Identifying the bond responsible for the fluorescence modulation in an amyloid fibril sensor

Article abstract of DOI:10.1002/chem.200902968

An ultrafast intramolecular bond twisting process is known to be the responsible mechanism for the sensing activity of the extensively used amyloid fibril sensor thioflavin T (ThT). However, it is not yet known which one of the two possible single bonds in ThT is actually involved in the twisting process. To resolve this fundamental issue, two derivatives of ThT have been designed and synthesized and subsequently their photophysical properties have been studied in different solvents. It is understood from the present study that the rotation around the central C-C single bond, and not that around the C-N single bond, is primarily responsible for the sensor activity of ThT. Detailed viscosity-dependent fluorescence studies revealed that the ThT derivative with restricted C-N bond rotation acts as a better sensor than the derivative with free C-N bond rotation. The better sensory activity is directly correlated with a shorter excited-state lifetime. Results obtained from the photophysical studies of the ThT derivatives have also been supported by the results obtained from quantum chemical calculations.

Full text of DOI:10.1002/chem.200902968

FULL PAPER
DOI: 10.1002/chem.200902968
Identifying the Bond Responsible for the Fluorescence Modulation
in an Amyloid Fibril Sensor
Anvita Srivastava,[a, c] Prabhat K. Singh,[a] Manoj Kumbhakar,[a] Tulsi Mukherjee,[a]
Subrata Chattopadyay,[b] Haridas Pal,[a] and Sukhendu Nath*[a]
Abstract: An ultrafast intramolecular
bond twisting process is known to be
the responsible mechanism for the
sensing activity of the extensively used
amyloid fibril sensor thioflavin T
(ThT). However, it is not yet known
which one of the two possible single
bonds in ThT is actually involved in
the twisting process. To resolve this
fundamental issue, two derivatives of
ThT have been designed and synthe-
sized and subsequently their photo-
physical properties have been studied
in different solvents. It is understood
from the present study that the rota-
cosity-dependent fluorescence studies
revealed that the ThT derivative with
ꢀ
restricted C N bond rotation acts as a
ꢀ
tion around the central C C single
better sensor than the derivative with
ꢀ
ꢀ
bond, and not that around the C N
free C N bond rotation. The better
single bond, is primarily responsible for
the sensor activity of ThT. Detailed vis-
sensory activity is directly correlated
with a shorter excited-state lifetime.
Results obtained from the photophysi-
cal studies of the ThT derivatives have
also been supported by the results ob-
tained from quantum chemical calcula-
tions.
Keywords: sensors · bond twisting ·
fluorescence · thioflavin T · time-
resolved spectroscopy
Introduction
mately leads to the fibril formation. Among several tech-
niques used to monitor and detect the amyloid fibril forma-
tion, the use of an extrinsic fluorescence probe is found to
be very extensive.[6–10]
Thioflavin T (ThT), a benzothiazole-based cationic dye
(see Scheme 1) is one of the frequently used extrinsic fluo-
rescent probes to study the formation and detection of amy-
The existence of protein molecules in their native structural
form is very essential for their normal biological activities.
A significant departure from their native structural forms
leads to several diseases. For example, misfolding of protein
molecules can lead to their association and ultimately for-
mation of insoluble protein aggregates. The presence of
such aggregated protein molecules in brain, known as amy-
loid fibrils, results in the several neurodegenerative disor-
ders, like Alzheimerꢀs and Parkinsonꢀs diseases.[1–5] To detect
and control such protein-related diseases, it is very essential
to monitor the progress of protein aggregation, which ulti-
[a] A. Srivastava, P. K. Singh, Dr. M. Kumbhakar, Dr. T. Mukherjee,
Dr. H. Pal, Dr. S. Nath
Radiation & Photochemistry Division
Bhabha Atomic Research Centre, Trombay
Mumbai-400 085 (India)
Scheme 1. Molecular structure of thioflavin T and its derivatives used in
the present study.
Fax : (+91)22-5505151
E-mail: snath@barc.gov.in
loid fibrils.[11–14] ThT is known to specifically bind to the pro-
teins when the latter is in the form of amyloid fibril, but not
in their native structural form.[15,16] The emission yield of
ThT in water is extremely low[17,18] but shows a large en-
hancement on binding with amyloid fibrils.[2,12,15,19–22] The
underlying mechanism for the extremely large fluorescence
enhancement for ThT associated with amyloid fibril is still
[b] Dr. S. Chattopadyay
Bio-Organic Division
Bhabha Atomic Research Centre, Trombay
Mumbai-400 085 (India)
[c] A. Srivastava
Department of Chemistry, University of Allahabad
Allahabad, Utter Pradesh-211 002 (India)
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9257

unknown. However, several hypotheses have been proposed
in the literature to explain this phenomenon. Formation of
ThT micelles in the environment of the amyloid fibril have
been proposed by Khurana et al.[23] Groenning et al.[24] have
proposed that the formation of ThT excimers in the excited
state is mainly responsible for the observed fluorescence en-
hancement, though the formation of excimers by a charged
species is always a topic of debate. Very recently, by using
optical microscopic measurements, Kitts and Vanden
Bout[25] have shown that the observed enhancement in the
emission of the ThT in amyloid fibril is due to the associa-
tion of the monomeric form of the dye to the protein rather
than the formation of their micelles or excimers.
Detailed studies in several confined environments, like in
amyloid fibril,[2,12,15,19–22] polymer,[26] glass matrix,[27] nano-
confined water pool,[18] etc., clearly indicate that the high
local viscosity of the microenvironment around the dye is di-
rectly related to the observed fluorescence enhancement of
the ThT dye in these systems. However, understanding the
basic molecular process that is associated with the observed
fluorescence enhancement of ThT in the above microenvir-
onments remains elusive. Considering the molecular struc-
ture of ThT, it is hypothesized that in bulk water, due to the
low viscosity of the medium, some intramolecular bond
twisting process takes place in the excited state of the ThT
molecules. This bond twisting process effectively introduces
a very fast nonradiative decay channel for the excited ThT
molecule, which leads to its extremely low emission yield. In
contrast, in a highly viscous medium, like in amyloid fibril,
the bond twisting process in the dye is substantially retard-
ed, reducing the nonradiative decay channel in its excited
state. This results in a remarkable increase in the emission
yield of ThT in a highly viscous medium. It is also proposed
that the bond twisting process is associated with the large in-
tramolecular charge transfer from the aniline moiety of ThT
to the benzothiazole moiety.[28,29] Such a charge transfer, as-
sociated with the bond twisting in the dye, effectively results
in the formation of a twisted intramolecular charge transfer
(TICT) state in the excited ThT molecules.
well as from theoretical calculations, it was shown that the
twisting of the amino group as well as of the anilino group
contribute significantly towards the nonradiative decay
channel of this molecule in its excited states.[32] Very recent-
ly, Saha et al.[33] have shown that in dimethyl-aminostyryl-
benzothiazole, having a close structural relation with ThT,
only the twisting of the amino group is responsible for the
fast nonradiative decay in its excited state.
Based on these literature precedents, it is expected that
for ThT twisting of either of these two bonds (see
Scheme 1) can be responsible for the ultrafast nonradiative
decay channel in its excited state. In order to use ThT or
some of its derivatives as an efficient sensor for different mi-
croenvironments, including that of amyloid fibril, it is very
essential to identify the bond, which is responsible for its ul-
trafast nonradiative decay channel. This information might
also help to develop much better sensor based on the ThT
structure. In order to disentangle this issue, we designed and
synthesized two ThT derivatives, Ia and Ib (Scheme 1), with
the premise that the lack of a methyl group in the benzo-
thiazole, as is present in ThT, would not change the fluores-
cence properties, compared to ThT. For the two dye mole-
cules, twisting around both bonds is possible in Ia, but the
presence of a julolidine group in Ib would prohibit twisting
ꢀ
of the Ph NR2 bond and allows twisting around the central
ꢀ
ꢀ
C C bond (benzothiazole aniline) only. Subsequently, we
carried out detailed steady-state and time-resolved photo-
physical studies of Ia and Ib in different solvents to identify
the bond that is responsible for the sensory activity of the
ThT class of molecules. The experimental results are also
substantiated by quantum chemical calculations.
Results and Discussion
Steady-state absorption and fluorescence studies: The
ground-state absorption and the steady-state fluorescence
spectra of the ThT derivatives Ia and Ib in aqueous solution
are shown in Figure 1. As indicated in the Figure, both ab-
sorption and emission spectra of Ib are significantly red
From the molecular structure of ThT (see Scheme 1), it is
clearly evident that there are two possible bond twistings,
ꢀ
one occurring around the C N single bond (i.e., twisting of
the amino (NR2) group) and/or the other occurring around
ꢀ
the central C C single bond (i.e., twisting of the anilino
(PhNR2) group). Twisting around any one of these bonds
can introduce the nonradiative decay channel in the excited
state of ThT, causing a very low emission yield. There are
several reports in the literature on organic dyes with an N-
alkylated anilino group where it is shown that the twisting
of either the N-alkylated amino group or of the anilino
group can cause the enhanced nonradiative decay of the ex-
cited dye that results in a large reduction of their emission
yields. For example, in several triphenyl dyes with amine
substitution, it is reported that the twisting of the anilino
group in their excited state is responsible for their low emis-
Figure 1. Ground-state absorption and steady-state fluorescence spectra
of Ia (c) and Ib (a) in aqueous solution. Absorption and emission
spectra of ThT in water (g) are also shown for comparison.
sion yield.[30,31] For para-(N,N-dialkylamino)benzylidene
ononitriles, however, based on experimental evidence as
ACHTUNGTNERmNUNG al-
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Fluorescence Modulation in an Amyloid Fibril Sensor
FULL PAPER
shifted with respect to those of Ia. This red shift is possibly
due to the fact that the difference in the ground-state and
the excited-state dipole moment is higher for the former dye
as compared to that in the latter. The more polar nature of
the dye Ib as compared to that of dye Ia is a result of the
presence of the julolidine group in the former. The red shift
of the absorption maxima in dye Ib compared to dye Ia is
also supported by the quantum chemical calculations (see
latter). Similar red shifts in the absorption and emission
spectra due to the introduction of the julolidine group have
also been observed in coumarin derivatives.[34] As anticipat-
ed, the absorption and the emission spectral characteristics
of Ia in water (and also in other solvents) were quite similar
to that of ThT (see Figure 1). This clearly supports our pro-
posed hypothesis on the inconsequential role of the methyl
substitution in the benzene ring of the benzothiazole unit in
the photophysical properties of the ThT molecules.
single bond (i.e., the twisting of the anilino group) is mainly
responsible for the fluorescence sensing activity of the ThT
dye.
As the sensing activity of ThT is related to the bond twist-
ing process, the emission quantum yield of the two ThT de-
rivatives Ia and Ib, is expected to be highly dependent on
the viscosity of the medium. To investigate this aspect, we
measured the fluorescence quantum yields of Ia and Ib in
solvent mixtures of acetonitrile–ethylene glycol in different
proportions so that the viscosity of the medium can be
changed over a wide range.[36] The reason for selecting ace-
tonitrile–ethylene glycol as solvent mixture is to maintain
the polarity of the medium while varying the composition of
the co-solvents. As charge separation takes place in the ex-
cited state of these molecules, the polarity of the medium
can affect its fluorescence quantum yield.[28] The dielectric
constant of acetonitrile and ethylene glycol are in the similar
range (eꢁ37).[37] Further, to see the fluorescence enhance-
ment at the higher viscosity region, the fluorescence yields
of the two dyes were also measured in glycerol at different
temperatures.[38,39] It is to be mentioned, that for ThT it is re-
ported that its fluorescence quantum yield in solvents with
high dielectric constants, like acetonitrile, methanol, and
water, is almost independent of the solvent polarity.[17] In
the present study, we also find similar fluorescence quantum
yields for Ia and Ib in water and acetonitrile (see Table 1).
Thus, it is expected that within this high solvent polarity
region the TICT-mediated nonradiative de-excitation of the
excited Ia and Ib molecules is not affected significantly by
the small changes in the solvent polarity. Thus, a marginally
higher dielectric constant of glycerol (eꢁ42.5) as compared
to that of acetonitrile and ethylene glycol should not affect
the fluorescence quantum yield of these dyes, rather the vis-
cosity of the solvent will play a significant role in modulat-
ing their photophysical behavior. The variation of the fluo-
rescence quantum yields (relative to that in acetonitrile) of
Ia and Ib with the viscosity of the medium is shown in
Figure 2. It is clearly indicated from the inset of Figure 2
Fluorescence quantum yield values have been measured
for both the ThT derivatives in different solvents and are
listed in Table 1. The quantum yield values of ThT in differ-
Table 1. Emission quantum yields (ff) of ThT, Ia, and Ib in different sol-
vents.
Solvent
Quantum yield (ff)ꢂ104
ThT
Ia
Ib
water
acetonitrile
ethylene glycol
1.0ꢂ0.2
1.42ꢂ0.15
17.5ꢂ0.5
1.0ꢂ0.2
1.45ꢂ0.2
16.1ꢂ0.4
0.84ꢂ0.2
0.84ꢂ0.15
10.1ꢂ0.5
ent solvents are also presented in Table 1 for comparison. It
is evident from Table 1 that the quantum yield of ThT and
Ia are quite comparable in all solvents studied. This result
indicates that ThT and its derivative Ia have similar photo-
physical behavior, which is consistent with our inference
from the absorption and emission spectral studies. Thus,
henceforth the dye Ia will be considered as a representative
of ThT and its photophysical properties will be compared
with those of Ib.
It is clearly indicated from Table 1 that the introduction
of the julolidine group in Ib to prevent the twisting around
ꢀ
the C N bond does not cause any appreciable changes in
the emission yield of the dye as compared to that of Ia and
ThT. Further, it can be seen that the emission yield of Ib is
always relatively less as compared to that of Ia and ThT in
all the solvents studied. As reported in the literature, the re-
striction of the bond rotation in ThT in viscous environment,
like in amyloid fibril, is the reason for the observed large
enhancement
(ꢁ1000 times)
in
the
fluorescence
yield.[2,12,15,19–22,35] If the restriction in the rotation around C
N bond is responsible for the sensing activity of ThT, one
would expect a large increase in the emission quantum yield
for the dye Ib in comparison to that of Ia and ThT. Lack of
any such enhancement in the fluorescence yield of Ib clearly
ꢀ
Figure 2. Variation of fluorescence quantum yield (relative to that in ace-
~
*
tonitrile) of Ia ( ) and Ib ( ) with the viscosity of the medium. The
inset shows the variation in the emission yield for the low solvent viscosi-
ty region. Open symbol corresponds to acetonitrile–ethylene glycol mix-
tures and filled symbol corresponds to glycerol solutions with different
viscosity (varied by changing the temperature).
ꢀ
suggests that the twisting around the C N bond is not re-
sponsible for the sensory activity of the ThT molecules. This,
ꢀ
in turn, revealed that the twisting around the central C C
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S. Nath et al.
that in the low-viscosity region (<20 cP) the fluorescence
enhancement due to the increase in the viscosity is very sim-
ilar for both of the dyes. However, a substantial difference
in the fluorescence enhancement was observed in the region
of higher solvent viscosity, where the rate of change in the
fluorescence quantum yield with the solvent viscosity is rela-
tively higher for Ib compared to that for Ia. This result
clearly demonstrates that Ib is a relatively better viscosity
sensor than Ia.
their estimated average fluorescence lifetimes in different
solvents are presented in Table 2. It is evident from Figure 3
and also from Table 2 that the average lifetime of Ia is
Table 2. Average excited state lifetime of Ia and Ib in different solvents.
Solvent
Average fluorescence lifetime [ps]
Ia
0.59
0.41
12.4
Ib
water
acetonitrile
ethylene glycol
0.48
0.30
11.8
Time-resolved fluorescence studies: The fundamental issue
that determines the sensing activity of a fluorescence dye is
the competition between the radiative and nonradiative
(due to the bond twisting) decay processes in the excited
state of the dye molecules. The fluorescence quantum yield,
ff, of a dye is defined by the following Equation (1),
always longer as compared to that of Ib in all the solvents
studied. The observed ultrafast decay is known to be due to
the torsional motion in the ThT dyes.[18] Thus, the present
results clearly indicate that the torsional motion is relatively
tor
nr
kf
faster in Ib compared to Ia. Accordingly, the k value is
ꢀf ¼
¼ tfkf
ð1Þ
kf þ knr þ ktnorr
always higher for Ib than that for Ia irrespective of the sol-
vent used. Because of the larger value of k and hence,
tor
nr
where kf is the radiative rate constant, knr is the nonradiative
rate constant for processes other than bond twisting process,
lower value of tf, the increase in fluorescence yield due to
an increase in the solvent viscosity is expected to be rela-
tively higher for Ib than for Ia. Hence, the observed larger
enhancement in fluorescence quantum yield of the dye Ib
with an increase in the solvent viscosity as compared to that
of dye Ia is attributed to the faster bond twisting process in
the former dye.
tor
nr
k
is the nonradiative rate constant due to the bond twist-
À
Á
ing process, and tf ¼ 1=kf þ knr þ ktnorr is the excited-state
lifetime of the dye. In a less viscous solvent, for example
water, the intramolecular bond twisting process is very effi-
tor
nr
cient for ThT and thus, leads to a higher value for k , re-
sulting in a very low tf value. Because of the very high kntorr
value, the quantum yield ff is also very low in low viscous
Quantum-chemical calculations: To understand the reason
behind the faster bond twisting dynamics in Ib as compared
to Ia, detailed quantum-chemical calculations were carried
out. The structure of both molecules were optimized by ap-
plying DFT using the B3LYP functional and are shown in
Figure 4.
From the optimized structures of both dyes it is evident
that these molecules are non-planner in nature. The plane
containing the benzothiazole moiety undergoes a twist of
solvent. However, in highly viscous media, the bond twisting
tor
nr
process is retarded significantly, resulting in a lower k
value and subsequently a higher ff value for the dye. Thus,
to understand the difference in the sensing activity of Ia
and Ib, time-resolved fluorescence measurements were car-
ried out in different solvents by using the fluorescence up-
conversion technique. Both dyes show multi-exponential
fluorescence decay behavior at their emission maxima in all
the solvents studied. Multi-exponential decay kinetics for
the barrierless bond twisting process is well known in the lit-
erature.[40,41] The representative fluorescence decays of Ia
and Ib in ethylene glycol are shown in Figure 3, whereas
Figure 3. Transient fluorescence decays of Ia (a, lex =410 nm, lem
=
490 nm) and Ib (c, lex =420 nm, lem =510 nm) in ethylene glycol solu-
tion. The instrument response function (IRF) is shown as the dotted
curve.
Figure 4. Optimized chemical structures of Ia (top) and Ib (bottom).
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Fluorescence Modulation in an Amyloid Fibril Sensor
FULL PAPER
about 408 for Ia and about 368 for Ib with respect to the
plane of anilino moiety. Vertical transition energies were
also calculated for the optimized geometry for these two
molecules and were found to be approximately 3.055 eV
(406 nm) and approximately 2.893 eV (428 nm) for Ia and
Ib, respectively. These values correspond reasonably well
with the experimentally observed absorption maxima
(412 nm and 440 nm for Ia and Ib, respectively).
To understand how the dihedral angle (qB) between the
benzothiazole and the anilino moieties affect the potential
energy surface of the two molecules, both ground-state and
excited-state potential energies were calculated for different
values of qB and are shown as in Figure 5. It is evident from
Figure 5 that for both dyes the locally excited state (with
compared to that in Ia. This lower energy gap between the
S1 and the S0 state also causes the nonradiative process to
be more efficient in Ib than in Ia. Thus, because of all
above factors, the fluorescence lifetime of Ib is expected to
be shorter than the one of Ia, which is also observed experi-
mentally. Therefore, the results of the quantum chemical
calculation are in good agreement with the observed experi-
mental results and support the role of the bond twisting pro-
ꢀ
cess around the central C C single bond to cause the ex-
tremely low fluorescence quantum yield of the ThT dyes
and its derivatives.
Conclusion
In conclusion, from detail photophysical studies of two
ꢀ
newly synthesized ThT derivatives it is inferred that the C
N bond twisting is not responsible for the sensing activity of
ꢀ
ThT and its derivatives. The twisting around the central C
C single bond between the benzothiazole and the anilino
moieties appears to be mainly responsible for the observed
fluorescence sensing activity of these dyes. Detailed viscosi-
ty-dependent studies show that dye Ib with a julolidine sub-
stituent acts as a better sensor than dye Ia. The better sens-
ing activity of Ib is found to be due to a faster nonradiative
decay rate of the excited state. The faster nonradiative pro-
cess in Ib is also predicted by the quantum chemical calcula-
tions.
~
*
Figure 5. Variation in the potential energy of Ia ( ) and Ib ( ) with the
dihedral angle qB in their ground state (S0) and the first excited state
(S1). The calculated energy is relative to the minimum of the ground-
state energy.
Experimental Section
Absorption and fluorescence spectral studies: Ground-state absorption
and steady-state fluorescence measurements were carried out with a Shi-
madzu (Japan, model UV-160A) spectrophotometer and Hitachi (Japan,
model 4010F) spectrofluorimeter, respectively. The fluorescence spectra
were corrected for the wavelength-dependent instrument responses by
measuring the spectrum of quinine sulfate and comparing it with the re-
ported standard spectrum.[42] The fluorescence quantum yield (ff) of Ia
and Ib were measured by comparison methods,[43] using coumarin-481 in
acetonitrile (ff =0.08[44]) as standard.
qB =408 for Ia and 368 for Ib), produced due to photoexci-
tation, does not correspond to the energetically minimum in
the potential energy surface. Thus, both molecules undergo
ꢀ
bond twisting around the C C single bond between the ben-
zothiazole and the anilino moiety to attain a qB value of
ꢁ908 in their excited state to attain the minimum in the po-
tential energy surface. This feature is qualitatively very simi-
lar to that obtained for the ThT molecules in the gas
phase.[28]
Time-resolved fluorescence studies: Time-resolved emission measure-
ments were carried out with a femtosecond Ti–sapphire laser-based fluo-
rescence upconversion instrument that has been described earlier.[45]
Briefly, a second harmonic laser pulse of Ti–sapphire laser (ꢁ50 fs at full
width at half maximum (FWHM)) was used for the excitation of the
sample. The fluorescence light from the sample was collected and focused
onto a 0.5 mm thick b-barium borate (BBO) crystal by using a pair of
parabolic mirrors. The fundamental laser pulse (gate pulse), after passing
through an optical delay rail, was mixed with the fluorescence light into
the BBO crystal to generate the sum frequency light, which was detected
by using a photon counter after passing through a double monochroma-
tor. To obtain the fluorescence decay of the sample, the sum frequency
light was detected as a function of the delay time between the gate and
the excitation pulse. The instrument response function (IRF) of the pres-
ent setup were measured through cross correlation between the gate
pulse and second harmonic excitation pulse and found to be 230 fs.
Decay traces were monitored at the emission maximum of the respective
dye, that is, 490 nm for Ia and 510 nm for Ib. It is to be mentioned that
the decay traces for both dyes are seen to be independent of the excita-
tion wavelength.
Although the qualitative nature of the potential energy
surfaces of Ia and Ib are very similar, there are distinct
quantitative differences between them, which can explain
the observed differences in the excited-state dynamics of
these two molecules. It is to be noted from Figure 5 that the
energy stabilization due to a bond twisting process in the ex-
cited state is relatively higher for Ib than for Ia, suggesting
that the twisting process occurs faster in the former. Further,
the excited-state potential energy surface in the region of
qB =40–908 is relatively steeper for Ib than that for Ia. This
feature also suggests a faster bond twisting process in Ib
than in Ia. In addition, it is also evident from the Figure 5
that the energy gap between the S1 and S0 states at the twist-
ed configuration (i.e., qB =908) is relatively small for Ib as
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S. Nath et al.
Quantum-chemical calculation: The ground-state geometry optimization
of Ia and Ib was performed by using the density functional theory
(DFT). Beckeꢀs three parameter hybrid exchange function with the Lee–
Yang–Parr gradient corrected correlated functional (B3LYP)[46, 47] was
CDCl3): d=3.05 (s, 6H), 6.74 (d, J=9.0 Hz, 2H), 7.25–7.34 (m, 1H),
7.40–7.48 (m, 1H), 7.84 (d, J=7.8 Hz, 1H), 7.95–8.01 ppm (m, 3H); 3b:
1H NMR (200 MHz, CDCl3): d=1.92–2.04 (m, 4H), 2.81 (t, J=6.2 Hz,
4H), 3.26 (t, J=5.8 Hz, 4H), 7.26–7.32 (m, 1H), 7.39–7.46 (m, 1H), 7.59
(s, 2H), 8.03 (d, J=8.0 Hz, 1H), 7.79 ppm (d, J=8.0 Hz, 1H).
used in conjunction with the 6-311+GACTHNUGRTNEUNG(d,p) basis set as implemented in
the Gaussian 03 software package.[48] The conductor-like polarizable con-
tinuum model (CPCM)[49] was used to incorporate the effect of the bulk
Synthesis of the dyes Ia and Ib: A mixture of 3a or 3b (each 0.2 g) and
MeI (2 equiv) was heated at 1208C in a sealed tube for 4 h. The mixture
was concentrated in vacuum, and the crude products were washed succes-
sively with acetone (3 mL) and Et2O (3 mL) and recrystallized from etha-
nol to give the desired products Ia (0.135 g, 63%) and Ib (0.125 g, 60%).
Ia: 1H NMR (200 MHz, [D6]DMSO): d=3.10 (s, 6H), 4.22 (s, 3H), 6.97
(d, J=8.4 Hz, 2H), 7.75–7.83 (m, 4H), 8.28–8.38 ppm (m, 2H); Ib:
1H NMR (200 MHz, [D6]DMSO): d=1.86–1.91 (m, 4H), 2.72–2.78 (m,
4H), 3.54 (m, 4H), 4.18 (s, 3H), 7.38 (s, 2H), 7.64–7.83 (m, 2H), 8.09–
8.12 (m, 1H), 8.26–8.30 ppm (m, 1H).
solvent (water). TDDFT method by using B3LYP/6–311+ GACTHNUTRGNEUG(N d,p) basis
set was used to calculate the energy in the excited state of the two dyes
at different dihedral angle. The energy of the first excited singlet state
(S1) was determined as the sum of the ground state (S0) energy and the
transition energy. The energy (expressed in eV) is relative to the mini-
mum of the ground-state energy.
Synthesis of Ia and Ib: The syntheses of Ia and Ib are outlined in
Scheme 2. All anhydrous reactions were carried out under an argon at-
mosphere by using freshly dried solvents. The organic extracts were dried
over anhydrous Na2SO4. The 1H NMR (200 MHz) spectra were recorded
with a Bruker AC-200 spectrometer.
Acknowledgements
The authors wish to thank Dr. S. K. Sarkar, Head, Radiation & Photo-
chemistry Division, for his constant encouragement and support during
this work and Prof. A. Dutta, Indian Institute of Technology, Bombay,
for his help in the quantum chemical calculations. One of the authors
(A.S.) is thankful to the Indian Science Academies (IASC, INSA &
NASI) for the summer research fellowship.
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9262
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Received: October 27, 2009
Revised: March 2, 2010
Published online: June 25, 2010
Chem. Eur. J. 2010, 16, 9257 – 9263
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9263