ESIPT-inspired benzothiazole fluorescein: Photophysics of microenvironment pH and viscosity

Article abstract of DOI:10.1016/j.dyepig.2013.03.019

A series of novel substituted ESIPT benzothiazole fluorescein derivatives were synthesized from the intermediate, (1H-benzo[d]thiazole-2-yl) benzene-1,3-diol and different anhydrides. The photophysical properties of these fluorescein derivatives were studied as a function of pH and viscosity. Enhancement in fluorescence emission was observed with increasing percentage of glycerin in water. The relative fluorescence quantum yields of these derivatives were determined by using anthracene and fluorescein as a reference standards. Density Functional Theory computations were performed to optimize the geometry at the ground state. The excited state geometry was optimized using the time dependent-DFT. The synthesized fluorescein derivatives were characterized by using elemental analysis, FT-IR and ¹H NMR spectroscopy.

Full text of DOI:10.1016/j.dyepig.2013.03.019

Dyes and Pigments 98 (2013) 507e517
Contents lists available at SciVerse ScienceDirect
Dyes and Pigments
journal homepage: www.elsevier.com/locate/dyepig
ESIPT-inspired benzothiazole fluorescein: Photophysics of
microenvironment pH and viscosity
*
Vikas S. Patil, Vikas S. Padalkar, Abhinav B. Tathe, N. Sekar
Tinctorial Chemistry Group, Institute of Chemical Technology (Formerly UDCT), N. P. Marg, Matunga, Mumbai 400 019, Maharashtra, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel substituted ESIPT benzothiazole fluorescein derivatives were synthesized from the
intermediate, (1H-benzo[d]thiazole-2-yl) benzene-1,3-diol and different anhydrides. The photophysical
properties of these fluorescein derivatives were studied as a function of pH and viscosity. Enhancement
in fluorescence emission was observed with increasing percentage of glycerin in water. The relative
fluorescence quantum yields of these derivatives were determined by using anthracene and fluorescein
as a reference standards. Density Functional Theory computations were performed to optimize the ge-
ometry at the ground state. The excited state geometry was optimized using the time dependent-DFT.
The synthesized fluorescein derivatives were characterized by using elemental analysis, FT-IR and 1H
NMR spectroscopy.
Received 6 March 2013
Accepted 18 March 2013
Available online 10 April 2013
Keywords:
ESIPT
Benzothiazole
Fluorescein
pH sensitive
Microenvironment
TD-DFT
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
earlier communication that incorporation of an ESIPT unit in fluo-
rescein causes a red shift in emission to the tune of 50 nm as
Synthesis and photophysical properties of 2-(20-hydroxyphenyl)
benzothiazole are well studied [1]. Their interactions with bio-
molecules and solvent-dependent emission properties are of in-
terest [2] and this area of research has been very active [3,4]. The
compounds showing ESIPT phenomenon are used as fluorescent
probes [5].
compared to that of the original fluorescein (570 nm) [18,19].
In continuation of our work in benzimidazole, oxazole and
thiazole chemistry [14,19,20] and to understand the effect of ESIPT
unit in the photophysical properties of fluorescein moiety, in this
paper, we report the synthesis and photophysical properties of
novel fluorescein molecules containing thiazole units. The photo-
physical properties are studied as a function of pH and viscosity.
The changes in the electronic transition, energy levels, and orbital
diagrams are studied using the Density Functional Theory (DFT)
and Time-Dependent Density Functional Theory (TD-DFT) com-
putations and are correlated with the experimental absorption and
emission.
Among the various ESIPT molecules, 2-(20- hydroxyphenyl)
benzoxazole (HBO), 2-(20- hydroxyphenyl) benzimidazole (HBI),
and 2-(20- hydroxyphenyl) benzothiazole (HBT) have been the
most often investigated compounds due to their structural
simplicity and the facile chemical modification that can be effec-
ted. HBO and HBI have emerged to be interesting, as they exhibit a
large Stokes shift (about 150 nm) arising from excited-state
intramolecular proton transfer (ESIPT). Utilization of this feature
has resulted in various applications including chemical sensors for
zinc (II) anions [6e8], electronic devices such as organic light-
emitting diodes [9], laser dyes [10e12], polymer modifiers and
polymeric solid materials [13]. HBT also exhibits properties similar
to HBI and HBO [14].
2. Experimental
2.1. Reagents and apparatus
All the commercial reagents and solvents were purchased from
S. D. Fine Chemicals (India) and were used without purification. The
reactions were monitored by thin layer chromatography using
0.25 mm silica gel 60 F254 precoated plates (Merck), which were
visualized under UV light. The FT-IR spectra were recorded on
PerkineElmer 257 spectrometer using KBr discs. 1H NMR spectra
were recorded on a VXR 400-MHz instrument using trimethylsi-
lane as an internal standard (VARIAN). Mass spectra were recorded
Fluorescein has a single emission at 520 nm and has wide ap-
plications as a probe in biology [15e17]. We have reported in our
* Corresponding author. Tel.: þ91 22 3361 1111, þ91 22 3361 2707; fax: þ91 22
3361 1020.
E-mail addresses: nethi.sekar@gmail.com, n.sekar@ictmumbai.edu.in (N. Sekar).
0143-7208/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.dyepig.2013.03.019

508
V.S. Patil et al. / Dyes and Pigments 98 (2013) 507e517
on Finnigan Mass spectrometer (EI). The visible absorption spectra
of the compounds were recorded on a Spectronic Genesys 2 UVe
visible spectrometer. DSC-TGA measurements were performed on
SDT Q 600 v8.2 Build 100 model of TA instruments Waters (India)
Pvt. Ltd.
in ethanol) and for long wavelength emission fluorescein (
10% NaOH) was used.
F
¼ 0.91
2.4. Synthesis of (1H-benzo[d]thiazole-2-yl) benzene-1,3-diol (3)
2,4-Dihydroxybenzoic acid 1 (10 g, 64.9 mmol), and o-amino
thiophenol 2 (7.07 g, 64.9 mmol) were mixed in polyphosphoric
acid (121.94 g). The mixture was stirred at 250 ꢁC for 4 h and then it
was allowed to cool to room temperature, poured into 1200 ml ice-
cold water with continuous stirring. A dark brown precipitate was
obtained. It was filtered and dissolved in a cold solution of 10%
Na2CO3 (140 mL) acidified with 10% HCl (40 mL) up to pH 2 at 10 ꢁC.
This solution was kept overnight at 0 ꢁC to give 3 with good yield
(Scheme 1).
2.2. Computational methods
The different fluorescein analogues 5ae5e were studied to
find out the effect of electron donor and acceptor on the rate of
excited state intramolecular proton transfer, electronic state and
energy level by ab initio computational methods. The ground
state geometry of the compounds 5ae5e in their Cs symmetry
were optimized using the tight criteria in the gas phase using DFT
[21]. The functional used was B3LYP. The B3LYP functional com-
bines Becke’s three parameter exchange functional (B3) [22] with
the nonlocal correlation functional by Lee, Yang and Parr (LYP)
[23]. The basis set used for all atoms was 6-31G(d) [24]. The
vibrational frequencies at the optimized structures were
computed using the same method to verify that the optimized
structures correspond to local minima on the energy surface. The
vertical excitation energies of the groundestate equilibrium ge-
ometries were calculated with TD-DFT [25]. The low-lying first
singlet excited state (S1) of each conformer was relaxed using the
TD-DFT to obtain its minimum energy geometry. The difference
between the energies of the optimized geometries in the first
singlet excited state and the ground state was used in computing
the emissions [26]. Frequency computations were also carried out
on Frank-Condon excited state of the conformers. All the com-
putations in solvents of different polarities were carried out using
the Polarizable Continuum Model (PCM) [27]. The electronic
structure computations were carried out using the Gaussian 09
program [28].
Yield: 61%, Melting point > 300 ꢁC.
1H NMR (CD3)2SO:
d
6.25 (m, 2H), 6.82 (d, H, J ¼ 9.3), 6.95 (d, H,
J ¼ 9.3), 7.59 (d, H, J ¼ 9.3), 7.82 (m, 2H, J ¼ 6.8, 7.4). 10.60 (s, 2H)
IR ( KBr: cmꢂ1) : 3341 (phenolic OeH stretching), 1696 (amide
C]O), 1651 (imine C]N), 1583 (aromatic C]C), 1501, 1448, 1422,
1215 (phenol CeO) cmꢂ1
.
MS m/z: 243 (Mþ), 244 (M þ 1).
2.5. General synthesis of fluorescein derivatives 5ae5e
2-(20,40-Dihydroxyphenyl) benzthiazole 3 (88 mmol) was mixed
with anhydrides 4ae4e (44 mmol) in H2SO4 at 160 ꢁC for 4 h and
was allowed to cool to room temperature. The reaction mixture was
poured into 100 ml ice-cold water and stirred for 15 min. The
precipitated product was filtered, and washed with cold water
(25 ml). The crude product was purified by dissolving into 10%
Na2CO3 solution (50 ml) and acidified with 10% HCl (13 mL) up to
pH 2 to give 5a to 5e (Scheme 2).
2.5.1. 20,70-Di(1H-benzo[d]thiazol-2-yl)-30,60-dihydroxy-3H-spiro
[isobenzofuran-1-90-xanthen]-3-one (5a)
2.3. Relative quantum yield calculations
Yield: 52%, Melting point > 300 ꢁC.
Quantum yield of the synthesized compounds in water were
calculated by using anthracene and fluorescein as reference stan-
dards using the comparative method [29]. The absorption and
emission characteristics of the standards and the compounds were
measured at different concentrations at (2, 4, 6, 8, and 10 ppm
level). The emission intensity values were plotted against the
absorbance values and linear plots were obtained. Gradients were
calculated for compounds in each solvent and for the standards. All
the measurements were done by keeping the parameters constant
such as solvent and slit width. The relative quantum yields of the
synthesized compounds in different solvents were calculated by
using the equation (1) [29]
1H NMR (DMSO- d6, 400 MHz):
d 6.35 (s, 2H), 6.93 (s, 2H), 7.51
(s, 2H), 7.63 (m, 4H, J ¼ 10.1, 9.7), 7.68 (m, 2H, J ¼ 10.1, 9.7), 7.71 (m,
2H, J ¼ 10.1, 9.7), 7.79 (m, 2H, J ¼ 11.2, 8.8), 7.93 (m, 2H, J ¼ 11.2, 8.8),
8.07 (m, 2H, J ¼ 11.2, 8.8).
FT-IR (KBr: cmL1): 3417 (OeH), 3315 (NeH), 1758 (lactone C]
O), 1731 (lactone C]O), 1689 (amide C]O), 1638 (C]N), 1578
(aromatic C]C),1541 (aromatic), 1515, 1441, 1402, 1319 (CeN), 1243
(phenol CeO), 1191 (lactone CeO), 1134 (ether linkage) cmꢂ1
Mass: m/z: 598 (Mþ), 599 (M þ 1).
2.5.2. 20,70-Di(benzo[d]thiazol-2-yl)-30,60-dihydroxy-3H-spiro
[furan-2,90-xanthen]-5(4H)-one (5b)
Yield: 48%, Melting point > 300 ꢁC.
Gradx h2x
Gradst
fx
¼
Fst
ꢀ
ꢀ
(1)
1H NMR (DMSO- d6, 400 MHz): :
d
2.45 (d, 2H, J ¼ 6.4), 3.50 (d,
h2st
2H, J ¼ 6.4), 6.00 (s, 2H), 7.11 (d, 2H, J ¼ 9.9), 7.11 (d, 2H, J ¼ 9.9), 7.39
(d, 2H, J ¼ 8.9) 7.67 (m, 4H, J ¼ 9.9), 7.79 (s, 2H), 7.97 (s, 2H).
FT-IR (KBr: cmL1): 3310 (phenolic OeH stretching),
2963(aliphatic CeH), 2924(aliphatic CeH),1770 (lactoneC]O),1689
where:
Fx ¼ Quantum yield of compound
Fst ¼ Quantum yield of standard sample
Gradx ¼ Gradient of compound
Gradst ¼ Gradient of standard sample
hx ¼ Refractive index of solvent used for synthesized compound
hst ¼ Refractive index of solvent used for standard sample
N
COOH
H2N
HO
PPA
HO
S
+
Since the compound 5a and 5d have two emissions, the fluo-
rescence quantum efficiency at each of the emissions was calcu-
lated using a standard having similar emission at the respective
HO
OH
OH
1
2
3
wavelength (For short wavelength emission anthracene (
F
¼ 0.27
Scheme 1. Synthesis of (1H-benzo[d]thiazole-2-yl) benzene-1,3-diol 3.

V.S. Patil et al. / Dyes and Pigments 98 (2013) 507e517
509
O
HO
O
O
S
O
N
N
OH
O
O
4a
S
5a
O
HO
O
O
O
N
N
N
OH
O
4b
S
S
5b
O
O
HO
OH
HO
O
O
O
N
N
S
O
OH
O
4c
S
S
3
5c
O
HO
O
O
O
N
N
HOOC
4d
OH
O
S
S
O
5d
O
HOOC
HO
O
O
O
N
S
N
O
NH
4e
2
OH
O
S
NH
5e
2
Scheme 2. Synthesis of fluorescein derivatives 5ae5e.
(amide C]O), 1658 (C]N), 1591 (aromatic C]C), 1504, 1448, 1406,
2.5.5. 4-Amino-20,70-di(benzo[d]thiazole-2-yl)-30,60-dihydroxy-3H-
spiro[isobenzofuran-1-90-xanthen]-3-one (5e) yield: 59%, Melting
point > 300 ꢁC
1348 (CeN), 1258 (phenol CeO), 1192 (lactone CeO), 1018 cmꢂ1
.
Mass: m/z: 548 (Mþ), 549 (M þ 1).
1H NMR (DMSO- d6, 400 MHz):
d 6.33 (s, 2H), 6.66 (s, 2H), 7.11-
2.5.3. 20,70-Di(benzo[d]thiazol-2-yl)-30,60-dihydroxy-5H-spiro
[furan-2,90-xanthen]-5-one (5c) yield: 59%, Melting point > 300 ꢁC
7.15 (d, 4H, J ¼ 8.5), 7.61 (s, 2H), 7.73 (s, 2H), 7.83 (s, 3H), 7.89 (s, 2H),
8.02 (s, 2H).
1H NMR (DMSO- d6, 400 MHz):
d
7.09 (d,1H, J ¼ 10.1), 7.21 (d,1H,
FT-IR (KBr: cmL1): 3523 (carboxylic acid OeH stretching), 3302
(phenol OeH stretching), 3119 (aromatic CeH), 1770 C]O),
1761 C]O), 1594 (aromatic C]C), 1507, 1406, 1301 CeO), 1235,
1122, 1024, 860, 733 cmꢂ1
J ¼ 10.1), 7.71 (dd, 2H, J ¼ 8.7, 6.9), 7.83 (d, 2H, J ¼ 8.9), 7.88 (s, 2H),
8.12 (s, 2H), 8.21 (s,2H), 8.29 (d, 2H, J ¼ 10.1), 8.31 (d, 2H, J ¼ 10.1).
FT-IR (KBr: cmL1): 3310 (phenolic OeH stretching), 2851
(aliphatic CeH), 1734 (lactone C]O), 1706 (amide C]O), 1658(C]
N), 1594 (aromatic C]C), 1504, 1448, 1406, 1348 (CeN), 1258
Mass: m/z: 613 (Mþ), 614 (M þ 1).
(phenol CeO), 1192 (lactone CeO), 1018 cmꢂ1
.
Mass: 550 (Mþ), 551 (M þ 1).
3. Result and discussion
2.5.4. 20,70-Di(benzo[d]thiazol-2-yl)-30,60-dihydroxy-3-oxo-3H-
spiro[isobenzofuran-1-90-xanthen]-5-carboxylic acid (5d)
Yield: 64%, Melting point > 300 ꢁC.
3.1. Synthesis and characterization of compounds
The reaction of 2, 4-dihydroxybenzoic acid 1 with o-amino-
thiophenol 2 in polyphosphoric acid at 150 ꢁC furnished the
required (1H-benzo[d]thiazole-2-yl) benzene-1,3-diol (3) with
good yield (Scheme 1). The reaction of (1H-benzo[d]thiazole-2-yl)
benzene-1,3-diol (3) with phthalic anhydride 4a, succinic anhy-
dride 4b, maleic anhydride 4c, phthalic anhydride 4-carboxylic acid
4d and 3-amino phthalic anhydride 4e in H2SO4 at 160 ꢁC for 4 h
furnished the expected fluorescein derivatives 5ae5e respectively
(Scheme 2).
1H NMR (DMSO- d6, 400 MHz):
d 6.47 (s, 2H), 7.11 (d, 2H,
J ¼ 11.2), 7.43 (d, 2H, J ¼ 11.2), 7.62 (dd, 3H, J ¼ 7.6, 6.8), 7.66 (dd, 2H,
J ¼ 7.6, 6.8), 7.81 (d, 2H, J ¼ 7.6), 8.09 (d, 2H, J ¼ 11.2), 8.23 (bs, 1H),
8.33 (bs, 1H), 8.41 (s, 1H).
FT-IR (KBr: cmL1): 3450, 3329 (phenol OeH stretching), 1703
(C]O), 1654 (C]O), 1589 (aromatic C]C), 1505, 1447, 1406, 1364,
1337 (CeN), 1258 (CeO), 1190, 906, 838 cmꢂ1
Mass: m/z: 642 (Mþ), 643 (M þ 1).

510
V.S. Patil et al. / Dyes and Pigments 98 (2013) 507e517
Table 1
UVevis absorption and emission maxima data of dyes 5ae5b from pH 6 to 13 at 1 ꢀ 10ꢂ6 mol Lꢂ1
.
pH
5a
Stoke shift
nm
5b
Stoke shift
nm
Absorption nm
330
Emission nm
cmꢂ1
Absorption nm
Emission nm
cmꢂ1
6
468
580
e
138
250
e
72463
40000
e
310
410
555
310
410
555
300
395
555
300
395
555
305
480
e
100
e
100000
e
550
330
e
e
e
7
468
578
e
138
248
e
72463
40322
e
481
e
171
e
58479
e
550
335
e
e
e
8
471
582
e
136
247
e
73529
40485
e
481
e
181
e
55248
e
550
335
e
e
e
9
465
577
e
130
242
e
76923
41322
e
481
e
181
e
57142
e
550
300
e
e
e
10
478
580
e
178
280
e
56179
35714
e
480
175
57142
405
550
310
385
555
305
e
e
e
e
e
e
e
e
e
11
478
580
e
168
270
e
59523
37037
e
471
166
60240
405
550
305
385
555
310
375
555
310
e
e
e
e
e
e
e
e
e
12
13
476
577
e
171
272
e
58479
36764
e
481
e
171
e
58479
e
550
305
e
e
e
473
576
e
168
271
e
59523
36900
e
468
158
63291
405
550
375
555
e
e
e
e
e
e
e
e
e
Absorption, emission and Stokes shift (Dl) were measured in nm.
3.2. Preparation of solutions
3.3. Photo-physical properties of compounds (5ae5e)
All the working solutions were prepared in distilled water of
required pH. pH of the solution was maintained by 5 N HCl and 10%
NaOH. Final concentrations of all the working solutions were kept
The fluorescein derivatives (5ae5e) contain two benzothia-
zole units each adjacent to the two hydroxyl groups of the
fluorescein nucleus causing an ESIPT. Unlike the known ESIPT
fluorophores these compounds (5ae5e) have a basic fluorophoric
constant to 1 ꢀ 10ꢂ6 mol Lꢂ1
.
Table 2
UVevis absorption and emission maxima data of dyes 5ce5d from pH 6 to 13 at 1 ꢀ 10ꢂ6 mol Lꢂ1
.
pH
5c
Stoke shift
5d
Stoke shift
nm
Absorption nm
295
Emission nm
458
nm
cmꢂ1
Absorption nm
305
Emission nm
cmꢂ1
6
163
61349
473
582
e
168
277
e
59523
36101
e
555
305
e
e
e
555
300
7
8
9
451
146
68493
495
580
e
195
280
e
51282
35714
e
555
305
e
e
e
555
300
448
143
69930
498
580
e
198
280
e
50505
35714
e
555
295
e
e
e
555
300
465
170
58823
480
580
e
180
280
e
55555
35714
e
555
305
e
e
e
555
285
456
151
66225
478
580
e
193
295
e
51813
33898
e
10
11
555
285
370
535
300
390
535
300
395
535
e
e
e
555
300
450
e
165
e
60606
e
480
580
e
180
280
e
55555
35714
e
e
e
e
555
300
12
13
456
e
156
e
64102
e
478
577
e
178
277
e
56179
36101
e
e
e
e
550
300
460
e
160
e
62500
e
495
582
e
195
282
e
51282
35460
e
e
e
e
555
Absorption, emission and Stokes shift (Dl) were measured in nm.

V.S. Patil et al. / Dyes and Pigments 98 (2013) 507e517
511
Table 3
dyes 5a, 5d and 5e contain phthalein unit while the dyes 5b and 5c
contain furan unit instead of phthalein unit. It is seen that 5b and 5c
do not have dual emission. Amongst the dyes containing phthalein
unit 5a, 5d and 5e, the dye 5e showed only one emission while the
other two showed dual emissions.
UVevis absorption and emission maxima data of dyes 5e from pH 6 to 13 at
1 ꢀ 10ꢂ6 mol Lꢂ1
.
pH
5e
Stoke shift
nm
Absorption nm
Emission nm
cmꢂ1
It was observed that the compounds 5a and 5d have a conju-
gated system to stabilize the electron flow at the central carbon
atom. The emission bands of 5a in the short and long wavelength
regions were located at 465e478 nm and 577e582 nm respectively
(Fig. 1, Table 1). The emission bands for 5d in the short and long
wavelength region were located at 473e498 nm and 578e582 nm
respectively (Fig. 4, Table 2). In the case of 5b and 5c there is no
additional conjugated phenyl ring on the central carbon atom of the
phthalein ring and has a weakly conjugated double bond. Due to
the lack of conjugated system, compounds 5b and 5c showed single
emission band in the region 468e481 nm and 450e465 nm
respectively (Figs. 2 and 3, Tables 1 and 2). Compound 5e has the
electron donating amino substituent at the phthalein ring and it
showed a single emission band at 406e476 nm (Fig. 5, Table 3).
6
7
8
305
550
305
550
305
406
e
101
e
99009
e
408
e
103
e
97087
e
408
593
e
103
288
e
97087
34722
e
550
305
550
305
550
300
550
300
550
300
550
9
408
e
103
e
97087
10
11
12
13
403
e
98
e
102040
e
410
e
110
e
90909
e
410
e
110
e
90909
e
476
e
176
e
56818
e
3.4. Effect of pH on photophysical properties of compounds (5ae5e)
unit (that is fluorescein molecule) interacting with the ESIPT
residue.
The UVevisible absorption emission characteristics of com-
pounds (5ae5e) were measured at different pH range from 6 to 13.
UVevisible absorption-emission spectra are shown in Figs. 1e5 and
values are described in Tables 1e3
The compounds 5ae5e show significant dual absorption bands,
the lower band is between 300 and 330 nm and the longer band is
The absorption-emission properties of all the dyes as a function
of pH are given in Tables 1e3. The dyes were not soluble below pH 6
and hence absorption-emission data could be obtained only using
alkaline solutions at physiological possible pH range. It can be seen
that all the dyes have the xanthene unit as the common feature. The
Fig. 1. Effect of pH on UVevisible absorption and emission spectra of 5a.
Fig. 2. Effect of pH on UVevisible absorption and emission spectra of 5b.

512
V.S. Patil et al. / Dyes and Pigments 98 (2013) 507e517
Fig. 4. Effect of pH on UVevisible absorption and emission spectra of 5d.
Fig. 3. Effect of pH on UVevisible absorption and emission spectra of 5c.
maxima. The emission of the compound 5d at short wavelength
showed red shift from 473 nm to 495 nm as pH values change
from 6 to 7. A blue shift from pH 7 to pH 12 was located at 478 nm;
further as pH value changed from 12 to 13, a red shift in the
emission was located from 478 nm to 495 nm. Long wavelength
emission of the compound 5d was found to be independent of the
pH values and was located near 580 nm (Fig. 4, Table 2). The
compound 5e showed two absorption bands at 305 nm and
550 nm which were found to be independent of pH. The com-
pound 5e on excitation at 305 nm gave emission in the region of
406e410 nm for pH 6 to 12. Bathocromic shift for pH 13 was
observed at 476 nm (additional emission band was observed for
pH 8 at 598 nm). (Fig. 5, Table 3). UVevisible absorption and
fluorescence emission photograph of compounds in water 5ae5e
is shown in Fig. 6.
around 550 nm for pH ranging from 6 to 9. From pH 10 onwards,
the absorption band experienced a blue shift and was located at
around 300 nm. For pH 12 and 13 one more extra absorption band
was located at 405 nm. The compound 5a showed two emission
bands on single excitation at 330 nm. These two emission bands
were found to be independent of the pH values. These emission
bands were located in the region of 468e478 nm and 576e580 nm
(Fig. 1, Table 1). The compound 5b showed three absorption bands
at 310, 410 and 555 nm for pH 6. Further from pH 7 onwards a blue
shift in short wavelength absorption band was observed in the
region of 300e310 nm and long wavelength absorption band
remained the same. The compound 5b on excitation at the cor-
responding short wavelength absorption maxima gave a single
emission band in the region of 481e468 nm (Fig. 2, Table 1). For
the compound 5c two absorption bands were located up to pH 11.
The short wavelength absorption band is between 295 and
305 nm; from pH 11 to pH 13 three absorption bands were located
in the region of 285e555 nm. The compound 5c on excitation at
the corresponding short wavelength absorption maxima gave
single emission band in the region of 448e465 nm (Fig. 3, Table 2).
The compound 5d showed two absorption bands, the short
wavelength absorption band was located in the region 285e
305 nm and the long wavelength absorption band was located at
555 nm. Absorption of the compound 5d was found to be inde-
pendent of the pH values. The compound 5d showed two emission
bands on excitation with the short wavelength absorption
3.5. Effect of viscosity on fluorescence intensity
Fluorescein is the most widely used label, due to its low cost,
high absorptivity and good quantum yield when conjugated to
biomolecules [30]. However the development of fluorescence la-
bels based on heterogeneous assay presented several problems
mainly low signal to noise ratio when proteins are labelled with
multiple fluorescence molecules [31]. This phenomenon has been
attributed to the small stokes shift of fluorescence molecule that
permits energy transfer from one fluorescent molecule to another
causing self quenching [32].

V.S. Patil et al. / Dyes and Pigments 98 (2013) 507e517
513
Fig. 6. The UVevis absorption and fluorescence emission photographs of compounds
5ae5e in water.
Table 5
Physical properties and relative fluorescence quantum yield of dyes 5ae5e.
Compound Mol. Wt. Thermal
Quantum yield at Quantum yield at
stability ꢁC (%) short wavelength long wavelength
5a
5b
5c
5d
5e
598.64
548.58
550.60
642.65
613.66
352(76.51)
295(78.09)
282(78.63)
280(78.00)
300(80.05)
ɸ1a ¼ 0.00574
ɸ1b ¼ 0.0126
ɸ1c ¼ 0.0100
ɸ1d ¼ 0.0074
ɸ1e ¼ 0.0159
ɸ1a ¼ 0.3027
ɸ1d ¼ 0.1134
Fig. 5. Effect of pH on UVevisible absorption and emission spectra of 5e>.
In order to overcome self quenching effect on conventional
fluorophores some specific dyes having similar absorbance char-
acteristics have been reported. The several substances such as
polyethylene glycol (PEG), triton X, and glycerin have been used as
fluorescence sensitivity enhancer by minimizing the fluorescence
self quenching phenomenon [31].
The effect of increasing the concentration of glycerin on the
fluorescence intensity of the novel synthesized substituted fluo-
rescein gains very high importance [31]. The reason behind the
increase in the fluorescence intensity is restricting the self
quenching of fluorophore. The concentration of glycerin increases
the viscosity which minimizes the speed of the charged species in
solution and ultimately loss of energy by collision. We have
reported here an interesting approach in order to overcome self-
quenching effects of newly synthesized fluorescent compounds.
An increasing concentration of glycerin up to 30% gives increasing
fluorescence intensity.
It has been observed that as concentration of glycerin increased,
percentage intensity of fluorescence was also increased by 16.21%,
7.19% and 16.98% for compound 5a, 5b and 5c respectively (Table 4).
An increase in the emission intensity for the compound 5d was
found to be the highest amongst all the dyes (25.83%) (Table 4). But
a different result was found in the case of the compound 5e that the
emission intensity decreased by 3.42% as the concentration of
glycerin increased (Table 4).
Table 4
Photophysical properties of fluorescein derivatives 5a-5e in water and glycerin.
% Of glycerin
Viscosity
centipoises/m Pa s
5a487 Emission
intensity (a.u.)
5b Emission
intensity (a.u.)
5c Emission
intensity (a.u.)
5d482 Emission
intensity (a.u.)
5e Emission
intensity (a.u.)
0
5
10
15
20
25
30
1.00
1.15
1.31
1.53
1.76
2.13
2.50
153
162
175
175
181
181
180
16.21%
643
662
666
670
679
684
691
7.19%
264
294
291
292
294
292
313
16.98%
118
134
141
146
148
150
153
25.83%
208
206
200
202
194
201
201
ꢂ3.42%
% Change intensity

514
V.S. Patil et al. / Dyes and Pigments 98 (2013) 507e517
Table 6
TD-DFT study of compounds 5ae5e.
Compounds
TD-DFT
lEmxapxt(nm)
Vertical
excitation
f
Assignment
Observed emission
Theoretical emission
Stokes shift
nm
nm
eV
nm
nm
5a Enol
5b Enol
330
550
310
410
555
305
555
305
555
305
550
496
2.497
1.154
1.213
H / L (0.692)
H / L (0.689)
468a 578b
565
138
248
171
480
2.577
481
550
5c Enol
5d Enol
5e Enol
498
482
473
2.489
2.567
2.493
1.009
1.190
1.145
H / L (0.691)
H / L (0.690)
H / L (0.653)
451
623
609
581
146
495a 580b
408
195
280
103
Analyses were carried out at room temperature (25 ꢁC); Experimentally observed lmax; TD-DFT computations were carried out with the use of optimized structures at B3LYP
method with 6e31G(d) basis set; f¼ Oscillator strength.
a
Emission at short wavelength.
Emission at longer wavelength.
b
3.7. Time Dependent Density Functional Theory (TD-DFT)
computation
Table 7
Computed absorption and emission of compounds 5a-5e in water (Keto Form).
Compounds TD-DFT
The compounds are highly fluorescent in solution under irra-
diation of UV-Light. Compounds 5ae5e shows two absorption
bands in UVevisible region. The experimental observed absorp-
tions of compounds were compared with the vertical excitations
obtained by TD-DFT method. Compounds emits in the range 451e
580 nm, Compounds 5a and 5d show dual emission and com-
pounds 5b, 5c and 5e shows single emission with large Stoke’s
shift value The DFT computation suggested only single emissions
for each of the compounds 5b, 5c and 5e. In the case of compound
5a longer emission 578 nm is well in agreement with the emis-
sion obtained by DFT 565 nm, the difference between the
observed and theoretical emissions is 13 nm, but a large differ-
ence was observed with the short emission of compound 5a
(103 nm). The compound 5d shows dual emission; the long
wavelength emission (580 nm) was in well agreement with
computational TD-DFT value (609 nm), the difference between
observed emission and theoretical emission is 29 nm, The dif-
ference between short emission obtained experimentally and
computationally is large (114 nm). In the case of compounds 5b,
5c and 5e the difference between the observed and computed
Vertical
excitation
f
Assignment
Computational Theoretical
emission (nm) stokes shift
(nm)
nm eV
5a Keto
5b Keto
5c Keto
5d Keto
5e Keto
543 2.279 1.307 H / L (0.689) 622
532 2.328 1.337 H / L (0.691) 603
534 2.321 1.338 H / L (0.690) 662
534 2.320 1.319 H / L (0.690) 678
79
71
128
144
71
532 2.327 1.164 H L (0.677)
603
3.6. Relative fluorescence quantum yield of compounds (5ae5e)
The relative quantum efficiency of the compound 5a at the short
and long wavelength emission is 0.00574 and 0.3027 respectively.
For the compounds 5b, 5c and 5e the relative quantum efficiency
values are 0.0126, 0.0100 and 0.0159 respectively. The relative
quantum efficiency values of the compound 5d at the short and
long wavelength emission are 0.0074 and 0.1134 respectively. The
relative fluorescence quantum yields are summarized for com-
pounds 5ae5e in Table 5.
Table 8
HOMO-LUMO orbital diagrams and energy of compound 5ae5e.
Compounds
HOMO
LUMO
5a Enol
e0.31 eV
e0.24 eV
5a Keto
e0.30 eV
e0.24 eV

V.S. Patil et al. / Dyes and Pigments 98 (2013) 507e517
515
Table 8 (continued )
Compounds
HOMO
LUMO
5b Enol
e0.31 eV
e0.24 eV
5b Keto
e0.30 eV
e0.24 eV
5c Enol
e0.31 eV
e0.24 eV
5c Keto
e0.30 eV
e0.24 eV
5d Enol
e0.30 eV
e0.25 eV
5d Keto
e0.30 eV
e0.24 eV
5e Enol
e0.31 eV
e0.4 eV
5e Keto
e0.30 eV
e0.23 eV

516
V.S. Patil et al. / Dyes and Pigments 98 (2013) 507e517
Acknowledgements
The authors Vikas Patil and Vikas Padalkar are thankful to
Institute of Chemical Technology, Mumbai India. AT thank to UGC
for JRF.
References
[1] Dey JK, Dograt SK. Electronic absorption and fluorescence spectral charac-
teristics and prototropic reactions of some hydroxy- and methoxy-substituted
derivatives of 2-(2’-hydroxyphenyl) benzothiazoles. J Photochem Photobio A
Chem 1992;66:15e31.
[2] Rodembusch FS, Leusin FP, Campo LF, Stefani V. Excited state intramolecular
proton transfer in amino 2-(2’-hydroxyphenyl) benzazole derivatives: ef-
fects of the solvent and the amino group position. Luminescence 2007;126:
728e34.
[3] LeGourriérec D, Vladimir AK, Brown RG, Rettig W. Excited-state intra-
molecular proton transfer (ESIPT) in 2-(2’-hydroxyphenyl)-oxazole and thia-
zole. J Photochem Photobioi A Chem 2000;130:101e11.
Fig. 7. Thermogravimetric analysis of compound 5ae5e.
[4] Soufi WA, Grellmaan KH, Nickel B. Triplet state formation and cis- trans
isomerization in the excited singlet state of the keto tautomer of 2-(2’-
hydroxyphenyl) benzothiazole. Chem Phys Lett 1990;174(6):609e16.
[5] Holler MG, Campoa LF, Brandelli A, Stefani V. Synthesis and spectroscopic
characterization of 2-(2’-hydroxyphenyl)benzazole isothiocyanates as new
fluorescent probes for proteins. J Photochem Photobio A Chem 2002;149:
217e25.
[6] Taki M, Wolford JL, O’Halloran TV. Emission ratiometric imaging of intracel-
lular zinc: design of a benzoxazole fluorescent sensor and its application in
two-photon microscopy. J Am Chem Soc 2004;126:712e3.
emissions are large. All compounds shows excitation from HOMO
to LUMO with almost same population. The energy gap between
HOMO to LUMO is also almost the same for all the compounds.
The theoretical vertical excitation, emission and excitation
pattern in both form are shown in Table 6 and Table 7. Orbital
diagram and energies for HOMO and LUMO orbital’s are sum-
marized in Table 8.
[7] Ohshima A, Momotake A, Arai T. A new fluorescent metal sensor with two
binding moieties. Tetrahedron Lett 2004;45:9377e81.
[8] Rodembusch FS, Brand FR, Correa DS, Pocos JC, Martinelli M, Stefani V.
Transition metal complexes from 2-(20-hydroxyphenyl)benzoxazole: a spec-
troscopic and thermogravimetric stability study. Mater Chem Phys 2005;92:
389e93.
3.8. Thermal stability of compounds
[9] Vazquez SR, Rodriguez MCR, Mosquera M, Rodriguez PF. Excited-state intra-
molecular proton transfer in 2-(30-Hydroxy- 20-pyridyl)benzoxazole, evidence
of coupled proton and charge transfer in the excited state of some o-
hydroxyarylbenzazoles. J Phys Chem A 2007;111:1814e26.
[10] Sakai K, Tsuzuki T, Itoh Y, Ichikawa M, Taniguchi Y. Using proton-transfer laser
dyes for organic laser diodes. Appl Phys Lett 2005;86:1e3.
[11] Kim S, Seo J, Jung HK, Kim JJ, Park SY. White luminescence from polymer thin
films containing excited-state intramolecular proton-transfer dyes. Adv Mater
2005;17:2077e82.
[12] Park S, Kwon JE, Kim SH, Seo J, Chung K, Park SY, et al. A white-light-emitting
molecule: frustrated energy transfer between constituent emitting centers.
J Am Chem Soc 2009;131:14043e9.
[13] Campo LM, Correa DS, Araujo MA, Stefani V. New fluorescent monomers and
polymers displaying an intramolecular proton-transfer mechanism in the
electronically excited state (ESIPT). 1 Synthesis of benzazolylvinylene de-
rivatives and its copolymerization with methyl methacrylate (MMA). Mac-
romol Rapid Commun 2000;21:832e6.
In order to give more insight into the dyes 5ae5e, the thermal
studies of the compounds have been carried out using thermal
gravimetric techniques (TGA). The thermogravimetric studies
have been carried out in the temperature range 50e600 ꢁC under
nitrogen gas at a heating rate of 10 ꢁC minꢂ1. The TGA results
indicated that the synthesized dyes are stable up to 300 ꢁC. TGA
revealed the onset decomposition temperature (Td) of dyes 5ae5e
at 352 ꢁC, 295 ꢁC, 282 ꢁC, 280 ꢁC and 300 ꢁC respectively as shown
in Fig. 7. Above 280 ꢁC the thermogravimetric curve of the syn-
thesized compounds show a major loss in weight. The compari-
sons of the Td (decomposition temperature) showed that the
thermal stability of the 5a-5e decreases in the order
5a > 5e > 5b > 5c > 5d. The results showed that synthesized dyes
have good thermal stability and completely decomposed beyond
450 ꢁC. However dyes 5a, 5b, 5c, 5d and 5e showed elongated
decomposition behaviours and completely decomposed beyond
600 ꢁC as shown in Fig. 7.
[14] Padalkar VS, Tathe AB, Gupta VD, Patil VS, Phatangare KR, Sekar N. Synthesis
and photo-physical characteristics of ESIPT inspired 2-substituted benzimid-
azole, benzoxazole and benzothiazole fluorescent derivatives.
2012;22:311e22.
J Fluoresc
[15] Haugland R, Spence M, Johnson I, Basey A. The handbook: a guide to fluo-
rescent probes and labeling technologies. 10th ed. Carlsbad: Molecular
Probes, Invitrogen Detection Technologies; 2005.
[16] Bernard V. Molecular fluorescence: principles and applications. Weinheim:
Wiley-VCH Verlag Gmbh; 2001.
[17] Duan Y, Liu M, Sun W, Wang M, Liu S, Li Q. Recent progress on synthesis of
fluorescein probes. Mini Rev Org Chem 2009;6:35e43.
4. Conclusion
In this paper we have reported new analogues of fluorescein
containing an inbuilt ESIPT residue and their photophysical prop-
erties as a function of pH and viscosity. The compounds 5a and 5b
show dual emission, their dual emission characteristics as a func-
tion of pH have been studied. It was also observed that with the
increase in viscosity by glycerin gives higher emission intensity. The
relative fluorescence quantum yields of compounds were deter-
mined by using suitable standards. Photophysical properties of the
synthesized compounds were supported by DFT and it was
observed that computational results are good agreement with the
theoretical observations. The synthesized compounds were
confirmed by IR, 1H NMR, mass spectral analysis. The thermal sta-
bility was determined by TGA analysis and it was found that the
compounds are thermally stable.
[18] Han J, Burgess K. Fluorescent indicators for intracellular pH. Chem Rev
2010;110:2709e28.
[19] Patil VS, Padalkar VS, Phatangare KR, Gupta VD, Umape PG, Sekar N. Synthesis
of new ESIPT-fluorescein: photophysics of ph sensitivity and fluorescence.
J Phys Chem A 2012;116:536e45.
[20] Padalkar VS, Gupta VD, Patil VS, Phatangare KR, Sekar N. Indion 190 resin:
efficient, environmentally friendly, and reusable catalyst for synthesis of
benzimidazoles, benzoxazoles, and benzothiazoles. Green Chem Lett Rev
2012;5(2):139e45.
[21] Treutler O, Ahlrichs R. Efficient molecular numerical integration schemes.
J Chem Phys 1995;102:346e54.
[22] Becke AD. A new mixing of Hartree-Fock and local density-functional theories.
J Chem Phys 1993;98:1372e7.
[23] Lee C, Yang W, Parr RG. Development of the colle-salvetti conelation energy
formula into a functional of the electron density. Phys Rev B 1988;37:785e9.
[24] (a) Kim CH, Park J, Seo J, Park SJ, Joo TJ. Excited state intramolecular proton
transfer and charge transfer dynamics of a 2-(20-hydroxyphenyl) benzoxazole
derivative in solution. Phys Chem A 2010;114:5618e29;

V.S. Patil et al. / Dyes and Pigments 98 (2013) 507e517
517
(b) Santra M, Moon H, Park MH, Lee TW, Kim Y, Ahn KH. Dramatic substituent
effects on the photoluminescence of boron complexes of 2-(benzothiazol-2-
yl)phenols. Chem Eur J. 10.1002/chem.201200726; 2012;
(b) Tomasi J, Mennucci B, Cammi. Quantum mechanical continuum solvation
models. R Chem Rev 2005;105:2999e3094.
[28] Frisch MJ, Trucks GW, Schlegel HB, Scuseria GE, Robb MA, Cheeseman JR, et al.
Gaussian 09, revision C.01. Wallingford, CT: Gaussian, Inc.; 2010.
[29] Padalkar VS, Patil VS, Sekar N. Synthesis and photophysical properties
of fluorescent 1,3,5-triazine styryl derivatives. Chem Cent J 2011;77(5):
1e7.
[30] Trevor AS, Lisa MB, David ED. Fluorescence polarization measurements of the
local viscosity of hydroxypropyl guar in solution. Macromolecules 2002;35(7):
2736e42.
(c) Li H, Niu L, Xu X, Zhang S, Gao F. Excited state proton transfer in guanine in
the gas phase and in water. J Fluorescence 2011;21:1721e8.
[25] Furche F, Rappaport D. Density functional theory for excited states: equilib-
rium structure and electronic spectra. In: Olivucci M, editor. Computational
photochemistry, 16. Amsterdam: Elsevier; 2005. [Chapter 3].
[26] (a) Lakowicz JR. Principles of fluorescence spectroscopy. 2nd ed. New York:
Kluwer Academic; 1999;
(b) Valeur B. Molecular fluorescence: principles and applications. Weinheim:
Wiley-VCH Verlag; 2001.
[27] (a) Cossi M, Barone V, Cammi R, Tomasi J. Ab initio study of solvated mole-
cules: a new implementation of the polarizable continuum model. J Chem
Phys Lett 1996;255:327e35;
[31] Patil VS, Padalkar VS, Phatangare KR, Gupta VD, Seregei E, Sekar N. Novel ESIPT
fluorescein: sensor for blood and viscosity. Bio Nano Sci 2012;2:259e65.
[32] Brey LA, Gary BS, Drickamer HG. High-pressure study of the effect of viscosity
on fluorescence and photoisomerization of trans-stilbene. J Am Chem Soc
1979;101(3):129e34.