Synthesis and characterization of near-infrared absorbing water soluble squaraines and study of their photodynamic effects in DLA live cells

Article abstract of DOI:10.1111/php.12236

Here, we report the synthesis, photophysical properties and photodynamic effects in DLA live cells of three water soluble squaraine dyes, viz. bisbenzothiazolium squaraine dyes SQMI and SQDI with iodine in one and both benzothiazolium units, respectively, and an unsymmetrical squaraine dye ASQI containing iodinated benzothiazolium and aniline substituents. The diiodinated SQDI showed an anomalous trend in both fluorescence and triplet quantum yields over the monoiodinated SQMI, with SQDI showing higher fluorescence and lower triplet quantum yields compared to SQMI. Nanosecond laser flash photolysis of SQDI and SQMI indicated the formation of triplet excited states with quantum yield of 0.19 and 0.26, respectively. On photoirradiation, both the SQDI and SQMI generate singlet oxygen and it was observed that both dyes undergoing oxidation reactions with the singlet oxygen generated. ASQI which exhibited a lower triplet quantum yield of 0.06 was, however, stable and did not react with the singlet oxygen generated. In vitro cytotoxicity studies of these dyes in DLA live cells were performed using Trypan blue dye exclusion method and it reflect an order of cytotoxicity of SQDI>SQMI>ASQI. Intracellular generation of the ROS was confirmed by dichlorofluorescein assay after the in vitro PDT. Synthesis, photophysical properties and photodynamic effects in DLA live cells of three water soluble squaraine dyes, SQDI, SQMI and ASQI, are presented. On photoirradiation, both the SQDI and SQMI generate singlet oxygen where both the dyes undergoing oxidation reactions with the singlet oxygen generated, whereas ASQI was stable and did not react with the singlet oxygen. In vitro cytotoxicity studies of these dyes in DLA cells were performed using Trypan blue dye exclusion method and intracellular generation of ROS was confirmed using dichlorofluorescein assay after the in vitro PDT.

Full text of DOI:10.1111/php.12236

Photochemistry and Photobiology, 20**, **: *–*
Synthesis and Characterization of Near-Infrared Absorbing Water Soluble
Squaraines and Study of their Photodynamic Effects in DLA Live Cells
Kulathinte M. Shafeekh1, Mohanannair S. Soumya2, Moochikkadavath A. Rahim1, Annie Abraham*2 and
Suresh Das*1
1Photosciences and Photonics Section, Chemical Sciences and Technology Division, National Institute for Interdisciplinary
Science and Technology (NIIST-CSIR), Trivandrum, India
2Department of Biochemistry, University of Kerala, Trivandrum, India
Received 20 June 2013, accepted 2 January 2014, DOI: 10.1111/php.12236
ABSTRACT
on relaxation to its ground state can initiate two types of cell
destruction pathways termed as Type I and Type II. Type-I path-
way involves the direct interaction of the sensitizer with cell sub-
strate or molecule by electron transfer resulting in the formation
of radical anion or cation of either the photosensitizer or the sub-
strate. Subsequent reaction of these species with oxygen can lead
to the formation of reactive oxygen species (ROS).The Type-II
pathway involves generation of reactive singlet oxygen from
ground state molecular oxygen through a triplet–triplet energy
transfer. The short lifetime and high reactivity of singlet oxygen
leads to reactions with biomolecules in close proximity of its
production (5–7). Thus, both singlet oxygen and the ROS gener-
ated in the Type-I process can initiate free radical chain reactions
which ultimately lead to cell death (8–10).
Most of the earlier photosensitizers clinically used and
reported for PDT were based on porphyrins which are known to
possess absorption mainly in the visible region. Although por-
phyrin-based molecules are being clinically used as PDT sensitiz-
ers (11–13), these dyes absorbing in the visible region suffer
from two major drawbacks, namely, scattering of most of the
excitation light in the inhomogeneous biological tissues by the
macromolecules present and competitive absorption by natural
biological absorbers. These two effects are minimal in the NIR
region (600–900 nm) resulting in higher penetration depth of the
NIR light hence better treatment of deeper lying tissues (14–19).
In view of this, research on synthesis of suitable sensitizer mole-
cules which absorb in NIR region is an active area of research
(20,21). In this approach, the use of cyanine- (22–24), squaraine-
(25–35), bodipy- (36–39) and azabodipy(40–42)-based dyes have
been explored. Among these, squaraines form an important class
of functional organic dyes having visible to NIR absorption. The
low-energy NIR absorption in these types of Donor–Acceptor–
Donor-type molecule is mainly due to the intramolecular charge
transfer whose range of absorption can be tuned either by chang-
ing the constituent aromatic/heteroaromatic substituents or by
extending the conjugation (43–45). In this study, we discuss the
synthesis, photophysical properties and photodynamic effects in
live cells of three water soluble squaraine dyes, viz., bis-
benzothiazolium squaraine dyes SQMI and SQDI with iodine
substitution in one and both benzothiazolium units, respectively,
and an unsymmetrical squaraine dye ASQI containing iodinated
benzothiazolium and aniline substituents (Chart 1). The dyes
were functionalized with sulphonyl group to make them water
soluble. The presence of iodine atoms in these dyes is expected
Here, we report the synthesis, photophysical properties and
photodynamic effects in DLA live cells of three water soluble
squaraine dyes, viz. bisbenzothiazolium squaraine dyes SQMI
and SQDI with iodine in one and both benzothiazolium units,
respectively, and an unsymmetrical squaraine dye ASQI con-
taining iodinated benzothiazolium and aniline substituents.
The diiodinated SQDI showed an anomalous trend in both
fluorescence and triplet quantum yields over the monoiodi-
nated SQMI, with SQDI showing higher fluorescence and
lower triplet quantum yields compared to SQMI. Nanosecond
laser flash photolysis of SQDI and SQMI indicated the for-
mation of triplet excited states with quantum yield of 0.19
and 0.26, respectively. On photoirradiation, both the SQDI
and SQMI generate singlet oxygen and it was observed that
both dyes undergoing oxidation reactions with the singlet
oxygen generated. ASQI which exhibited a lower triplet
quantum yield of 0.06 was, however, stable and did not react
with the singlet oxygen generated. In vitro cytotoxicity studies
of these dyes in DLA live cells were performed using Trypan
blue dye exclusion method and it reflect an order of cytotox-
icity of SQDI>SQMI>ASQI. Intracellular generation of the
ROS was confirmed by dichlorofluorescein assay after the
in vitro PDT.
INTRODUCTION
Photodynamic therapy (PDT) is a noninvasive clinical treatment
of diseased or malignant tissues which has been widely accepted
and used for many years (1). The administration of the drug, a
photosensitizer, either locally or systematically leads to the selec-
tive accumulation of the drug inside the tumor cells after a
defined time period. Excitation by light promotes the photosensi-
tizer to its singlet state from where it can undergo intersystem
crossing to reach the excited triplet state (2–4). The ease with
which a molecule undergoes intersystem crossing from the
excited singlet to triplet state defines the triplet efficiency of the
molecule, and good triplet efficiency and long triplet lifetimes
are considered as favorable properties for molecules to be effi-
cient sensitizers for PDT. A molecule in its excited triplet state
*Corresponding authors e-mail: sureshdas@niist.res.in (Suresh Das);
annieab2001@gmail.com (Annie Abraham)
© 2014 The American Society of Photobiology
1

2
Kulathinte M. Shafeekh et al.
output from a 200 W high-pressure Hg lamp filtered through a 590 nm
Oriel long pass filter.
Cytotoxicity studies. To maintain the cells for the cytotoxicity studies, the
DLA cells were injected into the intraperitoneal cavity of mice and main-
tained up to 3 weeks in aseptic conditions in the departmental animal
house (IAEC-KU-4/2010-11-BC-AA [15]). The matured cells were aspi-
rated from the peritoneum for in vitro studies. Cytotoxicity was evaluated
by trypan blue dye exclusion method where cells were subjected to PDT
treatment and trypan blue dye was added to the cells. The percentage of
the cell death was then calculated using a hemocytometer. Different dilu-
tions of DLA cells were made of 10À1, 10À2 and 10À3. The cells were
counted in the 10À3 dilution on hemocytometer and adjusted the cell
number to 1 9 106 cells mLÀ1. The cells were incubated at 37°C with
different concentrations of SQDI, SQMI and ASQI for different time
intervals, viz., 1, 2 and 3 h. Another set of cells, after treatment with dif-
ferent concentrations of squaraines were illuminated for 10 min with a
1000 W halogen lamp kept at a distance of 33 cm corresponding to a
total light dose of 46 J cmÀ2. A control was set with the cells, which
received the illumination alone, i.e. without treatment with the dye. A
quantity of 0.1 mL of 0.4% Trypan blue in phosphate-buffered saline
(PBS) was added and the number of dead cells was determined using
hemocytometer. Fractionated light delivery was used instead of delivering
the light at a single stretch. Short-term intervals (dark periods) was used
during the light irradiation to allow reoxygenation of tumor cells during
the treatment and thus enhances efficacy of PDT by preventing thermal
injury.
The DCFDA assay was used to measure the levels of ROS in cells
according to a reported procedure (52,53). Cells of 10% (w/v) were pre-
pared in PBS and aliquots were taken from this for ROS determination
and protein estimation. A quantity of 150 lL of 1 mM DCFDA in etha-
nol was added and incubated at 37°C for 30 min. Supernatant obtained
after centrifugation at 7826 g for 15 min was analyzed for fluorescence
at 523 nm with an excitation wavelength of 502 nm using a Perkin
Elmer-45 spectrofluorimeter.
Synthesis. Compounds 2-methyl-6-nitro-1,3-benzothiazole (1) (30),
6-iodo-2-methyl-1,3 benzothiazole (3) (30) and semisquaraine derivatives
5 (54) and 6 (55) were synthesized according to reported procedures.
Synthesis of 6-amino-2-methyl-1,3-benzothiazole (2). A solution of
2-methyl-6-nitro-1,3 benzothiazole (2.0 g; 10.3 mmol) in 3:1 THF: Etha-
nol was heated to about 60°C, 0.2 g iron oxide hydrate catalyst (56) was
added and the reaction was started by adding hydrazine hydrate
(0.75 mL; 15.4 mmol) drop by drop. It was then heated to 65°C and the
reaction was monitored by TLC. About 2 h later, all the starting material
was found to be consumed. The solution was then filtered and the sol-
vents were removed under vacuum to get 1.2 g of pure product.
Yield: 1.25 g (71%); m.p: 113°C; 1H NMR (500 MHz, CDCl3):
7.69 (d, J = 8.5 Hz, 1H); 7.05 (d, J = 2.0 Hz, 1H); 6.78 (dd,
J = 8.5 Hz, 2.0 Hz, 1H); 3.72 (br s, exchangeable with D2O, 2H); 2.74
(s, 3H) ppm; 13C NMR (125 MHz, CDCl3): 162.7, 146.7, 143.9, 137.2,
122.7, 115.1, 105.8, 19.8 ppm; FAB-MS: Calcd for (M+H)+: 165.23,
Found: 165.41.
Chart 1. Structures of the squaraine dyes used for this study.
to improve their intersystem crossing efficiency due to the heavy
atom effect (46). Photophysical studies showed that excitation of
these dyes led to the generation of their triplet state in good
yields and with very long triplet lifetime. Evaluation of their
cytotoxic behavior on Daltons Lymphoma Ascites (DLA) cells
was carried out with respect to both concentration and incubation
time. The percentage cell death was measured by Trypan blue
dye exclusion method (47,48). Cytotoxicity of these molecules
was observed to be through both Type-I ROS pathways and
Type-II singlet oxygen pathways wherein the generation of the
former was confirmed by using dichlorofluorescein (DCFDA)
assay.
MATERIALS AND METHODS
Squaric acid, 2-methyl-1,3-benzothiazole, 1,4-butanesultone and Hydra-
zine monohydrate were purchased from Sigma Aldrich. All other chemi-
cals and the solvents used were of highest available purity obtained from
local companies. 1H and 13C NMR spectra were recorded on Bruker 500/
300 MHz NMR spectrometer. FAB-MS were measured on a JEOL JMS
600H mass spectrometer and MALDI- TOF analysis was done using AX-
IMA CFR PLUS mass spectrometer from Shimadzu. Absorption mea-
surements were carried out in a Shimadzu UV-3101 PC UV–vis–NIR
scanning spectrophotometer and steady-state fluorescence measurements
were done in Spex Fluorolog 112X Spectrofluorimeter. Fluorescence
quantum yields were measured for SQDI and SQMI relative to Indicar-
bocyanine dye (/f = 0.36 in methanol) (49) and for ASQI relative to
Nileblue A perchlorate (/f = 0.23 in ethanol) (50) as standards. Fluores-
cence lifetime measurements were carried out in an IBH picosecond sin-
gle photon counting system using 590 nm (for ASQI) and 630 nm (for
SQMI and SQDI) nano-LEDs as the excitation source and a Hamamatsu
C4878-02 microchannel plate (MCP) as the detector. Nanosecond laser
flash photolysis was carried out using an applied photophysics model
LKS60 laser kinetic spectrometer by using the third harmonic (355 nm)/
second harmonic (532 nm) of an INDI-40-10-HG Quanta Ray Nd-YAG
laser in 1 cm pathlength quartz cell with a right-angle configuration
between the directions of laser and analyzing beams. The dye solutions
in methanol were deaerated by purging Argon for 75 min before the laser
flash photolysis measurements. All the experiments were carried out at
room temperature. The generation of singlet oxygen was investigated by
diphenylisobenzofuran (DPBF) as the singlet oxygen scavenger (51)
where the generation of the singlet oxygen induces photooxidation of the
furanose ring of DPBF leading to breaking of its conjugation, which can
be spectroscopically measured as bleach in the DPBF absorption without
affecting the dye absorption. Singlet oxygen generation studies of SQDI,
SQMI and ASQI were carried out in methanol and deuterated methanol
(CD3OD). The oxygen saturated solutions were irradiated by using the
Synthesis of N-sulphobutyl- 6-iodo -2-methyl-1, 3 benzothiazole (4). A
mixture of 6-iodo -2-methyl- 1,3 benzothiazole (1.0 g, 3.62 mmol) and
1,4 butanesultone (2.8 g, 20.5 mmol) were heated in a pressure tube at
120°C for 24 h. The solid obtained was washed thoroughly with diethyl
ether and then with acetone. Repeated precipitation from methanol using
acetone yielded 880 mg (59%) of product which was stored in vacuo and
used for the next step without further purification.
Yield: 880 mg (59%); 1H NMR (500 MHz, D2O): 8.47 (s, 1H); 8.09–
8.10 (d, J = 8.5 Hz,1H); 7.80–7.82 (d, J = 8.5 Hz,1H); 4.62(t, +N CH2,
2H); 3.06 (s, 3H); 2.86–2.89(t, CH2-SO3, 2H); 1.77–1.81(m, 4H) ppm.
FAB- MS: Calcd for M+: 411.28; Found: 411.57.
Synthesis of SQDI. The squaraine dye SQDI was synthesized by
refluxing a mixture of N-sulphobutyl- 6-iodo -2-methyl-1, 3 benzothiaz-
ole (4) (200 mg, 0.48 mmol) and squaric acid (28 mg, 0.24 mmol) in
2:1 butanol: benzene mixture in presence of 1 mL of quinoline fitted
with a Dean stark condenser for the azeotropic removal of water for
about 6 h. After cooling, the reaction mixture was concentrated to 2 mL,
and precipitated by adding approximately 20 times excess of hexane. It
was then filtered and the product was purified by column chromatography
using neutral alumina with 12:7:1 chloroform: methanol: triethyl amine
to yield 80 mg (37%) of the pure product.
Yield: 37%; m.p = 208–210°C (decomp.); UV kmax (MeOH)
À1
660 nm (e = 135 000 M cmÀ1) IR (in KBr): 2939.5, 2678.2, 1562.3,

Photochemistry and Photobiology
3
1474.6, 1270.1, 1202.6, 1040.6, 963.4, 811.0, 518.8 cmÀ1 1H NMR
;
ole on treatment with semisquaraine derived from N-ethyl-
benzothiazolium (5) (54) and N,N dimethylaniline (6) (55),
respectively.
(500 MHz, CD3OD): 8.06 (s, 2H); 7.73–7.75 (d, J = 8.5 Hz, 2H);
7.34–7.35 (d, J = 8.4 Hz, 2H); 6.61(s, olefinic, 2H); 4.25 (t, 4H); 2.89–
2.92 (t, 4H); 1.97 (m, 8H) ppm; 13C NMR (125 MHz, CD3OD):
172.61, 160.60, 142.38, 140.96, 139.22, 137.54, 136.83, 133.34,
115.33, 95.87, 62.55, 53.77, 30.15, 23.73 ppm. MALDI-TOF: Calcd:
900.02; Found: 900.94.
Absorption and emission properties
Synthesis of SQMI. The squaraine dye SQMI was synthesized by
refluxing a mixture of N-sulphobutyl- 6-iodo -2-methyl-1, 3 benzothiaz-
ole (4) (100 mg, 0.24 mmol) and semisquaraine of N-ethyl benzothiazole
(5) (66 mg, 0.24 mmol) in 2:1 butanol: benzene in presence of 1 mL
quinoline for 6 h with a Dean stark trap for the azeotropic removal of
water. It was then cooled and concentrated in vacuo to about 2 mL and
the product was precipitated by adding approximately 20 times excess of
hexane. It was further purified by column chromatography using silica
gel (100–200 mesh) with 25% methanol–chloroform to yield 60 mg
(38%) of pure product. Yield: 38%; m.p = 215–218°C (decomp.); UV
The normalized absorption and emission spectra of SQDI and
SQMI are shown in Figs. 1 and 2, respectively. Both SQDI and
SQMI possess strong absorption in the NIR region with maxima
centered in between 640 and 680 nm region in different solvents
with molar extinction coefficient values of 1.35 9 105 and
À1
1.52 9 105
M
cmÀ1, respectively, in methanol. The observed
mirror image relation of the emission spectra to that of absorp-
tion and the small Stokes shifts in all the solvents studied indi-
cated that during excitation the S0–S1 charge transfer was mainly
confined to the central squarate ring and negligible energy was
consumed for solvent and structural reorganization in the excited
state (57).
It was observed that in alcoholic solvents there is a hypso-
chromic shift in the absorption spectra with increase in solvent
polarity (Table 1). Alcoholic solvents were reported to form
hydrogen bonding with the central squarate oxygen atom and the
strength of which in turn depend on the relative acidity of the
alcohols (58–61). Strong hydrogen bonding stabilizes the ground
state more than the excited state resulting in a hypsochromic
shift in absorption maximum.
The absorption spectrum of SQDI in water showed maximum
at 598 nm with a shoulder at 651 nm, whereas the SQMI pos-
sess two different peaks at 643 and 590 nm. The peak at the
higher wavelength (643 nm) is attributed to the solvated mono-
mer and the peak at lower wavelength (590 nm) to that of aggre-
gates. Formation of aggregates with blueshifted absorption
relative to that of the monomer has been reported earlier for
squaraines (60–63). The increase in relative intensity of the blue-
shifted band with increasing concentration as well as decrease in
intensity with increasing temperature confirmed that the band
was due to formation of aggregates (see Fig. S1–S2 in Supple-
mentary information).
À1
kmax (MeOH) 655 nm (e = 152,000 M cmÀ1); IR (in KBr): 2918.3,
2850.8, 1587.4, 1491.0, 1268.2, 1105.2, 1044.4, 970.2, 743.5,
606.6 cmÀ1 1H NMR (500 MHz, CD3OD): 7.99 (s, 1H); 7.74–7.76 (d,
;
J = 8.0 Hz, 1H); 7.68–7.70(d, J = 8.5 Hz, 1H); 7.46–7.49(m, 2H); 7.27–
7.28(t, 2H); 5. 92 (br s, 1H, olefinic); 5.85 (br s, 1H, olefinic); 4.34 (m,
2H); 4.21(t, 2H); 2.92(t, 2H); 1.98 (m, 4H); 1.42–1.45 (t, 3H) ppm; 13C
NMR (125 MHz, CD3OD): 182.94, 163.63, 144.26, 141.74, 137.40,
131.40, 130.19, 129.12, 128.75, 125.78, 124.64, 123.88, 123.52, 118.62,
115.01, 114.35, 112.15, 71.49, 68.85, 62.56, 52.09, 32.74, 23.56,
22.66 ppm. MALDI-TOF: Calcd: 666.57, Found: 667.61
Synthesis of ASQI. The squaraine dye ASQI was synthesized by
refluxing a mixture of N-sulphobutyl-6-iodo-2-methyl-1, 3-benzothiazole
(4) (100 mg; 0.24 mmol) and semisquaraine of N,N-dimethylaniline (6)
(52 mg; 0.24 mmol) for 6 h in a 2:1 butanol: benzene in presence of
1 mL quinoline with a dean stark trap for the azeotropic removal of
water. It was then cooled and concentrated in vacuo to about to 2 mL
and the product was precipitated adding approximately 20 times excess
of hexane. It was further purified by column chromatography using silica
gel (100–200 mesh) with 10% methanol: chloroform to yield 80 mg
(55%) of pure product.
Yield: 55%; m.p = 182–184°C; UV kmax (MeOH) 605 nm
(e = 58 000 MÀ1 cmÀ1); IR (in KBr): 2933.7, 2677.2, 1583.5, 1410.9,
1369.4, 1294.2, 1191.0, 1048.3, 943.2, 825.5, 611.4 cmÀ1 1H NMR
.
(500 MHz, CD3OD): 8.23 (s, 1H); 7.83–7.86 (m, 3H); 7.58–7.59(d,
9.0 Hz, 1H); 6.67–6.68 (d, J = 7.5 Hz);6.34(br s, olefinic) 4.46 (t, 2H);
3.05(s, 6H); 2.92 (t, 2H);1.98–2.05 (m, 4H) ppm; 13C NMR (125 MHz,
CD3OD): 183.2, 166.9, 153.3, 141.6, 138.4, 132.6, 132.5, 132.4, 130.1,
120.0, 117.2, 114.7, 112.7, 91.0, 90.8, 47.9, 40.3, 30.7, 27.8, 23.4,
9.28 ppm; MALDI-TOF: Calcd: 610.48, Found: 611.44
In addition to the hypsochromic effect observed in absorption
and emission maxima, decrease in fluorescence lifetime and fluo-
rescent quantum yield was also observed in alcoholic solvents
with increase in solvent polarity. This can be attributed to spe-
cific solvent–solute interaction between the alcoholic solvents
and the central squarate oxygen leading to hydrogen-bonded
complex (60,61), which can provide a nonradiative decay path
for the excited state. The fluorescence quantum yield of SQDI
was significantly higher than that of the monoiodinated SQMI.
Similar observation was reported for halogenated squaraines by
RESULTS AND DISCUSSION
Synthesis
Solubility of the photosensitizer in water is an essential require-
ment for their cellular uptake. Most of the squaraine dyes which
have been investigated are poorly soluble in water. The presence
of sulphobutyl group on the nitrogen atom of benzothiazole
moiety of the squaraine dyes investigated in this study allows
the dyes to be slightly water soluble. 5-iodo-2-methyl-1,3-benzo-
thiazole was synthesized according to a reported procedure with
a slight modification (30). 2-methyl-1,3-benzothiazole on nitra-
tion followed by reduction with hydrazine hydrate in presence of
iron oxide hydroxide catalyst (56) gave the 5-amino-2-methyl-
1,3-benzothiazole (2). 5-amino-2-methyl-1,3-benzothiazole on diaz-
otization and subsequent iodination yielded 5-iodo-2-methyl-1,3-
benzothiazole (3) which on quarternization using 1,4-butanesul-
tone gave the corresponding N-sulphobutyl-5-iodo-2-methyl-1,3-
benzothiazole (4). SQDI was synthesized from squaric acid by
taking 2:1 equivalents of N-sulphobutyl-5-iodo-2-methyl-1,3-ben-
zothiazole and squaric acid (Scheme 1). SQMI and ASQI were
synthesized from N-sulphobutyl-5-iodo-2-methyl-1,3-benzothiaz-
€
Mayerhoffer et al. (66). The halogens can exhibit both the -I
(Inductive) and +M (Mesomeric) effect. The dominant would be
+M effect due to the increase in polarizabilty of the iodine sub-
stituent by the transfer of electron density from iodine to the
chromophore. The unsymmetrical iodo substituted squaraine dye
ASQI showed absorption above 600 nm with maxima centered
at 605–617 nm region in different solvents with a molar extinc-
À1
tion coefficient of 5.88 9 104
M
cmÀ1 in methanol. The dye
exhibited
a broad absorption and a large Stokes shift
(>1000 cmÀ1) as compared to the SQDI and SQMI indicating
considerable energy was utilized for the structural and solvent
reorganization in the excited state which is attributable to a

4
Kulathinte M. Shafeekh et al.
H2N
O2N
S
N
S
S
Conc: HNO3
Fe3O4
CH3
CH3
N
CH3
N2H4.H2O
N
Conc: H2SO4
(77%)
THF: EtOH; 650C; 2h
(71%)
(2)
(1)
O
S
O
O
(i) NaNO2/HCl; 00C
I
I
S
S
CH3
N
CH3
N
1200C; 24h
(59%)
(ii) KI; 00C
(25%)
(3)
(4)
SO3
I
S
N
O
HO
O
OH
O
I
I
S
S
N
Butanol: benzene
O
CH3
N
Quinoline; 900C; 6h
(37%)
HO3S
(4)
SQDI
SO3
SO3H
S
N
O
O
HO
I
S
N
O
I
CH3
S
N
S
Butanol: benzene
O
CH3
Quinoline; 900C; 6h
(38%)
N
SQMI
H3C
(4)
(5)
SO3H
SO3
H3C
N CH3
O
OH
O
I
S
N
H3C
Butanol: benzene
I
CH3
S
N
O
O
Quinoline; 900C; 6h
(55%)
H3C
N
(6)
(4)
ASQI
SO3
SO3H
Scheme 1. Synthesis of iodinated Squaraine dyes SQDI, SQMI and ASQI.
(a)
(b)
MeOH
BuOH
DMSO
Water
MeOH
BuOH
1.0
1.0
DMSO
Water
0.5
0.0
0.5
0.0
400
600
800
650
700
750
800
850
Wavelength, nm
Wavelength, nm
Figure 1. Normalized (a) absorption and (b) emission spectra of SQDI in different solvents. Temperature 25°C; Path length 1 cm.

Photochemistry and Photobiology
5
(a)
(b)
1.2
MeOH
BuOH
DMSO
Water
MeOH
BuOH
DMSO
Water
1.0
0.9
0.6
0.3
0.0
0.5
0.0
400
600
800
650
700
750
800
850
Wavelength, nm
Wavelength, nm
Figure 2. Normalized (a) absorption and (b) emission spectra of SQMI in different solvents. Temperature 25°C; Path length 1 cm.
Table 1. Photophysical data of SQDI, SQMI and ASQI.
SQDI
SQMI
ASQI
Stokes
shift,
Stokes
shift,
Em. Stokes
Abs.
Em.
Abs.
Em.
Abs.
kmax
shift,
Solvent kmax. (nm) kmax. (nm) (cmÀ1
)
/
s/ns kmax. (nm) kmax. (nm) (cmÀ1
)
/
s/ns kmax (nm) (nm) (cmÀ1
)
/
s/ns
MeOH
EtOH
PrOH
BuOH
DMSO
DMF
660
667
669
671
680
687
676
678
682
683
684
705
703
689
665
402
0.26 1.19
655
659
662
664
673
680
669
670
674
677
678
699
695
683
658
341.8 0.10 0.67
337.7 0.20 1.41
334.6 0.27 2.08
605
610
610
610
617
615
608
554
665 1491.3 0.20 0.64
667 1400.9 0.40 1.22
664 1333.2 0.48 1.67
655 1126.2 0.56 1.73
329.7 0.42 1.98
306.3 0.54 2.48
261
521
331
279.1 0.29 1.84
323.3 0.02 0.31
–
–
–
2.64
2.57
2.34
310.9
552.6
317.3
–
–
–
2.38
2.40
2.09
684 1587.5
680 1554.2
–
–
1.60
1.39
CAN
Water
306.3 0.17 1.30
354.5 0.01
670 1521.9 0.30 0.98
668 3080.4 0.02
598, 651
590,643
–
-
change in the dipole during excitation owing to the asymmetry
in molecular structure (Fig. 3). ASQI also showed a hypsochro-
mic shift in absorption and emission with alcoholic solvents with
increase in hydrogen bonding ability. ASQI showed a very high
fluorescence quantum yield in protic solvents. The tendency of
ASQI to form aggregates in water was significantly less as com-
pared to the symmetrical dyes (62–65). (see Fig. S3 in Supple-
mentary information)
squaraines have been reported to have a poor intersystem cross-
ing and in most of the cases the triplet excited states were gener-
ated through triplet–triplet sensitization (67–71). The transient
absorption spectra of SQDI, SQMI and ASQI were carried out
in deaerated methanol using nanosecond laser flash photolysis.
SQDI and SQMI were studied by excitation using the third har-
monic of the Nd: YAG at 355 nm, whereas ASQI was excited
by 532 nm as it possesses a broad absorption in the visible to
NIR region. As SQDI and SQMI have lower extinction coeffi-
cient at 355 nm, higher concentration in the range of 100 lM
and 50 lM, respectively, was required to generate a sufficient
excited state population. Concentration-dependent absorption
measurements of SQDI and SQMI in methanol showed no
aggregation tendency with increase in concentration (see Fig. S4
Properties of triplet excited state
The presence of both iodine and sulfur in SQDI, SQMI and
ASQI could facilitate the intersystem crossing from the singlet
to triplet state due to the heavy atom effect (46). Normally
(a)
(b)
MeOH
BuOH
DMSO
Water
MeOH
BuOH
1.0
1.0
0.5
0.0
DMSO
Water
0.5
0.0
Wavelengt6h0,0nm
Wavelen7g0t0h, nm
400
500
700
800
600
650
750
800
Figure 3. Normalized absorption (a) and emission (b) spectra of ASQI in different solvents. Temperature 25°C; Path length 1 cm.

6
Kulathinte M. Shafeekh et al.
in Supporting Information). Nanosecond laser flash photolysis of
ASQI was studied at a lower concentration of ~15 lM. Excita-
tion leads to formation of transient species, and the transient
absorption spectra of SQDI and SQMI are shown in Fig. 4. The
transient spectra exhibited ground state bleach in the 630–
690 nm region and absorption due to the transient species on
both sides of the bleach, with maxima at 580 nm for SQDI and
SQMI, respectively. The transient species were assigned to the
triplet excited state of the dyes based on the quenching experi-
ments with molecular oxygen and also by their ability to gener-
ate the triplet state of b-carotene through a triplet–triplet energy
transfer. As the intersystem crossing efficiency of the b-carotene
is negligible, formation of the triplet excited state can occur only
via energy transfer from the triplet excited state of the dyes
(Fig. 5). The triplet excited state of SQDI and SQMI was long
lived and the complete decay to the ground state was not
observed due to the instrument time limit which extended up to
400 ls (Insets Fig. 4a,b). Spectroscopic examination of the dye
solutions following the laser investigation studies indicated that
the dyes do not undergo photodegradation.
Tris(bipyridine)ruthenium(II)chloride, [Ru(bpy)3]Cl2 as the stan-
dard and growth of the triplet of b-carotene was monitored for
SQDI and SQMI (Fig. 5). The triplet quantum yields in metha-
nol were found to be 0.19 Æ 0.03, 0.26 Æ 0.04 and
0.06 Æ 0.01, respectively, for SQDI, SQMI and ASQI
(Table 2).
Singlet oxygen generation studies
The singlet oxygen generation experiments with SQDI and
SQMI using DPBF as the singlet oxygen scavenger in methanol
showed that along with bleach in DPBF absorption both SQDI
and SQMI absorptions were also decreased. This indicated that
both SQDI and SQMI compete with DPBF in reacting with sin-
glet oxygen (Fig. 6). In addition, this could also be due to a
quenching of the electronically excited states of the dyes by the
furan derivative. This was evidenced from the irradiation studies
of SQDI and SQMI in presence and absence of DPBF in argon
atmosphere, where a 5%–7% decrease in DPBF absorbance was
observed for a specific irradiation time compared to that in oxy-
gen atmosphere (Fig. 6 and S6 Supporting Information). Photo-
stability of the DPBF in presence of oxygen and argon was also
confirmed under similar irradiation conditions (Fig. S7 Support-
ing Information). The solvent isotope effect is a powerful tool to
confirm the generation of singlet oxygen and we have carried
out singlet oxygen measurement using DPBF in deuterated meth-
anol (CD3OD). In CD3OD the rate of decrease in absorbance of
DPBF was observed to be four times faster than that in methanol
Kinetic traces at 580 nm fitted with single exponential decay
indicated lifetimes of 115 Æ 9 and 101 Æ 2 ls, respectively, for
SQDI and SQMI. ASQI showed a transient spectrum with
ground state bleach in 520–640 nm region with small absorption
on both side (see Fig. S5 in Supporting Information) and the life-
time of the ground state recovery was observed to be
78 Æ 10 ls. The triplet quantum yield of these dyes were calcu-
lated by means of a previously described method (72,73) using
(b)
(a)
0.06
0.06
0.04
0.02
0.04
0.02
0.00
0.06
0.00
0.06
-0.02
-0.04
-0.06
-0.08
0.04
0.02
0.00
-0.02
0.03
-0.04
0.00
0
200
μ
300
400
1T00ime,
s
Time,
μ
200 s
-0.06
400
0
100
300 400
450
500
550
600
650
700
750
400
450
500
550
600
650
700
750
Wavelength, nm
Wavelength, nm
Figure 4. Transient absorption spectra obtained immediately after 355 nm laser pulse excitation of (a) SQDI (100 lM) and (b) SQMI (50 lM) in deaer-
ated methanol. Inset shows the corresponding kinetic traces at 580 nm.
(a)0.2
(b)
0.2
0.1
0.0
0.1
0.0
-0.1
0
3
6
9
0
3
6
9
Time, s
μ
Time, s
μ
Figure 5. Sensitized growth of the b-carotene triplet observed at 510 nm with (a) SQDI and (b) SQMI.

Photochemistry and Photobiology
7
Table 2. Triplet state properties.
of singlet oxygen which escapes via diffusion reacts with DPBF,
whereas the rest reacts with the dye to form the oxidized prod-
uct. Irradiation studies of an oxygen saturated solution of SQDI
and SQMI in the absence of DPBF also indicated the bleaching
of the dyes (Fig. S8 Supporting Information). Low singlet oxy-
gen generation and a large rate constant for the squaraine-
induced deactivation of singlet oxygen were reported by Salice
et al. attributed to substantial intramolecular charge transfer of
squaraine and intermolecular charge transfer in squaraine–oxygen
encounter complex (75).
Rappozi et al. recently reported that benzothiazole-based
squaraines undergo photooxidation in presence of the in situ gen-
erated singlet oxygen by trapping the oxygenated product using
limonene followed by analysis using GC-MS. Based on their
study, they have proposed that the squaraine dye undergoes a
charge transfer interaction with the singlet oxygen which was
followed by an attack on the exocyclic double bond of the ben-
zothiazole moiety resulting in the formation of oxygenated prod-
Dye
s (ls)
/
T
SQDI
SQMI
ASQI
115 Æ 9
101 Æ 2
78 Æ 10
0.19 Æ 0.03
0.26 Æ 0.04
0.06 Æ 0.01
which indicated that singlet oxygen is produced in the medium.
This solvent isotope effect is due to longer lifetime of singlet
oxygen in deuterated solvent which was attributed to the
decreased nonradiative electronic–vibronic-coupled transition by
the solvent (Fig. 6 and S7 in Supporting Information) (6,74). In
an attempt to reduce the competition between the DPBF and the
squaraine dyes for the singlet oxygen formed, irradiation studies
using solutions containing ~70-fold higher concentration of
DPBF were conducted (Fig. 7). As seen in Fig. 7a,b there was
no significant change in the rate of decay of the squaraine dyes
under the two conditions. A plot of the time course absorbance
change indicated both DPBF and the squaraine dyes followed
the same trend in absorbance bleaching (insets, Fig. 7).
The results clearly indicate that both SQDI and SQMI react
with singlet oxygen. It is interesting to note that squaraine dye
competes efficiently with DPBF even though their concentration
is several orders of magnitude lesser. This suggests that singlet
oxygen reacts with these dyes very efficiently and that the oxida-
tion process occurs between the singlet oxygen generated close
to the dye following collisional energy transfer. Only the fraction
ucts.
A
similar mechanism may be operating in the
photooxidation of SQDI and SQMI leading to the generation of
combination of peroxide, allyl and hydroperoxide radicals (34).
It is interesting that the unsymmetrical squaraine dye ASQI
was found to be stable and undergo little or no photooxidation
under similar conditions (Fig. 8). ASQI can exist in two differ-
ent canonical structures (Structures 7a and b; Scheme 2) with
the positive charge residing either on the aniline nitrogen or on
iodobenzothiazole nitrogen. As the exocyclic double bond is
absent in form 7a, this may be the preferred structure of ASQI.
(a)
(b)
0 S
0 s
0.6
20 S
40 S
70 S
100 S
20 s
50 s
0.4
0.2
0.0
80 s
110 s
140 s
200 s
230 s
260 s
320 s
350 s
0.4
0.2
0.0
130 S
160 S
190 S
220 S
280 S
340 S
400 S
460 S
520 S
300
450
600
750
900
300
450
600
750
900
Wavelength, nm
Wavelength, nm
Figure 6. Absorption spectra of (a) 4 lM SQDI with 20 lM DPBF and (b) 4 lM SQMI with 25 lM DPBF in methanol on irradiation. Path length
1 cm.
2.0
1.5
1.0
0.5
0 s
(a)
(b) 1.0
0.8
2.0
1.5
0 s
1.00
0.75
DPBF
SQMI
DPBF
SQDI
10 s
10 s
25 s
20 s
70 s
40 s
60 s
130 s
180 s
290 s
350 s
430 s
500 s
570 s
700 s
860 s
1020 s
1180 s
1300 s
0.08
0.04
0.00
0.15
0.10
0.05
90 s
120 s
150 s
190 s
230 s
290 s
350 s
410 s
485 s
545 s
605 s
0.6
0
300 600 900 1200
0
200
400
600
Time, s
Time, s
0.4
0.2
0.0
400
500
700
800
400
500
700
800
Wavelengt6h00, nm
Wavelengt6h00, nm
Figure 7. Absorption spectra of (a) 6 lM SQDI with 0.43 mM DPBF and (b) 4 lM SQMI with 0.22 mM DPBF in methanol on irradiation. Inset shows
time course absorbance changes of corresponding squaraines and DPBF. Path length of the cell is 0.2 cm.

8
Kulathinte M. Shafeekh et al.
Theoretical studies on related unsymmetrical dyes have shown
that form 7a is indeed the preferred structure for this class of
dyes (76).
0 s
10 s
20 s
30 s
40 s
55 s
75 s
95 s
125 s
155 s
185 s
245 s
305 s
365 s
0.6
0.4
0.2
0.0
Cytotoxicity studies
The in vitro cytotoxicity of the squaraine at different concentra-
tions was analyzed on DLA cells to evaluate the cytotoxicity in
the absence and presence of light (phototoxicity). Cytotoxicity of
the photosensitizer was checked by incubating the DLA cells
with different concentrations of the photosensitizer in the absence
of light and percentage of cell death was then analyzed. The
cytotoxicity assessment showed no toxicity on DLA cells in the
absence of light. The in vitro photodynamic effect of the squa-
raine was checked by exposing the squaraine-treated DLA cells
to visible light from a 1000 W halogen lamp for 10 min and the
percentage of cell death was then analyzed using trypan blue dye
exclusion method. Cytotoxicity was not observed with the sam-
ples subjected to either incubation with the dyes alone or light
treatment alone. Cytotoxicity was observed only after the com-
bined action of light and photosensitizer confirming that these
dyes are good PDT photosensitizers and cytotoxicity was pro-
duced by the synergistic effect of both the photosensitizer and
light.
300
450
600
750
900
Wavelength, nm
Figure 8. Absorption spectra of ASQI (11.3 lM) with DPBF (23.2 lM)
in methanol on irradiation. Path length 1 cm.
H3C
H3C
N CH3
N CH3
O
O
I
S
N
O
I
S
N
O
7b
7a
For 1 h dark incubation with a concentration of 0.6 mg mLÀ1
of SQDI and SQMI followed by light exposure resulted in
95%–100% cytotoxicity, whereas even with a concentration of
1 mg mLÀ1 ASQI could produce only 80% cytotoxicity under
the same condition (Fig. 9). On increasing the dark incubation
SO3H
SO3H
Scheme 2. Canonical structures of ASQI.
(a)
(b)
100
75
50
25
0
100
75
50
25
0
ASQI
SQMI
SQDI
ASQI
SQMI
SQDI
.1
.3
.5
.7
.9
.1
.3
.5
.7
.9
Concentration of the dye (mg/ml)
Concentration of the dye (mg/ml)
(c)
100
75
50
25
0
ASQI
SQMI
SQDI
.1
.3
.5
.7
.9
Concentration of the dye (mg/ml)
Figure 9. Cytotoxicity assessment of DLA cells with different concentrations of SQDI, SQMI and ASQI after in vitro PDT for (a) 1 h, (b) 2 h and (c)
3 h dark incubation time. Values are mean of 6 estimations per group Æ SD.

Photochemistry and Photobiology
9
400
300
200
100
0
time to 2 h, the required concentration for the complete
cytotoxicity could be reduced to 0.5 mg mLÀ1 for both SQDI
and SQMI, whereas ASQI required 1 mg mLÀ1 for the same
level of cytotoxicity (Fig. 9). Experiments were performed by
increasing the incubation time from 2 to 3 h and cell death was
monitored with different dye concentrations which showed com-
plete cell death with 0.2 mg mLÀ1 with SQDI, 0.4 mg mLÀ1
with SQMI and 0.6 mg mLÀ1 with ASQI (Fig. 9). Further
increase in the dark incubation time could not improve the level
of cytotoxicity.
1
2
3
4
5
6
7
8
Estimation of levels of ROS after PDT treatment
Groups
As the singlet oxygen generation experiments showed photooxi-
dation of SQDI and SQMI and the in vitro PDT treatment gave
very good cytotoxicity in DLA cells with low concentration, it
was expected that ROS might play a major role in the cytotoxic
mechanism. ROS can act as signaling molecules for the initiation
and execution of the apoptosis (77) wherein they can react with
and damage complex cellular molecules such as fats, proteins, or
DNA (78). To prove and confirm the generation and role of
ROS responsible for the cytotoxicity, the estimation of ROS was
carried out using dichlorofluorescein diacetate (DCFDA) assay.
DCFDA assay make use of the intracellular oxidation of the oxi-
dant sensitive probe 2′, 7′- dihydrodichlorofluorescein diacetate
(H2DCFDA) for the measurement of ROS, which will then
expressed as fluorescence intensity/mg protein (51,52). The ester-
ified form of the nonpolar H2DCFDA can diffuse the cell mem-
branes and can undergo deacetylation by intracellular esterases to
the nonfluorescent 2′,7′- dihydrodichlorofluorescein (H2DCF),
which is polar and gets trapped within the cell. H2DCF is sus-
ceptible to ROS-mediated oxidation to produce highly fluorescent
2′, 7′ dichlorofluorescein (DCF) whose emission intensity was
monitored at 523 nm.
Figure 10. Fluorescence intensity of Dichloroflourescein DLA cells after
ROS generation in different groups. Values are mean of 6 estimations
per group Æ SD.
improve its intersystem crossing efficiency to the triplet state as
refluxed from their good triplet quantum yield and a long triplet
lifetime. Good triplet excited properties combined with the pres-
ence of sulfonate groups allow to study the in vitro cytotoxic
effect in live cells by trypan blue dye exclusion method. At 3 h
dark incubation, 95%–100% cytotoxicity was observed with a
concentration of 0.2 mg mLÀ1 of SQDI, 0.4 mg mLÀ1 for
SQMI and 0.6 mg mLÀ1 for ASQI. Photodynamic effect with
these dyes was expected to be through the generation of both
singlet oxygen (Type II) and ROS (Type I) wherein the intracel-
lular generation of the ROS was confirmed by the DCFDA assay
Acknowledgements—This work was supported by Department of Science
and Technology and Council of Scientific and Industrial Research (CSIR,
Network Project NWP 0026), Government of India. K. M. S. and M. K.
A. R. thank UGC and M. S. S. thanks CSIR, Government of India for
the research fellowships. This is contribution no. PPD- 338 from RRLT-
PPG. We thank Dr. Karunakaran Venugopal for his help and valuable
discussions in laser flash photolysis studies.
The experimental design for the estimation of ROS was as
follows: The DLA cells were divided into eight major groups
Group 1- DLA cells alone
Group 2- DLA Cells + Light
Group 3- DLA Cells + SQMI
Group 4- DLA Cells + SQMI + Light
SUPPORTING INFORMATION
Group 5- DLA Cells + SQDI
Group 6- DLA Cells + SQDI + Light
Additional Supporting Information may be found in the online
version of this article:
Group 7- DLA Cells + ASQI
Group 8- DLA Cells + ASQI + Light
Figure S1. Concentration-dependent aggregation studies of
SQDI in water. Inset shows the zoomed spectra at lower concen-
tration
Figure S2. Concentration-dependent absorption changes of
SQMI in water (b) Temperature-dependent absorption changes of
25 M of SQMI in water. Inset in graph a shows the zoomed
spectra at lower concentration and inset in graph b shows
absorption spectra at 23°C, 55°C and 70°C.
Generation of ROS was evaluated by measuring the fluores-
cence intensity in the absence and presence of light after treat-
ment with SQDI, SQMI and ASQI. When the cells were treated
with squaraine dyes in presence of light, a significant increase in
fluorescence intensity was observed at 523 nm confirming ROS
generation, whereas no significant fluorescence enhancement was
observed in the dye alone treated group. The highest enhance-
ment in fluorescence intensity was observed with SQDI followed
by SQMI and the least with ASQI (Fig. 10). The decrease in
fluorescence intensity of ASQI may be due to the reabsorption
of the DCF fluorescence by ASQI. The overall result was found
to be in agreement with the earlier photophysical data and per-
centage cytotoxicity assessment.
Figure S3. Concentration-dependent aggregation studies of
ASQI in water.
Figure S4. Normalized concentration-dependent absorption
spectra of (a) SQDI and (b) SQMI in methanol. Insets show the
different concentrated spectra before normalization.
Figure S5. Transient spectra obtained for ASQI (15 M) at 1 s
(o), and 70 s (D) after 532 nm laser pulse excitation in deaerated
methanol. Inset shows the absorbance-time profiles at 590 nm.
Figure S6. Changes in absorbance on irradiation of a solution
of (a) 5 M SQDI and (b) 4.6 M SQDI +24 M DPBF (c) 4.2 M
SQMI and (d) 4 M SQMI +25 M DPBF in argon atmosphere. (e)
In summary, we have synthesized three water soluble iodin-
ated squaraine dyes having very good extinction coefficient rang-
ing in the order of 104–105 in the phototherapeutic window.
Presence of iodine and sulfur atoms in these molecules can

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and (f) are the absorption spectra of 32 M DPBF alone in argon
and oxygen atmosphere, respectively
Figure S7. Changes in absorbance of oxygen-saturated solu-
tion of (a) 4.0 M SQDI+ 25.0 M DPBF and (b) 5.0 M SQMI +
32.0 M DPBF on irradiation in deuterated methanol (CD3OD).
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Scheme SC1. Scheme showing the reaction of singlet oxygen
with DPBF
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