HORIBA JOBIN-YVON Fluoromas-4 spectro-
fluorometer with a 1 nm band-pass and a 1 cm cell length at room
temperature. The cellular fluorescence image was taken using a
Leica AF6000 fluorescence microscope with a DFC310 FX digital
color camera. The Xenon light source LAX-cute (Asahi Spectra) was
used to measure the singlet oxygen yield and PDT cell death.
method
using
4-((9-acridinecarbonyl)
amino)-2,2,6,6-
tetramethylpiperidin-1-oxyl (TEMPO-9-ac) as the radical detector.
For the fluorescence microscope observation, to collect blue emis-
sion of the compounds, a UV light cube, in which light passed
through a 370 ± 10 nm bp filter and the emission was collected
through a 440 nm lp filter.
2
.3. Cell culture conditions and compound incubation
The HeLa human cervical cancer cells were maintained in
Modified Eagle's Medium (MEM) containing non-essential amino
acids, Earle's salts, L-glutamine, 1 mM sodium bicarbonate, 1 mM
sodium pyruvate, 1% (penicillin þ streptomycin) and 10% fetal
2.6. Synthesis
2.6.1. 4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)aniline: (1a)
4-Bromoaniline (1 mmol) was added into a high pressure bottle
containing the catalyst of bis(triphenylphosphine) palladium(II)
chloride (5 mg, Strem Chemicals), followed by the addition of the
ꢀ
bovine serum (FBS). Cells were cultured at 37 C in a humid at-
2
mosphere with 95% air and 5% CO . Before the microscopic obser-
vation, cells were seeded onto coverslips and incubated for 24 h.
The next day, cells were incubated with various concentrations of
3
solvent pair (20 mL of dioxane/1 mL of Et N) and pinacoborane
2
H N
Br
I
NO
2
H
N
4
-bromoaniline
1-iodo-4-nitrobenzene
(iii)
(
i)
S
phenothiazine
O
O
O
B
NO
2
H
2
N
B
(ii)
O
2
a
1
a
H
N
(
iv)
(iv)
Br
S
Br
H
3
H
N
N
S
S
2
H N
NH
2
2
O N
NO2
1
2
Scheme 1. Synthetic route to produce phenothiazine-based photosensitizers: (i) pinacolborane, Pd(PPh)
bis(pinacolato)diboron, Pd(PPh) Cl , dioxane/Et N, N . (iv) K CO , (o-tol) P, Pd(OAc) , DME/H
O ¼ 5/1, N
3
Cl
, reflux.
2
, dioxane/Et
3 2
N, N . (ii) NBS (N-bromosuccinimide), THF, ice bath. (iii)
3
2
3
2
2
3
3
2
2
2

36
T.-S. Hsieh et al. / Dyes and Pigments 112 (2015) 34e41
(
1.5 mmol, Merck). The bottle was sealed after bubbling 10 min
ꢀ
with nitrogen. After maintaining the system under ~105 C over-
night, the system was cooled to room temperature and then
extracted twice with CH
MgSO and then evaporated in vacuum. The residue was chroma-
tographed on silica gel by hexane/acetone 5:1 (R ~0.4) and
recrystallized from acetone/hexane to produce a white solid (yield:
2 2 2
Cl /H O. The solvent was dried using
4
f
1
7
2
0%). Data for 1a: H NMR (400 Hz, DMSO-d6):
d
¼ 7.31(d, J ¼ 8 Hz,
H), 6.48 (d, J ¼ 8 Hz, 2H), 5.459 (s, 2H), 1.224 (s, 12H).
2.6.2. 4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)
nitrobenzene: (2a)
-Iodo-nitrobenzene (4 mmol) was added into a high pressure
4
bottle containing the catalyst of bis(triphenylphosphine) palladiu-
m(II) chloride (50 mg), followed by the addition of 35 mL of dioxane
3
12 mmol of CH COOK and bis(pinacolato)diboron (4.8 mmol). The
bottle was sealed after bubbling for 10 min with nitrogen. After
ꢀ
keeping the system under ~80 C for 48 h, the system was cooled to
room temperature and then extracted twice with CH
solvent was dried using MgSO and then evaporated in vacuum.
The residue was chromatographed on silica gel by hexane/ethyl
acetate 4:1 (R ~0.5) and recrystallized from acetone/hexane to
produce a yellow solid (yield: 45%). Data for 2a: H NMR (400 Hz,
CDCl ):
¼ 8.20 (d, J ¼ 8 Hz, 2H), 7.97 (d, J ¼ 8 Hz, 2H), 1.23 (s, 12H).
2
Cl
2
/H
2
O. The
Fig. 1. Absorption (left) and emission (right) spectra for neutral and protonated 1 in
DMSO and aqueous solutions, represented by the extinction coefficient and quantum
yield, respectively. The excited wavelengths are 350 nm, and the determination of the
4
quantum yields is relative to quinine sulfate. (Quantum yield
excitation 350 m)).
F
f
¼ 0.58 in DMSO
f
(
1
3
d
ꢀ
system under ~105 C for two days, the system was cooled to room
temperature and then extracted twice with CH Cl /H O. The sol-
2.6.3. 3,7-Dibromo-10H-phenothiazine (3)
2
2
2
A double-necked round bottomed flask was charged with
vent was dried by MgSO and evaporated in vacuum. The residue
4
10 mmol of phenothiazine in THF solution (20 mL). Then, NBS
was chromatographed on silica gel by hexane/acetone (1:1). The
(
22 mmol) was dissolved in 45 mL THF and was added dropwise
red compound was obtained by recrystallizing the residue with
acetone/hexane (yield: 60%). Data for compound 2: H NMR
1
over 1 h with an addition funnel. The reaction was stirred over an
ice bath until complete consumption by thin layer chromatography
(400 Hz, DMSO-d6):
d
¼ 9.15 (s, 1H), 8.32 (d, J ¼ 8.8 Hz, 4H), 7.89 (d,
(
TLC) monitoring. The solvent was evaporated in vacuum, and the
residue was purified via column chromatography (silica, ethyl ac-
etate/hexane. 1/8, v/v, R
¼ 0.4). The final light green products were
crystallized from acetone/ethyl acetate (yield: 74%). Data for 3: H
J ¼ 8.8 Hz, 4H), 7.48 (d, J ¼ 8.4 Hz, 2H), 7.41 (s, 2H), 6.78 (d,
þ
J ¼ 8.4 Hz, 2H). HRMS (ESI, m/z): [MþH] 442.08; found, 442.83;
f
24 15 3 4
Anal. Calcd % for C H N O S: C, 65.30; H, 3.42; N, 9.52; S, 7.26.
1
Found: C, 65.26; H, 3.49; N, 9.49.
NMR (400 Hz, DMSO-d6):
J ¼ 8 Hz, 2H) ppm.
d
¼ 8.848 (s, 1H), 7.122 (m, 4H), 6.590 (d,
2.6.6. 3,7-Bis(4-aminophenyl)phenothiazin-5-ium iodide (4)
The iodine (5 mmol) in chloroform (5 mL) was added dropwise
2
.6.4. 3,7-Bis (4-aminophenyl)phenothiazine (1)
Compound 3 (5 mmol) was added into a high pressure bottle
containing a mixture of palladium (II) acetate (8 mg, Strem
Chemicals), tri-o-tolyl phosphine (80 mg, Aldrich) and K CO (1.2
mmol), to which the solvent pair (5 mL of H O/25 mL of ethylene
to a solution of compound 1 (1 mmol) in chloroform (20 mL). The
ꢀ
temperature during the addition was kept under 20 C with
external cooling as required; the reaction mixture was then stirred
2
3
overnight. The slurry system was filtered, washed with chloroform
2
1
and dried in vacuum (yield: 82%). H NMR (400 Hz, DMSO-d6):
glycol dimethyl ether (DME)) and compound 1a (12.5 mmol) was
then added. The bottle was sealed after bubbling for 10 min with
nitrogen. After maintaining the system under ~105 C for three
days, the system was cooled to room temperature and then
extracted twice with CH Cl /H O. The solvent was dried by MgSO
2 2 2 4
and evaporated in vacuum. The residue was chromatographed on
silica gel by hexane/acetone (2:1). The light green compound was
d
¼ 7.062, 6.475 (b, 8H, aromatic aniline), 6.515, 6.427, 6.045 (b,
6
3
H, phenothiazinium), 4.467 (b, NH
2
of aniline). HRMS (ESI, m/z):
ꢀ
ꢁ
80.12(o-DAP), 507.03(o-DAP þ I ); found, 380.2, 507.20; Anal.
18
Calcd % for C24H N
3
IS: C, 56.81; H, 3.58; N, 8.28; I, 25.01, S, 6.32.
4
$(3H O): C24
2
H N
24 3
IO3S: C, 51.34; H, 4.31; N, 7.48; I, 22.60; O, 8.55;
S, 5.71. Found, C, 52.03; H, 4.33; N, 7.42.
obtained by recrystallizing the residue with acetone/hexane (yield:
1
5
(
6
(
7
5
5%). Data for compound 1: H NMR (400 Hz, DMSO-d6):
d
¼ 8.560
3. Results and discussion
s, 1H), 7.233 (d, J ¼ 8 Hz, 4H), 7.145 (d, J ¼ 8 Hz, 2H), 7.063 (s, 2H),
.662 (d, J ¼ 8 Hz, 2H), 6.568 (d, J ¼ 8 Hz, 4H), 5.141 (s, 4H). HRMS
ESI, m/z): 381.13; found, 381.30; Anal. Calcd % for C24 S: C,
OS: C, 72.15; H,
3.1. Molecular design and basic spectroscopic property
19 3
H N
5.56; H, 5.02; N, 11.01; S, 8.41. 1$(H
2
21
O): C24H N
3
In this investigation, the phenothiazine moiety was selected as the
electron donor because of its good chemical stability and the com-
mercial availability of various useful molecular building blocks. The
aniline moiety and nitrobenzene were selected as the electron
donating and electron withdrawing units to substitute on the 3,7-
positions of phenothiazine and become compound 1 and 2, respec-
tively. Here, we must claim that the pinacolborane was chosen to
synthesize the intermediates compounds 1a from 4-bromoaniline
while bis(pinacolato)diboron was suitable to synthesize the in-
.30; N, 10.52; S, 8.03. Found: C, 72.23; H, 5.28; N, 10.82.
2
.6.5. 3,7-Bis (4-nitrophenyl) phenothiazine (2)
Compound 3 (1.0 mmol) was added into a high pressure bottle
containing a mixture of palladium (II) acetate (10 mg), tri-o-tolyl
phosphine (100 mg) and K CO (2.1 mmol), to which the solvent
pair (5 mL of H O/25 mL of ethylene glycol dimethyl ether (DME))
2
3
2
and compound 2a (4.0 mmol) was then added. The bottle was
sealed after bubbling for 10 min with nitrogen. After keeping the
termediates
compounds
2a
from
4-bromonitrobenzene.

T.-S. Hsieh et al. / Dyes and Pigments 112 (2015) 34e41
37
Consequently, the Suzuki reaction synthesis of 3,7-diaryl substituted
PTZ derivatives was achieved by reacting 3,7-dibromophenothiazine
with phenylboronic pinacol ester, compounds 1a and 2a, in palla-
dium (II) acetate/tri-o-tolyl phosphine complex, exploiting the
organic coupling rearrangement (as Scheme 1). Fig. S1 showed basic
spectra studies; the final product 1 exhibited well-defined extinction
coefficients, quantum yields and unapparent solvatochromic effects,
while bathochromic absorptionwas observed in the compound 2 that
accompanied the dramatic decrease of quantum yields. This result
suggests that the compound 2 may mimic stronger pushepull effects
and electron transfer from the phenothiazine donor to the nitroben-
zene acceptor, which causes the absorption red shift and fluorescence
quench with respect to 1.
3.2. FONs character from cationic PTZ
Fig. 1 reveals the interesting result that the protonation of the
compound 1 resulted in its emission being enhanced, whether in
DMSO or aqueous condition. In most cases, once the electron
transfer occurs from the phenothiazine to the acceptor group, the
ground state intramolecular charge transfer (ICT) is enhanced
[23,24], and the excited state photo-induced electron transfer (PET)
is weakened [25,26]. That is, when the electronic pushepull effi-
ciency of organic fluorescent dye increases, the relative emission
should be quenched, especially under aqueous conditions. There-
fore, the unusual increase in the fluorescence of the protonated 1
(1-H) should be investigated using a special mechanism or model.
At first, the pKa values of 1 were determined by the fitting curves of
acid titration spectra, as shown in Fig. S2. The pKa value for the first
protonation (pKa ~5.12) is higher with respect to aniline (~4.68),
which means this compound is more easily protonated. The ex-
pected stabilization of the protonated species indicates that a
resonance effect between PTZ and protonated aniline moiety in-
creases the basicity of the first protonation site. Obviously, further
protonation at another aniline ring of 1-H (become 1-H2) occurs at
pKa ~2.1. Consequently, excess acids can lead to the protonation of
the 10-position N atom on the PTZ moiety of 1 (c.a. pKa <1). Here,
we want to claim that the first step of protonation did not induce
the fluorescence enhancement, and the protonation of the 10-
position N atom caused the fluorescence decreasing. That is, our
titration results indicated that the emission enhancement pH range
of the compound 1 was pH ¼ 1e3.5.
Fig. 2. Illustrations of AIEE phenomenon and FONs character for 1. (a) Emission
spectral variations under 0, 25, 50, 75 and 100 percent of pH ¼ 2 aqueous solutions
mixing ratios with THF. (The excitation wavelength for each solution is 350 nm). (b)
TEM images of the nanoparticles obtained under pH ¼ 2 aqueous solutions (top, scale
bar: 50 nm) or mixed with THF 25% v/v (bottom, scale bar: 20 nm). The preparation of
the mixed solution is described in experimental section. (c) Fluorescent microscopy
images of the nanoparticles obtained under pH ¼ 2 aqueous solutions (top, scale bar:
2.0 mm) or mixed with THF 25% v/v (bottom, scale bar: 1000 nm).
Additionally, as described in the introduction, one of the
mechanisms to explain the unusual emission enhancement phe-
nomenon is aggregation-induced emission enhancement (AIEE).
This effect involves the restriction of intramolecular rotation, which
leads to efficient non-radiative decay and fluorescence emission
being quenched. The AIEE effect can be easily verified by studying
molecule can aggregate to form micelles and that the AIEE phe-
nomenon is the cause of FONs. Consequently, we illustrated why 1
offered apparent emission enhancement under acidic aqueous and
were able to conveniently prepare solid fluorophore powder of this
compound by removing the solvent under vacuum from acid
the emission spectra of compounds in H
2
O and tetrahydrogenfuran
(
THF) solvent mixtures at various ratios [27]. Fig. 2 demonstrates
aqueous and/or mixing acidic H
2
O/THF solvent system. Alterna-
that the fluorescence exhibited presented interesting changes
when the compound 1 was dissolved in mixing volume ratios of
solutions containing acidic aqueous (pH ¼ 2) and THF. The emission
intensity increased drastically, and the quantum yield reached a
maximum value in 25% volume fractions of the THF. Meanwhile, the
particle character of FON can be examined using TEM and fluo-
rescence microscopy, regardless of whether an aqueous and mixing
solvent system was used. The insets of Fig. 2 reveal very fine
nanoparticles spheres with a mean diameter of 5e20 nm. Based on
the previous studies [20e22] and the above observation, we
considered that protonated 1 should act as a surfactant-like
molecule that can further self-assemble to form micelle-like
nanoparticles due to its amphiphilic characteristic. In the aggre-
gation state, intramolecular rotation is restricted and emission is
thus greatly enhanced. Overall, we propose that the protonated 1
tively, in neutral H O/THF mixed solutions, 1 did not exhibit AIEE.
2
Moreover, the compound 2 did not exhibit the AIEE phenomenon
under the same experimental conditions, whether in neutral or
acidic environments. Thus, we conclude that the FONs model that
was formed with protonation occurring on the aniline moiety of 1 is
appropriate. Nevertheless, the important issue is that the com-
pound can be an acidic fluorophore sensor both in aqueous and
organic solution systems.
3.3. Red fluorescence and NIR absorption from anionic PTZ
Fig. 3 exhibits the effect of deprotonation on the spectral shapes
and
lmax of compound 1 and 2. The lmax values of the molecular
absorptions showed significant bathochromic shifts when a large

38
T.-S. Hsieh et al. / Dyes and Pigments 112 (2015) 34e41
Fig. 4. Absorption spectra of iodine-oxidated compounds: (a) 1 and (b) 2 in DMSO
solution, represented by the extinction coefficient. (a) Also presents the spectrum of
the synthesized product from chloroform, compound 4.
The titration results of Fig. S3 indicated that the deprotonation
process was only one process due to the fine isosbestic point.
However, we assumed that there are two electronic configuration
structures for the deprotonated products, one located in the visible
absorption region and the other located in the NIR absorption re-
gion. These phenomena depended on the electron delocalization
between the 3,7-disubstiuted groups and phenothiazinate anion,
which originate from the deprotonation on 10NeH of phenothia-
Fig. 3. Spectral variations of (a) compound 1 and (b) compound 2 in DMSO in the
presence of NaOH, KOH, tBuOK, Na PO and K CO . The inserts in (a) show the
3
4
2
3
emission spectra of deprotonated 1 (the excited wavelengths was 535 nm) and pho-
tographs with visible emission under UV light (365 nm, top) and under 532 nm laser
light (bottom) of 1 and deprotonated 1. The insert photos in (b) show the white light
image of 2 and deprotonated 2. (c) Structure and dipole illustration of phenothiazinate
¡
¡
anion 1 (1 ) and phenothiazinate anion 2 (2 ).
¡
zine [21]. For the deprotonated 2 (2 of Fig. 3c) case, the electron-
withdrawing nitrobenzene groups can stabilize the phenothiazi-
ꢁ
nate anion (PTZ ) because the electron can delocalize between the
excess of base (~200 eq.) was added to each of the dye solutions in
DMSO. Uniform spectral shifts from 350 nm to 435, 535 and 740 nm
for 1 and red color emission bands were observed at 600 nm in
response to excitation at 535 nm, when were added to NaOH, KOH
and tBuOK the dye solutions. However, no spectral shifts were
phenothiazinate anion and nitrobenzene. Consequently, this effect
reduced the ionic property of the molecules and caused the high
extinction property of the NIR absorption species (825 nm) with
undetectable fluorescence emission. In the alternative case for
deprotonated 1 (1 of Fig. 3c), the electron-donating aniline groups
are not able to stabilize the phenothiazinate anion well; hence, the
ionic property of the 1 is retained. Furthermore, because the ab-
sorption and red emission signals of the
independent, we inferred that a separated ion-pair (SIP) but not a
contact ion-pair (CIP) [28,29] between the 1 and the positive
¡
3 4 2 3
observed when weaker organic bases such as Na PO and K CO
were used. Thus, the proton loss ability (10NeH) of 1 was estimated
to be approximately greater than that of these weak organic bases.
More apparent red shifts were observed for the deprotonation of 2
substituted with a stronger electron withdrawing nitrobenzene
¡
¡
1
were alkali-
¡
group. The
KOH, and tBuOK, while a partial degree of spectral change was
observed in Na PO and K CO . In this case, the strength of the
0NeH proton loss of the 2 was estimated to be roughly compa-
rable to that of Na PO and K CO , as indicated by ~50% of the
25 nm peak increasing at steady state. This result meant that the
lmax for 2 red shifted from 465 to 825 nm in NaOH,
þ
þ
metal ion of bases (Na , K ) was the main solvation form when in
DMSO solvent. Under this condition, the lifetime of PTZ anion may
ꢁ
3
4
2
3
be sufficiently long to observe red fluorescence [30,31]. Overall, the
ion pair-PTZ (535 nm) and delocalized-PTZ species (740 nm) co-
exist, and stable red emission is observed at 600 nm in response
to excitation at 535 nm. Nevertheless, these results indicate that
these compounds have the potential for use in alkaline sensor ap-
plications, whether fluorescence or NIR is used.
1
3
4
2
3
8
acidities of 10NeH for 2 should be stronger than those for 1. This
1
inference became more convincing when we checked the H NMR
6
spectra in DMSO-d solutions. The 10NeH proton on 1 was
shielding (8.548 ppm) with respect to phenothiazine (8.564 ppm),
while that on 2 was deshielding (9.15 ppm). That is, the enhance-
ment of dissociation for the proton on the nitrogen of PTZ was due
to the electron-withdrawing of nitrobenzene at the 3,7-positions of
the PTZs.
3.4. Stable NIR compound from cationic PTZ
Recently, methylene blue analogues have been conveniently
collected by using the oxidation of 10H-phenothiazine with iodine

T.-S. Hsieh et al. / Dyes and Pigments 112 (2015) 34e41
39
Fig. 5. Fluorescent and bright-field photodamage images from the photodamage assays of HeLa cancer cells incubated with (a)e(d) and without (e)e(h) 10
mM 1 for 8 h. (a), (e): the
fluorescent microscopy images (using a 370 ± 10 nm band pass filter as the exciting light source and collected through a 450 nm long pass filter); (b), (f): before irradiation; (c), (g):
irradiation (UV light source, 3 mW/cm2 and 20 min); (d), (h): incubation overnight.
[
32,33]. The oxidated intermediate, phenothiazinium salts, offer
oxidation products as the stable powder 3,7-bis(4-aminophenyl)
phenothiazinium tetraiodide (4), which can be stored for several
months. In addition, the oxidative spectrum exhibited no large
differences with respect to that of the original compound 2, even
active electrophiles and undergo amine nucleophilic substitution at
ring positions 3 and 7 to yield MB derivatives [34]. Furthermore,
these studies also reported that substitution of the phenothiazi-
nium with an electron donor group can lead to more active pho-
tosensitizers, whereas an electron acceptor group decreases the
photosensitizing effect [35,36]. In this investigation, both the
compound 1 and 2 were oxidated with iodine. Fig. 4 shows that the
NIR peaks at 957 nm were observed once the [I ] was added for 1 in
2
DMSO solution, which represents the absorption of oxidated 1
phenothiazinium salt. Furthermore, we successfully collected the
2
with the addition of excess [I ]. This result indicated that the
electron-donating aniline moiety of 1 can stabilize the phenothia-
zinium core; however, the electron withdrawing nitrobenzene
moiety of 2 cannot. Overall, we successfully expend the absorption
range to the NIR region base on di-aryl substituted phenothiazi-
nium and expect that this type of phenothiazinium derivative can
retain the photodynamic superiority of MB.

40
T.-S. Hsieh et al. / Dyes and Pigments 112 (2015) 34e41
progressed photosensitizer [38e40]. However, Fig. S4 presents a
complicated result. Because the absorption of 1 occurs in the UV
region, it is possible to excite the indicators (DPBF and TEMPO)
simultaneously when we irradiate the compound 1 with a UV-A
light source. Our control experiment demonstrates that these two
indicators were photo-responsive themselves to UV light (Fig. S4
(a) and (e)). Moreover, the control experiment also reveals that 1
is photo-active under UV light irradiation; this phenomenon may
be associated with the excited photo-induced proton or electron
transfer mechanism, which requires further study. However, based
on our results, both the disappearance rate of DPBF absorption and
the growth rate of TEMPO emission changed once the 1 was added.
This result indicated that it is very possible that this compound can
undergo the type I and/or type II PDT effect under UV-light illu-
mination. Nevertheless, the combination of the compound 1 dose
and light illumination resulted in quantitative cellular toxicity.
Fig. 6 reveals that the HeLa cell lines displayed no determinable
dark toxicity with the addition of 1 up to a concentration of 20 mM.
2
Fig. 6. Illustrations of dark-toxicity and phototoxicity of HeLa cancer cells when
treated with variable concentrations of 1 and 2 for 8 h before irradiation. The cell death
was monitored overnight and the irradiated conditions following the experimental
assays are described in Fig. 5. Each data point represents the average from four
separate experiments.
In contrast, irradiation with a 3.6 J/cm UV-A light dose resulted in a
significant light-induced toxicity with EC50 values of approxi-
mately 5.2 mM. Although the real photodamage mechanism was
unclear, we propose that 1 is an antibacterial and anticancer
photosensitizer candidate.
3.5. Phototoxicity from neutral PTZ
4. Conclusions
Here, we also examined whether the compounds 1 and 2 can be
We describe a convenient method to prepare novel aryl-
used as cell photosensitizers, Fig. 5 illustrates the photodamage
results obtained using a fluorescence microscope. It is clear that the
plasma membrane bleb formation and acute cell death were caused
by illumination with UV-A light for HeLa cells that were incubated
with the PTZ-A compound. Meanwhile, irradiating-time-
dependent fluorescent decay was also observed, which caused
unclear fluorescent images. In addition, the compound 2 did not
present any photodamage or dark-toxicity to HeLa cells under the
same experimental conditions. It is interesting that the PDT effect of
substituted phenothiazine derivatives with special optical dis-
tinguishing features. The electron rich phenothiazine core was
substituted with electron-donating aniline and electron-
withdrawing nitrobenzene at the 3,7-positions to yield com-
pounds 1 and 2, respectively. As summarized in Scheme 2, the
optical diversities of 1 included: (1) under the acidic aqueous
condition, the fluorescent emission of the compound was enhanced
due to the formation of FON; (2) red emission fluorescence
occurred in alkaline DMSO solution when exciting the visible ab-
sorption wavelength due to the deprotonation of 10NeH; (3) it is
conventional to manufacture a stable NIR dye with oxidation by
iodine; (4) photodamage to cancer cells was observed with low
dark-toxicity. Overall, 1 can be used as a pH sensor fluorophore,
precursor of NIR dye and potential PDT photosensitizer. However,
the control compound 2 enabled us to determine that an electron-
withdrawing group, when attaching on the phenothiazine, is un-
favorable for molecular design to manufacture a cation form of NIR
dye but is favorable to stabilize the phenothiazinate core and
manufacture an anion form of NIR dye.
1
is beyond our expectation because this compound is the pre-
cursor of MB analogues. Hence, the singlet oxygen acceptor 1,3-
diphenylisobenzofuran (DPBF) was used to detect the
photosensitizer-generated singlet oxygen, which belonged to the
type II mechanism of PDT. This reaction was achieved experimen-
tally by following the disappearance of the 410 nm absorbance
band of DPBF [37]. Meanwhile, the free radical probe 4-((9-
acridinecarbonyl)
amino)-2,2,6,6-tetramethylpiperidin-1-oxyl
(
TEMPO-9-ac), which can capture radicals and result in fluores-
cence (lex/em ¼ 358/440 nm), can be an indicator for a type I-
Red emission fluorophore
N
S
Stable NIR dye
(>950 nm)
Fluorescent Organic
Nanoparticle (FON)
1-
H
2
N
NH
2
-
H
I
N
-
N
+
S
[
OH ]/DMSO
S
H
N
H
3
+N
1- H2
N H
+
3
H
2
N
NH
2
4
S
+
[
H ]/H O
H
2
N
1
NH
2
[I ]/Oxidation
2
2
Incubated to cells
Photodamage to cancer cells
Scheme 2. Special optical properties and multiple applications of compound 1 in this manuscript.

T.-S. Hsieh et al. / Dyes and Pigments 112 (2015) 34e41
41
Acknowledgments
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